Grant Application for 2019-2021

ISP Uppsala Universitet Deadline Box 549 IPPS 15 August 2018 SE-751 21 Uppsala, Sweden Fax +46184713495 IPICS 2 September2018 [email protected]

I Grant application for 201I 9-2021 Research Groups and Scientific Networks Submit this application and enclosures by email attachment with a scanned/photographed copy of the first signed page. Read the separate document Guidelines for Enclosures and Budget for more information. Hover your pointer over theblueunderlinedwordsin this document for specific instructions.NOTE! ISP manages personal data (with care and only as long as necessary) provided on this form and in enclosures in order to honor the agreements with you and with Sida.

Program Activity code(if settled) Chemistry X Other ☐ ANRAP Mathematics ☐ Physics ☐ Applicant(Research group leader/Network coordinator: title, given name, family name) EMERITUS PROFESSOR MOHAMMED MOSIHUZZAMAN Deputy: Address Department/unit: ANRAP Secretariat University/institute: Bangladesh University of Health Sciences(BUHS), Room No:C-14 Street (visiting address): Darussalam, Mirpur P.O Box number: Post/zip code: Dhaka-1216 City:Dhaka Country: Bangladesh E-mail address(es):[email protected];[email protected] Website: www.anrap.org Telephone and telefax Office Mobile Fax 88-02-8035501-06 8801755654160 (Ext 1312) Name of Research Group/Network ASIAN NETWORK OF RESEARCH ON ANTIDIABETIC PLANTS (ANRAP) City: Approved by the Department: ………………………………………………………/ Date: Signature by Head of Department/Name in printing: Summary of budget request (SEK) 2019 2020 2021 Total Equipment/spare parts/service 18,000 29,000 58,000 105,000 Consumables/literature/field work 68,000 76,000 76,000 220,000 Conferences/workshops 231,000 170,000 229,000 630,000 Exchange visits by cooperating scientists 10,000 10,000 8,000 28,000 Fellowships for training: 70,000 70,000 75,000 215,000 Support to students 10,000 20,000 10,000 40,000 Costs for audit and RG meeting 10,000 10,000 30,000 50,000 Network (only) administration costs 53,000 65,000 64,000 182,000 Total 470,000 450,000 550,000 1,470,000 The following enclosures are submitted Yes / No City: Dhaka 1) Research plan/network program Yes Date: 01 August 2018 2) Applicant’s/Deputy’s CV Yes 3) Publications/Theses/Abstracts Yes 4) Application for improving gender balance 5) Fellowship application(s) Applicant’s signature 6) Other (Specify:…………………………………………...... ) Yes

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1. Summary of proposed research/network activity (Research plan/Network program) (Full details should be given in Enclosure 1Please, carefully read and follow guidelines)

Give a summary (half a page) of the research plan/network program by briefly describing the planned activity and its justification, and for scientific research clarify the justification in relation to previous research/literature,postulate the hypotheses to be tested/research questions to be addressed, and indicate which methods will be used.Please note that this is a summary of Enclosure 1, Sections a – c.

ANRAP was launched in Dhaka in 1994 as an outcome of collaborative research between BIRDEM and Dhaka University (DU) on antidiabetic plants. Since ANRAP was initiated in Dhaka, research activities on antidiabetic plants in the laboratories of BIRDEM and DU and now atBUHS became the focal point laboratory of the network. ANRAP acted as a planning coordinator and supervising organization to integrate the activities of various laboratories in the Asian Region.Through the course of time a number of important laboratories are now working synchronically under the umbrella of ANRAP. In the coming three years ANRAP will continue with its present activities of training Research Fellows from home and abroad, conduct National, Regional and International (every 3 years) Seminers and hold the hands-on-training workshop every year. Considering the practical use of herbal medicine specially in the poorer third world countries ANRAP has expanded its activityto scientifically evaluate antidiabetic herbal medicine (HM).HM, used for the treatment of other noncommunicable diseases will also be studied. In addition, in the next three years ANRAP will particularly give emphasis on the development of interlaboratory cooperation (NetHerb) for testing of herbal medicines. With this end in view ANRAP organized an International workshop on networking of ”Afro-Asian laboratories working on herbal medicine” in Dhaka, Bangladesh on 29-30 October, 2016 followed by a second meeting with representatives from laboratories in the Afro-Asian region in Kandy, Sri Lanka in January 2018.After detailed discussionsfour laboratories agreed to collaborate. An MOU will be signed among the collaborating laboratories in the upcoming 9th ANRAP International seminar that will be held in Karachi, Pakistan on 25-27 January, 2019. So the next target is to start in-depth research on herbal medicine incorporating Chemistry, Biochemistry, Pharmacology and ultimately preclinical toxicological studies and human trial.

2. Give an overview of objectives, planned outputs and expected outcomes Provide a summary of objectives, planned outputs and expected outcomes (and, if found convenient, formalized in the Logical Framework Matrix).Provide your answers under the below headings. This is a summary of Enclosure 1, Section d.

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Overall objective (impact) and relevance of the activity:

ANRAP is a network of natural product scientists and participating laboratories working on or are planning to work on antidiabetic plant materials.During the last three years, research capabilities of Bangladesh University of Health Sciences (BUHS) has been strengthened. Chemistry and Pharmacology laboratories of BUHSare capable of conducting research on antidiabetic HM as well as on HM for other noncommunicable diseases. ANRAP has the following overall objectives: • Compilation of information on antidiabetic plants and herbal medicines reported in scientific literature and including those from producers,traditional healers, formal practitioners and users. • Conduct research on the chemical constituents and biological activities to assess the safety, efficacy and standardization of antidiabetic plants and herbal medicines used by millions of people across the world, and bring them within acceptable limits. • Isolation, characterization and testing of biological activity of pure compounds or fractions. • Optimize, coordinate and utilize the facilities existing in various laboratories for the purpose of fruitful research on antidiabetic plant materials and herbal medicine used for the management of diabetes and other non-communicable diseases prevalent in the region. Specific objectives for the granting period:

• Training of young scientists and Fellowsincluding PhD studentsin the field of antidiabetic plant and HM research. • Holding of ANRAP Regional Workshop on Chemical Studies and Bioassay of antidiabetic plant materials (Participants will be from Bangladesh, Nepal, India, Pakistan, Sri Lanka, Malayasia and Mauritius) • Developing a network of herbal medicine(NetHerb) with representatives from different participating laboratories. • Evaluation of safety and efficacy of some antidiabetic HM and HM used for other diseases like hepatitis, hypertension and renal disorders.

• Conducting preclinical and toxicological studies and clinical trial of some antidiabetic and other HM.

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• Arrange exchange of visits of scientists working in the field by arranging National, Regional and International (every 3 years) seminars on antidiabetic plants &herbal medicines and those used for other diseases. Expected outcomes of the activity:

Scientific Results:

• Masters and PhD thesis will be produced. Papers will bepublished in national and international journals. Patents on new scientifically evaluated herbal medicine can be produced. • More collaborative research linkage will be established. • Safety and efficacy of antidiabetic and other herbal medicine for other diseases will be evaluated. • Development of new drugs from plant and commercial herbal medicine through collaboration in national and international level. • An arrangement has been done between GK Pharma (Unani) Ltd to carry out chemical & biological studies on hepatotoxic model animals with hepatoprotective herbal drug (Holy Liv) produced in Bangladesh. Graduations: A number of post graduate students will be trained up. They will be working to produce their thesis and publish papers.

Trained young scientists including post graduate students will be produced to work in the field of plant & herbal medicine research.

Dissemination(publications/conference contributions,etc):

• Training of young scientists and post graduate students. • National, Regional and International Workshops/Seminars on Chemistryand Biology of plant materials and herbal medicine for the management & treatment of diabetes and other diseases. • Publications of scientific articles in peer reviewed journals. • Conference reports/ Published Proceedings of Conferences.

ScientificExchange A good number of exchange visits within the region and organizations of different national, regional and international Seminars & Conferences will enhance collaborative research and exchange of knowledge and skill of the researchers working in this field.

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Interactions with government and society (outreach):

ANRAP has very good interaction with the Ministry of Science & Technology, Bangladesh Government and also with society in general. Interactions with traditional practioners, herbal medicine producers and medical professionals through its seminars and training workshops has widened the horizon of ANRAP to the society to a greater extent. Scientists and Fellows and students (post-graduate ) associated with ANRAP can apply for different grants through the Ministry of Scientific & Technology or other organizers and can get grant to work with antidiabetic plants/herbal medicine. For example: i) A grant of BDT 3 lac was received from the Ministry of Scientific & Technology in the year 2017-2018 for evaluation of antidiabetic herbal drugs. Project title is”chemical analysis of some active antidiabetic herbal preparations available in Bangladesh.” ii) A grant of BDT 5 lac was received from Bangladesh Medical and Research Council in the year 2017-2018 for evaluation of antidiabetic plant. Project title is ”Isolation, Chemical characterization and elucidation of mechanism of action of antidiabetic activity of different extracts and fractions of Psidium guajava on nSTZ-induced T2DM rats”. iii) A book in Bangla (local language) incorporating biological and chemical tests on 64 plants tested at BIRDEM & DU including there form of use, has been written. Thousands of copies of the book have already been sold. -Other:

As the chemistry and pharmacology labratory at BUHS hasimproved, service to herbal medicine producers can be provided. The process has already been started with one private company (herbal medicine producer, GK Pharma (Unani) Limited)who has agreed to work on antihepatic herbal drugs. We now wish to expandour activity with other companies producing herbal medicine.

a) Give a summary of the postgraduate education plan Give a maximum half apage summary of the proposed activity with emphasis on the education plan. This is a summary of Enclosure 1, Sectione.

ANRAP does not offer any postgraduate course. However, postgraduate students do attend the Fellowship programme and hands-on training workshops. A number of post graduate students will be trained up. They will be working to produce their thesis and publish papers.

b) Give a summary of the gender perspective on the research and education plans Summarize the information given on gender balance as well as strategies and measures to achieve gender balancegivenin Enclosure 1, section f. Provide your answers under the below headings. NOTE: If you are part of the mathematics or physics program and apply for

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extra gender funds (Enclosure 4) you don’t need to fill in this part, just refer to the separate gender application.

Comment on the current gender balance in perspective of previous years efforts:

Current gender balance in the ANRAP Board is better compared to previous years. There are 3 female members out of 5 members in the ANRAP Board(about 60% female member). In principle, ANRAP is trying to maintain a gender balance in awarding the Fellowship, selecting workshop participants and supporting students and young scientists from home and abroad. However, during the last 3 years female participation in the workshop and Fellowship programs were less than expected. In the context of Bangladesh perspectivenumber of women scientists are still limited. But recently the trend is changing and more & more female participants are attending Seminars & Workshops. Efforts are being made to have more female members in all activities.ANRAP will circulate its seminar & workshop brochures to more women scientists and young female postgraduate students in the future. It is interesting to note that in the 7th ANRAP workshop (2017) there were 12 female participants out of 19. The ratio of M:F was 7:12 which we think a good progress towards gender balance. In the upcoming 8th ANRAP workshop (from 16th to 20 September 2018) we are also expecting participation of more female participants. This is for the first time ANRAP is organizing accomodation for female participants from outside Dhaka city to facilitate their participation in the workshop.

Reasons for current gender distribution:

Bangladesh has relatively less number of female scientists and technical personnel, however this current trend is changing since female students are being enrolled in increasing number day by day in science faculties of universities and medical courses.

Planned strategy to achieve gender balance (if not already the case):

ANRAP gives priority to female applicants in its activities and hopefully ANRAP will get more and more applications from female candidates which will achieve gender balance in near future.

Concrete measures to achieve gender balance:

ANRAP specifically sends circulars of its activities to female faculty members, post graduate students, scientists and technical personnel working in educational and research organizations.

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c) Provide a number of keywords

Keywords: ANRAP, diabetes, research, antidiabetic plants, herbal medicine, noncommunicable deseases.

d) Give an overview (half a page) on the background and the development of the research group/network from the start of the ISP collaboration until present time. Please, include improvements/challenges in capacity building of higher education and research, and the needs to further develop (Please note that this is a summary of Enclosure 1, Section k)

Considering the importance of plant medicine in the treatment of diabetes mellitus (DM) fast increasing in the developing countries, BIRDEM (biological testing) and Dhaka University (chemical studies) started collaborating on the research of anti-diabetic plants in 1989. The collaboration was supported by ISP (IPICS). Realizing the interest in the field, the collaborating scientists organized a national seminar in 1992 and an International Seminar in January 1994. It was proposed at the discussion meeting of the International Seminar that a Network can be formed involving Asian laboratories for coordinated research on anti-diabetic plants. Accordingly, the Dhaka group presented a proposal for the formation of ”Asian Network of Research on Anti-diabetic Research (ANRAP) at the ACGC (Asian Coordinating Group in Chemistry) meeting in Melaka, Malaysia, in June 1994 where representatives of UNESCO, IFS, IPICS and FACS (Federation of Asian Chemical Societies) were present. The proposal was overwhelmly accepted and the Dhaka group launched ANRAP in July 1994 in Dhaka with full support of IPICS. Since ANRAP was initiated in Dhaka, research activities on antidiabetic plants in the laboratories of BIRDEM and DU and now at BUHS became the focal point laboratory of the network. ANRAP has been acting as a planning coordinator and supervising organization to integrate the activities of various laboratories in the Asian Region. ANRAP organizes National, Regional and International Seminars, arranges & supports Fellows in the region, and conducts hands-on Workshops on the biology & chemistry of anti-diabetic plants. Through the course of time ANRAP has trained a large number of Fellows, young scientists and post-graduate students while a number of important laboratories are now working synchronically under the umbrella of ANRAP. In the coming years ANRAP will give emphasis on Herbal Medicine (HM) used not only for diabetes but for other non-communicable diseases like hepatitis, hypertension and renal disorders. In the next three years ANRAP will pursue on the development of interlaboratory cooperation (NetHerb) to conduct research on the chemical and biological activities of herbal medicines for assessing the acceptable safety, efficacy and standardization.

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3. Specifications of costs 2019-2021 (Please, carefully read and follow guidelines for Budget, and, if applicable, conditions and instructions for Research Groups’ overhead costs)

3a) Specification of costs in 2019

Specification of Costs in 2019 Costs (SEK) Total (SEK) Equipment/spare parts/service Spare parts and instrument servicing & maintenance 8,000 18,000 Digital Balance 10,000 Consumables/literature/field work Books & Journals 7,000 Updating the website 8,000 68,000 Database for antidiabetic single plants and herbal medicine 5,000 Charges for publication results 8,000 Chemical, reagents and testing kits 40,000 Conferences/workshops a) To be visited i) One person to EASD in Barcelona, Spain 20,000 ii) Attendance to Regional Workshop 10,000 68,000 iii) One person to IDF in Busan, Korea 18,000 iv) One person to ADA Conference in USA 20,000 b) To be arranged i) International Seminar in Pakistan 68,000 ii) ANRAP Bangladesh National Seminar 20,000 163,000 iii)Workshop on development of Diabetic model rats and use them for evaluation of plant materials 25,000 iv) Afro-Asian Symposium with representatives of laboratories participating 50,000 in the proposal for the formation of NetHerb Exchange visits by cooperating scientists 10,000 10,000 Two to Three visits within the region

Fellowships for training/exchange 30,000 a) Local 70,000 2-3 Persons (Partial support) 40,000 b) Among participating laboratories Support to students A number of post graduate students will be trained up. They will be working to 10,000 10,000 produce their thesis and publish papers. Activities for improving gender balance ANRAP does not have any specific activities to improve ------gender balance but ANRAP gives priority to female applicants in its activities. Network administrative costs a. Board Meeting 20,000 b. Secretarial staff 10,000 53,000 c. Office stationeries and computer accessories 8,000 d. Conveyance & Communications 8,000 e. Contingency & Miscellaneous 7,000 Auditcosts: ANRAP accounts is being audited by internationally 10,000 10,000 certified Audit Firm TOTAL 470,000

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3b) Specifications of costs in 2020

Specification of Costs in 2020 Costs (SEK) Total (SEK) Equipment/spare parts/service Spare parts and instrument servicing & maintenance 9,000 29,000 Centrifuge Machine 20,000 Consumables/literature/field work Books & Journals 9,000 Updating the website 5,000 76,000 Database for antidiabetic single plants and herbal medicine 5,000 Charges for publication results 7,000 Chemical, reagents and testing kits 50,000 Conferences/workshops a) To be visited i) One person to EASD in Vienna, Austria 20,000 50,000 ii) Attendance to Regional Workshop 10,000 iii) One person to ADA Conference in USA 20,000 b) To be arranged i) Regional Seminar in Malaysia 75,000 ii) ANRAP Bangladesh National Seminar 20,000 120,000 iii)Workshop on development of Diabetic model 25,000 rats and use them for evaluation of plant materials Exchange visits by cooperating scientists 10,000 10,000 Two to Three visits within the region Fellowships for training/exchange a) Local 30,000 70,000 2-3 Persons (Partial support) b) Among participating laboratories 40,000 Support to students A number of post graduate students will be trained up. They will be working to 20,000 20,000 produce their thesis and publish papers. Activities for improving gender balance ANRAP does not have any specific activities to improve ------gender balance but ANRAP gives priority to female applicants in its activities. Network administrative costs a. Board Meeting 30,000 b. Secretarial staff 10,000 65,000 c. Office stationeries and computer accessories 10,000 d. Conveyance & Communications 8,000 e. Contingency & Miscellaneous 7,000 Auditcosts: ANRAP accounts is being audited by internationally 10,000 10,000 certified Audit Firm TOTAL 450,000 \ (Insert more rows as needed, or just write below the table)

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3c) Specifications of costs in 2021

Specification of Costs in 2021 Costs (SEK) Total (SEK) Equipment/spare parts/service Spare parts and instrument servicing & maintenance 8,000 58,000 Rotavapor 50,000 Consumables/literature/field work Books & Journals 9,000 Updating the website 4,000 Database for antidiabetic single plants and herbal medicine 5,000 76,000 Charges for publication results 8,000 Chemical, reagents and testing kits 50,000

Conferences/workshops a) To be visited i) One person to EASD 18,000 ii) Attendance to Regional Workshop 12,000 52,000 iii) One person to IDF Meeting 22,000 c) To be arranged i) ANRAP Bangladesh National Seminar 20,000 ii) Workshop on development of Diabetic model 25,000 177,000 rats and use them for evaluation of plant materials 50,000 v) Regional Seminar in India 82,000 vi) International Seminar

Exchange visits by cooperating scientists 8,000 8,000 Two to Three visits within the region Fellowships for training/exchange 25,000 a) Local 75,000 2-3 Persons (Partial support) 50,000 b) Among participating laboratories Support to students A number of post graduate students will be trained up. They will be working 10,000 10,000 to produce their thesis and publish papers. Activities for improving gender balance ANRAP does not have any specific activities to improve ------gender balance but ANRAP gives priority to female applicants in its activities. Network administrative costs a. Board Meeting 30,000 b. Secretarial staff 10,000 64,000 c. Office stationeries and computer accessories 9,000 d. Conveyance & Communications 8,000 e. Contingency & Miscellaneous 7,000 Auditcosts: ANRAP accounts is being audited by internationally 10,000 10,000 certified Audit Firm Reference group meeting participation costs Presentation of NITUB application, if invited for next 20,000 20,000 period, in reference group meeting TOTAL 550,000

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3d. Justification of budget items

List each budget item, and write a motivation to justify the need and the cost.

1) Equipment/spare parts, service:

Although ANRAP is a network of different laboratories, the Dhaka laboratory at BUHS is the focal point. The capacity of BUHS chemistry and pharmacology laboratories has been stregthenedand and is now able to meet up the requirment of participating Research Fellows and workshop participants. The expansion of the domain of ANRAP to include herbal medicine used for the management of diabetes and other non-communicable diseases will require addition of some small but essential instruments like digital balance, centrifuge machine etc. Chemicals, regents and testing kits will also be required to enchance the capability of the laboratories. Therefore, in the budget a distribution of instruments and spare parts are given. Other sophisticated instruments like NMR, Mass spectroscopy etc will be available in participating laboratories and also may be available in BUHS when International Center for Natural Product Research (ICNPR) will function . In the first year we have requesed for Digital Balance, Centrifuge Machine in the second year and a rotary evaporator in the third year.

2) Consumables / Literature / Field work One of the objectives of ANRAP is to build a comprehensive source of information on antidiabetic plants,antidiabetic herbal medicine and herbal medicines for other noncommunicable diseases. Fast retrieval of information is essential to achieve this goal. Subscriptions of online indexing services are necessary for this purpose. An amount of SEK 28,000 will cover the cost of updating the Website and database for antidiabetic single plant and herbal medicine. For supporting ANRAP Fellows some chemicals and reagents are to be bought. SEK 40,000 has been kept for that purpose in every year. Additional cost is met from BUHS.

Conferences / Workshops Organization of Local, Regional and International Seminars on antidiabetic plants is a continuous process for ANRAP. Three such Seminars are planned in three years. i) ANRAP Bangladesh National Seminar ANRAP Bangladesh National Seminar is planned to be held in July of 2019 in Dhaka, Bangladesh. An amount of SEK 20,000 has been reserved from the ANRAP Budget. Similar amount will be required in each year. ii) The 9th ANRAP International Seminar The 9th ANRAP International Seminar has been planned in Karachi, Pakistan on 25-27 January 2019. A An amount of SEK 68,000 has been allocated for the purpose. The 10thANRAP International Seminar will be held in 2021 at a venue to be decided in the general meeting in Karachi in January 2019. So an amount of SEK 82,000 has been reserved for this purpose.

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Deadline 2018 – see isp.uu.se/documents iii) Hands-on Training Workshop The most difficult part of research on antidiabetic plant materials is the lack of skilled manpower for developing appropriate diabetic animal models, primarily rats and mice. Over the years BIRDEM has excelled in this task and transferred knowledge and practical procedure to HEJ Research Institute of Chemistry, Karachi University. There is a growing demand locally and regionally to organize hands-on training programme for the development of diabetic rat (and mice) models. Hands-on Training Workshop on ‘development of diabetic model rats and their use for evaluation of plants materials’ have been organized in Dhaka, in 2010, 2011, 2012, 2013 2015, 2016-2018. BIRDEM and BUHS were the venue for all the workshops except 2015-2018 when the workshop was held exclusively in BUHS. A total number of 94 scientists have received the training so far. An affordable registration fee (SEK 530) was charged to partly compensate the costly training programme. An amount of SEK 25,000 has been reserved from the ANRAP Budget of 2019 for organizing the 9th Workshop on the same subject in BUHS. iv) Meeting for NetHerb

As an initiation process ANRAP organized an International workshop on networking of ”Afro- Asian laboratories working on herbal medicine” in Dhaka, Bangladesh on 29-30 October, 2016. Representatives from 8 laboratories (Asia and Africa) participated in that workshop. After that ANRAP has aranged the 2nd NetHerb meeting that was held in Kandy, SriLanka on 18th Januay, 2018 and as a result four laboratories agreed to collaborate. An MOU will be signed among the collaborative laboratories in the upcoming 9th ANRAP International seminar that will be held in Karachi, Pakistan on 25-27 January, 2019. In the next 2 years we plan to have a meeting in Dhaka where representatives from of at least ten laboratories will be invited. An amount of SEK 50,000 has been reserved from the ANRAP Budget .

Participation at the EASD, ADA and IDF meetings

The annual EASD (European Association for the Study of Diabetes) meeting is a highly reputed one in the field of diabetes and it will be held in Barcelona, Spain in 2019. Some of the works from the ANRAP supported labs will be presented in the meeting. The amount for one visit has been estimated to be SEK 20,000. Similar amounts will be required in 2020 & 2021. Similarly, participation in the IDF organized World Diabetes Congress, is very important for gaining up-to-date knowledge about the worldwide progress in diabetes research. One person will attend this conference to he held in Korea in 2019.The amount for the visit has been estimated to be SEK 18,000. The annual ADA (American Diabetic Association) is also a prestigous meeting dealing with endocrinology, metabolism and diabetes. We also wish to send abstracts from the ANRAP supported labs for presentation there. In 2019, ADA meeting will be held in Sunfrancisco, California. The amount for one visit has been estimated to be SEK 20,000.

4) Fellowships for training / exchange visits

From the very beginning ANRAP is providing Fellowship specially to postgraduate students who are working on antidiabetic plants and related areas. Training of younger scientists and postgraduate

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Deadline 2018 – see isp.uu.se/documents students are of great importance in achieving the objectives of ANRAP in enhancing research capacity and regional cooperation. Since 1998 ANRAP has focused its activities on Fellowship programmes to facilitate collaboration among participating laboratories. Every year 2-3 ANRAP Fellowships, both local and regional are planned. An amount of SEK 70,000 has been reserved from the ANRAP Budget for each year.

Exchange visit

Every year one or two senior level scientists will be expected to visit collaborating laboratories in the region for conducting training and/or for discussing research cooperation. A small amount of fund (SEK 10,000) is kept for covering the cost of the visit.

5) Network: administrative costs

Board meeting

ANRAP normally holds a Board meeting every year. The meeting also gives the opportunity of discussions about mutual research collaboration. To cover the travel expenses of the Board members from Dhaka, Karachi, Kuala Lumpur and Kolkata a sum of SEK 20,000-30,000 for each meeting.

Other administrative costs

ANRAP shares cost with BUHS for an Office Secretary. Office stationary, computer accessories, local travel, internet service charge and contingency & miscellaneous, Audit fee expenses are the other administrative costs. Altogether only a sum of SEK 30,000 is proposed in the budget for each year.

Audit costs

ANRAP accounts is being audited every year by an internationally certified Audit Firm. A small budget has been allocated for this purpose.

Reference group meeting participation costs

ANRAP hopes very much that its grant application to ISP for the next three years (2019 -2021) will be successful. ANRAP also expects that ISP will invite grant application from ANRAP in the year 2021 and with this anticipation a budget has been allocated to present the application in Reference Group Meeting.

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4. Staff and students in the group/network

List staff and students planned to be actively involved in the proposed activity.

a) List staff in the group/network Staff members who also are active students are to be listed ONLY under students. Networks list members of Board on Management Committee, node coordinators etc. Note that support staff (Supp.) comprises all non-academic and undergraduate staff engaged, e.g. laboratory assistants, technicians, secretaries, etc.

Gender Given name, family name Position held Staff qualifications F/M / Function1 PhD Other Supp. Ac. M Emeritus Prof M Mosihuzzaman Chairman PhD M Prof Begum Rokeya General Secretary PhD M Prof M Iqbal Choudhary Member PhD F Prof Khozirah Shaari Member PhD F Prof Banasri Hazra Member PhD F Prof Nilufar Nahar Working within the Group PhD M Prof Liaquat Ali Working within the Group PhD M Dr Md Shahinul Haque Khan Working within the Group PhD Dr Md Ranzu Ahmed Working within the Group PhD F Ms Nahida Islam Khan Office Secretary MSS

b) List students in the group/network (If alreay known) Students to be included should be those that benefit directly of the ISP support (fellowships) or indirectly by using consumables and equipment provided through the ISP support.

For each student, provide the following information: 1) Gender (female/male; F/M) 2) Full name 3) Target degree (PhD/MPhil/MSc) 4) Starting year on current degree study 5) Tentative title of thesis / subject 6) Expected year of graduation 7) Whether staff member or not 8) a) Local (L) or Sandwich (S)student;b) Direct or Indirect benefit of ISP support (D/I)? 1 2 3 4 5 6 7 8a 8b F/ Name Target Start Thesis/Subject Grad Staff L/S D/I M degree year year (Y/N ) F Amrita Bhowmik, PhD 2012 Association of TCF7L2, 2018- yes Local direct [email protected] KCNJ11 and SLC22A1 2019 benefit om genes polymorphism in type 2 diabetes mellitus in Bangladeshi population M Shamim Gazi MS 2017 Effect of selective Janury no Local direct shamimgazibge@gmail probiotics in 2018 benefit .com streptozotocin induced type 2 diabetics rats

1 Indicate deputy leader/coordinator, if applicable

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M MdMahabub Ali, PhD 2016 Chemical Composition of 2018- no Local direct [email protected] Juteleavesand its 2019 benefit m development as Herbal Tea F Arunima Biswas MS 2018 Effect of Ocimum sanctun 2019 no Local direct on renal glucose benefit absorption in nSTZT2DM model rats F Umma Kulsum Lucky MS 2018 Effect of Aegle marmelos 2019 no local direct on deodumal glucose benefit absorption in nSTZT2DM model rats F Madumita MPhil 2018 Glycemic and Insulin 2019 yes local direct response to major brands benefit of Bangladeshi honey among nondiabetic and diabetic M Md Shahajahan MS 2018 Effect of honey on serum 2019 no local direct paraoxonase1arylesterase benefit activity in expermental animal F Rasheda Akter MSc 2018 Evaluation of antidiabetic 2019 no local direct effect of plants from benefit chittagong hill tracts M Saiful Islam MSc 2018 Evaluation of antidiabetic 2019 no local direct effect of plants from benefit chittagong hill tracts

F Tania sharmin MSc 2018 Evaluation of antidiabetic 2019 no local direct effect of plants from benefit chittagong hill tracts

5. Scientific contacts/cooperation

Indicate which contacts with scientists working in the field of the proposed activities are established or planned(at your own university/institute, nationally, regionally, or internationally).The present and expected benefits of these contacts should be further described in the Research description (Enclosure 1).

a) List already established scientific contacts/collaborationthat were engaged in active collaboration with your research group/network in 2016-2018.

For each collaborator, provide the following: a) Gender (female/male; F/M) b) Title c) Full name d) Affiliation e) Country

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Gender Title Name Affiliation Country F/M M Prof Barry N Noller The University of Queensland Australia

F Prof Sadhana Amatya Dept of Pharmacy & Nepal Pharmacology, Tribhuvan University M Prof Bishnu Prasad Sharma GovtAyurvedic College & India Hospital, Guawahati, Assam

F Prof Khozirah Shaari Institute of Bioscience, Malaysia University Putra, Malaysia

F Prof Nor Hadiani Ismail Atta-ur-Rahman Institute for Malaysia natural Products Discovery, University Technology Mara

F Prof Banasri Hazra Research Scientist , Dept of India Pharmaceuticals Technology, Jadavpur University

M Prof Lalith Jayasinghe Institute of Fundamental Studies Sri Lanka

M Prof Vijaya Kumar Institute of Fundamental Sri Lanka Studies

F Prof Bilge Sener Gazi University, Faculty of Turkey Pharmacy, Dept of Pharmacognosy

F Dr Thushari Bandara University of Ruhuna Sri Lanka

a) List scientific contacts for collaboration you intend to establish with your research group/network in 2019-2021.

For each collaborator, provide the following: 1) Gender (female/male; F/M) 2) Title 3) Full name 4) Affiliation 5) Country

Gender Title Name Affiliation Country F/M M Hakim Md Yousuf Haroon Hamdard Laboratories Bangladesh Bhuiyan (Waqf) Ltd

M Mr Aminul Islam Hamdard Laboratories Bangladesh (Waqf) Ltd

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M Prof M A Rashid University of Dhaka Bangladesh

M Prof SiteshBachchar University of Dhaka Bangladesh

F Dr Ismet Ara Jahan BCSIR, Dhaka Bangladesh

M Dr Mohammed Mostafa BCSIR, Chittagong Bangladesh

F Prof Savitri Kumar University of Peradeniya, Sri Lanka Kandy

F Prof Bilge Sener Gazi University, Ankara Turkey

6. Other funding received (besides from ISP) 2016-2018, and available and foreseen funding for 2019-2021

Specify other funding obtained in 2016-2018, and other foreseen sources of funding for 2019-2021, from national as well as from other sources. List each source on a separate row, and give the amount for each year on separate rows. Estimate the amount in USD (currency conversion can be done at www.xe.com).

a) Other funding (besides from ISP) obtained in 2016-2018 Source Grant period/ Total Grant, mmyy-mmyy USD Sale of ANRAP Bengali Book 2016 593 USD Registration fee(Workshop and Seminar) 2016 1256 USD Registration fee(Workshop and Seminar) 2017 1197 USD Research Grant 2017 593 USD Bangladesh Medical Research Council (BMRC) 2018 5966 USD (Insert more rows as needed, or just write below the table)

b) Other available or foreseen funding for 2019-2021 Source Grant period/ lGrant, mmyy-mmyy USD Drug International 2019 5966 USD GK Pharma (Unani) Ltd 2019 3000 USD Federation of Asian Chemical Societies (FACS) 2019- 2020 1000 USD (Insert more rows as needed, or just write below the table)

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7. Main equipment available

a) List relevant facilities and infrastructure available to the group. For equipment, give type and name of the manufacture of main items and other articles. Start with your most important equipment. Please, note if the equipment is not functioning properly and why.

Equipment type, model, and name of manufacturer Year Fully functional, yes/no acquired If no, indicate why Centrifuge machine 1995 not functioning properly Gut perfusion set 2004 yes HPLC Rotavapor 2014 yes Refrigerator 2015 yes Freeze dryer 2015 yes Autoclave 2016 yes Elisa Reader 2018 yes (Insert more rows as needed, or just write below the table)

b) List below main equipment needed in the near future, with justification.

Our Digital Balance is not working. So, the equipment is crucial for us becauseit is being used very frequently in laboratories for every day research work.

8. Environmental impact

Information is required about measures taken to reduce environmental impacts following the Environmental Impact Assessment for ISP as submitted to Sida in August 2009 (available on request).

a) Indicate which of the following measures your group/network has already implemented to reduce negative environmental impact.

Does your group/organization: Yes No A strategy to reduce negative environmental impact caused by travelling and Yes transportation? The use e-meeting techniques? No A strategy to reduce the use of electric power? Yes Considering environment impact criteria in procurement? Yes Practicing sorting of waste categories for recycling? Yes A system for scrapping decommissioned equipment? Yes A management system for chemical and hazardous waste? Yes Internal discussion of how any negative environmental impact of your activities can Yes be reduced? Engagement in external activities – in research, dissemination and/or society Yes outreach – on how negative environmental impacts may be reduced?

b) Comment on the no-answers in the previous question, if any. What are the reasons why some measures have not been implemented? Are there plans to do it in the future? Are there structural obstacles, such as regulations or lack of

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regulation/technology, etc.? Also comment on the practicing of measures in general to reduce negative environmental impact.

9. Summary of results for previous agreement period

Provide a summary of the progress so far in the current agreement period (2016-2018), or latest years for new applicants.

a) Give a summary of the major achievements in the period, including results of scientific research activities. Place the achievements and results in relation to the objectives in the original proposal (if applicable). Not only major changes are interesting, but also small changes that may lead to larger changes over time. Summary: Fellowship Programme During the last two and half years 5fellows were supported by ANRAP. One fellow from India, four fellows (partial support) from BUHS have been supported by ANRAP.

Exchange Visits ANRAP sponsors /partially sponsors substantial number of exchange visits to promote regional collaboration. One scientist from India visited Bangladesh to discuss possible collaboration between Dhaka and India. Prof Peter Sundin and Dr Cecilia Öman from International Science Programme (ISP), Uppsala, Sweden visited Bangladesh University of Health Sciences (BUHS), Dhaka on 28 January 2016.

- Organization of Seminar at Home & Abroad i) The 11th and 12th ANRAP Bangladesh National Seminar were held at BUHS, respectively on 23July 2016 and 22July 2017. 1st Sri Lankan ANRAP Regional Seminar (ANRAPSL1) on “Herbal Approaches in Combating Diabetes and other Tropical Diseases” was held in National Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17-19 January 2018. The 13th ANRAP National Seminar will be held on 15 September 2018 in BUHS, Dhaka.

- - Workshop - The 6th ANRAP workshop on ‘Chemical studies and bioassay of antidiabetic plant materials’ was held in Research Division, BIRDEM during 24-28 July 2015. The 7th

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ANRAP workshop on ‘Chemical Studies and Bioassay of Antidiabetic Plant Materials’ was held in Bangladesh University of Health Sciences (BUHS), Mirpur, Dhaka, Bangladesh during 23-27 July2017. The 8th ANRAP Workshop will be held on 16-20 September 2018 in BUHS in Dhaka.

- ANRAP International Workshop - ANRAP Workshop on “Networking of Afro-Asian Laboratories Working on Herbal Medicine” was held in Hotel EmmaNuelle's Inn, Dhaka, Bangladesh on 29-30 October 2016.

- ANRAP Board Meeting The 23rd meetingof ANRAP Board were held in Dhaka, Bangladesh on 29October 2016. The 24th meeting of ANRAP Board was held in National Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka, on 18 January 2018

- A Book on the use of 62 antidiabetic plants tested in BIRDEM Published in Bengali

- ANRAP profile has been prepared and published.

b) Give a summary of the staff structure, students active and degrees awarding, dissemination, meetings, outreach, and visits to and from your group/network (scientific exchange) for the previous granting period.

Staff PhD or equivalent Other academic Technicians Female Male Female Male Female Male Number of staff 2018 Secretary (part time) 1 2

Students/degrees PhD or equivalent MSc/Mphil/Lic. BSc thesis Sandw Local Sandw Local students F M F M F M F M F M Number of students 2018 1 1 5 3 Number of degrees 2016-2018 1 6 3

Specification of awarded degrees 2016-June 2018

Gender, First name, Family name, Degree, Title of thesis (full abstract in Enclosure 3) (M), Md Ranzu Ahmed Choudhury,PhD, Chemical and Biological Studies on Antidiabetic Single Plants and Composite Herbal Preparations.

(M,) Md Asrafuzzaman MS, Anti-diabetic effect of oyster mushroom through activation of AMP-activated protein kinase (AMPK) pathway. (M), Md Nazmul Hasan, MSc, Effects of Enhydra Fluctuans Extract on Glycemic and Lipidemic Status and their Relation with Heavy Metal in Normal and Type-2 Diabetic Model Rats. (M), Ananta Kumar Das, Fazlay Rabbi, Polash Basak, BSc, “Studies on the antidiabetic activity of the alcoholic extracts of Glochidionvelutinum in streptozotocin induced type 2 diabetic model rats”.

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(M), Raj Kumar Bera, fellowship, “A short -term exposure on animal experimentation with animal model of diabetes using antidiabetic plant materials in the Pharmacology laboratory”. (M), Hafizur Rahman, MSc, National University (M), Sumon Kumar Roy, MS thesis, “Characterization of Streptozotocin induced Type 2 Diabetes Mellitus Long Evans Model Rats”. (M),Shamim Gazi, MS thesis, “Effect of selective probiotics in streptozotocin induced type 2 diabetes rats”.

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Publications in scientific journals 2016-June 2018

Author(s) (Year) Title. Journal, ISSN-number, Vol., Pages, DOI2 Was ISP Indicate how large a part of the work was performed at home University (<25%, 25-50%, support >50%) acknowledged (Yes/No) (Not filled by new applicants) Md Ranzu Ahmed, S. M. Z. H. Asna, M. S. H. Khan, Begum Rokeya, M. yes Mosihuzzaman and M. Abu Sayeed (2016). Microbial Contamination of Some Antidiabetic Herbal Preparations Available in Bangladesh.European Journal of Medicinal Plants, 13(1): 1-5.DOI: 10.9734/EJMP/2016/23556

Md Ranzu Ahmed, Tania Sultana, RayeshaRoutary, MD ShahinulHaque Khan, yes KhozirahShaari, M Abu Sayeed, M Mosihuzzaman and Begum Rokeya (2016). Chemistry and Antidiabetic Effects of PhlogacanthusthyrsiflorusNees Flowers. Natural Products Chemistry & Research. Ahmed et al., Nat Prod Chem Res 2016, 4:5. DOI:10.4172/2329-6836.1000229

Amrita Bhowmik, Mosihuzzaman M, Yearul Kabir and Begum Rokeya (2016). yes Glycemic, Insulinemic, Lipidemic and Antioxidant Status of nSTZ Rats after Chronic Administration of Cicerarietinum Extract. Metabolomics.ttp://dx.doi.org/10.4172/2153-0769.1000179

Shihab Uddin, Md. Mahmodul Islam, Md. Mynul Hassan, Amrita Bhowmik and yes Begum Rokeya (2016).Amaranthusviridis modulates anti-hyperglycemic pathways in hemi-diaphragm and improves glycogens is liver function in rats. Journal of Pharmacognosy and Phytotherapy.Vol. 8(10), pp.173-181. DOI: 10.5897/JPP2016.0406, http://www.academicjournals.org/JPP

Md Ranzu Ahmed, Ismet Ara Jahan, Khandoker Shahin Ahmed, M Abu Sayeed, yes Begum Rokeya, AK Azad Khan and M Mosihuzzaman (2016)“Antidiabetic activity of seven antidiabetic herbal preparations available in Bangladesh” Journal of Bangladesh Chemical Society, 28(1& 2): 1-8

Sonchita R. Mudi, Masfida Akhter, Subrata K Biswas, Mohammad A Muttalib, yes Subhagata Choudhury, Begum Rokeya, Liaquat Ali (2017). Effect of aqueous extract of Aeglemarmelos fruit and leaf on glycemic, insulinemic and lipidemic status of type 2 diabetic model rats.J Complement Integr Med. Mar 9; 14(2). . DOI: 10.1515/jcim-2016-0111.

2 Digital Object Identifier (www.doi.org) – to be provided if available

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Md. Asrafuzzaman. Yingnan Cao, Rizwana Afroz, Danielle Kamatoc, Susan Grayc, yes Peter J. Little. (2017). Animal models for assessing the impact of natural products on the aetiology and metabolic pathophysiology of Type 2 diabetes: Biomedicine & Pharmacotherapy 89 (2017) 1242–1251. Shahinul H Khan, Mahfuza Matin, Nasim Sultana, M Mosihuzzaman, Liaquat Ali, yes BegumRokeya, A K Azad Khan, Mohammad Shoeb and Nilufar Nahar.Hypoglycemicactivity of Scoparia dulcis linn extract and isolation of flavonoid glycosides: Journal of Bangladesh Chemical Society, Vol. 28(1&2), 36- 47, 2016.

Amrita Bhowmik, M. Mosihuzzaman, Yearul Kabir and Begum Rokeya, yes Antihyperglycemic Activity of Swertia chirata on nSTZ-T2DM Rats: A Chronic Study,Journal of Pharmaceutical Research International, 2018 - Volume 22 [Issue 5], Page 1-11 DOI : 10.9734/JPRI/2018/41878 M. Asrafuzzaman, M.M. Rahman, M. MandalM. Marjuque, A. Bhowmik, B. yes Rokeya, Z. Hassan, and M.O. Faruque, Oyster mushroom functions as an antihyperglycemic through phosphorylation of AMPK and increased expression of GLUT4 in type 2 diabetic model rats, Journal of Taibah University Medical Sciences (2018) -(-), 1e7 (Insert more rows as needed, or just write below the table)

List all contributions by your groupto conferences/workshops/courses/meetings 2016-June 2018, including invited lectures at external institutions/organizations

Presenter, Co-authors, Title of the presentation, Name of event, Venue, Date Form: Invited oral/poste yes/no r M Mosihuzzaman"Herbal medicine for health care-prospects and Oral yes challenges"International Seminar ‘Promotion, Prevention and Pacification: Ayurvedic Landscape’ Kolkata, India, 9-11 February 2016.

M. Mosihuzzaman, attend the 52nd EASD (European Association for the Study of Diabetes) meeting, Munich, Germany, 12-16 September 2016. No paper presented. Begum Rokeya,Combating diabetes with plant materials: scenario from Oral yes laboratory to clinical practice.Drug development from indigenous plants in developing countries" NASIC-UiTM Workshop, Malaysia, 3-7 October, 2016. B Rokeya, Clinical Pharmacology of Steroids Diabetes & Endocrine Conference, Oral yes BUHS, Dhaka, Bangladesh, 08-10 December 2016. MR Ahmed, Evaluation of antioxidant activity of seven antidiabetic Poly herbal Oral yes preparations (APHPS) available in Bangladesh. Diabetes & Endocrine Conference, BUHS, Dhaka, Bangladesh, 08-10 December 2016. F Sabrin, Effects of Enhydrafluctuans Extract on Glycemic and Lipidemic Status Oral yes and Their Relation with Heavy Metal in Normal and Type-2 Diabetic Model Rats.Diabetes& Endocrine Conference, BUHS, Dhaka, Bangladesh, 08-10 December 2016. B. Rokeya, M. Mosihuzzaman, M.S.H. Khan, L. Ali, "Preliminary exploratory Poster yes clinical trial with antidiabetic herbal medicine”, 23rdInternational Diabetic Federation (IDF) Meeting, 4 -8 December 2017, Abu Dhabi, UAE. M.R. Ahmed, B Rokeya, I. A. Jahan, M.S.H. Khan, M.A. Sayeed, L. Ali, A.K.A. Poster yes Khan, M. Mosihuzzaman, Hypoglycemic and antioxidant activities of some antidiabetic herbal Preparations (APHPS) Available in Bangladesh”, IDF Congress, 4-8 December 2017, Abu Dhabi, UAE.

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B. Amrita,B. Rokeya, Y. Kabir,”Association of SNP-KCNJ11 (E23K) gene with Poster yes susceptibility to type 2 diabetes mellitus in Bangladeshi population”, IDF Congress, 4-8 December 2017, Abu Dhabi, UAE.

MSH Khan, M Shahjalal, S Sultana, SK Roy, MA-Ud-Daula, M Akhter, L Ali: Poster yes Chemical studies on different parts of Farmed Tilapia Fish (Orechromisniloticus) and Their Feed. IDF Congress, Abu Dhabi, UAE, 4-8 December, 2017. P-0415.

MR Ahmed, Analysis of harmful constituents in some antidiabetic herbal Oral yes preparations (ADHPs) available in Bangladesh. Regional Public Health Conference, BUHS, Dhaka, Bangladesh, 29-30 December 2017

Prof M Mosihuzzamanfrom Bangladesh attended the 77th American Diabetes Association (ADA) Meeting that was held in June 09 - 13, 2017 at San Diego Convention Center, San Diego, California, United States of America.

B Rokeya,“Ethnopharmacology & Drug Development: Innovation meets Oral yes Tradition”18th International Congress of International Society for Ethnopharmacology & the 5th International Congress of the Society for Ethnopharmacology, India (ISE-SFEC 2018) congress, University of Dhaka, Bangladesh, 13-15 January 2018. Prof Begum Rokeya delivered Lectures in the “Workshop on Scientific Writing” Oral yes held from 24-28 June, 2018 in the Bangladesh Medical Research Council Bhabhan, Mohakhali, Dhaka. M Mosihuzzaman attended the 1st Sri Lankan ANRAP Regional Seminar (ANRAPSL1) on “Herbal Approaches in Combating Diabetes and other Tropical Diseases” Kandy, Sri Lanka from 17-19 January 2018. No paper presented.

Prof Begum Rokeya, 1st Sri Lankan ANRAPRegional Seminar (ANRAPSL1) on oral yes “Herbal Approaches inCombating Diabetes and other Tropical Diseases” held inNational Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17- 19 January 2018. She presented on“pathogenesis and management of postprandial hyperglycemia:current issues & emerging advances with plant materials”. M Mosihuzzaman, attended the 78th American Diabetes Association (ADA) meeting held at Forida, USA on June 22 - 26, 2018, No paper presented. Prof Begum Rokeya participated in the workshop on “Certification course on oral yes clinical research for study investigator” organized by Bangladesh Medical & Dental Council (BMRC) at BMRC Bhaban from 02-04 April, 2018. Prof M Iqbal Choudhary, 1st Sri Lankan ANRAP Regional Seminar oral yes (ANRAPSL1) on “Herbal Approaches inCombating Diabetes and other Tropical Diseases” held inNational Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17-19 January 2018. He presented on“ Discovery of Inhibitors of DPP-IV, α-glucosidase and Protein Glycation Inhibitors-Molecular Approached Towards the Treatment of Diabetes”. Prof Liaquat Ali, 1st Sri Lankan ANRAPRegional Seminar (ANRAPSL1) on oral yes “Herbal Approaches in Combating Diabetes and other Tropical Diseases” held inNational Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17- 19 January 2018. He presented on“ Sodium and Diabetes:An Update”. Prof Bilge Sener, the 1st Sri Lankan ANRAP Regional Seminar (ANRAPSL1) oral yes on “Herbal Approaches inCombating Diabetes and other Tropical Diseases” held inNational Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17-19 January 2018. She presented on“ Evaluation of Herbal Medicine for Rational Phytotherapy and Regulatory Issues”.

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Prof Banasri Hazra, 1st Sri Lankan ANRAPRegional Seminar (ANRAPSL1) on oral yes “Herbal Approaches in Combating Diabetes and other Tropical Diseases” held inNational Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17- 19 January 2018. She presented on“ New’ Surprises from ‘Old’ Plants:Valeriana wallichi-A Case in Point”.

Prof Khozirah Shaaari, 1st Sri Lankan ANRAPRegional Seminar (ANRAPSL1) oral yes on “Herbal Approaches inCombating Diabetes and other Tropical Diseases” held in National Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17- 19 January 2018. She presented on“ Applications of NMR- and MS-based Metabolomics in Natural Products Research”.

Prof Biswapati Mukherjee, 1st Sri Lankan ANRAP Regional Seminar oral yes (ANRAPSL1) on “Herbal Approaches inCombating Diabetes and other Tropical Diseases” held inNational Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17-19 January 2018. He presented on“ Current Awarness on the Development of Antidiabetic Drugs”.

Prof Jayasinghe L, 1st Sri Lankan ANRAP Regional Seminar (ANRAPSL1) on oral yes “Herbal Approaches inCombating Diabetes and other Tropical Diseases” held inNational Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17- 19 January 2018. He presented on“ Endophytic Fungi: An Ample Source of Structurally Diverse Bioactive Compounds”.

Prof AK Azad Khan, 1st Sri Lankan ANRAP Regional Seminar (ANRAPSL1) oral yes on “Herbal Approaches inCombating Diabetes and other Tropical Diseases” held inNational Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17- 19 January 2018. He presented on“ Problem and Prospects of Medicinal Plants Research”.

Prof Savitri Kumar, 1st Sri Lankan ANRAP Regional Seminar (ANRAPSL1) on oral yes “Herbal Approaches in Combating Diabetes and other Tropical Diseases” held in National Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17-19 January 2018. She presented on“ Bioactive Constituents from Fruits of some Indigenous Medicinal Plants of Sri Lanka”.

Prof Bisnu Prasad Sarma, 1st Sri Lankan ANRAP Regional Seminar oral yes (ANRAPSL1) on “Herbal Approaches in Combating Diabetes and other Tropical Diseases” held in National Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17-19 January 2018. He presented on“Anti-diabetic Activity of Aqueous Ethanolic Extract of Tamarindus Indica Fruit Pulp”.

Prof noor Hadiani Ismail, 1st Sri Lankan ANRAP Regional Seminar oral yes (ANRAPSL1) on “Herbal Approaches in Combating Diabetes and other Tropical Diseases” held in National Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17-19 January 2018. He presented on“ Ficus deltoidea, A Mysterious Antidiabetic Plants”.

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List conferences/workshops/courses/meetings organized by you 2016-June 2018

Name of event, Venue, Dates No. of participants 11th ANRAP National Seminar on “Antidiabetic Plant Materials 100 participants Separation Techniques and Biological Testing” BUHS, Dhaka, Bangladesh, 23 July2016 6th ANRAP Workshop on “Chemical Studies and Bioassay of 20 participants Antidiabetic Plant Materials, BUHS, Dhaka, Bangladesh, 24-28 July2016 ANRAP International Workshop on “Networking of Afro-Asian 18 participants Laboratories Working on Herbal Medicine” Hotel EmmaNuelle's Inn, Dhaka, Bangladesh , 29-30 October 2016 23rd Meeting of ANRAP Board, Hotel EmmaNuelle'sInn, Gulshan, 4 participants Dhaka, Bangladesh, 29October 2016 12th ANRAP National Seminar on Antidiabetic Plant Materials 80 participants Separation Techniques and Biological Testing, BUHS, Dhaka, Bangladesh, 22 July 2017 7thANRAP Workshop on Chemical Studies and Bioassay of 19 participants Antidiabetic Plant Materials, BUHS, Dhaka, Bangladesh, 23-27 July 2017 1st Sri Lankan ANRAP Regional Seminar on Herbal Approaches in 80 participants Combating Diabetes and other Tropical Diseases, Kandy, SriLanka, 17-19 January 2018 24th meeting of ANRAP Board was held in National Institute 6 of participants Fundamental Studies, (NIFS), Kandy, Sri Lanka, on 18 January 2018.

NetHerb meeting was held in Hotel Royal Mall, Kandy, SriLanka 12 participants on 18 January 2018.

Have regular, scientific seminars been held at the institution? If yes, state at which academic level (BSc, MSc, PhD) and how frequent. Name of seminar series Level and Frequency

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Describe any interaction (meetings, participation in committees, etc.) with government/society/industry/NGOs in the country, in the region or in global conventions, etc. Including unpublished reports to authorities, media exposure and public lectures etc. Were the outreach activities on your initiative or by invitation? Give account for any tangible or expected effects of outreach activities, including possibilities for policy influence.

ANRAP has interactions with the following government / society / private / learned bodies:

• Public and private universities and research organizations of Bangladesh • Public, private and industries of Bangladesh • Bangladesh Daibetic Somity (BADAS) • University of Dhaka • Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM) • Bangladesh University of Health Sciences (BUHS) • Hamdard Laboratories(waqf) Bangladesh • Bangladesh Academy of Sciences • Square Herbal & Nutraceuticals Ltd • Jayson Group of Companies • GK Pharma (Unani) Ltd • University Grants Commission (UGC), Bangladesh • Ministry of Science & Technology (Insert more rows as needed, or just write below the table)

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Enclosure 1) Research Plan/ Network Program

Overall objective (impact) and relevance of the activity:

In the coming three years ANRAP will continue with its present activities of training Research Fellows from home and abroad, conduct National, Regional and International (every 3 years) Seminers and hold the hands-on-training workshop every year. Considering the practical use of herbal medicine specially in the poorer third world countries ANRAP has expanded its activity to scientifically evaluate antidiabetic herbal medicine (HM).HM, used for the treatment of other noncommunicable diseases will also be studied.

In addition, in the next three years ANRAP will particularly give emphasis on the development of interlaboratory cooperation (NetHerb) for testing of herbal medicines. With this end in view ANRAP organized an International workshop on networking of ”Afro-Asian laboratories working on herbal medicine” in Dhaka, Bangladesh on 29-30 October, 2016 followed by a second meeting with representatives from laboratories in the Afro-Asian region in Kandy, Sri Lanka in January 2018 .After detailed discussions four laboratories agreed to collaborate. An MOU will be signed among the collaborating laboratories in the upcoming 9th ANRAP International seminar that will be held in Karachi, Pakistan on 25-27 January, 2019. So the next target is to start in-depth research on herbal medicine incorporating Chemistry, Biochemistry, Pharmacology and ultimately preclinical toxicological studies and human trial.

ANRAP is a network of natural product scientists and participating laboratories working on or are planning to work on antidiabetic plant materials. During the last three years, research capabilities of Bangladesh University of Health Sciences (BUHS) has been strengthened. Chemistry and Pharmacology laboratories of BUHS are capable of conducting research on antidiabetic HM as well as on HM for other noncommunicable diseases. ANRAP has the following overall objectives: • Compilation of information on antidiabetic plants and herbal medicines reported in scientific literature and including those from producers, traditional healers, formal practitioners and users. • Conduct research on the chemical constituents and biological activities to assess the safety, efficacy and standardization of antidiabetic plants and herbal medicines used by millions of people across the world, and bring them within acceptable limits. • Isolation, characterization and testing of biological activity of pure compounds or fractions. • Optimize, coordinate and utilize the facilities existing in various laboratories for the purpose of fruitful research on antidiabetic plant materials and herbal medicine used for the management of diabetes and other non-communicable diseases prevalent in the region.

Specific objectives for the granting period: • Training of young scientists and Fellows including PhD students in the field of antidiabetic plant and HM research. • Holding of ANRAP Regional Workshop on Chemical Studies and Bioassay of antidiabetic plant materials (Participants will be from Bangladesh, Nepal, India, Pakistan, Sri Lanka, Malayasia and Mauritius) • Developing a network of herbal medicine(NetHerb) with representatives from different participating laboratories. • Evaluation of safety and efficacy of some antidiabetic HM and HM used for other diseases like hepatitis, hypertension and renal disorders. • Conducting preclinical and toxicological studies and clinical trial of some antidiabetic and other HM. • Arrange exchange of visits of scientists working in the field by arranging National, Regional and International (every 3 years) seminars on antidiabetic plants &herbal medicines and those used for other diseases.

Expected outcomes of the activity:

Scientific Results: • Masters and PhD thesis will be produced. Papers will be published in national and international journals. Patents on new scientifically evaluated herbal medicine can be produced. • More collaborative research linkage will be established. • Safety and efficacy of antidiabetic and other herbal medicine for other diseases will be evaluated. • Development of new drugs from plant and commercial herbal medicine through collaboration in national and international level. • An arrangement has been done between GK Pharma (Unani) Ltd to carry out chemical & biological studies on hepatotoxic model animals with hepatoprotective herbal drug (Holy Liv) produced in Bangladesh.

Graduations: A number of post graduate students will be trained up. They will be working to produce their thesis and publish papers. Trained young scientists including post graduate students will be produced to work in the field of plant & herbal medicine research.

Dissemination (publications/conference contributions, etc): • Training of young scientists and post graduate students. • National, Regional and International Workshops/Seminars on Chemistry and Biology of plant materials and herbal medicine for the management & treatment of diabetes and other diseases. • Publications of scientific articles in peer reviewed journals. • Conference reports/ Published Proceedings of Conferences.

ScientificExchange

A good number of exchange visits within the region and organizations of different national, regional and international Seminars & Conferences will enhance collaborative research and exchange of knowledge and skill of the researchers working in this field.

Interactions with government and society (outreach): ANRAP has very good interaction with the Ministry of Science & Technology, Bangladesh Government and also with society in general. Interactions with traditional practioners, herbal medicine producers and medical professionals through its seminars and training workshops has widened the horizon of ANRAP to the society to a greater extent. Scientists and Fellows and students (post-graduate ) associated with ANRAP can apply for different grants through the Ministry of Scientific & Technology or other organizers and can get grant to work with antidiabetic plants/herbal medicine. For example: i) A grant of BDT 3 lac was received from the Ministry of Scientific & Technology in the year 2017- 2018 for evaluation of antidiabetic herbal drugs. Project title is ”chemical analysis of some active antidiabetic herbal preparations available in Bangladesh.” ii) A grant of BDT 5 lac was received from Bangladesh Medical and Research Council in the year 2017-2018 for evaluation of antidiabetic plant. Project title is”Isolation, Chemical characterization and elucidation of mechanism of action of antidiabetic activity of different extracts and fractions of Psidium guajava on nSTZ-induced T2DM rats”. iii) A book in Bangla (local language) incorporating biological and chemical tests on 64 plants tested at BIRDEM & DU including there form of use, has been written. Thousands of copies of the book have already been sold.

-Other: As the chemistry and pharmacology laboratory at BUHS has improved, service to herbal medicine producers can be provided. The process has already been started with one private company (herbal medicine producer, GK Pharma (Unani) Limited) who has agreed to work on antihepatic herbal drugs. We now wish to expand our activity with other companies producing herbal medicine.

Background Diabetes mellitus (DM) is a major metabolic disorder causing morbidity and mortality worldwide. The incidence of diabetes in developing countries have reached to epidemic proportions and International Diabetes Federation (IDF) has projected that in developing countries the number of people with diabetes would rise from 382 million in 2013 to 592 million in 2035. This diabetes epidemic in developing countries (mainly Type 2)occurs primarily due to urbanization, changing food habits and less physical activities.

It is well known that people with diabetes have multiple abnormalities in diverse tissues. Therefore, it is very difficult to get a single group of effective compound(s) to treat this complicated disease. There are several types of glucose-lowering drugs including insulin sensitizers, insulin secretagogues, GLP-1 analogs and α-glucosidase and DPP-IV inhibitors etc. Large sections of the populations are consuming daily synthetic hypoglycemic drugs of various types and doses. Most of them, however, have considerable side effects and are expensive. Hence, it is crucial to search for new drugs that would potentially have minimum side effects.

Indigenous medicinal treatment of diabetes in South Asia involves extensive use of herbs and plant parts either alone or as part of a mixture. Most of the countries also have a high biodiversity which could form a resource base for the discovery of new sources of anti-diabetic drugs. It is a fact that there is a long history of plant based treatment or herbal medicines used for diabetes in these countries, with some of the herbal constituents being common although the indigenous medical systems differ. However, much of the folklore knowledge incorporated in these treatments has not been subjected to experimental verification. Also very few herbal products are available in the market in a standardized form whose efficacy has been established, impacting on the popularity of herbal alternatives.

With the above perspective and realizing the fact that chemical and biological work have to be brought together collaborative research between the Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM), Dhaka and the Department of Chemistry, University of Dhaka, the University of Uppsala (UU) Sweden, and the Mahidol University (MU) Thailand, on antidiabetic plant materials was developing during 1990- 1991. At that time it was felt appropriate to organize a workshop in the field for exchanging views and expertise. With the financial assistance of the International Program in the Chemical Sciences (IPICS), and the International Foundation for Science (IFS), a workshop was held in January 1992 in Dhaka. The interest and enthusiasm seen at the workshop led to organize the First International Seminar in January 1994 on the same subject. The Seminar recognized the importance of a multidisciplinary concerted move for finding out remedy of an incurable disease like diabetes. Recommendation for the formation of ANRAP was made from the Seminar.

The Dhaka research group prepared a proposal for the formation of ANRAP. The proposal was placed at the ACGC (Asian Coordinating Group in Chemistry) meeting in Melaka, Malaysia in June 1994 by Prof M Mosihuzzaman and Prof Nilufar Nahar. Representatives of ISP, IFS, UNESCO and FACS, participated at the meeting and the ANRAP proposal was unanimously approved. The ANRAP network was launched in July 1994 with the Organizing Committee of the International Seminar as an ad-hoc committee. The First meeting of the Ad-hoc Committee held in Dhaka in January 1995 adopted the ANRAP constitution and appointed the first ANRAP Board.

ANRAP is a network of natural product scientists and participating laboratories working on or are planning to work on antidiabetic plant materials. These laboratories include Bangladesh University of Health Sciences (BUHS), BCSIR and, Chemistry/ Pharmacy departments of Dhaka, Jahangirnagar, Chittagong, Kustia, Hajee Danesh Science & Technology and Rajshahi Universities and Hamdard Laboratories (waqf) Bangladesh; HEJ Research Institute of Chemistry, Karachi University, Pakistan; Institute of Bioscience, University Putra Malaysia; National Institute of Fundamental Sciences, Sri Lanka; University of Santo Tomas, Philippines and Calcutta University, India.

In the upcoming year ANRAP will give emphasis on Herbal Medicine (HM) or Plant Medicine (PM) that are purported to have beneficial effects on not only diabetes but other various noncommunicable diseases. WHO reported that about 28 million people die worldwide each year from noncommunicable diseases. Rest of the people die from communicable and other diseases such as hepatitis and dysentery etc. To reduce thedeath rate from these diseases using modern medicine is not enough. So HM would be the alternative to manage and prevent these diseases.

In the coming three years ANRAP will continue with its present activities for examples training of Research Fellows from home and abroad, conduct National, Regional and International (every 3 years) Seminers and hold the hands-on-training workshop every year. Considering the practical use of herbal medicine specially in the poorer third world countries ANRAP has expanded its domain of activity to scientifically evaluate antidiabetic HM and now HM used for the treatment of other diseases, like hepatitis, hypertension and renal disorders etc. In addition to that ANRAP will particularly concentrate on the development of interlaboratory cooperation for testing of herbal medicines (NetHerb). To conduct research on the chemical and biological activities of herbal medicines for assessing the acceptable safety, efficacy and standardization, cooperation between various laboratories is required. As an initiation process ANRAP organized an International workshop on networking of ”Afro-Asian laboratories working on herbal medicine” in Dhaka, Bangladesh on 29-30 October, 2016. Representatives from 8 laboratories (Asia and Africa) participated in that workshop. After that ANRAP has arranged the 2nd NetHerb meeting that was held in Kandy, SriLanka on 18th January, 2018 and as a result four laboratories agreed to collaborate. An MOU will be signed among the collaborative laboratories in the upcoming 9th ANRAP International seminar that will be held in Karachi, Pakistan on 25-27 January, 2019. So the next target is to start in-depth research on herbal medicine incorporating Chemistry, Biochemistry, Pharmacology and ultimately preclinical toxicological studies and human trial.

Strategy and Plan

• Training of young scientists and Fellows in the field of antidiabetic plant research. • ANRAP Regional Workshop on Chemical Studies and Bioassay of Antidiabetic Plant Materials (Participants will be from Nepal, India, Pakistan, Malayasia, Sri Lanka and Mauritius) • Development of network of herbal medicine (NetHerb)with representation of laboratories from Afro-Asian rgion so that optimization, coordination and utilization of the facilities existing in various laboratories could be done for the purpose of fruitful research on antidiabetic plant materials and herbal medicine. • Compilation of the accumulated knowledge on herbal medicine from the practitioners. For this data collection sheet will be prepared and distributed among the practitioners. After a definite time period feedback will be received from the practioners.

Gender balance

Current gender balance in the ANRAP Board is better compared to previous years. There are 3 female members out of 5 members in the ANRAP Board (about 60% female members). In principle, ANRAP is trying to maintain a gender balance in awarding the Fellowship, selecting workshop participants and supporting students and young scientists from home and abroad. However, during the last 3 years female participation in the workshop and Fellowship programs were less than expected. In the context of Bangladesh perspective number of women scientists are still limited. But recently the trend is changing and more & more female participants are attending Seminars & Workshops. Efforts are being made to have more female members in all activities. ANRAP will circulate its seminar & workshop brochures to more women scientists and young female postgraduate students in the future. It is interesting to note that in the 7th ANRAP workshop (2017) there were 12 female participants out of 19. The ratio of M:F was 7:12 which we think a good progress towards gender balance. In the upcoming8th ANRAP workshop (16th September 2018) we are also expecting participation of more female participants.

Expected funding

International International Foundation for Sciences (IFS), Sweden Federation of Asian Chemical Societies (FACS)

National Diabetic Association of Bangladesh Bangladesh University of Health Sciences Hamdard Foundation Drug International GK Pharma (Unani) Ltd Training fee from participants

Logical framework Matrix

Overall objective (impact): Asian network of research on antidiabetic plants (ANRAP) has been supporting natural product scientists and participating laboratories working on antidiabetic plants throughout Bangladesh and Asian countries.

Types of Outcomes Performance Data source Data collection Assumptions Outputs indicator of Strategy Outcomes Spectific objective 1: Training of young scientists and Fellows including PhD students in the field of antidiabetic plant research. Training Training of During the period BSc, MSc & PhD Number of Post graduate students programme young 2016-2018 thesis produced, 25 thesis, papers will generate products scientists, ANRAP given conference reports and conference Fellows and training to 5 & 10 scientific reports PhD Fellows & post papers have been students graduate students published.

Specific objective 2: ANRAP Workshop on Chemical Studies and Bioassay of antidiabetic plant materials Workshop Young During the period Workshop report Number of Through this hands-on scientists, 2016-19 workshops and training participants Fellows and participants, 2017- number of become skilled and are PhD 20 participants, participants able to carry out students Total -39 research individually trained Participants have on antidiabetic plants. got hands-on training Specific objective 3: Exchange of visits of scientists working in the field by arranging National, Regional and International (every 3 years) seminars on antidiabetic plants Seminars Two About 80 in the Seminar reports number of Enhancement of Bangladesh National Seminars participants and collaborative research and one and 80 in the papers and exchange of Regional International presented knowledge and skill (1st Sri Seminar of the researchers Lankan participated. working in this field ANRAP Regional Seminar) and one International Workshop organized in Dhaka Specific objective 4: Optimization, coordination and utilization of the facilities existing in various laboratories for the purpose of fruitful research on antidiabetic plant materials and herbal medicine. Collaboration linkaged established.

Specific objective 5: ANRAP Regional Workshop on Chemical Studies and Bioassay of antidiabetic plant materials with participants from Asian countries (will be arranged in the coming 3 years).

Specific objective 6: Meeting for developing a network of herbal medicine (NetHerb) with representatives from different participating laboratories (at least ten laboratories will be invited). Leaders of collaborative laboratories will establish NetHerb. 2nd NetHerb meeting that was held in Kandy, SriLanka on 18th January, 2018 and as a result four laboratories agreed to collaborate

Specific objective 2: To attract sufficient financial support, other than from ISP Funding International National Amount of Applications (S & T); other than grant Seeking donations; from ISP FACS Hamdard Allocation/donation Service charge etc. Laboratories, Ministry of Science & Technology GK Pharma (Unani) Ltd Drug International

Activity Report of the previous grant period

Fellowship Programme During the last two and half years 5 fellows were supported by ANRAP. One fellow from India, came to BUHS, Dhaka, Bangladesh and four fellows (partial support) from BUHS have been supported by ANRAP.

Attendance in Seminar/Conferences/Workshops A good number of researchers in the region were supported, either fully or partially, by ANRAP to attend International or Regional scientific meetings related to diabetes and natural product chemistry. The meetings included an International Seminar on ‘Promotion, Prevention and Pacification: Ayurvedic Landscape on 9-11 February 2016. (Kolkata, India), The 52th Annual Meeting of European Association for the Study of diabetes (EASD) in 2016 (Munich, Germany), the NASIC-UiTM Workshop on "Drug development from indigenous plants in developing countries" held in Malaysia, during October 3-7, 2016. ANRAP International Workshop on “Networking of Afro-Asian Laboratories Working on Herbal Medicine” in Dhaka, Bangladesh on 29-30 October 2016. The 77th American Diabetes Association (ADA) Meeting that was held in June 09 - 13, 2017 at San Diego Convention Center, San Diego, California, United States of America. The 23rd International Diabetic Federation (IDF) Meeting in Abu Dhabi, UAE, from 4 to 8 December 2017. The 1st Sri Lankan ANRAP Regional Seminar (ANRAPSL1) on “Herbal Approaches in Combating Diabetes and other Tropical Diseases” held in National Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17-19 January 2018. The 77th American Diabetes Association (ADA) Meeting that was held in June 09 - 13, 2017 at San Diego Convention Center, San Diego, California, United States of America.

Organization of Seminar at Home & Abroad The 11th ANRAP National Seminar on ‘Antidiabetic Plant Materials Separation Techniques and Biological Testing’ was held in Bangladesh Institute of Health Sciences (BUHS), Darussalam, Mirpur, Dhaka, Bangladesh on 23 July 2016. The 12th ANRAP National Seminar on ‘Antidiabetic Plant Materials Separation Techniques and Biological Testing’ was held in Bangladesh Institute of Health Sciences (BUHS), Darussalam, Mirpur, Dhaka, Bangladesh on 22 July 2017. The 1st Sri Lankan ANRAP Regional Seminar (ANRAPSL1) on “Herbal Approaches in Combating Diabetes and other Tropical Diseases” held in National Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka from 17-19 January 2018.

Workshop The 6th ANRAP workshop on ‘Chemical studies and bioassay of antidiabetic plant materials’ was held in Research Division, BIRDEM during 24-28 July 2016. The 7th ANRAP workshop on ‘Chemical Studies and Bioassay of Antidiabetic Plant Materials’ was held in Bangladesh University of Health Sciences (BUHS), Mirpur, Dhaka, Bangladesh during 23-27 July 2017. ANRAP Workshop on “Networking of Afro-Asian Laboratories Working on Herbal Medicine” was held in Hotel EmmaNuelle's Inn, Dhaka, Bangladesh on 29-30 October 2016.

ANRAP Board Meeting The 23rd meeting of ANRAP Board was held in Hotel EmmaNuelle's Inn, Gulshan, Dhaka, Bangladesh on 29 October 2016. The 24th meeting of ANRAP Board was held in National Institute of Fundamental Studies, (NIFS), Kandy, Sri Lanka, on 18 January 2018. The NetHerb meeting was held in Hotel Royal Mall, Kandy, Sri Lanka on 18 January 2018.

A Book on the use of 62 antidiabetic plants tested in BIRDEM Published in Bengali

ANRAP profile has been prepared and published.

Thesis Paper (Completed in 2016) • Mr Ranzu Ahmed Choudhury, a faculty of BUHS has been awarded PhD degree on 30th June 2016. His thesis entitled “Chemical and Biological Studies on Antidiabetic Single Plants and Composite Herbal Preparations” • Md Nazmul Hasan, MSc (completed) thesis entitled “Effects of Enhydra Fluctuans Extract on Glycemic and Lipidemic Status and their Relation with Heavy Metal in Normal and Type-2 Diabetic Model Rats” Dept of Biotechnology & Genetic Engineering, Mawlana Bhashani Science and Technology University. • Ananta Kumar Das, Md Fazlay Rabbi, Polash Basak, BSc (completed) thesis entitled Studies on the antidiabetic activity of the alcoholic extracts of Glochidion velutinum in streptozotocin induced type 2 diabetic model rats. Dept of Biotechnology and Genetic Engineering, Gono University, Savar, Dhaka. • Sumon Kumar Roy, Dept of Applied Laboratory Sciences, Bangladesh University of Health Sciences (BUHS). The title of his MS thesis is “Characterization of Streptozotocin induced Type 2 Diabetes Mellitus Long Evans Model Rats”. • Shamim Gazi, Dept of Biotechnology and Genetic Engineering, Khulna University. The title of his MS thesis is “Effect of selective probiotics in streptozotocin induced type 2 diabetics rats”.

graduates supported by ANRAP

Graduatio ProgramCode Home Country n year Name Gender Thesis Type Present affiliation Email address Assistant Scientist, R&D Department, Square Pharmaceuticals Ltd. IPICS ANRAP Bangladesh 2014 Shihab Uddin M MSc LOC Approved for phD at Kyushu University, Japan [email protected] IPICS ANRAP Bangladesh 2015 Farjana Afrin F MSc LOC Research Trainee University of Newcastle, Callaghan NSW, 2308 [email protected]

IPICS ANRAP Bangladesh 2015 Farzana Akhter F MSc LOC Senior Officer, R&D analytical, Incepta Pharmaceuticals Ltd

IPICS ANRAP Bangladesh 2015 Manisha Das F PhD LOC Still awaiting for phD defense [email protected] PhD (2nd year) University of Tennessee Health Science Center, IPICS ANRAP Bangladesh 2015 Sanjana Haque F MSc LOC USA [email protected]

IPICS ANRAP Bangladesh 2015 Md Mahmodul Islam M MSc LOC Lecturer, Dept of Pharmacy, Daffodil Internartional University [email protected]

IPICS ANRAP Bangladesh 2016 Md Nazmul Hasan M MSc LOC Quality Control Officer, UniMed & UniHealth Manufactures [email protected] IPICS ANRAP Bangladesh 2016 Ranzu Ahmed Choudhury M PhD LOC Assistant Professor, Dept of Chemistry, Bangladesh University of Health [email protected] NSW, 2308

Dr. MOHAMMED MOSIHUZZAMAN Flat 5 B, House # 4, Road # 76 Gulshan-2, Dhaka-1212 Bangladesh

Date of birth: 06 August 1940 Place of birth: Jhenidah, Bangladesh Nationality: Bangladeshi Sex: Male Marital status: Married Tel:: (+880 2) 8837055 (Home) Mobile: (+88) 01755 654160 Email:: [email protected]

Education: 1959-1962 B.Sc.(Hons) in Chemistry, University of Dhaka, Dhaka, Bangladesh 1962-1963 M.Sc. in Chemistry, University of Dhaka 1965-1968 Ph.D. in Chemistry, Birmingham University, UK. 1968-1970 Post-doctoral Fellow, Ohio State University, Columbus, Ohio, USA 1977-1978 Research Fellow, Swedish University of Agricultural Sciences, Uppsala Sweden.

TEACHING/RESEARCH EXPERIENCE

Research Fellow, 1963-64 Chemistry Department, DU Senior Lecturer 1964-65 Chemistry Department, DU Commonwealth Scholar 1965-68 Birmingham University, UK Post Doctoral Fellow 1968-70 Ohio State University, USA Assistant Professor 1970-74 Chemistry Department, DU Associate Professor 1974-87 Chemistry Department, DU Professor 1987-2006 Chemistry Department, DU

Emeritus Professor 13 February 2015 – to date Department of Chemistry University of Dhaka Dhaka-1000, Bangladesh

Foreign Professor February 2007 – December 2008 H.E.J. Research Institute of Chemistry International Center for Chemical and Biological Sciences University of Karachi Karachi-75270, Pakistan Honorary Director Since January 2009 International Centre for Natural Product Research (ICNPR) Bangladesh University of Health Sciences of Health Sciences (BUHS) 125/1 Darussalam, Mirpur, Dhaka-1216 Bangladesh TEACHING

Successfully taught various courses of Organic Chemistry at the undergraduate and graduate levels for 40 years.

RESEARCH

Organized a research laboratory at the Department of Chemistry, University of Dhaka and led a research group producing more than 100 M. Sc., 5 M. Phil. and 19 Ph. D.s. Published more than 100 research papers. Research areas include plant and bacterial polysaccharides, natural products, medicinal plants specially anti-diabetic, anticancer and anti-HIV plants, and organic pollutants. Currently concentrating on scientific validation of Herbal Medicine.

RESEARCH GRANT/SUPPORT RECEIVED

International Foundation for Science (IFS), Sweden 1979-83 $ 40,000 (Project on Plant Polysaccharides) International Program in the Chemical Sciences (IPICS) 1980-07 $1100,000 Uppsala University, Sweden (Projects on Carbohydrates, Medicinal Plants and Organic Pollutants) Swedish Agency for Research Cooperation with 1982-86 $ 75,000 Developing Countries (SAREC), Sweden (Carbohydrates) Bose Centre for Advanced Research, Dhaka University 1982-88 $ 13,600 (Organic Pollutants and Fungi projects) 2002-07 $ 9,600 University Grants Commission, Bangladesh 1986-88 $ 3,000 Shaman Pharmaceuticals Inc., South San Francisco, USA 1997-98 $ 15,000 Natural Product Division, National Cancer Institute, 1996-07 $ 10,000 Frederick, Maryland, USA Ministry of Science and Information & Communication Technology, Government of Bangladesh 1999 & 2002 $ 12,000 Higher Education Commission, Pakistan 2007-08 $ 17,000

COLLABORATION

International

1. Department of Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden (1977-2006)

Collaborating Scientists : Prof . Olof Theander, Prof. Per Aman, Prof Lennart Kenne, Dr. Lennart Lundgren, Dr. Olle Larm and Engr Rolf Andersson.

Field of Study : Jute, Plant Polysaccharides, Dietary Fiber, Natural Products, Medicinal Plants.

2. Department of Organic Chemistry, Stockholm University, Stockholm, Sweden. (1985-1995) Collaborating Scientists : Prof. Bengt Lindberg and Dr. Per Eric Janssson

Field of Study : Bacterial Polysaccharides.

3. Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. (1989-1999) Collaborating Scientist : Prof. Bo Hellman and Eric Gylfe Field of Study : Mechanism of blood-sugar lowering action of anti-diabetic plant materials.

4. Department of Chemistry, Mahidol University, Bangkok, Thailand. (1985-1994) Collaborating Scientist : Prof. Vichai Reutrakul Field of Study : Medicinal Plants

5. Natural Products Division, National Cancer Institute, Frederick, Maryland, USA (1995-2006) Collaborating Scientists : Dr. Gordon Cragg and Mr. Tom McCloud Field of Study : Anticancer and Anti-HIV Plant Materials

6. Shaman Pharmaceuticals Inc., South San Francisco, California, USA (1995-1999) Collaborating Scientists : Dr. Steven King, Dr. Wayne Inman and Dr. Diana Fort Field of Study : Anti-diabetic Plant Materials

7. Department of Environmental Science, Stockholm University, Sweden (2005-2009). Collaborating Scientists : Prof Ake Bergman Field of Study : Organic Pollutants

8. HEJ Research Institute of Chemistry, University of Karachi, Pakistan (1988-2009) Collaborating Scientists : Prof Att-ur-Rahman Prof M Iqbal Choudhary Field of Study : Natural Product Chemistry

National

1. Bangladesh Institute of Research and Rehabilitation of Diabetic, Endocrine Metabolic Disorders (BIRDEM), Dhaka (1989-2009). Collaborating Scientists : Prof. A K Azad Khan, Dr. Liaquat Ali, Dr. Begum Rokeya Field of Study : Development of new anti-diabetic drugs from plant Sources

2. International Centre for Diarrheal Diseases Research, Bangladesh (ICDDR,B), Dhaka (1988-1996) Collaborating Scientists : Dr. Ferdousi Quadri and Dr. Khaleda Haider Field of Study : Structural and immunological studies of lipopolysaccharides isolated from new bacterial strains causing diarrhoea.

3. Bangladesh Agricultural Research Institute (BARI), Gazipur. (2003-2007) Collaborating scientists : Dr. M H Kabir Mr. Arifur Rahman Mr. Sultan Ahmed Field of study : Fate of pesticides used in crop protection

ORGANIZER

(A. Academic) Founder Chairman of the Asian Network of Research on Antidiabetic Plants (ANRAP). Functioning since 1994. Supported by IFS (International Foundation for Science), IPICS and UNESCO. Founder Coordinator of the Network of Instrument Technical personnel and user Scientists of Bangladesh (NITUB). Functioning since 1994. Supported by the IPICS, IFS and UNESCO. Chairman, National Organizing Committee, ASOMPS X (Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products) held in Dhaka, Bangladesh on 18-23 November 2000. Sheikh Hasina, the then Prime Minister inaugurated the Symposium. President, Bangladesh Chemical Society for the years 2001-2004. Chairman, AFASSA (Africa, Asia and South America) Coordinating Group in Natural Product Chemistry, 2004-2006. Founder-Director, OPRC (Organic Pollutants Research Centre), 2005-2007, Department of Chemistry, University of Dhaka, established in November 2005.

(B. Administrative) Senate member, DU for five terms Syndicate Member, DU for two terms, elected as Dean General Secretary, Dhaka University Teachers Association (DUTA), 1984 and 1985. Vice-President, DUTA 1988 President DUTA, 1989. President, Bangladesh Federation of University Teachers Associations, 1989-90. Founder Chairman, Dhaka University Teachers Co-operative Society 1989-2002

ADMINSTRATIVE EXPERIENCE

Dean 1991-95 Faculty of Science, DU Chairman 1991-93 Chemistry Department, DU Chairman (on Deputation) 1997- 2001 BCSIR, Dhaka Vice Chancellor 2012- 2015 Hamdard University Bangladesh (HUB)

PARTICIPATION IN INTERNATIONAL CONFERENCES

Attended conferences and presented papers (>100) in Australia, Botswana, Canada, Chile, China, Denmark, Ethiopia, Finland, France, Germany, Holland, India, Indonesia, Italy, Japan, Jordan, Malaysia, Nepal, Pakistan, Philippines, Poland, Sri Lanka, South Africa, South Korea, Sweden, Switzerland, Tanzania, Thailand, Turkey, UK, USA, Uruguay and Vietnam.

List of publications: Attached.

LIST OF PUBLICATIONS of Professor Dr. Mohammed Mosihuzzaman

1. Khundkar M H and Mosihuzzaman M; 1965, ‘Preparation of monochloroacetic acid’, Pakistan J. Sci. Ind. Res., 8(1), 285. 2. Khundkar M H, Mosihuzzaman M. and Bhattacharjee A K; 1965, ‘Studies on carboxymethylcellulose II. Use of organic alcohols as reaction media for carboxymethylation’, Pakistan J. Sci. Ind. Res., 8(3), 89. 3. Khundkar M H, Mosihuzzaman M. and Rahim Q A M A; 1966, ‘Phosphorylated jute cellulose’, Pakistan J. Sci. Ind. Res., 9(2), 117. 4. Mosihuzzaman M., Baggett N and Webber J M; 1969, ‘Benzylidenation in basic medium: reaction of methyl-2,3-di-O-methyl-6-O-toluene-p-sulphonyl-a-D- glucopyranoside and 3-chloro-1-propanol with benzaldehyde’, Carbohydrate Research, 11, 263. 5. Mosihuzzaman M., Salam M A and Ahmed M; 1974, ‘Sulphation of jute cellulose’, J. Ind. Chem. Soc., 51, 433. 6. Mosihuzzaman M. and Islam M R; 1976, ‘Homogeneous O-isopropylidenation of D- glucose’, Dhaka University Studies B, 24(2) 15. 7. Mosihuzzaman M and Roy J C; 1976, ‘Cellulose acetate from jute', Dhaka University Studies B, 24(1) 79. 8. Mosihuzzaman M., Roy J C and Ali M Z; 1980, Àcetylation of raw jute', Dhaka University Studies B, 28(1) 47-52. 9. Mosihuzzaman M., Theander O and Aman Per; 1982, Ànalysis of carbohydrates in jute plant', J. Sci. Food Agric., 33, 1207-1212. 10. Gouda I, Larm O and Mosihuzzaman M.; 1983, `The nitrous acid deamination of methyl 2-amino-2-deoxy-a-D-glucopyranoside, 2-amino-2-deoxy-1,3,4,6-tetra-O-acetyl-a-D- glucopyranosylamine in glacial acetic acid', Acta Chem. Scand., 37, 72-74. 11. Baggett N, Mosihuzzaman M and Webber J M; 1983, Synthesis of some monoacetals of 1,4-anhydro-D-mannitol, Carbohydrate Research, 116, 49-60. 12. Baggett N, Mosihuzzaman M and Webber J M; 1985, Kinetic control in the benzylidenation of some 1-alkylcyclohexane-cis-1,2-diols, Carbohydrate Research, 136, 347-355. 13. Mosihuzzaman M. and Chowdhury T A; 1986, `Cellulose acetate from unretted jute bark and stick (Corchorus capsularis, Linn)', Bangladesh J. Sci. Res., 4(1), 65-72. 14. Mosihuzzaman M, Mollah A H, Chowdhury T A, Theander O and Lundgren L N, 1986, `Phenolic acids in the jute plant (Corchorus capsularis)', J. Sci. Food Agric., 37, 955-960. 15. Nahar N, Rahman S M and Mosihuzzaman M; 1986, Ànalysis of water-soluble polysaccharides in retted jute fibre (Corchorus capsularis Linn)', Bangladesh J. Sci. Ind. Res., 21 (1-4), 238-245. 16. Haq Q N, Larm O, Mosihuzzaman M, Nahar N and Nizamuddin M; 1987, `Structural studies and chemical modifications of a xylan from unretted jute stem (Corchorus capsularis), Swedish J. Agric. Res., 17, 63-67. 17. Nahar N, Mosihuzzaman M, Larm O and Odlind B; 1987, `Fractionation and characterization of carbohydrates in uretral urine of White Leghorn hens', Swedish J. Agric. Res., 17, 9-12. 18. Nahar N, Mosihuzzaman M, Mian A J, Larm O and Hoffman J; 1987, Analysis of water-soluble polysaccharides from unretted jute plant (Corchorus capsularis), Carbohydrate Research, 163, 136-142. 19. Hoque A K M F, Mosihuzzaman M, Ahmed S A, Ahmed M G and Andersson R; 1987, `Synthesis of some substituted adamantane-2, 4-diones', J. Chem. Res., (S), 214: J. Chem. Res. (M), 1701. 20. Mosihuzzaman M, Hoque M F, Chowdhury T A, Theander O and Lundgren L N; 1988, `Phenolic acids in fresh and retted jute plants (Corchorus capsularis and Corchorus olitorius)', J. Sci. Food Agric., 42, 141-147.

1 21. Nahar N, Mosihuzzaman M, Chowdhury T A and Rahman S;1988, Ànalysis of water soluble carbohydrates in Abroma augusta', J. Bangladesh Acad. Sci., 12, 207-210. 22. Ahmed M G, Hoque A K M F, Ahmed S A, Mosihuzzaman M and Andersson R; 1988, `Reactions of 4,4-disubstituted cyclohexane enamines with unsaturated acid chlorides', J. Chem. Res. (S), 362; (M), 2815. 23. Rahman M M, Chowdhury T A and Mosihuzzaman M; 1988, Ànalysis of phenolic acids and low-molecular carbohydrates in Telakucha (Coccinia indica) plant', J. Bangladesh Chem. Soc., 1, 27-32. 24. Mosihuzzaman M, Islam M S and Nahar N; 1989, Ànalysis of in Dolichos biflorus seeds', Dhaka University Studies B, 37, 11-17. 25. Mosihuzzaman M, Quddus A, Nahar N and Theander O; 1989, `Comparative studies of carbohydrates in the two major species of jute (Corchorus capsularis and Corchorus olitorius)', J. Sci. Food Agric., 48, 305-310. 26. Mosihuzzaman M, Nahar N, Rahman S M and Mian A J; 1989, `Structural studies of hemicelluloses from retted jute bark and stem', Bangladesh J. Jute Fib. Res.,10, 7. 27. Nahar N, Mosihuzzaman M and Theander O; 1990, Ànalysis of phenolic acids and carbohydrates in pigeon pea (Cajanus cajan) plant', J. Sci. Food Agric., 50, 45-53. 28. Nahar N, Mosihuzzaman M and Rahman S; 1990, Ànalysis of carbohydrates in seven edible fruits of Bangladesh', J. Sci. Food Agric., 51, 185-192. 29. Lindberg B, Mosihuzzaman M, Nahar N, Abeysekara R M, Brown R G and Willison J H M; 1990, Àn unusual (4-O-methyl-b-D-glucurono)-D-xylan isolated from the mucilage of seeds of the quince tree (Cydonia oblonga)', Carbohydr. Res., 207, 307- 310. 30. Nahar N, Mosihuzzaman M, Ahmed S M and Rahman M R; 1990, `Structural studies of hemicellulose from pigeon pea (Cajanus cajan) stem', Dhaka University Studies B, 38 (2), 187-191. 31. Rahman M M, Chowdhury T A and Mosihuzzaman M; 1990, Ànalysis of water and alkali-soluble polysaccharide of Coccinia indica (Telakucha) plant', J. Bangladesh Chem. Soc., 3(2), 199-204. 32. Rahman S M, Mosihuzzaman M and Westerlund E; 1991, `Free sugars and dietary fibre in some fruits of Bangladesh', Food Chemistry, 42, 19-28. 33. Kenne L, Lindberg B, Rahman M M and Mosihuzzaman M; 1991, `Structural studies of the O-antigen-polysaccharide of Vibrio flubialis AA-18239', Carbohydr Res., 216, 145-149. 34. Rahman M O, Chowdhury T A and Mosihuzzaman M; 1991, Ànalysis of fatty acids and carbohydrates of Psidium guajava leaf', Dhaka Univ. Stud. B., 39, 159-164. 35. Nahar N, Rahman S and Mosihuzzaman M; 1991, Ànalysis of hemicelluloses from Psidium guajava fruit', J. Bangladesh Chem. Soc., 4(1), 31-34. 36. Rahman M M, Quadri F, Albert M J, Hussain A and Mosihuzzaman M; 1992, `Lipopolysaccharide composition and virulence properties of clinical strains of Vibrio fluvialis and Vibrio mimicus, Micriobiol '. Immunol., 36(4), 327-336. 37. Nahar N and Mosihuzzaman M; 1992, Ànalysis of carbohydrates in Sesbania cannabina (Dhanchi) Plant', J. Bangladesh Chem. Soc., 5(1), 37-43. 38. Shahjahan M, Mosihuzzaman M and Mian A J; 1992, `Phenolic acids in rice plant (Oryza sativa)', J. Bangladesh chem. Soc, 5(1), 59-63. 39. Roy J C, Rahman H, Ghani A, Mosihuzzaman M and Nahar N; 1992, `Steroid in a widely used folk medicine', Bangladesh J. Life Sci., 4, 59-63. 40. Hussain L, Chowdhury T A, Rahman M M and Mosihuzzaman M; 1992, Structural studies of the core-carbohydrate of Shigella dysenteriae type 1 mutant PSD-40,' J. bangladesh Chem. Soc., 5(2), 167-172. 41. Shahjahan M, Mosihuzzaman M. and Mian A J; 1992, Ànalysis of fatty acids in some local and high yielding varieties of rice straw (Oryza satiya)', Bangladesh J. Sci. Ind. Res., XXVII, Nos. (3-4), 148-153. 42. Dey S K, Nahar N and Mosihuzzaman M; 1993, Analysis of free sugar and dietary fibre of seven local vegetables', Food Chemistry., 48, 1-4.

43. Mosihuzzaman M, Murshed S and Mian A J; 1993, `Fatty acids in unretted jute (Corchorus capsularis and Corchorus olitorius) plant', Bangladesh J. Sci. Ind. Res., XXVIII, No. 1, 12-18. 44. Shahjahan M, Mosihuzzaman M and Mian A J; 1993, Ìn-vitro rumen digestibility of some local and high yielding varieties of rice straw', Anim. Feed Sci. Tech., 42 (1-2), 121-130. 45. Rahman M M, Kenne L, Lindberg B and Mosihuzzaman M; 1993, `Structural studies of the O-antigen polysaccharide of Vibrio flubialis M-940', Carbohydr. Res., 242, 181-189. 46. Rahman M M, Lindberg B, Kenne L and Mosihuzzaman M; 1993, `Studies of the O- antigen polysaccharide of Vibrio mimicus W-26768', Carbohydr. Res., 243, 131-138. 47. Ali L, Azad Khan A K, Mamun M I R, Mosihuzzaman M, Nahar N, Nur-e-Alam M, and Rokeya B; 1993, `Studies on hypoglycemic effects of fruit pulp, seed and whole plant of Momordica charantia on normal and diabetic model rats', Planta Medica, 59, 409-412. 48. Chowdhury T A, Hussain L, Mosihuzzaman M. and Haider K; 1993, Àssociation of plasmids with the carbohydrate composition of the O-antigen in Shigella dysenteriae type 1', J. Diarrhoeal Dis. Res., 11, 93-96. 49. Aman A M, Mosihuzzaman M and Nahar N; 1993, Ìsolation and characterization of some terpenoids and phenolic acids of Abroma augusta root bark', Dhaka Univ. Stud. B, 41(2), 119-126. 50. Paul G K, Mosihuzzaman M and Nahar N; 1993, Ànalysis of carbohydrates in green cocoanut (Cocos Nucifera) water,' Dhaka Univ. Stud. B, 41(2), 113-118. 51. Chowdhury T A, Mahmud H and Mosihuzzaman M; 1993, Ànalysis of fatty acids and carbohydrates of Litsea glutinosa leaf', J. Bangladesh Chem. Soc., 6(2), 183-189. 52. Mosihuzzaman M, Islam M S, Nahar N and Andersson R; 1993, Àn NMR study of a lignan glucoside form Carthamus tinctorius (Safflower seeds)', J. Bangladesh Chem. Soc., 6(2), 113-116. 53. Albert M J, Holme T, Lindberg B, Mosihuzzaman M, Qadri F and Rahman M M; 1994, `Structural studies of the Shigella boydii type 5-O-antigen polysaccharide', Carbohydr. Res., 265, 212-227. 54. Nahar N, Hazra B K, Mosihuzzaman M, Rahman M M. and Andersson R; 1994, `Structural studies of mucilage from Abroma augusta root bark', Carbohydrate Polymer., 24, 277-280. 55. Rahman S M M, Mosihuzzaman M, Rahman M M and Nahar N; 1994, `Structural studies of water-soluble dietary fibre of Psidium guajava fruit', J. Bangladesh Chem. Soc., 6(1), 120-126. 56. Quader M A, Khatun M and Mosihuzzaman M; 1994, Ìsolation of 4-hydroxy sesamin and ent-norescurinin from Phyllanthus niruri and their chemotaxonomic significance', J. Bangladesh. Acad. Sci., 18(2), 229-234. 57. Ali L, Azad Khan A K, Hasan Z, Mosihuzzaman M, Nahar N, Nasrin T, Nur-e-Alam M and Rokeya B; 1995, `Characterization of the hypoglycemic effects of Trigonella foenum-groecum seed', Planta Medica, 61(4), 358-360. 58. Mosihuzzaman M, Nahar N, Ali L, Rokeya B, Azad Khan A K, Nur-e-Alam M and Nandi R P; 1995, `Hypoglycemic effects of three plants from Eastern Himalayan belt', Diabetes Res., 26(3), 127-137. 59. Nahar N, Das R N, Shoeb M, Marma M S, Aziz M A and Mosihuzzaman M; 1997, `Four triterpenoids from the bark of Zizyphus rugosa and Zizyphus oenoplia', J. Bangladesh Acad.. Sci., 21(2), 151-158. 60. Nahar N, Mosihuzzaman M and Khan S H; 1997, `Determination of free sugars in plant materials having antidiabetic activity', Dhaka Univ. J. Sci., 46(1), 167-170. 61. Nahar N, Ahsan Habib A K M, Aziz M A and Mosihuzzaman M; 1998 ‘Free sugars and dietary fibres of some local vegetables’, Dhaka Univ. J. Sci., 46(2), 215-222.

3 62. Nahar N, Habib A K M, Ahsan Aziz M A and Mosihuzzaman M; 1998, ‘Free sugars and dietary fibres of some local vegetables’ Dhaka Univ. J. Sci., 46(2), 215-222. 63. Rahman M. A, Mian A J, Nahar N and Mosihuzzaman M; 1998, ‘Analysis of water- soluble polysaccharides of different parts of ripe jackfruit (Artocarpus heterophyllus)’ Daka Univ. J. Sci.46(2), 247-252. 64. Chowdhury T A, Halder S, Guha S K and Mosihuzzaman M; 1999, ‘Isolation and characterization of three triterpenoids and a dicarboxylic acid from fruit pulp of Dillenia indica’ Dhaka Univ. J. Sci., 47(2), 58-60. 65. Rahaman A M, Nahar N, Mosihuzzaman M, Mian A J; 1999 ‘Variation of carbohydrate composition of two forms of fruit from jack tree (Atrocarpus heterophyllus L.) with maturity and climatic conditions', Food Chemistry, 65, 91-97 . 66. Begum S A, Nahar N and Mosihuzzaman M; 2000, 'Chemical and Biological Studies of Scoparia dulcis L. Plant Extracts', J Bangladesh Acad. Sci., 24, 141-148. 67. Uddin M J, Khan S H, Mamun M I R, Nahar N, Aziz M A and Mosihuzzaman M; 2000, 'Four compounds form the anti-HIV active extracts of Fissistigma rubiginosum plant', J. Bangladesh Chem. Soc., 13 (1&2), 175-179. 68. Rahman M A, Nahar N, Mian A J, Mosihuzzaman M; 2000, 'Characterization of Aqueous 4% Alkali-Soluble Acidic Polysaccharides of Inner Stick of Ripe Jackfruit (Artocarpus heterophyllus, L)', J. Bangladesh Chem Soc., 13 (1 & 2), 53-61. 69. Nahar N, Rokeya B, Ali L, Hassan Z, Nur-e-Alam M, Chowdhury N S, Azad Khan A K and Mosihuzzaman M; 2000, 'Effects of Three Medicinal Plants on Blood Glucose Levels in Non-diabetic and Diabetic Model Rats', Diabetes Res., 35, 41-49. 70. Das R N, Nahar N and Mosihuzzaman M; 2001, 'Isolation and characterization of a 1,6-b-galactan from Momordica charantia fruit pulp', J. Bangladesh Acad. Sci., 25(1), 99-102. 71. Sultana N, Nahar N and Mosihuzzaman M; 2001, 'A Cucurbitane triterpenoid isolated from Momordica charantia (Karela) whole plant', Dhaka Univ. J Sci. 49(1), 21-25. 72. Mamun M I R, Rokeya B, Chowdhury N S, Moniruzzaman M, Nahar N, Ahmed M U, Mosihuzzaman M, Ali L, Azad Khan A K and Khan S H; 2001, 'Antihyperglycemic effect of Pterospermum acerifolium Willd. and Pterospermum semisagittatum Ham.', Diabetes Res., 35, 163-170. 73. Islam M S, Uddin M J, Khan S H, Nahar N and Mosihuzzaman M; 2001, 'Studies on the Extractives of Ficus Racemosa Fruits', J. Bangladesh Chem. Soc., 14 (1) 21- 26. 74. Nahar N, Uddin M J, Baroi M L, Khan S H and Mosihuzzaman M; 2001, 'Analysis of Phenolic acids from Fissistigma rubiginosum, 2001, J. Bangladesh Chem. Soc. 14(2) 207-211. 75. Nahar N, Hossain M A, Aziz M A, Das R N, Khan S H, Shoeb M. and Mosihuzzaman M; 2001, 'Studies of Oxalis corniculata Linn', J Bangladesh Chem. Soc. 14(2) 213-218. 76. Jahan I A, Nahar N, Mosihuzzaman M, Shaheen F, Parween Z, Atta-ur-Rahman and Choudhury M I; 2002, 'Novel Diterpene Lactones From Suregada multiflora', J Nat. Prod., 65, 932-934. 77. Rahman, S. M. M., Nahar N., Mosihuzzaman M and Andersson, R., 2002, Isolation of An Arabinogalactan from Elaeocarpus floribunda Blume (Jalpai) Fruit, J Bangladesh Acad. Sci. 26(1), 67-72. 78. Mamun, M. I.R., Nahar N., Mosihuzzaman M and Anderson, R., 2002, Constituents of Pterospermum acerifolium Willd. leaves and bark, J Bangladesh Chem. Soc., 15(1), 91-95.

4 79. Faruk, J A M, Nahar, N., Mosihuzzaman, M., and Rashid M. A., 2002, Two New Ellagic Acids from Lagerstroemia speciosa Linn. Plant, J Bangladesh Chem. Soc., 15(1), 73-78. 80. Rahman, S M. M, Nahar, N, Mosihuzzaman, M, Rahman, M M and Andersson, R, 2002, Structural studies of water soluble polysaccharides of Zizyphus mauritiana Lamk. Fruit, Dhaka Univ. J Sci, 50(2), 271-273. 81. Khan S H, Mosihuzzaman, M, Nahar, N, Rashid M A, 2003, 'Three magastigmane glycosides from the leaves of Pterospermum semisagitaitum', Pharmaceutical Biology, 41(7), 512. 82. Chakrabarti S, Biswas T K, Rokeya B, Ali L, Mosihuzzaman M, Nahar N, Azad Khan A K and Mukherjee B; 2003, 'Advanced studies on the hypoglycemic effect of Caesalpinia bonducella F. in type 1 and 2 diabetes in Long Evans rats', J. Ethnopharmcol., 84, 41-46. 83. Hannan J M A, Rokeya B, Farouque O, Nahar N, Mosihuzzaman M, Azad Khan A K and Ali L; 2003, 'Effect of soluble dietary fibre fraction of Trigonella foenumgraecum on glycemic, insuinemic, lipidemic and platelet aggregation status of Type 2 diabetic model rats', J. Ethnopharmcol., 88, 73-77. 84. Khan S H, Nahar N, Mosihuzzaman M and Rashid M A; 2003, 'Terpenoids and Benzofuran from Pterospermum semisagittatum, Dhaka Univ J. Biol. Sci., 12(2), 147- 152. 85. Mamun M I R, Nahar N,Mosihuzzaman M and Andersson R; 2003, 'Flavonoids, Lignan and their Glycosides from Pterospermum acerifolium Willd.', Dhaka Univ J. Sci., 51(2), 1-5. 86. Mostafa M, Dey S K, Nahar N and Mosihuzzaman M; 2003, 'Phytochemical Studies of Leucus indica (L)', J Bangladesh Chem Soc., 16(1), 1-5. 87. Dey S K, Islam M S, Mostafa M, Nahar N and Mosihuzzaman M; 2003, 'Some Secondary Metabolites from Cytotoxic Extract of Prismatomeris tetranda', J Bangladesh Chem.Soc., 16(1), 22-27. 88. Khan S H, Nahar N, Mosihuzzaman M and Rashid M A, 2003, 'A new Compound from Pterospermum semisagittatum', Dhaka Univ J Pharm. Sci., 2(1), 25-28. 89. Mostafa M, Nahar N and Mosihuzzaman M, 2003, 'Two acetylated Flavone Glycosides from Leucas indica, Dhaka Univ J Pharm. Sci., 2(1), 51-56. 90. Dey S K, Nahar N and Mosihuzzaman M, 2003, 'Studies of Calycopteris floribunda leaves', Dhaka Univ J Pharm. Sci., 2(1), 67-68. 91. Nahar N, Mosihuzzaman M, Mostafa M and Sokeng S D, 2003, 'Phytochemical investigation of Bridelia ndellensis, J Bangladesh Chem.Soc., 16(2), 165-169.

92. Jahan, I. A., Nahar, N., Mosihuzzaman, M., Shaheen, F., Atta-ur-Rahman, Choudhary, M. I. 2004, Six new diterpenoids from Suregada multiflora. J. Nat. Prod. 67, 1789- 1795 93. Khan, S.H., Nahar, N., Mosihuzzaman, M. and Rashid, M. A.; 2004, 'Neolignin and megastigmane glycosides from the leaves of Pterospermum semisagittatum', Pharmazie, 59, 12-15. 94. Mamun, M.I.R., Nahar, N., Rokeya, B., Ali, L., Azad Khan, A.K., Mosihuzzaman, M. 2004, Insulin releasing effect of Momordica charantia, J. Bangladesh Chem. Soc. 17(1), 59-64. 95. Anwar, K. J., Mosihuzzaman, M., Nahar, N. and Khan, S. H.; 2004, 'Chemical studies of anti-hyperglycemic active extract of Otostegia aucheri Bios', J. Bangladesh Chem. Soc., 17(1), 18-22. 96. Murshed, S., Rokeya, B., Nahar, N., Azad Khan, A.K., Mosihuzzaman, M., Banerji, A., Maiti, S. and Ali, L.; 2005, ‘Hypoglycemic and Hypolipidemic Effect of Hemidesmus indicus root on Diabetic Model Rats’, Diabetes Research, 39, 15-23. 97. Chakrabarti, S., Biswas, T.K., Seal, T., Rokeya, B., Ali, L., Azad Khan, A.K., Nahar, N., Mosihuzzaman, M. and Mukherjee, B., 2005. Antidiabetic activity of Caesalpinia bonducella F. in chronic type 2 diabetic model in Long-Evans rats and evaluation of

5 insulin secretagogue property of its fractions on isolated islets, J Ethnopharmacol, 97, 117-122. 98. Sokeng, S.D., Rokeya, B., Mostafa, M., Nahar, N., Mosihuzzaman, M., Ali, L., and Kamtchouing, P. 2005. Antihyperglycemic effect of Bridelia ndellensis ethanol extract and fractions in streptozotocin-induced diabetic rats. African Journal of Traditional, Complimentary and Alternative Medicines, 2 (2), 94-102. 99. Dey S K, Shoeb M, Rob T, Nahar N, Mosihuzzaman M and Sultana N, 2005. Biological and Chemical Studies of Calycopteris floribunda leaves, Dhaka Univ J Pharmaceutical Sci., 4(2), 103-106. 100. Haque M A, Hossain M S, Rahman M Z, Rahman M R, Hossain M S, Mosihuzzaman M, Nahar N and Khan S I, 2005. Isolation and Bioactive Secondary Metabolites from an Endophytic Fungus of Ocimum basilicus, Dhaka Univ J Pharmaceutical Sci., 4(2), 127-130. 101. Shoeb M, Mamun M I R, Nahar N and Mosihuzzaman M, 2005. Biological Screening of Zizyphus rugosa and Zizyphus oenoplia extractives, Dhaka Univ J of Pharmaceutical Sci., 4(2), 131-134. 102. Mostafa M, Nahar N, Mosihuzzaman M, Sokeng S D, Fatima N, Atta-ur-Rahman and Choudhary M I, 2006. Phosphodiesterase-I inhibitor quinovic acid glycosides from Bridelia ndellensis, Natural Product Research, 20(7), 686-692. 103. Nishat S, Nahar N, Mamun M I R and Mosihuzzaman M, 2006. Neolignans isolated from seeds of Myristica fragrans Houtt, Dhaka Univ J Sci., 54(2), 229-231 104. Mostafa M., Nahar N., Mosihuzzaman M., Makhmoor T, Choudhary M. I. and Atta- ur-Rahman; 2007, Free radical scavenging phenylethanoid glycosides from Leucas indica Linn, Natural Product Research 21 (4), 354-361. 105. Karsten Krohn, Dietmar Gehle, Sujit Kumar Dey, Nilufar Nahar, Mohammed Mosihuzzaman, Nasim Sultana, Hossain Sohrab, Philip J. Stephens, Jiang-Jung Pan and Florenz Sasse, 2007, Prismatomerin, a New Iridoid from Prismatomeris tetranda. Structure Elucidation, Determination of Absolute Configuration, and Cytotoxicity, J. Nat. Prod., 70 (8), 1339-1343. 106 Mamun M I R, Zamir R, Nahar N, Mosihuzzaman M, Linderholm L, Athanasiadou M and Bergman A, 2007, Traditional organochlorine pollutants in blood from humans living in the Bangladesh capital area, Organohalogen Compounds, 69, 2026- 2030. 107. Nahar N, Mamun M I R, Zamir R and Mosihuzzaman M, 2008. Analysis of pesticide residues in some local fish and vegetable, Dhaka Univ J Sci, 56, 1-4. 108. Mosihuzzaman M and Choudhary M I, 2008. Protocols on safety, efficacy, satandardization and documentation of herbal medicine (IUPAC Technical Report), Pure Appl Chem, 80, 2195-2230. 109. Zamir R, Athanasiadou, Nahar N, Mamun M I R, Mosihuzzaman M and Bergman A, 2009, Persistent organohalogen contaminants in plasma from groups of humans with different occupations in Bangladesh, Chemosphere, 74, 453-459. 110. Mosihuzzaman M and Sydnes LK, 2010, Herbal Products in Healthcare: Challenges and Potential in Comprehensive Bioactive Natural Products Vol I Potential and Challenges Ed. Gupta VK, pg 279-306. 111. Rokeya Begum, Mosihuzzaman M, Azad Khan AK, Nilufar Nahar, and Ali Liaquat, 2011, Emerging Challenge of Type 2 Diabetes: Prospects of Medicinal Plants in Recent Advances in the Pathogenesis Prevention AND Management of Type 2 Diabetes and its complications Ed. Mark Zimering, pg 375-390. 112. Mosihuzzaman M, 2012, Herbal Medicine in Healthcare-An Overview, Natural Product Communications, Vol. 7, No. 6 pg 807-812

6 CONFERENCE REPORTS

1. Nahar N, Rahman S, Mosihuzzaman M and Theander O; 1986, 'Carbohydrates in fruits', XIII International Carbohydrate Symposium Cornell University, Aug 10-15, Ithaca, New York, USA. 2. Mosihuzzaman M, Nahar N, Theander O and Larm O; 1986, 'Carbohydrates in Jute', XIII International Carbohydrate Symposium Cornell University, Aug 10-15, Ithaca, New York, USA. 3. Nahar N, Rahman S and Mosihuzzaman M; 1988, 'Analysis of hemicelluloses in Psidium guajava fruit', XIVth International Carbohydrate Symposium, Aug 14-19, Stockholm, Sweden. 4. Islam M S and Mosihuzzaman M; 1989, 'Isolation and characterization of chemical components of Carthamus tinctorius (Safflower) seeds', Sixth Asian Symposium on Medicinal Plants and Spices, Jan 24-28, Bandung, Indonesia. 5. Nahar N, Mollick R R and Mosihuzzaman M; 1989, 'Analysis of extractives of Cajanus cajan leaves', Sixth Asian Symposium on Medicinal Plants and Spices, Jan 24-28, Bandung, Indonesia. 6. Mosihuzzaman M, Nahar N, Mamun M I R, Nur-e-Alam M, Ali L and Azad Khan A K; 1990, 'Hypoglycemic activity of Momordica charantia fruit pulp', 17th IUPAC International Symposium on the Chemistry of Natural Products, Feb 4-9, New Delhi, India. 7. Chowdhury T A, Mosihuzzaman M and Lindberg B; 1990, 'Structural studies of the O- antigen polysaccharide of Vibrio mimicus', XV 36870, XVth International Carbohydrate Symposium, Aug 12-17, Yokohama, Japan. 8. Rahman S M M, Mosihuzzaman M and Nahar N; 1990, 'Structural studies of an arabinan from Flacaurtia indica fruit', XVth International Carbohydrate Symposium, Aug 12-17, Yokohama, Japan. 9. Nahar N, Hazra B K and Mosihuzzaman M; 1990, 'Structural studies of a mucilage from Abroma augusta root bark', XVth International Carbohydrate Symposium, Aug 12- 17, Yokohama, Japan. 10. Mosihuzzaman M, Shahjahan M and Mian A J; 1991, 'Phenolic acids in rice plant', Fourth Asian Chemical Congress, Aug 26-30, Beijing, China. 11. Nahar N, Mosihuzzaman M and Dey S K; 1991, 'Analysis of free sugar and dietary fibre of seven local vegetables', 28th Annual Conference of Chemists, Indian Chemical Soc., Dec 17-20, Calcutta, India. 12. Mosihuzzaman M, Rahman S M M, Nahar N and Andersson R; 1992, 'Arabinans and arabinogalactan from some fruits of Bangladesh', XVth International Carbohydrate Symposium, July 5-10, Paris. 13. Mahmud H, Chowdhury T A, Rahman M M and Mosihuzzaman M; 1992, 'Some structural aspects of the water-soluble polysaccharides isolated from the leaves of Litsea glutinosa (Lauraceae)', XVth International Carbohydrate Symposium, July 5-10, Paris. 14. Nahar N, Mosihuzzaman M, Nasreen T, Ali L, Azad Khan A K, Nur-e-Alam M., Rokeya B and Hasan Z; 1992, 'Studies of hypoglycemic effects of Trigenella foenumgraecum seeds'. Seventh Asian Symposium on Medicinal Plants and Spices, Feb 2-7, Manila, Philippines. 15. Nahar N, Mosihuzzaman M and Aman A M; 1992, 'Isolation and characterisation of some terpenoid and phenolic acids from Abroma augusta root bark'. Seventh Asian Symposium on Medicinal Plants and Spices, Feb 2-7, Manila, Philippines. 16. Mosihuzzaman M, Nahar N and Mamun M I R; 1993, 'Chemical studies on some plant materials having anti-diabetic activity', 5th Asian Chemical Congress, Nov 8-12, Kuala Lumpur, Malaysia.

7 17. Nahar N, Sultana N and Mosihuzzaman M; 1993, 'Isolation and Characterisation of terpenoids from Momordica charantia whole plant', 5th Asian Chemical Congress, Nov 8- 12, Kuala Lumpur, Malaysia. 18. Ali L, Azad Khan A K, Mamun M I R, Mosihuzzaman M, Nahar N, Nur-e-Alam M and Rokeya B; 1993, 'Insulin releasing properties of fractions from Momordica charantia fruit on isolated rat islets', EASD meeting, Sept 6-9, Istanbul, Turkey. 19. Mosihuzzaman M; 1994, 'Plant material as a source of antidiabetic agents-an overview of research at BIRDEM and Dhaka University', First International Seminar on Plant Material as a Source of Antidiabetic Agents, Jan 8-10, Dhaka, Bangladesh 20. Nahar N, Mamun M I R, Nasrin T and Mosihuzzaman M; 1994, 'Hypoglycemic agents from Momordica charantia fruit pulp', ASOMPS VIII, June 12-16, Melaka, Malaysia. 21. Nahar N, Begum S A, Hussain M and Mosihuzzaman M; 1994, 'Terpenoids from antidiabetic plants', ASOMPS VIII, June 12-16, Melaka, Malaysia. 22. Mosihuzzaman M, Ali L, Azad Khan A K, Nahar N, Nasreen T, Nur-e-Alam M and Rokeya B; 1994, 'Characterisation of the hypoglycemic effects of Trigonella foenumgraecum seed', 15th IDF Congress, Nov 6-11, Kobe, Japan. 23. Ali L, Azad Khan A K, Hasan Z, Mamun M I R, Mosihuzzaman M, Nahar N, Nasreen T, Nur-e-Alam M and Rokeya B; 1994, 'Cytoplasmic Ca2+ modulation by an insulin releasing fraction of Momordica charantia in pancreatic B cells', 15th IDF Congress, Nov 6-11, Kobe, Japan. 24. Rokeya B, Ali L, Azad Khan A K, Hassan Z, Mosihuzzaman M, Nahar N, Nasreen T and Nur-e-Alam M; 1994, 'Screening of plant materials for hypoglycemic activities', 15th IDF Congress, Nov 6-11, Kobe, Japan. 25. Mosihuzzaman M, Nahar N, Azad Khan A K, Ali L and Rokeya B; 1995, 'Hypoglycemic agents from some Bangladeshi plant materials', 6th Asian Chemical Congress, May 22-25, Manila, Philippines. 26. Nahar N, Mamun M I R and Mosihuzzaman M; 1995, 'Isolation and characterisation of terpenoids from Pterospermum acerifolium plant', 6th Asian Chemical Congress, May 22- 25, Manila, Philippines,. 27. Mosihuzzaman M, Nahar N, Azad Khan A K, Rokeya B and Shrestha T M; 1995, 'Hypoglycemic effects of some high altitude medicinal plants'. Regional Workshop on Phytochemistry of High Altitude Medicinal Plants, Aug 21-25, Kathmandu, Nepal. 28. Mosihuzzaman M; 1995, 'Problem and Prospects of Network in Third World Countries', National Workshop on Instrument Maintenance and Repair, Aug 27-30, Kathmandu, Nepal. 29. Murshed S, Azad Khan A K, Ali. L, Banik N G, Mosihuzzaman M, Nahar N and Rokeya B; 1995, 'Effect of Gymnama sylvestre leaves on plasma levels of vitamin E in streptozotocin induced IDDM model rats', EASD Meeting 746, Sept 12-16, Stockholm, Sweden. 30. Rokeya B, Ali L, Azad Khan A K, Mamun M I R, Mosihuzzaman M, Nahar N and Nur- e-Alam M; 1995, 'Insulin releasing effects of Momordica charantia fractions of isolated rat islets', EASD Meeting 747, Sept 12-16, Stockholm, Sweden. 31. Mosihuzzaman M, Nahar N, Ali L, Rokeya B and Azad Khan A K; 1996, 'Hypoglycemic effects of some tropical medicinal plants', 6th International Symposium on Natural Products Chemistry, Jan 4-8, Karachi, Pakistan. 32. Mosihuzzaman M, Nahar N, Azad Khan A K, Rokeya B, Ali L, Chowdhury N S and Hannan, J M A; 1996, 'Effects of various extracts from four plants on serum glucose of nondiabetic and diabetic model rats', 32nd EASD Conference, Sept 1-5, Vienna, Austria. 33. Murshed S, Sarkar S, Rokeya B, Ali L, Azad Khan A K, Nahar N and Mosihuzzaman M; 1996, 'Effects of Gymnema sylvestre and Momordica charantia on serum lipid levels of IDDM and NIDDM model rats', 32nd EASD Conference, Sept 1-5, Vienna, Austria.

8 34. Mosihuzzaman M, Nahar N, Azad Khan A K, Ali L and Rokeya B; 1996, 'Studies of hypoglycemic effects of some medicinal plants', IUPAC Natural Products Conference, Sep 15-20, Chicago, USA. 35. Mosihuzzaman M, Nahar N, Azad Khan A K, Ali L and Rokeya B; 1996, 'Hypoglycemic effects of some medicinal plants, Symposium on Bioactive Natural Products, Nov 11-15, Kandy, Sri Lanka. 36. Nahar N, Mamun M I R and Mosihuzzaman M; 1996, 'Isolation and characterisation of terpenoids and glycosides from Pterospermum acerifolium and Pterospermum semisagittatum plants', Symposium on Bioactive Natural Products, Nov 11-15, Kandy, Sri Lanka. 37. Nahar N, Mamun M I R, Mosihuzzaman M. and Khan S H; 1996, 'Isolation and characterisation of triterpenoids from Pterospermum acerifolium and Pterospermum semisagittatum, 6th International Symposium on Natural products Chemistry, Jan 4-8, Karachi, Pakistan. 38. Nur-e-Alam M, Rokeya B, Chowdhury N S, Ali L, Mosihuzzaman M, Nahar N and Azad Khan A K; 1997, 'Effects of three Nepalese plants on serum glucose levels of normal and diabetic model rats', XVI International Diabetes Federation Congress, July 20-25, Helsinki, Finland. 39. Chowdhury N S, Mosihuzzaman M, Azad Khan A K, Rokeya B, Ali L and Nahar N; 1997, 'Characterisation of the hypoglycemic effects of different extracts from five antidiabetic plant materials', XVI International Diabetes Federation Congress, July 20- 25, Helsinki, Finland. 40. Hannan J M A, Haque S, Ali L, Nahar N, Mosihuzzaman M and Azad Khan A K; 1997, 'Retardation of intestinal carbohydrate absorption by hot waters extract of Trigonella Foenum graecum in rats', XVI International Diabetes Federation Congress, July 20-25, Helsinki, Finland. 41. Rokeya B, Ali L, Azad Khan A K, Mosihuzzaman M, Mainuddin S M, Nahar N, Chowdhury N S and Hannan J M A; 1997, 'Insulin releasing effects of some pure compounds from Hemidesmus indicus on isolated rat islets', XVI International Diabetes Federation Congress, July, 20-25, Helsinki, Finland, 42. Mamun M I R, Mosihuzzaman M, Nahar N, Reutrakul V, Lundgren L and Andersson R; 1997-98, 'Isolation and characterization of three new iridoid glycosides from Gardenia erythroclada', 7th International Symposium on Natural products Chemistry, Dec 28 - Jan 8, Karachi, Pakistan. 43. Nur-e-Alam M, Rokeya B, Chowdhury N S, Ali L, Mosihuzzaman M, Nahar N and Azad Khan A K; 1997-98, 'Effect of three Nepalese plants on serum glucose levels of normal and diabetic model rats', 7th International Symposium on Natural products Chemistry, Dec 28 - Jan 8, Karachi, Pakistan.

44. Mosihuzzaman M, Nahar N, Rokeya B, Azad Khan A K and Ali L; 1997-98 'Antidiabetic, anticancer and anti-HIV activities of some tropical plant mat erials', 7th International Symposium on Natural Product Chemistry, Dec 28 – Jan 01, Karachi, Pakistan. 45. Nahar N, Mosihuzzaman M, Rokeya B , Azad Khan A K and Ali L; 1997-98, 'Studies of hypoglycemic agents from plant materials', 7th International Symposium on Natural Product Chemistry, Dec28 – Jan 01, Karachi, Pakistan. 46. Rokeya B, Mosihuzzaman M, Nahar N, Azad Khan A K and Ali L; 1997-98, ‘Antidiabetic effect of some plant materials through delayed carbohydrate absorption’, 7th International Symposium on Natural Product Chemistry', Dec 28 – Jan 01, Karachi, Pakistan. 47. Mosihuzzaman M; 1998, 'Antidibetic, anticancer and anti-HIV activities of some tropical plant materials', the Ninth Asian Symposium on Medicinal Plants, Spices and other Natural Products, Sept 24-28, Hanoi, Vietnam.

9 48. Nahar N, Mamun M I R, Mosihuzzaman M, Andersson R, Lundgren L and Reutrakul V; 1998, 'Studies of hypoglycemic extracts of Pterospermum acerifolium and Pterospermum semisagittatum', the Ninth Asian Symposium on Medicinal Plants, Spices and other Natural Products, Sept 24-28, Hanoi, Vietnam. 49. Nahar N, Rokeya B, Parveen S, Nur-e-Alam M, Ali L, Mosihuzzaman M and Azad Khan A K; 1999, 'Effect of a Natural compound on glucose level, lipid profile and total antioxidant status of type 1 diabetic rats', Diabetologia, 42, 875, (35th Annual Meeting of the EASD, Sept 28 - Oct 02, Brussels, Belgium. 50. Nahar N; 1999, 'Isolation and chracterization of low- and high molecular carbohydrates in plant materials' Regional Training on Chemistry of Natural Products and Related Fields, May 17-28, Kathmandu, Nepal. 51. Mosihuzzaman M; 1999, 'Traditional and Modern use of Plants for Healing', One day seminar of Bangladesh Association for the Scientists and Scientific Professions (BASSP), May, BCSIR, Dhaka, Bangladesh. 52. Mosihuzzaman M; 1999, 'Drugs from Medicinal Plants', One day seminar of Society for the Pharmaceutical Chemists (SPC), February, Dhaka, Bangladesh. 53. Mosihuzzaman M; 2000, 'Traditional and Modern Use of Plants', The 8th International Symposium on Natural Products Chemistry, Jan 18-22, Karachi, Pakistan. 54. Nahar N, Das R N, Mosihuzzaman M and Reutrakul V; 2000, 'Chemical Studies of Cytotoxic Extracts of Three Plant Materials', The 8th International Symposium on Natural Products Chemistry, Jan 18-22, Karachi, Pakistan. 55. Murshed S, Rokeya B, Ali L, Nahar N, Azad Khan A K and Mosihuzzaman, 2000, 'Chronic Effect of Pterospermum acerifolium Bark on Glycemic and Lipidemic Status of Type 2 Diabetic Model Rats', 3rd International Seminar on Plant Materials as a Source of Antidiabetic Agents, Nov 16-17, Dhaka, Bangladesh. 56. Rashid R, Rokeya B, Nahar N, Ali L, Mosihuzzaman M and Azad Khan A K; 2000, 'Evaluation of the Hypoglycemic Effect of Medicinal Plants from Balochistan', 3rd International Seminar on Plant Materials as a Source of Antidiabetic Agents, Nov 16- 17, Dhaka, Bangladesh. 57. Hannan J M A, Faruque M O, Bethe R A, Rokeya B, Ali L, Azad Khan A K, Mosihuzzaman M and Nahar N, 2000; 'Effect of an Antihyperglycemic Plant Fraction M001 on Carbohydrate and Lipid Metabolism in Rats', 3rd International Seminar on Plant Materials as a Source of Antidiabetic Agents, Nov 16-17, Dhaka, Bangladesh. 58. Mamun M I R, Mosihuzzaman M, Nahar N, Azad Khan A K, Ali L, Rokeya B, Lundgren L, Andersson R and Reutrakul V; 2000, 'New Hypoglycemic Agent from Pterospermum acerifolium', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 59. Das R N, Mosihuzzaman M, Nahar N and Reutrakul V; 2000, 'Chemical Investigation of Anticancer Plant Oroxylum indicum', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 60. Begum S A, Kenne L, Lundgren L, Andersson R, Mosihuzzaman M, Nahar N, Cragg G M and McCloud T G; 2000, 'Cytotoxic Bufadienolides from the Bulb of Urginea indica', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 61. Das R N, Mosihuzzaman M, Nahar N and Reutrakul V; 2000, 'Isolation and Characterization of Compounds from Calophyllum floribunda', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 62. Dey S K, Nahar N and Mosihuzzaman M; 2000, 'Biological and Chemical Studies of Two Anticancer Plant Extracts', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 63. Alam A B M M, Nahar N, Hussain M, Fazal M and Mosihuzzaman M; 2000,

10 'Constituents of the Essential Oil from Aquilaria agallocha Roxb', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 64. Abedin M Z U, Alam A B M M, Nahar N, Mosihuzzaman M, Andersson R and Mamun M I R; 2000, 'Phenylethanoid Glycoside and other Three Compounds from Leucas indica', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 65. Faruk M J A, Nahar N, Mosihuzzaman M and Rashid M A; 2000, 'Chemical Studies of Hypoglycemic and Anti-HIV Extracts of Lagerstroemia speciosa (L) Pers', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 66. Khan S H, Nahar N, Mosihuzzaman M and Reutrakul V; 2000, 'Chemical Constituents of an Antidiabetic Plant', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 67. Sultana A, Nahar N, Mosihuzzaman M and Andersson R; 2000, 'Studies of Anticancer Extractives of Streblus asper Leaves', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 68. Andersson R, Begum S A, Kenne L, Lundgren L, Nahar N and Mosihuzzaman M; 2000, 'A Strategy for Structural Elucidation by 1D and 2D NMR', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 69. Jahan I A, Mosihuzzaman M, Chaudhary M I, Nahar N, Shaheen F and Parveen Z; 2000, 'Bioactive Diterpenoids from Gelonium multiforum', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 70. Mostafa M, Mosihuzzaman M, Chaudhary M I, Nahar N and Ahmed S; 2000, 'Anti- oxidant Hydroxycinnamic Acid Ester of Phenethylalcohol Glycoside isolated with two Flavones from Leucas indica', Tenth Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS X), Nov 18-23, Dhaka, Bangladesh. 71. Mosihuzzaman M; 2001, 'Studies on Antidiabetic Plant Materials' Indian National Science Congress, Jan 03-07, New Delhi, India. 72. Mosihuzzaman M; 2001, 'Antidiabetic Agents from some Bangladeshi Plants', World Chemistry Congress, July 1-6, Brisbane, Australia. 73. Nahar N and Mosihuzzaman M; 2001, 'Some Novel Anticancer Agents from Bangladeshi Plants', World Chemistry Congress, July 1-6, Brisbane, Australia. 74. Nahar N; 2002, 'Some constituents of antidiabetic plants', 2nd National Seminar on Antidiabetic Plant Materials: Separation Techniques and Biological Testing, Feb 10-11, Chittagong, Bangladesh. 75. Matin M, Khan S H, Shoeb M, Sultana N, Nahar N, Mosihuzzaman M and Andersson R; 2002, 'Flavonoid glycosides from Scoparia dulcis', 2nd National Seminar on Antidiabetic Plant Materials: Separation Techniques and Biological Testing, Feb 10-11, Chittagong, Bangladesh. 76. Anwar K J, Mosihuzzaman M, Nahar N, Khan S H, Shoeb M, Rashid R, Azad Khan A K, Ali L, Rokeya B, and Sultana N; 2002, 'Biological and Chemical Studies of the aerial part of Otostegi aucheri', 2nd National Seminar on Antidiabetic Plant Materials: Separation Techniques and Biological Testing, Feb 10-11, Chittagong, Bangladesh 77. Begum S A, Kenne L, Lundgren L, Andersson R, Nahar N, and Mosihuzzaman M; 2002, 'Bioactivity and chemical studies of Urginea indica Kunth (Liliaceae)', 2nd National Seminar on Antidiabetic Plant Materials: Separation Techniques and Biological Testing, Feb 10-11, Chittagong, Bangladesh 78. Begum S A, Nahar N, and Mosihuzzaman M; 2002, 'Bioactivity and chemical studies of Scoparia dulcis', 2nd National Seminar on Antidiabetic Plant Materials: Separation Techniques and Biological Testing, Feb 10-11, Chittagong, Bangladesh.

11 79. Mosihuzzaman M; 2002, 'Biological and Chemical Studies on Medicinal Plants in Dhaka', June 28-39, Addis Ababa, Ethiopia. 80. Mosihuzzaman M; 2002, 'Studies of Anti-cancer, Anti-HIV and Antidiabetic Plants in Dhaka', SAARC Regional Workshop on Medicinal Plants, June 11-13, Dhaka, Bangladesh. 81. Mamun M I R, Matin M, Nahar N and Mosihuzzaman M; 2002, 'Biological and Chemical Studies of some Antidiabetic Plants', 7th Eurasia Conference on Chemical Sciences, March 8-12, Karachi, Pakistan. 82. Nahar N, Mamun M I R, Mosihuzzaman M and Nag S; 2002, 'Organochlorine pesticides in fresh water fishes of Bangladesh', 10th IUPAC International Congress on the Chemistry of Crop Protection' Aug 04-09, Basel, Switzerland. 83. Mamun M I R, Nahar N, Mosihuzzaman M and Nag S; 2002, 'Organochloripesticides in local vegetables of Bangladesh', 10th IUPAC International Congress on the Chemistry of Crop Protection' Aug 04-09, Basel, Switzerland. 84. Nahar N; 2002, 'Bioactive Compounds from some local plants', SAARC Regional Workshop on Medicinal Plants, June 11-13, Dhaka, Bangladesh. 84. Mosihuzzaman M; 2002, 'Studies of Anti-cancer, Anti-HIV and Antidiabetic Plants in Dhaka', SAARC Regional Workshop on Medicinal Plants, June 11-13, Dhaka, Bangladesh. 85. Mosihuzzaman M; 2003, 'Chemistry and Biology in Drug Discovery from Plant Sources', Conference on Drug Discovery and ICT, March 2-4, Kolkata, India. 86. Mosihuzzaman M.; 2003, IUPAC General Assembly, Council Meeting and Chemical Conference & World Chemistry Leadership Meeting (WCLM), August 15- 19, Ottawa, Canada. 87. Mosihuzzaman M, Rokeya B, Chakraborti S, Biswas T K, Ali L, Mukharjee B P, Nahar N and Azad Khan A K; 2003, 'Mechanism of hypoglycemic action of Caesalpinia bonducella in Type 2 diabetic Long-Evans rats', International Diabetic Federal Conference, August 26-31, Paris, France. 88. Rokeya B, Chakraborti S, Mukharjee B P, Nahar N and Azad Khan A K; 2003, 'Insulin releasing effects of some pure compounds from the Caesalpinia bonducella seeds on isolated rat islets', International Diabetic Federal Conference, August 26-31, Paris, France. 89. Murshed S, Rokeya B, Ali L, Nahar N, Mosihuzzaman M and Azad Khan A K; 2003, 'Effects of an antidiabetic plant extract GE007 on plasma free fatty acid composition in Type 1 diabetic rats ', International Diabetic Federal Conference, August 26-31, Paris, France. 90. Hannan J M A, Rokeya B, Khaleque J, Akhter M, Ali L, Nahar N, Mosihuzzaman M and Azad Khan A K; 2003, 'Hypoglycemic, hypolipidemic and hyperinsulinemic effect of a plant extract PE026 leaves in Long Evan rats', International Diabetic Federal Conference, August 26-31, Paris, France. 91. Mosihuzzaman M; 2003, FACS General assembly & 10th ACC Asian Chemical Congress, Oct 20-23, Hanoi, Vietnam. 92. Mosihuzzaman M; 2003, Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS XI), Oct 26-31, Kunming, China. 93. Rashid R, Rokeya B, Nahar N, Ali L, Mosihuzzaman M. and Azad Khan A K; 2004, 'Evaluation of the Hypoglycemic Effect of Medicinal Plants from Baluchistan', "4th International Seminar of Asian Network of Research on Antidiabetic Plants (ANRAP)", Jan 16-18, Kolkata, India. 94. Murshed R, Rokeya B, Ali L, Nahar N, Azad Khan A K and Mosihuzzaman M.; 2004, 'Chronic Effect of a Plant Extract E4b on Glycemic and Lipidemic Status of Type 2 Diabetic Model Rats', "4th International Seminar of Asian Network of Research on Antidiabetic Plants (ANRAP)", Jan 16-18, Kolkata, India. 95. Mostafa M, Skocng S D, Nahar N, and Mosihuzzaman M.; 2004, 'Chemical and Biological Studies of Bridelia ndellensis', "4th International Seminar of Asian

12 Network of Research on Antidiabetic Plants (ANRAP)", Jan 16-18, Kolkata, India. 96. Khan S H, Nahar N, and Mosihuzzaman M, Sultana N, Rokeya B, Ali L and Azad Khan A K ; 2004, 'Hypoglycemic Activity and Isolation of Some Chemical Constituents from Fissistigma rubiginosum (A.DC.Merr) (Anonaceae)', "4th International Seminar of Asian Network of Research on Antidiabetic Plants (ANRAP)", Jan 16-18, Kolkata, India. 97. Jahan I A, Nahar N, and Mosihuzzaman M, Rokeya B, Ali L, Azad Khan A K, Makhmur T, Atta-ur-Rahman and Choudhary M I ; 2004, 'Hypoglycemic and Antioxidant Agents from Ficus racemosa Fruits', "4th International Seminar of Asian Network of Research on Antidiabetic Plants (ANRAP)", Jan 16-18, Kolkata, India. 98. Bhagat M, Rokeya B, Jahan M, Purohit A, Nahar N, Mosihuzzaman M, Azad Khan A K and Ali L; 2004, 'Effect of Ethanol Extract of Capparis decidua on Serum Glucose Level of Type 1 and Type 2 Diabetic Model Rats, "4th International Seminar of Asian Network of Research on Antidiabetic Plants (ANRAP)", Jan 16-18, Kolkata, India. 99. Mosihuzzaman M; 2004, Chemistry and biology in Drug Discovery from Plant Sources', "IUPAC International Conference on Biodiversity and Natural Products: Chemistry and Medical Applications", Jan 26-31, New Delhi, India. 100. Jahan I A, Nahar N, Mosihuzzaman M, Shaheen F, Atta-ur-Rahman and Choudhary M I; 2004, 'Six New Diterpenoids from the Cytotoxic Extract of Suregada multiflora', "IUPAC International Conference on Biodiversity and Natural Products: Chemistry and Medical Applications", Jan 26-31, New Delhi, India 101. Mamun M. I. R., Nahar N., Nag S., Zamir R. and Mosihuzzaman M; 2004, 'Analysis of Pesticide Residues in Some Local Fish and Vegetables', "15th International Plant Protection Congress", May 11-16, Beijing, China. 102. Mamun M. I. R., Nahar N., Nag S., Zamir R. and Mosihuzzaman M; 2004, 'Analysis of Pesticide Residues in Some Local Fish and Vegetables', "5th European Pesticide Residue Workshop", June 13-16, Stockholm, Sweden. 103. Mosihuzzaman M; 2004, 'Chemical and Biological Studies of Some Medicinal Plants', "The 9th International Chemistry Conference in Africa", Aug 2-7, Arusha, Tanzania.

104. Mosihuzzaman M.; 2004, 'Studies of Persistance of Some Pesticides Used in Bangladesh', "Inaugural Conference of the African Network for Chemical Analysis of Pesticides", August 8-11, Arusha, Tanzania. 105. Mosihuzzaman M., 2006, “Studies of Organic Pollutants in some Food and Environmental samples”, 9th Eurasia Conference on Chemical Sciences, 8-15 September, Antalya, Turkey. 106. Mosihuzzaman M, 2007, “Prospects and Constraints of Developing Anti-Diabetic Drugs from Plant Materials”, 12th Asian Chemical Congress (12ACC), 23-25 August, Kuala Lumpur, Malaysia. 107. Mosihuzzaman M, 2008, “Research on Anti-Diabetic Plants---An Experience”, 11th International Symposium on Natural Product Chemistry, 29 October – 01 November, Karachi, Pakistan. 108. Mosihuzzaman M, 2008, “Research on Anti-Diabetic Plants---Challenges and Incentives” 13th Asian Symposium on Medicinal Plants, Spices and Other Natural Products (ASOMPS XIII), 3-6 November, Hyderabad, India. 109. Mosihuzzaman M, 2009, “Herbal Products for Healthcare”, International Herbal Conference: Herbal Medicine—Evaluation of Quality, Efficiency and Safety, 26-28 February, Bangalore, India. 110. Mosihuzzaman M, 2009, “Herbal Products in Healthcare”, 13th Asian Chemical Congress, 14-17 September 2009, Shanghai, China.

13 Effects of Enhydra Fluctuans Extract on Glycemic and Lipidemic Status and their Relation with Heavy Metal in Normal and Type-2 Diabetic Model Rats

Mohammad Nazmul Hasan*1, Salima Akter*2, Begum Rokeya3, Md Shahinul Haque Khan4, Mahtab Uddin Ahmed5, Liaquat Ali6, Md. Morsaline Billah7, Farah Sabrin1§,

1Department of Biotechnology and Genetic Engineering, Mawlana Bhashani Science and Technology University, Santosh, Tangail, Bangladesh; 2Department of Medical Biotechnology, Bangladesh University of Health Sciences, Dhaka, Bangladesh; 3Department of Pharmacology, Bangladesh University of Health Sciences, Dhaka, Bangladesh; 4Department of Chemistry, Bangladesh University of Health Sciences, Dhaka, Bangladesh; 5Department of Pathology, Bangladesh University of Health Sciences, Dhaka, Bangladesh; 6Department of Biochemistry and Cell Biology, Bangladesh University of Health Sciences, Dhaka, Bangladesh; 7Department of Biotechnology and Genetic Engineering, Khulna University, Khulna, Bangladesh

Abstract

The experimental studies regarding antidiabetic and antilipidemic activities of Enhydra fluctuans plant have not been investigated in isolated or cadmium induced condition. The aim of the study was to evaluate the potential effects of E fluctuans extract on diabetes and dyslipidemia and their relation with the heavy metal in normal and type 2 diabetic rats. A total of 48 Long-Evans rats were included in this study (32 were healthy control and 16 were type 2 diabetic rats). The rats were divided into the following six groups: Control (water with DMSO), Cd (Cdcl2 50 mg/kg), NP (Normal plant 200 mg/kg), NPCd (Normal plant with Cdcl2), DMP (Diabetes Mellitus with plant 200 mg/kg) and

DMPCd (Diabetes Melltus with plant and Cdcl2). Blood glucose and other biochemical parameters were estimated by enzymatic colorimetric method. Histological analysis of liver and heart were done by hematoxylin-eosin (H & E) method. Twenty one days treatment of Enhydra fluctuans extracts at a dose of 200 mg/kg resulted in a significant reduction of blood glucose level in the diabetic rats (p<0.05), (p<0.001) respectively. The plant extract did not have any direct effect on total blood lipids but, the ratio of TG to HDL-C showed the beneficial effects in NP, DMP and DMPCd groups (p<0.05). Cd induction significantly reduced body weight in the Cd, NPCd and DMPCd groups while liver dysfunction was significantly induced in NPCd groups. The histology of liver and arch of aorta of heart showed severe damage in the Cd group compared to other counterparts. Among the treatment groups, the NP group had the lowest steotasis, atherosclerotic change, inflammation and necrosis. The association analyses in the cadmium group revealed that body weight was associated with blood glucose, and hepatic damage with renal dysfunction (p<0.05). The data leads to the conclusion that E. fluctuans seems to have agents which may have potential antidiabetic and antilipidemic activities. The plant has ameliorating effects on cadmium toxicity which at least in part, is mediated by improving liver impairment. ANALYSIS OF HARMFUL CONSTITUENTS IN SOME ANTIDIABETIC HERBAL PREPARATIONS (ADHPS) AVAILABLE IN BANGLADESH

Md Ranzu Ahmed 1,2 Ismet Ara Jahan3, Badhan Shaha3, MA Sayeed2, B Rokeya4 and M Mosihuzzaman5

1Dept of Chemistry, Bangladesh University of Health Sciences (BUHS); 2Dept of Applied Chemistry & Chemical Engineering, University of Rajshahi; 3Bangladesh Council of Scientific and Industrial Research (BCSIR); 4Dept of Pharmacology, Bangladesh University of Health Sciences (BUHS; 5ICNPR, Bangladesh University of Health Sciences (BUHS), Bangladesh.

Background and Objectives: Since time immemorial human beings depend on trees/plants for preparing their foodstuffs, clothing, shelter and healthcare. It is our moral duty to find out whether these plant materials are safe to use. In Bangladesh a huge number of ADHPs are being used by the people without any scientific validity. Therefore, the present study was undertaken to make a preliminary assessment regarding the concentrations of some of the most common toxic metals in ADHPs.

Methods: Eleven ADHPs were bought from local herbal medicinal shops and analyzed for toxic metals by using a Shimadzu AA-6800 Atomic Absorption Spectrophotometer (AAS, Furnace system) controlled by Wizard Software. All the 11 ADHPs were analyzed for the presence of toxic metals like nickel (Ni), copper (Cu), manganese (Mn), chromium (Cr), cadmium (Cd), lead (Pb) and arsenic (As).

Results: The results showed that all the eleven ADHPs contained Ni, Cu, Mn, Cr, Cd, Pb, and As. Exceptions were with Cu in ADHP-5 and 6, Mn in ADHP-5 and 6, Cr in ADHP-2, 4 and 11, Cd in ADHP- 2, 4, 5, 6 and 7 and Pb in ADHP-1, 2, 4, 5, 6, 7, 9 and 11 that were below detection level. There are several regulatory bodies that set specific allowable limit for toxic metal content in herbal and traditional preparations based on different guidelines and this permissible limit varies among these regulatory bodies.

Conclusion: Presence of one or more of these metals in ADHPs may indicate potential risk of the accumulation of these toxic metals leading to toxicity. Most of the ADHPs contained small amount of toxic metals but some of ADHPs (ADHP-3, ADHP-8, ADHP-9 and ADHP-10) crossed the safety limit set by WHO, US FDA and HAS Singapore permissible limit. It may be concluded that the level of toxic metals found in herbal preparations are quite high, their long term use may cause serious harm for health.

Corresponding & Presenting Author: Dr Md Ranzu Ahmed, Dept of Chemistry, BUHS, Mobile: +88 01711 218760, Email: [email protected] Applications of Preclinical studies in combating diabetes with plant materials

Begum Rokeya

Dept of Pharmacology, Bangladesh University of Health Sciences, Dhaka, Bangladesh

Diabetes mellitus is a complex heterogenous metabolic disorder where multiple abnormalities can be detected in diverse tissues. Therefore, it is very difficult to get a single group of effective compound(s) to treat this complicated disease. Since existing antidiabetic medications have considerable side effects so, there is a continuous search for new drugs that would potentially have less side effects. Medicinal plant drug discovery provides important leads against various pharmacological targets. As more than 80% of the population of the developing countries depend on plant materials for their primary health care, it is imperative to scientifically evaluate the folkloric information on plant materials used for diabetes.

It is well known that animal models have enormously contributed to the study of diabetes. Use of good laboratory practices, is a prerequisite for preclinical laboratory studies which is the intermediate step in drug development process. In Preclinical studies the test materials/drugs undergo laboratory and animal testing to answer basic questions about safety. The two types of preclinical research are: In Vivo & In Vitro.

Research with antidiabetic plant materials have been started in Bangladesh Institute of Research and Rehabilitation in Diabetes and Endocrine and Metabolic Disorder (BIRDEM) since 1990 and it is now being continued in Bangladesh University of Health Sciences (BUHS). In BUHS lab series of experiments for screening the hypo-/antihyperglycemic activities of plant materials are being conducted in rodents model of diabetes including fasting and different prandial states. Morever, several in vivo and in vitro studies are being practiced with antidiabetic plant materials. In vivo studies include: i) measurement of unabsorbed sucrose content in the gut, ii) intestinal glucose absorption studies, iii) assessment of intestinal disaccharidase activity and iv) assessment of gastrointestinal motility. In vitro studies are: i) assessment of insulinotropic action in perfused rat pancreas, ii) assessment of insulin-releasing action in isolated islets from rodent pancreas with bioassay-guided isolation of compound(s)/fraction(s) from plants. The results of preclinical studies showed that some of the studied plants have potential effect in combating diabetes.

Herbal Medicine in Health Care----Prospects and Challenges

Mohammed Mosihuzzaman Department of Chemistry, University of Dhaka, Dhaka-1000, Bangladesh [email protected]

Abstract

It is now generally accepted that the modern pharmaceuticals will remain out of reach of many people and the dream of ‘health for all’ may only be realized by modernizing the use of herbal medicine. Although herbal medicine is increasingly being used worldwide, there are genuine concerns about the safety, claimed efficacy and quality of much of the herbal medical products. These issues have been reviewed and the salient features are included in this presentation. The historical background and the philosophy of herbal medical practice and its status at the present time science have been outlined. Although the general perception is that herbal medicines are safe due to their age-old usage, serious adverse effects have been reported for many herbal medicines. These are primarily due to intentional adulteration or accidental contamination. Methods of identifying contaminants and assuring safety by proper toxicity tests have been outlined. As herbal medicine has a holistic approach, assessment of efficacy is difficult. Adapting the methodologies used for modern medicine, practical ways of assessing claimed efficacy are described. Pragmatic approaches of assuring reproducible standard of herbal medicine by using modern tools of fingerprinting the chemical profile of herbal medicine have been discussed. As much of the traditional herbal medical knowledge is scattered around the world at the family & community levels and in tribal areas, there is the obvious danger of losing the knowledge unless they are quickly documented. Difficulties in documenting herbal medical knowledge specially due to concerns of Intellectual Property Rights (IPR), have been highlighted.

Key words:: Herbal medicine, safety, contamination, adulteration, adverse effects, efficacy, standardization, fingerprinting, documentation, intellectual property rights.

Hypoglycemic and antioxidant activities of some antidiabetic herbal preparations available in Bangladesh

M R Ahmed1, B Rokeya2, I A Jahan3, MA Sayeed4, L Ali5 and M Mosihuzzaman6

1Department of Chemistry, Bangladesh University of Health Sciences (BUHS),2Dept of Pharmacology, Bangladesh University of Health Sciences (BUHS), 3Chemical Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), 4Department of Applied Chemistry & Chemical Engineering, University of Rajshahi, 5Department of Biochemistry and Cell Biology, Bangladesh University of Health Sciences (BUHS), 6 ICNPR, Bangladesh University of Health Sciences (BUHS), Bangladesh.

Background: Diabetic Mellitus has turned to be a menace to public health because of unavailability of adequate drugs to manage this disorder. A scientific investigation of traditional antidiabetic herbal medicine may provide valuable leads for the development of alternative drugs.

Aims: The present study was undertaken to evaluate the hypoglycemic (in type 2 dibetic model rats) and antioxidant properties of five antidiabetic herbal preparations manufactured locally and readily available in Bangladesh.

Methods: Five Antidiabetic Herbal Preparations (ADHPs) (ADHP-1 Sijijium Jamb; ADHP-2 Syp ABC-3; ADHP-3 Cap Dicure; ADHP-4 Tab Alisa Garlitab and ADHP-5 Tab Ziabit) produced by different local herbal pharmaceutical companies, were collected from shops in Dhaka city. Long Evans rats were bred at animal house in Bangladesh University of Health Sciences (BUHS). Type 2 diabetes was induced by a single ip injection of STZ to 48 hours old pups bred at BUHS animal house 3 months later after confirming with an OGTT Type 2 rats were selected for experiment. All of the ADHPs at a dose of 2.5 g/kg bw were fed orally to T2DM model rats for 4 weeks with a single feeding daily. Blood samples were collected on 0 day and 14th day by cutting the tail tip and on 28th day by decapitation of rats. Serum fasting glucose, lipid profiles, insulin and liver glycogen content were measured by GOD-PAP, enzymatic colorimetric, ELISA and Anthrone-sulphuric acid method respectively; LDL level was calculated by Friedewald formula. The Methanol extracts of ADHPs were screened for total phenolic, flavonoids and tannin contents. ADHPs were also screened for free radical scavenging activity by using ABTS [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)]. Data were analyzed by unveriate and multivariates tools.

Result: Among all the 5 ADHPs tested only 2 were found to be effective in terms of glucose lowering effect. Treatment with ADHP-2 & 3 showed significant reduction in serum glucose on 28th day (mmol/L, M±SD; 0 day vs 28 day, 8.64±0.83 vs 7.48±1.27, p=0.029; ADHP-2) (8.53.±0.98 vs: 6.49±0.79, p=0.001, ADHP-3). Glibenclamide treatment group also significantly reduced the blood glucose level on 14th day and 28th day when it was compared to 0 day value (mmol/L, M±SD; 0 day vs 14 day, 8.05.±0.62 vs 7.02±1.02, p<0.05 and 0 day vs 28 day, 8.05.±0.62 vs: 6.77±1.52, p<0.025), respectively. Other ADHPs (ADHP-1, 4 & 5) did not show any significant blood glucose lowering effect. Fasting serum insulin levels were increased by 30% and 63% in ADHP-2 & 3 treated groups respectively on 28th day in comparison to baseline value. ADHP-1 decreased triglyceride level significantly (mg/dl, M±SD; 0 day vs 28 day, 80.±11 vs 61±7, p=0.014). LDL-C level decreased by 8% and 9% by the treatment of ADHP-2 & 3 group on final day. Liver glycogen content was increased by 5%, 57%, 27%, 45% and 9% after 28th days by the corresponding treatment with ADHPs-1, 2, 3, 4 & 5. Results of the phytochemical analysis revealed that phenolic content was highest in ADHP-1 (591.21±0.15 mg/g) compared to gallic acid (518.52±0.16 mg/g) equivalent, high amount of total flavonoid content was observed in ADHP-2 (27.39 ± 0.17 mg), and in ADHP-3 (30.67± 0.13 mg/g) compared to 30.67±0.13 mg/g of quercetin. The total tannin content was highest in ADHP-1 (40.10±0.13 mg/g) compared to 26.0±0.16 mg/g of tannic acid, equivalent. ADHP-1, 3 & 4 showed significant ABTS free radical scavenging activity (98.38±0.01, 96.03±0.01 & 96.93±0.02 %) in 400 ppm concentrations, respectively in comparison to the standard ascorbic acid 99.9±0.01 % inhibition at the same concentration.

Discussion: For the management of diabetes mellitus, herbal preparations are being used frequently in Bangladesh as well as worldwide. Previously we have screened antidiabetic effect of another six herbal medicines on type 2 diabetic model rats. As a continuation of this process we further included another five ADHPs. The result showed that studied ADHPs are marketed in Bangladesh but significant blood glucose lowering effect in type 2 diabetic rats were found in only in ADHP-2 & ADHP-3 . ADHP-1, 2 & 3 contain a number of secondary plant metabolites like flavonoid, alkaloids, tannins and ADHP-1, 3 & 4 showed significant antioxidant activity which is beneficial for type 2 diabetes management. Since dependence on herbal preparations for managing Diabetes is increasing, therefore, thorough scientific investigation of herbal medicines for both safety & efficacy is necessary. Abstract

In the management of diabetes mellitus by conventional synthetic drugs not use safely by the patients, due to their prominent side-effects, thus the search for natural anti-diabetic plant products for controlling diabetes is going on. Glochidion Velutinum (Euphorbiaceae) commonly known as “Matachhar” have been recognized to possess several pharmacological properties, i.e cancer, antidiabetic, and wound healing, the plant also contain antioxidant, antimicrobial, ant-diarrheal, antiurolithitic and anti-inflammatory activity. My previous study on phytochemical screening showed the presence of higher percentage of alkaloids and/or glycosides / polyphenols in the methanol and ethanol extract. The presence of alkaloids and/or glycosides / polyphenols responsible for anti-diabetic activity; hence the present study was carried out to investigate the effect of methanol and ethanol extract of the leaves of the Glochidion velutinum in Neonatal streptozotocin (N-STZ) rats, a good animal model for type 2 diabetes (T2DM) research.

In these study, the leaves of Glochidion Velutinum were defatted with n-hexane. Then the defatted powder was successively extracted with chloroform, methanol and ethanol respectively using soxhlet extractor. For anti-diabetic assay Long-Evans rats bred at BUHS animal house were used in the study. Type 2 diabetes was induced by a single ip injection of STZ to 48 hours old pups (N- STZ) and 3 months later after confirming with an OGTT (N-STZ) rats were selected for experiment. The N-STZ rats were divided into 6 groups: I) WC = Water Control 5ml/kg bw. ; II) Gliz = Gliclazide (20mg/5ml/kg bw.) treated group; III) GVM625 = Absolute Methanol extract of G velutinum leaf with a dose of 625mg; IV) GVM400 = Absolute Methanol extract of G velutinum leaf with a dose of 400mg; V) GVE625 = Absolute Ethanol extract of G velutinum leaf with a dose of 625mg and VI) GVE400 = Absolute Ethanol extract of G velutinum leaf with a dose of 400mg. For evaluation of the anti-diabetic activity, the methanol (GVM) and ethanol (GVE) extracts of the leaves of the G.Velutinum were administrated orally to the streptozotocin (STZ) induce type-2 diabetic rats for 14 days at a dose of 625 and 400 mg per kg body weight. To compare the pharmacological studies, the drug glicalzide administrated orally at a dose of 20mg/5 ml/kg body weight for Type 2 model rats. For the control groups, 5ml water was administrated per kg body weight. Blood was collected by cutting the tail tip on 0 and by decapitation on 14th day. The following parameters were measured to evaluate the activity, such as body weight, fasting serum glucose (FSG; GOD-POD method), serum lipid profile (enzymatic-colorimetric method) and liver glycogen (Anthron sulphuric acid Method).

We observed that the results of these study exhibit that 14 days feeding of the extract did not cause any significant change in body weight of the rats. Glochidion velutinum methanol (dose 625mg/kg bw) and ethanol extract (dose 400mg/kg bw) were significantly decreased serum fasting glucose level on 14 day in comparison to baseline values (M±SD, 0day 9.4±2.2 vs 14 day 6.6±1.4; p=0.057 and 0day 8.1±2.4 vs 14 day 5.9±1.5; p=0.058) respectively. Methanol (dose 625mg/kg bw, p=0.016) and ethanol extract (dose 400 and 625mg/kg bw) of G velutinum also significantly decreased serum triglycerides level (p=0.024 and p=0.022) on final day compare with 0 day values. Serum cholesterol was decreased (p=0.049) on 14 day in ethanol extract (dose 400mg/kg bw) group significantly. Liver glycogen was increased by 23% in ethanol extract (dose 400mg/kg bw) in comparison with water control group.

The findings of our study suggested that methanol and ethanol extract of the leaves of the Glochidion velutinum possess significant anti-diabetic properties. This plant may contain potential anti-hyperglycemic agents which possibly act through some extra pancreatic mechanism that include glucose uptake and/or increased glycogenesis by liver.

Chemical studies on different parts of Farmed Tilapia Fish (Orechromis niloticus) and Their Feed

MSH Khan1, M Shahjalal2, S Sultana2, SK Roy3, MA-Ud-Daula2, M Akhter3, L Al3 1Department of Chemistry, Bangladesh University of Health Sciences (BUHS); 2Department of Applied Nutrition and Food Technology, Islamic University; 3Department of Biochemistry and Cell Biology, Bangladesh University of Health Sciences (BUHS).

Aims & Objectives: To study the chemical composition such as the proximate composition & some toxic heavy metal analysis of different parts of tilapia (Oreochromis niloticus) fish such as head (with gill) & body’s edible part (with skin) that are consumed by the Bangladeshi people and their corresponding feeds.

Materials and Methods: Tilapia fishes were collected from selected commercial farms in Mymensingh area, Bangladesh and corresponding feeds were also collected from respective suppliers. The dried and powdered fish parts (head with gill and edible parts of the body including skin) and feed samples were separately analyzed for proximate composition and heavy metals (Cd, Cr and Pb). The proximate analysis was performed according to standard methods. For heavy metal analysis was determined by Flame Atomic Absorption Spectrophotometer (AAS-6800, Shimadzu, Japan).

Results: Proximate analysis revealed that all feed contained higher amount (8.96-27.28%) of proteins where three out of four feeds had substantially (28.8-52.20%) lower amount of fat when compared to standard suggested for fish feed. All feeds had higher amount of fiber 3.25 -81.88% except one that had 44% lower than standard. The body and head of Tilapia contained approximately 18.94-19.66% and 15.05-15.69% protein by fresh basis. On the other hand proportion of fat was only 2.45-2.77% in body and 10.38-10.84% in head on fresh basis. The results of heavy metal analysis showed that among the estimated heavy metal concentrations, the highest concentrations were for Cr in one feed sample (735.209 µg/g), and the lowest were for Cd in head sample (0.009µg/g) on dry weight basis. Heavy metals did not exceed the recommended limit in fish sample except Cr in head.

Conclusions: It may be concluded that a) Commercially available Tilapia fish feed have only minute amounts of sand/silica b) They are rich in protein (from unknown sources) and fiber but poor in fat contents c) Both body and head of Tilapia in general have high protein content, but again they have less proportion of fat d) Heavy metals contents within tolerable limit in farmed tilapia fish in Bangladesh.

Effect of aqueous extract of Aegle marmelos fruit and leaf on glycemic, insulinemic and lipidemic status of type 2 diabetic model rats.

Mudi SR, Akhter M, Biswas SK, Muttalib MA, Choudhury S, Rokeya B, Ali L.

Abstract

Background Aegle marmelos is a popular fruit plant in the Indian subcontinent, various parts of which are traditionally used against various illnesses including diabetes mellitus (DM). However, the underlying mechanisms of the antidiabetic effects of the plant are not clear, especially in type 2 DM. The present study was undertaken to investigate the effect of aqueous extracts of A. marmelos fruits (AMFE) and leaves (AMLE) on glycemic, lipidemic, insulinemic, insulin resistance and β-cell functional status of type 2 diabetic model rats.

Methods An interventional study was designed using 20 type 2 diabetic rats. Type 2 DM was induced in Long Evans rats by a single intra-peritoneal injection of streptozotocin (90 mg/kg body weight) to 48 h old pups. Three months after induction of diabetes, the rats were divided into three independent groups: water-treated control group (n=6), AMLE-treated group (n=7) and AMFE-treated group (n=7). The rats were fed with extracts or water for 21 consecutive days and blood samples were collected at days 0 and 21 after an overnight fast. Data were expressed as mean±SD and analyzed by paired t-test or ANOVA as appropriate. Results

There were significantly lower blood glucose values in AMLE and AMFE groups at Endpoint compared to Baseline (mmol/l, mean±SD, Baseline vs. Endpoint, 7.04±1.0 vs. 6.06±0.92; p=0.032 and 7.04±0.97 vs. 5.87±0.93; p=0.047). There were also significantly lower serum insulin levels in AMLE and AMFE groups at Endpoint compared to Baseline (µIU/mL, mean±SD, Baseline vs. Endpoint, 14.02±5.48 vs. 7.57±2.90; p=0.026 and 11.54±4.83 vs. 6.58±4.36; p=0.008). Insulin resistance (HOMA-IR) was significantly improved both in AMLE and AMFE groups at Endpoint compared to Baseline (mean±SD, Baseline vs. Endpoint, 4.22±1.68 vs. 2.05±0.90; p=0.021 and 3.69±1.79 vs. 1.69±1.61; p=0.013). However, β-cell function or lipid profile did not show any significant alteration at Endpoint compared to Baseline in AMLE and AMFE groups.

Conclusions Aqueous extracts of A. marmelos leaf and fruit have hypoglycemic property which seem to be mediated by lowering of insulin resistance. These findings highlight the therapeutic potential of the extracts of A. marmelos in human type 2 DM and provides strong impetus for further studies.

Keywords: Aegle marmelos; aqueous extract; fruit and leaf; HOMA; insulin resistance; type 2 diabetes mellitus

ics: O om pe ol n b A Bhowmik et al., Metabolomics (Los Angel) 2016, 6:3 a c t c e e

M s s Metabolomics: Open Access http://dx.doi.org/10.4172/2153-0769.1000179 ISSN: 2153-0769

Research Article Open Access Glycemic, Insulinemic, Lipidemic and Antioxidant Status of nSTZ Rats after Chronic Administration of Cicer arietinum Extract Amrita Bhowmik1, Mossihuzzaman M2,3, Yearul Kabir4 and Begum Rokeya5* 1Department of Applied Laboratory Sciences, Bangladesh University of Health Sciences (BUHS), Bangladesh 2Department of Chemistry, Bangladesh University of Health Sciences (BUHS), Bangladesh 3International Centre for Natural Product Research (ICNPR), Bangladesh 4Biochemistry and Molecular Biology, University of Dhaka, Bangladesh 5Department of Pharmacology, Bangladesh University of Health Sciences (BUHS), Bangladesh

Abstract Background: The aims of the study were to evaluate glycemic, insulinemic, lipidemic and antioxidant properties of C. arietinum in neonatal-streptozotocin (nSTZ) rats. Materials and methods: Seeds were collected from the commercially available sources of Dhaka city, identified from Bangladesh National Herbarium and absolute ethanol extract was prepared. A single iv injection of STZ were given to neonate rats of Long Evans strain and 12 weeks later an OGTT was done and rats with fasting glucose level above 7.5 mmol/L were selected. The rats were divided into four groups: i) Water control, ii) Glibenclamide (5 mg/kg bw), iii) C. arietinum 0.625 g/kg bw (CA Ext 1) and iv) 1.25 g/kg bw (CA Ext 2) treated. Body weight was measured weekly. Blood was collected by cutting the tail tip on 0 day and by decapitation on 28 day. Fasting serum glucose, insulin, lipid profiles, creatinine, ALT, MDA, GSH, hepatic glycogen were measured. HOMA B% and HOMA S% were calculated. The data were analyzed using appropriate tools. Results: A significant decrease of fasting glucose level was noticed on 28 day with CA Ext 2 compared to baseline (p<0.05); 26% and 18% decrease were found in comparison to water and glibenclamide treated groups respectively. Blood glucose lowering effect was associated with insulin lowering effect of CA Ext 2. Treatment with CA Ext 2 improved HOMAB%, and both treated groups improved HOMA IR of nSTZ diabetic rats. Total cholesterol was significantly decreased in comparison to water control on 28 day (p=0.014); triglycerides decreased by 11% and HDL increased by 4% respectively in CA Ext 2 group. Serum ALT and creatinine levels were remained unchanged by C. arietinum. A significant increase of reduced-GSH level was found in CA Ext 1 treated group (p=0.031). Conclusion: CA Ext 2 showed significant hypoglycemic and antilipidemic effects most likely through decreasing insulin resistance and improving insulin sensitivity. It also has antioxidant activity that reduces the oxidative changes induced by STZ administration.

Keywords: Hyperglycemia; Diabetes; Lipids; Antioxidant; nSTZ; been renewed interest in plant medicine for the treatment against Cicer arietinum different diseases as herbal drugs are generally believed to be less toxic as reported in different publications [2-6]. Many indeginous Abbreviations plant products, obtained from fruits, leaves, roots, bark etc. have been ALT: Amino Alanine Transferase; ANOVA: Analysis of Variance; shown to possess multiple therapeutic properties like antidiabetic, BIRDEM: Bangladesh Institute of Research and Rehabilitation in antihyperlipidemic, antihypertensive, antioxidant, anticancer, Diabetes, Endocrine and Metabolic Disorders; FSG: Fasting Serum antimicrobial, anti-inflammatory, analgesic and etc. [7-12]. Therefore, Glucose; GSH: Reduced Gluthathion; GOD-PAP: Glucose Oxidase; scientists are now focusing their attention on natural compounds to HOMA B%: B cell secretion; HOMA S%: Insulin sensitivity; HOMA find, at least a lead, for antidiabetic agents. In this regard, carefully IR: Insulin Resistance; HDL: High Density Lipoprotein; Nstz: planned scientific research to identify the hypoglycemic plants with neonatal Streptozotocin; OGTT: Oral Glucose Tolerance Test; MDA: true therapeutic efficacy and safety is utmost needed inorder to develop Malondialdehyde; TG: Triglyceride; LDL: Low Density Lipoprotein; them as new therapeutics. TBARS: Thiobarbituric Acid Reactive Substances; SPSS: Statistical Chickpea (Cicerarietinum L.) is an important pulse crop which is Package for Social Science consumed all over the world, especially in the Afro-Asian countries. It is Introduction

Hyperglycemia and hyperlipidemia are two important features *Corresponding author: Rokeya B, Department of Pharmacology, Bangladesh of diabetes mellitus, an endocrine and metabolic disorder that has University of Health Sciences (BUHS), Bangladesh, Tel: +088-01711811350; become the most challenging public health problem of the 21st century. E-mail: [email protected] In modern medicine, no satisfactory effective therapy is still available Received June 09, 2016; Accepted July 11, 2016; Published July 18, 2016 to cure diabetes mellitus [1]. Diabetes management studies show that Citation: Bhowmik A, Mossihuzzaman M, Kabir Y, Rokeya B (2016) Glycemic, conventional antidiabetic agents like sulfonylureas are the least durable Insulinemic, Lipidemic and Antioxidant Status of nSTZ Rats after Chronic agents followed by metformin and thiazolidinediones. Therefore, Administration of Cicer arietinum Extract. Metabolomics (Los Angel) 6: 179. doi:10.4172/2153-0769.1000179 search for improved antidiabetic drug has been continued. Over the last several years the incretin-based therapies have got significant Copyright: © 2016 Bhowmik A, et al. This is an open-access article distributed importance although they are very much expensive especially for the under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the people of the least developing countries. In recent years, there has original author and source are credited.

Metabolomics (Los Angel), an open access journal ISSN: 2153-0769 Volume 6 • Issue 3 • 1000179 Citation: Bhowmik A, Mossihuzzaman M, Kabir Y, Rokeya B (2016) Glycemic, Insulinemic, Lipidemic and Antioxidant Status of nSTZ Rats after Chronic Administration of Cicer arietinum Extract. Metabolomics (Los Angel) 6: 179. doi:10.4172/2153-0769.1000179

Page 2 of 7 considered as a good source of not only carbohydrates but also protein, dose of 10 ml/kg bw. and the quality of protein is better than other pulses. Chickpea has 2. Glibenclamide (positive) control group (n=7): Treated with several potential health benefits and, in combination with other pulses glibenclamide at a dose of 5 mg//kg bw. and cereals, it could have beneficial effects on some of the important human diseases like cardiovascular disease, type 2 diabetes, digestive 3. CA Ext 1 treated group (n=8): Fed with ethanol extract of C. diseases and some cancers [13]. A significant antihyperglycemic arietinum at a dose of 0.625 g/kg bw. activity of the chickpea have been reported in STZ induced diabetic rats 4. CA Ext 2 treated group (n=8): Fed with ethanol extract of C. [14,15]. The aims of the study were to evaluate glycemic, insulinemic, arietinum at a dose of 1.25 g/kg bw. Water, Glibenclamide and CA Ext lipidemic and antioxidant properties of C. arietinum in neonatal- 1 & 2 were administered intragastrically through metallic tubes to the streptozotocin (nSTZ) diabetic rats. corresponding group of rats after 12 hrs fast. Place of Study Collection of Blood Sample for Biochemical Analysis The study was conducted in the Department of Pharmacology, Blood samples were collected from rats kept under fasting Bangladesh Institute of Research and Rehabilitation in Diabetes, conditions (12 hours) by amputation of the tail tip under diethyl ether Endocrine and Metabolic Disorders (BIRDEM) and in the Department anesthesia on the 0 day. On the 28th day after 12 hours fast, blood of Biochemistry and Molecular Biology, University of Dhaka, was collected from the rats by cardiac puncture also under diethyl Bangladesh. ether anesthesia. The collected blood samples were centrifuged at Plant Material 2,500 rpm for 15 minutes and finally the serums were separated into another eppendorf tubes for biochemical analysis. Two mL of blood The dried matured seeds of C. arietinum were collected from the was collected in heparinized tubes and then packed red cells were used commercially available sources of Dhaka city. Seeds were identified for estimation of Malondialdehyde (MDA) and reduced Glutathione by the taxonomist of Bangladesh National Herbarium, Dhaka (DACB (GSH). Accession no 37757). Biochemical analysis Preparation of Ethanolic Extract of C. arietinum Serum glucose was measured by Glucose Oxidase (GOD- After collection, the matured dried seeds of C. arietinum were PAP) method using micro-plate reader (Bio-Tec, ELISA); total washed thoroughly and dried in the laboratory. Then the seeds were cholesteroland Triglyceride (TG) by enzymatic colorimetric grinded to make fine powder by a grinding machine. The grinded method (Randox Laboratories Ltd., UK), using autoanalyzer. powder was extracted by using absolute (96%) ethanolic solvent. LDL-cholesterol was calculated by Friedewald equation [17]. Following the completion of extraction, extract prepared from seeds of Serum insulin by (ELISA, Crystal Chem Inc., USA). HOMA B% C. arietinum was concentrated under reduced pressure using a rotary (Beta-cell function) and HOMA S% (Insulin Sensitivity) were evaporator (BUCHI R-114, Switzerland) maintained at 55°C. The semi- calculated by HOMA SIGMA Software [18]. HOMA IR (Insulin dried ethanolic extract was further dried in a freeze drier (HETOSICC, Resistance Index) were calculated by International Formula: Heto Lab Equipment, Denmark) at -55°C and stored in a reagent bottle fasting Glucose (mmol/L) × fasting Insulin (mU/L)/22.5. Creatinine at -8°C in a refregerator. and Amino Alanine Transferase (ALT) by Auto-analyzer. Hepatic glycogen was measured by Anthrone-sulphuric acid method. Reduced Animals Glutathione (GSH) and plasma Malondialdehyde (MDA) estimated by The Long Evans rats bred at Bangladesh Institute of Research using Ellman’s and Thiobarbituric Acid Reactive Substances (TBARS) and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders method respectively [19,20]. (BIRDEM) animal house, were used in the study. The animals were maintained at a constant room temperature of 23°C with humidity of Statistical analysis 40-70% and the natural 12 hours day-night cycle. The rats were fed Data from the experiments were analyzed using the Statistical on a standard laboratory pellet diet and water supplied ad libitum. Package for Social Science (SPSS) software for windows version The experiments were conducted according to the ethical guidelines 12 (SPSS Inc., Chicago, Illinois, USA). All the data were expressed approved by Bangladesh Association for Laboratory Animal Science. as Mean ± SD as appropriate. Statistical analysis of the results was performed by using the student’s t-test (paired and unpaired), ANOVA Preparation of Neonatal STZ (nSTZ) Diabetic Rats (Analysis of Variance) followed by Bonferroni post hoc test. The limit Diabetes was induced by a single intraperitoneal injection of of significance was set at p<0.05. streptozotocin (STZ) at a dose of 90 mg/kg body weight to the neonate rats (48 hours old) as described by Bonner-Weir et al. [16]. Following Results 3 months of STZ injection, rats were examined for their blood glucose Effect of Carietinu mextract on the body weight of nSTZ level by oral glucose tolerance test (OGTT, Glucose 2.5 g/kg bw). diabetic rats Diabetic model rats with blood glucose level >7.00 mmol/l, at fasting condition was selected for studying the effects of the extracts in chronic Table 1 shows the effect of C. arietinum seed extract on body weight studies. of type 2 diabetic model rats during 28 days of chronic administration. Body weight of each rat was taken at seven days interval. As it is seen The experiment was carried out for duration of 28 days on 30 rats. from the Figure 1 a gradualincrease of body weight was observed in CA Then STZ diabetic rats were divided into the following four groups: Ext 1 treated group. In all other groups i.e., water control, glibenclamide 1. Water Control group (n=7): Treated with deionized water at a and CA Ext 2 treated groups a nonsignificant fall in body weight was noticed in the 1st week of the experimental period and after that an

Page 3 of 7 increase was noticed in body weight which was also not significant in (HOMA B%) and 17% decrease in insulin resistance (HOMA IR) when any group. compared with the initial day value. HOMA B% remained unchanged in CA Ext 1 treated and Glibenclamide treated groups. Insulin Effect on glucose homeostasis sensitivity (HOMA S%) was found to be increased by 32% and 11% by Fasting Serum Glucose (FSG) levels of nSTZ diabetic rats of 4 the treatment of Ext 2 and glibenclamide treated groups respectively. experimental groups were almost similar on 0 day (Figure 2). After oral Insulin resistance index HOMA IRwas decreased by 30% and 70% in administration of respective treatment to the type 2 diabetic model rats CA Ext 2 (p<0.05) and glibenclamide treated groups respectively on of different groups for 28 days of experimental period, it was found that 28th day in comparison to 0 day value. the FSG level of all the groups of rats decreased however the decrease Effect of C. arietinum on the serum Lipid profile of nSTZ was not significant except for CA Ext 2 group. The nSTZ diabetic rats treated with CA Ext 2 (p=0.049) showed a significant decrease while diabetic rats comparing within group respectively. As expected, glibenclamide also Figure 3 depicts the effect of C. arietinum on the serum Lipid th ameliorated the diabetic condition on 28 day. profile of nSTZ diabetic rats. Effect on serum insulin level Treatment of diabetic model rats for 28 days with CA Ext 2 resulted Table 1 demonstrates the effect ofC. arietinum extract on fasting in a significant decrease (p=0.014) in serum cholesterol level when serum insulin level of nSTZ diabetic rats. It is seen that serum insulin compared with water control. A rapid decreased was shown in CA Ext level decreased in all the groups except CA Ext 2 treated group which 1 group on final day in comparison to water control but the level was showed 32% increase compared to baseline value. However the increase out of significant (p=0.007) and a significant (p=0.033) decrease was was not significant. noticed in glibenclamidetreated group on 28 day in comparison to water control value. The triglyceride levels were decreased by 11% in Effect of C. arietinum on HOMA B%, HOMA S% and HOMA CA Ext 1 group on the final day compared to baseline value, however IR of nSTZ diabetic rats the fall in TG level was insignificant. Serum HDL was increased by Table 2 represents the results of 28 days treatment of nSTZ diabetic 4% in CA Ext 1 treated group. LDL-cholesterol levels were remained with C. arietinum. CA Ext 1 showed a 30% increase in beta cell function unchanged among all ofthe groups under study. Chronic effect of Carietinum on Liver and kidney function Insulin (picomol/l) Groups 0 day 28 day As it is seen from Table 3 serum ALT level was increased by 32% and WC (n=7) 85.37 ± 53.23 (100%) 79.81 ± 71.46 (93%) decreased by 30% in CA Ext 1 and CA Ext 2 treated groups respectively Gliben (n=7) 78.87 ± 53.10 (100%) 67.59 ± 41.33 (85%) on final day incomparison to 0 day value, however, the change was not CA Ext 1 (n=8) 89.72 ± 68.37 (100%) 118.20 ± 71.51 (132%) statistically significant. Regarding serum creatinine level no significant CA Ext 2 (n=8) 77.68 ± 28.92 (100%) 39.13 ± 19.26 (50%) change was noticed in any group of nSTZ rats. Table 1: Chronic effect of C. arietinum extract on fasting serum insulin level of Effects on hepatic glycogen content nSTZ diabetic rats. Results are expressed as Mean ± SD. Between groups, comparison was done The chronic effects of Carietinum extract on hepatic glycogen using one way ANOVA with post hoc Bonferroni test and within groups, comparison was done using paired t test content (on fasting condition) of nSTZ diabetic rats after 28 days of

Figure 1: Effect of C. arietinum extract on the body weight of nSTZ diabetic rats.

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4 Water control Gliben CA Ext 1 CA Ext 2 Groups Figure 2: Chronic effect of C. arietinum extracts on Fasting Serum Glucose (FSG) level of nSTZ diabetic rats. Results are expressed as Mean ± SD. Between groups, comparison was done using one way ANOVA with post hoc Bonferroni test and within groups, comparison was done using paired t test. A=CA Ext 2, 0 day vs. 28 day

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Figure 3: Effects of C. arietinum on total serum lipid profiles of nSTZ diabetic rats. Results are expressed as Mean ± SD. Between groups, comparison was done using one way ANOVA with post hoc Bonferroni test and Within groups, comparison was done using paired t test. a=CA Ext 2, 0 day vs. 28 day chronic treatment is presented in Figure 4. It is clear from the Figure products i.e., Malondialdehyde (MDA) and reduced Glutathione 4 that there were no significant changes in hepatic glycogen content (GSH) in different groups of rats after 28 days of the study period. among glibenclamide and CA Ext 2 treated group; but 51% increased The levels of erythrocyte MDA were lowerd by 5% in CA Ext 2 in (p=ns) was shown in CA Ext 1 group after 28 days of oral administration comparison to water control group. CA Ext 1 showed a significant when it was compared with water control. increase in Reduced-GSH levels when it was compared with water Effect of C. arietinum on antioxidative enzymes control (p=0.031). However, there was an 8% increase in erythrocyte MDA level compared to control group. Figure 5 shows the concentration of erythrocyte lipid peroxidation

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) 40 G / g 35 ( m

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c 15 y l g

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Figure 4: Effect of C. arietinum on hepatic glycogen content of nSTZ diabetic rats. Results are expressed as Mean ± SD. Between groups, comparison was done using one way ANOVA with post hoc Bonferroni test and within groups, comparison was done using paired t test

HOMA B% HOMA S% HOMA IR Groups 0 day 28 day 0 day 28 day 0 day 28 day WC 48.02 ± 21.22 49.26 ± 19.10 121.09 ± 99.72 128.27 ± 118.00 4.7 ± 3.5 10.9 ± 13.9 (n=7) (100%) (102%) (100%) (105%) (100%) (231%) Gliben 39.98 ± 14.73 42.23 ± 22.69 92.41 ± 85.59 106.05 ± 79.11 5.5 ± 3.4 3.9 ± 2.8 (n=7) (100%) (105%) (100%) (111%) (100%) (70%) CA Ext 1 42.03 ± 9.24 52.00 ± 16.68 65.15 ± 45.16 64.90 ± 43.79 6.6 ± 4.3 5.5 ± 3.6 (n=8) (100%) (123%) (100%) (99%) (100%) (83%) 1.8 ± 1.3 CA Ext 2 43.15 ± 12.48 43.55 ± 43.78 74.45 ± 68.36 125.18 ± 54.65 6.0 ± 4.3 (30%) (n=8) (100%) (100%) (100%) (168%) (100%) p=0.052a

Table 2: Effect of C. arietinum on HOMA B%, HOMA S% and HOMA IR of nSTZ diabetic rats. Results are expressed as Mean ± SD. Between groups, comparison was done using one way ANOVA with post hoc Bonferroni test and within groups, comparison was done using paired t test. a=CA Ext 2, 0 day vs. 28 day.

ALT (U/L) S Creatinine (mg/dl) with lesser side effects. Though the development of modern medicine Groups 0 day 28 day 0 day 28 day results in the advent of modern pharmacotherapeutics (in additions to 64 ± 37 91 ± 51 WC (n=7) 0.77 ± 0.11 0.79 ± 0.09 insulin, sulphonylureas and biguanides, hiazolidinediones) like DPPIV (100%) (142%) inhibitors, Glucagon like peptides etcthere is still a need to look for 62 ± 22 93 ± 26 Gliben (n=7) 0.73 ± 0.14 0.83 ± 0.18 new drugs as the existing drugs do not modify the course of diabetic (100%) (147%) complications. Therefore, as the disease is progressing unabated, there 65 ± 15 86 ± 8 CA Ext 1 (n=8) 0.73 ± 0.09 0.81 ± 0.13 (100%) (132%) is an urgent need of identifying indigenous natural resources with 101 ± 84 71 ± 18 antidiabetic properties in order to develop them as new therapeutics. CA Ext 2 (n=8) 0.80 ± 0.08 0.76 ± 0.05 (100%) (70%) The present study was undertaken to assess the antidiabetic effect Table 3: Chronic effect of extract of C. arietinum on liver and kidney function of with underlying mechanism of action of C. arietinum extract on nSTZ nSTZ diabetic rats. Results are expressed as Mean ± SD. Between groups, comparison was done diabetic model rats. C. arietinum extract with two different doses were using one way ANOVA with post hoc Bonferroni test and within groups, comparison fed to diabetic rats for 28 consecutive days. It was found that the nSTZ was done using paired t test diabetic rats from all groups gained in body weight throughout the experimental period which was also observed by other investigators Discussion [14,22-24]. Oral hypoglycemic agents and insulin is the mainstay of treatment Ethanol extract of C. arietinum lowered serum glucose level of diabetes and are effective in controlling hyperglycemia, however, significantly on 28th day (p=0.049). The possible mechanism underlying they have prominent side effects and fail to significantly alter the course the hypoglycemic activity of extract may be potentiation of pancreatic of diabetic complications [21]. As the knowledge of heterogeneity of secretion of insulin from β-cell coupled with extra pancreatic this disorder increases, it is needed to look for more efficacious agents

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Figure 5: Effects of C. arietinum on serum Malondialdehyde (MDA) and reduced Glutathione (GSH) of nSTZ diabetic rats. Results are expressed as Mean ± SD. Between groups, comparison was done using one way ANOVA with post hoc Bonferroni test and within groups, comparison was done using paired t test. mechanisms like decreased glycogenolysis and enhanced glycogenesis in type 2 model rats is due to increased uptake of glucose for the by the liver. When serum insulin level was investigated it was found that formation of glycogen by enhanced glycogenesis. This may be one the obtained results showed that C. arietenum in the low dose (0.625 of the probable mechanisms for the hypoglycemic action. There is a mg/kw bw) increased serum insulin level but the higher dose (1.25 g/kg clearly documented link between diabetic complications and lipid bw) did the opposite effect. Interestingly, when B cell secretion (HOMA peroxidation. Hypoinsulinemia increases the activity of the enzyme, B%), insulin sensitivity (HOMA S%) and Insulin resistance (HOMA fatty acyl-CoA oxidase that initiates β-oxidation of fatty acids. This IR) were determined by using HOMA-SIGMA software it was found results in lipid peroxidation, which is determined by thiobarbituric that C. arietenum at higher dose (CA Ext 2) improve insulin sensitivity acid (TBAR) substances level. In our experiment, the MDA level was and insulin resistance. These effects in turn can lower blood glucose decreased when treated extract of C. aritenum compared with vehicle level and can thereby decrease the requirement for insulin. Therefore, group [26]. Our result showed a significant increase in the GSH level, it is speculated that the observed improvement in insulin sensitivity when treated with extract CA Ext 1. The obtained findings also correlate and insulin resistance was responsible for glucose and insulin lowering with the findings of other scientists [27-29]. effect of CA Ext 2 treated group. Conclusion Type 2 diabetes is associated with marked imbalance in lipid metabolism [25]. The association of hyperglycemia with an alteration of It may be concluded that ethanolic extract C. arietinum at higher lipid parameters presents a major risk for cardiovascular complications dose (1.25 g/kg bw) showed significant hypoglycemic and antilipidemic in diabetes. In the present study, effect of C. arietinum extract on lipid effects most likely through decreasing insulin resistance and improving profile was evaluated after 28 days of chronic administration and C. insulin sensitivity. It also has antioxidant activity that reduces the arietinum at 1.25 g/kg bw caused a significant decrease (p=0.014) in oxidative changes induced by STZ administration. serum cholesterol level when compared with water control. Serum Acknowledgement HDL-cholesterol increased nonsignificantlyin extract treated group in comparison to the base line level. The findings are in concordance with We gratefully acknowledge the financial support of International other investigators [26,27] Program for the Chemical Sciences (IPICS), Uppsala, Sweden; and Asian Network of Research on Antidiabetic plants (ANRAP) in Serum alanine amino transferase (ALT) is considered more specific conducting this study. for hapatocellular damage. Serum ALT level was decreased by 30% (nonsignificantly) in CA Ext 2 treated group on final day in comparison Authors’ Contributions to 0 day value. Renal excretory function can be assessed by measuring serum creatinine levels. In this study after 28 days consecutive feeding Authors’ contributions AB MM YK BR of C. arietinumserum creatinine level almost remained unchanged Research concept and design ✓ ✓ ✓ ✓ which means that C. arietinum has no toxic effect on liver and kidney Collection and/or assembly of data ✓ ------function. Data analysis and interpretation ✓ -- -- ✓ Writing the article ✓ ------Liver glycogen level may be considered as the best marker Critical revision of the article ✓ -- -- ✓ for assessing hypoglycemic activity of any drug. Increased liver ✓ Final approval of article ✓ ✓ ✓ ✓ glycogen level was observed in extract treated group. Therefore, it Financial and Logistic supports -- ✓ -- ✓ may be ascertained that the hypoglycemic activity of C. aritenum Statistical analysis ✓ -- -- ✓

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Metabolomics (Los Angel), an open access journal ISSN: 2153-0769 Volume 6 • Issue 3 • 1000179

Vol. 8(10), pp. 173-181, October 2016

DOI: 10.5897/JPP2016.0406 Article Number: EB9650660851 Journal of Pharmacognosy and ISSN 2141-2502 Copyright © 2016 Phytotherapy Author(s) retain the copyright of this article http://www.academicjournals.org/JPP

Full Length Research Paper

Amaranthus viridis modulates anti-hyperglycemic pathways in hemi-diaphragm and improves glycogenesis liver function in rats

Shihab Uddin1, Md. Mahmodul Islam2*, Md. Mynul Hassan3, Amrita Bhowmik4 and Begum Rokeya5

1Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Bangladesh. 2Department of Pharmacy, Dhaka International University, Banani, Dhaka, Bangladesh. 3Dept of Biotechnology and Genetic Engineering, Khulna University, Khulna, Bangladesh. 4Department of Applied Laboratory Sciences, Bangladesh University of Health Sciences, Dhaka, Bangladesh. 5Department of Pharmacology, Bangladesh University of Health Sciences, Dhaka, Bangladesh.

Received 13 June 2016; Accepted 5 September, 2016

Amaranthus viridis is an ecumenical species in the botanical family of Amaranthaceae, which has been traditionally used to treat several skin diseases along with some antilipidemic activities. The present study was carried out to investigate the anti-hyperglycemic effect of 75% ethanolic extract of A. viridis in Neonatal streptozotocin (N-STZ) induced rats’ hemi-diaphragm, including screening for secondary plant metabolites. Qualitative phytochemical studies were done by various conventional methods for the possible secondary metabolites. For antidiabetic assay via hemi-diaphragm, Long-Evan rats were used in the study. Type 2 diabetes was induced by a single ip injection of streptozotocin to 48 h old pups (N-STZ) and after 3 months, rats were confirmed by an oral glucose tolerance test and further selected for the experiment. Studies to evaluate the glucose utilization capacity of A. viridis in isolated rat hemi-diaphragm were done. The data were analyzed by appropriate statistical analysis. In vitro glucose uptake by hemi-diaphragm study showed glucose uptake increased significantly in left diaphragm of type 2 diabetes mellitus with insulin alone treated and A. viridis alone treated group, where A. viridis alone treated group showed very highly significance (p=0.000). Treatment with both insulin and A. viridis increased the glucose uptake also very significantly (p=0.004). A. viridis extract acted more significantly compared to insulin in T2DM rats. In the normal rats at left hemi diaphragm, A. viridis extract also increased glucose uptake more significantly (p=0.009) compared to insulin (p=0.013). At the right diaphragm, glucose uptake increased in all treated groups compared to control group but not significantly. This plant may contain potential anti-hyperglycemic agents which possibly act through some extra pancreatic mechanism that include glucose uptake by diaphragm and increased glycogenesis by liver.

Key words: Amaranthus viridis, antidiabetic, hemi-diaphragm, streptozotocin, glucose, Long-Evan rats.

INTRODUCTION

It is staggering to consider the threat that diabetes poses and therapeutical advancement in the management of to our current healthcare system. Recent technological diabetes mellitus includes pancreas regeneration, islet 174 J. Pharmacognosy Phytother.

transplantation, pancreas transplantation, glucose moni- a potent hepatoprotective and antioxidant plant (Kumar et toring at continuous basis, uninterrupted subcutaneous al., 2011). Studies also claimed that it is a good source of insulin infusion and assorted medication (George, 2009). anthelmintic and isoproterenol-induced cardiac toxicity For mortals with T2DM mellitus (T2DM), an assortment of inhibitory plant (Ashok et al., 2011; Kumar et al., 2012). treatments is available. Most of the pharmacological aid Plants that exhibit activity against hyperglycemia are schemes for T2DM are typically grounded on efficacy. mainly owing to their ability to bushel the function of Hence, prosperous responses to such therapeutics are pancreatic tissues by causing an alleviation in insulin frequently variable and unmanageable to predict. In this output or conquer the intestinal assimilation of glucose or circumstances, delineation of drug reaction is expected to aid of metabolites in insulin subordinate processes. Most considerably heighten our ability to provide patients with plants contain cartenoids, terpenoids, glycosides, the most effective treatment strategy given their individual flavonoids, alkaloids etc. that are usually entailed as backgrounds. Hence pharmacogenetic analysis of having antidiabetic effect (Jung et al., 2006). Type 2 medications against diabetes is still in its early stage. Up diabetes represents a progressing decline in beta-cell to date, major pharmacogenetic acquisitions have focused function. Regarding the restrictions of being therapies in on biguanides, TZDs and sulfonylureas (Distefano et al., fixing the quality of life to normal as well as reducing the 2010). Most recently researchers have focused on the risk of chronic diabetic complications by maintaining management diabetes and its associated complications. normal blood glucose level, the search for alternating A variety of approaches have been taken for this purpose. sources of oral hypoglycemic agents is a requirement. The plant is usually known as green amaranth or Due to the limitation of recent therapies to control all the slender amaranth. Possible origin is South America, metabolic defects of diabetes as well as their possible although widely distributed in tropical weed, foreign to pathological outcomes with the great expense, there is a hot-temperate regions and distributed in the tropical and clear need for the development of alternative strategies subtropical regions of the world. It is an annual herb with for diabetes treatment. erect or ascending habit, growing to 1 m tall. Leaves are There has been a possibility of anti-hyperglycemic light green and the fruit are obviously wrinkled. It has potentialities of A. viridis reported by Krishnamurthy et al. prominent axillary spines and its leaves can have an (2011). However, so far hemi-diaphragm pathways of A. obvious reddish or purplish tinge (Stanley et al., 1984). A. viridis on Long Evan rats against the hypoglycemic viridis is found to be a very common garden weed. Also it activities, has not been done. So in this study, an attempt is found in areas such as roadsides, parks, pastures and was made to evaluate the anti-hyperglycemic activity of other disturbed sites, but seldom cropped, often flattened ethanolic extract of A. viridis plant and also to find out the and prostrate, vacant lots, sometimes crevices of chemical factors present therein causative for the sidewalks and edge of asphalt strips, etc. (Stone, 1970), biological activity. The pharmacological study was carried casual in croplands and waste places too (Whistler, 1988). out on streptozotocin induced type 2 Neonatal model in The A. viridis is a good source of vitamins B and C, Long Evan rats. Glucose utilization capacity of bioactivity taken as vegetables (Sayed et al., 2007). Leaves and guided fractions of A. viridis in isolated rat hemi- seeds are also edible. Previous experiments ascertained diaphragm in both normal and N-STZ rats was performed it to be a superior source of protein (Macharla et al., and the observed activity was identiﬁed, characterized 2011). Traditionally it is used to cure eczema, psoriasis and quantiﬁed. and rashes including antinociceptive and antipyretic properties, reported by Kumar (Kumar et al., 2009). Besides these, it is reported by Krishnamurthy that A. MATERIALS AND METHODS viridis has anti-inflammatory, antihyperglycemic, hypolipidemic activity as well as acne and skin cleansing Chemicals and reagents property (Krishnamurthy et al., 2011). It has a wide Ethanol (PubChem CID: 702), Ferric chloride (PubChem CID: application over diuresis, for snake bites, scorpion stings, 24380), potassium ferrocyanide (PubChem CID: 11963580), dysentery, constipation, eczema, bronchitis, anemia, Chloroform (PubChem CID: 6212), sulphuric acid (PubChem CID: leprosy and stomach problems like many incidences 1118), sodium dihydrogen phosphate (PubChem CID: 23672064) (Pandhare et al., 2012; Macharla et al., 2011). According were purchased from Sigma-Aldrich Co (St. Louis, MO, USA). 4,6- to Syed et al. it is quite beneficial to pregnant women to Ethylidene glucose streptozotocin (PubChem CID: 3081692) were subside labor pains and diabetes (Syed et al., 2007). Its purchased from Merck (Darmstadt, Germany) and Human insulin (PubChem CID: 16131099) from Sanofi Bangladesh (Bangladesh). pharmacological study also reveals that it is antiviral (Obi All other chemicals used were from the laboratory stock of the et al., 2006). Meanwhile, its anti-allergenicity is claimed Department of Pharmacology, Bangladesh University of Health by Sayed et al. (2007). According to Kumar, A. viridis is Sciences, Dhaka, Bangladesh and were of the highest grade

*Corresponding author. E-mail: [email protected]. Tel: +8801824080725. Fax: +88-02-9871556.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Uddin et al. 175

available. distilled water for 5 min in a water bath and was filtered while hot. The extract sample or filtrate was taken for the experiments wherever applicable using standard protocols (Sharmistha et al., Plant mate rial collection and identification 2012) to test the presence of bioactive compounds.

In this study, whole plant part of A. viridis (family: amaranthaceae, local name: Notey shak) was used. The plants were collected from In vitro glucose uptake study of A. viridis by isolated rat hemi- Pabna, Bangladesh (Geographic coordinates: Latitude: 24°00′23″ diaphragm N, Longitude: 89°14′13″ E and elevation above sea level: 19 m = 62 ft) availablesources from the field in the month of June, 2015. The Glucose uptake by rat hemi-diaphragm was estimated by the plant was identified by the Bangladesh National Herbarium, Dhaka methods described elsewhere (Walaas and Walaas, 1952; (Accession No: DACB-38568). Chattopadhyay et al., 1992) with some modifications. 32 male and female Long Evans normal and Type 2 rats were weighed and rats weighing between 170 to 210 g were used in the study. The weight Preparation of ethanol extracts of Amaranthus viridis of the rats were measured before and after fasting. Four sets containing graduated test tubes (n=4) for each hemi-diaphragm Mature and fresh whole plants were washed thoroughly after were taken. Group I served as a control which contained 2 ml of collection and air dried. The weights of plants before and after dry Tyrode (NaCl (8 gm/L), KCl (0.20 gm/L), CaCl2 (0.20 gm/L), MgCl2 were recorded. After then, the whole plants were grinded and again (0.10 gm/L), NaH2PO4 (0.05 gm/L), NaHCO3 (1 gm/L), Glucose (1 weighed. Finally, these grind portions were extracted by using 75% gm/L) having pH 6.5) solution with 2% glucose, Group II contained ethanolic solvent. The ethanolic extract was prepared by using 2 ml Tyrode solution with 2% glucose and regular insulin (Novo Soxhlet (Beijing Getty glassware Co. Ltd. maintained at 70°C) and Nordisk) 0.62 ml of 0.4 units per ml solution. Group III contained 2 following the completion of extraction was concentrated by using ml Tyrode solution with 2% glucose and 1.38 ml of A. viridis extract water evaporator (Fujian Snowman Co. Ltd. 5 Litters, asserted at (30 mg extract dissolved in 3 ml H2O and adjust PH to 7.4) and the 80°C). The ethanolic extracts at semi dried state were encouraged Group IV contained 2 ml Tyrode solution with 2% glucose and to dry in a freeze drier (HETOSICC, Heto Lab Equipment, regular insulin 0.62 ml of 0.4 units per ml solution and 1.38 ml of A. Denmark) at -55°C temperature and preserved in a reagent bottle viridis extract. The volumes of all the test tubes were made up to 4 at -8°C in a freezer for analysis. ml with distilled water to match the volume of the test tubes of Group IV. Long Evans rats were tested overnight and killed by decapitation. The diaphragms were dissected out quickly with Preparation of animals for treatment minimal trauma and divided into two halves. Two diaphragms from the same animal were not used for the same set of experiment. Adult Long Evans rats weighting 160 to 220 g were included in the Four numbers of diaphragms were used for each group. The hemi- study. The animals were bred at Bangladesh Institute of Research diaphragms were placed in test tubes and incubated for 30 min at and Rehabilitation for Diabetes, Endocrine and Metabolic Disorders 37°C in an atmosphere of 100% oxygen with shaking at 140 (BIRDEM) animal house, Dhaka, Bangladesh, maintained at a cycles/min. Glucose uptake per gram of tissue was calculated as constant room temperature of 22±5°C with humidity of 40 to 70% the difference between the initial and final glucose content in the and the natural 12 h day-night cycle. Animal housing and handling incubated medium. were performed in accordance with Good Laboratory Practice (GLP) mentioned in US guidelines (NIH publication # 85-23, revised in 1985). The experimental protocols were critiqued and sanctioned by the Institutional Animal Ethics Committee prior to initiation of the Estimation of glucose level experiment. The rats were fed upon a stock lab pellet diet and water supplied ad libitum. Standard rat diet contained wheat (40%), The glucose oxidase (GOD-PAP) test was performed using the wheat bran (20%), fish meal (10%), germ (3.9%), oil cake (10%), established method published by Trinder (Trinder, 1969) without milk (3.8%), pulses (3.9%), soyabean oil (1.5%), rice polishing modification. (5%), molasses (0.95%) and salt (0.95%). Embavit GS (vitamin mixture) 250 g was added per 100 kg of rat food. The influence of circadian rhythm was avoided by starting all experiments at 7:30 Statistical analysis am. The experiments were conducted according to the ethical guidelines approved by Bangladesh Association for Laboratory Data from the experiments were analyzed using the Statistical Animal Science. Package for Social Science (SPSS) software for windows version 21 (SPSS Inc., Chicago, Illinois, USA). All the data were expressed as Mean ± SD or as Median (Range) as appropriate. Statistical Preparation of type 2 diabetes model rats analysis of the results was performed by using the student’s t-test (paired and unpaired), ANOVA (analysis of variance) followed by Type 2 diabetes was hastened by a single ip injection of Bonferroni and Dunnett post hoc test and Mann Whitney (u) test. streptozotocin (STZ) dissolved in citrate buffer (10 ml), at a dose of GraphPad Prism (Version 5) software was used for all statistical 90 mg/kg of body weight into the rat pups (48 h old, average weight analysis and P<0.05 was considered as significance. 7 g) as described by Weir and Bonner-Weir et al. (2013). Following 3 months of STZ injection, rats were examined by oral glucose tolerance test (OGTT) for their blood glucose level. Diabetic model rats with blood glucose level >7.00 mmol/L, at fasting condition RESULTS were selected for studying the effects of A. viridis extracts. Phytochemical screening of 75% ethanolic extract of A. viridis Phytochemical screening of A. viridis

Three (3) g of A. viridis 75% ethanolic extract was boiled with 30 ml The present investigation was carried out to assess the 176 J. Pharmacognosy Phytother.

Table 1. Phytochemical screening for the uptake was also increased significantly (glucose uptake possible secondary metabolites of the A. viridis m ± SD, mg/g/30 min, control 2.02 ± 0.62 vs plant extract extract. 6.93 ± 0.62; p = 0.009). When the hemi-diaphragm of

normal rats was exposed to both insulin and A. viridis Serial no Phytochemicals Results extract, it did not increase glucose uptake significantly. 1 Tannin ++ 2 Saponin ++ 3 Alkaloid ++ Effect of A. viridis extracts using glucose uptake by 4 Flavonoid ++ isolated rat-hemi (right) diaphragm on normal rats in 5 Phenol ++ vitro assay 6 Steroid - 7 Terpenoid + The results of glucose uptake by rat right hemi- 8 Carbohydrate - diaphragm in normal rats is presented in Table 4 and Figure 2. The results showed that glucose uptake was ++ = Presence; + = trace; - = absence. enhanced by hemi-diaphragm when the normal rats were treated with insulin alone (glucose uptake m ± SD, mg/g/30 min, control 0.568 ± 0.350 vs insulin 2.684 ± qualitative phytochemical analysis of 75% ethanolic 2.527; p = 0.148). When hemi-diaphragm was treated extract of A. viridis. The phytochemical screening reveals with A. viridis extract, glucose uptake was also increased the presence of various plants’ secondary metabolites as but not-significantly (glucose uptake m ± SD, mg/g/30 shown in Table 1. Tannin, saponin, alkaloid, flavonoid min, control 0.568 ± 0.350 vs plant extract 1.893 ± 1.704; and phenols were found in the extract of A. viridis. p = 0.178). When the hemi-diaphragm of normal rats was Terpenoid was in trace amount but carbohydrate and exposed to both insulin and A. viridis extract, it also did steroids were not found in our phytochemical studies. not increase glucose uptake significantly.

Check values of STZ induced Type 2 diabetic rats in Effect of A viridis extracts using glucose uptake by different groups isolated rat-hemi (left) diaphragm on STZ induced To generate a rat model mimicking human type 2 type 2 rats in vitro assay diabetes with impaired insulin secretion and insulin resistance, we used STZ injection (90 µg/kg, body The results of glucose uptake by rat left hemi-diaphragm weight) to 48 h old pulps. STZ injection to neonates led to in T2DM rats is presented in Table 5 and Figure 3. When the injury of the pancreas resulting in destruction of the the T2DM rats were treated with insulin alone the glucose functional β-cells. At the age of 3 months, when an oral uptake was significantly increased in compared to control glucose challenge (500 mg/kg, body weight) was done, (glucose uptake m ± SD, mg/g/30 min, control 2.55 ± 0.36 the remaining β cell could not cope with the load, which vs insulin 5.26 ± 1.21; p = 0.037). In the treatment with A. reflected in the postprandial rise of serum glucose level at viridis extract, glucose uptake was significantly higher in 30 min (Table 2). The rise was significant among all rats comparison to control (glucose uptake m ± SD, mg/g/30 compared to baseline value. On the basis of this min, control 2.55 ± 0.36 vs plant extract 9.74 ± 0.87; p = experiment these rats were selected and sorted into three 0.000) as well as insulin alone (glucose uptake m ± SD, distinct groups to carry out the experiments with feeding mg/g/30 min, insulin 5.26 ± 1.21 vs plant extract 9.74 ± of different extracts. 0.87; p = 0.002), respectively. Insulin with A. viridis extract was also exposed to a significant increased glucose uptake when it compared Effect of A viridis extracts using glucose uptake by with control (glucose uptake m ± SD, mg/g/30 min, isolated rat-hemi (left) diaphragm on normal rats in control 2.55 ± 0.36 vs insulin with plant extract 6.46 ± vitro assay 0.93; p = 0.004) as well as plant extract alone (glucose uptake m ± SD, mg/g/30 min, plant extract 9.74 ± 0.87 vs In order to explore the mechanism underlying the anti- insulin with plant extract 6.46 ± 0.93; p = 0.012), hyperglycemic activity of A. viridis in vitro glucose uptake respectively. study of rat hemi-diaphragm was done. The results of glucose uptake by the left hemi-diaphragm in normal rats are presented in Table 3 and Figure 1. The results Effect of A. viridis extracts using glucose uptake by showed that glucose uptake was enhanced by hemi- isolated rat-hemi (right) diaphragm on STZ induced diaphragm when the normal rats were treated with insulin type 2 rats in vitro assay alone (glucose uptake m ± SD, mg/g/30 min, control 2.02 ± 0.62 vs insulin 6.59 ± 2.01; p=0.013). When hemi- The results of glucose uptake by rat right hemi- diaphragm was treated with A. viridis extract, glucose diaphragm in T2DM rats is presented in Table 6 and Uddin et al. 177

Table 2. Check values of STZ induced Type 2 diabetic rats in different groups.

Groups Fasting (0 min) After OGTT (30 min) WC (n=6) 7.44±0.57 (100%) 14.62±1.92 (197%) Glc (n=6) 7.71±0.55 (100%) 14.82±1.35 (192%) AVEtE (n=6) 7.72±0.87 (100%) 14.73±1.39 (191%)

Paired samples T test Groups 0 min vs 30 min WC (n=6) 0.000 Glc (n=6) 0.000 AVEtE(n=6) 0.000

Results are expressed as Mean ±SD. OGTT= Oral Glucose Tolerance Test, STZ= Streptozotocin; WC = Type 2 Water Control; Glc = Type 2 Glibenclamide treated group and AVEtE= 75% ethanol extract of A. viridis.

Table 3. Effect of A. viridis extracts using glucose uptake by isolated rat-hemi (Left) diaphragm on normal rats in vitro assay.

Group Glucose uptake Treatment Incubation medium p value (G) (mg/g/30 min) G1 Control Tyrode solution with Glucose (2%) 2.020.62 (100%) -

Tyrode solution with Glucose and Insulin G2 Insulin 6.592.01 (326%) G1 Vs G2, p=0.013* (Actrapid 40 u/ml)

Tyrode solution with Glucose and plant extract G3 Plant extract 6.930.62 (343%) G1 Vs G3, p=0.009** (30 mg/3 ml H2O)

Insulin+ Tyrode solution with Glucose, Insulin and plant G4 4.381.25 (211%) G1 Vs G4, p= 0.021* plant extract extract

Results were expressed as Mean ±SD. Statistical analysis between group comparison was done by using one-way ANOVA with post hoc Bonferroni test. *= p<0.05; **=p<0.01.

Table 4. Effect of A viridis extracts using glucose uptake by isolated rat-hemi (Right) diaphragm on normal rats in vitro assay.

Group Glucose uptake Treatment Incubation medium p value (G) (mg/g/30 min) G1 Control Tyrode solution with Glucose (2%) 0.568±0.350 (100%)

Tyrode solution with Glucose and Insulin (Actrapid G2 Insulin 2.684±2.527 (471%) G1 Vs G2, p=0.148 40 u/ml)

Tyrode solution with Glucose and plant extract G3 Plant extract 1.893±1.704 (333%) G1 Vs G3, p=0.178 (30mg/3ml H2O)

Insulin+ Tyrode solution with Glucose, Insulin and. plant G4 1.898±0.977 (334%) G1 Vs G4, P=0.067 plant extract extract

Results were expressed as Mean ±SD. Statistical analysis between group comparison was done by using one-way ANOVA with post hoc Bonferroni test. *= p<0.05; **=p<0.01.

Figure 4. When the T2DM rats were treated with insulin treatment with A. viridis extract, glucose uptake was 84% alone the glucose uptake was increased by 59% higher in comparison to control group. Glucose uptake compared to control group but not significantly. In the was found to be increased up to 119% in case of 178 J. Pharmacognosy Phytother.

Table 5. Effect of A.viridis extracts using glucose uptake by isolated rat-hemi (Left) diaphragm on STZ induced type 2 rats in vitro assay.

Group Glucose uptake Treatment Incubation medium p value (G) (mg/g/30 min) G1 Control Tyrode solution with Glucose (2%) 2.55±0.36 (100%)

Tyrode solution with Glucose and G2 Insulin 5.26±1.21 (206%) G1 Vs G2, p=0.037* Insulin (Actrapid 40 u/ml)

Tyrode solution with Glucose and G1 Vs G3, p=0.000**, G2 Vs G3; G3 Plant extract 9.74±0.87 (381%) plant extract (30 mg/3 ml H2O) p=0.002**

Insulin+ Tyrode solution with Glucose, G1 Vs G4, p=0.004**, G3 Vs G4, G4 6.46±0.93 (253%) plant extract Insulin and plant extract p=0.012*

Results were expressed as Mean ±SD. Statistical analysis between group comparison was done by using one-way ANOVA with post hoc Bonferroni test. *= p<0.05; **=p<0.01.

Table 6. Effect of A viridis extracts using glucose uptake by isolated rat-hemi (right) diaphragm on STZ induced type 2 rats in vitro assay.

Group Glucose uptake Treatment Incubation medium p value (G) (mg/g/30 min) G1 Control Tyrode solution with Glucose (2%) 1.385±0.980 (100%)

Tyrode solution with Glucose and G2 Insulin 2.195±1.293 (159%) G1 Vs G2, p=0.359 Insulin (Actrapid 40 u/ml)

Tyrode solution with Glucose and G3 Plant Extract 2.548±1.423 (184%) G1 Vs G3, p=0.233 plant extract (30mg/3ml H2O)

Insulin+ Tyrode solution with Glucose, G4 3.033±0.823 (219%) G1 Vs G4, P=0.043* Plant Extract Insulin and. plant extract

Results were expressed as Mean ±SD. Statistical analysis between group comparison was done by using one-way ANOVA with post hoc Bonferroni test. *= p<0.05; **=p<0.01.

Figure 1. Effect of A viridis extracts using glucose uptake by isolated rat-hemi (Left) diaphragm on normal rats in vitro assay. *= p<0.05; **=p<0.01. Uddin et al. 179

Figure 2. Effect of A. viridis extracts using glucose uptake by isolated rat-hemi (Right) diaphragm on normal rats in vitro assay. *= p<0.05; **=p<0.01 and ns= not significance.

Figure 3. Effect of A. viridis extracts using glucose uptake by isolated rat-hemi (Left) diaphragm on STZ induced type 2 rats in vitro assay. *= p<0.05; **=p<0.01.

A. viridis extract with insulin, compared with control group polyphenols like phytoconstituents (Sharmistha et al., as well as plant extract and insulin individually. 2012). Therefore in the beginning of the study, preliminary phytochemical screening of the 75% ethanolic extract of DISCUSSION whole plant for secondary plant metabolites was performed. The results revealed the presence of saponin, Previous studies have shown that antidiabetic plants tannin, flavonoids, alkaloids, terpinoids, phenol (Table 1). possess the presence of alkaloids, glycosides and The presence of a significant number of secondary plant 180 J. Pharmacognosy Phytother.

Figure 4. Effect of A. viridis extracts using glucose uptake by isolated rat-hemi (right) diaphragm on STZ induced type 2 rats in vitro assay. ns = not significance.

metabolites in A. viridis might be responsible for the and 4). Hence, in case of T2DM rats p = 0.037 with biological activities observed later in this study. insulin alone and p = 0.000 with A. viridis extract (Table Type 2 diabetes was developed by injecting STZ (a 5). Moreover, treatment with both insulin and A. viridis pancreatic β cell toxin) to 48 h old pups, which inside the extract caused significantly much higher glucose uptake β-cell dissociates into glucose and methylnitrogenase. by rat left hemi-diaphragm (p = 0.004). Thus it can be The later alkaylates and modified biomolecules concluded that A. viridis improves hyperglycemia by breakdown DNA and destroys β cell, thereby causing extrapancreatic mechanism as the left diaphragm diabetes. Early injury of the β cells resulted in the partial improves glucose uptake more than right diaphragm. recovery of β cell leading to type 2 diabetes as a result of insulin resistance in target tissues and impaired insulin secretion, accompanied by increased adiposity. Conclusion When at the age of 3 months these rats have been challenged with an oral glucose load, all of them could The present study demonstrates that phytochemical not cope with the glucose load due to the defective β screening of 75% ethanol extract contains a number of cells. Although their fasting glucose values were a bit secondary plant metabolites including flavonoids, higher (ranging from 7.44 to 7.72), indicating the alkaloids which might be associated with the obtained presence of some functioning β cells but their post antidiabetic properties of A. viridis. In vitro glucose challenge glucose values were significantly higher which consumption by hemi-diaphragm study exhibited proved that these rats have developed type 2 diabetes increased state of the glucose by hemi-diaphragm in the (Table 2). presence of A. viridis extract. From the findings it can be Therefore, it may be assumed that the hypoglycemic concluded that different secondary metabolites of plant activity of A. viridis in type 2 model rats at least, may be materials had some extra pancreatic mechanism like partly due to increased uptake of glucose for the glucose consumption by peripheral tissues. Thus, the formation of glycogen by enhanced glycogenesis. To put plant might be considered for further chemical studies further insight regarding the mechanism of anti- and detailed toxicological studies for future drug hyperglycemic effect of A viridis extract in both normal development. and T2DM rats in vitro glucose uptake by hemi- diaphragm was performed. In this study, individual insulin (p = 0.013) and A. viridis Conﬂict of interest (p = 0.009) treated group showed a significant increment in glucose uptake in normal rats at left diagram (Tables 3 The authors have not declared any conflict of interest. Uddin et al. 181

ACKNOWLEDGEMENTS Kumar BSA, Lakshman K, Swamy VBN, Kumar PAA, Shekar DS, Manoj B, Vishwantha GL (2011). Hepatoprotective and Antioxidant Activities of Amaranthus viridis Linn. Maced. J. Med. Sci. 4(2):125-130. We gratefully acknowledge the logistic supports provided Kumar BSA, Lakshman K, Jayaveera KKN, Shekar DS, Muragan CSV, by the Asian Network of Research on Antidiabetic Plant Manoj B (2009). Antinociceptive and Antipyretic Activities of (ANRAP) and the study was conducted in the Amaranthus Viridis Linn in Different Experimental Models. Avicenna. Department of Pharmacology, Bangladesh University of J. Med. Biotechnol. 1(3):167-171. Macharla SP, Goli V, Bhasker KV, Devi PS, Dhanalakshmi C, Sanjusha Health Sciences, Dhaka Bangladesh; and the C (2011). Effects of antiinflammatory activity of Amaranthus viridis Department of Biochemistry and Molecular Biology, Linn. Ann. Biol. Res. 2:435-438. Jahangirnagar University, Dhaka, Bangladesh. Obi RK, Iroagba II, Ojiako OA (2006). Virucidal potential of some edible Nigerian vegetables. Afr. J. Biotechnol. 5(19):1785-1788. Pandhare R, Balakrishnan S, Popat M, Khanage S (2012). Antidiabetic and antihyperlipidaemic potential of Amaranthus viridis (L.) Merr. in Abbreviations streptozotocin induced diabetic rats. Asian Pacific J. Trop. Dis. 2:S180-S185. N-STZ, Neonatal streptozotocin; A. viridis, Amaranthus Sayed MA, Rumi MAK, Ali L, Banu A, Hussain ZA, Azad AK (2007). Effect of socioeconomic risk factors on the difference in prevalance of viridis; T2DM, type 2 diabetes mellitus; BIRDEM, diabetes between rural and urban population in Bangladesh. Biol. Bangladesh Institute of Research and Rehabilitation for Med. Res. Council 33(1):1-12. Diabetes, Endocrine and Metabolic Disorders; GLP, good Sharmistha C, Chandra KJ (2012). Preliminary Phytochemical laboratory practice; OGTT, oral glucose tolerance test; Screening and Acute Oral Toxicity Study of the Flower of Phlogacanthus thyrsiflorus Nees in Albino Mice. Int. Res. J. Pharm. GOD-PAP, glucose oxidase; SPSS, statistical package 3(4):2230-8407. for social science; ANOVA, analysis of variance; M±SD, Stanley TD, Ross EM (1984). Flora of South-eastern Queensland. mean ± standard deviation. Brittonia. 36(2): 205. Stone BC (1970). The Flora of Guam: A Manual for the Identification of the Vascular Plants of the Island. Micronesica. 6(1/2): 1-659. Trinder P (1969). Glucose: GOD-PAP method enzymatic colorimetric REFERENCES method. Ann. Clin. Biochem. 6:24-27. Walaas E, Walaas O (1952). Effect of insulin on rat diaphragm under Chattopadhyay RR, Sarkar SK, Ganguly S, Banerjee RN, Basu TK anaerobic conditions. J. Biol. Chem. 195:367-73. (1992). Effect of leaves of vinca rosen linn on glucose utilization and Whistler WA (1988). Checklist of the weed flora of western Polynesia: glycogen deposition by isolated rat hemi-diaphragm. India. J. Physiol. an annotated list of the weed species of Samoa, Tonga, Niue, and Pharmacol. 36:137-138. Wallis and Futuna, along with the earliest dates of collection and the Distefano JK, Watanabe RM (2010). Pharmacogenetics of Anti- local names, South Pacific Commission, Noumea, New Caledonia. Diabetes Drugs. Pharmaceuticals 3(8):2610-2646. Technical Paper No. 194. ISBN 9822030959. George CM (2009). Future trends in diabetes management. Nephrol. Weir GC, Bonner-Weir S (2013). Islet β cell mass in diabetes and how it Nurs. J. 36(5):477-483. relates to function, birth, and death. Ann. N. Y. Acad. Sci. 1281(1):92- Jung M, Park M, Lee HC, Kang YH, Kang ES, Kim SK (2006). Anti- 105. diabetic agents from medicinal plants. Curr. Med. Chem. 13(10):1203-1218. Krishnamurthy G, Lakshman K, Pruthvi N, Chandrika PU (2011). Antihyperglycemic and hypolipidemic activity of methanolic extract of Amaranthus viridis leaves in experimental diabetes. Indian. J. Pharmacol. 43(4):450-454. Kumar BSA, Lakshman K, Jayaveea KN, Shekar DS, Khan S, Thippeswamy BS (2012). Antidiabetic, antihyperlipidemic and antioxidant activities of methanolic extract of Amaranthus viridis Linn in alloxan induced diabetic rats. Exp. Toxicol. Pathol. 64:75-79.

European Journal of Medicinal Plants 13(1): 1-5, 2016, Article no.EJMP.23556 ISSN: 2231-0894, NLM ID: 101583475

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Microbial Contamination of Some Antidiabetic Herbal Preparations Available in Bangladesh

Md. Ranzu Ahmed 1,2* , S. M. Z. H. Asna 3, M. S. H. Khan 1, Begum Rokeya 4, M. Mosihuzzaman 5 and M. Abu Sayeed 2

1Department of Chemistry, Bangladesh University of Health Sciences (BUHS), Dhaka, Bangladesh. 2Department of Applied Chemistry and Chemical Engineering, University of Rajshahi, Rajshahi, Bangladesh. 3Department of Microbiology, Bangladesh University of Health Sciences (BUHS), Dhaka, Bangladesh. 4Department of Pharmacology, Bangladesh University of Health Sciences (BUHS), Dhaka, Bangladesh. 5Department of Chemistry, University of Dhaka, Dhaka, Bangladesh.

Authors’ contributions

This work was carried out in collaboration between all authors. Authors MRA and SMZHA wrote the protocol, managed the literature searches, wrote the first draft of the manuscript, designed the study, managed the experimental process of the study and statistical analysis of data of the study performed. Authors MRA and MSHK responded to the reviewers comments. Authors BR, MM and MAS corrected the manuscript and gave the valuable suggestions to write and perform the research. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/EJMP/2016/23556 Editor(s): (1) Marcello Iriti, Professor of Plant Biology and Pathology, Department of Agricultural and Environmental Sciences, Milan State University, Italy. Reviewers: (1) Alexander E. Berezin, Medical University of Zaporozhye, Ukraine. (2) Nayana Pinheiro Machado Freitas Coelho, Vale do Paraiba University (UNIVAP), Brazil. (3) Rebeca Garcia Macedo, Mexican Social Security Institute, Mexico. (4) Sema Kalkan Ucar, Ege University, Turkey. Complete Peer review History: http://sciencedomain.org/review-history/13513

Received 7th December 2015 nd Original Research Article Accepted 22 February 2016 Published 2nd March 2016

ABSTRACT

Herbal Medicines (HM) are being used in our country for a long time but the type and load of the microbial agents has not been isolated in locally produced finished products of HM. The present study was designed to assess the microbial load, genus & species of the microbes contaminating with HM. Seven different Antidiabetic Herbal Preparations (ADHPs) were purchased randomly and analyzed for microbial contaminants. Blood agar, Mac Conkey agar, Chocolate agar and Saboraud’s dextrose agar were used (Oxoid) for culturing and isolation of bacteria and fungus. ______

*Corresponding author: E-mail: [email protected], [email protected];

Ahmed et al.; EJMP, 13(1): 1-5, 2016; Article no.EJMP.23556

Identification of organisms were done as standard ways. Total aerobic bacterial plate count was done as per the method of Brown, Poxton and Wilkinson. Out of 07 antidiabetic solid and liquid samples, except ADHP-3, Bacillus subtilis (3.5 – 4.0 x 10 4 cfu/g) was isolated from solid ADHPs and Enterococcus spp. (1.0x10 4 cfu/ml) was isolated from liquid ADHP, but all samples were free from fungi (yeasts and moulds). However presence of bacteria in these samples indicates the possibility of increased number of bacteria. So, the sample should be handled in any step maintaining standard sterility of the environment, instrument and involved personnel. The result of present study showed the contamination rate within tolerable level but the presence of bacteria in these samples was not desirable.

Keywords: Herbal formulations; antidiabetic herbal preparations; microbial contamination; antibacterial assessment.

1. INTRODUCTION bacteria [9-13]. Primarily a good number of inhabitants in Bangladesh are believed to Herbal medicines embrace herbal materials, depend on herbal formulations for their medical herbs, herbal formulations and finished herbal needs. Unfortunately, no researches have been drugs. These types of herbal formulations have carried out to determine the microbiological been used since ancient times to treat a safety of these herbal products in Dhaka extensive range of diseases [1-3]. Many metropolis, Bangladesh to the best of our developing countries continued to get benefit knowledge. In this present study, the level of from the rich knowledge of medical herbalism. A contamination of powdered herbal formulations Good numbers of people are still being used marketed locally in Dhaka city with selected Ayurvedic medicine in India, Kampo medicine in pathogenic bacteria and the susceptibility of Japan, traditional Chinese medicine (TCM), and these contaminants. Unani medicine in the Middle East and South Asia [4]. About 70-80% of the world population 2. MATERIALS AND METHODS relies on non-conventional medicines mainly of herbal origins for their primary health care 2.1 Sample Collection and Study Area particularly in the developing countries, because herbal medicines are relatively accessible and A total of 07 different antidiabetic herbal cheaper than the synthetic drugs [5]. preparations (ADHPs) were purchased randomly from identified herbal shops and retail outlets in The quality and safety of herbal preparations are different parts of Dhaka city. All collected also of great concern. The efficacy and safety of samples were analyzed for microbial herbal drugs, and to ensure the standard of contaminations at the Department of research on herbal medicines, the quality of the Microbiology, Bangladesh University of Health plant materials or preparations is of supreme Sciences (BUHS), Mirpur, Dhaka. importance [6]. 2.2 Chemical and Reagent The quality criteria for herbal formulations are based on a clear scientific explanation of the All chemicals and Reagents were of analytical unprocessed materials. Comprehensive quality grade and procured from Oxoid ltd, UK. criteria for herbal drugs due to ‘professional Experiments were done carefully with secrecy’ of herbalists is difficult to establish, but appropriate control. Chemicals and reagents in order to improve the purity and safety of the used were: products, observation of basic hygiene during preparation, standardization of some physical For culture: Blood agar, Mac Conkey agar, characteristic such as moisture content, pH and Chocolate agar and Saboraud’s dextrose microbiological contamination levels are agar. desirable [7]. Earlier studies have established the presence of potential contaminants in herbal For Gram’s stain: 1% Crystal violet, Lugol’s preparations [8]. The presence contaminants that iodine, Acetone, Dilute carbol fuchsin. serious health hazard are pathogenic bacteria such as Salmonella, Escherichia coli, Others: Hydrogen peroxide, Bile esculin, Staphylococcus aureus, Shigella spp. and other 6.5% Sodium chloride in trypticase soy broth Gram positive and Gram negative strains of and Normal saline.

Ahmed et al.; EJMP, 13(1): 1-5, 2016; Article no.EJMP.23556

2.3 Bacteriological Analyses dilutions were made. For tablets and capsules 1 g was dissolved in 10 ml Normal saline and Blood agar, MacConkey agar, Chocolate agar then 1:10 dilutions were made. 100 µl of the and Saboraud’s dextrose agar were used (Oxoid) dilutions were placed on the surface of plates for culturing and isolation of bacteria and fungus and spread widely with sterile inoculation wire. [14]. Identification of organisms were done as The count was calculated from average colony standard ways [15,16]. count/plate [17].

2.4 Preparation of Media 3. RESULTS

All dehydrated media were prepared according to After overnight incubation, the colonies in each manufacturer's instructions. The sterile media plate were counted and the values were were dispensed or poured into sterilized Petri averaged. In case of no growth the culture plate dishes and allowed to cool. The sterility of the was incubated for up to 48 hours. If there is no prepared media was checked by incubation of growth after 48 hours the culture is taken as “No blindly selected plates at 37°C for 24 hrs. growth”. The result is given in Table 1. It was noted that “No growth” of any Bacteria was found 2.5 Total Aerobic Bacterial Plate Count in one drug and No growth of any fungi was found in all drugs. In one drug, Enterococcus The method as mentioned by Brown, Poxton and (Streptococcus faecalis ) was found and in others wilkinson was used. For liquid drugs 1:10 bacillus subtilis was isolated.

Table 1. Selected bacteria isolated from the antidiabetic herbal preparations (ADHPs)

Name of drug Name of bacteria isolated Colony count Name of fungus isolated ADHP–1 Bacillus Subtillis 3.5 x 10 4/g N/G ADHP–2 Enterococcus (Streptococcus faecalis ) 1.0 x 10 4/ml N/G ADHP–3 N/G 00 N/G ADHP–4 Bacillus Subtillis 3.7 x 10 4/g N/G ADHP–5 Bacillus Subtillis 3.2 x 10 4/g N/G ADHP–6 Bacillus Subtillis 3.8 x 10 4/g N/G ADHP–7 Bacillus Subtillis 4.0 x 10 4/g N/G

45000 40000 35000 Bacillus Subtillis 30000 25000 20000 Enterococcus (streptococcus

No. of No. Bacterias 15000 faecalis) 10000 5000 0 ADHP–1 ADHP–2 ADHP–3 ADHP–4 ADHP–5 ADHP–6 ADHP–7

Fig. 1. Average bacterial count of 07 ADHPs

Ahmed et al.; EJMP, 13(1): 1-5, 2016; Article no.EJMP.23556

4. DISCUSSION 5. CONCLUSION

Many people of Bangladesh rely upon the herbal Microbial contaminations of the experimental medicine for medication; the assessment of drugs were within the acceptable limit. Samples pathogens in these medicines is urgently should be randomly collected from market to required. The present study was attempted to maintain the quality of drugs for testing the identify and quantify the pathogenic microbial contaminations. In general, microbial microorganisms in samples randomly collected contamination can approach from raw materials, from local markets of Dhaka city. during processing of raw materials and manufacturing of finished products. The results if More than 650 medicinal plant species have goes outside the acceptable limit, it should be been identified to be in use with around 25 plants informed to the concerned manufactures. So having high medicinal value in Bangladesh. A that, they can take appropriate measures to standard guideline for manufacturing herbal maintain the quality. ADHPs should attain to medicines has been set by the “Drug consumers without any kind of contaminations; Administration” in Bangladesh in recent times. qualities have to be continued throughout the The regulators have also finalized the testing beginning of process from the selection of raw criteria to boost the herbal sectors but the material up to the finished product. Appealing progression was slow earlier in the lack of these particulars into our consideration, specific testing criteria [18]. The present regulatory agencies should keep regular study was performed to identify and monitoring to ensure the safety of herbal enumerate the microbial contaminations in medicines. commercial herbal medicines considering nearby situation. CONSENT

It is not applicable. In this study 7 ADHPs were studied of which one showed no growth of any bacteria or fungi. The ETHICAL APPROVAL organisms found in our study were Bacillus subtilis in 5 (71.43%) ADHPs and Enterococcus It is not applicable. in one (14.29%) and were free from Salmonella, Shigella, Escherichia coli , other coliforms and ACKNOWLEDGEMENTS fungi (yeast and mould). In the study of Abba, 2009, organisms were isolated from all herbal The International Science Program (ISP) in preparations [5]. Out 150 herbal preparations, Sweden, particularly the International Program in Salmonella typhi was found in 70 (46.67%), the Chemical Sciences (IPICS) and Ministry of Shigella spp . in 29 (19.33%), Escherichia coli Science and Technology, Government of the in 88 (58.67%), and Staphylococcus aurues in People’s Republic of Bangladesh are gratefully 98 (65.33%). However, Noor R, 2013 in acknowledged for financial support in conducting their study found coliform in one (1.18%), this study. fungus in10 (11.76%), no salmonella and shigella spp . in any sample [18]. Like present COMPETING INTERESTS study, Shah B and Pokhrel N, 2012 found predominantly Bacillus subtilis in samples Authors have declared that no competing [19]. interests exist.

Bacterial load of our study was Bacillus subtilis REFERENCES 3.5–4.0 × 10 4 cfu/g of solid ADHPs and Enterococcus spp . 1.0 × 10 4 cfu/ml of liquid 1. World Health Organization (WHO). WHO ADHP. However, this very much within the guidelines on safety monitoring of herbal standard limit of microbial contamination medicines in pharmacovigilance systems; according to British Pharmacopeia (2004). The 2004. limits of microbial contamination are: 10 5 cfu/g or Available:http://www.who.int/medicinedocs/ ml for total aerobic bacteria, 10 4 cfu/g or ml for index/assoc/s7148e/s7148e.pdf yeasts and moulds, 10 3 cfu/g for (Accessed November 20, 2011) Enterobacteriaceae and other gram negative 2. Khanyile ZC, Singh N, Smith M, Shode organism and E. coli and Salmonella should be FO, Mngomezulz S, Dewir YH. absent [20]. Comparative assessment of bacterial

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contamination in commercial herbal Lagos, Nigeria. J Health, Population and products of Lessertia futescens . American- Nutrition. 2005;23(3):296–297. [PubMed] Eurasian J Agric. & Environ Sci. 2009;5(4): 13. Okunlola A, Adewoyin AB, Odeku AO. 494–499. Evaluation of pharmaceutical and microbial 3. Kulkarni C, Deshpande A, More S. qualities of some herbal medicinal Assessment of microbial contamination in products in south western Nigeria. Trop J commercial herbal oral medicinal liquids. Pharmaceut Res. 2007;6(1):661–670. International Journal of Pharma Research 14. Prescott LM, Harley JP, Klein DA. Isolation and Development. 1999;2(9):191-194. of pure bacterial cultures from specimens: 4. Mosihuzzaman M, Choudhury MI. Microbiology International. 4th edition. Protocols on safety, efficacy, standardize- Boston: WCB McGraw's Hill Companies. tion, and documentation of herbal 1999;714–796. medicine. 2008 IUPAC, Pure and Applied 15. Lucia Martin Texeira, Maria DA Gloria, Chemistry . 2008;80:2195–2230. Siqueira Carvalho, Patricia Lynn 5. Abba D, Inabo HI, Yakubu SE, Olonitola Shewmaker, Richard R. Facklam, OS. Contamination of herbal medicinal Enterococcus . in : James Versalovic, products marketed in Kaduna metropolis Karen C Carroll, Guido Funke, James H with selected pathogenic bacteria. Afr J Jorgensen, Marie Louise Landry, David W Tradit Complement Altern Med. 2009;6(1): Warnock, (eds.). Manual of Clinical 70–77. Microbiology, 10 th edition, Washington DC, 6. World Health Organization (WHO), author 2011;1:350-364. Research guidelines for evaluating the 16. Niall A Logan, Alex R Hoffmaster, Sean V safety and efficacy of herbal medicines. Shadomy, Kendra E Stauffer. Bacillus and Manila: World Health Organization regional other Aerobic Endospore-Forming office for the western pacific; 1993. Bacteria . In: James Versalovic, Karen C 7. Bauer R. Quality criteria and standardize- Carroll, Guido Funke, James H Jorgensen, tion of phytopharmaceuticals: Can Marie Louise Landry, David W Warnock, acceptable drugs standard be achieved. (eds.). Manual of Clinical Microbiology, 10 th Drugs Information J. 1998;32:101–110. edition, Washington DC. 2011;1:381-402. 8. De Smet PAGM. Overview of herbal 17. Brown R, Poxton IR, Wilkinson JF. quality control. Drugs Information J. 1999; Centirfuges, colorimeters and bacterial 33:717–724. counts. In: Collee JG, Dgiud JP, Frase AG, 9. Arias ML, Chaves C, Alfaro D. Marmion BP, (eds). Mackie and Microbiological analysis of some herbal McCartney Practical Medical Microbiology. infusions used as medicines. Rev Biomed. Churchil Livingstone. 13 th edition. 1999;10(1):1–6. 1989;2:246. Available:http://www.imbiomed.com.max/u 18. Noor R, Huda N, Rahman F, Bashar T, ay. English/ZYu91-01.html Munshi SK, Microbial contamination in 10. Erich C, Wolfgang K, Brigitte K. herbal medicines available in Bangladesh. Microbiological status of commercially Bangladesh Med Res Counc Bull. available medicinal herbal drugs- A 2013;39: 124-129. screenings study. Planta Med. 2001;67: 19. Bibha Shah, Nabaraj Pokhrel. Microbial 263–269. [PubMed] quality and antibacterial activity of herbal 11. Wolfgang K, Erich C, Brigitte K. Microbial medicines. Nepal Journal of Science and contamination of medicinal plants- A Technology. 2012;13(2):191-196. review. Planta Medica. 2002;68:5–15. 20. British Pharmacopoeia Comission. [PubMed] Appendix XVI (A–D). In: British Pharma- 12. Adeleye IA, Okogi G, Ojo EO. Microbial copoeia, Volume IV. Wielka. Brytania, contamination of herbal preparations in Medicines Commission. 2004;331-351. ______© 2016 Ahmed et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Chem ts ist uc ry d & o r R

P e Ahmed et al., Nat Prod Chem Res 2016, 4:5

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a h http://dx.doi.org/10.4172/2329-6836.1000229

N Natural Products Chemistry & Research ISSN: 2329-6836 Research Article Open Access Chemistry and Antidiabetic Effects of Phlogacanthus thyrsiflorus Nees Flowers MD Ranzu Ahmed1,2, Tania Sultana3, Rayesha Routary4, MD Shahinul Haque Khan1, Khozirah Shaari5, M Abu Sayeed2, M Mosihuzzaman1,6 and Begum Rokeya7* 1Department of Chemistry, Bangladesh University of Health Sciences (BUHS), Dhaka, Bangladesh 2Department of Applied Chemistry and Chemical Engineering, University of Rajshahi, Rajshahi, Bangladesh 3Jahangirnagar University, Savar, Dhaka, Bangladesh 4Government Ayurvedic College and Hospital, Guwahati, Assam, India 5Laboratory of Natural Products, Institute of Bioscience, University Putra Malaysia, Selangor, Malaysia 6Department of Chemistry, University of Dhaka, Dhaka, Bangladesh 7Department of Pharmacology, Bangladesh University of Health Sciences (BUHS), Dhaka, Bangladesh

Abstract The present study was carried out to evaluate the chemical constituents and antidiabetic activity of flower extracts of Phlogacanthus thyrsiflorus Nees in streptozotocin induced diabetic Long-Evans rats by feeding for 28 days. The ethanolic extract (1.25 mg/kg bw) exhibited significant reduction of fasting serum glucose level in diabetic rats. The ethanolic extract also showed improvement in parameters of lipid profile. Five compounds namely, β- sitosterol (1), stigmasterol (2), 8(17),13-labdadien-15,16-olide-19-oic acid (3), 19-hydroxy-8(17),13- labdadien-15,16-olide (4) were isolated from the dichloromethane extract and 2-(3,4-dihydroxyphenyl)-5,7- dihydroxy-4-chromenone (Luteolin) (5) were isolated from the 1-butanol part of methanol extract of flowers. The structures of these compounds were elucidated by extensive spectroscopic studies. This is the first report of isolation of compounds (1-5) from flowers of this plant. The activities of the flower extract may be due to the presence of some of these compounds which demands further studies.

Keywords: Phlogacanthus thyrsiflorus Nees; Streptozotocin; Type 2 Experimental diabetic model rats; Hypoglycemic effect; Isolation of compounds General Introduction 1H and 13C-NMR spectra including DEPT, 1H-1H COSY and Medicinal plants from time immemorial have been used frequently HMBC spectra were recorded on BRUKER DBX-400 MHz NMR for the treatment of diabetes [1,2]. Dependence on plant materials for and VARIAN 500 MHz NMR spectrometers. The chemical shifts managing diabetes mellitus is increasing due to its easy availability and are reported in ppm with respect to residual non-deuterated solvent low cost with often wrongly perceived reduced toxicity [3,4]. Therefore, signals. The mass spectra were recorded at 70 ev with Finnigan 4021 with up surging interests in antidiabetic plant materials, there is an and GCMS- QP2010 Ultra instrument. The fast atom bombardment urgent need for thorough scientific investigations of many potential mass spectrum (FAB+MS) was recorded as a positive ion mode with plants for both efficacy and potential toxicities. However, the challenge m/z ranging between 0.0020-1000.0000. of determining specific active components with good pharmacological Plant material activities still remains [5]. Flowers of Phlogacanthus thyrsiflorus Nees were collected from Phlogacanthus thyrsiflorusNees (Acanthaceae) locally known as Dibrughar, Assam, India. The flower was identified by Taxonomist rambasak is a large shrub found usually in the sub-tropical Himalayas, of Bangladesh National Herbarium (Accession number 42992). The Bihar, North Bengal, Assam and Bangladesh [6]. The shrub grows collected materials were cleaned, air dried and finally dried in an oven during May to August. The leaves are elliptical. The flowers are yellow at 40°C. The dried flowers were ground to a course powder with a in colour. Flowers taste bitter when it is taken after frying. It grows in cyclotec grinder. forest and regenerated from seeds [7] and flowering occurs in the month of February to April [8]. Different parts of this plant are being used for Extraction the cure of different ailments like, fever, gastritis, pharyngitis, cough, The powdered flowers (300 g) ofPhlogacanthus thyrsiflorusNees bronchial asthma, rheumatism and many more [9]. This botanical were successively extracted at room temperature with dichloromethane herbaceous plant is widely used as verdant foods, medicine practice and traditional medicines. The aqueous extract of the flower ofP. *Corresponding author: Begum Rokeya, Department of Pharmacology, thyrsiflorushave been claimed to possess significant reduction of blood Bangladesh University of Health Sciences (BUHS), 125/1 Darus Salam, Mirpur-1, glucose level (p<0.0001), serum cholesterol (P<0.01) and increase in Dhaka-1216, Bangladesh, Tel: +8801711811350; E-mail: [email protected] liver glycogen (P<0.0001) on steptozotocin induced diabetic mice [10]. Received June 16, 2016; Accepted June 28, 2016; Published July 02, 2016

The leaves are reported to contain diterpene lactone, phlogantholide, Citation: Ahmed R, Sultana T, Routary R, Khan SH, Shaari K, et al. (2016) saponins and flavonoids and their glycosides [9]. The objective of this Chemistry and Antidiabetic Effects of Phlogocanthus thyrsiflorus Nees Flowers. work was to investigate the scientific basis for its use in the treatment Nat Prod Chem Res 4: 229. doi:10.4172/2329-6836.1000229 of diabetes mellitus. Therefore, the study was designed to evaluate the Copyright: © 2016 Ahmed R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted antidiabetic effect of P. thyrsiflorus flowers on type 2 diabetic model use, distribution, and reproduction in any medium, provided the original author and rats and isolate compounds from it. source are credited.

Nat Prod Chem Res Volume 4 • Issue 5 • 1000229 ISSN: 2329-6836 NPCR, an open access journal Citation: Ahmed R, Sultana T, Routary R, Khan SH, Shaari K, et al. (2016) Chemistry and Antidiabetic Effects of Phlogocanthus thyrsiflorus Nees Flowers. Nat Prod Chem Res 4: 229. doi:10.4172/2329-6836.1000229

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(DCM) (2 L × 4: 24 hrs) followed by ethyl acetate (2 L × 4: 24 hrs) compound was found to be 286 g/mol. From the 1H- and 13C-NMR and methanol (2 L × 4: 24 hrs). The extracts were filtered off and spectral data (CDCl3) are presented in Tables 1 and 2, respectively. the filtrates were evaporated to dryness at 40°C with rotary vacuum evaporator and finally freeze dried to afford 8.1 g of DCM, 3.0 g of ethyl Experimental animals acetate and 16 g of methanol extracts. The powdered flowers (800 g) The study was conducted on adult Long-Evans rats of both sexes were also successively extracted with aq 80% ethanol (2 L × 6 times) to (weighing 180-220 g). They were bred at the BIRDEM animal house and give 14 g of aq 80% ethanol extract. The residue (142 g) was suspended maintained at a constant room temperature of 22°C, 40-70% humidity in water (3 L) and partitioned with DCM (3 L × 3 times) followed by conditions and the natural day-night cycle with an ad libitum access to 1-butanol (3 L × 3 times). The DCM and 1-butanol soluble parts were food and water except the day of experimental procedure when animals evaporated to dryness to get DCM (36.59 g) and 1-butanol (54.0 g) were used after 12 hrs fasting. The rats had no access to food during the soluble extractives. whole period of blood sampling. The influence of circadian rhythms Isolation of compounds was avoided by starting all the experiments at 8.30 a.m. Experiments on the animals were performed following the guidelines approved by The first dichloromethane extract (8.1 g) was fractionated by Bangladesh Association for Laboratory Animal Science. column chromatography (silica gel) using gradient elution of n-hexane, ethyl acetate and methanol with 10% increment and fourteen fractions Induction of Type 2 diabetes to the rats

(1F1-1F14) were collected. Fraction 1F5 was again fractionated over a Type 2 diabetes was induced by a single intraperitoneal injection silica gel column and eluted gradually with n-hexane and the polarity of streptozotocin (STZ, Upjohn Company, Kalamazoo, MI, USA) at a was increased by adding ethyl acetate and methanol in different dose of 90 mg/kg body weight to 48 hrs old pups. proportion and six fractions (2F1-2F6) were collected. Compound 1 Three months later of STZ injection an oral glucose (2.5 g/kg bw) was purified as needle shaped crystal from fraction 2F5 by washing the tolerance test was performed and type 2 diabetic model rats (blood crystal with n-hexane and methanol. Fraction 2F6 was purified to give compound 2 over a silica gel column and eluted with the same mobile glucose level 7-9 mmol/L at fasting condition) were selected to carry out the experiment with ethanolic extract of P thyrsiflorus flowers. The phase. Compound 2, (Rf value of 0.54) was isolated as white needles, after washing the crystal with dichloromethane and methanol. length of experiment was 28 days. A total of 18 Type 2 rats were used in this experiment. The rats were divided into the following three groups Fraction 1F11 showed brownish crystals. Then the fraction was of six rats in each group: again fractionated over a silica gel column and eluted gradually with n-hexane and the polarity was increased by adding ethyl acetate and 1) Type 2 control group (fed with water) methanol in different proportion and fourteen fractions (3F1-3F14) were 2) Type 2 positive control group (fed with glibenclamide at a collected. Fraction 3F5 showed crystal and was purified to Compound dose of 5 mg/kg bw) 3 by washing the crystal with the same solvent n-hexane and methanol. 3) Type 2 treated group (fed with 80% ethanol extract of P. Fraction 3F8 also showed crystal. Compound 4 was isolated by washing the crystal with the same washing solvents. thyrsiflorus at a dose of 1.25 gm/kg bw/10mL of water). The butanol extract (52.0 g) was chromatographed over Sephadex The rats were fed consecutively for 28 days with a single feeding LH-20 column and eluted gradually with deionized water and every day. The body weight of each rat was measured on day 0, 7, 14, 21 and 28 day of experimental period. methanol mixtures with decreasing polarity and fractions (4F1-4F71) were collected. Fraction 4F35 was purified to compound5 (19.0 mg) by HPLC [Column RP-18 (250 mm × 4.6 mm i.d.), mobile phase 60% Collection of blood samples for biochemical procedures aqueous acetonitrile, flow rate: 1.0 mL/min, UV detection at 254 nm The rats were kept fasting for 12 hrs on the 0 day and blood was and column oven temperature 37°C]. The isolated compounds had the collected from the fasted rats by amputation of the tail tip under following characteristics: (diethyl ether) anesthesia. On the 29th day after 12 hrs fasting blood Compound 1 was needle shaped crystals with m.p. 137-38°C. The was collected from the rats by cardiac puncture. The collected blood 1 13 samples were centrifuged; the serum was separated and kept frozen at H- and C-NMR spectral data (CDCl3) are presented in Tables 1 and 2, respectively. -20°C until analysis of different biochemical parameters. Compound 2 was needle shaped crystals with m.p. 162.5°C. The Biochemical analysis crystal of the compound was soluble in CHCl . From the 1H- and 3 The parameters measured were: serum glucose (glucose-oxidase), 13C-NMR spectral data (CDCl ) are presented in Tables 1 and 2, 3 serum insulin (ELISA) and serum lipids by enzymatic-colorimetric respectively. method. Compound 3 was long fatty shaped crystals. It gave single spot 1 Statistical analysis on TLC with Rf value 0.53 (n-hexane : ethyl acetate; 3:7). The H- 13 and C-NMR spectral data (CDCl3) are presented in Tables 1 and 2, Data from the experiments were analyzed using the Statistical + respectively. GCMS m/z 332 [M+H ]. FTIR (KBr) νmax: 2940, 1691, Package for Social Science (SPSS) software for windows version 12 1528, 669 cm-1. (SPSS Inc., Chicago, Illinois, USA). All the data were expressed as Mean ± SD as appropriate. Statistical analyses of the results were performed Compound 4 was white powder. It gave single spot on TLC with R f by using the student’s t-test or ANOVA (analysis of variance) followed value 0.50 (n-hexane : ethyl acetate; 4:6). The1 H- and 13C-NMR spectral by Bonferroni post hoc test. The limit of significance was set at p<0.05. data (CDCl ) are presented in Tables 1 and 2, respectively. GCMS m/z 3 In addition, the percentage changes compared to the initial value of the 334 [M+H+]. FTIR (KBr) ν : 2935, 1714, 1528, 670 cm-1. max corresponding groups of rats were calculated to help the understanding Compound 5 was brownish powder. The molecular mass of this of the effect ofP. thyrsiflorus flowers on different parameters studied.

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δ , Multi (J in Hz) Position H 1 2 3 4 5 1 - - 1.78 (m) 1.78 (m) - 2 - - 1.63 (m) 1.65 (m) - 3 3.52 (1H, m) 3.52 (1H, m) 1.67 (m) 1.69 (m) (6.53, s) 4 - - - - - 5 - - 1.97 (d; J=11.65 Hz) 1.96 (d; J=11.65 Hz) - 1.53 (dddd/dq, J=4.15, 12.90, 1.53 (dddd/dq, J=4.15, 12.90, 6 5.35 (1H, brs) 5.35 (1H, brs) 25.65 Hz) 25.65 Hz) (6.43, br s) 1.41 (d; J=10.75 Hz) 1.40 (d?; J=10.75 Hz) 2.40 (d; J=12.75 Hz) 2.40 (d; J=12.75 Hz) 7 - - 2.08 (ddd/dt; J=4.12, 12.35, 16.80, 2.24 (ddd/dt; J=4.12, 12.35, - 24.7 Hz) 16.80, 24.7 Hz) 8 - - - - (6.2. br s) 9 - - 1.73 1.73 - 10 - - - - - 1.82 (m) 1.82 (m) 11 - - - 1.67 (m) 1.66 (m)

2.29 (m) 2.29 (m) 12 - - 2.59 (sept/m; J=3.35, 5.80, 8.65, 2.57 (sept/m; J=3.35, 5.80, - 14,45 Hz) 8.65, 14,45 Hz)

13 - - - - - 14 - - 5.86 (s) 5.85 (s) (6.89, d, J-12 Hz) 15 - - - - (7.38, d, J-12 Hz) 4.72 (s) 4.73 (s) 16 - - 4.70 (s) 4.71 (s) 4.90 (s) 4.88 (s) 17 - - 4.49 (s) 4.77 (s) 18 0.68 (3H,s) 0.70 (3H,s) 1.16 (s) 1.1 (s) 19 1.01(3H,s) 1.01(3H,s) - - 20 - - 0.74 (s) 0.69 (s) 21 0.92 (3H, d, 6.4) 1.02 (3H, d, 7.5) 22 - 5.14 (dd, 8.4, 8.8) 23 - 5.02 (dd, 8.4, 8.4) 24 - - 25 - - 26 0.81 (3H, d, 6.5) 0.79 (3H, d. 6.5) 27 0.83 (3H, d, 6.5) 0.85 (3H, d, 6.5) 28 - - 29 0.85 (3H, t, 7.5) 0.80 (3H, t, 7.5)

Table 1: 1H-NMR spectral data for Compounds (1-5).

δ , Multi Position c 1 2 3 4 5 1 37.3 t 37.3 t 38.0 t 39.2 t 2 31.7 t 31.5 t 18.3 t 21.5 t 123.8 3 71.8 d 71.8 d 37.0 t 35.4 t 103.8 4 42.3 t 42.3 t 47.4 s 56.3 s 183.9 5 140.8 s 140.8 s 49.4 d 56.4 s 166.7 6 121.7 d 121.7 d 26.8 t 27.2 t 100.3 7 31.9 t 31.9 t 37.7 t 38.6 t 166.4 8 31.9 d 31.7 d 147.1 d 115.3 s 95.1 9 51.2 d 51.3 d 56.2 d 65.1 s 159.4 10 36.5 s 36.5 s 39.0 s 39.7 s 105.2 11 21.1 t 21.1 t 21.2 t 24.5 s 114.1 12 39.8 t 39.7 t 27.5 t 27.5 t 147.0 13 42.2 s 42.2 s 170.9 s 147.5 s 151.0 14 56.8 d 56.8 d 115.2 s 115.3 t 116.8 15 24.3 t 24.3 t 174.2 s 171.0 s 120.4

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16 28.2 t 28.2 t 73.1 t 73.2 d 17 56.1 d 56.1 d 107.2 t 106.8 t 18 12.0 q 11.8 q 16.3 q 19.0 q 19 19.4 q 19.8 q 184.8 s 174.2 t 20 36.1 d 40.5 d 14.7 q 15.3 d 21 18.8 q 21.2 q 22 34.0 t 138.3 d 23 26.1 t 129.3 d 24 45.9 d 50.2 d 25 29.2 d 31.7 d 26 19.0 q 21.1 q 27 19.0 q 19.0 q 28 29.1 t 25.4 t 29 12.2q 12.0 q

Table 2: 13C-NMR spectral data of Compounds (1-5).

Glucose(mmol/L) Glucose(mmol/L) Glucose(mmol/L) Group 0 day 14 day 28 day T2WC 7.57 ± 0.52 8.30 ± 0.88 8.52 ± 1.43 (n=6) (100%) (110%) (113%) Gliben 7.99 ± 1.03 6.59 ± 1.30 6.39 ± 1.14* (n=6) (100%) (82%) (80%) Ethanol ext 8.86 ± 0.79 7.17 ± 1.22 6.42 ± 1.32* (n=6) (100%) (81%) (72%) Results are expressed as Mean ± SD; *p<0.05 vs Type 2 water control (ANOVA with post Hoc Bonferroni test). T2WC=Type 2 Water Control, Gliben=Glibenclamide. Table 3: Effect of P. thyrsiflorusflower extract on fasting blood glucose level of STZ-induced Type 2 diabetic model rats.

Results and Discussion (-OH), 1691 (>C=O), 1528, cm-1 spectrum indicated the presence of hydroxyl and carbonyl groups, and carbon carbon double bonds in the

Chemistry of P. thyrsiflorus compound 3. The molecular formula C20H28O4 of 3 was deduced from GCMS data. Three CH proton appears at δ 1.63 (m), 1.67(m), 1.78 Repeated chromatographic separation and purification of 2 C (m). Two H appears at δ 4.70 (s) and 4.72 (s) for the CH of furan dichloromethane and 1-butanol part of methanol extracts of flowers H 2 ring. Two olefinic H of 17 carbon appears at δ 4.90 (s) and 4.49 (s). of P. thyrsiflorus provided five compounds (1-5). The 13C-NMR (Table H Another olefinic H of furan ring comes atδ 5.86 (s). Carboxylic 2) of 1 showed 29 carbons including an oxymethine carbon signal at H functional group containing carbon appears at δ 184.8, furan ring δ 71.8 and two olefinic carbons atδ 140.8 and δ 121.7 characteristic C C C C containing quarternary carbon at δ 170.9 and cyclic carbonyl carbon of spirostene [11]. The multiplicities of the carbon signals were C at δ 174.2. The1 H-, 13C- and DEPT-NMR spectra of compound 3 are determined by DEPT 135 experiments which revealed the presence of C 6 methyls, 11 methylenes, 9 methines and 3 quaternary carbon atoms similar to the reported data of 8(17),13-labdadien-15,16-olide-19-oic in compound 1. The1 H- (Table 1) and 13C- (Table 2) NMR spectral data acid [9,14]. Therefore, the structure of compound3 is characterized of compound 1 are similar to the reported data for β-sitosterol [12,13]. as 8(17),13-labdadien-15,16-olide-19-oic acid. This is the first report Therefore, the structure of compound 1 is characterized as β-sitosterol of isolation of 8(17),13-labdadien-15,16-olide-19-oic acid from the (Figure 1). β-Sitosterol is a very common steroidal compound present flowers of this plant. in almost all kinds of plants. β -Sitosterol is reported for the first time The molecular formula 20C H30O4 of compound 4 was deduced from the flowers of this plant. from GCMS data. Compound 4 has three CH2 protons appearing at δ 1.65 (m), 1.69 (m) and 1.78 (m). Two H appears at δ 4.71 (s) and The 1H-NMR spectral data (Table 1) of compound 2 revealed H H 4.73 (s) for the CH2 of furan ring. Two olefinic H of 17 carbon appears multiplet signal for an oxymethine proton at δH 3.52. The olefin proton at δH 4.88 (s) and 4.77 (s). Another olefinic H of furan ring comes at resonating at δH 5.35 was charecterististic of 5-steroids [11]. The δH 5.85 (s). Hydroxyl functional group containing carbon appears at spectrum revealed signals at δH 0.68 and 1.01 (3H each) assignable to δC 174.2, furan ring containing quarternary carbon at δC 147.5 and two tertiary methyl groups at C-18 and C-19, respectively. The signals 1 13 cyclic carbonyl carbon at δC 171.0. The H- (Table 1), C- (Table 2) of two further secondary methyl groups at δH 0.84 (J=7.3 Hz) and δ 0.83 (J=7.3 Hz) could be attributed to two methyl groups at C-26 and DEPT-NMR spectra of compound 4 are similar to the reported H data of 8(17),13-labdadien-15,16-olide-19-oic acid [9,15] except the and C-27, respectively. The doublet at δH 0.92 (d, J=8.0 Hz, H-21) was demonstrative of a methyl group at C-21. On the other hand, the three- carboxylic acid functional group reduced to hydroxyl group. Therefore, the structure of compound 4 is characterized as 19-hydroxy-8(17),13- proton triplet at δH 0.81 could be assigned to the primary methyl group at C-29. The 1H- (Table 1), 13C- (Table 2) and DEPT-NMR spectra of labdadien-15,16-olide. This is first report of 19-hydroxy-8(17),13- compound 2 are similar to the reported data of stigmasterol [12,13]. labdadien-15,16-olide from the flowers of this plant. Therefore, the structure of compound 2 is characterized as stigmasterol. The molecular formula 15C H10O6 was determined from 1D and Stigmasterol is very common steroidal compound present in many 2D NMR data of compound 5. The13 C-NMR spectrum of 5 displayed plants but stigmasterol is the first report from the flowers of this plant. 15 carbon atoms, which is characteristic of a flavonoid type skeleton. 1 Compound 3 is a diterpenoid compound. Its IR (KBr) ν: 2940 The molecular mass of this compound is 286 g/mol. The H- (Table 1), 13C- (Table 2) and DEPT-NMR data of compound 5 are similar to

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HO HO

O O O O OH OH H H H H HO O H H

H H OH O O HO HO

iure Figure Stru c1:tu Structurere of is oofl aisolatedted 05 05 c compoundsompound sfrom fro P.m thyrsiflorus P. thyrs.iflorus. the reported data of luteolin [16,17]. On the basis of all the spectral Effect ofP. thyrsiflorus flower on serum insulin level of STZ- data, the compound 5 is characterized as luteolin. Luteolin is a induced Type 2 diabetic model rats: The effect of P. thyrsiflorus known compound and its IUPAC name is 2-(3,4-dihydroxyphenyl)- flowers extract on insulinemic status of type 2 diabetic model rats was 5,7-dihydroxy-4-chromenone. Compound 5 is isolated for the first observed (Figure 2). At the beginning of the study period, all groups time from this plant. showed almost similar serum insulin level. After 28 days consecutive feeding glibenclamide treated rats showed more than fivefold increase Antidiabetic effects ofP. thyrsiflorus flowers in serum insulin level which was highly significant (p<0.001). Ethanol Effect of P. thyrsiflorus on fasting serum glucose level of Type extract showed a 23% increase in serum insulin level at the end of the 2 diabetic model rats: Fasting serum glucose (FSG) levels of type 2 study period compared to baseline level, however the increase was not diabetic models rats of the three experimental groups were almost significant. As it is seen from the table, T2WC group showed a 48% similar on 0 day (Table 3). After oral administration of respective reduction compared to baseline value which is logical. Type 2 diabetic treatment to the type 2 diabetic model rats of different groups for 28 rat model those are produced in this study by injecting STZ to neonate rats have hypoinsulinemia which resemble beta cell secretory defect. days of experimental period, it was found that the FSG level of type Therefore, in this study the condition of the Type 2 water control group 2 rats treated with ethanol extract of P. thyrsiflorusflowers showed deteriorates with time which is manifested by hyperglycemia and a significant decrease compared to that of control group (p=0.042). decreased serum insulin level. Morever, It is seen from the Table 3 that ethanol extract treated group showed a 28% reduction of FSG level compared to 0 day value. Effect ofP. thyrsiflorus flowers extract on the body weight of Chakravaty et. al. found anti-hyperglycemic activity with P. thyrsiflorus Type 2 diabetic model rats: The effect ofP. thyrsiflorusflowers extract flower on STZ induced diabetic mice [10]. As expected, glibenclamide on body weight of type 2 diabetic model rats was observed during 28 also ameliorated the diabetic condition on 28th day. Glibenclamide days study period. Body weight of each rat was taken at seven days significantly (p=0.037) reduced fasting glucose on 28th day when interval. No significant change was found in body weight in any group compared to that of control group. On the contrary, there was a 13% after 28 days of study (Table 4). increase in the fasting glucose level of type 2 control group on 28th day Effects ofP. thyrsiflorus flowers extract on serum cholesterol of experimental period compared to the baseline. and triglyceride levels of Type 2 diabetic model rats: Effect ofP. thyrsiflorus flowers extract on serum cholesterol and triglyceride level

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level, there was a reduction of 23% in glibenclamide treated groups 1.6 after 28 days study period. Ethanol extract treated group also showed a 1.4 l reduction in serum TG level by 16% (Table 5), although the reduction

e 1.2 e

l * level was not significant.

i 1 l l

m Chronic effects of P. thyrsiflorus flowers extract on serum HDL-C s u 0.8 i 0 day and LDL-C levels of STZ-induced Type 2 diabetic model rats: The

m 0.6

u 28 day effects of chronic treatment with extract of P. thyrsiflorus flowers on r

e 0.4

the serum HDL- and LDL-cholesterol levels of type 2 diabetic model 0.2 rats are summarized in Table 6. It was found that at the end of the 0 28 days study period, HDL level increased significantly (p=0.031) in T2WC Gliben Ethanol ext ethanol extract of P. thyrsiflorustreated group compared to water rou control group. Glibenclamide treated group was unable to increase Figure 2: Effect of P. thyrsiflorus flower extract on the serum insulin level of serum HDL level at the end of 28 days study period when compared to STZ- induced Type 2 diabetic model rats. *p<0.001 compared water control the water control group. As it is seen from the table water control group group (ANOVA with post Hoc Bonferroni test). had a 17% decrease in HDL level compared to its baseline value. In case of atherogenic LDL-cholesterol, the level was decreased by 24% with the treatment of ethanol extract of P. thyrsiflorus flowers (25 mg/dL on Body weight (g) Group the 0 day to 19 mg/dL on the 28th day) at the end of the study period. 0 day 7 day 14 day 21 day 28 day Glibenclamide also decreased LDL level by 35% compared to the initial T2WC 192 ± 21 185 ± 28 188 ± 32 182 ± 29 193 ± 34 (n=6) (100%) (96%) (98%) (95%) (101%) value. However, the decrease was not statistically significant for both Gliben 182 ± 13 189 ± 25 183 ± 8 182 ± 7 182 ± 16 the cases. On the other hand, control group showed a 12% increase in (n=6) (100%) (104%) (101%) (101%) (100%) serum LDL which is harmful for health. Type 2 diabetes is associated Ethanol ext 202 ± 7 193 ± 19 200 ± 18 195 ± 20 198 ± 18 with marked imbalance in lipid metabolism [18]. As we know, the (n=6) (100%) (96%) (99%) (97%) (98%) most vulnerable problem in dyslipidemia is the increase in atherogenic Results are expressed as Mean ± SD. ANOVA with post Hoc Bonferroni test was lipids i.e., increase in serum LDL-C, Triglyceride and total cholesterol done. levels with the reduction in good cholesterol (HDL-C) level [19-21]. It Table 4: Effect of P. thyrsiflorus flowers extract on the body weight of Type2 is now well established that elevated triglycerides and low high-density diabetic model rats. lipoprotein cholesterol (HDL-C) levels contribute to cardiovascular disease risk. This abnormal high level of serum lipids is mainly due Cholesterol (mg/dL) TG (mg/dL) Group to the uninhibited actions of lipophytic hormones on the fat depots 0 day 28 day 0 day 28 day mainly due to the action of insulin. The role of hypolipidemic agents T2WC 79 ± 5.93 73 ± 8.28 70 ± 4.12 59 ± 8.56 is to correct dyslipidemia. In this study, at the end of the experimental (n=6) (100%) (92%) (100%) (84%) period, ethanol extract of P. thyrsiflorus flowers decreased Total Gliben 81 ± 8.87 65 ± 5.78 77 ± 7.53 59 ± 11.85 cholesterol, triglycerides and LDL-C level although non-significantly (n=6) (100%) (80%) (100%) (77%) and significantly improved HDL-C level (p=0.031) compared to water control group of Type 2 rats. This implies that ethanol extract Ethanol ext 80 ± 11.21 71 ± 8.96 70 ± 9.09 54 ± 7.52 of P. thyrsiflorus flowers can prevent or be helpful in reducing the (n=6) (100%) (89%) (100%) (77%) atherogenic lipids in diabetes. Results are expressed as Mean ± SD. Statistical analysis was done using ANOVA with post Hoc Bonferroni test. Conclusion Table 5: Effects of P. thyrsiflorus flowers extract on serum cholesterol and triglyceride levels of STZ-induced Type 2 diabetic model rats. From the obtained results it may be concluded that, flowers ofP. thyrsiflorus possesses hypoglycemic and to some extent hypolipidemic HDL (mg/dL) LDL (mg/dL) properties. The flowers of the plant contained 5 compounds which Group 0 day 28 day 0 day 28 day have been isolated and characterized. The obtained antidiabetic activity by the ethanolic extract of P. thyrsiflorus flowers may be due to the T2WC 40 ± 2.81 33 ± 3.25 25 ± 6.98 28 ± 10.62 (n=6) (100%) (83%) (100%) (112%) presence of these compounds, the activities of which remain to be explored in future. Gliben 40 ± 3.08 36 ± 6.26 26 ± 7.07 17 ± 9.70 (n=6) (100%) (90%) (100%) (65%) Acknowledgements Ethanol extract 41 ± 3.20 41 ± 3.39* 25 ± 13.60 19 ± 9.05 The International Science Program (ISP) in Sweden, particularly the (n=6) (100%) (100%) (100%) (76%) International Program in the Chemical Sciences (IPICS) and Ministry of Science Results are expressed as Mean ± SD. *p<0.05 vs Type 2 water control (ANOVA and Technology, Government of the People’s Republic of Bangladesh are with post Hoc Bonferroni test). gratefully acknowledged for financial support in conducting this study. The authors Table 6: Effect of P. thyrsiflorus flowers extract on the serum HDL-C and LDL-C are also grateful to Prof Dr BP Sharma, Assam, India for kindly providing the P. levels of STZ-induced Type 2 diabetic model rats. thyrsiflorus flowers. is presented in Table 5. Ethanol extract of P. thyrsiflorus flowers caused References non-significant reduction in total cholesterol level on 28th day [Serum 1. Patel DK, Prasad SK, Kumar R, Hemalatha S (2012) An overview on cholesterol (M ± SD) mg/dl, 0 day (80 ± 11.21) vs. 28 day (71 ± 8.96)] antidiabetic medicinal plants having insulin mimetic property. Asian Pac J Trop (p

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3. Mosihuzzaman M (2012) Herbal medicine in healthcare--an overview. Nat Prod 13. Chaturvedula VSP, Prakash I (2012) Isolation of Stigmasterol and ß-Sitosterol Commun 7: 807-812. from the dichloromethane extract of Rubus suavissimus. International Current Pharmaceutical Journal 1: 239-242. 4. Mosihuzzaman M, Sydnes LK (2010) Herbal products in healthcare-- challenges and potential. Comprehensive Bioactive Natural Products, Vols. I to 14. Singh SA, Singh NR (2010) Antimicrobial Activity of Cassia didymobotrya and VIII, Chapter 12, ISBN: 1933699507. Phlogacanthus thyrsiflorus. J Chem Pharm Res 2: 304-308.

5. Mosihuzzaman M, Choudhary MI (2008) Protocols on Safety, Efficacy, 15. Barua AK, Chowdhury MK, Biswas S, Gupta CD, Banerjee SK, et al. (1985) Standardization, and Documentation of Herbal Medicine. Pure Appl Chem 80: The structure and stereochemistry of phlogantholide-A, a diterpene from 2195-2230. Phlogacanthus thyrsiflorus. Phytochemistry 24: 2037-2039.

6. Khare CP (2007) Indian Medicinal Plants: An Illustrated Dictionary, USA. 16. Peters NK, Frost JW, Long SR (1986) A plant flavone, luteolin, induces Springer Science Business Media, p: 478. expression of Rhizobium meliloti nodulation genes. Science 233: 977-980.

7. Kar A, Bortakur SK (2008) Wild vegetables of karbi-Anglong district, Assam. 17. Ode OJ, Asuzu IU (2014) Luteolin isolate from the methanol extract identified Natural Product Radiance 7: 448-460. as the singlecarbon compound responsible for broad antiulcer activities of Cassia singueana Leaves. IOSR Journal of Pharmacy 4: 17-23. 8. Tamang JP, Thapa MP, Sharma RM, Rai AK, Rai P, et al. (2005) Carrying capacity study of Teesta Basin in Sikkim. Biological Environment Food 18. Gadi R, Samaha FF (2007) Dyslipidemia in type 2 diabetes mellitus. Curr Diab Resource, p: 8. Rep 7: 228-234.

9. Gogoi B, Kakoti BB, Bora NS, Goswami AK (2013) Phytochemistry and 19. Forrester JS, Makkar R, Shah PK (2005) Increasing high-density lipoprotein Pharmacology of Phlogacanthus Thyrsiflorus Nees: A Review. Int J Pharm Sci cholesterol in dyslipidemia by cholesteryl ester transfer protein inhibition: an Rev Res 23: 175-179. update for clinicians. Circulation 111: 1847-1854.

10. Chakravarty S, Kalita JC (2012) Antiheperglycemic effect of flower of 20. Milionis HJ, Kakafika AI, Tsouli SG, Athyros VG, Bairaktari ET, et al. (2004) Phlogacanthus thyrsiflorus Nees on streptozotocin induced diabetic mice. Effects of statin treatment on uric acid homeostasis in patients with primary Asian Pacific Journal of Tropical Biomedicine 2: S1357-S1361. hyperlipidemia. Am Heart J 148: 635-640.

11. Agrawal PK, Jain DC, Gupta RK, Thakur RS (1985) Carbon – 13 NMR 21. Hyeung SL, Sung JW, Sae KK (2008) Hypolipidemic and Hepatoprotective spectroscopy of steroidal saponins. Phytochemistry Reg 24: 2476-2496. effects of Picorrhiza Rhizome in high fat diet supplied mice. A prevention study. Biomolecules & therapeutics 16: 46-53. 12. Kamboj A, Saluja AK (2011) Isolation of Stigmasterol and ß-sitosterol From Petroleum Ether Extract of Aerial Parts of Ageratum Conyzoides (Asteraceae). International Journal of Pharmacy and Pharmaceutical Sciences 3: 94-96.

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Nat Prod Chem Res Volume 4 • Issue 5 • 1000229 ISSN: 2329-6836 NPCR, an open access journal

Biomedicine & Pharmacotherapy 89 (2017) 1242–1251

Available online at ScienceDirect

www.sciencedirect.com

Animal models for assessing the impact of natural products on the

aetiology and metabolic pathophysiology of Type 2 diabetes

a b c c c

Md. Asrafuzzaman , Yingnan Cao , Rizwana Afroz , Danielle Kamato , Susan Gray ,

,b,c

Peter J. Little*

Asian Network of Research on Antidiabetic Plants (ANRAP), Bangladesh University of Health Science, Mirpur, Dhaka 1216, Bangladesh

Department of Pharmacy, Xinhua College of Sun Yat-sen University,Tianhe District, Guangzhou 510520, China

School of Pharmacy, Pharmacy Australia Centre of Excellence, The University of Queensland, Woolloongabba, Queensland 4102 Australia

A R T I C L E I N F O A B S T R A C T

Article history:

Received 6 January 2017 Type 2 diabetes mellitus is a complex and heterogeneous disorder which in its most common

Received in revised form 28 February 2017 manifestation arises from insulin resistance and later insulin insufﬁciency. Type 2 diabetes is

Accepted 5 March 2017

characterised by impaired insulin sensitivity and diagnosed as hyperglycaemia. Because of its

cardiovascular consequences, Type 2 diabetes represents one of the world’s leading causes of mortality

Keywords: and morbidity. Drug discovery and development are required to produce better ways to prevent, treat and

Animal models

manage diabetes and its complications. Diabetes is a human, not an animal disease, so animals do not get

Genetics

Type 2 diabetes. However there are animal models which are variously suitable for the investigation of

Hyperglycaemia

new agents for the treatment of Type 2 diabetes. In this Review we have examined the various models

Streptozotocin,

that are available for the study of natural products with a focus on models (genetic, nutritional and

spontaneous) for the metabolic abnormities of diabetes. These models are also relevant to the

investigation of Western medicines for the treatment of diabetes. A suitable experimental model plays an

important role in drug discovery for translational studies leading to increased understanding of the

molecular basis and management of diabetes.

Contents

1. Introduction ...... 1243

2. Animal models for the study of the metabolic defects of type 2 diabetes ...... 1244

3. Spontaneous or genetically derived models of type 2

diabetes ...... 1244

4. Most common spontaneous animal models of type 2 diabetes ...... 1244

4.1. Ob/Ob (C57BL/6J-Lep ) mouse model ...... 1244

4.2. db/db mouse (C57BL/KsJ) model ...... 1244

4.3. KK mouse ...... 1244

4.4. KK/A mouse ...... 1244

4.5. NZO (New Zealand Obese) mouse ...... 1245

4.6. NONcNZO 10 mouse ...... 1245

4.7. TSOD (Tsumara Suzuki obese diabetic mice) mouse ...... 1245

4.8. M16 mouse ...... 1245

4.9. Zucker fatty rat ...... 1245

4.10. SHR/N-cp rat ...... 1245

4.11. JCR/LA-cp rat ...... 1245

4.12. OLETF rat ...... 1245

* Corresponding author at: School of Pharmacy, The University of Queensland, Pharmacy Australia Centre of Excellence, 20 Cornwall Street, Woolloongabba QLD 4102,

Australia.

E-mail address: [email protected] (P.J. Little).

http://dx.doi.org/10.1016/j.biopha.2017.03.010

M. Asrafuzzaman et al. / Biomedicine & Pharmacotherapy 89 (2017) 1242–1251 1243

4.13. Cohen diabetic rat ...... 1245

4.14. GK rat ...... 1246

4.15. Torri rat ...... 1246

4.16. TollyHo/Jng mouse ...... 1246

5. Diet or nutrition induced animal models of Type 2 diabetes ...... 1246

6. Common animal models of diet- or nutrition-induced Type 2 diabetes ...... 1246

6.1. C57BL/6 mice ...... 1246

6.2. Nagoya-Shibata-Yasuda (NSY) mice ...... 1246

7. Chemically induced models of hyperglycaemia/diabetes ...... 1246

7.1. STZ induced animal models ...... 1246

7.2. Neonatal STZ induced animal model of hyperglycaemia ...... 1246

7.3. STZ and nicotinamide induced animal model ...... 1247

7.4. STZ and High Fat Fed (HFD) animal model ...... 1248

7.5. Alloxan induced animal model ...... 1248

7.6. Goldthioglucose induced animal model ...... 1249

8. Surgically induced model of hyperglycaemia in multiple animals ...... 1249

8.1. Full and partial pancreatectomy models of hyperglycaemia ...... 1249

8.2. Genetically modiﬁed models of hyperglycaemia/diabetes: ...... 1249

8.2.1. Beta cell dysfunction animal model ...... 1249

8.2.2. Transgenic or knock-out animal model ...... 1249

9. Large animal models of hyperglycaemia and diabetes ...... 1249

9.1. Pigs ...... 1249

9.2. Rhesus monkey ...... 1249

10. Conclusions ...... 1250

References ...... 1250

1. Introduction addressed some conditions but did not reduce mortality and were

generally considered to have a low safety proﬁle with insulin

There has been an enormous increase in the number of people linked to hypoglycaemic episodes, sulfonylureas also associated

in the world with Type 2 diabetes in the last two decades [1]. In with hypoglycaemia and metformin with lactic acidosis. With the

Asia, the number of people with Type 2 diabetes has risen to be biggest rise in the number of people with Type 2 diabetes being in

well over 100 million people. This rise has mostly been driven by Asia, which is the home of natural product medicines such as

the rapid increase in community afﬂuence with levels of obesity Traditional Chinese Medicines (TCM), there has been an increase in

rising in breadth and extent – more individuals with higher body interest in the historical and future use of natural medicines for the

mass indices (BMI). Although Type 2 diabetes can occur in thin treatment of Type 2 diabetes. Interest in TCM extends to the

people, most patients with Type 2 diabetes have the form which is identiﬁcation and development of new Western drugs from

driven by insulin-resistance as a corollary to obesity [2–4]. Changes natural products. There is also a perception, not necessarily

in community behaviour and especially increasing economic strongly founded, that natural medicines are safer than Western

status has led to increased energy intake, changing employment medicines. So in its best outcome, natural product derived

characteristics have led to decreasing physical activity at work and medicines might be both efﬁcacious and safe.

life style factors related to technology have increased sedentary In all areas of therapeutic discovery the most important aspect

time and all of these components are independent predictors of is the availability of animal models which reproduce the human

diabetes [5,6]. Diabetes is diagnosed by hyperglycaemia although disease condition with high ﬁdelity. For Type 2 diabetes the three

the full metabolic pathophysiological proﬁle is much more leading parameters of these aetiologies are insulin resistance,

extensive. Hyperglycaemia has devastating effects on the cardio- pancreatic beta cell failure and dysfunction of the incretin system

vascular system precipitating both microvascular and macro- all of which lead to hyperglycaemia [19–21]. For the cardiovascular

vascular disease [7,8]. From a pathophysiological perspective, disease of diabetes the major pathology is atherosclerosis which is

diabetes is a cardiovascular disease although the pathophysiologi- markedly accelerated by the diabetic milieu; this is a very

cal consequences extend well beyond the cardiovascular system. important point but will not be directly addressed in this review

Atherosclerosis is the major underlying pathology of most whilst noting that essentially different models are required to

cardiovascular disease and it is accelerated by the metabolic study atherosclerosis from those which are suitable for the study of

milieu of diabetes but the pathological mechanism(s) of athero- the aetiology and pathology of diabetes [22,23]. The biggest

sclerosis and of the mechanism(s) of the accelerating effect of experimental issue is that the metabolic abnormalities of diabetes

diabetes remain largely unknown [9–13]. Drugs for treating and their consequential effects on the cardiovascular system have

hyperglycaemia/diabetes aim to prevent or reduce the impact of not been able to be reproduced with animal models that meet the

atherosclerosis by addressing various molecular mechanisms of required level of ﬁdelity. Similarly, there are issues with in vitro cell

disease [11,14–17]. Diabetes per se represents an independent risk models of hyperglycaemia and diabetes [22]. Various animal

factor for cardiovascular disease [18]. Cardiovascular disease is the models have been used to evaluate natural products and Western

largest single cause of mortality in the community and with the derived mechanistic medicines but in most cases the results have

superimposition of diabetes, cardiovascular disease becomes the not been translatable to the clinic because the models are not

majority cause of death in people with diabetes [18]. Rising adequate and the utility and extrapolation of the results have been

diabetes and CVD is having a major impact on human health and over estimated

also on the ﬁscal position of the budget of many countries. In this Review we have examined the various models that are

For many decades the treatment of diabetes, essentially the available for the study of natural products with a focus on models

treatment of hyperglycaemia, relied on insulin and several oral for the metabolic abnormities of diabetes; these models are also

agents, mostly sulfonylureas and metformin. These drugs relevant to the investigation of Western medicines for the

1244 M. Asrafuzzaman et al. / Biomedicine & Pharmacotherapy 89 (2017) 1242–1251

treatment of diabetes. We note that diabetes is a human, not an polygenic animal models symbolize more closely the human

animal disease, so animals do not get “Type 2 diabetes” however condition when compared to monogenic models [28].

this article is about animal models which are suitable for the

investigation of new agents for the treatment of Type 2 diabetes so 4. Most common spontaneous animal models of type 2 diabetes

we will refer to the models as being models of Type 2 diabetes

notwithstanding the obvious contradiction. 4.1. Ob/Ob (C57BL/6J-Lep ) mouse model

2. Animal models for the study of the metabolic defects of type 2 Ob/Ob mice are obtained from spontaneous mutations of the

diabetes leptin gene in the C57BL/J6 strain which shows a deterioration of

leptin signalling. Lep mutation shows obesity and hyperglycae-

In most cases, Type 2 diabetes begins from the development of mia with increasing insulin resistance [30,31]. Leptin deﬁciency or

obesity and increased insulin resistance due to unknown leptin resistance plays a key role in modulating energy intake and

mechanism(s) but most likely involving insulin signalling energy expenditure that result in obesity in ob/ob mice [29].

inhibition by lipophilic metabolites [9,20,24]. The most common Obesity functions like diabetes and the effects are characterised by

form of Type 2 diabetes is insulin resistance and later insulin hypertrophy and hyperplasia of pancreatic islets. Following on

insufﬁciency [25] noting that a sizable proportion of people with from increasing body weight, hyperglycaemia will develop,

Type 2 diabetes have a form which arises in thin subjects and probably due to the aforementioned hypertrophy and hyperplasia

arising from biochemically induced beta cell failure [2]. The of pancreatic islets. Human obesity due to monogenic mutations is

epidemiologically predominant form of Type 2 diabetes mellitus rare, though humans develop obesity due to leptin deﬁciency

is mainly characterised by peripheral insulin resistance, hyper- [27,28]. However monogenic (ob/ob mice) models of obesity are

glycaemia, hyperinsulinemia and later beta cell failure and hence commonly used for diabetes research [27,31]. Ob/ob mice have

insulin levels and glycaemic control differ depending upon the hyperglycaemia with insulin resistance when they gain weight and

stage of the disease and the capacity of the beta cells to secrete these characteristics reﬂect the condition of obesity in human [29].

sufﬁcient insulin (hyperinsulinemia) to maintain normoglycae- However hyperglycaemia in ob/ob mice is relatively mild and

mia [26]. Therefore, animal models of Type 2 diabetes should transient [30]. As well as hepatic glucose overproduction,

ideally mimic this pathway of insulin resistance and beta cell increased activity of gluconeogenic enzymes, decreased activity

failure [27]. of glycolytic and glycogen synthesis enzymes and increased

As described above, animal models do not, essentially, by lipogenesis in liver are developed from insulin resistance. On

deﬁnition, faithfully reproduce the human condition of Type 2 the molecular level insulin resistance develops in ob/ob mice due

diabetes. However, the availability of models with elements of to impaired insulin receptor autophosphorylation, decreased

Type 2 diabetes provides researchers with an opportunity to insulin receptor tyrosine kinase signal transduction and reduced

study speciﬁc metabolic and dysmetabolic parameters and the insulin binding to receptor. So ob/ob mice are severely obese,

impact of interventions which may provide insights which are as hyperinsulinaemic and insulin resistant throughout life. Any

applicable to the human condition as is possible at this time [26]. product which is purported to reduce the body weight, reduce

Animal models are useful and beneﬁcial in biomedical research hyperglycaemia and improve insulin sensitivity and to its

because they provide speciﬁc experimental opportunities and can antidiabetic effect could suitably be studied in this model [28].

thus provide new insights into the pathophysiology of human

diabetes. Rodents are the most suitable model because of their 4.2. db/db mouse (C57BL/KsJ) model

small size, amenability to genetic manipulations, short genera-

db/db

tion intervals and economic considerations [28] and thus rodents The Lep model is obtained from the C57BL/KsJ strain due to

are most thoroughly used animal to mimic human Type 2 autosomal recessive mutation of the leptin receptor on chromo-

diabetes mellitus [26]. Primates have been used successfully in some 4 [27,32]. The db/db mouse has been frequently used for the

studies of diabetes and cardiovascular disease but they provide study of Type 2 diabetes and particularly diabetic dyslipidaemia

special problems [26]. Thus, different types of animal models of and for agents such as insulin mimetics and insulin sensitizes [28].

diabetes can arise spontaneously or can be induced by treating db/db mice become hyperphagic, obese, hyperinsulinaemic and

animals with chemicals or by dietary or surgical interventions insulin resistant and consequently develop hyperglycaemia due to

and also as a result of genetic manipulations. The capability of an beta cell failure [26]. For the investigation of Type 2 diabetes and

animal model of Type 2 diabetes to develop ﬁrstly obesity and obesity, db/db mouse is an excellent model [28,33].

insulin resistance and then hyperglycaemia and ﬁnally frank

diabetes is the most important criteria for choosing an ideal 4.3. KK mouse

model [26].

The KK (Kuo Kondo) mouse is a mildly obese and hyper-

3. Spontaneous or genetically derived models of type 2 glycaemic strain obtained from wild-type ddY mice in Japan by

diabetes Kondo in 1957 [27,34]. The KK mouse is a polygenic model of

obesity and Type 2 diabetes. These mice develop hyperglycaemia

Spontaneous models of diabetes were ﬁrst identiﬁed in 1970s and insulin resistance in muscle and adipose tissue as well as

[29]. Spontaneous models of Type 2 diabetes may be obtained from diabetes with renal, retinal, and neurologic complications which is

animals with one or several genetic mutations transmitted from mirror human diabetes [27]. The KK mouse is an ideal genetic

generation to generation or from several generations which are model for the study of diabetes and its complications and the

selected from non-diabetic outbred animals by repeated breeding. evaluation of the impact of natural product preparations [28].

These animals generally carry inherited diabetes with single or

multigene defects. The majority of humans who develop Type 2 4.4. KK/A mouse

diabetes develop the form which arises from interaction between

y y

environmental (diet, exercise and sedentary behaviour) and KK/A mice carry both lethal yellow obese (A ) and diabetes

multiple gene defects (some protective and some causative) and susceptibility genes. They are heterozygous, exhibiting severe

only in a few cases do single gene defects lead toType 2 diabetes. So obesity, hyperglycaemia, hyperinsulinemia and glucose

M. Asrafuzzaman et al. / Biomedicine & Pharmacotherapy 89 (2017) 1242–1251 1245

intolerance and as such are a good model of obesity and Type 2 this model. The characteristics of this model are hyperphagia with

diabetes for screening various classes of antidiabetic agents early onset of obesity. The rats have leptin receptor deﬁciency that

[26,28]. The main cause of diabetes in this model is insulin produces the hyperphagia. The feature of the hyperphagia is

resistance which may be induced due to defects in insulin hypothalamic defect in leptin insulin signalling. This model also

receptors and the post receptor signalling system including defects develops mild hyperglycaemia, insulin resistance, mild glucose

in glucose uptake, pentose metabolic pathways and impaired intolerance, hyperlipidaemia, hyperinsulinemia, and moderate

insulin sensitive phosphodiesterase in fat cells [28]. hypertension. This model has been utilized for screening the action

of several agents for the treatment of obesity, insulin resistance

4.5. NZO (New Zealand Obese) mouse and also for examining agents which may potentiate insulin

secretion or incretin mimetics.

The NZO mouse is a polygenic model of obesity and diabetes

obtained by selective breeding [27,35,36]. This polygenic model 4.10. SHR/N-cp rat

shows a syndrome of hyperphagia, obesity, mild hyperglycaemia,

hyperinsulinemia, impaired glucose tolerance and insulin resis- SHR/N–cp is a genetic model of obesity, hypertension, and Type

tance [35,36]. In the early stage, NZO mice develop hepatic insulin 2 diabetes which is obtained from inbreeding of SHR/N strains.

resistance and hyperphagia that is due to leptin resistance as well SHR/N–cp male rats show hyperphagia, early onset of obesity,

as hyperleptinemia [26]. Due to a defect of leptin transporters, this hyperglycaemia, dyslipidaemia, hyperinsulinemia, hyperleptine-

model shows resistance to peripheral leptin but they remain mia, insulin resistance, impaired glucose tolerance and hyperten-

sensitive to central leptin [27]. This model also exhibits insulin sion if they are homozygous for the corpulent gene [45]. This

resistance and plasma glucose levels increase continuously in an model is this highly suitable to study Type 2 diabetes with obesity

age dependent manner. Glycogen synthesis activity in liver is and also to study the effects of carbohydrate diets on the

decreased and this is considered as the primary metabolic defect development of diabetes [28,45].

that causes the diabetes. They show increased glucogenesis,

hepatic glucose production and hepatic insulin resistance. This 4.11. JCR/LA-cp rat

model also shows an autoimmune disorder and as such is a good

model for studying the relationship between autoimmunity, To produce the JCR/LA-cp rat, backcrosses of LA/N-cp male and

obesity and diabetes. hooded rat species were undertaken. JCR/LA-cp rats show a severe

dysmetabolic proﬁle including insulin resistance, hyperinsuline-

4.6. NONcNZO 10 mouse mia, hyperphagia, obesity, glucose intolerance and hyperlipidae-

mia. The Cp gene is encoded by the stop codon of leptin receptor

This strain was obtained by linking independent diabetes risk and thus producing non-functional receptors. This model exhibits

alleles conferring quantitative trait loci from two different strains vasculopathy which progresses without any treatment interven-

of NZO mice with non-obese nondiabetic mice. This model shows tion.

insulin resistance in liver and skeletal muscle and consequently

chronic hyperglycaemia. This model has attracted a lot of attention 4.12. OLETF rat

in recent times because of its use in studies of the obesity diabetes

state “diabesity” a termed introduced by Etham Sims [37] and later In 1984, the OLETF rat model was produced from an outbred

used by Paul Zimmet [38,39]. colony of Long Evans rats [26,46]. This model is polygenic and

genetically biased (mostly male) and displays characteristics of

4.7. TSOD (Tsumara Suzuki obese diabetic mice) mouse diabetes later in life. They show polyphagia, obesity, hyper-

insulinemia, hypertriglyceridemia, hypocholesteremia and im-

The TSOD mouse is a polygenic model which is characterized by paired glucose intolerance. A defect of beta cell proliferation is

polydipsia with polyuria, followed by hyperglycaemia and hyper- considered responsible for the development of the diabetes. The

insulinemia [40,41]. Along with these symptoms obesity gradually pancreatic islets undergo three stage of histological changes.

develops [41]. Histopathological examinations of the pancreas Firstly, cellular inﬁltration and degeneration, secondly hyperplasia,

show severe hypertrophy of pancreatic islets due to proliferation and thirdly islets become ﬁbrotic and are replaced by connective

and swelling of beta cells [41]. Finally the mice develop reduced tissue. Many researchers consider that the lack of cholecystokinin-

insulin sensitivity and impaired insulin-stimulated glucose trans- A receptor with reduced number of GLUT4 transporters in muscle

porter translocation in muscle and adipocytes. The TSOD mouse is the chief characteristic of this model which is widely used for

exhibits human like Type 2 diabetes properties and it can be a good pharmacological research, speciﬁcally for testing antidiabetic and

model for the study of the complications of diabetes [41]. antihypertensive agents.

4.8. M16 mouse 4.13. Cohen diabetic rat

In addressing the obesity and Type 2 diabetes the M16 mouse Cohen diabetic rats are a genetically derived experimental

has become a new model [42]. The model shows early onset of model which exhibits diet-induced Type 2 diabetes that has

obesity and as well as hyperphagia, hyperinsulinemia, hyper- characteristics of human diabetes [47]. The important characteris-

leptinemia, hypercholesterolemia [42]. Diabetes and obesity in the tic of this model is that it expresses genetic susceptibility to a

M16 model occur at a young age that has some commonality with carbohydrate-rich diet. This experimental model is useful to

present trends in human population [43]. investigate interactions between nutritional-metabolic environ-

ment factors and as well as the genetic susceptibility for the

4.9. Zucker fatty rat development of Type 2 diabetes [47].

In 1961 after crossing of Mereck M-strain and Sherman rats, the

Zucker fatty rat model was discovered [27,44]. The autosomal

recessive (fa) gene on chromosome 5 is the cause of the diabetes in

1246 M. Asrafuzzaman et al. / Biomedicine & Pharmacotherapy 89 (2017) 1242–1251

4.14. GK rat 6.2. Nagoya-Shibata-Yasuda (NSY) mice

Goto-Kakizaki (GK) rat strain was established by a Japanese This animal is suitable for studies of Type 2 diabetes because it

group which sought animals with the poorest glucose tolerance has properties in common with the development of human Type 2

[48]. GK rats are a polygenic model of Type 2 diabetes and were diabetes [60]. The model develops age-dependent impaired

obtained by selective inbreeding of Wister rats in 1973 [26,28]. This glucose tolerance resulting from mild obesity [60]. In addition,

experimental model shows insulin resistance, normolipidemia and environmental nutritional factors such as high fat diet and sucrose

impaired insulin secretion because neonatal GK rats contain administration stimulate the development of a diabetic state in

decreased islet mass. Adult GK rats exhibit a 60 per cent reduction these animals [59].

in their total pancreatic beta cell mass. Thus, the GK rat model is

very useful for studying the mechanisms of beta cell destruction 7. Chemically induced models of hyperglycaemia/diabetes

and loss [48].

7.1. STZ induced animal models

4.15. Torri rat

STZ treatment represents by far the most commonly used

This model is a spontaneous non-obese diabetic rat which was models of diabetes. Streptozotocin (STZ) is a synthetic nitro-

derived from the Sprague-Dawley rat strain in 1997 [49]. Features soureido glucopyranose which is isolated from Streptomyces

of this Torri rat model are glucose intolerance hyperglycaemia, achromogenes with broad spectrum antibiotic and antineoplastic

hypertriglyceridemia and hyperinsulinemia. Even without insulin activity; the use of STZ was ﬁrst reported in 1963 [61]. When STZ is

treatment this diabetic rat model survives for a considerable injected intraperitoneally or intravenously it passes to the

duration, a feature that lends itself to the study of the pancreatic beta cells through the glucose transporter-2 (GLUT2)

complications of diabetes [28,49]. and causes alkylation of DNA [62]. The nicotinamide depletion,

cellular adenosine triphosphate reduction and inhibition of insulin

4.16. TollyHo/Jng mouse production occurs by activation of poly polymerase [62]. In

rodents, a single high dose of STZ induces hyperglycaemia because

TollyHo/Jng mice are a naturally occurring mice model of of the above effects [62]. In spite of its overall toxicity and potential

obesity and Type 2 diabetes which was obtained from selective wide-spread effects, STZ has a selectively toxic effect on beta cells

breeding in mice and these mice develop hyperglycaemia and [61]. High doses of STZ lead to insulin deﬁciency [63]. Beta cell

hyperinsulinemia [27,50]. Pancreatic islets of this model exhibit toxicity in animal models induced by STZ treatment has some

hypertrophy with degranulation and hyperglycaemia. Tolly/Jng characteristics comparable to Type 1 diabetes (Immune toxicity of

mice have not yet been completely characterised for the study of Type 1 diabetes). High dose STZ induced diabetes alone is not

the complications of diabetes. The model has used for some related to major and common form of (obesity-dependent) Type 2

investigations of the poor wound healing in diabetes. diabetes which depends on insulin resistance and later insulin

insufﬁciency [9]. Importantly, the use of STZ can be dose titrated to

5. Diet or nutrition induced animal models of Type 2 diabetes achieve different glycaemic outcomes. At lower doses of STZ, beta

cell mass is only partially reduced resulting in mild insulin

Food and beverages which are high in energy, fat and sugar are deﬁciency. STZ is useful due to its beta cell speciﬁc cytotoxicity but

overly consumed by modern societies [51]. Diet has become a sensitivity varies with strain, species, sex, nutritional state and

major cause of the so-called “obesogenic” environment in which dose and route of administration [63]. STZ treated rodent models

many humans live [51,52]. Prolonged consumption of such modern have stable long lasting hyperglycaemia and can be used for the

Western diets plays an initiating role in the development of Type 2 study of the complications of diabetes mostly those which are

diabetes [53]. Interestingly, European and Asian populations vary dependent upon hyperglycaemia such as renal and ophthalmic

in their diabetogenic response to the same level of obesity (BMI) pathophysiology. Due to the oncogenic effect of STZ, the STZ model

presumably due to genetic determinants [54,55]. Mimicking the can show kidney or liver tumours when used in chronic

diet induced obesity in humans by applying it to create diet experimental protocols. Multiple low dose STZ (20–40 mg/kg)

induced animals becomes a pathway for studying the origins of over 5 days induces insulitis. However it does not produce a model

human Type 2 diabetes [51]. So, diet composition has been studied like human diabetes but rather a spontaneous model of

as an important factor in the development of Type 2 diabetes in autoimmunity [62].

animals [51].

7.2. Neonatal STZ induced animal model of hyperglycaemia

6. Common animal models of diet- or nutrition-induced Type 2

diabetes This model exhibits a diabetes-like syndrome after injecting

STZ on day of birth or within 5 days of birth intravenously or

6.1. C57BL/6 mice intraperitoneally. Neonatal rats are relatively resistance to the

effects of streptozotocin. This model shows plasma glucose and

This mouse strain is most effective in developing diet-induced insulin values that are no longer different from those of controls

diabetes in contrast with other strains [56–58]. C57BL/6 mice are [63]. STZ administration to neonatal rats produce Type 2 diabetes

genetically predisposed to develop obesity and impaired glucose in adult animals. In this model, a peak of hyperglycaemia is seen

tolerance when fed a high fat diet. The hyperglycaemia/diabetes 2 days after STZ administration which is followed by regeneration

syndrome worsens with time and increasing obesity [59]. This of beta cell and normoglycemia will appear after 10 days. However

model exhibits two essential mechanistic characteristics of Type 2 after 6 weeks, due to the beta cell mass and beta cell dysfunction,

diabetes: insulin resistance and islet dysfunction and can be used hyperglycaemia again returns. So at adult age this model can be

for the study of diabetes and for the role of novel therapeutic used to study of Type 2 diabetes [27].

interventions in modulating the course of the condition [57,58].

M. Asrafuzzaman et al. / Biomedicine & Pharmacotherapy 89 (2017) 1242–1251 1247

7.3. STZ and nicotinamide induced animal model develop moderate and non-fasting hyperglycaemia without any

signiﬁcant change in plasma insulin levels with decreased

STZ with nicotinamide produces an animal model that develops pancreatic insulin store so it is a model of altered insulin

a diabetes-like condition which exhibits features similar to sensitivity [26,62,63,65]. Nicotinamide plays an important role

(human) Type 2 diabetes [64]. Nicotinamide administered to 3- as an antioxidant which provides the protective effects against

month old Wister rats 15 min before STZ produces animals which STZ-induced beta cytotoxicity by scavenging free radicals. This

Table 1

Spontaneous animal model of Type 2 diabetes.

Animal model Main characteristics Possible uses & Complexity Applicable natural product Reference

Ob/Ob (C57BL/6J-Lep ) Mutation of leptin gene and shows Any product that reduces the body X [27,28,30,31,78]

mouse obesity and hyperglycaemia with weight, antiobesity, antihyperglycemia

increasing insulin resistance [30,31,78] and improve insulin sensitivity [28].

Human obesity due to monogenic

mutation is rare, however ob/ob mouse

is a monogenic model of obesity [27]

bd/bd (C57BL/KsJ) Autosomal recessive mutation of the Any agent for the investigation of Type 2 Cinnamon, Inner part of Cinnamomum [26,33,79–82]

mouse leptin receptor on chromosome 4 and diabetes particularly diabetic verumexibit antihyperglycemic and

develop hyperphagic, obese, dyslipidaemia and for agents such as antihyperlipidemic action [81], Pueraria

hyperinsulinaemic and insulin insulin mimetics and insulin sensitizes (gegen), the root of Pueraria lobata

resistance and hyperglycaemia due to and obesity [28,33]. Uncontrollable reduces body weight, normalizes blood

beta cell failure [26,27,32,79]. hyperglycemia, they survive to only 10 glucose level, improves glucose

months of age [80]. tolerance and proliferate beta cells thus

increase beta cells by activating GLP-1R

signalling [82].

KK mouse Polygenic model of obesity and shows Evaluation of the impact of natural Maitake mushroom improve glucose [27,28,34,83]

hyperglycaemia and insulin resistance product preparations [28] and insulin metabolism [83]

in muscle and adipose tissue as well as

diabetes with renal, retinal, and

neurologic complications [27,28].

y y

KK/A mouse Carry both lethal yellow obese (A ) and Good model of obesity and Type 2 Tuberous root of Liriope spicata var [26,28,84,85]

diabetes susceptibility genes and diabetes for screening various classes of prolifera decrease fasting blood glucose

exhibit severe obesity, hyperglycaemia, antidiabetic agents]. Homozygous A and improve IR and lipid metabolism

hyperinsulinemia and glucose animal dies before implantation [26,28] [84]. Nitobegiku, the Herb Tithonia

intolerance [26,28] diversifolia improve glucose metabolism

by reducing insulin resistance [85]

NZO (New Zealand A polygenic model of obesity and shows Study of any agents showing X [27,28,35,36]

Obese) mouse hyperphagia, obesity, mild relationship between autoimmunity,

hyperglycaemia, hyperinsulinemia, obesity and diabetes [28]

impaired glucose tolerance and insulin

resistance [27,35,36],

NONcNZO 10 mouse A recombinant congenic new mouse Evaluation of the potential of any agent X [28]

shows hyperphagia, morbid obesity and as an antidiabetic drug. Female mice are

hyperglycaemia [28] diabetes resistant [28]

TSOD mouse A polygenic model which shows Studying of complications of diabetes Salacia reticulata shows potentiality of [40,41,86]

polydipsia with polyuria, followed by using different natural agent [41]. prevention obesity, metabolic disorder

hyperglycaemia and hyperinsulinemia Incidences of diabetes is very high in and development of metabolic

[40,41] male [41] syndromes [86]

M 16 mouse A polygenic model of obesity and shows Study about genomic, proteomic and X [42]

hyperphagia, hyperinsulinemia, metabolic analysis of diabesity using

hyperleptinemia, hypercholesterolemia different agent [42]

[42]

TallyHo/Jng mouse Naturally occurring polygenic model of Valuable for identiﬁcation of molecular X [27,49,87]

obese and develop hyperglycaemia, defect [87]. Female mice are not

hyperinsulinemia [27,49] hyperglycaemic [49]

Ins2Akita mouse Shows hyperglycaemia, Excellent model for islet X [28,88]

hypoinsulinaemia, polydipsia and transplantation studies. Female models

polyuria [28,88] are more suitable because they survive

longer period with chronic

hyperglycaemia without signiﬁcant

weight loss and without insulin therapy

[88]

ALS/Lt mouse A new mouse model with reduced Suitable model for free radical mediated X [28,88]

systemic ability to diffuse ROS [88] and damage and complication of diabetes

exhibit hyperinsulinaemia and (Type 1 & 2) [28,88]

impaired glucose tolerance [28]

Zucker fatty rat Autosomal recessive(fa) gene on Utilized for screening the action of Gynostemia Pentaphyllumshows [89,90]

chromosome 5 is the cause of diabetes several agents for the treatment of hypoglycemic and antihyperglycemic

and shows hyperphagia with early obesity, insulin resistance. Also suitable activity [90]

onset of obesity, mild hyperglycaemia, for the study of hypertension and

insulin resistance, mild glucose atherosclerosis [89]

intolerance, hyperlipidaemia,

hyperinsulinemia, and moderate

hypertension [89]

SHR/N-cp rat A genetic model show hyperphagia, Highly suitable to study Type 2 diabetes X [28,44]

early onset of obesity, hyperglycaemia, with obesity and also to study the

dyslipidaemia, hyperinsulinemia, effects of carbohydrate diets on the

1248 M. Asrafuzzaman et al. / Biomedicine & Pharmacotherapy 89 (2017) 1242–1251

Table 1 (Continued)

Animal model Main characteristics Possible uses & Complexity Applicable natural product Reference

hyperleptinemia, insulin resistance, development of diabetes [28,44]

impaired glucose tolerance and [27,43].Morphological changes are

hypertension [44] observed in SHR/N-cp rats [44]

JCR/LA-cp rat Shows obesity, hyperlipidemia and Suitable model for atherosclerosis study Chromium picolinate supplimentation [91,92]

hyperinsulinemia [91] [91]. Female mice are not useful. improves insulin action [92]

OLETF rat Show polyphagia, obesity, Potential model for analysis of Korean red ginseng (Panax ginseng) [45,93]

hyperinsulinemia, pathogenesis and Type 2 diabetes improves insulin resistance through

hypertriglyceridemia, complications and development of activation of AMPK pathway [93]

hypocholesteremia and impaired antidiabetic drug [45]

glucose intolerance [45]

Cohen diabetic rat A genetically obtained experimental Suitable model for studying the X [28,46]

model which exhibits diet induced Type interaction between nutritional-

2 diabetes [46] metabolic environmental factors and

genetic susceptibility for build-up of

type 2 diabetes [28]

GK rat A polygenic non obese model shows Useful for studying the mechanisms of Moringa oleifera shows better effect for [28,47,94]

insulin resistance, normolipidemia and beta cell destruction and occurrence of glucose intolerence [94]

impaired insulin secretion [28,47] diabetes, diabetic complications and

testing of antidiabetic drug [28,47]

Torri rat Shows glucose intolerance Useful model for study of diabetes X [28,48]

hyperglycaemia, hypertriglyceridemia complications. Depending on the age

and hyperinsulinemia [28,48] they show various clinical

characteristics among their gender. (At

the age of 40 weeks male rats become

100% diabetic model where only 33.3%

female rat at 65 weeks developed

diabetes [48].

anti-oxidant action of nicotinamide leads to only minor STZ- and dyslipidaemia [67]. The dyslipidaemia in this model is similar

induced damage to pancreatic beta cell cells. The outcome and to lipid abnormalities in Type 2 diabetes and results from

effectiveness of this dual action strategy depends on dosages of STZ combined administration of a HFD and STZ and it does not result

and nicotinamide [62,65]. Thus, the STZ plus nicotinamide induced when either STZ or a HFD is used alone [68]. Thus a HFD with low

animal is an attractive and useful model with pathophysiological dose STZ exhibits a pathophysiological state similar to Type 2

similarities to human diabetes and it is suitable for acute and diabetes. Speciﬁcally, short duration use of a HFD induces insulin

chronic studies. Pharmacological investigation of insulinotropic resistance and glucose intolerance which mimics human predia-

agents in this model have provided agents which are potentially betes whereas prolonged HFD produces a higher body fat

useful for the treatment of Type 2 diabetes (Tables 1–3 ). percentage and thus increasing obesity with increasing body

weight [66].

7.4. STZ and High Fat Fed (HFD) animal model

7.5. Alloxan induced animal model

The combined treatment of a HFD and STZ produces another

model of hyperglycaemia which shows a similar metabolic proﬁle The use of alloxan (2, 4, 5,6-tetraoxypyrimidine;5,6-dioxyur-

to Type 2 diabetes in human [66]. The combination of low dose STZ acil) was ﬁrst reported by Dunn and colleagues in 1943 (see [69])

and a HFD can alter the relevant gene expression levels in major and it is used for induction of hyperglycaemia/diabetes in animal

metabolic tissues and effectively induce a Type 2 diabetes-like models for experimental purposes. Alloxan (ALX) is rapidly taken

state. Insulin resistance induced by HFD feeding and low dose of up by pancreatic beta cells and the generation of free radicals that

STZ treatment produces initially beta cell dysfunction and a useful leads to fragmentation of the beta cell DNA. ALX has a narrow

prediabetes stage is apparent in this model [67]. The animals fed effective (for the induction of hyperglycaemia) dose range and

with a HFD develop obesity, hyperinsulinemia, insulin resistance kidney toxicity may arise from slight over dosing [62].

Table 2

Diet or Nutrition induced diabetic animal model.

Animal model Main characteristics Possible uses & Complexity Applicable natural product Reference

C57BL/6 mice Diet induced hyperglycaemia Suitable model for investigating the Cornelian cherries (Cornus mas) improve certain [55,95–97]

determined by the recessive pathogenesis of diabetes on molecular basis and metabolic parameters associated with diets high

gene and insulin resistance by also useful for testing the efﬁcacies of in saturated fats and obesity [97]

dominant gene [95] therapeutic measures [55]. Inﬂammatory lesions

is develop in multiple organs associated with

ageing process. Such as Salivary gland, kidney,

pancreas, lung and liver [96]

Nagoya-Shibata- Shows age dependent glucose Suitable model for studying human diabetes and X [59,98]

Yasuda (NSY) tolerance and mild obesity [59] also useful to identify genes conveying

mice susceptibility, especially whose expression

affected by ageing [59,98]

Sand rat A polygenic model and shows Excellent model for the study of diabesity Suaeda fruticosa extract showed hypoglycaemic [28,99,100]

hyperphagia, obesity, syndrome [28,99] and antihyperglycaemic effects. Furthermore,

hyperinsulinaemia, glucose the plant led to a decrease in plasma levels of

intolerance and insulin, total cholesterol, LDL cholesterol, VLDL

hyperglycaemia [28,99] cholesterol, oxidized LDL and triglycerides [100]

M. Asrafuzzaman et al. / Biomedicine & Pharmacotherapy 89 (2017) 1242–1251 1249

Table 3

Knockout diabetic animal model.

Animal model Main characteristics Possible uses & Complexity Reference

IR Knockout Lacking of IR gene [101] Double heterozygote IR/IRS-1 (1/2) mice model suitable for identifying [73,101–103]

and characterizing gene modelling of diabetic phenotype and thus

indicate new therapeutic approaches [Burning et al., 1997].Homozygous

À/À +/À

IR model die within one week after birth. Heterozygous IR model

do not die but only 10% develop Type 2 diabetes [73,102]

Muscle-speciﬁc IR Insulin resistance in muscle cell [104] Suitable model for studying role of insulin signalling and insulin [104]

Knockout resistance [104]

Beta cell-speciﬁc IR Shows selective loss of insulin secretion and impaired Good model to observe alteration in insulin secretion in Type 2 diabetes [73,105]

Knockout glucose tolerance [73] [73]

Liver-speciﬁc IR Shows severe insulin resistance associated with glucose Good model for studying genetic evidence of insulin signalling and [73,106]

Knockout tolerance [73] insulin sensitivity [106]

IRS-1 Knockout Exhibits insulin resistance, hypertriglyceridemia [73,107] This model can be used for investigating the role of IRS-1 in speciﬁc [73,107]

target organ [73]

IRS-2 Knockout This model exhibits insulin resistance and defects in Utilize to observe beta cell function and mass [108] [73,108]

insulin signalling pathway in liver and develop mild to

severe diabetes [73,108]

PI-3 kinase Show increased insulin sensitivity, hypoglycaemia and This model provides direct evidence that PI3K and its regulatory subunit [73,109]

Knockout adipocyte [73] have a role in glucose homeostasis in vivo [109]

+/À

GLUT4 Knockout Carry null mutation in GLUT4 gene and exhibits insulin Male GLUT4 is good model for studying the development of Type 2 [73]

resistance, hyperinsulinemia and hyperglycaemia [73] diabetes without obesity related complications. Homozygous null

À/À

mutant (GLUT ) generally live 5–7 month and did not show diabetic

phenotype [73]

7.6. Goldthioglucose induced animal model or (glucose kinase mutation) for understanding glucose sensor

function in beta cells. This model increases our knowledge about

Goldthioglucose can induce obesity and hyperglycaemia/ beta function and glycaemia [72].

diabetes [70]. This model gradually develops obesity, insulin

resistance, hyperinsulinemia, and hyperglycaemia and also shows 8.2.2. Transgenic or knock-out animal model

increased body lipid, hepatic lipogenesis, triglyceride secretion, Transgenic animals are helpful for providing insights into gene

increased adipose tissue lipogenesis and reduced glucose metabo- regulation and the development of the pathogenesis and treatment

lism. This treatment requires a long time to develop obesity and as of metabolic diseases and in searching for new targets and

well as diabetes and it is suitable for specialised studies. therapeutic entities. Transgenic animals are produced by transfer-

Goldthioglucose treated DBA/2, C57BLKs and DBF1 mice rapidly ring or altering the site or expression level of functional genes or by

develop body weight as well as obesity and increases in plasma removing speciﬁc endogenous genes or by placing them under the

glucose levels [70,71]. control of alternative promoters. These models have been

established in the search for the role of genes and also their

8. Surgically induced model of hyperglycaemia in multiple effects on peripheral insulin action which are related with the

animals development of Type 2 diabetes. Moreover combinations or double

knock-out models have produced different symptoms including

8.1. Full and partial pancreatectomy models of hyperglycaemia defects in insulin secretion and insulin action which clearly exhibit

the mechanisms which is related with insulin resistance develop-

These models involve a partial or complete pancreatectomy for ment and as well as beta cell dysfunctional and that are similar to

the purpose of inducing diabetes. In 90 per cent of situations human Type 2 diabetes [72]. h1APP is a transgenic mouse and

partial pancreatectomy is used and dissection of the pancreas is knockout mouse are IR knockout, muscle-speciﬁc knockout, beta-

performed in animals such as dogs, pigs, rabbits, and rats. The cell speciﬁc knockout, liver speciﬁc knockout, IRS-1, IRS-2,IRS-3

results of this procedure are characterised by moderate hyper- knockout mouse and double knockout mouse are GK/IRS-1, PDX/

glycaemia with neither reduction of body weight or decreased GLUT4 [68,73].

plasma insulin levels and it produces a mild form of diabetes. This

model exhibits impaired glucose stimulated insulin secretion but 9. Large animal models of hyperglycaemia and diabetes

maintains an intact response to other insulin secretogogues. The

surgically induced model of diabetes is utilized in the inves- 9.1. Pigs

tigations of transplantation pancreatic graft or islets and it is useful

in the identiﬁcation of factors related to islet regeneration and Pigs are very similar models for human physiology and

thus the study of potential treatments for speciﬁc types of diabetes pathophysiology. This model change their glucose and lipid proﬁle

[28]. and also induce aortic fatty streaks when fed with HFSD and thus

become a good of diabetes accelerated atherosclerosis. So the pigs

8.2. Genetically modiﬁed models of hyperglycaemia/diabetes: feeding with HFSD can produce insulin resistance, dysfunction of

beta cell and as well as diabetes [74].

8.2.1. Beta cell dysfunction animal model

Pancreatic beta cells and beta cell mass plays a major role in the 9.2. Rhesus monkey

aetiology and is thus a target for the treatment of Type 2 diabetes.

These models are suitable for understanding the pathway that Obese rhesus monkey is excellent non rodent model for

produces insufﬁcient beta cell function to secrete the appropriate developing obesity, hyperglycaemia and insulin resistance which

amount of insulin. This type of animal model is produced by normally leads to beta cell necrosis, decrease insulin level and

introducing genetic manipulation (Kir6.2 mutation) for k-channels increase hyperglycaemia [75]. This non rodent model shows a

1250 M. Asrafuzzaman et al. / Biomedicine & Pharmacotherapy 89 (2017) 1242–1251

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Antihyperglycemic Activity of Swertia chirata on nSTZ-T2DM Rats: A Chronic Study

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Journal of Pharmaceutical Research International

22(5): 1-11, 2018; Article no.JPRI.41878 ISSN: 2456-9119 (Past name: British Journal of Pharmaceutical Research, Past ISSN: 2231-2919, NLM ID: 101631759)

Antihyperglycemic Activity of Swertia chirata on nSTZ-T2DM Rats: A Chronic Study

Amrita Bhowmik1,2, M. Mosihuzzaman3,4, Yearul Kabir2 and Begum Rokeya5*

1Department of Applied Laboratory Sciences, Bangladesh University of Health Sciences (BUHS), Bangladesh. 2Department of Biochemistry and Molecular Biology, University of Dhaka, Bangladesh. 3Department of Chemistry, Bangladesh University of Health Sciences (BUHS), Bangladesh. 4International Centre for Natural Product Research (ICNPR), Bangladesh. 5Department of Pharmacology, Bangladesh University of Health Sciences (BUHS), Bangladesh.

Authors’ contributions

This work was carried out in collaboration between all authors. Author AB designed the proposal and protocol, performed the experiments, collection and assembly of data and analysis and wrote the first draft of manuscript. Author MM gave financial and logistic supports. Authors MM, BR and YK designed the protocol. Author BR corrected the protocol, managed the experiments and revised the manuscript. All authors approved the manuscript.

Article Information

DOI: 10.9734/JPRI/2018/41878 Editor(s): (1) Salvatore Chirumbolo, Clinical Biochemist, Department of Medicine, University of Verona, Italy. Reviewers: (1) Mohamed Ahmed Mohamed Nagy Mohamed, Egypt. (2) Dennis Amaechi, Veritas University, Nigeria. Complete Peer review History: http://www.sciencedomain.org/review-history/25088

Received 4th March 2018 th Original Research Article Accepted 16 May 2018 Published 11th June 2018

ABSTRACT

Aim: The present study aimed to investigate antidiabetic effects and to explore the underlying mechanism of S. chirata on neonatal-streptozotocin induced type 2 diabetic model (nSTZ-T2DM) rats. Methodology: Stem-barks were collected from local market, identified from Bangladesh National Herbarium; 96% ethanol extract and overnight soaked water solution were prepared. T2DM was induced by a single ip injection of STZ to 48 hours Long Evans neonatal pups. After 3 months, T2DM adult rats (confirmed by OGTT) were divided into 4 groups: i) water control; ii) Glibenclamide (5 mg/kg bw); iii) soaked water (SCWS, 0.25 g/10 ml/kg bw) and iv) ethanol extract (SCE, 0.25 g/kg bw)treated groups respectively. Blood was collected by cutting tail tip on 0 and 21 day; by cardiac ______

*Corresponding author: E-mail: [email protected];

Bhowmik et al.; JPRI, 22(5): 1-11, 2018; Article no.JPRI.41878

puncture on 28 day for measuring biochemical parameters. The data were analyzed using univariate & multivariate tools. Results: SCE (p=0.003) and SCWS (p<0.001) treated group significantly improved OGTT of T2DM rats after three weeks administration. A significant decrease of fasting glucose level (p=0.01) and HOMA IR (p=0.01) were noticed in SCWS treated group after 28th day in comparison to initial day value respectively. Cholesterol was significantly decreased in SCWS on 28th day (p=0.02 and p=0.03) in comparison to water control and initial day respectively. Triglycerides was decreased in both treated groups and LDL level decreased by 42% in SCWS group respectively. Hepatic glycogen content of SCE was significantly increased (p=0.05) compared to water control. Serum ALT and creatinine level almost remained unchanged. A gradual fall of glucose absorption in SCE- krebs-glucose solution through GIT after 30 minutes was shown in Gut Perfusion Technique. Conclusion: S. chirata stem-bark possesses significant antihyperglycemic activity in T2DM rats which may be improved glucose tolerance, increased glycogenesis, decreased insulin resistance and total cholesterol level that may provide a rationale for using it in diabetic treatment.

Keywords: S. chirata; antihyperglycemic; STZ; GI Tract; T2DM.

ABBREVIATIONS

BIRDEM= Bangladesh Institute of Research and Rehabilitation in Diabetes; Endocrine and Metabolic Disorders; DM =Diabetes Mellitus; FSG= Fasting serum glucose; GIT= Gastrointestinal tract; GOD-PAP = Glucose Oxidase; GSH = Reduced gluthathion; HDL= High density lipoprotein; HOMA B%= B cell secretion; HOMA IR= Insulin Resistance index; HOMA S%= insulin sensitivity; i.p = intraperitonial; LDL= Low density lipoprotein; MDA = Malondialdehyde; nSTZ = neonatal- streptozotocin; OGTT = Oral Glucose Tolerance Test; TBARS = Thiobarbituric acid reactive substances; TG = Triglycerides.

1. INTRODUCTION xanthone rich extract of this plant has shown significant anti-inflammatory, anti-platelet, anti- Diabetes Mellitus is a clinical syndrome cancer, CNS stimulant, anti-fungal and characterized by hyperglycemia caused by a antimalarial effects [13]. Swerchirin, a xanthone relative or absolute deficiency of insulin at the from S. chirata is a potent hypoglycaemic agent cellular level. It is the most common endocrine [14-17]. Mukherjee et al. reported significant disorder affecting mankind all over the world, blood sugar lowering effects of the 95% ethanolic prevalence of which is increasing day by day [1]. extract of S chirata in fed, fasted and glucose- Traditional preparations from plant sources are loaded albino rats [18]. The hypoglycaemic widely used almost everywhere in the world to activity of tolbutamide was increased in healthy treat this disease. Therefore, plant materials are albino rats by giving S chirata extract orally [19]. considered to be the alternative sources for Antidiabetic activity of swerchirin isolated from finding out new leads for hypoglycemic agents. A hexane fraction of S. chiratahas been reported total of more than 400 species were reported to by Bajpai et al. [20]. Except in rats with severe display hypoglycemic effects, but few of them pancreatic damage, swerchinin showed better have been investigated scientifically [2]. The glucose lowering effect compared to tolbutamide plant product undertaken in this study for anti- [20-22]. diabetic effect was S. chirata known as Chirota available in South Asian countries and much in Traditional healers in Bangladesh use most Indian subcontinent. S. chirata belongs to the commonly Stem-bark of S. chirata for antidiabetic family Gentianaceae [3,4]. It can be traced activity [23,24]. Although a huge number of through the medicinal history as a nontoxic and publications have been done with S. chirata, still safe ethnomedicinal herb utilized for its bitter the probable mechanisms of its antidiabetic bioactive compounds [5]. The chemical activity remained unclear. For this reason, we constituents of S. chirata include secoiridoid have tried to investigate some probable bitters, alkaloids, xanthones and triterpenoids [6, mechanisms of antidiabetic activity of S. chirata. 7,8]. Amarogentin, amaroswerin, gentiopicroside This study evaluated the glycemic, insulinemic, and swertiamarin are the reported bitter lipidemic and antioxidant properties of S. chirata secoiridoid glycosides of the plant [9-12]. A stem-bark in nSTZ-diabetic rats. Gut perfusion

Bhowmik et al.; JPRI, 22(5): 1-11, 2018; Article no.JPRI.41878

technique was also performed to study the upper experiments were conducted according to the intestinal glucose absorption by S. chirata stem- ethical guidelines approved by Bangladesh bark. Association for Laboratory Animal Science.

2. MATERIALS AND METHODS 2.5 Preparation of Type 2 Diabetes Model Rats 2.1 Place of Study Diabetes was induced by a single intraperitoneal The study was conducted in the Department of injection of streptozotocin (STZ) at a dose of 90 Pharmacology in Bangladesh Institute of mg/kg body weight to the neonate rats (48 hours Research and Rehabilitation in Diabetes, old) as described by Bonner-Weir et al. [25]. Endocrine and Metabolic Disorders (BIRDEM) Following 3 months of STZ injection, rats were and Bangladesh University of Health Sciences examined for their blood glucose level by oral (BUHS). glucose tolerance test (OGTT, Glucose 2.5 g/kg bw). Diabetic model rats in body weight 150-200 2.2 Plant Material gm with blood glucose level >7.00 mmol/l, at fasting condition was selected for studying the In this study, S. chirata stem-barkwas used. The effects of the extracts in chronic studies. stems were collected from the commercially available sources and identified by the 2.6 Experimental Groups of T2DM Rats Bangladesh National Herbarium, Dhaka (DACB Accession no 37789). The chronic experiment was carried out for duration of 28 days on 48 rats. Type 2 rats were 2.3 Preparation of S. chirata Extracts divided into four different groups. Those were followed as: After collection, S. chirata stem-barks were washed by fresh water thoroughly and sun dried 1) Water Control group (n = 7): Treated with in laboratory. The stem-barks were prepared for deionized water at a dose of 10 ml/kg body two types of extracts: 1) ethanolic extract and 2) weight (bw). water soaked solution. For preparation of 2) Glibenclamide (positive) control group ethanolic extract, stem-barks were grinded to (n = 7): Treated with glibenclamide at a make fine powder by a grinding machine. The dose of 5 mg//kg bw [26]. grinded powder was extracted by dissolving 3) Soaked water treated group SCWS (n = overnight absolute (96%) ethanolic solvent at - 8): Treated with 0.25 g stems overnight 8°C. Following the completion of extraction, soaked within distill water at a dose of 0.25 extract prepared from stem-barks was mg/ 10 ml/ kg bw. concentrated under reduced pressure using a 4) Extracttreated group SCE(n = 8): Fed rotary evaporator (BUCHI R-114, Switzerland) with ethanol extract at a dose of 0.25 mg maintained at 55°C. The semi-dried ethanolic /kg bw. extract was further dried in a freeze drier (HETOSICC, Heto Lab Equipment, Denmark) at - Water, Glibenclamide, soaked water solution and 55°C and stored in a reagent bottle at -8°C in a ethanol extract were administered intragastrically refrigerator for future use. The soaked water through metallic tubes to the corresponding solution of S. chirata was prepared dissolving by group of rats after 12 hrs fast. overnightdistilled water. 2.7 Collection of Blood Sample for 2.4 Animals Biochemical Analysis

The Long Evans rats bred at Bangladesh Blood samples were collected from rats kept Institute of Research and Rehabilitation in under fasting conditions (12 hours) by Diabetes, Endocrine and Metabolic amputation of the tail tip under diethyl ether st th Disorders(BIRDEM) animal house, were used in anesthesia on the 0 day and 21 day. On the 28 this study. The animals were maintained at a day, blood was collected from the rats by cardiac constant room temperature of 23°C with humidity puncture (diethyl ether anesthesia). The of 40-70% and the natural 12 hours day-night collected blood samples were centrifuged at cycle. The rats were fed on a standard laboratory 2,500 rpm for 15 minutes and finally the serums pellet diet and water supplied ad libitum. The were separated into another eppendorf tubes

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for biochemical analysis. Two (2) mL of blood (SPSS) software for windows version 16 (SPSS was collected in heparinized tubes and then Inc., Chicago, Illinois, USA).All the data were packed red cells were used for estimation of expressed as Mean ± SD or as Median (Range) Malondialdehyde (MDA) and reduced as appropriate. Statistical analysis of the results Glutathione (GSH). was performed by using the student’s t-test (paired and unpaired), ANOVA (analysis of 2.8 Effects on Intestinal Glucose variance) followed by Bonferroni post hoc test Absorption and Mann Whitney (u) test. The limit of significance was set at p<0.05. An intestinal perfusion technique [27] was used to study the effects of extract on intestinal 3. RESULTS absorption of glucose in type 2 diabetic rats fasted for 36 hours and anesthetized with sodium 3.1 Effect on the Body Weight of T2DM pentobarbital (50 mg/kg). The plant extract was Rats added to a kreb’s solution (g/L 1.02 CaCl2, 7.37 NaCl, 0.20 KCl, 0.065 NaH2PO4.6H2O, 0.6 Body weight of each rat was taken at seven days NaHCO3, pH 7.4), supplemented with glucose interval. As shown in Fig. 1 the gradual increase (54.0 g/L) and perfused at a perfusion rate of 0.5 ofbody weight was observed after 28 days in all mL/min for 30 min through the duodenum. The of the groups i.e. water control, glibenclamide perfusate was collected from a catheter set at 40 and both treated groups. cm. Both Extracts were added to Kreb’s solution to final conc. of 25 mg/mL so that the amount of 3.2 Effects on Glucose Homeostasis extract in the perfused intestine is equivalent to the dose of 1.25 g/kg. The control group was Effect of oral administration of S. chirata extracts perfused only with Kreb’s buffer supplemented on type 2 model rats on 30 min before with with glucose. The results were expressed as glucose load were observed on the 0 day and percentage of absorbed glucose, calculated from 21st day at four consecutive times (60, 90 and the amount of glucose in solution before and 120 min) respectively.On initial day, no after the perfusion. significant change was found in any of the

2.9 Biochemical Analysis treated groups.

st Serum glucose was measured by Glucose On 21 day, glucose level was lowered in SCWS Oxidase (GOD-PAP) method using micro-plate treated group when it compared among groups reader (Bio-Tec, ELISA); total cholesterol and (p=ns) at 60 min (Fig. 2). At 90 min after glucose Triglyceride (TG) by enzymatic colorimetric load, almost similar rise in serum glucose level method (Randox Laboratories Ltd., UK), using was found in glibenclamide and SCE treated autoanalyzer. LDL-cholesterol was calculated by groups, and a significantly reduced glucose was Friedewald equation [28]. Serum insulin was found in SCWS group in comparison to water estimated by ELISA (Crystal Chem Inc., USA); control (p=0.002). Moreover, at 120 min the and HOMA B% (Beta-cell function) and HOMA glucose level decreased in all treated group S% (Insulin sensitivity) were calculation by compared to 90 minutes value; and a significant HOMA SIGMA Software [29]. HOMA IR (Insulin decreased were found in SCWS (p<0.001) and Resistance Index) was calculated by SCE (p=0.003) treated groups when compared International Formula: fasting Glucose (mmol/L) with water control respectively. The positive × fasting Insulin (mU/L)/22.5. Serum Creatinine control glibenclamide showed a and Amino Alanine Transferase (ALT) by Auto- significant (p=0.03) decreased of glucose level at analyzer. Hepatic glycogen was measured by 120 min in comparison to water control, as Anthrone-sulphuric acid method. Reduced expected. Glutathione (GSH) and plasma Malondialdehyde (MDA) estimated by using Ellman’s [30] and Fasting serum glucose (FSG) levels of type 2 Thiobarbituric Acid Reactive Substances diabetic models rats for experimental groups (TBARS) method respectively [31]. were almost similar on 0 day (Table 1). After oral administration of respective treatment to T2DM 2.10 Statistical Analysis rat of different groups for 28 days of experimental period, it was found that FSG level of all the Data from the experiments were analyzed using groups of rats decreased on final day except the Statistical Package for Social Science water control. However, type 2 rats treated with

Bhowmik et al.; JPRI, 22(5): 1-11, 2018; Article no.JPRI.41878

SCWS (p=0.01) showed a significant decreased condition on 28th day. There was 17% deceased when compared within groups. As expected, level was found in SCE group on final day in glibenclamide also ameliorated the diabetic comparison to baseline value.

220

200 )

g 180 (

t Water control h g i e 160 Gliben W

d SCWS o

B 140 SCE 120

100 0 day 7 day 14 day 21 day 28 day

Fig. 1. Effect ofS. chirata extract on the body weight of type 2 diabetic model rats Results are expressed as Mean ±SD. Statistical analysis between group comparison was done by using one way ANOVA with post hoc Bonferroni test and Within groups, comparison was done using paired t test. WC = Water Control; Gliben = Glibenclamide treated group; SCWS = S. chirata water soaked group; SCE =S. chirata ethanol extract.

Fig. 2. Effect of S. chirata extracts on the blood glucose levels of T2DM rats in 30 min before with glucose load on 21st day Results are expressed as Mean ±SD. Statistical analysis between group comparison was done by using one way ANOVA with post hoc Bonferroni test and Within groups, comparison was done using paired t test. . WC = Water Control; Gliben = Glibenclamide treated group; SCWS = S. chirata water soaked group; SCE = S. chirata ethanol extract. *= p<0.05; **= p<0.005 ***= p<0.001

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Table 1. Chronic effects of S. chirata extracts (p=0.01) only in SCWS group on 28th day when it on fasting serumglucose (FSG) level of STZ- compared with baseline values. But the inducedT2DM rats decreased was significant (p=0.01) only for SCWS group. Groups Glucose (mmol/l) 0 day 28th day 3.4 Effect of S. chirata on the Serum WC (n=7) 8.28±1.62 8.27±1.91 Lipid Profiles of T2DM Rats (100%) (100%) Gliben 9.21±1.85 8.46±1.62 Chronic effects of S. chirata on serum lipid (n=7) (100%) (92%) profiles were presented in Table 3. Treatment of SCWS 9.24±0.92 7.11±1.11* diabetic model rats for 28 days with SCWS (n=8) (100%) (76%) (p=0.02) and SCE (p=0.01) showed a SCE (n=8) 9.52±1.76 7.93±0.94 significant decrease in serum cholesterol level (100%) (83%) when it compared with water control value Results are expressed as Mean±SD (percentage). respectively. In comparison to initial day, total Between groups, comparison was done using one-way cholesterol level was also decreased in SCWS ANOVA with post hoc Bonferroni test and Within group (p=0.03) on 28th day significantly. The groups, comparison was done using paired t test. triglyceride levels were decreased by 13% in *p=0.01, WC = Water Control; Gliben = Glibenclamide treated group; SCWS = S. chirata water soaked SCE group and LDL level deceased by 42% in group; SCE =S. chirata ethanol extract. SCWS treated rats on final day in comparison to baseline value (p=ns) respectively. Besides 3.3 Chronic effect of S. chirata extracts these all groups do not have any changes in the on β-cell function (HOMA B%), serum HDL- and LDL-cholesterol levels on final day. insulin sensitivity (HOMA S%) and insulin resistance HOMA IR 3.5 Effect on Liver and Kidney Function

There were no significant changed of HOMA S% As shown Table 4, the serum ALT level was and HOMA B% in SCWS and SCE treated group decreased by 18% and 8% in SCWS and SCE after 28 days in comparison to baseline value. treated group respectively on final day in Insulin resistance index HOMA IR decreased by comparison to initial day, which was not 30%, 72%, and 77% in glibenclamide, SCWS th statistically significant and serum creatinine level and SCE treated groups on 28 day in almost remained the same for all groups on final comparison to 0 day (mean of baseline) value day. respectively. But the decreased was significant

Table 2. Effect of S. chirata extracts on HOMA B%, HOMA S% and HOMA IR of STZ-induced T2DM rats

Groups HOMA B% HOMA S% HOMA IR 0 day 28 day 0 day 28 day 0 day 28 day WC 48.02±21.22 49.26±19.10 121.09±99.72 128.27±118.00 4.7±3.5 10.9±13.9 (n=7) (100%) (102%) (100%) (105%) (100%) (231%)

Gliben 39.98±14.73 42.23±22.69 92.41±85.59 106.05±79.11 5.5±3.4 3.9±2.8 (n=7) (100%) (105%) (100%) (111%) (100%) (70%)

SCWS 56.94±22.68 43.00±26.07 42.50±18.71 162.63±80.92 8.2±4.0 2.3±2.8 (n=8) (100%) (76%) (100%) (387%) (100%) (28%) p=0.01 SCE 40.24±13.00 30.97±13.56 78.40±65.91 164.95±69.41 6.4±4.8 2.1±1.8 (n=8) (100%) (77%) (100%) (211%) (100%) (33%) p=0.08 Results are expressed as Mean±SD (percentage). Between groups, comparison was done using one way ANOVA with post hoc Bonferroni p value and Within groups, comparison was done using paired t test. WC = Water Control; Gliben = Glibenclamide treated group; SCWS = S. chirata water soaked group; SCE =S. chirata ethanol extract.

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Table 3. Effect of S. chirata extracts on lipid profiles of STZ-induced T2DM rats

Groups Chol(mg/dL) TG (mg/dL) HDL (mg/dL) LDL (mg/dL) 0 day 28 day 0 day 28 day 0 day 28 day 0 day 28 day WC 67±7 72±5 83±11 89±14 43±2 46±5 7±6 8±7 (n=7) (100%) (107%) (100%) (107%) (100%) (106%) (100%) (114%)

Gliben 61±5 61±5 72±16 71±13 39±6 40±6 7±7 10±5 (n=7) (100%) (100%) (100%) (98%) (100%) (102%) (100%) (142%)

SCWS 68±9 61±5 68±11 66±18 41±5 41±6 12±7 7±9 (n=8) (100%) (89%) (100%) (97%) (100%) (100%) (100%) (58%) p =0.02a p= 0.03c SCE 62±6 59±6 86±21 75±17 39±4 38±5 5±4 9±8 (n=8) (100%) (95%) (100%) (87%) (100%) (97%) (100%) (180%) p= 0.01b Results are expressed as Mean±SD (percentage). Between groups, comparison was done using one way ANOVA with post hoc Bonferroni p value and Within groups, comparison was done using paired t test. WC = Water Control; Gliben = Glibenclamide treated group; SCWS = S. chirata water soaked group; SCE =S. chirata ethanol extract. a= WC Vs SCWS, b = WC Vs SCE, c= 0 day vs 28 day

Table 4. Effect on liver and Kidney function of STZ-induced T2DM rats

Groups ALT (U/L) S Creatinine (mg/dL) 0 day 28 day 0 day 28 day WC (n=7) 64±37 91±51 0.77±0.11 0.79±0.09 (100%) (142%) Gliben (n=7) 62±22 93±26 0.73±0.14 0.83±0.18 (100%) (147%) SCWS (n=8) 74±29 61±13 0.71±0.04 0.75±0.08 (100%) (82%) SCE (n=8) 76±25 70±21 0.73±0.07 0.71±0.08 (100%) (92%) Results are expressed as Mean±SD (percentage). Between groups, comparison was done using one-way ANOVA with post hoc Bonferroni test. Within groups, comparison was done using paired t test. WC = Water Control; Gliben = Glibenclamide treated group; SCWS = S. chirata water soaked group; SCE =S. chirata ethanol extract.

Fig. 3. Effect of S. chirata extracts on hepatic glycogen content of STZ-induced T2DM rats Results are expressed as Mean ± SD. Statistical analysis between group comparison was done by using Independent Sample T Test. WC = Water Control; Gliben = Glibenclamide treated group; SCWS = S. chirata water soaked group; SCE =S. chirata ethanol extract

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3.6 Effect on Hepatic Glycogen Content of experiment in type 2 rats. Fig. 4 depicts the of T2DM Rats gradual fall (p=ns) in glucose absorption during the whole perfusion period in type 2 rats The Fig. 3 showed that there were no significant compared to Krebs solution only as control. changed in hepatic glycogen content on Therefore, the obtained results suggest that both glibenclamide and SCWS treated group; but 48% extract delays glucose absorption in the upper increased was shown in SCE treated group after part of the gastrointestinal tract. 28th day of chronic oral administration when it compared to water control, which was statistically 4. DISCUSSION significant (p=0.05). The present study was undertaken to assess the 3.7 Investigation of Antioxidant Activities antidiabetic effect after chronic administration of S. chirata extract on T2DM rats and explore the Table 5shows the concentration of erythrocyte mode of antidiabetic action. At the beginning (0 lipid peroxidation products i.e. malondialdehyde day) extracts of S. chirata non-significantly (MDA) and reduced Glutathione (GSH) in opposed the rise of postprandial serum glucose level when fed 30 minutes before glucose load. different groups of rats after 28 days of the study st period. The levels of erythrocyte MDA was lower When OGTT was examined again at the 21 day by 10% in SCWS treated groups in comparison after chronic feeding of SCE and to water control group and GSH level did not SCWS,produced a significant antihyperglycemic showed any change in treated group effect in T2DM rats. Observed glucose lowering respectively. effect in T2DM rats after oral administration of extract of S. chirata with a simultaneous glucose Table 5. Effects of S. chirata extracts on load, indicated that S. chirata may interfere with serum malondialdehyde (MDA) and reduced the intestinal glucose absorption in the gut. It glutathione (GSH) of STZ-induced T2DM rats may also act by modifying the peripheral glucose uptake and probably increasing insulin sensitivity Groups MDA GSH (mg/g Hb) [32]. Our obtained results also indicate that in (μmol/ml) case of Type 2 diabetic rats both first as well as WC (n=7) 1.74±0.10 16.37±4.32 second phase of insulin response to glucose are (100%) (100%) impaired, whereas extracts treatment improved Gliben 1.78±0.37 16.52±6.40 glucose tolerance. It was, may be, and due to (n=7) (102%) (100%) restoration delayed insulin response. SCWS 1.57±0.81 14.75±8.39 (n=8) (90%) (90%) In the chronic study, the most important finding SCE 1.87±0.95 15.11±8.77 was that, after 28 days of consecutive feeding, (n=8) (107%) (92%) when the rats were sacrificed, a significant Results are expressed as Mean ±SD (percentage). reduction in the fasting glucose level was Statistical analysis between group comparison was observed in SCWS (p=0.01) fed group compared done by using Independent Sample T Test. WC = with baseline day and SCE group was decreased Water Control; Gliben = Glibenclamide treated group; by 17% respectively. In this experiment, SCWS = S. chirata water soaked group; g l i b enclamide treated group (positive control) SCE =S. chirata ethanol extract. also decreased fasting blood glucose level after

chronic feeding. This obtained result is supported 3.8 Effect of S. chirata on Upper by the finding of other investigators [14-16]. Intestinal Glucose Absorption

As shown in Fig. 4, the upper intestinal glucose It was explored whether the blood glucose absorption was almost constant during 30 lowering effect was due to reduction of food minutes of perfusion with glucose. When the in t a k e . This was done by comparing the body S. chirata extract and soaked water solution were weight between the control and treated groups. supplemented with the glucose solution, type 2 The result showed that there was an increasing model rats showed decrease in intestinal glucose tendency of body weight in control and treated absorption almost constantly during 30 min of (extract and glibenclamide) groups. The perfusion. tendencies were of similar proportion in the control and treated groups; and thus, they do not SCWS and SCE strongly affected the amount of responsible for the hypoglycemic effect found in absorbed glucose throughout the notable period the extract group. The findings also suggest that

Bhowmik et al.; JPRI, 22(5): 1-11, 2018; Article no.JPRI.41878

n 51 o i t p r

o 41 s b

A Krebs+glu

e 31 s

o Krebs+glu+ SCWS c u l

g 21

f Krebs+glu+ SCE O

% 11

1 5 min 10 min 15 min 20 min 25 min 30 min

Fig. 4. Effects of S. chirata soaked water solution and extract on upper intestinal glucose absorption on STZ-induced T2DM rats Results are presented as mean± SD (n=6). Between groups, comparison was done using one-way ANOVA with post hoc Bonferroni test. Within groups, comparison was done using paired t test. Rats were fasted for 36 hours and intestine was perfused with glucose solution (54 g/l) with or without SCWS/SCE (0.25mg/ml); K-g=Kreb’s solution +glucose; K-g-SCWS= Kreb’s solution + glucose + water shocked solution of S. chirata, K-g-SCE= Kreb’s solution + glucose + extract solution of S. chirata.

S. chirata extract does not alter normal metabolic Since dyslipidemia plays an important role in the parameters like food and water intake. pathogenesis of macro‐ and micro vascular complications of diabetes, hence, improvement A significant decrease of insulin resistance index in the lipid abnormalities must play beneficial role (HOMA IR) was shown in SCWS (p=0.01) after in inhibiting the complications of diabetes. In this 28th day in comparison to initial day and SCE study, SCWS (p=0.02) and SCE (p=0.01) groups group was decreased (p=ns) by 77% significantly decreased serum total cholesterol respectively. The result indicates that the level. Regarding triglyceride level, it was extracts affect the β‐cell of pancreas directly for decreased by 13% in SCE group and LDL level secretion of insulin by decreasing insulin was decreased by 42% in SCWS group on final resistance. Again, in our experiment, S. chirata day in comparison to initial day. After chronic extract showed a significantly higher (p=0.05) administration there was no significant change in glycogen content in the liver (Fig. 3). Synthesis of serum SGPT and creatinine level which indicates liver glycogen may be due to increased glycogen that there was no toxic effect in the liver or in the sythase activity. The levels of erythrocyte MDA kidney by the extracts of S. chirata. was lowered by 10% in SCWS treated groups in comparison with water control (Table 5), which In gut perfusion in situ experiment where both indicates that SCWS has antioxidant property. extract of S. chirata showed an inhibition of The extract may also improve insulin g lu c o s e absorption. The inhibition of intestinal sensitivity by reducing glucotoxicity which is one glucose absorption may contribute to the of the causes of insulin resistance in type2 rats reduction of postprandial glucose level which [33]. was observed in this study.

Apart from the blood sugar lowering effect, 5. CONCLUSION beneficial changes in lipid profile was also observed by S. chirata extract. It has been S. chirata extracts has potential demonstrated that postprandial hyperglycemia is antihyperglycemic effect on T2D model rats. The an important cardiovascular risk factors in Type 2 underlying mechanism may be at least partly due diabetic patients [34]. It has been claimed that to improved glucose tolerance, increased hypercholesterolemia and hypertriglyceridaemia glycogenesis, decreased insulin resistance and occurred in STZ induced diabetic rats [35]. improvement in lipid profile. Therefore, treatment Abnormalities in lipid profiles are one of the most with S. chirata provides a rationale for its use in common complications in diabetes mellitus. diabetic treatment.

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55 Taibah University 56 57 58 Journal of Taibah University Medical Sciences 59 60 61 www.sciencedirect.com 62 63 64 Original Article 65 1 66 2 67 3 Oyster mushroom functions as an anti-hyperglycaemic through 68 4 69 5 phosphorylation of AMPK and increased expression of GLUT4 in type 70 6 2 diabetic model rats 71 7 72 8 a b c b 73 9 Q6 M. Asrafuzzaman, MSc , M.M. Rahman, MSc , M. Mandal, MSc , M. Marjuque, MSc , 74 b d a e,* 10 Q1Q2 A. Bhowmik, MSc , B. Rokeya, PhD , Z. Hassan, PhD and M.O. Faruque, PhD 75 11 76 12 a Department of Physiology and Molecular Biology, Bangladesh University of Health Sciences, Dhaka, Bangladesh 77 13 b Department of Applied Laboratory Science, Bangladesh University of Health Sciences, Dhaka, Bangladesh 78 c 14 Q3 Department of Biochemistry and Molecular Biology, BSMRSTU, Gopalgonj, Bangladesh 79 15 d Department of Pharmacology, Bangladesh University of Health Sciences, Dhaka, Bangladesh 80 16 e Department of Nutrition and Food Technology, Jessore University of Science and Technology, Jessore, Bangladesh 81 17 82 18 Received 27 December 2017; revised 15 February 2018; accepted 19 February 2018; Available online --- 83 19 84 20 85 21 86 87 ﺍﻟﻨﺘﺎﺋﺞ: ﻣﻜﻤﻼﺕ ﻣﺴﺤﻮﻕ ﻓﻄﺮ ﺍﻟﻤﺤﺎﺭ ﻟﻤﺪﺓ ٨ ﺃﺳﺎﺑﻴﻊ ﻧﺘﺞ ﻋﻨﻪ ﺍﻧﺨﻔﺎﺽ ﻣﺴﺘﻮﻯ ﺍﻟﻤﻠﺨﺺ 22 88 ﺍﻟﺴﻜﺮ ﻓﻲ ﺩﻡ ﺍﻟﻔﺌﺮﺍﻥ ﺍﻟﻤﺼﺎﺑﺔ ﺑﺪﺍﺀ ﺍﻟﺴﻜﺮﻱ ﺍﻟﻨﻮﻉ ٢ ﻣﻦ ﺧﻼﻝ ﺗﺤﺮﻳﺾ 23 24 89 ﺍﻟﺴﺘﺮﺑﺘﻮﺯﻭﺗﻮﺳﻴﻦ ﺩﺍﺧﻞ ﺍﻟﺼﻔﺎﻕ. ﻣﺴﺘﻮﻳﺎﺕ ﻓﺴﻔﺮﺓ ﺃﺣﺎﺩﻱ ﻓﺴﻔﺎﺕ ﺍﻷﺩﻳﻨﻮﺯﻳﻦ ﺃﻫﺪﺍﻑ ﺍﻟﺒﺤﺚ: ﻳﺴﺘﺨﺪﻡ ﺍﻟﻔﻄﺮ ﻟﻠﺤﺪ ﻣﻦ ﺍﺭﺗﻔﺎﻉ ﺍﻟﺴﻜﺮ ﻓﻲ ﺍﻟﺪﻡ ﺗﻘﻠﻴﺪﻳﺎ. ﺑﺎﻟﺮﻏﻢ ﻣﻦ 25 90 ﺍﻟﻤﻨﺸﻂ ﺍﻟﺒﺮﻭﺗﻴﻦ ﻛﻴﻨﺎﺯ، ﻛﻨﺴﺒﺔ ﺇﻟﻰ ﺑﻴﺘﺎ ﺍﻻﻛﺘﻴﻦ، ﺍﺭﺗﻔﻌﺖ ﻓﻲ ﺍﻟﻌﻀﻼﺕ ﻭﺍﻷﻧﺴﺠﺔ ﺫﻟﻚ، ﻟﻢ ﻳﺘﻢ ﺍﺳﺘﻜﺸﺎﻑ ﺍﻵﻟﻴﺔ ﺍﻟﻜﺎﻣﻨﺔ ﻭﺭﺍﺀ ﻫﺬﺍ ﺍﻟﺘﺄﺛﻴﺮ ﻭﻣﻦ ﺍﻟﻤﻌﺮﻭﻑ ﺃﻥ ﺃﺣﺎﺩﻱ 26 . - 91 ﺍﻟﺪﻫﻨﻴﺔ ﻋﻨﺪ ﺍﻟﻔﺌﺮﺍﻥ ﺍﻟﻤﺼﺎﺑﺔ ﺑﺪﺍﺀ ﺍﻟﺴﻜﺮﻱ ﺍﻟﻨﻮﻉ ٢ ﺍﻟﻤﻌﺎﻟﺠﺔ ﺑﺎﻟﻔﻄﺮ، ﺑﺎﻟﻤﻘﺎﺭﻧﺔ ﻓﺴﻔﺎﺕ ﺍﻷﺩﻳﻨﻮﺯﻳﻦ ﺍﻟﻤﻨﺸﻂ ﺍﻟﺒﺮﻭﺗﻴﻦ ﻛﻴﻨﺎﺯ ﻳﺨﻔﺾ ﻣﻦ ﺍﺭﺗﻔﺎﻉ ﺍﻟﺴﻜﺮ ﻓﻲ ﺍﻟﺪﻡ ﻣﻦ 27 92 ﺑﻔﺌﺮﺍﻥ ﻣﺠﻤﻮﻋﺔ ﺍﻟﺘﺤﻜﻢ. ﺍﺭﺗﻔﻊ ﺍﺳﺘﺨﺮﺍﺝ ﻧﺎﻗﻞ ﺍﻟﺠﻠﻮﻛﻮﺯ ﺍﻟﻨﻮﻉ ﺍﻟﺮﺍﺑﻊ، ﻛﻨﺴﺒﺔ ﺇﻟﻰ ﺧﻼﻝ ﻣﺴﺎﺭ ﺍﻷﻧﺴﻮﻟﻴﻦ- ﺍﻟﻤﺴﺘﻘﻞ. ﺗﻬﺪﻑ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﻟﻤﺮﺍﻗﺒﺔ ﺗﺄﺛﻴﺮ ﻣﺴﺤﻮﻕ ﻓﻄﺮ 28 93 ﺟﻠﻴﺴﺮﺍﻟﺪﻳﻬﻴﺪ ٣-ﻓﻮﺳﻔﺎﺕ ﺩﻳﻬﻴﺪﺭﻭﺟﻴﻨﻴﺰ، ﺑﺸﻜﻞ ﻣﻠﺤﻮﻅ ﻓﻲ ﻛﻞ ﻣﻦ ﺍﻟﻌﻀﻼﺕ ﺍﻟﻤﺤﺎﺭ ﻋﻠﻰ ﻓﺴﻔﺮﺓ ﺃﺣﺎﺩﻱ ﻓﺴﻔﺎﺕ ﺍﻷﺩﻳﻨﻮﺯﻳﻦ ﺍﻟﻤﻨﺸﻂ ﺍﻟﺒﺮﻭﺗﻴﻦ ﻛﻴﻨﺎﺯ ﻭﺍﺳﺘﺨﺮﺍﺝ 29 94 ﻭﺍﻷﻧﺴﺠﺔ ﺍﻟﺪﻫﻨﻴﺔ ﻟﻠﻔﺌﺮﺍﻥ ﺍﻟﻤﺼﺎﺑﺔ ﺑﺪﺍﺀ ﺍﻟﺴﻜﺮﻱ ﺍﻟﻤﻌﺎﻟﺠﺔ ﺑﺎﻟﻔﻄﺮ. ﻣﺮﺍﺳﻞ ﺍﻟﺤﻤﺾ ﺍﻟﻨﻮﻭﻱ ﺍﻟﺮﻳﺒﻲ ﻟﻨﺎﻗﻞ ﺍﻟﺠﻠﻮﻛﻮﺯ ﺍﻟﻨﻮﻉ ﺍﻟﺮﺍﺑﻊ ﻓﻲ ﺍﻟﻔﺌﺮﺍﻥ ﺍﻟﻤﺼﺎﺑﺔ 30 95 ﺑﺪﺍﺀ ﺍﻟﺴﻜﺮﻱ 31 . 96 ﺍﻻﺳﺘﻨﺘﺎﺟﺎﺕ: ﻗﺪ ﻳﻘﻠﻞ ﻓﻄﺮ ﺍﻟﻤﺤﺎﺭ ﻣﻦ ﺍﺭﺗﻔﺎﻉ ﺍﻟﺴﻜﺮ ﻓﻲ ﺍﻟﺪﻡ ﻣﻦ ﺧﻼﻝ ﺯﻳﺎﺩﺓ 32 97 ﻓﺴﻔﺮﺓ ﺃﺣﺎﺩﻱ ﻓﺴﻔﺎﺕ ﺍﻷﺩﻳﻨﻮﺯﻳﻦ ﺍﻟﻤﻨﺸﻂ ﺍﻟﺒﺮﻭﺗﻴﻦ ﻛﻴﻨﺎﺯ ﻭﺃﻳﻀﺎ ﺍﺳﺘﺨﺮﺍﺝ ﻧﺎﻗﻞ ﻃﺮﻕ ﺍﻟﺒﺤﺚ: ﺗﻢ ﺍﺳﺘﺨﺪﺍﻡ ﻓﺌﺮﺍﻥ ﺍﻳﻔﺎﻧﺰ ﺍﻟﻄﻮﻳﻠﺔ ﻹﺳﺘﺤﺪﺍﺙ ﻓﺌﺮﺍﻥ ﻣﺼﺎﺑﺔ ﺑﺪﺍﺀ 33 98 ﺍﻟﺠﻠﻮﻛﻮﺯ ﺍﻟﻨﻮﻉ ﺍﻟﺮﺍﺑﻊ ﻓﻲ ﺍﻟﻌﻀﻼﺕ ﻭﺍﻷﻧﺴﺠﺔ ﺍﻟﺪﻫﻨﻴﺔ ﺍﻟﺴﻜﺮﻱ ﺍﻟﻨﻮﻉ ٢ ﻣﻦ ﺧﻼﻝ ﺗﺤﺮﻳﺾ ﺍﻟﺴﺘﺮﺑﺘﻮﺯﻭﺗﻮﺳﻴﻦ ﺩﺍﺧﻞ ﺍﻟﺼﻔﺎﻕ ﺗﻤﺖ 34 . . 99 100 ﺇﺿﺎﻓﺔ ٥٪ ﻣﺴﺤﻮﻕ ﻓﻄﺮ ﺍﻟﻤﺤﺎﺭ ﻣﻊ ﺍﻟﺘﻐﺬﻳﺔ ﺍﻟﻤﻌﺘﺎﺩﺓ ﻟﻠﻔﺌﺮﺍﻥ ﻟﻤﺪﺓ ٨ ﺃﺳﺎﺑﻴﻊ 35 101 ﺍﻟﻜﻠﻤﺎﺕ ﺍﻟﻤﻔﺘﺎﺣﻴﺔ: ﺃﺣﺎﺩﻱ ﻓﺴﻔﺎﺕ ﺍﻷﺩﻳﻨﻮﺯﻳﻦ ﺍﻟﻤﻨﺸﻂ ﺍﻟﺒﺮﻭﺗﻴﻦ ﻛﻴﻨﺎﺯ؛ ﻧﺎﻗﻞ ﻣﺘﻮﺍﻟﻴﺔ. ﺑﻌﺪ ﺫﻟﻚ، ﺗﻢ ﺫﺑﺢ ﺍﻟﻔﺌﺮﺍﻥ. ﻭﺍﺳﺘﺨﺮﺍﺝ ﺍﻟﺤﻤﺾ ﺍﻟﻨﻮﻭﻱ ﺍﻟﺮﻳﺒﻲ ﺑﻮﺍﺳﻄﺔ 36 102 ﺍﻟﺠﻠﻮﻛﻮﺯ ﺍﻟﻨﻮﻉ ﺍﻟﺮﺍﺑﻊ؛ ﺍﺭﺗﻔﺎﻉ ﺍﻟﺴﻜﺮ ﻓﻲ ﺍﻟﺪﻡ؛ ﻓﻄﺮ ﺍﻟﻤﺤﺎﺭ ﻛﺎﺷﻒ ﺗﺮﺍﻳﺰﻭﻝ، ﻭﺗﻢ ﺍﺳﺘﺨﻼﺹ ﺍﻟﺒﺮﻭﺗﻴﻨﺎﺕ ﻣﻦ ﺃﻧﺴﺠﺔ ﻣﺨﺘﻠﻔﺔ ﻣﻊ ﺗﺤﻠﻞ ﺍﻟﻌﺎﺯﻟﺔ 37 103 ﺭﻳﺒﺎ. ﻛﻤﺎ ﺗﻢ ﻗﻴﺎﺱ ﺍﺳﺘﺨﺮﺍﺝ ﻣﺮﺍﺳﻞ ﺍﻟﺤﻤﺾ ﺍﻟﻨﻮﻭﻱ ﺍﻟﺮﻳﺒﻲ ﻟﻨﺎﻗﻞ ﺍﻟﺠﻠﻮﻛﻮﺯ ﺍﻟﻨﻮﻉ 38 Abstract 104 ﺍﻟﺮﺍﺑﻊ ﻣﻦ ﺧﻼﻝ ﺗﻘﻨﻴﺎﺕ ﺗﻔﺎﻋﻞ ﺍﻟﺒﻮﻟﻴﻤﻴﺮﺍﺯ ﺍﻟﻤﺘﺴﻠﺴﻞ ﻟﻠﺤﻤﺾ ﺍﻟﻨﻮﻭﻱ ﺍﻟﺮﻳﺒﻮﺯﻱ 39 105 ﻣﻨﻘﻮﺹ ﺍﻷﻛﺴﺠﻴﻦ، ﻭﺗﻢ ﺗﺤﺪﻳﺪ ﻓﺴﻔﺮﺓ ﺃﺣﺎﺩﻱ ﻓﺴﻔﺎﺕ ﺍﻷﺩﻳﻨﻮﺯﻳﻦ ﺍﻟﻤﻨﺸﻂ ﺍﻟﺒﺮﻭﺗﻴﻦ 40 Objectives: Traditionally, mushrooms have been used to 106 ﻛﻴﻨﺎﺯ ﺑﻮﺍﺳﻄﺔ ﺍﻟﻨﺸﺎﻑ ﺍﻟﻐﺮﺑﻲ. ﺗﻢ ﻗﻴﺎﺱ ﻛﺜﺎﻓﺔ ﺷﺮﻳﻂ ﻣﻨﺘﺠﺎﺕ ﺗﻔﺎﻋﻞ ﺍﻟﺒﻠﻤﺮﺓ 41 42 reduce hyperglycaemia. However, the mechanism under- 107 ﺍﻟﻤﺘﺴﻠﺴﻞ ﻭﺍﻟﺒﺮﻭﺗﻴﻨﺎﺕ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺑﺮﻧﺎﻣﺞ ﺟﻴﻪ ﻟﻠﺼﻮﺭ. 43 lying this effect has not yet been explored. AMP- 108 44 activated protein kinase (AMPK) is known to reduce 109 45 * Corresponding address: Department of Nutrition and Food hyperglycaemia through an insulin-independent pathway. 110 46 Technology, Jessore University of Science and Technology, Jessore 111 47 This study aimed to observe the effect of oyster mush- 7408, Bangladesh. 112 48 room powder (OMP) on phosphorylation of AMPK (p- E-mails: [email protected], [email protected] 113 49 AMPK) and expression of GLUT4 mRNA in diabetic (M.O. Faruque) 114 50 model rats. Peer review under responsibility of Taibah University. 115 51 116 52 Methods: Long-Evans rats were used to develop type 2 117 53 diabetic model rats through intraperitoneal induction of 118 54 Production and hosting by Elsevier streptozotocin (STZ). OMP was supplemented at 5% 119 1658-3612 Ó 2018 Taibah University. Production and hosting by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). https://doi.org/10.1016/j.jtumed.2018.02.009 Please cite this article in press as: Asrafuzzaman M, et al., Oyster mushroom functions as an anti-hyperglycaemic through phosphorylation of AMPK and increased expression of GLUT4 in type 2 diabetic model rats, Journal of Taibah University Medical Sciences (2018), https://doi.org/10.1016/ j.jtumed.2018.02.009 JTUMED469_proof ■ 8 August 2018 ■ 2/7

2 M. Asrafuzzaman et al.

1 with the usual feed of rats for 8 consecutive weeks. Then, hypoglycaemic potential of the oyster mushroom has been 66 2 the rats were sacrificed. RNA was extracted by the already established in an animal model, the cellular 67 3 TRIzol reagent, and proteins were extracted from mechanism is still unknown. 68 4 different tissues with RIPA lysis buffer. Expression of In mammals, AMP-activated protein kinase (AMPK) is a 69 5 GLUT4 mRNA was measured through cDNA-PCR heterometric enzyme complex, which is activated by phos- 70 6 techniques, and p-AMPK was detected using western phorylation of threonine 172, due to a variety of metabolic 71 7 blotting. The band intensities of the PCR products and stressors.13 AMPK is activated in response to low levels of 72 8 73 proteins were measured using Image J software. ATP, which results in an increase in the AMP:ATP ratio 9 74 and also changes the cellular redox potential, resulting in a 10 75 Results: Supplementation of OMP for 8 weeks resulted in rise in the NAD/NADH ratio.13,14 In peripheral tissues, 11 76 a reduction of the serum glucose level in STZ-induced, AMPK maintains a number of metabolic processes, such 12 77 type 2 diabetic model rats. The levels of p-AMPK, as a as glucose and lipid metabolism.14 Moreover, AMPK 13 b 78 14 ratio relative to -actin, increased in the muscle and ad- serves as a fuel gauge that responds to fluctuations in 79 15 ipose tissues of mushroom-treated type 2 diabetic model cellular energy levels and extracellular nutrient levels, such 80 16 rats, compared to those in control diabetic model rats. as glucose, hormones, and fatty acids. AMPK plays an 81 17 Expression of GLUT4, as a ratio relative to GAPDH, important role in regulating whole body energy metabolism 82 18 increased significantly in both the muscle and adipose by responding to circulating hormones and by circulating 83 13 19 tissues of mushroom-treated diabetic rats. the food intake. 84 20 The glucose transporter 4 plays a key role in transporting 85 21 Conclusion: Oyster mushroom may decrease hyper- extracellular glucose into insulin sensitive muscles and adi- 86 22 glycaemia through increased p-AMPK and also expres- pose tissues in vivo. Besides, skeletal muscles and adipose 87 23 sion of GLUT4 in the muscle and adipose tissues. tissues are responsible for up to 50e80% of glucose trans- 88 24 portation in the body. GLUT4 expression in the skeletal 89 25 Keywords: AMP-activated protein kinase; Glucose trans- muscle and adipose tissues of type 2 diabetic patients is 90 26 porter 4; Hyperglycaemia; Oyster mushroom significantly reduced, indicating that such patients have a 91 27 lower capability to transport glucose.15 Therefore, the aim of 92 Ó 28 2018 Taibah University. this study was to observe the phosphorylation of AMPK and 93 Production and hosting by Elsevier Ltd. This is an open 29 the expression of GLUT4 mRNA in mushroom-treated type 94 access article under the CC BY-NC-ND license (http:// 30 2 diabetic model rats. 95 31 creativecommons.org/licenses/by-nc-nd/4.0/). 96 32 97 33 Materials and Methods 98 34 99 35 Animals 100 Q4 Introduction 36 101 37 e 102 Type 2 diabetes is a complex and heterogeneous disorder, Adult Long-Evans rats, weighing 170 220 g, were used 38 in this study. The animals were bred at the Bangladesh 103 39 which is characterized by impaired insulin sensitivity or 104 University of Health Sciences animal house, in Dhaka, 40 decreased insulin secretion and is diagnosed as hyper- 105 1 Bangladesh, and maintained at a constant room tempera- 41 glycaemia. A calorie-rich diet, obesity, and a sedentary 106 ture of 22 C, with a humidity of 40e70% and a natural 42 lifestyle contribute to the rising number of individuals with 107 e 43 type 2 diabetes worldwide.2 Insulin resistance and pancreatic 12 h day night cycle. The experiment was conducted ac- 108 44 b cell failure are defining metabolic parameters of type 2 cording to the ethical guidelines, approved by the 109 45 diabetes.3 Moreover, in the majority cases, type 2 diabetes Bangladesh University of Health Sciences. Type 2 diabetic 110 46 arises due to obesity and insulin resistance.4,5 However, model rats were created by a single intraperitoneal injection 111 47 type 2 diabetes is a chronic disease and leads to serious of streptozotocin (STZ) in citrate buffer (pH 4.5), at a dose 112 48 6 of 90 mg/kg of the body weight, into rat pups (48 h old; 113 health complications. Therefore, in developed and 16,17 49 developing countries, type 2 diabetes poses a major health average weight: 7 g). After 3 months, the STZ-injected 114 50 threat.7 The prevalence and complications of type 2 rats were examined for their blood glucose level by an 115 51 diabetes are aggrandizing every day. Furthermore, the use oral glucose tolerance test (OGTT), in which blood was 116 52 of conventional, pharmacological, anti-diabetic drugs can collected from the tail tips. Diabetic model rats with a blood 117 53 ˃ 118 sometimes increase the treatment complexity, due to drug glucose level 7.0 mmol/l under fasting conditions were 54 119 side effects and high costs.8 Accordingly, natural products selected to study the effect of white oyster mushroom 55 120 are alternatives, because these compounds are believed to powder. 56 121 57 have fewer side effects. 122 Preparation of rat feed, supplemented with 5% oyster 58 Mushrooms have been used as food and medicine for 123 mushroom (P. ostreatus) powder 59 thousands of years. The mushroom serves as a natural source 124 9 60 of medicine with antidiabetic potential. Oyster mushrooms 125 61 (Pleurotus ostreatus) possess many valuable food qualities, All of the standard rat pellet ingredients, i.e. flour, wheat 126 62 e.g., low in calories, fats, and essential fatty acids, but rich bran, maize bran, rice bran, fish meal, beshon, powder milk, 127 10,11 63 in proteins, vitamins, and minerals. The oyster salt, oil, vitamins, molasses, and oil cake, were purchased 128 64 mushroom has a promising hypoglycaemic potential in an from the market for poultry feed. Oyster mushrooms (P. 129 65 animal model.12 Although the acute and chronic, oral ostreatus) were collected from the National Mushroom 130

Please cite this article in press as: Asrafuzzaman M, et al., Oyster mushroom functions as an anti-hyperglycaemic through phosphorylation of AMPK and increased expression of GLUT4 in type 2 diabetic model rats, Journal of Taibah University Medical Sciences (2018), https://doi.org/10.1016/ j.jtumed.2018.02.009 JTUMED469_proof ■ 8 August 2018 ■ 3/7

Oyster mushroom stimulates p-AMPK and GLUT4 3

1 Development and Extension Center, Savar, Dhaka, and the changes in GLUT4 expression were normalized to 66 2 Bangladesh. Then, all of the ingredients were mixed in a bowl the housekeeping gene GAPDH. 67 3 with the following proportions: 40% flour, 15% wheat bran, 68 4 69 8% maize bran, 4% rice bran, 10% fish meal, 3% beshon, Western blotting 5 4% powder milk, 0.5% salt, 1% oil, 1% vitamins, 0.5% 70 6 molasses, 8% oil cake, and 5% oyster mushroom powder. 71 Proteins were extracted from the muscle and adipose tis- 7 Water was added to make dough, and then, the dough was 72 sues of all of the experimental rats using RIPA lysis buffer 8 placed in a thin layer on an oven tray and dried in the oven at 73 9 (200 mg tissue/1 ml buffer) and quantified by the Bradford 74 150 C for 30 min. This feed, supplemented with 5% OMP, 10 reagent. Western blotting for specific protein detection was 75 was prepared everyday throughout the experimental period. 11 carried out as described previously.14 Total cell lysates were 76 12 resolved by sodium dodecyl sulphate polyacrylamide gel 77 Experimental design 13 electrophoresis (SDS-PAGE) (12% separating and 4% 78 14 stacking gel). Protein was then transferred to a PVDF 79 15 A total of 21 rats (3 normal rats and 18 type 2 diabetic membrane in transfer buffer (Tris base, glycine, 20% 80 16 model rats) were used for 8 weeks in the chronic experimental methanol). Membranes were blocked in 3% BSA (bovine 81 17 study. serum albumin) and then, incubated with primary rabbit 82 18 The untreated control group (n ¼ 6) consisted of type 2 monoclonal antibodies (phospho-AMPK, Santa Cruz 83 19 diabetic model rats that were administered water as the Biotech. USA) (1:100), followed by a 1 h incubation with a 84 20 vehicle [7.5 g food/100 g body weight per day]. For the gli- 1:2000 dilution of the secondary horse-radish peroxide-anti 85 21 86 clazide-treated group (n ¼ 6), type 2 diabetic model rats were rabbit IgG (Santa Cruz Biotech. USA). Protein signals were 22 87 administered the standard drug gliclazide [20 mg/5 ml of detected using an ECL solution followed by autoradiog- 23 88 solvent (water with a few drops of 1 N sodium hydroxide) per raphy. Blots were imaged using autoradiography film. Equal 24 ¼ 89 kg of body weight]. For the mushroom-treated group (n 6), amounts of protein were also resolved for the detection of b- 25 white oyster mushroom powder (5%) was supplemented in 90 26 actin, which is ubiquitously expressed. After transfer of the 91 the normal feed. 27 protein to the PVDF membrane, the membrane was blocked 92 28 in 5% non-fat milk for 1 h at room temperature and then, 93 Biochemical analysis 29 incubated with primary mouse monoclonal antibody 94 30 (1:5000) b-actin for 1 h at room temperature, followed by 95 31 Serum glucose was measured by the glucose oxidase another 1 h incubation at room temperature with secondary 96 32 (GOD-PAP) method, using a micro-plate reader (Bio-Tec, horseradish peroxide anti-mouse IgG (1:2000). Protein was 97 33 ELISA). The serum lipid profile was measured by an enzy- detected by ECL solution on autoradiography film. The 98 34 matic colorimetric method (Randox Laboratories Ltd., UK), band intensities of this blot were determined by ImageJ 99 35 using a micro-plate reader. software, and changes in the p-AMK level were normalized 100 36 to the housekeeping protein b-actin. 101 37 Reverse-transcriptase PCR (RT-PCR) 102 38 103 Statistical analysis 39 104 mRNA was extracted using the TRIzol reagent (SIGMA, 40 105 USA) (1 ml/100 mg of tissue) from the muscle and adipose 41 Statistical analysis was performed using the Statistical 106 and liver tissues of all of the experimental rats. The con- 42 Package for Social Science (SPSS) software for Windows, 107 43 centration of the RNA in each solution was determined by version 12 (SPSS Inc., Chicago, Illinois, USA). Data were 108 44 measuring the absorbance at 260 nm, and the RNA solutions expressed as the mean SD, as a number (percentage) as 109 45 were stored at 80 C until assayed. For reverse transcrip- appropriate. The statistical difference between two groups 110 46 tion, mRNA was converted to a single stand of comple- was assessed by one way ANOVA paired t-tests. A two-tailed 111 47 mentary DNA (cDNA) using a high capacity cDNA reverse p value of <0.05 was considered statistically significant. 112 48 transcriptase kit (Promega Corp. USA), following the man- 113 ufacturer’s instructions and a previous study.18 Briefly, 1 mg 49 Results 114 50 of mRNA was used in a 10 ml reaction volume. The mixture 115 51 was incubated at 42 C for 1 h, snap-chilled on ice, and then 116 52 incubated at 70 C for 5 min. Reaction products were sepa- Effect of the oyster mushroom on the body weight of type 2 117 53 rated with a 3% agarose gel and visualized with a gel docu- diabetic model rats 118 54 mentation system. 119 55 Three ml of cDNA was used for PCR (35 cycles) to Changes in body weight for the different rat groups are 120 56 observe the expression of GLUT4 using the following depicted in Figure 1. The initial body weights (g) were 121 57 primers: Forward: 5ʹ-GGG CTG TGA GTG AGT GCT 176 12, 196 10, and 194 22 for the control diabetic 122 58 TTC-3ʹ and reverse: 5ʹ-CAG CGA GGC AAG GCT AGA- rats, the gliclazide-treated, and mushroom-treated diabetic 123 59 3ʹ. PCR for the housekeeping gene GAPDH was also con- model rats, respectively. Body weight was monitored every 124 60 ducted, but with the following primers: Forward: 5ʹ-TGC week, and after eight weeks, the bodyweight increases among 125 61 126 TGG GGC TGG CAT TGC TC-3ʹ and reverse: 5ʹ-TCC the groups were similar. Therefore, the oyster mushroom did 62 127 TTG CTG GGC TGG GTG GT-3ʹ. The band intensities of not show any effects on the body weight of the diabetic 63 128 the PCR products were measured using ImageJ software, model rats (Figure 1). 64 129 65 130

4 M. Asrafuzzaman et al.

1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 Figure 1: Body weights of the different groups of type 2 diabetic 79 15 model rats during the experimental periods: Groups CG, GT, and Figure 2: Effect of the oyster mushroom on the lipid profiles of 80 16 MT represent the control group diabetic model rat, gliclazide- T2DM model rats: Groups CG, GT, and MT represent the control 81 17 treated diabetic rat, and mushroom-treated diabetic rat, group diabetic model rat, gliclazide-treated diabetic rat, and 82 18 respectively. mushroom-treated diabetic rat, respectively. Data are presented as 83 19 the mean standard deviation (M SD). 84 20 85 21 Effect of the oyster mushroom on the serum glucose level of 86 22 the type 2 diabetic model rats Effect of the oyster mushroom on the phospho-AMPK (p- 87 23 AMPK) protein in the muscle and adipose tissue of type 2 88 24 To evaluate the effect of the oyster mushroom on glucose diabetic model rats 89 25 metabolism, fasting serum glucose (mmol/l) levels were 90 26 measured for the different experimental groups. The fasting As shown in Figures 3 and 4, p-AMPK from the muscle 91 27 blood glucose on the first day was considered 100%, and the and adipose tissue, respectively, of non-diabetic, diabetic 92 28 93 values on the 56th day were calculated based on the initial control, mushroom-treated, and gliclazide-treated rats was 29 94 day. The untreated control group did not have any signifi- detected using western blotting between the marker proteins 30 95 cant difference between day 0 and day 56. The fasting serum 85 kDa and 50 kDa in size. These values are presented as 31 ratios, compared to b-actin (a housekeeping protein) in bar 96 32 glucose level in the mushroom-treated (MT) group decreased 97 < diagrams. p-AMPK appeared to decrease more than 20% in 33 significantly (p 0.05; 29%) by the 56th day (day 0: 98 the STZ-induced, diabetic model rats (the water control), 34 8.97 1.31; day 56: 6.40 1.41). For the gliclazide-treated 99 compared to the non-diabetic rats. However, in the case of 35 (GT) group, the fasting glucose concentrations were 100 36 10.01 1.44 and 6.70 1.82 on day 0 and day 56, respec- the mushroom-treated diabetic rats, p-AMPK increased 101 37 tively, which indicates a 33% decline, compared to day about two-fold in the muscle tissues, compared to the control 102 38 0(Table 1). diabetic rats (Figure 3). Similar effects were also observed in 103 39 the muscle tissues of gliclazide-treated diabetic model rats 104 (Figure 3). In the case of the adipose tissue, p-AMPK seemed 40 Effect of the oyster mushroom on the serum lipid profile level 105 to increase more than three-fold in the mushroom-treated 41 of STZ-induced, type 2 diabetic model rats 106 42 group and more than two-fold in the gliclazide-treated rats, 107 43 compared to the control diabetic rats (Figure 4). 108 44 Chronic effects of the oyster mushroom on the lipid 109 45 profile were observed in the type 2 diabetic model rats. Both 110 the serum triglycerides (TG) and total cholesterol (Chol) Effect of the oyster mushroom on GLUT4 mRNA 46 expression in type 2 diabetic rats 111 47 were measured, but did not show any differences among the 112 48 control, the gliclazide-treated, or mushroom-treated groups 113 49 between day 0 and day 56 (Figure 2). GLUT4 mRNA was extracted immediately after the an- 114 50 imals were sacrificed using the TRIzol reagent. The cDNA 115 51 was prepared and amplified for the GLUT4 gene, using equal 116 52 Table 1: Fasting serum glucose level in different groups of type amounts of cDNA. Figure 5 shows the expression of GLUT4 117 53 2 diabetic model rats on day 0 and day 56 of the experiment. (glucose transporter type 4) as a ratio, relative to GAPDH 118 54 Groups Fasting glucose Fasting glucose (glyceraldehyde 3-phosphate dehydrogenase), which was 119 55 (mmol/l), day 0 (mmol/l), day 56 also similarly amplified. The ratio increased significantly in 120 56 121 CG (n ¼ 6) 9.15 1.24 (100%) 9.58 0.91 (106%) the mushroom-treated rats for the muscle and adipose tis- 57 GT (n ¼ 6) 10.01 1.44 (100%) *6.70 1.82 (67%) sues, compared to the control diabetic rats, but no significant 122 58 MT (n ¼ 6) 8.97 1.31 (100%) *6.40 1.41 (71%) difference was observed for the liver tissues. 123 59 124 60 Groups CG, GT, and MT represent the control group diabetic 125 Discussion 61 model rat, gliclazide-treated diabetic rat, and mushroom-treated 126 diabetic rat, respectively. Data are presented as the 62 127 mean standard deviation (M SD). Statistical comparison 63 Diabetes is a major public health problem worldwide, 128 between groups was performed using a paired sample t-test. 64 associated with serious complications and premature death, 129 *p < 0.05 compared to day 0 for the respective group. 65 due to the continuous damage, dysfunction, and failure of 130

Oyster mushroom stimulates p-AMPK and GLUT4 5

1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 79 15 80 Figure 3: Effect of oyster mushroom powder on p-AMPK in muscle tissues in type 2 diabetic model rats: M, marker; ND, non-diabetic rat 16 81 muscle; CG, control diabetic group; MT, mushroom-treated diabetic rat; GT, gliclazide-treated diabetic rat. Two hundred mg of total 17 82 protein from the muscle tissues was separated using SDS-PAGE, and p-AMPK was detected using western blot with a p-AMPK antibody. 18 83 Equal amounts of total protein were also analysed for the housekeeping protein b-actin. The band intensity of p-AMPK is expressed as a 19 b 84 20 bar diagram as ratios, relative to -actin. 85 21 86 22 87 23 88 24 89 25 90 26 91 27 92 28 93 29 94 30 95 31 96 32 97 33 98 34 99 35 100 36 Figure 4: Effect of oyster mushroom powder on p-AMPK in the adipose tissues of type 2 diabetic model rats: M, marker; ND, non- 101 37 diabetic rat; CG, diabetic control group; MT, mushroom-treated diabetic rat; GT, gliclazide-treated diabetic rat. Two hundred mgof 102 38 103 total protein from the adipose tissues of the different groups were separated using SDS-PAGE, and p-AMPK was detected using western 39 104 blot with a p-AMPK antibody. Equal amounts of total protein were also analysed for the housekeeping protein b-actin. The band in- 40 105 tensity of p-AMPK is expressed as a bar diagram as a ratio, relative to b-actin. 41 106 42 107 43 various organs. To prevent acute problems and to reduce the oyster mushroom powder, we attempted to analyse an 108 44 risk of long-term complications from diabetes, glycaemic intracellular protein (p-AMPK) and the mRNA of GLUT4. 109 45 monitoring, self-management education, support, and med- After 8 weeks of mushroom powder ingestion (5% of the 110 46 111 ications are often required.19 At present, there are no drugs usual feed), we sacrificed the animals in a fasting condition, 47 112 available that can cure the disease, and existing drugs are and the tissues and organs were immediately collected, 48 113 not complication-free for all individuals. Therefore, re- washed with ice-cold normal saline, and preserved at 70 C. 49 114 50 searchers are continuing their efforts to find new drugs for Tissue mRNA was extracted within a day, and proteins were 115 51 better management of the disease. However, diabetes is a extracted within the week of the sacrifice. Equal amounts of 116 52 metabolic disorder in humans, but not in animals. As such, extracted protein were analysed using SDS-PAGE and 117 53 development of a human-like, diabetic animal model for transferred to a PVDF membrane, and p-AMPK detected 118 54 experimental purposes is a major challenge. In this study, using western blotting. Equal amounts of protein were also 119 55 STZ-induced, type 2 diabetic model rats were developed, as analysed for the housekeeping protein b-actin, and the levels 120 16,17 56 previously described. As found in other studies in of p-AMPK were expressed as ratios, relative to b-actin, to 121 57 humans,20 we also found that the oyster mushroom minimize measuring or loading errors. This study showed 122 58 significantly reduced the blood glucose levels in model that p-AMPK was reduced in type 2 diabetic model rats, 123 59 diabetic rats. The study did not find any effects on body compared to non-diabetic animals, and that p-AMPK 124 60 weight or lipid profile levels with this regimen. In this increased significantly in the muscle and adipose tissues of 125 61 study, glycated haemoglobin, the lipid profile, and mushroom-treated diabetic model rats. As a positive control, 126 62 advanced glycation end-products were not measured, but we analysed the muscle and adipose tissues of gliclazide- 127 63 we plan to measure these in a future study. To obtain insights treated rats, and similar effects were observed. Existing 128 64 into the mechanisms behind the hyperglycaemic effects of literature suggests that activated AMPK releases GLUT4 129 65 130

6 M. Asrafuzzaman et al.

1 66 2 67 3 68 4 69 5 70 6 71 7 72 8 73 9 74 10 75 11 76 12 77 13 78 14 Figure 5: Effect of oyster mushroom powder on the expression of GLUT4 mRNA in type 2 diabetic model rats: 1, Marker; 2, Non- 79 15 80 diabetic rat muscle; 3, Control group rat muscle; 4, Mushroom-treated rat muscle; 5, Non-diabetic rat adipose tissue; 6, Water control 16 81 rat adipose tissue; 7, Mushroom-treated rat adipose tissue; 8, Non-diabetic rat liver; 9, Water control rat liver; 10, Mushroom-treated rat 17 82 liver; 11, Negative control. mRNA from different tissues was extracted using the TRIzol reagent, immediately after sacrificing the rats that 18 83 had been treated for 8 weeks with the mushroom powder. One mg of mRNA was used to prepare the cDNA using reverse transcriptase 19 m 84 20 PCR, and 3 l of cDNA was amplified by 35 cycles of PCR, using primers designed for both the GLUT4 and GAPDH genes. The band 85 21 intensity of GLUT4 is expressed as a ratio, relative to GAPDH. 86 22 87 23 88 24 from the micro-vesicles in the cytosol to organize in the Conflict of interest 89 25 membranes of muscle and adipose tissues, which helps 90 21 26 glucose enter the cells of those tissues. Therefore, it may be The authors have no conflict of interest to declare. 91 27 explained that the decreased hyperglycaemia in mushroom- 92 28 93 induced type 2 diabetic model rats could be due to Authors’ contributions 29 increased p-AMPK in the muscle and adipose tissues in these 94 30 animals. 95 MA: Animal experimentation, laboratory works (West- 31 This study also explored whether oyster mushroom 96 ern Blot) and Manuscript preparation. MMR: Lab works 32 ingestion affected the expression of the GLUT4 gene in the 97 specially in mRNA extraction, cDNA preparations and 33 muscle and adipose tissues of type 2 diabetic model rats. 98 34 PCR. MM: Band density of proteins and PCR products 99 Equal amounts of mRNA (1 mg in all cases) were used to 35 estimation and Result preparation. MM: Help in OGTT 100 prepare the cDNA; then, 3 ml of cDNA was used for 35 cycles 36 determination in model rats and tissue protein extraction. 101 of amplification using polymerase chain reaction (PCR). 37 AB: Rat feed management and performs OGTT experiments 102 Amplification of the housekeeping gene GAPDH was also 38 to select diabetic rats. BR: Diabetic model rat development 103 conducted, and expression of GLUT4 was expressed as a 39 and tissue collection from model rats. ZH: Experiment 104 ratio, relative to GAPDH. Our experiments showed that 40 design and Critical revision of the manuscript. MOF: Project 105 GLUT4 mRNA expression increased in both the muscle and 41 design, fund hunting, supervise all experiments and finalize 106 adipose tissues of oyster mushroom-treated type 2 diabetic 42 the manuscript. All authors have critically reviewed and 107 model rats. In a previous study, it was found that activation 43 approved the final draft and are responsible for the content 108 44 of AMPK regulates transcription of the GLUT4 gene in 109 22 and similarity index of the manuscript. 45 cultured human skeletal muscle cells. In another study, 110 46 mRNA expression of GLUT3, GLUT4, GLUT5, 111 47 GLUT10, and GLUT11 was poorly detected in different Acknowledgements 112 48 organs of pigs.23 However, in our study, we found ample 113 The authors greatly acknowledge the Ministry of Science 49 expression of GLUT4 in the livers of both model diabetic Q5 114 and Technology, Government of the People’s Republic of 50 and non-diabetic rats. Therefore, GLUT4 mRNA expres- 115 Bangladesh for their financial support and the Bangladesh 51 sion in rat liver tissues may not be affected by STZ or the 116 University of Health Sciences for providing the animals and 52 oyster mushroom; its expression is already in pick which may 117 laboratory space for this work. 53 explained that rat liver tissue may not be a problem for the 118 54 entry of glucose in diabetic condition. 119 55 Therefore, the hyperglycaemic effects of the oyster References 120 56 121 mushroom can be explained by the increased phosphoryla- 57 122 tion of AMPK and increased mRNA in muscle and adipose 1. Asrafuzzaman M, Cao Y, Afroz R, Kamato D, Gray S, 58 123 tissues. Little PJ. Animal models for assessing the impact of natural 59 products on the aetiology and metabolic pathophysiology of 124 60 e 125 Limitations Type 2 diabetes. Biomed Pharmacother 2017; 89: 1242 1251. 61 2. Hu FB. Globalization of diabetes: the role of diet, lifestyle and 126 62 genes. Diabetes Care 2011; 34: 1249e1257. 127 63 It would be better to understand the measured HbA1C, 3. Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking 128 64 glycation end-products, and the total antioxidant status of obesity to insulin resistance and type 2 diabetes. Nature 2006; 129 65 the animals. 444: 840e846. 130

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