Liver Diseases Are a Large Public Healthy Problem in the World(Guilluzo A

Evaluation of Antihyperlipidemic activity of Spilanthes radicans

M. PHARM DISSERTATION PROTOCOL SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

VIVEKANANDA S. B.Pharm.,

UNDER THE GUIDANCE OF

Mrs. N.C. NAGALAKSHMI M.Pharm, Assistant Professor MALLIGE COLLEGE OF PHARMACY, BANGALORE

MALLIGE COLLEGE OF PHARMACY #71 SILVEPURA, BANGALORE. 90

1 Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore. Annexure – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

Vivekananda S s/o Srikantaiah Y.L. Name and Address of the 01 Jaganatha Nilaya Candidate Agali(M), Madaksira(T), Ananthpur(D) Andrapradesh 515311 Mallige College Of Pharmacy #71 Silvepura, 02 Name of the Institution Chikkabanavara Post Bangalore 90

03 Course of the Study Branch M.Pharm (Pharmacology)

04 Date of Admission to course 23-11-2010

A Study on Antihyperlipidemic activity of 05 Title of the Topic Spilanthes radicans

Brief resume of the intended work Enclosure – I 6.1. Need for the Study 06 6.2. Review of the Literature Enclosure – II 6.3. Objective of the Study Enclosure – III

Materials and Methods 7.1. Source of data Enclosure – IV 7.2. Methods of collection of data Enclosure – V 7.3. Does the study require any 07 Investigations on animals? Enclosure – VI If yes give details 7.4. Has ethical clearance been obtained form your institution Yes in case of 7.3. 08 List of References Enclosure – VII

2 09 Signature of the Candidate (Vivekananda S.)

The present research work is original and not published in any of the journals with best of my 10 Remarks of the Guide knowledge upon extensive literature review. This work will be carried out in the Pharmacology laboratory by Mr. Vivekananda S. under my supervision. Name and Designation of (in Block Letters) Mrs. N.C.NAGALAKSHMI M. Pharm., 11.1. Guide Assistant professor Mallige College Of Pharmacy, Bangalore, Karnataka.

11.2.Signature

11 11.3.Co-Guide (if any)

11.4.Signature

11.5. Head of the Department Dr. SHIVAKUMAR SWAMY M. Pharm., Ph. D. Principal & HOD Mallige College Of Pharmacy, Bangalore, Karnataka

11.6.Signature

The present study is permitted to perform in the Remarks of the Principal Pharmacology laboratory of our institution and the study protocol has been approved by IAEC. 12

12.1. Signature (Dr. Shivakumar Swamy) Enclosure I

3 06. Brief resume of the intended work: 6.1 Need for the study: Hyperlipidemia is condition in which there is a elevated serum levels of one or more of total cholesterol, low-density lipoprotein cholesterol, triglycerides, or both total cholesterol and total triglycerides (combined Hyperlipidemia), very low density lipoprotein.1 Hyperlipidemia is a lifestyle disorder which seriously affects the human health.2 It leads to various cardiovascular disorders like angina pectoris, myocardial infraction, hypertension, atherosclerosis, congestive Heart failure.3 Hyperlipidemia always associated with diabetes mellitus.4 About 17 million people around the world die because of cardiovascular diseases every year.5 30% global deaths are due to cardio vascular disease of global deaths in which 80% deaths occur in low and middle income countries.6 The major treatment available for the hyperlipidemia includes statins, fibrates, bile acid sequestrents.7,8,9 The major side effects of antihyperlipidemic agents include muscle toxicity, rhabdomyolysis, psychiatric adverse reactions include depression, memory loss, confusion aggressive reactions.10,11These affect the lifestyle again. Hence it is the time to approach the hyperlipidaemia ethanopharmacologically.

Searching the safe and potent remedies from the herbal origin for the treatment of hyperlipidaemia and related cardiovascular disorders has become most fascinating and desired area of research for the pharmacologists. Literature review showed that some of the medicinal plants have been scientifically investigated and reported against various disorders.

Literature survey reveals that Spilanthes radicans is extensively used in ayurveda for variety of condition. However its antihyperlipidemic activity has not been investigated scientifically so far. Keeping in view of pathophysiological complications of hyperlipidaemia and therapeutic efficacy of herbal medicines, the plant Spilanthes radicans has been evaluated for antihyperlipidemic activity

Enclosure II

4 6.2 Review of literature:

Spilanthes radicans also known as Adormemuela, Indian food, quiebramuelas and is an indigenous herb of the family Asteraceae.12 It has been found that the other species of Spilanthes radicans like Spilanthes ciliate have hepatoprotective activity.13 Spilanthes acmella Murr. (Para-cress or toothache plant) is a medicinal plant of the Compositae family14 which is known in Thai as Phak-Kratt Huawaen and has long been used as a traditional medicine for toothache, headache and treatment of asthma, rheumatism, fever, sore throat and haemorrhoids.14,16 Its root decoction has been used as a laxative and diuretic drug15,17. It is an annual or short-lived herb, spreads in low open places and requires moist soil18. Its flowers and leaves have a pungent taste, accompanied with tingling and numbness15 and it is used as antiseptic, anti-bacterial, anti-fungal, anti-malarial and as remedy for toothache, flu, cough, rabies diseases and tuberculosis19. Other species like Spilanthes suffruticosa, Spilanthes ehrenbergii are used in ear ache, open fresh wounds, snake bites and ulcer 19, Spilanthes calva and Spilanthes paniculata have anti-malarial activity.20

Suja SR, Rajasekharan S, Pushpangadan P conducted the study on antihepatotoxic activity of Spilanthus ciliate. Pre-treatment of the rats with oral administration of the plant ethanolic extracts, Ixora coccinea, Rhinacanthus nasuta, Spilanthes ciliata prior to Aflatoxin B1 was found to provide signifcant protection against toxin-induced liver damage, determined 72 hours after the Aflatoxin B1 challenge (1.5 mg/kg, intraperitoneally) as evidenced by a signifcant lowering of the activity of the serum enzymes and enhanced hepatic reduced glutathione status. Pathological examination of the liver tissues supported the biochemical findings.12

Pandey V, Agrawal V, Raghavendra K, Dash AP conducted the study on antimalarial activity of three species of Spilanthes(Akarkara) against malaria (Anopheles stephensi Liston, Anopheles culicifacies, species C) and filaria vector .This is the first report of achieving cent percent lethality against these mosquito larvae using minimal doses of plant extracts from this or any other plant species.20

Chen Wu, Li-chen Wu, Nien-chu Fan, Ming-hui Lin et al. conducted the study on Anti-inflammatory Effect of Spilanthol from Spilanthes acmella on Murine Macrophage by Down-Regulating LPS-Induced Inflammatory Mediators Results of this study suggest that spilanthol, isolated from S. acmella, attenuates the lipopolysaccharide-induced inflammatory

5 responses in murine macrophages partly due to the inactivation of NF-κB, which negatively regulates the production of proinflammatory mediators.21

Ratnasooriya WD, Pierisb KPP, Samaratungac U and Jayakodya JRAC conducted a study on diuretic activity of Spilanthes acmella flowers in rats Spilanthes acmella also caused marked increase in urinary Na+ and K+ levels and a reduction in the osmolarity of urine suggesting that it is mainly acting as a loop diuretic. It may also inhibit ADH release and/or action. It is concluded that the Spilanthes acmella has strong diuretic action as is claimed.22

Ramesh Babu Kasettiet, Maddirala Dilip Rajasekhar, Vinay Kumar Kondeti, Shaik Sameena FatimaEthamakula conducted study on Antihyperglycemic and antihyperlipidemic activities of methanol:water (4:1) fraction isolated from aqueous extract of Syzygium alternifolium seeds in streptozotocin induced diabetic rats. A significant decrease in the activities of SGOT, SGPT, ALP and decreased levels of serum urea and creatinine in diabetic treated rats when compared to diabetic untreated rats, indicate the protective role against liver and kidney damage and non-toxic property of the fraction C. A comparison was made between the action of fraction C and antidiabetic drug glibenclamide (20 mg/kg.b.w). The effect of fraction C was more prominent when compared to that of glibenclamide.23

Tatiana Schoenfelder Pich CT, Geremias R, Avila S, Daminelli EN, Pedrosa RC, Bettiol J conducted a study on Antihyperlipidemic effect of methanolic extract of Casearia sylvestris. Casearia sylvestris methanolic extract was screened at doses of 125-500 mg/kg for its antihyperlipidemic activity. The antihyperlipidemic effect was evaluated in olive oil- loaded mice. Acute treatment caused inhibition in the triglyceride and serum lipase elevation- induced by 5 ml/kg of olive oil.24

Ebrahim Sajjadi S, Movahedian Atar A and Yektaian A conducted a study on Antihyperlipidemic effect of hydroalcoholic extract, and polyphenolic fraction from Dracocephalum kotschyi Boiss Blood lipid levels in rats with hyperlipidemia resulting from high-fat feeding were determined after oral administration of hydroalcoholic extract and polyphenolic fraction of Dracocephalum kotschyi leaves. Administration of the hydroalcoholic extract and polyphenolic fraction produced a significant decrease of blood triglyceride, total cholesterol and low density lipoprotien-cholesterol levels. High density lipoprotien-cholesterol level was significantly increased.25

6 Jeong-Sun Lee Song-Hae Bok, Seon-Min Jeon, Hye-Jin Kimet al. conducted a study on Antihyperlipidemic effects of buckwheat leaf and flower in rats fed a high-fat diet The result suggest that the beneficial effect of this buckwheat plant portion on plasma and hepatic lipid profiles in high-fat fed rats is partly mediated by higher excretion of faecal lipids and synergistic effect of phenolic compounds and fibre present in the Buck wheat Leaf and Flower.26

Distribution: It is widely found at elevations from 10 to 2,300 m, along the entire Pacific slope, and on both sides in the mountains of Tilarán, Central and Talamanca. Pacific Friend-ship, Arenal, Central Volcanic Cordillera, Guanacaste, She-bear,Central Pacific & Tempisque, from Mexico to Peru also.

Erect to prostrate herb, annual, up to 1 m. height, lower nodes often without roots. Sprigs with internodes 0.5 to 13 cm long. Leaves with petioles 0.2 to 2 cm. long, blade between 1 and 9 cm. long and 0.3 to 5.5 cm. wide, ovate to ovate-lanceolate, base obtuse, acuminate or acute at the apex, margin entire or short teeth.

Inflorescence terminal or auxiliary, solitary or paired heads. Heads discoid, with stems up to 7 cm. long, receptacle fusiform, up to 1.5 mm. diameter and 6 mm. high, flowers up to 2 mm. long, 5 lobed, achenes up to 1.5 mm. long, glabrous to hairy spread distally, ciliate on the margins, pappus consists of 2 fine bristles.

Acmella radicans is recognized for being an erect to prostrate herb with opposite leaves, veins or plinervada lost , heads discoid and solitary or pair of long stems, conical receptacle, paláceo and its achenes dimorphic, ciliate at the margin.12

Chemical Composition:

The composition of the essential oil of the fresh whole Acmella radicans(Jacq.) (Asteraceae) plant and two root-extracts from southern India were investigated by gas chromatogram phic–spectroscopic (GCFID and GC-MS) and olfactive methods to identify those volatiles responsible for the characteristic, pleasant aroma of these samples. The essential oil was found to be rich in 2- tridecanone (30.1%), 1-pentadecene (25.2%), trans-β- caryophyllene (12.8%), elemol (4.6%), guaiol (3.1%) and some further caryophyllene derivatives; the petroleum ether extract was rich in 1-pentadecene (54.1%), 2-tridecanone (10.3%), 2-pentadecanone (6.7%), 2-pentadecanol (4.1%), trans-β-caryophyllene (3.6%) and

7 1-tridecene (3.0%); and the petroleum ether–methanol–ethyl acetate extract was rich in 2- tridecanone (58.2%), 1-pentadecene (15.00%) and palmitic acid(16.6%).27

8 Enclosure III

6.3 Objectives of the study:

The present research work is an attempt to establish the curative antihyperlipidemic efficacy using 60% alcoholic extract of leaves and roots of Spilanthes radicans in rats with the following objectives.

1. To prepare 60% ethanolic extract of leaves and root of Spilanthes radicans.

2. To analyse the crude extract for the presence of phytoconstituents.

3. To carry out acute toxicity study of extract in mice.

4. To evaluate the antihyperlipidemic activity in animal model.

a) Diet induced hyperlipidemia

b) Fructose induced hyperlipidemia

9 Enclosure IV

7.1 Source of data:

1. Collection of raw material: For this study, the leaves and root of Spilanthes radicans will be collected from the surrounding gardens of Foundation for Revitalisation of Local Health Traditions. The sample will be identified by the botanist. Fresh leaves and root will be cleaned and dried at room temperature.

2. Pure drug Atorvostatin will be received as a gift sample by Microlabs Ltd.

Enclosure V

7.2 Method of collection of data:

1) Test for phytoconstituents: Then the powdered materials will be extracted with 60% hydro-alcohol by Soxhlet’s extraction method28. Thereafter, the extracts will be concentrated using rotary flash evaporator and percentage yield of the same will be recorded. The crude extracts thus obtained will be subjected to preliminary phytochemical screening following the standard procedures described in the literature.

2) Determination of acute toxicity: Fixed dose method29 (OECD guide line no. 420) of CPCSEA will be adapted to perform acute toxicity of the extracts. The Swiss Albino mice weighing between 20-25 g will be utilized for this purpose.

3) Screening of Antihyperlipidemic activity: Antihyperlipidemic activity of 60% hydro-alcoholic extract of leaves and root of Spilanthes radicans will be evaluated for the possible curative effects on fructose and diet induced in rats. Antihyperlipidemic efficacy of the test extracts will be assess by measuring the biochemical markers level such as serum triglycerides, serum total cholesterol, high density lipoproteins, low density lipoproteins, very low density lipoproteins will be determine in serum.

I Diet induced Hyperlipidemia30: High cholesterol diet produced a significant increase in the serum total cholesterol, low density lipoprotein, very low density lipoprotein, triglycerides. Healthy wister Albino rats of either sex weighing 180-220 g were divided into 7 groups each of six. Normal group receive standard pellet diet. All other groups received a high cholesterol diet along with respective treatments. Animals will be treated for seven days. Normal group receives standard chew diet and all other groups receive high cholesterol diet consisting of standard pellet diet 92%, cholesterol 2.0 %, cholic acid 1 % and coconut oil 5% for seven days.

11 Group 1: Normal control Group 2: Hyperlipidemic control Group 3: Standard drug Atorvastatin control Group 4: Administer root extract dose 1 Group 5: Administer root extract dose 2 Group 6: Administer leaf extract dose 1 Group 7: Administer leaf extract dose 2

II Fructose induced hyperlipidemia31: Forty two male Wistar rats weighing 150 to 200 gm were randomly divided into 7 groups of 6 each and kept in their cages for 5 days prior dosing to allow for acclimatization to the laboratory conditions. The chronic experimental hyperlipidemia will be produce by feeding fructose 66% once daily for 30 days. The drug is administered in constant volume of 1ml /100gm body weight. Normal group receive standard pellet diet and all other groups received a fructose along with respective treatments Group 1: Administer vehicle and serve as normal control. Group 2: Feed with fructose diet and serve as Fructose control. Group 3: Administer standard drug Atorvastatin orally Group 4: Administer root extract dose 1 orally Group 5: Administer root extract dose 2 orally Group 6: Administer leaf extract dose 1 orally Group 7: Administer leaf extract dose 2 orally

On day 31, animals are anaesthetized with pentobarbitone sodium (30 gm/kg i.p.) and blood is collect by cardiac puncture. The blood is subject to centrifugation to obtain serum. Serum will analyze for triglycerides, total cholesterol, high density lipoproteins, low density lipoproteins, very low density lipoproteins. Serum triglyceride Serum total cholesterol Serum High Density Lipoprotein cholesterol can be estimated by using Serum triglyceride diagnostic kit Serum total cholesterol diagnostic kit Serum High Density Lipoprotein cholesterol diagnostic kit respectively. Low density and very low density lipoproteins are estimated by Friede Wald equation32. Histopathological study of liver will be carried out33.

12 5) Statistical analysis: The data obtained from the above findings will be subjected to statistical analysis using two-way ANOVA followed by Turkey Kramer Multiple Comparison Test to assess the statistical significance of the results.

6) Work plan details: Total duration for the completion of proposed research work may be ten months 1. Collection of plant materials including authentication - One month. 2. Duration of experimentation on animals including - Five months. preparation of crude extracts 3. Literature collection - Two months. 4. Dissertation writing and communication of research -Two months.

Enclosure VI

7.3 Does the study require any investigation or interventions to be conducted on patients or other humans and animals? if so please describe briefly. The proposed study requires the investigation on albino rats of either sex (Wistar Strain) weighing 150 - 200 gm for the antihyperlipidemic activity. Whereas albino mice of Swiss Strain will be utilized for the acute toxicity study.

7.4 Has ethical clearance been obtained from your institution in case of 7.3? The present study protocol is applied for Institutional Animal Ethics Committee. On approval of study protocol IAEC certificate will be enclosed.

14 Enclosure VII References: 1. Prodigyguidance Online URL:http://www.prodigy.nhs.uk/guidance.asp? gtHyperlipidaemia: 12:2005.

2. Manjiri Arun, Nadkarni, Vyas SN, Baghel MS, Ravishankar B. Randomized placebo- controlled trial of Mustadi Ghanavati in Hyperlipidemia. An international quarterly journal of research in ayurveda 2010; 31(3): 287-293.

3. Grundy SM. Cholesterol and coronary heart disease a new era. Journal of American Medicine 1986; 256: 2849-2858.

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5. http://www.who.int/mediacentre/news/releases/2004/pr68/en/index.html Browsed on 14-5-2011 6. Naresh Terhan. Escort heart institute and research New Delhi India. www.chroniccareindia.org/document/NareshTarhan.pdf 7. Evans JR, Forland SC, Cutler RE. The effect of renal function on pharmacokinetics of gemfibrozil. J. Clin. Pharmacol 1987; 2:994-1000.

8. Lipid Research Clinics Program, The Lipid Research Clinics Coronary primary Prevention Trial results II, The relationship of reduction in incidencse of coronary heart disease to cholesterol lowering. JAMA 1984; 252:2545-2548

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15 12. Rodriguez A, Hammel B E, Zamora N, Grayum M H , Manual of Plants of Costa Rica. Missouri Bot. Louis and Inst Nac de Biodiversidad, Santo Domingo de Heredia: Costa Rica 2007; 4: 933

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14. Chopra RN, Nayara SL, Chopra IC. Glossary of Indian medicinal plants. Council of Scientific and Industrial Research, New Delhi 1956; 168–169.

15. Bunyapraphatsara N, Chokechareunporn O. Tradition medicinal plants. Prachachon; Bangkok: 1999.

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17. Perry LM, Metzger J. Medicinal plants of East and South-East Asia. MIT Press; Cambridge: 1980.

18. Wilkinson J. Herbs and flowers of the cottage garden. Book Builders: Melbourne 1989.

19. Ang Boon Haw, Chan Lai, Micropropagation of Spilanthes acmella L. a bio-insecticide plant, through proliferation of multiple shoots. Keng School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia. J Appl Hort 2003; 5:2: 65-68 .

20. Pandey V, Agrawal V, Raghavendra K, Dash AP. Strong larvicidal activity of three species of Spilanthes (Akarkara) against malaria (Anopheles stephensi Liston,Anopheles culicifacies, species C) and filaria vector. Parasitol Res 2007:102:171–174.

21. Chen Wu, Li-chen Wu, Nien-chu Fan, Ming-hui Lin, Inn-ray Chu, Shu-jung Huang, Ching-Yuan Hu et al. Anti-inflammatory Effect of Spilanthol from Spilanthes acmella on Murine Macrophage by Down-Regulating LPS-Induced Inflammatory Mediator. Agric Food Chem 2008; 56 (7): 2341–2349

22. WD Ratnasooriya K.P.PPierisb, U Samaratungac and J.R.A.C Jayakodya, Diuretic activity of Spilanthes acmella flowers in rats Journal of Ethnopharmacology 2004 ;91:(2- 3):317-32

16 23. Ramesh Babu Kasettiet Maddirala Dilip Rajasekhar, Vinay Kumar Kondeti, Shaik Sameena FatimaEthamakula, Guravaiah Tiruvenkata Kumar et al, Antihyperglycemic and antihyperlipidemic activities of methanol:water (4:1) fraction isolated from aqueous extract of Syzygium alternifolium seeds in streptozotocin induced diabetic rats. Food and Chemical Toxicology 2010;28:1078–1084. 24. Tatiana Schoenfelder Pich CT, Geremias R, Avila S, Daminelli EN, Pedrosa RC, Bettiol J, Antihyperlipidemic effect of methanolic extract of Casearia sylvestris Fitoterapia 2008;79; 465–467.

25. S Ebrahim Sajjadi, Movahedian Atar A and Yektaian A. Antihyperlipidemic effect of hydroalcoholic extract, and polyphenolic fraction from Dracocephalum kotschyi Boiss Pharmaceutica Acta Helvetiae 1998;73: 167–170. 26. Jeong-Sun Lee Song-Hae Bok, Seon-Min Jeon, Hye-Jin Kim, Kyung-Min Do, Yong-Bok Park and Myung-Sook Choi, Antihyperlipidemic effects of buckwheat leaf and flower in rats fed a high-fat diet Food Chemistry 2010;119: 235–240.

27. Leopold Jirovetz, Gerhard Buchbauer,George T Abraham, Mohamed P Shafi chemical composition and olfactoric characterization of Acmella radicans (Jacq.) RK Jansen var. radicans from southern India. Flavour and Fragrance Journal 2006;21:(1):88–91

28. William B, Jensen. The origin of Soxhelet Extractor. Journal of Chemical Education 2007; 12:1912-1984.

29. Veeraraghavan P. Expert consultant, CPCSEA, OECD Guideline no. 420.2000.

30. Yogendrasinh B Solanki, Rajendra V Bhatt. Effects of antioxidant vitamins along with atorvastatin and atorvastatin–niacin combination on diet-induced hypercholesterolemia in rats. Int J Physiol Pathophysiol Pharmacol 2010;2(1):57-63.

31. Mehdi Harati, Mohsen , Ani , Manoochehr , Messripour. Effect of Vandyl Sulfate on Fructose Induced Insulin Resistance Rat. Iranian biomedical journal 2003;7:4:179-182.

32. Venupamidiboina, Remarazdan, Hariprasad GM. Evaluation of the antihyperlipidemic, cardioprotective activity of a polyherbal formulation. www.ijppsjournal.com/Vol2Suppl1/385.pdf

17 33. Thamolwan Suanarunsawat, Watcharaporn Devakul N Ayutthaya, Thanapat Songsak, Suwan Thirawarapan and Somlak Poungshompoo Antioxidant Activity and Lipid- Lowering Effect of Essential Oils Extracted from Ocimum sanctum L. Leaves in Rats Fed with a High Cholesterol Diet Extracted from Ocimum sanctum L Leaves in Rats Fed with a High Cholesterol Diet. J Clin Biochem Nutr 2010;46:52–59.