Vidya (2018) Vol

Vidya (2018) Vol. No : 1

Prof. (Dr.) H. A. Pandya Vice-Chancellor Message

Holi, the festival of colours is here. It is a time, when people come together to clean their houses, streets, collect dry wood for Holika bonfire and then play together with colours on the next day - Dhuleti, leaving all their worries behind. It is also an occasion when people celebrate with energy irrespective of cast and creed, religion. Families come together, friends gather and they renew those dusted relationships. Holi gives us the message to forgive even our enemies and play with them, celebrate with them and make friends with them. Holika bonfire symbolizes an opportunity to burn all that is evil in us. People collect all the junk and put that into the bonfire that evening symbolizing that they are burning all that is eveil in their lives. As we celebrate Holi on March 1st this year, let's not just make it just another day to party and have fun. Let's take some of the life lessons Holi teaches us and enrich our lives with love, harmony and peace. Wishing all my students, staff, colleagues and their families a very joyful and colourful Holi.

1 Vidya (2018) Vol. No : 1

From the Editorial Desk

TOWARDS A BETTER ENVIRONMENT

Concern with the environment is imperative for us because we live from it; we live in it; and we live with it. By saying that ‘we live from it’ is meant that it provides us the means to our livelihood. It is the source of raw materials for productive activities. So much so that ‘we mine its resources; cultivate and harvest its fruits; shape the contours of the land for human habitation, roads, and dredge rivers for transport.We live in it means that ‘the environment is not just a physical precondition for human life and productive activity; it is where we lead our daily lives We live with the environment in the sense that the environment existed prior to our arrival and shall outlast us on earth. It is given to us, we have not created it. We are now realizing that the cause for environmental degradation is not need but greed, our natural and unnatural desire to accumulate more and more. According to experts the criterion of distinguishing between a natural or real, and a non-natural, artificial or contrived desire is that ‘natural desires are quenched easily: thirst by water, hunger by food. But the craving for possession is an artificial one; it goes on unceasingly and is never fully satisfied’. to revere traditional wisdom along with modern eco-friendly practices and learn to live in sync with the environment.

Prof. (Dr.) Meenu Saraf

"Sooner or later, we will have to recognise that the Earth has rights, too, to live without pollution. What mankind must know is that human beings cannot live without Mother Earth, but the planet can live without humans."

-Evo Morales

2 Vidya (2018) Vol. No : 1

Original Paper ISSN: 2321-1520

A record of White-rumpedvulture (Gyps bengalensis) nesting in Ahmedabad and Surendranagar districts of Gujarat.

Moradiya Mital and Jhala Devendrasinh*

*Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad. E-mail Id: [email protected]

*Corresponding Author

Received Date : 17-2-2018 Published Date : 7-3-2018

Abstract

Vultures are scavengers and flesh-eating raptors which soar at very high altitude as well as clean carcass dumping site efficiently. But unfortunately, vulture population have been declined globally as well as regionally due to the use of ‘diclofenac’ drug in veterinary. In India, after ‘diclofenac’ ban in year 2006 the declined of vulture population had slow down. On the other hand, to improve the breeding success and restore the damage that already been done to this K- selected population, it is very important to identify causes behind the breeding and nesting failure. In effort to identify reasons behind it we conducted this study and identify probable reasons of nesting failure. Moreover, tree preferences for nesting by White-rumped Vultures in Ahmedabad and Surendranagar were also evaluated and suggested that plantation of those trees near feeding sites may provide more nesting sites to these species. In this study we observed 58.69% nest failure at different stages and among these the nestling largely covered with white feathers/downs (Stage 3) was most vulnerable. Furthermore, during our repeated observations, it is inferred that the environmental and anthropogenic pressure have also induced nest failure during their growth so, consideration should also be given to it. Keyword:Vulture; Gyps bengalensis; Nesting success. Introduction

Vultures are one of the larger raptors that soar highest in the sky. They have coexisted with

3 Vidya (2018) Vol. No : 1 other species, including humans, since time immemorial. Congregating in large number over their feeding places, usually a dead carcass, vultures are known as efficient scavengers playing a crucial role in the recycling of nutrient in nature’s food webs (Pandey and Jethva, 2007). The White- rumped Vulture (WRV) Gyps bengalensis was once described as the commonest species of vulture found in the Indian sub-continent (Gilbert et al., 2006). The populations of vultures have been declined globally as well as regionally rapidly during the mid-1990s due to the contamination of ungulate carcasses with a veterinary drug ‘diclofenac’. But the decline of Gyps bengalensis in India has slowed after ban on veterinary use of drug ‘diclofenac’. Populations of Gyps bengalensis remained at a low level, but the decline had slowed and may even have reversed both in India and Nepal (Kamboj et al., 2016). However, estimates of the most recent population trends are imprecise, so it is possible that declines may be continuing, though at a significantly slower rate (Prakash et al., 2012; Kamboj et al., 2016). Hence, this study was carried out to know the nesting status of Gyps bengalensisin Ahmedabad and Surendranagar districts of Gujarat State.

Methodology and Study Area The observation of nesting of White-rumped Vulture was confined to Ahmedabad and Surendranagar districts of Gujarat State. The observations were made mainly during breeding season of the species. The nests of the species were surveyed extensively during October, 2014 to May, 2015 The colonies of WRV was located and identified. The coordinates of the location of each nest was recorded through GPS (Model: GARMIN eTrex Venture HC). Various parameters such as height of nests, tree girth and tree species were record using Bushnell (SIMMONS) Range Finder. Temperature and relative humidity measures by using Extech 45170 Hygro-Thermo- Anemometer-Light meter. Tree species used as substratum to build nest were also noted down. The observations were repeated at interval of 12 -15 days. To simplify the analysis of collected data, the nest was classified into 6 stages in this study. Stages of nests were coded as mentioned in the Table 1 and these coding was used to identify the stages at which nest failed. If nestling died in between any of the mentioned stages or if nest was destroyed then it is considered as nesting failure stage (NFS). Table 1: Proposed stages of nest in White-rumped Vulture.

Stage Proposed Stage Name Stage No. Code 1. Nest building stage NBS 2. Incubation stage INS 3. Nestling largely covered with white feathers/downs NWF 4. Nestling with dark wings but many white feathers visible on the NDW back and tail 5. Full grow n nestling FGN 6. Nestling flew out from the nest NFO

4 Vidya (2018) Vol. No : 1 Jhala & Moradiya Results

A total of 46 nests of White-rumped Vulture were recorded from the study area. Among them, 15 were in Ahmedabad district and 31 were in Surendranagar district. The nest building period was observed to be during October and November. The most used tree species as substratum for nest was Neem (Azadirachataindica) (21 nests), followed by Ambali (Tamarindusindica), Banyan (Ficusbenghalensis), Khijado (Prosopis cineraria), Peepal (Ficusreligiosa), Jamun (Syzygiumcumini), Palm (Borassusflabellifer) and Shirish (Albizialebbeck) (Fig. 1). Most of the nest build up on the edge of the tree and at top of the tree but very few were in the middle of the tree.

Figure 1: Tree species preferred for nest building by White-rumped Vulture in study area.

Out of the total 46 recorded nests, 27 nests were successful i.e. completed all six stages mentioned in Table 1, whereas reaming 19 nests were failed. In Ahmedabad, out of the total 15 nests only 8 nests showed successfully completion of all stages, whereas, 7 nests were failed. Likewise, in Surendrangar out of the total 31 nests, 19 nests were successful. It revealed that nesting success rate was 53% and 61% in Ahmedabadand Surendranagar respectively (Fig. 2). Beside nest failure and success record it was also noted that at which stage of nest the failure occurred. It was observed that the failure of nest was between stage 1 and stage 3 (i.e. NBS, INS and NWF). Among these three stages the maximum rate of failure (52.63%) was observed in stage 3 as compared to stage 1 (10.53%) and stage 2 (36.84%). On the contrary, none of the nest fail in stage 4 (NDW), 5 (FGN) and 6 (NFO) (Fig. 3).

5 Vidya (2018) Vol. No : 1

Figure 2: Nesting success ratio in Ahmedabad and Surendranagar.

Figure 3: Number of failure nest at particular stage (NBS = Nest building stage; INS = Incubation stage; NWF = Nestling largely covered with white feathers/downs). Discussion It is well-known that population of Gyps vultures in South Asia have been significantly decreased since the introduction of veterinary use of Diclofenac sodium in the 1990s (Prakash et al., 2003; Green et al., 2004; Prakash et al., 2007; Prakash et al., 2012). This is the single most important reason for the decline of vultures in India, Nepal and Pakistan. So, it is very important to know the current status and probable ways to increase the number of vultures. The data related to breeding and nest building in nature is also helpful to manage and improve the number of vultures. Among different vulture species, the nesting of Gyps bengalensis was evaluated during this study. 6 Vidya (2018) Vol. No : 1 Jhala & Moradiya

Present study divided the process of nestling development into six major stages to know the most fragile period during development. The present study recorded 46 nests of White-rumped Vulture built on 8 different tree species in Ahmedabad and Surendranagar districts of Gujarat. In these regions some dominating and tall trees are Neem, Ambali, Khiado, Banyan and Peepal on which White-rumped Vulture built nest. Generally, lofty and sparsely branched trees in the forest area were used by vultures for nesting and roosting. Such trees provide a better view of surroundings (Yamac, 2007). In our study most of the nests made up of dry twigs, sticks and leaves. White-rumped Vulture build nest during months of October and November on the edge or at the top of the tall trees like Neem, Ambali, Khiado, Banyan, Peepal, Jamun etc., because of their easy takeoff and landing flights. Hence,it is recommended that these tall trees should not be disturbed during months of October and November surrounding the feeding and traditional nesting sites. Moreover, trees of these species planted near feeding sites may provide habitat preference to the species which result into increase the number of White-rumped Vulture. By the observations of nesting success and failure, it was inferred that the maximum number of nests failed in the stage 3 (NWF) when nestling largely covered with white feathers/downs. It may be due to vigorous food consumption during this stage for their growth and less food availability in nearby area. So, parents were unable to fulfill the requirements of rapid growth of nestling during this stage leads to weakness and prone to infections of pathogens. As compared to previous records of Pandey and Jethva (2007) the number of nests decreased in Ahmedabad may be due to rapid urbanization and more human vulture conflict. On the other hand, number of nests increased in Surendrangar due to more food availability along with good vegetation of tall trees near feeding sites and less human vulture conflict. It was also possible that the fledging of White-rumped Vulture facing dehydration problem during summer (March to May) in the study area. Fledging which affected form dehydration they were weak and unable to fly easily and some had injuries as they fall down from their nests. Another important recommendation to prevent dehydration is to create a shallow artificial water bodies near vulture feeding and nesting sites. Conclusion Though the population of White-rumped Vulture is still declining since 1990s, the rate of decline is reduced. However, as the species is K-selected species, breeding success is also important for the recovery of the species in nature. The present study reveals that the nesting success was average about 57% collectively in Ahmedabad and Surendranagar districts, which means that it required species attention for the conservation of this scavenger. Among various stages the most important stage to take care was stage-3 i.e. Nestling largely covered with white feather/downs (NWF) due to high mortality was recorded in this stage. References Gilbert M, Watson RT, Virani MZ, et al. (2006) Rapid population declines and mortality 7 Vidya (2018) Vol. No : 1 cluster in three Oriental White-backed Vulture Gyps bengalensiscolonies due to diclophenac poisoning. Fauna and Flora International.40(4): 388–399. GreenRE, Newton I, Shultz S, et al. (2004) Diclofenac poisoning as a cause of vulture population declines across the Indian subcontinent. Journal of Applied Ecology.41(5): 793–800. Kamboj RD, Tatu K, Munjpara SB. (2016) Status of Vulture in Gujarat-2016.Gujarat Ecological Education and Research (GEER) Foundation, Gandhinagar. Pandey CN,Jethva B. (2007) Status of vultures in Gujarat state-2007. Gujarat Ecological Education and Research (GEER) Foundation, Gandhinagar. Prakash V, Pain DJ, Cunningham AA, et al. (2003) Catastrophic collapse of Indian white-backed Gyps bengalensis and long-billed Gyps indicus vulture populations. Biological Conservation. 109(3): 381–390. Prakash V, Green RE, Pain DJ, et al. (2007) Recent changes in populations of resident Gyps vultures in India. Journal of The Bombay Natural History Society. 104(2): 127–133. Prakash V, Bishwakarma MC, Chaudhary A, et al. (2012) The population decline of Gyps vultures in India and Nepal has slowed since veterinary use of diclofenac was banned. PLoS One. 7(11): e49118, 1-10. Yamac E. (2007) Roosting tree selection of Cinereous Vulture Aegypiusmonachusin breeding season in Turkey.Podoces. 2(1): 30–36. ❑

8 Vidya (2018) Vol. No : 1

Popular Article ISSN: 2321-1520

One Nation, One Tax – GST

Dr. H. C. Sardar

S D School of Commerce, Gujarat University, Ahmedabad. E-mail Id: [email protected] *Corresponding Author

Received Date : 10-2-2018 Published Date : 7-3-2018

Introduction Indian political system is three tier system i.e. Central government, State governments and local authority. At each level different types of taxes are levied. There are two types of taxes are being collected in India – Direct tax and Indirect Tax. Indirect taxes are collected by Central as well as State governments of the nation. Indirect taxes are those taxes which are indirectly levied on expenses incurred by the individual. Taxes like customs, excise, central sales tax, service tax were collected by central government and taxes like value added tax, professional tax, stamp duty tax, luxury tax were collected by state governments. Several Indirect taxes of central and state governments are subsumed in one tax i.e. Goods and services Tax. GST is emerged from limitations of indirect tax system. Indirect tax system had specific deficiencies like cascading effect, varied concessions and exemptions, lack of transparency multiple stages of tax collection, lack of uniformity, different provisions for goods and services in the context of taxes etc.To eliminate deficiencies of indirect tax system GST is introduced in place of several taxes of central and state governments so the uniformity can be maintained across the country. Benefits of GST are – reduction in multiplicity of taxes, relief in double taxation, efficient neutralization of taxes, development of common market, simple tax regime, simple tax mechanism, price reduction, uniform prices across the country, transparency in taxation system,benefit of input tax credit, benefits to small retailers, reduction in average tax burden, one nation one tax, strengthening economy of the country, uniform development of all states, reduction in accounting work etc. These benefits are availed by the traders and customers and assisting to establish good relation between central government and states and union territory governments. In GST different central and state level taxes are subsumed. It is very difficult task the conversion

21 Vidya (2018) Vol. No : 1 of several taxes of different governments in to one tax. There are several aspects of GST like history of indirect taxation, GST concept and origins, amendment of constitution for goods and services tax, GST council, definitions, administration, supply, registration, levy and collection of tax, composition levy, exemptions, input tax, output tax, input tax credit, rate of goods and services tax deducted at source, tax collected at source, refunds, assessment, compensation to states, accounts and records, offences and penalties, inspection, search, seizer, arrest and confiscation, reverse charge mechanism, export and import under GST and many more are carefully well designed. Only selected aspect of GST (i) concept of GST (ii) Rates of GST and Services and goods and services are covered under different tax rates (iii) Input tax credit (iv) Reverse charge mechanism and (V) GST council are discussed in brief in this paper. Good and Services Tax: "Goods and Services Tax "means any supply of goods or services or both except taxes on the supply of the alcoholic liquor for human consumption. Meaning of services here is anything other than goods, While goods has already been defined under article 366(12) in an inclusive manner, to provide that goods include all materials, commodities and articles. On 29-3-2017 Loksabha and on 6-4-2017 RajyaSabha passed the following four bills. 1. The Central Goods and Services Tax Bill, 2017 (CGST) 2. The Integrated Goods and Services Tax Bill, 2017 (IGST) 3. The Union Territories Goods and Services Tax Bill, 2017 (UTGST) 4. The Goods and Services Tax (compensation to states) Bill, 2017 (CGST) Goods and Services Tax of each state was to be passed by the respective state government. All state governments have passed their bills on different dates. Gujarat Government has passed GST Act on 9-5-2017. Rates of GST: An excellent arrangement of tax rates is made under GST regime. Keeping in mind nature, utility and need of goods and services these rates are determined by the concern authorities. Still process in transition period so on demand of public and government at its own is open to modify these rates. These tax rates determinations is based on two aspects (i) location of supplier and (ii) place of supply. With consideration of these two important factors the supply of goods and services are categorized in to two categories (i) Interstate supply and Intra State supply. Goods: In table Agoods related GST rates are shown.

22 Vidya (2018) Vol. No : 1 Sardar Table A Tax rates for Goods

Schedules Interstate supply of Intrastate supply of Goods and services and Goods and services Tax Rates (%) IGST (%) CGST (%) SGST/UTGST CGST + (%) SGST/UTGST (%) Schedule 1 5 5 2.5 2.5 5 Schedule 2 12 12 6 6 12 Schedule 3 18 18 9 9 18 Schedule 4 28 28 14 14 28 Schedule 5 3 3 1.5 1.5 3 Schedule 6 .25 .25 .125 .125 .25 Schedule 7 Nil Nil Nil Nil Nil

Following are tax rates and goods which covered under each tax rates. This list is not an exhaustive it is given for information purpose.

Tax Rates Goods 5% Meat/Fish (other than fresh/chilled) put up in a container and bearing a registered brand name, skimmed milk powder, chena or paneer put up in a container and bearing a registered brand name, singhada (dried whether or not shelled or peeled), mangoes (sliced, dried), grapes(dried and raisins), coffee(roasted), tea (whether or not flavoured, other than unprocessed green leaves of tea)mate, vanilla, cinnamon and cinnamon-tree flowers,cloves(whole fruit, cloves and steams) and many more. 12% Live horses, condensed milk, ghee/butter oil, branded cheese, brazil nuts, dates(soft or hard), citrus fruit, starches, pig fats, wool grease, edible mixtures, pasta, vegetables/fruits/ nuts/tomato/mushrooms(prepared or preserved by vinegar or acetic acid),jams, fruit juices, diabetic foods, soya milk drinks, printing/writing ink, tooth powder, candles, feeding bottles, plastic beads and many more.

23 Vidya (2018) Vol. No : 1

18% Malt(whether or not roasted), tendu leaves, Indian katha, glycerol, vegetable waxes, artificial honey, sugar confectionery, cocoa butter, cocoa powder, chocolates, malt extract, pastry, cakes, biscuits, ice cream, mineral water, non-alcoholic beverages, vinegar, marble/granite(other than blocks) and many more. 28% Pan masala, all goods(including aerated waters) containing added sugar/other sweetening matter/flavoured, cigars/cigarettes/manufactured tobacco substitutes, Portland cement, paints/varnishes/lacquers, glaziers’ putty, newpneumatictyres, air conditioning machines, refrigerators/freezers equipment, washing machines and many more. 3% Pearls(natural or cultured), diamonds, precious stones, gold, platinum, silver, jewellery (gold/silver)[not including bangles of lac/shellac] and others .25% Diamonds(industrial or non industrial, un-worked or simply sawn, cleaved or brutes, including unsorted diamonds) and others Nil Live sheep and goats, Fresh milk and pasteurized milk, including separated milk, milk and cream, not concentrated nor containing added sugar or other sweetening matter, excluding Ultra High Temperature (UHT) milk

Computation of GST: Preet limited of Pune, is a supplier of a X component within the state and outside the state. GST rate to his is 28%. Sales data for the month of January 2018 is as follows.

Date Recipient of supply Place of supply Amount of supply in Rupees January 5, 2018 A Jodhpur 12,00,000 January 12, 2018 B Nasik 15,00,000 January185, 2018 C Bombay 13,00,000 January 24,, 2018 D Delhi 28,00,000 January 30, 2018 E Chandiga rh 10,00,000

24 Vidya (2018) Vol. No : 1 Sardar Statement of GST liability of Preet limited for the month of January, 2018

Recipient Nature of supply (Place Taxable value IGST@ CGST @ SGST @ of of Supply) of goods 28% 14% 14% Supply A Jodhpur (Inter- state) 12,00,000 3,36,000 - - B Nasik (Intra State) 15,00,000 - 2,10,000 2,10,000 C Bombay (Intra State) 13,00,000 - 1,82,000 1,l82,000 D Delhi (Inter- state) 28,00,000 7,84,000 - - E Chandigarh (Interstate) 10,00,000 2,80,000 - - Total GST Liability 14,00,000 3,92,000 3,92,000

Note: Tax rate applicable to Preet limited is 28%. Irrespective of nature of supply company has to pay total tax @28%. If it is Interstate supply 28% IGST and if it is Intrastate than 14% CGST and 14% SGST are applicable. Services: In table B services related GST rates are shown. Table B Tax rates for Services

Categories Interstate supply of Intrastate supply of Goods and services and Goods and services Tax Rates (%) IGST (%) CGST (%) SGST/UTGST CGST + (% ) SGST/UTGST (%) Category 1 5 5 2.5 2.5 5 Category 2 12 12 6 6 12 Category 3 18 18 9 9 18 Category 4 28 28 14 14 28 Category 5 3 3 1.5 1.5 3 Category 6 .25 .25 .125 .125 .25 Category 7 Nil Nil Nil Nil Nil

25 Vidya (2018) Vol. No : 1 In case of services most of the services attracts 18% GST. A few services are taxable at the lower rate of 5% and 12%. Some services are taxable at a higher of 28%. These rates are briefly narrated as follows. In case of service tax for one service different tax rates are applicable. The reason behind this is that these kind services are further categorized into sub services by considering their nature. E.g. Accommodation, food and beverage service – Accommodation in a hotel/inns/guest house/club/campsites/other similar places for residential purposes having "declared tariff" a) Less than Rupees 1,000 per unit per day Nil Tax b) Rupees 1,000 or more but less than Rupees 2,500 per unit per day 12% c) Rupees 2,500 or more but less than Rupees 7,500 per unit per day 18% d) Rupees 7,500 or more per unit per day (Including 5 star accommodation) 28% Following are tax rates and services which covered under each tax rates. This list is not an exhaustive it is given for information purpose.

Tax Rates Services 5% Composite supply of works contract involving predominantly earth work (that is, constituting more than 75% of the value of the works contract) provided to the Central Government/State Government/Union Territory/Local authority/Government authority, Transport of passengers with or without accompanied belongings, by (a) rail in first class or air conditioned coach (b) air in economy class etc, Goods transport – transport of goods in vessels, 12% Composite supply of works contract (i.e. construction, repair, completion, maintenance, renovation etc) provided to Central Government/State Government/Union Territory/Local authority/Government authority, Goods transport – transport of goods in containers by rail any person other than rail, 18% Construction of complex, building, civil structure, services in retail trade, rental services of transport vehicles with or without operators, 28% Gambling, services provided by a race club by way of totalisator or a license to bookmaker in such club,

26 Vidya (2018) Vol. No : 1 Sardar

Nil Support services to agriculture, forestry, fishing, animal husbandry etc. Services provided by the Central Government, State Government, Union territory or local authority to another Central Government, State Government, Union territory or local authority: Provided that nothing contained in this entry shall apply to services(i) by the Department of Posts by way of speed post, express parcel post, life insurance, and agency services provided to a person other than the Central Government, State Government, Union territory; (ii) in relation to an aircraft or a vessel, inside or outside the precincts of a port or an airport; (iii) of transport of goods or passengers and many more.

GST council: GST council is a constitutional body to decide issued relating to GST. The union cabinet under the chairmanship of Prime Minister ShriNarendraModiapproved setting up of GST council on 12th September, 2016 and setting up the secretariat. The union finance minister who will be the chairman of the council. The union minister of state in charge of revenue or finance who will be the member of council. One member from each state who is minister in charge of finance or taxation or any other minister, any one of them will be vice chairman of the GST council who will be mutually elected by them. The secretary of revenue department will work as EX OFFICIO secretary to the GST council. The chairperson of central board of excise and custom will be the permanent invitee in all the proceedings of the GST council who will not have the voting rights. The role of GST council is very important because council brining all states on a common platform where issues pertaining to GST are to be resolved unanimously. GST council has to perform very crucial functions which would not create any kind of dissatisfaction amongst public, traders, central government and state governments including union territories.Article 279A (4) provides that GST council make recommendations to the central and state governments on: a) The taxes, cesses and surcharges levied by the union, the states and the local bodies which may be submitted in the goods and services tax, b) To decide exempted goods and services in the interest public, c) Formation of model tax laws for goods and services, principal of levy, apportionment of goods and services tax levied on applies in the course of inter-state trade or commerce, d) To decide the threshold limit of turnover below which goods and services may be exempted from this tax, e) To decide floor rated with bands of goods and services tax, f) To decide special provisions for stats – Arunachal Pradesh, Assam, J&K, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim, Tripura Himachal Pradesh and Uttrakhand,

27 Vidya (2018) Vol. No : 1 g) Any other matter relating to the goods and services tax, as council may decide. GST council has arranged several meetings after its formation to eliminate any kind of ambiguity, relaxation in GST and other related issues. So far 25 meetings are arranged by the council for hurdles free implementation of GST.Last 25th meeting of council was held at vigyanbhavan, New Delhi, on 18th January, 2018. Input Tax Credit: To eliminate the cascading effect of taxes and to minimize tax liability effectively, the concept of input tax credit is considered to be best mechanism to avoid double taxation on goods and services. Section 2(63) defines the expression ‘input tax credit’ as the credit of input tax. Section2(62) defines the expression ‘input tax’ in the relation to a registered person as the central tax, state tax, integrated tax or union territory tax charged on any supply of goods or services or both made to him. In brief the supplier of goods/services can avail credit of CGST/ SGST UTGST and IGST charge by input supplier of goods, services and capital goods. Utilization order of input tax credit of different GST is as under.

Sr. No. Balance of Utilization order of balance of GST GST 1 IGST (i) First will be utilized for payment of IGST (ii) any balance for payment of CGST and finally (iii) if any surplus remains can be used for payment of SGST/UTGST. 2 CGST (i) First will be utilized for payment of CGST (ii) any balance for payment of IGST and finally (iii) if any surplus remains cannot be used for payment of SGST/UTGST. 3 SGST/UTGST (i) First will be utilized for payment of SGST/UTGST (ii) any balance for pa yment of IGST and fi nal ly (i ii) if any surplus remains cannot be used for payment of CGST.

How and in which order input tax credit balance will be utilized by the company for eliminating cascading effect of taxes, is explained as follows. On 21st January, 2018 X company limited of Mumbai has supplied goods its customer of Pune for Rs. 53,60,000. On the same day sent goods of Rs. 10,00,000 to its customer of Chennai. Applicable GST rate is 18%. X Company limited has the following balances its electronic

28 Vidya (2018) Vol. No : 1 Sardar credit ledger. (i) IGST Rs. 1,84,000 , (ii) CGST Rs. 10,000 and (iii) SGST Rs. 18,00,000. Statement of Total GST (Company limited – Mumbai) (Tax paid on Input)

Particulars Taxable IGST @ CGST @ SGST @ Value (Rs) 18% 9% 9% a) Supply to Pune customer 53,60,000 --- 4,82,400 4,82,400 (Intrastate supply) b) Supply to Chennai customer 10,00,000 1,80,000 ------(Interstate supply) Total Payable GST 1,80,000 4,82,400 4,82,400

Statement of Utilization of Input tax credit and Net Payable GST

Particulars IGST (Rs) CGST(Rs) SGST(Rs) Tax on output supply 1,80,000 4,82,400 4,82,400 Less: IGST Balance (Utilization of Input tax Credit) 1,80,000 4,000 --- (See utilization order ) --- 4,78,400 4,82,400 Less: CGST Balance (Utilization of Input tax Credit) --- 10,000 ---- (See utilization order ) --- 4,68,400 4,82,400 Less: SGST Balance (Utilization of Input tax Credit) ------4,82,400 (See utilization order ) Net GST Payable --- 4,68,400 ---

X Company is liable to pay Output tax of Rs. 11,44,800 (IGST1,80,000+4,82,400+4,82,400) but input tax credit can used of Rs 6,76,400 (Rs.1,80,000+4,000+10,000+482400) (which are paid at the time acquisition of goods), so it will be deducted from Rs 11,44,000 net amount of payable GST will be Rs. 4,68,400. In brief to be for output Rs. 11,44,800 and paid at the time input acquisition Rs. 6,76,400

29 Vidya (2018) Vol. No : 1 (On the basis of conditions of input credit utilization) thus net payable amount is of Rs. 4,68,400. Reverse Charge Mechanism: Generally, supplier of goods and services is liable for payment of GST to the government. In a few cases, however, the recipient of goods/services is liable for payment of GST. There are two sections – section 9(3) and section 9(4) under which recipient are liable to pay GST. As per section 9(3) supply of cashew nuts, not shelled or peeled, Bidi wrappers leaves (tendu), Tobacco leaves, Silk Yarn, Raw Cotton, Supply of lottery, supply of services by goods transport agency(based on certain conditions), services provided by an individual advocate(including a senior advocate) or firm of the advocates by way of legal services, directly or indirectly, services provided by an arbitral tribunal, services provided by way of sponsorship, services supplied by government, services provided by the a director of a company or a body corporate to the said company or the body corporate, services supplies by an insurance agent to any person carrying on insurance business, services supplied by recovery agents, supply of services by an author, music composer, photographer, artist or the like by the way of transfer or permitting the use or enjoyment of a copy right relating to original literary, dramatic, musical or artistic works to a publisher, music company, produces or the like etc(Where nature of supply and recipient of supply for goods and services are specified.) As section 9(4) provides that GST in respect of the supply of taxable goods/services by a supplier, who is not registered, to a registered person shall be paid by the recipient under reverse charge mechanism and all GST provisions shall apply to such recipient as if he is the person liable for paying GST in relation to the supply of goods and services. So, whenever a registered person procures supplies from an unregistered supplier, he needs to pay GST as per reverse charge mechanism. Conclusion The elimination of different taxes and formation one tax in the nation is result of efficient and effective steps of central government and state governments. It is an achievement of the nation. GST is passing through transition period. It has certain limitations. In this regard GST council is working very sincerely and after full implementation of GST all concerned stakeholders will be happy with simple, easy and comfortable indirect taxation system. ❑

30 Vidya (2018) Vol. No : 1

Original Paper ISSN: 2321-1520

Health Risk Assessment on Women Rag Pickers of Ahmedabad

Divya Fulwani, Dr. Divya Chandel* Professor, Department of Zoology, BMT & Human Genetics, Gujarat University, Ahmedabad. E-mail Id: [email protected]

*Corresponding Author

Received Date :9-2-2018 Published Date : 7-3-2018

Abstract Waste is an unavoidable by-product of human activities.If accumulated, it leads to degradation of urban environment, stresses natural resources and leads to health problems.Rag pickers play an important but usually unrecognized role in the waste management system of Indian cities. They collect garbage in search of recyclable items that can be sold to scrap merchants (paper, plastic, tin, etc.).This paper presents a study on the women rag pickers of Ahmedabad city with focus on their socio-economic and occupational health aspects. The data has been developed through questionnaire survey by filling up of performa and hematological and oxidative stress analysis on peripheral blood. The results indicate health risks on women rag pickers at the dumping site of Ahmedabad city. Keywords:Rag pickers, Occupational health hazards, Complete Blood Count, Oxidative Stress Introduction Rag picking is one of the inferior economic activities in the urban informal sector, largely undertaken by Women and Children belonging to socio-economically backward sections of the society for their survival and for supplementing their family income. These individualsare not educationally equipped or skillful and thus by way of refuse collection contribute to household income or own survival.Rag pickers are subjected to various types of chemical poisons and infections. Because of malnutrition they suffer from retarded growth and anemia. The rag pickers are highly

31 Vidya (2018) Vol. No : 1 susceptible to diseases like tuberculosis and cancer due to their exposure to hazardous materials and also addiction to chewing and smoking low grade tobacco. Illiteracy and low social upbringing along with strive for survival make them alcoholics and then they switch to hard liquors and drugs. Inferior personal hygiene and street lives more often make them victims to HIV-AIDS. Hence, this study was undertaken to estimate the health risks in female rag pickers of Ahmedabad, Western Zone of Indian Subcontinent. Materials and Methods In Ahmedabad there are an estimated 30,000 waste pickers anda large proportion of them are women and children. Overall in the state of Gujarat there are estimated to be over 100,000 waste pickers.The presentstudy has been approved by Institutional Ethical Committee and only female rag pickers working on the dumping sites were enrolled after taking written informed consent.A total of 70 women exposed to various dumping sites over a period of three years were enrolled. Age matched women working around the Gujarat University area from similar socio- economic background were recruited as controls (50 individuals). All the subjects were explained purpose and nature of the study. Those who had any severe disease/infections were excluded. Medical examination was performed by doctor and specific points were noted in the Performa. Blood samples were collected by technical staff with no potential risk and were tested for various haemotologicaland oxidative stress parameters. In the present study, 7ml of blood sample was taken out of which 2ml was transferred in 2.0ml EDTA vial and samples were analyzed in fully automated Haemotolyzer. A copy of report was given to respective control and exposed subjects for their future treatment. Remaining 5ml of sample was used to separate serum from the sample for performing various oxidative stress parameters.All calculations were confirmed by software Graph-Pad prism-6. Statistical analysis was done using ‘Chi-square test’ and Student’s t-test. 1. Hematological Study: Haemoglobin, Red Blood Cell Count, Red Cell Distribution Width, White Blood Cell Count, Eosinophils, and Platelet count. 2. Oxidative stress analysis: Superoxide Dismutase (Sod), Kakkar et al. (1984). Catalase (CAT),Sinha and Ashru (1972). Glutathione Peroxidase (Gpx),Rotruck et al. (1973). Glutathione reductase (GR), Carlberg and Mannervik (1975). Glutathione-S-transferase (GST),Habig et al. (1974). Glutathione (GSH),Rotruck et al (1973). Lipid Peroxidation (LPO),Ohkawa et al. (1979). Total Protein, Lowry et al. (1951).

32 Vidya (2018) Vol. No : 1 Chandel & Fulwani Results Table 1 shows the family size of the subjects’ andreveals that 41% of exposed workers have large families as compared to Control group who majorly belong to small (54%) and medium (42%) families and the results were highly significant (P<0.0001).As per this study, 63% of the exposed women, had alcoholic father or husband or male member of the family which is significantly (P<0.01) above the Control women (38% have father/husband alcoholic). Many Exposed individuals (70%) had significantly (P<0.0001) higher number of injuries through needle/syringe, dog bites, harsh chemicals, metal scraps etc. as compared to Control subjects (26%). Only 23% of Exposed individuals use personal protective equipment like gloves, masks and tools for rag picking which is non-significant as compared to Control (38%) and this increases their chances of injuries and infections. Dietary habits reveal that 87% of Exposed subjects eat mixed food (vegetarian and non- vegetarian) as compared to Controls (28%).Results show that only 41% of Exposed subjects use Private toilet as compared to Control (68%). The rhythm of menstruation is significantly irregular (P<0.01) in Exposed (64%) as compared to Controls (30%). Table 2shows non-significant decrease in number of Exposed subjects (44%) having less than 12 g/dl of Haemoglobin (Hb) as compared to Control subjects (52%). Amongst the exposed subjects, 2% individuals showed abnormal increase in Hb (more than 15 g/dl) which was not observed in Control subjects. The results show 3% of Exposed subjects showing less than normal values of Red blood cell (RBC) count as compared to 6% Controls. Only 65% of Exposed showed normal RBC count as compared to Control 72%. The exposed subjects showed abnormally high RBC count (32%) as compared to Controls (22%).The Red cell distribution width (RDW) levels showed significantly increased values (P<0.05) in Exposed (48%) as compared to Controls (28%). The results showed non-significant increase in Exposed subjects (26%) having high values of White blood cell (WBC) count as compared to Controls (20%). 2% of Control subjects showed decrease in WBC count which is not observed in Exposed subjects.The Table 2 indicates highly significant increase (P < 0.0001) in Exposed individuals (46%) having high eosinophils as compared to Control subjects (10%).The table shows few numbers of subjects (2% Control and 2% Exposed) having low and other few (8% Control and 4% Exposed) having high platelet count which is non- significant (‘Chi-square test’). Table 3Shows results of Oxidative Stress Assay performed on Control and Exposed subjects with Mean ± Standard Error (S.E) for all parameters. These parameters were found to decrease significantly. Theactivity of enzymes like Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), Glutathione (GSH), Glutathione Reductase (GR)and Glutathione-S-Transferase (GST) in samples of Exposed subjects as compared to Controls were much lower. There was highly significant increase in Lipid Peroxidation (LPO) of Exposed subjects as compared to Controls. Total serum Protein (TP) was found to be decreased with high significance according to Student’s t-test in Exposed subjects as compared to Controls.

33 Vidya (2018) Vol. No : 1 Table 1: Showing General Characteristics of Control and Exposed Subjects (Chi- square test). Categories Control (n = 50) Exposed (n = 70) Size of family (***) Small (1-5) 27 (54%) 15 (22%) Medium (6-8) 21 (42%) 26 (37%) Large (9 and above) 2 (4%) 29 (41%) Father/Husband Alcoholic (***) Yes 19 (38%) 44 (63%) No 31 (62%) 26 (37%) Injury during work (***) Yes 13 (26%) 49 (70%) No 37 (74%) 21 (30%) Use of Masks/Gloves (ns) Yes 19 (38%) 16 (23%) No 31 (62%) 54 (77%) Dietary habits (***) Vegetarian 36 (72%) 9 (13%) Mixed 14 (28%) 61 (87%) Use of toilet (**) Public 16 (32%) 41 (59%) Private 34 (68%) 29 (41%) Rhythm of menstruation (**) Regular 35 (70%) 25 (36%) Irregular 15 (30%) 45 (64%)

P > 0.05 (ns), P < 0.05 (*), P < 0.01 (**), P < 0.001 (***)

34 Vidya (2018) Vol. No : 1 Chandel & Fulwani Table 2: Showing Haematological Parameters in Control and Exposed Subjects (Chi- square test). Complete Blood Count (CBC) Categories Control (n = 50) Exposed (n = 70) Haemoglobin (Hb) (ns) < 12 26 (52%) 31 (44%) 12 -15 (Normal range) 24 (48%) 38 (54%) > 15 0 1 (2%) Red blood cell (R.B.C) Count (ns) < 3.8 3 (6%) 2 (3%) 3.8 – 4.8 (Normal range) 36 (72%) 46 (65%) > 4.8 11 (22%) 22 (32%) Red cell distribution width (RDW) (*) 11.6 – 14 (Normal range) 14 (28%) 34 (48%) > 14 36 (72%) 36 (52%) White blood cell (WBC) Count (x 1000) (ns) < 4 1 (2%) 0 4 – 10 (Normal range) 39 (78) 52 (74%) > 10 10 (20%) 18 (26%) Eosinophils (***) 0 – 7 (Normal range) 45 (90%) 38 (54%) > 7 5 (10%) 32 (46%) Platelet Count (x 10000) (ns) < 15 1 (2%) 1 (2%) 15 – 40 (Normal range) 45 (90%) 66 (94%) > 40 4 (8%) 3 (4%)

P > 0.05 (ns), P < 0.05 (*), P < 0.001 (***)

35 Vidya (2018) Vol. No : 1 Table 3: Showing Oxidative stress analysis among Control and Exposed Subjects (Student’s t-test).

Sr. No. ROS Parameters Control (n=50) Exposed (n=70) 1. Superoxide Dismutase (SOD) 20.76 ± 0.85 17.69 ± 0.63*** 2. Catalase (CAT) 23.40 ± 0.63 20.44 ± 0.43*** 3. Glutathione Peroxidase (GPx) 58.98 ± 0.44 49.19 ± 0.32*** 4. Glutathione (GSH) 72.97 ± 0.72 57.49 ± 0.93*** 5. Glutathione Reductase (GR) 22.60 ± 0.41 18.07 ± 0.38*** 6. Glutathione-S-Transferase (GST) 21.10 ± 0.3 18.19 ± 0.26*** 7. Lipid Peroxidation (LPO) 14.78 ± 0.62 22.02 ± 0.53*** 8. Total Protein (TP) 1.20 ± 0.038 0.839 ± 0.033 ***

Values are Mean ± S.E. Protein content (mg protein/100μl serum) ; LPO, lipid peroxidation (NM MDA/100μl serum); GSH, total glutathione (nM total GSH/100μl serum); SOD, superoxide dismutase (units of SOD/100μl serum); CAT, catalase (nMH2O2/100μl serum); G-Px, glutathione peroxidase(mM GSH/100μl serum); G-Rd, glutathione reductase (nM NADPH oxidized/100μl serum); GST, Glutathione-s-Transferase (μMoles CDNB-GSH conjugate/100μl serum). ***p < 0.001 Discussion According to findings of present study, the rag pickers are exposed to harsh weather conditions surrounded by stray animals and infectious solid waste that may induce them to many diseases.Rag pickers (Control and Exposed group) develop addictions of chewing pan, tobacco and gutkha which act as confounding factors and could further aggravate the effects of occupational exposure.Most of them have alcoholic father/husband which gives family women more burden to earn for the family.Inhalation of noxious gases leads to cough, nasal irritation, dimness of vision and throat congestion amongst the rag pickers.The rag pickers do not use protective masks or gloves for waste- picking,instead, they use their bare hands and thus often get sick and injured. Lack of precautionary safety measures and unawareness regarding health were the main causes of deteriorated health conditions.As most of them use public toilets, they often get urinary tract infections (UTIs) and this may be also a reason for getting frequent fever. Also, most of rag pickers follow home remedies and do not consult doctor due to their ignorance of risks and low finances. Results show more irregular rhythm of menstruation in Exposed subjects. This may be due to hormonal imbalance, too much exertion, Polycystic ovary syndrome (PCOS), stress, Overactive thyroid (hyperthyroidism) or underactive thyroid (hypothyroidism), Uterine fibroids etc. Complete Blood Count analysis: Results showed more number of Exposed subjects having less than 12 g/dl of Hb. There are

36 Vidya (2018) Vol. No : 1 Chandel & Fulwani many reasons for anemia such as loss of blood, nutritional deficiency (iron, vitamin B12, and folate), kidney failure, and abnormal hemoglobin structure (sickle cell anemia or thalassemia).High Hb levels (as observed in exposed subjects) indicate conditions like advanced lung disease (eg. emphysema), certain tumors and premature RBC death. The reason for elevated Hb levels of exposed women rag pickers can be due to their mixed diet.Non-significant increase in RBC counts of subjects especially in Exposed individuals indicate conditions like poor heart or lung function, abnormal RBC breakdown (leads to elevated haemoglobin) and hypoxia.The results showed significant increase in number of Controls and Exposed having high RDW levelswhich could happen because of iron deficiency anemia or thalessemiaintermedia (Capanzana et al. 2017). Higher numbers in both Control and Exposed subjects indicate greater variation in cell size.High levels of WBC count in Exposed subjects may be due to leukocytosis which is triggered by infections, tissue damage, asthma, tuberculosis, smoking, leukemia, stress, immune system disorders.The result indicates highly significant increase in Exposed individuals having high eosinophils as compared to Control subjects. Exposed individuals showed higher-than-normal level of eosinophils which can lead to eosinophilia which is caused by allergies, asthma, inflammatory conditions, parasitic infections, reactions to medications etc. (Ayik et al. 2003).Elevated platelet count in few subjects (thrombocytosis) can be correlated with conditions like Cancer, Chronic Myeloid Leukemia (CML) along with multiple infections, while low platelet count (thrombocytopenia) in some others point towards conditions like decrease of platelets in bone marrow or platelets being destroyed in blood stream. Oxidative Stress Analysis: Oxidative stress and free radical production are involved in the etiology of various chronic conditions, including cancer, atherosclerosis, neurological and cardiovascular diseases and exposure to toxic conditions.The results showed increased Oxidative stress through significantly decreased activity of cellular anti-oxidant enzymes like Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), Glutathione (GSH), Glutathione Reductase (GR) and Glutathione- S-Transferase (GST) in serum samples of Exposed subjects as compared to Controls. There was highly significant increase in Lipid Peroxidation (LPO) of Exposed subjects as compared to Controls. Increased LPO is a clear indication of damaged membranous organelles of the cell. Total serum Protein (TP) was found to be increased significantly in serum samples of Exposed subjects as compared to Controls. Elevated total protein in Exposed samples may indicate conditions like inflammation or infections, such as viral hepatitis B or C, or HIV, bone marrow disorders, such as multiple myeloma or Waldenstrom’s disease. Further, occupational exposure can induce DNA damage through enhanced oxidative stress and free radical generation in the cells. Also, phagocytic cells associated with inflammation are a major source of oxidative stress to the biological system (Yang et al., 2001).Study on Municipal Solid Waste (MSW) handlers showed a significant decrease in the enzymatic antioxidants like SOD and CAT as well as the non-enzymatic redox sensitive thiol compound, reduced GSH (Odewabi and Ogundahunsi et al., 2012).The rag pickers deal with lot of plastic materials which can also increase the oxidative stress levels. Free radicals have been implicated in pathways of metabolism of drugs and environmental chemicals(Sati et al., 2011). Conclusion The results clearly showed the damage that has occurred to the women rag pickers due to

37 Vidya (2018) Vol. No : 1 the occupational exposure, which if not cured or taken care of can lead to various ailments in them and in their children as they are also present at dump site for long hours and exposed to unhealthy conditions. The CBC analysis showed high prevalence of Eosinophilia in Exposed subjects as compared to Controls which is caused by allergies, asthma, inflammatory conditions, parasitic infections and reactions to medications. The increased Oxidative stress through decreased activity of cellular anti-oxidant enzymes in serum samples of Exposed subjects as compared to Controls was observed, which is a clear indication of damaged membranous organelles of the cell.They were advised, counselled and provided medicines on basis of their reports at regular intervals of the study period. They were also given safety and precautionary measures (gloves/masks) at the health camps arranged by respective Municipal Counsellor of that area. Further research is needed to develop some natural/herbal antioxidant therapy which can reduce the long term harmful effects of high oxidative stress on these rag pickers. References Ashru, K. Sinha, (1971). Determination of catalase activity in white blood. AdvEnzymolRelat Areas MolBiol, 27:380. Ayik, S. Ö., BASO“LU, Ö. K., Erdinc, M., Bor, S., Veral, A., and Bílgen, C. (2003). Eosinophilic bronchitis as a cause of chronic cough. Resp med, 97(6):695-701. Capanzana, M. V., Mirasol, A., Smith, G., Angeles-Agdeppa, I., Perlas, L., de los Reyes, F., &Amarra, M. S. (2017).Thalassemia and other hemoglobinopathies among anemic individuals in Metro Manila, Philippines and their intake of iron supplements. Asia Pacific Journal of Clinical Nutrition. Carlberg, and Mannervik, (1975).Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem., 250(14):5475-5480. Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974).Glutathione S-transferases the first enzymatic step in mercapturic acid formation. J. Biol. Chem., 249(22):7130-7139. Kakkar, P., Das, B., and Viswanathan, P. N. (1984).A modified spectrophotometric assay of superoxide dismutase.NISCAIR Online Periodicals Repository.21(2):130-132. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951).Protein measurement with the Folin phenol reagent. J bio. Chem., 193(1):265-275. Odewabi, A. O., Ogundahunsi, O. A., Ebesunu, M. O., and Ekor, M. (2013). The levels of inflammatory markers and oxidative stress in individuals occupationally exposed to municipal solid waste in Ogun State, South West Nigeria. Toxicol Ind Health, 29(9):846-855. Ohkawa, H., Ohishi, N., and Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analybiochem., 95(2):351-358. Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G., & Hoekstra, W. (1973). Selenium: biochemical role as a component of glutathione peroxidase. Science, 179(4073), 588-590. Sati, P. C., Khaliq, F., Vaney, N., Ahmed, T., Tripathi, A. K., and Banerjee, B. D. (2011). Pulmonary function and oxidative stress in workers exposed to styrene in plastic factory: occupational hazards in styrene-exposed plastic factory workers. Hum ExpToxicol., 30(11): 1743-1750. Yang, C. Y., Chang, W. T., Chuang, H. Y., Tsai, S. S., Wu, T. N., and Sung, F. C. (2001). Adverse health effects among household waste collectors in Taiwan. Env. Res., 85(3):195-199.

38 Vidya (2018) Vol. No : 1

Review Article ISSN: 2321-1520

MurrayakoenigiiSpreng.- A plant with blessings

Jatav Rinki*, Ancy Fernandes and Archana Mankad

*Department of Botany, Gujarat University, Ahmedabad. E-mail Id: [email protected]

*Corresponding Author

Received Date : 26-12-2017 Published Date :7-3-2018

Abstract Medicinal plants are the nature’s gift for human beings. From ancient time plants have been utilized by humans for food as well as for healthcare though there are well developed technologies and synthetic drugs, the use of medicinal plants always remain at the top due to their safety, easy availability and low cost. MurrayakoenigiiSpreng., belongs to family Rutaceae, is one of the "Magical Plant of Indian spice" which has served not only as food enhancer but also as village or folk medication to cure many disorders. The tribal communities have reported to use many parts of the Murrayakoenigiito cure various ailments. A number of chemicals present inside the plant interact to elicit their pharmacodynamic response. The plant is highly valuable with various pharmacological activity and some of which have been scientifically proved. The compound carbazol alkaloids are responsible for substantial medicinal activities. Keywords- MurrayakoenigiiSpreng.,Pharmacodynamic responses, nature’s gift Introduction Since ancient times, plants are been utilized by humans for various purposes. The ability of plants to overcome various ailments has led human beings to use them as medicinal plants. In present era medicinal plants have become an enormous part in supporting healthcare system of world. According to a bulletin of World Health Organization (WHO, 2001) 80% the population in developing countries still rely upon medicinal plants for their primary healthcare needs (AK Shakya, 2016; AKMurugan. et. al., 2017 ). Among the mega biodiversity countries of the World, India possesses a historical track record of having made asignificant global contribution due to the traditionalknowledge of the medicinal plants (AKMurugan. et. al., 2017). Medicinal properties of plants does not restrict to a specific part; any part of the plant such as root, stem, bark, flower, leaves or whole plant etc. that can be used to cure diseases. They also form the essential raw 39

Vidya (2018) Vol. No : 1 Jatav et al

Morphological Characteristic Fig.1 Shows the whole plant of Murrayakoenigii(a) leaves (b) flower (c) and fruits (d) a) Stem:More or less deciduous shrub or small tree, growing 6m tall, with slender but strong woody stem having 14-42 cm diameter and covered with dark grey bark. The stem is dark green to brownish in color, with numerous dots on it (KK Chandruland B Singh, 2016; S Mandal, 2016; MMHossainet. al., 2016). It exposes white wood underneath upon peeling of the bark longitudinally (SC Saini and GBS Reddy, 2015). The diameter of main stem is 16cm (KK Chandrul and B Singh, 2016). b) Leaves:The leaves are bipinnately compound, 30 cm long, each bearing 24 leaflets alternate on rachis with reticulate venation. The leaflets are 4.9cm long, 1.8cm broad, ovate to lanceolate, short stalked, with dotted gland and having 0.5cm long petiole(KK Chandruland B Singh, 2016). c) Flower:From the mid-April plant starts flowering and ends in the middle of May. However, the peak flowering season has been observed in the last week of April (S Mandal, 2016).Inflorescence bears 60-90 flowers. The flowers are small, bisexual, white, funnel- shaped, sweetly scented, stalked, pentamerous, hypogynous, actinomorphic, ebracteate with average diameter of fully opened flower being 1.12 cm. The calyx is deeply lobed with five cleft, pubescent, persistent, inferior and green in color. Petals are five with free, whitish, inferior, lanceolate, length 5mm, glabrous with dotted glands. Androecium is polyandrous, inferior, with 10 stamens, arranged into circles of five each; smaller stamens are 4 mm long whereas the longer ones are 5 to 6 mm. Gynoecium 5 to 6 mm long stigma, bright, sticky; style, short; ovary, superior white small flower (KK Chandrul and B Singh, 2016;SC Saini and GBS Reddy, 2015).

41 Vidya (2018) Vol. No : 1 d) Fruit:Fruits occur in close clusters. They are small ovoid to sub-globose, glandular, with thin pericarp enclosing one or two seeds which are spinach green in color (SC Saini and GBS Reddy, 2015).Fruits are 1.4 to 1.6 cm long, 1 to 1.2 cm in diameter; fully ripe fruits, black with a very shining surface. Seed are 11 mm long and 8 mm in diameter (KK Chandrul and B Singh, 2016).The fruiting season has been observed in between the middle of July and the end of August. (S Mandal, 2016) Uses

a) Leaves: Curry leaves consist Vitamin A, Vitamin B and B2, Vitamin C and iron hence it is used as calcium source to those having calcium deficiency especially women suffering from osteoporosis.(MNishan and P Subramanian, 2015;KK Chandrul and B Singh, 2016;T Deepika and CMNoorjahan, 2016). People use the fresh leaves, dried leaf powder and essential oil for food industry. It can be eaten raw as a cure for dysentery and diarrhea. For stomach upsets, it is grounded to a fine paste and mixed with buttermilk and consumed orally and the infusion of roasted leaves is given to stop emesis. Fresh juice of its leaves alongwith lime juice and sugar is given for the treatment of morning sickness and for vomiting due to indigestion (T Deepika and CMNoorjahan, 2016; KKChandrul and B Singh, 2016). Fresh juice is also reported to prevent the progression of cataract (KK Chandrul and B Singh, 2016). The leaves are effective against diabetes Mellitus and are applied externally to bruises, eruption and on the boils for swift relief. They are extensively used in treatment of mucus in the stool and influenza (N Kamatet. al., 2015) aswell as tonic (P Mitraet. al., 2017). The blanked residue of boiled curry leaves along with coconut oil is used as natural hair toner and hair growth promoter and is known to retain the black color of the hair by preventing the premature graying of the hair. (H Dhongdeet. al., 2013; D Sharmaet. al., 2015; MNishan and P Subramanian, 2015).Leaves areused in fever, asthma and hypercholesteremia (CGalketiyaet. al., 2016). TheKurumba Communities (Thalamalai Hills, India) use the fried leaves by making chatnifor good hair growth (R Deepakkumaret. al., 2017).The incorporation of dried leaf powder in preparation of cookies increases the nutritional quality in dose dependent manner. (CRDrisyaet. al., 2015). b) Stem:The branches of Murryakoenigiiare used to strengthen gums, popularly used to clean teeth as datum (PR Bhandari, 2012; AKumaret. al., 2016). c) Fruits:Fruits are considered as astringent in Indo-China. d) Roots:The roots of the plant can be used for relieving pain of the kidney and removal of stains on teeth (KK Chandrul and B Singh, 2016; AKumar et. al., 2016). e) Bark and roots:The barks and roots are used as stimulant by the physicians. They are used externally to cure the bites of the poisonous animals and eruptions (TDeepika and CMNoorjahan, 2016). f) Leaves and roots:The leaves and roots are bitter in taste analgesic, acrid, cooling,stomachic, carminative, stimulant, anti-diarrhoeal, anti-dysenteric, anti-emetic, antipyretic, used in

42 Vidya (2018) Vol. No : 1 Jatav et al leucoderma, blood disordersa (AKumaret. al., 2016) and cure allays heat of the body, thirst and itching(SA James et. al., 2016). g) Essential oil:The aromatherapy industry uses the essential oil in the making of soaps and cosmetics. (HDhongdeet. al., 2013; D Sharmaet. al., 2015).The plant is useful in the treatment and prevention of diabetes, cancer, and cardiovascular diseases. The leaves of the plant are highly rich in antioxidants, such as tocopherol, â- carotene and lutein, and possess anti-lipid peroxidative activities and providing protection against oxidative stress (SMandal, 2016). Phytochemistry The plants are rich source of carbohydrates, proteins, amino acids; alkaloids as well as vitamin A, vitamin B, minerals etc. (P Mitraet. al., 2016) and leaves are rich in carotenoids, â- carotene, calcium and iron. (CRDrisyaet. al., 2015). The dried leaf powder contains higher amount of b-carotene (8.8 mg %) and polyphenols (2100 mg %) than other leafy vegetables. (CRDrisyaet. al., 2015).Numerous phytochemicals like koenigine, koenidine, koenine, mahanine, girinimibine ,girinimbiol, koenimbine, O-methyl murrayamine A, O-methyl mahanine, bismahanine, bispyrayafoline, isomahanine, scopotin, murrayanine were also isolated from the leaves (P Mitaraet. al.,2017). The plant also contains a rich amount of carbazole compounds which is found in every part from plant including leaves, roots, bark and stem etc. and responsible for various pharmacological activities. The alcoholic extract of stem bark has been reported to have some bioactive compounds, named as koenigine-quinone A, koeniginequinone B6, 9-carbethoxy-3-methylcarbazole, 9-formyl- 3-methylcarbazole7, methyl-2-methoxycarbazole-3-carboxylate and 1-hydroxy-3-methyl carbazole while petroleum ether extract consists of Marmesin-1´-O-â-D-galactopyranoside,osthol and umbelliferone10 and 3-(1,1-dimethylallyl) xanthyletin. Mukonal, a biogenetic intermediate of pyranocarbazole alkaloid was also detected in the stem bark of Murrayakoenigii. In addition to alkaloids, coumarin and cinnamic acid derivatives are found from the plant have also been previously isolated and characterized (MM Hossainet. al., 2016). Four coumarin derivatives have been isolated from stem bark named as, Meranzin hydrate, Epoxyosthol, Isomeranzin and Murracarpin by MM Hossainet. al., 2016. It also contains three cyclic monoterpenoidcarbazole alkaloids viz. murrayazolinol, murrayakoeninol,bicyclomahanimbine, affine, mahaminbineand girinimbine(K Ahmad, 2015; S Saiedet. al., 2015) The root bark contains monomeric and binary carbazole, quinone, mukoenine A, B and C, murrastifolin F, bis-2-hydroxy-3-methylcarbazole, bismahanine, biskoeniquinone A and bismurrayaquinone-A along with murrayanine, murrayastine, murrayatine, murrayacine and murrayazolinol(A Kumaret. al., 2016). The phytoconstituents P- gurjunene, P- caryophyllene, P- elemene and O- phellandrene are responsible for its characteristic aroma (PriyankaSangale and RupaliPatil, 2017) and the essential

43 Vidya (2018) Vol. No : 1 oil also contains 5ØüÞ-pinene, 5ØýÞ-phellandrene, (E) caryophyllene, 5ØüÞ-selinene, tetradecanoic acid, hexadecanoic acid, c-eudesmol, a-muurolol, (Z,E)-farnesol, and (Z,Z)-farnesol (NS Aniet. al., 2016). It possesses the ability to control the food spoilage due the presence of compounds like â- pinene, â- caryophyllene, â- phellandrene and á- pinene either alone or in form of combination with other metabolites. Protein Profiling:S. Satheshkumar and N. Punniamurthy, 2017investigated the effect of drying process on protein profile of its leaves by the standard SDS-PAGE procedure. Total 19 prominent bands were obtained, 14 (73.7%) with above 36kDa, in fresh curry leaves while in dried sample only 13 prominent bands were detected with an overall reduction of 31.6% of detectable proteins. The study, thus, revealed about the consumption of fresh herbs rather than dried and processed products for better results. Pharmacodynamic Properties Hyperlipidemia effect:Consumption of Curry leaf powder once daily for 45 consecutive days’ decreases the serum transaminase. A pre and post-test also showed the significant reduction in creatinine and urea and showed no harmful effect on liver and kidney function. (J Molly et. al., 2017). Antioxidant activity:The methanolic extract of plant is reported to have a remarkable scavenging activity along with higher amount of total phenolic content. In reducing power assay, Murrayakoenigii showed best reducing power among all the experimented plants of Sri Lanka (CGalketiyaet. al., 2016).DDPH radical scavenging of methanolic extract was shown to be decreased as leaf> stem> fruit while in lipid peroxidaion assay MDA level was reported to be higher in leaf extract compared to stem and fruit (P Vijayvergiya and R Vijayvergiya, 2016). Anti-canceractivity:Girimbine, a carbazole alkaloids found initsroots is reported to inhibit cancer cell proliferation and promote apoptosis in human cancer cell lines by many researchers. The compound was found to inhibited angiogen-esis both in vivo (zebrafish embryo model) and in vitro and subsequently induced apoptosis in a human colorectal adenocarcinoma cell line (HT-29) (V Imanet. al., 2016). Anti-inflammatory activity:Anti-inflammatory action was evidenced by the significant dose- dependent Girinimbineinhibition of nitric oxide production in lipopolysaccharide/interferon-gamma- induced cells along with significant inhibition of nuclear factor-kappa B translocation from the cytoplasm to nucleus in stimulated RAW 264.7 cells(V Imanet. al., 2016).In mice, with carrageenan- induced peritonitis, oral pretreatment with girinimbine helped limit total leukocyte migration (mainly of neutrophils), and reduced pro-inflammatory cytokine levels (interleukin-1beta and tumor necrosis factor-alpha) in the peritoneal fluid which had suggested that girinimbine could act as a chemopreventive and/or chemotherapeutic agent by inducing apoptosis while suppressing inflammation(V Imanet. al., 2016). Hydroalcoholic extract of itsfruits when administered orally at dose 100mg/kg and 200mg/ kg in male wistar albino rate, of which time dependently higher efficacy against carrageenan-

44 Vidya (2018) Vol. No : 1 Jatav et al induced edema was reported at the dose of 200mg/kg compared to standard drug, indomethacin (GPitchaiahet. al., 2016). Wound healing capacity:It was found that topical application of Murrayakoenigiihydroalcoholicfruit extract ointment (90.3%) shows significant wound closure and epithelialization in less time compared to control (59.7%)(G Pitchaiahet. al., 2016). Silver nano particle synthesis:CKamarajet. al., 2017 synthesized silver nano particles (size: 5-100nm, spherical) using b-caryophyllene isolated from the leaf extract of Murrayakoenigii.It has been reported that AgNPsexhibited promising activity on chloroquine-sensitive Plasmodium falciparum (3D7) (IC50: 2.34 ± 0.07 μg/ml), as well as significant cytotoxic activity on lung cancer cells (IC50: 9.39 ± 0.08 μg/ml) with reference to test agents: plant extract and purified b-caryophyllene. B-caryophyllene synthesized Ag NPs, thus, might be considered as a promising source for the development of cost effective and safer alternative drugs to treat malaria and cancer. Anti-thiamine activity:Compounds with anti-thiamine property were isolated from its leaves by solvent extraction, acid hydrolysis, chromatographic experiments followed by crystallization. It has been reported that 1g of the compound inactivated 31.8 mg of thiamine hydrochloride in 1 h which proved the anti-thiamine activity of curry leaves. Even with seasonal variation showed maximum in vitro anti-thiamine activity in the compound isolated from its leaves during the period of July – August (P Mitraet. al.,2017). Hepatoprotective activity:An alkaloid fraction isolated from its ethanolic leaf extracts at the dose of 30 mg/kg and 100 mg/kg, p.ohas shown decrease in the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) as a result of administration of carbon tetrachloride and paracetamol on adult wistar rats. The higher dose of Murrayakoenigii leaves (100 mg/kg, p.o.) was also reported to prevent the increase in liver weight compared to hepatotoxin treated animals.The cellular architecture of liver cells was also seen to be normal as compared to the standard drug silymarin, a well-known hepatoprotective agent. (PSangale and RPatil, 2017). Antimicrobial activity:The methanol, water, acetone and hexane extract of Murrayakoenigii leaves were tested against Methicillin Resistant Staphylococcus aureus, Micrococcus luteus, Bacillus subtilus, Psedomonasaeruginosa, Escherichia coli, Candida albicans and Aspergillus Niger and all the extracts were reported to have antimicrobial efficacy. (MV salomi and R Manimekalai, 2016). The ethanolic extract with DMSO of Murrayakoenigii was shown to be more effective against two bacterial strains viz. E.coliand P.aeroginosacompared to fungal strains of Candida albicansandAspergillusniger. The higher concentration (100μl) showed higher activity against bacterial strains as well as fungal strain compared to other concentration (25μl and 50μl) while for fungal strains both the concentration (25μl and 50μl) exhibited no effect (NS Kumar and N Simon, 2016). The methanolic extract of roots and leaves were examined against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Trichophytonrubrum, Pseudomonas aeruginosa, Salmonella 45 Vidya (2018) Vol. No : 1 typhi, Candida parapsilosisandAspergillusniger. Methanolic extract of both roots and leaves showed higher activity at very low concentration (2.5ìg/ml) against bacterial strain Staphylococcus aureusthanE.coli, Bacillus subtilisand fungal strain Candida parapsilosisthanAspergillusniger(NBRao, et. al., 2015). Anti-bacterial activity:The antibacterial activity of the ethanolic leaf extract of Murrayakoenigii, T. occidentalisand their synergy was tested at the concentration of 500mg/ml, 250mg/ml, 125mg/ ml, 62.5mg/ml and 31.25mg/ml. The higher concentration (500mg/ml) of ethanolic extract of Murrayakoenigii was more effective against all the tested strains with K. pneumonia as a most susceptible and S. dysenteriaeas a least susceptiblestrain. It was further reported that the synergy of Murrayakoenigiiand T. occidentalis gave higher zone of inhibition compare to individual extract of Murrayakoenigii and T. occidentalis at the same concentation (FI Akinnibosunand JA Umufo, 2015). Anti-listerial activity:A combination of pediocin (a natural antimicrobial) and Murrayakoenigii berries substantially reduced Listeria innocua count which was artificially inoculated in the raw goat meat emulsion at 4oC storage as compared to control and sodium nitrite and BT2 (Y Kumaret. al., 2017). Hence, this was suggested that a natural antioxidant and antimicrobial could be used to maintain the oxidative and microbial quality of minimally processed raw meat products at refrigerated temperature. Antinociceptive effects:Methanolic extract of Murrayakoenigii was evaluated for its antinociceptive potential by two methods, formalin-induced licking and acetic acid-induced writhing tests at the doses of 50, 100, and 200mg/kg .In both the test significant inhibition was observed in dose dependent manner. Furthermore, inhibition of glutamate induced pain was also reported and pretreatment with glibenclamide (an ATP-sensitive potassium channel blocker) at 10mg/kg significantly reversed the plant-mediated antinociception which suggest the possible antinociceptive mechanism including glutamatergic system and the ATP-sensitive potassium channels (NS Aniet. al., 2016). Anti-diabetic activity:Upon incubation of increasing concentration of hemoglobin plant extract inhibited hemoglobin glycosylation which was higher as compared to standard drug (alpha tocopherol). The glucose uptake was found to increase when yeast cells were treated with ethanolic plant extract compared to stand drug, Metformin. The higher activity was observed in 10mg/ml of plant extract. The plant showed concentration dependent reduction of alpha amylase enzyme compared to standard drug Metmorphin (VRMadhuriet.al, 2016). Antihelmintic effects:The ethanolic and methanolic extract of Murrayakoenigii fruits shows antihelmintic effects against the Indian earthworm (Pheretimaposthuma) in a dose dependent manner; Piperazine citrate (15mg/ml) was selected as standard drug. Ethanolic and petroleum ether extract were taken at 50 mg/ml, 100 mg/ml, 150 mg/ml and 200 mg/ml. Both the extract possesses significant activity while the ethanolic extract showed better anthelmintic activity than petroleum ether extract. The ethanolic extract (at 200mg/ml) causes the paralysis of Indian earth worm at 23.33 minutes and promotes lethal effect at 95.33 minutes (AN Waghmareet. al., 2015).

46 Vidya (2018) Vol. No : 1 Jatav et al Conclusion Since every part viz. root, stem, bark and leaves of the plant is useful, a wide range of study has been done from its stem up to its bark. This review compiles various aspects of Murrayakoenigiion which research has been done and provides a better awareness toward its therapeutic and non-therapeutic properties. Pharmacological studies, for further research towards knowing its unknown pharmacodynamicproperties. References A Kumar, J Yadav, A Kushwaha, Amardeep and A Sain (2016) In Vitro Analysis of Phytochemicals in Anti-diabetic Plant Murrayakoenigii,Asian Journal of Biochemical and Pharmaceutical Research, 6(2) AK Murugan, T Vaithiyanathan and P Sundaramoorthy (2017) Biodiversity of Medicinal Plants in Thudaripettai Village, Nagapattinam District, Tamil Nadu, India, International Journal of Environment, Agriculture and Biotechnology, 2 (1): 264 AK Shakya (2016) Medicinal Plants: Future source of new drugs, International Journal of Herbal Medicine, 4(4): 59-64 ANWaghmare, SV Tembhurne and DM Sakarkar (2015) Anthelmintic activity of Murrayakoenigii(L)Fruits Extract on Indian Earthworm, International Journal of Veterinary Science, 4(3): 148-151 C Galketiya, TS Weerarathna, JC Punchihewa, MN Wickramaratne and DBM Wickramaratne (2017) Screening of edible plants in Sri Lanka for antioxidant activity, Journal of Medicinal Plants Studies, 5(1): 91-95 C Kamaraj, G Balasubramani, C Siva, R Manickam, V Balasubramanian, RK Raja, S Tamilselvan, G Benelli and P Perumal (2017) Ag nano-particles synthesized using b-caryophyllene isolated from Murrayakoenigii: Antimalarial (Plasmodium falciparum 3D7) and Anticancer Activity (A549 and HeLa Cell Lines), J ClustSci, doi 10.1007/s10876-017-1180-6 CR Drisya, BG Swetha, V Velu, D Indrani and RP Singh (2015) Effect of dried Murrayakoenigii leaves on nutritional, textural and organoleptic characeteristics of cookies, Journal of Food Science and Technology, 52(1): 500-506 D Sharma, H Vashist and RB Sharma (2015) Pharmacological aspects on Murrayakoenigii –A review, European Journal of Biomedical and Pharmaceutical Sciences, 2(3): 664-678 FIAkinnibosun and JA Umufo(2015) Assessment of the phytochemical and antibacterial properties of the synergy of MurrayakoenigiiandTelfariaoccidentalisethanolic leaf extracts, International Journal of Research Studies in Biosciences,3(4): 80-89 G Singh, N Singh and V Dixit (2015) A review on Pharmacognostic studies on MurrayakoenigiiSpreng.,World Journal of Pharmaceutical Research, 5(1) H Dhongade, H Sawarkar, B Muley, V Deshmukh and A Pande (2013) Therapeutic potentials of MurrayakoenigiiSpreng (Rutaceae), Indo American Journal of Pharmaceutical Research, 3(9) 47 Vidya (2018) Vol. No : 1 J Molly, S Edison and R Vijajaraghavan (2017) Effect of Murrayakoenigii(Curry Leaves) Powder on the Liver and Renal Functions in Women with Hyperlipidemia, International Journal of Health Sciences and Research, 7(1) K Ahmad, SP Tan, H Hazni and MA Nafiah (2015) Cyclic monoterpenoidpyranocarbazole alkaloids from the bark of Murrayakoenigii(L.) Spreng, JurnalTeknologi, 77(2): 73-77 KK Chandrul and B Singh (2016) Botanical and Physicochemical standardization of aerial parts of Murrayakoenigii, Pharmacophore, 7 (3): 176-186 M Nishan and P Subramanian (2015) Murrayakoenigii (curry leave) - A review on its potential, International Journal of PharmTech Research, 7(4): 566-572 MM Hossain, F Tahia, MA Al-Mansur and MA Rashid (2016) 7-Methoxy-8-Prenylated Coumarins from Murrayakoenigii(Linn.)Spreng.,Dhaka Univ. J. Pharm. Sci., 15(2): 155-159 MV Salomi and R Manimekalai (2016) Phytochemical Analysis and Antimicrobial Activity of Four Different Extracts from the Leaves of Murrayakoenigii, International Journal of Current Microbiology and Applied Science, 5(7): 875-882 NKamat, DPearline and P Thiagarajan (2015) Murrayakoenigii(L.) (Curry Leaf): A Traditional Indian Plant,Research Journal of Pharmaceutical, Biological and Chemical Sciences, 6(5):691 NB Rao, OS Kumari and RG Gajula (2015) Phyto-chemical analysis and anti-microbial activity of Murrayakoenigii, Helix,3: 688-692 NS Ani, S Chakraborty and M Moniruzzama (2016) The Methanolic Extract from MurrayakoenigiiL. inhibits Glutamate-Induced Pain and Involves ATP-Sensitive K+ Channel as Antinociceptive Mechanism, Advances in Pharmacological Sciences, Volume 2016, Article ID 3790860, 6 pages NS Kumar and N Simon (2016) In vitro antimicrobial activity and phytochemical analysis of Murrayakoenigii (L.) leaf extracts, Global Journal of Science Frontier Research: C Biological Science, 16(1) Version 1.0 P Mitra, T Ghosh and PK Mitra (20) In vitro Anti Thiamine activity of an isolated Compound from Murryakoenigii(linn.) SprengWettst Leaves: Effect of Season, SMU Medical Journal, 4(1) P Sangale and R Patil (2017) Hepatoprotective activity of alkaloid Fractions from Ethanol Extract of Murrayakoenigii leaves in Experimental Animals, Journal of Pharmaceutical Sciences and Pharmacology, 3: 28-33 P Vijayvargia and R Vijayvergia (2016) Assesment of phytochemicals and antioxidant activity of MurrayakoenigiiLinn., International Journal of Pharmaceutical Sciences and Research, 7(5): 2163-2167. PR Bhandari (2012) Curry leaf (Murrayakoenigii) or Cure leaf: Review of its curative properties, Journal of Medical Nutrition and Nutraceuticals, 1(2) R Deepakkumar, E Sabari, M Karthick and KS Raysad (2017) Traditionally used Ethno-medicinal plants of the Kurumba communities surrounded in Thalamalai Hills, Namakkal District, Tamil Nadu, South Indian Journal of Biological Sciences, 3(1): 15-26 48 Vidya (2018) Vol. No : 1 Jatav et al S Mandal (2016) Curry plant, Murrayakoenigii L.: An indigenous spice plant with versatile medicinal property: A mini review, International Journal of Clinical and Experimental Physiology, 3: 59-65. S Saied, F Hassan, S Naz and H Siddiqi (2015) Carbazole alkaloids from stem bark of Murrayakoenigii(L.)Spreng, Journal of Basic & Applied Sciences, 11: 146-148 S Satheshkumar and N Punniamurthy (2017) Effect of drying on protein profile of Murrayakoenigiileaves, International Journal of Science, Environment and Technology, 6(1): 861- 865 SA James, RE MOmwirhiren, IA Joshua and I Dutse (2016) Anti-diabetic properties and phytochemical studies of ethanolic leaf extracts of Murrayakoenigii and Telfairiaoccidentalisalloxan-induced diabetic albino rats, Advances in Life Science and Technology, 49 SC Saini and GBS Reddy (2015) A Review on Curry Leaves (Murrayakoenigii): Versatile Multi- Potential Medicinal Plant, American Journal of Phytomedicine and Clinical Therapeutics, 3(4): 363-368 T Deepika and CM Noorjahan (2016) Phytochemical Screening and Thin Layer Chromatographic analysis for Antioxidant activity of Murrayakoenigii (Curry leaf) International Journal of Pharmacy & Life Sciences, 7(12): 5369-5374 V Iman, S Mohan, S Ibrahim, Abdelwahab, H Karimian, N Nordin, M Fadaeinasab, MI Noordin and SM Noor (2016) Anticancer and anti-inflammatory activities of Girinimbine isolated from Murrayakoenigii, Drug Design, Development and Therapy, 11: 103–121 VR Madhuri, SP Begum, HP Begum, PA Kumar, S Mahesh, K Shaik, G Nagarajan (2016) Evaluation of in vitro anti-diabetic activity on ethanolic extract of aerial parts of Murrayakoenigii, Indian Journal of Research in Pharmacy and Biotechnology, 4(3): 147 Y Kumar, K Kaur, AK Shahi, N Kairam and SK Tyagi (2017) Antilisterial, antimicrobial and antioxidant effects of pediocin and Murrayakoenigii berry extract in refrigerated goat meat emulsion, LWT - Food Science and Technology, 79: 135-144 ❑

49 Vidya (2018) Vol. No : 1

Research Paper ISSN: 2321-1520

Phytochemical screening and evaluation of leaves of Citrus limon (L.)Osbeck.

Palak Sapra*, Shirin Qureshi, Archana Mankad and Hitesh Solanki

*Department of Botany, Gujarat University, Navrangpura, Ahmedabad.

*Corresponding Author

Received Date : 20-12-2017 Published Date : 7-3-2018

Abstract Plants have some specific bioactive compounds which are called Phytochemicals. They usually don’t have any nutritional property but are used as defense by the plants and also to prevent many diseases. Human body can uptake only minimal amount but they are very useful to minimize the free radical damage caused to the cells, as a result of oxidative stress. Lemon is a citrus fruit considered to be rich in Phytochemicals. This study gives a brief perception of the Citrus limonumleaf, the bioactive compounds or Phytoconstituents present in them. Phytochemical screening was carried out in four extracts i.e. petroleum ether, acetone , methanol and chloroform, to identify the bioactive compounds such as alkaloids, carbohydrates, glycosides, steroids, Anthocyanins, Saponins, phenols, triterpenoids. Keywords:Citrus limon, Phytochemicals, extracts, medicinal properties. Introduction Phytochemicals are those compounds which are produced naturally in plants. Secondary metabolites are derived from primary metabolites and are used as drugs or to obtain medicines (Akerle Heywood and Synge, 1991). They play an important role in preventing number of diseases such as asthma, arthritis, cancer etc. These Phytochemicals do not have any side effects and are also called as ‘human friendly’ constituents.Some, particularly those belonging to the Rutaceae family have been found to be very effective to natural antioxidants and prevents many diseases. Our current study mainly deals with collection, extraction, qualitative and quantitative analysis of Phytochemicals or bioactive compounds which are present in the leaves of plant Citrus limon(L.)Osbeck. Nearly, all the Citrus fruits are considered to be rich in phytoconstituents. 50 Vidya (2018) Vol. No : 1 Sapra et al

Lemon is very famous in many parts of the world because of its unique and different flavor, taste, and aroma as well as multiple health benefits associated with it. Consumption of lemon according to recommended dietary allowances, they are very beneficial to maintain and improve health and alsoprovide prevention against many different kinds of diseases because of the presence of different kinds of Phytochemicals (Pellegriniet al., 2003; Peterson et al., 2006). The presence of phytochemicals is responsible for their therapeutic effects. The qualitative analysis of bioactive compounds of leaves of Citrus limon (L.) Osbeck for the four extracts i.e. Petroleum ether, acetone, chloroform and methanol have been analyzed in this study and there is wide range of phytochemical compounds present in the four extracts which are shown below in brief. Materials And Methods Materials The selected plant species for the experiment are Citrus limon of Rutaceae family. Plant part used are leaves. The selected solvents for extract preparation are Petroleum ether, Acetone, Methanol and chloroform. Plant collection: The leaves of plants species Citrus limon were collected from region of Ahmedabad city, Gujarat, India in the month of December-2016. Sample preparation: The leaves of Citrus limon were carefully separated, cleaned, washed well to remove all the dirt and are shade dried separately until all the water molecules were evaporated, mechanically grinded and coarsely powdered. The powder was subjected to solvent extraction with petroleum ether, acetone, methanol and chloroform. Extraction Technique: 10gm of each leaf powder was added to 100 ml of solvent (petroleum ether, acetone, methanol and chloroform) in a conical flask. They were kept in shaker for 24 hours at room temperature. All the extracts were filtered by using Whattman No.1 filter paper and the concentrated extracts were collected in the petridishes and allowed to evaporate properly. Petridishes were weighed before and after the evaporation of filtrate to know their weight of extracts and then stored in a cool and dry place. Phytochemical Screening Test for alkaloids: Extracts were mixed individually with a small amount of dilute HCland it wasfiltered. Thefiltrate was evaluated carefully with different alkaloidal reagents. Mayer’s test:2ml of Mayer’s reagent was added in 1 ml of filtrate. Formation of cream precipitate indicates the presence of alkaloids. 51 Vidya (2018) Vol. No : 1 Dragendroff’s test: 2ml of Dragendroff’s reagent was added in 1 ml of filtrate. Formation oforange brown color indicates the presence of alkaloids. Hager’s test: 2 ml of Hager’s reagent is added in 1 ml of filtrate. Formation of yellow precipitate indicates the presence of alkaloids. Wagner’s test:2ml of Wagner’s regent is added to 1 ml of filtrate. Formation of reddish brown indicates the presence of alkaloids. Test for Carbohydrate: The small amount of extract was dissolved in 5 ml of distilled water and it is filtered. The filtrate was subjected to analysis for the presence of carbohydrates. Molisch test: The filtrate was mixed with 2 to 3 drops of 1% alcoholic alpha napthol and along the sides of test tube; 2 ml of concentrated sulphuric acid was added and appearance of purple color ring at the junction of two liquids. Fehling’s test: Fehling A and Fehling B was mixed and few drops of extract are added and boiled. A brick red colored precipitate of cuprous oxide forms if reducing sugars are present. Benedicts test: 2 to 3 drops of benedicts regent was added to 1 ml extract and heat almost to boiling. Appearance of red, brown, orange or yellow color indicates the presence of carbohydrates. Test for glycosides:

Keller-Killiani test: To 2 ml extract, add glacial; acetic acid. 1 drop 5 % FeCl3 and conc.

H2SO4. Brown ring appears indicates the presence of glycosides Test for proteins and free amino acids: Small quantity of extracts were dissolved separately in a few ml of water and treated with following reagents: Millon’s test: Few mg of extract was added with millon’s reagent. Formation ofred color shows the presence of proteins and amino acids. Ninhydrin test: 0.1% ninhydrin solution was added to few mg of extract. Appearance of purple color shows the presence of protein and free amino acids. Biuret’s test: Equal volume of 5 % solution of sodium hydroxide and 1% solution of copper sulphate were added. Appearance of pink color shows the presence amino acids and proteins. Test for flavonoids: Few ml of extract was mixed with sulphuric acid. The formation of yellow orange indicates the presence of flavones, formation of yellowish orange color indicates the presence of anthocyanins and the formation of orange to crimson color indicates the presence of flavonones. Test for steroids:

Salkowski test: To 2 ml of extract add 2 ml chloroform and 2 ml conc. H2SO4 and was shaken for few minutes. Chloroform layer appeared red (upper layer) and acid layer showed greenish yellow fluorescence (lower layer) indicates the presence of steroids.

52 Vidya (2018) Vol. No : 1 Sapra et al Liebermann-Burchard test: Mix 2 ml extract with chloroform. Add 1 to 2 ml acetic anhydride and 2 drops conc. H2SO4from the side of test tube. First red, then blue and finally green color indicates the presence of steroids. Test for Triterpenoids: Liebermann-Burchard test: Mix 2 ml extract with chloroform. Add 1 to 2 ml acetic anhydride and 2 drops conc.H2SO4 from the side of test tube. First red, then blue and finally green color indicates the presence of steroids Test for Tannins: Gelatin test: to the extract, gelatin (dissolved in warm water immediately) solution was added. Formation of white precipitate indicates the presence of tannins. Lead acetate test: 10% of lead acetate solution was added in few ml of extract. Formation of white precipitate indicates the presence of tannins. Tests for Phenol: Ferric chloride test: Few mg of extract is dissolved in 5 ml of distilled water. 2 ml of 1 % solution of gelatin containing 10% NaCl is added to it. White precipitates indicate presence of phenolic compounds. Sodium hydroxide test: Five mg of extract was dissolved in 0.5 ml of 20% sulphuric acid solution. Add few drops of aqueous sodium hydroxide solution. Formation of blue color indicates the presence of phenols

Ellagic Acid Test: Add few drops of 5% glacial acetic acid and 5% NaNO2 solution was added in few ml of extracts . Muddy or Niger brown precipitate occurs. Tests for Saponins: Foam test: small amount of extract is shaken with little amount distilled water. Formation of foam indicates the presence of saponins. Result and Discussion The lemon leaf extractwas rich in Phytochemicals activity and constituents as shown in table 1. In present study, preliminary Phytochemical study was carried out to identify the bioactive compounds such as alkaloids, carbohydrates, glycosides, steroids, Anthocyanins, Saponins, phenols, triterpenoids were present.Phytochemical analysis of Citrus limon plant shows the presence of alkaloids, carbohydrates, glycosides and steroids in high amounts in all the four extracts. Anthocyanins are present in petroleum ether, acetone and chloroform extracts, while flavanones 53 Vidya (2018) Vol. No : 1 are present in methanolic extracts in moderate amount. Saponins, phenols, triterpenoids and tannins are also present in considerably moderate amount. Proteins and free amino acids are present in very few amounts in all the extracts except the chloroform extract (Prasad M. P. et al., 2014). Tannins, which were absent in methanolic and acetonic extracts (Saumendu Deb Roy et al., 2012). Phytochemical screening of Citrus limon(L.) Osbeck leaf extract

SRNO CONSTITUENTS TESTS RESULTS

PE AE ME CE A Alkaloids Mayer’s test + + - + Dragendroff’s test +++ ++ +++ ++ Hager’s test + + + ++ Wagner’s test - - - - B Carbohydrates Molisch test ++ +++ ++ +++ Fehling’s test - - - - Benedicts test ++ +++ ++ + C Glycosides Keller-killiani test ++ ++ +++ + D Proteins and free amino acids Millon’s test + ++ + - Ninhydrin test + + - - Biurets test - + + - E Flavonoids

H2SO4 test + + ++ + (antho (antho (flavo (antho cyanin) cyanin) nones) cyanin) F Steroids Salkowski test ++ +++ +++ - Liebermann- ++ ++ ++ + Burchard test G Triterpenoids Liebermann- ++ ++ ++ + Burchard test

54 Vidya (2018) Vol. No : 1 Sapra et al (-)Absent, (+) Very few, (++) Moderate, (+++)High Conclusion Plants are mostly used for medicinal purposes and considered as important source to obtain drugs in many different regions of the world. India is considered as one of the largest source and producer of many important medicinal herbs and plants. Many people still uses traditional methods to obtain drugs from different medicinally important plants. Drugs from the plants can be made easily available and can be isolated through different methods. They are also less expensive, more safe, and efficient and have negligible side effects in comparison to the pharmacological chemicals. The presence of bioactive constituents in the leaves of Citrus limoncan be used in a multitude of ways for obtaining medicines. Alkaloids are important compounds which have a metabolic role in the living systems. Flavonoids have been foundto prevent the cancer causing tumors. It also inhibits growth and progression of tumors.Phenols together with the flavonoids compounds in plants showmultiple activities like antioxidant, anti-carcinogenic, anti-inflammatory etc.Tannins acts against the pathogenic fungi and shows antimicrobial activity. Saponins are responsible for the leakage of proteins and degradation of cell wall .The plant based compounds which are naturally produced have the effective dosage response and minimal side effects when compared to the synthetic compounds with much more number of health benefits.It reflects a ray of hope for the development of many more new drugs which will prevent chemotherapeutic tumors.With the help from such plants in future they may serve for the production of drugs to cure the deadly diseases. Acknowledgement I would like to thank Department of Botany, Gujarat University for providing me the facilities for my research work. References Al-Anbari, A. K. H., &Hasan, M. A. (2015). Antioxidant activity in some citrus leaves and seeds ethanolic extracts. In International conference on advances in agricultural, biological & environmental sciences. Al-Juhaimi, F. Y., &Ghafoor, K. A. S. H. I. F. (2013). Bioactive compounds, antioxidant and physico-chemical properties of juice from lemon, mandarin and orange fruits cultivated in Saudi Arabia. Pak J Bot, 45, 1193-1196. Banu, K. S., &Cathrine, L. (2015). General Techniques Involved in Phytochemical Analysis. International Journal of Advanced Research in Chemical Science, 2(4), 25-32. Chede, P. S. (2012). Phytochemical Analysis of Citrus sinensis Pulp. International Journal of Pharmacognosy Phytochemical Research, 13(4), 4. Del Caro, A., Piga, A., Vacca, V., &Agabbio, M. (2004). Changes of flavonoids, vitamin C and antioxidant capacity in minimally processed citrus segments and juices during storage. Food Chemistry, 84(1), 99-105. Gayathri, V., &Kiruba, D. (2014). Preliminary phytochemical analysis of dry leaf powder extracts of Citrus aurantium. International Journal of Pharmacognosy and Phytochemical 55 Vidya (2018) Vol. No : 1 Research, 6, 332-334. Hsouna, A. B., Hamdi, N., Halima, N. B., &Abdelkafi, S. (2013). Characterization of essential oil from Citrus aurantium L. flowers: antimicrobial and antioxidant activities. Journal of oleo science, 62(10), 763-772. Mathew, B. B., Jatawa, S. K., &Tiwari, A. (2012).Phytochemical analysis of Citrus limonum pulp and peel. International Journal of Pharmacy and Pharmaceutical Sciences, 4(2), 369-371. Osarumwense, P. O., Okunrobo, L. O., &Uwumarongie-Ilori, E. G. (2013).Phytochemical screening, proximate and elemental analysis of Citrus sinensis peels (l.)Osbeck. Journal of Applied Sciences and Environmental Management, 17(1), 47-50. Penecilla, G. L., &Magno, C. P. (2011).Antibacterial activity of extracts of twelve common medicinal plants from the Philippines. Journal of Medicinal Plants Research, 5(16), 3975-3981. Rajeswari, A. (2015). Evaluation of phytochemical constituents, quantitative analysis and antimicrobial efficacy of potential herbs against selected microbes. Evaluation, 8(2). ❑

56 Vidya (2018) Vol. No : 1

Original Paper ISSN: 2321-1520

Estimation of Physico-Chemical Characterization of Forest Soil and Dominant Tree Species for Carbon Sequestration in Attersumba Range, Gandhinagar Forest Division, Gujarat, India

Dharmesh G. Jaiswal,Vijaybhai A. Khuman, and Hitesh A. Solanki*

*Professor of Botany, Department of Botany, Bioinformatics and Climate Change Impacts Managements, University School of Science,Gujarat University, Ahmedabad. E-mail Id: [email protected]

*Corresponding Author

Received Date : 9-1-2018

Published Date : 7-3-2018

Abstract This physico-chemical study of soil is based on various parameters like pH, Electrical Conductivity (EC), Total Organic Carbon, Available Nitrogen (N), Available Phosphorus and Available potassium. This study leads us to the conclusion of carbon stock and dominant plant species and inferior species of Attersumba range Gandhinagar Forest division. Soil sampling is the most vital step for any soil analysis. Soils in equilibrium with a natural forest ecosystem have high carbon (C) density. The rate of SOC sequestration, and the magnitude and quality of soil C stock depend on the complex interaction between climates, soils, treespecies. Climate change may also stimulate forest growth by enhancing availability ofmineral N and through the CO2 fertilization effect, which may partly compensate release of soil C in response to warming. There are significant advances in measurement of soil C stock. Keywords: Soil organic carbon, Sequestration, Carbon cycle, Climate change, Forest ecosystems, Quality of soil. Introduction Soil sampling is perhaps the most vital step for any soil analysis. As a very small fraction of the huge soil mass is used for analysis, it becomes extremely important to get a truly representative soil sample of the field. Soil test based nutrient management has emerged as a key issue in efforts

57 Vidya (2018) Vol. No : 1 to increase Forest plant growth and production of plant nutrients, based on soil analysis can improve forest productivity and minimize wastage of these nutrients, thus minimizing impact on environmental leading to bias through optimal production. Deficiencies of primary, secondary macronutrient have been observed in intensive forests areas.The forests areas falling in Gandhinagar Forest Division comprise of reserve, protected and unclassed forests. The Division is bounded by North: Banaskantha District East: Parts of Sabarkantha and Kheda Districts South: Parts of Kheda and Ahmedabad Districts West: Parts of Ahmedabad and Mehsana Districts The forests area lies between 2305’ and 23022’ north latitudes and 72047’ East longitudes. The total forest area covered by this Division is about 11263.31 ha, out of which 6390.11 ha is reserved forest, 44.92 ha is protected forests and the remaining 4828.28 ha forest areas is yet to be settled. The same forest area of 4828.28 ha Soil is important to everyone either directly or indirectly. It is natural body on which forest plant grow and it has fragile ecosystem. Carbon (C) storage in forest ecosystems involves numerous components including biomass C and soil C. The total ecosystem C stock is large and indynamic equilibrium with its environment. Because of the large areas involved at regional/global scale, forest soils play an important role in the global C cycle(Champion and Seth,1968,. C.Hontoria, et al. 1999 Detwiler and Hall, 1988; Bouwman and Leemans, 1995; Sedjo, 1992; Jaba´ggy and Jackson, 2000). Land use change causes perturbationof the ecosystem and can influence the C stocks andfluxis. The purpose of this paper is • To demonstrate carbon sequestrestation through soil in Attersumba range of Gandhinager forest division • Estimation of Dominant and inferior tree species in Attersumba range gandhinagar forest division • Determination of physical and chemical soil quality parameter analysis in Attersumba range gandhinagar forest division Materials And Methods The quality test survey of the soil was conducted in 2017. Twenty three villages from Attersumba range covering North, South, East and West were selected for this study. Representative soil samples were collected following standard quadric procedure and taken in polythene bags. In laboratory these samples were analyzed for different chemical parameters following standard methods. AR grade reagents and double distilled water were used for soil analysis. Results were compared with standard values to find out low, medium or high nutrient’s content and forest SOC. Physico - Chemical Analysis The collected samples were analyzed for major Physical and Chemical soil quality parameter like pH, Electrical Conductivity (EC), Organic Carbon (OC), Nitrogen (N), Organic matter is oxidized with chromic acid (Potassium Di-chromate, + H2SO4) . This method is widely used in Indian Laboratories. The K and P analysis by standard method.pH was measured using PH meter, EC was measured using a conductivity meter, OC was measured using colorimeter, Potassium

58 Vidya (2018) Vol. No : 1 Solanki et al was measured using Flame photometer, and Phosphorus was measured using Spectrophotometer. All apparatus are Systronic make. Examination of soil done by soil test laboratory, Gandhinagar, Gujarat. Result And Discussion Total 23 soil samples of Attersumba range, Gandhinagar forest division were collected in clean polythene bags and brought to the Laboratory Most of the parameters are fall under thepermissible standred according to soil standared. Experimental value of quality characteristic especially pH, EC,OC N,P,K, of soil of attersumba range are present in the table no1 and figure 1. Determination of Soil 1.pH:The soil reaction or pH is meant to express the acidity or alkalinity of the soil. The pH is very important property of the soil as it determines the capacity. The pH values fluctuated less than 8.5. The limit of pH value for soil acidic.< 6.5,Normal 6.5-7.8, Alkaline 7.8-8.5, Alkali > 8.5. 2.EC:Total soluble salts are estimated from electrical conductivity (EC) of aqueous soil extracts. Standard value of EC in soil- Normal < 0.8 dsm-1, critical for salt sensitive crops, critical for salt tolerant crops 1.6 -2.5 dsm-1, Injurious to most crops > 2.5 dsm-1. The EC value 04 to 1. 3.OC and Nitrogen (N):Soil organic carbon is the seat of nitrogen in soil and its determination is often carried out as an index of nitrogen availability. In the colorimeter method ( Dattaet al, 1962), Organic matter is oxidized with chromic acid. OC in Attarsumba range 0.23 to 0.85 .Standard value of OC low < 0.50, medium 0.50- 0.75 and high > 0.75. 4.Phosphorus:Phosphorus was found in the range of low, medium, high. Inorganic phosphorus as orthophosphate plays a dynamic role in aquatic ecosystem. Phosphorus, the most important micro nutrient, is utilized by plant in the form of H2PO4- & HPO4 2- species.

5.Potassium :Standard value of K as K2O in soil low < 140 kg K2O ha-1, medium 140-280 kg K2O ha-1 high > 280 kg K2O ha-1. Potassium was found in the range of low, medium , high. K though present in small amount in soil sample, plays a vital role in the metabolism of fresh water and considered to be an important micronutrient. The K is relatively abundant in the earth's crust, most of it is not accessible to plant. Table 1: Physico-chemical analysis of the soil of Attersumba rangeGandhinagar Forest Division.

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Sr.No. Name of EC pH OC P K Village 1 Apruji 0.16 6.8 0.31 23 450 2 Aral 0.17 6.4 0.45 27 450 3 Attersumba 0.12 6.0 0.50 23 450 4 Bardoli 0.09 6.3 0.22 23 450 5 Bariya 0.13 5.7 0.55 27 450 6 Bobha 0.08 7.4 0.15 12 93 7 Devkaran na 0.07 7.6 0.10 17 161 muvada 8 Fulji na muvada 0.08 7.4 0.20 12 206 9 Kakarkhad 0.07 7.3 0.25 19 198 10 Khadal 0.15 7.2 0.39 15 277 11 Kosam 0.08 7.7 0.28 8 198 12 Lal na muvada 0.07 7.6 0.28 6 198 13 Madadra 0.07 7.7 0.19 10 124 14 Mahudiya 0.08 7.8 0.13 17 134 15 Motipura 0.07 7.7 0.16 12 171 16 Punadra 0.16 7.8 0.21 6 114 17 Rampur 0.13 7.7 0.19 6 83 18 Sandesar 0.08 7.7 0.28 10 103 19 Shihor 0.08 7.8 0.31 12 124 20 Talpoda 0.09 7.6 0.25 12 124 21 Telnar 0.08 8.0 0.25 7 268 22 Vdhavat 0.11 7.6 0.31 10 450 23 Valjipur 0.08 7.9 0.21 11 445

Vidya (2018) Vol. No : 1 Detwiler R. P. and Hall C. A.(1988).Tropical forests and the global carbon cycle.Science. 239(4835):42-7. PMID: 17820628 DOI: 10.1126/science.239.4835.42 Jaba'ggy, E. G., and Jackson, R. B. (2000).The vertical distribution of soil organic carbon and its relation to climate and vegetation. Ecol. Appl. 10,423-436. Roger Sedjo (1992). Temperate Forest Ecosystems in the Global Carbon Cycle.Ambio (Sweden), AmbioISSN : 0044-7447 ❑

62 Vidya (2018) Vol. No : 1

Original Paper ISSN: 2321-1520

Effect of Copper on Soil, Growth and Metabolites in SpinaciaOleraceaL.

Vinal Patel*, Deepti Sharma, Suhani Parekh, Archana Mankad

*Department of Botany, University School of Sciences, Gujarat University, Ahmedabad. E-mail Id: [email protected]

*Corresponding Author

Received Date : 3-1-2018

Published Date :7-3-2018

Abstract This study explored the effects of foliar Spray of selected plant growth regulators (PGRs) on Spinaciaoleracea L. and it is used as Copper sulfate at 50,100,150,200,250 ppm.Two sets of selected plants were prepared, one of them was treated with different concentration of copper and another set was kept as control. The analysis of foliar treated plants revealed the content of Total sugar (TS), Reducing Sugar (RS), Totalprotein (TP), TotalPhenol (TPH), Totalchlorophyll (TC).The physiological parameters which weremeasured are Height of Plants, Fresh weight of Roots, Fresh weight of leaves, Number of leaves. Soil parameters (Soil pH, Electrical conductivity, Organic carbon and Nitrogen, Boron and Sulphur, Potassium, phosphorous, Zinc, Copper and Iron) were also measured. From the result it was observed that plant growth in treated plants was better than control plants. There was a positive change in plant Growth. Keywords:Foliar spray, Plant growth regulators, Copper sulfate, Physiological parameters, Soil parameters. Introduction Copper is an effectively biocide. Cu is a micronutrient for normal growth and development. Copper (Cu) is a heavy metal which in recent studies has been attributed an increasing role in metabolic processes of plant cells. Copper is taken up by higher plants largely in the form of Cu+2 due to the action of still not well-known mechanisms. For its absorption from the rhizophere, in

63 Vidya (2018) Vol. No : 1 which it is almost totally bound to various ligands. Copper sulfate are commonly applied to soil to provide copper. Copper is a redox active transition metal that is involved in many physiological processes in plants because Cu can exist in multiple oxidation states in vivo. Little work has been done in this field wherein the plants have been subjected to treat with copper sulfate in different concentration and another set was control which was without copper for this one plant was selected for experiments. Materials and Methods The selected plant was Spinaciaoleracea L.The plant seeds were grown in the pots in Botanical garden of Botany department, Gujarat University to investigate the effect of copper on plants, two sets with three replica of each were prepared for the experiment, one set was exposed to the copper sulfate and other set of plants was kept as control.i.e without any copper treatment. Plants were treated with copper for the 30 days of duration, in which copper was applied after each an interval of 5days.After treatment plants were harvested and washed thoroughly with distilled water. Harvested plants were used for analysis of soil, plant growth and various metabolites. The experiments were carried out in completely randomized design with three replicates. Chlorophyll were extracted from the leaves and estimated by the method of Arnon (1973), Estimation of Total Sugar& Reducing sugar was done in plant extracts by the protocol given by Nelson (1944), Starch was estimated by the method of Chinoy (1939), Estimation of protein was done in plant extracts by the method of Bradford (1976), Total phenol were estimated by using the method described by Bray and Thorpe (1954).In this experiment also performing soil parameters like Soil pH, Electrical Conductivity, Organic Carbon, Nitrogen,Sulphur, Boron, Phosphorous, Potassium, Zinc, Copper & Iron by using the standard methods. Result Copper and Plants are significantly related to eachother.When copper was applied to the plants, they showed some drastic responses and affect both morphologically and physiologically. Effect of Copper on Plant Growth

Fig 1.Effect of CuSO *5H O on plant height. Values represent mean ± S.E. (n=3) 4 2 64 Vidya (2018) Vol. No : 1 Patel et al

Fig 2.Effect of CuSO4*5H2O on fresh weight of roots. Values represent mean ±S.E. (n = 3)

Fig 3.Effect of CuSO4*5H2O on fresh weight leaves. Values represent mean ±S.E. (n = 3)

65 Vidya (2018) Vol. No : 1

Fig 4.Effect of CuSO4*5H2O on number of leaves. Values represent mean ±S.E. (n = 3) When plants were exposed to the copper sulfate then it showed noticeable changes in plant growth. The height of plants in treated set is more than the control one. In 100 ppm of concentration shows the best result (Fig 1).When copper applied to the plants fresh weight of roots and leaves also affected in plant growth in 50 ppm of concentration shows more weight than the other one. (Fig 2&3).Number of leaves also increased in treated plants as compare to the control plants.in 50,150,200 ppm of concentration shows more number of leaves. (Fig 4). Effect of Copper on plant metabolites

Fig 5.Concent ration of Total Sugar.Values represent mean ± S.E.(n=3)

66 Vidya (2018) Vol. No : 1 Patel et al

Fig 6.Concentration of Reducing Sugar. Values represent mean ± S.E. (n = 3)

Fig 7.Concentration of Total protein. Values represent mean ± S.E. (n = 3)

67 Vidya (2018) Vol. No : 1

Fig 8.Concentration of Total phenol. Values represent mean ± S.E. (n = 3)

Fig 9.Concentration of Total Starch. Values represent mean ± S.E. (n = 3)

68 Vidya (2018) Vol. No : 1 Patel et al

Fig 10.Concentration of Total Chlorophyll. Values represent mean ± S.E. (n =3) Result showed that copper not only affects the plantgrowth, but it also affects the concentration of various metabolites. When the copper was applied to the plants then there was increase in the concentration of total sugar & reducing sugar in treated plants as compare to the control one in concentration of 250 ppm shows the best result (Fig 5&6).when plants were exposed to the copper, it affects the concentration of protein.in this experiment also 250 ppm of concentration shows the best result (Fig 7).There was also increase in the concentration of total phenol content in the treated set. The concentration of 250 ppm shows best result as compare to control plant (Fig 8).there was no change in total starch content so result showed that starch content almost an equal in treated as well as control (Fig 9).In 250 ppm of concentration had the higher amount of chlorophyll in treated set as compare to control ones (Fig 10). Effect of copper on Soil

Fig 11.Soil pH.

69 Vidya (2018) Vol. No : 1

Fig 12.Elecrical conductivity.

Fig 13.Organic carbon and Nitrogen

Fig 14.Boron and Sulphur

70 Vidya (2018) Vol. No : 1 Patel et al

Fig 15.Phosphrous, Potassium, Zinc, Copper, Iron Result showed that pH was decreased in before treatment of soil sample as compared to other sets (Fig 11).In electrical conductivity result showed that in before treatment of soil electrical conductivity was decreased than the other sets, while in control set was increased. So, before treatment of soil was slightly saline and in control and other concentration of soil was moderately saline (Fig 12). When plants were exposed to the copper it affects the organic carbon and nitrogen, both were increased in 50 ppm of concentration (Fig 13). Result was also showed that sulphur was high amount in soil as compare to Boron in 250 ppm of concentration high amount of boron and sulphur were present (Fig 14). Graph showed that by copper treatment, among all the nutrients, significant nutrient was observed in potassium. Zinc was very low in this soil as compare to other elements. Discussion Our experiment shows that plant respond to copper treatment, it means in treated plants length of root increased and they become healthier. Copper also increases number of leaves. Copper effect actually influences the plant growth and in a similar manner, it also affecting the plants biochemically. If plants were exposed to the copper then there was also a change in the concentration of these metabolites in soil also affecting copper concentration. Conclusion When copper is applied to the plants, then plants shows positive results .There was a positive change in the plant growth. Plants grow faster when treated with copper. Copper also greatly influences the concentration of various metabolites. Hence this copper concept can be very useful in the field of Horticulture, Physiology, Ecology, Biochemistry and Soil sciences. Copper can be used in plant nurseries to speed-up seed germination and help us grow healthier plants. It concluded that plants grow faster in copper treated soil. The knowledge can be applied in agriculture to increase the yield. This idea may help to solve the problem of starvation and world hunger in the future.

71 Vidya (2018) Vol. No : 1 References Chinoy JJ, (1939): A new colorimetric method for the determination of starch applied to soluble starch, natural starches and flour, part-I, colorimetric determination of soluble starch. Mikrochemie; 26:132. . Weingerl, V. (2010). Copper and Zinc Accumulation in Vineyard soils Treated with Cu and Zn Containing Phytopharmaceuticals. International Journal of Sanitary Engineering Research, 4(1), 14–24 Nelson N (1944): A photometric adaption of the Somogyi’s method for the determination of glucose. Journal of Biological Chemistry; 153: 375-380. ❑

72 Vidya (2018) Vol. No : 1

Original Paper ISSN: 2321-1520

From Bench to Bedside: DNA Vaccines

Rachana Shah1, Bhoomika Vaghasiya1, Hir Patel2, Bhrugurajsinh Zhala1*

1. Clinical Research Programs, c/o Department of Microbiology, Gujarat University. 2. GCS Medical College, Naroda Road, Ahmedabad E-mail Id:[email protected]

*Corresponding Author

Received Date :22-12-2017

Published Date : 7-3-2018

Abstract DNA vaccines came into scientific limelight almost 25 years ago. Since that time, there have been many advancements in this field i.e. increased immunogenicity, use of improved formulations and improved physical methods of delivery. This review focuses on denoting the recent developments in the field of DNA Immunization. The recent studies demonstrated by a number of research centers showed that the DNA plasmid induced immune responses evoke protective immunity against several infectious diseases and cancers, which provides adequate support for the use of this approach. Four DNA vaccine products have recently been approved in the field of veterinary medicine indicating a constructive future for this technology. This review also highlights the advantages and disadvantages of DNA Vaccines. Introduction Currently, with the advance in the biotechnology and the utilization of novel techniques in molecular biology, it is possible to make new vaccines. The successful vaccine gives a fruitful opportunity to use it not just as a part of the term prophylaxis of infectious diseases but also to broaden their purposes in controlling existing and persisting infectious diseases. Currently, vaccines are being investigated as an approach to control HIV and other incessant viral infections as well as treatment of cancer and autoimmune ailments. In spite of all these accomplishment underway in production of vaccines, there are major challenges facing with difficulties, constraints and drawbacks confronting vaccination. Researchers were unable to produce a vaccine for pathogens with antigenic hyper variability including serogroup B meningococcus, HIV and HCV or against pathogens with 73 Vidya (2018) Vol. No : 1 an intracellular phase, causing infections that are transcendently controlled by T cells, such as tuberculosis and malaria.[1] Likewise, development of conventional vaccination can be time and labor intensive, not permitting a quick action to the need of a new vaccine, as in the occurrence of an influenza pandemics. Also there are likewise hypothetical safety concerns linked with the approaches of using both non-live and attenuated concepts.[2][3]To overcome all these challenges, new approaches amid the most recent 30 years have been applied to vaccine advancement. These updated approaches in vaccination technology include DNA vaccines, beforehand characterized as impossible to make.[4] Synthesis of DNA Vaccine[5] DNA vaccines are composed of bacterial plasmid. Expression plasmid of DNA-based vaccination contain two units: i. The antigen expression unit: contain promoter/enhancer sequence which is followed by antigen-encoding polyaldenylation sequences ii. The production unit: contain bacterial sequence which is necessary for plasmid amplication& selection

Benefits The ability of plasmid DNA to induce both cellular and humoral immune responses after inoculation has been demonstrated in several animal models, and hopes have been raised that its applications will lead to new therapies for a range of human diseases. [6]

74 Vidya (2018) Vol. No : 1 Zhala et al It is potentially cheaper to produce than recombinant protein vaccines. It is much easier to transport and use, especially in developing countries, DNA-based immunisation exhibits several important advantages over conventional immunisation strategies that involved live-attenuated or killed pathogens, proteins, or synthetic peptides. It incorporates many of the most attractive features of each approach. One of the important advantages of the DNA immunisation, is that the immune response to immunisation can be directed to elicit immune response without the need for live vectors or complex biochemical production techniques. [7] DNA Vaccines are heat stable and do not require refrigeration. They can be transported and administered at far off places. [8] Other advantages of DNA vaccines are that they are highly specific and the expressed immunizing antigen is subjected to the same glycosylation and post-translational modifications as natural viral infection. Moreover, it is relatively easier to insert multiple variants of an antigen into a single array of plasmid vaccine. Challenges DNA vaccines limited to protein immunogens (not useful for non-protein based antigens such as bacterial polysaccharides). Certain vaccines, such as those for pneumococcal and meningococcal infections, use protective polysaccharide antigens. The disadvantages of DNA vaccines are based mainly on health and safety issues. Most of the safety issues concerning the system are based on the activation of oncogenes as a result of genomic incorporation of immunising DNA, as well as eliciting anti-DNA antibodies; however, this has rarely been detected in experimental studies. [9] Other drawback of plasmid vaccines is the reduced level of immunogenicity. Insertion of foreign DNA into the host genome may cause the cell to become cancerous. Potential risks of integration of vaccine into cellular DNA of the host. A further concern is that an integrated vaccine might cause insertional mutagenesis through the activation of oncogenes or the inactivation of tumour suppressor genes. In addition, an integrated plasmid DNA vaccine could, in theory, result in chromosomal instability through the induction of chromosomal breaks or rearrangements. However, none of these concerns have been witnessed in the preclinical or clinical evaluation of DNA products. There is a chance of development of auto immunity. There is a possibility of development of antibiotic resistance. [6][10] Ongoing clinical trials A novel DNA vaccine has been developed the pre-membrane+ envelope proteins (prME) of ZIKA Virus. Mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. This study was partially successful, suggesting additional research for prevention of this disease in humans.[11] In 2017, Scientists at the Vaccine Research Center (VRC) of the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, developed the NIAID

75 Vidya (2018) Vol. No : 1 Zika virus investigational DNA vaccine. Vaccination have begun in multi-site phase 2/2b clinical trial testing an experimental DNA vaccine designed to protect against disease caused by Zika infection. [12] 20thCentuaryTimelines in DNA Vaccine Development

Year Accomplishment

2010 Recombinant hepatitis B surface antigen (HBsAg)-vectored DNA vaccine encoding the E7 and E6 tumor-associated oncoproteins of human papillomavirus (HPV) type 16 was constructed and it was shown that the use of a HBsAg-based DNA vaccine as a vehicle to elicit responses to coencodedtumor antigens, and have specific implications for the development of a therapeutic vaccine for HPV-associated squamous carcinomas [13] 2011 li-PADRE-E6 represents a novel DNA Vaccine for the treatment of HPV- associated head and neck cancer [14]

Pulmonary immunization with PEI-DNA is an efficient approach for inducing robust pulmonary CD8 + T-cell populations that are effective at protecting against respiratory pathogens.[15]

Major advancements have been made in the field of vector design hold considerable promise for rabies DNA vaccine development. [16]

Incorporation of TBK1 into a DNA vaccine was found to enhance the antigen-specific humoral immune responses targeting the Plasmodium falciparum serine repeat antigen (SERA), a candidate vaccine antigen expressed in the blood-stages of human malaria parasites. [17]

76 Vidya (2018) Vol. No : 1 Zhala et al

2013 Magnetic vectors comprising polyethylenimine (PEI)-coated superparamagnetic iron oxide nanoparticles were used for enhanced DNA Vaccine Delivery in combination with hyaluronic acid. [18]

A DNA vaccine was designed against apolipoproein thus opening a new dimension for the treatment of cardiovascular diseases related to high lipoprotein (a). [19]

An oral DNA vaccine against an Endoglin was developed and it showed significant inhibition of tumor growth. [20]

2014 Earlier it was reported that the short noncoding DNA fragments (sf-DNA) can significantly enhance EP-mediated transgene expression of reporter genes. Later a study showed that sf-DNA in EP-mediated HBV DNA vaccination leads to an enhanced expression of the HBV surface antigen, resulting in higher cellular and humoral responses. [21]

2015 Studies were conducted in order to improve the immunogenicity of DNA vaccines in humans. [22] 2016 ID injection of DNA vectors using an NF device (NF-ID) elicits a superior cell-mediated immune response, at much lesser DNA dosage, comparable in magnitude to the traditional intramuscular immunization. [23]

A novel DNA vaccine has been developed the pre-membrane+ envelope proteins (prME) of ZIKA Virus. Mice and non-human primates were immunised with this prME DNA-based immunogen through electroporation-mediated enhanced DNA delivery. This study was partially successful, suggesting additional research for prevention of this disease in humans.[11]

77 Vidya (2018) Vol. No : 1 DNA Vaccines Currently Being Used In the Market There are only 4 licensed DNA vaccines available in the market for animal use: 1. Vaccine against West Nile virus for horse. [24] 2. Against infectious haematopoietic necrosis virus.[25] 3. Against melanoma in dogs [26] 4. Growth hormone releasing hormone (GHRH).[27] Conclusion Since the last 2 decades DNA vaccine technologies have generated great deal of excitement as well as disappointment. DNA Vaccines are potentially cheaper to produce than recombinant protein vaccines. It is much easier to transport and use, especially in developing countries. There is a lot of room for improvement in this field regarding the issue of low levels of immunogenicity. This opinion is strengthened by recent licenses in the area of animal health and by the improvements in immune potency reported in the non-human primate model systems. However, the coming years of clinical testing of new and more complex DNA vaccines will be pivotal for either creating a true clinical success based on immune potency, or for telling us that we still have much further to go. A developing country such as India should channelize its efforts to develop novel DNA vaccines for a variety of diseases in which non-human DNA vaccine models have sufficiently proven themselves. Its success will be built on a high level of participation cooperation between industry, the regulatory authorities, funding by non-governmental organizations, the public and academicians. References Almeida RR, Raposo RAS, Coirada FC, da Silva JR, de Souza Ferreira LC, Kalil J, et al. Modulating APOBEC expression enhances DNA vaccine immunogenicity. Immunol Cell Biol. 2015 Nov;93(10):868–76. American Academy of Microbiology.The Scientific Future of DNA for Immunization.1996. [ONLINE] http://www.asmusa.org/acasrc/Colloquia/dnareprt.pdf Arunachalam PS, Mishra R, Badarinath K, Selvam D, Payeli SK, Stout RR, et al. Toll-Like Receptor 9 Activation Rescues Impaired Antibody Response in Needle-free Intradermal DNA Vaccination. Sci Rep. 2016 Sep 23;6:33564. Bergman PJ, Camps-Palau MA, McKnight JA, Leibman NF, Craft DM, Leung C, et al. Development of a xenogeneic DNA vaccine program for canine malignant melanoma at the Animal Medical Center.Vaccine. 2006 May 22;24(21):4582–5. Bivas-Benita M, Gillard GO, Bar L, White KA, Webby RJ, Hovav A-H, et al. Airway CD8+ T cells induced by pulmonary DNA immunization mediate protective anti-viral immunity. Mucosal Immunol. 2013 Jan;6(1):156–66. Coban C, Kobiyama K, Aoshi T, Takeshita F, Horii T, Akira S, et al. Novel strategies to improve DNA vaccine immunogenicity. Curr Gene Ther. 2011 Dec;11(6):479–84. Garver KA, LaPatra SE, Kurath G. Efficacy of an infectious hematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhynchustshawytscha and sockeye O. nerka salmon. Dis Aquat Org. 2005 Apr 6;64(1):13–22.

78 Vidya (2018) Vol. No : 1 Zhala et al Haigh O, Kattenbelt J, Cochrane M, Thomson S, Gould A, Tindle R. Hepatitis B surface antigen fusions delivered by DNA vaccination elicit CTL responses to human papillomavirus oncoproteins associated with tumor protection. Cancer Gene Ther. 2010 Oct;17(10):708–20. Immunologic Responses to West Nile Virus in Vaccinated and Clinically Affected Horses [Internet].PubMed Journals. [cited 2017 Mar 29]. Available from: https://ncbi.nlm.nih.gov/labs/ articles/15706975/ Khan KH. DNA vaccines: roles against diseases. Germs. 2013 Mar 1;3(1):26–35 Kyutoku M, Nakagami H, Koriyama H, Nakagami F, Shimamura M, Kurinami H, et al. Inhibition of neointima formation through DNA vaccination for apolipoprotein(a): a new therapeutic strategy for lipoprotein(a). Sci Rep. 2013;3:1600. Muthumani K, Griffin BD, Agarwal S, Kudchodkar SB, Reuschel EL, Choi H, et al.In vivo protection against ZIKV infection and pathogenesis through passive antibody transfer and active immunisation with a prMEnv DNA vaccine.npj Vaccines. 2016 Nov 10;1:16021. Nawwab Al-Deen FM, Selomulya C, Kong YY, Xiang SD, Ma C, Coppel RL, et al. Design of magnetic polyplexes taken up efficiently by dendritic cell for enhanced DNA vaccine delivery. Gene Ther. 2014 Feb;21(2):212–8 Phase 2/2b Trial Testing the NIAID Zika Virus Investigational DNA Vaccine [Internet] NIH NIAID; 2017 March 31 [cited 2018 January 11]. Available from: https://www.niaid.nih.gov/news-events/ phase-2-zika-vaccine-trial-begins-us-central-and-south-america Plasmid-Mediated Growth Hormone-Releasing Hormone Efficacy in Reducing Disease Associated With Mycoplasma Hyopneumoniae and Porcine Reproductive and Respiratory Syndrome Virus Infection [Internet]. PubMed Journals. [cited 2017 Mar 29]. Available from: https://ncbi.nlm.nih.gov/ labs/articles/16478966/ Plasmid-Mediated Growth Hormone-Releasing Hormone Efficacy in Reducing Disease Associated With Mycoplasma Hyopneumoniae and Porcine Reproductive and Respiratory Syndrome Virus Infection [Internet]. PubMed Journals. [cited 2017 Mar 29]. Available from: https://ncbi.nlm.nih.gov/ labs/articles/16478966/ QL Matthews, A Fatima, Y Tang, BA Perry, Y Tsuruta, S Komarova, et al.HIV antigen incorporation within adenovirus hexonhypervariable 2 for a novel HIV vaccine approachPLoS One, 5 (7) (2010), p. e11815 R RappuoliFrom Pasteur to genomics: progress and challenges in infectious diseasesNat Med, 10 (2004), pp. 1177-1185 Redding L, Werner DB. DNA vaccines in veterinary use. Expert Rev Vaccines. 2009 Sep;8(9):1251–76. S SadanandVaccination: the present and the futureYale J Biol Med, 84 (4) (2011), pp. 353-359 Sasaki S, Takeshita F, Xin KQ, Ishii N, Okuda K. Adjuvant formulations and delivery systems for DNA vaccines. Methods. 2003 Nov;31(3):243–54.

79 Vidya (2018) Vol. No : 1 Short noncoding DNA fragments improve the immune potency of electroporation-mediated HBV DNA vaccination - ProQuest [Internet]. [cited 2017 Mar 29]. Available from: http:// search.proquest.com/openview/6481cf24d10fa3e1c417c7520ac5e366/1.pdf?pq- origsite=gscholar&cbl=34384 The past, current and future trends in DNA vaccine immunisations (PDF Download Available) [Internet].ResearchGate. [cited 2017 Mar 30]. Available from: https://www.researchgate.net/ publication/275413281_The_past_current_and_future_trends_in_DNA_vaccine_immunisations. Therapeutic antitumor potential of endoglin-based DNA vaccine combined with immunomodulatory agents - ProQuest [Internet]. [cited 2017 Mar 29]. Available from: http://search.proquest.com/ openview/d36af0452fea517414bff7d750d96d43/1.pdf?pq-origsite=gscholar&cbl=34384 U Kumar, S Kumar, S Varghese, R Chamoli, P BarthwaDNA Vaccine: a modern biotechnological approach towards human welfare and clinical trialsInt J Res Biomed Biotechnol, 3 (1) (2013), pp. 17-20 Ullas PT, Desai A, Madhusudana SN. Rabies DNA Vaccines: Current Status and Future. World Journal of Vaccines. 2012 Feb 17;02(01):36. Wu A, Zeng Q, Kang TH, Peng S, Roosinovich E, Pai SI, et al. Innovative DNA Vaccine for Human Papillomavirus (HPV)-Associated Head and Neck Cancer. Gene Ther. 2011 Mar;18(3):304–12. ❑

80 Vidya (2018) Vol. No : 1

Original Paper ISSN: 2321-1520

A Retrospective Observational Study to Compare Efficacy of PleuroscopicPleurodesis (Talc Poudrage) and Conventional Pleurodesis via Tube Thoracostomy (Talc Slurry) In Patients with Pleural Disorders

Mukesh Patel1, Varun Patel1, Gaurav Gupta1, Hir Patel2 and Surabhi Saxena3*

1. Sparsh Hospital, Navrangpura, Ahmedabad 2. GCS Medical College, Naroda Road, Ahmedabad 3. Clinical Research Programs, c/o Department of Microbiology, Gujarat University E-mail Id:[email protected]

*Corresponding Author

Received Date: 1-2-2018

Published Date: 7-3-2018

Abstract The objectives of the study were to measure success rate of Pleurodesis& find better treatment option and to identify any complication & evaluate suffering related to pleuroscopicPleurodesis. This was a retrospective Observational Study to compare efficacy of Pleuroscopic Chemical Pleurodesis and pleurodesis by tube thoracostomy (ICD) in Case of Pleural Pathology was carried out at Pulmonology Department in Sparsh Hospital, Ahmedabad over a period of 4 months starting from December 2016 to March 2017. This study was aimed to compare the effect of the two treatment modalities which include pleurodesis through pleuroscopy and through Inter-costal drainage. All the patients with pneumothorax and rapidly refilling pleural effusion were included who visited site from January 2014 to December 2016. Data were collected of the patients who underwent either of the two procedures. Data was also collected of the follow up visit to identify any complication. In our study the success rate of pleuroscopicpleurodesis (Talc poudrage) in patients with pleural effusion was found to be 73.17% and pneumothorax was 84.61% whereas the success rate of pleurodesis by tube thoracostomy (Talc slurry) in patients with pleural effusion was 63.63%

81 Vidya (2018) Vol. No : 1 and pneumothorax was 78.57%. It was found that the p value is 0.692 which is not significant, that means there is not much significant difference in the success rate of pleurodesis by Pleuroscopy and ICD. Introduction Pleuroscopy is a percutaneous endoscopic procedure performed by pulmonologist to visualize pleural space under local anesthesia. It is used for both diagnostic and therapeutic indications like recurrent pleural effusion, pneumothorax, empyema etc. . [1 &2] It is also a less invasive procedure and maintains clear optical field, obtaining specimen for biopsy from regions that are not easily accessible. [2] In the past decades Pleuroscopy has been found to be almost 100% accurate for malignant and tuberculous effusions, safe and effective with very low complication rate (morbidity of 2-5% and mortality < 0.1%). [1] In the year 1910, Jacobaeus described rigid endoscope instruments made up of stainless steel. In the late 1990s Semi-rigid pleuroscope replaced rigid pleuroscope because of its greater advantage over the rigid pleuroscope. It allows physician to overcome a limited view by manoeuvring its flexible tip in different directions. It can be rotated 180 degrees anterior and 130 degrees posterior. [1 & 3] Pleurodesis is the instillation of an irritant into the pleural space to cause inflammatory changes between the visceral and parietal pleural surfaces, effectively obliterating the potential pleural space. Various agents are used like tetracycline derivatives, erythromycin, talc, silver nitrate, iodoprovidone, sodium hydroxide, quinacrine etc. Among them Talc pleurodesis is a specific form of chemical pleurodesis which gives better results comparatively. Talc has shown various advantage over other chemicals and is considered the most effective sclerosant available for pleurodesis in malignant non malignant pleural effusion[4] The chest tube drainage also called as pleural drain, is the procedure in which the trocar and cannula are inserted into the pleural space through the incision made in the chest wall. This procedure is carried out to drain blood, air, bile, pus or any other fluid. As this is the invasive procedure it should be carried out using aseptic techniques to avoid any chances of instillation of infection in the pleural cavity. The procedure is done under local anesthesia. The placement of a ICD tube allows for continuous, large volume drainage of fluid or air until the underlying pathology which can be addressed properly.[1]

Figure1.1 ICD Figure 1.2 Pleuroscope 82 Vidya (2018) Vol. No : 1 Saxena et al A Retrospective Observational Study to compare efficacy of Pleuroscopic Chemical Pleurodesis and Blind Procedure (ICD) in Care of Pleural Pathology was carried at Pulmonology Department in Sparsh Hospital, Ahmedabad over a period of 4 months starting from December 2016 to March 2017.  The two treatment modalities which included pleurodesis through pleuroscopy and through Inter-costal drainage. All the patient with Pneumothorax and rapidly refilling Pleural Effusion were included who visited site from January 2014 to December 2016. Data were collected of the patients who underwent either of the two procedures. Data was collected of the follow up visit to identify any complication. The follow up Visit of patients was conducted 1 month after the procedure. All the patients with pleural pathology, recurrent Pneumothorax, secondary Pneumothorax and rapidly refilling Pleural effusion of age 18 years and above were selected from the database.For analysis the follow up of patients was taken telephonically and their 1 month follow up X-ray were received. Other Data was collected from hospital database and patient’s medical records.  Study Population All the patient with Pneumothorax and rapidly refilling Pleural Effusion were included who visited site from January 2014 to December 2016. Recruitment Procedure • Gender: Both • Age : 18 and above • Patients were included in the study on the basis of Inclusion and Exclusion Criteria.  Inclusion Criteria • Recurrent Pneumothorax (PSP) and in Secondary Pneumothorax • Rapidly refilling Pleural Effusion  Exclusion Criteria • Patient who could not tolerate procedure were excluded from the study like patients with cardiac disorders and haemodynamically unstable patients Statistical Analysis • The data was collected from hospital database and medical records of the patient in the CRF and then entered into the excel sheet for analysis. The analysis was done using SPSS 20.0 software • Analysis was done with Chi square test. P value was obtained to determine P Value. Results & Discussion All the patient with Pleural Pathology and rapidly refilling Pleural Effusion were included who visited site from January 2014 to December 2016. Total number of patients was 79.

83 Vidya (2018) Vol. No : 1

1. Gender Gender N %

Female 23 29.11

Male 56 70.89

Total 79 100.00

Table 1 Gender Table 1 shows that the total number female patients who underwent either of the procedure that is Pleuroscopy or ICD are 23 and total number of male patients are 56. 1. Pleuroscopic findings

Sr. no Findings No of patients

1 Sago grain, multiple necrotic nodules (patient with 10 recurrent pleural effusion)

2 Polypoidal mass lesion , Multiple pinkish nodules 20

3 Plaque 8

4 Bullae/ Bleb/Normal 16

Table 2 Pleuroscopic findings The Table 2 shows various Pleuroscopic findings. E.g Sago grain, multiple necrotic nodules (patient with recurrent pleural effusion), Polypoidal mass lesion, Plaque, Bullae/ Bleb etc. Pleuroscopy helps to diagnose the underlying cause of the disease and then to address accordingly. 1. Habits

Habits Yes % No %

Smoking 30 37.97.% 49 62.03%

Alcohol 5 6.33% 74 93.67%

84 Vidya (2018) Vol. No : 1 Saxena et al Table 3 Habits Table 3 shows that 37.97% patients presenting with disease were smokers and 62.03% of patients were non smokers. And the percentage of patients taking alcohol is 6.33%. 1. Follow up X-ray of patients with Pneumothorax

Follow up (Pneumothorax)

Group success % Failure % p value

Pleuroscopy 11 84.61 2 15.39 0.692 ICD 11 78.57 3 21.43

Table 4 Follow up X-ray (Pneumothorax) The table 4 shows that in patients with pneumothorax who underwent Pleuroscopicpleurodesis success rate is 84.61% and in ICD success rate is 78.57%. As per p value there is no significant difference in both the treatments. 1. Follow up X-ray of patients with Pleural Effusion

Follow up (PE)

Group success % Failure % p value

Pluroscopy 30 73.17 11 26.83 0.708 ICD 7 63.63 4 36.36

Table 5 Follow up (PE) In table 5 it shows that the success rate of patients with Pleural Effusion who underwent pleuroscopicPleurodesis is 73.17% while in ICD success rate is found to be 63.63%. As per the p value there is not much significant difference in both the treatments. 1. Malignancy and non-malignancy

85 Vidya (2018) Vol. No : 1

Indication Procedure MPE / NMPE Success Rate

Malignant 72%

Non-malignant 63.63% Pleuroscopy Malignant 100% Pleural Effusion Non-malignant 57% ICD

Table 6 Efficacy of Pleuroscopy& ICD in Malignant and non-malignant PE The Table 6 shows that the success rate in Pleuroscopicpleurodesis in patients with malignant and non-malignant is 72% and 63.63% respectively and in pleurodesis via Pleuroscopic via ICD is 100% and 57% respectively. 1. Primary Spontaneous & Secondary Spontaneous (Pneumothorax)

Indication Procedure PS/SS Success Rate

Primary 100% Spontaneous Pleuroscopy Secondary 66.67% Spontaneous Pneum Primary 100% -othorax Spontaneous ICD Secondary 62.5% Spontaneous

Table 7 Efficacy of Pleuroscopy& ICD in PS and SS Pneumothorax The Table 7 shows that the success rate in PleuroscopicPleurodesis in patients with primary and secondary pneumothorax is 100% and 66.6% respectively and in pleurodesis via ICD success rate in primary andsecondary pneumothorax is 100% and 62.5% respectively. Complication was seen in 3.70% of patients who underwent pleuroscopy it included visceral pleura injury, subcutaneous emphysema whereas in patients with ICD the complication was found to be 12% (e.g bleeding, subcutaneous emphysema, lung injury. Mean age in females was found to be 53 and in males it was 57. Conclusion Pleuroscopy and ICD both are effective procedures for Pleurodesis with minimum complication

86 Vidya (2018) Vol. No : 1 Saxena et al rate. As per the p value, both the treatments, that is, ICD and Pleuroscopy are equally effective in the patients with Pleural Effusion and Pneumothorax, but Pleuroscopy is better option for diagnostic purpose. ICD procedure is used by many practitioners since many decades and can be done bedside, similarly Pleuroscopy is also becoming procedure of choice among the professionals as it is less invasive when compared to open thoracotomy and can be done in minor OT under short sedation and local anaesthesia. At the same time it gives more insight about the disease as pleural cavity is directly visible. Semi-flexible Pleuroscope has more advantages over Rigid because of its flexibility of handling. In terms of cost, ICD can be considered better option and is found to be affordable by any population, but Pleuroscopy can be considered to have additional benefit for taking the biopsy as it allows visual field and desired tissue can be taken which helps to investigate further. So advantageous in patients with malignancy Complication was seen in 3.70% of patients who underwent pleuroscopy it included visceral pleura injury, subcutaneous emphysema whereas in patients with ICD the complication was found to be 12% (e.g bleeding, subcutaneous emphysema, lung injury. References DK Muduly, SVS Deo, TS Subi, AA Kallianpur and NK Shukla, An update in the Management of Malignant Pleural Effusion, Indian J Palliat Care, 2011 May-Aug; 17(2): 98-103 Loddenkemper R, Lee P, Noppen M, Mathur PN. Medical thoracoscopy/pleuroscopy: step by step. Breathe. 2011 Dec 1;8(2):156–67 Loddenkemper R, Mathur PN, Lee P, Noppen M. History and clinical use of thoracoscopy/ pleuroscopy in respiratory medicine. Breathe. 2011 Dec 1;8(2):144–55. Stephanie K. McCarty, Kolene E. McDate, Gaetane C. Michaund, Pleuroscopy: indications and clinical considerations, ISSN: 2281-6550, 2014 July-September; (3): 97-101 Tube Thoracostomy: Overview, Indications, Contraindications. 2017 Jan 6; Available from: http:/ /emedicine.medscape.com/article/80678-overview ❑

87 Vidya (2018) Vol. No : 1

Review Article ISSN: 2321-1520

Biosurfactants: An Overview

Dimple Pardhi, Rakeshkumar Panchal and Kiransinh Rajput*

*Department of Microbiology and Biotechnology, School of Science, Gujarat University,Ahmedabad E-mail Id:[email protected]

*Corresponding Author

Received Date : 10-2-2018 Published Date : 1-3-2018

Abstract Biosurfactants are the biologically synthesized surface-active agents. They are amphiphilic compounds consisting of hydrophilic and hydrophobic domains, that award the organism the ability to accumulate between fluid phases thus reducing surface and interfacial tension. Biosurfactants are produced by a number of microorganisms which include Bacillus sp., Rhodococcussp., Candida sp., Pseudomonas aeruginosa. They are classified based on their molecular weight and chemical composition. Several advantages of biosurfactants over the chemical surfactants are biodegradability, low toxicity, simplicityand bioavailability, specificity of action, structural diversity and effectiveness at extreme conditions. The physiological functions of biosurfactant production in microorganisms includes activities like, antimicrobial, anti-adhesive, antioxidant and the capability to compose substrates readily accessible for uptake by the cells in unfavorable environmental circumstances. Biosurfactants have several applications in petroleum, agriculture, medicine and food sectors. Keywords:Biosurfactants, Amphiphilic compounds, Pseudomonas aeruginosa, Biodegradability, Antimicrobial activity. Introduction Surfactants are surface active agents that can reduce surface and interfacial tensions between two liquids. Biosurfactants are the biologically synthesized surface-active agents. They are amphiphilic compounds consisting of hydrophilic and hydrophobic domains. The hydrophilic domain can be carbohydrate, amino acid, phosphate group or some other compounds whereas the hydrophobic

88 Vidya (2018) Vol. No : 1 Rajput et al domain usually is a long chain fatty acid (Lang, 2002). They are extracellular secondary metabolites, their structure depends on carbon and nitrogen ratio and influences on total production (Janeket al., 2013). Synthetic surfactant causes environmental problems due to their toxicity and resistants to biodegradation in ecosystem. Biosurfactants are considered as one of the high values of microbial products, which have gained considerable interest in recent years that have become an important product of biotechnology for industrial and medical applications. The reasons for their publicity are lower toxicity, specificity of action, simplicity of preparation and extensive applicability (Kaskatepeet al., 2015). The bacterial strains are responsible for the type and production of biosurfactants produced, it also depends on the carbon source and the physico-chemical conditions provided for the production. Many researchers had reported production of these surface active agents from microorganisms. Most of the work was carried out with glucose, glycerol, crude oil, diesel, gas oil etc. as a carbon source. A wide range of microbial genera were reported as the biosurfactant producers. The characteristic of biosurfactant varies from strain to strain; therefore, it is essential to assess different available strains for their biosurfactant potential and their characterization(Panesaret al., 2011).Biosurfactants are widely used for industrial, agricultural, food, cosmetics and pharmaceutical applications (FakruddinMd, 2012). Properties and Classification of Biosurfactants: The studies and production of biosurfactants have been increased in recent years because of its different beneficial characters as compare to the chemical surfactans. They reduces the surface tension of water, have excellent capacity of forming critical micelle concentration (CMC) and good compatibility and digestibility.Moreover, they includes bioavailability, biodegradability, structural diversity (Dattaet al., 2011), effectiveness at extreme conditions of temperature, pH and salinity, the potential for in situ production, the ability to be produced from cheap raw materials and the organisms producing them can be modified genetically to overproduce these compounds (Bodouret al., 2003). In compliance with such properties the biosurfactants are classified in two different ways, based on their molecular weight and chemical composition. 1. Classification based on molecular weight: (i) Low molecular weight biosurfactants: These compounds lower the surface and interfacial tension at the air/water interfaces (Salihuet al., 2009). Glycolipids, lipopeptides, flavolipids, corynomycolic acids and phospholipids are several examples of such type of biosurfactants. (ii) High molecular weight biosurfactants: They are generally referred to as bioemulsans and reported as more effective in stabilizing oil in water emulsions. This includesemulsan, alasan, liposan, polysaccharides and protein complexes (Salihuet al., 2009). 2. Classification based on Chemical Structure: (i) Glycolipids: Carbohydrates are incorporated in these types of biosurfactants as a constituent

89 Vidya (2018) Vol. No : 1 of mono-, di-, tri and tetrasaccharides including glucose, mannose, galactose, rhamnose, galactosesulphate and glucoronic acid to a long-chain aliphatic acid or lipopeptide (Vijayakumar and Saravanan, 2015). (ii) Lipopeptides and Lipoproteins: They are consisting of a lipid attached to a polypeptide chain. A large number of cyclic lipopeptides including decapeptide antibiotics (gramicidin) and lipopeptide antibiotics (polymyxin) produced by Bacillus brevisand B. polymyxarespectivelyhad notable surface active properties (Desai and Banat, 1997, Muthusamyet al., 2008). (iii)Fatty Acids and Neutral lipids: Microbial oxidations of alkanes results in the production of straight chain fatty acids which have been considered as a surfactant. Complex fatty acids like corynomuolic acids containing –OH groups and alkyl branches are also produced by microorganisms (Rahman and Gakpe, 2008). (iv)Phospholipids: They are major components of microbial membranes, when certain hydrocarbon degrading bacteria or yeast are grown on alkane substrates their level increases greatly. For example, when Acinetobactersp. HOI-N utilizes Hexadecane as a substrate, it produces the phospholipids that are mainly phosphatidyl ethanolamine (Muthusamyet al., 2008). (v)Polymeric microbial surfactants: Mainly these are polymeric heterosaccharide containing proteins, including emulsan, liposan, mannoprotein and polysaccharide protein complexes (Desai and Banat, 1997). (vi)Particulate Biosurfactant: There are some bacteria that produce extracellular membrane vesicles partition hydrocarbons that form micro-emulsion, which play an essential role in uptake of alkane by microbial cells for example in case of Acinetobactersp. (Desai and Banat, 1997).

Figure 1: Chemical Structures of Major Classes of Biosurfactants(FakruddinMd, 2012): (a) Mannosylerythritol lipid (b) Surfactin (c) Trehalose lipid (d) Sophorolipid (e) Rhamnolipid (f) Emulsan.

90 Vidya (2018) Vol. No : 1 Rajput et al Microorganisms Producing Biosurfactants: A variety of microorganisms produce biosurfactants that are diverse in chemical composition. The composition and yield of the biosurfactant produced exclusively depends upon the site from where the microorganism is isolated and the various nutritional factors available for their growth (Mulligan, 2005). Many microorganisms have been isolated from different sources for industrial utilization of the various types of agro-industrial waste products (Table-1). Thus, these have an ability to grow on substrates considered potentially harmful for other non-producing microorganisms. Where the field of production of biosurfactants by bacterial species is well explored, relatively fewer fungi are also known to produce biosurfactants. Table 1: Microorganisms Producing Potential Biosurfactants (Bhardwajet al., 2013): Microorganism Sources of Isolation By-products/ Carbon Biosurfactant Sources Pseudomonas sp. Oil spilled soil Glucose/ Molasses/ Cheese Rhamnolipid whey Pseudomonas sp. Used edible oil Used edible oil/ Rice- Rhamnolipid water/ Diesel/Petrol/ Whey Bacill us subtilis Crude oil contaminated Glucose/ Rapeseed oil Iturin Localities supplementedwith crude oil Bordetellahinizi- Crude oil contaminated Sucrose/ Molasses Trehalose-2,3,4,2’- DAFI Localities supplementedwith crude Tetraester oil Pseudomonas LBI, Petroleum Soap-stock Rhamnolipids aeruginosa contaminated Soil Serratiamarcescens Petroleum Glycerol Lipopeptide contaminated Soil Candida sp. SY-16 Oil-containing soil Soybean oil and Glucose Mannosylerythritol sample (Glycolipid) Pseudomonas Petroleum Palm oil Rhamnolipid aeruginosaSP4 contaminated Soil Rhodococcussp. Oil contaminated soil Sucrose/ Kerosene/ n- Extra-cellular heptane/ n-octane/ n- Li pids and hexadecane/ n- Glycolipid paraffin/Gas oil Bacill us subtilis Oil contaminated soil Vegetable oil/ Kerosene/ Surfactin Petrol/ Diesel Pseudomonas Oil contaminated soil Vegetable oil/ Kerosene/ Rhamnolipid

91 Vidya (2018) Vol. No : 1 Applications: Biosurfactants have gained considerable interest in recent years as they have several commercial and potential applications in many industrial regions such as: petroleum, organic chemicals, pharmaceuticals and cosmetics, beverages and foods, metallurgy, mining, petrochemicals, biological control and management and many others because of their amphiphilic properties (Table- 2). Table-2: Potential biosurfactants and their Applications (Banat et al., 2000; Shah et al., 2016):

Biosurfactants Applications Lipopeptide Bioremediation of marine oil pollution Bioremediation of hydrocarbon contaminated sites Oil recovery Granulocyte colony stimulating factor (G-CSF) Granulocyte-macrophage colony stimulating factor (GM-CSF) inducing activity Rhamnol ipid Food processing Bioremediation of oil-contaminated sites Bioremediation of hydrocarbon-contaminated sites Bioremediation of marine and soil environments Insecticidal effect Waste water treatment Sophorolipids Oil recover Control of environmental oil pollution Removal of petroleum and motor oil Glycolipid Recovery of oil from an oil well or oil sands Bioremediation of marine environment Removal of petroleum deri vat e motor oil from sand Induction of cell differentiation in the human promyelocytic leukemia cell line HL60 and anti-tumor agents Surfactin Anti-mycoplasma agent Anti-tumor agents Anti-fungal agent Anti-viral agents Iturin Anti-fungal agent Mannosylerythritol lipid Oil removal Bioremediation of marine environment Pumilacidin Anti-viral agent Viscoelastic Microbial Enhanced Oil Recovery

92 Vidya (2018) Vol. No : 1 Rajput et al The range of their applications includes microbial enhanced oil recovery, cleaning of oil tankers, transportation of crude oil, soil/sand bioremediation, remediation of organics and metals, used as emulsifiers, moistening agents, dispersing agents, foaming agents, beneficial food elements and detergents. (Banat et al., 2000; Perfumoet al., 2010). They augment the biodegradation and removal of oil through mobilisation, solubilisation or emulsification. Recently they have been widely used in enhance oil recovery process by microbes which play an important role in clean-up of oil containers/storage tanks and formation of products from petrochemicals (Liu et al., 2011). Mulligan (2009) reported the application of biosurfactants in enhanced soil washing for hydrocarbon- and metal-contaminated soils. The miscellaneous structures of biosurfactants award them the capability to exhibit versatile performance. By its structure, biosurfactants wields its toxicity on the cell membrane permeability bearing the resemblance of a detergent like effect (Sridhar et al., 2015). El-Sheshtawy and Doheim(2014) reported that biosurfactant produced by Pseudomonas sp. have strapping antibacterial and antifungal activity; these surfactant play the role of anti-adhesive agents to pathogens making them useful for treating many diseases as well as its use as therapeutic agent. Biosurfactant produced by Stenotrophomonas sp. was accounted for the antimicrobial and antioxidant activity, and also employed in the solubilization of phenanthrene (Gargauriet al., 2017). Biosurfactants have been also used for diverse food processing application but they generally play a role as food formulation ingredient and anti-adhesive agents, as food formulation ingredient they support the formation and stabilization of emulsion due to their capability to reduce the surface and interfacial tension. Biosurfactants from Saccharomycescerevisiae andPseudomonasaeruginosa were used in the fruit salad for dressing (Sridhar et al., 2010). References Banat, I., A. Franzeti, Gandolfi, I., Bestetti, G., Martinotti, M., Fracchia, L., Smyth T., Marchant, R., 2010. Microbial Biosurfactants: Production, Applications and Future Potential. Applied Microbiology and Biotechnology, Vol. 87: 427-444. Banat, I., Makkar, R., Cameotra, S., 2000.Potential Commercial Applications of Microbial Surfactants.Applied Microbiology and Biotechnology, Vol. 53: 495-508. Bhardwaj, G., Cameotra, S., Chopra, H., 2013.Utilization of Oleo-Chemical Industry By-Products for Biosurfactant Production. Journal of Petrolium and Environmental Biotechnology, Vol. 4(6): 2157-7463. Bodour, A., Drees, K., Maier, R., 2003. Distribution of Biosurfactant-producing Bacteria in Undisturbed and Contaminated Arid Southwestern Soils. Applied and Environmental Microbiology, Vol. 69: 3280-3287. Datta, S., Sahoo, S., Biswas, D., 2011.Optimization of Culture Conditions for Biosurfactant Production from Pseudomonas aeruginosa OCD. Journal of Advanced Scientific Research, Vol. 2(3): 32-36. Desai J., Banat, I., 1997. Microbial Production of Surfactants and Their Commercial Potential.

93 Vidya (2018) Vol. No : 1 Microbiology and Molecular Biology Reviews, Vol. 61(1): 47-64. El-Sheshtawy, H., Doheim, M., 2014.Selection of Pseudomonas aeruginosa for Biosurfactant Production and Studies of its Antimicrobial Activity.Egyptian Journal of Petroleum, Vol. 23(1): 1- 6. Fakruddin, Md., 2012. Biosurfactant: Production and Application. Journal of Petroleum & Environmental Biotechnology, Vol. 3(4):1-5. Gargouri, B., Contreras, M., Ammar, S., Carretero, A., Bouaziz, M., 2017.Biosurfactant Production by the Crude Oil Degrading Stenotrophomonas sp. B-2: Chemical Characterization, Biological Activities and Environmental Applications. Environmental Science Pollution Research, Vol. 24: 3769-3779. Janek, T., Lukaszewicza, M., Krasowska, A., 2013.Identification and Characterization of Biosurfactants Produced by the Arctic Bacterium Pseudomonas putidaBD2. Colloids and Surfaces B: Biointerfaces, Vol. 110: 379– 386. Kaskatepe, B., Yildiz, S., Gumustas, M., Ozkam, S., 2015.Biosurfactant Production by Psedomonasaeruginosa in Kefir and Fish Meal. Brazilian Journal of Microbiology, Vol. 46(3): 855-859. Lang, Y., 2002. Biological Amphiphiles (Microbial Biosurfactants). Current Opinion Colloid Interface Science, Vol. 7: 12-20. Liu, T., Hou, J., Zuo, Y., Bi, S., Jing, J., 2011.Isolation and Characterization of a Biosurfactant- Producing Bacterium from Daqing Oil-Contaminated Sites. African Journal of Microbiology Research, Vol. 5(21): 3509-3514. Mulligan, C., 2005.Environmental Applications for Biosurfactants. Environment and Pollution, Vol. 133: 183-198. Mulligan, C., 2009.Recent Advances in the Environmental Applications of Biosurfactants. Current Opinion on Colloid Interface Science, Vol. 14: 372-378. Muthusamy, K., Gopalakrishnan, S., Ravi, T., Sivachidambaram, P., (2008). Biosurfactants: Properties, Commercial Production and Application. Current Science, Vol. 94: 736-747. Panesar, R., Panesar, P., Beta, M., 2011.Development of Low Cost Medium for the Production of Biosurfactants. Asian Journal of Biotechnology, Vol. 3(4): 388-396. Perfumo, A., Smyth, T., Marchant, R., Banat, I., 2010.Production and Roles of Biosurfactants and Bioemulsifiers in Accessing Hydrophobic Substrates. Handbook of Hydrocarbon and Lipid Microbiology, Chapter 47: 1502-1510. Rahman, K., Gakpe, E., 2008. Production, Characterization and Applications of Biosurfactants- review. Biotechnology, Vol. 7(2): 360–370. Salihu, A., Abdulkadir, I., Almustapha, M., 2009.An Investigation for Potential Development on Biosurfactants. Biotechnology and Molecular Biology Reviews, Vol. 3 (5): 111-117. Shah, N., Nikam, R., Gaikwad, S., Sapre, V., Kaur, J., 2016.Biosurfactant: Types, Detection

94 Vidya (2018) Vol. No : 1 Rajput et al Methods, Importance and Applications. Indian Journal of Microbiology Research, Vol. 3(1):5- 10. Sridhar, B., Karthik, R., Tamil Venthan M., Khanna, G., 2010.Production, Purification and Antimicrobial Activity of Biosurfactants from Saccharomyces cerevisiaeand Pseudomonas aeruginosa. International Journal of Advance Research in Pharmaceuticals and Bio Science, Vol. 3(4): 1-7. Sridhar, B., Karthik, R., Tamil Venthan, M., Khanna, G., 2015.Production, Purification and Antimicrobial Activity of Biosurfactants from Saccharomyces cerevisiaeand Pseudomonas aeruginosaand its Application on Fruit Salads. International Journal of Advance Research in Pharmaceuticals and Bio Science, Vol. 6(10): 97-104. Vijayakumar, S., Saravanan, V., 2015.Biosurfactants: Types, Sources and Applications. Research Journal of Microbiology, Vol. 10(5): 181-192. ❑

95 Vidya (2018) Vol. No : 1

Review Article ISSN: 2321-1520

Cumin:A Natural Drug

Shah Rupal K., Patel Priyanka J., Trivedi Goral and Saraf Meenu*

Department of Microbiology and Biotechnology ,School of Sciences, Gujarat University,Ahmedabad. E-mail Id:[email protected]

*Corresponding Author

Received Date : 11-1-2018

Published Date : 7-3-2018

Abstract Plants are considered as rich sources of phytochemical ingredients which enable to have medicinal value. Medicinal plants are a potential source for the development of new herbal drugs. Indian spices are used as a revitalizers and treating various diseases conditions.They can be used as tonics,antipyretics,diuretics,antiheumatic etc. In the 21st century, the pharmacological effects of medicinal plants have been considered as a promising future drug/medicine for the management of health care.Cumin,commonly known as ‘Jeera’ is a well known spice of Mediterranean areas.It ismostcommon ingredient of various cuisines.The ripened fruit of the cumin plant is known as Cumin seeds which is used in whole or ground dried form.It consist of essential oil which is volatile in nature have a potential biological activities.The major constituent of cumin is cuminaldehyde due to which it posseses strong,warm and spicy odor.Apart of being used as an important spice due to its aromatic property,it has various medicinal properties.hence it is classified under MAPs(Medicinal and Aromatic Plants).Cumin has reported to have antioxidant,antimicrobial,anti carcinogenic antiosteoporotic etc.properties. Therefore, the aim of the present review is to understand the knowledge of the medicinal plants as a future source of herbal drugs. Keywords:Cumin,volatileoil,cuminaldehyde,monoterpenes. Introduction Spices are the bionutrient used as both food flavor enhancer and nutrient supplements.As per AyurvedicPharmacoepia( Indian System of Medicine),spices also have various medicine

96 Vidya (2018) Vol. No : 1 Saraf et al properties and also believed to aid digestion.Cuminum cyminum L. is commonly known as Cumin,belongs to Apiaceae family.Cumin stem is slender with many small branches with purple and white flowers having seeds of green colour at the center of each flower.It is an annual herb grown in various areas of Mediterranean countries like Africa,Asia and Europe.In India,it is cultivated in the state of Gujarat(44%) and Rajasthan(56%).Various varieties of cumin is cultivated throughout the world(Rebey IB et.al,2012)There are varieties of names of cumin in various international and Indian languages.As per USFDA the nutritional values are mentioned in table 3 Table:1 Some International Names of Cumin Languages Names

Arabic Kammun

Chinese Machin

Dutch Komijn

French Cumin

German Romischer Kummel

Table:2 Indian Names of Cumin

Languages Names

Hindi Zeera,Safaid Jeera

Gujarati Jeeru

Punjabi Jira

Kashmiri Zyur

Oriya Jeera

Sanskrit Jirika

Marathi Jeregiri

97 Vidya (2018) Vol. No : 1 Table:3Nutritional values of cumin

NUTRIENTS Percent of RDA

Total F ats 33%

Cholesterol 0%

Sodium 38%

Potassium 11%

Total Carbohydrates 14%

Proteins 36%

Calcium 93%

Magnesium 91%

Iron 86%

Vitamin A 25%

Vitamin D 0%

Vitamin C 12%

Vitamin B6 20%

Vitamin B12 0%

It is one of the known spice since ancient time.It’s origin is Iran.the description regarding cumin is mentioned in both Old and New Testaments of Bible.It was the common spice for Greeks which they kept in a container on the dining table.It was used by Egyptians for preservation and mummification of dead bodies.Cumin is the most important flavor of spice in Europe.Today,it is mostly grown in various regions of Iran,India,Syria,Mexico,Chile and China.Interestingly,it was used by Romans and Greeks for making pale complexion.(Anshul et.al.,2014).It is also used as 98 Vidya (2018) Vol. No : 1 Saraf et al flavoring agent in commercial foods.The seeds are sprinkled over cakes and breads.The oil extracted by steam distillation is used in deserts.The fragrant compounds are used in creams,lotions etc. Botanical Interpretation: It is a dried seeds of plantCuminum cyminum L.of Apiaceae family which is known as parsley family or collection of typically aromatic family.Seeds of cumin are resemble to caraway seeds,plant of Umbellifereae family commonly known as Persian cumin or Meridian fennel(Anshul et.al.,2014) Scientific Classification of Cumin

Kingdom Plantae

Subkingdom Tracheobionta

Superdivision Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Subclass Rosidae

Order Apiales

Family Apiaceae

Genus Cuminum

Species cyminum

Description of cumin plants: Fruits:The fruits of cumin are ovate or fusiform, of a light brown or grayish color. The fruit resembles caraway, but is larger and about 2 lines in length, much longer than the pedicels, nearly tapering, but little contracted at the sides, fusiform, crowned by the short teeth of the calyx,densely covered with short rough hair upon the channels, and less densely upon the ridges, which are paler, filiform, and a little raised; The seeds or half fruits, 2 in number, are oblong; Plano convex, with the plane surfaces together (L.).The odor and taste of cumin fruit is similar to caraway, but it’s so warmer and not so agreeable.

99 Vidya (2018) Vol. No : 1 Seeds: The cumin seed is yellow to brownish-gray in color and is elongated in shape with nine protuberances that possesses numerous medicinal properties. The seeds of cumin are carminative, aromatic,stomachic, stimulant, astringent and cooling and synergistic in effect. Cultivation: It is one of the known cash crop of India. Cultivation of cumin requires fertile, well-drained soil with temperature 25°C-28°C. Seeds are sown in the first week of November and harvested in first week of March. Marketing of cumin starts in the month of April-May. In Gujarat, cumin variety Gujarat Cumin-1 and Gujarat Cumin-2 is cultivated ,harvested and marketed. (Hajlaouiet al.2010) Cumin seed oil is used as multifunctional luminescent paints or in topical ointment.Table 4 showing active component and uses of members of Apiaceae family.

Figure: 1 Cumin seeds

100 Vidya (2018) Vol. No : 1 Saraf et al

Table: 4 Appearance and properties of the Apiaceae family members Biological name Common name Active constituents uses

Carum carvi Caraway se ed, Carvone, limonene Antispasmodic, caraway fruit, carvacrol, antiseptic,

persian cumin, a-pinene, g-terpinene, antiparasitic, meridian fennel linalool, lactigenic,

carvenone, and p- hypolipidemic, cymene aromatic,

carminative, digestive, stomach-

calming and stimulant

Cuminum cyminum, Cumin, Jeera Safed b-pinene, p-cymene, Carminative, jeeraa eupeptic, astringent, g-terpinene, and cuminaldehyde antibacterial, cough remedy and

analgesic

Foeniculum vulgare Sweet cumin, sweet Anethole, α-pinene, Carminative, cold, fennel cough and cattle β-myrcene, β-pinene, fenchone, condiment

camphene, estragole,

fenchone,

limonene, p-cymen, and safrole

101

Vidya (2018) Vol. No : 1 Saraf et al expression.This activity is due to cuminaldehye.Streptococcus mutans was inhibited by biofilm formation preventive properties of limonene,pineneand other minor components.Antifungal activities of cumin were reported against various soil,food and human pathogens. 3.Anticarcinogenic Activity: Cumin oil possess potential anticarcinogenic activity.(Nalini N,2006) From a study carried out on rat with dietary supplementation of cumin was found to prevent the occurrence of rat colon cancer induced by mutagen 1,2-dimethylhydrazine(DMH).This leads to increase in excretion of bileacid and the decrease in activity of â-glucuronidase that causes the hydrolysis of glucuronide and produce toxins and mucinase enzyme that increases the hydrolysis of mucins in colon by dietary supplementation of cumin to mice inhibits the benzopyrene induced forestomach tumorigenesis,hepatomas etc.Cumin attributes the ability of modulating carcinogen metabolism by phase I and phase II metabolizing enzymes.the property of cumin is due to the presence of monoterpenes like anethofluran,carvone and limonene.with the anticarcinogenic property,cumin do have apoptotic,antimutagenic and antiproliferative properties. 4.Immunomodulatory Activity: In one of the dose dependent study,the oral treatment with cumin showed immunomodulatory activity in mormal and immunosuppressed animals by modulating T-lymphocytes,T-cells i.e.CD4+ and CD8+ ,Th1 cytokines expression in normal an cyclosporine-A in immunosuppressed animals(Chauhan PS,2010)In these animals,cumin compounds counters the depleted T- lymphocytes,decreases the weight of thymus and spleen. 5.Anti-Osteoporotic Activity: Various researches has reported the anti-osteoporotic activity of cumin seeds.(Malini been reported that cumin seeds reduces the urinary calcium excretion and leads to the augmentation of calcium.This increases the mechanical strength of bones. 6.Gastrointestinal: An aqueous extract of cumin was perfused in aspirin induced gastric mucosal injury of the rat leads to increase in the secreation of acid that heals the injury and prevent the reoccurring.Cumin extracts also increases the amylase,protease,lipases and phytaseactivities.(Johri RK,2011) 7. Drug Bioavailability Enhancing Activity: A pharmacokinetics interaction of herbal plants with various anti-tubercular drugs has reveal that the aqueous extract of cumin leads to the enhancement of rifampicin levels in rat plasma.it is due the unique novel component of cumin known as flavonoid glycoside.(Sachin BS,2009) Traditional Uses of Cumin asa Medicine: Cumin has been used from ancient times for healing the unhealthy body and making body healthy.It is a very good source of Iron that helps in transport of oxygen to all the cells of body.It increases the efficiency of nutrient absorption.it helps in relieving abdominal pain,muscles cramps and flatulence.It increases the milk production of mother during lactation period.it has culinary 103 Vidya (2018) Vol. No : 1 uses and it is an essential ingredient in all mixed spices for flavoring of soups,pickles,breadsetc Aroma Portrayal of Cumin: Cuminaldehyde (4-isopropylbenzaldehyde)is the major component of volatile essential oil of cumin which imparts strong ,pungent,spicy odor to cumin.the other componds are substituted pyrazines,2-ethoxy-3-isopropylpyrazine ,â-pinene and ã-terpines. Conclusion In short,it can be said that cumin is not only an important spices but also an important medicinal and aromatic plant.It is used as a flavoring agent in food and beverages industry.It is having various pharmacological activities that can make body healthy and heal various disorders efficiently.It has been used since ancient era but still it is a hidden chapter for the reseachers for various hidden activities of cumin as a drug. References Aggarwal, B. B. and Kunnumakkara, A. B. (2009).Molecular Targets and Therapeutic Uses of Spices - Modern Uses for Ancient Medicine. World Scientific Publishing Co. Pvt. Ltd. Singapore, 430 p [Different chapters written by experts compiling therapeutic uses of spices of selected spices, fenugreek in the present case] AICRPS (2010). All India Coordinated Research Project on Spices, 2009. Annual Report 2009- 10. Indian Institute of Spices Research, Calicut.131pp. Al-Hashemi FHY.( 2014) Chromatographic separation and identification of some volatile oils, organic acids and phenols from the seeds of cuminum cyminum growing in Iraq. IJRRAS; 19(1): 80 -90. AnshulBansal, VaibhavBansal, Rajeshwar Singh (2014) Cumin: A spice or a drug? World Journal of Pharmaceutical Sciences, ISSN (Print): 2321-3310 Bukhari SB, Iqbal S, Bhangar MI (2009). Antioxidant potential of commercially available cumin (CuminumcyminumLinn.). International Journal of Food Sciences and Nutrition;60: 240-247. Chauhan PS, Satti NK, Suri KA, Amina M, Bani S. (2010)Stimulatory effects of Cuminum cyminum and flavonoid glycoside on cyclosporine-A and restraint stressinduced immune- suppression in swiss albino mice. Chem Biol Interac 185: 66-72. Derakhshan S, Sattari M, Bigdeli M.(2008) Effect of sub-inhibitory concentrations of cumin (Cuminum cyminum L.) seed essential oil and alcoholic extract on the morphology, capsule expression and urease activity of Klebsiella pneumoniae. International Journal of Antimicrobial Agents;32: 432-436. El-Ghorab AH, Nauman M, Anjum FM, Hussain S, Nadeem M (2010).A comparative study on chemical composition and antioxidant activity ofginger (Zingiber officinale) and cumin (Cuminum cyminum). Journal of Agricultural and Food Chemistry;58: 8231-8233. El-Sawi SA, Mohamed MA (2002). Cumin herb as a new source of essential oils and its response to foliar spray with some micro-nutrients. Food Chemistry,;77: 75-80. 104 Vidya (2018) Vol. No : 1 Saraf et al

Hajlaoui H, Mighri H, Noumi E, Snoussi M, Trabelsi N, Ksouri R. (2010) Chemicalcomposition and biological activities of Tunisian Cuminum cyminum L. essential oil: A high effectiveness against Vibrio spp. strains. Food Chem Toxicol;48: 2186-92. Kitazima J, Ishikawa T, Fujimatu E, Kondho K, Takayanagi T. (2003)Glycosides of 2-C-methyl- D-erythritol from the fruits of anise, corianderand cumin. Phytochemistry ;62: 115-120. Nalini N, Manju V, Menon VP. (2006) Effect of spices on lipid metabolism in 1, 2-dimethylhydrazine-induced rat colon carcinogenesis. J Med Food 9: 237-45. Rakotonirainy MS, Lavédrine B.(2005) Screening for antifungal activity of essential oils and related compounds to control the biocontamination in libraries and archives storage areas. International Biodeterioration and Biodegradation. ;55(2):141–147. Rebey IB, Jabri-Karoui I, Hamrouni-Sellami I, Bourgou S, Limam F, Marzouk B.( 2012)Effect of drought on the biochemical composition and antioxidant activities of cumin (Cuminum cyminum L.) seeds. Industrial Crops and Products 36:238-245. Romagnoli C, Andreotti E, Maietti S, Mahendra R, Mares D. (2010)Antifungal activity of essential oil from fruits of Indian Cuminumcyminum. Pharmaceutical Biology;48: 834-848 Sachin BS, Monica P, Sharma SC, Satti NK, Tikoo MK, Tikoo AK. (2009)Pharmacokinetic interaction of some antitubercular drugs with caraway: Implications in the enhancement of drug bioavailability. Hum Exp Toxicol 28: 175–84. Shetty RS, Singhal RS, Kulkarni PR(1994). Antimicrobial properties of cumin. World Journal of Microbiology and Biotechnology,;10:232-233 Zaman U, Abbasi A.(2009) Isolation, purification and characterization of a nonspecific lipid transfer protein from Cuminumcyminum. Phytochemistry,;70: 979-987 ❑

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