Original Article Phytochemical Screening and GC-MS Analysis in Wild Variety of indica – An Future Promising Therapeutic Source V. Pushpa Rani*, B.B. PraneshMoorthy, K.Shanmuga Priya, A.Anitha Nancy and G.Meena Kumari

Department of Advanced Zoology & Biotechnology, Loyola College, Chennai-600 034, Tamil Nadu, India

ABSTRACT Objective: In current, attention has been reverting back to as

sources of therapeutic agents due to their superior properties. The

literature review exposed that there is no report on phytochemical

constituents of wild variety of Coccinia indica. As best of our

knowledge there is no such report on study on the fruits of wild

variety of Coccinia indica.

Methods: The phytochemical analysis of alcoholic extract of the

fruits of wild variety of Coccinia indica was done first time. Further

study has been preceded using Gas Chromatography – Mass

Spectrometry (GC-MS) from alcoholic extract of wild variety of

Coccinia indica fruit.

Result: Alcoholic extract of wild variety of Coccinia indica fruit

responded optimistically for Steroids, Terpenoids, Saponins,

Flavonoids Alkaloids, Tannins, Glycosides, Phenol and Mucilage Address for during qualitative phytochemical analysis. With reference to Library of Terpenoids, it is confirmed that the following compounds are Correspondence belongs to the group of Terpenoids and identified Terpineol which is Department of also belongs to the Terpinoid group. Advanced Zoology & Conculsion: Further investigation may lead to isolation of bio-active Biotechnology, Loyola compounds and their structural elucidation and screening of College, Chennai-600 pharmacological activity will be cooperative for further drug 034, Tamil Nadu, India development.

E-mail: push.rani76@ Keywords: Coccinia indica, Wild type, Phytochemical, Bio-active gmail.com compounds, Gas Chromatography – Mass Spectrometry and pharmacological

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INTRODUCTION Most of the modern researches on out on Gas Chromatography-Mass herbal medicine have hinged around Spectrometry traditional folklore medicine. The modern medicine has brought with it an array of METHODOLOGY drugs, none of which is non-toxic and quite safer for human consumption. There are Sample Collection hundreds of medicinal plants that have a Wild variety type of Coccinia indica long history of curative properties against (Wight and Arn) Family various diseases and ailments. However, fruits were collected from Alamadhiis screening of plants for their activity is very geographically located at latitude 13° 12′0'' essential and needs urgent attention in order and longitude 13° 12′0'', Chennai, to know the value of the . TamilNadu, India during the month of Plants have always been an January 2015. Two varieties of C. indica are exemplary source of drugs and many drugs recognized; tender fruits are bitter in one variety and not bitter in another, and the currently available have been derived 2-4 directly or indirectly from them. Indian latter is used in Asian cooking . system of traditional knowledge i.e. Morphologically no difference is evident Ayurvedic is well known for its effective between them, however; both varieties are herbal treatments. There are about 7000 invasive and are found to grow close to each plant species are found in India. Although other. most of them have a long history in folk medicine, there is lack of written data on Processing of Sample their efficacy and safety, especially from Mature unripe fruits were washed human studies. with running tap water and drained. The One such medicinal plant is fruits were sliced then shade dried in room Ivy Gourd (Syn. Coccinia indica Wight & temperature for 10 days and pulverized in Arnott.) is indigenous plant of Central mechanical grinder into coarse powder and Africa, India and Asia. Coccinia indica stored in airtight containers for further (Fig. 1) is belongs to the Family process. The extract was prepared using alcohol by Soxhlet apparatus. As per the Cucurbitaceae. It is found in climate that is 5 warm and humid. It is found in whole India protocol reported in was followed to obtain in wild. It is cultivated abundantly in India Coccinia indica mucilage extract. (Assam. Bihar, Orissa, Maharashtra, Andhra Pradesh, Tamil Nadu) as a vegetable and its Phytochemical Screening wild form is also found in many parts of Phytochemicals are the natural India. This plant has been widely used in bioactive compounds found in plants. The traditional Indian medicinal system1.The qualitative analysis of phytochemicals such phytochemistry and pharmacology of one as Steroids, Terpenoids, Saponins, popular anti-diabetic and hypoglycemic Flavonoids, Alkaloids, Carbohydrates, plant Coccinia indica. Tannins, Glycosides, Phenol and The present study focused to reveal Mucilagewere performed in the study. The Phytochemical analyses were carried out as phytochemical constituents from the 6 alcoholic extraction of wild variety Coccinia described by to screen the presence of the indica fruit and further investigation carried bioactive ingredients from the fruit extract.

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Gas Chromatography – Mass Spectrometry were visualized (Fig: 2). The particular The Gas Chromatography – Mass compounds present in the samples were Spectrometry (GC-MS) analysis was identified by matching their mass spectral performed on a combined GC-MS fragmentation patterns of the respective instrument (ITQ 900 Model of Thermo peaks in the chromatogram with those stored Fisher Scientific make) using a HP-5 fused in the National Institute of Standards and silica gel capillary column. The method to Technology Mass Spectral database (NIST- perform the analysis was designed for both MS, 1998) library. GC and MS. 1 µL aliquot of sample was The present study predicted the injected into the column using a PTV formula and structure and molecular weight injector whose temperature was set at 275ºC. of eight bio-molecules which has been The GC program was initiated by a column shown highest peak compare to the other temperature set at 60oC for 5 min, increased peaks. The name of the compounds and their to 300ºC at a rate of 8 °C/min, held for 10 molecular formula, molecular weight, peak min. Helium was used as the carrier gas (1.5 area (%) and retention time were tabulated mL/min). The mass spectrometer was in Table: 2. The GC-MS graph and their operated in EI mode with mass source was molecular structure of these eight identified set at 200oC. The chromatogram and compounds were illustrated. spectrum of the peaks were visualized. DISCUSSION RESULT There is no literature exist in wild Phytochemical Screening variety of Coccinia indica fruit so the In the present study preliminary present results were compared with reported phytochemical screening has done in the results of domesticated Coccinia indica fruit wild variety of Coccinia indica fruits. The and also to justify that wild variety Coccinia phytochemical screening from the fruits of indica fruit can be substitute domesticated wild variety of Coccinia indica revealed that Coccinia indica. Since wild variety Coccinia the presence of potential phytonutrients indica fruits are bitter in nature therefore present in the domesticated Coccinia indica they can be utilized for the therapeutic which has been reported in the previous. The properties which have been demonstrated by extract was subjected to qualitative chemical the researchers in domesticated Coccinia analysis. Alcoholic extract answered indica. The results of qualitative positively for steroids, terpenoids, saponins, phytochemical profiling were clearly flavonoids alkaloids, tannins, glycosides, revealed that the plant contained different phenol and mucilage compounds which bioactive compounds such as Steroids, were shown in Table 1. Terpenoids, Saponins, Flavonoids Alkaloids, Tannins, Glycosides, Phenol and Mucilage Gas Chromatography - Mass Spectrometry which are key compounds for the medical (GC-MS) properties as per the existing literature. The results of GC-MS analysis of the Earlier studies by various researchers also alcoholic extract of the fruits of wild variety supported that these compounds were of Coccinia indica lead to the identification identified for the medicinal value of the of a number of compounds. These plants. Also identified that these compounds compounds were identified through mass play role in curing the diseases (diabetic, spectrometry attached with GC. The cancer, malarial, inflammation and chromatogram and spectrum of the peaks

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tuberculosis) through animal studies using so this extraction also can be used as sewage different extracts of the plant. water treatment. The investigation by7demonstrated Phytochemical screening of Coccinia that Saponin and Flavonoid are found to be grandis fruit powder in organic solvent accountable for anti-diabetic activity of (methanol) revealed the presence of Coccina indica the present study of wild alkaloids (0.22±0.07mg), flavonoids variety Coccinia indica fruit also confirmed (1.09±0.00mg), saponin (0.05±0.01mg) and the presence of Saponin and Flavonoid. phenol (17.01±0.02mg). Anti-inflammatory The study carried out by8 activity was assessed by Human red blood demonstrates the significance of Coccinia cell membrane stabilization assay (HRBS) indica as anti-inflammatory agent. 60% by varying concentration of the fruit extract methanolic extract contained Flavonoid ranging from 100mg/ml to 500mg/ml. The which can be responsible for the inhibition results obtained in the study showed that the of prostaglandin synthesis which influenced fruit extract of Coccinia grandis showed anti-inflammatory activity in the tested rats. 44.46±0.09% and 84.63±0.04% anti- Therefore, the present study results inflammatory activity for 100mg/ml and represented that the presence of Flavoinds in 500mg/ml concentration respectively, and the alcoholic extract of wild variety of the activity was found to be concentration Coccinia indica fruit also can be utilized as dependent and proved folklore practice of anti-inflammatory agent. the fruit extracts in curing various The previous study by9showed that inflammatory responses11. wound healing potency of C. indicia be The growing interest in recent years attributed to the phytoconstituents such as in medicinal plant research has been to a Tannins, Flavonoids, Terpenoids, and greater extent due to rapid progress in Saponins present in it, which may be either experimental techniques, as well as to a due to their individual or additive effect that growing realization of the vast potential of fastens the process of wound healing. plant based resources. Correspondingly the results of From the GC –MS results indicated phytochemical screening of alcoholic that the presence of many bio-active extraction of wild variety Coccinia indica compounds in wild variety Coccinia indica fruit revealed the presence of these fruit. The GC- Mass Spectrometer phytochemicals which prominently shows investigated the compounds elute at the wound healing property of wild variety different times to identify the nature and Coccinia indica. structure of the compounds. The large The work results showed that the compound fragments into small compounds fruit extract of C. indica mucilage has higher giving rise to appearance of peaks at efficiency in removing high turbidity in different m/z ratios. These mass spectra are comparison with low turbidity10.The fingerprint of that compound which can be coagulation efficiency of C. indica mucilage identified from the data library. in the study was found to be dependent on With reference to Mass Finder initial turbidity of water samples. The Library of Terpenoids, it is confirmed that coagulation efficiency of C. indica mucilage the following compounds are belongs to the in that study was found to be dependent on group of Terpenoids. P-Methoxybenzoic initial turbidity of water samples. The acid, tetradecyl ester; Molecular Formula: current study of wild variety Coccinia indica C22H36O3; Molecular Weight: 348, (6Z)- fruit also proved the presence of mucilage, Nonen-1-ol; Molecular Formula: C9H18O;

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Molecular Weight: 142, 2-octyn-1-ol; potential. To establish a quantitative Molecular Formula: C8H14O; Molecular relationship between the species popularly Weight: 126. 2,4-pentadien-1-ol, 3-pentyl- considered to be anti-microbial, anti- (2Z); Molecular Formula: C10H18O; diabetic, and anti-diarrheal, the contents of Molecular Weight: 154 this is identified as tannins and flavonoids were determined14. Terpineol which is also belongs to the Despite the broad use of C. indica in Terpinoid group. traditional medicine, very few systematic Terpenoids are found to be pharmacological and phytochemical studies responsible for anti-diabetic activity7. Plant are reported till date assessing its therapeutic terpenoids are widely used as industrially properties. The present study is the relevant chemicals, including many inauguration for the further details of pharmaceuticals, flavours, fragrances, pharmacological study on wild variety of pesticides and disinfectants, and as large- Coccinia indica. volume feed stocks for chemical industries. Recently, there has been a renaissance of CONCLUSION awareness of plant terpenoids as a valuable biological resource for societies that will The current study reported the phyto- have to become less reliant on costituents of wild variety of Coccinia petrochemicals12. GC-MS results of wild indica fruit for the first time. Alcoholic variety C. indica fruit indicated the presence extract of wild variety of Coccinia indica of terpenoids from the bio-compound fruit answered positively in qualitative identified which confirms the various phytochemical analysis. Further Gas properties has been proven by the Chromatography – Mass Spectrometry (GC- researchers. MS) was used for identification of bio-active The carried out the study on compounds. The results of wild variety of Terpineol has important anti-inflammatory Coccinia indica fruit were judged against and anti hypernociceptive properties. These previous literatures of Coccinia indica to effects seem to be associated with the power substantiate that wild variety Coccinia of Terpineol to inhibit the cytokine indica has equivalent pharmacological cascadegenerated by carrageenan and/or properties. In view of the fact that wild decrease the production of inflammatory variety of Coccinia indica fruit is not mediators, as well as inhibit Nitric Oxide exposed or utilized for medicinal property release13. Thus, a-terpineol can be an hitherto. This study will be renaissance and interesting candidate for the development of ready to lend a hand for further detail new drugs for treating painful conditions analysis of wild variety of Coccinia indica associated with inflammation. GC-MS as medicinal plant. results of wild variety C. indica fruit indicated the presence of terpineol from the Conflict of Interests bio-compound identified which validate the The authors declare that there is no range of properties have been demonstrated conflict of interests. in the previous works. An approach used in the search for REFERENCES natural substances that possess therapeutic 1. GoldyYadav., (2010), Medical properties of value is ethnobotany or ethnopharmacology. ivy gourd (Cephalandra indica) : a review, Active substances that have phenolic groups International Journal of Pharmacology in their structure have great pharmacological

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Research and Development. vol-2,issue- 9. Mukharjee, P. K., (2002), Quality Control of 9;014. Herbal Drugs. Business Horizons.1st 2. Ramachandran, K., and Subramaniam, B., edition.New Delhi; 546-49. (1983), Scarlet gourd, Coccinia grandis, little- 10. VarshaPatale and Jay Pandya., (2012), known tropical drug plant, Economic Botany, Mucilage extract of Coccinia indica fruit as 37, 380–383. coagulant-flocculent for turbid water 3. Kunkel, G., (1984), Plants for Human treatment, Asian Journal of Plant Science and Consumption. Koenigstein, Germany: Koeltz Research, 2012, 2 (4):442-445. Scientific Books. 11. V.Selvam, R. Sumathy and 4. Manandhar, N. P., (2002), Plants and People R.Kumuthakalavalli., (2014), Phytochemical of Nepal. Portland, OR: Timber Press. constituents and invitro anti - inflammatory 5. Muyibi, S., Megat, M., and Loon, L., (2002), activity of an ivy gourd, Coccinia grandis. Effects of Oil Extraction from Moring olifera World Journal of Pharmacy and seeds on coagulation of turbid water, Journal Pharmaceutical Sciences. Vol 3(7), 1173- of Environmental Studies, 59(2): 243-254. 1179. 6. Harborne J.B., (1998), Phytochemical 12. JörgBohlmann and Christopher I. Keeling., methods: a guide to modern techniques of (2008), Terpenoid biomaterials, The Plant plant analysis. (3rd ed.) London: Chapman & Journal, 54, 656–669. Hall. ISBN: 0-412-57270-2, 302 p. 13. Gisele L. Oliveira, Davyson de L. 7. Deokate, U. A., and Khadabadi, S. S., (2011), Moreira,Aretusa Daniela R. Mendes, Elsie F. Pharmacology and phytochemistry of Guimarães, Lourdes S. Figueiredo , Maria Coccinia indica, Journal of Pharmacognosy Auxiliadora C. Kaplan , Ernane R. Martins., and Phytotherapy Vol.3 (11), pp.155-159. (2013), Growth study and essential oil 8. Aritra Chatterjee, Sumana Chatterjee., (2012), analysis of Piper aduncum from two sites of Proximate analysis, phyto-chemical screening Cerrado biome of Minas Gerais State, and anti-inflammatory activity of Coccinia Brazil.Rev Bras Farmacogn 23: 743-753. indica. Int. J.pharma, chemical & boil. Sci. 2(3): 299-304.

Table 1. Qualitative Analysis of the Phytochemical from the Alcoholic Extraction of Wild Variety of Coccinia indica fruit.

PHYTOCHEMICALS RESULT

STEROIDS +

TERPENOIDS +

SAPONINS + FLAVONOIDS + ALKALOIDS + CARBOHYDRATES - TANNINS + GLYCOSIDES + PHENOLS + MUCILAGE +

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+= Presence of Phytochemical; - = Absence of Phytochemical Table 2. Compounds Identified from GC –MS of the Alcoholic Extract from Wild Variety of Coccinia indica Fruit. Peak Compound Molecular Molecular Retention Components Area No. Formula Weight Time (%)

1 2-amniophenyl,N-(tert-butyldimethylsilyl) C18H25NSi 283 4.92 0.05

2 P-Methoxybenzoic acid, tetradecyl ester C22H36O3 348 6.18 0.07

3 (6Z)-Nonen-1-ol C9H18O 142 7.20 0.10

4 Benzene, 2-benzyl-1-methyl-3-nitro C14H13NO2 227 8.60 0.07 [1,1’-bicyclopropyl] 2-octanoic acid, 2’-hexyl- 5 C H O 322 9.81 0.10 methyl ester 2 38 2

6 2-octyn-1-ol C8H14O 126 10.88 0.06

7 2,4-pentadien-1-ol, 3-pentyl-(2Z) C10H18O 154 12.55 0.06 3-Buten-2-one,4-(2,2,6,7- tetramethyl-7- 8 C H NO 221 14.06 0.08 azabicyolo[4.10] heptan-1-yl) 14 23

Systematic Position of Coccina indica

Kingdom Plantae Division Magnoliophyta

Class Magnoliophyta

Order Family Cucurbitaceae

Genus Coccinia

Species indica

* Source: From the site of collection.

Fig: 1 Coccinia indica

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34 33.54 32.41

32

30.99 30

28.45 28 28.29

27.20 26 26.14 26.55 25.77 25.42 24 24.89 24.67

24.53 24.04 23.85 23.46 22 22.78

22.58 21.20 20 18.89 18.35

18 Time (min) Time 16.96 16 16.76

15.68 14 14.06

12.55 12

11.44 10.88 10 9.81 8.60 8 7.20 6 6.18 4.92

4 11G SM: 2

0 5 0 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 0.00 - 35.36 - 0.00 100

bundance A elative R RT:

Fig. 2 GC-MS Graph from the Alcoholic Extraction of Wild Variety Coccinia indica fruit.

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