Original Research Article

Antibacterial, antioxidant and cell proliferative properties of grandis fruits Prashant Sakharkar1, Balwantsinh Chauhan2,*

1Department of Clinical & Administrative Sciences, Roosevelt University College of Pharmacy, 1400 N. Roosevelt Blvd., Schaumburg, IL, 60173, USA 2Department of Bipoharmaceutical Sciences, Roosevelt University College of Pharmacy, 1400 N. Roosevelt Blvd., Schaumburg, IL, 60173, USA

Article history: Abstract Received: Jun22, 2016 Objective: Little knowledge is available on the antimicrobial and Received in revised form: Jan 16, 2017 antioxidant properties of Coccina grandis fruits and no study has Accepted: Jan 22, 2017 reported on its cell proliferative property. The aim of this study Vol. 7, No. 4, Jul-Aug 2017, was to examine the antimicrobial, antioxidant and cell proliferative 295-307. property of fruits of C. grandis. Materials and Methods: Fruits of C. grandis were extracted * Corresponding Author: using water; ethanol and acetone by cold and hot Soxhlet Tel: (+1) 847-330-4532 extraction. The antibacterial activities of the extracts were tested Fax:(+1) 847-330-4525 against Staphylococcus aureus, Enterococcus faecalis, Escherichia ‎[email protected] coli and Pseudomonas aeruginosa using the modified Kirby-Bauer diffusion method and compared against erythromycin. The Keywords: Coccinia grandis antioxidant property was determined using Cayman's antioxidant Antibacterial assay; whereas cell proliferation/cytotoxic properties were Antioxidant evaluated using the Cell Titer 96 Aqueous One Solution Cell MTS Cell proliferation assay with MDA-MB 321 breast cancer cells. Data were analyzed Ivy gourd for correlation and differences using unpaired student's t-test and one-way ANOVA. A p value of <0.05 was considered statistically significant. Results: Both cold and hot ethanol and acetone extracts of C. grandis fruits showed some degree of bacterial growth inhibition. Acetone extracts exhibited higher antibacterial activity. Both ethanol extracts showed antioxidant property when compared with standard Trolox. In contrary to cytotoxicity, all four extracts showed cell proliferation compared to controls at different concentrations. However, acetone extracts exhibited greater cell proliferation compared to ethanol extracts and cold extracts performed better than the hot extracts. Conclusion: C. grandis fruits exhibited some degree of antimicrobial, antioxidant and cell proliferative properties. Further investigation is warranted to isolate, confirm and characterize phytochemicals that are responsible for the medicinal properties observed. Please cite this paper as: Sakharkar P, Chauhan B. Antibacterial, antioxidant and cell proliferative properties of Coccinia grandis fruits. Avicenna J Phytomed, 2017; 7 (4): 295-307.

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Introduction has been considered of having some The concept of combining dietary medicinal value (Yadav et al., 2010; constituents to manage various illnesses is Nagare et al., 2015). Plant preparations historically part of most of the cultures. from C. grandis are indigenously used for Currently, there is wide spread use of various skin diseases, bronchitis, anorexia, complementary and alternative medicine cough, asthma, catarrh, and epilepsy. (CAM) globally. Of five categories of Moreover, in Unani systems of medicine it CAM identified, the herbal products are has been used for ringworm, psoriasis, the most abundantly used form of the small pox and (Kirtikar and Basu, therapy. The search for new herbal 1994; Philcox, 1997; Nagare et al., 2015). products/drugs for different human Plant preparations are also used for itchy ailments is increasing, as they are believed skin eruptions, wound healing, leprosy, to be less or non-toxic in nature. It is well gonorrhea, pyelitis, cystitis, snakebite, recorded that various belonging to malarial infection, infective hepatitis, and family are used as herbal jaundice; Also, it has been given as a medicine in most of the culture. Coccinia hepatoprotective remedy and for treating plants (Ivy) belonging to Cucurbitaceae renal calculi (Kirtikar and Basu, 1994; family have their own importance in Vadivu et al., 2008; Shaheen et al., 2009; traditional medicines, including Ayurveda, Deshpande et al., 2011a; Dnyaneshwar and practiced in India, Chinese herbal Patil, 2011; Ramakrishnan et al., 2011; medicines practiced in China and Unani Sood et al., 2012). C. grandis is also system of medicine or Greco-Arab known for its anti-diabetic, anti-obesity, medicine practiced in Iran (Khan et al., antimicrobial, antifungal, antileishmanic, 1979). antioxidant, antihypertensive, antitussive, Ivy gourd is known in India by various antiulcer, analgesic, antipyretic, vernacular names like tondi in Marathi, antianaphylactic, and anti-cancer Tindora, and Kundu in , properties (Kirtikar and Basu,1994; Yadav dondakaya in telugu, tomdekayi in et al., 2010; Tamilselvan et al., 2011; , etc. (Ali et al., 2005). In other Pekamwar et al., 2013; Gill et al., 2014; parts of the world, it is known as Hong gua Nagare et al., 2015). However, its in Chinese, Bat in Vietnam, Pepasan in therapeutic efficacy is yet not conclusive Malay, Yasai, Karasuuri in Japanese, due to the lack of carefully controlled Gourds ecarlate de l'Inde in France, pepino scientific investigations. Although, most of Cimarron in Spanish and Skariagenagurk. the studies have either used extract of Ivy gourd (Coccinia grandis) is found in stem, root and most often leaf alone or in tropical Asia (India, Pakistan, Bangladesh, combination, only few studies tested Sri Lanka, Indonesia, Malaysia, the medicinal properties of C. grandis fruits Philippines, and Thailand), and Africa (Vadivu et al., 2008; Shaheen et al., 2009; (Cooke, 1903). Coccinia indica Wright Deshpande et al., 2011a; Dnyaneshwar and and Arn., and Coccinia cordifolia (L.) Patil, 2011; Ramakrishnan et al., 2011; Cogn., Cephalandra indica, Naud., and Sood et al., 2012). To our knowledge, Bryonia cordifolia (L.) Voigt.( Kirtikar there are hardly any in-vitro studies testing and Basu, 1994; Philcox,1997; Nagare et cell proliferative property of C. grandis al., 2015) are the other names of C. fruit. The goal of this study was to conduct grandis which is a climber, trailer, preliminary phytochemical screening and dioecious, and perennial plant. Young, to evaluate the potential antimicrobial, tender and long slender stem tops, leaves, antioxidant and cell proliferative property and tuberous roots of C. grandis are of C. grandis fruit. cooked or used as a seasoning and young fruits are used in salads. Every part of this

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Materials and Methods (potassium mercuric iodide). Formation of Plant extract yellow creamy precipitates indicated the Un-ripened fresh fruits of C. grandis presence of alkaloids. were collected in June 2014 from a local c. Wagner’s reagent: Filtrate was Indian grocery store in Schaumburg, IL treated with 4-6 drops of Wagner’s reagent (USA) and authenticated at Department of (iodine in potassium iodide). Formation of Biology, Chicago State University in brown/reddish brown precipitation or Chicago, IL. Fresh fruits were washed with coloration indicated the presence of distilled water to clean any debris. Fruits alkaloids. were cut into small pieces and dried at room temperature away from direct Carbohydrates (Molisch’s test) sunlight. Dried material was weighed and Few drops of Molisch’s reagent were subjected to cold extraction using ethanol added to 2 mL of extract. This was (94-96 %, BDH) and acetone at 4oC, for 48 followed by gradual addition of 2 mL of hr with occasional stirring. Similarly, hot concentrated sulfuric acid down the side of extraction was carried out using a Soxhlet the test tube. The mixture was then apparatus (58oC; 7 cycles) with alcohol allowed to stand for two to three minutes. and acetone. Residues obtained on Formation of red or dull violet color at the extractions were evaporated to dryness interface of the two layers indicated the using ‘Rotavap’ and dried. All extracts presence of carbohydrates. were stored at 4oC until further use. Flavonoids Phytochemical screening: One mL of 10% lead acetate solution Dried fruit powder was extracted (at was added to 1 mL of the aqueous extract. 4oC) for 48 hr in MilliQ water and The formation of a yellow precipitate obtained residue was dissolved in small indicated the presence of flavonoids. volume of MilliQ water and labeled as “aqueous extract”. This aqueous extract Cardiac glycosides (Keller-Killiani test) along with ethanol and acetone extracts Crude extract (2-5 ml) was mixed with were subjected to preliminary 2 ml of glacial acid containing 1-2 drops of phytochemical screening using following 2% solution of ferric chloride. The mixture tests (Egwaikhide and Gimba, 2007; was then poured into another test tube Roopashree et al., 2008; Abba et al., 2009; containing 2 ml of concentrated sulfuric Njoku and Obi, 2009; Yadav and acid. A brown ring of a deoxy-sugar, Agarwala, 2011; Sood et al., 2012; characteristic of alcoholic cardenolides at Soloman et al., 2013). the interface indicated the presence of cardiac glycosides. Alkaloids Nine drops of 1% diluted hydrochloric Tannins (Braymer’s Test) acid was added to 6mL of extracts, mixed To 5ml of the extract, few drops of well and the mixture was left for some 0.1% ferric chloride were added. A time and filtered. The filtrate was divided brownish green or a blue-black coloration in three tubes (approx. 2 mL) and used for indicated the presence of tannins. alkaloid testing as follows: a. Draggendorff’s Reagent: Few drops Phlobatannins of this reagent were added to filtrate; Two ml of hydrochloric acid 1% was appearance of reddish brown/orange added to 2 ml of the aqueous extract, the precipitates indicated the presence of mixture was then boiled. Appearance of alkaloids. red precipitate indicated the presence of b. Mayer’s reagent: Filtrate was treated phlobatannins. with few drops of Mayer’s reagent

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Terpenoids (Salkowski test) strains, stored in Muller-Hinton broth, Five ml of the extract was mixed with were sub-cultured for testing in the same 2ml of chloroform and to that 3ml of medium and were grown at 37ºC for 48 hr. concentrated hydrochloric acid was Freshly cultured bacteria (100l, approx. carefully added to form a layer. Presence 105 bacteria) were used to inoculate the of terpenoids is indicated by interface culture plates. Wells of 5mm diameter forming a reddish brown color. were made using sterilized glass pipette end. Various concentrations of the extracts Proteins/Amino acids in triplicates were added to the wells. Two ml of the extract was treated with Ethanol and acetone controls and 2-5 drops of 1% ninhydrin solution in antibacterial drug erythromycin (5mg/ml) acetone in a test tube and placed in boiling used as a standard, were tested water bath for 1-2 min. Presence of amino simultaneously with different extracts. All acid was indicated by the formation of plates were incubated at 37ºC for 48 hr and purple color. diameter of ‘zone of inhibitions’ (ZOI) was measured using a transparent metric Resins ruler under magnifying glass. The lowest To 4mL of the extract, 4mL of 1% concentration of various extracts and aqueous hydrochloric acid was added. standard that inhibited the visible growth Formation of resinous precipitate indicated after 48 hr was considered as the minimum the presence of resins. inhibitory concentration (MIC).

Saponins Antioxidant activity assay Three mL of extract was shaken The antioxidant property of the extracts vigorously with 2mL of distilled water and compared to the standard (Trolox) was then, heated to boil; appearance of stable, measured using ‘Cayman’s Antioxidant persistent creamy miss of small bubbles Assay’ (Catalog No. 709001, Cayman indicated the presence of saponins. Chemical Company). Total antioxidant capacity of the sample was measured by Steroids (Liebermann Burchard Reaction) extracts ability of inhibiting oxidation of 2, To 10 mL of extract, 10 ml of 2’-azino-di-[3-ethylbenzthiazoline chloroform was added and filtered. In 2 sulfonate]. Sample’s antioxidant capacity mL of filtrate, 2 mL of acetic anhydride is compared with a water-soluble was added followed by few drops of tocopherol analogue (Tolox), and reported concentrated sulfuric acid. The appearance as molar Trolox equivalents. Only cold of blue, bluish-green or a rapid change and hot ethanol extracts were used for this from pink to blue color/ring indicated the assay. Assay procedure restricted the presence of steroids. testing of acetone extracts due to its interference with the assay reagents. The Antibacterial activity assay absorbance of the extracts was read at 405 Pure bacterial culture, nutrient broth, nm using ELISA plate reader (apDia, A.D. and culture plates were procured from Touch ELISA Reader). Different Carolina Biological Supply Company concentrations of Trolox as described in (USA). The antibacterial activities of the the assay procedure were used to generate cold and hot ethanol and acetone extracts the standard curve for comparison. The were tested against two Gram positive antioxidant property of the extracts was (Staphylococcus aureus and Enterococcus calculated using the following equation. faecalis) and two Gram-negative Antioxidant (mM) = Sample average microorganisms (Escherichia coli and absorbance – (y-intercept)/slope x dilution) Pseudomonas aeruginosa) using modified All samples were run as triplicates using Kirby-Bauer diffusion method. Bacterial 96 well plates provided with the assay kit.

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Cell proliferation assay correlation and differences using unpaired The Cell Titer 96® Aqueous One student's t-test and one way analysis of Solution Cell Proliferation Assay (MTS) variance (ANOVA) followed by post-hoc (Catalog No. 3582, Promega Corporation) analysis with GraphPad Prism Ver. 5 and a was used to study cell proliferation p<0.05 was considered statistically property with MDA-MB 321 breast cancer significant. cells. This is a colorimetric method for determining the number of viable cells in proliferation. Cells were cultured in Results DMEM (Dulbecco's Modified Eagle C. grandis fruits were extracted using Medium) with 4.5 g/L glucose without L- solvents of different polarity. Twenty-five Glutamine containing 10% FBS (Fetal grams of dried powder of C. grandis fruits Bovine Serum) and 1% PES yielded 3.8 g of residue on cold and 4 g (phenazineethosulfate). Cells were with Soxhlet extraction with ethanol. cultured in flask as adherent cells. Actively Similarly, 25 g of dried powder yielded growing cells (passage 2) were used and 2.75 g of residue with cold and 3.2 g of 5,000 cells (in 50 μl media) were seeded in residue on hot Soxhlet extraction with each well of 96 well plates and grown for acetone. The findings of preliminary 48 hr using only medium. After 48 hr, phytochemical screening are presented in medium was aspirated from each well and Table 1. Both aqueous and ethanol extracts cells were subjected to various tested positive for resins, saponins and concentrations of the extracts and controls terpenoids with the exception of aqueous (culture medium, ethanol and acetone extract, which tested positive for only) in triplicates. This well plate was carbohydrates, cardiac glycosides and kept at 37oC for another 48 hr in incubator proteins, whereas, ethanol extract tested having 5% CO , humidified atmosphere. 2 positive for alkaloids, flavonoids, tannins Then, 20 µl of MTS [3-(4, 5- and phlobatannins. Acetone extract tested dimethylthiazol-2-yl)-5-(3- positive for flavonoids. carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium] reagent was Table 1. Preliminary phytochemical screening of added to each well and cells were C. grandis fruit extracts. incubated for another 4 hr and absorbance was read at 490 nm using ELISA plate Tests Aqueous Ethanol Acetone reader. Cell proliferation property of the Extract Extract Extract extracts was determined by the quantity of Carbohydrates (+) (-) (-) ‘Formazan product’ measured by the Proteins (+) (-) (-) Alkaloids (-) (+) (-) absorbance at 490 nm, which was directly Cardiac (+) (-) (-) proportional to the number of living cells Glycosides in culture according to the assay protocol. Flavonoids (-) (+) (+) The Hemocytometer was used for cell Tannins (-) (+) (-) Phlobatannins (-) (+) (-) counting. Resins (+) (+) (-) Saponins (+) (+) (-) Statistical analysis Terpenoids (+) (+) (-) Results of this study are expressed as Steroids (-) (+) (-) means ± standard deviations where appropriate and in units suggested by the (+) Indicates Presence, (-) Indicates Absence assay procedures. Data were analyzed for

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Table 2. Zone of Inhibitions with different extracts of C. grandis fruit

Mean Zone of Inhibitions against S. aureus (mm ± SD)

Cold Ethanol Hot Ethanol Cold Acetone Hot Acetone (1) (2) (3) (4) A 12.5 ± 0.4 14.7 ± 0.2 15.2 ±0.3 16.5 ± 0.5 ***+++ ***++ *** B 10.7 ± 0.6 13.3 ± 0.6 13.0 ± 0.0 14.3 ± 0.6 *** ** *** C 6.5 ± 0.4 11.3 ± 0.2 12.0 ± 0.0 12.2 ± 1.0 *** *** *** D 5.8 ± 0.2 10.2 ± 0.2 7.2 ± 0.8 9.0 ± 0.5 ***### ***++ *** Mean Zone of Inhibitions against E. faecalis (mm ± SD) A 8.5 ± 0.0 9.2 ± 0.2 9.7 ± 0.6 11.2 ± 0.3 ++ ***++ *** B 7.5 ± 0.7 7.3 ± 0.2 8.3 ± 0.3 8.8 ± 0.3 ++ * C 6.0 ± 0.0 6.2 ± 0.2 6.8 ± 0.8 6.8 ± 0.3

D NZOI NZOI NZOI 5.7 ± 0.3 Mean Zone of Inhibitions against E. coli (mm ± SD)

A 9.1 ± 0.1 11.8 ± 0.2 12.2 ± 0.8 14.2 ± 0.8 ***++ ***++ *** B 8.5 ± 0.0 9.8 ± 0.2 10.7 ± 0.6 12.3 ± 0.6 *+++ ***++ *** C 7.0 ± 0.0 8.0 ± 0.0 7.8 ± 0.3 9.8 ± 0.3 **+++ ***+++ *** D NZOI NZOI NZOI 7.5 ± 0.5 Mean Zone of Inhibitions against P. aerugenosa (mm ± SD) A 8.2 ± 0.2 8.7 ± 0.2 9.0 ± 0.0 9.0 ± 1.0 B 7.7 ± 0.2 8.2 ± 0.2 7.8 ± 0.3 8.5 ± 0.5 C 6.2 ± 0.5 6.8 ± 0.6 7.3 ± 0.8 7.5 ± 0.5 D NZOI NZOI NZOI NZOI E 30.0±0.5 28.5±0.6 27.0±0.5 25.0±0.5

A: 29.60 g, B: 14.80 g, C: 7.40 g, D: 3.70 g, E: Erythromycin (300 g), NZOI = No Zone of Inhibition. Zone of inhibition differed significantly between all extracts and control (0 g/mL) and differences were significant at p value of <0.05 on ANOVA. *p<0.5, **p<0.01 and ***p<0.001 compared to cold ethanol and other extracts. ++p<0.01, +++p<0.001 compared between hot acetone and other extracts. ###p<0.001 compared between cold acetone and hot ethanol on post-hoc analysis and no significant differences were observed between various concentrations of different extracts and standard erythromycin. Controls were pure ethanol and acetone that showed no zone of inhibitions.

Table 3. MIC for different extracts of C. grandis fruit as compared to erythromycin as the standard

Minimum Inhibitory Concentration (MIC) (g/ml) Type of Extracts* S. aureus E. faecalis E. coli P. aerugenosa Cold Ethanol 56.7 ±0.71 86.67 ±1.0 80.0 ±1.62 108.3 ±1.9 Hot Ethanol 55.0 ±0.12 83.3 ±1.12 66.7 ±0.88 96.6 ±1.62 Cold Acetone 50.0 ±0.55 81.6 ±0.35 63.3 ±0.1 93.3 ±1.2 Hot Acetone 43.3 ±0.8 63.3 ±0.45 62.5 ±0.15 70.0 ±1.36

Erythromycin 36.6 ±0.5 38.3 ±0.72 43.0 ±1.0 63.3 ±1.554 (Standard)** *60 l loaded in each well; **Erythromycin=5g/ml, used as positive control

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Results of antibacterial activity are property measured by calculating Trolox presented in Table 2. Differences in zone equivalent of cold ethanol extract, was of inhibitions among various 0.197 mM for 1.315 mg/ml of the extract, concentrations of different extracts for S. 0.0263 mM for 0.329 mg/ml of the extract, aureus, E. Faecalis and E. coli were whereas, the antioxidant capacity of hot significant (p<0.05 to p<0.001) except for ethanol extract was 0.287 mM for 1.13 concentration of 7.40 g in case of E. mg/ml of the extract and 0.101 mM for faecalis, whereas, no significant 0.326 mg/ml of the extract (Table 4). The differences in zone of inhibitions were absorbance of cold and hot ethanol extracts observed against P. aerugenosa for various were found strongly negatively correlated concentrations of all four extracts. Hot with that of standard Trolox (r= -0.96 and ethanol and acetone extracts exhibited r= -0.99; p<0.05), respectively. Similarly, greater zone of inhibitions for all four concentrations of cold and hot ethanol microorganisms tested compared to cold extracts were found strongly, negatively extracts. No zones of inhibitions were correlated with that of standard Trolox (r= observed for concentration 3.70 g of cold -0.984 and r= -0.989; p<0.05) respectively. and hot ethanol, and cold acetone extract against E. faecalis, E. coli and P. aerugenosa whereas, for hot acetone extract against P. aerugenosa only. The four extracts showed different levels of antibacterial activity. Zones of inhibitions for the standard (erythromycin) were highly significant to that of various concentrations of different extracts tested (p<0.05 to p<0.001) and one and a half to two times greater, which may have resulted because of the higher concentrations of the standard used. The mean MIC values were higher for P. aerugenosa ranging from 4.2 to 6.5 g and Figure 1.Comparison of the absorbance of cold and lower for S. aureus ranging from 2.60 to hot ethanol extracts of C. grandis fruit with Trolox 3.4g for all four extracts tested. An *Significant strong negative correlation between increasing trend in mean MIC was the absorbance of cold and hot ethanol extracts and observed with S. aureus, E. Faecalis, E. concentration of cold and hot ethanol extracts with that of standard Trolox at p<0.05. coli, and P. aeurgenosa. All extracts showed similar trend of antibacterial Table 4. Antioxidant property of cold and hot activity. The MIC for erythromycin also ethanol extracts of C. grandis fruits expressed as showed similar antibacterial trend (Table Trolox Equivalent (TE). 3). Absorbance values of various Mean Trolox Type of Conc. concentration of Trolox, a known Equivalent Extract (mg/ml) antioxidant, were used to plot a standard Absorbance ±SD (TE)* curve. Mean absorbance values of various concentrations of cold and hot ethanol Cold 1.315 0.400 ± 0.024 0.197 Ethanol 0.329 0.605 ± 0.062 0.0263 extracts were compared to Trolox to Hot 1.13 0.293 ± 0.011 0.287 determine the relative strength of Ethanol 0.326 0.515 ± 0.044 0.101 antioxidant properties of the extracts *Calculated using Antioxidant (mM) = sample (Figure 1). A decrease in absorbance with average absorbance– (y-intercept)/slope x dilution). Trolox was considered as an evidence of increased antioxidant property indicating The results of cell proliferation assay their inverse relationship. The antioxidant are presented in Table 5. Mean absorbance

AJP, Vol. 7, No. 4, Jul-Aug 2017 301 Sakharkar et al. values for extracts were higher than any p<0.001). Cold extracts showed greater type of controls used. An increase in cell proliferation, suggesting increased cell absorbance was considered as an evidence growth compared to the hot extracts of absence of cytotoxic property indicating confirming the absence of any cytotoxic their inverse relationship. Significant properties in C. grandis fruits. Cold differences were found in cell proliferation acetone extract showed more marked measured by absorbance among different results as a cell proliferative treatment than concentrations of ethanol and acetone cold ethanol extract (Table 5 and Figure extracts compared to their control as well 2). as among different extracts (p<0.05 to

Table 5. Comparison of cell proliferation with different extracts of C. grandis fruit

Absorbance (Mean ± SD)

Conc. Cold Ethanol Hot Ethanol Control for Cold Acetone Hot Acetone Control for (1) (2) Ethanol (3) (4) (5) Acetone (6) A 1.302 ± 0.001 1.271 ± 0.010 0.789 ± 0.010 1.415 ±0.030 1.317 ± 0.018 0.770 ± 0.012 ***++ *** $$$## $$$ B 1.408 ± 0.028 1.300 ± 0.016 0.788 ± 0.013 1.458 ± 0.032 1.340 ± 0.020 0.772 ± 0.016 ***+++ *** $$$## $$$ C 1.500 ± 0.030 1.330 ± 0.019 0.778 ± 0.022 1.601 ± 0.022 1.400 ± 0.010 0.766 ± 0.028 ***+++ *** $$$### $$$

A. 4.94 g/10 µl+90 µL medium; B. 12.35g/25 µl+75 µL medium; C. 24.70 g/50 µl+50 µL medium Control A: 10 µL+ 90 µL medium; B: 25µL+75µL medium; C: 50 µL+50 µL medium Differences were significant at p value of <0.05 on ANOVA. ***p<0.001 compared to control for ethanol with cold and hot ethanol. ++p<0.01, +++p<0.001 compared to hot and cold ethanol. $$$p<0.001 compared to control for acetone with cold and hot acetone. ## p<0.01, ###p<0.001 compared to hot and cold acetone.

Figure 2. Cell proliferation with different extracts of C. grandis fruit. Data are presented as Mean ± SD; *Significant difference between extracts and controls on post-hoc analysis at p value of <0.05 and ** at p value of <0.001.

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Discussion been suggested as the major mechanism of It is well known that the various their antibacterial activity. This complex phytochemicals that are present in herbal induced by tannins, is considered being medicines and plants are responsible for toxic and inhibitory to microbial enzymes. their medicinal values. Nature and extent Studies have suggested that this inhibitory of phytochemicals present in these plants mechanism involves direct interaction with explain their medicinal values. membranes, extracellular proteins and cell Phytochemicals are present in all parts of walls. In this study, tannins may have also plants; however, it has been reported that played a major role in inhibiting the the leaves, roots, bark and stems have growth of microorganisms tested. Shaheen higher concentration of such et al. (2009) also reported similar phytochemicals compared to fruits and phytochemicals testing positive in their flowers (Siddiqui et al., 2009). study. Phytochemicals that we found in the fruits are also found in other parts like leaves, stem, and roots of C. grandis (Tamilselvan et al., 2012; Gautam et al., 2014; Hossain et al., 2014). The antibacterial, antioxidant and cell proliferative properties of C. grandis fruits observed in this study, could be attributable to the presence of phytochemicals that were identified during preliminary screening. Phytochemicals such as alkaloids, terpenoids, glycosides, flavonoids, and tannins are known to possess antibacterial and antioxidant Figure 3. Picture of C. grandis fruits properties. Flavonoids were also reported to have a broad spectrum of medicinal Antibacterial properties of C. grandis properties such as antioxidant, anti- leaves have been extensively studied inflammatory, antimicrobial, anti-cancer (Farrukh et al., 2008; Bhattacharya et al., and cardio-protective activities. Flavonoids 2010; Hussain et al., 2010; Bulbul et al., are hydroxylated polyphenols found in 2011; Satheesh and Murugan, 2011; Sivraj plants known for their wide range of et al., 2011; Khatun et al., 2012). antimicrobial activity in vitro. Similar to Moreover, study by Shaheen et al. (2009) tannins, flavonoids show their antibacterial has reported similar activity for fruits of C. activity by forming complex with grandis. In this study, hot petroleum ether, extracellular and soluble proteins. In this diethyl ether, chloroform, ethyl acetate, study, both ethanol and acetone extracts acetone, methanol and ethanol extracts tested positive for flavonoids. In addition, were used for screening the antibacterial ethanol extract tested positive for activity whereas, in our study, we used alkaloids, tannins, resins, saponins, both cold and hot extracts of ethanol and terpenoids and steroids. This may be the acetone. Antibacterial activities observed reason why ethanol extract exhibited in our study, were comparable to those of greater antibacterial activity compared to ethanol and acetone extracts studied by other extracts. Tannins are naturally Shaheen et al. (2009). However, in their occurring plant polyphenols, which can be study, petroleum ether and methanol classified into hydrolysable and non- extracts showed more pronounced hydrolysable (condensed) tannin. The antibacterial activity against gram-positive ability of plant polyphenols to form organisms including S. aureus, being more complex with minerals and polymers, has susceptible and S. paratyphi A, being more

AJP, Vol. 7, No. 4, Jul-Aug 2017 303 Sakharkar et al. resistant, whereas least activity was cardiovascular diseases, neurodegenerative observed with chloroform extract (Shaheen diseases, etc. Antioxidants are agents, et al., 2009). In contrast, in our study, which scavenge free radicals; thereby, they acetone extract exhibited higher prevent damages to proteins, enzymes, antibacterial activity than the ethanol carbohydrates, DNA and lipids (Halliwell extract and hot extracts were more and Gutterridge, 1999; Fang et al., 2002; effective than the cold ones. The choice of Lee et al., 2004). Lee et al. (2004) different solvents and bacteria used for classified antioxidants into two major antibacterial activity testing also limited categories namely, enzymatic and non- our ability to make appropriate enzymatic. The enzymatic antioxidants are comparisons for antibacterial activities. In produced endogenously and include a study by Sivaraj et al. (2010), ethanol superoxide dismutase, glutathione extract of leaves showed higher peroxidase and catalase. The non- antibacterial activity against E. coli enzymatic category includes some compared to acetone or methanol; phytochemicals such as curcumin, ascorbic however, we found acetone extract to be acid, carotenoids, tocopherols, tannins, more potent in inhibiting growth of all flavonoids that are mostly obtained from gram-positive and gram-negative bacteria plant sources. The results of our study (including E. coli), than the ethanol extract indicate that the fruits of C. grandis are a (Sivraj et al., 2010). The differences in potential source of natural antioxidants. MIC were profound for S. aureus and E. The antioxidant assay used in this study coli for fruit extracts, being lower measured the total antioxidant capacity of compared to MIC values of alcohol and the sample. Our extracts detected the acetone extracts of leaves. The MIC presence of more than one phytochemical reported by Sivaraj et al. (2010) for suggesting that this activity can be acetone extract against S. aureus and E. attributed to the presence of both tannins coli were 1000g/mL and 500 g/mL, and flavonoids. Our ethanol extract also respectively, in contrast to the ones that we exhibited antioxidant activity, showing observed for cold and hot acetone extract strong linear relationship with the against S. aureus (50 g/mL and 43.3 concentrations of extract tested, compared g/mL), and E. coli (63.3 g/mL and 62.5 to Trolox. Several studies have reported g/mL), respectively. These differences in antioxidant property of C. grandis leaves, MIC could be attributed to the differences stem and root extracts (Nanasombat and in plant material and the type and amount Teckchuen, 2009; Bulbul et al., 2011; of phytochemicals present in them. Higher Deshpande et al., 2011a; Moideen et al., MIC for ethanol extracts of C. grandis 2011; Ashwini et al., 2012; Bhaduria et al., leaves against S. aureus (1750 µg/mL), E. 2012; Maheshwari et al., 2015); however, coli (1500 µg/ml) and P. aeruginosa (1500 none of these studies tested this activity in µg/ml) reported by Bhattacharya et al. fruits. Antioxidants are known for being (2010). It would be difficult to compare able to reduce inflammation. C. grandis MIC values reported in other studies, as extracts are used as herbal medicine for they differ based on the solvent used as various illnesses including inflammation. well as the bacterium. Based on the above This further proves why C. grandis is used observations, we can safely presume that and known for its anti-inflammatory the fruits possess greater antibacterial properties in folklore and Indian traditional activity than the leaves. medicine, Ayurveda (Junaid et al., 2009; Free radicals are known to play a vital Niazi et al., 2009; Deshpande et al., 2011b; role as etiological factors in a wide variety Ashwini et al., 2012). of pathological conditions including Bulbul et al. (2011) has reported diabetes, autoimmune disorders, aging, adverse effects of various Coccinia

AJP, Vol. 7, No. 4, Jul-Aug 2017 304 Cell proliferative properties of Coccinia grandis extracts on cell survival. Studies have also property deserves further investigation. reported partial cure of gastric ulcer with Comparison of cell proliferation behavior leaf extract (Papiya et al., 2008; Preeth et among cancerous versus non-cancerous al., 2010) as well as its anticancer/anti- cells could be an interesting topic for tumor properties (Bhattacharya et al., future investigations. 2011; Girish et al., 2011; Behera and Dash, 2012). Bunkrongcheap et al. (2014) Acknowledgments conducted an in vitro cell culture study in We would like to thank Dr. 3T3-L1-cell using root extract of C. Venkateswara Potluri, Chair, Dept. of grandis. This study examined the effects Biology, Chicago State University, on differentiation of pre-adipocytes, in Chicago, IL, USA for authenticating the contrast to ours, where we examined the plant material; Ling Cheng and Bonnie cell proliferative property (cytotoxicity) Teng, second year PharmD students for using MDA-MB-231 breast cancer cells. their help with laboratory work. This In contrary to findings from other studies, research study was funded through we found that the number of cells intramural research grant provided by the increased following incubation with all authors’ institution to one of its author four extracts. Our results indicated that (BCC). cold extracts were better than the hot extracts and acetone extract was better Conflict of interest than ethanol in promoting cell Authors have no conflicts of interest to proliferation; however, it indicated the disclose. absence of any cytotoxic properties in C. grandis fruits. This cell proliferation property could be attributed to steroidal References hormone-like compound(s) present in Abba DI, Inabo HI, Yakubu SE, Olonitola OS. these fruits, which needs to be further 2009. Phytochemical analysis and investigated. It will be interesting to study antibacterial activity of some powdered the cell proliferation process in normal herbal preparations marketed in Kaduna (non-cancerous) cells using primary cell metropolis. Sci World J, 4: 23-26. culture method in future investigations. Ali AM, Pandey AK. 2005- 2006. Systematic Our study had some limitations. We did studies on the family cucurbitaceae of not use isolated and characterized eastern Bihar, India, In: Cucurbit Genetics phytochemicals from C. grandis fruits for cooperative Report. pp. 28-29 and 66-69. confirming its antibacterial, antioxidant Ashwini M, Lather N, Bole S, Vedamurthy and cell proliferative properties. Since, the AB, Balu S. 2012. In vitro antioxidant and anti-inflammatory activity of Coccinia extracts may have contained several grandis. Int J Pharm Pharm Sci, 4: 239- phytochemicals and were used for 242. screening of these potential activities, there Bandopadhyay U, Das A, Bannerjee RK. is a possibility that they may have 1999. Reactive oxygen species: oxygen interfered with the assay procedures used damage and pathogenesis. Curr Sci, 77: and the readings obtained. 658-666. The findings of this study suggest that Behera SK and Dash V. 2012. Some Indian C. grandis fruit contain alkaloids, vegetables used as an anticancer agent. Int flavonoids, tannins and steroids, which J Adv Pharm Res Bio Sci, 2: 250-264. perhaps responsible to some degree for its Bhaduria P, Arora B, Vimal B, Kulshrestha A. antibacterial, antioxidant and cell 2012. In vitro antioxidant activity of Coccinia grandis root extracts. Indo global proliferative properties as mentioned in J Pharm Sci, 2: 230-238. folklore and Indian traditional medicine, Bhattacharya B, Samantha M, Pal P, Ayurveda. However, its cell proliferative Chakraborty S, Samanta A. 2010. In-vitro

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