INTRODUCTION:

Plants play a vital significant role in the enhancement of new drugs. Phytoconstituents are considered to be a natural bioactive compound present in various parts of the plant that works along with nutrients and fibers to fight against diseases.¹ WHO (World Health Organization) estimated that more than 80% of the world’s population depends on traditional herbal medicine for their basic health care system. Various parts of the plants serve as a source for the development of new drugs and chemicals derived.²Cucurbits belonging to the family Cucurbitaceae are the vegetable crops, which are largely consumed by people worldwide. The previous study estimated that large group of medicinally valuable plants is found in the family Cucurbitaceae which consists of a number of bioactive compounds with cytotoxicity, anti-inflammatory, and cardiovascular effects.³

The family Cucurbitaceae are usually called as gourds that grow widely in tropical, subtropical, arid deserts and temperate locations. Several bioactive plants are present in this family so it is used widely from ancient time for their medicinal values.⁴These medicinal plants find its application in various industries like in agricultural, cosmetic, food and pharmaceutical. In the history of all civilizations, it has been documented that medicinal plants are helpful in curing diseases.^1,2 There are more than hundreds of medicinal plants available which are said to have a long history of medicinal properties against several disease and aliments.⁵ Majumder et.al., reported that in developing there are more than 3.3 billion people utilize medicinal plants for their daily use as it is considered to be a “backbone” of traditional medicine. In drug development and synthesis, medicinal plants are believed to be an excellent source of ingredients.⁶ Secondary metabolites like phenolic and flavanoids are extensively distributed in plants having a wide range of bioactivities such as anti-apoptosis, anti-aging, anti-carcinogen, anti-inflammation, anti-atherosclerosis and cell proliferation activities.⁷ In the 21^st century, the most significant challenges faced by developing countries are emerging and reemerging of infectious diseases. Worldwide estimation says that over 50% of all deaths are due to the cause of infectious diseases.⁸ The present study was thus designed to investigate the antimicrobial potential, antioxidant activity and analysis of secondary metabolites of most commonly available medicinal plants belonging to cucurbitaceae family. The plants studied are Momordica charantia (MC), Citrullus lanatus (CL), Coccinia grandis (CG) and Lagenaria siceraria (LS).

MATERIALS AND METHODS:

Collection and extraction of plant material:

Fresh leaves of Momordica charantia, Citrullus lanatus, Coccinia grandis and Lagenaria siceraria were collected from Vellore district, Tamil Nadu, India. The leaves were washed thoroughly with a distilled water to remove dust and foreign particles. Then the leaves were shade dried for 5-7 days. Dried leaves were milled to obtain a fine dry powder. About 10-15 grams of leaf powdered was dissolved in 200ml of methanol and petroleum ether solvent in an Erlenmeyer flask. Then the flask was sealed with parafilm and kept in a shaker for 48 hrs at 120 rpm. After a period of 48 hrs, the content was filtered using Whatman No: 1 filter paper. Then the filtrates were evaporated using a rotary vacuum evaporator. As the crude extract gets dried it is used for the further pharmacological study.

Phytochemical Screening:

The qualitative phytochemical screening was performed to analyze the presence of various phytoconstituents present in the plant leaf powder using the standard procedure.^9,10

Determination of total phenol content:

Total phenol content of two different extracts (Methanol and Petroleum ether) obtained from leaf powder was determined using Folin- Ciocalteau reagent based assay. 100 μL of each extract (1 mg/ml) was taken in a test tube and 500 μL of 1N Folin -Ciocalteau reagent was added. To this content, 1.5 ml of 20% of NaCO₃ was added and final volume (5 ml) was made by addition of distilled water. The mixture was kept for 30 min incubation at room temperature and absorbance was measured at 765 nm using UV-vis spectrophotometer. Gallic acid was used as a standard.¹¹

Antibacterial activity:

Agar well diffusion method was used to determine the antibacterial activity of methanol and petroleum ether extracts. Mueller-Hilton agar was prepared in a conical flask and sterilized in an autoclave. Then, the sterile medium was poured into Petri plates. After solidification, freshly prepared bacterial cultures (Bacillus cereus, Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Proteus mirabilis) were uniformly spread over the media using a cotton swab. Then, the well was filled with 100 µl of respective crude extract at various concentrations (100 µg, 75 µg, and 50 µg/ml dissolved in sterile distilled water) and streptomycin was used as positive control. The inoculated plates were then incubated at 37°C for about 24 h. The plates were observed for the presence of the inhibition zone around the wells. The size of zone obtained was measured, and the antibacterial activity obtained was measured in terms of the average diameter of inhibition zone in millimeter.¹²

Antifungal activity:

Antifungal activity was determined by using agar well diffusion method for methanol and petroleum ether leaf extracts. Sterile SDA media was poured into Petri plates, then the freshly prepared fungal strains was spread over the lawn using cotton swab. The pure cultures of the fungal strains used for the antifungal assay are Aspergillus niger and Aspergillus flavus. Wells were made using sterile cork borer and the wells were filled with 100 μl of leaf extracts and fluconazole was used as the positive control. Then the plates were incubated at room temperature for 3-5 days. After the incubation period, the zone of inhibition was measured in mm for the presence of inhibition zone around the wells.¹³

Antioxidant assay:

DPPH (2, 2-diphenyl-1-picrylhydrazyl) Assay:

Antioxidant activity through free radical scavenging was carried out for the test sample to monitor the difference in their optical density of DPPH radicals. 1 mg/ml concentration of leaf extract (Methanol and Petroleum ether) was used. DPPH produces violet color in methanol solution which is said to be stable and nitrogen-centered free radical. When a substrate is added in a DPPH solution, it was reduced to a yellow colored product (diphenyl picryl hydrazine) as a result of the donation of the hydrogen atom. The leaf extract (0.2 ml) was diluted using methanol and 2 ml of DPPH solution (0.5mmol/l) was added to the test sample. The mixture was incubated at room temperature for 30 min under dark condition. After the 30 min incubation period, the absorbance was measured at 517 nm.¹⁴

The following equation was used to calculate the percentage of DPPH radical scavenging activity: Inhibition of DPPH radical (%) = [(control absorbance-extract absorbance)/(control absorbance)]×100.

Reducing power assay:

Leaf extracts were taken in a test tube at different concentration (25, 50, 75,100 μg/ml) to that 2.5 ml of phosphate buffer and potassium ferricyanide was added. Then the mixture was kept in water bath at 50°c for 10 min. 2.5 ml of 10% TAA (Trichloroacetic acid) was added to the test tube once it gets cool, centrifugation was carried out at 3000 rpm for 10 min. After centrifugation 2.5 ml of supernatant was taken carefully and to this 2.5 ml of distilled water and 500 μl of ferric chloride solution was added. Standard was prepared at different concentration using gallic acid. The absorbance was taken at 700 nm in a UV visible spectrophotometry.¹⁵

RESULTS:

Preliminary phytochemical analysis:

The preliminary phytochemical screening of four medicinal plants leaf powder revealed the presence of secondary metabolites. In the present work Momordica charantia, Citrulluslanatus, Cocciniagrandis,and Lagenaria sicerariaconfirm the presence of phenol, flavonoids, saponins, tannins, anthraquinones whereas terpenoids are present only in c.grandis and for other plants, it is absent. The phytoconstituents alkaloids are absent in C.grandis and it is present in other three plants. Table 1 represents the qualitative phytochemical screening present in leaf powder of Momordica charantia, Citrullus lanatus, Coccinia grandis and Lagenari siceraria. The presence of various secondary metabolites suggests that plants have a broad range of biological applications.

Table 1 Result of qualitative phytochemical composition of various leaf powders

Phytoconstituents	MC	CL	CG	LS
Phenol	+	+	+	+
Flavonoids	+	+	+	+
Tannins	+	+	+	+
Saponins	+	+	+	+
Terpenoids	-	-	+	-
Anthraquinones	+	+	+	+
Alkaloids	+	+	-	+

‘+’ indicates presence of phytoconstituents

‘-’indicates absence of phytocontituents

Antibacterial activity:

The present study determined antibacterial activity for medicinal plants belonging to Cucurbitaceae family using agar well diffusion method. Methanolic and petroleum ether leaf extract of Momordica charantia, Citullus lanatus, Coccinia grandis and Lagenaria siceraria was analyzed for its activity against bacteria. Antibacterial activity was tested against three Gram-negative bacteria (E.coli, K.pneumoniae, P.mirabilis)and two Gram-positive bacteria (S.aureus and B.cereus). The antibacterial activity of methanol and petroleum ether leaf extract against tested pathogens was tabulated in the Table 2(a) and 2(b). The result revealed that, as the concentration of the extract increases, the resistant to pathogens also increased. Therefore the increase in sample concentration, the zone of inhibition also increases. Both the extract exhibited antibacterial activity with a maximum zone of inhibition against tested pathogens at 100 mg/ml concentration. Streptomycin was used as positive control.

In the present study, the highest zone of inhibition was exhibited by Momordica charantia methanol extract at 100 mg/ml, 50 mg/ml and 25 mg/ml concentrations. The zone of inhibition ranged from 10mm-19mm against the growth of bacteria. At lower concentration also the methanol extract exhibited antibacterial activity. Whereas for Citrullus lanatus, petroleum ether leaf extract exhibited a maximum zone of inhibition compared to methanol extract with the range of 10mm-18mm against bacteria. The plant Coccina grandis exhibited a maximum zone of inhibition in petroleum ether extracts compare to methanol extract against the tested pathogens. The zone of inhibition showed by petroleum ether extract ranged between 10mm-19mm and methanol extracts ranged between 10mm-17mm.Petroleum ether leaf extract of Lagenaria siceraria exhibited maximum antibacterial activity against tested pathogens compare to methanol extract.The present study estimated that, by comparing the antibacterial activity of methanol and petroleum ether leaf extracts, the highest zone of inhibition was found in the methanol extract of Momardica charantia and petroleum ether extract of other three plants. So the current study concludes that methanol leaf extract of bitter gourd as an excellent source of phytoconstituents to act against bacterial pathogens.

Tabel 2(a): Antibacterial activity for the methanol leaf extract

Plant name	Concentration 100mg/ml	*E. coli*	*S.aureus*	*P.mirabilis*	*B.cereus*	*K.pneumonia*
Plant name	Concentration 100mg/ml	Zone of inhibition - mm
Control		20	25	18	20	20
Momordica charantia	100	19	12	15	18	16
	50	16	10	13	13	14
	25	12	-	12	11	10
Citrullus lanatus	100	19	17	15	13	12
	50	-	12	12	11	-
	25	-	-	-	10	-
Coccinia grandis	100	17	-	12	12	14
	50	14	-	10	10	12
	25	10	-	-	-	-
Lagenaria siceraria	100	15	17	-	19	17
	50	11	-	-	12	12
	25	10	-	-	-	-

Table 2(b): Antibacterial activity of petroleum ether leaf extract

Plant name	Concentration 100mg/ml	*E. coli*	*S.aureus*	*P.mirabilis*	*B.cereus*	*K.pneumonia*
Plant name	Concentration 100mg/ml	Zone of inhibition - mm
Control		19	25	32	17	13
Momordica charantia	100	20	-	21	14	15
	50	-	-	20	10	12
	25	-	-	18	-	11
Citrullus lanatus	100	16	18	16	14	16
	50	13	16	13	12	12
	25	-	-	10	11	-
Coccinia grandis	100	16	16	20	17	19
	50	13	15	14	15	14
	25	-	13	10	12	11
Lagenaria siceraria	100	16	18	17	12	14
	50	13	12	12	11	10
	25	10	-	10	-	8

Escherichia coli (MTCC 1687), Staphylococcus aureus (MTCC 3160), Klebsiella pneumoniae (MTCC 7028), Proteus mirabilis (MTCC 3310), Bacillus cereus (MTCC 0430), Positive control- streptomycin disc (10mg)

Table 3: Antifungal activity of methanol and petroleum ether leaf extracts

Pathogen	*Momordica charantia*			*Citrullus lanatus*		*Coccinia grandis*		*Lagenaria siceraria*
	C	A	B	A	B	A	B	A	B
Aspergillus niger	8	18	-	7	-	-	-	10	-
Aspergillus flavus	7	17	-	-	-	-	-	-	-

‘C’ = Positive Control (Fluconazole disc); ‘A’= Methanol extract; ‘B’=Petroleum ether extract, Zone of inhibition- mm

Figure 2 – Zone of inhibition of Methanol and Petroleum ether leaf extracts

Table 4 Total phenolic content of Methanol and Petroleum ether leaf extracts

Botanical names	Methanol extract 1mg/ml	Petroleum ether extract 1mg/ml
Momordica charantia	11.06±0.001	11.34±0.002
Citrullus lanatus	11.26±0.002	11.37±0.001
Coccinia grandis	12.35±0.001	12.99±0.003
Lagenaria siceraria	10.22±0.001	11.55±0.001

Estimation of total phenol content:

The present study estimated the total phenol content of methanol and petroleum ether leaf extract of Momordica charatia, Citrullus lanatus, Coccinia grandis and Lagenaria siceraria. The highest amount of phenolic content was estimated in methanol and petroleum ether leaf extract of Coccinia grandis of about 12.35±0.001mgGAE/g and 12.99±0.003 mg GAE/g respectively as determined from the calibration curves of the gallic acid (Y =0.026x+0.025) the amount of phenol content present in leaf extracts is tabulated in Table 4.

Total phenol content of the extract were measured in terms of Gallic acid equivalent:

In the present study, it was found that petroleum ether extracts of tested plant contains the highest amount of phenol content compared to methanol extracts. So the current study predicts that the petroleum ether leaf extracts of these plants could be responsible for the pharmacological activities.

Antioxidant activity:

DPPH free radical scavenging assay:

Free radical scavenging assay was performed for methanol and petroleum ether leaf extracts of Momordica charantia, Citrullus lanatus, Coccinia grandis and Lagenariasiceraria. The result revealed that scavenging activity increases with increases in the concentration of the leaf extracts. In both extracts, the result showed maximum antioxidant activity in Lagenaria siceraria of about 60-72% inhibition at different concentrations 50, 100 and 150μg/ml. The lowest antioxidant activity was exhibited by methanol and petroleum ether leaf extracts of Momordica charantia. (Figure 3(a) and (b))

Reducing power assay:

Methanol and petroleum ether leaf extract of Momordica charantia, Citrullus lanatus, Cocinnia grandis and Lagenaria siceraria were investigated for its reducing power assay. This is the first report on the reducing power assay in leaf extracts of methanol and petroleum ether.

Figure 3(a) Free radical scavenging assay of methanol leaf extracts

Figure 3(b) Free radical scavenging assay of petroleum ether leaf extracts

The result (Figure 4(a) and 4(b)) shows the reducing power ability of the Momordica chranti, Citrullus lanatus, Cocinnia grandis and Lagenaria siceraria leaf extracts increases with increase in the concentration of the samples. The maximum reducing ability was examined in methanol and petroleum ether extract of Lagenaria siceraria compare to other leaf extracts. Citrullus lanatus, Cocinnia grandisand Momordica charantia exhibited a significant amount of reducing power ability. The result revealed that as the concentration of the extract increases, the reducing power ability also increases.

Figure 4(a) Reducing power assay of methanol leaf extract

Figure 4(b) Reducing power assay of petroleum ether extract

DISCUSSION:

Objective of this study was to analyze antimicrobial and antioxidant activity of methanol and petroleum ether leaf extracts of Momordica charantia, Citrullus lanatus, Cocinnia grandis and Lagenaria siceraria. Methanol and petroleum ether extract of these plants showed the presence of phenol, flavonoids, saponins, tannins, anthraquinones, terpenoids and alkaloids. These phytochemicals are similar to the previous report study of Muhammad Aslam et al.,2013; Alebiosu et al., 2015. The current findings reports that the highest amount of phenol content was showed by petroleum ether extracts compared to methanol extracts. The previous report study says that ethanol extracts showed high phenol content.¹⁶Antimicrobial activity of four medicinal plants has been estimated In Vitro against five bacterial and two fungi pathogens. These microbes cause some type of disease in humans. The result obtained from this study states that methanol and petroleum ether extract of four medicinal plant species, namely: Momordica charantia, Citrullus lanatus, Cocinnia grandis and Lagenaria siceraria exhibited activity against most of the tested pathogens. These findings are agreeable with those obtained in some previous studies.^17-19 The result obtained in the current study clearly reveals that antimicrobial activities vary with the species of the plants. Therefore, the antimicrobial activity of the plant extracts relays upon the plant species, the types of solvent and the types of microorganism tested. In general, out of five tested microbes nearly three microbes were inhibited by plant extracts of both methanol and petroleum ether solvent used in this study. Further studies on phytochemicals are invited to purify and characterize the active bioactive compounds of this plant species. In this study, Lagenaria siceraria exhibited the highest DPPH inhibitory activity and reducing power assay among the four plants, whilst Momordica charantia and citrullus lanatus were significantly less effective than the rest. The current report study is similar to the previous report for the antioxidant assays.^{20, 21} The study concludes that the comparison of medicinal plants belonging to cucurbitaceae family have found to have several pharmacological activities like antimicrobial, and antioxidants activity.Additional investigation on active principles and therapeutic values needs to be carried out on cucurbitaceae family, inorder to investigate the unknown biological active compounds and their medical application. Current study concludes that antimicrobial activity exhibited by this cucurbitaceae family would lead to the identification of antimicrobial drug molecule which helps in the treatment of microbial diseases in humans and animals.

CONFLICTS OF INTEREST:

No conflicts of interest.

ACKNOWLEDGEMENT:

The authors are express sincere thanks to the management of Vellore Institute of Technology, Vellore, India for providing necessary lab facilities to carry out this study.

REFERENCES:

1. Kumar KPS, Bhowmik D. Traditional medicinal uses and therapeutic benefits of Momordica charantia Linn. Int J Pharm Sci Rev Res. 2010, 4(3):23–8.

2. Raghavan Anilakumar K. Nutritional, Pharmacological and Medicinal Properties of Momordica Charantia. Int J Nutr Food Sci [Internet]. 2015, 4(1):75–83.

3. Yusuf AJ, Alebiosu C O et al. Efficacy of crude extract and fractions of Citrullus lanatus against some selected micro-organisms. Sky J Microbilogy Res. 2015, 3(5):67–72.

4. Amit Kumar, Sangh Partap, Neeraj K. Sharma KKJ. Phytochemical, Ethnobotanical and pharmacological profile of Lagenaria siceraria: An overview. J Pharmacogn Phytochem. 2012; 1(3):24–31.

5. Shaheen SZ, Bolla K, Vasu K, Charya M a. S. Antimicrobial activity of the fruit extracts of Coccinia indica. African J Biotechnol [Internet]. 2009; 8(24):7073–6.

6. T. Majumder. K.nagaraju, K.sreelekha M nikitha. Evaluation of Pharmacological Activity of Hydroethanolic Extract Coccinia grandis Linn. Leaves. Sch Acad J Pharm [Internet]. 2017; 6(4):149–57.

7. Rahman H, Manjula K, Anoosha T, Nagaveni K, Eswaraiah MC, Bardalai D. In-Vitro Anti-Oxidant Activity of Citrulus lanatus Seed Extracts. Asian J Pharm Clin Res. 2013; 6(3):152–7.

8. Bello, H.S. Ismail, H.Y. Goje, M.H. Mangga HK. Antimicrobial Activity of Citrullus Lanatus (Watermelon) Seeds on Some Selected Bacteria. J Biotechnol Res. 2016; 2(6):39–43.

9. Rahman Gul et al. Preliminary Phytochemical Screening, Quantitative Analysis of Alkaloids, and Antioxidant Activity of Crude Plant Extracts from Ephedra intermedia Indigenous to Balochistan. Sci World J. 2017; 7.

10. Gupta M, Thakur S, Sharma A, Gupta S. Qualitative and quantitative analysis of phytochemicals and pharmacological value of some dye yielding medicinal plants. Orient J Chem. 2013; 29(2):475–81.

11. Madhu M, Sailaja V, Satyadev T, Satyanarayana M V. Quantitative phytochemical analysis of selected medicinal plant species by using various organic solvents. J Pharmacogn Phytochem. 2016; 5(2):25–9.

12. Al-Mariri A, Safi M. In vitro antibacterial activity of several plant extracts and oils against some gram-negative bacteria. Iran J Med Sci. 2014; 39(1):36–43.

13. Mathur A, Singh R, Yousuf S, Bhardwaj A, Verma SK, Babu P, et al. Antifungal activity of some plant extracts against clinical pathogens. Adv Appl Sci Res [Internet]. 2011; 2(2):260–4.

14. Pisoschi AM, Negulescu GP. Methods for Total Antioxidant Activity Determination: A Review. Biochem Anal Biochem [Internet]. 2012; 01(01):1–10.

15. Reshma, Arun KP, Brindha P. In vitro anti-inflammatory, Antioxidant and nephroprotective studies on leaves of Aegle marmelos and Ocimum sanctum. Asian J Pharm Clin Res. 2014; 7(4).

16. Lalèyè OAF, Ahissou H, Assogba MF, Azando VBE, Olounladé AP, F ASK, et al. Research Article In vivo hypoglycemic activity and acute oral toxicity of ethanolic and aqueous leaves extract of Momordica charantia Linn (Cucurbitaceae) from Benin. J Chem Pharm Res. 2015; 7(6):376–85.

17. Adebola R, Akinwale Y, Ariyo J. Mormodica charantia Linn. A Potential Antibiotic and Anti-Fungal Drug. Int J Pharm Sci Invent. 2016; 5(2):21–7.

18. Y.P.Nagaraja KNG et al. Antimicrobial effect of Lagenaria siceraria(Mol.) standley, against certain bacteria and fungal strains. J Appl Nat Sci [Internet]. 2011; 3(1):124–7.

19. Hassan loiy elsir ahmed. In vitro Antimicrobial activities of chloroformic, hexane and ethanolic extracts of Citrullus lanatus var. citroides (Wild melon). J Med Plants Res [Internet]. 2011; 5(8):1338–44.

20. Sharma NK, Yadav P, Singh HK, Shrivastava AK. in Vitro Antioxidant Activity of Lagenaria Siceraria. Malaysian J Pharm Sci. 2013; 11(1):1–11.

21. Umamaheswari M, Chatterjee TK. In vitro antioxidant activities of the fractions of Coccinia grandis L. leaf extract. African J Tradit Complement Altern Med [Internet]. 2008; 5(1):61–73.

Received on 28.09.2018 Modified on 14.10.2018

Research J. Pharm. and Tech. 2019; 12(4):1541-1546.

DOI: 10.5958/0974-360X.2019.00255.5