INTRODUCTION: Since time immemorial, our traditional system of medicine and folklore medicine claim that medicinal plant as a whole or their parts are being used in all types of infectious diseases successfully.

Nowadays, Multiple Drug Resistance (MDR) has developed due to the indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of such infectious diseases.  Adding to this problem, antibiotics are sometimes associated with adverse effects on the host, including hypersensitivity, immune suppression and allergic response ¹.

Owing to the side effects and the resistance that pathogenic microorganisms build against antibiotics, many scientists have recently started paying attention to medicinal plants and biologically active compounds isolated from plant species used in herbal medicine.  Antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world ².  However, it is necessary to evaluate on a scientific base, the potential use of herbal medicine for the treatment of infectious disease caused by common pathogens.

Moringa oleifera belongs to the family Moringaceae. It is a highly valued plant, distributed in many countries.  It has an impressive range of medicinal uses with high nutritional value.  Various parts of the plant such as the leaves, roots, seeds, bark, fruits, flowers and immature pods act as cardiac and circulatory stimulants, possess antitumour, antipyretic, antiepileptic, anti-inflammatory, antiulcer property ³.  The seeds of this plant are used for their antibiotic and anti-inflammatory properties to treat arthritis, rheumatism, gout, cramp, sexually transmitted diseases and as a relaxant for epilepsy.

Momordica charantia belonging to the family Cucurbitaceae, is found in tropical and subtropical regions of the world such as India, Asia, South America and widely used as food and medicine.  It is said to possess innumerable biological activities like anthelminthic, antibacterial, antidiabetic, anti-inflammatory, antimutagenic, antioxidant, anti-tumour and antiulcer ⁴.  Traditionally it is said to possess astringent, aphrodisiac, carminative, cytostatic, cytotoxic, depressive, hypo-cholesterolemic, hypotriglyceridemic, hypo-glycaemic, insecticidal, laxative, purgative, stomachic, tonic and vermifuge properties.

The present study was undertaken to evaluate the antimicrobial activity of the methanol and aqueous extracts of the seeds of Moringa oleifera and Mormordica charantia. This study assesses the in vitro antimicrobial activity of the above plants against few common pathogenic microorganisms that cause the most common type of infectious disease in rural India.

MATERIALS AND METHODS:

Plant collection: Fresh vegetables of Moringa oleifera and Momordica charantia were collected from Vellore, Tamilnadu, India and their seeds were removed.  The taxonomic identities of these plants were made by the expertise of the Department of Botany, Voorhees College, Vellore.  The procured seeds were then washed with 70% alcohol and then rinsed with distilled water. It was shade-dried for two weeks at room temperature and stored in air-tight containers until further use.

Plant extraction: Collected, dried plant samples (seeds) in small pieces were crushed to powder form.  For aqueous extract, a lot of 5 g of the sample was soaked in 25 ml of sterile distilled water and kept in a rotary shaker for 12 hours at 30^oC. Thereafter, it was filtered with Whatmann No.1 filter paper.  The filtrate was concentrated in a rotary evaporator till a sticky mass was obtained.  The resultant M. oleifera aqueous extract (MOAE) and M. charantia aqueous extract (MCAE) were then weighed and stored at 4^oC.  For methanol extract, a lot of 5 g of the sample was soaked in 25 ml of methanol and kept in a rotary shaker for 12 hours at 30^oC. Thereafter it was filtered with Whatmann No.1 filter paper.  The filtrate was concentrated in a rotary evaporator till a sticky mass was obtained.  The resultant M.oleifera methanol extract (MOME) and M.charantia methanol extract (MCME) were then weighed and stored at 4^oC.

Test organisms: The bacterial strains used for the test were Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.  The fungal strains used for the test were Aspergillus niger, Candida albicans and Candida kefyr.  All the stock cultures were obtained from Microlabs, Institute of Research and Technology, Vellore, Tamilnadu.

Preliminary phytochemical screening: The extracts obtained were subjected to preliminary phytochemical analysis following standard methods ⁵. The extracts were screened to identify the presence or absence of various active principles like alkaloids, carbohydrates, saponins, glycosides, proteins, sterols, phenols, flavonoids, terpenoids and tannins.

Antibacterial activity: The extracts were screened for their antibacterial activity in comparison with the standard antibiotic ciprofloxacin (100 mg/ml) in vitro by agar-well diffusion method ⁶. Bacterial lawn was prepared using the test organisms on Mueller Hinton Agar (MHA).  Wells were punched for 8 mm deep in 30 min old bacterial lawn and each well was based by 50 ml molten MHA.  In each step of well cutting, the well cutter was thoroughly wiped with alcohol using sterile micropipettes. Further, wells were filled with 100 ml aliquots of 30 mg/ml solvent extract of the plants. Their respective solvents were used as a negative control. The plates were incubated at 37^oC for 24 hours. Antibacterial activities were evaluated by measuring the diameter values of zones of inhibition.

Antifungal activity: The extracts were also screened for their antifungal activity in comparison with the standard antibiotic fluconazole (10 mg/ml) in vitro by agar-well diffusion method ⁶.  Lawn culture was prepared using the fungal strains on Sabouraud’s Dextrose Agar (SDA).  The inoculated plates were kept for few minutes for solidification.  After solidifying, using well cutter, wells were made at a depth of 8 mm.  100 ml aliquots of the selected extracts were added into the well at a concentration of 30 mg/ml.  The plates were incubated at room temperature for 48 hours.  The activity of the extracts was determined by measuring the diameter of zones of inhibition.  Pure solvents were used as a negative control.

RESULTS:

Qualitative phytochemical analysis: Qualitative phytochemical analysis of M.oleifera seeds showed the presence of alkaloids, glycosides, flavanoids, terpenoids and tannins in both aqueous and methanol extract.  M.charantia seeds differed in their phytoconstituents in methanol and aqueous extracts.  While the aqueous extract contained saponins, flavonoids and sterols, the methanol extract contained saponins, flavonoids, alkaloids, glycosides and tannins.  The results are tabulated in Table 1.

TABLE 1: PHYTOCHEMICAL ANALYSIS OF M.OLEIFERA AND M.CHARANTIA.

The data in the table gives the phytochemicals that have been identified from the aqueous and methanol extracts of M.oleifera and M.charantia seeds.  ‘+’ indicates presence and ‘-’ indicates absence.

Antibacterial activity: The antibacterial activity of the plant extracts were variable according to various organisms and the solvent used.  The inhibition zones ranged from 5 mm to 8 mm diameter.   The results obtained in the present study revealed that M.oleifera methanol extract possessed potential antibacterial activity.  When tested, the zone of inhibition was maximum for S.aureus (8 mm) followed by E.coli (7 mm) and P.aeruginosa (5 mm), while the aqueous extract proved to have no effect on the test organisms.  Similarly the methanol extract of M.charantia exhibited potential bactericidal property showing maximum zone of inhibition for S.aureus (7 mm) followed by  E.coli (5 mm) and P.aeruginosa (5 mm).  Even here, the aqueous extract was not effective in controlling the growth of the bacteria.  The inhibition of S.aureus by MOME and MCME was comparable to the standard antibiotic ciprofloxacin very closely. The results are represented in Figure 1.

Antifungal activity: Antifungal activity of the seed extracts showed moderate activity when compared with the standard antibiotic flucanozole.  Among the four extracts, methanol extract of both M.oleifera and M.charantia showed a very good antifungal activity against all the three tested fungi as depicted in Figure 2.  However the aqueous extract of  M.oleifera was effective against C.kefyr (8 mm) and C.albicans (6 mm).  But the aqueous extract of M.charantia possessed activity against A.niger strain (5 mm) only. Interestingly, MOME significantly inhibited C.albicans similar to the standard fluconazole.

FIGURE 1: ANTIBACTERIAL ACTIVITY OF M.OLEIFERA AND M.CHARANTIA. Each data point represents the zone of inhibition formed by the extracts against three bacterial strains in comparison with the standard ciprofloxacin.  The methanol extracts showed significant activity against the tested organisms.

FIGURE 2: ANTIFUNGAL ACTIVITY OF M.OLEIFERA AND M. CHARANTIA. Each column represents the zone of inhibition formed by the extracts against three fungal strains.  The methanol extract possessed significant activity against all the three organisms, when compared to the aqueous extract.

DISCUSSION: It has been reported that higher plants show a promising potential source of new antimicrobial agents ⁷. Their activity is directly attributed to the presence of certain phytochemicals in such plants which contribute to its microbicidal property and inhibition of microbial growth by various mechanisms ⁸.  These botanical compounds extracted depend upon the type of solvent used in the extraction procedure.

In this study, methanol extract of the plants M.oleifera and M.charantia provided more consistent antimicrobial activity at 30 mg/ml concentration.  However, aqueous extract failed to show similar result.  It is clear that the methanol extract contain active phytoconstituents that gives the plant its antimicrobial property.

MOME had the broadest spectrum of activity on the tested bacterial and fungal strains.  This may be due to the presence of flavonoids, terpenoids and tannins.  The activity of flavonoids is probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell walls.  More lipophilic flavonoids may also disrupt microbial membranes ⁹.

The oil containing terpenoids has exerted a marked inhibition against multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, Enterococcus faecalis ¹⁰.  Terpenes and phenols from methanolic extract have been reported by different workers as effective antimicrobial compounds ^{11, 12}.  Possession of alkaloids, tannins and flavonoids has also been shown to enhance antimicrobial property of a plant ¹³.

MCME revealed the presence of alkaloids, glycosides and tannins which are absent in the corresponding MCAE.  Studies have shown that the bark of Terminalia arjuna contains glycosides and tannins which are antibacterial compounds ¹⁴.  Hence their presence may have added to its activity.  Another previous study proved that the aqueous extract of leaves of M.charantia was found to exhibit milder antimicrobial activity compared to methanolic extract which apparently indicates that the methanolic extract contains higher concentration of active antimicrobial phyto-compounds such as alkaloids and glycosides ^{15, 16}.

Both MOME and MCME were found to contain tannins.  Tannins are a group of compounds that has received a great deal of attention in the recent years.  One of their molecular actions is to complex with proteins through so-called nonspecific forces such as hydrogen bonding and hydrophobic effects, as well as by covalent bond formation ^{17, 18}. Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes and cell envelope transport proteins. They also complex with polysaccharides ¹⁹. Among the microorganisms tested, S. aureus and C. albicans are among the most commonly encountered pathogens in clinical practice and there has been an alarming increase in staphylococcal infections by strains with MDR ^{20, 21, 22}.

Here, both MOME and MCME have shown intrinsic bioactivity against S. aureus.  Therefore the result reconfirms that the methanolic extract of the plants have power over the microorganisms especially against S. aureus.  By further isolation and characterization of the methanol extracts of M. oleifera and M. charantia seeds, the exact active fraction and phytoconstituent responsible for their efficiency can be identified.

CONCLUSION: To conclude, this study evaluated the antimicrobial efficiency of the aqueous and methanol extracts of M.oleifera and M.charantia seeds and the methanol extract was found to possess significant antimicrobial activity comparable to the standard antibiotics.  This may be due to the presence of flavonoids, terpenoids and tannins in M.oleifera seeds and the presence of alkaloids, glycosides and tannins in M.charantia seeds.  These can be used to develop new drugs for the therapy of infectious diseases with particular reference to those caused by S. aureus and C. albicans.  However, further studies should be undertaken to elucidate the exact mechanism of action by which the extracts exert their antimicrobial effect.

ACKNOWLEDGEMENT: The authors thank the University Grant Commission - Major Research Project, Government of India for their funding.

REFERENCES:

1. Dey SK, Banerjee D, Chattapadhyay S and Karmakar B: Antimicrobial activities of some medicinal plants of West Bengal.  International Journal of Pharma and Bio Sciences 2010; 1(3): 1-10.
2. Bouabdelli F, Djelloul A, Kaid-Omar Z, Semmoud A and Addou A:  Antimicrobial activity of 22 plants used in urolithiasis medicinein western Algeria.  Asian Pacific Journal of Tropical Disease 2012; S530-S535.
3. Bukar A, Uba A and Oyeyi TI:  Antimicrobial profile of Moringa oleifera Lam. extracts against some food-borne microorganisms.  Bayero Journal of Pure and Applied Sciences 2010; 3(1): 43-48.
4. Leelaprakash G, Rose JC, Gowtham BM, Javvaji PK and Prasad SA:  In vitro antimicrobial and antioxidant activity of Momordica charantia leaves.  Pharmacophore 2011; 2(4): 244-252.
5. Harborne JB:  Phytochemical methods; Chapman and Hall, London. 1973.
6. Ahmad I and Beg AZ:  Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens.  Journal of Ethnopharmacology 2001; 74: 113-123.
7. Selvamohan T, Ramadas SV and Kishore SS:  Antimicrobial activity of selected medicinal plants against some selected human pathogenic bacteria.  Advances in Applied Science Research 2012; 3(5): 3374-3381.
8. Patra AK:  An overview of antimicrobial properties of different classes of phytochemicals.  In: Patra AK (Ed.), Dietary phytochemicals and microbes.  2012, XII; 1-32.
9. Jeyaseelan EC and Jashothan PTJ:  In vitro control of Staphylococcus aureus (NCTC 6571) and Escherichia coli (ATCC 25922) by Ricinus communis L.  Asian Pac J Trop Biomed 2012; 2(9): 717–721.
10. Mulyaningsih S, Sporer F, Zimmermann S, Reichling J and Wink M:  Synergistic properties of the terpenoids aromadendrene and 1,8-cineole from the essential oil of Eucalyptus globulus against antibiotic-susceptible and antibiotic-resistant pathogens.  Phytomedicine 2010; 17(13): 1061-6.
11. Sati SC and Joshi S: Antibacterial activities of Ginkgo biloba L. leaf extracts.  Scientific World Journal 2011; 11: 2237–2242.
12. Shabir G, Anwar F, Sultana B, Khalid ZM, Afzal M, Khan QM and Ashrafuzzaman M: Antioxidant and antimicrobial attributes and phenolics of different solvent extracts from leaves, flowers and bark of Gold Mohar [Delonix regia (Bojer ex Hook.) Raf].  Molecules 2011; 16(9): 7302-19.
13. Ruttoh EK, Tarus PK, Bii CC, Machocho AK, Karimie LK and Okemo PO: Antibacterial activity of Tabernaemontanastapfiana Britten (Apocynaceae) extracts.AfricanJournalofTraditionalComplementary and Alternative Medicines 2009; 6(2): 186-194.
14. Ramya S, Kalaivani T, Rajasekaran C, Jepachanderamohan P, Alaguchamy N, Kalayansundaram M and Jayakumararaj R: Antimicrobial activity of aqueous extracts of bark, root, leaves and fruits of Terminalia arjuna Wight & Arn. Ethnobotanical leaflets 2008; 12: 1192-97.
15. Zhang W, Hu J, Lv W, Zhao Q and Shi G: Antibacterial, antifungal and cytotoxic isoquinoline alkaloids from Litsea cubeba.  Molecules 2012; 17(11): 12950-12960.
16. Kouam J, Mabeku LBK, Kuiate JR, Tiabou AT and Fomum ZT: Antimicrobial glycosides and derivatives from roots of Picralima nitida.  International Journal of Chemistry 2011; 3(2): 23-31.
17. Haslam E: Natural polyphenols (vegetable tannins) as drugs: possible modes of action. J Nat Prod 1996; 59(2): 205–215.
18. Stern JL, Hagerman AE, Steinberg PD and Mason PK: Phlorotannin-protein interactions.  J Chem Ecol 1996; 22(10): 1877–1899.
19. Ya C, Gaffney SH, Lilley TH and Haslam E: Carbohydrate polyphenol complexation. In: Hemingway RW, Karchesy JJ (ed.), Chemistry and significance of condensed tannins. Plenum Press, New York, 1988: 553.
20. Prasad S, Nayak N, Satpathy G, Nag HL, Venkatesh P, Ramakrishnan S, Ghose S and Nag TC: Molecular & phenotypic characterization of Staphylococcus epidermidis in implant related infections.  Indian J Med Res 2012; 136: 483-490.
21. Onanuga A and Temedie TC:  Nasal carriage of multi-drug resistant Staphylococcus aureus in healthy inhabitants of Amassoma in Niger delta region of Nigeria.  Afr Health Sci2011; 11(2): 176-81.
22. Dubey D, Rath S, Sahu MC, Pattnaik L, Debata NK and Padhy RN: Surveillance of infection status of drug resistant Staphylococcus aureusin an Indian teaching hospital.  Asian Pac J Trop Dis 2013; 3(2): 133-142.
    

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    How to cite this article:

    Shoba FG, Babu VA, Parimala M and Sathya J: In vitro evaluation of antimicrobial activity of Moringa oleifera and Momordica charantia seeds. Int J Pharm Sci Res 2014; 5(5): 1988-93. doi: 10.13040/IJPSR.0975-8232.5 (5).1988-93

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