Antibacterial activity of Momordica charantia L. and Citrus limon L. on gram positive and gram negative bacteria

The uses of herbal medicines are well recognized since the advent of mankind having very little side effects. Their use is a cheaper and quite affordable source of antimicrobial particularly in the rural areas. Some of the microbial strains are drug resistant and are always been a threat to human life. The herbal therapy is the best choice of remedy against the drug-resistant strains, which had opened the new roads for conventional use due to several antimicrobial phytochemicals and essential oils. Momordica charantia Linnaeus (L). and Citrus limon L. contain phytochemicals, which affect the bacteria, fungi, viruses, and parasites The aim of this study is to examine the effects of Momordica charantia and Citrus limon peel extracts on gram-positive and gramnegative bacteria. Preparation of extracts in ethanol, methanol, and ether were prepared and activation of test cultures, antimicrobial assay by disc diffusion, well diffusion and spectrophotometry at 600 nm. The greater zones of inhibition were determined by ethanolic extract of Momordica charantia on the growth of Bacillus cereus and Pseudomonas aeruginosa whereas Citrus limon methanolic peel extract revealed the greater effects on Staphylococcus aureus, Streptococcus pyogenes, and Escherichia coli at 15 and 20 μl concentrations respectively. The observations concluded that both the plant extracts have effects on bacteria that indicate that the preparations of these extracts up to pharmaceutical standard may be effectively used as antibacterial therapy against gram-positive and gram-negative bacteria.


Introduction
Antibiotics are being used for the remedial purpose of the infections in the prescribed doses. As long as the use of antimicrobial substances got the great significance, there were the issues of the emergence of antimicrobial resistance [1, 2]. Since a long time, before Christ, men depend upon the use of natural products, which are known the important source of medicines. The long use of herbal extracts from medicinal plants and their products played a significant role to eliminate the infectious diseases and also possessing antioxidant, anti-inflammatory and anti-cancer potentials [3][4][5][6]. The use of allopathic and herbal medicines among the older populations was more important in chronic disease. It explored the great significance for clinical applications of the effectiveness and care of herbal medicines alone and also with the combination of traditional drugs. World Health Organization (WHO) reported the use of medicinal plants for treatment in the form of synthetic and semisynthetic drugs. WHO also reported that 80 % of the global population uses this form of medicines [7,8]. Momordica charantia, (Bitter gourd) a globally used vegetable is commonly called as Karela. It is significant tonic and laxative for remedy of the disorders of the gastrointestinal tract, disorders of skin, nose and bones and liver. Its effects are also known against blood cancer, malarial infection, dysmenorrhea, renal stone and anal hemorrhoids [9]. Bitter gourd as the food was used by people of tropical areas. Besides, the use as vegetable, the Karela leaves and fruits are being used in the form of tea, soups and also beer [10]. Citrus is a significant global fruit tree crop, belongs to the Rutaceae family possessing the phenolic compounds, vitamins, minerals and also carotenoids and have antioxidant activity [11][12][13][14][15]. Citrus fruits possess flavonoids that are considered as a protective agent against heart diseases and cancer and Ischemic stroke. Citrus limon L. is significantly used in small quantities, helps give taste to salads, various drinks and desserts. The health benefits of lemon include the maintenance of hypertension, iron absorption rising immunity, providing nutritional value including calories (17), 1.45 g of sugar, Iron (0.5g), folate (9µg), protein (0.64 g), fat (0.17g) potassium (116 mg), carbohydrate (5.41g), calcium (15 mg), zinc (0.05), magnesium (7 mg), phosphorus (13 mg) a rich source of vitamin C, which is required for the synthesis of collagen [16].

Materials and Methods Preparation of crude extract in water
Crude extract of bitter gourd and lemon peels was prepared by mixing ten grams of powdered extract in 40 ml of the warm distilled water. Each flask was allowed for 20 minutes time at 80°C in water bath. After mixing the test extracts were allowed to cool for 24 h. for percolation. Later on, the extracts were passed through double layered muslin cloth and the filtrate was allowed for centrifugation for thirty minutes at 5000 rpm. These extracts were again dissolved in 100 ml of distilled water mixed well and filtered by 0.2 mm size Millipore filter [17].

Preparation of aqueous extracts in solvents
The Momordica charantia L. and Citrus limon L. were purchased from local market of Jamshoro. Both vegetables were washed with tap water, surface was sterilized by 70% ethanol and rinsed with sterile double distilled water [18] and later on the vegetables were dried with Whattman filter paper and kept for one hour. The vegetables were peeled and allowed for 5-7 days in shadow to dry. The peeled skin of both vegetables were ground to powder and filtered to get fine powder and kept in freezer. The powdered samples (1 g) were added in 100 ml of ethanol, methanol and ether separately and extracted through the soxhlet apparatus for 24 h. at room temperature [19,20]. Later on the extracts were allowed at 150 rpm agitation in rotary incubator for 24 h. for evaporation then filtered through Whattman No. 1 filter paper [20]. The concentrations were made by mixing respective solvents (100 ml) volume / volume in the filtered extracts.

Antibacterial activity of test extracts Disc diffusion method
The antibacterial activity was performed by the method of [21]. The broth culture of the test bacteria was maintained at 10 6 cells /ml using hemocytometer (Quijing-China) The bacterial cultures (100 µL) were separately incubated at 37°C for 24 h to observe the colonies later on a small portion of colony was emulsified in nutrient broth and kept for 30 minutes to activate the test culture. Using sterile commercial swab the activated cultures were separately inoculated on Mueller Hinton agar plates (Oxoid). Filter paper discs of 6 mm size were prepared, sterilized and impregnated in crude extract and each aqueous extract for 5 minutes and later dried in air for 10 minutes and placed on the surface of the inoculated nutrient agar plates. Ampicillin disc of 10 µg was used a positive control and 5% DMSO as negative control.

Agar well diffusion method
The broth culture of the test bacteria were inoculated by swab and three wells of 6 mm size were bores by sterile cork borer. A small volume of (1µl) of molten nutrient agar was poured in each bore of each agar plate. After solidification of nutrient agar in bored wells 0.3 ml of aqueous extract of ethanol, methanol and ether) of bitter gourd and lemon was poured allowed the plates for few minutes at room temperature and the plates were incubated for 24 h at 37°C. The diameters of antibacterial activities of water, ethanol, methanol and ether extracts were measured by zone of inhibition in mm [22]. Spectroscopic studies Different volumes of aqueous extract of bitter gourd and lemon prepared in ethanol, methanol and ether were taken in 2, 10, 15, 20, 25 microliter (µl) concentrations and mixed in 1 ml of the culture broth of each test culture in separate flasks. This suspension was mixed gently and absorption was determined at 600 nm (A600) by Hitachi U-1800 spectrophotometer.

Results
The effects of Ampicillin 10 µg and Dimethysulfoxide (DMSO) 5% were observed and positive and negative control respectively. The findings indicated varying zones of growth inhibition in positive control. Ampicillin showed greater effect on Pseudomonas aeruginosa strains (22 mm) followed by Staphylococcus aureus, Bacillus cereus whereas no zone observed by DMSO as negative control (  (Table 17). Comparatively, the spectroscopic studies revealed the greater absorbance of Momordica charantia and Citrus limon at 15 and 20 µl concentration respectively at A600 (Fig. 1 & 2) on the test bacteria.

Discussion
All test strains were examined for their sensitivity against the positive control (Ampicillin) and negative control (5% DMSO) that revealed sensitivity of ally test strains to ampicillin antibiotic with varying zone sizes whereas DMSO showed no effect on all test strains (Table 1). Bitter gourd peels when dissolved in water revealed insignificant effect on test strains not more than 4-5 mm zone of inhibition by disc diffusion method and agar well diffusion methods (