Medicinal value of Azadirachta indica A Juss

Medicinal value of Azadirachta indica A Juss. and Mentha piperita L.
OR
Phytochemical screening and antimicrobial properties of Azadirachta indica A Juss. and Mentha piperita L.
OR
Antimicrobial potential of Azadirachta indica A Juss. and Mentha piperita L.
INTRODUCTION
It is established fact that; the various contagious diseases are treated with plants and these are the key medicinal source in some emerging countries (Mothana and Linclequist, 2005). A lot of pharmaceutical plants are considered to have potential antimicrobial crude drugs adding to a basis of new compounds with anti-microbial activity, with possibly new modes of action. This expectancy established that some naturally found plant compounds can kill antibiotic-resistant strains of bacteria such as E. coli, S. aureus, B. cereus and M. luteus (Friedman, 2006). Phytochemicals found in plants have shown great promise for the treatment of intractable infectious human diseases which also includes viral infections (Cowan, 1999). Further systematic screening of plants may result in the invention of new valued compounds (Nitta et al., 2002).
Neem (Azadirachta indica) which belongs to family Meliaceae is the most adaptable, diverse trees of tropics, with massive potential. Various compounds are synthesized during secondary metabolism which have antimicrobial properties due to which this plant has been used in the past. These properties are due to active substances, like phenolic compounds which are a part of the essential oils (Janssen et al., 1987). The natural activities are attributed to the occurrence of a large number of biologically active compounds in its different parts (Sonia and Srinivasan, 1999). The juice from the leaves is used as a tonic to increase hunger and to remove intestinal worms. It is also used for its hypoglycaemic, hypolipidemic, hepatoprotective and hypotensive activities and also to control fever. Therapeutically, the leaf extract is used for its antimicrobial activity against dental pathogens. In addition, in the Ayurvedic medicine system, the selected plant is used to treat malarial fever. Its formulated products include treatments for cancer, skin diseases, digestive disorders and AIDS. Aromatics plants including mentha, have traditionally been used in folk medicine as well as to extend the shelf life of foods, showing inhibition against bacteria, fungi and yeasts (Hulin et al., 1998). Menthol is prescribed as a medication for gastrointestinal disorders, common cold and musculoskeletal pain (Patel et al., 2007). As iron and magnesium play important role in human nutrition it is blessing that mint plants are wealthy resources of these component (Arzani et al., 2007). Moreover a large amount of literature is available on the therapeutic properties of essential oils present in Mentha spp. (Gulluce et al., 2007; Rasooli, 2008).
Materials and Methods
Preparation of plants extracts
The Azadirachta indica and Mentha piperita leaves were collected, dried and powdered using electrical grinder and taken in two different beakers with subsequent addition of 500 ml methanol (mother extractant) in each beaker with two to three times off and on shaking in a day. Then the filterate was obtained using Whatmann’s filter paper no. 1. Following condensation of both filtrates using hot plate magnetic stirrer until the filtrate turns to gummy solid. Then the gummy solid (methanolic extract) of each of plant was further processed by addition of 100 ml of distilled water and shaked to dissolve crude extract. The obtained solution was put to separating funnel and fractioned by 150 ml of ethyl acetate, chloroform and acetone respectively (Bempah et al., 2011).
Phytochemical Analysis
Phytochemical screening was carried out on all the extracts of both plants to check the presence of different phytochemicals using respective standard protocols such as, alkaloids (Evans, 1997), carbohydrates (Harborne, 1973), reducing sugars (Sofowora, 1993), tannins, phenols (Mace, 1963), flavonoids (Trease and Evans, 1989), terpenoids (Robinson, 1964), saponins, cardiac glycosides (Krisgnaveni et al., 2014) and fixed oils and fats (Adarsh et al., 2013).

Test microorganisms
S. haemolyticus, B. subtilis, K. pneumoniae, E. coli and P. aeruginosa were used to check antimicrobial activity of both plants extract in respective solvents. These all strains were obtained from Department of Microbiology and Molecular Genetics University of Punjab, Lahore.
Preparation of Bacterial Inoculum
0.5 McFarland inoculum was incubated onto LB (Luria Bertani), medium having 10g Bacto-tryptone, 5g yeast extract, 5g NaCl, 15g agar; pH 7 and autoclaved at 121oC, 15 lbs pressure for 15 minutes.
Determination of Antibacterial Activity
Well Diffusion Assay
10 ml of 0.5 McFarland solutions for all the five tested organisms was prepared separately in test tube. The test plates were prepared by pouring 20 ml of LB (Luria Bertani) and inoculated with 50µl of 0.5 McFarland inoculum of each test organism. Five wells (1 in centre and 4 on sides) were made with the help of sterile cork borer in each plate and filled with 50 µl of each extract (positive control) in side wells and 50 µl of pure solvent i.e. acetone, ethyl acetate and chloroform, in central well (negative control)). The plates were incubated at 37o C for 16-24 hours. The inhibition zones were appeared around each of well after incubation of 24 hours at 37ºC. Diameter of each zone was measured in “mm” with scale (Lertcanawanichakul et al., 2011).
Disk Diffusion Assay
The test plates were prepared for all tested organisms by adding16 ml of LB medium as base layer. After hardening, this layer was overlaid with 4 ml of molten LB agar media inoculated with 100 µl of 0.5 McFarland inoculum of test organism as seed layer. The sterilized discs of Whatman filter paper No. 1 (6 mm diameter) were prepared. Sterilized filter paper disks were then impregnated with 40 µl of crude extract solution and then were dried under sterile conditions and placed on the surface of test plates using sterile forceps. All test plates were incubated at 37ºC for 16–24 hours. After incubation, the diameter of inhibition zones was measured in ‘mm’ (Gerhardt et at., 1994).
Brine shrimps (Artemia Salina) microwell Cytotoxicity
Assay
Brine shrimp (Artemia salina) dried eggs were hatched in separating funnel (500ml) using artificial sea water (400ml). Bubbling air was used to oxygenate the suspension using an aquarium pump in the funnel. The aeration had been removed after 24-48 h incubation at room temperature, and kept undisturbed for 1 hour. One side of separating funnel was covered with aluminium foil while the other side was kept illuminated with a lamp and larvae were separated using a pipette. Then the larvae were transferred to a deep well micrititer plate (wells diameter 1.8cm, depth 2cm) filled with 0.2ml of artificial sea water. The dead larvae were counted (valued as N), 20 µg of crude extract was dissolved in 5-10 µl of dimethyl sulfoxide (DMSO) and was added in the plate which kept in the dark at room temperature. The number A of dead larvae was again counted after 24 h, under the microscope. To determine the total number G of the larvae, 0.5 ml methanol was added to kill the surviving larvae. Each test row was accompanied by a blind sample e pure dimethyl sulfoxide. For 100% mortality, actinomycin D (10 µg/ml) was used as a positive control (Solis et al., 1993). The mortality rate M was calculated using the following formula:
M= ((A-B-N))/((G-N) )×100
Where;
M = Percent of the dead larvae after 24 h.
A = Number of the dead larvae after 24 h.
B = Average number of the dead larvae in blind sample.
N = Number of the dead larvae before starting the test.
G = Total number of larvae.

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RESULTS AND DISCUSSION
1.1 Phytochemical Analysis
Preliminary phytochemical screening was carried out using evaporated extracts of both the plants. For qualitative screening of Azadirachta indica E. acetate and chloroform extracts showed the presence of all secondary metabolites except phenols while in acetone extract, tannins were found absent (table 1.1). Correspondingly, presence of tannins, alkaloids, saponins, flavonoids and few further active compounds such as saponins and steroids were also reported by earlier worker (Al-Hashemi and Hossain, 2016). For Mentha piperita in E. acetate, extract tannins and phenols were absent while in chloroform extract flavonoids and phenols were absent. Among three solvents, most of secondary metabolites were observed in acetone extract and only flavonoids were absent in acetone extract (table 1.1). The earlier reported results of phytochemical screening of mentha leaves showed the presence of carbohydrates, flavonoids, phenols while saponins, glycosides, steroids, alkaloids, reducing sugar and tannins was shown to be absent (Bansode and Chavan, 2014). Alkaloids were absent only in Azadirachta indicia chloroform layers (table 1.2).
Content Azadirachta indica Mentha piperita
E.A Chl Ace E.A Chl Ace
Carbohydrates + + + + + +
Tannins + – – – + +
Flavonoids + + + + – –
Phenols – – + – – +
Saponins + + + + + +
Terpenoids + + + + + +
Glycosides + + + + + +
Reducing Sugar + + + + + +
Oils + + + + + +
Table no. 1.1. Phytochemical constituent of various plant extracts prepared in different solvents
“+” indicating presence “-” indicating absence

Table no. 1.2 Phytochemical constituent of various plant extracts prepared in different solvents

Content Azadirachta indica Mentha piperita
Chl layer Org layer Chl layer Org layer
Alkaloids – + + +
“+” indicating presence “-” indicating absence
1.2 Determination of Antibacterial Activity
(a) Well-diffusion Assay
Azadirachta indica ethyl acetate extract gave maximum inhibition zone (10mm) against S. aureus, B. subtilis and P. aeruginosa while inactive against rest of strains (Fig.1) (plate no.1). The previous reported antimicrobial potential of Azadirachta indica leaves showed zone of inhibition of 20mm against E. faecalis and 7.1mm against C. albicans (Bohora et al., 2010). Zone of inhibition ranging from 0.8cm to 2cm was given by results of aqueous and alcoholic aleaf extract of Azadirachta indica against E. coli, S. aureus, S. Bacillus bacteria and Rhizopus fungi through cup-plate agar diffusion method (Gupta et al., 2013). The reason of difference between result of antibacterial activity of P. aeruginosa and E. coli was probably due to difference in method of solvent extraction (using soxhlet) and also due to variation in solvent used.
The Azadirachta indica chloroform extract was only active against S. aureus, giving 10mm inhibition zone around well while rest of all tested strains was seemed to be inactive (Fig.2) (plate no.2). The previously reported results were also same which was given by earlier workers for chloroform extract of Azadirachta indica against Pseudomonas sp., but the results deviated against E. coli and Klebsiella sp. (Shinde and Mulay, 2015; Zwetlana et al., 2014). The difference in results possibly due to the sources of microorganisms used.
The Azadirachta indica acetone extract gave 16mm maximum zone of inhibition against B. subtilis following 12mm against S. aureus, 11mm against E. coli, 10mm against P. aeruginosa, while the same extract was inactive against K. pneumoniae (Fig.3) (plate no.3). The similar result was also reported against Staphylococcus aureus Pseudomonas aeruginosa and Escherichia coli using acetone extract of Azadirachta indica (Shinde and Mulay, 2015; Edet, 2016).
The Mentha piperita E. acetate extract was inactive against all tested strain (Fig.1) (plate no.1) but the previously stated result of Mentha piperita L. indicated antibacterial activity against Bacillus sbubtilis, Streptococcus pneumonia, Staphylococcus aureus, Escherichia coli, Proteus vulgaris and Klebsiella pneumonia (Sujana et al., 2013). The variation from present results can be due to different extraction and experimental protocols.
The Mentha piperita chloroform extract was inactive against all tested strains of bacteria (Fig. 2) (plate no.2). Earlier reported study indicated that chloroform extract of Mentha piperita was active against Bacillus subtilis, Streptococcus pneumonia, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Klebsiella pneumonia (Sujana et al., 2013). The efficiency of plant extract against a particular pathogen is affected by various intrinsic and extrinsic factors may be the reason for the difference.
The acetone extract of Mentha piperita. gave 10mm inhibition zone against S. aureus but the respective was inactive against E. coli, K. Pneumonia, P. aeruginosa, B, subtilis (Fig.3) (plate no.3). The acetone extract of Mentha arvensis indicated the antimicrobial potential against B. cereus, Serratia sp., A. flavus and P. citrinum (Dhiman et al., 2016). The varying results between previous and present reported results may be due to the difference in tested species of plant and microorganisms employed in both study.
(b) Disc diffusion assay
Ethyl acetate extract of Azadirachta indica was found to be active against S. aureus (Fig. 4) (plate no.4). In previously reported result, respective test extracts of Azadirachta indica possesses significant antimicrobial activity against test microorganisms of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris and Klebsiella pneumonia (Latha et al., 2015). The reason of difference in results against E. coli and P. aeruginosa maybe the of difference in method used for preparation of plant extracts.
The chloroform extract of Azadirachta indica with comparison to pure solvent not show any obvious activity against all tested bacterial strains (Fig. 5) (plate no.5). Same results were also reported in previous study against P. aeruginosa and S. aureus (Al-Jadidi and Hossain, 2015).
Among acetone extracts, Azadirachta indicia gave 11mm and 10mm inhibition zone against B. subtilis and E. coli (Fig. 6) (plate no.6). The same results for such sample showed no antimicrobial activity against S. aureus and P. aeruginosa (Irshad et al., 2011). The understudy results also relate with earlier reported results against B. subtilis and E. coli. but as the results differed against S. aureus which is results of difference in experimental conditions.
Ethyl acetate extracted sample of Mentha piperita shown clear hallow against E. coli and K. pneumoniae but it was not active against P. aeruginosa, S. aureus and B. subtitles (Fig. 4) (plate no.4). Such result was also reported against E. coli and S. aureus but at 0.675 mg/ml concentration (Satmi and Hossain, 2016).
The chloroform sample from Mentha piperita was active against E. coli and K. pneumoniae while inactive against S. aureus, B. subtilis and against P. aeruginosa (Fig. 5) (plate no.5). In earlier reported results the activity was found against E. coli and S. aureus. The result was same against E. coli but the results deviated against S. aureus (Satmi and Hossain, 2016). Possibly the difference in result against S. aureus was due to difference in method of extraction of samples in previous (Soxhlet extractor) and present study.
The most obvious zone (14mm) was given by Mentha piperita. acetone extract against S. aureus (Fig. 6) (plate no.6). Same result was reported against Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli (Keskin and Toroglu, 2011).
1.3 Cytotoxicity potential of plants extracts against Brine shrimp larvae
Cytotoxicity potential of both plants were checked against brine shrimp larvae. Azadirachta indica plants showed maximum toxicity level (23.37%) with chloroform solvent while the Mentha piperita acetone extract showed least toxicity (9.15%) (Fig. 7). In reported results of water and methanol extracted samples of Azadirachta indica (Meliaceae) and Myrica salicifolia (Meliaceae) for brine shrimp the toxicity level was 101.26 ± 3.7 61.43 ± 2.9 (?g/ml) and 328.22 ± 10.9 320.17 ± 1.6 (?g/ml) respectively (Kirira et al., 2006). The toxicity study of under study Mentha piperita showed noticeable results among all extracts being tested.
CONCLUSION
Mostly biologically active compounds were found in E. acetate and acetone extracts of Azadirachta indica and acetone extract of Mentha. Using agar well diffusion assay chloroform and acetone samples of Azadirachta indica were found to be most active giving 16mm inhibition zone against B. subtilis. The disc diffusion assay was also used to determine the antimicrobial potential of respective plants in which ethyl acetate extracted sample Azadirachta indica indicated 12mm maximum zone of inhibition against B. subtilis. While acetone extracted sample of Mentha piperita exhibited 14mm maximum zone of inhibition. Brine shrimps microwell cytotoxicity assay was performed to check the cytotoxicity level of all the extracts. Mentha piperita acetone extract showed least toxicity (9.15%) while the Azadirachta indica plants showed maximum toxicity level (23.37%) with chloroform solvent.
RECOMMENDATION
The following recommendations could be made from the outcomes of this study. As only in vitro method was used in assessing the antimicrobial activity of the plant crude extracts, further investigations using bioassay guided fractionations are recommended to isolate and identify the pure compounds responsible for antibacterial activity of Azadirachta indica and Mentha piperita.
In addition, studies should be done in order to identify the active phytochemical constituents qualitatively in the respective plants extracts and also evaluate their efficiency in-vivo so that they can be made commercially. Furthermore, there is need for in-vivo trials to categorise those plants that are active and suitable for general use.

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