In Vitro Cytotoxic Activity Toward Anticancer and Antimicrobial of Azadirachta Indica, Aegle Marmelos, Ocimum Sanctum and Withania Somnifera Extracts

Background: Resistance to treatment represents the ‘big’ problem and the considerable improvement in survival rates still remains a researcher’s dream. Thus, continued research efforts are required to make treatments more personalized, to minimize side effects and improve overall survival and to also have an insight in toxico-genomics. Indeed proof of facts about medicinal plants worldwide and rich Indian medicinal flora, Objective: in the present investigation attempts to evaluate antimicrobials and anticancer therapeutic properties of isolated constituents from Withania somnifera (ASHWANGANDHA) Part Used Leaves, Aegle marmeleos (BEL) Part Used Leaves, Azadirachta indica (NEEM) Part Used Leaves, Ocimum sanctum (TULASI) Part Used Leaves, Method: Hydro alcoholic (1:1) Extracts were evaluated against cancer cell lines i.e. A549 (Lung carcinoma), PA-1 (Ovarian cancer) and MCF-7 (Breast cancer), with standard as Doxorubicin. Moreover the antimicrobial activity on Staphylococcus aureus, Bacillus coagulans and one Gram-negative—Escherichia coli, human pathogenic bacteria; and three fungal strains—Aspergillus niger, Tricoderma viride and Fusarium oxysporum. Result: The results showed significant association of phytochemicals on inhibition of test bacteria and fungi with significant p value p< 0.05, except For Tricoderma viride No Association Was Found P>0.05.


Introduction
The term "Cancer" is derived from the Greek word "Karkinos" coined by Hippocrates, a non-communicable disease (NCD) characterized by uncontrolled growth of cells [1,2]. It is the second leading cause of death after cardiovascular diseases amongst non-communicable disease and neither incidence, morbidity or mortality rate have declined much over the years [2]. Due to the serious side effects and resistance against cancerous cells with highly definite approved anticancer drugs, there is an urgent need to develop alternative approach toward the cancer anticancer drugs for the same context here we are approaching plant sources [3]. A lot of drawbacks are associated with conventional treatments like inter-patient heterogeneity, inter-tumor and intra-tumor heterogeneity, multidrug resistance (MDR) [4], cytotoxicity to nearby cells. As the effects of treatment are not targeted, they prove to be toxic to nontarget tissues and organs of the body, reducing the overall effectiveness of the treatment [5]. Because of the medicinal plants having an excellent work against cancerous cells, with high efficacy, some successful plants ingredients has been approved by FDA [6] moreover Recent research has suggested that phytochemicals target a number of pathways related to breast, lung and ovary cancer and mitigate the adverse effects of conventional treatment providing a positive feedback against malignancies and can also play a key role in preventing cancer [6][7][8][9]. Some of attempts are underway to work out the therapeutic and anti-neoplastic properties of medicinal plants [10,11]. Consequently, herbal medicines have received much attention as substitute anticancer drugs [12]. Many reasons have been given on why people use medicinal plants as therapy. Another big challenge in the treatment of breast cancer is the development of multiple drug resistance "MDR" in tumors via a number of complex mechanisms including modification of the drug efflux membrane transporters, alterations in beta-tubulin, and multidrug resistance protein (MRP) [13]. A lot of drugs which have been proved successful in the past can be resistant such as-anthracyclines (doxorubicin, daunorubicin, epirubicin, and mitoxantrone), taxanes (paclitaxel, docetaxel) and capecitabine [14,15]. On the basis of all these observations, it becomes a necessity to find out an alternative approach for cancer management that can minimize the risk and reduce the after-effects of the conventional treatment approaches. A lot of drugs derived from plants showing selective toxicity have led to clinical trials for therapeutic development [16]. Cell Culture & Freezing Lung cancer cell line A549, MCF-7 and PA-1 were procured from National Center for Cell Science (NCCS) Pune. Cells were routinely cultured in 25 cm2 flasks in Dulbecco's modified Eagle's Medium (DMEM) with high glucose (Lonza) supplemented with 10% fetal bovine serum (HyClone), 2 mM L-glutamine (Genetix Biotechnology) and penicillin (100 units/ml) streptomycin (100 µg/ml) (Invitrogen) at 37 °C in a CO2 incubator (Eppendorf).

Extraction, Fractionation and Isolation
Shade dried powdered material was extracted in Hydro alcoholic (1:1) by using soxhlet apparatus for Withania somnifera, Aegle marmeleos, Azadirachta indica, and Ocimum sanctum. The weight of the powder was 200 gm in 150 ml in solvent at 60°C for 96 hours for each sample. Then solution was filtered and concentrated under reduced pressure by rotator evaporator till constant mass is obtained at 40°C. Dark green/brown coloured semisolid crude was obtained. The herbal extract was dissolved in water and sterile filtered through 0.22 µm [17]. For all experiments, the sterile herbal Hydro alcoholic (1:1) extract was diluted with DMSO to the final concentrations are 10 µg/ml, 20 µg/ml, 40 µg/ml, 80 µg/ml and 100 µg/ml [18].

Phytochemical Analysis
Withania somnifera, Ocimum sanctum, Azadirachta indica and Aegle marmeleos were subjected to phytochemical tests for the identification of various constituents, such as alkaloid, flavonoids, phenolic compound and tannis, carbohydrate, steroid, glycosides, proteins, inorganic acid, oxalic acid, amino acid [19]. After phytochemical analysis bioactive compounds present in extract was separated out by column chromatography in a proper solvent system. Column chromatography was performed on a classic 20 cm long × 2 cm diameter glass column packed with 50 g Silica gel of 60-120 mesh size as stationary phase and crude drug were further subjected to column chromatography [CC] and eluted with specific solvent to obtain pure compounds. Silica gel for column chromatography was used as stationary phase [20]. The flow rate used was 5 ml/min. Three and four elutes for each solvent were taken. Further analysis were performed based on: TPC, TFC, antioxidant assay [21].

Total Phenolic Content (TPC)
The total phenolic content was determined by using calibration curve (5 to 10µg/ml). Four readings were taken for each solution for checking the reproducibility and to get accurate result. The intensity of the solution is proportional to the amount of tannins and can be estimated against standard tannic acid, the total phenolic content, expressed as mg tannic acid equivalents per 100 g dry weight of sample. The total phenolic content was measured by Folin-Ciocalteu reagent assay [22].

Total Flavonoid Content (TFC)
Total flavonoid contents were measured by Aluminum chloride colorimetric assay. Hydroalcoholic extracts that has been adjusted to come under the linearity range and different dilution of standard solution of Quercetin (10-100µg/ml) were added to 3ml of water. To the above mixture, 0.1ml of 5% C 4 H 4 O 6 KNa. 4 H 2 O (Potassium Sodium L-(+) -Tartrate Tetrahdrate) was added. After 5 minutes, 0.1ml of 10% AlCl 3 was added and the total volume was made up to 3 ml with distilled water. It was left at room temperature for 30 min after which the absorbance of the reaction mixture was measured at 430nm [23].

Free Radical Scavenging Activity
DPPH (2,2 -Diphenyl 1-Picryl Hydrazyl)-The free radical scavenging activity of aqueous and ethanolic extracts and the standard L-Ascorbic Acid (Vitamin C) was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH. Here, 0.1mM solution of DPPH in alcohol was prepared and was protected from light influence by maintaining the dark condition and was kept folded in aluminum foil and 3ml of this solution was added to 1ml various conc.(10 µg/ml) of extracts or standard solution of (10 µg/ml). Absorbance was taken after 30min at 550nm. The percentage inhibition activity was calculated from [(A0-A1)/A0] x 100, where A0 is the absorbance of the control and A1 is the absorbance of extract/standard taken as Ascorbic acid [24]. This is the most reported method for screening of antioxidant activity of many plants. DPPH assay method is based on the reduction of alcoholic solution of colored free radical DPPH by free radical scavenger. The procedure involves measurement of decrease in absorbance of DPPH at its absorption maxima of 516nm, which is proportional to concentration of free radical scavenger added to DPPH reagent solution. The activity is expressed as effective IC 50 [24].

Crude Drug Treatment
In Vitro Assay for Cytotoxic Activity A stock solution of each plant extracts was prepared by dissolving 30 mg of extract in 95 µl of dimethyl-sulfoxide (DMSO, Merck, Germany) and 2000 µl of cell culture medium to a final stock concentration of 10mg/ml and then diluted with complete culture medium to reach the desired concentrations [25].

McFarland Standard
McFarland standards are suspensions of either barium sulfate or latex particles that allow visual comparison of bacterial density. A 0.5ml McFarland standard is equivalent to a bacterial suspension containing between 1 x 108 and 2 x 108 CFU/ml of E. coli [26].
[28] The bacterial strains obtained from Institute of Microbial Technology, Chandigarh, were used for evaluating antimicrobial activity. In order to detect potential antimicrobial activity in the plant extracts, paper discs (diameter 12 mm) were soaked in an extract solution containing different concentration (40, 60, 80, and 100%). All plates were then incubated at 37°C for 24 hr and the zones of inhibition were subsequently measured in mm [17][18].

Phytochemical Analysis
The present work has undertaken phytochemical analysis which would show definite action value (IC 50 ) according to growth curve. Some of the parameters which were considered for the study of phytochemical analysis included Saponins, Flavonoids, Steroids, Terpenoids, Alkaloids and Glycoside. Table 1. Phytochemical screening of 4 selected medicinal plants. Test for chloride +ve +ve +ve +ve

FT-IR Analysis
FT-IR analysis of the extracts was done for the purpose of Aegle Marmelos, Ocimum Sanctum and Withania Somnifera Extracts detection of functional groups associated. The FT-IR spectrum of the plants extracts recorded the number of peaks lying between 3100-3400 respectively Figure 2. FTIR analysis was done by BRUKAR FTIR Spectroscopy.

Total Polyphenols Content (TPC)
Total polyphenols content (TPC) was determined by to the spectrophotometer method with Folin-Ciocalteu's reagent. Gallic acid was used as calibration standard and results were expressed as gallic acid equivalents µg per g dry weight. Total polyphenols content of the extracts was found: 200 µg/g, 120 µg/g, 66 µg/g and 36 µg/g Withania somnifera, Azadirachta indica, Aegle marmeleos, and Ocimum sanctum respectively. Absorbance was measured at 725 nm using a UV-Visible spectrophotometer.

Total Flavonoid Contents (TFC)
Total flavonoid contents (TFC) were measured by Aluminum chloride colorimetric assay. Hydro-alcoholic extracts that were adjusted to fall under the linearity range and different dilution of standard solution of Quercetin (10-100µg/ml). Total flavonoid content of the extracts was found : 4.78 µg/ml, 5.41 µg/ml, 4.77 µg/ml and 5.62 µg/ml. Withania somnifera, Aegle marmeleos, Azadirachta indica, and Ocimum sanctum respectively. Equivalents per dry weight of sample. Absorbance of the reaction mixture was measured at 430 nm with a single beam spectrophotometer (Systronic).

DPPH Radical Scavenging Assay
In the present work, Hydro alcoholic extracts was evaluated for antioxidant property by using 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity and Nitric oxide free radical scavenging activities. From the percentage inhibition calculated the Hydro alcoholic extracts possesses antioxidant properties. The significant antioxidant property showed by IC 50 values extracts is due to the presence of phenolic constituents. Hence it was used in the preparation of herbal formulation. DPPH Antioxidant IC 50 values of the extracts was found : 51.57 µg/ml, 40.0 µg/ml, 74.54 µg/ml and 91.52 µg/ml Withania somnifera, Ocimum sanctum, Azadirachta indica and Aegle marmeleos respectively.

Antimicrobial Assay
The antimicrobial activity was determined in the extracts using agar disc diffusion method. The antibacterial and antifungal activities of extracts (40, 60, 80, 100%) of Cassia fistula were tested against two Gram-positive Staphylococcus aureus, bacillus coagulus and one Gram-negative-Escherichia coli, human pathogenic bacteria; and three fungal strains-Aspergillus niger, tricoderma viride and fusarium oxysporum. Zone of inhibition of extracts were compared to that of different standards like Streptomycin for antibacterial activity and Griseofulvin for antifungal activity. The results showed that the remarkable inhibition of the bacterial growth was shown against the tested organisms. Tables 2 and 3.

Calculation of Cell Viability or IC 50 Values
For selected plant active molecules, (column chromatography extracts) IC 50 value were calculated and separately Mentioned (Figure 1) 50 of Doxorubicin are MCF-7 500nm, A549-550nm, PA-1-570nm as mentioned in Figure 1.

Statistical Analysis
The results of the inhibition percentage of growth of the three bacteria and three fungi as affected by the four concentrations (40%, 60%, 80%, 100%) of the Azadirachta indica, Aegle marmeleos, Ocimum sanctum and Withania somnifera extracts were statistically analyzed using One way analysis of variance (ANOVA) using Graph-Pad. The results showed significant association of phytochemicals on inhibition of test bacteria and fungi with significant p value p< 0.05, except For Tricoderma viride No Association Was Found P>0.05. The MTT assay indicated that Hydro alcoholic (1:1) through CC extracts of the plant exhibited significant cytotoxic effects on MCF-7, A549 and PA-1 cancer cell line (breast, lung and ovary respectively), This finding suggests that the reduction observed in the viable cells following treatment with Azadirachta indica, Aegle marmeleos, Ocimum sanctum and Withania somnifera. The further studies on the active components for proper assessment of their chemotherapeutic properties as well as their possible development as promising anticancer drugs in addition more research will be useful to investigate the unknown and unexplored potential of above mentioned plant and drug design analysis.

Discussion
In Plant chemistry considerable Contributions have been made by [1, 2, 6, 11-14, 18, 20]. They worked out the cytotoxic activity of medicinal plants and studied their antiproliferative activity against cancer. In this study we demonstrated that Withania somnifera, Aegle marmeleos, Azadirachta indica, and Ocimum sanctum might be a source of antibacterial, antifungal and antiproliferative substances/compounds. Optimization of culture conditions may accelerate their growth and stimulate the production of biologically active compounds. It may also limit the need to harvest Withania somnifera, Aegle marmeleos, Azadirachta indica, and Ocimum sanctum in the field. It is especially important for Withania somnifera. A high variety of biologically active substances present in Withania somnifera, extracts may provide new antibacterial and/or anticancer agents. As an urge to study the effect of the extracts of Withania somnifera as a novel therapeutic agent, they were characterized for their cytotoxic effects against all three study cell lines. To conclude, this article induces a concentration-dependent inhibition of cells. Based on these results, further studies could be carried out as a search for Withania somnifera compounds to develop alternative therapeutic measures against diseases.

Conflicts of Interest
The authors declare that there are no conflicts of interest.