Key Words

Tylophora indica, Curcuma amada, Urtica dioica, BHK-21 cell lines, soxhlet, phytochemical analysis.

Introduction

India is a continent with wide field of diversity. This diversity includes both flora as well as fauna. This variation is due to the varied climatic condition, vegetation, topography etc. resulting in enriched heterogeneity. As a result, many such herbs are present with increased medicinal value that is left unnoticed. These herbs may possess medicinal values, domestic values and therapeutic values.

It has been proved since ages the benefits of using these natural agents for curing various diseases. This property may be due to the presence of some active compounds that are different for each plant.

In the present study three plants were selected Curcuma amada, Tylophora indica and Urtica dioica. The three plants belonged to different family each belonging to Indian origin. These plants are used in daily household as spices, condiments and other primitive therapies. Based on the knowledge provided by the surrounding aged people these plants were selected for evaluating their antibacterial activity and cytopathic effects against some basic bacteria and fibroblast cell line. For the following analysis aqueous extracts and alcoholic extracts were chosen. Phytochemical screening was also carried out to determine the major photochemical present.

Materials and Methods

Plant materials

Three Indian origin plants viz., Curcuma amada (rhizome), Tylophora indica (leaves and stem) and Urtica dioica (leaves and stem) were collected from the polyhouse and natural environment at the Institute of biotechnology, Patwadangar, G.B.Pant University, Nainital.

Bacterial cultures

The normal strains of two gram negative bacteria Escherichia coli and Pseudomonas aeruginosa were isolated from throat and nasal swab of Indian rooster cock and human nasal secretion. Confirmatory tests for the respective bacteria’s were performed.

The multi drug resistant strains of one gram positive bacteria Staphylococcus aureus and two gram negative bacteria Escherichia coli and Pseudomonas aeruginosa were obtained from the microbiology department of the institute. All the isolates were subcultured at regular interval and stored at 4°C.

Preparation of plant extracts

Aqueous extraction

Cold extraction-0.50g of dried plant powder was added to 50ml of distilled water (15°C) and was mixed thoroughly for 30 minutes in rotary shaker at 110rpm. It was then filtered through muslin cloth/centrifuged at 5000g for 10 min. The supernatant was collected and stored at 4°C.

Warm extraction-0.50g of dried plant powder was added to 50ml of distilled water (40°C) and was mixed thoroughly for 30 minutes in rotary shaker at 110rpm, 40°C. It was then filtered through muslin cloth/centrifuged at 5000g for 10 min. The supernatant was collected and stored at 4°C.

Hot extraction-0.50g of dried plant powder was added to 50ml of distilled water (70°C) and was mixed thoroughly for 30 minutes in rotary shaker at 110rpm, 70°C. It was then filtered through muslin cloth/centrifuged at 5000g for 10 min. The supernatant was collected and stored at 4°C

Boiling water extraction-0.50g of dried plant powder was added to 50ml of distilled water (100°C) and was mixed thoroughly for 30 minutes by boiling. It was then filtered through muslin cloth/centrifuged at 5000g for 10 min. The supernatant was collected and stored at 4°C.

Organic solvent extraction

0.50g of dried plant powder was extracted for 8 hrs with (140ml) organic solvent (ethanol) in Soxhlet apparatus. It was then filtered through muslin cloth.

The supernatant was collected and stored at 4°C. It was then concentrated by evaporating the solvent using vacuum concentrator. The dried extract was then dissolved in PBS/DMSO. The process was repeated with other organic solvents (methanol, petroleum ether and chloroform).

Cell line

The cell line used was baby hamster kidney fibroblast cells (BHK-21). They were obtained from animal cell culture department, IVRI, Mukhteshwar. These cells were revived at regular interval and were subcultured daily for their efficient growth.

Phytochemical screening

The powdered seeds were evaluated for qualitative determination of major phytoconstituents i.e. Reducing sugar, non-reducing sugar, polysaccharides (starch), proteins, amino-acids, steroids, cardiac glycosides, anthraquinone glycosides, saponins, tannins and phenolic compounds, flavonoids and alkaloids (Manas boxi et.al., 2010).

Antibacterial assay by disc diffusion method

Antibacterial method of (Bauer et al., 1996) was adopted with slight modification. The bacterial cultures were grown in McConkey (E.coli) and nutrient broth (P.aeruginosa) at 37 °C. Muller Hinton agar sterile petriplates were used for the test cultures. 20ml of the media was poured on each petriplate. A loopful of the cultures (nearly 50µl) was uniformly spread over the surface of a sterile Muller

Reducing sugar

Non-reducing sugar

Non-reducing polysaccharides (starch)

Proteins

Amino acid

Steroid

Anthraquinone glycosides

Cardiac glycosides

Saponins

Flavonoids

Tannins and phenolic compounds

Alkaloids

Hilton agar with a sterile bent rod. Sterile discs (4.0mm in diameter) were dipped in solution of various extracts dissolved in 1ml of PBS and dried at 40°C for 30 minutes. The disc dipped in PBS was used as a negative control and standard antibacterial agent antibiotic (tetracycline-10μg/ disc) was used as positive control. The plates were incubated at 37°C for 24 hours and antibacterial activity was measured. After this period, it was possible to observe inhibition zone. The diameter of the Zone of Inhibition was measured in mm. Overall, cultured bacteria with halos equal to or greater than 7 mm were considered susceptible to either the tested extract or phytochemical. The antibacterial experiments were performed in triplicates.

The same antibacterial sensitivity test was performed even for MDR bacteria. Three strains of MDR bacteria was taken from the laboratory cultures of the institute namely of E.coli, S.aureus and P.aeruginosa. Plant extracts of same concentration were considered even for MDR bacteria.

Cell culture analysis

Sample preparation

Dilution of the plants extract in different concentration was prepared. Stock concentration of each plant was 0.20g/5ml DMSO. Serial dilution at varying concentration (20mg, 2mg, 200µg and 20µg) were carried in the ratio of 0.5:4.5ml media. Both cytopathic and cytotoxic effects of the different plants extract in varying concentration were carried out.

MTT assay (test for cytotoxicity)

96 well microtitre plates were used for MTT assay. MTT stock solution of 5mg/ml in phosphate buffer saline (PBS), pH=7.5 was prepared. It was filtered using 0.22µm syringe filter to sterilize and remove the small amount of insoluble residue. 96 well microtitre plates were prepared with 100µl of BHK-21 cell suspension in each well. After 24 hrs of growth, media was decanted and 100µl of varying concentration of different plants extracts were poured. 3hrs later 10µl of MTT stock solution was added in each well. Plates were incubated in humidified 5% CO2 incubator at 37°c for 3 hrs. 100µl of 0.04M HCl in propan-2-ol was added to each well and mixed thoroughly to dissolve the insoluble blue formazan crystals. The absorbance was read on micro-ELISA reader at 492nm. The plates were read within ½ an hour after adding the stopping reagent (Mosmann T et.al., 1983).

Data analysis

The 50% inhibition concentration (IC50) of the active substances was determined as the lowest concentration which reduced cell growth by 50% in treated. Dose-response curves between percentage of cell viability and concentrations of the extracts were constructed. The IC50 was determined from the plotted curve.

Result

Qualitative analysis of extracts for their phytoconstituents

Phytochemical analysis of plants extracts were carried out for both fresh plant and dry plant samples. Both showed nearly similar phytochemicals presence. However, in case of fresh plant samples the minute amount and sensitive phytochemicals were also detected. This could be because as time goes by few phytochemicals might get exhausted. The qualitative determination of these phytochemicals has been presented in table 1 & 2. The phytoconstituents detected in the plant materials could be responsible for their antimicrobial activity though their exact mode of action was not understood.

The extracts were tested for their antimicrobial activity against the pathogenic micro-organisms viz., E.coli a most common bacterium of which virulent strains can cause gastroenteritis, urinary tract infections and Pseudomonas aeruginosa which infects the pulmonary tract, urinary tract, burns and wounds. Same bacterial strains along with S. aureus were used in case of multi drug resistant bacteria. From the different range of temperatures selected for aqueous plants extract, hot water extracts (65-75°c) of U.dioica was found to be effective for the normal bacterial strain of P.aeruginosa. However, methanol extract showed antibacterial activity against E.coli, P.aeruginosa in most of the plants extract. When the plant extracts (alone) of aqueous and organic solvents were subjected to MDR bacterial strains none of them showed antibacterial activity towards any bacteria. The data has been reported in table 3 & 4, fig. 1 & 2.

Table 4: Antibacterial activity of plants extracts in terms of zone of inhibition (in mm)

Evaluation of the antimicrobial potential of plant extracts

Curcuma amada showed maximum antibacterial activity against E.coli with 9mm zone of inhibition for ethanol extract Urtica dioica showed maximum antibacterial activity against P.aeruginosa with 7mm zone of inhibition via methanol extract.

Synergistic effect of antibiotics and plant extracts on the bacteria samples

Upon the determination of the plant extracts individual effects, the combined effect of plant extract with antibiotic was determined. Tetracycline, the broad spectrum antibiotic was used. Different plants extract showed significant inhibitory zone but methanol extract in general exhibited antibacterial activity in all the plants. Hence, it can be considered that methanol is a good solvent for the extraction of various active compounds present although specificity in solvent could be exhibited. Also of all the plants extract Curcuma amada showed maximum antibacterial activity against E.coli and U.dioica against P.aeruginosa. This could be due to the phytoconstituents detected in the plant materials though their exact mode of action was not emphasized. The obtained results are presented in table 5, fig. 3 & 4.

Curcuma amada along with tetracycline showed maximum antibacterial activity against E.coli with 12mm zone of inhibition for ethanol extract Urtica dioica with tetracycline showed maximum maximum antibacterial activity against E.coli with 12mm zone of inhibition for ethanol extract Urtica dioica with tetracycline showed maximum antibacterial activity against P.aeruginosa with 11mm zone of inhibition for methanol extract.

Synergistic effect was observed in all the plants extracts. The plant extracts exhibited improved antibacterial activity in combination with the antibiotic tetracycline. But the antibacterial effect of the plant extracts with antibiotic was not determined against multidrug resistant bacteria.

Cell culture

In-vitro confirmation of the toxicity of the plant extracts Curcuma amada, Tylophora indica and Urtica dioica on BHK-21cell line was determined. Percentage of viable cell was obtained by performing trypan blue dye exclusion technique. The cytotoxicity activity was carried out by using MTT assay

Viability and characterization of cell lines

Cell lines derived from Indian Vetenary Research Institute, Mukhteshwar were free from any kind of bacterial and fungal contamination. Cell count was carried out by using hemocytometer with dilution factor 2 via Trypan blue dye exclusion technique. 74% cell viability was calculated with 2.16 X 104 viable cells in 100µl of cell suspension. These are most suitable to perform cytoxicity studies.

Determination of Cytotoxicity by MTT assay

All the three plant samples Curcuma amada, Tylophora indica and Urtica dioica were tested for cytotoxicity by MTT assay on BHK-21 cell lines. DMSO was considered as control for MTT assay. Percentage of cell viability was estimated. It was found that methanol extract of Tylophora indica showed least cell viability as compared to other two plants and their extracts. In terms of cytotoxicity, lower the IC50 value higher the cytotoxicity. According to the table 8, Tylophora indica recorded the least IC50 value (~20 µg/ml) indicating it being the most cytotoxic of all the three plants. Methanol and ethanol extracts of Tylophora indica was found to have IC50value ~20 µg/ml. Other plants were found to have IC50 value of (2- ~20mg/ml) indicating to be less cytotoxic. Thus it can be inferred that Tylophora indica (mainly alcoholic extracts) is cytotoxic even at lower concentration such as 20µg/ml. Thus, dosage for Tylophora indica should be considered at much lower concentration towards BHK-21 fibroblast cells for further studies.

Determination of percentage of cell viability

Percentage of cell viability was calculated by the following equation

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Fig. 5 Percentage of cell viability v/s concentration of different plants extracts by MTT assay

The 50% inhibition concentration (IC50) of the active substances was determined as the lowest concentration which reduced cell growth by 50% in treated. Dose-response curves between percentage of cell viability and concentrations of the extracts were constructed. The IC50 was determined from the plotted curve.

Table 8: IC50 value determined for each plants extracts according to the concentration gradient involved.

Tylophora indica was found to be very toxic at higher concentration. It has already been reported that this plant causes allergy to some people and sometimes may also be potentially serious. But the same allergen is reported to have anti-asthmatic effect. This plant being widely used to treat asthma contains the compound Tylophorine. Tylophorine is a Phenanthroindolizidine alkaloid which shows antitumor activity and anti-inflammatory effects. As in the present study too this plant was found to be most toxic of all for the BHK-21 fibroblast cells. It showed the maximum effect. Thus, not studied completely but it might be that this effect is too due to the higher concentration of the compound Tylophorine. As studied even in concentration as low as 20µg also the plant seemed to be toxic (Cheng-Wei Yang et,al. 2006).

Conclusion

In case of antibacterial effect Curcuma amada exhibited the maximum activity. Of the three plants the order of highest antibacterial activity was U.dioica>C.amada>T.indica for P.aeruginosa while for E.coli was C.amada> U.dioica>T.indica. All the plants showed comparable antibacterial activity which support their traditional use against infectious diseases. However in case of cytotoxicity T.indica showed maximum cytotoxic effects in comparison to other plants. The presence of general phytochemicals and specific active compounds might be responsible for their therapeutic effects. Further in depth study might reveal the key compounds present and their role in treating other many infectious diseases.

Acknowledgement

The IBT, Patwadangar. G.B.Pant University, Uttarakhand has provided their support, lab facility for this study.