Qualitative evaluation of antibacterial and DNA binding activity of four local ethnomedicinal plants available in Barishal, Bangladesh

Screening of antibacterial activities of plants due to secondary metabolites is an authentic basis to look for a new drug. As a result of increasing demand for traditional treatments, scientists have look forward to search the efficacy of these so called ethnomedicinal plants and have to exchange with pharmacologists. An initiative of this, the antibacterial activity of Eclipta prostrata (L.) Mant. Azadirachta indica A. Juss., Aloe vera (L.) Burm. f. and Catharanthus roseus (L.) G. Don was studied against Escherichia coli and Staphylococcus aureus. Among four ethnomedicinal plants subjected to test antibacterial activities, Eclipta prostrata was found to act against both strains of bacteria whereas Azadirachta indica was effective against Escherichia coli only. DNA binding affinity of these medicinal plants was also studied by viscometry. No affinity between each medicinal plant extracts and isolated human cheek cell’s DNA was observed.


Introduction
Bangladesh, specially Barishal has rich floral diversity of traditional ethnomedicinal plants and the traditional medical system which is several hundred years old including Unani and Ayurveda. Besides, from time immemorial primitive people living in close contact about utilization of ethnomedicinal plants by way of trial and error. World health organization (WHO) has estimated that 80% of the world population depends on traditional herbal medicines (WHO, 2001). Physiologically bioactive compounds are contributed from medicinal plant species for the remedy of numerous ailments (Adebanjo et al., 1983;Natarajan et al., 2005) which are infectious or non-infectious. As a consequence of sharing this resource of knowledge from one generation to next without any written documents, it is getting lost day by day. So, it is essential that the knowledge now available among local and tribal people to be documented and integrated with modern scientific values before it gets lost in Bangladesh. Synthetic antibiotics discovered in the twentieth century can treat only of the infectious diseases. However, due to the incessant use of these antibiotics over the years, pathogens have become resistant to synthetic medicines (Ahmad and Beg, 2001) as a result of bacteria's genetic ability and mutation. Thereby, herbs have been emphasized as traditional medicines containing medicinal ingredients because of their curative potentiality (Bhalodia and Shukla, 2011); thus they are used in the further development of drugs. Majority of plants species in different locations around the world have been examined for their pharmacological properties and they are distinguished to possess numerous secondary metabolites such as flavonoids, steroids, tannins, terpenes, saponins (Kamali and Myel, 2010;Lalitha et al., 2010;Sen and Batra, 2012) and different investigations have been published on describing the pharmacological properties. Antibacterials of plant origin have immense therapeutic potentials and are effective in the treatment of infectious diseases than synthetic antimicrobials which have adverse effects (Brandi et al., 2006). Out of the total 225000 plants species, only less than 10% have been studied so far for their medicinal properties (Widjaja and Lester, 1978;Kelmanson et al., 2000). The practice of complementary and alternative medicinal treatment is now increasing in developing countries in response to World Health Organization (WHO) directives and has provided the scientific basis for the effectiveness of traditional medicinal practices. The role of traditional medicines in the solution of health problems is invaluable on a global level and as a result, research on medicinal plants has intensified and information on these plants has been exchanged worldwide. Moussaoui et al. (2016) studied the antibacterial activity and synergistic effect between antibiotics and the essential oils of some medicinal plants. Furthermore, the combination of essential oils of plants with antibiotics was found effective against the emerging microbial drug resistance. Cagri-Mehmetoglu et al. (2017) studied the antibacterial properties and minimum inhibition concentration of Microdesmis puberula, Hypoestis verticillaris, Icacina tricantha, and Enterolobium cyclocarpum against 21 different bacteria and reported the antibacterial activity and phenolic contents of four medicinal plants of Nigeria. Meghla et al. (2016) studied the antibacterial activity of some medicinal plants against Escherichia coli, Salmonella spp. and Staphylococcus aureus. Valle et al. (2015) studied the antibacterial activities of ethanol extracts of Philippine medicinal plants against multi drug resistant bacteria. Valli et al., (2014) also studied the anti-inflammatory, analgesic and DNA binding studies of manjanathi fruit extract. They reported that manjanathi fruit extract have a binding affinity to calf thymus DNA. Therefore, in this study the antibacterial effect of the Ethanol extract of Eclipta prostrata, Azadirachta indica, Aloe vera and Catharanthus roseus were studied against Escherichia coli and Staphylococcus aureus as Uddin et al. (2017), Hossain and Rahman (2018), Alam and Haider (2018), Das et al. (2013) reported these species as ethnomedicinal plants along with high citation frequency, fidelity level and available in University of Barishal campus as wild, exotic and cultivated. So the objectives of present study are to find out the modern scientific basis of antibacterial properties by disk diffusion method in which the diameter of inhibition zone is directly proportional to bacterial susceptibility (Suhag et al., 2014). In addition, binding between DNA from human cheek cells and plant extracts was performed by viscometric analysis to find out the interaction.

Collection of plant specimens
The plants i.e. Eclipta prostrata, Azadirachta indica, Aloe vera and Catharanthus roseus had been collected from their natural habitat, from different locations of University of Barishal, Bangladesh. The plant specimens were identified and authenticated in the Department of Botany, University of Barishal, Bangladesh (Figure 1) using standard literature (Ahmed et al., 2008(Ahmed et al., -2009. Then the plant specimens had been subjected to ex-situ conservation at the net house of mentioned University for future uses.

Preparation of extraction
Collected medicinal plants leaves were washed with tap water, ethanol and distilled water; and dried in sunshine and at oven (50˚C temperature). Then the dried samples were ground by mortar and pestle (Meghla et al., 2016;Ramchandran et al., 2012). The conical flasks were sterilized in the oven for 1 hour. 5 g of powder from each of the samples was poured into the conical flask. Following this, 50 mL of ethanol (Merck, Germany) and 50 mL of methanol (Merck, Germany) was added to each flask separately and gently mixed it perfectly. Then solutions were shook for 3 days at room temperature at orbital shaker (Model No-JSOS-300). The preparation of each solution was repeated and kept for 3 days at static conditions at room temperature. The extracts were filtered, centrifuged and evaporated (Meghla et al., 2016;Ramchandran et al., 2012) and were kept for further uses.

Collection of bacterial strain
Isolated pathogenic bacterial strains of Escherichia coli and Staphylococcus aureus were collected form Popular Diagnostic Centre and Hospital, Sylhet. The collected strains were incubated in an incubator (Model No-Binder RL-53) and inoculated on sterile, analytical nutrient agar medium and stored at 4°C until further use.

Study of antibacterial activities
Antibacterial activity of medicinal plants was studied by the disk diffusion method (Suhag et al., 2014;Bell et al., 2009). At first nutrient broth medium was prepared by beef extract (Sigma-Aldrich, India), yeast extract (Sigma-Aldrich, India), peptone (Sigma-Aldrich, India) and NaCl (Sigma-Aldrich, India) in distilled water and autoclaved for 15 minutes. Then a single colony of Escherichia coli and Staphylococcus aureus bacteria was added in 20 mL broth separately and incubated at 37°C for 24 hours. After that, a stock solution of 50 mg mL −1 was made by dissolving ethanol extract of medicinal plants in distilled water. 10 μL of stock solution (500 μg sample) was socked in each sterilized filter paper (Whatman, 0.45 micro pore) disk of uniform diameter (5 mm) and thickness (1 mm). Then 25 μL of activated bacterial solution was placed onto the surface of an agar plate (agar plate was prepared by 35 gL -1 of bacteriological nutrient agar) with the help of micropipette with sterilized tips and spread evenly over the surface by using a sterilized bent glass rod. Finally, sample disks and antibiotic disks of ciprofloxacin (CONDA, Spain) were applied in freshly prepared agar plates and incubated at 37°C for 24 h. The zone of inhibition (diameter) was then measured (in mm) around the sample and standard antibiotic disks. Antibiotic ciprofloxacin (CIP) was used as a standard antibiotic disk to make a comparison with plants extracts. The antibacterial results of the extracts were compared with the standard antibiotic.

Isolation of DNA
DNA was isolated from human cheek cells by general salt extraction process (by using sodium chloride and detergent solution), purified by chloroform and precipitated in ice-cooled ethanol (Garbieri et al., 2017). Purity of DNA was evaluated by a UV-Vis spectrophotometer (Lamda-365).

DNA binding studies
The qualitative viscosity experiments were carried out by using Ostwald viscometer at room temperature. The relative viscosity of solutions was measured at a fixed concentration of DNA (25 μM) and different concentrations of plant extracts (50-500 ppm) with respect to pure DNA solution (25 μM) in water (Ramachandran et al., 2012 andPrasad et al., 2011).

Extraction of medicinal plants at different physico-chemical conditions
Medicinal plants were extracted by ethanol and methanol separately. It was observed that extracted ability of ethanol to medicinal plants is significantly higher than methanol. Plants were also extracted at static and shaking conditions separately. It was observed that negligible amount of extracts were found at static condition. So plant extracts by ethanol at shaking conditions have been chosen for antibacterial and DNA binding studies.

Antibacterial activity
Antibacterial activity of experimental medicinal plants was tested against Escherichia coli and Staphylococcus aureus and compare with antibacterial activity of antibiotics ciprofloxacin (CIP) against corresponding bacteria. Azadirachta indica showed highest zone of inhibition against E. coli among ethanol extract of Eclipta prostrata, Azadirachta indica, Aloe vera and Catharanthus roseus. Besides this, both of the bacterial strain showed highest susceptibility and resulted a landmark zone of inhibition against standard antibiotic disks named ciprofloxacin (Table 1; Figure 2A, 2B; and Figure 3). Detail description for each specific plant species are:

Eclipta prostrata
This medicinal plant species showed both gram-positive and negative reactions against Staphylococcus aureus and Escherichia coli respectively showing almost the same diameter of inhibition zone (Table 1 and Figure 2A, 2B; Figure 3A and 3B). Rahman and Rashid (2008), Kaisar et al. (2011), Ripa et al. (2012, Uddin et al. (2010) reported the antibacterial properties of this species against different of both gram-positive and gram-negative bacterial species including E. coli and S. aureus.

Azadirachta indica
Only gram-negative reaction has been observed against E. coli. But no antibacterial properties against S. aureus had been seen (Table 1; Figure 2A and Figure 3C). Islam et al. (2012) reported the antibacterial properties of this species against different of both gram-positive and gram-negative bacterial species including E. coli and S. aureus but the plant extraction was prepared in Hexane, Butanol.

Aloe vera
Both of the bacterial strains showed no antibacterial activity (Table 1). Molla et al. (2010) reported the antibacterial properties of this species against different strain of both gram positive and gram negative bacterial species including E. coli and S. aureus with ethanol extracts but minimum inhibitory concentration (MIC) was many times higher than ours. 3.2.4. Catharanthus roseus No antibacterial activity for both E. coli and S. aureus (Table 1) was observed. Molla et al. (2010) reported the antibacterial properties of this species against different of both gram-positive and gram-negative bacterial species including E. coli and S. aureus with ethanol extracts but MIC was many times higher than ours.

DNA binding studies
The binding of four experimental medicinal plants extract to the DNA was investigated by viscometric studies. It was observed that there is no significant increase in the viscosity of DNA on the addition of plants extracts, which indicates that there were no binding interactions of DNA with these medicinal plants.

Conclusions
Ethanol extract of Eclipta prostrata showed antibacterial activity against Escherichia coli and Staphylococcus aureus; whereas Azadirachta indica showed antibacterial activity against Escherichia coli only. Aloe vera and Catharanthus roseus did not show antibacterial activity against these subjected pathogens. Extract of Eclipta prostrata, Azadirachta indica, Aloe vera and Catharanthus roseus plants did not show any binding affinity to isolated DNA of human cheek cells. From this experimental result, E. prostrata and A. indica may provide an authentic basis to search for a new drug that will be used infectious ailments while remaining plant materials subjected to experiment will be investigated further for both extraction methodologies and antibacterial activities.