Identification of Phytoconstituents using GC-MS and Determination of Antimicrobial and Antimycobacterial Activity of Boerhaavia diffusa L. Leaves

Background: To identify bioactive phytoconstituents and evaluate antimicrobial and antimycobacterial potential of Boerhaavia diffusa leaves against selective human pathogens. Methods: The extract of Boerhaavia diffusa leaves was carried out by using methanol. Bioactive compounds was identified by GC-MS. Antimicrobial and antimycobacterial activity of methanolic extract Boerhaavia diffusa leaves was tested in vitro by Kirby-Bauer well diffusion method and rapid culture - MGIT TM DST method against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus & Pseudomonas aeruginosa and Candida, Aspergillus and M. tuberculosis (H37RV ) and Mycobacterium tuberculosis bacteria resistant to Isoniazid & Rifampicine. Results: The obtained results of the GC-MS of Boerhavia diffusa led to the identity of 16 bioactive compounds. The crude extract showed antimicrobial activity against E.coli (Sensitive), Pseudomonas aeruginosa Sensitive and Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (Sensitive) but extract did not show antimicrobial effect against, E.coli (ESBL), Klebsiella pneumonia (Sensitive), Klebsiella pneumonia (ESBL), Pseudomonas aeruginosa (Resistant), Candida albicans and Aspergillus fumigates . The result of anti-mycobacterial activity showed that extract not showed antimycobacterial activity against M. tuberculosis (H37RV) and Mycobacterium tuberculosis bacteria resistant to Isoniazid & Rifampicine. Conclusion: The study result proved that methanolic extract of Boerhavia diffusa leaves has antimicrobial potential due to the presence of the bioactive compounds.


INTRODUCTION
An infectious disease is a leading cause of morbidity and mortality in world. The clinical efficacy of leading antibiotics is suffered by the emergence of multi-drug resistant pathogens, so it needs to find out new drug molecule. The mechanisms of resistance are inactivation of antibiotics; reduce the membrane permeability, modification of target site, efflux or transport of antibiotic.
Escherichia coli are usually present in warm blooded organisms especially in the gastrointestinal tract. It commonly causes urinary tract infection and, Gastro-intestinal diseases [1]. Klebsiella pneumoniae, mostly present in the human gastro-intestinal, is a Gram-negative, non-sporulating, aerobic shaped rod bacterium. The development of an infection is contributed by adhesion to a mucosal surface [2]. Staphylococcus aureus generally lives in human skin and mucosa and it enters the body can led to disease spreading. It produces food poisoning, which gives to severe vomiting and cramps. Pseudomonas aeruginosa is a pathogen exploits break in the host defense system to start an infection [3]. Candida albicans lives in the human digestive tract, mouth, and genital region that contributes in fungal infections [4]. Aspergillus species is second most widespread systemic fungal infection. Aspergillus primarily affects lungs and causes bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, aspergilloma and invasive aspergillosis.
In recent decades bioactive compounds, previously with unknown biological activities, have been extensively investigated as a source of therapeutic active substances [5].

Preparation of Extract
Plant material leaves were collected from Amer forest region, Jaipur. The shade dried powder of leaves was used for the extraction with methanol for 24 hours by Soxhlet equipment and filtered through membrane filter. This filtrate was evaporated under reduced pressure and dried in a rotator evaporator at 55°C [19].

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
GC -MS analysis was carried out on an Agilent system equipped with Mass Spectrometer detector and split/splitless injection system. The GC was fitted with a HP-5MS capillary column (30m X 250m; film thickness: 0.25m). The temperature program was as follows: injector temperature 280°C, initial oven temperature at 50°C, then increased at 25°C/min to 300°C and was hold for 10 mins. Helium was used as carrier gas at 17.69 psi pressure with flow 2.1 ml/min. Samples were dissolved in methanol and 1 μl aliquot were injected automatically. The biological active compounds were identified through the comparison of their mass spectra with the reference mass spectra of library of the NIST (National Institute of Standards and Technology) [20,21].

Antimicrobial Study
Antimicrobial activity study of crude extract of Boerhaavia diffusa leaves including antibacterial and antifungal activity was carried by Kirby-Bauer well diffusion method at C.I.R.D (Centre for Innovation, Research & Development) Dr. B. Lal Institute of Biotechnology and Research Centre, Malviya Nagar, Jaipur, Rajasthan.

Selection of microorganisms
For the purpose of Antimicrobial evaluation, selection of Microorganisms were done by taking two different set (sensitive and resistant) for Gram positive, Gram negative and Mycobacterium. For Antifungal testing Candida albicans and Aspergillus fumigates were selected. All the pure microbial cultures utilized in this study were procured from Microbial Culture Collection Division (MCRD) established at CIRD.

Processing of samples
Two concentrations of the crude extract of Boerhaavia diffusa leaves under study were prepared from the stock solution (200 mg/ml and 100 mg/ml) and then the dilution series was prepared for the compound, out of which 50 μl was used in each well. Streptomycin was used as positive control (5 mg/ml concentration) for antibacterial activity and Itraconazole was used as positive control (5 mg/ml concentration) for antifungal activity.

Antimicrobial Susceptibility Testing
(Kirby-Bauer Well Diffusion Method)

Agar plates were prepared for the antibacterial activity
Mueller-Hinton agar medium and Sabouraud dextrose agar medium is the only susceptibility test medium that has been validated by CLSI for screening the antimicrobial activity by disk/ well diffusion susceptibility testing.

Inoculums size of bacteria was adjusted using McFarland turbidity standard as reference
The bacterial suspensions were compared to 0.5 McFarland Turbidity Standard.

Incubation
The bacterial and Candida plates were kept for incubation at 37°C for 24 hours and plates of Aspergillus were kept for incubation at 28°C for 7 days.

Antimycobacterial Susceptibility Test
The antimycobacterial activity of crude extract of Boerhaavia diffusa leaves was determined against M. tuberculosis (H37RV) and Mycobacterium tuberculosis bacteria resistant to Isoniazid & Rifampicine both by rapid culture -MGIT TM DST method.
The crude extract of Boerhaavia diffusa was diluted to the 10mg/ml concentrations. Total seven MGIT tubes were labeled and 0.8 ml supplement was added to each tube. 1 st tube was then kept a side and extract 100 μl from the stock of 100 mg / ml were added to the respective tubes. Tubes were mixed properly and kept a side. 1:100 dilution of DST inoculum (M. tuberculosis (H37RV) was prepared for the Growth control tube (1 st tube) and 1:5 dilution of DST inoculum ( M. tuberculosis (H37RV) & M. tuberculosis (MDR) were prepared for tubes. 0.5 ml of 1:100 dilution was added to the 1 st tube (Growth control tube). 0.5 ml of 1:5 dilution was added to other respective tubes. All tubes were incubated in MGIT-320 instrument at 37⁰C.

RESULTS
Due to emerging resistant it needs to find out alternative chemotherapeutic agents. Plants and herbs have rich amount of active phytoconstituents they are responsible for many pharmacological activities. The aims of this study find out bioactive compound which are responsible for antimicrobial activity. In additionally explore antimicrobial potential of Boerhavia diffusa leaves.

Antimicrobial Activity
The results of antibacterial activity of the Methanolic of Boerhavia diffusa are presented in

Antimycobacterial Activity
The antimycobacterial activity was evaluated by rapid culture -MGIT TM DST method. The result showed that methanolic extract of Boerhaavia diffusa leaves not inhabited growth of mycobecteria given in Table 3.

DISCUSSION
The potential of leaves extract of Boerhaavia diffusa as antimicrobial and antimycobial along with GC-MS profiling was investigated. The sixteen bioactive compounds ware identified using GC-MS in methanolic extract of Boerhaavia diffusa leaves.
From GC-MS analysis bioactive compounds like 9-Octadecenoic acid [22], phytol [23,24], 2, 4-Di tertbutylphenol [25] Myo-Inositol, 4-C-methyl, Hexadecanoic acid, methyl ester [26,27] have showed antimicrobial activity.  (Streptomycin). Additionally, the antimicrobial effect was found to totally depend upon concentration. The result was found that extract was not effective against Candida, Aspergillus. The antimycobacterial activity was evaluated by the MGIT TM DST method. Similarly, the extract has not inhibited the growth of M. tuberculosis (H37Rv) and, M. tuberculosis (MDR). The phytoconstituents could be responsible for antimicrobial activity and indicated plants could be used in the evolution of novel antimicrobial agents.

CONCLUSION
Inspired by the huge medicinal potential of the herbal wealth of Indigenous plants, the present study was conducted to find out the Antimicrobial and Antimycobacterial activity potential of Boerhavia diffusa leaves. Boerhavia diffusa is a famous herb used in the treatment of many ailments traditionally. The obtained result of the GC-MS showed the presence of many secondary metabolites or phytoconstituents in Boerhavia diffusa leaves. The study result showed that extract exhibited an antimicrobial effect against few pathogens. Further study will necessary to required to find out the molecular mechanism of the antimicrobial effect of the extract.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.