Phytochemical analysis and antimicrobial activities of Kochia indica (Wight), plant growing in District Karak Khyber Puhktunkhuwa, Pakistan

The medicinal plants play a key role for the treatment of various types of diseases because of bioactive compounds. Study aims, to investigate the antibacterial potency, phytochemical analysis and also perform gas chromatography and mass spectrometry (GC-MS) for isolations of bioactive compounds of Kochia indica Wight plant. In antibacterial activity the whole plant extracts of Kochia indica Wight, against S. typhi, Escherichia coli, Shigella flexneri, Pseudomonas aeroginosa and Staphylococcus aureus strains at different concentrations. According to the results obtained, the ethanol extract of Kochia indica was 40%20.37% (15 mg/ml), 13.11%-19.10% (10 mg/ml) and 11.20%-17. 44%, (5mg/ml) concentration over negative control. The best quantitative phytochemical analysis of Kochia indica Wight was performed using extracted optical density (OD) and mass chromatography-mass spectrometry (GC-MS) techniques. Results reveal the presence of important antibacterial and phytochemical compounds predominantly1-Propanol, 2methyl(63.27 %), 1,1-Diisobutoxy-isobutane (8.83%), Oleic Acid (7.54%), Cyclohexanone (6.60%), Acetone (3.57%), Hexanoic acid, 2-methylpropyl ester (2.75%), n-Hexadecanoic acid (1.47%), 2Butenedioic acid (Z)-, dibutyl ester (1.41%), Sebacic acid, ethyl hexyl ester (0.93%), Phytol (1.00%), Octadecanoic acid (0.78%), Cyclopentane carboxylic acid, butyl ester (0.65%), 1-Butanol (0.42%), Propanoic acid, 2-methyl, 2-methylpropyl ester (0.48%), Cyclohexene,1-(1,1-dimethylethoxy) (0.31%). To detect more effective process and compounds to initiate and develop the most effective approaches and methods to reduce the effectiveness of bacteria on environment as well as in human health.


Introduction
The medicine or drugs obtain from plants are safe and have no side effect as compared to the modern and synthetic medicine and drugs which are not safe having side effect and environment non-friendly. From the primitive times, plants keep the value medications, aromatic, nutritive as well as flavoring abilities or characters. Now a days, the advancement of modern synthetic drugs and medicine are fully compressed by the drugs and medicine obtained from plants i.e. herbal medicines or drugs [1].The 25% of drugs and medicine universally approved provide by the medicinal plants and 121 compounds are in active use and 252 prescriptions thought as elementary and important according to the World Health Organization (WHO).Medicinal plants are a rich source of antibacterial compounds that are effective against natural pathogens. Pakistan has many medicinal plants rich in antibacterial compounds with antibacterial activity. This plant has been used for decades in the production of various medicines [2]. Mainly the drugs which isolated from the medicinal plants worldwide preferred that of synthetic in medicine discipline. The remedial part of a therapeutic plants is used only fresh green leaves and stem, so the quality and quantity of the phytochemical present in that part of the medicinal plant. Such phytochemical have so many chemical constituents such as proteins, gums mucilage, carbohydrates, pectin's, different form of glycoside, phenol compounds, tannins, resins, lipids, fixed and volatile oils, different kinds of alkaloids etc [3]. Therefore, it is necessary and important not only to improve the quality of existing medicines (medicines), but also to find effective new medicines. Numerous studies have shown that herbal medicines have various antifungal and antibiotic properties. Not only are plant extracts cheap and have little impact on the environment, they also minimize synthetic health risks [4]. This study aims to investigate the phytochemical composition and antimicrobial activity of plant extracts. The plant was chosen because there is little scientific evidence for the plant and the need to discover the therapeutic value of herbs. In addition, various compounds found in the Kochia Indica plant were measured by gas chromatography and mass spectrometry (GC-MS) analysis.

Materials and Methods Preparation of plant extracts
Medicinal plants (Kochia indica Wight) were collected in district Karak, Khyber pukhtoonkhwa, Pakistan, dried in the shade and then homogenized with fine powder. Using this fine powder, methanol, ethanol, and water extracts were made from these plants according to the method described in [5]. Twenty-five grams of each plant powder was soaked in 100 ml of ethanol for 10 days. The extract was then passed through a muslin cloth, filtered and dried in a 45°C water bath. The resulting past was placed in an amber bottle and stored at 4 0 C until use.

Antibacterial activity
The Antibacterial activities was performed by using change of the method is clearly demonstrated and broadly used for activities test [6]. A lapful bacterium was point out from the solution and add o, 1 mili letter saline. After placing the discs, all tests were carried out on the surface of Mueller Hinton agar pre-inoculated with 10 liters of 10 liters of liquid medium MHA containing different crude solvent extracts than 9 mm in diameter. A known solvent without plant extract serves as a control. The standard antibiotics were chloramphenicol 30 mg: disc and streptomycin 30 mg: disc control +ve. The plate was heated at 37° C. for 24 hours and the formation of open areas around the disc was observed.

Phytochemical analysis
The dry powder and extract of Kochia indica plant were used for the evaluations of different types of phytoconstituents. The standard method was used for the evaluation of phenols and flavonoids [7]. Gas chromatography and mass spectrometry (GC-MS) analysis of plant extracts GC-MS analysis of Kochia indica Wight ethanol extract shows that the most active and promising antibacterial activity was achieved by Agilent 7890C gas chromatography in combination with 5975C mass selective detector (MSD) and hypersensitive panchromatic 5 (HP5), mass spectrometry imaging (MSI) separator columns. Here, the length is 30mm, the inside diameter is 0.250mm, and the film thickness is 0.25μm. The deposition, determination and quantity of compounds was carried out by accelerator mass spectrometry (AMDS) using the GCMS library with manual storage index, and the subsequent identification of the compounds was carried out using the NIST17 and Wiley 11 GC (Gas chromatography) spectral libraries.

Statistical analysis
The current analysis of variance was applied using statistical software 16.1. The mean and standard error of the triples were calculated. This solution was compared using the liquid crystal display (LSD) test at a significant level of ≤ 5%.  (Fig. 2). In ethanolic extract the maximum quantity of flavonoids substances were estimated by Kochia indica (2.80±0.02) at concentrations of 20ug/ml, while the ethanolic extract of the Kochia indica plants showed highest quantity of total phenol contents (1.30±0.5) at concentrations of 20ug/ml, and then followed by (1.19±0.05) and 1.09±0.05 at 10ug/ml and 30ug/ml. As compared to the other concentrations the total phenol and flavonoids contents shows more at concentration of 20ug/ml shown in (Table. 2; Fig. 3).      (Table. 3; Fig. 4). Different phenol compounds were detected from Kochia indica which proved their medicative values.

Discussion
The therapeutic medicative plants species are rich source of naturally accruing antimicrobial compounds which play an important role for the development of active, new and effective drugs and medicines. In the literature, the antibacterial activity of these plants has been reported against many plant and human pathogens according to the literature but difficult to study due to choice of solvent, fractionation or extraction method and use of microbes and antibacterial tests