Phytochemical Investigation and Antimicrobial Potential of Medicinal Plant Nepeta distans Royle ex Benth

Department of Botany and Microbiology, College of Science, King Saud University, P. No. 2455, Riyadh 11451, Saudi Arabia Department of Chemical and Life Sciences, Qurtuba University of Science & Information Technology Peshawar, Peshawar, KP, Pakistan Department of Zoology, Islamia College University, Peshawar, Pakistan Department of Pharmacy, Faculty of Medical Sciences, Aljanad University for Science and Technology, Taiz, Yemen Genetics and Bioengineering, Faculty of Engineering and Natural Sciences, International University of Sarajevo, Sarajevo 71210, Bosnia and Herzegovina Department of Clinical Pharmacology, Faculty of Medicine, Ain Shams University, Cairo 11566, Egypt


Introduction
Since time unknown, medicinal plants are used for the treatment of different infections. e World Health Organization reports that various plant fractions and their dynamic constituents are utilized as traditional medicines by 80% of the world population [1][2][3][4]. Herbal medicines or natural products and plants extract may exhibit promising alternatives or supplements for chemotherapy and antibiotic therapy [5,6]. Herbal medicines have astonishing potential such as high anticancer, antibacterial, antioxidant, and antifungal activities [7,8]. Plants offer an inexhaustible source of bioactive compounds and clinically useful drugs for infectious diseases, such as cancer and cardiovascular disorders [9][10][11]. Bioactive comp ounds play an important role in drug discovery [12]. However, the increasing antimicrobial resistance to the currently available antimicrobial agents demand intense investigations into the antimicrobial properties of the medicinal plants [13][14][15]. Regrettably, there has been limited pharmaceutical development of the plants with known bioactivity [16,17]. ere are few pharmaceutical products for infectious diseases that are of plant origin [18,19]. To fulfill the healthcare needs in Germany and Europe, herbal preparation got more attention, and nearly 1400 herbal preparations are presently used as per European Union [20,21]. e herbal preparation can also be utilized in various cosmetic industries (as antiwrinkling agents, skin tissue regenerators, and antiage creams) which can lead to an increase in the importance [22,23]. e genus Nepeta (Lamiaceae) is a large family which comprises about 400 species, most of which grow in the wild in the central and southern parts of Europe, North Africa, and central and southern Asia. A lot of species of this genus are used in the folk medicine for the antiseptic and astringent properties as topical remedies in children's cutaneous eruptions and snakes and scorpion bites; orally, they are utilized as antitussive, antispasmodic, antiasthmatic, febrifuge, and diuretic. Moreover, antibacterial, fungicidal, and antiviral activities have been attributed to Nepeta lactones and iridoids contained in several Nepeta species. Some endemic species in the southern Greece are utilized in traditional medicine. In particular, fresh leaves of some Nepeta species are chewed to alleviate toothache and a leaf alcoholic macerate is efficacious for treatment of contusions and rheumatic pains [24]. is genus is also reported to possess biological activities that help in reduction of serum lipids and possess anti-inflammatory, phytotoxic, platelet aggregation, antimicrobial, cytotoxic, and antiglycation properties [25].
e aim of the present study is to screen out the bioactive fractions of Nepeta distans, for isolation of the targeted compounds in future.

Plant Collection.
e plant Nepeta distans was collected from Darra Adam Khel district, Kohat (33.6945°N, 71.4959°E), Pakistan. e collected plants were then identified by a plant taxonomist at the Department of Chemical and Life Sciences, Qurtuba University of Science and Information Technology, Peshawar. It was then cleaned, washed, and dried in the shade. A specimen voucher number (ND-001) was deposited in the herbarium of the same department.

Extraction, Fractionation, and Isolation.
e whole plant of N. distans was grinded to a coarse powder. e powdered plant (2 Kg) was initially extracted with 20 L of 70% MeOH three times at room temperature. It was then filtered and methanol was evaporated under reduced pressure leaving behind a greenish, syrup residue that was dried and weighed. It was 100 g. is MeOH extract was then partitioned into various fractions through a separating funnel. It was partitioned into hexane, chloroform, ethyl acetate, butanol, and water fractions successively. e methanol extract was then subjected to column chromatography which afforded compounds (1)(2)(3)(4) from different subfractions at different polarities of the binary solvent system of chloroform/hexane and ethyl acetate/hexane.

Antimicrobial Activity.
e antifungal and antimicrobial activities of the methanol extract and chloroform fraction of N. distans were conducted as per available method in the literature [30][31][32][33][34].

Antifungal Activity.
Using the agar tube dilution method, the antifungal potential of the methanol extract and chloroform fraction of Nepeta distans was investigated. e stock solution was made with 24 mg extracts per ml (1.0 ml) of sterile DMSO (dimethyl sulfoxide). e Sabouraud dextrose agar medium was made by using a magnetic stirrer to thoroughly dissolve 4.0 g of agar, 4 percent of glucose agar, and 32.5 g of Sabouraud in distilled water (500 ml) to form a homogenous slurry. 4 ml of SDA (Sabouraud dextrose agar) was autoclaved at 120°C for 15 minutes before being chilled to 15°C. When each test tube was injected (4 mm diameter) with fungal strains of 6-7 days old culture for non-mycelia development, an agar surface was created by mixing the stock solution with the nonsolidified SDA medium, and solidifying it in a tilted position, streak was formed. Positive and negative controls were preserved for treatment evaluation by employing DMSO and antifungal medications, respectively (amphotericin B). Subsequent to 7 days of incubation at 27°C, the fungal growth reserve was measured.

Antibacterial Activity.
Using the well diffusion agar method, the antibacterial activity of methyl alcohol and chloroform extracts of Nepeta distans was investigated. Bacterial strains were activated by infusing the nutrient broth into the conical flasks and rearing the medium for 24 hours. e agar media was then transferred to Petri dishes containing the injected bacterial strains and solidified under the appropriate conditions. After the medium had solidified, the bores were made in the agar plate using a corkborer, and the extracts (50 l) were transferred to the bore and the plates were reared for 24 hours at 37°C. e results were expressed as the bacterial growth inhibition zone in millimeters. Amoxicillin and ciprofloxacin were employed as positive controls for Gram-positive and Gram-negative bacteria, respectively, and DMSO was utilized as the negative control retain for the purpose of determining the effect.

Isolation of Phytoconstituents.
e methanol extract of N. distans was subjected to column chromatography and the afforded four compounds were oleanolic acid [35], ursolic acid [36], β-sitosterol [37], and stigmasterol [37]. e structure of these compounds are given in Figure 1

Antibacterial Potential.
e result of the antibacterial activity shows that methanol extract of Nepeta distans shows the maximum region of inhibition (33 mm) against Klebsiella pneumoniae at 300 mg/ml, while the lowest inhibition of 22.5 mm was shown against Escherichia coli at 100 mg/ml (Table 1). Similarly, the chloroform extract of Nepeta distans showed the highest zone of inhibition (30 mm) against Staphylococcus aureus 300 mg/ml, while the lowest zone of inhibition (21 mm) was shown against E. coli at 100 mg/ml (Table 1). e methanol extract showed the lowest MIC values (2 μg/ml) against E. coli and pathovar, and the chloroform extract showed the lowest MIC values (2 μg/ml) against S. aureus (Table 2). e MIC values are shown in Table 2.

Antifungal Activity.
e result of the antifungal activity show that methanol extract of Nepeta distans show the highest zone of inhibition (35 mm) at 300 mg/ml, while the lowest zone of inhibition (22.5 mm) was shown by Fusarium solani at 100 mg/ml (Table 3). Similarly, the chloroform extract of the Nepeta distans showed the highest zone of inhibition (31 mm) against Penicillium chrysogenum at 300 mg/ml, while the lowest zone of inhibition ( (3) (4) Journal of Food Quality shown by Fusarium solani at 100 mg/ml (Table 3). e methanol extract showed the lowest MIC values (2 μg/ml) against Aspergillus niger, and the chloroform extract showed the lowest MIC values (2 μg/ml) against Aspergillus tamarii. Both the methanol extract and the chloroform fraction showed moderate activities against Aspergillus flavus and Aspergillus fumigatus at all three concentrations (Table 4).
e MIC values are displayed in Table 4.

Phytochemical Screening.
Phytochemical screening showed that both methanol extract and chloroform fraction showed the presence of alkaloids, flavonoids, saponins, glycosides, proteins, and oil (Table 5). Screening the crude extract for phytochemical analysis is an imperative means for finding the secondary metabolites that exist in the medicinal plant because these phytochemicals are counted responsible for therapeutic application. ese phytochemicals responsible for that it has been reported that saponins are among the most important compounds responsible for antidiabetic, antispasmodic, antitumor, anthelmintic, antimicrobial, phytotoxic, cytotoxic, and antioxidant potential [38].

Conclusion
e present study shows that Nepeta distans possess bioactive phytochemicals such as flavonoids, alkaloids, and tannins , and its methanol extract and chloroform fraction showed antimicrobial potential. e published literature showed multiple biological activities of Nepeta species. erefore, it is highly recommended to explore Nepeta distans for more phytochemical as well as biological investigation.

Data Availability
e data used to support this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.