Volatile Constituents Analysis and Antimicrobial Activity of Two Subspecies Ballota nigra L. From Iran

Background Ballota nigra L. is a perennial herb of the Lamiaceae family and it's native to the Mediterranean region and central Asia, also it can found throughout Europe. during this research, the volatile oil composition and antimicrobial activity of the methanol extract from owering aerial parts of two subspecies of B. nigra (B. nigra ssp. foetida and B. nigra ssp. anatolica) were evaluated. Plant extraction was prepared by the maceration method and the antibacterial activity of methanolic extracts against 12 microorganisms was investigated by disc diffusion and minimal inhibitory concentration (MIC) methods. Results: GC/MS analysis of the volatile oil conrms the presence of 25 constituents in B. nigra ssp. foetida and 27 constituents in B. nigra ssp. anatolica. the most constituents in these two subspecies were α- Terpineol, Eicosane, Hexadecanoic acid, Longipinene epoxide, and Ethyl linoleate, which had slight differences within the amount of total volatile oil. within the antibacterial and MIC tests, indicated that methanolic extract of two subspecies Ballota nigra L. had acceptable antibacterial activity and therefore the B. nigra ssp. anatolica had more antibacterial activity in compression to B. nigra ssp. foetida. Conclusion: Today the tendency of natural products due to antioxidant and antimicrobial properties is increasing, and plants are the main source of these safe materials. During this respect, our study is often considered because of the rst report on the volatile oil composition and antimicrobial activity of methanol extract of various subspecies of .B. nigra.


Introduction
Secondary metabolites and essential oils have wide applications in dietary regimens, food avoring and preservation, folk medicine, and the fragrance industry (Huang et al., 2005;Kalemba and Kunicka, 2003).
The application of plant products as dietary regimens and preservatives is especially due to their antioxidant, antimicrobial, and other biological potentials.
Tease natural antioxidants can inhibit or delay the oxidation of oxidizable substrates and this appears to be vital within the prevention of oxidative stress which is usually recommended because of the leading explanation for a good deal of oxidation-related diseases (Halliwell et al., 1992;Sarker et al., 2006).
Recently, thanks to undesirable side effects like toxicity and carcinogenicity of synthetic additives, interest has considerably increased for locating present antioxidant and antimicrobial compounds suitable to be used in food and medicine (Losso et al., 2007;Sachetti et al., 2005).
In this regard, a growing rate of research was conducted on many plant species so as to seek out new natural bioactive compounds in them. Ballota nigra L. (Lamiaceae) may be a perennial herb which is usually distributed in Western Europe where owering aerial parts are utilized in medicine for treating cough and more especially for neurobiological activities (Sokmen et al., 1999). Two subspecies of Ballota nigra were collected in 2019 from the natural habitat of Isfahan and Mazandaran province in Iran, subspecies of this plant species identi ed in Kashan arboretum (Isfahan/Iran). This study reported the chemical composition of the volatile oil and antimicrobial activity of the methanolic extract from owering aerial parts of those two subspecies of Ballota nigra L.

Plant materials
The owering aerial parts of B. nigra ssp. foetida were collected during the owering period in (April to May) 2019, from Qamsar, around Kashan (Isfahan province, Iran), and therefore the owering aerial parts of B. nigra ssp. anatolica were collected during the owering period in (May to June) 2019, from Kajoor, (Mazandaran province, Iran).
The voucher specimen of the plant was deposited within the herbarium of Research Institute of Forests and Rangeland, Kashan, Iran. The aerial parts of the plant were spread uniformly during a thin layer within the shade during a dry place with well air ventilation for drying, when the moisture content fell to a minimum suitable for grinding, the material was ground with a steel mill (Retschmühle, GmbH, 5657 HAAN, Germany), stored in well-closed glass containers, kept during a refrigerator and used within a couple of days. The aerial parts (50 g) of the crushed material were individually subjected to hydrodistillation for 3.5 hours using the modi ed simultaneous distillation and extraction (SDE) method.
(Likens and Nickerson, 1997; Anonymous, 1996;GRIN, 2009). After decanting and drying over anhydrous sodium sulfate, yellow-colored oils were recovered and stored at coldness (4 C °) under a nitrogen atmosphere in amber vials and were used for analyses within a couple of days. so as to gauge the antimicrobial effects, the methanolic extract (Methanol 70%), from aerial parts of the plant was prepared by maceration method (Anonymous, 1996

Gas chromatographic (GC/MS) analysis
Volatile oil samples obtained from the owering aerial parts of B. nigra L. were analyzed using an Agilent HP-6890 gas chromatograph (GC), (Agilent Technologies, Palo Alto, CA, USA) equipped with an FID detector using an HP-5MS 5% phenylmethyl siloxane capillary column (30 m × 0.25 mm, 0.25 µm lm thickness; Restek, Bellefonte, PA). Oven temperatures were programmed as follows: 50 C (2 min), 50-130 C (5 C/min), 130 C ° (2 min), and 130-200 C ° (3 C/min). Injector and detector temperatures were set at 220 C and 290 C °, respectively. Ultra-high purity helium ( ow rate: 1 ml/min), hydrogen ( ow rate: 40 ml/min), and nitrogen ( ow rate: 50 ml/min) were used as a carrier, fuel, and makeup gases respectively and compressed gas ( ow rate: 450 ml/min) was used for combustion. Diluted samples (1/1000 in n-pentane, v/v) of 1.0 µl were injected manually within the splitless mode. Peak area percent of every compound relative to the world percent of the whole spectrum (100%) were used for obtaining its quantitative data. The injection was repeated 3 times and therefore the peak area percent were reported as means ± SD of triplicates. Co-injection of selected commercially available components of the volatile oil were also administered and led to the enrichment of the respected picks within the spectrum and further con rmation of their identities (Bamoniri et al., 2010).
GC/MS analysis of the oil was administered on an Agilent HP-6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with an Agilent HP-5973 mass selective detector within the electron impact mode (ionization energy: 70 eV), operating under equivalent conditions as described above, using an HP-5MS 5% phenyl methyl siloxane capillary column (30 m × 0.25 mm, 0.25 µm lm thickness; Restek, Bellefonte, PA). Retention indices were calculated for all components employing a homologous series of n-alkanes injected in conditions adequate to the sample one. Identi cation of components of volatile oil was supported retention indices (RI) relative to n-alkanes and computer matching with the Wiley275.L and Wiley7n.L libraries, also as comparisons of the fragmentation pattern of the mass spectra with data published within the literature (Adams, 2001). Some commercially available components of the volatile oil were also Co-injected for further con rmation of their identi cation.

Antimicrobial activity
The methanolic extracts of two subspecies of B. nigra L. were individually tested against a panel of 12 microorganisms. Following microbial strains were provided by the Iranian Research Organization for Science and Technology (IROST) and utilized in this research: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 10536), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 29737), Klebsiella pneumoniae (ATCC 10031), Staphylococcus epidermidis (ATCC 12228), Shigella dysenteriae (PTCC 1188), Proteus vulgaris (PTCC 1182), Salmonella paratyphi-A serotype (ATCC 5702), Candida albicans (ATCC 10231), Aspergillus niger (ATCC 16404) and Aspergillus brasiliensis (PTCC 5011). Bacterial strains were cultured overnight at 37 C ° in agar (NA) and fungi were cultured overnight at 30 C °i n sabouraud dextrose agar (SDA).To prepare the methanolic extract was used the soaking (Maceration) method. After drying the aerial parts of the plant ( owers, leaves, and stems) under shade conditions and proximity to the air, samples powdered by an electrical mill, then the quantity of 15 g of the sample mixed with 200 ml of methanol 70%. After each day the organic phase was removed and methanol was added again. This action was repeated 3 times, nally, the entire plant extract was collected and used a rotary evaporator to evaporate the solvent, also to full water removal and prepare the plant's powder the freeze dryer was used. To investigate, the antibacterial activity of the extract, 0.2 g of methanolic extracts were weighed and dissolved using 10 ml DMSO. Finally, this stoke was wont to prepare different concentrations of extract within the disc diffusion and MIC methods (Bamoniri et al., 2010).
The Bacteria strains were grown in Muller-Hinton uid medium (Merck, Germany). Then a dense and uniform culture of bacteria was prepared with the bacterial suspension like half McFarland by sterile cotton swabs within the surface Müller-Hinton Agar (Merck, Germany). Sterile paper disks with a diameter of 6 mm (Padtan Teb, Iran) and impregnated with 30 µl of methanolic extract and dissolved in DMSO, were placed in agar separately. A disk impregnated with 30 µl DMSO solvent used as a negative control and therefore the antibiotic discs of Rifampin, Gentamicin, and Nystatin, was used as a positive control.
Plates incubated for twenty-four to 48 hours at 37 ° C. the antibacterial activity of plant extract was measured by the halo diameter without growth (mm).To determine the minimum inhibitory concentration of growth, the methanolic extracts of the aerial parts of the plant (leaf, ower, and stem) with 20 mg/li concentration in DMSO solvent was prepared. then was sterilized using the paper, with 0.22 µm in pore diameter, then the sterile extract was added to the LB liquid medium, to be achieved a concentration between 5 to 300 µg/ml. Also, the antibacterial activity of the methanolic extract was compared with Tetracycline antibiotic as a positive control and DMSO as a negative control within the DMSO in similar conditions. Then 0.5 percent of the fresh bacterial culture was added to tubes containing LB liquid medium and plant extract, separately. Pipes were placed at a warm home with Shaker at 37 ° C for twenty-four hours with 100 rounds per minute. Antibacterial activity of the methanolic extract was measured by the minimum inhibitory concentration (MIC) of bacteria growth. (NCCLS, 1997).  (Table 1). α-Terpineol (10.78%), Eicosane (9.11%), and palmitic acid (8.14%) were the most components within the volatile oil obtained from owering aerial parts of B. nigra ssp. foetida. Although these compounds were the main compounds within the B. nigra ssp. anatolica too, but their percentage was less within the volatile oil content. in order that α-Terpineol (9.50%), Eicosane (8.85%), and palmitic acid (7.20%) assigned the quantity of essential o ils.   . Its formula is CH3 (CH2)14COOH, and its C:D is 16:0. As its name indicates, it's a serious component of the oil from the fruit of oil palms (palm oil). hexadecanoic acid also can be found in meats, cheeses, butter, and dairy products. Palmitates are the salts and esters of hexadecanoic acid. The palmitate anion is that the observed sort of hexadecanoic acid at physiologic pH (7.4).

Results
Aluminum salts of hexadecanoic acid and naphthenic acid were combined during war II to supply napalm. The word "napalm" springs from the words naphthenic acid and hexadecanoic acid.

Antimicrobial activity
Results of antibacterial effects of methanolic extract of two subspecies Ballota nigra L. By existence or absence growth inhibition zone (halo diameter of without growth) in Table 3 and minimum inhibition concentration (MIC) showed in Table 4. consistent with Table 3, the very best halo diameter of without growth was observed within the effect of the methanolic extract of B. nigra ssp. anatolica. on the B. subtilis and E. coli strains with halo diameters of 11.63 ± 0.52 and 11.20 ± 0.80 respectively. Also, this two B. nigra subspecies showed a special reaction against S. dysenteriae and A. niger strains, that B. nigra ssp. anatolica had the effect on S. dysenteriae, while on the A. niger no effect was observed. The results of the MIC test indicated the lowest concentration of the extract regarding inhibiting the expansion of S. aureus and B. subtilis bacteria with 150 ug/ml concentration (Table 4).  As the results of Table 4 show, B. nigra ssp. anatolica has been ready to inhibit the expansion of two of these bacteria strains at lower concentrations. It is often said that the inhibitory power of the extract of those two subspecies was acceptable as compared with Tetracycline that used because of the control.  Table 3 and Table 4, the methanolic extract of owering aerial parts showed acceptable antimicrobial activities against almost quite half microorganisms, the results of this section of our research con rmed the previous study within the other species of B. nigra that reported by (Miraliakbari and Shahidi, 2008;Murray et al., 1999;Omidbaigi, 2005). It should be noted that the essential oils and therefore the antimicrobial effects of those two subspecies haven't been reported so far, and this study has addressed this issue for the primary time.

Conclusion
Different plant species and subspecies have differences in composition and percentage within the volatile oil. therefore the explanation of characteristics and perform studies on subspecies levels of the plants is extremely important and may use by researchers for proper selection of the plant for his or her future and supplementary research. Also, the Growing tendency for replacing synthetic additives with natural ones has emerged an excellent interest within the evaluation of antioxidant and antimicrobial properties of plant products in both research centers and industry. during this respect, our study is often considered because of the rst report on the volatile oil composition and antimicrobial activity of methanol extract of various subspecies of B. nigra. The rather good antimicrobial activity of the plant extract especially within the MIC test encourages more elaborate investigations in this respect. Ethics approval and consent to participate:

List Of Abbreviations
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