Introduction
Essential oils and plant extracts have antifungal, antibacterial, and cytotoxic activities [1]. Plants have been used for a thousand years as medicines for treating different diseases and medical complaints by most civilizations [2]. The advancement of pharmaceutical technology in the synthesis of chemical drugs and its widespread use has created the complex problem of side effects of drugs and resistance of pathogenic microorganisms against synthetic drugs. Secondary metabolites of medicinal plants have proved to be an excellent reservoir of new medicinal compounds [3] and directed scientists’ attention to natural and herbal medicines [4].
Pistacia atlantica is a tree belonging to the Anacardiaceae family. The most important compounds in the gum of Pistacia atlantica are turpentine oil and colophony. Turpentine is used as an herbicide and for the scent of soap, cleansers, and the production of wax [5]. Antibacterial activities of Pistacia atlantica, Pistacia chinensis, and Pistacia vera leaf extracts have been reported [5, 6, 7]. The antimicrobial activity of P. chinensis leaves methanol extract was more against Gram-positive than Gram-negative bacteria [7]. Generally, the higher resistance of Gram-negative bacteria could be related to the presence of their outer phospholipid membrane [8]. The antimicrobial activity of P. chinensis leaves methanol extract could be related to the presence of triterpenes and flavonoids [9]. The antimicrobial activity of P. lentiscus essential oil against microorganisms has been reported [10, 11]. 
Cassia absus is an annual plant belonging to the Fabaceae family [12]. C. absus has various chemical compounds, including various oils and alkaloids, minerals such as calcium, iron, and zinc, and thiamine and riboflavin vitamins [13]. Seeds of C. absus are used as an astringent in the bowel and abdomen and treat ocular diseases [14]. Anticancer activity of methanol extracts of Cassia fistula on human prostate cancer cell line has also been reported [15]. The flowers of C. singueana have long been used to treat typhoid, malaria, respiratory tract infections and as an antiulcer, antispasmodic, and anti-inflammatory agent [13].
Quercus persica is a tree with leather leaves from the Fagaceae family. This family includes nine genera, of which three (Fagus, Quercus, and Castanea) grow in Iran [16]. Approximately 3 million hectares of Iran’s forests are covered by various oak species, dominated by Quercus persica, Quercus infectoria, and Quercus libani, mostly in the west of Iran [17]. The medicinal importance of Quercus trees is mainly related to the tannins in its leaves [18]. Antibacterial activity of leaf extracts from Quercus persica and bioactivity of hydroalcoholic extract of Quercus brantii against palladium-induced oxidative stress in the male mice reproductive system has been proven [19]. 
Overall, the anticancer, antimicrobial, and antioxidant properties of plant extracts of Pistacia atlantica, Cassia absus, and Quercus persica have been reported. However, little or no work has been done on the seeds of these plants, which are growing in the western forests of Iran. Thus, this research aimed at the identification of Pistacia atlantica, Cassia absus, and Quercus persica seeds phytochemicals and also the evaluation of antibacterial and antioxidant activities of these plants in vitro. 

Materials and Methods 
Chemicals used
Nutrient Broth (NB), Mueller Hinton Agar (MHA) culture media, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Merck Co. (Darmstadt, Germany). Ciprofloxacin and gentamicin antibiotics were prepared from Paten Tab Co. (Tehran, Iran). 

Preparation of plant extracts
The seeds of Quercus persica var. ovoidea, Pistacia atlantica subsp. mutica and Cassia absus var. meonandra, with respective herbarium numbers of 37228, 37227, and 37222, were collected from Lorestan Province, Iran. The samples were dried at room temperature under the shadow (Figure 1).

The dried samples were broken into small pieces (2 mm) by a cylindrical crusher [20]. Ethanol (96%), methanol (80%), and distilled water extracts were obtained by the maceration method [21]. Accordingly, 25 g of dried powder was separately added to a volume of 250 mL of used solvents. The extracts were filtered through filter paper and centrifuged at 10000 rpm for 8 min [22]. The extract was evaporated by rotary and was transferred to an oven at 37˚C for complete drying. The residues were stored in the dark at -22˚C [23].

Tested bacterial strains
All bacteria were obtained from Tehran University of Medical Sciences, Iran. The antibacterial activity of the extracts was tested in vitro against the Gram-positive bacteria of Streptococcus pyogenes (PTCC-1447), Bacillus subtilis (PTCC-1156), Bacillus cereus (PTCC-1247), Micrococcus luteus (ATCC 10987), Enterococcus faecalis (PTCC-1195), and Staphylococcus aureus (PTCC-1189) as well as the Gram-negative bacteria of Escherichia coli (ATCC-25922), Shigella boydii (PTCC1744), Salmonella typhi (PTCC-1609), Pseudomonas aeruginosa (PTCC-1181), Enterobacter aerogenes (PTCC-1221), Proteus mirabilis (PTCC-1287), Neisseria meningitides (PTCC-4578), Acinetobacter baumannii (PTCC-4413), and Klebsiella pneumoniae (PTCC-1129). To prepare fresh bacterial cultures, a bacterial colony was transferred to MHA medium and incubated for 24 h at 37°C. Then a loop of the bacterial colony was transferred to NB medium and was incubated at 37˚C for 18 h [24]. The turbidity of suspension was adjusted to an equivalent of 0.5 McFarland standards (1.5×108 CFU). 

Disk diffusion test 
The antibacterial activity of plant extracts was done by disk diffusion test [25]. The ethanol (96%), methanol (80%), and distilled water extract (200 mg/mL) from Quercus persica, Pistacia atlantica, and Cassia absus seeds were prepared. A volume of 250 µL bacterial suspension (1.5×108 CFU) was poured onto the MHA medium and spread. A volume of 50 µL of the extract was poured on disks. Petri plates were incubated at 37˚C for 24 h [4]. Gentamicin (10 µg) and ciprofloxacin (0.005 µg) were used as positive controls [26]. The inhibitory zone around disks was measured (cm). 

Determination of DPPH for free radical scavenging activity 
The free radical activity was investigated according to the Stojicevic et al. [27] method. Different concentrations (0.2, 0.4, 0.6, 0.8, and 1 mg/mL) of methanol extract from Quercus persica, Pistacia atlantica, and Cassia absus seeds were prepared, and ascorbic acid was used as a reference standard. The samples were placed in darkness for 30 min, and then solvent absorption was recorded by spectrophotometer at 517 nm. The methanol (99%) was used as the blank. The Formula 1 calculated the free radical scavenging activity (%): 
1. Radical scavenging activity (%)= 100(1 - (As - Ab)/Ac
, where As refers to the sample, Ab denotes blank, and Ac refers to control.
Gas chromatography-mass spectrometry 
Gas Chromatography coupled with Mass Spectrometry (GC-MS) was applied to analyze the chemical compositions of the seeds methanol extracts (Tehran University, Iran). The GC-MS analysis was carried out using an Agilent 6890N coupled to Agilent 5973 mass detector, with HP-5, 30 m (length) × 0.25 mm (ID) × 0.25 µm column. The instrument was set to an initial temperature of 55˚C and maintained at this temperature for 2 min. The temperature was increased to 120°C, at the rate of 8°C/min and then to 200°C, at the rate of 3.5°C/min. Injection port temperature was ensured as 350°C and helium flow rate as 0.9 mL/min. The samples were injected in split/splitless mode. Solvent delay adjusted for 5 min, and 0.5 µL volume was injected.

Statistical analysis
The experiments were performed in a completely randomized design with a factorial test. The average comparison was analyzed by the Duncan test at P<0.05 in SPSS version 16. 

Results 
The methanol extract compounds of C. absus, P. atlantica and Q. persica seeds were determined by GC-MS. About 40, 31, and 8 compounds were identified in C. absus, P. atlantica and Q. persica, respectively. The dominant chemicals in C. absus included 2,4-di-tert-butylphenol (36.043%), tetradecanoic acid (4.92%), triacontane (4.102%), hexadecanoic acid (3.58%), octacosane (3.55%). However, the dominant chemicals in P. atlantica included germacyclopetene (38.119%), 1,2,3-benzenetriol (8.115%), 5-hydroxymethylfuraldehyde (HMF) (5.815%) and 2-cyclohexene-1-ol (4.701%). The most compounds found in Q. persica were 5H-tetrazole-5-thione, 1,4-dihydro-1,4-dimethy (38.505%), tetradecanoic acid (30.546%), benzyl (di-deuterated) methyl ether (9.631%), and 2-phenylcyclohexanone (5.914%) (Table 1). 

Antibacterial activity 
The inhibitory activity of alcoholic and aqueous extracts of C. absus, P. atlantica, and Q. persica was evaluated against some human pathogenic bacteria in vitro (Table 2).

Negative control (50 μL of used solvents) and positive controls (gentamicin and ciprofloxacin) were included. After incubation, the bacterial growth zone of inhibitions (diameter) around the wells was measured. The methanol extract of C. absus showed the highest inhibitory activity against M. luteus. The methanol extract had a more inhibitory effect than ethanol and aqueous extracts. Furthermore, E. coli and E. aerogenes had resistance against all extracts. The Gram-negative bacteria, including P. mirabilis and N. meningitides showed susceptibility on all of the tested extracts. Results indicated that the Gram-positive bacteria had more susceptibility than Gram-negative bacteria. S. pyogenes had resistance against ethanol and aqueous extracts of Q. persica. The inhibitory activity of C. absus methanol extract on B. cereus and M. luteus was more potent than gentamicin. In total, C. absus extracts showed a greater inhibitory effect than P. atlantica and Q. persica extracts (Table 2).

Antioxidant activity 
As seen in Table 3, the amount of DPPH free radicals inhibition was increased by increasing the concentration of plant extracts. A significant difference was observed between the IC50 values of methanol extract of C. absus compared with ascorbic acid as the control (Table 3).

The methanol extract of C. absus showed weak radical scavenging activities. 

Discussion
In recent years, several antibiotics have lost their effectiveness due to the development of resistant strains, mainly through the expression of resistance genes. Therefore, there is a need to develop alternative antimicrobial drugs from various sources, such as medicinal plants [28]. The antibacterial and antioxidant activity from 2,4-di-tert-butylphenol and the antioxidant, hypocholesterolemic, nematicide, pesticide, lubricant, and hemolytic inhibitor from hexadecanoic acid have been proven [29]. The 9-octadecanoic acid is a saturated fatty acid and has exhibited antimicrobial activity [30]. The human epidemiological studies have shown that tetradecanoic acid was the saturated fatty acid that is strongly related to the average serum cholesterol concentrations in humans [31].
C. fistula has been used as an Ayurvedic cure to treat heart disease, hematemesis, pruritus, leucoderma, abdominal lump, metabolic disorder, and purgative [32]. The anti-inflammatory, antioxidant, antimicrobial, wound healing properties, and anticancer activity on MCF-7 and SiHa cell lines of C. fistula has been reported [33]. The antioxidant activity of leaf hexane extract of C. fistula was increased by higher concentration [34]. The seed methanol extract of C. auriculata, C. absus, and C. fistula showed higher radical scavenging activity than ethyl acetate and hexane extracts [35], which was similar to our results. The methanol extract of Cassia fistula against S. faecalis showed a more inhibitory effect [36]. The results of this group, probably due to differences in the type of species and climate condition, are different from our results. The chemical compositions of C. fistula flower extract were identified as 4-dihydroxy-1-methoxyanthracene-9, 10-dione, methyl-16-ethylheptadecanoate, butyl hexadecanoate or butyl palmitate, parietin, methyl undecanoate, tetradecanoic acid, rhein, and butyl 2-butoxyhexadec-4-ynoate [37]. The tetradecanoic acid found in mentioned species was similar to our results. The chemical agents, including citronellol (17.24), isophytol (17.24), phytol (17.24), and linolenic acid (17.17), were identified from the methanol extract of C. fistula [15], which was not similar to our research. Some factors, such as plant species, organ types, extract solvents, time and stage of growth, and environmental conditions, are effective in the type and percentage of extracted compounds. The presence of 2-hydroxyethylhydrazine, phytol, n-hexadecanoic acid, oleic acid, cyclotrisiloxane, hexamethyl, di-ndecylsulfone alkaloids, anthraquinones, saponins, phenols, tannins, flavonoids, and terpenoids was confirmed in the leaves hexane extract of C. fistula [34]. Kuo et al. [38] reported oxyanthraquinones, chrysophanol, and chrysophanein in C. fistula seeds. Seeds and leaves of Cassia tora are used to treat itch, ringworm, and other skin diseases. 
The P. atlantica is used for the treatment of peptic ulcers [39]. The essential seed oil of P. chinensis is used for biodiesel production in China [40]. Antimicrobial activity of Pistacia species leaf extract against some plant pathogenic has been reported [5]. According to Mohammadi-Sichani et al. [41], the highest antibacterial activity and the minimum inhibitory concentration were observed from gall methanol extract. The compounds of β-sitosterol, luepol, flavonoids, quercetin, myricetin, quercetin 3-O-α-rhamnoside, quercetin 3-O-β-glucoside, myricetin 3-O-α-rhamnoside, and myricetin 3-O-β-glucuronide were identified in the leaves methanol extract of P. chinensis [7]. The antioxidant activity of quercetin has been proven and is used as a standard for the evaluation of secondary metabolites such as phenols and flavonoids [3]. The chemical compositions, including myrcene (19%-25%), a-pinene (16%), terpinen-4-ol (22%), d-3-carene (65%), myrcene, limonene, terpinen-4-ol, a-pinene, b -pinene, α -phellandrene, sabinene, para-cymene, and γ-terpinene were analyzed from P. atlantica essential oil [42]. 
The antibacterial activity of Q. persica ethanol extract has been reported [43]. The gall extracts of Q. infectoria have antibacterial, antiviral, antifungal, and anti-inflammatory activities [44]. The most susceptible and resistant bacteria were S. epidermidis and E. coli, respectively, against Quercus persica extract [45]. These findings were similar to our results about E. coli. Hussein et al. [46] determined the phytochemical compounds of cis-p-mentha-1(7), 8-dien-2-ol,3-Nonynoic acid, urea, N,N´-bis(2- hydroxyethyl)-, 3-trifluoroacetoxypentadecane, Pterin-6-carboxylic acid, 2,2-difluoroheptacosanoic acid, γ-sitosterol, spirost-8-en-11-one, 3-hydroxy in the gall methanol extract of Q. infectoria. These compounds, due to differences in species and organ types, were not similar to our results. Plants are the richest sources of secondary metabolites with various biological activities. Accordingly, differences in species and genus, extract, solvent, and different geographical locations, time and climate conditions, and so on could affect the type and content of extracted compounds and their antibacterial activities. 

Conclusion 
The chemical compositions of C. absus, P. atlantica and Q. persica were identified from seeds methanol extract by GC-MS. The mentioned plant extracts showed antibacterial activity against some human infectious bacteria in vitro. Also, the antioxidant activity and IC50 value were investigated in different extracts. Based on the findings, the extract compounds of C. absus, P. atlantica, and Q. persica seeds can be used to synthesize antibacterial drugs in pharmaceutical and medicinal science against some human pathogenic bacteria. 

Ethical Considerations
Compliance with ethical guidelines
There were no ethical considerations to be considered in this research.

Funding
This project has been derived from an empirical and research study in Biotechnology Department at Bu-Ali Sina University, Hamadan, Iran. 

Authors' contributions
Both authors equally contributed to preparing this article. 

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors would like to thank the responsible Biotechnology Laboratory. 


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