Phytochemical, Cytotoxic, and Antimicrobial Evaluation of the Fruits of Miswak Plant, Salvadora persica L

Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia Department Clinical Pharmacy, College of Pharmacy, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia Substance Abuse and Toxicology Research Centre, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia Faculty of Dentistry, Jazan University, P.O. Box 114, Jazan 54142, Saudi Arabia Department of Microbiology and Immunology, National Research Centre, 33 Bohouth St. Dokki, Affiliation I.D. 60014618, Giza 12311, Egypt Medicinal and Aromatic Plants Research Institute, National Center for Research, P.O. Box 2424, Khartoum 11111, Sudan Department of Pharmacology and Toxicology, Faculty of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia Department of Pharmacognosy, College of Pharmacy, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia


Introduction
Salvadora persica L. (family: Salvadoraceae), which has many local names (miswak, peelu, toothbrush tree, and mustard tree), is an ancient native plant found in Africa, Iran, Pakistan, India, Sri Lanka, and the Middle East countries including Saudi Arabia, Oman, Yemen, Jordan, and Syria [1,2]. It is an evergreen shrub with a wide crown of curved branches, slightly rough bark, greyish-brown stem, greenish-yellow flowers, and fleshy pink fruits [3].
ere are many traditional uses reported for the S. persica plant. It has mainly been used in dental care to cure toothache and gum diseases and for cleaning teeth. Additionally, it is used to relieve boils, chest diseases, gonorrhea, headache, spleen troubles, stomachache, and ulcers. e root infusion can also be used to increase milk production in lactating women. Leaves of the plant are being utilized as a mouthwash and in treatment of chest pain, cough, tooth and gum problems, body pain, backache, piles, stomachache, stomatitis, and wounds. e bark latex is beneficial to subside skin sores. e seeds are taken as a tonic, and the seed oil is rubbed to treat joint pain, lumbago, and rheumatism. It has also been used to relieve edema, fever, malaria, and worms. e plant juice is used by women as a female contraceptive [1,4]. e uses of stem and roots of S. persica in dentistry could be correlated to the presence of many phytochemical constituents such as tannins that inhibits glucosyltransferase enzyme to reduce plaque and gingivitis [5]. e resins of the chewing stick of the plant protect against dental caries by forming a layer on enamel [6]. Salvadorine isolated from the chewing stick of S. persica exerted bactericidal effects and stimulated the gingiva [7]. Recently, five α-amylases were isolated from the roots of the plant which showed good affinity towards the substrates such as starch and glycogen [8]. Moreover, S. persica sticks were found to stimulate the saliva flow in humans owing to the mildly bitter taste of essential oils present in it, which act as a buffer. Additionally, high chloride concentrations inhibit the calculus formation and removes stains from the tooth surfaces [7,9]. Moreover, the use of chewing sticks causes calcium saturation in human saliva leading to promotion in remineralization of tooth enamel [5]. e anticancer activity of S. persica was also investigated. In a previous study, S. persica aqueous root extract exhibited oral protective activity on the normal periodontal ligament fibroblast (PDL) cell lines, and notably it caused cytotoxicity on oral cancer cells: oral epithelial dysplasia (DOK) and oral squamous cell carcinoma (PE/CA-PJ15), all using the MTTassay [10]. e MTT assay was also used to investigate the cytotoxicity of S. persica sticks and bark extracts against HepG2, MCF7, A549, and HCT116 cancer cells. S. persica extracted with petroleum ether showed the highest cytotoxicity compared to the same plant extracted with aqueous alcohol, chloroform, and ethyl acetate. e petroleum ether extract yielded two triterpenes (ursolic and oleanolic acids) which showed IC 50 10.2-43.6 μg/ mL against the four cancer cell lines [11].
Studies related to the importance of S. persica fruits as nutraceutical are very scarce and less investigated [12]. e nutritional value of the S. persica fruits has been demonstrated by the presence of sugar metabolites, minerals, amino acids, vitamins (ascorbic acid and carotenoids), polyphenols, and flavonoids. Previously, these metabolites were proved to have considerable antimicrobial, antihyperglycemic, antitumor, and antioxidant properties. e fermented fruit juice is a general body tonic and acts as strong aphrodisiac [12][13][14].
In spite of the ethnopharmacological importance of S. persica, the research on this plant has been focusing only on the stem and root of the plant. To the best of our knowledge, this is the first study to emphasize the cytotoxicity and antimicrobial properties of the fruits of S. persica. is study was conducted to identify the constituents of the plant using GC-MS. e cytotoxic and antimicrobial properties of the fruit extract of S. persica were evaluated keeping in mind the high reputation and traditional ethnopharmacological uses of the stem and roots of the plant.

Plant Collection and Extraction.
Reddish purple fruits of S. persica were collected from Jazan region of Saudi Arabia in March 2018 and were identified by Dr. Yahiya Masrahi, Botany Department, Faculty of Science, Jazan University, Saudi Arabia. A voucher with specimen no. JU/COP/18-2 was deposited in the herbarium of the Department. e fruits of S. persica were extracted using previously described method by Harborne [15] with some modifications. Fresh fruits of S. persica (500 g) were grinded, macerated, and dipped in 2.5 liters of 80% ethanol and petroleum ether separately for 48 h at room temperature with constant shaking. e supernatant obtained was filtered using Whatman filter paper (0.45 μm). is process was repeated two times in the same conditions. Both extracts were mixed and dried at room temperature. e dry weight yield was calculated to be 10%. e extracts were then refrigerated at 4°C in dark bottles for consequent experiments.

GC-MS Analysis of the Extract.
e extract was diluted in methanol (1 : 10 v/v) and was analyzed using ermo Scientific GC-MS equipped with AS 3000 autosampler, Trace GC ultra, and ISQ detector. ermo Scientific TR 5MS with dimensions of 30 m × 0.25 mm (internal diameter) × 0.25 μm (film thickness) was used for separation of the components. Helium, at a flow rate of 1.2 mL/min (constant flow mode), was used as carrier gas. A volume of 2 μL of sample extracts was injected in splitless mode. e injection port was set at 320°C and temperature of oven was initially set at 70°C for 5 minutes, which was subsequently ramped to 205°C at rate of 5°C/min and held for 5 minutes, then increased to 280°C at rate of 5°C/min and held for 5 minutes, then to 290°C at rate of 5°C/min and again held for 5 minutes, and finally to 300°C at rate of 5°C/min and held for 5 minutes. e maximum oven temperature was set at 320°C. e mass spectrometer was operated in an electron ionization (EI) mode within the mass range of 60-900 amu with 0.6 scan times (min). e MS ion source temperature and transfer line temperature were set at 320°C and 350°C, respectively, with electron multiplier voltage of 1 Kv.

Identification of Phytochemical Constituents.
e mass spectra were interpreted using Xcalibur software, and the fragmentation patterns in the mass spectra obtained for all constituents were compared with the data stored in the instrument database using the NIST, MAINLIB, and REPLIB built-in libraries.
e constituent percentages were measured based on the peak area. e components were identified upon comparison with the structures available in the computer library. e reported biological activities of the constituents listed (Tables 1 and 2) are taken from Dr. Duke's Phytochemical and Ethnobotanical Databases [16].

Cytotoxicity Assay.
ree cancer cell lines, MCF7 (human breast carcinoma cells), A2780 (human ovary carcinoma cells), and HT29 (human colon carcinoma cells), were used in this study to evaluate the cytotoxic properties of the fruit ethanol extract in addition to the effects on MRC5 (normal human fibroblast) cells. e three tumor cell lines were subcultured in RPMI-1640 media (prepared in 10% Fetal Bovine Serum (FBS)), whereas MRC5 was subcultured in Eagle's Minimum Essential Medium (EMEM) (in 10% FBS at 37°C temperature, 5% CO 2 , and 100% relative humidity). e cytotoxic properties of the ethanol extract were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay according to previous report [17,18]. e three cancer cell lines as well as the normal human fibroblast were cultured separately in 96-well (3 × 10 3 /well) plates and placed for incubation at 37°C overnight.
ereafter, the final extract concentrations 0-100 μg/mL were prepared and added in triplicate to the wells. Plates were then incubated again for 72 h, followed by addition of MTT to each well. Finally, the plates were incubated for 3 h and DMSO was added to each well. Absorbance was measured at 550 nm using BIORAD, PR 4100 multiplate reader. e optical density was directly related to the number of viable cells. e cytotoxicity was indicated by the decrease in the optical density as compared to control. Extract concentration which causes 50% inhibition in comparison to control (cell growth � 100%) was determined as IC 50 .

Preparation of the Test Organisms.
A loopful of isolated bacterial colonies were inoculated into 4 mL peptone water and incubated at 37°C for 4 h. e turbidity of actively growing bacterial suspension was adjusted to match the turbidity standard of 0.5 McFarland units [19]. e fungal cultures were maintained on Sabouraud dextrose agar, incubated at 25°C for 4 days. e fungal growth was harvested and washed off with 100 mL sterile normal saline, and the suspension was stored in the refrigerator at 4°C until used.

In Vitro Antimicrobial Activity Testing of Extracts.
To test the antibacterial efficacy of the extract, a measured quantity (1 mL) of the standardized stock suspension of bacteria having 10 5 -10 6 CFU per milliliter was mixed with 100 mL of Mueller-Hinton agar medium maintained at 45°C. Aliquots (20 mL) of the resulting inoculated Mueller-Hinton agar medium were divided into previously sterilized plates. e agar medium was allowed to set at room temperature and 4 cups of 10 mm diameter were made in each of these plates using a sterilized cork borer (No. 4). Agar discs were removed and the hole was filled with different concentrations (100 μg/mL and 500 μg/mL) of ethanol extract and left undisturbed at room temperature to diffuse for 2 h. e resulting plates were allowed to incubate at 37°C overnight. e plates were then observed for any inhibition in the bacterial growth and the diameter of zone of inhibition was measured.
e method used for antibacterial study was also adopted for testing the extract for antifungal activity. e only difference in the method is that instead of Mueller-Hinton agar medium, another medium (Sabouraud dextrose agar medium) was used and the inoculated medium was maintained at 25°C [20].
Similarly, the total ion chromatogram of petroleum ether extract was obtained using GC-MS and is shown as Figure 2, whereas the chemical constituents and major biological activities of the petroleum ether extract of the S. persica fruits are summarized in Table 2.
e prominent constituents characterized in the petroleum ether were eicosamethylcyclodecasiloxane (23.81%), 1-monolinoleoylglycerol (11.78%), (Z,Z,Z)-9,12,15-octadecatrienoic acid ethyl ester derivative (10.56%), and tetracosamethyl-cyclododecasiloxane (9.91%). Four alkanes identified in the extract included 2,6,10,15-tetramethyl heptadecane (1.12%), 2methyl eicosane (1.7%), 2-methyl nonadecane (1.61%), and 3-ethyl-5-(2-ethylbutyl)-octadecane (2.53%). Figure 3 represents various important chemical constituents detected in ethanol and petroleum ether extracts of fruits of S. persica along with their structure. e previous phytochemical screening studies showed that the stem of S. persica yielded octacosanol, 1-triacantanol, β-sitosterol, 3-O-β-D-glucopyranoside salvadourea, m-anisic acid [22,23], pyrrolidine, pyrrole, and piperidine derivatives [24], salvadoside and salvadoraside glycosides [25], and flavonoids, which included quercetin, rutin, kaempferol, and quercetin glucoside [26], while the roots of S. persica yielded β-sitosteryl fatty esters and β-sitosteryl-3-vanilloyl-4′-fatty esters [27]. e roots, leaves, and stem contained ascorbic acid, butanediamide, salvadorine, salvadourea, lignans, fatty acids, trimethyl amine, cyanogenic glycosides, steroidal esters, tannins, aniline derivative, spiculesporic acid, methyl hexadecanoate, and homo-c-linolenic acid [28]. Additionally, the stem essential oil is reported to consist mainly of 1,8-cineole (eucalyptol) (46%), β-pinene (6.3%), and 9-epi-(E)-caryophyllene (13.4%) [29]. e roots and stems also possessed oleic,  Journal of Chemistry linoleic, and stearic acids as well as methyl and ethyl esters of fatty and other organic acids [30]. e ethanol fruit extract of S. persica was tested for cytotoxic effects using MTT assay (72 h), which showed activity against the breast, ovary, and colon cancer cells (IC50 : 5.12-17.50 μg/mL). However, it was observed to be more selective against the ovarian and the colon cancer cell lines compared to the normal cells MRC5 (Table 3, Figure 4). is result was in agreement with the previous reports showing the antitumor activity of S. persica root, sticks, and bark extracts against oral carcinoma, HepG2, MCF7, A549, and HCT116 cancer cells [31][32][33][34][35][36]. Both S. persica fruit extract used in this study and S. persica root extract previously tested have exhibited protection to the normal lung and oral cells, thus indicating the safety profile of this commonly used plant. Eicosane, psi-carotene, 3′,4′-didehydro-1′,2′-dihydro-1′,2′dihydroxy-(2′R)-9,12,15-octadecatrienoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester, (Z,Z,Z)-, and formyl colchicine were all identified in the GC-MS analysis of the ethanol extract of S. persica fruits in this study. ese constituents were previously reported to exert anticancer activities according to Dr. Duke's Phytochemical and Ethnobotanical Databases (Table 1) [16]. e antibacterial activity of the ethanol extract was determined using the disc diffusion method. e plant extract was found to possess selective antimicrobial activity, as it was ineffective at both the tested concentrations (100 μg/mL and 500 μg/mL) against the isolates of Staphylococcus aureus and Enterococcus faecalis as well as against the Gram-negative microorganisms, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Klebsiella pneumonia. Interestingly, it was found to be selectively effective against the isolates of Streptococcus mutans, which was resistant to the standard drug Ampicillin. e zone of inhibition at the higher concentration 500 μg/mL was found to be 25 ± 2.1 mm which was comparable to the zone of inhibition shown by ampicillin for other bacterial strains at the same concentration (Table 4). e MIC and MBC values of the extract were 3.12 and 6.25 mg/mL, respectively, whereas the standard drug ampicillin was found to have nonsignificant activity against the tested strain. is could mainly be due to the phytochemical differences between the constituents in the fruits of S. persica collected from this region or to the characteristic differences between S. mutans and other bacterial strains used in this study. is could also be attributed to any potential to new characters that could have been gained by the clinical strains that showed resistance against S. persica extracts. On the other hand, the sensitivity of S. mutans to the extract of S. persica supports the traditional use of this plant in oral health as it is considered one of the most common oral microbes that cause caries.
Interestingly, previous reports of in vitro antibacterial and antifungal studies on roots and stem extracts of S. persica on cariogenic bacteria and periodontal pathogens showed that the plant was effective against S. faecalis, S. aureus, S. pyogenes, S. mutans, P. aeruginosa, L. acidophilus, P. gingivalis, H. influenzae, A. actinomycetemcomitans, and C. albicans [37][38][39]. However, this study utilized the fruits of S. persica and included additional bacterial strains such as E. faecalis, E. coli, P. mirabilis, and K. pneumonia for antibacterial activity, but the fruits extract of the collected plant was ineffective against all these strains.
Previously, the roots and stem of S. persica used as toothbrushes showed antimicrobial, antiplaque, aphrodisiac, analgesic, alexiteric, antipyretic, anti-inflammatory, and astringent effects, which help in cleaning the teeth and decreasing tooth decay [40,41]. In another study, the bioactive compounds extracted from S. persica roots and stems showed wide scope of pharmacological activities including antimicrobial, antioxidant, antiulcer, anticonvulsant, sedative, antiplasmodia, antimalarial, analgesic, antitumor, scurvy, gonorrhea, boils, antiosteoporosis, enzyme inhibitory activity, anti-inflammatory, hypoglycemic,  hypolipidemic, anticonvulsion, diuretic, and bitter stomachic activities, in addition to antiurolithiatic properties [31-36, 42, 43]. Benzyl isothiocyanate and benzyl nitrile were major active compounds purified from S. persica with powerful antibacterial and antifungal activity [27,44,45]. Benzyl isothiocyanate is extracted from the mouth when chewing sticks were used more than once for 10 minutes. e essential oils present in the sticks showed positive effect on gingival fibroblasts and resistance towards oral keratinocytes [46]. N-Benzylbenzamide isolated from the roots of S. persica exhibited a considerable inhibitory activity against the collagen-induced platelet aggregation in human and a considerable antiseptic against E. coli [47]. S. persica stem was also reported to act as a protectant against the pentylenetetrazol-induced convulsions in humans by extending the sleeping period and decreasing the induction time induced by sodium pentobarbital [36]. Moreover, Persicaline, a sulphur-containing imidazoline alkaloid, and three furan derivatives with hydroxyl groups were identified from the root of S. persica, which showed promising antioxidant activity as compared to ascorbic acid [48,49]. e prevalence of dental caries was reported to be remarkably low in the users of S. persica stem due to the presence of strong antimicrobial thiocyanate component along with other substances such as potassium chloride, sodium chloride, tannins, and saponins [50]. e ethanol extract of S. persica stem exhibited substantial decrease in the cariogenic bacterial growth and dental decay [50]. Additionally, data obtained from controlled clinical trials

Conclusion
Forty-one phytochemicals were identified from the ethanol and petroleum ether extracts of the fruits of S. persica grown in the southern region of Saudi Arabia using GC-MS. e ethanol extract showed good cytotoxic properties against ovary and colon cancer cells and lesser cytotoxicity against the MRC5 normal cells. Further studies are required to establish the cytotoxic effects of the extract and identification of components acting as cytotoxic. e fruit extract of S. persica has also demonstrated potential antimicrobial activity against S. mutans which was resistant to the standard drug ampicillin. is study suggested a new potential ethnopharmacological use of S. persica fruits along with the roots and stem of the plant in dental care and for other related ailments.

Data Availability
All the data related to the study are available through the corresponding author and can be provided upon request.

Conflicts of Interest
e authors declare no conflicts of interest regarding the publication of this paper.