Metabolic profiling and biological activities of the aerial parts of Micromeria imbricata Forssk. growing in Saudi Arabia

The hydroalcoholic extract (MIT) of Micromeria imbricata (Forssk.) growing in Saudi Arabia in addition to the chloroform (MIC) and n-butanol (MIB) fractions were investigated for the first time using UPLC-ESI-MS/MS. The analysis revealed the tentative identification of fifty-eight compounds including three organic acids, twenty-five phenolic compounds, three coumarins, two anthocyanins, twenty-one flavonoids, three terpenes, and one miscellaneous. Moreover, the therapeutic potential of M. imbricata (MIT) and its fractions (MIC and MIB) were determined by in vitro evaluation of their cytotoxic, antioxidant, and anti-obesity characteristics. The MIT extract showed the highest phenolic (125.23 ± 0.87 mg gallic acid equivalent/100 gm extract) and flavonoid (112.24 ± 2.45 mg quercetin equivalent/100 gm extract) contents followed by n-butanol and chloroform fractions. The MIT extract revealed a potent cytotoxic activity against HepG-2 (Hepatocellular carcinoma) and MCF-7 (Breast carcinoma) with IC50 28.5 ± 2.0 and 35.2 ± 1.2 µg/mL, respectively. Additionally, the tested hydroalcoholic extract exhibited a significant DPPH scavenging activity with SC50 28.4 ± 1.2 µg/mL and a remarkable lipase inhibitory activity with IC50 54.2 ± 1.2 µg/mL. In conclusion, the current study presents the first insights into the phytochemical constituents and pharmacological properties of M. imbricata extract and its chloroform and n-butanol fractions. The results revealed that M. imbricata hydroalcoholic extract might be a prolific source of bioactive constituents with potent antioxidant, cytotoxic and anti-obesity potential. It might be a natural alternative therapy and nutritional strategy for obesity treatment.


Introduction
Genus Micromeria Benth. comprises 130 species belonging to Lamiaceae (mint) family. Plants of this genus are perennial herbs, shrubs and subshrubs (Brahmi et al., 2017). In traditional folk medicines, Micromeria species are used in different ailments like skin infections, wounds, colds, headache, fever, asthma, heart, respiratory and digestive systems disorders. They also have many biological actions including anti-inflammatory, antimicrobial, antifungal, antiseptic, antispasmodic, antioxidant and antitumor activities (Brahmi et al., 2017;Vladimir-Knežević et al., 2011;Azab, 2016). Essential oils, flavonoids and triterpenes are the main phytochemical constituents reported from the genus Micromeria (-Vladimir- Knežević et al., 2011;Slavkovska et al., 2005). Most of the studies on plants of this genus reported their volatile constituents and their antimicrobial properties (Marinković et al., 2002;Stojanović et al., 2006;Šavikin et al., 2010). Genus Micromeria is widespread in South Africa, West Asia, the Mediterranean region and the Canary Islands. Moreover, there is also a high similarity between the plants of this genus. M. imbricata is similar to M. fruticosa and M. graeca but they have elliptic not ovate leaves, longer calyces, and narrower calyx lobes (Ryding, 2007). To date, there is no report about the phytoconstituents and the biological activities of M. imbricata growing in Saudi Arabia. Therefore, the chemical composition of M. imbricata hydroalcoholic extract as well as the chloroform and n-butanol fractions were analyzed for the first time using UPLC-ESI-MS/MS to identify its chemical constituents.

Plant material
The aerial parts of M. imbricata (Forssk.) were collected from Abha, fertile Asir mountains of South-Western Saudi Arabia (18 o 13ʹ1 00 N 42 o 30ʹ19 00 E), in August 2008. Plant material was identified by Dr. Mohamed Yousef, professor of Pharmacognosy from the Pharmacognosy Department, College of Pharmacy of King Saud University, and a voucher specimen was deposited in the herbarium of the same department under registry number (MI-2543).

Preparation of M. Imbricata crude hydroalcoholic extract
Ethanol 95% was used for the extraction of the air-dried powdered aerial parts of M. imbricata (1.5 kg). The dried hydroalcoholic extract was defatted with petroleum ether (4 Â 1 L). The aqueous defatted hydroalcoholic extract (120 g) was fractionated using chloroform (CHCl 3 ) and n-butanol (n-BuOH), to give 30.1 g and 32.5 g of chloroform and n-butanol fractions respectively.

Quantitative measurement of the total phenolic and flavonoid contents of M. imbricata extract/ fractions
Total phenolic content was determined by the Folin-Ciocalteau method (Chen et al., 2014) and expressed in milligram gallic acid equivalents per gram of dried extract/fractions (mg GAE/g extract/fractions GAE). Total flavonoid content was determined using the aluminum chloride-potassium acetate colorimetric assay with quercetin as standard (Hossain and Rahman, 2011), and the total flavonoid content was expressed as mg of quercetin equivalents per gram of extract/fractions (mg QE/g extract or fractions).

UPLC-ESI-MS/MS instrument and separation technique
The hydroalcoholic extract of M. imbricata and its chloroform and n-butanol fractions were prepared as a solution of 100 lg/mL using HPLC grade methanol, filtered using a membrane disc filter (0.2 lm) then subjected to LC-ESI-MS analysis in negative and positive ion acquisition modes using a (XEVO TQD triple quadruple instrument) mass spectrometer. The UPLC system was a Waters Corporation, Milford, MA01757 U.S.A. The reversed-phase separations were performed on a (ACQUITY UPLC -BEH C 18 1.7 mm -2.1 Â 50 mm Column. (50 mm x 1.2 mm [inner diameter] and 1.7 mm particle size) and at 0.2 mL\min flow rate. The used analysis parameters and the gradient program were previously reported by (Hassan et al., 2019). The identification of the phytochemical constituents was done by their fragmentation patterns and their ESI-QqQLIT-MS/MS spectra. Peaks and spectra were processed using the Maslynx 4.1 software and tentatively identified by comparing their retention time (R t ), mass spectrum with the published data and Library search, such as ChemSpider (http://www.Chemspider.com), MassBank (http://www.massbank.eu), METLIN (https://metlin.scripps.edu/) and FooDB (http://www.Foodb.ca).

Antioxidant assay
The antioxidant activity of M. imbricata hydroalcoholic extract and its fractions was determined at the Regional Center for Mycology and Biotechnology (RCMB) at Al-Azhar University using the free radical 2,2-diphenyl-picrylhydrazyl (DPPH) scavenging assay (Al Khateeb et al., 2017).

Cytotoxicity assay
The cytotoxic effects of the hydroalcoholic extract of M. imbricata in addition to its chloroform and n-butanol fractions against HepG-2 and MCF-7 cells were carried out using the MTT cell viability assay (Ramos-Silva et al., 2017). HepG-2 (Human hepatocarcinoma) and MCF-7 (human breast carcinoma) cells were obtained from VACSERA Tissue Culture Unit and maintained in DMEM supplemented with 10% FBS and 100 lg/mL penicillin-streptomycinamphotericin B solutions.
2.7. In vitro anti-obesity activity using pancreatic lipase inhibitory assay The lipase inhibition activity of plant extract was determined as the method proposed by (Kim et al., 2010). Briefly, the porcine pancreatic lipase activity was measured using p-nitrophenyl butyrate (NPB) as a substrate. Lipase solution (100 mg/mL) was prepared in a 0.1 mM potassium phosphate buffer (pH 6.0). To determine the lipase inhibitory activity, samples with different concentrations (1000 to 7.81 lg/mL) were preincubated with 100 mg/mL of lipase for 10 min at 37°C. The reaction was then started by adding 0.1 mL NPB substrate after incubation at 37°C for 15. The amount of p-nitrophenol released in the reaction was measured using Multiplate Reader. Each experiment was performed in triplicates. The results were expressed as percentage inhibition, which was calculated using the formula; Inhibitory activity (%) = (1-As/Ac) Â 100, where is the absorbance in the presence of test substance and Ac is the absorbance of control. The IC 50 value is defined as the concentration of a-glucosidase inhibitor to inhibit 50% of its activity under the assay conditions.

Total phenolic and flavonoid contents
As shown in Table 1, the hydroalcoholic extract and n-butanol fraction of M. imbricata possessed the highest concentration of phenolic and flavonoid contents (125.23 ± 0.87 and 112.24 ± 2.45 (mg GAE/g extract) and 89.25 ± 1.75 and 81.15 ± 0.08 (mg QE/g extract) respectively. On the other hand, the chloroform fraction had the lowest concentration 20.43 ± 0.89 (mg GAE/g extract) and 6.35 ± 0.52 (mg QE/g extract).

Tentative identification of polyphenols and other constituents by UPLC-ESI-MS/MS
In this study, crude hydroalcoholic extract, chloroform and nbutanol fractions of M. imbricata were analyzed by UPLC-ESI-MS/ MS, operating in both positive and negative ionization modes. The identification of abundant compounds of these extracts was based on mass fragmentation patterns and the standards data reported in the literature and database. Fifty-eight compounds were tentatively identified in the three samples of M. imbricata including three organic acids, twenty-five phenolic compounds, three coumarins, two anthocyanins, twenty-one flavonoids, three terpenes, and one miscellaneous. Table 2 indicates all the identified compounds, their retention times, experimental m/z in positive and negative ionization mode and MS/MS fragments.

Antioxidant activity
In the present study, the antioxidant activity of the hydroalcoholic extract of M. imbricata (MIT) and its chloroform (MIC) and n-butanol (MIB) fractions were investigated, compared to ascorbic acid (a standard antioxidant) and summarized in Fig. 1 A and B. The MIT extract has the highest activity as indicated by its high percentage of DPPH scavenging (80%) at 320 lg/mL and low SC 50 (concentration of sample required to scavenge 50% of DPPH radicals) 28.4 ± 1.2 mg/mL compared to MIC and MIB with SC 50 429.3 ± 1.3 and 466.3 ± 0.8 mg/mL, respectively, with ascorbic acid SC 50 14.2 ± 0.5 lg/mL as standard..

Cytotoxicity
The cytotoxic activity M. imbricata extract and its fractions against HepG-2 (hepatocellular carcinoma) and MCF-7 (breast carcinoma) cell lines using MTT assay and cisplatin as a positive standard were studied. As shown in ( Fig. 1 C & D), all the tested samples showed dose-dependent cytotoxicity against HepG-2 and MCF-7 cells. However, the MIT extract showed potent cytotoxic activity against HepG-2 and MCF-7 cells with IC 50 28.5 ± 2.0 and 35.2 ± 1.2 mg/mL compared to cisplatin with IC 50 3.67 ± 1.70 and 5.71 ± 1.30 lg/mL, respectively.

The anti-obesity activity
The inhibition of lipase activity of the tested plant extracts is described in Fig. 1 E & F. The hydroalcoholic extract of M. imbricata exhibited higher inhibitory activity (IC 50 54.2 ± 1.2 mg/mL) than the chloroform and n-butanol fractions with IC 50 values 223.2 ± 1.7 and 120.7 ± 1.3 mg/mL, respectively which was compared with the positive control, orlistat (IC 50 23.8 ± 0.7 mg/mL).

Total phenolic and flavonoid contents
Most biological activities of plant extracts are associated with its phenolic and flavonoid contents. These secondary metabolites are widely distributed in several plant families. They play a crucial role in the management of various human disorders (Abbaszadeh et al., 2019). Total phenolic and flavonoid content values were observably high in MIT compared to MIB. The chloroform fraction had the lowest concentration. These results were in agreement with previous studies performed on other Micromeira species (Vladimir-Knežević et al., 2011;Abu-Reidah et al., 2019).

Tentative identification of polyphenols and other constituents by UPLC-ESI-MS/MS
Fifty-eight compounds were tentatively identified in the three samples of M. imbricata. These compounds comprised phenolic acids and their derivatives, flavonoids, terpenes and coumarins.

Organic acids
Succinic acid (16) and its isomers (46 and 55) were detected in MS spectra with a deprotonated molecular ion at m/z 117. The loss of water molecule was confirmed by an intense fragment at m/z 99 (Al Kadhi et al., 2017)
According to Lambert et al., 2015, precursor ion of tyrosol (27) and its isomers (36,50,52,57 and 58) at m/z 121 refer to [MÀH 2 -O + H] + , and they were characterized by two fragments at m/z 93 for the phenol group and m/z 77 for an aromatic ring.
The isomers 8, 22 and 38 showed the characteristic deprotonated molecular ion at m/z 359. These compounds were identified as rosmarinic acid isomers, based on their data obtained from MS 2 fragment ions at m/z 179 corresponding to caffeic acid and m/z 197 correlated to 2-hydroxy derivative of hydrocaffeic acid (Hossain et al., 2010). Different species of Micromeria and other Lamiaceae species were reported to contain rosmarinic acid, which is an ester of caffeic acid (2019 Abu

Coumarin derivatives:
Simple coumarins (1, 12 and 20) were found in the hydroalcoholic extract of M. imbricata and identified as umbelliferone. They showed a protonated molecular ion peak at m/z 163 and MS 2 fragments at m/z 135 and 107 as previously reported (Abu-Reidah et al., 2019;Tine et al., 2017).

Anthocyanins:
Two anthocyanin derivatives have been detected in hydroalcoholic extract of M. imbricata. Compound 10 was tentatively identified as malvidin derivative as it exhibits a molecular ion at m/z 621. In the MS 2 spectra, it showed a fragment ion at m/z 331, indicating the malvidin structure (Stein-Chisholm et al., 2017). Compound 13 was tentatively identified as peonidin derivative (peonidin-3-(pcoumaroylglucoside). It was characterized by a molecular ion peak at m/z 609 with a product ion at m/     Cirsilineol (15) is a flavonoid aglycone with a pseudomolecular ion peak at m/z 345 and daughter ions at m/z 329 and m/z 315 in the ESI-MS/MS analysis (2019 Abu-Reidah et al.). Compounds 33 and 37 exhibited a protonated molecular ion at m/z 345 and a deprotonated molecular ion at m/z 343, respectively. They were tentatively assigned as dihydroxy-trimethoxyflavone as previously reported (Abu-Reidah et al., 2019).
The aglycone apigenin has been detected in both positive and negative ionization modes. Compounds 29 and 35 exhibited a protonated molecular ion at m/z 271 while compound 48 showed a deprotonated molecular ion at m/z 269, which were tentatively assigned as apigenin isomers as previously reported (Abu-Reidah et al., 2019). Eriodictyol aglycone has been suggested for compound 56, based on the MS data ([MÀH]at m/z 287) and the MS 2 product ion at m/z 151 (Farag et al., 2016). Apigenin-7-O-glucuronide (3) and its isomer ( ]which was related to protonated acacetin. Therefore, they were identified as acacetin-7-O-rutinoside and its regioisomers (Abu-Reidah et al., 2019). Compound 53 exhibited a deprotonated molecular ion at m/z 517 and characteristic fragment ions at m/z 339 attributed to 8-prenylnaringenin that was detected in the MS 2 spectrum. Accordingly, it was identified as 8prenylnaringenin derivative (Quifer-Rada et al., 2015) 4.2.6. Terpenoid derivatives: A total of three terpenoid derivatives have been detected from the hydroalcoholic extract and chloroform fraction of M. imbricata.

Miscellaneous compounds
Compound 54 was suggested to be a saccharide derivative known as tuliposide B. The protonated pseudo molecular ion was observed at m/z 295 and MS 2 ions at m/z 591 (2 M + H) + and 143. Tuliposide B was previously identified in M. fruticosa (Abu-Reidah et al., 2019).

Antioxidant activity
Oxidative stress is the result of the inconsistency between free radicals and reactive metabolites production (oxidants) and their elimination by antioxidants. This inconsistency leads to the destruction of cells and organs with prospective effects on the whole organism. Antioxidants can decrease, delay or even prevent the oxidative damage through scavenging of free radicals. These reactive oxygen species and free radicals play a crucial role in the pathogenesis of different diseases such as hypertension, atherosclerosis, diabetes, cancer, and inflammatory diseases in addition to aging processes (Sharma et al., 2018;Vladimir-Kneže vić et al., 2011). Polyphenols are one of the most important natural antioxidants which are widely distributed in various plant families including Lamiaceae. This large family includes many species which are important sources of natural antioxidants (Vladimir-Kne žević et al., 2011). The antioxidant activity of the hydroalcoholic extract of M. imbricata (MIT) and its chloroform (MIC) and nbutanol (MIB) fractions showed a concentration-dependent antioxidant activity as demonstrated by an increase in their DPPH radical scavenging activity. There is a proportional correlation between radical scavenging (antioxidant) activity and the total phenolic contents (Gardner et al., 2000). The MIT extract has the highest activity compared to MIC and MIB. These results are in accordance with the previous reports about the antioxidant properties of various Micromeria species (Micromeria croatica, M. juliana, and M. thymifolia) (Brahmi et al., 2017;Vladimir-Knežević et al., 2011). The MIT antioxidant activity could be attributed to its contents of polyphenolic compounds such as tyrosol (Al-Yousef et al., 2020;Di Benedetto et al., 2007) salvianolic acid B (Zheng et al., 2020) in addition to flavonoid contents such as eupatorin (Shin et al., 2020) and acacetin-7-O-rutinoside (linarin) (Xie et al., 2020). During this work, tyrosol, salvianolic acid B, eupatorin and linarin were detected in LC-MS analysis of hydroalcoholic extract.

Cytotoxicity
The risk of neoplasia is increasing worldwide with higher mortality rates every year. Breast cancer is the leading cause of cancer death among females. It is responsible for 15-25% of all cancer cases and deaths. Liver cancer is much more common in males. Furthermore, it is the second major cause of cancer death in men all over the world, especially in underdeveloped countries (Torre et al., 2015). Recently, plant-derived compounds possess an attractive area for many researchers attention as a promising source of many cancer lead drugs (Al-Abbasi et al., 2016;Solowey et al., 2014;Wannes et al., 2017).
M. imbricata extract and its fractions showed dose-dependent cytotoxicity against HepG-2 and MCF-7 cells in comparison to cisplatin. The MIT extract showed potent cytotoxic activity against HepG-2 and MCF-7 cells. This activity may be attributed to the presence of high flavonoid content as apigenin (Abbaszadeh et al., 2019), apigenin-7-O-glucuronide (Mazumder et al., 2020), 8-prenyl naringenin (Bailly, 2020) and linarin (Xie et al., 2020). These compounds were the predominant flavonoids showing the highest content. In addition to the high presence of corosolic acid as a triterpenoid compound (Mazumder et al., 2020). The MIC fraction had weak cytotoxicity against HepG-2 and MCF-7 cells. Their broad range effects might be a result of multiple mechanisms such as modification or interaction with several enzymes, proteins and apoptosis induction (Abdal Dayem et al., 2016;Abbaszadeh et al., 2019).

The anti-obesity activity
The management of obesity by natural agents is not thoroughly investigated and might be a significant substituent for producing harmless and efficient anti-obesity drugs (Nderitu et al., 2017). To the best of our knowledge, there is no report on the anti-obesity activity of M. imbricata, so it was deemed of interest to investigate the anti-obesity activity of the M. imbricata fractions using in vitro pancreatic lipase inhibitory assay. The hydroalcoholic extract of M. imbricata showed higher inhibitory activity than the chloroform and n-butanol fractions. This significant activity of MIT could be assigned to the existence of corosolic acid (Stohs et al., 2012), 8prenyl naringenin (Paraiso et al., 2020), and eupatorin (Shin et al., 2020). 8-Prenyl naringenin diminishes the gain of body weight and enhances obesity-related metabolic parameters. (Paraiso et al., 2020).
Ultimately, the biological activity results indicated that the MIT extract of M. imbricata had the highest antioxidant, cytotoxic and lipase inhibitory activities compared to its chloroform and nbutanol fractions. This significant activity of MIT could be ascribed to the presence of polyphenolics like tyrosol, umbelliferone, salvianolic, rosmarinic, and corosolic acids in addition to the presence of apigenin glucuronide, acacetin-7-O-rutinoside and other flavonoids which have various therapeutic properties. It has been reported that flavonoids and other polyphenols have vital activities for human health like anticancer, antioxidant, anti-inflammatory effects in addition to their therapeutic actions in managing several obesity complications (Sekhon-Loodu and Rupasinghe, 2019; Rasheed and Azeez, 2019;Stohs et al., 2012;Di Benedetto et al., 2007).

Conclusion
The current study presents the first report about the phytochemical components and pharmacological activities of M. imbricata Forssk. growing in Saudi Arabia. The hydroalcoholic extract showed the highest antioxidant, cytotoxic and anti-obesity activities compared to the chloroform and n-butanol fractions. These activities of MIT extract could be attributed to the presence of a high percentage of flavonoids and other polyphenols. In summary, these findings revealed that the M. imbricata hydroalcoholic extract possessed potential antioxidant, cytotoxic and anti-obesity properties. Also, it brought to light a new lead to the limited therapeutic options of breast and liver cancers. Moreover, this work suggests that M. imbricata hydroalcoholic extract may be a promising candidate as anticancer, antioxidant and anti-obesity drugs. Further studies are warranted to isolate and identify the bioactive secondary metabolites from this extract using various spectroscopic and spectrometric techniques for future in vivo investigation.