A new diterpenoid from Salvia santolinifolia boiss

Abstract Phytochemical study of the Salvia santolinifolia root extract resulted in the isolation of one new quinone diterpenoid, aegyptinone E (1) as well as two known ones, aegyptinone A (2) and aegyptinone D (3). All the isolated compounds were reported for the first time from S. santolinifolia. Spectroscopic analyses including 1 D and 2 D NMR and HRESIMS were used to determine the chemical structures. Aegyptinone A (2) showed moderate antibacterial activity against Staphylococcus aureus, Staphylococcus epidermis, and Bacillus subtilis with MIC of 25 μg/mL. GRAPHICAL ABSTRACT


Introduction
Plants have long been a great source of natural compounds for sustaining human health, particularly in the past decade, when more rigorous investigations for natural medicines have been conducted (Cragg and Newman 2013).Salvia is one of the biggest genus in the Lamiaceae family, with over 900 species worldwide, and 58 found in Iran (Askari et al. 2021).Around 40 Salvia species have been utilized to treat coronary heart disease, and more than 700 pharmaceutical businesses in China create Danshen formulations (Meim et al. 2019).Diuretic (Peng et al. 2010;Bhadoriya et al. 2011), anti-HIV (Yamasaki et al. 1998;Zhang et al. 2008), antibacterial (Shakeri et al. 2018), antifungal , anti-depressant (Imanshahidi and Hosseinzadeh 2006), anti-oxidant (Kamatou et al. 2005;Wang 2010), antipyretic (Hern andez-P erez et al. 1995), antitumor, antispasmodic (Khan et al. 2011), cardiovascular (Wang et al. 2017), anticancer, and anti-inflammatory properties were found in plants belonging to the genus Salvia.Steroids, sesterterpenes, sesquiterpenoids, diterpenoids, flavonoids, triterpenesand, and caffeoyl related compounds are among the major non-volatile secondary metabolites biosynthesized by the members of the genus Salvia (Rajabi et al. 2014;Eghbaliferiz et al. 2018).Abietane and rearranged abietane diterpenoids have been reported as the largest class of compounds in the Salvia spp.It is worth mentioning that among 545 known Salvia diterpenoids, 365 of them are abietane diterpenoids.S. santolinifolia Boiss. is grown in sandy and rocky terrain in Pakistan, Afghanistan, and Iran which is historically consumed to cure hemorrhoids, hypercholesterolemia (seeds are rich in mucilage), inflammation (aerial parts), and diarrhea (Sefidkon and Khajavi 1999;Zaheer et al. 2008).Previously, three new 20,24-epoxydammarane triterpenes (santolins A-C), two novel amyrin type triterpenes (slavins A and B) (Sajid et al. 2006), a new ursanetype triterpene (santolinoic acid) (Zahher et al. 2007), along with few other compounds, have been isolated and identified from this species (Sajid et al. 2011;Jan et al. 2018).Due to the scarce phytochemical investigation on S. santolinifolia, as well as the ethnopharmacologic and chemotaxonomic relevance, its chemical profile is the aim of this study.Consequently, the isolation and characterization of one novel diterpenoid, aegyptinone E (1) along with two previously reported diterpenoids, aegyptinone A (2) and aegyptinone D (3), were described in this paper (Sabri et al. 1989;Aboul-Ela 2006).

Results and discussion
In this study, from the CH 2 Cl 2 extract of S. santolinifolia roots, one new diterpenoid, aegyptinone E (1) as well as two known ones, aegyptinone A (2) and aegyptinone D (3) were obtained (Figure 1).
Compound 1 was obtained as a red crystal.The HRESIMS gave an  The structure of 1 was similar to aegyptinone A which was isolated by Sabri et al. (1989) from S. aegyptiaca via comparison of their NMR spectroscopic.The presence of a furan ring instead of dihydrofuran ring, was the main difference in the structure of the new compound 1.Finally, the structure of compound 1 was elucidated and named aegyptinone E. Table S1 provide the full 1 H and 13 C NMR assignments for all compounds.Antimicrobial properties of the isolated compounds have shown in Table S2.The results indicated that compound 2 exhibited moderate antimicrobial activity.However, compounds 1 and 3 were demonstrated to have no antimicrobial activity (>100 lg/ mL).Moujir et al. (1993) reported that the quinone structure in abietane diterpenoids is responsible for the antibacterial action against Gram-positive bacteria.Yang et al. (2001) and R o_ zalski et al. ( 2007) also imply that the presence of the quinone group in abietane diterpenoids influences their antibacterial activity.They suggested that the molecule with p-naphthoquinone group is more effective than the one with isomeric o-naphthoquinone.For example, the antibacterial activity of salvipisone, which contains a p-naphthoquinone function at C-11 and C-14 and a hydroxyl group at C-12, is more effective than aethiopinone, which possesses an o-naphthoquinone group at C-11 and C-12 positions.Also, Honda et al. (1988) reported that the presence of a dihydrofuran ring in o-quinone diterpenoids is necessary for having the antibacterial activity.Our present observations are consistent with the above findings.Compound 2 with o-quinone group displayed better activity than the other two compounds.Although most of the previous phytochemical studies of S. santolinifolia have been performed on the essential oil, few papers have also reported its aerial part non-volatile secondary metabolites (Sefidkon and Khajavi 1999;Zaheer et al. 2008;Bahadori et al. 2016); however, no investigations on the roots have been published.Apart from diverse chemical classes of compounds which previously isolated from S. santolinifolia (Sajid et al. 2008;2011;Jan et al. 2018), two novel amyrin type triterpenes slavins A and B (Sajid et al. 2006), santolinoic acid, as a new ursane-type triterpene (Zahher et al. 2007), and three new 20,24-epoxydammarane triterpenes, santolins A-C (Zaheer et al. 2008) have been identified.Consequently, the present study is the first report on the isolation and characterization of diterpenoids from this species, nevertheless these bioactive compounds are considered as the main secondary metabolites in the Salvia genus (Cuvelier et al. 1994;Topcu and G€ oren 2007;Jassbi et al. 2016).

General procedures
All solvents used were analytical grade and purchased from Dr. Mojallali Industrial Chemical Complex Company.A KNAUER semi-preparative HPLC coupled to a DAD (diode array detector) using an Onyx TM Monolithic C18 100 Â 10 mm column (Phenomenex, Torrance, CA, USA) was implemented for the isolation procedure.The structure elucidation of the pure compounds was carried out by Bruker Avance II 600 spectrometer operating at 600.19 MHz ( 1 H) and 150.92 MHz ( 13 C) at 300 K. HRESIMS data were acquired on an Orbitrap Exploris TM 120 Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) operating in positive ion mode.The samples were dissolved in CDCl 3 for the spectroscopic analysis, and MestReNova v6.0.2e-5475 software was applied to perform the data processing.Mueller Hinton broth (MHB) obtained from Himedia (Mumbai, India).Tween 80 and 2,3,5-triphenyltetrazolium chloride (TTC) were provided from Merck (Darmstadt, Germany).

Plant materials
Salvia santolinifolia was collected during the flowering stage from Jiroft, the Kerman province, Iran, in April 2020.The plant was identified by Dr. Bibak and a voucher specimen (UJH802) was deposited in the herbarium of the Department of Plant Biology, University of Jiroft, Jiroft, Iran.

Extraction and isolation
The powdered roots of S. santolinifolia (900 g) were extracted with 90% MeOH, and the solvent was evaporated under reduced pressure using a rotary evaporator (Heidolph Instruments, Germany) to give a residue (7.5 g) and then suspended in distilled H 2 O and partitioned with dichloromethane (CH 2 Cl 2 ).The CH 2 Cl 2 extract (2.4 g) was subjected to column chromatography over silica gel (230-400 mesh) eluting with a gradient system of petroleum ether (PE), ethyl acetate (EtOAc), and MeOH, starting with 100% PE, then EtOAc (20 to 80%) in PE, afterwards increasing ratio of MeOH in EtOAc (50 to 100%).The obtained fractions were submitted to reversed-phase semipreparative HPLC using an Onyx TM Monolithic C18 100 Â 10 mm column (Phenomenex, Torrance, CA, USA) applying a gradient elution of solvent A (methanol) in solvent B (water) (0.1% TFA) at flow rate 5 mL/min.The gradient was the following: from 20 to 100% methanol in 9 min (Linear gradient mode), 100% methanol in 4 min (isocratic segment); from 100 to 20% methanol in 2 min and reequilibration at initial condition for 2 min.

Assessment of antibacterial activities
The antibacterial activity of the isolated compounds were evaluated via broth microdilution method, where minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were measured.The bacterial strains (Staphylococcus aureus (PTCC:1112), Staphylococcus epidermis (PTCC:1114), Bacillus subtilis (PTCC:1023), Escherichia coli (PTCC:1330), Pseudomonas aeruginosa (PTCC:1074)) were provided from Iranian Research Organization for Science and Technology (IROST).The microorganisms were cultivated on Mueller Hinton agar (MHA) at 37 C overnight.The insemination was posed to 10 6 CFU/mL sterile normal saline (0.9%).The compounds were dissolved in dimethyl sulfoxide (DMSO) and Mueller-Hinton broth (MHB) medium.Concentrations of each compounds were provided consecutively using two-fold dilution method (100-12.5 mg/mL).Thereafter, 180 mL of each concentration as well as 20 mL of bacterial suspensions (10 6 CFU/mL) were seeded in a 96-well culture plate, whereas MHB was added to a separate well as the negative control to examine the media sterility, cloxacillin and tobramycin were used as standard for Gram-positive and Gram-negative bacteria, respectively.The plates were incubated overnight at 37 C and the bacterial growth in the wells were evaluated by adding the colorimetric indicator 2,3,5-triphenyl tetrazolium chloride (TTC) (20 mL of the 5 mg/mL stock solution).The plates were then incubated at 37 C for 2 h.For each microorganism, the first concentration with no color change was accounted as MIC value.Culturing the wells comprising isolated compounds with concentrations higher or equal to MIC on the plates' surface comprising MHA medium were used to determine MBC.

Conclusion and perspectives
Chemical investigation on the CH 2 Cl 2 root extract of Salvia santolinifolia led to the isolation of three diterpenoids (1-3), identified for the first time from the genus.Among them, aegyptinone E (1) was characterized as a new diterpenoid.Compound 2 displayed moderate antimicrobial activity against S. aureus, S. epidermis, and B. subtilis with MIC values of 25 lg/mL.Considering the scarce studies of the species, further phytochemical and pharmacological analysis may lead to presenting promising bioactive compounds, potential candidates for further natural-based drug discovery procedures.