New Diterpenoids from the Aerial Parts of Salvia reuterana

authors:

avatar Mahdi Moridi Farimani a , * , avatar Mansour Miran b , avatar Samad Nejad Ebrahimi a

Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran, Iran.
Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
Iranian Journal of Pharmaceutical Research : IJPR: Vol.18, issue 1; 406-411
article type: Original Article
received: June , 2017
accepted: June , 2018
DOI: https://doi.org/10.22037/ijpr.2019.2314

how to cite: Moridi Farimani M, Miran M, Nejad Ebrahimi S. New Diterpenoids from the Aerial Parts of Salvia reuterana. Iran J Pharm Res. 2019;18(1):e126118. https://doi.org/10.22037/ijpr.2019.2314.

Abstract

The genus Salvia is a valuable origin of structurally diverse terpenoids. In a project directed at structurally interesting bioactive metabolites from Iranian Salvia species, we studied Salvia reuterana. Two new labdane diterpene, 6β, 14α-dihydroxy-15-acetoxysclareol (1), and 14α, 15- dihydroxy sclareol (2), were isolated from the aerial part of the plant. Their structures were established mainly by 1D and 2D NMR spectroscopic techniques, including 1H-1H COSY, HSQC and HMBC methods and HR-ESI-TOFMS spectral data. Compound 1 and 2 were tested for their inhibitory activity toward HeLa and MCF-7 cell lines. Our results showed that S. reuterana is a rich source of labdane diterpenoids. These compounds are rather rare in Salvia species, although they are frequently found in other genera of the Lamiaceae. S. reuterana is a new source of these diterpenoids.

Introduction

Salvia, consisting of about 1000 species, is the largest genus in the family Lamiaceae and widely distributed in various regions of the world, Namely, the Mediterranean area, south Africa, central and south America, and southeast Asia (1). Plant of the genus Salvia have attracted much attention owing a variety of medicinal properties and biological activities, such as antibacterial, antioxidant, to antitumor, anti-diabetic and anti-Inflammatory activities (1-3). The Iranian flora comprises 61Salvia species, 17of which are endemic (4). Some Iranian Salvia species have been investigated from a phytochemical viewpoint, and antibacterial, antiprotozoal, and anticancer compounds have been reported (5-12.). Salvia reuterana Boiss. is an endemic species which grows in the highlands of central Iran (13). Its common name in Persian is “Mariam Goli Esfahani” (14), and the aerial parts of the plant are traditionally used as sedative and anxiolytic herbal medicine. In addition, the antibacterial, antioxidant, free radical scavenging and anti-anxiety properties of this herb have been proved in recent studies (15–19). In a previous work, we have reported the presence of six labdane diterpenoids from this plant (20). Further examination of the n-hexane extract of S. reuterana led to the isolation of two new other labdane diterpenoids (1, 2).

Experimental

General

HR-ESI-MS was carried out on a Bruker 147 microTOF–ESIMS system. NMR spectra were recorded on Bruker AVANCE III-500 spectrometer operating at 500.13 MHz for 1H NMR and 125.77 MHz for 13C NMR with TMS as an internal standard. Silica gel (70-230 and 230-400mesh) used for column chromatography and silica gel F254 (20×20 cm) for TLC, were both supplied by the Merck company.

Plant material

The aerial parts of Salvia reuterana were collected from the northern hilly areas of Tehran in Juley 2011. A voucher specimen (MPH-1321) has been deposited in the herbarium of Medicinal Plants and Drugs Research Institute (MPH) of Shahid Behshti University, Tehran, Iran.

Extraction and Isolation

The air-dried aerial parts of S. reuterana (3.0 kg) was crushed and extracted with n-hexane (3 × 15 L( by maceration at room temperature. Extract was concentrated in vacuo, to afford 130g of a dark gummy residue. The residue was separated on a silica gel column (230-400 mesh, 850g) with a gradient of n-hexane-EtOAc (100/0 to 0/100) as eluent, followed by increasing concentration of MeOH (up to 25%) in EtOAc. On the basis of TLC analysis, fractions with similar composition were pooled to yield 27 combined fractions.

Fractions 22, 23 and 24 were combined (2g) and subjected to silica gel column chromatoghraphy (70-230 mesh), eluted with CHCl3-Me2CO-MeOH (77:13:10) to give six subfraction (C1-C6). Subfraction C2 was rechromatographed over silica gel (70-230), eluted with CHCl3-MeOH (1:1), to afford three subfraction (C21-C23). Subfraction C21 was recrystallized from CHCl3 to afford compound 1 (3.5mg). Fraction 25 (3g) was applied to a silica gel column chromatography (70-230, mesh), eluted with Me2CO-hexane (2:1) to separate eight subfraction (f1-f8). Subfraction f2 was recrystallized from hexane to afford compound 2 (8mg).

6β, 14α-dihydroxy-15-acetoxysclareol (1): White powder; [α] = -9.6 (c 0.2, CHCl3); for 1H and 13C NMR data, see Table 1; HR-ESI-TOFMS m/z423.2738 [M + Na] +, calcd for 400.2802).

14α, 15-dihydroxysclareol (2): White powder; [α]= -7.1(c 0.2, CHCl3); for 1H and 13C NMR data, see Table 1; HR-ESI-TOFMS m/z365.2712 [M + Na] +, calcd for 342.5107).

Table 1

1H and 13C NMR data of compounds 1 and 2 in CDCl /CD OD (500 MHz for δ ; 125 MHz for δ )

1
2
positionδCδH (J in Hz)δCδH(J in Hz)
1α 1β41.60.85b1.52b40.00.88b1.53b
2α 2β18.31.60b1.33b18.41.53b1.35b
3α 3β44.11.05b1.22b41.81.07b1.28b
4 33.4 32.9
5α56.70.83b56.70.83b
6α 6β67.64.39 brd (2.6)20.61.57b1.18b
7α 7β51.31.55b1.91 dd (13.5, 2.0)43.81.34b1.75 dt (12.5,3.0)
8 73.4 74.5
9α62.11.03b62.3 1.00 t (3.5)
10 38.9 39.2
11α 11β17.91.491.2818.01.46b1.26b
12α 12β40.91.60b1.41b40.81.60b1.38b
13 73.1 73.8
14β75.13.55 dd (8.5, 2.5)77.8 3.39 dd (7.5, 3.5)
15a 15b66.23.96 dd (11.5, 8.5)4.22 dd (11.5, 2.5)62.5 3.50 dd (11.5, 7.5) 3.65 dd (11.5, 3.5)
1622.61.10 s22.0 1.08 s
1724.81.32 s23.6 1.09 s
1832.80.89 s33.4 0.79 s
1924.21.10 s21.5 0.71 s
2016.11.08 s14.9 0.73 s
CH3CO CH3CO19.8171.31.99 s

δ values were extracted from COSY, HSQC and HMBC experiments.

Overlapping signals.

Table 2

Cytotoxic activities of compounds 1 and 2 and against two human tumor cell lines

CompoundHela (IC50 μM)MCF-7 (IC50μM)
1184.61 ± 1.67108.34 ± 2.07
2218.47 ± 2.77164.07 ± 3.09
Paclitaxelb0.0040.033

Data are expressed as mean ± standard deviation (n = 3).

Positive control.

Key H–H COSY and HMBC connectivities of compounds 1 (left) and 2 (right)

Key H–H COSY and HMBC connectivities of compounds 1 (left) and 2 (right)

Figure 1

Cytotoxicity assay

The human epithelioid cervix carcinoma (Hela) and human breast adenocarcinoma (MCF-7) cell lines were purchased from National Cell Bank of Iran (NCBI), Pasteur Institute of Iran (Tehran, Iran), and maintained in DMEM medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin. These cells were kept at 37 °C in a humidified atmosphere containing 5% CO2. Compounds 1-3 were dissolved in DMSO to make a stock of 1mg/mL and further diluted to final concentrations of 10-100 μg/mL with the serum free culture medium.

Cell viability assay

Cell viability was determined using the MTT assay. Briefly, 2.5×104 cells were seeded in 96-well plates at 37 °C with 5% CO2 for overnight incubation and treated with appropriate concentrations of compounds 1-3 for 24 h. The cells were then incubated with a serum-free medium containing MTT at a final concentration of 0.5 mg/mL for 4 h. The dark formazan crystals formed were dissolved in DMSO and the absorbance was measured at 570 nm.

Results and Discussion

Compound 1 was obtained as a white amorphous powder. Its HI-ESI-TOFMS exhibited a molecular ion peak at m/z 423.2738 [M + Na+], agreed with the molecular formula C22H40O6. The molecular formula accounted for three degrees of unsaturation. The 13C NMR spectrum (Table 1), showed 22 carbon resonances which were analyzed from the DEPT-HSQC spectra to consist of six methyls, seven methylens, four methines and four quaternary carbons. Two carbon signals at δC 73.1 and 73.4 indicated the presence of oxygen bearing sp3quaternary carbons. Another oxygenated carbons at δC 75.1 and 67.6 accounted for the carbons carrying two hydroxyl groups with the carbinolic methine protons coming at δH 3.55 (1H, dd, J = 2.5, 8.5 Hz) and δH 4.39 (1H, brd, J= 2.6 Hz), respectively. The signal at δC 66.2 accounted for amethylenic carbon attached to an acetoxy group, with the corresponding methylene protons coming at 3.96, 4.22 each as a doublet of doublet. According to the degree of unsaturation the molecule was bicyclic and appeared to be a labdane diterpenoid (21). The 1H and 13C NMR spectrum displayed features similar to those of 14α-hydroxy-15-acetoxysclareol, isolated previously from this plant by us (20). However, the 13C NMR spectrum of 1 showed the presence of an additional methine at δC 67.6 instead of the methylene group (C-6). Hence, the methylene was replaced by an oxygenated methine. The signals of C-7 (δC51.3) and C-5 (δC56.7) were paramagnetically shifted (Δδ +7.3 and +0.7 ppm, respectively) in comparison to those of 14α-hydroxy-15-acetoxysclareol. Also the resonances of neighboring H-7α (δH 1.55), H-7𝛽 (δH 1.91), and H-5α (δH 0.83) were shifted (Δδ= +0.2, +0.15, and -0.03ppm, respectively). HMBC correlations between H-5α, H-7α with C-6, and between H-6, C-8, and C-10 (Figure 1) confirmed the location of the hydroxyl group. Diagnostic COSY correlations were observed between H-6 and H-7αand H-7β, and between H-6 and H-5. The relative configuration of the hydroxyl group at C-6 was determined as β based on the magnitude of the vicinal coupling constants of the H-6 resonance (δH 4.39, brd, J = 2.6 Hz). NOESY contacts of H-6 with H-5 and H-7ax were observed. The 1H chemical shifts of CH3-17, CH3-19 and CH3-20 (δH 1.32, 1.10 and 1.08, respectively) appeared downfield (δH 0.21, 0.38, and 0.35) relative to those of 14α-hydroxy-15-acetoxysclareol. These differences were in agreement with an axial orientation of the hydroxyl group at C-6. Thus, the structure of 1 was established as 6β,14α-dihydroxy-15-acetoxysclareol.

Compound 2 was obtained as a white powder. The HR-ESI-TOFMS showed a peak at m/z 365.2712 [M+Na] +, in agreement with the elemental formula C20H33O4, which accounted for three degrees of unsaturation. The 1H and 13C NMR data (Table 1) strongly resembled those of 1, indicating that the two compounds were structurally related. Inspection of the 1H and 13C NMR spectra showed the lack of the signals belonges to the acetoxy group in compound 2. Also, the methine signals at67.6 ppm (δH 4.39) was replaced by a methylene group (δC 20.6 and δH 1.57, 1.18) in 2. Thus, compound 2 was established as 14α, 15- dihydroxy sclareol.

Compounds 1 and 2 were evaluated for their in vitro cytotoxic activity against Hela (human epitheloid cervix carcinoma) and MCF-7 (human breast adeno-carcinoma) cell lines. The results of the cytotoxicity studies were indicated in Table 2.

Conclusion

Our results showed that S. reuterana is a rich source of labdane diterpenoids. The most abundant diterpenoids in the genus are abietanes and rearranged abietanes. Labdane diterpenoids are rather rare in Salvia species although they are frequently found in other genera of the Lamiaceae. S. reuterana is a new source of these diterpenoids.

Acknowledgements

References

  • 1.

    Pan ZH, Wang YY, Li MM, Xu G, Peng LY, He J, Zaho Y, Li Y, Zaho QS. Terpenoids from Salvia trijuga. J. Nat. Prod. 2010;73:1146-50. [PubMed ID: 20515057].

  • 2.

    Nickavar B, Rezaee J, Nickavar A. Effect-directed analysis for the antioxidant compound in Salvia verticillata. Iran. J. Pharm. Res. 2016;15:241-6. [PubMed ID: 27610164].

  • 3.

    Firuzi OR, Miri R, Asadollahi M, Eslami S, Jassbi AR. Cytotoxic, Antioxidant and antimicrobial activities and phenolic contents of eleven Salvia species from Iran. Iran. J. Pharm. Res. 2013;12:801-10. [PubMed ID: 24523760].

  • 4.

    Jamzad Z, Assadi M, Maassoumi A, Mozaffarian V. Lamiaceae. Flora of Iran. 76. Tehran: Research Institute of Forest and Rangelands; 2012.

  • 5.

    Abedini A, Roumy V, Mahieux S, Gohari S, Moridi Farimani M, Riviere C, Samaillie J, Sahpaz S, Bailleul F, Neut C, Hennebelle T. Antimicrobial activity of selected Iranian medicinal plants against a broad spectrum of pathogenic and drug multi-resistant micro-organisms. Lett. Appl. Microbiol. 2014;59:412-21. [PubMed ID: 24888993].

  • 6.

    Ebrahimi SN, Moridi Farimani M, Mirzania F, Soltanipoor MA, De Mieri M, Hamburger M. Manoyloxide sesterterpenoids from Salvia mirzayanii. J. Nat. Prod. 2014;77:848-54. [PubMed ID: 24689905].

  • 7.

    Jassbi AR, Zare S, Firuzi OR, Xiao J. Bioactive phytochemicals from shoots and roots of Salvia species. Phytochem. Rev. 2016;15:829-67.

  • 8.

    Moridi Farimani M, Matloubi-Moghaddam F, Esmaeili MA, Amin GA. Lupane triterpenoid and other constituents of Salvia eremophila. Nat. Prod. Res. 2012;26:2045-9. [PubMed ID: 22103318].

  • 9.

    Moridi Farimani M, Mazarei Z. Sesterterpenoids and other constituents from Salvia lachnocalyx Hedge. Fitoterapia. 2014;98:234-40. [PubMed ID: 25128428].

  • 10.

    Moridi Farimani M, Abbas-Mohammadi M, Esmaeili MA, Salehi P, Ebrahimi SN, Sonboli A, Hamburger M. Seco-ursane-type triterpenoids from Salvia urmiensis with apoptosis-inducing activity. Planta Med. 2015;81:1290-5. [PubMed ID: 26252828].

  • 11.

    Moridi Farimani M, Taleghani A, Aliabadi A, Aliahmadi A, Esmaeili MA, Sarvestani NN, Khavasi HR, Smiesko M, Hamburger M, Ebrahimi SN. Labdane diterpenoids from Salvia leriifolia: Absolute configuration, antimicrobial and cytotoxic activities. Planta Med. 2016;82:1279-85. [PubMed ID: 27280932].

  • 12.

    Moridi Farimani M, Abbas-Mohammadi M. Two new polyhydroxylated triterpenoids from Salvia urmiensis and their cytotoxic activity. Nat. Prod. Res. 2016;30:2648-54. [PubMed ID: 30919695].

  • 13.

    Rechinger KH. Flora Iranica. Austria. Graz. 1987;150:403-76.

  • 14.

    Mozaffarian V. A dictionary of Iranian plant names. Tehran: Farhang Moaser; 1996. 477 p.

  • 15.

    Salimpour F, Mazooji A, Darzikolaei SA. Chemotaxonomy of six Salvia species using essential oil composition markers. J. Med. Plant Res. 2011;5:1795-805.

  • 16.

    Rabbani M, Sajjadi SE, Jafarian A, Vaseghi G. Anxiolytic effects of Salvia reuterana Boiss on the elevated plus-maze model of anxiety in mice. J. Ethnopharmacol. 2005;101:100-3. [PubMed ID: 15994042].

  • 17.

    Ghomi, JS, Masoomi R, Kashi FJ, Batooli H. In-vitro bioactivity of essential oils and methanol extracts of Salvia reuterana from Iran. Nat. Prod. Commun. 2012;7:651-4. [PubMed ID: 22799099].

  • 18.

    Esmaeili, MA, Kanani MR, Sonboli A. Salvia reuterana extract prevents formation of advanced glycation end products: An in-vitro study. Iran. J. Pharm. Sci. 2010;6:33-50.

  • 19.

    Esmaeili A, Rustaiyan A, Nadimi M, Larujani K, Nadjafi F, Tabrizi L, Chalabian F, Amiri H. Chemical composition and antibacterial activity of essential oils from leaves, stems and flowers of Salvia reuterana Boiss. grown in Iran. Nat. Prod. Res. 2008;22:516-20. [PubMed ID: 18415859].

  • 20.

    Moridi Farimani M, Miran M. Labdane diterpenoids from Salvia reuterana. Phytochemistry. 2014;108:264-9. [PubMed ID: 25236692].

  • 21.

    Su YA, Kazuo K, Dean G, Jia J, Liu J, Zheng J, Tamotso N. Three new labdane diterpenoid glycosides from Conyza blinii. Heterocycles. 2002;56:265-71.