New Diterpenoids from the Aerial Parts of Salvia reuterana

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).

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
position	δ C	δ H (J in Hz)	δ C	δ H (J in Hz)
1α 1β	41.6	0.85b 1.52b	40.0	0.88b 1.53b
2α 2β	18.3	1.60b 1.33b	18.4	1.53b 1.35b
3α 3β	44.1	1.05b 1.22b	41.8	1.07b 1.28b
4	33.4		32.9
5α	56.7	0.83b	56.7	0.83b
6α 6β	67.6	4.39 brd (2.6)	20.6	1.57b 1.18b
7α 7β	51.3	1.55b 1.91 dd (13.5, 2.0)	43.8	1.34b 1.75 dt (12.5,3.0)
8	73.4		74.5
9α	62.1	1.03b	62.3	1.00 t (3.5)
10	38.9		39.2
11α 11β	17.9	1.49 1.28	18.0	1.46b 1.26b
12α 12β	40.9	1.60b 1.41b	40.8	1.60b 1.38b
13	73.1		73.8
14β	75.1	3.55 dd (8.5, 2.5)	77.8	3.39 dd (7.5, 3.5)
15a 15b	66.2	3.96 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)
16	22.6	1.10 s	22.0	1.08 s
17	24.8	1.32 s	23.6	1.09 s
18	32.8	0.89 s	33.4	0.79 s
19	24.2	1.10 s	21.5	0.71 s
20	16.1	1.08 s	14.9	0.73 s
CH3CO CH3CO	19.8 171.3	1.99 s

^aδ values were extracted from COSY, HSQC and HMBC experiments.

^bOverlapping signals.

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).

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 δ_C 73.1 and 73.4 indicated the presence of oxygen bearing sp³quaternary carbons. Another oxygenated carbons at δ_C 75.1 and 67.6 accounted for the carbons carrying two hydroxyl groups with the carbinolic methine protons coming at δ_H 3.55 (1H, dd, J = 2.5, 8.5 Hz) and δ_H 4.39 (1H, brd, J= 2.6 Hz), respectively. The signal at δ_C 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 δ_C 67.6 instead of the methylene group (C-6). Hence, the methylene was replaced by an oxygenated methine. The signals of C-7 (δ_C51.3) and C-5 (δ_C56.7) 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 (δ_H 1.55), H-7𝛽 (δ_H 1.91), and H-5α (δ_H 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 (δ_H 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 (δ_H 1.32, 1.10 and 1.08, respectively) appeared downfield (δ_H 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.

Figure 1.

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

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 (δ_H 4.39) was replaced by a methylene group (δ_C 20.6 and δ_H 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.

Table 2.

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

Compound	Hela (IC 50 μM)	MCF-7 (IC 50 μM)
1	184.61 ± 1.67	108.34 ± 2.07
2	218.47 ± 2.77	164.07 ± 3.09
Paclitaxelb	0.004	0.033

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

^bPositive control.

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.