Two New Abietane Diterpenoids from Salvia yunnanensis

• Abstract
• Materials and Methods
• References

Abstract

Two new abietane diterpenoids, yunnannin A (1) and danshenol C (2), were isolated from Salvia yunnanensis together with ten known diterpenoids, danshenol A (3), przewalskin (4), tanshinone IIA, tanshinone I, crypotanshinone, 1,2-dihydrotanshinone, tanshinlactone, 5,6-dehydrosugiol, 12-hydroxy-6,7-seco-8,11,3-abietatriene-6,7-dial and phytol. Their structures were established based on spectroscopic data, chemical reactions and comparison with literature data. Compounds 1 - 3 were tested for their antitumor activity in T-24, QGY, K562, Me180 and BIU87 cell lines. Compound 3 showed inhibited growth of K562 (IC₅₀ = 0.53 μg/mL), T-24 (IC₅₀ = 7.94 μg/mL), QGY (IC₅₀ = 4.65 μg/mL) and Me180 (IC₅₀ = 6.89 μg/mL) cell lines while compound 2 was inactive. Compound 1 showed moderate inhibitory activity on QGY (IC₅₀ = 16.75 μg/mL) and Me180 (IC₅₀ = 5.84 μg/mL) cells.

Salvia miltiorrhiza Bunge (Labiatae) is a traditional Chinese herb used in the treatment of various cardiovascular diseases [1]. More than 30 abietane diterpenoids have been isolated from this plant [2], [3]. In the Southwest of China, S. yunnanensis C .H. Wright has been used as the surrogate of S. miltiorrhiza [4]. However, no chemical constituents have been reported from S. yunnanensis. In our continuous research analyzing the diterpenoids from Salvia species [5], we examined the constituents of S. yunnanensis collected in Eryuan county of the Yunnan province. Two new diterpenoids, named yunnannin A (1) and danshenol C (2), were isolated from this plant together with ten known diterpenoids, danshenol A (3) [6], przewalskin (4) [7], 12-hydroxy-6,7-seco-8,11,13-abietatriene-6,7-dial [8], tanshinlactone [9], phytol [10], 5,6-dehydrosugiol [11], 1,2-dihydrotanshinone [12], tanshinone I [13], crypotanshinone [10] and tanshinone IIA [15]. To the best of our knowledge, compound 2 was the third example of 21-carbon abietane-type diterpenoids with an additional CH₃-CO-CH₂ group at C-11 of the C-ring [6]. The major constituents of S. yunnanensis, tanshinone IIA, tanshinone I, and crypotanshinone were identical to the ones of S. miltiorrhiza, which supports the usage of S. yunnanensis as the surrogate of S. miltiorrhiza. We report here the isolation and structure elucidation of new compounds, as well as the cytotoxic activities against T-24, QGY, K562, Me180 and BIU87 cell lines of compounds 1 - 3.[*]

Compound 1 was isolated as a colorless oil. The formula of 1 was established as C₁₈H₂₄O₃ based on HR-ESI-MS ([M + H]⁺ m/z = 289.1802) and ¹³C-NMR spectral data (Table [1]). The IR spectrum displayed the absorption bands due to a hydroxy group at 3444 cm^-1 and a conjugated carbonyl group at 1671 cm^-1. Compound 1 can be ascribed to be an abietane type norditerpenoid according to the characteristic methine signal at δ _C = 38.1 due to C-5, two noticeable quaternary carbon signals at δ _C = 33.5 (C-4) and 40.2 (C-10) and two characteristic methyl carbon signals at δ _C = 32.6 (Me-18), 22.1 (Me-19) [6], [7], [14], [15]. Comparison of ¹H- and ¹³C-NMR spectral data of 1 and 4 indicated that the skeletons of the two compounds were the same and that the structures were also very similar [8]. The major differences in the ¹³C-NMR spectral data were the presence of a conjugated carbonyl group (δ _C = 188.3) and one oxygenated quaternary carbon (δ _C = 68.1) in 1 instead of two olefinic quaternary ones in 4. The HMBC correlations between the oxygenated quaternary carbon at δ _C = 68.1 and H-6, H-7, H-11, and H-14 suggested that the quaternary carbon could be ascribed to C-8 (Fig. [1]). The conjugated carbonyl group was assignable to C-12 by its HMBC correlations with H-11, H-14, and Me-15. The HMBC correlations of the methyl signal at δ _H = 1.85 with C-12, C-13 and C-14 indicated that the methyl can be ascribed to Me-15. The remaining fragment was confirmed to be identical to that of 4 upon analysis of HMQC, HMBC and COSY spectra.

The stereochemistry of 1 was established on the basis of a ROESY experiment (Fig. [2]). ROESY correlations of H-14 with Me-15, H-11 with H-1α and H-1β, H-20β with Me-19, H-7 with H-14 and of H-5α with Me-18 were observed, which supported the α-configuration of H-5 and H-7. The correlation of H-20α with H-14 indicated the α-configuration of OH-8. Therefore, the structure of yunnannin A was established as shown in 1.

Compound 2 was obtained as a red amorphous powder with a molecular ion peak at m/z = 336 in the EI-MS. It was deduced to have the molecular formula C₂₁H₂₀O₄ based on HR-ESI-MS ([M + H]⁺ m/z = 337.1439) and NMR spectral data (Table [1]). Its IR spectrum indicated the presence of hydroxy (ν_max = 3453 cm^-1) and ketone carbonyl groups (ν_max = 1706 cm^-1). The ¹³C-NMR spectra and the DEPT indicated the presence of three methyls, two methylenes (including an oxygenated one), six methines (including one sp ³ and five sp ² carbons) and nine quaternary carbons (including two ketone carbonyl carbons and seven olefinic ones). In the EI-MS of 2, the fragment ions at m/z = 293 and 279 corresponded to the loss of [CH₃COCH₂]⁺ and [CH₃CO]⁺ groups from the molecular ion, which implied the existence of an additional C₃ unit (CH₃COCH₂ group) [5]. The similarity of the 1D-NMR spectral characteristics of compounds 2 and 3 indicated that 2 was a stereoisomer of 3 differing in the stereochemistry at C-11 or C-15 [5]. This was supported by the HMBC correlations (Fig. [3]).

The ROESY spectra of 2 and 3 were very similar to each other. Therefore, it is difficult to distinguish the configuration of 2 from 3 by the ROESY experiment. To solve this problem, we carried out the retro-aldol reactions of 2 and 3 according to the method reported by Tezuka et al. [6]. After treatment with Eu(DPM)₃, both 2 and 3 were converted into dihydrotanshinone, which indicated that the configurations of C-11 were different for 2 and 3. Furthermore, the optical rotation value of 2 was at + 43.86 ° while that of 3 was -47.14 °, which confirmed the relative configuration difference at C-11 in 2 and 3. Accordingly, the structure of danshenol C can be represented as shown in 2.

Compounds 1 - 3 were tested for their cytotoxic activities against T-24 (urinary carcinoma), QGY (hepatocellular carcinoma), K562 (chronic myelogenous leukemia), Me180 (cervix epidermoid carcinoma) and BIU87 (transitional urinary bladder cell carcinoma) cell lines using the improved MTT method as reported previously [16], [17]. It is interesting that the cytotoxic activities of 2 and 3, being stereoisomers, differed significantly. Compound 3 was active in K562 (IC₅₀ = 0.53 μg/mL), T-24 (IC₅₀ = 7.94 μg/mL), QGY (IC₅₀ = 4.65 μg/mL), and Me180 (IC₅₀ = 6.89 μg/mL) cell lines (Table [2]). However, compound 2 was only weakly active in Me180 (IC₅₀ = 10.90 μg/mL) and K562 (IC₅₀ > 30 μg/mL) cell lines. Compound 1 showed moderate inhibitory activity in QGY and Me180 cell lines with IC₅₀ values of 16.75 and 5.84 (μg/mL), respectively.

Materials and Methods

Optical rotations were measured with a HORIBA SEPA-300 High Sensitive Polarimeter. UV spectra were obtained on a UV 2401 PC spectrometer. IR spectra were recorded on a Bio-Rad FTS-135 spectrometer with KBr pellets. ¹H- and ¹³C-NMR experiments were performed on a Bruker AM-400 spectrometer, while 2D-NMR spectra were recorded using a Bruker DRX-500 NMR instrument. EI-MS and ESI-MS were taken on VG Auto Spec-3000 and API Qstar Pulsar instruments, respectively. Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18, 9.4 mm × 25 cm column, a flow rate of 3 mL/min and detection with a UV detector at 272 nm. Silica gel for TLC and column chromatography was obtained from Qingdao Marine Chemical Inc., China.

The plant sample of Salvia yunnanensis was collected in Eyuan of the Yunnan province in July, 2000 and was identified by Prof. Xi-Wen Li of the Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. 2 000 215) was deposited in the Kunming Institute of Botany, Chinese Academy of Sciences, P. R. China.

The dried and powdered (4.7 kg) S. yunnanensis was extracted with Me₂CO (3 × 25 L) at room temperature. The solvent was removed under vacuum. The gummy residue (200 g) was subjected to column chromatography (Ø 12 × 150 cm) over DM-130 porous resin and eluted with MeOH-H₂O (50 % and 90 %, each 10 L). The residue of the 90 % MeOH-H₂O fraction was partitioned between H₂O (2.5 L) and EtOAc (2.5 L). The EtOAc part (65 g) was subjected to silica gel column chromatography (Ø 9 × 120 cm). Mixtures of petroleum-ether/EtOAc (1 : 0, 9 : 1, 8 : 2, 7 : 3, 6 : 4, 5 : 5, and 0 : 1, each 5 L) of increasing polarity were used as eluents. Seven fractions were collected, analyzed by TLC and combined. Tanshinone IIA (1.65 g) was crystallized from the second fraction (7 g). The remaining part of the second fraction was further subjected to silica gel column chromatography (Ø 7 × 90 cm) and was eluted with petroleum-ether:CHCl₃:EtOAc (75 : 20 : 5, 4 L) to give phytol (44 mg) and tanshinone I (258 mg). Crypotanshinone (100 mg) was crystallized from the third fraction. Tanshinlactone (5 mg), the mixture of 2 and 3 (20 mg) and 1,2-dihydrotanshinone (2 mg) were isolated from the third fraction (15 g) by silica gel column chromatography (Ø 7 × 120 cm) using petroleum-ether: CHCl₃:EtOAc (70 : 25 : 5, 10 L) as eluents. Compounds 2 (8 mg) and 3 (8 mg) were separated by semi-preparative HPLC using 85 % MeOH-H₂O as eluent. Compound 1 (6 mg) and 12-hydroxy-6,7-seco-8,11,13-abietatriene-6,7-dial (6 mg) were isolated using an RP-18 column (Ø 7 × 80 cm) (MeOH:H₂O, 8 : 2, 10 L) from the fourth fraction (14 g). The fifth fraction (9 g) was repeatedly chromotographed over RP-18 (Ø 7 × 80 cm) using MeOH:H₂O (8 : 2, 8 L) to give the crude 5,6-dehydrosugiol and przewalskin, which were subjected to Sephadex LH-20 (Ø 2 × 150 cm) (CH₃OH:CH₃Cl, 1 : 1, 0.5 L) to give pure 5,6-dehydrosugiol (7 mg) and przewalskin (50 mg). Compounds 1 - 3 were developed on silica gel TLC (petroleum-ether: EtOAc, 8 : 2) with Rf values of 0.64, 0.55, and 0.50, respectively.

Eu(DPM)₃ treatment of 2 and 3: Eu(DPM)₃ (10 mg) was added to the CHCl₃ solution (0.6 mL) of 2 (2 mg) or 3 (2 mg) and incubated at room temperature overnight. The solution was detected by TLC (silica gel) developed in several solvent systems and compared to dihydrotanshinone. Both 2 and 3 were converted to the same compound, dihydrotanshinone (1.5 mg).

Yunnannin A (1): colorless oil; [α]_D ^19.1: + 25.19° (c 0.60, MeOH); UV (MeOH): λ_max (log ε) = 297.2 (2.97), 237.0 (3.82), 204.0 (3.94) nm; IR (KBr): ν_max = 3444, 2931, 1671, 1636, 1367, 1162, 1034, 856 cm^-1; ¹H- and ¹³C-NMR data: see Table [1]; EI-MS: m/z = 288 [M]⁺ (22), 273 (95), 272 (80), 255 (37), 215 (38), 201 (41), 151 (56), 138 (100), 137 (81), 109 (25), 95 (13); positive HR-ESI-MS: m/z = 289.1802 [M + H]⁺ (calcd. for C₁₈H₂₅O₃ : 289.1804).

Danshenol C (2): red amorphous powder; [α]_D ^19.1:+ 43.86° (c 0.20, CHCl₃); UV (MeOH): λ_max (log ε) = 339 (3.09), 299 (3.54), 287 (3.54), 276 (3.58), 260 (3.56), 234.2 (4.04), 205.2 (4.25) nm; IR (KBr): ν_max = 3453, 2927, 1706, 1629, 1340, 1184, 780 cm^-1; ¹H- and ¹³C-NMR: see Table [1]; EI-MS: m/z = 336 [M]⁺ (67), 293 (17), 279 (100), 261 (17), 250 (76), 235 (94), 179 (30), 139 (14); positive HR-ESI-MS: m/z = 337.1439 [M + H]⁺ (calcd. for C₂₁H₂₁O₄ : 337.1440).

References

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Prof. Dr. Qin-Shi Zhao

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

Chinese Academy of Sciences

Heilongtan

Kunming 650204

People’s Republic of China

Phone: +86-871-522-3254

Fax: +86-871-521-6317

Email: qinshizhaosp@ yahoo.com

References

• 1 Liu X L, Wang X M. The development and utilization of the Salvia species in Anhui province.  Zhongguozhongyaozazhi. 1996;  21 73-5
  PubMedSearch in Google Scholar
• 2 Wu C Y, Li H W. On the evolution and distribution in Labiatae.  Acta Bot Yunnan. 1982;  4 97-118
  PubMedSearch in Google Scholar
• 3 Chang H M, Cheng K P, Choang T F, Chow H F, Chui K Y, Hon P M. et al . Structure elucidation and total synthesis of new tanshinones isolated from Salvia miltiorrhiza Bunge (Danshen).  J Org Chem. 1990;  55 3537-43
  CrossrefPubMedSearch in Google Scholar
• 4 Kunming Institute of B otany, Chinese Academy of S ciences. Flora Yunnannica. Beijing; Science Press 1977: p 672-3
  Search in Google Scholar
• 5 Xu G, Peng L Y, Niu X M, Zhao Q S, Li R T, Sun H D. Novel diterpenoids from Salvia dugesii .  Helv Chim Acta. 2004;  87 949-55
  CrossrefPubMedSearch in Google Scholar
• 6 Tezuka Y, Kasimu R, Basnet P, Namba T, Kadota S. Aldose reductase inhibitory constituents of the roots of Salvia miltiorrhiza Bunge.  Chem Pharm Bull. 1997;  45 1306-11
  PubMedSearch in Google Scholar
• 7 Li B, Niu F D, Lin Z W, Zhang H J, Wang D Z, Sun H D. Diterpenoids from the roots of Salvia przewalskii .  Phytochemistry. 1991;  30 3815-7
  CrossrefPubMedSearch in Google Scholar
• 8 Su W C, Fang J M, Cheng Y S. Abietanes and kauranes from leaves of Cryptomera japonica .  Phytochemistry. 1994;  35 1279-84
  CrossrefPubMedSearch in Google Scholar
• 9 Luo H W, Ji J, Wu M Y. Tanshinlactone, a novel seco-abietanoid from Salvia miltiorrhiza .  Chem Pharm Bull. 1986;  34 3166-8
  PubMedSearch in Google Scholar
• 10 Rasool N, Ahmad V U, Malik A. Terpenoids from Pentatropis spiralis .  Phytochemistry. 1991;  30 1331-42
  CrossrefPubMedSearch in Google Scholar
• 11 Gil R R, Cordell G A, Topcu G, Ulubelen A. Montbretol and salvinolone are identical.  J Nat Prod. 1994;  57 181-5
  CrossrefPubMedSearch in Google Scholar
• 12 Ryu S Y, Lee C O, Choi S. In vitro cytotoxicity of tanshinones from Salvia miltiorrhiza .  Planta Med. 1997;  63 339-42
  Search in Google Scholar
• 13 Zhang K Q, Bao Y D, Wu P, Rosen R T, Ho C T. Antioxidative components of tanshen (Salvia miltiorrhiza Bung).  J Agric Food Chem. 1990;  38 1194-7
  CrossrefPubMedSearch in Google Scholar
• 14 Lius J G, Scanadres L. C-16 hydroxylated abietane diterpenes from Salvia mellifera .  Phytochemistry. 1993;  33 635-8
  CrossrefPubMedSearch in Google Scholar
• 15 Ryu S Y, No Z, Kim S H, Ahn J W. Two novel abietane diterpenes from Salvia miltiorrhiza .  Planta Med. 1997;  63 44-6
  Thieme ConnectPubMedSearch in Google Scholar
• 16 Andreescu S, Sadik O A, McGee D W. Effect of nature and synthetic estrogens on A549 lung cancer cells: correlation of chemical structures with cytotoxic effects.  Chem Res Toxicol. 2005;  18 466-74
  CrossrefPubMedSearch in Google Scholar
• 17 Zhou J J, Yu X F, Han J X, Yang W Y. Improved MTT assay for activity of antitumor agents.  Chin J Pharmacol. 1993;  24 455-7
  PubMedSearch in Google Scholar

Prof. Dr. Qin-Shi Zhao

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

Chinese Academy of Sciences

Heilongtan

Kunming 650204

People’s Republic of China

Phone: +86-871-522-3254

Fax: +86-871-521-6317

Email: qinshizhaosp@ yahoo.com