Journal of Medicinal Materials, 2016, VoL 21, No. 5 (pp. 334 - 337}
CYTOTOXIC ACTIVITY OF ABIETANE-TYPE DITERPENES FROM SALVIA MILTIORRHIZA BUNGE CULTIVATED IN HA GIANG PROVINCE
Pham Thanh Binh', Bui Thi Mal Anh', Nguyen HaiDang^, Pham Van Cuong''^, Nguyen Van Hung', Pham Thi Thu Hanh', Nguyen The Cuong', Luong Trieu Vung*, Giang Loc Thang^,
Nguyen Tien Dat'' *
'institute of Marine Biochemtstry (IMBC), Vietnam Academy ofScience and Technology (VAST) Hanoi, Vietnam;
^Advanced center for Bio-organic Chemistry, IMBC, VAST;
'institute ofEcology and Biological Resources. VAST;
''Department ofAgricuIture and Rural Development of Ha Giang;
Pho Bang Centre for Plant and Animal Varieties, Dong Van. Ha Giang
•Corresponding author: [email protected] (Received September, 04"', 2016)
Summary
Cytotoiie Aclivity of Abietane-type Dilerpenes from Salvia miltiorrltiza Bunge Cullivated in Ha Giang Province A phytochemical fraciionalion of the melhanol exIraci of the roots of Salvia milliorrhiza cultivated in Ha Giang province led to the jsolation of four abietane-type dilerpenes, which were identined as 6.7-didehydroferruginol (I), ferraginol (2).
lanshjnone llA (3), and sibiriquinone B (4). Compound 1 was identified for the first time from S miltiorrhiza. All compounds were evaluated for their cytotoxicity against human lung carcinoma (A549, EGFR-TKI resistanl, wild type), human breast carcinoma (MDA-MB.231). and human prostate carcinoma CDU145) cell lines. Compound I showed potem cytotoxic effec! against EGFR-TKI resisiani A549 cells with the ICso value of 0 68 pM
Keywords: Salvia miltiorrhiza, Abietane-type diterpene. 6.7-didehydro/erruginol. EGFR-TKI resistant.
1. Introduction pigments as main constituents [3]. Other compounds such as salvianolic acid, alkaloids, and essential oi! have been reported [2]. In our study, a methanol extract of the roots of S.
miltiorrhiza cultivated in Ha Giang province d.sorders,_and hepatitis [I]. Although a large was found to exhibit strong cytotoxicity against human lung cancer A549 cells (ICjo 22.1 Salvia milliorrhiza Bunge (Lamiaceae) 1
been used in oriental medicine for a long time to treat various diseases, especially coronary heart diseases and cerebrovascular diseases, menstrual number of studies on S miltiorrhiza have been reported, lhe plant still draws attention of scientists due to its various biological and pharmaceutical activities such as anticancer, antioxidant, anti-inflammation, Alzheimer's amelioration, cardiovascular and cerebrovascular protection [2]. Chemical investigations have revealed the presence of abietane-type diterpene
Mg/ml). A phytochemical fractionation led to the isolation of four abietane-type diterpenes including 6,7-didehydroferruginol (1), ferruginol (2), tanshinone IIA (3), and sibiriquinone B (4) (Fig. 1). The present paper deals with the isolation and cytotoxic evaluation of these compounds.
334
Figure 1. Stmcture of compounds 1-4 isolated from roots of Salvia milliorrhiza
Journal of Medicinal Materials, 2016, Vol. 21, No, 5
2. Materials and Methods 21. General procedures
TLC was perfonned using precoated Kiesel
>t gel 60 F254 and visualized by UV light 254 nm f, and 10% HjSO, reagent with heat. Column i chromatography was performed using silica gel
^ 60 (Merck, 70-230 mesh). 'H-NMR (500 MHz) '' and "C-NMR (125 MHz) expermients were
catried out on a Bruker AM500 FT-NMR spectrometer (Bruker, Rheinstetten, Germany) using tetramcthylsilane (TMS) as intemal standard. ESI-MS spectra were obtained from an Agilent 1260 Series Single Quadrupole LC/MS Systems (Agilent Technologies, USA).
2.2. Plant materials
_ The roots of Salvia miillorrhlza were , collected in Oct. 2014 at Pho Bang, Dong Van district, Ha Giang province and identified by one
• of the authors, Dr. Nguyen The Cuong. The 1 voucher specimens (HaQiang-10) were deposited at the Department of Bioactive Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology.
2.3. Exiraclion and isolalion
The air-dried and powdered materials (900 g) were extracted with methanol (3L x 3 times) in a sonic bath at 40 "C in 15 min. The combined extracts were concentrated unin vacuo to obtain a crude residue (150 g), which was then re- suspended in" water (IL), and successively extracted by dichloromethane (each 0.5 L x 3 times). The organic layers were concentrated to give 22.1 g of residue. The dichloromethane residue was submitted to a siiica gel column chromatography (CC) eluted with a gradient of «- hexane-acetone (100:0-0:100 v/v) to obtain tlve fraction SEB4.I-5. Fraction SEB4.2 was chromatographed on d siiica gel column (n- hexane-acetone 80:1, v/v) to afford twelve sub fractions SEB4.2.I-12. Compounds 1 (6.0 mg) and 2 (187.2 mg) were isolated from SEB4.2.7 by using a RPI 8 column eluted with acetone-water
(3:1, v/v). Compounds 4 (62.2 mg) was purified from SEB4.2.9 by siiica gel column chromatography (n-hexane-acetone 50:1, v/v).
Fraction SEB4.4 was fractionated on a column eluted with «-hexane-ethyl acetate (15:1 v/v) to obtain 3 (11.2 mg)
2.4. Cylotoxic assay
A549 (Human lung carcinoma, wild type of EGFR and TKI-resistance), MDA-MB-231 (human breast carcinoma) and DUI45 (human prostate carcinoma) cell lines were kindly provided by Prof Jeong-Hyung Lee, Department of Biochemistry, College of Namral Sciences, Kangwon National University, Korea. The cells were cultured at 37 °C in RMPI-1640 (A549) or DMEM (MDA-MB-231 and DUI45) medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 ng/ml streptomycin in 5% COj incubator. The cytotoxic assay was perfomied using the 3-(4,5- dimethylthiazoI-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The cells were seeded in 96-well plates at a concentration of 2 x 10*
cells/well and treated with various concentrations of test compounds and incubated in a humidified 5% CO2 atmosphere at 37 "C. Af^er 48 h incubation, 20 nL of 5 mg/mL MTT was added to each well and incubated for another 4 h. Afler removing the supematant, fonnazan crystals were dissolved in isopropanol and the OD values were measured at 570 nm using a microplate reader.
The antitumor agent etoposide was used as a positive control.
3. Results and Discussion
By means of MS and NMR spectroscopic data (Table I) and in comparison with reported values, the isolated compounds were identified to be 6 7- didehydroferruginol (1) [4], fem,gi„o| (2) [5]
tanshmone IIA (3) [6], and sibiriquinone B (4) [7]
Although compound 1 was isolated from several Salvia plants such as S aplana, S munzli [4] [8]
it IS found for the first time from,?. mllltorrliiza
Journal of Medicinal Materials, 2016, VoL 21, No. 5
No
2 3 4 5 6 7 1 8
9
! 10 II 12 13 14 15 16 17 IS 19 20 ESI-MS
Table 1. NMR and ESI-MS data 1 (CDCljl
1 5 H ( y i i i H z ) I 76, m j 2 07. m ] 1.25, m 1 1 55, m 1 67. m
! 2.06.m 5.87. dd (9.5,2.0) 6.49, dd (9 5. 2 0)
6c
! 38.5 1
19.1 42.3
• 3 1 7 , 53.4 ' 127.5 127 9 1 125.2 1 148 5
• 38 9 6.57. s
6.88,s 3.15. m 1 26. d (7 0) 1.22, d (7,0) 0 9 6 , s 1.03, s 1,01,5 m ' ; 2 8 5 . 1 | M +
1 1 0 8 152 7 132.9 126 8 27.5 22,5 22 3 32.6 20,1 22.9 H]-
2 (CDjOD) ' S u f y i n H z )
1.71-1.83, m
1.55, m 1,45, m
2 2 0 , m 1.63, m 2.66-2 76, m
6 64.S
6.74, s 3.19, m 1.19, d (7.0) 1 1 7 . d ( 7 0) 0-91, s 0 9 2 , s 1.14, s m/i 287.4 [M
Sc 40.0
20.4 42.8 34.2 51.9 20.3 30.9 126,7 149.0 3S.5 111.5 153.0 133.0 127.1 27 6 23.3 23 2 33.9 22 1 25 3 +Hr
of compounds 1-4 3 (CDCIj) SH (J in Hz) 3.18,t{6.5)
1,78, m 1.65. m
7 55. d (8.5) 7.63, d (8.5)
7.26, s
2.26, s 1 3 l , s 1.31,s
m/z 95 2 [M Sc 29.8
19.1 3 7 8 34.6 144.4 133.4 120,2 1 2 7 4 126.4 150 1 183.6 175.7 121.1 161 7 141.2 1 1 9 8 8.7 31.8 31.8
f H f
4 (CDCIO S„(J in tiz) 3.17.1(6.5)
!.78, m 1.64, m
7.60, d (8.0) 7.12, d (8.0)
7.08, s
3.01. m 1.16, d (7.0) l . l 6 , d ( 7 0)
5c 29.8
19.0 37.8 34.4 149.6 133.7 ' 127.9 128.1 134.4 144.4 [ 182,3 139.9
1814
m/j 283.1 [M + H ] ' The cytotoxicity of the four isolated
compounds was evaluated on human lung careinoma (A549), human breast carcinoma (MDA-MB-231), and human prostate carcinoma (DU14S) cell lines. 6,7-Didehydrofennginol (1) showed the mosl cytotoxic effect against A549 cells with the IC„ value of 0.68 pM. The breast Table 2. Cytotoxicity (IC.
cancer cells MDA-MB-231 was sensitive to compounds 1, 2 (fenuginol) and 4 (sibiriquinone B), while prostate cancer cells DU145 was sensitive to 2, 3 (tanshinone ILA) and 4. Notably, the effects of all compounds (except for 3 on MDA-MB-231 cells) were stronger than the reference control, etoposide (Table 2).
in |jM) of compounds 1-4
• ' • " p T J r c ! ! ? " ' " " " " " " ' ^ * ' " ^ ' S " - " ' ) '•^'P'-O'-' "Periments and values „ e = x p , ^ as men 1 SD
Journal of Medicinal Materials, 2016, VoL 21, No. 5
g cancer is the leading cause of cancer 4. Conclusion
death worldwide with up to 8 5 % related to non- Four abietane-type diterpenes including 6,7- small cell lung cancer (NSCLC) [9]. tn the past didehydroferrugino] (1), femiginol (2),
ew years, the discovery of the pivotal role in tanshinone I M (3), and sibiriquinone B (4) were tumongenesis of the epidennal growth factor isolated from roots of Salvia miltiorrhiza receptor (EGFR) provided a new class of targeted cuhivated in Ha Giang province. Compound I therapeutic agents: the EGFR-tyrosine kinase was reported for the first time from S F r ™ ™ / " ' " ' ' ' ' ™ ' ' ' " " • " ° ™ ' " ' •"= " " • ' " " " • t e " - A l l compounds showed cytotoxicity EUI-K-IIU resistance has recently been detected against different human lung AS49 breast MDA- in some NSCLC patients [11]. Interestingly, MB.23I and prostate DU145 'cancer cells compound 1 potently inhibited the growth of Compound 1 showed potent cytotoxic effect
> r T ' ' " ' ' " ' ' " " " ' ' " " ' ^ ° ° ^8"'"=' EGFR-TKI resismn, A549 cells This w,th etoposide. Further smdies are needed to resuh suggested that 6,7-didehydrofe,ruginol lartfy the mode of actton of this compound on might be a promising candidate for treatmem of dt^g-resrstant cancer cells. Ferruginol (2) and EGFR-TKI-resistam lung cancer
si^inqmnone B (4) showed remarkable Acknowledgment,: r / , „ „„r* „ „ , , „ „ , , , c ^ t o x i c , ^ agamst MDA-MB-231 and D U I 4 5 . t,e Vietnam Academy ofScience and Lnaio^
Th„ effect mrght be related to the activation of igram code VASTUDCN.05m.,6, We titank 1 caspase-assocated apoptosis or inhibition of ,„slltale of Ciiemlstry (VAST, for iHe NMR
hypoxia-inducible factor-1 [12, 13]. y , , j me experiments.
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Journal of Medicinal Materials, 2016, VoL 21, No. 5
