TP CH KHOA HC V CNG NGH, I HC NNG - S 4(45).

2011

CHEMICAL CONSTITUENTS OF ETHYL ACETATE EXTRACT FROM SCHEFFLERA HYPOLEUCA (KURZ) HAMRS LEAVES
THNH PHN HO HC CA DCH CHIT ETHYL AXETATE T L CY CHN CHIM DI TRNG (SCHEFFLERA HYPOLEUCA (KURZ) HAMRS) Giang Th Kim Lin
i hc Nng

Nguyn Th Hong Anh

Vin Ha hc - Vin Khoa hc v Cng ngh Vit Nam

o Hng Cng
Trng i hc S phm, i hc Nng

SUMMARY Schefflera hypoleuca (Kurz) Hamrs (Chan chim duoi trang) belongs to family Araliaceae. The leaves of this plant were collected in Sapa, Laocai Province. The ethyl acetate extract of it's leaves was purified by using silicagel column with solvent system of dichlormetan:methanol (99:1 to 70:30) as eluate to yield 3 compounds S1, S2 and S3. The structures of S1 and S3 were determined as -sitosterol and -sitosterol glucoside, respectively by direct comparision with authentic samples. Compound S2 was elucidated as 3-hydroxy-20demethylisoaleuritolic-14(15)-en-28,30-dioic acid by analysis of MS, NMR spectra and comparison with the published data. Compound S2 exhibited cytotoxic activity against four human cancer cell lines (KB, HepG2, Lu and MCF7). This is the first report about chemical constituents of Schefflera hypoleuca growing in Vietnam. Keywords: Schefflera hypoleuca, Araliaceae, triterpene, 14-taraxeren-28-oic acid TM TT Cy Chn chim di trng (Schefflera hypoleuca (Kurz) Hamrs) thuc h Ng gia b (Araliaceae) c thu hi ti Sa Pa, tnh Lo Cai. Tinh ch cn chit etyl axetat ca l cy bng sc k ct trn cht hp ph silicagel, h dung mi ra gii diclometan : metanol vi t l metanol tng dn t 1 n 30% thu c 3 cht S1, S2 v S3. Cu trc ca hai cht S1 v S3 c xc nh -sitosterol v -sitosterol glucosid, tng ng bng cch so snh trn bn mng phn tch v ph IR vi cht chun. Cu trc cht S2 c xc nh l 3-hydroxy-20demethylisoaleuritolic-14(15)-en-28,30-dioic nh phn tch cc s liu ph khi MS, cng hng t ht nhn NMR kt hp so snh vi ti liu cng b. Kt qu th hot tnh sinh hc ca cht S2 cho thy cht ny c hot tnh gy c vi 4 dng t bo ung th: biu m (KB), gan (HepG2), phi (Lu) v v (MCF7). y l ln u tin thnh phn ha hc ca l cy Schefflera hypoleuca Vit Nam c nghin cu. T kho: Chn chim di trng, Ng gia b, triterpen khung 14-taraxeren-28-oic acid

1. Introduction The genus Schefflera which belongs to the Araliaceae family with 25 species is a large genus of many species widely distributed in Vietnam [1]. The previous studies showed that many species of Schefflera have beneficial effects in treating diseases of internal medicine. In Oriental medicine, it is a precious medicine which has the effect
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that remains strong tendons and bones, expels rheumatism, osteoarthritis pain, colic, child advocacy muscle weakness, difficulty walking, fights depression, increases the intellectual memory, anti-inflammatory, analgesic, antipyretic...[2,3]. The previous research showed that the compounds which have biological activity isolated from the genus Schefflera were mainly triterpenes and triterpene glycoside [4-6]. Although the genus Schefflera have multiple values for use, in our country there are few researches on this genus. Schefflera hypoleuca which has a local name of Chan chim duoi trang and grows much in some northern mountainous provinces in Vietnam. In some reference materials we have had so far in Vietnam, the world has not dealt with any work published on the chemical composition of the tree S. hypoleuca. growing wild in Sonla Province. This article will present some first results on the chemical constituents of ethyl acetate extract from the leaves of Schefflera hypoleuca harvested in Sapa - Laocai, Vietnam. 2. Experimental 2.1. General Experimental Procedures ESI-MS: Agilent 6310. HR-ESI-MS: FT-ICR-MS Varian. NMR: BRUKER Avance 500 spectrometer [499.8 MHz (1H) and 125 MHz (13C, DEPT)]. Chemical shifts were recorded in DMSO-d6 and the value are in (ppm) based on residual of DMSOd6 2.49 and 39.5 for 1H and 13C-NMR, respectively; CDCl3 ( = 77.0 ppm) and CD3OD ( = 49.0 ppm). The sephadex LH-20 or silicagel 60, 0.06-0.2mm (Merck) used for the first columm chromatography; silicagel 60, 40-63m (Merck) used for the next columms. TLC: silicagel 60 F-254 (Merck). 2.2. Plant Material Samples were collected in Sapa, Lao Cai. The species was identified at the Institute of Ecology and Biological Resources with the scientific name of Schefflera hypoleuca (Kurz) Hamrs. A voucher specimen (TAB 0001) was deposited in the Institute of Ecology and Biological Resources. 2.3. Extraction and isolation The leaves of Schefflera hypoleuca La., after harvest, are washed, dried naturally, then dried in an oven at 400C and minced. (1.9 kg powdered leaves were extracted with the solvent n-hexane, ethyl acetate and methanol, respectively). Evaporation of solvent in vacuo (450C) gave extracts, respectively. The EtOAc extract (SHE9, 25g) was chromatographed over silicagel eluted with dichlomethane, then the system of solvent dichlomethane/methanol gradient from 99:1 to 70:30 to give 6 fractions (SHE9.1SHE9.6). Fraction SHE9.3 (1.2 g) was further separated over silicagel with CH2Cl2-MeOH (99:1) to afford 3 fractions. Fr. 2 was recrystallized with CH2Cl2-MeOH to give
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compound S1 (25mg)-white needle solid, Rf = 0,65 (n-hexane/ethyl acetate= 75:25); IR (KBr) (cm-1): 3454.47; 2944.36; 2866.43; 1709.53; 1641.85; 1464.20; 1376.79; 1052.51; 959.46; 733.87; 595.70; 465.99. Fraction SHE9.4 (8.5 g) was purified by silicagel column chromatography with CH2Cl2-MeOH (95:5) to afford 7 fractions. Fr. 2 was recrystallized with CH2Cl2-MeOH (80:20) to give compound S2 (125 mg)-white amorphous powder. 1H-NMR (500 MHz, DMSO): 5.52 m, 1H (H-15); 4.16 br s, 1H (H-3); 3.17 br s, 1H (H-13), 0.87; 0.86; 0.82; 0.79; 0.78; 0.73 (each s signal, 3H of 6 methyl groups). Fraction SHE9.5 (3.5 g) was purified by silicagel column chromatography with CH2Cl2-MeOH (95:5) to afford 4 fractions (SHE9.5.1 SHE9.5.4). Evaporation of solvent SHE9.5.4 in vacuo (450C), dissolving in EtOAc gives the white precipitate (300 mg). Recrystallizing it with CH2Cl2-MeOH (50:50) to give compound S3 (125 mg). Rf = 0.67 (dichlometane/methanol = 85:15). IR (KBr) (cm-1): 3430.40; 2939.11; 1644.07; 1463.78; 1370.90; 1029.44; 999.39; 830.03; 769.93; 663.39; 603.29; 433.93. 3. Results and Discussion The ethyl acetate extracts from leaves of Schefflera hypoleuca we have isolated three substances: S1, S2 and S3. Compounds S1 and S3 were identified as -sitosterol and -sitosterol glucosid by a comparison on TLC and infrared data with standard compound [7].

S2: 3-hydroxy-20-demethylS3: R = Glucose: -sitosterol glucosid isoaleuritolic-14(15)-en-28,30-dioic acid Compound S2 obtained as white amorphous powder. ESI-MS for the molecular ion peak at m/z = 486 [M]+. The molecular formula of S2 was determined as C30H46O5 from its 1H-, 13C-NMR and ESI-MS data. The 13C-NMR and 1H-NMR spectra indicated signals six singlet signals at H 0.87; 0.86; 0.82; 0.79; 0.78; 0.73 of six quaternary carbons attached with the methyl groups; a three-substituted double bond was revealed by signals at H 5.52 m and C 125.08; 136.6; a methin group linked hydroxy group at H 4,16 (br s) v C 73,56. Signals at C 176,54 and 178,47 also showed the presence of two carboxyl groups. In addition, the NMR spectra indicated S2 had 10 CH2 groups, 3 CH groups and 6 quaternary carbons. By the analysing of spectra suggested the structure of S2 was a triterpene with frame 14-taraxeren-28-oic acid.
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S1: R = OH: -sitosterol

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The chemical shift of the hydroxy group linked to the methin group at C 73.56 proved hydroxy group in position 3 by a comparison with other substances 3-OH groups [8]. Carbon signal at C 178.47 characteristic carboxylic group attached to C-17, while signals in C 176.54 assigned to C-30 by comparison with the document [9]. The spectral data of S2 completely similar to the compound 3 -hydroxy-20demethylisoaleuritolic-14 (15)-en-28,30-dioic acid. This substance was the first time isolated from the leaves of Schefflera bodinieri in 1996 by Min Zhu et al. [9]. 13C- and 1 H-NMR spectral data of S2 and the document [9] were given in Table 1.
Table 1. 13C- and 1H-NMR spectral data of S2 [DMSO, 125MHz)

C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

S2 (DMSO) 32.88 t 23.65 t 73.56 d 36.90 s 46.21 d 25.22 t 33.01 t 36.67 s 48.18 d 36.67 s 17.74 t 43.16 t 46.21 d 136.60 s 125.08 d

[9] (DMSO) 33.11 23.98 73.76 37.11 46.40 25.45 33.54 36.90 48.40 36.90 18.00 43.01 46.40 136.80 125.40

C 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

S2 (DMSO) 23.76 t 55.18 s 40.00 s 31.64 t 30.34 s 35.93 t 22.37 t 33.32 q 17.96 q 15.83 q 22.39 q 23.34 q 178.47 s 28.58 q 176.54 s

[9] (DMSO) 23.90 55.35 40.18 31.85 30.57 36.17 22.58 33.25 18.20 16.10 22.50 23.60 178.70 28.80 176.80

The results of cytotoxic activity and antimicrobial activity of S2 at Department of Biological activity. Vietnam Academy of Science and Technology showed the good inhibition activity with the 4 cell lines cancer: KB, HepG2, Lu and MCF7 with the IC50 value is 17.88; 22.15; 8.0 and 3, 5g/ml, respectively. But it has no activity against the microorganisms the available Gram (+), Gram (-) and fungi.
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4. Conclusion From the ethyl acetate extract of the leaves of Schefflera hypoleuca (Kurz) Hamrs three substances S1, S2 and S3 were isolated. By combining spectral methods and comparing with the standard substances, we had identified the structure of the three substances: - S1 was -sitosterol - S2 was 3-hydroxy-20-demethylisoaleuritolic-14(15)-en-28,30-dioic acid. - S3 was -sitosterol glucosid. Compound S2 showed that it has good activity with all the four cancer cell lines: KB, HepG2, Lu and MCF7. REFERENCES [1] Yaplan Li, Paul P.H. But, Vincent E.C. Ooi, (2005), Antiviral activity and mode of action of caffeoylquinic acids from Schefflera Heptaphylla (L.) Frodin, Antiviral research 68, 1-9. [2] T.V. Sung, C. Lavaud, A. Porzel, W. Steglich, G. Adam, (1992), Triterpenoids and their Glycosides from the Bark of Schefflera Octophylla, Phytochemistry, 31 (1), 227-231. [3] T. V. Sung, W. Steglich, G. Adam, (1991), Triterpene Glycosides from Schefflera octophylla, Phytochemistry, 30 (7), 2349-2356. [4] T.V. Sung, J. Peter Katalinic, G. Adam, (1991), A Bidesmosidic Triterpenoid Saponin from Schefflera Octophylla, Phytochemistry, 30 (11), 3717-3720. [5] Goad J. L. and Akihisa T, (1997), Analysis of Sterols, Blackie Academic and Professional Pub., First edition. [6] Tanaka R. and Matsunaga S., (1991), Fernane and Multiflorane Triterpenes Ketols from Euphorbia supina, Phytochemistry, 30 (12), 4093-4097. [7] Min Zhu, J. David Phillipson, Pam M. Greengrass, Norman G. Bowery, (1996), Triterpenoids and a Triterpene Gycoside from Schefflera Bodinieri Leavess, Phytochemistry, 43 (6), 1307-1311. (BBT nhn bi: 10/05/2011, phn bin xong: 13/06/2011)

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