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7/27/2019 ChanChim-DuoiTrangn

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TP CH KHOA HC V CNG NGH, I HC NNG - S 4(45).2011

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CHEMICAL CONSTITUENTS OF ETHYL ACETATE EXTRACT

FROM SCHEFFLERA HYPOLEUCA (KURZ) HAMRS LEAVES

THNH PHN HO HC CA DCH CHIT ETHYL AXETATE TL CYCHN CHIM DI TRNG (SCHEFFLERA HYPOLEUCA (KURZ) HAMRS)

Giang ThKim Lini hc Nng

Nguyn ThHong AnhVin Ha hc - Vin Khoa hc

v Cng ngh Vit Nam

o Hng CngTrngi hc Sphm,

i hc Nng

SUMMARY

Schefflera hypoleuca (Kurz) Hamrs (Chan chim duoi trang) belongs to family

Araliaceae. Theleaves of this plantwere collected in Sapa, Laocai Province. The ethyl acetateextract of it's leaves was purified by using silicagel column with solvent system ofdichlormetan:methanol (99:1 to 70:30) as eluate to yield 3 compounds S1, S2 and S3. Thestructures of S1 and S3 were determined as -sitosterol and -sitosterol glucoside, respectivelyby direct comparision with authentic samples. Compound S2 was elucidated as 3-hydroxy-20-demethylisoaleuritolic-14(15)-en-28,30-dioic acid by analysis of MS, NMR spectra andcomparison with the published data. Compound S2 exhibited cytotoxic activity against fourhuman cancer cell lines (KB, HepG2, Lu and MCF7). This is the first report about chemicalconstituents of Schefflera hypoleucagrowing in Vietnam.

Keywords: Schefflera hypoleuca, Araliaceae, triterpene, 14-taraxeren-28-oic acid

TM TTCy 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 bngsc k ct trn cht hp ph silicagel, h dung mi ra gii diclometan : metanol vi t lmetanol tng dn t 1 n 30% thu c 3 cht S1, S2 v S3. Cu trc ca hai cht S1 v S3c xc nh -sitosterol v -sitosterol glucosid, tng ng bng cch so snh trn bnmng phn tch v ph IR vi cht chun. Cu trc cht S2c xc nh l 3-hydroxy-20-demethylisoaleuritolic-14(15)-en-28,30-dioic nh phn tch cc s liu ph khi MS, cnghng t ht nhn NMR kt hp so snh vi ti liu cng b. Kt qu th hot tnh sinh hcca 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 whichbelongs to the Araliaceae family with 25 species is alarge genus of many species widely distributed in Vietnam [1]. The previous studiesshowed that many species of Schefflera have beneficial effects in treating diseases ofinternal 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 theintellectual memory, anti-inflammatory, analgesic, antipyretic...[2,3].

The previous research showed that the compounds which have biologicalactivity isolated from the genus Schefflera were mainly triterpenes and triterpeneglycoside [4-6].

Although the genus Schefflera have multiple values for use, in our country thereare few researches on this genus. Schefflera hypoleuca which has a local name ofChan chim duoi trang and grows much in some northern mountainous provinces inVietnam. In some reference materials we have had so far in Vietnam, the world has notdealt 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 thechemical 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: BRUKERAvance 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 DMSO-

d6 2.49 and 39.5 for1H 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 nextcolumms. TLC: silicagel 60 F-254 (Merck).

2.2. Plant Material

Samples were collected in Sapa, Lao Cai. The species was identified at theInstitute of Ecology and Biological Resources with the scientific name of Schefflerahypoleuca (Kurz) Hamrs. A voucher specimen (TAB 0001) was deposited in theInstitute of Ecology and Biological Resources.

2.3. Extraction and isolation

The leaves of Schefflera hypoleuca La., after harvest, are washed, driednaturally, then dried in an oven at 400C and minced. (1.9 kg powdered leaves wereextracted 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 withdichlomethane, then the system of solvent dichlomethane/methanol gradient from 99:1to 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 withCH2Cl2-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 withCH2Cl2-MeOH (95:5) to afford 4 fractions (SHE9.5.1 SHE9.5.4). Evaporation ofsolvent SHE9.5.4 in vacuo(450C), dissolving in EtOAc gives the white precipitate (300mg). 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 ofSchefflera hypoleuca we have isolatedthree substances: S1, S2 and S3. Compounds S1 and S3 were identified as -sitosteroland -sitosterol glucosid by a comparison on TLC and infrared data with standardcompound [7].

S1: R = OH: -sitosterol

S3: R = Glucose: -sitosterol glucosid

S2: 3-hydroxy-20-demethyl-

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 C30H46O5from its 1H-, 13C-NMR and ESI-MS data. The 13C-NMR and 1H-NMR spectra indicatedsignals six singlet signals at H 0.87; 0.86; 0.82; 0.79; 0.78; 0.73 of six quaternarycarbons attached with the methyl groups; a three-substituted double bond was revealedby signals at H 5.52 m and C 125.08; 136.6; a methin group linked hydroxy group at H4,16 (br s) v C 73,56. Signals at C 176,54 and 178,47 also showed the presence of twocarboxyl groups. In addition, the NMR spectra indicated S2 had 10 CH2 groups, 3 CHgroups and 6 quaternary carbons. By the analysing of spectra suggested the structure ofS2 was a triterpene with frame 14-taraxeren-28-oic acid.


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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-20-

demethylisoaleuritolic-14 (15)-en-28,30-dioic acid. This substance was the first timeisolated from the leaves ofSchefflera bodinieri in 1996 by Min Zhu et al. [9]. 13C- and1H-NMR spectral data ofS2 and the document [9] were given in Table 1.

Table 1.13C- and1H-NMR spectral data ofS2 [DMSO, 125MHz)

CS2

(DMSO)

[9]

(DMSO)C

S2

(DMSO)

[9]

(DMSO)

1 32.88 t 33.11 16 23.76 t 23.90

2 23.65 t 23.98 17 55.18 s 55.35

3 73.56 d 73.76 18 40.00 s 40.18

4 36.90 s 37.11 19 31.64 t 31.85

5 46.21 d 46.40 20 30.34 s 30.57

6 25.22 t 25.45 21 35.93 t 36.17

7 33.01 t 33.54 22 22.37 t 22.58

8 36.67 s 36.90 23 33.32 q 33.25

9 48.18 d 48.40 24 17.96 q 18.20

10 36.67 s 36.90 25 15.83 q 16.10

11 17.74 t 18.00 26 22.39 q 22.50

12 43.16 t 43.01 27 23.34 q 23.60

13 46.21 d 46.40 28 178.47 s 178.70

14 136.60 s 136.80 29 28.58 q 28.8015 125.08 d 125.40 30 176.54 s 176.80

The results ofcytotoxicactivityandantimicrobial activityofS2 at Departmentof Biological activity. Vietnam Academy of Science and Technology showed thegood inhibition activity with the 4 cell lines cancer: KB, HepG2, Lu and MCF7with the IC50 value is 17.88; 22.15; 8.0 and 3, 5g/ml, respectively. But it has noactivity againstthemicroorganisms the available Gram(+), Gram(-) and fungi.


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4. Conclusion

From the ethyl acetate extract of the leaves of Schefflera hypoleuca (Kurz)HamrsthreesubstancesS1, S2andS3 wereisolated. Bycombining spectral methods

and comparing with the standard substances, we had identified the structure of thethreesubstances:

- 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 withall the fourcancercelllines: KB, HepG2, Luand MCF7.

REFERENCES

[1] Yaplan Li, Paul P.H. But, Vincent E.C. Ooi, (2005), Antiviral activity and mode ofaction of caffeoylquinic acids from Schefflera Heptaphylla (L.) Frodin, Antiviralresearch 68, 1-9.

[2] T.V. Sung, C. Lavaud, A. Porzel, W. Steglich, G. Adam, (1992), Triterpenoids andtheir Glycosides from the Bark ofSchefflera 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 andProfessional Pub., First edition.

[6] Tanaka R. and Matsunaga S., (1991), Fernane and Multiflorane Triterpenes KetolsfromEuphorbia 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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