Journal of Medicinal Materials, 2014, VoL 19, No. 3 (ppJ68 -171)
PRELIMINARY STUDY ON THE CHEMICAL CONSTITUENTS FROM THE RHIZOMA OF POLYGONUMBARBATUM L.
COLLECTED IN VIETNAM
Tran Thanh Ha'\ Nguyen Van Dai^, Nguyen ThiDung^, Nguyen Minh Khoi' 'Vietnam National Institute of Medicinal Materials
•^University of Natural Sciences - Vietnam National University, Hanoi
•Corresponding autiior: [email protected] (Received March 12*, 2014)
Summary
Preliminary Study on the Chemical Constituents from the Rhizoma of Polygonum barbatum L. in Vietnam The first study on the phytochemistry of Polygonum barbatum L. growing in Vietnam has been done. Four compounds p-sitosterol (l),ursolic acid (2), oleanoUc acid (3) and daucosterol (4) were isolated from the n- hexane fraction of 90% aqueous ethanol extract of the rhizoma of Polygonum barbatum L.. The structures of these compounds were identified on the basis of NMR, MS spectral analysis and in comparison with the published NMR data.
Keywords: Polygonum barbatum L., Ursolic acid, OleanoUc acid.
I. Introduction screetiing revealed the presence of alkaloids, Polygonum barbatum L. common name flavanoids, carbohydrates, phenols and saponins
"nghS trSng" or "ngh^ rau, ngh^ dai" is [5]. Sitosterone, viscozulenic acid, acetophenone perennial herbs of the genus Polygonum, were isolated from the leaves of this plant.
family Polygonaceae. This species grows widely in marshy and aquatic places, by the sides • of the rivers, seasonally flooded roadsides ditches and small ponds throughout Vietnam and also in many other countries in the South-east Asia. In Malabar and Canara, the seeds are used to relieve the griping pains of colic. In North India the roots are used as an astringent and cooling remedy. In Chinese medicine, decoction of the leaves and stalks are used to treat ulcers. But in Vietnam folk medicine. Polygonum barbatum L. has not ever been used as medicines [1]. Recent studies of the biological activity showed antimicrobial effects of 95 % ethanol exfract of the leaves on 4 bacterial sfrains of gram (+), 4 bacterial strains of gram (-) and 2 fungal strains [2]; antispasmodic, larvicidal activity, antinociceptive, wound healing, antioxidant [3], antitumor [4]. The phytochemical
Viscozulenic acid was assessed for its analgesic, anticholinergic, anti-infiammatory, CNS depressant, cytotoxic and anti-HFV-l effects [ 6]
Although Polygonum barbatum L. has been studied in the world, to the best of our knowledge, no thorough phytochemical and biological study have ever been carried out on this plant in Vietnam. In this report we present the results of our study on the chemical composition of the w-hexane extracts fix)m Polygonum barbatum L. rhizoma.
2. Experimental 2.1. Plant material
The rhizoma of Polygonum barbatum L.
was collected in Nam Dinh, Vietnam in September 2013. Ailer collecting, the rhizoma was air-dried imder the shade and dried at 45*'C, then groimd into fine powder. A voucher specimen of the plant material is maintained 168 Journal of Medicinal Materials, 2014, Vol.l9,No. 3
in tne Vietnam National Institute of Medicinal Materials (NIMM), 3B Quang Trung road, Hoan Kiem District, Ha Noi, Vietnam.
2.2. General experimental procedures H- and '^C-NMR were measured in CDCI3 & MeOD-di, on a Bruker 500 (500 MHz for ' H - N M R and 125 MHz for "C- NMR) NMR spectrometer. Chemical shifts are in ^ (Ppm) with tetramethylsilane as an mtemal standard. Electron Ionization Mass Spectra (EI-MS) were recored on a Hewlett Packard HP 5890, Serie II Mass Spectrometer operating at 70 eV. Silica gel (Merck, Darmstadt, Germany, particle size 63-200 and 40-63 jun was used for column chromatography. TLC was performed on precoated silica gel 60 F2S4 plates (Merck);
spots were detected by UV or by spraying with 10% aq. H2SO4 followed by heating.
2.3. Extraction and isolation
Air-dried and pulverized rhizoma of F.
barbatum L. (5 kg) were extracted with ethanol 90% (EtOH) by maceration for 4 days. The extraction was repeated three times. The extracts were combined and evaporated at 50''C under low pressure to obtain ethanol crude residue. The total residue (207,26 g) was suspended in water and successively extracted with n-hexane, ethyl acetate, n-butanol. Solvent was then evaporated in vaccuo to obtain the corresponding n-hexane (40,29 g), ethyl acetate (50,97 g), and n-butanol extracts (35,76 g).
The n-hexane extract was separated by silica gel (Merck 0.063 - 0.2mm) colunm chromatography (CC) using n-hexan/ aceton gradient solvent system (100: 0; 50:1; v/v 1:1, 0:1) to give thirty-one fractions (denoted as PBH-1-»31). Fraction PBH-2 was decolorized and re-crystallized in n-hexane/ethyl acetate (8:2, v/v) to furnish compound 1 (140 mg).
Fraction PBH-26 was purified by silica gel flash cfaiomatogiqihy eluting with dichlomethane/
aceton (6:1) to afford two compounds 2 (15 mg) and 3 (35 mg). Fraction PBH-30 was decolorized and re-crystallized in dichlomethane^
methanol (9:1) to give compound 4 (30 mg).
Compound 2 (Ursolic add): white amorphous solid, mp. 290-291°C
EI-MS (70 eV), m/z (%): 456 (M*, 4), 438, 284, 248 (100), 219 (5), 203 (45), 189 (5), 161 (4), 133,119 (5), 95 (9), 69 (8), 55 (9).
' H - N M R (CDCls&MeOD, 500 MHz), S (ppm) 5.24 (IH, t, y = 7.5 Hz, H-12); 3.20 (IH, t, . ^ 16.5 Hz, H-3); 2.19 (IH, d, >11.0 Hz, H-18); 1.09 (3H. s. H-27); 0,98 (3H, s, H-26); 0.95 (3H, s, H-25); 0.93 (3H, s, H- 30); 0.86 (3H, d, .^6.5 Hz, H-29); 0.81 (3H, s, H-23); 0.77 (3H, s, H-24).
"C-NMR (CDCb&MeOD, 125 MHz), S (ppm) 180.7 (C-28), 138.0 (C-13), 125.4 (C- 12), 78.8 (C-3), 55.1 (C-5), 52.7 (C-18), 47.7 (C-17), 47.4 (C-9), 41.9 (C-14), 39.4 (C-8), 38.9 (C-19), 38.8 (C-20), 38.6 (C-1), 38.5 (C-4), 36.8 (C-22), 36.7 (C-10), 32.9 (C-7), 30.6 (C-21), 29.6 (C-15), 27.9 (C-23), 26.7. (C-2). 24.1 (C-16), 23.4 (C-27), 23.2 (C-11), 21.01 (C-30), 18.2 (C-6), 16.9 (C- 26), 16.8 (C-29), 15.5 (C-24), 15.3 (C-25).
Compound 3 (Oleanolic acid): white amorphous solid, mp. 307-308''C.
EI-MS (70 eV), m/z (%): 456 (M*, 4), 248 (100), 203 (79), 133 (45), 119 (20), 69 (24), 55 (28).
' H - N M R (500MHZ, CDCI3& MeOD), S (ppm) 5.26 (IH, t,J= 3.5 Hz, H-12); 3.17 (IH, dd, J= 11.5; 5.0 Hz, H-3); 2.85 (IH, dd, J= 14.0; 4.0 Hz, H-18); 1.17 (3H, s, CH3-27);
0.98 (3H, s, CH3-23); 0.81 (3H, s, CH3-24);
0.96 (3H, s, CH3-3O); 0.94 (3H, s, CH3-29);
0.94 (3H, s, CH3-29); 0.91 (3H, s, CH3-25);
0.78 (3H, s, CH3-26).
"C-NMR (125MHz, CDCI3& MeOD), S (ppm) 39.6 (C-1), 27.6 (C-2), 79.5 (C-3), 37.9 (C-4), 56.5 (C-5), 19.3 (C-6), 33.5 (C- 7), 40.3 (C-8), 48.7 (C-9), 37.9 (C-10), 23.8 Journal of Medicinal Materials, 2014, Vol .19, No.3 169
(C-U), 123.4 (C-12), 144.9 (C-13), 42.7 (C- 14), 28.6 (C-15), 24.3 (C-16), 47.4 (C-17), 42.4 (C-18), 46.9 (C-19), 31.4 (C-20), 34.7 (C-21), 33.8 (C-22), 28.6 (C-23), 15.8 (C- 24), 16.2 (C-25), 17.6 (C-26), 26.4 (C-27), 181.6 (C-28), 33.5 (C-30), 23.9 (C-29).
3. Result and discussion
Compound 1 and compound 4 were identified as /^-sitosterol, daucosterol by comparison of TLC characteristics under the same conditions and specfral data with authentic samples.
Compoimd 2 was obtained as a white amorphous solid. The mass spectrum (EI- MS) shows a molecular ion peak at rrt/z 456 M ^ (C3oH4g03) and Augments decay at 248, 203,189 corresponding to the refro Diels- Alder decay from urs-12-en skeleton of triterpen with OH group in ring A.
Its 'H-NMR showed the proton signal of hydroxymethin group [5H 3,20 ppm (IH, t, J
= 16.5 Hz, H-3)] corresponding to the carbon signal at 6c 78.8 (C - 3) in die '^C NMR spectrum. It also has six singlet methyls 5H 1.09 (H-27); 0.98 (H-26); 0.95 (H-25); 0.93 (H-30); 0.81 (H-23); 0.77 (H-24) and one doublet methyl 6H 0.86 (J = 6.5 Hz, H-29).
The presence of methin olefinic group was reflected by proton signal at .5H 5.24 (IH, t, J
= 7.5 Hz, H - 12) attached to atom C-12 (5c 125.4). The '^C-NMR indicated thuty carbon
signals in the broad band decoupled spectrum, seven methyl, nine methylene, seven methine and seven quartery carbon atoms from DEPT experiments. The chemical shifts at 5c 180.7 (C-28); 138.0 (C-13); 125.4 (C-12) were the characteristic peaks for ursan type of skeleton. From the above spectral data and comparison with published spectral data [7], compoimd 2 was identified as a triterpen named ursolic acid
Compound 3: The 'H- NMR and '"'C- NMR spectra of 3 were very similar to those of 2 with the spectral signals charaterized of triterpene ursan skeleton. The difference between compoimds 2 and 3 is the signal of the methyl group at position 29. In ^H NMR spectra of 2, this methyl group was a doublet signal at 8H 0.86 (J = 6.5 Hz) be due to its direct link witii the C-19 (5c 38.9) and interaction with the proton at position C-20 (5c 38.8). While die specfra of this methyl group of 3 was only singlet signals 5H 0.94.
This can be explained by the fact that the methyl group has moved into position and directly associated with C-20 (5c 31.4), therefore, C-20 appeared as a quartery carbon and C-19 (5c 46.9) as a methylene. Thus, compound 3 was identified as oleanolic acid, a structural isomer of compound 2, by comparison of then- spech^ data with those reported in literature [7].
170 Journal of Medicinal Materials, 2014, Vol .19, No. 3
R 1 H 4 Glu
Figure 1. The compounds isolated from the n- hexan extract of Polygonum barbatum L.
4. Conclusion (2), oleanoUc acid (3) and daucosterol (4) on the Two steroids and two triterpens were isolated basis of NMR, MS spectral analysis and m from the ihizoma of Polygonum barbatum L. comparison with the published NMR data. All collected from Nam Dinh, Vidnam. Thdr structures the compounds were reported for die first time were identified as ^sitosterol (1), ursolic acid &om±ePolygonum barbatum L.
References
1. Viehiam National Institute of Medicmal Materials (2006), Medicinal plants and animals medicine in Vietnam, vol 2, Publishers Science and Technology, pp. 382. 2. Queen, Rosaiy Sheela X, Alex, Ramani V (2011),
"Antimicrobial screening of Polygonum barbatum leaf extract", InternationalJournal of Current Pharmaceutical Research, 3(3), 117-US. 3. Hitesh Kumar Kinger, Mahesh Kumar Gupta (2012), "Wound healing activity of Polygonum barbatum Linn, (whole plant)", World journal of pharmacy and pharmaceutical sciences, 1(3), 1084- 1091. 4. Md. Abdul Mazid, Bidyut K. Datta, Lutfim Nahar and Satyajit D. Sarker. (2011), "Assessment of anritumour activity of two Polygonum species using potato disc assay", Bangladesh PharmaceuticalJoumal, 14(1), 37-40. 5. Queen, Rosary Sheela X, Alex, Ramani V (2011), "Pharmacognositic and phytochemical investigation on Itaf of Polygonum barbatum", International Journal of Current Pharmaceutical Research, 2(2), 174-179. 6. M.
Abdul Mazid, Bidyut K. Datta, Lutfim Nahar, S. A. M. Khairul Bashar, Sitesh C. Bachar and Satyajit D. SaHcer (2011), "Phytochemical Studies on Polygonum barbatum (L.) Hara var. barbata (Polygonaceae)", Rec. Nat. Prod, 5(2). 143-146. 7. Werner Seebacher, Nebojsa Simic, Robert Weis, Robert Saf, Olaf Kunert (2003), "Complete assignments of 'H and " C - NMR resonances of oleanolic acid, 18a-oleanoIic acid, ursolic acid and their 11-oxo derivatives", Magnetic Resonance in Chemistry, 41(8), 636-638.
Journal of Medicinal Materials, 2014, VoL 19, No, 3 (pp, 171-177)
PHARMACOGNOSTIC STUDY OF APIUM GRA VEOLENS L. SEEDS Nguyin Thu HSng'*, Ph^m Thiiy L'lnh^, HoangPhir&ng Thao',
Nguyin Nggc Chu', BUi Dinh Vi^^
' Department of Pharmacognosy, Hanoi University of Pharmacy, Ha Noi
^Department of Pharmacognosy, Thai Nguyen University of Medicine and Pharmacy, Thai Nguyen
* Corresponding author; [email protected] (Received April 25*, 2014)
Summary
Pharmacognostic Study of Apium graveolens L. Seeds
The Celery (Apium graveolens L.) belongs to family Apiaceae. It was cultivated through out of Vietnam as a vegetable. Celery seeds are used as a herbal medicine because of many important biological activities such as antihypertensive, anri-inflammatory, anticancer, antibiotic effects. The objective of current study was to carry
