Three natural compounds including betulin (1), betulinic acid (2) and lupeolactone (3) were isolated from the dichloromethane extract of Helicteres hirsuta Lour. for the first time. Their structures have been elucidated by modern spectroscopic methods as IR, 1H-NMR, 13C-NMR, DEPT, HSQC, HMBC, MS and by comparison with those of previously reported data.
AGU International Journal of Sciences – 2019, Vol (4), 12 – 19 DERIVATIVES OF TRITERPENE ISOLATED FROM THE DICHLOROMETHANE EXTRACT OF HELICTERES HIRSUTA LOUR Nguyen Van Ky1, Nguyen Huu Duyen1, Le Thanh Phuoc1 College of Natural Sciences, Can Tho University, Vietnam Information: Received: 07/08/2018 Accepted: 03/01/2019 Published: 11/2019 Keywords: Betulin, betulinic acid, lupeolactone, Helicteres hirsuta Lour ABSTRACT Three natural compounds including betulin (1), betulinic acid (2) and lupeolactone (3) were isolated from the dichloromethane extract of Helicteres hirsuta Lour for the first time Their structures have been elucidated by modern spectroscopic methods as IR, 1H-NMR, 13C-NMR, DEPT, HSQC, HMBC, MS and by comparison with those of previously reported data INTRODUCTION Lour was resistant to some experimental cancer cells (Chin YW et al., 2006) Helicteres hirsuta Lour was introduced in the previous paper of the research group (Duyen, N.H and Phuoc, L.T., 2016; Ky, N.V., et al., 2018) Regarding the bioactivities, the results of cytotoxic activity on Hep-G2 cell line of four extracts including petroleum ether, dichloromethane, ethyl acetate and methanol, showed that two extracts of petroleum ether and dichloromethane gave positive test results with the IC50 values being 28.29 µg/mL and 30.30 µg/mL, respectively Regarding the chemical constituents, the research team isolated six compounds including stigmasterol; lupeol; apigenin; tiliroside; α-p-hydroxy truxillic acid; 7,4’-di-O-methyl-8-O-sulphate isoscutellarein With the above-mentioned biological activities, Helicteres hirsuta Lour is potentially useful in cancer treatment Therefore, in this paper, the research team continues to publish results of a chemical composition survey to contribute to a more scientific explanation of the pharmaceutical application of this herb MATERIALS AND METHODS Material: The specimens were stems, leaves and flowers of Helicteres hirsuta Lour collected in Hon Son, Lai Son commune, Kien Hai district, Kien Giang province and identified by Mr Dang Minh Quan, Director of Department of Education, Can Tho Universit They were then washed, chopped, dried in shade, and ground to fine powder as raw materials used in the study In 2006, another research group announced the isolation of six compounds: (±)-pinoresinol, (±)-medioresinol, (±)-syringaresinol, (-)boehmenan, (-)-boehmenan H and (±)-transdihydrodiconiferyl alcohol It also stated that the extract from the trunk of Helicteres hirsuta Method for extraction: the dried powder was exhaustively extracted with methanol, then 12 AGU International Journal of Sciences – 2019, Vol (4), 12 – 19 proceeded to liquid-liquid extraction with solvents of increasing polarity, and obtains petroleum ether extract (PE), dichloromethane extract (DC), ethyl acetate extract (EA) and methanol extract (ME) Dried powder of the aerial parts of the Helicteres hirsuta Lour (12.5 kg) was put into a cloth bag and exhaustively extracted with methanol at room temperature After 24 hours, the crude extract was filtered with a filter paper and evaporated the solvent (3-4 times) to obtain ethanol extract (200 g) Then, this extract was suspended in distilled water (approximately 1:1) and partitioned with petroleum ether (PE), dichloromethane (DC), ethyl acetate (EA), and methanol (Me), respectively The partitioned solutions were removed solvent to give four extracts: PE (105.3 g), DC (20.85 g), EA (18.35 g) and Me (15.6 g) Method for isolation: Column chromatography was used to isolate compounds with silica gel (stationary phase) and elution solvent (mobile phase), and to monitor column chromatography and purity of compounds by thin layer chromatography (fractions with the similar characteristic on TLC were combined) Recrystallization was used for purifying the compound; the elution process started with petroleum ether (PE), followed by polarization by adding ethyl acetate (EA) in an appropriate ratio Detection was achieved by spraying the mixture of vanillin, methanol and 10% H2SO4, followed by heating, iron chloride (III) salt solution, UV light with 254 nm wavelength Silica gel (230-400 mesh, India, Germany) was used for column chromatography, Thin-layer chromatography was performed on pre-coated TLC F254 60 G (Merck, Germany) Isolation The DC extract was repeatedly subjected to silica gel column, eluted with petroleum solvent (PE) and the mixture of petroleum and ethyl acetate with increasing polarity to yield 12 fractions, DC1-12 Two fractions DC5 (PE: EA 9:1) and DC6 (PE: EA 9:1) were dissolved in pure solvent respectively in ascending order from petroleum, ethyl acetate to methanol; then recrystallized A white power was obtained in methanol from the fraction DC5, then checked by TLC (the mixture solvent of petroleum and ethyl acetate, PE: EA 4:1) with the show-up of a dark purple mark after spraying the mixture of vanillinmethanol-10% H2SO4 followed by heating (Rf = 0.37) This compound is denoted as PKD01 (1) Similarly, a white powder was obtained in methanol from the fraction DC6 with the Rf value being 0.33 in the same elution solvent This compound is denoted as PKD03 (3) Method for structural analysis: the structures of the compounds were determined by the spectroscopic methods: IR, 1H-NMR, 13CNMR, DEPT, HSQC, HMBC, MS and related documents to identify the chemical structure of the isolated substances The 1H- and 13C-NMR spectra were measured by Bruker 500 MHz equipment and the chemical shifts were given on a δ (ppm) scale with tetramethylsilane (TMS) as an internal standard, ESI-MS was recorded with a VG 7070 Mass spectrometer operating at 70 eV All spectra were recorded at Institute of Chemistry, Vietnam Academy of Science and Technology, Hanoi The fraction DC8 (PE: EA 7: 1) was chromatographed to silica gel column, eluted with the mixture of petroleum and ethyl acetate (PE: EA) being gradually increased in the polarity to obtain fractions DC8a (PE: EA 7:1), DC8b (PE: EA 5:1) and DC8c (EA 100%) The fraction DC8a was further purified by column chromatography to afford RESULT AND DISCUSSION 3.1 Extraction and isolation Extraction 13 AGU International Journal of Sciences – 2019, Vol (4), 12 – 19 subfractions: DC8a1 (PE: EA 10: 1), DC8a2 (PE: EA 7:1), DC8a3 (PE: EA 5:1) and DC8a4 (EA 100%) In the subfraction DC8a2, a pure needle-shaped crystalline compound was formed; then it was checked by TLC with the mixture of petroleum ether and ethyl acetate (PE: EA 2:1), showing a dark purple mark after spraying the mix of vanillin-methanol-10% H2SO4 following by heating (Rf = 0.15) This compound is denoted as PKD02 (2) Table The 1H-NMR, 13C-NMR data of compound 1, and COMPOUND No δH1 ppm (J in Hz) COMPOUND δC1 ppm COMPOUND δH2 ppm δC2 ppm (J in Hz) δH3 ppm (J in Hz) δC3 ppm 38.2 38.8 40.0 26.6 27.9 27.0 2.97 (1H, t, 5) 76.8 3.16 (1H, 5.5, 11.0) 38.5 0.62 (1H, s) 54.8 dd, 78.9 3.15 (1H, dd, 4.3, 7.0) 38.7 0.67 (1H, d, 9.5) 55.4 79.7 57.9 0.73 (1H, d, 10.0) 56.9 17.9 18.3 19.5 33.8 34.5 35.7 40.4 40.7 42.0 49.8 50.5 52.1 10 36.6 37.2 38.4 11 20.3 20.7 22.1 12 24.8 25.5 27.0 13 36.7 38.3 39.6 14 42.2 42.2 43.6 15 27.1 30.6 31.8 16 29.3 32.2 33.7 17 47.3 56.2 50.6 18 48.1 46.9 48.8 19 2.38 (1H, m) 47.3 3.0 (1H, td, 4.5) 49.4 3.09 (1H, td, 4.0) 48.5 20 150.3 150.7 152.3 21 29.0 29.7 30.9 22 33.8 37.0 38.4 23 0.87 (3H, s) 28.1 0.97 (3H, s) 27.1 14 1.95 (3H, s) 24.0 AGU International Journal of Sciences – 2019, Vol (4), 12 – 19 COMPOUND COMPOUND δH2 ppm (J in Hz) δC1 ppm (J in Hz) δC2 ppm 24 0.66 (3H, s) 15.7 0.95 (3H, s) 15.3 25 0.77 (3H, s) 15.9 0.82 (3H, s) 15.9 0.88 (3H, s) 16.7 26 0.98 (3H, s) 14.5 0.75 (3H, s) 16.0 1.00 (3H, s) 16.8 27 0.93 (3H, s) 15.7 0.94 (3H, s) 14,6 1.02 (3H, s) 15.1 28 3.52 (1H, 10.5) d, 179.2 0.77 (3H, s) 16.1 3.09 (1H, 10.0) d, 4.66 2.5) d, 109.4 4.72 (H, s) 110.0 No 29 δH1 ppm COMPOUND (1H, 57.9 109.5 4.73 (1H, s) 4.60 (1H, s) δH3 ppm (J in Hz) δC3 ppm 181.0 4.58 (H, d, 17.0) 4.53 (1H, dd, 1.0) 30 1.63 (3H, s) 18.7 1.69 (3H, s) 19.3 3.2 Structural identification 1.71 (3H, s) 19.6 methylene carbons and methyl carbons which were characterized for a lupeane-type triterpene In the HMBC, the correlative signals between the protons of methyl group δH 1.63 (δC 18.7, C-30) and double-bonded methylene group at δH 4.66 and 4.53 (δC 109.5, C-29) with the double-bonded quaternary carbon at δC 150.3 (C-20) and tertiary carbon of the cyclopentane ring of lupane frame δC 47.3 (C17) showed the presence of isopropyl group The characteristic carbon signals of the oxymethine group at δC 76.8 (C-3) and oxymethylene group at δC 57.9 (C-28) are also consistent with their proton signals on the 1HNMR spectrum and the correlation between the protons of methyl group δH 0.87 (δC 28.1, C23), 0.66 (δC 15.7, C-24) with the carbon of oxymethine group at δC 76.8 (C-3) and the tertiary carbon of the first cyclohexane ring of lupane frame at δC 38.5 (C-4), the correlation between the two protons of oxymethylene group at δH 3.52, 3.09 (δC 57.9, C-28) with the methylene carbon at δC 33.8 (C-22), 29.3 (C- Compound The ESI-MS spectrum indicated a molecular ion peak at m/z 443.1 [M]+, corresponding to the molecular formula C30H50O2 (M = 442.73 đvC) The 1H-NMR spectrum (500 MHz, MeOD, δH ppm) showed the signal of an oxymethin group at δH 2.97 (1H, t, 5.0); two signals of doublebonded methylene group at δH 4.66 (1H, d, 2.5 Hz) and 4.53 (1H, dd, 1.0 Hz); two signals of oxymethylene group at δH 3.52 (1H, d, 10.5 Hz) and 3.09 (1H, d, 10.0 Hz); six signals of methyl group in the range δH 0.66-1.63 There were also some signals of other methine and methylene groups The 13C-NMR spectrum (125 MHz, MeOD, δC ppm) in combination with DEPT-90 and DEPT-135 spectra presented the 30 signals of carbon atoms in total Among them, quaternary carbons, methine carbons, 12 15 AGU International Journal of Sciences – 2019, Vol (4), 12 – 19 same IC50 value of 40 μg/mL Betulin also exhibits anti-HIV activity with the IC50 value of 6.1 μg/mL (Deed K.S.El et al., 2003) In addition, betulin has a protective effect on the liver and reduces the toxicity of CdCl2 at low concentration of 0.1 μg/mL The mechanism can be explained that betulin promotes the synthesis of proteins, which protect cells from the effects of CdCl2 (Nobuhiko Muira et al., 1999) 16) At the same time, with two oxygenated groups, it is allowed to indicate the presence of -CH2-OH and >CH-OH groups in the molecule of the compound From the analysis of the above data combined with published data of betulin (Khanh, T.C., et al., 2007), compound was identified as betulin (Figure 1) Betulin shows that there is a cytotoxic activity on two cell lines (HeLa and Hep-2) with the Figure Structure and HMBC correlations of betulin Comparing data with Betulinic acid–IR (KBr, νmax cm-1) 3484 (–OH), 1686 (C=O), 1362 (CH2=CH-CH3), 1156 (C–O) (E KovacBesovic et al., 2009), it can be concluded that compound has an oxymethine group and a carboxylic functional group (–COOH) Compound Infrared spectrum (IR), (KBr, νmax cm-1), the signal at 3432.04 cm-1 is the characteristic oscillation of the OH bond, 1688.90 cm-1 (C=O bond), 1642.64 cm-1 (C=C bond), 1378.35 cm-1 (CH3 group), 1235.85 cm-1 (C–O bond) 16 AGU International Journal of Sciences – 2019, Vol (4), 12 – 19 The 1H-NMR spectra (500 MHz, CDCl3 and MeOD, δH ppm) of compound also appeared similar proton signals but less than compound by one oxymethylene group From the analysis of the above data combined with the published data of betulinic acid (Enamul Haque Md et al., 2006), compound was identified as betulinic acid (Figure 2) The 13C-NMR spectrum (125 MHz, CDCl3 and MeOD, δC ppm) combined with DEPT-90, DEPT-135 spectrum of compound also showed the total signals of 30 carbon atoms similar to compound 1, but one oxymethylene group less and one carboxylic carbon more at δC 179.2 Betulinic acid is a substance commonly found in plants that has antimicrobial, antiinflammatory, anti-malarial and anti-cancer properties (Perumal Yogesswari & Dharmarajan Sriam, 2005) Figure Structure of betulinic acid secondary carbon at δC 57.9 (C-4) The presence of isopropenyl group is similar to compound 1, base on the HMBC correlations between the protons of methyl group at δH 1.71 (δC 19.6, C-30) and double-bonded methylene group at δH 4.72 and 4.58 (δC 110.0, C-29) with the double-bonded quaternary carbon at δC 152.3 (C-20) and tertiary carbon of cyclopentane ring of lupane frame at δC 50.6 (C-17) Compound The ESI-MS spectrum showed a pseudomolecular ion peak at m/z 439.2 [M+H]+, corresponding to the molecular formula C30H46O2 (M = 438.3 đvC) The 1H-NMR spectrum (500 MHz, MeOD, δH ppm) of compound also appeared proton signals similar to compound In particular, the proton signal of methyl group at δH 1.95 is different from compound From the analysis of the above data combined with the data of compound (betulinic acid), compound was identified as lupeolactone in comparison with published data (Kikuchi Hiroyuki et al., 1983) (Figure 3) The 13C-NMR spectrum (125 MHz, MeOD, δC ppm) combined with spectrum of DEPT-90, DEPT-135 of compound showed the signals of a total of 30 carbon atoms similar to compound But compound has the presence of the lactone ring identified by the correlation in HMBC spectrum between the proton of methyl group at δH 1.95 (δC 24.0, C-23) with the carbonyl carbon at δC 181.0 (C-24), oxymethine carbon at δC 79.7 (C-3) and Lupeolactone was found to lower cholesterol levels in normal and hypercholesterolemic rats by oral administration (Serina L Robinson, et al., 2018) 17 AGU International Journal of Sciences – 2019, Vol (4), 12 – 19 Figure Structure and HMBC correlations of lupeolactone CONCLUSION Forsskaoliana Saudi Pharmaceutical Journal, 11(4), 184-191 From dichloromethane extract, three triterpene derivatives were isolated and identified: betulin, betulinic acid and lupeolactone All of them were the first compounds isolated from Helicteres hirsuta Lour This study contributes to a more scientific explanation of the medicinal properties of this herb (in which betulin exhibits cytotoxic activity on the HeLa and Hep-2 cell lines, which protect the liver and reduce the toxicity of CdCl2; betulinic acid has antibacterial, anti-inflammatory, antimalarial and anti-cancer activity, according to previous studies) and increases the phytochemistry data of Helicteres hirsuta Lour to 15 compounds Duyen, N.H., & Phuoc, L.T., (2016) Study on chemical constituents and cytotoxic activity on Hep-G2 cell lines of Helicteres hirsuta L Can Tho University Journal of Science, Part A: Natural Science, Technology and Environment, 47, 93-97 E Kovac-Besovic, K Duric, Z Kaloðera & E Sofic (2009) Identification and isolation of betulin, betulinic acid and lupeol from birch bark Planta Medica, 75(09), 126-133 http://dx.doi.org/10.1055/s-0029-1234938 Enamul Haque Md, Hussain Uddin Shekhar, Akim Uddin Mohamad, Hafizur Rahman, Mydul Islam AKM & Sabir Hossain M (2006) Triterpenoids from the stem bark of Avicennia officinalis Dhaka Univ.J Pharm.Sci., 5(1-2), 53-57 https://doi.org/10.3329/dujps.v5i1.229 REFERENCES Chin YW, Jones WP, Rachman I, Riswan S, Kardono LB, Chai HB, Farnsworth NR, Cordell GA, Swanson SM, Cassady JM & Kinghorn AD (2006) Cytotoxic lignans from the stems of Helicteres hirsuta collected in Indonesia Phytotherapy Research, 20(1), 6265 https://doi.org/10.1002/ptr.1806 Khanh, T.C., Hang, N.T.M., Huong, D.T.M & Hung, N.V., (2007) Study on the chemical composition of roots of Pseuderanthenum palatiferum Vietnamese Journal of Science and Technology, (DB), 309-314 Deed K.S.El, Al-Haidari RA, Mossa J.S & Abdel Monem A (2003) Phytochemical and Pharmacological studies of Maytenus 18 AGU International Journal of Sciences – 2019, Vol (4), 12 – 19 Kikuchi Hiroyuki, Tensho Akira, Shimizu Iwao, Shiokawa Hideaki, Kuno Atsushi, Yamada Seiichiro,… Tomita Kenichi (1983) Lupeolactone, a new βlactone from Antidesma pentandrum Merr Chemistry Letters 1983 (4), 603606 https://doi.org/10.1246/cl.1983.603 triterpene compounds against the cytotoxicity of Cadmium in Hep-G2 Cells Molecular Pharmacology, 56, 1324-1328 https://doi.org/10.1124/mol.56.6.1324 Perumal Yogesswari & Dharmarajan Sriam (2005) Betulinic acid and its derivatives: A review on their biological properties Current Medicinal Chemistry, 12, 657666 https://doi.org/10.2174/092986705320 2214 Ky, N.V., Phuoc, L.T & Duyen, N.H., (2018) Sulphated and α-truxillic-acid derivatives of flavonoids isolated from the dichloromethane extracts of Helicteres hirsuta Lour Vietnamese Pharmaceutical Journal, Research-Technoques, 505 (58), 57-60 Serina L Robinson, James K Christenson & Lawrence P Wackett, (2018) Biosynthesis and chemical diversity of β-lactone natural products Natural Product Reports, View Article Online https://doi.org/10.1039/c8np00052b Nobuhiko Muira, Yoko Matsumoto, Shinichi Miyairi, Shoji Nishiyama & Akira Naganuma (1999) Protective effects of 19 ... extract, three triterpene derivatives were isolated and identified: betulin, betulinic acid and lupeolactone All of them were the first compounds isolated from Helicteres hirsuta Lour This study... Duyen, N.H., (2018) Sulphated and α-truxillic-acid derivatives of flavonoids isolated from the dichloromethane extracts of Helicteres hirsuta Lour Vietnamese Pharmaceutical Journal, Research-Technoques,... (DC), ethyl acetate extract (EA) and methanol extract (ME) Dried powder of the aerial parts of the Helicteres hirsuta Lour (12.5 kg) was put into a cloth bag and exhaustively extracted with methanol