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.
Trang 1DERIVATIVES OF TRITERPENE ISOLATED FROM THE DICHLOROMETHANE
EXTRACT OF HELICTERES HIRSUTA LOUR
Nguyen Van Ky1, Nguyen Huu Duyen1, Le Thanh Phuoc1
1
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
1 INTRODUCTION
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
In 2006, another research group announced the
isolation of six compounds: (±)-pinoresinol,
(±)-medioresinol, (±)-syringaresinol,
(-)-boehmenan, (-)-boehmenan H and
(±)-trans-dihydrodiconiferyl alcohol It also stated that
the extract from the trunk of Helicteres hirsuta
Lour was resistant to some experimental
cancer cells (Chin YW et al., 2006)
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
2 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
Method for extraction: the dried powder was
exhaustively extracted with methanol, then
Trang 2proceeded 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)
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)
Method for structural analysis: the structures
of the compounds were determined by the
spectroscopic methods: IR, 1H-NMR, 13
C-NMR, 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
3 RESULT AND DISCUSSION
3.1 Extraction and isolation
Extraction
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)
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 vanillin-methanol-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)
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 3 fractions DC8a (PE: EA 7:1), DC8b (PE: EA 5:1) and DC8c (EA 100%) The fraction DC8a was further purified
Trang 3subfractions: 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 1 The 1H-NMR, 13C-NMR data of compound 1, 2 and 3
No
δH1 ppm
(J in Hz)
δC1
ppm
δH2 ppm
(J in Hz)
δC2
ppm
δH3 ppm
(J in Hz)
δC3
ppm
3 2.97 (1H, t, 5) 76.8 3.16 (1H, dd,
5.5, 11.0)
78.9 3.15 (1H, dd, 4.3,
7.0)
79.7
5 0.62 (1H, s) 54.8 0.67 (1H, d, 9.5) 55.4 0.73 (1H, d, 10.0) 56.9
19 2.38 (1H, m) 47.3 3.0 (1H, td, 4.5) 49.4 3.09 (1H, td, 4.0) 48.5
Trang 4No
δH1 ppm
(J in Hz)
δC1
ppm
δH2 ppm
(J in Hz)
δC2
ppm
δH3 ppm
(J in Hz)
δC3
ppm
28 3.52 (1H, d,
10.5)
3.09 (1H, d,
10.0)
29 4.66 (1H, d,
2.5)
4.53 (1H, dd,
1.0)
109.5 4.73 (1H, s)
4.60 (1H, s)
109.4 4.72 (H, s)
4.58 (H, d, 17.0)
110.0
3.2 Structural identification
Compound 1
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 1 H-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
double-bonded 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 13 C-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, 6
quaternary carbons, 6 methine carbons, 12
methylene carbons and 6 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 (C-17) 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 1 H-NMR spectrum and the correlation between the
protons of methyl group δH 0.87 (δC 28.1, C-23), 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
Trang 516) 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 1 was identified as
betulin (Figure 1)
Betulin shows that there is a cytotoxic activity
on two cell lines (HeLa and Hep-2) with the
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)
Figure 1 Structure and HMBC correlations of betulin
Compound 2
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)
Comparing data with Betulinic acid–IR (KBr,
νmax cm-1) 3484 (–OH), 1686 (C=O), 1362 (CH2=CH-CH3), 1156 (C–O) (E
Kovac-Besovic et al., 2009), it can be concluded that
compound 2 has an oxymethine group and a
carboxylic functional group (–COOH)
Trang 6The 1 H-NMR spectra (500 MHz, CDCl3 and
MeOD, δH ppm) of compound 2 also appeared
similar proton signals but less than compound 1
by one oxymethylene group
The 13 C-NMR spectrum (125 MHz, CDCl3 and
MeOD, δC ppm) combined with DEPT-90,
DEPT-135 spectrum of compound 2 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
From the analysis of the above data combined with the published data of betulinic acid
(Enamul Haque Md et al., 2006), compound 2
was identified as betulinic acid (Figure 2)
Betulinic acid is a substance commonly found
in plants that has antimicrobial, anti-inflammatory, anti-malarial and anti-cancer properties (Perumal Yogesswari &
Dharmarajan Sriam, 2005)
Figure 2 Structure of betulinic acid
Compound 3
pseudomolecular ion peak at m/z 439.2
[M+H]+, corresponding to the molecular
formula C30H46O2 (M = 438.3 đvC)
The 1 H-NMR spectrum (500 MHz, MeOD, δH
ppm) of compound 3 also appeared proton
signals similar to compound 2 In particular, the
proton signal of methyl group at δH 1.95 is
different from compound 2
The 13 C-NMR spectrum (125 MHz, MeOD, δC
ppm) combined with spectrum of DEPT-90,
DEPT-135 of compound 3 showed the signals
of a total of 30 carbon atoms similar to
compound 2 But compound 2 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),
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)
From the analysis of the above data combined
with the data of compound 2 (betulinic acid), compound 3 was identified as lupeolactone in
comparison with published data (Kikuchi
Hiroyuki et al., 1983) (Figure 3)
Lupeolactone was found to lower cholesterol levels in normal and hypercholesterolemic rats
by oral administration (Serina L Robinson, et al., 2018)
Trang 7Figure 3 Structure and HMBC correlations of lupeolactone
4 CONCLUSION
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
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