Chemical Composition of Vietnamese Black Lingzhi Amauroderma subresinosum Murr
Fatty acid composition of Vietnamese black lingzhi, Amauroderma subresinosum was investigated by GC (Gas chromatography) with authentic samples. Fourteen fatty acids were identified, in which the concentration of unsaturated fatty acid, 11-octadecaenoic is the highest (around 51.01% of total fatty acids). Furthermore, two fatty acids, lignoceric acid and 11-octadecaenoic together with three esters of cholesta-7,22-dien-3β-ol with three fatty acids 14- methylpentadecanoic, 8,11-octadecaenoic and 8-octadecenoic with the ratio 1/1/1 were isolated and structural elucidated. These results support a new evidence for classification of Ganodermataceae family, in which A. subresinosum is placed in the distinct genus Amauroderma, isolated from Ganoderma, since it does not produce any triterpenoid, typically elaborated from Ganoderma species, particularly G. lucidum.
Received: June 14, 2011;
Accepted: July 28, 2011;
Published: October 29, 2011
The fungal family Ganodermataceae is represented by about 322 species (Foroutan
and Vaidya, 2007), which mostly occur in subtropical and tropical regions.
The fruiting bodies of Ganoderma species have been widely used in traditional
Chinese, Japanese, Korean and Vietnamese medicine to treat a variety of conditions
from ancient time (Kleinwachter et al., 2001).
In addition, Ganoderma species could also be used as feed supplement
in animal diet since it produced various compounds such as steroids, cardiac
glycosides, saponins, resins and amino acids (Ogbe et
al., 2009). Interesting biological activities have been observed in
these mushrooms; Ganoderma lucidum showed cytotoxic (Min
et al., 2000) and antiviral activities (El-Mekkawy
et al., 1998), Ganoderma colossum showed anti-inflammatory,
cytotoxic (Kleinwachter et al., 2001) and antimicrobial
activities (Ofodile et al., 2005), Ganoderma
applanatum (Lee et al., 2006) revealed antibacterial
and aldose reductase inhibitory activities, Ganoderma concinna induced
apoptosis in human promyelocytic leukemia HL-60 cells (Gonzalez
et al., 2002) and Ganoderma pfeifferi showed antimicrobial
activity (Mothana et al., 2000). These basidiomycetes
are known to be prolific producers of lanostane type triterpenoids and over
130 such compounds have been recognized from the genus Ganoderma. However,
chemical investigation on Vietnamese Ganoderma is very poor. So far,
only isolation of colossolactones from G. colossum was reported (Kleinwachter
et al., 2001). In the course of our investigation on Vietnamese fungi,
we study the chemical constituents of A. subresinosum in order to get
more evidence for its classification as well as to look for novel bioactive
substances. This study reports the fatty acid composition as well as the isolation
of two fatty acids and ester of sterol from fruit bodies of A. subresinosum.
MATERIALS AND METHODS
Material: Fruit bodies of A. subresinosum was collected in Cattien
National Park in November 2008 and identified by L.X. Tham. Voucher specimens
were deposited at faculty of Chemistry, Hanoi University of Education (ND0801).
General: NMR spectra were recorded on Varian Brucker Avance 500 MHZ,
using CDCl3 as solvent. Chemical shifts are given with TMS used as
internal standard (1H-NMR) and δ 77.03 (ppm) from CDCl3
as a standard (13C-NMR). Mass spectra were recorded on a JOEL JMS
AX-500 spectrometer. Fatty acid content was analyzed by using a Gas chromatography
(Finnigan Trace GC) with an ultra-column BPX70. The temperature programming
of GC analysis was performed from 50°C isothermal for 3 min, then 50-250°C
at 4°C min-1 and finally isothermal at 250°C for 10 min.
TLC was performed on silica gel plates (Kieselgel 60 F254, Merck) and reversed
phase C18 silica gel plates (Merck).
Extraction and isolation: Dried A. subresinosum (230 g) was extracted with methanol. The methanolic extract was concentrated to give a residue (6.8 g) which was further divided into four fractions. Fraction 2 (278 mg) was purified by reversed phase RP-18 column, using MeOH as solvent to afford compound 1 (30 mg). Compounds 2 (40 mg) and 3 (52 mg) were obtained from fraction 3 (1068 mg) by silica gel column, eluting with CHCl3.
Hydrolysis of 3: The mixture 3 (10 mg) in MeOH (2 mL) was added KOH (5 mg) and the mixture was stirred at room temperature for 3 h. Then, the reaction mixture was concentrated and water (2 mL) was added and extracted with n-hexane (2 mL) for 2 times. The n-hexane extract was further purified by silica gel column, using n-hexane: EtOAc = 4:1 (v/v) as eluent to yield compound 4 (2.5 mg). The water phase was neutralized and methylated as the same method as mentioned below and analyzed by GC to determine the fatty acid content.
Methylation of crude extract: Crude extract of A. subresinosum (50 mg) was added 5 mL H2SO4 in methanol in a flask and the mixture was stirred at 80°C for 4 h. The reaction mixture was concentrated, then added 2 mL of water and finally extracted with n-hexane (2 mL). The n-hexane extract was directly injected to GC for fatty acid analysis.
||Compound 1: 1H NMR (CDCl3): δ
0.88 (3H, t, J = 6.5 Hz, H-24), 1.28 (m, from H-5 to H-22), 1.30 (2H, m,
H-23), 1.66 (4H, m, H-3 and H-4), 2.34 (2H, t, J = 7.5 Hz, H-2)
||Compound 2: 1H NMR (CDCl3): δ 0.88 (3H,
t, J = 6.4 Hz, H-18), 1.40 (m, from H-4 to H-9 and H-14 to H-17), 2.34 (2H,
m, H-13), 2.78 (2H, m, H-10), 5.30 (2H, m, H-11 and H-12).
||Compound 2: 13C NMR (CDCl3): δ 14.0
(C-18), 29.3 (C-3), 29.4 (C-14 to C-17), 29.6 (C-4 to C-9), 31.9 (C-10,
C-13), 34.0 (C-2), 129.7 (C-12), 130.0 (C-11), 179.8 (C-1)
||Compound 4: EI-MS: m/z 398. 1H NMR (CDCl3):
δ 0.54 (6H, s, H-18 and H-19), 0.82 (3H, d, J = 6.9 Hz, H-27), 0.84
(3H, d, J = 6.9 Hz, H-26), 0.91 (3H, d, J = 6.9 Hz, H-28), 1.02 (3H, d,
J = 6.6 Hz, H-21), 3.60 (1H, m, H-3), 5.15 (1H, m, H-7), 5.17 (1H, m, H-23),
5.20 (1H, m, H-22). 13C NMR (CDCl3): δ 11.8 (C-18),
12.1 (C-19), 17.9 (C-28), 19.6 (C-26), 20.0 (C-27), 21.1 (C-21), 22.7 (C-15),
23.0 (C-11), 27.6 (C-6), 28.1 (C-2), 28.3 (C-16), 33.1 (C-25), 34.5 (C-10),
37.0 (C-1), 39.4 (C-4, C-12), 40.0 (C-20), 42.8 (C-24), 42.9 (C-5), 43.3
(C-13), 49.4 (C-9), 55.8 (C-14), 56.8 (C-17), 71.1 (C-3), 117.5 (C-7), 131.9
(C-23), 135.6 (C-22), 139.6 (C-8)
RESULTS AND DISCUSSION
Table 1 shows that fatty acid composition of A. subresinosum.
Accordingly, fourteen major fatty acids were found. The concentration of unsaturated
fatty acid, 11-octadecaenoic is highest (51.077%), followed by 9,12- octadecadienoic
(22.979%) which is dominant in Rumen liquor (Achenef et
al., 2009) and hexadecanoic (19.05%). Consequently, total unsaturated
fatty acids in A. subresinosum are about 80% of total fatty acid. Analysis
of fatty acid composition of several Ganoderma sp. also has revealed
that they possess mainly polyunsaturated fatty acids (PUFAs), especially 18-carbon
unsaturated fatty acids (Martinez et al., 1991).
Previous experimental suggest that one of the food components that reduces serum
cholesterol and high blood pressure is PUFAs content (Bavelaar
and Beynen, 2004; Abd El-Baky et al., 2004).
PUFAs also are anticancer (Ndem et al., 2008)
and antioxidant agents (Ogbuewu et al., 2010).
In addition, repeated column chromatographs resulted in the isolation of three
compounds. Compound 1 was obtained as an oil. Analysis of its 1H
NMR spectrum suggests that it contains one methyl group (δH
0.88 ppm) and no proton signal at low field, thus, it should be a saturated
fatty acid. By comparing its NMR data with those of previous publication, compound
1 is characterized as lignoceric acid (Kang et al.,
2011; Onwuliri et al., 2004). Compound 2
was also obtained as white powder. Interpretation of its 1H and 13C
NMR revealed that it is an unsaturated fatty acid with one double bond at C11-12
in its molecule. Thus, compound 2 was found to be 11-octadecaenoic as shown
in Fig. 1.
Compound 3 was obtained a white powder. From TLC (both silica gel and reversed
phase) analysis, only one spot was detected. However, analysis of its 1H
NMR suggests that it has two moieties, fatty acid and sterol parts attached
together by an ester linkage. Thus, EI-MS was recorded and several peaks at
m/z 600-700 were observed. Then, compound 3 was treated with KOH and extracted
|| Fatty acid compositions (percentage of total fatty acids)
of A. subresinosum
|| Structures of compounds 1-4
Compound 4 was obtained from n-hexane extract. Its EI-MS shows the molecular
peak at m/z 398. The 1H and 13C-NMR spectral data of 4
showed the presence of four tertiary methyls, one tertiary alcohol (δC
71.1), three olefinic protons (δH 5.15, 5.17 and 5.20). On the
basis of the above spectral data, 4 was deduced to be a sterol (Reich
et al., 1969). The structure of 4 was then determined by 2D NMR spectra.
The position of the first double bond was found at C7-8 by the HMBC
correlation between H-6, H-9 and H-14 to C-7 and C-8. There are HMBC correlations
between H-21, H-24, H-28 to C-22 and C-23, thus, the second double bond was
decided at C22-23. Therefore, compound 4 was characterized as cholesta-7,22-dien-3β-ol.
The fatty acid moiety was later analyzed by GC analysis. The result shows that it is a mixture of three fatty acids, 14- methylpentadecanoic, 8,11-octadecaenoic and 8-octadecenoic with the ratio 1/1/1. Consequently, compound 3 is a mixture of three esters (3A-C) of cholesta-7,22-dien-3β-ol with three fatty acids 14- methylpentadecanoic, 8,11-octadecaenoic and 8-octadecenoic with the ratio 1/1/1.
Even, Ganoderma sp. has already been characterized by a combination
of morphological, chemotaxonomy and sequences of rDNA 26S. But, the morphological
concepts for identification of Ganoderma sp. in tropical area is not
well-established (Zakaria et al., 2009). Therefore,
these above results play a very important role in classification and support
a new and further taxonomy of the Ganodermataceae family. A. subresinosum
is now isolated from Ganoderma genus and created a distinct genus Amauroderma,
since it does not produce any lanostane triterpenoid, typically elaborated from
Ganoderma species, particularly G. lucidum (Tham,
2005). This classification is in good agreement with that by examining its
basidiospore structure as well as complete sequencing of D1, D2 regions (26S)
of A. subresinosum (Tham, 1998).
Chemical composition of black lingzhi A. subresinosum collected from Cattien National Park has been investigated. Fourteen fatty acid are found in its crude extract. Fruit bodies of A. subresinosum mainly produce unsaturated fatty acids which cover about 80% of total fatty acids. In addition, from its MeOH extract, two fatty acids, lignoceric acid and 11-octadecaenoic together with three esters of cholesta-7,22-dien-3β-ol with three fatty acids 14- methylpentadecanoic, 8,11-octadecaenoic and 8-octadecenoic with the ratio 1/1/1 were purified and structural elucidated.
Financial supports of Ministry of Education and Training (project No. B2010-17-239) and International Foundation of Science, IFS (Sweden) (project No. F/4896-1) are greatly acknowledged.
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