a study on some chemical constituents of ganoderma lucidum
TRANSCRIPT
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MM0900109
Jour. Myatu Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany
A Study on Some Chemical Constituents of Ganodermalucidum Leyss. Ex. Fr. p. Karst (vifZD;rId) from
Pyin-Oo-Lwin AreaKhine Kyi OO1, Aye Aye Tun2
AbstractA study on some chemical constituents of G.lucidum (vifZD;) which growsthroughout Myanmar has been undertaken. Two compounds, Ergosterol andits ester from mushroom body have been isolated by solvent extractionfollowed by column chromatography, Isolated compounds were identifiedby spectroscopic techniques. Lingzhi is traditionally utilized for loweringblood cholestrol, regulate binod pressure, prevent heart disease, andregulate blood sugar in diabetic patients and increase body immunity.
Key words: Medicinal Plants, Chemical Studies, G.lucidum (lingzhi)
Introduction
G.lucidum leyss. Ex.Fr.p. Karst is the most famous medicinal fungusall over the world. It was discovered in China two thousand years ago and inJapan it is known as "Man Nen Take" (10,000 year mushroom). Collectiontrips on Ganoderma species gave the knowledge that this mushroom wasknown in Myanmar for a long time as "Ut-Htoo-Hmo (or) Ut-Site-Hmo."
G.lucidum is a species of Basidiomycetes which belongs to the familyPolyporaceae (Ganodermataceae) and the order Aphyllophorales. Its fruitingbodies are called "Reishi" in Japanese, "Lingzhi" in Chinese, "Chaga" inRussian and "Lingchich" in American.
The whole plant of G.lucidum (Lingzhi) grows in densely woodedmountains of high humidity and dim lighting in China and Japan. It growsmainly on the dried trunks of dead maples and other decaying hard woodsrather than on conifers.
In Myanmar, G.lucidum was found to the growing wild in moist, darkforest area, attached close to rotten tree logs and decaying woods.
'. Assistant Lecturer and Ph.D Candidate Department of Botany, YU.2. Dr. Associate Professor, Department of Chemistry, YU.
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238 Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany
In Chinese, always kept Lingzhi (Red) as a symbol of "Long - life theKing", upper class hung it on their doors for good luck and Health (WillardTerry, 1990).
Present work, involves extraction, isolation and characterization of twocompounds from the mushroom body of G.lucidum which is growing wild inMandalay Division from Pyin-Oo-Lwin area.
Extraction was carried out by using Soxhlet apparatus with petroleumether (PE) (60-80 C) for 8 hours. The crude extract after evaporation was thensubjected to column Chromatographic separations followed by crystallization.
Ergosterol and its ester have been isolated and reported in this paper.The purity of isolated compounds was investigated by TLC and melting pointsdetermination. Their structures have been characterized by UV and FT-IRspectroscopy.
Materials and Methods
General Procedures
UV: Shimadzu UV-240, Cyclohexane; FT-IR: Perkin Elmer GXsystem, KBr; CC: Merck silica gel 60 (70-730 mesh), eluents: Petroleumether: ethyl acetate (PE:EtoAc); TLC: 0.25 mm precoated silica gel (60 F254,Merck), solvent system PE: EtoAc, spots were detected by inspection underUV light (254 nm or 365 nm) or by colour developed with spraying 10%sulphuric acid (or) anisaldehyde, Libermann - Burchard followed by heating.
Plant Materials
A Lingzhi mushroom used in this study was collected during Augustto September 2003, from Pyin-Oo-Lwin Township, Mandalay Division. Thesemushrooms were identified as G.lucidum in Department of Botany, YangonUniversity. These mushrooms were crushed, carefully washed with water andallowed to dry in shady place. After being dried in air for 3 weeks, it wasground to powder by using grinding mill and then stored in glass bottles.
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Jour. My an. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany 239
Extraction and Isolation of Compounds
The dried powder lingzhi fungus 100.0 g were extracted withpetroleum ether (500 cm3) for 8 hours in Soxhlet extractor.
Removal of the solvent provided crude extract (1.1 g), which waschromatographed on silica gel (50 g) in Petroleum ether (100 ml).
The column was eluted consecutively with petroleum Ether 100 cm3,column size 2.0 cm in diameter, 1% Pet-EtoAc (100 cm3), 2% Pet-EtoAc(100 cm3), 5% Pet-EtoAc (100 cm3), 10% Pet-EtoAc (100 cm3), 15% Pet-EtoAc (100 cm3), 20% Pet-EtoAc (100 cm3), 30% Pet-EtoAc (100 cm3) and40% Pet-EtoAc (100 cm3). A quantity of (5 cm3) was collected for eachfraction and the chromatography was monitored by TLC. The fractions thatgave similar TLC pattern were combined together and concentrated. In thisway, four combined fractions were obtained.I.e. Fri to
From Friv (95 mg) of sterol compound I (Si) was isolated as crystal of
colourless needles with melting point (164C) and the yield percent was found
to be 0.09%.
From Fri (27 mg) of sterol compound II (S2) was isolated as colourlessplates with melting point 108C and the yield percent was found to be 0.03%.The crystals of isolated Ergosterol and its ester are shown in Figure I.
Respective Rf values on TLC were 0.23 (Si) and 0.86 (S2) inPet:EtoAc (4:1) when the chromatogram was treated with anisaldehydereagent followed by heating the spot having Rf value of 0.23 was found tochange to pink purple, indicating the characteristic of ergosterol (or) steroid.
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Result and Discussion
Identification of St as Ergosterol by Thin Layer Chromatography (TLC)method
The isolated compound Si was checked by TLC to determine itspurity. It appeared as a single spot on thin layer chromatogram and Rf valuescoincide with authentic ergosterol in all solvent systems. Therefore, S| wascharacterized as ergosterol.
Melting Point Determination
The melting point of isolated compound (Si) was found to be 164°C(Merck Index 1996} comparable with the melting point of ergosterol fromliterature. (Ref: 163-164°C, Phytochemistry, 1984)
Identification of Isolated compound Si by spectral data
After being identified, the isolated constituent of G.lucidum as 1ergosterol by column chromatographic data and crystal form, it was confirmed ;by UV spectral data. Spectral maxima of ergosterol (X max) in cyclohexane ;were (X 218,270, 280, and 295) indicating it consists of diene chromophore.
Identification of Isolated compound Si by FT-IR spectrum i
The FT-IR spectrum of isolated compound Si is shown in Figure II. j
A strong and broad band centered at 3434 cm"1 was attributed to OHstretching vibration band. Asymmetric and symmetric u C-H stretching bandsappeared at 2954 and 2870 cm'1. A medium band at 1643 was due to c = c Istretching vibration bands. Symmetric and asymmetric bending vibration of-CH3 and -CH2- groups absorbed at 1458, 1382 and 1369 cm"1. In addition, a Istrong band at u 1039 cm"1 was attributed to the c - o stretching vibration Ibands. \
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Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany 241
All these bands assignment support the identity of isolated compoundas ergosterol with the following molecular structure.
HO \
Characterization of isolated compound S2 as fatty acid ester of ergosterol
Rf values of isolated compound S2 has higher than that of compoundSi (identified as ergosterol). The melting point of S2 was measured tabe lowerthan that of Si. Bothcompounds gave colour reaction when treating withLibermann Burchard reagent followed by heating.
The X max values of S2 in cyclohexane is (211, 226, 271,280, 294 nm)which were found to be very similar with S1.
The main differences in their FT-IR spectra is that compound S2revealed the presence of c = o stretching band at u 1740 cm*1 which did notappear in S\. In addition, the c-o stretching vibration band of S2 absorbed at1178 cm"1 which is higher when comparing with the c - o stretching vibrationband of Si (1039). From these observations, isolated compound S2 could becharacterized as fatty acid ester of ergosterol, most of probably, (literature mp.107-108°C) when comparing with melting point reported in literature.
Conclusion
Two compounds, (0.09% and 0.03 % in yield) have been isolated frompetroleum ether extract of lingzhi. The isolated compounds have beencharacterized by melting point examination and spectroscopic measurement(UV and FT-IR). Thus, the two compounds are ergosterol and fatty acid esterof ergosterol.
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242 Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany
AcknowledgementsThe authors wish to thank Department of Higher Education, Ministry of Education,
and Yangon, Myanmar for provision of opportunity to do this research.
I wish to express my heartfelt thanks to Dr. Daw Aye Kyi, Professor& Head, Dr.Aung Aung Min, Professor, Department of Botany, University of Yangon, for permitting meto undertake this research in the department.
My particular thanks to Dr. Maung Maung Htay, Professor& Head, Department ofChemistry, Yangon University, for the permission to use their laboratory facilities.
I would like to thanks to my supervisor Dr. Daw Aye Aye Tun, Associate Professor,Department of Chemistry, Yangon University for supervisions this research work withconstructive criticisms and invaluable advice.
I also wish to thank all of my friends and colleagues from Department of Chemistryfor their help and cooperation.
Fractionation of Phytoconstituents using PE solvent
Extracted with PE (60-80°C)(In Soxhlet; 8h)
PE Layer Residue
evapd. To dryness
PE Extract
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Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany 243
Isolation of compound I and II from PE Extract
PE Extract
(1-1 g)
CC (SiO2 gel) 50 gi. PE(lOOml)
ii. 1 % EtoAc in PE (100 ml)iii. 2% EtoAc in PE (100 ml)iv. 5% EtoAc in PE (100 ml)v. 10% EtoAc in PE (100 ml)
vi. 20% EtoAc in PE (100 ml)vii. 25% EtoAc in PE (100 ml)
viii. 40% EtoAc in PE (100 ml)
rF2F,
R f=0.86
20% EtoAc in PE
Crystallization in
MeOH: CHC13
F3 F4
Rf=0.23
20% EtoAc in PE
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Some Properties of Isolated Compounds
Compound I.
(S.)
Compound II.
(S2)
MeltingPoint
164C
108C
UV (?.max) (nm)(cyclohexane)
218,270
280,295
211,266,271
280, 294
IR (iW) (cm'1)
(KBr)
3434,2954,2870,1643,
1458,1369,1039,967
3439,2850, 1740, 1638,
1459_1383, 1178,720
Fig. I The UV spectrum of isolated copound I.ergosterol (cyclohexane)
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Fig. II The UV spectrum of isolated copound II.fatty acid ester of ergosterol(cyclohexane)
%T
49.7
4 5 .
•SO.
3s
30.
25.
204
V
. / •
1117; \ I'M sh\ 5 "
: 2m ' MOO 600 400.0
Fig. Ill The FT-IR spectrum of isolated compound I ergosterol (KBr)
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246 Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany
Fig. IV The FT- IR spectrum of isolated copound Il.fatty acid ester ofergosterol (KBr)
Fig. V the Crystal form of compound I. Ergosterol
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Fig. VI, Detail the needle-shaped Crystal form of Ergostero!
Fig. VII.The Crystal.form of-compound II Fatty acid ester of Ergosterol
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248 Jour.Myan. Acad. Arts & Sc. 2005 Vol. III. .No. 4(ii) Botany
Fig. VIII. Detail the plate-shaped Crystal form of Fatty acid ester ofErgosterol ' . . '•• •• ' "
References \ • '
AlexopoulosC.J (1996) Introductory Mycology. JohnWiley & Sons New York.
Chang Huang K. (1999). "The Pharmacology of Chinese Herbs," CRS press, 2nd Ed, BocaRaton London, New York. Washington D.C.
Jones K. (1990). "Reishi": Ancient Herb of Modern Times, Issaquah, WA: Sylvan Press.
Kammatsuse K, Kajiware N, Hayashi K. (1985) Studies on Ganoderma liicidum: LEtficacyagainst hypertension and side effects, Yakugaku Zasshi; 105:531-3.
Me Guffin M, Hobbs C, Upton R,. Goldberg A (Eds) (1997). "American Herbal productsAssociation's Botanical Safety Handbook. Boca Raton, FL: CRS Press, 55.
Richard M.Lucas (1991). "Miracle Medicine Herbal", London New York, 1st Ed, Stuttgart.
Willard T (1990) "Reishi Mushroom": Herb of Spiritual Potency and Wonder, Issaquah; WA:Sylvan press: 6. .
The Merck Index (1996) An Encyclopedia of Chemicals Drugs, Add Biologicals, Publishedby Merck Research, printed in USA, 12th Ed. Number 3701.
Phytochemistry. Vol. 23, No. 3, pp. 686-687, 1984.