MM0900109

Jour. Myatu Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany

A Study on Some Chemical Constituents of lucidum Leyss. Ex. Fr. p. Karst (vifZD;rId) from Pyin-Oo-Lwin Area Khine Kyi OO1, Aye Aye Tun2

Abstract A study on some chemical constituents of G.lucidum (vifZD;) which grows throughout Myanmar has been undertaken. Two compounds, Ergosterol and its ester from mushroom body have been isolated by solvent extraction followed by column chromatography, Isolated compounds were identified by spectroscopic techniques. is traditionally utilized for lowering blood cholestrol, regulate binod pressure, prevent heart disease, and regulate 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 all over the world. It was discovered in China two thousand years ago and in Japan it is known as "Man Nen Take" (10,000 year mushroom). Collection trips on Ganoderma species gave the knowledge that this mushroom was known 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 family (Ganodermataceae) and the order Aphyllophorales. Its fruiting bodies are called "Reishi" in Japanese, "Lingzhi" in Chinese, "Chaga" in Russian and "Lingchich" in American. The whole plant of G.lucidum (Lingzhi) grows in densely wooded mountains of high humidity and dim lighting in China and Japan. It grows mainly on the dried trunks of dead maples and other decaying hard woods rather than on conifers. In Myanmar, G.lucidum was found to the growing wild in moist, dark forest 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. 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 the King", upper class hung it on their doors for good luck and Health (Willard Terry, 1990). Present work, involves extraction, isolation and characterization of two compounds from the mushroom body of G.lucidum which is growing wild in Mandalay Division from Pyin-Oo-Lwin area. Extraction was carried out by using Soxhlet apparatus with petroleum ether (PE) (60-80 C) for 8 hours. The crude extract after evaporation was then subjected 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 points determination. Their structures have been characterized by UV and FT-IR spectroscopy.

Materials and Methods General Procedures UV: Shimadzu UV-240, Cyclohexane; FT-IR: Perkin Elmer GX system, KBr; CC: Merck silica gel 60 (70-730 mesh), eluents: Petroleum ether: ethyl acetate (PE:EtoAc); TLC: 0.25 mm precoated silica gel (60 F254, Merck), solvent system PE: EtoAc, spots were detected by inspection under UV 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 August to September 2003, from Pyin-Oo-Lwin Township, Mandalay Division. These mushrooms were identified as G.lucidum in Department of Botany, Yangon University. These mushrooms were crushed, carefully washed with water and allowed to dry in shady place. After being dried in air for 3 weeks, it was ground to powder by using grinding mill and then stored in glass bottles. 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 with petroleum ether (500 cm3) for 8 hours in Soxhlet extractor. Removal of the solvent provided crude extract (1.1 g), which was chromatographed 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) and 40% Pet-EtoAc (100 cm3). A quantity of (5 cm3) was collected for each fraction and the chromatography was monitored by TLC. The fractions that gave similar TLC pattern were combined together and concentrated. In this way, 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 colourless plates 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) in Pet:EtoAc (4:1) when the chromatogram was treated with anisaldehyde reagent followed by heating the spot having Rf value of 0.23 was found to change to pink purple, indicating the characteristic of ergosterol (or) steroid. 240 Jour. Myan. Acad. Arts & Sc. 2005 Vol. 111. No. 4(ii) Botany

Result and Discussion Identification of St as Ergosterol by Thin Layer Chromatography (TLC) method The isolated compound Si was checked by TLC to determine its purity. It appeared as a single spot on thin layer chromatogram and Rf values coincide with authentic ergosterol in all solvent systems. Therefore, S| was characterized 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 from literature. (Ref: 163-164°C, Phytochemistry, 1984)

Identification of Isolated compound Si by spectral data After being identified, the isolated constituent of G.lucidum as 1 ergosterol 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 OH stretching vibration band. Asymmetric and symmetric u C-H stretching bands appeared at 2954 and 2870 cm'1. A medium band at 1643 was due to c = c I stretching vibration bands. Symmetric and asymmetric bending vibration of 1 -CH3 and -CH2- groups absorbed at 1458, 1382 and 1369 cm" . In addition, a I strong band at u 1039 cm"1 was attributed to the c - o stretching vibration I bands. \ Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany 241

All these bands assignment support the identity of isolated compound as 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 compound Si (identified as ergosterol). The melting point of S2 was measured tabe lower than that of Si. Bothcompounds gave colour reaction when treating with Libermann 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 S2 revealed the presence of c = o stretching band at u 1740 cm*1 which did not appear in S\. In addition, the c-o stretching vibration band of S2 absorbed at 1178 cm"1 which is higher when comparing with the c - o stretching vibration band of Si (1039). From these observations, isolated compound S2 could be characterized 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 from petroleum ether extract of lingzhi. The isolated compounds have been characterized by melting point examination and spectroscopic measurement (UV and FT-IR). Thus, the two compounds are ergosterol and fatty acid ester of ergosterol. 242 Jour. Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany

Acknowledgements The 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 me to undertake this research in the department. My particular thanks to Dr. Maung Maung Htay, Professor& Head, Department of Chemistry, 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 with constructive criticisms and invaluable advice. I also wish to thank all of my friends and colleagues from Department of Chemistry for 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 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 g i. 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) r viii. 40% EtoAc in PE (100 ml) F, F2 F3 F4

Rf=0.86 Rf=0.23 20% EtoAc in PE 20% EtoAc in PE Crystallization in

MeOH: CHC13 244 Jour, Myan. Acad. Arts & Sc. 2005 Vol. III. No. 4(ii) Botany

Some Properties of Isolated Compounds 1 Melting UV (?.max) (nm) IR (iW) (cm' ) Point (cyclohexane) (KBr) Compound I. 164C 218,270 3434,2954,2870,1643, (S.) 280,295 1458,1369,1039,967 Compound II. 108C 211,266,271 3439,2850, 1740, 1638,

(S2) 280, 294 1459_1383, 1178,720

Fig. I The UV spectrum of isolated copound I.ergosterol (cyclohexane) Jour. Myan. Acad, Arts & Sc. 2005 Vol. III. No. 4(ii)-Botany 245

Fig. II The UV spectrum of isolated copound II.fatty acid ester of ergosterol (cyclohexane)

49.7

./•

45.

•SO.

1117; \ I'M sh\ 5" 3s

30. %T V

25.

204

: 2m ' MOO 600 400.0

Fig. Ill The FT-IR spectrum of isolated compound I ergosterol (KBr) 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 of ergosterol (KBr)

Fig. V the Crystal form of compound I. Ergosterol Jour. Myan.-Acud, Arts & Sc. 2005 Vol. III. No. 4{ii) Botany 247

Fig. VI, Detail the needle-shaped Crystal form of Ergostero!

Fig. VII.The Crystal.form of-compound II Fatty acid ester of Ergosterol 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 of Ergosterol ' . . '•• •• ' "

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