Isolation, Purification, Structure Elucidation Of Triterpenoids From The Fruiting Bodies Of Ganoderma Lucidum And Antitumor Activity In Vitro

Ganoderma lucidum (Curtis:Fr.) P. Karst. is a well-known mushroom in China and hasbeen used to treat various human ailments for over 2,000 years. Triterpenoids are one ofseveral important bioactive constituents of G lucidum and have been reported to exhibitvarious pharmaceutical effects including antitumor and hepatoprotective activity, as well asinhibiting both the release of anti-histamines and the action of angiotensin-convertingenzyme. Although G. lucidum triterpenoids have been extensively research worldwide,investigations have concentrated mainly on isolation and purification. Bioactivitiesassociated with some purified constituents have been determined, but the pharmaceuticaleffects of fractions obtained at every phase of isolation and purification have not beenascertained.In this study, G. lucidum fruit bodies were extracted with 95% (v/v)aqueous ethanol.Combined ethanolic extracts were evaporated to dryness, redissolved in water andextracted with chloroform. After addition of a saturated NaHCO₃ solution, the chloroformlayer containing non-acidic triterpenoids was collected. The aqueous layer was acidifedwith HCl and re-extracted with chloroform to obtain the acidic triterpenoid fraction. Crudeextracts were purified using silica gel, MCI and ODS column chromatography andpreparatory HPLC, and a total of six compounds were obtained.Four compounds were obtained from the non-acidic triterpenoid fraction and theirstructures elucidated from ¹H-NMR and ¹³C-NMR spectral data. These were as follows:Compoundâ… , methyl 7β-hydroxy-3, 11, 15, 23-tetraoxo-5α-lanost-8-en-26-oate (methylganoderate D); Compoundâ…¡, methyl 12β-acetoxy-3, 7, 11, 15-tetraoxo-5α-lanost-8-en-24-oate (methyl lucidenate D); Compoundâ…¢, 7β-hydroxy-4, 4, 14α-trimethyl-3, 11,15-trioxo-5α-lanost-8-en-24-oic acid (lucidenic acid A). Compoundsâ… andâ…¡are newnatural products identified for the first time in fruit bodies of Ganoderma spp., andcompoundâ…¢was a known triterpenoid. The structure of compoundâ…£has not yet beenelucidated. Two compounds, Compoundsâ…¤andâ…¥, were obtained from the acidic triterpenoid fraction but their structures were not identified. The sub-fractions contained98% of Compoundâ…¤and 99.54% of Compoundâ…¥, respectively as determined by HPLC.At concentrations of 200μg/mL, most fractions strongly inhibited the growth of L1210tumor cells and inhibition rates exceeded 80% except in the case of fraction A12M5 wherethe growth inhibition rate was only 30.69%. Similar L1210 inhibition rates were observedat sample concentrations of 50μg/mL, with most fractions inhibiting tumor cell growth bymore than 60% and some by 80%. However, fractions N7M1, N7M4, N8, N8M1, N8M2and A12M4-A12M7 were less inhibitory. Most fractions also strongly inhibited the growthof L1210 cells even at 10μg/mL concentrations. In the case of fractions N1, N6, N7, N7M6,N7M9, A8, A10, A10sl, A10s2, A10s4-A10s8, A11s2, A11s4, A11s8, A11s9, A12M12,A13s3-A13s6, the inhibition rate exceeded 60%.