the present invention will now be described more specifically with reference to the following embodiments . it is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for purpose of illustration and description only ; it is not intended to be exhaustive or to be limited to the precise form disclosed . for conveniently describing the ergostane triterpenoid compositions e1 to e 12 extracted in the present invention , compositions e1 to e12 , the corresponding structural formulas ( formulas i to x ) and the corresponding peaks in the hplc spectra were detailedly illustrated as follows . for conveniently describing the lanostane triterpenoid compositions l1 to l6 extracted in the present invention , compounds l1 to l6 , the corresponding structural formulas ( formulas xi to xvi ) and the corresponding peaks in the hplc spectra were detailedly illustrated as follows . please refer to preparation method 10 in fig1 , the dried fruiting body of ac was ground as fine powder ( step 12 ), which was heated at reflux in ethanol ( etoh ) solution at 75 ° c . at a ratio of 1 / 10 ( weight / volume ) for 2 hours ( step 14 ). the extract was cooled , and then was precipitated at 4 ° c . overnight . furthermore , the supernatant of the extract was filtered with filter paper , and the precipitate was removed by centrifuging at 3 , 000 rpm for 30 min . the extract , which was the etoh extract of the fruiting body of ac , was lyophilized and stored at − 70 ° c . ( step 16 ). the etoh extract was extracted with n - hexane to obtain the n - hexane extract of the fruiting body of ac ( step 18 ) and the first debris of the fruiting body of ac ( step 20 ). next , the first debris ( step 20 ) was extracted with ethyl acetate ( ea ) to obtain the ea extract of the fruiting body of ac ( hereinafter “ the ea extract ”, step 22 ) and the second debris of the fruiting body of ac ( step 24 ). the ea extract ( 6 . 8 g ) was chromatographed in gradient with n - hexane - etoac - methanol ( meoh ) ( sequentially 10 : 1 : 0 , 5 : 1 : 0 , 1 : 1 : 0 , 0 : 1 : 0 , 0 : 40 : 1 , 0 : 30 : 1 , 0 : 20 : 1 , 0 : 10 : 1 ) with silica gel 60 ( merck , 230 - 400 mesh ) to obtain 17 fractions . ( 1 ) isolation of antcin k : fraction 15 ( 245 . 7 mg ) was purified with ods hplc column ( 250 × 10 mm , hypersil ods , acetonitrile ( ch 3 cn )— h 2 o ( 0 ˜ 2 min ( 35 % ch 3 cn ˜ 45 % ch 3 cn ); 20 ˜ 25 min ( 45 % ch 3 cn ˜ 100 % ch 3 cn )) to afford antcin k ( retention time of 14 . 7 min , flow rate of 3 ml / min ). ( 2 ) isolation of antcin c : fraction 10 ( 132 . 6 mg ) was isolated using thin layer chromatography ( tlc ) with dichloromethane ( ch 2 cl 2 )- meoh ( 15 : 1 ), and the chromatographic band with rf value of 0 . 31 was harvested and then purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 70 : 30 )) to afford antcin c ( retention time of 10 min , flow rate of 2 ml / min ). ( 3 ) isolation of zhankuic acid c : fraction 13 ( 100 . 0 mg ) was isolated using tlc with ch 2 cl 2 - meoh ( 15 : 1 ), and the chromatographic band with rf value of 0 . 18 was harvested and then purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 70 : 30 )) to afford zhankuic acid c ( retention time of 10 min , flow rate of 2 ml / min ). ( 4 ) isolation of zhankuic acid b : fraction 10 ( 132 . 6 mg ) was isolated using tlc with ch 2 cl 2 - meoh ( 15 : 1 ), and the chromatographic band with rf value of 0 . 31 was harvested and then purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 50 : 50 )) to afford zhankuic acid b ( retention time of 50 min , flow rate of 2 ml / min ). ( 5 ) isolation of zhankuic acid a : fraction 6 ( 100 . 0 mg ) was isolated using tlc with ch 2 cl 2 - meoh ( 15 : 1 ), and the chromatographic band with rf value of 0 . 42 was harvested and then purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 75 : 25 )) to afford zhankuic acid a ( retention time of 12 min , flow rate of 2 ml / min ). ( 6 ) isolation of antcin a : fraction 6 ( 100 . 0 mg ) was isolated using tlc with ch 2 cl 2 - meoh ( 15 : 1 ), and the chromatographic band with rf value of 0 . 42 was harvested and then purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 75 : 25 )) to afford antcin a ( retention time of 19 min , flow rate of 2 ml / min ). ( 1 ) isolation of dehydrosulphurenic acid : fraction 13 ( 200 . 0 mg ) was isolated twice using tlc with ch 2 cl 2 - meoh ( 15 : 1 ), and the chromatographic band with rf value of 0 . 36 was harvested and then purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 60 : 40 )) to afford dehydrosulphurenic acid ( retention time of 22 min , flow rate of 2 ml / min ). ( 2 ) isolation of sulphurenic acid : fraction 10 ( 132 . 6 mg ) was isolated using tlc with ch 2 cl 2 - meoh ( 15 : 1 ), and the chromatographic band with rf value of 0 . 31 was harvested and then purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 50 : 50 )) to afford sulphurenic acid ( retention time of 53 min , flow rate of 2 ml / min ). ( 3 ) isolation of 15α - acetyl - dehydrosulphurenic acid : fraction 6 ( 100 . 0 mg ) was isolated using tlc with ch 2 cl 2 - meoh ( 15 : 1 ), and the chromatographic band with rf value of 0 . 42 was harvested and then purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 75 : 25 )) to afford 15α - acetyl - dehydrosulphurenic acid ( retention time of 20 min , flow rate of 2 ml / min ). ( 4 ) isolation of versisponic acid d : fraction 6 ( 100 . 0 mg ) was isolated using tlc with ch 2 cl 2 - meoh ( 15 : 1 ), and the chromatographic band with rf value of 0 . 42 was harvested and then purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 75 : 25 )) to afford versisponic acid d ( retention time of 22 min , flow rate of 2 ml / min ). ( 5 ) isolation of dehydroeburicoic acid : fraction 5 ( 100 . 0 mg ) was purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 90 : 10 )) to afford dehydroeburicoic acid ( retention time of 27 min , flow rate of 2 ml / min ). ( 6 ) isolation of eburicoic acid : fraction 5 ( 100 . 0 mg ) was purified with ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 90 : 10 )) to afford eburicoic acid ( retention time of 31 min , flow rate of 2 ml / min ). at present , there is no prior art or literature to disclose the absolute stereo structure of ergostane triterpenoid compositions , and no pure compound is obtained . based on the following descriptions , the present invention is the first technical literature in the world to disclose the asymmetrical center at c - 25 position of ergostane triterpenoid composition , and pure compounds were obtained . taking the isolation of stereoisomeric mixture of zhankuic acid a as the example , the zhankuic acid a standard obtained in experiment 2 showed a spot in the positive phase tlc ( solvent system is ch 2 cl 2 - meoh ( 20 : 1 )) for one separation . however , other closer spots were found after a various of separations , and the phenomenom that stereoisomeric mixtures were separated was observed . please refer to fig2 , which illustrates the recycle chromatography spectrum using reverse phase hplc . purification was performed using ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 55 : 45 ), flow rate of 4 . 3 ml / min ). after the eighth recycle separation , the stereoisomeric mixtures of zhankuic acid a were separated at retention time of 416 and 447 min , and pure compounds e9 and e10 were afforded respectively . the stereoisomeric mixtures of other ergostane triterpenoids in experiment 2 could be separated using the same method . please refer to fig3 , in addition to the above method , the stereoisomeric mixtures of zhankuic acid a were separated using hplc cosmosil 5c - 18 - ms column ( 250 × 10 . 0 mm ) at retention time of 42 and 43 min at the conditions that solvents a and b respectively were ch 3 cn and h 2 o ( containing 0 . 05 % acetic acid ) in the mobile phase and solvent system was ch 3 cn — h 2 o ( 50 : 50 ) at the flow rate of 3 . 0 ml / min , and pure compounds e9 and e10 were afforded respectively . the stereoisomeric mixtures of other ergostane triterpenoids also were separated at the condition of mobile phase containing acid . the stereoisomeric mixtures of antcin k were purified using ods hplc column ( 250 × 10 mm , hypersil ®, ch 3 cn — h 2 o ( 0 ˜ 2 min ( 35 % ch 3 cn ˜ 45 % ch 3 cn ); 20 ˜ 25 min ( 45 % ch 3 cn ˜ 100 % ch 3 cn ) at retention time of 14 . 5 and 15 . 3 min , and pure compounds e1 and e2 were afforded respectively . please refer to fig4 , antcin c was purified using cosmosil hplc column ( 250 × 10 mm , ch 3 cn — h 2 o ( 50 : 50 ), flow rate of 3 . 0 ml / min ), the stereoisomeric mixtures of antcin c were separated at retention time of 27 and 29 min , and pure compounds e3 and e4 were afforded respectively . please refer to fig5 , zhankuic acid c was purified using cosmosil hplc column ( 250 × 10 mm , ch 3 cn — h 2 o ( 50 : 50 ), flow rate of 3 . 0 ml / min ), the stereoisomeric mixtures of zhnakuic acid c were separated at retention time of 31 and 33 min , and pure compounds of e5 and e6 were afforded respectively . zhnakuic acid b was purified using cosmosil hplc column ( 250 × 10 mm , ch 3 cn — h 2 o ( 0 ˜ 20 min ( 55 % ch 3 cn ˜ 60 % ch 3 cn ); 20 ˜ 25 min ( 60 % ch 3 cn ˜ 100 % ch 3 cn ), flow rate of 3 . 0 ml / min ), the stereoisomeric mixtures of zhnakuic acid b were separated at retention time of 19 . 84 and 20 . 29 min , and pure compounds e7 and e8 were afforded respectively . antcin a was purified using cosmosil hplc column ( 250 × 10 mm , ch 3 cn — h 2 o ( 60 : 40 ), flow rate of 3 . 0 ml / min ), the stereoisomeric mixtures of antcin a were separated at retention time of 32 . 73 and 33 . 83 min , and pure compounds e11 and e12 were afforded respectively . based the separation method in experiment 4 , six ergostane triterpenoid stereoisomeric mixtures were separated and purified and 12 pure compounds e1 to e12 were afforded . taking structural identification of the afforded compounds e9 and e10 separated from zhankuic acid a as the example , zhankuic acid a is a stereoisomeric mixture having an asymmetrical center at c - 25 position at its structure . please refer to fig6 ( a ), zhankuic acid a showed two sets of ch 3 - 27 signal at δ h 1 . 521 ( 3h , d , j = 7 . 2 hz ) and 1 . 528 ( 3h , d , j = 7 . 2 hz ) in 1 h nmr spectrum . please refer to fig7 ( a ), zhankuic acid a significantly showed two sets of signals at δ c 34 . 242 and 34 . 342 ( ch 2 - 22 ), 31 . 575 and 31 . 766 ( ch 2 - 23 ), 46 . 558 and 46 . 793 ( ch - 25 ), and 17 . 003 and 17 . 179 ( ch 3 - 27 ) at side chains in 13 c nmr spectrum ( 150 mhz in c 5 d 5 n ) due to the c - 25 asymmetrical center . two sets of signals at δ c 27 . 960 and 27 . 997 ( ch 2 - 16 ), 53 . 937 and 53 . 986 ( ch - 17 ), 35 . 847 and 35 . 885 ( ch - 20 ), and 18 . 519 and 18 . 564 ( ch 3 - 21 ) also could be observed . please refer to fig6 ( b ), 6 ( c ), 7 ( b ) and 7 ( c ), the afforded compounds e9 and e10 separated from zhankuic acid a only showed one set of signal in nmr spectrum , but there was no two - set characteristic signal of stereoisomer mixture presented . with the comparisons of the above nmr spectra , it was proved that the stereoisomeric mixtures of zhankuic acid a were respectively separated and purified , and pure compounds were afforded . please refer to fig8 , c - 26 carboxylic group of compound e9 formed an ester with c - 1 position ( r - form ) of ( 1r )- 1 -( 9 - anthryl )- 2 , 2 , 2 - trifluoroethanol ( 1rat ). please refer to fig8 ( b ), compound e9 formed an ester with c - 1 position ( s - form ) of ( 1s )- 1 -( 9 - anthryl )- 2 , 2 , 2 - trifluoroethanol ( 1sat ). then , the absolute stereo structure of compound e9 at c - 25 position was determined by the difference of 1 h nmr chemical shifts ( δδ rs values in fig9 ) between the synthetic compounds e9 - 1rat and e9 - 1sat . please refer to fig9 , it was supposed that group l1 which had a negative signal difference value between the 1rat and 1sat synthetic esters was disposed over a horizontal plane and group l2 which a positive signal difference value therebetween was disposed below the horizontal plane . the absolute stereo structure of compound e9 at c - 25 position then was determined by cahn - ingold - prelog priority rules . the experimental method was described as follows . compound e9 ( 6 . 42 mg ) was mixed with 1rat ( 1 equivalent ) and dissolved in tetrahydrofuran ( thf ) to obtain solution a . 1 - ethyl - 3 -( 3 - dimethylaminopropyl ) carbodiimide ( edc - hcl , 3 equivalents ) was mixed with 4 - dimethylaminopyridine ( dmap , 1 . 5 equivalents ) and dissolved in ch 2 cl 2 to obtain solution b . solutions a and b were mixed , and then triethylamine ( et 3 n , 2 equivalents ) was added to react for 12 hours . partition extraction was performed with h 2 o and ch 2 cl 2 , and the obtained organic layer was analyzed with pre - tlc and separated with ch 2 cl 2 , to obtain compound e9 - 1rat ( 3 . 43 mg ). the 13 c nmr signals of compounds e9 and e9 - 1rat at c - 26 position respectively were δ c 176 . 900 and 172 . 774 , indicating that compound e9 successfully formed an ester bond with 1rat at c - 26 position . compound e9 - 1sat was also obtained with the similar reaction steps . compound e9 ( 11 . 15 mg ) was mixed with 1sat ( 1 equivalent ) to cooperatively dissolved in thf to obtain solution a . edc - hcl ( 3 equivalents ) was mixed with dmap ( 1 . 5 equivalents ) to cooperatively dissolved in ch 2 cl 2 to obtain solution b . solutions a and b were mixed , and then et 3 n ( 2 equivalents ) was added to react for 12 hours . partition extraction was performed with h 2 o and ch 2 cl 2 , and the obtained organic layer was analyzed with pre - tlc and separated with ch 2 cl 2 , to obtain an ester compound e9 - 1sat ( 9 . 01 mg ), which showed a c - 26 ester signal of δ c 172 . 681 . please refer to table 7 , the difference value of 1 h nmr chemical shifts between compounds e9 - 1rat and e9 - 1sat was positive ( δδ rs & gt ; 0 ) at c - 27 position and was negative ( δδ rs & lt ; 0 ) at c - 28 position . it was determined that c - 25 position of compound e9 was s form . compound e9 was nominated as 4α - methylergosta - 8 , 24 ( 28 )- dien - 3 , 7 , 11 - trion - 25s - 26 - oic acid , and its nmr data was referred to table 4 . compound e10 of 7 . 73 mg and 9 . 17 mg respectively were esterified with 1rat and 1sat , and partition extraction was performed with h 2 o and ch 2 cl 2 beyond the reaction . the obtained organic layer was analyzed with pre - tlc and separated with ch 2 cl 2 , to obtain compounds e10 - 1rat ( 5 . 44 mg ) and e10 - 1sat ( 9 . 86 mg ). please refer to table 7 , the difference value of 1 h nmr chemical shifts between compounds e10 - 1rat and e10 - 1sat was negative ( δδ rs & lt ; 0 ) at c - 27 position and was positive ( δδ rs & gt ; 0 ) at c - 28 position . it was determined that c - 25 position of compound e10 was r form . compound e10 was nominated as 4α - methylergosta - 8 , 24 ( 28 )- dien - 3 , 7 , 11 - trion - 25r - 26 - oic acid , and its nmr data was referred to table 4 . after determining the absolute configuration , we tried to measure the optical rotation aiming to complete the spectral profile for the isolated compounds . in a previous report , the mixture form of zhankuic acid a was dissolved in methanol to measure its optical rotation . however , the solubility of e9 ( 25s ) and e10 ( 25r ) was not sufficient in single solvent ( methanol or ethanol ). the solubility of compounds e9 and e10 improved through using acetone - methanol mixture . considering compounds solubility and the convenience of collecting physical and spectral data using single solvent , pyridine was used in optical rotation experiment . the optical rotation of compound e9 was [ α ] d 25 + 32 . 1 ( c 0 . 70 , pyridine ), and that of compound e10 was [ α ] d 25 + 9 . 0 ( c 0 . 84 , pyridine ). the optical rotations of compounds e3 and e4 , which were obtained by isolating from stereoisomeric mixture of antcin c , respectively were [ α ] d 25 + 124 . 8 ( c 0 . 81 , pyridine ) and [ α ] d 25 + 79 . 9 ( c 0 . 47 , pyridine ). 1 h nmr spectrum characteristic signals of 1rat and 1sat eater compounds of compounds e3 and e4 were described in table 5 . based on the difference value of 1 h nmr chemical shifts at c - 27 and c - 28 positions beyond reaction , it was determined that c - 25 position of compound e3 was s form . compound e3 was nominated as 7β - hydroxy - 4α - methylergosta - 8 , 24 ( 28 )- dien - 3 , 11 - dion - 25s - 26 - oic acid , and its nmr data was referred to table 2 . c - 25 position of compound e4 was r form . compound e4 was nominated as ( 7β - hydroxy - 4α - methylergosta - 8 , 24 ( 28 )- dien - 3 , 11 - dion - 25r - 26 - oic acid , and its nmr data was referred to table 2 . compounds e5 and e6 were obtained by isolating from stereoisomeric mixture of zhankuic c , and their optical rotations were [ α ] d 25 + 82 . 0 ( c 0 . 64 , pyridine ) and [ α ] d 25 + 110 . 6 ( c 0 . 70 , pyridine ). 1 h nmr spectrum characteristic signals of 1rat and 1sat ester compounds of compounds e5 and e6 were described in table 6 . based on the difference value of 1 h nmr chemical shifts at c - 27 and c - 28 positions beyond reaction , it was determined that c - 25 position of compound e5 was r form . compound e5 was nominated as 3α , 12α - dihydroxy - 4α - methylergosta - 8 , 24 ( 28 )- dien - 7 , 11 - dion - 25r - 26 - oic acid , and its nmr data was referred to table 3 . c - 25 position of compound e6 was s form . compound e6 was nominated as 3α , 12α - dihydroxy - 4α - methylergosta - 8 , 24 ( 28 )- dien - 7 , 11 - dion - 25s - 26 - oic acid , and its nmr data was referred to table 3 . compounds e1 and e2 were obtained by isolating from stereoisomeric mixture of antcin k , and their optical rotations respectively were [ α ] d 25 + 61 . 0 ( c 0 . 42 , pyridine ) and [ α ] d 25 + 71 . 8 ( c 0 . 27 , pyridine ). since the amount of the obtained compound e1 was insufficient , only compound e2 was used to proceed esterification of 1rat and 1sat . the characteristic signals of 1 h nmr for compound e2 - 1rat was δ h 1 . 342 ( ch 3 - 27 , d , j = 7 . 2 hz ) and 5 . 170 , 5 . 118 ( ch 2 - 28 ), and those for compound e2 - 1sat was δ h 1 . 387 ( ch 3 - 27 , d , j = 7 . 2 hz ) and 4 . 903 , 5 . 005 ( ch 2 - 28 ). the difference value of 1 h nmr chemical shifts at c - 27 position was negative ( δδ rs & lt ; 0 ) and that at c - 28 position was positive ( δδ rs & gt ; 0 ), and it was determined that c - 25 position of compound e2 was r form . compound e2 was nominated as 3α , 4α , 7α - trihydroxy - 4α - methylergosta - 8 , 24 ( 28 )- dien - 11 - on - 25r - 26 - oic acid , and its nmr data was referred to table 1 . c - 25 position of compound e1 was s form . compound 1 was nominated as 3α , 4α , 7α - trihydroxy - 4α - methylergosta - 8 , 24 ( 28 )- dien - 11 - on - 25s - 26 - oic acid , and its nmr data was referred to table 1 . in addition to the above major ergostane triterpenoid components , the minor ergostane triterpenoid stereoisomeric mixtures also were isolated and purified . the optical rotations of compounds e7 and e8 , which were isolated from stereoisomeric mixture of zhankuic acid b , respectively were [ α ] d 25 + 11 . 9 ( c 0 . 57 , pyridine ) and [ α ] d 25 + 36 . 4 ( c 0 . 49 , pyridine ). the optical rotations of compounds e11 and e12 , which were isolated from stereoisomeric mixture of antcin a , respectively were [ α ] d 25 + 146 . 9 ( c 0 . 69 , pyridine ) and [ α ] d 25 + 117 . 2 ( c 0 . 34 , pyridine ). since the amounts of the obtained compounds e7 , e8 , e11 and e12 were insufficient , their 1rat and 1sat esterifications were not proceeded . it could be known from the hplc spectrum and optical rotation data that the stereoisomeric mixtures with asymmetrical centers at c - 25 position have been isolated , and was obtained in form of pure compounds . cytotoxicity test of ergostane triterpenoid stereoisomeric pure compounds on cancer cells the cytotoxicity test of the obtained major ergostane triterpenoid compounds ( zhankuic acid a , zhankuic acid c , antcin c and antcin k ) and their stereoisomeric pure compounds ( compounds e1 to e6 and e9 to e10 ) were performed on three leukemia cell lines using mtt ( 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ) colorimetric method , and the results were referred to table 8 . the component analysis of the ea extract was further performed using hplc method to set up the optimal analytic conditions , and ergostane triterpenoid stereoisomeric mixtures could be completely isolated and lanostane triterpenoid compounds could be detected simultaneously . what is mainly discussed in this experiment is described as follows . ( 1 ) the baseline stability and resolution relationship between the ergostane and lanostane acidic compounds in the chromatographic spectra are compared by adding different types of organic acids in the water mobile phase , and the most appropriate organic acid for adding in the water mobile phase is selected . ( 2 ) the acidity coefficient ( pka ) of the test compound structure was calculated using analytic software , and average acidity coefficient of ergostane and lanostane acidic compounds are determined . furthermore , the ph values of the mobile phase are monitored using ph meter to make the ph values of the mobile phase approach the average acidity coefficient of the compound , and thus the optimal separation effect can be achieved . hplc method 1 . the detection method was described as follows . the ea extract ( 1 . 0 mg ) was dissolved in 1 ml meoh to be the sample for hplc analysis . the conditions of hplc were described as follows . hplc apparatus was shimadzu lc - 10at , detector was shimadzu spd - m10a photodiode array detector , auto sampler was shimadzu sil - 20a prominence auto sampler , and hplc column was cosmosil 5c - 18 - ms ( 250 × 4 . 6 mm , 5 μm ). solvents a and b in the mobile phase respectively were ch 3 cn and pure water ( hplc grade h 2 o ), and the various organic acids , 0 . 1 % trifluoacetic acid ( tfa , ph 2 . 20 ), 0 . 1 % formic acid ( ph 2 . 80 ) and 0 . 1 % acetic acid ( ph 3 . 30 ), were added respectively . flow rate was 1 ml / min . the temperature of column was room temperature , and detection wavelength was uv 254 nm . the conditions of solvent system were described as follows . mobile phase included solvents a and b , linear gradients were 0 ˜ 30 min ( 45 % a ˜ 50 % a ), 30 ˜ 35 min ( 50 % a ˜ 55 % a ), 35 ˜ 45 min ( 55 % a ˜ 60 % a ), 45 ˜ 55 min ( 60 % a ˜ 70 % a ), 55 ˜ 60 min ( 70 % a ˜ 85 % a ) and 60 ˜ 100 min ( 85 % a ˜ 100 % a ). flow rate and column temperature were the same as above . hplc method 2 . the detection method was described as follows . the ea extract ( 1 . 0 mg ) was dissolved in 1 ml meoh to be the sample for hplc analysis . the conditions of hplc were described as follows . hplc apparatus was shimadzu lc - 10at , detector was shimadzu spd - m10a photodiode array detector , auto sampler was shimadzu sil - 20a prominence auto sampler , and hplc column was agilent poroshell 120 sb - c18 ( 150 × 4 . 6 mm , 2 . 7 μm ). solvents a and b in the mobile phase were ch 3 cn and pure water ( hplc grade h 2 o , contain 0 . 1 % acetic acid ( ph 3 . 30 )). flow rate was 1 . 3 ml / min . the temperature of column was room temperature , and detection wavelength was uv 254 nm . the conditions of solvent system were described as follows . mobile phase included solvents a and b , linear gradients were 0 ˜ 15 min ( 44 % a ˜ 49 % a ), 15 ˜ 17 . 5 min ( 49 % a ˜ 54 % a ), 17 . 5 ˜ 22 . 5 min ( 54 % a ˜ 59 % a ), 22 . 5 ˜ 27 . 5 min ( 59 % a ˜ 69 % a ), 27 . 5 ˜ 30 min ( 69 % a ˜ 84 % a ) and 30 ˜ 50 min ( 84 % a ˜ 100 % a ). flow rate and column temperature were the same as above . please refer to fig1 , which is the hplc spectra of ea extract at a wavelength of 254 nm in the different organic acids ( 0 . 1 % tfa , 0 . 1 % formic acid and 0 . 1 % acetic acid ) as the mobile phase . the results indicated that the more stable baseline and resolution were obtained when 0 . 1 % acetic acid ( ph 3 . 30 ) was the supplemented organic acid in the water mobile phase . thus , 0 . 1 % acetic acid was chosen to be the organic acid supplemented in the water mobile phase for the analytic conditions , and the compounds represented in each peaks in hplc spectra were referred to fig1 ( a ) and 11 ( b ). optimization the mobile phase condition in hplc analysis . however , although the more stable chromatographic baseline could be obtained at the analytic condition that 0 . 1 % acetic acid was contained in the mobile phase , ergostane triterpenoid stereoisomeric mixtures could not be completely isolated . thus , the acidity coefficient of each ergostane and lanostane triterpenoid compounds were calculated using the online chemical algorithm software “ sparc ( sparc performs automated reasoning in chemistry )”. please refer to table 9 , the acidity coefficients of these two types of acidic compounds were ranged in 4 . 30 ˜ 4 . 60 . next , ammonium acetate ( 10 mm ) then was added , and ph value of water mobile phase was adjusted with ph meter . five solutions with different ph values were prepared , and the respective ph value was 3 . 75 , 4 . 00 , 4 . 25 , 4 . 50 and 5 . 00 . hplc analyses for these five solutions and the original solution ( 0 . 1 % acetic acid , ph 3 . 30 ) were compared . the conditions for hplc were listed as follows . hplc apparatus was shimadzu lc - 10at , detector was shimadzu spd - m10a photodiode array detector , auto - sampler was shimadzu sil - 20a prominence auto sampler , and hplc column was cosmosil 5c - 18 - ms 250 × 4 . 6 mm solvents a and b in mobile phase respectively were ch 3 cn and pure water , and 0 . 1 % acetic acid was added to mix with 10 mm ammonium acetate . ph values were adjusted to 3 . 75 , 4 . 00 , 4 . 25 , 4 . 50 and 5 . 00 . flow rate was 1 ml / min , the temperature of column was room temperature , and detection wavelength was uv 254 nm . the conditions of solvent system were described as follows . mobile phase included solvents a and b , the linear gradients were 0 ˜ 30 min ( 45 % a ˜ 50 % a ), 30 ˜ 35 min ( 50 % a ˜ 55 % a ), 35 ˜ 45 min ( 55 % a ˜ 60 % a ), 45 ˜ 55 min ( 60 % a ˜ 70 % a ), 55 ˜ 60 min ( 70 % a ˜ 85 % a ) and 60 ˜ 100 min ( 85 % a ˜ 100 % a ). flow rate and column temperature were the same as above . please refer to fig1 ( a ) and 12 ( b ), which are the hplc results of ea extract at a wavelength of 254 nm at the water mobile phase ( 0 . 1 % acetic acid mixed with 10 mm ammonium acetate ) with different ph values . the results revealed that ergostane triterpenoid stereoisomers ( compounds e1 to e12 ) had better resolution and isolation at ph 4 . 25 ˜ 4 . 50 . thus , it could be determined that the optimal isolation effect could be achieved in the chromatographic spectrum when ph value of mobile phase approached or equaled to the average acidity coefficient of the analytic sample . please refer to fig1 , it could be known from the above experiment that the optimal hplc conditions for detecting ergostane triterpenoid stereoisomers of fruiting body of ac needed to maintain ph value of mobile phase at 4 . 25 . another major component is lanostane triterpenoid compound . compounds l1 and l2 are similar in structure , compounds l3 and l4 are similar in structure , and compounds l5 and l6 are similar in structure . the structural differences lies in two sets of double bond ( at c7 - c8 and c9 - c11 ) and one set of double bond ( at c8 - c9 ). although peaks of compounds l1 and l2 are overlapped and peaks of compounds l3 and l4 are overlapped at the eluting gradient condition of this experiment , molecular weights ( mw ) of compounds l1 and l2 and mw of compounds l3 and l4 are not identical . the qualitative and quantitative determinations of lanostane triterpenoid compounds can be performed by using hplc - mass spectrometry ( ms , e . g . triple quadrupole mass spectrometry ) at the above hplc conditions according to the property of different mw of compounds . the compounds represented in each peaks at the optimal analytic conditions in hplc spectra were referred to fig1 . ergostane triterpenoid stereoisomeric pure compounds e1 to e12 obtained in experiment 4 and their stereoisomeric mixtures ( antcin k , antcin c , zhankuic acid c , zhankuic acid b , zhankuic acid a and antcin a ) before purification and isolation were analyzed with hplc . please refer to fig1 ( a ) to 14 ( f ), it was shown on the chromatographic spectra that purities of compounds e1 to e12 achieved more than 95 % at the optimal hplc conditions . in the isolation procedure of stereoisomeric mixtures of ergostane triterpenoids with asymmetrical centers in experiment 4 , solvents a and b used in mobile phase were ch 3 cn and water ( containing 0 . 05 % acetic acid ) respectively , and 0 . 05 % acetic acid was added in its water phase solvent to reach ph 3 . 53 . it was again proved in the above experiment that the optimal isolation effect could be achieved using hplc when ph value of mobile phase approached or equaled to the average acidity coefficient of the analytic sample , and thus the experiment provided a method for isolating and analyzing ergostane triterpenoid stereoisomeric pure compounds of fruiting body of ac . it was known from the above experiment that the major component of ea extract was triterpenoid compound which further was divided as two groups , ergostane and lanostane . furthermore , the absolute content analyses of the total ergostane triterpenoid compounds and the total lanostane triterpenoid compounds were performed using nmr spectrum analysis method . the detection experiment procedure was described as follows . first , the appropriate deuterium solvent was selected , and then the standards for these two groups of compounds were respectively chosen to prepare the calibration standard curves with different concentrations . the certain amount of internal standard was added in the standard that was examined , and the integral area ratio of the characteristic signal of the respective standard to target signal of the internal standard was calculated . this integral value and concentration was plotted using linear regression , and thus the calibration curves for standards of two groups of compounds were obtained . next , ea extract with specific concentration was prepared , and the equal amount of deuterium solvent and the internal standard were added to proceed nmr spectrum analysis . the characteristic signals of two groups of compounds and the target signals of internal standard were detailedly integrated to calculate the integral ratios , and the absolute contents of two groups of compounds in ea extract were obtained based on the calibration curves . the quantitative analysis of the total ergostane triterpenoid compounds and the total lanostane triterpenoid compounds in ea extract was proceeded using nmr spectrum analysis method in the present invention . the experimental conditions were listed as follows . the standards of two groups of compounds with different concentrations were prepared , which respectively were zhankuic acid a of ergostane triterpenoid and dehydroeburicoic acid of lanostane triterpenoid . the internal standard ( pyrazine , 0 . 132 mg ) was added and dissolved in dmso - d6 solution ( 0 . 6 ml ), which was the test solvent for nmr spectrum analysis ( cdcl 3 and c 5 d 5 n also could be chosen , but they had problems such as signal interference and solubility ; data not shown ). nmr apparatus was varian unity plus 400 mhz spectrometer , the scanning times was 10 ( 7 min ), the spectrum width was 6002 . 4 hz , and width for impulse strength was 6 . 3 μs . please refer to tables 10 and 11 , the start point and end point for c - 28 methylene characteristic signal of the standard for two groups of compounds were further manually selected to calculate peak integral area , and integral area ratio of the peak integral area to the target signal of internal standard pyrazine ( δ h 8 . 66 ) was calculated . the characteristic proton absorption signal of the standard zhankuic acid a was situated at δ h 4 . 82 ( 2h , br d ), and that of the standard dehydroeburicoic acid was situated at δ h 4 . 63 ( 1h , s ) and 4 . 70 ( 1h , s ). the whole experiment was made in triplicate and the relative standard deviation value ( rsd %) was determined . please refer to table 12 , the integral ratio and concentration further was plotted using linear regression , and calibration curve ( standard curve and the determination coefficient of regression analysis ) for the standard of two groups of compounds could be obtained and to be the basis of this quantitative analysis method . after obtaining the calibration curves for standards of two groups of compounds , ea extract ( 20 . 12 mg ) was further prepared , the equal amount of dmso - d6 and internal standard pyrazine were added , and nmr spectrum analysis was proceeded . please refer to fig1 and table 13 , the c - 28 methylene characteristic signals for standards of two groups of compounds in ea extract and the target signals of the internal standard in the nmr spectrum were detailedly integrated to calculate the integral ratios . the whole experiment was made in triplicate and rsd % was determined . the absolute contents for two groups of compounds in ea extract were obtained based on the calibration curves for standards of above obtained two groups of compounds . it could be known from the results that the absolute content of total ergostane triterpenoid compound was 5 . 67 mg and that of total lanostane triterpenoid compound was 2 . 71 mg in 20 . 12 mg of ea extract . in accordance with the calibration curve for standard of two groups of compound obtained from nmr spectrum and the rsd values within the acceptable range , this method not only is rapid but also has reproducibility . while the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments , it is to be understood that the invention needs not be limited to the disclosed embodiments . on the contrary , it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims , which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures .