TW201217782A - Method for HPLC analysis of triterpenoids from Antrodia camphorata - Google Patents

Abstract

The present invention relates to a method for high performance liquid chromatography (HPLC) analysis of triterpenoids from Antrodia camphorata, which is characterized in the specific conditions of the mobile phase and UV wavelengths of the HPLC. The analysis under the specific conditions obtains specific peak areas at specific retention time points for various triterpenoids from Antrodia camphorata, and those peaks do not overlap with each other and are easy to distinguish.

Description

201217782 VI. Description of the invention: [Technical field to which the invention pertains] The technical field to which the present invention pertains relates to a method for analyzing Antrodia camphorata using high performance liquid chromatography: a method of moving liquid chromatography with high performance liquid chromatography And specific conditions for UV detection wavelengths. [Prior Art] Cangzhi Secret Theory), folk alias Niuzhizhi, burdock mushroom, red oyster mushroom, etc. It is unique to Taiwan, because it grows, and it only grows in the decaying inner wall of Taiwan’s old-aged cows. Because of its small amount, it is difficult to collect, and it is not easy to collect, but the pathogenicity does not seem strong, so it is not easy to cause The living tree is dead. The reason why burdock

It has excellent (four) rot, mainly due to the fact that the tree is rich in sputum and contains the extracted ingredients. The cow machine extracts the oysters but promotes the growth of zhizhi. In addition, 樟芝 can also secrete anti-biomass and inhibit other _ It grows, so it can't grow on the burdock tree except for the cockroach. Burdock tree can be used as an insect repellent because of its strong aroma. In the natural world, the ethnic group is scarce. Because the amount of Antrodia camphorata in the burdock tree is rare, it is regarded as the most precious medicine of the people. In oral or financial, it has long been aboriginal hangover, antidote to health and food _ return, such as acupuncture, food poisoning, treatment of abdominal abdomen pain, vomiting, blood pressure, skin disease, liver cancer and other symptoms, have a role. Taiwan's aborigines are the small petals of the burdock (4), and the tree is turned into a fine weizhi. Take the chew of 201217782 or cook it. Due to the aboriginal life type __, usually the physical exertion is very large, after serving Xiao Yuzhi, 'the function of strong liver detoxification, strong S body and hangover, by word of mouth, 'Shuhe Taiwan special agency to transfer duty is regarded as "Ganoderma lucidum Wang, is a unique silk in Taiwan. Because of the special identity and efficacy of Antrodia, many studies have also been carried out like fire. As evidenced by the scientific approach, the burdock fungus is not much hiding in the wild, and it is a single-hosted burdock tree, which causes its price to rise. The market price is more than 100,000 to hundreds of thousands per kilogram, and it is regarded as the most expensive in the world. Wild money is also known as the (four) stone of forest towels. The burdock ^ fruiting body is longer than the old cows to touch the dry face, the first lining is good, before reading _ from the beginning, there is a cow suffocating taste 'taste it is extremely bitter. The shape of the fruit body varies, and it has a plate shape, a bell shape, a horseshoe shape or a tower shape, and has no solid shape. Fresh red at the beginning of life 'gradual change to self-color, light reddish brown, light brown or light yellow brown. The child's body is born with a sterile handle. The surface is orange-red when fresh, brown to dark brown when aging, and the top is smooth' with concentric rings. The secret is round to polygonal, with a scorpion. _, generative hyphae, clamp connecti〇ns and ske]letal hyphae 'The sexual organ is basidi〇sp〇re, the shape is microbend Cylindrical, and the handle of the stalk is ciavate. The mycelium is orange-red when it is active and reddish brown after aging. The analysis of the composition of Antrodia sinensis has been reported in the literature since 1990 and is known by many research institutes for separation and analysis. The wild amaranth fruit body has a dry weight of about 30%, which contains about 32% crude fat, 23% crude fiber, 37% carbohydrate, and about 7% protein. The main bioactive components of Antrodia can be divided into polysaccharides, triterpenoids and steroids, in which polysaccharides can enhance human immunity and inhibit hepatitis B virus, while triterpenoids Related to anti-cancer, 201217782 liver protection, 'bribery has anti-inflammatory effect ^ can be different from the physical and metabolic factors of the body of the body of the money body', resulting in decreased biochemical synthesis of diols and sterols. However, there is currently no accurate and practical analytical method for the detection of triterpenoids. The amaranth fruit body contains a variety of three footwear compounds. After the smashing of the body of the scorpion scorpion, the triterpenoids can be prepared by heating and refluxing with ageing, and the three kinds of products have the functions of anti-cancer and liver protection. With the commercial riding, it is necessary to develop an effective purification method and accurate analytical method for the triterpenoids of Antrodia camphorata. SUMMARY OF THE INVENTION The object of the present invention is high performance liquid chromatography (DO) identification and content analysis of the indicator triterpenoids of the body composition of Antrodia camphorata, and the triterpenoids of the Antrodia camphorata product are determined by using the prepared standard of the Antrodia camphorata triterpenoid compound. content. The present invention relates to a method for analyzing Astragalus triterpenoids (this is called ruthenium) by high performance liquid chromatography, wherein the high performance liquid chromatography conditions are as follows: the mobile phase linear gradient program is: 1) initial self-sufficiency Starting with 60% acetonitrile (ACN) and 40% water (containing 0.1% to 0.2% formic acid or acetic acid), after 60 minutes 'to 2') 90% acetonitrile and 10% water (containing 0.1% to 0.2% formic acid or acetic acid); It is 〇8 to L0 ml/min; and the side detector: the ultraviolet wavelength is between 235 and 255 nm; there is a specific wave peak when it is between (4).

In a preferred embodiment of the present invention, the triterpenoids of Antrodia camphorata include methyl anthraquinone B 201217782 (methyl antcinate B), dehydroeburicoic acid, 15α-ethyl dehydrogenated polyporogen Acid (15 α-acety 1 dehydrosulphurenic acid ), 3β, 15α-di-lane 甾-7,9 (11), 24-trisole-21 g complex (3β,15a-dihydroxy lanosta-7,9 (11), 24-triene-21-oic acid), zhankuic acid A, zhankuic acid C, sulphurenic acid, and antcin A. In a preferred embodiment of the invention, methyl antcinate B has a specific peak value at a residence time of about 35 minutes. In a preferred embodiment of the invention, dehydroeburicoic acid has a specific peak value at a residence time of about 53 minutes. In a preferred embodiment of the invention, the 15a-acetamidine dehydrochromophoric acid (1) (tetra) dehydrated sulphurenic acid has a specific peak value at a residence time of about 28 minutes. In a preferred embodiment of the invention, Deng, "worker wool" _7,9 (11), 24_three thin _21 β, 15a dihydroxy lanosta-7,9 (11), 24-triene-21 -oic acid) has a specific peak value when the residence time is about 16 minutes. In a preferred embodiment of the present month, an acid a (acid a ) has a specific peak value at a residence time of about 18 minutes. In a preferred embodiment of the invention, zhankuic acid C is The residence time has a specific peak value of about Π minutes. 201217782 In a preferred embodiment of the invention, the sulphurenic acid has a specific peak value at a residence time of about 5 minutes. In a preferred embodiment of the invention, Anthraquinone A (antcinA) has a specific peak value at a residence time of about 27 minutes. In a preferred embodiment of the invention, the mobile phase linear gradient procedure for high performance liquid chromatography conditions is: 1) initially starting with 60% acetonitrile (ACN) and 40% water (containing 0.2% acetic acid), after 60 Minute ' to 2' 90% acetonitrile and 10% water (containing 0.2% acetic acid in a preferred embodiment of the invention) The mobile phase flow rate under high performance liquid chromatography conditions is 0.8 ml/min. In a preferred embodiment, the ultraviolet light wavelength of the detector of high performance liquid chromatography is 248 nm.

[Examples] Example 1. Separation of 8 triterpenoids from Antrodia camphorata extract The present invention uses an air-dried Antrodia camphorata fruit powder (1〇1 9 g) in the form of n-hexane, chloroform and methanol under heating. The extraction device was extracted (3 χ 1 〇〇〇 mL). After the complete extraction step, the extracts were concentrated under reduced pressure, respectively, to obtain 3.02 g (2.96% by dry weight § 10) of the residue, 46.3 g (45.430 / 〇) of trichloromethane. 201217782 Residue and 2.7 g (2.64%) of methanol residue. The polarity was increased by the calcined/acetic acid-acetic acid mixture, and the residue was extracted by triturated gas to be subjected to repeated stone chromatography (5 x 9 〇). After thin layer chromatography (TLC) analysis, the eluate with her chromatographic results was combined to obtain a 6-component solution (F1-F6). The liquid fraction F2 was re-chromatized by calcination/ethyl acetate on a silica gel column gradient elution method to produce methyl antdnate B (three-factor hydrazine, dehydroeburicoic add). (triterpenoid 2) and 15α-acetyl dehydrosulphurenic acid (triterpenoid 3). Dispensing F3 with hexane/ethyl acetate in a latex column gradient Purification method was carried out to obtain the compound Deng, 15α_di-lane 甾-7,9 (11),24-diene-21 acid (3β,15a-dihydroxy lan〇sta-7,9 (11), 24-triene -21-oic acid) (diterpenoid 4) and zhankuic acid A (triterpenoid 5). Separation of F4 and F5 with positive hexane/ethyl acetate gradient column chromatography Purification, yielding 4 sub-liquids (D-1 to D-4), zhankuic acid C (triterpenoid 6) and sulphurenic acid (triterpenoids) 7) From the secondary liquid separation 1 > 3 and D~4 respectively, using a mixture of 100% tri-gas methane to 20% methanol in three gas-burning/methanol, and performing liquid separation F6 矽 rubber column chromatography to purify 'output 樟 樟 甾 甾a (antcinA) (triterpenoid 8). The structure of triterpenoid 1-8 is determined by A and 13C nuclear magnetic resonance (NMR) spectroscopy, and the obtained spectral data is compared with published values (Shen, CC, Kuo, YC, Huang , RL, Lin, LC, Don, MJ, Chang, TT, Chou, CT, (2003). New ergostane and lanostane from Antrodia camphorata. Journal of Chinese Medicine, 14, 247-258 ; Male, KB, Rao, YK, Tzeng, YM, Montes, J., Kamen, A., Luong, JH, (2008). Probing inhibitory effects of Antrodia camphorata isolates using insect cell-based impedance spectroscopy: inhibition vs chemical structure. Chemical 201217782

Research in Toxicology 21, 2127-2133 i Yeh, CT, Rao, YK, Yao, CJ, Yeh, CF, Li, CH, Chuang, SE, Luong, JH, Lai, GM, Tzeng, YM, (2009). Cytotoxic Triterpenes from Antrodia camphorata and their mode of action in HT-29 human colon cancer cells. Cancer Letters 285, 73-79 ; Geethangili, M.,

Fang, SH, Lai, CH, Rao, YK, Lien, HM, Tzeng, YM, (2010). Inhibitory effect of Antrodia camphorata constituents on the Helicobacter p_y/on'-associated gastric inflammation. Food Chemistry 119, 149-153 . The molecular weights of the eight triterpenoids are listed in Table 1.

Table 1. Molecular weights of three kinds of three kinds of compounds isolated from Antrodia camphorata extracts Triterpenoids 1 to 3 compounds 8 Molecular weight (MW) 1 · Methyl antcinate B 482 2. Dehydroporous acid (dehydroeburicoic acid) 468 3· 15α-ethyl dehydrosulphate acid (15a-acetyl dehydrosulphurenic acid) 526 4· 3β,15α-dihydroxylane-7,9 (11),24-triene-21 Acid (3β, 15a-dihydroxy lanosta-7,9 (11), 24-triene-21-oic acid) 470 5. zhankuic acid A 468 6. zhankuic acid C 486 7. Sulfuric acid sulphatenic acid 466 8 · Anthraquinone A (antcinA) 454 201217782 Figures 1 to 8 are the chemical formulas of triterpenoids 1 to triterpenoids 8, respectively. Example 2. Analysis of Antrodia camphorata by High Performance Liquid Chromatography Triterpenoids 1 to triterpenoids 8 were analyzed by high performance liquid chromatography. The solvents for high performance liquid chromatography are: Ethanol (ECHO, Miaoli, Taiwan), Acetonitrile (ECHO 'Miaoli, Taiwan), Capric acid (Formic acid) (ECH〇, Miaoli, Taiwan) and Acetic acid (ECHO, Miaoli, Taiwan). The high-performance liquid chromatograph equipment is: detector: Diode Array UV Detector· L-7400 (Hitachi ' Tokyo 'Japan); pump: L-7100 (Hitachi, Tokyo, 曰本) and analytical column : J 'sphere ODS-M80 C18 column (25 〇 x 4_6 mm, 4 nm particle size) (YMC Sep, Technol, Japan). After extracting the extract of A. annuum L. from ethanol, it was analyzed by high performance liquid chromatography to analyze the triterpenoids contained therein. The high performance liquid chromatograph analysis conditions are: mobile phase acetonitrile and water (containing 0.2% acetic acid); mobile phase linear gradient program: 1) initial self-contained 60% acetonitrile (ACN) and 40% water (including 0.2%) Starting with acetic acid, after 60 minutes, to 2) 90% acetonitrile and 10% water (containing 0.2% acetic acid); the flow rate is 0.8 ml/min; the detector sets the ultraviolet wavelengths of 235 nm, 248 nm and 255 nm; The injection volume was 5 microliters. Figure 9 shows the results of analyzing the triterpenoids of Antrodia camphorata by high performance liquid chromatography. The analysis conditions of the liquid chromatograph in 201217782 are: mobile phase acetonitrile and water (containing 〇.2〇/. acetic acid) ); mobile phase linear gradient procedure: 1) initial from 60% acetonitrile (ACN) and 40% water (containing 0.2% acetic acid), after 60 minutes 'to 2') 90% acetonitrile and 1% water (including 0.2%) Acetic acid); flow rate 〇 8 ml / min. (A) indicates that the side detector sets the ultraviolet wavelength of 235 nm, (B) indicates that the side detector sets the ultraviolet wavelength of 248 nm, and (C) indicates that the side detector sets the ultraviolet wavelength of 255 nm, and the result shows that the ultraviolet wavelength is 248 nm. The most obvious peak obtained at a residence time of about 35 minutes represents the triterpenoid compound 1 of Antrodia camphorata. Figure 10 shows the results of analyzing Astragalus triterpenoids 2 by high performance liquid chromatography. The high performance liquid chromatograph analysis conditions are: mobile phase acetonitrile and water (containing 〇·2〇/. acetic acid); mobile phase linear gradient program: 1) initial self-contained 60% acetonitrile (ACN) and 40% water Starting with 0.2% acetic acid, after 60 minutes 'to 2' 90% acetonitrile and 1% water (containing 0.2% acetic acid); the flow rate was 〇8 ml/min. (Α) indicates that the side detector sets the ultraviolet wavelength of 235 nm, (Β) indicates that the side detector sets the ultraviolet wavelength of 248 nm, and (C) indicates that the side detector sets the ultraviolet wavelength of 255 nm, and the result shows that the ultraviolet wavelength is 248 nm. The most obvious peak obtained at about 53 minutes of residence time represents the Antrodia camphorata compound 2. Figure 11 shows the results of analyzing Astragalus triterpenoids 3 by high performance liquid chromatography. The high performance liquid chromatograph analysis conditions are: mobile phase acetonitrile and water (containing 〇.2〇 / 0 acetic acid); mobile phase linear gradient program: 1) initial 60% acetonitrile (ACN) and 40% water (containing 0.2% acetic acid) Start 'after 60 minutes' to 2) 90% acetonitrile and 1% water (containing 0.2% acetic acid); flow rate is 〇8 ml/min. (Α) indicates that the side detector sets the ultraviolet wavelength of 235 nm, (Β) indicates that the side detector sets the ultraviolet wavelength of 248 nm, and (C) indicates that the side detector sets the ultraviolet wavelength of 255 nm, and the result 201217782 shows the ultraviolet wavelength of 248 nm. The most obvious peak can be obtained at a residence time of about 28 minutes to represent the compound 3 of the burdock. Figure 12 shows the results of analyzing Astragalus triterpenoids 4 by high performance liquid chromatography. The high performance liquid chromatograph analysis conditions are: mobile phase acetonitrile and water (containing 〇 2% acetic acid); mobile phase linear gradient program: 1) initial self-contained 60% acetonitrile (ACN) and 40% water (including 0.2% acetic acid) Start 'after 60 minutes' to 2) 90% acetonitrile and 1% water (containing 0.2% acetic acid); flow rate is 〇8 ml/min. (A) indicates that the side detector sets the ultraviolet wavelength of 235 nm, (B) indicates that the debt side sets the ultraviolet wavelength of 248 nm '(C) indicates that the side detector sets the ultraviolet wavelength of 255 nm, and the result shows that the ultraviolet wavelength is 248 nm. The most obvious peak can be obtained at a residence time of about 6 minutes to represent the triterpenoid compound 4 of the burdock. Figure 13 shows the results of analyzing the anthraquinone triterpenoid 5 by high performance liquid chromatography. The high performance liquid chromatograph analysis conditions are: mobile phase acetonitrile and water (containing 〇2% acetic acid); mobile phase linear gradient program: 1) initial self-contained 60% acetonitrile (ACN) and 4% water (including 〇·2. /. Acetic acid) Start, after 60 minutes, to 2) 90% acetonitrile and 10% water (containing 〇·2% acetic acid); the flow rate is 〇8 spring ML/min. (A) indicates that the side detector sets the ultraviolet wavelength of 235 nm, (B) indicates that the side detector sets the ultraviolet wavelength of 248 nm, and (C) indicates that the side detector sets the ultraviolet wavelength of 255 nm, and the result shows that the ultraviolet wavelength is 248 nm. The most obvious peak was obtained at a residence time of about 8 minutes, representing the Niu Zhuang Zhi Sanzhuang compound 5. Figure 14 shows the results of analyzing the Antrodia camphorata III compound 6 by a liquid chromatograph. The analysis conditions of the South Performance Liquid Chromatograph are: mobile phase 6 water (including 02% 6 acid); mobile 12 201217782 phase linear gradient program: 1) initial 60% acetonitrile (ACN) and 40% water ( Starting with 0.2% acetic acid, after 60 minutes, to 2) 90% acetonitrile and 10% water (containing 0.2% acetic acid); the flow rate was 0.8 ml/min. (A) indicates that the side detector sets the ultraviolet wavelength of 235 nm, (B) indicates that the side detector sets the ultraviolet wavelength of 248 nm, and (C) indicates that the side detector sets the ultraviolet wavelength of 255 nm, and the result shows that the ultraviolet wavelength is 248 nm. The most obvious peak obtained at a residence time of about 11 minutes represents the triterpenoid compound 6 of Antrodia camphorata. Figure 15 shows the results of analyzing Astragalus triterpenoids 7 by high performance liquid chromatography. The high performance liquid chromatograph analysis conditions are: mobile phase acetonitrile and water (containing 0.2% acetic acid); mobile phase linear gradient program: 1) initial self-contained 60% acetonitrile (ACN) and 40% water (including 0.2%) Acetic acid) starts 'after 60 minutes, to 2) 90% acetonitrile and 10% water (containing 0.2% acetic acid); the flow rate is 0 8 ml/min. (A) indicates that the side detector sets the ultraviolet wavelength of 235 nm, (B) indicates that the side detector sets the ultraviolet wavelength of 248 nm, and (C) indicates that the side detector sets the ultraviolet wavelength of 255 nm, and the result shows that the ultraviolet wavelength is 248 nm. The most obvious peak obtained when the residence time is about 5 minutes represents the bovine 樟 萜 萜 triterpenoid compound 7. Figure 16 shows the results of analyzing Astragalus triterpenoids 8 by high performance liquid chromatography. The high performance liquid chromatograph analysis conditions are: mobile phase acetonitrile and water (containing 0.2% acetic acid); mobile phase linear gradient program: 1) initial self-contained 60% acetonitrile (ACN) and 40% water (including 0.2%) Acetic acid) starts 'after 60 minutes, to 2) 90% acetonitrile and 1% water (containing 0.2% acetic acid); the flow rate is 〇8 ml/min. (A) indicates that the side detector sets the ultraviolet wavelength of 235 nm, (B) indicates that the side detector sets the ultraviolet wavelength of 248 nm, and (C) indicates that the side detector sets the ultraviolet wavelength of 255 nm, and the result shows that the ultraviolet wavelength is 248 nm. The most obvious peak obtained at the residence time of about 27 minutes represents the bovine 13 201217782 Antrodia sinensis compound 8. Example 3: Preparation of Antrodia camphorata indicator III_Standard and calibration line The standard product purified by the present invention, a bovine stick, Sanzhuang compound i to 8, is re-dissolved in a concentration of milligrams per milliliter for efficient analysis and analysis. Genealogy. The concentration of the calibration line ranges from about (four) mg/ml to about 0.80 mg/ml. Figure 17 shows the results of analyzing the purified standard of the present invention by high performance liquid chromatography. The experiment includes the results of the analysis of the 樟 三 三 丨 丨 , , , , , , , , , , , , , , , , , , , , 高效Ethyl acetonitrile and water (containing 0.2% acetic acid); mobile phase linear gradient procedure: 1) Initially starting with 60% acetonitrile (ACN) and 40% water (containing 0.2% acetic acid), after 6 minutes, to 2) 90% Acetonitrile and 1% water (containing 〇.2〇/. acetic acid); flow rate was 0 8 ml/min. (A) indicates a concentration of the trivalent compound of 0.05 mg/ml; (B) indicates a concentration of the triterpenoid compound of 0.10 mg/ml; (C) indicates a concentration of the trizate compound of 0.20 mg/ml; indicating a triterpenoid The concentration was 0.40 mg/ml; (E) indicates the concentration of the triterpenoid was 〇8〇mg/ml. The results show that the high-performance liquid chromatogram peaks are not analyzed in the concentration range of about 0.05 mg/ml to about 0.80 mg/ml using the above high performance liquid chromatography conditions. Will overlap and can be easily interpreted. Figure 18 shows the quantitative standard curve of the solution of the Antrodia camphorata triterpenoid 1 to 8 standard, wherein the standard curve parameters are shown in Table 2 below. Table 2, Antrodia camphora triterpenoids 1 to 8 standard solution quantitative standard curve parameters 201217782 Figure 18 (A) Antrodia camphora triterpenoid 1, Υ = A + B xX Parameter value error A -5849.58333 5213.08255 B 443345.10753 12625.02977 Correlation coefficient ( R) Standard deviation (SD) Number of samples (N) P-value, significant value (P) 0.99879 7700.23325 5 <0.0001 Figure 18 (B) Antrodia camphorata compound 2, Y = A + B xX Parameter value error A - 290.8333 2428.65835 B 622663.97849 5881.71848 Correlation coefficient (R) Standard deviation (SD) Number of samples (N) P-value, significant value (P) 0.99987 3587.36613 5 <0.0001 Figure 18 (C) Astragalus triterpenoid 3, Y = A + B xX Parameter value error A 378.75 2366.80029 B 604933.70968 5731.91078 Correlation coefficient (R) Standard deviation (SD) Number of samples (N) P-value, significant value (P) 0.99987 3495.99571 5 <0.0001 Figure 18(D) Antrodia camphorata Compound 4, Y = A + B xX Parameter value error A 2138.91667 2134.67482 B 659597.04301 5169.74993 Correlation coefficient (R) Standard deviation (SD) Number of samples (N) P-value, significant value (P) 15 201217782

0.99991 3153.12368 5 <0.0001 Fig. 18(E) Astragalus triterpenoids 5, Y = Α + ΒχΧ Parameter error A -2493 4327.43363 B 391379.35484 10480.16751 Correlation coefficient (R) Standard deviation (SD) Number of samples (N) P- Value, significant value (P) 0.99893 6392.04309 5 <0.0001 Fig. 18(F) Astragalus triterpenoid 6, Y = Α + ΒχΧ Parameter error A -6640.125 1576.83011 B 210549.43548 3818.76306 Correlation coefficient (R) Standard deviation (SD) Number of samples (N) P-value, significant value (P) 0.99951 2329.13243 5 <0.0001 Figure 18 (G) Astragalus triterpenoids 7, Y = Α + ΒχΧ Parameter error A -4845.25 7024.60664 B 996276.93548 17012.1741 Correlation coefficient ( R) Standard deviation (SD) Number of samples (N) P-value, significant value (P) 0.99956 10376.03166 5 <0.0001 Figure 18 (H) Astragalus triterpenoids 8, Y = Α + ΒχΧ Parameter error A -6841.83333 7106.24023 B 724671.72043 17209.87411 16 201217782 Correlation coefficient (R) Standard deviation (SD) Number of samples (N) P-value, significant value (P) 0.99916 10496.61246 5 <0.0001 Example 4, use high Liquid chromatography can be used formic acid or acetic acid phase flow and different flow rates analytical standard I Analysis of triterpenoids 1-3 terpenoid 8 by high performance liquid chromatography. The solvents for high performance liquid chromatography were: Ethanol (ECHO, Miaoli, Taiwan), Acetonitrile (ECHO, Miaoli, Taiwan), Formic acid (ECHO, Miaoli, Taiwan) and acetic acid ( Aceticacid) (ECHO, Miaoli, Taiwan). The high performance liquid chromatography equipment is: debt detector: Diode Array UV Detector L-7400 (Hitachi, Tokyo, Japan); pump: L-7100 (Hitachi, Tokyo, Japan) and analytical column: J , sphere ODS-M80C18 column (250 x 4.6 mm, 4 nm grain diameter) (YMC Sep, Technol, Japan). The purified standard of the present invention, N. sinensis, was dissolved back at a concentration of 0.80 mg/ml, and its spectrum was analyzed by high performance liquid chromatography. The high performance liquid chromatograph analysis conditions are: mobile phase acetonitrile and water (containing 0.1% to 0.2% formic acid or acetic acid); mobile phase linear gradient program: 1) initial 60% acetonitrile (ACN) and 40% Starting with water (containing 0.1% to 0.2% citric acid or acetic acid), after 60 minutes, to 2) 90% acetonitrile and 10% water (containing 0.1% to 0.2% formic acid or acetic acid); the flow rate is 0.8 to 1.0 ml/min; The detector is set to UV wavelength 248 奈 17 201217782 m; the injection volume is 5 μl. Figure 19 shows the mobile phase containing formic acid or acetic acid using a high performance liquid chromatograph, and analyzing the triterpenoids of the burdock with different mobile flow rates. Results for 1 to 8 standard solutions. Figure 19 (A), (B) high performance liquid chromatography analysis conditions: mobile phase acetonitrile and water ((A), containing 0.1% acetic acid; (B), containing 2% acetic acid); mobile phase Linear gradient procedure.1), starting from 60% acetonitrile (ACN) and 4% water ((A), containing 1.1% acetic acid; (6), containing 〇2〇/acetic acid), after 60 minutes, To 2) 90% acetonitrile and 1% water ((eight), containing 〇ι% acetic acid; (9), containing 0.2% acetic acid: K flow rate is 〇. 8 ml / min; gamma setting ultraviolet wavelength _ nanometer; injection volume is 5 Microliters. The results of Fig. 19(A) and (8) show that under the above conditions, shifting her to B guess and containing (U% to 0.2% acetic acid water can be specific to the burdock three _ compounds i to 8 between specific stagnations (four) The peak value of the wave, and the peaks of the high-performance liquid chromatograms of the various 4 compounds do not overlap and can be easily interpreted. Under the condition of Fig. 19(A), the most obvious peak can be obtained at a residence time of about % minutes. Three-sided compound 1, the most obvious peak obtained when the residence time is about 53 minutes, represents the burdock three _ compound 2 'on time _ 28 minutes face _ scales represent burdock three _ compound 3, in __ about 15 points _ The most _ wave peak silk burdock three _ chemical substance 4 in the retention time _ 18 minutes to obtain the most _ wave peak representative of burdock three _ compound 5 'when the residence time is about 11 minutes, the ^ sensation peak represents the burdock triterpenoid compound 6, in When the residence time is about 5 minutes, the most obvious, the ancient road a main & skin peak represents the burdock triterpenoid compound 7, and the most obvious peak symplectic time is about 26 minutes to represent the burdock three _ compound 8. In Figure 19 ( B) Under the condition of 201217782, the most obvious wave ridge can be obtained at the time of residence time of about 34 minutes, and the yoghurt is found in the stagnation of the stagnation of the stagnation of the stagnation of the stagnation of the stagnation of the stagnation. When the most observational peak is obtained, it represents the triterpenoid compound 3, and the most obvious wave-receiving time is about 15 minutes. The representative time is about 18 minutes. The retention time is about 18 minutes. The most __ silk is more than the three fine compound. When the residence time is about 11 minutes, the most _ peak is obtained, representing the burdock three _ compound 6, and when the residence time is about $ minutes, the county is riding the squad to identify the 樟 三 _ _ compound 7, and when it is staying _ % minutes, the most peak snail is obtained. Compared with the three ugly compounds 8. The high-performance liquid chromatograph of graphs and (D) is characterized by: mobile phase acetonitrile and water (linear gradient program containing w❹ mobile phase: υ initial self-transfer acetonitrile (acn) and Water (including (U% formic acid) starts, after (four) minutes, to 2) 9 rounds of guessing secret water (including (4) formic acid); 嶋 (Q, Q·8 _ min class, 1 practice clock; (4) The ejection volume is 5 μL. The results of Figure 19 (Q, (D) show that in the case of mobile phase acetonitrile and water (containing 〇·1% formic acid), the flow rate is 88 ml/min to L〇 ml. /min can be used to obtain a higher peak value between the 樟 三 三 _ 丨 丨 于 于 于 于 于 于 于 于 于 , , , , , , , , , , , , , , , , , , , , , , , , In Fig. 19 ((10), _日_(10) 糊 显 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表 代表Niu Zhi Zhi San _ compound 3, in the 5 points of the 麟 最 最 最 鹤 波 波 化合物 化合物 化合物 化合物 化合物 ' ' ' ' ' ' 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约 约樟 樟 _ _ compound 6, 'Take the temple 1 about 5 knives when the most obvious peaks represent the burdock three _ compound 7, in the retention ' _ ^ ^ _ face slant compared to the four types of compounds 8. In Figure 19 (D) , °; retention time of about 3G minutes to obtain the most distinct New Zealand peak represents Niuzizhi three boxes of compounds 1 'at the retention time of about 47 minutes to obtain the most obvious peaks representing the Antrodia camphora III compound

2 M__ 25 ^ when the most brilliant peaks represent the three ugly compounds 3 of the burdock, in the retention _ 13 minutes __ wave peaks represent the burdock three _ compound 4, the most obvious peaks when the knives of the spoons are retained during the detention Zhuang compound 5, the most duck peak in the retention time of about 9 minutes, represents the burdock triaxial compound 6, and the most _ peak on the _ about 4 minutes to represent the burdock three _ compound 7, the most lag at about 23 minutes _ wave peak represents burdock three _ compound 8. Figure 19 (E), (F) high performance liquid chromatography analysis conditions: mobile phase B guess water (including 0 · 2 〇 / 〇 formic acid); mobile phase linear gradient program: υ initial self-contained 〇%B guess (acn) and · water (containing 0.2% formic acid) start 'after 60 minutes, to 2) 9〇% acetonitrile and i 〇% water (containing hydrazine formic acid flow rate of (6), 0.8 ml / min and (F ), L 〇 ml / min; side device set UV wavelength 248 nm, injection volume is 5 μl. The results of Figure 19 (6), (F) show in the mobile phase acetonitrile and water (including 0.2% citric acid) 'The flow rate is 毫升8 ml/min to 1 〇ml/min. The peak of the mosquito wave can be obtained for the burdock three _ compound 1 to 8 in a specific __, and the peaks of the south-effect liquid chromatograms of various bis-zinc compounds do not overlap. It can be easily interpreted. Under the condition of Fig. 19(E), the most obvious peak can be obtained at about 35 minutes, which means that the scorpion triterpenoid compound 1 is obtained. When the residence time is about 53 minutes, the most obvious peak is obtained. 201217782 Zhisan fine compound 2, __28 minutes __ wave peak represents burdock three = compound 3, 15 minutes domain the most squama芝三三式化"4 in 18 reward Lin her shirt tender three fine compound 5 'in stagnation coffee, _ touch _ scale silk tender three _ compound 6, in the detention _ 5 ___ scaly oblique key three coffee _, When the residence time is about 26 minutes, the most obvious peak is obtained, which represents the burdock three _ compound 8. Under the condition of Fig., the _ _ 3Q arying the most crane scale 牛牛 观三议 compound 卜湾留时_ 47 minutes When the most peaks are obtained, the bovine 樟 扇 扇 fan compound 2 'when the residence time is about 24 minutes, the most _ peak represents the burdock three _ compound 3, when the stagnation is about I2 read shouting He Bofeng represents the burdock three (four) 贞 compound *, in the detention When the time is about 15 ′′, the simplest peak is the representative of Niu Zhizhi tri-small compound $. When the residence time is about 9 o'clock, the most obvious peak is obtained. It represents the three-sided compound of Niu Zhizhi. When the residence time is about * minutes, the most Congbo peak is represented by Niobium. Fine compound 7, (4) When the retention time is about η minutes, the most _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ l), using the above high efficiency liquid Chromatographic conditions analysis of the liquid extraction of the material, its "process phase chromatograph" parts: mobile phase acetonitrile and water (including 0.2% acetic acid); mobile phase linear gradient program:]) initial self-contained 6〇% B guess (ACN) and 40/. water (containing 0.2% acetic acid) starts 'after 6 minutes, to 2) 9% acetonitrile and 1% water (including 21 201217782 0.2% acetic acid); the flow rate is 0.8 liters / The minute κ is set to an ultraviolet wavelength of 248 nm; the injection volume is 5 μl. The analysis results are shown in Fig. 2. From the g 2 〇, the high-performance liquid chromatograph analysis can be applied to the B. The bovine scorpion striata compound 2 to 8 obtained a specific wave peak at a specific residence time, and the peaks of the high-performance liquid chromatograms of the various three (five) compounds do not overlap and can be easily interpreted. Using the quantitative standard curve of the Antrodia camphora III compound to 8 standard solution (Example 3), the concentrations of the diceramide compounds 2 to 8 in the ethanol extract of the Antrodia camphorata fruit body were obtained, and are listed in Table 3 below. Table 3. Concentrations of Sanzhuang Compounds 2 to 8 in the ethanol extract of Antrodia camphorata fruit body Antrodia camphorata triterpenoids --2- Concentration of three bribes in the ethanol extract of Antrodia camphorata fruit extract (μg/ml) ~ ~~ TL5 3 154.7 " - 4 _ 233.1 " —-- 5 7Q0 S --- 6 L 655.1 "-- 7 772.2 " 8 249.3 " A *- 22 201217782 [Simple diagram] Figure 1, Niu Zhizhi Sancha Chemical formula of compound 1; chemical formula of triterpenoid 2 of Antrodia camphorataFig. 3 Chemical formula of triterpenoid 3 of Antrodia camphorataFig. 4 Chemical formula of triterpenoid compound 4 of Antrodia camphorata

Figure 5. Chemical formula of the triterpenoid compound 5 of Antrodia camphorata. Figure 6. Chemical formula of the triterpenoid compound 6 of Antrodia camphorata. Figure 7. Chemical formula of the triterpenoid compound 7 of Antrodia camphorata. Figure 8. Chemical formula of three boxes of compound 8 of Niobium sinensis. Figure 9. Analysis of the results of the triterpenoid compound of Niobium lucidum by 1% performance liquid chromatography. Figure 丨〇, analysis of the results of the triterpenoids 2 of O. chinensis by high performance liquid chromatography. Round 11. The results of analyzing the triterpenoids 3 of Antrodia camphorata by high performance liquid chromatography. Figure 12. Analysis of the results of Astragalus triterpenoids 4 by high performance liquid chromatography. Figure 13. Analysis of the results of the Astragalus triterpenoids 5 by high performance liquid chromatography. Figure 14. Analysis of the results of Astragalus triterpenoids 6 by high performance liquid chromatography. Figure 15. Results of analysis of Astragalus triterpenoids 7 by high performance liquid chromatography. Figure 16. Analysis of the results of three boxes of compound 8 of Antrodia camphorata by high performance liquid chromatography. Circle 17. The results of the purified standard of the present invention were analyzed by high performance liquid chromatography. Figure 18. Quantitative standard curve of the solution of the triterpenoid triterpenoids 1 to 8 standard solution. Fig. 19 shows the results of using a mobile phase containing formic acid or acetic acid in a high performance liquid chromatography instrument and analyzing the solutions of the 1 to 8 standard solutions of the Antrodia camphorata triterpenoids at different flow rates in the mobile phase. Figure 20. Analysis of the ethanol extract of Antrodia camphorata fruit body by high performance liquid chromatography. 23 201217782 [Description of main component symbols] No 0

twenty four

Claims (1)

201217782 VII. Patent application scope: 1. A method for analyzing triterpen〇ids from Antrodia camp/zorato by high performance liquid chromatography, in which the conditions of high performance liquid chromatography are as follows: (A), The mobile phase linear gradient procedure is: 1) Initially starting with 6〇% acetonitrile (ACN) and 40% water (containing 1.1% to 〇.2% citric acid or acetic acid), after 6 , to 2) 90 % acetonitrile and 1% water (containing 〇.1% to 〇2% citric acid or acetic acid); flow rate of 0.8 to 1.0 ml / min; and (8), side detector · UV wavelength between 235 and 255 nm; It has a specific wave + value at a specific residence time, in which the Astragalus membranaceus includes methyl antcinate B, dehydr〇eburicoic acid, 15α-acetamidine sulphate polysporic acid ( 15a-acetyl dehydrosulphurenic acid), 3β, 15α-di-lane 甾-7,9 (11),24-diluted--21 acid (3β,15a-dihydroxy lanosta-7,9 (11), 24-triene-21 -oiC acid), zhankuic acid A, zhankuic acid C, sulphurenic acid, and burdock (antcinA). 2. The method according to claim 1, wherein the methyl antcinate B has a specific peak value when the residence time is from about 30 to about 35 minutes. 3. The method according to claim 1 wherein dehydroeburicoic acid has a specific peak value when the residence time is from about 47 to about 53 minutes. 4. The method according to claim 1, wherein the ΐ5α-ethyl dehydrosulphurenic acid (15a-acetyl dehydrosulphurenic acid) has a specific wave when the residence time is from about 24 to about 28 minutes. Peak. 5. The method described in the first paragraph of the patent, wherein 3β, 15α-two fresh hair (4) (11), ^-^2^(3M5a.dihy^〇xyW retention time is about 12 to about 16 minutes There is a peak of the wave. 6. The method according to the scope of the patent application, wherein the baric acid a ("pass a) has a specific peak value when the residence time is from about 15 to about 18 minutes. The method of claim 2, wherein the oxalic acid c Μ - C) has a specific peak value when the residence time is from about 9 to about 11 minutes. 8. The method of claim i, wherein the sulphurenic acid has a specific peak value when the residence time is from about 4 to about 5 minutes. 9. According to the scope of the patent application! The method of the present invention, wherein the cow has a specific peak value when the residence time is from about 22 to about 27 minutes. 10. The method according to claim i, wherein the mobile phase linear gradient program is (1) Initially starting with 60% acetonitrile (ACN) and 40% water (containing 〇2% acetic acid), after 6 minutes, to 2) 90% acetonitrile and 10% water (containing 〇.2% acetic acid); 8 ml / min] 贞 side device UV wavelength is 248 nm. 26