CN104177240B - Compounds, extracts and uses isolated from Antrodia camphorata - Google Patents

Abstract

The invention discloses a compound separated from antrodia camphorata, which is represented by a formula I:

Description

Compounds, extracts and uses isolated from Antrodia camphorata

technical field

The present invention relates to a compound isolated from Antrodia camphorata, its extract and its use, especially the use of the compound and its extract to inhibit tumor growth.

Background technique

Antrodia camphorata (Antrodia camphorata), also known as Antrodia camphorata, Antrodia camphorata or Red Antrodia, belongs to the perennial fungi of the order Aphyllophorales and the family Polyporaceae. On the inner wall of the hollow decaying heartwood of Taiwan's conservation tree species - Cinnamoum kanehirai Hay. Due to the extremely rare distribution of Cinnamomum camphora and human-made poaching, the number of wild Antrodia camphorata parasitic in it is even rarer, and because its fruiting body grows very slowly, the growth period is only between June and October. , and therefore very expensive.

The fruiting body of Antrodia cinnamomea is perennial, sessile, corky to woody, with a strong camphor tree aroma, and its shape is varied, with plate, bell, horseshoe or tower shape. It is flat and bright red when it is born, and then its periphery will show a radial and reverse roll shape, and it will expand and grow around, and the color will also change to light reddish brown or light yellowish brown, with many pores, and it is the medicine of Antrodia camphorata. Use the most valuable parts.

In Taiwanese folk medicine, Antrodia Cinnamomea has the functions of detoxification, alleviating diarrhea symptoms, anti-inflammation, treating liver-related diseases and anti-cancer. Antrodia camphorata, like common edible and medicinal mushrooms, has many complex components. The known physiologically active components include: triterpenoids, polysaccharides (such as β-D-glucan), Adenosine, vitamins (such as vitamin B, niacin), protein (including immunoglobulin), superoxide dismutase (superoxide dismutase, SOD), trace elements (such as calcium, phosphorus, germanium), nucleic acid, Sterols and blood pressure stabilizing substances (such as antodia acid), etc. These physiologically active ingredients are considered to have various effects such as anti-tumor, increasing immunity, anti-allergy, anti-bacteria, anti-hypertension, lowering blood sugar and lowering cholesterol.

Among the many components of Antrodia camphorata, triterpenoids have been studied the most. Triterpenoids are a general term for natural compounds that combine 30 carbon elements into hexagonal or pentagonal shapes. The bitter taste of Antrodia camphorata mainly comes from triterpenes. In 1995, Cherng et al. found that the fruiting body extract of Antrodia camphorata contained three new triterpenoids with ergostane as the skeleton: antcin A, antcin B and antcin C (Cherng, I.H., and Chiang, H.C. 1995. Three new triterpenoids from Antrodiacinnamomea. J. Nat. Prod. 58:365-371). Chen et al found three triterpenoids such as zhankuic acid A, zhankuic acid B and zhankuic acid C after extracting the fruiting bodies of Antrodia camphorata with ethanol (Chen, C.H., and Yang, S.W.1995. New steroid acids from Antrodiacinnamomea,-a fungus parasitic on Cinnamomum micranthum. J. Nat. Prod. 58:1655-1661). In addition, in 1995, Chiang et al. also found three other new triterpenoids, which were sesquiterpene lactone and two bisphenol derivatives, namely antrocin,4, 7-dimethoxy-5-methyl-1,3-benzodioxole (4,7-dimethoxy-5-methy-1,3-benzodioxole) and 2,2',5,5'-tetra Methoxy-3,4,3',4'-bis-methylenedioxy-6,6'-dimethylbiphenyl (2,2',5,5'-teramethoxy-3,4,3 ',4'-bi-methylenedioxy-6,6'-dimethylbiphenyl) (Chiang,H.C.,Wu,D.P.,Cherng,I.W.,and Ueng,C.H.1995.Asesquiterpene lactone,phenyl and biphenyl compounds from Antrodiacinnamomea.Phytochemistry.39:613- 616). In 1996, Cherng et al. used the same analysis method to discover four new triterpenoids: antcin E, antcin F, methyl antcinate G, methylantcinate H (Cherng, I.H., Wu, D.P., and Chiang, H.C.1996. Triteroenoids fromAntrodia cinnamomea.Phytochemistry.41:263-267); and Yang et al. discovered two new compounds zhankuic acid D and zhankuic acid E with ergosterane as the backbone, and three with lanostane as the backbone New compounds of 15α-acetyl-dehydrosulphurenic acid (15α-acetyl-dehydrosulphurenic acid), dehydroeburicoic acid and dehydrasulphurenic acid (Yang, S.W., Shen, Y.C., and Chen, C.H. 1996. Steroids and triterpenoids of Antrodiacinnamomea—a fungus parasitic on Cinnamomum micranthum. Phytochemistry. 41:1389-1392). Among them, some components have been discovered one after another and may play an important role in the AMPK and TOR signal transmission pathways. Through the activation of AMPK and the inhibition of the mTOR translation pathway, good control of the G1 phase of the cancer cell cycle can be achieved, and cancer cells can be completely blocked. As the cycle progresses, a series of cancer cells dies.

Although it can be known from many current experiments that the extract of Antrodia Cinnamomea has the aforementioned effects, and its components have been analyzed one after another, but whether there are still other compounds with anticancer activity or other medical uses in the extract of Antrodia Cinnamomea has not been found out , yet to be clarified by further experimental studies.

Contents of the invention

Accordingly, one object of the present invention is to provide a compound isolated from Antrodia camphorata, represented by formula I:

Wherein R1 is hydrogen or acetyl.

Preferably, R of the compound is hydrogen, represented by formula (II):

Preferably, R1 of the compound is acetyl, represented by formula (III):

Another object of the present invention is to provide a use of the above compound for the preparation of a drug for inhibiting tumor growth, wherein the tumor is one of lung adenocarcinoma, colon cancer, prostate cancer, and liver cancer.

Another object of the present invention is to provide an extract extracted from Antrodia cinnamomea, which is extracted by the following steps: take the mycelia of Antrodia cinnamomea, fruiting bodies or a mixture of the two and extract twice with 10 times the amount of alcohol Combined and concentrated afterward to obtain a crude extract, the crude extract was subjected to distribution extraction with dichloromethane/water (1:1) for 3 times to be divided into a dichloromethane layer and a water layer, and the dichloromethane The layers were separated by silica gel column chromatography through the solvents of n-hexane/dichloromethane (1:4), dichloromethane and methanol/dichloromethane (5:95) to obtain the extract.

Description of drawings

Figure 1-Figure 3 is the structural identification of Antrocamol LT1 provided by the embodiment of the present invention;

Figure 4-Figure 6 is the identification of the structure of Antrocamol LT2 provided by the embodiment of the present invention.

detailed description

Take Antrodia camphorata (Antrodia camphorata) mycelia, fruiting bodies or a mixture of the two (1.0kg) and extract twice with 10 times the amount of alcohol, combine and concentrate to obtain about 230g of crude extract (LT-E), crude extract Dichloromethane/water (1:1) was used for partition extraction three times, divided into about 102.6g of dichloromethane layer (LT-E-D) and about 127.4g of water layer (LT-E-W), and 6.0 g of dichloromethane layer was taken g is separated into ANCA-E-D-1, ANCA-E-D-1, ANCA-E-D- 2. Four floors including ANCA-E-D-3 and ANCA-E-D-4.

Anticancer Activity of Antrodia Antrodia Extract

A549 cell line (lung adenocarcinoma), CT26 cell line (colon cancer), DU145 cell line (prostate cancer), HepG2 cell line (liver cancer), MDCK cell line (kidney cancer), PC3 cell line (prostate cancer) For the cell viability assay (MTT cell viability assay), see the results in Tables 1-6 below.

The above cell lines were respectively cultured in appropriate culture medium for 24 hours. The proliferated cells were washed once with PBS, and the cells were treated with 1 times trypsin-EDTA, then centrifuged at 1,200 rpm for 5 minutes, the cells were pelleted and the supernatant was discarded. Then add 10ml of new culture medium, shake slightly to resuspend the cells, and then divide the cells into 96-well microplates. During the test, 0.01-200 μg/ml Antrodia camphorata extract was added to each well, and cultured at 37°C and 5% CO2 for 48 hours. Thereafter, 2.5 mg/ml of MTT was added to each well in a dark environment, and after 4 hours of reaction, 100 μl of lysis buffer was added to each well to terminate the reaction. Finally, the absorbance value was measured with an enzyme immunoassay analyzer at a wavelength of 570nm to calculate the survival rate of the cells, and calculate the concentration required for the half-inhibition rate of growth (ie, the IC50 value). All experimental data are expressed as mean ± standard error . Experimental data were statistically analyzed by paired-t test. A p value less than 0.05 was considered to be statistically different.

Table 1: A549 cell line (lung adenocarcinoma)

Table 3: DU145 cell line (prostate cancer)

Table 4: HepG2 cell lines (liver cancer)

Table 5: MDCK cell lines (kidney cancer)

Table 6: PC3 Cell Lines (Prostate Cancer)

In Table 1~6, 0~25% cell survival: +/-, 25~50% cell survival: +, 50~75% cell survival: ++, 75~100% cell survival: +++, >100 % Cell Survival: ++++. The solvent is DMSO, and its IC50 is 2.34%, which means that when the drug is diluted to a DMSO content of 2.34%, 50% of the cells will die. Here, when the drug is diluted to 100 μg/ml, the DMSO content is 0.5%. ANCA-E, ANCA-E-D, ANCA-E-W, ANCA-E-D-1, ANCA-E-D-2, ANCA-E-D-3, ANCA-E-D-4, LT-80 and LT-C80 are different extracts of Antrodia camphorata .

According to the results in the above tables, ANCA-E-D-2, ANCA-E-D-3, and ANCA-E-D-4 have better inhibitory effects on the growth of various cancer cells. For example, ANCA-E-D-2 and ANCA-E-D-3 among them are for A549 cell line (lung adenocarcinoma), CT26 cell line (colon cancer), DU145 cell line (prostate cancer), HepG2 cell line (liver cancer), MDCK cell line cell line (kidney cancer) and PC3 cell line (prostate cancer), compared with other groups, it has a relatively better inhibitory effect. Although ANCA-E-D-4 is slightly lower than ANCA-E-D-2 and ANCA-E-D-3, it also has a certain inhibitory effect on various tumor cells. Accordingly, the extracts can be used to prepare drugs for inhibiting tumor growth, including one of lung adenocarcinoma, colon cancer, prostate cancer, and liver cancer, and the effective single components can be further purified.

Preparation of Antrocamol LT1 and Antrocamol LT2 from Antrodia Antrodia Extraction

According to the above results, ANCA-E-D-2 and ANCA-E-D-3 were further purified, and the ANCA-E-D-3 layer was purified by preparative reverse phase column chromatography (C-18 reverse phase chromatography column) with 80% MeOH/H20 can obtain about 150 mg of a new compound Antrocamol LT1 around 18.75 minutes, and the LT-E-D-2 layer is prepared by preparative reverse phase column chromatography (C-18 reverse phase chromatography column) with 80% MeOH/H20 About 170 mg of another new compound, Antrocamol LT2, can be obtained around 25.10 minutes. The structural identification results of AntrocamolLT1 are as follows:

Antrocamol LT1 is a colorless liquid product. After analysis, the molecular formula of the compound is C24H38O5, the molecular weight is 406, and the complete Chinese and English name is 4-hydroxy-2,3-dimethoxy-6-methyl-5-(9- Hydroxy-3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone, 4-hydroxy-5-[9-hydroxy-3,7,11-trimethyldodeca -2,6,10-trienyl]-2,3-dimethoxy-6-methyl-cyclohex-2-enone.

LT1 structure identification data (see Figure 1-Figure 3): 1H-NMR (400MHz, CDCl3): 1.12 (3H, d, J=7.2Hz,), 1.61 (3H, s), 1.64 (3H, s), 1.66(3H,s),1.68(3H,s),1.72(1H,m),1.98–2.30(8H),2.51(1H,dq,J=11.6,7.2Hz),3.62(3H,s),4.02 (3H,s),4.33(1H,d,J=2.8Hz),4.35(1H,dt,J=9.2,4.0Hz),5.09(1H,d,J=8.4Hz),5.14(1H,t, J=7.2Hz),5.15(1H,t,J=7.2Hz);13C-NMR(100MHz,CDCl3):12.17(q),15.95(q),16.19(q),18.13(q),25.72(q ), 25.93(t), 26.78(t), 39.41(t), 39.98(d), 43.29(d), 47.94(t), 58.81(q), 60.48(q), 65.35(d), 67.24(d ), 121.64(d), 127.64(d), 128.42(d), 132.03(s), 134.99(s), 135.97(s), 137.42(s), 160.82(s), 197.15(s).

Antrocamol LT2 (refer to accompanying drawing 4-Fig. 6): It is a colorless liquid product, and the molecular formula of this compound is C26H40O6 after analysis; The molecular weight is 448, and the complete Chinese and English name is 4-acetylcarboxy-2,3-dimethoxy -6-methyl-5-(9-hydroxy-3,7,11-trimethyl-2,6,10-dodecatriene)-2-cyclohexenone, 4-acetoxy-5-[ 9-hydroxy-3,7,11-trimethyldodeca-2,6,10-trienyl]-2,3-dimethoxy-6-methyl-cyclohex-2-enone.

LT2 structure identification data: 1H-NMR (400MHz, CDCl3): 1.18 (3H, d, J=7.2Hz,), 1.54 (3H, s), 1.64 (3H, s), 1.67 (3H, s), 1.69 ( 3H,s),1.72(1H,m),1.80–2.40(8H),2.50(1H,dq,J=11.6,7.2Hz),3.65(3H,s),3.98(3H,s),4.36(1H ,m),5.10(1H,t,J=6.8Hz),5.12(1H,d,J=8.0Hz),5.20(1H,t,J=6.4Hz),5.72(1H,t,J=3.2Hz );13C-NMR (100MHz, CDCl3): 12.80(q), 15.96(q), 16.09(q), 18.14(q), 20.93(q), 25.72(q), 26.19(t), 26.76(t) ,39.47(t),41.25(d),42.98(d),48.12(t),59.65(q),60.67(q),65.53(d),68.98(d),120.74(d),127.42(d) , 128.25(d), 131.74(s), 134.70(s), 137.31(s), 137.56(s), 158.21(s), 169.73(s), 196.84(s).

Carry out MTT assay (not repeated here) according to the above-mentioned experimental method, use the analyzer to measure the absorbance value at 570nm absorbance wavelength, so as to calculate the survival rate of the cells, and calculate the concentration required for the half-inhibition rate of its growth (ie IC50 value ), tested the anticancer activity of Antrocamol LT1 and Antrocamol LT2, and compared with ANCA-E, ANCA-E-D and ANCA-E-D-3. All experimental data are expressed as mean ± standard error. Experimental data were statistically analyzed by paired-t test. A p value less than 0.05 was considered to be statistically different. And its half-inhibitory concentration (IC50) is organized into the following table (Table 7).

Table 7

IC50 refers to the concentration of the inhibitor at which it is half-inhibited. IC50 values are usually used to measure the ability of drugs to induce cell death, that is, the stronger the induction ability, the lower the value. It can be seen from the results in the table that the new compounds Antrocamol LT1 and Antrocamol LT2 and the extracts of ANCA-E, ANCA-E-D and ANCA-E-D-3 all have good anticancer activity. Accordingly, these compounds can be used to prepare drugs for inhibiting tumor growth, including one of lung adenocarcinoma, colon cancer, prostate cancer, and liver cancer, and can be further developed into anticancer drugs.

The compounds, extracts and uses isolated from Antrodia cinnamomea provided by the present invention do have industrial utilization value, but the above description is only a description of the preferred embodiments of the present invention, and those who are proficient in this art can rely on the above-mentioned The explanation and make other various improvements, but these changes still belong to the spirit of the present invention and in the scope of the patent defined below.

Claims (6)

1. A compound isolated from Antrodia camphorata, represented by formula I: Wherein R1 is hydrogen or acetyl. 2. The compound as claimed in claim 1, wherein R of the compound is hydrogen, represented by formula (II): 3. The compound as claimed in claim 1, wherein R of the compound is an acetyl group, represented by formula (III): 4. A use of the compound as claimed in claim 1 in the preparation of a drug for inhibiting tumor growth, wherein the tumor is one of lung adenocarcinoma, colon cancer, prostate cancer, and liver cancer. 5. An extract extracted from Antrodia camphorata, which is obtained by extracting the following steps: take the mycelium of Antrodia camphorata, the fruiting body or the mixture of the two, extract twice with 10 times the amount of alcohol, combine and concentrate to obtain a crude extract, the crude extract was subjected to partition extraction with dichloromethane/water 1:1 for 3 times to be divided into a dichloromethane layer and a water layer, and the dichloromethane layer was subjected to silica gel column chromatography The extract was obtained by solvent separation of n-hexane/dichloromethane 1:4, dichloromethane, and methanol/dichloromethane 5:95. 6. A use of the extract as claimed in claim 5 in the preparation of drugs for inhibiting tumor growth, wherein the tumor is one of lung adenocarcinoma, colon cancer, prostate cancer, and liver cancer.