Triterpenoids as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a triterpenoid compound obtained from edible and medicinal fungus Ganoderma Shandong as well as a preparation method and application of the triterpenoid compound in the aspect of resisting diabetes mellitus. The triterpenoid compound is shown as a structural formula I. The triterpenoid compound provided by the invention has remarkable alpha-glucosidase inhibitory activity, is an ideal candidate compound of an alpha-glucosidase inhibitor which is novel in structure and efficient in activity, and has a good application prospect in the aspect of treatment of diabetes mellitus.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a triterpenoid compound obtained from the edible and medicinal fungus Ganoderma lucidum (Shandong Ganoderma), its preparation method, and its application in antidiabetic treatment. Background Technology

[0002] Ganoderma is a fungus belonging to the genus Ganoderma. Ganoderma The collective term for spp., belonging to the Basidiomycota ( Basidiomycetes ), Polyporeles ( Polyporales ), Ganoderma ( Ganodermataceae ), Ganoderma ( Ganoderma As a traditional Chinese medicinal herb, Ganoderma lucidum has a history of medicinal research spanning over a thousand years, and its dried fruiting bodies (such as Ganoderma lucidum var. rubrum) have been studied. G. lingzhi Ganoderma lucidum G. sinense (The following text appears to be a separate, unrelated sentence: "Ganoderma has been included in the Pharmacopoeia of the People's Republic of China. However, current research on the Ganoderma genus mainly focuses on the aforementioned common species, while systematic research on other species remains relatively scarce. Among them, Shandong Ganoderma (...") G. shandongense Discovered and identified as a new species by Zhao Jiding et al. in 1982, there are currently few reports on its chemical composition and biological activities. Ganoderma triterpenes are one of the active components isolated from Ganoderma lucidum. They have complex chemical structures, a bitter taste, high lipid solubility, and broad medicinal value. Modern research has confirmed that the antitumor activity of Ganoderma triterpenes is widely recognized, exerting its effects through multiple mechanisms such as inhibiting tumor cell proliferation, inducing apoptosis, and blocking angiogenesis. Furthermore, some Ganoderma triterpenes have been found to inhibit... α - Pathways such as glucosidase and regulation of glucose metabolism have shown potential anti-diabetic activity, but related research is still in the exploratory stage and a research system of the same scale as that for anti-tumor activity has not yet been formed.

[0003] Diabetes mellitus is a chronic disease characterized by systemic metabolic dysfunction, which can lead to a range of complications, such as atherosclerosis. Data from the International Diabetes Federation (IDF) shows that there are over 463 million people with diabetes worldwide, and the prevalence continues to rise. Currently, first-line treatments for diabetes include insulin injections and oral hypoglycemic agents (such as metformin, acarbose, DDP-4 inhibitors, and SGLT-2 inhibitors), but these therapies are often accompanied by unavoidable side effects. Therefore, developing safer and more effective hypoglycemic drugs and exploring radical treatments have become the focus of current research. Summary of the Invention

[0004] The primary objective of this invention is to provide a triterpenoid compound obtained from the edible and medicinal fungus Ganoderma lucidum, its preparation method, and its application in the treatment of diabetes.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A triterpenoid compound, as shown in structural formula 1.

[0006] In the formula, R1 represents the structure shown in A, B, and C;

[0007] R2 and R3 can be the same or different and are selected from H or hydroxyl groups.

[0008] The triterpenoid compounds are compounds 1-5, and their structures are shown below:

[0009] A method for preparing the aforementioned triterpenoid compound: (1) Preparation of seed culture medium: Shandong Ganoderma mycelium ( Ganoderma shandongense Inoculate with malt culture medium and incubate at 28°C for 5–7 days; (2) Fermentation: The mycelium in the above seed culture medium was inoculated into the rice solid fermentation culture medium and fermented for 40 days using solid fermentation. (3) Extraction: The culture obtained from fermentation is extracted with ethyl acetate at room temperature 3-5 times, and the extracts are combined and concentrated under reduced pressure to obtain crude extract; (4) Chromatographic separation: The crude extract was separated by silica gel, HP20 and ODS column chromatography to obtain the compound shown in Formula 1.

[0010] In step (4), the crude extract is separated by vacuum silica gel column elution using a dichloromethane-methanol (50:1 to 1:1) system with gradient elution at volume ratios to obtain two crude fractions: Fr. A (dichloromethane-methanol 50:1 to 10:1) and Fr. B (dichloromethane-methanol 8:1 to 1:1). Component Fr. A was subjected to gradient elution with 10%–100% ethanol-water solution via HP20 macroporous resin, and 40%–70% of the fraction was collected, which was designated as Fr. A2. The collected fraction was then separated by ODS column chromatography, and gradient elution was performed with 20%–100% ethanol-water solution, and 20%–50% of the fraction was collected, designated as Fr. A2-2, 50%–75% of the fraction was designated as Fr. A2-3, and 75%–100% of the fraction was designated as Fr. A2-4. The collected Fr. A2-2 fraction was subjected to silica gel column chromatography with gradient elution of dichloromethane-methanol at a volume ratio of 20:1 to 1:1, and the fractions at 20:1 to 15:1 were collected and designated as Fr. A2-2-1 and 15:1 to 10:1, respectively. The fraction Fr. A2-2-2 was subjected to preparative HPLC with methanol-water (v / v=60:40), and the chromatographic peak fractions at 22.8 to 25.5 min (a) and 75.2 to 77.8 min (b) were collected. The resulting sample a was subjected to semi-preparative HPLC with acetonitrile-water (44:56), and the chromatographic peak at 39 min was collected to obtain compound 1. The resulting sample b was subjected to semi-preparative HPLC with acetonitrile-water (40:60), and the chromatographic peak at 46.0 min was collected to obtain compound 3. The collected Fr. A2-2-1 fraction was subjected to preparative HPLC, eluted with methanol-water (v / v=58:42), and the chromatographic peak fraction (c) from 49.2 to 54.1 min was collected. The obtained sample c was subjected to semi-preparative HPLC, eluted with acetonitrile-water (44:56), and the chromatographic peak from 27.0 min was collected to obtain compound 2. The collected fraction Fr. A2-3 was eluted by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 10:1, and the collected fraction was Fr. A2-3-1. The fraction was then eluted by preparative HPLC with methanol-water (v / v=60:40), and the chromatographic peak fraction (d) from 59.0 to 63.4 min was collected. The fraction was then eluted by semi-preparative HPLC with acetonitrile-water (39:61), and the chromatographic peak at 30.9 min was collected to obtain compound 4. The collected Fr. A2-4 fraction was eluted by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 5:1. The collected fraction Fr. A2-4-1 was then subjected to preparative HPLC (250×20 mm, 5 μm) with methanol / water at a volume ratio of 65:35 at a flow rate of 7 mL / min. The chromatographic peak fraction (e) from 40.2 to 44.3 min was collected. The obtained sample e was subjected to semi-preparative HPLC (250×20 mm, 5 μm) with acetonitrile-water (30:70) and the chromatographic peak at 34.7 min was collected to obtain compound 5.

[0011] The malt extract culture medium consists of: 20 g / L malt extract, 12 g / L agar, 1 L distilled water, and natural pH; the rice culture medium consists of: 35 g / bottle of rice, 45 mL / bottle of distilled water, and natural pH.

[0012] A pharmaceutical composition comprising one or more of the triterpenoid compounds shown in Formula 1.

[0013] The use of the triterpenoid compound or the composition described herein, specifically the use of the triterpenoid compound of Formula 1 or the pharmaceutical composition described herein, in the preparation of an antidiabetic drug. Compounds 1-5 provided in this invention are all discovered for the first time, and there are no related reports on the bioactivity of these 5 compounds; The compounds 1-5 obtained in this invention were evaluated for their α-glucosidase inhibitory activity. The results showed that, compared with the positive control drug acarbose compound (IC50), their inhibitory activity was significantly lower. 50 Compared to (value of 0.98 µmol / L), both 1 and 4 showed better inhibition. α -Glucosidase activity (IC) 50 The values ​​were 1.26 and 1.28 µmol / L, respectively. Compounds 2, 3, and 5 did not show significant differences. α -Glucosidase inhibitory activity. Therefore, the triterpenoid compounds 1 and 4 described in this invention have the potential to be developed into antidiabetic drugs, paving the way for the development of innovative drugs for the treatment of diabetes. α - This class of innovative drugs, including glucosidase inhibitors, provides a new material basis and a scientific foundation for the development and utilization of natural active substances derived from macrofungi. Furthermore, these compounds were isolated from well-known edible and medicinal macrofungi in my country, thus their safety is preliminarily assured. Detailed Implementation

[0014] The embodiments listed below are intended to help those skilled in the art better understand the present invention, but do not limit the invention in any way.

[0015] The following examples are examples of Ganoderma lucidum from Shandong. G. shandongense The strain used in this invention is a well-known strain recorded in the Journal of Mycology 1986(02):86-92. The strain used in this invention was provided by Professor Dai Yucheng of Beijing Forestry University.

[0016] Example 1: Preparation of triterpenoid compounds 1-5 from Ganoderma lucidum from Shandong 1. Fermentation conditions Strain activation: Shandong Ganoderma lucidum G. shandongense Mycelium was inoculated onto malt extract medium and cultured at 28°C in the dark for 5-7 days. Then, the mycelium on the plate was evenly cut into 0.5 cm pieces. 2 Small pieces are to be used later; the components of the malt extract culture medium are: 10 g malt extract, 500 mL distilled water, 7.5 g agar, and natural pH. Large-scale culture of the strain: Take 10 g of malt extract powder, 500 mL of distilled water, and 7.5 g of agar, and autoclave at 121℃ for 30 min. Set aside. Cut the activated strain into pieces... G. shandongense Inoculate into the above culture medium and incubate at 28°C for 5-6 days.

[0017] Fermentation: 100g of rice was placed in a 500mL Erlenmeyer flask, 100mL of water was added, and the mixture was autoclaved at 121℃ for 30 minutes. The resulting culture was expanded by adding 1 / 4 volume dish to each flask to obtain Shandong Ganoderma lucidum. G. shandongense The filaments were inoculated and cultured statically at 28°C for 40 days.

[0018] 2. Extraction and Separation The fermented culture was extracted four times with ethyl acetate at room temperature. The extracts were combined and concentrated under reduced pressure at 40°C to obtain an extract. The obtained extract (350g) was mixed with 100-200 mesh silica gel and separated by a silica gel column (25×15 cm) under reduced pressure. Gradient elution was performed sequentially by volume ratio using a dichloromethane-methanol (50:1 to 1:1) system. The fractions were identified and combined by thin-layer chromatography and high-performance liquid chromatography to obtain two components: Fr.A (dichloromethane-methanol 50:1 to 10:1) and Fr.B (dichloromethane-methanol 8:1 to 1:1).

[0019] Fraction Fr. A was subjected to gradient elution with 10%–100% ethanol-water solution via HP20 macroporous resin, and the fraction collected from 40% to 70% was designated as Fr. A2. The obtained fractions were then separated by ODS column chromatography, and gradient elution was performed with 20%–100% ethanol-water solution, and the fractions collected from 20% to 50% were designated as Fr. A2-2, 50% to 75% as Fr. A2-3, and 75% to 100% as Fr. A2-4. The collected Fr. A2-2 fraction was subjected to silica gel column chromatography with gradient elution of dichloromethane-methanol at a volume ratio of 20:1 to 1:1. The fractions of 20:1 to 15:1 were collected and designated as Fr. A2-2-1 and the fractions of 15:1 to 10:1 were designated as Fr. A2-2-2.

[0020] Fraction Fr. A2-2-2 was subjected to preparative HPLC, eluted with methanol-water (v / v=60:40), and the chromatographic peak fractions (a) from 22.8 to 25.5 min and (b) from 75.2 to 77.8 min were collected. Sample a was subjected to semi-preparative HPLC, eluted with acetonitrile-water (44:56), and the chromatographic peak at 39 min was collected to obtain compound 1. Sample b was subjected to semi-preparative HPLC, eluted with acetonitrile-water (40:60), and the chromatographic peak at 46.0 min was collected to obtain compound 3.

[0021] Fr. A2-2-1 was subjected to preparative HPLC, eluted with methanol-water (v / v=58:42), and the chromatographic peak (c) from 49.2 to 54.1 min was collected. Then, the component was subjected to semi-preparative HPLC, eluted with acetonitrile-water (44:56), and the chromatographic peak at 27.0 min was collected to obtain compound 2.

[0022] The collected fraction Fr. A2-3 was eluted by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 10:1, and the collected fraction was Fr. A2-3-1. This fraction was then eluted by preparative HPLC with methanol-water (v / v=60:40), and the chromatographic peak fraction (d) from 59.0 to 63.4 min was collected. Subsequently, the fraction was subjected to semi-preparative HPLC chromatography with acetonitrile-water (39:61), and the chromatographic peak at 30.9 min was collected to obtain compound 4.

[0023] The Fr. A2-4 fraction was eluted by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 5:1, and the fraction Fr. C2-4-1 was collected. The fraction was then subjected to preparative HPLC (250×20 mm, 5 μm) with methanol / water at a volume ratio of 65:35 at a flow rate of 7 mL / min, and the chromatographic peak fraction (e) from 40.2 to 44.3 min was collected. The obtained sample e was subjected to semi-preparative HPLC (250×20 mm, 5 μm) with acetonitrile-water (30:70), and the chromatographic peak at 34.7 min was collected to obtain compound 5.

[0024] The results of systematic structural identification of compounds 1-5 are as follows: Compound 1: White powder (methanol), HRESIMS gives quasi-molecular ion peaks m / z 521.2882 [M + Na] + (calcd for C 30 H 42 O6Na (521.2879), its molecular formula was determined to be C based on proton and carbon spectral data. 30 H 42 O6, with a calculated unsaturation of 10. The proton spectrum shows four singlet methyl proton signals in the high-field region. d H 1.40 (3H, s), 1.05 (3H, s), 0.95 (3H, s), 0.71 (3H, s), two bimodal methyl proton signals. d H 1.29 (3H, d, J = 6.5 Hz), 1.15 (3H, d, J = 6.9 Hz), showing three oxygen proton signals in the low field region. d H 4.88(1H, dd, J = 8.9, 3.1Hz), 3.69 (1H, d, J = 11.4 Hz), 3.35 (1H, d, J = 11.4 Hz); combined with carbon spectrum data, 30 carbon signals were given, and in the low field region, there were 4 ketone carbonyl carbon signals. d C 215.9, 211.0, 197.4, 179.0; a pair of double-bonded carbon signals. d C 162.9, 139.5; two oxygen-carbon signals, d C 79.0, 66.0; six methyl carbon signals d C 25.2, 18.7, 17.0, 16.8, 16.4, 15.6; four methylene carbon signals d C 45.7, 44.8, 43.5, 33.4; eight methylene carbon signals d C 36.8, 35.6, 34.9, 32.7, 32.2, 30.2, 28.7, 24.1; four seasonal carbon signals d C 52.4, 47.6, 45.1, 39.2. The above NMR data were compared with (+)-(5 α , twenty three R , twenty four Z Comparison of α-lanosta-8, 24-dien-3, 7-dioxo-23, and 26-γ-lactone revealed that both compounds share the same skeleton. However, compound 1 exhibits an additional carbonyl carbon signal and lacks a double bond signal, suggesting that compound 2 also contains a furan ring in its side chain. The planar structure of compound 2 is shown in the figure. 1 H- 1 HCOSY, HMBC, and HSQC spectra were determined. H-1 / H-2, H-11 / H-12, H-15 / H-16 / H-17 / H-20 / H-21, and H-23 / H-24 / H-25 / H-27 were respectively... 1 H- 1 Four spin-coupled systems are shown in H COSY; the planar structure of 2 is determined by HSQC and HMBC spectra, Me-29 and d C 215.9, 43.5, 66.0, and 52.4 are related; H-28 and d C 215.9 is relevant, confirming that the methyl group at C-29 and the hydroxyl group at C-28 form a substitution; Me-19 and d C 162.9 is relevant; Me-30 and d C The correlation between 139.5 and 47.6 confirms the double bond at positions C-8 and C-9; Me-21 and d C The correlation between 211.0 and 45.7 indicates the presence of a carbonyl substitution at C-22; Me-27 and d C 32.7 and 179.0 are related, H-23 and d C The correlations at 211.0, 179.0, and 32.7 indicate that the branched chain of compound 1 does indeed form a furan ring, and C-26 is a carbonyl ester signal. The configuration of compound 1 was determined by NOESY spectroscopy. Correlation exists between H-28 and Me-29, H-5 and Me-30, and H-17 and Me-21 and Me-30, proving that Me-21, Me-28, Me-30, H-5, and H-17 are... α Configuration; there is a correlation between Me-18 / Me-19 / Me-29 / H-20, indicating that Me-29, Me-18, Me-19, and H-20 are... β The correlation between H-23 and H-25 is not obvious in the NOESY spectrum, so the chiral carbon on the furan ring in the side chain is determined by calculated NMR, and the correlation is obtained by comparing R. 2 Based on the DP4+ value, the configuration of compound 1 was finally determined to be 23. S , 25 S A Scifinder search revealed the compound to be a novel compound, named ganoshandongone Q.

[0025] Compound 2: White powder (methanol), HRESIMS gives quasi-molecular ion peaks m / z 521.3247 [M + Na]+, (calcd for C 31 H 46 O5Na, 521.3243), its molecular formula is C 31 H 46 O5, the calculated degree of unsaturation is 9. 1 ¹H-NMR (600MHz, CDCl₃) showed a bimodal methyl proton signal in the high-field region. d H 0.94 (3H, d, J= 6.8 Hz), 6 single-peak methyl proton signals, respectively d H The values ​​of 1.70 (3H, s), 1.34 (3H, s), 1.12 (3H, s), 1.10 (3H, s), 0.97 (3H, s), and 0.69 (3H, s) are consistent with the characteristics of triterpenoids, suggesting that this is a triterpenoid compound. A proton signal of an olefin was observed in the low-field region. d H 5.47 (1H, t, J = 6.9 Hz); two oxygen-linked proton signals, d H 3.75 (1H,dd, J = 8.5, 5.1 Hz), 4.04 (1H, s). In 13 In the C-NMR (150 MHz), 30 carbon signals were given, with two ketone carbonyl carbon signals present in the low-field region. d C 214.8, 198.2; two pairs of double-bonded carbon signals, d C 162.9, 139.6 and d C 137.6, 122.4, two oxygen-carbon signals. d C 73.4, 68.8. d C 25.5, 25.2, 21.5, 18.1, 16.0, 14.1, 12,1 represent the seven methyl carbon signals; d C 37.3, 35.5, 34.5, 34.1, 32.0, 30.3, 28.3, and 24.0 represent eight methylene carbon signals; d C 50.5, 45.0, and 41.0 represent three methylene carbon signals; d C 48.0, 47.4, 45.7, and 39.6 represent four quaternary carbon signals. These data suggest that compound 2 may be a lanostane-type triterpene. Furthermore, combining the HSQC spectrum, the carbon signals were assigned to their corresponding hydrogen signals, and the hydrogen spectrum... d H 5.47 (1H, t, J = 6.9 Hz) indicates that one olefin carbon is bonded to a methylene group; the two oxygen-containing carbon signals, d C 73.4, 68.8, corresponding to 1H NMR data. d H 3.75 (1H, dd, J = 8.5, 5.1 Hz), 4.04 (1H, s); H-1 / H-2, H-11 / H-12, H-15 / H-16 / H-17 / H-20 / H-21 / H-22 / H-23 / H-24 are respectively 1 H- 1 The three spin-coupling systems in H COSY; the planar structure of compound 2 was determined based on the HMQC and HMBC spectra. In the HMBC spectrum, Me-19 and d C 162.9 is relevant, Me-30 and d C 139.6 is related, thus confirming a double bond between C-8 and C-9; Me-27 and d C 137.6 and 122.4 are related; H-24 and d C 14.1 is relevant, proving that another double bond is located between C-24 and C-25; the hydroxyl substitution formed at C-22 is through Me-21 and d C The correlation between 41.0 and 73.4 is used to prove this; Me-26 and d C The correlations at 137.6, 122.4, and 14.1 indicate that a hydroxyl substitution has also formed at C-26. The stereoconfiguration of compound 2 was determined by NOESY spectroscopy. The correlations between H-5 and Me-28 and Me-30, and between H-17 and Me-21 and Me-30, indicate that Me-21, Me-28, Me-30, H-5, and H-17 are... α Configuration; there is a correlation between Me-18 / Me-19 / Me-29 / H-20, indicating that Me-29, Me-18, Me-19, and H-20 are... β Type 1; due to the large spatial distance between the H-22 position on the side chain and the parent nucleus, no relevant signal was observed. Therefore, the two possible configurations were determined by calculating NMR, and the correlation coefficient R between the NMR data and the carbon spectrum data was used to determine the configuration. 2 Analysis of the probability analysis results DP4+ revealed that compound 2 showed the highest agreement with the calculated results of A, R. 2 The value was 0.9973, and DP4+(all date) was 100%, thus determining that the configuration at position C-22 of the compound was 22S. After searching with SciFinder, compound 2 was identified as a new compound and named ganoshandongone R.

[0026] Compound 3: White powder (methanol), HRESIMS gives quasi-molecular ion peaks m / z 509.3240 [M + Na] + (calcd for C 30 H 46 O5Na, 509.3243), its molecular formula is C 30 H 46 O5, calculated degree of unsaturation is 7. Compound 3... 1 In the H-NMR (600 MHz, CDCl3), seven singlet methyl proton signals were observed in the high-field region, namely... d H The values ​​of 1.70 (3H, s), 1.34 (3H, s), 1.29 (3H, s), 1.12 (3H, s), 1.10 (3H, s), 0.97 (3H, s), and 0.88 (3H, s) are consistent with the characteristics of triterpenoids, leading to a preliminary inference that the compound is a triterpenoid. A proton signal of an olefin was observed in the low-field region. d H 5.52 (1H, t, J = 7.4 Hz); Two oxygen-bound proton signals d H 3.40 (1H,dd, J =10.3, 2.3 Hz), 4.04 (1H, s); in 13 In the 150 MHz C NMR spectrum, 30 carbon signals were obtained. By combining the HSQC spectrum and assigning the carbon signals to their corresponding hydrogen signals, two ketone carbonyl carbon signals were found in the low-field region. d C 214.8, 198.2; d C 162.8, 139.0 and d C 138.1 and 122.6 represent two pairs of double-bonded carbon signals. d C 79.0, 76.5, and 68.7 represent three oxygen-bound carbon signals; the chemical shifts of the remaining 21 carbon signals are all below 51. Combined with the proton NMR spectrum... d H 5.52 (1H, t, J (7.4 Hz) It was learned that one of the methylene groups is bonded to an olefin carbon atom; d C 79.0 and 68.7 are two oxygen-bound carbon signals, corresponding to the following proton spectrum data: d H 3.40 (1H,dd,J = 10.3, 2.3 Hz), 4.04 (1H, s); 1 H- 1 H COSY analysis revealed four spin-coupled systems: H-1 / H-2, H-11 / H-12, H-15 / H-16 / H-17, and H-22 / H-23 / H-24. Combined with HMQC and HMBC spectra, the planar structure of compound 3 was determined. In the HMBC spectrum, Me-19 and... d C 162.8, 35.5, 39.6, and 50.5 are related; Me-30 and d C The values ​​139.0, 47.9, and 31.7 are related, thus determining the location of the double bonds at C-8 and C-9; Me-27 and d C 122.6 is relevant; H-24 and d C 14.2 and 68.7 are related, H-26 and d C 138.1 and 122.6 are related, proving that another double bond lies between C-24 and C-25; Me-21 and d C The correlation between 76.5, 47.4, and 79.0 proves that a hydroxyl substitution has formed at C-22; Me-26 and... d C 138.1, 122.6, and 14.2 are related; H-22 and d C A correlation exists at 47.4, proving that hydroxyl substitutions have formed at C-20, C-22, and C-26. In the NOESY spectrum, H-24 and H-26 are correlated, confirming... D 24, 25 The configuration is trans; there is a correlation between H-17 / Me-30 / Me-21 / H-22 and Me-30 / H-5 / Me-28, proving that Me-30, Me-28, Me-21, H-5, H-17, and H-22 are... α Configuration; there is a correlation between Me-18 / Me-19 / Me-29, indicating that Me-29, Me-18, and Me-19 are... β The final determination was that compound 3 was a new compound, named ganoshandongone S.

[0027] Compound 4: White powder (methanol), HRESIMS gives a quasi-molecular ion peak. m / z 525.3237 [M + Na] +(calcd for C 30 H 46 O6Na, 525.3243), its molecular formula is C 30 H 46 O6, the calculated degree of unsaturation is 7. In compound 4... 1 In the H-NMR (600 MHz, CDCl3), seven singlet methyl proton signals were observed in the high-field region, namely... d H 1.71 (3H, s), 1.37 (3H, s), 1.25 (3H, s), 1.13 (6H, s), 1.10 (3H, s), 0.92 (3H, s); The low-field region shows a proton signal of an olefin. d H 5.56 (1H, t, J = 6.9 Hz); Three oxygen proton signals d H 3.70 (1H, dd, J =10.6, 2.1 Hz), 4.05 (1H, s), 4.79 (1H, m). In 13 The 150 MHz C NMR spectrum yielded 30 carbon signals. Combined with the HSQC spectrum, the carbon signals were assigned to their corresponding hydrogen signals, indicating the presence of two ketone carbonyl carbon signals in the low-field region. d C 214.6, 198.1; two pairs of double-bonded carbon signals, d C 163.6, 138.4 and d C 138.2, 122.7, four oxygen-carbon signals, d C The chemical shifts for the remaining 21 carbon signals are all below 50.6, at 80.6, 78.3, 75.2, and 68.6, respectively. Combined with the proton NMR spectrum... d H 5.56 (1H, t, J = 6.9 Hz) indicates that one olefin carbon is bonded to a methylene group; the three oxygen-containing carbon signals d C 80.6, 75.2, 68.6, corresponding to the proton NMR data are: d H 3.70 (1H, dd, J = 10.6, 2.1 Hz), 4.79(1H,m), 4.05 (1H, s); 1 H- 1H COSY analysis revealed three spin-coupled systems: H-1 / H-2, H-11 / H-12, and H-22 / H-23 / H-24. Furthermore, combining HMQC and HMBC spectra, the planar structure of compound 4 was determined. In the HMBC spectrum, Me-19 and... d C 163.7, 35.3, 39.7, and 50.5 are related; Me-30 and d C The correlation between 138.4, 44.1, and 46.1 indicates that one of the double bonds is located between C-8 and C-9; Me-27 and d C 122.7, 138.2, and 68.6 are related; H-26 and d C 14.2, 122.7, and 138.2 are related, proving that another double bond is located between C-24 and C-25 and that a hydroxyl substitution is formed at C-26; Me-21 and d C 78.3, 80.6, and 48.9 are related; H-22 and d C 78.3, 23.6, and 122.7 are related, proving that hydroxyl substitutions are formed at C-20 and C-22; H-16 and H-17 are related to... d C A correlation exists at 46.0, proving that a hydroxyl group has been substituted at C-16. In the NOESY spectrum, H-24 and H-26 are correlated, confirming... D 24, 25 The configuration is trans; there is a correlation between H-17 / Me-30 / Me-21 / H-22, and H-17 ( J = 7.8 Hz), there is a correlation between Me-30 / H-5 / Me-28, proving that Me-30, Me-28, Me-21, H-17, and H-22 are α Configuration, H-16 is β The type; related to Me-18 / Me-19 / Me-29 indicates that Me-29, Me-18, and Me-19 are... β In conclusion, compound 5 is a new compound, named ganoshandongone T.

[0028] Compound 5: White powder (methanol), HRESIMS gives quasi-molecular ion peaks m / z 567.3303 [M + Na] + (calcd for C 32 H 48O7Na, 567.3298), its molecular formula is C 32 H 48 O7, calculated unsaturation degree is 8. In 1 In the 1H-NMR (600MHz, CDCl3) spectrum, compound 5 showed 8 methyl signals. d H 2.08 (3H, s), 1.70 (3H, s), 1.37 (3H, s), 1.24 (3H, s), 1.13 (6H, s), 1.10 (3H, s), 0.91 (3H, s); three hydrogen-oxygen signals. d H 4.80(1H, m), 4.49(2H, s), 3.72 (1H, dd, J = 10.3, 2.2 Hz); a olefin proton signal d H 5.60 (1H, t, J = 7.4 Hz). Combined with the carbon spectrum data, 32 carbon signals were obtained, suggesting that this compound is a triterpenoid. Further analysis of its carbon spectrum revealed two ketone carbonyl carbon signals and one ester carbonyl carbon signal in the low-field region. d C 214.6, 198.1, 171.2; two pairs of double-bonded carbon signals. d C 163.7, 138.4 and d C 133.4, 126.5. H-1 / H-2, H-11 / H-12, H22 / H23 / H24 are respectively 1 H- 1 Three spin-coupled systems in H COSY; combining HMQC and HMBC spectra, the planar structure of 5 was determined, and in the HMBC spectrum, Me-19 and d C 163.7, 50.5, 39.7, and 35.3 are related; Me-30 and d C 138.4 is relevant, thus identifying one of the double bonds located between C-8 and C-9; Me-28 and d C 214.6, 47.4, and 21.6 are related; Me-29 and d C 214.6, 47.4, and 25.5 are related, confirming that two methyl groups are attached at the C-4 position; H-24 is related to... d C126.5 connected, Me-23 and d C 126.5 is relevant; Me-27 and d C 126.5 and 133.4 are related, confirming that another double bond lies between C-24 and C-25; Me-22 and d C 78.3, 48.9, and 23.3 are related; Me-17 and d C 78.3 and 75.2 are related, proving that hydroxyl substitutions are formed at positions C-16, C-20, and C-22; Me-27 is related to... d C 70.1 is relevant. d H 4.49, Me-32 and d C A correlation was found at 171.2, confirming that the acetoxy group is located at C-26, thus determining the planar structure of compound 5. In the NOESY spectrum, a correlation was found between H-24 and H-26, confirming... D 24, 25 The configuration is trans; the H-17 is related to the Me-30 and Me-21, and the H-17 ( J = 7.8Hz), H-5 and Me-28 are correlated, proving that Me-28, Me-30, Me-21, H-5, H-17, and H-22 are α Configuration, H-16 is β The type; related to Me-18 / Me-19 / Me-29 indicates that Me-29, Me-18, and Me-19 are... β Compound 5 is a new compound, named ganoshandongone U.

[0029] The NMR signals of compounds 1-5 are assigned as follows: Table 1 1 H NMR (600 MHz) data for compounds1-5(CDCl3)

[0030] Table 2 13 C NMR(150 MHz) data for compounds1-5(CDCl3)

[0031] Example 2: Compound α -Glucosidase inhibitory activity The obtained compounds and the positive control acarbose were first dissolved in DMSO and then diluted with PBS to concentrations of 200 µmol / L, 100 µmol / L, 50 µmol / L, 10 µmol / L, and 5 µmol / L, respectively, for later use. 25 µL of the sample (or acarbose) solution was added to each well of a 96-well plate. α 25 µL of glucosidase solution (0.4 U / mL) and 175 µL of PBS were added in triplicate and incubated at 37 °C for 10 min. Then, 25 µL of pNPG substrate solution (5 mmol / L) was added, and the reaction was continued at 37 °C for 30 min. The absorbance A1 was measured at 405 nm. An equal volume of PBS was then used to replace the previous incubation. α - Glucosidase solution, measured in triplicate, absorbance A2 at 405 nm; replace the sample with an equal volume of PBS, and measure absorbance A3 at 405 nm; replace the sample with an equal volume of PBS and... α - Glucosidase was measured at 405 nm, and the absorbance A4 was determined. Acarbose diluted to concentrations of 200 µmol / L, 100 µmol / L, 50 µmol / L, 10 µmol / L, and 5 µmol / L served as positive controls. Triple replicates were performed, and the average value was calculated. α -Glucosidase inhibition rate, the formula is: Inhibition rate (%) = [1] - (A) 样品组 - A 样品空白组 ) / (A 空白组 - A 空白对照组 []×100. Finally, the half-inhibition concentration (IC50) of each sample was calculated using GraphPad Prism 9.5.1 software. 50 )value.

[0032] Experiments have confirmed that the positive control acarbose is effective against... α -Glucosidase IC 50 The value was 0.98 ± 0.03 µmol / L, for compounds 1 and 4. α -Glucosidase IC 50 The values ​​were 1.26 ± 0.07 and 1.28 ± 0.12 µmol / L, respectively, indicating that compounds 1 and 4 have... α - It inhibits glucosidase activity, and its extraction and separation methods are simple. At the same time, the obtained compound is a natural compound and safe, which facilitates further pharmacological research and development of its application in the preparation of hypoglycemic or antidiabetic drugs.

[0033] Table 3 Inhibition of compounds 1 and 4 α -IC50 of glucosidase 50 value

Claims

1. A triterpenoid compound, characterized in that: Triterpenoid compounds are shown in structural formula 1. In the formula, R1 represents the structure shown in A, B, and C; R2 and R3 can be the same or different and are selected from H or hydroxyl groups.

2. The triterpenoid compound according to claim 1, characterized in that: The triterpenoid compounds are compounds 1-5, and their structures are shown below: 。 3. A method for preparing the triterpenoid compound according to claim 1, characterized in that: (1) Preparation of seed culture medium: Shandong Ganoderma lucidum mycelium ( Ganoderma shandongense Inoculate with malt culture medium and incubate at 28°C for 5–7 days; (2) Fermentation: The mycelium in the above seed culture medium was inoculated into the rice solid fermentation culture medium and fermented for 40 days using solid fermentation. (3) Extraction: The culture obtained from fermentation is extracted with ethyl acetate at room temperature 3-5 times, and the extracts are combined and concentrated under reduced pressure to obtain crude extract; (4) Chromatographic separation: The crude extract was separated by silica gel, HP20 and ODS column chromatography to obtain the compound shown in Formula 1.

4. The method for preparing triterpenoid compounds according to claim 3, characterized in that: In step (4), the crude extract is separated by vacuum silica gel column elution using a dichloromethane-methanol (50:1 to 1:1) system with gradient elution at volume ratios to obtain two crude fractions: Fr. A (dichloromethane-methanol 50:1 to 10:1) and Fr. B (dichloromethane-methanol 8:1 to 1:1). Component Fr. A was subjected to gradient elution with 10%–100% ethanol-water solution via HP20 macroporous resin, and 40%–70% of the fraction was collected, which was designated as Fr. A2. The collected fraction was then separated by ODS column chromatography, and gradient elution was performed with 20%–100% ethanol-water solution, and 20%–50% of the fraction was collected, designated as Fr. A2-2, 50%–75% of the fraction was designated as Fr. A2-3, and 75%–100% of the fraction was designated as Fr. A2-4. The collected Fr. A2-2 fraction was subjected to silica gel column chromatography with gradient elution of dichloromethane-methanol at a volume ratio of 20:1 to 1:1, and the fractions at 20:1 to 15:1 were collected and designated as Fr. A2-2-1 and 15:1 to 10:1, respectively. The fraction Fr. A2-2-2 was subjected to preparative HPLC with methanol-water (v / v=60:40), and the chromatographic peak fractions at 22.8 to 25.5 min (a) and 75.2 to 77.8 min (b) were collected. The resulting sample a was subjected to semi-preparative HPLC with acetonitrile-water (44:56), and the chromatographic peak at 39 min was collected to obtain compound 1. The resulting sample b was subjected to semi-preparative HPLC with acetonitrile-water (40:60), and the chromatographic peak at 46.0 min was collected to obtain compound 3. The collected Fr. A2-2-1 fraction was subjected to preparative HPLC, eluted with methanol-water (v / v=58:42), and the chromatographic peak fraction (c) from 49.2 to 54.1 min was collected. The obtained sample c was subjected to semi-preparative HPLC, eluted with acetonitrile-water (44:56), and the chromatographic peak from 27.0 min was collected to obtain compound 2. The collected fraction Fr. A2-3 was eluted by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 10:1, and the collected fraction was Fr. A2-3-1. The fraction was then eluted by preparative HPLC with methanol-water (v / v=60:40), and the chromatographic peak fraction (d) from 59.0 to 63.4 min was collected. The fraction was then eluted by semi-preparative HPLC with acetonitrile-water (39:61), and the chromatographic peak at 30.9 min was collected to obtain compound 4. The collected Fr. A2-4 fraction was eluted by silica gel column chromatography with dichloromethane-methanol at a volume ratio of 5:

1. The collected fraction Fr. A2-4-1 was then subjected to preparative HPLC (250×20 mm, 5 μm) with methanol / water at a volume ratio of 65:35 at a flow rate of 7 mL / min. The chromatographic peak fraction (e) from 40.2 to 44.3 min was collected. The obtained sample e was subjected to semi-preparative HPLC (250×20 mm, 5 μm) with acetonitrile-water (30:70) and the chromatographic peak at 34.7 min was collected to obtain compound 5.

5. The method for preparing triterpenoid compounds according to claim 3, characterized in that: The malt extract culture medium consists of: 20 g / L malt extract, 12 g / L agar, 1 L distilled water, and natural pH; the rice culture medium consists of: 35 g / bottle of rice, 45 mL / bottle of distilled water, and natural pH.

6. A pharmaceutical composition, characterized in that, The composition contains one or more of the triterpenoid compounds represented by Formula 1 as described in claim 1.

7. The use of a triterpenoid compound according to claim 1 or a composition according to claim 6, characterized in that: The use of the triterpenoid compound of claim 1 or the pharmaceutical composition of claim 6 in the preparation of an antidiabetic drug.

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