Sesquiterpenoids Oxyphyllene F and Oxyphyllene G separated from fructus alpiniae oxyphyllae and application of sesquiterpenoids Oxyphyllene F and Oxyphyllene G

Abstract

The invention relates to sesquiterpenoids Oxyphyllene F and Oxyphyllene G which are separated from fructus alpiniae oxyphyllae and application of the sesquiterpenoids Oxyphyllene G. Experimental research finds that the two compounds can inhibit the expression of fibronectin, type I collagen and alpha-smooth muscle actin in rat renal tubular duct epithelial cells induced by TGF-beta1, have anti-renal fibrosis activity, and can be used for preparing anti-renal fibrosis drugs. The fructus alpiniae oxyphyllae can be effectively used for preparing kidney protection drugs, the preparation method is good in reproducibility, the purity of the obtained compound is high, further pharmacological and clinical research on the compound is facilitated, the medicinal value of the fructus alpiniae oxyphyllae is expanded, and the economic and social benefits are huge.

Description

I. TECHNICAL FIELD

[0001] The present application relates to the technical field of extraction and separation of natural active ingredients, in particular to the sesquiterpenes compounds Oxyphyllerene F (compound III) and Oxyphyllerene G (compound IV) separated from Fructus Alpiniae Oxyphyllae and the application thereof. II. BACKGROUND

[0002] Chronic kidney disease (CKD) is one of the major public health problems worldwide, with a global prevalence of 9.1% (approximately 69.75 million cases) and 1.2 million deaths per year. According to the statistics from 1997 to 2017, the prevalence rate increased by 29.3%, while the incidence of dialysis and kidney transplantation increased by 32.8% and 21.6%, respectively. These results indicate the urgent need to improve CKD and the importance of finding targeted therapies. The development of renal fibrosis is one of the important features of the biological and pathological changes of CKD patients. Certain fibrosis molecules, such as fibronectin (Fibronectin), transforming growth factor (TGF-β1), and collagen I, III, IV (collagen I, III, IV), are important indicators of the progression of renal fibrosis. Fibroblasts can also proliferate and express alpha-smooth muscle actin (alpha-SMA), which is identified as a marker of renal fibrosis. In addition, during the progression of progressive tubulointerstitial nephropathy, damaged or apoptotic cells attract inflammatory cells such as macrophages and granulocytes to infiltrate to induce an inflammatory response, leading to the progression of kidney damage. Therefore, anti-fibrosis has recently been considered as a new strategy for the treatment of CKD.

[0003] Traditional Chinese medicine often has the characteristics of multiple effects, bidirectional regulation, and fewer adverse reactions, and is rich in sources, and is increasingly valued by people. Finding and developing traditional Chinese medicine and its effective components with kidney protection have gradually become one of the research hotspots in modern medicine. Fructus Alpiniae Oxyphyllae is the dried mature fruit and seed of Alpinia oxyphylla Miq. of Zingiberaceae, which is a natural plant with medicinal and edible properties announced by the Ministry of Health in 1998, mainly produced in the mountainous areas of Hainan Island, China, and is one of the four major southern medicines in China. Fructus Alpiniae Oxyphyllae is warm in nature and pungent in taste, and belongs to the heart, spleen, and kidney channels, and has the effects of warming and tonifying, warming the spleen to stop diarrhea, and warming the kidney to astringe the essence and shrink the urine. Fructus Alpiniae Oxyphyllae mainly contains terpenes, diphenyl heptanes, flavonoids, sterols, and phenolic acids. However, how to extract sesquiterpenes compounds Oxyphyllerene F (compound III) and Oxyphyllerene G (compound IV) from Fructus Alpiniae Oxyphyllae and their application in the preparation of anti-renal fibrosis drugs have not been reported in patents or documents to date.

[0004] Oxyphyllerene G (compound IV) from Fructus Alpiniae Oxyphyllae and their application in the preparation of anti-renal fibrosis drugs have not been reported in patents or documents to date. III. SUMMARY

[0005] In view of the above, in order to solve the defects of the prior art, the purpose of the present application is to provide a sesquiterpene compound Oxyphyllerene F and Oxyphyllerene G isolated from Fructus Alpiniae Oxophyllae and applications thereof, novel compounds are discovered from Fructus Alpiniae Oxophyllae, and the application of the novel compounds in the development of kidney protection drugs is realized.

[0006] One of the technical solutions provided by the present application is that two new sesquiterpene compounds, Oxyphyllerene F (compound III) and Oxyphyllerene G (compound IV), are isolated and identified from the n-butanol part of Fructus Alpiniae Oxophyllae. The two compounds are a group of epimers, and the difference between them is the configuration of the 11th carbon atom. Their structural formulas are as follows:

[0007]

[0008] The second technical solution provided by the present application is an extraction and separation method of two new sesquiterpene compounds, Oxyphyllerene F (compound III) and Oxyphyllerene G (compound IV), which comprises the following steps:

[0009] 1) After Fructus Alpiniae Oxophyllae is dried and crushed, it is extracted with 6-10 times the amount of 70% ethanol by refluxing for 3 times, each time for 2 hours. The extraction liquid is combined and concentrated under reduced pressure to obtain a decoction. The decoction is suspended in 2-4 times the amount of water, and then extracted with ethyl acetate and n-butanol in sequence until the extraction liquid is colorless (10 times or more). The ethyl acetate part, the n-butanol part and the water part are obtained by concentrating;

[0010] 2) The n-butanol part in step 1) is subjected to MCI Gel CHP-20 column chromatography, and gradient elution is carried out with an ethanol-water system (0:100-95:5). The water part, 10% ethanol part, 30% ethanol part, 50% ethanol part, 70% ethanol part and 95% ethanol part are obtained in sequence;

[0011] 3) The 30% ethanol part in step 2) is subjected to Sephadex LH-20 column chromatography, and gradient elution is carried out with a methanol-water system (0:100-95:5) as eluent. Fr.C.1-Fr.C.7 are obtained in sequence;

[0012] 4) The component Fr.C.1.5 in step 3) is subjected to silica gel column chromatography (200-300 mesh) with dichloromethane-methanol gradient elution (40:1-1:1). Six components Fr.C.1.5.1-Fr.C.1.5.6 are obtained in sequence;

[0013] 5) The components in Fr.C.1.5.5 in step 4) are purified by semi-preparative high performance liquid chromatography to obtain compound III*, which is a mixture of two optical isomers;

[0014] 6) Compound III* in step 5) is separated by semi-preparative high performance liquid chromatography (HPLC) on an AD-H column to obtain compound III and compound IV.

[0015] The third technical solution of the present application provides application of the sesquiterpenes Oxyphyllerene F (compound III) and Oxyphyllerene G (compound IV) separated from Alpiniae Oxyphyllae Fructus in preparation of an active drug for resisting renal fibrosis.

[0016] The present application has the following beneficial technical effects:

[0017] The present application relates to extraction and separation of two novel sesquiterpenes Oxyphyllerene F (compound III) and Oxyphyllerene G (compound IV) from Alpiniae Oxyphyllae Fructus medicinal materials, and it is found through experimental research that the two compounds can inhibit expression of Fibronectin, Collagen I and α-SMA in rat renal tubular epithelial cells (NRK 52E cells) induced by TGF-β1, have anti-renal fibrosis activity, can be effectively used for preparation of a kidney protection drug, and the preparation method has good reproducibility, the obtained compounds have high purity, are beneficial to further pharmacological and clinical research, expand the medicinal value of Alpiniae Oxyphyllae Fructus, and have huge economic and social benefits. IV. DESCRIPTION OF DRAWINGS

[0018] Figure 1 The HMBC (H→C, ) of the compounds III and IV in the present application, 1 H- 1 H COSY and NOE correlation spectrum.

[0019] Figure 2 The experimental and calculated ECD diagrams of the compound III in the present application.

[0020] Figure 3 The experimental and calculated ECD diagrams of the compound IV in the present application.

[0021] Figure 4 The CCK-8 method is used to detect the influence of different concentrations of the compounds on cell proliferation.

[0022] Figure 5The expression levels of Fibronectin, Collagen I and a-SMA at 20 μM concentration of compound III, IV were determined by Western blot for the present invention.

[0023] Figure 6 The effects of compound III of the present invention on Fibronectin, Collagen I and a-SMA in three kidney cells (NRK 52E, NRK 49F and HKC-8) induced by TGF-β1.

[0024] Figure 7 is the H-NMR spectrum of compound III of the present invention. 1 H-NMR spectrum.

[0025] Figure 8 is the C-NMR spectrum of compound III of the present invention. 13 C-NMR spectrum.

[0026] Figure 9 is the COSY spectrum of compound III of the present invention.

[0027] Figure 10 is the HSQC spectrum of compound III of the present invention.

[0028] Figure 11 is the HMBC spectrum of compound III of the present invention.

[0029] Figure 12 is the NOESY spectrum of compound III of the present invention.

[0030] Figure 13 is the HR-ESI-MS spectrum of compound III of the present invention.

[0031] Figure 14 is the UV spectrum of compound III of the present invention.

[0032] Figure 15 is the IR spectrum of compound III of the present invention.

[0033] Figure 16 is the CD spectrum of compound III of the present invention.

[0034] Figure 17 is the calculated NMR chart of compound III of the present invention.

[0035] Figure 18 is the H-NMR spectrum of compound IV of the present invention. 1 H-NMR spectrum.

[0036] Figure 19 is a COSY spectrum of the compound IV according to the present application. 13 C-NMR spectrum.

[0037] Figure 20 is a COSY spectrum of the compound IV according to the present application.

[0038] Figure 21 is a HSQC spectrum of the compound IV according to the present application.

[0039] Figure 22 is a HMBC spectrum of the compound IV according to the present application.

[0040] Figure 23 is a NOESY spectrum of the compound IV according to the present application.

[0041] Figure 24 is a CD spectrum of the compound IV according to the present application.

[0042] Figure 25 is a calculated NMR spectrum of the compound IV according to the present application. V. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0044] Embodiment 1

[0045] In the specific implementation of the present application, the extraction and separation method of two new sesquiterpenes, Oxyphyllerene F (compound III) and Oxyphyllerene G (compound IV), includes the following steps:

[0046] 1) After 40.00 kg of Fructus Schisandrae is dried and crushed, it is extracted with 8 times of 70% ethanol by reflux for 3 times, each time for 2 h. The extraction liquid is combined and concentrated under reduced pressure to obtain 8.30 kg of extract. The extract is suspended in 3 times of water, and then extracted with 30 L of ethyl acetate and n-butanol in sequence until the extraction liquid is colorless (more than 10 times) and concentrated to obtain the ethyl acetate fraction (1.45 kg), the n-butanol fraction (628.33 g) and the water fraction (6.60 kg);

[0047] 2) The MCI Gel CHP-20 column (Mitsubishi Chemical Corporation, Japan) in the n-butanol fraction in step 1) was eluted with a gradient of ethanol-water system (0:100-95:5) to obtain water fraction (371.10 g), 10% ethanol fraction (8.43 g), 30% ethanol fraction (33.36 g), 50% ethanol fraction (49.82 g), 70% ethanol fraction (20.73 g) and 95% ethanol fraction (105.06 g) in sequence;

[0048] 3) The 30% ethanol fraction (33.36 g) in step 2) was eluted with a gradient of methanol-water (0:100-95:5) as eluent using Sephadex LH-20 column to obtain Fr.C.1-Fr.C.7 in sequence;

[0049] 4) The component Fr.C.1.5 (2.88 g) in step 3) was eluted with a gradient of dichloromethane-methanol (40:1-1:1) using silica gel column chromatography (200-300 mesh) to obtain six components Fr.C.1.5.1-Fr.C.1.5.6 in sequence;

[0050] 5) The component Fr.C.1.5.5 in step 4) was purified by semi-preparative high performance liquid chromatography to obtain compound III*, which was a mixture of two optical isomers;

[0051] 6) The compound III* in step 5) was separated by semi-preparative high performance liquid chromatography (HPLC) on an AD-H column (n-hexane / isopropyl alcohol, 94:6) to obtain compound III (1.25 mg; t R = 19.7 min) and compound IV (0.95 mg; t R = 22.7 min).

[0052] The high resolution mass spectrometry was measured by TripleTOF 6600 high performance liquid chromatography-high resolution mass spectrometry combination instrument (AB SCIEX); the compound was purified by semi-preparative liquid chromatograph (Shimadzu Beijing Technology Co., Ltd.); the specific optical rotation was measured by Anton Paar MCP 5100 polarimeter (Anton Paar, Austria); the NMR spectrum was measured by Bruker AM-500MHz or Bruker AM-600MHz nuclear magnetic resonance spectrometer (TMS as internal standard, Germany Bruker company); the ultraviolet spectrum was measured by Thermo EVO300 ultraviolet spectrophotometer, and the infrared spectrum was measured by Thermo Nicolet IS10 infrared spectrometer (Thermo Scientific, USA).

[0053] I. Structure identification of the compound

[0054] 1. Structure identification of compounds III and IV

[0055]

[0056] Both compounds III and IV were colorless oils. The m / z in HR-ESI-MS was 437.2152 [M+Na]+ (calc. for C 21 H 34 O8Na 437.2146), which indicated that the molecular formula of compounds III and IV was C 21 H 34 O8. The1H NMR spectrum of compound III showed a characteristic olefinic hydrogen δ 1 H H 5.74 (1H, s, H-1) and three methyl signals δ H 1.14 (3H, s, H-14), 1.05 (3H, s, H-13), 1.01 (3H, d, J = 6.8 Hz, H-15). In addition, signals of the β-glucose moiety were observed at δ H 4.27 (1H, d, J = 7.8 Hz, H-1') and 3.25-3.87 (6H, H-2'-6'). The1H NMR spectrum of compound IV showed a characteristic olefinic hydrogen δ 13 C NMR (CD3OD, 150 MHz) spectrum in combination with HSQC spectrum, a total of 21 carbons were observed, including 15 signals of eriophonan sesquiterpenoids, one carbonyl signal δ C 202.5 (C-2), one carbon-carbon double bond signal [δ C 124.7 (C-1), 175.2 (C-10)], two quaternary carbon signals [δ C 40.7 (C-5), 75.2 (C-11)], two methine signals [δ C 42.0 (C-4), 40.0 (C-7)], five methylene signals [δ C 42.8 (C-3), 40.0 (C-6), 29.1 (C-8), 34.1 (C-9), 76.8 (C-12)] and three methyl signals [δ C 20.0 (C-13), 17.2 (C-14), 15.2 (C-15)], and six carbon signals of glucose [δ C105.0 (C-1'), 74.7 (C-2'), 78.0 (C-3'), 71.7 (C-4'), 78.1 (C-5') and 62.7 (C-6'). After acid hydrolysis and derivatization, the compound was analyzed by liquid chromatography-mass spectrometry (LC-MS) and identified as D-glucose. According to the HMBC correlation signals of H-1 with C-2 / C-3 / C-5 / C-10, H-6a with C-4 / C-5 / C-7 / C-8, long-range HMBC correlation signals of H-6a / H2-12 / H3-13 with C-11 and H-1' with C-12, in combination with a series of COSY correlation signals of H2-6 / H-7 / H2-8 / H2-9 and H2-3 / H-4 / H3-15, the planar structure of the compound was determined. Compound III, NOESY spectrum of H-7 (δ 1 H- 1 H COSY correlation signals of H-7 (δ H 2.20) / H3-15 (δ H 1.01) and H-7 (δ H 2.20) / H3-14 (δ H 1.14) indicated that H-7, H3-14 and H3-15 were in the same orientation. Compound IV and compound III had the same molecular formula, and their 1 H NMR, 13 C NMR and HSQC spectra were almost identical. In addition, in the NOESY spectrum of compound IV, H-7 (δ H 2.07) / H3-15 (δ H 1.00), H-7 (δ H 2.07) / H3-14 (δ H 1.13) also indicated that H-7, H3-14 and H3-15 had the same orientation. By comparing with the reported 11S- and 11R-nootkatone-11,12-diol eremophilane sesquiterpene structure, the absolute configuration of C-11 position in compounds III and IV was determined. Compared with compound IV, the signals of C-5, C-6 and C-7 positions of compound III appeared at lower field (0.4, 0.9, 0.8 ppm higher, respectively), while the signal of C-8 position appeared at higher field (1.0 ppm higher), which indicated that compounds III and IV were 11R- and 11S-configuration, respectively. In addition, the final DP4+ scores were calculated using the improved DP4+ probability method: 99.98% for 11R-III and 100.00% for 11S-IV. Therefore, the TDDFT-ECD method ( Figure 4) clearly demonstrated that the absolute configuration of III is 4R, 5S, 7R, 11R, and that of IV is 4R, 5S, 7R, 11S, and the compounds III and IV were named as oxyphyllerene F and oxyphyllerene G, respectively.

[0057] Table 1. The chemical structures of compounds III and IV 1 H-NMR (600MHz) and 13 C-NMR data (150MH) in CD3OD

[0058]

[0059] Secondly, the kidney protective activity of the two new sesquiterpenes compounds of the present application was studied.

[0060] 2.1 Cell lines

[0061] NRK 52E (rat kidney tubular duct epithelial cells), NRK 49F (rat normal kidney fibroblasts) and HKC-8 (human embryonic kidney tubular epithelial cells): rat normal kidney tubular epithelial cells.

[0062] 2.2 Experimental instruments, consumables and reagents

[0063] Table 2. List of instrument names and manufacturers

[0064]

[0065]

[0066] Table 3. List of reagent names and manufacturers

[0067]

[0068] 2.3 Experimental methods

[0069] (1) Cell culture and treatment: NRK 52E, NRK 49F and HKC-8 cells were cultured in DMEM medium containing 10% fetal bovine serum. The cells were seeded into 12-well plates (2x10 4 cells / mL) until they reached 70%-80%, TGF-β1 (10 ng / mL) was added to induce fibrosis of the cells, and compounds III and IV were added at the same time. The cells were collected for Western Blot after 48 h. The compounds used in the cell experiment were dissolved in DMSO.

[0070] (2) Cell viability test: NRK 52E cells (2x10 4cells / mL) seeded in 96-well cell culture plates. After overnight incubation, cells were treated with 20 mM of different compounds or DMSO for 48 h. The supernatant was removed and 100 mL of medium (V 空培养基 / V Cell C ount Kit-8 =10 / 1) to each well. After 1 h, the absorbance of each well was recorded at 450 nm using a microplate reader.

[0071] (3) Western blot: After treatment of cells with compounds and TGF-β1, total protein of cell lines was extracted using 100 mL RIPA (RIPA) lysis buffer (1 mM PMSF + protease inhibitors + nucleases), and the protein sample was quantified using BCA assay. Equal amounts of protein extracts were separated by 8% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated with the indicated antibodies overnight at 4°C, and then incubated with secondary antibodies at room temperature for 2 h. The bands were visualized and measured by ECL kit and developer. The results of immunoblotting were analyzed by gray scale using ImageJ software.

[0072] (4) Data analysis: All experimental data obtained in this study were independently repeated three times. The data were provided in the form of mean standard error (SEM). Statistical analysis was performed using Graphpad Prism 8 (Graphpad Software, San Diego, CA, USA) and Excel (Microsoft). Differences were considered significant when * P≤0.05, ** P≤0.01, *** P≤0.001,and **** P≤0.0001.

[0073] 2.4 Experimental results

[0074] First, the anti-renal fibrosis activity of the two compounds at a concentration of 20 mM was evaluated by a single indicator of fibronectin. Before activity evaluation, the effect of Compound III and IV on the proliferation of NRK 52E cells at this concentration was detected by CCK-8 method (A) to investigate their cytotoxicity. As shown in Figure 2, the proliferation of NRK 52E cells was inhibited by Compound III and IV at a concentration of 20 mM, and the inhibition rate was 18.6% and 22.3%, respectively. Therefore, the anti-renal fibrosis activity of the two compounds at this concentration was evaluated by a single indicator of fibronectin. Figure 4As shown in Figure A, there was no significant decrease in cell viability after the addition of compounds III and IV at a concentration of 20 μM. Next, the effect of compounds III and IV on the expression of fibronectin in TGF-β1 -stimulated NRK 52E cells was determined by Western blotting at a concentration of 20 μM. Cells were treated with 10 ng / mL of TGF-β1 for 48 h after the addition of 20 μM of compounds III and IV. β-tubulin was used as an internal control, GW (GW788388) was used as a positive control, and nintedanib (NKT, 20 μM) was used as another control sample (B). The results of Western blotting showed that Figure 4 B), at a concentration of 20 μM, compounds III and IV significantly reduced the expression of fibronectin. Therefore, the effect of compounds III and IV on the expression levels of fibronectin, collagen I, and a-SMA at a concentration of 20 μM was determined by Western blotting to accurately evaluate the renal protective biological activity of compounds III and IV at this concentration. The results showed that Figure 5 ), compounds III and IV significantly inhibited the expression of fibronectin and collagen I, indicating that compounds III and IV can reduce TGF-β1 -induced renal fibrosis in NRK 52E cells and have renal protective activity. Moreover, the renal protective activity of compound III was more significant than that of compound IV.

[0075] To further verify the renal protective activity and effective dose of compound III, three renal cell lines were used to investigate the dose-effect relationship of compound III. First, the CCK-8 method was used to rule out possible cytotoxicity at 40 μM (Figure C). Figure 4 The results of the dose-effect relationship (Figure D) showed that compound III exhibited a dose-effect relationship on the expression levels of fibronectin and collagen I in the above three renal cell lines (NRK 52E, NRK 49F, and HKC-8) at test concentrations of 10 μM, 20 μM, and 40 μM, respectively. Moreover, compound III significantly inhibited the expression of these two proteins in NRK 52E and NRK 49F cell lines even at a concentration of 10 μM. However, the effect of compound III on the expression level of a-SMA did not exhibit a clear dose-effect curve in the three renal cells. Figure 6

[0076] 2.5 Conclusion

[0077] ​The anti-renal fibrosis activity research on three kinds of renal cells shows that the compounds in the Fructus Alpiniae Oxphyllae have inhibitory activity on fibronectin, collagen I and alpha-smooth muscle actin (α-SMA) in the cell lines induced by TGF-β1.

[0078] The two structurally novel sesquiterpenes Oxyphyllerene F (compound III) and Oxyphyllerene G (compound IV) extracted and separated from the Fructus Alpiniae Oxphyllae are identified, and it is found through test research that the two compounds both have the anti-renal fibrosis effect, can be used for preparing renal protection drugs, develop the medicinal value of the Fructus Alpiniae Oxphyllae, have great development prospects, and have good economic and social benefits.

Claims

1. Oxyphyllerene F and Oxyphyllerene G, sesquiterpenoid compounds isolated from Alpinia oxyphylla, characterized in that, The structural formulas are as follows:

2. The method for extracting and separating sesquiterpenoid compounds Oxyphyllerene F and Oxyphyllerene G from Alpinia oxyphylla according to claim 1, characterized in that, Includes the following steps: 1) After drying and pulverizing Alpinia oxyphylla, extract it three times by reflux with 6-10 times the amount of 70% ethanol for 2 hours each time. Combine the extracts and concentrate them under reduced pressure to obtain an extract. Suspend the extract with 2-4 times the amount of water and extract it sequentially with ethyl acetate and n-butanol until the extract is colorless. Then concentrate the extract to obtain the ethyl acetate fraction, n-butanol fraction and water fraction. 2) The n-butanol fraction from step 1) was loaded onto an MCI Gel CHP-20 column and eluted with an ethanol-water system in a gradient of 0:100 to 95:5 to obtain the water fraction, 10% ethanol fraction, 30% ethanol fraction, 50% ethanol fraction, 70% ethanol fraction and 95% ethanol fraction in sequence. 3) The 30% ethanol fraction from step 2) was eluted using a Sephadex LH-20 column with methanol-water 0:100 to 95:5 as eluent to obtain Fr.C.1 to Fr.C.7 sequentially. 4) The fraction Fr.C.1.5 from step 3) was eluted by silica gel column chromatography with a gradient of dichloromethane-methanol at a ratio of 40:1 to 1:1 to obtain six fractions Fr.C.1.5.1 to Fr.C.1.5.

6. 5) The Fr.C.1.5.5 component from step 4) was purified by semi-preparative high-performance liquid chromatography to obtain compound III*, which is a mixture of two optical isomers; 6) Compound Ⅲ* from step 5) was separated by semi-preparative high performance liquid chromatography on an AD-H column to obtain compound Ⅲ and compound Ⅳ.

3. The method for extracting and separating sesquiterpenoid compounds Oxyphyllerene F and Oxyphyllerene G from Alpinia oxyphylla according to claim 2, characterized in that, Includes the following steps: 1) 40.00 kg of Alpinia oxyphylla was dried and pulverized. It was then extracted three times with 8 times the amount of 70% ethanol, each time for 2 hours. The extracts were combined and concentrated under reduced pressure to obtain an extract. The extract was suspended with 3 times the amount of water and then extracted with 30 L of ethyl acetate and n-butanol in sequence until the extract was colorless. The extract was then concentrated to obtain the ethyl acetate fraction, n-butanol fraction and water fraction. 2) The n-butanol fraction from step 1) was loaded onto an MCI Gel CHP-20 column and eluted with an ethanol-water system in a gradient of 0:100 to 95:5 to obtain the water fraction, 10% ethanol fraction, 30% ethanol fraction, 50% ethanol fraction, 70% ethanol fraction and 95% ethanol fraction in sequence. 3) The 30% ethanol fraction from step 2) was eluted using a Sephadex LH-20 column with methanol-water 0:100 to 95:5 as eluent to obtain Fr.C.1 to Fr.C.7 sequentially. 4) The fraction Fr.C.1.5 from step 3) was eluted by silica gel column chromatography with a gradient of dichloromethane-methanol at a ratio of 40:1 to 1:1 to obtain six fractions Fr.C.1.5.1 to Fr.C.1.5.

6. 5) The Fr.C.1.5.5 component from step 4) was purified by semi-preparative high-performance liquid chromatography to obtain compound III*, which is a mixture of two optical isomers; 6) Compound Ⅲ* from step 5) was separated by semi-preparative high performance liquid chromatography on an AD-H column to obtain compound Ⅲ and compound Ⅳ.

4. The use of the sesquiterpenoid compounds Oxyphyllerene F and Oxyphyllerene G isolated from Alpinia oxyphylla according to claim 1 in the preparation of an active drug for preventing renal fibrosis.

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