A tomentosane type diterpene compound in chinese toon seed and application thereof

Abstract

The application discloses a toonasinensin type diterpene compound in Chinese toona seeds and application thereof, and belongs to the technical field of biological medicines. 22 H 38 O4, the toonasinensin type diterpene compound is extracted from Chinese toona seeds by means of backflow extraction, solvent extraction, normal phase and reverse phase silica gel column chromatography separation and the like, and has a molecular formula of C The diterpene new compound toonasinensin F can inhibit the proliferation of glomerular mesangial cells in a high-sugar environment, can improve the SOD activity, and can reduce the MDA, TNF-alpha and IL-6 levels, so as to play an antioxidation and anti-inflammatory role, and can be applied to the prevention and treatment or auxiliary medicine of diabetic nephropathy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a retinane-type diterpenoid compound in Toona sinensis seeds and its application. Background Technology

[0002] Chinese toon Toona sinensis (A. Juss.) Roem is a tree belonging to the genus *Toona* in the family Meliaceae. It is widely distributed in my country and has high edible, medicinal, and economic value. The leaves, seeds, bark, buds, and roots of *Toona sinensis* can all be used medicinally, making it a well-known plant with both medicinal and edible properties. Plants in the *Toona* genus possess antioxidant, hypoglycemic, antibacterial, anti-inflammatory, analgesic, and anticancer bioactivities, showing great potential for development and application. *Toona sinensis* seeds are the seeds of the fruit and have the effects of dispelling wind, cold, and relieving pain.

[0003] The term "terpene" originally referred to unsaturated hydrocarbons found in turpentine and many volatile oils. Currently, over 20,000 terpenes have been discovered, making them the most abundant type of natural compound. Terpenes are a general term for polymers and derivatives of isoprene or isopentane structural units. Based on the "isoprene rule," terpenes are classified according to the number of isoprene units in their structure. For example, compounds containing one isoprene unit are called hemiterpenes, those containing two are called monoterpenes, and therefore diterpenes are compounds containing four isoprene units. Diterpenes play a crucial regulatory role in the life activities of plants and animals, and some can be directly used to treat diseases, such as anti-tumor agents. There are few reports on the extraction of terpenes from Toona sinensis (Chinese mahogany). Patent application CN105481876A discloses a diterpenes compound for treating ovarian cancer, using diterpenes extracted from Toona sinensis leaves for this purpose.

[0004] Diabetic nephropathy (DN) is a common microvascular disease in diabetic patients and one of the leading causes of death in clinical diabetic patients. The proliferation of glomerular mesangial cells (GMCs) plays a crucial role in the occurrence and development of glomerular sclerosis. High glucose is a risk factor for abnormal expression of reactive oxygen species (ROS) and oxidative stress damage. A high-glucose environment can trigger GMC activation and abnormal proliferation, induce excessive ROS production and antioxidant enzyme inactivation within GMCs, reduce the antioxidant capacity of GMCs, and cause thickening of the GMC basement membrane, all of which contribute to the occurrence and development of DN. Currently, there are few reports on the use of terpenoid compounds extracted from Toona sinensis to treat diabetic nephropathy. Patent application number CN111471079A discloses a novel triterpenoid compound of the Euphorbia kansui type from Toona sinensis pericarp, along with its extraction and separation method and application, for the treatment of diabetic nephropathy. Therefore, further research on Toona sinensis is needed to extract more terpenoid compounds for better treatment of diabetic nephropathy. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a retinane-type diterpenoid compound from Toona sinensis seeds and its application. This invention extracts retinane-type diterpenoid compounds from Toona sinensis fruit seeds using reflux extraction, solvent extraction, and normal-phase and reversed-phase silica gel column chromatography, which can be used as an adjunct treatment for diabetic nephropathy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a retinane-type diterpenoid compound from Toona sinensis seeds, wherein the molecular formula of the retinane-type diterpenoid compound from Toona sinensis seeds is C1. 22 H 38 O4, the structural formula is:

[0008] .

[0009] Preferably, the retinane-type diterpenoid compound in the Chinese toon seed is prepared by the following method:

[0010] (1) The dried Chinese toon seeds were crushed and sieved, ethanol was added, and the mixture was heated and refluxed for extraction. The ethanol extract was then concentrated under reduced pressure.

[0011] (2) Add water to the ethanol extract to obtain a suspension, and extract it with petroleum ether, ethyl acetate and n-butanol respectively. Collect the extract and concentrate it under reduced pressure to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively.

[0012] (3) Take the ethyl acetate extract and place it on a normal phase silica gel column. First, elute with petroleum ether-ethyl acetate, then elute with dichloromethane-methanol. Combine the results with thin-layer chromatography and combine them into 9 components, which are denoted as Fr. A to Fr. I.

[0013] (4) Take the third component Fr.C onto a reversed-phase silica gel chromatography column, elute with methanol-water, and obtain 13 components based on the thin-layer chromatography results, denoted as Fr.C1~Fr.C13;

[0014] (5) Take the 6th component Fr. C6 and place it on a normal phase silica gel chromatography column. Elute with dichloromethane-methanol. According to the results of thin-layer chromatography, 10 components are obtained and recorded as Fr.C6.1~Fr.C6.10;

[0015] (6) The fifth component Fr.C6.5 was subjected to ODS column chromatography, separated by methanol-water, and then eluted with petroleum ether-ethyl acetate to obtain the retinane-type diterpenoid compound in Toona sinensis.

[0016] Preferably, in step (1), the volume concentrations of the ethanol are 55%, 75% and 95%, respectively; the heating reflux temperature is 80°C and the time is 2.5 h, 2 h and 1.5 h, respectively.

[0017] Preferably, in step (2), the extraction is performed 3 times, and the extraction time is 12 h, 6 h, and 6 h.

[0018] Preferably, in step (3), the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixture is 30 to 1:1; and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 20 to 1:1.

[0019] Preferably, in step (4), the volume ratio of methanol to water in the methanol-water mixture is 65~100:0~35.

[0020] Preferably, in step (5), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 50~100:0~1.

[0021] Preferably, in step (6), the volume ratio of methanol to water in the methanol-water mixture is 55~80:20~45; and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate mixture is 3~5:1.

[0022] A second aspect of the present invention provides the use of retinane-type diterpenoid compounds from Toona sinensis in the preparation of a medicament for treating diabetic nephropathy.

[0023] The beneficial effects of this invention are:

[0024] (1) The present invention extracts retinane-type diterpenoid compounds from the seeds of Toona sinensis fruit—Toona sinensis seeds—using reflux extraction, solvent extraction, normal phase and reverse phase silica gel column chromatography, etc., which can be used as an adjunct treatment for diabetic nephropathy.

[0025] (2) The retinane-type diterpenoid compounds extracted in this invention can inhibit the proliferation of glomerular mesangial cells under high glucose conditions and effectively reduce the levels of high glucose-induced oxidative stress and inflammation. Through research on the protective effects against oxidative stress damage and inflammatory response of glomerular mesangial cells, the new diterpenoid compound toonasinensin F can inhibit the proliferation of glomerular mesangial cells under high glucose conditions, increase SOD activity, and reduce the levels of MDA, TNF-α and IL-6, thus exerting antioxidant and anti-inflammatory effects. It can be used as a preventive or adjuvant drug for diabetic nephropathy. Attached Figure Description

[0026] Figure 1 HR-ESI-MS spectrum of the product in Experimental Example 1;

[0027] Figure 2 The product in Experiment Example 1 1 H-NMR spectrum;

[0028] Figure 3 The product in Experiment Example 1 13 C-NMR spectrum;

[0029] Figure 4 : The DEPT135° NMR spectrum of the product in Experimental Example 1;

[0030] Figure 5 Nuclear magnetic resonance of the product in Experiment Example 1 1 H- 1 H COSY spectrum;

[0031] Figure 6 : The HSQC nuclear magnetic resonance spectrum of the product in Experimental Example 1;

[0032] Figure 7 : The nuclear magnetic resonance HMBC spectrum of the product in Experimental Example 1;

[0033] Figure 8 : The NMR NOESY spectrum of the product in Experiment Example 1;

[0034] Figure 9 Histogram of MTT colorimetric assay results; compared with the normal group, ## P <0.01; compared with the high glucose group, * P <0.05,** P <0.01;

[0035] Figure 10 (a) Histogram of SOD measurement results, (b) Histogram of MDA measurement results; compared with the normal group, ## P <0.01; compared with the high glucose group, * P <0.05,** P <0.01;

[0036] Figure 11 (a) Histogram of TNF-α measurement results, (b) Histogram of IL-6 measurement results; compared with the normal group, ## P <0.01; compared with the high glucose group, * P <0.05,** P <0.01. Detailed Implementation

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0038] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0039] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0040] Example 1: Preparation of the novel diterpenoid compound toonasinensin F

[0041] (1) Weigh 55 kg of dried Chinese toon seeds, crush and sieve them, add a certain volume of 55%, 75% and 95% ethanol respectively, heat and reflux for 2.5 h, 2 h and 1.5 h, repeat 3 times, combine the extracts obtained from the 3 times, concentrate under reduced pressure to obtain ethanol extract.

[0042] (2) The ethanol extract was suspended in water to obtain a suspension. The suspension was extracted three times with petroleum ether, ethyl acetate and n-butanol respectively, with extraction times of 12 h, 6 h and 6 h respectively. After each extraction, the extracts were collected and combined, and concentrated under reduced pressure to obtain extracts from the petroleum ether, ethyl acetate and n-butanol fractions respectively.

[0043] (3) Take 528.8 g of the extract obtained from the ethyl acetate fraction and load it onto a normal phase silica gel column. First, use the petroleum ether-ethyl acetate system as the mobile phase and elute with solvents at volume ratios of 30:1, 20:1, 10:1, 7:1, 5:1, and 1:1. Then, use the dichloromethane-methanol system as the mobile phase and elute with solvents at volume ratios of 20:1, 15:1, 10:1, 5:1, 3:1, and 1:1. Combine the results with thin layer chromatography (TLC) and combine the components with similar ratio shift values ​​into 9 components, denoted as Fr. A to Fr. I.

[0044] (4) Take the third component Fr. C, load it onto a reversed-phase silica gel chromatography column, use methanol-water system as mobile phase, and perform gradient elution in the order of volume ratios of 65:35, 75:25, 85:15 and 100:0. According to the TLC results, the components with similar specific shift values ​​are combined to obtain 13 components, which are denoted as Fr.C1 to Fr.C13.

[0045] (5) Take the 6th component Fr. C6 (3.2 g), load it onto a normal phase silica gel chromatography column, use dichloromethane-methanol system as mobile phase, and elute with solvent in volume ratios of 100:0, 100:1, 80:1, 70:1 and 50:1 in sequence. According to the TLC results, the components with similar specific flotation values ​​are combined to obtain 10 components, which are denoted as Fr.C6.1 to Fr.C6.10.

[0046] (6) The fifth component Fr. C6.5 was subjected to ODS column chromatography and separated by methanol-water in volume ratios of 55:45, 60:40, 65:35, 70:30, 75:25 and 80:20. Then, it was eluted by petroleum ether-ethyl acetate in volume ratios of 5:1, 4:1 and 3:1 to obtain the new diterpenoid compound toonasinensin F.

[0047] Example 2: Characterization of toonasinensin F

[0048] Using nuclear magnetic resonance spectroscopy (NMR) 1 H-NMR, 13 C-NMR, DEPT-135° 1 H- 1 The structure of this compound was identified by HCl (COSY, HSQC, HMBC, NOESY) and high-resolution mass spectrometry (HR-ESI-MS), and the results are as follows: Figure 1~8 And as shown in Table 1.

[0049] The compound toonasinensin F is a colorless, oily liquid, according to Figure 1 HR-ESI-MS m / z389.26550 [M+Na] + (Calculated value 389.26623), the molecular formula is determined to be C. 22 H 38 O4.

[0050] exist Figure 2 of 1 In the 1H NMR (400 MHz, CDCl3) spectrum, two olefin hydrogen proton signals are observed in the low field region. d H 5.34 (1H, t, J = 6.6 Hz, H-2), 5.12 (1H, t, J = 6.4 Hz, H-6), suggesting the structure may contain two double bonds and one oxidized methylene hydrogen proton signal. d H 4.58 (2H, d, J = 7.1 Hz, H-1), a signal of one oxidized methylene hydrogen proton. d H 3.32 (1H, dd, J = 11.2, 4.0 Hz, H-5′), the high field region exhibits 6 methylene hydrogen proton signals. d H 2.12 (2H, d, J = 7.7 Hz, H-5), 2.09 (2H, d, J = 10.2 Hz, H-8), 2.07 (2H,d, J = 8.5 Hz, H-4), 1.76 (3H, ddd, J = 13.0, 6.7, 3.1 Hz, H-3′a, H-4′), 1.54 (1H, m, H-9a), 1.47 (1H, m, H-3′b), 1.44 (1H, m, H-9b), 6 methyl hydrogen proton signals d H 2.05 (3H, s, 1-OCO CH 3 ), 1.70 (3H, s, H-10), 1.62 (3H, s, H-11), 1.16 (3H, s, H-9′), 1.03 (3H, s, H-8′), 0.79 (3H, s, H-7′), 1 methylene hydrogen proton signal d H 1.10 (1H,t, J= 4.2 Hz, H-1′).

[0051] Figure 3 of 13 The C-NMR (100 MHz, CDCl3) spectrum showed a total of 22 carbon signal peaks, including one carbonyl carbon signal. d C 171.3 (1-O CO CH3), 2 groups of olefin carbon signals d C 142.3 (C-3), 136.5 (C-7), 124.1 (C-6), 118.5 (C-2), three signals of oxidized carbon. d C 78.4 (C-5′), 73.6 (C-2′), 61.6 (C-1), 6 methyl carbon signals d C 28.2 (C-8′), 23.2 (C-9′), 21.2 (1-OCO CH 3 ), 16.6 (C-10), 16.3 (C-11), 15.0 (C-7′), 1 methylene carbon signal, 1 quaternary carbon signal, 6 methylene carbon signals. Figure 4 The DEPT135° readings showed a total of 6 methyl carbon signals, 7 methylene carbon signals, and 4 methine carbon signals. Figure 5 of 1 H- 1 H COSY showed that H2-3′ / H2-4′ and H2-4′ / H-5′ were present. 1 H- 1 H COSY association. Figure 6 HSQC display d C 42.9 (C-8), d C 24.5 (C-9), d C 55.9 (C-1′) and respectively d H 2.09 (H-8), d H 1.54, 1.44 (H-9), d H 1.10 (H-1′) related. Figure 7 The HMBC shows that H-1′ ( d H 1.10) and C-2′ ( d C73.6), C-6′ ( d C 40.6) related; H-4′ ( d H 1.76) and C-5′ ( d C 78.4) related; H-7′ ( d H 0.79), H-8′ ( d H 1.03) are both similar to C-6′ ( d C 40.6) related; H-9′ ( d H 1.16) and C-2′( d C 73.6), C-1′( d C 55.9), C-3′ ( d C 41.1) Related. Figure 8 NOESY data showed spatial NOESY correlations between H-1′ and H-5′, H-5′ and H3-8′, and H3-7′ and H3-9′.

[0052] HMBC shows that 1-OCO CH 3 ( d H 2.05), H-1 ( d H 4.58) and 1-O CO CH3( d C 171.3) related; d H 4.58(H-1) and d C 118.5 (C-2) and 142.3 (C-3) are relevant; d H 5.34 (H-2) and d C 16.6 (C-11), 39.6 (C-4) are relevant; H-6 ( d H 5.12) and C-10 ( d C 16.3), C-5 ( d C 26.3), C-8 ( d C 42.9) related; H-10 ( dH 1.62) and C-8 ( d C 42.9) related; H-11 ( d H 1.70) and C-4 ( d C 39.6) related. HMBC shows that H-8 ( d H 2.09) and C-9 ( d C 24.5), C-1′ ( d C 55.9) related, H-1′ ( d H 1.10) and C-9 ( d C 24.5), C-2′ ( d C 73.6), C-6′ ( d C (40.6) Related, proving that the chain structure of the compound is attached at position 1′. In summary, the compound prepared in Example 1 was identified as toonasinensin F, and the NMR data are shown in Appendix Table 1.

[0053] Table 1. NMR data of the product (400 / 100 MHz, CD3Cl3)

[0054]

[0055] Test case

[0056] (1) MTT assay for cell viability

[0057] Rat glomerular mesangial cells (GMCs) (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were seeded in 96-well culture plates at a cell density of 5 × 10⁶ cells / well. 3Cells were cultured overnight at 37°C with 100 μL of medium per well in 5% CO2. The following groups were established: a normal group (5.6 μM DMEM, NG group), a high glucose group (25 μM DMEM, HG group), a positive control group (epalrestat EPA, manufacturer: Beijing Solarbio Science & Technology Co., Ltd.) (10 μM, EPA group), and an intervention group (high glucose + toonasinensin F, high glucose concentration same as HG group, HG + 5 μM, HG + 10 μM, HG + 20 μM). Different concentrations of toonasinensin F (5, 10, 20 μM) were added to the intervention groups, with three replicates per group to investigate the effect of the drug on cells. After culturing the cells in each group for 48 h, 10 μL of 5 mg / mL MTT was added to each well, and the cells were incubated at 37 ℃ under 5% CO2 conditions. The culture was terminated after 4 h, the liquid in the wells was aspirated, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The cells were shaken for 15 min to fully dissolve the intracellular crystals. The optical density (OD) of each well was measured at 490 nm using a microplate reader. The results are shown below. Figure 9 .

[0058] A high-sugar environment can trigger the activation and abnormal proliferation of glucose-containing macrophages (GMCs), induce excessive production of reactive oxygen species and inactivation of antioxidant enzymes within GMCs, reduce the antioxidant capacity of GMCs, and cause thickening of the GMC basement membrane, thus participating in the occurrence and development of diabetic nephropathy. Figure 9 As shown, compared with the NG group, high glucose can promote the proliferation of glomerular mesangial cells in the HG group; the intervention group has an inhibitory effect on the proliferation of glomerular mesangial cells, and the inhibitory effect increases with the increase of the concentration of the intervention drug.

[0059] (2) Determination of SOD activity and MDA content

[0060] GMCs are divided into 10×10 4Cells were seeded at a density of 10 cells / mL in 6-well plates, with 2 mL of cell suspension added to each well. Cells were incubated at 37 °C and 5% CO2 for 24 h. The following groups were established: NG group, HG group, positive control group (epalrestat EPA, manufacturer: Beijing Solarbio Science & Technology Co., Ltd.) (10 μM, EPA group), and HG+toonasinensin F group (HG+5 μM, HG+10 μM, HG+20 μM). After culturing for 48 h, wash the 6-well plates with an appropriate amount of PBS. Add 400 μL of trypsin-EDTA digestion solution (without phenol red) to each well and digest until the cells become rounded. For the NG group, add 1 mL of DMEM low-glucose complete medium (purchased from Wuhan Pronosai Biotechnology Co., Ltd.) to terminate digestion. For the HG group and the HG+toonasinensin F group, add 1 mL of DMEM high-glucose complete medium (purchased from Wuhan Pronosai Biotechnology Co., Ltd.) to terminate digestion. Transfer the cells to 1.5 mL centrifuge tubes, centrifuge, discard the supernatant, add 1 mL of PBS, centrifuge again, discard the supernatant, add an appropriate amount of PBS, and mix well by pipetting. Repeat the freeze-thaw cycle 3 times, centrifuge at 4 ℃ and 5000 rpm for 5 min, collect the supernatant, and measure SOD activity and MDA content according to the kit instructions. The results are shown in the figure. Figure 10 .

[0061] like Figure 10 As shown in (a), after intervention with toonasinensin F, SOD levels increased compared to HG, and the effect was more significant with increasing drug concentration. Figure 10 As shown in (b), MDA levels decreased, with the most significant reduction observed at a drug concentration of 20 μM. This indicates that intervention with toonasinensin F effectively alleviated cellular oxidative stress damage, achieving effects comparable to, or even slightly better than, the positive control.

[0062] (3) Measure the levels of TNF-α and IL-6.

[0063] GMCs are divided into 10×10 4 Cells were seeded at a density of 10 cells / mL in 6-well plates, with 2 mL of cell suspension added to each well. Cells were incubated at 37 °C and 5% CO2 for 24 h. The following groups were established: NG group, HG group, positive control group (epalrestat EPA, manufacturer: Beijing Solarbio Science & Technology Co., Ltd.) (10 μM, EPA group), and HG+toonasinensin F group (HG+5 μM, HG+10 μM, HG+20 μM). After 48 h of incubation, the supernatant was collected, centrifuged at 4 °C and 1000 rpm for 3 min, and transferred to new EP tubes and labeled. Following the ELISA kit operation guide provided by Nanjing Jiancheng Biotechnology Research Institute, the levels of TNF-α and IL-6 were detected. The results are shown below. Figure 11 .

[0064] like Figure 11 As shown, compared with HG, drug intervention reduced the levels of TNF-α and IL-6 induced by HG, and the inhibitory effect became more significant with increasing drug concentration. The drug showed strong inhibitory activity at 20 μM, significantly reducing the levels of inflammatory factors TNF-α and IL-6. P <0.01), its effect is comparable to that of the positive control.

[0065] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A xanthorrhyl-type diterpene compound in Chinese toona seed, characterized in that, The molecular formula of the xanthanol type diterpene compound in the Chinese toon seed is C 22 H 38 O4, and the structural formula is 。 2. The method for preparing the meso-rondanin type diterpene compound in Japanese pagodatree fruit according to claim 1, characterized in that, It comprises the following steps: (1) crushing and sieving dry Chinese toona seed, adding ethanol, extracting by heating reflux, and obtaining ethanol extract after concentration under reduced pressure; (2) obtaining suspension by adding water to the ethanol extract, extracting with petroleum ether, ethyl acetate and n-butanol respectively, collecting the extract and concentrating under reduced pressure to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively; (3) taking the ethyl acetate extract on a normal phase silica gel chromatography column, eluting with petroleum ether-ethyl acetate first and then with dichloromethane-methanol, combining 9 fractions according to the detection results of thin layer chromatography, and marking them as Fr. A~Fr. I; (4) taking the 3rd fraction Fr. C on a reversed phase silica gel chromatography column, eluting with methanol-water, and obtaining 13 fractions according to the detection results of thin layer chromatography, and marking them as Fr. C1~Fr. C13; (5) taking the 6th fraction Fr. C6 on a normal phase silica gel chromatography column, eluting with dichloromethane-methanol, and obtaining 10 fractions according to the detection results of thin layer chromatography, and marking them as Fr. C6.1~Fr. C6.10; (6) taking the 5th fraction Fr. C6.5 on an ODS column, eluting with methanol-water first and then with petroleum ether-ethyl acetate, and obtaining the xanthanol-type diterpene compound in Chinese toona seed.

3. The method of claim 2, wherein, In step (1), the volume concentrations of the ethanol are 55%, 75% and 95% respectively; and the heating reflux temperature is 80℃, and the time is 2.5 h, 2 h and 1.5 h in turn.

4. The preparation method according to claim 2, characterized in that, In step (2), the extraction is performed for 3 times, and the time is 12 h, 6 h and 6 h in turn.

5. The preparation method according to claim 2, characterized in that, In step (3), the volume ratio of petroleum ether to ethyl acetate in petroleum ether-ethyl acetate is 30~1:1; and the volume ratio of dichloromethane to methanol in dichloromethane-methanol is 20~1:

1.

6. The preparation method according to claim 2, characterized in that, In step (4), the volume ratio of methanol to water in methanol-water is 65~100:0~35.

7. The preparation method according to claim 2, characterized in that, In step (5), the volume ratio of dichloromethane to methanol in dichloromethane-methanol is 50~100:0~1.

8. The preparation method according to claim 2, characterized in that, In step (6), the volume ratio of methanol to water in methanol-water is 55~80:20~45; and the volume ratio of petroleum ether to ethyl acetate in petroleum ether-ethyl acetate is 3~5:

1.

9. The xanthanol-type diterpene compound in Chinese toona seed of claim 1 in the preparation of a medicament for treating diabetic nephropathy.

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