A dihydrophenanthrene compound isolated from Panlong ginseng and its application

Abstract

The present invention belongs to the field of natural medicines, and specifically relates to a dihydrophenanthrene compound isolated from Panlong ginseng and its application. The present invention uses a variety of separation methods to separate a new compound from the petroleum ether extraction part of the 95% ethanol extract of the dried root of Panlong ginseng. The compound is identified as a dihydrophenanthrene compound. Experimental verification shows that the compound can enhance the vitality of MIN6 cells after PA-induced damage, antagonize the damaging effect of PA on MIN6 cells, and has a protective effect on MIN6 cells. At the same time, the compound can significantly reduce the intracellular TG content, improve lipid accumulation, and has a potential therapeutic effect on fatty liver.

Description

Technical Field

[0001] The invention belongs to the field of natural medicines, and specifically relates to a dihydrophenanthrene compound separated from Panlong ginseng and application thereof. Background Art

[0002] Panlong ginseng is a plant of the genus Spiranthes in the family Orchidaceae. It is also known as pig Liao ginseng, dragon holding pillar, dragon wrapping pillar, pig tooth ginseng, etc. Panlong ginseng is used as medicine with its root or whole plant, which has the effects of nourishing yin and qi, cooling blood and detoxifying, moistening lungs and relieving cough. Panlong ginseng is widely distributed throughout the country, mainly produced in Hainan, Guizhou, Yunnan, Sichuan and other places. It is commonly found under forests, under shrubs, grasslands or riverbed swamp meadows at an altitude of 200 to 3400 meters. Panlong ginseng was first recorded in "The Book of Songs·Chen Feng". As a medicinal material, it was first recorded in "Southern Yunnan Materia Medica" and it was described as: sweet in taste and warm in nature. It enters the lung, liver and kidney meridians. "Tianbao Materia Medica" records that Panlong ginseng "replenishes essence and strengthens yang, treats dizziness, back pain and weakness". "Hunan Pharmacopoeia" records that Panlong ginseng "stops deficiency heat and thirst, and coughs and blood due to lung tumors". It is also used in Tibetan medicine and Mongolian medicine in my country. Panlong ginseng has a rich variety of chemical components, including dihydrophenanthrene, flavonoids, phenylpropanoids, etc. Among them, dihydrophenanthrene is its characteristic component. At present, more than 20 dihydrophenanthrene compounds have been isolated. Therefore, further research on the chemical components of Panlong ginseng has positive significance for the in-depth development of the effects of Panlong ginseng. Summary of the invention

[0003] The present invention provides a dihydrophenanthrene compound separated from Panlong ginseng and its application. The compound can significantly enhance the activity of MIN6 cells, produce a protective effect on MIN6 cells and has potential therapeutic activity for fatty liver.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A dihydrophenanthrene compound isolated from Panlong ginseng has a structure as shown in Formula I:

[0006]

[0007] The preparation method of the dihydrophenanthrene compound comprises the following steps:

[0008] (1) The dried Panlongshen medicinal material is crushed, extracted with 95% ethanol under reflux for 2 hours each time, extracted twice, filtered and concentrated to obtain a total extract;

[0009] (2) After the total extract is dispersed evenly with water, it is extracted three times with petroleum ether, ethyl acetate, and n-butanol in sequence, and the petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction are concentrated respectively;

[0010] (3) The petroleum ether extraction part of Panlong ginseng was separated by silica gel column, and the mobile phase of petroleum ether-ethyl acetate system was used for elution, and a total of 14 components A to N were obtained;

[0011] (4) The FG components were combined, concentrated, dried, and separated by silica gel column chromatography using a mobile phase of petroleum ether-ethyl acetate to separate FG1 to FG 12 There were 12 fractions in total;

[0012] (5) The FG5 fraction separated in step (4) was separated using a silica gel column to obtain 23 samples, and the 11th to 16th fractions were combined to obtain FG 5-11 ~FG 5-16 , separated by high performance liquid chromatography (HPLC), with methanol: water = 85:15 as eluent, detection wavelength 254nm and 210nm, flow rate 2mL / min, elution time 15min, to obtain compound 1

[0013] Furthermore, in the step (3), the silica gel particle size used for silica gel column chromatography is 200-300 mesh; in the step (4), the concentration is carried out by reduced pressure concentration, and the drying is carried out by vacuum freeze drying.

[0014] Further, in the step (3), the volume ratio of petroleum ether and ethyl acetate used for separating components A to N is 100:0, 100:1, 100:3, 100:5, 100:7, 100:9, 100:10, 100:15, 100:20, 100:25, 100:40, 100:50, 100:75, 100:100; in the step (4), FG1 to FG2 are separated. 12 The volume ratios of petroleum ether and ethyl acetate used in the distillation are 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:12.5, 100:15, 100:25, 100:50, 100:100, and 0:100.

[0015] Furthermore, in the step (5), the FG5 fraction is separated by a silica gel column, and the volume ratio of the eluent used is petroleum ether-ethyl acetate = 4:1; the high performance liquid chromatography is a semi-preparative high performance liquid chromatography.

[0016] The dihydrophenanthrene compound is used in preparing a preparation having pancreatic islet protection effect.

[0017] Furthermore, the insulin cells are MIN6 cells.

[0018] The dihydrophenanthrene compound is used in preparing a preparation for treating fatty liver.

[0019] The dihydrophenanthrene compound is used in preparing a preparation having the effect of improving lipid accumulation.

[0020] Furthermore, the dihydrophenanthrene compound can reduce the intracellular TG content; in the application, the concentration of the dihydrophenanthrene compound is 0.39-25 μmol / L.

[0021] Beneficial effects of the present invention:

[0022] The present invention uses a variety of chromatographic methods to separate a new compound from the 95% ethanol extract and petroleum ether extraction part of the dried root of Panlongshen. The compound is identified as a dihydrophenanthrene compound. Experimental verification shows that at a concentration of 0.39 to 25 μmol / L, the compound shows an enhanced activity of MIN6 cells after PA-induced damage, antagonizes the damage of PA to MIN6 cells, and has a protective effect on MIN6 cells. At the same time, the compound can significantly reduce the intracellular TG content, improve lipid accumulation, and has a potential therapeutic effect on fatty liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0024] Figure 1 The structure and number of compound 1.

[0025] Figure 2 The main 1H-1H COSY, HMBC and NOESY correlations of compound 1.

[0026] Figure 3 Effect of compound 1 on PA-induced MIN6 cell injury (x±s, n=5), compared with the control group, ***P<0.001; compared with the PA group, # P<0.05, ## P<0.01.

[0027] Figure 4 Compound 1 improves lipid accumulation in vitro (x±s, n=5), compared with the control group, ***P<0.001, compared with the PA group, # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1 Isolation of Compounds

[0030] 1. Instruments and Materials

[0031] SB-1100 rotary evaporator (Shanghai Ailang Instrument Co., Ltd.), BRUKERAVⅢ600 nuclear magnetic resonance instrument (Bruker, Germany), four-hole water bath (Jiangsu Zhongda Instrument Technology Co., Ltd.), ZF-20D dark box UV analyzer (Shanghai Baoshan Gucun Electro-Optical Instrument Factory), KQ5200DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), semi-preparative high performance liquid chromatograph (Shimadzu, Japan), column chromatography silica gel (200-300 mesh, Qingdao Ocean Chemical Co., Ltd.), silica gel GF254 prefabricated plate (Qingdao Ocean Chemical Co., Ltd.), semi-preparative chromatographic column is ODS-A semi-preparative chromatographic column (YMC, 250 mm × 10 mm, 5 μM), electronic balance (Hangzhou Wante Weighing Instrument Co., Ltd.), precision balance (Shimadzu, Japan), Thermo Fisher LTQ liquid-mass ion trap mass spectrometer liquid-mass spectrometer (Thermo Fisher), enzyme marker (Model 680) (Bio-Rad, USA), methanol, n-butanol, dichloromethane, ethyl acetate, petroleum ether (analytical grade, Foshan Xilong Chemical Co., Ltd.), methanol (chromatographic grade) (Tianjin Concord Technology Co., Ltd.), RPMI Medium 1640 culture medium, fetal bovine serum, trypsin solution, and PBS were all purchased from Gibco, CCK8 (Biyuntian Biotechnology Co., Ltd.), palmitic acid (PA) (Hainan Hongke Biotechnology Co., Ltd.), and mouse pancreatic islet cells MIN6 (Anhui Differential Gene Technology Co., Ltd.).

[0032] Panlong ginseng is produced in Hezhang County, Bijie City, Guizhou Province. It was picked in August 2021 and identified by Professor Zeng Niankai of Hainan Medical College as the dried root of Panlong ginseng (Spiranthes sinensis), a plant of the genus Spiranthes in the Orchidaceae family. The plant specimen is preserved in the Natural Medicinal Chemistry Laboratory of Hainan Medical College, and the sample number is No.SS202108.

[0033] 2 Extraction and separation

[0034] The dried Panlongshen medicinal material (20 kg) was crushed and extracted with 95% ethanol reflux for 2 hours each time, extracted twice, filtered and concentrated to obtain 1203.06g of total extract. After the total extract was evenly dispersed with water, it was extracted with petroleum ether, ethyl acetate and n-butanol three times in sequence, and concentrated to obtain petroleum ether part (169.4g), ethyl acetate part (80.2g), and n-butanol part (168.3g). The petroleum ether extraction part of Panlongshen was separated on a silica gel column (200-300 mesh), and the mobile phase of petroleum ether-ethyl acetate system was selected for elution, and a total of 14 components A to N were obtained, and the volume ratio of petroleum ether and ethyl acetate is shown in Table 1.

[0035] Table 1 Nomenclature of components obtained from different gradient eluents of silica gel column

[0036]

[0037] The FG fractions were combined after TLC identification and separated by silica gel column chromatography (200-300 silica gel) using petroleum ether: ethyl acetate = 100: 0-100% as the eluent to obtain 12 fractions FG1-FG 12 , the ratio of the eluent is shown in Table 2.

[0038] Table 2 Nomenclature of fractions obtained from different gradient eluents of silica gel column

[0039]

[0040]

[0041] The FG5 fraction was further separated by silica gel column chromatography (200-300 silica gel) with petroleum ether-ethyl acetate as the eluent, and the volume ratio of the eluent was petroleum ether:ethyl acetate = 4:1. A total of 23 samples were received during the elution process and combined to obtain FG 5-11 ~FG 5-16 .

[0042] FG 5-11 ~FG 5-16 The product was separated by high performance liquid chromatography (HPLC) with methanol:water=85:15 as the eluent, detection wavelengths of 254 nm and 210 nm, and an eluent flow rate of 2 mL / min to obtain compound 1 (2.7 mg) with a retention time of 15 min.

[0043] TLC was performed using silica gel GF254 prefabricated plates, and the mixture was developed with petroleum ether: dichloromethane: ethyl acetate = 7:1:1; the color developing agent was 10% sulfuric acid in methanol.

[0044] The HPLC used was a semi-preparative high performance liquid chromatography with the following specific configuration: Shimadzu liquid chromatography LC-16 system, equipped with SPD-16 detector and YMC column (ODS, 2.5 mm×250 mm, 5 μm) (Shimadzu Corporation, Japan).

[0045] 3 Structure identification

[0046] Compound 1, white powdery solid; the spectral data are mainly as follows:

[0047] [α] D 20 =+70.0°(c 0.10,CH3OH); IR(KBr)v max : 3419, 1635, 1577, 1463, 1384, 1154, 1080cm -1 ; UV(CH3OH)λ max nm(logε): 211.6(5.86), 282.4(5.98), 297.2(6.00), 311.4(6.03); HR-ESI-MS m / z 363.1577([M+Na] + , calculated value 363.1572), the molecular formula is determined to be C 21 H 24 O4. 1 H-NMR (600MHz, DMSO-d6) gives two groups of benzene ring hydrogen signals: δ H 7.89 (1H, d, J = 8.8 Hz, H-5), 6.59 (1H, d, J = 8.8 Hz, H-6), 6.52 (1H, d, J = 2.4 Hz, H-3), 6.50 (1H, d, J = 2.4 Hz, H-1); 2 methoxy hydrogen signals: δ H 3.80 (3H, s, 4-OCH3), 3.78 (3H, s, 2-OCH3); 2 methoxy hydrogen signals: δ H 1.30 (3H, s, -CH3), 1.14 (3H, s, -CH3); 1 methine hydrogen signal: δ H 3.67 (1H, dd, J = 8.0, 5.7 Hz, H-2'); 3 methylene hydrogen signals: δ H 2.88 (1H, dd, J=5.6, 16.3Hz, H-3′), 2.65 (2H, m, H-10), 2.57 (2H, m, H-9), 2.06 (1H, m, H-3′); 13 The C-NMR (150 MHz, DMSO-d6) spectrum gives two groups of benzene ring carbon signals: δ C158.7 (C-2), 157.7 (C-4), 151.2 (C-7), 140.3 (C-10a), 137.3 (C-8a), 127.2 (C-5), 125.0 (C-4b), 117.7 (C-8), 116.5 (C-4a), 114.0 (C-6), 105.4 (C-1), 98.1 (C-3), 2 oxygen-linked carbon signals: δ C 76.6 (C-4′), 69.1 (C-2′), 2 methoxy carbon signals: δ C 56.0 (2-OCH3), 55.6 (4-OCH3), 3 methylene carbon signals: δ C 30.2 (C-10), 29.9 (C-3′), 24.5 (C-9), 2 methyl carbon signals: δ C 26.1(-CH3), 20.3(-CH3). Based on the above analysis, there are 2 benzene rings and 2 methylene groups in the structure of this compound, which is a typical feature of 9,10-dihydrophenanthrene compounds. In addition to the 2 benzene rings and 2 methylene groups, there are 2 methoxy groups and 5 carbon atoms in the structure, indicating that there is an isoprene-derived fragment in the structure. In-depth analysis revealed that the isoprene-derived fragment consists of 1 methylene (C-3′), 1 oxygen-linked methyl group, 1 oxygen-linked quaternary carbon and 2 methyl groups, indicating that the structural fragment is connected to the benzene ring to form a furan ring. The above analysis suggests that this compound is a 9,10-dihydrophenanthrene compound containing 1 furan ring, and its structure is as follows: Figure 1 shown.

[0048] 1 H- 1 H COSY shows δ H 7.89(H-5) and δ H 6.59(H-6) related, δ H 3.67(H-2′) and δ H 2.88(H-3′) correlation, δ H 2.57(H-9) and δ H 2.65 (H-10), indicating the presence of three structural fragments (C-5-C-6, C-2′-C-3′, C-9-C-10) (e.g. Figure 2 ). The HMBC spectrum shows that H-3 is associated with C-1, C-2 and C-4; 2-OCH3 and 4-OCH3 are associated with C-2 and C-4. Therefore, the connection positions of the two methoxy groups are determined; at the same time, the two methyl hydrogen H1.30 (-CH3), 1.14 (-CH3) and 69.1 (C-2′), 76.5 (C-6′) are related, confirming that the isopentenyl fragment in the structure is cyclized to form a furan ring, and the cyclization position is at C-7 and C-8. At the same time, H-3′ (δ H 2.88, 2.06) are correlated with C-7 (151.2), C-8 (117.7) and C-8a (137.3), confirming the above speculation. In the NOESY spectrum, 4-OCH3 and H-5 are correlated, further verifying the above speculation. In summary, the compound was identified as 2-(7,9-dimethoxy-2,3,4,5-tetrahydrophenanthro[2,1-b]furan-2-yl)propan-2-ol. After searching, the compound was found to be a new compound.

[0049] Effects of 4 new compounds on palmitic acid-induced MIN6 cell injury

[0050] method:

[0051] (1) MIN6 cell recovery and culture: Take out the cells from the -80℃ freezer, centrifuge and discard the upper layer of cryopreservation solution, add 1640 medium containing 10% fetal bovine serum and 1% double antibody to resuspend the cells, and culture them in an incubator with a relative humidity of 95%, 37℃, and 5% CO2; depending on the cell growth, replace the fresh medium every 24 to 36 hours, and when the cells grow to 75% to 85%, subculture or cryopreservation. (2) Evenly inoculate the well-growing MIN6 cells into a 96-well plate at 5×10 5 100 μL of complete medium (medium containing 10% fetal bovine serum) was added to 100 μL of culture medium (medium containing 10% fetal bovine serum) per mL. After culturing for 24 hours, the original culture medium was removed from the experimental group and the model group, and 100 μL of culture medium containing compound 1 and PA (300 μM) was added to the experimental group, and 100 μL of 300 μM (300 μmol / L) of PA was added to the model group. The blank control group did not receive any treatment. The concentrations of compound 1 in the experimental group were set at 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 μM, totaling 8 concentrations. After different concentrations of compound 1 and palmitic acid (PA) co-intervention cells for 24 hours, the proliferation activity of MIN6 cells was detected by CCK8 method.

[0052] The specific steps of CCK8 are as follows: After MIN6 cells were treated with the above conditions, 100 μL of CCK-8 solution was added to each well of 96-well cells. After incubation at 37°C, 5% CO2 incubator for 1 hour, the optical density (OD) was measured at 450 nm using an enzyme label, and the cell viability was calculated.

[0053] The results showed that after palmitic acid (PA) induced MIN6 cell damage, the reproductive activity of the model group was significantly lower than that of the control group (P<0.001), indicating that PA can induce MIN6 cell damage and the model was successfully established. Compound 1 was given for intervention. Compared with the model group (PA), the cell proliferation activity of each group treated with compound 1 at a concentration of 0.39-25 μM and palmitic acid was enhanced to varying degrees (P<0.05, P<0.01), and at a concentration of 1.56-25 μM, the cell proliferation activity was significantly enhanced (P<0.01), with the highest concentration at 3.125 μM. Compared with the PA group, the cell proliferation activity value reached 18.58±2.735. When the concentration reached 50 μM, there was no significant difference in cell proliferation activity from that of the model group. The results are as follows Figure 3 shown.

[0054] The above results show that compound 1 can antagonize the damaging effect of palmitic acid on MIN6 cells and promote cell growth and proliferation, and the effect is greatly affected by the concentration. 5 New compounds improve lipid accumulation in vitro

[0055] method:

[0056] (1) HL-7702 cell recovery and culture: Take out the cells from the -80°C freezer, centrifuge and discard the upper layer of freezing solution, add DMEM medium containing 20% ​​fetal bovine serum and 1% double antibody to resuspend the cells, and culture them in an incubator with a relative humidity of 95%, 37°C, and 5% CO2; depending on the cell growth, replace the fresh medium every 24 to 36 hours, and when the cells grow to 80% to 90%, subculture or cryopreservation is performed.

[0057] (2) A 300 μM free fatty acid (PA-BSA)-induced hyperlipidemia model was established with HL-7702 cells, and compound 1 was administered. HL-7702 cells were cultured (RPMI1640+10% FBS+1% P / S) and used for experimental studies after two stable passages. The cell suspension was incubated at 2.0×10 5 The cells / mL density was evenly inoculated in a 12-well plate, 1 mL per well, and cultured in a cell culture incubator. Each well was divided into the following groups: blank control group (10% BSA), model group (300μM PA-BSA), and drug group (300μM PA-BSA + different concentrations of compound 1), with 5 replicates in each group. After the inoculated 12-well plate was cultured for 24 hours, the original culture medium was removed and replaced with a mixture of BSA, PA-BSA and compound 1 with the target concentration, and the culture treatment was carried out for 24 hours. The cell culture medium was discarded, and the cells were digested with trypsin and collected by centrifugation.

[0058] Determination of intracellular TG levels: The tissue cell triglyceride (TG) enzymatic assay kit (Beijing Pulilai Gene Technology Co., Ltd., E1013) was used to determine the intracellular TG levels of each treatment group: (1) Differentiation of adipocytes: The cells were collected and then lysed, and the ratio of 1x10 6 Add 0.1 mL of lysis buffer to each cell, mix well and let stand at room temperature for 10 minutes. (2) Lysis buffer treatment: Transfer an appropriate amount of supernatant to a 1.5 mL centrifuge tube and heat at 70°C for 10 minutes. Flocculent precipitation may appear when the amount of tissue is large. Centrifuge at 2000 rpm for 5 minutes at room temperature, and the supernatant can be used for enzymatic assays. Then add the sample to be tested to a 96-well plate with a starting volume of 10 μL. React at 37°C for 15 minutes. After the reaction is balanced, the color is stable within 60 minutes. First, adjust the blank tube to zero with distilled water + working solution, and then measure the OD value of each well. Finally, correct the triglyceride content by mg protein concentration or cell number.

[0059] The results showed that the concentration of TG in the PA group was significantly higher than that in the control group (P < 0.001), indicating that the palmitic acid-coupled bovine serum albumin (PA-BSA) was used to induce lipid accumulation in the hepatocyte HL-7702 cells, and the model was successfully established. After intervention treatment with compound 1 at a concentration of 0.39-25 μM, the intracellular triglyceride (TG) content was reduced compared with the model group (PA) (P < 0.05, P < 0.01), and when the concentration was 1.56-25 μM, the TG content decreased significantly (P < 0.01), reaching the lowest level at a concentration of 3.125 μM, which was 88.58 ± 12.34 nmol / ng lower than that of the PA group. When the concentration reached 50 μM, there was no significant difference in cell proliferation activity compared with the model group. The results are as follows Figure 4 shown.

[0060] The above results indicate that compound 1 can significantly reduce the intracellular TG content, improve lipid accumulation, and has a potential therapeutic effect on fatty liver, and the effect is greatly affected by concentration.

[0061] 5 Conclusion

[0062] In this study, a new compound was isolated from the 95% ethanol extract and petroleum ether extract of the dried root of Panlongshen using a variety of chromatographic methods. The new compound showed that it could enhance the vitality of MIN6 cells after PA-induced injury at a certain concentration, antagonize the damage of PA to MIN6 cells, and have a protective effect on MIN6 cells. At the same time, the compound can significantly reduce the intracellular TG content, improve lipid accumulation, and has a potential therapeutic effect on fatty liver.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A dihydrophenanthrene compound isolated from Panlong ginseng, characterized in that: The structure is shown in Formula I: 。 2. The method for preparing a dihydrophenanthrene compound according to claim 1, wherein The following steps are involved: (1) The dried Panlongshen medicinal material was crushed, extracted with 95% ethanol under reflux for 2 hours each time, and extracted twice, and filtered and concentrated to obtain the total extract; (2) After the total extract is dispersed evenly with water, it is extracted three times with petroleum ether, ethyl acetate, and n-butanol in sequence, and the petroleum ether fraction, ethyl acetate fraction, and n-butanol fraction are concentrated respectively; (3) The petroleum ether extraction part of Panlong ginseng was separated by silica gel column, and the mobile phase of petroleum ether-ethyl acetate system was used for elution, and a total of 14 components A~N were obtained; (4) The FG components were combined, concentrated, dried, and separated by silica gel column chromatography using a mobile phase of petroleum ether-ethyl acetate to separate FG1~FG 12 There were 12 fractions in total; (5) The FG5 fraction obtained in step (4) was separated using a silica gel column to obtain 23 samples, and the 11th to 16th fractions were combined to obtain FG 5-11 ~FG 5-16 , separated by high performance liquid chromatography (HPLC), with methanol: water = 85:15 as eluent, flow rate 2 mL / min, elution time 15 min, to obtain the compound of formula I.

3. The preparation method according to claim 2, characterized in that: In the step (3), the silica gel particle size used for silica gel column chromatography is 200-300 mesh; in the step (4), the concentration is carried out by reduced pressure concentration, and the drying is carried out by vacuum freeze drying.

4. The preparation method according to claim 2, characterized in that: In the step (3), the volume ratios of petroleum ether and ethyl acetate used to separate components A to N are 100:0, 100:1, 100:3, 100:5, 100:7, 100:9, 100:10, 100:15, 100:20, 100:25, 100:40, 100:50, 100:75, and 100:100, respectively; in the step (4), FG1 to FG2 are separated. 12 The volume ratios of petroleum ether and ethyl acetate used in the distillation are 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:12.5, 100:15, 100:25, 100:50, 100:100, and 0:

100.

5. The preparation method according to claim 2, characterized in that: In the step (5), the FG5 fraction is separated by a silica gel column, and the volume ratio of the eluent used is petroleum ether-ethyl acetate = 4:1; the high performance liquid chromatography is a semi-preparative high performance liquid chromatography.

6. Use of the dihydrophenanthrene compound as claimed in claim 1 in preparing a preparation having an islet protective effect.

7. The use according to claim 6, characterized in that The cells of the pancreatic islets are MIN6 cells.

8. Use of the dihydrophenanthrene compound according to claim 1 in preparing a preparation for treating fatty liver.

9. Use of the dihydrophenanthrene compound as claimed in claim 1 in preparing a preparation having an effect of improving lipid accumulation.

10. The use according to claim 9, characterized in that The dihydrophenanthrene compound can reduce the intracellular TG content; in the application, the concentration of the dihydrophenanthrene compound is 0.39-25 μmol / L.

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