A method for preparing oleanane-type compound pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge. and its application

Abstract

A method for preparing oleanane-type compound pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge., the steps are as follows: Cut Stellaria dichotoma var. lanceolata Bge. into sections, extract and extract, concentrate under reduced pressure and then evaporate to dryness to obtain an ethyl acetate part extract; Purify, activate, wash away impurities, suspend, load the column by wet method, slowly add the column bed, close or reduce the opening at the lower end of the column to make it slowly adsorb in the macroporous resin, elute with gradient, collect the eluate, and concentrate under reduced pressure; Load the column by wet method, load the sample by dry method, elute with gradient, collect and combine; Load the column by wet method, load the sample by dry method, elute, collect and combine; Load the column by wet method, load the sample by dry method, elute, collect and combine; Elute, collect fractions to obtain the compound Stellaria dichotoma var. lanceolata Bge. pentacyclic triterpenoid A. Its raw materials are rich, and the preparation method is easy to operate. It can be effectively used to prepare oleanane-type compound pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge., realizing its application in the preparation of drugs for treating allergic diseases, opening up the medicinal value of Stellaria dichotoma var. lanceolata Bge., and having significant economic and social benefits.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically to a method for preparing oleanane-type compound pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge. and its application. Background Art

[0002] The root of Stellaria dichotoma var. lanceolata Bge. and Gypsophila licentiana Hand.-Mazz. of the genus Gypsophila L. in the family Caryophyllaceae is sweet in taste, slightly cold in nature, and belongs to the lung and stomach meridians. It has the functions of clearing away deficiency-heat and cooling blood, and is used for symptoms such as consumptive fever due to yin deficiency, chronic malaria due to yin deficiency, infantile malnutrition, hepatitis, etc. Stellaria dichotoma var. lanceolata Bge. has a wide history of folk medicine use and broad prospects for development and utilization. Research has found that Stellaria dichotoma var. lanceolata Bge. contains many good medicinal active ingredients.

[0003] Allergic diseases are one of the diseases that the World Health Organization has listed as key research and prevention diseases in the 21st century. It is mainly a type I allergic reaction mediated by IgE, which can affect multiple organs and systems throughout the body, including diseases such as urticaria, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic asthma, etc. RBL-2H3 cell degranulation has been widely used in allergy and immunology research, especially in screening the anti-allergic properties of extracted and separated compounds and natural extracts. At present, the long-term curative effect of treating allergic diseases clinically is still not ideal. Therefore, finding effective anti-allergic drugs remains an urgent technical problem for pharmaceutical researchers. Then, what about extracting the active ingredient compound from Stellaria dichotoma var. lanceolata Bge. to prepare drugs for treating allergic diseases? However, there has been no public report so far. Summary of the Invention

[0004] In view of the above situation, in order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing oleanane-type compound pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge. and its application, which can effectively solve the preparation of oleanane-type compound pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge. and realize its application in the preparation of drugs for treating allergic diseases.

[0005] The technical solution solved by the present invention is: A method for preparing oleanane-type compound pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge., the chemical molecular formula of the compound pentacyclic triterpenoid A is:

[0006]

[0007] The preparation method is:

[0008] The Radix Stellariae dichotomae with residual above-ground stems was cut into sections and refluxed with 70% ethanol by volume three times for 2 hours each time. The ethanol dosages were 10, 8, and 6 times the weight-volume of Radix Stellariae dichotomae, where the weight-volume means the solid is in kg and the liquid is in L. It was centrifuged at 12,000 rpm for 10 minutes and concentrated under reduced pressure until the alcohol smell disappeared. It was successively extracted repeatedly with equal volumes of petroleum ether, ethyl acetate, and n-butanol until the color of the upper-phase solution no longer became lighter. After the extraction solutions were concentrated under reduced pressure, they were transferred to an evaporating dish for drying to obtain the petroleum ether fraction extract, ethyl acetate fraction extract, and n-butanol fraction extract respectively;

[0009] The ethyl acetate fraction extract was purified by macroporous resin DM-130. The resin was activated in 95% ethanol by volume (abbreviated as 95% ethanol, the same below) for 24 hours before use to wash away impurities, suspended in 95% ethanol, and packed into a column by the wet method. 1 BV = 20 L. Then it was rinsed with 95% ethanol. When the ethanol rinsed out and mixed with an equal volume of water without becoming turbid, it was successively gradient-transitioned from 95% ethanol to 10% ethanol by volume (abbreviated as 10% ethanol, the same below). The ethyl acetate fraction extract was dissolved in 10% ethanol and slowly added to the column bed. The opening at the lower end of the column was closed or reduced to allow the sample to slowly adsorb onto the macroporous resin. Gradient elution was carried out using ethanol solutions with different volume concentrations of 10%, 30%, 50%, 70%, and 95%. Each gradient was rinsed with 4 column volumes, and the elution solutions of each gradient were collected and concentrated under reduced pressure to obtain five components Fr1 - Fr5;

[0010] Component Fr5 was purified by silica gel column chromatography: packed into a column by the wet method and loaded with the sample by the dry method. It was eluted with a dual system of petroleum ether - ethyl acetate with a volume ratio of 10:1 and dichloromethane - methanol with a volume ratio of 1 - 100:1. 1 BV = 1500 mL, and collected once every 1 / 3 column volume. It was spotted on a plate and inspected under an ultraviolet lamp. Six fractions were combined, namely: fraction Fr5-1 of petroleum ether - ethyl acetate 10:1, fraction Fr5-2 of dichloromethane - methanol 100:1 and 80:1, fraction Fr5-3 of dichloromethane - methanol 60:1 and 40:1, fraction Fr5-4 of dichloromethane - methanol 20:1, fraction Fr5-5 of dichloromethane - methanol 10:1 and 5:1, and fraction Fr5-6 of dichloromethane - methanol 1:1;

[0011] Purification of fraction Fr5-3, silica gel column chromatography: The column was packed with the wet method, and the sample was loaded with the dry method. It was eluted with a dichloromethane-methanol system with a volume ratio of 40-100:1. 1 BV = 450 mL, and fractions were collected every 1 / 3 column volume. The plates were spotted and examined under ultraviolet light. Five fractions were obtained by combination, namely: fraction Fr5-3-1 with dichloromethane-methanol 1:0, fraction Fr5-3-2 with dichloromethane-methanol 100:1, fraction Fr5-3-3 with dichloromethane-methanol 80:1, fraction Fr5-3-4 with dichloromethane-methanol 60:1, and fraction Fr5-3-5 with dichloromethane-methanol 40:1;

[0012] Purification of fraction Fr5-3-2, silica gel column chromatography: The column was packed with the wet method, and the sample was loaded with the dry method. It was eluted with a dichloromethane-methanol system with a volume ratio of 30-100:1. 1 BV = 314 mL, and fractions were collected every 1 / 3 column volume. The plates were spotted and examined under ultraviolet light. Three fractions were obtained by combination, namely: combining the fractions of dichloromethane-methanol 100:1 and 80:1 into Fr5-3-2-1, combining the fractions of dichloromethane-methanol 60:1 and 40:1 into Fr5-3-2-2, and fraction Fr5-3-2-3 with dichloromethane-methanol 30:1;

[0013] Semi-preparative purification of fraction Fr5-3-2, eluted with methanol water with a volume concentration of 83%, flow rate 3 mL / min, dual-channel wavelengths: 210 nm, 254 nm. The fraction with a retention time T = 20 min was collected to obtain the compound shanyinchaihu pentacyclic triterpenoid A.

[0014] Application of the compound shanyinchaihu new pentacyclic triterpenoid A prepared by the above method in the preparation of drugs for treating allergic diseases, wherein the allergic diseases are diseases such as urticaria, atopic dermatitis, allergic conjunctivitis, allergic rhinitis or allergic asthma.

[0015] The raw materials of the present invention are rich, and the preparation method is easy to operate. It can be effectively used to prepare the oleanane-type compound pentacyclic triterpenoid A from shanyinchaihu, and realize its application in the preparation of drugs for treating allergic diseases, expanding the medicinal value of shanyinchaihu, and having significant economic and social benefits. Description of the Drawings

[0016] Figure 1 It is the chemical molecular formula structure diagram of the compound shanyinchaihu pentacyclic triterpenoid A of the present invention;

[0017] Figure 2 It is the HMBC correlation diagram of the compound shanyinchaihu pentacyclic triterpenoid A of the present invention;

[0018] Figure 3 It is the NOE correlation diagram of the compound shanyinchaihu pentacyclic triterpenoid A of the present invention;

[0019] Figure 41H-NMR spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention;

[0020] Figure 5 13C-NMR spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention;

[0021] Figure 6 DEPT spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention;

[0022] Figure 7 HSQC spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention;

[0023] Figure 8 HMBC spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention;

[0024] Figure 9 NOE spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention;

[0025] Figure 10 H-H COSY spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention;

[0026] Figure 11 IR spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention;

[0027] Figure 12 UV spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention;

[0028] Figure 13 Mass spectrum of the compound Ophiopogon japonicus (Thunb.) Ker-Gawl. pentacyclic triterpenoid A of the present invention. Detailed Description of the Invention

[0029] The following is a detailed description of the specific embodiments of the present invention in combination with examples and specific situations.

[0030] The specific implementation of the present invention is given by the following examples:

[0031] The present invention relates to a method for preparing oleanane-type compound pentacyclic triterpenoid A from Ophiopogon japonicus (Thunb.) Ker-Gawl., and the chemical formula of the compound pentacyclic triterpenoid A is:

[0032]

[0033] The preparation method is as follows:

[0034] Chop 20 kg of Stellaria dichotoma var. lanceolata with remaining aerial stems into sections, and reflux extract three times with ethanol at a volume concentration of 70% for 2 h each time. The ethanol dosages are 10, 8, and 6 times the weight-to-volume ratio of Stellaria dichotoma var. lanceolata, where the weight-to-volume ratio means the solid is in kg and the liquid is in L. Centrifuge at 12,000 rpm for 10 min, and concentrate under reduced pressure to 50 L until there is no alcohol smell. Repeatedly extract with equal volumes of petroleum ether, ethyl acetate, and n-butanol in sequence until the color of the upper-phase solution no longer fades. After concentrating each extract under reduced pressure, transfer it to an evaporating dish for drying to obtain the petroleum ether fraction extract, ethyl acetate fraction extract, and n-butanol fraction extract respectively;

[0035] The ethyl acetate fraction extract (266.57 g) is purified by macroporous resin DM-130. Before use, the resin is activated in ethanol with a volume concentration of 95% (abbreviated as 95% ethanol, the same below) for 24 h to wash away impurities, suspended in 95% ethanol, and packed into a column by the wet method. 1 BV = 20 L. Then rinse with 95% ethanol. When the ethanol rinsed out is mixed with an equal amount of water and does not become turbid, gradually transition from 95% ethanol to ethanol with a volume concentration of 10% (abbreviated as 10% ethanol, the same below) in sequence. Dissolve the ethyl acetate fraction extract in 10% ethanol, slowly add it to the column bed, close or reduce the opening at the lower end of the column to allow the sample to slowly adsorb onto the macroporous resin. Use ethanol solutions with different volume concentrations of 10%, 30%, 50%, 70%, and 95% for gradient elution. Rinse each gradient with 4 column volumes, collect the eluates of each gradient, and concentrate under reduced pressure to obtain five components Fr1 - Fr5;

[0036] Component Fr5 (49.81 g) is purified by silica gel column chromatography: Pack the column by the wet method and load the sample by the dry method. Elute with a dual system of petroleum ether - ethyl acetate with a volume ratio of 10:1 and dichloromethane - methanol with a volume ratio of 1 - 100:1. 1 BV = 1500 mL, collect once every 1 / 3 column volume, check the plates under ultraviolet light, and combine to obtain 6 fractions, namely: fraction Fr5-1 of petroleum ether - ethyl acetate 10:1, fractions Fr5-2 of dichloromethane - methanol 100:1 and 80:1, fractions Fr5-3 of dichloromethane - methanol 60:1 and 40:1, fraction Fr5-4 of dichloromethane - methanol 20:1, fractions Fr5-5 of dichloromethane - methanol 10:1 and 5:1, and fraction Fr5-6 of dichloromethane - methanol 1:1;

[0037] The fraction Fr5-3 (11.046 g) was purified by silica gel column chromatography. The column was packed with the wet method and the sample was loaded with the dry method. It was eluted with a dichloromethane-methanol system with a volume ratio of 40 - 100:1. 1 BV = 450 mL, and fractions were collected every 1 / 3 column volume. The plates were spotted and examined under ultraviolet light. Five fractions were obtained by combining, namely: fraction Fr5-3-1 with dichloromethane-methanol 1:0, fraction Fr5-3-2 with dichloromethane-methanol 100:1, fraction Fr5-3-3 with dichloromethane-methanol 80:1, fraction Fr5-3-4 with dichloromethane-methanol 60:1, and fraction Fr5-3-5 with dichloromethane-methanol 40:1;

[0038] The fraction Fr5-3-2 (2.68 g) was purified by silica gel column chromatography. The column was packed with the wet method and the sample was loaded with the dry method. It was eluted with a dichloromethane-methanol system with a volume ratio of 30 - 100:1. 1 BV = 314 mL, and fractions were collected every 1 / 3 column volume. The plates were spotted and examined under ultraviolet light. Three fractions were obtained by combining, namely: fraction Fr5-3-2-1 by combining the fractions with dichloromethane-methanol 100:1 and 80:1, fraction Fr5-3-2-2 by combining the fractions with dichloromethane-methanol 60:1 and 40:1, and fraction Fr5-3-2-3 with dichloromethane-methanol 30:1;

[0039] The fraction Fr5-3-2-2 (563.4 mg) was semi-preparatively purified, eluted with 83% methanol water solution at a flow rate of 3 mL / min. The dual-channel wavelengths were 210 nm and 254 nm. The fraction with a retention time T = 20 min was collected to obtain compound Oleanane-type pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge. (5 mg).

[0040] According to the compound Oleanane-type pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge. given in the above example, any amount of Chinese medicine can be prepared as needed. The given example is only used to illustrate the specific implementation manner of the present invention, rather than to limit the protection scope of the present invention. All technical solutions that are essentially the same as the present invention by using equivalent substitution or transformation means belong to the protection scope of the present invention.

[0041] The raw materials of the present invention are rich, and the preparation method is easy to operate. It can be effectively used to prepare Oleanane-type compound pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata Bge. This compound has the function of reducing the IgE level and can be effectively used to prepare drugs for treating allergic diseases. Very good beneficial technical effects have been obtained through experiments. The relevant data are as follows:

[0042] I. Instruments and reagents

[0043] Bruker AVANCE III 500 nuclear magnetic resonance spectrometer for nuclear magnetic resonance (with TMS internal standard) (Bruker), Nicolet is 10 Microscope Spectrometer for infrared spectroscopy (Thermo Scientific, USA), Agilent6546 UPLC-Q-TOF-MS system for ultra-high performance liquid chromatography - quadrupole time-of-flight tandem mass spectrometry (Agilent Technologies, USA), Shimadzu UV-2401PC apparatus for ultraviolet spectroscopy, FDU-2110 freeze dryer (Shanghai Ailan Instrument Co., Ltd.), carbon dioxide incubator (Thermo Fisher Scientific Inc.), 3020 multi-functional microplate reader (Thermo Fisher Scientific Inc.), AE2000 inverted microscope (Motic Group Co., Ltd.), superconducting nuclear magnetic resonance spectrometer (Bruker, Switzerland, DPX-500), semi-preparative high performance liquid chromatography (Beijing Sapphire Technology Co., Ltd., LC-52 type), full-preparative high performance liquid chromatography (Shanghai Wufeng Science Co., Ltd., LC100), rotary evaporator (EYELA Tokyo Rika Kogyo Co., Ltd., Japan, N-1000 type, BSA124S-CW), condensing water circulation device (EYELA Tokyo Rika Kogyo Co., Ltd., Japan, N-1111), Agilent high performance liquid chromatograph (Agilent Technologies, USA, Agilent 1260 Infinity II).

[0044] MEM medium (Gibco Invitrogen), fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., Four Seasons Green), trypsin digestion solution (Beijing Solarbio Science & Technology Co., Ltd.), DMSO (Maclean Biochemical Co., Ltd.), PBS buffer (Wuhan Sevier Biotechnology Co., Ltd.), C48 / 80 (Sigma, USA), Na2CO3, NaHCO3, NaOH (Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.). Radix Stellariae was collected from Tianshui County, Gansu Province in August 2022, and identified by Professor Chen Suiqing of Henan University of Chinese Medicine as the underground root of Gypsophila licentiana Hand.-Mazz. of the genus Gypsophila, and is stored in the Key Laboratory of Traditional Chinese Medicine Resources and Traditional Chinese Medicine Chemistry of Henan Province.

[0045] II. Structural identification

[0046] Using the nuclear magnetic resonance spectrometer and liquid chromatograph given in the above item I, the spectra were measured. After identification and testing:

[0047] Radix Stellariae pentacyclic triterpenoid A: white crystal, soluble in chloroform. ESI-QTOF-MS m / z : 485.3262[M+H] +, its molecular formula was determined to be: C 30 H 44 O5. UV (MeOH) λ max (logε) / nm: 205 (1.876); In the IR spectrum: 3660 cm- 1 was the absorption peak of the stretching vibration of the hydroxyl group, 1770 cm- 1 and 1727 cm- 1 indicated that there were two carbonyl groups in this compound, and among them, 1770 cm- 1 it was speculated that there was a lactone ring structure in this compound. 1 Six methyl hydrogen signals were given in 1H-NMR (500 MHz, CDCl3), δ H : 1.28 (3H, d) 1.08 (3H, s), 1.02 (6H, s), 0.99 (3H, d), 0.78 (3H, s), one double bond hydrogen signal 5.56 (1H, s); it was speculated to be the hydrogen signal of an oleanolic acid-type compound; 4.52 (1H, t, J = 6.6 Hz,), 4.13 (1H, d, J = 5.9 Hz) were the hydrogen signals on two oxygen-bearing carbons; From 13 13C-NMR (125 MHz, CDCl3) and DEPT, it was found that there were a total of 30 carbon signals, including 6 methyl carbon signals (27.69, 27.09, 23.68, 19.67, 16.14, 13.54); 9 methylene carbon signals (36.81, 36.77, 34.66, 31.57, 29.67, 26.24, 24.35, 24.02, 21.27); 6 methine carbon signals (121.98, 84.42, 75.12, 52.86, 45.61, 39.99); the rest were quaternary carbon signals (182.19, 175.82, 140.86, 43.50, 42.26, 39.21, 38.96, 93.91, 33.73); among them, δ C : 182.19, 175.82 were two carbonyl carbon signals; 140.86, 121.96 were the carbon signals of the double bond. It was speculated that this compound might be a pentacyclic triterpene of olean-12-ene, 84.42, 75.12 were oxygen-bearing carbon signals; there was a lactone ring, but there was a double bond in this compound. The methyl carbon signals at positions 29 and 30 were BC-correlated with 84.42. It was speculated that the lactone ring structure was in ring E. Through H-H COSY, it was found that 4.13 was correlated with 1.82. Therefore, it was speculated that the lactone ring site was at position 21. The relevant parameters of the NMR data are shown in Table 1.

[0048] Table 1 NMR data attribution table of the compound shanyinchaihu new pentacyclic triterpene A of the present invention

[0049] The chemical molecular structure is:

[0050]

[0051] Specific 13 C-NMR, 1 H-NMR and HMBC related peak assignments are shown in Figure 2 - 13 .

[0052] From the above, it can be clearly seen that the new pentacyclic triterpene A from Bupleurum chinense is a new compound, and there has been no public report on this compound before.

[0053] 3. Activity Detection

[0054] 1. Cell Culture

[0055] Rat basophil leukemia cell line (RBL-2H3) was cultured in MEM medium containing 10% fetal bovine serum and penicillin-streptomycin solution for one week and then placed in an incubator at 37°C and 5% CO2. RBL-2H3 cells in the logarithmic growth phase were selected for the experiment. Fresh culture medium was replaced every 48 hours and the cells were passaged at a ratio of 1:2-1:3.

[0056] 2. Reagent Preparation

[0057] Take C48 / 80 out of the -20℃ refrigerator and place it at room temperature for 0.5h, dissolve it with PBS in the clean bench, and prepare a solution with a concentration of 20mg / mL for use; in the clean bench, take 100μL of Triton X-100, add 9.9mL of benchtop solution to dissolve, and prepare 1% Trinton X-100, which is prepared before use; 4-nitrophenyl-N-acetyl- β -D-glucosamine was prepared into a substrate solution with a concentration of 1mmol / L using 1mol / L citrate buffer (pH4.5); 4.00g of NaOH particles were weighed and dissolved in 100mL of Wahaha purified water to adjust the pH value of the solution; 2.65g of Na2CO3 and 2.1g of NaHCO3 were weighed and dissolved in 250mL of Wahaha purified water, and the solution was adjusted to a pH of 10.7 using 1mol / L NaOH.

[0058] 3. Activity screening method

[0059] Cells in logarithmic growth phase were taken and 2×10 4200 μL of cell suspension was inoculated in a 96-well plate at a density of 1 cell / mL, with 4 replicates in each group. After 24 hours, the plates were divided into a blank control group, a model group (C48 / 80, 20 μg / mL), a group of new pentacyclic triterpenoids from Bupleurum chinense A + C48 / 80 (10 μM), and a total enzyme group (1% Trinton X-100). Both the drug and C48 / 80 solutions were prepared with benchtop solution. In addition, 4 blank wells containing only benchtop solution without cells were set up. After 30 minutes of stimulation, 50 μL of cell supernatant was taken, and 50 μL of substrate solution with a concentration of 1 mmol / L was added. The plates were incubated at 37°C for 1 hour, and 150 μL of Na2CO3 / NaHCO3 stop solution was added to terminate the reaction. The absorbance was detected at 405 nm by a microplate reader, and the value was calculated. β -Hex release rate. The experiment was repeated three times in parallel.

[0060] The formula is: β -Hex release rate = (OD value of supernatant in experimental group - OD value of supernatant in blank well) / (OD value of supernatant in total enzyme well - OD value of supernatant in blank well) × 100%. Table 2 shows that the compound can significantly inhibit β -Hex Release ( P <0.01)

[0061] Table 2 Effect of the compound of the present invention, Bupleurum chinense, new pentacyclic triterpenoid A, on C48 / 80-induced degranulation of RBL-2H3 cells

[0062] ( ±sd, n=4)

[0063] Group -Hex Release Rate Blank Control Group 0.651±0.036 Model Group <![CDATA[1.000±0.046 ## > Neo-pentacyclic Triterpenoid A <![CDATA[0.706±0.025 ** >

[0064] Note: Compared with the normal group, ## express P <0.01, compared with the model group, ** express P <0.01.

[0065] 4. Immunoglobulin E (IgE) ELISA kit to measure IgE content

[0066] RBL-2H3 cells were cultured at 2×10 4 The cells were seeded at a density of 10 cells / mL in a 96-well plate, 200 μL per well, and the IgE content was determined using an IgE kit. After 24 hours, the cells were divided into a blank control group, a model group (C48 / 80, 20 μg / mL), and a new pentacyclic triterpene A group + C48 / 80 (10 μM). After 24 hours of action, the supernatant of each group of cells was collected, centrifuged at 3000 rpm for 20 minutes at 4°C, and the supernatant was collected. The operation steps were strictly performed according to the instructions of the kit.

[0067] Table 3 shows that the pentacyclic triterpenoid A in Stellaria dichotoma var. lanceolata can significantly reduce the level of IgE.

[0068] ( ±s, n = 4)

[0069] Group IgE Content (μg / ml) Blank Control Group 0.711±0.095 Model Group <![CDATA[0.969±0.074 ## > Neo-pentacyclic Triterpenoid A <![CDATA[0.802±0.080 * >

[0070] Experiments show that the pentacyclic triterpenoid A can significantly reduce the level of IgE and can be effectively used for the preparation of drugs for treating allergic diseases.

[0071] In conclusion, it can be clearly seen from the above that the raw materials of the present invention are abundant, the preparation method is easy to operate, and it can be effectively used for the preparation of the oleanane-type compound pentacyclic triterpenoid A from Stellaria dichotoma var. lanceolata, realizing its application in the preparation of drugs for treating allergic diseases, expanding the medicinal value of Stellaria dichotoma var. lanceolata, and having significant economic and social benefits.

Claims

1. An oleanane-type compound pentacyclic triterpene A prepared from Bupleurum chinense, the molecular formula structure of the compound is:

2. The method for preparing the oleanane-type compound pentacyclic triterpene A according to claim 1, characterized in that: The Bupleurum chinense with residual stems above ground was cut into sections, and extracted with 70% ethanol by volume for three times, each time for 2 hours, with the amount of ethanol being 10, 8, and 6 times the weight volume of the Bupleurum chinense, respectively. The weight volume refers to the solid in kg and the liquid in L. The extract was centrifuged at 12000 rpm for 10 min, and concentrated under reduced pressure until there was no alcohol taste. The extract was repeatedly extracted with equal volumes of petroleum ether, ethyl acetate, and n-butanol in sequence until the color of the upper phase solution no longer became lighter. After the extracts were concentrated under reduced pressure, they were transferred to an evaporating dish and dried to obtain a petroleum ether extract, an ethyl acetate extract, and an n-butanol extract, respectively. The ethyl acetate extract was purified by macroporous resin DM-130. The resin was activated in 95% ethanol for 24 hours before use, and impurities were washed away. It was suspended in 95% ethanol and wet loaded into the column, 1BV=20L, and then washed with 95% ethanol. When the washed ethanol was mixed with an equal amount of water and remained turbid, it was gradually transferred from 95% ethanol to 10% ethanol by volume concentration. The ethyl acetate extract was dissolved in 10% ethanol and slowly added to the column bed. The opening at the lower end of the column was closed or reduced to allow the sample to slowly adsorb on the macroporous resin. Different ethanol solutions with different volume concentrations of 10%, 30%, 50%, 70%, and 95% were used for gradient elution. Each gradient was washed with 4 column volumes. The gradient eluents were collected and concentrated under reduced pressure to obtain five components Fr1-Fr5. Purification of component Fr5, silica gel column chromatography: wet column packing, dry sample loading, elution with a dual system of petroleum ether-ethyl acetate with a volume ratio of 10:1 and dichloromethane-methanol with a volume ratio of 1-100:1, 1BV=1500mL, 1 / 3 column volume is collected once, the plate is inspected under ultraviolet light, and the components of dichloromethane-methanol 60:1 and 40:1 are combined to obtain component Fr5-3; Purification of component Fr5-3, silica gel column chromatography: wet column packing, dry sample loading, elution with dichloromethane-methanol system with a volume ratio of 40-100:1, 1BV=450mL, 1 / 3 column volume collected once, spot plate inspected under ultraviolet light, and combined dichloromethane-methanol 100:1 components to obtain component Fr5-3-2; Purification of component Fr5-3-2, silica gel column chromatography: wet column packing, dry sample loading, elution with dichloromethane-methanol system with a volume ratio of 30-100:1, 1BV=314mL, 1 / 3 column volume collected once, spot plate inspected under ultraviolet light, and combined dichloromethane-methanol 60:1, 40:1 components to obtain component Fr5-3-2-2; The component Fr5-3-2-2 was semi-preparatively purified and eluted with methanol water with a volume concentration of 83%, a flow rate of 3 mL / min, and dual-channel wavelengths: 210 nm and 254 nm. The components with a retention time of T=20 min were collected to obtain the compound Bupleurum pentacyclic triterpene A.

3. The method for preparing the oleanane-type compound pentacyclic triterpene A according to claim 2, characterized in that: 20 kg of Bupleurum chinense with residual stems above ground were cut into sections, and extracted with 70% ethanol by volume for three times, each time for 2 hours, with the amount of ethanol being 10, 8, and 6 times the weight volume of Bupleurum chinense, respectively. The weight volume refers to the solid in kg and the liquid in L. The extract was centrifuged at 12000rpm for 10 min, and concentrated under reduced pressure to 50L without alcohol taste. The extract was repeatedly extracted with equal volumes of petroleum ether, ethyl acetate, and n-butanol in sequence until the upper phase solution no longer became lighter. After the extracts were concentrated under reduced pressure, they were transferred to an evaporating dish and dried to obtain petroleum ether extract, ethyl acetate extract, and n-butanol extract, respectively. 266.57 g of ethyl acetate extract was purified by macroporous resin DM-130. The resin was activated in 95% ethanol by volume for 24 h before use, impurities were washed away, and it was suspended in 95% ethanol. The column was wet loaded, 1BV=20L, and then washed with 95% ethanol. When the washed ethanol was mixed with an equal amount of water and did not become turbid, it was gradually transferred from 95% ethanol to 10% ethanol by volume concentration. The ethyl acetate extract was dissolved in 10% ethanol and slowly added to the column bed. The opening at the lower end of the column was closed or reduced to allow the sample to slowly adsorb on the macroporous resin. Different ethanol solutions with different volume concentrations of 10%, 30%, 50%, 70%, and 95% were used for gradient elution. Each gradient was washed with 4 column volumes. Each gradient eluate was collected and concentrated under reduced pressure to obtain five components Fr1-Fr5. Component Fr5 49.81 g was purified by silica gel column chromatography: wet column loading, dry loading, eluted with a dual system of petroleum ether-ethyl acetate in a volume ratio of 10:1 and dichloromethane-methanol in a volume ratio of 1-100:1, 1BV=1500mL, 1 / 3 column volume was collected once, the plate was inspected under ultraviolet light, and the components of dichloromethane-methanol 60:1 and 40:1 were combined to obtain component Fr5-3; Fraction Fr5-3 11.046 g was purified by silica gel column chromatography: wet column loading, dry loading, elution with a dichloromethane-methanol system with a volume ratio of 40-100:1, 1BV=450mL, 1 / 3 column volume was collected once, the plate was inspected under ultraviolet light, and the components of dichloromethane-methanol 100:1 were combined to obtain component Fr5-3-2; Fraction Fr5-3-2 2.68g Silica gel column chromatography: wet packing, dry loading, elution with dichloromethane-methanol system with a volume ratio of 30-100:1, 1BV=314mL, 1 / 3 column volume collected once, spot plate inspected under ultraviolet light, and combined dichloromethane-methanol 60:1, 40:1 components to obtain component Fr5-3-2-2; The fraction Fr5-3-2-2 (563.4 mg) was semi-preparatively purified and eluted with 83% volume concentration of methanol water at a flow rate of 3 mL / min. The dual-channel wavelengths were 210 nm and 254 nm. The fraction with a retention time of T = 20 min was collected to obtain 5 mg of the compound Bupleurum chinense pentacyclic triterpene A.

4. Use of the oleanane-type compound pentacyclic triterpene A according to claim 1 in the preparation of drugs for treating allergic diseases.

5. The use of the oleanane-type compound pentacyclic triterpene A in the preparation of therapeutic drugs according to claim 4, characterized in that: The allergic disease is urticaria, atopic dermatitis, allergic conjunctivitis, allergic rhinitis or allergic asthma.

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