CN115141245A - Cucurbitane tetracyclic triterpenoid extracted from Chinese hemsleya amabilis and having anti-mastitis activity, and preparation method and application thereof - Google Patents
Cucurbitane tetracyclic triterpenoid extracted from Chinese hemsleya amabilis and having anti-mastitis activity, and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
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Abstract
The invention relates to a cucurbitane tetracyclic triterpenoid compound with anti-mastitis activity extracted from Chinese snow gall, a preparation method and application thereof, and can effectively prepare a new cucurbitane tetracyclic triterpenoid compound from the Chinese snow gall, and realize the application problem in preparation of medicaments for treating mastitis.
Description
Technical Field
The invention relates to the field of medicines, in particular to a cucurbitane tetracyclic triterpene compound with anti-mastitis activity extracted from Chinese snowflake, a preparation method and application thereof.
Background
The Hemsleya chinensis is a tuber of Hemsleya chinensis (Hemsleya chinensis) of Hemsleya of Cucurbitaceae, and is currently collected in quality standards of Chinese medicinal materials of Guizhou and Hubei. Is bitter in taste and cold in nature, has the effects of clearing away heat and toxic materials, relieving pain, stopping bleeding, eliminating swelling, and promoting diuresis, and can be used for treating bacillary dysentery, hepatitis, enteritis, etc. Chinese hemsleya amabilis is widely distributed in the mountainous areas in the southwest of China, such as Yunnan province, sichuan province, guizhou province and the like, and has large yield and low price. For a long time, chinese hemsleya amabilis is used as a common folk medicine in southwest areas and has obvious curative effect on mastitis of livestock such as cattle and sheep. The hemsleyadin tablet is developed and marketed by taking extract of Hemsleya plant such as Hemsleya chinensis as a raw material, is used for treating bacillary dysentery, enteritis, bronchitis, acute tonsillitis and the like, and comprises cucurbitane tetracyclic triterpenes such as hemsleyadin A and hemsleyadin B as main active ingredients. However, there has been no report on how to extract a cucurbitane-type tetracyclic triterpene component having an anti-mastitis activity from the Chinese snowflake gallbladder (hemchinin E).
Disclosure of Invention
In view of the above situation, in order to solve the defects of the prior art, the invention aims to provide a preparation method and application of a cucurbitane tetracyclic triterpene compound extracted from a Chinese snowflake liner, which can effectively solve the application problem of extracting the cucurbitane tetracyclic triterpene compound from the Chinese snowflake liner in preparing a medicament for treating mastitis.
The technical scheme for solving the problem is that the cucurbitane tetracyclic triterpenoid hemichinin E extracted from the Hemsleya amabilis has a molecular formula of C 30 H 44 O 6 The unsaturation degrees are all 9, and the structural formula is as follows:
the preparation method comprises the following steps:
(1) Taking 30kg of dried hemsleya amabilis root tuber, adding 150-300L of water each time, extracting for 2-5 times by refluxing, extracting for 2-4 h each time, combining the extracting solutions, and concentrating under reduced pressure to obtain a sample solution with the amount of crude drugs being 1.0-3.0 g/mL;
(2) And (2) loading the sample solution obtained in the step (1) onto a D101 macroporous adsorption resin column with the diameter-height ratio of 1-1. 70％EtOAc ；
(3) And (5) obtaining an extract Fr. 70％EtOAc Performing silica gel column chromatography of 80-300 meshes, performing gradient elution by using a silica gel column d = 6-12 cm and H = 25-60 cm by using dichloromethane-methanol in a volume ratio of 1. 70％EtOAc -3(15.9～23.7g)；
(4) Component Fr. 70％EtOAc -3, performing medium-pressure FlashODS column chromatography (220-330 g), performing gradient elution with methanol-water in a volume ratio of 45. 70％EtOAc -3-G(1.2～2.3g)；
(5) And the component Fr. 70％EtOAc -3-G was dissolved in methanol to a sample solution of 50mg/mL and further separated on semi-preparative HPLC using a C18 column eluting with a solvent of 35% by volume in acetonitrile-water, 3mL/min,the chromatographic peak was collected for 42.80min, and the solvent was recovered under reduced pressure to obtain a dry powdery monomer compound (135.7 to 185.4 mg).
The invention relates to application of cucurbitane tetracyclic triterpenoids extracted from Chinese snowflake gall in preparing medicaments for treating mastitis.
The invention is identified to be a new cucurbitane tetracyclic triterpenoid compound hemchinin E extracted from the Chinese hemsleya amabilis, the preparation method is easy to operate, the guidance quality is strong, the product purity is high, the compound can be effectively used for preparing the medicine for treating mastitis, the medicinal value and the commercial value of the Chinese hemsleya amabilis are exploited, and the economic benefit and the social benefit are huge.
Drawings
FIG. 1 is a chemical structural diagram of hemchinin E of the present invention.
FIG. 2 shows the key of hemchinin E of the present invention 1 H- 1 H COSY and HMBC correlation diagram.
FIG. 3 is a key NOESY correlation diagram of hemchinin E of the present invention.
FIG. 4 shows experimental and calculated ECD spectra of hemchinin E of the present invention.
FIG. 5 is a process flow diagram of the present invention.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples.
Example 1
In specific implementation, the preparation method of the invention is shown in fig. 5, and comprises the following steps:
(1) Taking 30kg of dried hemsleya amabilis root tuber, adding 250L of water each time, extracting under reflux for 3 times, extracting for 3h each time, mixing the extracting solutions, and concentrating under reduced pressure to obtain a sample solution with the amount of crude drug of 1.5 g/mL;
(2) And (2) loading the sample solution obtained in the step (1) onto a D101 macroporous adsorption resin column with the diameter-height ratio of 1:8, performing gradient elution by using 40L of ethanol with the volume concentration of 30%, 50% and 70% in sequence at the flow rate of 4mL/min, collecting 70% ethanol part eluent, and performing reduced pressure concentration to obtain an extract Fr with the relative density of 1.3 at 50 ℃. 70％EtOAc ；
(3) Extract of Chinese medicinal materialsFr. 70％EtOAc After 200 mesh silica gel column chromatography, silica gel column d =8cm, H =45cm, gradient elution was performed with dichloromethane-methanol at volume ratio 1. 70％EtOAc -3(18.5g)；
(4) Component Fr. 70％EtOAc -3, by medium pressure FlashODS column chromatography (220 g), gradient elution with methanol-water in volume ratios of 45, 55, 60, 40, 70, flow rate of 20mL/min, elution of 10 column volumes per ratio, collection of 60. 70％EtOAc -3-G(1.7g)；
(5) Component Fr. 70％EtOAc -3-G was dissolved in methanol to give a sample solution of 50mg/mL, further separated on semi-preparative HPLC using a C18 column eluting with a solvent of acetonitrile-water at a volume ratio of 35:65, 3mL/min, collecting the chromatographic peak at 42.80min, and recovering the solvent under reduced pressure to give the monomeric compound (152.3 mg) as a dry powder.
Example 2
In specific implementation, the preparation method of the invention is shown in fig. 5, and comprises the following steps:
(1) Taking 30kg of dried root tuber of Hemsleya chinensis, adding 150L of water each time, reflux-extracting for 5 times, extracting for 2h each time, mixing the extractive solutions, and concentrating under reduced pressure to obtain sample solution with amount of crude drug of 3.0 g/mL;
(2) And (2) loading the sample solution obtained in the step (1) onto a D101 macroporous adsorption resin column with the diameter-height ratio of 1:12, performing gradient elution by sequentially using 60L of ethanol with the volume concentration of 30%, 50% and 70% at the flow rate of 6mL/min, collecting the eluent of the 70% ethanol part, and performing reduced pressure concentration to obtain an extract Fr with the relative density of 1.2 at 50 ℃. 70％EtOAc ；
(3) And (5) obtaining an extract Fr. 70％EtOAc After 80-mesh silica gel column chromatography, silica gel column d =10cm, H =50cm, and gradient elution is performed with dichloromethane-methanol at a volume ratio of 1. 70％EtOAc -3(23.7g)；
(4) Component Fr. 70％EtOAc -3, by medium pressure FlashODS column chromatography (330 g), gradient elution with methanol-water in volume ratios of 45, 55, 60, 40, 70, flow rate 25mL/min, elution of 5 column volumes per ratio, collection of 60. 70％EtOAc -3-G(2.3g)；
(5) Component Fr. 70％EtOAc -3-G was dissolved in methanol to give a sample solution of 50mg/mL, further separated on semi-preparative HPLC using a C18 column eluting with a solvent of acetonitrile-water at a volume ratio of 35:65, 3mL/min, collecting the chromatographic peak at 42.80min, and recovering the solvent under reduced pressure to give the monomeric compound (185.4 mg) as a dry powder.
Example 3
In specific implementation, the preparation method of the invention is shown in fig. 5, and comprises the following steps:
(1) Taking 30kg of dried root tuber of Hemsleya chinensis, adding 300L of water each time, reflux-extracting for 2 times, extracting for 4h each time, mixing the extractive solutions, and concentrating under reduced pressure to obtain sample solution with amount of crude drug of 1.0 g/mL;
(2) And (2) loading the sample solution obtained in the step (1) onto a D101 macroporous adsorption resin column with the diameter-height ratio of 1:4, performing gradient elution by using 30L of ethanol with the volume concentration of 30%, 50% and 70% in sequence at the flow rate of 2mL/min, collecting 70% ethanol part eluent, and performing reduced pressure concentration to obtain an extract Fr with the relative density of 1.4 at 50 ℃. 70％EtOAc ；
(3) And (5) obtaining an extract Fr. 70％EtOAc After 300-mesh silica gel column chromatography, silica gel column d =6cm, H =25cm, and gradient elution is performed with dichloromethane-methanol at a volume ratio of 1. 70％EtOAc -3(15.9g)；
(4) Component Fr. 70％EtOAc -3 by medium pressure FlashODS column chromatography (220 g), gradient elution with methanol-water in volume ratio 45, 55, 60, 70, flow rate 20mL/min, elution of 6 column volumes per ratio, collection of 60. 70％EtOAc -3-G(1.2g)；
(5) Component Fr. 70％EtOAc -3-G was dissolved in methanol to give a sample solution of 50mg/mL, which was further separated by semi-preparative HPLC using a C18 column eluting with 35% by volume acetonitrile-water (3 mL/min) of a solvent, collecting the chromatographic peak at 42.80min, and recovering the solvent under reduced pressure to obtain a monomeric compound (135.7 mg) in the form of a dry powder.
Example 4
In specific implementation, the preparation method of the invention is shown in fig. 5, and comprises the following steps:
(1) Taking 30kg of dried root tuber of Hemsleya chinensis, adding 200L of water each time, reflux-extracting for 4 times, extracting for 2h each time, mixing the extractive solutions, and concentrating under reduced pressure to obtain sample solution with crude drug amount of 2.0 g/mL;
(2) And (2) loading the sample solution obtained in the step (1) onto a D101 macroporous adsorption resin column with the diameter-height ratio of 1:6, performing gradient elution by sequentially using 50L of ethanol with the volume concentration of 30%, 50% and 70% at the flow rate of 4mL/min, collecting the eluent of the 70% ethanol part, and performing reduced pressure concentration to obtain an extract Fr with the relative density of 1.2 at 50 ℃. 70％EtOAc ；
(3) And (5) obtaining an extract Fr. 70％EtOAc After 200-mesh silica gel column chromatography, silica gel column d =12cm, H =25cm, and gradient elution is performed with dichloromethane-methanol at a volume ratio of 1. 70％EtOAc -3(20.9g)；
(4) And the component Fr. 70％EtOAc -3 through medium pressure FlashODS column chromatography (330 g), gradient elution with methanol-water in volume ratio 45, 55, 60, 70, flow rate 25mL/min, eluting 3 column volumes per ratio, collecting eluent of 60. 70％EtOAc -3-G(2.0g)；
(5) Component Fr. 70％EtOAc -3-G was dissolved in methanol to give a sample solution of 50mg/mL, which was further separated by semi-preparative HPLC using a C18 column eluting with 35% by volume acetonitrile-water (3 mL/min) of a solvent, collecting the chromatographic peak at 42.80min, and recovering the solvent under reduced pressure to obtain a monomeric compound (168.6 mg) in the form of a dry powder.
It is to be noted that the above examples are only intended to illustrate specific embodiments of the present invention, and the detailed description of the cucurbitane tetracyclic triterpenoid compound with anti-mastitis activity and the extraction method thereof from the cucurbita maxima is illustrative and not intended to limit the scope of the present invention, and all changes and modifications that do not depart from the general concept of the present invention shall fall within the scope of the present invention, and the experimental results show that the relevant experimental data are as follows:
1. structural identification
The compound prepared by the invention is identified as a novel cucurbitane tetracyclic triterpenoid hemchinin E extracted from Hemsleya chinensis by determination, and the molecular structural formula is shown in figure 1.
Hemchinin E: white amorphous powder, which is easily soluble in organic solvents such as methanol. Optical rotation
(c 0.1,CH 3 OH); the ultraviolet spectrum shows a maximum absorption peak at 203 nm; infrared spectroscopy indicated the presence of a hydroxyl group (3469 cm) in the compound -1 ) Carbonyl group (1695 cm) -1 ) (ii) a HR-ESI-MS spectrum showing excimer ion peak m/z 501.32132[ m ] +H] + (C 30 H 45 O 6 Calculated 501.32107), binding 1 H-NMR、 13 C-NMR spectrum to confirm that the molecular formula is C 30 H 44 O 6 The unsaturation number was 9.
Three ketocarbonyl delta s are shown by 1D NMR and HSQC spectra C 214.7 (C-22), 212.7 (C-11), 209.9 (C-2); two groups of double bonds (one group of terminal double bonds) delta H 5.85(d,J＝6.2Hz,H-6),4.68(s,H-26),4.65(s,H-26)；δ C 144.9 (C-25), 138.8 (C-5), 120.2 (C-6), 109.9 (C-26); one quaternary carbon with oxygen delta C 79.2 (C-20); two vicinal oxymethylene delta H 3.83(s,H-3),4.34(t,J＝8.0Hz,H-16)；δ C 79.5 (C-3), 69.1 (C-16); seven methyl groups delta H 1.68(s,H-27),1.23(s,H-21),1.19(s,H-28),1.17(s,H-30),1.01(s,H-19),0.75(s,H-29),0.79(s,H-18)；δ C 24.1 (C-28), 25.0 (C-21), 21.3 (C-29), 22.6 (C-27), 18.3 (C-30), 19.6 (C-19), 19.8 (C-18), in detailSee table 1 for information. The data above contain 5 unsaturations, suggesting that the compound is a tetracyclic triterpenoid.
Bonding of 1 H- 1 The H COSY spectrum shows that there are four spin-coupled fragments H-10/H 2 -1、H-6/H 2 -7/H-8、H 2 -15/H-16/H-17 and H 2 -23/H 2 -24.HMBC spectra (see FIG. 2) shows H 3 18 is related to C-12, C-13, C-14, C-17, H 3 -19 is related to C-8, C-9, C-10, C-11, H 3 -21 is related to C-17, C-20, C-22, H 3 -28/29 is related to C-3, C-4, C-5, H 3 -27 is related to C-24, C-25, C-26, presumably the compound is cucurbitane tetracyclic triterpenoid; olefinic hydrogen proton H-6 is related to C-4, C-5, C-7, C-8 and C-10, and terminal double bond hydrogen proton H 2 -26 is related to C-24, C-25, C-27, indicating the presence of two double bonds Δ 25,26 And Δ 5,6 ；H 2 -1, H-3, H-10 are respectively related to C-2 ketocarbonyl, H-8, H-10, H 3 -19、H 2 -12 is independently related to the C-11 ketocarbonyl group, H-17, H 3 -21、H 2 -23、H 2 -24 is respectively related to the C-22 ketocarbonyl group, indicating that the substitution positions of 3 ketocarbonyl groups are respectively at the C-2, C-11 and C-22 positions; two hydrogen-bonded protons H-3 are related to C-1, C-2, C-4, C-5, me-28 and Me-29, H-16 is related to C-14, C-15, C-13, C-17 and C-20, and the molecular formula of the compound shows that the positions C-3 and C-16 are respectively substituted by free hydroxyl. Combining the above information, the planar structure of the compound was determined.
The relative configuration of the compound was determined by NOESY analysis (see figure 3). NOESY spectra show H-3 and H 3 -28 correlation, H-10 and H 3 Correlation at-29, suggesting that H-3 and H-10 are in the opposite side, and are tentatively in the beta, alpha configuration, respectively. From H 3 10 is related to H-30 and H-30 is related to H-17, suggesting that both H-17 and H-30 are in the alpha configuration. From H-8 and H 3 -18、H 3 -19 correlation, H 3 -19 is related to H-1 beta, H 3 18 is related to H-16, suggesting that H-8, H-16, me-18 and Me-19 are all in the beta configuration. The ECD spectra of the compounds showed positive Cotton effects at 201nm and 300 nm. Through comparing the calculated and actually measured ECD spectra, the calculated curve is well matched with the experimental curveBut with slight peak shifts (see FIG. 4), the absolute configurations of C-3, C-8, C-9, C-10, C-13, C-14, C-16, C-17 of the compound were determined to be S, S, R, R, R, S, R, R, respectively. The compound is determined to be 2 beta, 3 alpha, 16 alpha, 20-tetrahydroxyucurbita-5, 25-dien-2,11,22-trione, and the molecular structural formula is as follows:
TABLE 1 Hemchinin E 1 H and 13 C-NMR data (500 and 125MHz in MeOD, delta in ppm, J in Hz)
2. In vivo Activity assay
A medicine for treating mastitis is prepared by taking the compound hemchinin E as a main component, and an in vivo activity experiment is carried out.
The test method comprises the following steps: kunming mice 6-8 weeks old, after being adaptively fed for one week, are female: male ratio was 3:1, breeding in a cage, and giving sufficient diet and drinking water every day, wherein the female mouse after delivery is used for constructing an animal model of staphylococcus aureus mastitis. 30 female mice bearing 5-7 d births were randomly divided into 5 groups: blank control group, model group, positive control group (antibiotic group), high dose administration group (150 mg/Kg), low dose administration group (75 mg/Kg). Except for the blank control group, the other groups were subjected to the construction of a staphylococcus aureus mouse mastitis model. Injecting 0.05 mL/mouse of anesthetic Shutai into abdominal cavity of a mother mouse which is born for 5-7 days, after anesthesia, stretching limbs of the mouse to lie supinely and fixing, wiping the periphery of the 3 rd and 4 th pairs of mammary glands of the mouse by 70% ethanol, fixing the nipples by holding a sterilized forceps with the left hand, holding a microinjector with the right hand, injecting bacterial suspension into the basal part of the mammary glands from the 4 th pair of mammary glands of the mouse, wherein the concentration of staphylococcus aureus liquid is 2 multiplied by 10 8 CFU/. Mu.L. Model setThe positive control group and each administration group are respectively infused with stomach physiological saline, ciprofloxacin (130 mg/Kg) and medicines prepared by high and low doses (150 and 75 mg/Kg), the medicines are continuously administered for 7 days 2 times a day and 0.3 mL/time, and the blank control group is not treated. At the time of 3,5,7d, the mental state, diet and water intake of each group of mice were observed. After the end of the 7d dosing period, fasting was performed for 16h and the mice were sacrificed by decapitation. Taking mammary gland tissue, adding sterilized normal saline according to the mass volume ratio of 1.
The prepared medicine for treating mastitis has obvious mastitis resistance in high and low dosage groups. In the administration process, the mice in the blank control group eat and drink water normally and have good spirit; the mice in the model group are listless, the exercise, the food intake and the drinking water are reduced, and mammary swelling and ulceration are caused; mice in each administration group and the positive control group gradually recover mental state, drinking water and ingestion, and the mammary swelling degree is less changed and the festering degree is less compared with that in the model group. After 7d of administration, the number of staphylococcus aureus (4.87 +/-0.04) lg CFU/g in mammary gland tissues of model mice is remarkably increased compared with that of a blank control group; compared with the model group, the quantity of staphylococcus aureus (3.77 +/-0.05), (3.55 +/-0.03) and (4.02 +/-0.06) lg CFU/g in mammary gland tissues of mice in the positive control group and the high and low dose administration group show a remarkable reduction trend. Therefore, the prepared medicament for treating mastitis can obviously inhibit the mastitis of mice caused by staphylococcus aureus under the dosage of 150mg/kg and 75mg/kg, and has good mastitis treatment activity.
The invention is identified to be a new cucurbitane tetracyclic triterpenoid hemchinin E extracted from the Chinese snowflake, the preparation method is easy to operate, the guidance is strong, the product purity is high, the cucurbitane tetracyclic triterpenoid hemchinin E is used as a lead compound for treating mastitis, is effectively used for preparing the medicine for treating mastitis, has huge development prospect, and has huge economic and social benefits.
Claims (6)
1. A Chinese medicinal composition for treating cholelithiasisTaking cucurbitane tetracyclic triterpenoid compounds, wherein the molecular formula of the compounds is C 30 H 44 O 6 All the unsaturation degrees are 9, and the structural formula is as follows:
2. the method for extracting cucurbitane tetracyclic triterpenoids from Chinese snowflake, according to claim 1, is characterized by comprising the following steps:
(1) Taking 30kg of dried hemsleya amabilis root tuber, adding 150-300L of water each time, extracting for 2-5 times by refluxing, extracting for 2-4 h each time, combining the extracting solutions, and concentrating under reduced pressure to obtain a sample solution with the amount of crude drugs being 1.0-3.0 g/mL;
(2) And (2) loading the sample solution obtained in the step (1) onto a D101 macroporous adsorption resin column with the diameter-height ratio of 1-1. 70％EtOAc ；
(3) And (5) obtaining an extract Fr. 70％EtOAc Performing silica gel column chromatography of 80-300 meshes, performing gradient elution by using a silica gel column d = 6-12 cm and H = 25-60 cm by using dichloromethane-methanol in a volume ratio of 1. 70％EtOAc -3；
(4) And the component Fr. 70％EtOAc -3, performing medium-pressure FlashODS column chromatography, performing gradient elution with methanol-water in volume ratios of 45, 55, 60, and 70, eluting 3 to 10 column volumes per ratio at a flow rate of 20 to 25mL/min, collecting eluent of 60. 70％EtOAc -3-G；
(5) Component Fr. 70％EtOAc -3-G was dissolved in methanol to a sample solution of 50mg/mL and further separated on semi-preparative HPLC using a C18 column eluting with a solvent of 35% by volumeAcetonitrile-water, 3mL/min, collecting 42.80min chromatographic peak, and recovering solvent under reduced pressure to obtain dry powdered monomer compound.
3. The method for extracting cucurbitane tetracyclic triterpenoids from Chinese Symphytum officinale according to claim 2, which comprises the following steps:
(1) Taking 30kg of dried hemsleya amabilis root tuber, adding 250L of water each time, extracting under reflux for 3 times, extracting for 3h each time, mixing the extracting solutions, and concentrating under reduced pressure to obtain a sample solution with the amount of crude drug of 1.5 g/mL;
(2) And (2) loading the sample solution obtained in the step (1) onto a D101 macroporous adsorption resin column with the diameter-height ratio of 1:8, performing gradient elution by using 40L of ethanol with the volume concentration of 30%, 50% and 70% in sequence at the flow rate of 4mL/min, collecting 70% ethanol part eluent, and performing reduced pressure concentration to obtain an extract Fr with the relative density of 1.3 at 50 ℃. 70％EtOAc ；
(3) And (5) obtaining an extract Fr. 70％EtOAc After 200-mesh silica gel column chromatography, silica gel column d =8cm, H =45cm, and gradient elution is performed with dichloromethane-methanol at a volume ratio of 1. 70％EtOAc -3；
(4) Component Fr. 70％EtOAc -3, by medium pressure FlashODS column chromatography, gradient elution with methanol-water in volume ratios of 45, 55, 60, 70, flow rate 20mL/min, 10 column volumes per ratio, collecting 60 eluates of 40, recovering solvent under reduced pressure to obtain dry powder component Fr. 70％EtOAc -3-G；
(5) Component Fr. 70％EtOAc Dissolving 3-G in methanol to obtain a sample solution of 50mg/mL, further separating on semi-preparative HPLC by using a C18 column, eluting with a solvent of acetonitrile-water with a volume ratio of 35:65 and 3mL/min, collecting a chromatographic peak of 42.80min, and recovering the solvent under reduced pressure to obtain a dry powdery monomeric compound.
4. The method for extracting cucurbitane tetracyclic triterpenoids from Chinese Symphytum officinale according to claim 2, which comprises the following steps:
(1) Taking 30kg of dried root tuber of Hemsleya chinensis, adding 150L of water each time, reflux-extracting for 5 times, extracting for 2h each time, mixing the extractive solutions, and concentrating under reduced pressure to obtain sample solution with amount of crude drug of 3.0 g/mL;
(2) And (2) loading the sample solution obtained in the step (1) onto a D101 macroporous adsorption resin column with the diameter-height ratio of 1:12, performing gradient elution by using 60L of ethanol with the volume concentration of 30%, 50% and 70% in sequence at the flow rate of 6mL/min, collecting 70% ethanol part eluent, and performing reduced pressure concentration to obtain an extract Fr with the relative density of 1.2 at 50 ℃. 70％EtOAc ；
(3) And (5) obtaining an extract Fr. 70％EtOAc After 80-mesh silica gel column chromatography, silica gel column d =10cm, H =50cm, and gradient elution is performed with dichloromethane-methanol at a volume ratio of 1. 70％EtOAc -3；
(4) Component Fr. 70％EtOAc -3, by medium pressure FlashODS column chromatography, gradient elution with methanol-water in volume ratios of 45, 55, 60, 70, flow rate 25mL/min, 5 column volumes per ratio, collecting 60 eluates of 40, recovering solvent under reduced pressure to obtain dry powder component Fr. 70％EtOAc -3-G；
(5) Component Fr. 70％EtOAc Dissolving the-3-G in methanol to obtain a sample solution of 50mg/mL, further separating by using a C18 column on semi-preparative HPLC, eluting with a solvent of acetonitrile-water with a volume ratio of 35.
5. The preparation method for extracting the cucurbitane tetracyclic triterpenoid from the Chinese snowflake liner according to claim 2, is characterized by comprising the following steps:
(1) Taking 30kg of dried root tuber of Hemsleya chinensis, adding 300L of water each time, reflux-extracting for 2 times, extracting for 4h each time, mixing the extractive solutions, and concentrating under reduced pressure to obtain sample solution with amount of crude drug of 1.0 g/mL;
(2) Mixing the sample of step (1)Loading the solution on a D101 macroporous adsorption resin column with the diameter-height ratio of 1:4, performing gradient elution by using 30L of ethanol with the volume concentration of 30%, 50% and 70% in sequence at the flow rate of 2mL/min, collecting 70% ethanol part eluent, and performing reduced pressure concentration to obtain an extract Fr with the relative density of 1.4 at 50 ℃. 70％EtOAc ；
(3) And (5) obtaining an extract Fr. 70％EtOAc After 300-mesh silica gel column chromatography, silica gel column d =6cm, H =25cm, and gradient elution is performed with dichloromethane-methanol at a volume ratio of 1. 70％EtOAc -3；
(4) Component Fr. 70％EtOAc -3, by medium pressure FlashODS column chromatography, eluting with methanol-water in a volume ratio of 45, 55, 60, 70, in a gradient manner at a flow rate of 20mL/min for 6 column volumes per ratio, collecting the eluate of 60. 70％EtOAc -3-G；
(5) Component Fr. 70％EtOAc Dissolving the-3-G in methanol to obtain a sample solution of 50mg/mL, further separating by using a C18 column on semi-preparative HPLC, eluting with a solvent of acetonitrile-water with a volume ratio of 35.
6. The use of the cucurbitane-type tetracyclic triterpenoid extracted from the Chinese Symphytum officinale according to claim 1 in the preparation of a medicament for treating mastitis.
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