WO2019213807A1 - 一种波棱瓜子的木脂素有效成分组合物及其制备方法、应用和剂型 - Google Patents

一种波棱瓜子的木脂素有效成分组合物及其制备方法、应用和剂型 Download PDF

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WO2019213807A1
WO2019213807A1 PCT/CN2018/085862 CN2018085862W WO2019213807A1 WO 2019213807 A1 WO2019213807 A1 WO 2019213807A1 CN 2018085862 W CN2018085862 W CN 2018085862W WO 2019213807 A1 WO2019213807 A1 WO 2019213807A1
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blg
acetonitrile
preparation
content
fraction
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PCT/CN2018/085862
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English (en)
French (fr)
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袁海龙
申宝德
刘园
刘娟
邓莉
沈成英
宋青
吴燕
张娴勰
钱毓洲
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袁海龙
钱毓洲
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Priority to CN201880081675.XA priority Critical patent/CN112020355A/zh
Priority to PCT/CN2018/085862 priority patent/WO2019213807A1/zh
Publication of WO2019213807A1 publication Critical patent/WO2019213807A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems

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  • the invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a lignan active ingredient composition of a sea cucumber and a preparation method, application and dosage form thereof.
  • Hepatitis B is aggravated by hepatic fibrosis, hepatic ascites, hepatic palliative and liver cancer.
  • Liver fibrosis is the common pathological basis of various chronic liver diseases, and is an important stage in the process of cirrhosis. It is often caused by chronic inflammation, cholestasis, immune damage and other causes in the liver. There is data showing that the incidence of liver fibrosis in chronic hepatitis is 85.1%, and the progression of liver fibrosis will develop from 25% to 40% to cirrhosis. Studies have confirmed that liver fibrosis is a reversible disease, so it is meaningful to develop drugs that inhibit chronic liver fibrosis.
  • Pleurotus ostreatus has the functions of protecting liver and reducing enzyme, resisting hepatitis B and resisting liver fibrosis.
  • Substance basic research shows that the anti-hepatitis and anti-liver injury active ingredients are mainly concentrated in the ethyl acetate fraction, mainly lignans such as dehydrodiconiferylalcohol, herpetotriol I, herpetolide A, herpetrione, herpetin, and these components are resistant.
  • Hepatitis B and anti-fibrosis are significant.
  • a second object of the present invention is to disclose a process for preparing a lignan active ingredient composition of the above-mentioned sea cucumber.
  • a third object of the present invention is to disclose the use of the lignan active ingredient composition of the above-mentioned sea cucumber.
  • a fourth object of the present invention is to disclose a dosage form of the lignan active ingredient composition of the above-mentioned sea cucumber.
  • a lignan active ingredient composition of the sea cucumber wherein the lignan active ingredient composition of the sea cucumber comprises the following weight percentage components: dehydrodiconiferyl alcohol content is 0-10%, and herpetotriol I content is 0-10%, herpetolide A content is 0-10%, herpetrione content is 5-70%, herpetin content is 0-40%, herpetetrone content is 0-20%, herpetone content is 0-30%, herpetenone content is 0 -20%.
  • the lignan active ingredient composition of the sea cucumber according to the above aspect, wherein the lignan active ingredient composition of the sea cucumber has a dehydrodiconiferyl alcohol content of 4-7% and a herpetotriol I content of 3-6%.
  • Herpetolide A content is 2-5%, herpetrione content is 15-30%, herpetin content is 10-20%, herpetetrone content is 0-20%, herpetone content is 20-30%, and herpetenone content is 8-15%.
  • the components of the lignan active ingredient composition of the sea cucumber are prepared by the following steps:
  • the fraction is extracted by ethyl acetate.
  • the specific steps are as follows: the fraction is decanted and steamed at 10-15 ° C to recover acetonitrile, and the distilled liquid is added to ethyl acetate, and extracted three times. The extract is combined, and the acetic acid is recovered by vacuum distillation at 10-15 ° C. Ethyl ester, steamed to dryness, dissolved in methanol, transferred to a glass bottle and blown dry with nitrogen; wherein the fraction of ethyl acetate added during the extraction of the BLG-A to BLG-G fractions is the fraction volume 13% of the BLG-H segment was purified and purified to 10% of the fraction volume;
  • BLG-E, BLG-F and BLG-G have the same composition, and only the content of each component is different.
  • the secondary preparation method of BLG-F, the BLG-E part and the BLG-G part are respectively subjected to secondary treatment.
  • BLG-C 6 section contained BLG-3, and the same fractions were combined and treated according to the method of 2.3, wherein the BLG-C 6 fraction was 138.4 mg 74.0%;
  • the BLG-D part is the same as the BLG-C part sample dissolution and preparation method
  • BLG-12 A total of 2 needles were received, and one fraction was received: BLG-12, and the same fraction was combined to obtain one monomer BLG-12, ie, herpetenone.
  • the dosage form prepared by the lignan active ingredient composition of the squamous melon seed according to the above technical solution, wherein the lignan active ingredient composition of the porphyrin seed of the above technical solution and the pharmaceutically acceptable carrier;
  • the dosage form is a capsule, a tablet, a pill, a dropping pill, a granule, a soft capsule, a film, a plaster, a spray, an aerosol, a patch, a gel or an injection.
  • the pharmaceutically acceptable carrier is selected from the group consisting of starch, microcrystalline cellulose, lactose, sucrose, powdered sugar, dextrin, cellulose derivative, sodium carboxymethyl starch, low substitution Hydroxypropyl cellulose, stearic acid and its salts, talc, polyethylene glycol, beeswax, vegetable oil, animal glue, vegetable gum, carbomer resin, sodium carboxymethyl cellulose, polyvinyl alcohol, acrylate One or more of a polymer, polyacrylic acid, sodium polyacrylate, polyvinylpyrrolidone, glycerin, propylene glycol, sorbitol, triethylamine, azone, hydroxypropyl- ⁇ -cyclodextrin and Tween-80 The combination.
  • the invention adopts the liquid phase technology to separate and prepare eight lignan active ingredients in the squash seed, and can make the purity of each component more than 90%, and the prepared lignan lignin active ingredient composition and the dosage form thereof are prepared. Clear, quality control, to meet the medication requirements.
  • the granule lignan active ingredient composition prepared by the invention and the dosage form thereof can be used for the treatment of liver injury, hepatitis B and liver fibrosis, and has good therapeutic effect; the invention is tested by B in type B The therapeutic effects of hepatitis and liver fibrosis were initially evaluated.
  • Figure 1 is a representative chromatogram of the preparation of the extract of the seeds.
  • Figure 2 is a representative chromatogram of the secondary preparation of BLG-F fraction.
  • Figure 3 is a representative chromatogram of the second preparation of BLG-C fraction.
  • Figure 4 is a representative chromatogram of the second preparation of BLG-D fraction.
  • Figure 5 is a representative chromatogram of the second preparation of BLG-H fraction.
  • Figure 6 is a representative chromatogram of the preparation of the BLG-C 6 fraction.
  • Figure 7 is a representative chromatogram of the BLG-H 3 fraction obtained in three preparations.
  • Figure 8 is a representative chromatogram of the BLG-H 4 partial fraction prepared in triplicate.
  • Figure 9 is a representative chromatogram of the BLG-H 5 partial fraction prepared in triplicate.
  • the lignan active ingredient composition of the sea cucumber seed of the present invention a preparation method and application thereof will be further described below in conjunction with specific test examples.
  • Agilent 1100 High Performance Liquid Chromatograph (Agilent, USA); High Pressure Preparation Machine (Tianjin Bona Aijieer Technology Co., Ltd.); EXRE-20L Constant Temperature Water Oil Bath (Shanghai Zhenjie Experimental Equipment Co., Ltd.); RE-2000A Rotary evaporator (Shanghai Yarong Biochemical Instrument Factory); SHB-B95A circulating water multi-purpose vacuum pump (Shanghai Yukang Science and Education Equipment Co., Ltd.); SHB-III circulating water multi-purpose vacuum pump (Shanghai Zhenjie Experimental Equipment Co., Ltd.) ; nitrogen blower (Tianjin Bona Aijieer Technology Co., Ltd.); FD8-8 freeze dryer; FA1004 electronic balance (Shanghai Yueping Scientific Instrument Co., Ltd.); EX125DZH electronic balance (Ohaus Instruments (Changzhou) Co., Ltd.) .
  • the fraction is extracted by ethyl acetate.
  • the specific steps are as follows: the fraction is decanted and steamed at 10-15 ° C to recover acetonitrile, and the rotary liquid is added with a certain proportion of ethyl acetate (BLG-A to BLG-G fraction is prepared and purified to receive the fraction extracted.
  • the ratio of ethyl acetate added is 13% of the volume of the fraction, and the purified fraction of each of the fractions is 10% of the fractional volume.
  • the extract is combined, and the extract is combined, and the ethyl acetate is recovered by steaming at 10-15 ° C under reduced pressure. It was evaporated to dryness, dissolved in methanol, and transferred to a glass bottle and dried with nitrogen.
  • BLG-E, BLG-F and BLG-G have the same composition, and only the content of each component is different.
  • the secondary preparation method of BLG-F, the BLG-E part and the BLG-G part are respectively subjected to secondary treatment.
  • 200 mg of the treated powder was prepared in one portion, ultrasonically dissolved in 1 ml of methanol, and filtered.
  • a total of 3 needles were prepared.
  • the chromatogram of the preparation process is shown in Figure 3.
  • the 9 fractions were received.
  • 6 is BLG-C 6
  • the BLG-C 6 section contains BLG-3.
  • the same fractions are combined and processed according to the method of 3.3.
  • the fraction was worked up with a BLG-C 6 portion of 138.4 mg 74.0%.
  • the BLG-D fraction is the same as the BLG-C fraction sample dissolution and preparation method.
  • the chromatogram of the preparation process is shown in Figure 4. It receives 8 fractions.
  • 5 is BLG-D 5
  • BLG-D 5 is BLG-4, ie, heretotriol I.
  • the same fraction is combined and processed according to the method of 3.3.
  • the treatment, specific purity and quality are shown in Table 1.
  • the powder of the treated powder was twice prepared, dissolved in 1 ml of methanol, and filtered.
  • Example 2 Lignipin active ingredient composition of Poring melon seeds:
  • the lignan active ingredient composition of the squamous melon is obtained by weighing 65 mg of stellate and 35 mg of angolin.
  • Example 3 Lignipin active ingredient composition of Poring melon seeds:
  • Example 4 Lignipin active ingredient composition of Poring melon seeds:
  • Example 5 Lignipin active ingredient composition of Poring melon seeds:
  • Example 6 Lignipin active ingredient composition of Poring melon seeds:
  • Example 7 Lignipin active ingredient composition of Poring melon seeds:
  • the cell line HBV was transfected into the 2.2.15 cell line of human hepatoma cells (HepG2), constructed by the Mount Sinai Medical Center in the United States, and subcultured by the virus chamber of the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences.
  • HepG2 human hepatoma cells
  • the virus strain duck hepatitis B virus DNA (DHBV-DNA) strong positive serum, collected from Shanghai duck, stored at -70 °C.
  • Test drug (8 components in Table 2, prepared by "Example 1"); Lamivudine (3TC, produced by British Glaxo Wellcome Co., Ltd.) was provided by the Air Force General Hospital outpatient pharmacy; HBsAg, HBeAg enzyme
  • the immunoassay kit is provided by Huamei Biotech Co., Ltd.; ⁇ - 32 P-dCTP is provided by Beijing Yahui Biomedical Engineering Co., Ltd.; the random primer kit for probe labeling is provided by Promega; SePhadex G-50, Ficoll PVP by Swedish Pharmacia Provided by the company; nitrocellulose membrane (0.45 ⁇ m) supplied by Amersham; fetal bovine serum supplied by Hyclone Lab, USA; other reagents are commercially available analytical grades.
  • PHOMO microplate reader (Antu experimental instrument (Zhengzhou) Co., Ltd.); BH2 inverted microscope (Olympus optical instrument company); 800D carbon dioxide incubator (Thermo, USA); MSC-Advantag biosafety cabinet (Thermo, USA) L-550 low speed desktop large capacity centrifuge (Hunan Xiangyi Group); BSA124S electronic balance (Sedoli Scientific Instrument (Beijing) Co., Ltd.).
  • 2.2.15 cells in DMEM medium 0.03% glutamine, 380 ⁇ g/mL G-418, 100 IU/mL penicillin, 100 IU/mL streptomycin) containing 10% fetal bovine serum at 37 ° C, 5% CO 2 Incubate under conditions.
  • the F1-F6 lignan composition and the sea cucumber extract (T) ("2, preparation of the extract of the sea cucumber seed” in Example 1) were formulated into a solution having a concentration of 4000 ⁇ g/mL in DMEM medium. Then, the medium was diluted by a 2-fold gradient to 7 mass concentrations of 4000, 2000, 1000, 800, 400, 200, and 100 ⁇ g/mL for use.
  • the 96-well culture plate was inoculated with 2 ⁇ 10 4 cells/mL, 100 ⁇ L per well, cultured at 37° C. 5% CO 2 for 24 hours, and the supernatant was aspirated, and 100 ⁇ L of the culture solution containing different concentrations of the drug was added, respectively, at a concentration of 3 wells.
  • a drug-free cell control group and a blank well were set. After incubation at 37 ° C for 5% CO 2 for 72 h, 10 ⁇ L of MTT (5 mg/mL) was added to each well for 4 h, the culture was terminated, the supernatant was discarded, and 150 ⁇ L of DMSO was added and shaken for 10 min. The OD value at 570 nm was measured with a microplate reader. The inhibition rate of the drug to 2.2.15 cells was calculated, and the half toxic concentration (TC 50 ) and the maximum non-toxic concentration (TC 0 ) were calculated.
  • the degrees were 400, 200 , 100, 50, 25 ⁇ g / mL, 3 wells per concentration, 37 ° C 5% CO 2 culture, every 4 days for the same concentration of drug solution, set the drug-free cell control group.
  • the drug-containing culture solution on the 8th day was collected and stored frozen at -20 °C.
  • the supernatants were assayed for HBsAg and HBeAg by ELISA. According to the instructions of the kit, the OD value of each well was measured by a microplate reader, and the inhibition rate was calculated.
  • One-year-old Peking duck was injected with DHBV-DNA positive duck serum from Shanghai ducks, and 0.3 mL each, and blood was taken 7 days after infection. The serum was separated and the serum DHBV-DNA content was detected. After the duck serum was tested positive for DHBV, the ducks were randomly divided into 9 groups: virus control group, 3TC group (50mg/kg), F1-F6 lignan composition group and porphyrin extract group (T group). The doses of F1-F6 and T were 100 mg/kg. Administered by gavage twice a day. The virus control group was given the same volume of physiological saline for 10 days.
  • Blood was taken from the duck iliac vein before the administration (T0), 5 days after the drug treatment (T5), 10 days (T10) and 3 days after the withdrawal (P3), and the serum was separated and stored at -70 ° C for examination.
  • the DHBV-DNA probe was labeled with 32 P, and the duck serum spots were hybridized, and the auto-developed membrane spots were spotted.
  • the OD value (filter was 490 nm) was measured by a microplate reader to calculate the serum DHBV-DNA light. Density, using the hybrid spot OD value as the sample DHBV-DNA level value.
  • the mean values of serum DHBV-DNA before and after administration of the same group of ducks were compared, and the inhibition rate of serum DHBV-DNA at different times of each administration group was calculated.
  • T test was used to compare the means between the two groups.
  • One-way ANOVA was used to compare the mean between groups.
  • the statistical analysis was performed by SPSS 17.0 software. P ⁇ 0.05 was considered to be significant.
  • the cytotoxic effects of the F1-F6 lignan composition and the scutellaria extract (T) on 2.2.15 cells are shown in Table 3. It can be seen from Table 3 that the F1-F6 lignan composition and the scutellaria extract have a half-toxic concentration of 2.2.15 cells in the range of 924-1324 ⁇ g/mL, and the maximum non-toxic dose is greater than 400 ⁇ g/mL.
  • 3TC has only one dose group, 200 ⁇ g/mL.
  • 3TC has only one dose group, 200 ⁇ g/mL.
  • DHBV-DNA was positive in ducklings infected with hepatitis B virus. There was no significant change in serum DHBV-DNA levels in the virus control group during the experiment.
  • the positive drug 3TC significantly decreased the level of DHBV-DNA in serum on the 5th and 10th day after administration (P ⁇ 0.01), but the inhibition rate decreased to 24.98% after 3 days of drug withdrawal.
  • F1-F6 lignan composition and B. chinensis extract can significantly reduce the level of DHBV-DNA in serum on the 5th day, 10th day after drug administration and 3d stop. ⁇ 0.01), and the F1-F6 lignan composition was not significantly different from the sea cucumber extract (T group), and the results are shown in Table 6.
  • hepatitis B is caused by hepatitis B virus HBV, which is an infectious disease mainly composed of hepatic inflammatory lesions, which is easily transformed into chronic, and even develops into cirrhosis and liver cancer.
  • HBV hepatitis B virus
  • the continuous replication of hepatitis B virus in hepatocytes causes the host to produce an immune response, causing immunopathological damage to the cells, which is the pathological basis for chronic hepatitis. Therefore, antiviral treatment for chronic infection of hepatitis B virus is the main measure to block the development of chronic hepatitis B.
  • ALT Alanine aminotransferase
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase assay kits
  • ALT aspartate aminotransferase
  • ALP alkaline phosphatase assay kits
  • HA hydroxyproline
  • TGF- ⁇ 1 transforming growth factor- ⁇ 1
  • PHOMO microplate reader (Antu experimental instrument (Zhengzhou) Co., Ltd.); BH2 inverted microscope (Olympus optical instrument company); 800D carbon dioxide incubator (Thermo, USA); MSC-Advantag biosafety cabinet (Thermo, USA) L-550 low speed desktop large capacity centrifuge (Hunan Xiangyi Group); BSA124S electronic balance (Sedoli Scientific Instrument (Beijing) Co., Ltd.); automatic biochemical analyzer (Shenzhen Leidu Life Technology Co., Ltd.).
  • HSC-T6 cells were cultured in a DMEM medium containing 10% fetal calf serum under the conditions of 37 ° C and 5% CO 2 .
  • CCK8 method for cell proliferation The HSC-T6 cells in logarithmic growth phase were digested with trypsin, inhaled into a 15 mL plastic centrifuge tube, centrifuged at 1000 r/min for 5 min, blown into a single cell suspension, discarded, and gravity. Suspended cells. The cell concentration was adjusted to approximately 3 ⁇ 10 4 /mL, and the cell suspension was seeded in a 96-well cell culture plate at 100 ⁇ L/well, and the 96-well plate was incubated at 37 ° C in a 5% CO 2 incubator overnight.
  • the experiment was divided into a blank group (only the culture solution was added), a control group, a F1-F6 and a sea cucumber extract (T) ("2, preparation of the extract of the sea cucumber seed extract” in the example 1) (5, 10). , 25, 50, 100 ⁇ g / mL), the positive drug colchicine (0.1 ⁇ g / mL) group. After the cells were attached to the wall, the old solution was discarded, and the serum culture medium containing different drugs and DMEM were separately added to the group for culture, and each group had 6 duplicate wells. After 24 and 48 hours of drug treatment, 10 ⁇ L of CCK8 solution was added to each well, and the plate was incubated in an incubator for 2.5 hours.
  • the F1-F6 lignan composition of the striata has a significant inhibitory effect on the proliferation of HSC-T6 cells in the range of 10-100 ⁇ g/mL, and is concentration-dependent, and each composition and wave There was no significant difference in the inhibitory effect on the proliferation of HSC-T6 cells compared with the extract of squamosa seeds (T), indicating that each composition has the same anti-hepatic effect as the extract of Cyclobalanopsis ssp.
  • Rats were weighed and randomly divided into control group, model group, F1-F6 administration group (200 mg/kg, suspended in 0.5% CMC-Na solution), and colchicine group (positive control, 0.1 mg). /kg), 8 in each group.
  • Addition to the control group, other groups of rats first were injected subcutaneously (sc) 5mL / kg 40% CCl 4 solution soybean oil, 3mL / kg40% CCl 4 solution soybean sc 3d, 2 times a week (Wednesday, Friday ), while the control group sc the same dose of soybean oil solution for 6 weeks.
  • the control group and the model group were intragastrically administered (ig) the same dose of 0.5% CMC-Na solvent solution daily, the drug volume was 10 mL/kg, and the remaining drug-administered group was given the corresponding dose of the test solution every day.
  • the model was administered for a total of 12 weeks.
  • the rats were fasted for 24 hours. The rats were weighed and the blood was taken from the abdominal aorta. The supernatant was centrifuged and stored at -20 °C. Fresh liver was taken, weighed, and a small portion of 10% Fulma was cut. Immersed in the forest for inspection.
  • liver index liver mass (g) / body weight (g) x 100. The results are shown in Table 10.
  • the liver weight and liver index of the model control group were significantly increased (P ⁇ 0.05 or P ⁇ 0.01); compared with the model control group, the F1-F6 group and the extract group (T group)
  • the hepatic weight and liver index of the colchicine group were significantly decreased (P ⁇ 0.01 or P ⁇ 0.05), and there was no significant difference between the F1-F6 group and the extract group (T group).
  • the results showed that each administration group can significantly reduce liver swelling caused by liver fibrosis and reduce liver index.
  • liver function indexes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in rat serum were detected by automatic biochemical analyzer. The results are shown in Table 11.
  • the serum levels of ALT, AST, and ALP in the model group were significantly higher than those in the control group (P ⁇ 0.05 or P ⁇ 0.01).
  • the levels of ALT, AST, and ALP in the F1-F6 group, the extract group (T group), and the colchicine group were significantly lower (P ⁇ 0.05 or P ⁇ 0.01), and the F1-F6 group and T. There was no significant difference compared to the group.
  • HA hyaluronic acid
  • Hyp hydroxyproline
  • TGF- ⁇ 1 transforming growth factor- ⁇ 1
  • hepatic fibrosis is a common disease caused by chronic liver diseases.
  • the mechanism of HF is similar to that of other chronic disease injuries, and the occurrence of fibrosis is a dynamic reversible process.
  • HF hepatic fibrosis
  • Hepatic fibrosis is a compensatory response caused by inflammation of the liver or tissue repair after injury. It is characterized by massive deposition of extracellular matrix (ECM) in liver tissue.
  • ECM extracellular matrix
  • HSCs hepatic stellate cells
  • the activation and proliferation of HSC promotes liver fibrosis, inhibits its proliferation, induces apoptosis, and can alleviate or even reverse liver fibrosis.
  • This study found that different doses of P. squamosa extract and active ingredient composition can effectively inhibit the proliferation of HSC-T6 cells, and the dose is lazy. With the increase of drug concentration and the development of culture time, HSC-T6 The inhibition rate of cell proliferation was significantly increased; the results showed that the extract of Viburnum chinensis and its active ingredient composition had anti-fibrosis effect.
  • CCl 4 induced liver fibrosis model rats were used to evaluate the anti-hepatic fibrosis effect of the extract of the seeds and the active ingredient composition.
  • the results showed that the extract of squamosa seeds and the active ingredient composition can significantly reduce the levels of serum ALT, AST, ALP and HA, Hyp and TGF- ⁇ 1 in liver fibrosis rats, indicating that the extract of squamosa seeds and the composition of active ingredients against CCl 4 liver fibrosis rats have obvious protective effects on liver tissue and liver function damage, and have obvious anti-liver fibrosis effect.
  • the anti-hepatic fibrosis mechanism may be related to down-regulation of TGF- ⁇ 1 protein expression.

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Abstract

一种波棱瓜子的木脂素有效成分组合物及其制备方法、应用和剂型，属于中医药技术领域。其中波棱瓜子的木脂素有效成分组合物由下列重量百分比的成分组成：去氢双松柏醇含量为0-10%、波棱醇I含量为0-10%、波棱内酯A含量为0-10%、波棱甲素含量为5-70%、波棱素含量为0-40%、herpetetrone含量为0-20%、波棱酮含量为0-30%、波棱乙素含量为0-20%。具有以下优点：采用制备液相色谱技术分离制备波棱瓜子中8种木脂素有效成分，能够使各成分纯度达90%以上；组合物及其剂型可用于肝损伤、乙型肝炎及肝纤维化的治疗，具有较好的治疗效果。

Description

一种波棱瓜子的木脂素有效成分组合物及其制备方法、应用和剂型 技术领域
本发明属于中医药技术领域，具体涉及一种波棱瓜子的木脂素有效成分组合物及其制备方法、应用和剂型。
背景技术
我国一直是乙肝大国，多年来一直通过乙肝疫苗防止其蔓延，但部分地区普及程度较低，原有HBV携带人群基数大等原因，在未来的数十年内它仍是危害我国人民健康的疾病之一。在治疗方面，现有的干扰素、核苷类化学药物，由于存在肾毒性、病毒性变异等不良反应，前景不容乐观。加上慢性乙型肝炎病程长、不易控制、预后差、发病机理复杂等特点，所以其相关的治疗仍为当前肝病领域中的难题之一。
乙型肝炎病情加重则发展到肝纤维化，肝腹水，肝腹化和肝癌。肝纤维化是各种慢性肝病的共同病理基础，是肝硬化发生过程的重要阶段，常由肝内慢性炎症、胆汁淤积、免疫损伤等原因引起。有资料显示慢性肝炎肝纤维化的发病率为85.1％，肝纤维化持续发展将有25％-40％发展为肝硬化。研究证实肝纤维化是可逆性病变，所以研制抑制慢肝纤维化的药物很有意义。
藏医药在肝胆疾病等优势病种方面有独特疗效，且疗效稳定，毒副作用小，民间运用广泛。常用抗肝胆疾病药波棱瓜子(葫芦科植物波棱瓜Herpetospermum caudigerum Wall的干燥成熟种子)，收载于1995年版《中华人民共和国卫生部药品标准·藏药标准》，其味苦，性寒，能清腑热，胆热，具有清热解毒，去火降热，助消化的作用，用于治疗肝热、黄疸性传染型肝炎等症。现代药理研究表明波棱瓜子提取物具有保肝降酶、抗乙型肝炎及抗肝纤维化作用。物质基础研究表明，波棱瓜子抗肝炎，抗肝损伤的活性成分主要集中在乙酸乙酯部位，主要为木脂素类成分，如dehydrodiconiferylalcohol、herpetotriol I、herpetolideA、herpetrione、herpetin，且该类成分抗乙型肝炎及抗肝纤维化作用显著。
发明内容
本发明的目的在于公开了一种波棱瓜子的木脂素有效成分组合物。
本发明的第二个目的在于公开了上述波棱瓜子的木脂素有效成分组合物的制备方法。
本发明的第三个目的在于公开了上述波棱瓜子的木脂素有效成分组合物的应用。
本发明的第四个目的在于公开了上述波棱瓜子的木脂素有效成分组合物的剂型。
本发明的目的是通过以下技术方案实现的：
一种波棱瓜子的木脂素有效成分组合物，其中，所述波棱瓜子的木脂素有效成分组合物由下列重量百分数的成分组成：dehydrodiconiferyl alcohol含量为0-10％、herpetotriol I含量为0-10％、herpetolide A含量为0-10％、herpetrione含量为5-70％、herpetin含量为0-40％、herpetetrone含量为0-20％、herpetone含量为0-30％、herpetenone含量为0-20％。
上述技术方案所述的波棱瓜子的木脂素有效成分组合物，其中，波棱瓜子的木脂素有效成分组合物中，dehydrodiconiferyl alcohol含量为4-7％、herpetotriol I含量为3-6％、herpetolide A含量为2-5％、herpetrione含量为15-30％、herpetin含量为10-20％、herpetetrone含量为0-20％、herpetone含量为20-30％、herpetenone含量为8-15％。
上述技术方案所述波棱瓜子的木脂素有效成分组合物的制备方法，其中，称取上述技术方案所述重量百分数的各成分，混匀后即得波棱瓜子的木脂素有效成分组合物；
波棱瓜子的木脂素有效成分组合物中的各成分由下述步骤制备：
(1)、波棱瓜子提取物的制备：取波棱瓜子药材粗粉，加80％乙醇回流提取3次，每次溶剂量5倍，每次1小时，合并提取液，静置24小时后，滤过，滤液回收乙醇至无醇味，静置12小时，分去油层。水层以碳酸钠固体调pH值10-11使溶解，离心，上清液加浓盐酸调pH值2-3，静置1小时，离心，取沉淀，用石油醚反复洗至滤液无色，减压干燥，得波棱瓜子提取物粉末；
(2)、一次制备：
(2.1)、样品溶液的配制：
取波棱瓜子提取物600mg，加12ml甲醇超声溶解，再加入3ml蒸馏水混匀，过滤。
(2.2)、制备方法：
色谱柱：Innoval ODS-2 10μm 100A；上样量：600mg；流速：80mL/min；检测波长：280nm/254nm；流动相：水-乙腈，梯度洗脱：0～25min，21％～24％乙腈；25～50min，24％～25％乙腈；50～75min，25％乙腈；75min～90min，95％乙腈；90～100min，21％乙腈；
共进50针，得到共8个馏分：BLG-A、BLG-B、BLG-C、BLG-D、BLG-E、BLG-F、BLG-G、BLG-H，相同馏分合并处理；
(2.3)、馏分后处理：
馏分采用乙酸乙酯萃取的方法，具体步骤：馏分减压旋蒸10-15℃回收乙腈，旋蒸液 加入乙酸乙酯，萃取3次，合并萃取液，减压旋蒸10-15℃回收乙酸乙酯，旋蒸至干，再用甲醇溶解，转移至玻璃瓶中氮气吹干；其中BLG-A～BLG-G部分的制备纯化接收的馏分萃取时加入的乙酸乙酯的比例均为馏分体积的13％，BLG-H段制备纯化的均为馏分体积的10％；
一次制备馏分处理后得：BLG-A部分130mg，BLG-B部分210mg，BLG-C部分600mg，BLG-D部分600mg，BLG-E部分1.6g，BLG-F部分2.4g，BLG-G部分500mg；
(3)、二次制备：
(3.1)、BLG-E、BLG-F、BLG-G部分：
(3.1.1)、样品溶液的配制：
取一次制备处理后的BLG-F粉末130mg，加入1ml甲醇超声溶解，过滤。
(3.1.2)、BLG-F部分二次制备方法：
色谱柱：Innoval ODS-2 10μm 100A；上样量：130mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈为78％:22％，等度洗脱；60min进一针，收集上一针的馏分；
共进19针，分别接收BLG-5、BLG-6、BLG-7共3个馏分，相同馏分合并处理，得BLG-5即herpetolideA、BLG-6即herpetrione、BLG-7即herpetin共3个单体或组分；
BLG-E、BLG-F、BLG-G三部分馏分所含成分一致，仅各成分含量不同，按照BLG-F部分二次制备的方法分别对BLG-E部分和BLG-G部分分别进行二次制备并得到BLG-5即herpetolideA、BLG-6即herpetrione、BLG-7即herpetin共3个单体或组分；
(3.2)、BLG-C部分：
(3.2.1)、样品溶液的配制：
一次制备处理后的BLG-C粉末200mg，加入1ml甲醇超声溶解，过滤；
(3.2.2)、二次制备方法：
色谱柱:Innoval ODS-2 10μm 100A；上样量：200mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～30min，23％～28％乙腈；30～35min，28％乙腈；35～45min，100％乙腈；45～55min，23％乙腈；
共进3针，接收9个馏分，其中BLG-C 6段含BLG-3，相同馏分合并处理，按2.3中方法进行馏分后处理，其中BLG-C 6部分138.4mg 74.0％；
(3.3)、BLG-D部分：
BLG-D部分与BLG-C部分样品溶解及制备方法相同；
接收8个馏分，其中BLG-D 5段为BLG-4即herpetotriol I，相同馏分合并处理，按2.3中方法进行馏分后处理；
(3.4)、BLG-H段二次制备方法：
(3.4.1)、样品溶液的配制：
500mL乙酸乙酯层萃取液，旋蒸至60mL，超声混匀。
色谱柱:Innoval ODS-2 10μm 100A；上样体积：3mL；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈，梯度洗脱：0～80min，26％～41％乙腈；80～90min，100％乙腈；90～96min，26％乙腈；
共进7针，接收9个馏分：其中含BLG-10的BLG-H 3部分307.5mg 59.9％、含BLG-11的BLG-H 4部分1085.1mg 89.2％、含BLG-12的BLG-H 5部分993.2mg 76.1％；
(4)、三次制备：
(4.1)、BLG-C 6部分：
(4.1.1)、样品溶解：
二次制备处理后的BLG-C 6粉末138mg，加入1ml甲醇超声溶解，过滤；
(4.1.2)、制备方法：
色谱柱:Innoval ODS-2 10μm 100A；上样量：138mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～50min，20％乙腈；50～56min，100％乙腈，56～62min，20％乙腈；
共进1针，接收1个馏分：BLG-3，得1个单体BLG-3即dehydrodiconiferyl alcohol；
(4.2)、BLG-H 3部分：
(4.2.1)、样品溶解：
二次制备处理后的BLG-H 3粉末300mg，加入1ml甲醇超声溶解，过滤；
(4.2.2)、制备方法：
色谱柱:Innoval ODS-2 10μm 100A；上样量：100mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～62min，24％乙腈；62～92min，25％～26％乙腈，92～98min，100％乙腈，98～104min，24％乙腈；
共进1针，接收1个馏分：BLG-10，得1个单体BLG-10即herpetetrone；
(4.3)、BLG-H 4部分：
(4.3.1)、样品溶解：
二次制备处理后的BLG-H 4粉末220mg，加入1ml甲醇超声溶解，过滤；
(4.3.2)、制备方法：
色谱柱：Innoval ODS-2 10μm 100A；上样量：100mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～40min，30％～35％乙腈，40～46min，100％乙腈，46～52min，30％乙腈；
共进1针，接收1个馏分：BLG-11，得1个单体BLG-11即herpetone；
(4.4)、BLG-H 5部分：
(4.4.1)、样品溶解：
二次制备处理后的BLG-H 5粉末990mg，加入1.5ml甲醇超声溶解，过滤；
(4.4.2)、制备方法：
色谱柱：Innoval ODS-2 10μm 100A；上样量：100mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～40min，30％～35％乙腈，40～57min，35％～38％乙腈，57～63min，100％乙腈，63～69min，30％乙腈；
共进2针，接收1个馏分：BLG-12，相同馏分合并处理，得1个单体BLG-12即herpetenone。
上述技术方案所述的波棱瓜子的木脂素有效成分组合物制备的剂型，其中，它由上述技术方案的波棱瓜子的木脂素有效成分组合物和药学上可接受的载体组成；所述剂型为胶囊剂、片剂、丸剂、滴丸、颗粒剂、软胶囊、膜剂、膏剂、喷雾剂、气雾剂、贴剂、凝胶剂或注射剂。
上述技术方案所述的剂型，其中：所述药学上可接受的载体选自淀粉、微晶纤维素、乳糖、蔗糖、糖粉、糊精、纤维素衍生物、羧甲基淀粉钠、低取代羟丙基纤维素、硬脂酸及其盐、滑石粉、聚乙二醇、蜂蜡、植物油、动物胶、植物胶、卡波姆树脂、羧甲基纤维素钠、聚乙烯醇、丙烯酸酯类聚合物、聚丙烯酸、聚丙烯酸钠、聚乙烯吡咯烷酮、甘油、丙二醇、山梨醇、三乙胺、氮酮、羟丙基-β-环糊精和吐温-80中的一种或两种以上的组合。
上述技术方案所述的波棱瓜子的木脂素有效成分组合物在制备治疗乙肝药物中的应用。
本发明具有以下有益效果：
1、本发明采用制备液相技术分离制备波棱瓜子中8种木脂素有效成分，能够使各成分纯度达90％以上，所制备的波棱瓜子木脂素有效成分组合物及其剂型成分明确，质量可控，能够满足用药要求。
2.本发明制备的波棱瓜子木脂素有效成分组合物及其剂型可用于肝损伤、乙型肝炎及肝纤维化的治疗，具有较好的治疗效果；本发明通过实验对其在乙型肝炎及肝纤维化的治疗作用进行了初步评价。
附图说明：
1、图1为波棱瓜子提取物一次制备时代表性色谱图。
2、图2为BLG-F部分馏分二次制备时代表性色谱图。
3、图3为BLG-C部分馏分二次制备时代表性色谱图。
4、图4为BLG-D部分馏分二次制备时代表性色谱图。
5、图5为BLG-H部分馏分二次制备时代表性色谱图。
6、图6为BLG-C 6部分馏分三次制备时代表性色谱图。
7、图7为BLG-H 3部分馏分三次制备时代表性色谱图。
8、图8为BLG-H 4部分馏分三次制备时代表性色谱图。
9、图9为BLG-H 5部分馏分三次制备时代表性色谱图。
具体实施方式：
为使本发明的技术方案便于理解，以下结合具体试验例对本发明一种波棱瓜子的木脂素有效成分组合物及其制备方法和应用作进一步的说明。
实施例1：波棱瓜子木脂素化合物的制备：
1、仪器：
Agilent 1100高效液相色谱仪(美国Agilent公司)；高压制备一体机(天津博纳艾杰尔科技有限公司)；EXRE-20L恒温水油浴锅(上海振捷实验设备有限公司)；RE-2000A旋转蒸发器(上海亚荣生化仪器厂)；SHB-B95A型循环水式多用真空泵(上海豫康科教仪器设备有限公司)；SHB-Ⅲ循循环水式多用真空泵(上海振捷实验设备有限公司)；氮吹仪(天津博纳艾杰尔科技有限公司)；FD8-8冻干机；FA1004电子天平(上海越平科学仪器有限公司)；EX125DZH电子天平(奥豪斯仪器(常州)有限公司)。
2、波棱瓜子提取物的制备：
取波棱瓜子药材粗粉，加80％乙醇回流提取3次，每次溶剂量5倍，每次1小时， 合并提取液，静置24小时后，滤过，滤液回收乙醇至无醇味，静置12小时，分去油层。水层以碳酸钠固体调pH值10-11使溶解，离心，上清液加浓盐酸调pH值2-3，静置1小时，离心，取沉淀，用石油醚反复洗至滤液无色，减压干燥，得波棱瓜子提取物粉末。
3、一次制备：
3.1、样品溶液的配制：
取波棱瓜子提取物600mg，加12ml甲醇超声溶解，再加入3ml蒸馏水混匀，过滤。
3.2、制备方法：
色谱柱：Innoval ODS-2 10μm 100A；上样量：600mg；流速：80mL/min；检测波长：280nm/254nm；流动相：A(水)-B(乙腈)，梯度洗脱：(0～25min，21％B～24％B；25～50min，24％B～25％B；50～75min，25％B；75min～90min，95％B；90～100min，21％B)。
共进50针，接收图1中的8个馏分：BLG-A、BLG-B、BLG-C、BLG-D、BLG-E、BLG-F、BLG-G、BLG-H，相同馏分合并处理；图1中红线为280nm检测波长，蓝线为254nm检测波长(图2～图9中红线与蓝线与此相同)，由图1可知BLG-H是75min后用高比例乙腈冲下来的馏分，富集后萃取，浓缩，待制备。
3.3、馏分后处理：
馏分采用乙酸乙酯萃取的方法，具体步骤：馏分减压旋蒸10-15℃回收乙腈，旋蒸液加一定比例的乙酸乙酯(BLG-A～BLG-G部分的制备纯化接收的馏分萃取时加入的乙酸乙酯的比例均为馏分体积的13％，H段制备纯化的均为馏分体积的10％)萃取3次，合并萃取液，减压旋蒸10-15℃回收乙酸乙酯，旋蒸至干，再用甲醇溶解，转移至玻璃瓶中氮气吹干。
一次制备馏分处理后得：BLG-A部分130mg，BLG-B部分210mg，BLG-C部分600mg，BLG-D部分600mg，BLG-E部分1.6g，BLG-F部分2.4g，BLG-G部分500mg。
4、二次制备：
4.1、BLG-E、BLG-F、BLG-G部分：
4.1.1、样品溶液的配制：
取一次制备处理后的BLG-F粉末130mg，加入1ml甲醇超声溶解，过滤。
4.1.2、BLG-F部分二次制备方法：
色谱柱：Innoval ODS-2 10μm 100A；上样量：130mg；流速：80ml/min；检测波长：280nm/254nm；流动相：A(水)-B(乙腈)(78％：22％)等度洗脱。60min进一针，收集上 一针的馏分。
共进19针，制备过程色谱图如图2所示，接收3个馏分BLG-5(即herpetolideA)、BLG-6(即herpetrione)、BLG-7(即herpetin)，相同馏分合并处理，得3个单体或组分，具体纯度与质量等见表1，图2中1为BLG-5，2为BLG-6，3为BLG-7。
BLG-E、BLG-F、BLG-G三部分馏分所含成分一致，仅各成分含量不同，按照BLG-F部分二次制备的方法分别对BLG-E部分和BLG-G部分分别进行二次制备并得到BLG-5即herpetolideA、BLG-6即herpetrione、BLG-7即herpetin共3个单体或组分；
4.2、BLG-C部分：
4.2.1、样品溶液的配制：
一次制备处理后的粉末200mg，加入1ml甲醇超声溶解，过滤。
4.2.2、二次制备方法：
色谱柱:Innoval ODS-2 10μm 100A；上样量：200mg；流速：80ml/min；检测波长：280nm/254nm；流动相A(水)-B(乙腈)，梯度洗脱(0～30min，23％～28％B；30～35min，28％B；35～45min，100％B；45～55min，23％B)。
共进3针，制备过程色谱图如图3所示，接收9个馏分，图3中6为BLG-C 6，BLG-C 6段含有BLG-3，相同馏分合并处理，按3.3中方法进行馏分后处理，其中BLG-C 6部分138.4mg 74.0％。
4.3、BLG-D部分：
BLG-D部分与BLG-C部分样品溶解及制备方法相同。
制备过程色谱图如图4所示，接收8个馏分，图4中5为BLG-D 5，BLG-D 5段为BLG-4即herpetotriol I，相同馏分合并处理，按3.3中方法进行馏分后处理，具体纯度及质量等见表1。
4.4、BLG-H段二次制备方法：
4.4.1、样品溶液的配制：
500mL乙酸乙酯层萃取液，旋蒸至60mL，超声混匀。
色谱柱:Innoval ODS-2 10μm 100A；上样体积：3mL；流速：80ml/min；检测波长：280nm/254nm；流动相：A(水)-B(乙腈)梯度洗脱(0～80min，26％～41％B；80～90min，100％B；90～96min，26％B)。
共进7针，制备过程色谱图如图5所示，接收9个馏分：图5中3为BLG-H 3，4为 BLG-H 4，5为BLG-H 5；其中BLG-H 3(含BLG-10)部分307.5mg 59.9％、BLG-H 4(含BLG-11)部分1085.1mg 89.2％、BLG-H 5(含BLG-12)部分993.2mg 76.1％。
5、三次制备：
5.1、BLG-C 6部分：
5.1.1、样品溶解：
二次制备处理后的粉末138mg，加入1ml甲醇超声溶解，过滤。
5.1.2、制备方法：
色谱柱:Innoval ODS-2 10μm 100A；上样量：138mg；流速：80ml/min；检测波长：280nm/254nm；流动相：A(水)-B(乙腈)梯度洗脱(0～50min，20％B；50～56min，100％B，56～62min，20％B)。
共进1针，制备过程色谱图如图6所示，箭头所指为接收目标馏分，接收1个馏分：BLG-3，得1个单体BLG-3即dehydrodiconiferyl alcohol，纯度及质量见表1。
5.2、BLG-H 3部分：
5.2.1、样品溶解：
二次制备处理后的粉末300mg，加入1ml甲醇超声溶解，过滤。
5.2.2、制备方法：
色谱柱:Innoval ODS-2 10μm 100A；上样量：100mg；流速：80ml/min；检测波长：280nm/254nm；流动相：A(水)-B(乙腈)，梯度洗脱(0～62min，24％B；62～92min，25％～26％B，92～98min，100％B，98～104min，24％B)。
共进1针，制备过程色谱图如图7所示，箭头所指为接收目标馏分，接收1个馏分：BLG-10，得1个单体BLG-10即herpetetrone，具体纯度及质量见表1。
5.3、BLG-H 4部分：
5.3.1、样品溶解：
二次制备处理后的粉末220mg，加入1ml甲醇超声溶解，过滤。
5.3.2、制备方法：
色谱柱:Innoval ODS-2 10μm 100A；上样量：100mg；流速：80ml/min；检测波长：280nm/254nm；流动相：A(水)-B(乙腈)梯度洗脱(0～40min，30％～35％B，40～46min，100％B，46～52min，30％B)。
共进1针，制备过程色谱图如图8所示，箭头所指为接收目标馏分，接收1个馏分： BLG-11，得1个单体BLG-11即herpetone，具体纯度及质量见表1。
5.4、BLG-H 5部分：
5.4.1、样品溶解：
二次制备处理后的粉末990mg，加入1.5ml甲醇超声溶解，过滤。
5.4.2、制备方法：
色谱柱:Innoval ODS-2 10μm 100A；上样量：100mg；流速：80ml/min；检测波长：280nm/254nm；流动相：A(水)-B(乙腈)梯度洗脱(0～40min，30％～35％B，40～57min，35％～38％B，57～63min，100％B，63～69min，30％B)。
共进2针，制备过程色谱图如图9所示，箭头所指为接收目标馏分，接收1个馏分：BLG-12，相同馏分合并处理，得1个单体BLG-12即herpetenone，具体纯度及质量见表1。
表1 8种木脂素化合物样品纯度及制备量
编号 纯度 制备量
BLG-3 95.2％ 14.2mg
BLG-4 93.1％ 120.2mg
BLG-5 93.9％ 323.1mg
BLG-6 97.0％ 2339.2mg
BLG-7 95.9％ 261.7mg
BLG-10 95.4％ 18.7mg
BLG-11 98.6％ 935.1mg
BLG-12 96.5％ 251.2mg
注：纯度为HPLC面积归一化法计算
6、化合物结构解析：
1、化合物BLG-3：ESI-MS(C 20H 22O 6)m/z 381.13[M+Na] +；UVλ max(CH 3OH)nm:219,276；IR(KBr)cm -1：3430,2924,1649,1605,1519,1498,1463,1331,1275,1212,1143,1030,964,854,812； 1H-NMR(600MHz,CD 3OD)δ:6.94(1H,d,J＝2.1Hz,H-2),6.77(1H,d,J＝8.1Hz,H-5),6.82(1H,dd,J＝8.2,1.9Hz,H-6),5.52(1H,d,J＝6.3Hz,H-7),3.49(1H,dd,J＝12.3,6.2Hz,H-8),3.78(2H,dd,J＝11.1,7.0,H-9),6.94(1H,d,J＝2.1Hz,H-2’),6.97(1H,s,H-6’),6.54(1H,d,J＝15.8Hz,H-7’),6.22(1H,dt,J＝15.8,5.9Hz,H-8’),4.19(2H,dd,J＝5.9,1.4Hz,H-9’),3.87(3H,s,3-OCH 3),3.81(3H,s,3’-OCH 3)； 13C-NMR(150MHZ,CD 3OD)δ:134.56(C-1),110.58(C-2),149.28(C-3),147.60(C-4),116.17(C-5).119.75(C-6),89.32(C-7),55.17(C-8),64.90(C-9),132.58(C-1’),112.17(C-2’),145.49(C-3’),149.12(C-4’),130.36(C-5’),116.53(C-6’),132.01(C-7’)127.56(C-8’),63.86(C-9’),56.40(3-OCH 3),56.78(3’-OCH 3)。
以上数据与文献In S J,Seo K H,Song N Y,et al.Lignans and neolignans from the stems  of Vibrunum erosum and their neuroprotective and anti-inflammatory activity[J].Archives of Pharmacal Research,2015,38(1):26-34.基本一致，鉴定该化合物为dehydrodiconiferylalcohol，(去氢双松柏醇)。
2、化合物BLG-5：ESI-MS(C 16H 14O 6)m/z 301.07[M-H] -；UVλ max(CH 3OH)nm:260,302,338；IR(KBr)cm -1:3386,2927,1680,1612,1522,1455,1291,1153,1005； 1H-NMR(600MHz,CD 3OD)δ:6.93(1H,d,J＝2.8Hz,H-6),6.67(1H,d,J＝2.8Hz,H-8),7.89(1H,s,H-1’),7.51(1H,s,H-4’),3.83(3H,s,7-OCH 3),4.00(3H,s,2’-OCH 3),9.69(1H,s,3’-OH)； 13C-NMR(150MHz,CD 3OD)δ:162.98(C-2),114.75(C-3),130.59(C-4),139.86(C-5),116.37(C-6),160.76(C-7),102.14(C-8),65.35(C-9),112.25(C-4a),153.92(C-8a),109.63(C-1’),155.28(C-2’),147.71(C-3’),115.41(C-4’),56.02(7-OCH 3),56.56(2’-OCH 3)。
以上数据与文献Mei Z,Yun D,Zhang H,et al.Two new coumarins from Herpetospermum caudigerum.[J].Chemical&Pharmaceutical Bulletin,2008,56(2):192.基本一致，鉴定该化合物为herpetolideA(波棱内脂A)。
3、化合物BLG-6：ESI-MS(C 30H 32O 10)m/z 575.19[M+Na] +；UVλ max(CH 3OH)nm:230,280；IR(KBr)cm -1:3400,2936,1663,1601,1518,1278,1031； 1H-NMR(600MHz,CD 3OD)δ7.66(1H,d,J＝2.0Hz,H-2),6.83(1H,d,J＝2.0Hz,H-5),7.63(1H,dd,J＝8.3,2.0Hz,H-6),5.31(1H,dd,J＝8.9,4.7Hz,H-8),4.26(2H,dd,J＝10.6,9.1Hz,H-9),6.83(1H,d,J＝2.0Hz,H-2’),6.74(1H,d,J＝3.0Hz,H-6’),4.65(1H,d,J＝5.3Hz,H-7’),3.03(1H,m,H-8’),6.91(1H,s,H-2”),6.76(1H,d,J＝2.8Hz,H-5”),6.83(1H,d,J＝2.0Hz,H-6”),4.60(1H,d,J＝5.2Hz,H-7”),3.03(1H,m,H-8”),4.15(2H,m,H-9’a,9”a),3.77(2H,m,H-9’b,9”b),3.82(3H,s,3-OCH 3),3.86(3H,s,3’-OCH 3),3.83(3H,d,J＝2.1Hz,3”-OCH 3)； 13C-NMR(150MHz,CD 3OD)δ:133.31(C-1),110.98(C-2),149.07(C-3),153.01(C-4),115.76(C-5),124.63(C-6),199.82(C-7),49.84(C-8),64.09(C-9),133.76(C-1’),116.07(C-2’),149.34(C-3’),144.38(C-4’),130.18(C-5’),124.48(C-6’),87.39(C-7’),55.35(C-8’),72.57(C-9’),133.39(C-1”),118.61(C-2”),148.70(C-3”),147.27(C-4”),120.04(C-5”),112.81(C-6”),87.39(C-7”),55.23(C-8”),72.38(C-9”),56.40(3-OCH3),56.57(3’-OCH3),56.40(3”-OCH3)。
以上数据与文献Kaouadji M,Favrebonvin J,Sarrazin F,et al.Herpetetrone,Another Tetrameric Lignoid from Herpetospermum caudigerum Seeds[J].Journal of Natural Products,1987,50(6):1089-1094.基本一致，鉴定该化合物为herpetrione(波棱甲素)。
4、化合物BLG-7：ESI-MS(C 30H 34O 9)m/z 561.21[M+Na] +；UVλ max(CH 3OH)nm:228,280；IR(KBr)cm -1:3435,1614,1604,1518,1276,1212,1031(文献最大)； 1H-NMR(600MHz,CD 3OD)δ:6.95(1H,d,J＝1.9Hz,H-2),6.75(1H,d,J＝8.0Hz,H-5),6.83(2H dd,J＝8.2,1.9Hz,H-6),5.49(1H,d,J＝6.4Hz,H-7),3.48(1H,dd,J＝12.3,6.3Hz,H-8),6.78(1H,d,J＝1.5Hz,H-2’),6.77(1H,d,J＝1.3Hz,H-6’),2.95(1H,dd,J＝13.5,4.7Hz,H-7’a),2.52(1H,dd,J＝13.4,11.3Hz,H-7’b),2.74(1H,m,H-8’),4.00(1H,dd,J＝8.3,6.4Hz,H-9’),6.91(1H,d,J＝1.5Hz,H-2”),6.75(1H,d,J＝8.0Hz,H-5”),6.83(2H dd,J＝8.2,1.9Hz,H-6”),4.75(1H,d,J＝ 6.9Hz,H-7”),2.38(1H,p,J＝7.1Hz,H-8”),3.84(2H,d,J＝4.1Hz,H-9a,9”a),3.80(1H,d,J＝3.7Hz,H-9b),3.63(1H,dd,J＝11.0,6.4Hz,H-9”b),3.86(3H,s,3-OCH 3),3.87(3H,s,3’-OCH 3),3.89(3H,s,4”-OCH 3)； 13C-NMR(151MHz,CD 3OD)δ:134.66(C-1),110.68(C-2),148.98(C-3),147.51(C-4),116.13(C-5),119.80(C-6),89.03(C-7),55.32(C-8),64.88(C-9),135.48(C-1’),114.38(C-2’),147.04(C-3’),130.15(C-4’),147.77(C-5’),118.29(C-6’),33.91(C-7’),43.94(C-8’),73.52(C-9’),135.74(C-1”),110.59(C-2”),145.34(C-3”),149.07(C-4”),115.99(C-5”),119.80(C-6”),84.05(C-7”),54.05(C-8”),60.48(C-9”),56.40(3-OCH 3),56.78(3’-OCH 3),56.37(4”-OCH 3)。
以上数据与文献Yuan H L,Liu Y,Zhao Y L,et al.Herpetin,a new bioactive lignan isolated from Herpetospermum caudigerum[J].Journal of Chinese Pharmaceutical Sciences,2005,14(3):140-143.基本一致，鉴定该化合物为herpetin(波棱素)。
5、化合物BLG-10：ESI-MS(C 40H 42O 13)m/z 753.25[M+Na] +；UVλ max(CH 3OH)nm:230,282,306；IR(KBr)cm 1:3429,2933,1661,1603,1518,1463,1427,1278,1213,1147,1031； 1H-NMR(600MHz,CD 3OD)δ:7.66(1H,d,J＝2.0Hz,H-2),6.83(1H,d,J＝1.8,H-5),7.62(1H,dd,J＝8.4,2.0Hz,H-6),5.30(1H,m,H-8),4.25(2H,dd,J＝10.6,9.1,H-9),6.88(1H,s,H-2’),6.75(1H,d,J＝3.3,H-6’),4.68(1H,d,J＝5.4Hz,H-7’),3.05(1H,m,H-8’),4.17(2H,m,H-9’),,6.75(1H,d,J＝3.3,H-2”),6.76(1H,d,J＝3.1,H-6”),4.61(1H,d,J＝5.2Hz,H-7”),3.05(1H,m,H-8”),4.17(2H,m,H-9”),6.94(1H,d,J＝1.8Hz,H-2’”),6.76(1H,d,J＝3.1,H-5’”),6.81(1H,dd,J＝8.2,1.8,H-6’”),5.51(1H,d,J＝6.3Hz,H-7’”),3.48(1H,m,H-8’”),3.82(2H,dd,J＝7.5,3.1,H-9’”),3.86(3H,s,3-OCH 3),3.85(3H,s,3’-OCH 3),3.84(3H,s,5”-OCH 3),3.80(3H,s,3’”-OCH 3)； 13C-NMR(151MHz,CD 3OD)δ:130.19(C-1),119.70(C-2),149.09(C-3),153.03(C-4),112.81(C-5),110.58(C-6),199.81(C-7),49.48(C-8),64.08(C-9),133.36(C-1’),109.48(C-2’),149.36(C-3’),144.39(C-4’),124.63(C-5’),124.47(C-6’),87.59(C-7’),56.39(C-8’),72.68(C-9’),133.36(C-1”),116.03(C-2”),135.88(C-3”),149.09(C-4”),147.56(C-5”),115.77(C-6”),87.32(C-7”),56.39(C-8”),72.58(C-9”),134.56(C-1’”),112.09(C-2’”),148.71(C-3’”),145.47(C-4’”),116.16 (C-5’”),118.59(C-6’”),89.18(C-7’”),55.31(C-8’”),64.83(C-9’”),56.39(3-OCH 3),56.57(3’-OCH 3),56.82(3”-OCH 3),56.57(3’”-OCH 3)。
以上数据与文献Kaouadji M,Favrebonvin J,Sarrazin F,et al.Herpetetrone,Another Tetrameric Lignoid from Herpetospermum caudigerum Seeds[J].Journal of Natural Products,1987,50(6):1089-1094.基本一致，鉴定该化合物为herpetetrone。
6、化合物BLG-11：ESI-MS(C 29H 30O 9)m/z 545.18[M+Na] +；UVλ max(CH 3OH)nm:230,278,304；IR(KBr)cm -1:3391,1667,1592,1517,1463,1426,1274,1030； 1H-NMR(600MHz,CD 3OD)δ:7.61(1H,d,J＝2.0Hz,H-2),6.82(1H,d,J＝8.3Hz,H-5),7.65(1H,dd,J＝8.3,2.0Hz,H-6),4.22(2H,s,H-8),6.85(1H,d,J＝1.8Hz,H-2’),6.75(1H,d,J＝2.6Hz,H-6’),4.67(1H,d,J＝4.7Hz,H-7’),3.10(2H,m,H-8’),6.93(1H,d,J＝1.8Hz,H-2”),6.76(1H,s,H-5”),6.79(1H,dd,J＝8.2,1.8Hz,H-6”),4.67(1H,d,J＝4.7Hz,H-7”),3.10(1H,m,H-8”),4.19(2H,m,H9’a,9”a),3.80(2H,dt,J＝9.2,3.8Hz,H-9’b,9”b),3.82(3H,s,3-OCH 3),3.86(3H,s,3’-OCH 3),3.84(3H,s,3”-OCH 3)； 13C-NMR(151MHz,CD 3OD)δ:130.01(C-1),112.64(C-2),149.09(C-3),153.18(C-4),115.79(C-5),124.94(C-6),199.40(C-7),40.21(C-8),133.09(C-1’),109.19(C-2’),148.86(C-3’),144.96(C-4’),123.22(C-5’),121.49(C-6’),87.53(C-7’),55.30(C-8’),72.69(C-9’),133.09(C-1”),110.99(C-2”),149.09(C-3”),147.29(C-4”),116.07(C-5”),120.06(C-6”),87.42(C-7”),55.30(C-8”),72.53(C-9”),56.53(3’-OCH 3),56.41(3-OCH 3,3”-OCH 3)。
以上数据与文献张梅,董小萍,邓赟,等.藏药波棱瓜子中一个新的倍半降木脂素[J].药学学报,2006,41(7):659-661.基本一致，鉴定该化合物为herpetone(波棱酮)。
7、化合物BLG-12：ESI-MS(C 30H 30O 9)m/z 557.18[M+Na] +；UVλ max(CH 3OH)nm:282,308；IR(KBr)cm 1:3396,2937,2852,1650,1591,1515,1463,1427,1273,1157,1032； 1H-NMR(600MHz,CD 3OD)δ:7.45(1H,s,H-2),6.76(1H,d,J＝1.8Hz,H-5),7.44(1H,d,J＝2.0Hz,H-6),5.96(1H,s,H-9a),5.66(1H,s,H-9b),6.94(1H,d,J＝3.1Hz,H-2’),6.94(1H,d,J＝3.1Hz,H-6’),4.73(1H,d,J＝4.9Hz,H-7’),3.13(2H,m,H-8’),4.22(2H,dd,J＝15.9,6.9Hz,H-9’),6.94(1H,d,J＝3.1Hz,H-2”),6.78(1H,d,J＝2.2Hz,H-5”),6.80(1H,dd,J＝8.2,1.8Hz,H-6”),4.70(1H,d,J＝5.0Hz,H-7”),,3.13(2H,m,H-8”)4.22(2H,dd,J＝15.9,6.9Hz,H-9”),3.84(3H,s,3-OCH 3),3.82(3H,s,5’-OCH 3),3.79(3H,s,3”-OCH 3)； 13C-NMR(151MHz,CD 3OD)δ:130.15(C-1),113.76(C-2),148.55(C-3),152.92(C-4),116.09(C-5),126.42(C-6),198.37(C-7),127.19(C-8),123.30(C-9),133.60(C-1’),120.20(C-2’),148.37(C-3’),144.57(C-4’),149.22(C-5’),110.52(C-6’),87.48(C-7’),55.34(C-8’),72.64(C-9’),133.80(C-1”),111.00(C-2”),149.10(C-3”),147.30(C-4”),115.45(C-5”), 120.05(C-6”),87.33(C-7”),55.43(C-8”),72.64(C-9”),56.43(3-OCH 3),56.58(5’-OCH 3),56.32(3”-OCH 3)。
以上数据与文献Yu J Q,Hang W,Duan W J,et al.Two new anti-HBV lignans from Herpetospermum caudigerum[J].Phytochemistry Letters,2014,10:230-234.基本一致，鉴定该化合物为herpetenone(波棱乙素)
8、化合物BLG-4：ESI-MS(C 30H 32O 10)m/z 551.19[M-H] -；UVλ max(CH 3OH)nm:276；IR(KBr)cm -1:3430,2923,1605,1519,1498,1429,1331,1275,1212,1143,1030,
964,812； 1H-NMR(600MHz,CD 3OD)δ:7.64(1H,d,J＝2.0Hz,H-2),6.75(1H,dd,J＝8.3,6.0Hz,H-5),7.61(1H,dd,J＝4.8,2.0Hz,H-6),5.41(1H,m,H-8),6.69(1H,dd,J＝6.9,1.9Hz,H-2’),6.84(1H,d,J＝1.8Hz,H-5’),5.28(1H,dd,J＝8.9,4.7Hz,H-7’),3.65(1H,m,H-8’),3.72(2H,m,H-9’),6.92(1H,s,H-2”),6.93(1H,s,H-5”),6.52(1H,d,J＝15.8Hz,H-7”),6.21(1H,dt,J＝15.8,5.9Hz,H-8”),4.27–4.15(4H,m,H-9,9”),3.82(3H,s,3-OCH 3),3.84(3H,s,3’-OCH 3),3.85(3H,s,3”-OCH 3)； 13C-NMR(150MHz,CD 3OD)δ:124.68(C-1),112.30(C-2),148.68(C-3),152.97(C-4),115.74(C-5),124.60(C-6),199.68(C-7),49.84(C-8),64.69(C-9),133.98(C-1’),109.03(C-2’),149.14(C-3’),144.73(C-4’),130.21(C-5’),116.49(C-6’),89.30(C-7’),54.95(C-8’),64.04(C-9’),130.40(C-1”),112.81(C-2”),145.43(C-3”),149.34(C-4”),131.99(C-5”),118.63(C-6”),132.58(C-7”),127.56(C-8”),63.96(C-9”),56.44(3-OCH 3),56.81(3’-OCH 3),56.91(3”-OCH 3)。
根据以上数据，结合文献Fan Y,Zhang H J,Zhang Y Y,et al.A hepatitis B virus inhibitory neolignan from Herpetospermum caudigerum.[J].Chemical&Pharmaceutical Bulletin,2010,41(33):no-no.和周雪杉.藏药波棱瓜子木脂素类成分系统研究[D].西南交通大学,2014.，初步分析推断得出结构，经查新，初步推断为新化合物，命名为herpetotriol I(波棱醇I)。
实施例2：波棱瓜子的木脂素有效成分组合物：
称取波棱甲素65mg和波棱素35mg，混匀即得波棱瓜子的木脂素有效成分组合物。
实施例3：波棱瓜子的木脂素有效成分组合物：
称取波棱甲素30mg、波棱素20mg、herpetetrone15mg、波棱酮20mg和波棱乙素15mg，混匀即得波棱瓜子的木脂素有效成分组合物。
实施例4：波棱瓜子的木脂素有效成分组合物：
称取去氢双松柏醇5mg、波棱醇I 5mg、波棱内脂A 5mg、波棱甲素15mg、波棱素 15mg、herpetetrone 15mg、波棱酮15mg和波棱乙素15mg，混匀即得波棱瓜子的木脂素有效成分组合物。
实施例5：波棱瓜子的木脂素有效成分组合物：
称取去氢双松柏醇7mg、波棱醇I3mg、波棱内脂A2mg、波棱甲素23mg、波棱素12mg、herpetetrone20mg、波棱酮23mg和波棱乙素10mg，混匀即得波棱瓜子的木脂素有效成分组合物。
实施例6：波棱瓜子的木脂素有效成分组合物：
称取去氢双松柏醇5mg、波棱醇I5mg、波棱内脂A5mg、波棱甲素20mg、波棱素10mg、herpetetrone15mg、波棱酮25mg和波棱乙素15mg，混匀即得波棱瓜子的木脂素有效成分组合物。
实施例7：波棱瓜子的木脂素有效成分组合物：
称取去氢双松柏醇6mg、波棱醇I5mg、波棱内脂A4mg、波棱甲素25mg、波棱素12mg、herpetetrone13mg、波棱酮25mg和波棱乙素10mg，混匀即得波棱瓜子的木脂素有效成分组合物。
以下通过具体实验例来说明本发明一种波棱瓜子的木脂素有效成分组合物所具有的有益效果。
实验例1：波棱瓜子的木脂素有效成分组合物体内外抗乙肝作用：
一、材料：
1、细胞、病毒株及动物：
细胞HBV转染人肝癌细胞(HepG2)的2.2.15细胞系，美国Mount Sinai医学中心构建，中国医学科学院医药生物技术所病毒室传代培养。
病毒株鸭乙型肝炎病毒DNA(DHBV-DNA)强阳性血清，采自上海麻鸭，-70℃保存。
动物 1日龄北京鸭由北京前进种鸭场提供。
2、药品与试剂：
供试药物(表2中的8个成分，由“实施例1”制备)；拉米夫定(3TC，英国葛兰素威康有限公司产)由空军总医院门诊药房提供；HBsAg、HBeAg酶联免疫试剂盒由华美生物公司提供；α- 32P-dCTP由北京亚辉生物医学工程公司提供；探针标记用的随机引物试剂盒由Promega公司提供；SePhadex G-50、Ficoll PVP由瑞典Pharmacia公司提供；硝酸纤维素膜(0.45μm)由Amersham公司提供；胎牛血清由美国Hyclone Lab公司提供； 其它试剂均为市售分析纯。
3、仪器：
PHOMO酶标仪(安图实验仪器(郑州)有限公司)；BH2倒置显微镜(奥林巴斯光学仪器公司)；800D二氧化碳培养箱(美国Thermo公司)；MSC-Advantag生物安全柜(美国Thermo公司)；L-550低速台式大容量离心机(湖南湘仪集团)；BSA124S电子天平(赛多利科学仪器(北京)有限公司)。
二、方法：
1、波棱瓜子不同木脂素类成分组合物的制备：
称取不同量的木脂素类成分按表2处方混合均匀，制成不同脂素类成分的组合物，F1-F6共6种，备用。
表2 处方设计
Figure PCTCN2018085862-appb-000001
注：“-”代表处方中不含该成分。
2、F1-F6木脂素组合物体外抗乙肝活性评价：
2.1、细胞培养：
2.2.15细胞用含有10％胎牛血清的DMEM培养基(0.03％谷氨酰胺，380μg/mL G-418，100IU/mL青霉素，100IU/mL链霉素)，在37℃、5％CO 2条件下孵箱培养。
2.2、细胞毒性试验：
将F1-F6木脂素组合物及波棱瓜子提取物(T)(实施例1中的“2、波棱瓜子提取物的制备”)用DMEM培养基配制成浓度为4000μg/mL的溶液，然后用培养基2倍梯度稀释至4000、2000、1000、800、400、200、100μg/mL 7个质量浓度备用。以2×10 4个/mL细胞接种96孔培养板，每孔100μL，37℃ 5％CO 2培养24小时，吸去上清，分别加入含不同浓度药物的培养液100μL，每浓度3孔，设无药细胞对照组和空白孔。37℃ 5％CO 2培养72h后，于各孔中加入10μL MTT(5mg/mL)孵育4h，终止培养，弃去上清液，加入DMSO 150μL振荡10min。用酶标仪测定570nm处OD值。计算药物对2.2.15细胞的抑制率，计算半数有毒浓度(TC 50)和最大无毒浓度(TC 0)。
2.3、HBsAg、HBeAg抑制试验：
以8×10 4个/mL细胞接种24孔培养板，每孔1mL，37℃ 5％CO 2培养24小时，吸去上清，加无毒浓度以下2倍稀释试验药液1mL，5个稀释度分别为400、200、100、50、25μg/mL，每浓度3孔，37℃ 5％CO 2培养，每4天换同浓度药液，设无药细胞对照组。收集第8天含药培养液，-20℃冰冻保存。采用ELISA法检测上清HBsAg、HBeAg。按照试剂盒说明书操作，酶标仪测定各孔OD值，计算抑制率。
2.4、F1-F6木脂素组合物在鸭体内对鸭乙型肝炎病毒的影响：
1龄北京鸭，经鸭腿胫静脉注射上海麻鸭DHBV-DNA阳性鸭血清，每只0.3mL,感染后7天取血,分离血清，检测血清中DHBV-DNA含量。雏鸭血清经检测DHBV呈阳性后，将鸭随机分为9组：病毒对照组、3TC组(50mg/kg)、F1-F6木脂素组合物组及波棱瓜子提取物组(T组)，F1-F6及T组给药剂量均为100mg/kg。灌胃给药，每天2次。病毒对照组给予同体积生理盐水，连续给药10天。分别于给药前(T0)、药物治疗后5天(T5)、10天(T10)和停药后3天(P3)自鸭胫静脉取血，分离血清，-70℃保存待检。取上述待检鸭血清，每批同时点膜，测定鸭血清中DHBV-DNA水平的动态。按试剂盒说明书方法，用 32P标记DHBV-DNA探针，并作鸭血清斑点杂交，放射自显影膜片斑点，酶标仪测定OD值(滤光片为490nm)，计算血清DHBV-DNA光密度，以杂交斑点OD值作为标本DHBV-DNA水平值。将同组鸭用药前后血清DHBV-DNA的平均值进行比较，计算各给药组的不同时间血清DHBV-DNA的抑制率。
2.5、数据分析：
所得数据以
Figure PCTCN2018085862-appb-000002
表示，采用t检验作两组间均数比较，单因素方差分析作多组间均数比较，数据统计分析采用SPSS17.0软件进行，P<0.05认为有显著性差异。
三、结果：
3.1、F1-F6木脂素组合物的细胞毒性：
F1-F6木脂素组合物及波棱瓜子提取物(T)对2.2.15细胞的细胞毒作用结果见表3。由表3可知，F1-F6木脂素组合物及波棱瓜子提取物对2.2.15细胞半数有毒浓度在924-1324μg/mL范围内，其最大无毒剂量均大于400μg/mL。
表3 F1-F6木脂素组合物对2.2.15细胞的细胞毒性作用
Figure PCTCN2018085862-appb-000003
Figure PCTCN2018085862-appb-000004
3.2、F1-F6木脂素组合物对HBsAg、HBeAg抑制作用：
由表4和表5可知，F1-F6木脂素组合物及波棱瓜子提取物对2.2.15细胞分泌的HBsAg和HBeAg有较强的抑制作用，且呈现一定的浓度依赖性。F1-F6木脂素组合物与提取物组(T组)相比，对2.2.15细胞分泌的HBsAg和HBeAg抑制作用无明显差异，表明各组合物与波棱瓜子提取物具有相同的抗乙肝作用。
表4 F1-F6组合物对2.2.15细胞HBeAg抑制作用
Figure PCTCN2018085862-appb-000005
Figure PCTCN2018085862-appb-000006
注：3TC只有一个剂量组，200μg/mL。
表5 F1-F6组合物对对2.2.15细胞HBsAg抑制作用
Figure PCTCN2018085862-appb-000007
Figure PCTCN2018085862-appb-000008
注：3TC只有一个剂量组，200μg/mL。
3.3、F1-F6木脂素组合物对DHBV感染鸭血清DHBV-DNA的影响：
表6 F1-F6组合物对DHBV感染鸭血清DHBV-DNA抑制作用
Figure PCTCN2018085862-appb-000009
Figure PCTCN2018085862-appb-000010
Figure PCTCN2018085862-appb-000011
注：与对照组比较： **P＜0.01；
雏鸭感染乙肝病毒后DHBV-DNA全部阳性，实验过程中病毒对照组血清DHBV-DNA的水平无明显变动。阳性药3TC在给药后第5天及第10天可以显著的降低血清中DHBV-DNA的水平(P＜0.01)，但停药3d，抑制率下降至24.98％。与自身给药前比较，F1-F6木脂素组合物及波棱瓜子提取物在给药后第5天、第10天以及停药3d，均可显著降低血清中DHBV-DNA的水平(P＜0.01)，且F1-F6木脂素组合物与波棱瓜子提取物(T组)相比无显著性差异，结果见表6。
由本实验例可知，乙型肝炎是由乙型肝炎病毒HBV引起的，是以肝脏炎性病变为主的感染性疾病，易转化为慢性，甚至发展为肝硬化、肝癌。乙型肝炎病毒在肝细胞内持续复制会引起宿主产生免疫应答，造成细胞的免疫病理损害，是导致肝炎慢性化的病理学基础。因此针对乙肝病毒的慢性感染的抗病毒治疗，是阻断慢性乙型肝炎发展的主要措施。
2.2.15细胞及鸭乙肝病毒感染模型是目前公认的研究乙肝发病机制、病毒复制过程及筛选有效药物的理想模型。本研究选择这两种模型对F1-F6木脂素组合物及波棱瓜子提取物的抗乙肝病毒活性进行评价。相同剂量下，F1-F6木脂素组合物的体内外抗乙肝作用与波棱瓜子提取物(T组)无显著差异，表明F1-F6木脂素组合物与波棱瓜子提取物(T组)具有一致的体内外抗乙肝作用。
实验例2：波棱瓜子的木脂素有效成分组合物抗肝纤维化作用：
一、材料与仪器：
1、试剂：
丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)测定试剂盒均购自深圳迈瑞生物医疗电子股份有限公司；羟脯胺酸(Hyp)、透明质酸(HA)试剂盒，购自华美生物工程有限公司；转化生长因子-β 1(TGF-β 1)、由Life Technology提供合成。
2、仪器：
PHOMO酶标仪(安图实验仪器(郑州)有限公司)；BH2倒置显微镜(奥林巴斯光学仪 器公司)；800D二氧化碳培养箱(美国Thermo公司)；MSC-Advantag生物安全柜(美国Thermo公司)；L-550低速台式大容量离心机(湖南湘仪集团)；BSA124S电子天平(赛多利科学仪器(北京)有限公司)；全自动生化分析仪(深圳雷杜生命科技有限公司)。
3、实验动物：
SD大鼠，体重(200±20)g，雌雄各半，SPF级，由中国人民解放军军事医学科学院实验动物中心提供，生产许可证号SCXK(军)2012-0004。于中国人民解放军第三〇二医院实验动物房中适应性饲养3天，观察无异常后开始试验，实验动物使用许可证号SYXK(军)2012-0010。
二、方法与结果：
1、波棱瓜子不同木脂素类成分组合物的制备：
称取不同量的木脂素类成分按表7处方混合均匀，制成不同脂素类成分的组合物，F1-F6共6种，备用。
表7 处方设计
Figure PCTCN2018085862-appb-000012
2、体外抗肝纤维化活性评价：
2.1、细胞培养：HSC-T6细胞用含有10％胎牛血清的DMEM培养基，在37℃、5％CO2条件下孵箱培养。
2.2、CCK8法检测细胞增殖取对数生长期的HSC-T6细胞用胰酶消化，吸入15mL塑料离心管中，1000r/min，离心5min，吹打成单细胞悬液，弃上清，重力重悬细胞。调节细胞浓度约为3×104/mL，将细胞悬液接种于96孔细胞培养板中，100μL/孔，将96孔板置37℃、5％CO2孵箱孵育过夜。实验分为空白组(仅加入培养液)，对照组，F1-F6及波棱瓜子提取物(T)(实施例1中的“2、波棱瓜子提取物的制备”)组(5、10、25、50、100μg/mL)，阳性药秋水仙碱(0.1μg/mL)组。待细胞贴壁后，吸弃旧液，分别依组加入含不同药物的血清培养液及DMEM进行培养，每组6个复孔。药物处理24、48h后，向每孔加入10μL CCK8溶液，将培养板在培养箱内孵育2.5h后，用酶标仪测定在450nm处的吸光度(A)值，按照公式计算细胞增殖抑制率。细胞增殖抑制率＝1 －(A 实验－A 空白)/(A 对照－A 空白)。结果见表7和表8。
由表8和表9可知，波棱瓜子F1-F6木脂素组合物在10-100μg/mL范围内对HSC-T6细胞增殖具有显著的抑制作用，且呈现浓度依赖性，各组合物与波棱瓜子提取物(T)相比，对HSC-T6细胞增殖的抑制作用均无显著差异，表明各组合物与波棱瓜子提取物具有相同的抗肝纤作用。
表8 F1-F6组合物对HSC-T6细胞培养24h增殖抑制率
Figure PCTCN2018085862-appb-000013
Figure PCTCN2018085862-appb-000014
注：秋水仙碱只有一个剂量组，0.1μg/mL。
表9 F1-F6组合物对HSC-T6细胞培养48h增殖抑制率
Figure PCTCN2018085862-appb-000015
Figure PCTCN2018085862-appb-000016
注：秋水仙碱只有一个剂量组，0.1μg/mL。
3、体内抗肝纤维化活性评价：
3.1、分组及给药：
大鼠称重后随机分为空白对照组、模型组、F1-F6给药组(200mg/kg，混悬于0.5％CMC-Na溶液中给药)、秋水仙碱组(阳性对照，0.1mg/kg)，每组8只。除空白对照组外，其他各组大鼠首次皮下注射(sc)5mL/kg 40％CCl 4大豆油溶液，3d后sc 3mL/kg40％CCl 4大豆油溶液，每周2次(周三、周五)，同时对照组sc同等剂量的大豆油溶液，连续6周。从第7周开始，对照组及模型组每天灌胃(ig)同等剂量的0.5％CMC-Na溶媒 溶液，给药容量为10mL/kg，剩余给药组每天均ig对应剂量的供试品溶液，共给药6周，造模加给药总计12周。末次给药后禁食不禁水24h，大鼠称体重，腹主动脉取血，离心取上层血清，-20℃保存待检，取新鲜肝脏，称重，切下一小部分10％福尔马林浸泡，待检。
3.2、肝脏指数：
处死大鼠前称体重，取出肝脏，滤纸拭干后立即称质量，计算肝脏指数。肝脏指数＝肝脏质量(g)/体重(g)×100。结果见表10。
表10 F1-F6组合物对CCl 4致肝纤维化大鼠肝脏指数的影响(±s,n＝8)
Figure PCTCN2018085862-appb-000017
注：与空白对照组比较： **P＜0.01；与模型对照组比较 #P＜0.05， ##P＜0.01。
由表10可知，与对照组比较，模型对照组的肝重和肝脏指数均显著性增加(P＜0.05或P＜0.01)；与模型对照组比较，F1-F6组、提取物组(T组)及秋水仙碱组的肝重和肝脏指数均显著性降低(P＜0.01或P＜0.05)，且F1-F6组与提取物组(T组)相比无显著性差异。结果表明各给药组均能显著性减轻肝纤维化引起的肝肿胀，降低肝脏脏指数。
3.3、肝功能：
应用自动生化仪检测大鼠血清中肝功能指标丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)水平，结果见表11。
由表11可知，与对照组比较，模型组大鼠血清中ALT，AST，ALP水平显著升高(P＜0.05或P＜0.01)。与模型对照组比较，F1-F6组、提取物组(T组)及秋水仙碱组的ALT，AST，ALP水平均显著降低(P＜0.05或P＜0.01)，且F1-F6组与T组相比无显著性差异。
表11 F1-F6组合物对肝纤维化大鼠血清ALT、AST和ALP含量的影响
Figure PCTCN2018085862-appb-000018
Figure PCTCN2018085862-appb-000019
注：与空白对照组比较： *P＜0.05， **P＜0.01；与模型对照组比较 #P＜0.05， ##P＜0.01。
3.4肝纤维化指标检测：
按ELISA试剂盒说明书操作，酶标仪检测大鼠血清中透明质酸(HA)、羟脯胺酸(Hyp)和转化生长因子-β 1(TGF-β 1)的水平，结果见表12。
表12 F1-F6组合物对肝纤维化大鼠血清HA、Hyp和TGF-β 1含量的影响
Figure PCTCN2018085862-appb-000020
注：与空白对照组比较： *P＜0.05， **P＜0.01；与模型对照组比较 #P＜0.05， ##P＜0.01。
由表12可知，与对照组比较，模型组大鼠血清中HA、Hyp和TGF-β 1的水平明显升高(P＜0.01)。与模型对照组比较，F1-F6组、提取物组(T组)及秋水仙碱组的HA、Hyp和TGF-β 1水平均显著降低(P＜0.05或P＜0.01)，且F1-F6组与T组相比无显著性差异。
由本实验例可知，肝纤维化(hepatic fibrosis，HF)是由慢性肝疾病引发的一种常见的疾病。HF发生的机制与其他慢性疾病损伤的机制相似，纤维化的发生是一个动态的可逆过程。在肝纤维化阶段只要进行合理治疗，完全可以逆转肝纤维化进程，进而缓解乃至治愈肝纤维化。
肝纤维化(hepatic fibrosis，HF)是由肝脏炎症或损伤后组织修复所引起的代偿反应，以细胞外基质(extracellular matrix，ECM)在肝组织内大量沉积为主要特征。肝星状细胞(HSCs)的激活和凋亡为肝纤维化发生及发展的关键，HSC的激活和增殖促进肝纤维化，抑制其增殖、诱导其凋亡，可减轻甚至逆转肝纤维化。本研究发现，不同剂量的波棱瓜 子提取物及有效成分组合物均可以有效抑制HSC-T6细胞的增殖，并呈现剂量依懒性，随着药物浓度的增高及培养时间的研制，HSC-T6细胞的增殖抑制率明显增加；结果表明波棱瓜子提取物及有效成分组合物具有抗肝纤维化作用。
本研究采用CCl 4诱导肝纤维化模型大鼠评价波棱瓜子提取物及有效成分组合物的体内抗肝纤维化作用。结果显示棱瓜子提取物及有效成分组合物能够显著降低肝纤维化大鼠血清ALT，AST，ALP水平及HA、Hyp和TGF-β 1的水平，表明棱瓜子提取物及有效成分组合物对CCl 4致肝纤维化大鼠的肝组织及肝功能损伤具有明显的保护作用，具有明显的抗肝纤维化作用，其抗肝纤维化机制可能与下调TGF-β 1蛋白表达有关。
本研究结果显示，相同剂量下，波棱瓜子提取物及有效成分组合物的体内外抗肝纤维化作用无显著差异，表明波棱瓜子有效成分组合物能够达到提取物的抗肝纤维化水平，因其成分清楚，更有利于制剂的开放与质量控制。
以上所述，仅为本发明的较佳实施例，并非对本发明作任何形式上和实质上的限制，凡熟悉本专业的技术人员，在不脱离本发明技术方案范围内，当可利用以上所揭示的技术内容，而作出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对以上实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (6)

  1. 一种波棱瓜子的木脂素有效成分组合物，其特征在于，所述波棱瓜子的木脂素有效成分组合物由下列重量百分数的成分组成：dehydrodiconiferyl alcohol含量为0-10％、herpetotriol I含量为0-10％、herpetolide A含量为0-10％、herpetrione含量为5-70％、herpetin含量为0-40％、herpetetrone含量为0-20％、herpetone含量为0-30％、herpetenone含量为0-20％。
  2. 根据权利要求1所述的波棱瓜子的木脂素有效成分组合物，其特征在于，波棱瓜子的木脂素有效成分组合物中，dehydrodiconiferyl alcohol含量为4-7％、herpetotriol I含量为3-6％、herpetolide A含量为2-5％、herpetrione含量为15-30％、herpetin含量为10-20％、herpetetrone含量为0-20％、herpetone含量为20-30％、herpetenone含量为8-15％。
  3. 权利要求1或2所述波棱瓜子的木脂素有效成分组合物的制备方法，其特征在于：称取权利要求1或2所述重量百分数的各成分，混匀后即得波棱瓜子的木脂素有效成分组合物；
    波棱瓜子的木脂素有效成分组合物中的各成分由下述步骤制备：
    (1)、波棱瓜子提取物的制备：取波棱瓜子药材粗粉，加80％乙醇回流提取3次，每次溶剂量5倍，每次1小时，合并提取液，静置24小时后，滤过，滤液回收乙醇至无醇味，静置12小时，分去油层。水层以碳酸钠固体调pH值10-11使溶解，离心，上清液加浓盐酸调pH值2-3，静置1小时，离心，取沉淀，用石油醚反复洗至滤液无色，减压干燥，得波棱瓜子提取物粉末；
    (2)、一次制备：
    (2.1)、样品溶液的配制：
    取波棱瓜子提取物600mg，加12ml甲醇超声溶解，再加入3ml蒸馏水混匀，过滤；
    (2.2)、制备方法：
    色谱柱：Innoval ODS-2 10μm 100A；上样量：600mg；流速：80mL/min；检测波长：280nm/254nm；流动相：水-乙腈，梯度洗脱：0～25min，21％～24％乙腈；25～50min，24％～25％乙腈；50～75min，25％乙腈；75min～90min，95％乙腈；90～100min，21％乙腈；
    共进50针，得到共8个馏分：BLG-A、BLG-B、BLG-C、BLG-D、BLG-E、BLG-F、BLG-G、BLG-H，相同馏分合并处理；
    (2.3)、馏分后处理：
    馏分采用乙酸乙酯萃取的方法，具体步骤：馏分减压旋蒸10-15℃回收乙腈，旋蒸液 加入乙酸乙酯，萃取3次，合并萃取液，减压旋蒸10-15℃回收乙酸乙酯，旋蒸至干，再用甲醇溶解，转移至玻璃瓶中氮气吹干；其中BLG-A～BLG-G部分的制备纯化接收的馏分萃取时加入的乙酸乙酯的比例均为馏分体积的13％，BLG-H段制备纯化的均为馏分体积的10％；
    一次制备馏分处理后得：BLG-A部分130mg，BLG-B部分210mg，BLG-C部分600mg，BLG-D部分600mg，BLG-E部分1.6g，BLG-F部分2.4g，BLG-G部分500mg；
    (3)、二次制备：
    (3.1)、BLG-E、BLG-F、BLG-G部分：
    (3.1.1)、样品溶液的配制：
    取一次制备处理后的BLG-F粉末130mg，加入1ml甲醇超声溶解，过滤；
    (3.1.2)、BLG-F部分二次制备方法：
    色谱柱：Innoval ODS-2 10μm 100A；上样量：130mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈为78％:22％，等度洗脱；60min进一针，收集上一针的馏分；
    共进19针，分别接收BLG-5、BLG-6、BLG-7共3个馏分，相同馏分合并处理，得BLG-5即herpetolideA、BLG-6即herpetrione、BLG-7即herpetin共3个单体或组分；
    BLG-E、BLG-F、BLG-G三部分馏分所含成分一致，仅各成分含量不同，按照BLG-F部分二次制备的方法分别对BLG-E部分和BLG-G部分分别进行二次制备并得到BLG-5即herpetolideA、BLG-6即herpetrione、BLG-7即herpetin共3个单体或组分；
    (3.2)、BLG-C部分：
    (3.2.1)、样品溶液的配制：
    一次制备处理后的BLG-C粉末200mg，加入1ml甲醇超声溶解，过滤；
    (3.2.2)、二次制备方法：
    色谱柱:Innoval ODS-2 10μm 100A；上样量：200mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～30min，23％～28％乙腈；30～35min，28％乙腈；35～45min，100％乙腈；45～55min，23％乙腈；
    共进3针，接收9个馏分，其中BLG-C 6段含BLG-3，相同馏分合并处理，按2.3中方法进行馏分后处理，其中BLG-C 6部分138.4mg 74.0％；
    (3.3)、BLG-D部分：
    BLG-D部分与BLG-C部分样品溶解及制备方法相同；
    接收8个馏分，其中BLG-D 5段为BLG-4即herpetotriol I，相同馏分合并处理，按2.3中方法进行馏分后处理；
    (3.4)、BLG-H段二次制备方法：
    (3.4.1)、样品溶液的配制：
    500mL乙酸乙酯层萃取液，旋蒸至60mL，超声混匀；
    色谱柱:Innoval ODS-2 10μm 100A；上样体积：3mL；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈，梯度洗脱：0～80min，26％～41％乙腈；80～90min，100％乙腈；90～96min，26％乙腈；
    共进7针，接收9个馏分：其中含BLG-10的BLG-H 3部分307.5mg 59.9％、含BLG-11的BLG-H 4部分1085.1mg 89.2％、含BLG-12的BLG-H 5部分993.2mg 76.1％；
    (4)、三次制备：
    (4.1)、BLG-C 6部分：
    (4.1.1)、样品溶解：
    二次制备处理后的BLG-C 6粉末138mg，加入1ml甲醇超声溶解，过滤；
    (4.1.2)、制备方法：
    色谱柱:Innoval ODS-2 10μm 100A；上样量：138mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～50min，20％乙腈；50～56min，100％乙腈，56～62min，20％乙腈；
    共进1针，接收1个馏分：BLG-3，得1个单体BLG-3即dehydrodiconiferyl alcohol；
    (4.2)、BLG-H 3部分：
    (4.2.1)、样品溶解：
    二次制备处理后的BLG-H 3粉末300mg，加入1ml甲醇超声溶解，过滤；
    (4.2.2)、制备方法：
    色谱柱:Innoval ODS-2 10μm 100A；上样量：100mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～62min，24％乙腈；62～92min，25％～26％乙腈，92～98min，100％乙腈，98～104min，24％乙腈；
    共进1针，接收1个馏分：BLG-10，得1个单体BLG-10即herpetetrone；
    (4.3)、BLG-H 4部分：
    (4.3.1)、样品溶解：
    二次制备处理后的BLG-H 4粉末220mg，加入1ml甲醇超声溶解，过滤；
    (4.3.2)、制备方法：
    色谱柱：Innoval ODS-2 10μm 100A；上样量：100mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～40min，30％～35％乙腈，40～46min，100％乙腈，46～52min，30％乙腈；
    共进1针，接收1个馏分：BLG-11，得1个单体BLG-11即herpetone；
    (4.4)、BLG-H 5部分：
    (4.4.1)、样品溶解：
    二次制备处理后的BLG-H 5粉末990mg，加入1.5ml甲醇超声溶解，过滤；
    (4.4.2)、制备方法：
    色谱柱：Innoval ODS-2 10μm 100A；上样量：100mg；流速：80ml/min；检测波长：280nm/254nm；流动相：水-乙腈；梯度洗脱：0～40min，30％～35％乙腈，40～57min，35％～38％乙腈，57～63min，100％乙腈，63～69min，30％乙腈；
    共进2针，接收1个馏分：BLG-12，相同馏分合并处理，得1个单体BLG-12即herpetenone。
  4. 权利要求1或2所述的波棱瓜子的木脂素有效成分组合物制备的剂型，其特征在于：它由1或2所述的波棱瓜子的木脂素有效成分组合物和药学上可接受的载体组成；所述剂型为胶囊剂、片剂、丸剂、滴丸、颗粒剂、软胶囊、膜剂、膏剂、喷雾剂、气雾剂、贴剂、凝胶剂或注射剂。
  5. 根据权利要求4所述的剂型，其特征在于：所述药学上可接受的载体选自淀粉、微晶纤维素、乳糖、蔗糖、糖粉、糊精、纤维素衍生物、羧甲基淀粉钠、低取代羟丙基纤维素、硬脂酸及其盐、滑石粉、聚乙二醇、蜂蜡、植物油、动物胶、植物胶、卡波姆树脂、羧甲基纤维素钠、聚乙烯醇、丙烯酸酯类聚合物、聚丙烯酸、聚丙烯酸钠、聚乙烯吡咯烷酮、甘油、丙二醇、山梨醇、三乙胺、氮酮、羟丙基-β-环糊精和吐温-80中的一种或两种以上的组合。
  6. 权利要求1或2所述的波棱瓜子的木脂素有效成分组合物在制备治疗乙肝药物中的应用。
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