CN112020355A

CN112020355A - Lignan effective component composition of herpetospermum pedunculosum seeds, and preparation method, application and dosage form thereof

Info

Publication number: CN112020355A
Application number: CN201880081675.XA
Authority: CN
Inventors: 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
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-05-07
Filing date: 2018-05-07
Publication date: 2020-12-01
Also published as: WO2019213807A1

Abstract

A lignan effective component composition of Herpetospermum seeds, and a preparation method, application and dosage form thereof belong to the technical field of traditional Chinese medicine. The composition of the lignan effective components of the herpetospermum pedunculosum seeds comprises the following components in percentage by weight: 0-10% of dehydrodiconiferyl alcohol, 0-10% of herpetiformyl alcohol I, 0-10% of herpesvilide A, 5-70% of herpetiformyl A, 0-40% of herpetrone, 0-20% of herpetiformyl ketone and 0-20% of herpetiformyl ethyl alcohol. Has the following advantages: the preparation liquid chromatography technology is adopted to separate and prepare 8 lignan active ingredients in the herpetospermum pedunculosum seeds, and the purity of each ingredient can reach more than 90 percent; the composition and the dosage form thereof can be used for treating liver injury, hepatitis B and hepatic fibrosis and have better treatment effect.

Description

Lignan effective component composition of herpetospermum pedunculosum seeds, and preparation method, application and dosage form thereof

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a lignan active ingredient composition of herpetospermum pedunculosum seeds, and a preparation method, application and dosage form thereof.

Background

China is always the major country of hepatitis B, and hepatitis B vaccines are used for preventing the spread of hepatitis B for years, but the popularization degree of partial areas is low, the number of original HBV carrying people is large, and the like, so that the HBV vaccine is still one of diseases harmful to the health of people in China in the coming decades. In the aspect of treatment, the existing chemical drugs of interferon and nucleoside have poor prospects due to adverse reactions such as nephrotoxicity, viral variation and the like. In addition, chronic hepatitis B has the characteristics of long course, difficult control, poor prognosis, complex pathogenesis and the like, so that the related treatment is still one of the problems in the field of the current liver diseases.

The aggravation of hepatitis B progresses to hepatic fibrosis, hepatic ascites and liver cancer. Hepatic fibrosis is the common pathological basis of various chronic liver diseases, is an important stage of the liver cirrhosis occurrence process, and is often caused by chronic inflammation in the liver, cholestasis, immune injury and other reasons. The data show that the incidence rate of chronic hepatitis and hepatic fibrosis is 85.1 percent, and 25 to 40 percent of the hepatic fibrosis continuously develops into cirrhosis. Research proves that hepatic fibrosis is reversible lesion, so that the research on the medicine for inhibiting the chronic hepatic fibrosis is significant.

The Tibetan medicine has unique curative effect on dominant diseases such as liver and gall diseases, stable curative effect, small toxic and side effect and wide application in folk. The common drug for resisting liver and gall diseases, namely the Herpetospermum pedunculosum seeds (dried mature seeds of Herpetospermum pedunculosum Wall of cucurbitaceae), is recorded in 1995 edition ' drug Standard, Tibetan drug Standard of Ministry of public health of the people's republic of China ', has bitter taste and cold property, can clear internal heat and gall-bladder heat, has the effects of clearing heat and detoxicating, reducing pathogenic fire and lowering heat, and helping digestion, and is used for treating diseases such as liver heat, icteric infectious hepatitis and the like. Modern pharmacological research shows that the pedunculate herpetospermum seed extract has the functions of protecting liver, reducing transaminase, resisting hepatitis B and resisting hepatic fibrosis. Substance basic research shows that the active ingredients of the herpetospermum pedunculosum seeds for resisting hepatitis and liver injury are mainly concentrated at the ethyl acetate part, and mainly comprise lignanoid ingredients, such as dihydrodiferuloyl alcohol, herpetriol I, herpetolide A, herpetrione and hepetin, and the ingredients have obvious effects of resisting hepatitis B and hepatic fibrosis.

Disclosure of Invention

The invention aims to disclose a lignan active ingredient composition of pedunculate herpetospermum seeds.

The second purpose of the invention is to disclose a preparation method of the composition of the lignan active ingredients of the herpetospermum pedunculosum seeds.

The third purpose of the invention is to disclose the application of the composition of the lignan effective components of the herpetospermum pedunculosum seeds.

The fourth purpose of the invention is to disclose the dosage form of the composition of the lignan active ingredients of the herpetospermum pedunculosum seeds.

The purpose of the invention is realized by the following technical scheme:

the lignan effective component composition of the herpetospermum pedunculosum seeds comprises the following components in percentage by weight: 0-10% of dehydrodiferucyl alcohol, 0-10% of hepetotriol I, 0-10% of hepetolide A, 5-70% of hepetotriol, 0-40% of hepetin, 0-20% of hepetine, 0-30% of hepetine and 0-20% of hepetinone.

The lignan active ingredient composition of herpetospermum pedunculosum seeds in the technical scheme comprises 4-7% of dehydrodiferulyl alcohol, 3-6% of herpetospermol I, 2-5% of herpetospermol A, 15-30% of herpetrione, 10-20% of herpetin, 0-20% of heretrone, 20-30% of heretrone and 8-15% of hereptone.

The preparation method of the lignan active ingredient composition of herpetospermum pedunculosum seeds in the technical scheme comprises the following steps of weighing the components in percentage by weight in the technical scheme, and uniformly mixing to obtain the lignan active ingredient composition of herpetospermum pedunculosum seeds;

the components in the lignan active ingredient composition of the pedunculate herpetospermum seeds are prepared by the following steps:

(1) and preparing the herpetospermum pedunculosum seed extract: extracting coarse powder of Herpetospermum planch seed with 80% ethanol under reflux for 3 times, each time with 5 times of solvent amount and each time for 1 hr, mixing extractive solutions, standing for 24 hr, filtering, recovering ethanol from filtrate until no alcohol smell exists, standing for 12 hr, and separating oil layer. Adjusting pH of water layer to 10-11 with sodium carbonate solid to dissolve, centrifuging, adjusting pH of supernatant with concentrated hydrochloric acid to 2-3, standing for 1 hr, centrifuging, collecting precipitate, repeatedly washing with petroleum ether until filtrate is colorless, and drying under reduced pressure to obtain Herpetospermum Herpetersum seed extract powder;

(2) and one-time preparation:

(2.1) preparation of sample solution:

taking 600mg of the herpetospermum pedunculosum seed extract, adding 12ml of methanol for ultrasonic dissolution, adding 3ml of distilled water for mixing uniformly, and filtering.

(2.2) the preparation method comprises the following steps:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 600 mg; flow rate: 80 mL/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile, gradient elution: 0-25 min, 21-24% acetonitrile; 25-50 min, 24% -25% acetonitrile; 50-75 min, 25% acetonitrile; 75-90 min, 95% acetonitrile; 90-100 min, 21% acetonitrile;

a total of 50 needles were run to give a total of 8 fractions: BLG-A, BLG-B, BLG-C, BLG-D, BLG-E, BLG-F, BLG-G, BLG-H, and combining the same fractions;

(2.3) fraction post-treatment:

the method for extracting the fraction by adopting ethyl acetate comprises the following specific steps: distilling the distillate under reduced pressure, rotary-steaming at 10-15 deg.C for recovering acetonitrile, adding ethyl acetate into the rotary-steaming solution, extracting for 3 times, mixing the extractive solutions, rotary-steaming at 10-15 deg.C for recovering ethyl acetate, rotary-steaming to dry, dissolving with methanol, and transferring into a glass bottle for drying with nitrogen; wherein the proportion of ethyl acetate added during the extraction of the fractions received by the preparation and purification of the BLG-A to BLG-G parts is 13 percent of the volume of the fractions, and the proportion of ethyl acetate added during the preparation and purification of the BLG-H section is 10 percent of the volume of the fractions;

the first preparation fraction is treated to obtain: 130mg of BLG-A fraction, 210mg of BLG-B fraction, 600mg of BLG-C fraction, 600mg of BLG-D fraction, 1.6G of BLG-E fraction, 2.4G of BLG-F fraction, 500mg of BLG-G fraction;

(3) and secondary preparation:

(3.1), BLG-E, BLG-F, BLG-G moiety:

(3.1.1) preparation of sample solution:

130mg of the BLG-F powder after the primary preparation treatment is taken, 1ml of methanol is added for ultrasonic dissolution, and the mixture is filtered.

(3.1.2) BLG-F part of secondary preparation method:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 130 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile is 78%: 22%, isocratic elution; feeding one needle at 60min, and collecting the fraction of the previous needle;

feeding 19 needles together, respectively receiving 3 fractions of BLG-5, BLG-6 and BLG-7, combining the same fractions, and obtaining 3 monomers or components of BLG-5, namely, heretolideA, BLG-6, namely heretriptione and BLG-7, namely heretriptin;

the components of the three fractions of BLG-E, BLG-F, BLG-G are consistent, only the contents of the components are different, the BLG-E fraction and the BLG-G fraction are respectively prepared twice according to the method for secondarily preparing the BLG-F fraction, and 3 monomers or components of BLG-5, namely, heretolide A, BLG-6, namely heretrione, and BLG-7, namely heretin are obtained;

(3.2), BLG-C part:

(3.2.1) preparation of sample solution:

adding 1ml of methanol into 200mg of BLG-C powder subjected to primary preparation treatment, ultrasonically dissolving, and filtering;

(3.2.2) and a secondary preparation method:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 200 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-30 min, 23-28% acetonitrile; 30-35 min, 28% acetonitrile; 35-45 min, 100% acetonitrile; 45-55 min, 23% acetonitrile;

all go into 3 needles to receive 9 fractions of which BLG-C₆The fractions containing BLG-3 are combined and treated, and the fraction is worked up according to the method of 2.3, wherein BLG-C₆Fraction 138.4mg 74.0%;

(3.3), BLG-D moiety:

the sample dissolving and preparing method of the BLG-D part and the BLG-C part is the same;

receiving 8 fractions of which BLG-D₅The segment is BLG-4, namely, heretocotriol I, the same fractions are combined and processed, and the fraction post-processing is carried out according to the method in 2.3;

(3.4) BLG-H stage secondary preparation method:

(3.4.1) preparation of sample solution:

and (3) performing rotary evaporation on 500mL of ethyl acetate layer extract until the volume is 60mL, and performing ultrasonic mixing.

Chromatographic column Innoval ODS-210 μm 100A; sample loading volume: 3 mL; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile, gradient elution: 0-80 min, 26-41% acetonitrile; 80-90 min, 100% acetonitrile; 90-96 min, 26% acetonitrile;

a total of 7 needles received 9 fractions: BLG-H containing BLG-10 therein₃Fraction 307.5mg 59.9%, BLG-H containing BLG-11₄Fraction 1085.1mg 89.2%, BLG-H containing BLG-12₅Fraction 993.2mg 76.1%;

(4) and preparing for three times:

(4.1)、BLG-C ₆the method comprises the following steps:

(4.1.1), sample dissolution:

secondary preparation of treated BLG-C₆Adding 138mg of powder into 1ml of methanol, ultrasonically dissolving, and filtering;

(4.1.2), the preparation method comprises the following steps:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 138 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-50 min, 20% acetonitrile; 50-56 min, 100% acetonitrile, 56-62 min, 20% acetonitrile;

1 needle was shared, receiving 1 fraction: obtaining 1 monomer BLG-3, namely, dihydrodiferucyl alcohol;

(4.2)、BLG-H ₃the method comprises the following steps:

(4.2.1), sample dissolution:

secondary preparation of treated BLG-H₃Adding 300mg of powder into 1ml of methanol, ultrasonically dissolving, and filtering;

(4.2.2), the preparation method comprises the following steps:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 100 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-62 min, 24% acetonitrile; 62-92 min, 25-26% acetonitrile, 92-98 min, 100% acetonitrile, 98-104 min and 24% acetonitrile;

1 needle was shared, receiving 1 fraction: BLG-10 to obtain 1 monomer BLG-10, namely herecotrone;

(4.3)、BLG-H ₄the method comprises the following steps:

(4.3.1), sample dissolution:

secondary preparation of treated BLG-H₄Adding 220mg of powder into 1ml of methanol, ultrasonically dissolving, and filtering;

(4.3.2), the preparation method comprises the following steps:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 100 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-40 min, 30-35% acetonitrile, 40-46 min, 100% acetonitrile, 46-52 min, 30% acetonitrile;

1 needle was shared, receiving 1 fraction: BLG-11, obtaining 1 monomer BLG-11, namely, the monomer hepetone;

(4.4)、BLG-H ₅the method comprises the following steps:

(4.4.1), sample dissolution:

secondary preparation of treated BLG-H₅990mg of powder, adding 1.5ml of methanol, ultrasonically dissolving, and filtering;

(4.4.2), the preparation method comprises the following steps:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 100 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-40 min, 30-35% acetonitrile, 40-57 min, 35-38% acetonitrile, 57-63 min, 100% acetonitrile, 63-69 min, 30% acetonitrile;

2 needles were co-fed, receiving 1 fraction: BLG-12, the same fractions are combined and processed to obtain 1 monomer BLG-12, namely, heretenone.

The dosage form prepared from the lignan effective component composition of herpetospermum pedunculosum seeds in the technical scheme comprises the lignan effective component composition of herpetospermum pedunculosum seeds in the technical scheme and a pharmaceutically acceptable carrier; the dosage form is capsule, tablet, pill, dripping pill, granule, soft capsule, pellicle, unguent, spray, aerosol, patch, gel or injection.

The dosage form of the above technical scheme, wherein: the pharmaceutically acceptable carrier is selected from one or a combination of more than two of starch, microcrystalline cellulose, lactose, sucrose, powdered sugar, dextrin, cellulose derivatives, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, stearic acid and salts thereof, talcum powder, polyethylene glycol, beeswax, vegetable oil, animal glue, vegetable glue, carbomer resin, sodium carboxymethyl cellulose, polyvinyl alcohol, acrylate polymers, polyacrylic acid, sodium polyacrylate, polyvinylpyrrolidone, glycerol, propylene glycol, sorbitol, triethylamine, azone, hydroxypropyl-beta-cyclodextrin and tween-80.

The application of the lignan effective component composition of herpetospermum pedunculosum seeds in the technical scheme in preparing the medicine for treating hepatitis B.

The invention has the following beneficial effects:

1. the invention adopts the preparation liquid phase technology to separate and prepare 8 lignans active ingredients in the herpetospermum pedunculosum seeds, the purity of each ingredient can reach more than 90 percent, and the prepared herpetospermum pedunculosum seed lignans active ingredient composition and the dosage form thereof have definite ingredients and controllable quality, and can meet the medication requirements.

2. The prepared herpetospermum pedunculosum seed lignan effective component composition and the preparation thereof can be used for treating liver injury, hepatitis B and hepatic fibrosis and have better treatment effect; the invention carries out preliminary evaluation on the treatment effect of the traditional Chinese medicine composition on hepatitis B and hepatic fibrosis through experiments.

Description of the drawings:

1. FIG. 1 is a typical chromatogram of a single preparation of an extract of Herpetospermum seeds.

2. FIG. 2 is a representative chromatogram of a second preparation of a partial fraction of BLG-F.

3. FIG. 3 is a representative chromatogram of a second preparation of a partial fraction of BLG-C.

4. FIG. 4 is a representative chromatogram of a second preparation of a partial fraction of BLG-D.

5. FIG. 5 is a representative chromatogram of a second preparation of a partial fraction of BLG-H.

6. FIG. 6 shows BLG-C₆Representative chromatograms of three fractional preparations.

7. FIG. 7 shows BLG-H₃Representative chromatograms of three fractional preparations.

8. FIG. 8 shows BLG-H₄Representative chromatograms of three fractional preparations.

9. FIG. 9 shows BLG-H₅Representative chromatograms of three fractional preparations.

the specific implementation mode is as follows:

In order to facilitate understanding of the technical scheme of the invention, the following provides a composition of lignan active ingredients of pedunculate herpetospermum seeds, a preparation method thereof and application thereof as further description.

Example 1:preparation of herpetospermum pedunculosum lignan compound:

1. the instrument comprises the following steps:

agilent 1100 high performance liquid chromatograph (Agilent corporation, usa); high pressure preparation integrated machine (Tianjin Bonnajiel technologies, Inc.); EXRE-20L constant temperature water oil bath (Shanghai Shajie experiment equipment Co., Ltd.); RE-2000A rotary evaporator (Shanghai Yangrong Biochemical Instrument factory); model SHB-B95A circulating water type multipurpose vacuum pump (shanghai yukang scientific instruments ltd); an SHB-III circulating water type multipurpose vacuum pump (Shanghai Shajie laboratory Equipment Co., Ltd.); nitrogen blowing instruments (Tianjin Bora Aijiel technologies, Inc.); FD8-8 freeze dryer; FA1004 electronic balance (shanghai yueping scientific instruments ltd); EX125DZH electronic balance (aohaus instruments (chang.)) inc.

2. Preparing the herpetospermum pedunculosum seed extract:

extracting coarse powder of Herpetospermum planch seed with 80% ethanol under reflux for 3 times, each time with 5 times of solvent amount and each time for 1 hr, mixing extractive solutions, standing for 24 hr, filtering, recovering ethanol from filtrate until no alcohol smell exists, standing for 12 hr, and separating oil layer. Adjusting pH of water layer to 10-11 with sodium carbonate solid to dissolve, centrifuging, adjusting pH of supernatant with concentrated hydrochloric acid to 2-3, standing for 1 hr, centrifuging, collecting precipitate, repeatedly washing with petroleum ether until filtrate is colorless, and drying under reduced pressure to obtain Herpetospermum Herpetersum seed extract powder.

3. Primary preparation:

3.1, preparation of a sample solution:

3.2, the preparation method comprises the following steps:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 600 mg; flow rate: 80 mL/min; detection wavelength: 280nm/254 nm; mobile phase: a (water) -B (acetonitrile), gradient elution: (0-25 min, 21-24% B, 25-50 min, 24-25% B, 50-75 min, 25% B, 75-90 min, 95% B, 90-100 min, 21% B).

A total of 50 needles received the 8 fractions in fig. 1: BLG-A, BLG-B, BLG-C, BLG-D, BLG-E, BLG-F, BLG-G, BLG-H, and combining the same fractions; in the graph 1, the red line is 280nm detection wavelength, the blue line is 254nm detection wavelength (the red line and the blue line are the same as those in the graphs 2-9), and as can be seen from the graph 1, BLG-H is a fraction washed off by high proportion acetonitrile after 75min, and the fraction is extracted and concentrated after enrichment to prepare the product.

3.3, fraction post-treatment:

the method for extracting the fraction by adopting ethyl acetate comprises the following specific steps: distilling the distillate at reduced pressure and rotary steaming at 10-15 ℃ to recover acetonitrile, adding ethyl acetate in a certain proportion into the rotary steaming liquid (the proportion of the ethyl acetate added during the extraction of the distillate received by the preparation and purification of the BLG-A-BLG parts is 13% of the volume of the distillate, and the proportion of the ethyl acetate added during the preparation and purification of the H section is 10% of the volume of the distillate) to extract for 3 times, combining the extract liquids, reducing the pressure and rotary steaming at 10-15 ℃ to recover the ethyl acetate, rotary steaming until the ethyl acetate is dried, dissolving the ethyl acetate by using methanol, and transferring the ethyl acetate.

The first preparation fraction is treated to obtain: 130mg of BLG-A fraction, 210mg of BLG-B fraction, 600mg of BLG-C fraction, 600mg of BLG-D fraction, 1.6G of BLG-E fraction, 2.4G of BLG-F fraction, 500mg of BLG-G fraction.

4. Secondary preparation:

4.1, BLG-E, BLG-F, BLG-G moiety:

4.1.1, preparation of sample solution:

4.1.2 BLG-F part of the secondary preparation method:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 130 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: a (water) -B (acetonitrile) (78%: 22%) isocratic elution. After 60min, one needle is injected, and the fraction of the previous needle is collected.

19 needles are added together, the chromatogram in the preparation process is shown in figure 2, 3 fractions BLG-5 (namely, hetetolide A), BLG-6 (namely, hetetrenone) and BLG-7 (namely, hetetin) are received, the same fractions are combined and processed to obtain 3 monomers or components, the specific purity, the quality and the like are shown in table 1, 1 in figure 2 is BLG-5, 2 is BLG-6, and 3 is BLG-7.

4.2, BLG-C part:

4.2.1, preparation of sample solution:

the powder after the primary preparation treatment was 200mg, dissolved in 1ml of methanol by sonication, and filtered.

4.2.2, a secondary preparation method:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 200 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; and (3) carrying out gradient elution on the mobile phase A (water) -B (acetonitrile) (0-30 min, 23-28% of B, 30-35 min, 28% of B, 35-45 min, 100% of B, 45-55 min and 23% of B).

3 needles are added in total, the chromatogram in the preparation process is shown in figure 3, 9 fractions are received, 6 in figure 3 is BLG-C₆，BLG-C ₆The stage contains BLG-3, the same fractionCombining, and performing fraction post-treatment according to 3.3, wherein BLG-C₆Portion 138.4mg 74.0%.

4.3, BLG-D part:

the sample dissolution and preparation methods of the BLG-D part and the BLG-C part are the same.

The chromatogram obtained in the preparation process is shown in FIG. 4, and 8 fractions are received, 5 in FIG. 4 is BLG-D₅，BLG-D ₅The segment is BLG-4, namely, heretocotriol I, the same fractions are combined and processed, and the fraction post-processing is carried out according to the method in 3.3, and the specific purity, the quality and the like are shown in the table 1.

4.4, BLG-H section secondary preparation method:

4.4.1, preparation of sample solution:

Chromatographic column Innoval ODS-210 μm 100A; sample loading volume: 3 mL; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: gradient elution of A (water) -B (acetonitrile) (0-80 min, 26-41% B, 80-90 min, 100% B, 90-96 min, 26% B).

A total of 7 needles were run and the preparative process chromatogram is shown in fig. 5, receiving 9 fractions: in FIG. 5,3 is BLG-H₃And 4 is BLG-H₄And 5 is BLG-H₅(ii) a Wherein BLG-H₃(containing BLG-10) portion 307.5mg 59.9%, BLG-H₄(containing BLG-11) fraction 1085.1mg 89.2%, BLG-H₅(containing BLG-12) portion 993.2mg 76.1%.

5. And (3) preparation for three times:

5.1、BLG-C ₆the method comprises the following steps:

5.1.1, sample dissolution:

138mg of the twice-prepared powder was dissolved in 1ml of methanol by ultrasonic wave, and filtered.

5.1.2, the preparation method comprises the following steps:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 138 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: gradient elution of A (water) -B (acetonitrile) (0-50 min, 20% B; 50-56 min, 100% B, 56-62 min, 20% B).

The chromatogram of the preparation process is shown in fig. 6, and the arrows indicate that the target fraction is received, and 1 fraction is received: BLG-3, 1 monomer BLG-3, namely, dihydrodiferucyl alcohol, is obtained, and the purity and the quality are shown in table 1.

5.2、BLG-H ₃The method comprises the following steps:

5.2.1, sample dissolution:

300mg of the powder after the secondary preparation treatment was added with 1ml of methanol to be ultrasonically dissolved and filtered.

5.2.2, the preparation method comprises the following steps:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 100 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: and (3) carrying out gradient elution on the A (water) -B (acetonitrile) (0-62 min, 24% of B, 62-92 min, 25-26% of B, 92-98 min, 100% of B, 98-104 min and 24% of B).

The chromatogram of the preparation process is shown in fig. 7, and the arrows indicate that the target fraction is received, and 1 fraction is received: BLG-10 to obtain 1 monomer BLG-10, i.e. hereetrone, and the specific purity and quality are shown in Table 1.

5.3、BLG-H ₄The method comprises the following steps:

5.3.1, sample dissolution:

the powder after the secondary preparation treatment was 220mg, dissolved in 1ml of methanol by ultrasonic wave, and filtered.

5.3.2, the preparation method comprises the following steps:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 100 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: gradient elution of A (water) -B (acetonitrile) (0-40 min, 30-35% of B, 40-46 min, 100% of B, 46-52 min and 30% of B).

The chromatogram of the preparation process is shown in fig. 8, and the arrows indicate that the target fraction is received, and 1 fraction is received: BLG-11, 1 monomer BLG-11, namely, the herethone, and the specific purity and quality are shown in Table 1.

5.4、BLG-H ₅The method comprises the following steps:

5.4.1, sample dissolution:

990mg of the twice-prepared powder was dissolved in 1.5ml of methanol by ultrasonic wave, followed by filtration.

5.4.2, the preparation method comprises the following steps:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 100 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: gradient elution of A (water) -B (acetonitrile) (0-40 min, 30-35% of B, 40-57 min, 35-38% of B, 57-63 min, 100% of B, 63-69 min and 30% of B).

2 needles were added together, and the chromatogram of the preparation process is shown in FIG. 9, with the arrows indicating the reception of the target fraction, and 1 fraction: BLG-12, the same fractions are combined and processed to obtain 1 monomer BLG-12, namely, heretenone, and the specific purity and quality are shown in Table 1.

Table 18 lignan compound sample purities and preparation amounts

Numbering	Purity of	Preparation amount
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

Note: the purity is calculated by HPLC area normalization method

6. Structural analysis of the compound:

1. compound 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):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):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)。

The above data are In substantial agreement with the documents In S J, Seo K H, Song N Y, et al, Lignans and neolignes from the series of Vibrunum erosum and the first neuroprotection and anti-inflammatory activity [ J ]. Archives of pharmaceutical Research,2015,38(1):26-34. the compound was identified as dehydrobiconical alcohol (dehydroconiferyl alcohol).

2. Compound 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):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):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)。

The above data are essentially in agreement with the references Mei Z, Yun D, Zhang H, et al, two new coumarins from Herpetospermum caudigerum [ J ]. Chemical & Pharmaceutical Bulletin,2008,56(2):192. this compound was identified as heretolideA (Boragin A).

3. Compound 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)7.66(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):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)。

The above data are essentially in agreement with the documents Kaouadji M, Favrebonvin J, Sarrazin F, et al Herpettron, Another quaternary Ligniod from Herpetospermum caudigerum Seeds [ J ]. Journal of Natural Products,1987,50(6): 1089-.

4. Compound BLG-7: ESI-MS (C)₃₀H ₃₄O ₉)m/z 561.21[M+Na] ⁺；UVλ _max(CH ₃OH)nm:228,280；IR(KBr)cm ^-13435,1614,1604,1518,1276,1212,1031 (largest literature);¹H-NMR(600MHz,CD ₃OD):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):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)。

the above data are substantially in agreement with the documents Yuan H L, Liu Y, ZHao Y L, et al, hereptin, a new biologically active lipid isolated from heretospermum caudigerum [ J ]. Journal of Chinese Pharmaceutical Sciences,2005,14(3):140-143. the compound was identified as hereptin.

5. Compound 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):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):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)。

The above data are in substantial agreement with the documents Kaouadji M, Favrebonvin J, Sarrazin F, et al Herpettron, Another quaternary Ligniod from Herpetospermum caudigerum Seeds [ J ]. Journal of Natural Products,1987,50(6): 1089-.

6. Compound 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):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):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)。

The data are basically consistent with the data in the documents Zhang Mei, Dong Xiao Lian, Deng Ying 36191, and the like, a new sesquinorlignan [ J ] in Tibetan medicine Herpesone seeds, the Proc. of pharmacy 2006,41(7):659-661, and the compound is identified as herpesone.

7. Compound 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):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):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)。

The above data are substantially in agreement with the references Yu J Q, Hang W, Duan W J, et al, two new anti-HBV lignans from Herpesspermum caudigerum [ J ]. Phytochemistry Letters,2014,10:230-

8. Compound 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):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):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)。

Based on the above data, in combination with the documents Fan Y, Zhang H J, Zhang Y, et al, a hepatitis B virus inhibition, neolignan from heretospermum caudigerum [ J ]. Chemical & Pharmaceutical Bulletin,2010,41(33): no-No. and sequoia, tibetans poliumoside component system research [ D ]. southwest traffic university, 2014.

Example 2:the composition of the lignan effective components of the herpetospermum pedunculosum seeds comprises the following components:

weighing 65mg of herpetospermum pedunculosum A and 35mg of herpetospermum pedunculosum L, and mixing to obtain the lignan effective component composition of herpetospermum pedunculosum seeds.

Example 3:the composition of the lignan effective components of the herpetospermum pedunculosum seeds comprises the following components:

weighing herpetospermum elegans A30 mg, herpetospermone 20mg, herpetospermone 15mg, herpetospermone 20mg and herpetospermum elegans 15mg, and mixing to obtain the lignan effective component composition of herpetospermum elegans seeds.

Example 4:the composition of the lignan effective components of the herpetospermum pedunculosum seeds comprises the following components:

weighing 5mg of dehydrodiconiferol, 5mg of herpetospermum pedunculatum L, 15mg of herpetrone, 15mg of herpetospermum pedunculatum L and 15mg of herpetospermum pedunculatum L, and mixing uniformly to obtain the lignan effective component composition of herpetospermum pedunculatum L.

Example 5:the composition of the lignan effective components of the herpetospermum pedunculosum seeds comprises the following components:

weighing 7mg of dehydrodiconiferol, herpetospermum pedunculatum I3mg, herpetospermum pedunculatum lactone A2mg, herpetospermum pedunculatum A23 mg, herpetospermum pedunculatum element 12mg, herpetospermum pedunculatum 20mg, herpetospermum pedunculatum ketone 23mg and herpetospermum pedunculatum element 10mg, and mixing uniformly to obtain the lignan effective component composition of herpetospermum pedunculatum seeds.

Example 6:the composition of the lignan effective components of the herpetospermum pedunculosum seeds comprises the following components:

weighing 5mg of dehydrodiconiferol, 5mg of herpetospermum pedunculatum L, 5mg of herpetospermum pedunculatum L, 20mg of herpetospermum pedunculatum L, 10mg of herpetrone 15mg, 25mg of herpetospermum pedunculatum L and 15mg of herpetospermum pedunculatum L, and mixing uniformly to obtain the lignan effective component composition of herpetospermum pedunculatum L.

Example 7:the composition of the lignan effective components of the herpetospermum pedunculosum seeds comprises the following components:

weighing 6mg of dehydrodiconiferol, I5mg, A4mg, 25mg of herpetospermum pedunculatum element A, 12mg of herpetrolene 13mg, 25mg of herpetone and 10mg of herpetospermum pedunculatum element B, and mixing uniformly to obtain the lignan effective component composition of herpetospermum pedunculatum seeds.

The beneficial effects of the lignan active ingredient composition of pedunculate herpetospermum seeds according to the present invention will be described below by specific experimental examples.

Experimental example 1:the lignan effective component composition of herpetospermum pedunculosum seeds has the in vivo and in vitro anti-hepatitis B effect:

firstly, materials:

1. cells, viral strains and animals:

a2.2.15 cell line of HBV transfected human hepatoma cells (HepG2) was constructed by the U.S. Mount Sinai medical center and subcultured in the virus chamber of the institute of Chinese medical science and Biotechnology.

The virus strain duck hepatitis B virus DNA (DHBV-DNA) strong positive serum is collected from Shanghai Ma duck and preserved at-70 deg.C.

The Beijing duck of 1 day old animal is provided by Beijing advancing breeding duck farm.

2. Drugs and reagents:

test drugs (8 ingredients in table 2, prepared from "example 1"); lamivudine (3TC, produced by glanruikang gmbh, uk) is supplied from an outpatient pharmacy of general air force hospitals; the HBsAg and HBeAg enzyme linked immunosorbent assay kit is provided by Huamei biology company; alpha-³²P-dCTP was supplied by Beijing Yahui biomedical engineering Inc.; a random primer kit for probe labeling was supplied by Promega; SePhadex G-50, Ficoll PVP supplied by Pharmacia, Sweden; nitrocellulose membrane (0.45 μm) was supplied by Amersham; fetal bovine serum was supplied by Hyclone Lab corporation, usa; other reagents were all commercially available analytical grade.

3. The instrument comprises the following steps:

PHOMO microplate reader (angstrom laboratory instruments (zheng zhou) ltd); BH2 inverted microscope (olympus optics); 800D carbon dioxide incubator (Thermo corporation, USA); MSC-Advantag Biosafety Cabinet (Thermo corporation, USA); l-550 low speed desk high capacity centrifuges (Hunan instruments group); BSA124S electronic balance (sedoli scientific instruments (beijing) ltd).

Secondly, the method comprises the following steps:

1. preparing different lignanoid component compositions of pedunculate herpetospermum seeds:

weighing different amounts of lignans, mixing uniformly according to the prescription in Table 2, and making into composition of different lignans, F1-F6 for use in total of 6.

Table 2 prescription design

Note: "-" indicates that the formulation does not contain the ingredient.

2. F1-F6 lignan composition in vitro anti-hepatitis B activity evaluation:

2.1, cell culture:

2.2.15 cells were cultured in DMEM medium (0.03% glutamine, 380. mu.g/mL G-418, 100IU/mL penicillin, 100IU/mL streptomycin) containing 10% fetal bovine serum at 37 ℃ with 5% CO₂Culturing in an incubator under the condition.

2.2, cytotoxicity test:

F1-F6 lignan composition and Herpetospermum pedunculatum seed extract (T) (example 1, "2, preparation of Herpetospermum pedunculatum seed extract") were prepared into 4000. mu.g/mL solution in DMEM medium, and then diluted with medium 2-fold gradient to 7 mass concentrations of 4000, 2000, 1000, 800, 400, 200, 100. mu.g/mL for use. At 2X 10⁴cells/mL were seeded in 96-well plates at 100. mu.L/well with 5% CO at 37 ℃₂After 24 hours of culture, the supernatant was aspirated, and 100. mu.L of each of the culture solutions containing drugs at different concentrations was added to each of 3 wells, and a drug-free cell control group and a blank well were placed. 5% CO at 37 ℃₂After 72h incubation, 10. mu.L MTT (5mg/mL) was added to each well and incubated for 4h, the incubation was terminated, the supernatant was discarded, and 150. mu.L DMSO was added and shaken for 10 min. OD at 570nm was measured with a microplate reader. Calculating the inhibition rate of the drug on 2.2.15 cells, and calculating the median Toxic Concentration (TC)₅₀) And maximum non-Toxic Concentration (TC)₀)。

2.3 HBsAg and HBeAg inhibition test:

at 8X 10⁴Each/mL cell was inoculated into 24-well plates, 1mL per well, 5% CO at 37 ℃₂Culturing for 24 hr, removing supernatant, diluting with 1mL of test liquid 2 times of nontoxic concentration, adding 5 dilutions of 400, 200, 100, 50, and 25 μ g/mL, each concentration being 3 wells, and adding 5% CO at 37 deg.C₂Culturing, changing the same concentration liquid medicine every 4 days, and setting drug-free cell control group. Collecting the culture solution containing medicine on day 8, and freezing and storing at-20 deg.C. And detecting supernatant HBsAg and HBeAg by an ELISA method. And (4) measuring the OD value of each hole by using an enzyme-labeling instrument according to the operation of the kit specification, and calculating the inhibition rate.

2.4 effects of F1-F6 lignan composition on duck hepatitis B virus in duck:

injecting DHBV-DNA positive duck serum of Shanghai Ma duck (0.3 mL each) into Beijing duck of 1 year via leg and shin vein of duck, collecting blood 7 days after infection, separating serum, and detecting DHBV-DNA content in serum. After the serum of the duckling is detected to be positive by DHBV, the ducks are randomly divided into 9 groups: the virus control group, 3TC group (50mg/kg), F1-F6 lignan composition group and Herpetospermum bracteatum extract group (T group), F1-F6 and T group all administered at a dose of 100 mg/kg. The administration is performed by gavage 2 times a day. The virus control group was administered with the same volume of physiological saline for 10 consecutive days. Blood was taken from the duck tibia vein before administration (T0), 5 days after drug treatment (T5), 10 days after drug treatment (T10) and 3 days after drug withdrawal (P3), and serum was isolated and stored at-70 ℃ for testing. And (3) taking the duck serum to be detected, and measuring the dynamic state of the DHBV-DNA level in the duck serum at the same time point of each batch of time films. According to the kit instructions, using³²P marks DHBV-DNA probe, and makes duck serum spot hybridization, autoradiography film spot, enzyme labeling instrument measures OD value (filter is 490nm), calculates serum DHBV-DNA optical density, and uses hybridization spot OD value as specimen DHBV-DNA horizontal value. And comparing the average value of the serum DHBV-DNA of the ducks of the same group before and after the administration, and calculating the inhibition rate of the serum DHBV-DNA of each administration group at different time.

2.5, data analysis:

the obtained data are

Showing that the mean comparison between two groups is carried out by adopting t test, the mean comparison between multiple groups is carried out by adopting one-factor variance analysis, the statistical analysis of data is carried out by adopting SPSS17.0 software, P<0.05 was considered to have a significant difference.

Thirdly, obtaining a result:

3.1 cytotoxicity of F1-F6 lignan composition:

the cytotoxic effect of the F1-F6 lignan composition and the pedunculate herpetospermum seed extract (T) on 2.2.15 cells is shown in Table 3. As shown in Table 3, the toxic concentration of F1-F6 lignan composition and the pedunculate herpetospermum seed extract on the 2.2.15 cell half is in the range of 924-1324 μ g/mL, and the maximum non-toxic dose is more than 400 μ g/mL.

TABLE 3 cytotoxic Effect of F1-F6 lignan composition on 2.2.15 cells

3.2, the F1-F6 lignan composition has the effect of inhibiting HBsAg and HBeAg:

as is clear from tables 4 and 5, the F1-F6 lignan composition and the Aleuritopteris pedunculata seed extract have strong inhibitory effects on HBsAg and HBeAg secreted from 2.2.15 cells, and show certain concentration dependence. Compared with the extract group (T group), the F1-F6 lignan composition has no obvious difference on the inhibitory action of HBsAg and HBeAg secreted by 2.2.15 cells, and shows that each composition and the pedunculate herpetospermum seed extract have the same anti-hepatitis B effect.

TABLE 4 inhibition of HBeAg by F1-F6 compositions on 2.2.15 cells

Note: 3TC has only one dose group, 200. mu.g/mL.

TABLE 5 inhibition of HBsAg in 2.2.15 cells by F1-F6 compositions

Note: 3TC has only one dose group, 200. mu.g/mL.

3.3 effects of F1-F6 lignan composition on DHBV infection of Duck serum DHBV-DNA:

TABLE 6 inhibition of DHBV-DNA in duck serum infected with DHBV by F1-F6 composition

Note: comparison with the control group:^**P＜0.01；

DHBV-DNA is all positive after the duckling is infected by hepatitis B virus, and the level of the DHBV-DNA in the serum of a virus control group has no obvious change in the experimental process. The positive drug 3TC can obviously reduce the level of DHBV-DNA in serum (P is less than 0.01) on the 5 th and 10 th days after the administration, but the inhibition rate is reduced to 24.98% when the drug is stopped for 3 d. Compared with the composition before self administration, the F1-F6 lignan composition and the herpetospermum pedunculosum seed extract can obviously reduce the level of DHBV-DNA (P is less than 0.01) in serum on the 5 th day, the 10 th day and the 3d after the administration, and the F1-F6 lignan composition has no obvious difference compared with the herpetospermum pedunculosum seed extract (T group), and the results are shown in the table 6.

As can be seen from the experimental examples, hepatitis B is caused by the hepatitis B virus HBV, is an infectious disease mainly caused by hepatitis B virus, and is easily converted into chronic hepatitis, even into cirrhosis and liver cancer. The continuous replication of hepatitis B virus in liver cells can cause the host to generate immune response, which causes the immunopathological damage of cells and is the pathological basis for chronic hepatitis. Therefore, the antiviral treatment aiming at the chronic infection of the hepatitis B virus is a main measure for blocking the development of the chronic hepatitis B.

2.2.15 cell and duck hepatitis B virus infection model is the currently accepted ideal model for studying hepatitis B pathogenesis, virus replication process and screening effective drugs. The study selects the two models to evaluate the anti-hepatitis B virus activity of the F1-F6 lignan composition and the herpetospermum pedunculosum seed extract. Under the same dosage, the in vitro and in vivo anti-hepatitis B effect of the F1-F6 lignan composition is not obviously different from that of the herpetospermum pedunculosum seeds extract (group T), which shows that the F1-F6 lignan composition and the herpetospermum pedunculosum seeds extract (group T) have consistent in vitro and in vivo anti-hepatitis B effect.

Experimental example 2:the lignan effective component composition of the herpetospermum pedunculosum seeds has the effect of resisting hepatic fibrosis:

firstly, materials and instruments:

1. reagent:

alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and alkaline phosphatase (ALP) determination kits were purchased from Shenzhen Merrier biomedical electronics, Inc.; hydroxyproline acid (Hyp), Hyaluronic Acid (HA) kit, purchased from Huamei bioengineering, Inc.; transforming growth factor-beta₁(TGF-β ₁) Synthesis provided by Life Technology.

2. The instrument comprises the following steps:

PHOMO microplate reader (angstrom laboratory instruments (zheng zhou) ltd); BH2 inverted microscope (olympus optics corporation); 800D carbon dioxide incubator (Thermo corporation, USA); MSC-Advantag Biosafety Cabinet (Thermo corporation, USA); l-550 low speed desk high capacity centrifuges (Hunan instruments group); BSA124S electronic balance (sedoli scientific instruments (beijing) ltd); full-automatic biochemical analyzer (Shenzhen Redu Life technologies, Inc.).

3. Experimental animals:

SD rat with weight of 200 + -20 g, half of male and female, SPF grade, provided by laboratory animal center of military medical academy of sciences of people liberation military, production license number SCXK (military) 2012-0004. The experimental animals are adaptively raised in experimental animal rooms of a third good and two hospital of China people liberation army for 3 days, the experiment is started after no abnormity is observed, and the experimental animals use license numbers SYXK (army) 2012-0010.

II, the method and the result are as follows:

weighing different amounts of lignans, mixing uniformly according to the prescription in Table 7, and making into composition of different lignans, F1-F6 for use in total of 6.

Table 7 prescription design

2. Evaluation of in vitro anti-hepatic fibrosis activity:

2.1, cell culture: HSC-T6 cells were incubated in DMEM medium containing 10% fetal bovine serum at 37 ℃ in 5% CO 2.

2.2, detecting cell proliferation by a CCK8 method, taking HSC-T6 cells in a logarithmic growth phase, digesting the cells by pancreatin, sucking the cells into a 15mL plastic centrifuge tube, centrifuging the cells for 5min at a speed of 1000r/min, blowing the cells into a single cell suspension, removing supernatant, and suspending the cells by gravity. The cell concentration was adjusted to about 3X 104/mL, the cell suspension was seeded into a 96-well cell culture plate at 100. mu.L/well, and the 96-well plate was incubated overnight at 37 ℃ in a 5% CO2 incubator. The experiment was divided into a blank group (culture medium only added), a control group, F1-F6 and Herpetospermum seeds extract (T) (fruit)"2, preparation of herpetospermum pedunculosum seed extract" in example 1) group (5, 10, 25, 50, 100. mu.g/mL), positive drug colchicine (0.1. mu.g/mL). And after the cells adhere to the wall, the old solution is sucked away, and serum culture solution containing different drugs and DMEM are added according to groups for culture, wherein each group comprises 6 multiple wells. After 24 and 48 hours of drug treatment, 10 microliter of CCK8 solution was added to each well, the culture plate was incubated in an incubator for 2.5 hours, and then the absorbance (A) at 450nm was measured with a microplate reader, and the cell proliferation inhibition rate was calculated according to the formula. Cell proliferation inhibition rate of 1- (A)_{Experiment of}－A _{Blank space})/(A _Control－A _{Blank space}). The results are shown in tables 7 and 8.

As can be seen from tables 8 and 9, the Herpetospermum pedunculatum seeds F1-F6 lignan composition has a significant inhibitory effect on the proliferation of HSC-T6 cells within the range of 10-100 μ g/mL and shows concentration dependence, and the inhibitory effect on the proliferation of HSC-T6 cells of each composition is not significantly different from that of the Herpetospermum pedunculatum seed extract (T), indicating that each composition has the same anti-hepatic fibrosis effect as the Herpetospermum pedunculatum seed extract.

TABLE 8 inhibition of HSC-T6 cell culture for 24h proliferation by F1-F6 compositions

Note: colchicine had only one dose group, 0.1. mu.g/mL.

TABLE 9 inhibition of HSC-T6 cell culture for 48h proliferation by F1-F6 compositions

Note: colchicine had only one dose group, 0.1. mu.g/mL.

3. Evaluation of in vivo anti-hepatic fibrosis activity:

3.1, grouping and administration:

the rats were weighed and randomly divided into a blank control group, a model group, an F1-F6 administration group (200mg/kg, administered suspended in 0.5% CMC-Na solution), a colchicine group (positive control, 0.1mg/kg), and 8 rats per group. Except for the blank control group, rats in each group were first injected subcutaneously (sc) with 5mL/kg of 40% CCl₄Soybean oil solution, sc 3mL/kg 40% CCl after 3d₄The soybean oil solution was administered 2 times per week (Wednesday and Friday), and the same dosage of the control group sc was administered for 6 weeks. Starting from week 7, the control group and the model group were administered with the same dose of 0.5% CMC-Na solvent solution per day by gavage (ig) with a dose of 10mL/kg, the remaining administration groups were administered with the test solution per day at a dose corresponding to ig for 6 weeks, and the total of 12 weeks were administered by molding and adding. Fasting for 24h after the last administration, weighing the rat, taking blood from abdominal aorta, centrifuging to obtain upper layer serum, storing at-20 deg.C for detection, taking fresh liver, weighing, cutting off a small part of 10% formalin, and soaking for detection.

3.2, liver index:

before sacrifice, the weight of the rat is weighed, the liver is removed, the weight is weighed immediately after wiping the rat with filter paper, and the liver index is calculated. Liver index is liver mass (g)/body weight (g) × 100. The results are shown in Table 10.

TABLE 10F 1-F6 composition vs CCl₄Influence of liver index of hepatic fibrosis rat (± s, n ═ 8)

Note: comparison with blank control:^**p is less than 0.01; comparison with model control group^#P＜0.05， ^##P＜0.01。

As can be seen from Table 10, the liver weight and liver index of the model control group were significantly increased (P < 0.05 or P < 0.01) compared to the control group; compared with the model control group, the liver weights and liver indexes of the F1-F6 group, the extract group (T group) and the colchicine group are all significantly reduced (P < 0.01 or P < 0.05), and the F1-F6 groups have no significant difference compared with the extract group (T group). The results show that each administration group can remarkably reduce the liver swelling caused by the hepatic fibrosis and reduce the liver index.

3.3, liver function:

the results of the measurement of the levels of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and alkaline phosphatase (ALP), which are liver function indices, in rat serum by using an automatic biochemical analyzer are shown in table 11.

As can be seen from Table 11, the serum levels of ALT, AST and ALP in the model rats were significantly increased (P < 0.05 or P < 0.01) compared with the control group. ALT, AST, ALP levels were significantly reduced (P < 0.05 or P < 0.01) in the F1-F6, extract (T) and colchicine groups compared to the model control group, and there was no significant difference between the F1-F6 groups and the T group.

TABLE 11 influence of F1-F6 compositions on serum ALT, AST and ALP levels in liver fibrosis rats

Note: comparison with blank control:^*P＜0.05， ^**p is less than 0.01; comparison with model control group^#P＜0.05， ^##P＜0.01。

3.4 hepatic fibrosis index detection:

according to the operation of an ELISA kit specification, an enzyme-linked immunosorbent assay (ELISA) instrument is used for detecting Hyaluronic Acid (HA), hydroxyproline acid (Hyp) and transforming growth factor-beta in rat serum₁(TGF-β ₁) The results are shown in the table12。

TABLE 12F 1-F6 compositions on liver fibrosis rat serum HA, Hyp and TGF-. beta.₁Influence of the amount

As can be seen from Table 12, HA, Hyp and TGF-. beta.in the serum of the model group rats compared with the control group₁The level of (P < 0.01) is significantly increased. HA, Hyp and TGF-beta of F1-F6, extract (T) and colchicine groups compared to model controls₁The levels were all significantly reduced (P < 0.05 or P < 0.01), and there was no significant difference between the F1-F6 groups compared to the T group.

It is known from this experimental example that Hepatic Fibrosis (HF) is a common disease caused by chronic liver disease. The mechanism of HF generation is similar to that of other chronic disease lesions, and fibrosis occurs as a dynamic, reversible process. In the hepatic fibrosis stage, the hepatic fibrosis process can be completely reversed as long as reasonable treatment is carried out, so that the hepatic fibrosis is relieved and even cured.

Hepatic Fibrosis (HF) is a compensatory response caused by inflammation of the liver or tissue repair following injury, and is characterized primarily by massive deposition of extracellular matrix (ECM) within the liver tissue. The activation and apoptosis of Hepatic Stellate Cells (HSCs) are the key to the occurrence and development of hepatic fibrosis, and the activation and proliferation of HSCs promote hepatic fibrosis, inhibit the proliferation and induce the apoptosis of hepatic fibrosis, and can alleviate or even reverse the hepatic fibrosis. The research finds that the extracts of the seeds of the pedunculate herpetospermum fruit with different doses and the composition of the effective components can effectively inhibit the proliferation of the HSC-T6 cells, the dose dependence is presented, and the proliferation inhibition rate of the HSC-T6 cells is obviously increased along with the increase of the drug concentration and the development of the culture time; the results show that the herpetospermum pedunculosum seed extract and the effective component composition have the effect of resisting hepatic fibrosis.

CCl was used in this study₄Induced liver fibrosis model rats evaluate the in vivo anti-hepatic fibrosis effect of the herpetospermum pedunculosum seed extract and the effective component composition. The results show that the extract of the cucurbita pepo seeds and the composition of the effective components can obviously reduce the serum ALT, AST and ALP levels and HA, Hyp and TGF-beta of rats with hepatic fibrosis₁Indicating that the composition of the semen melo extract and the effective components has a positive effect on CCl₄The liver tissue and liver function damage of the rat with hepatic fibrosis have obvious protective action and obvious anti-hepatic fibrosis action, and the anti-hepatic fibrosis mechanism of the rat with hepatic fibrosis is possible to reduce TGF-beta₁Protein expression is relevant.

The research result shows that the in-vivo and in-vitro anti-hepatic fibrosis effects of the herpetospermum pedunculosum seed extract and the effective component composition have no obvious difference under the same dosage, and the result shows that the herpetospermum pedunculosum seed effective component composition can reach the anti-hepatic fibrosis level of the extract, and is more favorable for the opening and quality control of the preparation due to clear components.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims; meanwhile, any equivalent changes, modifications and variations of the above embodiments according to the essential technology of the present invention are within the scope of the technical solution of the present invention.

Claims

The lignan effective component composition of herpetospermum pedunculosum seeds is characterized by comprising the following components in percentage by weight: 0-10% of dehydrodiferucyl alcohol, 0-10% of hepetotriol I, 0-10% of hepetolide A, 5-70% of hepetotriol, 0-40% of hepetin, 0-20% of hepetine, 0-30% of hepetine and 0-20% of hepetinone.
The composition of lignan-containing active ingredient of herpetospermum pedunculosum seeds of claim 1, wherein the composition of lignan-containing active ingredient of herpetospermum pedunculosum seeds has a dihydrodiferulyl alcohol content of 4-7%, a heretoliol I content of 3-6%, a heretolide A content of 2-5%, a hererone content of 15-30%, a hereptin content of 10-20%, a heretrone content of 0-20%, a herethone content of 20-30%, and a heretenone content of 8-15%.
A method for preparing a lignan active ingredient composition of pedunculate herpetospermum seeds of claim 1 or 2, wherein the method comprises the steps of: weighing the components in the weight percentage of claim 1 or 2, and uniformly mixing to obtain the lignan effective component composition of the herpetospermum pedunculosum seeds;

the components in the lignan active ingredient composition of the pedunculate herpetospermum seeds are prepared by the following steps:

(1) and preparing the herpetospermum pedunculosum seed extract: extracting coarse powder of Herpetospermum planch seed with 80% ethanol under reflux for 3 times, each time with 5 times of solvent amount and each time for 1 hr, mixing extractive solutions, standing for 24 hr, filtering, recovering ethanol from filtrate until no alcohol smell exists, standing for 12 hr, and separating oil layer. Adjusting pH of water layer to 10-11 with sodium carbonate solid to dissolve, centrifuging, adjusting pH of supernatant with concentrated hydrochloric acid to 2-3, standing for 1 hr, centrifuging, collecting precipitate, repeatedly washing with petroleum ether until filtrate is colorless, and drying under reduced pressure to obtain Herpetospermum Herpetersum seed extract powder;

(2) and one-time preparation:

(2.1) preparation of sample solution:

taking 600mg of the herpetospermum pedunculosum seed extract, adding 12ml of methanol for ultrasonic dissolution, adding 3ml of distilled water for mixing uniformly, and filtering;

(2.2) the preparation method comprises the following steps:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 600 mg; flow rate: 80 mL/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile, gradient elution: 0-25 min, 21-24% acetonitrile; 25-50 min, 24% -25% acetonitrile; 50-75 min, 25% acetonitrile; 75-90 min, 95% acetonitrile; 90-100 min, 21% acetonitrile;

a total of 50 needles were run to give a total of 8 fractions: BLG-A, BLG-B, BLG-C, BLG-D, BLG-E, BLG-F, BLG-G, BLG-H, and combining the same fractions;

(2.3) fraction post-treatment:

the method for extracting the fraction by adopting ethyl acetate comprises the following specific steps: distilling the distillate under reduced pressure, rotary-steaming at 10-15 deg.C for recovering acetonitrile, adding ethyl acetate into the rotary-steaming solution, extracting for 3 times, mixing the extractive solutions, rotary-steaming at 10-15 deg.C for recovering ethyl acetate, rotary-steaming to dry, dissolving with methanol, and transferring into a glass bottle for drying with nitrogen; wherein the proportion of ethyl acetate added during the extraction of the fractions received by the preparation and purification of the BLG-A to BLG-G parts is 13 percent of the volume of the fractions, and the proportion of ethyl acetate added during the preparation and purification of the BLG-H section is 10 percent of the volume of the fractions;

the first preparation fraction is treated to obtain: 130mg of BLG-A fraction, 210mg of BLG-B fraction, 600mg of BLG-C fraction, 600mg of BLG-D fraction, 1.6G of BLG-E fraction, 2.4G of BLG-F fraction, 500mg of BLG-G fraction;

(3) and secondary preparation:

(3.1), BLG-E, BLG-F, BLG-G moiety:

(3.1.1) preparation of sample solution:

taking 130mg of BLG-F powder subjected to primary preparation treatment, adding 1ml of methanol, ultrasonically dissolving, and filtering;

(3.1.2) BLG-F part of secondary preparation method:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 130 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile is 78%: 22%, isocratic elution; feeding one needle at 60min, and collecting the fraction of the previous needle;

feeding 19 needles together, respectively receiving 3 fractions of BLG-5, BLG-6 and BLG-7, combining the same fractions, and obtaining 3 monomers or components of BLG-5, namely, heretolideA, BLG-6, namely heretriptione and BLG-7, namely heretriptin;

the components of the three fractions of BLG-E, BLG-F, BLG-G are consistent, only the contents of the components are different, the BLG-E fraction and the BLG-G fraction are respectively prepared twice according to the method for secondarily preparing the BLG-F fraction, and 3 monomers or components of BLG-5, namely, heretolide A, BLG-6, namely heretrione, and BLG-7, namely heretin are obtained;

(3.2), BLG-C part:

(3.2.1) preparation of sample solution:

adding 1ml of methanol into 200mg of BLG-C powder subjected to primary preparation treatment, ultrasonically dissolving, and filtering;

(3.2.2) and a secondary preparation method:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 200 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-30 min, 23-28% acetonitrile; 30-35 min, 28% acetonitrile; 35-45 min, 100% acetonitrile; 45-55 min, 23% acetonitrile;

all go into 3 needles to receive 9 fractions of which BLG-C₆The fractions containing BLG-3 are combined and treated, and the fraction is worked up according to the method of 2.3, wherein BLG-C₆Fraction 138.4mg 74.0%;

(3.3), BLG-D moiety:

the sample dissolving and preparing method of the BLG-D part and the BLG-C part is the same;

receiving 8 fractions of which BLG-D₅The segment is BLG-4, namely, heretocotriol I, the same fractions are combined and processed, and the fraction post-processing is carried out according to the method in 2.3;

(3.4) BLG-H stage secondary preparation method:

(3.4.1) preparation of sample solution:

rotatably steaming the 500mL ethyl acetate layer extract liquid to 60mL, and ultrasonically mixing uniformly;

chromatographic column Innoval ODS-210 μm 100A; sample loading volume: 3 mL; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile, gradient elution: 0-80 min, 26-41% acetonitrile; 80-90 min, 100% acetonitrile; 90-96 min, 26% acetonitrile;

a total of 7 needles received 9 fractions: BLG-H containing BLG-10 therein₃Fraction 307.5mg 59.9%, BLG-H containing BLG-11₄Fraction 1085.1mg 89.2%, BLG-H containing BLG-12₅Fraction 993.2mg 76.1%;

(4) and preparing for three times:

(4.1)、BLG-C ₆the method comprises the following steps:

(4.1.1), sample dissolution:

secondary preparation of treated BLG-C₆Adding 138mg of powder into 1ml of methanol, ultrasonically dissolving, and filtering;

(4.1.2), the preparation method comprises the following steps:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 138 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-50 min, 20% acetonitrile; 50-56 min, 100% acetonitrile, 56-62 min, 20% acetonitrile;

1 needle was shared, receiving 1 fraction: obtaining 1 monomer BLG-3, namely, dihydrodiferucyl alcohol;

(4.2)、BLG-H ₃the method comprises the following steps:

(4.2.1), sample dissolution:

secondary preparation of treated BLG-H₃Adding 300mg of powder into 1ml of methanol, ultrasonically dissolving, and filtering;

(4.2.2), the preparation method comprises the following steps:

chromatographic column Innoval ODS-210 μm 100A; sample loading amount: 100 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-62 min, 24% acetonitrile; 62-92 min, 25-26% acetonitrile, 92-98 min, 100% acetonitrile, 98-104 min and 24% acetonitrile;

1 needle was shared, receiving 1 fraction: BLG-10 to obtain 1 monomer BLG-10, namely herecotrone;

(4.3)、BLG-H ₄the method comprises the following steps:

(4.3.1), sample dissolution:

secondary preparation of treated BLG-H₄Adding 220mg of powder into 1ml of methanol, ultrasonically dissolving, and filtering;

(4.3.2), the preparation method comprises the following steps:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 100 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-40 min, 30-35% acetonitrile, 40-46 min, 100% acetonitrile, 46-52 min, 30% acetonitrile;

1 needle was shared, receiving 1 fraction: BLG-11, obtaining 1 monomer BLG-11, namely, the monomer hepetone;

(4.4)、BLG-H ₅the method comprises the following steps:

(4.4.1), sample dissolution:

secondary preparation of treated BLG-H₅990mg of powder, adding 1.5ml of methanol, ultrasonically dissolving, and filtering;

(4.4.2), the preparation method comprises the following steps:

a chromatographic column: innoval ODS-210 μm 100A; sample loading amount: 100 mg; flow rate: 80 ml/min; detection wavelength: 280nm/254 nm; mobile phase: water-acetonitrile; gradient elution: 0-40 min, 30-35% acetonitrile, 40-57 min, 35-38% acetonitrile, 57-63 min, 100% acetonitrile, 63-69 min, 30% acetonitrile;

2 needles were co-fed, receiving 1 fraction: BLG-12, the same fractions are combined and processed to obtain 1 monomer BLG-12, namely, heretenone.
A dosage form of the composition of lignan active ingredients of pedunculate herpetospermum seeds of claim 1 or 2, wherein: the composition consists of 1 or 2 of the lignan effective component composition of the herpetospermum pedunculosum seeds and a pharmaceutically acceptable carrier; the dosage form is capsule, tablet, pill, dripping pill, granule, soft capsule, pellicle, unguent, spray, aerosol, patch, gel or injection.
The dosage form of claim 4, wherein: the pharmaceutically acceptable carrier is selected from one or a combination of more than two of starch, microcrystalline cellulose, lactose, sucrose, powdered sugar, dextrin, cellulose derivatives, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, stearic acid and salts thereof, talcum powder, polyethylene glycol, beeswax, vegetable oil, animal glue, vegetable glue, carbomer resin, sodium carboxymethyl cellulose, polyvinyl alcohol, acrylate polymers, polyacrylic acid, sodium polyacrylate, polyvinylpyrrolidone, glycerol, propylene glycol, sorbitol, triethylamine, azone, hydroxypropyl-beta-cyclodextrin and tween-80.
Use of the lignan-containing active ingredient composition of pedunculate herpetospermum seeds of claim 1 or 2 for the preparation of a medicament for treating hepatitis B.