CN103417644B - A kind of Heiguteng exract compound extract and its production and use - Google Patents

Abstract

The present invention provides a preparation method of the compound extract of Teng chinensis, which comprises the following steps: (1) Weighing raw materials according to the weight ratio of the compound: 2.5 to 10 parts of Teng chinensis, 1 to 4 parts of Gentiana chinensis, and 1 part of Rhizoma chinensis ~4 parts; after extracting with 40~60% v/v ethanol, combine the extracts, recover the ethanol until there is no alcohol smell, and obtain the crude extract; (2) take the crude extract, put it on a non-polar macroporous resin column, After washing with water to remove impurities, elute with 20-60% v/v ethanol, collect the ethanol eluate, concentrate, and dry to obtain the compound extract of Teng chinensis. The present invention also provides a compound extract of the black bone vine and its application. The research of the present invention shows that the compound extract of Heiguteng compound obtained by the preparation method of the present invention has good pharmacodynamic activity on rheumatoid arthritis, and the effect is better than that of other extraction parts, further clarifying the effective parts of the compound of Heiguteng , Provided a guarantee for the safety monitoring of preparations and drugs.

Description

A kind of black bone rattan compound extract and its preparation method and application

technical field

The present invention relates to a kind of compound extract of Teng chinensis and its preparation method and application.

Background technique

Rheumatoid arthritis (RheumatioidArthritis, RA) is an autoimmune deficiency disease characterized by chronic inflammation of the joint synovium. In traditional Chinese medicine, it belongs to the category of "arthritis syndrome". Infection, immune disorders and other factors are related. The incidence of RA in my country is about 0.4%, mostly concentrated in southern provinces. Therapeutic methods include chemical medicine, traditional Chinese medicine, biological agents and other drug treatments and physical therapy.

Black bone vine is the dry root or whole plant of Periploca forrestii Schltr, a plant of the genus PeriplocaLinn. in the family Asclepiasaceae. Commonly used medicine for treating rheumatism and rheumatoid, closed soft tissue injury and other diseases.

Gentiana macrophylla Pall, Gentiana macrophylla Pall, G. straminea Maxim, G. crassicaulis Duthieex Burk or G. dahurica Fisch have the functions of protecting the liver, treating rheumatoid arthritis, clearing away dampness and heat , anti-allergic, anti-arthritis and other effects.

Yuanhu, also known as Yanhusuo, is the dry tuber of the poppy plant Corydalis yanhusuo (Corydalisyanhusuo W.T.Wang). It has the effects of promoting blood circulation and removing blood stasis, regulating qi and relieving pain.

Patent application number: 201210094512.x, discloses a pharmaceutical composition for treating rheumatoid arthritis, the formula is 2.5 to 10 parts of Rheumatoid Artemisiae, 1 to 4 parts of Gentiana, and 1 to 4 parts of Rhizoma Rhizoma. The 50% ethanol extract of the compound can effectively treat rheumatoid arthritis, and has low toxic and side effects. Its ethanol extract contains 2-4% of total flavonoids, 3-6% of total steroidal saponins, and 0.1-0.3% of total alkaloids , gentiopicroside 1-3%, tetrahydropalmatine 0.04-0.06%, and glucoside 0.1-0.2%.

In the above-mentioned patents, the medicinal effect of the compound is better, but the content of active ingredients in its extract is relatively low. If it is prepared into a prescription, the amount of excipients will be large, which is not conducive to the saving of industrial production costs, and its dosage is relatively large. , not convenient for patients to use. If a kind of compound extract with higher active ingredient content and significant medicinal activity can be provided, it will be beneficial to the popularization of the above-mentioned compound medicine.

Contents of the invention

The object of the present invention is to provide a compound extract of Teng japonica and its preparation method and application.

The present invention provides the preparation method of the compound extract of Teng chinensis, which comprises the following steps:

(1) Weigh the raw materials according to the weight ratio of the compound: 2.5 to 10 parts of Rhododendron chinensis, 1 to 4 parts of Gentiana chinensis, and 1 to 4 parts of Rhizoma chinensis; after extracting with 40 to 60% v/v ethanol, combine the extracts, After recovering the ethanol until it has no alcohol smell, the crude extract is obtained;

(2) Take the crude extract, put it on a non-polar macroporous resin column, wash it with water to remove impurities, and then elute it with 20-60% v/v ethanol, collect the ethanol eluate, concentrate and dry it, and then you can get S. Compound extract.

Further, the weight ratio of the compound recipe is: 2.5 parts of Heliconia chinensis, 1 part of Gentiana chinensis, and 1 part of Yuanhu.

Further, in step (1), the ethanol concentration is 50% v/v.

Further, in step (2), the non-polar macroporous resin is D101.

Furthermore, in step (2), the specific elution operation is one of the following schemes:

Scheme A: After washing with water to remove impurities, elute with 20% v/v ethanol, and collect the ethanol eluate;

Scheme B: After washing with water to remove impurities, elute with 20% v/v and 40% v/v ethanol in sequence, and collect the 40% v/v ethanol eluate;

Scheme C: After washing with water to remove impurities, elute with 40% v/v and 60% v/v ethanol in sequence, and collect the 60% v/v ethanol eluate.

Wherein, in scheme C, before elution with 40% v/v ethanol, elute with 20% v/v ethanol first.

The present invention also provides the compound extract of Teng japonica prepared by the above method. In the extract obtained in Scheme A, the content of gentiopicroside is 7-9% w/w, and the content of glucoside is 0-1% w/w. The content of tetrahydropalmatine is 0-1%w/w, the content of total flavonoids is 11-15%w/w, and the content of total saponins is 7-9%w/w;

In the extract obtained in scheme B, the content of gentiopicroside is 0.4-0.6%w/w, the content of glucoside is 3-5%w/w, the content of tetrahydropalmatine is 0-1%w/w, and the content of total flavonoids 16-19%w/w, total saponin content 15-18%w/w;

In the extract obtained in scheme C, the content of gentiopicroside is 0.01-0.2%w/w, the content of glucoside is 1-3%w/w, the content of tetrahydropalmatine is 2-4%w/w, and the content of total flavonoids 8~12%w/w, total saponin content 15~18%w/w.

Further, in the extract obtained in scheme A, the content of gentiopicroside is 8% w/w, the content of glucoside is 0% w/w, the content of tetrahydropalmatine is 0% w/w, and the content of total flavonoids is 13- 14%w/w, the total saponin content is 8-9%w/w;

In the extract obtained in scheme B, the content of gentiopicroside is 0.5-0.6%w/w, the content of glucoside is 4%w/w, the content of tetrahydropalmatine is 0%w/w, and the content of total flavonoids is 17-18% %w/w, the total saponin content is 16~17%w/w;

In the extract obtained in scheme C, the content of gentiopicroside is 0.1-0.2%w/w, the content of glucoside is 2%w/w, the content of tetrahydropalmatine is 2-3%w/w, and the content of total flavonoids is 10% ~11%w/w, total saponin content is 16~18%w/w.

The present invention also provides the application of the compound extract of the above-mentioned vine radix in preparing anti-inflammatory or treating rheumatoid arthritis medicines.

The present invention also provides a pharmaceutical composition for treating rheumatoid arthritis, which uses one of the extracts obtained in the above schemes A to C or the composition of the extracts obtained in schemes A and B as active ingredients, plus Preparations prepared from excipients or auxiliary ingredients commonly used in pharmacy.

In the composition of the extracts obtained in Scheme A and Scheme B, the ratio of the two is 1:1w/w.

Further, the preparation is an oral preparation, such as tablet, capsule, pill, powder, granule and the like.

The research of the present invention shows that the extract of the compound recipe of Heiguteng obtained by the preparation method of the present invention has good pharmacodynamic activity on rheumatoid arthritis, and the effect is better than that of other extraction parts. Further formulation and drug safety monitoring provides assurance.

detailed description

Embodiment 1 The preparation of the compound extract of Teng japonica of the present invention

prescription:

Black bone vine 50g, Gentiana 20g, Yuanhu 20g

Preparation:

(1) Weigh the raw material drug by weight, ultrasonically extract it with 50% v/v ethanol, combine the extracts, recover the ethanol until it has no alcohol smell, and obtain the crude extract;

(2) Take the crude extract, put it on a D101 macroporous resin column, first elute with water to remove impurities (20 times column volume), then elute with 20 times column volume 20% v/v ethanol, collect the ethanol eluate, reduce After being concentrated under pressure, freeze-dried to obtain the compound extract of Teng Teng.

For the determination method of total flavonoids and total saponins in the present invention, please refer to the ultraviolet spectrophotometry commonly used in this field; for the detection methods of gentiopicroside, glucoside and tetrahydropalmatine, please refer to Chinese patent application number: 201210094512.x.

Embodiment 2 The preparation of the compound extract of Teng japonica of the present invention

prescription:

Black bone vine 50g, Gentiana 20g, Yuanhu 20g

Preparation:

(1) Weigh the raw material drug by weight, ultrasonically extract it with 50% v/v ethanol, combine the extracts, recover the ethanol until it has no alcohol smell, and obtain the crude extract;

(2) Take the crude extract, put it on a D101 macroporous resin column, first elute with water to remove impurities (20 times column volume), and then use 20 times column volume 20%v/v, 40%v/v, 60% Eluted with v/v ethanol, collected 60% ethanol eluate, concentrated under reduced pressure, and freeze-dried to obtain the compound extract of Teng Teng.

Embodiment 3 The preparation of the compound extract of Teng japonica of the present invention

prescription:

Black bone vine 50g, Gentiana 20g, Yuanhu 20g

Preparation:

(1) Weigh the raw material drug by weight, ultrasonically extract it with 50% v/v ethanol, combine the extracts, recover the ethanol until it has no alcohol smell, and obtain the crude extract;

(2) Take the crude extract, put it on a D101 macroporous resin column, first elute with water to remove impurities (20 times column volume), and then use 20 times column volume 20%v/v, 40%v/v ethanol to elute successively , collected 40% ethanol eluate, concentrated under reduced pressure, and freeze-dried to obtain the compound extract of S.

Embodiment 4 The preparation of the compound extract of Teng japonica of the present invention

prescription:

Black bone vine 50g, Gentiana 20g, Yuanhu 20g

Preparation:

(1) Weigh the raw material drug by weight, ultrasonically extract it with 50% v/v ethanol, combine the extracts, recover the ethanol until it has no alcohol smell, and obtain the crude extract;

(2) Take the crude extract, put it on a D101 macroporous resin column, first elute with water to remove impurities (20 times column volume), then elute with 20 times column volume 40% v/v ethanol, collect 40% ethanol eluate , concentrated under reduced pressure, and then freeze-dried to obtain the compound extract of Teng Teng.

Embodiment 5 Preparation of the pharmaceutical composition of the present invention

Take the compound extract of Caulis japonica prepared in Example 1 or Example 2, add appropriate amount of starch and dextrin, and granulate to obtain granules.

Embodiment 6 Preparation of the pharmaceutical composition of the present invention

Take the compound extracts of Teng japonica prepared in Example 1 and Example 3, mix them at a ratio of 1:1 w/w, add appropriate amount of starch and dextrin, granulate, and compress into tablets to obtain tablets.

The extract that embodiment 7 different eluent concentration prepares

1 Preparation of crude extract

Take 500g of Rhizoma chinensis, 200g of Gentiana chinensis, and 200g of Rhizoma chinensis, crush them, mix well, add 50% v/v ethanol for ultrasonic extraction, combine the extracts, recover ethanol, concentrate, freeze-dry, and obtain the crude extract for purification .

2 Effect of eluent concentration on purification effect

Weigh clean macroporous resin D1012kg and put it on the chromatographic column, weigh 200g of the dry powder of the crude extract of Caulis sinensis and add 1000ml of distilled water to dissolve it ultrasonically, and then load the sample with concentrations of 0%, 20%, 40%, 60%, 80% % and 95% ethanol solutions were eluted with 20 times the column volume each, and the eluents were collected and spun and concentrated, then freeze-dried for later use, and the contents of total saponins, total flavonoids, glucoside, tetrahydropalmatine, and gentiopicroside were determined. The detection method can refer to the prior art.

2.1 Contents of gentiopicroside, glucoside and tetrahydropalmatine

See the table below for the results.

Contents of gentiopicroside, baricolidin and tetrahydropalmatine in each concentration ethanol solution of table 1 ( n=3)

Group A: 0% ethanol eluate; B group: 20% ethanol eluate; C group: 40% ethanol eluate; D group: 60% ethanol eluate; E group: 80% ethanol eluate ; Group F: 95% ethanol eluate; Group G: samples before purification.

It can be seen from the above table that the content of chopicroside is the highest in group B, and the enrichment factor is about 7 times; the content of baricolidin is the highest in group C, and the enrichment factor is about 14 times; the content of tetrahydropalmatine is the highest in group D , the enrichment factor is about 24 times. The overall effect is obvious.

2.2 Content of total flavonoids and total saponins

The results are shown in the table below.

The content of table 2 total flavonoids and total saponins ( n=3)

Group A: 0% ethanol eluate; B group: 20% ethanol eluate; C group: 40% ethanol eluate; D group: 60% ethanol eluate; E group: 80% ethanol eluate ; Group F: 95% ethanol eluate; Group G: samples before purification.

The content of total flavonoids was the highest in group B, the content of total saponins was the highest in group C, and the total flavonoids and total saponins were enriched in groups B, C, D, and E.

The beneficial effects of the present invention will be specifically described below through test examples.

Test example 1 drug efficacy experiment

1.1 Mouse ear swelling test of each extraction site

1.1.1 Experimental animals

Normal-grade Kunming healthy mice, weighing 18-22 g, 60 days old, all male, were reared in separate cages, and were purchased from Chengdu Dashuo Animal Breeding Center.

1.1.2 Experimental materials, reagents and instruments

Samples after drying of each gradient eluent in Example 7.

The positive control drug prednisone (Zhejiang Xianju Pharmaceutical Co., Ltd.), sodium carboxymethylcellulose and xylene were purchased from Chengdu Kelong Chemical Reagent Factory, and the water was distilled water.

ESJ120-4/10,000 electronic balance (Shenyang Longteng), surgical scissors, puncher, tweezers.

1.1.3 Drug configuration method

Weigh 5g of sodium carboxymethylcellulose into a 1000mL beaker, add distilled water to dissolve it, add distilled water to the mark after cooling to room temperature to obtain 100mL of 0.5% sodium carboxymethylcellulose solution, weigh 25mg of prednisone acetate Tablet dry powder was dissolved in a 100mL volumetric flask with 0.5% sodium carboxymethylcellulose to obtain 100mL of 0.25mg/ml prednisone acetate solution.

Weigh 1.25 g of 20%, 40%, and 60% v/v ethanol eluted products into a 50mL volumetric flask, and then add 0.5% sodium carboxymethylcellulose to dissolve them to obtain 20%, 40% ethanol with a concentration of 25mg/ml , 60% v/v elution product solution 50mL.

Pipette 10 mL of 20% v/v ethanol elution product solution at a concentration of 25 mg/mL and mix with 10 mL of 40% v/v ethanol elution product solution at a concentration of 25 mg/mL to obtain a mixture of 20% and 40% ethanol elution products solution.

Pipette 10 mL of 20% v/v ethanol elution product solution with a concentration of 25 mg/mL and mix with 10 mL of 60% v/v ethanol elution product solution with a concentration of 25 mg/mL to obtain 20% and 60% v/v ethanol elution products. De-product mixed solution.

Pipette 10 mL of 40% v/v ethanol elution product solution with a concentration of 25 mg/mL and mix with 10 mL of 60% v/v ethanol elution product solution with a concentration of 25 mg/mL to obtain 40% and 60% v/v ethanol elution products. De-product mixed solution.

1.1.4 Mouse ear swelling test

After purchasing 18-22g healthy mice, they were adaptively fed for three days, and then divided into 8 groups with 10 mice in each group.

Group A was the blank group, which was given 0.5% sodium carboxymethylcellulose solution by intragastric administration, 0.2 mL each time.

Group B was the positive group, and 0.25 mg/ml prednisone acetate solution was administered intragastrically, 0.2 mL each time.

Group C was 20% ethanol group, and 25 mg/mL 20% v/v ethanol elution product solution was administered intragastrically, 0.2 mL each time.

Group D was 40% ethanol group, and 25 mg/mL40% v/v ethanol elution product solution was administered intragastrically, 0.2 mL each time.

Group E was the 60% ethanol group, and 25 mg/mL 60% v/v ethanol elution product solution was administered intragastrically, 0.2 mL each time.

Group F is the 20% and 40% ethanol mixed group, and 25 mg/mL 20% and 40% v/v ethanol eluted product mixed solution is administered intragastrically, 0.2 mL each time.

Group G was a mixed group of 20% and 60% ethanol, and 25 mg/mL 20% and 60% v/v ethanol eluted product mixed solution was administered intragastrically, 0.2 mL each time.

Group H was the 40% and 60% ethanol mixed group, and 25 mg/mL 40% and 60% v/v ethanol eluted product mixed solution was administered intragastrically, 0.2 mL each time.

Gastrointestinal administration once every 12 hours, 6 times in a row. One hour after the last administration, the right ear of the mouse was evenly coated with xylene, half an hour later, the vertebrae were cut off, the left and right ears were cut off, and equal-sized ear pieces were punched at the same position of the left and right ears with a punch and weighed.

Calculated as follows:

1.2 Experimental results

1.3.1 Experimental results of mouse ear swelling

See Table 3 for the experimental results analyzed by SPSS13.0.

Table 3 mouse ear swelling test ( n=10)

Note: *: P<0.05 compared with the model group **: P<0.01 compared with the model group

The results in Table 3 show that the swelling rates of administration groups C and F are significantly different from those of the model group, and have anti-inflammatory effects. The experimental results show that the swelling rate of the administration group D and E is significantly different from that of the model group, and has a good anti-inflammatory effect. It can be seen that the elution site of 20% v/v ethanol has the best drug effect, followed by the elution site of 40% and 60% v/v ethanol; at the same time, the test also found that after mixing the elution site of 20% and 40% ethanol , its pharmacodynamic effect has also been enhanced, but after mixing 20% and 60%, 40% and 60% ethanol eluting parts, the medicinal activity decreases instead.

The research of the present invention shows that the extract of the compound recipe of Helicoptera fragrans obtained by the preparation method of the present invention has good pharmacodynamic activity on rheumatoid arthritis, and the effect is better than that of other extraction parts. It provides a guarantee for the safety monitoring of preparations and drugs.

Claims (10)

1. the preparation method of Heiguteng exract compound extract, is characterized in that: it comprises following operating procedure:

(1) by compound recipe weight proportion weighting raw materials: Heiguteng exract 2.5 parts, Radix Gentianae Macrophyllae 1 part, Rhizoma Corydalis 1 part; After 40 ~ 60%v/v ethanol extraction, merge extractive liquid, reclaims ethanol to without after alcohol taste, obtains crude extract;

(2) get crude extract, upper non-polar macroporous resin post, after ion, with 20 ~ 60%v/v ethanol elution, collect ethanol elution, after concentrated, drying, obtain Heiguteng exract compound extract; Described non-polar macroporous resin is D101;

Concrete elution action is one of following scheme:

Option A: after ion, uses 20%v/v ethanol elution, collects ethanol elution;

Option b: after ion, uses 20%v/v, 40%v/v ethanol elution successively, collects 40%v/v ethanol elution;

Scheme C: after ion, uses 40%v/v, 60%v/v ethanol elution successively, collects 60%v/v ethanol elution.

2. preparation method according to claim 1, is characterized in that: in step (1), and described concentration of alcohol is 50%v/v.

3. preparation method according to claim 1, is characterized in that: in scheme C, before 40%v/v ethanol elution, first uses 20%v/v ethanol elution.

4. the Heiguteng exract compound extract prepared of method described in claim 1-3 any one, it is characterized in that: during option A is extract obtained, gentiopicroside in different morphological is 7 ~ 9%w/w, periplocoside content is 0 ~ 1%w/w, Content determination of dl-tetrahydropalmatine is 0 ~ 1%w/w, general flavone content is 11 ~ 15%w/w, and total saponin content is 7 ~ 9%w/w;

During option b is extract obtained, gentiopicroside in different morphological is 0.4 ~ 0.6%w/w, and periplocoside content is 3 ~ 5%w/w, and Content determination of dl-tetrahydropalmatine is 0 ~ 1%w/w, and general flavone content is 16 ~ 19%w/w, and total saponin content is 15 ~ 18%w/w;

During scheme C is extract obtained, gentiopicroside in different morphological is 0.01 ~ 0.2%w/w, and periplocoside content is 1 ~ 3%w/w, and Content determination of dl-tetrahydropalmatine is 2 ~ 4%w/w, and general flavone content is 8 ~ 12%w/w, and total saponin content is 15 ~ 18%w/w.

5. Heiguteng exract compound extract according to claim 4, is characterized in that:

During option A is extract obtained, gentiopicroside in different morphological is 8%w/w, and periplocoside content is 0%w/w, and Content determination of dl-tetrahydropalmatine is 0%w/w, and general flavone content is 13 ~ 14%w/w, and total saponin content is 8 ~ 9%w/w;

During option b is extract obtained, gentiopicroside in different morphological is 0.5 ~ 0.6%w/w, and periplocoside content is 4%w/w, and Content determination of dl-tetrahydropalmatine is 0%w/w, and general flavone content is 17 ~ 18%w/w, and total saponin content is 16 ~ 17%w/w;

During scheme C is extract obtained, gentiopicroside in different morphological is 0.1 ~ 0.2%w/w, and periplocoside content is 2%w/w, and Content determination of dl-tetrahydropalmatine is 2 ~ 3%w/w, and general flavone content is 10 ~ 11%w/w, and total saponin content is 16 ~ 18%w/w.

6. the purposes of Heiguteng exract compound extract in the medicine preparing antiinflammatory described in claim 4 or 5.

7. the purposes of Heiguteng exract compound extract described in claim 4 or 5 in the medicine of preparation treatment rheumatoid arthritis.

8. an anti-inflammatory pharmaceutical compositions, is characterized in that: it be with the extract obtained one of the option A ~ C described in claim 4 or 5 for active component, add the preparation that pharmaceutically conventional adjuvant prepares.

9. an anti-inflammatory pharmaceutical compositions, is characterized in that: it be with the option A described in claim 4 or 5 and the extract obtained compositions of option b for active component, add the preparation that pharmaceutically conventional adjuvant prepares.

10. pharmaceutical composition according to claim 9, is characterized in that: in described option A and the extract obtained compositions of option b, both proportionings are 1:1w/w.