CN101244220A - Black thorn smilax total saponin extract and preparation method thereof - Google Patents

Abstract

The present invention discloses a total saponin extract extracted from the Chinese medicine Smilax china and a preparation method thereof. The extract mainly comprises Smilax china saponin a, Smilax china saponin b and analogs thereof. The extract can be prepared by any one of solvent extraction, solvent extraction, macroporous adsorption resin, supercritical fluid extraction, column chromatography, liquid-liquid countercurrent distribution chromatography, or any combination of these methods. The total content of total saponin components in the obtained Smilax china total saponin extract is 5-100% (W/W), wherein the total content of Smilax china saponin a, Smilax china saponin b and analogs thereof accounts for 5-100% of the total saponin content by weight.

Description

Black thorn smilax total saponin extract and preparation method thereof

technical field

The invention relates to an extract of total saponins from Smilax chinensis, a preparation method, a quality control method and its application in the field of medicine and health food, belonging to the technical field of medicine.

Background technique

Black thorn smilax (Smilax scobinicaulis C.H.Wright), also known as short-stalked smilax, is a plant of the genus Smilax in the family Smilax. It grows in low mountain shrubs or valley riverbanks. It is mainly produced in Shaanxi and Gansu provinces, and is widely distributed in northern regions. The resources are very rich. Its rhizome is used as medicine instead of Clematis in northern my country, and it is called "wire clematis", "King Kong thorn", "King Kong vine" and so on. Decoction or soaked in wine for oral administration; grinding into powder, grinding juice for application or decocting for external use; it has the functions of eliminating rheumatism, promoting blood circulation and detoxification, calming convulsions and wind and anti-cancer, etc. It is clinically used to treat rheumatic lumbar and leg diseases and infantile convulsions Enteritis, scrofula, boils, cancer and other diseases.

Studies have found that Smilax nigra plant mainly contains steroidal saponins, phenols and sterols, among which saponins are the main active ingredients, and the main saponins are steroidal saponins containing laxogenin Laxogenin and its analogs are excellent raw materials for the preparation of Laxogenin (patent "a method for preparing Laxogenin from Smilax nigra plant", application number 200710199224.X). These saponin components contain active functional groups such as carbonyl, hydroxyl, methoxy, double bonds, epoxy ether bonds, etc. in their structures at the same time or respectively. Structural transformation is easy to occur under the action of S. blackthorn, so it is of great significance to study the preparation method of total saponins of Smilax nigra.

Contents of the invention

The object of the present invention is to provide a kind of total saponin extract of Smilax chinensis and its preparation method. The total saponins extract of Smilax nigrum provided by the present invention is a combination containing various saponins active ingredients extracted from the plant of Chinese medicine Smilax nigrum, wherein the structure of the main compound is as follows:

compound R English name Blackthorn smilax saponin a α-L-Ara-(1→6)-β-D-glu- smilaxin A Smilax saponin b β-D-Glu-(1→4)-[α-L-ara-(1→6)]-β-D-glu- smilaxin B

The material of the present invention is derived from Smilax scobinicaulis C.H.Wright, a plant of the genus Smilax in the family Liliaceae. As the raw material for extracting the total saponins of Smilax smilax, it can be commercially available Smilax smilax decoction pieces, or any part or all plants of Smilax smilax tuberous roots, fibrous roots, rhizomes, stems, tendrils and leaves, among which the preferred The part of the medicinal material is the dried root and rhizome of Smilax blackthorn. The above-mentioned Smilax blackthorn includes raw medicinal materials and decoction pieces without any processing, and also includes various processed products.

The total saponins extract of Smilax nigra in the present invention refers to a composition obtained by extracting from any part of the above-mentioned plants and containing various saponins active ingredients. These saponins mainly include a series of derivatives with the structure shown in the figure below as the core:

As the total saponin extract of Smilax chinensis, the total percentage content of each saponin component is 5-100% (W/W), and preferably 50-100% (W/W).

Among the various active ingredients of saponins covered by the total saponin extract of Smilax nigrum according to the present invention, the most important ones are smilax saponin a, smilax saponin b and their analogs.

The present invention also proposes the preparation process of the total saponin extract of Smilax nigrum, which can be prepared by any one of the following methods, or any combination of these methods: (1) solvent extraction method; (2) solvent extraction method; ( 3) macroporous adsorption resin method; (4) supercritical fluid extraction method; (5) column chromatography; (6) liquid-liquid countercurrent partition chromatography. Among them, the preferred method is the macroporous adsorption resin method.

When these methods are used for preparation, the following steps are generally included:

(1) Extraction: the solvent used can be water or any alcohol, ketone or ester solvent, or a mixed solvent composed of these solvents in a certain proportion, or an acidic or Alkaline solvent. Extraction methods can be decoction, heating reflux, ultrasonic extraction, cold soaking, percolation, microwave extraction, high-pressure extraction, etc.

The preferred extraction process is: Add 0-80% ethanol or alkaline solvent made of these solvents and alkaline substances to the medicinal material of Smilax chinensis, and reflux extraction for 2-4 times, each time for 1-2 hours, and the solvent consumption is 5 ~15 times the amount (L/kg).

(2) Filtration: including centrifugation, suction filtration, ultrafiltration, pressure filtration, etc., with or without the use of any of the following clarifiers or their combination: alcohol precipitation agent, gelatin, kaolin, various resins, polyethylene glycol, Polyethylene glycol, chitosan and natural clarifier products such as 101 juice clarifier, ZTC+1 natural clarifier, etc.

(3) Concentration: including thin film evaporation, rotary evaporation and decoction concentration under normal pressure or reduced pressure.

(4) Drying: including vacuum drying, spray drying, freeze drying, etc.

When the extraction method is used for preparation, the extract is generally suspended in water first, and then used with low-polarity esters, alkanes or ether solvents (such as petroleum ether, ether, hexane, gasoline, ethyl acetate, etc.) ) extraction to remove fat-soluble impurities, and then use a suitable polar solvent, such as chloroform, ethyl acetate, acetone, n-butanol, etc., or a mixture of these solvents to extract the total saponin components therein to obtain a total saponin extract.

When the macroporous adsorption resin method is used for preparation, the macroporous resin used can be any type of non-polar, weak polar, medium polar, weakly alkaline or weakly acidic, such as D101, D4020, D3500, D941, D21, HP-20, HP-20, XDA-1, AB-8, HPD400, S-8, HZ-806, etc. Among them, weak or medium polar resins are preferred, such as AB-8, HPD400, D101 etc. The eluent used is water and ethanol containing water, methanol, acetone, etc., wherein the preferred ethanol is 70-95%.

The preferred resin purification process of the total saponins extract of Smilax nigra is as follows: select D101, AB-8, HPD400 and other medium weak polarity or medium polar resins as the resin for purification, and the dilution ratio of the sample solution of the Smilax nigra extract 1:5~1:18 (based on the crude drug amount), the adsorption flow rate is 2~9BV/h, the resin column diameter-to-height ratio is 1:3~1:10, and the sample loading is 0.1~0.5g/mL (based on the crude drug amount ), 0-10% ethanol elutes 1-5 times the resin volume for impurity removal, the impurity removal flow rate is 2-7BV/h, and 3-10 times the resin volume is eluted with 60-95% ethanol, and the elution flow rate is 2-7BV/h 9BV/h.

When supercritical fluid extraction is used for preparation, the raw material of Smilax nigra can be directly extracted, or the product obtained by any of the above methods and steps can be extracted. Extraction can be performed with or without any of the following solvents and solvent mixtures: water, alcohols, ketones, and esters.

When column chromatography is used for preparation, the object of treatment may be the product obtained in the above extraction step, or the product preliminarily purified by the above solvent extraction method, macroporous adsorption resin method or supercritical fluid extraction method. The stationary phase used can be silica gel, polyamide, aluminum oxide, dextran (Sephadex series or Sephadex-20 series), C-8, C-18, activated carbon, cellulose, etc., and the eluent used varies depending on the stationary phase. It varies with different solvents, and it is generally a mixed solvent composed of water, methanol, ethanol, acetone, chloroform, ethyl acetate, petroleum ether, etc.

When the liquid-liquid countercurrent extraction method is used for preparation, the object of treatment can be the product of the above extraction step, or the product that has been preliminarily purified by the above solvent extraction method, macroporous adsorption resin method or supercritical fluid extraction method. Generally, the extract is suspended in water, and then extracted with low-polarity esters, alkanes or ether solvents (such as petroleum ether, ether, hexane, gasoline, ethyl acetate, etc.) to remove fat-soluble impurities, and then use A suitable polar solvent, such as chloroform, ethyl acetate, acetone, n-butanol, etc., or a mixture of these solvents, is extracted to obtain the total saponin components therein to obtain a total saponin extract.

The extract can be used alone or in combination with any other Chinese and Western medicine or food in any proportion to prepare medicine or functional food. The obtained medicine or functional food can be in the form of capsules, tablets, pills, granules, oral liquid, Syrups, granules, liquors, injections, ointments, powders, beverages, etc.

The quality control method of the present invention may include one or both of the following assay methods:

1. Determination of saponin content by hemolytic index method

Preparation of 0.5% medicinal extract: the medicinal powder of Smilax blackthorn is precisely prepared with isotonic phosphate buffer (or physiological saline) to a concentration of 0.5%.

Determination of hemolytic index: Take 9 small test tubes with the same diameter and length, accurately inhale 0.1, 0.2, 0.3, ... 0.9mL of Smilax japonica extract, add buffer to make up the volume to 1mL, and add each Accurately add 1mL of 2% blood cell suspension, shake each tube well and calculate the hemolysis index from the lowest concentration of Smilax nigra extract that can produce complete hemolysis.

Determination of saponin content: use the standard saponin Smilax saponin a or Smilax saponin b to prepare a solution of appropriate concentration, measure the hemolytic index with the same method as above, and calculate the black thorn The percentage content of smilax saponins.

2. HPLC-ELSD analysis of the content of smilax saponins in blackthorn

Chromatographic conditions: evaporative light scattering detector, chromatographic column: reversed-phase C18 (4.6×150mm, 5μm); mobile phase: acetonitrile-water gradient elution, 40-80% (0-70min); flow rate: 1.0mL/Min; Column temperature: 25°C.

Standard curve drawing: 0, 2, 5, 10, 15, 20 μL of reference substance solution (1.0 μg/μL) of Smilax saponin standard saponin Smilax saponin a or Smilax saponin b (1.0 μg/μL) was drawn precisely and injected into liquid chromatography Instrument, measure the peak area of each chromatographic peak, take the control injection volume (μg) as the abscissa, and the chromatographic peak area as the ordinate, draw the standard curve.

Content determination: Accurately weigh 3 batches of 3 batches of Smilax nigra extract samples, each about 20mg, put them in a 10mL measuring bottle, add 70% ethanol to ultrasonically dissolve, dilute to the scale, shake well, and use as Smilax nigrum For the test sample solution for the determination of the saponin content, 20 μL of the above-mentioned test sample solution was precisely drawn and injected into the liquid chromatograph, and the area of each chromatographic peak was measured to calculate the content.

Detailed ways:

Example 1: Preparation process of total saponins extract of Smilax chinensis

Take 1kg of the rhizome of Smilax chinensis, dry it, grind it into a coarse powder, add 4L 70% ethanol (V/V) solution at room temperature to extract 4 times, 4 hours each time, combine the extracts, concentrate the filtrate into an extract, add water to disperse and dissolve , so that the dilution ratio of the aqueous solution is 1:15 (based on the amount of crude drug), it is extracted 5 times with an equal volume of petroleum ether and n-butanol successively, and the solvent is recovered under reduced pressure to obtain the extract. The n-butanol extract was dissolved in deionized water, and purified on D-101 macroporous resin according to the ratio of sample to filler of 1:7. Until negative, the impurity removal flow rate is 3BV/h, then 8 times the resin volume is eluted with 90% ethanol, the elution flow rate is 2BV/h, the 90% ethanol eluate is collected, the ethanol is recovered, and dried under reduced pressure to obtain Smilax chinensis Total Saponin Extract. The content of total saponins in the total saponin extract of Smilax nigra was determined to be 54%, and the sum of the content of the two components of Smilax nigra saponin a and Smilax nigra saponin b was determined to be 40%.

Example 2: Preparation process of total saponins extract of Smilax chinensis

Take 1kg of the stem of Smilax chinensis, crush it, add water to boil and extract three times and control the pH, the first time is 8 times the amount of water and 0.7% NaCO ₃ of the amount of the drug, and the second time is 5 times the amount of water and 0.3% of the amount of the drug The amount of NaCO ₃ in the amount of medicinal materials, the third time is 4 times the amount of water; countercurrent circulation extraction, after boiling, keep for 1.5, 1, and 1 hour respectively; the medicinal liquids are combined and cooled to room temperature, filtered, and the treated macropores on the filtrate Type weakly basic anion D392 exchange column, rinse with 2% NaOH solution 2 times the volume of the resin bed, discard the rinse solution, then rinse with 3 times pure water, discard the water rinse solution; use 70% of the resin bed volume 7 times eluate with alcohol, collect the eluate, concentrate to a thick paste (relative density at 50°C is 1.28-1.33) and dry to obtain the total saponin extract of Smilax nigra. The total saponin content in the total saponin extract of Smilax nigra was determined to be 64%, and the sum of the content of the two components of Smilax nigra saponin a and Smilax nigra saponin b was determined to be 45%.

Example 3: Preparation process of total saponins extract from Smilax chinensis

Get 1 kg of Smilax nigra root powder, add 12 times the amount of 95% ethanol, reflux extraction 3 times, extract 3 hours each time, combine the ethanol extract, reclaim the ethanol, concentrate to dryness, then add 8 times the amount of deionized water Stir and dissolve, the solution is passed through the AB-8 type macroporous adsorption resin chromatography column, the adsorption flow rate is 4BV/h, the numerical column diameter-to-height ratio is 1:9, and the sample loading is 0.1g/mL (based on crude drug amount); Water lotion resin 12BV, the elution flow rate is 6BV/h, the washing solution is discarded; 8 times the resin volume is eluted with 90% ethanol, the elution flow rate is 5BV/h, the ethanol eluate is collected, the ethanol is recovered, and dried under reduced pressure to obtain Blackthorn Smilax total saponin extract. The content of total saponins in the total saponin extract of Smilax nigra was determined to be 65%, and the sum of the contents of the two components of Smilax nigra saponin a and Smilax nigra saponin b was determined to be 53%.

Example 4: Preparation of Smilax chinensis Total Saponins Tablets

Blackthorn Smilax total saponins extract 10g

Starch 10g

The above components are mixed evenly, and an appropriate amount of talcum powder is added, and pressed into 100 tablets.

Example 5: Preparation of the compound preparation of total saponins of Smilax chinensis

Blackthorn Smilax total saponins extract 30g

Banji lotus water extract 30g

Hedyotis diffusa water extract 30g

The above components are mixed evenly, and packed into hard gelatin capsules, 360 capsules in total.

Claims (9)

1. black thorn chinaroot greenbrier total saponin extract is characterized in that this extract by obtaining in the Chinese medicine black thorn chinaroot greenbrier plant, and contains following saponin component: black thorn chinaroot greenbrier saponin a, black thorn chinaroot greenbrier saponin b and analog thereof.

2. black thorn chinaroot greenbrier total saponin extract as claimed in claim 1, it is characterized in that black thorn chinaroot greenbrier is arbitrary position or whole plant such as commercially available black thorn chinaroot greenbrier decoction pieces or Liliaceae smilax plant black thorn chinaroot greenbrier tuber, fibrous root, rhizome, stem, tendril and leaf, comprise crude drug and the decoction pieces handled without any process of preparing Chinese medicine, also comprise various processed products.

3. black thorn chinaroot greenbrier total saponin extract as claimed in claim 1 is characterized in that above-mentioned total saponins constituents content summation is 5～100% by weight.

4. black thorn chinaroot greenbrier total saponin extract as claimed in claim 3 is characterized in that in each saponin component that it is 5～100% by weight that the summation of the content of composition such as black thorn chinaroot greenbrier saponin a, black thorn chinaroot greenbrier saponin b and analog thereof accounts for whole total saponin contents.

5. as the described black thorn chinaroot greenbrier total saponin extract of claim 1～4, it is characterized in that adopting solvent extraction method, solvent extraction, macroporous adsorbent resin method, supercritical fluid extraction, column chromatography, liquid-any one methods such as liquid adverse current partography, or the combination in any of these methods is prepared.

6. black thorn chinaroot greenbrier total saponin extract preparation method as claimed in claim 5 is characterized in that comprising following one or several step when these methods of use are prepared:

Extract: solvent for use can be water or any one alcohols, ketone or esters solvent, or the mixed solvent formed by a certain percentage of these solvents, or the acidity or the basic solvent that are made into by these solvents and acid, alkali, salt.Extracting method can be decoction, reflux, supersound extraction, merceration, seepage, microwave extraction, high pressure extract etc.

Filter: comprise methods such as centrifugal, sucking filtration, ultrafiltration, filter pressing, use or do not use following any one clarifier or its combination: precipitate with ethanol agent, gelatin, Kaolin, various resin, Polyethylene Glycol, poly-second triol, chitosan and natural clarifying agent finished product are as 101 fruit juice clarifiers, ZTC+1 natural clarifying agent etc.

Concentrate: comprise thin film evaporation, rotary evaporation and decocting and concentrating etc. under normal pressure or the reduced pressure.Dry: as to comprise vacuum drying, spray drying, lyophilization etc.

7. black thorn chinaroot greenbrier total saponin extract preparation method as claimed in claim 6, it is characterized in that when using the macroporous adsorbent resin method to be prepared, used macroporous resin can be any one types such as nonpolar, low pole, middle polarity, alkalescence or faintly acid, as D101, D4020, D3500, D941, D21, HP-20, XDA-1, AB-8, HPD-400, S-8, HZ-806 etc., the resin of low pole or middle polarity preferably wherein is as AB-8, HPD400, D101 etc.Used eluant is water and aqueous ethanol, methanol, acetone etc.

8. black thorn chinaroot greenbrier total saponin extract preparation method as claimed in claim 7, it is characterized in that selecting for use D101, AB-8, the resin of medium low pole such as HPD400 or middle polarity is as the purification resin, black thorn chinaroot greenbrier extract sample solution extension rate 1: 5～1: 18 (in the crude drug amount), absorption flow velocity 2～9BV/h, resin column blade diameter length ratio 1: 3～1: 10, applied sample amount is 0.1～0.5g/mL (in the crude drug amount), 1～5 times of resin volume of 0～10% ethanol elution carries out remove impurity, the remove impurity flow velocity is 2～7BV/h, with 3～10 times of resin volumes of 60～95% ethanol elutions, elution flow rate is 2～9BV/h.

9. as the application of the described black thorn chinaroot greenbrier total saponin extract of claim 1～4, it is characterized in that this extract can be separately or with other any Chinese medicine and western medicine or food by the arbitrary proportion compatibility, be used to prepare medicine or functional food, the medicine of gained or functional food can be capsule, tablet, pill, granule, oral liquid, syrup, electuary, medicated wine, injection, unguentum, powder, beverage etc.