CN101856435B - A kind of preparation method and content determination method of Zushima total coumarin extract - Google Patents

Abstract

The invention provides a preparation method and a content determination method of a Daphne giraldii Nitsche total coumarin extract, wherein the preparation method comprises the following steps: prepared from cortex Daphne Giraldii NitscheAs raw material, water and/or C₁～C₅Extracting with lower alcohol water solution as solvent to obtain extract; mixing the extract with a solid diluent, adding an organic solvent for extraction, and recovering the solvent to obtain a solid extract, or adding water for suspension to the extract, adding the organic solvent for extraction, and recovering the solvent to obtain the solid extract; suspending the solid extract in water, and performing chromatography with macroporous adsorbent resin, and adding water or C₁～C₅Eluting with lower alcohol aqueous solution as mobile phase to remove impurities, eluting with lower alcohol aqueous solution, and collecting eluate; recovering the first half eluate to obtain solid, i.e. part of Daphne giraldii Nitsche total coumarin extract; the second half of the eluate is recovered with solvent to obtain solid, and mixed with water to obtain suspension, and the organic layer is recovered with solvent to obtain solid, i.e. another part of Daphne giraldii Nitsche total coumarin extract.

Description

A kind of preparation method and content determination method of Zushima total coumarin extract

technical field

The invention belongs to the field of natural medicinal chemistry, and mainly relates to a preparation method and a content determination method of a total coumarin extract of Zushima.

Background technique

Zushi Ma is the root bark and stem bark of Daphne giraldii Nitsche, a plant belonging to the genus Daphne in the family Thymelaeaceae. Folks are used to treat headaches, toothaches, rheumatoid arthritis, bruises, stomach pains, liver pains, and bronchitis. Modern pharmaceutical research shows that Zushima contains a variety of chemical components, such as coumarins (daphnetin and daphnetin, etc.), diterpenoids (daphnetin, 12-hydroxy daphnein, daphnetin A, daphnetin B, daphnetin, etc.) Daphnein, etc.), lignins ((±)-syringaresinol, (-)-pineresinol, (-)-dihydrosesamin, (-)-larixisinol, etc.), flavonoids, anthracene Chemical components such as quinones and sterols. Among them, coumarin components have good analgesic, anti-acute inflammation and anti-adjuvant arthritis effects, and can be widely used to relieve and treat various pains, rheumatic rheumatoid arthritis, joint sprains and contusions and other diseases.

Chinese invention patent application CN1883618A discloses an effective part of Zushima and its preparation method and application. The content is 50% to 90%. However, the impurity content in the extract prepared in this patent application is high and mostly flavonoids, when the content of coumarins is measured by spectrophotometry alone, because flavonoids are effective under the wavelength of total coumarin determination Absorbed, resulting in inaccurate detection of the content of coumarin compounds, the actual content of active ingredients coumarin compounds is low.

Contents of the invention

Therefore, the object of the present invention is to provide a kind of preparation method of total coumarin extract of Zushima, the extraction process of the preparation method is further optimized, and the prepared total coumarin extract of Zushi has clear composition, low impurity content, The content of active ingredients is higher, and the drug effect is stronger, thereby reducing the dosage and reducing side effects.

The technical scheme for realizing the above-mentioned purpose of the present invention is as follows:

A preparation method of total coumarin extract of Zushi Ma, comprising the following steps:

(1) Extracting with the medicinal material of zumima as raw material and water or C ₁ -C ₅ lower alcohol aqueous solution as solvent to obtain the extract;

(2) mixing the extract with a solid diluent, adding an organic solvent for extraction, recovering the solvent to obtain a solid extract; or adding water to suspend the extract, adding an organic solvent for extraction, recovering the solvent to obtain a solid extract;

(3) Suspend the solid extract with water, use macroporous adsorption resin chromatography, first use water or C ₁ ~ C ₅ lower alcohol aqueous solution as the mobile phase to remove impurities, and then elute with C ₁ ~ C ₅ lower alcohol aqueous solution , to collect the eluate;

(4) The first half of the eluent recovers the solvent to obtain a solid, that is, a part of Zushima total coumarin extract; the solid obtained by recovering the solvent from the second half of the eluent is suspended in water, then extracted with an organic solvent, and the organic layer recovers the solvent to obtain Solid, that is another part of Zushi Ma total coumarin extract.

In step (1) of the above preparation method, the extraction method is preferably reflux or percolation; the lower alcohol concentration of the C ₁ to C ₅ lower alcohol aqueous solution can be 0 to 95% (volume/volume), preferably 30 to 70% % (vol/vol).

In step (2) of the above preparation method, the organic solvent is preferably ethyl acetate, n-butanol and/or water-saturated n-butanol. Preferably, step (2) of the above preparation method is to mix the extract with a solid diluent, add an organic solvent for extraction, and recover the solvent to obtain a solid extract; wherein, the solid diluent can be selected from starch, micronized silica gel, diatomaceous earth One or more of , kaolin, activated carbon and kaolin.

In step (3) of the above preparation method, the macroporous adsorption resin can be selected from one or more of styrene, styrene-acrylate, stilbene, acrylate or methacrylate resins, such as D101 , D201, DA201, HPD300, HPD400, HPD800, FL-1, FL-2 and/or AB-8 resin, preferably styrene type or styrene-acrylate type macroporous adsorption resin; The lower alcohol concentration of the alcohol aqueous solution can be 0～50% (volume/volume), preferably 0～20% (volume/volume); The lower alcohol concentration of the lower alcohol aqueous solution that eluting resin uses can be 20～90% (volume /volume), preferably 20-50% (volume/volume).

In the step (4) of the above preparation method, the organic solvent used for the extraction of the second half of the eluent is preferably ethyl acetate and/or n-butanol.

The present invention also provides the total coumarin extract of Zushima which is prepared by the above preparation method. Wherein, based on the total weight of the extract, the content of the total coumarin is not less than 50% (weight), such as 50%-90% (weight). Specifically, the total coumarin extract comprises one or more of daphnetin, daphnetin, daphnetin-8-O-glucoside and 7-OH-coumarin. Preferably, based on the total weight of the extract, the total coumarin extract comprises 0% to 50% (weight) of daphnetin, 0% to 50% (weight) of daphnetin, daphnetin-8-O- Glucoside 0%-50% (weight), 7-OH-coumarin 0%-30% (weight).

The present invention also provides a method for assaying the content of the above-mentioned total coumarin extract of Zushi Ma, which includes the following steps:

(1) HPLC determination of daphnetin, daphnetin, daphnetin-8-O-glucoside and 7-OH-coumarin:

Accurately weigh 50mg of the total coumarin extract of Zushima, put it in a 50mL measuring bottle, add an appropriate amount of 50% (volume/volume) methanol aqueous solution to dissolve and dilute to the scale, shake well, and precisely draw 2mL into a 10mL measuring bottle In, add 50% (volume/volume) methanol aqueous solution and dilute to scale, shake up, as need testing solution; Respectively daphnetin, Zuma B, daphnetin-8-O-glucoside and 7-OH- The methanol solution of bean element is the reference substance solution; the chromatographic column uses octadecylsilane bonded silica gel as the filler, and the methanol-0.5% acetic acid aqueous solution with a volume ratio of 25:75 is the mobile phase; the detection wavelength is 327nm;

(2) Determination of the total coumarin content of Zushi hemp:

Dilute the above-mentioned test solution and Zuma B reference substance solution to an appropriate volume respectively, and measure the absorbance at a wavelength of 310nm according to the ultraviolet-visible spectrophotometry, and calculate.

In the preparation method of Zushi Ma total coumarin extract of the present invention, except adopting some conventional methods, also adopted some comparatively important advanced technological steps to reduce the content of impurity flavonoids, to improve the content of total coumarin content. These include: (1) the use of solid-liquid extraction or liquid-liquid extraction can greatly enrich the active ingredients (i.e. coumarin components), while increasing the extraction rate and the content of active ingredients, while more targeted removal of Toxic diterpenes. Among them, the solid diluent used in the solid-liquid extraction method is porous, has a strong adsorption force to impurities, and can also adsorb pigments; the contact surface between the extract and the organic solvent is increased, and heating or percolation methods can be used at the same time The extraction is carried out, thereby effectively avoiding problems such as emulsification and precipitation blocking the liquid outlet during the extraction process, and further improving the extraction efficiency. (2) Not only further removed toxic components such as diterpenes by resin adsorption process, but also greatly improved the content of total coumarin components; The content of total coumarin is further improved, and at the same time, the content of flavonoid impurities is effectively reduced. (3) Correspondingly, the method for assaying the content of total coumarin extract of Patriarch hemp of the present invention combines the HPLC method with the spectrophotometric method, which overcomes the defects of poor specificity, low accuracy and serious interference of impurities in the original spectrophotometric method . (4) At the same time, from the point of view of the overall action intensity of the pharmacological action, the extract prepared by the present invention has significantly stronger drug efficacy, and the administration dose is obviously reduced.

Detailed ways

The present invention will be further described below through embodiment. It should be understood that the following examples are only used to illustrate the present invention, but not to limit the scope of the present invention.

Example 1

Take 1.0 kg of Zumima medicinal material, extract 3 times with 20% (volume/volume) ethanol aqueous solution under reflux, filter, combine the filtrate to recover ethanol, mix into starch, reflux extract with water-saturated n-butanol, recover solvent, add water to absorb solid matter In D201 resin, wash with water to remove impurities, then elute with 20% (volume/volume) ethanol aqueous solution, collect eluate, recover ethanol in the first half, and use the solid for later use; recover ethanol in the second half, add water to suspend the solid, and wash Ester extraction, recovering the solvent from the organic layer to obtain a solid, combining the two parts of the eluate to obtain the solid substance after treatment, and drying to obtain the effective fraction of the total coumarin of Zumima.

Determination according to high performance liquid chromatography:

Accurately weigh 50 mg of the effective fraction of Zushima total coumarin extract, put it in a 50mL measuring bottle, add an appropriate amount of 50% (volume/volume) methanol aqueous solution to dissolve it and dilute it to the scale, shake well, and precisely draw 2mL into 10mL In the measuring bottle, add 50% (volume/volume) methanol aqueous solution and dilute to scale, shake up, as need testing solution; The methanol solution of -OH-coumarin is the reference substance solution; The chromatographic column uses octadecylsilane bonded silica gel as the filler, and the methanol-0.5% acetic acid aqueous solution of 25:75 (volume/volume) is the mobile phase; the detection wavelength 327nm.

According to the total weight of the extract, 3.8% (weight) of ancestralin A, 40.6% (weight) of ancestralin B, 18.4% (weight) of daphnetin-8-O-glucoside and 2.4% (weight) of 7-OH-coumarin %(weight).

Determined by UV spectrophotometry:

Dilute the above-mentioned test solution and Zuma B reference substance solution to an appropriate volume respectively, and measure the absorbance at a wavelength of 310nm according to the ultraviolet-visible spectrophotometry, and calculate.

According to the total weight of the extract, the content of the total coumarin of Zushima is 73.6% (weight).

Example 2

Take 1.0 kg of zushima medicinal material, extract by percolation with 30% (volume/volume) ethanol aqueous solution, recover ethanol from the percolation liquid under reduced pressure, mix it into micropowder silica gel, extract by percolation with ethyl acetate, recover the solvent, and add water to absorb the solid matter In the D201 resin, wash away impurities with 10% (volume/volume) ethanol aqueous solution, then elute with 30% (volume/volume) ethanol aqueous solution, collect the eluate, recover ethanol under reduced pressure at 60°C in the first half, and save the solid matter for later use; In the second half, ethanol is recovered under reduced pressure at 60°C, the solid substance is suspended with water, extracted with n-butanol, the solvent is recovered from the organic layer to obtain a solid, and the solid substance obtained after the treatment of the two parts of the eluent is combined, dried in vacuo to obtain Zushima total coriander The effective part of the element.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, ancestralin 5.2% (weight), ancestralin 28.6% (weight), daphnetin-8-O-glucoside 20.4% (weight) and 7-OH-coumarin 3.0 %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 65.5% (weight).

Example 3

Take 1.0 kg of zushima medicinal material, extract with 40% (volume/volume) ethanol aqueous solution under reflux, recover ethanol from the extract under reduced pressure, mix it into diatomaceous earth, extract with n-butanol percolation, recover the solvent, and add water to absorb the solid matter in D101 resin, wash away impurities with 20% (volume/volume) ethanol aqueous solution, then elute with 40% (volume/volume) ethanol aqueous solution, collect the eluate, reclaim ethanol in the first half, and solid matter for standby; reclaim ethanol in the second half , the solid substance is suspended with water, extracted with ethyl acetate, the solvent is recovered from the organic layer to obtain a solid, the solid substance obtained after the two parts of the eluent are combined, and vacuum-dried to obtain the effective fraction of total coumarin of Zushima.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, 10.5% (weight) of ancestralin A, 14.6% (weight) of ancestralin B, 48.6% (weight) of daphnetin-8-O-glucoside and 4.4% (weight) of 7-OH-coumarin %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 87.4% (weight).

Example 4

Take 1.0 kg of zushima medicinal material, extract by percolation with 50% (volume/volume) ethanol aqueous solution, recover ethanol from the percolation solution under reduced pressure, mix it into activated carbon, extract with ethyl acetate reflux, recover the solvent, and add water to absorb the solid matter on FL -2 Resin, wash away impurities with 30% (volume/volume) ethanol aqueous solution, then elute with 50% (volume/volume) ethanol aqueous solution, collect the eluate, recover ethanol under reduced pressure at 60°C in the first half, and save the solid matter for later use; In the second half, ethanol is recovered under reduced pressure at 60°C, the solid substance is suspended with water, extracted with n-butanol, the solvent is recovered from the organic layer to obtain a solid, and the solid substance obtained after the treatment of the two parts of the eluent is combined, dried in vacuo to obtain Zushima total coriander The effective part of the element.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, 14.2% (weight) of ancestralin A, 32.3% (weight) of ancestralin B, 20.1% (weight) of daphnetin-8-O-glucoside and 8.5% (weight) of 7-OH-coumarin %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 90.6% (weight).

Example 5

Take 1.0kg of zushima medicinal material, reflux extraction with 60% (volume/volume) ethanol aqueous solution, recover ethanol from the extract under reduced pressure, mix it into kaolin, extract with n-butanol reflux, recover solvent, add water to absorb the solid matter on AB-8 Resin, wash away impurity with 40% (volume/volume) ethanol aqueous solution, then elute with 60% (volume/volume) ethanol aqueous solution, collect eluate, first half reclaims ethanol, and solid matter is standby; Second half reclaims ethanol, Suspend the solid matter with water, extract with ethyl acetate, recover the solvent from the organic layer to obtain a solid, combine the solid matter obtained after treatment with two parts of the eluent, and dry it in vacuum to obtain the effective fraction of total coumarin of Zushima.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, 41.7% (weight) of ancestralin A, 5.6% (weight) of ancestralin B, 5.1% (weight) of daphnetin-8-O-glucoside and 20.5% (weight) of 7-OH-coumarin %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 85.4% (weight).

Example 6

Take 1.0 kg of zushima medicinal material, extract by percolation with 70% (volume/volume) ethanol aqueous solution, recover ethanol from the percolation liquid under reduced pressure, mix it into kaolin, reflux extraction with ethyl acetate, recover the solvent, and add water to absorb the solid matter in AB-8 and D101 composite resin, wash away impurities with 50% (volume/volume) ethanol aqueous solution, then elute with 70% (volume/volume) ethanol aqueous solution, collect the eluate, and recover ethanol under reduced pressure at 60°C in the first half, The solid matter is reserved; the second half of the ethanol is recovered under reduced pressure at 60°C, the solid matter is suspended in water, extracted with n-butanol, the organic layer is recovered from the solvent to obtain a solid, and the solid matter obtained after the two parts of the eluent is combined and vacuum-dried to obtain Zushi The active part of hemp total coumarin.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, 47.8% (weight) of ancestralin A, 3.5% (weight) of ancestralin B, 3.8% (weight) of daphnetin-8-O-glucoside and 28.5% (weight) of 7-OH-coumarin %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 93.5% (weight).

Example 7

Take 1.0 kg of zushima medicinal material, extract it with 80% (volume/volume) ethanol aqueous solution under reflux, recover ethanol from the extract under reduced pressure, mix it into diatomaceous earth: kaolin (weight ratio=1:1), and reflux with n-butanol Extract, recover solvent, add water to solid matter and adsorb on D101 and D201 composite resin, wash away impurities with 20% (volume/volume) ethanol aqueous solution, then elute with 90% (volume/volume) ethanol aqueous solution, collect eluate, first half Partial recovery of ethanol, the solid matter is used for later use; the second half of the recovery of ethanol, the solid matter is suspended in water, extracted with ethyl acetate, the organic layer is recovered from the solvent to obtain a solid, and the solid matter obtained after the two parts of the eluent is combined and vacuum-dried to obtain Zushi The active part of hemp total coumarin.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, 15.2% (weight) of ancestralin A, 10.1% (weight) of ancestralin B, 8.6% (weight) of daphnetin-8-O-glucoside and 10.6% (weight) of 7-OH-coumarin %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 67.6% (weight).

Example 8

Take 1.0kg of zushima medicinal material, extract by percolation with 40% (volume/volume) ethanol aqueous solution, recover ethanol from the extract under reduced pressure, mix it into kaolin, extract by percolation with ethyl acetate, recover the solvent, and add water to absorb the solid matter on AB -8 and D201 composite resin, wash away impurities with 20% (volume/volume) ethanol aqueous solution, then elute with 40% (volume/volume) ethanol aqueous solution, collect the eluate, reclaim ethanol in the first half, and solid matter for subsequent use; Ethanol is recovered in half, the solid matter is suspended with water, extracted with n-butanol, the solvent is recovered from the organic layer to obtain a solid, and the solid matter obtained after the treatment of the two eluents is combined and vacuum-dried to obtain the effective fraction of the total coumarin of zushima.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, 6.8% (weight) of ancestralin A, 25.2% (weight) of ancestralin B, 38.5% (weight) of daphnetin-8-O-glucoside and 5.6% (weight) of 7-OH-coumarin %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 85.2% (weight).

Example 9

Take 1.0 kg of zushima medicinal material, reflux extraction with 50% (volume/volume) ethanol aqueous solution, recover ethanol from the extract under reduced pressure, mix it into diatomaceous earth, and reflux extraction with water-saturated n-butanol, recover the solvent, and add water to absorb the solid matter in FL-1 resin, washed with water to remove impurities, then eluted with 30% (volume/volume) ethanol aqueous solution, collected the eluent, the first half of the ethanol was recovered under reduced pressure at 60°C, and the solid matter was used for later use; the second half was recovered under reduced pressure at 60°C Add ethanol and water to suspend the solid substance, extract it with n-butanol, recover the solvent from the organic layer to obtain a solid, combine the two parts of the eluate to obtain the solid substance after treatment, and dry it in vacuum to obtain the effective fraction of total coumarin.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, 3.6% (weight) of ancestralin A, 48.2% (weight) of ancestralin B, 20.4% (weight) of daphnetin-8-O-glucoside and 2.8% (weight) of 7-OH-coumarin %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 82.6% (weight).

Example 10

Take 1.0kg of zushima medicinal material, extract by percolation with 60% (volume/volume) ethanol aqueous solution, extract the ethanol under reduced pressure, mix it into kaolin, extract by percolation with ethyl acetate, recover the solvent, and add water to absorb the solid matter on AB -8 resin, wash away impurities with 10% (volume/volume) ethanol aqueous solution, then elute with 50% (volume/volume) ethanol aqueous solution, collect eluate, first half reclaims ethanol, and solid matter is standby; Second half reclaims Add ethanol and water to suspend the solid substance, extract it with n-butanol, recover the solvent from the organic layer to obtain a solid, combine the two parts of the eluate to obtain the solid substance after treatment, and dry it in vacuum to obtain the effective fraction of total coumarin.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, Zushimadin 14.3% (weight), Zushimabin 20.6% (weight), daphnetin-8-O-glucoside 25.2% (weight) and 7-OH-coumarin 5.7% %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 90.2% (weight).

Example 11

Take 1.0 kg of zushima medicinal material, reflux extract 3 times with 20% (volume/volume) ethanol aqueous solution, filter, combine the filtrate to recover ethanol, extract with water-saturated n-butanol, recover the solvent, and add water to absorb the solid substance on AB-8 and D201 Composite resin, washed with water to remove impurities, then eluted with 30% (volume/volume) ethanol aqueous solution, collected the eluate, recovered the ethanol in the first half, and used the solid for later use; recovered the ethanol in the second half, added water to suspend the solid material, and used ethyl acetate Extraction, recovery of the solvent in the organic layer to obtain a solid, combining the two parts of the eluent to obtain the solid substance after treatment, and drying to obtain the effective fraction of the total coumarin of Zumima.

Determination according to high performance liquid chromatography:

According to the total weight of the extract, 10.8% (weight) of ancestralin A, 30.5% (weight) of ancestralin B, 25.2% (weight) of daphnetin-8-O-glucoside and 1.5% (weight) of 7-OH-coumarin %(weight).

Determined by UV spectrophotometry:

According to the total weight of the extract, the content of the total coumarin of Zushima is 75.5% (weight).

Example 12

Take 1.0 kg of the medicinal material of zushima, extract by percolation with 30% (volume/volume) ethanol aqueous solution, recover the ethanol from the percolation liquid under reduced pressure, extract with ethyl acetate, recover the solvent, add water to the solid matter and absorb it on HPD-300 resin, and use 10 % (volume/volume) ethanol aqueous solution to wash away impurities, then elute with 40% (volume/volume) ethanol aqueous solution, collect eluate, first half 60 ℃ decompression recovery ethanol, solid matter for subsequent use; second half 60 ℃ decompression The ethanol is recovered under pressure, the solid substance is suspended with water, extracted with n-butanol, the solvent is recovered from the organic layer to obtain a solid, and the solid substance obtained after the treatment of the two parts of the eluent is combined, dried in vacuo to obtain the effective fraction of total coumarin.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, ancestralin 8.5% (weight), ancestralin 28.2% (weight), daphnetin-8-O-glucoside 22.1% (weight) and 7-OH-coumarin 4.3 %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 76.5% (weight).

Example 13

Take 1.0kg of zushima medicinal material, extract by percolation with 70% (volume/volume) ethanol aqueous solution, recover ethanol from the percolation liquid under reduced pressure, extract with ethyl acetate, recover the solvent, add water to the solid matter and absorb it on HPD-400 resin, and use 20 % (volume/volume) ethanol aqueous solution washes away impurities, then elutes with 50% (volume/volume) ethanol aqueous solution, collects the eluent, the first half 60 ℃ depressurizes and recovers ethanol, and the solid matter is standby; The ethanol is recovered under pressure, the solid substance is suspended with water, extracted with n-butanol, the solvent is recovered from the organic layer to obtain a solid, and the solid substance obtained after the treatment of the two parts of the eluent is combined, dried in vacuo to obtain the effective fraction of total coumarin.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, 25.2% (weight) of ancestralin A, 10.1% (weight) of ancestralin B, 8.6% (weight) of daphnetin-8-O-glucoside and 10.6% (weight) of 7-OH-coumarin %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 68.6% (weight).

Example 14

Take 1.0kg of zushima medicinal material, reflux extraction with 80% (volume/volume) ethanol aqueous solution, extract the ethanol under reduced pressure, extract with water-saturated n-butanol, recover the solvent, add water to the solid matter and absorb it on HPD-800 resin, and add water to the HPD-800 resin. (volume/volume) ethanol aqueous solution washes away impurity, then elutes with 60% (volume/volume) ethanol aqueous solution, collects eluate, first half reclaims ethanol, and solid matter is standby; Second half reclaims ethanol, and solid matter adds water suspension, Extracting with ethyl acetate, recovering the solvent from the organic layer to obtain a solid, combining the two parts of the eluate to obtain the solid substance after treatment, and drying in vacuum to obtain the effective fraction of the total coumarin of Zuma.

Determine according to the above-mentioned high-performance liquid chromatography:

According to the total weight of the extract, 30.8% (weight) of ancestralin A, 10.2% (weight) of ancestralin B, 10.5% (weight) of daphnetin-8-O-glucoside and 15.3% (weight) of 7-OH-coumarin %(weight).

According to the above-mentioned UV spectrophotometric method for determination:

According to the total weight of the extract, the content of the total coumarin of Zushima is 78.2% (weight).

Table 1 Components in Zushi Hemp Total Coumarin Extract

As can be seen from the above table, the total coumarin content measured by UV spectrophotometry and the sum of the 4 known components measured by HPLC method only differ by 7.4 to 24.4% (weight/weight), and this part includes unknown fragrance Soybeans, flavonoids and other impurities that absorb at this wavelength. This result shows that the content of flavonoids and other impurities in the extract prepared by the present invention is greatly reduced.

Pharmacodynamic test:

The present invention has carried out following pharmacodynamics test to the total coumarin extract of the ancestor that obtains:

(1) Effects on tail flicking of mice in water bath

Compared with the control group ^** p<0.01 ^** p<0.01

(2) Effects on photothermal tail flick of mice

Compared with the control group ^** p<0.01 ^*** p<0.001

(3) Effects on mouse acetic acid writhing

Compared with the control group ^** p<0.01 ^*** p<0.001

(4) Effect on mouse ear swelling

Compared with control group ^* p<0.05 ^*** p<0.001

(5) Effect of total coumarin of Zushi hemp on cotton ball granuloma in rats (X±SD)

Compared with the control group ^* P<0.05 ^** P<0.01 ^*** p<0.001

(6) Effect of total coumarin of Zushi hemp on carrageenan foot swelling in rats

The above experiments show that the effective fraction of total coumarin of Zushima has more significant anti-inflammatory and analgesic effects.

Claims (18)

1. a preparation method of Zushi Ma total coumarin extract, which comprises the following steps: (1) Extracting with the medicinal material of zumima as raw material and water or C ₁ -C ₅ lower alcohol aqueous solution as solvent to obtain the extract; (2) mixing the extract with a solid diluent, adding an organic solvent for extraction, recovering the solvent to obtain a solid extract, or adding water to suspend the extract, adding an organic solvent for extraction, recovering the solvent to obtain a solid extract; (3) Suspend the solid extract with water, use macroporous adsorption resin chromatography, first use water or C ₁ ~ C ₅ lower alcohol aqueous solution as the mobile phase to remove impurities, and then elute with C ₁ ~ C ₅ lower alcohol aqueous solution , to collect the eluate; (4) The first half of the eluent recovers the solvent to obtain a solid, that is, a part of Zushima total coumarin extract; the solid obtained by recovering the solvent from the second half of the eluent is suspended in water, then extracted with an organic solvent, and the organic layer recovers the solvent to obtain Solid, that is another part of Zushi Ma total coumarin extract. 2. The preparation method according to claim 1, characterized in that, the method of extracting in the step (1) is reflux or percolation. 3. The preparation method according to claim 1 or 2, characterized in that the lower alcohol concentration of the C ₁ -C ₅ lower alcohol aqueous solution in the step (1) is 0-95% (volume/volume). 4. The preparation method according to claim 3, characterized in that, in the step (1), the lower alcohol concentration of the C ₁ -C ₅ lower alcohol aqueous solution is 30-70% (volume/volume). 5. The preparation method according to claim 1 or 2, characterized in that, in the step (2), the organic solvent is ethyl acetate, n-butanol and/or water-saturated n-butanol. 6. The preparation method according to claim 1 or 2, characterized in that, the step (2) is mixing the medicinal extract with a solid diluent, adding an organic solvent for extraction, and recovering the solvent to obtain a solid extract. 7. according to the described preparation method of claim 1 or 2, it is characterized in that, in described step (2), solid diluent is selected from one or the other in starch, micronized silica gel, diatomite, kaolin, gac and kaolin Various. 8. according to the described preparation method of claim 1 or 2, it is characterized in that, in described step (3), macroporous adsorption resin is selected from styrene, styrene-acrylic acid ester, totilbene, acrylate or methacrylic acid One or more of ester resins. 9. The preparation method according to claim 1 or 2, characterized in that, the macroporous adsorption resin in the step (3) is a styrene type or a styrene-acrylate type macroporous adsorption resin. 10. The preparation method according to claim 1 or 2, characterized in that the lower alcohol concentration of the lower alcohol aqueous solution used for rinsing to remove impurities in the step (3) is 0-50% (volume/volume). 11. The preparation method according to claim 10, characterized in that the lower alcohol concentration of the lower alcohol aqueous solution used for rinsing and removing impurities in the step (3) is 0-20% (volume/volume). 12. The preparation method according to claim 1 or 2, characterized in that the lower alcohol concentration of the lower alcohol aqueous solution used to elute the resin in the step (3) is 20-90% (volume/volume). 13. The preparation method according to claim 12, characterized in that the lower alcohol concentration of the lower alcohol aqueous solution used to elute the resin in the step (3) is 20-50% (volume/volume). 14. The preparation method according to claim 1 or 2, characterized in that, the organic solvent used in the second half of the eluent extraction of the step (4) is ethyl acetate and/or n-butanol. 15. according to the preparation method described in any one in the claim 1 to 14 the total coumarin extract of ancestral hemp. 16. The total coumarin extract of Zushima according to claim 15, wherein the total coumarin content is not less than 50% by weight of the extract. 17. according to claim 15 or 16 described Zushima total coumarin extract, it is characterized in that, described total coumarin extract comprises daphnetin, Zushima B, daphnetin-8-O-glucoside and 7-OH-coumarin. 18. according to claim 15 or 16 or the content assay method of total coumarin extract of Zu Shimao described in 17, comprise the following steps: (1) HPLC determination of daphnetin, daphnetin, daphnetin-8-O-glucoside and 7-OH-coumarin: Accurately weigh 50mg of the total coumarin extract of Zushima, put it in a 50mL measuring bottle, add an appropriate amount of 50% (volume/volume) methanol aqueous solution to dissolve and dilute to the scale, shake well, and precisely draw 2mL into a 10mL measuring bottle In, add 50% (volume/volume) methanol aqueous solution and dilute to scale, shake up, as need testing solution; Respectively daphnetin, Zuma B, daphnetin-8-O-glucoside and 7-OH- The methanol solution of bean element is the reference substance solution; the chromatographic column uses octadecylsilane bonded silica gel as the filler, and the methanol-0.5% acetic acid aqueous solution with a volume ratio of 25:75 is the mobile phase; the detection wavelength is 327nm; (2) Determination of the total coumarin content of Zushi Ma: Dilute the above-mentioned test solution and Zuma B reference substance solution to an appropriate volume respectively, and measure the absorbance at a wavelength of 310nm according to the ultraviolet-visible spectrophotometry, and calculate.