CN101481398A - Method for preparing high-purity 5-hydroxy-lamiophlomiol A glycoside and lamiophlomiol A glycoside extract from lamiophlomiol at the same time - Google Patents

Abstract

The invention provides a method for simultaneously preparing high-purity sesamoside and phlorigidoside C extracts. The method adopts solitary as a raw material, carries out warm immersion extraction with ethanol solution, and after concentration, is purified by polyacyl column and macroporous resin in turn, and finally Purified by a liquid chromatography preparation system to obtain high-purity sesamoside and phlorigidoside C extracts, the concentrations of the two reached 98.85% and 99.37% respectively, meeting the quality requirements of the national reference substance for content determination. The production cycle of the present invention saves the steps of silica gel column chromatography, is simple to operate, significantly reduces the loss of sesamoside and phlorigedoside C, and is suitable for large-scale industrial production.

Description

A method for simultaneously preparing high-purity 5-hydroxy-monopsin A glycoside and unique monosourdin A glycoside extract from Du Yiwei

technical field

The invention belongs to the technical field of medicines, and in particular relates to a method for simultaneously separating and purifying monomeric compounds sesamoside and phlorigidoside C as a reference substance from a plant.

Background technique

Duyiwei [lamiophlomis rotata (benth.) kudo] is a folk herbal medicine of Tibetan, Mongolian, Naxi and other ethnic groups. It has been recorded in the famous Tibetan medical works "Four Medical Codes" and "Jingzhu Materia Medica" more than 1,000 years ago. It grows alone in alpine meadows and river beaches at an altitude of 2700-4500m. It is mainly produced in Tibet, Qinghai, Yunnan, Sichuan, Gansu and other provinces and regions. Tibetan medicine uses it for osteomyelitis, joint jaundice, fractures, falls, gunshot wounds, etc.

The 2005 edition of Chinese Pharmacopoeia includes the unique medicinal material, but the content determination item, which is a key indicator of the quality control of medicinal materials, uses the method of measuring the content of luteolin in medicinal materials. Flavonoids have various physiological activities, but whether the flavonoids in Duyiwei are the main pharmaceutical ingredients for hemostasis and analgesia remains to be further studied. Studies have shown that the iridoids in Duyiwei are the main medicinal components for its hemostatic and analgesic effects. The iridoid glycosides in Duyiwei include at least 26 compounds with similar structures, among which sesamoside and phlorigidoside C are the two iridoid glycosides with higher content in Duyiwei.

The aerial part of Duyiwei was extracted with ethanol reflux, and the obtained ethanol liquid extract was extracted with acetone. The acetone extract was subjected to polyamide chromatography, and the water-eluted part was subjected to repeated silica gel column chromatography to obtain sesamoside. See Zhang Chengzhong et al., Chinese herbal medicine , Vol23 (1992), 509-510. The method uses conventional silica gel column chromatography to separate and purify the large polar iridoid glycoside component sesamoside, the separation effect is not good, the product purity is low, and there is often a large loss; Ultrasonic extraction of ethanol and 50% ethanol, 50% ethanol extract was purified with D-1400 macroporous resin, 10% ethanol eluted part was purified by silica gel column chromatography, and then purified by ODS-A reverse phase packing to prepare sesamoside and Phlorigidoside C, see Jun-Jie Tan et al., Helv. Chim. Acta, Vol 90 (2007), 143-148. This method has the following limitations: the iridoid glycosides are highly polar and have good water solubility, and they also have great solubility in ethanol-water mixed solvents. Studies have shown that: 95% ethanol has a good effect on the extraction of iridoid glycosides, and the yield of the dry paste can reach 13%, and the contents of iridoid glycosides sesamoside and phlorigidoside C in the dry paste can respectively reach 4.7% and 3.1%. Therefore, extracting the unique medicinal material with 95% ethanol first will cause a large loss of sesamoside and phlorigidoside C.

Chinese patent CN 1660163A discloses a method for extracting total iridoid glycosides from solitary flavor. The method uses solitary flavor as a raw material, extracts by warm soaking in 0-95% ethanol solution at 20-70°C, and concentrates the extract to In the form of extract, add 10-15 times of water at 30-90°C to dissolve, let cool, filter, remove fat-soluble components to obtain an extract, add the extract to a polyamide column, combine the effluent and washing liquid onto a macroporous resin column, 10 -95% ethanol elutes the macroporous resin column to obtain the total iridoid glycosides. The total iridoid glycosides obtained by this method is a complex mixture system, and whether it contains sesamoside and phlorigidoside C is not reported.

Sesamoside is also found in other plants: Crab chinensis, crushed and extracted with 95% ethanol, the extract is recovered by vacuum distillation to obtain a crude extract. After the crude extract was treated, it was extracted with petroleum ether, chloroform, ethyl acetate and n-butanol, and the n-butanol extracted part was subjected to silica gel column chromatography to obtain the compound sesamoside respectively. See Gao Yongli et al., Chinese Materia Medica, Vol 30( 2007), 1239-1242; take naturally dried Rhizoma Rhizoma Rhizoma Rhizome root, grind it and extract it with 95% ethanol under reflux for 3 times, each time for 6h. The extract was concentrated under reduced pressure to obtain the extract, which was suspended with water and then extracted with petroleum ether, ethyl acetate, and n-butanol respectively, and the ethyl acetate extract was separated repeatedly through a silica gel column and gel permeation chromatography to prepare a high-pressure Purified by liquid chromatography to obtain the compound sesamoside, refer to people such as Liu Pu, Acta Pharmaceutica Sinica, Vol 42 (2007), 401-404; Gentiana radish root coarse powder, reflux extraction with 95% ethanol, each 2h, combined extracts, reduced Press and concentrate to obtain the extract. After the extract is dissolved and diluted with water, it is adsorbed by D101 macroporous adsorption resin and eluted with 10%, 40%, and 95% ethanol respectively. The extract extracted from 40% ethanol was subjected to vacuum silica gel column chromatography and chloroform/methanol gradient elution, and the obtained parts were separated by repeated silica gel column chromatography and preparative high-performance liquid chromatography to obtain the compound sesamoside. See Yu Zhenxi et al., China Journal of Traditional Chinese Medicine, Vol 31 (2006), 656-658; the powder of the underground part of Rhizoma Rhizome, first leached 4 times with 95% ethanol in a slightly boiling state, and after concentrated under reduced pressure, the extract was suspended in water and washed with ethyl acetate successively. The ester and n-butanol were extracted separately, and the n-butanol extract was subjected to silica gel column chromatography, eluted with a gradient of chloroform/methanol, and detected by TCL, and combined into 9 fractions (Fr1-9). Fr-3 was subjected to silica gel column chromatography and chloroform/methanol gradient elution to obtain sesamoside, see Yang Yongli et al., Journal of Lanzhou University, Vol 40 (2004), 67-71.

The above preparation process adopts phytochemical research methods, and the extraction and purification processes of sesamoside and phlorigidoside C have not been studied in depth, and the research level is also limited to the laboratory scale, which is not suitable for industrial production. Repeat above-mentioned method, the purity of sesamoside and phlorigidoside C that separation obtains is checked, can not reach national standard. At the same time, there are no reference substances of sesamoside and phlorigidoside C that meet the national standards on the market.

On the basis of previous studies, the inventor aimed at the shortcomings of the current technology, and after repeated research and exploration, finally invented a simple and industrially feasible method for simultaneously separating and purifying sesamoside and phlorigidoside C from the unique aerial part. The chemical structural formulas of sesamoside and phlorigidoside C are as follows:

Contents of the invention

The purpose of the present invention is to provide a method for simultaneously extracting and purifying sesamoside and phlorigidoside C which can be used as reference substances from Duyiwei.

The present invention is achieved through the following technical solutions:

(1) Extraction: Add 6-12 times of the unique coarse powder, 50-80% ethanol, soak at 50-80°C, extract 3 times, each time for 1-3 hours, combine the extracts, concentrate under reduced pressure, and recover ethanol to obtain a concentrated solution.

(2) Purification of polyamide: add the concentrate obtained in step (1) to a polyamide column, elute with 3-5BV of water, collect the effluent from the upper column and the water eluate to obtain a mixed solution.

(3) macroporous resin purification: add the mixed solution obtained in step (2) to the macroporous resin column, first elute with 5-10BV of water, then elute with 3-5BV30-80% ethanol, collect the ethanol for elution liquid, concentrated to dryness under reduced pressure to obtain a crude product.

(4) Purification by liquid chromatography preparation system: the crude product obtained in step (3) is purified by liquid chromatography preparation system RP-C18 column, methanol-water (20-35%) is eluted, the detection wavelength is 237nm, and the retention times are collected respectively Fractions of 9-17min and 11-19min were concentrated under reduced pressure and freeze-dried to obtain the obtained product.

The liquid chromatography preparation system purification described in the present invention includes a medium pressure or high pressure liquid chromatography preparation system.

The medium-pressure or high-pressure liquid chromatography preparation system of the present invention adopts RP-C18 column.

The advantages of the present invention are:

(1) The sesamoside and phlorigidoside C prepared by this method have a purity of 98.85% and 99.37% respectively. This method was proposed for the first time, and the product obtained by using this method met the quality requirements of the national content determination reference substance for the first time;

(2) the method adopts a medium-pressure or high-pressure liquid chromatography preparation system, which saves the step of silica gel column chromatography, is simple to operate, and significantly reduces the loss of sesamoside and phlorigedoside C;

(3) The method can simultaneously separate and purify two substances, sesamoside and phlorigidoside C, from Duyiwei. This process greatly saves costs and is suitable for large-scale industrial production.

Attachment: The carbon spectrum and hydrogen spectrum data of sesamoside and plorigidoside C are as follows:

Carbon spectrum data of sesamoside and plorigidoside C

Proton spectrum data of sesamoside and plorigidoside C

Description of drawings

Fig. 1 is a high performance liquid phase purity inspection diagram of Sesamoside of the present invention, and through high performance liquid phase detection, the purity of Sesamoside reaches 99.85%.

Fig. 2 is the HPLC purity inspection diagram of Phlorigidoside C of the present invention, through HPLC detection, the purity of Phlorigidoside reaches 99.37%.

Detailed ways

Embodiment one

(1) Extraction: Duyiwei coarse powder 100g, add 6 times the amount, 50% ethanol, soak at 50°C, extract 3 times, each time for 1 hour, combine the extracts, concentrate under reduced pressure, reclaim ethanol, concentrate, filter After that, 300ml of concentrated solution with a relative density of 1.05 was obtained.

(2) Purification of polyamide: add the concentrate to the polyamide column, elute with 3BV of water, collect the effluent from the upper column and the water eluate to obtain 600ml of mixed solution.

(3) Macroporous resin purification: the mixed solution is added to the AB-8 macroporous resin column, first eluted with 5BV of water, and then 3BV of 30% ethanol, and the alcohol eluate is collected and concentrated to dryness under reduced pressure to obtain crude Extract 10.3g.

(4) Purification by liquid chromatography preparation system: the crude extract is purified by medium-pressure liquid chromatography preparation system RP-C18 column, methanol-water (25%) is eluted, the detection wavelength is 237nm, and the retention time is 14min and 16min respectively. The fractions were concentrated under reduced pressure and freeze-dried to obtain sesamoside, 312mg and phlorigidoside C, 285mg.

Embodiment two

(1) Extraction: Add 100 g of Duyiwei coarse powder, add 8 times the amount, 60% ethanol, soak at 70 ° C, extract 3 times, each time for 1 hour, combine the extracts, concentrate under reduced pressure, reclaim ethanol, and obtain a relative density of 1.05 concentrate 300ml.

(2) Purification of polyamide: add the concentrate to the polyamide column, elute with 5BV of water, collect the effluent from the upper column and the water eluate to obtain 800ml of mixed solution.

(3) Macroporous resin purification: the mixed solution is added to the XAD-1600 macroporous resin column, first eluted with 8BV of water, then 5BV of 70% ethanol, and the alcohol eluate is collected and concentrated to dryness under reduced pressure to obtain crude Extract 11.9g.

(4) Purification by liquid chromatography preparation system: the crude extract is purified by medium-pressure liquid chromatography preparation system RP-C18 column, methanol-water (20%) is eluted, and the detection wavelength is 237nm, and the retention time is 11min and 13min respectively. The fractions were concentrated under reduced pressure and freeze-dried to obtain sesamoside, 345mg and phlorigidoside C, 296mg.

Embodiment three

(1) Extraction: 100 g of Duyiwei coarse powder, add 10 times the amount, 80% ethanol, soak at 80 ° C, extract 3 times, each time for 2 hours, combine the extracts, concentrate under reduced pressure, reclaim ethanol, and obtain a relative density of 1.10 Concentrate 140ml.

(2) Purification of polyamide: Add the crude extract 1 to the polyamide column, elute with 5BV of water, collect the effluent from the upper column and the water eluate to obtain 640ml of the mixed solution.

(3) macroporous resin purification: the concentrated solution is added to the XAD-16 macroporous resin column, first eluted with 10BV of water, and then eluted with 4BV of 80% ethanol, the alcohol eluate is collected, and concentrated to dryness under reduced pressure to obtain Crude extract 11.9g.

(4) Purification by liquid chromatography preparation system: the crude extract is purified by medium-pressure liquid chromatography preparation system RP-C18 column, methanol-water (35%) is eluted, the detection wavelength is 237nm, and the retention time is 6min and 9min respectively. The fractions were concentrated under reduced pressure and freeze-dried to obtain sesamoside, 345mg and phlorigidoside C, 296mg.

Embodiment four

(1) Extraction: Add 100 g of Duyiwei coarse powder, add 8 times the amount, 60% ethanol, soak at 70 ° C, extract 3 times, each time for 1 hour, combine the extracts, concentrate under reduced pressure, reclaim ethanol, and obtain a relative density of 1.05 concentrate 300ml.

(2) Purification of polyamide: add the concentrate to the polyamide column, elute with 5BV of water, collect the effluent from the upper column and the water eluate to obtain 800ml of mixed solution.

(3) Macroporous resin purification: add the mixed solution to the D101 macroporous resin column, elute with 5BV of water first, then elute with 4BV of 70% ethanol, collect the alcohol eluate, concentrate to dryness under reduced pressure, and obtain a crude extract 10.8g.

(4) Purification by liquid chromatography preparation system: the crude extract is purified by the RP-C18 column of the high-pressure liquid chromatography preparation system, eluting with methanol-water (20%), and the detection wavelength is 237nm, and the streams with retention times of 6min and 9min are collected respectively. Parts, concentrated under reduced pressure, freeze-dried to obtain sesamoside, 365mg and phlorigidoside C, 294mg.

Claims (4)

1, a kind of method for preparing high purity sesamoside and phlorigidosideC extract from Root of Common Lamiophlomis simultaneously may further comprise the steps:

(1) extract: the Root of Common Lamiophlomis meal, add the ethanol of 6-12 times of 50-80%, 50-80 ℃ of warm lixiviate got 3 times, each 1-3 hour, united extraction liquid, concentrating under reduced pressure reclaims ethanol, concentrated solution;

(2) polyamide purifying: the concentrated solution that step (1) is obtained adds on the polyamide column, uses the 3-5BV water elution, collects upper prop effluent liquid and water elution liquid, gets mixed solution;

(3) macroporous resin purification: the mixed solution that step (2) is obtained adds on the macroporous resin column, use the 5-10BV water elution earlier, uses the ethanol elution of 3-5BV30-80% again, collects alcohol eluen, be evaporated to dried, must crude product;

(4) liquid chromatography preparation system purifying: the crude product that step (3) is obtained is through liquid chromatography preparation system purifying, carries out gradient elution with the methanol-water of 20-35%, detects wavelength 237nm, collecting retention time respectively is stream part of 9-17min and 11-19min, concentrating under reduced pressure, lyophilize, promptly.

2, the method for preparing high purity sesamoside and phlorigidosideC extract simultaneously as claimed in claim 1 is characterized in that: the liquid chromatography preparation system that (4) step wherein adopts comprises medium-pressure or high pressure liquid chromatography preparation system.

3, the method for preparing high purity sesamoside and phlorigidosideC extract simultaneously as claimed in claim 2 is characterized in that: described medium-pressure or high pressure liquid chromatography preparation system adopts the RP-C18 post.

4, the method for preparing high purity sesamoside and phlorigidosideC extract simultaneously as claimed in claim 1, it is characterized in that: the sesamoside and the phlorigidoside C purity of preparation gained have reached 98.85% and 99.37% respectively, meet the specification of quality of national assay with reference substance.

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