CN104163755B - A kind of method preparing magnolol and Honokiol from Leaf of Magnolia officinalis - Google Patents

Abstract

The invention relates to a method for preparing magnolol and honokiol from magnolia leaves, belonging to the field of traditional Chinese medicine modernization. A method for preparing magnolol and honokiol from magnolia leaves, using methods such as organic solvent extraction, small-pore resin separation preparation, and organic solvent crystallization to prepare high-purity magnolol and honokiol from magnolo leaves Compared with the existing technological methods, the phenol monomer component has the advantages of simple process, low cost of crude extraction solvent, low cost of column chromatography, high separation precision of packing, reusable, low cost of chromatographic solvent, and relative environmental pollution. It has the advantages of light weight, high purity of magnolol and honokiol, and is suitable for industrial preparation.

Description

A method for preparing magnolol and honokiol from magnolol leaves

technical field

The invention relates to a method for preparing magnolol (Magnolol) and honokiol (Honokiol) from magnolia leaves, belonging to the field of traditional Chinese medicine modernization.

Background technique

Magnolol and honokiol are isomers, and they are the antibacterial active components of Magnolia officinalis commonly used in traditional Chinese medicine, and they are also the main index components of the quality control of Magnolia officinalis. It has been reported in the literature that in addition to antibacterial effects, the two also have anti-inflammatory, anti-anxiety, anti-depressant, anti-tumor, anti-aging and cardioprotective effects; in addition, there are obvious and long-lasting central muscle relaxation and central nervous inhibition effects; recently Pharmacodynamic studies have confirmed that magnolol and honokiol also exhibit hypoglycemic activity.

Magnolia officinalis is a commonly used traditional Chinese medicine, derived from the dried dry bark, branch bark and root bark of Magnolia officinalis Rehd et Wils. and Magnolia officinalis Rehd.et Wils.var.biloba Rehd.et Wils. It is mainly produced in Sichuan, Hubei, Zhejiang, Fujian, Anhui and other places. It has the effects of aromatizing dampness, promoting qi and eliminating accumulation, drying dampness and eliminating fullness, reducing adverse events and relieving asthma. Symptoms such as abdominal distension, vomiting and diarrhea, phlegm stagnation and qi reversal, chest fullness, wheezing and coughing. Magnolia officinalis is used as medicine in many commonly used prescriptions and Chinese patent medicine preparations.

With the expansion of the application range of magnolia officinalis, its demand is constantly increasing. In order to meet the increasing demand, magnolia officinalis production bases have been established in major production areas such as Sichuan, Hubei, Zhejiang, and Fujian. Develop Magnolia officinalis planting. According to preliminary statistics, the area of Magnolia officinalis and Magnolia officinalis planted in the country is not less than 700,000 mu, and most of the cultivated Magnolia officinalis forests are pure Magnolia officinalis forests, with about 200 to 300 Magnolia officinalis cultivated per mu, and the average annual dry leaves of Magnolia officinalis per tree Calculated at 1.5-2.5kg, the reserve of Magnolia officinalis in the whole country can reach more than 300,000 tons per year. Magnolia officinalis is a deciduous tree. Its leaves are 25-50cm long and 15-25cm wide. They will naturally wither in November and produce new leaves in the next spring. Therefore, Magnolia officinalis leaves are easy to obtain, rich in resources and can be regenerated every year. At present, a large number of Magnolia officinalis leaves are not utilized in China, and they are allowed to fall and rot. If they can be developed and utilized as new medicinal materials, it will not only benefit the comprehensive utilization of Magnolia officinalis resources, but also produce greater economic and social benefits. The income increase of drug farmers and pharmaceutical farmers also played a certain supporting role.

Modern analytical chemistry studies have found that Magnolia officinalis leaves also contain the same lignans as Magnolia officinalis, which can be used as raw plant parts of magnolol and honokiol. Existing patents report magnolol and honokiol extraction methods in Magnolia officinalis leaves include organic solvent extraction, temperature control curve control of heating rate and constant temperature extraction. These extraction methods have too many impurities in the target substance, and the purity of magnolol and honokiol is low, so they are not suitable for large-scale industrial production.

Chinese patent application CN 102304027 A discloses a method for extracting magnolol from Magnolia officinalis leaves, the steps are: 1. Magnolia officinalis leaf powder is soaked in methanol for 24h, suction filtered, repeated leaching three times, and the filtrate is concentrated to an extract ;2. Add saturated and clarified lime water to the extract, stir for 1 hour, centrifuge, and repeat three times; 3. Acidify the supernatant to pH=2-3, stir for 30 minutes, centrifuge, mix the solid with silica gel, and then heat with petroleum ether Bring to a slight boil, keep warm for 10 minutes, heat filter, and dry the solution with anhydrous Na ₂ SO ₄ ; 4. Concentrate the dry petroleum ether solution, cool and crystallize to obtain the magnolol product. In this method, it is necessary to use concentrated acid to adjust the pH value of the supernatant to 2-3, and to use petroleum ether to heat to a slight boil. Petroleum ether is flammable and explosive, and its vapor and air can form explosive mixtures, which can cause combustion and explosion when exposed to open flames and high heat. These factors increase the risk of operation, which is not conducive to the protection of laborers, nor to the implementation of industrialization. The cost of petroleum ether is higher.

Contents of the invention

The purpose of the present invention is to address the shortcomings of the existing industrialized separation of magnolol and honokiol technology, to provide a process with simple equipment, short production cycle, small investment scale, safe operation, relatively light environmental pollution, and low production cost. High-purity magnolol with low yield and high yield and a preparation method of honokiol.

The present invention realizes object of the present invention by following method:

A method for preparing magnolol and honokiol from magnolia leaves, the steps are as follows:

(1) get dried Magnolia officinalis and pulverize;

(2) Magnolia officinalis leaf powder is reflux extracted 2-3 times with the pH value of 4～6 times of amount being 9-10 volume percentage concentration of 60-95% alkaline methanol or ethanol solution, and extraction temperature is 60～70 ℃, Reflux extraction for 0.5-3 hours each time, combine the extracts, adjust the pH value to 6-7 with acid, and recover the solvent to obtain the extract;

(3) After dissolving the extract with 9-10 times the amount of water, extract 2-3 times with 1-2 times the amount of dichloromethane, or use alumina to absorb and enrich, collect the effluent, evaporate to dryness, and set aside;

(4) Take the small-pore separation resin and pressurize the column, balance the column with methanol or ethanol or acetone aqueous solution with a concentration of 35-50% by volume, then slowly add the sample solution dropwise, and let it stand for 1-2 hours after loading the sample. After 1.5h, use 1 to 2 times the volume percentage concentration of 50%, 55%, 60%, 65%, 70% methanol, ethanol or acetone aqueous solution to elute under pressure, and collect magnolol and honokiol Section eluate, concentrate recovery solvent, obtain magnolol and honokiol mixed component;

(5) Take magnolol and honokiol mixed components, dissolve with anhydrous methanol, or ethanol, or acetone, mix the sample with 4 to 6 times the amount of silica gel, use silica gel as a filler, and use 18 to 20 times the amount The volume ratio of petroleum ether-acetone is 6:1-8:1 as the eluent, chromatography, detection, crystallization twice with 2 to 4 times the amount of acetone-methanol mixture to obtain magnolol and honokiol monomer.

The alkaline methanol or ethanol solution described in step (2) adopts sodium bicarbonate, sodium hydroxide or ammonia water to adjust its pH value; the acid adjusted to neutrality with acid is hydrochloric acid.

The medium and small pore separation resin in step (4) is Chroma-R204. The high-fine separation and small-pore-diameter adsorption resin adopted in the present invention is cheap, uniform in pore size distribution, high in separation precision, does not cause dead adsorption to samples, can be reused, has low cost of elution solvent, and large sample loading.

The acetone-methanol mixed solution described in step (5) is prepared by mixing acetone and methanol at a volume ratio of 15:1.

A magnolia bark leaf extract is prepared by the following method:

(1) get dried Magnolia officinalis and pulverize;

(2) Magnolia officinalis leaf powder is reflux extracted 2-3 times with the pH value of 4～6 times of amount being 9-10 volume percentage concentration of 60-95% alkaline methanol or ethanol solution, and extraction temperature is 60～70 ℃, Reflux extraction for 0.5-3 hours each time, combine the extracts, adjust the pH value to 6-7 with acid, and recover the solvent to obtain the extract;

(3) After dissolving the extract with 9-10 times the amount of water, extract 2-3 times with 1-2 times the amount of dichloromethane, or use alumina to absorb and enrich, collect the effluent, evaporate to dryness, and set aside;

(4) Take the small-pore separation resin and pressurize the column, balance the column with methanol or ethanol or acetone aqueous solution with a concentration of 35-50% by volume, then slowly add the sample solution dropwise, and let it stand for 1-2 hours after loading the sample. After 1.5h, use 1 to 2 times the volume percentage concentration of 50%, 55%, 60%, 65%, 70% methanol, ethanol or acetone aqueous solution to elute under pressure, and collect magnolol and honokiol Section eluate, concentrate recovery solvent, obtain magnolol and honokiol mixed component;

(5) Take magnolol and honokiol mixed components, dissolve with anhydrous methanol, or ethanol, or acetone, mix the sample with 4 to 6 times the amount of silica gel, use silica gel as a filler, and use 18 to 20 times the amount The volume ratio of petroleum ether-acetone is 6:1-8:1 as the eluent, chromatography, detection, crystallization twice with 2 to 4 times the amount of acetone-methanol mixture to obtain magnolol and honokiol monomer.

The alkaline methanol or ethanol solution described in step (2) adopts sodium bicarbonate, sodium hydroxide or ammonia water to adjust its pH value; the acid adjusted to neutrality with acid is hydrochloric acid.

The medium and small pore separation resin in step (4) is Chroma-R204.

The acetone-methanol mixed solution described in step (5) is prepared by mixing acetone and methanol at a volume ratio of 15:1.

The content of magnolol and honokiol in the extract is >98%.

The Magnolia officinalis extract is used to prepare antibacterial drugs, anti-inflammatory drugs, anti-anxiety drugs, antidepressants, anti-tumor drugs, anti-aging drugs, cardioprotective drugs or hypoglycemic drugs.

The advantages of the method of the invention are as follows: the invention provides a method for industrially producing magnolol and honokiol, which are active characteristic components, using the non-medicinal parts of Magnolia officinalis as raw materials. Magnolol and honokiol are less polar and weakly acidic, so extraction with a high concentration of alkaline alcohol can make the dissolution rate of the two higher. A single spot of magnolol and honokiol on TLC can be better separated by using different concentrations of acetone, ethanol or methanol as eluents on the extract to separate the resin under pressure. It can effectively separate impurities such as chlorophyll and long-chain fatty acids. The high-fine separation and small-pore-diameter adsorption resin will not cause dead adsorption to the sample, and it can be reused. It has the advantages of low cost of elution solvent and large sample loading. At present, the small-pore semi-gel type adsorption resin developed by some domestic manufacturers has uniform pore size distribution, and the price of 40-100um is relatively low, about 300 yuan per kilogram. Series separation function.

The method of the invention has simple process equipment, short production cycle, small investment scale, relatively light environmental pollution, low preparation cost, high purity of magnolol and honokiol, simple and safe operation, and is suitable for industrial production.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, which are not intended to limit the present invention. All equivalent replacements in the field made according to the disclosure of the present invention belong to the protection scope of the present invention.

Description of drawings

Fig. 1 is the honokiol HPLC collection of patterns of embodiment 1

Fig. 2 is the honokiol HPLC collection of illustrative plates of embodiment 2

Fig. 3 is the magnolol HPLC collection of patterns of embodiment 1

Fig. 4 is the magnolol HPLC collection of illustrative plates of embodiment 2

Detailed ways

The method of the present invention is described in further detail below by way of examples, but protection scope of the present invention is not limited to described content, if no special instructions in the examples, use reagent is routine commercially available reagent, or the reagent that makes by conventional method.

Example 1:

1. Take 96g of Magnolia officinalis leaves and grind them to a particle size of 40 mesh;

2. Magnolia officinalis leaf powder is reflux extracted three times with ammonia water to adjust the pH value of 10 95% ethanol solution, the extraction temperature is 60 ° C, wherein the first 500ml solution is reflux extracted for 2 hours and then filtered, and the filter residue is reflux extracted for the second time with 500ml solution for 1 hour After filtering, the filter residue was refluxed for the third time with 500ml of solution and filtered for 0.5 hours, and the three extracts were combined; the pH value was adjusted to 6 with hydrochloric acid, and the solvent was recovered to obtain a crude extract (28.8g);

3. Dissolve the crude extract in 300ml of water, extract 3 times with dichloromethane, 300ml each time, combine the dichloromethane solution, and evaporate to dryness to obtain extract (14.2g);

4. Take 500g of small-pore separation resin Chroma-R204 and pressurize the column, equilibrate the column with acetone aqueous solution with a volume percentage concentration of 50%, and then slowly add the sample solution dropwise. Concentration by volume is 50%, 55%, 60%, 65%, 70% acetone aqueous solution pressurized elution (7bar), TLC distinguishes and collects magnolol and honokiol section eluate, reclaims acetone water, obtains the composition Point A (8.3g);

5. Take component A, dissolve it with 100ml of acetone, mix the sample with 50g of silica gel, use silica gel as a filler, elute with 800ml of petroleum ether—acetone (8:1), concentrate to obtain component B (2.2g) and Part C (1.8g) was crystallized twice with 7ml of acetone-methanol mixture (acetone and methanol were mixed at a volume ratio of 15:1) and 6ml of acetone-methanol mixture (acetone and methanol were mixed at a volume ratio of 15:1), which could be separated Purification afforded compound 1 (0.24 g) and compound 2 (0.13 g). HPLC detection content >98% (see Figure 1 and Figure 3)

Compound monomer 1 and compound 2 obtained above were identified as Magnolol and Honokiol respectively by mass spectrometry, H-NMR spectrum and C-NMR spectrum.

Example 2:

1. Take 95g of Magnolia officinalis leaves and crush until the particle size is 40 mesh;

2. Magnolia officinalis leaf powder is reflux extracted three times with ammonia water to adjust the pH value of 95% ethanol solution, the extraction temperature is 60 ° C, wherein the first 500ml solution is reflux extracted for 2 hours and then filtered, and the filter residue is reflux extracted for the second time with 500ml solution for 1 hour After filtering, the filter residue was refluxed for the third time with 500ml solution and filtered for 0.5 hours, and the three extracts were combined; the pH value was adjusted to 6 with hydrochloric acid, and the solvent was recovered to obtain a crude extract (28.2g);

3. Dissolve the crude extract in 300ml of water, filter, dissolve the residue in 100ml of acetone, add 400ml of water to form a suspension, use 100g of alumina to absorb and enrich, collect the effluent, evaporate to dryness, and obtain an extract (13.3g);

4. Take 500g of small-pore separation resin Chroma-R204 and pressurize the column, equilibrate the column with acetone aqueous solution with a volume percentage concentration of 50%, and then slowly add the sample solution dropwise. Concentration by volume is 50%, 55%, 60%, 65%, 70% acetone aqueous solution pressurized elution (7bar), TLC distinguishes and collects magnolol and honokiol section eluate, reclaims acetone water, obtains the composition Point A (9.2g);

5. Take component A, dissolve it with 100ml of acetone, mix the sample with 50g of silica gel, use silica gel as a filler, elute with 900ml of petroleum ether—acetone (8:1), concentrate to obtain component B (2.4g) and Part C (1.6g) was crystallized twice with 7.5ml of acetone-methanol mixture (acetone and methanol were mixed at a volume ratio of 15:1) and 5ml of acetone-methanol mixture (acetone and methanol were mixed at a volume ratio of 15:1). Compound 1 (0.73g) and compound 2 (0.49g) were obtained by separation and purification. HPLC detection content > 98% (see Figure 2 and Figure 4).

Compound monomer 1 and compound 2 obtained above were identified as Magnolol and Honokiol respectively by mass spectrometry, H-NMR spectrum and C-NMR spectrum.

Example 3:

1. Take 98g Magnolia officinalis leaves and grind them to a particle size of 40 mesh;

2. Magnolia officinalis leaf powder is reflux extracted twice with ammonia water to adjust the pH value of 9 60% methanol solution, the extraction temperature is 70 ° C, wherein the first 500ml solution is reflux extracted for 3 hours and then filtered, and the filter residue is reflux extracted for the second time with 500ml solution 2 Filtrate after 1 hour, combine two extracts; Regulate pH value 7 with hydrochloric acid, reclaim solvent, obtain crude extract (29.4g);

3. Dissolve the crude extract in 300ml of water, extract twice with dichloromethane, 300ml each time, combine the dichloromethane solution, evaporate to dryness, and obtain extract (15.2g);

4. Take 500g of small pore size separation resin Chroma-R204 and pressurize the column, equilibrate the column with methanol aqueous solution with a concentration of 35% by volume, and then slowly add the sample solution dropwise. 500ml volume percentage concentration is 50%, 55%, 60%, 65%, 70% methanol aqueous solution pressurized elution (7bar), TLC distinguishes and collects magnolol and honokiol section eluate, reclaims methanol water, obtains Component A (7.8 g);

5. Take component A, dissolve it with 100ml of methanol, mix the sample with 50g of silica gel, use silica gel as a filler, elute with 800ml of petroleum ether—acetone (6:1), concentrate to obtain component B (3.3g) and Part C (1.9g) was crystallized twice with 10ml of acetone-methanol mixture (acetone and methanol mixed at a volume ratio of 15:1) and 6ml of acetone-methanol mixture (acetone and methanol were mixed at a volume ratio of 15:1), which could be separated Purification afforded Compound 1 (0.22 g) and Compound 2 (0.12 g). HPLC detection content>98%.

Compound monomer 1 and compound 2 obtained above were identified as Magnolol and Honokiol respectively by mass spectrometry, H-NMR spectrum and C-NMR spectrum.

Example 4:

1. Take 97g of Magnolia officinalis leaves and grind them to a particle size of 40 mesh;

2. Magnolia officinalis leaf powder is reflux extracted three times with ammonia water to adjust the pH value of 9 70% ethanol solution, the extraction temperature is 65 ° C, wherein the first 500ml solution is reflux extracted for 3 hours and then filtered, and the filter residue is reflux extracted for the second time with 500ml solution for 2 hours After filtering, the filter residue was refluxed for the third time with 500ml solution and filtered for 0.5 hours, and the three extracts were combined; the pH value was adjusted to 7 with hydrochloric acid, and the solvent was recovered to obtain a crude extract (28.7g);

3. Dissolve the crude extract in 300ml of water, filter, dissolve the residue in 100ml of acetone, add 400ml of water to form a suspension, use 100g of alumina to absorb and enrich, collect the effluent, evaporate to dryness, and obtain an extract (13.2g);

4. Take 500g of small pore size separation resin Chroma-R204 and pressurize the column, equilibrate the column with 40% ethanol water solution by volume, then slowly add the sample solution dropwise, and then use the 500ml volume percentage concentration is 50%, 55%, 60%, 65%, 70% acetone aqueous solution pressurized elution (7bar), TLC differentiates and collects magnolol and honokiol section eluate, reclaims ethanol water, obtains Component A (6.6 g);

5. Take component A, dissolve it with 100ml ethanol, mix the sample with 50g silica gel, use silica gel as filler, elute with 1000ml petroleum ether—acetone (7:1), concentrate to obtain component B (2.8g) and Part C (1.4g) was crystallized twice with 9ml of acetone-methanol mixture (acetone and methanol were mixed at a volume ratio of 15:1) and 4.5ml of acetone-methanol mixture (acetone and methanol were mixed at a volume ratio of 15:1), and Compound 1 (0.63g) and compound 2 (0.21g) were obtained by separation and purification. HPLC detection content>98%.

Compound monomer 1 and compound 2 obtained above were identified as Magnolol and Honokiol respectively by mass spectrometry, H-NMR spectrum and C-NMR spectrum.

Claims (3)

1. a method for preparing magnolol and honokiol from Magnolia officinalis leaves, is characterized in that the steps are as follows: (1) get dried Magnolia officinalis and pulverize; (2) Magnolia officinalis leaf powder is reflux extracted 2-3 times with the pH value of 4～6 times of amount being 9-10 volume percentage concentration of 60-95% alkaline methanol or ethanol solution, and extraction temperature is 60～70 ℃, Reflux extraction for 0.5-3 hours each time, combine the extracts, adjust the pH value to 6-7 with acid, and recover the solvent to obtain the extract; (3) After dissolving the extract with 9-10 times the amount of water, extract 2-3 times with 1-2 times the amount of dichloromethane, or use alumina to absorb and enrich, collect the effluent, evaporate to dryness, and set aside; (4) Take the small-pore separation resin Chroma-R204 and pressurize the column, equilibrate the column with methanol or ethanol or acetone aqueous solution with a concentration of 35-50% by volume, then slowly add the sample solution dropwise, and wait until the sample is loaded. After standing for 1-1.5h, use 1-2 times the volume percentage concentration of 50%, 55%, 60%, 65%, 70% methanol, ethanol or acetone aqueous solution for pressure elution, and collect magnolol and The magnolol segment eluate is concentrated and recovered to obtain a mixed component of magnolol and honokiol; (5) Take magnolol and honokiol mixed components, dissolve with anhydrous methanol, or ethanol, or acetone, mix the sample with 4 to 6 times the amount of silica gel, use silica gel as a filler, and use 18 to 20 times the amount The volume ratio of petroleum ether-acetone is 6:1-8:1 as the eluent, chromatography, detection, crystallization twice with 2 to 4 times the amount of acetone-methanol mixture to obtain magnolol and honokiol monomer. 2. the method extracting magnolol and honokiol from Magnolia officinalis leaves according to claim 1, is characterized in that: the alkaline methanol or ethanol solution described in step (2) adopts sodium bicarbonate, sodium hydroxide Or ammonia water to adjust its pH value; the acid adjusted to a pH value of 6-7 with acid is hydrochloric acid. 3. the method for extracting magnolol and honokiol from Magnolia officinalis leaves according to claim 1, is characterized in that: the acetone-methanol mixed solution described in step (5) is acetone and methyl alcohol by volume ratio 15:1 Made by mixing.