KR20080016269A - Manufacturing process of high purity honokiol, magnolol and extract - Google Patents

Abstract

A method for preparing a highly pure honokiol, magnolol, or a mixture thereof from a lowly pure Magnoliae Cortex or an extract thereof is provided to mass-produce effectively the honokiol, magnolol, or the mixture thereof having high medicinal activities by recrystallizing the lowly pure Magnoliae Cortex or an extract thereof using a non-polar solvent or solubility. A method for preparing a highly pure honokiol comprises the steps of: (a) adding a non-polar solvent having a polarity index of 0-3, which is more than 5 times of a weight of a sample and is selected from the group consisting of hexane, petroleum ether, trichloroethylene, ethyl ether, cyclohexane, and benzene, to a lowly pure Magnoliae Cortex or an extract thereof; and (b) after warming up the mixture of the step(a) in a double boiler at a temperature of more than 40 deg.C, cooling it to obtain the highly pure honokiol as a precipitate. A method for preparing a highly pure magnolol comprises the steps of: (a) adding 0.1-2.0% of a solvent having a polarity index of 3-6, which is selected from the group consisting of acetone, acetonitrile, chloroform, methylene chloride, ethanol, ethyl acetate, ethyl ether, and methanol, to a lowly pure Magnoliae Cortex or an extract thereof; and (b) after warming up the mixture of the step(a) in a double boiler at a temperature of more than 40 deg.C, cooling it to obtain the highly pure magnolol as a precipitate. A method for preparing a mixture having a magnolol and a honokiol with high content comprises the steps of: (a) adding 0.1-2.0% of at least one of the solvent having a polarity index of 3-6 to a lowly pure Magnoliae Cortex or an extract thereof and dissolving it by bathing; (b) adding at least one of the polar solvent to the solution of the step(a) and warming up the mixture at a temperature of more than 40 deg.C; and (c) cooling the mixture of the step(b) to obtain the mixture as a precipitate.

Description

Manufacturing Process of High Purity Honokiol, Magnool and Extract Containing the Same {Manufacturing Process of High Purity Honokiol, Magnolol and Extract}

Figure 1 is a schematic diagram showing a method of producing a high purity honokiol, magnol and extract containing the same of the present invention.

Figure 2 is a 40-fold micrograph showing the high purity honokiol crystals prepared according to the present invention.

FIG. 3 is a 40-fold micrograph showing high purity magnol crystals prepared according to the present invention. FIG.

The present invention relates to a method for effectively separating and purifying high-purity honokiol, magnol or a high-purity extract containing the same from the groin or the groin muscle, and more specifically, the thick-bag containing low-purity honokiol and magnool ) A method of producing high purity honokiol by dissolving the extract using a nonpolar solvent and adjusting the solubility using temperature difference, ii) a high purity mag using solubility after saturating the extract The present invention relates to a method for producing nool, i) a method of preparing an extract comprising high purity honokiol and magnool by dissolving a sample through a minimum solvent for dissolving the extract, and then adding a solvent for decreasing solubility.

Magnolia bark and Magnoliae radix are deciduous trees belonging to the Magnoliaceae family, and their origin is divided into Chinese Magnolia officinalis Rehder et Wilson and Japanese Magnolia obovata Thunberg. In addition, it has the effects of hygroscopic hyphae and Hagijeman, so it is wet, dry soil, and food gas. , Abdominal constipation (담), Dammamcheonpo (痰 飮 喘咳) to treat.

The essential ingredients of bakbak are essential oils such as alpha, beta, gamma-oidesmol (α, β, γ-eudesmol), magnool, honokiol, tetrahydromagnolol and magnocuralin. contains magnocurarine, magnoflorine, obovatol, obovataldehyde, saponin, etc. (Quality Standardization Research Report on Herbal Medicine and Herbal Medicine, 1996, Korea Food and Drug Administration, Seoul)

Studies on the pharmacological effects of hoobac have shown that the water extract of hoobak is anti-allergic (Shin, TY et al., Arch. Pharm., Res., 2001, 24: 249-255), and apoptosis Effect (Park, HJ, et al., Arch. Pharm., Res., 2001, 24: 342-348), NO synthesis inhibitory effect, TNF-α expression inhibitory effect (Son, HJ, et al., Planta med. , 2000, 66: 467-471), antifungal effect (Bang, KH, et al., Arch. Pharm, Res., 2000, 23: 46-49), psychostable effect (Kuribara, H., et al, J Pharm.Pharmacol., 1999, 51: 97-103) and skin cancer inhibitory effects (Komoshima, T. et al., J. Nat. Prod., 1991, 54: 816-822). It has been.

In particular, honokiol and magnool inhibit smooth muscle relaxation, central inhibitory effect, anti-helicobacter pylori activity, anti-allergic effect, antibacterial effect, antibacterial effect, anti-inflammatory and neuroprotective effect, nitric oxide production and TNF-α expression inhibition Effects, anticancer effects and the like are known. In addition, honokiol and magnool inhibit the damage of the small intestine due to ischemic-reperfusion and prevent liver damage during peroxidation damage caused by ischemic-reperfusion. A paper has been published that reports its use as a deposit.

However, studies on the high-purity manufacturing process of honokiol and magnolol having such an effect are insufficient. The conventional general method extracts the thick foil by cooling with methanol for 24 hours at room temperature, and extracts the filtrate, and the filtrate is concentrated under reduced pressure to obtain a dark brown methanol. The methanol extract is solvent fractioned with benzene and water to obtain a benzene fraction. The benzene fraction was suspended on a silica gel column and eluted with benzene-ethyl acetate (1: 0 to 5: 1) to obtain a fraction. The fraction was suspended on a silica gel column and eluted with benzene-ethyl acetate (20: 1) to obtain a fraction containing magnool. This was again suspended on a silica gel column and purified with benzene-ethyl acetate (20: 1) to obtain crude crystals. Then, the crude crystals are recrystallized with ethyl acetate to obtain pure magnool (herbal medicine, herbal medicine quality standardization study, 1996, Ministry of Health and Welfare, p. 201-202). However, the above method is difficult to apply to mass production because it requires expensive equipment and several steps such as undergoing column chromatography and complex recrystallization steps.

In addition, Chinese Patent Application Publication Nos. 1444785 and 1583700 also extract dried hubak with alcohol and separate Honokiol and Magnol by column chromatography and recrystallization, but the process is complicated and the ingredient content is not standardized evenly. Most of the methods that rely on column chromatography have a lot of facility investment and high production cost, making it difficult to apply to mass production.

Meanwhile, Korean Patent Publication No. 10-2006-0046286 discloses a manufacturing process by recrystallization of magolol according to temperature and RPM using hexane without using expensive equipment. There are technical problems such as a lot of repetitive steps to be adjusted.

Accordingly, the present inventors have repeatedly conducted extensive studies to develop an economical mass production process of high purity honokiol, magnool or high purity extracts containing the high purity from the thick and thick roots, and by recrystallization using a nonpolar solvent, High purity honokiol, magnol or extract containing the same can be obtained to complete the present invention.

After all, it is an object of the present invention to provide a process for the preparation of high purity honokiol, magnol or high purity thick extracts under conditions which significantly shorten the separation process step and are suitable for high volume manufacturing facilities.

The present invention relates to a method for effectively separating and purifying high-purity honokiol, magnol or high purity extract containing the same from the groin or the groin muscle, and more particularly, the thick extract of the honokiol and the magnool with low purity. From, dissolving the extract through a non-polar solvent and lowering the solubility using a temperature difference to produce a high purity honokiol, after saturating the extract to form a recrystallization using solubility to produce a high purity magnool The present invention relates to a method for preparing a high purity extract containing honokiol and magnool by dissolving a sample through a minimum amount of soluble solvents and then adding a solvent to lower the solubility.

Hereinafter, the present invention will be described in more detail.

The polarity index used in the present invention is Richard J.P. Data reported in Natural Products Isolation, Humana Press, New Jersey, page 63, by Richard J. P. Cannell, were used.

Honokiol or magnool according to the present invention is a neolignan (neolignans) -based component isolated from Magnolia obovata Thunberg and M. officinalis Rehder et Wilson or Magnoliae radix, isomer (isomer) component. They are hardly soluble in water and hexane, and have the property of being soluble in solvents such as chloroform, dichloromethane, ethyl acetate, ethanol and methanol.

The method for preparing a thick leaf extract containing low-purity honokiol and magnolol is obtained by pulverizing, drying, and extracting a thick leaf or a thin leaf root with an alcohol or an aqueous alcoholic solution, and then suspending the dried extract in distilled water, followed by hexane, petroleum ether, chloroform, System fractional fractionation using non-polar solvents such as ethyl acetate, and the fractions are dried and prepared by column chromatography, and the carbon dioxide liquefied at high pressure and high temperature is mixed with a co-solvent and extracted, and the resultant is subjected to column chromatography. There are two ways to make them.

From the thick gourd extract obtained through the above method, after dissolving the extract through a non-polar solvent to adjust the temperature to lower the solubility to produce a honokiol, saturation of the extract and to prepare a magnool using solubility and extract After dissolving the sample through a minimum solvent to dissolve the high purity extract was prepared using the method of producing a homonool and magnool crystals by adding a solvent to lower the solubility.

The solvent used in the present invention used a solvent having a polarity index of 0 to 3 for separation of high purity honokiol, and a solvent having a polarity index of 3 to 6 for the preparation of high purity magnool. In the preparation of high-purity extract containing nool, the same polarity index as in the preparation of the magnool was used by mixing a solvent corresponding to 3-6.

Hereinafter, the present invention will be described in more detail by the following examples.

However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention.

Example 1 Preparation of Low Purity Thick Extract

A. Using Solvent Low purity  Thick pepper extract manufacturer

10-15 times of ethanol, methanol, distilled water, and 70% ethanol were added to the powder produced by grinding the dried hubak or hugh root, and then extracted at 80 ° C. for 5 hours. The results of confirming the content and yield of the honokiol and magnool for the extracts are shown in Table 1 below, the highest yield in 70% ethanol extract.

TABLE 1

Extraction solvent Honokiol content (%) Magnool Content (%) Honokiol + Magnool Content (%)  yield(%) ethanol 1.84 16.29 18.13 11.79 Methanol 2.98 14.39 17.37 19.60 Distilled water 0.27 9.45 9.72 1.23 70% ethanol 3.36 15.52 18.88 21.26

The 70% ethanol extract was filtered, concentrated under reduced pressure and suspended in distilled water 10 times the weight of the extract, and the same amount of nonpolar solvents, hexane (hexane), chloroform (chloroform) and ethyl acetate (ethyl acetate), were added to the non-polarity. After obtaining the fractions, the solutions are combined and dried under reduced pressure. After drying, a low purity thick extract is prepared by column chromatography or crystallization with a solvent such as ethyl acetate, hexane, methylene chloride, and the like.

The results of confirming the content and the yield of each of the non-polar fractions honokiol and magnool are shown in Table 2 below, showing the highest yield in the ethyl acetate fraction.

TABLE 2

Fraction solvent Honokiol content (%) Magnool Content (%) Honokiol + Magnool Content (%) yield(%) 70% Ethanol Extract 3.36 15.52 18.88 21.26 Hexane fraction 4.58 26.69 31.27 8.10 Chloroform fraction 4.83 26.97 31.8 6.88 Ethyl acetate fraction 5.50 29.23 34.73 8.95

B. Supercritical  By extraction Low purity  Thick pepper extract manufacturer

Carbon dioxide is liquefied under the conditions of 50 ~ 60 ℃, 300 ~ 500bar, then mixed with a co-solvent (methanol, ethanol, ethyl acetate, chloroform) is used as an extraction solvent. The extracted sample is dried and used as a low purity thick extract.

Example 2 Preparation of High Purity Honokiol and Magnool

A. Hexane  High purity Honokiol  Produce

To the low-purity thick extract of Example 1 was added hexane which is a solvent corresponding to the polarity index of 0 to 3, and only the supernatant was taken by ultrasonic decomposition / stirring at 40 to 60 ° C., to obtain high purity honokiol crystals formed by cooling down. Table 3 below shows the content and yield of the honokiol according to the bath temperature, it was confirmed that can obtain a high purity honokiol of at least 95% at 55 ℃.

TABLE 3

Bath temperature Honokiol content (%) yield(%) 40 ℃ 90.1 57.9 50 ℃ 90.3 69.3 55 ℃ 96.0 53.2 60 ℃ 95.3 61.3

B. Using high solubility Magnool  Produce

To the low-purity thick extract of Example 1, ethyl acetate, which is a solvent having a polarity index corresponding to 3 to 6, was added so as to be 0.1 to 2.0% based on the sample volume. Obtaining high-purity magnool crystals produced by cold cooling. Table 4 below shows the content and yield of magolol according to the amount of ethyl acetate, and it was confirmed that 95% or more of high purity magnol could be obtained when the final concentration was added to 0.3% or more.

TABLE 4

Amount of ethyl acetate Magnool Content (%) yield(%) 0.1% 70.1 77.4 0.2% 74.2 85.4 0.4% 95.1 53.1 0.8% 96.1 41.0 1.4% 98.8 39.5

Example 3 Preparation of High Purity Honokiol and Magnool Complexes

A small amount of a solvent such as acetone, ethyl acetate, and methylene chloride was added to the low purity thick gourd extract of Example 1, followed by dissolution in a water bath at 40 ° C. or higher, and then hexane was added until precipitation occurred, and again at 40 ° C. or higher. When completely dissolved in a water bath, the mixture was slowly cooled down to obtain a precipitate containing 90% or more of honokiol and magnol.

Table 5 below shows the content and yield of the honokiol and magnool complexes depending on the solvent used for recrystallization. When ethyl acetate was used, it was confirmed that high purity extract of more than 95% could be obtained.

TABLE 5

Recrystallized solvent Honokiol content (%) Magnool Content (%) Magnool + Honokiol Content (%) yield(%) Ethyl acetate 37.82 59.76 97.58 87.3 Acetone 32.46 58.02 90.48 65.7 Methylene chloride 25.84 64.54 90.38 89.4

The hexane ratio of Table 6 indicates the volume ratio of hexane used to the volume of the solvent added in Table 5, and indicates the content and yield of the honokiol and magnool complexes according to the amount of hexane.

TABLE 6

Hexane Honokiol content (%) Magnool Content (%) Magnool + Honokiol Content (%) yield(%) 10 times 48.7 49.2 97.9 32.3 15 times 47.2 51.5 98.7 40.5 17 times 47.2 51.3 98.5 75.5 20 times 38.3 59.8 98.1 87.5 30 times 32.7 62.5 95.2 81.1 40 times 30.3 64.9 95.2 79.2

<Content analysis>

The HPLC used for the analysis of the compound according to the present invention used a water system (waters, 600 controller pump, 2996 Photodiode array detector, 717 plus autosampler) system, the analytical conditions were a column (Phenomenex Luna (5㎛) stationary phase ), C18 (2) 100 (A) 4.6 × 250mm) and acetonitrile: (distilled water) water: acetic acid = 50: 50: 1, and constant solvent (isocratic) ), The flow rate was 1.0 ml / min, the total analysis time was 60 minutes, the temperature of the column oven was set to 25 ° C., the injection amount of the sample was 10 μl, and detected at 289 nm with a UV detector. Magnool is detected at 17 minutes and honokiol is detected at 27 minutes.

Samples for the analysis of thick foil prepared from the above items were prepared in methanol at a concentration of 1 mg / ml. Honokiol and magnool standard samples were prepared at a concentration of 0.2 mg / ml, and the standard samples and the prepared samples were added together with an automatic sampler for analysis.

As described above, the present invention is a simple process step of the high purity of the thick extract and high purity magnool and honokiol by recrystallization from a low-purity thick or thick root extract, using a non-polar solvent or solubility such as ethyl acetate, hexane Produced via By using the production method of the present invention, it is possible to mass-produce economically and effectively the honokiol and magnolol or mixtures thereof having high pharmacological action.

Claims (6)

Claims 1.The one selected from non-polar solvents such as hexane, petroleum ether, trichloroethylene, ethyl ether, cyclohexane, benzene and more than five times the weight of the sample corresponding to the polarity index 0 to 3 in the low purity thick or thick root extract A method for producing a high purity honokiol, which is added to a solvent and heated at 40 ° C or higher, and then cooled down to obtain a precipitate. 2. The method for producing high-purity honokiol according to claim 1, wherein hexane is added at least 5 times the weight of the sample, followed by hot water at 40 ° C or higher, and then cooled down to obtain a precipitate. The low purity thick or thick root extract contains 0.1 to 1 solvent selected from solvents such as acetone, acetonitrile, chloroform, methylene chloride, ethanol, ethyl acetate, ethyl ether, and methanol having polar indices of 3-6. A method for producing a high purity magnool, which is obtained by adding 2.0%, dissolving in hot water, dissolving it, and cooling down to obtain a precipitate. The method of claim 3, wherein ethyl acetate having a final concentration of 0.1% to 1.0% or more is added, dissolved in a water bath at 40 ° C or higher, and then cooled down to obtain a precipitate. 0.1 to 2.0% of one solvent selected from acetone, acetonitrile, chloroform, methylene chloride, ethanol, ethyl acetate, ethyl ether, and methanol was added from the low purity thick or thick root extract, and then dissolved in a hexane. , A solvent selected from petroleum ether, trichloroethylene, ethyl ether, cyclohexane, and benzene is added until a suspension is formed in the solution. A process for preparing a mixture comprising a content of magnool and honokiol. The method of claim 5, wherein the acetone, ethyl acetate or methylene chloride of claim 3 is preferably added to the water bath and dissolved, and after dissolving, the hexane of claim 1 is added until a float is formed in the solution. Method of producing a mixture comprising a high content of magnool and honokiol, characterized in that it is obtained by cooling after cooling.

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