KR20060084083A - Method for Purifying Painol Intradermal - Google Patents

Abstract

The present invention relates to a method for enhancing the content of paeonol in the neck skin and to a method for purifying paeonol content enhanced by the above method,

There is provided a method for enhancing the content of paeonol in the endothelial skin by heating the neck skin, and a method for purifying paonol by using ethanol extraction, solubility difference and various chromatography in the neck skin with the content of paeonol obtained in this way.

According to the present invention, it is possible to extract by increasing the content of paeonol active compounds such as antioxidant, anti-inflammatory, anti-cancer, sedation, inhibiting atherosclerosis.

Bark Peel, Heating, Paeonol, Content, Extract, Purification

Description

Method for increasing paeonol content in pelvic dermal skin and method for purifying paeonol content increased by the above method {Methods for increasing paeonol content in Moutan Cortex, and the method for extraction of paeonol having increased contents obtained by the method}

1 is a flow chart illustrating a method of extracting paeonol from the extract after enhancing the content of paeonol as an active compound in the dermis as an embodiment of the present invention,

Figure 2 is an HPLC chromatogram measuring the change in the content of paeool in the neck skin before and after heat treatment of the present invention,

3 is a hydrogen nuclear magnetic resonance spectrum of paeonol purified according to an embodiment of the present invention,

4 is a carbon nuclear magnetic resonance spectrum of paeonol purified according to an embodiment of the present invention,

5 is a graph showing the measurement of the change in paeonol content for each temperature during the heat treatment of the present invention, and

6 is an antioxidant activity comparison graph showing the DPPG radical scavenging ability of the ethanol extract of the bark skin after heat treatment of the present invention.

The present invention relates to a method of enhancing the content of paeonol in the neck skin and to a method of purifying paeonol content enhanced by the above method, more specifically, antioxidant, anti-inflammatory, anti-cancer, The present invention relates to a method for enhancing the content of paeonol, an active compound such as sedation and inhibiting atherosclerosis, and effectively extracting paonol, an active compound, from the extract.

Generally, mokdanpi (Moutan Cortex) is galip bush coronary into root bark of peony (Paeonia moutan Sims, Paeonia suffruticosa Andrews .) Belongs to the tree - form the half-tubular outer surface is dark brown - represents a purplish brown long horizontally, a small circle It is known to have side marks and vertical wrinkles, and its inner surface has a light grayish brown color. Paeonol, which is known to be contained in the bark skin, is reported in the literature to exhibit various physiological activities such as antioxidant, anti-inflammatory, anticancer, sedative, and atherosclerosis inhibition.

The chemical formula of such paeonol is as follows.

As reported in the literature, literature on the antioxidant activity of paeonol includes Tang, Jingrong; Shi, Lin; Li, Shengting. “Effects of Paeool on Lipid Peroxidase in Normal and Calcium Paradox Cultured Neonatal Myocardial Cells” Zhongcaoyao (1990), 21 (12), 547-549 ”and Tang, J; "Protective Effect of Paeonol on New Rat Cardiac Cells Cultured During Calcium Paradox" by Shi, L ;. Zhongguo zhong yao za zhi (1991), 16 (9), 557-569, and the literature on anti-inflammatory action is described by Chou, Tz-Chong. Inflammatory and analgesic effects. ”British Journal of Pharmacology (2003), 139 (6), 1146-1152 and Wu, Guanzhong; Hang, Bingqian; Hang, Jingxia; Ling, Gengxin. “Anti-inflammatory Effects of Paeonol and Its Mechanisms.” Zhongguo Yaoke Daxue Xuebao (1989), 20 (3), 147-150, et al., Sun, Guoping; Shen, Yuxian; Zhang, Lingling; Zhou, Aiwu; Wei, Wei; Xu, Shuyun. “Study on the immunomodulation and antitumor activity of paeonol in HepA tumor rats.” Zhongguo Yaolixue Tongbao (2003), 19 (2), 160-162, etc. The literature on sedation is Kim Seung-hoon, Kim Sung-ae, Park Mi-kyung, By Kim, Sung-Hyung, Young-Doo Park, Ho-Jung Na, Hyung-Min Kim, Min-Kyu Shin, and Kyu-Seok Ahn, “Paeonol inhibits anaphylactic action by regulating histamine and TNF-α.” International immunopharmacology (2004), 4 (2), 279-287 and Wu, Guanzhong; Hang, Bingqian; Hang, Jingxia; Lin, Gengxin. “Anti-sensitizing effect of paeolol.” Zhongguo Yaoke Daxue Xuebao (1990), 21 (2), 103-106, et al., Dai, Min; Zhi, Xiaomei; Peng, Daiyin; Liu, Qungyun. "The inhibitory effect of paeonol on experimental atherosclerosis in quails." Zhongguo Zhongyao Zazhi (1999), 24 (8), 488-490).

Meanwhile, prior arts related to intradermal paeolol have been disclosed in Chinese Patent Publication Nos. 1331968 (20020123) and 1142338 (19970212), but the patents are for the manufacture of toothpaste containing paeonole and for preventing heart disease containing paeonole, respectively. It is just a method of manufacturing health tobacco. No technique has been found to increase the content of paeonol in the skin of both neck and neck, and to extract paeonol with such enhanced content.

Accordingly, an object of the present invention is to provide a method for increasing the content of paeonol, the active compound in the bark skin.

Another object of the present invention is to provide a method for purifying paeonol from bark skins treated to increase the paeonol content.

According to one aspect of the invention,

Provided is a method for enhancing the content of paeonol in the endothelial skin, wherein the neck skin is heated at 50-210 ° C. for 5-60 minutes.

According to the second aspect of the present invention,

Extracting the bark skin with increased content of paeolol by ethanol according to the first method;

Dispersing the extract in water to extract one to five portions of a solvent selected from the group consisting of dichloromethane, chloroform, ethyl acetate, ether and ethanol, and then concentrating to obtain an organic solvent soluble fraction; And

Silica gel, reverse phase silica gel, fast liquid, and the organic solvent soluble fraction using a solvent selected from the group consisting of methanol, hexane, acetic acid, ethyl acetate, formic acid, chloroform, benzene and a solvent mixed with two or more selected from them. Purifying paeonol using a chromatography selected from the group consisting of Sephadex (Sephadex) using paeolol; there is provided a method for purifying intradermal paeool.

Hereinafter, with reference to the accompanying Figure 1 with respect to the present invention will be described in detail.

Paeonol, the active compound in bark dermis, maximizes its content by heating the bark. The heating temperature is 5 to 60 minutes at 50 to 210 ° C, preferably 20 to 60 minutes at 140 to 200 ° C, and most preferably 20 to 60 minutes at 180 to 200 ° C. Outside the above range, it is not preferable because the content of paeonol cannot be sufficiently enhanced as intended.

In this case, any kind of machine for heating can be used as long as the machine can properly heat the wood skin such as a heater, a pressurizer, as well as a self-cooking machine.

Thus, ethanol extract is obtained by extracting the bark skin with increased paeonol content with ethanol. As the ethanol concentration, it is preferable to use 50 to 100%, preferably 80 to 95%, and most preferably 95%, and to dry the obtained extract by a known method such as distillation, distillation under reduced pressure, or spray drying. More preferred.

The obtained extract is dispersed by adding an appropriate amount, preferably about 3 times, of water, and then one solvent selected from the group consisting of dichloromethane, chloroform, ethyl acetate, ether and ethanol, preferably ethyl acetate or chloroform, or Dichloromethane, most preferably dichloromethane, is 0.5 to 3 times the amount of water used for dispersing, preferably 1 to 5 times, preferably 2 to 3 times, and most preferably twice . The partition extract thus obtained is concentrated to obtain an organic solvent soluble fraction.

The organic solvent soluble fraction is a silica gel, reverse phase silica gel, high-speed liquid using a solvent selected from the group consisting of methanol, hexane, acetic acid, ethyl acetate, formic acid, chloroform, benzene and a solvent mixed with two or more selected from them. And Sepadex (Sephadex) is selected from the group consisting of a column chromatography using a carrier, preferably silica gel using a silica gel to obtain a pure content of paeonol content is enhanced. In case of using silica gel, it is simple and economical, and it is effective because of its excellent resolution. On the other hand, the organic solvent soluble fraction is more preferably used by drying and then concentrated using a known method such as distillation, distillation under reduced pressure, or a spray dryer prior to separation.

According to this method, it is possible to obtain paeonol per 45 g of ethanol extract of the bark skin by increasing the range to 454.3 ± 5.7 mg, and the above-described steps are sequentially performed on not only silk bark but also similar plants containing paonol. The same or similar effects can be obtained by applying.

On the other hand, the obtained paeool may be formulated according to a known method using commercially available ingredients such as excipients, carriers, diluents and the like which are acceptable as pharmaceutical, cosmetic or food composition.

Carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, cellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil . The usable content is not limited thereto, but may be used in an amount of 100 mg to 10 g.

In addition to the above components, commercially available lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like may further be included.

On the other hand, as an example of the formulation, the active ingredient may be mixed or diluted with the carrier, or may be contained in a capsule, sachet, paper or other kind of carrier. The carrier can then be a solid, semisolid, or liquid substance that acts as a vehicle, excipient or medium for the active ingredient to serve as a diluent. Thus, the resulting formulation may be in the form of tablets, pills, dispersants, granules, elixirs, suspensions, emulsifiers, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders and ointments, etc. It may be administered orally or parenterally.

Paeonol preparations obtained in this way can release the active ingredient rapidly, continuously or delayed as necessary after administration, more specifically in adult diseases such as antioxidant, anti-inflammatory, anti-cancer, sedative, and atherosclerosis. It can be used as a health functional food composition and a pharmaceutical composition, which are effective for the prevention and treatment of pneumonia.

In addition, the daily dosage of paeonol as the active compound is usually within the range of about 0.1-20 mg per kg of body weight, and when used as a functional food or pharmaceutical composition for health supplements, it is converted into water or ethanol extract. In the range of 100 mg to 10 g per kg of body weight is sufficient.

Example

EMBODIMENT OF THE INVENTION Hereinafter, embodiment of this invention is described in detail with reference to drawings. However, the following examples are illustrative of the present invention, and the present invention is not limited thereto.

Example 1 Effect of Heat Treatment of Bark Bark on Paeonol Content

This embodiment refers to Figure 1 for the effect on the content of paeonol by heat treatment of the bark skin.

First, as shown in the flowchart of FIG. 1, the bark skin is collected to remove foreign matter, and then finely chopped or crushed, the 500g is heated at 190 ° C for 30 minutes using an electric roasting machine, and then refluxed with 95% ethanol at 80 ° C. The filtrate was concentrated under reduced pressure to give 84.56 g of extract.

The extract thus obtained was dispersed in 1 L of distilled water and then partitioned and extracted three times with the same amount of dichloromethane, followed by concentrating by mixing only the soluble portion in dichloromethane to obtain 10.46 g as the dichloromethane soluble fraction.

Then, silica gel column chromatography (Merck, No.7734, column size 4, 54.5 cm, eluent: nucleic acid-ethyl acetate-acetic acid = 100-1-1%) was used for the dichloromethane soluble fraction. 13 fractions were obtained by eluting with a slope of 1-1%, and 6.23 g of paeonol having 95% purity on HPLC was obtained from the third fraction.

Using paeonol (95% purity over HPLC UV 280 nm) isolated from this experiment as a reference material, the calibration curve [Y (peak area) was calculated using the correlation between peak area and concentration in HPLC. = 0.0469 × X (concentration in μg) + 0.0014625, r ² = 0.9948] and the paeonol content was quantified by substituting the peak area of unknown concentration into the correlation. After roasting, 10 mg of ethanol extract of bark ethanol was dissolved in 1 ml of ethanol, and then filtered through a 4.5 μm membrane filter after roasting. Μl was analyzed by HPLC. HPLC conditions were analyzed by giving a concentration gradient of 60% so that acetonitrile containing 1% acetic acid (acetonitrile) from 0% to 100%. The column used was ZORBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 μm, Agilent, USA), flow rate 0.8 ml / min, detection at 280 nm.

In addition, the process was repeated five times, and the converted value thereof based on 1 g of ethanol extract is shown in Table 1 together.

Experimental Example 1 Experimental Example 2 Experimental Example 3 Experimental Example 4 Experimental Example 5 Paeonol content (g) 6.23 6.02 5.96 6.15 6.33 Paeonol equivalent ((1 g of ethanol extract, mg) 448.6 450.3 454.3 455.4 463.1

As shown in the table above, the conversion value of paonol content to 1 g of ethanol extract is 454.3 ± 5.7 mg.

Comparative Example-Content of Paeonol from Unheated Bark Bark

Except for the heat treatment step, the paeonol content obtained by repeating the same process as in Example 1 was 2.00 g.

The above process was repeated four times, and the paeonol content obtained and the converted value thereof based on 1 g of ethanol extract of bark skin are shown in Table 2 together.

Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 Paeonol content (g) 2.00 1.98 2.21 2.36 1.88 Paeonol equivalent ((1 g of ethanol extract, mg) 138.4 139.9 142.3 144.5 146.8

As shown in the table above, the conversion value of paonol content to 1 g of ethanol extract is 142.4 ± 3.3 mg. Therefore, as a result of comparing the Tables 1 and 2, it can be seen that by performing the method of the present invention can increase the paeonol content up to three times as compared to the prior art.

Example 2- Identification of Paeonol

The present embodiment was tested to determine whether the substance enhanced in the neck dermal content after heat treatment is pure paeonol, see FIGS. 2 to 4.

HPLC chromatogram

First, the change in the content of the ethanol extract of the bark skin obtained from Example 1 and the ethanol extract of the bark skin obtained from the comparative example (control) was measured using HPLC chromatogram, and the results are shown in FIG. 2.

As can be seen in Figure 2, the upper control shows a very small paeonol peak, while in the lower example it was confirmed that the peak was increased very much.

Magnetic resonance spectrum

In addition, the hydrogen nuclear magnetic resonance spectra and the carbon nuclear magnetic resonance spectra for the substances having a very high content of the bark skin extract were measured and the results are shown in FIGS. 3 and 4, respectively.

As shown in Figs. 3 and 4, the results of the respective nuclear magnetic resonance spectra and the characteristics exhibited by paeool were exactly the same, and this compound was identified as paeonol.

Example 3 Confirmation of Optimal Heating Temperature of Bark Bark

This embodiment refers to Figure 5 as to determine the optimum temperature during heat treatment of the neck skin.

The change in paeonol content according to heating temperature for the extract obtained in Example 1 was measured and the results are shown in FIG. 5. As shown in Figure 5, it was confirmed that the content of paeonol in the range of 50 ~ 210 ℃ was improved, the production of paeonol was significantly increased at 140 ~ 200 ℃, maximizing the production of paeonol around 180 ~ 200 ℃ It could be confirmed.

Example 4- Confirmation of Optimal Heating Time of Bark Skin

This embodiment is for confirming the optimum time during heat treatment of the neck skin.

In consideration of the optimum heating temperature obtained in Example 3, the change in paeonol content by heating time for the extract of wood bark obtained in Example 1 was measured and the results are shown in Table 3 below.

Paeonol content change 5 minutes 20 minutes 30 minutes 40 minutes 60 minutes 50 ℃ 145.3 150.3 172.5 176.3 166.9 150 ℃ 143.9 175.6 200.1 199.3 200.0 170 ℃ 146.3 188.6 206.3 208.3 205.9 190 ℃ 142.5 440.3 454.3 449.9 430.0 210 ℃ 145.9 165.3 275.3 255.6 230.6

As shown in the above table, it was confirmed that the content of paeonol was improved by heating for 5 to 60 minutes at a temperature of 50 to 210 ° C, and the production of paonol was significantly increased by heating for 20 to 60 minutes at a temperature of 140 to 200 ° C. When heated for 20 to 60 minutes in the vicinity of 180 ~ 200 ℃ it was confirmed that the production of paeonol is maximized.

Example 5- Antioxidant Activity Measurement

This example refers to FIG. 6 for measuring DPPH antioxidant activity.

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was carried out according to the method of Brois. That is, 800 μl of ⁴ × 10 ⁻⁴ M DPPH solution was added to 200 μl of ethanol solution containing samples having 25 and 100 ppm concentrations obtained in Example 1, mixed for 10 seconds, left for 10 minutes, and absorbance was measured at 525 nm. Next, the scavenging activity was shown based on the following formula.

Scavenging activity = {1- (S / C)} X100

Where S is the absorbance value when the sample is added and C is the absorbance value of the 200 μl ethanol solution containing no sample.

The experiment was repeated twice and the average value was calculated and shown in FIG. 6, where BHA (butylated hydroxyanisole) was used as a positive control. As can be seen from the graph shown in Figure 6, it was confirmed that the antioxidant activity increased by about 10% by heat treatment.

On the other hand, not only the change in antioxidant activity after heat treatment but also the activity changes on ACE (angiotensin converting enzyme) and TNF-α (tumor necrosis factor-α) were measured, but no significant change was observed.

It is apparent to those skilled in the art that the present invention is not limited to the above-described embodiments, and that various modifications and changes can be made without departing from the spirit of the present invention.

That is, in the present invention, for example, the content of paeonol is increased by taking the bark of the bark, but the same or similar effects may be obtained through the same heat treatment on the similar plants containing paeonol. Therefore, such modifications or variations are intended to fall within the claims of the present invention.

 According to the present invention, it is possible to extract by increasing the content of paeonol active compounds such as antioxidant, anti-inflammatory, anti-cancer, sedation, inhibiting atherosclerosis.

Claims (8)

How to enhance the content of paeonol in the neck skin, characterized in that the heat treatment for 5 to 60 minutes at 50-210 ℃ The method of claim 1, wherein the heat treatment is a method for enhancing the content of palonol in the neck skin, characterized in that for 20 to 60 minutes heating at 140 ~ 200 ℃ According to claim 2, wherein the heat treatment is a method for enhancing the content of paeolol in the neck skin, characterized in that for 20 to 60 minutes heating at 180 ~ 200 ℃ Extracting by extracting the bark skin enriched in paeonol content by ethanol by the method of any one of claims 1 to 3; Dispersing the extract in water to extract one to five portions of a solvent selected from the group consisting of dichloromethane, chloroform, ethyl acetate, ether and ethanol, and then concentrating to obtain an organic solvent soluble fraction; And Silica gel, reverse phase silica gel, fast liquid, and the organic solvent soluble fraction using a solvent selected from the group consisting of methanol, hexane, acetic acid, ethyl acetate, formic acid, chloroform, benzene and a solvent mixed with two or more selected from them. Purifying paeonol using chromatography using a carrier selected from the group consisting of Sephadex; The method of claim 4, wherein the partition extraction solvent is dichloromethane using dichloromethane twice or three times. The method for purifying intradermal paeonol according to claim 4, wherein silica chromatography is used as the carrier for chromatography. The method of claim 4, further comprising the step of drying prior to dispersing the extract in water. 8. The method of claim 4 or 7, further comprising drying and concentrating prior to purifying the organic soluble fraction.

Images