KR20130093372A - Preparation method of oxyresveratrol, t-resveratrol, and moracin having anti-inflammatory and anti-aging function from mulberry twig extract - Google Patents

Abstract

PURPOSE: A method for preparing a mulberry branch extract is provided to produce a large amount of purified powder containing oxyresveratrol with anti-inflammatory and antiaging functions and to use trans-rasveratrol and moracin as a novel material for functional foods and cosmetic products. CONSTITUTION: A method for preparing a mulberry branch extract containing oxyresveratrol comprises the steps of: steaming mulberry branches for one hour and drying; adding 650 kg of a 75-95% ethanol solution to 100 kg of the dried mulberry branches and extracting by ultrasonic wave at 40-50°C for 12 hours; concentrating the extract by reduced pressure; dissolving the concentrate in a 80% ethanol solution and incubating at room temperature; removing precipitate, adding normal-hexane, and fractioning; removing an upper layer oil and concentrating the lower layer; adding a 20% ethanol solution to the defatted ethanol extract of mulberry branches and suspending; treating the defatted ethanol extract with 10% pectinase and hydrolyzing at 30-40°C for 12 hours to obtain a hydrolysate; fractioning the hydrolysate with an ethyl acetate solvent and concentrating the upper layer by reduced pressure; dissolving the concentrate using the 20% ethanol solution and adsorbing onto diaion HP-20 adsorption ion exchange column; washing the resultant with the 20% ethanol solution; and eluting the resultant with a 40% ethanol solution and concentrating. [Reference numerals] (AA) Dried mulberry branches(100kg); (BB) CNCNF(650kg, 80% ethanol solution, ultrasonic extractor, 50°C, 12 hours, twice); (CC) Filter; (DD) Filtered liquid; (EE, LL) Concentrate by reduced pressure; (FF) Ethanol extract of the mulberry branches(10kg); (GG) Re-dissolve(50kg, ethanol solution); (HH) Incubate day and night; (II) Supernatant; (JJ) Defatting(100kg, normal-hexane); (KK) Lower layer; (MM) Defatted ethanol extract of mulberry branches(8.5kg); (NN) Suspend(50kg, 20% ethanol solution); (OO) Enzyme treatment(4L, pectinex 5 XL, 30~40°C, 12 hours); (PP) Cooling; (QQ) Fraction with a solvent(100kg, ethyl acetate, twice); (RR) Dehydrate(10kg, sodium sulfate anhydrous); (SS) Filter and concentrate by reduced pressure; (TT) Ethyl acetate extract(2.1kg); (UU) Diaion HP-20 column chromatography; (VV) Fraction with 40% ethanol solution; (WW) Fraction with 60% ethanol solution; (XX) Mulberry branch extract I(1.2kg); (YY) Mulberry branch extract II(110g)

Description

The present invention relates to a method for preparing a mulberry twig extract comprising an anti-inflammatory, anti-aging functional oxyreservatrol, trans-resveratrol and morasin. The preparation method of oxyresveratrol, t-resveratrol, and moracin having anti-

The present invention relates to a method for producing high-quality topical extracts containing anti-inflammatory and anti-oxidizing agents, such as oxirebazelor, trans-resveratrol and morasin.

Plant taxonomic Mulberry is an arboreal plant represented by Morus alba L. Mulberry is a kind of mulberry tree which is grown from a mulberry tree, a mulberry tree, a root bark, a mistletoe parasitic on a mulberry tree, a mulberry tree,蠶), and Dongchuhecho (冬蟲 夏草) as one of the 'Hyeonbokbo' and 'Hoseo gangmok' is a new breed of high blood pressure, arteriosclerosis, paralysis, edema by each other, neuralgia and arthritis, cold cough and sputum is effective (Tsuneo Namba: 百 百 百 鑑 鑑 鑑 鑑 鑑 鑑............................).

It is said that the upper tea made by drying the mulberry branches (upper body) is used to eliminate the customs, to help the joints, to bite the bones with the handwheel, to treat the symptoms caused by the abdominal torsion, limb twist, cough and paralysis. A person who is bloated is said to have the effect of losing flesh. In addition, the upper body is said to rule all the winds, to promote digestion, to lower the flame, and to control the dryness of the mouth with the edgewise. It is said that it is good for the diabetic mats who put up the upper part in the Japanese coffee cup curing period. In addition, it is said that the sangju (가지 가지 술 술)), which is made from the concentrated juice made from the sweet potatoes and the sesame oil, is effective for the paralysis, especially the paralysis, the otic paralysis, and the cough (Tsuneo Namba 和 漢 药 百科 圖書 I, 1980).

So far, there is a physiological action of the known upper extremity have been reported include hanggan toxic, anti-oxidation, anti-obesity, anti-inflammatory, anti-aging, anti-arthritic action, known are such prenylflavonoids, coumarin, stilbene as the main bioactive components (Oh et al ., Planta Med 68: 932-934, 2002; Hu et al ., J Nat Prod 74: 816-824, 2011; Kim et al ., J Ethnopharmacol 136: 392-398, 2011; Chang et al ., Food Chem Toxicol 49: 785-790, 2011).

Stilbene (stilbene) compound and the like of the upper extremity of the new Mora oxy resveratrol and resveratrol, such as are present in many products, such as latent image mulberries audio as well as reconstituted tobacco, Mori Cortex (Kim et al ., J Korean Soc Food Sci Nutr 37: 124-128, 2008; Kim et al ., Korean J Seric Sci 47: 51-55, 2005; Piao et al ., Phytochem Anal 22: 230-235, 2011, Kim et al ., J Biol Chem 277: 16340-16344, 2002). In particular, the topical oxir reseratrol is a powerful inhibitor of tyrosinase, which promotes the production of melanin pigment in skin, as well as a source of whitening cosmetics (Shin et al ., Biochem Biophys Res Commun 243: 801-803,1998; Oh et al ., Arch Pharm Res 21: 703-706,1998; Shimizu meat al ., Planta Med 66: 11-15, 2000; Chung et al ., < / RTI > J Pharm Pharm 55: 1695-1700, 2003; Kim et al, J Biol Chem 277:. 16340-16344, 2002) Inflammatory mediators in enoh (NO) synthase in inducible nitric oxide synthase (iNOS inhibiting) and inflammation promoting material NF- k B and cyclooxygenase-2 (COX -2) activity (Chung et < RTI ID = 0.0 > al ., J Pharm Pharm 55: 1695-1700, 2003). In addition, Trans-Resveratrol is a major component of the 'French Paradox' of grape wines (Ren Lorgeril, The Lancet 339: 1523-1526, 1992), as well as anti-hypertensive, anti-inflammatory, anti- (Fremont Life Sci 66: 663-673, 2000), and has recently been reported to have an extended life span. Moratin derivatives are well known as antidiabetic as well as antiinflammatory and antioxidative substances (Basnet et al., Chem Pharm Bull 41: 1238-1243, 1993; Lee et al., Biol Pharm Bull 25: 1045-10488 , 2002), in particular as an anti-tyrosinase substance having a whitening effect (Lee et al., Biol Pharm Bull 25: 1045-10488, 2002).

On the other hand, the physiologically active substances such as oxyreservatol, trans-resveratrol and morazin in the upper limbs exist as almost all the glycosides in the latent products, and their contents vary depending on the kind of the plant, variety, mountain area, season and processing meat al ., J Korean Soc Food Sci Nutr 37, 124-128, 2008; Kim et al ., Korean J Seric Sci 47, 51-55, 2005; Piao et al ., Phytochem Anal 22, 230-235, 2011). Oxy-resveratrol in the upper limb is mostly present as a glycoside, and is known as the main physiologically active substance of the alfalfa, but it hardly exists in the upper leaves or the oti (Kim et al ., J Korean Soc Food Sci Nutr 37, 124-128, 2008; Kim et al ., Korean J Seric Sci 47, 51-55, 2005; Piao et al ., Phytochem Anal 22, 230-235, 2011). On the other hand, the upper extremity of trans-resveratrol (trans -resveratrol) is present, as well as mulberry mulberry, Morus, its isomers the cis-glycosides present in nearly all with resveratrol (cis -resveratrol) (Kim et al ., J Biol Chem 277, 16340-16344, 2002). Morraccal constituents are also present in the upper extremities, as well as in the oti, upper leaves and mantis (Basnet et al., Chem Pharm Bull 41: 1238-1243, 1993).

On the other hand, since most of the components of oxir reseratrol, trans-resveratrol and morasin contained in the upper limb are present as inactive glycosides, when they are used as such, the absorption ability of human body is low and the effect of inhibiting aging is very low. Therefore, in order for these physiologically active substances to fully exhibit anti-aging function, it is necessary to develop a bioconversion technology that converts existing inactive components into active ingredients.

Biotransformation technology is a method of producing a new active derivative of an active substance by an enzyme produced by a microorganism or by converting an inactive polymer having a plant into an active low molecular substance by fermentation, It not only doubles the amount of ingredients but also absorbs more quickly into deep areas of the skin, which greatly reduces skin aging. In recent years, development of anti-aging functional materials using anti-aging technology has been actively developed in Korea. For example, isoflavone of soybean, rhapontigenin of rhubarb, and ginsenoside derivatives of ginseng have been widely recognized as functional cosmetic materials.

So far, a number of studies have been reported on the isolation and identification of oxyreservatol, trans-resveratrol and morazin derivatives from the latent images including the upper limb and their physiological actions (Shin et al ., Biochem Biophys Res Commun 243, 801-803, 1998; Oh et al, Arch Pharm Res 21, 703-706, 1998;.. Shimizu et al, Planta Med 66, 11-15, 2000) , but a bar-type anti-inflammatory activity than the inactive form and the chemical conversion hangno oxy resveratrol, resveratrol And mora-containing high-value upper limb extracts.

It is an object of the present invention to provide a method for producing mulberry tree branch extract containing oxyreservatrol, trans-resveratrol and morasin having anti-inflammatory, anti-aging function.

The above object of the present invention can be accomplished by a method for preparing a pharmaceutical composition comprising a step of steam treatment of the upper part, an ethanol extraction step of the dry upper part, a hydrolysis treatment step with pectinex enzyme, an ethylacetate fraction step and a Diaion HP-20 adsorption ion resin column chromatography to obtain ox res reseratrol, ≪ / RTI > and morragin-containing topical extracts.

INDUSTRIAL APPLICABILITY As described above, the separation and purification method of the present invention is capable of mass-producing purified powder containing anti-inflammatory and anti-aging functional oxyreservatol from mulberry branches, as well as being capable of mass production of anticancer, antihypertensive and anti-aging functional trans- It has an excellent effect in providing the component as a new material for functional foods and cosmetics.

Figure 1 shows a method for preparing anti-inflammatory and anti-oxidizing ox resveratrol, trans-resveratrol and topical extracts containing morasin.
FIG. 2 shows the results of high-performance liquid chromatography of oxyreservatrol, trans-resveratrol, and morassin-containing upper limb extract.

The present invention relates to a method for the treatment of ovari resveratrol, resveratrol and morazine by steam treatment of the upper limb, ethanol extraction of the dry upper part, hydrolysis treatment with pectinex enzyme, ethyl acetate fractionation, Diaion HP-20 adsorption ion resin column chromatography And a purification step.

The present invention provides a method for the isolation and purification of oxiresteral, resveratrol and morazine from the steaming tops comprising the steps of: The upper limb was placed in a steamer, steamed for 1 hour, cooled, and dried in a hot air drier to obtain a dry upper limb. An 80% aqueous ethanol solution (650 kg / 100 kg sample) , Followed by filtration and concentration under reduced pressure to obtain a topical ethanol extract.

The upper limestone extract was dissolved again with 80% ethanol aqueous solution. The upper layer obtained by allowing to stand overnight was added with 2-fold amount of 100% n-hexane, followed by fractionation. The ethanol extract of the upper layer of the lower layer obtained above was partially concentrated under reduced pressure and then suspended in an aqueous 20% ethanol solution of about 5 times that of the extract. 10% pectinase enzyme was added to the defatted ethanol extract, Gt; 40 C < / RTI > for 12 hours. The hydrolyzate obtained from the above was allowed to cool at room temperature, and ethyl acetate was added to the reaction mixture. The resulting upper layer was concentrated under reduced pressure and subjected to ion-adsorption resin column chromatography.

The ethylacetate fractions obtained above were first dissolved in a 20% aqueous ethanol solution and adsorbed on a Diaion HP-20 ion resin. The column was washed thoroughly with 20% aqueous ethanol and then eluted with 40% and 60% aqueous ethanol And concentrated to separate and purify the anti-inflammatory and anti-oxidizing oxiresteral, resveratrol and morasin.

In the method of separation and purification of oxyreservatrol, resveratrol and morasin from the steamed upper part of the present invention, the preferred concentration of the aqueous ethanol solution is from 75% to 95%, most preferably from 80% to 90% aqueous ethanol solution. Methanol may be used instead of ethanol as an extraction solvent at this time.

The preferred temperature for the heat extraction in the separation and purification method of oxyreservatrol, resveratrol and morasin components in the steamed upper part of the present invention is 40 캜 to 60 캜, and most preferably 50 캜.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example  One : Steam treatment , Enzyme treatment, solvent fraction and Adsorption ion resin  From the upper ethanol extract by column chromatography Oxyresveratrol , Resveratrol  And Moral  Separation and purification

In order to separate and purify the anti-inflammatory and antinospermic oxys resveratrol, resveratrol and morasin from the upper limb, the ethanol extract of the steamed upper limb was subjected to enzyme treatment, ethyl acetate fractionation, and Diaion HP-20 adsorption ion resin column chromatography. First, the upper limb was steamed for 1 hour, and then dried. After 100 kg of Morus Twig was added with 650 kg of an aqueous 80% ethanol solution, the mixture was sonicated for 12 hours at 50 ° C in an ultrasonic extractor and filtered. The above extraction procedure was repeated twice, and the resulting mixed ethanol extract was partially decompressed and concentrated to obtain an upper ethanol extract (10 kg).

Next, the ethanol extract was dissolved in an 80% ethanol aqueous solution (50 kg) and allowed to stand overnight at room temperature. The resulting supernatant was separated, and then normal-hexane (100 kg) was added thereto. Was removed and the lower layer was concentrated under reduced pressure to obtain an upper ethanol extract (8.5 kg).

4 L of pectinase 5 XL (Novozymes, Denmark) was added to the suspension, and the mixture was hydrolyzed with stirring at 30 to 40 ° C for 12 hours. The mixture was allowed to cool, and then ethyl Acetate (100 kg) was added and the mixture was repeated twice. The upper layer was dehydrated with anhydrous ganoderma (10 kg), filtered and concentrated under reduced pressure to obtain ethyl acetate extract (2.1 kg). The suspension was suspended in a 20% aqueous ethanol solution (5 kg) and filled in a Diaion HP-20 adsorption ion resin (Mistubishi Chem. Co., Japan) column (10 cm × 50 cm) previously equilibrated with an aqueous 20% The cells were sufficiently washed with 20% ethanol aqueous solution (40 L) and eluted with a 40% aqueous ethanol solution (20 L) and a 60% aqueous ethanol solution (20 L). At this time, the fraction eluted with the aqueous 40% ethanol solution was concentrated under reduced pressure at 50 ° C or lower using a rotary evaporator to obtain an upper limb extract I (1.2 kg, yield: 1.2%) containing oxyreservatrix (purity: 60% .

(110 g, yield: 0.11%) containing trans-resveratrol (purity: 70%) and morasin (purity: 80%) were obtained from the aqueous 60% ethanol solution in the same manner .

Example  2: Preparation of < RTI ID = 0.0 > Upper limb extract  I and II Oxyresveratrol , Trans - Resveratrol  And Moral  Quantitative analysis

Quantitative analysis of oxreservatol, trans-resveratrol and morasin in the upper limb extracts I and II prepared according to Example 1 was performed as follows using high performance liquid chromatography (HPLC). First, 100 g of methanol was appropriately dissolved in 1 g of the top-purified powder, and 100 mL of the same solution was used. The solution was diluted 10-fold with the same solvent, and 1 mL of the solution was passed through a 0.45 ㎛ membrane filter (gelman Sci, USA) and 20 μL was injected into the HPLC to quantitatively analyze the HPLC analysis. 5 high performance liquid chromatography, column; YMC-Pack C ₁₈ (5 μm, 250 × 4.6 mm ID, YMC Inc., USA), mobile phase (concentration gradient elution for 60 min with solvent A -> solvent B); Solvent A (0.05% phosphoric acid) & Solvent B (100% methanol), ultraviolet detector; UV _{320 nm} , flow rate; 0.8 mL / min.

The high-performance liquid chromatogram of oxyreservatrix (compound 1), trans-resveratrol (compound 2) and morazine (compound 3) isolated from the upper limb prepared according to Example 1 is shown in FIG. 2, As follows.

(Compound 1)

(Compound 2)

(Compound 3)

In order to identify the chemical structure of oxyreservatol, trans-resveratrol, and morasin isolated from the upper limb, ultraviolet and nuclear magnetic resonance spectra of the three compounds were first measured. Shin et al ., Biochem Biophys Res Commun 243: 801-803 , UV and nuclear magnetic resonance spectra (MICs) already reported by Oh et al ., Arch Pharm Res 21: 703-706, 1998, Kim et al ., J Korean Soc Food Sci Nutr 37, 124-128, And each compound was identified by comparative analysis.

Table 1 shows the results of measurement of the content of oxyreservatol, trans-resveratrol and morasin in the extracts I and II prepared from the upper limb prepared according to Example 1 above.

[Table 1] Ethanol treatment of skim dipping ethanol treatment, ethyl acetate fraction and Diaion HP -20 Adsorption ion resin  Column chromatography to give < RTI ID = 0.0 > Upper limb extract I and II Oxyresveratrol , Trans - Resveratrol  And morashine content

Trans resveratrol, trans-resveratrol and morazine in tablets Powder I and II prepared by sequential separation of the upper part defatted ethanol extracts with enzyme treatment, ethyl acetate fraction and Diaion HP-20 column chromatography as in the above Table 1 The contents were 60.50%, 40.64%, and 45.16%, respectively. The yields of purified powder I and purified powder II were 1.2% and 0.11%, respectively.

Example  3: presence or absence of enzyme treatment Topical extract Oxyresveratrol , Trans - Resveratrol  And Moratin

To quantify the four physiologically active substances (oxyreservatrol 3- O -beta-diglycoside, ox resveratrol, trans-resveratrol and morasin) contained in the upper limb extract according to the presence or absence of the enzyme treatment according to Example 2, As shown in FIG. 2, four kinds of physiologically active substances were confirmed by HPLC analysis of the degreased ethanol extract of the non-enzyme treated group according to the conditions. At this time, a standard calibration curve prepared using four physiologically active substances, oxy resveratrol 3- O-beta-D glycoside: y = 10.12x + 0.19 (r 2 = 0.997), oxy resveratrol: y = 10.17x + 0.12 (r 2 = 0.998), trans-resveratrol: y = 6.78x + The results of measuring the contents of the four physiologically active substances in the upper ethanol extract according to the presence or absence of the enzyme treatment on the basis of 0.08 (r ² = 0.997) and morasin: y = 6.89x + 0.10 (r ² = 0.998) 2].

[Table 2] Upper limb extract  Comparison of the contents of four physiologically active substances

As shown in Table 2, the contents of the four physiologically active substances of the ethanol extract of the non-enzyme-treated upper limb were 43.82 mg / 100 g of oxyresperatoric 3- O -beta-diglycoxide, ox resveratrol, Oxyreservatrol 3- O -beta-diglycoxide was the major component of resveratrol (0.54 mg / 100 g) and morasin (trace amount), while the enzyme treated group contained the majority of the oximes resveratrol aglycone component as 42.93 mg / 100 g, Some trans-resveratrol and mora were present. Thus, it was confirmed that the oxyreservatrix glycoside components present in the upper extremities were almost converted into aglycon components by the enzyme treatment.

Claims (3)

The upper limb was steamed for 1 hour in a steamer and then dried. Ultrasonic wave was extracted for 12 hours at 40 to 50 ° C by adding 650 kg of a 75 to 95% ethanol aqueous solution to 100 kg of a dry upper limb (Morus Twig)
The concentrate was dissolved again in an aqueous 80% ethanol solution, and then allowed to stand at room temperature overnight to remove the precipitate, which was then fractionated by adding normal-hexane to remove the oil in the upper layer, and the lower layer was concentrated again.
Suspension was added by adding about 5 times of 20% ethanol aqueous solution to the obtained defatted ethanol extract, and then hydrolyzed at 30-40 ° C. for 12 hours by treating 10% pectinase enzyme with respect to the extract.
The hydrolyzate obtained above was fractionated with ethyl acetate solvent, the upper layer was concentrated under reduced pressure, dissolved in 20% ethanol solution, and then adsorbed on a DIION HP-20 adsorption ion resin column. After elution with 40% ethanol aqueous solution and oxyresveratrol containing upper limb extract characterized in that it comprises the step of concentrating.
The method according to claim 1, further comprising the step of eluting and concentrating the adsorbent ion resin with a 60% aqueous ethanol solution, and further comprising the step of concentrating the transabdominal upper limb extract.
An anti-skin inflammation and skin whitening functional cosmetic composition comprising the extract of the upper limbs prepared by the method according to claim 1 or 2 as an active ingredient.

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