TWI632919B - Crude extract of red djulis with anti-skin wrinkling activity, method of making the same and applications thereof - Google Patents

Abstract

The invention relates to a crude red scorpion liquid with skin anti-wrinkle activity and a preparation method thereof, which utilizes an aqueous extraction solution of at least 85 ° C, soaks red glutinous grains or chaff for no more than 40 minutes, and then removes insoluble matter. , get the crude extract of red peony. The obtained crude extract of red peony has been proved to have skin anti-wrinkle activity for biological materials by in vitro experiments, and can be applied to the external composition for skin.

Description

Red scorpion crude extract with skin anti-wrinkle activity and its manufacture Method and application

The invention relates to a plant extract and a preparation method thereof, in particular to a crude red scorpion liquid having skin anti-wrinkle activity, a preparation method and application thereof.

Red quinoa (Djulis;. Chenopodium formosanum Koidz) was born kinds of excellent drought tolerance Chenopodiaceae (Chenopodiaceae) lambsquarters (Chenopodium) Taiwan original annual herb, plant growth robust. Traditionally known as Hung Hom, it was renamed Taiwanese in December 2008.

In Taiwan, Hung Hom is a traditional food crop of the aborigines, mostly cooked with rice or steamed bread, or used for brewing millet wine. At present, indigenous people in Pingtung, Taitung and Hualien areas have been planted.

Red peony is rich in dietary fiber, polysaccharides, phenols and other ingredients, and is known to have antioxidant effects. In the application, the red glutinous grains are generally dried, processed and ground into powder to be further added to various products. However, the red enamel raw material is lost in the grinding process, and the components extracted by the ground powder are more complex. Miscellaneous, if further purification of specific components for a specific use is required, the process steps are cumbersome, the process time is long, and the cost is high.

In view of this, there is a need to provide a method for reusing red glutinous grains or chaffs to simplify the conventional process steps and shorten the processing time, thereby enhancing the economic value of Chenopodium formosanum Koidz.

Accordingly, one aspect of the present invention provides a method for producing a crude red mash extract having skin anti-wrinkle activity, which utilizes an aqueous extraction solution of at least 85 ° C to soak a grain or valley of Chenopodium formosanum Koidz. The immersion treatment liquid obtained by the shell can be obtained by removing the insoluble matter, and the crude extract of red peony can be used to promote the proliferation of animal cells, inhibit the activity of elastin and inhibit the saccharification of protein in vitro.

Next, another aspect of the present invention provides a crude extract of red peony having skin anti-wrinkle activity, which is obtained by a drying step of a crude extract of red peony obtained by the above method at a temperature lower than 85 ° C. Made with a solvent.

Still further, another aspect of the present invention provides a method of treating a biological material in vitro, which utilizes a composition comprising the above-described crude extract of red peony to treat the biological material in vitro. In an example, when the biological material is an animal cell, the composition containing the above-mentioned crude extract of red peony can promote proliferation of animal cells, inhibit elastin-degrading enzyme, and inhibit protein saccharification in vitro.

According to the above aspect of the present invention, a method for producing a crude red scorpion extract having skin anti-wrinkle activity is proposed. In one embodiment, the method comprises providing a grain or chaff of Chenopodium formosanum Koidz., wherein the red dragonfly can be, for example, a red variety, an orange variety, a brown variety, or any combination of the foregoing. Next, an extraction step is performed to soak the above-mentioned grain or chaff with an aqueous extraction solution of at least 85 ° C to obtain an immersion treatment liquid, wherein the soaking time of the extraction step may be, for example, not more than 40 minutes. Thereafter, a solid-liquid separation step is performed to remove the insoluble matter of the above-mentioned immersion treatment liquid, thereby obtaining a crude red sorghum extract. In one example, the crude red sorghum extract has an activity of promoting proliferation of animal cells, inhibiting elastin-degrading enzyme, and inhibiting protein saccharification in vitro.

According to an embodiment of the invention, the moisture content of the grain or chaff may be, for example, from 8 weight percent to 15 weight percent.

According to an embodiment of the invention, the weight ratio of the grain or chaff to the extraction solution may be, for example, 1:20 to 1:5.

According to an embodiment of the invention, the animal cells described above may be, for example, fibroblasts.

According to another aspect of the present invention, a crude red peony extract having skin anti-wrinkle activity is provided, which is obtained by removing the extraction solvent by a drying step at a temperature lower than 85 ° C by using the crude red sorghum extract obtained by the above method. be made of.

According to an embodiment of the invention, the drying step may include, but is not limited to, a vacuum drying step, a reduced pressure drying step, a freeze drying step, or a vacuum freeze drying step.

According to still another aspect of the present invention, a method for treating a biological material in vitro is provided, characterized in that the biological material is treated in vitro by using a composition containing the above-mentioned crude extract of red peony, wherein an effective dose of the crude extract of red peony can be exemplified. Such as 0.01mg/mL to 10.0mg/mL.

According to an embodiment of the invention, the biological material may include, but is not limited to, animal cells, enzymes or proteins, and the animal cells may be, for example, fibroblasts. In order to promote the proliferation of animal cells, inhibit the elastin-degrading enzyme and inhibit protein saccharification in vitro. In one example, the above-mentioned crude extract of red peony can promote the proliferation of animal cells, inhibit the activity of elastin-degrading enzyme, and inhibit the saccharification of proteins in vitro.

The invention relates to a crude red scorpion liquid with skin anti-wrinkle activity of the invention and a preparation method thereof, which utilizes an aqueous extraction solution of at least 85° C., soaks red glutinous grains or chaff for no more than 40 minutes, and then obtains insoluble matter, and then obtains Red sorghum crude extract. The obtained crude extract of red peony and the crude extract of red peony have been proved to have skin anti-wrinkle activity by in vitro experiments, and can be applied to the external composition for skin.

100‧‧‧ method

101‧‧‧Provide the steps of grain or chaff of red glutinous rice

103‧‧‧ Carry out the extraction step, soak the grains or chaff of the red peony for at least 40 minutes with an aqueous extraction solution of at least 85 ° C to obtain the immersion treatment solution

105‧‧‧The solid-liquid separation step of the immersion treatment liquid to obtain the crude red sorghum extract

107‧‧‧Selective drying step for the crude red sorghum extract

109‧‧‧Get the red extract

The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Part of the process flow chart.

Fig. 2 is a high performance liquid chromatography (HPLC) spectrum showing a crude extract of red peony according to an embodiment of the present invention and a comparative example.

As stated above, the present invention provides a skin anti-wrinkle activity The red sorghum crude extract liquid and the preparation method thereof, the aqueous extracting solution of at least 85 ° C is used, the red glutinous rice grains or the chaff are immersed for no more than 40 minutes, and after the insoluble matter is removed, the red sorghum crude extract liquid is obtained.

The term "red sorghum crude extract" as used herein refers to a grain of Chenopodium formosanum Koidz. which is native to Taiwan or a husk that has been discarded during processing, and is soaked in a short time by an aqueous extraction solution. The solution is obtained by immersing the treatment liquid and then removing the insoluble matter. In one embodiment, the red mullet used in the present invention is a red cultivar, an orange cultivar, a brown cultivar, or any combination thereof, and is substantially free of the above-described cultivars. In another embodiment, the sorghum grain or chaff used in the present invention is not subjected to any grinding treatment to avoid subsequent extraction of other undesirable ingredients in the seed kernel. In still another embodiment, the moisture content of the sorghum grain or chaff used in the present invention may be, for example, from 8 weight percent to 15 weight percent.

Here, it is explained that if the above-mentioned variety or the red glutinous grain or chaff having a water content outside the above range is used, the crude extract of the red mash obtained by the subsequent extraction cannot achieve the desired skin anti-wrinkle activity.

The aforementioned crude red sorghum extract can be obtained by the following method. Please refer to FIG. 1 , which is a partial flow chart showing a method for manufacturing a red sorghum crude extract according to an embodiment of the present invention. In one embodiment, the method 100 can provide a grain or chaff of red sorghum as shown in step 101 of FIG. 1, wherein the red mullet is a red cultivar, an orange cultivar, a brown cultivar, or any combination thereof. In one example, the moisture content of the aforementioned red glutinous grains or husks may be, for example, 8 to 15 weight percent, and the water content of the sorghum grain or husk may be dried by a conventional drying process, such as natural drying. Or in an oven of, for example, about 50 ° C, Dry to adjust to the desired moisture content.

Next, as shown in step 103 of FIG. 1, an extraction step is performed, and the grain or chaff of the red peony is soaked with an aqueous extraction solution of at least 85 ° C to obtain an immersion treatment liquid. In one embodiment, the soaking time of the above extraction step is shorter, preferably no more than 40 minutes. It is worth mentioning that the above extraction step uses a whole grain or chaff of red peony, excluding any grinding treatment of red glutinous grains or chaff. If the above-mentioned sorghum grain or chaff is ground, the crude extract of the red sorghum obtained by subsequent extraction may contain other undesirable sub-components and fail to achieve the desired skin anti-wrinkle activity.

In one example, the aqueous extraction solution may be, for example, water, and the weight ratio of the above grains or chaff to the aqueous extraction solution may be, for example, 1:20 to 1:5.

In the above extraction step, the temperature of the aqueous extraction solution is preferably, for example, 85 ° C to 100 ° C. In an example, the soaking time of the above extraction step may be, for example, 5 minutes to 40 minutes. The above extraction step only requires static soaking without the need to incorporate other auxiliary means (such as stirring, ultrasonic vibration).

It is stated here that if the weight ratio of the above grain or chaff to the aqueous extraction solution is less than 1:20 or higher than 1:5, or the temperature of the above aqueous extraction solution is lower than 85 ° C or exceeds 100 ° C, or If the soaking time is less than 5 minutes or exceeds 40 minutes, the yield and biological activity of the subsequently obtained crude extract of red peony are not good, and the animal cells can not be promoted in an effective dose of 0.01 mg/mL to 10.0 mg/mL in vitro. Proliferation, inhibition of elastin and inhibition of protein saccharification.

After step 103, as shown in step 105, the immersion treatment The liquid is subjected to a solid-liquid separation step to remove the insoluble matter of the above-mentioned immersion treatment liquid, thereby obtaining a crude red sorghum extract, wherein the solid content of the obtained red sorghum crude extract liquid is generally not more than 3.3% by weight, but 0.8 weight A percentage to 3.3 weight percent is preferred.

After step 105, the above-mentioned red sorghum crude extract liquid is selectively subjected to a drying step, and the extraction solvent contained in the crude red sorghum crude extract liquid is removed by a drying step at a temperature of, for example, less than 85 ° C to obtain a red sorghum crude extract. As shown in step 107.

In one example, the drying step may be performed in any conventional manner, such as a vacuum drying step, a reduced pressure drying step, a freeze drying step, a vacuum freeze drying step, a spray drying step, etc., or other suitable temperatures below 85 ° C. The way to dry. It should be additionally noted that the solid-liquid separation and drying steps and the like may be carried out by any conventional means, and are not limited to the above, and are well known to those of ordinary skill in the art to which the present invention pertains, so that they are not separately Narration.

In one embodiment, when the biological material is treated in vitro by using the composition containing the above-mentioned red peony crude extract, the effective dose of the crude red peony extract may be, for example, 0.01 mg/mL to 10.0 mg/mL for providing in vitro. The anti-wrinkle effect of the skin. In one example, the biological material can be, for example, an animal cell, an enzyme, or a protein, and the animal cell can be, for example, a fibroblast. In another example, the above-mentioned crude extract of red peony does have an anti-wrinkle effect on the skin, and can promote proliferation of animal cells, inhibit activity of elastin-degrading enzyme, and inhibit glycation of proteins in vitro.

In other embodiments, the above-mentioned red sorghum crude extract may have an anti- Oxidation (removal of DPPH free radicals, ability to chelate ferrous ions) activity.

In addition, if the crude extract of red peony is subjected to a drying step at a temperature of 85 ° C or higher, the yield of the subsequently obtained crude extract of red peony and the anti-wrinkle activity of the skin are not good enough to be 0.01 mg/mL. An effective dose of up to 10.0 mg/mL promotes proliferation of animal cells, inhibits elastin-degrading enzymes, and inhibits glycosylation of proteins in vitro.

It is explained that since the above-mentioned crude extract of red peony and the crude extract of red peony have excellent biological activity and are not cytotoxic, they can be applied to preparation of various skin external compositions, for example, cosmetics, emulsions, masks. , medicated soap, body wash, ointment, spray, etc., but the above applications are merely illustrative, and the invention is not limited thereto.

The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Preparation of red sorghum crude extract

Example 1

First, as shown in Table 1, the red glutinous grains of the red variety harvested in the current year were collected, and after natural air drying, the moisture content of the grain was controlled to be 8 to 15 weight percent. Next, using a sorghum grain and water having a weight ratio of 1:10, immersing at a temperature of 95 ° C for 10 minutes to obtain an immersion treatment liquid, and then performing a solid-liquid separation step using a filter paper to remove the immersion treatment liquid. Insoluble matter, thereby obtaining a crude extract of red peony. The crude red sorghum extract obtained in Example 1 The content of the solid matter was 1.0% by weight, and various characteristics were measured in each evaluation manner described later.

Examples 2 to 17 and Comparative Example 2

Examples 2 to 14 and Comparative Example 2 were extracted in the same manner as in Example 1 according to the conditions listed in Table 1, except that the difference was in the variety and location of the red peony used, the extraction conditions, and the time from the extraction to the solid-liquid separation. Wait. The solids of the crude red sorghum extracts obtained in Examples 2 to 14 and Comparative Example 2 were subjected to various evaluation methods described later.

Further, the crude red sorghum extract obtained in Example 14 was further subjected to a spray drying step to obtain a crude extract of red peony, wherein the spray drying inlet temperature was 180 ° C and the outlet temperature was 80 ° C. A spray dried powder was obtained.

Comparative Examples 1 and 3 to 5

Comparative Example 1 collects red glutinous grains of the red variety harvested in the current year, and after natural air drying, the moisture content of the grain is controlled to be 8 to 15 weight percent. Next, according to the conditions listed in Table 1, using a weight ratio of 1:10 sorghum grain and water, combined with ultrasonic shock assisted extraction at 30 ° C, 35 ° C or 95 ° C for 20 minutes to obtain impregnation After the treatment liquid, a solid-liquid separation step was carried out by using a filter paper to remove the insoluble matter of the above-mentioned immersion treatment liquid, thereby obtaining a crude red sputum extract. Each of the evaluation methods described in Comparative Examples 1 and 3 to 5 was subjected to various evaluation methods described later.

Evaluation method

The efficacy of the crude red sorghum extract or the crude red peony extract of Examples 1 to 17 and Comparative Examples 1 to 5 was evaluated by several tests, wherein the test group represented The groups of the crude red sorghum extracts of Examples 1 to 17 and Comparative Examples 1 to 5 were added, and the blank group represented the group to which no crude sorghum crude extract was added.

1. Assess anti-wrinkle effect

The anti-wrinkle effect of the crude red peony extract was evaluated based on the fibroblast proliferation test, inhibition of the activity of the elastase, and anti-glycation ability.

1.1 fibroblast proliferation test

This test was evaluated using human/murine fibroblasts (BCRC 60031). First, fibroblasts (cell density: 2000 cells/well) (cell culture medium per well and the crude red sputum extract of Example 1 in total, 200 μL) were reacted with 20 μL of the MTT solution in a 37 ° C incubator for 4 hours. Remove the cell culture medium containing MTT, add 100 μL of DMSO to avoid shaking for 5 minutes, and then read the absorbance at 550 nm with an enzyme immunoassay analyzer (ELISA reader) to compare the OD value of the experimental group and the control group with the control group (100%). In comparison, the cell survival rate (%) was obtained, and the results are shown in Table 2 and Table 3.

From the results of Table 2, it can be seen that the red variety of red peony is used as an example, and the composition containing the crude extract of red peony of Examples 1, 15 to 17 has an anti-wrinkle effect on the skin when it is treated in vitro. Promotes the anti-wrinkle effect of animal cell proliferation. In contrast, Comparative Examples 3 to 5 have a poor effect of promoting animal cell proliferation in vitro because of the different sites or extraction conditions used.

Referring to FIG. 2, there is shown a high performance liquid chromatography (HPLC) spectrum of a crude red sorghum extract according to an embodiment of the present invention and a comparative example, which uses a commercially available HPLC instrument (for example, Hitachi). Commercially available tubing columns (e.g., RH18 tubing (4.6 mm x 250 mm; Biosil 5u Pro-ODS-U)) were run at a flow rate of 1 mL/min. The mobile phase of the gradient electrolysis was water and methanol. In HPLC, 0 to 3 minutes is 2% methanol (solution), 3 to 7 minutes is 2% to 22% methanol (solution), 7 to 13 minutes is 22% to 34% methanol solution, 13 to 34% to 69% methanol in 20 minutes, 69% to 71% methanol in 20 to 26 minutes, 71% to 80% methanol in 26 to 27 minutes, and 80% to 80% methanol in 27 to 29 minutes; The detection wavelength was 254 nm.

The curve 201 of Fig. 2 represents the result of the HPLC chart of the crude red sorghum extract of Example 1 of the present invention, and the curve 203 represents the result of the HPLC chart of the crude sorghum extract of Comparative Example 3 of the present invention. As is apparent from the results of Fig. 2, the crude red sorghum extract of Example 1 of the present invention has a plurality of peaks, and a peak at about 22.6 minutes to about 22.8 minutes, which is not in Comparative Example 3.

1.2 inhibiting the activity of elastase

Inhibition of elastase can reduce the breakdown of elastin. In this test, the elastin hydrolase and the fluorescently labeled elastase in the test group and the enzymatic hydrolase assay reagent set (EnZChek Elastase Assay Kit, Molecular Probes, USA) were subjected to room temperature (10 ° C to 40 ° C) from the light to 2 hours. If the test substance cannot inhibit the elastase, an enzymatic reaction will be carried out to cut the substrate, and the converted value can be detected by a fluorescence analyzer (excition/emission 485/530 nm), and the following formula (I) is used. The inhibition rate was calculated, and the results are shown in Table 1: Inhibition rate (%) = [1 - (test group / blank group)] × 100% (I).

1.3 Anti-glycation ability

Collagen in the skin is susceptible to saccharification, causing wrinkles, sagging, and reducing skin elasticity causing skin aging. Therefore, in this test, glucose and calf serum albumin (BSA) were co-cultured with different groups for 1 day, and then the fluorescence value of 370/440 nm was detected by optical analyzer and calculated by the following formula (II). The anti-glycation ability (%) was obtained, and the results are shown in Table 1.

The relevant groups are as follows. Test group: 20 μL of 10 mg/mL BSA, 20 μL of 50 mg/mL glucose aqueous solution, and 20 μL of the crude red sputum extract of Example 1 into a 1.5 mL test tube, and cultured at 60 ° C day. Control group: 20 μL of 10 mg/mL BSA, 20 μL of 50 mg/mL aqueous glucose solution, and 20 μL of sodium phosphate buffer were added to a 1.5 mL test tube, and cultured at 60 ° C for 1 day. Blank group: 20 μL of 10 mg/mL BSA and 40 μL of sodium phosphate buffer solution were taken to a 1.5 mL test tube, and cultured at 60 ° C for 1 day. Background group: 20 μL of 10 mg/mL BSA, 20 μL of sodium phosphate buffer solution, and 20 μL of the crude red sputum extract of Example 1 were placed in a 1.5 mL test tube, and cultured at 60 ° C for 1 day.

Anti-glycation ability (%) = [1-(OD (test group) / OD (control group)) × 100% (II).

2. Antioxidant effect

The antioxidant efficacy of the crude red sorghum extract was evaluated based on DPPH free radical scavenging ability and chelating ferrous ion capacity.

3.1 DPPH free radical scavenging ability

DPPH can form stable free radicals at normal temperature, and its ethanol solution has strong absorption at 517 nm. Therefore, the sample can reduce the absorption value of DPPH ethanol solution at 517 nm as an indicator to eliminate the free-machine capability. In this test, 1.2 mL of Tris-HCl buffer (0.1 M, pH 7.4), 1.5 mL of 0.5 mM DPPH ethanol solution, and 300 μL of the sample were added to a glass test tube, uniformly mixed, and allowed to stand at room temperature in the dark. Minutes and the OD value at 517 nm was measured with a spectrophotometer. In this test, 300 μL of secondary water was used as a negative control group, and an ethanol solution of butylhydroxytoluene (BHT, antioxidant) was used as a positive control group. According to the percentage decrease of OD value relative to the negative control group, it can be known that each test sample clears DPPH self. Based on the ability of the base, the results are shown in Table 3.

In the above embodiment, the number of samples per set of data is at least three (n ≧ 3).

From the results of Table 3, it is known that the composition containing the crude extract of the red peony of Examples 1 to 14 has an anti-wrinkle effect on the skin when it is treated in vitro, and can have wrinkle resistance in vitro (promotes proliferation of animal cells and inhibits elasticity). The activity of proteolytic enzymes and the inhibition of protein saccharification) and the antioxidant effect. In contrast, Comparative Examples 1 to 2 have poor antioxidant effects because of the different sites or extraction conditions used. In addition, the crude extract of red peony of Example 14 was tested for tyrosinase activity, and its IC ₅₀ was 3.31 mg/mL, which was lower than that of commercially available arbutin (5 mg/mL), and the crude extract of red peony represented by Example 14 had Better whitening ability.

It should be added that the present invention is based on a specific variety and location of red sorghum grain or chaff, specific extraction equipment and conditions, specific concentration of extract (object), specific test cells, specific analytical methods or specific instruments. The present invention is not limited thereto, and the present invention is not limited thereto, and the present invention is not limited thereto. Within the spirit and scope, the crude red scorpion extract having skin anti-wrinkle activity of the present invention and the method for producing the same may also use other concentrations of red peony extract (object), other extraction equipment and conditions, other test cells, and others. Analytical methods or other instruments.

It can be seen from the above embodiments of the present invention that the crude red scorpion liquid having the anti-wrinkle activity of the present invention and the method for producing the same have the advantages of effectively utilizing the aqueous extraction solution of at least 85 ° C, soaking the red glutinous grains or the chaff not exceeding 40 After a minute, the insoluble matter was removed, and a crude extract of red peony was obtained. The crude red sorghum extract and the red sorghum crude extract thus obtained have been proved to have skin anti-wrinkle activity by in vitro experiments and can be applied to the external composition for skin, but are merely illustrative here, and the present invention is not limited thereto.

Although the present invention has been disclosed above in several embodiments, It is not intended to limit the invention, and any one of ordinary skill in the art to which the invention pertains can make various changes and modifications without departing from the spirit and scope of the invention. The scope of the patent application is subject to change.

Claims (12)

A method for producing a crude red scorpion extract having skin anti-wrinkle activity, comprising: providing a grain of red carp ( Chenopodium formosanum Koidz.), wherein the red peony is a red variety; performing an extraction step, using at least 85 ° C Soaking the grain with water to obtain an immersion treatment liquid, wherein one of the extraction steps is immersed for no more than 40 minutes, and the grain is not subjected to any grinding treatment; and a solid-liquid separation step is performed to remove the immersion treatment The crude inoculum is obtained by insoluble matter, wherein the crude extract of red sorghum has an activity of promoting proliferation of an animal cell, inhibiting an elastin-degrading enzyme, and inhibiting protein saccharification in vitro. The method for producing a crude red mash extract having skin wrinkle-reducing activity according to claim 1, wherein the grain has a water content of from 8 to 15% by weight. The method for producing a crude red mash extract having skin anti-wrinkle activity according to claim 1, wherein the weight ratio of the aqueous extraction solution to the grain is from 5:1 to 20:1. The method for producing a crude red scorpion extract having skin wrinkle resistance according to the first aspect of the invention, wherein the temperature of one of the waters is from 85 ° C to 100 ° C. A method for producing a crude red scorpion extract having skin anti-wrinkle activity according to claim 1, wherein the soaking time is from 5 minutes to 40 minutes. Skin anti-resistance according to item 1 of the patent application scope A method for producing a wrinkle-active red mash crude extract, wherein the solid content of the crude red sorghum extract is not more than 3.3% by weight. A method for producing a crude red scorpion extract having skin anti-wrinkle activity according to claim 1, wherein the animal cell is a fibroblast. A crude extract of red scorpion having a skin anti-wrinkle activity, which is obtained by using a method of any one of the methods of claim 1 to claim 7, wherein the crude red sorghum extract is at a temperature lower than 85 ° C. It is obtained by removing an extraction solvent through a drying step. The crude red peony extract having skin anti-wrinkle activity according to claim 8 wherein the drying step comprises a vacuum drying step, a reduced pressure drying step, a freeze drying step or a vacuum freeze drying step. A method for treating a biological material in vitro, characterized in that a biological material is treated in vitro using a composition comprising a crude extract of red peony as described in any one of claims 8 to 9 wherein the red One of the effective doses of the crude extract is from 0.01 mg/mL to 10.0 mg/mL. A method for treating a biological material in vitro according to claim 10, wherein the biological material is an animal cell, an enzyme or a protein, and the animal cell is a fibroblast. The method for treating a biological material in vitro according to the invention of claim 11, wherein the crude red peony extract promotes proliferation of the animal cell, inhibits the activity of the elastin enzyme, and inhibits saccharification of the protein in vitro.