TWI625125B - Preparing method of chenopodium formosanum extracts and dressing composition - Google Patents

Abstract

The invention discloses a preparation method of the Taiwan cockroach extract, comprising the steps of: extracting the scorpion seed from the first solvent to obtain a crude extract; and extracting the crude extract with the second solvent to obtain 藜Extracts. The first solvent is ethanol, and the second solvent is at least one selected from the group consisting of water, n-hexane, ethyl acetate, and n-butanol. The present invention further provides a dressing composition comprising the Taiwan cockroach extract.

Description

Preparation method of Taiwan cockroach extract and dressing composition

The invention relates to the application of a Taiwan cockroach extract, in particular to a preparation method of a Taiwan cockroach extract capable of promoting wound healing and a dressing composition comprising the same.

At present, most of the wound healing materials, dressings and wound care markets are concentrated in the United States, Europe and Japan. Among the global product sales, the total is over 80%, and the global market value is expected to be US$12-15 billion, but in recent years, consumption People are paying more and more attention to the health of their bodies, and they are increasingly demanding home care, medical beauty and health care. Therefore, there is still a lot of room for development and application of materials for wound healing. Finding new natural materials, verifying their efficacy scientifically, and combining modern processing technology to develop wound healing materials that meet the needs of Chinese people, has great market potential. However, at this stage, the research and development of domestic raw materials for wound healing relies on the transfer of foreign research results, and the direct import of foreign ingredients is often added. The lack of screening materials, collecting active ingredients, determining product forms, constructing appropriate processes, and even tracking The technology and experience of the product during storage and distribution. The demand for wound healing materials is increasing. At this stage, the research and development of wound healing can extend foreign creative or research concepts to develop local raw materials and select appropriate product projects. Develop specific ingredient blending and performance enhancement technologies, and combine functional verification to develop wound healing materials that will enable immediate market demand and benefits.

Chenopodium formosanum is rich in nutrients and is even superior to common food crops. Its starch content is about 55.7%; protein content is 17.7%; dietary fiber is also as high as 17.5%, which is three times that of oats and more than six times that of sweet potatoes. Many studies have also pointed out that increasing dietary fiber intake can improve intestinal function and many diseases. happened. In the trace elements, such as calcium, iron, zinc, sodium, etc. are better than the average cereal. Taiwan has a considerable amount of essential amino acids, such as threonine, isoleucine, and lysine. In particular, isoleucine and lysine which are often lacking in cereals. These two amino acids are more abundant in Taiwan than in other cereals. In recent years, it has been pointed out that the main pigment of Taiwanese alfalfa seeds extracted by water is betanin, isobetanin, aramantine, isoamaranthine (in terms of content) in betaine. This pigment is quite unstable and is very susceptible to damage from light, oxygen, temperature and pH, but it also has good oxidation resistance. Studies have shown that the amount of betaine in Taiwan's sorghum has a significant relationship with its antioxidant capacity, and it has the best antioxidant power and stability at pH 5. Polyphenolic compounds in Taiwan's scorpion also play an important role in antioxidants. Rutin is the main component, followed by chlorogenic acid and catechin. In addition to beet pigments and polyphenolic compounds, Taiwanese glutinous rice is also rich in many functional components, such as superoxide dismutase, γ-aminobutyric acid (GABA), β-glucan. (β-glucan), lactoperoxidase and lysozyme have such diverse active substances that they have anti-oxidation, anti-atherosclerosis, anti-mutagenicity, etc. Physiological activity. Studies have shown that the shelled Taiwanese carp is extracted with different solvents and subjected to antioxidant tests. The total polyphenols and total flavonoids are the highest in the 50% ethanol extraction group, and the antioxidant capacity is determined (DPPH free radical scavenging ability, Both the reducing power and the ability to chelate ferrous ions show good results. The nano-induced scorpion seed of Taiwan was fed a high-fat diet for 4 weeks, and its low-density lipoprotein (LDL-cholesterol) and total cholesterol in plasma were significantly decreased, indicating that it is beneficial for the prevention of cardiovascular disease. In terms of anti-mutagenicity, the anti-mutagenic force of the unsealed Taiwanese ethanol extract at 0.01 mg/plate can reach more than 60%, and it has a concentration effect. However, this local plant has been paid more and more attention in recent years. At present, most of the emphasis is on the health effects and physiological activities of the human body, and little research and literature on promoting wound healing are rarely discussed.

In view of the foregoing, the inventors of the present invention have painstakingly studied, thought about, and designed a preparation method and a dressing composition of a Taiwanese cockroach extract to improve the lack of the prior art, thereby enhancing the industrial use and utilization.

In view of the above-mentioned problems, the object of the present invention is to provide a preparation method of a Taiwan cockroach extract and a dressing composition for solving the conventional defects.

Based on the above object, the present invention provides a method for preparing a Taiwan cockroach extract, comprising the steps of: extracting a sorghum seed from a first solvent to obtain a crude extract. And extracting the crude extract with a second solvent to obtain a Taiwan cockroach extract. The first solvent may be ethanol, and the second solvent may be at least one selected from the group consisting of water, n-hexane, ethyl acetate, and n-butanol.

Preferably, the step of obtaining a crude extract further comprises removing the first solvent in the crude extract to obtain a powder of the crude extract, and the second solvent extracting the powder.

Preferably, the extraction step using the second solvent may be carried out by sequentially extracting with water, n-hexane, ethyl acetate and n-butanol to obtain a corresponding solvent-differentiated Taiwan 藜 extract.

Preferably, the second solvent is ethyl acetate.

In view of the above, the present invention further provides a dressing composition comprising a Taiwanese cockroach extract, which can be obtained by any of the above.

Preferably, the Taiwanese cockroach extract comprises Rutin.

Preferably, the dressing composition comprises a gel.

Based on the above object, the present invention further provides a dressing composition comprising the Taiwanese cockroach extract, which can be extracted by extracting 95% ethanol at a solid-liquid ratio of 1:10 to 1:20, and removing the ethanol after extraction. And made.

Preferably, the dressing composition comprises a gel.

Preferably, the content of the Taiwanese extract in the dressing composition is from 4% to 20% by weight based on the total weight of the dressing composition.

As described above, the Taiwanese cockroach extract according to the present invention has the characteristics of promoting wound healing under the use of a specific extraction method, and thus can be used for preparing a dressing to enhance the utilization value of the cockroach extract in Taiwan.

In order to make the above-mentioned objects, technical features, and gains after actual implementation more obvious, a more detailed description will be given below with reference to the corresponding drawings in the preferred embodiments.

S10~S40‧‧‧Steps

Figure 1 is a flow chart showing the preparation of the Taiwan cockroach extract of the present invention.

Figure 2 is a graph showing the results of cell viability of sputum extracts from Taiwan to dermal fibroblasts (CCD966SK).

Figure 3 is a graph showing the results of cell viability of different concentrations of Taiwan 藜 ethanol extract (DEE). Part (A) acts on human fibroblasts, and part (B) acts on skin keratinocytes (HaCaT).

Figure 4 shows the finer aqueous extracts (DWE) of different concentrations of ethanol. Cell survival rate results. Part (A) acts on human fibroblasts, and part (B) acts on skin keratinocytes.

Figure 5 is a graph showing the cell viability results for different concentrations of crude ethanol extract n-butanol extract (DBE). Part (A) acts on human fibroblasts, and part (B) acts on skin keratinocytes.

Figure 6 is a graph showing the cell viability results for different concentrations of ethanol crude extract n-hexane extract (DHE). Part (A) acts on human fibroblasts, and part (B) acts on skin keratinocytes.

Figure 7 is a graph showing the results of cell viability of the ethyl acetate layer extract (DEAE) of different concentrations of ethanol extract. Part (A) acts on human fibroblasts, and part (B) acts on skin keratinocytes.

Figure 8 is a record of the healing process of the crude extract of ethanol from Taiwan in the abdominal wound of mice.

Figure 9 is a graph showing the area change of the abdominal wound healing process of rats with crude ethanol extracts from Taiwan.

Please refer to Figure 1. The Taiwanese cockroach extract in this case can be obtained by the following methods.

In step S10, the sorghum seed can be extracted with an ethanol solution to obtain a crude ethanol extract. In this step, a solvent having a concentration of 90% to 100% (for example, ethanol) can be used, and the solid-liquid ratio (solid: liquid) is 1:10 to 1:20, and the extraction time is 48 hours to 80 hours. extraction.

In step S20, the solvent in the crude ethanol extract can be removed to obtain a powder of the crude ethanol extract.

In step S30, the powder of the crude ethanol extract may be extracted sequentially with water, n-hexane, ethyl acetate and n-butanol to obtain a corresponding solvent-separated extract.

In step S40, the solvent can be removed to distinguish the solvent in the extract to obtain the Taiwan cockroach extract.

In practice, in step S30, since the characteristics of the four solvents are different and incompatible with each other, in this step, any one selected from the group consisting of water, n-hexane, ethyl acetate, and n-butanol may be separately used according to requirements. A solvent extracts a powder of a crude ethanol extract.

In practice, the method of removing the solvent of the crude extract or the extract may be used in the step S20 or the step S40 to obtain a powder product. For example, at least one of concentration under reduced pressure, room temperature drying, and freeze drying may be used.

The crude ethanol extract of the sputum extract of Taiwan obtained by the above method, or the solvent distinguishing layer obtained by further using ethyl acetate, has the best effect for promoting cell survival rate or promoting wound healing, and thus can be applied to preparation promotion. A topical dressing for wound healing.

Preparation:

1. Taiwanese cockroach sample: Taiwanese cockroach ( Chenopodium formosanum ) was planted in Pingtung. After harvesting, the Taiwanese glutinous seeds were beaten from the plants by tapping, and the mature and immature Taiwanese glutinous seeds were separated by running water precipitation method. After drying for three days, the dried Taiwanese glutinous rice is ground and passed through a 100mesh sieve and stored at room temperature for later use.

2. Sample extraction: The extracts were extracted with n-hexane, ethyl acetate, ethanol, acetone, water and methanol, respectively, and concentrated under reduced pressure and dried to obtain six crude extracts, extraction conditions and extraction of each crude extract. The rate is shown in Table 1.

In the preparation of the sample, the crude ethanol extract powder was further subjected to solvent differentiation, and 60 g of the ethanol extract powder was sequentially subjected to solvent differentiation with water, n-hexane, ethyl acetate and n-butanol, and concentrated under reduced pressure. After the solvent was drained, a distinguishing layer of four solvents was obtained, and the extraction conditions and extraction rates of the respective solvent-separating layer extracts are shown in Table 2.

3. Cell proliferation and toxicity assay: Human skin fibroblast (CCD966SK, BCRC 60153) was purchased from the Center for Bioresource Conservation and Research of the Food Industry Development Research Institute (Hsinchu, Taiwan). The skin keratinocyte (HaCaT) cell line was purchased from Cell Line Service, Germany. CCD-966SK and HaCaT cell lines were cultured in DMEM medium containing 10% FBS. All cells were cultured at 37 ° C with 5% CO ₂ .

The cells were planted in a 24-well microassay plate at a cell density of 1×10 ⁵ cells/well (cytotoxicity assay) and ⁴ × ^{10 4} cells/well (cell proliferation assay). After 24 hours of culture, the original medium was aspirated and replaced with no addition. Serum medium, add 100μL of various concentrations of the sample, to 37 ° C, 5% CO ₂ incubator, after 3 days of culture, the supernatant was removed, then add 200 μL MTT, then placed at 37 ° C, 5% CO ₂ After 4 hours in the incubator, the MTT was aspirated, 200 μL of DMSO was added, and after shaking for 15 minutes, the absorbance at 570 nm was measured by an enzyme immunoassay analyzer, and the cell survival rate was calculated by the following formula: cell survival rate (%) = (absorbance of sample / absorbance of control group) x100

4. Animal experiment of wound healing: Male mice of the 6-week-old ICR strain (purchased from Lesco Co., Ltd.) were weighed between 30 and 35 grams. The rats were housed in an animal room with darkness and light cycle for 12 hours and no restriction on eating or drinking. The room temperature was maintained at 25 ± 1 °C by air conditioning. The mice were anesthetized using an air-analyzer, and the mice were anesthetized and then the hair was removed by an electric shaver. After disinfection with 70% alcohol, the full thickness was created using the surgical scissors and forceps after sterilization. The wound area was measured on the skin to open a wound of about 8 mm. The skin healing was observed on days 1, 4, 6, 8, 10, 12, 14, and 16 respectively, and the tissue was sliced in the wound tissue. In addition, the other groups were treated with a blank gel in the wound, containing different concentrations of Taiwan cockroach separation and purification, rubbing the drug twice a day, twice a day.

Take a photo with a digital camera on the skin wound and record the wound recovery until the wound is completely healed. Scale wound photos to the same using Adobe Photoshop and Adobe Illustrator software The ratio is then cropped as a continuous record of the healing process of the wound appearance, assessing the change in the healing area of the wound by the sample treatment, and observing the health of the animal and the health of the appearance.

On the observation day, the wound size was drawn on a transparent paper, and the thick cardboard of the same area was cut, and the results of the weighing were recorded, and the gel was continuously applied until the wound completely healed. The wound area was calculated using Image Pro Plus software, with the original wound area being 100%, which translates the area percentage of each period during wound healing.

W _f : the weight of the original wound area; W _t : the weight of the unhealed wound area

Experimental results: Please refer to Fig. 2, which is a graph showing the results of cell survival of skin fibroblasts from Taiwan crude extract. The crude extract powder was configured into a sample with a concentration of 1000 ppm for cytotoxicity analysis. The results showed that the six extracts of Taiwan carp had no toxic effect on skin fibroblasts, and the crude extract of ethanol promoted human skin fibroblasts. The effect of hyperplasia was 3 times higher than that of the control group in stimulating human skin fibroblasts at 1000 ppm.

Figures 3 through 7 below are for cell proliferation assays using different extracts. Please refer to Figure 3 for the cell viability results of different concentrations of Taiwan 藜 ethanol extract (DEE). Further diluting the crude ethanol extract sample to different concentrations of 5 ppm to 1000 ppm, and evaluating the crude ethanol extract to skin fibroblasts (part (Fig. 3 (A)) and skin keratinocytes at different concentrations (Fig. 3) (B) Part) The impact. The results showed that the crude extract of ethanol from Taiwan had the effect of promoting the proliferation of human skin fibroblasts at various concentrations. In addition, the crude extract of ethanol from Taiwan had no toxic effect on skin keratinocytes at various concentrations, and the concentration was about 50 ppm. Have Significantly promote the proliferative effect of skin keratinocytes. After screening by two kinds of cell experiments, it was confirmed that the crude extract of ethanol in Taiwan has the potential to become a material for promoting wound healing. Therefore, a sample was further prepared in a large amount, and ethanol extraction was carried out with 2 kg of Taiwanese mash to obtain about 524.22 g of a crude extract, which was further subjected to solvent discrimination.

Referring to Table 2, the results of the extraction of the different layers of the solvent showed that the highest recovery rate of the aqueous layer (DWE) in all solvents (up to 41.33%) indicates that most of the material is concentrated in this layer. While the aqueous layer becomes very viscous during the concentration process, the dried sample is easily hygroscopic, so it is speculated that the water layer may contain sugars and substances with sugar groups. In addition, the recovery of the n-hexane layer (DHE) was second, at 26.40%.

On the other hand, in the outer shell of the plant buckwheat ( Chenopodium quinoa Willd.) of the same genus of Taiwan, the main substance and bitter taste source were found to be saponin, which was mainly distributed in the n-butanol layer and the water layer after being separated by the solvent. After analysis, it mainly includes triterpenoid saponins containing one to three sugar chains, and can carry up to four glycosyl groups (Mizui et al., 1990; Kuljanabhagavad and Wink, 2009). The substances in the alcohol layer (DBE) and the water layer (DWE) may also contain triterpenoid saponins. The recovery of ethyl acetate layer (DEAE) is only 11.13%.

The above solvent-layered extract powder was further configured into a sample having a concentration of 1000 ppm, and diluted to different concentrations, and the effects of the samples on skin fibroblasts and skin keratinocytes at different concentrations were evaluated.

Please refer to Fig. 4, which is a graph showing the cell viability of different concentrations of ethanol extract aqueous extract (DWE). The results in part (A) of Figure 4 show that the aqueous extract of ethanol extract from Taiwan (DWE) has a significant effect on the proliferation of fibroblasts at a concentration of 5-250 ppm, with a high concentration of 500-1000 ppm. Significantly poisoning the effect of skin fibroblasts. The results of part (B) of Fig. 4 show that the aqueous layer extract has no significant effect on stimulating skin keratinocyte proliferation, and it has obvious toxicity to skin keratinocytes at a high concentration of 1000 ppm. situation.

Please refer to Fig. 5, which is a graph showing the cell viability results of different concentrations of ethanol crude extract n-butanol extract (DBE). The results of part (A) of Fig. 5 show that the extract of the distinguishing layer has the effect of stimulating the proliferation of skin fibroblasts at a low concentration (5-50 ppm), but at high concentrations (500-1000 ppm), it is toxic to kill cells. Ability. The results in part (B) of Figure 5 show that the extract of this distinguishing layer inhibits skin keratinocyte growth as the concentration increases.

Please refer to Fig. 6, which is a graph showing the cell viability results of different concentrations of ethanol crude extract n-hexane extract (DHE). The results of part (A) and (B) of Fig. 6 show that the extract of the distinguishing layer has no obvious proliferative effect on skin fibroblasts and skin keratinocytes at different concentrations.

Please refer to Fig. 7, which is a graph showing the cell viability of the ethyl acetate layer extract (DEAE) of different concentrations of ethanol extract. The results of part (A) of Fig. 7 show that the extract of the distinguishing layer has an effect of promoting skin fibroblast proliferation significantly at a concentration of 250-1000 ppm. The results of part (B) of Fig. 7 show that the distinguishing layer also significantly promotes the effect of skin keratinocyte proliferation at a concentration of 100-250 ppm. Further, since the component having a lower polarity is distributed to the ethyl acetate layer, the active ingredient in the ethyl acetate layer is excellent in lipophilicity, and is more likely to enter the cell and has good usability. Further, by preliminary HPLC component analysis, Rutin was contained in the ethyl acetate layer extract of the crude ethanol extract, but no chlorogenic acid was contained.

Based on the results of Figures 4 to 7 above, the extract of the ethyl acetate layer of the ethanol extract of Taiwan can exhibit better ability to promote skin cell proliferation.

Please refer to Fig. 8 and Fig. 9, which are the record and area change diagrams of the crude extract of ethanol from Taiwan in the abdominal wound healing process. The crude ethanol extract of Taiwan 藜 was prepared into 5% and 10% gels, and the gel samples were administered daily to evaluate the condition of promoting wound healing. Such as the first As shown in Fig. 8, wounds were produced on day 0, and 5% and 10% of the wounds of the crude ethanol extract of Taiwan 藜 ethanol were completely healed on the 13th day compared to the control group (carbopol 940 gel), only The remaining hair has not yet fully grown to the same length. Therefore, Taiwan's crude ethanol extract has the potential to promote wound healing.

Please refer to Figure 9, from the results of the wound area, the wound healing area in the control group was 0.3mm on the 0th day to 0.08mm on the 18th day, and the 5% Taiwan 藜 ethanol extract gel was used. The wound healing area of the group was 0.3mm on the 0th day to 0.05mm on the 18th day, and the wound healing area of the 10% Taiwanese sputum ethanol extract extract group was 0.3mm on the 0th day. On the 18th day, it was 0.02 mm. From this result, it can be known that the crude extract of ethanol in Taiwan promotes wound healing at a faster rate than that of the control group, and therefore can be used as a plant dressing or novel material for promoting wound healing.

The above results confirmed that Taiwan's crude ethanol extract has the potential to promote wound healing. In addition, further purification of the skin fibroblasts and keratinocytes can be further obtained by further separating the crude ethanol extract with ethyl acetate. The extract of the active ingredient can be used as a dressing composition for promoting wound healing.

The above is intended to be illustrative only and not limiting. Any equivalent modifications or alterations to the spirit and scope of the invention are intended to be included in the scope of the appended claims.

Claims (6)

A method for preparing a Taiwan cockroach extract, comprising the steps of: extracting a sorghum seed from a first solvent to obtain a crude extract; and extracting the crude extract with a second solvent to obtain the a cerium extract of Taiwan; wherein the first solvent is ethanol, and the second solvent is at least one selected from the group consisting of water, n-hexane, ethyl acetate and n-butanol; wherein the ceramium seed and the first solvent are Extracting the solid-liquid ratio from 1:10 to 1:20; wherein the step of obtaining the crude extract further comprises removing the first solvent in the crude extract to obtain a powder of the crude extract, and the first The second solvent extracts the powder; wherein the extraction step using the second solvent is followed by extraction of the crude extract with water, n-hexane, ethyl acetate and n-butanol to obtain a crude ethanol extract. The aqueous layer extract, the ethanol crude extract n-butanol extract and the ethanol crude extract ethyl acetate layer extract were used as the Taiwan cockroach extract. A dressing composition comprising a Taiwanese cockroach extract obtained by the preparation method as described in claim 1 of the patent application. The dressing composition of claim 2, wherein the dressing composition comprises a gel. A dressing composition comprising a Taiwanese cockroach extract, the cockroach extract is extracted with 95% ethanol at a solid-liquid ratio of 1:10 to 1:20, and the ethanol is removed after extraction. And made. The dressing composition of claim 4, wherein the dressing composition comprises a gel. The dressing composition of claim 4, wherein the Taiwanese extract is present in the dressing composition in an amount of from 4% to 20% by weight based on the total weight of the dressing composition.