TWI653056B - Use of sorghum distiller's extract as whitening skin care product - Google Patents

Abstract

The invention discloses a sorghum distiller's grains extract, which uses Jinmen sorghum distiller's grains as raw materials, and extracts sorghum distiller's grains with pure water, alcohol solvents and supercritical fluid; Ability and ability to reduce melanin content, which can be used as an additive for preparing a beauty or cosmeceutical composition; wherein the sorghum lees extract is from 0.001% by weight to 0.003 weight based on the total weight of the beauty or cosmeceutical composition The amount of% has better cytotoxicity, anti-oxidation ability and whitening ability.

Description

Use of sorghum lees extract as whitening care product

The invention relates to a sorghum lees extract, in particular to a sorghum lees extract which can be used as an additive for preparing a beauty or medicinal cosmetic composition.

Sorghum distillery residue (SDR) is a by-product produced after brewing sorghum. 25 kg of sorghum can produce about 8.2 kg of sorghum distiller's grains. The daily output of sorghum lees from Golden Gate Winery is up to 250 metric tons. Currently, it is mainly provided as Kinmen makes livestock feed locally, or provides ordinary family or small farms as organic fertilizer. However, the amount mentioned above is always too small, and the sorghum lees that can be digested are quite limited. Therefore, most of the sorghum lees are still unused and treated as industrial waste, and the general method of sorghum lees is three steaming and three brewing. , Its biomass utilization rate is about 90%, and the sorghum liquor process of Jinmen Winery is two-steaming and two-brew, and its biomass utilization rate is less than 70%, so it is hoped that this substance will be used to extract its functional ingredients using a green non-toxic process. Apply to beauty care products.

According to the study of lees byproducts fermented by sorghum in Jinmen Distillery in 2001, the starch content can still be as high as 25% (dry weight), and the literature indicates that sorghum has rich raw and pharmacologically active polyphenol compounds. For example, tannins, flavonoids, phenolic acid, and other functional ingredients are long-chain fats. Alcohols (policosanols) and phytochemicals (phytochemicals). Studies have confirmed that these functional ingredients have the effects of scavenging free radicals in the body, anti-lipid oxidation, preventing cardiovascular disease, reducing human serum cholesterol, anti-cancer and anti-microbial. Studies have also shown that feeding of mullet with sorghum distiller's grains can enhance the antioxidant properties in the blood, reduce blood coagulation, make it resistant to cold, and increase the growth rate in winter.

In previous literature publications, there have been some research outputs on sorghum distiller's grains. Republic of China Patent I430755, an antibacterial sorghum lees extract and its manufacturing method: the following extraction methods are used in this patent, (a) cold water extraction: sorghum lees are distilled at 0 ~ 25 ℃ with 1/10 ~ 1 / 100w / v ratio stirring; (b) alcohol extraction: sorghum distiller's grains is stirred at 0 ~ 25 ℃ with 5 ~ 95% alcohol solution in 1/10 ~ 1 / 100w / v ratio range; (c) Hot water extraction: Add sorghum lees to distilled water at a ratio of 1/10 ~ 1 / 100w / v and heat-treat at 100 ~ 135 ° C. After selecting from the extracts obtained by the above method, filter it, remove water, and grind. The sorghum lees powder is obtained because the powder is not directly ground without fiber materials such as rice and sorghum hulls. The purpose is to inhibit bacteria and inhibit Bacillus cereus , Escherichia coli O157: H7 , Salmonella spp., And Staphylococcus aureus . The effectiveness of the strain can therefore be used as a food additive.

Taiwan Agricultural Chemistry and Food Science (October 1999, Volume 48, Issue 5: Pages 233 to 238), extraction of functional components of sorghum lees, this document is intended for use in the extraction process The effect of different solvents on the extraction components, it uses (1) methanol, isopropanol, n-hexane, acetone, ethyl acetate, different ratios of ethanol aqueous solution and 90 ° C hot water for stirring extraction (2) supercritical carbon dioxide fluid Sorghum lees was extracted at an extraction temperature of 35 to 80 ° C and an extraction pressure of 172 to 483 bar. The extraction was performed for 3 hours.

Republic of China Patent Publication No. 201000628, with antioxidant and tyrosine inhibition A method of enzymatically active health raw materials. The main purpose of this patent is to develop a process for extracting rice wine meal using a carbon dioxide supercritical fluid separation procedure. The operating temperature is 40 ° C and the pressure is 350 bar. , Can be obtained with antioxidant and inhibit tyrosinase activity of health raw materials.

Republic of China Patent No. 104142021, distiller's grains extract and its manufacturing method, using acid and alkali hydrolysis to destroy plant cell wall, and then purifying and separating through ethanol. In addition to extracting free phenolic acid, it can increase the binding phenolic acid. Content is obtained to increase yield. The distiller's grains extract was identified by High-Performance Liquid Chromatography (HPLC), and then structurally identified by gas chromatography-mass spectrometry (GC-MS). Contains a variety of phenolic acid functional ingredients such as ferulic acid. These compounds have anti-oxidant ability and inhibit the whitening effect of tyrosinase, and can be used in beauty care composition.

The invention further adopts an extraction process method of all-green, non-toxic, and no harmful chemical solvents, and discloses that under this invention process, the most suitable addition ratio of the additives most suitable for preparing beauty or medicinal cosmetic compositions.

In addition to the solvent extraction method (using ethanol in different ratios of ethanol aqueous solution and pure water at different temperatures) for extraction of sorghum lees, the invention also uses supercritical carbon dioxide fluid extraction equipment for extraction. The process method of the present invention is applied to the extraction of functional ingredients in skin care products, so the composition requirements of skin care products can be considered. When using the solvent extraction method, the use of organic solvents that are harmful to the environment and the human body must be avoided, such as : Methanol (methanol), n-hexane (hexane), etc., the selected ones are ethanol and pure water, which are extracted by adjusting the proportion and temperature, and the raw material development is based on the higher specifications of the USP 467 solvent residues. For self-examination, ethanol is the standard class III residual solvent. Generally, class III residual solvent is considered to be less harmful to humans than class I and class II residual solvents.

In addition to using solvent extraction as a common method, supercritical carbon dioxide fluid extraction equipment is also used. Carbon dioxide is used as the fluid for supercritical extraction technology, which meets the standards of green industrial processes. It also fulfills the "Corporate Social Responsibility CSR (Corporate Social Responsibility) ", and in accordance with the health regulations of various countries, makeup and maintenance products must not remain harmful to human substances, coupled with low extraction temperature, can better retain the biological activity of the extract and other ingredients. Coupled with the use of carbon dioxide supercritical fluid extraction, the supercritical fluid has selectivity. After experimental design verification of the present invention, a high-purity, low-toxic lees functional ingredient active can be obtained, which is suitable for application as a cosmetic product.

The present invention makes good use of the waste generated after sorghum steaming of sorghum wine, and it is expected that the sorghum lees waste is transformed into a usable substance, which not only reduces environmental pollution but also improves economic value. In recent years, the awareness of environmental protection has risen, and the problem of unscrupulous goods has become increasingly serious. The safety of skin care products and makeup products that directly contact human skin cannot be ignored. In view of the fact that most of the commercial care products and makeup products contain preservatives and artificial flavors and pigments If the extraction and purification in the process of raw material synthesis is not accurate, long-term use may cause the body to accumulate many chemical components and cause skin damage such as sensitivity and irritation. Therefore, consumers prefer natural maintenance products, so the present invention hopes to develop Makeup products for consumers' peace of mind.

The invention researches and develops a more natural and environmentally friendly process for extracting functional components in sorghum lees, and it is verified through experiments that the total polyphenol content of the extract obtained by the solvent extraction method is about 207.46 mg / 100g SDR to 298.87mg / 100g SDR, and the total polyphenol content of the extract obtained by the supercritical carbon dioxide fluid extraction method is about 27.22mg / 100g SDR. Previous studies have confirmed that these polyphenol compounds have anti-oxidant ability and anti-oxidant properties. Rhenamine has inhibitory effect, and there is no precedent for use in cosmetics. Therefore, this extract is used to find the concentration most suitable for cosmetics, and it is the most suitable rosacea in the cosmetic composition. The total polyphenol content is 0.001%. To 0.02%, and when the content in the cosmetic composition is 0.02%, it has an antioxidant capacity of more than 80%.

Figure 1 is the analysis result of DPPH (1,1-Diphenyl-2-picrylhydrazyl) free radical scavenging ability measurement; Figure 2 is the analysis result of MTT cell survival rate; Figure 3 is the analysis result of intracellular tyrosinase activity; The fourth is the analysis result of the reduction of melanin content.

In the following, the preparation of sorghum lees extract and its use in whitening skin care products are discussed through specific examples. Those with ordinary knowledge in the technical field to which the present invention pertains can understand other advantages and effects of the present invention from the contents disclosed in this specification, and modify and change the embodiments discussed in the present invention without departing from the scope of the present invention, and still It belongs to the scope of the present invention.

Example 1 Preparation of Sorghum Distillers Grains Extract

(1) Sorghum distiller's grains: The test samples are subjected to continuous drying equipment using a fluidized bed at 100 ° C to 120 ° C, and then ground using a grinder.

(2) Solvent extraction of sorghum distiller's grains: Weigh 20g of sorghum distiller's grains in a triangular conical flask, each with 200ml The 75 ℃ pure water, 90 ℃ pure water, 75% ethanol and 90% ethanol aqueous solution were stirred and extracted for 1 hour. After the extraction, the volume of the filtrate was quantified after filtering with filter paper.

(3) Supercritical carbon dioxide fluid extraction of sorghum lees: The supercritical carbon dioxide extraction used in the present invention is performed using a 250ml extraction tank. Three extractions were 93.51g, 98.85g, and 98.85g of sorghum lees powder. The carbon dioxide was opened to enter the pipeline, and the back pressure regulaor was adjusted to control the system pressure (the three extraction pressures were 170bar, 250bar, and 300bar), and The temperature in the extraction tank (this time the temperature is 40 ° C). The co-extraction solvent used is ethanol. After the pressure and temperature reach the experimental set value, the time is counted. The sample is first immersed in a static extraction with ethanol / carbon dioxide supercritical fluid. In the extraction tank for 2 hours, the ethanol / carbon dioxide supercritical fluid was dynamically extracted for 2 hours with carbon dioxide flow rate of 0.2 L / hr and ethanol flow rate of 0.4 L / hr. The samples collected in the two separation tanks were mixed uniformly and stored. spare.

Example 2 Analysis of Total Polyphenolic Compounds (TPC)

(1) Preparation of standards and reagents: Preparation of standards: This experiment can use conventional methods, using gallic acid (Arcos Organics) as the standard, weigh 0.1g of anhydrous gallic acid and add 100ml pure After the water is uniformly dissolved by ultrasonic vibration, the concentration of the standard is 1000 ppm, and then according to the volume indicated in the table below, add pure water to continuously dilute the standards of different concentrations:

The experimental reagents need to be prepared (a) Folin-ciocalteus phenol reagent, diluted 10 times with pure water, and (b) 7.5% sodium carbonate (Na ₂ CO ₃ ).

(2) Experimental steps: Standards and samples are prepared with pure water to an appropriate concentration, then 1ml is added to 5ml of Fulin reagent diluted 10 times. After mixing, it is left in the dark for 5 minutes, and then 4ml of 7.5% sodium carbonate is added. The aqueous solution was mixed uniformly and left in the dark for 1 hour, and then centrifuged, and the absorbance was measured at 765 nm with a spectrophotometer. Linear regression analysis was performed with the concentration of the standard as the abscissa and the absorbance value on the ordinate. The correlation coefficient (R ² ) of the standard curve was 0.999 to 1 before use. Then the sample absorbance value was brought into the standard curve to calculate its total polyphenol compound content. The extraction amount of total polyphenol compounds is expressed in milligrams per 100 grams of sorghum distiller's grains (mg / 100g SDR).

(3) Experimental results:

Table 1 shows the analysis results of total polyphenol content. When using pure water for extraction, temperature has no significant effect on it, but when using 75% ethanol for extraction, its total polyphenol content is significantly higher than that for 90% ethanol extract. In the critical extraction method, the total polyphenol content is significantly lower than the other two extraction methods. Because carbon dioxide is used as the solvent, its polarity is low, while polyphenol compounds contain hydroxyl groups, and most of them have higher polarity. Carbon dioxide with lower polarity is less compatible.

Example 3 Determination of DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging ability

(1) Reagent preparation: Take 0.004g of DPPH and add 100ml of ethanol (concentration: 40ppm), dissolve it evenly and store it in the dark, and it should not be stored for more than half a day.

(2) Experimental steps: formulate each extract to 200ppm, and then sequentially dilute with the solvent 100ppm, 50ppm, 25ppm and 12.5ppm, take 6ml of DPPH and add to 1ml sample. After the configuration is completed, the final sample conversion concentration is 28.6ppm, 14.3ppm, 7.1ppm, 3.6pm, 1.8ppm. After mixing, stand in the dark for 40 minutes. Measure its absorbance at 517 nm with a spectrophotometer, and calculate the scavenging effects (scavenging effects;%) of DPPH radicals of each extract according to the following formula

Scavenging effect of DPPH free radicals (%)

(3) Experimental results:

The substances contained in the extract are relatively complicated, and the phenolic compounds have a high correlation with the antioxidant capacity. Therefore, the determination of the DPPH free radical scavenging capacity test is based on the total polyphenol concentration. Figure 1 is the analysis result of the DPPH free radical scavenging capacity measurement experiment, and the abscissa is the content of total polyphenol compounds; Figure 1 shows that its scavenging capacity decreases with the concentration of the extraction solution, and 75% ethanol extraction solution and 90% The ethanol extract is the best. It has a removal ability of about 85.3% and 85.2% at a concentration of 200 ppm of total polyphenols in distiller's grains (final concentration of 28.6 ppm).

Since the main free radical scavenging substance in sorghum distiller's grains is total polyphenol compounds, the experimental results also prove that the DPPH free radical scavenging ability of the extract is positively correlated with the total polyphenol compounds. From the experimental results, it can be known that the samples of lees extract can be used for anti-aging and anti-oxidation applications. .

Example 4 Analysis of cell survival rate

(1) Experimental principle

Tetrazolium in MTT reagent is used in living cells to be reduced by dehydrogenase (Dehydrogenase) in mitochondria to produce blue-purple Formazan crystals to detect the relative proportion of viable cells. Formazan crystals have a maximum absorbance at a wavelength of 570nm, so the survival rate of the cells is calculated by measuring the absorbance.

(2) Experimental method

Melanoma cells (5 × 104 cells / well) were implanted into 24-well culture plates, and after 24 hours of culture, the cells were treated with lees extraction solution samples with a final concentration of 10, 20, and 30 ppm for 24 hours. Each well was treated with PBS (phosphate buffer saline). ) Wash once, add MTT solution and place at 37 ° C for 4 hours, then use Dimethyl Sulphoxide (DMSO) to dissolve the formazan product formed by MTT reduced by dehydrogenase in the cytoplasmic glands, and use an immunoenzyme analyzer ( ELISA) was used to determine the OD ₅₇₀ absorbance of the product. The more cells survive, the darker the product becomes.

Cell survival rate formula (%) = 100-[(A-B) / A × 100%]

A: OD ₅₇₀ absorbance of the control group

B: OD ₅₇₀ absorbance of sample group

(3) Experimental results:

Figure 2 shows the cell viability analysis of water extraction, alcohol extraction, and supercritical extraction. The final concentrations of total polyphenols in distiller's grains in the solution were 10 ppm, 20 ppm, and 30 ppm, respectively. From the experimental results, it can be seen that the supercritical fluid has better cell survival rate in the same distillers grains extraction solution, and the distillers grains extract solution has a cell survival rate of more than 70% except for 30 ppm alcohol. From the experimental results, we can know that the sample of lees extract will not have toxic effect on the cells.

Example 5 Analysis of intracellular tyrosinase activity

(1) Analysis principle of intracellular tyrosinase activity

Using the freeze-thaw cycle method, the cells are frozen at -80 ° C for a period of time (30 minutes), so that the water in the cells forms ice crystals; the cells are taken out and thawed at room temperature (30 minutes), so that The ice crystals return to water, and by this process, the cells are ruptured, and then the intracellular material is released, and the tyrosinase we want to measure is also in it. Adding a tyrosinase to a catalytic substrate (such as L-dopa), and analyzing the amount of product produced, we can infer the relative activity of its tyrosinase.

(2) Analysis method of intracellular tyrosinase activity

By measuring the inhibitory rate of dopaquinone (dopachrome) auto-oxidation, to understand whether lees extract can inhibit tyrosinase activity and reduce melanin formation. B16F10 melanoma cells with a density of 5 × 10 ⁴ cells / ml were implanted in a 24-well culture plate, cultured in a medium containing 100nM α-MSH, placed in a 37 ° C, 5% CO2 incubator, and added for 24 hours. The distiller's grains SFE was further processed for 24 hours, and then 100 μl of 1% (V / V) Triton X-100 / 50mM sodium phosphate buffer (pH 6.8) and 10mM PMSF were added to each hole at -80 ° C for 30 minutes, and then removed at room temperature for 30 minutes. After centrifugation at 12000 r.pm for 30 minutes, remove 80 μl of the upper suspension and place it in a 96-well plate. Add 1 mg / ml dopa solution to each well, and measure the OD ₄₉₀ absorbance. Measure every 10 minutes for one hour. Finally, calculate the tyrosinase inhibition rate.

Inhibition rate formula (%) = (A-B) / A × 100%

A: OD ₄₉₀ absorbance of the control group

B: OD ₄₉₀ absorbance of sample group

(3) Experimental results

Figure 3 shows the analysis results of intracellular tyrosinase activity. The supercritically extracted lees extract has a better tyrosinase inhibition rate. When the concentration of total polyphenols in the lees is 20ppm and 30ppm, the inhibition rate reaches More than 70%. From the experimental results, it can be known that the sample of lees extract can be used for the whitening effect.

Example 6 Analysis of Melanin Content

(1) Analysis principle of melanin content

High temperature and strong alkali were used to dissolve the melanin in the cells, and the specific absorption wavelength was measured at 405nm to analyze the effects of various conditions on the melanin content in B ₁₆ F ₁₀ melanoma cells.

(2) Analysis method of melanin content

5 × 104 cells were implanted in a 24-well culture plate, cultured in a medium containing 100 nM α- MSH, and cultured for 24 hours (in a 37 ° C., 5% CO 2 incubator). Thereafter, the medium containing distiller's grains SFE or osmic acid with different concentrations was replaced (the control group only changed the medium), and cultured for another 24 hours. Then remove the medium, wash with PBS twice, add 100 μl 1N NaOH (10% DMSO) and react at 60 ° C for 1 hour. Finally, take 80 μl to a 96-well plate to measure the absorbance at 405nm and calculate the melanin Relative content.

(3) Experimental results

Figure 4 is the analysis result of melanin content. Water extract, alcohol extract and supercritical extraction of distiller's grains extraction liquid samples all have the effect of reducing melanin production. Among them, supercritical extraction of lees total polyphenols at 30ppm final concentration has better results. . From the experimental results, it can be known that the sample of lees extract can be used for the application of scars and spots.

Claims (7)

An extraction method of sorghum lees extract, using Jinmen sorghum lees as raw material, using ethanol / carbon dioxide supercritical fluid as a co-solvent, first statically extracting sorghum lees raw materials with ethanol / carbon dioxide supercritical fluid for 2 hours, and then using ethanol / The carbon dioxide supercritical fluid is continuously and dynamically extracted for 2 hours. The moisture content of the dried sorghum grains used is less than 30%, the operating pressure is between 150 bar and 350 bar, and the temperature is between 40 ° C and 60 ° C. For example, the extraction method of sorghum distiller's grains extract according to item 1 of the patent scope, wherein the optimal conditions for the supercritical fluid extraction are an operating pressure of 170 bar, a temperature of 40 ° C, an operating pressure of 170 bar, a temperature of 50 ° C, an operating pressure of 170 bar, and a temperature of 65 ° C. The operating pressure is 250 bar and the temperature is 40 ° C, and the operating pressure is 300 bar and the temperature is 40 ° C. For example, the extraction method of the sorghum lees extract of the scope of patent application, wherein the extract has antioxidant activity, ability to inhibit tyrosine glutamate, and ability to reduce melanin content, which can be used as a cosmetic or medicinal combination Additive uses. For example, the method for extracting sorghum lees extract from patent application No. 3, wherein the extract is used as an additive for preparing a cosmetic or medicinal composition to prevent skin aging, whitening, whitening, pale spots or bright skin . For example, the extraction method of sorghum lees extract of the scope of patent application No. 4, wherein the extract is used as an additive for preparing a beauty or medicinal composition based on 0.001 weight of the total polyphenols based on the lees of the composition It is present in an amount from% to 0.02% by weight. For example, the extraction method of sorghum lees extract of the scope of patent application No. 5, wherein the extract is used as an additive for preparing a beauty or cosmeceutical composition, wherein the extract is based on the total weight of the total polyphenols in the lees of the composition The amount of 0.001% to 0.003% by weight has better cytotoxicity, anti-oxidation ability and whitening ability. For example, the extraction method of sorghum lees extract according to item 6 of the patent application, wherein the extract is used as an additive for preparing a beauty or cosmeceutical composition, and the extract extracted with supercritical fluid is based on the total lees of the composition An amount of 0.003% by weight of the total polyphenol has the best cytotoxicity, anti-oxidation ability and whitening ability.