TW201912177A - Taiwan clam shell extracting substance with whitening and anti-aging function and its extraction and separation method - Google Patents

Abstract

The present invention relates to an extraction method of Chenopodium formmosanum hull beneficial for whitening and anti-aging, in which a composition of the extract obtained by extraction and purification can be used in anti-oxidation, whitening or anti-aging application. The method includes the following steps: obtaining a Chenopodium formmosanum hull, and sequentially performing alcohol extraction, filtration and drying so as to produce a preliminary extract; and selectively performing a purification step and a separation step on the aforementioned preliminary extract to separate the components of different nature of the preliminary extract or to directly apply the extract to the product.

Description

Taiwan quinoa shell extract with whitening and anti-aging functions and its extraction method

The invention is a method for extracting Taiwan quinoa shell, especially a Taiwan quinoa shell extract obtained by using alcohol extraction for concentration and purification. The extract can be subsequently separated by chromatography, which can be helpful by applying to the skin or eating the extract. For whitening and anti-aging.

Chenopodium formmosanum, also known as red quinoa, red quinoa, purple quinoa, and rainbow rice, is a Taiwan native plant of the genus Chenopodium and subfamily Chenopodiaceae. It is an annual herb and is a Taiwanese aboriginal tradition for more than 100 years. The growth characteristics of crops are drought tolerance and high salt tolerance. Traditionally, the aborigines traditionally used hulled Taiwan quinoa grains and cooked them with rice and other miscellaneous grains to cook rice dumplings, bamboo rice, and rice wine. Taiwan quinoa plants are edible, and their seedlings, tender leaves and flower spikes are used in vegetables. In addition, in terms of Chinese herbal medicine, Taiwan quinoa plants can be used as medicines, which have functions of dehumidifying, clearing heat, detoxifying, stopping diarrhea and improving skin itching. Taiwan quinoa is rich in excellent and comprehensive nutrients, minerals and various antioxidants. It is a nutrient-rich food and is known as the "grain ruby".

The functional components of Chenopodium sibiricum are mainly flavonoids, phenols, and beet pigments, and they all have antioxidant, anti-inflammatory, and anti-cancer physiological activities. It has many anti-oxidant effects, can resist inflammation, inhibit arteriosclerosis and thrombosis, lower blood pressure, maintain vascular elasticity, promote insulin secretion, inhibit cancer cell proliferation, etc., and can prevent aging and maintain skin elasticity. Rich in quinoa in Taiwan Flavonoids also help to scavenge free radicals, prevent oxidation, help to soften blood vessels, and promote sugar, lipid metabolism and insulin secretion.

Most of the grains of Taiwan Chenopodium will remove the reddish-brown shell after harvesting. Because the Taiwan Chenopodium shell contains the slightly toxic "saponin", it will cause the Taiwanese Chenopodium to be eaten with a bitterness and poor taste. There are restrictions on the intake of Taiwan quinoa, so the use of Taiwan quinoa is very limited. In the past, it was found that Taiwan quinoa husk accounts for about 27% of the total seeds. Removing Taiwan quinoa husk will lose nearly 30% of agricultural output. How to effectively use Taiwan quinoa has become an important issue to be solved.

It is inherited that Taiwan quinoa is an excellent miscellaneous food product. It is a waste to extract and use edible parts. If the inedible Taiwan quinoa shell can be used alone, it will be able to use resources more effectively. Therefore, the present invention uses Taiwan quinoa shell to The method of alcohol extraction purifies the extract of Taiwan quinoa which helps skin whitening, anti-aging and anti-wrinkle.

Skin In order to protect the skin from ultraviolet rays in the sun, melanocytes in the skin will synthesize melanin to resist ultraviolet rays and prevent ultraviolet rays from causing damage to cells and genetic material. Past research has pointed out that tyrosinase is a key enzyme that regulates melanin production, not only participates in many reactions in the process of melanin production, but also is a key enzyme that catalyzes the rate limiting step in the reaction. Therefore, the content of tyrosinase can be used as melanin Generate an indicator of the response. As the increase in melanin production will lead to dull skin tone and make people look lacking in spirit, how to maintain skin to slow down melanin production or restore fair skin has become the direction pursued by many consumers.

There are many whitening agents used in cosmetics and medicine (such as hydroquinone and mercury), which can cause abnormal skin pigmentation after use, such as allergic reactions, redness of the skin, skin rash, discoloration, and reduction of bacteria and fungi. Side effects such as the resistance of infected skin can even cause kidney toxicity, so researchers are constantly looking for safe and effective natural products to develop whitening products. Natural substances such as kojic acid, ellagic acid, and arbutin, which are confirmed to have whitening effects through research, are all tyrosinase inhibitors. Although these substances do have significant effects on whitening, they each have their use restrictions. For example, osmic acid is very unstable and easily oxidized to cause discoloration and skin irritation. And long-term excessive use of osmic acid products can cause cytotoxicity and occur. Lesions; in addition, the role of some whitening agents is to directly destroy melanocytes, which is likely to cause cell toxicity. Therefore, how to find effective ingredients that can suppress melanin and have safety has become an important issue.

Aging is a phenomenon in which the physiological state deteriorates over time, and aging is a process of biological aging. The occurrence of aging can be observed from changes in molecules such as genetic material, proteins, lipids and other molecules. This kind of molecular damage causes many diseases in old age. In fact, there are many self-repairing mechanisms of free radical damage in the body. However, when the repair speed is not as fast as the free radical damage, the damaged molecules will easily accumulate and further cause disease. As the age increases, the probability of occurrence increases. Symptoms, including skin aging, arthritis, cancer, Alzheimer's disease, etc. Regarding aging, the most popular theory is the "free radical theory". The theory assumes that aging results from the accumulation of changes in the body caused by reactions initiated by highly active molecules called "free radicals." It is now widely believed that changes induced by free radicals are the main cause of aging and disease development. During normal physiological metabolism, free radicals are mostly generated from the physiological upper mitochondrial electron transport chain, peroxidase, and cytochrome P-450, and energy is produced by metabolism. Provide biological applications; in fact, there are many self-repairing mechanisms for free radical damage in the body to maintain metabolic balance. However, when the repair speed is not as fast as the free radical damage, the damaged molecules will easily accumulate and further cause aging. Or disease. Increasing age, decreased metabolism, or affected by abnormal environments, such as ultraviolet rays, smoking, air pollution, and even psychological stress and abnormal life may tilt the balance scale of free radicals toward aging.

It further illustrates that free radicals cause damage to biological genetic material and increase the chance of genetic mutation, which in turn increases the incidence of cancer. Similarly, the damage of free radicals to lipid molecules can lead to the over-oxidation of low-density cholesterol (LDL) to produce arterial plaques, which in turn may cause atherosclerosis and other cardiovascular diseases. In addition, free radicals can cause damage to protein molecules resulting in cross linking, amyloid formation, and changes in brain cells, which in turn can cause Alzheimer's disease or increase the incidence of Alzheimer's disease. Damage of free radicals to protein molecules can also cause changes in the tissue structure of arthritis and autoimmune disorders, as well as damage to collagen and elastic fibers that affect skin function and appearance. Therefore, how to prevent oxidation, reduce free radicals in the body, achieve disease prevention, and delay cell aging is a major development focus of contemporary medical biotechnology.

Glycation, on the other hand, refers to the attachment of glucose or fructose to a protein, which changes the structure and properties of the glycosylated protein, making it easier to aggregate and cross-link. Saccharification occurs randomly, and it is generally believed that a high concentration of free radicals will cause a higher probability of saccharification. At present, it is believed that the final glycated proteins (Advanced Glycation End-products, AGEs) generated by excessive saccharification of proteins are closely related to aging and some diseases relationship. When glycosylation in the body produces too much final glycated protein, it is easy to cause different lesions in various parts, such as eyes may be easily fatigued, dry and even increase the risk of cataract and retinopathy; the final glycated protein produced by glycation will also accumulate on the vessel wall It induces the generation of free radicals in the body, accelerates the hardening of arteries, and the thickening of the tube wall. When the final glycated protein accumulates in the bones, the bones become fragile, the cell regeneration mechanism is inhibited, and the condition of osteoporosis is accelerated. In addition, according to research, the protein in the frontal lobe of the brain of Alzheimer's patients is more than three times the final glycated protein compared to healthy elderly, indicating that the accumulation of final glycated protein in the body is relevant for many diseases.

Glycation occurs on proteins in the dermal layer of the skin and is considered to be one of the important mechanisms for skin aging and relaxation. Collagen (Collagen) is an important protein in the human body, accounting for about 25 to 35% of the total protein. It is mainly present in connective tissue, is a scaffold between cells, has a strong ability to stretch, and is widely present in skin, cornea, joints, Cartilage and other tissues. Especially in the skin, collagen is the main supporting protein, keeping the skin firm and plump. The saccharification of collagen will make the collagen fibrosis and become stiff, the skin will lose its filling and accelerate the generation of skin wrinkles.

In addition, elastin (Elastin) is a protein constituting elastic fibers, which is widely distributed in various tissues and organs in the body, mainly in elastic tissues such as skin, ligaments, blood vessels, lungs, and skin. Not only does this type of tissue require elasticity, but tensile strength is also essential, and elastin plays an important role in morphological composition and maintenance. Elastin is also an important component of skin composition. Elastin contains only 5% in the skin, but it is the key to producing elastic fibers and supporting collagen. Elastin molecules can be curled arbitrarily. Under the pull of an external force, the curled elastin molecules stretch and stretch. After removing the external force, the elastin molecules return to a curled state, just like a spring. Human elastin is mainly synthesized from the late embryo to the early neonatal period, and almost no new elastin is produced in the adult stage. Collagen, which accounts for 70% of the skin, determines the suppleness and fullness of the skin, while elastic fibers determine the firmness and elasticity of the skin. The same saccharification and photoaging processes will also cause the elastic fibers to undergo qualitative changes and lose their original elasticity. Elastase is an enzyme that can break down elastin in connective tissue. Decreased elastin can cause signs of aging such as wrinkles and sagging. Therefore, how to reduce the reduction and destruction of elastin is an anti-aging, anti-wrinkle, and maintaining skin health and firmness, which is a topic worthy of attention.

In summary, supplementing collagen or enhancing the collagen production capacity of cells, and inhibiting the degradation of elastin are important methods to prevent or slow skin aging, and can prevent related diseases derived from the accumulation of glycated proteins. At present, there are collagen-related products on the market, and the main function is to directly supplement collagen. However, the large molecular weight of collagen makes it difficult to apply collagen through the stratum corneum of the skin to the skin fibers of the dermis. Mother cells; if collagen is directly replenished by injection, although the fine lines of the skin can be reduced immediately, the collagen injected into the body is easily broken down by enzymes in the body, which requires regular application. The process is complicated and costly, and The immune rejection that can be caused by collagen injection is a big risk. If collagen is supplemented by eating foods rich in collagen, collagen enters the stomach and intestines, is cut into small molecules through digestive juices, and finally breaks down into amino acids. Although the body uses these amino acids to synthesize proteins, it is not limited to collagen, so the actual effect of collagen supplementation is limited. On the other hand, elastin products can not be replaced by the same method of elastin or fiber; the study found that the elastin in the body can hardly be regenerated, and the existing in vitro culture technology that is still in the research stage produces elastin structure and then implants it. In vivo, but not yet technically mature. Therefore, inhibition of elastase activity has become one of the few important ways to avoid elastin reduction and skin sag.

The main feature of the invention is that the composition obtained after the extract is extracted and purified can be used for anti-oxidation, whitening or anti-aging of the skin.

Another main feature of the present invention is that it comprises the following steps: taking Taiwan quinoa shell, sequentially performing alcohol extraction, filtering, and drying to produce a preliminary extract; the preliminary extract may be further purified or differentiated to make the preliminary extract Ingredients with different properties are used separately or directly in the product.

Take the Taiwan quinoa shell, clean it, dry it and grind it into powder physically; take the Taiwan quinoa shell powder into alcohol and concentrate to obtain a preliminary extract.

More preferably, after taking Taiwan quinoa powder and adding alcohol to concentrate, the initial extract is dissolved in water, and the organic solvent is added for layered extraction several times to remove the interferences in the oil phase; then the aqueous phase extract is taken. Concentrate to reduce the organic solvents that may remain in the extract to obtain a purified extract. The purified and extracted Taiwan quinoa alcohol extract (EEDH) can be used directly, or the chromatography can be differentiated according to the requirements, and the different conditions can be used according to the requirements. The method of differentiation can be, but not limited to, chromatography. The organic solvent for the layered extraction may be ether, dichloromethane, chloroform, carbon tetrachloride, benzene, isoamyl alcohol, n-butanol, n-pentane, and n-hexane.

For a preferred embodiment, take Taiwan quinoa powder, dissolve it in 95% alcohol, and concentrate it four times to obtain a preliminary extract. After filtering the preliminary extract and removing alcohol, re-dissolve the extract with secondary water; N-hexane was added for layered extraction several times to remove oil phase material. Take the aforementioned phase-extracted aqueous phase extraction solution (EEDH) for column chromatography and obtain the water (EEDH-W), 50% alcohol (EEDH-50), and 95% alcohol (EEDH-95) ), And subsequent experiments and analysis with Taiwan quinoa alcohol extract (EEDH) without column chromatography, the results are shown in Figure 2-1

Figure 2-1 is an extracellular elastase activity inhibition test. The test was performed with the 95% alcohol fraction of Taiwan quinoa, and the results showed that the 95% alcohol fraction of Taiwan quinoa was at a concentration of 0.25-4.0 mg / mL. It has significant ability to inhibit elastase activity with a dose effect. IC50 is about 1.0 mg / mL, and its inhibitory effect at 4.0 mg / mL is equivalent to 0.66 mg / mL (3.75 mM) ascorbic acid (inhibition rate is about 95%). It shows that the 95% alcohol fraction of Taiwan quinoa has the potential to develop whitening and anti-wrinkle compositions.

From the study results in Figure 2-2, it was found that in the system that induced skin fibroblasts (HaCat) with ultraviolet light (UVB), at a concentration of 1000 μg / mL, it was found that Taiwan quinoa alcohol extract (EEDH), 50% Ethanol classifiers (EEDH-50) and 95% alcohol classifiers (EEDH-95) have less than 40% inhibition of elastin degrading enzyme activity, while the water classifier (EEDH-W) of Taiwan quinoa alcohol extract inhibits It has the best ability and has a significant dose effect. It has an inhibitory capacity of about 81% and an IC50 of about 500 μg / mL.

Culture melanoma cells (B16F10) to test the melanin inhibition ability by applying different reagents to evaluate the whitening ability of the present invention, Taiwan quinoa alcohol extract and its differentiation, and induce melanoma cells with 1 μM α-melanocyte stimulating hormone (α-MSH) Influence of the ability to form melanin. The test results are shown in Figure 3 with a microscope magnification of 100X.

The results in Figure 3 show that culture of α-melanocyte stimulating hormone and melanoma cells for six days significantly promoted melanin formation, and 200 μM Kojic acid had a significant tendency to inhibit melanin formation. Taiwan quinoa wine extract (EEDH) at a concentration of 50-200 μg / mL, the amount of melanin formation significantly decreased, and its distinguishing 50% alcohol (EEDH-50) and 95% alcohol (EEDH-95) There is also a significant downward trend.

Based on the concentration of the Taiwan quinoa alcohol extract and its differentiators, it was divided into five groups: 50, 100, 200, 500, and 1000 ug / mL to test the cell survival rate of melanocytes. The safety of the extract is shown in Figure 4, which shows that Taiwan Quinoa wine extract (EEDH), water (EEDH-W) and 50% alcohol (EEDH-50) have no significant cytotoxicity at 200 μg / mL, and the cell survival rate is> 85%. In the subsequent experiments, the sample was selected to be under 200 μg / mL, and the 95% alcohol fraction (EEDH-95) had very significant cytotoxicity at 200 μg / mL. Therefore, the concentration below 100 μg / mL was selected to inhibit melanin Tumor cells for tyrosinase activity and melanin formation test.

Next, the effects of alcohol extracts from Taiwan quinoa and their differentiators on the tyrosinase activity and melanin formation of melanoma cells induced by 1 μM α-melanocyte stimulating hormone were further analyzed. Figures 5 and 6 show that Taiwan quinoa extract (EEDH) at a concentration of 200 μg / mL inhibits tyrosinase activity and melanin formation ability of approximately 60% and 50%, respectively. All three distinguishers have significant inhibition of tyrosinase activity and melanin formation ability. , Its effect is better than 200 μM of osmic acid. Among them, the inhibitory effect of the 50% alcohol classifier (EEDH-50) is the best, showing a significant dose effect. The inhibition of tyrosinase activity and melanin formation ability at a concentration of 200 μg / mL can reach about 84% and 86% inhibition effect.

To further analyze the anti-aging and anti-glycation ability of Taiwan quinoa alcohol extract and its fractions, the effects of free radical scavenging ability and final glycosylated protein (AGEs) formation ability were tested in two experiments. Evaluation of the antioxidant power and anti-glycation ability of the material.

The ability to scavenge free radicals, as measured by the hydrogen supply capacity, is expressed by the scavenging effect of 1,1-diphenyl-2-trinitrophenylhydrazine (abbreviated as DPPH). Take 4 ml of the extract, add 1 mL of freshly prepared methanol solution of 1.0 × 10-4 mM DPPH, and mix uniformly. After standing for 30 minutes, check the absorbance at 517 nm. The results are shown in Figure 7.

The results shown in Figure 7 show that the water and 50% alcohol fractions of Taiwan quinoa alcohol extract and Taiwan quinoa alcohol extract have a very good ability to scavenge DPPH free radicals, except for the 95% alcohol fraction, both present Significant concentration inhibition effect. Among them, the 50% alcohol fraction (EEDH-50) has the best effect (IC50 is about 1.5 mg / mL), followed by the water fraction (EEDH-W), At a concentration of 2 mg / mL, the Taiwanese quinoa alcohol extract (EEDH) had DPPH clearance rates of 66.7, 45.6, and 33.5% in order.

The effect of Taiwan quinoa shell alcohol extract and its fraction on the final glycosylated protein formation ability, 10 μL of the extract was added to 740 μL of 50 mg / mL bovine serum albumin BSA (dissolved in phosphate buffer containing 0.05% sodium azide Normal saline) and 250 μL of 3M glucose were mixed uniformly. After reacting at 37 ° C for 3 weeks, the fluorescence value was measured using a fluorescence detector at an excitation wavelength of 330 nm and an emission wavelength of 410 nm. The results are shown in FIG. 8.

From the results in Figure 8, it can be seen that the alcohol extract of Taiwan quinoa has only about 30% ability to inhibit the formation of the final glycosylated protein at a concentration of 1000 μg / mL, and the 50% alcohol classifier (EEDH-50) has the best performance. The inhibitory effect is about 65%, and its ability to inhibit the final glycosylated protein formation at 500 μg / mL is equivalent to 1 mM of the known glycosylation inhibitor Aminoguanidine. However, the other differentiations did not significantly inhibit the final glycated protein formation ability.

Based on the above results, Taiwan quinoa alcohol extract (EEDH) has significant inhibitory effects in scavenging free radicals, inhibiting saccharification, and evaluating the activity of extracellular tyrosinase and extracellular elastin degrading enzymes. The fractions obtained after column chromatography were significantly improved in various evaluations of antioxidant, whitening and anti-aging capabilities. Among them, the 50% alcohol classifier (EEDH-50) has the best antioxidant and anti-glycation and whitening ability; however, it is distinguished by 95% alcohol classifier (EEDH-95) and water in different models of extracellular and skin cell systems. (EEDH-W) is the best. Subsequent products can be selected as suitable raw materials according to the use and efficacy requirements.

Claims (8)

A Taiwan quinoa alcohol extract, the composition obtained after extraction and purification can be used for anti-oxidation, whitening or anti-aging applications. As described in claim 1 of the scope of patent application, the alcohol extract of Chenopodium taiwanensis, wherein the composition is applicable to the raw materials for external wipes, external medicines, external care products or cosmetics. As described in item 1 of the patent application scope, the Taiwan quinoa alcohol extract, wherein the composition is applicable to the raw materials of food or medical care products. As described in the first patent application scope, the Taiwan quinoa shell alcohol extract, wherein the extraction is from Taiwan quinoa shell, followed by alcohol extraction, filtration and drying to produce a preliminary extract. As described in item 1 of the patent application scope, the Taiwan quinoa alcoholic extract, wherein the purification is a step of further purification or differentiation after extraction, so that components of different properties of the preliminary extract are separated or used directly in the product. A method for extracting Taiwanese quinoa shell alcohol extract includes the following steps: Extraction step: taking Taiwan quinoa shell, sequentially performing alcohol extraction, filtering and drying to produce a preliminary extract. Purification step: The aforementioned preliminary extract can be optionally subjected to further purification or differentiation steps to separate or directly use different components of the preliminary extract from the product. The method for extracting alcohol extract from Taiwan quinoa, as described in item 4 of the scope of patent application, wherein the purification step is to separate unwanted interferences through layered extraction. The method for extracting the alcohol extract of Taiwan quinoa, as described in item 4 of the scope of patent application, wherein the distinguishing step is separating and concentrating the effective ingredients by chromatography, and the chromatography liquid of the column chromatography is alcohol. Its distinguishing concentration is 50-95%.