Application of stevia rebaudiana extract

Abstract

The invention provides application of a stevia rebaudiana extract, and belongs to the technical field of pure natural plant extracts, the stevia rebaudiana extract at least contains phenols, flavones and potassium ions, and when a health care product and / or a composition are given to a subject, at least one of the following components is achieved: antioxidant activity, antioxidant activity, antioxidant activity, antioxidant activity, antioxidant activity, antioxidant activity, antioxidant activity and the like. The expression of IRS protein, tyrosine phosphorylation of IRS and the content of GLUT4 protein are increased; bacteria are inhibited, and viruses and allergy are resisted; the intraocular pressure is reduced; the appetite is promoted; the hair loss is prevented or the hair growth is promoted. The application comprises application of the stevia rebaudiana extract in preparation of hair loss preventing or hair growth promoting products. The health-care product containing the stevia rebaudiana extract can prevent or treat type 2 diabetes mellitus, inhibit bacteria or resist viruses or allergy, and can be used as auxiliary food and / or sports food for sub-health people, type 2 diabetes mellitus patients, patients receiving glaucoma treatment, cancer patients, hepatitis patients, overweight people, picky and anorexia children or patients during general disease rehabilitation. The hair loss can be prevented or the hair growth is promoted.

Description

[0001] This case is a divisional application of the Chinese invention patent application with the application date of "2019.09.16", application number "201910872847.1", and invention name "Health products containing stevia extract and their uses". Technical Field

[0002] The invention belongs to the technical field of pure natural plant extracts, and in particular relates to the use of stevia extracts. Background Art

[0003] Stevia( Stevia rebaudiana ) belongs to the Asteraceae family. Its species name is Stevia, also known as Stevia leaf and sweet grass. It is called sugar grass in Brazil and sweet grass and sweet tea in Paraguay. It is one of the sweetest sugar plants known so far. Stevia is native to eastern Paraguay in South America. It is a natural herb with high sweetness, low calories, non-toxicity and no side effects. Stevioside extracted from its leaves has long been widely used as a natural sweetener. Its sweetness is 300 times that of sucrose, and its heat energy is only 1 / 90 of that of sucrose. It is safe to eat and is a highly respected natural sugar source. Stevia is sweet and flat in nature. It is not only rich in steviol glycosides, but also contains sterols, flavonoids, alkaloids, water-soluble chlorophyll, lutein, o-hydroxycinnamic acid, neutral water-soluble oligosaccharides, free sugars, amino acids, lipids, volatile oils and trace elements. These ingredients make the extract of stevia leaves have multiple biological activities such as antioxidant (anti-free radical), anti-allergic, antibacterial (anti-inflammatory), analgesic and liver protection. It has been widely used in various industries such as food, beverages, and medicine. Summary of the invention

[0004] One object of the present invention is to provide a health product and / or composition, which can improve the body's immunity and improve the potential pre-type 2 diabetes population and the population that has already become type 2 diabetes patients; has antibacterial, antiviral and anti-allergic effects on a variety of bacteria, viruses or histamine; and has obvious effects of weight loss, refreshing, promoting normalization of gastrointestinal function and increasing appetite.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A health product and / or composition comprises a stevia extract, wherein the stevia extract comprises at least phenols, flavonoids, and potassium ions, wherein when the health product and / or composition is administered to a subject, at least one of the following is achieved: antioxidant activity; increased expression of IRS protein, tyrosine phosphorylation of IRS, and GLUT4 protein content; inhibition of bacteria, antiviral, and anti-allergy; reduction of intraocular pressure; increased appetite; and hair growth.

[0006] According to one embodiment of the present invention, the stevia extract has a phenol content of 0.46-126.82 mg / g, a flavonoid content of 0.08-1.56 mg / g, and a potassium ion content of 0.77-37.24 mg / g.

[0007] According to one embodiment of the present invention, the stevia extract contains 0.073-4.17 mg / g 3-allyl-6-methoxyphenol.

[0008] According to one embodiment of the present invention, the stevia extract further contains crude polysaccharides with a concentration of 5 to 4650 ppm.

[0009] According to one embodiment of the present invention, the concentration of the stevia extract is ≥ Brix5.

[0010] According to one embodiment of the present invention, the health care product is prepared into a concentrated or ordinary drink; or, the health care product is prepared into a jelly; or, the health care product is prepared into a powder, and the powder is prepared into an instant tea, a hard capsule or a soft capsule.

[0011] The present invention also provides use of the health care product and / or the composition in preparing a product for preventing or treating type 2 diabetes.

[0012] The present invention also provides the use of the health care product and / or the composition in preparing a product for inhibiting bacteria, resisting viruses or resisting allergies.

[0013] The present invention also provides the use of the health care product and / or the composition in preparing supplementary food and / or sports food for sub-healthy people, type 2 diabetes patients, patients receiving glaucoma treatment, cancer patients, hepatitis patients, overweight people, picky and anorexic children, or patients recovering from general diseases.

[0014] The present invention also provides use of the health care product and / or the composition in preparing a hair loss prevention or hair growth product.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The stevia extract of the present invention has super strong antioxidant activity, can improve the immunity of the body, promote the health of the body, meet the health needs of the body, especially has a significant improvement effect on the health of people in oxidative stress state; can improve the resistance to insulin, by increasing the expression of IRS protein, strengthening IR, improving the tyrosine phosphorylation of IRS, strengthening GLUT4 translocation and activating the transmission of insulin signals in cells, thereby effectively reducing blood sugar, and then improving the potential pre-type 2 diabetes population and the type 2 diabetes population; has antibacterial and antiviral effects on a variety of bacteria or viruses or histamine; the polysaccharides, polyphenols and trace elements contained are essential elements for human health; has obvious weight loss, refreshing, promoting the normalization of gastrointestinal function and increasing appetite at the same time. The health care product of the present invention can be used for sub-healthy people, type 2 diabetes patients, patients receiving glaucoma treatment, cancer patients, hepatitis patients, overweight people, picky and anorexic children, and supplementary foods and / or sports foods for patients during the recovery period of general diseases.

[0016] The present invention adopts the above technical scheme to provide a health product containing stevia extract and its use, which makes up for the shortcomings of the prior art, has reasonable design and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a comparison of the DPPH free radical scavenging ability of the four components I, II, III, and IV in Example 3 of the present invention; Figure 2 is the GC-MS characteristic spectrum of component IV in Example 3 of the present invention; Figure 3 is the measurement result of the fasting blood glucose value of rats in Example 5 of the present invention; Figure 4 is the measurement result of glucose tolerance of rats in Example 5 of the present invention; Figure 5 is the measurement result of the IRS-2 protein expression in Example 5 of the present invention; Figure 6 is the measurement result of IRS-2 protein phosphorylation in Example 5 of the present invention; Figure 7 is the measurement result of GLUT4 protein content in Example 5 of the present invention; Figure 8 It is the score of the growth of new hair in the depilatory area of ​​the mouse in Example 8 of the present invention. DETAILED DESCRIPTION

[0018] The embodiments of the present invention are described in detail below.

[0019] The present invention allows various modifications and variations, and its specific embodiments are given as examples and described in detail below. However, it is not intended to limit the present invention to the specific form disclosed. On the contrary, the present invention includes all modifications, equivalents and substitutions consistent with the concept of the present invention defined by the claims.

[0020] These embodiments are only used to illustrate the present invention in more detail. According to the gist of the present invention, the scope of the present invention is not limited to these embodiments, which is self-evident to those skilled in the art.

[0021] One embodiment of the present invention provides a health product and / or composition, comprising a stevia extract, wherein the stevia extract contains at least phenols, flavonoids, and potassium ions, wherein when the health product and / or the composition is administered to a subject, at least one of the following is achieved: antioxidant activity; increased expression of IRS protein, tyrosine phosphorylation of IRS, and GLUT4 protein content; inhibition of bacteria, antiviral, and anti-allergic effects; lowering of intraocular pressure; increased appetite; and hair growth. The stevia extract used in this embodiment has the following effects: it has super strong antioxidant activity, can improve the body's immunity, promote the body's health, meet the body's health needs, and especially has a significant improvement effect on the health of people in a state of oxidative stress; it can improve insulin resistance by increasing the expression of IRS protein, strengthening IR, improving IRS tyrosine phosphorylation, strengthening GLUT4 translocation and activating the transmission of insulin signals in cells, thereby effectively lowering blood sugar, and thus improving the potential pre-type 2 diabetes population and the population that has already become type 2 diabetes patients; it has antibacterial and antiviral effects on a variety of bacteria, viruses or histamine; the polysaccharides, polyphenols and trace elements contained are essential elements for human health; it has obvious effects of weight loss, refreshing, promoting normalization of gastrointestinal function and increasing appetite, which can help children improve picky eating habits, increase nutrition and promote development; in addition, the increase in appetite and the improvement of immunity are beneficial to tumor treatment and have an inhibitory effect on the growth of residual cancer tissue.

[0022] In one embodiment of the present invention, the stevia extract has a phenol content of 0.46-126.82 mg / g (e.g., 4.6 mg / g, 10.55 mg / g, 22.00 mg / g, 30.47 mg / g, 38.75 mg / g, 46.02 mg / g, 50.00 mg / g, 70.50 mg / g, 95.00 mg / g, 105.23 mg / g, 120.38 mg / g, etc.), and a flavonoid content of 0.08-1.56 mg / g (e.g., 0.10 mg / g, 0.14 mg / g, 0.4 The content of potassium ion is 0.77~37.24mg / g (for example, 2.27mg / g, 5.15mg / g, 12.40mg / g, 17.24mg / g, 22.71mg / g, 23.00mg / g, 25.03mg / g, 30.00mg / g, 33.20mg / g, 35.48mg / g, etc.). Preferably, the phenolic component is one or more of 3-allyl-6-methoxyphenol, 2,4-bis(1,1-dimethylethyl)phenol, catechol, thymol, 4-chloro-2,6-bis(1,1-dimethylethyl)phenol, 4-ethylphenol, propofol, 4-(1-methyl-1-cyclobutyl)phenol, 3-methyl-4-isopropylphenol, and 2,5-bis(1-methylpropyl)phenol.

[0023] In one embodiment of the present invention, the stevia extract contains 0.073-4.17 mg / g 3-allyl-6-methoxyphenol, such as 0.12 mg / g, 0.57 mg / g, 0.73 mg / g, 1.02 mg / g, 1.64 mg / g, 1.86 mg / g, 2.32 mg / g, 3.07 mg / g, 3.75 mg / g, 3.98 mg / g, etc. 3-allyl-6-methoxyphenol has strong antioxidant activity, and can play a complementary role with catechol in the antioxidant active ingredient, further improving the antioxidant activity of the obtained stevia extract.

[0024] In one embodiment of the present invention, the stevia extract also contains crude polysaccharides at a concentration of 5 to 4650 ppm. For example, 228 ppm, 534.2 ppm, 840 ppm, 1148 ppm, 1683.4 ppm, 2280 ppm, 2295.5 ppm, 2300 ppm, 2875 ppm, 3000 ppm, 3260 ppm, 4500 ppm, etc. Stevia polysaccharides have six isomers, and crude sugar contains any type of stevia polysaccharides, and stevia polysaccharides have almost no antioxidant activity. However, as long as the content of this crude polysaccharide reaches more than 5 ppm, it will work together with the antioxidant active substances phenols, flavonoids and potassium ions in this antioxidant, and can show an inhibitory effect on type 2 diabetes.

[0025] In one embodiment of the present invention, the concentration of stevia extract is ≥ Brix5. The above stevia extract also has the effect of lowering blood sugar. More preferably, when the concentration Brix of the above stevia extract is 5-8, it has the effect of lowering blood sugar, but it is not very obvious and has a Chinese medicine taste; when the concentration Brix of the above stevia extract is 8-12, it has the effect of lowering blood sugar, and the individual difference between people is obvious, the overall effect is good, and the Chinese medicine taste is slightly strong; when the concentration Brix of the above stevia extract is 12-16, the effect of lowering blood sugar is obvious, and the Chinese medicine taste is strong; when the concentration Brix of the above stevia extract is 17-23, the effect of lowering blood sugar is obvious, and the Chinese medicine taste is very strong; generally, drinking 20-40mL a day, blood sugar can be restored from 10.8 or higher to about 7 in 3 months (there are individual differences), the effect is obvious, the effect is better than metformin and there is no side effect.

[0026] In one embodiment of the present invention, the preparation method of stevia extract comprises the following steps: 1) adding stevia raw material into the extraction solution to perform extraction; 2) mixing and concentrating the extracts obtained after step 1); c) fermenting the concentrated liquid obtained after step 2); and 4) Purifying and concentrating the fermentation liquid obtained after step 3) to obtain stevia extract.

[0027] In one embodiment of the present invention, the stevia raw material is in powder or granular form or in truncated rod or original branch form.

[0028] In one embodiment of the present invention, the stevia raw material contains 0-100wt% of stevia stems and 0-50wt% of stevia leaves, for example, 80wt% of stevia stems and 20wt% of stevia leaves, 95wt% of stevia stems and 5wt% of stevia leaves, 99.5wt% of stevia stems and 0.05wt% of stevia leaves, 100wt% of stevia stems, etc.

[0029] In one embodiment of the present invention, the extraction in step 1) is performed by hot water ultrasonic extraction, and the extraction process conditions are: the solid-liquid ratio is 1:6-30 (for example, 1:7, 1:8.5, 1:15, 1:20.6, 1:28, etc.), the water bath extraction temperature is above 90˚C (for example, 91˚C, 95.5˚C, 97˚C, 98.5˚C, etc.), the water bath extraction time is 100-160min (for example, 110min, 125min, 135.5min, 150min, etc.), and the ultrasonic-assisted extraction time is 15-25m in (e.g. 16min, 18min, 20.5min, 24min, etc.), the ultrasonic frequency is 15kHz-60kHz (e.g. 18kHz, 35.5kHz, 58kHz, etc.), the ultrasonic intensity is 80-200W (e.g. 85W, 120W, 185W, etc.); stevia waste is rich in bioactive substances with strong antioxidant ability, and ultrasound assistance can improve the extraction rate of antioxidant active substances. Under this condition, the scavenging rate of the obtained extract for DPPH is more than 90%, and the antioxidant stability is good.

[0030] In one embodiment of the present invention, hot water ultrasonic extraction is carried out in stages: The material-liquid ratio in the first stage is 1:12-16, and the boiling is maintained at a slight boil for 90 minutes; The material-liquid ratio in the second stage is 1:10, and the boiling is maintained at a slight boil for 45 minutes; The solid-liquid ratio in the third stage is 1:6-8, and the mixture is kept at a slight boil for 20 minutes after boiling. Ultrasonic assisted extraction is used in this stage, and the ultrasonic intensity is around 53kHz. The phased implementation of hot water ultrasonic extraction can improve the extraction rate and extraction rate.

[0031] In one embodiment of the present invention, the extraction in step 1) can also be carried out by alcohol extraction or hot water pressure extraction. The extraction solvent for alcohol extraction is 30-80% ethanol by volume, the ultrasonic power is 300-550W, and the ultrasonic time is 10-60min. When hot water pressure extraction is performed, pressurization can improve the extraction rate. Generally, 1.25 atmospheric pressure can be considered for pressurization, and the extraction temperature is selected to be 105˚C. The hot water pressure extraction process can reduce the number of extractions.

[0032] In one embodiment of the present invention, the concentration in step (ii) is heated open concentration or heated vacuum concentration, and the concentration is to a Brix of more than 20, that is, the water-soluble solid content is more than 20%.

[0033] In one embodiment of the present invention, the fermentation strains in step 3) are derived from the effective lactic acid bacteria spores naturally attached to the stems of stevia. Hot water ultrasonic extraction kills other miscellaneous bacteria with poor heat resistance, leaving lactic acid bacteria spores that can withstand high temperatures. When the temperature, humidity and oxygen content conditions are appropriate, they are revived and reproduced. At the same time, the macromolecular substances in the extract are fully decomposed to obtain small molecular active polyphenols, achieving extremely strong antioxidant activity. The fermentation conditions are controlled as follows: avoid light, control oxygen, and avoid the presence of yeast; in principle, low-temperature anaerobic fermentation is based on lactic acid bacteria fermentation, and the presence of yeast is taboo.

[0034] In one embodiment of the present invention, the fermentation time in step 3) is 6 to 12 months. The longer the fermentation time, the higher the content of the active ingredient; the higher the Brix, the better the antioxidant effect.

[0035] In one embodiment of the present invention, the fermentation conditions in step 3) are controlled as follows: avoid light and avoid the presence of yeast; in order to further improve the antioxidant effect of the antioxidant, the fermentation is low-temperature anaerobic fermentation, with lactic acid bacteria fermentation as the main line, and avoid the presence of yeast.

[0036] In one embodiment of the present invention, the purification step (iv) uses an M500 dialysis molecular membrane for one night of stirring dialysis. After the extramembrane liquid is concentrated, an extract with a molecular weight less than 500M can be obtained, and inorganic potassium salts, small molecules such as benzyl alcohol, etc. are all concentrated therein. After the intramembrane liquid is concentrated, a mixture with a molecular weight greater than 500M is obtained, which contains a variety of macromolecular substances such as macromolecular polysaccharides, polyphenols, pigments, chlorophyll, etc. It also has a relatively strong antioxidant activity. Generally, no separation is required. Because the antioxidant activity of the mixed extract is higher than the activity of any antioxidant active substance after separation. In addition, purification can also be carried out by reverse phase column separation and ion resin chromatography.

[0037] In one embodiment of the present invention, the health care product is prepared into a concentrated or ordinary beverage; or, the health care product is prepared into a jelly; or, the health care product is prepared into a powder, and the powder is prepared into an instant tea, a hard capsule or a soft capsule. The prepared stevia extract is directly filled or concentrated or diluted and then filled to prepare a concentrated or ordinary beverage, and the dosage is adjusted as needed. The prepared stevia extract is prepared into a jelly product for easy consumption by children. The prepared stevia extract is spray-dried to prepare a powder, or a pharmaceutical excipient is added to the stevia extract, and a certain amount of CO2 is charged, and the powder is prepared by spray drying.

[0038] This embodiment also provides the use of health products and / or compositions in the preparation of products for preventing or treating type 2 diabetes. In this embodiment, stevia extract can significantly reduce fasting blood sugar and blood sugar peak of diabetes, stabilize blood sugar, improve glucose tolerance, and has a certain blood sugar regulation function for type 2 diabetes. After oral administration of the health product of this embodiment, blood sugar can be significantly reduced in humans, especially for some patients who are currently using metformin for poor treatment effects. After using this product, blood sugar can be significantly reduced and controlled to a certain extent; and it has the effect of improving the three highs that plague adults, namely metabolic syndrome.

[0039] This embodiment also provides the use of the health care product and / or the composition in the preparation of a product that inhibits bacteria, fights viruses or fights allergies.

[0040] The present embodiment also provides the use of health care products and / or compositions in preparing supplementary foods and / or sports foods for patients with sub-healthy people, type 2 diabetes patients, patients receiving glaucoma treatment, cancer patients, hepatitis patients, overweight people, picky eaters, or patients during the recovery period of general diseases. It can be used as appropriate according to personal circumstances, and 20-60mL can be consumed every night before going to bed, regardless of time, or during exercise rest time. The maximum daily drinking amount is preferably no more than 500ml. More preferably, the above-mentioned patients during the recovery period of general diseases do not include patients with renal insufficiency. Because the health care products of this embodiment contain more potassium ions.

[0041] This embodiment also provides the use of health products and / or compositions in the preparation of anti-hair loss or hair growth products. In this embodiment, stevia extract can effectively inhibit 5α-reductase activity, improve scalp blood circulation, promote metabolism, accelerate hair growth, prolong growth period and increase the number of hair follicles, thereby promoting hair growth and effectively improving hair loss symptoms.

[0042] The following further describes the implementation of the present invention in conjunction with specific examples.

[0043] Embodiment 1: A health product comprising stevia extract, wherein the preparation steps of the stevia extract are as follows: 1) adding stevia powder raw material into water at a material-liquid ratio of 1:30, wherein the stevia powder raw material contains 100 wt% of stevia stems, and extracting, wherein the water bath extraction temperature is above 98˚C, the water bath extraction time is 160 minutes, the ultrasonic assisted extraction time is 25 minutes, and the ultrasonic intensity is 60kHz; 2) mixing the extracts obtained in step 1), heating and vacuum concentrating to Brix 22; 3) The concentrated liquid obtained after step 2) is fermented for 12 months to obtain an antioxidant stock solution.

[0044] Embodiment 2: A health product comprising stevia extract, wherein the preparation steps of the stevia extract are as follows: A stevia powder raw material is put into water at a material-liquid ratio of 1:16, wherein the stevia powder raw material contains 80wt% of stevia stems and 20wt% of stevia leaves, and extraction is performed, wherein the water bath extraction temperature is above 95˚C, the water bath extraction time is 107min, the ultrasonic assisted extraction time is 20min, and the ultrasonic intensity is 53kHz; the obtained extract is mixed, heated, vacuumed and concentrated to Brix 25 to obtain a stevia extract concentrate; the obtained concentrate is fermented for 15 months to obtain a stevia extract.

[0045] Embodiment 3: A health product comprises a stevia extract, wherein the preparation steps of the stevia extract are as follows: a stevia powder raw material is added into water at a material-liquid ratio of 1:16, wherein the stevia powder raw material contains 95wt% of stevia stems and 5wt% of stevia leaves, and extraction is performed, wherein the water bath extraction temperature is above 95˚C, the water bath extraction time is 107min, the ultrasound-assisted extraction time is 20min, and the ultrasound intensity is 53kHz; the extract is mixed, heated, vacuumed and concentrated to Brix 25, and then fermented for 9 months to obtain the stevia extract.

[0046] Stock solution - stevia extract; high-efficiency solution - refined sample of stock solution (solid content above 18%); unsterilized filtered - unsterilized sample filtered during the refining process of stock solution; unsterilized filtered - unsterilized sample unfiltered during the refining process of stock solution; sterilized filtered - filtered and sterilized sample during the refining process of stock solution; stems - raw powder of stevia stems; stems + leaves - 95% stevia stems + 5% mixed raw powder of leaves. Determination of the basic components and content of some active substances in stevia extract: The moisture content was determined according to GB 5009.3-2016 National Food Safety Standard; The protein content was determined according to GB 5009.5-2016 National Food Safety Standard; The crude fat content was determined in accordance with GB 5009.6-2016 National Food Safety Standard; Ash content determination refers to GB 5009.4-2016 National Food Safety Standard; The carbohydrate determination refers to GB 5009.1-2016 National Food Safety Standard.

[0047] The basic components and some active substance contents of each sample are shown in Tables 1 and 2.

[0048] Table 1 Basic ingredients of stevia preparations (%, n=2)

[0049] Note: Different letters in the same row indicate significant differences ( P <0.05).

[0050] Table 2 Antioxidant activity-related and other indicators of stevia preparations (n=3)

[0051] Note: Different letters in the same row indicate significant differences ( P <0.05).

[0052] Antioxidant activity of 3-allyl-6-methoxyphenol among antioxidants: Accurately measure 50mL of stevia extract in a 250mL separatory funnel, add 100mL of petroleum ether, shake well, and let stand. After the solution is completely separated, release the lower biological agent aqueous solution, retain the upper petroleum ether, and extract the lower biological agent aqueous solution twice by the same method. Combine the three petroleum ether extracts, and recover the petroleum ether under reduced pressure at 40°C to obtain a colorless viscous oil, which is the petroleum ether part of the stevia extract. Then continue to extract with ether for 3 times, combine the three upper organic solvent extracts, and recover the organic solvent under reduced pressure at 40-50°C to obtain the ether extraction part. The ether part sample was vacuum concentrated to prepare a 40% ether solution. 10 preparative silica gel thin layer plates were used for strip spotting with a spotting capillary, and the spotting width was controlled at about 2mm. Each plate was spotted 8 times, and the total spotting volume was 1mL. The experiment selected V (toluene): V (methyl formate): V (formic acid) = 5.5: 4.0: 0.5 as the developing solvent. After developing in the chromatography tank for about 50 minutes, the solvent was evaporated and the color bands and the separation of each peak were observed. Then the silica gel thin layer plate was placed in an ultraviolet analyzer at 365nm for color development. According to the ultraviolet color development positioning, the recognizable color bands were scraped off (there were 4 clear and easy-to-scrape color bands from top to bottom), and the R f The color bands with the same value were placed in a 50mL centrifuge tube, dissolved with an appropriate amount of methanol, centrifuged (2000r / min, 20min), and eluted three times. The supernatant after elution was collected and diluted to 10mL with methanol to obtain four components I, II, III, and IV, which were stored at -18°C filled with nitrogen. Then the scavenging ability of the four components I, II, III, and IV on DPPH free radicals was compared (such as Figure 1 ). As can be seen from the figure, the four components separated all have a certain ability to scavenge DPPH free radicals, and the order of their anti-free radical activity is IV>III>I>II. Therefore, component IV with the strongest free radical scavenging ability was selected for structural identification using gas chromatography-mass spectrometry. Component IV was detected by GC-MS, and the GC-MS characteristic spectrum of component IV was given by the chemical workstation (such as Figure 2). The figure shows that IV is a main component chromatographic peak with a retention time of 29.636min, and the impurity interference is small. According to the retention time of the IV component, it can be known that the IV component is 3-allyl-6-methoxyphenol. In addition, the mass spectrum of the IV component is measured, and then through computer retrieval and comparison with the structure library, it is concluded that the component is 3-allyl-6-methoxyphenol, with a similarity of 61%. Therefore, it is approximately inferred that the component is 3-allyl-6-methoxyphenol, which has strong antioxidant properties.

[0053] Embodiment 4: A health product comprises a stevia extract, wherein the preparation steps of the stevia extract are as follows: a stevia powder raw material is added into water at a material-liquid ratio of 1:16, wherein the stevia powder raw material contains 99.5wt% of stevia stems and 0.05wt% of stevia leaves, and extraction is performed, wherein the water bath extraction temperature is above 95˚C, the water bath extraction time is 107min, the ultrasonic assisted extraction time is 20min, and the ultrasonic intensity is 53kHz; the extract is mixed, heated, vacuumed and concentrated to Brix 25, and fermented for 9 months to obtain the stevia extract.

[0054] Embodiment 5: 1. Effects of stevia extract on type 2 diabetes 1.1 Experimental animal husbandry and model establishment Experimental SD rats, male, SPF grade, weighing 80±5g, were acclimatized for 1 week in an environment with a light-dark cycle of 12h, a constant temperature of 20~22℃, and a relative humidity of 65~70% before the experiment began. They were given standard feed and free drinking water.

[0055] The rats with type 2 diabetes model were induced by high-sugar and high-fat diet combined with streptozotocin (STZ). After 6 weeks of high-sugar and high-fat diet, the rats were fasted for 12 hours and injected with STZ (30 mg / kg, dissolved in 0.1 mmol / L citric acid buffer, pH 4.4, ice bath, freshly prepared and used within 5 minutes) intraperitoneally. The normal group was injected with citric acid buffer (0.1 mmol / L, pH 4.4) intraperitoneally at a dose of 1 mL / kg body weight. Fasting blood glucose was measured after 72 hours, and rats with fasting blood glucose greater than 11.1 mmol / L were diabetic models.

[0056] 1.2 Grouping and dosing method After the type 2 diabetes rat model was successfully established, the rats were randomly divided into four groups according to blood sugar and body weight: the experimental group (after modeling, the rats were treated with the stevia extract of Example 3), the model group, the normal control group 1, and the control group 2 (the normal rat group was treated with the stevia extract of Example 1), with 15 rats in each group. The stevia extract of Example 1 was used for intervention for 8 weeks, and the administration method of each group of rats during the period was as follows: Experimental group: high-fat diet, free diet and water, daily gavage with 30 mL of Stevia extract from Example 3; Comparative group 1: high-fat diet, free diet and water, daily gavage of 30 mL of the stevia extract concentrate of Example 3; Control group 2: high-fat diet, free diet and water, daily gavage of 30 mL of the stevia extract of Example 3 (excluding crude polysaccharides); Model group: high-fat diet, free diet and water, daily intragastric administration of the same volume of normal saline; Normal control group 1: normal feed, free diet and water, daily intragastric administration of the same volume of normal saline; Control group 2: Ordinary feed, free diet and water, daily gavage with 30 mL of the stevia extract of Example 1.

[0057] During the experiment, the general conditions of the rats in each group, such as spirit, nutrition, activity, diet, excretion, and body weight, were observed. Blood samples were collected from the tail tip every two weeks to measure the fasting blood glucose level after fasting for 12 hours. The results were as follows: Figure 3 At the same time, the rats that had been given the drug for 4 weeks were fasted for 12 h, anesthetized, blood was collected from the abdominal aorta and the animals were killed. The liver, fat, muscle and pancreas tissues were quickly separated, collected and stored at -80°C for future use.

[0058] Glucose tolerance test: After 8 weeks of the experiment, the rats were fasted for 12 hours but not water, and then gavaged with 2 g / kg of 50% glucose solution. The changes in blood sugar were measured before gavage (0 min) and 30, 60, 90 and 120 mm after gavage. The results were as follows: Figure 4 shown.

[0059] from Figure 3 It can be seen that the fasting blood glucose of the normal group control group 1 and the control group 2 remained at a stable level during the experiment, the rats in the comparison group and the model group always maintained a high blood glucose level, and the blood glucose level of the rats in the test group showed a rapid downward trend over time. Compared with the model group, the fasting blood glucose of the rats in the test group decreased significantly, and the blood glucose value decreased by 32.75% at the 8th week, but it was still significantly higher than that of the normal control group (p<0.05). As can be seen from Figure 2, the intervention treatment with stevia extract in Example 3 can reduce the peak value of fasting blood glucose and maintain a relatively stable blood glucose value, but it has no obvious effect on the blood glucose levels of normal rats. Figure 4It can be seen that compared with the model group, the area under the glucose tolerance curve of the test group was significantly lower than that of the model group. The above results show that the intervention of stevia extract in Example 3 can significantly reduce the fasting blood sugar and blood sugar peak of diabetic rats, stabilize blood sugar, improve glucose tolerance, and has a certain blood sugar regulation function for type 2 diabetic rats. At the same time, the presence of crude polysaccharides in stevia extract will work together with the antioxidant active substances phenols, flavonoids and potassium ions in this stevia extract, and can show an inhibitory effect on type 2 diabetes. In addition, after oral administration of the stevia extract in Example 1 to the human body, blood sugar can be significantly reduced, especially for some patients who are currently using metformin for poor treatment effects. After using this product, blood sugar can be significantly reduced and controlled to a certain extent; and stevia extract has the effect of improving the three highs that plague adults, namely metabolic syndrome.

[0060] 2. Effects of stevia extract on the insulin signal transduction pathway After insulin (INS) enters the cell and binds to the insulin receptor (IR), the β-subunit of IR is phosphorylated in the proximal membrane region of tyrosine (Tyr) and binds to IRS-2, providing sites for downstream proteins containing SH2 regions to form a signal protein complex to mediate further signal transduction, ultimately stimulating the displacement of GLUT4 on the cell membrane to transport glucose. This process is abnormal in type 2 diabetes. This part intends to detect the effect of stevia extract on the insulin signal transduction pathway by Western blot immunoblotting.

[0061] 2.1 Determination of IRS-2 protein expression Western blot was used. Samples containing equal amounts of total protein (50 μg) were separated by 7.5% SDSePAGE, electrotransferred to the membrane, blocked with 5% skim milk powder, and then reacted with rabbit anti-human IRS-1 and IRS-2 polyclonal antibodies, followed by goat anti-rabbit secondary antibodies labeled with horseradish peroxidase to display protein bands by enhanced chemiluminescence (ECL), and the optical density of protein bands was measured by a laser optical density scanner.

[0062] 2.2 Determination of IRS-2 protein phosphorylation Immunoprecipitation and enhanced chemiluminescence were used. Samples containing an equal amount of total protein (500 μg) were taken and incubated with anti-IRS-2-antibody 4e overnight. After the antigen-antibody complex was precipitated by immobilized protein G, it was redissolved in Laemmli loading buffer, heated to 95e5min, and subjected to 7.5% polyacrylamide gel electrophoresis for 1.5h. For anti-phosphotyrosine immunoblotting, the cellulose acetate membrane was directly incubated with the primary antibody APY20H conjugated to horseradish peroxidase for 1.5h. After thorough washing, it was reacted with ECL for 1min, immediately exposed to X-ray film, and scanned with an image analyzer after washing to calculate the optical density. The IRS-2 protein content and phosphorylation of the normal control group 1 were used as standards to calculate the relative amounts of the other groups.

[0063] 2.3 GLUT4 protein content determination Western blot was used to separate proteins from 50 μg of skeletal muscle cell membrane samples in each group by SDS-PAGE. The proteins were then electrotransferred to nitrocellulose (NC) membranes. The membranes were then blocked with TBS containing 5% skim milk powder for 4e overnight, and anti-GLUT4 monoclonal antibodies were added for 1h at room temperature. After washing the membranes, horseradish peroxidase-labeled secondary antibodies were added, and the membranes were gently shaken for 1h at room temperature. After being fully washed, the membranes were reacted with ECL for 1min, and immediately exposed to X-ray film. After washing, the membranes were scanned with an image analyzer, and the optical density was calculated for quantitative analysis. The GLUT4 protein content in the normal control group 1 was used as the standard to calculate the relative content of the other groups.

[0064] Figure 5-7 The results of the determination of IRS-2 protein expression, IRS-2 protein phosphorylation and GLUT4 protein content were respectively. Compared with the model group, the IRS-2 protein expression, IRS-2 protein phosphorylation and GLUT4 protein content in the test group were increased. This shows that stevia extract increases the expression of IRS protein, tyrosine phosphorylation of IRS and GLUT4 protein content, enhances GLUT4 translocation and activates the transmission of insulin signals in cells, thereby effectively lowering blood sugar, and thus plays an improvement role in potential pre-type 2 diabetes patients and people who have already become type 2 diabetes patients.

[0065] Embodiment 6: Antibacterial and antiviral properties of stevia extract 1. Stevia extract detoxifies histamine allergy During the breeding process of rainbow trout, compound feed containing fish meal may contain excessive histamine due to quality problems of raw fish, which can easily cause fish to have symptoms such as growth stunting, deformity, and gastric mucosal erosion due to histamine allergy.

[0066] This embodiment selects rainbow trout fry with a weight of about 5g after hatching for experiment. The experiment is divided into groups: the basic feed feeding group is the blank group, the basic feed is added with 1% histamine as the control group, and the basic feed is added with 1% histamine and 0.2% embodiment 3 stevia extract as the test group. Taking satiation as the benchmark, the feed fed is measured every day. It is observed that there is no difference between the three test groups when the feeding is two weeks. When continuing to feed, the seedlings of the histamine group show obvious growth retardation. At the end of the 4-week experiment, the average weight of each group of rainbow trout is as shown in Table 3. It can be seen that the average weight of the blank group rainbow trout is 11g, and the average weight of the test group rainbow trout is 10g, which is not much different from the weight of the blank group, while the control group rainbow trout is obviously thin, with an average of only 7g, which is significantly different from the weight of the blank group. At the same time, the control group is subjected to gastric tissue microscopic observation and it is found that the gastric mucosal damage is obvious, which is significantly different from the other two groups. This shows that adding 0.2% embodiment 3 stevia extract in fish farming feed can effectively prevent the negative impact of fish allergy to histamine in feed fish meal on fish growth.

[0067] Table 3 Average weight of rainbow trout (g)

[0068] 2. Bactericidal effect of stevia extract on enterohemorrhagic Escherichia coli O-157:H7 and other food poisoning pathogens 2.1 Determination of antibacterial effect of stevia extract by punch method Use a sterile inoculation loop to pick up the colonies on the activated test tube slope and inoculate them into a test tube containing 10 mL of nutrient broth. Mix well with a vortex mixer and shake and culture at 37°C for 12 h. Adjust the concentration of the test bacterial suspension to 10 6 ~10 7 / mL. Then take 0.1mL of various test bacterial suspensions on the corresponding plates, spread the bacterial suspensions evenly with an L stick, and use a sterilized puncher to punch holes in the culture dish in a cross-symmetric manner. The test group is injected with 50μL of the stevia extract of Example 3 with a pH of 4.2 (the pH is adjusted with acetic acid or lactic acid); the comparison group 1 is injected with 50μL of the stevia extract of Example 3 with a pH of 7.0; the comparison group 2 is injected with 50μL of the stevia extract concentrate of Example 1 with a pH of 4.2; the comparison group 3 is injected with 50μL of the stevia extract concentrate of Example 1 with a pH of 7.0; the comparison group 4 is injected with 50μL of stevia sugar with a pH of 4.2; the comparison group 5 is injected with 50μL of stevia sugar with a pH of 7.0; the comparison group 6 is injected with 50μL of lactic acid; the comparison group 7 is injected with 50μL of acetic acid; and a sterile water negative control group is set up at the same time. The plate was then placed in a 37°C constant temperature incubator and cultured for 24 hours before the diameter of the inhibition zone was measured. The results are shown in Table 4. It can be seen that the stevia extract of Example 3 with a pH of 4.2 has a significant bactericidal effect on food poisoning pathogens such as enterohemorrhagic Escherichia coli, Salmonella, Staphylococcus aureus, Ceresella, Eilsonia and Vibrio enteritidis. In the stevia extract of Example 3 with a bactericidal effect and a pH of 4.2, probiotic lactic acid bacteria and bifidobacteria still survive, which shows that the stevia extract can protect the body's intestinal flora, promote the normalization of gastrointestinal function, and increase appetite, thereby improving digestion and appetite. Improve the body's immunity and promote the body's health; by comparing the test group and the comparison group 1, it can be seen that when the pH of the stevia extract in Example 3 with a pH of 4.2 is adjusted to 7.0, the bactericidal effect disappears; the pH value of the stevia extract concentrate is adjusted to 4.2, which has a more obvious bactericidal effect, but its bactericidal effect is worse than that of the test group. At the same time, the pH value of the stevia extract concentrate is adjusted to 7.0, and its bactericidal effect disappears; low-energy stevioside has no bactericidal effect whether the pH is adjusted to 4.2 or 7.0; and when lactic acid or acetic acid is used alone to identify the bactericidal effect, it is almost ineffective.

[0069] Table 4 Diameter of inhibition zone of stevia extract (mm)

[0070] Note: “-” indicates no inhibition zone 2.2 LD50 acute toxicity test of stevia extract The stevia extract of Example 3, which originally had a pH of 4.2, was orally administered to test mice once a day at a dose of 50 cc / kg body weight for one week according to the test regulations, and it was confirmed to be non-toxic.

[0071] The above results indicate that if humans drink stevia fermented liquid, it can not only quickly inhibit the reproduction of various food pathogens in the intestines, but also restore the beneficial flora in the intestines. Therefore, stevia fermented liquid may become a good medicine for treating intestinal infections during food poisoning.

[0072] 3. Inhibitory effect of stevia extract on rotavirus Rotavirus (RV) is a double-stranded RNA virus belonging to the Reoviridae family. Rotavirus gastroenteritis is common in children, 5-10% of whom require hospitalization. Moreover, it is reported that hundreds of thousands of infants and young children die from this disease every year in Asia and Africa. The United States has approved a weak rotavirus vaccine, but it is only a symptomatic treatment, not a cure.

[0073] 3.1 Toxicity assay of stevia extract on MA104 cells MA104 cells were cultured at 2×10 5 / well was inoculated in a 96-well plate, and the cell culture medium was discarded after culturing for 24 hours, and washed with DMEM. 100 μL of the prepared stevia extract dilution solution of Example 3 (diluted with cell maintenance medium to 50%, 40%, 30%, 20%, 10%) was added to each well, and 7 replicates were made in parallel for each drug at each dilution, and a blank control was set. After culturing at 37°C and 5% CO2 for 72 hours, the cell pathological changes were observed. The cytotoxicity of the drug to the cells was manifested as rounding of the cells, increase in intracellular particles, and cell shedding at high concentrations. The cell barrier concentration CC50 of the stevia extract in Example 3 (the drug concentration that causes 50% of the cells to develop pathological changes compared with the cell control) was: dilution 105 times was effective.

[0074] 3.2 Inhibition test of stevia extract on RV biosynthesis In a 96-well plate with MA104 cells grown in monolayer, 100 μL of 1000 TCID 50 / mL RV SA11 strain virus solution, adsorb at 37℃ for 1.5h, and discard the virus supernatant. 50 Value, dilute the health product to 20%, 10%, 5%, 2.5% with serum-free DMEM medium. 100μL per well, 4 replicates per concentration, and set up normal cell control wells and virus control wells. Observe the cytopathic effect (CPE) daily, discard the culture supernatant when the virus control CPE is above 90%, and detect the OD value by MTT method. The proliferation inhibition concentration EC50 (the drug concentration that reduces cytopathic effect by 50% compared with the cell control and rotavirus infection control under the combined action of drugs and viruses) is: dilution 220 times is effective.

[0075] The above results indicate that the stevia extract of Example 3 may become an oral solution for inhibiting or controlling rotavirus infection.

[0076] Embodiment 7: Effects of stevia extract on glaucoma patients Under ketamine and toluidine intraperitoneal anesthesia, 48 mice were successfully established with a high intraocular pressure model by unilateral intracameral microsphere injection. After the microspheres were injected into the right eye, the intraocular pressure gradually increased. Between 8 and 10 am on the 8th day, the right eye of each group of mice was treated with the stevia extract of Example 3. Each dose was 4 μL, which was dropped into the conjunctival sac of the right eye of the mouse. The intraocular pressure was measured at multiple time points before and after medication, 1h, 2h, 6h and 24h. The results showed that the intraocular pressure can be effectively reduced in the mouse high intraocular pressure model induced by stevia extract microspheres in Example 3, and it has a relatively longer intraocular pressure reduction time, which can reach 24h, and the maximum pressure reduction amplitude is 8.2±0.3mmHg. This shows that the stevia extract of Example 3 has a clear optic nerve protective effect in the microsphere-induced glaucoma model.

[0077] Embodiment 8: Effects of stevia extract on hepatitis C patients The case data and serum specimens of 68 patients with chronic hepatitis C were collected. The diagnosis was in accordance with the 2004 hepatitis C prevention and treatment guidelines. All cases were excluded from other hepatitis, autoimmune liver disease, alcoholic liver disease, etc., and none of them received antiviral treatment. All patients were divided into two groups according to ALT levels. The 36 cases with ALT greater than 40U / L and less than 200U / L were group A, and the 32 cases with normal ALT were group B. There was no statistically significant difference in gender, age, and HCV RNA load between the two groups (P>0.05).

[0078] All patients took 20 mL of the stevia extract of Example 3 every day for a course of 48 weeks, and were followed up to 24 weeks after the end of treatment. Fluorescence quantitative PCR reagents were used to detect the hepatitis C virus HCV RNA load at the 4th, 12th, 24th, 36th, and 48th weeks of treatment. Hitachi 7600 fully automatic biochemical analyzer and supporting reagents were used to detect serum alanine aminotransferase ALT. ALT<40U / L is normal, and HCVRNA is negative when it is below the detection limit. The average load of HCV RNA was (6.74±1.03)log10 copies / mL, and the ALT negative conversion rate was 92.7% at the end of the 48-week antiviral treatment. This shows that after taking the stevia extract of Example 3, the activity of HCV can be controlled and the resistance can be enhanced. Patients with chronic hepatitis C can achieve good therapeutic effects, and because the mental state and appetite have been significantly improved, they can maintain a fairly good physical state.

[0079] Embodiment 8: Effects of stevia extract on hair growth 1. Inhibitory effect of stevia extract on 5α-reductase Blank control group: 1.5 mL 5α-reductase reaction system: 0.5 mL 5α-reductase preparation solution with an added concentration of 4.0 mg / mL; 0.5 mL 20 μM phosphate buffer solution at pH 6.5; 150 μL 0.5 mg / mL testosterone; 250 μL 2.0 mg / mL NADPH.

[0080] Experimental group: 1.5 mL 5α-reductase reaction system: 0.5 mL 5α-reductase preparation solution with an added concentration of 4.0 mg / mL; 0.5 mL 20 μM phosphate buffer solution at pH 6.5; 150 μL 0.5 mg / mL testosterone; 250 μL 2.0 mg / mL NADPH; 0.1 mL 50% ethanol dissolved stevia extract of Example 3.

[0081] Determination of blank control group: After incubation at 37℃ for 30min, measure the absorbance change value △A0.

[0082] Determination of positive control group, experimental group and comparison group: incubate at 37℃ for 30min, and measure the change value of each absorbance at 340nm △A n The inhibition rate was calculated according to the formula. The experiment was repeated 3 times and the average value was taken.

[0083] The calculation formula is: I (%) = (△A0-ΔA n ) / △A0×100%.

[0084] The inhibition rate of 5α-reductase in the experimental group was determined to be 84.76%, which can inhibit the conversion of testosterone to dihydrotestosterone and can slow down or treat the symptoms of androgenic alopecia.

[0085] 2. The hair growth effect of stevia extract Take 60 clean-grade mice, half male and half female, with a body weight of (18±2g). Cut the back hair short, apply 6% sodium sulfide depilatory agent on the back, and the depilatory area is 3cm×3cm. Wash with warm water after 3 minutes (the mouse back is smooth, without damage and residual hair). The next day, the mice were randomly divided into 6 groups, 10 in each group. The depilatory area of ​​the mice was massaged with 5mL of Example 3 stevia extract (with a concentration of Brix 16.8%) for 20-30min, left to stand for 10min, and then washed with clean water. Apply 1mL twice a day for 1 month. The growth of new hair in the depilatory area of ​​the mice was scored every 5 days. The scoring criteria are: no hair growth is 0 points, light hair growing all over the depilatory area is 1 point, the length and density of new hair is about half of the non-depilatory area is 2 points, the length of new hair is no different from that of the non-depilatory area is 3 points, and mice with irregular hair growth are calculated according to the area ratio. The average score of each group was calculated and a curve was drawn. The results are as follows: Figure 8 As shown. Figure 8 It can be seen that the stevia extract in Example 3 has a very good hair growth effect, effectively improves the symptoms of hair loss, and is suitable for people who have signs of hair loss or have already lost their hair.

[0086] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0087] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. A person skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the patent protection scope of the present invention shall be defined by the claims.

Claims

1. Use of stevia extract in preparing anti-hair loss or hair growth products, characterized in that: The stevia extract contains at least phenols, flavonoids, and potassium ions. The phenolic component is one or more of 3-allyl-6-methoxyphenol, 2,4-bis(1,1-dimethylethyl)-phenol, catechol, thymol, 4-chloro-2,6-bis(1,1-dimethylethyl)-phenol, 4-ethyl-phenol, propofol, 4-(1-methyl-1-cyclobutyl)phenol, 3-methyl-4-isopropylphenol, and 2,5-bis(1-methylpropyl)-phenol. The stevia extract contains 0.46-126.82 mg / g of phenols, 0.08-1.56 mg / g of flavonoids, and 0.77-37.24 mg / g of potassium ions. The stevia extract contains 0.073-4.17 mg / g of 3-allyl-6-methoxyphenol, the stevia extract also contains crude polysaccharides with a concentration of 5 to 4650 ppm; The preparation method of the stevia extract is: 1) putting stevia raw material into the extraction solution and performing hot water ultrasonic extraction, wherein the stevia raw material comprises 80-100wt% stevia stems and 0-20wt% stevia leaves, and the extraction process conditions are: solid-liquid ratio of 1:6-30, water bath extraction temperature of above 90°C, water bath extraction time of 100-160min, ultrasonic assisted extraction time of 15-25min, ultrasonic frequency of 15kHz-60kHz, and ultrasonic intensity of 80-200W; 2) mixing and concentrating the extracts obtained in step 1) to a concentration of Brix 20 or more; 3) fermenting the concentrated solution obtained after step 2) in a dark and anaerobic manner for 6 to 12 months to obtain a stevia extract, wherein the concentration of the stevia extract is ≥ Brix5; When the stevia extract is administered to a subject, at least one of the following is achieved: antioxidant activity; increased expression of IRS protein, tyrosine phosphorylation of IRS and GLUT4 protein content; bacterial inhibition, antiviral, anti-allergic; lowering intraocular pressure; increasing appetite; preventing hair loss or promoting hair growth.

2. The use according to claim 1, characterized in that: The invention discloses a use of the stevia extract in inhibiting the activity of 5α-reductase.

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