Anti-aging Chinese herbal medicine extract and its extraction method and use

Abstract

The invention discloses an anti-aging Chinese medicine extract, an extraction method and a use thereof. The ginseng and astragalus extract of the invention contains 20-30% of astragaloside IV, 1-5% of ginsenoside Rg1, 2-6% of ginsenoside Re and 0.2-0.5% of ginsenoside Rb1, and has 17 characteristic peaks in its high performance liquid chromatography fingerprint. The extract can effectively increase the activity of telomerase, delay cell aging and can be used in the preparation of anti-aging products.

Description

Technical Field

[0001] The present invention relates to the field of Chinese herbal medicine extracts and extraction methods, in particular to anti-aging Chinese herbal medicine extracts and extraction methods and uses thereof. Background Art

[0002] Cells are the basic units of structure and function of organisms, and are also the basic units of aging. Cell aging refers to the process in which cells gradually decline in their proliferation and differentiation abilities and physiological functions over time as they perform their life activities. The life course of a cell goes through several stages: undifferentiation, differentiation, growth, maturity, aging, and death.

[0003] Although aging and death are inevitable natural laws, delaying aging, especially trying to avoid pathological aging, is possible. It is reported that by 2050, the number of people over 60 years old in the world will reach 2 billion, accounting for about 20-30% of the total population. Faced with the trend of accelerated population aging and generally increased life expectancy, ensuring that the elderly enjoy good health and a higher quality of life has become a major issue of common concern to social sciences and life sciences. In-depth research on more effective anti-aging products will certainly be conducive to the realization of my country's goal of "healthy aging". Therefore, conducting research on delaying aging and developing products that delay aging have important scientific significance and social value. Summary of the invention

[0004] For a long time, people have been constantly exploring ways to delay aging and prolong life. Traditional Chinese medicine has a long history of research on prolonging life, with precious literature records and rich practical experience. It also records that many Chinese medicinal materials have anti-aging effects. In the process of developing anti-aging products, developers have found that although many Chinese medicinal materials have anti-aging effects, their anti-aging effects are not very ideal or significant. Even if extracts with higher content of active substances are used, the anti-aging activity of Chinese medicinal materials is still difficult to further enhance.

[0005] Therefore, further improving the anti-aging activity of traditional Chinese medicines and obtaining extracts that can more effectively delay aging have always been a problem to be solved in the field.

[0006] The purpose of the present invention is to provide an anti-aging Chinese medicine extract and an extraction method thereof, so as to obtain a product with better anti-aging effect.

[0007] In order to solve the above problems, the present invention provides a composition comprising the following components in parts by weight: 20-30 parts of astragaloside IV, 1-5 parts of ginsenoside Rg1, 2-6 parts of ginsenoside Re, and 0.2-0.5 parts of ginsenoside Rb1.

[0008] Furthermore, it contains the following components in parts by weight: 22-28 parts of astragaloside IV, 1.8-4 parts of ginsenoside Rg1, 3-5 parts of ginsenoside Re, and 0.2-0.3 parts of ginsenoside Rb1.

[0009] The present invention also provides an extract, whose raw materials include the following raw materials by weight: 1 part of ginseng and / or American ginseng, 4-8 parts of astragalus, and further 4-6 parts of astragalus; the extract contains 20-30% of astragaloside IV, 1-5% of ginsenoside Rg1, 2-6% of ginsenoside Re, and 0.1-0.5% of ginsenoside Rb1.

[0010] Furthermore, the extract contains 22-28% of astragaloside IV, 1.8-4% of ginsenoside Rg1, 3-5% of ginsenoside Re, and 0.2-0.3% of ginsenoside Rb1.

[0011] Furthermore, the total saponin content in the extract is 75-95%, and the total polysaccharide content is 3-10%.

[0012] Furthermore, the total saponin content is 80-90%, and the total polysaccharide content is 5-8%.

[0013] Furthermore, the ginseng is ginseng and / or processed ginseng.

[0014] Furthermore, the processed ginseng product is selected from one or more of raw sun-dried ginseng, red ginseng and active ginseng.

[0015] The present invention also provides an extract comprising ginsenoside Re; the high performance liquid chromatography fingerprint of the extract comprises at least 6 characteristic peaks, the characteristic peak of ginsenoside Re is used as a reference peak, and the relative retention times of the 6 characteristic peaks and the reference peak are: 0.95, 1.00, 1.80, 2.13, 2.36, and 3.20.

[0016] Furthermore, it includes 11 characteristic peaks, and the relative retention times of the 11 characteristic peaks and the reference peak are: 1.66, 1.69, 1.73, 1.86, 1.88, 1.94, 1.99, 2.02, 2.28, 2.40, and 2.45.

[0017] Furthermore, the raw materials include the following raw materials in parts by weight: 1 part of ginseng and / or American ginseng, 4 to 8 parts of astragalus, and further 4 to 6 parts of astragalus.

[0018] Furthermore, the ginseng is ginseng and / or processed ginseng.

[0019] Furthermore, the processed ginseng product is selected from one or more of raw sun-dried ginseng, red ginseng and active ginseng.

[0020] Furthermore, the relative peak areas of the characteristic peaks with relative retention times of 0.95, 1.00, 1.80, 2.13, 2.36 and 3.20 are 48.41%, 100.00%, 107.04%, 48.70%, 340.51% and 54.83%, respectively.

[0021] Furthermore, the relative peak areas of the characteristic peaks with relative retention times of 1.66, 1.69, 1.73, 1.86, 1.88, 1.94, 1.99, 2.02, 2.28, 2.40 and 2.45 are 9.52%, 17.21%, 2.61%, 4.16%, 22.88%, 10.92%, 16.34%, 8.33%, 3.37%, 5.14% and 14.59%, respectively.

[0022] The above relative retention time and relative peak area may fluctuate within a reasonable range. For example, if they fluctuate within 5% above or below their values, they should be considered to fall within the scope of protection claimed by the present invention.

[0023] The present invention also provides a method for detecting the HPLC fingerprint of the above extract, using HPLC for qualitative and / or quantitative detection, and the chromatographic conditions include:

[0024] Chromatographic column: C18 silica gel column;

[0025] Mobile phase: water and acetonitrile, using the following gradient elution program:

[0026] Time (min) Water (V%) Acetonitrile (V%) 0 80～90 10～20 30～35 60～70 30～40 70～85 10～30 70～90 ;

[0027] Further, the following elution procedure was adopted:

[0028] Time (min) Water (V%) Acetonitrile (V%) 0 85 16 33 66 34 78.5 20 80 .

[0029] Furthermore, the detection method also includes the following conditions:

[0030] ①Chromatographic column specifications: 150*3.9mm, 5μm;

[0031] ② Column temperature: 25-35°C, preferably 30°C;

[0032] ③ Flow rate: 0.8~1.2mL / min, preferably 1.00mL / min;

[0033] ④ Detection wavelength: 200-205nm, preferably 203nm.

[0034] The present invention also provides a method for preparing a natural plant extract, comprising the following steps: (1) extracting the raw material with water; (2) enriching with a macroporous adsorption resin; (3) enzymatic hydrolysis; (4) membrane filtration with a molecular weight cutoff of 200 to 1000; (5) solid-liquid separation;

[0035] The plant is selected from one or two of ginseng, American ginseng, and / or astragalus; that is, the plant can be one of ginseng, American ginseng, and astragalus; it can also be: ① ginseng and astragalus, ② American ginseng and astragalus, ③ ginseng and American ginseng, a mixed extraction of two plants; it can also be a mixed extraction of ginseng, American ginseng, and astragalus.

[0036] Furthermore, the ginseng is ginseng and / or processed ginseng.

[0037] Furthermore, the processed ginseng product is selected from one or more of raw sun-dried ginseng, red ginseng and active ginseng.

[0038] Raw sun-dried ginseng is a commercial ginseng, also known as white ginseng. It is a product made by digging out the ginseng roots, washing them, and drying them.

[0039] Red ginseng is the steamed dried root and rhizome of the cultivated product of Araliaceae ginseng, which is dug in autumn, washed, steamed and then dried. Common red ginseng is used in the present invention.

[0040] Active ginseng is low-temperature freeze-dried ginseng. Because this product can quickly absorb water when placed in water and its appearance can be restored to a state similar to fresh ginseng, it is called "active ginseng".

[0041] Raw sun-dried ginseng, red ginseng and active ginseng are all common ginseng processed products in the field, which can be purchased from the market or prepared by existing technologies. The obtained raw sun-dried ginseng, red ginseng and active ginseng can be applied to the present invention, and raw sun-dried ginseng, red ginseng and active ginseng that meet national standards are preferred (national standard documents: GB / T 22536-2018 Raw sun-dried ginseng quality classification; GB / T 22538-2018 Red ginseng quality classification; GB / T 22535-2018 Active ginseng quality classification).

[0042] In an embodiment of the present invention, in the water extraction step, the liquid-to-material ratio of water to raw material is 5 to 15:1 mL / g;

[0043] Furthermore, the water extraction is performed 1 to 4 times; furthermore, 2 times.

[0044] In an embodiment of the present invention, the macroporous adsorption resin enrichment is to adsorb the extract obtained by water extraction with a macroporous adsorption resin, and then wash it with water, 10-40% ethanol, and 60-80% ethanol in sequence, and collect the part washed with 60-80% ethanol;

[0045] Further, washing with water, 25-30% ethanol, and 70-80% ethanol respectively, and collecting the part obtained by washing with 70-80% ethanol;

[0046] Furthermore, the macroporous adsorption resin is a non-polar or weakly polar macroporous adsorption resin; preferably, the macroporous adsorption resin is selected from D-101, HPD-10, X-5, and AB-8.

[0047] In an embodiment of the present invention, the enzymatic hydrolysis is to enzymatically hydrolyze the substance obtained after enrichment with the macroporous adsorption resin;

[0048] Enzymatic hydrolysis can be performed by using one or more of glycosidase, pectinase and amylase.

[0049] Furthermore, enzymatic hydrolysis may use one or more of β-glucosidase, xylanosidase, lactase, arabinofuranosidase, pectin lyase, and amylase.

[0050] In a specific embodiment of the present invention, the enzyme is a composite enzyme composed of β-glucosidase and xylanosidase in a weight ratio of 2 to 4:1.

[0051] Furthermore, the enzymatic hydrolysis conditions are: enzymatic hydrolysis under the conditions of 0.2-1.0% composite enzyme by weight of raw materials, pH 5-6, and 40-55° C.;

[0052] Furthermore, the dosage of the complex enzyme is 0.4-0.6%;

[0053] Furthermore, the enzymolysis time is 3 to 8 hours; further, the enzymolysis time is 4 to 6 hours.

[0054] The present invention also provides one of the following extracts prepared by the above method:

[0055] (1) Ginseng extract; (2) American ginseng extract; (3) Astragalus extract; (4) Mixed extract of ginseng and American ginseng; (5) Mixed extract of ginseng and astragalus; (6) Mixed extract of American ginseng and astragalus; (7) Mixed extract of ginseng, American ginseng and astragalus.

[0056] The present invention also provides a composition, comprising the following raw materials in parts by weight: 1 part of the above-mentioned ginseng extract and / or American ginseng extract, and 5 to 14 parts of the above-mentioned astragalus extract; further, comprising 1 part of the above-mentioned ginseng extract and / or American ginseng extract, and 5.5 to 11 parts of the above-mentioned astragalus extract.

[0057] The present invention also provides the use of one or more of the above-mentioned extracts, extraction methods, and compositions in the preparation of products that can enhance telomerase activity and / or increase telomere length; the extracts described herein can be selected from all the extracts mentioned above, and are not limited to extracts defined by components or fingerprints.

[0058] Further, the product is a telomerase activator;

[0059] Further, the product is an anti-aging product;

[0060] Furthermore, the product is a product for delaying cell aging.

[0061] The present invention also provides a product, comprising one or more of the above-mentioned extracts and compositions; the extracts described herein can be selected from all the extracts mentioned above, and are not limited to the extracts defined by components or fingerprints.

[0062] The products mentioned in the present invention include but are not limited to food, medicine, and health care products.

[0063] In addition to the aforementioned extracts or compositions, the above-mentioned products may also include auxiliary materials and auxiliary ingredients.

[0064] The pharmaceutically acceptable excipients described in the present invention are a general term for all additional materials in the drug except the main drug. The excipients should have the following properties: (1) no toxic effects on the human body and almost no side effects; (2) stable chemical properties, not easily affected by temperature, pH, storage time, etc.; (3) no incompatibility with the main drug, and no impact on the efficacy and quality inspection of the main drug; (4) no interaction with the packaging material. The excipients in the present invention include but are not limited to fillers (diluents), lubricants (glidants or anti-adhesive agents), dispersants, wetting agents, adhesives, regulators, solubilizers, antioxidants, antibacterial agents, emulsifiers, disintegrants, etc. Binders include syrup, gum arabic, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methyl cellulose, etc.), gelatin slurry, syrup, starch slurry or polyvinyl pyrrolidone, etc.; fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; lubricants include micropowder silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate , pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinyl pyrrolidone or microcrystalline cellulose, etc.; wetting agents include sodium dodecyl sulfate, water or alcohol, etc.; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutyl benzoic acid, etc.; antibacterial agents include 0.5% phenol, 0.3% cresol, 0.5% trichlorobutanol, etc.; regulators include hydrochloric acid, citric acid, potassium hydroxide (sodium), sodium citrate and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; emulsifiers include polysorbate-80, sorbitan monophosphate, Pluronic F-68, lecithin, soybean lecithin, etc.; solubilizers include Tween-80, bile, glycerol, etc.

[0065] The auxiliary ingredients may be Chinese medicinal materials or extracts thereof, chemicals and the like having the same effects or functions as the extract or composition of the present invention, or may be active substances having other effects or functions.

[0066] The beneficial effects of the present invention are:

[0067] (1) The ginseng extract, astragalus extract, and the extract obtained by the extraction method of the present invention can effectively enhance the activity of telomerase, have high anti-aging activity, and can be used to prepare anti-aging products.

[0068] (2) The ginseng and astragalus extract of the present invention has better anti-aging activity than any other extract at the same concentration, indicating that the ginseng and astragalus extract of the present invention has a synergistic effect after combination, so that its anti-aging performance is significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the fingerprint of the ginseng and astragalus extract of Example 1 of the present invention. DETAILED DESCRIPTION

[0070] The present invention will be further described below through specific examples and specific tests.

[0071] Example 1

[0072] Weigh 10g of medicinal powder (1.67g ginseng, 8.33g astragalus), add 100mL water to soak for 3 hours, then heat to reflux extraction for 2 hours, filter; the filter residue is refluxed with 80mL water for 2 hours, filtered, the filtrate is combined, adsorbed with macroporous adsorption resin D101, and then washed with water, 20% ethanol, and 70% ethanol respectively; collect the 70% ethanol part, remove the ethanol, add 0.5% of the medicinal powder mass of composite enzyme (β-glucosidase and xylanase weight ratio 2:1), adjust the pH to 5-6, stir at 40-55°C for 4 hours, and filter. Membrane filtration is performed through a ceramic nanofiltration membrane (molecular weight cutoff 200-1000Da), concentrated, and spray-dried to obtain 11.0mg of the ginseng astragalus extract of the present invention (yield 0.110%).

[0073] The ginseng and astragalus extract obtained in Example 1 was subjected to chromatographic detection. The chromatographic conditions for the detection are shown in Table 1. The fingerprint spectrum obtained by the detection is shown in Figure 1 , the fingerprint data are shown in Table 2 (the relative retention time and relative peak area are calculated with Peak 2 as the characteristic peak, and the content is determined by the external standard method).

[0074] Table 1

[0075]

[0076]

[0077] Table 2

[0078] Peak number Retention time Relative retention time Peak area Relative peak area area% content% 1(Ginsenoside Rg1) 15.943 0.95 1365658 48.41％ 5.943％ 2.806％ 2(Ginsenoside Re) 16.780 1.00 2821205 100.00％ 12.277％ 4.213％ 3 27.838 1.66 268658 9.52％ 1.169％ 4 28.399 1.69 485506 17.21％ 2.113％ 5 29.050 1.73 73623 2.61％ 0.320％ 6(Astragaloside I) 30.193 1.80 3019883 107.04％ 13.141％ 25.322％ 7(Ginsenoside Rb1) 31.203 1.86 117290 4.16％ 0.510％ 0.232％ 8 31.548 1.88 645376 22.88％ 2.808％ 9 32.524 1.94 308034 10.92％ 1.340％ 10 33.406 1.99 461005 16.34％ 2.006％ 11 33.915 2.02 235129 8.33％ 1.023％ 12 35.731 2.13 1373974 48.70％ 5.979％ 13 38.200 2.28 94937 3.37％ 0.413％ 14 39.545 2.36 9606382 340.51％ 41.803％ 15 40.254 2.40 145125 5.14％ 0.632％ 16 41.063 2.45 411738 14.59％ 1.792％ 17 53.758 3.20 1546825 54.83％ 6.731％

[0079] Example 2

[0080] Weigh 10g of ginseng powder, add 100mL of water to soak for 3 hours, then heat to reflux extraction for 2 hours, filter; extract the residue with 80mL of water for 2 hours, filter, combine the filtrate, adsorb with macroporous adsorption resin D101, and then rinse with water, 20% ethanol, and 70% ethanol respectively; collect the 70% ethanol part, remove the ethanol, add 0.5% of the compound enzyme (β-glucosidase and xylanase weight ratio 2:1) of the medicinal material powder, adjust the pH to 5-6, stir at 40-55℃ for 4 hours, and filter. Membrane filtration (molecular weight cutoff 200-1000Da) was performed through a ceramic nanofiltration membrane, concentrated, and spray-dried to obtain 7.5mg of ginseng extract (yield 0.075%).

[0081] Example 3

[0082] Weigh 10g of Astragalus powder, add 100mL of water to soak for 3 hours, then heat to reflux extraction for 2 hours, filter; extract the residue with 80mL of water for 2 hours, filter, combine the filtrate, adsorb with macroporous adsorption resin D101, and then rinse with water, 20% ethanol, and 70% ethanol respectively; collect the 70% ethanol part, remove the ethanol, add 0.5% of the compound enzyme (β-glucosidase and xylanase weight ratio 2:1) of the medicinal material powder, adjust the pH to 5-6, stir at 40-55℃ for 4 hours, and filter. Membrane filtration (molecular weight cutoff 200-1000Da) was performed through a ceramic nanofiltration membrane, concentrated, and spray-dried to obtain 11.8mg of Astragalus extract (yield 0.118%).

[0083] In practice, the extract yield in the above examples may fluctuate within a certain reasonable range, such as within 5% above or below its value.

[0084] Example 4

[0085] Weigh 10g of medicinal powder (1.67g ginseng, 8.33g astragalus), add 120mL water to soak for 3 hours, then heat to reflux extraction for 1.5 hours, and filter; the filter residue is reflux extracted with 100mL water for 1.5 hours, filtered, the filtrate is combined, adsorbed with macroporous adsorption resin D101, and then rinsed with water, 30% ethanol, and 80% ethanol respectively; collect the 80% ethanol part, remove the ethanol, add 0.4% of the weight of the medicinal powder complex enzyme (β-glucosidase and xylanosidase weight ratio 4:1), adjust the pH to 5-6, stir at 40-55℃ for 6 hours, and filter. The extract was filtered through a ceramic nanofiltration membrane (molecular weight cutoff 200-1000Da), concentrated, and spray-dried to obtain the ginseng astragalus extract of the present invention; HPLC analysis showed that the ginseng astragalus extract prepared in this example contained 23.456% of astragaloside IV, 1.985% of ginsenoside Rg1, 3.669% of ginsenoside Re, and 0.209% of ginsenoside Rb1.

[0086] Example 5

[0087] Weigh 10g of medicinal powder (1.67g ginseng, 8.33g astragalus), add 100mL water to soak for 4 hours, then heat to reflux extraction for 1.5 hours, and filter; reflux extraction of the residue with 100mL water for 1.5 hours, filter, combine the filtrate, adsorb with macroporous adsorption resin D101, and then rinse with water, 25% ethanol, and 70% ethanol respectively; collect the 70% ethanol part, remove the ethanol, add 0.5% of the weight of the medicinal powder complex enzyme (β-glucosidase and xylanosidase weight ratio 3:1), adjust the pH to 5-6, stir at 40-55℃ for 5 hours, and filter. The extract was filtered through a ceramic nanofiltration membrane (molecular weight cutoff 200-1000Da), concentrated, and spray-dried to obtain the ginseng astragalus extract of the present invention; HPLC analysis showed that the ginseng astragalus extract prepared in this example contained 22.498% astragaloside IV, 1.906% ginsenoside Rg1, 3.235% ginsenoside Re, and 0.217% ginsenoside Rb1.

[0088] Example 6

[0089] Weigh 10g of medicinal powder (1.67g ginseng, 8.33g astragalus), add 100mL water to soak for 3 hours, then heat to reflux extraction for 2 hours, and filter; the filter residue is reflux extracted with 100mL water for 2 hours, filtered, the filtrate is combined, adsorbed with macroporous adsorption resin D101, and then rinsed with water, 25% ethanol, and 80% ethanol respectively; collect the 80% ethanol part, remove the ethanol, add 0.6% of the weight of the medicinal powder complex enzyme (β-glucosidase and xylanosidase weight ratio 4:1), adjust the pH to 5-6, stir at 40-55℃ for 4.5 hours, and filter. The ginseng astragalus extract of the present invention was obtained by membrane filtration through a ceramic nanofiltration membrane (molecular weight cutoff 200-1000Da), concentration, and spray drying. HPLC detection showed that the ginseng astragalus extract prepared in this example contained 22.956% of astragaloside IV, 2.085% of ginsenoside Rg1, 3.726% of ginsenoside Re, and 0.213% of ginsenoside Rb1.

[0090] Example 7

[0091] Different batches of ginseng and astragalus were extracted according to the ratio and method in Example 1, and the contents of astragaloside IV, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in the extracts were detected, and the contents of total saponins and total polysaccharides were also determined. The test results are shown in Table 2. The contents of total saponins and total polysaccharides were detected using existing conventional methods.

[0092] Table 2

[0093]

[0094] Example 8

[0095] A composition: obtained by mixing 2 mg of the ginseng extract prepared in Example 2 and 14.2 mg of the astragalus extract prepared in Example 3.

[0096] Example 9

[0097] A composition: obtained by mixing 2 mg of the ginseng extract prepared in Example 2 and 17.0 mg of the astragalus extract prepared in Example 3.

[0098] The beneficial effects of the extract of the present invention are demonstrated by the following test examples:

[0099] Test Example 1

[0100] 1. Main Reagents

[0101] Telomerase activity detection kit (Roche, Switzerland);

[0102] CCL137 normal human fibroblast cell line (American Type Culture Collection, ATCC);

[0103] Ginseng and Astragalus extract: the extract prepared in Example 1 of the present invention;

[0104] Astragalus extract: the Astragalus extract prepared in Example 3 of the present invention;

[0105] Ginseng extract: the ginseng extract prepared in Example 2 of the present invention.

[0106] 2. Methods

[0107] PCR-ELISA method to detect the effects of different extracts on telomerase activity

[0108] Principle: This method is based on the Telomeric Repeat Amplification Protocol (TRAP) method. It amplifies telomerase-mediated derivative products by PCR and combines it with the subsequent ELISA method.

[0109] Experimental steps:

[0110] (1) Preparation of cell extracts

[0111] Prepare cell suspension by conventional method, then centrifuge at 3000g for 10 min at 2-8℃ to form cell pellet, carefully remove supernatant, resuspend cell pellet with PBS and repeat centrifugation at 3000g for 10 min, carefully remove supernatant, add 200uL lysis reagent and resuspend cell pellet, precool on ice and extract at least 3 times, incubate on ice for 30 min, centrifuge at 16000g at 2-8℃ for 20 min, carefully remove supernatant and transfer to new EP tube, store the extract at -80℃.

[0112] (2) Telomerase repeat amplification step (TRAP reaction)

[0113] All operation steps were carried out on ice, and an ice box was prepared in advance. For each group of samples to be tested, 25uL of reaction complex was first added to the PCR amplification tube, 2uL of cell extract was added, and sterile double-distilled water was added to make up the total volume to 50uL. The primers were amplified and extended in a PCR instrument. The PCR reaction conditions were: 94°C inactivation for 5min, 94°C for 30s, 55°C for 30s, for a total of 33 cycles, 72°C extension for 10min, and termination of the reaction.

[0114] (3) ELISA assay

[0115] Take 20uL of denaturing reagent from each group of samples and add it to the EP tube for marking, add 5uL of the product obtained after PCR amplification, incubate at 20℃ for 10min, add 225uL of hybridization buffer to each EP tube and mix well, take out 100uL of the mixture from each group and add it to the microtiter plate, then incubate it in an oscillator at 37℃ and 300rpm for 2h, then pour out all the hybridization solution, wash 3 times, add 100uL of anti-DIG-POD working solution to each well, cover the lid, incubate on a shaker at 20℃ and 300rpm for 30min, remove the supernatant, add 250uL of cleaning solution to each well, rinse 5 times, 1min each time, add 100uLTMB fifth solution to each well to preheat to room temperature, then incubate on a shaker at 300rpm for 12min, then add 100uL of stop reagent to each well to terminate the reaction. Then, the absorbance value was measured at 450nm using a microplate reader and A690nm was used as a reference. The absorbance value of each group of samples was subtracted from the value of the negative control. If the difference in absorbance value (△A) was higher than 0.2 (A450nm-A690nm), it was considered telomerase positive.

[0116] The experimental results were analyzed and processed using SPSS18.0 statistical software. Variance analysis was used for statistics, and the SNK method was used to compare the data of each group pairwise. The difference was considered statistically significant when P<0.05.

[0117] Samples to be tested (all aqueous solutions):

[0118] Low concentration group:

[0119] A1: Ginseng and Astragalus extract solution, concentration 25ug / ml;

[0120] B1: Astragalus extract solution, concentration 25ug / ml;

[0121] C1: ginseng extract solution, concentration 25ug / ml;

[0122] Medium concentration group:

[0123] A2: Ginseng and Astragalus extract solution, concentration 50ug / ml;

[0124] B2: Astragalus extract solution, concentration 50ug / ml;

[0125] C2: ginseng extract solution, concentration 50ug / ml;

[0126] High concentration group:

[0127] A3: Ginseng and Astragalus extract solution, concentration is 100ug / ml;

[0128] B3: Astragalus extract solution, concentration 100ug / ml;

[0129] C3: ginseng extract solution, concentration 100ug / ml;

[0130] The effects of each group of samples on the telomerase activity of normal human fibroblasts were tested respectively. The results are shown in Table 3:

[0131] Table 3 Effect of the extract of the present invention on telomerase activity of normal human fibroblasts (x±s, n=9)

[0132]

[0133] * indicates P < 0.05 compared with the control group;

[0134] # indicates that compared with ginseng and astragalus extract solution A, P<0.05

[0135] Both programmed aging and exogenous aging of cells depend on the effective regulation between telomerase and telomeres. Studies have found that the telomere length of cells with negative telomerase expression is shortened, and the activity expression of cell aging-related indicators is enhanced, while the telomere length of cells with positive telomerase expression is elongated, the cell division and proliferation ability is strong, and the activity expression of cell aging-related indicators is reduced. It is precisely because the effective regulation between telomerase and telomeres controls the occurrence and development of intrinsic aging and exogenous aging. Therefore, the present invention observes the effects of different extracts on the telomere length enzyme activity of normal human fibroblasts to investigate their physiological activity that affects telomere length.

[0136] It can be seen from the measurement results of Table 3 that the telomerase activity of the cells treated with the homemade ginseng and astragalus extract, astragalus extract, ginseng extract and astragaloside IV in the high, medium and low concentration groups is higher than that of the control group, indicating that the samples tested can improve the activity of intracellular telomerase, delay the occurrence and development of cell aging, and with the increase of concentration, the more the activity of telomerase is improved, the difference is statistically significant (P<0.05). At the same concentration, the increase in telomerase activity in the ginseng and astragalus extract group of the present invention is significantly greater than that in other extract groups. And the greater the concentration, the greater the difference in telomerase activity, which can fully explain that the components in the ginseng and astragalus extract of the present invention play a synergistic effect, and its effect is significantly better than the effect of using ginseng extract or astragalus extract alone.

[0137] Test Example 2

[0138] The ginseng and astragalus extract prepared in Example 1 of the present invention was made into a preparation for a 9-month trial (one tablet of the preparation (each tablet contains 10 mg of the ginseng and astragalus extract of the present invention) was taken after meals twice a day). Each participant underwent a comprehensive physical examination and telomere length test before and after taking the preparation (in order to prevent the measurement results from being affected by personal information, the information of all samples was blinded). There were a total of 45 trial participants (23 females and 22 males. Before the trial, 25 participants were aged 40-55 years old, and 20 participants were aged 55-80 years old). The dates for physical examinations and peripheral blood sampling before the trial were from December 2016 to April 2017 (specific dates were December 16, December 23, and December 29, 2016, January 3, April 20, and April 24, 2017). The dates for physical examinations and peripheral blood sampling after the trial were from December 2017 to May 2018 (specific dates were December 8, 2017, January 26, 2018, March 12, 2018, March 30, April 13, and May 30, 2018).

[0139] The peripheral blood leukocytes of the test subjects were extracted before and after taking the ginseng and astragalus extract, and the telomere length was tested by LIFELENGTH. The main indicators in the test were: the average length of 20% short telomeres, the median telomere length, the average telomere length and the corresponding biological age. The test results are as follows:

[0140] (1) The average length of the 20% short telomeres before and after taking ginseng and astragalus extract was analyzed: among the 45 subjects, 38 subjects showed different degrees of growth, accounting for 84.44%; the average growth was 0.73Kb, and the growth rate was 15.61%;

[0141] (2) The median telomere length data of the subjects before and after taking ginseng and astragalus extract were analyzed: among the 45 subjects, 42 subjects showed different degrees of growth, accounting for 93.33%; the average growth was 0.86Kb, and the growth rate was 8.95%;

[0142] (3) The average telomere length data of the subjects before and after taking ginseng and astragalus extract were analyzed: among the 45 subjects, 43 subjects showed different degrees of growth, accounting for 95.56%; the average growth was 0.63Kb, and the growth rate was 4.51%;

[0143] (4) The biological age data of the subjects before and after taking ginseng and astragalus extract were analyzed: among the 45 subjects, 41 subjects showed a corresponding decrease in biological age, accounting for 91.11%; the average decrease was 4.34 years; among the remaining 4 cases, 2 had a biological age increase that was lower than their actual age.

[0144] From the above results, it can be seen that the ginseng astragalus extract of the present invention can increase the telomere length of the users, reduce their biological age, and effectively resist aging. Even though the biological age of 2 users did not decrease after taking the medicine compared with before taking it, the increase in their biological age was smaller than the increase in their actual age, which also shows that the ginseng astragalus extract of the present invention has a role in delaying aging for them.

[0145] The physical examination reports of the test subjects before and after the test were analyzed again. According to the main positive results of the physical examinations of 44 test subjects (no data was available for the 45th test subject due to failure in peripheral blood leukocyte extraction) before and after taking the preparation of the ginseng and astragalus extract of the present invention, by comparing and counting the main positive results before and after taking the preparation, it can be known that the preparation has the effects of lowering systolic blood pressure of blood vessels (effectiveness 85%) and lowering serum triglycerides (effectiveness 73%), and the side effects (increased positive results) are not obvious for the time being.

[0146] Test Example 3

[0147] To explore the effects of different ginseng preparations, American ginseng and commercially available extracts on telomerase activity:

[0148] Main reagents:

[0149] Telomerase activity detection kit (Roche, Switzerland);

[0150] CCL137 normal human fibroblast cell line (American Type Culture Collection, ATCC);

[0151] Ginseng (raw sun-dried) and Astragalus extract D (raw medicinal material dosage: Astragalus: raw sun-dried ginseng = 5:1): homemade;

[0152] Ginseng (red ginseng) astragalus extract E (raw material dosage: astragalus: red ginseng = 5:1): homemade;

[0153] Ginseng (active ginseng) Astragalus extract F (raw material dosage: Astragalus: active ginseng = 5:1): homemade;

[0154] American ginseng and astragalus extract G (raw material dosage: astragalus: American ginseng = 5:1): homemade;

[0155] Extract H: commercially available astragalus extract (extraction ratio 25:1 Chengdu Jintaihe Biotechnology Co., Ltd.): commercially available ginseng extract (extraction ratio 25:1 Shandong Tangzheng Biotechnology Co., Ltd.) = 8:1;

[0156] Extract J: commercially available astragalus extract (extraction ratio 20:1 Xi'an Qingzhi Biotechnology Co., Ltd.): commercially available ginseng extract (extraction ratio 25:1 Jilin Ruilong Pharmaceutical Co., Ltd.) = 8:1;

[0157] Extract K: astragaloside IV (specification 98%) 24.00%, ginsenoside Rg1 (specification 98%) 2.50%, ginsenoside Re (specification 98%) 4.00% and ginsenoside Rb1 (specification 98%) 0.25%, and the remaining ingredients are corn dextrin;

[0158] Astragaloside IV (specification 98%), L: purchased from Chengdu Jintaihe Biotechnology Co., Ltd.;

[0159] Ginsenoside Rg1 (specification 98%), ginsenoside Re (specification 98%), and ginsenoside Rb1 (specification 98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0160] The homemade extracts are all prepared by the following method:

[0161] Weigh 10g of medicinal powder, add 100mL of water to soak for 3 hours, then heat to reflux extraction for 2 hours, filter; extract the residue with 80mL of water for 2 hours, filter, combine the filtrate, adsorb with macroporous adsorption resin D101, and then rinse with water, 20% ethanol, and 70% ethanol respectively; collect the 70% ethanol part, remove the ethanol, add 0.5% of the compound enzyme (β-glucosidase and xylanase weight ratio 3:1) of the medicinal powder, adjust the pH to 5-6, stir at 40-55℃ for 5 hours, filter. Membrane filtration (molecular weight cutoff 200-1000Da) is performed through a ceramic nanofiltration membrane, concentrated, and spray-dried to obtain the corresponding extract.

[0162] Liquid chromatography was performed on extracts D to F, H, and J to detect the content of each component therein, and the content of astragaloside IV, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 was as follows:

[0163] Table 4

[0164] Extract D Extract E Extract F Extract H Extract J Astragaloside I (%) 25.372 24.965 26.598 1.236 1.069 Ginsenoside Rg1(%) 2.328 2.268 3.012 0.365 0.289 Ginsenoside Re (%) 3.891 3.681 3.692 0.489 0.426 Ginsenoside Rb1(%) 0.257 0.289 0.286 0.127 0.096

[0165] The test method and steps for detecting the effect on telomerase activity are the same as those in Test Example 1, and the measured data are shown in Table 5.

[0166] Table 5 Effects of different extracts on telomerase activity in normal human fibroblasts (x±s, n=9)

[0167]

[0168]

[0169] *P<0.05 compared with the control group

[0170] # indicates that compared with raw sun-dried ginseng and astragalus extract D, P<0.05

[0171] It can be seen from the test results in Table 5 that the extracts D, E and F obtained by extracting different ginseng products, raw sun-dried ginseng, red ginseng, active ginseng and astragalus, all significantly improved the telomerase activity of normal human fibroblasts, and the effect was equivalent to that of the extract directly mixed with ginseng and astragalus.

[0172] When ginseng was replaced with American ginseng and mixed with astragalus for extraction, the extract could increase the telomerase activity of human fibroblasts, but the effect was relatively poor compared with ginseng astragalus extract.

[0173] The extract obtained by combining commercially available astragalus extract and ginseng extract in a certain proportion only slightly increases the telomerase activity of human fibroblasts and has weak anti-aging activity.

[0174] Extract K obtained by combining the finished products of astragaloside IV, ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1 according to the content range of the extract determined by the present invention has a certain degree of improvement effect on the telomerase activity of normal human fibroblasts, and is significantly better than astragaloside IV at high concentrations, with a better effect.

[0175] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An extract, characterized in that The weight proportions of the raw materials are as follows: 1 part of ginseng, 4-8 parts of astragalus, the extract contains 20-30% of astragaloside IV, 1-5% of ginsenoside Rgl, 2-6% of ginsenoside Re, and 0.1-0.5% of ginsenoside Rb1; the total saponin content of the extract is 75-95%, and the total polysaccharide content is 3-10%.

2. The extract according to claim 1, characterized in that 4-6 portions of Astragalus.

3. The extract according to claim 2, characterized in that The extract contains 22-28% of astragaloside IV, 1.8-4% of ginsenoside Rg1, 3-5% of ginsenoside Re, and 0.2-0.3% of ginsenoside Rb1.

4. The extract according to claim 1, characterized in that The extract has a total saponin content of 80-90% and a total polysaccharide content of 5-8%.

5. The extract according to claim 1, characterized in that Containing ginsenoside Re; the extract includes at least 6 characteristic peaks in the high performance liquid chromatography fingerprint, with the characteristic peak of ginsenoside Re as the reference peak, and the relative retention time of the 6 characteristic peaks and the reference peak is: 0.95、1.00、1.80、2.13、2.36、3.20； The chromatographic conditions for HPLC include: Chromatographic column: C18 silica gel column; Mobile phase: water and acetonitrile, using the following gradient elution program: 。 6. The extract according to claim 5, characterized in that The following elution program was used: 。 7. The extract according to claim 5, characterized in that It also includes 11 characteristic peaks, and the relative retention times of the 11 characteristic peaks and the reference peak are: 1.66、1.69、1.73、1.86、1.88、1.94、1.99、2.02、2.28、2.40、2.45。 8. The extract according to claim 1, characterized in that The ginseng is a processed ginseng product.

9. The extract according to claim 8, characterized in that The processed ginseng product is selected from one or more of raw sun-dried ginseng, red ginseng and active ginseng.

10. The extract according to claim 5, characterized in that The relative peak areas of the characteristic peaks with relative retention times of 0.95, 1.00, 1.80, 2.13, 2.36 and 3.20 are 48.41%, 100.00%, 107.04%, 48.70%, 340.51% and 54.83%, respectively.

11. The extract according to claim 7, characterized in that The relative peak areas of the characteristic peaks with relative retention times of 1.66, 1.69, 1.73, 1.86, 1.88, 1.94, 1.99, 2.02, 2.28, 2.40 and 2.45 are 9.52%, 17.21%, 2.61%, 4.16%, 22.88%, 10.92%, 16.34%, 8.33%, 3.37%, 5.14% and 14.59%, respectively.

12. The method for preparing the extract according to any one of claims 1 to 11, characterized in that: It includes the following: (1) extracting the raw material with water; (2) enriching with a macroporous adsorption resin, which is a non-polar or weakly polar macroporous adsorption resin; (3) enzymatic hydrolysis; (4) membrane filtration with a molecular weight cutoff of 200 to 1000 Da; (5) solid-liquid separation.

13. The preparation method according to claim 12, characterized in that: In the water extraction step, water: liquid material of the raw material The specific dosage is 5-15:1 mL / g.

14. The preparation method according to claim 12, characterized in that: The water extraction is performed 1 to 4 times.

15. The preparation method according to claim 14, characterized in that: The number of water extractions was 2 times.

16. The preparation method according to claim 12, characterized in that: The macroporous adsorption resin enrichment is to adsorb the extract obtained by water extraction with a macroporous adsorption resin, and then wash it with water, 10-40% ethanol, and 60-80% ethanol in sequence, and collect the part washed with 60-80% ethanol.

17. The preparation method according to claim 16, characterized in that: The samples were washed with water, 25-30% ethanol and 70-80% ethanol respectively, and the fraction obtained by washing with 70-80% ethanol was collected.

18. The preparation method according to claim 12, characterized in that: The macroporous adsorption resin is selected from D-101, HPD-10, X-5, and AB-8.

19. The preparation method according to claim 12, characterized in that: The enzymatic hydrolysis is to enzymatically hydrolyze the material obtained after enrichment with the macroporous adsorption resin; the enzyme is a composite enzyme composed of beta-glucosidase and xylanosidase in a weight ratio of 2 to 4:

1.

20. The preparation method according to claim 19, characterized in that: The enzymatic hydrolysis conditions are: enzymatic hydrolysis under the conditions of 0.2-1.0% composite enzyme by weight of raw materials, pH of 5-6, and 40-55°C.

21. The preparation method according to claim 20, characterized in that: The dosage of the complex enzyme is 0.4-0.6%.

22. The preparation method according to claim 20, characterized in that: The enzymatic hydrolysis time is 3 to 6 hours.

23. The preparation method according to claim 22, characterized in that: The enzymatic hydrolysis time is 4h.

24. Use of the extract according to any one of claims 1 to 11 in the preparation of a product capable of enhancing telomerase activity and / or increasing telomere length.

25. The use according to claim 24, characterized in that The product is a telomerase activator.

26. The use according to claim 24, characterized in that The product described is an anti-aging product.

27. The use according to claim 24, characterized in that The product is a product for delaying cell aging.

28. A product, characterized in that The invention comprises the extract according to any one of claims 1 to 11.

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