Lijiang arenaria kansuensis extract as well as preparation method and application thereof
By extracting saponin compounds from Lijiang Snow Ganoderma lucidum and preparing high-content extracts, the pharmacological and drug efficacy in the application of Lijiang Snow Ganoderma lucidum was solved, and effective skin care and anti-aging effects were achieved.
Patent Information
- Application Number
- CN202411901633.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-02-25
AI Technical Summary
The existing technology has not fully explained the pharmacological effects of Lijiang Snow Ganoderma lucidum, and how to scientifically apply Lijiang Snow Ganoderma lucidum is still a problem to be solved.
By extracting saponin compounds of Lijiang Snow Ganoderma lucidum, an extract with a total saponin content of no less than 70g/kg was prepared, and the content and quality of the extract were improved by the steps of crushing, extraction, separation and purification.
Lijiang Snow Ganoderma lucidum Extract can effectively inhibit the activities of collagenase and hyaluronidase, achieve firming, soothing, moisturizing, and repairing effects, and have excellent anti-aging effects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant extracts, and in particular to a Lijiang Snow Ganoderma extract and a preparation method and application thereof. Background Art
[0002] Lijiang Snow Ganoderma (Arenaria lichiangensis W.W.Smith), also known as mountain fortune grass (ArenariaoreophilaHook.f.).
[0003] At present, people know very little about Lijiang Snow Ganoderma, and only a few papers have reported some chemical components of Lijiang Snow Ganoderma. Some papers reported that Lijiang Snow Ganoderma contains a variety of cyclopeptides, namely cyclo(PPLLFSGT), cyclo(SC), cyclo(IGVALIIVP) and cyclo(PPGIVLGATG) (see New Minor Cyclopeptides from Arenaria oreophila (Caryophyllaceae) [J]. Chinese Chemical Letters, 2006, (04): 477-480. and Cyclopeptidic Constituents from Arenaria oreophila JD, Hooker (Caryophyllaceae) [J]. Journal of Integrative Plant Biology, 2006, 48 (06): 740-744.). In addition, some literature also reported that Lijiang Snow Ganoderma contains flavonoids, glycosides, triterpenoids and carbaryl alkaloids (see Chang Zhongyi, Gong Weihong. Chemical components of Lijiang Fleas [J]. Guangxi Plants, 2005, 25(3): 278-280. and Wang Mingming, Li Haitao, Cao Fang. Study on the chemical components of Lijiang Fleas [J]. West China Journal of Pharmacy, 2020, 35(02): 122-126.). However, the above reports did not clarify the pharmacological and pharmacodynamic effects of Lijiang Snow Ganoderma. How to scientifically apply Lijiang Snow Ganoderma is still a problem to be solved in this field. Summary of the invention
[0005] The present invention relates to a Lijiang snow ganoderma extract and a preparation method and application thereof. The Lijiang snow ganoderma (Arenaria lichiangensis WWSmith) is a plant of the genus Arenaria of the family Caryophyllaceae. The Lijiang snow ganoderma extract has the effects of caring for or treating skin or mucous membranes.
[0006] In one aspect, the present disclosure provides a Lijiang Snow Ganoderma extract, wherein the total saponin content of the extract is not less than 70 g / kg based on dry powder.
[0007] Lijiang Snow Ganoderma Extract has a variety of active ingredients that can care for the skin, which can inhibit the activity of collagenase and hyaluronidase, thereby achieving the effects of firming and soothing, moisturizing, repairing, etc., and has excellent anti-aging effects. After further research on the ingredients of Lijiang Snow Ganoderma, the inventors found that the main active ingredient of Lijiang Snow Ganoderma that exerts the above-mentioned effects is saponin compounds. The higher the total saponin content, the better the effect of inhibiting the activity of collagenase and hyaluronidase. Therefore, in some embodiments, the total saponin content of Lijiang Snow Ganoderma Extract is not less than 90g / kg in terms of dry powder.
[0008] The present disclosure explains the term "measured on a dry powder basis": the content of the Lijiang Snow Ganoderma extract in any form is calculated based on the weight after deducting the water content. Those skilled in the art know that the Lijiang Snow Ganoderma extract can exist in the form of extracts, thick extracts, clear pastes, powders, etc., and the content of total saponins in extracts in different forms is not the same. For example, the total saponin content of the Lijiang Snow Ganoderma extract can be adjusted by drying or diluting with water. In order to unify the content standard of total saponins in the Lijiang Snow Ganoderma extract described in the present disclosure, the present disclosure defines the content standard as "measured on a dry powder basis", but it does not mean that it constitutes a limitation on the Lijiang Snow Ganoderma extract disclosed in the present disclosure.
[0009] On the other hand, in order to increase the content of total saponins in the Lijiang Snow Ganoderma extract, the present disclosure also provides a method for preparing the Lijiang Snow Ganoderma extract, comprising four steps of crushing, extraction, separation and purification.
[0010] In the pulverizing step, a ball mill, a mortar grinder, ultrasonic pulverization, a mixer or a filter can be used for pulverization, and the above pulverizing methods are conventional pulverizing methods that can be selected by those skilled in the art. The purpose of pulverizing is to improve the extraction efficiency, but whether or not pulverizing does not change the composition of the extract, so the pulverizing step is not necessary, and the preparation process that omits the pulverizing step should be regarded as falling within the scope of protection claimed by the present disclosure.
[0011] The extraction step is one of the necessary steps, including the step of extracting the desired useful ingredients from Lijiang Snow Ganoderma using a specific extraction solvent. Extraction is a process of transferring the target component from the plant to the solvent using a solvent of similar polarity, using the principle of like dissolves like. Due to the different polarities of different target components, the selection of solvents of different polarities is one of the most critical factors. Some polar solvents and some non-polar solvents can effectively extract saponin compounds, so the extraction solvent disclosed in the present invention can choose to use a polar solvent or a non-polar solvent. In order to further increase the content of total saponins in the extract, it is possible to consider using solvents with high solubility for saponin compounds, including but not limited to alcohols, esters, alkanes, etc., such as methanol (and its aqueous solution), ethanol (and its aqueous solution), n-propanol (and its aqueous solution), ethyl acetate, n-hexane, etc. In some embodiments, the polar solvent used in the present disclosure is 70-95% ethanol; in some embodiments, the polar solvent used in the present disclosure is 95% ethanol.
[0012] The purpose of the separation step is to separate the solvent containing the target component from the plant body for use. The separation method is well known to those skilled in the art, including but not limited to normal pressure filtration, reduced pressure filtration and centrifugal filtration.
[0013] In the purification step, macroporous resin method, acid-base method, salting-out method, dialysis method, water precipitation method, etc. can be used. The above purification methods are all conventional purification methods that can be selected by those skilled in the art. The purpose of purification is to further enrich the target component to increase the content of the target component. Those skilled in the art can decide whether to perform further purification steps on the extract according to the actual production situation. Therefore, the purification step is not necessary, and the preparation process that omits the purification step should be regarded as falling within the protection scope required by the present disclosure.
[0014] Another aspect of the present disclosure provides use of the above-mentioned Lijiang Snow Ganoderma extract in preparing cosmetics.
[0015] The Lijiang Snow Ganoderma extract disclosed in the present invention refers to an extract obtained by extracting Lijiang Snow Ganoderma by any extraction method, and also includes a mixture of Lijiang Snow Ganoderma extracts obtained by different preparation methods, as long as the total saponin content is not less than 70g / kg. Efficacy experiments have shown that the Lijiang Snow Ganoderma extract with a total saponin content of not less than 70g / kg can inhibit the activity of collagenase and inhibit the activity of hyaluronidase, thereby exerting the effects of firming, soothing, moisturizing and repairing, having excellent anti-aging effects, and can be used as one of the active ingredients to make cosmetics.
[0016] In addition, the present disclosure also provides a use of the Lijiang Snow Ganoderma extract prepared by the above preparation method in preparing cosmetics.
[0017] Cosmetics are mostly applied topically, and the applicable sites include skin, mucous membranes, etc. In some embodiments, the cosmetics prepared from the extract of Lijiang Snow Ganoderma can be topically applied to the skin and / or mucous membranes to care for the skin or mucous membranes.
[0018] On the other hand, the present disclosure also provides a use of the above-mentioned Lijiang Snow Ganoderma extract, or the Lijiang Snow Ganoderma extract prepared by the above-mentioned preparation method in preparing an anti-aging composition.
[0019] On the other hand, the present disclosure also provides a use of the above-mentioned Lijiang Snow Ganoderma extract, or the Lijiang Snow Ganoderma extract prepared by the above-mentioned preparation method, in preparing a composition for inhibiting collagenase activity, increasing skin elasticity and / or improving skin firmness.
[0020] On the other hand, the present disclosure also provides a use of the above-mentioned Lijiang Snow Ganoderma extract, or the Lijiang Snow Ganoderma extract prepared by the above-mentioned preparation method, in preparing a composition for soothing, moisturizing and / or repairing.
[0021] On the other hand, the present disclosure also provides a use of the above-mentioned Lijiang Snow Ganoderma extract, or the Lijiang Snow Ganoderma extract prepared by the above-mentioned preparation method, in preparing a composition for inhibiting hyaluronidase activity and / or repairing the skin barrier.
[0022] In the present disclosure, the term "skin" is understood to refer to the multiple layers that make it up, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both ends are included. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and / or adipocytes, etc. In the present disclosure, the term "skin" includes the scalp.
[0023] The term "skin care" refers to the maintenance and care of the skin, improving the condition of the skin, making it delicate, smooth, tender and healthy.
[0024] The present disclosure has the following advantages and effects:
[0025] 1. The Lijiang Snow Ganoderma extract disclosed in the present invention can effectively inhibit the activity of collagenase. Inhibiting the activity of collagenase can inhibit the hydrolysis of collagen and increase the content level of collagen in skin tissue. The lack of collagen is the main reason for the loosening, sagging and fine wrinkles of skin aging. Therefore, the Lijiang Snow Ganoderma extract disclosed in the present invention can inhibit the activity of collagenase, thereby increasing skin elasticity, improving skin firmness and delaying aging.
[0026] 2. The Lijiang Snow Ganoderma extract disclosed in the present invention can effectively inhibit the activity of hyaluronidase. Inhibiting the activity of hyaluronidase can reduce the decomposition of hyaluronic acid in cells, increase the content of hyaluronic acid in cells, help increase the hydration of skin or mucous membranes, repair the skin barrier, and have moisturizing, soothing, and repairing effects. Therefore, the Lijiang Snow Ganoderma extract disclosed in the present invention can exert moisturizing, soothing, and repairing effects by inhibiting the activity of hyaluronidase. DETAILED DESCRIPTION
[0027] In order to make the objects, features and advantages of the present disclosure more obvious and understandable, the present disclosure is further described in detail below in conjunction with embodiments. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the claims attached to the present disclosure.
[0028] Example 1
[0029] Lijiang snow ganoderma extract is prepared by the following steps:
[0030] The dried Lijiang Snow Ganoderma was crushed, 100g of powder was taken into a flask, 1000mL of 95% ethanol was added, refluxed for 2h, and filtered to obtain a filtrate; the residue was added with 500mL of 95% ethanol and refluxed for 2h, and the filtrate was filtered. The filtrates obtained twice were combined, concentrated to an extract by a rotary evaporator, and dried in a vacuum oven to obtain Lijiang Snow Ganoderma Extract A.
[0031] Example 2
[0032] Lijiang snow ganoderma extract is prepared by the following steps:
[0033] Crush the dried Lijiang Snow Ganoderma lucidum, take 100g of powder into a flask, add 1000mL 75% ethanol, reflux extract for 2h, and filter to obtain a filtrate; add 1000mL 75% ethanol to the filter residue again, reflux extract for 2h, and filter to obtain a filtrate. Combine the filtrates obtained twice, and after the filtrate is concentrated to be alcohol-free, add an equal amount of water to the concentrated solution, stir at 60°C for one hour, centrifuge and take the supernatant. Add the supernatant to a chromatographic column for adsorption (the chromatographic column filler is AB-8 macroporous resin), first wash with water, and then elute with 80% ethanol. The obtained eluate is concentrated to a relative density of 1.05 and then spray-dried to obtain Lijiang Snow Ganoderma lucidum extract B.
[0034] Example 3
[0035] Lijiang snow ganoderma extract is prepared by the following steps:
[0036] The dried Lijiang Snow Ganoderma lucidum was crushed, 100g of the powder was taken into a flask, 1000mL of water was added, refluxed for 2h, and the filtrate was obtained by filtering; the filter residue was added with 1000mL of water again, refluxed for 2h, and the filtrate was obtained by filtering. The filtrates obtained twice were combined, concentrated by rotary evaporation, and the concentrated solution was added with 95% ethanol for alcohol precipitation. After standing, the concentrated solution was centrifuged and filtered, and the supernatant was concentrated to an extract, and dried in a vacuum oven to obtain Lijiang Snow Ganoderma lucidum extract C.
[0037] The contents of total saponins, total polyphenols, total proteins and total flavonoids in each sample of Example 1, Example 2 and Example 3 were determined.
[0038] The determination method of total saponins is as follows:
[0039] Weigh diosgenin, add chloroform to dissolve, and prepare a 0.2mg / mL reference solution. Pipette 0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5mL of the reference solution into a 10mL test tube, evaporate the solvent, add 10mL of concentrated sulfuric acid, place in a 50℃ water bath for 30min, take out and cool in an ice water bath for 4min, let stand for 150min, and compare the color at 410nm, with concentrated sulfuric acid as the blank control. The color development stability time is within 150-250min. Draw a standard curve with the reference concentration as the ordinate and the absorbance value as the abscissa. Weigh 0.4g of the sample into a conical flask, add 20mL of methanol, and filter after ultrasonic treatment for 30min. Wash the filter residue with 8mL of methanol, combine the methanol solution, and recover the methanol and evaporate to dryness. Add 8mL 3mol / L hydrochloric acid solution (added in 3 times, 4mL, 2mL, 2mL) to the residue after evaporation to dissolve, transfer to a conical flask in sequence, heat and hydrolyze in a water bath for 30min, take out, and cool. Add 12mL chloroform, heat and reflux for 15min, and then use 12mL chloroform to treat in sequence, filter, and then use 12mL chloroform to wash the container and residue, combine the chloroform solution 3 times, concentrate and transfer to a 10mL volumetric flask, dilute to the scale with chloroform, shake well, and obtain the sample extract. Take 0.5-1mL of the sample extract in a 10mL test tube, evaporate the solvent, add 10mL concentrated sulfuric acid, operate the same as the standard curve, measure its absorbance at 410nm, and convert it into the content of diosgenin in the sample according to the regression equation.
[0040] The total polyphenols were determined as follows:
[0041] Weigh monohydrated gallic acid, add distilled water to dissolve, and prepare 0, 10, 20, 30, 40, 50 mg / L reference solution. Take 1.0 mL of each concentration of monohydrated gallic acid standard solution, add 1 mL of FC color developer and 3 mL of 7.5% sodium carbonate solution, dilute to 10 mL with distilled water, mix, color at room temperature (30-60 min), measure the absorbance at 765 nm, and draw a standard curve. Weigh 1.75 g of sample in a 25 mL volumetric flask, add 20 mL of distilled water to dissolve completely, take 1.0 mL of sample solution, add 1 mL of FC color developer and 3 mL of 7.5% sodium carbonate solution, dilute to 10 mL with distilled water, mix, color at room temperature (30-60 min), measure the absorbance at 765 nm, and calculate the polyphenol content of the sample according to the dilution multiple and the content found from the standard curve.
[0042] The total protein was determined as follows:
[0043] Weigh 1.000g sample, transfer to a dry 300mL digestion tube, first drip a little water to wet the sample, then add Kjeldahl nitrogen determination catalyst tablets and 5mL concentrated sulfuric acid, digest in a 400℃ digestion furnace for 1h, take out after cooling, dilute to 50mL, obtain the test solution, and set aside. Take another dry 300mL digestion tube, drip a little water, then add Kjeldahl nitrogen determination catalyst tablets and 5mL concentrated sulfuric acid, digest in a 400℃ digestion furnace for 1h, take out after cooling, dilute to 50mL, obtain blank solution, and set aside. Before distillation, add sodium hydroxide (40% (m / V)), sulfuric acid standard solution (c(1 / 2H2SO4)=0.02mol / L), and methyl red-bromocresol green mixed indicator to the nitrogen analyzer, and fully preheat the nitrogen analyzer, perform empty distillation to clean the pipeline until the reading is stable. Then, 5 mL of the test solution or blank solution was drawn and injected into the distillation tube of the Kjeldahl nitrogen analyzer. After the parameters were set, the test solution was measured, and the alkali addition time was set to 3 s.
[0044] The determination method of total flavonoids is as follows:
[0045] Dissolve rutin with 30% ethanol to prepare a rutin reference solution with a concentration of 0.28 mg / mL. Accurately measure 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the rutin reference solution into a 10 mL volumetric flask, add 30% ethanol to 1.0 mL each, add 0.5 mL of 5% NaNO2 solution, let stand for 6 minutes, then add 0.3 mL of 10% Al(NO3)3 solution, shake well, let stand for 6 minutes, add 3 mL of 4% NaOH solution, dilute to the mark with water, shake well, let stand for 15 minutes, measure the absorbance at 510 nm, and draw a standard working curve with absorbance as the horizontal coordinate and rutin content as the vertical coordinate. Take the sample to be tested, grind it finely, accurately weigh about 0.3 g, extract it with 80% ethanol reflux twice (30 mL each time), filter, combine the filtrates, evaporate to dryness, dissolve it with 30% ethanol and dilute to 10 mL. Accurately measure 1.0 mL of the sample solution and place it in a 10 mL volumetric flask. According to the method of drawing the standard working curve, measure the absorbance from the time 0.5 mL of 5% NaNO2 solution is added. The rutin content in the sample solution can be found from the standard working curve, and the total flavonoid content in the sample can be calculated.
[0046] The results are shown in Table 1.
[0047] Table 1 Content of each ingredient
[0048] Lijiang Snow Ganoderma Extract A Lijiang Snow Ganoderma Extract B Lijiang Snow Ganoderma Extract C Total saponins (g / kg) 91.09 73.68 36.70 Total polyphenols (g / kg) 8.49 19.90 18.79 Total protein (g / kg) 33.35 55.25 31.95 Total flavonoids (g / kg) 35.74 43.07 26.06
[0049] Example 4 Hyaluronidase activity assay experiment
[0050] 4.1 Reagents and Materials
[0051] Sodium acetate buffer (pH=5.6), hyaluronidase (500U / mL), calcium chloride, sodium hyaluronate (0.5mg / mL), sodium hydroxide solution (5mol / L), acetylacetone, P-DAB color developer (obtained by uniformly mixing p-dimethylaminobenzaldehyde (0.8g) and concentrated hydrochloric acid (15mL) and an equal amount of glacial acetic acid), and anhydrous ethanol.
[0052] 4.2 Instruments
[0053] ELISA reader, electronic balance.
[0054] 4.3 Samples to be tested and grouping
[0055] 4.3.1 Samples to be tested
[0056] The test concentration of Lijiang Snow Ganoderma Extract A, Lijiang Snow Ganoderma Extract B and Lijiang Snow Ganoderma Extract C are all 20ppm.
[0057] 4.3.2 Grouping
[0058] Sample group: test sample, hyaluronidase, sodium hyaluronate;
[0059] Sample zeroing group: test sample, sodium acetate buffer, sodium hyaluronate;
[0060] Blank control group: distilled water, hyaluronidase, sodium hyaluronate;
[0061] Blank zero adjustment group: distilled water, sodium acetate buffer, sodium hyaluronate.
[0062] 4.4 Experimental methods
[0063] Hyaluronidase and sodium hyaluronate were dissolved in sodium acetate buffer.
[0064] Take a 96-well plate, add 25μL of the sample to be tested and 25μL of hyaluronidase (500U / mL) to the sample group; add 25μL of the sample to be tested and 25μL of sodium acetate buffer to the sample zeroing group; add 25μL of distilled water and 25μL of hyaluronidase (500U / mL) to the blank control group; add 25μL of distilled water and 25μL of sodium acetate buffer to the blank zeroing group. After shaking in a 37℃ constant temperature air bath for 20min, add 5μL of calcium chloride solution (2.5mol / L) to each well, and shake in a 37℃ constant temperature air bath for 20min. Add 25μL of sodium hyaluronate to the sample group, sample zeroing group, blank control group and blank zeroing group, shake in a 37℃ constant temperature air bath for 40min, and then leave at room temperature for 10min. Add 25 μL of distilled water, 5 μL of sodium hydroxide solution (5 mol / L), and 25 μL of acetylacetone solution to each well and place in a boiling water bath for 15 min, then in an ice bath for 10 min, and finally place at room temperature for 10 min. Add 50 μL of P-DAB to each well, then add 100 μL of anhydrous ethanol, place at room temperature for 30 min, and measure OD at 570 nm. 570 value.
[0065]
[0066] Where: A1 is the OD of the sample zeroing group 570 value, A2 is the sample group OD 570 A3 is the OD of the blank zero adjustment group. 570 A4 is the OD of the blank control group. 570 value.
[0067] 4.5 Experimental Results
[0068] Hyaluronidase activity is closely related to type I allergic reactions. Inhibiting hyaluronidase activity can play a soothing and anti-allergic role. Therefore, inhibiting hyaluronidase activity is often used as an evaluation indicator for studying soothing and anti-allergic effects. In addition, hyaluronidase is a hydrolase that degrades hyaluronic acid. Inhibiting hyaluronidase activity can reduce the degradation of hyaluronic acid in cells and increase the content of hyaluronic acid in cells, which is beneficial to increase the hydration of the skin or mucous membranes, repair the skin barrier, and play a moisturizing, soothing, and repairing role.
[0069] Sodium hyaluronate is a substrate of hyaluronidase. Under the catalytic action of hyaluronidase, sodium hyaluronate is degraded to generate glucuronic acid and N-acetylglucosamine, which develop color and absorb visible light of 570nm wavelength under the action of P-DAB. In this experiment, the test sample is used to treat hyaluronidase, and the reaction amount of sodium hyaluronate is detected to determine whether the relevant extracts of Lijiang Snow Ganoderma disclosed in the present invention can inhibit the activity of hyaluronidase, so as to evaluate whether they have moisturizing, soothing and repairing effects.
[0070] The results of the effects of the test samples on the inhibition of hyaluronidase activity are shown in Table 2.
[0071] Table 2 Inhibition rate of different Lijiang Snow Ganoderma extracts on hyaluronidase activity
[0072] Lijiang Snow Ganoderma Extract Sample Hyaluronidase activity inhibition rate (Mean±SD) Extract A 46.15％±4.19％ Extract B 30.77％±5.54％ Extract C 7.69％±5.54％
[0073] The results showed that Lijiang Snow Ganoderma Extract A had the strongest inhibitory effect on hyaluronidase, followed by Lijiang Snow Ganoderma Extract B, and Lijiang Snow Ganoderma Extract C had the weakest inhibitory effect on hyaluronidase. The total saponin contents of Lijiang Snow Ganoderma Extract A, Lijiang Snow Ganoderma Extract B, and Lijiang Snow Ganoderma Extract C were 91.09g / kg, 73.68g / kg, and 36.70g / kg, respectively. It can be seen that there is a significant gradient difference between the total saponin contents of Lijiang Snow Ganoderma Extract A, Lijiang Snow Ganoderma Extract B, and Lijiang Snow Ganoderma Extract C, and the inhibition rate of hyaluronidase activity of the three at the same concentration also has a significant gradient difference, proving that the effective ingredients that inhibit hyaluronidase are mainly saponin compounds in Lijiang Snow Ganoderma Extract. In addition, by comparing Lijiang Snow Ganoderma Extract A with Lijiang Snow Ganoderma Extract B, it can be seen that among the various components of Lijiang Snow Ganoderma Extract A, only the content of total saponins is higher than that of Lijiang Snow Ganoderma Extract B, and the contents of the other components are lower than those of Lijiang Snow Ganoderma Extract B. However, the inhibitory effect of Lijiang Snow Ganoderma Extract A on hyaluronidase is stronger than that of Lijiang Snow Ganoderma Extract B, which further proves that the effective ingredients that inhibit hyaluronidase are mainly saponin compounds in Lijiang Snow Ganoderma Extract. Therefore, the Lijiang Snow Ganoderma Extract with a total saponin content of not less than 70g / kg disclosed in the present invention has a significant ability to inhibit hyaluronidase activity, can be used to repair the skin barrier, and has the effects of moisturizing, soothing and repairing.
[0074] Example 5 Collagenase Activity Determination Experiment
[0075] 5.1 Reagents and Materials
[0076] 0.2 mg / mL collagenase IV working solution (freshly prepared, prepared by dissolving 2 mg collagenase IV in 1 mL TESCA buffer and diluting to 10 mL with PBS buffer 1), 0.8% gelatin solution, PBS buffer 1 (pH=7.5, containing 0.36 mmol / L CaCl2), PBS buffer 2 (pH=7.5, containing 4 mmol / L CaCl2), ninhydrin reaction solution (freshly prepared, prepared by mixing 5 mL 1% ninhydrin solution with 0.5 mL 0.1% Vc solution and then diluting to 10 mL with secondary water).
[0077] 5.2 Instruments
[0078] ELISA reader, electronic balance.
[0079] 5.3 Samples to be tested and grouping
[0080] 5.3.1 Samples to be tested
[0081] The test concentration of Lijiang Snow Ganoderma Extract A and Lijiang Snow Ganoderma Extract B are both 1000ppm.
[0082] 5.3.2 Grouping
[0083] Sample group: test sample, collagenase IV, gelatin, PBS buffer 2;
[0084] Sample zeroing group: test sample, PBS buffer 1, gelatin, PBS buffer 2;
[0085] Blank control group: PBS buffer 1, collagenase IV, gelatin, PBS buffer 2;
[0086] Blank zero adjustment group: PBS buffer 1, gelatin, PBS buffer 2.
[0087] 5.4 Experimental methods
[0088] The sample to be tested was dissolved in PBS buffer 1.
[0089] Take a 96-well plate, add 25μL sample and 25μL collagenase IV working solution (test final concentration is 0.05mg / mL, i.e.: 10.5U / mL) to each well of the sample group; add 25μL PBS buffer 1 and 25μL collagenase IV working solution (test final concentration is 0.05mg / mL, i.e.: 10.5U / mL) to each well of the blank control group; add 25μL sample and 25μL PBS buffer 1 to each well of the sample zero adjustment group; add 50μL PBS buffer 1 to each well of the blank zero adjustment group. After incubating each well at 37℃ for 5min, add 20μL PBS buffer 2 and 30μL gelatin solution, and incubate at 37℃ for 60min. Finally, add 25μL 0.01mol / L HCl solution to terminate the enzyme reaction.
[0090] Add 50 μL of ninhydrin reaction solution to each well, mix thoroughly, heat at 100°C for 60 min, cool to room temperature, add 75 μL of anhydrous ethanol, mix thoroughly, and use an enzyme-linked immunosorbent assay to detect at 570 nm to measure the absorbance of each well.
[0091]
[0092] Where: A2 is the OD of the sample group 570 Value, A1 is the sample zeroing group OD 570 A4 is the OD of the blank control group. 570 A3 is the OD of the blank zero adjustment group. 570 value.
[0093] 5.5 Experimental Results
[0094] Collagenase IV can degrade gelatin and decompose it into small molecules such as peptides and amino acids. The degradation products react with ninhydrin to form a colorimetric reaction. The colorimetric analysis at a wavelength of 570 nm can determine the amino acid content in the sample, thereby reflecting the activity of collagenase IV. In this experiment, the test sample was treated with collagenase and the OD was detected. 570 Values were used to determine whether the relevant extracts of the Lijiang Snow Ganoderma disclosed in the present invention could inhibit the activity of collagenase.
[0095] The results of the effects of the test samples on the inhibition of collagenase activity are shown in Table 3.
[0096] Table 3 Inhibition rate of different Lijiang Snow Ganoderma extracts on collagenase activity
[0097] Lijiang Snow Ganoderma Extract Sample Collagenase activity inhibition rate (Mean±SD) Extract A 60.72％±0.49％ Extract B 21.56％±0.68％
[0098] The results show that both Lijiang Snow Ganoderma Extract A and Lijiang Snow Ganoderma Extract B have an inhibitory effect on collagenase. The experimental results show that the Lijiang Snow Ganoderma Extract disclosed in the present invention has a significant ability to inhibit collagenase activity, has the effect of increasing skin elasticity and skin firmness, and can be used for anti-aging.
[0099] In the present disclosure, relational terms such as first and second, etc. are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the statement "comprise one..." do not exclude the presence of other identical elements in the process, method, article or terminal device including the elements.
[0100] Although the specific embodiments of the present disclosure are described for the purpose of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the present disclosure. These modifications or improvements should all fall within the scope of the appended claims of the present disclosure.
Claims
1. A Lijiang Snow Ganoderma extract, characterized in that: Calculated on the basis of dry powder, the total saponin content of the extract is not less than 70 g / kg.
2. The Lijiang Snow Ganoderma extract according to claim 1, characterized in that Calculated on the basis of dry powder, the total saponin content of the extract is not less than 90 g / kg.
3. The method for preparing the Lijiang Snow Ganoderma extract according to claim 1 or 2, comprising an extraction step and a separation step, characterized in that: In the extraction step, 70-95% ethanol is used to extract the Lijiang Snow Ganoderma.
4. The preparation method according to claim 3, characterized in that: In the extraction step, 95% ethanol is used to extract the Lijiang Snow Ganoderma.
5. Use of the Lijiang Snow Ganoderma extract according to claim 1 or 2, or the Lijiang Snow Ganoderma extract prepared by the preparation method according to claim 3 or 4 in the preparation of cosmetics.
6. The use according to claim 5, characterized in that The cosmetics are used for caring for the skin or mucous membranes.
7. Use of the Lijiang Snow Ganoderma extract according to claim 1 or 2, or the Lijiang Snow Ganoderma extract prepared by the preparation method according to claim 3 or 4 in preparing an anti-aging composition.
8. Use of the Lijiang Snow Ganoderma extract according to claim 1 or 2, or the Lijiang Snow Ganoderma extract prepared by the preparation method according to claim 3 or 4, in the preparation of a composition for inhibiting collagenase activity, increasing skin elasticity and / or improving skin firmness.
9. Use of the Lijiang Snow Ganoderma extract according to claim 1 or 2, or the Lijiang Snow Ganoderma extract prepared by the preparation method according to claim 3 or 4 in preparing a composition for soothing, moisturizing and / or repairing.
10. Use of the Lijiang Snow Ganoderma extract according to claim 1 or 2, or the Lijiang Snow Ganoderma extract prepared by the preparation method according to claim 3 or 4 in preparing a composition for inhibiting hyaluronidase activity and / or repairing the skin barrier.