CN115957164A - Compound extract, application thereof and cosmetics - Google Patents
Compound extract, application thereof and cosmetics Download PDFInfo
- Publication number
- CN115957164A CN115957164A CN202211647277.4A CN202211647277A CN115957164A CN 115957164 A CN115957164 A CN 115957164A CN 202211647277 A CN202211647277 A CN 202211647277A CN 115957164 A CN115957164 A CN 115957164A
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- CN
- China
- Prior art keywords
- extract
- sample
- ginseng
- extraction
- ganoderma lucidum
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Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of plant extraction, and particularly provides a compound extract, application thereof and cosmetics. Compared with the prior art, the compound extract obtained according to the specific proportion has antioxidant and anti-aging activities; compared with a single moutan bark extract, a ginseng extract or a ganoderma lucidum extract, the synergistic effect on the antioxidant activity is remarkable.
Description
Technical Field
The invention relates to the technical field of plant extraction, and particularly relates to a composite extract, application thereof and cosmetics.
Background
In recent years, cosmetics of a plant concept have been popular, and development thereof have become a mainstream trend in the cosmetics industry. 168 Chinese herbal medicines for beautifying in the traditional Chinese herbal medicines are summarized in Ben Cao gang mu, and at present, plant raw materials are mostly applied in cosmetics in the form of extracts. For color cosmetics, products with skin nourishing and caring functions will make the products have a qualitative leap.
Cortex moutan is dried root bark of Paeonia suffruticosa Andr. Produced in Anhui, sichuan, henan, shandong, etc. Bitter and pungent, slightly cold, enter heart, liver and kidney meridians, have low toxicity, and have the efficacies of clearing heat and cooling blood, promoting blood circulation and removing blood stasis, and relieving deficiency heat and the like.
Ganoderma lucidum is a precious Chinese medicinal material called 'strengthening body resistance, consolidating constitution, nourishing and strengthening', belongs to a medicinal fungus of Polyporaceae, and has been used for treating diseases such as liver disease, high cholesterol, hyperlipemia and the like. The main antioxidant active ingredients in ganoderma lucidum are considered as ganoderma triterpene and ganoderma polysaccharide. The ganoderma lucidum is divided into three growth stages: spore, hypha, fruiting body, and Ganoderma as the second growth stage.
Ginseng is a perennial herb of the genus Panax of the family Araliaceae. The stems, leaves, flowers, fruits and processing by-products of ginseng are light industrial materials, and can be processed into commercial products such as cigarettes, wines, teas, crystals, pastes, etc., containing ginseng components.
Related to the compounding of the moutan bark extract, the lucid ganoderma extract and the ginseng extract, and the application of the compounded product in an antioxidant product, no relevant research is available in the prior art.
Disclosure of Invention
In view of this, the invention aims to provide a composite extract, application thereof and cosmetics, wherein the composite extract has antioxidant and anti-aging activities; compared with a single moutan bark extract, a ginseng extract or a ganoderma lucidum extract, the synergistic effect on the antioxidant effect is remarkable.
In order to achieve the purpose, the invention adopts the following technical scheme:
a compound extract comprises cortex moutan extract, ginseng radix extract and Ganoderma extract.
The main component of the tree peony bark extract is paeonol, and the paeonol can obviously absorb UVB ultraviolet rays, has wide wavelength coverage and can be used as a sun-screening agent of cosmetics; the wide pharmacological action of paeonol is mainly based on the comprehensive action of antioxidant and anti-inflammatory mechanisms of the paeonol, and the application of the paeonol in clinical practice is mainly focused on two parts, wherein one part is paeonol which is used as ointment and is used for treating skin inflammation, and the anti-inflammation of the paeonol has the effect of preventing and treating various skin diseases such as dampness and the like. Considering that lip products can meet the requirement of improving aging caused by lip inflammation for consumers, paeonol is taken as a main considered active substance in the cortex moutan raw material in subsequent experiments.
The composite extract comprises the following components in parts by weight:
10-75 parts of moutan bark extract;
5-60 parts of ginseng extract;
4 to 80 parts of ganoderma lucidum extract.
The composite extract disclosed by the invention preferably comprises the following components in parts by weight:
35-72 parts of moutan bark extract;
12-46 parts of ginseng extract;
4 to 49 parts of ganoderma lucidum extract.
In one embodiment of the invention, the composite extract comprises the following components in parts by weight:
71 parts of moutan bark extract;
12 parts of ginseng extract;
and 17 parts of ganoderma lucidum extract.
In one embodiment of the invention, the composite extract comprises the following components in parts by weight:
50 parts of moutan bark extract;
46 parts of ginseng extract;
4 parts of ganoderma lucidum extract.
In the present invention, the preparation method of the moutan bark extract comprises: extracting the tree peony bark by using an extraction solvent, wherein the extraction temperature is 20-30 ℃, the material-liquid ratio is 1 (10-15), and the extraction time is 50-80 min;
the extraction solvent consists of 25wt% red rice oil, 25wt% ethanol and 50wt% gtcc;
the frequency of the ultrasonic wave is 20-30 kHz.
The preparation method of the moutan bark extract preferably comprises the following steps: extracting cortex moutan with an extraction solvent at normal temperature at a material-liquid ratio of 1;
the frequency of the ultrasound was 25.6kHz.
The preparation method of the moutan bark extract of the present invention preferably comprises: taking cortex moutan powder, adding mixed extracting agents (25% red rice oil, 25% ethanol and 50% GTCC) into the powder at normal temperature according to the proportion of the material-liquid ratio of 1 to 15, simultaneously carrying out ultrasonic treatment at 25.6kHz, keeping the initial temperature consistent, stirring at 60r/min, carrying out ultrasonic extraction for 60min, carrying out suction filtration on filter paper to remove medicine residues, carrying out rotary evaporation to remove ethanol, and complementing the loss weight of each step with mixed oil (red rice oil: GTCC = 1:2) to obtain the cortex moutan extract.
GTCC in the invention is high-purity grease formed by esterifying caprylic/capric acid and glycerol, has good spreadability and enables the skin to have a smooth but not greasy feeling; is easily absorbed by the skin; the skin care cream plays a good role in the uniformity and fineness of cosmetics and enables the skin to be smooth and glossy; meanwhile, the product can be used as a base material of moisturizing factors, a stabilizing agent of cosmetics, an antifreezing agent and a homogenizing agent. The red rice belongs to a medicine and food dual-purpose resource, is rich in protein, amino acid, vitamin, trace elements and functional active ingredients, and reportedly has good anti-inflammatory and antioxidant effects. The oryzanol in the red rice has good effect of stabilizing fat and grease, and can accelerate blood circulation and metabolism of skin; absorbing ultraviolet rays, preventing erythema caused by ultraviolet rays, and inhibiting tyrosinase activity; can be used as moisturizer, skin conditioner, whitening and freckle removing agent and antioxidant in cosmetics, and has the functions of nourishing circulation, nourishing skin and preventing skin aging. The red rice oil as natural vegetable oil has stable quality and low viscosity, and is not easy to cause greasy feeling when being smeared; has good affinity and permeability with skin; the product is rich in natural active components such as oryzanol, vitamin E, sitosterol, etc. In addition, the red rice oil can be extracted from various plant raw materials to obtain abundant liposoluble active ingredients with different polarities, such as paeonol, ganoderma lucidum triterpene, ginsenoside and the like, so that the oil extract has remarkable effect.
In the invention, the ginseng extract is a red ginseng rootlet extract. Ginseng radix Rubri has antioxidant, antiaging, and antifatigue effects. The ginsenoside is the main active component in Ginseng radix Rubri, is amphoteric substance, and has antioxidant and antiaging biological activity.
In the present invention, the preparation method of the ginseng extract comprises: extracting ginseng by using an extraction solvent, wherein the extraction temperature is 20-30 ℃, the material-liquid ratio is 1 (10-15), and the extraction time is 50-80 min;
the extraction solvent consists of 25wt% red rice oil, 25wt% ethanol and 50wt% gtcc;
the frequency of the ultrasonic wave is 20-30 kHz.
The preparation method of the ginseng extract preferably comprises: extracting ginseng with an extraction solvent at normal temperature in a material-liquid ratio of 1;
the frequency of the ultrasound was 25.6kHz.
The preparation method of the ginseng extract of the present invention preferably includes: taking ginseng powder, adding a mixed extractant (25% red rice oil, 25% ethanol and 50% GTCC) into the powder at normal temperature according to the proportion of the material-liquid ratio of 1 to 15, simultaneously carrying out ultrasonic extraction at 25.6kHz while keeping the initial temperature consistent, stirring at 60r/min, carrying out ultrasonic extraction for 60min, carrying out suction filtration on filter paper to remove medicine residues, carrying out rotary evaporation to remove ethanol, and supplementing the loss weight of mixed oil (red rice oil: GTCC = 1:2) in each step to obtain the ginseng extract.
In the invention, the ganoderma lucidum extract is ganoderma lucidum fruiting body extract. Ganoderma lucidum is recorded in Shen nong Ben Cao Jing and has the effects of strengthening body resistance, consolidating constitution, prolonging life and the like, and the modern medical research reveals that the Ganoderma lucidum has the pharmacological effects of resisting tumor, enhancing immunity, reducing blood sugar and the like, and mainly contains active ingredients such as triterpenes, polysaccharides, nucleosides, alkaloids and the like. The ganoderma triterpene has high lipid solubility, has activities of oxidation resistance, antibiosis, antivirus and the like, and is one of important indexes for identifying the quality of the ganoderma lucidum.
In the invention, the preparation method of the ganoderma lucidum extract comprises the following steps: extracting the lucid ganoderma by using an extraction solvent, wherein the extraction temperature is 20-30 ℃, the material-liquid ratio is 1 (10-15), and the extraction time is 50-80 min;
the extraction solvent consists of 25wt% red rice oil, 25wt% ethanol and 50wt% gtcc;
the frequency of the ultrasonic wave is 20-30 kHz.
The preparation method of the ganoderma lucidum extract preferably comprises the following steps: extracting Ganoderma with extraction solvent at normal temperature at a ratio of 1:15 for 60min;
the frequency of the ultrasound was 25.6kHz.
The preparation method of the ganoderma lucidum extract preferably comprises the following steps: taking ganoderma lucidum powder, adding a mixed extracting agent (25% red rice oil, 25% ethanol and 50% GTCC) into the powder at normal temperature according to the proportion of 1 to 15, simultaneously carrying out ultrasonic treatment at 25.6kHz, keeping the initial temperature consistent, stirring at 60r/min, carrying out ultrasonic extraction for 60min, carrying out suction filtration on filter paper to remove medicine residues, carrying out rotary evaporation to remove ethanol, and supplementing the loss weight of mixed oil (red rice oil: GTCC = 1:2) in each step to obtain the ganoderma lucidum extract.
The invention also provides application of the composite extract in preparing an antioxidant product.
In the present invention, the oxidation resistance is to scavenge DPPH radicals.
The invention also provides cosmetics comprising the compound extract and cosmetically acceptable auxiliary materials.
The cosmetic of the present invention is preferably a lotion, an emulsion, a skin base, essence, a mask, a sunscreen cream, a cream, an eye cream, a hand cream or a body cream; the facial mask is preferably a patch type facial mask or a smearing type facial mask; the essence is preferably essence water or essence oil.
The auxiliary material is a cosmetically acceptable auxiliary material, preferably one or more of a solvent, a thickening agent, a humectant, a film forming agent, a liposome, a preservative, a colorant, a pH regulator and essence; the solvent is preferably water or an alcohol solvent.
The invention adopts grease to extract fat-soluble active ingredients in natural products (ganoderma lucidum, red ginseng, tree peony bark and red rice oil) with homology of medicine and food, compounds the extract and evaluates the antioxidant and anti-aging effects, calculates the degree of coordination of compound samples by an isoradiometric analysis method, inspects the synergistic effect among single samples, provides a method and a basis for the activity evaluation of oil extracts of the compound natural products and the feasibility of the oil extracts in color cosmetics application, and has the following beneficial effects:
(1) Most of the traditional oil extraction methods for medicinal plants are that vegetable oil is used for frying and extracting raw materials at high temperature, and the high-temperature condition of the traditional process not only can easily damage active ingredients in the plants, but also can easily cause rancidity of the vegetable oil. Compared with the traditional frying process, the composite extract sample has the advantages of less loss of fat-soluble active ingredients, good sample stability, simple operation process and low equipment requirement, and the oil extract can be directly used in color cosmetics products after ethanol is removed by rotary evaporation. In addition, the oryzanol rich in the red rice oil improves the oxidation resistance and the moistening degree of the sample; GTCC is high-purity grease formed by esterifying caprylic/capric acid and glycerol, has good spreadability, and can enhance the stability and solubility of a sample.
(2) The invention utilizes a biochemical method to respectively evaluate the antioxidant and anti-aging activities of the compound extracts of the ganoderma lucidum, the ginseng and the tree peony bark from three indexes of DPPH free radical scavenging capacity, ABTS free radical reducing capacity and elastase inhibiting capacity, and simultaneously, calculates the degree of synergy of compound samples by an equal radiation analysis method to investigate the synergistic effect among single oil extracts.
Experiments prove that the synergy of the combined K2# in the compound combination is the highest and 2.535 in DPPH free radical scavenging capacity, and the corresponding combination proportion is 50% of peony oil extract, 45.8% of ginseng oil extract and 4.17% of ganoderma oil extract; the synergy of the combination K1# is 2.236, the corresponding combination proportion is 71 percent of tree peony bark oil extract, 12 percent of ginseng oil extract and 17 percent of ganoderma lucidum oil extract, and the synergy of the K1# and the K2# has no statistical difference.
Drawings
FIG. 1 is a graph comparing data from pre-experiments using different extraction agents to extract paeonol;
FIG. 2 is a graph comparing experimental data of paeonol extraction rate under different extraction conditions in an orthogonal experiment;
FIG. 3 is a graph comparing the activity of extracts of a single plant;
FIG. 4 is a graph comparing DPPH free radical scavenging IC50 values for extracts from single plants and for reconstituted samples;
FIG. 5 is a graph comparing VC equivalent values for extracts from single plants and reconstituted samples;
FIG. 6 is a front view of an equivalent surface of DPPH radical scavenging ability of a compounded sample;
FIG. 7 is a left side view of the equivalent surface of DPPH radical scavenging ability of the compounded sample;
FIG. 8 is a top view of the equivalent surface of the DPPH radical scavenging capability of the compounded sample;
FIG. 9 is a graph comparing DPPH radical scavenging ability and elastase inhibitory ability of the formulated samples.
FIG. 10 is a graph comparing elastase inhibitory activity of the compounded samples with that of the single samples.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below with reference to embodiments of the present invention, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
To further illustrate the present invention, the following examples are provided for illustration. The starting materials used in the following examples of the present invention are all commercially available products. The raw materials used in the examples of the present invention were commercially available products, with GTCC purchased from IOI eschecem (M) sdn.
1. Preparation of extracts of tree peony bark, red ginseng rootlets and red ganoderma sporophore and analysis of main active ingredients
1.1 optimization of the extraction Process
Because of the excellent lipid solubility and anti-inflammation of paeonol and the relatively low cost of raw materials of cortex moutan, the extraction agent and the extraction process are optimized by considering the target substance which takes paeonol as an active ingredient.
Comparative test
Ultrasonic extracting with oleum Olivarum, red rice oil, GTCC, and 2EHP; and respectively fusing olive oil, red rice oil, GTCC (gas chromatography) and 2EHP (ethanol-ethanol) with the volume ratio of 1:1 to perform auxiliary extraction of paeonol, and taking the extraction rate of the paeonol and the physical state of a sample as evaluation indexes (the extraction process is not optimized, namely the ultrasonic time, the ultrasonic frequency and the like are not considered). The results are shown in FIG. 1.
As can be seen from FIG. 1, the extraction yield under ethanol-assisted extraction is higher than that of single extraction solvent, wherein GTCC-ethanol and red rice oil-ethanol are higher. The extract obtained by extracting cortex moutan after compounding olive oil and ethanol is viscous and has strange odor, and 2EHP is limited in use in cosmetic industry. The red rice oil is rich in oryzanol, has good oxidation resistance, stable GTCC and strong fluidity, so the red rice oil and the GTCC are finally selected as main components of an extractant, and the absolute ethyl alcohol is used for auxiliary extraction, so the usage amount of the absolute ethyl alcohol is reduced as much as possible under the condition of improving the extraction rate of paeonol as much as possible.
1.1.1 preparation of the extract
The moutan bark powder was placed in a 250mL beaker and orthogonal experiments were performed according to Table 1 and Table 2 (the total amount of extractant was kept the same). Each group of experiments is repeated for 3 times, the initial temperature is kept consistent, stirring is carried out at 60r/min, filter paper is filtered (centrifugation is carried out at 4000-5000 rpm if turbidity exists), medicine residues are removed, ethanol is removed by rotary evaporation (the rotary evaporation temperature is 40 ℃, the rotating speed is constant, the liquid level in a water bath kettle exceeds the liquid level of a flask, the time used by rotary evaporation in different ethanol proportions is recorded, different parallel intervals in the same level are kept consistent), and the filtrate is weighed to correspond to the weight loss of the oil compensation.
Taking about 0.25g of oil sample into a 10mL centrifuge tube, dissolving with 5mL of methanol, centrifuging at 8000r/min for 10min, taking supernatant, storing in a liquid storage bottle, and measuring.
The low frequency was 25.6kHz, the medium frequency was 45.8kHz and the high frequency was 69.8kHz.
TABLE 1 levels of factors
TABLE 2 orthogonal experimental design sheet
1.1.2 detection of Paeonol content in extract
The liquid chromatography conditions were as follows. And (3) chromatographic column: eritt C18 Hypersil ODS 5 μm (4.60 mm. Times.250 mm); mobile phase: b =0.1% phosphoric acid: acetonitrile, isocratic elution a: B = 25; analysis time: 5min; the flow rate is 1.0mL/min; the column temperature is 30 ℃; the sample volume is 10 mu L; detection wavelength: 274nm.
0.2mg/mL paeonol standard stock solution: accurately weighing 1mg of paeonol standard substance, placing in a 5mL brown volumetric flask, dissolving in methanol, diluting to constant volume, shaking up, and storing at 4 deg.C.
Evaluation of the method: using standard paeonol stock solution to prepare standard paeonol solutions with the concentrations of 10ng/mL, 50ng/mL, 500ng/mL, 1 mu g/mL, 10 mu g/mL and 50 mu g/mL respectively. Using HPLC analysis, each concentration is injected for 6 times, the peak area average value is taken, a standard curve is drawn, and the precision is calculated. And calculating the detection limit and the quantification limit according to the chromatographic data of the paeonol standard solution with the concentration of 10 ng/mL.
The sample solution in the volumetric flask was taken in an amount of 1mL, and the liquid phase vial was filled with a filtration membrane (0.45 μm organic filter). And (4) carrying out HPLC sample injection for 3 times, taking an average value, calculating the concentration of a sample solution by using a standard curve, and calculating the content of paeonol in the sample.
1.1.3 results
The results of the extraction rate of paeonol by the orthogonal experiment are shown in the following table 3, and the influence on the extraction rate is as follows: extracting agent > ultrasonic frequency > material-liquid ratio > ultrasonic time. And the optimal extraction process comprises the following steps: extracting with mixed extractant of 25% red rice oil, 25% absolute ethyl alcohol, 50% GTCC at low frequency, material-liquid ratio of 1. In the experiment, the extraction process also obtains the highest extraction rate. The results are shown in FIG. 2.
TABLE 3 Paeonol extraction yield results table
1.2 preparation of cortex moutan extract and detection of paeonol content
1.2.1 preparation of moutan cortex Radicis extract
The moutan bark powder was placed in a 500mL beaker, and the mixed extractant (25% red rice oil, 50% gtcc,25% absolute ethanol) was added in a ratio of 1 to 15, and each experiment was repeated 3 times. And (3) carrying out low frequency, keeping the initial temperature consistent, stirring at 60r/min, carrying out ultrasonic extraction for 60min, carrying out suction filtration by using filter paper to remove medicine residues, carrying out rotary evaporation to remove ethanol, and weighing the filtrate to correspond to the weight loss of the oil supplement foot. The final extract was opaque oil, light yellow-brown, with a moutan bark smell.
And (3) putting about 0.25g of oil sample into a 10mL centrifuge tube, adding 5mL of methanol for dissolving, centrifuging for 10min at 8000r/min, taking supernatant, storing in a liquid storage bottle, and detecting.
1.2.2 method for detecting content of paeonol
Same as 1.1.2.
1.3 preparation of Leptoradix Ginseng extract and detection of ginsenoside content
1.3.1 preparation of Leptoradix Ginseng extract
The powder of Leptoradix Ginseng Rubra was placed in a 500mL beaker and 3 replicates of each experiment were performed by adding the mixed extractant (25% Red Rice oil, 50% GTCC,25% absolute ethanol) at 1. And (3) carrying out low frequency, keeping the initial temperature consistent, stirring at 60r/min, carrying out ultrasonic extraction for 60min, carrying out suction filtration by using filter paper to remove medicine residues, carrying out rotary evaporation to remove ethanol, and weighing the filtrate to correspond to the weight loss of the oil supplement foot. The final extract was a clear oil, light yellow-brown, characteristic odor of the plant.
And (3) putting about 0.25g of oil sample into a 10mL centrifuge tube, adding 5mL of methanol for dissolving, centrifuging for 10min at 8000r/min, taking supernatant, storing in a liquid storage bottle, and detecting.
1.3.2 method for detecting content of ginsenoside
The liquid chromatography conditions were as follows. A chromatographic column: elite C18 Hypersil ODS 5 μm (Φ 4.60 mm. Times.250 mm); mobile phase: b = water: acetonitrile, gradient elution procedure see table 4; analysis time: 80min; the flow rate is 1.0mL/min; the column temperature is 30 ℃; the sample volume is 10 mu L; detection wavelength: 203nm.
Note that the oil samples should be enriched for a low level of the target active.
Table 4 HPLC gradient elution procedure for detecting ginsenoside
Ginsenoside mixed standard stock solution: precisely weighing 10mg of ginsenoside Rg1 standard substance, 10mg of ginsenoside Re standard substance and 10mg of ginsenoside Rb1 standard substance respectively, placing all in a 5mL brown volumetric flask, dissolving with methanol, and metering to obtain 2mg/mL ginsenoside mixed standard stock solution.
The evaluation method comprises the following steps: ginsenoside standard-mixing stock solutions are used to prepare ginsenoside standard-mixing solutions with the concentrations of 1 mug/mL, 10 mug/mL, 50 mug/mL, 0.1mg/mL, 0.5mg/mL and 2mg/mL respectively. Using HPLC analysis, injecting sample for 3 times for each concentration, taking peak area average value, drawing standard curve, calculating precision. And calculating the detection limit and the quantification limit according to chromatographic data of the paeonol standard solution with the concentration of 1 mu g/mL.
The sample solution in the volumetric flask was taken in an amount of 1mL, and the liquid phase vial was filled with a filtration membrane (0.45 μm organic filter). Sampling by HPLC for 3 times, taking an average value, calculating the concentration of the sample solution by using a standard curve, and calculating the content of ginsenoside in the sample.
1.4 preparation of Ganoderma lucidum fruiting body extract and detection of Ganoderma lucidum triterpene content
1.4.1 preparation of Ganoderma lucidum fruiting body extract
Ganoderma lucidum fruit body powder was placed in a 500mL beaker and 3 replicates of each experiment were performed with 1. Low frequency, keeping the initial temperature consistent, stirring at 60r/min, ultrasonic extracting for 60min, filtering with filter paper to remove residue, rotary steaming to remove ethanol, and weighing filtrate to correspond to weight loss of oil supplement. The obtained extract is transparent oil, reddish brown, and has special sweet taste of Ganoderma.
Other fat-soluble active ingredients in the sample can perform other color reaction with perchloric acid-vanillin, so that the absorbance of the sample is greatly changed. Therefore, the normal hexane is taken as a simulated extractant of the mixed grease extractant, the extraction of the ganoderma lucidum triterpenes is carried out according to the same extraction conditions, and the normal hexane-ganoderma lucidum extract is taken as a sample to detect the triterpenoid content in the sample.
1.4.2 method for detecting content of ganoderma triterpene
Oleanolic acid standard solution: precisely weighing 2mg of oleanolic acid standard, placing the standard in a 5mL brown volumetric flask, dissolving by using methanol and carrying out volume metering to obtain 0.4mg/mL oleanolic acid standard solution.
Vanillin glacial acetic acid solution: accurately weighing 0.5g of vanillin, placing the vanillin in a 10mL brown volumetric flask, adding glacial acetic acid to dissolve, fixing the volume and oscillating to obtain a vanillin glacial acetic acid solution with the concentration of 50 mg/mL.
Preparation of a standard curve: precisely measuring 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL and 0.8mL of oleanolic acid standard solution, respectively putting the standard solution into 15mL test tubes with plugs, volatilizing, cooling, precisely adding 0.2mL of newly prepared vanillin glacial acetic acid solution and 0.8mL of perchloric acid, shaking up, heating in 70 ℃ water bath for 15 minutes, immediately putting in ice bath for cooling for 5 minutes, taking out, precisely adding 4mL of ethyl acetate, and shaking up. According to the above operation, the absorbance is measured at the wavelength of 546nm by using a spectrophotometer without adding oleanolic acid standard solution as a blank, and a standard curve is drawn by taking the absorbance as the ordinate and taking the concentration as the abscissa.
Precisely measuring 0.2mL of the test solution, placing the test solution in a 15mL test tube with a plug, measuring the absorbance by the same method from the 'volatilizing', reading the content of oleanolic acid in the test solution from the standard curve, and calculating to obtain the oleanolic acid test solution.
1.5 analysis results of the content of the Main active ingredient
The paeonol content of the moutan bark extract, the ginsenoside content of the red ginseng rootlet extract, and the ganoderma triterpene content of the ganoderma lucidum fruiting body extract are shown in Table 5.
TABLE 5 content (mg/g) of major active ingredients in extracts of moutan cortex, ginseng radix Rubri and Ganoderma lucidum fruit body
2. Research on activity of three extracts and research on compounding and synergy of three extracts
2.1 Single Activity Capacity determination of three extracts
2.1.1DPPH radical scavenging Capacity determination
DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) is a relatively stable lipid radical with a free electron on N, its ethanol solution is purple in color, and has a maximum absorption peak at 517 nm. After the antioxidant is added, DPPH captures an electron to be paired with a free electron, the purple color fades, the DPPH becomes a colorless substance, the absorption at 517nm disappears, and the fading degree is in a quantitative relation with the number of the accepted electrons. The change of the light absorption value after the reaction of DPPH free radical and sample liquid is detected by a spectrophotometer according to the principle, and the capability of the sample for providing hydrogen atoms and eliminating free radical oxidation resistance can be detected.
Respectively diluting cortex moutan extract, leptoradix Ginseng Rubra extract, and Ganoderma fruiting body extract with n-hexane ethanol solution (n-hexane: ethanol = 1:1) at different concentrations.
In a 96-well plate, 100 μ L of sample solution with different concentrations and 100 μ L of 0.25mmol/L DPPH-ethanol solution are respectively added by a pipette, mixed evenly, and after standing for 30min, the light absorption value of the sample is measured at 517 nm. Each sample was subjected to 3 replicates, the mean value was taken as A1, and VC was used as a positive control. DPPH clearance was calculated as follows:
wherein A0 is the absorbance at 517nm of 100. Mu.L of sample solvent + 100. Mu.L of DPPH-ethanol; a1 is the absorbance at 517nm of 100. Mu.L of sample solution + 100. Mu.L of DPPH-ethanol; a2 is the absorbance at 517nm of 100. Mu.L of the sample solution + 100. Mu.L of ethanol. Each set was in triplicate (only one sample was made in one 96-well plate).
2.1.2ABTS radical reduction Capacity determination
ABTS is oxidized into green ABTS under the action of proper oxidant + And measuring the absorbance of ABTS at the wavelength of 734nm to obtain the total antioxidant capacity of the sample.
Preparation of ABTS stock solution (7 mmol/L): ABTS 0.09602g was taken and 25mL of distilled water was added. (MW = 548.7). K 2 S 2 O 8 Stock solution (140 mmol/L): get K 2 S 2 O 8 0.94612g, 25mL distilled water was added. (M) W = 270.32). And (3) dissolving and diluting the G1#, G2#, and G3# oil extract samples by using absolute ethyl alcohol. Exactly measure 5mL 7mmol/LABTS solution and 88 μ L140 mmol/L (final concentration 2.45 mmol/L) potassium persulfate (K) 2 S 2 O 8 ) And mixing the solution uniformly to prepare ABTS working mother liquor. Storing the mixture for 12-16h at room temperature in dark. Before use, the samples were diluted with absolute ethanol (or PBS solution with pH = 7.4) and the absorbance of ABTS working solution minus the blank was required to be 0.700 ± 0.020 for a 734. Accurately measuring 10 μ L of sample solution (positive control solution) with each concentration gradient and 200 μ L of ABTS working solution in a 96-well plate, mixing gently, standing at room temperature for 6min, and measuring absorbance at 734nm with an enzyme-linked immunosorbent assay. The total antioxidant capacity was calculated as follows:
wherein A is 0 The reaction system does not contain a sample, namely, absolute ethyl alcohol is used for replacing the absorbance value of the sample; a. The 1 The absorbance value of a sample contained in a reaction system; a. The 2 The absorbance value of the reaction system containing the sample, but replacing ABTS with absolute ethyl alcohol.
A 0 : sample lysis reagent 10. Mu.L + ABTS 200. Mu.L;
A 1 sample 10 uL + ABTS 200 uL
A 2 Sample 10. Mu.L + ethanol 200. Mu.L
2.1.3 Elastase inhibitory Capacity assay
Elastase is one of the most important enzymes in the matrix metalloprotease family, is synthesized and secreted by fibroblasts, and can degrade elastin in skin to cause skin aging.
Method for determination of Porcine Pancreatic Elastase (PPE) inhibitory activity: using AAANA as a substrate, 100. Mu.L of 50mmol/L Tris-HCl buffer (pH 8.0) was mixed with 50. Mu.L of sample solutions of different mass concentrations, 25. Mu.L of 1.015mmol/L AAANA solution was added, incubation was carried out at 37 ℃ for 15min, followed by addition of 25. Mu.L elastase solution (0.3U/mL), absorbance value was measured at 410nm after 15min, elastase solution and AAANA solution were each formulated with 50mmol/L Tris-HCl buffer (pH 8.0), and EGCG was used as a positive control, and 3 replicates were measured. The inhibition rate of elastase by the sample was calculated according to the following formula.
In the formula: a is the absorbance of the reaction solution containing no sample; b is the absorbance of the reaction solution containing the sample.
2.2 compounding ternary extracts and measuring antioxidant capacity
2.2.1 sample compounding
Mixing the cortex moutan extract obtained in 1.2, the red ginseng rootlet extract obtained in 1.3 and the ganoderma lucidum fruiting body extract obtained in 1.4 according to the proportion in a table 6, and taking DPPH free radical scavenging capacity as an index for evaluating the compounding effect. Table 6 shows the formulation ratios obtained by designing the formulation ratios using a uniform design recipe, and analyzing each formulation ratio by using the calculation of the degree of synergy and the equivalent surface model.
TABLE 6 Single extract sample combination table (volume fraction)
2.2.2 measurement of DPPH free radical scavenging Capacity of Compound sample
The same as 2.1.1.
2.2.3 calculation of degree of synergy of Compound sample
Some compounds have weaker activity in a single state and show stronger antioxidant effect in a mixed state, namely, positive synergistic effect, namely, synergistic effect for short, exists among the components. Such as VE and VC. At present, an analysis method of antioxidant synergistic effect mainly depends on the research of pharmacodynamics in the field of medicine, and comprises a simple effect addition model, an independent model, an isoradiometric analysis method and a response surface method. The cooperation degree of the compound sample is analyzed by the equal radiation analysis method for the experiment.
The DPPH radical scavenging ability measurement IC50 values of the samples described in 2.1.1 and 2.2.2 are expressed as the amount of VC with equal effect. The Theoretical Antioxidant Capacity (TAC) of the composite sample is related to the single antioxidant capacity, and the calculation formula is as follows:
TAC＝W a ×T a +W b ×T b +W c ×T C
t in the formula a 、T b 、T C Respectively representing the antioxidant capacity (TE), W of cortex moutan extract, ganoderma lucidum fruiting body extract, and Ginseng radix Leptoradix extract a 、W b 、W c Respectively represents the weight (%) of the antioxidant capacity of the single component with equal dosage in the compound sample of the cortex moutan extract, the ganoderma lucidum fruiting body extract and the red ginseng rootlet extract.
Synergistic Effect (SE) is the ratio of the actual effect to the theoretical effect.
Exhibits synergy when SE is greater than 1; antagonism was observed when SE was less than 1.
And an independent sample T test was performed using SPSS 26.
2.2.4 equivalent surface construction
And constructing an equivalent surface by referring to a Concentration Addition (CA) model in a chemical combined toxicity model. Mathematically the CA model is expressed as:
wherein n is the number of components of the mixture, C i Is the concentration of component i, EC, in the mixture which produces the effect x% x,i Is the concentration at which component i alone causes the x% effect.
And after the equivalent surface is constructed, multiplying the proportion of each compound sample by the IC50 value of the corresponding sample to obtain the corresponding scatter point coordinate of each sample. On both sides of the plane, one side containing the origin of the coordinate system is defined as under the plane, and the other side is on the plane. If the data point is below the plane, the compound sample generates a synergistic effect; data points represent the composite CA model of the composite sample on a plane; the data points are shown on the plane to produce an antagonistic effect.
2.2.5 data statistics
All experiments were repeated at least three times, IC50 values were determined by linear fit, and significance analysis was performed on Theoretical (TAC) and actual (EAC) values using independent sample T-tests, with p <0.05 indicating a statistical difference, p <0.01 indicating a significant statistical difference, and p <0.001 indicating a very significant statistical difference.
2.2.6 measurement of Elastase inhibition ability of Compound sample
Same as 2.1.3.
2.3 results of the experiment
2.3.1 Activity assay results for Single oil extracts
Linear fit was performed on the DPPH radical clearance at different concentrations of the samples to obtain IC50 values for the samples. The IC50 values for the G1#, G2#, and G3# samples are summarized in Table 7. The DPPH free radical scavenging capacity of three single extraction samples is relatively close, the effect is obvious, and the effect is better by taking the DPPH free radical scavenging capacity as a compound assessment index.
Linear fits were made for the clearance of ABTS radicals at different concentrations of the sample to obtain IC50 values for the sample. The IC50 values for the G1#, G2#, and G3# samples are summarized in Table 7. The ABTS free radical reducing capability of the moutan bark extract is more than that of the ginseng extract is more than that of the ganoderma lucidum extract. But the ABTS free radical reducing ability of the three is lower than the DPPH free radical scavenging ability.
Because the elastase measuring system is a water-soluble system, the grease system can not be effectively detected, 95% ethanol is used for replacing grease, the same raw materials are extracted by the same extraction process, H1#, H2#, and H3# samples are obtained, and the elastase inhibition capability of the G1#, G2#, and G3# samples can be simulated and equivalently detected.
And performing linear fitting according to the inhibition rate of the elastase at different concentrations of the sample to obtain the IC50 value of the sample. The IC50 values for the H1#, H2#, and H3# samples are summarized in Table 7. The elastase inhibition ability of the ganoderma lucidum extract is larger than that of the moutan bark extract and that of the ginseng extract.
TABLE 7 IC50 value of antioxidant capacity of sample # G
A comparison of the activity of the extracts of a single plant is shown in FIG. 3. In conclusion, the activities of three oil extracts of ganoderma lucidum, ginseng and peony: DPPH free radical scavenging > ABTS free radical reducing > elastase inhibitory. Specifically, the DPPH free radical scavenging capacity of the three oil extracts is similar; ABTS radical reducing power: peony extract, ginseng extract, ganoderma extract; elastase inhibitory ability: the ganoderma lucidum extract, the peony extract and the ginseng extract. Furthermore, the results of the elastase inhibitory activity measured on each sample extracted with 95% ethanol do not fully represent the elastase inhibitory activity of each oil extract. Therefore, the three oil extracts have the optimal effect of eliminating DPPH free radicals, and the optimal formula optimization is finally carried out by taking the measurement of the DPPH free radical eliminating capacity as an index for evaluating the antioxidant capacity of the compound sample. In FIG. 3, the left Y-axis (0.55-0.75) is the DPPH radical scavenging IC50 value for D-G1#, D-G2#, and D-G3#, i.e., to the left of the dashed line; the right Y axis (0-160) is ABTS free radical scavenging ability and elastase inhibition ability IC50 value of A-G1#, A-G2#, A-G3#, H1#, H2#, and H3 #. D-G1# indicates DPPH radical scavenging ability of peony extract; A-G1# represents ABTS free radical reducing ability of peony extract; h1# indicates the elastase inhibitory activity of the peony extract. G2# and H2# are ginseng extracts, and G3# and H3# are ganoderma lucidum extracts.
2.3.2 DPPH radical scavenging Capacity of Complex oil extracts
G1# is a tree peony bark extract, G2# is a red ginseng rootlet extract, G3# is a ganoderma lucidum fruit body extract, and from the numerical value of the IC50 value of the DPPH free radical scavenging capacity of each compound sample and a single sample, the IC50 values of the DPPH free radical scavenging capacity of the K1#, K2# and K3# samples after compound are all lower than that of each single sample, which shows that the DPPH free radical scavenging capacity of the compound sample is obviously improved, and shows that the DPPH free radical scavenging capacity of the three combinations possibly generates a synergistic effect compared with the single oil extract; the DPPH free radical scavenging capacity IC50 values of the K4# and K5# samples are slightly lower than those of the single samples but have little difference; the DPPH free radical scavenging ability IC50 values of the samples K6# and K7# are slightly higher than those of the single samples. The comparison graph of DPPH free radical scavenging IC50 values for each sample is shown in fig. 4, where: p <0.001 indicates a very significant statistical difference; * ***: statistical differences were more significant for P < 0.0001 than for P < 0.001.
2.3.3 calculation of degree of synergy of Compound sample
The composite and single samples were expressed separately in equal effect VC (how much VC is needed to achieve the same DPPH clearance under the same operating conditions), and the VC equivalent value pairs for the single and composite samples are shown in fig. 5, where: p <0.001 indicates a very significant statistical difference. Theoretical values and degrees of synergy were calculated, and the results are shown in table 8. The K2# has the highest synergy and is 2.535; the K1# synergy is 2.236, the difference is 0.299, and the two have significant difference.
TABLE 8 summary of theoretical values and degrees of synergy for samples
Note: * : p <0.05, indicating a statistical difference; * *: p <0.01, indicating significant statistical difference; * **: p <0.001 indicates a very significant statistical difference.
2.3.4 equivalent plane
The equivalent surface equation is constructed according to the CA model as follows:
wherein x is the G1 sample concentration (%); y is G2 sample concentration (%); z is the G3 sample concentration (%).
Equivalent surfaces are shown in fig. 6-8. On the plane formed by points G1#, G2#, and G3#, the points of the compounded sample below the plane are indicated as synergistic effects, the points above the plane are indicated as antagonistic effects, and the points falling within the plane are indicated as additive effects.
As shown in the following figures, K1# -K5# all showed synergistic effects below the plane, K6# antagonism, and K7# additive effects. Substantially the same as the synergy calculation result. K1# and K2# are similar in color and have the same degree of synergy.
Therefore, in all the combinations, the combination K2# has the highest synergy degree which is 2.535, and the corresponding combination proportion is 50 percent of peony oil extract, 45.8 percent of ginseng oil extract and 4.17 percent of ganoderma lucidum oil extract; the combination K1# synergy, second, was 2.236, which differed from K2# by 0.299 (K1 # did not statistically differ from K2# in synergy). Compared with a single sample, the antioxidant capacity of the K1# and the K2# is remarkably different.
2.3.5 Elastase inhibitory Activity of Compound sample
The elastase inhibition abilities of the K1# and K2# samples were tested, and the results are shown in fig. 9, where the elastase inhibition abilities of the K1# and K2# samples are significantly different. The elastase inhibition abilities of the K1# and K2# samples are greatly different, the elastase inhibition ability of the K1# sample is strong, and in fig. 9, the ratio of: p <0.05, indicating a statistical difference; * *: p <0.01, indicating a significant statistical difference. The elastase inhibition ability of each single sample and the compound sample is compared as shown in fig. 10, and it can be seen that the elastase inhibition ability of the compound sample is not significantly different from that of the single sample.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A composite extract is characterized by comprising a moutan bark extract, a ginseng extract and a ganoderma lucidum extract.
2. The composite extract according to claim 1, wherein the composite extract comprises the following components in parts by weight:
10-75 parts of moutan bark extract;
5-60 parts of ginseng extract;
4 to 80 parts of ganoderma lucidum extract.
3. The composite extract according to claim 2, characterized in that it comprises the following components in parts by weight:
35-72 parts of moutan bark extract;
12-46 parts of ginseng extract;
4-49 parts of ganoderma lucidum extract.
4. The complex extract as claimed in any one of claims 1 to 3, wherein the preparation method of the moutan bark extract comprises: extracting the tree peony bark by using an extraction solvent, wherein the extraction temperature is 20-30 ℃, the material-liquid ratio is 1 (10-15), and the extraction time is 50-80 min;
the extraction solvent consists of 25wt% red rice oil, 25wt% ethanol and 50wt% gtcc;
the frequency of the ultrasonic is 20-30 kHz.
5. The composite extract as claimed in claim 1, wherein the ginseng extract is an extract of red ginseng rootlets.
6. The composite extract as claimed in claim 1 or 5, wherein the preparation method of the ginseng extract comprises: extracting ginseng by using an extraction solvent, wherein the extraction temperature is 20-30 ℃, the material-liquid ratio is 1 (10-15), and the extraction time is 50-80 min;
the extraction solvent consists of 25wt% red rice oil, 25wt% ethanol and 50wt% gtcc;
the frequency of the ultrasonic wave is 20-30 kHz.
7. The composite extract as claimed in claim 1, wherein the ganoderma lucidum extract is a ganoderma lucidum fruiting body extract.
8. The composite extract as claimed in claim 1 or 7, wherein the preparation method of the ganoderma lucidum extract comprises: extracting the lucid ganoderma by using an extraction solvent, wherein the extraction temperature is 20-30 ℃, the material-liquid ratio is 1 (10-15), and the extraction time is 50-80 min;
the extraction solvent consists of 25wt% red rice oil, 25wt% ethanol and 50wt% gtcc;
the frequency of the ultrasonic is 20-30 kHz.
9. Use of a complex extract as defined in any one of claims 1 to 8 for the preparation of an antioxidant product.
10. Cosmetic product, characterized in that it comprises a complex extract according to any one of claims 1 to 8, together with cosmetically acceptable adjuvants.
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