CN116492256A - Paeonia ostii extract and preparation method and application thereof - Google Patents
Paeonia ostii extract and preparation method and application thereof Download PDFInfo
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- CN116492256A CN116492256A CN202310499181.6A CN202310499181A CN116492256A CN 116492256 A CN116492256 A CN 116492256A CN 202310499181 A CN202310499181 A CN 202310499181A CN 116492256 A CN116492256 A CN 116492256A
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- paeonia ostii
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- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 description 1
- 229960004555 rutoside Drugs 0.000 description 1
- 239000009565 shengxuening Substances 0.000 description 1
- 230000037394 skin elasticity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000007103 stamina Effects 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000003041 virtual screening Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
-
- 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 provides a paeonia ostii extract and a preparation method and application thereof, and relates to the technical field of separation and purification. The preparation method of the paeonia ostii extract provided by the invention comprises the following steps: leaching paeonia ostii with ethanol solution, performing solid-liquid separation on the leaching solution after leaching to obtain clear liquid, and drying the clear liquid to obtain the paeonia ostii extract; wherein the volume concentration of the ethanol solution is 10% -70%; the ratio of the leached liquid to the leached liquid is 1:5-1:25g/mL; the leaching temperature is 30-80 ℃. The preparation method is simple, low in cost and high in yield, and the prepared paeonia ostii extract is high in polyphenol, stauroside, coside and flavone, and has good effects of resisting saccharification, allergy, whitening, wrinkle and blood and the like, and can be used as a functional raw material of foods and cosmetics for product development.
Description
Technical Field
The invention relates to the technical field of separation and purification, in particular to a paeonia ostii extract and a preparation method and application thereof.
Background
Paeonia ostii (Paeonia ostii T. Hong et J. X. Zhang), which is large in flower and bright in color, has high ornamental value, and is rich in starch, fat, vitamins, various essential amino acids and trace elements of human body. Dan Feng mu is mostly distributed in Yangtze river and yellow river basin, such as Henan, shandong and Anhui, etc., 2013/10/30 is approved as a new food raw material.
According to the description of Ben Cao gang mu, peony is a traditional medicinal material for clearing heat and detoxicating, has bitter taste and flat nature, has the effects of activating blood and generating blood, and mainly treats the internal heat of blood and dysphoria heat. The paeonia ostii is not only used for ornamental, medical and edible purposes, but also has research and application in the aspect of skin care, and is mainly concentrated on cortex moutan radicis, paeonia suffruticosa seeds and paeonia suffruticosa. The peony root (also called moutan bark and moutan bark) is a traditional Chinese medicine, and a great deal of researches show that the peony root is rich in various active substances such as paeonol, paeonol glycoside, paeoniflorin and the like, and has good antioxidant and anti-aging effects. The peony seed oil produced from peony seeds has good antioxidant capacity, and can be used as base oil or functional components in skin care products. The peony is a characteristic natural biological resource, has long medical and edible history in China, is a good natural nutrition and health care resource, but the preparation and application of the peony extract are not systematically and deeply studied.
The invention patent CN202210082325.3 discloses a preparation method of a peony flavone extract, which adopts an ultrasonic wave and water extraction mode to examine the content of total flavone. The invention patent CN202110917299.7 discloses a preparation method of a peony extract, which adopts a juice squeezing mode by taking fresh petals as raw materials. However, the specific active monomers are not known, and the structure-activity relationship of the extract is not known.
In view of this, the present invention has been made.
Disclosure of Invention
The invention provides a preparation method of a paeonia ostii extract, which is simple, has high content of effective substances, has the effects of resisting saccharification, resisting allergy, whitening, resisting wrinkles and enriching blood, and can be used as an efficacy raw material of foods and cosmetics for product development.
The second object of the present invention is to provide an extract of Paeonia ostii.
A third object of the present invention is to provide an application of the paeonia ostii extract.
In a first aspect, the present invention provides a method for preparing an extract of paeonia ostii, comprising the steps of:
leaching paeonia ostii with ethanol solution, performing solid-liquid separation on the leaching solution after leaching to obtain clear liquid, and drying the clear liquid to obtain the paeonia ostii extract;
the volume concentration of the ethanol solution is 10% -70%;
the ratio of the leached liquid to the leached liquid is 1:5-1:25g/mL;
the leaching temperature is 30-80 ℃.
As a further technical scheme, the volume concentration of the ethanol solution is 30% -70%, preferably 53%.
As a further technical scheme, the ratio of the leaching liquid to the leaching liquid is 1:10-1:20g/mL, preferably 1:10g/mL.
As a further technical scheme, the leaching temperature is 40-60 ℃, preferably 60 ℃.
As a further technical scheme, the leaching time is 30-150min.
As a further technical scheme, the leaching times are 1-3 times, preferably 2 times.
As a further technical scheme, the method further comprises the step of sequentially drying and crushing the paeonia ostii.
As a further technical scheme, the solid-liquid separation method comprises centrifugation or filtration.
In a second aspect, the invention provides a paeonia ostii extract, which is prepared by the preparation method.
In a third aspect, the present invention provides the use of the above-described Paeonia ostii extract in any one of the following a-e:
a. preparing an anti-saccharification product;
b. preparing an antiallergic product;
c. preparing a whitening product;
d. preparing an anti-wrinkle product;
e. preparing the blood replenishing product.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method of the paeonia ostii extract provided by the invention is simple, low in cost and high in yield, and the prepared paeonia ostii extract is high in polyphenol, stauroside, cosmosiin, paeoniflorin and flavone, and the inventor researches show that the paeonia ostii extract has good effects of resisting saccharification, allergy, whitening, wrinkle resistance, enriching blood and the like, and can be used as a raw material with efficacy of foods and cosmetics for product development.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of the technical scheme of the invention;
FIG. 2 is an HPLC chromatogram of the blank, extract, and incubation groups;
FIG. 3 is a total ion flow diagram of 3 components;
FIG. 4 shows the peak 1 identification results;
FIG. 5 shows the peak 2 identification results;
FIG. 6 shows the peak 3 identification results;
FIG. 7 is a graph showing the effect of ethanol concentration on the content of polyphenols, rhoifolin, coside and paeoniflorin in Danfeng peony;
FIG. 8 is a graph showing the effect of ethanol concentration on the flavone content of Paeonia ostii;
FIG. 9 is a graph showing the effect of feed liquid comparison on the levels of paeoniflorin, and polyporus umbellatus;
FIG. 10 shows the effect of feed liquid ratio on the flavone content of Paeonia ostii;
FIG. 11 is a graph showing the effect of extraction time on the content of polyphenols, rhoifolin, coside and paeoniflorin in Paeonia suffruticosa;
FIG. 12 is a graph showing the effect of extraction time on the flavone content of Paeonia ostii;
FIG. 13 is a graph showing the effect of extraction temperature on the content of polyphenols, rhoifolin, coside and paeoniflorin in Paeonia suffruticosa;
FIG. 14 is a graph showing the effect of extraction temperature on the flavone content of Paeonia ostii;
FIG. 15 is a graph showing the effect of the number of extractions on the content of polyphenols, rhoifolin, coside and paeoniflorin in;
FIG. 16 shows the effect of the number of extractions on the flavone content of Paeonia ostii.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In a first aspect, the present invention provides a method for preparing an extract of paeonia ostii, comprising the steps of:
leaching paeonia ostii with ethanol solution, performing solid-liquid separation on the leaching solution after leaching to obtain clear liquid, and drying the clear liquid to obtain the paeonia ostii extract;
the volume concentration of the ethanol solution may be, for example, but not limited to, 10%, 30%, 50%, or 70%;
the feed ratio of the leaching may be, for example, but not limited to, 1:5g/mL, 1:10g/mL, 1:15g/mL, 1:20g/mL, or 1:25g/mL;
the temperature of the leaching may be, for example, but not limited to, 30 ℃,40 ℃,50 ℃, 60 ℃, or 80 ℃.
The preparation method of the paeonia ostii extract provided by the invention is simple, low in cost and high in yield, and the prepared paeonia ostii extract is high in polyphenol, stauroside, cosmosiin, paeoniflorin and flavone, and the inventor researches show that the paeonia ostii extract has good effects of resisting saccharification, allergy, whitening, wrinkle resistance, enriching blood and the like, and can be used as a raw material with efficacy of foods and cosmetics for product development.
In some preferred embodiments, the ethanol solution has a volume concentration of 30% to 70%;
the ratio of the leached liquid to the leached liquid is 1:10-1:20g/mL;
the leaching temperature is 40-60 ℃;
the time of the leaching may be, for example, but not limited to, 30min, 60min, 90min, 120min, or 150min.
In a further preferred embodiment, the volume concentration of the ethanol solution is 53%;
the ratio of the leached liquid to the leached liquid is 1:10g/mL;
the temperature of the leaching is 60 ℃.
By further optimizing and adjusting each condition parameter in the preparation method, the prepared extract has higher contents of polyphenol, rhoifolin, coside, paeoniflorin and flavone.
In some preferred embodiments, the number of leaches may be, for example, but not limited to, 1, 2, or 3, preferably 2.
The extraction times refer to the times of extracting the same raw material by the same extraction method, and the extraction rate of effective substances in the raw material is improved by multiple times of extraction, and the 2 times of extraction times are optimal in comprehensive consideration.
In some preferred embodiments, the leaching further comprises a step of sequentially drying and pulverizing the paeonia ostii, wherein the drying step may be, for example: drying Paeonia suffruticosa at 50-60deg.C; the pulverizing step may be, for example: pulverizing dried Paeonia ostii and sieving with 40 mesh sieve.
In some preferred embodiments, the method of solid-liquid separation comprises centrifugation or filtration, wherein centrifugation may be, for example: the extract was centrifuged at 4000rpm for 10min and the supernatant was taken.
In a second aspect, the invention provides a paeonia ostii extract, which is prepared by the preparation method.
The paeoniflorin extract provided by the invention has high content of polyphenol, rhoifolin, cosmosiin, paeoniflorin and flavone, and has good effects of resisting saccharification, allergy, whitening, wrinkle resistance, enriching blood and the like.
In a third aspect, the present invention provides the use of the above-described Paeonia ostii extract in any one of the following a-e:
a. preparing an anti-saccharification product;
b. preparing an antiallergic product;
c. preparing a whitening product;
d. preparing an anti-wrinkle product;
e. preparing the blood replenishing product.
In some preferred embodiments, the product comprises a pharmaceutical or food or cosmetic product.
The invention is further illustrated by the following specific examples and comparative examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and should not be construed as limiting the invention in any way.
The technical scheme of the invention is shown in fig. 1, and comprises the following steps:
1.3 modes of preparing the extract of the paeonia ostii are selected; the method comprises water extraction, alcohol precipitation and alcohol extraction, wherein the content of components and the activity in vitro are used as indexes, and the optimal extraction method, namely the alcohol extraction, is selected.
2. The method adopts affinity chromatography and molecular docking technology, and has the following definite characteristic components: rhus verniciflua, cosmosiin and paeoniflorin.
3. The ethanol extraction process is optimized in response surface, and the optimal process is determined by taking the content of the components as an index; and controlling the quality of the extract.
4. Preparing the paeonia ostii extract by an optimal process, and evaluating sugar resistance, wrinkle resistance and allergy resistance; meanwhile, a zebra fish model is adopted to evaluate the whitening, anti-wrinkle and blood-replenishing effects of the extract; providing data support for its use.
Test example 1
1. Preparation method of paeonia ostii extract
The preparation method of the paeonia ostii aqueous extract comprises the following steps:
drying Paeonia suffruticosa at 50-60deg.C, pulverizing, and sieving with 40 mesh sieve; accurately weighing 50.00g of paeonia ostii, adding 500mL of pure water, and uniformly mixing; leaching at 50deg.C for 2 hr; centrifuging at 4000rpm for 10min, collecting supernatant, concentrating, and drying to obtain powdery Paeonia ostii extract.
The preparation method of the paeonia ostii by water extraction and alcohol precipitation comprises the following steps:
drying Paeonia suffruticosa at 50-60deg.C, pulverizing, and sieving with 40 mesh sieve; accurately weighing 50.00g of paeonia ostii, adding 500mL of pure water, and uniformly mixing; leaching at 50deg.C for 2 hr; centrifuging at 4000rpm for 10min, collecting supernatant, concentrating to 100mL, adding 400mL of absolute ethanol, precipitating with ethanol at room temperature for 4h, centrifuging, collecting supernatant, concentrating, and drying to obtain powdery Paeonia ostii extract.
The preparation method of the paeonia ostii ethanol extract comprises the following steps:
drying Paeonia suffruticosa at 50-60deg.C, pulverizing, and sieving with 40 mesh sieve; accurately weighing 50.00g of paeonia ostii, adding 500mL of 50% ethanol, and uniformly mixing; leaching at 50deg.C for 2 hr; centrifuging at 4000rpm for 10min, collecting supernatant, concentrating, and drying to obtain powdery Paeonia ostii extract.
The content of total polyphenol and total flavone in the dansheng flower petals, the dansheng flower aqueous extract and the dansheng flower alcohol extract were measured respectively, and the anti-saccharification, hyaluronidase inhibition rate and elastase inhibition rate of the dansheng flower aqueous extract, the dansheng flower aqueous extract and the dansheng flower alcohol extract were measured, and the results are shown in tables 1 to 4.
TABLE 1 total polyphenol and total flavone content
Note that: the method for detecting total polyphenol comprises the following steps: spectrophotometry for determining total polyphenol content in T/AHFIA 005-2018 plant extract and products thereof; the detection method of total flavonoids comprises the following steps: determination of total flavonoids in SNT4592-2016 outlet food.
Anti-glycation assay method:
the fructose and BSA were dissolved in phosphate buffer at concentrations of 1.5mol/L and 60mg/mL, respectively. 60. Mu.L of BSA and 60. Mu.L of fructose solution were added to a black 96-well ELISA plate with 60. Mu.L of the test substance, and incubated at 50℃for 24 hours. The blank was replaced with 60 μl deionized water. After the incubation, fluorescence intensity is detected at the excitation wavelength of 360nm and the emission wavelength of 460nm by using a fluorescence microplate reader, and the generation amount of AGEs is calculated. Since the sample itself is fluorescent, background absorption needs to be detected and the final result corrected. The glycation inhibition rate was calculated as follows: inhibition ratio (%) = [1- (sample fluorescence intensity/blank fluorescence intensity) ]×100%.
TABLE 2 anti-glycation IC50 values of different Paeonia ostii extracts
Elastase inhibition rate determination method:
after mixing 85. Mu.L of 50mmol/LTris-HCl buffer (pH 8.0) with 15. Mu.L of sample solution with different mass concentrations, 25. Mu.L of elastase solution (60 mU/mL) was added, incubated at 25℃for 15min, then 25. Mu.L of 1.015mmol/L.AAAPAN solution was added, absorbance at 410nm was measured after 15min, and the elastase solution and AAAPAN solution were both prepared with 50mmol/LTris-HCl buffer (pH 8.0) and the inhibition of elastase by the sample was calculated according to the formula in parallel for 3 times.
Inhibition ratio (%) = [1- (C-D)/(a-B) ]x100;
a is the absorbance of the reaction solution without the sample; b is the absorbance of the reaction solution without sample and enzyme; c is the absorbance of the reaction solution containing the sample and the enzyme; d is the absorbance of the reaction solution without enzyme.
TABLE 3 anti-wrinkle IC50 values (elastase inhibition ratio) for different Paeonia ostii extracts
The method for measuring the inhibition rate of hyaluronidase comprises the following steps:
the experimental steps are as follows:
and (3) calculating:
hyaluronidase inhibition ratio X (%) = [ (C-D) - (a-B)/(C-D) ]x100%;
in the test: a 0D value of a- (hyaluronidase + sample + sodium hyaluronate) sample solution;
b- (acetate buffer + sample + acetate buffer) sample blank 0D value;
c- (hyaluronidase + deionized water + sodium hyaluronate) control solution 0D straight;
d- (acetate buffer + deionized water + acetate buffer) versus the blank 0D value.
TABLE 4 antiallergic IC50 values (hyaluronidase inhibition ratio) of different Paeonia ostii extracts
According to the results of anti-glycation, hyaluronidase inhibition rate, elastase inhibition rate, etc., ethanol is selected as solvent to prepare extract.
2. Characteristic component of paeonia ostii extract
1.1 affinity chromatography fast screening method (anti-wrinkle)
The targeted affinity technology is an effective method for efficiently finding small molecule ligands with strong affinity to target proteins, and ligand molecules with strong binding to target proteins can be efficiently found from complex mixtures.
1.1.1 affinity chromatography method-incubation
(1) Preparation of elastase and extract
Elastase (26 mg/ml, 30U/mg), 50mmol/L Tris-HCl buffer (pH 8.0) was diluted 10-fold and used.
The extract is fully dissolved by adopting buffer solution, and filtered by a 0.45 mu m filter, and the concentration is 0.5mg/ml, 1.0mg/ml and 2.0mg/ml for standby.
(2) Elastase-extract reaction system
Incubation group (0.5 mL extract and 0.5mL elastase);
blank (0.5 mL buffer and 0.5mL elastase);
extract set (0.5 mL buffer and 0.5mL extract);
positive control group (0.5 mL buffer and 0.5mL catechin solution (0.2 mg/mL)).
Respectively reacting at 37 ℃, vibrating for 30min in a gas bath, transferring into an ultrafiltration centrifuge tube, centrifuging for 5min at 10000r/min, discarding filtrate, adding 0.5mL of 90% methanol, centrifuging for 5min at 10000r/min after vibrating, repeating for 3 times, collecting filtrate, filtering with a 0.45 μm filter membrane, and measuring chromatographic peak change condition by using a high performance liquid chromatograph.
Note that: according to the experimental results, the concentration of elastase is higher than that of the extract, so that the active ingredient can be fully combined with the enzyme.
HPLC chromatograms of the blank, extract and incubation groups are shown in FIG. 2, and it can be seen that the chromatographic peaks at 1, 2 and 3 can be specifically combined with elastase to influence the activity of the elastase.
1.1.2 preliminary identification of target Components
The identification of peaks 1, 2 and 3 in FIG. 2 shows (FIG. 3-FIG. 6), that peak 1 is paeoniflorin, 2 is rhoifolin and peak 3 is cosmosiin.
1.1.3 elastase Activity assay
TABLE 5 inhibition of elastase Activity
| Sample of | Concentration (0.1 mg/ml) | Concentration (1 mg/ml) |
| Paeonia suffruticosa extract | 18.3％ | 36.3％ |
| Rhus verniciflua Stokes glycoside | 37.3％ | 52.8％ |
| Cosmosiin (DABES chrysanthemum glycoside) | 39.0％ | 69.5％ |
| Paeoniflorin | 41.8％ | 72.2％ |
| Catechin (positive control) | 43.2％ | 76.4％ |
The extract of Paeonia ostii has the lowest inhibition rate on elastase due to complex and various components and activity difference. Referring to the inhibition activity of catechin (positive control), it is known that the binding ability of the rhoifolin, the coside and the paeoniflorin with elastase is strong, and the rhoifolin has a strong elastase inhibition activity.
1.2 molecular Butt screening method (antiallergic)
Computer virtual screening method research of active ingredients is carried out by taking hyaluronidase and G protein coupled receptor-X2 (Mrgprx 2/MRGX 2) as receptors (antiallergic effect).
ADT analyzes the docking result, and firstly deletes all molecules in the current window. Binding energies less than-1.2 kcal/mol or less than-5 kj/mol are considered feasible with the lowest possible choice.
And optimizing the visual docking result (marking information such as docking amino acid, bond energy and the like) by using PyMOL software.
The current results are as follows:
TABLE 6 antiallergic results
The molecular docking technique is adopted to virtually screen active ingredients, and the molecular docking result shows that: the hyaluronidase is used as a target and the G protein coupled receptor X2 is used as a target, and it can be determined that the rhoifolin, paeoniflorin and cosis are active ingredients with antiallergic effect, and the other ingredients have weaker effects.
The characteristic components of the paeonia ostii extract are as follows: rhus verniciflua Stokes glycoside, paeoniflorin, and cosmosiin.
TABLE 7 content of Each component of Paeonia ostii extract
| Sample of | Content of Rhus verniciflua Stokes glycoside mg/g | Cosmosiin content mg/g | Paeoniflorin content mg/g |
| Paeonia suffruticosa extract | 81.9 | 41.2 | 12.1 |
Note that: the content of each substance in the table refers to the content of each substance in the paeonia ostii extract.
3. Optimization of preparation process of paeonia ostii extract
1 Experimental materials and methods
1.1 Experimental materials
1.1.1 Experimental raw materials
And the paeonia suffruticosa is sold in the market.
1.1.2 reagents and pharmaceutical products
Absolute ethyl alcohol, pure water, formic acid, acetonitrile, aluminum nitrate, potassium acetate, rutin standard, gallic acid standard, fu Lin Fen and sodium carbonate.
1.1.3 instruments and apparatus
Thermostatic water bath, ultrasonic cleaner, centrifuge, multifunctional enzyme-labeled instrument Tecan infinite M1000 pro, liquid chromatograph Waters e2695.
1.2 Experimental methods
1.2.1 extraction Process
Accurately weighing 2.00g of paeonia ostii, adding 20mL of 50% ethanol, and uniformly mixing; leaching at 50deg.C for 2 hr; centrifuging at 4000rpm for 10min, and collecting supernatant.
1.2.2 Single factor test
The influence of each factor on the content of the effective components of the paeonia ostii extract is studied by taking the concentration of ethanol, the feed-liquid ratio, the extraction time, the extraction temperature and the extraction times as factor elements, and the evaluation is carried out by taking the content of 5 indexes of polyphenol, flavone, rhoifolin, coside and paeoniflorin.
1.2.2.1 ethanol concentration
Weighing 2.00g of paeonia ostii according to the method in 1.2.1, adding 20mL of ethanol (10%, 30%, 50% and 70%) with different concentrations, and uniformly mixing; leaching at 50deg.C for 2 hr; centrifuging at 4000rpm for 10min, collecting supernatant, and examining the influence of ethanol concentration on the content of effective components of flos moutan extract.
1.2.2.2 feed to liquid ratio
On the basis of optimizing the concentration of ethanol, 2.00g of paeonia ostii is weighed, 50% ethanol with different volumes is added, so that the feed liquid ratio is 1:5, 1:10, 1:15, 1:20 and 1:25, and the materials are uniformly mixed; leaching at 50deg.C for 2 hr; centrifuging at 4000rpm for 10min, collecting supernatant, and examining the influence of liquid-liquid ratio on the content of effective components of flos Paeonia ostii extract.
1.2.2.3 extraction time
On the basis of optimizing the concentration of ethanol and the feed liquid ratio, weighing 2.00g of paeonia ostii, adding 40ml of 50% ethanol, and uniformly mixing; leaching at 50deg.C for 0.5 hr, 1.0 hr, 1.5 hr, 2.0 hr, 2.5 hr; centrifuging at 4000rpm for 10min, collecting supernatant, and examining the influence of extraction time on the content of effective components of flos Paeonia ostii extract.
1.2.2.4 extraction temperature
On the basis of optimizing the concentration of ethanol, the feed-liquid ratio and the extraction time, weighing 2.00g of paeonia ostii, adding 40ml of 50% ethanol, and uniformly mixing; leaching at 30deg.C, 40deg.C, 50deg.C, 60deg.C and 80deg.C for 1.5 hr; centrifuging at 4000rpm for 10min, collecting supernatant, and examining the influence of extraction temperature on the content of effective components of flos moutan extract.
1.2.2.5 times of extraction
On the basis of optimizing the concentration of ethanol, the feed-liquid ratio, the extraction time and the extraction temperature, weighing 2.00g of paeonia suffruticosa, adding 40ml of 50% ethanol, and uniformly mixing; leaching at 40 ℃ for 1.5 hours; extracting for different times (1 time, 2 times and 3 times), centrifuging at 4000rpm for 10min, collecting supernatant, and examining the influence of the times of extraction on the content of effective components of the Paeonia ostii extract.
1.2.3 response surface optimization test
Based on a single-factor optimization test, taking the ethanol concentration (A), the feed-liquid ratio (B) and the extraction temperature (C) as independent variables, and carrying out a three-factor and three-level response surface analysis test according to a Box-Behnken design principle. The contents of flavone, rhoifolin, coside and paeoniflorin are taken as response values, and the extraction process parameters are optimized; the factor level design is shown in Table 8.
TABLE 8 response surface test factor level Table
1.3 data processing
Data analysis was performed using SPSS 26.0, origin Pro2018 and Excel 2016 software, all data expressed as mean.+ -. Standard deviation (X.+ -. S), one-way analysis of variance (One way ANOVA), tukey test and Dunnet' S multiple comparisons, with P <0.05 statistically significant differences.
Analysis of the results of two experiments
2.1 Single factor test results
2.1.1 influence of ethanol concentration on content of effective ingredient of Paeonia ostii
The influence of ethanol concentration on the content of the effective components of Paeonia ostii is shown in FIG. 7 and FIG. 8.
As can be seen from fig. 7 and 8, the content of the active ingredient in the paeonia ostii rose with increasing ethanol concentration, and reached a higher level at 50% concentration, and no significant rising trend was observed at 70%, and the ethanol concentration was selected to be 50%.
In fig. 7, 9, 11, 13 and 15, four columns at each detection point were the results of polyphenol, rhoifolin, coside and paeoniflorin in this order from left to right.
2.1.2 influence of feed liquid ratio on the content of the effective ingredient of Paeonia ostii
The effect of the liquid-liquid ratio on the content of the effective components of the paeonia ostii is shown in figures 9 and 10.
As can be seen from fig. 9 and 10, the contents of polyphenol, flavone, stamina and paeoniflorin in the paeonia suffruticosa flower are highest at 1:20, the contents of the cosmos die glycoside are highest at 1:15, and the ratio of feed to liquid is selected to be 1:20.
2.1.3 influence of extraction time on the content of the effective ingredient of Paeonia ostii
The effect of extraction time on the content of effective components of Paeonia ostii is shown in FIG. 11 and FIG. 12.
As can be seen from fig. 11 and 12, the influence fluctuation of the extraction time on each component in the paeonia ostii is small, the change of each component along with the extraction time is not obvious, and the extraction time is selected to be 1.5 hours in combination with the content change condition and the cost of each component.
2.1.4 influence of extraction temperature on content of effective ingredient of Paeonia ostii
The influence of the extraction temperature on the content of the effective components of the Paeonia ostii is shown in fig. 13 and 14.
As can be seen from fig. 13 and 14, the contents of polyphenols and flavones increase with increasing temperature, but too high a temperature is unfavorable for the stabilization of polyphenols and flavones, and the contents reach an inflection point at 40 ℃; comprehensively considering, the extraction temperature was chosen to be 40 ℃.
2.1.5 influence of the number of extractions on the content of the active ingredient of Paeonia ostii
The effect of the number of extraction on the content of the effective components of Paeonia ostii is shown in FIG. 15 and FIG. 16.
As can be seen from fig. 15 and 16, the content increases as the number of extractions increases, and most of the components are highest when extracted 1 time, and the number of extractions is selected to be 2 times in consideration of the extraction yield and yield.
2.2 response surface test results
2.2.1Box-Behnken test design and results
Response surface test design and results on the basis of single factor test, the effect on the content of the effective components is obvious in 3 factors: the ethanol concentration, the feed-liquid ratio and the extraction temperature are independent variables, and the flavone content, the rhoifolin, the coside and the paeoniflorin are respectively response values Y 1 、Y 2 、Y 3 、Y 4 Three-factor three-level response surface tests were performed according to the Box-Behnken model, and the test designs and results are shown in Table 9.
TABLE 9 response surface analysis experiment design and results
2.2.2 modeling and significance testing
TABLE 10 analysis of variance of flavone regression model
Performing quadratic multiple regression fitting on the table 9 data by using Design Expert 8.0 software to obtain a regression equation:
Y 1 ＝4.4-0.0026A-1.06B+0.3222C+0.2534AB-0.0975AC-0.2709BC+0.1281A2+0.3653B2-0.3812C2
wherein Y is 1 Is the predicted value of flavone; A. b, C the ethanol concentration, the feed-liquid ratio and the extraction temperature are respectively. The equation was subjected to significance testing and analysis of variance, and the results are shown in Table 10. From the ANVOA analysis of Table 10, it is seen that: p of model<0.01 (extremely remarkable), the mismatch term P>0.05 (not significant), the decision coefficient r2= 0.9240 of the equation, the correction coefficient r2adj= 0.8262, indicates that the equation has a better fit to the experiment.
The influence of the respective variable factors on the flavone content of the paeonia ostii extract is as follows: the primary factors B (feed to liquid ratio) and C (extraction temperature) have significant effects (P < 0.05).
Table 11 analysis of variance of the Rhus verniciflua Stokes regression model
Performing quadratic multiple regression fitting on the table 9 data by using Design Expert 8.0 software to obtain a regression equation:
Y 2 ＝12.95-0.121A+1.3B-1.7C+2.98AB-1.45AC-2.33BC-2.21A 2 +0.5676B 2 +2.1C 2
wherein Y is 2 Is the predicted value of the rhoifolin; A. b, C the ethanol concentration, the feed-liquid ratio and the extraction temperature are respectively. The equation was subjected to significance testing and analysis of variance, and the results are shown in Table 11. From the ANVOA analysis of table 11, it can be seen that: p of AB model<0.05 (significant), mismatch term P>0.05 (insignificant) the coefficient of determination R of the equation 2 =0.8193, correction factor R 2 Adj = 0.5869, indicating that this equation fits well to the experiment.
The influence of the respective variable factors on the rhoifolin in the paeonia ostii extract is as follows: the interaction term factor AB (ethanol concentration to feed ratio) affects significantly (P < 0.05).
TABLE 12 regression model analysis of variance for cosmosiin
Performing quadratic multiple regression fitting on the table 9 data by using Design Expert 8.0 software to obtain a regression equation:
Y 3 ＝7.56-0.4932A+0.6478B+0.8017C+1.61AB-0.8303AC-0.7804BC-1.36A2+0.4261B2+2.88C2
wherein Y is 3 Is the predicted value of the cosmosiin; A. b, C the ethanol concentration, the feed-liquid ratio and the extraction temperature are respectively. The regression equation was subjected to significance testing and analysis of variance, and the results are shown in Table 12. From the ANVOA analysis of table 12, it can be seen that: p-average of model<0.05 (extremely remarkable), the mismatch term P>0.05 (insignificant) the coefficient of determination R of the equation 2 = 0.8454, correction coefficient R 2 Adj = 0.6467, indicating that this equation fits well to the experiment.
The influence of the respective variable factors on the cosmosiin in the paeonia ostii extract is as follows: the interaction term factor AB (ethanol concentration to feed ratio) affects significantly (P < 0.05).
TABLE 13 analysis of variance of paeoniflorin regression model
Performing quadratic multiple regression fitting on the table 9 data by using Design Expert 8.0 software to obtain a regression equation of Y 4 ＝3.47-0.295A+0.2698B+0.2436C+0.7577AB-0.2901AC-0.2094BC-0.5995A2+0.2761B2+1.24C2。
Wherein Y is 4 Is the predicted value of paeoniflorin; A. b, C the ethanol concentration, the feed-liquid ratio and the extraction temperature are respectively. The equation was subjected to significance testing and analysis of variance, and the results are shown in Table 13. From the ANVOA analysis of table 13, it follows that: p of model<0.01 (electrode)Significant), dislike term P>0.05 (insignificant) the coefficient of determination R of the equation 2 = 0.8309, correction coefficient R 2 Adj = 0.6136, indicating that this equation fits well to the experiment.
The influence of the respective variable factors on the flavone of the paeonia ostii is as follows: the primary factors B (feed to liquid ratio) and C (extraction temperature) have significant effects (P < 0.05).
2.3.4 response surface interaction analysis and optimization
Response surface analysis is carried out on interaction among all factors through Design Expert 8.0.6 software, and a response surface graph is drawn. The three-dimensional response surface diagram can intuitively reflect interaction among all factors; the steeper the response surface, the more pronounced the pairwise interaction between the individual factors is exhibited. The contour plot may intuitively reflect the degree of significance of interaction between 2 variables, where a circle indicates that the two-factor interaction is not significant and an oval indicates that the two-factor interaction is significant. The higher the curve density of the contour diagram along the direction of a certain factor axis, the more sensitive the response value is to the change of the factor, the steeper the gradient of a curved surface is reflected on a response surface, the more the contour shape tends to be elliptical, and the larger the angle between the axis of the ellipse and the coordinate axis is, the more obvious the interaction is.
2.3.4.1 verification experiment
Solving a quadratic polynomial regression equation of the paeonia ostii extract to obtain the optimal extraction process conditions: the ethanol concentration is 53.2746%, the feed-liquid ratio is 1:10.0869, the extraction temperature is 59.6393 ℃, and under the optimal condition, the flavone content, the rhoifolin content, the cosmos and the paeoniflorin content can reach 6.3857%, 20.1992mg/g, 12.981mg/g and 5.7366mg/g theoretically.
Considering experimental operability, the optimal extraction process conditions are modified as follows: the ethanol concentration is 53%, the feed-liquid ratio is 1:10, the extraction temperature is 60 ℃, under the condition, after 3 parallel experiments, the measured flavone content, the rhoifolin content, the Daboshi content and the paeoniflorin content (the content of each component accounts for the paeonia suffruticosa) are respectively 7.67+/-0.87%, 19.79+/-0.69 mg/g, 11.94+/-0.54 mg/g and 5.30+/-0.36 mg/g, which shows that the fitting degree of the model is better, and the reliability of the model is verified.
Conclusion 3
The research adopts a single factor to research and develop and optimize the extraction process of the effective components of the paeonia ostii, and examines the influence of five factors of ethanol concentration, feed-liquid ratio, extraction time, extraction temperature and extraction times on the contents of polyphenol, flavone, rhoifolin, coside and paeoniflorin. Experimental results show that the optimal extraction process conditions are as follows: the concentration of ethanol is 53%, the feed-liquid ratio is 1:10, the extraction time is 1.5 hours, the extraction temperature is 60 ℃, and the extraction is carried out for 2 times.
Evaluation of Activity of 4-danfeng peony extract
The anti-glycation, hyaluronidase inhibition rate and elastase inhibition rate of the paeonia ostii extract prepared under the optimal process conditions are measured, and the results are shown in the following table:
TABLE 14 evaluation of Activity
4. Efficacy evaluation
The efficacy of the paeonia ostii extract prepared under the optimal process condition is evaluated by taking the zebra fish as an experimental animal.
1.1 blood replenishing efficacy evaluation of zebra fish
Zebra fish strain: wild type AB strain zebra fish.
Model control group: zebra fish were induced with phenylhydrazine. Phenylhydrazine acts on erythrocyte membranes to accelerate the hydrolysis of leucine, lysine and histidine on the membrane surfaces, simultaneously selectively oxidizes membrane frameworks and alpha globin, and translocates phosphatidylserine to the surfaces of erythrocytes, so that the deformability of the erythrocytes is reduced, the ability of the erythrocytes to adhere to extracellular matrixes is enhanced, mature erythrocytes in circulating blood are destroyed, hemoglobin is denatured and aggregated to form Heinz corpuscles, and the erythrocyte has no destructive effect on myelogenic erythrocytes.
Normal control group: normal zebra fish.
Positive control group: and (5) carrying out shengxuening tablet treatment on the modeling module.
Treatment group: and (3) carrying out the treatment of the paeonia ostii extract on the molding module.
Zebra fish were treated as in table 15, and after 48 hours each, the mortality and phenotype of zebra fish were observed to determine the maximum detected concentration of the dansheng peony extract.
Table 15 sample blood replenishing efficacy concentration fumbling experimental results (n=30)
The normal control group, the model control group, the positive control group and the treatment group were set as in Table 16, and after 10 pieces each for 48 hours, the number of red blood cells (staining intensity) was measured by a staining method.
Table 16 blood replenishing efficacy evaluation results of zebra fish
1.2 evaluation of whitening efficacy of zebra fish (external use)
Zebra fish strain: wild type AB strain zebra fish.
Normal control group: zebra fish that were not treated. The skin whitening effect evaluation method has the advantages that the body surface of the zebra fish has melanin precipitation, and the skin whitening effect evaluation method can be directly used through the inhibition effect of tyrosinase activity and melanin synthesis. Zebra fish were transparent throughout their body at the beginning of development, and melanin began to grow from retinal epithelium when embryos developed to 24h. Pigment cells originate from a population of cells differentiated by the dorsal ectoderm-neural crest cells, and then proliferate, migrate, differentiate into pigment blast cells. Intervention during melanin formation can inhibit melanin formation.
Positive control group: and carrying out arbutin treatment on the zebra fish.
Treatment group: and (5) carrying out the treatment of the danfeng peony extract on the zebra fish.
Zebra fish were treated as in table 17, and after 48 hours each, the mortality and phenotype of zebra fish were observed to determine the maximum detected concentration of the dansheng peony extract.
Table 17 sample whitening efficacy concentration fumbling experimental results (n=30)
Conclusion of experiment: under the experimental conditions, the maximum detection concentration (MTC) of the paeonia ostii extract is 1000 mug/mL.
The normal control group, the positive control group and the treatment group were set as in Table 18, and after 10 pieces of each group for 48 hours, the melanin signal intensity of the head of the zebra fish was detected.
Table 18 evaluation results of whitening efficacy
1.3 evaluation of anti-wrinkle efficacy of zebra fish
Zebra fish strain: wild type AB strain zebra fish.
Normal control group: zebra fish that were not treated. The contents of collagen, elastin and active oxygen in the skin are key to determining the elasticity of the skin. When collagen and elastin are deficient or active oxygen metabolism is unbalanced in the body, skin elasticity will be reduced and wrinkles will be generated. The anti-wrinkle effect of the test sample can be evaluated by detecting the content of skin active oxygen.
Positive control group: and (5) performing reduced glutathione treatment on the zebra fish.
Treatment group: and (5) carrying out the treatment of the danfeng peony extract on the zebra fish.
Zebra fish were treated as in table 19, and after 48 hours each, the mortality and phenotype of zebra fish were observed to determine the maximum detected concentration of the dansheng peony extract.
Table 19 anti-wrinkle efficacy concentration of samples fumbling experimental results (n=30)
Conclusion of experiment: under the experimental conditions, the maximum detection concentration (MTC) of the paeonia ostii extract is 1000 mug/mL.
The normal control group, the positive control group and the treatment group were set as in Table 20, 10 pieces each, and after 48 hours, the active oxygen content was measured.
TABLE 20 evaluation results of anti-wrinkle efficacy
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A preparation method of a paeonia ostii extract is characterized by comprising the following steps:
leaching paeonia ostii with ethanol solution, performing solid-liquid separation on the leaching solution after leaching to obtain clear liquid, and drying the clear liquid to obtain the paeonia ostii extract;
the volume concentration of the ethanol solution is 10% -70%;
the ratio of the leached liquid to the leached liquid is 1:5-1:25g/mL;
the leaching temperature is 30-80 ℃.
2. The preparation method according to claim 1, wherein the volume concentration of the ethanol solution is 30% -70%, preferably 53%.
3. The method of claim 1, wherein the leaching liquor ratio is 1:10-1:20g/mL, preferably 1:10g/mL.
4. The method of preparation according to claim 1, characterized in that the leaching temperature is 40-60 ℃, preferably 60 ℃.
5. The method of claim 1, wherein the time of leaching is 30-150min.
6. The method of claim 1, wherein the number of leaches is 1-3, preferably 2.
7. The method of claim 1, further comprising the step of sequentially drying and pulverizing the paeonia ostii prior to the leaching.
8. The method of claim 1, wherein the solid-liquid separation method comprises centrifugation or filtration.
9. An extract of paeonia ostii, characterized in that the extract is prepared by the preparation method of any one of claims 1-8.
10. Use of the paeonia ostii extract of claim 9 in any one of the following a-e:
a. preparing an anti-saccharification product;
b. preparing an antiallergic product;
c. preparing a whitening product;
d. preparing an anti-wrinkle product;
e. preparing the blood replenishing product.
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| CN117045564B (en) * | 2023-10-11 | 2024-01-26 | 北京世纪伟信医药科技有限公司 | Skin care product containing peony extract and preparation method thereof |
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