CN115006305B - Composition with antioxidation effect and preparation method and application thereof - Google Patents
Composition with antioxidation effect and preparation method and application thereof Download PDFInfo
- Publication number
- CN115006305B CN115006305B CN202210561603.3A CN202210561603A CN115006305B CN 115006305 B CN115006305 B CN 115006305B CN 202210561603 A CN202210561603 A CN 202210561603A CN 115006305 B CN115006305 B CN 115006305B
- Authority
- CN
- China
- Prior art keywords
- ethanol extract
- ethanol
- composition
- rehmannia root
- prepared rehmannia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 239000000203 mixture Substances 0.000 title claims abstract description 80
- 238000002360 preparation method Methods 0.000 title claims abstract description 40
- 230000000694 effects Effects 0.000 title claims abstract description 34
- 230000003064 anti-oxidating effect Effects 0.000 title claims abstract description 18
- 239000000469 ethanolic extract Substances 0.000 claims abstract description 147
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 99
- 241000405414 Rehmannia Species 0.000 claims abstract description 67
- 241000382455 Angelica sinensis Species 0.000 claims abstract description 55
- 239000000243 solution Substances 0.000 claims abstract description 49
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- DQNGMIQSXNGHOA-UMEXKXKESA-N (3e,6s,7s)-3-butylidene-6,7-dihydroxy-4,5,6,7-tetrahydro-2-benzofuran-1-one Chemical compound C1([C@@H]([C@@H](O)CC2)O)=C2C(=C/CCC)\OC1=O DQNGMIQSXNGHOA-UMEXKXKESA-N 0.000 description 2
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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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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Abstract
The invention discloses a composition with an antioxidation effect and a preparation method thereof; the composition with the antioxidation effect comprises ethanol extract of angelica sinensis and ethanol extract of prepared rehmannia root. The preparation method comprises the following steps: the preparation method of the ethanol extract of Chinese angelica comprises the following steps: reflux-extracting radix Angelicae sinensis with ethanol under heating, and collecting extractive solution; the preparation method of the ethanol extract of the prepared rehmannia root comprises the following steps: taking prepared rehmannia root, performing cold soaking with ethanol, then performing heating reflux extraction, and obtaining ethanol extract of prepared rehmannia root after the extraction is finished; mixing the extracting solution: mixing radix Angelicae sinensis ethanol extract with radix rehmanniae Preparata ethanol extract to obtain mixed solution; concentrating and drying: concentrating and drying the mixed solution to obtain the composition with the antioxidation effect. Studies have shown that: the composition comprising ethanol extract of Angelica sinensis and ethanol extract of rehmanniae radix Preparata has excellent antioxidant effect and cell injury resisting effect.
Description
Technical Field
The invention relates to the technical field of natural pharmaceutical chemistry, in particular to a composition with an antioxidant effect, and a preparation method and application thereof.
Background
The rapid increase of the aging population and the improvement of the living standard promote the consumption trend of functional health care products, foods and cosmetics to change, and people pay more attention to the health care and disease prevention of the people, and the anti-aging health care products, foods and cosmetics are concerned by people. It is well known that the efficacy of many herbal compounds has proven to be superior to single drugs, and that the high efficacy of compounds may result from the synergistic effect between the herbs in the formulation, but information about the interactions between the herbs in the compound, the mechanisms of disease prevention or treatment are limited. Along with the increasing attention of the traditional Chinese medicine in treating and preventing chronic diseases and the modern development requirement of traditional Chinese medicines, the research on the interaction relation of each component in the traditional Chinese medicine compound and the elucidation of the action mechanism are necessary.
Angelica sinensis [ Angelica sinensis (Oliv) Diels ] is a traditional plant used as both medicine and food, has warm nature, sweet and pungent taste, and belongs to the dry root of Angelica sinensis (Oliv.) Diels of Umbelliferae. The Chinese angelica is mainly distributed in southeast part of Gansu province, yunnan province, sichuan province, shanxi province, hubei province and the like in China. The medical application of the plant is recorded in the Shennong herbal medicine Jing in the pharmaceutical literature of China at the earliest, and has the effects of enriching blood, activating blood, relaxing bowel, regulating menstruation and relieving pain. Qianjin Fang also describes the treatment of stroke diseases, aging-related diseases and gynecological diseases with Dang Gui. The main active ingredients of the angelica are water-soluble ingredients and fat-soluble ingredients. The water-soluble component contains organic acids such as ferulic acid and chlorogenic acid, amino acids, polysaccharides and polyphenols, and the fat-soluble component contains volatile oil and butenyl phthalide compounds such as ligustilide and senkyunolide I. The active ingredients of Angelica sinensis have wide pharmacological activities including anti-inflammatory activity, anti-fibrosis activity, spasmolytic activity, antioxidant activity, neuroprotection, etc.
Prepared rehmannia root [ Rehmannia glutinosa (Gaertn.) DC ], mainly produced in Henan, shanxi, shandong and Hebei provinces of China. Prepared rehmannia root is recorded in Shen nong Ben Cao Jing and Ben Cao gang mu, and has the actions of replenishing blood and nourishing yin, replenishing essence and nourishing marrow, and is also used as a medicine for nourishing yin and tonifying kidney and replenishing blood in clinic. The main chemical components of radix rehmanniae Preparata include iridoid glycoside (such as catalpol), polysaccharides, flavonoids, polyphenols, phenethyl alcohol glycoside (such as acteoside), amino acids, etc. The prepared rehmannia root is prepared from rehmannia root by steaming or stewing with wine, and the longer the rehmannia root is processed, the more times are processed. The amino acid content is reduced, catalpol, sucrose, raffinose and stachyose are decomposed, and the content of 5-HMF (5-hydroxymethylfurfural), glucose, fructose and mannose is increased. Research reports that 5-HMF is generated during the processing of radix rehmanniae into mature radix rehmanniae, and the content of 5-HMF in radix rehmanniae Preparata is increased to about 20 times after processing. The research reports that the biological activity shown by the 5-HMF has antioxidant activity, antiallergic activity, antihypertensive activity, neuroprotective activity and the like. 5-HMF by scavenging ABTS and DPPH free radicals and reducing ROS and MDA content, 5-HMF shows a novel natural antioxidant with potential application value. Acteoside has antioxidant, cholinergic and anti-inflammatory effects on neurons, thus has neuroprotective function and is expected to be a candidate for neuroprotective application. The prepared rehmannia root polysaccharide is one of the main active ingredients in prepared rehmannia root. Pharmacological research shows that the prepared rehmannia root polysaccharide has powerful immunological regulation, nerve protecting activity, antitumor activity, antioxidant activity and other physiological functions, and may be used in developing food and health product.
However, is the combination of angelica and prepared rehmannia root to have synergistic antioxidant effect or anti-cell damage effect? Is it able to exert better antioxidant or anti-cell damaging effects? What are the ways to combine them to exert better antioxidant or anti-cell damage effects? Those skilled in the art are unaware and are left to be further investigated.
Disclosure of Invention
In order to overcome at least one technical problem existing in the prior art, the invention firstly provides a composition with an antioxidation effect; research shows that the composition has excellent antioxidation and cell injury resisting effects.
The technical scheme of the invention is as follows:
the invention firstly provides a composition with an antioxidant effect, which comprises ethanol extracts of angelica sinensis and ethanol extracts of prepared rehmannia root.
The inventors have surprisingly found in the study that a composition composed of ethanol extract of Angelica sinensis and ethanol extract of rehmanniae radix Preparata has excellent antioxidant effect and cell injury resisting effect.
Preferably, the weight ratio of the ethanol extract of Chinese angelica to the ethanol extract of prepared rehmannia is 1-5:1-5;
most preferably, the weight ratio of the ethanol extract of angelica sinensis to the ethanol extract of radix rehmanniae preparata is 3:2.
The invention also provides a preparation method of the composition with the antioxidation effect, which comprises the following steps:
the preparation method of the ethanol extract of Chinese angelica comprises the following steps: reflux-extracting radix Angelicae sinensis with ethanol under heating, and collecting extractive solution;
the preparation method of the ethanol extract of the prepared rehmannia root comprises the following steps: taking prepared rehmannia root, performing cold soaking with ethanol, then performing heating reflux extraction, and obtaining ethanol extract of prepared rehmannia root after the extraction is finished;
mixing the extracting solution: mixing radix Angelicae sinensis ethanol extract with radix rehmanniae Preparata ethanol extract to obtain mixed solution;
concentrating and drying: concentrating and drying the mixed solution to obtain the composition with the antioxidation effect.
Preferably, the ethanol in the step of preparing the ethanol extract of angelica is ethanol aqueous solution with the volume fraction of 65-75%.
The inventor finds in research that the extract prepared by extracting angelica sinensis and prepared rehmannia root by the method, and then mixing the angelica sinensis ethanol extract obtained by the method and the prepared rehmannia root ethanol extract has excellent antioxidant effect and cell injury resistance.
Most preferably, the ethanol in the step of preparing the ethanol extract of angelica is ethanol aqueous solution with the volume fraction of 70%;
preferably, in the step of preparing the ethanol extract of the angelica, the volume ratio of the weight of the angelica to the ethanol is 1 g:13-15 mL.
Optimally, in the preparation step of the ethanol extract of the angelica, the volume ratio of the weight of the angelica to the ethanol is 1g to 14mL.
Preferably, in the step of preparing the ethanol extract of the angelica, the temperature of heating reflux extraction is 81-83 ℃ and the extraction time is 1-3 h.
Most preferably, in the step of preparing the ethanol extract of angelica sinensis, the temperature of heating reflux extraction is 83 ℃, and the extraction time is 2 hours.
Preferably, the ethanol in the preparation step of the ethanol extract of the prepared rehmannia root refers to an ethanol water solution with the volume fraction of 55-65%.
Most preferably, the ethanol in the preparation step of the ethanol extract of the prepared rehmannia root refers to an ethanol aqueous solution with the volume fraction of 60%;
preferably, in the preparation step of the ethanol extract of the prepared rehmannia root, the volume ratio of the weight of the prepared rehmannia root to the ethanol is 1 g:8-12 mL.
Optimally, in the preparation step of the ethanol extract of the prepared rehmannia root, the volume ratio of the weight of the prepared rehmannia root to the ethanol is 1g and 10mL.
Preferably, the cold soaking time in the preparation step of the ethanol extract of the prepared rehmannia root is 16-30 hours.
Most preferably, the cold soaking time in the ethanol extract of radix rehmanniae Preparata preparation step is 24 hr.
Preferably, in the preparation step of the ethanol extract of the prepared rehmannia root, the temperature of heating reflux extraction is 78-80 ℃ and the extraction time is 1-1.5 h.
Most preferably, in the preparation step of the ethanol extract of the prepared rehmannia root, the temperature of the reflux extraction by heating is 80 ℃ and the extraction time is 1h.
Preferably, in the step of mixing the extracting solutions, the volume ratio of the ethanol extracting solution of the angelica to the ethanol extracting solution of the prepared rehmannia is 2-4:1-3.
Most preferably, in the step of mixing the extracting solutions, the volume ratio of the ethanol extracting solution of the angelica to the ethanol extracting solution of the prepared rehmannia is 3:2.
The inventors have surprisingly found in the study that: the angelica sinensis and the prepared rehmannia root are extracted by the method, and then the angelica sinensis ethanol extract and the prepared rehmannia root ethanol extract obtained by the method are mixed according to the volume ratio of 3:2 to prepare the extract, wherein the antioxidant effect and the cell injury resisting effect of the extract are far higher than those of the single angelica sinensis ethanol extract and the single prepared rehmannia root ethanol extract; the Chinese angelica ethanol extract and the prepared rehmannia root ethanol extract are mixed according to the volume ratio of 3:2 to generate obvious synergistic effect; the prepared composition has excellent antioxidation and cell injury resisting effects.
The inventors herein have emphasized that the methods of preparing the ethanol extract of Angelica sinensis and the ethanol extract of radix rehmanniae Preparata, and the volume ratio of the ethanol extract of Angelica sinensis and the ethanol extract of radix rehmanniae Preparata play a decisive role in whether a synergistic antioxidant effect can be produced between the ethanol extract of Angelica sinensis and the ethanol extract of radix rehmanniae Preparata, or whether a remarkable synergistic antioxidant effect can be produced. Studies have shown that: the composition prepared by mixing the angelica ethanol extract prepared by the method and the prepared rehmannia root ethanol extract according to the volume ratio of 3:2 has significantly higher antioxidation effect than the composition prepared by other volume ratios; the synergistic effect of the antioxidant effect is obviously higher than that of the composition prepared according to other volume ratios.
The invention also provides a composition with an antioxidation effect, which is prepared by the preparation method.
The invention also provides application of the composition with the antioxidant effect in preparing products with DPPH free radical scavenging effect and/or hydroxyl free radical scavenging effect.
The invention also provides application of the composition with the antioxidation effect in preparing a product with the antioxidation effect or with the cell injury resistance;
the product is a cosmetic, food or medicine.
The beneficial effects are that: the invention provides a brand new composition with an antioxidation effect; studies have shown that: the composition comprising ethanol extract of radix Angelicae sinensis and ethanol extract of radix rehmanniae Preparata has excellent antioxidant effect and cell injury resisting effect; in particular, the composition prepared by mixing the ethanol extract of Chinese angelica and the ethanol extract of prepared rehmannia root according to the volume ratio of 3:2 has significantly higher antioxidation than the composition prepared according to other volume ratios; the synergistic effect of the antioxidant effect is obviously higher than that of the composition prepared according to other volume ratios. Therefore, the composition provided by the invention has important application value in preparing cosmetics, foods or medicines.
Drawings
FIG. 1 shows the scavenging effect of the compositions of the present invention on DPPH radicals; FIG. 1A is a graph showing the effect of DPPH radical scavenging effect of the composition of the present invention; FIG. 1B is a graph showing the IC50 value test results of DPPH radical scavenging by the composition of the present invention.
FIG. 2 is a graph showing the scavenging effect of the composition of the present invention on hydroxyl radicals; FIG. 2A is a graph showing the effect of scavenging hydroxyl radicals in a composition of the present invention; FIG. 2B is a schematic diagram of an IC with hydroxyl radical scavenging composition of the present invention 50 And (5) a value test result graph.
FIG. 3 is a graph showing the results of the apoptosis and mitochondrial membrane potential measurements of SHSY-5Y cells by the compositions of the present invention; wherein, fig. 3A is a flow chart of apoptosis; FIG. 3B is a graph of apoptosis analysis; FIG. 3C shows the composition of the present invention vs. H 2 O 2 Mitochondrial membrane potential analysis of induced SHSY-5Y cell injury; FIG. 3D is a graph of the composition of the present invention versus H 2 O 2 Mitochondrial membrane potential flow pattern of induced SHSY-5Y cell injury.
Detailed Description
The present invention is further explained below with reference to specific examples, which are not intended to limit the present invention in any way.
Example 1 preparation of a composition having antioxidant effect
(1) Preparing ethanol extract of Chinese angelica: pulverizing radix Angelicae sinensis, sieving with 60 mesh sieve, weighing 40g radix Angelicae sinensis crude herb powder, adding radix Angelicae sinensis powder into round bottom flask of condensing reflux device, adding 70% ethanol water solution at a weight-volume ratio of 1:14, and reflux extracting at 83 deg.C for 2 hr to obtain radix Angelicae sinensis ethanol extractive solution;
(2) Preparing ethanol extract of prepared rehmannia root: pulverizing radix rehmanniae Preparata herbs, sieving with 60 mesh sieve, weighing 70g radix rehmanniae Preparata crude herb powder, adding radix rehmanniae Preparata crude herb powder into round bottom flask, adding 60% ethanol water solution at a weight-volume ratio of 1:10 times, cold soaking for 24 hr, reflux extracting at 80deg.C in a condensing reflux device for 1 hr to obtain radix rehmanniae Preparata ethanol extractive solution;
(3) Mixing the ethanol extract of Chinese angelica and the ethanol extract of prepared rehmannia root according to a volume ratio of 3:2 to obtain a mixed solution; concentrating the mixed solution in a rotary evaporator at 55deg.C, and freeze drying to obtain antioxidant composition.
Example 2 preparation of a composition having antioxidant effect
Steps (1) and (2) are the same as in example 1;
step (3) is different from example 1 in that the ethanol extract of Angelica sinensis and the ethanol extract of rehmannia glutinosa Libosch are mixed in a volume ratio of 1:4, and the rest is the same as example 1.
Example 3 preparation of a composition having antioxidant effect
Steps (1) and (2) are the same as in example 1;
step (3) is different from example 1 in that the ethanol extract of Angelica sinensis and the ethanol extract of rehmannia glutinosa Libosch are mixed in a volume ratio of 2:3, and the rest is the same as example 1.
Example 4 preparation of a composition having antioxidant effect
Steps (1) and (2) are the same as in example 1;
step (3) is different from example 1 in that the ethanol extract of Angelica sinensis and the ethanol extract of rehmannia glutinosa Libosch are mixed in a volume ratio of 4:1, and the rest is the same as example 1.
Example 5 preparation of composition with antioxidant effect
Steps (1) and (2) are the same as in example 1;
step (3) is different from example 1 in that the ethanol extract of Angelica sinensis and the ethanol extract of rehmannia glutinosa Libosch are mixed in a volume ratio of 5:5, and the rest is the same as example 1.
Comparative example 1 preparation of ethanol extract of Angelica sinensis
Pulverizing radix Angelicae sinensis, sieving with 60 mesh sieve, weighing 40g radix Angelicae sinensis crude herb powder, adding radix Angelicae sinensis powder into round bottom flask of condensing reflux device, adding 70% ethanol water solution at a weight-volume ratio of 1:14, and reflux extracting at 83 deg.C for 2 hr to obtain radix Angelicae sinensis ethanol extractive solution; concentrating and drying the ethanol extract of the angelica sinensis to obtain the ethanol extract of the angelica sinensis.
Comparative example 2 preparation of ethanol extract of rehmanniae radix Preparata
Pulverizing radix rehmanniae Preparata herbs, sieving with 60 mesh sieve, weighing 70g radix rehmanniae Preparata crude herb powder, adding radix rehmanniae Preparata crude herb powder into round bottom flask, adding 60% ethanol water solution at a weight-volume ratio of 1:10 times, cold soaking for 24 hr, reflux extracting at 80deg.C in a condensing reflux device for 1 hr to obtain radix rehmanniae Preparata ethanol extractive solution; concentrating and drying the ethanol extract of the prepared rehmannia root to obtain the ethanol extract of the prepared rehmannia root.
Experimental example 1 the antioxidant composition of the present invention has the effect of scavenging DPPH free radical
Accurately measuring 8mg of DPPH solid powder, dissolving the powder with absolute ethyl alcohol, and keeping the solution in a volumetric flask of 100mL, and refrigerating the solution in a dark place. The ethanol extract of Angelica sinensis, the ethanol extract of rehmannia glutinosa, or the composition prepared in examples 1 to 5, each 0.250mg, was precisely weighed, and dissolved in distilled water to prepare a sample solution having a concentration of 10 mg/mL. The accurate sample solution is prepared into concentration gradient solutions of 0.125, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 8.0mg/mL respectively. Adding 2mL of DPPH solution into 1mL of various samples with different concentrations (0.125-8.0 mg/mL), shaking, standing at room temperature in the dark for 30 min, using distilled water as blank control, measuring absorbance of the obtained solution at 517nm to obtain a 1 . Adding 2mL of DPPH solution into 1mL of distilled water, shaking uniformly, standing at room temperature in the dark for 30 minutes, and measuring the absorbance of the obtained solution at 517nm to be A 0 . Adding 2mL of absolute ethanol solution into 1mL of various samples with different concentrations (0.0125-0.8 mg/mL), shaking, standing at room temperature in the dark for 30 min, and measuring absorbance of the obtained solution at 517nm to obtain A 2 . The DPPH radical scavenging rate was calculated as follows:
DPPH radical clearance (%) = [1- (a) 1 -A 2 )/A 0 ] (1)
Wherein A is 1 Is the absorbance of the sample, A 2 Absorbance without DPPH, A 0 Is the absorbance without the sample.
Examples 1-5 the DPPH radical scavenging activity of the compositions prepared from the ethanol extract of Angelica sinensis and the ethanol extract of rehmannia glutinosa at different volume ratios is shown in figure 1. The dose response curve for DPPH radical scavenging of the compositions of the present invention was obtained by CompuSyn software processing (see FIG. 1A). Compared with single ethanol extract of angelica sinensis, single ethanol extract of prepared rehmannia root with concentration of 0.125-8.0mg/mL has obvious DPPH free radical scavenging capability.
As shown in FIG. 1B, the composition prepared by mixing the angelica ethanol extract and the prepared rehmannia root ethanol extract according to the volume ratio of 3:2 has a DPPH free radical scavenging effect which is obviously higher than that of a single angelica ethanol extract and a single prepared rehmannia root ethanol extract, and is also obviously higher than that of a composition prepared by mixing the angelica ethanol extract and the prepared rehmannia root ethanol extract according to other volume ratios; this illustrates: the angelica ethanol extract and the prepared rehmannia root ethanol extract prepared by the method of the invention are mixed according to the volume ratio of 3:2 to generate obvious synergistic DPPH free radical scavenging effect, and the synergistic DPPH free radical scavenging effect is obviously higher than that of the composition prepared by the angelica ethanol extract and the prepared rehmannia root ethanol extract according to other volume ratios. Since the composition of the invention has excellent DPPH free radical scavenging effect; therefore, the composition of the invention has excellent oxidation resistance caused by DPPH free radical.
Experimental example 2 the antioxidant composition of the present invention has the effect of scavenging hydroxyl radicals
The ethanol extract of Angelica sinensis, the ethanol extract of rehmannia glutinosa, or the composition prepared in examples 1 to 5, each 0.250mg, was precisely weighed, and dissolved in distilled water to prepare a sample solution having a concentration of 10 mg/mL. The accurate sample solution is prepared into concentration gradient solutions of 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0mg/mL respectively. 1mL of samples of various concentrations (0.25-6.0 mg/mL) were combined with 1mL of H 2 O 2 Solution (8.8 mM) and 1mL of ferrous sulfate (FeSO) 4 ) The solutions (1.8 mM) were mixed. Then, after adding 1mL of salicylic acid-ethanol solution (9 mM), the mixture was incubated at 37℃for 60 minutes in a constant temperature water bath, distilled water was used as a control, and the absorbance A of the sample was measured at 510nm 1 . 1mL of samples of various concentrations (0.25-6.0 mg/mL) were mixed with 1mL of distilled water and 1mL of ferrous sulfate solution. Then, after adding 1mL of salicylic acid-ethanol solution, the absorbance A of the sample was measured at 510nm in a constant temperature water bath at 37℃for 60 minutes 2 . 1mL distilled water was combined with 1mL H 2 O 2 Absorbance A measured after mixing the solution, 1mL of ferrous sulfate solution and 1mL of salicylic acid-ethanol solution 0 . The formula for the hydroxyl radical scavenging rate is as follows:
hydroxyl radical clearance (%) = [1- (a) 1 -A 2 )/A 0 ] (2)
Wherein A is 1 Is the absorbance of the sample, A 2 Is free of H 2 O 2 Absorbance of A 0 Is the absorbance without the sample.
Examples 1-5 the hydroxyl radical scavenging activity of the compositions prepared from the ethanol extract of Angelica sinensis and the ethanol extract of rehmannia glutinosa at different volume ratios is shown in figure 2. The dose response curve for scavenging hydroxyl radicals of the compositions of the present invention was obtained by CompuSyn software processing (see FIG. 2A). Compared with single ethanol extract of Chinese angelica and single ethanol extract of prepared rehmannia root, the composition prepared by mixing the ethanol extract of Chinese angelica and the ethanol extract of prepared rehmannia root according to the volume ratio of 3:2 has the strongest capability of scavenging hydroxyl radicals.
As shown in fig. 2B, the composition prepared by mixing the ethanol extract of angelica sinensis and the ethanol extract of radix rehmanniae preparata according to the volume ratio of 3:2 has a significantly higher effect of scavenging hydroxyl radicals than the single ethanol extract of angelica sinensis and the single ethanol extract of radix rehmanniae preparata, and is also significantly higher than the composition prepared by mixing the ethanol extract of angelica sinensis and the ethanol extract of radix rehmanniae preparata according to other volume ratios; this illustrates: the angelica ethanol extract and the prepared rehmannia root ethanol extract prepared by the method of the invention are mixed according to the volume ratio of 3:2 to generate obvious synergistic hydroxyl radical scavenging effect, and the synergistic hydroxyl radical scavenging effect is obviously higher than that of the composition prepared by the angelica ethanol extract and the prepared rehmannia root ethanol extract according to other volume ratios. Since the composition of the invention has excellent effect of scavenging hydroxyl free radicals; therefore, the composition of the invention has excellent oxidation resistance caused by hydroxyl radicals.
Experimental example 3 analysis of apoptosis and mitochondrial membrane potential
SHSY-5Y apoptosis was detected with Annexin V/Propidium Iodide (PI) probe. The experiment is divided into blank control group and H 2 O 2 Injury group and drug intervention group. The digested SHSY-5Y cells were inoculated into 6-well cell culture plates and placed in an incubator for incubation for 24 hours. In the blank group and 80 mu M H 2 O 2 Adding 1mL of DEME culture medium without serum into injured group cells, adding 1mL of 100 μg/mL single ethanol extract of radix Angelicae sinensis, 100 μg/mL single ethanol extract of radix rehmanniae Preparata and 100 μg/mL of the composition prepared in example 1 (composition prepared by mixing radix Angelicae sinensis ethanol extract and radix rehmanniae Preparata ethanol extract at volume ratio of 3:2), respectively, and adding at 37deg.C and 5% CO 2 Pre-incubation for 12 hours in the incubator of (c). Except for the blank cells, 1mL of cells containing 80 mu M H were added to each well 2 O 2 Is used to stimulate cells for 24 hours. Cells will be digested with pancreatin without EDTA and collected in EP tubes, corresponding tags made, cells washed 2 times with PBS and resuspended in 100 μl binding buffer. Then Annexin V and PI dye (2.5. Mu.L each) were added to the binding buffer, mixed well, and after incubation of the cells in the dark for 15 minutes, 200. Mu.L of binding buffer was added. Cells were transferred into flow tubes and flow cytometers were used to obtain early apoptotic cell populations, late apoptotic cell populations, and normal cell populations.
Mitochondrial membrane potential of SHSY-5Y cells can be measured using a flow cytometer and a 5,5', 6' -tetrachloro-1, 1', 3' -tetraethylbenzimidazolyl carbocyanine iodide (JC-1) probe. The experiment is divided into blank control group and H 2 O 2 Injury group and drug intervention group. Cell culture plate with 6 holesSHSY-5Y cells were cultured to the logarithmic growth phase of the cells. The cells of the pharmaceutical intervention group were pre-incubated with a single ethanol extract of Angelica sinensis, a single ethanol extract of radix rehmanniae Preparata, and the composition prepared in example 1 (composition prepared by mixing Angelica sinensis ethanol extract and radix rehmanniae Preparata ethanol extract at a volume ratio of 3:2) (100. Mu.g/mL) for 12h, and then incubated in a solution containing 80. Mu. M H 2 O 2 Incubation was performed for 24h in serum-free medium. SHSY-5Y cells were digested and the collected cells were labeled in EP tubes. 300. Mu.L of JC-1 staining solution was added to each EP tube for 15 minutes in the dark. After centrifugation and washing with 500. Mu.L of washing solution, the washing solution was discarded and the cells were resuspended in 300. Mu.L of washing solution for flow cytometry analysis.
As can be seen from FIGS. 3A-D, only H is exposed 2 O 2 The experimental group of SHSY-5Y cells after 24 hours had a relatively high apoptosis rate and a low mitochondrial membrane potential level. Exposure to H compared to the normal control 2 O 2 The apoptosis rate of SHSY-5Y cells increased from 3.84+ -1.76% to 36.3+ -3.95% and the mitochondrial membrane potential of the cells decreased to 42.34+ -3.09%. After the cells are pretreated by angelica and prepared rehmannia root, the apoptosis rate is respectively reduced to 20.81+/-0.02% and 22.32+/-0.87%, and the mitochondrial membrane potential is respectively restored to 56.68+/-8.47% and 46.37 +/-9.75%. Compared with the pretreatment of single angelica ethanol extract and single prepared rehmannia root ethanol extract, 100 mug/mL of the composition prepared in the example 1 (the composition prepared by mixing the angelica ethanol extract and the prepared rehmannia root ethanol extract according to the volume ratio of 3:2) reduces the apoptosis rate to 17.8+/-2.03% after pretreatment of cells, and the mitochondrial membrane potential is restored to 65.20 +/-12.84% from 42.34+/-3.09%, which shows that the composition prepared in the example 1 (the composition prepared by mixing the angelica ethanol extract and the prepared rehmannia root ethanol extract according to the volume ratio of 3:2) has significantly better effects of preventing apoptosis and improving mitochondrial membrane potential than the single angelica ethanol extract and the single prepared rehmannia root ethanol extract. This also demonstrates that the composition of the present invention has significantly better anti-cell damage effect than the ethanol extract of Angelica sinensis alone and rehmannia glutinosa Libosch aloneYellow ethanol extract.
Claims (7)
1. A composition with antioxidant effect is characterized by comprising ethanol extract of Angelica sinensis and
ethanol extract of radix rehmanniae Preparata;
the preparation method of the composition with the antioxidation effect comprises the following steps:
the preparation method of the ethanol extract of Chinese angelica comprises the following steps: reflux-extracting radix Angelicae sinensis with ethanol under heating, and collecting extractive solution;
the preparation method of the ethanol extract of the prepared rehmannia root comprises the following steps: taking prepared rehmannia root, performing cold soaking with ethanol, then performing heating reflux extraction, and obtaining ethanol extract of prepared rehmannia root after the extraction is finished;
mixing the extracting solution: mixing radix Angelicae sinensis ethanol extract with radix rehmanniae Preparata ethanol extract to obtain mixed solution;
concentrating and drying: concentrating and drying the mixed solution to obtain the composition with the antioxidation effect;
in the step of mixing the extracting solutions, the volume ratio of the ethanol extracting solution of the angelica to the ethanol extracting solution of the prepared rehmannia is 3:2;
the ethanol in the step of preparing the angelica ethanol extract is ethanol aqueous solution with the volume fraction of 65-75%;
in the preparation step of the ethanol extract of Chinese angelica, the volume ratio of the weight of Chinese angelica to the ethanol is 1 g:13-15 mL;
in the preparation step of the ethanol extract of Chinese angelica, the temperature of heating reflux extraction is 81-83 ℃ and the extraction time is 1-3 h;
the ethanol in the preparation step of the ethanol extract of the prepared rehmannia root refers to ethanol water solution with the volume fraction of 55-65%;
in the preparation step of the ethanol extract of the prepared rehmannia root, the volume ratio of the weight of the prepared rehmannia root to the ethanol is 1 g:8-12 mL;
the cold soaking time in the preparation step of the ethanol extract of the prepared rehmannia root is 16-30 hours;
in the preparation step of the ethanol extract of the prepared rehmannia root, the temperature of heating reflux extraction is 78-80 ℃ and the extraction time is 1-1.5 h.
2. The composition having an antioxidant effect according to claim 1, wherein the ethanol in the step of preparing the ethanol extract of Angelica sinensis is an aqueous ethanol solution having a volume fraction of 70%;
in the preparation step of the ethanol extract of Chinese angelica, the volume ratio of the weight of Chinese angelica to the ethanol is 1g to 14mL.
3. The composition with antioxidant effect according to claim 1, wherein in the step of preparing the ethanol extract of Angelica sinensis, the temperature of the reflux extraction by heating is 83 ℃ and the extraction time is 2 hours.
4. The composition having an antioxidant effect according to claim 1, wherein the ethanol in the step of preparing the ethanol extract of radix rehmanniae Preparata is an aqueous ethanol solution having a volume fraction of 60%;
in the preparation step of the ethanol extract of the prepared rehmannia root, the volume ratio of the weight of the prepared rehmannia root to the ethanol is 1g to 10mL.
5. The composition with antioxidant effect according to claim 1, wherein the cold soaking time in the preparation step of the ethanol extract of radix rehmanniae Preparata is 24 hours.
6. The composition with antioxidant effect according to claim 1, wherein in the step of preparing the ethanol extract of radix rehmanniae Preparata, the temperature of the reflux extraction under heating is 80℃and the extraction time is 1h.
7. Use of the composition having an antioxidant effect according to claim 1 for the preparation of a product having an antioxidant effect or having an anti-cell damage effect;
the product is a cosmetic or a medicine.
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