CN116870064B - Traditional Chinese medicine composition with antioxidant activity and preparation method thereof - Google Patents
Traditional Chinese medicine composition with antioxidant activity and preparation method thereof Download PDFInfo
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Classifications
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- 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
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
-
- 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
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
-
- 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
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/61—Myrtaceae (Myrtle family), e.g. teatree or eucalyptus
-
- 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
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/738—Rosa (rose)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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Abstract
The invention discloses a traditional Chinese medicine composition with antioxidant activity and a preparation method thereof, wherein the traditional Chinese medicine composition is prepared from, by mass, 10% -15% of cinnamon, 20% -25% of rose, 25% -30% of fructus chebulae immaturus and 35-40% of clove, and the raw materials are extracted, concentrated, purified and dried. The composition obtained by the invention has better free radical inhibition effect and obvious antioxidation effect.
Description
Technical Field
The invention relates to the technical field of preparation of traditional Chinese medicine compositions, in particular to a traditional Chinese medicine composition with antioxidant activity and a preparation method thereof.
Background
Antioxidant refers to the abbreviation of antioxidant free radical, english Anti-oxidation. Antioxidant is a relatively broad concept of metabolic substance changes in the human body, free radical metabolism occurs in body tissues, and when the free radicals are excessively generated (during intense exercise) or the body scavenging capacity is reduced, the free radicals can cause damage to a plurality of biological molecules such as proteins and nucleic acids, and cause super-oxidation reaction, so that the cell structure and function are widely damaged. Antioxidant includes peroxy radical scavenging, superoxide radical scavenging, hydroxyl radical scavenging, etc., which maintain dynamic balance of radical generation and scavenging in vivo through their respective routes of action. The oxidation resistance is any substance which can effectively inhibit the oxidation reaction of free radicals when being present in low concentration, and the action mechanism of the oxidation resistance can be directly acting on the free radicals or indirectly consuming substances which are easy to generate the free radicals to prevent further reaction. The human body inevitably generates free radicals and simultaneously naturally generates antioxidant substances resisting the free radicals so as to counteract the oxidative attack of the free radicals on human cells.
At present, there is a need to develop a pure natural antioxidant Chinese medicinal composition with high activity and few side effects, and no additional auxiliary medicaments are required.
Disclosure of Invention
The invention starts from the problem of natural antioxidant demand of people, and screens natural components by taking DPPH, ABTS, SORAC antioxidant high-throughput screening model as a key means, so as to obtain the traditional Chinese medicine composition of rose, fructus chebulae immaturus, cinnamon and clove with high antioxidant activity.
The technical scheme adopted for solving the technical problems is as follows:
a preparation method of a traditional Chinese medicine composition with antioxidant activity comprises the following steps:
(1) Extracting: respectively weighing 10-15% of cinnamon, 20-25% of rose, 25-30% of fructus chebulae immaturus and 35-40% of clove according to mass percentage, crushing, adding 10-15 times of 35% ethanol solution, ultrasonically extracting for 2-3 times at 450-500W for 60-80min each time, cooling to room temperature after extraction, filtering, and combining filtrates;
(2) Concentrating and drying: concentrating the filtered extractive solution under reduced pressure and vacuum drying;
(3) And (3) preparation and purification: dissolving the extract in ethanol solution, adsorbing with AB-8 resin column for 2 hr, eluting with 6 times of ultra-pure water for 2 hr, eluting with 6 times of low concentration ethanol solution for 2 hr, eluting with high concentration ethanol solution for 2 hr, and collecting eluate;
(4) Concentrating and drying: the collected 35% ethanol and ultrapure water eluates were concentrated under reduced pressure and dried.
Cinnamon (Cinnamomum CASSIA PRESL) is a tree species of the genus Cinnamomum of the family Lauraceae, and is mainly distributed in Guangdong provinces and Guangxi provinces of China. The chemical components of the cinnamon comprise polysaccharide compounds, sesquiterpenes, diterpene and glycoside compounds, flavonoid compounds and the like, and have the functions of resisting oxidation, resisting bacteria and tumors, enhancing digestion, easing pain and the like.
Roses (Rosa rugosa Thunb) are small-sized deciduous shrubs, high in stem height and growing thorns and naps, and the veins are obviously wrinkled. Different flowering periods are mainly opened in 4-7 months. The color is mostly red and deep red, and a few are white or yellow. The rose contains a large amount of polyphenols, polysaccharides and flavonoids, and also contains a large amount of dietary fibers, sugar, amino acids, linolenic acid and the like, and has high nutritional value and functional activity.
Fructus Canarii albi is also called "Tibetan fructus Canarii albi", and is dry young fruit of fructus Chebulae (TERMINALIA CHEBULA Retz.) of Combretaceae, and can be used as heat clearing and toxic substances removing medicine in clinical application of traditional Chinese medicine for treating pneumonia, laryngitis, tonsillitis, pharyngitis, bacillary dysentery, etc. The fructus Chebulae Immaturus contains abundant tannins and phenolic acids, and has main chemical components such as chebula acid, chebular acid, gallic acid, etc.
Flos Caryophylli (Syzygium aromaticum) is plant of Syzygium (Syzygium) of Myrtaceae (Myrtales), is evergreen arbor, and is a perfume native to Indonesia. The clove contains various chemical components, has the effects of resisting bacteria, diminishing inflammation, relieving fever, easing pain, resisting cancer, resisting oxidation, resisting depression, reducing blood sugar and the like, and has various applications in industries such as food, essence, spice, medicine and the like.
Preferably, the drying temperature of step (2) and step (4) is 45-50 ℃.
Preferably, the step (3) specifically comprises: the extract was dissolved by adding 6 times of 50vol% ethanol solution, adsorbed on AB-8 resin column for 2 hours, eluted with 6 times of ultra pure water for 2 hours, eluted with 6 times of 35vol% ethanol solution for 2 hours, eluted with 95vol% ethanol solution for 2 hours, and the eluate was collected.
Preferably, the raw materials are: 10% of cinnamon, 20% of rose, 30% of fructus chebulae immaturus and 40% of clove.
Preferably, the raw materials are: cinnamon 12%, rose 22%, fructus chebulae immaturus 28% and clove 38%.
Preferably, the raw materials are: 15% of cinnamon, 25% of roses, 25% of fructus chebulae immaturus and 35% of clove.
As another aim of the invention, the invention also discloses a traditional Chinese medicine composition with antioxidant activity, which is prepared by adopting the method.
The invention has the following beneficial effects:
1. simple process and small side effect: compared with other antioxidant products, the combination of natural products is adopted, synthesis and structural modification are not needed, and the generation of side effects of human bodies is reduced. Meanwhile, auxiliary medicines are not required to be added additionally, so that the pure nature of the component sources is maintained.
2. High inhibition rate: compared with other antioxidant products, the traditional Chinese medicine composition disclosed by the invention has a better free radical inhibition effect and an obvious antioxidant effect by utilizing DPPH, ABTS, SORAC antioxidant high-throughput screening experiments.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
The following examples prepared an antioxidant Chinese medicinal composition according to the following method:
(1) Extracting: respectively weighing 10-15% of cinnamon, 20-25% of rose, 25-30% of fructus chebulae immaturus and 35-40% of clove according to mass percentage, crushing, adding 10-15 times of 35% ethanol solution, ultrasonically extracting for 2-3 times at 450-500W for 60-80min each time, cooling to room temperature after extraction, filtering, and merging filtrate.
(2) Concentrating and drying: concentrating the filtered extractive solution under reduced pressure and vacuum drying at 45-50deg.C.
(3) And (3) preparation and purification: dissolving the above extract with 6 times of 50% ethanol, loading on AB-8 resin column for adsorption for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35% ethanol for 2 hr, eluting with 95% ethanol for 2 hr, and collecting eluate.
(4) Concentrating and drying: concentrating and drying the collected 35% ethanol and ultrapure water eluent under reduced pressure and vacuum at 45-50deg.C.
(5) Carrying out DPPH, ABTS, SORAC antioxidation high-throughput screening on the prepared traditional Chinese medicine composition:
1. Based on DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) free radical is a synthetic stable free radical taking nitrogen as a center, because unpaired electrons on nitrogen atoms have a strong absorption peak (an alcohol solution of the unpaired electrons is dark purple) at 517nm, when an antioxidant exists, DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) captures one electron to be paired with free electrons so that the absorption of the DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) gradually disappears, the fading degree of the DPPH (1, 1-trinitrophenylhydrazine) is quantitatively related to the number of the electrons received by the DPPH, thus the change of the absorbance value after the DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) free radical reacts with a sample can be detected by an enzyme-labeled instrument and the like, and the free radical scavenging capability of the sample can be detected. 2. ABTS is a substrate of catalase, the oxidation-reduction potential of ABTS/ABTS+ is 0.68V, and electron transfer is easy to occur, so that stable green free radical ABTS+ is generated. The prepared ABTS solution is subjected to electron transfer under the catalysis of potassium peroxodisulfate to generate stable ABTS+ free radicals. The ability of the sample to scavenge ABTS free radicals (the free radicals are scavenged, the reduction in the number can lighten the color of the solution, thereby leading to the reduction in absorbance at 734 nm) is detected, so that the ability of the sample to scavenge ABTS+ free radicals can be judged, 3, xanthine generate superoxide free radicals under the catalysis of xanthine oxidase, and dihydroethidium is oxidized to generate 2-dihydroethidium, and the fluorescence intensity is increased.
Example 1
A preparation method of a traditional Chinese medicine composition with antioxidant activity comprises the following steps:
(1) Extracting: respectively weighing 10% of cinnamon, 20% of roses, 30% of fructus chebulae immaturus and 40% of flos caryophylli according to mass percentage, crushing, adding 10 times of 35% ethanol solution, carrying out ultrasonic extraction for 2 times at 450W for 60min each time, cooling to room temperature after the extraction is finished, filtering, and combining the filtrates.
(2) Concentrating and drying: concentrating and drying the collected fractions under reduced pressure at 45 ℃;
(3) And (3) preparation and purification: and (3) preparation and purification: dissolving the above extract with 6 times of 50% ethanol, loading on AB-8 resin column for adsorption for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35% ethanol for 2 hr, eluting with 95% ethanol for 2 hr, and collecting eluate.
(4) The 35% ethanol and ultrapure water eluates collected above were concentrated and dried under vacuum at 45 ℃.
(5) DPPH antioxidation high-flux detection is carried out on the prepared traditional Chinese medicine composition: the absorbance of the DPPH solution in a 96-well plate was set to 0.7.+ -. 0.02 (100. Mu.l DPPH+100. Mu.l ethanol), the samples were diluted 1000-fold with ethanol and divided into four groups, 10, 20, 30 and 40. Mu.l were added to the plate, and 100. Mu.l were made up with ethanol, 3 groups of parallel samples were made for each group, and after 100. Mu.l of DPPH solution was added to each well, the samples were rapidly placed in an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.033mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as the abscissa, the clearance was taken as the ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e., the IC 50 value).
(6) Performing ABTS antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: the ABTS solution and potassium peroxodisulfate were reacted for 18 hours in a buffer solution of 0.75M ph=7.4 with sodium dihydrogen phosphate and dipotassium hydrogen phosphate, diluted to an absorbance of 0.7±0.02 in 96-well plates (100 μl ABTS reaction solution+100 μl buffer solution), the samples were diluted 1000-fold with the buffer solution to four groups, 10, 20, 30, 40 μl respectively were added to the well plates, 100 μl was made up with the buffer solution, 3 groups of replicates were made for each group, and after 100 μl ABTS reaction solution was added to each well, the samples were rapidly put into an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.0133mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as the abscissa, the clearance was taken as the ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e., the IC 50 value).
(7) Performing SORAC antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: sodium dihydrogen phosphate, dipotassium hydrogen phosphate and diethylenetriamine pentaacetic acid are prepared into a buffer solution with the pH value of 0.75M and 7.4, HE fluorescein working solution with the pH value of 19.08 mu M is prepared, xanthine solution is prepared (7.5 mg of xanthine is accurately weighed in a 50mL volumetric flask, 2.5mL of 0.1M sodium hydroxide is added for complete dissolution, and then the buffer solution is used for volume fixation), xanthine oxidase (working concentration: 0.1U/mL) is prepared, and SOD working solution (300 units/mL) is prepared. After 25. Mu.L of each of a blank (buffer solution), a sample (diluted 1000-fold) and a standard antioxidant (SOD) was mixed with 150. Mu.L of a dihydroethidium fluorescein HE (xanthine-containing) solution, the well plate was covered with a cover and incubated at 37℃for 10 minutes, 25. Mu.L of a xanthine oxidase solution was immediately added, and the inhibition was traced by using an enzyme-labeled instrument, to obtain an inhibition of 84.8%.
Example 2
A preparation method of a traditional Chinese medicine composition with antioxidant activity comprises the following steps:
(1) Extracting: respectively weighing 12% of cinnamon, 22% of roses, 28% of fructus chebulae immaturus and 38% of flos caryophylli according to mass percentage, crushing, adding 12 times of 35% ethanol solution, performing ultrasonic extraction for 2 times at 480W for 70min each time, cooling to room temperature after extraction, filtering, and combining filtrates.
(2) Concentrating and drying: concentrating and drying the collected fractions under reduced pressure at 45 ℃;
(3) And (3) preparation and purification: and (3) preparation and purification: dissolving the above extract with 6 times of 50% ethanol, loading on AB-8 resin column for adsorption for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35% ethanol for 2 hr, eluting with 95% ethanol for 2 hr, and collecting eluate.
(4) The 35% ethanol and ultrapure water eluates collected above were concentrated and dried under vacuum at 45 ℃.
(5) DPPH antioxidation high-flux detection is carried out on the prepared traditional Chinese medicine composition: the absorbance of the DPPH solution in a 96-well plate was set to 0.7.+ -. 0.02 (100. Mu.l DPPH+100. Mu.l ethanol), the samples were diluted 1000-fold with ethanol and divided into four groups, 10, 20, 30 and 40. Mu.l were added to the plate, and 100. Mu.l were made up with ethanol, 3 groups of parallel samples were made for each group, and after 100. Mu.l of DPPH solution was added to each well, the samples were rapidly placed in an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.034mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as the abscissa, the clearance was taken as the ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e., the IC 50 value).
(6) Performing ABTS antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: the ABTS solution and potassium peroxodisulfate were reacted for 18 hours in a buffer solution of 0.75M ph=7.4 with sodium dihydrogen phosphate and dipotassium hydrogen phosphate, diluted to an absorbance of 0.7±0.02 in 96-well plates (100 μl ABTS reaction solution+100 μl buffer solution), the samples were diluted 1000-fold with the buffer solution to four groups, 10, 20, 30, 40 μl respectively were added to the well plates, 100 μl was made up with the buffer solution, 3 groups of replicates were made for each group, and after 100 μl ABTS reaction solution was added to each well, the samples were rapidly put into an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.0134mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as abscissa, the clearance was taken as ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e. IC 50 value).
(7) Performing SORAC antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: sodium dihydrogen phosphate, dipotassium hydrogen phosphate and diethylenetriamine pentaacetic acid are prepared into a buffer solution with the pH value of 0.75M and 7.4, HE fluorescein working solution with the pH value of 19.08 mu M is prepared, xanthine solution is prepared (7.5 mg of xanthine is accurately weighed in a 50mL volumetric flask, 2.5mL of 0.1M sodium hydroxide is added for complete dissolution, and then the buffer solution is used for volume fixation), xanthine oxidase (working concentration: 0.1U/mL) is prepared, and SOD working solution (300 units/mL) is prepared. After 25. Mu.L of each of a blank (buffer solution), a sample (diluted 1000-fold) and a standard antioxidant (SOD) was mixed with 150. Mu.L of a dihydroethidium fluorescein HE (xanthine-containing) solution, the well plate was covered with a cover and incubated at 37℃for 10 minutes, 25. Mu.L of a xanthine oxidase solution was immediately added, and the inhibition was traced by using an enzyme-labeled instrument, thereby obtaining an inhibition ratio of 83.5%.
Example 3
A preparation method of a traditional Chinese medicine composition with antioxidant activity comprises the following steps:
(1) Extracting: respectively weighing 15% of cinnamon, 25% of roses, 25% of fructus chebulae immaturus and 35% of flos caryophylli according to mass percentage, crushing, adding 12 times of 50% ethanol solution, carrying out ultrasonic extraction for 3 times at 500W for 80min each time, cooling to room temperature after the extraction is finished, filtering, and combining the filtrates.
(2) Concentrating and drying: concentrating and drying the collected fractions under reduced pressure at 45 ℃;
(3) And (3) preparation and purification: and (3) preparation and purification: dissolving the above extract with 6 times of 50% ethanol, loading on AB-8 resin column for adsorption for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35% ethanol for 2 hr, eluting with 95% ethanol for 2 hr, and collecting eluate.
(4) The 35% ethanol and ultrapure water eluates collected above were concentrated and dried under vacuum at 45 ℃.
(5) DPPH antioxidation high-flux detection is carried out on the prepared traditional Chinese medicine composition: the absorbance of the DPPH solution in a 96-well plate was set to 0.7.+ -. 0.02 (100. Mu.l DPPH+100. Mu.l ethanol), the samples were diluted 1000-fold with ethanol and divided into four groups, 10, 20, 30 and 40. Mu.l were added to the plate, and 100. Mu.l were made up with ethanol, 3 groups of parallel samples were made for each group, and after 100. Mu.l of DPPH solution was added to each well, the samples were rapidly placed in an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.038mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as the abscissa, the clearance was taken as the ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e., the IC 50 value).
(6) Performing ABTS antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: the ABTS solution and potassium peroxodisulfate were reacted for 18 hours in a buffer solution of 0.75M ph=7.4 with sodium dihydrogen phosphate and dipotassium hydrogen phosphate, diluted to an absorbance of 0.7±0.02 in 96-well plates (100 μl ABTS reaction solution+100 μl buffer solution), the samples were diluted 1000-fold with the buffer solution to four groups, 10, 20, 30, 40 μl respectively were added to the well plates, 100 μl was made up with the buffer solution, 3 groups of replicates were made for each group, and after 100 μl ABTS reaction solution was added to each well, the samples were rapidly put into an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.0154mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as abscissa, the clearance was taken as ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e. the IC 50 value).
(7) Performing SORAC antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: sodium dihydrogen phosphate, dipotassium hydrogen phosphate and diethylenetriamine pentaacetic acid are prepared into a buffer solution with the pH value of 0.75M and 7.4, HE fluorescein working solution with the pH value of 19.08 mu M is prepared, xanthine solution is prepared (7.5 mg of xanthine is accurately weighed in a 50mL volumetric flask, 2.5mL of 0.1M sodium hydroxide is added for complete dissolution, and then the buffer solution is used for volume fixation), xanthine oxidase (working concentration: 0.1U/mL) is prepared, and SOD working solution (300 units/mL) is prepared. After 25. Mu.L of each of a blank (buffer solution), a sample (diluted 1000-fold) and a standard antioxidant (SOD) was mixed with 150. Mu.L of a dihydroethidium fluorescein HE (xanthine-containing) solution, the well plate was covered with a cover and incubated at 37℃for 10 minutes, 25. Mu.L of a xanthine oxidase solution was immediately added, and the inhibition was traced by using an enzyme-labeled instrument, thereby obtaining an inhibition rate of 82.6%.
Comparative example 1
The preparation method of the traditional Chinese medicine composition with the antioxidant activity of the comparative example comprises the following steps:
(1) Respectively weighing 30% of cinnamon, 30% of rose and 40% of fructus chebulae immaturus according to mass percentage, crushing, adding 15 times of 35% ethanol solution, carrying out ultrasonic extraction for 3 times at 500W for 80min each time, cooling to room temperature after extraction, filtering, and merging filtrate.
(2) Concentrating and drying: concentrating the filtered extractive solution under reduced pressure and vacuum drying at 45-50deg.C.
(3) And (3) preparation and purification: dissolving the above extract with 6 times of 50% ethanol, loading on AB-8 resin column for adsorption for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35% ethanol for 2 hr, eluting with 95% ethanol for 2 hr, and collecting eluate.
(4) Concentrating and drying: concentrating and drying the collected 35% ethanol and ultrapure water eluent under reduced pressure and vacuum at 45-50deg.C.
(5) DPPH antioxidation high-flux detection is carried out on the prepared traditional Chinese medicine composition: the absorbance of the DPPH solution in a 96-well plate was set to 0.7.+ -. 0.02 (100. Mu.l DPPH+100. Mu.l ethanol), the samples were diluted 1000-fold with ethanol and divided into four groups, 10, 20, 30 and 40. Mu.l were added to the plate, and 100. Mu.l were made up with ethanol, 3 groups of parallel samples were made for each group, and after 100. Mu.l of DPPH solution was added to each well, the samples were rapidly placed in an enzyme-labeled instrument for testing. The IC 50 value was 0.062mg/mL.
(6) Performing ABTS antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: the ABTS solution and potassium peroxodisulfate were reacted for 18 hours in a buffer solution of 0.75M ph=7.4 with sodium dihydrogen phosphate and dipotassium hydrogen phosphate, diluted to an absorbance of 0.7±0.02 in 96-well plates (100 μl ABTS reaction solution+100 μl buffer solution), the samples were diluted 1000-fold with the buffer solution to four groups, 10, 20, 30, 40 μl respectively were added to the well plates, 100 μl was made up with the buffer solution, 3 groups of replicates were made for each group, and after 100 μl ABTS reaction solution was added to each well, the samples were rapidly put into an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.057mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as the abscissa, the clearance was taken as the ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e., the IC 50 value).
(7) Performing SORAC antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: sodium dihydrogen phosphate, dipotassium hydrogen phosphate and diethylenetriamine pentaacetic acid are prepared into a buffer solution with the pH value of 0.75M and 7.4, HE fluorescein working solution with the pH value of 19.08 mu M is prepared, xanthine solution is prepared (7.5 mg of xanthine is accurately weighed in a 50mL volumetric flask, 2.5mL of 0.1M sodium hydroxide is added for complete dissolution, and then the buffer solution is used for volume fixation), xanthine oxidase (working concentration: 0.1U/mL) is prepared, and SOD working solution (300 units/mL) is prepared. After 25. Mu.L of each of a blank (buffer solution), a sample (diluted 1000-fold) and a standard antioxidant (SOD) was mixed with 150. Mu.L of a dihydroethidium fluorescein HE (xanthine-containing) solution, the well plate was covered and incubated at 37℃for 10 minutes, 25. Mu.L of xanthine oxidase solution was immediately added, and the mixture was subjected to trace recording using an enzyme-labeled instrument.
In contrast to example 1, in comparative example 1, in which the clove herb was absent, DPPH antioxidant activity IC 50 was increased from 0.033mg/mL to 0.062mg/mL, ABTS antioxidant activity IC 50 was increased from 0.0133mg/mL to 0.057mg/mL (the smaller the value of IC 50 was, the greater the antioxidant activity was), and the SORAC antioxidant activity (xanthine oxidase inhibition rate) of comparative example 1 was decreased from 84.8% to 62.4%.
Comparative example 2
The preparation method of the traditional Chinese medicine composition with the antioxidant activity of the comparative example comprises the following steps:
(1) Respectively weighing 40% of cinnamon, 40% of rose and 20% of fructus chebulae immaturus according to mass percentage, crushing, adding 15 times of 35% ethanol solution, carrying out ultrasonic extraction for 3 times at 500W for 80min each time, cooling to room temperature after extraction, filtering, and merging filtrate.
(2) Concentrating and drying: concentrating the filtered extractive solution under reduced pressure and vacuum drying at 45-50deg.C.
(3) And (3) preparation and purification: dissolving the above extract with 6 times of 50% ethanol, loading on AB-8 resin column for adsorption for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35% ethanol for 2 hr, eluting with 95% ethanol for 2 hr, and collecting eluate.
(4) Concentrating and drying: concentrating and drying the collected 35% ethanol and ultrapure water eluent under reduced pressure and vacuum at 45-50deg.C.
(5) DPPH antioxidation high-flux detection is carried out on the prepared traditional Chinese medicine composition: the absorbance of the DPPH solution in a 96-well plate is set to be 0.7+/-0.02 (100 microliters of DPPH+100 microliters of ethanol), samples are diluted 1000 times by ethanol and divided into four groups, 10, 20, 30 and 40 microliters of the samples are respectively added into the well plate, the samples are supplemented to 100 microliters by ethanol, 3 groups of samples are prepared in parallel, 100 microliters of the DPPH solution is added into each well, and the samples are quickly placed into an enzyme-labeled instrument for testing, so that the IC 50 value of the samples is 0.067mg/mL.
(6) Performing ABTS antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: the ABTS solution and potassium peroxodisulfate were reacted for 18 hours in a buffer solution of 0.75M ph=7.4 with sodium dihydrogen phosphate and dipotassium hydrogen phosphate, diluted to an absorbance of 0.7±0.02 in 96-well plates (100 μl ABTS reaction solution+100 μl buffer solution), the samples were diluted 1000-fold with the buffer solution to four groups, 10, 20, 30, 40 μl respectively were added to the well plates, 100 μl was made up with the buffer solution, 3 groups of replicates were made for each group, and after 100 μl ABTS reaction solution was added to each well, the samples were rapidly put into an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.064mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as abscissa, the clearance was taken as ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e., the IC 50 value).
(7) Performing SORAC antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: sodium dihydrogen phosphate, dipotassium hydrogen phosphate and diethylenetriamine pentaacetic acid are prepared into a buffer solution with the pH value of 0.75M and 7.4, HE fluorescein working solution with the pH value of 19.08 mu M is prepared, xanthine solution is prepared (7.5 mg of xanthine is accurately weighed in a 50mL volumetric flask, 2.5mL of 0.1M sodium hydroxide is added for complete dissolution, and then the buffer solution is used for volume fixation), xanthine oxidase (working concentration: 0.1U/mL) is prepared, and SOD working solution (300 units/mL) is prepared. After 25. Mu.L of each of a blank (buffer solution), a sample (diluted 1000-fold) and a standard antioxidant (SOD) was mixed with 150. Mu.L of a dihydroethidium fluorescein HE (xanthine-containing) solution, the well plate was covered with a cover and incubated at 37℃for 10 minutes, 25. Mu.L of a xanthine oxidase solution was immediately added, and the inhibition was traced by using an enzyme-labeled instrument, thereby obtaining an inhibition of 66.7%.
Compared with example 1, in comparative example 2, after the clove medicinal material is absent, the DPPH antioxidant activity IC 50 value is increased from 0.033mg/mL to 0.067mg/mL, the ABTS antioxidant activity IC 50 value is increased from 0.0133mg/mL to 0.064mg/mL, and the SORAC antioxidant activity (xanthine oxidase inhibition rate) is reduced from 84.8% to 66.7%.
Comparative example 3
The preparation method of the traditional Chinese medicine composition with the antioxidant activity of the comparative example comprises the following steps:
(1) Respectively weighing 40% of cinnamon, 30% of rose and 30% of clove according to the mass percentage, crushing, adding 15 times of 35% ethanol solution, carrying out ultrasonic extraction for 3 times at 500W for 80min each time, cooling to room temperature after the extraction is finished, filtering, and combining the filtrates.
(2) Concentrating and drying: concentrating the filtered extractive solution under reduced pressure and vacuum drying at 45-50deg.C.
(3) And (3) preparation and purification: dissolving the above extract with 6 times of 50% ethanol, loading on AB-8 resin column for adsorption for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35% ethanol for 2 hr, eluting with 95% ethanol for 2 hr, and collecting eluate.
(4) Concentrating and drying: concentrating and drying the collected 35% ethanol and ultrapure water eluent under reduced pressure and vacuum at 45-50deg.C.
(5) DPPH antioxidation high-flux detection is carried out on the prepared traditional Chinese medicine composition: the absorbance of the DPPH solution in a 96-well plate was set to be 0.7.+ -. 0.02 (100. Mu.l DPPH+100. Mu.l ethanol), the samples were diluted 1000-fold with ethanol and divided into four groups, 10, 20, 30 and 40. Mu.l were added to the plate, and 100. Mu.l were made up with ethanol, 3 groups of parallel samples were made for each group, and after 100. Mu.l of DPPH solution was added to each well, the mixture was rapidly placed in a microplate reader and tested, to obtain an IC 50 value of 0.043mg/mL.
(6) Performing ABTS antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: the ABTS solution and potassium peroxodisulfate were reacted for 18 hours in a buffer solution of 0.75M ph=7.4 with sodium dihydrogen phosphate and dipotassium hydrogen phosphate, diluted to an absorbance of 0.7±0.02 in 96-well plates (100 μl ABTS reaction solution+100 μl buffer solution), the samples were diluted 1000-fold with the buffer solution to four groups, 10, 20, 30, 40 μl respectively were added to the well plates, 100 μl was made up with the buffer solution, 3 groups of replicates were made for each group, and after 100 μl ABTS reaction solution was added to each well, the samples were rapidly put into an enzyme-labeled instrument for testing. The IC 50 value was 0.021mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as the abscissa, the clearance was taken as the ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to obtain the corresponding abscissa value, namely the IC 50 value).
(7) Performing SORAC antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: sodium dihydrogen phosphate, dipotassium hydrogen phosphate and diethylenetriamine pentaacetic acid are prepared into a buffer solution with the pH value of 0.75M and 7.4, HE fluorescein working solution with the pH value of 19.08 mu M is prepared, xanthine solution is prepared (7.5 mg of xanthine is accurately weighed in a 50mL volumetric flask, 2.5mL of 0.1M sodium hydroxide is added for complete dissolution, and then the buffer solution is used for volume fixation), xanthine oxidase (working concentration: 0.1U/mL) is prepared, and SOD working solution (300 units/mL) is prepared. After 25. Mu.L of each of a blank (buffer solution), a sample (diluted 1000-fold) and a standard antioxidant (SOD) was mixed with 150. Mu.L of a dihydroethidium fluorescein HE (xanthine-containing) solution, the well plate was covered and incubated at 37℃for 10 minutes, 25. Mu.L of xanthine oxidase solution was immediately added, and the mixture was subjected to trace recording using an enzyme-labeled instrument.
Compared with example 1, in comparative example 3, after the fructus chebulae immaturus is absent, the DPPH antioxidant activity IC 50 value is increased from 0.033mg/mL to 0.043mg/mL, the ABTS antioxidant activity IC 50 value is increased from 0.0133mg/mL to 0.021mg/mL, and the SORAC antioxidant activity (xanthine oxidase inhibition rate) is reduced from 84.8% to 78.4%.
Comparative example 4
The preparation method of the traditional Chinese medicine composition with the antioxidant activity of the comparative example comprises the following steps:
(1) Respectively weighing 40% of cinnamon, 30% of fructus chebulae immaturus and 30% of flos caryophylli according to mass percentage, pulverizing, adding 15 times of 35% ethanol solution, performing ultrasonic extraction for 3 times at 500W for 80min each time, cooling to room temperature after extraction, filtering, and combining filtrates.
(2) Concentrating and drying: concentrating the filtered extractive solution under reduced pressure and vacuum drying at 45-50deg.C.
(3) And (3) preparation and purification: dissolving the above extract with 6 times of 50% ethanol, loading on AB-8 resin column for adsorption for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35% ethanol for 2 hr, eluting with 95% ethanol for 2 hr, and collecting eluate.
(4) Concentrating and drying: concentrating and drying the collected 35% ethanol and ultrapure water eluent under reduced pressure and vacuum at 45-50deg.C.
(5) DPPH antioxidation high-flux detection is carried out on the prepared traditional Chinese medicine composition: the absorbance of the DPPH solution in a 96-well plate was set to 0.7.+ -. 0.02 (100. Mu.l DPPH+100. Mu.l ethanol), the samples were diluted 1000-fold with ethanol and divided into four groups, 10, 20, 30 and 40. Mu.l were added to the plate, and 100. Mu.l were made up with ethanol, 3 groups of parallel samples were made for each group, and after 100. Mu.l of DPPH solution was added to each well, the samples were rapidly placed in an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.047mg/mL.
(6) Performing ABTS antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: the ABTS solution and potassium peroxodisulfate were reacted for 18 hours in a buffer solution of 0.75M ph=7.4 with sodium dihydrogen phosphate and dipotassium hydrogen phosphate, diluted to an absorbance of 0.7±0.02 in 96-well plates (100 μl ABTS reaction solution+100 μl buffer solution), the samples were diluted 1000-fold with the buffer solution to four groups, 10, 20, 30, 40 μl respectively were added to the well plates, 100 μl was made up with the buffer solution, 3 groups of replicates were made for each group, and after 100 μl ABTS reaction solution was added to each well, the samples were rapidly put into an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.029mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as abscissa, the clearance was taken as ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e., IC 50 value).
(7) Performing SORAC antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: sodium dihydrogen phosphate, dipotassium hydrogen phosphate and diethylenetriamine pentaacetic acid are prepared into a buffer solution with the pH value of 0.75M and 7.4, HE fluorescein working solution with the pH value of 19.08 mu M is prepared, xanthine solution is prepared (7.5 mg of xanthine is accurately weighed in a 50mL volumetric flask, 2.5mL of 0.1M sodium hydroxide is added for complete dissolution, and then the buffer solution is used for volume fixation), xanthine oxidase (working concentration: 0.1U/mL) is prepared, and SOD working solution (300 units/mL) is prepared. After 25. Mu.L of each of a blank (buffer solution), a sample (diluted 1000-fold) and a standard antioxidant (SOD) was mixed with 150. Mu.L of a dihydroethidium fluorescein HE (xanthine-containing) solution, the well plate was covered and incubated at 37℃for 10 minutes, 25. Mu.L of xanthine oxidase solution was immediately added, and the mixture was subjected to trace recording using an enzyme-labeled instrument. The inhibition rate was found to be 72.5%.
Compared with example 1, after the rose medicinal material is removed in comparative example 4, the DPPH antioxidant activity IC 50 value is increased from 0.033mg/mL to 0.047mg/mL, the ABTS antioxidant activity IC 50 value is increased from 0.0133mg/mL to 0.029mg/mL, and the SORAC antioxidant activity (xanthine oxidase inhibition rate) is reduced from 84.8% to 72.5%.
Comparative example 5
The preparation method of the traditional Chinese medicine composition with the antioxidant activity of the comparative example comprises the following steps:
(1) Respectively weighing 40% of fructus Canarii albi, 30% of flos Rosae Rugosae and 30% of flos Caryophylli according to mass percentage, pulverizing, adding 15 times of 35% ethanol solution, extracting with 500W ultrasound for 3 times each for 80min, cooling to room temperature after extraction, filtering, and mixing filtrates.
(2) Concentrating and drying: concentrating the filtered extractive solution under reduced pressure and vacuum drying at 45-50deg.C.
(3) And (3) preparation and purification: dissolving the above extract with 6 times of 50% ethanol, loading on AB-8 resin column for adsorption for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35% ethanol for 2 hr, eluting with 95% ethanol for 2 hr, and collecting eluate.
(4) Concentrating and drying: concentrating and drying the collected 35% ethanol and ultrapure water eluent under reduced pressure and vacuum at 45-50deg.C.
(5) DPPH antioxidation high-flux detection is carried out on the prepared traditional Chinese medicine composition: the absorbance of the DPPH solution in a 96-well plate was set to 0.7.+ -. 0.02 (100. Mu.l DPPH+100. Mu.l ethanol), the samples were diluted 1000-fold with ethanol and divided into four groups, 10, 20, 30 and 40. Mu.l were added to the plate, and 100. Mu.l were made up with ethanol, 3 groups of parallel samples were made for each group, and after 100. Mu.l of DPPH solution was added to each well, the samples were rapidly placed in an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.039mg/mL.
(6) Performing ABTS antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: the ABTS solution and potassium peroxodisulfate were reacted for 18 hours in a buffer solution of 0.75M ph=7.4 with sodium dihydrogen phosphate and dipotassium hydrogen phosphate, diluted to an absorbance of 0.7±0.02 in 96-well plates (100 μl ABTS reaction solution+100 μl buffer solution), the samples were diluted 1000-fold with the buffer solution to four groups, 10, 20, 30, 40 μl respectively were added to the well plates, 100 μl was made up with the buffer solution, 3 groups of replicates were made for each group, and after 100 μl ABTS reaction solution was added to each well, the samples were rapidly put into an enzyme-labeled instrument for testing. The IC 50 value was found to be 0.017mg/mL (IC 50: the experimentally obtained antioxidant concentration was taken as the abscissa, the clearance was taken as the ordinate, and then a linear regression equation was calculated. The clearance 50% was substituted into the equation to find the corresponding abscissa value, i.e., the IC 50 value).
(7) Performing SORAC antioxidation high-throughput detection on the prepared traditional Chinese medicine composition: sodium dihydrogen phosphate, dipotassium hydrogen phosphate and diethylenetriamine pentaacetic acid are prepared into a buffer solution with the pH value of 0.75M and 7.4, HE fluorescein working solution with the pH value of 19.08 mu M is prepared, xanthine solution is prepared (7.5 mg of xanthine is accurately weighed in a 50mL volumetric flask, 2.5mL of 0.1M sodium hydroxide is added for complete dissolution, and then the buffer solution is used for volume fixation), xanthine oxidase (working concentration: 0.1U/mL) is prepared, and SOD working solution (300 units/mL) is prepared. After 25. Mu.L of each of a blank (buffer solution), a sample (diluted 1000-fold) and a standard antioxidant (SOD) was mixed with 150. Mu.L of a dihydroethidium fluorescein HE (xanthine-containing) solution, the well plate was covered and incubated at 37℃for 10 minutes, 25. Mu.L of xanthine oxidase solution was immediately added, and the mixture was subjected to trace recording using an enzyme-labeled instrument.
Compared with example 1, after the cinnamon medicinal material is removed in comparative example 5, the DPPH antioxidant activity IC 50 value is increased from 0.033mg/mL to 0.039mg/mL, the ABTS antioxidant activity IC 50 value is increased from 0.0133mg/mL to 0.017mg/mL, and the SORAC antioxidant activity (xanthine oxidase inhibition rate) is reduced from 84.8% to 81.2%.
Claims (5)
1. The preparation method of the traditional Chinese medicine composition with the antioxidant activity is characterized by comprising the following steps:
(1) Extracting: respectively weighing 10-15% of cinnamon, 20-25% of rose, 25-30% of fructus chebulae immaturus and 35-40% of clove according to mass percentage, crushing, adding 10-15 times of 35% ethanol solution, ultrasonically extracting for 2-3 times at 450-500W for 60-80min each time, cooling to room temperature after extraction, filtering, and combining filtrates;
(2) Concentrating and drying: concentrating the filtered extractive solution under reduced pressure and vacuum drying;
(3) And (3) preparation and purification: dissolving the extract in 6 times of 50vol% ethanol solution, adsorbing with AB-8 resin column for 2 hr, eluting with 6 times of ultrapure water for 2 hr, eluting with 6 times of 35 vol% ethanol solution for 2 hr, eluting with 95 vol% ethanol solution for 2 hr, and collecting eluate;
(4) Concentrating and drying: concentrating and drying the collected 35% ethanol and ultrapure water eluent under reduced pressure and vacuum;
the drying temperature of the step (2) and the step (4) is 45-50 ℃.
2. The preparation method according to claim 1, wherein the raw materials are: 10% of cinnamon, 20% of rose, 30% of fructus chebulae immaturus and 40% of clove.
3. The preparation method according to claim 1, wherein the raw materials are: cinnamon 12%, rose 22%, fructus chebulae immaturus 28% and clove 38%.
4. The method of claim 1, wherein the cinnamon is 15%, the rose is 25%, the fructus chebulae immaturus is 25% and the clove is 35%.
5. A Chinese medicinal composition having antioxidant activity prepared by the method of any one of claims 1 to 4.
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