CN103940926A - Method for rapidly detecting antioxidant ingredients in rhubarb seeds - Google Patents
Method for rapidly detecting antioxidant ingredients in rhubarb seeds Download PDFInfo
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- CN103940926A CN103940926A CN201410170083.9A CN201410170083A CN103940926A CN 103940926 A CN103940926 A CN 103940926A CN 201410170083 A CN201410170083 A CN 201410170083A CN 103940926 A CN103940926 A CN 103940926A
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Abstract
The invention relates to a method for rapidly detecting antioxidant ingredients in rhubarb seeds. The method comprises the following steps: (1) crushing the rhubarb seeds to obtain rhubarb seed powder; (2) extracting the rhubarb seed powder by using methanol, and filtering to obtain a rhubarb seed extracting solution; (2) performing chromatographic column separation on the rhubarb seed extracting solution by using a high performance liquid chromatography, enabling the separated ingredients to flow through a first ultraviolet detector to serve as a sample, deriving the sample and an antioxidant, namely 1,1-biphenyl-2trinitrobenzene hydrazine which is pumped, serves as a mobile phase in a second pump and has the mass concentration of 10-200mg/L in a reactive loop, enabling the product to enter a second ultraviolet detector, and comparing chromatogram maps recorded by the first ultraviolet detector and the second ultraviolet detector, wherein when a compound with a negative peak occurs in the chromatogram map recorded by the second ultraviolet detector, the compound is the antioxidant ingredient in the extract. The method is simple and rapid, and the antioxidant ingredients in rhubarb seeds can be effectively evaluated.
Description
Technical field
The present invention relates to a kind of method of fast detecting antioxidant content, relate in particular to the method for antioxidant content in a kind of fast detecting rheum officinale seed.
Background technology
Chinese herb rhubarb is the dry root and rhizome of the ancient especially big Huang (Rheum tanguticum Maxim. ex Balf .) of polygonaceae Rheum plant Tang, sorrel (Rheum p almatum Linn.) or Rheum officinale (Rh. of f icinale Baill.).The ancient especially big Huang of Tang has another name called monarch's wood and pricks (hiding name), rheum tanguticum Tschircs, belongs to Qinghai Province's genunie medicinal materials, is born in border, sylvan life cheuch or shrubbery, and height above sea level 2300 ~ 4200m, separately also has distribution in SOUTH OF GANSU, Eastern Tibet the north, Northwest Sichuan etc.Sorrel main product in Gansu, the ground such as Qinghai, Tibet, Sichuan, be mainly cultivation, output is maximum.Rheum officinale main product in Sichuan, the ground such as Guizhou, Yunnan, Hubei, Shaanxi, cultivation or wild.
Medicinal material seed is the source of production of crude drugs and development, is the internal factor that determines Chinese crude drug quality, is the science prerequisite that development high-quality Chinese crude drug is produced.Medicinal plant is of a great variety, and its seed is because the reasons such as heredity, habitat there will be the form varying.
At present, for the research of rheum officinale seed, mainly concentrate in the quality inspection indexs such as germination percentage, mass of 1000 kernel, cleanliness, water cut, rheum officinale seed resource is abundant, and wherein anti-oxidant aspect has no report.
1,1-diphenyl-2-trinitrophenyl-hydrazine (DPPH) free radical method has become in the world a kind of Antioxidative Activity Determination method of comparatively generally acknowledging, by continually for the antioxidation activity in vitro evaluation study method of bioactivator.DPPH is a kind of stable free radical in organic solvent, its lone electron has strong absorption (being darkviolet) near 517nm, when free radical scavenger exists, lone electron is paired, absorb and disappear or weaken, therefore by mensuration, absorb the degree weakening, can evaluate the activity of this free radical scavenger.But utilize DPPH method only can analyze total antioxidant activity in extract, cannot know oxidation-resistant active ingredient, cannot compare the size of each active substance antioxidation activity.
Summary of the invention
Technical matters to be solved by this invention be to provide a kind of simply, the method for antioxidant content in fast detecting rheum officinale seed fast and accurately.
For addressing the above problem, the method for antioxidant content in a kind of fast detecting rheum officinale seed of the present invention, comprises the following steps:
(1) rheum officinale seed meal is broken to after 40 ~ 60 orders, obtains rheum officinale seed powder;
(2) ultrasonic extraction 20 ~ 50min under the condition that is 200 ~ 300W with the methyl alcohol that the volumetric concentration of 50 ~ 100 times of its quality is 50% ~ 100% at power by described rheum officinale seed powder, obtains rheum officinale seed extract after filtration;
(3) described rheum officinale seed extract is separated through high performance liquid chromatography chromatographic column, separated composition is first flowed through after first UV-detector as sample, the mass concentration pumping into as mobile phase in this sample and second pump is the antioxidant 1 of 10 ~ 200 mg/L, after 1-diphenyl-2 trinitrophenyl-hydrazine is derivative in reaction ring, enter in second UV-detector, then the chromatogram described first UV-detector and described second UV-detector being recorded is compared, while there is the compound of negative peak in the chromatogram that described second UV-detector records, this compound is the antioxidant content in extract.
Described step (3) middle high-efficiency liquid chromatography method for detecting refers to that mobile phase A is methyl alcohol, the acetic acid aqueous solution that Mobile phase B volumetric concentration is 0.1%; Flow velocity is 0.6 ~ 1.2 mL/min, and mobile phase condition is: 0 min ~ 28 min, 35%A ~ 43%A, 65%B ~ 57%B; 28 min ~ 50 min, 43%A ~ 60%A, 57%B ~ 30%B.
The described step (3) length of middle reaction ring is 5 ~ 25 meters.
Described step (3) in first UV-detector to detect wavelength be 254 nm; It is 517 nm that second UV-detector detects wavelength.
The present invention compared with prior art has the following advantages:
1, because the present invention adopts antioxidant content in DPPH-HPLC method on-line determination rheum officinale seed, and this method has that agents useful for same amount is few, high flux detects, automaticity is high, detection time is short, sensitivity and specificity be compared with high, therefore, antioxidant content in suitable mensuration rheum officinale seed, thus realize the efficiency evaluation that rheum officinale seed is carried out to pharmacological action.
2, because the present invention adopts antioxidant content in DPPH-HPLC method on-line determination rheum officinale seed, and the chromatogram that this method only need relatively enter after first and second detecting device can judge antioxidant content.As Fig. 1.Each chromatographic peak in comparison colours spectrogram, the chromatogram entering after the second detecting device that wherein in chromatogram, the peak of chromatographic peak A, B, C, D, E, F, G, H is corresponding is negative peak, illustrates that compd A, B, C, D, E, F, G, H are antioxidant content.
3, the present invention is simple, quick, is easy to promote.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is chromatogram that first detecting device of the present invention and second detecting device the record figure that compares.
Embodiment
embodiment 1a method for antioxidant content in fast detecting rheum officinale seed, comprises the following steps:
(1) rheum officinale seed meal is broken to after 40 orders, obtains rheum officinale seed powder.
(2) ultrasonic extraction 50min under the condition that is 200W with the methyl alcohol that the volumetric concentration of 50 times of its quality is 50% at power by rheum officinale seed powder, obtains rheum officinale seed extract after filtration.
(3) rheum officinale seed extract is separated through high performance liquid chromatography chromatographic column, separated composition is first flowed through after first UV-detector as sample, the mass concentration pumping into as mobile phase in this sample and second pump is the antioxidant 1 of 10 mg/L, after 1-diphenyl-2 trinitrophenyl-hydrazine is derivative in reaction ring, enter in second UV-detector, then the chromatogram first UV-detector and second UV-detector being recorded is compared, while there is the compound of negative peak in the chromatogram that second UV-detector records, this compound is the antioxidant content in extract.
Wherein:
High-efficiency liquid chromatography method for detecting refers to that mobile phase A is methyl alcohol, the acetic acid aqueous solution that Mobile phase B volumetric concentration is 0.1%; Flow velocity is 0.6 mL/min, and mobile phase condition is: 0 min ~ 28 min, 35%A ~ 43%A, 65%B ~ 57%B; 28 min ~ 50 min, 43%A ~ 60%A, 57%B ~ 30%B.
The length of reaction ring is 5 meters.
It is 254 nm that first UV-detector detects wavelength; It is 517 nm that second UV-detector detects wavelength.
embodiment 2a method for antioxidant content in fast detecting rheum officinale seed, comprises the following steps:
(1) rheum officinale seed meal is broken to after 60 orders, obtains rheum officinale seed powder.
(2) ultrasonic extraction 30min under the condition that is 300W with the methyl alcohol that the volumetric concentration of 100 times of its quality is 80% at power by rheum officinale seed powder, obtains rheum officinale seed extract after filtration.
(3) rheum officinale seed extract is separated through high performance liquid chromatography chromatographic column, separated composition is first flowed through after first UV-detector as sample, the mass concentration pumping into as mobile phase in this sample and second pump is the antioxidant 1 of 100 mg/L, after 1-diphenyl-2 trinitrophenyl-hydrazine is derivative in reaction ring, enter in second UV-detector, then the chromatogram first UV-detector and second UV-detector being recorded is compared, while there is the compound of negative peak in the chromatogram that second UV-detector records, this compound is the antioxidant content in extract.
Wherein:
High-efficiency liquid chromatography method for detecting refers to that mobile phase A is methyl alcohol, the acetic acid aqueous solution that Mobile phase B volumetric concentration is 0.1%; Flow velocity is 0.8 mL/min, and mobile phase condition is: 0 min ~ 28 min, 35%A ~ 43%A, 65%B ~ 57%B; 28 min ~ 50 min, 43%A ~ 60%A, 57%B ~ 30%B.
The length of reaction ring is 10 meters.
It is 254 nm that first UV-detector detects wavelength; It is 517 nm that second UV-detector detects wavelength.
embodiment 3a method for antioxidant content in fast detecting rheum officinale seed, comprises the following steps:
(1) rheum officinale seed meal is broken to after 40 orders, obtains rheum officinale seed powder.
(2) ultrasonic extraction 30min under the condition that is 300W with the methyl alcohol that the volumetric concentration of 100 times of its quality is 100% at power by rheum officinale seed powder, obtains rheum officinale seed extract after filtration.
(3) rheum officinale seed extract is separated through high performance liquid chromatography chromatographic column, separated composition is first flowed through after first UV-detector as sample, the mass concentration pumping into as mobile phase in this sample and second pump is the antioxidant 1 of 200 mg/L, after 1-diphenyl-2 trinitrophenyl-hydrazine is derivative in reaction ring, enter in second UV-detector, then the chromatogram first UV-detector and second UV-detector being recorded is compared, while there is the compound of negative peak in the chromatogram that second UV-detector records, this compound is the antioxidant content in extract.
Wherein:
High-efficiency liquid chromatography method for detecting refers to that mobile phase A is methyl alcohol, the acetic acid aqueous solution that Mobile phase B volumetric concentration is 0.1%; Flow velocity is 1.2 mL/min, and mobile phase condition is: 0 min ~ 28 min, 35%A ~ 43%A, 65%B ~ 57%B; 28 min ~ 50 min, 43%A ~ 60%A, 57%B ~ 30%B.
The length of reaction ring is 25 meters.
It is 254 nm that first UV-detector detects wavelength; It is 517 nm that second UV-detector detects wavelength.
embodiment 4a method for antioxidant content in fast detecting rheum officinale seed, comprises the following steps:
(1) rheum officinale seed meal is broken to after 60 orders, obtains rheum officinale seed powder.
(2) ultrasonic extraction 20min under the condition that is 300W with the methyl alcohol that the volumetric concentration of 80 times of its quality is 80% at power by rheum officinale seed powder, obtains rheum officinale seed extract after filtration.
(3) rheum officinale seed extract is separated through high performance liquid chromatography chromatographic column, separated composition is first flowed through after first UV-detector as sample, the mass concentration pumping into as mobile phase in this sample and second pump is the antioxidant 1 of 100 mg/L, after 1-diphenyl-2 trinitrophenyl-hydrazine is derivative in reaction ring, enter in second UV-detector, then the chromatogram first UV-detector and second UV-detector being recorded is compared, while there is the compound of negative peak in the chromatogram that second UV-detector records, this compound is the antioxidant content in extract.
Wherein:
High-efficiency liquid chromatography method for detecting refers to that mobile phase A is methyl alcohol, the acetic acid aqueous solution that Mobile phase B volumetric concentration is 0.1%; Flow velocity is 1.0 mL/min, and mobile phase condition is: 0 min ~ 28 min, 35%A ~ 43%A, 65%B ~ 57%B; 28 min ~ 50 min, 43%A ~ 60%A, 57%B ~ 30%B.
The length of reaction ring is 20 meters.
It is 254 nm that first UV-detector detects wavelength; It is 517 nm that second UV-detector detects wavelength.
embodiment 5a method for antioxidant content in fast detecting rheum officinale seed, comprises the following steps:
(1) rheum officinale seed meal is broken to after 50 orders, obtains rheum officinale seed powder.
(2) ultrasonic extraction 45min under the condition that is 250W with the methyl alcohol that the volumetric concentration of 80 times of its quality is 80% at power by rheum officinale seed powder, obtains rheum officinale seed extract after filtration.
(3) rheum officinale seed extract is separated through high performance liquid chromatography chromatographic column, separated composition is first flowed through after first UV-detector as sample, the mass concentration pumping into as mobile phase in this sample and second pump is the antioxidant 1 of 50 mg/L, after 1-diphenyl-2 trinitrophenyl-hydrazine is derivative in reaction ring, enter in second UV-detector, then the chromatogram first UV-detector and second UV-detector being recorded is compared, while there is the compound of negative peak in the chromatogram that second UV-detector records, this compound is the antioxidant content in extract.
Wherein:
High-efficiency liquid chromatography method for detecting refers to that mobile phase A is methyl alcohol, the acetic acid aqueous solution that Mobile phase B volumetric concentration is 0.1%; Flow velocity is 0.8 mL/min, and mobile phase condition is: 0 min ~ 28 min, 35%A ~ 43%A, 65%B ~ 57%B; 28 min ~ 50 min, 43%A ~ 60%A, 57%B ~ 30%B.
The length of reaction ring is 10 meters.
It is 254 nm that first UV-detector detects wavelength; It is 517 nm that second UV-detector detects wavelength.
Claims (4)
1. a method for antioxidant content in fast detecting rheum officinale seed, comprises the following steps:
(1) rheum officinale seed meal is broken to after 40 ~ 60 orders, obtains rheum officinale seed powder;
(2) ultrasonic extraction 20 ~ 50min under the condition that is 200 ~ 300W with the methyl alcohol that the volumetric concentration of 50 ~ 100 times of its quality is 50% ~ 100% at power by described rheum officinale seed powder, obtains rheum officinale seed extract after filtration;
(3) described rheum officinale seed extract is separated through high performance liquid chromatography chromatographic column, separated composition is first flowed through after first UV-detector as sample, the mass concentration pumping into as mobile phase in this sample and second pump is the antioxidant 1 of 10 ~ 200 mg/L, after 1-diphenyl-2 trinitrophenyl-hydrazine is derivative in reaction ring, enter in second UV-detector, then the chromatogram described first UV-detector and described second UV-detector being recorded is compared, while there is the compound of negative peak in the chromatogram that described second UV-detector records, this compound is the antioxidant content in extract.
2. the method for antioxidant content in a kind of fast detecting rheum officinale seed as claimed in claim 1, is characterized in that: described step (3) middle high-efficiency liquid chromatography method for detecting refers to that mobile phase A is methyl alcohol, the acetic acid aqueous solution that Mobile phase B volumetric concentration is 0.1%; Flow velocity is 0.6 ~ 1.2 mL/min, and mobile phase condition is: 0 min ~ 28 min, 35%A ~ 43%A, 65%B ~ 57%B; 28 min ~ 50 min, 43%A ~ 60%A, 57%B ~ 30%B.
3. the method for antioxidant content in a kind of fast detecting rheum officinale seed as claimed in claim 1, is characterized in that: the described step (3) length of middle reaction ring is 5 ~ 25 meters.
4. the method for antioxidant content in a kind of fast detecting rheum officinale seed as claimed in claim 1, is characterized in that: described step (3) in first UV-detector to detect wavelength be 254 nm; It is 517 nm that second UV-detector detects wavelength.
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| CN201410170083.9A CN103940926B (en) | 2014-04-25 | 2014-04-25 | A kind of method of antioxidant content in quick detection Radix Et Rhizoma Rhei seed |
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| CN201410170083.9A CN103940926B (en) | 2014-04-25 | 2014-04-25 | A kind of method of antioxidant content in quick detection Radix Et Rhizoma Rhei seed |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108918437A (en) * | 2018-05-16 | 2018-11-30 | 天津中医药大学 | A kind of the antioxidant activity detection method and application of blood-activating and stasis-removing preparation |
| CN110108808A (en) * | 2019-04-12 | 2019-08-09 | 广西壮族自治区兽医研究所 | Method that is a kind of while evaluating oxidation-resistant active ingredient and content in downy rosemyrtle root |
| CN111423487A (en) * | 2020-03-31 | 2020-07-17 | 东北林业大学 | Method for separating and purifying antioxidant active protein components guided by on-line free radical scavenging method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108918437A (en) * | 2018-05-16 | 2018-11-30 | 天津中医药大学 | A kind of the antioxidant activity detection method and application of blood-activating and stasis-removing preparation |
| CN110108808A (en) * | 2019-04-12 | 2019-08-09 | 广西壮族自治区兽医研究所 | Method that is a kind of while evaluating oxidation-resistant active ingredient and content in downy rosemyrtle root |
| CN111423487A (en) * | 2020-03-31 | 2020-07-17 | 东北林业大学 | Method for separating and purifying antioxidant active protein components guided by on-line free radical scavenging method |
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Effective date of registration: 20220725 Address after: No.1, Tongrentang Road, Hexi street, Delingha City, Haixi Mongolian and Tibetan Autonomous Prefecture, Qinghai Province Patentee after: BEIJING TONGRENTANG HEALTH PHARMACEUTICAL (QINGHAI) Co.,Ltd. Address before: 810001 No. 59 Xiguan Street, Xining, Qinghai Patentee before: Northwest Institute of Plateau Biology, Chinese Academy of Sciences Patentee before: QINGHAI ZHONGKE PLATEAU BIOLOGICAL TECHNOLOGY DEVELOPMENT Co.,Ltd. |