CN105116092A - HPLC determination method for shikimic acid and 3-dehydrogenated shikimic acid in pomegranate peels - Google Patents
HPLC determination method for shikimic acid and 3-dehydrogenated shikimic acid in pomegranate peels Download PDFInfo
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Abstract
The invention discloses an HPLC determination method for shikimic acid and 3-dehydrogenated shikimic acid in pomegranate peels. The method disclosed by the invention comprises the following steps: taking methanol-phosphoric acid water solution with the volume percent of 1% as a mobile phase and carrying out isocratic elution to simultaneously determine the content of shikimic acid and 3-dehydrogenated shikimic acid in the pomegranate peels, so as to pointedly detect shikimic acid and 3-dehydrogenated shikimic acid components in pomegranate fruits. The determination method disclosed by the invention is simple to operate, has short elution time, can detect comprehensively and has high detection efficiency, so that a theoretical foundation is laid for disclosing a metabolism mechanism of gallic acid in the pomegranate fruits.
Description
Technical field
The present invention relates to the HPLC assay method of shikimic acid and 3-dehydroshikimate in pericarpium granati, belong to medical art.
Background technology
Pomegranate (PunicagranatumL.) fruit is rich in the bioactivators such as solubility aldehydes matter, steroids and alkaloid, there is anti-oxidant, many nutritive values and the health care such as anticancer antibiotic and prevention cardiovascular and cerebrovascular disease, more and more be subject to consumers, development prospect is wide.Gallic acid is one of aldehydes matter main in pomegranate fruit, and biological effect is extensive, and the health care stronger with it is closely related.The metabolic pathway of gallic acid is substantially clear, and the 3-dehydroshikimate that shikimic acid pathway generates generates gallic acid under shikimate dehydrogenase effect.Separately have report, shikimic acid generates 3-dehydroshikimate under shikimate dehydrogenase effect, also can generate gallic acid after 3-dehydroshikimate enolization under the effect of dehydroshikimate dehydrogenasa.
Shikimic acid (Shikimicacid) is the synthesis material that anti-avian influenza medicine Oseltamivir Phosphate (trade name: Tamiflu) and antineoplastic two dislike mycin, aldoketonutase inhibitor etc.3-dehydroshikimate (3-dehydroshikmiicacid, DHS) be the intermediate product of aromatic amino acid biosynthesis pathway, it is a kind of important renewable resource, can the important chemicals such as synthesizing gallic acid, protocatechuic acid, vanillic aldehyde and catechol, also be a kind of highly effective antioxidant, there is important commercial value.Plant containing shikimic acid and 3-dehydroshikimate is screened, is set up the foundation of science by the determination being quantitatively detected as shikimic acid and 3-dehydroshikimate extraction raw material, there is meaning and the positive role of reality.Be rich in gallic acid in pomegranate fruit, also should contain shikimic acid and 3-dehydroshikimate, be the metabolic mechanism of gallic acid in clear and definite pomegranate fruit, set up a kind of fast, accurately, the method for Simultaneously test shikimic acid and 3-dehydroshikimate content is very necessary.
At present, to the method mainly high performance liquid chromatography of shikimic acid assay, only on the plants such as coniferale plant, Chinese anise, there is a small amount of report, but the method for 3-dehydroshikimate assay so far there are no both at home and abroad relevant report.Therefore, a kind of method that set up science, that be suitable for measuring shikimic acid and 3-dehydroshikimate in pomegranate fruit is necessary.
Summary of the invention
For the deficiencies in the prior art, the invention provides the HPLC assay method of shikimic acid and 3-dehydroshikimate in a kind of pericarpium granati.The inventive method can the content of shikimic acid and 3-dehydroshikimate in Simultaneously test pericarpium granati, detects the shikimic acid in pomegranate fruit and 3-dehydroshikimate composition pointedly, for the metabolic mechanism disclosing gallic acid in pomegranate fruit has established theoretical foundation.
Technical scheme of the present invention is as follows:
In pericarpium granati, the HPLC assay method of shikimic acid and 3-dehydroshikimate, comprises the steps:
(1) pre-treatment of pomegranate fruit sample
Pomegranate fruit is cleaned and dries, pericarp is cut freezing 5 ~ 10min in liquid nitrogen, pulls out in double dish afterwards, after-20 DEG C of refrigerator freezing 24 ~ 36h, then put into freeze drier freeze drying 36 ~ 48h; It is for subsequent use that pericarp after freeze drying puts into-20 DEG C of refrigerators after pulverizing;
(2) preparation of need testing solution
Take the pericarp 0.1g that step (1) is handled well, put in tool plug conical flask, add ultrapure water 40mL, ultrasonic process 90-120min, filter, be settled to 50mL, with 0.22 μm of filtering with microporous membrane, put into-20 DEG C of refrigerators and preserve, measure for HPLC;
(3) assay of shikimic acid and 3-dehydroshikimate
High performance liquid chromatography experiment condition is as follows: Agilent ZorbaxXDB-C18 chromatographic column, length × wide=150mm × 4.6mm, thickness is 5 μm, take methyl alcohol as mobile phase A, percent by volume 1% phosphate aqueous solution is Mobile phase B, isocratic elution, mobile phase ratio is: A:B=5:95 volume ratio, elution time: 10-15min, UV-detector determined wavelength is 200 ~ 260nm, flow velocity is 0.8-1ml/min, and column temperature is 30 DEG C, sample feeding amount 10 μ l;
Shikimic acid and 3-dehydroshikimate content assaying method as follows: get shikimic acid and 3-dehydroshikimate standard items, be made into the standard solution of every 1ml containing 0.25mg, 0.50mg, 0.75mg, 1.00mg, 1.25mg, 1.50mg, 1.75mg, 2.00mg respectively, after the two mixing, be standard items mixed liquor;
High-performance liquid chromatogram determination: under the chromatographic condition determined above, the standard items mixed liquor of above-mentioned variable concentrations, respectively with 10 μ l sample introductions, measures the high performance liquid chromatography peak area of shikimic acid and 3-dehydroshikimate standard solution; With each standard items peak area of gained for ordinate, corresponding standard concentration is that ordinate does typical curve, calculates equation of linear regression respectively; Then high-performance liquid chromatogram determination is carried out to 10 pomegranate kind need testing solutions, obtain the peak area of shikimic acid and 3-dehydroshikimate, to calculate in test sample the content of the two according to typical curve, to obtain final product.
Preferred according to the present invention, in the pre-treatment of described step (1) pomegranate fruit sample, pericarp directly cuts freezing 5min in liquid nitrogen, prevents Tissue Browning, pulls out in double dish afterwards again.
Preferred according to the present invention, described step (3) medium ultraviolet determined wavelength is 214nm.
Preferred further, in pericarpium granati, the HPLC assay method of shikimic acid and 3-dehydroshikimate, comprises the steps:
(1) pre-treatment of pomegranate fruit sample
Pomegranate fruit is cleaned and dries, pericarp is directly cut freezing 5min in liquid nitrogen, pulls out in double dish afterwards again, after-20 DEG C of refrigerator freezing 24h, then put into freeze drier freeze drying 48h; It is for subsequent use that pericarp after freeze drying puts into-20 DEG C of refrigerators after pulverizing;
(2) preparation of need testing solution
Take the pericarp 0.1g that step (1) is handled well, put in tool plug conical flask, add ultrapure water 40mL, ultrasonic process 90min, power 200W, frequency 33kHz; Filter, be settled to 50mL, with 0.22 μm of filtering with microporous membrane, put into-20 DEG C of refrigerators and preserve, measure for HPLC;
(3) assay of shikimic acid and 3-dehydroshikimate
High performance liquid chromatography experiment condition is as follows: Agilent ZorbaxXDB-C18 chromatographic column, length × wide=150mm × 4.6mm, thickness is 5 μm, take methyl alcohol as mobile phase A, percent by volume 1% phosphate aqueous solution is Mobile phase B, isocratic elution, mobile phase ratio is: A:B=5:95 volume ratio, elution time: 10min, UV-detector determined wavelength is 214nm, flow velocity is 0.8ml/min, and column temperature is 30 DEG C, sample feeding amount 10 μ l;
Shikimic acid and 3-dehydroshikimate content assaying method as follows: get shikimic acid and 3-dehydroshikimate standard items, be made into the standard solution of every 1ml containing 0.25mg, 0.50mg, 0.75mg, 1.00mg, 1.25mg, 1.50mg, 1.75mg, 2.00mg respectively, after the two mixing, be standard items mixed liquor;
High-performance liquid chromatogram determination: under the chromatographic condition determined above, the standard items mixed liquor of above-mentioned variable concentrations, respectively with 10 μ l sample introductions, measures the high performance liquid chromatography peak area of shikimic acid and 3-dehydroshikimate standard solution; With each standard items peak area of gained for ordinate, corresponding standard concentration is that ordinate does typical curve, calculates equation of linear regression respectively; Then high-performance liquid chromatogram determination is carried out to 10 pomegranate kind need testing solutions, obtain the peak area of shikimic acid and 3-dehydroshikimate, the content of the two is calculated in test sample according to typical curve, obtain, to calculate shikimic acid content be 1.028 ~ 2.316mg/g, 3-dehydroshikimate content is 1.139 ~ 2.529mg/g.
Beneficial effect of the present invention is as follows:
(1) pericarp is directly cut in liquid nitrogen freezing, prevents Tissue Browning.Very easily there is brown stain in pericarpium granati, for ensureing the accuracy of experimental result, pericarp, directly through liquid nitrogen frozen, can prevent brown stain preferably.
(2) in chromatographic condition, mobile phase is methyl alcohol: 1% phosphate aqueous solution=5:95.By comparing methanol-water, acetonitrile-water, methyl alcohol-phosphoric acid, acetonitrile-phosphoric acid finds as the separating effect of mobile phase, with methyl alcohol: 1% phosphoric acid=5:95 system is mobile phase, and detect shikimic acid and 3-dehydroshikimate under isocratic condition, separating effect is best.
(3) UV detect wavelength of the present invention is 214nm, and the optimum absorb wavelength of shikimic acid and 3-dehydroshikimate is 214nm, and under this wavelength, separating effect and peak shape are all better.
(4) detection method is simple to operate, and elution time is short, and detect comprehensively, detection efficiency is high.
Following experimental example is used for further illustrating but is not limited to the present invention.
Experimental example 1:
1. sample-pretreating method
Because pomegranate fruit is rich in solubility aldehydes matter, easy generation brown stain, for preventing pericarpium granati brown stain, affect the mensuration of shikimic acid and 3-dehydroshikimate content in the present invention, compare pericarpium granati directly to cut double dish, cut in 3% citric acid solution, cut the impact on pericarp browning in liquid nitrogen, find after deliberation, pericarpium granati is cut in liquid nitrogen and can be made its tissue freezing, reduce brown stain odds, treatment effect is best.Percent by volume 3% citric acid can prevent pericarp browning to a certain extent, but after soaking 10 ~ 20min, brown stain in various degree also can occur different cultivars, therefore the effect of 3% citric acid treatment is taken second place.The most easily there is brown stain in the pericarpium granati without any process.
2. best detection wavelength is determined
Utilize ultraviolet spectrophotometer to scan shikimic acid and the maximum absorption wavelength of 3-dehydroshikimate standard items in the whole ultraviolet wavelength range of 200nm ~ 300nm, thus obtain the highest detection sensitivity.Finally determine that shikimic acid and 3-dehydroshikimate optimum absorb wavelength are 214nm.
3. mobile phase is selected
The present invention compares vol/vol methanol: water=90:10, acetonitrile: water=95:5, acetonitrile: 1% phosphoric acid=94:6, acetonitrile: 2% phosphoric acid=90:10, methyl alcohol: 0.1% phosphoric acid=90:10, methyl alcohol: 1% phosphate aqueous solution=3:97, methyl alcohol: 1% phosphate aqueous solution=5:95 is as the separating effect of mobile phase shikimic acid and 3-dehydroshikimate, find through test, with methyl alcohol: 1% phosphoric acid=5:95 system is mobile phase, wherein the concentration of phosphoric acid is percent by volume, shikimic acid and 3-dehydroshikimate is detected under isocratic condition, peak shape is symmetrical, good separating effect.
4. standard specimen chromatogram
Under fixed chromatographic condition, get 10 μ L standard items sample introductions respectively, shikimic acid and 3-dehydroshikimate are standard items mixed liquor, obtain standard items liquid chromatogram.As can be seen from Figure 1, the retention time of shikimic acid and 3-dehydroshikimate is respectively 3.045 and 3.971min, and the separating effect of each standard items and peak shape are all better.
5. typical curve and detection line measure
Configure shikimic acid and the 3-dehydroshikimate standard solution of a series of variable concentrations, sample introduction analysis under the chromatographic condition determined, take peak area as ordinate, take mass concentration as horizontal ordinate, curve plotting carries out linear regression, obtains regression equation and the related coefficient (table 1) of each material.According to signal to noise ratio (S/N ratio) RSN=3, record the lowest detection mass concentration of each material.The peak area of each material in range of liner and mass concentration are good linear relationship, and related coefficient is all greater than 0.99.Lowest detectable limit shows simultaneously, and the method can detect the material that content is lower.Therefore the method can be carried out accurate analysis to sample and have higher sensitivity.
The regression equation of table 1 shikimic acid and 3-dehydroshikimate, related coefficient and lowest detectable limit
6. recovery of standard addition and precision measure
Take two parts of identical samples, a certain amount of various standard items are added respectively in a copy of it sample, according to need testing solution, preparation method processes, the extract of two increment product is carried out HPLC analysis at identical conditions, typical curve is utilized to calculate the concentration of each sample, according to formula: recovery of standard addition (%)=(measured value-sample size)/add value × 100, calculate the recovery of standard addition of each component.Same increment product repeat sample introduction and carry out Precision Experiment 6 times, calculate the relative standard deviation (RSD) of each material.As shown in Table 2,4 kinds of materials recovery of standard addition be 98.9% ~ 99.4%, RSD be 1.2% ~ 1.6%, this illustrates that this method is reproducible, and accuracy is high, meets testing requirement.
Table 2 recovery and precision measure
7. the assay of fruit sample
The method adopting the present invention to set up has carried out quantitative and qualitative analysis detection to shikimic acid, 3-dehydroshikimate in the pomegranate kind maturity stage pericarp of 10, Shandong.As can be seen from Table 3, in 10 pomegranate kind pericarps, shikimic acid content is 1.028 ~ 2.316mg/g, 3-dehydroshikimate content is 1.139 ~ 2.529mg/g, and in tree peony pericarp, two kinds of acid contents are the highest, and in the pomegranate pericarp of hilllock, content is minimum.
Shikimic acid and 3-dehydroshikimate content in table 3 different pomegranate kind pericarp
Accompanying drawing explanation
Fig. 1 is the high-efficient liquid phase chromatogram of shikimic acid and 3-dehydroshikimate standard solution;
Wherein, horizontal ordinate is retention time, unit: minute, ordinate is voltage, unit: millivolt.
Embodiment
Following embodiment is used for further illustrating but is not limited to the present invention.
Embodiment 1:
In pericarpium granati, the HPLC assay method of shikimic acid and 3-dehydroshikimate, comprises the steps:
(1) pre-treatment of pomegranate fruit sample
Pomegranate fruit is cleaned and dries, pericarp is directly cut freezing 5min in liquid nitrogen, pulls out in double dish afterwards again, after-20 DEG C of refrigerator freezing 24h, then put into freeze drier freeze drying 48h; It is for subsequent use that pericarp after freeze drying puts into-20 DEG C of refrigerators after pulverizing;
(2) preparation of need testing solution
Take the pericarp 0.1g that step (1) is handled well, put in tool plug conical flask, add ultrapure water 40mL, ultrasonic process 90min, power 200W, frequency 33kHz; Filter, be settled to 50mL, with 0.22 μm of filtering with microporous membrane, put into-20 DEG C of refrigerators and preserve, measure for HPLC;
(3) assay of shikimic acid and 3-dehydroshikimate
High performance liquid chromatography experiment condition is as follows: Agilent ZorbaxXDB-C18 chromatographic column, length × wide=150mm × 4.6mm, and thickness is 5 μm.Take methyl alcohol as mobile phase A, percent by volume 1% phosphate aqueous solution is Mobile phase B, isocratic elution, mobile phase ratio is: A:B=5:95 volume ratio, elution time: 10min, and UV-detector determined wavelength is 214nm, flow velocity is 0.8ml/min, and column temperature is 30 DEG C, sample feeding amount 10 μ l;
Shikimic acid and 3-dehydroshikimate content assaying method as follows: get shikimic acid and 3-dehydroshikimate standard items, be made into the standard solution of every 1ml containing 0.25mg, 0.50mg, 0.75mg, 1.00mg, 1.25mg, 1.50mg, 1.75mg, 2.00mg respectively, after the two mixing, be standard items mixed liquor.
High-performance liquid chromatogram determination: under the chromatographic condition determined above, the standard items mixed liquor of above-mentioned variable concentrations, respectively with 10 μ l sample introductions, measures the high performance liquid chromatography peak area of shikimic acid and 3-dehydroshikimate standard solution; With each standard items peak area of gained for ordinate, corresponding standard concentration is that ordinate does typical curve, calculates equation of linear regression respectively; Then high-performance liquid chromatogram determination is carried out to 10 pomegranate kind need testing solutions, obtain the peak area of shikimic acid and 3-dehydroshikimate, the content of the two is calculated in test sample according to typical curve, obtain, to calculate shikimic acid content be 1.028 ~ 2.316mg/g, 3-dehydroshikimate content is 1.139 ~ 2.529mg/g.
Embodiment 2:
In pericarpium granati, the HPLC assay method of shikimic acid and 3-dehydroshikimate, comprises the steps:
(1) pre-treatment of pomegranate fruit sample
Pomegranate fruit is cleaned and dries, pericarp is directly cut freezing 10min in liquid nitrogen, pulls out in double dish afterwards again, after-20 DEG C of refrigerator freezing 24h, then put into freeze drier freeze drying 48h; It is for subsequent use that pericarp after freeze drying puts into-20 DEG C of refrigerators after pulverizing;
(2) preparation of need testing solution
Take the pericarp 0.1g that step (1) is handled well, put in tool plug conical flask, add ultrapure water 40mL, ultrasonic process 110min, power 200W, frequency 33kHz; Filter, be settled to 50mL, with 0.22 μm of filtering with microporous membrane, put into-20 DEG C of refrigerators and preserve, measure for HPLC;
(3) assay of shikimic acid and 3-dehydroshikimate
High performance liquid chromatography experiment condition is as follows: Agilent ZorbaxXDB-C18 chromatographic column, length × wide=150mm × 4.6mm, and thickness is 5 μm.Take methyl alcohol as mobile phase A, percent by volume 1% phosphate aqueous solution is Mobile phase B, isocratic elution, mobile phase ratio is: A:B=5:95 volume ratio, elution time: 15min, and UV-detector determined wavelength is 240nm, flow velocity is 1ml/min, and column temperature is 30 DEG C, sample feeding amount 10 μ l;
Shikimic acid and 3-dehydroshikimate content assaying method as follows: get shikimic acid and 3-dehydroshikimate standard items, be made into the standard solution of every 1ml containing 0.25mg, 0.50mg, 0.75mg, 1.00mg, 1.25mg, 1.50mg, 1.75mg, 2.00mg respectively, after the two mixing, be standard items mixed liquor.
High-performance liquid chromatogram determination: under the chromatographic condition determined above, the standard items mixed liquor of above-mentioned variable concentrations, respectively with 10 μ l sample introductions, measures the high performance liquid chromatography peak area of shikimic acid and 3-dehydroshikimate standard solution; With each standard items peak area of gained for ordinate, corresponding standard concentration is that ordinate does typical curve, calculates equation of linear regression respectively; Then high-performance liquid chromatogram determination is carried out to 10 pomegranate kind need testing solutions, obtain the peak area of shikimic acid and 3-dehydroshikimate, the content of the two is calculated in test sample according to typical curve, obtain, to calculate shikimic acid content be 1.030 ~ 2.300mg/g, 3-dehydroshikimate content is 1.099 ~ 2.422mg/g.
Claims (4)
1. the HPLC assay method of shikimic acid and 3-dehydroshikimate in pericarpium granati, comprises the steps:
(1) pre-treatment of pomegranate fruit sample
Pomegranate fruit is cleaned and dries, pericarp is cut freezing 5 ~ 10min in liquid nitrogen, pulls out in double dish afterwards, after-20 DEG C of refrigerator freezing 24 ~ 36h, then put into freeze drier freeze drying 36 ~ 48h; It is for subsequent use that pericarp after freeze drying puts into-20 DEG C of refrigerators after pulverizing;
(2) preparation of need testing solution
Take the pericarp 0.1g that step (1) is handled well, put in tool plug conical flask, add ultrapure water 40mL, ultrasonic process 90 ~ 120min, filter, be settled to 50mL, with 0.22 μm of filtering with microporous membrane, put into-20 DEG C of refrigerators and preserve, measure for HPLC;
(3) assay of shikimic acid and 3-dehydroshikimate
High performance liquid chromatography experiment condition is as follows: Agilent ZorbaxXDB-C18 chromatographic column, length × wide=150mm × 4.6mm, thickness is 5 μm, take methyl alcohol as mobile phase A, percent by volume 1% phosphate aqueous solution is Mobile phase B, isocratic elution, mobile phase ratio is: A:B=5:95 volume ratio, elution time: 10 ~ 15min, UV-detector determined wavelength is 200 ~ 260nm, flow velocity is 0.8 ~ 1ml/min, and column temperature is 30 DEG C, sample feeding amount 10 μ l;
Shikimic acid and 3-dehydroshikimate content assaying method as follows: get shikimic acid and 3-dehydroshikimate standard items, be made into the standard solution of every 1ml containing 0.25mg, 0.50mg, 0.75mg, 1.00mg, 1.25mg, 1.50mg, 1.75mg, 2.00mg respectively, after the two mixing, be standard items mixed liquor;
High-performance liquid chromatogram determination: under the chromatographic condition determined above, the standard items mixed liquor of above-mentioned variable concentrations, respectively with 10 μ l sample introductions, measures the high performance liquid chromatography peak area of shikimic acid and 3-dehydroshikimate standard solution; With each standard items peak area of gained for ordinate, corresponding standard concentration is that ordinate does typical curve, calculates equation of linear regression respectively; Then high-performance liquid chromatogram determination is carried out to 10 pomegranate kind need testing solutions, obtain the peak area of shikimic acid and 3-dehydroshikimate, to calculate in test sample the content of the two according to typical curve, to obtain final product.
2. the HPLC assay method of shikimic acid and 3-dehydroshikimate in pericarpium granati as claimed in claim 1, it is characterized in that, in the pre-treatment of described step (1) pomegranate fruit sample, pericarp directly cuts freezing 5min in liquid nitrogen, prevent Tissue Browning, pull out in double dish afterwards again.
3. the HPLC assay method of shikimic acid and 3-dehydroshikimate in pericarpium granati as claimed in claim 1, it is characterized in that, described step (3) medium ultraviolet determined wavelength is 214nm.
4. the HPLC assay method of shikimic acid and 3-dehydroshikimate in pericarpium granati as claimed in claim 1, comprises the steps:
(1) pre-treatment of pomegranate fruit sample
Pomegranate fruit is cleaned and dries, pericarp is directly cut freezing 5min in liquid nitrogen, pulls out in double dish afterwards again, after-20 DEG C of refrigerator freezing 24h, then put into freeze drier freeze drying 48h; It is for subsequent use that pericarp after freeze drying puts into-20 DEG C of refrigerators after pulverizing;
(2) preparation of need testing solution
Take the pericarp 0.1g that step (1) is handled well, put in tool plug conical flask, add ultrapure water 40mL, ultrasonic process 90min, power 200W, frequency 33kHz; Filter, be settled to 50mL, with 0.22 μm of filtering with microporous membrane, put into-20 DEG C of refrigerators and preserve, measure for HPLC;
(3) assay of shikimic acid and 3-dehydroshikimate
High performance liquid chromatography experiment condition is as follows: Agilent ZorbaxXDB-C18 chromatographic column, length × wide=150mm × 4.6mm, thickness is 5 μm, take methyl alcohol as mobile phase A, percent by volume 1% phosphate aqueous solution is Mobile phase B, isocratic elution, mobile phase ratio is: A:B=5:95 volume ratio, elution time: 10min, UV-detector determined wavelength is 214nm, flow velocity is 0.8ml/min, and column temperature is 30 DEG C, sample feeding amount 10 μ l;
Shikimic acid and 3-dehydroshikimate content assaying method as follows: get shikimic acid and 3-dehydroshikimate standard items, be made into the standard solution of every 1ml containing 0.25mg, 0.50mg, 0.75mg, 1.00mg, 1.25mg, 1.50mg, 1.75mg, 2.00mg respectively, after the two mixing, be standard items mixed liquor;
High-performance liquid chromatogram determination: under the chromatographic condition determined above, the standard items mixed liquor of above-mentioned variable concentrations, respectively with 10 μ l sample introductions, measures the high performance liquid chromatography peak area of shikimic acid and 3-dehydroshikimate standard solution; With each standard items peak area of gained for ordinate, corresponding standard concentration is that ordinate does typical curve, calculates equation of linear regression respectively; Then high-performance liquid chromatogram determination is carried out to 10 pomegranate kind need testing solutions, obtain the peak area of shikimic acid and 3-dehydroshikimate, the content of the two is calculated in test sample according to typical curve, obtain, to calculate shikimic acid content be 1.028 ~ 2.316mg/g, 3-dehydroshikimate content is 1.139 ~ 2.529mg/g.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112546079A (en) * | 2021-01-28 | 2021-03-26 | 广西壮族自治区中医药研究院 | Preparation and quality detection method of total organic acid extract in Chinese fir leaves |
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