CN102854268B - Detection method of authentic rheum officinale seeds - Google Patents
Detection method of authentic rheum officinale seeds Download PDFInfo
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Abstract
The invention discloses a detection method of authentic rheum officinale seeds, which adopts a high-efficient liquid-phase chromatography to detect, and the detection method comprises the following steps of (1) taking seeds to be detected, grinding the seeds, extracting the seeds through methanol, filtering to obtain the filter liquid, removing methanol, hydrolyzing with acid, extracting with organic solvent to obtain an organic solvent layer, recycling the solvent until being dried, and re-dissolving to prepare sample solution; (2) taking aloe-emodin reference substances to be dissolved to prepare reference substance solution; (3) utilizing the high-efficient liquid-phase chromatography to detect, wherein the colored peak in chromatogram map of the sample solution is maintained consistent with the colored peak of the aloe-emodin reference substances on the aspect of the time, and determining whether the authentic rheum officinale seeds are contained in the seeds to be detected or not, wherein the chromatography condition is as follows: solid phase: octadecylsilane chemically bonded silica, flow phase: methanol-0.1 percent phosphoric acid solution, the volume ratio of the solid phase and the flow phase is 80 to 90: 10 to 20; and the detection wavelength: 254nm. The detection method of the authentic rheum officinale seeds is high in accuracy, convenient in operation, low in cost and good in market application prospect.
Description
Technical field
The detection method that the present invention relates to genuine rhubarb seed, belongs to the field of Chinese medicines.
Background technology
Rheum officinale is the dry root and rhizome of the ancient especially big yellow Rheum tanguticum Maxim.ex Balf. of polygonum rheum palmatum Rheum palmatum L., Tang or Rheum officinale Rheum officinale Baill..There are the logical intestines that purge heat, removing pattogenic heat from the blood and toxic material from the body, the effect of stimulating the menstrual flow by the stasis of blood.For real hot constipation, stagnant stomachache, rushes down dysentery not well, jaundice with damp-heat pathogen, and blood-head is told nosebleed, hot eyes, pharynx is swollen, abdominalgia with intestinal abscess, the carbuncle furunculosis that swells, hemostasis is through closing, traumatic injury, upper gastrointestinal bleeding; Control scald outward.Sorrel is distributed in the southeast of Gansu, Qinghai, western Sichuan, northwestern Yunnan Province and Eastern Tibet; The ancient especially big Huang of Tang is distributed in Gansu, Qinghai, Sichuan and Eastern Tibet the north; Rheum officinale is distributed in the ground such as Southern Shaanxi, West Henan, West of Hubei Province, Sichuan, Yunnan.North China rheum officinale, Rheum hotaoense C. Y. Cheng et C. T. Kao are common adulterant rheum officinales, easily obscure with the genuine rhubarb such as Tang ancient especially big Huang.
In recent years, due to the excavating without plan of wild Plant Resources of Rheum, cause wild Plant Resources of Rheum to reduce year by year, national wild Plant Resources of Rheum has been on the verge of exhaustion.For meeting the market demand growing to high-quality rheum officinale, rheum officinale artificial growth scale constantly expands, and the pure seed of quality is the prerequisite of the planting of medicinal materials, is the basic guarantee that improves root yield and quality.
At present, China's rheum officinale seed does not have test stone and quality standard, is mainly to differentiate to have certain limitation by proterties such as shape, size, color, epidermis, decorations.Application number: 201110337787.7, denomination of invention: a kind of Patent Application Publication of the method for identifying authenticity of Chinese rhubarb seed a kind of method of the rheum officinale seed of discerning the false from the genuine, it comprises the steps: that (1) get seed to be checked, extracts Chrysophanol and archen in seed to be checked; (2) content of Chrysophanol and archen in mensuration rheum officinale seed; (3) ratio of calculating Determination of chrysophanol/emodin content; (4) ratio in judgement of calculating according to step (3): if ratio is greater than 1, judge that this seed is genuine rhubarb seed, if ratio is less than or equal to 1, judge that this seed is adulterant rheum officinale seed.The method determines by the ratio of Chrysophanol in seed and emodin content whether rheum officinale seed is certified products, and more difficult because of Accurate Measurement component content, error is larger, causes the method to use inconvenient, and accuracy is not high enough.Especially for the situation of doped portion adulterant in genuine rhubarb seed, easily get the wrong sow by the ear, be difficult to substitute traditional detection method.
Need to find more easy, accurate, the reliable method qualification of one certified products, adulterant, and the method for the positive pseudo-rheum officinale seed mixing.
Summary of the invention
In order to address the above problem, the invention provides a kind of detection method of genuine rhubarb seed.
The detection method of genuine rhubarb seed of the present invention,, it is to adopt high performance liquid chromatography to detect, and comprises the steps:
(1) get seed to be checked, pulverize, with methyl alcohol extraction, filter, obtain filtrate, remove methyl alcohol, acid hydrolysis, with organic solvent extraction, obtains organic solvent layer, reclaims solvent to doing, then dissolves, and makes need testing solution;
(2) get aloe-emodin reference substance, dissolve, preparation reference substance solution;
(3) detect by high performance liquid chromatography, in the chromatogram of need testing solution, have chromatographic peak consistent with aloe-emodin reference substance chromatographic peak retention time, identify in seed to be checked and contain genuine rhubarb seed, chromatographic condition is as follows: fixing phase: octadecylsilane chemically bonded silica; Mobile phase: methyl alcohol-0.1% phosphoric acid solution, the volume ratio of the two is (80 ~ 90): (10 ~ 20); Detect wavelength: 254nm.
Wherein, the described seed to be checked of step (1) is the seed of sorrel Rheum palmatum L., Tang ancient especially big yellow Rheum tanguticum ' Maxim.ex Balf, North China rheum officinale Rheum franzenbachiiMunt. or Rheum hotaoense C. Y. Cheng et C. T. Kao Rhum hotaoense C.Y.Cheng et C.T.Kao.
Wherein, when the described acid hydrolysis of step (1), in solution, hydrionic concentration is 2 ~ 3mol/L; Described organic solvent is methenyl choloride.
Preferably, in described step (1), seed to be checked is pulverized, cross sieve No. four, obtain powder, add methyl alcohol, the mass volume ratio of powder and methyl alcohol is 1:150 ~ 185, add hot reflux 30 ~ 90min, cooling, weigh, supply the weight of less loss with methyl alcohol, shake up, filter, obtain subsequent filtrate, fling to solvent, add concentration and be 6 ~ 10% hydrochloric acid, ultrasonic processing 1 ~ 3min, add isopyknic methenyl choloride, add hot reflux 60 ~ 80min, use again chloroform extraction 3 times, each methenyl choloride volume used is with hydrochloric acid volume, obtain methenyl choloride layer, reclaim solvent to dry, add methyl alcohol and make need testing solution.
Wherein, in described step (3), the volume ratio of methyl alcohol and 0.1% phosphoric acid solution is 85:15.
It also comprises qualitative checking method, and step is as follows:
1) get seed to be checked, pulverize, with methyl alcohol extraction, filter, obtain filtrate, fling to solvent, acid solution hydrolysis, with organic solvent extraction, obtains water layer;
2) get step 1) gained water layer, add the strong aqua of 1 ~ 3 times of volume, jolt, without rufous to sepia flocculent deposit;
3) identify that seed to be checked is genuine rhubarb seed.
Wherein, acid hydrolysis described in step 1), in solution, hydrionic concentration is 0.52 ~ 2mol/L; Organic solvent is ether.
Preferably, in described step 1), seed meal to be checked is broken into fine powder, add methyl alcohol, the mass volume ratio of fine powder and methyl alcohol is 1:15 ~ 25, soaks 30min ~ 90min, filter, obtain filtrate, evaporate to dryness, add water, then add concentrated hydrochloric acid, hydrochloric acid volume is water volume 1/20 ~ 1/5, add hot reflux 20 ~ 40min, cooling, by extracted with diethyl ether, extract 2 times, each ether volume used is water volume 3 ~ 5 times, obtain water layer.
The detection method of genuine rhubarb seed of the present invention can accurately detect in seed to be checked, whether to contain genuine rhubarb seed, by the preferred detection method of the present invention, can further determine whether seed to be checked is also mixed with adulterant seed, positive pseudo-seed melange is got rid of from certified products, accuracy is high, and detection method is simple, quick, can substitute traditional detection method, has a good application prospect.
Obviously,, according to foregoing of the present invention, according to ordinary skill knowledge and the customary means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make amendment, replacement or the change of other various ways.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as to the scope of the above-mentioned theme of the present invention only limits to following example.All technology realizing based on foregoing of the present invention all belong to scope of the present invention.
Brief description of the drawings
Fig. 1 high performance liquid chromatography dissociated anthraquinone reference substance collection of illustrative plates, wherein, 1 is that aloe-emodin, 2 is that Rhein, 3 is that archen, 4 is that Chrysophanol, 5 is Physcion;
Fig. 2 high performance liquid chromatography DH10 collection of illustrative plates, wherein, 3 is archen, 4 is that Chrysophanol, 5 is Physcion;
Fig. 3 high performance liquid chromatography DH13 collection of illustrative plates, wherein, 1 is that aloe-emodin, 3 is that archen, 4 is that Chrysophanol, 5 is Physcion.
Fig. 4 high performance liquid chromatography DH15 collection of illustrative plates, wherein, 1 is that aloe-emodin, 3 is that archen, 4 is that Chrysophanol, 5 is Physcion.
Fig. 5 high performance liquid chromatography DH26 collection of illustrative plates, wherein, 3 is that archen, 4 is that Chrysophanol, 5 is Physcion.
Fig. 6 precipitates testing result, and wherein, the left side is DH10, DH26, and the right is DH13, DH15.
Embodiment
Embodiment 1 detection method of the present invention
1, detection method
(1) get seed to be checked;
(2) get seed powder to be checked (cross No. four sieve) 0.15g, accurately weighed, to put in tool plug conical flask, precision adds methyl alcohol 22.5ml, and weighed weight adds hot reflux 30min, lets cool, more weighed weight, supplies the weight of less loss with methyl alcohol, shakes up, and filters.Precision measures subsequent filtrate 5ml, puts in flask, flings to solvent, add 6% hydrochloric acid solution 5ml, ultrasonic processing 1 minute, then add methenyl choloride 5ml, add hot reflux 30min, let cool, put in separating funnel, with a small amount of methenyl choloride washing container, be incorporated in separating funnel, divide and get methenyl choloride layer, acid solution is extracted 3 times with methenyl choloride again, each 5ml, merge methenyl choloride liquid, decompression and solvent recovery is to dry, and residue adds methyl alcohol to be made to dissolve, be transferred in 10ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain need testing solution.
Get aloe-emodin reference substance, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.
The detection of high performance liquid chromatography: taking octadecylsilane chemically bonded silica as fixing phase; Taking methyl alcohol-0.1% phosphoric acid solution (80:10) as mobile phase; Detection wavelength is 254nm.Accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures.
(3) if in step (2), in the chromatogram of need testing solution, there is chromatographic peak consistent with aloe-emodin reference substance chromatographic peak retention time, illustrate and in seed to be checked, contain genuine rhubarb seed.
Embodiment 2 detection method of the present invention
1, detection method
(1) get seed to be checked;
(2) get seed powder to be checked (cross No. four sieve) 0.15g, accurately weighed, to put in tool plug conical flask, precision adds methyl alcohol 25ml, and weighed weight adds hot reflux 1 hour, lets cool, more weighed weight, supplies the weight of less loss with methyl alcohol, shakes up, and filters.Precision measures subsequent filtrate 5ml, put in flask, fling to solvent, add 8% hydrochloric acid solution 10ml (in 8% hydrochloric acid solution, hydrionic concentration is 2.5mol/L), ultrasonic processing 2 minutes, add again methenyl choloride 10ml, add hot reflux 1 hour, let cool, put in separating funnel, with a small amount of methenyl choloride washing container, be incorporated in separating funnel, divide and get methenyl choloride layer, acid solution is extracted 3 times with methenyl choloride again, each 10ml, merge methenyl choloride liquid, decompression and solvent recovery is to dry, residue adds methyl alcohol to be made to dissolve, be transferred in 10ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain need testing solution.
Get aloe-emodin reference substance, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.
The detection of high performance liquid chromatography: taking octadecylsilane chemically bonded silica as fixing phase; Taking methyl alcohol-0.1% phosphoric acid solution (85:15) as mobile phase; Detection wavelength is 254nm.Accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures.
(3) if in step (2), in the chromatogram of need testing solution, there is chromatographic peak consistent with aloe-emodin reference substance chromatographic peak retention time, illustrate and in seed to be checked, contain genuine rhubarb seed.
Embodiment 3 detection method of the present invention
1, detection method
(1) get seed to be checked;
(2) get seed powder to be checked (cross No. four sieve) 0.15g, accurately weighed, to put in tool plug conical flask, precision adds methyl alcohol 27.75ml, and weighed weight adds hot reflux 90min, lets cool, more weighed weight, supplies the weight of less loss with methyl alcohol, shakes up, and filters.Precision measures subsequent filtrate 5ml, puts in flask, flings to solvent, add 10% hydrochloric acid solution 15ml, ultrasonic processing 3 minutes, then add methenyl choloride 15ml, add hot reflux 90min, let cool, put in separating funnel, with a small amount of methenyl choloride washing container, be incorporated in separating funnel, divide and get methenyl choloride layer, acid solution is extracted 3 times with methenyl choloride again, each 15ml, merge methenyl choloride liquid, decompression and solvent recovery is to dry, and residue adds methyl alcohol to be made to dissolve, be transferred in 10ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain need testing solution.
Get aloe-emodin reference substance, add methyl alcohol and make the solution of every 1ml containing 16 μ g, product solution in contrast.
The detection of high performance liquid chromatography: taking octadecylsilane chemically bonded silica as fixing phase; Taking methyl alcohol-0.1% phosphoric acid solution (90:20) as mobile phase; Detection wavelength is 254nm.Accurate reference substance solution and the each 20 μ l of need testing solution of drawing respectively, injection liquid chromatography, measures.
(3) if in step (2), in the chromatogram of need testing solution, there is chromatographic peak consistent with aloe-emodin reference substance chromatographic peak retention time, illustrate and in seed to be checked, contain genuine rhubarb seed.
Embodiment 4 detection method of the present invention
1, detection method
(1) get seed to be checked;
(2) detect according to the detection method of embodiment 1, in the chromatogram of need testing solution, have chromatographic peak whether consistent with aloe-emodin reference substance chromatographic peak retention time;
(3) get seed fine powder 1.0g to be checked, add methyl alcohol 15ml, soak 30min, filter, get filtrate 5ml, evaporate to dryness, residue add water 10ml make dissolve, adding hydrionic concentration in concentrated hydrochloric acid 0.5ml(gained dilute hydrochloric acid solution is 0.52mol/L again), add hot reflux 20 minutes, cooling immediately, divide 2 extractions with ether, each 15ml, obtains water layer;
Water intaking layer, adds the strong aqua of 1 times of volume, jolts, and observes and has or not rufous to sepia flocculent deposit;
(4) if in step (2), in the chromatogram of need testing solution, there is chromatographic peak consistent with aloe-emodin reference substance chromatographic peak retention time, and.In step (3), water layer to sepia flocculent deposit, is genuine rhubarb seed by Seed Identification to be checked without rufous.
Embodiment 5 detection method of the present invention
1, detection method
(1) get seed to be checked;
(2) detect according to the detection method of embodiment 2, in the chromatogram of need testing solution, have chromatographic peak whether consistent with aloe-emodin reference substance chromatographic peak retention time;
(3) get seed fine powder 1.0g to be checked, add methyl alcohol 20ml, soak 1 hour, filter, get filtrate 5ml, evaporate to dryness, residue add water 10ml make dissolve, adding hydrionic concentration in concentrated hydrochloric acid 1ml(gained dilute hydrochloric acid solution is 1.09mol/L again), add hot reflux 30 minutes, cooling immediately, divide 2 extractions with ether, each 20ml, obtains water layer;
Water intaking layer, adds the strong aqua of 2 times of volumes, jolts, and observes and has or not rufous to sepia flocculent deposit;
(4) if in step (2), in the chromatogram of need testing solution, there is chromatographic peak consistent with aloe-emodin reference substance chromatographic peak retention time, and.In step (3), water layer to sepia flocculent deposit, is genuine rhubarb seed by Seed Identification to be checked without rufous.
Embodiment 6 detection method of the present invention
1, detection method
(1) get seed to be checked;
(2) detect according to the detection method of embodiment 3, in the chromatogram of need testing solution, have chromatographic peak whether consistent with aloe-emodin reference substance chromatographic peak retention time;
(3) get seed fine powder 1.0g to be checked, add methyl alcohol 25ml, soak 90min, filter, get filtrate 5ml, evaporate to dryness, residue add water 10ml make dissolve, adding hydrionic concentration in concentrated hydrochloric acid 1.5ml(gained dilute hydrochloric acid solution is 1.09mol/L again), add hot reflux 40 minutes, cooling immediately, divide 2 extractions with ether, each 25ml, obtains water layer;
Water intaking layer, adds the strong aqua of 3 times of volumes, jolts, and observes and has or not rufous to sepia flocculent deposit;
(4) if in step (2), in the chromatogram of need testing solution, there is chromatographic peak consistent with aloe-emodin reference substance chromatographic peak retention time, and.In step (3), water layer to sepia flocculent deposit, is genuine rhubarb seed by Seed Identification to be checked without rufous.
Experimental example 1 use detection method of the present invention detects seed to be checked
1, experiment material
| Seed numbering |
| DH10 |
| DH13 |
| DH15 |
| DH26 |
2, experimental technique
(1) with traditional proterties discrimination method qualification:
(2) detection method of the present invention detects: detect according to embodiment 2 methods, wherein, get aloe-emodin, Rhein, archen, Chrysophanol, Physcion reference substance, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.
3, experimental result
(1) traditional proterties is differentiated
DH10: fruit is brown to bright orange brown, long 5.5 ~ 10mm, wide 5 ~ 8mm, the less oval that is of seed, close with fruit color, puckery, is accredited as the seed of North China rheum officinale Rheum franzenbachii Munt., is adulterant rheum officinale seed;
DH13: fruit yellowish-brown is to tawny, and long 7 ~ 9mm(fruit top is to base portion), the ultimate range of two fruit wing wing edge on wide 5.5 ~ 7.5mm(fruit square section), the less oval that is of seed, color is slightly darker than fruit, and tawny is to brown; Bitter, after plantation, according to the character of medicinal material plant, is accredited as the seed of sorrel Rheum palmatum L., is genuine rhubarb seed;
DH15: fruit yellowish-brown is to tawny, and long 6 ~ 9mm(fruit top is to base portion), the ultimate range of two fruit wing wing edge on wide 4.5 ~ 7mm(fruit square section), the less oval that is of seed, color is slightly darker than fruit, and tawny is to brown; Bitter, after plantation, according to the character of medicinal material plant, is accredited as the seed of ancient sub-Rheum tanguticum ' the Maxim.ex Balf of the especially big yellow race of Tang, is genuine rhubarb seed;
DH26: fruit is brown to bright orange brown, long 7 ~ 10.5mm, wide approximately 5.5 ~ 9mm, the less oval that is of seed, close with fruit color, puckery, after plantation, being the seed of Rheum hotaoense C. Y. Cheng et C. T. Kao Rheum hotaoense C.Y.Cheng et C.T.Kao according to the Property Identification of medicinal material plant, is adulterant rheum officinale seed;
(2) detection method of the present invention
Aloe-emodin, Rhein, archen, Chrysophanol, Physcion reference substance: as shown in Figure 1;
DH10: as shown in Figure 2;
DH13: as shown in Figure 3;
DH15: as shown in Figure 4;
DH26: as shown in Figure 5;
Result is as shown in Fig. 1 ~ 5: in DH10, DH26 rheum officinale seed need testing solution chromatogram, without identical peak value,, containing aloe-emodin, be not accredited as adulterant rheum officinale seed in the position consistent with aloe-emodin reference substance chromatogram peak value retention time; In DH13, DH15 rheum officinale seed need testing solution chromatogram, there is identical peak value in the position consistent with aloe-emodin reference substance chromatogram peak value retention time, containing aloe-emodin, be accredited as genuine rhubarb seed.
Experimental result explanation, high performance liquid chromatography testing result of the present invention is consistent with the result of traditional discrimination method, can whether have genuine rhubarb seed by precise Identification seed to be checked.
Experimental example 2 use detection method of the present invention detects seed to be checked
1, experiment material
| Seed numbering |
| DH10 |
| DH13 |
| DH15 |
| DH26 |
2, experimental technique
(1) with traditional proterties discrimination method qualification:
(2) detection method of the present invention detects: detect according to embodiment 5 methods, wherein, get aloe-emodin, Rhein, archen, Chrysophanol, Physcion reference substance, add methyl alcohol and make the solution of every 1ml containing 1mg, product solution in contrast.
3, experimental result
(1) traditional proterties discrimination method qualification
With experimental example 1;
(2) detection method of the present invention
The testing result of a, step (2) is with experimental example 1;
The testing result of b, step (3) is as shown in Figure 6:
DH10 and DH26 occur that rufous is to sepia flocculent deposit, are accredited as adulterant rheum officinale seed; DH13 and DH15 do not occur that rufous, to sepia flocculent deposit, is accredited as genuine rhubarb seed.
Experimental result explanation, whether the testing result of the inventive method is consistent with the result of traditional discrimination method, can precise Identification seed to be checked be genuine rhubarb seed.
Use detection method of the present invention, high efficiency liquid phase detecting step can accurately determine in seed to be checked, whether there is genuine rhubarb, precipitation detecting step can accurately determine in seed to be checked, whether there is adulterant rheum officinale, the two joint-detection, can be divided into seed to be checked the melange of certified products, adulterant and certified products and adulterant exactly:
If seed to be checked detects by high-efficient liquid chromatography, have identical peak in the position consistent with aloe-emodin reference substance chromatogram peak value retention time, and the precipitation method detect without precipitation, are accredited as genuine rhubarb seed;
If seed to be checked detects by high-efficient liquid chromatography, have identical peak in the position consistent with aloe-emodin reference substance chromatogram peak value retention time, and the precipitation method detect there is precipitation, is accredited as the melange of certified products and adulterant rheum officinale seed;
If seed to be checked detects by high-efficient liquid chromatography, in the position consistent with aloe-emodin reference substance chromatogram peak value retention time, without identical peak, and the precipitation method detect precipitation, is accredited as adulterant rheum officinale seed.
To sum up, whether genuine rhubarb seed detection method of the present invention can have genuine rhubarb seed by precise Identification rheum officinale seed, by the preferred detection method of the present invention, positive pseudo-melange can be got rid of from certified products, improve accuracy in detection, easy and simple to handle, with low cost, have a good application prospect and economic benefit.
Claims (2)
1. a detection method for genuine rhubarb seed, is characterized in that:
Described detection method is to adopt high performance liquid chromatography to detect, and comprises the steps:
(1) get seed to be checked, pulverize, with methyl alcohol extraction, filter, obtain filtrate, remove methyl alcohol, acid hydrolysis, with organic solvent extraction, obtains organic solvent layer, reclaims solvent to doing, then dissolves, and makes need testing solution;
(2) get aloe-emodin reference substance, dissolve, preparation reference substance solution;
(3) whether detect by high performance liquid chromatography, in the chromatogram of need testing solution, in qualification need testing solution, have chromatographic peak consistent with aloe-emodin reference substance chromatographic peak retention time, chromatographic condition is as follows: fixing phase: octadecylsilane chemically bonded silica; Mobile phase: methyl alcohol-0.1% phosphoric acid solution, the volume ratio of the two is (80 ~ 90): (10 ~ 20); Detect wavelength: 254nm;
Acid hydrolysis described in step (1) is 2 ~ 3mol/L for the hydrionic concentration of acid-hydrolyzed solution; Described organic solvent is methenyl choloride;
In described step (1), seed to be checked is pulverized, cross sieve No. four, obtain powder, add methyl alcohol, the mass volume ratio of powder and methyl alcohol is 1:150 ~ 185, add hot reflux 30 ~ 90min, cooling, weigh, supply the weight of less loss with methyl alcohol, shake up, filter, obtain subsequent filtrate, fling to solvent, add concentration and be 6 ~ 10% hydrochloric acid, ultrasonic processing 1 ~ 3min, add isopyknic methenyl choloride, add hot reflux 60 ~ 80min, use again chloroform extraction 3 times, each methenyl choloride volume used is with hydrochloric acid volume, obtain methenyl choloride layer, reclaim solvent to dry, add methyl alcohol and make need testing solution, described mass volume ratio refers to g:ml,
Described detection method also comprises qualitative checking method, and step is as follows:
1) get seed to be checked, pulverize, with methyl alcohol extraction, filter, obtain filtrate, fling to solvent, acid solution hydrolysis, with organic solvent extraction, obtains water layer;
2) get step 1) gained water layer, add the strong aqua of 1 ~ 3 times of volume, jolt, detect whether there is rufous to sepia flocculent deposit;
Acid hydrolysis described in step 1) is 0.52 ~ 2mol/L for the hydrionic concentration of acid-hydrolyzed solution; Organic solvent is ether
In described step 1), seed meal to be checked is broken into fine powder, adds methyl alcohol, the mass volume ratio of fine powder and methyl alcohol is 1:15 ~ 25, soaks 30min ~ 90min, filters, obtain filtrate, evaporate to dryness, adds water, add concentrated hydrochloric acid, hydrochloric acid volume is water volume 1/20 ~ 1/5 again, adds hot reflux 20 ~ 40min, cooling, by extracted with diethyl ether, extract 2 times, each ether volume used is water volume 3 ~ 5 times, obtain water layer; Described mass volume ratio refers to g:ml;
If in abovementioned steps (3), in need testing solution, there is chromatographic peak consistent with aloe-emodin reference substance chromatographic peak retention time, and, abovementioned steps 2) in, to sepia flocculent deposit, identify that seed to be checked is genuine rhubarb seed without rufous.
2. method according to claim 1, is characterized in that: in described step (3), the volume ratio of methyl alcohol and 0.1% phosphoric acid solution is 85:15.
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