CN106680403A - Method for detecting dendrophenol in Dendrobium officinale - Google Patents
Method for detecting dendrophenol in Dendrobium officinale Download PDFInfo
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Abstract
The invention discloses a method for detecting dendrophenol in Dendrobium officinale. The method comprises the following steps: (1) preparation of standard substance liquid; (2) preparation of test sample liquid: weighing a Dendrobium officinale sample, extracting the Dendrobium officinale sample by using a methanol water liquid as an extraction solvent to obtain a Dendrobium officinale extracting liquid, removing the solvent in the Dendrobium officinale extracting liquid, redissolving in chromatographic-grade methanol, standing for 12-24 hours, and finally carrying out filtration treatment to obtain the filtrate which is the test sample liquid; and (3) determination of test sample liquid: detecting the standard substance liquid and test sample liquid by an HPLC-DAD (high performance liquid chromatography-diode array detection) process. The method is simple in technique and convenient to operate, and has the advantages of high efficiency, low application cost, and high detection sensitivity and accuracy. The methanol water liquid used as the extracting agent can efficiently extract dendrophenol and avoid the interference of too many large polar impurities. The method confirms that the dendrophenol has the maximum absorption peak under the conditions of 24.107-24.352 minutes and 230-300nm.
Description
Technical field
The present invention relates to the Extraction and enrichment and detection field of compound, relate in particular to stone in a kind of detection Herba Dendrobii
The method of dry measure used in former times phenol.
Background technology
Herba Dendrobii is orchid family Dendrobium Sw, is a kind of conventional rare Chinese medicine.China there are about 76 kinds of Dendrobium Sw, supply at present
Make medicinal former plant for 45 kinds.The primary medicinal component of Herba Dendrobii includes polysaccharide, alkaloid and Stilbene class.
Wherein, dendrophnol is about 0.001% as its content of distinctive stilbenes compound in Herba Dendrobii, to stomach cancer cell, liver
Cancerous cell has inhibitory activity, is the focus of Herba Dendrobii medicinal ingredient research.Dendrophnol (gigantol) 4,4 '-dihydroxy -2 ', 3-
Dimethoxy bibenzyl, its chemical structural formula are as follows：
At present for the research of dendrophnol focuses mostly in pharmacological effect, and detection deeply the grinding in dendrophnol to its content
Study carefully and the field plot test of Herba Dendrobii during be all it is very important, therefore develop it is a kind of can efficiently separate detection dendrophnol point
Analysis method is very important.
The content of the invention
The method that the technical problem to be solved is to provide dendrophnol in a kind of detection Herba Dendrobii.
In order to solve above-mentioned technical problem, the present invention is adopted the following technical scheme that：Dendrophnol in a kind of detection Herba Dendrobii
Method, comprise the following steps：
(1) preparation of standard solution
Dendrophnol standard substance being weighed respectively, being dissolved in hplc grade methanol, vortex 30s~60s makes 200 μ g/mL, 20 μ g/
ML, 10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, the standard solution of 0.625 μ g/mL；
(2) preparation of need testing solution
Herba Dendrobii sample is weighed, then Herba Dendrobii sample is extracted as Extraction solvent with methanol aqueous solution,
Herba Dendrobii extracting solution is obtained, the solvent in Herba Dendrobii extracting solution is removed into dry afterwards, then is redissolved in hplc grade methanol, then
Place 12~24 hours, finally carry out filtration treatment, the filtrate of gained is need testing solution；
(3) measure of need testing solution
Standard solution and need testing solution are detected using HPLC-DAD detection methods, the chromatographic condition of application
For：Chromatograph column parameter C18Column 250 × 4.6mm 5um, 20 DEG C~60 DEG C of column temperature, 1 μ L~50 μ L of sample size, flow velocity
1.0mL/min, mobile phase are acetonitrile and aqueous formic acid, and condition of gradient elution is that organic faciess are in mobile phase in 0min~40min
In ratio rise to 80%~90% from 5%~20%.
Further, in step (2), the volumetric concentration of the methanol aqueous solution is 60%~80%.Implementing the present invention
During, inventor has found, the extraction effect using this Extraction solvent is preferable, so as to ensure the standard of the detection to dendrophnol
True property.
Further, in step (2), the detailed process of extraction is：According to the ratio of 0.5g/1ml~2ml by Herba Dendrobii
Sample is dipped in methanol aqueous solution, then supersound process 15min~60min at a temperature of 40 DEG C~60 DEG C, then in 4000rpm
Centrifugal treating 15min~20min under the rotating speed of~6000rpm, takes supernatant, repeats above procedure 2~4 times, merges supernatant,
As described Herba Dendrobii extracting solution.In implementing the present invention, it may, inventor has found, and using above extraction process, can
The dendrophnol in Herba Dendrobii is made fully to separate out, so as to ensure the accuracy of the detection to dendrophnol.
Further, in step (2), by the solvent in Herba Dendrobii extracting solution except dry mode is to be dried up with nitrogen.
Further, in step (2), during redissolution, 200 μ L~500 μ L chromatographic grade first are used per 0.5g Herba Dendrobii sample
Alcohol.
Further, in step (2), filtration treatment adopts filtering with microporous membrane, and the aperture of filter membrane is 0.22 μm~0.45 μ
m。
Further, in step (3), the volumetric concentration of the aqueous formic acid of mobile phase is 0.01%~0.1%.
Further, the Detection wavelength of diode array detector is 190nm~400nm.
Further, dendrophnol has maximum absorption band at 24.107min~24.352min, 230nm~300nm.
Beneficial effects of the present invention are embodied in：
1. present invention process is simple, and easy to operate, efficient, application cost is low, and detection sensitivity, degree of accuracy and accurately
Degree is higher, and the response rate is also very high.
2. the present invention using methanol aqueous solution as extractant, can high efficiency extraction dendrophnol, be avoided that much pole again
The interference of property impurity.
3. present invention determine that dendrophnol 24.107min~24.352min, has maximum absorption band at 230nm~300nm.
4. the extraction process of the present invention using ultrasound can smudge cellses, make extraction more abundant, repeat extraction can make stone
Dry measure used in former times phenol is completely dissolved in methanol, and as dendrophnol content is relatively low, the concentration that can improve dendrophnol in solution is redissolved in concentration, just
In detection.
Description of the drawings
Ultra-violet absorption spectrums of the Fig. 1 for dendrophnol standard substance.
Liquid chromatograms of the Fig. 2 for dendrophnol standard substance.
Fig. 3 is the standard curve that embodiment 1 is obtained.
Specific embodiment
To make those skilled in the art more fully understand technical scheme, in the following detailed description, it is only logical
The mode for crossing explanation describes some one exemplary embodiments of the present invention.Undoubtedly, one of ordinary skill in the art can be with
Recognize, in the case of without departing from the spirit and scope of the present invention, can with a variety of modes to described enforcement
Example is modified.
The dendrophnol standard substance used by following examples are purchased from Nat'l Pharmaceutical ＆ Biological Products Control Institute.
Embodiment 1
In a kind of detection Herba Dendrobii, the method for dendrophnol, comprises the following steps：
(1) preparation of standard solution
Weigh dendrophnol standard substance respectively, be dissolved in hplc grade methanol, vortex 45s, make 200 μ g/mL, 20 μ g/mL, 10
μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, the standard solution of 0.625 μ g/mL；
(2) preparation of need testing solution
Weigh 0.5g Herba Dendrobii samples, then with the methanol aqueous solution that volumetric concentration is 80% as Extraction solvent to ferrum
Skin Herba Dendrobii sample is extracted, and the detailed process of extraction is：Herba Dendrobii sample is dipped in methanol by the ratio according to 0.5g/2ml
In aqueous solution, then supersound process 30min at a temperature of 50 DEG C, then centrifugal treating 20min under the rotating speed of 6000rpm takes
Supernatant, repeats above procedure 2 times, for the first time during ultrasonic centrifugal treating, to the Herba Dendrobii sample being dipped in methanol aqueous solution
Process is ground, Herba Dendrobii extracting solution is dried up by merging supernatant, as Herba Dendrobii extracting solution afterwards with nitrogen, then
Redissolve in 300 μ L hplc grade methanols, then place 18 hours, finally carry out filtration treatment, filtration treatment adopts microporous filter membrane
Filter, the aperture of filter membrane is 0.22 μm, and the filtrate of gained is need testing solution；
(3) measure of need testing solution
Standard solution and need testing solution are detected using HPLC-DAD detection methods, the chromatographic condition of application
For：Chromatograph column parameter C18Column 250 × 4.6mm 5um, 25 DEG C of column temperature, sample size 20 μ L, flow velocity 1.0mL/min, flowing
It is mutually acetonitrile and aqueous formic acid that volumetric concentration is 0.1%, condition of gradient elution is that, in 0min~40min, organic faciess are in stream
The ratio of dynamic Xiangli rises to 80% from 5%；
The Detection wavelength of diode array detector is 279nm；
(4) result parsing
Before testing, ultraviolet-visible analysis of spectrum is carried out to dendrophnol standard substance first, obtaining the ultraviolet of dendrophnol can
See spectrogram, referring to Fig. 1, the liquid chromatogram of 200 μ g/mL standard solutions learns by analysis that referring to Fig. 2 dendrophnol exists
There is maximum absorption band at 24.352min, 279nm.
By the measure to standard solution, standard curve is drawn out, referring to Fig. 3, obtain dendrophnol cubage formula
It is as follows：Y=3.7649x+0.0481, y are concentration (μ g/mL), x is peak area；
Embodiment 2
In a kind of detection Herba Dendrobii, the method for dendrophnol, comprises the following steps：
(1) preparation of standard solution
Weigh dendrophnol standard substance respectively, be dissolved in hplc grade methanol, vortex 30s, make 200 μ g/mL, 20 μ g/mL, 10
μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, the standard solution of 0.625 μ g/mL；
(2) preparation of need testing solution
Weigh 0.5g Herba Dendrobii samples, then with the methanol aqueous solution that volumetric concentration is 60% as Extraction solvent to ferrum
Skin Herba Dendrobii sample is extracted, and the detailed process of extraction is：Herba Dendrobii sample is dipped in methanol by the ratio according to 0.5g/1ml
In aqueous solution, then supersound process 60min at a temperature of 40 DEG C, then centrifugal treating 15min under the rotating speed of 4000rpm takes
Supernatant, repeats above procedure 3 times, for the first time during ultrasonic centrifugal treating, to the Herba Dendrobii sample being dipped in methanol aqueous solution
Process is ground, Herba Dendrobii extracting solution is dried up by merging supernatant, as Herba Dendrobii extracting solution afterwards with nitrogen, then
Redissolve in 200 μ L hplc grade methanols, then place 24 hours, finally carry out filtration treatment, filtration treatment adopts microporous filter membrane
Filter, the aperture of filter membrane is 0.45 μm, and the filtrate of gained is need testing solution；
(3) measure of need testing solution
Standard solution and need testing solution are detected using HPLC-DAD detection methods, the chromatographic condition of application
For：Chromatograph column parameter C18Column 250 × 4.6mm 5um, 20 DEG C of column temperature, 1 μ L of sample size, flow velocity 1.0mL/min, mobile phase
For acetonitrile and aqueous formic acid that volumetric concentration is 0.01%, condition of gradient elution is that, in 0min~40min, organic faciess are in flowing
The ratio of Xiangli rises to 85% from 10%；
The Detection wavelength of diode array detector is 190nm；
(4) result parsing
After measure, spectrogram is analyzed, it is as a result similar to Example 1, dendrophnol 24.107min~
24.352min, have maximum absorption band at 230nm~300nm, obtain dendrophnol cubage formula as follows：Y=3.7653x+
0.0481, y is concentration (μ g/mL), x is peak area.
Embodiment 3
In a kind of detection Herba Dendrobii, the method for dendrophnol, comprises the following steps：
(1) preparation of standard solution
Weigh dendrophnol standard substance respectively, be dissolved in hplc grade methanol, vortex 60s, make 200 μ g/mL, 20 μ g/mL, 10
μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, the standard solution of 0.625 μ g/mL；
(2) preparation of need testing solution
Weigh 0.5g Herba Dendrobii samples, then with the methanol aqueous solution that volumetric concentration is 70% as Extraction solvent to ferrum
Skin Herba Dendrobii sample is extracted, and the detailed process of extraction is：Herba Dendrobii sample is dipped in first by the ratio according to 0.5g/1.5ml
In alcohol-water solution, then supersound process 15min at a temperature of 60 DEG C, then centrifugal treating 18min under the rotating speed of 5000rpm,
Supernatant is taken, repeats above procedure 4 times, for the first time during ultrasonic centrifugal treating, to the Herba Dendrobii sample being dipped in methanol aqueous solution
Product are ground process, and Herba Dendrobii extracting solution is dried up by merging supernatant, as Herba Dendrobii extracting solution afterwards with nitrogen,
Redissolved in 500 μ L hplc grade methanols again, then placed 12 hours, finally carry out filtration treatment, filtration treatment is filtered using micropore
Membrane filtration, the aperture of filter membrane is 0.35 μm, and the filtrate of gained is need testing solution；
(3) measure of need testing solution
Standard solution and need testing solution are detected using HPLC-DAD detection methods, the chromatographic condition of application
For：Chromatograph column parameter C18Column 250 × 4.6mm 5um, 60 DEG C of column temperature, sample size 50 μ L, flow velocity 1.0mL/min, flowing
It is mutually acetonitrile and aqueous formic acid that volumetric concentration is 0.06%, condition of gradient elution is that, in 0min~40min, organic faciess are in stream
The ratio of dynamic Xiangli rises to 90% from 20%；
The Detection wavelength of diode array detector is 400nm；
(4) result parsing
After measure, spectrogram is analyzed, it is as a result similar to Example 1, dendrophnol 24.107min~
There is maximum absorption band at 24.352min, 230nm~300nm, obtain dendrophnol cubage formula as follows：Y=3.7649x+
0.0481, y is concentration (μ g/mL), x is peak area.
Embodiment 4
Precision test
6 parts of dendrophnol standard solution are taken, the middle dendrophnol content of dendrophnol standard solution is detected as described in Example 1,
Testing result see the table below 1.
Table 1
Conclusion (of pressure testing)：The RSD of 6 parts of dendrophnol standard solution contents is 1.2%, shows that the present invention has preferable precision,
Meet GB/T 27404-2008 quality control specifications food Physico-chemical tests》Requirement【GB/T 27404-2008 require RSD (%)
≤ 1.3%】.
Embodiment 5
Recovery test
Precision weighs 6 groups of samples, has the Herba Dendrobii sample of the quality such as two parts, respectively to 6 groups of samples in every group of sample
In a copy of it Herba Dendrobii sample in add dendrophnol reference substance, detect that 6 groups of dendrophnols contain as described in Example 1 respectively
Amount, calculates per group of the response rate, measures reference substance input amount=mark-on sample measured amount-non-mark-on sample measured amount；The response rate
(%)=and reference substance input amount/actual input amount of reference substance is measured, testing result see the table below 2.
Table 2
Conclusion (of pressure testing)：Dendrophnol mark-on average recovery rate is：96.408%, relative standard deviation (RSD) is respectively
0.32%, meet GB/T 27404-2008《Good Laboratory controls specification food Physico-chemical tests》Requirement【GB/T 27404-
2008 require that the response rate is 95%~105%】
In addition, also testing respectively to embodiment 2 and 3, as a result the precision for embodiment 2 is 1.37, and the response rate is
96.261%, the precision of embodiment 3 is 1.35, and the response rate is 95.873%.
It should be understood that example as herein described and embodiment are not limited to the present invention, this area only for explanation
Technical staff can make various modifications or change according to it, all any modifications within the spirit and principles in the present invention, made,
Equivalent, improvement etc., should be included within the scope of the present invention.
Claims (6)
1. it is a kind of detection Herba Dendrobii in dendrophnol method, it is characterised in that comprise the following steps：
(1) preparation of standard solution
Weigh dendrophnol standard substance respectively, be dissolved in hplc grade methanol, vortex 30s~60s, make 200 μ g/mL, 20 μ g/mL,
10 μ g/mL, 5 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, the standard solution of 0.625 μ g/mL；
(2) preparation of need testing solution
Herba Dendrobii sample is weighed, then Herba Dendrobii sample is extracted as Extraction solvent with methanol aqueous solution, is obtained
Solvent in Herba Dendrobii extracting solution is removed dry, then is redissolved in hplc grade methanol, then placed by Herba Dendrobii extracting solution afterwards
12~24 hours, filtration treatment is finally carried out, the filtrate of gained is need testing solution；
(3) measure of need testing solution
Standard solution and need testing solution are detected using HPLC-DAD detection methods, the chromatographic condition of application is：Color
Spectrum column parameter C18 Column 250 × 4.6mm 5um, 20 DEG C~60 DEG C of column temperature, 1 μ L~50 μ L of sample size, flow velocity 1.0mL/
Min, mobile phase are acetonitrile and aqueous formic acid, and condition of gradient elution is ratio of the organic faciess in mobile phase in 0min~40min
Example rises to 80%~90% from 5%~20%.
2. the method for detecting dendrophnol in Herba Dendrobii as claimed in claim 1, it is characterised in that：In step (2), the first
The volumetric concentration of alcohol-water solution is 60%~80%.
3. the method for detecting dendrophnol in Herba Dendrobii as claimed in claim 1 or 2, it is characterised in that：In step (2), carry
The detailed process for taking is：Ratio according to 0.5g/1ml~2ml is dipped in Herba Dendrobii sample in methanol aqueous solution, then 40
DEG C~60 DEG C at a temperature of supersound process 15min~60min, then the centrifugal treating under the rotating speed of 4000rpm~6000rpm
15min~20min, takes supernatant, repeats above procedure 2~4 times, merges supernatant, as described Herba Dendrobii extracting solution.
4. the method for detecting dendrophnol in Herba Dendrobii as claimed in claim 1 or 2, it is characterised in that：It is in step (2), multiple
200 μ L~500 μ L hplc grade methanols are used per 0.5g Herba Dendrobii sample when molten.
5. the method for detecting dendrophnol in Herba Dendrobii as claimed in claim 1 or 2, it is characterised in that：In step (3), stream
The volumetric concentration of the aqueous formic acid of dynamic phase is 0.01%~0.1%.
6. the method for detecting dendrophnol in Herba Dendrobii as claimed in claim 1 or 2, it is characterised in that：Dendrophnol exists
There is maximum absorption band at 24.107min~24.352min, 230nm~300nm.
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| CN111929396A (en) * | 2020-07-24 | 2020-11-13 | 杭州师范大学 | On-line enrichment method of dendrobium |
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| CN111929396A (en) * | 2020-07-24 | 2020-11-13 | 杭州师范大学 | On-line enrichment method of dendrobium |
| CN114660205A (en) * | 2022-04-12 | 2022-06-24 | 江苏北环生物科技有限公司 | High-precision dendrophenol detection device and method |
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Application publication date: 20170517 |