CN111220719A - Method for evaluating quality of ginseng medicinal material by using fingerprint spectrum - Google Patents
Method for evaluating quality of ginseng medicinal material by using fingerprint spectrum Download PDFInfo
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Abstract
The invention provides a characteristic map evaluation method of ginseng medicinal material quality, which comprises the steps of preparing a test sample solution and a reference sample solution, detecting a liquid phase and establishing an evaluation standard, wherein the conditions of HPLC detection are preferably acetonitrile and water gradient elution, the conditions of the liquid phase are simple, the establishment of the evaluation standard is the key point of the method, the characteristic map is obtained through the liquid phase, the stability of the method, the Rf content of ginsenoside and the content comparison of Rg1 and Re are concerned, so that the quality information of the ginseng medicinal material is obtained, and the method plays a vital role in evaluating the quality of the ginseng medicinal material.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine quality evaluation, and particularly relates to a method for evaluating the quality of a ginseng medicinal material by combining a fingerprint spectrum and ginsenoside content.
Background
Ginseng radix is dried root and rhizome of Panax ginseng C.A.Mey. Collected in autumn, washed, dried in the sun or baked. The cultivation is commonly called as 'Yuanshen'; the seeds planted in mountain forest are called forest ginseng and seed sea.
Ginsenoside is the main active component of ginseng, has the functions of resisting tumor, resisting radiation, resisting aging, detoxifying and the like, and has earlier and deeper chemical research due to the biological activity in multiple aspects. To date, more than 50 ginsenosides have been isolated and identified by Chinese scholars from sun-dried ginseng, white ginseng and red ginseng. Due to the higher health care and treatment value, but the growth years are long, and the growth region is limited, the price of the wild ginseng is high, and even though the garden ginseng and the ginseng under forest recorded in the 'Chinese pharmacopoeia' of 2015 edition supplement the source of the wild ginseng, the price is still high. Therefore, lawbreakers feel favorable, ginseng with uneven quality is filled in the market, and in order to increase the content and add more fibrous roots, some lawbreakers smash ginseng, add American ginseng leftovers or spray the extract of overground parts and the like to meet the content requirement.
In 2015 edition "Chinese pharmacopoeia", for content determination of ginseng medicinal materials, only the ginsenoside Rg1+ Re should not be less than 0.3%, and the ginsenoside Rb1 should not be less than 0.2% (HPLC method); the United states pharmacopoeia USP41-NF36 specifies Rg1 of not less than 0.2%, Rb1 of not less than 0.1% (HPLC method), the European pharmacopoeia 7.0 edition specifies Rg1+ Rb1 of not less than 0.4% (HPLC method) and the Japanese pharmacopoeia 16 edition specifies Rg1 of not less than 0.1%, Rb of not less than 0.2% (UV method). Through research, the ginseng with more fibrous roots can be found to improve the content of Re, so that the ginseng with more fibrous roots can easily meet the requirements of Chinese pharmacopoeia, but is not the source of high-quality ginseng.
The characteristic fingerprint spectrum is a spectrum capable of identifying common peaks of group characteristics of various components of the traditional Chinese medicine, is a comprehensive and simultaneously qualitative and quantitative method, can be used for identifying the truth of the traditional Chinese medicine and evaluating the stability of the quality of the traditional Chinese medicine, and is an effective analysis method. The evaluation of the quality of ginseng using a fingerprint has not been studied because ginseng contains many specific components and has many similar components to other related adulterants, and if the characteristic components are not clear, the quality of ginseng cannot be reflected only by the fingerprint or the index of a single content without analyzing a large amount of congeneric and similar products, and there is adulteration.
Disclosure of Invention
Based on the technical problems, the invention provides a fingerprint spectrum evaluation method of ginseng medicinal material quality, which comprises the steps of sample injection liquid preparation of a sample injection liquid and a reference sample injection liquid, HPLC detection of the sample injection liquid and the reference sample injection liquid and fingerprint spectrum analysis,
the sample injection liquid preparation of the sample injection liquid and the reference sample comprises that the sample is ginseng, the sample injection liquid preparation comprises that ginseng or ginseng powder is heated and refluxed by aqueous alcohol or lower alcohol, and is filtered to obtain filtrate, the upper layer solution is precisely taken and dried to obtain residue, the residue is dissolved by methanol to obtain the sample injection liquid of the sample, and the reference sample is a solution containing ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rf;
in the HPLC detection steps of the test sample and the reference sample: gradient eluting with water and acetonitrile as mobile phase at 20-40 deg.C and flow rate of 0.8-1.2ml/min, using octadecylsilane chemically bonded silica packed column as chromatographic column, and detecting UV wavelength of 200-210 nm;
after the fingerprint of the test sample is obtained, the analysis step of the fingerprint comprises the following qualified medicinal material standards, and simultaneously the following A, B and C are met:
a, the ginseng fingerprint spectrum formed by A needs to comprise characteristic peaks of 8 ginsenosides, wherein the characteristic peaks comprise ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rc and ginsenoside Rd;
the content of ginsenoside Rf in the sample fingerprint is more than or equal to 0.05% (experimental data verifies that the commonly used ginseng stems and leaves do not contain Rf in adulteration, the American ginseng does not contain Rf or the content of Rf is less than 0.05%, and the non-ginseng product does not contain Rf);
c, the content of ginsenoside Rg1+ Re in the fingerprint of the test sample is not less than 0.3%, and the content of ginsenoside Rb1 is not less than 0.1%. (if other impurities are incorporated, the contents of corresponding Rg1+ Re and Rb1 decrease)
Wherein the aqueous alcohol is water saturated n-butanol, aqueous ethanol, methanol, ethanol, and other alcohols containing no more than 5 carbons.
Further, in the above rating method, the Rg1: re is set at 1: 1-3: 1 (experimental data verifies that according to a common method of adulteration or counterfeiting, the Rg1 content of the American ginseng leftovers is particularly low, the growth life of the ginseng is not long enough or the ginseng or ginseng rootlets cut by counterfeiting are obtained, the adulterated and counterfeit products all accord with pharmacopoeia standards, but the Rg1 content is lower than Re, even the content is 0).
The indication reference substance comprises specific components of ginseng and other standard substances required by quantification, and the reference substance is a solution at least containing ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rf;
the inventor respectively optimizes the existing extraction process and mobile phase of ginseng test sample, ginsenoside has weak ultraviolet absorption due to single double bond in most of its structure, and weak absorption at 200-210nm, and the ELSD detector is used to replace the detector with weak ultraviolet absorption in the existing high performance liquid chromatography, but the ELSD sensitivity is one order of magnitude lower than that of the ultraviolet detector, so if the ultraviolet detector can be used, the ultraviolet detector is used as far as possible, but the requirement on solvent is high, and the solvent with terminal absorption at 200-210nm, such as methanol, is not suitable. When the ginsenoside fingerprint in the ginseng is measured, the pretreatment occupies an important position because the treatment of a test sample directly causes the quality of the fingerprint, and the method adopts the ultrasonic treatment of water saturated alcohol or lower alcohol to effectively transfer the ginsenoside.
The mobile phase ensures that more control peaks achieve better separation.
More importantly, by the method, an evaluation standard is obtained, and the ginsenoside is generally considered as a main active ingredient in the ginseng, so that more researches are carried out. However, studies have shown that different ginseng parts contain different types and contents of ginsenosides. Especially, after the study on the stems and leaves of Panax quinquefolium and Panax ginseng belonging to the same genus, it was found that the stems and leaves of Panax quinquefolium and Panax ginseng contain ginsenoside, but each of them has unique components and different contents. The specific component of the ginseng is ginsenoside Rf which is almost absent in stems and leaves, the Rg1 content in the ginseng is higher than Re, and the Rg1 content in the stems and leaves of the ginseng is lower than Re and almost does not contain Rb 1; the pseudo-ginsenoside F11 in the American ginseng is a special component, but the content is extremely low, the pseudo-ginsenoside F11 is difficult to detect at the wavelength of 203, but the American ginseng does not contain ginsenoside Rf, the Rg1 is far lower than Re, and the Rb1 content is higher than that of the ginseng.
In the fingerprint spectrum evaluation method for the quality of the ginseng medicinal material, according to the standard of qualified medicinal materials, the medicinal material standards A, B and C must be the same, and any one of the medicinal material standards A, B and C can not be met, so that the medicinal material can be judged to be unqualified.
Further, in the above method for evaluating the fingerprint of the quality of the ginseng medicinal material, the step of HPLC detection of the test sample and the reference sample comprises: octadecylsilane chemically bonded silica packed column; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and the gradient elution procedure comprises the following steps of 0-35 minutes, mobile phase A19% and mobile phase B81%; 35-55 minutes, 19% → 29% of mobile phase A, and 81% → 71% of mobile phase B; 55-70 minutes, wherein the mobile phase A29% and the mobile phase B71%; 70-100 minutes, mobile phase A29% → 40%, mobile phase B71% → 60%, column temperature 30 ℃, and flow rate 1 ml/min. The octadecylsilane chemically bonded silica packed column was an Agilent ZORBAX SB-C184.6 x 250mm, 5 μm or Inertsil ODS-3.5 μm4.6 x 250mm chromatography column.
The fingerprint of the test article comprises 8 common peaks, wherein the No. 5 peak is a ginsenoside Rb1 control peak, and the relative retention time of each characteristic peak is as follows: peak 1, peak 0.42 + -10%, peak 2, peak 0.44 + -10%, peak 3, peak 0.86 + -10%, peak 4, peak 0.90 + -10%, peak 5, peak 1.00, peak 6, peak 1.06 + -10%, peak 7, peak 1.13 + -10%, peak 8, and peak 1.25 + -10%. Six peaks (1, 2, 3, 5, 6, 8) were identified from the 8 selected common peaks by purchasing standards, comparing the retention time of the HPLC chart with the PDA profile, and sampling and recovering.
The ginseng medicinal material fingerprint comprises the following characteristic peaks: the peak 1 is ginsenoside Rg1, the peak 2 is ginsenoside Re, the peak 3 is ginsenoside Rf, the peak 5 is ginsenoside Rb1, the peak 6 is ginsenoside Rb2+ ginsenoside Rc, and the peak 8 is ginsenoside Rd.
After obtaining the corresponding test sample fingerprint, the inventor further provides the evaluation standard of the fingerprint, and the theoretical plate number of the ginsenoside Rg1 in the obtained fingerprint is not lower than 6000.
The components of the ginseng medicinal material can be better reflected by constructing the fingerprint of the ginsenoside in the ginseng medicinal material, and the quality of the ginseng medicinal material can be well judged by judging whether the ginseng medicinal material contains the ginsenoside Rf, the absolute contents of the ginsenosides Rg1+ Re and Rb1 and the relative content relationship of the ginsenoside Rg1 and Re. The applicant also carries out relevant literature search, and the search finds that (1) the American ginseng does not contain ginsenoside Rf and contains pseudoginsenoside F11; the ginseng contains no pseudo-ginsenoside F11 and contains Rf, and can be used as the identification basis of American ginseng and ginseng; (2) the contents of Rb1, Rc, Rb2 and Rd in the ginseng fibrous root and the branch root are higher than those in other parts, and the content of Re in the leaves is higher. (3) The relative content of Rg1 in the ginseng fibrous root is lower than Re. According to comparative studies we also found (1) that the relative content of Rg1 in ginseng fibrous root is lower than Re; rg1 in Ginseng: re is between 1:1 and 1:3, and Rg1: Re in American ginseng is less than 1: 8. (3) The fingerprint of ginseng, American ginseng and ginseng stem and leaf can be obtained by using the same sample treatment condition and the same high performance liquid separation condition, and the difference of the three medicinal materials can be clearly seen through the fingerprint. The applicant carries out relevant summary through a plurality of batches of experiments, further simplifies the evaluation standard and obtains the evaluation standard.
According to the method, the inventor further provides a specific ginseng quality fingerprint spectrum evaluation method, which comprises the following steps: establishing a feature map, comprising: a) preparation of a test solution: taking about 1g of ginseng powder, precisely weighing, placing in a Soxhlet extractor, adding 50ml of precisely water-saturated n-butyl alcohol, heating and refluxing for 1 hour, filtering, discarding the primary filtrate, precisely measuring 25ml of the subsequent filtrate, placing in an evaporating dish, evaporating to dryness, dissolving the residue with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, shaking uniformly, filtering, and taking the subsequent filtrate. b) Preparation of control solutions: weighing 8 reference substances including 1) ginsenoside Rg1, 2) ginsenoside Re, 3) ginsenoside Rf, 4) ginsenoside Rb1, 5) ginsenoside Rb2, 6) ginsenoside Rc, 7) ginsenoside Rb3 and 8) ginsenoside Rd, adding methanol to dissolve to a constant volume, making into a reference solution with a certain concentration, and filtering with a microporous membrane at 0.45 μm for later use; c) chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and the gradient elution procedure is as follows: 0-35 minutes, mobile phase A19%, mobile phase B81%; 35-55 minutes, wherein the mobile phase A is 19% → 29%, and the mobile phase B is 81% → 71%; 55-70 minutes, mobile phase A29%, and mobile phase 71%; 70-100 minutes, wherein the mobile phase A is 29% → 40%, and the mobile phase B is 71% → 60%; the detection wavelength is 200-210nm, preferably 201-204nm, most preferably 203 nm. The column temperature is 20-40 ℃, and the flow rate is 0.8-1.2 ml/min. The number of theoretical plates with the sample amount of 1-5 μ L is not less than 6000 according to the peak of ginsenoside Rg 1.
The qualified medicinal materials need to satisfy the following three at the same time, and one of the qualified medicinal materials does not satisfy the requirement, and the qualified medicinal materials are judged as unqualified medicinal materials.
A, the ginseng fingerprint spectrum formed by A needs to comprise characteristic peaks of 8 ginsenosides, wherein the characteristic peaks comprise ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rc and ginsenoside Rd;
the content of ginsenoside Rf in B medicinal material fingerprint is not less than 0.05%
The content of ginsenoside Rg1+ Re in the fingerprint of the medicinal material C is not less than 0.3%, and the content of Rg1: re is between 1 and 3; the content of ginsenoside Rb1 should be not less than 0.1%.
The method adopts various evaluation modes for evaluation, and the visual fingerprint similarity is combined with the absolute content of the main ginsenoside and the relative content of the specific components to evaluate the quality of the ginseng medicinal material.
According to the evaluation method, the inventor provides a reliable evaluation method, which can accurately detect the quality of the ginseng, integrate the correlation peak and content and direct ratio relation, quickly judge the quality of the ginseng and improve the detection level in the quality control field of the ginseng product.
Description of the drawings:
FIG. 1: ginsenoside fingerprint of Ginseng radix reference medicinal material.
Detailed Description
The following preferred embodiments are merely illustrative of the technical solutions of the present invention and are not restrictive, and although the present invention has been described in detail with reference to the following preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the present invention as defined by the appended claims.
First, research on ginsenoside fingerprint spectrum method in ginseng medicinal material
1. Instrument and reagent
Agilent 1200 type high performance liquid chromatograph, Shimadzu LC-2010AHT, one hundred thousand electronic balance AY220 type 1/10 ten thousand electronic analytical balance (Shimadzu), AS series ultrasonic cleaner (250W, 50Hz/20Hz dual frequency) (Tianjin Orthon instruments, Inc.). Agilent ZORBAX SB-C18(5 μm 4.6X 250mm) column.
Ginseng radix control medicinal materials (China food and drug assay research institute, batch No. 120917-201211), ginsenoside Rg1 (China food and drug assay research institute, batch No. 11073-201530, ginsenoside Re (China food and drug assay research institute, batch No. 110754-201626), ginsenoside Rf (China food and drug assay research institute, batch No. 111719-201505), ginsenoside Rb1 (China food and drug assay research institute, batch No. 110704-201625), ginsenoside Rb2 (Standard genetic chemicals Inc. part Number RSS-411868, Lot Number MK180418-06), ginsenoside Rc (Standard chemical Inc. part Number RSD-152027, Lot Number PR180325-18), ginsenoside Rg (Standard genetic chemicals Inc. part Number RSD-387: RS 2-contained 5) (GSPR 3907-343635), lot Number: PR180222-05), ginsenoside Rf: (Stanfordalytic chemicals Inc. part Number: GSS-107769, Lot Number: PF180626-08) acetonitrile was chromatographically pure, water was water for injection, and the other reagents were analytically pure.
2. Selection of chromatographic conditions
When the chromatographic condition of ginsenoside in ginseng medicinal materials is selected, a mobile phase system is selected particularly. The chromatographic conditions finally determined were: a chromatographic column which takes octadecylsilane chemically bonded silica as a filler and octadecylsilane chemically bonded silica as a filler; mobile phase a was acetonitrile and mobile phase B was water, the gradient elution procedure was as follows: 0-35 minutes, mobile phase A19%, mobile phase B81%; 35-55 minutes, wherein the mobile phase A is 19% → 29%, and the mobile phase B is 81% → 71%; 55-70 minutes, mobile phase A29%, and mobile phase 71%; 70-100 minutes, wherein the mobile phase A is 29% → 40%, and the mobile phase B is 71% → 60%; the detection wavelength is preferably 203 nm. The column temperature was 30 ℃ and the flow rate was 1 ml/min. The sample amount is 1 μ L, and the number of theoretical plates is not less than 6000 calculated according to the peak of ginsenoside Rg 1.
3. Selection of conditions for extraction of test sample
(1) Selection of extraction method
For the ginseng medicinal material test sample: preparation of a test solution: precisely weighing about 1g of Ginseng radix powder, precisely adding 50ml of water saturated n-butanol, heating and refluxing for 1 hr, removing solvent, volatilizing solvent from residue, filtering, discarding primary filtrate, precisely weighing 25ml of subsequent filtrate, evaporating to dryness in evaporating dish, dissolving residue with methanol, transferring to 5ml measuring flask, diluting with methanol to scale, shaking, filtering, and collecting subsequent filtrate.
(2) Selection of extraction time
After the ginseng medicinal material is crushed, precisely weighing 1g, adding 50ml of water saturated n-butyl alcohol, heating and refluxing for 45min, 60min, 90min and 120min, and comparing the extraction rates, wherein the extraction rates are basically consistent between 60min, 90min and 120 min.
Finally, the extraction method of the test sample is selected, and the water saturated n-butanol is heated and refluxed for 60 min.
4. Chromatographic column selection
The separation effect of the columns of Inertsil ODS-3(5 μm 4.6X 250mm) and Agilent ZORBAX SB-C18(5 μm 4.6X 250mm) was examined, and the columns of Inertsil ODS-3 were not as good as Agilent ZORBAX SB-C18 in terms of separation, and Agilent ZORBAX SB-C18 was also preferable from the viewpoint of one-test-and-many-evaluation and convenience.
5. Wavelength selection
The ginsenoside compounds mostly have only one isolated double bond, and the maximum absorption wavelength is 200-210 nm. The pharmacopoeia also uses the wavelength of 203nm as the wavelength for measuring the content of the ginsenoside Rg1, Re and Rb1 in the ginseng, and in order to keep consistent with the content measurement and also achieve the effect of one measurement and multiple evaluations, the fingerprint spectrum follows the wavelength for measuring the content, and uses 203nm as the detection wavelength of the fingerprint spectrum of the ginsenoside components in the ginseng medicinal material.
6. Selection of reference peaks
The ginsenoside Rb1 content in the ginsenoside extract is high, and the ginsenoside extract is well separated from other ginsenosides, so the relative chemical shifts of other ginsenosides are calculated by taking the ginsenoside Rb1 as a reference peak.
(I) ginsenoside component fingerprint spectrum methodology in ginseng medicinal material
Preparation of a test solution: preparation of a test solution: taking about 1g of ginseng powder, precisely weighing, precisely adding 50ml of water saturated n-butyl alcohol, heating and refluxing for 1 hour, filtering, discarding the primary filtrate, precisely measuring 25ml of the subsequent filtrate, evaporating to dryness in an evaporating dish, dissolving the residue with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, shaking uniformly, filtering, and taking the subsequent filtrate. The amount of sample was 1. mu.l.
1. Precision (Precision):
and continuously and repeatedly injecting the sample for 6 times, and carrying out similarity evaluation on the obtained result.
TABLE 1 finger print precision test of ginseng medicinal materials
The results show that: the mutual similarity results of 6 needles are all larger than 0.9
2. And (3) repeatability test:
precisely weighing about 1.0g of the same batch of ginseng, weighing 6 parts, processing according to the method, carrying out parallel operation on 6 parts, injecting 1 mu l of sample, detecting the fingerprint spectrum, and carrying out similarity evaluation on the obtained result. The results are shown in Table 2
TABLE 2 repeatability test of finger print of ginseng
3. Stability of solution
Sampling the same sample solution for 0, 4, 8, 12 and 24h respectively, measuring, recording 8 characteristic peak areas, detecting the fingerprint, and performing similarity evaluation on the obtained result. The results are shown in Table 3
TABLE 3 stability test of finger print of ginseng
The results show that: the mutual similarity results of 6 needles are all larger than 0.9
4. Sample assay
30 batches of ginseng medicinal materials are taken, the fingerprint spectrum is detected according to the method, and the result is shown in table 4.
TABLE 4 content of four ginsenosides in 30 batches of ginseng material
5. Limit of
Taking 30 batches of ginseng medicinal materials, measuring according to a fingerprint method, and calculating the relative retention time of each characteristic peak and a reference substance (Rb1), wherein the results are shown in a table 5:
TABLE 5 relative retention time of each characteristic peak with reference (Rb1)
The result shows that the retention time RSD% of each characteristic peak and a reference peak (reference peak 5, corresponding substance is ginsenoside Rb1) is less than 2%, so the fingerprint standard is formulated as follows:
in the fingerprint of the test article, 8 characteristic peaks are needed, the peak corresponding to the reference substance peak is an S peak, the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within +/-5% of a specified value. The specified values were 0.469 (Peak 1), 0.506 (Peak 2), 0.846 (Peak 3), 0.890 (Peak 4), 1.000[ Peak 5(s) ], 1.076 (Peak 6), 1.141 (Peak 7), 1.230 (Peak 8)
Example 1
1. Instrument and reagent
An Agilent 1200 type high performance liquid chromatograph, a one hundred thousand electronic balance AY220 type 1/10 ten thousand electronic analytical balance (shimadzu), an AS series ultrasonic cleaner (250W, 50Hz/20Hz dual frequency) (tianjin ott sains instruments ltd). Agilent ZORBAX SB-C18(5 μm 4.6X 250mm) column.
Ginseng radix control medicinal materials (China food and drug testing institute, batch No. 120917-201211), radix Panacis Quinquefolii control medicinal materials (China food and drug testing institute, batch No. 120997-201309), caulis Et folium Ginseng (China food and drug testing institute, batch No. 121586-201202), ginsenoside 1 (China food and drug testing institute, batch No. 11073-201530, ginsenoside Re (China food and drug testing institute, batch No. 110754-201626), ginsenoside Rf (China food and drug testing institute, batch No. 111719-201505), ginsenoside Rb1 (China food and drug testing institute, batch No. 110704-201625), ginsenoside Rb2 (Standard Analytical chemicals Inc.partnumber: RSS-411868, Lot Number MK: 180418-06), ginsenoside Rc (Standard Analytical chemicals Inc.sub-Number: PR 2-539: RSRc 325), ginsenoside Rd: (Stanford Analytical chemicals Inc. part Number: RSD-152938, Lot Number: PR180620-07) ginsenoside Rb 3: (Stanford Analytical chemicals Inc. part Number: GSB-343635, Lot Number: PR180222-05), ginsenoside Rf: (Stanford Analytical chemistry Inc. part Number: GSS-107769, Lot Number: PF180626-08), acetonitrile was chromatographically pure, water was water for injection, and the other reagents were analytically pure.
2. Selection of chromatographic conditions
When the chromatographic condition of ginsenoside in ginseng medicinal materials is selected, a mobile phase system is selected particularly. The chromatographic conditions finally determined were: a chromatographic column which takes octadecylsilane chemically bonded silica as a filler and octadecylsilane chemically bonded silica as a filler; agilent ZORBAX SB-C18, 5 μm 4.6X 250mm, detection wavelength 203nm, flow rate: 1.0ml/min, mobile phase a acetonitrile, mobile phase B water, gradient elution procedure as follows: 0-35 minutes, mobile phase A19%, mobile phase B81%; 35-55 minutes, wherein the mobile phase A is 19% → 29%, and the mobile phase B is 81% → 71%; 55-70 minutes, mobile phase A29%, and mobile phase 71%; 70-100 minutes, wherein the mobile phase A is 29% → 40%, and the mobile phase B is 71% → 60%; the detection wavelength is preferably 203 nm. The column temperature was 30 ℃ and the flow rate was 1 ml/min. The sample amount is 1 μ L, and the number of theoretical plates is not less than 6000 calculated according to the peak of ginsenoside Rg 1.
3. Preparation of a test solution:
taking about 1g of ginseng powder, precisely weighing, placing in a Soxhlet extractor, adding 50ml of precisely water-saturated n-butyl alcohol, heating and refluxing for 1 hour, filtering, discarding the primary filtrate, precisely measuring 25ml of the subsequent filtrate, placing in an evaporating dish, evaporating to dryness, dissolving the residue with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, shaking uniformly, filtering, and taking the subsequent filtrate.
4. Preparation of control solutions:
weighing 8 reference substances including 1) ginsenoside Rg1, 2) ginsenoside Re, 3) ginsenoside Rf, 4) ginsenoside Rb1, 5) ginsenoside Rb2, 6) ginsenoside Rc, 7) ginsenoside Rb3 and 8) ginsenoside Rd, adding methanol to dissolve to a constant volume, making into a reference solution with a certain concentration, and filtering with a microporous membrane at 0.45 μm for later use;
5. the determination method comprises the following steps: precisely sucking 10 μ L of the reference solution and 10-20 μ L of the test solution, respectively, injecting into a liquid chromatograph, and measuring.
The HPLC chromatogram of the Ginseng radix control material is shown in FIG. 1.
Example 2
Chromatographic conditions and system applicability test: a chromatographic column which takes octadecylsilane chemically bonded silica as a filler and octadecylsilane chemically bonded silica as a filler; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and the gradient elution procedure is as follows: 0-35 minutes, mobile phase A19%, mobile phase B81%; 35-55 minutes, wherein the mobile phase A is 19% → 29%, and the mobile phase B is 81% → 71%; 55-70 minutes, mobile phase A29%, and mobile phase 71%; 70-100 minutes, wherein the mobile phase A is 29% → 40%, and the mobile phase B is 71% → 60%; the detection wavelength is preferably 203 nm. The column temperature was 30 ℃ and the flow rate was 1 ml/min. The number of theoretical plates is not less than 6000 according to the peak calculation of ginsenoside Rg 1.
Preparation of control solutions:
weighing 4 kinds of reference substances including 1) ginsenoside Rg1, 2) ginsenoside Re, 3) ginsenoside Rf, 4) ginsenoside Rb1, dissolving in methanol to desired volume, making into reference solution with certain concentration, and filtering with microporous membrane of 0.45 μm;
preparing a test solution: taking about 1g of ginseng powder (passing through a No. four sieve), precisely weighing, placing in a Soxhlet extractor, adding chloroform, heating and refluxing for 3 hours, discarding chloroform liquid, volatilizing solvent from medicine residues, transferring into a 100ml conical flask together with a filter paper cylinder, precisely adding 50ml of water-saturated n-butanol, sealing, standing overnight, performing ultrasonic treatment (power 250W and frequency 50kHz) for 30 minutes, filtering, discarding primary filtrate, precisely weighing 25ml of secondary filtrate, placing in an evaporating dish, evaporating to dryness, dissolving residues in methanol, transferring to a 5ml volumetric product, adding methanol to dilute to scale, shaking uniformly, filtering, and taking secondary filtrate to obtain the final product.
The determination method comprises the following steps: precisely sucking 10 μ L of the reference solution and 10-20 μ L of the test solution, respectively, injecting into a liquid chromatograph, and measuring.
The HPLC of the ginseng control material is similar to that of FIG. 1 and is not shown.
Example 3
Measuring fingerprint and content of Ginseng radix, Ginseng radix stem and leaf, and radix Panacis Quinquefolii as reference materials
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system adaptability tests; acetonitrile was used as mobile phase a and water as mobile phase B, and gradient elution was performed as specified in the following table: the detection wavelength was 203 nm. The theoretical plate number is not lower than 6000 calculated according to the peak of the ginsenoside Rg 1. The chromatographic conditions are shown in Table 6
TABLE 6 chromatographic conditions
Preparing reference solution by accurately weighing ginsenoside Rg1 reference, ginsenoside Re reference, ginsenoside Rb1 reference, and ginsenoside Rf reference, adding methanol to obtain mixed solution containing 0.2mg of ginsenoside Rg1, ginsenoside Re reference, and ginsenoside Rf reference, and shaking.
Preparing a test solution, namely taking ginseng reference medicinal material powder (screened by a sieve of four), American ginseng reference medicinal material powder (screened by a sieve of four), about 1g of ginseng stem leaf reference medicinal material powder (screened by a sieve of four), precisely weighing, placing in a Soxhlet extractor, adding trichloromethane, heating and refluxing for 1 hour, discarding trichloromethane liquid, volatilizing a solvent from dregs, moving into a 100ml conical flask together with a filter paper cylinder, precisely adding 50ml of water-saturated n-butyl alcohol, sealing, placing overnight, carrying out ultrasonic treatment (power of 250W and 50KHz) for 30 minutes, filtering, discarding a primary filtrate, precisely measuring 25ml of a subsequent filtrate, placing in an evaporating dish, evaporating, dissolving residues with methanol, transferring into a 5ml measuring flask, adding methanol to dilute to scale, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution.
The determination method comprises precisely sucking 10 μ l of control solution and 10-20 μ l of test solution, injecting into liquid chromatograph, and determining.
The results are shown in Table 7:
TABLE 7
The results show that:
1. the content of ginsenoside Rg1 in Ginseng radix is higher than that of ginsenoside Re, Rg1 in radix Panacis Quinquefolii is far lower than Re, and Rg1 in caulis Et folium Ginseng is higher than Re.
2. The ginseng contains ginsenoside Rf, and the ginseng stem and leaf and American ginseng do not contain ginsenoside Rf.
3. The content of ginsenoside Rb1 in Ginseng radix is lower than that in radix Panacis Quinquefolii, and the stem and leaf of Ginseng radix do not contain Rb 1.
Example 4
Determining fingerprint and content data of Ginseng radix added with radix Panacis Quinquefolii at different ratio
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system adaptability tests; acetonitrile was used as mobile phase a and water as mobile phase B, and gradient elution was performed as specified in the following table: the detection wavelength was 203 nm. The theoretical plate number is not lower than 6000 calculated according to the peak of the ginsenoside Rg 1. The liquid phase conditions are shown in Table 8
TABLE 8
Preparing reference solution by accurately weighing ginsenoside Rg1 reference, ginsenoside Re reference, ginsenoside Rb1 reference, and ginsenoside Rf reference, adding methanol to obtain mixed solution containing 0.2mg of ginsenoside Rg1, ginsenoside Re reference, and ginsenoside Rf reference, and shaking.
Preparing test solution by respectively taking ginseng medicinal material powder (sieved by a sieve of No. four), and American ginseng medicinal material powder (sieved by a sieve of No. four) taking ginseng powder: mixing radix Panacis Quinquefolii powder 1g each at 9:1, 4:1 and 1:1, precisely weighing, placing in a Soxhlet extractor, adding chloroform, heating and refluxing for 1 hr, discarding chloroform solution, volatilizing solvent from residue, transferring into a 100ml conical flask together with a filter paper cylinder, precisely adding water saturated n-butanol 50ml, sealing, standing overnight, ultrasonic treating (power 250W, 50KHz) for 30 min, filtering, discarding primary filtrate, precisely weighing secondary filtrate 25ml, evaporating to dryness in an evaporating dish, dissolving residue with methanol, transferring into a 5ml measuring flask, adding methanol to dilute to scale, shaking, filtering, and taking secondary filtrate.
The determination method comprises precisely sucking 10 μ l of control solution and 10-20 μ l of test solution, injecting into liquid chromatograph, and determining.
The results are shown in Table 9:
TABLE 9
The results show that: 1. the content of 10 percent of American ginseng Rg1 doped in ginseng is not equal to or higher than that of Re, and the ratio of Rg1 to Re is lower with the increase of the added amount.
2. The content of ginsenoside Rf is lower with the more radix Panacis Quinquefolii.
Example 5 Ginseng radix fibril content 30%
Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system adaptability tests; acetonitrile was used as mobile phase a and water as mobile phase B, and gradient elution was performed as specified in the following table: the detection wavelength was 203 nm. The theoretical plate number is not lower than 6000 calculated according to the peak of the ginsenoside Rg 1. The liquid phase conditions are shown in Table 10.
Preparing reference solution by accurately weighing ginsenoside Rg1 reference, ginsenoside Re reference, ginsenoside Rb1 reference, and ginsenoside Rf reference, adding methanol to obtain mixed solution containing 0.2mg of ginsenoside Rg1, ginsenoside Re reference, and ginsenoside Rf reference, and shaking.
Preparation of test solution Ginseng radix powder is respectively taken (sieved with No. four sieve)1Precisely weighing, placing in a Soxhlet extractor, adding chloroform, heating and refluxing for 1 hour, discarding chloroform liquid, volatilizing solvent from residues, transferring into a 100ml conical flask together with a filter paper cylinder, precisely adding 50ml of water-saturated n-butanol, sealing, standing overnight, performing ultrasonic treatment (power of 250W and 50KHz) for 30 minutes, filtering, discarding primary filtrate, precisely measuring to obtain 25ml of subsequent filtrate, evaporating to dryness in an evaporating dish, dissolving residues with methanol, transferring into a 5ml measuring flask, adding methanol to dilute to scale, shaking uniformly, filtering, and obtaining subsequent filtrate.
The determination method comprises precisely sucking 10 μ l of control solution and 10-20 μ l of test solution, injecting into liquid chromatograph, and determining. The results are shown in Table 11.
TABLE 11
The results show that: 1. the content of 30 percent of fibrous root Rg1 doped in the ginseng is not equal to or higher than that of Re, and the ratio of Rg1 to Re is lower along with the increase of the added amount; 2. the ginsenoside Rf content is also lower as the ginseng fibrous root is more incorporated.
Therefore, in order to prevent the ginseng from being doped with common impurities such as fibrous roots, ginseng leaves, American ginseng and the like, the evaluation method provides a standard, and related researches and verifications are carried out on Rg1, Re, Rb1 and Rf, corresponding standards are provided and must be met simultaneously, namely 1. the formed ginseng fingerprint spectrum needs to comprise 8 characteristic peaks of ginsenoside, wherein the characteristic peaks comprise ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rc and ginsenoside Rd; (must contain the corresponding ginseng must peak);
1. the content of ginsenoside Rf in the test sample fingerprint is not less than 0.05%; (even if 30% of fibrous roots are blended, Rf is only 0.045%, and if American ginseng is blended, Rf is lower)
2. The content of ginsenoside Rg1+ Re in the fingerprint of the test sample is not less than 0.3% (see fibrous root data), and the content of ginsenoside Rb1 is not less than 0.1% (preventing the ginseng from being mixed into ginseng stems and leaves).
Claims (10)
1. A fingerprint evaluation method for ginseng medicinal material quality is characterized by comprising the steps of sample injection liquid preparation of a sample injection liquid and a reference sample injection liquid, HPLC detection of the sample injection liquid and the reference sample injection liquid and fingerprint analysis,
the sample injection solution preparation of the sample injection solution and the reference sample comprises that the sample is ginseng, the sample injection solution preparation of the sample comprises that the ginseng is heated and refluxed by aqueous alcohol or lower alcohol, and is filtered to obtain filtrate, the upper solution is accurately taken and dried to obtain residue, the residue is dissolved by methanol to obtain the sample injection solution of the sample, and the reference sample is a solution containing ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rf;
in the HPLC detection steps of the test sample and the reference sample: gradient eluting with water and acetonitrile as mobile phase at 20-40 deg.C and flow rate of 0.8-1.2ml/min, using octadecylsilane chemically bonded silica packed column as chromatographic column, and detecting UV wavelength of 200-210 nm;
after the fingerprint of the test sample is obtained, the analysis steps of the fingerprint comprise the following qualified medicinal material standards, and simultaneously meet the following A, B and C:
the Ginseng radix fingerprint obtained by A at least comprises characteristic peaks of 8 ginsenosides, wherein the ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rc and ginsenoside Rd should be contained;
the content of ginsenoside Rf in the sample fingerprint is not less than 0.05%;
c, the content of ginsenoside Rg1+ Re in the fingerprint of the test sample is not less than 0.3%, and the content of ginsenoside Rb1 is not less than 0.1%.
2. The method for evaluating the fingerprint of the quality of the ginseng medicinal material according to claim 1, wherein the Rg1 is satisfied simultaneously in the step C: re is 1: 1-3: 1.
3. The method of claim 1, wherein the aqueous alcohol is water-saturated n-butanol, aqueous ethanol, methanol, ethanol, or other alcohols containing no more than 5 carbons.
4. The method for evaluating the fingerprint of the quality of the ginseng medicinal material according to claim 1, wherein the UV wavelength is 202-204 nm.
5. The method for evaluating the fingerprint of the quality of the ginseng medicinal material according to any one of claims 1 to 4, wherein in the HPLC detection steps of the test sample and the reference sample: octadecylsilane chemically bonded silica packed column; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and the gradient elution procedure comprises the following steps of 0-35 minutes, mobile phase A19% and mobile phase B81%; 35-55 minutes, 19% → 29% of mobile phase A, and 81% → 71% of mobile phase B; 55-70 minutes, wherein the mobile phase A29% and the mobile phase B71%; 70-100 minutes, mobile phase A29% → 40%, mobile phase B71% → 60%, column temperature 30 ℃, flow rate 1ml/min, and detection UV wavelength 203 nm.
6. The method for evaluating the quality of a ginseng medicinal material according to any one of claims 1-5, wherein the octadecylsilane chemically bonded silica packed column is an Agilent ZORBAX SB-C184.6 x 250mm, 5 μm or InertsilODS-3.5 μm4.6 x 250mm chromatographic column.
7. The method for evaluating the fingerprint of the quality of the ginseng medicinal material according to claim 1, wherein the sample fingerprint comprises 8 ginsenoside peaks, wherein the No. 5 peak is a ginsenoside Rb1 control peak, and the relative retention time of each characteristic peak is as follows: peak 1, peak 0.42 + -10%, peak 2, peak 0.44 + -10%, peak 3, peak 0.86 + -10%, peak 4, peak 0.90 + -10%, peak 5, peak 1.00, peak 6, peak 1.06 + -10%, peak 7, peak 1.13 + -10%, peak 8, and peak 1.25 + -10%.
8. The method for evaluating the quality of the ginseng medicinal material according to claim 1, wherein the theoretical plate number of the ginsenoside Rg1 in the obtained fingerprint is not less than 6000.
9. The method for evaluating the fingerprint of the quality of the ginseng medicinal material according to claims 1-8, wherein the fingerprint of the test sample comprises 8 ginsenoside peaks: the peak 1 is ginsenoside Rg1, the peak 2 is ginsenoside Re, the peak 3 is ginsenoside Rf, the peak 5 is ginsenoside Rb1, the peak 6 is ginsenoside Rb2+ ginsenoside Rc, and the peak 8 is ginsenoside Rd.
10. The method for evaluating the fingerprint of the quality of the ginseng medicinal material according to claims 1 to 8, wherein the test sample is preferably ginseng powder.
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