CN109991330A - A kind of detection method of the finger-print of ginseng under forest - Google Patents
A kind of detection method of the finger-print of ginseng under forest Download PDFInfo
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- CN109991330A CN109991330A CN201910272398.7A CN201910272398A CN109991330A CN 109991330 A CN109991330 A CN 109991330A CN 201910272398 A CN201910272398 A CN 201910272398A CN 109991330 A CN109991330 A CN 109991330A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
Abstract
The present invention provides a kind of detection method of the finger-print of ginseng under forest, by the test solution containing ginseng under forest and containing the reference product solution with reference to product, it is measured using high performance liquid chromatography, by the test solution of acquisition and referring to the fingerprint chromatogram of product solution, it is compared with the standard fingerprint spectrogram of ginseng under forest, according to relative retention time, common characteristic peaks are identified, to obtain the finger-print of ginseng under forest in test solution.The present invention further provides application of the detection method in the quality testing of ginseng under forest and component content measurement.A kind of detection method of the finger-print of ginseng under forest provided by the invention, the finger-print of ginseng under forest is established, and can be identified with garden ginsent, quantitative analysis is carried out to Multiple components in ginseng under forest, the status of reflection ginseng under forest comprehensively, the Quality Control for improving ginseng under forest are horizontal.
Description
Technical field
The invention belongs to traditional Chinese medicine ingredients technical field of analysis and detection, are related to a kind of detection side of the finger-print of ginseng under forest
Method.
Background technique
Ginseng under forest is the dry root and rhizome of Araliaceae ginseng (Panax ginseng C.A.Mey.), is simply
Common rare Chinese medicine, Shennong's Herbal are classified as top grade, it is believed that it, which has, reinforces vital energy, veins takes off admittedly, reinforces the spleen to benefit the lung, pacify
The effect of refreshing intelligence development.Modern clinic pharmacological research shows that the main pharmacodynamics ingredient of ginseng is ginsenoside, with many aspects
Drug effect, as Chen Jie et al. thinks that ginseng is able to ascend the immunity (Shanxi progress [J] of ginsenoside immunoregulation effect
College of traditional Chinese medicine's journal, 2014 (05): 75-77), Jiang Weiwei et al. thinks that ginseng being capable of antitumor (ginsenoside Rb1, Rg3, Rh2
Antitumor action and mechanism overview [J] World Science technology-TCM Modernization, 2013 (07): 1634-1637), Cai Xiaoyue
Et al. think that ginseng can improve that cardiovascular (pharmaceutical research [J] Changchun University of Traditional Chinese Medicine that ginseng treats cardiovascular disease is learned
Report, 2012 (01): 158-159), Wu Xiaomin et al. thinks that ginseng can anti-oxidant (pharmacological action of panaxan be ground with clinic
Study carefully the research of progress [J] ginseng, 2016 (05): 40-46), Li Chengpeng et al. thinks that ginseng being capable of anti-aging (Ginsenoside Rg 1
Delay brain aging Mechanism Study [J] CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2014 (22): 4442-4447), Zheng Yousheng et al. thinks that ginseng can
Anti-senile dementia (experimental study [J] world's combination of Chinese tradiational and Western medicine magazine of ginsenoside Rg1's promotion nerve stem cell proliferation, 2011
(12): 1021-1024 it), opens coloured silk et al. and thinks that ginseng being capable of hypoglycemic (Chemical Constituents of Panax Ginseng and Advance on Pharmacological Activities [J]
Food and drug, 2016 (04): 300-304), Peng Xue et al. thinks that ginseng being capable of antibacterial anti-inflammatory (ginseng essential oil research [J]
Jilin traditional Chinese medicine, 2017 (01): 71-74).
In recent years, with ginseng under forest food, pharmaceutical preparations apply gradually extensively, quality become further people pass
The focus of note, due to ginseng under forest scarcity of resources, the expensive and market demand is big, palms off ginseng under forest and to mix pseudo- phenomenon continuous
Increase, formulates the accurate quality control method of ginseng under forest science, improve ginseng under forest quality standard, it appears particularly important.In " China
Pharmacopeia " 2015 years versions (one), limited with the minimum content of ginsenoside Rg1 and Rb1 and Re to control its quality, but only with
These three ginsenosides are that index is difficult to comprehensively judge the quality of medicinal material.In addition, ginseng under forest is as herbaceos perennial,
Harvesting Years are an important factor for influencing its quality, even more to formulate the main foundation of its market price.Because either in clinic
In curative effect or in the cognition of most of consumer, ginseng age high ginseng under forest its drug effect is better than ginseng age low ginseng under forest.
Currently identify the micro-judgment for joining agriculture always with old medicine work mainly by character to the identification method in ginseng age, inevitably there is a degree of master
The property seen, with the diversification that commodity are processed, the limitation that character identifies is more obvious.Thus, Vehicles Collected from Market is to ginseng under forest visitor
Seeing accurate growth year identification and quality evaluating method has urgent need.
Traditional Chinese medicine fingerprint has the characteristics that whole, macroscopic view, fuzzy analysis, can by the description to Chinese medicine global feature,
Using appropriate Fuzzy Processing mode, achieve the purpose that global quality control, therefore becomes the effective means of traditional Chinese medicine quality control.Its
Middle chromatographic fingerprinting analysis can be such that the global feature of a variety of chemical constituents contained by Chinese medicine visualizes, to disclose out conventional inspection
Test indiscoverable quality problems.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of finger-prints of ginseng under forest
Detection method, the finger-print of ginseng under forest is established using the efficient liquid-phase chromatography method of optimal conditions, and at the same time once
Quantitative analysis is carried out to Multiple components in ginseng under forest, and similarity-rough set and orthogonal Partial Least Squares discriminant analysis can be passed through
(OPLS-DA), Liaoning Province Huan Renxian life in 10 years, 15-year-old and 20 years raw ginseng under forest growth years are identified, with garden ginsent
Sample is identified, and than the status for more comprehensively reflecting ginseng under forest, the Quality Control for improving ginseng under forest is horizontal.
In order to achieve the above objects and other related objects, first aspect present invention provides a kind of finger-print of ginseng under forest
Detection method, by the test solution containing ginseng under forest and containing with reference to product reference product solution, using high-efficient liquid phase color
Spectrometry is measured, and by the test solution of acquisition and referring to the fingerprint chromatogram of product solution, is composed with the standard fingerprint of ginseng under forest
Figure is compared, and according to relative retention time, identifies common characteristic peaks, to obtain the finger of ginseng under forest in test solution
Line map.
Preferably, the test solution is that ginseng under forest is added to ultrasonic extraction after methanol solution, after filtering for the first time
Take subsequent filtrate, rotary evaporated to dryness, be added after methanol solution dissolution constant volume carry out second of filtering to get.
It is obtained it is highly preferred that the ginseng under forest is ground at powdered rear sieving.
It is further preferred that the screen-aperture of the sieving is 75-85 mesh.Most preferably, the screen-aperture of the sieving
For 80 mesh.
It is highly preferred that the ratio between the volume mL that weight g and the methanol solution that the ginseng under forest is added are added is 2:15-
25.It is further preferred that the ratio between the volume mL that weight g and the methanol solution that the ginseng under forest is added are added is 2:20.
It is highly preferred that needing precise weighing after methanol solution is added in the ginseng under forest.
It is highly preferred that the temperature of the ultrasonic extraction is 45-55 DEG C.Most preferably, the temperature of the ultrasonic extraction is 50
℃。
It is highly preferred that the ultrasonic extraction time is 90-110min.Most preferably, the ultrasonic extraction time is
100min。
It is highly preferred that needing to be re-weighed after the ultrasonic extraction, and weightlessness is supplied with methanol solution.
The subsequent filtrate refers to: when filtering, the solution after primary filtrate is subsequent filtrate.
It is highly preferred that the sampling amount of the subsequent filtrate is 8.0-12.0mL.It is further preferred that the sampling of the subsequent filtrate
Amount is 10.0mL.
The rotary evaporated to dryness refers to subsequent filtrate rotary evaporated to dryness using Rotary Evaporators.
It is highly preferred that the subsequent filtrate before rotary evaporation is 8- with the ratio between volume that methanol solution is added after rotary evaporation
12:2.It is further preferred that the subsequent filtrate before rotary evaporation is with the ratio between the volume that methanol solution is added after rotary evaporation
10:2。
It is highly preferred that the first time is filtered into filter paper filtering.The filter paper is qualitative filter paper (middling speed).
It is highly preferred that described be filtered into membrane filtration for the second time.The filter membrane is 0.22 μm of filter membrane.
It is highly preferred that the methanol solution is the methanol aqueous solution of percent by volume 65-75%.It is highly preferred that the first
Alcoholic solution is the methanol aqueous solution of percent by volume 70%.
Preferably, it is described referring to product solution be will referring to product be added methanol after dissolution constant volume be made.
It referring to product include ginsenoside Rg1's (No. CAS be 22427-39-0), ginsenoside Re it is highly preferred that described
(No. CAS is 51542-56-4), ginsenoside Rb1's (No. CAS is 41753-43-9), (No. CAS is 11021- to Ginsenoside Rc
14-0), ginsenoside Rd (No. CAS is 52705-93-8).
It is highly preferred that described referring to ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginsenoside in product solution
Rc, ginsenoside Rd concentration be 0.5-2.0mg/mL.It is further preferred that it is described referring to ginsenoside Rg1 in product solution,
Ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd concentration be 1.0mg/mL.
Preferably, the chromatographic condition of the high performance liquid chromatography are as follows: chromatographic column: C18 column；Column temperature: 20-40 DEG C；Detection
Wavelength: 200-205nm；Flow velocity: 0.5-2.0ml/min；Sample volume: 1-10 μ L；Mobile phase: 0.05-0.15v/v% formic acid is water-soluble
Liquid -0.05-0.15v/v% formic acid acetonitrile solution, wherein A phase is 0.05-0.15v/v% aqueous formic acid, and B phase is 0.05-
0.15v/v% formic acid acetonitrile solution；Analysis time: 60min；Gradient elution.
It is highly preferred that the chromatographic condition of the high performance liquid chromatography are as follows: chromatographic column: Waters CORTECS C18 column
(150mm × 4.6mm, 2.7 μm)；Column temperature: 30 DEG C；Detection wavelength: 203nm；Flow velocity: 1.0ml/min；Sample volume: 5 μ L；Flowing
Phase: 0.1v/v% aqueous formic acid -0.1v/v% formic acid acetonitrile solution, wherein A phase is 0.1v/v% aqueous formic acid, and B phase is
0.1v/v% formic acid acetonitrile solution；Analysis time: 60min；Gradient elution.
It is further preferred that the specific procedure of the gradient elution are as follows:
0-20min, A phase: B phase volume ratio is 85:15-75:25；
20-37min, A phase: B phase volume ratio is 75:25-60:40；
37-45min, A phase: B phase volume ratio is 60:40-40:60；
45-49min, A phase: B phase volume ratio is 40:60-21:79；
49-58min, A phase: B phase volume ratio is 21:79-5:95；
58-60min, A phase: B phase volume ratio is 5:95-85:15.
The standard fingerprint spectrogram of the ginseng under forest refers to according to State Food and Drug Administration on August 15th, 2000
The regulation in " technical requirements (provisional) of traditional Chinese medicine finger-print research " promulgated, takes same at least ten batch
Ginseng under forest, prepares test solution and referring to product solution respectively, then after being measured by above-mentioned HPLC condition, obtains respectively
Finger-print, then finger-print is imported into " chromatographic fingerprints of Chinese materia medica similarity evaluation software 2012 editions ", control map is generated,
The final standard fingerprint spectrogram for determining ginseng under forest.When actually detected, test sample and standard finger-print carry out similarity
Compare, it is as qualified when similarity >=0.9, illustrate the full spectrogram of fingerprint for obtaining ginseng under forest in test sample.
Preferably, the test solution and referring to product solution fingerprint chromatogram and ginseng under forest standard fingerprint spectrogram into
When capable comparison, carried out using " the chromatographic fingerprints of Chinese materia medica similarity evaluation software 2012 editions " of Chinese Pharmacopoeia Commission's publication
Similarity-rough set is referred to as reference peak according to relative retention time using the reference product chromatographic peak in the fingerprint chromatogram referring to product solution
Recognize the individual features peak in the finger-print of test solution, thus to the index components in test solution finger-print into
Row ownership positioning, to identify the common characteristic peaks in the fingerprint chromatogram of test solution.
Second aspect of the present invention provides a kind of detection method of the finger-print of ginseng under forest ingredient in ginseng under forest
Purposes in quality testing.
Third aspect present invention provides a kind of quality determining method of ginseng under forest, including the finger using aforementioned ginseng under forest
The detection method of line map obtains ginseng under forest finger-print, and obtained ginseng under forest finger-print and identical fingerprints map are examined
The standard finger-print of the ginseng under forest obtained under the conditions of survey carries out similarity-rough set.
Preferably, when by ginseng under forest finger-print compared with the standard finger-print of ginseng under forest carrying out similarity,
It is compared using " similarity evaluation " 2012 editions softwares that Chinese Pharmacopoeia Commission issues.More
Preferably, similarity >=0.90 of the standard finger-print of the finger-print and ginseng under forest for the ginseng under forest that the present invention measures.
Preferably, the mark of ginseng under forest is obtained using condition identical with the detection method of aforementioned ginseng under forest finger-print
Quasi- finger-print, the standard finger-print of the ginseng under forest include 33 shared fingerprint peaks, with No. 2 peaks be positioning peak (peak S,
Relative retention time is that 1.000), the relative retention time of other 32 shared fingerprint peaks is followed successively by No. 1 peak (0.9793-
0.9803), No. 3 peaks (1.0949-1.1049), No. 4 peaks (1.1408-1.1433), No. 5 peaks (1.1870-1.2747), No. 6 peaks
(1.2704-1.2824), No. 7 peaks (1.5468-1.5613), No. 8 peaks (1.5962-1.6072), No. 9 peak (1.6678-
1.6879), No. 10 peaks (1.7159-1.7742), No. 11 peaks (1.8003-1.8793), No. 12 peaks (1.8369-1.8513), 13
Number peak (1.8524-1.8680), No. 14 peaks (1.8579-1.8852), No. 15 peaks (1.8708-1.9028), No. 17 peaks
(1.9075-1.9584), No. 18 peaks (1.9424-1.9729), No. 19 peaks (1.9575-1.9801), No. 20 peak (2.0217-
2.0361), No. 21 peaks (2.0600-2.0752), No. 22 peaks (2.0798-2.0968), No. 23 peaks (2.1182-2.1326), 24
Number peak (2.7044-2.7358), No. 25 peaks (2.8304-2.8658), No. 26 peaks (2.8561-2.8918), No. 27 peaks
(2.9297-2.9679), No. 28 peaks (2.9865-3.0260), No. 29 peaks (3.1100-3.1501), No. 30 peak (3.1208-
3.1620), No. 31 peaks (3.1371-3.1778), No. 32 peaks (3.2340-3.2767), No. 33 peaks (3.2628-3.3052).
Preferably, the finger-print of the ginseng under forest is compared with the finger-print referring to product solution, and positioning determines
No. 1 peak is the fingerprint peaks of ginsenoside Rg1, and No. 2 peaks are the fingerprint peaks of ginsenoside Re, and No. 11 peaks are ginseng
The fingerprint peaks of saponin(e Rb1, No. 13 peaks are the fingerprint peaks of Ginsenoside Rc, and No. 20 peaks are the fingerprint of ginsenoside Rd
Peak.
Fourth aspect present invention provides a kind of detection method of the finger-print of ginseng under forest ginseng soap in ginseng under forest
Application in the measurement of glycosides component content.
Fifth aspect present invention provides a kind of measuring method of ginsenoside component content in ginseng under forest, including following step
It is rapid:
A it the) preparation of test solution: prepares and supplies by the same steps of the detection method of the finger-print of above-mentioned ginseng under forest
Test sample solution；
B) the preparation of reference substance solution: by the reference substance of one or more ginsenoside ingredients, being added methanol and dissolve constant volume,
Up to reference substance solution；
C it) measures: using identical high performance liquid chromatography (HPLC) in the detection method of the finger-print of above-mentioned ginseng under forest
Condition, respectively determination step A) in test solution and step B) in reference substance solution, and using external standard method calculate for examination
The content of ginsenoside ingredient in product solution.
Preferably, step B) in, the reference substance solution be selected from ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1,
One of reference substance solution of Ginsenoside Rc, ginsenoside Rd is a variety of.
It is highly preferred that the concentration of ginsenoside Rg1 is 0.59~3.90mg/mL, ginsenoside in the reference substance solution
The concentration of Re is 0.58~3.89mg/mL, and the concentration of ginsenoside Rb1 is 0.61~4.06mg/mL, the concentration of Ginsenoside Rc
For 0.58~3.86mg/mL, the concentration of ginsenoside Rd is 0.40~2.67mg/mL.
The external standard method refers to: pipetting the step B of certain volume respectively) reference substance solution, it is made into certain density
Reference substance standard solution, using high performance liquid chromatograph, sample introduction is analyzed, obtains ginsenoside ingredient in reference substance standard solution and contains
The linear relationship of amount and peak area corresponds to its corresponding content with each ginsenoside ingredient chromatographic peak area, draws corresponding
Standard working curve, the regression equation of each standard working curve is calculated.Step A) the test solution is used again
High performance liquid chromatograph detection, by the chromatographic peak area of ginsenoside ingredient in the test solution of acquisition, respectively described in substitution
In the regression equation of each standard working curve, the content of corresponding ginseng saponin constituent in ginseng under forest can be obtained.
Heretofore described ginseng under forest is the Ginseng under Forest that wild environment is grown naturally, and the garden ginsent is the flat of artificial growth
Ground plants ginseng.
As described above, a kind of detection method of the finger-print of ginseng under forest provided by the invention, using optimal conditions
Efficient liquid-phase chromatography method establishes the finger-print of ginseng under forest, carries out quantitative analysis to Multiple components in ginseng under forest, and can
By similarity-rough set and orthogonal Partial Least Squares discriminant analysis (OPLS-DA), than more comprehensively reflecting showing for ginseng under forest
Shape is identified with garden ginsent, and the Quality Control for improving ginseng under forest is horizontal, to scientific and effective evaluation ginseng under forest quality, improves market
Environment, it is scientific and effective to distinguish different ginseng age ginseng under forest and have the certain significance.The finger-print for the ginseng under forest that the present invention establishes
Detection method, precision, stability and repeatability, sample recovery rate are good, it is determined that 33 shared peaks, methodological study are each
The relative retention time at shared peak and RSD≤3% of relative peak area, the similarity evaluation result of multiple batches of ginseng under forest sample
It is all larger than 0.90.
Detailed description of the invention
Fig. 1 is shown as the finger-print of 26 batches of ginseng under forest samples in the present invention.
Fig. 2 is shown as the finger-print of the ginseng under forest in the present invention.
Fig. 3 is shown as the cluster tree graph of 34 batches of samples in the present invention.
Fig. 4 is shown as the OPLS-DA shot chart of 34 batches of samples in the present invention.
Specific embodiment
The present invention is further explained combined with specific embodiments below, it should be appreciated that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
The reagent and instrument that following embodiment uses are as follows:
1, reagent
Acetonitrile, methanol (chromatographically pure, Merck company)；(analysis is pure, and Chinese medicines group chemical reagent Limited Liability is public for formic acid
Department)；Water (pure water)；Ginsenoside Rg1's (HPLC chromatogram pure >=99.7%), ginsenoside Re (HPLC chromatogram is pure >=
99.2%), ginsenoside Rb1's (HPLC chromatogram pure >=99.9%), Ginsenoside Rc's (HPLC chromatogram pure >=99.6%), ginseng soap
Glycosides Rd (HPLC chromatogram pure >=99.6%), above-mentioned reference substance is all from Shanghai Yuan Ye Biotechnology Co., Ltd.
Ginseng under forest and garden ginsent Shanghai medicine-feeding mind as healthy pharmaceutcal corporation, Ltd provide, totally 26 parts of ginseng under forest medicinal material, garden
Totally 8 parts of sample of ginseng, concrete condition see the table below 1.
Table 1
2, instrument
Waters Acquity ARC high performance liquid chromatograph (Waters, US)；KQ5200DE type numerical control supersonic
Washer (Kunshan Ultrasonic Instruments Co., Ltd.)；EYELA N-1100 type Rotary Evaporators (the limited public affairs of Shanghai Ai Lang instrument
Department)；EYELA CCA-1112 type cooling water circulating device (Shanghai Ai Lang Instrument Ltd)；Sartorius BS 124S electricity
Sub- balance (Sai Duolisi scientific instrument (Beijing) Co., Ltd)；Ten a ten thousandth of Sartorius SQPQUINTIX65-1CN point
It analyses balance (Sai Duolisi scientific instrument (Beijing) Co., Ltd).
Embodiment 1
1, sample pre-treatments
The preparation of test solution: ginseng under forest was ground into powder 75-85 mesh, accurately weighed, and 65- is added
75% methanol aqueous solution, the ratio between the volume mL that the weight g and methanol solution that ginseng under forest is added are added is 2:15-25.After weighing,
Ultrasonic extraction 90-110min, is re-weighed at 45-55 DEG C, and supplies weightlessness with 65-75% methanol aqueous solution.Using qualitative filter
Paper carries out first time filtering, takes 8-12mL subsequent filtrate, and rotary evaporated to dryness is added 65-75% methanol aqueous solution and dissolves constant volume, rotation
It is 8-12:2 that the ratio between volume of methanol is added after the subsequent filtrate and rotary evaporation before turning evaporation.Again using 0.22 μm of filter membrane into
Second of filtering of row is to get test solution.
Referring to the preparation of product solution: dissolving constant volume after methanol being added referring to product, be made referring to product solution.Include referring to product
There are ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd.Referring to ginseng soap in product solution
Glycosides Rg1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd concentration be 0.5-2.0mg/mL.
2, chromatographic condition
The chromatographic condition of the high performance liquid chromatography are as follows: chromatographic column: C18 column；Column temperature: 20-40 DEG C；Detection wavelength:
200-205nm；Flow velocity: 0.5-2.0ml/min；Sample volume: 1-10 μ L；Mobile phase: 0.05-0.15v/v% aqueous formic acid-
0.05-0.15v/v% formic acid acetonitrile solution, wherein A phase is 0.05-0.15v/v% aqueous formic acid, and B phase is 0.05-
0.15v/v% formic acid acetonitrile solution；Analysis time: 60min；Gradient elution.
The specific procedure of the gradient elution are as follows:
0-20min, A phase: B phase volume ratio is 85:15-75:25；
20-37min, A phase: B phase volume ratio is 75:25-60:40；
37-45min, A phase: B phase volume ratio is 60:40-40:60；
45-49min, A phase: B phase volume ratio is 40:60-21:79；
49-58min, A phase: B phase volume ratio is 21:79-5:95；
58-60min, A phase: B phase volume ratio is 5:95-85:15.
3, it measures
Test solution and reference in above-mentioned 1 are measured using the high performance liquid chromatography of the chromatographic condition in above-mentioned 2 respectively
Product solution, the test solution of acquisition and the fingerprint chromatogram referring to product solution are compared with the standard fingerprint spectrogram of ginseng under forest
Compared with according to relative retention time, common characteristic peaks being identified, to obtain the finger-print of ginseng under forest in test solution.
Wherein, the standard fingerprint spectrogram progress of the test solution and fingerprint chromatogram and ginseng under forest referring to product solution
Comparison when, using Chinese Pharmacopoeia Commission publication " chromatographic fingerprints of Chinese materia medica similarity evaluation software 2012 editions " carry out phase
Compare like degree, is pointed out as reference peak according to relative retention time using the reference product chromatographic peak in the fingerprint chromatogram referring to product solution
Individual features peak in the finger-print of test solution out, to be carried out to the index components in test solution finger-print
Ownership positioning, to identify the common characteristic peaks in the fingerprint chromatogram of test solution.
Embodiment 2
1, sample pre-treatments
The preparation of test solution: crossing the powder 2.0g of 80 meshes after taking ginseng under forest to grind, accurately weighed, sets 25mL tool
It fills in conical flask, after adding 70% methanol aqueous solution 20.0mL to weigh, ultrasound 100min, is re-weighed at 50 DEG C, and with 70% first
Alcohol solution supplies weightlessness.First time filtering is carried out using qualitative filter paper, takes 10.0mL subsequent filtrate, rotary evaporated to dryness is used
70% methanol aqueous solution is transferred in 2mL volumetric flask after redissolving, and dissolves constant volume with 70% methanol aqueous solution, then using 0.22 μm of filter
Film carries out second of filtering to get test solution.
Referring to the preparation of product solution: being being set in 5mL volumetric flask referring to product for 5.0mg by various contents, after methanol is added
Constant volume is dissolved, is made referring to product solution.It include ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, ginseng soap referring to product
Glycosides Rc, ginsenoside Rd.Referring to ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc, people in product solution
The concentration for joining saponin(e Rd is 1.0mg/mL.
2, chromatographic condition
The chromatographic condition of the high performance liquid chromatography are as follows: chromatographic column: Waters CORTECS C18 column (150mm ×
4.6mm, 2.7 μm)；Column temperature: 30 DEG C；Detection wavelength: 203nm；Flow velocity: 1.0ml/min；Sample volume: 5 μ L；Mobile phase: 0.1v/
V% aqueous formic acid -0.1v/v% formic acid acetonitrile solution, wherein A phase is 0.1v/v% aqueous formic acid, and B phase is 0.1v/v%
Formic acid acetonitrile solution；Analysis time: 60min；Gradient elution.
The specific procedure of the gradient elution are as follows:
0-20min, A phase: B phase volume ratio is 85:15-75:25；
20-37min, A phase: B phase volume ratio is 75:25-60:40；
37-45min, A phase: B phase volume ratio is 60:40-40:60；
45-49min, A phase: B phase volume ratio is 40:60-21:79；
49-58min, A phase: B phase volume ratio is 21:79-5:95；
58-60min, A phase: B phase volume ratio is 5:95-85:15.
3, it measures
Test solution and reference in above-mentioned 1 are measured using the high performance liquid chromatography of the chromatographic condition in above-mentioned 2 respectively
Product solution, the test solution of acquisition and the fingerprint chromatogram referring to product solution are compared with the standard fingerprint spectrogram of ginseng under forest
Compared with, according to relative retention time, common characteristic peaks are identified, so that the finger-print of ginseng under forest in test solution is obtained,
Specific finger-print is shown in Fig. 2.
Wherein, the standard fingerprint spectrogram progress of the test solution and fingerprint chromatogram and ginseng under forest referring to product solution
Comparison when, using Chinese Pharmacopoeia Commission publication " chromatographic fingerprints of Chinese materia medica similarity evaluation software 2012 editions " carry out phase
Compare like degree, is pointed out as reference peak according to relative retention time using the reference product chromatographic peak in the fingerprint chromatogram referring to product solution
Individual features peak in the finger-print of test solution out, to be carried out to the index components in test solution finger-print
Ownership positioning, to identify the common characteristic peaks in the fingerprint chromatogram of test solution.
Embodiment 3
Using the ginseng under forest established in above-described embodiment 2 finger-print detection method to 26 batches of ginseng under forest samples
It is detected, ginseng under forest finger-print is obtained, by obtained ginseng under forest finger-print and identical fingerprints map testing conditions
The standard finger-print of the ginseng under forest of lower acquisition carries out similarity evaluation, and finger-print stacking chart is as shown in Figure 1.26 batches of hayashishitas
The similarity of the standard finger-print of the finger-print and ginseng under forest of wild ginseng sample is all larger than 0.90.
Meanwhile as shown in Fig. 2, ginseng under forest standard finger-print include 33 shared fingerprint peaks, with No. 2 peaks be positioning
Peak (peak S, relative retention time 1.000), the relative retention time of other 32 shared fingerprint peaks are followed successively by No. 1 peak
(0.9793-0.9803), No. 3 peaks (1.0949-1.1049), No. 4 peaks (1.1408-1.1433), No. 5 peak (1.1870-
1.2747), No. 6 peaks (1.2704-1.2824), No. 7 peaks (1.5468-1.5613), No. 8 peaks (1.5962-1.6072), No. 9 peaks
(1.6678-1.6879), No. 10 peaks (1.7159-1.7742), No. 11 peaks (1.8003-1.8793), No. 12 peak (1.8369-
1.8513), No. 13 peaks (1.8524-1.8680), No. 14 peaks (1.8579-1.8852), No. 15 peaks (1.8708-1.9028), 17
Number peak (1.9075-1.9584), No. 18 peaks (1.9424-1.9729), No. 19 peaks (1.9575-1.9801), No. 20 peaks
(2.0217-2.0361), No. 21 peaks (2.0600-2.0752), No. 22 peaks (2.0798-2.0968), No. 23 peak (2.1182-
2.1326), No. 24 peaks (2.7044-2.7358), No. 25 peaks (2.8304-2.8658), No. 26 peaks (2.8561-2.8918), 27
Number peak (2.9297-2.9679), No. 28 peaks (2.9865-3.0260), No. 29 peaks (3.1100-3.1501), No. 30 peaks
(3.1208-3.1620), No. 31 peaks (3.1371-3.1778), No. 32 peaks (3.2340-3.2767), No. 33 peak (3.2628-
3.3052)。
Wherein, No. 1 peak is the fingerprint peaks of ginsenoside Rg1, and No. 2 peaks are the fingerprint peaks of ginsenoside Re, institute
The fingerprint peaks that No. 11 peaks are ginsenoside Rb1 are stated, No. 13 peaks are the fingerprint peaks of Ginsenoside Rc, and No. 20 peaks are ginseng
The fingerprint peaks of saponin(e Rd.
Embodiment 4
1 part of ginseng under forest sample of number A10 is taken, using the finger-print for the ginseng under forest established in above-described embodiment 2
Detection method, continuous sample introduction 5 times, investigate finger-print in each main chromatographic peak retention time and its consistency of peak area,
Concrete outcome is shown in Table 2,3, specified in share peak relative retention time RSD≤3.0%, relative peak area RSD≤
3.0%.The result shows that the precision of this method is good.
2 retention time of table
3 peak area of table
Embodiment 5
1 part of ginseng under forest sample of number A10 is taken, using the finger-print for the ginseng under forest established in above-described embodiment 2
Detection method, prepare and place 0h, 2h respectively after test solution, 4h, 10h, 12h, detected for 24 hours, is calculated each total
There is the RSD of peak relative retention time and relative peak area, concrete outcome is shown in Table 4,5, the results showed that when each shared peak retains relatively
Between RSD≤3.0%, RSD%≤3.0% of relative peak area.Show that sample is good in interior detection stability for 24 hours in the present invention
It is good.
4 retention time of table
5 peak area of table
Embodiment 6
5 parts of ginseng under forest sample of number A10 are taken, using the finger-print for the ginseng under forest established in above-described embodiment 2
Detection method detected, calculate the relative retention time at each shared peak and the RSD of relative peak area, concrete outcome is shown in Table 6,
7, specified in share peak relative retention time RSD≤3.0%, RSD≤3.0% of relative peak area.Show the present invention
The repeatability of middle method is good, and accuracy is higher.
6 retention time of table
7 peak area of table
Embodiment 7
By the same steps preparation in the detection method of the finger-print for the ginseng under forest established in above-described embodiment 2 for examination
Product solution.The reference substance of 5 kinds of ginsenoside ingredients is taken, methanol is added and dissolves constant volume, obtains reference substance solution.5 kinds of ginsenosides
Ingredient is respectively ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd.Reference substance solution
In, the concentration of ginsenoside Rg1 is 0.59~3.90mg/mL, and the concentration of ginsenoside Re is 0.58~3.89mg/mL, ginseng
The concentration of saponin(e Rb1 is 0.61~4.06mg/mL, and the concentration of Ginsenoside Rc is 0.58~3.86mg/mL, ginsenoside Rd's
Concentration is 0.40~2.67mg/mL.
By the identical high-efficient liquid phase color in the detection method of the finger-print for the ginseng under forest established in above-described embodiment 2
(HPLC) condition of composing measures test solution and control solution respectively, and calculates ginseng in test solution using external standard method
The content of saponin constituent.
Embodiment 8
Precision weighs that reference substance solution is appropriate, by being prepared into pair containing different quality concentration components in above-described embodiment 7
It is measured according to product solution, and by high performance liquid chromatography (HPLC) condition in above-described embodiment 7, calculates and obtain 5 kinds of ginseng soaps
Standard regressive method, related coefficient and the range of linearity of glycosides ingredient, concrete outcome are shown in Table 8.
Table 8
| Compound | Standard curve | r | The range of linearity (mg/mL) |
| Ginsenoside Rg1 | Y=0.8331x-245500 | 0.9991 | 0.59~3.90 |
| Ginsenoside Re | Y=0.8678x-215454 | 0.9994 | 0.58~3.89 |
| Ginsenoside Rb1 | Y=0.9941x-161639 | 0.9995 | 0.61~4.06 |
| Ginsenoside Rc | Y=0.8835x-153894 | 0.9998 | 0.58~3.86 |
| Ginsenoside Rd | Y=0.83x-103987.71 | 1.0000 | 0.40~2.67 |
According to table 8 it is found that standard regressive method is using chromatographic peak area as ordinate (Y), compound quality is abscissa
(X), meanwhile, 5 kinds of ingredients are in good linear relationship in range of liner, and the related coefficient of standard regressive method is equal
Greater than 0.9990.
Meanwhile detection limit is determined with S/N=3, quantitative limit is determined with S/N=10, concrete outcome is shown in Table 9.
Table 9
| Ginsenoside Rg1 | Ginsenoside Re | Ginsenoside Rb1 | Ginsenoside Rc | Ginsenoside Rd | |
| Sampling volume (μ L) | 5 | 5 | 5 | 5 | 5 |
| Sample introduction concentration (mg/mL) | 0.116 | 0.116 | 0.116 | 0.116 | 0.08 |
| S/N | 10 | 10 | 10 | 10 | 10 |
| Sample introduction concentration (mg/mL) | 0.042 | 0.042 | 0.03 | 0.03 | 0.0336 |
| S/N | 3 | 3 | 3 | 3 | 3 |
| Quantitative limit (μ g) | 580 | 580 | 625 | 625 | 400 |
| Detection limit (μ g) | 210 | 210 | 150 | 150 | 168 |
Embodiment 9
Precision weighs that reference substance solution is appropriate, 5 kinds of ginsenoside ingredients of certain content is accurately added respectively, by above-mentioned reality
High performance liquid chromatography (HPLC) condition applied in example 7 is measured, and the results are shown in Table 10-14.By table 10-14 it is found that 5 kinds of ginseng soaps
The sample recovery rate of the content assaying method of glycosides ingredient is between 90-110%, and RSD is respectively less than 3.0%, measurement result
The rate of recovery is good.
10 ginsenoside Rg1 of table
11 ginsenoside Re of table
12 ginsenoside Rb1 of table
13 Ginsenoside Rc of table
14 ginsenoside Rd of table
Embodiment 10
By the measuring method of ginsenoside component content in the ginseng under forest in above-described embodiment 7, to 26 batches in table 1
Ginseng under forest sample is detected, and assay the results are shown in Table 15.As shown in Table 15, the measuring method in the present invention can have
The content of ginsenoside ingredient in effect measurement ginseng under forest.
Table 15
Embodiment 11
By the detection method of the finger-print for the ginseng under forest established in above-described embodiment 2, to 34 lot sample as shown in Table 1
Product are detected, and finger-print is obtained.The peak area at 33 shared peaks in the finger-print of acquisition is imported into SIMCA-P
In 14.0 softwares, clustering is carried out, obtained cluster tree graph is as shown in Figure 3.It is two big groups that 34 batches of samples, which gather, as the result is shown, one
Group is garden ginsent, and one group is ginseng under forest.Show that there are quality differences between garden ginsent and ginseng under forest.Ginseng under forest is divided into three again
Group, one group is 10 years, and one group is 15 years, and one group is 20 years.Illustrate that quality has differences between the ginseng under forest in different ginseng ages.This
Method can identify Liaoning Province Huan Renxian life in 10 years, 15-year-old and 20 years raw ginseng under forest growth years in invention.
Embodiment 12
By the detection method of the finger-print for the ginseng under forest established in above-described embodiment 2, to 34 lot sample as shown in Table 1
Product are detected, and the chromatogram of ginseng under forest and garden ginsent is obtained.34 batches of sample chromatographic datas in the map of acquisition are imported
SIMCA-P14.0 software carries out orthogonal Partial Least Squares discriminant analysis (OPLS-DA) analysis processing, and OPLS-DA shot chart is such as
Shown in Fig. 4.As can be seen from Figure 4 sample is obviously divided into ginseng under forest and garden ginsent two major classes, and ginseng under forest is divided into again
Life in 10 years, 15-year-old and 20 years raw three classes, it is consistent with cluster analysis result.Method can be 10 years to Liaoning Province Huan Renxian in the present invention
Raw, 15-year-old and 20 years raw ginseng under forest growth years are identified.
In conclusion a kind of detection method of the finger-print of ginseng under forest provided by the invention, establishes ginseng under forest
Finger-print carries out quantitative analysis to Multiple components in ginseng under forest, reflects the status of ginseng under forest comprehensively, improve ginseng under forest
Quality Control it is horizontal.So the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (10)
1. a kind of detection method of the finger-print of ginseng under forest, by the test solution containing ginseng under forest and containing with reference to product
Reference product solution, be measured using high performance liquid chromatography, by the test solution of acquisition and referring to the fingerprint of product solution
Spectrogram is compared with the standard fingerprint spectrogram of ginseng under forest, according to relative retention time, identifies common characteristic peaks, thus
Obtain the finger-print of ginseng under forest in test solution.
2. a kind of detection method of the finger-print of ginseng under forest according to claim 1, which is characterized in that described for examination
Product solution is that ginseng under forest is added to ultrasonic extraction after methanol solution, takes subsequent filtrate after filtering for the first time, rotary evaporated to dryness adds
Enter to carry out after methanol solution dissolution constant volume second filtering to get.
3. a kind of detection method of the finger-print of ginseng under forest according to claim 2, which is characterized in that described for examination
Product solution includes any of the following conditions or multinomial:
A1 the ratio between the volume mL that the weight g and the methanol solution that) ginseng under forest is added are added is 2:15-25；
A2) temperature of the ultrasonic extraction is 45-55 DEG C；
A3) the ultrasonic extraction time is 90-110min；
A4 the ratio between volume of methanol solution) is added as 8-12:2 after the subsequent filtrate before rotary evaporation and rotary evaporation；
A5) first time is filtered into filter paper filtering；
A6) described to be filtered into membrane filtration for the second time；
A7) methanol solution is the methanol aqueous solution of percent by volume 65-75%.
4. a kind of detection method of the finger-print of ginseng under forest according to claim 1, which is characterized in that the reference
Product solution is that dissolution constant volume be made after methanol will be added referring to product, and described referring to product includes ginsenoside Rg1, ginsenoside
Re, ginsenoside Rb1, Ginsenoside Rc, ginsenoside Rd.
5. a kind of detection method of the finger-print of ginseng under forest according to claim 1, which is characterized in that described efficient
The chromatographic condition of liquid chromatography are as follows: chromatographic column: C18 column；Column temperature: 20-40 DEG C；Detection wavelength: 200-205nm；Flow velocity: 0.5-
2.0ml/min；Sample volume: 1-10 μ L；Mobile phase: 0.05-0.15v/v% aqueous formic acid -0.05-0.15v/v% formic acid second
Nitrile solution, wherein A phase is 0.05-0.15v/v% aqueous formic acid, and B phase is 0.05-0.15v/v% formic acid acetonitrile solution；Point
Analyse the time: 60min；Gradient elution.
6. a kind of detection method of the finger-print of ginseng under forest according to claim 5, which is characterized in that the gradient
The specific procedure of elution are as follows: 0-20min, A phase: B phase volume ratio is 85:15-75:25；20-37min, A phase: B phase volume ratio is
75:25-60:40；37-45min, A phase: B phase volume ratio is 60:40-40:60；45-49min, A phase: B phase volume ratio is 40:
60-21:79；49-58min, A phase: B phase volume ratio is 21:79-5:95；58-60min, A phase: B phase volume ratio is 5:95-85:
15。
7. according to claim 1 a kind of detection method of the finger-print of -6 any ginseng under forest in ginseng under forest at
The purposes in quality testing divided.
8. a kind of quality determining method of ginseng under forest, including the fingerprint using any ginseng under forest of claim 1-6
The detection method of map obtains ginseng under forest finger-print, and obtained ginseng under forest finger-print and identical fingerprints map are detected
Under the conditions of the standard finger-print of ginseng under forest that obtains carry out similarity-rough set.
9. the detection method of the finger-print of -6 any a kind of ginseng under forest people in ginseng under forest according to claim 1
Join the purposes in saponin constituent assay.
10. the measuring method of ginsenoside component content in a kind of ginseng under forest, comprising the following steps:
1) preparation of test solution: using the detection method of the finger-print of any ginseng under forest of claim 1-6
Same steps prepare test solution；
2) preparation of reference substance solution: by the reference substance of one or more ginsenoside ingredients, be added methanol dissolution constant volume to get
Reference substance solution；
3) it measures: identical efficient in the detection method using the finger-print of any ginseng under forest of claim 1-6
Liquid phase chromatogram condition, respectively determination step 1) in test solution and step 2) in reference substance solution, and use external standard method
Calculate the content of ginsenoside ingredient in test solution.
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