CN112946145B - Content determination method of dendrobium luminous pills - Google Patents
Content determination method of dendrobium luminous pills Download PDFInfo
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- CN112946145B CN112946145B CN202110331995.XA CN202110331995A CN112946145B CN 112946145 B CN112946145 B CN 112946145B CN 202110331995 A CN202110331995 A CN 202110331995A CN 112946145 B CN112946145 B CN 112946145B
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Abstract
The invention discloses a content determination method of dendrobium noctilucent pills, which uses naringin, neohesperidin and ginsenoside Rg 1 Ginsenoside Re and ginsenoside Rb 1 Determining Ginseng radix (ginsenoside Rg) in herba Dendrobii luminous pill by high performance liquid chromatography as reference 1 Ginsenoside Re and ginsenoside Rb 1 ) And fructus Aurantii (neohesperidin and naringin) parched with bran. The quality conditions of ginseng and bran-fried bitter orange in the dendrobium luminous pills are inspected by adopting the content determination method, and whether the enterprise has the actions of no feeding or less feeding and feeding of fake medicinal materials can be quickly and accurately checked.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine quality detection, in particular to a content determination method of dendrobium luminous pills.
Background
Herba Dendrobii herba luminous pill comprises herba Dendrobii, ginseng radix, rhizoma Dioscoreae, poria, glycyrrhrizae radix, cistanchis herba, fructus Lycii, semen Cuscutae, rehmanniae radix, radix rehmanniae Preparata, fructus Schisandrae chinensis, radix asparagi, radix Ophiopogonis, semen Armeniacae amarum, radix Saposhnikoviae, rhizoma Ligustici Chuanxiong, bran-parched fructus Aurantii, coptidis rhizoma, achyranthis radix, flos Chrysanthemi, fructus Tribuli, semen Celosiae, semen Cassiae, cornu Bubali concentrated powder, and cornu Naemorhedi. Grinding goat horn into fine powder, and grinding the rest twenty-three medicinal materials such as dendrobium into fine powder; grinding pulvis Cornus Bubali Concentratus and the above powders, sieving, and mixing. Adding an appropriate amount of water into 35-50 g of refined honey for every 100g of powder, and preparing into pills, drying and preparing into water-honeyed pills; or adding 95-120 g of refined honey to prepare small honeyed pills or big honeyed pills. The herba Dendrobii luminous pill has effects of nourishing yin, invigorating kidney, removing liver fire and improving eyesight; the traditional Chinese medicine composition is clinically used for treating liver and kidney deficiency, yin deficiency and fire hyperactivity, cataract and dim eyesight. The index controlled by the current standard [ content determination ] of the dendrobium noctilucent pill is berberine hydrochloride in coptis, so that the quality of the dendrobium noctilucent pill can not be effectively and integrally evaluated.
Gu Yuanyin et al, in which 7 Dendrobium noctilucent pills from Beijing, jinan, guangzhou, etc. were used as samples, and the content of Panaxadiol and Panaxatriol in the samples was determined by thin-layer scanning, which established a determination method and provided a reference for establishing the quality control standard of Dendrobium noctilucent pills (Gu Yuanyin, zhao Bo, liu Qing, liu Yongjun. Content determination of Panaxadiol and Panaxatriol in Dendrobium noctilucent pills, chinese journal of Chinese medicine, 1995,20 (12): 732-735.). However, the thin-layer scanning method is easily affected by factors such as temperature, thin-layer plates and sample application amount, the accuracy of a content measurement result is unstable, the main effective component of ginseng is ginsenoside, hydrolysis products of the ginsenoside, namely panaxadiol and panaxatriol, are used as indexes for content measurement in the article, and various methods are tested to show that the content of the main saponin in a sample cannot be directly measured due to serious component interference, so that the method cannot accurately reflect the real quality of the ginseng in the dendrobium luminous pill, and still needs to be deeply researched to find a method suitable for establishing a content detection standard of the dendrobium luminous pill.
Disclosure of Invention
The invention provides a content determination method of dendrobium luminous pills, which establishes naringin, neohesperidin and ginsenoside Rg by high performance liquid chromatography 1 Ginsenoside Re and ginsenoside Rb 1 The content determination method is used for inspecting the quality conditions of the ginseng and the bran-fried bitter orange in the dendrobium noctilucent pills, and can quickly and accurately check whether the enterprises have the behaviors of no feeding or less feeding and feeding of fake and inferior medicinal materials.
A content determination method of herba Dendrobii luminous pill comprises the following steps:
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a packed column; acetonitrile is taken as a mobile phase A, water is taken as a mobile phase B, and gradient elution is carried out according to the following table 1; the flow rate is 1.0mL/min; detecting with high temperature type evaporative light scattering detector, and counting theoretical plates according to ginsenoside Rg 1 The peak count should not be below 3000.
(2) Preparation of mixed control solution: precisely weighing naringin, neohesperidin, and ginsenoside Rg 1 Ginsenoside Re and ginsenoside Rb 1 Adding 70% methanol to 1mL of the mixture containing naringin and neohesperidin0.5mg each, and ginsenoside Rg 1 Mixing with ginsenoside Re 0.1mg and ginsenoside Rb 1 0.2mg of the control solution was mixed.
(3) Preparation of a test solution: taking herba dendrobii noctilucent pill water-honeyed pill, grinding, and taking 6g; or cutting herba Dendrobii luminous pill into small honeyed pill or big honeyed pill, and collecting 9g; precisely weighing, placing in a conical flask with a plug, adding 100mL of 70% methanol solution, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss with 70% methanol solution, shaking up, filtering, precisely absorbing 50mL of secondary filtrate, evaporating to dryness, adding 20mL of water into residue to dissolve, shaking up and extracting with water saturated n-butanol for 4 times, each time extracting 25mL, taking n-butanol solution, evaporating to dryness, adding methanol into residue to dissolve and transferring to a 5mL measuring flask, adding methanol to dilute to scale, shaking up, filtering, and taking secondary filtrate.
(4) And (3) determination: precisely sucking 5 μ L and 15 μ L of mixed reference solution and 10 μ L of test solution, respectively, injecting into liquid chromatograph, measuring, and calculating content by external standard two-point method logarithmic equation.
The herba Dendrobii luminous pill contains fructus Aurantii parched with bran and naringin (C) 27 H 32 O 14 ) And neohesperidin (C) 28 H 34 O 15 ) The water-honeyed pill contains naringin not less than 0.42mg per 1g, and neohesperidin not less than 0.31mg per 1 g; the small honeyed pill contains naringin not less than 0.28mg per 1g, and neohesperidin not less than 0.21mg per 1 g; the large honeyed pill should contain naringin not less than 1.57mg per pill, and neohesperidin not less than 1.18mg per pill; ginsenoside Rg containing ginseng 1 (C 42 H 72 O 14 ) Ginsenoside Re (C) 48 H 82 O 18 ) Ginsenoside Rb 1 (C 54 H 92 0 23 ) Measuring the content of ginsenoside Rg in the water-honeyed pill 1 And ginsenoside Re should not be less than 0.13mg per 1g, and contains ginsenoside Rb 1 Each 1g should not be less than 0.084mg; small honeyed pill containing ginsenoside Rg 1 And ginsenoside Re should not be less than 0.086 mg/1 g, and contains ginsenoside Rb 1 Each 1g should not be less than 0.057mg; big honeyed pill containing ginsenoside Rg 1 And soap of ginsengThe total amount of glycoside Re should not be less than 0.47mg, and ginsenoside Rb is contained 1 The dosage should not be less than 0.31mg.
The invention has the beneficial effects that:
the invention establishes naringin, neohesperidin and ginsenoside Rg by high performance liquid chromatography 1 Ginsenoside Re and ginsenoside Rb 1 The content determination method is used for inspecting the quality conditions of the ginseng and the bran-fried bitter orange in the dendrobium noctilucent pills, and can quickly and accurately check whether the enterprises have the behaviors of no feeding or less feeding and feeding of fake and inferior medicinal materials. In the content determination method, a high-temperature evaporation light scattering detector is adopted for detection, the interference peak is small, and the ginsenoside Rg is detected 1 Ginsenoside Re and ginsenoside Rb 1 The response value is higher, and the separation degree is good. By adding the content measurement method disclosed by the invention as a supplement to the existing standard (content measurement) of the dendrobium luminous pill, the evaluation standard of the dendrobium luminous pill can be more comprehensive, the drug safety of the dendrobium luminous pill can be effectively ensured, the phenomenon of no feeding or adulteration of feeding is reduced, and a scientific basis is provided for the quality standard research and quality control of the dendrobium luminous pill.
Drawings
FIG. 1 is a high performance liquid chromatogram of a mixed control solution.
FIG. 2 is a high performance liquid chromatogram of a sample of herba Dendrobii luminous pill (B pharmaceutical factory, lot number 18013508).
FIG. 3 is a high performance liquid chromatogram of a sample of herba Dendrobii luminous pill (company I, lot number 191004).
FIG. 4 is a HPLC chromatogram of a sample of herba Dendrobii herba Euphorbiae Humifusae pill (N corporation, lot 191001).
FIG. 5 is a high performance liquid chromatogram of a negative sample of herba Dendrobii luminous pill lacking Ginseng radix.
FIG. 6 is a high performance liquid chromatogram of a negative sample of herba Dendrobii luminous pill fructus Aurantii stir-fried with bran.
Detailed Description
In order to describe the present invention in more detail, the present invention will be further described with reference to the following examples.
Examples
Instruments and reagents:
waters hplc, high temperature evaporative light scattering detector; model ML204 electronic analytical balance (mertler-toledo instruments shanghai ltd).
Naringin (supplied by China institute for testing food and drug, lot number: 110722-201815, content 91.7%)
Neohesperidin (supplied by China institute for testing food and drug) with content of 99.4%: in batch number 11857-201804
Ginsenoside Rg 1 ( The Chinese food and drug testing research institute provides the following tests for the test, batch number: 110703-201933, content 93.4% )
Ginsenoside Re (for assay provided by Chinese food and drug testing institute, batch number: 110754-202028, content 93.9%)
Ginsenoside Rb 1 ( The Chinese food and drug testing research institute provides the following tests for the test, batch number: 110704-202028, content 93.1% )
Acetonitrile is chromatographically pure, and other reagents are analytically pure.
The 91 batches of dendrobium luminous pills (74 batches of water-honeyed pills, 15 batches of large honeyed pills and 2 batches of small honeyed pills) are all samples tested in 2020 national evaluative test.
Method survey the dendrobium luminous pill (water honeyed pill) used is manufactured by R company, batch number: 1904030; herba Dendrobii luminous pill (big honeyed pill) is prepared by pharmaceutical factory B, lot number: 18013507.
a content determination method of herba Dendrobii luminous pill comprises the following steps:
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a packed column; acetonitrile is taken as a mobile phase A, water is taken as a mobile phase B, and gradient elution is carried out according to the following table 1; the flow rate is 1.0mL/min; detecting with high temperature type evaporative light scattering detector, and counting theoretical plates according to ginsenoside Rg 1 The peak calculation should be not less than 3000.
(2) Preparation of mixed control solution: precisely weighing naringin, neohesperidin, and ginsenoside Rg 1 Ginsenoside Re and ginsenoside Rb 1 Adding 70% methanol to 1mL of the mixture containing naringin, neohesperidin 0.5mg, and ginsenoside Rg 1 Mixing with ginsenoside Re 0.1mg and ginsenoside Rb 1 0.2mg of the control solution was mixed.
(3) Preparing a test solution: taking herba dendrobii noctilucent pill water-honeyed pill, and grinding to obtain about 6g; or cutting herba Dendrobii luminous pill into small honeyed pill or big honeyed pill, and collecting about 9g; precisely weighing, placing in a conical flask with a plug, adding 100mL of 70% methanol solution, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss with 70% methanol solution, shaking up, filtering, precisely absorbing 50mL of secondary filtrate, evaporating to dryness, adding 20mL of water into residue to dissolve, shaking up and extracting with water saturated n-butanol for 4 times, each time extracting 25mL, taking n-butanol solution, evaporating to dryness, adding methanol into residue to dissolve and transferring to a 5mL measuring flask, adding methanol to dilute to scale, shaking up, filtering, and taking secondary filtrate.
(4) And (3) determination: precisely sucking 5 μ L and 15 μ L of mixed reference solution and 10 μ L of test solution, respectively, injecting into liquid chromatograph, measuring, and calculating content by external standard two-point method logarithmic equation.
The applicant adopts the method of the invention to perform content determination on 91 batches of dendrobium noctilucent pills after the duplicate number of 18 production enterprises is removed, the determination result is shown in tables 2-4, the high performance liquid chromatogram of the mixed reference solution is shown in figure 1, and typical high performance liquid chromatograms of 3 production enterprise samples are shown in figures 2-4.
The detection result shows that the naringin content in 13 batches of samples does not meet the specification, and the reject ratio is 85.7%; the neohesperidin content in 15 batches of samples does not meet the specification, and the reject ratio is 83.5%; ginsenoside Rg in 1 batch of samples 1 The total content of the ginsenoside Re does not meet the specification, and the reject ratio is 98.9%;1 batch of samples ginsenoside Rb 1 The content does not meet the specification, and the reject ratio is 98.9 percent. Wherein naringin and neohesperidin in samples produced by 3 production enterprises of company I, company K and company H are not detected, which indicates that the 3 production enterprises have problems in feeding the fructus aurantii stir-fried with bran in the production of the dendrobium noctilucent pills; ginsenoside Rb of 8 production enterprises of D company, G company, H company, L company, N company, I company, J company, Q company 1 The relative content is higher, which indicates that the feeding of the ginseng has problems in the production of the dendrobium noctilucent pills.
Statistical analysis is carried out on the content measurement results of all production enterprises by using SPSS data analysis software, and the results show that the p values of the content measurement results of all the components in the pharmaceutical factory B are all larger than 0.05, which indicates that the content measurement results of all the batches have no significant difference, the enterprises have strict quality control on the raw medicinal materials, and the production process is stable. Herba Dendrobii luminous pill naringin, neohesperidin, and ginsenoside Rg of other production enterprises 1 Ginsenoside Re and ginsenoside Rb 1 The content determination results show that the p values are all less than 0.05, which indicates that 5 components of each production enterprise have significant differences, and the enterprises have poor quality control on the raw medicine and have unstable production process.
In addition, the applicant also conducts special and methodological investigation on the content determination method of the invention, and the specific steps are as follows:
1. specialization inspection
Preparing a certain amount of negative sample of absent ginseng according to the prescription proportion of herba Dendrobii luminous pill, grinding, taking about 6g, precisely weighing, placing in a conical bottle with a plug, adding into the conical bottleWeighing 100mL of 70% methanol solution, heating and refluxing for 1h, taking out, cooling, weighing again, supplementing the weight loss by using 70% methanol solution, shaking up, filtering, precisely absorbing 50mL of subsequent filtrate, evaporating to dryness, dissolving the residue in 20mL of water, shaking and extracting for 4 times by using water-saturated n-butyl alcohol, taking 25mL of n-butyl alcohol solution each time, evaporating to dryness, dissolving the residue in methanol, transferring to a 5mL measuring flask, adding methanol to dilute to a scale, shaking up, filtering, and taking subsequent filtrate to obtain the ginseng-deficient negative sample solution. Sucking 10 μ L of the ginseng-deficient negative sample solution, injecting into a liquid chromatograph, measuring, analyzing, and obtaining the result that the ginseng-deficient negative sample is in ginsenoside Rg 1 Ginsenoside Re and ginsenoside Rb 1 The corresponding positions of the chromatographic peaks of the 3 reference substance components have no corresponding chromatographic peak, which indicates that the product is negative and has no interference, and the method of the invention has good specificity and is shown in figure 5.
Weighing a certain amount of bran-fried fructus aurantii negative sample according to the formula proportion of the dendrobium noctilucent pill, grinding, taking about 6g, precisely weighing, placing in a conical flask with a plug, adding 100mL of 70% methanol solution, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss by 70% methanol solution, shaking up, filtering, precisely sucking 50mL of subsequent filtrate, evaporating to dryness, adding 20mL of water to dissolve residues, shaking up and extracting by using water-saturated n-butyl alcohol for 4 times, 25mL each time, taking n-butyl alcohol solution, evaporating to dryness, adding methanol to dissolve residues, transferring to a 5mL measuring flask, adding methanol to dilute to scale, shaking up, filtering, taking subsequent filtrate, and obtaining the bran-fried fructus aurantii negative sample solution. And (3) sucking 10 mu L of bran-deficient fried fructus aurantii negative sample solution, injecting the solution into a liquid chromatograph, measuring and analyzing, wherein the result shows that the bran-deficient fried fructus aurantii negative sample has no corresponding chromatographic peak at the corresponding position of the chromatographic peaks of the ingredients of the 2 reference substances of naringin and neohesperidin, which indicates that the method is negative and free of interference, and the method has good specificity and is shown in figure 6.
2. Methodology survey
1. Investigation of Linear relationships
(1) Accurately weighing ginsenoside Rg 1 Adding 10.73mg of reference substance into 10mL measuring flask, diluting with methanol to scale, and shaking to obtain ginsenoside Rg 1 Stock solution (concentrated)The degree is 1.0022 mg/mL).
(2) Accurately weighing ginsenoside Re reference substance 9.82mg, placing in 10mL measuring flask, adding methanol to dilute to scale, and shaking to obtain ginsenoside Re stock solution (with concentration of 0.9221 mg/mL).
(3) Precisely weighing ginsenoside Rb 1 Adding 10.93mg of reference substance into 5mL measuring flask, diluting with methanol to scale, and shaking to obtain ginsenoside Rb 1 Stock solution (concentration 2.0352 mg/mL).
(4) Precisely weighing naringin reference substance 9.99mg and neohesperidin reference substance 11.53mg, placing into the same 20mL measuring flask, and precisely adding ginsenoside Rg 1 Ginsenoside Re and ginsenoside Rb 1 Diluting the stock solutions to scale with methanol 2.00mL each, and shaking to obtain reference mixed solution (naringin reference 0.4580mg/mL, neohesperidin reference 0.5730mg/mL, ginsenoside reference Rg 1 0.1002mg/mL, ginsenoside reference Re 0.09221 mg/mL, and ginsenoside reference Rb 1 0.2035mg/mL）。
(5) Precisely sucking 2 μ L, 5 μ L, 10 μ L, 15 μ L and 20 μ L of the control mixed solution, respectively, injecting into a liquid chromatograph, measuring, and drawing a standard curve with the peak area logarithmic value as ordinate and the control sample amount logarithmic value as abscissa.
The results are shown in Table 5 and show that: naringin control substance is 0.9161 g-9.161 mug, neohesperidin control substance is 1.146 g-11.46 mug, and ginsenoside Rg 1 0.2004-2.004 μ g of reference substance, 0.1844-1.844 μ g of ginsenoside Re reference substance, and Rb of ginsenoside 1 The linear relationship of the control samples was good in the range of 0.4070g to 4.070 μ g.
2. Repeatability survey
Taking a proper amount of dendrobium noctilucent pills (water-honeyed pills), grinding, taking about 6g, precisely weighing, taking 6 parts in total, respectively placing in conical flasks with stoppers, adding 100mL of 70% methanol solution by volume concentration, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the lost weight with 70% methanol solution by volume concentration, shaking up, filtering, precisely sucking 50mL of subsequent filtrate, evaporating to dryness, adding 20mL of water to dissolve residues, shaking up and extracting with water-saturated n-butanol for 4 times, 25mL each time, taking n-butanol solution, evaporating to dryness, adding methanol to dissolve residues, transferring to a 5mL measuring flask, adding methanol to dilute to scales, shaking up, filtering, taking subsequent filtrate to obtain 6 parts of dendrobium noctilucent pills (water-honeyed pills) sample solution, precisely sucking 10 μ L of each, injecting into a liquid chromatograph, measuring peak areas, calculating content, and obtaining the results shown in Table 6.
Taking herba dendrobii noctilucent pills (big honeyed pills), cutting into pieces, taking about 9g, taking 6 parts in total, precisely weighing, placing in a conical flask with a plug, adding 100mL of methanol solution with the volume concentration of 70%, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, supplementing the weight loss by using methanol solution with the volume concentration of 70%, shaking up, filtering, precisely sucking 50mL of subsequent filtrate, evaporating to dryness, adding 20mL of water into residues to dissolve, shaking up and extracting for 4 times by using water-saturated n-butanol, taking n-butanol liquid, evaporating to dryness, adding methanol into residues to dissolve, transferring to a 5mL measuring flask, adding methanol to dilute to a scale, shaking up, filtering, taking subsequent filtrate to obtain 6 parts of herba dendrobii noctilucent pills (big honeyed pills) sample solution, precisely sucking 10 mu L of each time, injecting into a liquid chromatograph, measuring peak areas, calculating the content, and obtaining the result shown in Table 7.
The results show that naringin, neohesperidin and ginsenoside Rg are measured in 6 samples of each dendrobium luminous pill (water-honeyed pill) and dendrobium luminous pill (big honeyed pill) 1 Ginsenoside Re and ginsenoside Rb 1 The RSD content is less than 3 percent, and the method meets the requirement of methodology, thereby showing that the method has good repeatability.
3. Precision survey
Taking the same dendrobium luminous pill (water-honeyed pill) sample solution in the repeatability investigation, repeatedly injecting the sample for 6 times, measuring the peak area, and calculating RSD; and additionally, taking the same dendrobium luminous pill (large honeyed pill) to-be-tested sample solution in the repeatability investigation, repeatedly injecting the sample for 6 times, measuring the peak area, and calculating the RSD. The results show that the naringin, the neohesperidin and the ginsenoside Rg 1 Ginsenoside Re and ginsenoside Rb 1 The peak areas RSD are all less than 3 percent, which accords with the requirement of methodology, and shows that the method of the invention has good precision, and the results are shown in tables 8 and 9.
4. Stability survey
Taking the same dendrobium luminous pill (water-honeyed pill) sample solution in the repeatability inspection, respectively injecting samples for 0h, 4h, 8h, 16h, 20h and 24h, measuring peak areas, and calculating RSD; and taking the same dendrobium luminous pill (big honeyed pill) test solution in the repeatability investigation, respectively injecting samples for 0h, 4h, 8h, 16h, 20h and 24h, measuring peak areas, and calculating RSD. The results show that the ginsenoside Rg in the dendrobium luminous pill (water-honeyed pill) test solution and the dendrobium luminous pill (big honeyed pill) test solution 1 Ginsenoside Re and ginsenoside Rb 1 The peak areas RSD of the neohesperidin and the naringin measured within 24h are all less than 3 percent, which shows that the test solution is stable within 24h and is shown in tables 10 and 11.
6. Recovery test
(1) Luminous dendrobium pill (Water-honeyed pill)
1) Precisely weighing 21.37mg ginsenoside Rg 1 Placing in a 10mL measuring flask, adding 70% methanol to dissolve, and diluting to scale as ginsenoside Rg 1 Control stock solution (concentration 1.996 mg/mL).
2) Accurately weighing 21.79mg of ginsenoside Re, placing in a 10mL measuring flask, adding 70% methanol to dissolve, and diluting to scale to obtain ginsenoside Re stock solution (with concentration of 2.046 mg/mL).
3) Precisely weighing naringin 27.08mg, neohesperidin 20.95mg, and ginsenoside Rb 10.97mg 1 Placing in a 500mL measuring flask, and precisely adding ginsenoside Rg 1 Dissolving the reference stock solution and ginsenoside Re reference stock solution in 70% methanol 3.00mL respectively, diluting to scale, and adding the mixed solution (1) (naringin: 49.665 μ g/mL; neohesperidin: 41.649 μ g/mL; ginsenoside Rg 1 :11.976 μ g/mL; ginsenoside Re:12.276 μ g/mL; ginsenoside Rb 1 ：20.426μg/mL）。
4) Taking a proper amount of herba dendrobii noctilucent pills (water-honeyed pills), grinding, taking about 3g, precisely weighing, precisely adding the sample adding reference substance mixed solution (1).
5) Precisely absorbing 10 μ L of herba Dendrobii luminous pill (water-honeyed pill) sample solution, injecting into liquid chromatograph, measuring peak area, calculating recovery rate, and adding naringin, neohesperidin and ginsenoside Rg in herba Dendrobii luminous pill (water-honeyed pill) sample solution 1 Ginsenoside Re and ginsenoside Rb 1 The recovery was good, meeting the methodology requirements, see table 12.
(2) Luminous dendrobium pill (big honeyed pill)
1) Precisely weighing 21.37mg ginsenoside Rg 1 Placing in a 10mL measuring flask, adding 70% methanol to dissolve, and diluting to scale as ginsenoside Rg 1 Control stock solution (concentration 1.996 mg/mL).
2) Accurately weighing 21.79mg of ginsenoside Re, placing in a 10mL measuring flask, adding 70% methanol to dissolve, and diluting to scale to obtain ginsenoside Re stock solution (with concentration of 2.046 mg/mL).
3) Precisely weighing 33.07mg naringin, 20.07mg neohesperidin, 16.19mg ginsenoside Rb 1 Placing into a 500mL measuring flask, and precisely adding ginsenoside Rg 1 4.00mL of each of the stock solutions of the reference substance and the ginsenoside Re reference substance, dissolving with 70% methanol, diluting to scale, and using as a sample application reference substance solution (2) (naringin: 56.982 μ g/mL; neohesperidin: 39.899 μ g/mL; ginsenoside Rg 1 :15.968 μ g/mL; ginsenoside Re:16.368 mug/mL; ginsenoside Rb 1 ：30.146μg/mL）。
4) Grinding a proper amount of dendrobium noctilucent pills (big honeyed pills), taking 4.5g, precisely weighing, precisely adding a sample-adding reference substance solution (2) to 50mL, precisely adding 70% methanol, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss by a methanol solution with the volume concentration of 70%, shaking uniformly, filtering, precisely sucking 50mL of subsequent filtrate, evaporating to dryness, adding 20mL of water to the residue to dissolve, shaking and extracting by water-saturated n-butyl alcohol for 4 times, 25mL each time, taking n-butyl alcohol solution, evaporating to dryness, adding methanol to the residue to dissolve, transferring to a 5mL measuring flask, adding methanol to dilute to a scale, shaking uniformly, filtering, and taking subsequent filtrate to obtain a sample solution of the sample-adding dendrobium noctilucent honeyed pills (big honeyed pills).
5) Precisely absorbing 10 mu L of sample solution of herba Dendrobii luminous pill (big honeyed pill), injecting into liquid chromatograph, measuring peak area, calculating recovery rate, and sampling herba Dendrobii luminous pill (big honeyed pill) sampleThe product solution contains naringin, neohesperidin, and ginsenoside Rg 1 Ginsenoside Re and ginsenoside Rb 1 The recovery of (D) was good and met the methodology requirements, see Table 13.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. A content determination method of dendrobium luminous pills is characterized by comprising the following specific steps:
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a packed column; performing gradient elution by taking acetonitrile as a mobile phase A and water as a mobile phase B; the elution procedure was: 0 to 25min,14% A,25 to 45min,14% → 20% A,45 to 63min,20% → 24A, 63 to 70min,24% → 28% A,70 to 82min,28% → 33% A,82 to 95min,33% A,95 to 102min,33% → 38% A,102 to 105min,38% A; the flow rate is 1.0mL/min; detecting with high temperature type evaporative light scattering detector, and counting theoretical plates according to ginsenoside Rg 1 Peak calculation should be no less than 3000;
(2) Preparation of mixed control solution: precisely weighing naringin, neohesperidin, and ginsenoside Rg 1 Ginsenoside Re and ginsenoside Rb 1 Adding 70% methanol to 1mL of the mixture containing naringin and neohesperidin 0.5mg, and ginsenoside Rg 1 Mixing with ginsenoside Re 0.1mg and ginsenoside Rb 1 0.2mg of a mixed control solution;
(3) Preparation of a test solution: taking herba dendrobii noctilucent pill water-honeyed pill, and grinding to obtain about 6g; or cutting herba Dendrobii luminous pill into small honeyed pill or big honeyed pill, and collecting about 9g; precisely weighing, placing in a conical flask with a plug, adding 100mL of 70% methanol solution, weighing, heating and refluxing for 1h, taking out, cooling, weighing again, complementing the weight loss with 70% methanol solution, shaking up, filtering, precisely absorbing 50mL of secondary filtrate, evaporating to dryness, adding 20mL of water into residue to dissolve, shaking up and extracting with water-saturated n-butanol for 4 times, each time extracting 25mL, taking n-butanol solution, evaporating to dryness, adding methanol into residue to dissolve and transferring to a 5mL measuring flask, adding methanol to dilute to scale, shaking up, filtering, and taking secondary filtrate;
(4) And (3) determination: precisely sucking 5 μ L and 15 μ L of mixed reference solution and 10 μ L of test solution, respectively, injecting into liquid chromatograph, measuring, and calculating content by external standard two-point method logarithmic equation;
according to the content determination method of the dendrobium luminous pill, the bran-fried fructus aurantii contained in the dendrobium luminous pill is calculated by naringin and neohesperidin, the naringin contained in a water-honeyed pill is not less than 0.42mg per 1g, and the neohesperidin contained in the water-honeyed pill is not less than 0.31mg per 1 g; the small honeyed pill contains naringin not less than 0.28mg per 1g, and neohesperidin not less than 0.21mg per 1 g; the large honeyed pill should contain naringin not less than 1.57mg per pill, and neohesperidin not less than 1.18mg per pill; ginsenoside Rg containing ginseng 1 Ginsenoside Re and ginsenoside Rb 1 Measuring the content of ginsenoside Rg in the water-honeyed pill 1 And ginsenoside Re is not less than 0.13mg per 1g, and contains ginsenoside Rb 1 Each 1g should not be less than 0.084mg; small honeyed pill containing ginsenoside Rg 1 And ginsenoside Re should not be less than 0.086 mg/1 g, and contains ginsenoside Rb 1 Each 1g should not be less than 0.057mg; big honeyed pill containing ginsenoside Rg 1 And ginsenoside Re should not be less than 0.47 mg/pill, and contains ginsenoside Rb 1 The dosage should not be less than 0.31mg.
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