CN109270203B - Construction method of characteristic spectrum of active ingredients of Jingyaokang capsule and quality detection method of Jingyaokang capsule - Google Patents
Construction method of characteristic spectrum of active ingredients of Jingyaokang capsule and quality detection method of Jingyaokang capsule Download PDFInfo
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Abstract
The invention relates to the field of medicines, in particular to a method for constructing a characteristic spectrum of an active ingredient of a Jingyaokang capsule and a method for detecting the quality of the Jingyaokang capsule. The invention defines the effective components through the basic research of the effective substances, establishes the quality standard of the effective components, provides a construction method of the characteristic map of the active components of the Jingyaokang capsule and a quality detection method of the Jingyaokang capsule, improves the quality monitoring level of the active components of the Jingyaokang capsule by enterprises, and ensures the safe, effective and uniform and stable quality of the product.
Description
Technical Field
The invention relates to the field of medicines, in particular to a method for constructing a characteristic spectrum of an active ingredient of a Jingyaokang capsule and a method for detecting the quality of the Jingyaokang capsule.
Background
The Jingyaokang capsule is made up by using 10 Chinese medicinal materials of prepared nux vomica, lycopodium clavatum, Chinese silkvine root bark, frankincense, myrrh, carthamus flower, earthworm, scalded drynaria rhizome, tetrandra root and achyranthes root, and possesses the functions of relaxing muscles and tendons, promoting blood circulation and removing blood stasis, reducing swelling and stopping pain. Can be used for treating fracture blood stasis swelling pain, fracture convalescence, and arthralgia due to kidney deficiency with blood stasis (proliferative spondylitis, lumbar disc herniation).
The complexity of the traditional Chinese medicine compound components increases the difficulty for quality control, and the characteristic spectrum technology can comprehensively present the complexity of the sample on the whole, and is widely applied to the quality evaluation system of the traditional Chinese medicine in recent years. The Chinese medicine characteristic map takes holistic concept and fuzzy concept as principles, and provides a new idea for Chinese medicine variety identification and quality evaluation. The Chinese medicine characteristic map is a common peak map which is obtained by properly processing the Chinese medicinal materials and detecting the Chinese medicinal materials by adopting a certain analysis means and an instrument and can identify the group characteristics of various components in the Chinese medicinal materials. The Chinese medicine characteristic map is a comprehensive and quantifiable identification means and can be divided into a chemical (component) characteristic map and a biological characteristic map. The chemical (component) characteristic spectrum is mostly measured by adopting the chromatographic and spectroscopic techniques, and reflects the characteristics of the composition and the variety of the chemical components of the traditional Chinese medicine.
The existing method uses two ingredients to characterize the internal quality of the Jingyaokang capsule, and has certain one-sidedness. To control the quality of the Jingyaokang capsule, it is not enough to characterize and control only one or two chemical components, and it is necessary to control the whole substance group. Therefore, in addition to microscopic analysis, a certain macroscopic analysis method is used to effectively characterize the quality of the traditional Chinese medicine on the whole, which has important practical significance.
Disclosure of Invention
In view of the above, the invention provides a method for constructing a characteristic spectrum of an active ingredient of a Jingyaokang capsule and a quality detection method of the Jingyaokang capsule. The method defines effective components through basic research of effective substances, establishes quality standard for the effective components, improves quality monitoring level of neck and waist health capsule active components, and ensures safe and effective product effect and uniform and stable quality.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for constructing a characteristic spectrum of an active ingredient of a Jingyaokang capsule, which comprises the following steps:
step 1: extracting the Jingyaokang capsule to obtain an extract, and obtaining an active ingredient of the Jingyaokang capsule through a pharmacodynamic test;
step 2: preparing a test solution from the active ingredients;
and step 3: obtaining a reference solution;
and 4, step 4: respectively taking the test solution and the reference solution, and measuring by high performance liquid chromatography to obtain characteristic spectrums of the active ingredients of the Jingyaokang capsule;
the active ingredients are strychnine and/or naringin;
the sequence of the step 2 and the step 3 is not sequential.
In some embodiments of the invention, the active ingredient is strychnine.
In some embodiments of the invention, the extraction is: mixing the contents of the Jingyaokang capsule with petroleum ether, carrying out ultrasonic extraction, filtering, collecting dregs, mixing with water, heating and refluxing, filtering, collecting filtrate for later use, mixing the dregs with 95% ethanol, heating and refluxing, filtering, combining the filtrate and the dregs, centrifuging, concentrating supernate, adjusting the pH value to 0.5-4, adjusting the pH value to 10-13, extracting for 2-4 times by using chloroform, and combining chloroform extract.
In some embodiments of the invention, the extraction is: precisely weighing 1-10g of neck and waist health capsule content, placing the neck and waist health capsule content in a conical flask with a plug, adding 5-100mL of petroleum ether (30-60 ℃), carrying out ultrasonic treatment (power 250W and frequency 50kHz) for 20-60min, filtering, drying medicine residues, adding 5-100mL of water, heating and refluxing for 0.5-3h, filtering, and concentrating the filtrate to about 2-20mL for later use. Adding 10-100mL of 95% ethanol into the residue after water extraction, heating and refluxing for 0.5-3h, filtering, combining the filtrate with the water extract, stirring, centrifuging, concentrating the supernatant to about 2-20mL, adding a proper amount of concentrated hydrochloric acid to adjust the pH to 0.5-4, adding a 5mol/L NaOH solution to adjust the pH to 10-13, extracting for 2-4 times by shaking with chloroform, 10-50mL each time, combining chloroform solutions, evaporating to dryness, dissolving the residue with methanol, placing in a 5-50mL measuring flask, adding methanol to the scale, and shaking uniformly to obtain a chloroform layer sample.
In some embodiments of the present invention, the method for preparing the test solution comprises: and (3) evaporating the chloroform extracting solution to dryness, adding methanol into the obtained residue for dissolving, and fixing the volume.
In some embodiments of the present invention, the control solution is prepared by: dissolving strychnine reference substance, tetrandrine reference substance and fangchinoline reference substance in methanol respectively, and diluting to desired volume.
In some embodiments of the invention, the chromatographic strip of the high performance liquid chromatography assayThe parts are as follows: octadecylsilane chemically bonded silica is used as a filling agent; methanol is used as a mobile phase A, 0.3% formic acid is used as a mobile phase B, the detection wavelength is 250-300 nm, and the flow rate is 0.5-1.5 mL/min-1The sample injection amount is 5-20 mu l, and the column temperature is 20-40 ℃; preferably, the detection wavelength is 275nm, and the number of theoretical plates is not less than 3000 calculated according to the strychnine peak;
gradient elution was performed according to the following elution procedure:
in some embodiments of the invention, the signature comprises 9 characteristic peaks, wherein peak No. 2: strychnine base; peak No. 5: strychnine; peak No. 7: fangchinoline; peak No. 8: tetrandrine;
and the No. 5 peak corresponding to the peak of the characteristic map and the reference substance is an S peak, and the relative retention time is obtained, wherein the relative retention time is within +/-5% of a specified value, and the specified value is peak 1: 0.26, peak 2: 0.69, peak 3:0.89, peak 4:0.91, peak S:1.00, peak 6:1.90, peak 7:1.97, peak 8:2.10, peak 9: 2.37.
In other embodiments of the present invention, the active ingredient is naringin.
In other specific embodiments of the present invention, the extraction is: mixing the contents of the Jingyaokang capsule with petroleum ether, carrying out ultrasonic extraction, filtering, collecting dregs, mixing with water, carrying out heating reflux, filtering, collecting filtrate for later use, mixing the dregs with 95% ethanol, carrying out heating reflux, filtering, combining with the filtrate, centrifuging, taking supernate, adjusting the pH value to 0.5-4, adjusting the pH value to 10-13, carrying out shake extraction with chloroform, combining the upper-layer aqueous solution, passing through a D101 type macroporous adsorption resin column, eluting with water, discarding a water washing solution, eluting with 20-100 mL of 60% ethanol, and collecting the eluent.
In other specific embodiments of the present invention, the extraction is: precisely weighing 1-10g of neck and waist health capsule content, placing in a conical flask with a plug, adding 5-100mL of petroleum ether (30-60 ℃), carrying out ultrasonic treatment (power 250W and frequency 50kHz) for 20-60min, filtering, drying the medicine residue, adding 5-100mL of water, heating and refluxing for 0.5-3h, filtering, and concentrating the filtrate to about 2-20mL for later use. Adding 10-100mL of 95% ethanol into the water-extracted residues, heating and refluxing for 0.5-3h, filtering, combining the residues with the water extract, stirring, centrifuging, concentrating the supernatant to about 2-20mL, adding a proper amount of concentrated hydrochloric acid to adjust the pH to 0.5-4, adding 5mol/L NaOH solution to adjust the pH to 10-13, extracting for 2-4 times with chloroform and 10-50mL each time, combining the upper aqueous solution, concentrating to about 2mL, passing through a D101 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 10cm), eluting with 60mL of water, discarding the aqueous solution, eluting with 20-100 mL of 60% ethanol, and collecting the eluent. Evaporating to dryness, dissolving the residue with 60% ethanol, placing in a 5-50mL measuring flask, adding 60% ethanol to the scale, and shaking to obtain 60% ethanol layer sample.
In other embodiments of the present invention, the test solution is prepared by: and mixing the eluent with ethanol, and fixing the volume.
In other embodiments of the present invention, the test solution is prepared by: evaporating the 60% ethanol layer extractive solution to dryness, dissolving the obtained residue with 60% ethanol, and diluting to desired volume.
In other embodiments of the present invention, the control solution is prepared by: mixing naringin reference substance with methanol, and metering volume.
In other embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography assay are: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, and 0.1% formic acid is taken as a mobile phase B; the detection wavelength is 250-300 nm, the column temperature is 20-40 ℃, and the volume fraction of the detection solution is 0.5-1.5 mL/min-1The sample injection amount is 5-20 mu l; preferably, the detection wavelength is 280nm, the column temperature is 30 ℃, and the flow rate is 1.0 mL/min; the number of theoretical plates is not less than 2000 calculated according to naringin peak;
gradient elution was performed according to the following elution procedure:
time (min) Mobile phase A (%) Mobile phase B (%)
0～60 5→29 95→71
60～70 29→40 71→60。
In other specific embodiments of the present invention, the characteristic pattern comprises 13 characteristic peaks, wherein peak No. 8: naringin;
the No. 8 peak corresponding to the characteristic map and the reference peak is an S peak, and the relative retention time of each characteristic peak and the S peak is obtained, wherein the relative retention time is within +/-5% of a specified value: peak 1: 0.15, peak 2: 0.19, peak 3: 0.24, peak 4: 0.41, peak 5: 0.69, Peak 6: 0.79, peak 7: 0.86, peak S:1.00, peak 9: 1.32, peak 10: 1.37, peak 11: 1.39, peak 12: 1.47, Peak 13: 1.50.
on the basis of the technical scheme, the invention also provides a quality detection method of the Jingyaokang capsule, the characteristic spectrum of the active ingredients of the Jingyaokang capsule is obtained according to the construction method, the similarity evaluation is carried out on the characteristic spectrum of the sample to be detected and the characteristic spectrum of the active ingredients of the Jingyaokang capsule, and the qualified product is obtained when the relative retention time is +/-5% of a specified value.
Olibanum and Myrrha in the JINGYAOKANG Capsule contain liposoluble components such as terpenes, volatile oil and resin, and can be extracted with petroleum ether; the more polar component in the capsule can be extracted with water, and the less polar component can be extracted with 95% ethanol; wherein radix Stephaniae Tetrandrae and Achyranthis radix contain polysaccharide components, and can be separated by water extraction and ethanol precipitation; wherein the semen Strychni preparata, herba Lycopodii, radix Stephaniae Tetrandrae, etc. contain alkaloid components, and can be prepared by extracting with water, precipitating with ethanol, mixing the supernatant with the ethanol extractive solution, concentrating, dissolving with acid, extracting with alkaline chloroform to obtain alkaline chloroform component and water component, and separating the water component with macroporous adsorbent resin to obtain 60% ethanol eluate.
According to the physicochemical properties of different components of each medicinal material of the Jingyaokang capsule, a solvent extraction method is adopted for carrying out component separation preparation; and (3) screening drug effects: the analgesic effect of different extraction parts of the Jingyaokang capsule is inspected by adopting a mouse hot plate experiment and an acetic acid writhing experiment, and an active part equivalent to the analgesic effect of the product is screened out. The pharmacodynamic test in animal body is carried out by adopting a component elimination method, and the pharmacodynamic active site for resisting inflammation, reducing swelling, promoting blood circulation and removing blood stasis is determined.
Through the basic research of the drug effect substances, the drug effect ingredients are determined, the quality standards of the drug effect ingredients are established, the quality monitoring level of the active ingredients of the Jingyaokang capsule by an enterprise is improved, and the safety, effectiveness and uniform and stable quality of the drug effect of the product are ensured.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a schematic diagram of a separation technique for neck and waist health capsule components;
FIG. 2 shows a schematic diagram of separation technique for component D of Jingyaokang capsule;
FIG. 3 shows an HPLC chromatogram of a mixed control; wherein, peak 2: strychnine base; peak 5 (S): strychnine; peak 7: fangchinoline; peak 8: tetrandrine;
FIG. 4 shows a control feature map; among the 9 characteristic peaks, peak 2: strychnine base; peak 5 (S): strychnine; peak 7: fangchinoline; peak 8: tetrandrine;
FIG. 5 shows an HPLC chromatogram of naringin control;
FIG. 6 shows a control feature map; wherein, the peak 8(S) in the 13 characteristic peaks: naringin;
FIG. 7 shows the total ion flow diagram of the component C positive ion mode (A) and negative ion mode (B);
fig. 8 shows a total ion flow diagram of the G components positive ion mode (a) and negative ion mode (B).
Detailed Description
The invention discloses a construction method of a neck and waist health capsule active ingredient characteristic spectrum and a quality detection method of the neck and waist health capsule. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The preparation method of the active ingredients provided by the invention comprises the following steps:
degreasing with petroleum ether
Taking 500g of neck and waist health capsule content, adding 700mL petroleum ether, ultrasonic defatting for 3 times, each time for 20min, filtering, and volatilizing solvent from the filtrate to obtain component A. And drying the medicine residues for later use.
Water extraction
Adding 12 times of water into degreased petroleum ether dregs, carrying out reflux extraction for 3h, filtering, adding 8 times of water, carrying out reflux extraction for 3h, filtering, combining two supernatants, concentrating to 500mL, namely the crude drug concentration of 1g/mL, adding a proper amount of 95% ethanol until the final concentration is 80% ethanol, standing at 4 ℃ overnight (12h), carrying out ethanol precipitation, centrifuging, respectively collecting precipitate and supernatant, and drying the precipitate to obtain the component B. The supernatant was stored at 4 ℃ until use (within 2 days).
Ethanol extraction
Adding 6 times of 95% ethanol into water extraction residues, carrying out reflux extraction at 80 ℃ for 2 times, each time for 1h, combining two extracting solutions, combining the extracting solutions with the water extraction and alcohol precipitation supernatant, concentrating to remove the ethanol to obtain a component E, adding water to 1.25g/mL crude drug (namely 400mL), adding a proper amount of concentrated hydrochloric acid to adjust the pH value to 1-2 (the concentration of the 1.25g/mL crude drug), adding 5mol/L NaOH to adjust the pH value to 11-12, carrying out shake extraction on chloroform for 3 times, each time for 400mL, and combining chloroform layers. Volatilizing the solvent from the chloroform layer, and drying to obtain component C. Concentrating the water layer, and drying to obtain component D.
Separation by macroporous adsorbent resin
Dissolving the component D sample in a proper amount of water, sequentially performing gradient elution by using 3 times of column volume of water, 60% and 95% ethanol, collecting eluates, respectively concentrating, and drying to obtain a component F (water eluent), a component G (60% ethanol eluent) and a component H (95% ethanol eluent) sample. All samples were stored at-20 ℃ in the dark for later use, and were prepared as they were before use.
The invention carries out drug effect screening on the obtained components: the analgesic effect of different extraction parts of the Jingyaokang capsule is inspected by adopting a mouse hot plate experiment and an acetic acid writhing experiment, and an active part equivalent to the analgesic effect of the product is screened out. The pharmacodynamic test in animal body is carried out by adopting a component elimination method, and the pharmacodynamic active site for resisting inflammation, reducing swelling, promoting blood circulation and removing blood stasis is determined.
Aiming at active ingredients, the invention provides a method for constructing an HPLC (high performance liquid chromatography) characteristic spectrum of a Jingyaokang capsule, which comprises the following specific steps:
a method for constructing an HPLC characteristic spectrum I of a Jingyaokang capsule comprises the following steps:
(1) preparation of test solution 1-10g of neck and waist health capsule content is precisely weighed, placed in a conical flask with a plug, added with 5-100mL of petroleum ether (30-60 ℃), ultrasonically treated (power 250W, frequency 50kHz) for 20-60min, filtered, dregs of a decoction are dried, added with 5-100mL of water, heated and refluxed for 0.5-3h, filtered, and the filtrate is concentrated to about 2-20mL for later use. Adding 10-100mL of 95% ethanol into the residue after water extraction, heating and refluxing for 0.5-3h, filtering, combining the residue with the water extract, stirring, centrifuging, concentrating the supernatant to about 2-20mL, adding a proper amount of concentrated hydrochloric acid to adjust the pH to 0.5-4, adding a 5mol/L NaOH solution to adjust the pH to 10-13, extracting for 2-4 times with chloroform by shaking, 10-50mL each time, combining chloroform solutions, evaporating to dryness, dissolving the residue with methanol, placing in a 5-50mL measuring flask, adding methanol to the scale, and shaking uniformly to obtain a chloroform layer sample.
(2) Preparation of control solution A proper amount of strychnine control, tetrandrine control and fangchinoline control is precisely weighed, placed in a 10mL measuring flask, dissolved in methanol and diluted to scale, shaken well, and prepared into mixed control solution containing 55 μ g of strychnine, 35 μ g of fangchinoline and 30 μ g of tetrandrine per 1 mL.
(3) And (3) determination: precisely sucking 5 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and recording chromatogram. 9 characteristic peaks should be presented in the characteristic map of the test sample, the peak corresponding to the reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated and should be within +/-5% of the specified value. The specified values of the relative retention time were 0.26 (peak 1), 0.69 (peak 2), 0.89 (peak 3), 0.91 (peak 4), 1.00 (peak S), 1.90 (peak 6), 1.97 (peak 7), 2.10 (peak 8), and 2.37 (peak 9).
The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filling agent; methanol is taken as a mobile phase A, and 0.3% formic acid is taken as a mobile phase B; the detection wavelength is 250-300 nm, and the flow rate is 0.5-1.5 mL/min-1The sample injection amount is 5-20 mu l, and the column temperature is 20-40 ℃; preferably, the detection wavelength is 275 nm. The number of theoretical plates is not less than 3000 calculated according to the specnuezhenide peak.
Gradient elution was performed according to the following elution procedure:
the invention also provides a method for constructing the neck and waist health capsule HPLC characteristic spectrum II, which comprises the following steps:
(1) preparation of test solution 1-10g of neck and waist health capsule content is precisely weighed, placed in a conical flask with a plug, added with 5-100mL of petroleum ether (30-60 ℃), ultrasonically treated (power 250W, frequency 50kHz) for 20-60min, filtered, dregs of a decoction are dried, added with 5-100mL of water, heated and refluxed for 0.5-3h, filtered, and the filtrate is concentrated to about 2-20mL for later use. Adding 10-100mL of 95% ethanol into the water-extracted residues, heating and refluxing for 0.5-3h, filtering, combining the water-extracted solution, stirring, centrifuging, concentrating the supernatant to about 2-20mL, adding a proper amount of concentrated hydrochloric acid to adjust the pH to 0.5-4, adding a 5mol/L NaOH solution to adjust the pH to 10-13, extracting for 2-4 times by shaking with chloroform, 10-50mL each time, combining the upper-layer aqueous solution, concentrating to about 2mL, passing through a D101 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 10cm), eluting with 60mL of water, discarding the aqueous solution, eluting with 20-100 mL of 60% ethanol, collecting the eluate, evaporating to dryness, dissolving the residue in 60% ethanol, placing in a 5-50mL measuring flask, adding 60% ethanol to the scale, and shaking uniformly to obtain a 60% ethanol layer active component supply sample solution.
(2) Preparation of reference solution A proper amount of naringin reference is precisely weighed, and methanol is added to obtain reference solution containing naringin 0.5mg per 1 mL.
(3) And (3) determination: precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, measuring, and recording chromatogram. 13 characteristic peaks should be presented in the characteristic map of the test article, the peak corresponding to the reference peak is the 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 the specified value. The predetermined values were 0.15 (peak 1), 0.19 (peak 2), 0.24 (peak 3), 0.41 (peak 4), 0.69 (peak 5), 0.79 (peak 6), 0.86 (peak 7), 1.00 (peak S), 1.32 (peak 9), 1.37 (peak 10), 1.39 (peak 11), 1.47 (peak 12), and 1.50 (peak 13).
The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, and 0.1% formic acid is taken as a mobile phase B; the detection wavelength is 250-300 nm, the column temperature is 20-40 ℃, and the volume fraction of the detection solution is 0.5-1.5 mL/min-1The sample injection amount is 5-20 mu l; preferably, the detection wavelength is 280nm, the column temperature is 30 ℃ and the flow rate is 1.0 mL/min. The number of theoretical plates is not less than 2000 calculated according to naringin peak.
Gradient elution was performed according to the following elution procedure:
time (min) Mobile phase A (%) Mobile phase B (%)
0～60 5→29 95→71
60～70 29→40 71→60。
The invention defines the effective components through the basic research of the effective substances, establishes the quality standard of the effective components, improves the quality monitoring level of the active components of the Jingyaokang capsule by enterprises, and ensures the safe and effective effect and uniform and stable quality of the product.
The raw materials and reagents used in the construction method of the characteristic spectrum of the active ingredients of the Jingyaokang capsule and the quality detection method of the Jingyaokang capsule provided by the invention are all available in the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 component separation
Degreasing with petroleum ether
Taking 500g of neck and waist health capsule content, adding 700mL petroleum ether, ultrasonic defatting for 3 times, each time for 20min, filtering, and volatilizing solvent from the filtrate to obtain component A. And drying the medicine residues for later use.
Water extraction
Adding 12 times of water into degreased petroleum ether dregs, carrying out reflux extraction for 3h, filtering, adding 8 times of water, carrying out reflux extraction for 3h, filtering, combining two supernatants, concentrating to 500mL, namely the crude drug concentration of 1g/mL, adding a proper amount of 95% ethanol until the final concentration is 80% ethanol, standing at 4 ℃ overnight (12h), carrying out ethanol precipitation, centrifuging, respectively collecting precipitate and supernatant, and drying the precipitate to obtain the component B. The supernatant was stored at 4 ℃ until use (within 2 days).
Ethanol extraction
Adding 6 times of 95% ethanol into water extraction residues, carrying out reflux extraction at 80 ℃ for 2 times, each time for 1h, combining two extracting solutions, combining the extracting solutions with the water extraction and alcohol precipitation supernatant, concentrating to remove the ethanol to obtain a component E, adding water to 1.25g/mL crude drug (namely 400mL), adding a proper amount of concentrated hydrochloric acid to adjust the pH value to 1-2 (the concentration of the 1.25g/mL crude drug), adding 5mol/L NaOH to adjust the pH value to 11-12, carrying out shake extraction on chloroform for 3 times, each time for 400mL, and combining chloroform layers. Volatilizing the solvent from the chloroform layer, and drying to obtain component C. Concentrating the water layer, and drying to obtain component D.
Separation by macroporous adsorbent resin
Dissolving the component D sample in a proper amount of water, sequentially performing gradient elution by using 3 times of column volume of water, 60% and 95% ethanol, collecting eluates, respectively concentrating, and drying to obtain a component E (water eluent), a component G (60% ethanol eluent) and a component H (95% ethanol eluent). All samples were stored at-20 ℃ in the dark for later use, and were prepared as they were before use.
Example 2 model for screening analgesic drug substance
2.1 materials of the experiment
The ICR mice, female, have the weight of 18-22 g and are provided by Yisi laboratory animal technology, Inc. of Changchun city, and the production license of the laboratory animals is SCXK (Ji) -2011-. The Jingyaokang capsule is provided by modified pharmaceutical industry group, Inc.
2.2 instrumentation
YLS-6B Intelligent hotplate instrument (Shanghai precision instruments Co., Ltd.); JY2003 electronic balance (Shanghai Shunhui scientific instruments, Inc.).
2.3 Experimental methods
2.3.1 routine raising and management of laboratory animals
And carrying out daily feeding and management on the experimental animals according to the animal feeding principle.
2.3.2 doses administered
The clinical dose of aspirin (0.03g/kg) was 0.27g/kg (equivalent to 5.46mg/20g) in terms of mouse administration. The clinical dose of the Jingyaokang capsule (0.05g/kg) is 0.45g/kg (equivalent to 9mg/20g) in terms of mouse administration. For comparison among groups, the dose of each isolated part was the same as that of the capsule group of Jingyakang (0.45 g/kg). Wherein the C component (chloroform layer) contains more alkaloid components, such as strychnine, etc., the minimum 100% death amount of the prepared nux vomica is 90.0mg/kg according to literature reports, and 0.45g/kg of administration can cause animal poisoning death, so that the neck and waist health capsule content with equal dosage is obtained according to the alkaloid extraction yield, namely 0.01g/kg of dry extract is administered, namely the C component (chloroform layer) amount obtained by extracting and separating 0.45g/kg of capsule content is equivalent. The gavage volume of the mice was 20mL/kg, and the blank control group was given an equal volume of physiological saline once a day.
2.3.3 pharmaceutical formulation
Because the petroleum ether layer fat-soluble component of the component A is difficult to dissolve in 0.9 percent normal saline solution, and edible soybean oil is selected as a solvent to dissolve the medicine, an edible soybean oil control group is additionally arranged. Group B, group C and group D are prepared by using 0.5% sodium carboxymethylcellulose in normal saline as solvent.
2.3.4 mouse Hot plate test
After the mice are adaptively fed for 3D, female mice with sensitive reaction (10s < pain threshold <30s) are randomly divided into a normal saline control group, a soybean oil control group, an aspirin group, a neck and waist capsule group, an A group, a B group, a C group and a D group, and each group is 12 mice. Continuously administering for 5 days, placing on hot plate at 55 + -0.5 deg.C 3h after the last administration, and determining pain threshold value with the latency of foot reaction after licking of mouse as pain threshold index.
2.3.5 mouse acetic acid writhing test
The mice are randomly divided into a normal saline control group, a soybean oil control group, an aspirin group, a neck and waist capsule group, a group A, a group B, a group C and a group D, wherein each group contains 12 mice, and each group contains half of a male mouse and a female mouse. After 5 days of continuous administration, 0.2 mL/mouse of 0.7% acetic acid solution was injected into the abdominal cavity 30min after the last administration, and then the number of writhing of the mice and the average time (latency) for starting writhing reaction within 15min were observed and recorded.
2.3.6 statistical processing
SPSS 17.0 software was used for statistical analysis of data in this study, and a two-sided independent sample t-test was used for comparisons between groups.
2.4 results and discussion
2.4.1 mouse Hot plate drug substance screening test
After 5 days of gastric administration, the animals in each group were in good condition, the hair was smooth, and the drinking, diet and defecation were all normal. The pain threshold time(s) of each group of mice is as follows
Shown, the data between groups was analyzed using independent sample t-test, and the results are shown in table 1.
TABLE 1 Hot plate pain threshold time test results for mice at different extraction sites: (
n＝12)
Note: comparison with the control group:###p<0.001、##p<0.01、#p<0.05; compared with the neck and waist capsule group:***p<0.001、**p<0.01、*p<and 0.05, the component A takes soybean oil as a drug solvent, the soybean oil is compared with a soybean oil control group during pharmacodynamic analysis, and the other administration groups are compared with a normal saline control group.
As shown in table 1, there was no significant difference (p >0.05) in group a compared to the soybean oil control group, indicating that group a had no analgesic effect; compared with the normal saline control group, the B group, the C group and the D group have significant difference (p is less than 0.001), compared with the neck and waist capsule group, the analgesic effect of the B, D group is not as good as that of the neck and waist capsule group (p is less than 0.001), and the analgesic effect of the C group and the neck and waist capsule group is not significantly different (p is more than 0.05). The test result shows that the component C is an analgesic and pharmacodynamic active substance of the Jingyaokang capsule, and the pharmacodynamic effect of the Jingyaokang capsule is equivalent to that of the Jingyaokang capsule.
2.4.2 results of acetic acid writhing test
After 5 days of gastric administration, the animals in each group were in good condition, the hair was smooth, and the drinking, diet and defecation were all normal. The number of writhing times (within 15 min) and the incubation period(s) of each group of mice
Shown, the data between groups was analyzed using independent sample t-test, and the results are shown in table 2.
TABLE 2 test results of the effect of different extraction sites on the number of acetic acid writhing and incubation period of mice: (
n＝12)
| Group of | Dosage (g/kg) | Number of wriggling (within 15 min) | Incubation period(s) |
| Physiological saline control group | —— | 32.083±2.712 | 213.750±14.252 |
| Soybean oil control group | —— | 33.833±2.980 | 208.417±15.951 |
| Aspirin group | 0.27 | 20.000±3.977### | 315.083±15.762### |
| Capsule set for neck and waist health | 0.45 | 18.500±3.177### | 299.583±15.132### |
| Group A | 0.45 | 32.167±2.250 | 207.917±12.573 |
| Group B | 0.45 | 29.167±1.946##*** | 219.167±8.430 |
| Group C | 0.01 | 20.917±2.503### | 303.917±9.120### |
| Group D | 0.45 | 27.750±2.179###*** | 215.917±10.431 |
Note: comparison with the control group:###p<0.001、##p<0.01、#p<0.05; compared with the neck and waist capsule group:***p<0.001、**p<0.01、*p<and 0.05, the component A takes soybean oil as a drug solvent, the soybean oil is compared with a soybean oil control group during pharmacodynamic analysis, and the other administration groups are compared with a normal saline control group.
As shown in table 2, there was no significant difference (p >0.05) in group a compared to the soybean oil control group, indicating that group a had no analgesic effect; compared with a normal saline control group, the times of writhing of mice in 15min can be obviously reduced in the group B and the group D (p is less than 0.01, p is less than 0.001), but the times are very different from those in the cervical and lumbar capsule group (p is less than 0.001), and the times have no influence on the latency period of writhing reaction (p is more than 0.05); the C group can obviously reduce the times of writhing (p is less than 0.001), improve the latency period of writhing reaction (p is less than 0.001), and has no significant difference with the analgesic effect of the Jingyaokang capsule group (p is more than 0.05). The test result shows that the component C is an analgesic and pharmacodynamic active substance of the Jingyaokang capsule, and the pharmacodynamic effect of the component C is equivalent to that of the Jingyaokang capsule.
EXAMPLE 3 anti-inflammatory and repercussive drug substance screening
3.1 Experimental materials
The ICR mice, male, have the weight of 18-22 g and are provided by Yisi laboratory animal technology, Inc. of Changchun city, and the production license of the laboratory animals is SCXK (Ji) -2011-.
Jingyaokang capsules (batch number: 160913) were provided by modified pharmaceutical group, Inc.; dexamethasone acetate (batch No. 160313) was purchased from Tianjin Tianyao pharmaceutical industries, Inc.; xylene (batch number: 20161020) was purchased from Beijing chemical plant.
3.2 instrumentation
JY2003 electronic balance (Shanghai Shunhui scientific instruments, Inc.).
3.3 Experimental methods
(1) Experimental animal daily feeding and management
And carrying out daily feeding and management on the experimental animals according to the animal feeding principle.
(2) Dosage to be administered
According to the yield of each extracted and separated component, the content of the Jingyaokang capsule with the same dosage is converted, and the stomach irrigation administration is carried out on the mouse.
Mice were randomly divided into a blank control group, a model group, a dexamethasone acetate group (150mg/kg, a cervicodynia and lumbosacral capsule group (450mg/kg), an E group (according to the extraction yield of the E group, the content of the cervicodynia and lumbosacral capsule with equal dosage is 450 x 17.21% ═ 77mg/kg), a C group (15mg/kg), a D group (63mg/kg), a G group (13mg/kg) and a C + G group (27 mg/kg).
The intragastric volume of the mice is 20mL/kg, and the blank control group and the model group are given physiological saline with the same volume once a day.
(3) Mouse ear swelling test
After the male mice are adaptively fed for 3D, the male mice are randomly divided into a blank control group, a model group, a dexamethasone group, a neck and waist capsule group, a C group, a D group, an E group, a G group and a C + G group, wherein each group contains 12 mice. Continuously administering for 5 days, and administering for 3d, performing first sensitization with xylene, and applying 100% xylene 20 μ L/on the inner and outer surfaces of the right ear; after 1h of the last administration, each animal was anesthetized with ether, and then applied to both the inside and outside of the right ear with 20 μ L/100% xylene, the left ear was left untreated, after 30min the animal was sacrificed, the ears were cut off, round ears were punched at the same position with a punch, and weighed with an electronic analytical balance.
Swelling degree of mouse ear-right ear weight-left ear weight
Swelling rate ═ mass of right ear-mass of left ear)/mass of left ear × 100%
The inhibition rate is (average swelling degree of model group-average swelling degree of administration group)/average swelling degree of model group x 100%
(5) Statistical treatment
SPSS 17.0 software was used for statistical analysis of data in this study, and a two-sided independent sample t-test was used for comparisons between groups.
3.4 results and discussion
After 5 days of gastric administration, the animals in each group were in good condition, the hair was smooth, and the drinking, diet and defecation were all normal. Ear swelling degree (mg) of each group of mice
Shown, the data between groups was analyzed using independent sample t-test, and the results are shown in table 3.
As shown in table 3, the model group showed a significant increase in swelling (p <0.01) compared to the blank control group, indicating successful replication of the model. Compared with the model group, the dexamethasone acetate group and the Jingyaokang capsule group remarkably reduce mouse ear swelling caused by dimethylbenzene (p is less than 0.01), and the Jingyaokang capsule has remarkable anti-inflammatory and detumescence effects. Compared with the model group, the C group, the D group, the E group, the G group and the C + G group have significant difference (p is less than 0.01), compared with the Jingyakang capsule group, the C group, the D group and the G group have significant difference (p is less than 0.01), the E group and the C + G group have no significant difference (p is more than 0.05), the test result shows that the C component and the G component have synergistic action, the components after the C component and the G component are mixed can improve the anti-inflammatory and detumescent effects when the C component and the G component are independently applied, the Jingyakang capsule is an anti-inflammatory and detumescent drug effect active substance, and the drug effect is equivalent to that of the Jingyakang capsule.
TABLE 3 swelling test results of mouse auricles at different extraction sites: (
n＝12)
Note: comparison with blank control:##p<0.01、#p<0.05; comparison with model groups:**p<0.01、*p<0.05; neck and waist harmonizing capsuleGroup comparison:△△p<0.01、△p<0.05；
example 4 blood-activating drug substance screening
4.1 Experimental materials
The ICR mouse is half male and female, has the weight of 18-22 g, and is provided by Yise laboratory animal technology, Limited liability company of Changchun city, and the production license of the laboratory animal is SCXK (Ji) -2011-.
Jingyaokang capsules (batch number: 160913) were provided by modified pharmaceutical group, Inc.; red tablets (batch No. 160805) were purchased from Shenyang Red medicine group, Inc.
4.2 instrumentation
JY2003 electronic balance (Shanghai Shunhui scientific instruments, Inc.).
4.3 Experimental methods
(1) Experimental animal daily feeding and management
And carrying out daily feeding and management on the experimental animals according to the animal feeding principle.
(2) Dosage to be administered
The administration dose of the compound was 0.16g/kg (equivalent to 3.15mg/20g) in terms of red tablets; the other groups were dosed as under item 3.3 (2).
(3) Tail shearing test for mice
The mice were randomly divided into a blank control group, a red tablet group, a neck and waist capsule group and an E group, each group had 12 mice, and each group had half male and female. Continuously administering for 7d, cutting the tip of the mouse with scissors for 2mm after last administration for 3h, counting with stopwatch when blood overflows, sucking off blood drop with filter paper every 30s until blood does not overflow, and calculating bleeding time.
Mice were randomly divided into a blank control group, a red tablet group, a neck and waist capsule group, a group C, a group D, a group E, a group G and a group C + G, each group had 12 mice each and half of males and females. Continuously administering for 7d, cutting the tip of the mouse with scissors for 2mm after last administration for 3h, counting with stopwatch when blood overflows, sucking off blood drop with filter paper every 30s until blood does not overflow, and calculating bleeding time.
(4) Statistical treatment
SPSS 17.0 software was used for statistical analysis of data in this study, and a two-sided independent sample t-test was used for comparisons between groups.
4.4 results and discussion
After the intragastric administration for 7 days, the animals in each group are in good condition, the hair is smooth, and the drinking, eating and urination are all normal. Bleeding time (min) of each group of mice
Shown, the data between groups was analyzed using independent sample t-test, and the results are shown in table 4.
As shown in table 4, compared with the blank control group, the red tablet group and the cervicobrachial capsule group significantly prolong the tailcut bleeding time (p is less than 0.01), which indicates that the cervicobrachial capsule has significant effects of promoting blood circulation and removing blood stasis. Compared with a blank control group, the group C, the group D, the group E, the group G and the group C + G all have significant difference (p is less than 0.01), compared with the neck and waist rehabilitation capsule group, the group C, the group D and the group G have significant difference (p is less than 0.01), the group E and the group C + G have no significant difference (p is more than 0.05), test results show that the component C and the component G have synergistic effect, the components formed by mixing the component C and the component G can improve the blood circulation promoting and blood stasis removing effects when the components are independently applied, the neck and waist rehabilitation capsule is a blood circulation promoting and blood stasis removing pharmacodynamic active substance, and the pharmacodynamic effect of the neck and waist rehabilitation capsule is equivalent to that of the neck and waist rehabilitation capsule.
TABLE 4 bleeding time results of tail-shearing test of mice at different extraction sites: (
n＝12)
Note: comparison with blank control:##p<0.01、#p<0.05; compared with the neck and waist capsule group:△△p<0.01、△p<0.05
in conclusion, the neck and waist rehabilitation capsule contains C component and G component as the drug effect substances for easing pain, resisting inflammation, reducing swelling and promoting blood circulation, and the invention establishes the characteristic maps of the drug effect components of the two components.
Example 5 construction of neck and waist health capsules HPLC characteristic map I
1. Neck and waist recovery capsule HPLC characteristic spectrum I detection method
(1) Preparation of test solution 5g of neck and waist health capsule content is precisely weighed, placed in a conical flask with a plug, added with 50mL of petroleum ether (30-60 ℃), ultrasonically treated (power 250W, frequency 50kHz) for 30min, filtered, dried dregs of a decoction, added with 50mL of water, heated and refluxed for 2h, filtered, and concentrated to about 10mL of filtrate for later use. Adding 50mL of 95% ethanol into the residue after water extraction, heating and refluxing for 1h, filtering, combining the filtrate with the water extract, stirring, centrifuging, taking the supernatant, concentrating to about 10mL, adding a proper amount of concentrated hydrochloric acid to adjust the pH to 1-2, adding a 5mol/L NaOH solution to adjust the pH to 11-12, shaking and extracting for 3 times with chloroform, each time extracting for 20mL, combining chloroform solutions, evaporating to dryness, dissolving the residue with methanol, placing the solution in a 25mL measuring flask, adding methanol to the scale, and shaking up to obtain a chloroform layer sample.
(2) Preparation of control solution A proper amount of strychnine control, tetrandrine control and fangchinoline control is precisely weighed, placed in a 10mL measuring flask, dissolved in methanol and diluted to scale, shaken well, and prepared into mixed control solution containing 55 μ g of strychnine, 35 μ g of fangchinoline and 30 μ g of tetrandrine per 1 mL.
(3) And (3) determination: precisely sucking 5 μ L of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and recording chromatogram.
The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filling agent; gradient elution was performed with methanol as mobile phase a and 0.3% formic acid as mobile phase B according to the elution procedure in table 5:
TABLE 5
The detection wavelength was 275nm, and the flow rate was 1.0mL/min-1The column temperature was 30 ℃. The number of theoretical plates is not less than 3000 calculated according to the specnuezhenide peak.
9 characteristic peaks should be presented in the characteristic map of the test sample, the peak corresponding to the reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated and should be within +/-5% of the specified value. The specified values of the relative retention time were 0.26 (peak 1), 0.69 (peak 2), 0.89 (peak 3), 0.91 (peak 4), 1.00 (peak S), 1.90 (peak 6), 1.97 (peak 7), 2.10 (peak 8), and 2.37 (peak 9).
2. Methodology investigation
(1) Precision test
Taking the same batch of samples, preparing the sample solution according to the sample solution preparation method, continuously injecting samples for 6 times, and recording the characteristic map. The experimental result shows that the relative retention time RSD of each chromatographic peak is within 5 percent.
TABLE 6 precision test-relative Retention time
(2) Repeatability survey
Taking the same batch of samples, preparing 6 test sample solutions in parallel, sequentially injecting samples, and recording a characteristic spectrum. The experimental result shows that the RSD of the relative retention time of each chromatographic peak is within 5 percent, and the reproducibility is good.
TABLE 7 repeatability tests-relative retention time
(3) Stability survey
Sampling the same sample and prepared test solution at 0, 2, 8, 12, 18 and 24h, and recording the characteristic map. The experimental result shows that the RSD of the relative retention time of each chromatographic peak is within 5 percent, which indicates that the components in the test solution are stable within 24 hours.
TABLE 8 stability test-relative Retention time
3. Establishment of feature map common mode and identification of partial common peak
According to the preparation method of the test solution, 10 batches of test solution are prepared, 5 mu L of sample is injected according to the chromatographic conditions, and the chromatogram is measured. The spectra of 10 batches of samples are introduced into the Chinese medicine chromatogram fingerprint similarity evaluation system software (2012 edition) of the national pharmacopoeia committee to generate a common pattern of characteristic spectra, wherein 9 characteristic peaks are included. By comparing and analyzing the retention time with a reference substance, the peak 2 is determined to be strychnine, the peak 5 is determined to be strychnine, the peak 7 is determined to be fangchinoline, and the peak 8 is determined to be tetrandrine.
4. 10 batches of chloroform layer active component characteristic spectrum relative retention time comparison
A common mode is established for 10 chloroform layer active component maps of neck and waist health finished products in different batches by adopting an average vector method, the relative retention time is calculated, a relative retention time analysis table of each map and a comparison map is obtained, and the analysis results are shown in table 9.
Therefore, the relative retention time specification values of the characteristic peaks were determined to be 0.26 (peak 1), 0.69 (peak 2), 0.89 (peak 3), 0.91 (peak 4), 1.00 (peak S), 1.90 (peak 6), 1.97 (peak 7), 2.10 (peak 8), and 2.37 (peak 9).
TABLE 910 relative retention time of characteristic peaks of chloroform layer of batches
Example 6 construction of neck and waist health capsules HPLC characteristic map II
1. Neck and waist health capsule HPLC characteristic spectrum II detection method
Preparation of test solution 5g of the content of the product is precisely weighed, placed in a conical flask with a plug, added with 50mL of petroleum ether (30-60 ℃), ultrasonically treated (power 250W and frequency 50kHz) for 30min, filtered, dried in dregs, added with 50mL of water, heated and refluxed for 2h, filtered, and concentrated to about 10mL of filtrate for later use. Adding 50mL of 95% ethanol into the water-extracted medicine residue, heating and refluxing for 1h, filtering, combining with the water-extracted liquid, stirring, centrifuging, taking the supernatant, concentrating to about 10mL, adding a proper amount of concentrated hydrochloric acid to adjust the pH to 1-2, adding a 5mol/L NaOH solution to adjust the pH to 11-12, shaking and extracting for 3 times with chloroform, 20mL each time, combining the upper-layer aqueous solution, concentrating to about 2mL, passing through a D101 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 10cm), eluting with 60mL of water, discarding the water solution, eluting with 60mL of 60% ethanol, collecting the eluent, evaporating to dryness, dissolving the residue with 60% ethanol, placing in a 25mL measuring flask, adding 60% ethanol to the scale, shaking uniformly, and obtaining a sample solution of 60% ethanol layer active component.
Preparation of reference solution A proper amount of naringin reference is precisely weighed, and methanol is added to obtain reference solution containing naringin 0.5mg per 1 mL.
And (3) determination: precisely sucking 10 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, measuring, and recording chromatogram.
The chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filling agent; gradient elution was performed according to the elution procedure in table 10 using acetonitrile as mobile phase a and 0.1% formic acid as mobile phase B;
watch 10
The detection wavelength was 280nm, the column temperature was 30 ℃ and the flow rate was 1.0 mL/min. The number of theoretical plates is not less than 2000 calculated according to naringin peak.
13 characteristic peaks should be presented in the characteristic map of the test article, the peak corresponding to the reference peak is the 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 the specified value. The predetermined values were 0.15 (peak 1), 0.19 (peak 2), 0.24 (peak 3), 0.41 (peak 4), 0.69 (peak 5), 0.79 (peak 6), 0.86 (peak 7), 1.00 (peak S), 1.32 (peak 9), 1.37 (peak 10), 1.39 (peak 11), 1.47 (peak 12), and 1.50 (peak 13).
2. Methodology investigation
(1) Precision test
Taking the same batch of samples, preparing the sample solution according to the sample solution preparation method, continuously injecting samples for 6 times, and recording the characteristic map. The experimental results show that the relative retention time RSD of 13 chromatographic peaks is within 5 percent.
TABLE 11 precision test-relative Retention time
(2) Repeatability survey
Taking the same batch of samples, preparing 6 test sample solutions in parallel, sequentially injecting samples, and recording a characteristic spectrum. The experimental result shows that the RSD of the relative retention time of 13 chromatographic peaks is within 5 percent, and the reproducibility is good.
TABLE 12 repeatability tests-relative retention time
(3) Stability survey
Taking the same sample, preparing a sample solution, injecting samples for 0, 4, 8, 12, 18 and 24 hours respectively, and recording a characteristic map. The experimental result shows that the RSD of the relative retention time of each 13 chromatographic peaks is within 5 percent, which indicates that the components in the test solution are stable within 24 hours.
TABLE 13 stability test-relative Retention time
3. Establishment of characteristic map common mode and identification of part common peaks
According to the preparation method of the test solution, 10 batches of finished test solutions are prepared, 10 mu L of sample is injected according to the chromatographic conditions, and the characteristic spectrum is recorded. And (3) introducing the characteristic spectrums of 10 batches of samples into traditional Chinese medicine chromatogram fingerprint similarity evaluation system software (2012 edition) of the national pharmacopoeia committee, generating a characteristic spectrum common mode, calibrating 13 characteristic peaks, and determining that the peak No. 8 is naringin by comparing and analyzing retention time of a reference substance.
4. Comparison of 10 batches of 60% ethanol layer active component characteristic spectrum relative retention time
A common mode is established for 60% ethanol layer active component spectrums of 10 different batches of neck and waist health finished products by adopting an average vector method, relative retention time is calculated, a relative retention time analysis table of each spectrum and a control spectrum is obtained, and analysis results are shown in a table 14.
Therefore, the relative retention time specified values of the characteristic peaks were determined to be 0.15 (peak 1), 0.19 (peak 2), 0.24 (peak 3), 0.41 (peak 4), 0.69 (peak 5), 0.79 (peak 6), 0.86 (peak 7), 1.00 (peak S), 1.32 (peak 9), 1.37 (peak 10), 1.39 (peak 11), 1.47 (peak 12), and 1.50 (peak 13).
TABLE 1410 batch 60% ethanol layer profile relative retention time
Example 7 mass spectrometric analysis of pharmacodynamic Components
C component mass spectrometry
The chloroform layer component-C component positive ion mode (A) and negative ion mode (B) total ion flow chart is shown in figure 7, and the analysis result is shown in table 7. Through the analysis of a primary mass spectrum, a secondary mass spectrum and related references, structural analysis and identification are carried out on each peak, and 13 components are identified in total. Wherein, the No. 1 is strychnine, the No. 2 is strychnine, the No. 3 is pseudo-strychnine, the No. 4 is brucine, and belongs to nux vomica medicinal materials; number 5 is phellodendrine, number 6 is fangchinoline, number 7 is tetrandrine, belongs to radix Stephaniae Tetrandrae medicinal material; no. 8 is periplogenin belonging to cortex Periplocae Radicis medicinal material; no. 9 is lycopodiol, belonging to herba Lycopodii medicinal material; no. 10 is 11-carbonyl-beta-boswellic acid, No. 11 is 11-carbonyl-beta-acetyl boswellic acid, No. 12 is elemenone acid, No. 13 is acetyl-alpha-boswellic acid, and belongs to boswellia medicinal materials.
Table 15 chemical composition identification results of component C
Mass spectrometric analysis of G Components
The total ion flow diagram of the G component positive ion mode (A) and negative ion mode (B) is shown in FIG. 8, and the analysis result is shown in Table 8. And analyzing the structure of each peak through a primary mass spectrum, a secondary mass spectrum and related references, and identifying 7 components in total. Wherein No. 1 is adenine, belonging to Lumbricus medicinal material; no. 2 is hydroxysafflor yellow A, No. 3 is benzoic acid, belongs to safflower medicinal materials; no. 5 is phellodendrine belonging to radix Stephaniae Tetrandrae; neoeriocitrin No. 6 and naringin No. 7 belong to rhizoma Drynariae; 9 is notoginsenoside R1Belonging to achyranthes root.
Table 16 chemical composition identification results of the components
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. A method for constructing a characteristic spectrum of an active ingredient of a Jingyaokang capsule is characterized by comprising the following steps:
step 1: extracting a neck and waist rehabilitation capsule to obtain an extract, and determining active components of the neck and waist rehabilitation capsule through a pharmacodynamic test, wherein the active components comprise strychnine or naringin;
the preparation method of the active component comprises the following steps:
degreasing with petroleum ether
Taking 500g of neck and waist health capsule content, adding 700mL of petroleum ether, ultrasonic degreasing for 3 times, each time for 20min, filtering, volatilizing solvent from filtrate to obtain component A, and drying medicinal residues for later use;
water extraction
Adding 12 times of water into degreased petroleum ether dregs, performing reflux extraction for 3 hours, filtering, adding 8 times of water, performing reflux extraction for 3 hours, filtering, combining two supernatants, concentrating to 500mL, namely the crude drug concentration of 1g/mL, adding a proper amount of 95% ethanol until the final concentration is 80% ethanol, standing at 4 ℃ overnight for 12 hours for ethanol precipitation, centrifuging, respectively collecting precipitate and supernatant, drying the precipitate to obtain a component B, and storing the supernatant at 4 ℃ for later use within 2 days;
ethanol extraction
Adding 95% ethanol in an amount which is 6 times that of the water extraction residue, performing reflux extraction at 80 ℃ for 2 times, each time for 1h, combining the two extracting solutions with the water extraction and alcohol precipitation supernatant, concentrating to remove the ethanol to obtain a component E, adding water to 1.25g/mL crude drug, namely 400mL, adding a proper amount of concentrated hydrochloric acid to adjust the pH value to 1-2, adding 5mol/L NaOH to adjust the pH value to 11-12, performing shake extraction on chloroform for 3 times, each time for 400mL, combining chloroform layers, volatilizing the solvent of the chloroform layers, drying to obtain a component C, and concentrating and drying the water layers to obtain a component D;
separation by macroporous adsorbent resin
Dissolving the component D sample in a proper amount of water, sequentially carrying out gradient elution by using 3 times of column volume of water, 60 percent and 95 percent ethanol, collecting each eluent, respectively concentrating and drying to obtain a component F eluent, a component G eluent, a component H eluent, a component F eluent, a component G eluent and a component H eluent, storing all the samples at-20 ℃ in a dark place for later use, and preparing the samples before use;
the neck and waist rehabilitation capsule is composed of C component and G component as drug effect substances for easing pain, diminishing inflammation and swelling and promoting blood circulation;
step 2: preparing a sample solution from the component C or the component G;
and step 3: obtaining a reference solution;
and 4, step 4: respectively taking the test solution and the reference solution, and measuring by high performance liquid chromatography to obtain characteristic spectrums of the active ingredients of the Jingyaokang capsule;
the sequence of the step 2 and the step 3 is not sequential;
for component C the active ingredient comprises strychnine;
the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filling agent; methanol is used as a mobile phase A, 0.3% formic acid is used as a mobile phase B, the detection wavelength is 250-300 nm, the flow rate is 0.5-1.5 mL/min < -1 >, the sample injection amount is 5-20 mu l, the column temperature is 20-40 ℃, and the number of theoretical plates is not lower than 3000 calculated according to the strychnine peak;
gradient elution was performed according to the following elution procedure:
For the G component, the active ingredient comprises naringin;
the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, and 0.1% formic acid is taken as a mobile phase B; the detection wavelength is 250-300 nm, the column temperature is 20-40 ℃, the flow rate is 0.5-1.5 mL/min < -1 >, and the sample injection amount is 5-20 mu l; the number of theoretical plates is not less than 2000 calculated according to naringin peak;
gradient elution was performed according to the following elution procedure:
。
2. The construction method according to claim 1, wherein the characteristic pattern of the C component comprises 9 characteristic peaks, wherein the ratio of the No. 2 peak: strychnine base; peak No. 5: strychnine; peak No. 7: fangchinoline; peak No. 8: tetrandrine;
and the No. 5 peak corresponding to the peak of the characteristic map and the reference substance is an S peak, and the relative retention time is obtained, wherein the relative retention time is within +/-5% of a specified value, and the specified value is peak 1: 0.26, peak 2: 0.69, peak 3:0.89, peak 4:0.91, peak S:1.00, Peak 6:1.90, peak 7:1.97, Peak 8:2.10, peak 9: 2.37.
3. the construction method according to claim 1, wherein the characteristic pattern of the G component comprises 13 characteristic peaks, wherein the ratio of No. 8 peak: naringin;
the No. 8 peak corresponding to the characteristic map and the reference peak is an S peak, and the relative retention time of each characteristic peak and the S peak is obtained, wherein the relative retention time is within +/-5% of a specified value: peak 1: 0.15, peak 2: 0.19, peak 3: 0.24, peak 4: 0.41, peak 5: 0.69, Peak 6: 0.79, peak 7: 0.86, peak S:1.00, peak 9: 1.32, peak 10: 1.37, peak 11: 1.39, peak 12: 1.47, Peak 13: 1.50.
4. the quality detection method of the Jingyaokang capsule is characterized in that the characteristic spectrum of the active ingredients of the Jingyaokang capsule is obtained according to the construction method of any one of claims 1 to 3, the similarity evaluation is carried out on the characteristic spectrum of a sample to be detected and the characteristic spectrum of the active ingredients of the Jingyaokang capsule, and the qualified product is obtained when the relative retention time is +/-5% of a specified value.
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