CN109270203A - The construction method of neck waist recovering capsule active constituent characteristic spectrum and the quality determining method of neck waist recovering capsule - Google Patents
The construction method of neck waist recovering capsule active constituent characteristic spectrum and the quality determining method of neck waist recovering capsule Download PDFInfo
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Abstract
The present invention relates to field of medicaments, the in particular to quality determining method of the construction method of neck waist recovering capsule active constituent characteristic spectrum and neck waist recovering capsule.The present invention is studied by effective substance, specify effective component, and effective component is established into quality standard, provide the construction method of neck waist recovering capsule active constituent characteristic spectrum and the quality determining method of neck waist recovering capsule, it is horizontal to the quality-monitoring of neck waist recovering capsule active constituent to improve enterprise, it is ensured that product drug effect safely and effectively, quality stable homogeneous.
Description
Technical field
The present invention relates to field of medicaments, the in particular to construction method and neck waist of neck waist recovering capsule active constituent characteristic spectrum
The quality determining method of recovering capsule.
Background technique
Neck waist recovering capsule is by Semen Strychni (processed), lycopodium calvatum, cortex periplocae, olibanum, myrrh, safflower, pheretima, Rhizoma drynariae preparata, prevents
Oneself and 10 taste medicinal material of radix achyranthis bidentatae form, and have the effect of channels sootheing and network vessel quickening, activating microcirculation and removing stasis medicinal, swelling and pain relieving.For blood stasis pain of fracturing
Bitterly, fracture convalescence and kidney deficiency hold numbness pain caused by the stasis of blood (hyperplastic spondylitis, prolapse of nucleus pulposus of lumbar spine) under the arm.
The complexity of Chinese medicine compound prescription ingredient is that the control of its quality increases difficulty, and characteristic spectrum technology can be on the whole
The complexity that sample is presented comprehensively, is widely used in recent years in the quality evaluation system of Chinese medicine.Chinese medicine characteristic spectrum is with whole
Body idea and fuzzy theory are principle, identify for herbal species and quality evaluation provides a new approaches.Chinese medicine characteristic spectrum
Refer to Chinese medicine after processing appropriate, it is wherein various using can be identified for that of detecting of certain analysis means and instrument
The shared peak figure of component population characteristic is composed.Chinese medicine characteristic spectrum is a kind of synthesis, quantifiable identification means, can be divided into chemistry
(ingredient) characteristic spectrum and biological characteristic map.Chemical (ingredient) characteristic spectrum mostly uses chromatography, spectrum technology determining, in reflection
Feature on medicine chemical component composition and type.
Current existing method is all that the inherent quality of neck waist recovering capsule is characterized with two taste ingredients simply, is had centainly
One-sidedness.The quality for controlling neck waist recovering capsule, it is inadequate for being characterized and controlled just for wherein one or two of chemical component,
Must the substance group to it integrally controlled.So other than micro-analysis, with the method for certain macroscopic analysis, from entirety
Upper effective characterization traditional Chinese medicine quality has important practical significance.
Summary of the invention
In view of this, the present invention provides the construction method and neck waist health glue of a kind of neck waist recovering capsule active constituent characteristic spectrum
The quality determining method of capsule.This method is studied by effective substance, specifies effective component, and effective component is established matter
Amount standard it is horizontal to the quality-monitoring of neck waist recovering capsule active constituent to improve enterprise, it is ensured that product drug effect is safe and effective, quality is equal
One stablizes.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of construction methods of neck waist recovering capsule active constituent characteristic spectrum, include the following steps:
Step 1: taking neck waist recovering capsule to extract and obtain extract, the active constituent of neck waist recovering capsule is obtained through pharmacodynamic test；
Step 2: the active constituent being taken to prepare test solution；
Step 3: obtaining reference substance solution；
Step 4: taking the test solution and the reference substance solution through high effective liquid chromatography for measuring respectively, obtain neck
The characteristic spectrum of waist recovering capsule active constituent；
The active constituent is strychnia and/or aurantiin；
The sequence of step 2 and step 3 is in no particular order.
In some specific embodiments of the invention, the active constituent is strychnia.
In some specific embodiments of the invention, the extraction are as follows: take neck waist recovering capsule content and petroleum ether mixed
It closes, ultrasonic extraction, filtering collects the dregs of a decoction, mixes, be heated to reflux with water, filters, and collects filtrate, and it is spare, by the dregs of a decoction and 95% second
Alcohol mixing, is heated to reflux, and filters, merges with the filtrate, is centrifuged, supernatant is taken to be concentrated, adjusting pH value to 0.5~4, then adjusts
PH value is extracted 2~4 times with chloroform shaking to 10~13, merges chloroform extracted solution.
In some specific embodiments of the invention, the extraction are as follows: neck waist recovering capsule 1~10g of content is taken, it is accurate
It is weighed, it sets in stuffed conical flask, petroleum ether (30~60 DEG C) 5~100mL is added, be ultrasonically treated (power 250W, frequency 50kHz)
20~60min, filtration, the dregs of a decoction are dry, and 5~100mL of water is added, is heated to reflux 0.5~3h, filter, and filtrate is concentrated into about 2~
20mL, it is spare.Water is mentioned into the dregs of a decoction, 95% 10~100mL of ethyl alcohol is added, is heated to reflux 0.5~3h, is filtered, and mentioned with above-mentioned water
Liquid merges, and stirs, and centrifugation takes supernatant to be concentrated into about 2~20mL, and appropriate concentrated hydrochloric acid is added and adjusts pH to 0.5~4, adds
5mol/L NaOH solution adjusts pH to 10~13, is extracted 2~4 times with chloroform shaking, and 10~50mL, merges chloroform solution every time, steams
Dry, residue adds methanol to dissolve, and sets in 5~50mL measuring bottle, adds methanol to scale, shakes up to get chloroform layer sample.
In some specific embodiments of the invention, the test solution the preparation method comprises the following steps: the chloroform is taken to mention
Liquid is taken to be evaporated, the dissolution of residue obtained plus methanol, constant volume.
In some specific embodiments of the invention, the reference substance solution the preparation method comprises the following steps: strychnine is taken to compare
Product, strychnia reference substance, tetrandrine reference substance and tetrandrine reference substance, respectively plus methanol dissolves, constant volume.
In some specific embodiments of the invention, the chromatographic condition of the high performance liquid chromatography measurement are as follows: with 18
Alkyl silane bonded silica gel is filler；Using methanol as mobile phase A, using 0.3% formic acid as Mobile phase B, Detection wavelength be 250~
300nm, flow velocity are 0.5~1.5mLmin-1, sample volume be 5~20 μ l, 20~40 DEG C of column temperature；It is preferred that Detection wavelength is
275nm, number of theoretical plate is calculated by strychnia peak should be not less than 3000；
Gradient elution is carried out according to following elution program:
In some specific embodiments of the invention, the characteristic spectrum includes 9 characteristic peaks, wherein No. 2 peaks: scholar
Peaceful alkali；No. 5 peaks: strychnia；No. 7 peaks: fangchinoline；No. 8 peaks: tetrandrine；
The characteristic spectrum No. 5 peaks corresponding with reference substance peak are the peak S, obtain relative retention time, the opposite reservation
Time should be within ± the 5% of 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 specific embodiments of the invention, the active constituent is aurantiin.
In other specific embodiments of the invention, the extraction are as follows: take neck waist recovering capsule content and petroleum ether
Mixing, ultrasonic extraction, filtering collect the dregs of a decoction, mix, be heated to reflux with water, filter, and collect filtrate, spare, by the dregs of a decoction and 95%
Ethyl alcohol mixing, is heated to reflux, and filters, merges with the filtrate, is centrifuged, and takes supernatant, adjusting pH value to 0.5~4, then adjust pH
Value is shaken with chloroform and is extracted, merged upper layer aqueous solution and be eluted with water by D101 type large pore resin absorption column, abandoned to 10~13
Water lotion is removed, then is eluted with 60% 20~100mL of ethyl alcohol, eluent is collected.
In other specific embodiments of the invention, the extraction are as follows: take neck waist recovering capsule content 1-10g, essence
It is close weighed, it sets in stuffed conical flask, petroleum ether (30~60 DEG C) 5-100mL is added, be ultrasonically treated (power 250W, frequency
50kHz) 20-60min, filtration, the dregs of a decoction are dry, and water 5-100mL is added, is heated to reflux 0.5-3h, filter, and filtrate is concentrated into about 2-
20mL, it is spare.Water is mentioned into the dregs of a decoction, 95% ethyl alcohol 10-100mL is added, is heated to reflux 0.5-3h, is filtered, and with above-mentioned Aqueous extracts
Merge, stir, centrifugation takes supernatant to be concentrated into about 2-20mL, and appropriate concentrated hydrochloric acid is added and adjusts pH to 0.5~4, adds
5mol/L NaOH solution adjusts pH to 10~13, is extracted 2-4 times, each 10-50mL with chloroform shaking, merges upper layer aqueous solution,
It is concentrated into about 2mL, by D101 type large pore resin absorption column (internal diameter 1.5cm, pillar height 10cm), is eluted, is abandoned with water 60mL
Aqueous is gone, then is eluted with 60% 20~100mL of ethyl alcohol, eluent is collected.It is evaporated, residue adds 60% ethyl alcohol to dissolve, and sets 5-50mL
In measuring bottle, adds 60% ethyl alcohol to scale, shake up to get 60% alcohol layer sample.
In other specific embodiments of the invention, the test solution the preparation method comprises the following steps: taking the elution
Liquid is mixed with ethyl alcohol, constant volume.
In other specific embodiments of the invention, the test solution the preparation method comprises the following steps: taking described 60%
Alcohol layer extracting solution is evaporated, the dissolution of residue obtained plus 60% ethyl alcohol, constant volume.
In other specific embodiments of the invention, the reference substance solution the preparation method comprises the following steps: taking aurantiin pair
It is mixed according to product with methanol, constant volume.
In other specific embodiments of the invention, the chromatographic condition of the high performance liquid chromatography measurement are as follows: with ten
Eight alkyl silane bonded silica gels are filler；Using acetonitrile as mobile phase A, using 0.1% formic acid as Mobile phase B；Detection wavelength is 250
~300nm, column temperature are 20~40 DEG C, 0.5~1.5mLmin-1, sample volume is 5~20 μ l；It is preferred that Detection wavelength is 280nm,
Column temperature is 30 DEG C, flow velocity 1.0mL/min；Number of theoretical plate is calculated by aurantiin peak should be not less than 2000；
Gradient elution is carried out according to following elution program:
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 invention, the characteristic spectrum includes 13 characteristic peaks, wherein No. 8 peaks:
Aurantiin；
The characteristic spectrum No. 8 peaks corresponding with reference substance peak are the peak S, obtain each characteristic peak and when retaining relatively of the peak S
Between, the relative retention time should be within ± the 5% of specified value, the specified value are as follows: 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.
Based on the above technical solution, the present invention also provides the quality determining methods of neck waist recovering capsule, according to institute
The construction method stated obtains neck waist recovering capsule active constituent characteristic spectrum, to the characteristic spectrum and the neck waist health glue of sample to be tested
Capsule active constituent characteristic spectrum carries out similarity evaluation, and relative retention time is qualified products ± the 5% of specified value.
Olibanum, myrrh contain the liposoluble constituents such as terpene, volatile oil and resin in neck waist recovering capsule, can be selected petroleum ether into
Row extracts；Water extraction can be used in the biggish component of polarity in capsule, and the extraction of 95% ethyl alcohol can be used in the relatively small component of polarity；
Wherein the root of fangji and radix achyranthis bidentatae contain polysaccharide component, and the method that water extract-alcohol precipitation can be used realizes the separation of polysaccharide component；Wherein Strychnos nux-vomica processed
The components containing alkaloids such as son, lycopodium calvatum and the root of fangji, can be by merging concentration, sour water with alcohol extract for water extract-alcohol precipitation supernatant
After dissolution, alkaline chloroform recovery is divided into alkaline chloroform layer component and water layer component, and it is isolated that water layer crosses macroporous absorbent resin
60% ethanol elution component.
According to the physicochemical property of each medicinal material different component of neck waist recovering capsule, component separation system is carried out using solvent extraction
It is standby；Medicine efficacy screening: it is tested using mouse hot-plate experiment with acetic acid twisting, investigates the antalgesic of neck waist recovering capsule the different extracted parts
Effect effect, filters out and the comparable active site of product analgesia effect.Pharmacodynamic test in animal body is carried out using component scalping method,
Determine the drug activity position of anti-inflammation detumescence and activating microcirculation and removing stasis medicinal.
It is studied by effective substance, specifies effective component, and effective component is established into quality standard, improve enterprise
It is horizontal to the quality-monitoring of neck waist recovering capsule active constituent, it is ensured that product drug effect safely and effectively, quality stable homogeneous.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows neck waist recovering capsule component isolation technics route map；
Fig. 2 shows neck waist recovering capsule D component isolation technics route map；
Fig. 3 shows the HPLC chromatogram of mixing reference substance；Wherein, peak 2: strychnine alkali；Peak 5 (S): strychnia；Peak 7: anti-
Own promise woods alkali；Peak 8: tetrandrine；
Fig. 4 shows compare feature map；Wherein, in 9 characteristic peaks, peak 2: strychnine alkali；Peak 5 (S): strychnia；Peak 7:
Fangchinoline；Peak 8: tetrandrine；
Fig. 5 shows the HPLC chromatogram of aurantiin reference substance；
Fig. 6 shows compare feature map；Wherein, 13 feature coneincones 8 (S): aurantiin；
Fig. 7 shows the total ion current figure of component C positive ion mode (A) and negative ion mode (B)；
Fig. 8 shows the total ion current figure of G component positive ion mode (A) and negative ion mode (B).
Specific embodiment
The invention discloses the matter of a kind of construction method of neck waist recovering capsule active constituent characteristic spectrum and neck waist recovering capsule
Quantity measuring method, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular
It is that all similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this
Invention.Method and application of the invention is described by preferred embodiment, and related personnel can obviously not depart from this
Method described herein and application are modified or appropriate changes and combinations in summary of the invention, spirit and scope, realizing and
Using the technology of the present invention.
Active constituent provided by the invention the preparation method comprises the following steps:
Petroleum ether degreasing
Neck waist recovering capsule content 500g is taken, 700mL petroleum ether is added, ultrasonic degreasing 3 times, each 20min, filters, filter
Liquid volatilizes solvent, obtains component A.The dregs of a decoction are dry, for use.
Water extracts
By the petroleum ether degreasing dregs of a decoction, 12 times of amount water, refluxing extraction 3h are added, filtration adds 8 times of amount water, refluxing extraction
3h, filtration merge supernatant twice, are concentrated into 500mL, i.e. 1g/mL crude drug concentration, and appropriate 95% ethyl alcohol is added to final concentration of
80% ethyl alcohol, 4 DEG C stand overnight (12h) alcohol precipitation, and precipitating and supernatant are collected in centrifugation respectively, precipitate drying, obtain B component.Supernatant
4 DEG C of liquid storages, stand-by (being used in 2 days).
Ethyl alcohol extracts
Water is mentioned into the dregs of a decoction, is added 6 times of 95% ethyl alcohol of amount, 80 DEG C refluxing extraction 2 times, each 1h, merge extracting solution twice, and
Merge with above-mentioned water extract-alcohol precipitation supernatant, concentration removes ethyl alcohol, obtains component E, adds water to 1.25g/mL crude drug (i.e. 400mL), add
Enter appropriate concentrated hydrochloric acid and adjust pH to 1~2 (1.25g/mL crude drug concentration), 5mol/L NaOH is added and adjusts pH to 11~12, chloroform
Shaking out 3 times, each 400mL, merge chloroform layer.Chloroform layer volatilizes solvent, dry, obtains component C.Water layer is concentrated, dry,
Obtain D component.
Macroporous absorbent resin separation
It is dissolved in D component sample in suitable quantity of water, successively with the water of 3 times of column volumes, 60%, 95% ethanol gradient elution, receives
Collect each eluent, is concentrated respectively, it is dry, obtain F component (water elution), G component (60% ethanol eluate), H component (95% second
Alcohol eluen) sample.Spare, matching while using before use is kept in dark place in all samples in -20 DEG C.
The present invention carries out medicine efficacy screening to the component of acquisition: being tested using mouse hot-plate experiment with acetic acid twisting, investigates neck
The analgesia effect of waist recovering capsule the different extracted parts acts on, and filters out and the comparable active site of product analgesia effect.Using group
Divide scalping method to carry out pharmacodynamic test in animal body, determines the drug activity position of anti-inflammation detumescence and activating microcirculation and removing stasis medicinal.
For active constituent, the present invention provides the construction method of neck waist recovering capsule HPLC characteristic spectrum, method particularly includes:
A kind of I construction method of neck waist recovering capsule HPLC characteristic spectrum, comprising the following steps:
(1) preparation of test solution takes neck waist recovering capsule content 1-10g, accurately weighed, sets in stuffed conical flask, adds
Entering petroleum ether (30~60 DEG C) 5-100mL, is ultrasonically treated (power 250W, frequency 50kHz) 20-60min, filtration, the dregs of a decoction are dry,
Water 5-100mL is added, is heated to reflux 0.5-3h, filters, filtrate is concentrated into about 2-20mL, spare.Water is mentioned into the dregs of a decoction, is added 95%
Ethyl alcohol 10-100mL is heated to reflux 0.5-3h, filtration, and merges with above-mentioned Aqueous extracts, stirs, and centrifugation takes supernatant to be concentrated into
About 2-20mL is added appropriate concentrated hydrochloric acid and adjusts pH to 0.5~4, adds 5mol/LNaOH solution and adjusts pH to 10~13, uses chlorine
Imitative shaking is extracted 2-4 times, each 10-50mL, is merged chloroform solution, is evaporated, residue adds methanol to dissolve, and sets in 5-50mL measuring bottle, adds
Methanol shakes up to scale to get chloroform layer sample.
(2) preparation of reference substance solution takes strychnine reference substance, strychnia reference substance, tetrandrine reference substance and the root of fangji
Promise woods alkali reference substance is appropriate, accurately weighed, is placed in 10mL measuring bottle, adds methanol to dissolve and is diluted to scale, shakes up, and is made every
The 1mL mixed reference substance solution containing 55 μ g of strychnine, 35 μ g of strychnia, 35 μ g of fangchinoline, 30 μ g of tetrandrine respectively.
(3) it measures: accurate absorption reference substance solution and each 5 μ L of test solution respectively, injection liquid chromatograph, measurement,
Record chromatogram to get.9 characteristic peaks should be presented in test sample characteristic spectrum, peak corresponding with object of reference peak is the peak S, is calculated
The relative retention time of each characteristic peak and the peak S, should be within the scope of ± the 5% of specified value.Relative retention time specified value is
0.26 (peak 1), 0.69 (peak 2), 0.89 (peak 3), 0.91 (peak 4), 1.00 (peak S), 1.90 (peaks 6), 1.97 (peaks 7), 2.10 (peaks
8), 2.37 (peak 9).
The chromatographic condition of the high performance liquid chromatography measurement are as follows: using octadecylsilane chemically bonded silica as filler；With first
Alcohol is mobile phase A, using 0.3% formic acid as Mobile phase B；Detection wavelength is 250~300nm, and flow velocity is 0.5~1.5mLmin-1,
Sample volume be 5~20 μ l, 20~40 DEG C of column temperature；It is preferred that Detection wavelength is 275nm.Number of theoretical plate should not by the calculating of Specnuezhenide peak
Lower than 3000.
Gradient elution is carried out according to following elution program:
The present invention also provides a kind of II construction methods of neck waist recovering capsule HPLC characteristic spectrum, comprising the following steps:
(1) preparation of test solution takes neck waist recovering capsule content 1-10g, accurately weighed, sets in stuffed conical flask, adds
Entering petroleum ether (30~60 DEG C) 5-100mL, is ultrasonically treated (power 250W, frequency 50kHz) 20-60min, filtration, the dregs of a decoction are dry,
Water 5-100mL is added, is heated to reflux 0.5-3h, filters, filtrate is concentrated into about 2-20mL, spare.Water is mentioned into the dregs of a decoction, is added 95%
Ethyl alcohol 10-100mL is heated to reflux 0.5-3h, filtration, and merges with above-mentioned Aqueous extracts, stirs, and centrifugation takes supernatant to be concentrated into
About 2-20mL is added appropriate concentrated hydrochloric acid and adjusts pH to 0.5~4, adds 5mol/LNaOH solution and adjusts pH to 10~13, uses chlorine
Imitative shaking is extracted 2-4 times, each 10-50mL, is merged upper layer aqueous solution, is concentrated into about 2mL, passes through D101 type macroporous absorbent resin
Column (internal diameter 1.5cm, pillar height 10cm) is eluted with water 60mL, is discarded aqueous, then eluted with 60% 20~100mL of ethyl alcohol, is received
Collect eluent, be evaporated, residue adds 60% ethyl alcohol to dissolve, and sets in 5~50mL measuring bottle, adds 60% ethyl alcohol to scale, shake up to get
60% alcohol layer active component test solution.
(2) preparation of reference substance solution takes aurantiin reference substance appropriate, accurately weighed, adds methanol that every 1mL is made containing shaddock ped
The reference substance solution of glycosides 0.5mg to get.
(3) it measures: accurate absorption reference solution and each 10 μ L of test solution respectively, injection liquid chromatograph, measurement,
Record chromatogram to get.13 characteristic peaks should be presented in test sample characteristic spectrum, peak corresponding with object of reference peak is the peak S, is calculated
The relative retention time of each characteristic peak and the peak S, relative retention time should be within ± the 5% of specified value.Specified value is 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 (peaks 9), 1.37 (peaks 10), 1.39 (peaks 11), 1.47 (peaks 12), 1.50 (peaks 13).
The chromatographic condition of the high performance liquid chromatography measurement are as follows: using octadecylsilane chemically bonded silica as filler；With second
Nitrile is mobile phase A, using 0.1% formic acid as Mobile phase B；Detection wavelength be 250~300nm, column temperature be 20~40 DEG C, 0.5~
1.5mL·min-1, sample volume is 5~20 μ l；It is preferred that Detection wavelength is 280nm, column temperature is 30 DEG C, flow velocity 1.0mL/min.Reason
2000 should be not less than by calculating by plate number by aurantiin peak.
Gradient elution is carried out according to following elution program:
Time (min) mobile phase A (%) Mobile phase B (%)
0~60 5 → 29 95 → 71
60~70 29 → 40 71 → 60.
The present invention is studied by effective substance, is specified effective component, and effective component is established quality standard, is mentioned
High enterprise is horizontal to the quality-monitoring of neck waist recovering capsule active constituent, it is ensured that product drug effect safely and effectively, quality stable homogeneous.
The quality inspection of the construction method and neck waist recovering capsule of neck waist recovering capsule active constituent characteristic spectrum provided by the invention
Raw materials used and reagent is available on the market in survey method.
Below with reference to embodiment, the present invention is further explained:
Example 1 group point separation
Petroleum ether degreasing
Neck waist recovering capsule content 500g is taken, 700mL petroleum ether is added, ultrasonic degreasing 3 times, each 20min, filters, filter
Liquid volatilizes solvent, obtains component A.The dregs of a decoction are dry, for use.
Water extracts
By the petroleum ether degreasing dregs of a decoction, 12 times of amount water, refluxing extraction 3h are added, filtration adds 8 times of amount water, refluxing extraction
3h, filtration merge supernatant twice, are concentrated into 500mL, i.e. 1g/mL crude drug concentration, and appropriate 95% ethyl alcohol is added to final concentration of
80% ethyl alcohol, 4 DEG C stand overnight (12h) alcohol precipitation, and precipitating and supernatant are collected in centrifugation respectively, precipitate drying, obtain B component.Supernatant
4 DEG C of liquid storages, stand-by (being used in 2 days).
Ethyl alcohol extracts
Water is mentioned into the dregs of a decoction, is added 6 times of 95% ethyl alcohol of amount, 80 DEG C refluxing extraction 2 times, each 1h, merge extracting solution twice, and
Merge with above-mentioned water extract-alcohol precipitation supernatant, concentration removes ethyl alcohol, obtains component E, adds water to 1.25g/mL crude drug (i.e. 400mL), add
Enter appropriate concentrated hydrochloric acid and adjust pH to 1~2 (1.25g/mL crude drug concentration), 5mol/L NaOH is added and adjusts pH to 11~12, chloroform
Shaking out 3 times, each 400mL, merge chloroform layer.Chloroform layer volatilizes solvent, dry, obtains component C.Water layer is concentrated, dry,
Obtain D component.
Macroporous absorbent resin separation
It is dissolved in D component sample in suitable quantity of water, successively with the water of 3 times of column volumes, 60%, 95% ethanol gradient elution, receives
Collect each eluent, is concentrated respectively, it is dry, obtain component E (water elution), G component (60% ethanol eluate), H component (95% second
Alcohol eluen) sample.Spare, matching while using before use is kept in dark place in all samples in -20 DEG C.
2 analgesia effect screening substances model of embodiment
2.1 experimental material
ICR mouse, female, 18~22g of weight are provided by this experimental animal technology Co., Ltd of Changchun hundred million, real
Testing animal productiong licensing is SCXK (Ji) -2011-0004.Neck waist recovering capsule is mentioned by Xiuzheng Pharmaceutical Group Co., Ltd
For.
2.2 instrument and equipment
YLS-6B intelligence hot-plate instrument (Shanghai precision instrumentation Co., Ltd)；(Shanghai Shun's space is permanent for JY2003 electronic balance
Flat scientific instrument Co., Ltd).
2.3 experimental method
2.3.1 the daily raising of experimental animal and management
The daily raising of experimental animal and management are carried out according to animal feeding principle.
2.3.2 dosage
Aspirin clinical medicine dose (0.03g/kg), conversion mouse dosage are that 0.27g/kg (is equivalent to
5.46mg/20g).Neck waist recovering capsule clinical medicine dose (0.05g/kg), conversion mouse dosage are 0.45g/kg (suitable
In 9mg/20g).It is each to extract separated part dosage and neck waist recovering capsule group dosage to make to be comparable between each group
(0.45g/kg) is same.Wherein component C (chloroform layer) contains more alkaloidal constituent, such as strychnine, strychnia, according to document
100% mortality of minimum for reporting Semen Strychni (processed) is 90.0mg/kg, and administration 0.45g/kg may cause animal poisoning death, because
This extracts yield according to alkaloid and is converted into isodose neck waist recovering capsule content, that is, gives 0.01g/kg dry cream, i.e., quite
In extracted isolated component C (chloroform layer) amount of 0.45g/kg capsule 's content.Intragastric administration on mice volume is 20mL/kg, empty
White control group gives isometric physiological saline, once a day.
2.3.3 drug is prepared
Since component A petroleum ether layer fat-soluble ingredient is insoluble in 0.9% normal saline solution, it is molten for selecting edible soybean oil
Drug is dissolved in agent, therefore separately sets up edible soybean oil control group.B group, C group and D group are with the physiology salt of 0.5% sodium carboxymethylcellulose
Aqueous solution is solvent.
2.3.4 mouse hot-plate is tested
After mouse adaptable fed 3d, the female mice for reacting sensitive (10s < pain threshold < 30s) is randomly divided into physiology salt
Water control group, soybean oil control group, aspirin group, neck waist recovering capsule group, A group, B group, C group and D group, every group each 12.Even
Continuous administration 5d is placed on 55 ± 0.5 DEG C of hot plates after last dose 3h, is referred to using the incubation period that mouse licks metapedes reaction as the threshold of pain
Mark measures pain threshold.
2.3.5 mouse acetic acid twisting is tested
Mouse is randomly divided into saline control group, soybean oil control group, aspirin group, neck waist recovering capsule group, A
Group, B group, C group and D group, every group each 12, half male and half female.Successive administration 5d is injected intraperitoneally 0.7% after last dose 30min
Acetum 0.2mL/ only, then observes and records the writhing number of mouse in 15min and starts being averaged for writhing response occur
Time (incubation period).
2.3.6 statistical procedures
17.0 software of this research and utilization SPSS carries out data statistic analysis, and comparison among groups are examined using bilateral independent sample t
It tests.
2.4 results and discussion
2.4.1 mouse hot-plate effective substance screening test
After gastric infusion 5d, groups of animals is in good condition, and hair is smooth, and drinking water diet and stool and urine all go well.Respectively
Group mouse threshold of pain time (s) withIt indicates, data compare between group is analyzed using independent samples t test, the results are shown in Table 1.
1 the different extracted parts of table to mouse hot-plate threshold of pain time test result (N=12)
Note: compared with the control group:###p<0.001、##p<0.01、#p<0.05；Compared with neck waist recovering capsule group:***p<
0.001、**p<0.01、*P < 0.05.A component using soybean oil as drug solvent, compared with soybean oil control group by when Toxicity Analysis
Compared with remaining each administration group is compared with saline control group.
As shown in table 1, compared with soybean oil control group, there was no significant difference for A group (p > 0.05), shows the town component A Zu Wu
Pain acts on；Compared with saline control group, B group, C group, D group all have significant difference (p < 0.001), with neck waist recovering capsule
Group is compared, and the analgesic effect of B, D group is not as good as neck waist recovering capsule group (p < 0.001), the analgesic activity of C group and neck waist recovering capsule group
There was no significant difference (p > 0.05).Test result shows that component C is neck waist recovering capsule analgesia effect active material, and drug effect is made
With suitable with neck waist recovering capsule.
2.4.2 acetic acid twisting test result
After gastric infusion 5d, groups of animals is in good condition, and hair is smooth, and drinking water diet and stool and urine all go well.Respectively
Group mouse writhing number (in 15min) and incubation period (s) withIt indicates, data compare using independent samples t test point between group
Analysis, the results are shown in Table 2.
2 the different extracted parts of table on mouse acetic acid twisting number and incubation period influence test result (N=12)
| Group | Dosage (g/kg) | Writhing number (in 15min) | Incubation period (s) |
| 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### |
| Neck waist recovering capsule group | 0.45 | 18.500±3.177### | 299.583±15.132### |
| A group | 0.45 | 32.167±2.250 | 207.917±12.573 |
| B group | 0.45 | 29.167±1.946##*** | 219.167±8.430 |
| C group | 0.01 | 20.917±2.503### | 303.917±9.120### |
| D group | 0.45 | 27.750±2.179###*** | 215.917±10.431 |
Note: compared with the control group:###p<0.001、##p<0.01、#p<0.05；Compared with neck waist recovering capsule group:***p<
0.001、**p<0.01、*P < 0.05.A component using soybean oil as drug solvent, compared with soybean oil control group by when Toxicity Analysis
Compared with remaining each administration group is compared with saline control group.
As shown in table 2, compared with soybean oil control group, there was no significant difference for A group (p > 0.05), shows the town component A Zu Wu
Pain acts on；Compared with saline control group, B group, D group can obviously reduce mouse writhing number in 15min (p < 0.01, p <
0.001) with extremely significant difference (p<0.001), but compared with neck waist recovering capsule group, on writhing response incubation period without influence (p>
0.05)；Component C group can substantially reduce writhing number (p < 0.001), improve the incubation period (p < 0.001) of writhing response, and and neck
There was no significant difference (p > 0.05) for the analgesic activity of waist recovering capsule group.Test result shows that component C is neck waist recovering capsule antalgesic
Active material is imitated, drug action is suitable with neck waist recovering capsule.
The screening of 3 anti-inflammation detumescence effective substance of embodiment
3.1 experimental material
ICR mouse, male, 18~22g of weight are provided by this experimental animal technology Co., Ltd of Changchun hundred million, real
Testing animal productiong licensing is SCXK (Ji) -2011-0004.
Neck waist recovering capsule (lot number: 160913) is provided by Xiuzheng Pharmaceutical Group Co., Ltd；Dexamethasone acetate (batch
Number: 160313) it is purchased from Tianjin Tianyao Pharmaceutical Co., Ltd.；Dimethylbenzene (lot number: 20161020) is purchased from Beijing Chemical Plant.
3.2 instrument and equipment
JY2003 electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd.).
3.3 experimental method
(1) the daily raising of experimental animal and management
The daily raising of experimental animal and management are carried out according to animal feeding principle.
(2) dosage
It is converted into isodose neck waist recovering capsule content according to each extraction separation component yield, intragastric administration on mice is carried out and gives
Medicine.
Mouse is randomly divided into blank control group, model group, dexamethasone acetate tablets group (150mg/kg, neck waist recovering capsule group
(450mg/kg), E group (extract yield according to component E, are converted into isodose neck waist recovering capsule content 450*17.21%=
77mg/kg), C group (15mg/kg), D group (63mg/kg), G group (13mg/kg) and C+G group (27mg/kg).
Intragastric administration on mice volume is 20mL/kg, and blank control group and model group give isometric physiological saline, daily one
It is secondary.
(3) mouse ear swelling test
After male mice adaptable fed 3d, it is randomly divided into blank control group, model group, Dexamethasone group, neck waist health glue
Capsule group, C group, D group, E group, G group, C+G group, every group each 12.Successive administration 5d, administration 3d, dimethylbenzene first time sensitization,
100% dimethylbenzene, 20 μ L/ is only applied to two sides inside and outside auris dextra；After last dose 1h, every animal etherization, then with 100% 2
20 μ L/ of toluene is only applied to two sides inside and outside auris dextra, and left ear with no treatment, puts to death animal after 30min, cuts ears punching
Device lays round auricle, electronic analytical balance weighing at same position respectively.
Mouse ear swelling degree=auris dextra sheet weight-left auricle weight
Swelling rate=(auris dextra quality-left ear quality)/left ear quality × 100%
Inhibiting rate=(model group be averaged swelling-administration group be averaged swelling)/model group is averaged swelling × 100%
(5) statistical procedures
17.0 software of this research and utilization SPSS carries out data statistic analysis, and comparison among groups are examined using bilateral independent sample t
It tests.
3.4 results and discussion
After gastric infusion 5d, groups of animals is in good condition, and hair is smooth, and drinking water diet and stool and urine all go well.Respectively
Group mouse ear swelling degree (mg) withIt indicates, data compare between group is analyzed using independent samples t test, the results are shown in Table 3.
As shown in table 3, compared with blank control group, model group swelling significantly increases (p < 0.01), shows model copy
Success.Compared with model group, dexamethasone acetate group and neck waist recovering capsule group significantly reduce dimethylbenzene induced mice ear swelling (p <
0.01), show that neck waist recovering capsule has significant anti-inflammation detumescence effect.Compared with model group, C group, D group, E group, G group and C+G
Group all has significant difference (p < 0.01), compared with neck waist recovering capsule group, C group, D group and G group have significant difference (p <
0.01), without significant difference (p > 0.05), test result shows that component C and G component have synergistic effect for E group and C+G group, secondly
Anti-inflammation detumescence effect when being respectively used alone can be improved in the mixed component of person, is neck waist recovering capsule anti-inflammation detumescence drug activity
Substance, drug action are suitable with neck waist recovering capsule.
3 the different extracted parts mouse auricle swelling test result of table (N=12)
Note: compared with blank control group:##p<0.01、#p<0.05；Compared with model group:**p<0.01、*p<0.05；With neck
Waist recovering capsule group compares:△△p<0.01、△p<0.05；
The promoting blood circulation drug effect screening substances of embodiment 4
4.1 experimental material
ICR mouse, half male and half female, 18~22g of weight, by Changchun hundred million, this experimental animal technology Co., Ltd is mentioned
For experimental animal production licence is SCXK (Ji) -2011-0004.
Neck waist recovering capsule (lot number: 160913) is provided by Xiuzheng Pharmaceutical Group Co., Ltd；Red tablet (lot number:
160805) it is purchased from Shenyang Hongyao Pharmaceutical Co., Ltd..
4.2 instrument and equipment
JY2003 electronic balance (Shanghai Sunny Hengping Scientific Instrument Co., Ltd.).
4.3 experimental method
(1) the daily raising of experimental animal and management
The daily raising of experimental animal and management are carried out according to animal feeding principle.
(2) dosage
Mouse dosage is 0.16g/kg (being equivalent to 3.15mg/20g) by weight of red tablet；Other groups of dosages
With (2) under 3.3.
(3) mouse cuts tail test
Mouse is randomly divided into blank control group, red tablet group, neck waist recovering capsule group, component E group, it is every group each 12, female
It is male fifty-fifty.Mouse Tail-tip is cut 2mm with scissors after last dose 3h by successive administration 7d, when blood from overflowing, is opened with stopwatch
Beginning timing sucks drop of blood until blood no longer overflows with filter paper every 30s, calculates the bleeding time.
Mouse is randomly divided into blank control group, red tablet group, neck waist recovering capsule group, C group, D group, E group, G group and C+G
Group, every group each 12, half male and half female.Mouse Tail-tip is cut 2mm with scissors after last dose 3h, to blood by successive administration 7d
When hydrorrhea goes out, start timing with stopwatch, suck drop of blood until blood no longer overflows with filter paper every 30s, when calculating bleeding
Between.
(4) statistical procedures
17.0 software of this research and utilization SPSS carries out data statistic analysis, and comparison among groups are examined using bilateral independent sample t
It tests.
4.4 results and discussion
After gastric infusion 7d, groups of animals is in good condition, and hair is smooth, and drinking water diet and stool and urine all go well.Respectively
Group mouse bleeding time (min) withIt indicates, data compare between group is analyzed using independent samples t test, the results are shown in Table 4.
As shown in table 4, compared with blank control group, red tablet group significantly extends with neck waist recovering capsule group cuts the tail bleeding time
(p < 0.01) shows that neck waist recovering capsule has significant blood circulation invigorating efficacies.Compared with blank control group, C group, D group, E group, G
Group and C+G group group all have significant difference (p < 0.01), and compared with neck waist recovering capsule group, C group, D group and G group have conspicuousness
Without significant difference (p>0.05), test result shows that component C has with G component and cooperates with work for difference (p<0.01), E group and C+G group
With blood circulation invigorating efficacies when being respectively used alone can be improved in the mixed component of the two, are the recovering capsule activating microcirculation and removing stasis medicinal of neck waist
Drug activity substance, drug action are suitable with neck waist recovering capsule.
4 the different extracted parts mouse of table cut tail test bleeding time result (N=12)
Note: compared with blank control group:##p<0.01、#p<0.05；Compared with neck waist recovering capsule group:△△p<0.01、△p<0.05
In conclusion the effective substance of the analgesia of neck waist recovering capsule, anti-inflammation detumescence and promoting blood circulation includes component C and G component, this hair
The more than bright foundation characteristic spectrum of two component effective components.
The building of 5 neck waist recovering capsule HPLC characteristic spectrum I of embodiment
1, I detection method of neck waist recovering capsule HPLC characteristic spectrum
(1) preparation of test solution takes neck waist recovering capsule content 5g, accurately weighed, sets in stuffed conical flask, is added
Petroleum ether (30~60 DEG C) 50mL is ultrasonically treated (power 250W, frequency 50kHz) 30min, and filtration, the dregs of a decoction are dry, and water is added
50mL is heated to reflux 2h, and filtration, filtrate is concentrated into about 10mL, spare.Water is mentioned into the dregs of a decoction, 95% ethyl alcohol 50mL is added, heats back
1h, filtration are flowed, and is merged with above-mentioned Aqueous extracts, is stirred, centrifugation takes supernatant to be concentrated into about 10mL, appropriate concentrated hydrochloric acid tune is added
PH to 1~2 is saved, 5mol/L NaOH solution is added and adjusts pH to 11~12, is extracted 3 times, each 20mL, is closed with chloroform shaking
And chloroform solution, it is evaporated, residue adds methanol to dissolve, and sets in 25mL measuring bottle, adds methanol to scale, shakes up to get chloroform layer sample.
(2) preparation of reference substance solution takes strychnine reference substance, strychnia reference substance, tetrandrine reference substance and the root of fangji
Promise woods alkali reference substance is appropriate, accurately weighed, is placed in 10mL measuring bottle, adds methanol to dissolve and is diluted to scale, shakes up, and is made every
The 1mL mixed reference substance solution containing 55 μ g of strychnine, 35 μ g of strychnia, 35 μ g of fangchinoline, 30 μ g of tetrandrine respectively.
(3) it measures: accurate absorption reference substance solution and each 5 μ L of test solution respectively, injection liquid chromatograph, measurement,
Record chromatogram to get.
The chromatographic condition of high performance liquid chromatography measurement are as follows: using octadecylsilane chemically bonded silica as filler；It is with methanol
Mobile phase A carries out gradient elution by the elution program in table 5 using 0.3% formic acid as Mobile phase B:
Table 5
Detection wavelength is 275nm, flow velocity 1.0mLmin-1, 30 DEG C of column temperature.Number of theoretical plate is answered by the calculating of Specnuezhenide peak
Not less than 3000.
9 characteristic peaks should be presented in test sample characteristic spectrum, peak corresponding with object of reference peak is the peak S, calculates each characteristic peak
It, should be within the scope of ± the 5% of specified value with the relative retention time at the peak S.Relative retention time specified value be 0.26 (peak 1),
0.69 (peak 2), 0.89 (peak 3), 0.91 (peak 4), 1.00 (peak S), 1.90 (peaks 6), 1.97 (peaks 7), 2.10 (peaks 8), 2.37 (peaks 9).
2, methodological study
(1) precision test
Same batch of sample is taken, test solution is made by sample solution preparation method, continuous sample introduction 6 times, records feature
Map.The experimental results showed that the relative retention time RSD of each chromatographic peak is within 5%.
6 precision tests of table-relative retention time
(2) repeatability is investigated
Same batch of sample is taken, prepares 6 parts of test solutions in parallel, successively sample introduction, records characteristic spectrum.The experimental results showed that
For the RSD of the relative retention time of each chromatographic peak within 5%, reproducibility is good.
7 repetitive tests of table-relative retention time
(3) study on the stability
Same sample is taken, the test solution of preparation, respectively 0,2,8,12,18, sample introduction, records characteristic spectrum for 24 hours.It is real
Test the result shows that the RSD of the relative retention time of each chromatographic peak is within 5%, illustrate in test solution ingredient for 24 hours with
Interior stabilization.
8 stability tests of table-relative retention time
3, the foundation of characteristic spectrum common pattern and part share pointing out for peak
According to the preparation method of test solution, 10 batch test solutions are prepared, according to above-mentioned 5 μ of chromatographic condition sample introduction
L measures map.The map of 10 batch samples is imported to " the chromatographic fingerprints of Chinese materia medica similarity evaluation of Chinese Pharmacopoeia Commission
System software (2012 editions) " generates characteristic spectrum common pattern, wherein characteristic peak 9.By being compared with retention time of reference substance
Analysis determines that No. 2 peaks are strychnine alkali, and No. 5 peaks are strychnia, and No. 7 peaks are fangchinoline, and No. 8 peaks are tetrandrine.
4,10 batch chloroform layer active component characteristic spectrum relative retention times compare
Common pattern is established to 10 different batches neck waist health finished product chloroform layer active component maps using mean vector method,
And relative retention time is calculated, obtaining each map with the relative retention time analytical table of map, analysis is compareed the results are shown in Table 9.
Accordingly, it is determined that the relative retention time specified value of characteristic peak be 0.26 (peak 1), 0.69 (peak 2), 0.89 (peak 3),
0.91 (peak 4), 1.00 (peak S), 1.90 (peaks 6), 1.97 (peaks 7), 2.10 (peaks 8), 2.37 (peaks 9).
9 10 batch chloroform layer characteristic peak relative retention time of table
The building of 6 neck waist recovering capsule HPLC characteristic spectrum II of embodiment
1, II detection method of neck waist recovering capsule HPLC characteristic spectrum
The preparation of test solution takes this product content 5g, accurately weighed, sets in stuffed conical flask, and petroleum ether (30 is added
~60 DEG C) 50mL, it is ultrasonically treated (power 250W, frequency 50kHz) 30min, filtration, the dregs of a decoction are dry, and water 50mL is added, heats back
2h is flowed, filtration, filtrate is concentrated into about 10mL, spare.Water is mentioned into the dregs of a decoction, 95% ethyl alcohol 50mL is added, is heated to reflux 1h, is filtered,
And merge with above-mentioned Aqueous extracts, it stirs, centrifugation takes supernatant to be concentrated into about 10mL, and appropriate concentrated hydrochloric acid is added and adjusts pH to 1~2,
It adds 5mol/L NaOH solution and adjusts pH to 11~12, extracted 3 times, each 20mL with chloroform shaking, it is water-soluble to merge upper layer
Liquid is concentrated into about 2mL, by D101 type large pore resin absorption column (internal diameter 1.5cm, pillar height 10cm), is eluted with water 60mL,
Aqueous is discarded, then is eluted with 60% ethyl alcohol 60mL, eluent is collected, is evaporated, residue adds 60% ethyl alcohol to dissolve, and sets 25mL measuring bottle
In, add 60% ethyl alcohol to scale, shakes up to get 60% alcohol layer active component test solution.
The preparation of reference substance solution takes aurantiin reference substance appropriate, accurately weighed, adds methanol that every 1mL is made containing aurantiin
The reference substance solution of 0.5mg to get.
Measurement: it is accurate respectively to draw reference solution and each 10 μ L of test solution, liquid chromatograph is injected, is measured, note
Record chromatogram to get.
The chromatographic condition of high performance liquid chromatography measurement are as follows: using octadecylsilane chemically bonded silica as filler；It is with acetonitrile
Mobile phase A carries out gradient elution by the elution program in table 10 using 0.1% formic acid as Mobile phase B；
Table 10
Detection wavelength is 280nm, and column temperature is 30 DEG C, flow velocity 1.0mL/min.Number of theoretical plate should not by the calculating of aurantiin peak
Lower than 2000.
13 characteristic peaks should be presented in test sample characteristic spectrum, peak corresponding with object of reference peak is the peak S, calculates each characteristic peak
With the relative retention time at the peak S, relative retention time should be within ± the 5% of specified value.Specified value 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 (peaks
9), 1.37 (peaks 10), 1.39 (peaks 11), 1.47 (peaks 12), 1.50 (peaks 13).
2, methodological study
(1) precision test
Same batch of sample is taken, test solution is made by sample solution preparation method, continuous sample introduction 6 times, records feature
Map.The experimental results showed that the relative retention time RSD of 13 chromatographic peaks is within 5%.
11 precision tests of table-relative retention time
(2) repeatability is investigated
Same batch of sample is taken, prepares 6 parts of test solutions in parallel, successively sample introduction, records characteristic spectrum.The experimental results showed that
For the RSD of the relative retention time of 13 chromatographic peaks within 5%, reproducibility is good.
12 repetitive tests of table-relative retention time
(3) study on the stability
Same sample is taken, test solution is prepared, respectively 0,4,8,12,18, sample introduction, records characteristic spectrum for 24 hours.Experiment
The result shows that the RSD of the relative retention time of each 13 chromatographic peaks illustrates that ingredient is for 24 hours in test solution within 5%
Within stablize.
13 stability tests of table-relative retention time
3, the foundation of characteristic spectrum map common pattern and part share pointing out for peak
According to the preparation method of test solution, the test solution of 10 batch finished products is prepared, according to above-mentioned chromatographic condition
10 μ L of sample introduction records characteristic spectrum.The characteristic spectrum of 10 batch samples is imported to " the Chinese medicine chromatographic fingerprint of Chinese Pharmacopoeia Commission
Map similarity evaluation system software (2012 editions) ", generate characteristic spectrum common pattern, demarcate 13 characteristic peaks, by with it is right
According to product retention time comparative analysis, determine that No. 8 peaks are aurantiin.
4,10 batch, 60% alcohol layer active component characteristic spectrum relative retention time compares
10 60% alcohol layer active component maps of different batches neck waist health finished product are established using mean vector method shared
Mode, and relative retention time is calculated, obtain each map the results are shown in Table with the relative retention time analytical table of map, analysis is compareed
14。
Accordingly, it is determined that the relative retention time specified value of characteristic peak 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 (peaks 9), 1.37 (peaks 10), 1.39
(peak 11), 1.47 (peaks 12), 1.50 (peaks 13).
14 10 batch of table, 60% alcohol layer characteristic spectrum relative retention time
7 composition and effectiveness interpretation of mass spectra of embodiment
Component C interpretation of mass spectra
Chloroform layer component-component C positive ion mode (A) and negative ion mode (B) total ion current figure are shown in Fig. 7, analyze result
It is shown in Table 7.It is analyzed by first mass spectrometric, second order ms and relevant references, structure elucidation and identification has been carried out to each peak, altogether
Identify 13 ingredients.Wherein No. 1 is strychnine, and No. 2 are strychnia, and No. 3 are pseudobrucine, and No. 4 are vomicine, is returned
Belong to Semen Strychni (processed) medicinal material；No. 5 are phellodendrine, and No. 6 are fangchinoline, and No. 7 are tetrandrine, belong to root of fangji medicinal material；No. 8 are thick stick
Willow aglycon belongs to cortex periplocae medicinal material；No. 9 are four keto-alcohol of lycopod, belong to lycopodium calvatum medicinal material；No. 10 are 11- keto-β-boswellic acid,
No. 11 are 11- carbonyl-beta-acetyl masticinic acid, and No. 12 are elemonic acid, and No. 13 are acetyl group-α-masticinic acid, belong to olibanum medicinal material.
The chemical component qualification result of 15 component C of table
G component interpretation of mass spectra
G component positive ion mode (A) and negative ion mode (B) total ion current figure are shown in Fig. 8, and analysis the results are shown in Table 8.Pass through one
Grade mass spectrum, second order ms and relevant references are analyzed, and are carried out structure elucidation and identification to each peak, are identified 7 ingredients altogether.Its
In No. 1 be adenine, belong to pheretima medicinal material；No. 2 are hydroxyl radical carthamin yellow carthamus A, and No. 3 are benzoic acid, belong to flos carthami；No. 5
For phellodendrine, belong to root of fangji medicinal material；No. 6 are Neoeriocitrin, and No. 7 are aurantiin, belong to rhizome of davallia medicinal material；No. 9 are ginger shape
Panax Notoginseng saponin R1, belong to radix achyranthis bidentatae medicinal material.
The chemical component qualification result of 16 G component of table
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of construction method of neck waist recovering capsule active constituent characteristic spectrum, which comprises the steps of:
Step 1: taking neck waist recovering capsule to extract and obtain extract, the active constituent of neck waist recovering capsule is obtained through pharmacodynamic test；
Step 2: the active constituent being taken to prepare test solution；
Step 3: obtaining reference substance solution；
Step 4: taking the test solution and the reference substance solution through high effective liquid chromatography for measuring respectively, obtain neck waist health
The characteristic spectrum of Capsule Active ingredient；
The active constituent is strychnia and/or aurantiin；
The sequence of step 2 and step 3 is in no particular order.
2. construction method as described in claim 1, which is characterized in that the extraction are as follows: take neck waist recovering capsule content and stone
The mixing of oily ether, ultrasonic extraction, filtering collect the dregs of a decoction, mixs, be heated to reflux with water, filter, and collect filtrate, spare, by the dregs of a decoction and
The mixing of 95% ethyl alcohol, is heated to reflux, and filters, merges with the filtrate, is centrifuged, supernatant is taken to be concentrated, adjusting pH value to 0.5~4,
PH value is adjusted again to 10~13, is extracted 2~4 times with chloroform shaking, is merged chloroform extracted solution.
3. construction method as claimed in claim 2, which is characterized in that the active constituent is strychnia.
4. construction method as described in any one of claims 1 to 3, which is characterized in that the color of the high performance liquid chromatography measurement
Spectral condition are as follows: using octadecylsilane chemically bonded silica as filler；Using methanol as mobile phase A, using 0.3% formic acid as Mobile phase B,
Detection wavelength is 250~300nm, and flow velocity is 0.5~1.5mLmin-1, sample volume is 5~20 μ l, 20~40 DEG C of column temperature, theoretical
Plate number is calculated by strychnia peak should be not less than 3000；
Gradient elution is carried out according to following elution program:
5. such as the described in any item construction methods of Claims 1-4, which is characterized in that the characteristic spectrum includes 9 features
Peak, wherein No. 2 peaks: strychnine alkali；No. 5 peaks: strychnia；No. 7 peaks: fangchinoline；No. 8 peaks: tetrandrine；
The characteristic spectrum No. 5 peaks corresponding with reference substance peak are the peak S, obtain relative retention time, the relative retention time
Should be within ± the 5% of specified value, 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.
6. construction method as described in claim 1, which is characterized in that the active constituent is aurantiin.
7. construction method as claimed in claim 6, which is characterized in that the extraction are as follows: take neck waist recovering capsule content and stone
The mixing of oily ether, ultrasonic extraction, filtering collect the dregs of a decoction, mixs, be heated to reflux with water, filter, and collect filtrate, spare, by the dregs of a decoction and
The mixing of 95% ethyl alcohol, is heated to reflux, and filters, merges with the filtrate, is centrifuged, and takes supernatant, adjusting pH value to 0.5~4, then adjust
PH value is saved to 10~13, is shaken and is extracted with chloroform, merged upper layer aqueous solution and be washed with water by D101 type large pore resin absorption column
It is de-, water lotion is discarded, then eluted with 60% 20~100mL of ethyl alcohol, collects eluent.
8. construction method as claimed in claims 6 or 7, which is characterized in that the chromatographic condition of the high performance liquid chromatography measurement
Are as follows: using octadecylsilane chemically bonded silica as filler；Using acetonitrile as mobile phase A, using 0.1% formic acid as Mobile phase B；Detect wave
A length of 250~300nm, column temperature are 20~40 DEG C, 0.5~1.5mLmin-1, sample volume is 5~20 μ l；Number of theoretical plate presses shaddock ped
Glycosides peak, which calculates, should be not less than 2000；
Gradient elution is carried out according to following elution program:
Time (min) mobile phase A (%) Mobile phase B (%)
0~60 5 → 29 95 → 71
60~70 29 → 40 71 → 60.
9. such as the described in any item construction methods of claim 6 to 8, which is characterized in that the characteristic spectrum includes 13 features
Peak, wherein No. 8 peaks: aurantiin；
The characteristic spectrum No. 8 peaks corresponding with reference substance peak are the peak S, obtain the relative retention time of each characteristic peak Yu the peak S, institute
Stating relative retention time should be within ± the 5% of specified value, the specified value are as follows: 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.
10. the quality determining method of neck waist recovering capsule, which is characterized in that according to building as described in any one of claim 1 to 9
Method obtains neck waist recovering capsule active constituent characteristic spectrum, to the characteristic spectrum of sample to be tested and the neck waist recovering capsule activity at
Characteristic spectrum is divided to carry out similarity evaluation, relative retention time is qualified products ± the 5% of specified value.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811243104.XA CN109270203B (en) | 2018-10-24 | 2018-10-24 | Construction method of characteristic spectrum of active ingredients of Jingyaokang capsule and quality detection method of Jingyaokang capsule |
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| CN110988159A (en) * | 2019-11-25 | 2020-04-10 | 通化卫京药业股份有限公司 | Method for measuring contents of multiple components of Jingyaokang capsule |
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