CN113933445A

CN113933445A - A kind of Dendrobium standard decoction quality control method

Info

Publication number: CN113933445A
Application number: CN202111296922.8A
Authority: CN
Inventors: 何述金; 周代俊; 黄黎明; 周雪生; 周乐学; 喻艳; 朱美成
Original assignee: Changsha Xinlin Pharmaceutical Co ltd; HUNAN XINHUI PHARMACEUTICAL CO Ltd
Current assignee: Changsha Xinlin Pharmaceutical Co ltd; HUNAN XINHUI PHARMACEUTICAL CO Ltd
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2022-01-14

Abstract

The invention provides a quality control method for dendrobium standard decoction. For content determination, the content of schaffotaside in the proposed standard decoction is in the range of 0.4-1.20 mg/g; among them, the extraction rate of dry extract is determined by decoction method; TLC is used for identification; Determination by heat soaking method; characteristic map and determination of schaffoside content were determined by liquid chromatography. The properties of Dendrobium standard decoction, the extraction rate of dry extract, the identification of thin layers, the extracts, the characteristic map and the content determination were studied, which can effectively control the quality of Dendrobium standard decoction.

Description

Quality control method for dendrobium standard decoction

Technical Field

The invention relates to the technical field of quality control of traditional Chinese medicinal materials, in particular to a quality control method of a dendrobium standard decoction.

Background

Dendrobium fimbriatum Hook is a fresh or dry stem of Dendrobium plant of Orchidaceae, mainly produced from southwest to northwest of China, and distributed in India, Nipol, Centario, Plumbum preparatium, Burma, Thailand, Vietnam, etc. Dendrobium fimbriatum is one of medicinal dendrobium nobile recorded in 2010 version of Chinese pharmacopoeia, also called as Dendrobium fimbriatum, has the effects of benefiting stomach, promoting the production of body fluid, nourishing yin and clearing heat, and can be used for treating fever body fluid impairment, anorexia, retching, persistent asthenic fever after illness, hyperactivity of fire due to yin deficiency, bone steaming, fatigue heat, dim eyesight and flaccidity of bones and muscles. The chemical components of the composition mainly comprise polysaccharide, bibenzyl, phenanthrene, emodin anthraquinone, coumarin, steroid and the like, wherein the polysaccharide is one of the main components. Modern pharmacological research shows that the polysaccharide has the functions of enhancing the immunity of organisms, reducing blood sugar, resisting tumors, resisting aging, resisting oxidation and the like, and has higher medicinal value.

At present, the dendrobium fimbriatum medicinal material has not established relevant standards. However, the existing liquid chromatography method is only used for detecting that the dendrobium decoction is defective, and the quality control requirement of the traditional Chinese medicine formula granules cannot be met. Therefore, the establishment of a quality control method of the dendrobium standard decoction is necessary, and is more beneficial to the quality control of the dendrobium medicinal material.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art. Therefore, the invention provides a quality control method of dendrobe standard decoction, and aims to conveniently and effectively evaluate and control the internal quality of dendrobe medicinal materials.

Based on the aim, the invention provides a quality control method of a dendrobium standard decoction, which is characterized in that the charaftoside content range of the standard decoction is drawn up to be 0.4-1.20mg/g by the characteristics of the dendrobium standard decoction, the dry extract yield, the thin layer identification, the extract, the characteristic spectrum and the charaftoside content measurement; wherein, the extraction rate of the dry extract is measured by adopting a decoction method; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; the characteristic spectrum and the content of schaftoside are measured by liquid chromatography.

The limiting range of the dry extract yield is 8.5-15.0%.

The thin layer chromatography comprises the following steps:

a1, adding methanol into herba Dendrobii decoction, performing ultrasonic treatment, filtering, evaporating filtrate, and dissolving residue with methanol to obtain sample solution a;

a2 ultrasonic processing herba Dendrobii with methanol, filtering, evaporating filtrate, and dissolving residue with methanol to obtain control solution a;

a3, setting thin-layer chromatography conditions: silica gel G thin layer plate; sample amount of spotting: the test solution a and the reference solution a are both 10 ul; developing agent: mixing petroleum ether and ethyl acetate in a volume ratio of 3: 2; color development: heating the mixture to a 10% sulfuric acid ethanol solution at 105 ℃ until spots are clearly developed.

The hot dipping method uses ethanol as solvent, and the extract content measured by the hot dipping method in the alcohol-soluble extract measuring method is not less than 23.5%.

The characteristic spectrum and schaftoside content determination method comprises respectively sucking herba Dendrobii reference substance solution b and test solution b, injecting into liquid chromatograph, and determining; wherein, the adopted chromatographic conditions are as follows: a chromatographic column: shim-pack GIST; mobile phase: performing gradient elution by using acetonitrile as a mobile phase A and using 0.1% phosphoric acid solution as a mobile phase B; flow rate: 0.8 mL/min; column temperature: 40 ℃; detection wavelength: 340 nm.

The test solution b is prepared by collecting herba Dendrobii standard decoction sample, adding 25 times of 50% methanol, sealing, subjecting to ultrasound, adding methanol to reduce weight loss, shaking, and filtering.

The method for measuring the characteristic spectrum by adopting the liquid chromatography further comprises the steps of injecting the test samples of the same batch into the liquid chromatograph, measuring and evaluating the similarity of the established common characteristic peaks.

The method for measuring the characteristic spectrum and the content of schaftoside by adopting the liquid chromatography further comprises the steps of feeding samples of the test samples of the same batch for 0h, 2h, 4h, 8h, 12h and 24h respectively, measuring the peak shape and the peak number, and evaluating the similarity of the common characteristic peaks for testing the stability of the test sample solution.

The method for measuring the characteristic spectrum and the content of schaftoside by adopting the liquid chromatography further comprises the steps of continuously feeding 6-needle samples into the samples to be measured in the same batch to measure the peak shape and the peak number, and evaluating the similarity of the common characteristic peaks for testing whether the precision of a chromatograph is good or not.

The method for measuring the content of schaftoside by adopting the liquid chromatography also comprises the step of weighing a test sample, adding the test sample into a schaftoside reference solution, and testing the average sampling recovery rate of schaftoside.

The invention has the beneficial effects that:

1. according to the invention, the characteristics of the dendrobium standard decoction, the dry extract yield, the thin-layer identification, the extract, the characteristic map and the content measurement are researched, so that the quality of the dendrobium standard decoction can be effectively controlled.

2. The dendrobium standard decoction is prepared by ultrasonically extracting dendrobium, the average content of schaftoside is 0.74mg/g, the measured content range is 0.537-0.919 mg/g, and SD is 0.13; calculated according to the mean value +/-3 SD, the allowable range of the content of schaftoside is 0.35-1.13 mg/g. Therefore, the content range of schaftoside in the standard decoction is drawn as follows: 0.40 mg/g-1.20 mg/g. The results show that schaftoside and the transfer rate thereof in 15 batches of standard decoction are within the allowable range, and can provide reference basis for the quality research of dendrobium (dendrobium fimbriatum) formula granules.

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, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a TLC pattern of a 15 lot standard decoction of the present invention; wherein, A, negative control; s, comparing the medicinal materials; 1-15, a test article;

FIG. 2 is a linear survey of schaftoside.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.

It is to be noted that technical terms or scientific terms used in the embodiments of the present invention should have the ordinary meanings as understood by those having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined.

The invention provides a quality control method of a dendrobium standard decoction, which is characterized in that the content range of schaftoside of the standard decoction is drawn up to be 0.4-1.20mg/g through the characteristics of the dendrobium standard decoction, the extraction rate of dry extract, thin layer identification, extract, characteristic spectrum and schaftoside content measurement; wherein, the extraction rate of the dry extract is measured by adopting a decoction method; thin-layer identification is carried out by adopting thin-layer chromatography; measuring the extract by adopting a hot dipping method; the characteristic spectrum and the content of schaftoside are measured by liquid chromatography. The following is a description by specific examples.

The preparation method comprises the following steps: referring to the management standard of traditional Chinese medicine decoction rooms of medical institutions (No. 2009 of the State administration of traditional Chinese medicine) relevant parameters such as a pretreatment method, decoction times, water addition amount, decoction time and the like are fixed for decoction, and then solid-liquid separation, concentration and drying are carried out.

The paste yield is as follows: taking 15 batches of dendrobium fimbriatum (dendrobium fimbriatum) decoction pieces, preparing 15 batches of standard decoction dry extract powder according to the preparation method, calculating the dry extract yield (see table 1 below) by using the dry extract powder, calculating the average yield to be 11.23%, calculating according to the standard limit allowable range (the average value is 70% -130%), wherein the allowable range of the plaster yield is 7.861% -14.599%, so that the allowable range of the standard decoction of the dendrobium fimbriatum (dendrobium fimbriatum) decoction pieces is drawn up to be 8.5% -15.0%.

TABLE 1 standard decoction yield of herba Dendrobii (herba Dendrobii) decoction pieces

The results show that the cream yield of the 15 batches of standard decoction is 8.9-14.8 percent and all the standard decoction meet the set limit range of 8.5-15.0 percent.

The characteristics are as follows: the 15 batches of standard decoction were described as a grayish yellow to yellowish brown powder according to their physical characteristics; light smell, slightly bitter and sweet taste.

Thin-layer identification: the product is a dry extract of single-flavor decoction piece dendrobium (dendrobium fimbriatum), the thin-layer identification method of dendrobium fimbriatum is established by referring to the method for identifying the thin-layer of dendrobium fimbriatum under the identification item of dendrobium in Chinese pharmacopoeia and taking dendrobium (dendrobium fimbriatum) as a reference, and the thin-layer identification method is established by testing 15 batches of samples, so that the spot of the test sample is clear, and the negative reference sample is free of interference, thus the product identification item is drawn up. The test methods and results are as follows:

the test method comprises the following steps: performing thin layer chromatography (China pharmacopoeia 2020 edition four-part general rule 0502)

Preparing a test solution: taking about 0.5g of the product, adding 25ml of methanol, carrying out ultrasonic treatment for 45 minutes, filtering, evaporating filtrate to dryness, and adding 5ml of methanol into residues for dissolving to obtain the product.

Control solution: collecting 1.5g of radix Cudraniae (fructus Broussonetiae) reference medicinal material, and making into reference medicinal material solution by the same method.

Thin-layer chromatography conditions: thin-layer plate: silica gel G thin layer plate; sample amount of spotting: the test solution is 10ul, and the reference solution is 10 ul; developing agent: petroleum ether (60-90 ℃) -ethyl acetate (3: 2); color development: heating the mixture to a 10% sulfuric acid ethanol solution at 105 ℃ until spots are clearly developed.

As a result: in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution. The TLC pattern of the 15 batches of standard decoction is shown in FIG. 1.

And (3) extract determination: taking 15 batches of standard decoction, taking ethanol as solvent, and performing hot-dipping assay under alcohol-soluble extract assay (China pharmacopoeia 2020 Ed. 2201), and the results are shown in Table 2 below.

TABLE 2 extract measurement results

The results show that the mean value of 15 batches of standard decoction extract is 33.44%, and the lower limit of the reference standard limit allowable range (mean value is 70-130%), the alcohol-soluble extract of the product is determined to be not less than 23.5%. The measurement results of 15 batches of standard decoction all meet the requirement of a set limit.

Testing of feature maps

Determination of measurement wavelength: the reference determines a detection wavelength of 340 nm.

Test method

Chromatographic conditions are as follows: a chromatographic column: (Shim-pack GIST 4.6mm 250mm,5.0 um); mobile phase: acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; flow rate: 0.8ml per minute; column temperature: 40 ℃; detection wavelength: 340 nm.

TABLE 3

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0～10	10→15	90→85
10～18	15	85
			18～35	15→14	85→86
35～50	14	86

Preparation of reference solutions: precisely weighing 1g of herba Dendrobii control material, precisely adding 10ml of 50% methanol, weighing, performing ultrasonic treatment for 1 hr, taking out, cooling, weighing again, supplementing the lost weight with 50% methanol, shaking, filtering, and collecting the filtrate. Precisely weighing appropriate amount of schaftoside reference substance, and adding methanol to obtain 70ug/ml solution as reference substance solution.

Preparation of a test solution: precisely taking 1.0g of the product, placing the product in a sealed conical flask, precisely adding 25ml of 50% methanol, weighing the weight, ultrasonically treating for 30 minutes, complementing the weight loss by methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product.

The determination method comprises the following steps: precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and measuring.

Methodology investigation

Investigation of extraction method: preparing test solution by different extraction methods (ultrasonic and reflux), and determining according to the test method in the characteristic spectrum test. The result shows that the number of the main peaks is consistent, the sample is subjected to ultrasonic treatment for 30min, and the total peak area of the main peaks is larger, so that the sample extraction mode is selected to be ultrasonic treatment.

And (3) extracting time investigation: the test solutions were prepared at different sonication times (20 min, 30min, 40 min) and tested as described above. The result shows that the number of main peaks extracted at different extraction time is consistent, and when the extraction time is 30min, the total peak area of the main peaks is the largest, so that the extraction time is determined to be 30 min.

Investigation of extraction solvent: the test solutions were prepared with different extraction solvents (methanol, 50% methanol, water) and tested according to the test methods described above. The result shows that the number of the main peaks is consistent, the total peak area of the main peaks is not greatly different, but the content is the highest when the solvent is 50% methanol, so that the 50% methanol is determined to be used as the extraction solvent.

Sample sampling amount investigation: test solutions were prepared in sample amounts (0.5g,1.0g,1.5g), respectively, and measured according to the above test methods. The result shows that when the sample amount is 1.0g, the ratio of the total area of the main peak to the sample amount is the largest, so that the sample amount of the sample is determined to be 1.0 g.

In summary, the main parameters for determining the preparation method of the test solution are as follows: precisely weighing about 1g of dendrobe standard decoction sample, placing in a conical flask with a plug, precisely adding 25mL of 50% methanol, sealing, weighing, ultrasonically treating for 30min, cooling, weighing again, supplementing with 50% methanol to reduce weight loss, shaking, and filtering.

Verification of characteristic spectrum analysis method

And (3) special investigation: taking 10ul of the sample with 50% methanol alcohol as solvent, and determining according to the chromatographic conditions in the above characteristic spectrum test. The test shows that: the blank solvent is free of interference.

And (3) repeatability test: taking about 0.1g and 6 parts of samples in the same batch, and determining according to a method in a characteristic spectrum test, wherein the result shows that 4 common peaks exist in the characteristic spectrum of the 6 test samples, performing similarity flattening on the specified 4 common characteristic peaks by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), and indicating that the method has good reproducibility, wherein the relative retention time and the relative peak area RSD are less than 5% (see the following tables 4 and 5).

TABLE 4 relative retention time of feature profile for reproducibility test

Peak number	S1	S2	S3	S4	S5	S6	RSD(％)
								1	0.725	0.727	0.727	0.728	0.727	0.727	0.135
2	0.782	0.783	0.783	0.783	0.783	0.783	0.052
								3(S)	1	1	1	1	1	1	0.000
4	1.568	1.563	1.563	1.559	1.559	1.564	0.217

TABLE 5 relative peak area of characteristic spectrum for repeatability test

And (3) precision test: about 1g of samples in the same batch are taken, 6 needles are continuously injected for measurement according to the test method in the characteristic spectrum test, the peak shape and the peak number are basically consistent, a similarity evaluation system (2012 edition) of a traditional Chinese medicine chromatogram fingerprint image is adopted to evaluate the similarity of specified 4 common characteristic peaks, and the relative retention time and the RSD of the relative peak area are in a qualified range (see the following tables 6 and 7), which indicates that the method has good precision.

TABLE 6 relative retention time of the feature profile for the precision test

Peak number	S1	S2	S3	S4	S5	S6	RSD(％)
								1	0.727	0.727	0.727	0.727	0.727	0.727	0.000
2	0.783	0.783	0.783	0.783	0.784	0.783	0.052
								3(S)	1	1	1	1	1	1	0.000
4	1.564	1.564	1.564	1.564	1.565	1.564	0.026

TABLE 7 relative peak area of characteristic spectrum for precision test

Peak number	S1	S2	S3	S4	S5	S6	RSD(％)
								1	0.787	0.793	0.792	0.793	0.813	0.816	1.508
2	1.234	1.243	1.244	1.246	1.241	1.242	0.317
								3(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.000
4	0.798	0.803	0.804	0.810	0.813	0.813	0.759

And (3) stability test: taking about 1g of a batch of samples, respectively injecting samples at 0h, 2h, 4h, 8h, 12h and 24h according to the test method in the characteristic spectrum test to determine, wherein the peak shape and the peak number are basically stable, and performing similarity evaluation on the specified 4 common characteristic peaks by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2012 edition), wherein RSD of relative retention time and retention area is in a qualified range (see Table 8 and Table 9), which indicates that the sample solution is stable within 24 hours.

TABLE 8 stability test feature profiles relative retention time

Peak number	0	2h	4h	8h	12h	24h	RSD(％)
								1	0.725	0.726	0.728	0.727	0.727	0.727	0.130
2	0.782	0.782	0.783	0.783	0.783	0.783	0.060
								3(S)	1	1	1	1	1	1	0.000
4	1.568	1.565	1.56	1.563	1.563	1.564	0.001

TABLE 9 relative peak area of characteristic spectra for stability test

Peak number	0h	2h	4h	8h	12h	24h	RSD(％)
								1	0.709	0.729	0.696	0.696	0.728	0.745	2.793
2	1.151	1.177	1.173	1.222	1.234	1.195	2.645
								3(S)	1.000	1.000	1.000	1.000	1.000	1.000	0.000
4	0.714	0.724	0.719	0.725	0.742	0.739	1.560

Characterization and analysis of standard decoction characteristic spectrum

Determination of standard decoction characteristic map

According to the drawn characteristic spectrum analysis method, the characteristic spectrums of 15 batches of dendrobium standard decoction and 15 batches of Chinese herbal pieces used for preparation are measured, and the result shows that the characteristic chromatogram of the standard decoction and the Chinese herbal pieces used for preparation have 4 common peaks and correspond to the retention time of 4 characteristic peaks in the chromatogram of a reference substance of a reference medicinal material, wherein the peak corresponding to the reference substance of the schaftoside is peak 3, and the common peak characteristic spectrum is obtained.

And (3) evaluating the similarity of the characteristic chromatograms: the similarity evaluation system (2012 edition) is adopted to evaluate the similarity of the selected 4 common characteristic peaks, and the result shows that the similarity of the characteristic chromatograms of the standard decoction pieces of the dendrobium 15 batches is more than 0.9, which indicates that the quality of the standard decoction is relatively stable. The peak (3) corresponding to the schaftoside reference peak was taken as the S peak, and the relative retention times of the common peak and the S peak were calculated, and the relative retention times and ranges thereof are shown in table 10 below.

TABLE 1015 Standard decoction batches shared peak relative retention time

In conclusion, the method for determining the standard decoction characteristic spectrum established by the high performance liquid chromatography is adopted, and the established method is verified in terms of precision, repeatability and stability according to the analysis method verification guiding principle (general rule 9101) of the four parts of the Chinese pharmacopoeia 2020 edition, and meets the requirements. The similarity evaluation is carried out on the characteristic spectrums of 15 batches of standard decoction samples by adopting a traditional Chinese medicine chromatogram fingerprint image similarity evaluation system (2012 edition), and 4 common characteristic peaks are calibrated, wherein the peak 3 is schaftoside. Taking the peak corresponding to the schaftoside reference substance as an S peak, calculating the relative retention time of the other 3 characteristic peaks, and drawing up the average value of the relative retention time of the peaks of 15 batches of samples as specified values: 0.72 (peak 1), 0.78 (peak 2), 1.57 (peak 4), considering the error of experiment operation, instrument, reagent and other multifactor, the relative retention time allowed range is defined as + -10%.

Content determination: the content of dendrobium huoshanense is not determined by referring to 2020 edition of Chinese pharmacopoeia, and schaftoside is referred to as a peak of a dendrobium huoshanense characteristic spectrum. Through tests, the schaftoside has obvious peak shape, so that the dendrobium (dendrobium fimbriatum) formula granules select schaftoside as a content measurement component.

Test method

Preparation of control solutions: precisely weighing appropriate amount of schaftoside reference substance, and adding methanol to obtain 70ug/ml solution as reference substance solution.

Preparation of a test solution: precisely taking 1g of the product, placing the product in a sealed conical flask, precisely adding 25ml of 50% methanol, weighing the weight, performing ultrasonic treatment for 30 minutes, complementing the weight loss by 50% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product.

The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

Methodology investigation

Investigation of extraction method: preparing test solution by different extraction methods, and determining according to the test method in the standard decoction characteristic spectrum determination. The results show that the sample ultrasound (power 250W, frequency 25KHZ) has no significant difference from the reflux, and the RSD value is 0.153%. (see table 11 below), the sample extraction was chosen to be a more convenient sonication.

TABLE 11 comparison of different extraction methods

Examination of extraction time: preparing test solution at different extraction time, and determining according to the test method in the standard decoction characteristic spectrum determination. The results show that the sample content is relatively high for 30min of sonication (see table 12 below) and the RSD value is 12.462%, so the sonication time is chosen to be 30 min.

TABLE 12 comparison of different extraction times

Investigation of extraction solvent: preparing test solution with different extraction solvents, and determining according to the test method in the standard decoction characteristic spectrum determination. The results showed that schaftoside content was highest when the extraction solvent was methanol (see table 13 below), and RSD value was 12.441% so that 50% methanol was determined as the extraction solvent.

TABLE 13 comparison of different extraction solvents

And (3) sample quantity investigation: the test solutions are prepared in different sample weighing amounts (0.5g,1.0g and 1.5g) respectively, and the determination is carried out according to the test method in the standard decoction characteristic spectrum determination. The results show that the sample content was slightly higher for a sample size of 1.0g (see Table 14 below), so that the sample size was 1.0 g.

TABLE 14 comparison of sample volumes taken from different samples

Verification of content determination methodology

And (3) repeatability test: taking about 1g of standard decoction samples of the same batch, and taking 6 parts in total, determining according to the test method in the standard decoction characteristic map determination, wherein the measured average value of the content of schaftoside in the samples is 0.944mg/g, and the RSD value is 0.93%, and the test shows that the method has good reproducibility (see table 15 below).

TABLE 15 repeatability tests

And (3) precision test: taking a sample solution in the standard decoction characteristic spectrum determination, continuously injecting a sample of 6 needles, determining the peak area according to the test method of 9.1, and calculating the RSD value of the schaftoside peak area in the sample to be 0.646%, which indicates that the precision of the instrument is good (see the following table 16).

TABLE 16 precision test

And (3) stability test: about 1g of a batch of decoction samples are taken, sample injection is carried out for 0h, 2h, 4h, 8h, 12h and 24h respectively according to the test method of 9.1, the peak areas are measured, the RSD value of the peak areas is calculated to be 1.416%, and tests show that the solution of the test sample is stable within 24 hours (see table 17 below).

TABLE 17 stability test

Linear range test: taking schaftoside reference solution (concentration of 69.5ug/ml), injecting sample (1ul, 5ul, 10ul, 15ul, 20ul, 25ul) at different injection volumes. The measurement was carried out under the chromatographic conditions described above. Taking the peak area of schaftoside as ordinate and the sample injection quality as abscissa, drawing a standard curve, performing linear regression,

the schaftoside equation is: y 3.0E +6X +11826

R²＝1.0000

It can be seen that schaftoside has a good linear relationship with its peak area in the range of 0.00695mg/ml to 0.17375mg/ml (see table 18 below and fig. 2 below).

TABLE 18 Linear test results of schaftoside

Test solution	Quality of sample introduction	Peak area
			Linear
1	0.0695	206254
			Linearity 2	0.3475	1032445
Line 3	0.695	2082237
			Linearity 4	1.0425	3107439
Linear 5	1.39	4110789
			Linear 6	1.7375	5135836

Sample recovery rate test: precisely weighing 6 parts of sample (the content is 0.994mg/g) of about 0.5g, adding 10ml of schaftoside reference solution (0.0695mg/ml) with known concentration, preparing test solution according to the above method, and measuring according to chromatographic conditions, wherein the calculated average sample adding recovery rate is 93.66%, and the RSD is 0.54%.

TABLE 19 sample recovery test

Measuring the contents of the standard decoction and the traditional Chinese medicinal materials: the dendrobium medicinal material is processed into dendrobium decoction pieces after being cleaned after being initially processed in the producing area, and the content of schaftoside of the dendrobium medicinal material is not changed.

According to the proposed content analysis method, the content of schaftoside in 15 batches of dendrobe (dendrobium fimbriatum) standard decoction pieces and the 15 batches of dendrobe (dendrobium fimbriatum) decoction pieces prepared by the same and the medicinal materials is determined, and the result is shown in the following table 20.

Table 2015 batch schaftoside medicinal material determination results

TABLE 2115 determination of dried dendrobe decoction pieces

TABLE 2215 measurement results of Dendrobium Standard decoction

Content transfer rate: according to the detection method determined by standard decoction methodology research, the content transfer rate of schaftoside is calculated for 15 batches of standard decoction and the determination results of prepared traditional Chinese medicine decoction pieces thereof, the quality transfer condition of schaftoside is mastered, and a basis is provided for formulating the internal control standard of materials and the allowable range of characterization parameters. The standard decoction is prepared by decocting herba Dendrobii decoction pieces in water for 2 times, concentrating the filtrate, and freeze drying. The schaftoside content transfer rate (see table 23 below).

TABLE 2315 Shibataside content transfer rate of standard decoction of herba Dendrobii

According to the data, the dendrobium (dendrobium fimbriatum) decoction pieces are decocted according to the scheme to prepare the dendrobium (dendrobium fimbriatum) decoction piece standard decoction, the schaftoside average transfer rate of the dendrobium (dendrobium fimbriatum) decoction piece standard decoction is 32.45%, the measured transfer rate range is 23.61% -47.25%, and the SD is 6.57. According to technical requirements for quality control and standard formulation of traditional Chinese medicine formula granules, the allowable range of the schaftoside content transfer rate is 22.72-42.18% calculated according to 70-130% of the mean value of the transfer rate; the content is 12.74-52.16% calculated according to +/-3 SD. Therefore, the standard decoction is drawn up to have a schaftoside content transfer rate range: 12.74-52.16%. The results show that the schaftoside transfer rate in the 15 batches of standard decoction is within the allowable range of +/-3 SD.

The average content of the schaftoside of the product is 0.74mg/g, the measured content range is 0.537-0.919 mg/g, and SD is 0.13; calculated according to the mean value +/-3 SD, the allowable range of the content of schaftoside is 0.35-1.13 mg/g. Therefore, the content range of schaftoside in the standard decoction is drawn as follows: 0.40 mg/g-1.20 mg/g. The results show that schaftoside and the transfer rate thereof in 15 batches of standard decoction are within the allowable range, and can provide reference basis for the quality research of dendrobium (dendrobium fimbriatum) formula granules.

Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the idea of the invention, also features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

The embodiments of the invention are intended to embrace all such alternatives, modifications and variances that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements and the like that may be made without departing from the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. a dendrobium standard decoction quality control method, is characterized in that, described quality control method is to pass through to the character of dendrobium standard decoction, dry extract extract rate, thin layer identification, leachate, characteristic atlas and Schaffer Determination of taboside content, the content range of schaffoside in the proposed standard decoction is 0.4-1.20mg/g; among them, the extraction rate of dry extract is determined by decoction method; TLC identification is carried out by TLC method; The extracts were determined by hot soaking method; the characteristic map and the content of schaffatin were determined by liquid chromatography.

2. The quality control method for dendrobium standard decoction according to claim 1, characterized in that, the limited range of the dry extract extraction rate is 8.5-15.0%.

3. according to the described dendrobium standard decoction quality control method of claim 1, it is characterized in that, described TLC comprises the steps:

A1, take Dendrobium decoction and add methanol to ultrasonically treat, filter, evaporate the filtrate to dryness, and dissolve the residue in methanol to obtain test solution a;

A2. Take the reference medicinal material of Dendrobium officinale, ultrasonically treat it with methanol, filter the filtrate, evaporate the filtrate to dryness, and dissolve the residue in methanol to obtain the reference medicinal material solution a;

A3. Set TLC conditions: silica gel G thin-layer plate; Spotting volume: both the test solution a and the reference medicinal material solution a are 10ul; the developing agent: petroleum ether and ethyl acetate are obtained by mixing with a volume ratio of 3:2; Color development: 10% sulfuric acid ethanol solution, heated at 105 ℃ until the spot color is clear.

4. according to the described dendrobium standard decoction quality control method of claim 1, it is characterized in that, described hot soaking method is solvent with ethanol, adopts the hot soaking method in alcohol-soluble extract assay method to measure leachate and must not be less than 23.5% .

5. according to the described dendrobium standard decoction quality control method of claim 1, it is characterized in that, the method that described characteristic spectrum and schifftar glycosides content determination adopts liquid chromatography to measure comprises drawing respectively dendrobium reference substance solution b and test The product solution b was injected into the liquid chromatograph, and the measurement was obtained; wherein, the chromatographic conditions used: chromatographic column: Shim-pack GIST; mobile phase: acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B for gradient washing flow rate: 0.8 mL/min; column temperature: 40 °C; detection wavelength: 340 nm.

6. according to the described dendrobium standard decoction quality control method of claim 5, it is characterized in that, the preparation method of described need testing solution b is to take the dendrobium standard decoction sample, add the 50% methyl alcohol of 25 times of amounts, and seal, After that, it is sonicated, supplemented with methanol to reduce weight loss, shaken well, and filtered to obtain.

7. according to the described dendrobium standard decoction quality control method of claim 5, it is characterized in that, the method that described characteristic spectrum adopts liquid chromatography to measure also comprises injecting liquid chromatograph to the test sample of same batch, measures, And evaluate the similarity of the developed common characteristic peaks.

8. according to the described dendrobium standard decoction quality control method of claim 5, it is characterized in that, the method that described characteristic spectrum and schiffroside content measure adopt liquid chromatography to measure also comprise to the same batch of need testing respectively in Samples were injected at 0h, 2h, 4h, 6h, 12h, and 24h, the peak shape and number of peaks were determined, and the similarity of the common characteristic peaks was evaluated to test the stability of the test solution.

9. according to the described dendrobium standard decoction quality control method of claim 5, it is characterized in that, the method that described characteristic spectrum and schifftar glycosides content determination adopt liquid chromatography to measure also comprise to the same batch of need testing continuous feeding. 6 samples were used to measure the peak shape and number of peaks, and the similarity of the common characteristic peaks was evaluated to test whether the precision of the chromatograph was good.

10. according to the described dendrobium standard decoction quality control method of claim 5, it is characterised in that the method for measuring the content of schaffoside using liquid chromatography also comprises taking by weighing test sample and adding schiffoside reference substance The solution was subjected to the steps of testing the average spike recovery of schaffaroside.