CN112666280B - Method for measuring main components of indigo naturalis before and after irradiation - Google Patents
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Abstract
The invention provides a method for measuring main components of indigo, which comprises three steps of measuring the content of indigo and indirubin before and after irradiation of the indigo, measuring the whole chemical components before and after irradiation of the indigo and investigating the stability of the indigo before and after irradiation of the indigo, wherein the measuring of the content of indigo and indirubin before and after irradiation of the indigo comprises the preparation of a reference substance solution and a test substance solution, and the calculation and analysis of the content of indigo and indirubin in a sample by adopting an external standard one-point method under a certain chromatographic condition. The method for measuring the main components of the indigo naturalis before and after irradiation is adopted to research the quality of the indigo naturalis before and after irradiation, and has important significance for ensuring the quality and the clinical curative effect of the product.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a method for measuring main components of indigo naturalis before and after irradiation.
Background
Indigo naturalis indigowurlis, namely indigo naturalis, clam powder, honeysuckle flower, blue dew, lawsonia inermis and indigo foam flower, and the quality standard is collected in Chinese pharmacopoeia, and the source of the quality standard is dry powder, lumps or particles prepared by processing leaves or stems and leaves of Baphicacanthesis cushia (Nees) Bremek. Has the effects of clearing away heat and toxic materials, cooling blood, removing speckle, purging pathogenic fire, and arresting convulsion. It can be used for treating macula due to toxic heat, hematemesis, pharyngalgia, aphtha, pyocutaneous disease, cough, chest pain, bloody sputum, epilepsy due to summer-heat, and convulsion.
The main chemical components are as follows: contains indigo, indirubin, indigo brown, indigo yellow, tannic acid, beta-sitosterol, protein and a large amount of inorganic salt.
The pharmacological action is as follows: indigo naturalis has effects of inhibiting Staphylococcus aureus, Bacillus anthracis, Shigella shigella and Vibrio cholerae. The indirubin is the effective anticancer component and has moderate inhibiting effect on transplanted tumor.
The processing method comprises the following steps: collecting fallen leaves, soaking in water until the leaves are rotten and peeled, removing the fallen leaves, adding appropriate amount of lime milk, stirring thoroughly until the steep turns dark red from dark green, taking out the foam on the liquid surface, and drying in the sun.
Indigo naturalis is widely used in clinical treatment of traditional Chinese medicine, and is often used in powder and pills. Due to the characteristics of the traditional Chinese medicinal materials, the powder can contain a large amount of microorganisms in the harvesting, processing and production processes, and the powder has the requirement of controlling the microbial limit according to the requirement of the appendix of the Chinese pharmacopoeia, so that the indigo is used for the powder preparation and the microorganisms in the indigo need to be treated and researched. Enterprises generally adopt cobalt 60 irradiation for sterilization treatment, but the history of sterilization of the traditional Chinese medicines by irradiation sterilization is short, basic research is incomplete, and the necessity of the traditional Chinese medicines by irradiation sterilization is fully explained according to the traditional Chinese medicine irradiation sterilization technical guide principle issued by the State food and drug administration. The quality research before and after the irradiation of the indigo naturalis has important significance for ensuring the quality and the clinical curative effect of the product.
Disclosure of Invention
The invention aims to provide a method for measuring main components of indigo naturalis before and after irradiation, which is used for researching the quality of the indigo naturalis before and after irradiation and has important significance for ensuring the quality and clinical curative effect of products.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for measuring main components of indigo naturalis before and after irradiation comprises the following steps:
(1) determining the content of indigo and indirubin before and after indigo naturalis irradiation:
preparation of control solutions: 2.5mg of an indigo or indirubin reference substance is precisely weighed, placed in a 250mL volumetric flask, added with about 220mL of 2% chloral hydrate trichloromethane solution, ultrasonically treated for 1.5 hours, cooled, added with 2% chloral hydrate trichloromethane solution to scale, and shaken up to obtain the indigo or indirubin reference substance solution containing 10 mu g of indigo or indirubin in each liter of mL;
preparation of a test solution: taking 50mg of fine powder of a indigo naturalis sample, precisely weighing, placing in a 250mL volumetric flask, adding about 220mL of N, N-dimethylformamide, carrying out ultrasonic treatment for 30 minutes, cooling, adding the N, N-dimethylformamide to a scale, shaking up, filtering, and taking a subsequent filtrate to obtain the indigo naturalis extract;
and (3) sample analysis: taking 3 batches of samples S1-S3 before and after irradiation, respectively preparing 2 parts of reference substance and sample solution in parallel, measuring for 2 times in parallel under chromatographic conditions, recording peak areas, and calculating the content of the indigo and the indirubin in the samples by adopting an external standard one-point method;
(2) measuring the whole chemical components before and after irradiation of indigo naturalis:
preparation of control solutions: respectively taking 1.0mg of indigo blue and indirubin as reference substances, precisely weighing, and adding methanol to obtain solution containing 0.1mg per 1 mL;
preparation of a test solution: taking 250mg of indigo naturalis powder, accurately weighing, placing in a conical flask with a plug, accurately adding 25mL of methanol, weighing, ultrasonically extracting for 30min, cooling, weighing again, adding methanol to complement weight loss after weighing, shaking up, filtering, and taking subsequent filtrate to obtain the final product;
fingerprint spectrum determination: precisely absorbing 20 mu L of each of the reference solution and the test solution, injecting into a high performance liquid chromatograph, measuring to obtain the chromatogram and each chromatogram peak area of each batch of samples, and comparing the difference between the samples irradiated in the same batch and the samples not irradiated;
establishing a fingerprint spectrum: taking 6 samples in total from S1-S3 of indigo naturalis samples before and after 3 batches of irradiation, determining a fingerprint, setting S1 as a reference spectrum, setting the time width to be 0.10, generating the reference spectrum by adopting a median method, determining 14 chromatographic peaks as fingerprint common peaks of the indigo naturalis, establishing the fingerprint, taking indirubin with a peak at about 39.99min as a reference peak S, taking the samples before and after 3 batches of indigo naturalis medicinal materials are irradiated and the reference medicinal material fingerprint, calculating the retention time of each chromatographic peak and the ratio of the retention peak area to the retention time of the S peak and the retention peak area in the same spectrum, obtaining the relative retention time and the relative peak area, and comparing the characteristic peaks, namely the similarity of the spectrums;
(3) examining stability before and after irradiation of indigo naturalis:
investigating the stability of the content of the indigo and the indirubin in the sample before and after irradiation of the indigo naturalis: taking indigo naturalis samples S1-S3 before and after 3 batches of irradiation, standing for 0, 6, 7 and 8 months, and determining the content (%) of indigo blue and indirubin before and after the irradiation by adopting the indigo naturalis content determination method in the step (1) so as to judge the influence of the irradiation on the stability of the content (%) of the indigo blue and the indirubin in the indigo naturalis medicinal material;
and (3) investigating the stability of the whole chemical components in the sample before and after irradiation of indigo naturalis: taking 3 batches of indigo naturalis samples S1-S3 before and after irradiation, standing for 0, 6, 7 and 8 months, and performing fingerprint analysis on the indigo naturalis samples before and after irradiation by adopting the indigo naturalis fingerprint analysis method in the step (2) so as to judge the influence of irradiation on the stability of the whole chemical components of the indigo naturalis samples.
Further, in the determination of the content of the indigo and the indirubin before and after the indigo naturalis irradiation, the preparation process of the trichloromethane solution is as follows: placing water-containing chloral in a silica gel dryer for 24 hours, weighing 2.0g, adding chloroform to 100mL, placing until turbidity appears, dehydrating with anhydrous sodium sulfate, and filtering to obtain the final product.
Furthermore, in the determination of the content of the indigo and the indirubin before and after the indigo naturalis is irradiated, the power of ultrasonic treatment is 250W, and the frequency is 33 kHz.
Further, in the determination of the content of indigo and indirubin before and after irradiation of indigo naturalis, the chromatographic conditions are as follows: methanol-water (70: 30) is used as a mobile phase, the detection wavelength is 292nm, the column temperature is 25 ℃, and the sample injection amount is 10 mu L.
Further, in the measurement of the whole chemical components before and after the irradiation of the indigo naturalis, in the fingerprint spectrum measurement, the detection wavelength of a chromatograph is 242 nm; the mobile phase was acetonitrile (B) -0.02% phosphoric acid water (pH ═ 3) (a); the gradient of the mobile phase is 0-5 min, 20% B, 5-35 min, 20-55% B, 35-45 min, 55-66% B, 45-60 min and 66-95% B; the flow rate was 0.8 mL/min.
Further, the irradiation dose is 8 kGy.
Has the advantages that: the method for determining the main components of the indigo naturalis before and after irradiation has important significance for researching the quality of the indigo naturalis before and after irradiation and ensuring the quality and the clinical curative effect of the product.
Drawings
FIG. 1 is a chromatogram of the HPLC fingerprint chromatogram of indigo naturalis at different detection wavelengths in example 2 of the present invention;
FIG. 2 is a chromatogram of the HPLC fingerprint of indigo naturalis under different mobile phase systems in example 2 of the present invention;
FIG. 3 is a chromatogram of the HPLC fingerprint chromatogram of indigo naturalis under different mobile phase gradients in example 2 of the present invention;
FIG. 4 is a chromatogram of the HPLC fingerprint chromatogram of indigo naturalis at different flow rates in example 2 of the present invention;
FIG. 5 is an HPLC fingerprint chromatogram of samples before and after irradiation of 3 batches of indigo naturalis in example 2 of the present invention;
FIG. 6 is the HPLC control fingerprint spectrum of indigo naturalis in example 2 of the present invention;
FIG. 7 is HPLC fingerprint for overall chemical stability examination after 6 months of natural indigo sample placement in example 4 of the present invention;
FIG. 8 is HPLC fingerprint for whole chemical composition stability investigation after 7 months of natural indigo sample placement in example 4 of the present invention;
FIG. 9 is HPLC fingerprint for overall chemical stability examination after 8 months of natural indigo sample placement in example 4 of the present invention.
Detailed Description
The apparatus used in the present invention comprises: agilent 1260 high performance liquid chromatograph (quaternary pump, autosampler, column oven, DAD detector) (Agilent, usa); KQ-5200 ultrasonic cleaner (ultrasonic instruments, Inc., Kunshan); HH digital display constant temperature water bath (Jincheng Guosheng laboratory plant, Jiangsu Jintan city); one hundred thousand electronic balances (mettler-toledo instruments ltd) type AT 201; precision balances (model XP6, mettler-toledo instruments ltd); FA2104 analytical electronic balance (shanghai liangping instruments ltd).
The reagent and the medicinal materials used in the invention comprise: acetonitrile (HPLC, Tedia); methanol (HPLC, Tedia); phosphoric acid (chromatographically pure, Nanjing chemical reagents Ltd.); the water was Millipore ultrapure water.
The reference substances used in the invention: indigo (China food and drug testing institute, lot number 110716 one 201612, content 98.7%); indirubin (China institute for food and drug testing, lot No. 110717-201805, content in 99.6%). The indigo naturalis samples before and after irradiation are provided by Jiangsu seven good quality natural pharmaceutical Co. The irradiation dose was 8 kGy. Sample information is shown in table 1. The abbreviation bi (before irradiation) and ai (after irradiation) are used, and after the sample lot number, the abbreviation is used for all samples in the present invention.
TABLE 1 indigo sample information
Example 1
Determination of content of indigo and indirubin before and after indigo irradiation
Preparation of control solutions: 2.5mg of an indigo or indirubin reference substance is taken and precisely weighed, the reference substance is placed in a 250mL volumetric flask, a trichloromethane solution containing 2% chloral hydrate (the chloral hydrate is taken and placed in a silica gel drier for 24 hours, 2.0g is weighed, the trichloromethane is added to 100mL and placed, turbidity appears, dehydration is carried out by anhydrous sodium sulfate and filtration is obtained) about 220mL, ultrasonic treatment (power 250W and frequency 33kHz) is carried out for 1.5 hours, the reference substance is cooled, the trichloromethane solution containing 2% chloral hydrate is added to the scales, and the mixed solution is shaken up to obtain the compound (each mL contains 10 mu g of indigo or indirubin);
preparation of a test solution: taking 50mg of a natural indigo sample, precisely weighing, placing in a 250mL volumetric flask, adding about 220mL of N, N-dimethylformamide, carrying out ultrasonic treatment (power 250W and frequency 33kHz) for 30 minutes, cooling, adding the N, N-dimethylformamide to a scale, shaking up, filtering, and taking a subsequent filtrate to obtain the natural indigo;
chromatographic conditions are as follows: a chromatographic column: agilent ZORBAX SB-C18(4.6 mm. times.250 mm, 5 μm), guard column: agilent C18 ODS (4.6 mm. times.12.5 mm, 5 μm), with methanol-water (70: 30) as the mobile phase, detection wavelength 292nm, column temperature, 25 ℃, sample loading: 10 mu L of the solution;
and (3) sample analysis: taking 3 batches of samples S1-S3 before and after irradiation, respectively preparing 2 parts of reference substance and sample solution in parallel, measuring 2 times in parallel under chromatographic conditions, recording peak areas, and calculating the content of the indigo and the indirubin in the samples by adopting an external standard one-point method.
The results of the content of indigo and indirubin before and after irradiation of 3 batches of indigo naturalis medicinal materials are shown in table 2.
TABLE 2 indigo naturalis content before and after irradiation
In Table 2, the relative content changes are such that + represents an increase in the content after irradiation, and-represents a decrease in the content after irradiation, as compared with the result before irradiation.
M in the formula After irradiation The content of indigo blue and indirubin in the natural indigo medicinal material after irradiation, M Before irradiation The content of indigo and indirubin in the natural indigo medicinal material is not irradiated.
The content determination requirement of indigo naturalis medicinal materials in 2015 edition of Chinese pharmacopoeia shows that the content of indigo blue and indirubin in medicinal materials before and after irradiation of each batch is in accordance with the specification of the pharmacopoeia according to the calculation of dry products, wherein the content of indigo blue is not less than 2.0% and the content of indirubin is not less than 0.13%. Comparing the content results of the indigo and the indirubin in the indigo sample before and after the irradiation of the same batch, calculating the relative content change percentage of the indigo and the indirubin before and after the irradiation of the indigo according to a formula 1, wherein the results are shown in a table 2, and the results show that the content of the indirubin is unchanged and the relative content change of the indigo is between 3 and 8 percent before and after the irradiation of 3 batches of samples; further carrying out paired sample t test on the indigo content before and after irradiation of 3 batches of samples to obtain p Indigo blue ＝0.068>0.05. According to the results, under the irradiation dose of 8kGy, the content of the indigo and the indirubin after the indigo medicinal material is irradiated has no significant difference with that before the irradiation.
Example 2
Determination of whole chemical components before and after irradiation of indigo naturalis
Preparation of control solutions: respectively taking 1.0mg of indigo blue and indirubin as reference substances, precisely weighing, and adding methanol to obtain solution containing 0.1mg per 1 mL;
preparation of a test solution: taking 250mg of indigo naturalis powder, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of methanol, weighing, ultrasonically extracting for 30min, cooling, weighing again, supplementing methanol to zero gravity, shaking up, filtering, and taking the subsequent filtrate;
chromatographic conditions are as follows: a chromatographic column: agilent ZORBAX SB-C18(4.6 mm. times.250 mm, 5 μm), guard bar Agilent ZORBAX SB-C18(4.6 mm. times.12.5 mm, 5 μm); mobile phase: acetonitrile (B) - -0.02% phosphoric acid in water (pH ═ 3) (a), gradient elution: 0-5 min, 20% B; 5-35 min, 20% -55% of B; 35-45 min, 55% -66% B; 45-60 min, 66-95% B; the flow rate was 0.80 mL/min -1 (ii) a The detection wavelength is 242 nm; the column temperature is 30 ℃; the sample volume is 20 mu L;
fingerprint spectrum determination: precisely absorbing 20 mu L of each of the reference solution and the test solution, injecting into a high performance liquid chromatograph, measuring to obtain the chromatogram of each batch of samples and the peak area of each chromatogram peak, and comparing the difference between the irradiated samples and the non-irradiated samples in the same batch;
establishing a fingerprint spectrum: taking 6 samples in total from S1-S3 of indigo naturalis samples before and after 3 batches of irradiation, determining a fingerprint, setting S1 as a reference spectrum, setting the time width to be 0.10, generating a reference spectrum by adopting a median method, determining 14 chromatographic peaks as common peaks of the indigo naturalis fingerprint, establishing the fingerprint as shown in figure 5, taking indirubin which appears at the peak about 39.99min as a reference peak S, taking the samples before and after 3 batches of indigo naturalis medicinal materials are irradiated and the reference medicinal material fingerprint, calculating the retention time of each chromatographic peak and the ratio of the retention peak area to the retention time and the retention peak area of the S peak in the same spectrum, obtaining the relative retention time and the relative peak area, and comparing the similarity of characteristic peaks, namely the spectrums.
The result shows that the relative retention time RSD value of each common peak is less than 5%, which indicates that the peak emergence time of the common peak is relatively stable, the relative peak area result is shown in Table 3, the relative peak area RSD value of each common peak is between 5.77% and 41.95%, which indicates that the content of each component of indigo naturalis samples of different production batches has certain difference. The similarity of each map and the control map is respectively 0.994, 0.986, 0.997, 0.999, 0.995 and 0.999, which are all larger than 0.95, and the similarity is higher.
Establishing fingerprints by using 3 batches of samples before irradiation, wherein the similarity of each batch of samples to the reference fingerprint is 0.988, 0.998 and 1.000 respectively; establishing fingerprints by using 3 batches of irradiated samples, wherein the similarity of each batch of samples and the comparison fingerprints is 0.985, 0.999 and 1.000 respectively; the similarity of the fingerprint spectra of the 3 batches of irradiated samples and the samples before irradiation is respectively 0.986, 0.998 and 0.999; the similarity between the sample comparison fingerprint before irradiation and the sample comparison fingerprint after irradiation is 0.985.
The similarity of the samples before and after irradiation of 3 batches is respectively 0.988, 0.998 and 0.996 which are all more than 0.95.
From the results, no matter the fingerprint spectrum common mode is established by the samples before and after 3 batches of irradiation or the fingerprint spectrum common mode is established by the samples before and after irradiation respectively, the similarity between each batch of samples and the comparison fingerprint spectrum is more than 0.95, and the similarity between the samples before irradiation and the samples after irradiation is also more than 0.95, which shows that the component types in the samples before and after irradiation are basically unchanged, the irradiation has little influence on the whole chemical components of the indigo naturalis, and the whole consistency of the indigo naturalis samples of different batches is better.
Table 33 Total peak relative peak areas of fingerprint spectra of indigo naturalis crude drugs before and after irradiation
Example 3
Investigation of stability of content of indigo blue and indirubin in sample before and after irradiation of indigo naturalis
Taking indigo naturalis samples S1-S3 before and after 3 batches of irradiation, standing for 0, 6, 7 and 8 months, adopting the indigo naturalis content measuring method in the embodiment 1 to measure the content (%) of indigo blue and indirubin before and after the irradiation, so as to judge the influence of the irradiation on the stability of the content (%) of the indigo blue and the indirubin in indigo naturalis medicinal materials, and the results are respectively shown in tables 4 and 5.
TABLE 4 stability study of indigo content (%) before and after irradiation
TABLE 5 stability study of indirubin content (%) before and after irradiation of indigo naturalis
The results show that after 3 batches of samples S1, S2 and S3 are irradiated and placed for 6, 7 and 8 months, the RSD value of the change of the indigo content is between 3.9 and 9.5 percent, and the RSD value of the change of the indirubin content is between 2.1 and 3.5 percent; paired sample t test is carried out on the content of the indigo and the indirubin in the samples at 0 month, 6 months, 7 months and 8 months before and after irradiation, and the result is shown in a table 6. As can be seen from the table, when the indigo blue is placed for 6 months, 7 months and 8 months, the p value of the indigo blue content t test is more than 0.05, which shows that the indigo blue content is changed but has no obvious difference; p values of t tests of indirubin content at 6 months and 7 months are both larger than 0.05, and t tests cannot be carried out due to the standard error of the content difference value being 0 at 8 months. In conclusion, the irradiation has no obvious influence on the stability of the natural indigo medicinal material.
TABLE 6 stability of indigo content before and after irradiation
In Table 6, "-" indicates that t-test could not be performed because the standard error of the difference was 0.
Example 4
Investigation of stability of overall chemical components of sample before and after irradiation of indigo
Taking 3 batches of indigo naturalis samples S1-S3 before and after irradiation, standing for 0, 6, 7 and 8 months, and performing fingerprint analysis on the indigo naturalis samples before and after irradiation by using the indigo naturalis fingerprint analysis method in the embodiment 2 so as to judge the influence of irradiation on the stability of the whole chemical components of the indigo naturalis samples. For convenience of fingerprint stability analysis, wherein S1-S6 are 3 batches of samples before and after irradiation when the month is 0, S7-S12 are numbers of the samples before and after irradiation of the 3 batches after being placed in different months (6, 7 and 8), odd numbers represent the samples before irradiation, even numbers represent the samples after irradiation, and the sample numbers are detailed in Table 7.
TABLE 7 indigo stability survey sample information
After the indigo naturalis sample is placed for 6 months, the analysis result of the sample is shown in figure 7, similarity calculation is carried out on spectrograms of the sample placed for 0 month and 6 months according to a traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004A edition), the similarity of each batch of the sample placed for 0 month and 6 months is respectively 0.974, 0.992 and 0.989, and is more than 0.95, which shows that the whole chemical composition of the sample is not obviously changed after the sample is placed for 6 months.
After the indigo naturalis sample is placed for 7 months, the analysis result of the sample is shown in figure 8, similarity calculation is carried out on the spectrogram of the sample placed for 0 month and 7 months according to the similarity evaluation system of traditional Chinese medicine chromatogram fingerprint (2004A edition), the similarity of each batch of the sample placed for 0 month and 7 months is respectively 0.977, 0.989 and 0.990 which are all more than 0.95, and the result shows that the whole chemical composition of the sample is not obviously changed after the sample is placed for 7 months.
The analytical results of the indigo naturalis sample after being left for 8 months are shown in figure 9. Similarity calculation is carried out on the spectrogram of the sample placed for 0 month and 8 months according to the similarity evaluation system of the chromatographic fingerprint of traditional Chinese medicine (2004 edition), the similarity of each batch of samples placed for 0 month and 8 months is respectively 0.976, 0.989 and 0.989 which are all more than 0.95, and the result shows that the whole chemical components of the samples are not obviously changed after the samples are placed for 8 months.
The research data show that the indigo naturalis is irradiated by cobalt 60, so that the microbial limit of the indigo naturalis is ensured to meet the requirement of powder for medicine taking, the effective components of the medicine are retained, and the clinical use curative effect is ensured.
Claims (5)
1. A method for measuring main components of indigo naturalis before and after irradiation is characterized by comprising the following steps: the method comprises the following steps:
(1) determining the content of indigo and indirubin before and after indigo naturalis irradiation:
preparation of control solutions: 2.5mg of an indigo or indirubin reference substance is precisely weighed, placed in a 250mL volumetric flask, added with about 220mL of 2% chloral hydrate trichloromethane solution, ultrasonically treated for 1.5 hours, cooled, added with 2% chloral hydrate trichloromethane solution to scale, and shaken up to obtain the indigo or indirubin reference substance solution containing 10 microgram of indigo or indirubin in each liter of mL;
preparation of a test solution: taking 50mg of fine powder of a indigo naturalis sample, accurately weighing, placing in a 250mL volumetric flask, adding about 220mL of N, N-dimethylformamide, carrying out ultrasonic treatment for 30 minutes, cooling, adding N, N-dimethylformamide to scale marks, shaking up, filtering, and taking a subsequent filtrate to obtain the natural indigo naturalis extract;
and (3) sample analysis: taking 3 batches of samples S1-S3 before and after irradiation, respectively preparing 2 parts of reference substance solution and 2 parts of test solution in parallel, measuring 2 times in parallel under chromatographic conditions, recording peak areas, and calculating the content of the indigo and the indirubin in the samples by adopting an external standard one-point method;
(2) measuring the whole chemical components before and after irradiation of indigo naturalis:
preparation of control solutions: respectively taking 1.0mg of indigo blue and indirubin as reference substances, precisely weighing, and adding methanol to obtain solution containing 0.1mg per 1 mL;
preparation of a test solution: taking 250mg of indigo naturalis powder, accurately weighing, placing in a conical flask with a plug, accurately adding 25mL of methanol, weighing, ultrasonically extracting for 30min, cooling, weighing again, adding methanol to complement weight loss after weighing, shaking up, filtering, and taking subsequent filtrate to obtain the final product;
fingerprint spectrum determination: precisely absorbing 20 mu L of each of the reference solution and the test solution, injecting into a high performance liquid chromatograph, measuring to obtain the chromatogram of each batch of samples and the peak area of each chromatogram peak, and comparing the difference between the irradiated samples and the non-irradiated samples in the same batch;
establishing a fingerprint spectrum: taking 6 samples in total from S1-S3 of indigo naturalis samples before and after 3 batches of irradiation, determining a fingerprint, setting S1 as a reference spectrum, setting the time width to be 0.10, generating the reference spectrum by adopting a median method, determining 14 chromatographic peaks as fingerprint common peaks of the indigo naturalis, establishing the fingerprint, taking indirubin with a peak at about 39.99min as a reference peak S, taking the samples before and after 3 batches of indigo naturalis medicinal materials are irradiated and the reference medicinal material fingerprint, calculating the retention time of each chromatographic peak and the ratio of the retention peak area to the retention time of the S peak and the retention peak area in the same spectrum, obtaining the relative retention time and the relative peak area, and comparing the characteristic peaks, namely the similarity of the spectrums;
(3) examining stability before and after irradiation of indigo naturalis:
investigating the stability of the content of the indigo and the indirubin in the sample before and after irradiation of the indigo naturalis: taking 3 batches of indigo naturalis samples S1-S3 before and after irradiation, standing for 0, 6, 7 and 8 months, and determining the content% of indigo blue and indirubin before and after irradiation by adopting the indigo naturalis content determination method in the step (1) so as to judge the influence of irradiation on the stability of the content of the indigo blue and the indirubin in the indigo naturalis medicinal material;
and (3) investigating the stability of the whole chemical components in the sample before and after irradiation of indigo naturalis: taking 3 batches of indigo naturalis samples S1-S3 before and after irradiation, standing for 0, 6, 7 and 8 months, and performing fingerprint analysis on the indigo naturalis samples before and after irradiation by adopting the indigo naturalis fingerprint analysis method in the step (2) so as to judge the influence of irradiation on the stability of the whole chemical components of the indigo naturalis samples;
in the measurement of the whole chemical components before and after the irradiation of the indigo naturalis, the detection wavelength of a chromatograph is 242nm in the fingerprint spectrum measurement; the mobile phase is acetonitrile B-0.02% phosphoric acid water pH = 3A; the gradient of the mobile phase is 0-5 min, 20% of B, 5-35 min, 20% -55% of B, 35-45 min, 55% -66% of B, 45-60 min and 66% -95% of B; the flow rate was 0.8 mL/min.
2. The method for determining the main components of indigo naturalis before and after irradiation according to claim 1, which is characterized in that: in the determination of the content of the indigo and the indirubin before and after the irradiation of the indigo naturalis, the preparation process of the trichloromethane solution is as follows: placing water-containing chloral in a silica gel dryer for 24 hours, weighing 2.0g, adding chloroform to 100mL, placing until turbidity appears, dehydrating with anhydrous sodium sulfate, and filtering to obtain the final product.
3. The method for determining the main components of indigo naturalis before and after irradiation according to claim 1, which is characterized in that: in the determination of the content of the indigo and the indirubin before and after the indigo naturalis is irradiated, the power of ultrasonic treatment is 250W, and the frequency is 33 kHz.
4. The method for measuring the main components of indigo naturalis before and after irradiation according to claim 1, which is characterized by comprising the following steps: in the determination of the content of the indigo and the indirubin before and after the irradiation of the indigo naturalis, the chromatographic conditions are as follows: with a methanol-water ratio of 70: 30 is mobile phase, the detection wavelength is 292nm, the column temperature is 25 ℃, and the sample injection amount is 10 mu L.
5. The method for determining the main components of indigo naturalis before and after irradiation according to claim 1, which is characterized in that: the irradiation dose was 8 kGy.
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