Quality detection method of strobilanthes cusia medicine
By determining moisture, total ash, acid-insoluble ash, and extractives, combined with thin-layer chromatography, the problem of incomplete quality standards for *Indigofera tinctoria* medicinal materials was solved, enabling clear identification of indirubin, indigofera tinctoria, and indole glycosides, thus ensuring the accuracy and reliability of the medicinal material quality.
Patent Information
- Application Number
- CN202311809562.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-26
- Publication Date
- 2026-01-23
AI Technical Summary
The existing quality standards for Indigofera tinctoria are incomplete, and there is a lack of thin-layer chromatography identification methods for Indirubin, Indigofera tinctoria, and Indoleglycosides, resulting in inconsistent quality of the medicinal materials and making it difficult to effectively control their quality.
A quality detection method for strychnos nucifera was established. By determining moisture, total ash, acid-insoluble ash and extracts, and combining thin-layer chromatography, indirubin, indigo and indole glycosides were simultaneously identified. Ethyl acetate extraction was used with cyclohexane-chloroform-methanol as the developing solvent, and the spots were examined under ultraviolet light.
It enables clear identification of indigo, indigo and indole glycosides, provides a basis for qualitative identification of strychnine, and ensures the accuracy and reliability of the quality standards for medicinal materials.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of quality detection of traditional Chinese medicinal materials, and particularly relates to a quality detection method of Baphicacanthus cusia medicine. BACKGROUND
[0002] Baphicacanthus cusia (Nees) Bremek. is a plant of Baphicacanthus in Acanthaceae, and roots and leaves thereof are commonly used traditional Chinese medicines. South Baphicacanthus root, which is derived from dried roots and rhizomes of Baphicacanthus cusia, has the effects of clearing heat and resolving toxicity, and cooling blood and removing macula. The aboveground part of Baphicacanthus cusia is the main source of traditional Chinese medicine indigo, and traditional indigo dyeing technology is to use indigo medicine. Baphicacanthus cusia is widely used for treating colds including influenza, mumps, epidemic encephalitis B, hepatitis and the like.
[0003] The quality of Baphicacanthus cusia medicine on the market is uneven, and there are serious quality problems. Research shows that indigo and indigo thin layer chromatography has important guiding significance for rapid identification of south Baphicacanthus root mixed products. However, the current research on Baphicacanthus cusia is still insufficient: 1. The quality standard related to Baphicacanthus cusia is not perfect, and the current research does not match the demand of the developing quality control, and there is still a big gap. 2. The existing research on a single component does not match the complex characteristics of Baphicacanthus cusia in playing its mechanism, and there is a lack of perfect quality standard. 3. For the thin layer identification of Baphicacanthus cusia, the pharmacopoeia only has the thin layer identification method for indigo and indigo, and lacks the thin layer identification method for other components such as indole glycoside.
[0004] Therefore, the application determines the moisture, total ash, acid insoluble ash and extract of Baphicacanthus cusia medicine, and provides a reference for improving the quality standard of Baphicacanthus cusia medicine. A thin layer chromatography for simultaneously identifying indigo, indigo and indole glycoside is preliminarily established, and the thin layer identification method of Baphicacanthus cusia is optimized and improved, thereby providing a reference for subsequent in-depth research on the identification method of Baphicacanthus cusia. SUMMARY
[0005] The purpose of the application is to provide a quality detection method of Baphicacanthus cusia medicine, which can simultaneously identify the thin layer chromatography of indigo, indigo and indole glycoside, the spots are clear and visible, and there is no obvious tailing phenomenon, and the components of Baphicacanthus cusia medicine can be better reflected, so that the Baphicacanthus cusia medicine can be qualitatively identified.
[0006] The application is realized by adopting the following technical scheme: The quality detection method of Baphicacanthus cusia medicine provided by the application is characterized by comprising: determining the moisture, total ash, acid insoluble ash and extract content of Baphicacanthus cusia medicine; and identifying indigo, indigo and indole glycoside in Baphicacanthus cusia medicine.
[0007] The thin layer identification of indigo, indigo blue and indoxyl in the indigofera tinctoria medicinal material is disclosed in the present application, and the specific method is as follows: Take 1-3g of the powder of the product, add 10-30mL of ethyl acetate, heat and reflux for 0.5-1.5h, filter, take the filtrate, and concentrate to 1-3mL as the test sample solution; take the indigo red control sample and the indigo blue control sample, respectively add chloroform to prepare a solution containing 0.1mg per 1mL, and take the indoxyl control sample, add water to prepare a solution containing 0.1mg per 1mL as the control sample solution; according to the test of thin layer chromatography in the general rule 0502, take the test sample solution, the indigo blue control sample solution, the indigo red control sample solution and the indoxyl control sample solution and point them on the same silica gel G thin layer plate, use cyclohexane-chloroform-methanol as the developing agent, pre-saturate in the developing cylinder for 20-40min, develop, take out, dry, and immediately inspect the indigo blue and indigo red, and inspect the indoxyl under the ultraviolet lamp. The test sample chromatogram shows the same color spots or fluorescent spots at the positions corresponding to the chromatogram of the control medicinal material.
[0008] Preferably, the thin layer identification method disclosed in the present application is as follows: Take 1-3g of the powder of the product, add 10-30mL of ethyl acetate, heat and reflux for 0.5-1.5h, filter, take the filtrate, and concentrate to 1-3mL as the test sample solution; take the indigo red control sample and the indigo blue control sample, respectively add chloroform to prepare a solution containing 0.1mg per 1mL, and take the indoxyl control sample, add water to prepare a solution containing 0.1mg per 1mL as the control sample solution; according to the test of thin layer chromatography in the general rule 0502, take the test sample solution, the indigo blue control sample solution, the indigo red control sample solution and the indoxyl control sample solution and point them on the same silica gel G thin layer plate, use cyclohexane-chloroform-methanol as the developing agent, pre-saturate in the developing cylinder for 20-40min, develop, take out, dry, and immediately inspect the indigo blue and indigo red, and inspect the indoxyl under the ultraviolet lamp. The test sample chromatogram shows the same color spots or fluorescent spots at the positions corresponding to the chromatogram of the control medicinal material.
[0009] The ratio of the developing agent is cyclohexane-chloroform-methanol=4-6:3.5-4.5:2.
[0010] Preferably, the ratio of the developing agent is cyclohexane-chloroform-methanol=5:4:2.
[0011] The wavelength of the ultraviolet light for inspection is 302nm.
[0012] The moisture inspection method of the present application is as follows: 24 batches of Banlangen medicinal material powder (passing No. 2 sieve) 2g are precisely weighed, spread in a flat-shaped weighing bottle dried to constant weight, precisely weighed, dried in a 105°C oven for 5h, moved to a desiccator, cooled for 30min, precisely weighed, dried at the above temperature for 1h, cooled, weighed, until the difference between the two consecutive weighings is not more than 5mg; each batch of medicinal material is determined in triplicate, and the water content (%) in the test sample is calculated according to the weight loss.
[0013] The total ash inspection method of the present application is as follows: 24 batches of Banlangen medicinal material powder (passing No. 2 sieve) 5g are precisely weighed, placed in a crucible ignited to constant weight, weighed (accurate to 0.01g), slowly heated, pay attention to avoid burning, when completely carbonized, gradually increase the temperature to 500-600°C, make complete ashing and reach constant weight; according to the weight of the residue, the content of total ash in the test sample (%) is calculated.
[0014] The acid-insoluble ash inspection method of the present application is as follows: the ash obtained in the experiment of claim 8 is added with 10mL of dilute hydrochloric acid in the crucible, the crucible is covered with a watch glass, heated in a water bath for 10min, the watch glass is washed with 5mL of hot water, the washing liquid is added to the crucible, filtered with ashless filter paper, the residue in the crucible is washed on the filter paper with water until the washing liquid shows no chloride reaction; the filter residue together with the filter paper is moved to the same crucible, dried, ignited to constant weight; according to the weight of the residue, the content of acid-insoluble ash in the test sample (%) is calculated.
[0015] The extract inspection method of the present application is as follows: 24 batches of Banlangen medicinal material powder (passing No. 2 sieve) 3g are precisely weighed, two portions of each batch of medicinal material are determined in parallel, placed in a 150mL conical flask, precisely added with 75mL of dilute ethanol, tightly sealed, weighed, after standing for 1h, connected with a reflux condenser, heated to boiling and kept at a slight boil for 1h; after cooling, the conical flask is removed, tightly sealed, weighed again, the weight loss is made up with dilute ethanol, shaken uniformly, filtered with a dry filter, 25mL of the filtrate is precisely measured, placed in an evaporating dish dried to constant weight, evaporated on a water bath, dried at 105°C for 3h, placed in a desiccator to cool for 30min, weighed quickly and precisely; unless otherwise specified, the content of water-soluble extract in the test sample is calculated based on the dried product.
[0016] The present application has the following advantages: 1. The thin layer chromatography identification method of the present application is simple, accurate and reliable, and provides a basis for revising the quality standard of Banlangen. The Rf values of each component obtained by thin layer chromatography are moderate, the Rf value of indigo is 0.70; the Rf value of indigo is 0.62; the Rf value of indole is 0.31, the spots are clear and visible without obvious tailing phenomenon, which can better reflect the components of Banlangen medicinal material, and can be used for qualitative identification of Banlangen medicinal material.
[0017] 2, The application determines the contents of 24 batches of indigofera jahoei, and the results show that the moisture content of indigofera jahoei is between 7.68% and 9.37%, the total ash content is between 7.32% and 9.94%, the acid insoluble ash content is between 0.51% and 1.07%, and the extract content is between 14.46% and 26.76%. The experimental results meet the requirements. BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1 : The average moisture content of indigofera jahoei is shown in the column chart; Figure 2 : The average total ash content of indigofera jahoei is shown in the column chart; Figure 3 : The average acid insoluble content of indigofera jahoei is shown in the column chart; Figure 4 : The extract content of indigofera jahoei is shown in the column chart; Figure 5 : The chromatogram of the identification method (1: indigofera jahoei leaf sample (batch number: S1); 2: indigofera jahoei stem sample (batch number: S1); D1: indigoferon reference; D2: indigo reference; D3: indole glycoside reference); Figure 6 : The chromatogram of indigofera jahoei medicinal materials under the developing agent of benzene-chloroform-ethyl acetate-isopropyl alcohol (5:4:2:3) (1: indigofera jahoei leaf sample (batch number: S1); 2: indigofera jahoei stem sample (batch number: S1); D1: indigoferon reference; D2: indigo reference; D3: indole glycoside reference); Figure 7 : The chromatogram of indigofera jahoei medicinal materials under the developing agent of cyclohexane-chloroform-ethyl acetate-isopropyl alcohol (5:4:1:2) (1: indigofera jahoei leaf sample (batch number: S1); 2: indigofera jahoei stem sample (batch number: S1); D1: indigoferon reference; D2: indigo reference; D3: indole glycoside reference); Figure 8 : The chromatogram of indigofera jahoei medicinal materials under the developing agent of cyclohexane-chloroform-methanol (5:4:1) (1: indigofera jahoei leaf sample (batch number: S1); 2: indigofera jahoei stem sample (batch number: S1); D1: indigoferon reference; D2: indigo reference; D3: indole glycoside reference); Figure 9 : The chromatogram of indigofera jahoei medicinal materials under the developing agent of cyclohexane-chloroform-methanol (5:4:2) (1: indigofera jahoei leaf sample (batch number: S1); 2: indigofera jahoei stem sample (batch number: S1); D1: indigoferon reference; D2: indigo reference; D3: indole glycoside reference); Figure 10Chromatograms of solvent analysis for the extraction of *Indigofera tinctoria* (1-3: *Indigofera tinctoria* stem sample S7 (1: chloroform, 2: ethyl acetate, 3: methanol); 4-6: *Indigofera tinctoria* leaf sample S7 (4: chloroform, 5: ethyl acetate, 6: methanol); D1: indirubin reference standard; D2: indigo reference standard; D3: indole glycoside reference standard). Figure 11 Chromatograms for the extraction method of *Indigofera tinctoria* (1-2: *Indigofera tinctoria* stem sample S7 (1 sonication, 2 reflux); 3-4: *Indigofera tinctoria* leaf sample S7 (3 sonication, 4 reflux); D1: Indirubin reference standard; D2: Indigo reference standard; D3: Indole glycoside reference standard). Figure 12 Chromatograms for the extraction time of *Indigofera tinctoria* (1-3: *Indigofera tinctoria* stem sample S7 (sample 1 refluxed for 0.5 h, sample 2 refluxed for 1 h, sample 3 refluxed for 1.5 h); 4-6: *Indigofera tinctoria* leaf sample S7 (sample 1 refluxed for 0.5 h, sample 2 refluxed for 1 h, sample 3 refluxed for 1.5 h); D1: Indirubin reference standard; D2: Indigo reference standard; D3: Indole glycoside reference standard). Figure 13 Thin-layer chromatograms of *Indigofera tinctoria* stems (1-12: *Indigofera tinctoria* stem samples (batch: S1-S12); D1: Indirubin reference standard; D2: Indigo reference standard; D3: Indole glycoside reference standard). Figure 14 Thin-layer chromatograms of *Indigofera tinctoria* stems (13-24: *Indigofera tinctoria* stem samples (batch: S13-S24); D1: Indirubin reference standard; D2: Indigo reference standard; D3: Indole glycoside reference standard). Figure 15 Thin-layer chromatograms of Indigofera tinctoria leaves (1-12: Indigofera tinctoria leaf samples (batch: S1-S12); D1: Indirubin reference standard; D2: Indigo reference standard; D3: Indole glycoside reference standard). Figure 16 Thin-layer chromatograms of Indigofera tinctoria leaves (13-24: Indigofera tinctoria leaf samples (batch: S13-S24); D1: Indirubin reference standard; D2: Indigo reference standard; D3: Indole glycoside reference standard). Detailed Implementation
[0019] Example 1 Thin-layer chromatography identification of indirubin, indigofera tinctoria, and indole glycosides in *Indigofera tinctoria*: Take 2g of the powder, add 20 mL of ethyl acetate, heat and reflux for 1 h, filter, take the filtrate, concentrate to 2 mL, and use as the test solution; take indirubin reference standard and indigo reference standard, add chloroform to prepare a solution containing 0.1 mg per mL, and take indole glycoside reference standard, add water to prepare a solution containing 0.1 mg per mL, and use as the reference solution; perform the thin-layer chromatography test according to General Rule 0502, take the test solution, indigo reference standard solution, indirubin reference standard solution, and indole glycoside reference standard solution and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-chloroform-methanol (5:4:2) as the developing solvent, place in the developing tank for pre-saturation for 30 min, develop, remove, and air dry. Indigo and indirubin should be examined immediately, and indole glycoside should be examined under ultraviolet light (302 nm). In the chromatogram of the test sample, spots or fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference medicinal material.
[0020] Example 2 Thin-layer chromatography identification of indirubin, indigofera tinctoria, and indole glycosides in *Indigofera tinctoria*: Take 1g of the powder, add 10mL of ethyl acetate, heat and reflux for 0.5h, filter, take the filtrate, concentrate to 1mL, and use as the test solution; take indirubin reference standard and indigo reference standard, add chloroform to prepare a solution containing 0.1mg per 1mL, and take indole glycoside reference standard, add water to prepare a solution containing 0.1mg per 1mL, and use as the reference solution; perform the thin-layer chromatography test according to General Rule 0502, take the test solution, indigo reference standard solution, indirubin reference standard solution, and indole glycoside reference standard solution and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-chloroform-methanol (5:4:2) as the developing solvent, place in the developing tank for pre-saturation for 20min, develop, remove, and air dry. Indigo and indirubin are examined immediately, and indole glycoside is examined under ultraviolet light (302nm). In the chromatogram of the test sample, spots or fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.
[0021] Example 4 Thin-layer chromatography identification of indirubin, indigofera tinctoria, and indole glycosides in *Indigofera tinctoria*: Take 3g of the powder, add 30 mL of ethyl acetate, heat and reflux for 1.5 h, filter, take the filtrate, concentrate to 3 mL, and use as the test solution; take indirubin reference standard and indigo reference standard, add chloroform to prepare a solution containing 0.1 mg per mL, and take indole glycoside reference standard, add water to prepare a solution containing 0.1 mg per mL, and use as the reference solution; perform the thin-layer chromatography test according to General Rule 0502, take the test solution, indigo reference standard solution, indirubin reference standard solution, and indole glycoside reference standard solution and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-chloroform-methanol (5:4:2) as the developing solvent, place in the developing tank for pre-saturation for 40 min, develop, remove, and air dry. Indigo and indirubin should be examined immediately, and indole glycoside should be examined under ultraviolet light (302 nm). In the chromatogram of the test sample, spots or fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference medicinal material.
[0022] Example 4 Thin-layer chromatography identification of indirubin, indigofera tinctoria, and indole glycosides in *Indigofera tinctoria*: Take 2g of the powder, add 20 mL of ethyl acetate, heat and reflux for 1 h, filter, take the filtrate, concentrate to 2 mL, and use as the test solution; take indirubin reference standard and indigo reference standard, respectively add chloroform to prepare a solution containing 0.1 mg per 1 mL, and take indole glycoside reference standard, add water to prepare a solution containing 0.1 mg per 1 mL, as the reference solution; perform the thin-layer chromatography test according to General Rule 0502, take the test solution, indigo reference standard solution, indirubin reference standard solution, and indole glycoside reference standard solution and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-chloroform-methanol (6:4.5:2) as the developing solvent, place in the developing tank for pre-saturation for 30 min, develop, remove, and air dry. Indigo and indirubin are examined immediately, and indole glycoside is examined under ultraviolet light (302 nm). In the chromatogram of the test sample, spots or fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.
[0023] Example 5 Thin-layer chromatography identification of indirubin, indigofera tinctoria, and indole glycosides in *Indigofera tinctoria*: Take 2g of the powder, add 20 mL of ethyl acetate, heat and reflux for 1 h, filter, take the filtrate, concentrate to 2 mL, and use as the test solution; take indirubin reference standard and indigo reference standard, respectively add chloroform to prepare a solution containing 0.1 mg per 1 mL, and take indole glycoside reference standard, add water to prepare a solution containing 0.1 mg per 1 mL, as the reference solution; perform the thin-layer chromatography test according to General Rule 0502, take the test solution, indigo reference standard solution, indirubin reference standard solution, and indole glycoside reference standard solution and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-chloroform-methanol (4:3.5:2) as the developing solvent, place in the developing tank for pre-saturation for 30 min, develop, remove, and air dry. Indigo and indirubin should be examined immediately, and indole glycoside should be examined under ultraviolet light (302 nm). In the chromatogram of the test sample, spots or fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference medicinal material.
[0024] Example 6 Moisture content test: Accurately weigh 2 g of Malan herb powder, spread it evenly in a flat weighing bottle that has been dried to constant weight, accurately weigh it, dry it in an oven at 105 °C for 5 h, transfer it to a desiccator, cool it for 30 min, accurately weigh it, dry it again at the above temperature for 1 h, cool it, weigh it, until the difference between two consecutive weighings does not exceed 5 mg; perform three parallel determinations, and calculate the moisture content in the test sample based on the weight loss.
[0025] Example 7 Total ash content test: Accurately weigh 5 g of indigo powder, place it in a crucible heated to constant weight, weigh it, heat it slowly, taking care to avoid combustion, and when it is completely carbonized, gradually increase the temperature to 500-600 ℃ to completely ashed and reach constant weight; calculate the total ash content in the sample based on the weight of the residue.
[0026] Example 8 Test for acid-insoluble ash: Take the ash obtained in the experiment described in Example 7 above, carefully add 10 mL of dilute hydrochloric acid to the crucible, cover the crucible with a watch glass, place it on a water bath and heat for 10 min, rinse the watch glass with 5 mL of hot water, add the washing liquid to the crucible, filter it with ashless filter paper, wash the residue in the crucible with water onto the filter paper, and wash until the washing liquid does not show a chloride reaction; transfer the filter residue and filter paper to the same crucible, dry, and ignite to constant weight; calculate the content of acid-insoluble ash in the test sample based on the weight of the residue.
[0027] Example 9 Extractives test: Accurately weigh 3g of *Indigofera tinctoria* powder, perform two parallel tests, place in a 150 mL Erlenmeyer flask, accurately add 75 mL of dilute ethanol, seal tightly, weigh, let stand for 1 h, connect a reflux condenser, heat to boiling, and maintain a gentle boil for 1 h; after cooling, remove the Erlenmeyer flask, seal tightly, weigh again, replenish the lost weight with dilute ethanol, shake well, filter through a dry filter, accurately measure 25 mL of the filtrate, place in an evaporating dish that has been dried to constant weight, evaporate to dryness on a water bath, dry at 105℃ for 3 h, cool in a desiccator for 30 min, and quickly and accurately weigh; unless otherwise specified, calculate the content of alcohol-soluble extractives in the test sample based on the dried product.
[0028] To demonstrate the scientific validity and reproducibility of the detection method of this invention, the following methodological experimental studies were conducted: I. Determination of the extract, moisture, ash and acid-insoluble ash of *Indigofera tinctoria* 1. Instruments and Materials 1.1 Instruments Some of the experimental instruments are shown in Table 1.
[0029] 1.2 Materials and Reagents The experimental reagents are shown in Table 2.
[0030] 1.3 Sample Source Twenty-four different species of *Indigofera tinctoria* were collected. The samples were freshly harvested, air-dried, pulverized, sieved through a No. 2 sieve, and then placed in a desiccator for later use. Specific information is shown in Table 3.
[0031] 2. Methods and Results 2.1 Moisture content determination Accurately weigh 2 g of each of 24 batches of *Indigofera tinctoria* powder (passed through a No. 2 sieve), spread it evenly in a flat weighing bottle dried to constant weight, accurately weigh it, dry it in an oven at 105 °C for 5 h, transfer it to a desiccator, cool it for 30 min, accurately weigh it, dry it again at the above temperature for 1 h, cool it, and weigh it until the difference between two consecutive weighings does not exceed 5 mg. Each batch of medicinal material was tested in triplicate, and the water content (%) in the sample was calculated based on the weight loss. The results are shown in Table 4.
[0032] Table 4 shows that the moisture content of *Indigofera tinctoria* ranges from 7.68% to 9.37%. The average moisture content of *Indigofera tinctoria* is 8.39%. Figure 1It can be seen that the average moisture content of different batches of Indigofera tinctoria varies to some extent. Among them, Indigofera tinctoria S18 has the highest moisture content at 9.37%, while Indigofera tinctoria S6 has the lowest moisture content at 7.68%, which is lower than the average content of Indigofera tinctoria.
[0033] 2.2 Determination of total ash and acid-insoluble ash The test sample was pulverized to pass through a No. 2 sieve. After thorough mixing, 5g of each of 24 batches of medicinal powder (passed through a No. 2 sieve) was accurately weighed and placed in a crucible heated to constant weight. The weight was then determined (accurate to 0.01g). The crucible was slowly heated, taking care to avoid combustion, until complete carbonization. At this point, the temperature was gradually increased to 500-600℃ to ensure complete ashing and constant weight. Based on the weight of the residue, the total ash content (%) in the test sample was calculated. The results are shown in Table 5.
[0034] Determination of Acid-Insoluble Ash: Take the ash obtained in the above experiment, carefully add about 10 mL of dilute hydrochloric acid to the crucible, cover the crucible with a watch glass, place it in a water bath and heat for 10 min. Rinse the watch glass with 5 mL of hot water, and add the washing solution to the crucible. Filter through ashless filter paper. Wash the residue in the crucible with water onto the filter paper, and continue washing until the washing solution does not show a chloride reaction. Transfer the filter residue and filter paper to the same crucible, dry, and ignite to constant weight. Calculate the acid-insoluble ash content (%) in the sample based on the weight of the residue. The results are shown in Table 5.
[0035] Table 5 shows that the total ash content of *Indigofera tinctoria* ranges from 7.32% to 9.94%. The average total ash content of *Indigofera tinctoria* is 8.74%. Figure 2 It can be seen that there are certain differences in the average total ash content of different batches of Strobilanthes. Among them, Strobilanthes S2 has the highest total ash content at 9.94%, while Strobilanthes S3 has the lowest total ash content at 7.32%, which is lower than the average total ash content of Strobilanthes.
[0036] Table 6 shows that the insoluble ash content of strobilurinary acid ranges from 0.51% to 1.07%, with an average insoluble ash content of 0.80%. Figure 3It can be observed that the average content of acid-insoluble ash in *Indigofera tinctoria* varies from batch to batch. The highest acid-insoluble content was found in *Indigofera tinctoria* S10 at 1.07%, while the lowest was in *Indigofera tinctoria* S19 at 0.51%, both lower than the average content. Acid-insoluble ash reflects the amount of impurities such as soil and sand present in the medicinal material; therefore, it affects the purity of the medicinal material. It is recommended that the acid-insoluble ash content of *Indigofera tinctoria* should not exceed 1.0%.
[0037] 2.3 Determination of Leachate Accurately weigh 3g of each of 24 batches of medicinal powder (passed through a No. 2 sieve). Perform two parallel tests on each batch. Place the samples in a 150mL Erlenmeyer flask, accurately add 75mL of dilute ethanol, seal tightly, weigh, and let stand for 1 hour. Then connect a reflux condenser, heat to boiling, and maintain a gentle boil for 1 hour. After cooling, remove the Erlenmeyer flask, seal tightly, weigh again, and replenish the lost weight with dilute ethanol. Shake well, filter through a dry filter, and accurately measure 25mL of the filtrate. Place the filtrate in an evaporating dish that has been dried to constant weight, evaporate to dryness on a water bath, dry at 105℃ for 3 hours, cool in a desiccator for 30 minutes, and quickly and accurately weigh. Unless otherwise specified, calculate the content (%) of water-soluble extract in the test sample based on the dried product. The results are shown in Table 7.
[0038] Table 7 shows that the extract content of *Indigofera tinctoria* ranges from 14.46% to 26.76%, with an average extract content of 19.15%. Figure 4 It can be seen that the content of extract from different batches of Indigofera tinctoria varies. Among them, the extract content of Indigofera tinctoria S10 is the highest at 26.76%, while the extract content of Indigofera tinctoria S1 is the lowest at 14.46%, which is lower than the average content of extract from Indigofera tinctoria.
[0039] 3. Summary In the storage of Chinese medicinal herbs, moisture content is a crucial quality indicator, playing a dominant role in changes in quality. Excessive moisture not only degrades the appearance of the herbs but also leads to mold, insect infestation, and other problems. Therefore, the study and management of moisture content must be given sufficient attention. Ash content is also an important indicator, reflecting the degree of adulteration and contamination in Chinese medicinal herbs, thus assessing their quality level. Acid-insoluble ash reflects the content of contaminated sand and soil, as well as trace amounts of silica naturally present. The presence of these impurities significantly impacts the quality and stability of the herbs. Extracts from medicinal herbs are water-soluble substances other than proteins, salts, and vitamins; the quality standards for medicinal herbs are primarily determined based on their extract content.
[0040] The moisture content of 24 different batches of Baphicacanthus cusia measured in this experiment was 7.68% - 9.37%, the total ash content was 7.32% - 9.94%, the acid-insoluble ash was 0.51% - 1.07%, and the extract content was between 14.45% - 26.87%. All four test results met the requirements. It was found that there were certain differences between different batches, which might be related to factors such as the origin, storage environment, and altitude of Baphicacanthus cusia. Further research on Baphicacanthus cusia should be carried out to better develop and utilize it rationally.
[0041] II. Improvement of the TLC Identification Method for the Stems and Leaves of Baphicacanthus cusia 1 Instruments and Materials 1.1 Instruments See Table 8.
[0042] 1.2 Materials and Reagents See Table 9.
[0043] 1.3 Sample Sources The sample sources were the same as the Baphicacanthus cusia medicinal materials in the first section.
[0044] 2 Methods and Results Precisely weigh 2.0000 g each of the medicinal material powders of the stems and leaves of Baphicacanthus cusia (batch number: S1), add 20 mL of chloroform respectively, heat under reflux for 1 h, filter, take the subsequent filtrate, and concentrate it to 2 mL to obtain the test solution. Take indirubin reference substance and indigo reference substance, and add chloroform respectively to prepare reference substance solutions containing 0.1 mg per 1 mL. Additionally, take indoxyl glucoside reference substance and make a reference substance solution containing 0.1 mg per 1 mL with water. Refer to the thin-layer chromatography method (General Principles 0502) for testing. Absorb the above three solutions and spot them on the same silica gel G thin-layer plate respectively. Use petroleum ether (60 - 90 °C) - chloroform - ethyl acetate (1:8:1) as the developing agent, develop, take out, dry in the air, and immediately examine. The results are shown in Figure 5 . The results showed that indoxyl glucoside had a relatively large polarity and did not move at the starting line, and a relatively serious tailing phenomenon was visible in the sample of the stems of Baphicacanthus cusia.
[0045] 2.2 TLC Methodology Investigation 2.2.1 Investigation of the Developing Agent Using indigo, indirubin, and indole glycosides as controls, and benzene-chloroform-ethyl acetate-isopropanol (5:4:2:3), cyclohexane-chloroform-ethyl acetate-isopropanol (5:4:1:2), cyclohexane-chloroform-methanol (5:4:1), and cyclohexane-chloroform-methanol (5:4:2) as developing solvents, the samples were developed, removed, and air-dried. Indigo and indirubin were examined under daylight, and indole glycosides were examined under ultraviolet light (302 nm). The results are shown in [Figure number missing]. Figures 6 to 9 The results showed that cyclohexane-chloroform-methanol (5:4:2) as the developing solvent had a moderate Rf value, no tailing phenomenon, good separation effect, and low toxicity. Therefore, cyclohexane-chloroform-methanol (5:4:2) was selected as the developing solvent.
[0046] 2.2.2 Investigation of Extraction Solvents Accurately weigh three portions (2.0000 g each) of powdered stems and leaves of *Indigofera tinctoria*, and place them separately in 100 mL round-bottom flasks. Add 20 mL each of chloroform, ethyl acetate, and methanol to each flask, heat under reflux for 1 h, filter, and collect the filtrate, concentrating it to 2 mL as the test solution. Chromatographic conditions are as follows: Apply the test solution, indirubin reference solution (0.1 mg / mL), indigo reference solution (0.1 mg / mL), and indole glycoside reference standard (0.1 mg / mL) separately to the same silica gel G thin-layer plate. Develop with cyclohexane-chloroform-methanol (5:4:2) as the developing solvent. Remove the plate, air dry, and examine indigo and indirubin under fluorescent light, and indole glycoside under ultraviolet light (302 nm). The thin-layer chromatographic results are shown below. Figure 10 The results showed that the effects of chloroform and ethyl acetate were not significantly different, but because ethyl acetate had the lowest toxicity, it was chosen as the extraction solvent.
[0047] 2.2.3 Examination of Extraction Methods Accurately weigh three portions (2.0000 g each) of powdered indigo stems and leaves, and place them separately in 100 mL round-bottom flasks. Add 20 mL of ethyl acetate and perform ultrasonic extraction and reflux extraction for 1 h each, respectively. Filter the extracts, collect the filtrates, and concentrate them to 2 mL to obtain the test solution. Conduct the experiment according to the method described in "2.2.2". Figure 11 It can display the detection results of thin-layer chromatography. The results show that the spots in the test solution prepared by heating and reflux are clearer, thus confirming that the extraction method is heating and reflux.
[0048] 2.2.4 Examination of extraction time Accurately weigh three portions (2.0000 g each) of powdered indigo stems and leaves, and place them separately in 100 mL round-bottom flasks. Heat under reflux for 0.5 h, 1 h, and 1.5 h respectively. Filter the mixture, collect the filtrate, and concentrate it to 2 mL as the solvent for the test sample. Perform the experiment according to the method described in "2.2.2". The results of the thin-layer chromatography detection are as follows. Figure 12 As shown in the figure. The experimental results show that a reflux time of 1 h is just right, and the spots in the test sample solution are clearer. Therefore, a reflux time of 1 h was ultimately chosen as the extraction time.
[0049] 2.3 Thin-layer chromatography identification method for Strychnos nucifera 2.3.1 Preparation of reference standards Accurately weigh indirubin and indigo reference standards, and add chloroform to prepare solutions containing 0.1 mg of each per mL. Separately, take indole glycoside reference standard, add water to prepare solutions containing 0.1 mg per mL, and use these as reference solutions.
[0050] 2.3.2 Preparation of the test sample Accurately weigh 2.0000 g each of powdered stems and leaves from 24 batches of *Indigofera tinctoria*, add 20 mL of ethyl acetate, heat under reflux for 1 h, filter, take the filtrate, concentrate to 2 mL, and use as the test solution.
[0051] 2.3.3 Thin-layer chromatography identification conditions Spot the test solution, indigo reference solution, indirubin reference solution, and indole glycoside reference solution onto the same silica gel G thin-layer plate. Use cyclohexane-chloroform-methanol (5:4:2) as the developing solvent, pre-saturate the plate in the developing tank for 30 min, develop, remove, and air dry. Examine the indigo and indirubin immediately, and examine the indole glycoside under a UV lamp (302 nm).
[0052] 2.4 Thin-layer chromatographic identification of Indigofera tinctoria stem Weigh 24 batches of powdered *Indigofera tinctoria* stems as described in "2.3.2" to prepare the test solution. Separately prepare the reference solution as described in "2.3.1". Perform the experiment according to the thin-layer chromatography identification conditions described in "2.3.3". The results are as follows: Figure 13 , Figure 14 As shown, the test sample chromatogram and the reference sample chromatogram show spots of the same color at corresponding positions.
[0053] 2.5 Thin-layer chromatographic identification of Indigofera tinctoria leaves Weigh 24 batches of *Indigofera tinctoria* leaf powder and prepare the test solution as described in "2.3.2". Separately prepare the reference solution as described in "2.3.1". Perform the experiment according to the thin-layer chromatography identification conditions described in "2.3.3". The results are shown in [Figure 1]. Figure 15 , Figure 16In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference sample.
[0054] 3. Summary The experimental results showed that all 24 batches of *Indigofera tinctoria* stems and leaves contained three key components: indirubin, indigofera tinctoria, and indole glycosides, but the content varied. The indole glycoside and indigofera tinctoria stems contained higher levels than the leaves. Figures 10-13 It is known that only substances near the solvent front can be seen under fluorescent light, but when examined under ultraviolet light (302nm), other chemical components can be seen in its stems and leaves, the specifics of which still need to be verified.
[0055] The thin-layer chromatography identification method established in this experiment is simple and easy to prepare the test solution. The Rf values of each component are moderate: the Rf value of indigo is 0.70; the Rf value of indirubin is 0.62; and the Rf value of indole glycoside is 0.31. The spots are clearly visible and there is no obvious tailing phenomenon. It can well reflect the components of Indigofera tinctoria and can be used for qualitative identification of Indigofera tinctoria.
[0056] 4. Summary This experiment determined the content of various components in 24 batches of *Indigofera tinctoria* (Malan) medicinal materials. The results showed that the moisture content ranged from 7.68% to 9.37%, the total ash content from 7.32% to 9.94%, the acid-insoluble ash content from 0.51% to 1.01%, and the extract content from 14.46% to 26.76%. All results met the requirements. The experimental data revealed some differences between different batches, which may be related to factors such as harvesting time, growing environment, and altitude. The quality of traditional Chinese medicine is affected by many factors; ensuring the quality of traditional Chinese medicine and perfecting quality standards are crucial for improving clinical efficacy and ensuring the safety of clinical medication.
[0057] For qualitative identification, a cyclohexane-chloroform-methanol (5:4:2) development system was used, with silica gel G plates as the support. Indigo and indirubin were examined under fluorescent light, while indole glycosides were examined under 302 nm light. This method showed good resolution, with an Rf value of 0.70 for indigo, 0.62 for indirubin, and 0.31 for indole glycosides. The spots were also relatively clear, indicating that the experimental procedure was accurate and reliable, and can provide a basis for revising the quality standard of Strobilanthes bidentata.
[0058] The embodiments described herein are merely preferred embodiments of the invention and are not intended to limit the concept and scope of the invention. Any modifications and improvements made by those skilled in the art to the technical solutions of the invention without departing from the design concept of the invention should fall within the protection scope of the invention.
Claims
1. A method for quality testing of *Indigofera tinctoria*, characterized in that, include: Determination of moisture, total ash, acid-insoluble ash and extract content in *Indigofera tinctoria* (a type of medicinal herb); Thin-layer chromatography identification of indigo, indigo and indole glycosides in *Indigofera tinctoria*.
2. The method for quality testing of *Indigofera tinctoria* according to claim 1, characterized in that, The specific method for thin-layer chromatography identification of indirubin, indigofera tinctoria, and indole glycosides in *Indigofera tinctoria* is as follows: Take 1-3g of the powder, add 10-30 mL of ethyl acetate, heat and reflux to extract for 0.5-1.5 h, filter, take the filtrate, concentrate to 1-3 mL, and use as the test solution; take indirubin reference standard and indigo reference standard, add chloroform to prepare a solution containing 0.1 mg per mL, and take indole glycoside reference standard, add water to prepare a solution containing 0.1 mg per mL, and use as the reference solution; perform the thin-layer chromatography test according to General Rule 0502, take the test solution, indigo reference standard solution, indirubin reference standard solution, and indole glycoside reference standard solution and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-chloroform-methanol as the developing solvent, place in the developing tank for pre-saturation for 20-40 min, develop, remove, and air dry. Indigo and indirubin should be examined immediately, and indole glycoside should be examined under ultraviolet light. In the chromatogram of the test sample, spots or fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference medicinal material.
3. The method for quality testing of *Indigofera tinctoria* according to claim 2, characterized in that, The specific method for thin-layer chromatography identification of indirubin, indigofera tinctoria, and indole glycosides in *Indigofera tinctoria* is as follows: Take 2g of the powder, add 20 mL of ethyl acetate, heat and reflux for 1 h, filter, take the filtrate, concentrate to 2 mL, and use as the test solution; take indirubin reference standard and indigo reference standard, respectively add chloroform to prepare a solution containing 0.1 mg per 1 mL, and take indole glycoside reference standard, add water to prepare a solution containing 0.1 mg per 1 mL, as the reference solution; perform the thin-layer chromatography test according to General Rule 0502, take the test solution, indigo reference standard solution, indirubin reference standard solution, and indole glycoside reference standard solution and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-chloroform-methanol as the developing solvent, place in the developing tank for pre-saturation for 30 min, develop, remove, and air dry. Indigo and indirubin should be examined immediately, and indole glycoside should be examined under ultraviolet light. In the chromatogram of the test sample, spots or fluorescent spots of the same color should appear at the corresponding positions as in the chromatogram of the reference medicinal material.
4. The method for quality testing of *Indigofera tinctoria* according to any one of claims 2 or 3, characterized in that, The ratio of the developing agent is: cyclohexane-chloroform-methanol = 4-6:3.5-4.5:
2.
5. The method for quality testing of *Indigofera tinctoria* according to claim 4, characterized in that, The ratio of the developing solvent is: cyclohexane-chloroform-methanol = 5:4:
2.
6. The method for quality testing of *Indigofera tinctoria* according to any one of claims 2 or 3, characterized in that, The wavelength of the ultraviolet light used for inspection is 302 nm.
7. The method for quality testing of *Indigofera tinctoria* according to claim 1, characterized in that: The method for determining moisture content is as follows: Accurately weigh 2 g of *Indigofera tinctoria* powder, spread it evenly in a flat weighing bottle that has been dried to constant weight, accurately weigh it, dry it in an oven at 105 ℃ for 5 h, transfer it to a desiccator, cool it for 30 min, accurately weigh it, dry it again at the above temperature for 1 h, cool it, weigh it, until the difference between two consecutive weighings does not exceed 5 mg; perform three parallel determinations, and calculate the moisture content in the sample based on the weight loss.
8. The method for quality testing of *Indigofera tinctoria* according to claim 1, characterized in that: The method for checking total ash content is as follows: accurately weigh 5 g of indigo powder, place it in a crucible heated to constant weight, weigh it, heat it slowly, taking care to avoid combustion, and when it is completely carbonized, gradually increase the temperature to 500-600 ℃ to completely ashed and reach constant weight; calculate the total ash content in the sample based on the weight of the residue.
9. The method for quality testing of *Indigofera tinctoria* according to claim 1, characterized in that: The method for testing acid-insoluble ash is as follows: Take the ash obtained in the experiment described in claim 8 above, carefully add 10 mL of dilute hydrochloric acid to the crucible, cover the crucible with a watch glass, place it on a water bath and heat for 10 min, rinse the watch glass with 5 mL of hot water, add the washing liquid to the crucible, filter it with ashless filter paper, wash the residue in the crucible with water onto the filter paper, and wash until the washing liquid does not show a chloride reaction; transfer the filter residue and filter paper to the same crucible, dry it, and ignite it to constant weight; calculate the content of acid-insoluble ash in the test sample based on the weight of the residue.
10. The method for quality testing of *Indigofera tinctoria* according to claim 1, characterized in that: The method for testing the extract is as follows: Accurately weigh 3g of *Indigofera tinctoria* powder, perform two parallel tests, place it in a 150 mL conical flask, accurately add 75 mL of dilute ethanol, seal tightly, weigh, let stand for 1 h, connect a reflux condenser, heat to boiling, and maintain a gentle boil for 1 h; after cooling, remove the conical flask, seal tightly, weigh again, replenish the lost weight with dilute ethanol, shake well, filter through a dry filter, accurately measure 25 mL of the filtrate, place it in an evaporating dish that has been dried to constant weight, evaporate to dryness on a water bath, dry at 105℃ for 3 h, cool in a desiccator for 30 min, and quickly and accurately weigh; unless otherwise specified, calculate the content of water-soluble extract in the test sample based on the dried product.