Quality detection method for indigofera pseudotinctoria
Through UPLC method and multiple identification methods, the comprehensive quality inspection of horse thorn medicinal materials was solved, and the problem of lack of horse thorn quality standards and detection methods in the existing technology was achieved, and simple and reliable quality control and detection of horse thorn medicinal materials was achieved.
Patent Information
- Application Number
- CN202411547318.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-01
- Publication Date
- 2025-04-15
AI Technical Summary
In the prior art, there is a lack of quality standards and quality detection methods for horse thorn medicinal materials, making it difficult to achieve comprehensive quality control and testing of horse thorn medicinal materials.
The UPLC method was used to determine the content of monocaffeyl tartaric acid, gentilic acid and chicolic acid in horse spines, and comprehensive quality testing was carried out in combination with trait identification, micro identification and thin-layer identification.
It realizes the comprehensive quality control of Mazha medicinal materials, provides a simple and reliable detection method, can measure the content of multiple ingredients at the same time, and supports the formulation of quality standards.
Smart Images
Figure CN119827647A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of quality analysis and control of traditional Chinese medicines, and in particular relates to a quality detection method for horsethorn. Background Art
[0002] Horsethorn is the fresh or dried aerial part of the horsethorn Indigofera pseudotinctoria Matsum. (IpMatsum.) of the genus Indigofera in the Leguminosae family. It is a variety recorded in the third edition of the "Guizhou Flora" and the "National Compendium of Chinese Herbal Medicines". It will also be included as a new variety in the 2019 edition of the "Guizhou Provincial Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials". Horsethorn is grown in most parts of my country. At the same time, horsethorn is also a good feed raw material, mostly used as green fodder for cattle, sheep and other animals. Horsethorn has the effects of clearing heat, detoxifying, reducing swelling and dispersing knots. It can be used to treat colds and coughs, snake bites, hemorrhoids, and malnutrition in children. It is used as an ethnic medicine in ethnic minority areas and has a good effect on the treatment of burns and scalds. It has the effect of stopping bleeding quickly and promoting rapid wound healing in knife wounds. Its compound horsethorn medicine has a certain effect on chronic liver fibrosis; the ointment "one medicine" with the whole herb of horsethorn as the main ingredient has a significant effect on burns. Studies have found that horsethorn has pharmacological effects such as anti-atherosclerosis, antioxidant, and anti-inflammatory. The wild resources of horsethorn are rich and it is a special medicinal material with broad development prospects. Because its quality standard and related literature and books are rarely reported, the present invention provides a quality detection method. Summary of the invention
[0003] In view of the lack of quality standards and quality inspection methods for horse thorn in the prior art, the present invention provides a quality inspection method, which conducts comprehensive quality control and inspection of the medicinal material from the aspects of properties, microscopic identification, inspection items, content determination, etc., and has great innovation and economic value.
[0004] The technical scheme of the present invention comprises: a quality detection method of horse thorn, using UPLC method to determine the content of monocaffeoyltartaric acid, gentisic acid and chicoric acid in horse thorn, then performing property identification, microscopic identification and thin layer identification, and determining the moisture, total ash and extract.
[0005] The steps for determining the content of monocaffeoyltartaric acid, gentisic acid and chicoric acid in horse thorn using UPLC are as follows:
[0006] (1) Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column: 2.1×100 mm1.8 m, volume flow rate 0.45 mL / min, column temperature 45°C, detection wavelength 325 nm, injection volume 1 μL, 0.1% formic acid aqueous solution A and acetonitrile B as mobile phases, gradient elution, 0-6 min, A changes from 91 to 80%; 6-10 min, A changes from 80 to 50%; 10-12 min, A remains unchanged at 50%; 12-13 min, A changes from 50 to 91%; 13-15 min, A remains unchanged at 91%;
[0007] (2) Preparation of reference solution: Accurately weigh the reference substances gentisic acid, monocaffeoyltartaric acid, and chicoric acid, dissolve them in methanol, and make up to volume to prepare 1 mg / mL single reference solutions, which are then stored in a -20°C refrigerator for future use;
[0008] (3) Preparation of test solution: The horse thorn medicinal material was crushed, passed through a 60-mesh sieve, and stored at room temperature. Accurately weigh 1.0 g of horse thorn medicinal material powder, add 45% ethanol mass fraction, 15% K2HPO4 mass fraction, and a solid-liquid ratio of 1:10 g / mL. The ultrasonic time was 15 min, and the solution was centrifuged at 8600×g for 5 min. The supernatant was diluted 2 times and filtered through a 0.22 μm microporous filter membrane before sampling and analysis.
[0009] The specific parameters for trait identification are: measuring the length of the horse thorn, surface color, shape and diameter of the main branch, and the state of branching; the shape, color and size of the seeds; observing the shape and surface color of the leaves, the color of the cross section, the smell and taste.
[0010] The specific steps of microscopic identification are to observe the color of the powder; surface morphology; the number and morphology of non-glandular hairs; and the morphology of mesophyll cells and seed coat palisade cells.
[0011] The steps of thin layer identification include the following steps
[0012] (1) Preparation of test solution Take 2 g of the powder of the product, add 25 mL of methanol:25% hydrochloric acid solution, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 10 mL of water to dissolve it, then extract it twice with ethyl acetate, each time with 10 mL, combine the ethyl acetate solution, evaporate to dryness, and add 1 mL of methanol to dissolve it, which is used as the test solution;
[0013] (2) Preparation of reference solution Accurately weigh the quercetin reference substance, dissolve it in methanol and prepare a solution containing 0.1 mg per 1 mL as the reference solution;
[0014] (3) TLC results of horsetail herb were developed with a developing agent and observed at a wavelength of 365 nm.
[0015] The preferred developing solvent is toluene-ethyl acetate-formic acid, with a volume ratio of 8:4:0.5.
[0016] The moisture and total ash content shall not exceed 13% and 9% respectively, and the extract content shall not be less than 16%.
[0017] Compared with the prior art, the present invention has the following beneficial effects: This study studies the quality control method of horse thorn, establishes a qualitative identification method, and stipulates the limits of each inspection item. The above method is simple to operate, accurate and reliable, and can provide a reference for the formulation of its quality standards. At the same time, this study establishes a method for simultaneously determining the contents of three components, monocaffeoyltartaric acid, gentisic acid and chicoric acid, in horse thorn medicinal materials. The method is simple, reliable and low in cost.
[0018] The present invention performs thin layer identification on quercetin contained in horse thorn medicinal materials. In the process of selecting a developing agent, according to the physical and chemical properties of horse thorn and quercetin, the influence of factors such as the composition and ratio of the developing agent are respectively investigated, and toluene-acetone-formic acid (10:3:0.5), toluene-ethyl acetate-formic acid (7:3:0.5), and toluene-ethyl acetate-formic acid (8:4:0.5) are respectively used as developing agents, and the samples are developed, taken out, dried, sprayed with aluminum chloride test solution, and inspected under a 365nm ultraviolet light. When toluene-acetone-formic acid (10:3:0.5) is used as the developing agent, the spot separation is poor and unclear, so this developing agent is not selected. When toluene-ethyl acetate-formic acid (7:3:0.5) is used, the result reproducibility is poor and the spot separation is not very good, so it is not selected. When toluene-ethyl acetate-formic acid (8:4:0.5) was used as the developing agent, the results were reproducible, the spots were clear, and the separation was good, so this developing agent was selected as the final condition. Through the extraction conditions and the determination of the developing agent, the temperature, humidity, and different thin layer silica gel G plates of horse thorn were investigated. The results showed that the chromatographic development of horse thorn was good at three different temperatures: low temperature (5℃), room temperature (25℃) and high temperature (40℃); it could also be well separated under low humidity (32%), medium humidity (56%) and high humidity (72%); in addition, this experiment investigated two kinds of thin layer silica gel G plates, homemade and prefabricated, and the results showed that the separation effect of the sample was good. This proves that the thin layer chromatography method of horse thorn is stable, feasible and reliable.
[0019] Under the above chromatographic conditions, the three compounds have good separation effect and stable baseline, which can meet the requirements of quantitative analysis. According to the content determination results, the contents of the main active ingredients in the 10 batches of horse thorn medicinal materials vary greatly, which is related to the different origins and harvesting periods of horse thorn. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Attached Figure 1 Characteristic diagram of horsethorn;
[0021] Attached Figure 2 Microscopic characteristics of powder: 1. Non-glandular hairs; 2. Seed coat cells; 3. Leaf surface cells; 4. Vessels; 5. Calcium oxalate clusters; 6. Fibers;
[0022] Attached Figure 3 TLC of horsethorn;
[0023] Attached Figure 4 Chromatogram of the sample (A) and the reference substance (B) (1-monocaffeoyltartaric acid; 2-gentisic acid; 3-chicoric acid. DETAILED DESCRIPTION
[0024] Example 1
[0025] 1 Materials
[0026] 1.1 Instruments The optical biological microscope (DM2500) and image analysis system (DFC500) were purchased from Leica Instruments, Germany; the ME104E electronic balance (0.0001 mg) was purchased from Mettler Toledo, Switzerland; the Milli-Q ultrapure water preparation instrument was purchased from Merck Millipore, USA; the Waters H-Class ultra-high performance liquid chromatograph (Waters, USA); and the silica gel G plate was purchased from Qingdao Ocean Chemical Co., Ltd.
[0027] 1.2 Reagents Quercetin reference substance (batch number: 100081-201910, purity: 98%) was purchased from China Food and Drug Inspection Institute; gentisic acid (batch number C14068945), monocaffeoyltartaric acid (batch number S123062751), and chicoric acid (S122081796) were all purchased from Shanghai Shifeng Biotechnology Co., Ltd., and their purities were all greater than 98%; methanol, hydrochloric acid, toluene, formic acid, acetone, ethyl acetate, aluminum chloride, etc. were purchased from Komiou Chemical Reagent Co., Ltd. All reagents used were analytical grade, and water was ultrapure water.
[0028] 1.3 Medicinal Materials The source information of 10 batches of horsetail samples is shown in Table 1. They were identified by Professor Wang Xiangpei of Guizhou Minzu University as the dried aerial parts of horsetail of the plant Indigofera genus of the Leguminosae family.
[0029] Table 1 Information of 10 batches of horsethorn samples
[0030]
[0031] 2 Methods and Results
[0032] 2.1 Identification of properties The horsethorn medicinal materials from different origins have slight differences in size, but other characteristics are relatively stable. The identification characteristics of the medicinal material are summarized as follows: This product is in the shape of branches of varying lengths, 10 to 15 cm long, with a light green or gray-brown surface, and the main branches are cylindrical, 0.5 to 2.0 cm in diameter, with thin branches and sparse longitudinal wrinkles. The pods are opposite on the branches, long cylindrical, with a sharp tip, slightly curved, 2-5 cm long, yellowish brown, and about 3 mm in diameter. There are transverse septa between the seeds, which are nearly square, and some are slightly concave on one side, gray-green or brown, and about 1 mm in size. The leaves are mostly curled, light brown on the upper surface, and light green on the lower surface. The texture is hard, not easy to break, the cross section is yellowish-white, and fibrous. The smell is slight, the taste is bitter, see attached Figure 1 .
[0033] 2.2 Microscopic identification of powder The powder of this product is brown to gray-brown. The surface view is polygonal or irregular in shape, and the stomata are indefinite and quasi-circular. There are many non-glandular hairs, including T-shaped non-glandular hairs, which are relatively thick and thick-walled. Some have nearly equal lengths at both ends, and some have one end that is shorter, spindle-shaped, and slightly curved. The calcium oxalate clusters are small and scattered in the mesophyll cells. The seed coat is palisade-shaped, with the radiant band at the top; polygonal in top view, with thick walls; the supporting cells are dumbbell-shaped. The fibers are bundled, relatively long, and some have septa. Trapezoidal vessels are visible, and occasionally there are fragments of spiral vessels and vessels with margins and pits, see attached. Figure 2 .
[0034] 2.3 Thin layer identification experiments and methodological investigations were carried out in accordance with the relevant requirements of the "Implementation Rules of the Guidelines for Validation of Traditional Chinese Medicine Analytical Methods" of the State Pharmacopoeia Committee.
[0035] 2.3.1 Preparation of test solution Take 2 g of the powder of the product, add 25 mL of methanol:25% hydrochloric acid solution, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 10 mL of water to dissolve it, then extract with ethyl acetate twice, 10 mL each time, combine the ethyl acetate, evaporate to dryness, and add 1 mL of methanol to the residue to dissolve it, which is used as the test solution.
[0036] 2.3.2 Preparation of reference solution Accurately weigh the quercetin reference substance, dissolve it in methanol and prepare a solution containing 0.1 mg per 1 mL as the reference solution.
[0037] 2.3.3 TLC results of horse thorn medicinal materials Toluene-ethyl acetate-formic acid (8:4:0.5) was used as the developing solvent for development. When observed at a wavelength of 365 nm, the TLC spots were clear and obvious in color, with good separation. The TLC spots obtained by this method had a moderate retention factor value (Rf). Therefore, this method was used as the TLC method for horse thorn, among which the S position quercetin reference substance, No. 1 to No. 5 were horse thorn samples, see the attached Figure 3 .
[0038] 2.4 Inspection
[0039] 2.4.1 The moisture content was determined according to the second method of General Rules 0832 of the 2020 edition of the Chinese Pharmacopoeia. The sample measurement results were 10.28-10.62%. The measurement results are shown in Table 2. The average value was 10.51%. The tentative moisture content should not exceed 13.0%. 2.4.2 The total ash content was determined according to General Rules 2302 of the 2020 edition of the Chinese Pharmacopoeia. The sample measurement results were 7.25-7.83%. The measurement results are shown in Table 2. The average value was 7.52%. The tentative total ash content should not exceed 9.0%.
[0040] 2.4.3 Extracts The extracts of a batch of horsethorn were determined by cold leaching and hot leaching using ethanol and water as solvents, respectively. The results are shown in Table 2. According to the results, water was used as the solvent and the hot leaching method was used to determine the extracts of 5 batches of samples. The results of the samples were 19.08-22.31%, with an average value of 20.23%. The results are shown in Table 2. According to the results, the tentative extract standard shall not be less than 16.0%.
[0041] Table 2 Determination results of moisture, total ash and extract
[0042]
[0043] 2.5 Content determination
[0044] 2.5.1 Chromatographic conditions: A Waters ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 m) was used, with a volume flow rate of 0.45 mL / min, a column temperature of 45°C, a detection wavelength of 325 nm, an injection volume of 1 μL, 0.1% formic acid aqueous solution (A) and acetonitrile (B) as the mobile phases, and a gradient elution. From 0 to 6 min, the change in A% was 91 to 80%; from 6 to 10 min, the change in A% was 80 to 50%; from 10 to 12 min, A% remained unchanged at 50%; from 12 to 13 min, A% changed from 50 to 91%; from 13 to 15 min, A% remained unchanged at 91%.
[0045] 2.5.2 Preparation of reference solution Accurately weigh the reference substances gentisic acid, monocaffeoyltartaric acid and chicoric acid respectively, dissolve them in methanol and make up to volume to prepare 1 mg / mL single reference solution, and store them in a -20℃ refrigerator for later use.
[0046] 2.5.3 Preparation of test solution The horse thorn medicinal material was crushed, passed through a 60-mesh sieve, and stored at room temperature. Accurately weigh 1.0 g of horse thorn medicinal material powder, add 45% ethanol mass fraction, 15% K2HPO4 mass fraction, solid-liquid ratio of 1:10 g / mL, ultrasonic time for 15 min, centrifuge at 8600×g for 5 min, take the supernatant and dilute it 2 times, filter it through a 0.22μm microporous filter membrane, and then inject it for analysis.
[0047] 2.5.4 Results of standard curve, detection limit and quantitation limit After extraction and determination according to the above conditions, the regression equations and linear ranges of the three components were obtained. The linear regression equations and correlation coefficients and other parameter results are shown in Table 3. The mass concentrations of each reference substance when the signal-to-noise ratio was 3 and 10 were used as the detection limit (LOD) and quantitation limit (LOQ), respectively.
[0048] Table 3 Regression equation, linear range, correlation coefficient, detection limit and quantification limit of the three target components
[0049]
[0050] 2.5.5 Results of precision, repeatability, stability and sample recovery The above method was used to determine the precision, repeatability, stability and sample recovery of the method. The results are shown in Table 4. By calculating the peak area of each compound, the RSD of the peak area of the precision, repeatability and stability experiments was less than ≤2.07%, indicating that the instrument precision, repeatability and stability of the method were good; by calculating the sample recovery, the average spike recovery was between 96.90% and 106.16%, and the RSD value was between 0.22% and 2.38%. The results are shown in Table 4.
[0051] Table 4. Test results of recovery rate of horse thorn sample addition
[0052]
[0053]
[0054] 2.5.6 Sample content determination Horsethorn samples were prepared according to the above conditions and injected for analysis. The content of each component was calculated using the external standard method. The results of the determination and calculation are attached. Figure 4 and Table 5.
[0055] Table 5 External standard method content determination results (x±s, n=3, mg / g)
[0056]
[0057]
Claims
1. A method for detecting the quality of horse thorns, characterized in that: The contents of monocaffeoyltartaric acid, gentisic acid and chicoric acid in horse thorn were determined by UPLC method, and then the properties, microscopic identification and thin layer identification were carried out, and the moisture, total ash and extract were determined.
2. A method for detecting the quality of horse thorns according to claim 1, characterized in that: The steps for determining the content of monocaffeoyltartaric acid, gentisic acid and chicoric acid in horse thorn using UPLC are as follows: (1) Chromatographic conditions: Waters ACQUITY UPLC HSS T3 column: 2.1×100mm 1.8m, volume flow rate 0.45 mL / min, column temperature 45℃, detection wavelength 325 nm, injection volume 1 μL, 0.1% formic acid aqueous solution A and acetonitrile B as mobile phases, gradient elution, 0-6 min, A changes from 91 to 80%; 6-10 min, A changes from 80 to 50%; 10-12 min, A remains unchanged at 50%; 12-13 min, A changes from 50 to 91%; 13-15 min, A remains unchanged at 91%; (2) Preparation of reference solution: Accurately weigh the reference substances gentisic acid, monocaffeoyltartaric acid, and chicoric acid, dissolve them in methanol, and make up to volume to prepare 1 mg / mL single reference solutions. Store in a -20°C refrigerator for later use. (3) Preparation of test solution: Pulverize the horse thorn medicinal material, pass through a 60-mesh sieve, and store at room temperature. Accurately weigh 1.0 g of horse thorn medicinal material powder, add 45% ethanol mass fraction, 15% K2HPO4 mass fraction, and a solid-liquid ratio of 1:10 g / mL. Ultrasonic time is 15 min, and centrifuge at 8600×g for 5 min. Take the supernatant and dilute it 2 times. Pass it through a 0.22 μm microporous filter membrane and then inject it for analysis.
3. A method for detecting the quality of horse thorns according to claim 1, characterized in that: The specific parameters for trait identification are: measuring the length of the horse thorn, surface color, shape and diameter of the main branch, and the state of branching; the shape, color and size of the seeds; observing the shape and surface color of the leaves, the color of the cross section, the smell and taste.
4. A method for detecting the quality of horse thorns according to claim 1, characterized in that: Microscopic identification includes observing the color of the powder; surface morphology; observing the number and morphology of non-glandular hairs; and observing the morphology of mesophyll cells and seed coat palisade cells.
5. The method for detecting the quality of horse thorn according to claim 1, characterized in that: The steps of thin layer identification include the following steps (1) Preparation of test solution Take 2 g of the powder of the product, add 25 mL of methanol:25% hydrochloric acid solution, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, add 10 mL of water to dissolve, extract with ethyl acetate twice, 10 mL each time, combine the ethyl acetate solution, evaporate to dryness, and add 1 mL of methanol to the residue to dissolve it, which is used as the test solution; (2) Preparation of reference solution Accurately weigh the quercetin reference substance, dissolve it in methanol and prepare a solution containing 0.1 mg per 1 mL as the reference solution; (3) The TLC results of the horsethorn medicinal material were developed with a developing solvent and observed at a wavelength of 365 nm.
6. A method for detecting the quality of horse thorns according to claim 5, characterized in that: The developing solvent was toluene-ethyl acetate-formic acid with a volume ratio of 8:4:0.
5.
7. The method for detecting the quality of horse thorn according to claim 1, characterized in that: The moisture and total ash content shall not exceed 13% and 9% respectively, and the extract content shall not be less than 16%.