A method for determining the content of gallic acid and (+)-catechin in sesame medicinal material.

Abstract

This invention discloses a method for determining the content of gallic acid and (+)-catechin in *Hedysarum heterotropoides* medicinal materials. After reflux treatment with 60% methanol solution in a water bath, the *Hedysarum heterotropoides* medicinal materials are subjected to high-performance liquid chromatography (HPLC) with octadecyl-bonded silica gel as the stationary phase and acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) as the mobile phase. The peak area values ​​of gallic acid and (+)-catechin showed good linearity in the injection amounts of 10.00–100.0 μg and 200.0–2000 μg, respectively, with average recoveries of 98.31% and 97.68%, and RSD values ​​of 1.2% and 1.2%, respectively. The method is simple and accurate, and can be used for the determination of gallic acid and (+)-catechin content, providing a theoretical basis for the quality control of *Hedysarum heterotropoides* medicinal materials.

Description

Technical Field

[0001] This invention relates to a method for determining the content of gallic acid and (+)-catechin in sesame seed, belonging to the field of medicine. Background Technology

[0002] Purple sesame (Ampelopsis humulifolia Bunge. var. heterophylla (Thunb.) K. Koch. and Ampelopsis delavayana Planch., belonging to the genus Ampelopsis Michx. of the Vitaceae family, is derived from the dried roots of these plants. It possesses properties of clearing heat and tonifying deficiency, dispersing blood stasis and promoting blood circulation, and detoxifying. It is commonly used to treat postpartum irritability and thirst, hemiplegia due to stroke, and injuries from falls. However, its quality standards are relatively low, hindering quality control of the medicinal material and related preparations. Modern research indicates that purple sesame contains phenolic, flavonoid, terpene, steroidal, and tannin chemical components. Among these, gallic acid and (+)-catechin are known to be major components, exhibiting antioxidant, anti-inflammatory, analgesic, hepatoprotective, and hypoglycemic effects. Therefore, using gallic acid and (+)-catechin as indicators, a high-performance liquid chromatography method was established to determine the content of sesame seeds, providing experimental basis for the quality standard research of sesame seeds.

[0003] This invention establishes a high-performance liquid chromatography (HPLC) method to more precisely and accurately determine the content of gallic acid and (+)-catechin, key components in sesame seed medicinal materials. Summary of the Invention

[0004] The present invention aims to provide a method for determining the content of gallic acid and (+)-catechin in sesame medicinal materials. The method uses high performance liquid chromatography to determine the content of gallic acid and (+)-catechin in sesame medicinal materials. The method is simple, easy to operate, and has strong specificity and reliability.

[0005] The technical solution of this invention:

[0006] A method for determining the content of gallic acid and (+)-catechin in hemp seed medicinal material, wherein the method employs high performance liquid chromatography and includes the following steps:

[0007] (1) Preparation of mixed reference solution: Accurately weigh gallic acid and (+)-catechin, place them in volumetric flasks, add 50-70% methanol to dissolve them and prepare single reference solutions with mass concentrations of 0.10 mg / mL and 2.00 mg / mL respectively. Accurately pipette different volumes of single reference solutions to prepare 1 mL of mixed reference solution containing 0.035-0.045 mg gallic acid and 0.75-90 mg (+)-catechin;

[0008] (2) Preparation of test solution: Take 0.9-1.1g of purple hemp powder, accurately weigh it, place it in a stoppered conical flask, add 15-25mL of 50-70% methanol solution, weigh it, extract by reflux in a water bath for 1.5-2.5h, take it out, cool it, add the corresponding amount of methanol solution to make up the lost weight, shake well, filter, evaporate the filtrate to dryness in a water bath, dissolve the residue in 50-70% methanol in a 5mL volumetric flask, and make up to the mark, shake well, and the test solution is ready;

[0009] (3) Chromatographic conditions and system suitability test: Column: Diamonsil-C18 column (250mm×4.6mm, 5μm); Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution is performed; Detection wavelength is 271-280nm; Column temperature is 20-30℃; Flow rate is 0.8-1.2mL / min; Injection volume is 10-15μL;

[0010] (4) Accurately pipette 10 μL of the mixed reference solution and the test solution into the high performance liquid chromatograph and perform the determination according to the chromatographic conditions in step (3).

[0011] In step (1) above, the preparation of the mixed reference solution is as follows: Gallic acid and (+)-catechin are accurately weighed and placed in volumetric flasks. They are dissolved in 60% methanol and prepared as single reference solutions with mass concentrations of 0.10 mg / mL and 2.00 mg / mL, respectively. Different volumes of single reference solutions are accurately pipetted to prepare 1 mL of mixed reference solution containing 0.04 mg gallic acid and 0.83 mg (+)-catechin.

[0012] In step (2) above, take 0.95-1.05g of purple hemp powder, weigh it accurately, place it in a stoppered conical flask, add 15-20mL of 55-65% methanol solution, weigh it, extract by reflux in a water bath for 2-3h, take it out, cool it, add the corresponding amount of methanol solution to make up the lost weight, shake it well, filter it, evaporate the filtrate to dryness in a water bath, dissolve the residue in 55-65% methanol in a 5mL volumetric flask, and make up to the mark, shake it well, and the test solution is ready.

[0013] Specifically, in step (2) above, take 1.0g of purple hemp powder, weigh it accurately, place it in a stoppered conical flask, add 20mL of 60% methanol solution, weigh it, reflux in a water bath for 2h, take it out, cool it, add the corresponding amount of methanol solution to make up the lost weight, shake it well, filter it, continue to evaporate the filtrate in a water bath, add 60% methanol to dissolve the residue in a 5mL volumetric flask, and make up to the mark, shake it well, and the test solution is ready.

[0014] In step (3) above, the detection wavelength is 271nm and the column temperature is 25℃.

[0015] In step (3) above, the gradient elution conditions are as follows: the elution program is 0-22 min, 0%-5% B; 22-25 min, 5%-8% B; 25-40 min, 8% B; 40-50 min, 8%-8.5% B; 50-75 min, 8.5%-40% B.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. This invention establishes an HPLC method for the determination of gallic acid and (+)-catechin in sesame seed by studying the content determination method of gallic acid and (+)-catechin in sesame seed. The method is simple, easy to operate, and produces accurate results.

[0018] 2. This invention optimizes extraction and chromatographic conditions by using peak shape, resolution, and peak area as indicators during the optimization process to obtain the optimal extraction and chromatographic conditions.

[0019] 3. This invention provides a theoretical basis for the study of quality standards for sesame medicinal materials by measuring the content of gallic acid and (+)-catechin in sesame medicinal materials. Attached Figure Description

[0020] Figure 1 Specificity assessment HPLC chromatograms (A: blank solution; B: test solution; C: reference solution; 1: gallic acid in the test solution; 2: (+)-catechin in the test solution; 3: gallic acid in the reference solution; 4: (+)-catechin in the reference solution);

[0021] Figure 2 UV spectra of gallic acid in the test solution and the mixed reference solution (a is the UV spectrum of gallic acid in the mixed reference solution; b is the UV spectrum of gallic acid in the test solution).

[0022] Figure 3 UV spectra of (+)-catechins in the test solution and the mixed reference solution (a is the UV spectrum of (+)-catechins in the mixed reference solution; b is the UV spectrum of (+)-catechins in the test solution).

[0023] Figure 4 Gallic acid standard curve;

[0024] Figure 5 (+)-Catechin standard curve;

[0025] Figure 6 Precision test HPLC chromatogram overlay;

[0026] Figure 7HPLC chromatogram for repeatability testing;

[0027] Figure 8 Stability test HPLC chromatogram overlay;

[0028] Figure 9 HPLC chromatogram of recovery test;

[0029] Figure 10 HPLC chromatograms of 11 batches of purple hemp medicinal materials samples. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0031] Example 1:

[0032] Preparation of mixed reference solution: Accurately weigh gallic acid and (+)-catechin, place them in volumetric flasks, dissolve them in 60% methanol, and prepare single reference solutions with mass concentrations of 0.10 mg / mL and 2.00 mg / mL, respectively. Accurately pipette different volumes of the single reference solutions to prepare 1 mL of mixed reference solution containing 0.04 mg gallic acid and 0.83 mg (+)-catechin.

[0033] Preparation of the test solution: Accurately weigh 1.0 g of purple hemp powder, place it in a stoppered conical flask, add 20 mL of 60% methanol solution, weigh it, reflux in a water bath for 2 h, remove it, cool it, add the corresponding amount of methanol solution to make up the weight loss, shake well, filter, evaporate the filtrate to dryness in a water bath, dissolve the residue in 60% methanol in a 5 mL volumetric flask, and dilute to the mark, shake well, and the test solution is ready.

[0034] Chromatographic conditions and system suitability test: Column: Diamonsil-C18 column (250mm×4.6mm, 5μm); Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution is performed; Detection wavelength is 271nm; Column temperature is 5℃; Flow rate is 1mL / min; Injection volume is 10μL.

[0035] The gradient elution conditions are as follows: elution program is 0-22 min, 0%-5% B; 22-25 min, 5%-8% B; 25-40 min, 8% B; 40-50 min, 8%-8.5% B; 50-75 min, 8.5%-40% B.

[0036] Accurately pipette 10 μL each of the mixed reference solution and the test solution into the high-performance liquid chromatograph, and perform the determination according to the chromatographic conditions described above.

[0037] Example 2:

[0038] Preparation of mixed reference solution: Accurately weigh gallic acid and (+)-catechin, place them in volumetric flasks, dissolve them in 50-70% methanol, and prepare single reference solutions with mass concentrations of 0.10 mg / mL and 2.00 mg / mL, respectively. Accurately pipette different volumes of the single reference solutions to prepare 1 mL of mixed reference solution containing 0.035 mg gallic acid and 0.75 mg (+)-catechin.

[0039] Preparation of the test solution: Accurately weigh 0.9 g of purple hemp powder, place it in a stoppered conical flask, add 15 mL of 50% methanol solution, weigh it, reflux in a water bath for 1.5 h, remove it, cool it, add the corresponding amount of methanol solution to make up the weight loss, shake well, filter, evaporate the filtrate to dryness in a water bath, dissolve the residue in 50% methanol in a 5 mL volumetric flask, and dilute to the mark, shake well, and the test solution is ready.

[0040] Chromatographic conditions and system suitability test: Column: Diamonsil-C18 column (250mm×4.6mm, 5μm); Mobile phase: Mobile phase A is acetonitrile, Mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution; Detection wavelength: 271nm; Column temperature: 20℃; Flow rate: 0.8mL / min; Injection volume: 10μL.

[0041] The gradient elution conditions are as follows: elution program is 0-22 min, 0%-5% B; 22-25 min, 5%-8% B; 25-40 min, 8% B; 40-50 min, 8%-8.5% B; 50-75 min, 8.5%-40% B.

[0042] Accurately pipette 10 μL each of the mixed reference solution and the test solution into the high-performance liquid chromatograph, and perform the determination according to the chromatographic conditions described above.

[0043] Example 3:

[0044] Preparation of mixed reference solution: Accurately weigh gallic acid and (+)-catechin, place them in volumetric flasks, dissolve them in 50-70% methanol, and prepare single reference solutions with mass concentrations of 0.10 mg / mL and 2.00 mg / mL, respectively. Accurately pipette different volumes of the single reference solutions to prepare 1 mL of mixed reference solution containing 0.045 mg gallic acid and 90 mg (+)-catechin.

[0045] Preparation of the test solution: Accurately weigh 1.1g of purple hemp powder, place it in a stoppered conical flask, add 25mL of 70% methanol solution, weigh it, reflux in a water bath for 2.5h, remove it, cool it, add the corresponding amount of methanol solution to make up the lost weight, shake well, filter, evaporate the filtrate to dryness in a water bath, dissolve the residue in 70% methanol in a 5mL volumetric flask, and dilute to the mark, shake well, and the test solution is ready.

[0046] Chromatographic conditions and system suitability test: Column: Diamonsil-C18 column (250mm×4.6mm, 5μm); Mobile phase: Mobile phase A is acetonitrile, Mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution is performed; Detection wavelength is 280nm; Column temperature is 30℃; Flow rate is 1.2mL / min; Injection volume is 15μL.

[0047] The gradient elution conditions are as follows: elution program is 0-22 min, 0%-5% B; 22-25 min, 5%-8% B; 25-40 min, 8% B; 40-50 min, 8%-8.5% B; 50-75 min, 8.5%-40% B.

[0048] Accurately pipette 10 μL each of the mixed reference solution and the test solution into the high-performance liquid chromatograph, and perform the determination according to the chromatographic conditions described above.

[0049] Example 4:

[0050] Preparation of mixed reference solution: Accurately weigh gallic acid and (+)-catechin, place them in volumetric flasks, dissolve them in 65% methanol, and prepare single reference solutions with mass concentrations of 0.10 mg / mL and 2.00 mg / mL, respectively. Accurately pipette different volumes of the single reference solutions to prepare 1 mL of mixed reference solution containing 0.04 mg gallic acid and 0.83 mg (+)-catechin.

[0051] Preparation of the test solution: Accurately weigh 0.95 g of purple hemp seed powder, place it in a stoppered conical flask, add 20 mL of 65% methanol solution, weigh it, reflux in a water bath for 2.5 h, remove it, cool it, add the corresponding amount of methanol solution to make up the weight loss, shake well, filter, evaporate the filtrate to dryness in a water bath, dissolve the residue in 65% methanol in a 5 mL volumetric flask, and dilute to the mark, shake well, and the test solution is ready.

[0052] Chromatographic conditions and system suitability test: Column: Diamonsil-C18 column (250mm×4.6mm, 5μm); Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution is performed; Detection wavelength is 271nm; Column temperature is 5℃; Flow rate is 1mL / min; Injection volume is 10μL.

[0053] The gradient elution conditions are as follows: elution program is 0-22 min, 0%-5% B; 22-25 min, 5%-8% B; 25-40 min, 8% B; 40-50 min, 8%-8.5% B; 50-75 min, 8.5%-40% B.

[0054] Accurately pipette 10 μL each of the mixed reference solution and the test solution into the high-performance liquid chromatograph, and perform the determination according to the chromatographic conditions described above.

[0055] The inventor conducted numerous experiments; the following are some of the experimental studies.

[0056] 1. Instruments and reagents

[0057] 1.1 Instruments

[0058] Thermo UltiMate-3000 high-performance liquid chromatograph (Thermo Scientific, USA); DAD detector; Diamonsil-C18 column (250mm×4.6mm, 5μm); AG135 electronic balance (Mettler-Toledo, Switzerland); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), etc.

[0059] 1.2 Reagents

[0060] Methanol (Tenet, Inc., chromatographic grade; Sinopharm Chemical Reagent Co., Ltd., analytical grade); acetonitrile (Tenet, Inc., chromatographic grade); ethanol (Tianjin Fuyu Fine Chemical Co., Ltd., analytical grade); phosphoric acid (Tianjin Kemeio Chemical Reagent Co., Ltd., chromatographic grade); redistilled water; gallic acid (batch number: 110831-200302), (+)-catechin (batch number: 110877-201604), all purchased from the National Institutes for Food and Drug Control.

[0061] 1.3 Medicinal Materials

[0062] The experimental samples were collected from Guiyang City, Anshun City, and Huishui County in Guizhou Province. They were identified by Professor Sun Qingwen of Guizhou University of Traditional Chinese Medicine as roots of *Ampelopsis humulifolia* Bunge. var. *heterophylla* (Thunb.) K. Koch. and *Ampelopsis delavayana* Planch. ex Franch., both belonging to the genus *Ampelopsis* of the family Vitaceae. The roots were washed, dried at 50°C, pulverized, and passed through a 60-mesh sieve for later use. Detailed sample information is shown in Table 6.

[0063] 2. Methods and Results

[0064] 2.1 Chromatographic conditions

[0065] Chromatographic column: Diamonsil-C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution, gradient elution (0–22 min, 5% A; 22–25 min, 5%–8% A; 25–40 min, 8% A; 40–50 min, 8%–8.5% A); detection wavelength: 271 nm; column temperature: 25 °C; flow rate: 1 mL / min -1 Injection volume: 10 μL.

[0066] 2.2 Solution Preparation

[0067] (1) Preparation of reference solution

[0068] Accurately weigh appropriate amounts of gallic acid and (+)-catechin, place them in volumetric flasks, dissolve them in 60% methanol, and prepare solutions with mass concentrations of 0.10 and 2.00 mg·mL, respectively. -1 Prepare single reference solutions. Accurately pipette different volumes of the single reference solution to prepare 1 mL mixed reference solutions containing 0.04 mg and 0.83 mg, respectively.

[0069] (2) Preparation of the test solution

[0070] Accurately weigh approximately 1.0 g of the medicinal powder and place it in a stoppered conical flask. Add 20 mL of 60% methanol solution, weigh the flask, and extract by reflux in a water bath for 2 hours. Remove the flask, cool it, add the corresponding amount of methanol solution to make up the weight loss, shake well, filter, and evaporate the filtrate to dryness in a water bath. Dissolve the residue in 60% methanol in a 5 mL volumetric flask and dilute to the mark. Shake well to obtain the test solution.

[0071] 2.3 Specificity Examination

[0072] Accurately pipette 10 μL each of the reference solution, test solution, and blank solution, and determine the concentration according to the chromatographic conditions described in step 2.1 above. The results show that the retention time and UV spectrum of the target peak in the chromatogram of the test solution are similar to those of the gallic acid and (+)-catechin reference standards. Figure 1 The results showed that the resolution was greater than 1.5 and baseline separation was achieved, with a purity factor of 1000 for all peaks, indicating good peak purity and strong specificity of the established method. Figure 1-3 As shown.

[0073] 2.4 Examination of the linear range

[0074] Take 1, 2, 4, 6, 8, and 10 mL of the above gallic acid and (+)-catechin reference solutions respectively and place them in 10 mL volumetric flasks. Dilute to the mark with 60% methanol solution, shake well, and prepare a series of reference solutions of varying concentrations. Accurately pipette 10 μL of each solution and inject it into the high-performance liquid chromatograph under the planned chromatographic conditions. Plot a standard curve with the injection volume (X) as the abscissa and the peak area (Y) as the ordinate. The regression equation for gallic acid is Y = 30.172X + 0.0922, r = 0.9999; the regression equation for (+)-catechin is Y = 6.2559X + 0.8829, r = 0.9999. The injection volume shows a good linear relationship with the peak area in the ranges of 10.00–100.0 μg and 200.0–2000 μg, as shown in Table 1. Figure 4-5 As shown.

[0075] Table 1. Results of the linear range investigation (n=6)

[0076]

[0077] 2.5 Precision Test

[0078] Accurately pipette the gallic acid and (+)-catechin reference solutions, and inject 10 μL six times consecutively under the prescribed chromatographic conditions. Measure the peak areas of gallic acid and (+)-catechin components (see [reference]). Figure 6 The calculated peak area RSD values ​​were 0.24% and 0.11%, respectively, which meet the technical requirements for the validation of the quality standard analytical method (RSD within 2.0%), indicating that the instrument has good precision. The results are shown in Table 2.

[0079] Table 2. Precision test results (n=6)

[0080]

[0081] 2.6 Repeatability Test

[0082] Six samples of the same batch of medicinal materials were taken, and test solutions were prepared according to the proposed test solution preparation method. The solutions were then injected and analyzed under the proposed chromatographic conditions, with 10 μL injected each time. The results showed that the average contents of gallic acid and (+)-catechin were 0.029% and 0.346%, respectively (see...). Figure 7 The RSD values ​​of the contents were calculated to be 1.7% and 1.9%, respectively, which meet the technical requirements for the validation of analytical methods in quality standards (RSD within 3.0%), indicating that the repeatability of the method is good. The results are shown in Table 3.

[0083] Table 3. Results of repeatability tests (n=6)

[0084]

[0085] 2.7 Stability Test

[0086] Take one sample of the powdered medicinal material from the same batch and prepare one test solution according to the proposed method. Inject 10 μL of the solution at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h under the proposed chromatographic conditions, and determine the peak area values ​​of gallic acid and (+)-catechin components (see...). Figure 8 The RSD values ​​of the peak areas of gallic acid and (+)-catechin were calculated to be 0.29% and 0.15%, respectively, indicating that the test solution was stable within 48 h. The results are shown in Table 4.

[0087] Table 4. Stability test results (n=8)

[0088]

[0089] 2.8 Recovery Test

[0090] Nine portions of the medicinal material to be tested, each approximately 0.5 g, from the same batch as the repeatability test were accurately weighed. Gallic acid and (+)-catechin reference standards were added at ratios of 1:0.5, 1:1, and 1:1.5, respectively. The test solutions were prepared according to the prescribed methods. The solutions were injected and analyzed under the prescribed chromatographic conditions, and the peak areas of gallic acid and (+)-catechin were recorded (see...). Figure 9 The average recoveries of gallic acid and (+)-catechin were calculated to be 98.31% and 97.68%, respectively, with RSD values ​​of 1.2% and 1.2%, respectively.

[0091] The recovery rate was 1.2%, which meets the technical requirements for the validation of analytical methods according to quality standards (recovery limit of 90-108%), indicating that the method has high accuracy. The results are shown in Table 5.

[0092] Table 5 Results of the recovery rate test (n=9)

[0093]

[0094]

[0095] 2.9 Sample Determination

[0096] Eleven batches of purple hemp medicinal material, each approximately 1.0 g, were accurately weighed. Test solutions were prepared according to the prescribed method, and the solutions were injected and analyzed under the prescribed chromatographic conditions. Each sample was analyzed three times, and the peak areas of gallic acid and (+)-catechin were recorded (see...). Figure 10 The percentage contents of gallic acid and (+)-catechin in the dried medicinal materials were calculated using the standard curve method. The results are shown in Table 6.

[0097] Table 6. Test results of 11 batches of dried purple hemp medicinal materials (n=5)

[0098]

[0099] 3 Discussion

[0100] 3.1 Selection of chromatographic conditions

[0101] In this experiment, the detection wavelength was screened using 3D spectra obtained from a DAD detector. The results showed that gallic acid and (+)-catechin had maximum absorption at 271 nm. Different solvent systems were used, including acetonitrile-water, acetonitrile-0.1% phosphoric acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution, and methanol-acetonitrile-0.1% phosphoric acid aqueous solution. The results showed that the acetonitrile-0.1% phosphoric acid aqueous solution system provided symmetrical peaks, good resolution, and a stable baseline during gradient elution. Under other conditions, the chromatographic peaks were tailed and the resolution did not meet the requirements. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution was selected as the elution solvent. Chromatographic columns including Dimonsil-C18, Thermo-Hytimate-C18, Phenomenex Synergi 4u Hydro-RP 80A-C18, and Diamonsil Spursil-C18 were investigated. The results showed that Diamonsil Spursil-C18 exhibited high sensitivity for the detection of gallic acid and (+)-catechin peaks, with good peak shape and resolution; therefore, it was selected. Column temperatures of 20℃, 25℃, and 30℃ were set. The results indicated that at a column temperature of 25℃, the retention times of gallic acid and (+)-catechin peaks were moderate, and the peak shape and separation were good; therefore, this column was selected.

[0102] 3.2 Examination of Extraction Conditions

[0103] This experiment compared ultrasonic extraction and reflux extraction. The results showed that the contents of gallic acid and (+)-catechin obtained by reflux extraction (0.025% and 0.318%) were higher than those obtained by ultrasonic extraction (0.015% and 0.252%). Therefore, reflux extraction was selected. 20%, 40%, 60%, 80%, and 100% methanol solutions were used as extraction solvents. The results showed that when using 60% methanol, the average extraction rate of the measured components was relatively high, and the contents of gallic acid and (+)-catechin were (0.024% and 0.302%). The chromatographic peak resolution was better, and the peak shape was symmetrical. Therefore, reflux extraction was selected. Extraction times were set to 60 min, 120 min, and 180 min, respectively. The results showed that the contents of gallic acid and (+)-catechin were 0.025% and 0.314% at 60 min, 0.029% and 0.371% at 120 min, and 0.027% and 0.361% at 180 min. The overall extraction rate of the components measured at 120 min was generally higher, and the peak shape was symmetrical; therefore, it was selected. The above is a preferred embodiment of this experiment. For those skilled in the art, various corresponding changes and modifications can be made based on the above technical solutions and concepts, and all such changes and modifications should be included within the protection scope of the claims of this invention.

Claims

1. A method for determining the content of gallic acid and (+)-catechin in Kandelia candel, characterized in that: The content determination method employs high-performance liquid chromatography (HPLC) and includes the following steps: (1) Preparation of mixed reference solution: accurately weigh gallic acid and (+)-catechin respectively, place them in volumetric flasks, add 50-70% methanol to dissolve them and prepare single reference solutions with mass concentrations of 0.10 mg / mL and 2.00 mg / mL respectively. Accurately pipette different volumes of single reference solutions to prepare 1 mL of mixed reference solution containing 0.035-0.045 mg gallic acid and 0.75-90 mg (+)-catechin; (2) Preparation of test solution: Take 0.95-1.05g of purple hemp powder, weigh accurately, place in a stoppered conical flask, add 15-20mL of 55-65% methanol solution, weigh, reflux in water bath for 2-3h, take out, cool, add the corresponding methanol solution to make up the lost weight, shake well, filter, continue to evaporate the filtrate in water bath, add 55-65% methanol to the residue in a 5mL volumetric flask, and make up to the mark, shake well, and the test solution is obtained; (3) Chromatographic conditions and system suitability test: Column: Diamonsil-C18 column, size 250mm×4.6mm, 5μm; Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution is performed; The gradient elution conditions are as follows: Elution program is 0-22 min, 0%-5%B; 22-25 min, 5%-8%B; 25-40 min, 8%B; 40-50 min, 8%-8.5%B; 50-75 min, 8.5%-40%B; Detection wavelength is 271nm; Column temperature is 25℃; Flow rate is 0.8-1.2mL / min; Injection volume is 10-15. (4) Accurately pipette 10 μL of the mixed reference solution and the test solution into the high performance liquid chromatograph and perform the determination according to the chromatographic conditions in step (3).

2. The method for determining the content of gallic acid and (+)-catechin in the purple perilla medicinal material according to claim 1, characterized in that: In step (1), the preparation of the mixed reference solution is as follows: Gallic acid and (+)-catechin are accurately weighed and placed in volumetric flasks. They are dissolved in 60% methanol and prepared as single reference solutions with mass concentrations of 0.10 mg / mL and 2.00 mg / mL, respectively. Different volumes of single reference solutions are accurately pipetted to prepare 1 mL of mixed reference solution containing 0.04 mg gallic acid and 0.83 mg (+)-catechin.

3. The method for determining the content of gallic acid and (+)-catechin in the purple perilla medicinal material according to claim 1, characterized in that: In step (2), take 1.0g of purple hemp powder, weigh it accurately, place it in a stoppered conical flask, add 20mL of 60% methanol solution, weigh it, reflux in a water bath for 2h, take it out, cool it, add the corresponding amount of methanol solution to make up the lost weight, shake it well, filter it, continue to evaporate the filtrate in a water bath, add 60% methanol to dissolve the residue in a 5mL volumetric flask, and make up to the mark, shake it well, and the test solution is ready.

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