A one-test-multiple-evaluation method for testing Chinese medicine ointments

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to a one-test-multiple-evaluation method for traditional Chinese medicine ointment, comprising an S1 thin layer chromatography step and an S2 high performance liquid chromatography step. The invention adopts a high performance liquid chromatography method to quantitatively determine the quantitative requirements of the preparation, and adopts a gradient wavelength to determine the mobile phase as follows: 0-25min, the concentration of the mobile phase A increases from 5-10% to 10-20%, and the concentration of the mobile phase B decreases from 90-95% to 80-90%; 25-45min, the concentration of the mobile phase A increases from 10-20% to 15-30%, and the concentration of the mobile phase B decreases from 80-90% to 75-85%; 45-60min, the concentration of the mobile phase A increases from 15-30% to 40-60%, and the concentration of the mobile phase B decreases from 75-85% to 40-60%; three wavelengths are used for determination: 0-25min (206nm), 25-45min (328nm), and 45-60min (223nm); the column temperature is 25°C, and after gradient elution and detection, the contents of the four components can be accurately detected, and compared with the single gradient and single wavelength detection in the prior art, the result is more accurate.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to a one-test-multiple-evaluation method for traditional Chinese medicine ointments. Background Art

[0002] The selection of materials for Chinese herbal ointments is particular and the production process is strict, which also makes it have more significant advantages. Although Chinese herbal ointments have significant effects, people should still pay attention to the method when taking them to achieve the best therapeutic effect of the ointment. Due to its lipid characteristics, the quality control of Chinese herbal ointments has always been a problem.

[0003] The external extract prepared by extracting and concentrating Coptis chinensis, Rhubarb, Scutellaria baicalensis and Phellodendron amurense has the effects of detoxification, swelling reduction, analgesia and antibacterial when used for external application. It is mostly used clinically for sores, swelling, external blepharitis, etc.; this external ointment has made progress in the treatment of inflammatory diseases such as blepharitis and blepharitis caused by excess heat syndrome.

[0004] According to the search, the following two prior art documents were found, and the specific disclosure contents are as follows:

[0005] CN115024977A discloses a quality control method and quality control equipment for Chinese medicine ointment, which belongs to the technical field of Chinese medicine, including the preparation of Chinese medicine ointment, the processing of special medicinal materials and auxiliary materials, and the determination of relevant quality indicators of Chinese medicine ointment. It includes the following steps: soaking, decoction, precipitation, filtration, concentration, ointment collection and drying. The invention aims at the problems existing in the quality control of existing Chinese medicine ointments, treats different medicinal materials and auxiliary materials in different ways, avoids the quality problems of Chinese medicine ointment during the processing, and effectively improves its quality level; detects the Chinese medicine ointment, measures the relative density, water content, dynamic viscosity and other related indicators of the prepared Chinese medicine ointment, establishes the quality control standard of Chinese medicine ointment, thereby helping to effectively control the quality of finished products in production; provides a method to solve the problem of easy to stick to the bottom of the decoction, avoids the quality problems of Chinese medicine ointment during the processing, and improves the qualified rate of finished products in production.

[0006] CN101676717A discloses a method for detecting a traditional Chinese medicine product, comprising the following steps: obtaining a fingerprint spectrum of a target traditional Chinese medicine product; calculating the degree of identity E according to the obtained fingerprint spectrum and a standard fingerprint spectrum of the traditional Chinese medicine product according to formula I; and then measuring the traditional Chinese medicine product according to the degree of identity E.

[0007] The above two methods do not fundamentally solve the detection method of traditional Chinese medicine ointments and have no direct guiding significance for the calibration of active ingredients. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a one-test-multiple-evaluation method for detecting Chinese medicine ointments. The method uses thin-layer chromatography to identify the components of the Chinese medicine ointment for qualitative analysis, and then uses high-performance liquid chromatography to quantitatively determine the preparation for quantitative analysis. The detection result is accurate and the method is highly convenient.

[0009] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is as follows:

[0010] A one-test-multiple-evaluation method for Chinese medicine ointment detection, comprising an S1 thin layer chromatography step and an S2 high performance liquid chromatography step,

[0011] S1: Using thin layer chromatography to identify the ingredients of the Chinese medicine ointment:

[0012] ⑴Test sample processing:

[0013] Take 1-3g of the Chinese medicine ointment, add 10-30ml of a mixed solution of ethyl acetate:methanol in a volume ratio of 2-3.5:1-1.5, ultrasonicate for 20-30min, filter, evaporate the filtrate to dryness, and dissolve the residue with 0.5-2ml of methanol as the test solution;

[0014] ⑵Control medicinal material treatment:

[0015] Take 2-6g of each of the control medicinal materials of rhubarb, coptis root, phellodendron, and scutellaria baicalensis and prepare control medicinal material solutions in the same manner as the test samples;

[0016] (3) Chromatographic conditions

[0017] ① Use a mixed solution of toluene-ethyl formate-formic acid with a volume ratio of 10-20:1-10:0.5-2 as the developing agent, and a high-efficiency silica gel G plate as the stationary phase. Take cyclohexane solution to fumigate the high-efficiency silica gel G plate for 30-60 minutes, and pre-saturate the developing agent for 30-60 minutes;

[0018] ② Spotting: Pipette 3-5 μl of the test solution, rhubarb, phellodendron, coptis chinensis and scutellaria baicalensis control drug solution and spot them on the above-mentioned high-efficiency silica gel G plate, and develop with the above-mentioned developing agent;

[0019] ③ Color development: Take out, dry, and immediately inspect under 365nm ultraviolet light;

[0020] S2: Quantitative determination of coptis root, phellodendron amurense, rhubarb and scutellaria baicalensis in the preparation by high performance liquid chromatography, comprising the following steps:

[0021] ⑴ Preparation of test solution: Take 1-2g of Chinese medicine ointment, weigh accurately, place in a stoppered conical flask, accurately add 25-50ml of 70-100% methanol to dissolve, take 1-2ml and dilute 5-10 times to make the test solution;

[0022] (2) Preparation of reference solution: Take appropriate amount of phellodendron hydrochloride reference substance, baicalin reference substance, berberine hydrochloride reference substance, and aloe-emodin reference substance, accurately weigh them, and add methanol to prepare reference solution containing 5-20 μg of phellodendron hydrochloride, 50-300 μg of baicalin, 10-50 μg of berberine hydrochloride, and 0.5-1.5 μg of aloe-emodin per 1 ml;

[0023] ⑶Chromatographic conditions:

[0024] The chromatographic column was C18 (250 mm × 4.6 mm, 5 μm); acetonitrile was used as mobile phase A, and 0.2-0.4% phosphoric acid was used as mobile phase B; gradient mobile phase and three gradient wavelengths were used for detection;

[0025] Column temperature 20-35°C, flow rate 0.8-1.2 ml / min;

[0026] (4) Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0027] The thin layer chromatography developing agent is a mixed solution of toluene-ethyl formate-formic acid in a volume ratio of 10-20:1-10:0.5-2.

[0028] The mobile phase of the liquid chromatography is as follows:

[0029] From 0 to 25 min, the concentration of mobile phase A increased from 5-10% to 10-20%, and the concentration of mobile phase B decreased from 90-95% to 80-90%;

[0030] 25-45 min, the concentration of mobile phase A increased from 10-20% to 15-30%, and the concentration of mobile phase B decreased from 80-90% to 75-85%;

[0031] At 45-60 min, the concentration of mobile phase A increased from 15-30% to 40-60%, and the concentration of mobile phase B decreased from 75-85% to 40-60%.

[0032] The three wavelength ranges of the liquid chromatography are: 0-25min (200-230nm), 25-45min (270-380nm), 45-60min (200-254nm).

[0033] The beneficial effects of the present invention are as follows:

[0034] 1. The present invention adopts thin layer chromatography to simultaneously identify coptis chinensis, phellodendron chinense, rhubarb and scutellaria baicalensis in traditional Chinese medicine preparations. The test sample is added into an ethyl acetate-methanol mixture, and the residue is dissolved with methanol to serve as the test sample solution. The method can remove the lipid components of the traditional Chinese medicine ointment and separate the effective ingredients coptis chinensis, phellodendron chinense, rhubarb and scutellaria baicalensis, thereby ensuring the detection accuracy.

[0035] 2. The quantitative determination of the preparation by high performance liquid chromatography in the present invention uses a gradient wavelength for the determination of the mobile phase as follows:

[0036] 0-25min, the concentration of mobile phase A increases from 5-10% to 10-20%, and the concentration of mobile phase B decreases from 90-95% to 80-90%; 25-45min, the concentration of mobile phase A increases from 10-20% to 15-30%, and the concentration of mobile phase B decreases from 80-90% to 75-85%; 45-60min, the concentration of mobile phase A increases from 15-30% to 40-60%, and the concentration of mobile phase B decreases from 75-85% to 40-60%; three wavelengths of measurement: 0-25min (206nm), 25-45min (328nm), 45-60min (223nm); column temperature is 25°C, after gradient elution and detection, the contents of four components can be accurately detected, and compared with the single gradient and single wavelength detection of the prior art, the results are more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a thin layer chromatogram at 365 nm in Example 1 (thin layer chromatogram after cyclohexane fumigation pretreatment). In the figure, 1 is a reference medicinal material of Rheum officinale, 2 is an extract sample, 3 is a reference medicinal material of Phellodendron chinense, 4 is a reference medicinal material of Coptis chinensis, and 5 is a reference medicinal material of Scutellaria baicalensis.

[0038] Figure 2 It is a segmented wavelength chromatogram; A is sample; B is mixed standard; 1 is phellodendron hydrochloride; 2 is baicalin; 3 is berberine hydrochloride; 4 is aloe-emodin.

[0039] Figure 3 The single wavelength chromatogram at 206 nm is shown in Figure 1. Sample A; B is mixed standard, 1 is phellodendron hydrochloride; 2 is baicalin; 3 is berberine hydrochloride; 4 is aloe-emodin.

[0040] Figure 4 The chromatogram is a single wavelength chromatogram at 328 nm. Sample A; Standard B: 1: phellodendron hydrochloride; 2: baicalin; 3: berberine hydrochloride; 4: aloe-emodin.

[0041] Figure 5 It is a single wavelength chromatogram at 223nm; A is sample; B is mixed standard, 1 is phellodendron hydrochloride; 2 is baicalin; 3 is berberine hydrochloride; 4 is aloe-emodin.

[0042] Figure 6This is the thin layer chromatogram at 365 nm in Example 1, without cyclohexane fumigation. 1 Rheum officinale reference medicinal material; 2 Scutellaria baicalensis reference medicinal material; 3 Phellodendron chinense reference medicinal material; 4 Coptis chinensis reference medicinal material; 5 Sample. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the implementation case and the accompanying drawings. This implementation case is implemented based on the technology of the present invention, and a detailed implementation method and specific operation process are now given to illustrate that the present invention is creative, but the protection scope of the present invention is not limited to the following implementation case.

[0044] The detection method provided in the present application is suitable for traditional Chinese medicine ointments, granular preparations or other forms of medicines containing rhubarb, phellodendron, coptis chinensis and scutellaria baicalensis. The following embodiments take traditional Chinese medicine ointments as an example to illustrate the detection method of the present invention. In addition to the above four ingredients, other ingredients may be contained, and the present invention is not limited thereto.

[0045] Example 1

[0046] A one-test-multiple-evaluation method for detecting a traditional Chinese medicine ointment, wherein the traditional Chinese medicine ointment is prepared from four main ingredients: rhubarb, phellodendron, coptis chinensis, and scutellaria baicalensis. The detection method comprises the following steps:

[0047] S1: Thin layer chromatography was used to simultaneously identify Rhizoma Coptidis, Phellodendron Amurense, Radix Rhei and Radix Scutellariae in traditional Chinese medicine preparations, as follows:

[0048] ⑴ Sample treatment: Take 2g of traditional Chinese medicine preparation, add 20ml of ethyl acetate-methanol mixture with a volume ratio of 3:1, ultrasonicate for 20min, filter, evaporate the filtrate to dryness, and dissolve the residue with 1ml of methanol as the sample solution;

[0049] ⑵Control medicinal material processing method: Take 4g of each of rhubarb, coptis root, phellodendron, and scutellaria baicalensis, and prepare 4 kinds of control medicinal material solutions in the same way as the test samples;

[0050] ⑶ Use thin layer chromatography to qualitatively test the ingredients of the preparation, as follows:

[0051] ① Use a mixed solution of toluene-ethyl formate-formic acid in a volume ratio of 15:5:1 as the developing agent and a high-efficiency silica gel G plate as the stationary phase. Take cyclohexane solution to fumigate the high-efficiency silica gel G plate for 30 minutes and pre-saturate the developing agent for 30 minutes.

[0052] ② Spotting: Pipette 3 μl of the test solution, rhubarb, phellodendron, coptis chinensis and scutellaria baicalensis control drug solution, respectively, and spot them on the above-mentioned high-efficiency silica gel G plate, and develop with the above-mentioned developing agent;

[0053] ③ Color development: Take out, dry, and immediately inspect under ultraviolet light (365nm);

[0054] ④Results: In the test solution, main spots of the same color appear at the corresponding positions of the control medicinal materials, such as Figure 1 As shown, it is proved that the traditional Chinese medicine ointment contains four ingredients: rhubarb, phellodendron, coptis chinensis and scutellaria baicalensis.

[0055] S2: Quantitative determination of coptis root, phellodendron amurense, rhubarb and scutellaria baicalensis in the preparation was performed by high performance liquid chromatography. The specific steps are as follows:

[0056] (1) Preparation of test solution: take 1g of the Chinese medicine preparation, weigh it accurately, put it in a stoppered conical flask, add 25ml of 70% methanol accurately, sonicate, take 2ml and dilute it in a 10ml volumetric flask to prepare the test solution;

[0057] (2) Preparation of reference substance (B mixed standard) solution: accurately weigh 1 mg of phellodendron hydrochloride reference substance, 13 mg of baicalin reference substance, 4 mg of berberine hydrochloride reference substance, and 0.09 mg of aloe-emodin reference substance, add 70% methanol to prepare a standard solution containing 10 μg of phellodendron hydrochloride, 130 μg of baicalin, 40 μg of berberine hydrochloride, and 0.9 μg of aloe-emodin per 1 ml;

[0058] Element Concentration μg / ml Phellodendron hydrochloride 10 Baicalin 130 Berberine Hydrochloride 40 Aloe-emodin 0.9

[0059] ⑶Chromatographic conditions:

[0060] The chromatographic column was C18 (250 mm × 4.6 mm, 5 μm); acetonitrile was used as mobile phase A, and 0.3% phosphoric acid was used as mobile phase B;

[0061] (4) Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0062] The mobile phase is as follows:

[0063] From 0 to 25 min, the concentration of mobile phase A increased from 10% to 20%, and the concentration of mobile phase B decreased from 90% to 80%;

[0064] 25-40 min, the concentration of mobile phase A increased from 20% to 30%, and the concentration of mobile phase B decreased from 80% to 70%;

[0065] From 40 to 60 min, the concentration of mobile phase A increased from 30% to 51%, and the concentration of mobile phase B decreased from 70% to 49%;

[0066] Three wavelength measurements: 0-25min (206nm), 25-45min (328nm), 45-60min (223nm);

[0067] The column temperature was 25°C, and the chromatogram was obtained, such as Figure 2 shown.

[0068] The theoretical plate number is 18,000 based on phellodendron chinense hydrochloride.

[0069] The results of the spectrum analysis are as follows:

[0070] Element Extract content mg / g Separation Phellodendron hydrochloride 1.05 4.79 Baicalin 21.88 4.55 Berberine Hydrochloride 2.17 3.10 Aloe-emodin 0.05 ＞1.5

[0071] The converted original concentration is as follows:

[0072] Element Concentration μg / ml Phellodendron hydrochloride 7.20 Baicalin 121.43 Berberine Hydrochloride 19.40 Aloe-emodin 0.37

[0073] The mixed standard and samples were analyzed at wavelengths of 206nm, 328nm, and 223nm, respectively. The results are as follows:

[0074]

[0075]

[0076] from Figure 3-Figure 5 Conclusion: At 206nm, the response value of peak 4 is low, and there are impurity peaks near peak 3 that affect the analysis; at 328nm, peak 1 has no response and the response value of peak 4 is low; at 223nm, the response values ​​of peaks 1 and 4 are low and are easily interfered by impurities; at segmented wavelengths, peaks 1, 2, 3, and 4 all have high response values, and there are no impurity peaks nearby to interfere, so the segmented wavelength method is adopted.

[0077] The segmented wavelengths used were 0-25min (206nm), 25-45min (328nm), and 45-60min (223nm). Under this condition, the chromatographic peak separations of berberine hydrochloride, baicalin, berberine hydrochloride, and aloe-emodin were all greater than 1.5, with good separations and accurate and reliable analysis results.

Claims

1. A one-test-multiple-evaluation method for Chinese medicine ointment detection, comprising S1 thin layer chromatography step and S2 high performance liquid chromatography step, characterized in that: S1: Using thin layer chromatography to identify the ingredients of the Chinese medicine ointment: ⑴Test sample processing: Take 1-3 g of the Chinese medicine ointment, add 10-30 ml of a mixture of ethyl acetate and methanol in a volume ratio of 2-3.5:1-1.5, ultrasonicate for 20-30 min, filter, evaporate the filtrate to dryness, and dissolve the residue with 0.5-2 ml of methanol as the test solution; ⑵Control medicinal material treatment: Take 2-6 g of each of the control herbs of Radix Rhei, Rhizoma Coptidis, Cortex Phellodendri, and Radix Scutellariae and prepare control herbal solutions in the same manner as the test samples; (3) Chromatographic conditions ① Use a mixed solution of toluene-ethyl formate-formic acid with a volume ratio of 10-20:1-10:0.5-2 as the developing agent, and a high-efficiency silica gel G plate as the stationary phase. Take cyclohexane solution to fumigate the high-efficiency silica gel G plate for 30-60 minutes, and pre-saturate the developing agent for 30-60 minutes; ② Spotting: Pipette 3-5 μl of the test solution, rhubarb, phellodendron, coptis chinensis and scutellaria baicalensis control drug solution and spot them on the above-mentioned high-efficiency silica gel G plate, and develop with the above-mentioned developing agent; ③ Color development: Take out, dry, and immediately inspect under 365nm ultraviolet light; S2: Quantitative determination of coptis root, phellodendron amurense, rhubarb and scutellaria baicalensis in the preparation by high performance liquid chromatography, comprising the following steps: ⑴ Preparation of test solution: Take 1-2g of Chinese medicine ointment, weigh accurately, put it in a stoppered conical bottle, accurately add 25-50ml of 70-100% methanol to dissolve, take 1-2ml and dilute 5-10 times to make the test solution; (2) Preparation of reference solution: Take appropriate amount of phellodendron hydrochloride reference substance, baicalin reference substance, berberine hydrochloride reference substance, and aloe-emodin reference substance, accurately weigh them, and add methanol to prepare reference solution containing 5-20 μg of phellodendron hydrochloride, 50-300 μg of baicalin, 10-50 μg of berberine hydrochloride, and 0.5-1.5 μg of aloe-emodin per 1 ml; ⑶Chromatographic conditions: The chromatographic column was C18, 250 mm × 4.6 mm, 5 μm; acetonitrile was used as mobile phase A, and 0.2-0.4% phosphoric acid was used as mobile phase B; gradient mobile phase and three gradient wavelengths were used for detection; Column temperature 20-35°C, flow rate 0.8-1.2 ml / min; (4) Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine; The three wavelength ranges of the liquid chromatography are: 200-230 nm for 0-25 min, 270-380 nm for 25-45 min, and 200-254 nm for 45-60 min; The mobile phase of the liquid chromatography is as follows: From 0 to 25 min, the concentration of mobile phase A increased from 5-10% to 10-20%, and the concentration of mobile phase B decreased from 90-95% to 80-90%; 25-45 min, the concentration of mobile phase A increased from 10-20% to 15-30%, and the concentration of mobile phase B decreased from 80-90% to 75-85%; At 45-60 min, the concentration of mobile phase A increased from 15-30% to 40-60%, and the concentration of mobile phase B decreased from 75-85% to 40-60%.

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