Method for identifying angelica sinensis and atractylodes macrocephala koidz in depression-resolving and nerve-soothing granules

Abstract

The invention discloses a method for identifying angelica sinensis and rhizoma atractylodis macrocephalae in depression-resolving and nerve-soothing granules, which adopts a thin-layer chromatography identification method and comprises the following steps of: developing by taking toluene-ethyl acetate-methanol-formic acid in a volume ratio of 18: 5: 2: 0.6 as a developing solvent, and enabling the developing distance to be more than 16cm. The method can be used for simultaneously identifying the Chinese angelica and the bighead atractylodes rhizome in the depression-resolving and nerve-soothing granules, and has the advantages of good thin-layer chromatography separation effect, high development rate, obvious color development, clear and round spots, strong specificity and no interference in negative control. The method is simple and convenient, high in detection efficiency, accurate in detection result and low in cost, the quality conditions of angelica sinensis and rhizoma atractylodis macrocephalae in the depression-resolving and nerve-soothing granules can be quickly and accurately inspected by adopting the method, and the medication safety of the depression-resolving and nerve-soothing granules is further guaranteed.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine detection, and in particular to a method for identifying angelica and atractylodes macrocephala in Jieyu Anshen granules. Background Art

[0002] Jieyu Anshen Granules are composed of 16 Chinese herbs, including Semen Ziziphi Spinosae, Acorus Tatarinowii, Atractylodes Macrocephala (fried), Polygala Tenuifolia (processed), Gardenia Fructus (fried), Arisaema Cistanche, Dragon Tooth, Poria, Jujube, Pinellia Rhizome, Floating Wheat, Roasted Licorice, Lilium Bulb, Curcuma Radix, Semen Ziziphi Spinosae (fried) and Angelica Sinensis. It has the functions of soothing the liver and relieving depression, calming the mind and settling the spirit. It is used for mental stimulation caused by emotional discomfort, liver depression and qi stagnation, anxiety, insomnia, forgetfulness, menopausal syndrome, neurosis, etc.

[0003] Angelica sinensis in the prescription is a perennial herb of the Umbelliferae family Angelica sinensis The dried root of (Oliv.) Diels has the effects of nourishing blood and activating blood circulation, regulating menstruation and relieving pain, and moistening the intestines and facilitating bowel movements. It is mainly used for blood deficiency and chlorosis, dizziness and palpitations, irregular menstruation, amenorrhea and dysmenorrhea, abdominal pain due to deficiency and cold, rheumatism and pain. The Atractylodes macrocephala (fried) in the prescription is the dried rhizome of the Atractylodes macrocephala Koidz. of the Asteraceae family. It has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and calming the fetus. In clinical applications, it is mainly used for the conditioning and treatment of clinical manifestations of spleen deficiency, poor appetite, abdominal distension and diarrhea, phlegm and fluid, dizziness and palpitations, edema and spontaneous sweating, and restless fetuses. At present, the identification of Angelica and Atractylodes in Jieyu Anshen Granules generally uses different methods for identification. In actual testing, there are many steps, which is relatively cumbersome and has low testing efficiency. Therefore, developing a simple, accurate and simultaneous method for identifying Angelica and Atractylodes in Jieyu Anshen Granules is conducive to more easily and quickly assessing the quality of Angelica and Atractylodes in Jieyu Anshen Granules and ensuring the safety of Jieyu Anshen Granules. Summary of the invention

[0004] The purpose of the present invention is to provide a method for identifying angelica and atractylodes in Jieyu Anshen granules. The method can be used to simultaneously perform thin layer chromatography identification on the angelica and atractylodes in the Jieyu Anshen granules, and the thin layer chromatography separation effect is good, the color development is obvious, the spots are clear and round, and the specificity is strong. The method is simple and convenient, the inspection efficiency is high, the detection results are accurate, and the cost is low. The method can be used to quickly and accurately inspect the quality of the angelica and atractylodes in the Jieyu Anshen granules.

[0005] The technical solution adopted by the present invention is as follows: A method for identifying angelica and atractylodes in Jieyu Anshen granules, comprising the following steps: (1) Take 15g or 8g of this product (without sucrose), grind it into powder, add 60ml of hot water to dissolve it, shake and extract it twice with ethyl acetate, 30ml each time, combine the ethyl acetate extracts, evaporate to dryness, and add 1ml of methanol to dissolve the residue as the test solution; (2) Take 1 g of Atractylodes macrocephala and Angelica sinensis as control medicinal materials, add 50 ml of water to each, heat and reflux for 1 hour, cool, filter, add 50 ml of ethyl acetate to the filtrate and shake to extract, separate the ethyl acetate layer, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the control medicinal material solution; take an appropriate amount of Atractylodes lactone III reference substance, add methanol to make a reference substance solution containing 0.5 mg per ml; (3) According to the thin layer chromatography method (General Rule 0502), 5 μl of the test sample solution and 5 μl of each of the two control medicinal material solutions and the control substance solution are respectively spotted on the same Merck silica gel G thin layer plate, and the plate is developed with toluene-ethyl acetate-methanol-formic acid in a volume ratio of 18:5:2:0.6. The plate is developed with a development distance of more than 16 cm. The plate is removed, air-dried, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, a spot of the same color is visible at the corresponding position in the chromatogram of the control medicinal material Angelica sinensis. Spray with 10% ethanolic sulfuric acid solution, heat at 105°C until the spot is clear, and examine under ultraviolet light (365 nm). In the chromatogram of the control medicinal material, a fluorescent spot of the same color is visible at the corresponding position in the chromatogram of the control medicinal material and the chromatogram of the control medicinal material Atractylodes macrocephala.

[0006] The beneficial effects of the present invention are: The present invention establishes a thin layer chromatography identification method of angelica and atractylodes in Jieyu Anshen granules, which is simple to operate, has good thin layer chromatography separation effect, fast development rate, obvious color development, clear and round spots, strong specificity, no interference with negative control, and changes in temperature, humidity, and spotting mode have no obvious effect on the identification method of the present invention, and has strong applicability. The method of the present invention can be used to identify the angelica and atractylodes in Jieyu Anshen granules at the same time, with high inspection efficiency, accurate test results, simple and convenient, time-saving and labor-saving, and low cost. The method of the present invention can be used to quickly and accurately investigate the quality of the angelica and atractylodes in Jieyu Anshen granules, thereby ensuring the medication safety of Jieyu Anshen granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1Thin layer chromatogram of the specificity test results, in the figure, 1, 5μl of negative sample lacking Angelica sinensis, 2, 5μl of Angelica sinensis control medicinal material, 3, 5μl of negative sample lacking Atractylodes macrocephala, 4, 5μl of Atractylodes macrocephala control medicinal material, 5. Jieyu Anshen Granules (Siping Zhenghe Pharmaceutical Co., Ltd. 210504) 5ul, 6. Jieyu Anshen Granules (Jilin Songliao Pharmaceutical Co., Ltd. 20221001) 5ul, 7. Jieyu Anshen Granules (Shenwei Pharmaceutical Group Co., Ltd. 22040841) 5ul, A is a green fluorescent spot, B is a blue fluorescent spot, silica gel G thin layer plate, provided by Merck, batch number: HX16958826, temperature 28℃, humidity 70%RH.

[0008] Figure 2 Thin layer chromatograms of test results at different temperatures. In the figure, 1. Angelica sinensis control medicinal material 5μl, 2. Jieyu Anshen Granules (Shenwei Pharmaceutical Group Co., Ltd., batch number: 22040841) 5μl, 3. Jieyu Anshen Granules (Siping Zhenghe Pharmaceutical Co., Ltd., batch number: 210503) 5μl, 4. Jieyu Anshen Granules (Jilin Songliao Pharmaceutical Co., Ltd., batch number: 20220408) 5μl, 5. Atractylodes macrocephala control medicinal material 5μl, 6. Atractylodes lactone III reference substance (purchased from China National Institute for Food and Drug Control, batch number: 111978-202302) 5μl, A is a blue-green fluorescent spot, B is a blue fluorescent spot, prefabricated silica gel G thin layer plate, provided by Merck, batch number: HX16958826.

[0009] Figure 3 Thin layer chromatograms of test results at different humidity levels, in the figure, 1. Angelica sinensis control medicinal material 5μl, 2. Jieyu Anshen Granules (Shenwei Pharmaceutical Group Co., Ltd., batch number: 22040841) 5μl, 3. Jieyu Anshen Granules (Siping Zhenghe Pharmaceutical Co., Ltd., batch number: 210503) 5μl, 4. Jieyu Anshen Granules (Jilin Songliao Pharmaceutical Co., Ltd., batch number: 20220408) 5μl, 5. Atractylodes macrocephala control medicinal material 5μl, 6. Atractylodes lactone III reference substance 5μl, A is a blue-green fluorescent spot, B is a blue fluorescent spot, prefabricated silica gel G thin layer plate, provided by Merck, batch number: HX16958826.

[0010] Figure 4Thin layer chromatograms of the test results of different sampling methods, in the figure, 1. Angelica sinensis control medicinal material 5μl, 2. Jieyu Anshen Granules (Shenwei Pharmaceutical Group Co., Ltd., batch number: 22040841) 5μl, 3. Jieyu Anshen Granules (Siping Zhenghe Pharmaceutical Co., Ltd., batch number: 210503) 5μl, 4. Jieyu Anshen Granules (Jilin Songliao Pharmaceutical Co., Ltd., batch number: 20220408) 5μl, 5. Atractylodes macrocephala control medicinal material 5μl, 6. Atractylodes lactone III reference substance 5μl, A is a blue-green fluorescent spot, B is a blue fluorescent spot, prefabricated silica gel G thin layer plate, provided by Merck, batch number: HX16958826. DETAILED DESCRIPTION Example 1

[0011] A method for identifying angelica and atractylodes in Jieyu Anshen granules, comprising the following steps: (1) Take 15g or 8g of this product (without sucrose), grind it into powder, add 60ml of hot water to dissolve it, shake and extract it twice with ethyl acetate, 30ml each time, combine the ethyl acetate extracts, evaporate to dryness, and add 1ml of methanol to dissolve the residue as the test solution; (2) Take 1 g of Atractylodes macrocephala and Angelica sinensis as control medicinal materials, add 50 ml of water to each, heat and reflux for 1 hour, cool, filter, add 50 ml of ethyl acetate to the filtrate and shake to extract, separate the ethyl acetate layer, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the control medicinal material solution; take an appropriate amount of Atractylodes lactone III reference substance, add methanol to make a reference substance solution containing 0.5 mg per ml; (3) According to the thin layer chromatography method (General Rule 0502), 5 μl of the test sample solution and 5 μl of each of the two control medicinal material solutions and the control substance solution are respectively spotted on the same Merck silica gel G thin layer plate, and the plate is developed with toluene-ethyl acetate-methanol-formic acid in a volume ratio of 18:5:2:0.6. The plate is developed with a development distance of more than 16 cm. The plate is removed, air-dried, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, a spot of the same color is visible at the corresponding position in the chromatogram of the control medicinal material Angelica sinensis. Spray with 10% ethanolic sulfuric acid solution, heat at 105°C until the spot is clear, and examine under ultraviolet light (365 nm). In the chromatogram of the control medicinal material, a fluorescent spot of the same color is visible at the corresponding position in the chromatogram of the control medicinal material and the chromatogram of the control medicinal material Atractylodes macrocephala. Example 2

[0012] 1. Materials and Reagents Prefabricated silica gel G thin layer plates were purchased from Merck; other reagents were of analytical grade.

[0013] Angelica sinensis reference medicinal materials (batch number 120927-202118); Atractylodes macrocephala reference medicinal materials (batch number 120925-202114), and Atractylodes lactone III reference material (111978-202302) were all purchased from China Food and Drug Inspection Institutes.

[0014] The manufacturers and batch numbers are: Diaoyutai Pharmaceutical Group Jilin Tianqiang Pharmaceutical Co., Ltd., batch number: 221106; Jilin Jinghui Pharmaceutical Co., Ltd., batch number: 211203; Jilin Kangfu Pharmaceutical Co., Ltd., batch number: 20220609; Jilin Luobang Pharmaceutical Co., Ltd., batch number: 2110001; Jilin Songliao Pharmaceutical Co., Ltd., batch number: 20210504; Jilin Xinhui Pharmaceutical Co., Ltd., batch number: 20220601; Siping Zhenghe Pharmaceutical Co., Ltd., batch number: 211006; Yangling Biotechnology Co., Ltd., batch number: 20220602; Jilin Jierji Pharmaceutical Co., Ltd., batch number: 2302202, 2201201 (no sucrose); Shenwei Pharmaceutical Group Co., Ltd., no sucrose, batch number: 22112612.

[0015] 2 Test log 2.1 Preparation of test solution Take 15g or 8g of Jieyu Anshen Granules (without sucrose), grind them into powder, add 60ml of hot water to completely dissolve them, then shake and extract them with ethyl acetate twice, 30ml each time, combine the ethyl acetate extracts, evaporate to dryness, add 1ml of methanol to dissolve them, and use this as the test solution.

[0016] 2.2 Preparation of reference medicinal material solution Take 1g each of Angelica sinensis and Atractylodes macrocephala, add 60ml of water respectively, heat and reflux for 60 minutes, cool, filter, and extract the filtrate by shaking with ethyl acetate twice, 30ml each time. Combine the ethyl acetate extracts, evaporate to dryness, and add 1ml of methanol to the residue to dissolve it, as the Angelica sinensis and Atractylodes macrocephala control medicinal material solutions.

[0017] 2.3 Preparation of negative sample solution Weigh the remaining ingredients except Angelica sinensis and Atractylodes macrocephala according to the prescription ratio, prepare an appropriate amount of negative samples lacking Angelica sinensis and Atractylodes macrocephala according to the preparation method, and prepare a negative sample solution in the same way as the planned test sample solution preparation method.

[0018] 2.4 Chromatographic conditions Chromatography plate: silica gel G thin layer plate; developing solvent: toluene-ethyl acetate-methanol-formic acid (18:5:2:0.6).

[0019] Inspection method: Air-dry, inspect under ultraviolet light (365nm), and fluorescent spots of the same color will appear at the corresponding positions of the Angelica sinensis control medicinal material; then spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored, and inspect under ultraviolet light (365nm), and fluorescent spots of the same color will appear at the corresponding positions of the Atractylodes lactone III reference substance and the Atractylodes macrocephala control medicinal material.

[0020] The sample volume was 5 μl each of the test solution and the control medicinal material solution.

[0021] 2.5 Results The test was carried out according to the proposed method, and the results were as follows: in the chromatogram of the test sample, a fluorescent spot of the same color appeared at the corresponding position in the chromatogram of the control medicinal material (product); in the chromatogram of the negative samples of Angelica sinensis and Atractylodes macrocephala, a fluorescent spot of the same color did not appear at the corresponding position in the chromatogram of the control medicinal material (product), indicating that the method is negative and has no interference, and meets the methodological requirements.

[0022] 3 Methodological investigation 3.1 Specificity test The test was carried out according to the proposed method. The results showed that in the chromatograms of the negative samples of Angelica sinensis and Atractylodes macrocephala, no fluorescent spots of the same color appeared at the corresponding positions of the chromatograms of the control medicinal materials of Angelica sinensis and Atractylodes macrocephala, indicating that the negative samples of Angelica sinensis and Atractylodes macrocephala had no interference (see Figure 1 ).

[0023] 3.2 Investigation at different temperatures Take the silica gel G thin layer plate after spotting, unfold it at 26℃ and 4℃ respectively, dry it, and examine it under ultraviolet light (365nm). At the corresponding position of the angelica reference medicinal material, a fluorescent spot of the same color appears; then spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spot is clearly colored, and examine it under ultraviolet light (365nm). At the corresponding position of the Atractylodes lactone III reference substance and the Atractylodes reference medicinal material, a fluorescent spot of the same color appears. The results show that the change of temperature has no obvious effect on the identification method, (see Figure 2 ).

[0024] 3.3 Investigation of different humidity Take the silica gel G thin layer plate after spotting, unfold it in the environment of relative humidity of 32% and 70%, dry it, and examine it under ultraviolet light (365nm). At the corresponding position of the angelica reference medicinal material, fluorescent spots of the same color appear; then spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spots are clearly colored, and examine it under ultraviolet light (365nm). At the corresponding position of the Atractylodes lactone III reference substance and the Atractylodes reference medicinal material, fluorescent spots of the same color appear. The results show that the change of humidity has no obvious effect on this identification method (see Figure 3 ).

[0025] 3.4 Investigation of different sampling methods Take the silica gel G thin layer plate after strip spotting and dot spot spotting, dry it, and examine it under ultraviolet light (365nm). At the corresponding position of the angelica reference medicinal material, a fluorescent spot of the same color appears; then spray it with 10% sulfuric acid ethanol solution, heat it at 105℃ until the spot is clearly colored, and examine it under ultraviolet light (365nm). At the corresponding position of the Atractylodes lactone III reference substance and the Atractylodes reference medicinal material, a fluorescent spot of the same color appears. The results show that the change of spotting method has no obvious effect on the identification method (see Figure 4 ).

Claims

1. A method for identifying angelica and atractylodes in Jieyu Anshen granules, characterized in that: The following steps are involved: (1) Take 15g or 8g of this product (without sucrose), grind it into powder, add 60ml of hot water to dissolve it, shake and extract it twice with ethyl acetate, 30ml each time, combine the ethyl acetate extracts, evaporate to dryness, and add 1ml of methanol to dissolve the residue as the test solution; (2) Take 1 g of Atractylodes macrocephala and Angelica sinensis as control medicinal materials, add 50 ml of water to each, heat and reflux for 1 hour, cool, filter, add 50 ml of ethyl acetate to the filtrate and shake to extract, separate the ethyl acetate layer, evaporate to dryness, add 1 ml of methanol to dissolve the residue, and use it as the control medicinal material solution; take an appropriate amount of Atractylodes lactone III reference substance, add methanol to make a reference substance solution containing 0.5 mg per ml; (3) Perform the test in accordance with the general rules of thin layer chromatography 0502. Take 5 μl of the test sample solution and 5 μl of each of the two reference medicinal material solutions and the reference substance solution, and spot them on the same Merck silica gel G thin layer plate. Use toluene-ethyl acetate-methanol-formic acid with a volume ratio of 18:5:2:0.6 as the developing solvent. Develop the plate with a development distance of more than 16 cm. Remove the plate, dry it in the air, and examine it under ultraviolet light at 365 nm. In the chromatogram of the test sample, a spot of the same color as that of the chromatogram of the reference medicinal material of Angelica sinensis will appear. Spray it with 10% ethanolic sulfuric acid solution, heat it at 105°C until the spot is clear, and examine it under ultraviolet light at 365 nm. In the chromatogram of the reference substance, a fluorescent spot of the same color will appear at the corresponding position of the chromatogram of the reference medicinal material and the chromatogram of the reference medicinal material of Atractylodes macrocephala.

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