Identification method for whole prescription medicine flavor of classic famous prescription peach kernel and safflower four-ingredient decoction

Abstract

The invention discloses a thin-layer identification method for the whole formula medicine flavor of peach kernel and safflower four-ingredient decoction, and belongs to the field of traditional Chinese medicines. According to the method, the same sample is used for preparing a test solution, the test solution is applied to three thin-layer plates, and the six medicines of radix paeoniae alba, peach kernel, rehmannia, safflower, angelica sinensis and ligusticum wallichii in the peach kernel and safflower four-ingredient decoction can be simultaneously identified under three inspection conditions. The method has the advantages of clear spots, good separation degree and strong specificity, can reflect the quality information of the peach kernel and safflower decoction by one time, and has high inspection efficiency.

Description

Technical Field

[0001] The present invention relates to a method for identifying all the medicinal herbs in the classical famous prescription Taohong Siwu Decoction, belonging to the field of traditional Chinese medicine. Background Art

[0002] Taohong Siwu Decoction originated from "Gynecological Ice Mirror" by Chai Dehua in the Qing Dynasty. Taohong Siwu Decoction takes promoting blood stasis removal as the core, supplemented by nourishing blood and promoting qi movement. In the formula, the powerful blood-breaking herbs, peach kernel and safflower, are the main ingredients, mainly aiming at promoting blood circulation and removing blood stasis; the sweet and warm rehmannia root and angelica are used to nourish yin and liver, and regulate menstruation by nourishing blood; paeony root nourishes blood and harmonizes nutrient qi to enhance the power of nourishing blood; ligusticum wallichii promotes blood circulation and qi movement, and regulates qi and blood to assist the effect of promoting blood circulation. The whole formula is properly formulated, so that blood stasis is removed, new blood is generated, qi movement is smooth, and the significant feature of this formula is promoting blood stasis and generating new.

[0003] The thin-layer identification method is the main means for qualitative identification in the quality standard of traditional Chinese medicine compound preparations. In the "Drafting Instructions for the Quality Standard of Traditional Chinese Medicine Compound Preparations" under [Identification] in the "Technical Requirements for the Research of New Traditional Chinese Medicines", it is required that: "Corresponding identification items can be determined according to the prescription composition and research data. In principle, each medicinal herb in the prescription should be tested and studied, and selected to be included in the standard according to the test results." At present, the settings of [Identification] items in the quality standards of traditional Chinese medicine compound preparations, including the Chinese Pharmacopoeia, are mostly one method for one component, which is time-consuming and laborious. Summary of the Invention

[0004] In view of the problems existing in the prior art, the inventor uses a method to identify the six medicinal herbs in Taohong Siwu Decoction. Specifically as follows: A method for identifying all the medicinal herbs in the classical famous prescription Taohong Siwu Decoction, characterized by comprising the following steps: Step 1: Preparation of the test solution Take 2 g of the dried powder of the Taohong Siwu Decoction reference sample, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract with ether by shaking for 3 times, 20 ml each time, combine the ether solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution A; take the aqueous solution after extraction with ether by shaking, take 1 / 2 of the aqueous solution, extract with ethyl acetate by shaking for 4 times, 10 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution B; take another 1 / 2 of the aqueous solution, extract with n-butanol saturated with water by shaking for 2 times, 20 ml each time, combine the n-butanol solutions, wash once with 20 ml of water saturated with n-butanol, discard the aqueous solution, evaporate the n-butanol solution to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution C; Step 2: Preparation of the reference medicinal herb and reference substance solutions Take 0.5g of Angelica sinensis and Chuanxiong rhizome control medicinal materials, add 20ml of ether respectively, ultrasonically treat for 10 minutes, filter, evaporate the filtrate, add 1ml of methanol to the residue to dissolve, and use them as Angelica sinensis and Chuanxiong rhizome control medicinal material solutions; take paeoniflorin reference substance, add ethanol to make a solution containing 1mg per 1ml, as paeoniflorin reference substance solution; take amygdalin reference substance, add methanol to make a solution containing 2mg per 1ml, as amygdalin reference substance solution; take 0.5g of safflower reference medicinal material, add 5ml of 80% acetone solution, plug, shake for 15 minutes, let stand, and take the supernatant as safflower reference medicinal material solution; take 0.5g of Rehmannia glutinosa control medicinal material, add 20ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 10ml of water to the residue to dissolve, shake and extract with ethyl acetate 4 times, 10ml each time, combine the ethyl acetate solution, evaporate to dryness, and add 1ml of methanol to the residue to dissolve, as Rehmannia glutinosa control medicinal material solution; Step 3: Thin layer chromatography test According to the thin layer chromatography test in General Rule 0502 of Appendix IV of the 2020 edition of the Chinese Pharmacopoeia, 1 to 3 μl of each of the test solution A, the Angelica sinensis control medicinal material solution, and the Chuanxiong control medicinal material solution were respectively taken and spotted on the same silica gel G thin layer plate, and a mixed solution of n-hexane-ethyl acetate with a volume ratio of 9:1 was used as the developing agent. The plate was developed, taken out, dried, and examined under an ultraviolet lamp at a wavelength of 365 nm. As a result, in the chromatogram of the test solution A, at the corresponding position of the chromatogram of the Angelica sinensis control medicinal material and the Chuanxiong control medicinal material, a main fluorescent spot of the same color appeared; 1 to 3 μl of each of the test solution C, safflower control medicinal material solution, and peony glycoside reference solution were respectively taken and spotted on the same silica gel G thin layer plate, and a mixed solution of n-butanol-glacial acetic acid-water with a volume ratio of 4:1:1 was used as a developing agent. The plate was developed, taken out, dried, and examined under a UV lamp at a wavelength of 365 nm. As a result, in the chromatogram of the test solution C, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the safflower control medicinal material. The thin layer plate was then sprayed with 5% vanillin sulfuric acid solution and heated at 105°C until the spots were clearly colored. As a result, in the chromatogram of the test solution C, a spot of the same color appeared at the corresponding position of the chromatogram of the peony glycoside reference. 1 to 3 μl of each of the test solution B, the Rehmannia glutinosa control drug solution, the test solution C, and the amygdalin reference solution were respectively taken and spotted on the same silica gel G thin layer plate, and a mixed solution of ethyl acetate-methanol-water with a volume ratio of 20:5:3 was used as the developing agent. The plate was developed, taken out, dried, and examined under an ultraviolet lamp at a wavelength of 365 nm. As a result, in the chromatogram of the test solution B, a main fluorescent spot of the same color appeared at the corresponding position of the chromatogram of the Rehmannia glutinosa control drug. The thin layer plate was then sprayed with a phosphomolybdic acid-sulfuric acid solution and heated at 105°C until the spots were clearly colored. As a result, in the chromatogram of the test solution C, a spot of the same color appeared at the corresponding position of the chromatogram of the amygdalin reference drug.

[0005] The preparation method of the above-mentioned reference sample dry powder of Taohong Siwu Decoction is as follows: Take Rehmanniae Radix, Angelicae Sinensis Radix, Paeoniae Radix Alba, Chuanxiong Rhizoma, Persicae Semen, and Carthami Flos pieces according to the weight ratio of 11.19:14.92:5.60:3.73:3.78:3.73, add an appropriate amount of water, soak for 30 minutes, heat to boiling on an electric ceramic stove at 2200W, then decoct at 400W for 30 minutes, filter while it is hot, add water to the medicinal residues, decoct in the same way for 20 minutes, filter while it is hot, combine the filtrates, and obtain the reference sample dry powder of Taohong Siwu Decoction by freeze-drying or spray-drying or vacuum-drying methods.

[0006] The above-mentioned phosphomolybdic acid sulfuric acid solution is prepared by dissolving 2g of phosphomolybdic acid in 20ml of water, and then slowly adding 30ml of sulfuric acid and mixing well. Specific implementation manners

[0007] The following examples are listed to further illustrate the present invention. Each example is only used to illustrate the present invention and does not limit the present invention: Take 11.19g of Rehmanniae Radix (washed with wine), 14.92g of Angelicae Sinensis Radix (washed with wine), 5.60g of Paeoniae Radix Alba (fried with wine), 3.73g of Chuanxiong Rhizoma, 3.5g of Persicae Semen, and 3.73g of Carthami Flos (washed with wine). Mash the Persicae Semen, and place it together with Rehmanniae Radix, Angelicae Sinensis Radix, Paeoniae Radix Alba, Chuanxiong Rhizoma, and Carthami Flos in a casserole, decoct twice with water. For the first time, add 9 times the amount of water (385ml), soak for 30 minutes, cover the lid, use an electric ceramic stove as the heat source, first heat to boiling with strong fire (600W), and then keep it slightly boiling with slow fire (150W - 200W) for 30 minutes. For the second time, add 7 times the amount of water (300ml) and decoct for 20 minutes. Filter while it is hot (through a 150-mesh nylon filter cloth). Concentrate the filtrate under reduced pressure (60°C, -0.1MPa) to a relative density of 1.09 - 1.17 (40°C), pre-freeze until it freezes, freeze-dry, pulverize, mix well, and divide into portions to obtain the reference sample dry powder of Taohong Siwu Decoction.

[0008] Take 2g of the reference sample dry powder of Taohong Siwu Decoction, add 20ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water, extract with ether by shaking 3 times, 20ml each time. Combine the ether solutions, evaporate to dryness, dissolve the residue in 1ml of methanol to obtain the test solution A; Take the aqueous solution after extraction with ether by shaking, take 1 / 2 of the aqueous solution, extract with ethyl acetate by shaking 4 times, 10ml each time. Combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1ml of methanol to obtain the test solution B; Take another 1 / 2 of the aqueous solution, extract with n-butanol saturated with water by shaking 2 times, 20ml each time. Combine the n-butanol solutions, wash once with 20ml of water saturated with n-butanol, discard the aqueous solution, evaporate the n-butanol solution to dryness, dissolve the residue in 1ml of methanol to obtain the test solution C; Take 0.5 g each of the reference crude drugs Angelicae Sinensis Radix and Chuanxiong Rhizoma, add 20 ml of diethyl ether to each, ultrasonically treat for 10 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the reference crude drug solution of Angelicae Sinensis Radix and the reference crude drug solution of Chuanxiong Rhizoma; take paeoniflorin reference substance, add ethanol to make a solution containing 1 mg per 1 ml as the paeoniflorin reference substance solution; take amygdalin reference substance, add methanol to make a solution containing 2 mg per 1 ml as the amygdalin reference substance solution; take 0.5 g of the reference crude drug Carthami Flos, add 5 ml of 80% acetone solution, tightly stopper, shake for 15 minutes, let stand, take the supernatant as the reference crude drug solution of Carthami Flos; take 0.5 g of the reference crude drug Rehmanniae Radix, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, extract with ethyl acetate by shaking 4 times, 10 ml each time, combine the ethyl acetate layers, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the reference crude drug solution of Rehmanniae Radix; According to the test of thin-layer chromatography method in Appendix General Principles 0502 of the Fourth Part of Chinese Pharmacopoeia 2020 Edition, respectively draw 1 - 3 μl of the test solution A, the reference crude drug solution of Angelicae Sinensis Radix, and the reference crude drug solution of Chuanxiong Rhizoma, and respectively spot them on the same silica gel G thin-layer plate. Use a mixed solution with a volume ratio of n-hexane - ethyl acetate of 9:1 as the developing agent, develop, take out, dry in air, and examine under ultraviolet light at a wavelength of 365 nm. As a result, in the chromatogram of the test solution A, at the positions corresponding to the chromatograms of the reference crude drugs Angelicae Sinensis Radix and Chuanxiong Rhizoma, there are main fluorescent spots of the same color; Respectively draw 1 - 3 μl of the test solution C, the reference crude drug solution of Carthami Flos, and the paeoniflorin reference substance solution, and respectively spot them on the same silica gel G thin-layer plate. Use a mixed solution with a volume ratio of n-butanol - glacial acetic acid - water of 4:1:1 as the developing agent, develop, take out, dry in air, and examine under ultraviolet light at a wavelength of 365 nm. As a result, in the chromatogram of the test solution C, at the position corresponding to the chromatogram of the reference crude drug Carthami Flos, there is a main fluorescent spot of the same color. Then spray the thin-layer plate with 5% vanillin sulfuric acid solution and heat at 105 °C until the spots are clearly developed. As a result, in the chromatogram of the test solution C, at the position corresponding to the chromatogram of the paeoniflorin reference substance, there are spots of the same color; Respectively draw 1 - 3 μl of the test solution B, the reference crude drug solution of Rehmanniae Radix, the test solution C, and the amygdalin reference substance solution, and respectively spot them on the same silica gel G thin-layer plate. Use a mixed solution with a volume ratio of ethyl acetate - methanol - water of 20:5:3 as the developing agent, develop, take out, dry in air, and examine under ultraviolet light at a wavelength of 365 nm. As a result, in the chromatogram of the test solution B, at the position corresponding to the chromatogram of the reference crude drug Rehmanniae Radix, there is a main fluorescent spot of the same color. Then spray the thin-layer plate with phosphomolybdic acid sulfuric acid solution and heat at 105 °C until the spots are clearly developed. As a result, in the chromatogram of the test solution C, at the position corresponding to the chromatogram of the amygdalin reference substance, there are spots of the same color.

Claims

1. A method for identifying all the medicinal flavors of the classical famous prescription Taohong Siwu Decoction, characterized in that ,Thin-layer identification was performed on all of the Angelica sinensis, Ligusticum wallichii, Paeonia lactiflora, Prunus persica, Carthamus tinctorius, and Rehmannia glutinosa in the classic famous prescription Taohong Siwu Decoction.

2. The method for identifying all the medicinal flavors of the classical famous prescription Taohong Siwu Decoction according to claim 1, characterized in that, It includes the following steps: Step 1: Preparation of the test solution Take 2 g of the dry powder of the Taohong Siwu Decoction reference sample, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20 ml of water, extract with ether by shaking 3 times, 20 ml each time, combine the ether solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution A; take the aqueous solution after extraction with ether by shaking, take 1 / 2 of the aqueous solution, extract with ethyl acetate by shaking 4 times, 10 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution B; take another 1 / 2 of the aqueous solution, extract with n-butanol saturated with water by shaking 2 times, 20 ml each time, combine the n-butanol solutions, wash once with 20 ml of water saturated with n-butanol, discard the aqueous solution, evaporate the n-butanol solution to dryness, dissolve the residue in 1 ml of methanol to obtain the test solution C; Step 2: Preparation of the reference medicinal materials and reference substance solutions Take 0.5 g each of the reference medicinal materials of Angelica sinensis and Ligusticum wallichii, add 20 ml of ether respectively, ultrasonically treat for 10 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 1 ml of methanol to obtain the reference medicinal material solution of Angelica sinensis and the reference medicinal material solution of Ligusticum wallichii; take paeoniflorin reference substance, add ethanol to make a solution containing 1 mg per 1 ml as the paeoniflorin reference substance solution; take amygdalin reference substance, add methanol to make a solution containing 2 mg per 1 ml as the amygdalin reference substance solution; take 0.5 g of the reference medicinal material of Carthamus tinctorius, add 5 ml of 80% acetone solution, tightly stopper, shake for 15 minutes, let stand, take the supernatant as the reference medicinal material solution of Carthamus tinctorius; take 0.5 g of the reference medicinal material of Rehmannia glutinosa, add 20 ml of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of water, extract with ethyl acetate by shaking 4 times, 10 ml each time, combine the ethyl acetate solutions, evaporate to dryness, dissolve the residue in 1 ml of methanol to obtain the reference medicinal material solution of Rehmannia glutinosa; Step 3: Thin-layer chromatography test According to the thin-layer chromatography method in Appendix General Rules 0502 of the Fourth Part of the Chinese Pharmacopoeia 2020 Edition, respectively absorb 1 - 3 μl of the test solution A, the reference medicinal material solution of Angelica sinensis, and the reference medicinal material solution of Ligusticum wallichii, and spot them on the same silica gel G thin-layer plate respectively. Use a mixed solution with a volume ratio of n-hexane - ethyl acetate of 9:1 as the developing agent, develop, take out, dry in air, and examine under an ultraviolet lamp at a wavelength of 365 nm. As a result, in the chromatogram of the test solution A, at the positions corresponding to the chromatograms of the reference medicinal materials of Angelica sinensis and Ligusticum wallichii, there are fluorescent main spots of the same color; Respectively absorb 1 - 3 μl of the test solution C, safflower reference crude drug solution, and paeoniflorin reference substance solution, and spot them on the same silica gel G thin layer plate respectively. Use a mixed solution with a volume ratio of n - butanol - glacial acetic acid - water of 4:1:1 as the developing agent, develop, take out, dry in the air, and examine under an ultraviolet lamp at a wavelength of 365 nm. As a result, in the chromatogram of the test solution C, at the position corresponding to the chromatogram of the safflower reference crude drug, there is a main fluorescent spot of the same color. Then spray the thin layer plate with 5% vanillin sulfuric acid solution and heat at 105 °C until the spots are clearly developed. As a result, in the chromatogram of the test solution C, at the position corresponding to the chromatogram of the paeoniflorin reference substance, there is a spot of the same color; Respectively absorb 1 - 3 μl of the test solution B, rehmannia root reference crude drug solution, test solution C, and amygdalin reference substance solution, and spot them on the same silica gel G thin layer plate respectively. Use a mixed solution with a volume ratio of ethyl acetate - methanol - water of 20:5:3 as the developing agent, develop, take out, dry in the air, and examine under an ultraviolet lamp at a wavelength of 365 nm. As a result, in the chromatogram of the test solution B, at the position corresponding to the chromatogram of the rehmannia root reference crude drug, there is a main fluorescent spot of the same color. Then spray the thin layer plate with phosphomolybdic acid sulfuric acid solution and heat at 105 °C until the spots are clearly developed. As a result, in the chromatogram of the test solution C, at the position corresponding to the chromatogram of the amygdalin reference substance, there is a spot of the same color.

3. The method for identifying all the medicinal flavors of the classical famous prescription Taohong Siwu Decoction according to claim 2, characterized in that , The preparation method of the reference dry powder of Taohong Siwu Decoction is as follows: Take rehmannia root, angelica, white peony root, chuanxiong rhizome, peach kernel, and safflower pieces according to the weight ratio of 11.19:14.92:5.60:3.73:3.78:3.73, add an appropriate amount of water, soak for 30 minutes, heat to boiling with an electric ceramic stove at 2200 W, and then decoct for 30 minutes at 400 W. Filter while it is hot. Add water to the residue and decoct in the same way for 20 minutes, filter while it is hot. Combine the filtrates and obtain the reference dry powder of Taohong Siwu Decoction by freeze - drying or spray - drying or vacuum - drying.

4. The method for identifying all the medicinal flavors of the classical famous prescription Taohong Siwu Decoction according to claim 2, characterized in that , The phosphomolybdic acid sulfuric acid solution is prepared by dissolving 2 g of phosphomolybdic acid in 20 ml of water, and then slowly adding 30 ml of sulfuric acid and mixing well.