A detection method for a traditional Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris

Abstract

The invention discloses a detection method for a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, wherein the Chinese medicine preparation comprises the following raw materials in parts by weight: 5-15 parts of red peony root, 10-20 parts of smilax glabra, 3-9 parts of zedoaria, 10-20 parts of scutellaria baicalensis and 5-15 parts of ebony, and the detection method comprises using thin layer chromatography to identify the red peony root, smilax glabra, scutellaria baicalensis and ebony in the Chinese medicine preparation, and using high performance liquid chromatography to determine the content of paeoniflorin in the red peony root. The invention has the characteristics that the detection method is simple, convenient, stable and reliable, and can effectively ensure the quality of the product.

Description

Technical Field

[0001] The invention relates to a detection method for a traditional Chinese medicine preparation, in particular to a detection method for a traditional Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris. Background Art

[0002] Traditional Chinese medicine has a long history of treating psoriasis. Psoriasis has been recorded in ancient books for many times, such as "dry psoriasis" and "silver money madness". The currently recognized name is "white scalp". Blood stasis runs through psoriasis, and the treatment principle of promoting blood circulation and removing blood stasis to treat psoriasis is established based on this. The "Silver Psoriasis" prescription was created in the prior art. However, the original prescription of Silver Psoriasis is large, has many ingredients, and is difficult to control in quality, making it difficult to develop. The inventor used three animal models that are closely related to the pathogenesis and clinical manifestations of psoriasis to carry out drug efficacy screening and evaluation, optimized the "Silver Psoriasis" prescription, and obtained the "Shaoling prescription".

[0003] Shaoling Fang is made of five herbs, namely, red peony root, wild smilax glabra, zedoaria, black plum and swollen knotweed, which are extracted, concentrated, dried and pressed. Red peony root and wild smilax glabra are the main herbs in the formula. Red peony root is sour, bitter and cold in nature and belongs to the liver meridian. It can remove blood stasis, cool blood and eliminate spots. Wild smilax glabra is sweet, light and neutral in nature and belongs to the liver and stomach meridians. It can strengthen the spleen and eliminate dampness. It is good at removing skin dampness and toxins and relieving itching. It is the main herb together with red peony root. Curcuma zedoaria is bitter and warm in nature, and belongs to the liver and spleen meridians. It can enter the blood and break blood. Its fragrance and warm nature can help to promote qi circulation. It can treat both the result of blood stasis and the cause of blood stasis. It is a special medicine for treating blood stasis. In this prescription, it not only helps red peony root to remove blood stasis and eliminate spots, but also restrains its cold nature as a minister medicine; black plum tastes sour and astringent in nature and is flat in nature. It belongs to the liver, spleen, lung, and large intestine meridians. It is used to assist the monarch and minister medicines to solve the secondary symptoms of dryness and itching caused by blood stasis and dryness caused by psoriasis; swollen knotweed, also known as nine-section tea, tastes bitter and pungent in nature and is flat in nature. It belongs to the heart and liver meridians, and has the effect of promoting blood circulation, eliminating spots and unblocking collaterals. This medicine is good at entering the blood and promoting blood circulation and eliminating spots. Its bitter taste is pungent, so it has the ability to move to the surface, so it can be used to lead all medicines directly to the skin disease site, which is the guiding medicine. The whole prescription has the effects of promoting blood circulation, removing blood stasis and eliminating spots, removing dampness, detoxifying and relieving itching. It is characterized by "removing blood stasis without consuming blood, nourishing blood without promoting dampness", and is suitable for patients with common psoriasis.

[0004] Shaoling Fang is mainly used to treat moderate psoriasis vulgaris, and can improve and alleviate the patient's skin lesions, affected area, and itching symptoms; in terms of safety, no adverse events were found in the patient's blood and urine routine, liver function, kidney function, and coagulation medical examinations, and no serious adverse events related to the drug were found in the adverse event analysis. Therefore, "Shaoling Fang" has a good effect on improving psoriasis lesions and reducing inflammatory states in imiquimod-type psoriasis models. The action links involve multiple links such as anti-keratinocyte hyperproliferation, anti-inflammatory, and immune regulation. Under the condition of equivalent drug efficacy, "Shaoling Fang" selectively inhibits Th1 cells, indicating that compared with traditional immunosuppressants, "Shaoling Fang" has fewer toxic side effects; compared with the already marketed Chinese patent medicine Yu Jin Psoriasis Tablets, Shaoling Tablets targets the core pathological link of blood stasis, does not contain toxic drugs, has better efficacy, and is safer and more reliable.

[0005] However, at present, there is no scientific and reasonable quality standard for "Shaoling Fang". The prescription is complex, and the quality of medicinal materials seriously affects the efficacy of compound preparations. How to quickly identify the quality of medicinal materials in compound preparations plays an important role in improving product quality. Summary of the invention

[0006] The purpose of the present invention is to provide a detection method for a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris. The present invention has the characteristics of being simple, convenient, stable and reliable, and being able to effectively ensure product quality.

[0007] The technical solution of the present invention is a detection method for a traditional Chinese medicine preparation for treating psoriasis vulgaris blood stasis syndrome, wherein the traditional Chinese medicine preparation comprises the following raw materials in parts by weight: 5-15 parts of red peony root, 10-20 parts of rhizoma smilax, 3-9 parts of zedoaria, 10-20 parts of rhizoma smilax and 5-15 parts of ebony. The detection method comprises using thin layer chromatography to identify the red peony root, rhizoma smilax, rhizoma smilax and ebony in the traditional Chinese medicine preparation, and using high performance liquid chromatography to determine the content of paeoniflorin in the red peony root.

[0008] In the aforementioned detection method of a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the identification method of the red peony root is as follows: take 1g of the ground Chinese medicine preparation, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in water, add it to a polyamide column, wash with water until the effluent is colorless, combine the effluent with the eluent, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution; elute the polyamide column with ethanol, collect 20ml of the eluent, evaporate to dryness, and add 1ml of ethanol to the residue. ml dissolved, the solution for later use; take another 0.5g of red peony root reference medicinal material, add 10ml of ethanol, shake for 5 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2ml of ethanol as the reference solution; according to the thin layer chromatography test, take 4μl of the test solution and the reference solution, respectively, and spot them on the same silica gel G thin layer plate, use chloroform-methanol-formic acid with a volume ratio of 7:2:0.5 as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat until the spots are clearly colored.

[0009] In the aforementioned detection method of a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the identification method of the Chinese smilax glabra is as follows: take 1g of Chinese smilax glabra reference medicinal material, add 20ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of ethanol as the reference solution. According to the thin layer chromatography test, take 5μl of the test solution and 5-10μl of the Chinese smilax glabra reference solution, and spot them on the same silica gel G thin layer plate, respectively, with petroleum ether: ethyl acetate: methanol: formic acid in a volume ratio of 3: 6: 2: 1 as the developing solvent, develop, take out, dry, spray with aluminum chloride test solution, leave for 5 minutes, and then inspect under ultraviolet light.

[0010] In the aforementioned detection method for a traditional Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the identification method of the Sarcandra sarcandra is specifically as follows: take 2 g of Sarcandra sarcandra control medicinal material, add 30 ml of petroleum ether, ultrasonically treat for 30 minutes, filter, evaporate to dryness, and dissolve the residue in 1 ml of ethanol as a reference solution; according to the thin layer chromatography test, aspirate 2 to 4 μl of the reference solution and the test solution, respectively, and spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-formic acid with a volume ratio of 6:2:1 as the developing agent, develop, take out, dry, and inspect under ultraviolet light.

[0011] In the aforementioned detection method for a traditional Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the test solutions of Smilax glabra and Sarcandra chinensis are both standby solutions for identification of red peony root, or the preparation method of the test solutions of Smilax glabra and Sarcandra chinensis is: take 1g of the ground traditional Chinese medicine preparation, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in water, add it to a polyamide column, wash with water until the effluent is colorless, and then elute with ethanol, collect 20ml of the eluate, evaporate to dryness, dissolve the residue in 1ml of ethanol as the test solution.

[0012] In the aforementioned detection method of a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the identification method of the black plum is as follows: take 1g of the ground Chinese medicine preparation, add 15ml of water, ultrasonically treat for 15 minutes, centrifuge, take the supernatant, concentrate to 9ml, add 1ml of formic acid, mix well, add ethyl acetate to the solution and shake and extract twice, 10ml each time, combine the ethyl acetate solution, evaporate to dryness, and dissolve the residue in 1ml of ethanol as the test solution; take another 1g of black plum reference medicinal material, add 25ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2ml of ethanol as the reference solution. According to the thin layer chromatography test, take 5μl of the test solution and 2μl of the reference solution, respectively, and spot them on the same silica gel G thin layer plate, use the upper layer solution of butyl acetate-formic acid-water with a volume ratio of 4:2:2 as the developing agent, develop, take out, dry, spray with 0.04% bromophenol blue ethanol solution, and inspect under sunlight.

[0013] In the aforementioned detection method for a traditional Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the method for determining the content of paeoniflorin in red peony root is: using paeoniflorin reference substance as a reference, octadecylsilane bonded silica gel as a filler, acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B for gradient elution, within 0 to 20 minutes, mobile phase A increases from 14% to 20%, and mobile phase B increases from 86% to 80%; within 20 to 30 minutes, mobile phase A increases from 20% to 80%, and mobile phase B increases from 80% to 20%; within 30 to 40 minutes, the proportion of mobile phase A is 80%, and the proportion of mobile phase B is 20%; the detection wavelength is 230 nm; 10 μl of the reference solution and the test solution are accurately aspirated respectively, injected into a liquid chromatograph for determination.

[0014] In the aforementioned detection method of a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the preparation method of the reference solution is: take the paeoniflorin reference substance dried in a phosphorus pentoxide vacuum dryer for 36 hours, add methanol to make a solution containing 0.3 mg per 1 ml, and obtain the solution; the preparation method of the test solution is: take 0.5g of the ground Chinese medicine preparation, add 30ml of 80% methanol, seal, weigh, heat and reflux for 1 hour, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate. Each tablet of this product contains red peony root with paeoniflorin (C 23 H 28 O 11 ) shall not be less than 4mg.

[0015] In the aforementioned detection method of a traditional Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the traditional Chinese medicine preparation comprises the following raw materials in parts by weight: 15 parts of red peony root, 20 parts of wild yam, 9 parts of curcuma, 20 parts of sarmentosa and 15 parts of ebony.

[0016] In the aforementioned detection method of a traditional Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the traditional Chinese medicine preparation comprises the following raw materials in parts by weight: 9 parts of red peony root, 15 parts of rhizoma smilax, 6 parts of zedoaria, 15 parts of radix polygoni multiflori and 6 parts of ebony.

[0017] In the aforementioned detection method of a traditional Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, the preparation method of the traditional Chinese medicine preparation is: take Smilax glabra and add 50% ethanol to heat and extract twice, add 10 times the amount of ethanol of Smilax glabra for the first time, and add 8 times the amount of ethanol of Smilax glabra for the second time, each extraction is 1 hour, filtered, combined with the alcohol solution, decompressed to recover the ethanol and concentrate to a clear paste with a relative density of 1.12 to 1.14, filtered, and the clear paste and the residue are reserved; take red peony root, zedoary, swollen knotweed and ebony, add water and decoct twice, add 1 0 times the amount of water, soak for 1 hour, and boil for 1 hour; for the second time, combine it with the residue after alcohol extraction of Smilax glabra, add 10 times the amount of water, boil for 1 hour, filter, combine the filtrate, and concentrate under reduced pressure to a clear paste with a relative density of 1.12-1.14, filter, combine it with the Smilax glabra clear paste, add silicon dioxide, mix well, spray dry, and obtain dry paste powder; add pregelatinized starch and sodium carboxymethyl starch to the dry paste powder, mix well, granulate with 90% ethanol, dry at 70℃, granulate, add pregelatinized starch, sodium carboxymethyl starch, silicon dioxide and magnesium stearate to obtain.

[0018] In the aforementioned method for detecting a Chinese medicine preparation for treating blood stasis syndrome in psoriasis vulgaris, the Chinese medicine preparation can be prepared into any conventional preparation, including tablets, pills, capsules, granules, oral liquids, sprays, injections, suspensions and microsphere preparations.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides the Poria prescription, including red peony root, smilax glabra, zedoaria, swollen joint herb and ebony, can treat blood stasis syndrome of psoriasis vulgaris, and limit and optimize the identification method of red peony root, smilax glabra, swollen joint herb, ebony and the content determination method of red peony root. Wherein the identification method of red peony root, smilax glabra, swollen joint herb and ebony has the advantages of high accuracy, high precision, high recovery rate, high stability, strong specificity, good repeatability and no interference; The content determination method of red peony root has simple operation, low cost, short inspection time, and has the advantages of good separation effect, sensitivity and accuracy. The present invention can effectively detect whether the product is qualified and whether the quality is good or bad, ensure the consistency of drug effect and the stability of quality, and further ensure the stability of product quality and the safety and effectiveness of clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the ultraviolet absorption of paeoniflorin reference substance determined by HPLC;

[0022] Figure 2 This is the HPLC chart of the paeoniflorin reference substance;

[0023] Figure 3 This is the HPLC graph of the negative control sample lacking red peony root;

[0024] Figure 4 This is the effect diagram of the specificity investigation test of red peony root thin layer identification;

[0025] Figure 5 This is the identification effect diagram of the red peony root thin layer of the prefabricated thin layer plate;

[0026] Figure 6 This is a picture of the identification effect of red peony root thin layer plate laid by hand;

[0027] Figure 7 This is the effect picture of thin layer identification of red peony root at 10℃;

[0028] Figure 8 This is the effect picture of thin layer identification of red peony root at 25℃;

[0029] Fig. 9 This is the effect picture of thin layer identification of red peony root under the condition of 18% humidity;

[0030] Fig.10 This is the effect picture of thin layer identification of red peony root under the condition of 88% humidity;

[0031] Fig.11 This is the effect diagram of the color development conditions and specificity investigation test of Smilax glabra thin layer identification;

[0032] Fig.12 This is the effect picture of the identification of the Smilax glabra thin layer of the prefabricated thin layer plate;

[0033] Fig.13 This is the effect picture of the identification of Smilax glabra thin layer on hand-laid thin layer board;

[0034] Fig.14 This is the effect picture of TLC identification of Smilax glabra at 10℃;

[0035] Fig.15 This is the effect picture of TLC identification of Smilax glabra at 25℃;

[0036] Fig.16 This is the effect picture of TLC identification of Smilax glabra under the condition of 18% humidity;

[0037] Fig.17 This is the effect picture of thin layer identification of red peony root under the condition of 88% humidity;

[0038] Fig.18 It is the color development conditions and specificity test effect diagram of thin layer identification of Radix Achyranthis Bidentatae;

[0039] Fig.19 This is the identification effect diagram of the swollen and thin layer of the prefabricated thin layer board;

[0040] Fig. 20 This is the identification effect diagram of the thin layer of swollen knotweed of hand-laid thin layer board;

[0041] Fig.21 This is the effect diagram of thin layer identification of Sargassum sutchuenensis at a temperature of 10℃;

[0042] Fig. 22 This is the effect diagram of thin layer identification of Sargassum sutchuenensis at 25℃;

[0043] Fig.23 This is the effect diagram of thin layer identification of Scutellaria baicalensis under the condition of 18% humidity;

[0044] Fig.24 This is the effect diagram of thin layer identification of Scutellaria baicalensis under the condition of 88% humidity;

[0045] Fig.25 This is the color development condition and specificity test effect diagram of Wumei thin layer identification;

[0046] Fig.26 It is the identification effect diagram of the ebony thin layer of prefabricated thin layer board;

[0047] Fig. 27 This is the effect picture of the identification of the black plum thin layer on the hand-laid thin layer board;

[0048] Fig.28 This is the effect picture of the identification of black plum thin layer under the temperature of 10℃;

[0049] Fig.29 This is the effect picture of the thin layer identification of black plum at a temperature of 25℃;

[0050] Fig.30 This is the effect picture of the identification of black plum thin layer under the condition of 18% humidity;

[0051] Fig.31 This is a picture of the identification effect of black plum thin layer under 88% humidity conditions. DETAILED DESCRIPTION

[0052] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0053] Example:

[0054] A traditional Chinese medicine preparation (Fuling prescription) for treating blood stasis syndrome of psoriasis vulgaris, the prescription of Fuling prescription is: 600g of red peony root, 1000g of wild yam, 400g of Curcuma, 1000g of Sarcandra chinensis, and 400g of Prunus mume.

[0055] The preparation method of Fuling Fang is as follows: take the above five flavors, add 50% ethanol to the smilax glabra and heat and extract twice, add 10 times the amount of ethanol to the smilax glabra for the first time, add 8 times the amount of ethanol to the smilax glabra for the second time, extract for 1 hour each time, filter, combine the ethanol solution, recover the ethanol under reduced pressure and concentrate to a clear paste with a relative density of 1.12-1.14 (60°C), filter, and use the clear paste and the residue for later use. The remaining four flavors such as red peony root are decocted with water twice, add 10 times the amount of water for the first time, soak for 1 hour, and decoct for 1 hour; combine with the residue after alcohol extraction of the smilax glabra for the second time, add 10 times the amount of water, decoct for 1 hour, filter, combine the filtrate, concentrate under reduced pressure to a clear paste with a relative density of 1.12-1.14 (60°C), filter, combine with the smilax glabra clear paste, add appropriate amount of silicon dioxide, mix, spray dry, and obtain dry paste powder. Add appropriate amount of pregelatinized starch and sodium carboxymethyl starch to the dry paste powder, mix well, granulate with 90% ethanol, dry at 70°C, granulate, add appropriate amount of pregelatinized starch, sodium carboxymethyl starch, silicon dioxide and magnesium stearate, mix the whole, press into 1000 tablets, and film-coat to obtain the product.

[0056] The detection method of Fuling Fang is:

[0057] (1) The identification method of red peony root is as follows: take an appropriate amount of this product, grind it into powder, take 1g, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, add an appropriate amount of water to dissolve the residue, add it to a treated polyamide column (80-100 mesh, 5g, inner diameter 1cm, wet column loading with water), wash with water until the effluent is colorless, combine the effluent with the eluent, evaporate to dryness, and dissolve the residue in 1ml of ethanol as the test solution. Elute the polyamide column with ethanol again, collect 20ml of the eluent, evaporate to dryness, and dissolve the residue in 1ml of ethanol. The solution is used for later use. Take another 0.5g of red peony root control medicinal material, add 10ml of ethanol, shake for 5 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2ml of ethanol as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0502), 4 μl of the test solution and the reference solution were taken and spotted on the same silica gel G thin layer plate, and chloroform-methanol-formic acid (7:2:0.5) was used as the developing agent. After development, the plate was taken out, dried, sprayed with 5% vanillin sulfuric acid solution, and heated until the spots were clearly colored. In the chromatogram of the test sample, spots of the same color appeared at the corresponding position of the chromatogram of the reference medicinal material.

[0058] (2) The identification method of Smilax glabra is as follows: take an appropriate amount of this product, grind it into powder, take 1g, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of water, add it to a treated polyamide column (80-100 mesh, 5g, inner diameter 1cm, wet column loading with water), wash with water until the effluent is colorless, then elute with ethanol, collect 20ml of the eluate, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution. Alternatively, take the spare solution in the identification method of Radix Paeoniae Rubra as the test solution.

[0059] Take 1g of Smilax glabra reference medicinal material, add 20ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate, and dissolve the residue in 1ml of ethanol as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Four General Rules 0502), take 5μl of the test solution and 5-10μl of the Smilax glabra reference solution, and spot them on the same silica gel G thin layer plate, respectively, with petroleum ether: ethyl acetate: methanol: formic acid (3:6:2:1) as the developing agent, develop, take out, dry, spray with aluminum chloride test solution, leave for 5 minutes, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference medicinal material, a fluorescent spot of the same color appears.

[0060] (3) The identification method of Radix Salviae Miltiorrhizae is as follows: take an appropriate amount of this product, grind it into powder, take 1g, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of water, add it to a treated polyamide column (80-100 mesh, 5g, inner diameter 1cm, wet column loading with water), wash with water until the effluent is colorless, then elute with ethanol, collect 20ml of the eluate, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution. Alternatively, take the spare solution in the identification method of Radix Paeoniae Rubra as the test solution.

[0061] Take 2g of Radix Sarcosinae control medicinal material, add 30ml of petroleum ether, ultrasonically treat for 30 minutes, filter, evaporate to dryness, and dissolve the residue in 1ml of ethanol as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Four General Rules 0502), take 2-4μl of the reference solution and the test solution, respectively, and spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-formic acid (6:2:1) as the developing agent, develop, take out, dry, and examine under ultraviolet light (365nm). In the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference medicinal material, a fluorescent spot of the same color appears.

[0062] (4) The identification method of black plum is as follows: take an appropriate amount of this product, grind it into powder, take 1g, add 15ml of water, ultrasonically treat for 15 minutes, centrifuge, take the supernatant, concentrate it to 9ml, add 1ml of formic acid, mix it, add ethyl acetate to the solution and shake it for extraction twice, 10ml each time, combine the ethyl acetate solution, evaporate to dryness, and dissolve the residue in 1ml of ethanol as the test solution. Take another 1g of black plum reference medicinal material, add 25ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 2ml of ethanol as the reference solution. According to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Four General Rules 0502), take 5μl of the test solution and 2μl of the reference solution, and spot them on the same silica gel G thin layer plate, use butyl acetate-formic acid-water (4:2:2) upper layer solution as the developing agent, develop, take out, dry, spray with 0.04% bromophenol blue ethanol solution, and inspect under sunlight. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the control medicinal material.

[0063] (5) The method for detecting the content of paeoniflorin in red peony root is: determining the content according to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512).

[0064] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the detection wavelength was 230nm. The theoretical plate number calculated based on the paeoniflorin peak should be no less than 3000;

[0065]

[0066] Preparation of reference solution: Take an appropriate amount of paeoniflorin reference substance dried in a phosphorus pentoxide vacuum dryer for 36 hours, weigh accurately, and add methanol to make a solution containing 0.3 mg per 1 ml to obtain the reference solution;

[0067] Preparation of test solution: Take the product under the weight difference item, grind it into powder, take 0.5g, weigh it accurately, put it in a stoppered conical flask, add 30ml of 80% methanol accurately, stopper it tightly, weigh it, heat and reflux for 1 hour, cool it, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to get the test solution;

[0068] Determination method: Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0069] Each tablet of this product contains red peony root and paeoniflorin (C 23 H 28 O 11 ) shall not be less than 4mg.

[0070] In order to ensure that the detection method of the present invention is scientific, reasonable and feasible, the detection method of the present invention was studied and investigated.

[0071] 1. Investigation on the content determination methodology of red peony root:

[0072] Red peony root is the main drug in Shaoling tablets (preparation of the present invention), and paeoniflorin is the main effective ingredient of red peony root. Therefore, paeoniflorin is selected as the indicator component of the intrinsic quality of this preparation, and high performance liquid chromatography is used for sample determination. The results of the experiment show that the method is simple to operate, low in cost, and short in inspection time, and has the advantages of good separation effect, sensitivity, and accuracy.

[0073] 1. Test materials

[0074] Agilent Technologies 1200 series high performance liquid chromatograph, 1 / 100,000 electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.), methanol (analytical grade 20130411, Shanghai Shenbo Chemical Co., Ltd.), and paeoniflorin reference substance (batch number 110736-201337 / 110736-201438) were provided by the China Food and Drug Administration. All reagents were analytical grade. Shaoling Tablets (20160306, 20160310, 20160316, 20190118, 20190123, 20190126).

[0075] 2. The chromatographic conditions were prepared according to those in the examples.

[0076] 3. Preparation of reference solution

[0077] Prepared according to the example.

[0078] 4. Preparation of test solution

[0079] Prepared according to the example.

[0080] 5. Preparation of negative control solution (lacking red peony root): Take the remaining prescribed amount of medicinal materials except red peony root, prepare a negative sample according to the preparation process, and prepare a negative control solution lacking red peony root according to the above test solution preparation method.

[0081] 6. Methodological Validation

[0082] 6.1 System suitability test According to the above chromatographic conditions and sample processing methods, the system suitability test was carried out. Paeoniflorin and other components were baseline separated and the peak shape was good. The negative sample test results showed that other components had no interference with the determination of paeoniflorin. Results are shown in Figure 1 to Figure 3 .

[0083] 6.2. Investigation of linearity and range Investigation of linearity and range of paeoniflorin: Accurately draw an appropriate amount of paeoniflorin reference solution (C = 0.51246 mg / ml), add methanol to dilute to concentrations of 0.128 mg / ml, 0.192 mg / ml, 0.256 mg / ml, 0.320 mg / ml, 0.384 mg / ml, then accurately draw 10 μl and inject into liquid chromatograph to measure the peak area integral value. Draw a standard curve with the concentration of the reference solution as the abscissa and the peak area integral value as the ordinate. The regression equation is: y = (2E+07) x-51326, r = 0.9998. The results show that paeoniflorin has a good linear relationship in the range of 0.128-0.384 mg / ml.

[0084] 6.3 Precision test

[0085] Paeoniflorin precision test: From the same batch of samples (batch number 20160223), samples were taken at three different concentrations: low, medium, and high (80%, 100%, and 120%, respectively). Three test solutions were prepared for each concentration and measured by different liquid chromatographs operated by different personnel. The measurement results of 9 samples were evaluated, and the RSD was 1.76%, indicating that the instrument had good precision.

[0086] 6.4 Repeatability test

[0087] The same batch of samples (batch number 20160223) were taken at three different concentrations: low, medium and high (80%, 100% and 120% respectively). Three test solutions were prepared for each concentration for measurement. The measurement results of 9 samples were used for evaluation, and the RSD was 0.88%, indicating that the method had good repeatability.

[0088] 6.5 Stability test

[0089] 10 μl of the test solution was accurately pipetted and injected at 0, 2, 4, 8, and 12 hours, and the peak area integral value was measured. The RSD value was 2.21%, indicating that the paeoniflorin in the test solution had good stability within 12 hours.

[0090] 6.6. Sample recovery test

[0091] The same batch of samples (batch number 20160223, content of 11.20 mg / g) were taken, and three different concentrations of high, medium and low were designed (the three concentrations were 80%, 100% and 120% respectively). The ratio of the amount of the medium concentration of paeoniflorin reference substance added to the amount of the component to be determined in the sample was controlled at 1:1. Three test sample solutions were prepared for determination, and 9 determination results were used for evaluation and calculation of the recovery rate. The results are shown in Table 1.

[0092] Table 1 Paeoniflorin recovery test results (n = 9)

[0093]

[0094] According to the results in Table 1, the average recovery rate was 98.84% and the RSD was 1.85%, indicating that the method had high accuracy.

[0095] 6.7 Durability test

[0096] The comparison of different chromatographic columns and column temperatures in the selected experiments was processed according to the treatment methods under items 3 and 4 to investigate the above factors respectively.

[0097] 6.7.1. Investigation of different chromatographic columns

[0098] Two samples from the same batch (batch number 20160223) were taken and determined according to the content determination method using three brands of chromatographic columns: A: TECHIC18 (250mm×4.6mm, 5μm, column number: 1000-15), B: Elite C18 (250mm×4.6mm, 5μm, column number: E2622802), and C: Sepax C18 (250mm×4.6mm, 5μm, column number: 12301470737). The results are shown in Table 2.

[0099] Table 2 Determination results of different chromatographic columns

[0100]

[0101] The results in Table 2 show that there is no significant difference in the results when using three brands of chromatographic columns.

[0102] 6.7.2. Investigation of different column temperatures

[0103] Take the same batch of samples and prepare the test solution according to the content determination method. The test was carried out at column temperatures of 30℃, 20℃ and 10℃ respectively. The results are shown in Table 3.

[0104] Table 3 Investigation of different column temperatures

[0105]

[0106] The results in Table 3 show that there is no significant difference in the measurement results when the column temperature is between 30 and 10°C.

[0107] 6.8. Content determination results of 6 batches of samples: The content of paeoniflorin in six batches of samples was determined according to the above content determination method. The results are shown in Table 4.

[0108] Table 4 Determination results of paeoniflorin content in 6 batches of Shaoling tablets

[0109]

[0110] According to the results in Table 4, the average content of paeoniflorin in the six batches of samples is 6.48 mg / tablet. Considering the loss problem in large-scale production, according to 70% of the average content of paeoniflorin, it is tentatively estimated that each tablet of this product contains paeoniflorin (C 23 H 28 O 11 ) shall not be less than 4mg.

[0111] 2. Identification of Red Peony Root:

[0112] Red peony root is the dried root of Paeonia veitchii Lynch, a plant of the Ranunculaceae family, and is included in the 2020 edition of the Chinese Pharmacopoeia (Part I). Red peony root is the main drug in the prescription of Shaoling Tablets. This experiment will select red peony control medicinal materials as a control. Ultrasonic extraction is first used, and then the characteristic components of red peony in Shaoling Tablets are extracted by elution and impurity removal with a polyamide column. Thin layer chromatography identification of red peony medicinal materials is performed, and the effects of specificity, different thin layer plates, different temperatures, and different humidity on the thin layer chromatography of red peony medicinal materials in the preparation are investigated. The experimental results show that: using red peony control medicinal materials as the reference substance and chloroform-methanol-formic acid (7:2:0.5) as the development system, the thin layer identification characteristics of red peony medicinal materials are obvious, the specificity is strong, and it can be used as a thin layer identification method for red peony medicinal materials in Shaoling Tablets, so it is included in the detection items of the present invention.

[0113] 2.1 Instruments, reagents and samples

[0114] KQ-250DB numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), DGG-9246A electric constant temperature blast drying oven (Shanghai Qixin Scientific Instrument Co., Ltd.), 1 / 100,000 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), all reagents were analytically pure, red peony root reference drug (National Institute for the Control of Pharmaceutical and Biological Products, No. 121092-201003), Shaoling tablets (20160223, 20160306, 20160310, 20160316, the preparation of the present invention, provided by Guizhou Bailing Enterprise Group Pharmaceutical Co., Ltd.). Sources of thin layer chromatography plates: (1) homemade thin layer chromatography silica gel G (Qingdao Hailang Silica Gel Desiccant Factory, batch number: 1506011), chemically pure; sodium carboxymethyl cellulose (CMC-Na, Jiangsu Wuxi Drug and Equipment Purchasing and Supply Station Chemical Reagent Branch, batch number: 20110112); distilled water: homemade. G-0.5% CMC.Na board, specification: 10×20 cm, thickness about 0.3 mm. (2) Prefabricated board: silica gel G thin layer board (Qingdao Hailang Silica Gel Desiccant Co., Ltd., batch number: 20150312), specification: 10×20 cm, thickness about 0.2-0.25 mm.

[0115] 2.2 Preparation of test solution

[0116] Take an appropriate amount of this product, grind it into powder, take 1g, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate, dissolve the residue in an appropriate amount of water, add it to a treated polyamide column (80-100 mesh, 5g, inner diameter 1cm, wet column loading with water), wash with water until the effluent is colorless, combine the effluent with the eluent, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as the test solution.

[0117] 2.3 Preparation of reference solution

[0118] Take 0.5 g of red peony root reference medicinal material, add 10 ml of ethanol, shake for 5 minutes, filter, evaporate the filtrate to dryness, add 2 ml of ethanol to dissolve the residue, and use it as the reference solution.

[0119] 2.4 Preparation of negative samples

[0120] According to the prescription ratio and preparation process, prepare a negative sample without red peony root in the same way as "Item 2.2".

[0121] 2.5 Expand the system

[0122] Chloroform-methanol-formic acid (7:2:0.5).

[0123] 2.6 Color developer

[0124] 5% vanillin sulfuric acid solution.

[0125] 2.7 Investigation of TLC conditions

[0126] 2.7.1 Specificity Investigation

[0127] Take 4 μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use chloroform-methanol-formic acid (volume ratio of 7:2:0.5) as the developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatogram of the reference solution, and no interference with the identification by the negative control solution is observed. Results are shown in Figure 4 .

[0128] Depend on Figure 4 It can be seen that the polyamide film plate did not separate the test solution; the silica gel G plate had a good separation effect, the spots were round, and the same color spots appeared at the corresponding positions of the chromatogram of the reference solution, indicating that the method has strong specificity and the spots are clearly colored. The methodological verification was also carried out.

[0129] 2.8 Methodological Validation

[0130] 2.8.1 Durability test: Different thin layer boards were compared respectively, with different temperature and humidity as variable factors, and the experiment was carried out according to the above identification method.

[0131] 2.8.1.1 Tests on different thin layers

[0132] Take the homemade silica gel G-CMC-Na plate and the prefabricated silica gel G plate and test them according to the proposed method. Figure 5-6 Results There was no significant difference between the separation effect of the homemade silica gel G-CMC-Na plate and the prefabricated silica gel G plate. The results showed that the main spots of the thin layer identification map of red peony root in Shaoling tablets had good separation effect, the spots were round, different thin layer plates had little effect on the main spots, and the repeatability was good.

[0133] 2.8.2.2 Different temperatures

[0134] Take the thin layer plate after spotting, develop it at 10℃ and 25℃ respectively, and inspect it. The results show that the test in the range of 10~25℃ has no obvious effect on identification. Figure 7-8 .

[0135] 2.8.2.3 Tests at different humidity levels

[0136] Take the sampled thin layer plate and unfold it in a chromatography cylinder with relative humidity adjusted to 18% and 88% with sulfuric acid, and inspect it. The results show that the test between relative humidity of 18 and 88% has no significant effect on identification. Figure 9-10 .

[0137] Through the experimental spectrum of different temperatures and relative humidity, it can be seen that in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, there are fluorescent spots of the same color, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control has no interference. It can be seen that the change of temperature and relative humidity within the normal environmental range does not affect the effectiveness of the identification method.

[0138] The results of the above methodological verification test showed that: in terms of thin layer plates, there was no significant difference in the separation effect between the hand-laid silica gel G-CMC-Na plate and the prefabricated silica gel G plate; changes in temperature and humidity had little effect on the development effect. Therefore, it is believed that this method has good durability.

[0139] 3. Identification of Smilax glabra:

[0140] Smilax glabra is the dried rhizome of Smilax glabra Roxb., a plant of the Liliaceae family. It is included in the 2020 edition of the Chinese Pharmacopoeia (Part I). Smilax glabra is the main drug in the prescription of Shaoling Tablets. This experiment will select Smilax glabra control medicinal materials as the indicator component for the experiment, and use Smilax glabra control medicinal materials as the control for thin layer identification, and investigate the effects of specificity, different thin layer plates, different temperatures and different humidity on the thin layer chromatography of Smilax glabra medicinal materials in Shaoling Tablets. The experimental results show that using Smilax glabra control medicinal materials as the control, petroleum ether: ethyl acetate: methanol: formic acid (3: 6: 2: 1) as the development system, the thin layer identification characteristics of Smilax glabra medicinal materials are obvious and the specificity is strong. Therefore, it can be used as a thin layer identification method for Smilax glabra medicinal materials in Shaoling Tablets, so it is included in the detection items of the present invention.

[0141] 3.1 Instruments, reagents and samples

[0142] KQ-250DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), DGG-9246A electric constant temperature blast drying oven (Shanghai Qixin Scientific Instrument Co., Ltd.), 1 / 100,000 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), all reagents were of analytical grade, Smilax glabra reference medicinal material (China Food and Drug Inspection Institutes, for content determination, No. 121439-201102), Shaoling tablets (20160223, 20160306, 20160310, 20160316, the preparation of the present invention, provided by Guizhou Bailing Enterprise Group Pharmaceutical Co., Ltd.).

[0143] 3.2 Preparation of test solution

[0144] Take an appropriate amount of this product, grind it into powder, take 1g, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of water, add it to a treated polyamide column (80-100 mesh, 5g, inner diameter 1cm, wet column loading with water), wash with water until the effluent is colorless, then elute with ethanol, collect 20ml of the eluate, evaporate to dryness, dissolve the residue in 1ml of ethanol as the test solution.

[0145] 3.3 Preparation of reference solution

[0146] Take 1 g of Smilax glabra reference medicinal material, add 20 ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, add 1 ml of ethanol to dissolve the residue, and use it as the reference solution.

[0147] 3.4 Preparation of negative samples

[0148] According to the prescription ratio and preparation process, prepare the negative sample without Smilax glabra medicinal material, and prepare the negative sample solution in the same way as "Item 3.2".

[0149] 3.5 Expand the system

[0150] Petroleum ether: ethyl acetate: methanol: formic acid (volume ratio is 3:6:2:1).

[0151] 3.6 Color developer

[0152] Aluminum chloride test solution.

[0153] 3.7 Study on color development conditions

[0154] Take 5 μl of each of the above solutions and spot them on the same silica gel G thin layer plate, develop, take out, dry, spray with aluminum chloride test solution, leave for 5 minutes, and then examine under ultraviolet light (365nm). In the chromatogram of the test sample, a fluorescent spot of the same color appears at the corresponding position of the chromatogram of the control medicinal material. Fig.11 .

[0155] Depend on Fig.11 It can be seen that after the wild yam is sprayed with aluminum chloride test solution, the color of the spots is directly related to the temperature and time. If the temperature is high, the spots will appear yellow after 3-5 minutes, and if the temperature is low, the spots will appear brown. In the experiment, after spraying aluminum chloride test solution, heating at 105℃ for 3-5 minutes can achieve better color development effect.

[0156] 3.8 Methodological Validation

[0157] 3.8.1 Specificity inspection

[0158] Take 5 μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use petroleum ether: ethyl acetate: methanol: formic acid (volume ratio of 3:6:2:1) as the developing agent, develop, take out, dry, spray with aluminum chloride test solution, leave for 5 minutes, and then examine under ultraviolet light (365nm). Fig.11 .

[0159] Depend on Fig.11 It can be seen that the negative sample has no interference; at the corresponding position of the chromatogram of the reference substance, clear spots of the same color are shown, the spots are round, and the separation effect is good. This shows that the method has strong specificity and the spots are clearly colored.

[0160] 3.8.2 Durability test: Different thin layer boards were compared respectively, with different temperature and humidity as variable factors, and the experiment was carried out according to the above identification method.

[0161] 3.8.2.1 Tests on different thin layers

[0162] Take the homemade silica gel G-CMC-Na plate and the prefabricated silica gel G plate and test them according to the proposed method. Figure 12-13 .

[0163] Figure 12-13The results showed that the TLC identification spots of Smilax glabra in Shaoling tablets showed good separation effect, the spots were round, and the separation effect of the homemade silica gel G-CMC-Na plate was not significantly different from that of the prefabricated silica gel G plate, with good repeatability.

[0164] 3.8.2.2 Tests at different temperatures

[0165] Take the spotted thin layer plate, develop it at 10℃ and 25℃ respectively, and inspect it. Figure 14-15 .

[0166] Figure 14-15 The results show that testing in the range of 10-25°C has no significant effect on identification.

[0167] Through the experimental spectrum of different temperatures and relative humidity, it can be seen that in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, there are fluorescent spots of the same color, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control has no interference. It can be seen that the change of temperature and relative humidity within the normal environmental range does not affect the effectiveness of the identification method.

[0168] 3.8.2.2 Tests at different humidity levels

[0169] Take the spotted thin layer plate and develop it in a chromatography cylinder with relative humidity adjusted to 18% and 88% with sulfuric acid, and inspect it. Figure 16-17 .

[0170] Figure 16-17 The results show that the test between relative humidity 18% and 88% has no significant effect on identification. Through the experimental spectrum of different temperatures and relative humidity, it can be seen that in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, there are fluorescent spots of the same color, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control has no interference. It can be seen that the change of temperature and relative humidity within the normal environmental range does not affect the effectiveness of the identification method. Therefore, it is believed that the durability of this method is good.

[0171] 4. Identification of Sarcandra serrata

[0172] It is included in the 2020 edition of the Chinese Pharmacopoeia (Part I). Radix Sarcandrae is the adjuvant drug of the Shaoling Tablets prescription. In this experiment, Radix Sarcandrae control medicinal materials will be selected as the control, and petroleum ether will be used to fully ultrasonically extract the characteristic components, and the Radix Sarcandrae medicinal materials will be identified by thin layer chromatography, and the effects of different spot amounts, thin layer plates from different manufacturers, different temperatures, and different humidities on the thin layer chromatography of Radix Sarcandrae medicinal materials in the preparation will be investigated. The experimental results show that: using the Radix Sarcandrae control medicinal materials as the reference substance and cyclohexane-ethyl acetate-formic acid (volume ratio of 6:2:1) as the development system, the thin layer identification characteristics of Radix Sarcandrae medicinal materials are obvious and the specificity is strong. It can be used as a thin layer identification method for Radix Sarcandrae medicinal materials in Shaoling Tablets, so it is included in the detection items of the present invention.

[0173] 4.1 Instruments, reagents and samples

[0174] KQ-250DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), DGG-9246A electric constant temperature blast drying oven (Shanghai Qixin Scientific Instrument Co., Ltd.), 1 / 100,000 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), all reagents were of analytical grade, Radix Sarcandrae reference medicinal materials (China Food and Drug Inspection Institutes, for content determination, No. 121048-201205), Shaoling tablets (20160223, 20160306, 20160310, 20160316, the preparation of the present invention, provided by Guizhou Bailing Enterprise Group Pharmaceutical Co., Ltd.).

[0175] 4.2 Preparation of test solution

[0176] Take an appropriate amount of this product, grind it into powder, take 1g, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in an appropriate amount of water, add it to a treated polyamide column (80-100 mesh, 5g, inner diameter 1cm, wet column loading with water), wash with water until the effluent is colorless, then elute with ethanol, collect 20ml of the eluate, evaporate to dryness, dissolve the residue in 1ml of ethanol as the test solution.

[0177] 4.3 Preparation of reference solution

[0178] Take 2 g of Radix Sarcandrae as a reference medicinal material, add 30 ml of petroleum ether, treat with ultrasound for 30 minutes, filter, evaporate to dryness, add 1 ml of ethanol to dissolve the residue, and use it as the reference solution.

[0179] 4.4 Preparation of negative samples

[0180] According to the prescription ratio and preparation process, prepare the negative sample without Sarcandra sylvestris medicinal materials, and prepare the negative sample solution in the same way as "Item 4.2".

[0181] 4.5 Expand the system

[0182] Cyclohexane-ethyl acetate-formic acid (volume ratio is 6:2:1).

[0183] 4.6 Examination of color development conditions

[0184] Take 2-4 μl of each of the above solutions and spot them on the same silica gel G thin layer plate, develop, take out, dry, and examine under ultraviolet light (365 nm). In the chromatogram of the test sample, at the corresponding position of the chromatogram of the control medicinal material, a fluorescent spot of the same color appears. Fig.18 .

[0185] 4.7 Methodological Validation

[0186] 4.7.1 Specificity inspection

[0187] Take 2-4 μl of each of the above solutions and spot them on the same silica gel G thin layer plate, use cyclohexane-ethyl acetate-formic acid (volume ratio of 6:2:1) as the developing solvent, develop, take out, dry, and examine under ultraviolet light (365nm). Fig.18 .

[0188] Depend on Fig.18 It can be seen that the negative sample has no interference; at the corresponding position of the chromatogram of the reference substance, clear spots of the same color are shown, the spots are round, and the separation effect is good. This shows that the method has strong specificity and the spots are clearly colored.

[0189] 4.8.2 Durability test

[0190] Different thin layer plates were compared respectively, with different temperatures and humidity as variable factors, and the experiment was carried out according to the above identification method.

[0191] 4.8.2.1 Tests on different thin layers

[0192] Take the homemade silica gel G-CMC-Na plate and the prefabricated silica gel G plate and test them according to the proposed method. Figure 19-20 .

[0193] Figure 19-20 The results showed that the TLC identification spots of Smilax glabra in Shaoling tablets showed good separation effect, the spots were round, and the separation effect of the homemade silica gel G-CMC-Na plate was not significantly different from that of the prefabricated silica gel G plate, with good repeatability.

[0194] 4.8.2.2 Tests at different temperatures

[0195] Take the spotted thin layer plate, develop it at 10℃ and 25℃ respectively, and inspect it. Figure 21-22 . Figure 21-22 The results show that testing in the range of 10-25°C has no significant effect on identification.

[0196] Through the experimental spectrum of different temperatures and relative humidity, it can be seen that in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, there are fluorescent spots of the same color, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control has no interference. It can be seen that the change of temperature and relative humidity within the normal environmental range does not affect the effectiveness of the identification method.

[0197] 4.8.2.2 Tests at different humidity levels

[0198] Take the spotted thin layer plate and develop it in a chromatography cylinder with relative humidity adjusted to 18% and 88% with sulfuric acid, and inspect it. Figure 23-24 .

[0199] Figure 23-24 The results show that the test between relative humidity 18% and 88% has no significant effect on identification. Through the experimental spectrum of different temperatures and relative humidity, it can be seen that in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, there are fluorescent spots of the same color, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control has no interference. It can be seen that the change of temperature and relative humidity within the normal environmental range does not affect the effectiveness of the identification method. Therefore, it is believed that the durability of this method is good.

[0200] 5. Identification of Wumei

[0201] It is included in the 2020 edition of the Chinese Pharmacopoeia (Part I). Wumei is used as the drug in the prescription of Shaoling Tablets. In this experiment, Wumei control medicinal materials will be selected as the control. The characteristic components of Wumei in Shaoling Tablets will be extracted by ultrasonic extraction, followed by formic acid and then ethyl acetate shaking extraction. Thin layer identification of Wumei medicinal materials will be carried out, and the effects of thin layer plates from different manufacturers, different temperatures, and different humidity on the thin layer chromatography of Wumei medicinal materials in the preparation will be investigated. The experimental results show that: using Wumei control medicinal materials as the reference substance and the upper layer solution of butyl acetate-formic acid-water (volume ratio of 4:2:2) as the development system, the thin layer identification characteristics of Wumei medicinal materials are obvious and the specificity is strong. It can be used as a thin layer identification method for Wumei medicinal materials in Shaoling Tablets, so it is included in the detection items of the present invention.

[0202] 5.1 Instruments, reagents and samples

[0203] KQ-250DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), DGG-9246A electric constant temperature blast drying oven (Shanghai Qi Xin Scientific Instrument Co., Ltd.), 1 / 100,000 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), all reagents were of analytical grade, black plum reference medicinal materials (China Food and Drug Inspection Institutes, for content determination, batch number 121208-201305), Shaoling tablets (20160223, 20160306, 20160310, 20160316, the preparation of the present invention, provided by Guizhou Bailing Enterprise Group Pharmaceutical Co., Ltd.).

[0204] 5.2 Preparation of test solution

[0205] Take an appropriate amount of this product, grind it into powder, take 1g, add 15ml of water, ultrasonically treat for 15 minutes, centrifuge, take the supernatant, concentrate it to 9ml, add 1ml of formic acid, mix well, add ethyl acetate to the solution and shake it to extract twice, 10ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1ml of ethanol to dissolve the residue, and use it as the test solution.

[0206] 5.3 Preparation of reference solution

[0207] Take 1 g of black plum reference medicinal material, add 25 ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 2 ml of ethanol to dissolve the residue, and use it as the reference solution.

[0208] 5.4 Preparation of negative samples

[0209] According to the prescription ratio and preparation process, prepare the negative sample without black plum medicinal materials, and prepare the negative sample solution in the same way as "Item 5.2".

[0210] 5.5 Expand the system

[0211] Butyl acetate-formic acid-water (volume ratio of 4:2:2) upper layer solution.

[0212] 5.6 Examination of color development conditions

[0213] 5 μl of the test solution and 2 μl of the reference solution were respectively spotted on the same silica gel G thin layer plate, and the upper layer solution of butyl acetate-formic acid-water (volume ratio of 4:2:2) was used as the developing agent. The plate was developed, taken out, dried, sprayed with 0.04% bromophenol blue ethanol solution, and examined under sunlight. In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions of the chromatogram of the reference medicinal material. Fig.25 .

[0214] 5.7 Color Developer

[0215] 0.04% bromophenol blue solution in ethanol.

[0216] 5.8 Methodological Validation

[0217] 5.8.1 Specificity inspection

[0218] 5 μl of the test solution and 2 μl of the reference solution were respectively spotted on the same silica gel G thin layer plate, and the upper layer solution of butyl acetate-formic acid-water (volume ratio of 4:2:2) was used as the developing agent. The plate was developed, taken out, dried, and sprayed with 0.04% bromophenol blue ethanol solution. The plate was examined under sunlight. Fig.25 .

[0219] Depend on Fig.25 It can be seen that the negative sample has no interference; at the corresponding position of the chromatogram of the reference substance, clear spots of the same color are shown, the spots are round, and the separation effect is good. This shows that the method has strong specificity and the spots are clearly colored.

[0220] 5.8.2 Durability test

[0221] Different thin layer plates were compared respectively, with different temperatures and humidity as variable factors, and the experiment was carried out according to the above identification method.

[0222] 5.8.2.1 Tests on different thin layers

[0223] Take the homemade silica gel G-CMC-Na plate and the prefabricated silica gel G plate and test them according to the proposed method. Figure 26-27 .

[0224] Figure 26-27 The results showed that the TLC identification spots of Wumei in Shaoling Tablets showed good separation effect, the spots were round, and the separation effect of the homemade silica gel G-CMC-Na plate was not significantly different from that of the prefabricated silica gel G plate, with good repeatability.

[0225] 5.8.2.2 Tests at different temperatures

[0226] Take the spotted thin layer plate, develop it at 10℃ and 25℃ respectively, and inspect it. Figure 28-29 . Figure 28-29 The results show that testing in the range of 10-25°C has no significant effect on identification.

[0227] Through the experimental spectrum of different temperatures and relative humidity, it can be seen that in the chromatogram of the test sample, at the corresponding position of the chromatogram of the reference sample, there are fluorescent spots of the same color, the spots are clear, the separation effect is good, the Rf value is moderate, and the negative control has no interference. It can be seen that the change of temperature and relative humidity within the normal environmental range does not affect the effectiveness of the identification method.

[0228] 5.8.2.2 Test at different humidity

[0229] Take the spotted thin layer plate and develop it in a chromatography cylinder with relative humidity adjusted to 18% and 88% with sulfuric acid, and inspect it. Figure 30-31 .

[0230] Figure 30-31 The results show that the test between relative humidity 18% and 88% has no significant effect on identification. Through the experimental spectrum of different temperatures and relative humidity, it can be seen that in the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the reference sample. The spots are clear, the separation effect is good, the Rf value is moderate, and the negative control has no interference. It can be seen that the change of temperature and relative humidity within the normal environmental range does not affect the effectiveness of the identification method. Therefore, it is believed that the durability of this method is good.

Claims

1. A method for detecting a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris, characterized in that: The Chinese medicine preparation comprises the following raw materials in parts by weight: 5-15 parts of red peony root, 10-20 parts of smilax glabra, 3-9 parts of zedoaria, 10-20 parts of smilax glabra and 5-15 parts of ebony. The detection method comprises using thin layer chromatography to identify the red peony root, smilax glabra, smilax glabra and ebony in the Chinese medicine preparation, and using high performance liquid chromatography to determine the content of paeoniflorin in the red peony root; The identification method of red peony root is as follows: take 1g of the ground Chinese medicine preparation, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate, dissolve the residue in water, add it to a polyamide column, wash with water until the effluent is colorless, combine the effluent with the eluent, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as a test solution; elute the polyamide column with ethanol again, collect 20ml of the eluent, evaporate to dryness, dissolve the residue in 1ml of ethanol, and use it as a standby solution; take another 0.5g of red peony root reference medicinal material, add 10ml of ethanol, shake for 5 minutes, filter, evaporate the filtrate, dissolve the residue in 2ml of ethanol, and use it as a reference solution; according to the thin layer chromatography test, take 4μl of the test solution and the reference solution, respectively, and spot them on the same silica gel G thin layer plate, use chloroform-methanol-formic acid with a volume ratio of 7:2:0.5 as a developing agent, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, and heat until the spots are clearly colored.

2. The detection method of a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris according to claim 1, characterized in that: The identification method of the Chinese smilax glabra is as follows: take 1g of Chinese smilax glabra reference medicinal material, add 20ml of ethanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, and dissolve the residue in 1ml of ethanol as a reference solution; according to the thin layer chromatography test, take 5μl of the test solution and 5-10μl of the Chinese smilax glabra reference solution, respectively spot them on the same silica gel G thin layer plate, use petroleum ether: ethyl acetate: methanol: formic acid with a volume ratio of 3:6:2:1 as the developing agent, develop, take out, dry, spray with aluminum chloride test solution, leave for 5 minutes, and then inspect under ultraviolet light; The test solution of the wild smilax glabra uses the spare solution in the identification of red peony root, or the test solution of the wild smilax glabra is prepared by the following preparation method: take 1g of the ground traditional Chinese medicine preparation, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in water, add it to a polyamide column, wash with water until the effluent is colorless, and then elute with ethanol, collect 20ml of the eluate, evaporate to dryness, and dissolve the residue in 1ml of ethanol as the test solution.

3. The detection method of a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris according to claim 1, characterized in that: The identification method of Sarcandra glabra is specifically as follows: 2g Sarcandra glabra reference medicinal material is taken, 30ml petroleum ether is added, ultrasonic treatment is performed for 30 minutes, filtration is performed, and 1ml ethanol is added to the residue to dissolve the solution as a reference solution; according to the thin layer chromatography test, 2 to 4μl of the reference solution and the test solution are respectively taken and spotted on the same silica gel G thin layer plate, cyclohexane-ethyl acetate-formic acid with a volume ratio of 6:2:1 is used as a developing agent, the plate is developed, taken out, dried, and inspected under an ultraviolet lamp; The test solution of Sarcandra glabra uses the reserve solution in the identification of Paeonia lactiflora, or the test solution of Sarcandra glabra is prepared by the following preparation method: take 1g of the ground traditional Chinese medicine preparation, add 20ml of ethanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in water, add it to a polyamide column, wash with water until the effluent is colorless, then elute with ethanol, collect 20ml of the eluate, evaporate to dryness, dissolve the residue in 1ml of ethanol as the test solution.

4. The method for detecting a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris according to claim 1, characterized in that: The identification method of the black plum is as follows: take 1g of the ground Chinese medicine preparation, add 15ml of water, ultrasonically treat for 15 minutes, centrifuge, take the supernatant, concentrate to 9ml, add 1ml of formic acid, mix well, add ethyl acetate to the solution and shake to extract twice, 10ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1ml of ethanol to dissolve the residue, and use it as the test solution; take another 1g of black plum reference medicinal material, add 25ml of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 2ml of ethanol to dissolve the residue, and use it as the reference solution; according to the thin layer chromatography test, absorb 5μl of the test solution and 2μl of the reference solution, respectively spot them on the same silica gel G thin layer plate, use the upper layer solution of butyl acetate-formic acid-water with a volume ratio of 4:2:2 as the developing agent, develop, take out, dry, spray with 0.04% bromophenol blue ethanol solution, and inspect under sunlight.

5. The method for detecting a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris according to claim 1, characterized in that: The method for determining the content of paeoniflorin in red peony root is as follows: using paeoniflorin reference substance as reference, using octadecylsilane bonded silica gel as filler, using acetonitrile as mobile phase A, using 0.1% phosphoric acid solution as mobile phase B for gradient elution, within 0 to 20 minutes, the mobile phase A increases from 14% to 20%, and the mobile phase B increases from 86% to 80%; within 20 to 30 minutes, the mobile phase A increases from 20% to 80%, and the mobile phase B increases from 80% to 20%; within 30 to 40 minutes, the proportion of mobile phase A is 80%, and the proportion of mobile phase B is 20%; the detection wavelength is 230 nm; 10 μl of the reference substance solution and the test solution are respectively accurately aspirated, injected into a liquid chromatograph, and determined.

6. The method for detecting a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris according to claim 5, characterized in that: The preparation method of the reference solution is as follows: take the paeoniflorin reference substance dried in a phosphorus pentoxide reduced pressure dryer for 36 hours, add methanol to prepare a solution containing 0.3 mg per 1 ml, and obtain the solution; the preparation method of the test solution is as follows: take 0.5 g of the ground Chinese medicine preparation, add 30 ml of 80% methanol, seal it, weigh it, heat it under reflux for 1 hour, cool it, weigh it again, make up the lost weight with 80% methanol, shake it evenly, filter it, and take the filtrate to obtain the solution.

7. The method for detecting a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris according to claim 1, characterized in that: The traditional Chinese medicine preparation comprises the following raw materials in parts by weight: 15 parts of red peony root, 20 parts of rhizoma smilax, 9 parts of zedoaria, 20 parts of rhizoma schizonepetae and 15 parts of ebony.

8. The method for detecting a Chinese medicine preparation for treating blood stasis syndrome of psoriasis vulgaris according to claim 1, characterized in that: The preparation method of the traditional Chinese medicine preparation comprises the following steps: taking smilax glabra and adding 50% ethanol to heat and extract twice, adding 10 times the amount of ethanol to smilax glabra for the first time and 8 times the amount of ethanol to smilax glabra for the second time, extracting for 1 hour each time, filtering, combining the alcohol solution, recovering the ethanol under reduced pressure and concentrating to a clear paste with a relative density of 1.12 to 1.14, filtering, and using the clear paste and the residue for later use; taking red peony root, zedoaria, swollen knotweed and ebony, adding water and decocting twice, adding 10 times the amount of water for the first time, soaking for 1 hour, decocting for 1 hour hours; the second time, combine it with the residue after alcohol extraction of Smilax glabra, add 10 times the amount of water, boil for 1 hour, filter, combine the filtrate, and concentrate under reduced pressure to a clear paste with a relative density of 1.12-1.14, filter, combine it with the clear paste of Smilax glabra, add silicon dioxide, mix well, spray dry to obtain a dry paste powder; add pregelatinized starch and sodium carboxymethyl starch to the dry paste powder, mix well, granulate with 90% ethanol, dry at 70°C, granulate, add pregelatinized starch, sodium carboxymethyl starch, silicon dioxide and magnesium stearate to obtain.

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