CN115963192A - Quality control method of muscle and bone pain relieving pills - Google Patents
Quality control method of muscle and bone pain relieving pills Download PDFInfo
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- CN115963192A CN115963192A CN202211174915.5A CN202211174915A CN115963192A CN 115963192 A CN115963192 A CN 115963192A CN 202211174915 A CN202211174915 A CN 202211174915A CN 115963192 A CN115963192 A CN 115963192A
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- solution
- tanshinone
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- methanol
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- HYXITZLLTYIPOF-UHFFFAOYSA-N 1,6,6-trimethyl-8,9-dihydro-7H-naphtho[1,2-g]benzofuran-10,11-dione Chemical compound O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1C(C)=CO2 HYXITZLLTYIPOF-UHFFFAOYSA-N 0.000 claims abstract description 75
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- 208000024891 symptom Diseases 0.000 description 1
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Abstract
The invention relates to a quality control method of a muscle and bone pain relieving pill, belonging to the technical field of traditional Chinese medicines. The quality control method comprises improving thin layer identification of rhizoma Cyperi, ramulus Cinnamomi, radix Paeoniae alba, radix Cyathulae and radix Gentianae Marcrophyllae by adding thin layer of Glycyrrhrizae radix on the basis of current quality standardIdentifying, and simultaneously measuring cryptotanshinone, tanshinone I and tanshinone II by HPLC A The chromatographic conditions are optimized, and the technical aim of better controlling the product quality is fulfilled.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, relates to a quality standard of a muscle and bone pain relieving pill, and particularly relates to a quality control method of the muscle and bone pain relieving pill.
Background
The muscle and bone pain relieving pill is a new traditional Chinese medicine developed according to a Ping Leguo's bone setting representative prescription, consists of eleven traditional Chinese medicines, namely salvia miltiorrhiza, suberect spatholobus stem, rhizoma cyperi, combined spicebush root, medicinal cyathula root, cassia twig, clematis root, large-leaved gentian, white paeony root, rehmannia root and liquorice, has the functions of promoting blood circulation and activating qi, warming and activating meridians and relieving swelling and pain, is used for treating symptoms such as knee joint pain, swelling, limited activity and the like caused by blood stasis and congealing cold and knee joint hyperosteogeny, is a national traditional Chinese medicine protection variety, and is received in volume 28 of new medicine transformation standard. This standard was revised once in 2009. In 2009, the promulgated article obtained from national drug standards (revision), lot number: ZGB2009-19, standard No.: WS 3 423 (Z-067) -2000 (Z) -2009, started on 1/10 of 2010.
Over the years of practice, there are still some problems with this standard, including: (1) in the aspect of thin-layer chromatography identification, the identification method of the white paeony root is not stable enough, the preparation process of a test solution contains an elution link of a neutral alumina column, the adsorption capacities of neutral alumina of different brands are greatly different, the methanol elution capacities of different brands are also different, and the condition that target components cannot be eluted frequently occurs; (2) thin-layer identification of rhizoma Cyperi, wherein the chromatographic spots of the test sample and the reference medicinal material are weak, which affects the judgment of the result; (3) thin-layer identification of ramulus Cinnamomi, wherein the main spot of chromatogram of the sample is weak, and the information provided by the reference medicinal material and the reference substance is repeated; (4) thin-layer identification of radix Cyathulae, wherein the contrast of the chromatographic spot intensity of the test product and the reference medicinal material is large; (5) thin-layer identification of radix Gentianae Marcrophyllae, selecting radix Gentianae Marcrophyllae reference medicinal material as reference, and radix Gentianae Marcrophyllae is multi-gene variety, so thatThe spot of the sample chromatogram cannot completely correspond to the spot of the reference medicinal material; (6) thin layer identification without licorice; (7) in the aspect of content determination, only tanshinone II is used A The control component is comparatively limited as an index. The existence of the problems is very unfavorable for controlling the quality of the muscle and bone pain relieving pill product.
Disclosure of Invention
In order to overcome the defects, the invention aims to provide a quality control method of a muscle and bone pain relieving pill, which is characterized in that the thin-layer identification of liquorice is added and the content determination item is optimized by improving the thin-layer identification of rhizoma cyperi, cassia twig, white paeony root, medicinal cyathula root and large-leaf gentian root, so that the quality of a product can be better controlled by the quality control method.
The invention adopts the specific scheme that:
a quality control method of JINGUTONGXIAO pill comprises identification item and content determination item;
the identification item comprises thin-layer chromatography identification of rhizoma cyperi, cassia twig, radix paeoniae alba, radix cyathulae, radix gentianae macrophyllae and liquorice, and specifically comprises the following steps:
(1) The thin-layer chromatography identification method of the rhizoma cyperi comprises the following steps: taking 10g of the product, grinding, placing the product in a 500ml round-bottom flask, adding 200ml of water and a plurality of glass beads, uniformly mixing, connecting a volatile oil tester, adding water from the upper end of the tester to a scale, overflowing the water into the flask, adding 2ml of petroleum ether with the boiling range of 60-90 ℃, connecting a reflux condenser, heating to boil, keeping slightly boiling for 1 hour, cooling, and taking a petroleum ether layer as a test solution; preparing another rhizoma Cyperi control 1g, and making into control solution by the same method; adding ethyl acetate into alpha-cyperone reference substance to obtain a solution containing 1mg per 1ml as a reference substance solution; performing thin layer chromatography by sucking sample solution, control solution and control solution respectively 8 μ l and 1 μ l, respectively dropping on the same silica gel GF 254 Spreading dichloromethane-ethyl acetate-glacial acetic acid with volume ratio of 80: 1 as developing agent on the thin layer plate, taking out, air drying, and inspecting under 254nm ultraviolet lamp; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference materials and the reference solution; spraying a dinitrophenylhydrazine test solution, standing for a moment, and gradually changing the spots into orange red;
(2) The thin-layer chromatography identification method of the cassia twig comprises the following steps: taking the sample solution of the identification item of the rhizoma cyperi in the step (1) as a sample solution; taking cinnamaldehyde as a reference substance, and adding petroleum ether with a boiling range of 60-90 ℃ to prepare a solution containing 1 microlitre of each 1ml, wherein the solution is used as the reference substance solution; according to a thin-layer chromatography test, sucking 4 mu l of each of the two solutions, respectively dropping the two solutions on the same silica gel G thin-layer plate, developing by using petroleum ether-ethyl acetate with a boiling range of 60-90 ℃ and a volume ratio of 17; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control solution;
(3) The thin-layer chromatography identification method of the white paeony root comprises the following steps: grinding 3g of the product, adding 30ml of ethanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, cooling, adding 30ml of water into the residue, stirring, filtering, extracting the filtrate with water-saturated n-butanol for 2 times, 20ml each time, mixing the n-butanol solutions, evaporating to dryness, and adding 1ml of methanol into the residue to dissolve the residue to obtain a sample solution; adding methanol into penoniflorin control to obtain 2mg solution per 1ml as control solution; performing thin layer chromatography test, sucking 5 μ l of sample solution and 2 μ l of reference solution, respectively dropping on the same silica gel G thin layer plate, developing with ethyl acetate-acetone-formic acid-water at volume ratio of 15: 6: 2: 0.5 as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution containing 5% vanillin, and heating at 105 deg.C until the spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control solution;
(4) The thin-layer chromatography identification method of the medicinal cyathula root comprises the following steps: grinding 3g of the product, adding 40ml of 75% ethanol, reflux extracting for 1 hr, filtering, evaporating filtrate, dissolving residue with 10ml of water, shaking and extracting with 20ml of diethyl ether, evaporating diethyl ether solution, dissolving residue with 1ml of anhydrous ethanol to obtain sample solution; taking another radix Cyathulae control medicinal material 0.5g, and making into control medicinal solution by the same method; performing thin layer chromatography test, sucking the two solutions, respectively dropping on the same silica gel G thin layer plate, developing with toluene-chloroform-acetone as developing agent at volume ratio of 8: 4: 1, taking out, air drying, and inspecting under 365nm ultraviolet lamp; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the reference solution;
(5) The thin-layer chromatography identification method of gentiana macrophylla comprises the following steps: grinding 1g of the product, adding 20ml of methanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and dissolving residue with 1ml of methanol to obtain sample solution; adding methanol into gentiopicroside control substance to obtain 1mg solution per 1ml as control solution; subjecting to thin layer chromatography, sucking the above two solutions 2 μ l each, and spotting on the same silica gel GF 254 Spreading on a thin layer plate with ethyl acetate-methanol-water as developing agent at volume ratio of 10: 2: 1, taking out, air drying, and inspecting under 254nm ultraviolet lamp; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution;
(6) The thin-layer chromatography identification method of the liquorice comprises the following steps: taking 4g of the product, grinding, adding 30ml of methanol, heating and refluxing for 1 hour, filtering, evaporating filtrate to dryness, adding 30ml of 5% sodium carbonate solution into residues for dissolving, washing with ethyl acetate for 2 times, 30ml each time, adjusting the pH value of a water layer to 2-3 with 10% hydrochloric acid, shaking and extracting with ethyl acetate for 2 times, 30ml each time, combining ethyl acetate solutions, evaporating to dryness, adding 1ml of methanol into residues for dissolving to obtain a test solution; preparing 0.2g of Glycyrrhrizae radix control medicinal material, and preparing control medicinal solution by the same method; adding methanol into liquiritin reference substance to obtain solution containing 0.5mg per 1ml as reference substance solution; performing thin-layer chromatography test, sucking the three solutions 1-2 μ l respectively, dropping on the same silica gel G thin-layer plate, developing with ethyl acetate-formic acid-glacial acetic acid-water as developing agent at volume ratio of 15: 1: 2, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under sunlight and 365nm ultraviolet lamp respectively; in the chromatogram of the test solution, main spots or fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material; spots or fluorescent spots of the same color appear at the positions corresponding to the color spectrum of the reference substance;
the content determination items are as follows: controlling cryptotanshinone, tanshinone I and tanshinone II A The total amount of (A) and the content limit are as follows: each 1g of the composition contains cryptotanshinone, tanshinone I and tanshinone II A Should not be less than 0.42mg in total.
The content determination item is determined by adopting a liquid chromatography;
the chromatographic conditions are as follows: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent and acetonitrile-0.02% phosphoric acid solution as a mobile phase, wherein the detection wavelength is 270nm, and the sample injection amount is 10 mu l; theoretical plate number according to tanshinone II A Peak count should not be less than 20 000; preparation of control solutions: collecting cryptotanshinone, tanshinone I and tanshinone II A Accurately weighing appropriate amount of reference substance, placing in brown measuring flask, adding methanol to obtain a solution containing cryptotanshinone and tanshinone II each in 1ml A 10 mu g and 5 mu g of tanshinone I to obtain a mixed solution;
preparing a test solution: taking a proper amount of the product, grinding, precisely weighing about 1.0g, placing into a conical flask with a plug, precisely adding 50ml of methanol, sealing the plug, weighing, placing on a water bath oscillator at 60 ℃ for 1 hour, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking the subsequent filtrate;
the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the test solution, injecting into a liquid chromatograph, and measuring;
based on dry product, each 1g of Saviae Miltiorrhizae radix contains cryptotanshinone, tanshinone I and tanshinone II A Should not be less than 0.42mg in total.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention revises and improves the quality standard of the pill for relieving the pain of muscles and bones, adds the thin layer identification of liquorice, improves the thin layer identification of nutgrass galingale rhizome, cassia twig, white paeony root, medicinal cyathula root and large-leaved gentian on the basis of the existing quality standard, and simultaneously measures cryptotanshinone, tanshinone I and tanshinone II by using an HPLC method A The chromatographic conditions are optimized, and the quality of the product can be better controlled by adopting the quality standard of the invention. Through the revision, the quality standard of the pill for relieving the muscle and bone pain is greatly improved, and the pill has important significance for controlling the product quality.
2. In the thin-layer identification of the rhizoma cyperi, a method for extracting volatile oil is adopted for preparing a test solution, so that impurity interference is avoided, dichloromethane-ethyl acetate-glacial acetic acid (80: 1) is used as a developing agent, the effect is better, the test solution can be shared with the cassia twig identification, and the operation is simplified.
3. In the thin-layer identification of the white paeony root, the preparation method of a test solution is changed, and the reproducibility is better than that of a method using a neutral alumina column for elution; ethyl acetate-acetone-formic acid-water (15: 6: 2: 0.5) is used as a developing agent, the developing speed is high, and the chromatographic separation effect of the test sample is better.
4. The thin-layer identification of the radix cyathulae and the large-leaf gentian root has better chromatographic effect than the original method. Wherein, the TLC of radix cyathulae increases the sample volume of the sample, changes the preparation method of the sample solution, and reduces the sample volume of radix cyathulae reference drug; TLC for radix Gentianae Marcrophyllae, adopting gentiopicroside reference substance as reference, deleting radix Gentianae Marcrophyllae reference medicinal material as reference, and changing and preferably selecting preparation method and developing agent of test solution.
5. Thin layer identification of Glycyrrhrizae radix as new item, and its sample solution is prepared by using liquiritin with 7% of content / The flavone with-OH structure can be dissolved in 5% sodium carbonate solution, and has better extraction effect than traditional n-butanol, and research shows that the effect of using licorice as reference medicinal material and liquiritin as reference is better than that of using ammonium glycyrrhizinate.
6. The main functions of the pill are activating blood circulation, anti-inflammation and relieving pain, and the effect is related to the fat-soluble component represented by tanshinone. Saviae Miltiorrhizae radix is a principal drug in the prescription, cryptotanshinone, tanshinone I and tanshinone II A All are fat-soluble active ingredients, so the measurement of the total amount of the three kinds of tanshinone can better control the product quality than the measurement of only a single ingredient. Refer to the relevant literature (Chinese pharmacopoeia [ S ]]2020 edition, one part, 78.) chromatography conditions, and gradient optimization, the target peak can achieve baseline separation, each chromatographic parameter meets the requirements, and the negative is free from interference. The method for preparing the test solution in the content measurement is also the optimum condition for repeated comparison of each parameter. The invention controls tanshinone II A Content change control of cryptotanshinone, tanshinone I and tanshinone II A The content limit of the total amount of the tanshinone II is that each 1g of the tanshinone II is contained A Not less than 0.34mg is changed to: 1g of the composition contains cryptotanshinone (C) 19 H 20 O 3 ) Tanshinone I (C) 18 H 12 O 3 ) And tanshinone II A (C 19 H 18 O 3 ) Should not be less than 0.42mg in total.
Drawings
FIG. 1 is a TLC identification chart of Cyperus rotundus at 254nm (left) and visible light (right); wherein, 1-3 are samples (batch numbers: 201101, 201102 and 201103), 4 are rhizoma cyperi reference medicinal materials, 5 are alpha-cyperone reference substances, and 6 are negative references; FIG. 2 is a TLC identification chart of cinnamon twig; wherein, 1-3 are samples (batch numbers: 201101, 201102, 201103), 4 are cinnamaldehyde reference substances, and 5 are negative references;
FIG. 3 is a TLC identification chart of white peony root; wherein, 1-3 are samples (batch number: 201101, 201102, 201103), 4 are paeoniflorin reference substances, and 5 are negative references;
FIG. 4 is TLC identification chart of radix Cyathulae; wherein, 1-3 are samples (batch numbers: 201101, 201102 and 201103), 4 are medicinal cyathula root control materials, and 5 are negative controls;
FIG. 5 is a TLC identification chart of Gentiana macrophylla; wherein, 1-3 are samples (batch numbers: 201101, 201102 and 201103), 4 are gentiopicrin reference substances, and 5 are negative references;
FIG. 6 is a TLC identification chart of licorice under visible light (left) and 365nm (right); wherein, 1-3 are samples (batch numbers: 201101, 201102 and 201103), 4 are licorice control medicinal materials, 5 are liquiritin control substances, and 6 are negative controls;
FIG. 7 is a chromatogram of a control (A), a test sample (B), and a negative control solution (C); wherein 1 is cryptotanshinone, 2 is tanshinone I, and 3 is tanshinone II A 。
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
The embodiment is as follows: a quality control method of JINGUTONGXIAO pill is provided.
1 Instrument and reagent
1.1 instruments
LC-20AD high performance liquid chromatograph (Shimadzu, japan); AB135-S electronic analytical balance (METTLER, switzerland); SHA-B dual-function water bath constant temperature oscillator (Hangzhou brand instruments science and technology Co., ltd.).
1.2 reagent
Tanshinone II A (batch No. 110766-202022), cryptotanshinone (batch No. 110852-201807), tanshinone I (batch No. 110867-201607), alpha-cyperone (batch No. 110748-201312), cinnamaldehyde (batch No. 110710-201821), paeoniflorin (batch No. 110736-201842), gentiopicroside (batch No. 110770-201918) reference substances, rhizoma cyperi (batch No. 121059-201407), radix cyathulae (batch No. 121065-201707) and liquorice (batch No. 120904-202021) reference medicinal materials which are all from China food and drug testing institute; glycyrrhizin control (batch: MUST-19033010), dowman bioscience, inc.; pill for relieving pain of bones and muscles (batch number: 201101, 201102, 201103), produced by Luzheng pharmaceutical industry, inc. of Henan province; silica gel G and silica gel GF 254 Precast slabs, tabacco chemical industry institute; acetonitrile and phosphoric acid are chromatographically pure, and the others are analytically pure; purified water, wahaha group.
2 methods and results
2.1 thin layer chromatography identification
2.1.1 thin-layer chromatography identification of rhizoma Cyperi.
In the thin-layer chromatography identification method of rhizoma cyperi in the existing standard, the sample sampling amount and the sample loading amount of a sample are small, the sample loading amount of a reference medicinal material is also small, spots corresponding to the chromatographic spot points of an alpha-cyperone reference substance in the chromatograms of a test sample and the reference medicinal material are weak, and the judgment of the result is influenced. The invention revises the thin-layer chromatography identification method of rhizoma cyperi, optimizes the sample volume of the sample, the preparation method of the sample, the sample loading volume and the developing agent, greatly improves the chromatogram effect, can share one sample solution with the cassia twig identification, and is simpler and more convenient.
The identification method comprises the following steps: grinding 10g of the product, placing in 500ml round bottom flask, adding 200ml of water and several glass beads, mixing, and connecting with volatile oil detector (d)<1) Adding water from the upper end of the tester to the scale, adding 2ml of petroleum ether (60-90 ℃) until the water overflows into the flask, connecting a reflux condenser, heating to boil, keeping slightly boiling for 1 hour, cooling, taking outThe petroleum ether layer is used as a test solution. Taking 10g of the rhizoma cyperi-lacking negative sample, and preparing the rhizoma cyperi-lacking negative sample solution by the same method. Taking another 1g of rhizoma Cyperi as control material, and making into control material solution by the same method. And adding ethyl acetate into alpha-cyperone control to obtain a solution containing 1mg of alpha-cyperone per 1ml, wherein the solution is used as a control solution. Performing thin layer chromatography (2020 version of Chinese pharmacopoeia, general rules of four parts 0502) test by sucking 8 μ l each of test solution, control solution, 1 μ l control solution and 8 μ l rhizoma Cyperi negative sample solution, and respectively dropping on the same silica gel GF 254 Spreading with dichloromethane-ethyl acetate-glacial acetic acid (80: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). Spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; spraying dinitrophenylhydrazine test solution, standing for a moment, and allowing the spots to gradually change into orange red without interference. See fig. 1.
According to the invention, the sample sampling amount is increased, and the sample solution is prepared by extracting volatile oil, so that compared with the existing standard method, the interference of other fat-soluble components can be effectively removed; and the current standard developing agent petroleum ether (60-90 ℃) -ethyl acetate (15: 1) and the developing agent dichloromethane-ethyl acetate-glacial acetic acid (80: 1) are compared, and as a result, the dichloromethane-ethyl acetate-glacial acetic acid (80: 1) is used as the developing agent, and the separation effect of each spot of the sample chromatogram is better. The effect is good when the film is inspected directly under an ultraviolet lamp (254 nm) and after color development.
2.1.2 thin-layer chromatography identification of ramulus Cinnamomi.
In the thin-layer chromatography identification method of cassia twig in the existing standard, the main spot of the chromatogram of a test solution is weaker, and the chromatogram of a reference medicinal material only contains cinnamaldehyde one corresponding spot, so that the thin-layer chromatography identification method cannot provide more valuable information compared with the thin-layer chromatography identification method only using cinnamaldehyde as a reference. The method improves the sample sampling amount, the sample concentration and the sample loading amount, and deletes the cassia twig reference medicinal material as a reference, so that the chromatographic spot of the test sample is clearer and the operation is simpler.
The identification method comprises the following steps: taking a test solution of the identification item of the rhizoma cyperi as a test solution. Taking another 10g negative sample lacking ramulus Cinnamomi, and preparing into ramulus Cinnamomi negative sample solution by the same method. Taking cinnamaldehyde as a reference substance, adding petroleum ether (60-90 ℃) to prepare a solution containing 1 mu l of cinnamaldehyde per 1ml, and using the solution as a reference substance solution. Performing thin-layer chromatography (general rule of four parts 0502 of 2020 edition in Chinese pharmacopoeia), sucking the three solutions 4 μ l each, respectively dropping on the same silica gel G thin-layer plate, developing with petroleum ether (60-90 deg.C) -ethyl acetate (17: 3) as developing agent, taking out, air drying, and spraying with dinitrophenylhydrazine ethanol test solution. Spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution, and the negative result is free from interference. See fig. 2.
2.1.3 identifying radix Paeoniae alba by thin layer chromatography.
In the existing quality standard, the thin-layer chromatography identification of the white paeony root has some problems, which are mainly shown in the aspect of the preparation method of a test solution. The preparation process of the test solution contains an elution link using a neutral alumina column, and the neutral alumina of different manufacturers has great difference in adsorption capacity due to quality problems or quality differences of neutral alumina, so that the situation that target components cannot be eluted frequently occurs, the detection result has problems, the result judgment is influenced, and the product quality control is seriously disturbed. To avoid this, the present invention improves the preparation method of the test solution and the developing agent, and the result is satisfactory.
The identification method comprises the following steps: grinding 3g of the product, adding 30ml of ethanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, cooling, adding 30ml of water into the residue, stirring, filtering, extracting the filtrate with water saturated n-butanol for 2 times, 20ml each time, mixing the n-butanol solutions, evaporating to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution. 3g of the negative sample lacking the white paeony root is taken, and the white paeony root negative sample solution is prepared by the same method. Adding methanol into penoniflorin control to obtain solution containing 2mg per 1ml as control solution. Performing thin layer chromatography (general rule of four parts 0502 of 2020 edition of Chinese pharmacopoeia), sucking 5 μ l of test solution, 2 μ l of control solution and 5 μ l of radix Paeoniae alba negative sample solution, respectively dropping on the same silica gel G thin layer plate, developing with ethyl acetate-acetone-formic acid-water (15: 6: 2: 0.5) as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution containing 5% vanillin, and heating at 105 deg.C until the spots are clearly developed. Spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution, and the negative result is free from interference. See fig. 3.
In the research, the preparation methods of various test solutions are compared, and the preparation methods comprise the steps of eluting the sample solution with methanol after extraction, eluting the sample solution with methanol and then 40% methanol, ultrasonically extracting the sample solution with ethanol, extracting the sample solution with n-butyl alcohol, and then washing the sample solution with ethyl ether, ammonia test solution/0.5% sodium hydroxide/n-butyl alcohol saturated water. By adopting the method, most impurities can be removed by a method of evaporating the ethanol filtrate to dryness, cooling, adding water, stirring (without heating) and filtering, the paeoniflorin loss is less, the method is simple, and the chromatographic background is clear, so that the preparation method of the test solution is finally determined and the method is drawn up by the invention. In addition, in the aspect of reference substance selection, the radix paeoniae alba reference medicinal material and the paeoniflorin reference substance are used as reference substances, and the two reference substances can provide the same valuable information, so that only the paeoniflorin is selected as the reference substance.
In this study, reference is also made to the literature to compare a plurality of developing systems, including chloroform-n-butanol-glacial acetic acid (3: 0.2) (the current standard), a lower layer of chloroform-n-butanol-glacial acetic acid (13: 7: 2), chloroform-n-butanol (1: 1), ethyl acetate-formic acid-glacial acetic acid-water (15: 1: 2) and (8: 1: 2), ethyl acetate-acetone-formic acid-water (10: 6: 2: 0.5) and (15: 6: 2: 0.5), etc., wherein chloroform-n-butanol-glacial acetic acid (3: 0.2), ethyl acetate-formic acid-glacial acetic acid-water (8: 1: 2) and ethyl acetate-acetone-formic acid-water (15: 6: 2: 0.5) as developing agents are all effective and have no interference with negative effect. But takes ethyl acetate-acetone-formic acid-water (15: 6: 2: 0.5) as developing agent, the developing speed is fast, the separation effect of each spot of the sample is good, R is f The value is appropriate.
2.1.4 thin-layer chromatography identification of radix cyathulae.
In the existing quality standards, the thin-layer chromatography identification method of radix cyathulae has some problems, which are mainly reflected in that the chromatographic spot of a test sample is weak, the chromatographic spot of a radix cyathulae reference medicinal material is too strong, and the contrast between the chromatographic spot and the chromatographic spot is large, so that the result judgment is influenced. The invention increases the sampling amount of the test sample, reduces the sampling amount of the radix cyathulae reference medicinal material, and ensures that the chromatographic spots of the radix cyathulae reference medicinal material are proper in strength and satisfactory in result.
The identification method comprises the following steps: grinding 3g of the product, adding 40ml of 75% ethanol, reflux extracting for 1 hr, filtering, evaporating filtrate to dryness, dissolving residue with 10ml of water, shaking and extracting with 20ml of diethyl ether, evaporating diethyl ether solution to dryness, and dissolving residue with 1ml of absolute ethanol to obtain sample solution. Taking 3g of radix Cyathulae-lacking negative sample, and preparing radix Cyathulae negative sample solution by the same method. Taking radix Cyathulae 0.5g as reference material, and making into reference material solution by the same method. Performing thin layer chromatography (general rules of the four kingdoms 0502 of 2020 edition of Chinese pharmacopoeia), sucking the three solutions 4 μ l each, respectively dropping on the same silica gel G thin layer plate, developing with toluene-chloroform-acetone (8: 4: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, spots with the same color appear at the corresponding positions of the chromatogram of the reference solution, and the negative side is not interfered. See fig. 4.
2.1.5 thin-layer chromatography identification of gentiana macrophylla.
In the existing quality standard, a small gentiana macrophylla contrast medicinal material is selected as a contrast for identifying the gentiana macrophylla by thin layer chromatography, and as the gentiana macrophylla is a multi-base variety, the condition that chromatographic spots of a sample cannot completely correspond to the contrast medicinal material can occur, so that the specificity is poor, and the result judgment is often influenced. In order to improve the situation, a gentiopicroside reference substance is adopted as a reference in an essential quantity standard draft, and a preparation method and a developing agent of a test solution are optimized, so that the method is simple and has strong specificity.
The identification method comprises the following steps: grinding 1g of the product, adding 20ml of methanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, and dissolving residue with 1ml of methanol to obtain test solution. Preparing 3g of negative sample lacking radix Gentianae Marcrophyllae, and preparing into radix Gentianae Marcrophyllae negative sample solution by the same method. Taking gentiopicroside reference substance, adding methanol to obtain solution containing 1mg per 1ml, and making into reference substance solution. Performing thin layer chromatography (China pharmacopoeia 2020 edition four-part general rule 0502) test by sucking 2 μ l of each of the three solutions, and respectively dropping on the same silica gel GF 254 Spreading on thin layer plate with ethyl acetate-methanol-water (10: 2: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm)And (6) viewing. In the chromatogram of the test solution, spots of the same color appear at the corresponding positions of the chromatogram of the control solution, and the negative result is not interfered. See fig. 5.
In the present study, various comparisons were made on the preparation methods of the test samples, including ethanol-10% ammonia solution (50: 7) soaking method (current method), ultrasonic method, methanol ultrasonic method, and the like, in which the ethanol ultrasonic extraction method is performed after removing impurities by extraction, and then methanol ultrasonic extraction method is performed.
In the research, chromatograms of three basic-source reference medicinal materials of small gentiana macrophylla, large-stem gentiana macrophylla and twisted gentiana macrophylla are compared, and the result shows that the chromatographic spots of the three basic-source gentiana macrophylla are greatly different, so that the reference medicinal materials are not suitable for being used as reference in finished product inspection.
In this study, the use of gentiopicroside and roburic acid controls as controls was compared, and roburic acid could not be shown under ultraviolet light (254 nm), so the gentiopicroside control was chosen as the control in the present invention.
In this study, a number of deployment systems were compared, including: toluene-chloroform-acetone-isopropanol (8: 4: 1) (current standard), ethyl acetate-methanol-water (10: 2: 1)/(14: 2: 1), chloroform-methanol-formic acid (50: 1: 0.5), lower layer of chloroform-methanol-water (30: 10: 3), etc., and the results showed that ethyl acetate-methanol-water (10: 2: 1) was the most effective as a developing agent. The lower layer of chloroform-methanol-water (30: 10: 3) also works well. As trichloromethane in the developing solvent has certain toxicity and needs to be layered, ethyl acetate-methanol-water (10: 2: 1) is selected as the developing solvent in the invention.
2.1.6 thin-layer chromatography identification of licorice.
In the existing quality standard, the thin-layer chromatography identification of liquorice is not available. In the research process of the product quality control method, the thin-layer chromatography identification of the liquorice is correspondingly researched, the preparation method of the test solution is compared, the effects of three basic source reference medicinal materials and liquiritin and ammonium glycyrrhizinate reference substances serving as the reference are compared, the developing agent is preferably selected, and finally the liquorice reference medicinal material and the liquiritin reference substance are selected as the reference, so that the method is simple and has strong specificity.
The identification method comprises the following steps: taking 4g of the product, grinding, adding 30ml of methanol, heating and refluxing for 1 hour, filtering, evaporating filtrate to dryness, adding 30ml of 5% sodium carbonate solution into residues for dissolving, washing with ethyl acetate for 2 times, 30ml each time, adjusting the pH value of a water layer to 2-3 with 10% hydrochloric acid, shaking and extracting with ethyl acetate for 2 times, 30ml each time, combining ethyl acetate solutions, evaporating to dryness, adding 1ml of methanol into residues for dissolving to obtain a test solution. 4g of negative sample without liquorice is prepared into a liquorice negative sample solution by the same method. Another control medicinal material 0.2g is prepared, and the same method is used to prepare control medicinal solution. Taking liquiritin reference substance, adding methanol to obtain solution containing 0.5mg per 1ml as reference substance solution. Performing thin-layer chromatography (general rules of the four kingdoms 0502 of 2020 edition of Chinese pharmacopoeia), sucking 1-2 μ l of each of the above four solutions, respectively dropping on the same silica gel G thin-layer plate, developing with ethyl acetate-formic acid-glacial acetic acid-water (15: 1: 2) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the color of spots is clear, and respectively inspecting under sunlight and ultraviolet lamp (365 nm). In the chromatogram of the test solution, main spots or fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material; spots of the same color or fluorescent spots appear at positions corresponding to the color spectrum of the control. And negative without interference. See fig. 6.
In the research process, corresponding research is also carried out by referring to a thin-layer identification method of liquorice in 'Chinese pharmacopoeia' of 2020 edition, and the selection of a reference substance and the preparation of a test solution in the method are different from the method adopted by the invention, wherein the reference substance is ammonium glycyrrhizinate, and the preparation method of the test solution specifically comprises the following steps: taking 4g of the product, grinding, adding 40ml of diethyl ether, heating and refluxing for 1 hour, filtering, discarding the ethyl ether solution, adding 30ml of methanol into the medicine residue, heating and refluxing for 1 hour, filtering, evaporating the filtrate to dryness, adding 40ml of water into the residue to dissolve, shaking and extracting with n-butanol for 3 times, 20ml each time, combining the n-butanol solutions, washing with n-butanol saturated water for 3 times, 20ml each time, evaporating the n-butanol solution to dryness, and adding 1ml of methanol into the residue to dissolve to obtain a sample solution. As a result, the interference was severe at the position corresponding to the chromatogram of the ammonium glycyrrhizinate control. Therefore, liquiritin can be used as a reference, but ammonium glycyrrhizinate is not suitable for the reference. In addition, the resultsThe chromatographic background of the test solution is found to be large, so the method of the invention is selected for preparing the test solution. The preparation method of the test solution mainly comprises the following steps of preparing a solution containing 7 parts of liquiritin and the like / the-OH structure flavone can be separated and purified by an alkali-soluble acid precipitation method due to the property of being soluble in a 5% sodium carbonate solution.
There are three sources of licorice, including licorice, glycyrrhiza glabra and Glycyrrhiza inflata, and in this study, the chromatographic characteristics of three control medicinal materials of licorice were also investigated. The three reference medicinal materials are all prepared by the method, and after development and color development, the main chromatographic spots of the three reference medicinal materials are completely consistent, so that the liquorice identification method selects the liquiritin reference substance as a reference and also selects the liquorice reference medicinal material as a reference, and the chromatographic information is richer.
In the present study, a plurality of development systems are compared with the related literature, and the development agent used in the present invention comprises toluene-ethyl acetate-formic acid (20: 4: 0.5), chloroform-acetone-ethyl acetate-formic acid (5: 2: 1), ethyl acetate-methanol-water (20: 3: 2), ethyl acetate-acetone-water (6: 1), and the like, and the development agent used in the present invention, ethyl acetate-formic acid-glacial acetic acid-water (15: 1: 2), has the best effect, the development speed is high, the chromatographic background is clear, and the negative effect is not interfered.
2.1.7 thin-layer chromatography identification of other medicinal materials.
In the research process, referring to relevant documents, a great deal of research is also carried out on the thin-layer chromatography identification of clematis root, suberect spatholobus stem, combined spicebush root and rehmannia root in the sinew and bone pain relieving pill, and the result is serious interference, so that an ideal effect cannot be obtained, and therefore, the quality control method of the sinew and bone pain relieving pill is not adopted.
2.2 measurement of content
2.2.1 chromatographic conditions and system applicability octadecylsilane chemically bonded silica is used as a filler; acetonitrile is taken as a mobile phase A, 0.02 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the conditions in the table 1; the column temperature is 20 ℃; the flow rate is 1 ml/min -1 The detection wavelength is 270nm, the column temperature is 20 ℃, and the sample injection amount is 10 mu l. Number of theoretical plates based on tanshinone II A Peak calculation should be noBelow 20000.
TABLE 1HPLC gradient elution conditions
2.2.2 preparation of reference solution cryptotanshinone, tanshinone I and tanshinone II A Accurately weighing appropriate amount of reference substance, placing in brown measuring flask, adding methanol to obtain a solution containing cryptotanshinone and tanshinone II per 1ml A 10 mu g and 5 mu g of tanshinone I.
2.2.3 preparation of test solution A proper amount of the product is taken, ground, taken about 1.0g, precisely weighed, placed in a conical flask with a plug, precisely added with 50ml of methanol, tightly plugged, weighed, placed on a water bath oscillator at 60 ℃ for 1 hour, cooled, weighed again, supplemented with methanol to the loss weight, shaken well, filtered, and a subsequent filtrate is taken, thus obtaining the test solution.
2.2.4 preparation of negative sample solutions: taking 1.0g of the preparation lacking the salvia miltiorrhiza, grinding, and preparing a negative sample solution according to the preparation method of the test sample solution.
The chromatograms of the control, test sample and negative sample are shown in FIG. 7.
2.2.5 Linear relationship examination and preparation of a series of cryptotanshinone, tanshinone I and tanshinone II A The mixed solution (each concentration is 2 to 20. Mu.g/ml) -1 ). Sucking 10 μ l of each of the above mixed solutions, measuring peak area under the above chromatographic conditions, and taking peak area integral value as ordinate and sample concentration (μ g/ml) -1 ) Drawing a standard curve for the abscissa, and calculating a regression equation as follows:
cryptotanshinone Y = 50.9X-232.4, R 2 ＝0.999 4。
Tanshinone IY = 43.3X-4 647.3 2 ＝0.999 4。
Tanshinone II A Y＝57 180.1X+2 686.6，R 2 ＝0.999 6。
The results show that: cryptotanshinone, tanshinone I and tanshinone II A The analysis concentration is 2-20 mu g/ml -1 Within the range, exhibits good linearity with the peak area integral valueAnd (4) relationship.
2.2.6 precision test by precisely sucking 10 μ l of the same sample solution, repeating sample injection for 6 times, measuring peak area, and calculating cryptotanshinone, tanshinone I and tanshinone II A The peak areas RSD of the cells were 0.47%, 0.40%, and 0.25%, respectively, indicating that the precision of the instrument was good.
2.2.7 detection limit and quantitative limit were determined by signal-to-noise ratio method for cryptotanshinone, tanshinone I and tanshinone II A The detection limits of (A) are respectively: 0.301 5ng, 0.301 2ng and 0.308 ng, and the quantitative limits are respectively as follows: 1.005ng, 1.004ng and 1.028ng.
2.3.8 stability test accurately extracts 10 μ l of the same test solution, and measures cryptotanshinone, tanshinone I and tanshinone II in 0, 2, 4, 8, 12, 18 and 24h respectively A The peak areas RSD of the samples were 0.65%, 0.30%, and 0.41%, respectively. The results show that: the test solution is stable within 24 h.
3238 Zxft 3238 repeatability test six parts of the same test sample (lot No. 210103) are extracted according to the preparation method of test sample solution, and analyzed according to the proposed chromatographic conditions, the total tanshinone amount is 0.852mg g -1 And RSD is 0.44%. The results show that: the method has good repeatability.
2.2.10 sample recovery the sample (lot number 210103, 0.852mg. G.g) was sampled at a known concentration -1 ) 9 portions of each portion of which is about 0.50g and is precisely weighed, and cryptotanshinone, tanshinone I and tanshinone II with different concentrations are precisely added into each 3 portions of samples respectively A The reference solution is prepared into a test solution according to the preparation method of the test solution, and the recovery rates of the three kinds of tanshinone are respectively calculated according to the determination of the established chromatographic conditions. The results are shown in tables 2 to 4. The results show that: the recovery rates of the three tanshinones are all between 95 and 105 percent, which shows that the method has good recovery rates.
TABLE 2 cryptotanshinone recovery test results
TABLE 3 tanshinone I recovery test results
TABLE 4 tanshinone II A Results of recovery test
According to the content determination results of 20 batches of products, the content limit of the product is specified by combining the content regulation and the process transfer rate of the salvia miltiorrhiza medicinal material in 'Chinese pharmacopoeia' (one part) of 2020 edition: based on the dry product, each 1g of the product contains cryptotanshinone (C) in radix Salviae Miltiorrhizae 19 H 20 O 3 ) Tanshinone I (C) 18 H 12 O 3 ) And tanshinone II A (C 19 H 18 O 3 ) Should not be less than 0.42mg in total.
Test example: the test example optimizes the measurement conditions of the HPLC method adopted by the content measurement items, and the optimization process is as follows: 1 selection of chromatographic conditions
Preparation of reference solution for cryptotanshinone, tanshinone I and tanshinone II A An appropriate amount of reference substance is precisely weighed, and methanol is added to prepare mixed solutions each containing 10 μ g of reference substance per 1 ml.
The preparation method of the test solution comprises grinding 0.4g of the product, precisely weighing, placing in a conical flask with a plug, precisely adding 30ml of methanol, weighing, ultrasonically treating (power 600W, frequency 40 kHz) for 45min, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate.
1.1 selection of detection wavelength
Collecting cryptotanshinone, tanshinone I and tanshinone II A The reference solution was extracted with 10 μ l of the reference solution and injected into a liquid chromatograph for analysis according to a method for measuring the tanshinone content in salvia miltiorrhiza medicinal material in the first part of the China pharmacopoeia, 2020 edition.
Liquid chromatograph (LC-20AD, SPD-M20A diode array detector, chromatographic column: shimadzu WondaSIL TM C18 Superb 5μm 4.6×250mm)。
Generated by diode array detectorsThe maximum absorption wavelength was determined from the spectrogram and the results showed that: in the wavelength range of 200-800 nm, cryptotanshinone has maximum absorption at 263nm, tanshinone I has maximum absorption at 244nm, and tanshinone II A There is a maximum absorption at 268nm.
A sample solution of 10. Mu.l was injected into a liquid chromatograph and analyzed, and the results of the degrees of separation of the target components in the sample at each wavelength are shown in Table 5.
TABLE 5 degree of separation of target component in test sample at each wavelength
The results show that: the resolution was better at detection wavelengths 268nm and 270nm. Meanwhile, 270nm is selected by referring to the detection wavelength in the measurement of the content of tanshinone of a salvia miltiorrhiza medicinal material in China pharmacopoeia of 2020 edition.
1.2 selection of the Mobile phase
Using chromatographic conditions for measuring tanshinone content in salvia miltiorrhiza medicinal material in the 2020 edition of Chinese pharmacopoeia, sucking 10 mul of test solution, and respectively using acetonitrile-0.01% phosphoric acid, acetonitrile-0.02% phosphoric acid and acetonitrile-0.03% phosphoric acid as mobile phase analysis samples, and the results are shown in table 6.
TABLE 6 separation of target component in test sample at different mobile phases
The results show that: taking acetonitrile-0.01% phosphoric acid, acetonitrile-0.02% phosphoric acid, and acetonitrile-0.03% phosphoric acid as mobile phase, and collecting cryptotanshinone, tanshinone I and tanshinone II in the sample A The separation degree of the extract has no obvious difference, and acetonitrile-0.02 percent phosphoric acid is selected as a mobile phase by referring to the mobile phase in the content measurement of salvia miltiorrhiza in pharmacopoeia.
1.3 selection of elution gradient
A tanshinone gradient elution method for measuring the content of red sage root in the first part of China pharmacopoeia of 2020 edition is used for detecting cryptotanshinone, tanshinone I and tanshinone II in a test sample A In which the separation degree of cryptotanshinone is slightly lowerAnd there are more interference peaks around the target peak. On the basis, the gradient is tried to be changed to ensure that the cryptotanshinone, the tanshinone I and the tanshinone II in the test sample A The chromatographic parameters such as retention time, separation degree, tailing factor and the like are in a better level.
In the study, various gradient elution methods were selected and compared, and are shown in tables 7 to 9, respectively.
TABLE 7 gradient elution method 1
TABLE 8 gradient elution method 2
TABLE 9 gradient elution method 3
The results show that: cryptotanshinone, tanshinone I and tanshinone II using gradient elution method 3 A All the components are effectively separated, and the separation degree, the tailing factors and the like meet the requirements.
On the basis of the elution methods, various elution methods such as changing initial gradient concentration and changing change rate are tried, and the effect is not better than that of the gradient elution method 3. Method 3 was therefore selected as the final gradient elution method.
1.4 selection of column temperature
The column temperatures were set at 20, 25, 30 and 40 ℃ respectively, 10. Mu.l of the sample solution was taken and injected into a liquid chromatograph (LC-20AD, SPD-M20A diode array detector, column: nacalai tesque COSMOSIL 5C 18-MS-II 4.6 ID. Times.250 mm) under the prescribed chromatographic conditions, and as a result: the column temperature can be 20-30 ℃, wherein the effect is the best at 20 ℃.
1.5 determination of the number of theoretical plates
Of the three components to be measured, tanshinone II A The content of tanshinone II is relatively high, therefore, the theoretical plate number of the chromatographic condition of the method is also tanshinone II A And (4) peak counting. In the experiment, the chromatographic conditions of acetonitrile-0.02% phosphoric acid as a mobile phase, the column temperature of 20 ℃, the elution method 3 are adopted, the detection wavelength is 270nm, a plurality of samples are analyzed on a plurality of chromatographic columns, and tanshinone II is used A When the number of the peak theoretical plates is more than 2 ten thousand, the target peak separation degree can reach more than 1.5, so the tanshinone II is used for determining the number of the theoretical plates under the chromatographic condition of the method A The peak calculation should be no less than 20000.
2 selection of preparation method of test solution
2.1 examination of extraction solvent
Taking 0.4g of a sample (batch No. 210103), grinding, precisely weighing, placing in a conical flask with a plug, respectively adding 30ml of methanol and ethanol, weighing, performing ultrasonic treatment (600W, 40kHz, normal temperature) for 45 minutes, cooling, weighing again, respectively supplementing the lost weight with methanol or ethanol, filtering, taking the subsequent filtrate, and analyzing according to the set chromatographic conditions. The results are shown in Table 10.
TABLE 10 selection of extraction solvents
The results show that: the methanol is used as a solvent, and the extraction effect is good. Methanol was therefore chosen as the extraction solvent.
2.2 examination of the extraction method
Taking 0.4g of the same test sample (batch No. 210103), grinding, precisely weighing, placing in a conical flask with a plug, adding 30ml of methanol, weighing, heating and refluxing for 45 minutes, supplementing the lost weight with methanol, filtering, taking the subsequent filtrate, and analyzing according to the preset chromatographic conditions.
As a result, the total tanshinone amount in the test sample was found to be 0.8943mg/g. The heating reflux method is superior to the normal temperature ultrasonic method. The proper temperature is favorable for extracting the total tanshinone, and the temperature can not be too high or too low.
2.3 examination of sample volume of sample and bath temperature under reflux
In the existing quality standard, the sample weight of the sample is 0.4g, 30ml of methanol is added, and the common pipette is 25 or 50ml, so that the practical operation is inconvenient. The study was intended to examine the extraction effect of different sample weights and different water bath temperatures in 50ml of methanol. The test conditions set for this were as follows:
condition (1) 0.4g,50ml methanol, 70 ℃ heating reflux 45min;
condition (2) 0.6g,50ml methanol, heated to reflux at 70 ℃ for 45min;
condition (3) 0.8g,50ml methanol, 70 degrees C heating reflux 45min;
condition (4) 1.0g,50ml methanol, heating reflux at 70 ℃ for 45min;
condition (5) 0.8g,50ml methanol, 80 ℃ heating reflux 45min;
condition (6) 1.0g,50ml methanol, 80 ℃ heating reflux 45min.
The results of the analysis according to the proposed chromatographic conditions are shown in Table 11.
TABLE 11 examination of sample size and bath temperature under reflux conditions
As a result: the results of several sample weighing measurements are close, and the water bath temperature of 80 ℃ is slightly better than 70 ℃; considering that the chromatographic peak area of the test sample and the chromatographic peak area of the reference sample are closer when the sample is weighed to 1.0g, the sample is weighed to 1.0g.
In the research, the fact that the chromatogram of the test sample obtained by the reflux method has the condition that the leading edge of the tanshinone I chromatographic peak and part of tailing factors are less than 0.95 is found, although the extraction efficiency of the reflux method is superior to that of the ultrasonic method. Therefore, an extraction method which can ensure both the extraction efficiency and the chromatographic parameters needs to be explored.
2.4 review of extraction methods
The ultrasonic extraction is tried at a certain temperature, and experiments show that the temperature is increased within a certain range to facilitate the extraction of the total tanshinone, but the conditions are severe when the extraction temperature is 60-70 ℃, more unstable factors exist, and the tested sample is easy to splash out of a bottle to cause unreliable experimental results; and when the temperature is lower than 50 ℃, the extraction is not complete. In the research, the water bath oscillation method is good, the conditions are mild, a certain extraction temperature can be ensured, the sample is not easy to lose, the operation is easy, and the result is stable.
2.5 Water bath Oscillating temperature investigation
The temperature is a key parameter for extracting tanshinone. The oscillating temperature of the water bath is set to be 40 ℃,50 ℃ and 60 ℃ (the temperature is higher, the situation similar to the backflow can occur), the oscillating speed is 150r/min, the extraction time is 60min, and the experimental results are shown in table 12.
TABLE 12 Water bath Oscillating temperature investigation
The results show that: the extraction effect is better at 60 ℃.
2.6 Water bath Oscillating time survey
Under the experimental conditions of water bath oscillation speed of 150r/min and 60 ℃, the water bath oscillation time is respectively set to be 30 minutes, 45 minutes, 60 minutes and 90 minutes, extraction tests are carried out, and the experimental results are shown in Table 13.
TABLE 13 examination of Water bath oscillation time
The results show that: under the condition of 60 ℃, the content homogeneous phases are not very different under four extraction times, and the effects of 45 minutes and 60 minutes are better. Considering that 60 minutes is a good operation, 60 minutes is chosen for the experiment.
2.7 Water bath Oscillating speed inspection
Under the experimental conditions of water bath oscillation for 60 minutes and 60 ℃, the water bath oscillation rotating speeds are respectively set to be 100r/min, 150r/min and 200r/min, and the experimental results are shown in Table 14.
TABLE 14 Water bath Oscillating speed survey
The results show that: the measured result of the rotating speed of 100-200r/min is approximate, and the 100r/min effect is better. Under the condition of 60 ℃, the rotating speed has a limited influence on the experimental result, but the liquid level is rotated at a higher rotating speed, such as 200r/min, during the experiment, so that the sample may be splashed out, and the result is easy to be unstable. Since the influence of the water bath oscillation speed is small, the speed is not specified in the method for preparing the test solution.
2.8 examination of the amount of solvent in Water bath oscillation
Taking a sample, grinding, precisely weighing about 1.0g, placing in a conical flask with a plug, precisely adding 40/50/60ml of methanol, sealing the plug, weighing, placing in a water bath environment at 60 ℃ for shaking at 100r/min for 60 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking a subsequent filtrate. The analytical results are shown in Table 15.
TABLE 15 investigation of the amount of solvent in the Water bath
The results show that: the amounts of methanol of 1.0g of the sample were 40ml, 50ml and 60ml, and the results were not very different. Since 50ml of methanol was more conveniently measured, 50ml of methanol was determined.
Through the research, the preparation method of the test solution is determined as follows:
taking a proper amount of the product, grinding, precisely weighing about 1.0g, placing into a conical flask with a plug, precisely adding 50ml of methanol, sealing the plug, weighing, placing on a water bath oscillator at 60 ℃ for 1 hour, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering, and taking the subsequent filtrate.
3 negative test
Considering the test sample with low content of tanshinone I, the reference solution concentration is adjusted to cryptotanshinone and tanshinone II A 10 mu g/ml of each and 5 mu g/ml of tanshinone I, and the peak areas of the test sample and the reference sample are approximate.
Preparing a reference substance solution: collecting cryptotanshinone, tanshinone I and tanshinone II A Adding appropriate amount of reference substance, precisely weighing, and adding methanol to obtain a solution containing cryptotanshinone and tanshinone II per 1ml A Respectively 10 μ g and 5 μ g of tanshinone I (reference solution concentration: cryptotanshinone 10.44 μ g/ml, tanshinone I5.07 μ g/ml and tanshinone II) A 10.01μg/ml)。
The test solution is prepared according to the test solution preparation method.
Preparation of negative sample solution about 1.0g of the preparation lacking Salvia miltiorrhiza is taken, ground and prepared into the negative sample solution according to the preparation method of the test sample solution.
A high performance liquid chromatograph: LC-20AD; a detector: an SPD-M20A diode array detector; column oven: CTO-20AC.
A chromatographic column: nacalai tesque COSMOSIL 5C 18-MS-II 4.6ID × 250mm
And sucking 10 mu l of each of the reference solution, the test solution and the negative sample solution, injecting the solution into a liquid chromatograph, and analyzing according to the established chromatographic conditions.
The results show that: the negative sample did not interfere with the three controls.
Comparative example: the invention relates to a muscle and bone pain relieving pill, which has the current standard number of: WS 3 423 (Z-067) -2000Z-2009, the details referred to under this standard are as follows:
1. and (7) identifying by TLC.
(1) Grinding 3g of the product, adding diethyl ether 30ml, shaking, standing for 1 hr, filtering, volatilizing the filtrate, and dissolving the residue with ethyl acetate 1ml to obtain a sample solution. Taking 0.5g of radix Salviae Miltiorrhizae as control, and making into control solution by the same method. Collecting tanshinone II A As a control, 2mg of ethyl acetate was added to 1ml of the solution to prepare a control solution. Thin layer chromatography (attached to the first edition of Chinese pharmacopoeia 2005)Recording VIB) test, sucking the three solutions, respectively dropping the solutions on the same silica gel G thin layer plate, developing with toluene-ethyl acetate (19: 1) as developing agent, taking out, and air drying. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.
(2) Taking 3g of the product, grinding, adding 30ml of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, adding 1ml of methanol into the residue to dissolve the residue, adding a proper amount of neutral alumina, stirring uniformly, loading onto a pre-filled neutral alumina small column (the inner diameter is about 1cm, and the weight of alumina is about 4 g), eluting with 40ml of methanol, discarding the former 2ml of eluent, receiving the subsequent eluent, evaporating to dryness, and adding 1ml of methanol into the residue to dissolve the residue to obtain a sample solution. Adding methanol into penoniflorin control to obtain solution containing 2mg per 1ml as control solution. Performing thin-layer chromatography (appendix VI B of the first edition of Chinese pharmacopoeia 2005), sucking the two solutions, respectively dripping the two solutions on the same silica gel G thin-layer plate, developing with chloroform-n-butanol-glacial acetic acid (3: 0.2) as developing agent, taking out, air drying, spraying with 5% vanillin sulfuric acid solution, and heating until the spots are clearly developed. The same bluish purple spots appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control solution.
(3) Grinding 5g of the product, soaking in 20ml of diethyl ether for 1 hr, shaking, filtering, evaporating the filtrate, and dissolving the residue in 2ml of ethyl acetate to obtain a sample solution. Taking another 1g of rhizoma Cyperi as control material, and making into control material solution by the same method. Then taking alpha-cyperone as a reference substance, adding ethyl acetate to prepare a solution containing 1 mul of alpha-cyperone per 1ml, and taking the solution as a reference substance solution. According to the test of thin-layer chromatography (appendix VI B of the first edition of Chinese pharmacopoeia 2005), 2 mul of each of the three solutions is absorbed and respectively spotted on the same piece of silica gel GF 254 On the thin layer plate, petroleum ether (60-90 deg.C) -ethyl acetate (15: 1) is used as developing agent, and the developed thin layer plate is taken out, dried and placed under the UV lamp (254 nm) for inspection. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference materials and the reference solution.
(4) 2g of the product is taken, ground and added with 20ml of mixed solution of ethanol and 10 percent ammonia solution (50: 7) to be soaked for 3 hours, and then the mixture is shaken. Filtering, recovering ethanol from the filtrate, evaporating to dryness, dissolving the residue with 5ml methanol, filtering, and collecting the filtrate as sample solution. Preparing 1g of radix Gentianae Marcrophyllae reference medicinal material, and preparing reference medicinal material solution by the same method. Performing thin-layer chromatography (appendix VI B of the first edition of Chinese pharmacopoeia 2005), sucking the two solutions 2 μ l each, respectively dropping on the same silica gel G thin-layer plate, developing with toluene-chloroform-acetone-isopropanol (8: 4: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The same green fluorescent spot appears on the chromatogram of the test solution at the position corresponding to the chromatogram of the reference solution.
(5) Grinding 2g of the product, adding 40ml of 75% ethanol, reflux extracting for 1 hr, filtering, evaporating filtrate to dryness, adding 10ml of water to dissolve residue, shaking and extracting water solution with 20ml of diethyl ether, evaporating extractive solution to dryness, and dissolving residue with 1ml of anhydrous ethanol to obtain test solution. Taking 1g radix Cyathulae as reference material, and making into reference material solution by the same method. Performing thin-layer chromatography (appendix VI B of the first edition of Chinese pharmacopoeia 2005), sucking the two solutions, respectively dripping the two solutions on the same silica gel G thin-layer plate, developing with toluene-chloroform-acetone (8: 4: 1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The same blue fluorescent spot appears on the chromatogram of the test solution at the position corresponding to the chromatogram of the control solution.
(6) Taking 5g of the product, grinding, transferring into a 500ml round bottom flask, adding 200ml of water and a plurality of glass beads, uniformly mixing, connecting with a volatile oil tester (d < 1), adding water from the upper end of the tester to the scale, overflowing into the flask, adding 5ml of petroleum ether (60-90 ℃), connecting with a reflux condenser, heating to boil, keeping slightly boiling for 1 hour, cooling, and taking a petroleum ether layer as a test solution. And preparing 2g of cassia twig control medicinal material into a control medicinal material solution by the same method. Taking cinnamaldehyde as a reference substance, adding petroleum ether (60-90 ℃) to prepare a solution containing 1 mu l of cinnamaldehyde per 1ml, and using the solution as a reference substance solution. According to a thin-layer chromatography test (appendix VI B of the first part of Chinese pharmacopoeia 2005), sucking 2 mu l of each of the three solutions, respectively dropping the solution on the same silica gel G thin-layer plate, taking petroleum ether (60-90 ℃) and ethyl acetate (17: 3) as a developing agent, developing, taking out, airing, and spraying dinitrophenylhydrazine ethanol test solution. In the chromatogram of the test solution, at the position corresponding to the chromatogram of the control solution, an orange-red spot of the same color appears.
2. And (4) measuring the content.
Measured according to high performance liquid chromatography (appendix VI D of the first edition of Chinese pharmacopoeia 2005).
(1) The chromatographic condition and the system applicability test use octadecylsilane chemically bonded silica as a filler; methanol-water (75: 25) is used as a mobile phase; the detection wavelength was 268nm. Theoretical plate number according to tanshinone II A The peak calculation should not be below 2000.
(2) Preparation of reference solution tanshinone II A Accurately weighing appropriate amount of reference substance, and adding methanol to obtain solution containing 10 μ g per 1 ml.
(3) Preparation of test solution the product under the condition of different loading amount is taken, ground, taken about 0.4g, precisely weighed, placed in a conical flask with a plug, precisely added with 30ml of methanol, weighed, ultrasonically treated (power 600W, frequency 40 kHz) for 45 minutes, cooled, weighed again, supplemented with methanol to the loss weight, shaken, filtered, and a subsequent filtrate is taken, thus obtaining the test solution.
(4) The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
(5) The product contains tanshinone II per 1g of Saviae Miltiorrhizae radix A (C 19 H 18 O 3 ) Calculated, the content of the active ingredient should not be less than 0.34mg.
It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.
Claims (2)
1. A quality control method of JINGUTONGXIAO pill comprises identification item and content determination item; the method is characterized in that:
the identification item comprises thin-layer chromatography identification of rhizoma cyperi, cassia twig, radix paeoniae alba, radix cyathulae, gentiana macrophylla and liquorice, and specifically comprises the following steps:
(1) The thin-layer chromatography identification method of the rhizoma cyperi comprises the following steps: taking 10g of the product, grinding, placing in a 500ml round-bottom flask, adding 200ml of water and glass beads, mixing uniformly, connecting with a volatile oil tester, adding water from the upper end of the tester to a scale, overflowing into the flask, adding petroleum ether 2ml with a boiling range of 60-90 ℃, connecting with a reflux condenser, heating to boil, keeping slight boiling for 1 hour, cooling, and taking a petroleum ether layer as a test solution; preparing rhizoma Cyperi reference material 1g, and preparing reference material solution by the same method; adding ethyl acetate into alpha-cyperone reference substance to prepare a solution containing 1mg per 1ml as a reference substance solution; performing thin layer chromatography by sucking sample solution, control solution and control solution respectively 8 μ l and 1 μ l, respectively dropping on the same silica gel GF 254 Spreading dichloromethane-ethyl acetate-glacial acetic acid with volume ratio of 80: 1 as developing agent on the thin layer plate, taking out, air drying, and inspecting under 254nm ultraviolet lamp; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; spraying a dinitrophenylhydrazine test solution, standing for a moment, and gradually changing the spots into orange red;
(2) The thin-layer chromatography identification method of cassia twig comprises the following steps: taking the test solution of the identification item of the rhizoma cyperi in the step (1) as a test solution; taking cinnamaldehyde as a reference substance, and adding petroleum ether with the boiling range of 60-90 ℃ to prepare a solution with the concentration of 1 mu l in 1ml as a reference substance solution; performing a thin-layer chromatography test, sucking 4 mu l of each of a test solution and a reference solution, respectively dropping the solutions on the same silica gel G thin-layer plate, developing by using petroleum ether-ethyl acetate with a boiling range of 60-90 ℃ and a volume ratio of 17; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution;
(3) The thin-layer chromatography identification method of white paeony root comprises the following steps: taking 3g of the product, grinding, adding 30ml of ethanol, performing ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, cooling, adding 30ml of water to residue, stirring, filtering, extracting filtrate with water saturated n-butanol for 2 times, each time 20ml, mixing n-butanol solutions, evaporating to dryness, adding 1ml of methanol to residue, dissolving, and making into sample solution; adding methanol into penoniflorin reference substance to obtain solution containing 2mg per 1ml as reference substance solution; performing thin layer chromatography test, sucking 5 μ l of test solution and 2 μ l of reference solution, respectively dropping on the same silica gel G thin layer plate, developing with ethyl acetate-acetone-formic acid-water at volume ratio of 15: 6: 2: 0.5 as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution containing 5% vanillin, and heating at 105 deg.C until the spots are clearly developed; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution;
(4) The thin-layer chromatography identification method of radix cyathulae comprises the following steps: taking 3g of the product, grinding, adding 75% ethanol 40ml, extracting under reflux for 1 hour, filtering, evaporating filtrate to dryness, adding water 10ml into residues for dissolving, shaking and extracting with diethyl ether 20ml, evaporating diethyl ether solution to dryness, adding absolute ethanol 1ml into residues for dissolving to obtain a sample solution; preparing radix Cyathulae reference medicinal material 0.5g, and preparing reference medicinal solution by the same method; performing thin-layer chromatography test, sucking sample solution and control solution, respectively dispensing 4 μ l each on the same silica gel G thin-layer plate, developing with toluene-chloroform-acetone as developing agent at volume ratio of 8: 4: 1, taking out, air drying, and inspecting under 365nm ultraviolet lamp; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the reference solution;
(5) The thin-layer chromatography identification method of gentiana macrophylla comprises the following steps: taking the product 1g, grinding, adding methanol 20ml, ultrasonic treating for 30 min, filtering, evaporating filtrate, dissolving residue in methanol 1ml to obtain sample solution; adding methanol into gentiopicrin control to obtain solution containing 1ml and 1mg as control solution; performing thin layer chromatography by sucking 2 μ l of each of the sample solution and the reference solution, and respectively dropping on the same silica gel GF 254 Spreading on a thin layer plate with ethyl acetate-methanol-water as developing agent at volume ratio of 10: 2: 1, taking out, air drying, and inspecting under 254nm ultraviolet lamp; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution;
(6) The thin-layer chromatography identification method of the liquorice comprises the following steps: taking 4g of the product, grinding, adding 30ml of methanol, heating and refluxing for 1 hour, filtering, drying the filtrate to dryness, adding 5% sodium carbonate solution 30ml of residues for dissolving, washing with ethyl acetate for 2 times, 30ml each time, adjusting the pH value of a water layer to be 2-3 with 10% hydrochloric acid, shaking and extracting with ethyl acetate for 2 times, 30ml each time, combining ethyl acetate solutions, drying to dryness, adding 1ml of methanol to dissolve the residues to be used as a sample solution; preparing Glycyrrhrizae radix control 0.2g, and preparing control solution by the same method; then taking liquiritin reference substance, adding methanol to prepare solution containing 0.5mg per 1ml as reference substance solution; performing thin-layer chromatography, namely sucking 1-2 mu l of each of a test solution, a reference medicinal material solution and a reference solution, respectively dropping the solutions on the same silica gel G thin-layer plate, developing by using ethyl acetate-formic acid-glacial acetic acid-water as a developing agent in a volume ratio of 15: 1: 2, taking out, drying in the air, spraying 10% sulfuric acid ethanol solution, heating at 105 ℃ until spots are clearly developed, and respectively inspecting under sunlight and 365nm ultraviolet lamps; in the chromatogram of the test solution, main spots or fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution; spots or fluorescent spots of the same color appear at the positions corresponding to the color spectrum of the reference substance;
the content determination items are as follows: controlling cryptotanshinone, tanshinone I and tanshinone II A The total amount of (A) and the content limit are as follows: every 1g contains Saviae Miltiorrhizae radix extract containing cryptotanshinone, tanshinone I and tanshinone II A Should not be less than 0.42mg in total.
2. The quality control method of the muscle and bone pain relieving pill according to claim 1, which is characterized in that: the content determination item is determined by adopting a liquid chromatography;
the chromatographic conditions are as follows: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent and acetonitrile-0.02% phosphoric acid solution as a mobile phase, wherein the detection wavelength is 270nm, and the sample injection amount is 10 mu l; theoretical plate number according to tanshinone II A Peak count should not be less than 20 000;
preparation of control solutions: collecting cryptotanshinone, tanshinone I and tanshinone II A Accurately weighing appropriate amount of reference substances, placing in brown measuring flask, adding methanol to obtain a solution containing cryptotanshinone and tanshinone II in each 1ml A 10. Mu g and 5 mu g of tanshinone I;
preparation of a test solution: taking a proper amount of the product, grinding, taking about 1.0g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of methanol, sealing the plug, weighing, placing on a water bath oscillator at 60 ℃ for 1 hour, cooling, weighing again, complementing the lost weight with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the product;
the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the test solution, injecting into a liquid chromatograph, and measuring;
calculated according to the dry product, each 1g contains cryptotanshinone, tanshinone I and tanshinone II A Should not be less than 0.42mg in total.
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