CN110464825B

CN110464825B - Rehmannia root potion pharmaceutical composition, preparation method and detection method

Info

Publication number: CN110464825B
Application number: CN201910804769.1A
Authority: CN
Inventors: 郁华军
Original assignee: Guizhou Jingcheng Pharmaceutical Co ltd
Current assignee: Guizhou Jingcheng Pharmaceutical Co ltd
Priority date: 2019-08-28
Filing date: 2019-08-28
Publication date: 2021-10-08
Anticipated expiration: 2039-08-28
Also published as: CN110464825A

Abstract

The invention relates to a rehmannia root potion pharmaceutical composition, a preparation method and a detection method thereof, wherein the pharmaceutical composition comprises the following components: the traditional Chinese medicine composition is prepared by water extraction, freeze drying and other methods, and solves the technical problems that in the prior art, decompression drying causes loss of morroniside, loganin and schisandrin, and dry paste powder is granular and hard; the invention determines the specific detection methods of qualitative identification, fingerprint spectrum and content measurement, a good detection method effectively reflects the quality of the material reference quality, and solves the technical problem that the quality detection method in the prior art is difficult to reflect the quality of the material reference quality.

Description

Rehmannia root potion pharmaceutical composition, preparation method and detection method

Technical Field

The invention relates to the field of pharmacy, and in particular relates to a rehmannia root potion pharmaceutical composition, a preparation method and a detection method.

Background

The drink of rehmannia is the 53 th prescription in the catalog of ancient classical famous prescriptions (first batch) published by the State administration of traditional Chinese medicine in 2018, 4 months. The rehmannia root potion comes from jin, Liu Jing Su (Huangdi Su Xuan Ming Lun Fang). The original prescription is as follows: cooked rehmannia root, morinda root (with the core removed), cornus officinalis, dendrobium, cistanche deserticola (soaked and baked in wine), prepared aconite root (processed), schisandra fruit, cinnamon bark, white poria cocos, ophiopogon root (with the core removed), calamus and polygala root (with the core removed) are respectively divided into equal parts. The right part is powder, three coins are taken each time, one cup and half water, five pieces of ginger, one jujube and mint are decocted to eight minutes, except for the time. The original one copy is 200-300 ml, and the original one money is used together with 3.75g, so the original dose is converted into the modern dose: prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, cistanche deserticola, prepared aconite root, schisandra chinensis, cinnamon, poria cocos, ophiopogon root, grassleaf sweelflag rhizome and polygala root each 1g, ginger 5g (cut into thick slices when using), Chinese date 4.6g (broken when using) and mint 1 g. According to the examination results, the decoction to eighth is the liquid level height, so the decoction method of the formula is as follows: adding 300-450 ml of water, and decocting to eighty percent (liquid level height). The composition has effects in nourishing kidney yin, invigorating kidney yang, inducing resuscitation and eliminating phlegm, and can be used for treating aphonia syndrome due to deficiency of lower-jiao and up-diffusion of phlegm, manifested by failure of tongue function, foot deficiency, dry mouth, thirst, cold foot, flushed face, and deep thready and weak pulse.

The rehmannia root drink has certain curative effect, but has the following defects: firstly, as the decocting end point is described as 'decocting to eighth', the liquid level height is 'eighth' according to the examination result, but when the extracting solution height is taken as the decocting end point, the extracting solution height needs to be measured in real time in the test process, so that the extracting solution height needs to be left from a fire source for a long time to cause discontinuous decocting, and under the conditions of a fixed heating mode (open fire) and fire control, the time node reaching the decocting end point extracting solution 'eighth' height is taken as the decocting time by utilizing the correlation between the extracting solution height change and the decocting time. ② the rehmannia potion is a powder boiling agent and is taken after dregs are removed in the original text, so the situations of dregs and silt exist. The third step of concentration is that the mass ratio of the fed amount to the concentrated solution is not determined, so that the dry powder is foamed and not melted, and the powder is not loosened, and the fourth step of concentration is that the wall is hung during the concentration process, the total extraction amount of the morroniside and the loganin and the total extraction amount of the schizandrol A are influenced, so that the concentration efficiency is low. Fifthly, drying under reduced pressure, wherein the dry paste powder is granular and hard, and the active ingredients of morroniside, loganin and schizandrol A are lost. Sixthly, the prepared rehmannia root is only definitely crushed but is not definitely crushed into coarse powder or fine powder, so that the phenomenon of difficult operation is caused during pretreatment. The existing quality detection method is difficult to reflect the quality of the material reference quality.

In order to solve the problems, the invention carries out material reference process and quality detection research on the rehmannia root drink. In the formula, iridoid glycoside components (morroniside and loganin) in fructus Corni as principal active ingredients, and lignanoid components (schizandrol A) in fructus Schisandrae as ministerial drug are related to their effectiveness, and are the basis of main drug effect substance, and can be used as index component of radix rehmanniae potion; therefore, the method takes the morroniside and the loganin in the monarch drug cornus pulp and the schisandrin in the ministerial drug schisandra fruit as content determination indexes, and inspects the controllability of the prescription process and quality. Decocting according to the method recorded in the list of classical famous prescriptions, in combination with the Ministry of health and the administration of Chinese medicine and drug administration of the State administration of traditional Chinese medicine and drug administration of medical institutions (the State administration of traditional Chinese medicine [ 2009 ] 3). Decocting in a casserole, concentrating the extracting solution, freeze-drying to prepare dry paste, and researching relevant parameters such as decocting time, filtering, concentrating, drying and the like in the material-based corresponding object preparation process by taking the paste yield, the total extraction amount of morroniside and loganin, the total extraction amount of schisandrin and a fingerprint as indexes so as to determine the rehmannia drink substance reference process. According to the control requirements of the conventional research on the quality standard and the preparation process of the product, the conventional quality standard control item of the product is established: including name, prescription, preparation method, properties, identification, diester alkaloid limit, extract, fingerprint, content, etc. Meanwhile, in order to ensure the storage stability of the product, the water content of the corresponding substance (dry paste powder) is brought into the standard. In order to better control the quality of all the components, the fingerprint spectrum of the product is established to ensure the stability of the preparation process of the product and provide a foundation for the development of subsequent compound preparations.

Disclosure of Invention

The invention aims to provide a rehmannia root potion pharmaceutical composition.

The invention aims to provide a preparation method of a rehmannia root potion pharmaceutical composition.

The invention aims to provide a detection method of a rehmannia root potion pharmaceutical composition.

The invention aims to provide application of a rehmannia root potion pharmaceutical composition in the aspects of nourishing kidney yin, tonifying kidney yang, inducing resuscitation and reducing phlegm.

The rehmannia root potion pharmaceutical composition provided by the invention comprises the following formula: 0.5-1.5 parts of prepared rehmannia root, 0.5-1.5 parts of morinda officinalis, 0.5-1.5 parts of cornus officinalis, 0.5-1.5 parts of dendrobe, 0.5-1.5 parts of cistanche salsa, 0.5-1.5 parts of sliced aconite, 0.5-1.5 parts of schisandra chinensis, 0.5-1.5 parts of cinnamon, 0.5-1.5 parts of poria cocos, 0.5-1.5 parts of radix ophiopogonis, 0.5-1.5 parts of rhizoma acori graminei, 0.5-1.5 parts of polygala tenuifolia, 4-6 parts of ginger, 3-6 parts of Chinese date and 0.5-1.5 parts of mint.

Preferably, the rehmannia root potion pharmaceutical composition disclosed by the invention comprises the following formula: 1 part of prepared rehmannia root, 1 part of morinda officinalis, 1 part of cornus officinalis, 1 part of dendrobium, 1 part of wine cistanche, 1 part of processed aconite, 1 part of schisandra chinensis, 1 part of cinnamon, 1 part of poria cocos, 1 part of radix ophiopogonis, 1 part of rhizoma acori graminei, 1 part of polygala tenuifolia, 5 parts of ginger, 4.6 parts of Chinese date and 1 part of mint.

The preparation method of the rehmannia root potion pharmaceutical composition comprises the following steps:

(1) weighing prepared rehmannia root, morinda officinalis, cornus officinalis, dendrobium, cistanche deserticola, processed aconite, schisandra chinensis, cinnamon, poria cocos, radix ophiopogonis, rhizoma acori graminei and polygala tenuifolia, and crushing into coarse powder for later use respectively; the ginger is cut into 2-4 mm thick slices when being used, and the Chinese date is broken when being used; standby;

(2) decocting, namely putting the pretreated medicines and the mint into a 600ml casserole, adding 200-400 ml of water, heating by open fire, covering the casserole for decocting, boiling by strong fire, and decocting by slow fire for 15-25 minutes to obtain a decoction;

(3) filtering the filtered decoction by using 200-300-mesh filter cloth under normal pressure while the decoction is hot to obtain filtrate;

(4) concentrating the concentrated filtrate under reduced pressure at the temperature of 60-70 ℃ and the pressure of-0.08 to-0.1 MPa until the mass ratio of the fed amount of the decoction pieces to the concentrated solution is 1g:3 g-1 g:4 g;

(5) freeze drying the concentrated solution by freeze drying under the following conditions: pre-freezing temperature: -20 to-48 ℃, cold trap temperature: -40 to-80 ℃, time: at least 30 minutes; and (3) drying: cold trap temperature: -60 to-70 ℃, vacuum degree: < 100Pa, lyophilization time: at least 17 hours to obtain dry paste powder;

(6) adding pharmaceutically acceptable adjuvants into the dry extract powder to make into pharmaceutically acceptable dosage form.

Preferably, the preparation method of the rehmannia root potion pharmaceutical composition comprises the following steps:

(1) weighing prepared rehmannia root, morinda officinalis, cornus officinalis, dendrobium, cistanche deserticola, prepared aconite, schisandra chinensis, cinnamon, poria cocos, radix ophiopogonis, rhizoma acori graminei and polygala tenuifolia, crushing into coarse powder, cutting ginger into 2-4 mm thick slices when in use, and breaking Chinese dates when in use;

(2) decocting the pretreated medicines and the mint in a 600ml casserole, adding 300ml of water, heating by open fire, covering and decocting, boiling by strong fire, and decocting by slow fire for 20 minutes to obtain a decoction;

(3) filtering the decoction with 300 mesh filter cloth under normal pressure; obtaining filtrate;

(4) concentrating the concentrated filtrate under reduced pressure at 70 ℃ and under the pressure of-0.08 to-0.1 MPa until the mass ratio of the fed amount of the decoction pieces to the concentrated solution is 1g:4 g;

(5) the conditions of freeze drying and freeze drying are as follows: pre-freezing temperature: -20 to-48 ℃, cold trap temperature: -40 to-80 ℃, time: at least 30 minutes; and (3) drying: cold trap temperature: -60 to-70 ℃, vacuum degree: < 100Pa, lyophilization time: at least 17 hours to obtain dry paste powder;

The pharmaceutical composition also comprises a pharmaceutically acceptable dosage form prepared by pharmaceutically acceptable auxiliary materials.

The dosage form of the invention is granules, capsules, tablets, pills, powder, injection, effervescent tablets, paste and the like.

The detection method of the pharmaceutical composition comprises qualitative identification, fingerprint spectrum and content measurement.

The specific detection method for qualitative identification, fingerprint spectrum and content measurement comprises the following steps:

[ IDENTIFICATION ] taking 4g of the product, adding 40-60 ml of water, dissolving by ultrasonic, shaking and extracting with dichloromethane for 1-3 times, 20-40 ml each time, shaking and extracting with n-butanol for 1-3 times, 20-40 ml each time, combining n-butanol solutions, washing with ammonia solution (1 → 10) for 1-3 times, 20-30 ml each time, discarding ammonia solution, evaporating n-butanol solution to dryness, adding 1ml of methanol to the residue to dissolve, as sample solution, taking 1g of dogwood control drug, adding 50ml of water to 2015, decocting for 15-25 minutes, filtering, shaking and extracting the filtrate with dichloromethane for 2 times, 30ml each time, discarding dichloromethane solution, preparing a control drug solution by the same method from "shaking and extracting 2 times with n-butanol", taking a morroniside control, a loganin control, adding 80% methanol to prepare a mixed solution containing 1mg each 1ml, as a control solution, testing by thin layer chromatography (0502 of pharmacopoeia of the four editions of the year), sucking 5 μ l of test solution, 1 μ l of control solution and 2 μ l of control solution, respectively dropping on the same silica gel GF254 thin layer plate, developing with chloroform-methanol (4: 1-3: 1) as developing agent, taking out, air drying, inspecting under ultraviolet lamp (254nm), and displaying fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatogram of the control solution and the chromatogram of the control solution;

(2) taking 1g of the product, adding 25-35 ml of 70% methanol, heating and refluxing for 0.5-1.5 hours, filtering, evaporating filtrate to dryness, dissolving residues by using 15-25 ml of 0.1mol/L sodium hydroxide solution, shaking and extracting for 1-3 times by using ethyl acetate, 15-25 ml each time, combining ethyl acetate solutions, evaporating to dryness, dissolving residues by adding 1ml of methanol to serve as a sample solution, taking 1g of 15vb control medicinal material or adding 50ml of water to the 1g of 15vb control medicinal material, decocting for 20 minutes, filtering, starting from the step of shaking and extracting for 2 times by using ethyl acetate, preparing a control medicinal material solution by the same method, taking a schizandrol methanol control, adding 80% methanol to prepare a solution containing 1mg of each 1ml serving as a control solution; performing thin-layer chromatography (according to the general rule of Chinese pharmacopoeia 2015) 0502 test, respectively dropping 5 μ l of test solution, 1 μ l of control solution and 2 μ l of control solution on the same silica gel GF254 thin-layer plate, developing with petroleum ether (60-90 deg.C) -ethyl acetate-methanol (5:3: 0.2-15: 5:2) as developing agent, 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;

[ fingerprinting ] measuring by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China);

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.3 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 254 nm; the number of theoretical plates is not less than 3000 calculated according to schizandrol A;

preparing reference solution by precisely weighing appropriate amount of schizandrol A reference substance, and adding 80% methanol to obtain solution containing 0.15mg per 1 ml;

preparing a test solution, precisely weighing 2.0g of the product, placing the product in a conical flask with a plug, precisely adding 20-30 ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power of 250W and frequency of 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, extracting the filtrate by using an ethyl acetate-n-butyl alcohol (1:1) mixed solution for 2 times, 25ml each time, combining the ethyl acetate-n-butyl alcohol (1:1) mixed solution, evaporating to dryness, adding methanol into residues to dissolve, transferring the residues to a 5ml measuring flask, diluting to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution;

the determination method comprises precisely sucking reference solution and sample solution 5 μ l each, injecting into liquid chromatograph, determining, and recording chromatogram;

the sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 14 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90. The control fingerprint is shown in FIG. 23.

[ CONTENT DETERMINATION ] the pulp of Corni fructus is determined by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia of China;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking acetonitrile as a mobile phase A, taking a 0.3% phosphoric acid solution as a mobile phase B, and carrying out gradient elution according to a specified mobile phase gradient elution program; the detection wavelength is 240 nm; the number of theoretical plates is not less than 3000 calculated according to the morroniside peak; the mobile phase gradient elution procedure was:

preparing a reference solution by precisely weighing a proper amount of morroniside and loganin, and adding 80% methanol to obtain a mixed solution containing 40 μ g of morroniside and 20 μ g of loganin per 1 ml;

preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 20-30 ml of 80% methanol, sealing the plug, weighing, ultrasonically treating the product for 30 minutes at the power of 250W and the frequency of 40kHz, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution;

the determination method comprises precisely sucking 5 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and determining;

the product contains morroniside (C) per 1g of Corni fructus₁₇H₂₆O₁₁) And loganin (C)₁₇H₂₆O₁₀) The total amount is 1.5 mg-3.0 mg;

fructus Schisandrae chinensis is measured by high performance liquid chromatography (0502 of the four ministerial rules of the design reside in the Chinese pharmacopoeia 2015);

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water (45:55) is used as a mobile phase; the detection wavelength is 250nm, and the number of theoretical plates is not less than 3000 calculated according to the schizandrol A peak;

preparing reference solution by precisely weighing appropriate amount of schizandrol A reference, and adding 80% methanol to obtain solution containing 20 μ g of schizandrol A per 1 ml;

preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 20-30 ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power of 250W and frequency of 40kHz) for 25-35 minutes, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution;

the determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and determining;

the product contains Schisandra chinensis fruit and schizandrol A (C) in an amount of 1g per dried product₂₄H₃₂O₇) The dosage is 0.4 mg-0.8 mg.

Preferably, the specific detection method for qualitative identification, fingerprint spectrum and content determination comprises the following steps:

[ IDENTIFICATION ] collecting 4g of the product, adding 50ml of water, dissolving by ultrasonic wave, extracting with dichloromethane for 2 times, 30ml each time, extracting with n-butanol for 2 times, 30ml each time, mixing n-butanol solutions, washing with ammonia solution (1 → 10) for 2 times, 25ml each time, discarding ammonia solution, evaporating n-butanol solution to dryness, dissolving residue with 1ml of methanol to obtain a sample solution, collecting 1g of Corni fructus control drug, adding 50ml of water, decocting for 20 minutes, filtering, extracting filtrate with dichloromethane for 2 times, 30ml each time, discarding dichloromethane solution, collecting water solution from the step of extracting with n-butanol for 2 times, preparing control drug solution by the same method, collecting morroniside control and loganin control, adding 80% methanol to obtain mixed solution each 1ml containing 1mg, as control solution, performing thin layer chromatography with 0502 in accordance with the four-part rule of the pharmacopoeia 2015 edition, sucking 5 μ l of sample solution, 1 μ l of control solution, and 2 μ l of control solution, respectively dropping on the same silica gel GF254 thin layer plate, developing with chloroform-methanol (3:1) as developing agent, taking out, air drying, and inspecting under 254nm ultraviolet lamp to show fluorescent spots of the same color in the sample chromatogram at the positions corresponding to the control chromatogram and the control chromatogram;

(2) taking 1g of the product, adding 30ml of 70% methanol, heating and refluxing for 1 hour, filtering, evaporating filtrate to dryness, dissolving residues by using 20ml of 0.1mol/L sodium hydroxide solution, shaking and extracting for 2 times by using ethyl acetate, 20ml each time, combining ethyl acetate solutions, evaporating to dryness, dissolving residues by adding 1ml of methanol to serve as a test solution, taking 1g of schisandra chinensis contrast medicinal material, adding 50ml of water, decocting for 20 minutes, filtering, preparing the contrast medicinal material solution by the same method from the 'shaking and extracting for 2 times by using ethyl acetate', further taking a schisandrin contrast product, adding 80% methanol to prepare a solution containing 1mg of schisandra chinensis per 1ml, and taking the contrast product solution; performing thin-layer chromatography test according to 0502 of general Law of Chinese pharmacopoeia 2015, sucking 5 μ l of sample solution, 1 μ l of control solution, and 2 μ l of control solution, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (60-90 deg.C) -ethyl acetate-methanol (5:3:0.2) 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;

[ fingerprinting ] is determined according to 0512 of the four Ministry rules of the Chinese pharmacopoeia 2015 edition of high performance liquid chromatography;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.3 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the procedures specified in the following table; the detection wavelength is 254 nm;

the number of theoretical plates is not less than 3000 calculated according to schizandrol A;

preparing a test solution, precisely weighing 2.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, treating for 30 minutes by using ultrasound (power is 250W and frequency is 40kHz), cooling, weighing again, complementing the weight loss by using 80% methanol, shaking up, filtering, extracting the filtrate by using an ethyl acetate-n-butyl alcohol (1:1) mixed solution for 2 times, each time is 25ml, combining the ethyl acetate-n-butyl alcohol (1:1) mixed solution, evaporating to dryness, adding methanol into residues for dissolving, transferring the residues to a 5ml measuring flask, diluting to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution;

a chromatographic peak with the same retention time as that of a chromatographic peak of a reference substance is required to be presented in the sample fingerprint, the sample chromatogram is basically consistent with the reference fingerprint and has 14 corresponding common peaks, the similarity is calculated by the common peaks according to a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint is not less than 0.90; the control fingerprint is shown in FIG. 23.

[ MEASUREMENT ] Corni fructus is measured by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia of China);

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.3 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 240 nm; the number of theoretical plates is not less than 3000 calculated according to the morroniside peak;

preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power of 250W and frequency of 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss by 80% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the product;

preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, ultrasonically treating the product for 30 minutes at the frequency of 40kHz and the power of 250W, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product;

The weight parts may be those known in the medical field such as μ g, mg, g, kg, etc.

The pharmaceutically acceptable auxiliary material refers to a conventional medicine carrier in the field of pharmacy, and is selected from one or more of a filling agent, a lubricating agent, an adhesive, a disintegrating agent, a surfactant or a flavoring agent.

Wherein the filler is selected from dextrin, starch, sorbitol, lactose, mannitol, xylitol, microcrystalline cellulose, etc.;

the adhesive is selected from cellulose derivatives, vinyl acetate resin, polyvinyl alcohol, gelatin, alginate and the like;

the disintegrating agent is selected from dry starch, sodium bicarbonate, low-substituted cellulose, microcrystalline cellulose, sodium carboxymethyl starch, polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose or croscarmellose sodium and the like;

the lubricant is selected from stearic acid, polyethylene glycol, calcium carbonate, sodium bicarbonate, silicon dioxide, talcum powder or magnesium stearate and the like;

the surfactant is selected from sodium dodecyl benzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, sorbitan fatty acid or polysorbate (Tween) and the like;

the correctant is selected from glycerol, sorbitol, mannitol, aspartame, sucralose, fructus Citri Limoniae, fructus Foeniculi, oleum Menthae Dementholatum, etc.

The dosage form of the invention is granules, capsules, tablets, pills, powder, injection, effervescent tablets and paste.

The medicine composition is applied to the aspects of nourishing kidney yin, tonifying kidney yang, inducing resuscitation and reducing phlegm. It is especially suitable for treating aphonia due to deficiency of lower-jiao and up-diffusion of phlegm, failure of tongue to speak, failure of foot to be used, dry mouth without desire for drinking, cold foot, reddish face, and deep, thready and weak pulse.

Has the advantages that:

1. compared with the prior art, the invention has the following beneficial effects:

in the 53 th prescription rehmannia potion in the ancient classic famous prescription catalog (first batch) published by the State administration of traditional Chinese medicine in 2018 in 4 months, the following defects exist: firstly, as the decocting end point in the prescription original text is described as 'decocting to eight minutes', the liquid level height is 'eight minutes' according to the examination result, but when the extracting solution height is taken as the decocting end point, the extracting solution height needs to be measured in real time in the test process, so that the operation of leaving the fire source for a long time is needed, and the decocting is not continuous. ② the rehmannia potion is a powder boiling agent and is taken after dregs are removed in the original text, so the situations of dregs and silt exist. The third step of concentration is that the mass ratio of the fed amount to the concentrated solution is not determined, so that the dry powder is foamed and not melted, and the powder is not loosened, and the fourth step of concentration is that the wall is hung during the concentration process, the total extraction amount of the morroniside and the loganin and the total extraction amount of the schizandrol A are influenced, so that the concentration efficiency is low. Fifthly, drying under reduced pressure, wherein the dry paste powder is granular and hard, and the active ingredients of morroniside, loganin and schizandrol A are lost. Sixthly, the prepared rehmannia root is only definitely crushed but is not definitely crushed into coarse powder or fine powder, so that the phenomenon of difficult operation is caused during pretreatment. The existing quality detection method is difficult to reflect the quality of the material reference quality.

The invention overcomes the defects and solves the corresponding technical problems, and specifically comprises the following steps:

under the conditions of fixed heating mode (open fire) and fire control, the invention utilizes the correlation between the height change of the extracting solution and the decocting time to reach the time node of the height of 'eight minutes' of the extracting solution at the end point of decocting as the decocting time, and the preferred decocting time is determined to be 20 minutes; the technical problem of discontinuous decoction caused by the fact that the height of the extracting solution needs to be measured in real time in the test process and the extracting solution needs to leave a fire source for operation for a long time when the height of the extracting solution is taken as the decoction endpoint in the original prescription of the prior art is solved;

the invention defines that the decoction is preferably filtered by 300-mesh filter cloth when the decoction is hot and at normal pressure, and solves the technical problems of dregs and silt caused by removal of dregs in the prior art of powder boiling preparation;

the invention defines that the mass ratio of the feeding amount of the decoction pieces to the concentrated solution is preferably 1g to 4g, the dry powder is melted and unfoamed, and the powder is loose, thereby solving the technical problems that the dry powder is not foamed and melted and the powder is not loose because the mass ratio of the feeding amount of the concentrated solution to the concentrated solution is not defined in the prior art;

the invention clearly defines the preferable concentration conditions as follows: the concentration is carried out under the conditions that the temperature is 70 ℃ and the pressure is-0.08 to-0.1 MPa, the wall is not hung in the concentration process, the total extraction amount of the morroniside and the loganin and the total extraction amount of the schizandrol A are not influenced, and the concentration efficiency is high; the technical problems that the concentration temperature is not determined, wall hanging occurs in the concentration process, the total extraction amount of the morroniside and the loganin and the total extraction amount of the schizandrol A are influenced, and the concentration efficiency is low in the prior art are solved;

the invention adopts freeze drying and defines the freeze drying conditions as follows: pre-freezing temperature: -20 to-48 ℃, cold trap temperature: -40 to-80 ℃, time: at least 30 minutes; and (3) drying: cold trap temperature: -60 to-70 ℃, vacuum degree: < 100Pa, lyophilization time: at least 17 hours to obtain dry paste powder; compared with the method before freeze drying, the total amount of the morroniside and the loganin is not greatly different, the schizandrol A is slightly lost, and the dried paste is loose in powder state; solves the technical problems that the morroniside, loganin and schizandrol A are lost, and dry paste powder is granular and harder in the prior art when the pressure reduction drying (60 ℃) is compared with the prior art.

The invention definitely pulverizes the prepared rehmannia root into coarse powder, and solves the technical problem that the operation is difficult to be carried out during pretreatment because the coarse powder or the fine powder is not definitely pulverized in the prior art.

The invention determines the specific detection methods of qualitative identification, fingerprint and content measurement, and the good detection method effectively reflects the quality of the material reference quality, thereby solving the technical problem that the quality detection method in the prior art is difficult to reflect the quality of the material reference quality.

2. The thin-layer chromatography method established by the invention is identified, and the result durability is good and the main spot is clear through tests on silica gel G thin-layer plates of different manufacturers and influence factors such as different development temperatures, different relative humidities and the like.

3. The liquid phase identification method established by the invention inspects the durability of different chromatographic columns at different column temperatures and different flow rates, and the result shows that the echinacoside peak in the chromatogram under different conditions is sharp and symmetrical, the separation degree is good, and the negative is free of interference, thus the method has good durability to different flow rates, different column temperatures and different chromatographic columns.

4. The invention establishes an integral quality determination method mainly based on a fingerprint, and solves the technical problem that the quality of the reference quality of a substance is difficult to reflect by the qualitative identification and the quantitative analysis of a single index component.

5. The established fingerprint spectrum is shown by methodology investigation, and has good specificity, integrity, instrument precision and repeatability; the measurement results are basically consistent under the conditions of different flow rates, different column temperatures, different chromatographic columns, different acid proportions and different instruments, the common peak peaks in the chromatogram are sharp and symmetrical, the separation degree is good, the similarity is not lower than 0.90, the fingerprint of the sample shows 14 main chromatographic peaks and corresponds to the characteristic peak in the reference substance chromatographic peak of the reference substance, and the method is proved to have good durability to different flow rates, different column temperatures, different chromatographic columns, different acidity and different instruments; the solution was stable well within 48 hours.

6. The high performance liquid chromatography established by the invention has specificity, and peak purity inspection shows that the purity of the morroniside and loganin chromatographic peaks meets the requirement; the examination of the linear relation shows that the loganin has good linearity in the range of 0.02036 mug-2.0360 mug; the precision of the instrument is good through an instrument precision test instrument; the repeatability is good; the test solution has good stability and accuracy within 48 hours.

Drawings

FIG. 1 shows the fingerprint of the standard process for verifying the quality of drink with rehmannia root (S1. extractive solution 1, S2. extractive solution 2, S3. extractive solution 3, S4. concentrated solution 1, S5. concentrated solution 2, S6. concentrated solution 3, S7. dried paste powder 1, S8. dried paste powder 2, S9. dried paste powder 3, S10. Chinese magnoliavine fruit decoction pieces, S11. polygala root decoction pieces, S12. dogwood fruit decoction pieces, S13. cistanche decoction pieces, S14. mint decoction pieces)

FIG. 2 identification of fructus Corni thin layer [ silica gel GF254 plate (100 x 100mm) RT:23.5 deg.C RH: 37%; 1. loganin reference substance solution (batch No. 11640-201707, China food and drug testing institute, 5. mu.l), 2, morroniside reference substance solution (batch No. 111998-201702, China food and drug testing institute, 2. mu.l), 3, dogwood reference drug solution (batch No. 121495-201303, China food and drug testing institute, 2. mu.l), 4, test sample solution (D-181120-02, 2. mu.l)

5. Test solution (D-181120-02, 5. mu.l), 6, test solution (D-181120-02, 10. mu.l), 7, pulp of fructus Corni negative sample (D-181126-Y-SZY-02, 10. mu.l)

FIG. 3 is a schematic diagram of Schisandra chinensis thin layer identification (Qingdao silica gel GF254 plate (100 x 100mm) RT:23.4 ℃ RH: 39%; 1. schisandrin solution (batch No. 110857-

Figure 4 finger print searching

FIG. 5 shows the method for preparing the test substance [1, 80% methanol; 2. extracting with ethyl acetate by shaking; 3. ethyl acetate-n-butanol (1:1) shaking extraction ]

FIG. 6 HPLC comparison chart of herb attribution

FIG. 7 HPLC comparison chart for confirming related components

FIG. 8 UPLC-UV chromatogram and UPLC-MS total ion flow chart of rehmanniae radix drinker reference test solution

FIG. 9 HPLC comparative chromatogram of test sample, control sample and blank solvent

FIG. 10 HPLC finger print for precision test of instrument

FIG. 11 stability test HPLC fingerprint

FIG. 12 repeatability test HPLC fingerprint

FIG. 13 comparative graph of different flow rates

FIG. 14 comparison of durability at different column temperatures

FIG. 15 comparative graph of durability of different acid ratios

FIG. 16 comparison of durability of different instruments

FIG. 17 common mode of fingerprint of drink substance of rehmannia glutinosa Libosch-1 [ R. reference map, S2.D-181212-01, S3.D-181212-03, S4.D-181212-05, S5.D-181212-07, S6.D-181213-09, S7.D-181213-11, S8.D-181213-13, S9.D-181213-15, S10.D-181214-17, S11.D-181214-19, S12.D-181214-21, S13.D-181214-23, S14.D-181215-25, S15.D-181215-27, S16.D-181215-29]

FIG. 18 common mode figure of DIHUANGDINGYINZI material reference fingerprint map-2 [ R. reference map, S2.D-181212-03, S3.D-181212-04, S4.D-181212-06, S5.D-181212-08, S6.D-181213-10, S7.D-181213-12, S8.D-181213-14, S9.D-181213-16, S10.D-181214-18, S11.D-181214-20, S12.D-181214-22, S13.D-181214-24, S14.D-181215-26, S15.D-181215-28, S16.D-181215-30]

Figure 19 control fingerprint [14 consensus peaks peak 1: gallic acid, peak 4: loganin, peak 5: echinacoside, peak 7: 3, 6' -erucyl sucrose, Peak 9: rosmarinic acid, peak 11 (S): schisandrin A)

FIG. 20 determination of morroniside and loganin content

FIG. 21 HPLC comparative chart of control, test sample, negative sample and blank solvent

FIG. 22 full wavelength scan of schizandrin A

Fig. 23 compares the fingerprint (determination of the range of key mass attributes corresponding to the physical object), [14 common peaks in peak 1: gallic acid, peak 4: loganin, peak 5: echinacoside, peak 7: 3, 6' -erucyl sucrose, Peak 9: rosmarinic acid, peak 11 (S): schisandrin A

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples.

Example 1 recipe: 0.5g of prepared rehmannia root, 0.5g of morinda officinalis, 0.5g of cornus officinalis, 0.5g of dendrobium, 0.5g of wine cistanche, 0.5g of prepared aconite root, 0.5g of schisandra chinensis, 0.5g of cinnamon, 0.5g of poria cocos, 0.5g of radix ophiopogonis, 0.5g of rhizoma acori graminei, 0.5g of polygala tenuifolia, 4g of ginger, 3g of Chinese date and 0.5g of mint.

The preparation method comprises the following steps: (1) weighing prepared rehmannia root, morinda officinalis, cornus officinalis, dendrobium, cistanche deserticola, processed aconite, schisandra chinensis, cinnamon, poria cocos, radix ophiopogonis, rhizoma acori graminei and polygala tenuifolia, and crushing into coarse powder for later use respectively; the ginger is cut into 2-4 mm thick slices when being used, and the Chinese date is broken when being used; standby;

(2) decocting the pretreated medicines and the mint in a 600ml casserole, adding 200ml of water, heating with open fire, covering and decocting, boiling with strong fire, and decocting with slow fire for 15 minutes to obtain a decoction;

(3) filtering the decoction with 200 mesh filter cloth under normal pressure;

(4) concentrating the concentrated filtrate under reduced pressure at 60 deg.C and-0.08 MPa until the mass ratio of the decoction pieces to the concentrated solution is 1g:3 g;

(6) adding 1/10 starch into the dry extract powder, mixing, and granulating to obtain granule.

Example 2 recipe: 0.5g of prepared rehmannia root, 0.5g of morinda officinalis, 0.5g of cornus officinalis, 0.5g of dendrobium, 0.5g of wine cistanche, 0.5g of prepared aconite root, 0.5g of schisandra chinensis, 0.5g of cinnamon, 0.5g of poria cocos, 0.5g of radix ophiopogonis, 0.5g of rhizoma acori graminei, 0.5g of polygala tenuifolia, 4g of ginger, 3g of Chinese date and 0.5g of mint.

(2) decocting the pretreated medicines and the mint in a 600ml casserole, adding 400ml of water, heating with open fire, covering and decocting, boiling with strong fire, and decocting with slow fire for 25 minutes to obtain a decoction;

(6) adding starch of 1/12 into the dry extract powder, mixing, pulverizing into fine powder, and encapsulating to obtain capsule.

Example 3 recipe: 0.5g of prepared rehmannia root, 0.5g of morinda officinalis, 0.5g of cornus officinalis, 0.5g of dendrobium, 0.5g of wine cistanche, 0.5g of prepared aconite root, 0.5g of schisandra chinensis, 0.5g of cinnamon, 0.5g of poria cocos, 0.5g of radix ophiopogonis, 0.5g of rhizoma acori graminei, 0.5g of polygala tenuifolia, 4g of ginger, 3g of Chinese date and 0.5g of mint.

The preparation method comprises the following steps: (1) weighing prepared rehmannia root, morinda officinalis, cornus officinalis, dendrobium, cistanche deserticola, prepared aconite, schisandra chinensis, cinnamon, poria cocos, radix ophiopogonis, rhizoma acori graminei and polygala tenuifolia, crushing into coarse powder, cutting ginger into 2-4 mm thick slices when in use, and breaking Chinese dates when in use;

Example 4 prescription: 1.5g of prepared rehmannia root, 1.5g of morinda officinalis, 1.5g of cornus officinalis, 1.5g of dendrobium, 1.5g of wine cistanche, 1.5g of prepared aconite root, 1.5g of schisandra fruit, 1.5g of cinnamon, 1.5g of poria cocos, 1.5g of ophiopogon root, 1.5g of grassleaf sweelflag rhizome, 1.5g of polygala root, 6g of ginger, 6g of Chinese date and 1.5g of mint.

(3) filtering the decoction with 200 mesh filter cloth under normal pressure;

Example 5 recipe: 1.5g of prepared rehmannia root, 1.5g of morinda officinalis, 1.5g of cornus officinalis, 1.5g of dendrobium, 1.5g of wine cistanche, 1.5g of prepared aconite root, 1.5g of schisandra fruit, 1.5g of cinnamon, 1.5g of poria cocos, 1.5g of ophiopogon root, 1.5g of grassleaf sweelflag rhizome, 1.5g of polygala root, 6g of ginger, 6g of Chinese date and 1.5g of mint.

Example 6 prescription: 1.5g of prepared rehmannia root, 1.5g of morinda officinalis, 1.5g of cornus officinalis, 1.5g of dendrobium, 1.5g of wine cistanche, 1.5g of prepared aconite root, 1.5g of schisandra fruit, 1.5g of cinnamon, 1.5g of poria cocos, 1.5g of ophiopogon root, 1.5g of grassleaf sweelflag rhizome, 1.5g of polygala root, 6g of ginger, 6g of Chinese date and 1.5g of mint.

Example 7 recipe: 1g of prepared rehmannia root, 1g of morinda officinalis, 1g of cornus officinalis, 1g of dendrobium, 1g of cistanche salsa, 1g of processed aconite, 1g of schisandra chinensis, 1g of cinnamon, 1g of poria cocos, 1g of radix ophiopogonis, 1g of rhizoma acori graminei, 1g of polygala tenuifolia, 5g of ginger, 4.6g of Chinese date and 1g of mint.

(3) filtering the decoction with 200 mesh filter cloth under normal pressure;

Example 8 prescription: 1g of prepared rehmannia root, 1g of morinda officinalis, 1g of cornus officinalis, 1g of dendrobium, 1g of cistanche salsa, 1g of processed aconite, 1g of schisandra chinensis, 1g of cinnamon, 1g of poria cocos, 1g of radix ophiopogonis, 1g of rhizoma acori graminei, 1g of polygala tenuifolia, 5g of ginger, 4.6g of Chinese date and 1g of mint.

Example 9 prescription: 1g of prepared rehmannia root, 1g of morinda officinalis, 1g of cornus officinalis, 1g of dendrobium, 1g of cistanche salsa, 1g of processed aconite, 1g of schisandra chinensis, 1g of cinnamon, 1g of poria cocos, 1g of radix ophiopogonis, 1g of rhizoma acori graminei, 1g of polygala tenuifolia, 5g of ginger, 4.6g of Chinese date and 1g of mint.

Example 10

Prescription: 1g of prepared rehmannia root, 1g of morinda officinalis, 1g of cornus officinalis, 1g of dendrobium, 1g of cistanche salsa, 1g of processed aconite, 1g of schisandra chinensis, 1g of cinnamon, 1g of poria cocos, 1g of radix ophiopogonis, 1g of rhizoma acori graminei, 1g of polygala tenuifolia, 5g of ginger, 4.6g of Chinese date and 1g of mint.

Preparation method

(6) adding the dry extract powder into 1/7 dextrin, mixing, drying, and making into pill.

Example 11

(6) adding 1/6 dextrin, tabletting, and drying to obtain tablet.

Example 12

(6) adding 1/12 sodium bicarbonate, granulating, tabletting, and making into effervescent tablet.

Example 13 prescription: 1g of prepared rehmannia root, 1g of morinda officinalis, 1g of cornus officinalis, 1g of dendrobium, 1g of cistanche salsa, 1g of processed aconite, 1g of schisandra chinensis, 1g of cinnamon, 1g of poria cocos, 1g of radix ophiopogonis, 1g of rhizoma acori graminei, 1g of polygala tenuifolia, 5g of ginger, 4.6g of Chinese date and 1g of mint.

(6) pulverizing the dry extract powder, and mixing with 0.8 times of ethanol to obtain unguent.

Example 14 prescription: 1g of prepared rehmannia root, 1g of morinda officinalis, 1g of cornus officinalis, 1g of dendrobium, 1g of cistanche salsa, 1g of processed aconite, 1g of schisandra chinensis, 1g of cinnamon, 1g of poria cocos, 1g of radix ophiopogonis, 1g of rhizoma acori graminei, 1g of polygala tenuifolia, 5g of ginger, 4.6g of Chinese date and 1g of mint.

(6) pulverizing the dry extract powder, adding 6 times of water for injection, filtering, and sterilizing to obtain injection.

EXAMPLES 1-14 assays according to any of the assays of examples 15-17

Example 15 detection method

[ IDENTIFICATION ] collecting 4g of the product, adding 40ml of water, dissolving by ultrasonic wave, extracting with dichloromethane for 1 time, 20ml each time, extracting with n-butanol for 1 time, 2ml each time, mixing n-butanol solutions, washing with ammonia solution (1 → 10) for 1 time, 20ml each time, discarding ammonia solution, evaporating n-butanol solution, dissolving residue with 1ml of methanol to obtain a sample solution, collecting 1g of Corni fructus control drug, adding 50ml of water, decocting for 15 minutes, filtering, extracting filtrate with dichloromethane for 2 times, 30ml each time, discarding dichloromethane solution, collecting water solution from the step of extracting with n-butanol for 2 times, preparing control drug solution by the same method, collecting morroniside control and loganin control, adding 80% methanol to obtain mixed solution each 1ml containing 1mg, using as control solution, testing by thin layer chromatography (Pharmacopeia 2015, four ministerial Processations 0502), sucking 5 μ l of test solution, 1 μ l of control solution, and 2 μ l of control solution, respectively dropping on the same silica gel GF254 thin layer plate, developing with chloroform-methanol (4: 1-3: 1) as developing agent, taking out, air drying, inspecting under ultraviolet lamp (254nm), and displaying fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatogram of the control solution and the chromatogram of the control solution.

(2) Taking 1g of the product, adding 25ml of 70% methanol, heating and refluxing for 0.5 hour, filtering, evaporating filtrate to dryness, dissolving residues by using 15ml of 0.1mol/L sodium hydroxide solution, shaking and extracting for 1 time by using ethyl acetate, 15ml each time, combining ethyl acetate solutions, evaporating to dryness, dissolving residues by adding 1ml of methanol to serve as a test solution, taking 1g of schisandra chinensis contrast medicinal material, adding 50ml of water, decocting for 20 minutes, filtering, performing the same method on the contrast medicinal material solution from the step of shaking and extracting for 2 times by using ethyl acetate, taking a schizandrol A contrast product, adding 80% methanol to prepare a solution containing 1mg in each 1ml, and taking the contrast product solution; performing thin-layer chromatography (0502 of the general Law of the national pharmacopoeia 2015), respectively dropping 5 μ l of a test solution, 1 μ l of a reference medicinal material solution and 2 μ l of a reference solution on the same silica gel GF254 thin-layer plate, developing with petroleum ether-ethyl acetate-methanol (5:3: 0.2-15: 5:2) at 60-90 deg.C as a developing agent, taking out, air drying, and inspecting under an ultraviolet lamp (254 nm); 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.

[ fingerprinting ] was measured by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).

preparation of reference solution A proper amount of schizandrol A reference substance is precisely weighed, and 80% methanol is added to make into solution containing 0.15mg per 1 ml.

Preparing a test solution, precisely weighing 2.0g of the product, placing the product in a conical flask with a plug, precisely adding 20-30 ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, extracting the filtrate by using an ethyl acetate-n-butyl alcohol mixed solution in a ratio of 1:1 for 2 times, 25ml each time, combining the ethyl acetate-n-butyl alcohol (1:1) mixed solution, evaporating to dryness, dissolving the residue by adding methanol, transferring to a 5ml volumetric flask, diluting to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution.

The determination method comprises precisely sucking reference solution and sample solution 5 μ l each, injecting into liquid chromatograph, determining, and recording chromatogram.

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking acetonitrile as a mobile phase A and 0.3 percent phosphoric acid solution as a mobile phase B, and carrying out gradient elution according to a specified gradient elution program; the detection wavelength is 240 nm; the number of theoretical plates is not less than 3000 calculated according to the morroniside peak; the mobile phase gradient elution procedure was:

preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 20-30 ml of 80% methanol, sealing the plug, weighing, performing ultrasonic treatment (power of 250W and frequency of 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the product;

preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 20-30 ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 25 minutes, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution;

Example 16 detection method

[ IDENTIFICATION ] A thin-layer chromatography (thin-layer chromatography) (according to the pharmacopoeia of the four ministry of the pharmacopoeia of China 0502, the four ministry of the Japan) is performed by taking 4g of the product, adding 60ml of water, dissolving by ultrasonic, extracting 3 times by shaking dichloromethane, 40ml each time, extracting 1-3 times by shaking n-butanol, 20-40 ml each time, combining n-butanol solutions, washing 3 times by ammonia solution (1 → 10), 30ml each time, discarding ammonia solution, evaporating n-butanol solution, dissolving the residue by adding 1ml of methanol, using as a sample solution, taking 1g of a dogwood fruit control drug, adding 50ml of water, decocting for 25 minutes, filtering, extracting the filtrate by shaking dichloromethane for 2 times, 30ml each time, discarding dichloromethane solution, preparing a control solution by the same method from "extracting 2 times by shaking n-butanol", taking a morroniside control and a loganin control, adding 80% methanol to prepare a mixed solution each 1mg per 1ml, using as a control solution, sucking 5 μ l of test solution, 1 μ l of control solution and 2 μ l of control solution, respectively dropping on the same silica gel GF254 thin layer plate, developing with chloroform-methanol (4: 1-3: 1) as developing agent, taking out, air drying, inspecting under 254nm ultraviolet lamp, and displaying fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatogram of the control solution and the control solution.

(2) Taking 1g of the product, adding 25-35 ml of 70% methanol, heating and refluxing for 1.5 hours, filtering, evaporating filtrate to dryness, dissolving residues by using 15-25 ml of 0.1mol/L sodium hydroxide solution, shaking and extracting by using ethyl acetate for 3 times, 25ml each time, combining ethyl acetate solutions, evaporating to dryness, adding 1ml of methanol to dissolve residues to serve as a sample solution, taking 1g of a schisandra chinensis control medicinal material, adding 50ml of water, decocting for 20 minutes, filtering, starting from the step of shaking and extracting by using ethyl acetate for 2 times, carrying out the same method to obtain a control medicinal material solution, further taking a schizandrol A control product, adding 80% methanol to prepare a solution containing 1mg of each 1ml, and taking the solution as a control solution; performing thin-layer chromatography (0502 of the general Law of the national pharmacopoeia 2015), respectively dropping 5 μ l of a test solution, 1 μ l of a reference medicinal material solution and 2 μ l of a reference solution on the same silica gel GF254 thin-layer plate, developing with petroleum ether-ethyl acetate-methanol (5:3: 0.2-15: 5:2) at 60-90 deg.C as a developing agent, taking out, air drying, and inspecting under an ultraviolet lamp (254 nm); 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.

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.3 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 254 nm;

Preparing a test solution, precisely weighing 2.0g of the product, placing the product in a conical flask with a plug, precisely adding 30ml of 80% methanol, sealing the plug, weighing, performing ultrasonic treatment (power is 250W and frequency is 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss by 80% methanol, shaking up, filtering, extracting the filtrate by using an ethyl acetate-n-butyl alcohol mixed solution in a ratio of 1:1 for 2 times, 25ml each time, combining the ethyl acetate-n-butyl alcohol (1:1) mixed solution, evaporating to dryness, adding methanol to dissolve residues, transferring the residues to a 5ml measuring flask, diluting to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution.

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking acetonitrile as a mobile phase A, taking a 0.3% phosphoric acid solution as a mobile phase B, and carrying out gradient elution according to a specified mobile phase gradient elution program; the detection wavelength is 240 nm; the number of theoretical plates is not less than 3000 calculated according to the morroniside peak; the gradient elution procedure was:

preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 20-30 ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 25-35 minutes, cooling, weighing again, complementing the lost weight with 80% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution;

the product contains Schisandra chinensis fruit and schizandrol A (C) in an amount of 1g per dried product₂₄H₃₂O₇) The counting is carried out by the following steps of,should be between 0.4mg and 0.8 mg.

Example 17 detection method

[ IDENTIFICATION ] collecting 4g of the product, adding 50ml of water, dissolving by ultrasonic wave, extracting with dichloromethane for 2 times, each for 30ml, extracting with n-butanol for 2 times, each for 30ml, mixing n-butanol solutions, washing with ammonia solution (1 → 10) for 2 times, each for 25ml, discarding ammonia solution, evaporating n-butanol solution, dissolving residue with 1ml of methanol to obtain a sample solution, collecting 1g of Corni fructus control drug, adding 50ml of water, decocting for 20 minutes, filtering, extracting filtrate with dichloromethane for 2 times, each for 30ml, discarding dichloromethane solution, collecting water solution from the step of extracting with n-butanol for 2 times, preparing control drug solution by the same method, collecting morroniside control and loganin control, adding 80% methanol to obtain mixed solution each 1ml containing 1mg, and using as control solution, testing by thin layer chromatography (2015 in accordance with 0502 of the four ministerial rules of pharmacopoeia 2015 of China), sucking sample solution 5 μ l, control solution 1 μ l, and control solution 2 μ l, respectively dropping on the same silica gel GF₂₅₄Developing with chloroform-methanol (3:1) as developing agent, taking out, air drying, inspecting under 254nm ultraviolet lamp, and displaying fluorescent spots of the same color in the chromatogram of the sample at the positions corresponding to the chromatogram of the reference material and the chromatogram of the reference material.

(2) Taking 1g of the product, adding 30ml of 70% methanol, heating and refluxing for 1 hour, filtering, evaporating filtrate to dryness, dissolving residues by using 20ml of 0.1mol/L sodium hydroxide solution, shaking and extracting for 2 times by using ethyl acetate, 20ml each time, combining ethyl acetate solutions, evaporating to dryness, dissolving residues by adding 1ml of methanol to serve as a test solution, taking 1g of schisandra chinensis contrast medicinal material, adding 50ml of water, decocting for 20 minutes, filtering, preparing the contrast medicinal material solution by the same method from the 'shaking and extracting for 2 times by using ethyl acetate', further taking a schisandrin contrast product, adding 80% methanol to prepare a solution containing 1mg of schisandra chinensis per 1ml, and taking the contrast product solution; performing thin layer chromatography (0502 of the general Law of the national pharmacopoeia 2015), sucking 5 μ l of sample solution, 1 μ l of control solution, and 2 μ l of control solution, respectively dropping on the same silica gel GF₂₅₄Spreading petroleum ether (60-90 deg.C) -ethyl acetate-methanol (5:3:0.2) as developing agent on the thin layer plate, taking out, and air dryingDrying, and inspecting under ultraviolet lamp (254 nm); 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.

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.3 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength was 254 nm. The number of theoretical plates is not less than 3000 calculated according to schizandrol A;

preparing a test solution, precisely weighing 2.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, treating with ultrasound (power 250W and frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 80% methanol, shaking up, filtering, extracting the filtrate with an ethyl acetate-n-butanol (1:1) mixed solution for 2 times, 25ml each time, combining the ethyl acetate-n-butanol (1:1) mixed solution, evaporating to dryness, dissolving the residue with methanol, transferring to a 5ml measuring flask, diluting with methanol to scale, shaking up, filtering, and taking the subsequent filtrate;

[ CONTENT DETERMINATION ] Corni fructus is determined by high performance liquid chromatography (0512 in the four Ministry of communications in 2015 pharmacopoeia of China).

preparation of reference solution A proper amount of morroniside and loganin reference substances are precisely weighed, and 80% methanol is added to prepare a mixed solution containing 40 μ g of morroniside and 20 μ g of loganin per 1 ml.

Preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power of 250W and frequency of 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 80% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution.

The determination method comprises precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

The product contains morroniside (C) per 1g of Corni fructus₁₇H₂₆O₁₁) And loganin (C)₁₇H₂₆O₁₀) The total amount is 1.5 mg-3.0 mg.

Fructus Schisandrae chinensis is determined by high performance liquid chromatography (0502 of the four ministerial rules of the book 2015 pharmacopoeia).

preparation of control solution A proper amount of schizandrol A control is precisely weighed, and 80% methanol is added to make into solution containing 20 μ g per 1 ml.

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.

To further verify the feasibility of the present invention, the inventors performed a series of experiments.

Experimental example 1: pretreatment study

As the rehmannia root potion is a boiling powder, twelve flavors of prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, cistanche deserticola, prepared aconite root, schisandra chinensis, cinnamon, tuckahoe, radix ophiopogonis, rhizoma acori graminei and polygala tenuifolia need to be crushed according to the original record, and the examination result shows that the crushed rehmannia root, the morinda officinalis, the fructus schisandrae, the cinnamon, the tuckahoe, the radix ophiopogonis, the rhizoma acori graminei and the polygala tenuifolia need to be crushed by a third sieve (50 meshes), and the crushing into coarse powder is determined according to the test that the crushing by the third sieve is difficult to operate. The ginger is a massive root and is not suitable for being boiled thoroughly, so the ginger is cut into thick slices when in use according to a decoction piece processing method under the item of ginger in 2015 edition of Chinese pharmacopoeia, so the ginger is cut into the thick slices of 2mm to 4mm for later use; the Chinese dates are fruit type traditional Chinese medicines and are not suitable for being boiled thoroughly, so the Chinese dates are broken when being used for standby by referring to a processing method of the Chinese dates under the item of the 2015 edition of Chinese pharmacopoeia; the mint decoction pieces are irregular short sections, and the original text has no pretreatment requirement, so the mint decoction pieces can be directly fed. The decoction pieces obtained were pulverized and the results are shown in Table 1.

TABLE 1 decoction pieces crushing results table

And (3) test results: the twelve medicines of prepared rehmannia root, morinda officinalis, cornus officinalis, dendrobium, cistanche deserticola, prepared aconite, schisandra chinensis, cinnamon, tuckahoe, dwarf lilyturf tuber, grassleaf sweelflag rhizome and thinleaf milkwort root are crushed into coarse powder, and the yield is more than 80 percent.

Experimental example 2 decoction study

1. Observation of decoction endpoint and determination of decoction time

Because the decoction endpoint in the prescription original text is described as 'decocting to eight minutes', the liquid level height is 'eight minutes' according to the examination result, but when the extraction liquid height is taken as the decoction endpoint, the extraction liquid height needs to be measured in real time in the test process, so that the operation of leaving the fire source for a long time is needed, the decoction is discontinuous, and under the conditions of fixed heating mode (open fire) and fire control, the time node for reaching the decoction endpoint extraction liquid height 'eight minutes' is taken as the decoction time by utilizing the correlation between the extraction liquid height change and the decoction time. The process is as follows: weighing 3 parts of each dose of decoction pieces (prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, cistanche salsa, prepared aconite root, schisandra fruit, cinnamon, poria cocos, ophiopogon root, grassleaf sweelflag rhizome, polygala coarse powder each 1g, fresh ginger (cut into thick pieces when using) 5g, Chinese date (broken when using) 4.6g and mint 1g), putting into a 600ml casserole, adding 300ml of water, weighing the liquid level to the pot bottom height H₁Heating with open fire (gas stove), decocting with cover, boiling with strong fire, decocting with slow fire, measuring height every 5 min after boiling until liquid level reaches 0.8H₁Recording the liquid level height and the decoction time, respectively measuring the content of morroniside and loganin, the content of schizandrin A and the extraction rate, and calculating the total amount of morroniside and loganin and the total amount of schizandrin A. The results are shown in Table 2.

The method for measuring the cream yield of the extracting solution comprises the following steps: precisely measuring 20ml of extractive solution, placing in evaporating dish W1 dried to constant weight, evaporating in water bath, drying at 105 deg.C for 3 hr, cooling in drier for 30 min, and rapidly precisely weighing W₂。

TABLE 2 ingredient-based decoction endpoint examination table for rehmanniae radix

The test result shows that: in the rehmannia decoction process, the decoction end point is eight times of the height of an extracting solution, the liquid level height is 1.46cm, and the optimal decoction time is 20 minutes.

2. In summary, the process for decocting the temporary rehmannia root drink material comprises the following steps: taking prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, cistanche salsa, prepared aconite root, schisandra chinensis, cinnamon, poria cocos, radix ophiopogonis, rhizoma acori graminei and polygala tenuifolia coarse powder 1g respectively, ginger (sliced into thick slices when used), Chinese date (broken) 4.6g and mint 1g, putting the raw materials into a 600ml casserole, adding 300ml of water, heating by adopting open fire (a gas stove), covering and decocting, boiling with strong fire, and decocting with slow fire for 20 minutes to obtain a decoction.

Experimental example 3 study of filtration Process

Taking the extract under the items of 'observation of the decocting end point and determination of the decocting time', filtering the extract by 200-mesh filter cloth under normal pressure, filtering the extract by 300-mesh filter cloth, filtering the extract by 200-mesh filter cloth, centrifuging the extract for 5 minutes (the rotating speed is 4000 revolutions per minute) after filtering the extract by the 300-mesh filter cloth, and observing the clarity of the filtrate and the state of the filtrate after standing the filtrate for 30 minutes.

And (3) test results: filtering and clarifying at normal pressure of 200 mesh and 300 mesh, standing for 30 min, and centrifuging to obtain precipitate of 200 mesh and 300 mesh, wherein the precipitate is not residue and silt, and may be liposoluble component. Because the rehmannia drink is a powder boiling agent and is taken after removing dregs in the original text, the optimal filtration mode is determined by 300-mesh filter cloth and normal pressure filtration when the drink is hot in comprehensive consideration.

Experimental example 4 research on concentration Process

Since the volume of the extract liquid is large, the concentration ratio, the concentration temperature, and the concentration method were examined.

(1) Investigation of concentration ratio

Weighing 2 parts of each dose of decoction pieces (prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, herba cistanche, prepared aconite root, schisandra chinensis, cinnamon, tuckahoe, dwarf lilyturf tuber, grassleaf sweelflag rhizome, polygala coarse powder 1g each, ginger 5g (thick slices when used), Chinese date 4.6g (broken) and mint 1g), respectively placing in a 600ml casserole, adding 300ml of water, heating by open fire, covering and decocting, boiling by strong fire, decocting for 20 minutes by slow fire, filtering by 300-mesh filter cloth while hot and normal pressure, respectively concentrating the filtrate under reduced pressure (60 ℃) until the mass ratio of the decoction piece to the concentrated solution is about 1:3(g: g) and 1:4(g: g), and recording the concentration time. Pre-freezing the concentrated solution in a low-temperature cold trap (the temperature is between minus 20 and minus 48 ℃), observing the bottle hanging state of the materials during pre-freezing, and recording the pre-freezing time. Drying the pre-frozen concentrated solution on a freeze dryer, and observing the state of dry paste powder. The results are shown in Table 3.

TABLE 3 examination result table of reference concentration ratio of rehmanniae radix drink

Test results show that when the mass ratio of the feeding amount of the filtrate to the concentrated solution is about 1:3(g: g) and 1:4(g: g), pre-freezing and hanging bottles are good, freeze-dried powder is not foamed and melted, the texture is loose, and the feeding amount and the concentrated solution are selected to be concentrated until the mass ratio of the feeding amount to the concentrated solution is about 1:4(g: g) because the concentration time is shorter when the mass ratio of the feeding amount to the concentrated solution is about 1:4(g: g) and comprehensive consideration is taken.

(2) Investigation of concentration temperature

Weighing 4 parts of each dose of decoction pieces (prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, herba cistanches, radix aconiti preparata, schisandra chinensis, cinnamon, poria cocos, radix ophiopogonis, rhizoma acori graminei, polygala tenuifolia coarse powder, 1g of each 1g of ginger (cut into thick slices when used), 4.6g of jujube (broken when used) and 1g of mint), respectively placing the decoction pieces in a 600ml casserole, adding 300ml of water, covering and decocting the decoction pieces by open fire, boiling the decoction pieces by strong fire, decocting the decoction pieces by slow fire for 20 minutes, filtering the decoction pieces by 300-mesh filter cloth when the decoction pieces are hot and under normal pressure, combining the filtrates, mixing the filtrates, keeping samples (the feeding amount needs to be reduced) to carry out the determination of the content of the morroniside and the content of the loganin A, averagely dividing the rest filtrates into four parts, carrying out the reduced pressure concentration of 60 ℃, carrying out the reduced pressure concentration of the two parts at 70 ℃, respectively concentrating the feeding amount and the mass of the concentrated solution to be 1:4(g: g), carrying out the determination of the content of the morroniside and the loganin A in the concentrated solution sample, and calculating the total amount of morroniside and loganin and schizandrol A. The results are shown in Table 4.

TABLE 4 examination table of reference concentration temperature of rehmannia root drink

Test results show that the total extraction amounts of the morroniside and the loganin and the total extraction amount of the schizandrol A are basically consistent when the rehmannia root drink seed substance is subjected to different concentration temperatures, and the concentration temperature is determined to be 70 ℃ due to the fact that the concentration time at 70 ℃ is short and the concentration efficiency is considered.

(3) Comparison and investigation of single-pot concentration and combined concentration

Weighing 4 parts of each dose of decoction pieces (prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, herba cistanches, radix aconiti preparata, schisandra chinensis, cinnamon, poria cocos, radix ophiopogonis, rhizoma acori graminei, polygala tenuifolia coarse powder, 1g of each 1g of ginger (cut into thick slices when used), 4.6g of jujube (broken when used) and 1g of mint), respectively placing the decoction pieces in a 600ml casserole, adding 300ml of water, heating by open fire, covering and decocting, boiling by strong fire, decocting by slow fire for 20 minutes after boiling, filtering at hot normal pressure, mixing the filtrates, uniformly mixing, dividing into four parts, combining the two parts, concentrating under reduced pressure (70 ℃), separately concentrating the two parts under reduced pressure (70 ℃), respectively concentrating until the mass of the decoction pieces is 1:4(g: g) and observing the difference of the total amount of morroniside and loganin in the concentrated solution when the decoction pieces are combined in a plurality of pots and concentrated in one pot (daily prescription). The results are shown in Table 5.

TABLE 5 rehmannia root drink substance reference concentration mode investigation result table

Test results show that the total amount of the morroniside and the loganin and the total amount of the schizandrol A in the concentrated solution are basically consistent when the single-pot (daily prescription) concentration and the multi-pot combined concentration are carried out, and the multi-pot combined concentration has no influence on index content. Therefore, the concentration can be carried out by single-pot concentration or multi-pot combined concentration, so that the process verification and the reference preparation of different batches of substances can adopt a multi-pot combined concentration mode.

Experimental example 5 drying Process study

Two single-pot concentrated solutions obtained in the experimental example 4 are uniformly mixed, samples are left (the amount of the concentrated solution needs to be reduced), the concentrated solution is averagely divided into two parts, and freeze drying (the pre-freezing temperature is between-20 and-48 ℃, the cold trap temperature is between-40 and-80 ℃, the cold trap temperature is between-60 and-70 ℃, the vacuum degree is less than 100Pa, and the freeze drying time is at least 17 hours) and reduced pressure drying (60 ℃) are respectively carried out. The prefreezing time and the drying time were recorded. Respectively measuring water content (rapid water content measuring instrument), morroniside and loganin content, and schizandrol content, and calculating the total amount of morroniside and loganin, and the total amount of schizandrol. The results are shown in Table 6.

TABLE 6 rehmanniae radix drink material benchmark drying process research results table

Test results show that the total amount of morroniside and loganin is not much different and the schizandrol A is slightly lost when the freeze drying is compared with the pre-drying; the reduced pressure drying (60 deg.C) has the loss of morroniside, loganin and schizandrol A compared with the drying method, so the drying method is determined to be freeze drying.

Through the research, the standard preparation process of the tentative rehmannia root drink substance comprises the following steps: weighing 1g of prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, cistanche salsa, prepared aconite root, schisandra chinensis, cinnamon, poria cocos, ophiopogon root, grassleaf sweelflag rhizome and polygala root coarse powder, 5g of ginger (thick slices in use), 4.6g of Chinese date (broken in use) and 1g of mint, putting the raw materials into a 600ml casserole, adding 300ml of water, heating by open fire, covering and decocting, boiling by strong fire, decocting by slow fire for 20 minutes, filtering by 300-mesh filter cloth at normal pressure while hot, concentrating the filtrate under reduced pressure (70 ℃ and-0.1 MPa) until the mass ratio of the fed amount to the concentrated solution is about 1:4(g: g), freeze-drying (pre-freezing temperature is-20 to-48 ℃, cold trap temperature is-40 ℃ to-80 ℃, drying for at least 30 minutes, freeze-drying at cold trap temperature of-60 ℃ to-70 ℃, vacuum degree of less than 100Pa, freeze-drying for at least 17 hours), and (5) obtaining the product.

Experimental example 6 Key Process Steps and intermediates

1. Material reference process verification

According to the determined process, three batches of process verification are carried out, wherein each batch comprises 14 parts per dose: weighing 42 parts of each dose of decoction pieces (prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, herba cistanche, radix aconiti preparata slice, schisandra chinensis, cinnamon, poria cocos, radix ophiopogonis, rhizoma acori graminei, polygala tenuifolia coarse powder, 1g of each, 5g of ginger (sliced into thick slices when used), 4.6g of jujube (broken when used) and 1g of mint), putting the decoction pieces into a 600ml casserole, adding 300ml of water, heating by open fire, covering and decocting, boiling by strong fire, decocting for 20 minutes by slow fire, filtering by 300-mesh filter cloth when hot and normal pressure is high, uniformly mixing 14 parts of extracting solution in each batch, respectively reserving 100ml of the extracting solution, averagely dividing the extracting solution into 2 parts, respectively concentrating under reduced pressure (70 ℃ and-0.09 MPa to-0.1 MPa) until the mass ratio of the feeding amount to the concentrating solution is about 1:4(g: g), uniformly mixing the concentrating solutions, respectively reserving 50ml of the extracting solution, and dividing the extracting solution into 10 parts, freeze drying (the pre-freezing temperature is-48 ℃ and the cold trap temperature is-40 ℃ to-80 ℃, time: at least 30 minutes; and (3) drying: cold trap temperature: -60 to-70 ℃, vacuum degree: less than 100Pa), collecting corresponding substance (dry paste powder), and calculating the paste yield. And measuring the contents of the morroniside and the loganin, the content of the schizandrol A and the fingerprint in the extracting solution, the concentrated solution and the dry extract powder, and the water content and the extract of the dry extract powder, and respectively calculating the total amount of the morroniside and the loganin and the total amount of the schizandrol A in the extracting solution, the concentrated solution and the dry extract powder. Taking other medicinal materials and decoction pieces, and performing fingerprint spectrum determination. The results are shown in tables 7 and 8, FIG. 1.

TABLE 7 Standard Process verification results of rehmannia glutinosa Libosch substance

TABLE 8 DIHUANGDINGYIZI SUBSTANCE REFERENCE PROCESS VERIFICATION RELATIVE RETENTION TIME RESULTS

From the above results, it can be seen that the mean volume value of three batches of the substance (each comprising 14 parts per dose) was 2187ml, and 156ml per dose, which is close to "decocting to eight minutes" (160 ml of liquid medicine with eight minutes height) in the original text, according to the analysis of the volume of the intermediate (extract), and the determination of the process decocting time was reasonable.

Analyzing the total extraction amount of the morroniside and the loganin and the total extraction amount of the schizandrol A, and verifying that the data of the intermediate (extracting solution) and the corresponding real object (dry paste powder) are basically parallel, and the material transfer loss in the technical process is small; three batches verify that corresponding objects (dry paste powder) have no obvious difference by analyzing the paste yield, extract and water.

From fingerprint analysis, peaks 1, 4, 5, and 10 are fructus Corni chromatogram peaks,

peaks

3, 16, 18, and 19 are fructus Schisandrae chromatogram peaks, peak 6 is herba cistanches Deserticolae chromatogram peak, peaks 7, 8, 9, 11, 12, and 15 are radix Polygalae chromatogram peaks, and peak 14 is herba Menthae chromatogram peak. And three batches verify that corresponding positions of the extracting solution, the concentrated solution and the dry paste powder have the same common peak, the relative retention time is basically consistent, and the preliminarily determined substance reference preparation process is stable and feasible.

In summary, the preferred process for determining the material basis is as follows: weighing 1g of prepared rehmannia root, morinda officinalis, dogwood fruit, dendrobium, cistanche salsa, prepared aconite root, schisandra chinensis, cinnamon, poria cocos, ophiopogon root, grassleaf sweelflag rhizome and polygala root coarse powder, 5g of ginger (prepared into thick slices), 4.6g of Chinese date (prepared into broken pieces), 1g of mint, putting the raw materials into a 600ml casserole, adding 300ml of water, heating by open fire, covering and decocting, boiling by strong fire, decocting by slow fire for 20 minutes, filtering by 300-mesh filter cloth under hot normal pressure, concentrating the filtrate under reduced pressure (70 ℃ to-0.1 MPa) until the mass ratio of the fed amount to the concentrated solution is about 1:4(g: g), freeze-drying (pre-freezing temperature is-20 to-48 ℃, cold trap temperature is-40 to-80 ℃, drying for at least 30 minutes, freeze-drying at cold trap temperature of-60 ℃ to-70 ℃, vacuum degree of vacuum is less than 100Pa, and the time is at least 17 hours), obtaining dry extract powder, packaging to obtain corresponding substance, or adding pharmaceutically acceptable adjuvants to make into pharmaceutically acceptable dosage forms.

Preparation of Multi-batch Material benchmarks

2-3 production places are collected for each medicinal material, including a genuine production area and a main production area, wherein each production place is not less than 3 batches, and 15 total collection batches are obtained, wherein the dogwood pulp is collected from 4 batches of southwestern Xixia, 7 batches of Chunan Jiang and 3 batches of Shanxi danfeng; collecting 5 batches of Liaoning Qingyuan, Jilin flood and Heilongjiang magnolia respectively by using the schisandra chinensis; the decoction pieces collected at present are sorted according to the sequence of the extract from high to low, and are correspondingly combined from low to high one by one to prepare 15 batches of substance standards. The results of the 15 batches of decoction pieces ordered and combined are shown in Table 9 below.

Preparing 15 batches of substance-based corresponding real object (dry extract powder) according to the above determination process, wherein decoction piece information of each batch is 9, each group of experiment designs two parallel samples, each parallel sample comprises 10 parts per dose, mixing concentrated solutions, freeze drying, and packaging. The results of the 15 batch material basis cream yields are shown in table 10.

TABLE 9 Standard combination of the results for the drink of rehmannia glutinosa Libosch

TABLE 1015 batch Digitalis decoction basis cream Rate results

The test result shows that 15 batches of rehmannia root drink seed substances are temporarily corresponding to the real object (dry paste powder) in the paste yield range of between 23.69 and 31.12 percent and the average value is 27.30 percent in consideration of various influence factors and the like in the actual production process, and the paste yield range of the real object (dry paste powder) is temporarily determined to be between 20.0 and 40.0 percent.

Experimental example 7 qualitative identification method

The product is prepared by decocting fifteen traditional Chinese medicines with water, selects a thin-layer identification method with strong specificity, rapidness, simplicity and good reproducibility and a liquid-phase identification method with strong specificity, rapidness, high detection sensitivity and good reproducibility, carries out identification test research on main characteristic components of the dogwood fruit and the Chinese magnoliavine fruit, and lists the identification method into the text of the detection method. Furthermore, the differentiation test research of rehmanniae radix, rhizoma acori graminei, cinnamon, poria cocos, radix ophiopogonis, polygala tenuifolia and Chinese date in the prescription is not included in the text of the detection method due to poor specificity, and the specific research process is as follows.

1. Summary of safety-related chemical compositions and their limits

In the prescription, monarch drug prepared rehmannia root, dogwood fruit, wine desert cistanche, morinda root, ministerial drug prepared aconite, cinnamon, dwarf lilyturf tuber, dendrobium, Chinese magnoliavine fruit, adjuvant drug grassleaf sweelflag rhizome, thinleaf milkwort root-bark and Indian buead, and messenger drug ginger, Chinese date and mint are used, except the prepared aconite, thinleaf milkwort root-bark and Chinese date, no safety-related chemical components are found at present, and the content limit of endogenous toxic components of the prepared aconite and the content limit of exogenous components of thinleaf milkwort root-bark and Chinese date are detailed in the following table 11.

TABLE 11 safety-related chemical composition and limits thereof

Except for the requirement of limited amount of the processed radix aconiti lateralis, the other medicines have no endogenous toxic components and have good safety.

2. The Corni fructus contains polysaccharides, iridoid glycosides, organic acids (oleanolic acid), tannin, etc. Wherein the iridoid glycosides comprise loganin, morroniside and cornuside, and morroniside and loganin are main effective components. The following thin-layer grope experiments were performed on cornus based on these chemical components.

Firstly, a thin-layer chromatography method is established, and a thin-layer identification method of dogwood is referred to in the 'Chinese pharmacopoeia' 2015 edition.

Taking 4g of the product, adding 50ml of water, dissolving by ultrasonic, extracting with dichloromethane for 2 times, 30ml each time, extracting with n-butanol for 2 times, 25ml each time, combining n-butanol solutions, washing with ammonia solution (1 → 10) for 2 times, 30ml each time, discarding the ammonia solution, combining the n-butanol solutions, evaporating to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution. Taking another 4g of pulp of dogwood fruit negative sample, adding 50ml of water to dissolve, shaking and extracting for 2 times by using dichloromethane, 30ml each time, discarding dichloromethane liquid, and preparing the water liquid into a negative sample solution by the same method from the step of shaking and extracting for 2 times by using n-butyl alcohol. And taking 1g of the dogwood control medicinal material, adding 50ml of water, decocting for 20 minutes, filtering, extracting the filtrate for 2 times by shaking with dichloromethane, 30ml each time, discarding dichloromethane liquid, and preparing the control medicinal material solution by the same method from the step of extracting the water solution for 2 times by shaking with n-butanol. The morroniside control was added with 80% methanol to make a solution containing 0.5mg per 1 ml. Adding loganin control, and adding 80% methanol to obtain control solution containing 1mg per 1 ml. Testing by thin layer chromatography (0502 of the four ministry of the national pharmacopoeia 2015), collecting appropriate amount of the above five solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol (4:1) as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet lamp (365 nm).

And (3) test results: in the chromatogram of the test solution, fluorescent spots of the same color appear at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution, but background interference exists.

The second method comprises the following steps: on the basis of the method I, the thin layer plate is replaced by a GF254 silica gel plate, the developing agent is adjusted to chloroform-methanol (3:1), the thin layer plate is developed, taken out, dried and placed under an ultraviolet lamp (254nm) for inspection, and the method is shown in figure 2:

and (3) test results: in the chromatogram of the test solution, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution, and the negative result is free from interference. Determining the sample amount as follows: 5 mul of test solution, 5 mul of loganin reference solution, 2 mul of morroniside reference solution and 1 mul of reference solution.

The preliminary identification method for determining the optimized thin layer of the dogwood fruit comprises the following steps: taking 4g of the product, adding 50ml of water, dissolving by ultrasonic, extracting with dichloromethane for 2 times, 30ml each time, extracting with n-butanol for 2 times, 30ml each time, combining n-butanol solutions, washing with ammonia solution (1 → 10) for 2 times, 25ml each time, discarding the ammonia solution, combining the n-butanol solutions, evaporating to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution. Taking 1g of Corni fructus as reference material, adding 50ml of water, decocting for 20 min, filtering, shaking and extracting the filtrate with dichloromethane for 2 times (30 ml each time), discarding dichloromethane solution, and preparing into reference material solution by the same method from the step of shaking and extracting the water solution with n-butanol for 2 times. The morroniside control solution is prepared by adding 80% methanol to 1ml solution containing 0.5mg of morroniside as control solution. Subsequently, loganin control solution was added with 80% methanol to make 1mg solution per 1ml, and used as control solution. Performing thin-layer chromatography (0502 of the four ministry of the university in the pharmacopoeia of China 2015), collecting sample solution 5 μ l, control solution 1 μ l, loganin control solution 5 μ l and morroniside control solution 2 μ l, respectively dropping on the same silica gel GF254 thin-layer plate, developing with chloroform-methanol (3:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution.

Durability examination

The durability test is carried out according to a determined method, and different manufacturers of silica gel GF254 thin-layer plates (Nicoti chemical industry institute, Qingdao ocean chemical plant and Germany Macherey-Nagel (MN)) and different development temperatures (23.5 ℃ and 6.6 ℃), different relative humidities (38% and 87%) and other influence factors result in good durability of the development conditions and clear main spots.

Third, the method determines

Taking 4g of the product, adding 50ml of water, dissolving by ultrasonic, extracting with dichloromethane for 2 times, 30ml each time, extracting with n-butanol for 2 times, 30ml each time, combining n-butanol solutions, washing with ammonia solution (1 → 10) for 2 times, 25ml each time, discarding the ammonia solution, evaporating the n-butanol solution to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution. Taking 1g of Corni fructus as reference material, adding 50ml of water, decocting for 20 min, filtering, shaking and extracting the filtrate with dichloromethane for 2 times (30 ml each time), discarding dichloromethane solution, and preparing into reference material solution by the same method from the step of shaking and extracting the water solution with n-butanol for 2 times. And adding 80% methanol into the morroniside control and loganin control to obtain mixed solutions each containing 1mg per 1ml as control solutions. Performing thin layer chromatography (0502 of the four ministerial general rules of the design of Chinese pharmacopoeia 2015), collecting sample solution 5 μ l, control solution 1 μ l, and control solution 2 μ l, respectively dropping on the same silica gel GF254 thin layer plate, developing with chloroform-methanol (3:1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the positions corresponding to the chromatogram of the reference solution and the chromatogram of the reference solution.

(2) Schisandra chinensis

The main chemical components of fructus Schisandrae include lignanoids, triterpenes, volatile oil, polysaccharide, etc. Wherein the lignanoid comprises schizandrin A, schizandrin B, etc. The following thin layer groping experiments were performed on schisandra chinensis based on these chemical components.

Firstly, establishing thin-layer chromatography

The method comprises the following steps: thin layer identification method of schisandra chinensis in the first part of 2015 year referred to Chinese pharmacopoeia

Collecting 1g of the product, adding 70% methanol 30ml, heating and refluxing for 1 hr, filtering, evaporating the filtrate to dryness, dissolving the residue with 0.1mol/L sodium hydroxide solution 20ml, extracting with ethyl acetate under shaking for 2 times, each time 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 1ml to obtain sample solution. Taking another negative sample 1g lacking fructus Schisandrae chinensis, and preparing a negative sample solution by the same method. Taking 1g of fructus Schisandrae chinensis as reference material, adding 50ml of water, decocting for 20 min, filtering, and preparing the reference material solution by the same method from 'extracting with ethyl acetate for 2 times'. Collecting deoxyschizandrin reference substance, and adding 80% methanol to obtain solution containing 1mg per 1ml as reference substance solution. Performing thin-layer chromatography (0502 of the general Law of the national pharmacopoeia of 2015), collecting the sample solution, the negative sample solution, the control solution, and 2 μ l of the control solution, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (60-90 deg.C) -ethyl formate-methanol (15:5:0.3) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm).

And (3) test results: in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material, but the Rf value is lower; the spots at the locations corresponding to the control chromatogram were lighter and the control needed to be replaced.

The second method comprises the following steps: the reference substance is replaced by schisandrin, and the developing agent is adjusted to petroleum ether (60-90 ℃) -ethyl formate-methanol (15:5: 2).

And (3) test results: in the chromatogram of the test solution, fluorescent spots with the same color appear at the positions corresponding to the chromatogram of the control solution and the chromatogram of the control solution, but negative interference exists at the positions corresponding to negativity.

The third method comprises the following steps: on the basis of the second method, the developing agent is adjusted to be petroleum ether (60-90 ℃) -ethyl acetate-methanol (5:3:0.2), and the test results are shown in the following figure 3:

and (3) test results: in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution and the chromatogram of the reference solution, and the method is feasible and has no interference to the negative.

The preliminary determination method comprises the following steps: collecting 1g of the product, adding 70% methanol 30ml, heating and refluxing for 1 hr, filtering, evaporating the filtrate to dryness, dissolving the residue with 0.1mol/L sodium hydroxide solution 20ml, extracting with ethyl acetate under shaking for 2 times, each time 20ml, mixing ethyl acetate solutions, evaporating to dryness, and dissolving the residue with methanol 1ml to obtain sample solution. Decocting fructus Schisandrae 1g in water 50ml for 20 min, filtering, and making into control solution by the same method from "extracting with ethyl acetate for 2 times". Then adding 80% methanol into the schizandrol A control to obtain a solution containing 1mg of schizandrol A per 1ml as control solution. Performing thin-layer chromatography (0502 of the general Law of the national pharmacopoeia of 2015), collecting 5 μ l of sample solution, 1 μ l of control solution, and 2 μ l of control solution, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (60-90 deg.C) -ethyl acetate-methanol (5:3:0.2) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). 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.

Durability examination

The durability test is carried out according to a determined method, and different manufacturers of silica gel GF254 thin-layer plates (Nicoti chemical industry institute, Qingdao ocean chemical plant and Germany Macherey-Nagel (MN)) and different development temperatures (22.7 ℃ and 2.6 ℃), different relative humidities (50% and 90%), and other influencing factors result in good durability of the development conditions and clear main spots.

③ the method is to determine to take 1g of the product, add 30ml of 70 percent methanol, heat and reflux for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue by 20ml of 0.1mol/L sodium hydroxide solution, shake and extract by ethyl acetate for 2 times, 20ml each time, combine the ethyl acetate solution, evaporate to dryness, add 1ml of methanol to dissolve the residue to be used as the test solution. Decocting fructus Schisandrae 1g in water 50ml for 20 min, filtering, and making into control solution by the same method from "extracting with ethyl acetate for 2 times". Then adding 80% methanol into the schizandrol A control to obtain a solution containing 1mg of schizandrol A per 1ml as control solution. Performing thin-layer chromatography (0502 of the general Law of the national pharmacopoeia 2015), collecting 5 μ l of sample solution, 1 μ l of reference medicinal material solution and 2 μ l of reference solution, respectively dropping on the same silica gel GF254 thin-layer plate, developing with petroleum ether (60-90 deg.C) -ethyl acetate-methanol (5:3:0.2) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). 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.

Experimental example 8 finger print method

Due to the simple qualitative identification and quantitative analysis of index components, the quality of the reference quality of the substance is difficult to reflect. As a standard reference substance for determining whether the substance standard substance (dry extract powder) is basically consistent with the classical famous decoction, the quality should be enhanced with specificity identification and multi-component and overall quality control. Therefore, the quality control based on material quality control emphasizes the overall quality control mainly based on fingerprint.

(1) Instrument and reagent liquid chromatograph: shimadzu LC-20A, Agilent 1260 II; a chromatographic column: GL Sciences Wondasil C18 (250X 4.6mm, 5 μm); electronic analytical balance: double jie test instrument factory JJ1000 (hundredth), sydow scientific instruments (beijing) ltd BSA224S-CW (one in ten thousand) mettler-tollim instruments (shanghai) ltd ME503T (one in thousand), acetonitrile is chromatographically pure, produced by OCEANPAK; phosphoric acid is chromatographically pure, produced by western reagent limited; the methanol is analytically pure and is produced by Guangzhou chemical reagent factories; formic acid is chromatographically pure, Tianjin Kemi European chemical reagent Co., Ltd; the water is ultrapure water, Shanghai and Tai instruments Inc. The morroniside reference substance (batch number: 111998-.

(2) Selection of mobile phase

The method comprises the following steps: the reference literature, namely a middle fingerprint method for researching chemical components of DACHAIHU decoction and GUIFUDIHUANG prescription, regulates the mobile phase proportion on the basis of the method:

the chromatographic conditions were gradient elution as specified in Table 12 using GL Sciences Wondasil C18 (4.6X 250mm, 5 μm) as a column, acetonitrile as a mobile phase A, and 0.1% formic acid as a mobile phase B; the detection wavelength was 254 nm.

TABLE 12 mobile phase gradient elution procedure

Preparing a test sample solution, precisely weighing 2g of freeze-dried powder, placing the freeze-dried powder into a conical flask with a plug, precisely weighing 50ml of 70% methanol, weighing the weight, carrying out ultrasonic treatment (power is 250W and frequency is 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 70% methanol, shaking up, filtering, evaporating the filtrate, adding 25ml of water into residues to dissolve, extracting the solution by shaking with an ethyl acetate-n-butyl alcohol (1:1) mixed solution for 3 times, 25ml each time, combining the ethyl acetate-n-butyl alcohol (1:1) mixed solution, evaporating to dryness, adding methanol into the residues to dissolve, transferring the residues into a 5ml measuring flask, diluting the residues to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test sample solution.

The determination method comprises precisely sucking 5 μ l of each sample solution, injecting into liquid chromatograph, determining, and recording chromatogram.

And (3) test results: peak resolution was poor between 20 minutes and 48 minutes, and between 54 minutes and 56 minutes.

The second method comprises the following steps: the mobile phase gradient elution procedure was adjusted on a "method one" basis, see table 13:

TABLE 13 procedure for mobile phase gradient elution

And (3) test results: the chromatographic peak at the arrow has poor resolution and needs to be optimized.

The third method comprises the following steps: mobile phase example optimization was performed on a "method two" basis while mobile phase B was changed to 0.3% phosphoric acid and mobile phase gradient elution program was adjusted as in table 14:

TABLE 14 mobile phase gradient elution procedure

And (3) test results: the chromatographic peak pattern is good under the condition, and the separation degree is better. Can be used for subsequent studies, see fig. 4.

Selection of test article preparation method

Ultrasonically taking about 2g of the product by 80% methanol, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 80% methanol, weighing, ultrasonically treating (with the power of 250W and the frequency of 40kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 80% methanol, shaking up, filtering, evaporating to dryness, dissolving the residue by adding methanol, transferring to a 5ml measuring flask, diluting to scale by using methanol, shaking up, filtering, and taking the subsequent filtrate.

Shaking and extracting the product by ethyl acetate to obtain about 2g, precisely weighing, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, weighing the weight, carrying out ultrasonic treatment (the power is 250W and the frequency is 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, steaming to near dryness, adding 25ml of water to the residue to dissolve, shaking and extracting by ethyl acetate for 2 times, each time 25ml, combining ethyl acetate solutions, evaporating to dryness, adding methanol to the residue to dissolve, transferring to a 5ml measuring flask, diluting to a scale by methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product.

Shaking and extracting a mixed solution of ethyl acetate and n-butyl alcohol (1:1) to obtain about 2g of the product, precisely weighing, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, weighing, ultrasonically treating (the power is 2500W, the frequency is 40kHz) for 30 minutes, cooling, weighing again, complementing the loss weight by 80% methanol, shaking up, filtering, steaming to be nearly dry, adding 25ml of water into residues to dissolve, shaking and extracting for 2 times by using the mixed solution of ethyl acetate and n-butyl alcohol (1:1) with 25ml each time, combining the mixed shaking and extracted solution of ethyl acetate and n-butyl alcohol (1:1), drying by distillation, adding methanol into residues to dissolve, transferring to a 5ml measuring flask, diluting to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the product.

The test results are shown in fig. 5:

and (3) test results: the peak information of the ethyl acetate sample is less, the peak information of the ethyl acetate-n-butyl alcohol (1:1) and the peak information of the 80% methanol sample are more, the two are basically consistent, but the viscosity of the test sample after being treated by the 80% methanol is larger than that after being treated by the ethyl acetate-n-butyl alcohol (1:1), and the ethyl acetate-n-butyl alcohol (1:1) is selected and preferably selected for preparing the test sample in comprehensive consideration.

(3) Selection of detection wavelength

A diode array detector is adopted, a sample solution is scanned at the wavelength of 190-400 nm, an equal absorption spectrum is used for analysis, the result shows that the chromatographic peak is in the wavelength range of 230-280 nm in 50 minutes, and the information content is large. The wavelengths of 230nm, 254nm and 280nm are selected for analysis, and under the wavelength of 254nm, the information content of each spectral peak is large, the response value is moderate, the separation degree is good, and the base line is stable. Therefore, 254nm is selected as the detection wave.

(4) Peak assignment and selection of reference

Attribution of medicinal flavor peaks

Preparation of decoction piece sample solution about 0.5g of each medicinal powder (sieved by a No. two sieve) according to the prescription is precisely weighed, placed in an erlenmeyer flask, added with 50ml of water, decocted for 30 minutes, cooled, filtered, the filtrate is shaken and extracted for 2 times with a mixed solution of ethyl acetate-n-butyl alcohol (1:1) and 25ml of each time, an ethyl acetate-n-butyl alcohol (1:1) mixed solution layer is combined, evaporated to dryness, the residue is dissolved by adding methanol, transferred to a 5ml measuring flask, diluted to the scale by methanol, shaken uniformly, filtered, and the subsequent filtrate is taken, thus obtaining the decoction piece.

The measurement was carried out according to the determined chromatographic conditions, and the assignment of each peak in the substance reference fingerprint was determined by mass spectrometric detection (see fig. 6 and table 15). Wherein peaks 1, 3, 11, 13, and 14 are chromatographic peaks of fructus Schisandrae, peaks 2 and 4 are chromatographic peaks of Corni fructus, peak 5 is chromatographic peak of Cistanchis herba, peaks 6, 7, 8, and 10 are chromatographic peaks of cortex et radix Polygalae, and peaks 9 and 12 are chromatographic peaks of herba Menthae.

TABLE 15 attribution list of herbs and flavors

Identification of chromatographic peaks

The relevant main components of each medicinal flavor are selected through literature research: the peak localization studies were performed on morroniside, loganin, echinacoside, 3, 6-dipalmitoyl sucrose, rosmarinic acid, and schizandrol A. (see FIG. 7).

The analysis result is consistent with the mass spectrum result by comparing 7 known components (gallic acid, morroniside, loganin, echinacoside, 3, 6' -dibapinyl sucrose, and schizandrol A) with the control product. Of the 19 peaks, 7 of the known components were finally identified together. The correspondence between the mass spectrometry results and the reference identification results is shown in Table 16.

Simultaneously, the mass spectrum is used for detecting the chromatographic peak in the fingerprint of the material standard corresponding to the real object (dry extract powder) of the rehmannia root drinkers for preliminary identification, the UPLC-UV chromatogram and the total ion flow diagram (positive mode) are shown in figure 8, the liquid phase part of the liquid chromatograph-mass spectrometer is UPLC, the chromatogram is slightly different from the chromatogram of the HPLC fingerprint, and the corresponding relation of the specific peak is shown in the following table 16

TABLE 16 correspondence between mass spectrometry results and common peak assignment results

Selection of reference object

The retention time of the chromatographic peak of the schizandrol A in the chromatogram is moderate, the response value is high, and the baseline separation is achieved, so the schizandrol A is selected as the reference peak of the reference substance, the schizandrol A is marked as the peak S, and the schizandrol A reference substance is used as the reference substance at the same time.

(5) Preliminary determination of the method

The measurement is carried out by high performance liquid chromatography (0512 in the four-department general regulation of the 2015 edition in China pharmacopoeia).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile was used as mobile phase a, 0.3% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in table 17; the detection wavelength is 254 nm; the number of theoretical plates is not less than 3000 calculated according to schizandrol A.

TABLE 17 procedure Table for gradient elution of mobile phase

Preparing a test solution, precisely weighing about 2.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power is 250W and frequency is 40kHz) for 30 minutes, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, extracting the filtrate by using an ethyl acetate-n-butyl alcohol (1:1) mixed solution for 2 times, each time 25ml, combining ethyl acetate-n-butyl alcohol (1:1) mixed solution layers, evaporating to dryness, adding methanol into residues to dissolve, transferring to a 5ml measuring flask, diluting to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution.

(6) Methodology investigation

First, special property test and integrity investigation

Through the investigation of specificity and integrity tests, the blank solvent has no interference and good specificity; no chromatographic peak was present for the extended 30 minutes, and the integrity was good. See in particular fig. 9.

Instrument precision test

About 2.0g of this product (lot number: D-181120-03) was weighed precisely, and the preparation and measurement of the test solution under the term of "preliminary determination of method" were carried out by continuous sampling 6 times, and the relative retention time and similarity between each characteristic peak and the S peak were calculated, and the measurement results are shown in tables 18 and 19, and FIG. 10.

Table 18 fingerprint precision test relative retention time results (n ═ 6)

TABLE 19 fingerprint Instrument precision test similarity results (n ═ 6)

R control, S2 Instrument precision 1, S3 Cry precision 2, S4 Instrument precision 3, S5 Instrument precision 4, S6 Instrument precision 5, S7 Instrument precision 6

The result shows that the fingerprint of the test sample presents chromatographic peaks with the same retention time as the chromatographic peaks of the reference substance and presents 14 main chromatographic peaks. The relative retention time is less than 3.0%, the similarity is more than 0.90, and the precision of the instrument is good.

Stability test

Taking about 2.0g of the product (batch number: D-181112-03), precisely weighing, preparing and measuring the sample solution according to the 'method preliminary determination', standing for 0, 3,6, 9, 12, 15, 24, 36, and 48 hours after preparation, respectively, injecting sample, measuring, calculating the relative retention time of each characteristic peak and S peak, and similarity with reference spectrum, and obtaining the measurement results shown in tables 20-21, FIG. 11

Table 20 stability test relative retention time results (n ═ 6)

TABLE 21 stability test similarity results

The result shows that the relative retention time RSD of the fingerprint is less than 3.0%, the similarity is more than 0.90, and the solution has good stability within 48 hours.

(iv) repeatability test

About 2.0g (total 6 parts) of this product (lot number: D-181120-03) was weighed out precisely, and the relative retention time and similarity between each characteristic peak and the S peak were calculated according to the preparation and measurement procedures of the test solution under the "preliminary determination by method", and the measurement results are shown in Table 22, Table 23, and FIG. 12.

Table 22 repeatability test relative retention time results (n ═ 6)

Table 23 repeatability test similarity results (n ═ 6)

R control map, S2 repeat 1, S3 repeat 2, S4 repeat 3, S5 repeat 4, S6 repeat 5, S7 repeat 6

The result shows that the relative retention time RSD of the fingerprint is less than 3.0%, the similarity is greater than 0.90, and the repeatability of the fingerprint method is good.

Durability test

The durability of different flow rates, different column temperatures, different chromatographic columns, different acid ratios and different instruments on the chromatographic conditions is considered, the durability is automatically matched with a reference spectrum R (a repetitive HPLC spectrum peak is automatically matched by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation software system 2012 edition issued by the State pharmacopoeia Committee to form a common mode diagram and establish the reference spectrum R), the similarity is calculated, and the measurement result is shown in the diagrams 13-16 and tables 24-27.

TABLE 24 durability similarity results for different flow rates

R control map S21.1ml/min S31.0ml/min S40.9ml/min

TABLE 25 results of similarity in durability of different columns

R control map, S2 Agient 5TC, S3 GL Sciences Wondasil C18, S4 xtate C18, S5 Inerstil ODS-SP

TABLE 26 different Instrument durability similarity results

R control spectrum S20.25% phosphoric acid S30.3% phosphoric acid S40.35% phosphoric acid

TABLE 27 results of similarity in durability for different instruments

R control map S2 Agilent 1260II S3 Shimadzu LC20A

The result shows that the measurement results of different flow rates, different column temperatures, different chromatographic columns, different acid ratios and different instrument conditions are basically consistent, the common peak peaks in the chromatogram are sharp and symmetrical, the separation degree is good, and the similarity is not lower than 0.90. The fingerprint of the sample shows 14 main chromatographic peaks, and the main chromatographic peaks correspond to characteristic peaks in the chromatographic peaks of the reference substance. The method is proved to have good durability to different flow rates, different column temperatures, different chromatographic columns, different acidity and different instruments.

(8) Determination of fingerprint determination method

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.3 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 254 nm; the number of theoretical plates is not less than 3000 calculated according to schizandrol A.

TABLE 28 procedure for mobile phase gradient elution

Preparing a test solution, precisely weighing about 2.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power is 250W and frequency is 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss by 80% methanol, shaking up, filtering, extracting the filtrate by using an ethyl acetate-n-butyl alcohol (1:1) mixed solution for 2 times, 25ml each time, combining the ethyl acetate-n-butyl alcohol (1:1) mixed solution, evaporating to dryness, adding methanol into residues to dissolve, transferring the residues to a 5ml measuring flask, diluting to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution.

(7) Establishment of fingerprint

Calibration of common peaks

The 15 batches (30 parts) of test articles are measured according to a determined method, the obtained fingerprints are analyzed, chromatographic peaks with better stability and proper response value in the 15 batches (30 parts) of sample fingerprints are selected as common peaks, 14 common peaks are calibrated, and the result is shown in figures 17-18.

Establishment of contrast atlas

The method comprises the steps of automatically matching 15 batches (30 parts) of rehmannia potion substance standard corresponding to physical object (dry paste powder) HPLC chromatogram peaks by adopting 'traditional Chinese medicine chromatogram fingerprint similarity evaluation software system 2012 edition' issued by the State pharmacopoeia Committee to form a common pattern, establishing a reference spectrum, and obtaining a result shown in figure 19.

Calculating similarity

A traditional Chinese medicine chromatogram fingerprint similarity evaluation software system is adopted, 15 batches (30 parts) of substance reference corresponding real objects (dry paste powder) and the similarity of the comparison fingerprint are calculated by common peaks, and the result is shown in tables 29-30. According to the similarity calculated by the fingerprint, the similarity of 15 batches (30 parts) of substance reference and the rehmannia glutinosa drinker reference fingerprint is compared, the actual measurement range is 0.902-0.997, and the actual measurement ranges are all more than 0.90, so the fingerprint is selected as the evaluation standard of the rehmannia glutinosa drinker substance reference. The sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 14 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90.

TABLE 29 results of similarity-1

R, reference substance atlas S2.D-181212-01, S3.D-181212-03, S4.D-181212-05, S5.D-181212-07, S6.D-181213-09, S7.D-181213-11, S8.D-181213-13, S9.D-181213-15, S10.D-181214-17, S11.D-181214-19, S12.D-181214-21, S13.D-181214-23, S14.D-181215-25, S15.D-181215-27, S16.D-181215-29

TABLE 30 results of similarity-2

R, contrast map S2.D-181212-03, S3.D-181212-04, S4.D-181212-06, S5.D-181212-08, S6.D-181213-10, S7.D-181213-12, S8.D-181213-14, S9.D-181213-16, S10.D-181214-18S11.D-181214-20, S12.D-181214-22, S13.D-181214-24, S14.D-181215-26, S15.D-181215-28S16.D-181215-3

Experimental example 9 measurement of content

In the formula of the rehmannia drink, cooked rehmannia, morinda officinalis, cornus officinalis and cistanche salsa are used as monarch drugs, dendrobium, prepared aconite, schisandra chinensis, cinnamon, radix ophiopogonis are used as ministerial drugs, poria cocos, rhizoma acori graminei and polygala tenuifolia are used as adjuvant drugs, and ginger, Chinese date and mint are used as conductant drugs. In the formula, iridoid glycoside (morroniside and loganin) in fructus Corni as principal drug is its main active ingredient, and lignanoid (schizandrol A) in fructus Schisandrae as ministerial drug is related to its effectiveness, is the basis of its main drug effect substance, and can be used as index ingredient of radix rehmanniae potion; therefore, the total amount of the morroniside and the loganin in the monarch drug cornus pulp and the content of the schizandrol in the ministerial drug schisandra are selected for research.

(1) The pulp of Corni fructus is determined by high performance liquid chromatography (0512 in the four ministry of communications in 2015 pharmacopoeia of China).

Instrument and reagent

② selection of chromatographic conditions

a selection of mobile phase and mobile phase ratio

The method comprises the following steps: referring to a method for measuring the contents of 467 morroniside and loganin under the item of dogwood in the first part of 'Chinese pharmacopoeia' 2015 edition, the method is searched:

the chromatographic conditions were performed using Kromasil 100-5-C18(4.6 x 250mm, 5um) as a column, acetonitrile as mobile phase a, and 0.3% phosphoric acid as mobile phase B, with a gradient as specified in table 31; the detection wavelength is 240 nm; the column temperature was 35 ℃.

TABLE 31 procedure for mobile phase gradient elution

Preparing a reference solution by precisely weighing a proper amount of morroniside reference substance and loganin reference substance, and adding 80% methanol to obtain mixed solutions each containing 50 μ g of morroniside and loganin per 1 ml.

Preparing a test solution, precisely weighing about 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power of 250W and frequency of 40kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 80% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution.

And (4) analyzing results: under the condition, the loganin has poor separation degree and negative interference, and the proportion of a mobile phase needs to be optimized. The difference between the peak areas of the loganin reference substance and the test substance is large, and the peak area of the test substance and the peak area of the reference substance need to be adjusted to be about 1: 1.

The second method comprises the following steps: based on "method one", the mobile phase gradient elution procedure was adjusted as in table 32 and the results of the experiment are shown in fig. 20.

TABLE 32 mobile phase gradient elution procedure

And (4) analyzing results: negative without interference, and good peak type and peak separation degree of morroniside and loganin, and can be used for subsequent research.

Therefore, the determination method for primarily determining the content of morroniside and loganin comprises the following steps:

chromatographic conditions and system applicability test Kromasil 100-5-C18(4.6 x 250mm, 5um) as a chromatographic column, acetonitrile as mobile phase A, 0.3% phosphoric acid as mobile phase B, and elution in a gradient as specified in Table 33, with a detection wavelength of 240 nm; the column temperature was 35 ℃.

TABLE 33 mobile phase gradient elution procedure

Preparing a reference solution by precisely weighing a proper amount of morroniside and loganin, and adding 80% methanol to obtain a mixed solution containing 40 μ g of morroniside and 20 μ g of loganin per 1 ml.

b determination of the detection wavelength

Taking a proper amount of a morroniside reference substance and a loganin reference substance, precisely weighing, adding 80% methanol to prepare a mixed solution containing 40 mu g of morroniside and 20 mu g of loganin in each 1ml, scanning at the wavelength of 190-400 nm, and obtaining a full-wavelength scanning chart shown in 6.3-132-6.3-133, wherein the maximum absorption peak of the morroniside at 240nm is free from impurity interference, the maximum absorption peak of the loganin at 238nm is free from impurity interference, and the wavelength is basically consistent with the 240nm wavelength of the content determination of dogwood in 2015 edition of Chinese pharmacopoeia, so 240nm is selected as the detection wavelength.

Investigation of preparation method of test solution

a examination of extraction solvent

Precisely weighing about 1.0g (3 groups) of the product (batch number: D-181120-01), placing in a conical flask with a plug, precisely adding 80% ethanol, 80% methanol and 25ml methanol respectively, sealing the plug, weighing, performing ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, supplementing lost weight with 80% ethanol, 80% methanol and methanol respectively, shaking up, filtering, taking out subsequent filtrate, precisely sucking 5 μ l of subsequent filtrate, injecting into a liquid chromatograph, and measuring. The results are shown in Table 34.

Table 34 results of the test with the extraction solvent

The results show that the extraction rates of different extraction solvents are basically consistent, and are consistent with the content determination item of dogwood in the '2015 version in Chinese pharmacopoeia', so that 80% methanol is selected as the extraction solvent.

b examination of extraction mode

Precisely weighing about 1.0g (total 3 groups) of the product (batch number: D-181120-01), placing in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, respectively performing ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, performing reflux extraction for 30 minutes, performing shaking extraction for 30 minutes, taking out, cooling, weighing again, complementing the weight loss with 80% methanol, shaking up, filtering, taking out the subsequent filtrate to obtain the final product, precisely absorbing 5 μ l of the subsequent filtrate, injecting into a liquid chromatograph, and measuring. The results are shown in Table 35.

TABLE 35 examination of extraction modes

The experimental results are as follows: the contents of morroniside and loganin are basically consistent in different extraction modes, and ultrasonic is selected as the extraction mode in consideration of convenient operation.

c investigation of extraction time

Precisely weighing about 1.0g (3 groups) of the product (batch number: D-181120-01), placing in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, respectively performing ultrasonic treatment (power 250W and frequency 40kHz) for 15 min, 30 min and 45 min, taking out, cooling, weighing again, supplementing lost weight with 80% methanol, shaking up, filtering, and taking out the subsequent filtrate to obtain the final product, precisely sucking 5 μ l of the subsequent filtrate, injecting into a liquid chromatograph, and measuring. The results are shown in Table 36.

Table 36 extracts temporal findings

The results show that the content difference of the morroniside and the loganin is not large at different extraction time, and the extraction time is selected to be 30 minutes in consideration of the content difference of different batches of decoction pieces.

The chromatographic condition for preliminary determination of the content determination method and the system applicability test use octadecylsilane chemically bonded silica as a filling agent; gradient elution was performed as specified in table 37 using acetonitrile as mobile phase a and 0.3% phosphoric acid solution as mobile phase B; the detection wavelength was 240 nm. The number of theoretical plates should not be less than 3000 calculated by the morroniside peak.

TABLE 37 procedure for mobile phase gradient elution

Methodology investigation

a specificity test

The dogwood fruit contains morroniside and loganin, in order to examine whether other medicines interfere the determination of morroniside and loganin, decoction pieces are weighed according to the prescription proportion to prepare a dogwood fruit negative sample by the same method, a sample of the dogwood fruit negative sample solution is prepared according to the treatment method of a test sample, and a chromatogram is recorded. The result shows that the chromatographic peak separation effect of the morroniside and the loganin is good, and no chromatographic peak exists in the negative chromatogram in the retention time corresponding to the morroniside and the loganin, which indicates that other decoction pieces except for cornus are not interfered for the measurement of the morroniside and the loganin, and the method has specificity in simultaneously measuring the content of the morroniside and the loganin in the product. The results are shown in FIG. 21.

b peak purity check

Injecting the sample solution into a liquid chromatograph, performing full-wavelength detection by a PDA (personal digital Assistant) detector under the chromatographic conditions, and calculating peak purity, wherein impurity peaks are not detected in the sample chromatogram in the result; the minimum peak purity thresholds were 995.03 and 992.00, respectively, indicating that the purities of the morroniside and loganin chromatographic peaks were satisfactory under the chromatographic conditions.

c investigation of linear relationship

Precisely weighing 10.29mg of the morroniside reference substance (batch number: 111998-201703, purity: 97.4%), 10.18mg of the loganin reference substance (batch number: 111640-201707, purity: 99.2%), putting the morroniside reference substance into a 10ml measuring flask, adding 80% methanol for dissolving, fixing the volume to a marked line, shaking up, and respectively preparing a morroniside reference substance solution (i) with the concentration of 1.0022mg/ml and a loganin reference substance solution (i) with the concentration of 1.0180 mg/ml; precisely measuring the comparison products (i.e. 2ml and ② 1 ml), placing the comparison products in a 5ml measuring flask, diluting the comparison products to scale with 80% methanol, shaking up the comparison products to prepare solution of the morroniside with the concentration of 0.4009mg/ml and the loganin comparison product with the concentration of 0.2036 mg/ml; precisely measuring the comparison products (i.e. 2ml and (ii) 1ml, placing the comparison products in a 100ml measuring flask, diluting the comparison products to a scale with 80% methanol, shaking up, and preparing the comparison product solution (iv) of the morroniside with the concentration of 20.04 mu g/ml and the loganin with the concentration of 10.18 mu g/ml. Precisely sucking the

above control solutions

2, 5, 10, and 15 μ l, respectively, and injecting the

control solutions

2, 5, and 10 μ l into a liquid chromatograph, and measuring by liquid chromatography (0512 in the four ministry of the general rules of the national pharmacopoeia 2015). The sample amount (μ g) was taken as the abscissa and the peak area as the ordinate, and a standard curve was drawn. See tables 38-39.

TABLE 38 Monoside Linear relationship

The results showed that the morroniside was well linear in the range of 0.04008. mu.g to 4.0090. mu.g.

TABLE 39 loglinear loganin relationships

The results show that loganin is linear well in the range of 0.02036-2.0360. mu.g.

d measurement of precision

Taking the morroniside and loganin mixed reference solution, wherein the morroniside concentration is 40.09 μ g/ml, the loganin concentration is 20.36 μ g/ml, continuously injecting samples for 6 times, and the determination results are shown in Table 40.

TABLE 40 Instrument precision test results (n ═ 6)

The results show that the RSD is less than 2.0%, which indicates that the precision of the instrument is good.

e repeatability test

The experimenter (A) performs the operation, and takes about 1.0g (total 6 parts) of the product (batch number: D-181120-01), precisely calls it, and performs the operation according to the preparation and determination method of the test solution under the item of 'preliminary determination of the method'. The results are shown in Table 41.

Table 41 repeatability test results (n ═ 6)

The result shows that the RSD is less than 3.0 percent, which shows that the repeatability of the method is good.

f stability test

About 1g of this product (lot: D-181120-01) was weighed precisely, and subjected to the preparation and measurement of the test solution under the item "preliminary determination of method", and the samples were respectively placed after the preparation for 0, 2, 4, 8, 12, 24, 28, 36, and 48 hours for measurement, and the measurement results are shown in Table 42.

TABLE 42 stability test results

The result shows that RSD is less than 3 percent, and the test solution has good stability within 48 hours.

g accuracy test

Precisely weighing 11.49mg of morroniside (batch number: 111998-; accurately weighing 12.14mg of loganin reference substance (batch number: 111640-201707, purity: 99.2%) in a 20ml measuring flask, adding 80% methanol solution to dissolve and dilute to scale, and shaking up to obtain 0.6070mg/ml solution (II); precisely measuring the comparison solution (i) and (ii) respectively in 4-200 ml measuring bottles, adding 80% methanol solution to dilute to scale, and shaking up to obtain the mixed comparison solution (iii) with concentration of 22.38 μ g/ml morroniside and 12.14 μ g/ml loganin.

Taking about 0.5g (batch number: D-181120-01) (6 parts in total), precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of reference solution, sealing the plug, weighing, performing ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the lost weight with 80% methanol, shaking up, filtering, precisely absorbing 5 μ l of test solution, injecting into a liquid chromatograph, and measuring to obtain the results shown in tables 43-44. Corresponding real object (dry extract powder) (batch No. D-181120-01) of rehmanniae radix drinker substance has morroniside content of 1.1135mg/g and loganin content of 0.5863 mg/g.

TABLE 43 Monoside accuracy test results (n ═ 6)

TABLE 44 loganin accuracy test results (n ═ 6)

The result shows that the average recovery rate of morroniside is 101.26%, RSD is less than 3%, the average recovery rate of loganin is 97.10%, and RSD is less than 3%, which indicates that the method has better accuracy.

h intermediate precision test

Selecting different measurement time, different HPLC, and different experimenters (B), taking about 1.0g of the product (batch number: D-181120-01), precisely weighing, and performing preparation and measurement according to the method of preliminary determination. The results are shown in tables 45 to 46.

TABLE 45 intermediate precision test results for morroniside (n ═ 6)

TABLE 46 tramadol intermediate precision test results (n ═ 6)

As a result, the average content of the morroniside in the samples determined by A and B is 1.0948mg/g, and RSD is less than 2.0%; the average loganin content is 0.5798mg/g, RSD is less than 2.0%, which shows that the method has good intermediate precision.

i durability test

The durability of the chromatographic column to the conditions of the spectrum was examined for different flow rates, different column temperatures, different acid ratios, and different chromatographic columns, and the results are shown in Table 47.

TABLE 47 Monoloside and loganin durability test results

The results show that the determination results under various conditions are basically consistent, the RSD is less than 5%, the morroniside and loganin peaks in the chromatogram are sharp and symmetrical, and the separation degree is good, thus the method has good durability at different flow rates, different chromatographic columns, different column temperatures and different acidity.

The research results show that the method for measuring the content of the dogwood fruit in the substance (dry extract powder) corresponding to the radix rehmanniae potion basis has specificity, the stability, the repeatability, the intermediate precision, the accuracy and the like all accord with the regulations, and the durability of the chromatographic condition is better, so the liquid chromatographic condition can be used for measuring the content of the dogwood fruit in the substance (dry extract powder) corresponding to the radix rehmanniae potion basis.

Determination of content determination method

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile was used as mobile phase a, 0.3% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in table 48; the detection wavelength was 240 nm. The number of theoretical plates should not be less than 3000 calculated by the morroniside peak.

TABLE 48 procedure for mobile phase gradient elution

Seventhly, determining the content of corresponding real objects (dry paste powder) in different batches

The contents of morroniside and loganin in 15 batches (30 parts) of corresponding real substances (dry extract powder) were measured according to the preparation and measurement procedures of the test sample solution under the term "determination of content measurement method", and the results are shown in Table 49.

TABLE 4915 determination results of morroniside and loganin content in corresponding substance (dried powder) of 30 batches (parts)

And (4) conclusion: the content of morroniside and loganin in 15 batches (30 parts) of corresponding real objects (dry paste powder) is 1.8189 mg/g-6614 mg/g, the average value is 2.21mg/g, and considering various influence factors in the actual production process, the product is provisionally calculated according to the dry product, and the content of 1.5 mg-3.0 mg of the total amount of morroniside (C17H26O11) and loganin (C17H26O10) in every 1g of cornus officinalis containing product is calculated.

(2) Schisandra chinensis

Instrument and reagent

② selection of chromatographic conditions

a selection of mobile phase and mobile phase ratio

The method comprises the following steps: referring to a method for measuring the content of [467] schizandrol A under the item of "schisandra chinensis" in the first edition of Chinese pharmacopoeia 2015, the method is characterized by comprising the following steps:

chromatographic conditions and System suitability test by Ultimate LP C18 (4.6X 250mm, 5. mu.l); methanol-water (65:35) is used as a mobile phase; the detection wavelength was 250 nm.

Preparing reference solution by precisely weighing appropriate amount of schizandrol A reference, and adding methanol to obtain solution containing 0.3mg per 1 ml.

Preparing a test solution, precisely weighing 1g of the product, placing the product in a conical flask with a plug, precisely adding 10ml of methanol, weighing, ultrasonically treating for 20 minutes (250W and 40kHZ), taking out, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and taking a subsequent filtrate.

The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, determining, and recording chromatogram.

And (3) test results: negative interference and the method is not feasible.

The second method comprises the following steps: on the basis of the first method, the mobile phase is replaced by acetonitrile-water (45:55),

and (3) test results: has no interference to negative, and good schizandrol A peak type and peak separation degree.

b determination of the detection wavelength

Taking a proper amount of a schizandrol A reference substance, precisely weighing, adding 80% methanol to prepare a solution containing 20 mug of the schizandrol A per 1ml, scanning at the wavelength of 190-400 nm, and obtaining a full-wavelength scanning graph shown in figure 22, wherein the schizandrol A has a maximum absorption peak at 252nm and has no impurity interference, and the wavelength is basically consistent with 250nm of the content measurement wavelength of the schisandra chinensis in 2015 edition of Chinese pharmacopoeia, so 250nm is selected as the detection wavelength.

Investigation of preparation method of test solution

a examination of extraction solvent

Precisely weighing about 1.0g (3 groups) of the product (batch number: D-181120-01), placing in a conical flask with a plug, precisely adding 80% ethanol, 80% methanol and 25ml methanol respectively, sealing the plug, weighing, performing ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, supplementing lost weight with 80% ethanol, 80% methanol and methanol respectively, shaking up, filtering, taking out subsequent filtrate, precisely sucking 10 μ l of subsequent filtrate, injecting into a liquid chromatograph, and measuring. The results are shown in Table 50.

Table 50 results of solvent extraction

The experimental results are as follows: the contents of schizandrin A in different extraction solvents are basically consistent, and one part of test solution is used for measuring the contents of the schizandrin A, the morroniside and the loganin A, so 80% methanol is selected as the extraction solvent.

b, considering the extraction mode, precisely weighing 1g (3 groups) of the product (batch number: D-181120-01), placing the product into a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing the weight, respectively carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, carrying out reflux extraction for 30 minutes, shaking the extract for 30 minutes, taking out the extract, cooling the extract, weighing the weight again, complementing the lost weight with 80% methanol, shaking the extract uniformly, filtering the extract, taking the subsequent filtrate to obtain the final product, precisely sucking 10 mu l of the subsequent filtrate, injecting the final product into a liquid chromatograph, and measuring the final product. The results are shown in Table 51.

Table 51 examination results of extraction methods

The experimental results are as follows: the contents of the schizandrol A in different extraction modes are basically consistent, and the ultrasonic is selected as the extraction mode considering the convenient operation.

c investigation of extraction time

Precisely weighing about 1.0g (3 groups) of the product (batch number: D-181120-01), placing in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, respectively performing ultrasonic treatment (power 250W and frequency 40kHz) for 15 minutes, 30 minutes and 45 minutes, taking out, cooling, weighing again, supplementing lost weight with 80% methanol, shaking up, filtering, and taking out the subsequent filtrate, precisely sucking 10 μ l of the subsequent filtrate, injecting into a liquid chromatograph, and measuring. The results are shown in Table 52.

Table 52 extracts temporal findings

The results show that the content of the schizandrol A is not greatly different at different extraction times, and the extraction time is selected to be 30 minutes in consideration of the content difference of different batches of decoction pieces.

Preliminary determination of content determination method

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water (45:55) is used as a mobile phase; the detection wavelength was 250 nm. The number of theoretical plates is not less than 3000 calculated according to schizandrol A peak. Preparation of control solution A proper amount of schizandrol A control is precisely weighed, and 80% methanol is added to make into solution containing 20 μ g per 1 ml.

Methodology investigation

a specificity test

The schisandra chinensis contains schizandrol A, in order to investigate whether other medicines interfere the determination of the schizandrol A, the decoction pieces are weighed according to the prescription proportion to prepare the negative sample lacking the schisandra chinensis by the same method, the negative sample solution lacking the schisandra chinensis is prepared according to the treatment method of the test sample, and the chromatogram is recorded. The result shows that no chromatographic peak exists in the negative chromatogram in the retention time corresponding to the schizandrol A, which indicates that other decoction pieces except the schisandra chinensis have no interference to the determination of the schizandrol A, and the method has specificity in determining the content of the schizandrol A in the product.

b peak purity check

Injecting the test solution into a liquid chromatograph, performing full-wavelength detection by a PDA detector under the chromatographic conditions, and calculating peak purity, wherein no impurity peak is detected in schizandrol A in the test chromatogram; the minimum peak purity threshold value is 999.99, which indicates that the schizandrol A chromatographic peak purity meets the requirement under the chromatographic condition.

c investigation of linear relationship

Accurately weighing 9.96mg of a schizandrol A reference substance (batch number: 110857-201714, purity: 99.9%), putting the schizandrol A reference substance into a 10ml measuring flask, adding a proper amount of methanol, carrying out ultrasonic treatment to dissolve the schizandrol A reference substance, taking out the solution, cooling the solution, adding methanol to dilute the solution to a scale, and shaking the solution uniformly to obtain 0.9960mg/ml of a schizandrol A reference substance solution I; precisely measuring 1ml of the reference substance solution, placing the reference substance solution in a 5ml measuring flask, diluting the reference substance solution to a scale with methanol, shaking up the reference substance solution to prepare a schizandrol A reference substance solution (II) with the concentration of 0.1992 mg/ml; precisely measuring 1ml of the reference substance solution, placing the reference substance solution in a 50ml measuring flask, diluting the reference substance solution to scale with methanol, shaking up, and preparing the schisandrin reference substance solution with the concentration of 19.92 mu g/ml. Precisely sucking 2, 5, 10 and 15 μ l of the above control solution, respectively, injecting 2, 5 and 10 μ l of the control solution into a liquid chromatograph, and measuring by liquid chromatography (0512 in the four ministry of communications in the pharmacopoeia of China 2015). The sample amount (μ g) was taken as the abscissa and the peak area as the ordinate, and a standard curve was drawn. See table 53.

TABLE 53 Linear relationship of Schizandrol A

The results show that the schizandrol A has good linearity in the range of 0.03984-1.9920 mug.

d measurement of precision

Taking the schizandrol A control solution with the concentration of 19.92 μ g/ml, continuously injecting samples for 6 times, and determining results are shown in Table 54.

TABLE 54 Instrument precision test results (n ═ 6)

And (3) test results: RSD is less than 2.0%, and the precision of the instrument is good.

e repeatability test

The experimenter (A) performs the operation, and takes about 1.0g (total 6 parts) of the product (batch number: D-181120-01), precisely calls it, and performs the operation according to the preparation and determination method of the test solution under the item of 'preliminary determination of the method'. The results are shown in Table 55.

Table 55 repeatability test results (n ═ 6)

f stability test

About 1g of this product (lot: D-181120-01) was weighed precisely, and subjected to sample injection measurement for 0, 2, 4, 8, 12, 24, 28, 36, and 48 hours after preparation and measurement according to the test solution preparation and measurement method under the section of "preliminary determination of method", and the measurement results are shown in Table 56.

TABLE 56 stability test results

The result shows that RSD is less than 3.0%, and the stability of the test solution is good within 48 hours.

g accuracy test

Accurately weighing 10.37mg of schisandrin (batch number: 110857-201714, purity: 99.9%) in a 20ml measuring flask, adding 80% methanol solution for dissolving and diluting to scale, and shaking up to obtain a first reference substance solution with the concentration of 0.5185 mg/ml; precisely measuring a reference substance solution (1 ml to 100 ml) in a measuring flask, adding 80% methanol solution to dilute to a scale, and shaking up to obtain a reference substance solution (5.19 mug/ml); precisely measuring a comparison product solution (a measuring flask of 2ml to 100 ml), adding 80% methanol solution to dilute to a scale, and shaking up to obtain a comparison product solution (c) with the concentration of 10.37 mug/ml; precisely measuring the reference substance solution (3 ml to 100 ml) in a measuring flask, adding 80% methanol solution to dilute to scale, and shaking up to obtain the reference substance solution (15.56 μ g/ml)

Taking about 0.5g (batch number: D-181120-01) (9 parts in total), precisely weighing, placing in a conical flask with a plug, precisely adding reference substance solutions (25 ml each) respectively, sealing the plug, weighing, ultrasonically treating (power 250W, frequency 40kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with 80% methanol, shaking up, filtering, precisely absorbing 10 μ l of sample solution, injecting into a liquid chromatograph, and measuring to obtain the final product, wherein the results are shown in Table 57.

In the rehmanniae radix drink material standard (batch number: D-181120-01): the content of schisandrin glycoside is 0.4937 mg/g.

TABLE 57 accuracy test results for schizandrol A (n ═ 9)

The result shows that the average recovery rate of the schizandrol A is 98.44 percent, and the RSD is less than or equal to 3.0 percent, which shows that the method has better accuracy.

h intermediate precision test

Selecting different measurement time, different HPLC, and different experimenters (B), taking about 1.0g of the product (batch number: D-181120-01), precisely weighing, and performing preparation and determination according to the method of preliminary determination of the method. The results are shown in Table 58.

TABLE 58 schizandrin intermediate precision test results (n ═ 6)

As a result, the average content of the schizandrol A in the samples measured by A and B is 0.4888mg/g, and the RSD is less than 2.0 percent, which indicates that the method has good intermediate precision.

j durability test

The durability of the chromatographic conditions for different flow rates, different chromatographic columns, different column temperatures, different mobile phase ratios were examined and the results are shown in table 59.

TABLE 59 schizandrol A durability test results

The results show that the determination results under various conditions are basically consistent, the RSD is less than 5.0%, the schizandrol A peak in the chromatogram is sharp and symmetrical, and the separation degree is good, thus the method has good durability at different flow rates, different chromatographic columns, different column temperatures and different mobile phase proportions.

The research results show that the method for measuring the content of the schisandrin in the substance (dry extract powder) corresponding to the radix rehmanniae potion basis has specificity, the stability, the repeatability, the intermediate precision, the accuracy and the like all accord with the regulations, and the durability of the chromatographic condition is better, so the liquid chromatographic condition can be used for measuring the content of the schisandrin in the substance (dry extract powder) corresponding to the radix rehmanniae potion basis.

Determination of content determination method

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water (45:55) is used as a mobile phase; the detection wavelength was 250 nm. The number of theoretical plates is not less than 3000 calculated according to schizandrol A peak.

According to the preparation and measurement operation of the test solution under the item of "determination of content measurement method", the content of schisandrin in 15 batches (30 parts) of the corresponding real object (dry extract powder) was measured, and the results are shown in Table 60.

TABLE 6015 lots (30 parts) of the measurement results of schizandrol A content in corresponding real substance (dry extract powder)

And (4) conclusion: 15 batches (30 parts) of corresponding real objects (dry paste powder) have the schizandrol A content of 0.5307 mg/g-0.6776 mg/g and the average value of 0.58mg/g, and considering various influence factors and the like in the actual production process, the temporary product is calculated according to the dry product, and each 1g of the temporary product contains 0.4 mg-0.8 mg of the schizandrol A (C24H32O 7).

Quality analysis of k-corresponding species

15 batches of material reference corresponding physical result summary

The results of the primary tests are summarized in table 61 for 15 batches of material.

Table 6115 data summarization of material reference corresponding to real object

Determination of corresponding object key quality attribute range

Table 62 material reference to physical key mass attribute range

(1) Content of index component

15 batches (30 parts) of corresponding real object (dry paste powder) of morroniside and loganin with the content of 1.8189 mg/g-2.6614 mg/g and the average value of 2.21 mg/g; the schizandrol A content is 0.5307 mg/g-0.6776 mg/g, the average value is 0.58mg/g, considering various influence factors in the actual production process, etc., the product is tentatively calculated according to the dry product, and every 1g of the cornus officinalis is calculated by the total amount of morroniside (C17H26O11) and loganin (C17H26O10), and the content is 1.5 mg-3.0 mg; every 1g of the Chinese magnoliavine fruit contains 0.4mg to 0.8mg of Chinese magnoliavine fruit calculated by schisandrin (C24H32O 7).

(2) Finger print

The chromatogram of 15 batches (30 parts) of corresponding real object (dry paste powder) is adopted to generate a control fingerprint (see figure 23). The sample fingerprint should present a chromatographic peak with the same retention time as the reference chromatographic peak, and the similarity is calculated according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity is calculated by using the common peak, and the similarity between the sample fingerprint and the reference fingerprint is not less than 0.90.

The invention is obtained through experiments: 15 batches (30 parts) of corresponding real object (dry paste powder) of morroniside and loganin with the content of 1.8189 mg/g-2.6614 mg/g and the average value of 2.21 mg/g; the schizandrol A content is 0.5307 mg/g-0.6776 mg/g, the average value is 0.58mg/g, considering various influence factors in the actual production process, the product is tentatively calculated by morroniside (C) per 1g of fructus Corni based on the dry product₁₇H₂₆O₁₁) And loganin (C)₁₇H₂₆O₁₀) The total amount is 1.5 mg-3.0 mg; every 1g contains Schisandra chinensis and schizandrol A (C)₂₄H₃₂O₇) The dosage is 0.4 mg-0.8 mg.

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The rehmannia root potion pharmaceutical composition is characterized by comprising the following components in percentage by weight: 0.5-1.5 parts of prepared rehmannia root, 0.5-1.5 parts of morinda officinalis, 0.5-1.5 parts of cornus officinalis, 0.5-1.5 parts of dendrobe, 0.5-1.5 parts of cistanche salsa, 0.5-1.5 parts of sliced aconite, 0.5-1.5 parts of schisandra chinensis, 0.5-1.5 parts of cinnamon, 0.5-1.5 parts of poria cocos, 0.5-1.5 parts of radix ophiopogonis, 0.5-1.5 parts of rhizoma acori graminei, 0.5-1.5 parts of polygala tenuifolia, 4-6 parts of ginger, 3-6 parts of Chinese date and 0.5-1.5 parts of mint;

the preparation method of the composition comprises the following steps:

(1) pretreatment

Weighing radix rehmanniae Preparata, radix Morindae officinalis, Corni fructus, herba Dendrobii, Cistanchis herba, radix Aconiti lateralis Preparata, fructus Schisandrae, cortex Cinnamomi, Poria, radix Ophiopogonis, rhizoma Acori Graminei, and cortex et radix Polygalae, respectively pulverizing into coarse powder; the ginger is cut into 2-4 mm thick slices when being used, and the Chinese date is broken when being used; standby;

(2) decocting and decocting

Putting the pretreated medicines and the mint into a 600ml casserole, adding 200-400 ml of water, heating by open fire, covering and decocting, boiling by strong fire, and decocting for 15-25 minutes by slow fire to obtain a decoction;

(3) filtration

Filtering the decoction liquid by using 200-300-mesh filter cloth under normal pressure while the decoction liquid is hot to obtain filtrate;

(4) concentrating

Concentrating the filtrate under reduced pressure at the temperature of 60-70 ℃ and the pressure of-0.08 to-0.1 MPa until the mass ratio of the fed amount of the decoction pieces to the concentrated solution is 1g:3 g-1 g:4 g;

(5) freeze drying

Freeze-drying the concentrated solution under the following conditions: pre-freezing temperature: -20 to-48 ℃, cold trap temperature: -40 to-80 ℃, time: at least 30 minutes; and (3) drying: cold trap temperature: -60 to-70 ℃, vacuum degree: < 100Pa, lyophilization time: at least 17 hours to obtain dry paste powder;

2. The composition of claim 1, wherein the composition is prepared by a process comprising the steps of:

(1) pretreatment

Weighing prepared rehmannia root, morinda officinalis, cornus officinalis, dendrobium, cistanche deserticola, prepared aconite, schisandra chinensis, cinnamon, poria cocos, radix ophiopogonis, rhizoma acori graminei and polygala tenuifolia, crushing into coarse powder, cutting ginger into thick slices of 2-4 mm when in use, and breaking Chinese dates when in use;

(2) decocting and decocting

Placing the pretreated medicines and the mint in a 600ml casserole, adding 300ml of water, heating by open fire, covering and decocting, boiling by strong fire, and decocting by slow fire for 20 minutes to obtain a decoction;

(3) filtration

Filtering the decoction with 300 mesh filter cloth under normal pressure; obtaining filtrate;

(4) concentrating

Concentrating the filtrate under reduced pressure at 70 ℃ and under the pressure of-0.08 to-0.1 MPa until the mass ratio of the fed amount of the decoction pieces to the concentrated solution is 1g:4 g;

(5) freeze drying

The freeze-drying conditions were: pre-freezing temperature: -20 to-48 ℃, cold trap temperature: -40 to-80 ℃, time: at least 30 minutes; and (3) drying: cold trap temperature: -60 to-70 ℃, vacuum degree: < 100Pa, lyophilization time: at least 17 hours to obtain dry paste powder;

3. The composition according to claim 1 or 2, wherein the pharmaceutical composition consists of the following formulation: 1 part of prepared rehmannia root, 1 part of morinda officinalis, 1 part of cornus officinalis, 1 part of dendrobium, 1 part of wine cistanche, 1 part of processed aconite, 1 part of schisandra chinensis, 1 part of cinnamon, 1 part of poria cocos, 1 part of radix ophiopogonis, 1 part of rhizoma acori graminei, 1 part of polygala tenuifolia, 5 parts of ginger, 4.6 parts of Chinese date and 1 part of mint.

4. The pharmaceutical composition according to any one of claims 1 to 2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable formulation prepared from pharmaceutically acceptable excipients.

5. The pharmaceutical composition of claim 4, wherein the dosage form is a granule, a capsule, a tablet, a pill, a powder, an injection, or an ointment.

6. The detection method for the pharmaceutical composition of any one of claims 1-2, wherein the detection method for the pharmaceutical composition comprises qualitative identification, fingerprint spectrum and content measurement, and comprises the following specific detection methods:

identifying (1) taking 4g of the product, adding 40-60 ml of water, dissolving by ultrasonic, shaking and extracting by dichloromethane for 1-3 times, 20-40 ml each time, shaking and extracting by n-butanol for 1-3 times, 20-40 ml each time, combining n-butanol liquid, washing by 1 → 10 ammonia solution for 1-3 times, 20-30 ml each time, discarding ammonia solution, evaporating n-butanol liquid to dryness, adding 1ml of methanol to dissolve residues, taking 1g of dogwood control medicinal material, adding 50ml of water, decocting for 15-25 minutes, filtering, shaking and extracting filtrate by dichloromethane for 2 times, 30ml each time, discarding dichloromethane liquid, shaking and extracting water liquid 2 times by n-butanol, preparing control medicinal material solution by the same method, taking morroniside control and loganin control, adding 80% methanol to prepare mixed solution containing 1mg of each 1ml, taking control solution as 2015, conducting thin layer chromatography test according to 0502 in the four parts of pharmacopoeia 2015 edition, sucking sample solution 5 μ l, control solution 1 μ l, and control solution 2 μ l, respectively dropping on the same silica gel GF₂₅₄Developing on a thin-layer plate by using chloroform-methanol as a developing agent in a ratio of 4: 1-3: 1, taking out, drying, and inspecting under an ultraviolet lamp with a wavelength of 254nm, wherein fluorescent spots with the same color appear in the chromatogram of the test sample at positions corresponding to the chromatograms of the reference medicinal material and the reference substance;

(2) taking 1g of the product, adding 25-35 ml of 70% methanol, heating and refluxing for 0.5-1.5 hours, filtering, evaporating filtrate to dryness, dissolving residue by using 15-25 ml of 0.1mol/L sodium hydroxide solution, shaking and extracting for 1-3 times by using ethyl acetate, 15-25 ml each time, combining ethyl acetateEvaporating ester solution to dryness, dissolving residue in 1ml methanol to obtain sample solution, collecting fructus Schisandrae control 1g, adding water 50ml, decocting for 20 min, filtering, shaking with ethyl acetate for 2 times, making into control solution by the same method, collecting fructus Schisandrae control, adding 80% methanol to obtain solution containing 1mg of fructus Schisandrae control per 1ml, and using as control solution; according to the test of thin layer chromatography of 0502 in 2015 th edition of Chinese pharmacopoeia, 5 μ l of sample solution, 1 μ l of control solution, and 2 μ l of control solution are respectively spotted on the same silica gel GF₂₅₄On the thin-layer plate, developing with petroleum ether-ethyl acetate-methanol at 60-90 ℃ in a ratio of 5:3: 0.2-15: 5:2 as a developing agent, taking out, drying in the air, and inspecting under an ultraviolet lamp with the wavelength of 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;

the fingerprint spectrum is measured by high performance liquid chromatography according to 0512 of the general rules of four departments of 2015 edition of Chinese pharmacopoeia;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking acetonitrile as a mobile phase A, taking a 0.3% phosphoric acid solution as a mobile phase B, and carrying out gradient elution according to a specified mobile phase gradient elution program; the detection wavelength is 254 nm; the number of theoretical plates is not less than 3000 calculated according to schizandrol A; the mobile phase gradient elution procedure is specifically as follows:

preparing a test solution, precisely weighing 2.0g of the product, placing the product in a conical flask with a plug, precisely adding 20-30 ml of 80% methanol, sealing the plug, weighing, ultrasonically treating the product for 30 minutes at the power of 250W and the frequency of 40kHz, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, extracting the filtrate by using an ethyl acetate-n-butyl alcohol mixed solution with the ratio of 1:1 for 2 times, shaking up 25ml each time, combining the ethyl acetate-n-butyl alcohol mixed solution with the ratio of 1:1, evaporating to dryness, dissolving the residue by adding methanol, transferring the residue into a 5ml measuring flask, diluting the residue to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution;

the sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 14 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatogram fingerprint similarity evaluation system, the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90, the reference fingerprint is shown in figure 23,

content measurement of Corni fructus by high performance liquid chromatography according to 0512 of the general rules of four parts of the 2015 version of Chinese pharmacopoeia;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking acetonitrile as a mobile phase A, taking a 0.3% phosphoric acid solution as a mobile phase B, and carrying out gradient elution according to a specified mobile phase gradient elution program; the detection wavelength is 240 nm; the number of theoretical plates is not less than 3000 calculated according to the morroniside peak; the mobile phase gradient elution procedure is specifically as follows:

the fructus Schisandrae is determined by high performance liquid chromatography according to 0502 of four general rules of the pharmacopoeia 2015 edition;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water in a ratio of 45:55 is taken as a mobile phase; the detection wavelength is 250nm, and the number of theoretical plates is not less than 3000 calculated according to the schizandrol A peak;

preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 20-30 ml of 80% methanol, sealing the plug, weighing, ultrasonically treating the product with 250W of power and 40kHz of frequency for 25-35 minutes, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution;

7. The method for detecting the pharmaceutical composition according to claim 6, wherein the specific detection methods of qualitative identification, fingerprint and content determination are as follows:

identifying (1) taking 4g of the product, adding 50ml of water, dissolving by ultrasonic, extracting with dichloromethane for 2 times, 30ml each time, extracting with n-butanol for 2 times, 30ml each time, combining n-butanol solutions, washing with 1 → 10 ammonia solution for 2 times, 25ml each time, discarding ammonia solution, evaporating n-butanol solution to dryness, adding 1ml of methanol to residue to dissolve to obtain test solution, taking 1g of Corni fructus as control material, adding 50ml of water, decocting for 20 minutes, filtering, extracting filtrate with dichloromethane for 2 times, 30ml each time, discarding dichloromethane solution, extracting water solution with n-butanol for 2 times, preparing control solution by the same method, taking morroniside control and loganin control, adding 80% methanol to obtain mixed solution containing 1mg each 1ml, taking control solution as control solution, performing thin layer chromatography test according to 2015 edition of pharmacopoeia of China general rules 0502, sucking 5 μ l of the test solution, Respectively dropping 1 μ l of control solution and 2 μ l of control solution on the same silica gel GF254 thin layer plate, developing with 3:1 chloroform-methanol as developing agent, taking out, air drying, inspecting under 254nm ultraviolet lamp, and displaying fluorescent spots of the same color in the chromatogram of the test solution at the positions corresponding to the chromatogram of the control solution and the control solution;

(2) taking 1g of the product, adding 30ml of 70% methanol, heating and refluxing for 1 hour, filtering, evaporating filtrate to dryness, dissolving residues by using 20ml of 0.1mol/L sodium hydroxide solution, shaking and extracting for 2 times by using ethyl acetate, 20ml each time, combining ethyl acetate solutions, evaporating to dryness, dissolving residues by adding 1ml of methanol to serve as a test solution, taking 1g of a schisandra chinensis control medicinal material, adding 50ml of water, decocting for 20 minutes, filtering, shaking and extracting for 2 times by using ethyl acetate, preparing the control medicinal material solution by the same method, taking a schizandrol A control product, adding 80% methanol to prepare a solution containing 1mg of the schisandra chinensis in each 1ml to serve as a control solution; performing thin-layer chromatography test according to 0502 of general Law of Chinese pharmacopoeia 2015, respectively dropping 5 μ l of test solution, 1 μ l of control solution and 2 μ l of control solution on the same silica gel GF254 thin-layer plate, developing with 60-90 deg.C petroleum ether-ethyl acetate-methanol at a ratio of 5:3:0.2 as developing agent, 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;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking acetonitrile as a mobile phase A, taking a 0.3% phosphoric acid solution as a mobile phase B, and carrying out gradient elution according to a specified mobile phase gradient elution program; the detection wavelength is 254 nm; the number of theoretical plates is not less than 3000 calculated according to schizandrol A; the mobile phase gradient elution procedure was:

preparing a test solution, precisely weighing 2.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, ultrasonically treating the product for 30 minutes at the power of 250W and the frequency of 40kHz, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, extracting the filtrate by using an ethyl acetate-n-butyl alcohol mixed solution with the ratio of 1:1 for 2 times, shaking up for 25ml each time, combining the ethyl acetate-n-butyl alcohol mixed solution with the ratio of 1:1, evaporating to dryness, dissolving the residue by adding methanol, transferring the residue into a 5ml measuring flask, diluting the residue to a scale by using methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the test solution;

a chromatographic peak with the same retention time as that of a chromatographic peak of a reference substance is required to be presented in the sample fingerprint, the sample chromatogram is basically consistent with the reference fingerprint and has 14 corresponding common peaks, the similarity is calculated by the common peaks according to a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint is not less than 0.90; the reference fingerprint is shown in figure 23;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; taking acetonitrile as a mobile phase A, taking a 0.3% phosphoric acid solution as a mobile phase B, and carrying out gradient elution according to a specified mobile phase gradient elution program; the detection wavelength is 240 nm; the number of theoretical plates is not less than 3000 calculated according to the morroniside peak;

the gradient elution procedure was:

preparing a test solution, precisely weighing 1.0g of the product, placing the product in a conical flask with a plug, precisely adding 25ml of 80% methanol, sealing the plug, weighing, ultrasonically treating the product for 30 minutes at the power of 250W and the frequency of 40kHz, cooling, weighing again, complementing the weight loss by 80% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the product;

fructus Schisandrae chinensis is measured according to high performance liquid chromatography in 2015 edition of Chinese pharmacopoeia 0502;