CN106728651B

CN106728651B - Preparation method and quality detection method of rhizoma cyperi four-ingredient granules

Info

Publication number: CN106728651B
Application number: CN201710156960.0A
Authority: CN
Inventors: 段金廒; 钱大玮; 刘培; 宿树兰
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2017-03-16
Filing date: 2017-03-16
Publication date: 2020-10-30
Anticipated expiration: 2037-03-16
Also published as: CN106728651A

Abstract

The invention discloses a process for preparing cyperus tuber four-component granules and its quality control method, wherein the granules are prepared from Chinese angelica root, Ligusticum wallichii, cyperus tuber and banksia rose through steps of extracting volatile oil by wet distillation, grinding and clathrating the volatile oil with beta-cyclodextrin, charging prepared rhizome of rehmannia, root of herbaceous peony and corydalis tuber into residue after extracting volatile oil, charging water, decocting and extracting, precipitating the extract with 95% alcohol, stewing, filtering, concentrating the supernatant fluid under reduced pressure, drying to obtain dry extract, charging sugar powder and dextrin, mixing, granulating, drying, finishing granules, charging inclusion compound and mixing. The quality control method comprises thin layer qualitative identification and HPLC content determination. The volatile oil part is separately extracted and included, so that the invention has double effects of reducing the dosage of auxiliary materials and playing a role in treatment, can fully play the advantages of compatibility of the traditional Chinese medicines compared with the prior single-ingredient formula granules when being taken, establishes perfect quality standard and can effectively control the quality of the compound granules.

Description

Preparation method and quality detection method of rhizoma cyperi four-ingredient granules

Technical Field

The invention belongs to the technical field of traditional Chinese medicine formula granules, and particularly relates to a preparation method and a quality control method of cyperus rotundus granule.

Background

The cyperus rotundus decoction is prepared from seven medicines of cyperus rotundus, elecampane, corydalis tuber, angelica, ligusticum wallichii, prepared rehmannia root, white paeony root and the like, is one of typical formulas for promoting qi circulation and removing blood stasis, is from volume four of Qing Dynasty Lianfu (unknown medical essences), has the effects of nourishing blood, regulating blood, promoting qi circulation and relieving pain, and is mainly used for treating dysmenorrheal, irregular menstruation and other symptoms caused by qi stagnation and blood stasis.

Dysmenorrhea is mainly related to the increase of synthesis and release of prostaglandin in endometrium during menstrual period, uterine muscle is hyperreactive, and pain is caused by secondary uterine muscle ischemia. The traditional Chinese medicine considers that the main pathogenesis of dysmenorrhea is unsmooth qi and blood circulation, qi movement blockage and obstruction leading to pain caused by emotional injury, carelessness in daily life or six excesses. Unsmooth circulation of qi and blood is the most important pathological basis, and clinically, dysmenorrhea caused by qi stagnation and blood stasis is common.

The cyperus rotundus decoction is prepared from rhizoma cyperi, elecampane and rhizoma corydalis, has the effects of nourishing blood, regulating blood, promoting qi circulation and relieving pain, and is mainly used for treating dysmenorrheal, irregular menstruation and other symptoms caused by qi stagnation and blood stasis. Is one of the typical recipes for promoting the circulation of qi and removing blood stasis. The nutgrass galingale rhizome is pungent, sweet and slightly bitter in taste, has mild and warm nature, mainly enters liver meridian, is pungent and warm in nature, is capable of entering liver meridian, can pass qi and blood, can condition depression of liver qi and can regulate stagnation of menstrual blood, so that the nutgrass galingale rhizome has the functions of regulating liver, regulating qi, regulating menstruation and relieving pain. It is usually combined with Dang Gui to play the role of regulating qi and promoting blood circulation, with the actions of governing qi, blood and qi. Radix aucklandiae is pungent and bitter in flavor and warm in nature, mainly enters spleen, stomach, large intestine and triple energizer meridians, is pungent and fragrant in flavor and capable of dispersing, bitter in flavor and cooling, can ascend and descend, and clear triple energizer, has the effects of promoting qi circulation and relieving pain, and invigorating spleen and helping digestion, and is often combined with rhizoma cyperi, rhizoma corydalis and the like to sooth liver, activate blood and regulate menstruation and relieve pain. Corydalis tuber, pungent and bitter in flavor and warm in property, mainly enters heart, liver, spleen and lung meridians, and is pungent, bitter in flavor, pungent, capable of purging and warming and unblocking, enters both heart and liver meridians and spleen and lung meridians to circulate blood and qi, so as to activate blood, circulate qi in blood and alleviate pain, so it is the essential herb for activating blood, moving qi and alleviating pain, and all the meridians in the upper and lower parts of the body belong to qi stagnation and blood stasis. It is often combined with Dang Gui to dredge meridians, activate stagnation and relieve pain.

Biological effect research shows that the cyperus rotundus decoction has obvious inhibition effect on the whole mouse dysmenorrhea model, can obviously inhibit the contraction frequency and the contraction activity of the isolated uterus of the mouse, and obviously enhances the inhibition effect on COX-2 enzyme; can obviously improve the hemorheology index of a qi stagnation and blood stasis model rat, has unobvious effect of improving peripheral hemogram of an intervened blood deficiency model rat, and has main analgesic effect in the function of inhibiting dysmenorrhea. Clinical researches show that the total effective rate of the formula for treating qi stagnation and blood stasis type primary dysmenorrhea exceeds 80%, and larger varieties of Yueyueshutongjinbao granules and ibuprofen sustained-release capsules can better improve the symptoms of dysmenorrhea of patients.

The traditional Chinese medicine decoction is used as a main dosage form of traditional Chinese medicine clinical medication, has the advantages of addition and subtraction according to symptoms, flexible formula, easy absorption, quick response and the like, and is deeply trusted by patients. But can not meet the living requirements of modern people due to the defects of easy mildew, spoilage, bitter taste, large amount and the like of blending, carrying, temporary decoction and long-term storage. The traditional Chinese medicine formula particles keep the multi-drug effect of a single medicine and retain the active ingredients of the medicine to the maximum extent, but the traditional Chinese medicine formula particles are extracts of the single medicine at present, and have the difference problem of 'co-decoction' and 'separate decoction' when the traditional Chinese medicine formula particles are used, namely 'single-medicine extraction and mixed taking with water'. The compound formula granule inherits the advantages of the single traditional Chinese medicine formula granule, considers the interaction of decoction pieces in the decoction process, accords with the traditional Chinese medicine theory, and has great value.

Disclosure of Invention

The invention aims to provide a preparation method and a quality control method of cyperus rotundus four-substance particles.

The invention is realized by the following technical scheme:

a preparation method of rhizoma cyperi four-ingredient granules is prepared from rhizoma cyperi, elecampane, rhizoma corydalis, angelica sinensis, ligusticum wallichii, radix rehmanniae preparata and radix paeoniae alba according to a weight ratio of 3:2:3:6:3:8:3, and specifically comprises the following steps:

a. weighing angelica, ligusticum wallichii, rhizoma cyperi and costustoot decoction pieces according to the weight ratio, adding water with the weight 5-15 times of the total weight of the medicinal materials, soaking for 10-15 hours, and then extracting for 8-12 hours by steam distillation to extract volatile oil;

b. adding beta-cyclodextrin and water into the volatile oil, fully grinding, and freeze-drying to obtain a volatile oil clathrate;

c. taking the residue obtained after the volatile oil extraction, adding the prepared rehmannia root, the white paeony root and the corydalis tuber according to the weight ratio, adding water, decocting for 2-3 times, wherein the water adding amount is 6-14 times of that of the total medicinal materials each time, decocting for 1-3 hours, combining the medicinal liquids, filtering, drying under reduced pressure to obtain a concentrated solution with the relative density of 1.00-1.15, adding ethanol with the volume concentration of 95% until the ethanol concentration in the concentrated solution is 80-85%, standing, carrying out suction filtration, taking the filtrate, concentrating under reduced pressure, and drying to obtain dry extract powder;

d. and (c) adding dextrin and sugar powder into the dry paste powder according to a certain proportion, uniformly mixing, granulating, drying, grading, and finally adding the volatile oil inclusion compound prepared in the step (b).

As a preferable scheme, the preparation method of the cyperus rotundus four-substance particles specifically comprises the following steps:

a. weighing angelica, ligusticum wallichii, rhizoma cyperi and costustoot decoction pieces according to the weight ratio, adding water with the weight being 8 times of the total weight of the medicinal materials, soaking for 12 hours, and then carrying out steam distillation extraction for 10 hours to extract volatile oil;

c. adding radix rehmanniae Preparata, radix Paeoniae alba and rhizoma corydalis in the above weight ratio into the residue obtained after volatile oil extraction, decocting with water for 2 times, each time adding water amount 10 times of the total medicinal materials, decocting for 3 hr, mixing the medicinal liquids, filtering, drying under reduced pressure to obtain concentrated solution with relative density of 1.05-1.08, adding 95% ethanol until the ethanol concentration in the concentrated solution is 80%, standing for 24 hr, vacuum filtering, concentrating the filtrate under reduced pressure, and drying to obtain dry extract powder;

d. and (c) adding dextrin and sugar powder into the dry paste powder, uniformly mixing, granulating, drying, grading, and finally adding the volatile oil inclusion compound prepared in the step (b).

The angelica, the ligusticum wallichii, the rhizoma cyperi and the elecampane contain volatile oil, and in order to extract effective components to the maximum extent, the volatile oil is obtained by adopting a volatile oil collecting device and is subjected to inclusion treatment. In the preferable step b, the addition amount of the beta-cyclodextrin is 5-20 times, particularly preferably 8-15 times of the volume of the volatile oil; the amount of water added is 10-50 times, especially 20-40 times of the volume of the volatile oil.

Preferably, in step d, the dextrin is added in an amount of 0-2 times, particularly preferably 1-2 times, the weight of the dry extract powder, and the sugar powder is added in an amount of 0-2 times, particularly preferably 1-2 times, the weight of the dry extract powder.

The invention also provides a quality control method of the cyperus rotundus granule prepared by the preparation method, which adopts thin-layer chromatography qualitative identification and high performance liquid chromatography content determination.

The method specifically comprises one or more of the following methods:

the quality control method of the cyperus rotundus granule is characterized by comprising one or more of the following methods:

(1) qualitative identification by thin layer chromatography

Taking rhizoma cyperi four-substance particles, grinding, adding a sodium bicarbonate solution, carrying out ultrasonic treatment and centrifugation, taking supernate, adjusting the pH value to 2-3 by using dilute hydrochloric acid, shaking and extracting for 2-3 times by using ether, combining ether solutions, volatilizing, and adding methanol into residues to dissolve the residues to obtain a ligustilide test solution;

taking nutgrass galingale rhizome four-substance particles, adding ethanol for ultrasonic dissolution, placing the nutgrass galingale rhizome four-substance particles on a water bath for concentration, shaking and extracting the nutgrass galingale rhizome four-substance particles for 2 to 3 times by using ether, combining ether extract, adding sodium carbonate solution into the ether solution for shaking and extracting, removing the ether solution, adjusting the pH value of the extract to 2 to 3 by using dilute hydrochloric acid, shaking and extracting the extract by using ether, combining the ether extract, volatilizing the ether extract, and adding methanol to dissolve residues to obtain ferulic acid test solution;

taking angelica and ligusticum wallichii contrast medicinal materials, adding ethanol for ultrasonic treatment, concentrating on a water bath, shaking and extracting for 2-3 times by using ether, combining ether extract, adding sodium carbonate solution into the ether solution for shaking and extracting, removing the ether solution, adjusting the pH value of the extract to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ether, combining the ether extract, volatilizing, and dissolving residues by adding methanol to prepare a contrast medicinal material solution;

taking rhizoma cyperi, elecampane, rhizoma corydalis, radix rehmanniae preparata and radix paeoniae alba, adding ethanol for ultrasonic treatment, placing the mixture on a water bath for concentration, extracting the mixture for 2 to 3 times by shaking with diethyl ether, combining diethyl ether extracting solutions, adding a sodium carbonate solution into the diethyl ether solution for extracting by shaking, removing the diethyl ether solution, adjusting the pH value of the extracting solution to 2 to 3 by using dilute hydrochloric acid, extracting by shaking with diethyl ether, combining the diethyl ether extracting solutions, volatilizing the solution, and dissolving residues by adding methanol to prepare a double-negative control solution;

taking ferulic acid and ligustilide reference substances, and adding methanol to obtain reference substance solution;

performing thin-layer chromatography test, respectively dropping the above 5 solutions on the same silica gel G thin-layer plate, developing with benzene-glacial acetic acid-methanol at volume ratio of 30:1:3 as developing agent, taking out, air drying, placing under ultraviolet lamp, and inspecting at 365nm to obtain results, wherein spots of the same color are displayed at corresponding positions of the sample and the reference, and the negative reference has no interference;

(2) qualitative identification by thin layer chromatography

Grinding rhizoma Cyperi granule, adding diethyl ether, heating under reflux, filtering, volatilizing the filtrate, and dissolving the residue with ethyl acetate to obtain sample solution;

collecting rhizoma Ligustici Chuanxiong powder, adding diethyl ether, heating under reflux, filtering, volatilizing the filtrate, dissolving the residue with ethyl acetate to obtain control solution;

heating and refluxing rhizoma Cyperi, radix aucklandiae, rhizoma corydalis, radix rehmanniae Preparata, radix Angelicae sinensis and radix Paeoniae alba with diethyl ether, filtering, volatilizing the filtrate, dissolving the residue with ethyl acetate to obtain negative control solution;

taking an angelica lactone A reference substance, and adding ethyl acetate to prepare a reference substance solution;

performing thin-layer chromatography test, dropping the above 4 solutions on the same silica gel GF254 thin-layer plate, developing with n-hexane-ethyl acetate at volume ratio of 3:1 as developing agent, taking out, air drying, and inspecting under ultraviolet lamp at 254nm to obtain the result that spots of the same color appear at corresponding positions of the sample and the reference, and the negative reference has no interference;

(3) qualitative identification by thin layer chromatography

Taking cyperus rotundus four-substance particles, grinding, adding diethyl ether, standing for 1-2 hours, shaking constantly, filtering, volatilizing filtrate, adding ethyl acetate into residues for dissolving to obtain a test solution;

taking rhizoma cyperi reference medicinal material powder, adding diethyl ether, standing for 1-2 hours, shaking constantly, filtering, volatilizing filtrate, adding ethyl acetate into residues to dissolve to prepare reference medicinal material solution;

taking radix aucklandiae, rhizoma corydalis, angelica, ligusticum wallichii, radix rehmanniae preparata and radix paeoniae alba, adding diethyl ether, standing for 1-2 hours, shaking constantly, filtering, volatilizing filtrate, and adding ethyl acetate into residues to dissolve to prepare a negative control solution;

adding ethyl acetate into alpha-cyperone reference substance to obtain a reference substance solution;

performing thin-layer chromatography test, respectively dropping the above 4 solutions on the same silica gel GF254 thin-layer plate, developing with dichloromethane-ethyl acetate-glacial acetic acid at volume ratio of 80:1:1 as developing agent, taking out, air drying, inspecting under ultraviolet lamp 254nm, spraying 2, 4-dinitrophenylhydrazine ethanol solution, and inspecting under visible light; as a result, spots with the same color appear at the corresponding positions of the test sample and the reference sample, and the negative reference sample has no interference;

(4) qualitative identification by thin layer chromatography

Grinding rhizoma cyperi four-substance particles, adding petroleum ether, cold soaking for 30-60 minutes, shaking constantly, filtering, volatilizing filtrate, and adding ethyl acetate into residues to dissolve the residues to obtain a test solution;

taking powder of a costustoot reference medicinal material, adding petroleum ether, cold soaking for 30-60 minutes, shaking constantly, filtering, volatilizing filtrate, and adding ethyl acetate into residues to dissolve the residues to prepare a reference medicinal material solution;

taking rhizoma cyperi, rhizoma corydalis, angelica sinensis, ligusticum wallichii, radix rehmanniae preparata and radix paeoniae alba, adding petroleum ether, cold soaking for 30-60 minutes, shaking constantly, filtering, volatilizing filtrate, and adding ethyl acetate into residues to dissolve to prepare a negative control solution;

taking dehydrocostuslactone reference substance and costunolide reference substance, and adding methanol to obtain reference substance solution;

performing thin-layer chromatography test, respectively dropping the 4 solutions on the same silica gel G thin-layer plate, developing with cyclohexane-ethyl acetate-formic acid as developing agent at volume ratio of 15:5:1, taking out, air drying, spraying 5% vanillin-sulfuric acid ethanol solution, heating until the spots are clearly developed, inspecting with sunlight, and the result shows that the spots of the same color appear at the corresponding positions of the sample and the reference, and the negative reference has no interference;

(5) qualitative identification by thin layer chromatography

Taking 15g of particles, grinding, adding 100mL of water, fully dissolving by warming, cooling, filtering with absorbent cotton, shaking and extracting the filtrate with diethyl ether for 2 times, 25mL each time, discarding the ethyl ether solution, shaking and extracting with ethyl acetate for 2 times, 25mL each time, combining the ethyl acetate solutions, evaporating to dryness, and adding 1mL of methanol to dissolve the residue to obtain a sample solution. Preparing 1g of radix rehmanniae Preparata powder into control medicinal solution by the same method; 15g of the sample without the rehmannia glutinosa is prepared into a negative control solution by the same method. Taking a 5-hydroxymethylfurfural reference substance, and adding methanol to prepare a solution containing 0.290mg per 1mL as a reference substance solution. Performing thin layer chromatography (general rule 0502) test, sucking the above 4 solutions, respectively dropping on the same silica gel G thin layer plate, developing with xylene-ethyl acetate (1:1) as developing agent, taking out, air drying, spraying 2, 4-dinitrophenylhydrazine ethanol solution, and inspecting with sunlight. As a result, spots of the same color appear on the corresponding positions of the test sample and the control sample. The negative control product has no interference.

(6) Qualitative identification by thin layer chromatography

Taking nutgrass galingale rhizome four-substance particles, grinding, adding water, heating for dissolving, extracting with water saturated n-butyl alcohol for 2-3 times, combining n-butyl alcohol solutions, washing with n-butyl alcohol saturated water, combining the n-butyl alcohol solutions, volatilizing in a water bath, adding methanol for dissolving, adopting a silica gel column, eluting with chloroform and methanol in a volume ratio of 10:1, then eluting with chloroform and methanol in a volume ratio of 9:1, preferably eluting with chloroform and methanol in a volume ratio of 8:1, collecting subsequent 8:1 eluent, evaporating to dryness, and dissolving residues with methanol to obtain a sample solution;

taking radix paeoniae alba reference medicinal material powder, adding water, carrying out ultrasonic treatment, extracting for 2-3 times by using water saturated n-butyl alcohol, combining n-butyl alcohol solutions, washing by using n-butyl alcohol saturated water, combining the n-butyl alcohol solutions, volatilizing in a water bath, adding methanol for dissolving, adopting a silica gel column, eluting by using chloroform and methanol with the volume ratio of 10:1, then eluting by using chloroform and methanol with the volume ratio of 9:1, preferably by using chloroform and methanol with the volume ratio of 8:1, collecting subsequent 8:1 eluent, evaporating to dryness, and dissolving residues by adding methanol to prepare a reference medicinal material solution;

taking rhizoma cyperi, elecampane, rhizoma corydalis, angelica sinensis, ligusticum wallichii and radix rehmanniae preparata, adding water, carrying out ultrasonic treatment, extracting for 2-3 times by using water saturated n-butanol, combining n-butanol liquid, washing by using n-butanol saturated water, combining the n-butanol liquid, volatilizing in a water bath, adding methanol for dissolving, adopting a silica gel column, eluting by using chloroform and methanol with a volume ratio of 10:1, then eluting by using chloroform and methanol with a volume ratio of 9:1, preferably eluting by using chloroform and methanol with a volume ratio of 8:1, collecting subsequent 8:1 eluent, evaporating to dryness, dissolving residues by adding methanol, and preparing a negative control solution;

accurately weighing penoniflorin reference substance, and adding methanol to obtain reference substance solution;

performing thin-layer chromatography test, respectively dropping the above 4 solutions on the same silica gel G thin-layer plate, developing with chloroform-ethyl acetate-methanol-formic acid as developing agent at volume ratio of 40:5:10:0.2, taking out, air drying, spraying 5% vanillin-sulfuric acid ethanol solution, and observing with sunlight to obtain no interference of negative control.

(7) Qualitative identification by thin layer chromatography

Grinding rhizoma Cyperi granule, adding methanol, ultrasonic treating, filtering, evaporating filtrate, dissolving residue in water, adding concentrated ammonia solution to adjust to alkalinity, shaking and extracting with diethyl ether, mixing diethyl ether solutions, evaporating, and dissolving residue in methanol to obtain sample solution;

collecting rhizoma corydalis reference material powder, adding methanol, ultrasonic treating, filtering, evaporating filtrate to dryness, dissolving residue in water, adding concentrated ammonia solution to adjust to alkalinity, shaking and extracting with diethyl ether, mixing diethyl ether solution, evaporating to dryness, and dissolving residue in methanol to obtain reference material solution;

taking rhizoma Cyperi, radix aucklandiae, radix Angelicae sinensis, rhizoma Ligustici Chuanxiong, radix rehmanniae Preparata and radix Paeoniae alba, adding methanol, ultrasonic processing, filtering, evaporating filtrate to dryness, dissolving residue in water, adding concentrated ammonia solution to adjust to alkalinity, shaking and extracting with diethyl ether, mixing diethyl ether solutions, evaporating to dryness, and dissolving residue in methanol to obtain negative control solution;

accurately weighing tetrahydropalmatine reference substance, and adding methanol to obtain reference solution;

and (3) performing a thin-layer chromatography test, sucking the 4 solutions, respectively dropping the solutions on the same silica gel G thin-layer plate, developing by taking toluene-acetone with the volume ratio of 9:2 as a developing agent, taking out, drying in the air, spraying an improved bismuth potassium iodide solution, and observing by sunlight, wherein spots with the same color appear on corresponding positions of the test sample and the reference sample, and the negative reference sample does not interfere with the test sample.

The quality control method of the cyperus rotundus granule comprises one or more of the following methods:

(1) the content of the cyperus rotundus granule is determined by adopting a high performance liquid chromatography, and the method comprises the following specific steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system adaptability tests; when detecting ferulic acid, a glacial acetic acid solution with the volume ratio of 20:80 acetonitrile-1% is used as a mobile phase; when detecting vanillic acid, taking acetonitrile-1% glacial acetic acid solution with the volume ratio of 12:88 as a mobile phase; the detection wavelengths are 323nm and 260nm respectively; the theoretical plate number is not less than 3000 calculated according to ferulic acid and vanillic acid;

preparing reference substance solution by accurately weighing appropriate amount of ferulic acid and vanillic acid reference substances, and adding methanol to obtain reference substance solution containing 0.0316mg and 0.0295mg per 1mL respectively;

preparing a test solution by precisely weighing rhizoma Cyperi granule, placing in a conical flask with a plug, precisely adding methanol, weighing, standing overnight, ultrasonically treating, weighing again, supplementing lost mass with methanol, shaking, filtering, precisely weighing subsequent filtrate, and evaporating in water bath; dissolving the residue with sodium hydroxide solution, transferring into a reflux flask, refluxing in water bath, and cooling; transferring into a separating funnel, adjusting pH to 2 with hydrochloric acid, extracting with diethyl ether, mixing diethyl ether solutions, recovering diethyl ether, dissolving with methanol, and shaking;

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

the content of radix Angelicae sinensis and rhizoma Ligustici Chuanxiong in each 15g granule is not less than 0.714mg and 0.439mg calculated by ferulic acid and vanillic acid;

(2) the content of the cyperus rotundus granule is determined by adopting a high performance liquid chromatography, and the method comprises the following specific steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system adaptability tests; taking 18:82 acetonitrile-0.1% acetic acid water in volume ratio as a mobile phase; the detection wavelength is 230nm, and the theoretical plate number is not less than 2000 calculated according to paeoniflorin and albiflorin;

preparation of control solution A proper amount of penoniflorin and albiflorin as control is precisely weighed, and added with methanol to obtain control solutions containing 0.300mg and 0.200mg per 1 mL.

Preparing a test solution, precisely weighing rhizoma cyperi four-substance particles, placing the particles into a conical flask with a plug, precisely adding methanol, precisely weighing, ultrasonically treating, cooling, weighing again, supplementing the loss mass with methanol, shaking up, filtering, and taking a subsequent filtrate as the test solution;

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.

The product contains radix Paeoniae alba (calculated as paeoniflorin and albiflorin) per 15g, and is not less than 52.5mg and 17.9 mg;

(3) the content of the cyperus rotundus granule is determined by adopting a high performance liquid chromatography, and the method comprises the following specific steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system adaptability tests; using 55:45 acetonitrile-0.1% acetic acid water in volume ratio, and using triethylamine to adjust pH to 6.0 as mobile phase; the detection wavelength is 280 nm; the theoretical plate number is not less than 3000 calculated according to tetrahydropalmatine and berberine hydrochloride;

preparing reference substance solution by accurately weighing appropriate amount of tetrahydropalmatine and berberine hydrochloride, and adding methanol to obtain reference substance solutions containing 0.246mg and 0.297mg per 1mL respectively;

preparing a test solution, precisely weighing 15g of rhizoma cyperi four-substance particles, placing the particles into a flat-bottomed flask, precisely adding a concentrated ammonia test solution-methanol mixed solution with the volume ratio of 1:20, weighing, cold soaking, heating, refluxing, cooling, weighing again, complementing the weight loss by the concentrated ammonia test solution-methanol mixed solution with the volume ratio of 1:20, shaking uniformly, and filtering;

precisely measuring 75mL of subsequent filtrate, evaporating to dryness, dissolving the residue with methanol, transferring to a 10mL measuring flask, diluting to scale, shaking, filtering, and collecting subsequent filtrate;

the content of rhizoma corydalis in 15g granule is not less than 2.67mg and 1.22mg calculated by tetrahydropalmatine and berberine hydrochloride.

Compared with the prior art, the invention has the following beneficial effects:

(1) compared with the existing single formula granule, the traditional Chinese medicine compatibility advantage can be fully exerted, the concept of the whole traditional Chinese medicine is reflected, the aim of reducing toxicity and improving efficacy is ensured, and a new choice is provided for clinical medication.

(2) The invention collects the effective part of the volatile oil in the process of decoction and extraction, and the volatile oil is coated by beta-cyclodextrin and then is prepared into particles, thus reducing the volatility, increasing the utilization rate, promoting the absorption of non-volatile components and improving the bioavailability of the medicine.

(3) The invention adopts chromatography, namely thin layer chromatography and high performance liquid chromatography, to carry out qualitative and quantitative analysis on the effective components, and carry out quality control, thereby well ensuring the product quality.

(4) The invention establishes a quality control method for simultaneously determining the contents of six main index components of ferulic acid, vanillic acid, paeoniflorin, albiflorin, tetrahydropalmatine and berberine hydrochloride in the granules by utilizing a high performance liquid chromatography, realizes the simultaneous control of the contents of four decoction pieces of radix paeoniae alba, angelica sinensis, ligusticum wallichii and rhizoma corydalis in a prescription by the quality standard, has stronger specificity and good reproducibility, and truly reflects the safety, effectiveness and controllable quality of the medicine.

Drawings

FIG. 1 is a thin layer chromatogram of example 1, wherein 1 is a sample lacking both negative properties of radix Angelicae sinensis and rhizoma Ligustici Chuanxiong, 2-4 are 3 batches of samples, 5 is radix Angelicae sinensis reference drug, 6 is rhizoma Ligustici Chuanxiong reference drug, and 7 is ferulic acid reference;

FIG. 2 is a thin layer chromatogram of example 1, wherein 1 is a sample lacking both yin of Angelica sinensis and Ligusticum chuanxiong, 2-4 are 3 batches of samples, 5 is an Angelica sinensis reference drug, 6 is an Ligusticum chuanxiong reference drug, and 7 is a ligustilide reference;

FIG. 3 is a thin layer chromatogram of example 2, wherein 1 is a rhizoma Ligustici Chuanxiong-deficient negative sample, 2-4 are 3 batches of samples, 5 is a rhizoma Ligustici Chuanxiong reference material, and 6 is an levistilide A reference material;

FIG. 4 is a thin layer chromatogram of example 3, wherein 1 is a Cyperus rotundus negative sample, 2-4 are 3 batches of test samples, 5 is a Cyperus rotundus reference drug, and 6 is an alpha-cyperone reference;

FIG. 5 is a thin layer chromatogram of example 4, wherein 1 is a costus root-deficient negative sample, 2-4 are 3 batches of the test sample, 5 is a costus root control drug, 6 is a dehydrocostus lactone control, and 7 is a costunolide control;

FIG. 6 is a thin layer chromatogram of example 5, wherein 1 is a negative sample lacking radix rehmanniae Preparata, 2-4 are 3 batches of the test sample, 5 is a radix rehmanniae Preparata reference drug, and 6 is a 5-hydroxymethylfurfural reference;

FIG. 7 is a thin layer chromatogram of example 6, wherein 1 is a negative sample lacking white peony root, 2-4 are 3 batches of the test sample, 5 is a white peony root control drug, and 6 is a paeoniflorin control;

FIG. 8 is a thin layer chromatogram of example 7, wherein 1 is a rhizoma corydalis-deficient negative sample, 2-4 are 3 batches of the test sample, 5 is a rhizoma corydalis control drug, and 6 is a tetrahydropalmatine control;

FIG. 9 is the HPLC chromatogram of example 8, wherein A is ferulic acid control, B is the test solution, C is a sample lacking both anions of Angelica sinensis and Ligusticum chuanxiong, and 1 is ferulic acid;

FIG. 10 is the HPLC chromatogram of example 8, wherein A is vanillic acid control, B is the test solution, C is the sample lacking both anions of Dang Gui and Chuan Xiong, and 1 is vanillic acid;

FIG. 11 is the HPLC chromatogram of example 8, wherein A is a control of paeoniflorin and albiflorin, B is the test solution, C is a negative sample lacking white peony root, 1 is albiflorin, and 2 is paeoniflorin;

FIG. 12 is the HPLC chromatogram of example 8, wherein A is tetrahydropalmatine and berberine hydrochloride control, B is the sample, C is a rhizoma corydalis-deficient negative sample, 1 is berberine hydrochloride, and 2 is tetrahydropalmatine.

Detailed Description

The present invention is further illustrated by the following specific embodiments, which are not intended to limit the scope of the invention.

Example 1:

a method for preparing rhizoma cyperi Siwu granules comprises the following steps:

a. taking 300g of rhizoma cyperi, 200g of costustoot, 600g of angelica and 300g of ligusticum wallichii, adding 14kg of water, soaking for 12 hours, and then extracting for 10 hours by steam distillation to extract volatile oil.

b. Adding beta-cyclodextrin (the addition amount is 10 times of the volume amount of the volatile oil) and water (the addition amount is 30 times of the volume amount of the volatile oil) into the volatile oil, fully grinding for 2 hours, and freeze-drying to obtain the volatile oil inclusion compound.

c. Adding 800g of prepared rehmannia root, 300g of white paeony root and 300g of corydalis tuber according to the corresponding proportion into the residue obtained after the volatile oil is extracted, adding water for decocting for 2 times, adding 30kg of water for each time, decocting for 2 hours, extracting the liquid medicine, combining the liquid medicine, filtering, drying under reduced pressure to obtain concentrated solution with the relative density of 1.05, adding 95 percent ethanol until the ethanol concentration in the system is 80 percent (v/v), standing for 24 hours, carrying out suction filtration, concentrating the filtrate under reduced pressure, and drying to obtain dry paste powder.

d. Adding dextrin (equivalent to dry extract powder) and sugar powder (equivalent to dry extract powder) into the dry extract powder, mixing, granulating, drying, grading, and adding the volatile oil clathrate to obtain rhizoma Cyperi granule.

The method for qualitatively identifying rhizoma cyperi four-substance particles by adopting thin-layer chromatography comprises the following steps:

1. preparation of samples

1.1 preparation of control solutions

Taking ferulic acid and ligustilide as reference substances, adding methanol to obtain solution containing 2.05mg and 2.00mg per 1mL as reference substance solution.

1.2 preparation of reference drug solution

Taking 3g of angelica and ligusticum wallichii reference medicinal material powder respectively, adding 150mL of 1% sodium bicarbonate solution, carrying out ultrasonic treatment for 10min, centrifuging, taking supernate, adjusting the pH value to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ether for 2 times, wherein 40mL of the solution is obtained each time, combining ether solutions, volatilizing the ether solutions, and adding 1mL of methanol to dissolve residues to obtain the reference medicinal material solution.

1.3 method for treating test solution

Test solution 1: taking 15g of the rhizoma cyperi four-substance particles, adding 150mL of 1% sodium bicarbonate solution, carrying out ultrasonic treatment for 10min, centrifuging, taking supernate, adjusting the pH value to 2-3 by using dilute hydrochloric acid, shaking and extracting for 2 times by using ether, wherein 40mL of ether is used for each time, combining ether solutions, volatilizing, adding 1mL of methanol into residues for dissolving, and taking the residues as a sample solution 1;

sample solution 2: taking 15g of the cyperus rotundus four-substance particles into a beaker, adding 150mL of 80% ethanol for ultrasonic dissolution, putting the mixture on a water bath to evaporate the ethanol to about 30mL, shaking and extracting the mixture for 2 times by using ether, 40mL each time, combining ether extract, adding 1% sodium carbonate solution into the ether solution, shaking and extracting the mixture for 2 times by using 40mL each time, discarding the ether solution, adjusting the pH value of the extract to 2-3 by using dilute hydrochloric acid, shaking and extracting the extract for 2 times by using ether, 60mL each time, combining the ether extract, volatilizing the mixture, and dissolving residues by adding 1mL of methanol to obtain a sample solution 2.

1.4 preparation of negative control solution

According to the prescription proportion and the preparation method, a double-negative control sample of the angelica and ligusticum wallichii lacking medicinal materials is prepared. A double negative control solution was prepared according to the preparation method of the

test solutions

1 and 2.

2. Condition optimization of developing agent

In order to optimize the developing agent, the solution is absorbed and respectively spotted on the same silica gel G thin layer plate, two developing systems are selected, developed, taken out, dried and inspected under an ultraviolet lamp (365nm), and the result shows that ferulic acid and ligustilide have obvious spots in the developing system A, and ferulic acid spots are not obvious and the separation effect is not good in the developing system B, so that benzene-glacial acetic acid-methanol (30:1:3) is selected as the developing agent of ferulic acid and ligustilide.

3. Determination of the amount of spotting

3.1 determination of the amount of spotting of control solutions

Respectively sucking 1, 2,4 and 6 mu L of ferulic acid reference substance solution to be sequentially spotted on the same silica gel G thin layer plate, respectively sucking 1, 2, 3 and 4 mu L of ligustilide reference substance solution to be sequentially spotted on the same silica gel G thin layer plate, and analyzing by using a determined TLC analysis development condition, wherein the two reference substances have obvious spots in different sample quantities, the spots of ferulic acid are clearest at 4 mu L, and the spots of ligustilide are brighter at 2 mu L. Therefore, 4. mu.L and 2. mu.L were selected as spot amounts of ferulic acid and ligustilide, respectively.

3.2 determination of sample application amount of sample solution

Respectively sucking 4, 6, 8 and 10 mu L of the test solution 1, respectively sucking 2,4, 6 and 8 mu L of the test solution 2, respectively and sequentially and respectively spotting on the same silica gel G thin-layer plate, and analyzing under a determined TLC condition, wherein spots of the test solution 1 in 4-10 mu L are clear, and the spot amount can be 4 mu L; the spot of the test solution 2 is most obvious when the volume is 8 mu L, and the spot size can be selected from 8 mu L.

4. Sample testing

According to the determined TLC identification condition, carrying out TLC identification on ferulic acid and ligustilide on the cyperus rotundus L.Sichuan particles of 3 batches, wherein the results are shown in figures 1 and 2, and spots with the same color appear on the corresponding positions of the test product and the reference product. The negative control product has no interference.

Example 2:

a. taking 300g of rhizoma cyperi, 200g of costustoot, 600g of angelica and 300g of ligusticum wallichii, adding 16.8kg of water, soaking for 15 hours, and then extracting for 8 hours by steam distillation to extract volatile oil.

b. Adding beta-cyclodextrin (the addition amount is 8 times of the volume amount of the volatile oil) and water (the addition amount is 20 times of the volume amount of the volatile oil) into the volatile oil, fully grinding for 2 hours, and freeze-drying to obtain the volatile oil inclusion compound.

c. Adding 800g of prepared rehmannia root, 300g of white paeony root and 300g of corydalis tuber according to the corresponding proportion into the residue obtained after the volatile oil is extracted, adding water for decocting for 2 times, adding 28kg of water for each time, decocting for 2 hours, extracting liquid medicine, combining the liquid medicine, filtering, drying under reduced pressure to obtain concentrated solution with the relative density of 1.13, adding 95 percent ethanol until the ethanol concentration in the system is 80 percent (v/v), standing for 24 hours, carrying out suction filtration, concentrating the filtrate under reduced pressure, and drying to obtain dry paste powder.

1. preparation of samples

1.1 preparation of control solutions

A proper amount of the levistilide A as a control was weighed, and ethyl acetate was added to make a solution containing 0.116mg per 1mL (in a brown measuring flask) as a control solution.

1.2 preparation of reference drug solution

Taking 1g of rhizoma Ligustici Chuanxiong powder, adding 100mL of diethyl ether, heating and refluxing l h, filtering, volatilizing the filtrate, and dissolving the residue with 2mL of ethyl acetate to obtain a control solution.

1.3 method for treating test solution

Taking 15g of the rhizoma cyperi four-substance granules, adding 100mL of diethyl ether, heating and refluxing to l h, filtering, volatilizing the filtrate, adding 2mL of ethyl acetate into the residue to dissolve the residue to obtain a test solution.

1.4 preparation of negative control solution

Preparing a negative control sample lacking rhizoma Ligustici Chuanxiong according to the above formula proportion and preparation method, and preparing into negative control solution according to the preparation method of the test solution under item 1.3.

2. Condition optimization of developing agent

Comparing three development systems of n-hexane-ethyl acetate (3:1), n-hexane-ethyl acetate-formic acid (3:1:0.05) and n-hexane-ethyl acetate-formic acid (7:1:0.05), selecting n-hexane: ethyl acetate (3:1) is the developing agent of angelicin A.

3. Determination of the amount of spotting

3.1 determination of the amount of spotting of control solutions

Respectively dripping 1, 2,4 and 5 μ L of the reference solution of levisticide A on the same silica gel GF254 plate. The control sample has obvious spots at different spot sizes, the spots are clear at 4 μ L, and the spot size of the angelicin A is selected as 4 μ L.

3.2 determination of sample application amount of sample solution

Respectively sucking 2,4, 8 and 10 mu L of test solution to analyze under the determined TLC condition, wherein the spot of the test solution is more clear and regular at 8 mu L, so the sample application amount is selected to be 8 mu L.

4. Sample testing

According to the determined TLC condition, the thin-layer identification of levistilide A was carried out on the cyperus rotundus four-substance granules of 3 batches, and the results are shown in figure 3, and spots with the same color appear on the corresponding positions of the test product and the reference product. The negative control product has no interference.

Example 3:

1. preparation of samples

1.1 preparation of control solutions

Taking alpha-cyperone as a reference substance, adding ethyl acetate to prepare a solution containing 2.2mg per 1mL, and taking the solution as a reference substance solution.

1.2 preparation of reference drug solution

Taking 1g of rhizoma Cyperi control material powder, adding 75mL of diethyl ether, standing for 1h, shaking constantly, filtering, volatilizing the filtrate, adding 1mL of ethyl acetate into the residue to dissolve, and using as the control material solution.

1.3 method for treating test solution

Taking 15g of the rhizoma cyperi four-substance granules, adding 75mL of diethyl ether, standing for 1h, shaking constantly, filtering, volatilizing the filtrate, adding 1mL of ethyl acetate into the residues for dissolving to obtain a test solution.

1.4 preparation of negative control solution

Preparing a negative control sample lacking rhizoma Cyperi according to the proportion and preparation method of the formula, and preparing a negative control solution according to the preparation method of the test solution.

2. Optimization of developing agent and developing conditions

Dichloromethane-ethyl acetate-glacial acetic acid (80:1:1) is the most suitable developing agent for alpha-cyperone. The spots obtained by the silica gel GF254 plate and the silica gel G plate have equivalent effects, and for the purpose of accuracy, the silica gel GF254 is selected to be developed, an ultraviolet lamp (254nm) is used for inspection, and then 2, 4-dinitrophenylhydrazine ethanol test solution is sprayed to observe spot color development.

3. Determination of the amount of spotting

3.1 determination of the amount of spotting of control solutions

Respectively sucking 1, 2,4 and 5 mu L of alpha-cyperone reference substance solution, sequentially dropping on the same silica gel GF254 thin-layer plate, analyzing by determined TLC condition, wherein the spot is most regular at 2 mu L, and selecting 2 mu L as spot sample amount.

3.2 determination of sample application amount of sample solution

Respectively sucking 4, 6, 8 and 10 mu L of test solution to analyze under the determined TLC condition, wherein the spot of the test solution is clear and regular at 10 mu L, so the spot sample amount is selected to be 10 mu L.

4. Sample testing

According to the determined TLC condition, thin-layer identification of alpha-cyperone is carried out on the cyperus rotundus four-substance particles of 3 batches, and the results are shown in figure 4, and spots with the same color are displayed on the corresponding positions of the test sample and the control sample. The negative control product has no interference.

Example 4:

1. preparation of samples

1.1 preparation of control solutions

Taking appropriate amount of dehydrocostuslactone reference substance and costunolide reference substance, and adding methanol to make into solutions containing 2.28mg and 1.85mg per 1mL respectively as reference substance solutions.

1.2 preparation of reference drug solution

Collecting 0.5g radix aucklandiae reference medicinal material powder, adding 75mL petroleum ether (30-60 deg.C), cold soaking for 30min, shaking constantly, filtering, evaporating filtrate to dryness, dissolving residue with 1mL ethyl acetate, and collecting filtrate as reference medicinal material solution.

1.3 method for treating test solution

Taking 15g of the rhizoma cyperi four-substance particles, adding 75mL of petroleum ether (30-60 ℃), cold soaking for 30min, shaking constantly, filtering, volatilizing the filtrate, and adding 1mL of ethyl acetate into residues to dissolve the residues to obtain a sample solution.

1.4 preparation of negative control solution

Preparing a negative control sample of the costus root-lacking medicinal material according to the formula proportion and the preparation method. Preparing double negative control solution according to the preparation method of the test solution.

2. Condition optimization of developing agent

In order to optimize the developing agent, the solution is absorbed and respectively spotted on the same silica gel G thin-layer plate, three developing systems are selected, the developing system is developed, taken out and dried, spots obtained by the developing agents B and C are fuzzy, the spreading distance is close to the front edge, the spots obtained by the developing agent A are clear and regular, and the spreading distance is proper, so cyclohexane-ethyl acetate-formic acid (15:5:1) is selected as the developing agent of costunolide and dehydrocostuslactone.

3. Determination of the amount of spotting

3.1 determination of the amount of spotting of control solutions

Respectively sucking 1, 2, 3 and 4 mu L of dehydrocostuslactone reference substance solution and 2,4, 6 and 8 mu L of costuslactone reference substance solution, sequentially and respectively dropping on the same silica gel G thin layer plate, analyzing by determined TLC condition, wherein the dehydrocostuslactone reference substance has obvious spots at different spot sample amounts, and the spots are most regular at 2 mu L, so 2 mu L is selected as the spot sample amount. The costunolide control was the most marked at 8. mu.L, so 8. mu.L was chosen as the spotting amount.

3.2 determination of sample application amount of sample solution

Sucking sample solution of 4, 6, 8 and 10 μ L, sequentially and respectively dropping on the same silica gel G thin layer plate, and analyzing under determined TLC condition, wherein the sample solution of 4-10 μ L shows spots, and the dropping amount can be 10 μ L.

4. Sample testing

Thin-layer identification of dehydrocostuslactone and costunolide was performed on 3 batches of cyperus rotundus four-substance particles according to the determined TLC identification conditions, and the results are shown in FIG. 5, and spots with the same color were displayed on the corresponding positions of the test sample and the control sample. The negative control product has no interference.

Example 5:

1. preparation of samples

1.1 preparation of control solutions

Taking a 5-hydroxymethylfurfural reference substance, and adding methanol to prepare a solution containing 0.29mg per 1mL as a reference substance solution.

1.2 preparation of reference drug solution

Taking 1g of radix rehmanniae Preparata powder, adding 100mL of water, heating to fully dissolve, cooling, filtering with absorbent cotton, shaking and extracting the filtrate with diethyl ether for 2 times, 25mL each time, discarding the diethyl ether solution, shaking and extracting with ethyl acetate for 2 times, 25mL each time, combining the ethyl acetate solutions, evaporating to dryness, and dissolving the residue with 1mL of methanol to obtain a control solution.

1.3 method for treating test solution

Taking 15g of the granules, adding 100mL of water, heating to fully dissolve the granules, cooling, filtering with absorbent cotton, shaking and extracting the filtrate with diethyl ether for 2 times, 25mL each time, discarding the ethyl ether solution, shaking and extracting with ethyl acetate for 2 times, 25mL each time, combining the ethyl acetate solutions, evaporating to dryness, and adding 1mL of methanol to dissolve the residue to obtain a sample solution.

1.4 preparation of negative control solution

Preparing a negative control sample of the radix rehmanniae Preparata-deficient medicinal material according to the prescription proportion and the preparation method. Preparing double negative control solution according to the preparation method of the test solution.

2. Condition optimization of developing agent

Different optimization conditions are as follows: inspecting the GF254 plate and developing agent A by ultraviolet (254 nm); spraying a color developing agent on the G plate and the developing agent B, and observing in sunlight; g plate and developing agent C, spraying color developing agent, and inspecting by sunlight. Color developing agent: 2, 4-dinitrophenylhydrazine ethanol solution. The three development and color development systems were compared. The results show that the Rf of developer a is small and the span of developer B is close to the front, so developer C is selected: xylene-ethyl acetate (1:1) is a developing agent of 5-hydroxymethyl furfural, and 2, 4-dinitrophenylhydrazine ethanol solution is a color developing agent.

3. Determination of the amount of spotting

3.1 determination of the amount of spotting of control solutions

Respectively sucking 6, 8, 10 and 12 mu L of 5-hydroxymethylfurfural reference substance solution, sequentially dropping on the same silica gel G thin-layer plate, and analyzing by determined TLC conditions, wherein the reference substance has obvious spots at different sample amounts, and the spots are regular at 8 mu L, so that 8 mu L is selected as the sample amount.

3.2 determination of sample application amount of sample solution

Respectively sucking 1, 2, 3 and 4 mu L of the test solution 2, sequentially dropping on the same silica gel G thin layer plate, analyzing under a determined TLC condition, wherein the test solution shows obvious spots when being 1-4 mu L, and the dropping amount can be 2 mu L.

4. Sample testing

According to the determined TLC identification conditions, thin-layer identification of 5-hydroxymethylfurfural was carried out on the cyperus rotundus four-substance particles of 3 batches, and the results are shown in figure 6, and spots of the same color were displayed on the corresponding positions of the test sample and the reference sample. The negative control product has no interference.

Example 6:

1. preparation of samples

1.1 preparation of control solutions

Weighing appropriate amount of penoniflorin as control, adding methanol to obtain solution containing 1.03mg per 1mL as control solution.

1.2 preparation of reference drug solution

0.5g of white peony root control drug powder is taken, dissolved by 1mL of methanol, and mixed with silica gel column (300 meshes 400, about 20g, inner diameter about 2-2.5cm) and chloroform: methanol (10:1, 44 mL; 9:1, 50 mL; 8:1, 45mL), discarding the first 15mL of eluate, and collecting the subsequent 8:1 eluate. Evaporating to dryness, and dissolving the residue with 1mL of methanol to obtain a control solution.

1.3 method for treating test solution

Taking 15g of rhizoma cyperi four-substance particles, adding 50mL of water, heating to dissolve, extracting with water saturated n-butyl alcohol for 2 times, 25mL each time, combining n-butyl alcohol solutions, washing with n-butyl alcohol saturated water for 2 times, 25mL each time, combining the n-butyl alcohol solutions, and volatilizing in water bath to obtain residues. The residue was dissolved in 1mL of methanol, purified by column chromatography on silica gel (300 mesh 400, about 20g, inner diameter about 2-2.5cm), purified by chloroform: methanol (10:1, 44 mL; 9:1, 50 mL; 8:1, 45mL) was eluted, the first 15mL of the eluate was discarded, the subsequent 8:1 eluate was collected and evaporated to dryness, and 1mL of methanol was added to the residue to dissolve it as a sample solution.

1.4 preparation of negative control solution

Preparing a negative control sample lacking the white paeony root according to the proportion and the preparation method of the prescription, and preparing a negative control solution according to the preparation method of the test solution.

2. Determination of the amount of spotting

2.1 determination of the amount of spotting of control solutions

Respectively sucking 4, 6, 8 and 10 mu L of paeoniflorin reference substance solution, sequentially dropping on the same silica gel G plate, analyzing by determined TLC condition, wherein the paeoniflorin reference substance has obvious spots at different spot sample amounts, the spots are regular at 10 mu L, and the color is clear, so 10 mu L is selected as the spot sample amount of paeoniflorin.

2.2 determination of sample application amount of sample solution

3. Sample testing

Thin-layer identification of paeoniflorin was performed on 3 batches of cyperus rotundus four-substance granules according to the determined TLC conditions, and the results are shown in FIG. 7, wherein spots with the same color were displayed on the corresponding positions of the test sample and the control sample. The negative control product has no interference.

Example 7:

1. preparation of samples

1.1 preparation of control solutions

Weighing appropriate amount of tetrahydropalmatine control, and adding methanol to obtain 1.25mg solution per 1mL as control solution.

1.2 preparation of reference drug solution

Taking 1g rhizoma corydalis reference medicinal material powder, adding 100mL methanol, ultrasonic treating for 30min, filtering, evaporating filtrate to dryness, dissolving residue with 20mL water, adding concentrated ammonia solution to adjust to alkalinity, shaking with diethyl ether for 3 times, extracting 20mL each time, mixing ethyl ether solution, evaporating to dryness, dissolving residue with 1mL methanol to obtain reference medicinal material solution.

1.3 method for treating test solution

Taking 15g of the granules, adding 100mL of methanol, carrying out ultrasonic treatment for 30min, filtering, evaporating the filtrate to dryness, adding 20mL of water into the residue to dissolve the residue, adding concentrated ammonia test solution to adjust the pH to be alkaline, shaking and extracting with diethyl ether for 3 times, 20mL each time, combining the diethyl ether solutions, evaporating to dryness, and adding 1mL of methanol into the residue to dissolve the residue to obtain a test solution.

1.4 preparation of negative control solution

According to the prescription proportion and the preparation method, a negative control sample lacking the rhizoma corydalis medicinal material is prepared. Then, a negative control solution was obtained according to the method for preparing the test solution.

2. Optimization of color development method

In order to optimize the color development method, 10 μ L of each solution is absorbed and respectively spotted on the same silica gel G thin layer plate, and the method I comprises the following steps: spraying improved bismuth potassium iodide solution with toluene-acetone (9:2) as developing agent, and observing with sunlight; the second method comprises the following steps: taking out toluene-acetone (9:2) as developing agent, volatilizing, placing in an iodine jar for about 1min, taking out, volatilizing iodine adsorbed on the board, placing under an ultraviolet lamp (365nm) for inspection, spraying the improved bismuth potassium iodide solution to obtain obvious spots, and under the inspection of the ultraviolet lamp, the spots of tetrahydropalmatine in the test solution are not easy to be distinguished and are covered by other spots, so a color developing agent color development method is selected for analysis.

3. Determination of the amount of spotting

3.1 determination of the amount of spotting of control solutions

Respectively sucking 4, 6, 8 and 10 mu L of tetrahydropalmatine reference substance solution, sequentially dropping on the same silica gel G plate, and analyzing under determined conditions, wherein the tetrahydropalmatine reference substance has speckles with different spot sample amounts, and the speckles are regular at 8 mu L, so 8 mu L is selected as the spot sample amount of tetrahydropalmatine.

3.2 determination of sample application amount of sample solution

The sample solutions were aspirated at 4, 6, 8, and 10 μ L each, and sequentially spotted on the same silica gel G plate for analysis under defined conditions by TLC. The sample solution has spots at 4-10 μ L, and the sample application amount can be 6 μ L.

4. Sample testing

Thin-layer identification of tetrahydropalmatine was performed on 3 batches of cyperus rotundus four-substance granules according to the determined TLC identification conditions, and the results are shown in FIG. 8, and spots of the same color were displayed on the corresponding positions of the test sample and the control sample. The negative control product has no interference.

Example 8:

The content of the cyperus rotundus granule is measured by adopting a high performance liquid chromatography, and the specific method comprises the following steps:

1. determination of content of ferulic acid and vanillic acid

1.1 instruments and reagents

WatersAlliance 2695 high performance liquid chromatography system (quaternary gradient pump, autosampler, column temperature system, 2998 photodiode array detector and Empower 2 chromatography workstation, Waters corporation, USA);

model KH-500 ultrasonic cleaner (kunshan grass ultrasonic instrument ltd);

HH-S type digital display constant temperature water bath (medical instrument factory of gold jar city);

MS105 analytical balance (Mettler-Toledo instruments, Inc.);

EPED ultrapure water system (Nanjing Yipu Yi Da science and technology development Co., Ltd.).

Samples of rhizoma Cyperi granule (prepared by the above method of this example, lot numbers: 20151101, 20151102, 20151103);

the control ferulic acid (batch No. 110773-201313) and the vanillic acid control (batch No. 110776-200402) were purchased from the institute of food and drug assay in China. The other reagents are analytically pure.

1.2 methods and results

1.2.1 chromatographic conditions

Alltima^TMC₁₈Chromatography columns (250 mm. times.4.6 mm, 5 μm, GRACE);

mobile phase: ferulic acid (acetonitrile-1% glacial acetic acid solution volume ratio is 20:80), vanillic acid (acetonitrile-1% glacial acetic acid solution volume ratio is 12: 88);

the volume flow is 1.0 mL/min; the column temperature is 30 ℃; the sample volume is 10 mu L;

detection wavelength: ferulic acid of 323nm and vanillic acid of 260 nm;

the peak time: ferulic acid for 16min, and vanillic acid for 18 min.

1.2.2 preparation of the solution

Preparation of reference solution A proper amount of ferulic acid and vanillic acid reference substances are precisely weighed, methanol is respectively added to prepare reference solution containing 0.0316mg and 0.0295mg per 1mL, and the reference solution is gradually diluted to prepare 6 reference solutions with different concentrations.

Preparing a test solution, precisely weighing 15g of rhizoma cyperi four-substance particles, placing the particles into a conical flask with a plug, precisely adding 150mL of methanol, weighing, standing overnight, carrying out ultrasonic treatment for 1h, weighing again, complementing the lost mass with methanol, shaking up, filtering, precisely weighing 50mL of subsequent filtrate, and evaporating in a water bath to dryness. The residue was dissolved in 50mL of 0.5mol/L sodium hydroxide solution 3 times, transferred to a reflux flask, refluxed in a water bath for 2 hours, and allowed to cool. Transferring into a separating funnel, adjusting pH to 2 with hydrochloric acid, extracting with diethyl ether for 4 times (25 mL each time), mixing diethyl ether solutions, recovering diethyl ether, dissolving with methanol to 10mL, and shaking.

Preparation of negative control solution according to the prescription and preparation process of rhizoma Cyperi granule, radix Angelicae sinensis and rhizoma Ligustici Chuanxiong negative control samples are prepared. Then preparing double negative solution according to the processing method of the test solution.

1.3 assay method

The control solution, the test solution and the negative control solution were each pipetted at 10. mu.L, and analyzed under the chromatographic conditions of item 1.2.1, and the results are shown in FIGS. 9 and 10. As can be seen, the chromatogram of the sample has a chromatographic peak with the same retention time at the position corresponding to the chromatogram of the ferulic acid control and the vanillic acid control, and the solution of the negative control has no interference at the retention time.

1.4 methodological considerations

1.4.1 Standard Curve and Linear Range

Precisely sucking 10 μ L of 6 control substances with different concentrations, respectively, injecting into liquid chromatograph, and measuring according to chromatographic conditions of item 1.2.1. Taking the peak area value Y (mu V.s) of the reference substance as a vertical coordinate, and taking the mass concentration X (mu g/mL) as a horizontal coordinate, and performing linear regression to obtain a regression equation.

Ferulic acid: y is 5.04 × 10⁷X-5185.4, wherein r is 0.9999, and has good linear relation with peak area within the mass concentration range of 6.32-47.4 mug/mL;

vanillic acid: y is 3.83 × 10⁴X +12993, r is 0.9999, and has good linear relation with peak area within the mass concentration range of 5.90-29.5 mu g/mL.

1.4.2 precision test

The same control solution was sampled for 6 times, 10. mu.L each time, under the chromatographic condition of 1.2.1, and the reproducibility was observed. The results show that the RSD of the peak areas of the ferulic acid and the vanillic acid reference products are 0.620 percent and 0.560 percent respectively, and the precision of the display instrument is good. The results are shown in Table 1.

TABLE 1 results of precision tests of ferulic acid and vanillic acid

1.4.3 reproducibility test

Taking 6 parts of cyperus rotundus four-substance particles (batch number 20151101), accurately weighing the particles respectively, preparing a test solution according to a preparation method of the test solution, accurately sucking 10 mu L of the test solution, injecting the test solution into a liquid chromatograph, measuring according to chromatographic conditions under 1.2.1, calculating the RSD of ferulic acid and vanillic acid in the test solution to be 0.363% and 0.844% respectively, and having good method reproducibility. The results are shown in Table 2.

TABLE 2 results of reproducibility tests of ferulic acid and vanillic acid

1.4.4 stability test

Precisely absorbing ferulic acid and vanillic acid reference substance solutions respectively at 10 μ L for 0, 2,4, 6, 8, 10, 12, and 24h, injecting into liquid chromatograph, and measuring according to conditions of item 1.2.1. The results show that the RSD of the peak area of the ferulic acid control solution is 1.27%, and the RSD of the peak area of the vanillic acid control solution is 2.66%, which indicates that the two control solutions are stable within 24 h. The results are shown in Table 3.

TABLE 3 stability test results for ferulic acid and vanillic acid

1.4.5 sample recovery test

Precisely weighing 6 parts of the determined rhizoma cyperi four-substance particle sample (batch number: 20151101), respectively adding appropriate amounts of ferulic acid and vanillic acid reference substances, preparing a test solution according to the treatment method of the test solution, determining according to the conditions under the item 1.2.1, and calculating the sample adding recovery rate. The results show that the average recovery rates of ferulic acid and vanillic acid are 99.6% and 101%, respectively, and the RSD is 0.822% and 1.07%, respectively, and both have good recovery rates. The results are shown in tables 4 and 5.

TABLE 4 sample recovery test results for ferulic acid

TABLE 5 Vanillic acid sample recovery test results

1.5 sample determination

Precisely weighing 3 batches of rhizoma cyperi four-substance particle samples (batch numbers: 20151101, 20151102 and 20151103), preparing a test sample solution according to the preparation method of the test sample solution, measuring according to chromatographic conditions under 1.2.1, and calculating the content, wherein the results are shown in table 6.

TABLE 6 determination of the content of ferulic acid and vanillic acid

2. Content determination of paeoniflorin and albiflorin

2.1 instruments and reagents

2.1.1 instruments

As described in item 1.1.1.

2.1.2 reagent

a reference substance paeoniflorin (batch number: 110736-201438) purchased from China institute for food and drug assay;

a control product, albiflorin (batch No. 39011-90-0), was purchased from Nanjing spring and autumn bioengineering Co., Ltd. The other reagents are analytically pure.

2.2 methods and results

2.2.1 chromatographic conditions

Alltima^TMC₁₈Chromatography columns (250 mm. times.4.6 mm, 5 μm, GRACE);

mobile phase: acetonitrile-0.1% acetic acid (18: 82);

the volume flow is 1.0 mL/min;

the column temperature is 30 ℃;

the detection wavelength is 230 nm;

the sample volume is 10 mu L;

the peak time of paeoniflorin and albiflorin is about 12min and 10 min.

2.2.2 preparation of the solution

Preparation of control solution A proper amount of penoniflorin and albiflorin is precisely weighed, and methanol is added to prepare control solutions containing 0.300mg and 0.200mg per 1mL, and the control solutions are gradually diluted to prepare 6 control solutions with different concentrations.

Preparation of test solution 15g of rhizoma cyperi four-substance particles are precisely weighed, placed in a conical flask with a plug, and precisely added with 75mL of methanol for precise weighing. Ultrasonic treating for 25min, cooling, weighing, adding methanol to reduce loss, shaking, filtering, and collecting filtrate as sample solution.

Preparation of negative sample solution according to the prescription and preparation process of rhizoma cyperi four-ingredient granules, a negative preparation lacking white paeony root is prepared. Then preparing a negative solution according to the preparation method of the test solution.

2.3 assay

The control solution, the test solution and the negative control solution were each pipetted 10. mu.L each, and analyzed under the chromatographic conditions of item 2.2.1, and the results are shown in FIG. 11. In the chromatogram of the sample, there is a chromatographic peak with the same retention time at the position corresponding to the chromatogram of the control substance of paeoniflorin and albiflorin. While the negative control solution had no peak at this retention time.

2.4 methodological considerations

2.4.1 Standard Curve and Linear Range

Precisely sucking 10 μ L of 6 control substances with different concentrations, respectively, injecting into liquid chromatograph, and measuring according to chromatographic conditions of 2.2.1 items. Taking the peak area value Y (mu V.s) of the reference substance as a vertical coordinate, and taking the mass concentration X (mu g/mL) as a horizontal coordinate, and performing linear regression to obtain a regression equation.

Paeoniflorin: y is 1.39X 10⁴X +98826, r is 0.9992, at 150-750 μ g/mL massHas good linear relation with the peak area within the range of quantitative concentration.

Albiflorin: y is 1.24X 10⁴X +48408 and r-0.9996, which has a good linear relationship with the peak area in the mass concentration range of 100-500. mu.g/mL.

2.4.2 precision test

The same control solution was sampled for 6 times, 10. mu.L each time, under 2.2.1 chromatographic conditions, and the reproducibility was observed. The results show that the RSD of the peak areas of the paeoniflorin reference substance and the albiflorin reference substance are 1.19 percent and 0.820 percent respectively, and the precision of the instrument is good. The results are shown in Table 7.

TABLE 7 precision test results of paeoniflorin and albiflorin

2.4.3 reproducibility test

6 parts of cyperus rotundus granule (batch number: 20151101) are taken, precisely weighed and prepared into a test solution according to the preparation method of the test solution. Accurately sucking 10 μ L of the test solution, injecting into a liquid chromatograph, and measuring according to the conditions of 2.2.1, wherein the RSD of the paeoniflorin and the albiflorin in the test solution are respectively 1.43 percent and 1.24 percent, which indicates that the method has good reproducibility. The results are shown in Table 8.

TABLE 8 reproducibility test results of paeoniflorin and albiflorin

2.4.4 stability test

Precisely sucking 10 μ L of penoniflorin and albiflorin reference solution at 0, 2,4, 6, 8, 10, 12, and 24 hr respectively, injecting into liquid chromatograph, and measuring under 2.2.1. The results show that the RSD of the peak areas of the control solutions of paeoniflorin and albiflorin is 1.05% and 1.48%, which indicates that the two control solutions are stable within 24 h. The results are shown in Table 9.

TABLE 9 stability test results of paeoniflorin and albiflorin

2.4.5 sample recovery test

Taking 6 parts of the determined rhizoma Cyperi granule sample (batch number: 20151101), precisely weighing, adding appropriate amount of penoniflorin and albiflorin reference substances, and preparing into test solution according to the preparation method of the test solution. The recovery rate was calculated by measuring under the conditions of item 2.2.1. The results showed that the recovery rates of paeoniflorin and albiflorin were 101% and 102%, and RSD were 1.74% and 1.52%, both of which had good recovery rates. The results are shown in tables 10 and 11.

TABLE 10 Paeoniflorin sample recovery test results

TABLE 11 Paeoniflorin sample recovery test results

2.5 sample determination

Precisely weighing 3 batches of rhizoma cyperi four-substance particle samples (batch numbers: 20151101, 20151102 and 20151103), preparing a test sample solution according to the preparation method of the test sample solution, measuring according to the chromatographic conditions under the item 2.2.1, and calculating the content, wherein the results are shown in Table 12.

TABLE 12 measurement results of paeoniflorin and albiflorin

3. Content determination of tetrahydropalmatine and berberine hydrochloride

3.1 instruments and reagents

3.1.1 instruments

As described in item 1.1.1.

3.1.2 reagent

the reference tetrahydropalmatine (batch number: 110726-201414) and the reference berberine hydrochloride (batch number: 110713-201212) are purchased from China food and drug testing institute. The other reagents are analytically pure.

3.2 methods and results

3.2.1 chromatographic conditions

Alltima^TMC₁₈Chromatography columns (250 mm. times.4.6 mm, 5 μm, GRACE);

the mobile phase is acetic acid water with the volume ratio of 55:45 acetonitrile-0.1%, and the pH value is adjusted to 6.0 by triethylamine;

the volume flow is 1.0 mL/min;

the column temperature is 30 ℃; the detection wavelength is 280 nm; the sample volume is 10 mu L;

the peak-off time of tetrahydropalmatine and berberine hydrochloride is about 11min and 7 min.

3.2.2 preparation of the solution

Preparation of control solutions: taking appropriate amount of tetrahydropalmatine and berberine hydrochloride reference substances, precisely weighing, adding methanol to obtain reference substance solutions containing 0.246mg and 0.297mg per 1mL, diluting step by step, and making into 6 reference substance solutions with different concentrations.

Preparation of a test solution 15g of rhizoma cyperi four-substance particles are precisely weighed, placed in a flat-bottomed flask, precisely added with 150mL of a concentrated ammonia test solution-methanol (1:20) mixed solution, weighed, heated and refluxed for 1h after being immersed for 1h, cooled, weighed again, added with the concentrated ammonia test solution-methanol (1:20) mixed solution to supplement the weight loss, shaken uniformly and filtered. Accurately measuring 75mL of subsequent filtrate, evaporating to dryness, dissolving the residue with methanol, transferring to a 10mL measuring flask, diluting to scale, shaking, filtering, and collecting subsequent filtrate.

The preparation of the negative sample solution is prepared into a negative preparation without rhizoma corydalis medicinal materials according to the prescription and the preparation process of the rhizoma cyperi four-ingredient particles. Then preparing a negative solution according to the preparation method of the test solution.

3.3 assay methods

The control solution, the test solution and the negative control solution were each pipetted at 10. mu.L, and analyzed under the conditions of item 3.2.1, and the results are shown in FIG. 12. In the chromatogram of the sample, there is a chromatographic peak with the same retention time at the corresponding position of the chromatogram of the reference substance of tetrahydropalmatine and berberine hydrochloride. While the negative control solution had no peak at this retention time.

3.4 methodological considerations

3.4.1 Standard Curve and Linear Range

Precisely sucking 10 μ L of 6 control substances with different concentrations, respectively, injecting into liquid chromatograph, and measuring under the conditions of item 3.2.1. Taking the peak area value Y (mu V.s) of the reference substance as a vertical coordinate, and taking the mass concentration X (mu g/mL) as a horizontal coordinate, and performing linear regression to obtain a regression equation.

Tetrahydropalmatine: y is 8.42 × 10³And (3) X-12481, wherein r is 0.9999, and the linear relation with the peak area is good within the mass concentration range of 98.4-492 mu g/mL.

Berberine hydrochloride: y is 3.08X 10⁴And (3) X-134654, wherein r is 0.9999, and the linear relation with the peak area is good in the mass concentration range of 59.4-446 mu g/mL.

3.4.2 precision test

The same control solution was sampled for 6 times, 10. mu.L each time, under the chromatographic condition of 3.2.1, and the reproducibility was observed. The results show that the RSD of the peak areas of the tetrahydropalmatine reference substance and the berberine hydrochloride reference substance are 1.52 percent and 1.00 percent respectively, and the instrument precision is good. The results are shown in Table 13.

TABLE 13 precision test results of tetrahydropalmatine and berberine hydrochloride

3.4.3 reproducibility test

6 parts of cyperus rotundus granule (batch number 20151101) are taken, precisely weighed and prepared into a test solution according to the preparation method of the test solution. Precisely sucking 10 μ L, and measuring according to the conditions of item 3.2.1, calculating RSD of tetrahydropalmatine and berberine hydrochloride in the sample solution to be 0.943% and 1.14%, respectively, which indicates that the method has good reproducibility. The results are shown in Table 14.

TABLE 14 reproducibility test results of tetrahydropalmatine and berberine hydrochloride

3.4.4 stability test

Precisely absorbing tetrahydropalmatine and berberine hydrochloride reference solutions 10 μ L respectively at 0, 2,4, 6, 8, 10, 12, and 24 hr, and determining under 3.2.1 conditions. The results show that the RSD of the peak areas of the tetrahydropalmatine and berberine hydrochloride reference substance solutions are 0.830% and 1.09%, respectively, which indicates that the two reference substance solutions are stable within 24 h. The results are shown in Table 15.

TABLE 15 stability test results for tetrahydropalmatine and berberine hydrochloride

3.4.5 sample recovery test

Taking 6 parts of the tested rhizoma Cyperi granule sample (batch number: 20151101), precisely weighing, adding appropriate amount of tetrahydropalmatine and berberine hydrochloride reference substances, respectively, preparing into test solution according to the preparation method of the test solution, testing under the condition of 3.2.1, and calculating the recovery rate. The results show that the average recovery rates of tetrahydropalmatine and berberine hydrochloride are 99.8% and 100%, and the RSD is 0.454% and 0.835%, and both have good recovery rates. The results are shown in tables 16 and 17.

TABLE 16 sample recovery test results for tetrahydropalmatine

TABLE 17 sample recovery test results for berberine hydrochloride

3.5 sample determination

Precisely weighing 3 batches of rhizoma Cyperi granule samples (batch numbers: 20151101, 20151102, 20151103), preparing into test solution according to the preparation method of the test solution, measuring according to the chromatographic conditions under item 3.2.1, and calculating the content, the results are shown in Table 18.

TABLE 18 determination of tetrahydropalmatine and berberine hydrochloride content

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A quality detection method of rhizoma cyperi four-substance particles is characterized by comprising the following steps:

(1) qualitative identification by thin layer chromatography

taking angelica and ligusticum wallichii as reference medicinal materials, adding ethanol for ultrasonic dissolution, concentrating on a water bath, shaking and extracting for 2-3 times by using ether, combining ether extract, adding sodium carbonate solution into the ether solution for shaking and extracting, removing the ether solution, adjusting the pH value of the extract to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ether, combining the ether extract, volatilizing, adding methanol for dissolution of residues, and preparing a reference medicinal material solution;

taking rhizoma cyperi, elecampane, rhizoma corydalis, radix rehmanniae preparata and radix paeoniae alba, adding ethanol for ultrasonic dissolution, placing the mixture on a water bath for concentration, shaking and extracting the mixture for 2 to 3 times by using ether, combining ether extract, adding sodium carbonate solution into the ether solution for shaking and extracting, removing the ether solution, adjusting the pH value of the extract to 2 to 3 by using dilute hydrochloric acid, then shaking and extracting by using ether, combining the ether extract, volatilizing the ether extract, and dissolving residues by adding methanol to prepare a double-negative control solution;

(2) qualitative identification by thin layer chromatography

(3) qualitative identification by thin layer chromatography

(4) qualitative identification by thin layer chromatography

(5) qualitative identification by thin layer chromatography

Taking 15g of rhizoma cyperi four-substance particles, grinding, adding 100mL of water, heating to fully dissolve, cooling, filtering by absorbent cotton, extracting the filtrate for 2 times by shaking with diethyl ether, 25mL each time, discarding the ethyl ether solution, extracting for 2 times by shaking with ethyl acetate, 25mL each time, combining the ethyl acetate solutions, evaporating to dryness, and adding 1mL of methanol to the residues to dissolve to obtain a sample solution; preparing 1g of radix rehmanniae Preparata powder into control medicinal solution by the same method; preparing 15g of a sample without the rehmannia glutinosa into a negative control solution by the same method; taking a 5-hydroxymethylfurfural reference substance, and adding methanol to prepare a solution containing 0.290mg per 1mL as a reference substance solution; testing by adopting thin-layer chromatography general rule 0502, sucking the 4 solutions, respectively dropping on the same silica gel G thin-layer plate, developing by taking xylene-ethyl acetate as a developing agent in a volume ratio of 1:1, taking out, drying, spraying 2, 4-dinitrophenylhydrazine ethanol solution, inspecting by sunlight, wherein spots with the same color are displayed on corresponding positions of the sample and the reference, and the negative reference is not interfered;

(6) qualitative identification by thin layer chromatography

Taking nutgrass galingale rhizome four-substance particles, grinding, adding water, heating for dissolving, extracting with water saturated n-butyl alcohol for 2-3 times, combining n-butyl alcohol solutions, washing with n-butyl alcohol saturated water, combining the n-butyl alcohol solutions, volatilizing in a water bath, adding methanol for dissolving, adopting a silica gel column, eluting with chloroform and methanol in a volume ratio of 10:1, then eluting with chloroform and methanol in a volume ratio of 9:1, finally eluting with chloroform and methanol in a volume ratio of 8:1, collecting subsequent 8:1 eluent, evaporating to dryness, and dissolving residues with methanol to obtain a sample solution;

taking radix paeoniae alba reference medicinal material powder, adding water, performing ultrasonic treatment, extracting with water saturated n-butyl alcohol for 2-3 times, combining n-butyl alcohol solutions, washing with n-butyl alcohol saturated water, combining the n-butyl alcohol solutions, volatilizing in a water bath, adding methanol for dissolving, adopting a silica gel column, eluting with chloroform and methanol in a volume ratio of 10:1, eluting with chloroform and methanol in a volume ratio of 9:1, eluting with chloroform and methanol in a volume ratio of 8:1, collecting subsequent 8:1 eluent, evaporating to dryness, and dissolving residues with methanol to prepare a reference medicinal material solution;

adding water into rhizoma cyperi, elecampane, rhizoma corydalis, angelica sinensis, ligusticum wallichii and radix rehmanniae preparata, performing ultrasonic treatment, extracting for 2-3 times by using water saturated n-butanol, combining n-butanol liquid, washing by using n-butanol saturated water, combining the n-butanol liquid, volatilizing in a water bath, adding methanol for dissolving, adopting a silica gel column, eluting by using chloroform and methanol with the volume ratio of 10:1, then eluting by using chloroform and methanol with the volume ratio of 9:1, finally eluting by using chloroform and methanol with the volume ratio of 8:1, collecting the subsequent 8:1 eluent, evaporating to dryness, and dissolving residues by adding methanol to prepare a negative control solution;

performing thin-layer chromatography test, respectively dropping the 4 solutions on the same silica gel G thin-layer plate, developing with chloroform-ethyl acetate-methanol-formic acid as developing agent at volume ratio of 40:5:10:0.2, taking out, air drying, spraying 5% vanillin-sulfuric acid ethanol solution, and inspecting with sunlight to obtain interference-free negative control substance with spots of the same color at corresponding positions of the sample and the control substance;

(7) qualitative identification by thin layer chromatography

performing thin-layer chromatography test, respectively dropping the 4 solutions on the same silica gel G thin-layer plate, developing with toluene-acetone as developing agent at volume ratio of 9:2, taking out, air drying, spraying improved bismuth potassium iodide solution, and observing with sunlight to obtain a result that spots of the same color appear at corresponding positions of the sample and the reference substance, and the negative reference substance has no interference;

the cyperus rotundus four-ingredient particles are prepared from cyperus rotundus, elecampane, corydalis tuber, angelica, ligusticum wallichii, prepared rhizome of rehmannia and white paeony root according to the weight ratio of 3:2:3:6:3:8:3 by the following steps:

d. and (c) taking dry paste powder, adding dextrin and sugar powder, mixing uniformly, granulating, drying, grading, and finally adding the volatile oil clathrate compound prepared in the step (b).

2. A quality detection method of rhizoma cyperi four-substance particles is characterized by comprising the following steps:

preparing a test solution by precisely weighing rhizoma Cyperi granule, placing in a conical flask with a plug, precisely adding methanol, weighing, standing overnight, ultrasonically treating, weighing again, supplementing lost mass with methanol, shaking, filtering, precisely weighing subsequent filtrate, and evaporating in water bath; dissolving the residue with sodium hydroxide solution, transferring into a reflux flask, refluxing in water bath, and cooling; transferring into a separating funnel, adjusting pH to =2 with hydrochloric acid, extracting with diethyl ether, mixing diethyl ether solutions, recovering diethyl ether, dissolving with methanol, and shaking;

the content of radix Angelicae sinensis and rhizoma Ligustici Chuanxiong in per 15g rhizoma Cyperi granule is not less than 0.714mg and 0.439mg calculated by ferulic acid and vanillic acid;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system adaptability tests; acetonitrile-0.1% acetic acid water with the volume ratio of 18:82 is taken as a mobile phase; the detection wavelength is 230nm, and the theoretical plate number is not less than 2000 calculated according to paeoniflorin and albiflorin;

preparing control solution by accurately weighing appropriate amount of penoniflorin and albiflorin, and adding methanol to obtain control solution containing 0.300mg and 0.200mg per 1 mL;

the content of radix Paeoniae alba (calculated as paeoniflorin and albiflorin) in 15g of rhizoma Cyperi granule should not be less than 52.5mg and 17.9 mg;

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system adaptability tests; using 55:45 acetonitrile-0.1% acetic acid water in volume ratio, and using triethylamine to adjust pH =6.0 as a mobile phase; the detection wavelength is 280 nm; the theoretical plate number is not less than 3000 calculated according to tetrahydropalmatine and berberine hydrochloride;

rhizoma Cyperi granule contains rhizoma corydalis (calculated by tetrahydropalmatine and berberine hydrochloride) no less than 2.67mg and 1.22mg per 15g granule;

3. The quality detection method of cyperus rotundus four-substance particles according to claim 1 or 2, characterized in that the cyperus rotundus four-substance particles are prepared by the following steps:

4. The quality detection method for cyperus rotundus four-substance granules according to claim 3, characterized in that in step b, the addition amount of beta-cyclodextrin is 5-20 times of the volume of volatile oil; the addition amount of water is 10-50 times of the volume of the volatile oil.