CN112630370B - Method for detecting active ingredients of infantile stomach-invigorating syrup - Google Patents

Abstract

The invention relates to a method for detecting effective components of a children's stomach strengthening syrup, which comprises the following identification items: comprises the thin-layer chromatography identification of lotus leaves, dried orange peels and hawthorn; the content measurement comprises the content of paeoniflorin and hesperidin; the method can perfect and comprehensively construct a quality detection system of the infantile stomach strengthening syrup, and provides guarantee for medicine safety; the feasibility of the quality standard is evaluated scientifically and objectively, and the method is suitable for daily production detection.

Description

Method for detecting active ingredients of infantile stomach-invigorating syrup

Technical Field

The invention relates to the field of medicines, in particular to a method for detecting effective components of a pediatric stomach strengthening syrup.

Background

The infantile stomach strengthening syrup is a traditional Chinese medicine preparation consisting of 11 medicinal materials such as hawthorn, dwarf lilyturf tuber, tangerine peel, white paeony root, tree peony bark and the like, has the effects of strengthening spleen and promoting digestion, clearing heat and nourishing yin, and is mainly used for treating anorexia and dyspepsia caused by deficiency of spleen-yin and stomach-yin. The prescription is collected in the tenth volume of the Chinese patent preparation in the drug Standard of the Ministry of health in 1995, and the quality standard only contains the conventional examination item of syrup, which does not meet the requirements of the current drug standard. The quality standard of the children's stomach-invigorating syrup is perfected in 2014, and comprises thin-layer chromatography identification of hawthorn and ophiopogon root medicinal materials, a high performance liquid chromatography method adopts double wavelengths, gradient elution is adopted, and the content of paeoniflorin in radix paeoniae alba and moutan bark and the content of hesperidin in dried orange peel medicinal materials are simultaneously measured (quality standard research of children's stomach-invigorating syrup, No. 21, No. 7, No. 8-11 of 3 months of the contemporary Chinese medicine 2014). The standard improvement in 2014 only identifies the hawthorn and the dwarf lilyturf root, and cannot comprehensively monitor the quality of the medicine.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for detecting the effective components of the children's stomach strengthening syrup, which can perfect and comprehensively construct a quality detection system of the children's stomach strengthening syrup and provide guarantee for the safety of medicines; scientifically and objectively evaluate the feasibility of the quality standard, and is suitable for daily production detection.

The invention is realized by the following technical scheme:

the children's stomachic syrup of the invention is prepared from the following raw medicinal materials in parts by weight: 10-20 parts of radix glehniae, 10-20 parts of rice sprout, 2-10 parts of radix paeoniae alba, 10-20 parts of radix polygonati officinalis, 10-20 parts of fried malt, 5-15 parts of hawthorn, 5-15 parts of radix ophiopogonis, 2-10 parts of dried orange peel, 10-20 parts of lotus leaf, 2-10 parts of cortex moutan and 10-20 parts of Chinese yam.

Preferably, the children's stomach strengthening syrup is prepared from the following raw medicinal materials in parts by weight: 15 parts of radix glehniae, 15 parts of rice sprouts, 4 parts of radix paeoniae alba, 15 parts of radix polygonati officinalis, 15 parts of roasted malt, 10 parts of hawthorn, 10 parts of radix ophiopogonis, 4 parts of pericarpium citri reticulatae, 15 parts of lotus leaves, 4 parts of cortex moutan and 15 parts of Chinese yam.

The preparation method of the children's stomach strengthening syrup comprises the following steps:

decocting the eleven materials in water for 1-3 times, each time for 1-3 hours, mixing decoctions, filtering, concentrating the filtrate, concentrating to a relative density of 1.20-1.25 (55-60 ℃), adding ethanol to make the ethanol content 40-80%, standing, filtering, and recovering ethanol from the filtrate for later use. Adding sucrose into the above solution, adding water to adjust total amount, stirring, adding caramel, filtering, packaging, and sterilizing.

Further preferably, the preparation method of the children's stomach-invigorating syrup comprises the following steps:

decocting the eleven materials in water for 2 times, each time for 2 hours, mixing decoctions, filtering, concentrating the filtrate, concentrating to the relative density of 1.20-1.25 (55-60 ℃), adding ethanol to ensure that the ethanol content is 60%, standing, filtering, and recovering the ethanol from the filtrate for later use. Adding sucrose into the above solution, adding water to adjust total amount, stirring, adding caramel, filtering, packaging, and sterilizing.

A method for detecting active ingredients of a pediatric stomach strengthening syrup comprises the following steps:

step 1, authentication item: wherein, the method comprises the thin-layer chromatography identification of lotus leaves and dried orange peel;

step 2, content determination: including paeoniflorin and hesperidin.

The thin-layer chromatography identification method of the lotus leaves comprises the following steps:

taking infantile stomach strengthening syrup, adding concentrated ammonia water, shaking, extracting with dichloromethane for several times, mixing dichloromethane layers, evaporating to dryness, and dissolving residue with dichloromethane to obtain test solution;

adding water into folium Nelumbinis reference material, standing in boiling water bath, shaking, cooling, filtering, and preparing filtrate from concentrated ammonia water and the sample solution to obtain reference material solution;

performing thin-layer chromatography, respectively dropping the above solutions on the same silica gel G thin-layer plate, developing with dichloromethane-acetone-concentrated ammonia water as developing agent, taking out, air drying, spraying diluted bismuth potassium iodide solution, and inspecting under sunlight;

spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

Preferably, the thin layer chromatography identification method of lotus leaves comprises the following steps:

taking 50ml of infantile stomach strengthening syrup, adding 10-30ml (optimally 20ml) of concentrated ammonia water, shaking, extracting with dichloromethane for 1-3 times (optimally 3 times), each time 20-50ml (optimally 30ml), mixing dichloromethane layers, evaporating to dryness, and dissolving residue with dichloromethane to 1ml to obtain test solution;

adding 50ml of water into 1g of lotus leaf reference medicinal material, placing in boiling water bath for 1-3h (optimally 2h), shaking once every 30-60min (optimally 30min), cooling, filtering, and preparing filtrate from "adding concentrated ammonia water 10-30 ml" with the sample solution to obtain reference medicinal material solution;

performing thin layer chromatography test, sucking 10 μ l of each solution, respectively dropping on the same silica gel G thin layer plate, developing with dichloromethane-acetone-concentrated ammonia water (6: 5: 0.1) as developing agent, taking out, air drying, spraying diluted bismuth potassium iodide solution, and inspecting under sunlight;

spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

The thin-layer chromatography identification method of the dried orange peel comprises the following steps:

extracting infantile stomach invigorating syrup with ethyl acetate, evaporating ethyl acetate layer to dryness, and dissolving residue with 1ml of ethyl acetate to obtain sample solution;

taking pericarpium Citri Tangerinae as reference material, adding methanol, ultrasonic treating, filtering, evaporating filtrate, and dissolving residue with 1ml ethyl acetate to obtain reference material solution;

performing thin layer chromatography test, sucking the above solutions, respectively dropping on the same silica gel G thin layer plate prepared with sodium hydroxide solution, spreading to about 3cm with ethyl acetate-methanol-water as developing agent, taking out, air drying, spreading to about 8cm with upper layer solution of toluene-ethyl acetate-formic acid-water as developing agent, taking out, air drying, spraying with aluminum trichloride test solution, and inspecting under ultraviolet lamp (365 nm);

in the chromatogram of the test solution, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference solution.

Preferably, the thin-layer chromatography identification method of dried orange peel comprises the following steps:

extracting 50ml of infantile stomach invigorating syrup with ethyl acetate for 1-3 times (preferably 3 times), each time 10-30ml (preferably 30ml), mixing ethyl acetate layers, evaporating to dry, and dissolving the residue with 1ml of ethyl acetate to obtain test solution;

taking pericarpium Citri Tangerinae as control material 1g, adding methanol 10-30ml (optimally 10ml), performing ultrasonic treatment for 10-30min (optimally 20min), filtering, evaporating filtrate to dryness, and dissolving residue with ethyl acetate 1ml to obtain control material solution;

performing thin layer chromatography test, taking 10 μ l of each solution, respectively dropping on the same silica gel G thin layer plate prepared from 0.5% sodium hydroxide solution, developing to about 3cm with ethyl acetate-methanol-water (100:17:13) as developing agent, taking out, air drying, developing to about 8cm with upper layer solution of toluene-ethyl acetate-formic acid-water (20:10:1:1) as developing agent, taking out, air drying, spraying aluminum trichloride test solution, and inspecting under ultraviolet lamp (365 nm);

in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.

The invention also comprises the identification of the hawthorn by thin-layer chromatography, and the method comprises the following steps:

extracting infantile stomach invigorating syrup with ethyl acetate, evaporating ethyl acetate layer to dryness, and dissolving residue with 1ml of ethyl acetate to obtain sample solution;

adding water into fructus crataegi control material, standing in boiling water bath, shaking, cooling, filtering, extracting the filtrate with ethyl acetate, evaporating ethyl acetate layer, and dissolving the residue with 1ml ethyl acetate to obtain control solution;

performing thin layer chromatography test, respectively dropping the above solutions on the same silica gel GF254 thin layer plate, developing with toluene-ethyl acetate-formic acid as developing agent, taking out, air drying, and inspecting under 254nm ultraviolet lamp; or sucking the above solutions, respectively dropping on the same silica gel G thin layer plate, spreading with cyclohexane-ethyl acetate-formic acid as developing agent, taking out, air drying, spraying 5% FeCl3 solution, and drying at 105 deg.C until spots are clear;

spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

Preferably, the hawthorn thin-layer chromatography provided by the invention is used for identification:

extracting 50ml of infantile stomach invigorating syrup with ethyl acetate for 1-3 times (preferably 3 times), each time 10-30ml (preferably 30ml), mixing ethyl acetate layers, evaporating to dry, and dissolving the residue with 1ml of ethyl acetate to obtain test solution; taking 2g of fructus crataegi as reference material, adding 50ml of water, placing in boiling water bath for 1-3h (best 2h), shaking once every 30-60min (best 30min), cooling, filtering, extracting the filtrate with ethyl acetate for 1-3 times (best 3 times), 10-30ml (best 30ml) each time, combining ethyl acetate layers, evaporating to dryness, and dissolving the residue with 1ml of ethyl acetate to obtain reference material solution;

performing thin layer chromatography test by taking 10 μ l of each of the above solutions, respectively dropping on the same silica gel GF254 thin layer plate, developing with toluene-ethyl acetate-formic acid (6: 2: 0.5) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm); or sucking the above solutions, respectively dropping on the same silica gel G thin layer plate, spreading with cyclohexane-ethyl acetate-formic acid (7: 4: 1) as developing agent, taking out, air drying, spraying 5% FeCl3 solution, and drying at 105 deg.C until spots are clear; spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

[ EXAMINATION ] the relative density should not be less than 1.16 (general rule 0601).

The pH should be 4.0-6.0 (general rule 0631)

Others should comply with the regulations in the syrup section (general rule 0116).

[ PROPERTIES ] the product is a brown yellow viscous liquid; sweet and slightly astringent.

The method for measuring the content of paeoniflorin and hesperidin in step (2) comprises the following steps:

preparation of a test solution: precisely sucking 5ml of the product, placing in a 10ml measuring flask, diluting with methanol to scale, shaking, and separating

Centrifuging, collecting supernatant, filtering with 0.45um microporous membrane, and collecting filtrate;

preparation of control solutions: accurately weighing appropriate amount of penoniflorin and hesperidin, and adding methanol to obtain mixed control solution containing 50 μ g and 30 μ g per 1 ml;

high performance liquid chromatography conditions: octadecylsilane chemically bonded silica is used as a filling agent; gradient elution was performed as specified in table 1 below using acetonitrile as mobile phase a and 0.05-0.1% phosphoric acid solution (preferably 0.1%) as mobile phase B; detection wavelength of 230nm, column temperature: 30 ℃ or 25 ℃; the number of theoretical plates is not less than 2000 calculated according to paeoniflorin peak;

TABLE 1

The determination method comprises the following steps: respectively taking the test solution and the reference solution, injecting into high performance liquid chromatograph, and measuring.

Advantageous effects

1. The detection method of the invention adds the thin layer identification of the lotus leaves and the dried orange peels on the basis of the prior art, and the product has comprehensive quality control.

2. The developing agent, the extraction solvent and the like in the identification item are obtained by screening a large amount, and the chromatogram obtained by the screened solvent has clear map and moderate Rf value, and spots have no trailing phenomenon and the like.

3. The preparation method of the content determination test sample is simple, and the detection of the two components under the same wavelength is simple, convenient and quick.

4. The invention proves that the method is accurate and precise through methodology verification, and the real quality of the product can be reliably reflected.

5. The detection method is simple and easy to operate, saves energy, reduces consumption and is more suitable for industrial daily inspection.

Drawings

FIG. 1 is a thin-layer chromatogram of lotus leaves at 27 ℃ and RH 67%, wherein, the lotus leaf negative control comprises 180901, 180902, 180903, lotus leaf control medicinal material, (+) -lotus leaf negative control; wherein A, B, C shows a thin layer chromatogram obtained using a precast plate and a Tezhou plate T, NM plate T.

FIG. 2 is a thin layer chromatogram of lotus leaf for tolerance test, named Qingdao plate, wherein, the materials of (i) 180901, (ii) 180902, (iii) 180903, (iii) lotus leaf contrast medicine, (< v > -lotus leaf negative contrast); wherein, A, B, C is a thin layer chromatogram at 32%, 72% relative humidity and under refrigerator conditions, respectively.

FIG. 3 is a thin-layer chromatogram of dried orange peel at 27 deg.C and RH 67%, wherein, the dried orange peel is negative control of dried orange peel 180901, 180902, 180903, dried orange peel control medicinal material, dried orange peel; wherein A, B, C shows a thin layer chromatogram obtained using a precast plate and a Tezhou plate T, NM plate T.

FIG. 4 is a thin-layer chromatogram of dried orange peel in tolerance test, named Qingdao plate, wherein, the first is 180901, the second is 180902, the third is 180903, the third is dried orange peel contrast medicinal material, the fifth is dried orange peel negative contrast; wherein, A, B, C is a thin layer chromatogram at 32%, 72% relative humidity and under refrigerator conditions, respectively.

FIG. 5 shows a thin layer chromatogram of fructus crataegi-Qingdao plate at 27 deg.C and RH 67%, and is examined under ultraviolet lamp (254nm) in graph A, and oven-dried at 105 deg.C in graph B. Wherein 180901, 180902, 180903, hawthorn control medicine, hawthorn negative control.

FIG. 6, control profiles of paeoniflorin and hesperidin.

FIG. 7, sample high performance liquid chromatography.

FIG. 8, control spectrum of double negative of Paeonia lactiflora and moutan cortex Radicis.

FIG. 9, pericarpium Citri Tangerinae negative control map.

FIG. 10, linear plot of paeoniflorin.

Fig. 11, linear graph of hesperidin.

FIG. 12 shows ultraviolet spectrum of control, wherein A is paeoniflorin ultraviolet spectrum, and B is hesperidin ultraviolet spectrum.

FIG. 13 is a thin layer chromatogram of lotus leaf at 27 ℃ and RH 67%, wherein ethyl acetate, n-butanol, dichloromethane, chloroform, cyclohexane

FIG. 14 shows a selective chromatogram of a lotus leaf thin-layer detection developing agent, in which A is dichloromethane-acetone-concentrated ammonia (6: 5: 0.1), and B is chloroform-ethyl acetate-methanol-water (3: 4: 2: 1); c is chloroform-ethyl acetate-concentrated ammonia (10: 5: 0.1).

Detailed Description

The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

Example 1 authentication item

Instruments and reagents

DigiStore 3 digital imaging system (CAMAG, switzerland); double-tank deployment cylinders (Shanghai Xin Yi Instrument factory); .

Silica gel G precast slabs (Qingdao oceanic factory), silica gel G precast slabs (tetramethylbiochemical plastics factory, Taizhou city road, Zhejiang province); TLC Silica 60(Merck common Silica gel G plate); a self-prepared plate (silica gel G plate (thickness: 500 μm) using 0.3% sodium carboxymethylcellulose as a binder; a self-prepared plate (silica gel G plate containing 1% sodium hydroxide).

Chemical reagents: cyclohexane, ethyl acetate, methanol, glacial acetic acid, 95% ethanol and sodium hydroxide, which are analytically pure.

Reference substance and reference medicinal materials: a lotus leaf reference drug (batch number: 121008-; pericarpium Citri Tangerinae control drug (batch No. 120904-200512); a hawthorn control drug (hawthorn, with the batch number of 121180-; . The above are provided by the verification of Chinese medicine biological products.

The traditional Chinese medicinal materials for experiments: the quality of the traditional Chinese medicinal materials used in the method is tested according to the regulations of the first edition of Chinese pharmacopoeia 2005, provided by the pharmaceutical industry of Guangzhou WangLaoji, and all the quality of the traditional Chinese medicinal materials meets the quality standard regulations.

The infantile stomach strengthening syrup sample and the infantile stomach strengthening syrup negative sample are provided by research institute of the company and prepared according to proportion of the infantile stomach strengthening syrup prescription and the preparation method.

15g of radix glehniae, 15g of rice sprout, 4g of white peony root, 15g of polygonatum odoratum, 15g of roasted malt, 10g of hawthorn, 10g of radix ophiopogonis, 4g of dried orange peel, 15g of lotus leaf, 4g of moutan bark and 15g of Chinese yam; decocting the eleven ingredients in water twice, each time for 2 hours, mixing decoctions, filtering, concentrating the filtrate, concentrating to the relative density of 1.20-1.25 (55-60 ℃), adding ethanol to ensure that the ethanol content is 60%, standing, filtering, and recovering the ethanol from the filtrate for later use. And preparing 450g of sucrose into simple syrup, adding the simple syrup into the standby liquid, adding water to adjust the total amount to 1000ml, uniformly stirring, adding a proper amount of caramel, filtering, filling and sterilizing to obtain the caramel beverage.

[ IDENTIFICATION ] folium Nelumbinis thin layer chromatography identification

Taking 50ml of the product, adding 20ml of concentrated ammonia water, shaking up, extracting with dichloromethane for 3 times, 30ml each time, combining dichloromethane layers, evaporating to dryness, and dissolving the residue with dichloromethane by 1ml to obtain a test solution. Taking 50ml of a sample lacking the lotus leaf medicinal material, preparing the sample according to a sample to be tested, and using the sample as a lotus leaf negative sample. Taking 1g of lotus leaf control drug, adding 50ml of water, placing in boiling water bath for 2h, shaking once every 30min, cooling, filtering, and preparing the control drug solution by the same method from 'adding 20ml of concentrated ammonia water'. Performing thin layer chromatography (general rule 0502) test, sucking 10 μ l of the above solutions, respectively dropping on the same silica gel G thin layer plate, developing with dichloromethane-acetone-strong ammonia water (6: 5: 0.1) as developing agent, taking out, air drying, spraying diluted bismuth potassium iodide solution, and inspecting under sunlight. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution. No interference was observed in the negative, see FIG. 1.

(2) Thin-layer chromatography identification of dried orange peel

Taking 50ml of the product, extracting with ethyl acetate for 3 times, 30ml each time, combining ethyl acetate layers, evaporating to dryness, and dissolving the residue with 1ml of ethyl acetate to obtain a test solution. Taking 50ml of a sample without pericarpium citri reticulatae medicinal materials, preparing the sample according to a test sample, and using the sample as a pericarpium citri reticulatae negative sample. Collecting pericarpium Citri Tangerinae control material 1g, adding methanol 10ml, subjecting to ultrasound for 20min, filtering, evaporating filtrate, and dissolving residue with ethyl acetate 1ml to obtain control solution. Performing thin layer chromatography (general rule 0502) test, sucking 10 μ l of each of the above solutions, dropping on the same silica gel G thin layer plate prepared with 0.5% sodium hydroxide solution, spreading to about 3cm with ethyl acetate-methanol-water (100:17:13) as developing agent, taking out, air drying, spreading to about 8cm with upper layer solution of toluene-ethyl acetate-formic acid-water (20:10:1:1) as developing agent, taking out, air drying, spraying with aluminum trichloride test solution, and inspecting under ultraviolet lamp (365 nm). In the chromatogram of the test solution, fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the reference solution. Negatives were not interfered, as shown in FIG. 3.

(3) Thin-layer chromatography identification of hawthorn

The method comprises the following steps: taking 50ml of the product, extracting with ethyl acetate for 3 times, each time 30ml, combining ethyl acetate layers, evaporating to dryness, and dissolving the residue with 1ml of ethyl acetate to obtain a test solution. Taking 50ml of a sample lacking the hawthorn medicinal material, preparing the sample according to a sample to be tested, and using the sample as a hawthorn negative sample. Taking 2g of fructus crataegi control material, adding 50ml of water, standing in boiling water bath for 2h, shaking once every 30min, cooling, filtering, extracting the filtrate with ethyl acetate for 3 times, 30ml each time, mixing ethyl acetate layers, evaporating to dryness, and dissolving the residue with 1ml of ethyl acetate to obtain control solution. Performing thin layer chromatography (general rule 0502) test, sucking 10 μ l of the above solutions, respectively dropping on the same silica gel GF254 thin layer plate, developing with toluene-ethyl acetate-formic acid (6: 2: 0.5) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (254 nm). In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material, and the negative control has interference. The results are shown in Panel A of FIG. 5. The second method comprises the following steps: sucking 10 μ l of the solution in the first method, respectively dropping on the same silica gel G thin layer plate, developing with cyclohexane-ethyl acetate-formic acid (7: 4: 1) as developing agent, taking out, air drying, spraying 5% FeCl3 solution, and drying at 105 deg.C until the spots are clear. In the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material, and the negative control has interference. The results are shown in panel B of FIG. 5.

Example two [ content determination ] content determination of paeoniflorin and hesperidin

1 Instrument and reagent

A high performance liquid chromatograph: agilent 1200.

Acetonitrile is chromatographically pure, water is purified water, and other reagents are analytically pure.

The paeoniflorin reference substance (the batch number is 110736-201135, the content is 96.5 percent, the content of 110736-201136 is 96.0 percent, the content of 110736-201842 is 97.4 percent), and the hesperidin reference substance (the batch number is 110721-201818, the content is 96.2 percent) are provided by China institute for testing food and drug. The infantile stomach-invigorating syrup sample and the negative sample are provided by Guangzhou Wanglaoji pharmaceutical industry GmbH.

2 chromatographic conditions

A chromatographic column: welch ultimate XB-C18 (4.6X 250mm, 5 μm)

Kromasil 100-5 C18(4.6×250mm，5μm)

Capcell Pak MG S5 C18(4.6×250mm，5μm)

Mobile phase: acetonitrile (A) -0.1% phosphoric acid (B) was subjected to gradient elution as specified in Table 1 below

TABLE 1

Detection wavelength: 230nm

Column temperature: 30 ℃ or 25 DEG C

Sample injection volume: 10 μ l (control) or 20 μ l (test)

3. And (4) detecting a plurality of batches of samples, and determining 12 batches of infantile stomach strengthening syrup samples, wherein the samples are shown in a table 2.

TABLE 2 determination of the content of the pediatric stomach-invigorating syrup sample

Example three methodological validation

5.1 preparation of the solutions

Preparation of control solutions: accurately weighing penoniflorin and hesperidin, respectively placing into a measuring flask, adding appropriate amount of methanol to dissolve and dilute to scale, and making into penoniflorin reference preparation stock solution (about 0.25mg/ml) and hesperidin reference preparation stock solution (about 0.4 mg/ml).

Precisely sucking the above two reference stock solutions respectively, diluting with methanol, and diluting to desired volume to obtain penoniflorin reference solution (about 50 μ g/ml) and hesperidin reference solution (about 30 μ g/ml).

Preparing a test solution: precisely sucking 5ml of the product, placing in a 10ml measuring flask, diluting with methanol to scale, shaking, centrifuging, collecting supernatant, filtering with 0.45um microporous membrane, and collecting filtrate.

Preparation of negative control solution: taking a double-negative sample of the white paeony root and the tree peony bark and a negative sample of the tangerine peel, and preparing the same with the preparation method of the test solution.

5.2 accuracy test

5.2.1 paeoniflorin

Precisely measuring 3ml of the same batch of samples (batch No. 180903-1: 53.1. mu.g/ml repeatability test result) with known concentration in a 10ml measuring flask, sampling 6 parts in parallel, respectively adding paeoniflorin reference solution, preparing according to the preparation method of the test solution, measuring according to the chromatographic conditions, and calculating the recovery rate, which is shown in Table 5.

TABLE 5 recovery test results of paeoniflorin

5.2.2 hesperidin production

Precisely measuring 3ml of the same batch of samples (batch number 180903-1: 27.7 mu g/ml repeatability test result) with known concentration in a 10ml measuring flask, sampling 6 parts in parallel, respectively adding hesperidin reference solution, preparing according to the preparation method of the test solution, measuring according to the chromatographic conditions, and calculating the recovery rate, which is shown in Table 6.

TABLE 6 hesperidin recovery test results

5.3 precision test

5.3.1 repeatability test

6 portions of the same batch (batch No. 180903-1) of samples were precisely measured, prepared according to the method for preparing the test sample solution, measured according to the above chromatographic conditions, and the RSD% was calculated from the content, as shown in Table 7.

TABLE 7 results of the repeatability tests

5.3.2 intermediate precision

The same batch of samples (batch No. 180903-1) was tested at different times in the same laboratory by 3 different analysts, the results of which are given in Table 8.

TABLE 8 intermediate precision results

5.4 specificity test

Preparing double negative sample without radix Paeoniae alba and cortex moutan and pericarpium Citri Tangerinae negative sample according to prescription, preparing negative control solution according to the method for preparing test solution, and determining according to the above chromatographic conditions. Results negative controls were not interfered with, see FIGS. 6-9. 5.5 Linearity

5.5.1 paeoniflorin

Accurately sucking penoniflorin reference substance stock solution, diluting with methanol, and metering to 6 reference substance solutions with different concentrations. Taking the reference substance solution, determining the peak area of paeoniflorin according to the chromatographic conditions, and drawing a standard curve by taking the sample injection concentration (mu g/ml) of the reference substance as the abscissa and the peak area as the ordinate. And (4) carrying out regression analysis on the sample concentration x of the control by using the peak area y to obtain a regression equation y which is 11.60530x-3.69493, and a correlation coefficient r which is 0.99991. The results are shown in Table 9 and FIG. 10.

TABLE 9 Paeoniflorin standard curve data sheet

The results show that: under the chromatographic condition, the paeoniflorin has good linear relation within the concentration range of 5.0372 mu g/ml-125.9325 mu g/ml.

5.5.2 hesperidin

And precisely sucking the hesperidin reference product stock solution, diluting with methanol, and fixing the volume to 6 parts of reference product solutions with different concentrations. Taking the reference substance solution, determining the peak area of paeoniflorin according to the chromatographic conditions, and drawing a standard curve by taking the sample injection concentration (mu g/ml) of the reference substance as the abscissa and the peak area as the ordinate. And (4) carrying out regression analysis on the sample injection concentration x of the control by using the peak area y to obtain a regression equation y which is 21.97821x-6.54722, and a correlation coefficient r which is 0.99999. The results are shown in Table 10 and FIG. 11.

TABLE 10 data sheet of standard curve for hesperidin

The results show that: under the chromatographic conditions, the hesperidin has a good linear relation within the concentration of 3.134 mu g/ml-195.850 mu g/ml.

5.6 durability test

5.6.1 investigation of different columns

The results of examining 3 chromatographic columns of different specifications and determining the reference substance and the sample according to the above chromatographic conditions are shown in table 11.

Table 113 chromatographic column examination results

5.6.2 sample stability Studies

The same sample solution was taken and left at room temperature for 0h, 4h, 8h, 12h, 24h after preparation, and measured according to the above chromatographic conditions, and the RSD% was calculated from the measured peak area, see Table 12.

Table 12 stability test results

Test example I screening test of thin layer chromatogram of Lotus leaf

1. Preparation of test solution screening test

Taking 50ml of the infantile stomach strengthening syrup, adding 20ml of concentrated ammonia water, shaking, extracting with ethyl acetate, n-butanol, dichloromethane, chloroform, cyclohexane for 3 times, 30ml each time, mixing organic solvent layers, evaporating to dryness, and dissolving the residue with corresponding solvent to 1ml to obtain sample solution; performing thin layer chromatography test, sucking 10 μ l of each solution, respectively dropping on the same silica gel G thin layer plate, developing with dichloromethane-acetone-concentrated ammonia water (6: 5: 0.1) as developing agent, taking out, air drying, spraying diluted bismuth potassium iodide solution, and inspecting under sunlight; the results are shown in FIG. 13:

the results show that the dichloromethane and chloroform have the best extraction effect, and the dichloromethane has lower toxicity than the chloroform, so the dichloromethane is selected as the extraction solvent

2. Screening test for developing Agents

According to a thin-layer chromatography test, 10 mu l of the test solution is respectively spotted on the same silica gel G thin-layer plate, dichloromethane-acetone-concentrated ammonia water (6: 5: 0.1), an upper solution of chloroform-ethyl acetate-methanol-water (3: 4: 2: 1) and chloroform-ethyl acetate-concentrated ammonia water (10: 5: 0.1) are respectively selected as developing agents, the developing agent is developed, taken out and dried in the air, diluted bismuth potassium iodide test solution is sprayed, and the test solution is inspected in the sunlight; the results are shown in FIG. 14:

the result shows that the dichloromethane-acetone-strong ammonia water (6: 5: 0.1) is used as the developing solvent, the RF value is moderate, and the spots are clear.

3. Comparing the chromatographic effects of different thin-layer plates

Panels T were operated as in example 1 using a prefabricated panel, taizhou panel T, NM. Test results show that the chromatographic effect is ideal by adopting the prefabricated plate, and the expanded system has stronger adaptability, and the results are shown in figure 1.

4. Durability examination:

when the Qingdao plate is used for being unfolded in a refrigerator under the condition of 32% relative humidity and 72% relative humidity, the thin layer separation conditions of main spots are basically consistent, and the influence of temperature and humidity on the thin layer chromatography is small, so that the thin layer separation system is high in durability, free of the influence of the environment and suitable for daily industrial inspection, and the result is shown in figure 2.

Test example II screening test of dried orange peel thin-layer chromatogram

1. Comparing the chromatographic effects of different thin-layer plates

Panels T were operated as in example 1 using prefabricated panels, taizhou panels T, NM. Test results show that the precast slab is adopted, the chromatographic effect is ideal, and the developed system has stronger adaptability. The results are shown in FIG. 3.

2. Durability examination:

the Qingdao plate is used for being unfolded in a refrigerator under the condition of 32% relative humidity and 72% relative humidity, the thin layer separation conditions of main spots are basically consistent, and the influence of temperature and humidity on the thin layer chromatography is small. The results are shown in FIG. 4.

Test example three, selection of conditions for high performance liquid chromatography

1. Selection of mobile phase

Combining the previous research, and modifying according to a mobile phase acetonitrile-0.1% phosphoric acid solution (14:86) for measuring the content of paeoniflorin in the section of a white paeony root medicinal material of the 2015 version of pharmacopoeia, the paeoniflorin and the hesperidin are detected under the same condition, the experimental result is better, and the system adaptability meets the requirements.

2. Selection of detection wavelength

Ultraviolet spectrum detection shows that paeoniflorin has maximum ultraviolet absorption at 230 nm; the hesperidin has an obvious absorption peak at 283nm, and also has an absorption peak at 230nm, and the ultraviolet absorption is stronger than that at 283 nm. The content of the paeoniflorin and the hesperidin is determined under the same condition, so that 230nm is selected as the detection wavelength, as shown in figure 12, the reason that the invention does not select the double wavelength is as follows: the hesperidin has absorption peaks at 230nm and 283nm, but the absorption at 230nm is stronger than that at 283nm, so that the two components can be more conveniently measured by using the same wavelength, the requirement on an instrument is not so high, and the liquid phase with an ultraviolet absorption detector can be generally operated; the hesperidin prepared by the test method of the invention has no interference at 230nm, and is shown in figure 9 in detail.

3. Examination of test article preparation method

Comparative group 1: (comparison document 1, zhengrongbao, etc., quality standard research of the syrup for invigorating stomach of children, contemporary medicine of China, 2014, 21(7), 8-11) precisely measures 10ml of the product, sequentially elutes the product through a processed C18 small column by using 15ml of water, 5% methanol and 60 methanol respectively, collects 60% methanol eluent, evaporates the eluent, dissolves residues by using 60% methanol and transfers the dissolved residues to a 5ml volumetric flask, dilutes the residues to scale by using 60% methanol, and shakes the residues evenly.

Comparative group 2: (comparison document 2, Yuanhong et al, study on improvement of quality standard of children's stomachic syrup, Yuanhong and the like, medicine evaluation and analysis in Hospital, 2016,16(5), 630-632), precisely sucking 20ml of the product, placing the product in a separating funnel, sequentially adding ethyl ether and ethyl acetate to wash for 2 times, 25ml each time, extracting residual water with water-saturated n-butanol for 5 times, 25ml each time, combining the n-butanol solutions, evaporating to dryness, dissolving residues with a mobile phase, quantitatively transferring to a 10ml volumetric flask, adding the mobile phase to scale, shaking up, filtering with a microporous filter membrane (0.45 μm), and preparing a test solution; taking appropriate amount of penoniflorin as control, precisely weighing, adding mobile phase to obtain solution containing 40 μ g per 1ml, and filtering with microporous membrane (0.45 μm) to obtain control solution.

Comparative group 3: (comparison document 3, Lianghuiming et al, HPLC method for determining paeoniflorin content in the children's stomach-invigorating syrup, pharmaceutical today, 2008,18(3), 30-31). Precisely measuring 20ml of the product, extracting with n-butanol for 4 times (40,30,30,20ml), standing for more than 20min each time, mixing n-butanol extractive solutions, evaporating to dryness, dissolving the residue with 80% methanol 5ml under heating, loading onto pretreated neutral alumina column (3.0g, column packed by dry method, column inner diameter 10mm), eluting with 60% methanol, collecting eluate about 30ml, evaporating to dryness, dissolving the residue with mobile phase, transferring to 5ml volumetric flask, and adding the mobile phase to desired volume.

The invention comprises the following steps: example 2 test article solutions were prepared.

The conditions and measurement method of the high performance liquid chromatography were the same as in example 2.

The results are shown in Table 13, the preparation method of the test sample is superior to the comparison group, the operation method is simple, the operation is convenient, the time is saved, the content of the effective components is improved, and the product quality can be truly reflected.

TABLE 13 examination of the preparation methods of the test articles

3. Investigation of sample extraction solvent

The same batch of infant stomach strengthening syrup samples were taken, 50% methanol, 70% methanol and methanol were selected to examine the results of the determination of paeoniflorin and hesperidin, and the experimental data are shown in table 14. Three solvents have little influence on the dissolution measurement of the two components, and methanol is selected as the solvent for preparing the sample.

TABLE 14 solvent examination results

The foregoing description of specific exemplary embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims (5)

1. A method for detecting active ingredients of a child stomach-invigorating syrup comprises the following steps:

step 1, authentication item: comprises the thin-layer chromatography identification of lotus leaves and dried orange peel;

step 2, content determination: including paeoniflorin and hesperidin content;

in the step (1), the thin-layer chromatography identification method of the lotus leaves comprises the following steps:

taking 50ml of the infantile stomach-invigorating syrup, adding 10-30ml of concentrated ammonia water, shaking, extracting with dichloromethane for 1-3 times, each time 20-50ml, mixing dichloromethane layers, evaporating to dryness, and dissolving the residue with dichloromethane to obtain 1ml as sample solution;

taking 1g of folium Nelumbinis reference material, adding 50ml of water, standing in boiling water bath for 1-3h, shaking once every 30-60min, cooling, filtering, and preparing the filtrate from "adding concentrated ammonia water 10-30 ml" with the sample solution to obtain reference material solution;

absorbing 10 mu l of each solution by thin layer chromatography, respectively dropping the solution on the same silica gel G thin layer plate, taking dichloromethane-acetone-strong ammonia water as a developing agent, wherein the volume ratio of dichloromethane-acetone-strong ammonia water is 6: 5: 0.1, developing, taking out, airing, spraying a dilute bismuth potassium iodide test solution, and inspecting in the sunlight;

spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the control solution;

in the step (1), the thin-layer chromatography identification method for the dried orange peel comprises the following steps:

extracting infantile stomach invigorating syrup 50ml with ethyl acetate for 1-3 times (10-30 ml each time), mixing ethyl acetate layers, evaporating to dry, and dissolving residue with ethyl acetate 1ml to obtain sample solution;

taking pericarpium Citri Tangerinae as control material 1g, adding methanol 10-30ml, ultrasonic treating for 10-30min, filtering, evaporating filtrate, and dissolving residue with ethyl acetate 1ml to obtain control material solution;

taking thin-layer chromatography, sucking 10 μ l of each of the above solutions, respectively dropping on the same silica gel G thin-layer plate prepared with 0.5% sodium hydroxide solution, developing to about 3cm with ethyl acetate-methanol-water as developing agent and the volume ratio of ethyl acetate-methanol-water being 100:17:13, taking out, air drying, developing to about 8cm with the upper layer solution of toluene-ethyl acetate-formic acid-water as developing agent and the volume ratio of toluene-ethyl acetate-formic acid-water being 20:10:1:1, taking out, air drying, spraying aluminum trichloride test solution, and inspecting under 365nm ultraviolet lamp;

in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution;

in the step (2), the content determination of paeoniflorin and hesperidin comprises the following steps:

preparation of a test solution: precisely absorbing the product, diluting with methanol to scale, shaking, centrifuging, collecting supernatant, filtering with microporous membrane, and collecting filtrate;

preparation of control solutions: accurately weighing appropriate amount of penoniflorin and hesperidin, and adding methanol to obtain mixed control solution containing 50 μ g and 30 μ g per 1 ml;

high performance liquid chromatography conditions: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is taken as a mobile phase A, 0.05-0.1% phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; detection wavelength 230nm, column temperature: the number of theoretical plates is not less than 2000 calculated according to paeoniflorin peak at 30 deg.C or 25 deg.C;

the determination method comprises the following steps: respectively injecting the test solution and the reference solution into a high performance liquid chromatograph, and measuring.

2. The detection method of claim 1, wherein the pediatric stomach strengthening syrup is prepared from the following raw materials in parts by weight: 10-20 parts of radix glehniae, 10-20 parts of rice sprouts, 2-10 parts of radix paeoniae alba, 10-20 parts of radix polygonati officinalis, 10-20 parts of roasted malt, 5-15 parts of hawthorn, 5-15 parts of radix ophiopogonis, 2-10 parts of pericarpium citri reticulatae, 10-20 parts of lotus leaves, 2-10 parts of cortex moutan and 10-20 parts of Chinese yam.

3. The detection method of claim 2, wherein the pediatric stomach strengthening syrup is prepared from the following raw materials in parts by weight: 15 parts of radix glehniae, 15 parts of rice sprout, 4 parts of radix paeoniae alba, 15 parts of radix polygonati officinalis, 15 parts of fried malt, 10 parts of hawthorn, 10 parts of radix ophiopogonis, 4 parts of dried orange peel, 15 parts of lotus leaf, 4 parts of cortex moutan radicis and 15 parts of Chinese yam.

4. The detection method according to claim 2, wherein the pediatric stomach strengthening syrup is prepared by the following method: decocting the eleven materials in water for 1-3 times, each time for 1-3 hours, mixing decoctions, filtering, concentrating the filtrate, concentrating to the relative density of 1.20-1.25 at 55-60 ℃, adding ethanol to ensure that the ethanol content is 40-80%, standing, filtering, and recovering ethanol from the filtrate for later use; and preparing sucrose into simple syrup, adding into the above stock solution, adding water to adjust total amount, stirring, adding appropriate amount of caramel, filtering, bottling, and sterilizing.

5. The assay of claim 4, wherein the pediatric stomach syrup is prepared by: decocting the eleven materials in water for 2 times, each time for 2 hours, mixing decoctions, filtering, concentrating the filtrate, concentrating to the relative density of 1.20-1.25 at 55-60 ℃, adding ethanol to ensure that the ethanol content is 60%, standing, filtering, and recovering the ethanol from the filtrate for later use; and preparing 450 parts of simple syrup from cane sugar, adding the prepared solution into water to adjust the total amount, uniformly stirring, adding a proper amount of caramel, filtering, filling and sterilizing to obtain the syrup.

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