A method for detecting a yichengling composition

Abstract

The application discloses a detection method of Yichangleng composition, and the method comprises the following steps: S1, preparation of reference solution; S2, preparation of test solution; S3, preparation of mixed control solution; S4, respectively, precisely sucking Yichangleng test solution of S2 and mixed control solution of S3; injecting into a liquid chromatograph; S5, exporting Yichangleng characteristic spectrum obtained in S4, selecting chromatographic peaks existing in chromatograms of Yichangleng solution as characteristic peaks; calculating relative peak area and relative retention time of each characteristic peak; and according to the retention time of the chromatogram of the mixed control solution, labeling the components of the peaks in the control characteristic spectrum, the method can be used for detecting gallic acid, vanillic acid, paeoniflorin, uncarine, thalictrifolin, 5-O-methylvisamminol glycoside, quercetin, kaempferol and atractylochide II, and can realize overall quality control of Yichangleng solution.

Description

TECHNICAL FIELD

[0001] The present application relates to the quality detection technical field of traditional Chinese medicine, and particularly relates to a detection method of Yichangling composition. BACKGROUND

[0002] Yichangling composition is an empirical prescription from the Department of Gastroenterology of Lianyungang City Hospital of Traditional Chinese Medicine, which is composed of six medicines, namely, Punica granatum seeds, Saposhnikovia divaricata, Uncaria, Paeonia lactiflora, Bupleurum farrum and Poria cocos. Among them, Paeonia lactiflora is the monarch drug, Saposhnikovia divaricata and Uncaria are the ministerial drugs, and Bupleurum farrum, Poria cocos and Punica granatum seeds are the auxiliary drugs. Yichangling is a classic prescription for treating liver stagnation and spleen deficiency syndrome, which has the effects of soothing liver and extinguishing wind, invigorating spleen and eliminating dampness, astringing intestine and stopping diarrhea, and relieving acute and stopping pain. Diarrhea-predominant irritable bowel syndrome (IBS-D) belongs to the category of liver stagnation and spleen deficiency syndrome in traditional Chinese medicine. Modern clinical and pharmacological studies have found that there are few reports on the use of Yichangling decoction for the treatment of irritable bowel syndrome. The pathogenesis of IBS-D is not yet clear.

[0003] Yichangling simple prescription has good therapeutic effect on diarrhea-predominant irritable bowel syndrome in clinic, and the main components can be released only by decoction to exert the therapeutic effect. Gallic acid (with the effects of anti-inflammatory and antibacterial) and paeoniflorin (with the effects of anti-inflammatory and analgesic) are derived from Paeonia lactiflora. The epimedoside (with the effects of anti-inflammatory, antibacterial and immune regulation) and 5-O-methylvisamminol glycoside (with the effects of anti-inflammatory and antiviral) are derived from Saposhnikovia divaricata. Uncarine (with the effect of sedation) is derived from Uncaria. Bupleurum farrum is derived from Bupleurum farrum. The atractyloside II (with the effect of anti-inflammatory) is derived from Poria cocos. Vanillic acid (with the effects of anti-inflammatory and antibacterial), kaempferol (with the effects of anti-inflammatory and antioxidant) and quercetin (with the effects of anti-inflammatory and antioxidant) are derived from Bupleurum farrum. At present, there are few reports on the material basis of Yichangling decoction, and it is very important to detect the nine components by HPLC at the same time. The existing detection methods cannot effectively control the quality of Yichangling solution.

[0004] Traditional Chinese medicine characteristic spectrum is a comprehensive and quantifiable identification method. Traditional Chinese medicine characteristic spectrum has the characteristics of large information quantity, strong characteristic, integrity and fuzziness. It includes the analysis of known components and unknown components, and the chemical component information (specifically represented by relative retention time and relative peak area) has high specificity and selectivity, and the distribution of chemical components and content. It can combine various chromatography, spectroscopy and spectrum methods to characterize the authenticity and quality of traditional Chinese medicine, and become the “chemical barcode” of traditional Chinese medicine itself.

[0005] Therefore, it is of great significance to establish a Yichangling composition characteristic spectrum by high performance liquid chromatography for accurately controlling the quality of Yichangling solution. SUMMARY

[0006] The technical problem to be solved by the present invention is to provide a detection method for a Yichangling composition. This method measures multiple components, has a simple detection procedure, and low detection cost. The method of the present invention can ensure the quality stability, consistency and controllability of the Yichangling composition, thereby ensuring the safety and effectiveness of the Yichangling solution.

[0007] To address the aforementioned technical problems, this invention provides a method for detecting the Yichangling composition. The method employs high-performance liquid chromatography (HPLC), and the steps are as follows:

[0008] S1: Preparation of reference solutions: Gallic acid, vanillic acid, paeoniflorin, rhizoma lycoside, cimicifugain, 5-O-methylvisamidolol, quercetin, kaempferol and atractylodes lactone II reference standards were dissolved in 60% methanol to prepare reference solutions.

[0009] S2: Preparation of test solution: Take the Yichangling composition, decoct and extract, filter, add methanol, mix well, centrifuge, take the supernatant and filter it through a 0.45μm organic microporous membrane, take the filtrate, and the Yichangling test solution is obtained.

[0010] S3: Preparation of mixed reference solution: Accurately pipette appropriate amounts of reference solutions of gallic acid, vanillic acid, paeoniflorin, rhizoma lycoside, cimicifugain, 5-O-methylvisamidol, quercetin, kaempferol and atractylodes lactone II, and add methanol to make up to 5 ml to obtain mixed reference solution.

[0011] S4: Accurately pipette the intestinal-protecting test solution (S2) and the mixed reference solution (S3) into the liquid chromatograph, respectively.

[0012] Chromatographic column: octadecylsilane-bonded silica gel as the packing material; mobile phase: 0.1% formic acid aqueous solution as mobile phase A, methanol as mobile phase B, gradient elution, detection wavelength 254 nm; column temperature 30℃; mobile phase flow rate 1 mL / min; injection volume 10 μl; gradient elution curve as follows:

[0013] From 0 to 10 minutes, mobile phase A decreased from 95% to 95%, and mobile phase B decreased from 5% to 5%.

[0014] Over 10-25 minutes, mobile phase A decreased from 95% to 80%, and mobile phase B decreased from 5% to 20%.

[0015] Over 25-45 minutes, mobile phase A decreased from 80% to 60%, and mobile phase B decreased from 20% to 40%.

[0016] Over 45-60 minutes, mobile phase A decreased from 60% to 40%, and mobile phase B decreased from 40% to 60%.

[0017] Over 60-70 minutes, mobile phase A decreased from 40% to 20%, and mobile phase B decreased from 60% to 80%.

[0018] Over 70-80 minutes, mobile phase A decreased from 20% to 20%, and mobile phase B decreased from 80% to 80%.

[0019] S5: Export the characteristic chromatogram of Yichangling obtained in S4, select the chromatographic peaks that are present in all the chromatograms of Yichangling solution as characteristic peaks; calculate the relative peak area and relative retention time of each characteristic peak; and label the components of the peaks in the characteristic chromatogram of the mixed reference solution according to the retention time of the chromatogram.

[0020] Furthermore, the specific method for preparing the intestinal health test solution described in S2 is as follows:

[0021] Take the following ingredients for the Yichangling formula: pomegranate seeds: 6g; Saposhnikovia divaricata: 6g; Uncaria rhynchophylla: 6g; Paeonia lactiflora: 12g; Atractylodes macrocephala stir-fried with wheat bran: 12g; Atractylodes lancea stir-fried with wheat bran: 12g. Add 12 times the amount of purified water, soak for 1 hour, heat and extract for 1 hour, extract once, filter with 3 layers of gauze, shake well, take 4ml of the test solution, add 6ml of methanol, mix well, centrifuge at 5000r / min for 10min, take the supernatant and filter it through a 0.45μm organic microporous membrane, take the filtrate, and that is the product.

[0022] Furthermore, the preparation method of the mixed reference solution in S3 is as follows:

[0023] Accurately weigh appropriate amounts of gallic acid, vanillic acid, paeoniflorin, rhubarb alkaloid, cimicifugain glycoside, 5-O-methylvisamilol glycoside, quercetin, kaempferol, and atractylodes lactone II reference standards, and place them in 10 ml volumetric flasks. Dissolve in 60% methanol to prepare the following concentrations: gallic acid 20 μg / ml, vanillic acid 1 μg / ml, paeoniflorin alkaloid 230 μg / ml, rhubarb alkaloid 3 μg / ml, cimicifugain glycoside 2 μg / ml, 5-O-methylvisamilol glycoside 2.5 μg / ml, quercetin 5.5 μg / ml, kaempferol 1 μg / ml, and atractylodes lactone II 20 μg / ml. Prepare a standard stock solution of 1 ml; accurately pipette appropriate amounts of standard solutions of gallic acid, vanillic acid, paeoniflorin, rhubarb, cimicifugain, 5-O-methylvisamiloside, quercetin, kaempferol, and atractylodes lactone II, and dilute to 5 ml with 60% methanol to prepare a mixed standard solution containing 4 μg gallic acid, 0.2 μg vanillic acid, 4.6 μg paeoniflorin, 0.6 μg rhubarb, 0.4 μg cimicifugain, 0.5 μg 5-O-methylvisamiloside, 1.1 μg quercetin, 0.2 μg kaempferol, and 4 μg atractylodes lactone II per ml.

[0024] Furthermore, the characteristic spectrum of Yichangling obtained in S5 contains 9 characteristic peaks, and the retention times of each characteristic peak are as follows: characteristic peak 1, retention time is 8.651 min; characteristic peak 2, retention time is 29.694 min; characteristic peak 3, retention time is 37.559 min; characteristic peak 4, retention time is 41.154 min; characteristic peak 5, retention time is 43.771 min; characteristic peak 6, retention time is 52.900 min; characteristic peak 7, retention time is 59.379 min; characteristic peak 8, retention time is 60.272 min; characteristic peak 9, retention time is 71.780 min.

[0025] Furthermore, among them, characteristic peak 1 is gallic acid, characteristic peak 2 is vanillic acid, characteristic peak 3 is paeoniflorin, characteristic peak 4 is rhynchophylline, characteristic peak 5 is cimicifugin glycoside, characteristic peak 6 is 5-O-methylvisamidol glycoside, characteristic peak 7 is quercetin, characteristic peak 8 is kaempferol, and characteristic peak 9 is atractylodes lactone II.

[0026] The above technical solution can achieve the following beneficial effects:

[0027] The characteristic spectrum of Yichangling solution established by this invention can effectively characterize the quality of Yichangling solution, which is beneficial for comprehensive monitoring of drug quality. The characteristic spectrum established by this invention can focus on the order and interrelationship of each constituent characteristic peak, and focus on the overall appearance characteristics. It can avoid the one-sidedness of judging the quality of Yichangling solution based on the determination of individual chemical components, and reduce the possibility of artificial manipulation to meet quality standards. The method of this invention has the advantages of being simple, stable, highly precise and reproducible. Attached Figure Description

[0028] Figure 1 Characteristic chromatograms matched to the test solution of Yichangling.

[0029] Figure 2 This is the HPLC chromatogram of the mixed reference solution.

[0030] Figure 3 To compare the spectra under the mobile phases (a: pure water-methanol, b: 0.1% formic acid water-acetonitrile, c: 0.1% formic acid water-methanol).

[0031] Figure 4 Comparison of spectra at detection wavelengths (a: 200nm, b: 254nm, c: 280nm).

[0032] Figure 5 To compare the spectra at different injection volumes (2μL, 6μL, 10μL).

[0033] Figure 6 This is a linear graph of gallic acid.

[0034] Figure 7 This is a linear graph of vanillic acid.

[0035] Figure 8 This is a linear graph of paeoniflorin.

[0036] Figure 9 The graph shows the linearity of rhynchophylline.

[0037] Figure 10 The graph shows the linearity of cimicifugoside.

[0038] Figure 11 The graph shows the linearity of 5-O-methylvisamidol glycoside.

[0039] Figure 12 This is a linear graph of quercetin.

[0040] Figure 13 The graph shows the linearity of kaempferol.

[0041] Figure 14 The graph shows the linearity of atractylodes lactone II. Detailed Implementation

[0042] The following is in conjunction with the appendix Figures 1-14 The present invention will be further illustrated by the following examples:

[0043] The following examples illustrate a detection method for a probiotic composition. Unless otherwise specified, conditions used in these examples should be performed under standard conditions or those recommended by the manufacturer. Reagents and instruments used, unless otherwise specified, are commercially available products.

[0044] Instruments used in the examples: Shimadzu LC-20AD high-performance liquid chromatography system, including an online degasser, Prominence SIL-20A autosampler, SPD-M20A diode array detector, and CTO-20A column oven; MS105DU / A electronic analytical balance (Mettler-Toledo International Co., Ltd., accurate to 0.0001%); BSM-120.4 analytical balance (Shanghai Zhuojing Co., Ltd., accurate to 0.0001%); KQ2200DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0045] The reagents used in the examples were: gallic acid reference standard (batch number PS012548, purity 100.00%), vanillic acid reference standard (batch number PS010559, purity 99.94%), paeoniflorin reference standard (batch number PS000826, purity 99.17%), rhynchophylline reference standard (batch number PS101020, purity 98.75%), and cimicifugin reference standard (batch number PS011294, purity 99%). The following standards were purchased from Chengdu Pusi Biotechnology Co., Ltd.: 0.70% , 5-O-methylvisamidol glycoside reference standard (batch number PS011860, purity 100.00%), quercetin reference standard (batch number PS012150, purity 99.94%), kaempferol reference standard (batch number PS001201, purity 98.54%), and atractylodes lactone II reference standard (batch number PS013629, purity 99.86%).

[0046] Methanol was of chromatographic grade, water was ultrapure water, and formic acid was of analytical grade.

[0047] Yichangling Composition: Pomegranate seeds (batch number: 241101), Saposhnikovia divaricata (batch number: 240701), Uncaria rhynchophylla (batch number: 240701), Paeonia lactiflora (batch number: 240501), Atractylodes macrocephala stir-fried with wheat bran (batch number: 2408100), Atractylodes lancea stir-fried with wheat bran (batch number: 2411126), and other reagents (analytical grade).

[0048] Example 1:

[0049] This embodiment provides an optimization experiment for the optimal chromatographic conditions of the present invention, which includes the following steps:

[0050] Step 1: Selection of different mobile phases

[0051] This embodiment uses experiments to screen three mobile phase systems for detection: pure water-methanol, 0.1% formic acid-acetonitrile, and 0.1% formic acid-methanol. The results show that the pure water-methanol mobile phase exhibits disordered peaks and poor separation between peaks. Figure 3 As shown in (a); no peak or insignificant peak appears under the 0.1% formic acid-acetonitrile mobile phase, such as... Figure 3 As shown in (b); the peaks were significant and the separation of each component was good under the 0.1% formic acid-water-methanol mobile phase, as shown in (b). Figure 3 As shown in (c), a water-methanol mixture containing 0.1% formic acid was chosen as the mobile phase, resulting in the best system baseline stability, peak shape, and peak resolution. Therefore, water containing 0.1% formic acid (by volume) was selected as mobile phase A, and methanol as mobile phase B as the optimal mobile phase. Based on the determined mobile phases, to achieve better resolution, this invention screened gradient elution methods. Experimental results showed that different gradient elution methods had a significant impact on the resolution of each compound. This invention optimized the gradient elution curve by adjusting the resolution and the number of chromatographic peaks at different time points.

[0052] From 0 to 10 minutes, mobile phase A decreased from 95% to 95%, and mobile phase B decreased from 5% to 5%.

[0053] Over 10-25 minutes, mobile phase A decreased from 95% to 80%, and mobile phase B decreased from 5% to 20%.

[0054] Over 25-45 minutes, mobile phase A decreased from 80% to 60%, and mobile phase B decreased from 20% to 40%.

[0055] Over 45-60 minutes, mobile phase A decreased from 60% to 40%, and mobile phase B decreased from 40% to 60%.

[0056] Over 60-70 minutes, mobile phase A decreased from 40% to 20%, and mobile phase B decreased from 60% to 80%.

[0057] Over 70-80 minutes, mobile phase A decreased from 20% to 20%, and mobile phase B decreased from 80% to 80%.

[0058] Step 2: Selection of different detection wavelengths

[0059] This embodiment experimentally screened three wavelengths: 200nm, 254nm, and 280nm for chromatographic detection. At 200nm wavelength, the chromatogram showed poor peak resolution at 40-45 minutes, making component identification impossible. Figure 4 As shown in (a), under a wavelength of 280 nm, the chromatogram shows poor peak resolution at 40-45 min, making it impossible to determine the components. Figure 4 As shown in (c), the detection results indicate that the intensity of each characteristic peak at 254 nm is good, and the separation is also good, allowing for the determination of the components. Figure 4 As shown in (b), 254nm was therefore chosen as the detection wavelength. Figure 1 As shown.

[0060] Step 3: Selection of different injection volumes

[0061] This embodiment tested three injection volumes: 2 μL, 6 μL, and 10 μL for chromatographic detection. Under the 2 μL injection condition, the chromatogram showed fewer peaks, and even no peaks at certain times. Figure 5 As shown in (a); under the condition of 6 μL injection, the chromatogram shows poor peak resolution, with peaks not appearing even after 60 min of detection time. Figure 5 As shown in (b), under the condition of 10 μL injection, it can be seen from the chromatogram that the separation of each characteristic peak is good, so the injection volume of 10 μL was selected.

[0062] Based on steps 1, 2, and 3 above, the optimal chromatographic detection method for the Yichangling composition was determined as follows: mobile phase: 0.1% formic acid-methanol; optimal gradient elution mode: 0~10 min, 5%~5% methanol; 10~25 min, 5%~20% methanol; 25~45 min, 20%~40% methanol; 45~60 min, 40%~60% methanol; 60~70 min, 60%~80% methanol; 70~80 min, 80%~80% methanol; detection wavelength: 254 nm; injection volume: 10 μL; considering the research group's previous studies and literature on chromatographic detection conditions of related traditional Chinese medicine compositions, the optimal flow rate was determined to be 1 mL / min, and the optimal column temperature was determined to be 30℃.

[0063] Example 2:

[0064] Based on the optimal chromatographic detection conditions of the Yichangling composition in Example 1, a method for detecting the Yichangling composition includes the following steps:

[0065] Step 1: Preparation of the test solution

[0066] Take the Yichangling composition (pomegranate seed: 6g; Saposhnikovia divaricata: 6g; Uncaria rhynchophylla: 6g; Paeonia lactiflora: 12g; Atractylodes macrocephala stir-fried with wheat bran: 12g; Atractylodes lancea stir-fried with wheat bran: 12g), add 12 times the amount of purified water, soak for 1 hour, heat and extract for 1 hour, extract once, filter with 3 layers of gauze, shake well, take 4ml of the test solution, add 6ml of methanol, mix well, centrifuge at 5000r / min for 10min, take the supernatant and filter it through a 0.45μm organic microporous membrane, take the filtrate, and that is the product.

[0067] Step 2: Preparation of the reference solution

[0068] Accurately weigh appropriate amounts of gallic acid, vanillic acid, paeoniflorin, rhubarb alkaloid, cimicifugain glycoside, 5-O-methylvisamilol glycoside, quercetin, kaempferol, and atractylodes lactone II reference standards, and place them in 10 ml volumetric flasks. Dissolve in 60% methanol to prepare the following concentrations: gallic acid 20 μg / ml, vanillic acid 1 μg / ml, paeoniflorin alkaloid 230 μg / ml, rhubarb alkaloid 3 μg / ml, cimicifugain glycoside 2 μg / ml, 5-O-methylvisamilol glycoside 2.5 μg / ml, quercetin 5.5 μg / ml, kaempferol 1 μg / ml, and atractylodes lactone II 20 μg / ml. Prepare a standard stock solution of 1 ml; accurately pipette appropriate amounts of standard solutions of gallic acid, vanillic acid, paeoniflorin, rhubarb, cimicifugain, 5-O-methylvisamiloside, quercetin, kaempferol, and atractylodes lactone II, and dilute to 5 ml with 60% methanol to prepare a mixed standard solution containing 4 μg gallic acid, 0.2 μg vanillic acid, 4.6 μg paeoniflorin, 0.6 μg rhubarb, 0.4 μg cimicifugain, 0.5 μg 5-O-methylvisamiloside, 1.1 μg quercetin, 0.2 μg kaempferol, and 4 μg atractylodes lactone II per ml.

[0069] Step 3: Accurately pipette the Yichangling test solution from Step 1 and the mixed reference solution from Step 2, inject them into the high-performance liquid chromatograph, and record the chromatogram.

[0070] Chromatographic conditions and system suitability

[0071] Chromatographic column: YMC-Pack ODS-A C18-001 (batch number: 116FB00163), 250mm×4.6mm, 5.0μm.

[0072] Mobile phase: Pump A: 0.1 vol% formic acid; Pump B: methanol; Detection wavelength: 254 nm; Flow rate: 1.0 mL / min; Column temperature: 30°C; Gradient elution mode: The proportion of mobile phase B changes as follows: 0~10 min, 5%~5% methanol; 10~25 min, 5%~20% methanol; 25~45 min, 20%~40% methanol; 45~60 min, 40%~60% methanol; 60~70 min, 60%~80% methanol; 70~80 min, 80%~80% methanol.

[0073] The optimal gradient elution curve is:

[0074] From 0 to 10 minutes, mobile phase A decreased from 95% to 95%, and mobile phase B decreased from 5% to 5%.

[0075] Over 10-25 minutes, mobile phase A decreased from 95% to 80%, and mobile phase B decreased from 5% to 20%.

[0076] Over 25-45 minutes, mobile phase A decreased from 80% to 60%, and mobile phase B decreased from 20% to 40%.

[0077] Over 45-60 minutes, mobile phase A decreased from 60% to 40%, and mobile phase B decreased from 40% to 60%.

[0078] Over 60-70 minutes, mobile phase A decreased from 40% to 20%, and mobile phase B decreased from 60% to 80%.

[0079] Over 70-80 minutes, mobile phase A decreased from 20% to 20%, and mobile phase B decreased from 80% to 80%.

[0080] Step 4: Export the characteristic chromatogram of the Yichangling test solution obtained in Step 3, and select the chromatographic peaks present in all chromatograms of the Yichangling solution as characteristic peaks. Calculate the relative retention time and relative peak area of ​​each characteristic peak; and label the chemical composition of the peaks in the characteristic chromatogram of the mixed reference solution according to the retention time of the chromatogram.

[0081] Example 3:

[0082] Methodological research

[0083] 1. Methodological Examination

[0084] 1.1 Examination of Linear Relationships

[0085] Accurately pipette appropriate amounts of each reference solution under "Preparation of Reference Solution S1" and add 60% methanol aqueous solution to prepare a series of reference solutions of various concentrations. Perform chromatographic determination according to the chromatographic conditions under "Chromatographic Conditions S4" and record the chromatograms. Perform linear regression with the mass concentration of the reference solution as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 1.

[0086] Table 1. Results of the linear relationship examination (n=5)

[0087]

[0088] 1.2 Precision Examination

[0089] Each medicinal material in the same batch of Yichangling compound was accurately weighed, and the test solution was prepared according to the test sample preparation method in Example 2 above. The solution was injected 6 times consecutively, with an injection volume of 10 μL each time. The HPLC chromatogram was determined according to the chromatographic conditions in Example 2 above. The 9 characteristic peaks in the chromatogram were examined. With the peak area and retention time of cimicifugain as a reference, the RSD of the relative peak area of ​​each characteristic peak was calculated to be <4.90%, and the RSD of the relative retention time was <0.06%, indicating that the instrument precision was good. The results of the precision test of the relative peak area and relative retention time of gallic acid, vanillic acid, paeoniflorin, rhubarb, cimicifugain, 5-O-methylvisamidolol, quercetin, kaempferol, and atractylodes lactone II are shown in Tables 2 and 3.

[0090] Table 2. Relative peak areas in precision tests (n=6)

[0091]

[0092] Table 3. Relative retention times in precision tests (n=6)

[0093]

[0094] 1.3 Stability Test

[0095] Each herb in the same batch of Yichangling compound was accurately weighed, and a test solution was prepared according to the test solution preparation method. The solution was injected at 0, 2, 4, 6, 12, and 24 h according to the chromatographic conditions in Example 2 above, and the chromatograms were recorded. The nine characteristic peaks in the chromatograms were examined. With the peak area and retention time of cimicifugain as a reference, the RSD of the relative peak area of ​​each characteristic peak was calculated to be <4.43%, and the RSD of the relative retention time was <0.06%, indicating that the test solution had good stability within 24 h. The results of the precision test of the relative peak area and relative retention time of gallic acid, vanillic acid, paeoniflorin, rhizoma lucida, cimicifugain, 5-O-methylvisamidolol, quercetin, kaempferol, and atractylodes lactone II are shown in Tables 4 and 5.

[0096] Table 4. Relative peak areas in stability tests (n=6)

[0097]

[0098] Table 5. Relative retention times in stability tests (n=6)

[0099]

[0100] 1.4 Repeatability Test

[0101] Each herb in the same batch of Yichangling compound was accurately weighed. Six test solutions were prepared in parallel according to the test sample preparation method in Example 2 above. The solutions were injected and measured separately, and the chromatograms were recorded. The nine characteristic peaks in the chromatograms were examined. With the peak area and retention time of cimicifugain as a reference, the relative peak area RSD of each characteristic peak was calculated to be <3.17%, and the relative retention time RSD was <0.05%, indicating that the method has good repeatability. The results of the precision test of relative peak area and relative retention time of gallic acid, vanillic acid, paeoniflorin, rhizoma lucida, cimicifugain, 5-O-methylvisamidolol, quercetin, kaempferol, and atractylodes lactone II are shown in Tables 6 and 7.

[0102] Table 6. Relative peak areas in repeatability tests (n=6)

[0103]

[0104] Table 7. Relative retention times in repeatability tests (n=6)

[0105]

[0106] 1.4 Recovery Test

[0107] Accurately pipette an appropriate amount of the mixed standard solution from section "Preparation of S3 Mixed Standard Solution," and add 15 μL, 10 μL, and 5 μL of the mixed standard solution respectively. Inject each volume three times. Perform chromatographic determination according to the chromatographic conditions under section "S4 Chromatographic Conditions," and record the chromatograms. Perform linear regression with the mass concentration of the reference solution as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 8.

[0108] Table 8 Results of the recovery rate test (n=9)

[0109]

[0110]

[0111]

[0112]

[0113] The experimental results above show that the characteristic spectrum detection method for Yichangling solution established in this invention has good precision, stability and repeatability, and can effectively characterize the quality of Yichangling solution, which is beneficial for comprehensive quality monitoring.

[0114] The above descriptions are all preferred embodiments of the present invention. For those skilled in the art, any modifications to the present invention in various equivalent forms without departing from the principle of the present invention shall fall within the protection scope of the appended claims.

Claims

1. A method for detecting a probiotic composition, characterized in that: The method uses high-performance liquid chromatography (HPLC) for detection, and the steps are as follows: S1: Preparation of reference solutions: Gallic acid, vanillic acid, paeoniflorin, rhizoma lycoside, cimicifugain, 5-O-methylvisamidolol, quercetin, kaempferol and atractylodes lactone II reference standards were dissolved in 60% methanol to prepare reference solutions. S2: Preparation of test solution: Take the Yichangling composition, decoct and extract, filter, add methanol, mix well, centrifuge, take the supernatant and filter it through a 0.45μm organic microporous membrane, take the filtrate, and the Yichangling test solution is obtained. S3: Preparation of mixed reference solution: Accurately pipette appropriate amounts of reference solutions of gallic acid, vanillic acid, paeoniflorin, rhizoma lycoside, cimicifugain, 5-O-methylvisamidol, quercetin, kaempferol and atractylodes lactone II, and add methanol to make up to 5 ml to obtain mixed reference solution. S4: Accurately pipette the intestinal cleansing test solution of S2 and the mixed reference solution of S3, respectively, and inject them into the liquid chromatograph. The chromatographic conditions are as follows: Chromatographic column: octadecylsilane-bonded silica gel as the packing material; mobile phase: 0.1% formic acid aqueous solution as mobile phase A, methanol as mobile phase B, gradient elution, detection wavelength 254 nm; column temperature 30℃; mobile phase flow rate 1 mL / min; injection volume 10 μl; gradient elution curve as follows: From 0 to 10 minutes, mobile phase A decreased from 95% to 95%, and mobile phase B decreased from 5% to 5%. Over 10-25 minutes, mobile phase A decreased from 95% to 80%, and mobile phase B decreased from 5% to 20%. Over 25-45 minutes, mobile phase A decreased from 80% to 60%, and mobile phase B decreased from 20% to 40%. Over 45-60 minutes, mobile phase A decreased from 60% to 40%, and mobile phase B decreased from 40% to 60%. Over 60-70 minutes, mobile phase A decreased from 40% to 20%, and mobile phase B decreased from 60% to 80%. Over 70-80 minutes, mobile phase A decreased from 20% to 20%, and mobile phase B decreased from 80% to 80%. S5: Export the characteristic chromatogram of Yichangling obtained in S4, select the chromatographic peaks that are present in all the chromatograms of Yichangling solution as characteristic peaks; calculate the relative peak area and relative retention time of each characteristic peak; and label the components of the peaks in the characteristic chromatogram of the mixed reference solution according to the retention time of the chromatogram.

2. The detection method according to claim 1, characterized in that: The specific method for preparing the Yichangling test solution described in S2 is as follows: Take the following ingredients for the Yichangling formula: pomegranate seeds: 6g; Saposhnikovia divaricata: 6g; Uncaria rhynchophylla: 6g; Paeonia lactiflora: 12g; Atractylodes macrocephala stir-fried with wheat bran: 12g; Atractylodes lancea stir-fried with wheat bran: 12g. Add 12 times the amount of purified water, soak for 1 hour, heat and extract for 1 hour, extract once, filter with 3 layers of gauze, shake well, take 4ml of the test solution, add 6ml of methanol, mix well, centrifuge at 5000r / min for 10min, take the supernatant and filter it through a 0.45μm organic microporous membrane, take the filtrate, and that is the product.

3. The detection method according to claim 1, characterized in that: The preparation method of the mixed reference solution in S3 is as follows: Accurately weigh appropriate amounts of gallic acid, vanillic acid, paeoniflorin, rhubarb alkaloid, cimicifugain glycoside, 5-O-methylvisamilol glycoside, quercetin, kaempferol, and atractylodes lactone II reference standards, and place them in 10 ml volumetric flasks. Dissolve in 60% methanol to prepare the following concentrations: gallic acid 20 μg / ml, vanillic acid 1 μg / ml, paeoniflorin alkaloid 230 μg / ml, rhubarb alkaloid 3 μg / ml, cimicifugain glycoside 2 μg / ml, 5-O-methylvisamilol glycoside 2.5 μg / ml, quercetin 5.5 μg / ml, kaempferol 1 μg / ml, and atractylodes lactone II 20 μg / ml. Prepare a standard stock solution of 1 ml; accurately pipette appropriate amounts of standard solutions of gallic acid, vanillic acid, paeoniflorin, rhubarb, cimicifugain, 5-O-methylvisamiloside, quercetin, kaempferol, and atractylodes lactone II, and dilute to 5 ml with 60% methanol to prepare a mixed standard solution containing 4 μg gallic acid, 0.2 μg vanillic acid, 4.6 μg paeoniflorin, 0.6 μg rhubarb, 0.4 μg cimicifugain, 0.5 μg 5-O-methylvisamiloside, 1.1 μg quercetin, 0.2 μg kaempferol, and 4 μg atractylodes lactone II per ml.

4. The detection method according to claim 1, characterized in that: The characteristic spectrum of Yichangling obtained in S5 has 9 characteristic peaks, and the retention times of each characteristic peak are as follows: characteristic peak 1, retention time is 8.651 min; characteristic peak 2, retention time is 29.694 min; characteristic peak 3, retention time is 37.559 min; characteristic peak 4, retention time is 41.154 min; characteristic peak 5, retention time is 43.771 min; characteristic peak 6, retention time is 52.900 min; characteristic peak 7, retention time is 59.379 min; characteristic peak 8, retention time is 60.272 min; characteristic peak 9, retention time is 71.780 min.

5. The detection method according to claim 4, characterized in that: Among them, characteristic peak 1 is gallic acid, characteristic peak 2 is vanillic acid, characteristic peak 3 is paeoniflorin, characteristic peak 4 is rhynchophylline, characteristic peak 5 is cimicifugin glycoside, characteristic peak 6 is 5-O-methylvisamidol glycoside, characteristic peak 7 is quercetin, characteristic peak 8 is kaempferol, and characteristic peak 9 is atractylodes lactone II.

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