Method for determining content of active ingredients in gardenia-rhubarb decoction

Abstract

The invention provides a method for measuring the content of active ingredients in gardenia-rhubarb decoction. The method comprises the following operation steps: 1) preparing a reference solution; (2) preparing a test solution; and (3) detecting by adopting an HPLC (High Performance Liquid According to the method for determining the content of the active ingredients in the gardenia-rhubarb decoction, the reference sample of the rhubarb decoction is obtained on the basis of the original record of Jingui Litu and is used for constructing the method for detecting the active ingredients in the gardenia-rhubarb decoction; according to the method, high-resolution detection of seven key functional components including genipin gentiobioside, geniposide, narirutin, hesperidin, daidzein, emoditin-7-O-beta-D-glucoside and genistein is realized, the specificity is high, the repeatability is good, a reliable technical support is provided for quality standard improvement and clinical precise medication of the gardenia-rhubarb decoction, and the method has a wide application prospect. The method has important significance on guaranteeing the stability of the gardenia-rhubarb decoction reference sample.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine preparation component detection, and particularly relates to a method for determining the content of active ingredients in Zhizi Dahuang Decoction. BACKGROUND

[0002] Zhizi Dahuang Decoction is first recorded in Synopsis of Golden Chamber, and is a classic prescription for treating jaundice due to alcohol, which is composed of four ingredients of Gardenia, Fructus Aurantii Immaturus, Rhizoma et Radix Rhei and Semen Sojae Praeparatum, and has the effects of clearing heat and dampness, relieving depression and relieving restlessness, and promoting bile discharge and relieving jaundice. The original record states that "for jaundice due to alcohol, with depression in the heart or heat and pain, Zhizi Dahuang Decoction is used for treatment", and the pathogenesis is that dampness and heat are accumulated in the middle jiao, heat is more serious than dampness, and the upper part of the heart is scalded, which causes depression and restlessness, and heat pain is caused by the obstruction of Qi. In the prescription, Gardenia is the monarch drug, which has the effects of clearing heat and reducing fire, and cooling blood, and when combined with Semen Sojae Praeparatum, it can clear and reduce dampness and heat, and relieve restlessness, and promote Qi circulation and relieve fullness; Fructus Aurantii Immaturus is the minister drug, which has the effects of breaking Qi and dispersing mass, and reducing phlegm and eliminating accumulation; Rhizoma et Radix Rhei is the assistant drug, which can clear heat in the Qi aspect, and disperse stasis and heat, and can be used to enhance the effect of Rhizoma et Radix Rhei, and Fructus Aurantii Immaturus and Rhizoma et Radix Rhei can break Qi and eliminate accumulation, and reduce heat. In traditional Chinese medicine, it is mainly used for the treatment of liver and gall diseases with heat being more serious than dampness or heart heat, such as acute and chronic hepatitis, acute jaundice hepatitis, infectious hepatitis, non-jaundice hepatitis, alcoholic hepatitis, acute pancreatitis, cholecystitis, and recurrent oral ulcer, and can reduce the condition of patients to a certain extent, and has a significant effect.

[0003] The results of chemical component research show that Zhizi Dahuang Decoction mainly contains iridoid glycosides (geniposide, genipin 3-O-β-D-glucopyranoside), flavonoids (naringin, hesperidin, daidzein), anthraquinones (emodin-7-O-β-D-glucoside), polyphenols (genistein), coumarins, monoterpenes and organic acids. Among them, geniposide and genipin 3-O-β-D-glucopyranoside are effective components in the monarch drug Gardenia in Zhizi Dahuang Decoction, geniposide is recorded in the content determination item of Chinese Pharmacopoeia, and is a mandatory detection item for the detection of Gardenia medicinal materials or decoction pieces, which has the biological activities of liver protection, anti-inflammatory analgesia, neuroprotection, anti-depression, and inhibition of ischemia-reperfusion injury, genipin 3-O-β-D-glucopyranoside has important significance in pharmacological activity and quality control, and also has the biological activities of liver protection, anti-inflammatory, anti-oxidation, and anti-thrombosis, and is a commonly used index for evaluating the quality of Gardenia; naringin and hesperidin are the main active ingredients in Fructus Aurantii Immaturus, which have the effects of liver protection, liver detoxification, anti-fungus, treatment of acute and chronic hepatitis, liver cirrhosis, and anti-free radical and anti-oxidation; emodin-8-O-β-D-glucoside has obvious antibacterial and anti-inflammatory activities, and is an important chemical marker in Rhizoma et Radix Rhei, and also contributes to various pharmacological activities, and pharmacological research shows that it has the effects of anti-bile stasis and liver protection; genistein and daidzein are one of the main active ingredients in Semen Sojae Praeparatum, which also has the effects of liver protection, anti-inflammatory and anti-oxidation, and are often listed as key indicators for the quality evaluation of Semen Sojae Praeparatum.

[0004] Currently, there is limited research on Gardenia and Rhubarb Decoction. Tang Zheng et al., in their study "UFLC Fingerprint Study of Gardenia and Rhubarb Decoction," proposed a fingerprint detection method for Gardenia and Rhubarb Decoction, which could identify 51 common chromatographic peaks in the decoction and pinpoint 14 of them through comparison with reference standards. However, the resolution of the characteristic peaks failed to reach above 1.5, making it difficult to meet the precise quality control requirements of the active ingredients in actual production. Summary of the Invention

[0005] To address the above problems, this invention establishes a method for determining the content of active ingredients in Gardenia and Rhubarb Decoction, which includes the following steps: 1) Preparation of reference solutions: Take reference standards of iridoids, flavonoids, anthraquinones, and polyphenols, and prepare reference solutions by adding methanol; 2) Preparation of the test solution: Take the Gardenia and Rhubarb Decoction preparation, extract it with methanol solution to obtain the test solution; 3) Detection: HPLC was used for detection. The chromatographic conditions were as follows: Column: Octadecylsilane-bonded silica gel; Mobile phase: Acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B; Gradient elution program was as follows:

[0006] Further, in step 1), each 1 mL of the reference solution contains 50-100 μg of each reference standard.

[0007] Furthermore, the iridoid compounds in the reference standard are geniposide and / or genipin gentiopicroside; the flavonoid compounds are rutin, hesperidin and / or daidzein; the anthraquinone compounds are emodin-7-O-β-D-glucoside; and the polyphenolic compounds are genistein.

[0008] Further, in step 2), the mass-to-volume ratio of the Gardenia and Rhubarb Decoction preparation to the methanol solution is 0.1~0.5g:10~50ml.

[0009] Furthermore, the Gardenia and Rhubarb Decoction preparation includes Gardenia and Rhubarb Decoction decoction, Gardenia and Rhubarb Decoction granules, Gardenia and Rhubarb Decoction pills, Gardenia and Rhubarb Decoction tablets, and Gardenia and Rhubarb Decoction material standard; the concentration of the methanol solution is 70%.

[0010] Further, in step 2), the extraction is ultrasonic extraction, with a time of 20-60 min, a power of 400-800 W, and a frequency of 20-60 kHz.

[0011] Further, in step 3), the chromatographic column is an Agilent ZORBAX SB-Aq 250×4.6mm, 5um.

[0012] Further, in step 3), the chromatographic conditions are as follows: column temperature is 25–35°C; flow rate is 0.8 mL / min–1.2 mL / min; injection volume is 5–15 μL; and detection wavelength is 250–270 nm.

[0013] Furthermore, in step 3), the chromatographic conditions are as follows: column temperature is 30°C; flow rate is 1.00 mL / min; injection volume is 10 μL; and detection wavelength is 260 nm.

[0014] Furthermore, it uses the external standard method to calculate the content of active ingredients in Gardenia and Rhubarb Decoction based on peak area.

[0015] The material standard for Gardenia and Rhubarb Decoction used in this invention is based on the preparation method of Gardenia and Rhubarb Decoction recorded in the ancient medical book *Synopsis of Prescriptions of the Golden Chamber*. Except for the molding process, the other preparation methods are basically consistent with those recorded in *Synopsis of Prescriptions of the Golden Chamber*. The specific preparation method is as follows: According to the formula of Gardenia and Rhubarb Decoction, weigh out 12.6g of Gardenia, 13.8g of Rhubarb, 69.0g of Fructus Aurantii Immaturus and 69.0g of Fermented Soybean. Boil with 1200ml of water to obtain 600ml of liquid. Remove the dregs, collect the liquid, concentrate it, freeze-dry it, and pulverize it to obtain the physical dry extract powder corresponding to the material basis of Gardenia and Rhubarb Decoction.

[0016] Based on the original text of "Synopsis of Prescriptions of the Golden Chamber" and the quantitative research of ancient and modern literature, this invention establishes a standardized decoction method for the reference sample of Gardenia and Rhubarb Decoction. Based on this, a method for simultaneously determining the content of seven key active ingredients, namely genipin gentiopicroside, gardenoside, rutin naringin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein, is constructed. This method achieves high resolution detection with a resolution of over 1.5 for each characteristic peak and a theoretical plate number of not less than 5000 based on gardenoside.

[0017] The method of this invention has strong specificity and good repeatability. It not only makes up for the shortcomings of existing fingerprinting technology, such as insufficient separation and difficulty in precise quality control, but also provides reliable technical support for improving the quality standard of Gardenia and Rhubarb Decoction, precise clinical medication, and the stability of reference materials for modern preparations. It is of great significance for ensuring the overall quality of Gardenia and Rhubarb Decoction.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 Mobile phase selection diagram (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin naringin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Figure 2 Chromatograms of elution gradients (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Figure 3 Results of different extraction methods (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Figure 4 Results of different extraction solvents (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Figure 5 Results of different solvent dosages (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Figure 6 Results of different extraction times (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin naringin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein); Figure 7 HPLC chromatograms of mixed reference standard and test sample (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Figure 8 Results of specificity study (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin naringin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Figure 9Results of different flow rates (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin naringin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Figure 10 Results of different column temperatures (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin naringin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Figure 11 Results of different injection volumes (1 is genipin gentiopicroside, 2 is geniposide, 3 is rutin, 4 is hesperidin, 5 is daidzein, 6 is emodin-7-O-β-D-glucoside, 7 is genistein). Detailed Implementation

[0021] Example 1: Method for determining the content of active ingredients in Gardenia and Rhubarb Decoction of the present invention 1. Detection Method (1) Preparation of reference solution Take genipin gentiopicroside, geniposide, rutin naringin, hesperidin, daidzein, emodin-7-O-β-D-glucoside and genistein reference standards, add methanol to prepare a mixed solution containing 50-100 μg of each reference standard per ml.

[0022] (2) Preparation of the test solution Take 0.2g of Gardenia and Rhubarb Decoction preparation, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 70% methanol, weigh it, sonicate it (power 600W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product. (3) HPLC chromatographic detection The chromatographic column was an Agilent ZORBAX SB-Aq 250×4.6mm, 5µm; mobile phase A was acetonitrile, and mobile phase B was 0.1% phosphoric acid aqueous solution; the flow rate was 1.0 mL / min; the column temperature was 30℃; the detection wavelength was 260 nm; the injection volume was 10µL; and the gradient elution program was as follows: (4) The contents of genipin, geniposide, rutin, hesperidin, daidzein, emodin-7-O-β-D-glucoside and genistein in Gardenia and Rhubarb Decoction were calculated by peak area using the external standard method.

[0023] The following experimental examples further illustrate the beneficial effects of the present invention. Experimental Example 1: Quantitative Study of Components in Gardenia and Rhubarb Decoction 1. Experimental instruments and materials High-performance liquid chromatograph: Agilent 1260 Infinity II high-performance liquid chromatograph; Electronic balance: ME204E, MS205DM, XPE26 (Mettler-Toledo Instruments Co., Ltd.); Ultrapure water system: Cellular 1810A (Shanghai Moller Scientific Instruments Co., Ltd.); Ultrasonic cleaner: KQ5200DB (600W, 40KHz; Kunshan Ultrasonic Instruments Co., Ltd.); Chromatographic column: Agilent ZORBAX SB-Aq 250×4.6mm, 5µm; Naringin reference standard (Shanghai Yuanye Biotechnology Co., Ltd., batch number: J06HB172580, content calculated as 98%); geniposide reference standard (batch number: 111749-201718, content calculated as 97.6%), hesperidin reference standard (batch number: 111721-202220, content calculated as 97.2%), daidzein reference standard (batch number: 111502-202003, content calculated as 99.3%) and genistein reference standard (batch number: 111704-20170). 3. All samples (content calculated as 99.5%) were provided by the China National Institutes for Food and Drug Control; the reference standard of rhein-8-O-β-D-glucoside (batch number: wkq18082803, content calculated as 98%) and the reference standard of gentiopicrin disaccharide (batch number: wkq18010504, content calculated as 98%) were provided by Sichuan Weikeqi Biotechnology Co., Ltd.; the medicinal materials of gardenia, rhubarb, immature bitter orange, fermented soybean, and the reference sample of Gardenia and Rhubarb Decoction were provided by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd.

[0024] The preparation method for the Gardenia and Rhubarb Decoction reference sample is as follows: The original formula for Gardenia and Rhubarb Decoction states: "Fourteen gardenia fruits, one ounce of rhubarb, five immature bitter oranges, and one liter of fermented soybean. Boil these four ingredients in six liters of water until two liters remain. Divide into three warm doses." Based on a quantitative analysis of ancient and modern literature, we find that one ounce in the Han Dynasty was 13.8g, each gardenia fruit was 0.9g, one immature bitter orange was approximately 18.0g, and one liter of fermented soybean was approximately 90.0g. Therefore, the calculated dosage of Gardenia and Rhubarb Decoction in the *Synopsis of Prescriptions of the Golden Chamber* is: 12.0g gardenia, 13.8g rhubarb, 69.0g immature bitter orange, and 69.0g fermented soybean.

[0025] (1) Decoction: Based on the investigation, the decoction method was determined as follows: Take 12.6g of gardenia, 13.8g of rhubarb, 69.0g of immature bitter orange and 69.0g of fermented soybean, boil with 1200ml of water to obtain 600ml, remove the residue, and the water extract is obtained.

[0026] (2) Concentration: Take the water extract and concentrate it to a density of 1.01 to 1.03 under vacuum conditions of -0.050 to -0.080 MPa and temperature of 50 to 70℃ to obtain the concentrated solution.

[0027] (3) Drying: Take the concentrated liquid and prepare freeze-dried powder according to the freeze-drying parameters below. The freeze-drying program of the vacuum freeze dryer is as follows: ① Pre-freezing: Temperature: -50℃, holding temperature: 6h; Pressure: atmospheric pressure; ② Sublimation drying: Temperature: -40℃, holding temperature: 4h; Pressure: 0.0pa; Temperature: -30℃, temperature maintenance: 4h; Pressure: 0.0pa; Temperature: -20℃, temperature maintenance: 4h; Pressure: 0.0pa; Temperature: -10℃, temperature maintenance: 3h; Pressure: 0.0pa; Temperature: 0℃, temperature maintenance: 3h; Pressure: 0.0pa; ③ Drying: Temperature: 10℃, holding temperature: 3h; Pressure: 0.0pa; Temperature: 20℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 30℃, temperature maintenance: 2h; Pressure: 0.0pa; Temperature: 40℃, temperature maintenance: 2h; pressure: 0.0pa.

[0028] Six batches of Gardenia and Rhubarb Decoction reference samples were prepared, numbered ZZDHT-JZYP-01 to ZZDHT-JZYP-06. The yield of each batch of samples (sample powder yield = mass of lyophilized powder obtained / total mass of raw herbs) was calculated to be 30.53%, 30.85%, 33.9%, 32.38%, 30.02%, and 31.21%, respectively. The average yield of the six batches of Gardenia and Rhubarb Decoction reference samples was 31.48%.

[0029] 2. Experimental conditions 2.1 Investigation of chromatographic conditions 2.1.1 Investigation of the mobile phase The Gardenia and Rhubarb Decoction reference sample was extracted with 70% methanol using ultrasound. The filtrate was then analyzed using the following method: The chromatographic column was an Agilent ZORBAX SB-Aq 250×4.6mm, 5µm; the mobile phases were acetonitrile (A)-0.1% phosphoric acid (B), methanol (A)-0.1% phosphoric acid (B), acetonitrile (A)-0.1% formic acid (B), and acetonitrile (A)-0.1% glacial acetic acid (B); the flow rate was 1.0 mL / min; the column temperature was 30℃; the detection wavelength was 260 nm; the injection volume was 10 μL; and the gradient elution program was as follows: See results Figure 1The results showed that the mobile phase of acetonitrile-0.1% phosphoric acid resulted in better peak separation and a more stable baseline.

[0030] 2.1.2 Gradient Investigation Based on the determined mobile phase, the gradient elution program in the chromatographic conditions was adjusted (Table 1) and examined together with the elution program (proposed gradient) under "2.1.1 Mobile Phase Investigation".

[0031] Table 1. Elution gradient investigation - Method 1 Results under different gradient elution programs are shown below Figure 2 The results showed that only when a predetermined gradient elution was used and the elution time was 50 minutes could the target component chromatographic peaks be completely eluted, the resolution of each chromatographic peak be greater than 1.5, the separation effect be good, and the peak shapes of each component be good.

[0032] The chromatographic conditions determined based on the above findings are as follows: The chromatographic column was an Agilent ZORBAX SB-Aq 250×4.6mm, 5µm; mobile phase A was acetonitrile, and mobile phase B was 0.1% phosphoric acid aqueous solution; the flow rate was 1.0 mL / min; the column temperature was 30℃; the detection wavelength was 260 nm; the injection volume was 10µL; and the gradient elution program was as follows: 2.2 Sample Pretreatment Optimization 2.2.1 Examination of Extraction Methods Method 1: Take 0.2g of the freeze-dried powder of Gardenia and Rhubarb Decoction reference sample, add 20mL of 70% methanol, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, take out, cool to room temperature, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the product. Method 2: Take 0.2g of the lyophilized powder of Gardenia and Rhubarb Decoction reference sample, add 20mL of 70% methanol, seal tightly, weigh, heat under reflux for 30 minutes, remove, cool to room temperature, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate. The active ingredients in the Gardenia and Rhubarb Decoction reference sample were quantified using the reference standard under the determined chromatographic conditions. The results of the extraction method investigation are shown in Table 2. Figure 3 .

[0033] Table 2 Content results for different extraction methods The results showed that, under conditions where the chromatograms were not significantly different, the contents of each indicative component, namely genipin, geniposide, rutin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein, were higher during ultrasonic extraction than during reflux extraction. Therefore, reflux was chosen as the extraction method for preparing the test solution in the determination of the content of Gardenia and Rhubarb Decoction reference sample.

[0034] 2.2.2 Investigation of Extraction Solvents Take 0.2 g of the lyophilized powder of Gardenia and Rhubarb Decoction reference sample, add water, 30% methanol, 70% methanol, and 20 mL of methanol respectively, seal tightly, weigh, and sonicate (600 W power, 40 kHz frequency) for 30 minutes. Remove, cool to room temperature, weigh again, replenish the lost weight with the appropriate extraction solvent, shake well, filter, and collect the filtrate. Quantify the active ingredients in the Gardenia and Rhubarb Decoction reference sample using the reference standard under the determined chromatographic conditions. The results of the extraction solvent evaluation are shown in Table 3. Figure 4 .

[0035] Table 3 Results for different extraction solvent contents The results showed that when water, 30% methanol, 70% methanol, and methanol were used as extraction solvents, the content of various index components extracted by 70% methanol was higher than that of genipin, gentiopicrin, rutin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein. Therefore, 70% methanol was used as the extraction solvent for preparing the test solution in the determination of the content of Gardenia and Rhubarb Decoction reference sample.

[0036] 2.2.3 Investigation of Solvent Usage Take 0.2g of the lyophilized powder of Gardenia and Rhubarb Decoction reference sample, add 10ml, 20ml, and 50ml of 70% methanol respectively, seal tightly, weigh, and sonicate (600W power, 40kHz frequency) for 30 minutes. Remove, cool to room temperature, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. The active ingredients in the Gardenia and Rhubarb Decoction reference sample were quantified using the reference standard under the determined chromatographic conditions. The results of solvent dosage investigation are shown in Table 4. Figure 5 .

[0037] Table 4 Results of different solvent dosages The results showed that when 10 ml, 20 ml, and 50 ml of 70% methanol were added as solvents, 20 ml resulted in higher contents of the index components extracted, including genipin, geniposide, rutin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein. Therefore, 20 ml of 70% methanol was used as the extraction solvent for preparing the test solution in the determination of the content of Gardenia and Rhubarb Decoction reference sample.

[0038] 2.2.4 Examination of extraction time Take 0.2g of the lyophilized powder of Gardenia and Rhubarb Decoction reference sample, add 25ml of 70% methanol, seal tightly, weigh, and sonicate (600W power, 40kHz frequency) for 20 minutes, 30 minutes, and 40 minutes respectively. Remove, cool to room temperature, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. The active ingredients in the Gardenia and Rhubarb Decoction reference sample were quantified using the reference standard under the determined chromatographic conditions. The results of solvent dosage investigation are shown in Table 5. Figure 6 .

[0039] Table 5. Content results at different extraction times The results showed that when the extraction time was 30 minutes, the contents of each index component, genipin, geniposide, rutin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein, were relatively high. Therefore, 30 minutes was used as the extraction time for preparing the test solution in the determination of the content of Gardenia and Rhubarb Decoction reference sample.

[0040] 2.2.5 Determine the preparation method of the test sample Take 0.2g of the freeze-dried powder of Gardenia and Rhubarb Decoction reference sample, add 20ml of 70% methanol, seal tightly, weigh, sonicate (power 600W, frequency 40kHz) for 30 minutes, take out, cool to room temperature, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the final product.

[0041] 2.3 Methodological Examination 2.3.1 System Applicability Assessment Mixed reference solution: Take appropriate amounts of genipin gentiopicroside, geniposide, rutin naringin, hesperidin, daidzein, emodin-7-O-β-D-glucoside and genistein reference standards, add methanol to prepare a mixed solution containing 50-100 μg of each reference standard per ml. Test solution: Prepared according to the method under "2.2.5 Determining the preparation method of the test solution"; Blank control solution: 70% methanol; Take appropriate amounts of the mixed reference solution, the test solution, and the blank control solution, and inject them under the determined chromatographic conditions. Record the chromatograms as shown in the figure. Figure 7 As shown in the figure, under these chromatographic conditions, the resolution of each chromatographic peak is greater than 1.5, the theoretical plate number is greater than 5000, the elution time of each chromatographic peak in the chromatogram of the test solution is consistent with that of the mixed reference solution, and there is no interference from the blank control solution.

[0042] 2.3.2 Specificity Examination Take 0.2g each of the following samples: lyophilized powder of Gardenia and Rhubarb Decoction reference sample, negative samples of fermented soybean, negative samples of immature bitter orange, negative samples of gardenia, and negative samples of rhubarb. Add 20ml of 70% methanol to each sample, seal tightly, weigh, and sonicate (600W, 40kHz) for 30 minutes. Remove, cool to room temperature, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, and collect the filtrate. Detect under the determined chromatographic conditions. Results are shown in the figure. Figure 8 .

[0043] 2.3.3 Investigation of different flow velocities Take an appropriate amount of the test solution prepared by the method described in section "2.2.5 Determining the Preparation Method of the Test Sample," and inject it under the determined chromatographic conditions. Record the chromatograms at flow rates of 0.9, 1.0, and 1.1 ml / min. The results are shown in [Figure Number]. Figure 9 .

[0044] The results showed that the resolution of each chromatographic peak was greater than 1.5 at a flow rate of 1.0 ml / min. Therefore, 1.0 ml / min was used as the flow rate for the chromatographic conditions in the determination of the content of Gardenia and Rhubarb Decoction reference sample.

[0045] 2.3.4 Investigation at different column temperatures Take an appropriate amount of the test solution prepared according to the method described in section "2.2.5 Determining the Preparation Method of the Test Sample", inject it under the determined chromatographic conditions, and record the chromatograms at column temperatures of 25, 30, and 35℃. The results are shown in [Figure Number]. Figure 10 .

[0046] The results showed that the resolution of each chromatographic peak was greater than 1.5 at a column temperature of 30℃. Therefore, 30℃ was used as the column temperature for the determination of the content of Gardenia and Rhubarb Decoction reference sample.

[0047] 2.3.5 Investigation of different injection volumes Take an appropriate amount of the test solution prepared according to the method described in section "2.2.5 Determining the Preparation Method of the Test Sample", inject it under the determined chromatographic conditions, and record the chromatograms for injection volumes of 5, 10, and 20 μl. The results are shown in [Figure Number]. Figure 11 .

[0048] The results showed that when the injection volume was 10 μl, the resolution of each chromatographic peak was greater than 1.5, indicating good separation. Therefore, 10 μl was used as the injection volume for the chromatographic conditions in the determination of the content of Gardenia and Rhubarb Decoction reference sample.

[0049] 2.3.6 Examination of Linear Relationships Take appropriate amounts of genipin gentiopicroside, geniposide, rutin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein, respectively, accurately weigh them, and add them to methanol to prepare a solution containing 19.8352 μg / ml of genipin gentiopicroside, 19.1296 μg / ml of geniposide, 33.5160 μg / ml of rutin, 194.2360 μg / ml of hesperidin, 167.817 μg / ml of daidzein, 39.7880 μg / ml of emodin-8-O-β-D-glucoside, and 274.0712 μg / ml of geniposide, as a mixed reference stock solution. The mixed reference stock solution was diluted 0, 2, 3.33, 6.67, 10, and 20 times respectively to prepare standard curve solutions I to VI. 10 μl of each solution was taken and determined according to the chromatographic conditions in section 2.2. Linear regression was performed with the reference mass (ng) as the abscissa (x) and the peak area as the ordinate (y). The results are shown in Table 5.

[0050] Table 5 Results of Linearity Examination The results showed that the correlation coefficient R of the standard curve fitting equations for each index component—genipin gentiopicroside, geniposide, rutin naringin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein—was relatively high. 2 All values ​​are greater than 0.999, indicating a good linear relationship.

[0051] 2.3.7 Precision Test Take the mixed reference solution and inject it 6 times consecutively, 10 μl each time. Record the peak area of ​​each component and calculate the RSD value of the peak area. The results are shown in Table 6.

[0052] Table 6 Precision Test Results The results showed that the RSD values ​​of the peak areas of each index component, genipin, geniposide, rutin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein, were 0.27%, 0.16%, 0.32%, 0.22%, 0.2%, 0.26%, and 0.21%, respectively, indicating good instrument precision.

[0053] 2.3.8 Repeatability Test Six test solutions were prepared from the same batch of Gardenia and Rhubarb Decoction reference samples according to the method described in section "2.2.5 Determining the Preparation Method of Test Samples". These solutions were injected for analysis, and the peak areas of each component were recorded. The content and RSD values ​​of each component were calculated. The results are shown in Table 7.

[0054] Table 7 Repeatability Test Results The results showed that the contents of genipin, geniposide, rutin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein were 1.725, 15.192, 0.752, 4.397, 4.667, 0.835, and 6.751 mg / g, respectively. The contents of these five components in the sample were all within the range of the standard curve. The RSD values ​​were 0.47%, 0.39%, 0.80%, 0.80%, 1.79%, 0.89%, and 1.45%, respectively, indicating that the method had good repeatability.

[0055] 2.3.9 Stability Test The same batch of Gardenia and Rhubarb Decoction reference samples were used as test samples. The test sample solutions were prepared according to the method in section "2.2.5 Determining the Test Sample Preparation Method". The samples were injected and analyzed at 0, 2, 4, 8, 12 and 24 h respectively. The peak area and RSD value of each component were recorded. The results are shown in Table 8.

[0056] Table 8 Stability Test Results The results showed that the RSD values ​​of genipin gentiopicroside, geniposide, rutin naringin, hesperidin, daidzein, emodin-7-O-β-D-glucoside and genistein were 0.81%, 0.99%, 1.09%, 1.08%, 1.03%, 1.70% and 1.17%, respectively, indicating that the test solution was stable within 24 hours.

[0057] 2.3.10 Spiking Recovery Test Accurately weigh 0.08 g of the Gardenia and Rhubarb Decoction reference sample with known contents of 7 components, accurately weigh 6 portions, add an equal amount of reference standard, and prepare the test solution according to the method under "2.2.5 Determining the preparation method of the test sample", and inject for analysis. The results are shown in Table 9.

[0058] Table 9 Results of the spiking recovery test The results showed that the average recoveries of genipin gentiopicroside, geniposide, rutin, hesperidin, daidzein, emodin-7-O-β-D-glucoside, and genistein were 99.27%, 97.31%, 97.21%, 96.52%, 97.49%, 98.59%, and 96.65%, respectively, with RSD values ​​of 1.44%, 1.87%, 1.32%, 1.23%, 1.18%, 1.60%, and 1.72%, respectively, indicating that the method had good accuracy.

[0059] 2.3.11 Intermediate Precision Six test solutions were prepared by different personnel at different times using the same batch of Gardenia and Rhubarb Decoction reference samples, following the method described in section "2.2.5 Determining the Preparation Method of Test Samples". These solutions were then analyzed using different instruments. The peak areas of each component were recorded, and the content and RSD values ​​of each component were calculated. The results are shown in Table 10.

[0060] Table 10 Intermediate Precision Results The results showed that the RSD values ​​of genipin gentiopicroside, geniposide, rutin naringin, hesperidin, daidzein, emodin-7-O-β-D-glucoside and genistein were 0.53%, 0.02%, 0.41%, 0.56%, 0.52%, 0.70% and 0.28%, respectively, indicating that the method had good intermediate precision.

[0061] 2.4 Sample content determination Six batches of Gardenia and Rhubarb Decoction reference samples were taken. Two test solutions were prepared for each batch according to the method under "2.2.5 Determining the Preparation Method of the Test Sample". The samples were injected for analysis, and the content of each analyte was calculated. The results are shown in Table 11.

[0062] Table 11 Content determination results As can be seen from the above, the method for determining the content of Gardenia and Rhubarb Decoction can effectively detect the reference sample of Gardenia and Rhubarb Decoction, and the method is stable and feasible.

[0063] In summary, the method of this invention has strong specificity and good reproducibility, providing reliable technical support for improving the quality standards of Gardenia and Rhubarb Decoction, precise clinical medication, and the stability of reference materials for modern preparations, and has practical application value.

Claims

1. A method for determining the content of active ingredients in Zhizi Dahuang Decoction, characterized by: It comprises the following operation steps: 1) Preparation of reference solution: take reference substances of iridoid compounds, flavonoids, anthraquinones and polyphenols, and prepare a reference solution by adding methanol; 2) Preparation of test solution: take Gardenia and Rhubarb Decoction preparation, extract with methanol solution to obtain a test solution; 3) Detection: detect by HPLC chromatography, and the chromatographic conditions are as follows: chromatographic column: octadecylsilane bonded silica gel as the filler; mobile phase: acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; the gradient elution program is as follows: 。 2. The method of claim 1, wherein: In step 1), the reference solution contains 50-100 μg of each reference substance per 1 mL.

3. The method according to claim 1 or 2, characterized in that: The iridoid compounds in the reference substance are gardenoside and / or genipin gentiopicroside; the flavonoids are naringin, hesperidin and / or daidzein; the anthraquinones are emodin-7-O-β-D-glucoside; and the polyphenols are genistein.

4. The method of claim 1, wherein: In step 2), the mass-volume ratio of Gardenia and Rhubarb Decoction preparation to methanol solution is 0.1-0.5 g: 10-50 ml.

5. The method of claim 4, wherein: The Gardenia and Rhubarb Decoction preparation includes Gardenia and Rhubarb Decoction, Gardenia and Rhubarb Decoction granules, Gardenia and Rhubarb Decoction pills, Gardenia and Rhubarb Decoction tablets, and Gardenia and Rhubarb Decoction substance reference; and the concentration of the methanol solution is 70%.

6. The method of claim 1, wherein: In step 2), the extraction is ultrasonic extraction, with a time of 20-60 min, a power of 400-800 W, and a frequency of 20-60 kHz.

7. The method of claim 1, wherein: In step 3), the chromatographic column is Agilent ZORBAX SB-Aq 250×4.6 mm, 5 um.

8. The method of claim 1, wherein: In step 3), the column temperature of the chromatographic conditions is 25-35℃; the flow rate is 0.8 mL / min-1.2 mL / min; the injection amount is 5-15 μL; and the detection wavelength is 250-270 nm.

9. The method of claim 8, wherein: In step 3), the column temperature of the chromatographic conditions is 30℃; the flow rate is 1.00 mL / min; the injection amount is 10 μL; and the detection wavelength is 260 nm.

10. The method of claim 1, wherein: It is an external standard method for calculating the content of active ingredients in Gardenia and Rhubarb Decoction.

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