A method for constructing a characteristic map of Sophora japonica buds and a method for distinguishing Sophora japonica buds and their adulterants.

Abstract

This invention discloses a method for constructing a characteristic spectrum of Sophora japonica buds and a method for distinguishing Sophora japonica buds from their adulterants. The method involves performing high-performance liquid chromatography (HPLC) on the test sample solution and the reference solution under specific chromatographic conditions to obtain the characteristic spectrum. This method establishes a characteristic spectrum of saponin components in Sophora japonica buds to distinguish them from adulterants such as Sophora alopecuroides and Sophora oleifera. HPLC is used to compare and contrast standard decoctions of Sophora japonica buds and their adulterants, allowing for control of the intrinsic quality of the standard decoctions. This method comprehensively reflects the characteristics of the standard decoctions and provides a new analytical tool for the quality control of standard decoctions and granules of Sophora japonica buds, achieving the goal of identifying standard decoctions and granules of Sophora japonica buds and their adulterants.

Description

Technical Field

[0001] This invention relates to a method for constructing a characteristic spectrum of Sophora japonica buds and a method for distinguishing Sophora japonica buds and their adulterants, belonging to the field of traditional Chinese medicine identification technology. Background Technology

[0002] Sophora japonica flowers are the dried flowers and buds of the legume Sophora japonica L. They are harvested in summer when the flowers open or the buds form, dried promptly, and the branches, stems, and impurities removed. The former is commonly known as "Sophora japonica flowers," and the latter as "Sophora japonica buds." They are slightly cold in nature and bitter in taste; they enter the liver and large intestine meridians; they have the effects of cooling the blood and stopping bleeding, clearing liver heat and purging fire; they are used for hematochezia, tuberculosis, bloody dysentery, metrorrhagia, hematemesis, epistaxis, red eyes due to liver heat, headache, and dizziness.

[0003] Modern research indicates that Sophora japonica buds possess various pharmacologically active ingredients, with flavonoids, triterpenoid saponins, fatty acids, polysaccharides, and volatile components being the most studied. The main pharmacologically active components are flavonoids and triterpenoid saponins. Sophora japonica buds exhibit hemostatic, hypoglycemic, antioxidant, gastrointestinal protective, antibacterial, immune-enhancing, antiviral, blood pressure-lowering, and antitumor pharmacological activities.

[0004] Literature reports indicate that sophora flower saponin III possesses anti-aging, hypoglycemic, and hypolipidemic effects; soybean saponin Bb exhibits effects such as inhibiting adrenaline vasodilation, lowering blood pressure, anti-inflammation, nerve protection, and regeneration; and sophora flower saponin I has anticoagulant properties. Saponins are an important component of the pharmacological activity of Sophora japonica buds, and sophora flower saponin I, sophora flower saponin II, and sophora flower saponin III are specific components of Sophora japonica buds.

[0005] Traditional Chinese medicine (TCM) mainly originates from plants and animals in nature. Some of these medicines have multiple origins, often leading to confusion about their origins and difficulty in distinguishing genuine from counterfeit products. This results in highly unstable quality and significantly limits their clinical application. Common adulterants in the market include the flowers and buds of the legume *Robinia pseudoacacia* L., commonly known as locust flowers, and the dried buds of the legume *Albizia kalkora* (Roxb.) Prain, commonly known as mountain locust rice.

[0006] Currently, there are many methods for comparative studies of Sophora japonica buds and their adulterants. Research on adulterants mainly focuses on morphological and chromatographic aspects. Xu Fei et al. compared and identified Sophora japonica buds and Robinia pseudoacacia buds based on morphology, thin-layer chromatography, and ultraviolet spectroscopy. Fu Fengping et al. used rutin as a control to conduct qualitative comparisons using thin-layer chromatography, colorimetric determination of total flavonoid content, and high-performance liquid chromatography (HPLC) determination of rutin content between Sophora japonica buds and Robinia pseudoacacia buds. The chromatograms of Sophora japonica buds and Robinia pseudoacacia buds showed significant differences. Rutin was the main chemical component in Sophora japonica buds, while Robinia pseudoacacia buds contained main components different from rutin. The morphological comparisons of the above studies are not applicable to standard decoctions and granules. Thin-layer chromatography, ultraviolet spectrophotometry, and HPLC mainly analyze flavonoids commonly found in traditional Chinese medicine, such as rutin, naringin, and quercetin, resulting in poor specificity and susceptibility to adulteration. Therefore, it is necessary to establish more specific characteristic chromatograms to distinguish genuine from counterfeit products. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for constructing a characteristic map of Sophora japonica buds and a method for distinguishing Sophora japonica buds and their counterfeits.

[0008] Technical Solution: To solve the above technical problems, this invention provides a method for constructing a feature map of Sophora japonica buds, comprising the following steps:

[0009] (1) Preparation of test solution: Take Sophora japonica standard decoction, add extraction solvent for extraction, cool, weigh again, replenish the lost weight with appropriate reagents, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in water, extract with water-saturated n-butanol, combine the n-butanol solutions, wash with ammonia test solution, take the n-butanol solution and evaporate to dryness, dissolve the residue in methanol, shake well, filter, take the filtrate to obtain the test solution;

[0010] (2) Preparation of reference solution: Take soybean saponin Bb reference standard, add solvent to dissolve it, and the solution is obtained.

[0011] (3) Ultra-high performance liquid chromatography (UHPLC) detection: The test solution and reference solution were injected into the UHPLC chromatograph and detected using an electro-fogging detector to obtain characteristic spectra. The characteristic spectra have 10 common peaks. The peak corresponding to the soybean saponin Bb reference is designated as peak S. The relative retention times of the remaining characteristic peaks and peak S are calculated. The relative retention times are within ±10% of the specified values. The specified values ​​for peaks 1, 2, 3, 4, 6, 7, 8, 9, and 10 are as follows: : 0.47, 0.57, 0.84, 0.97, 1.05, 1.15, 1.21, 1.22, 1.28; The chromatographic conditions for the ultra-high performance liquid chromatography are: chromatographic column: HPH-C18, EC-C18 or SB-C18; acetonitrile as mobile phase A, 0.01 mol / L ammonium formate aqueous solution as mobile phase B; flow rate: 0.28–0.32 ml / min; column temperature: 38–42 °C; elution gradient: 0–1 min, mobile phase A The volume fraction of mobile phase A changed from 21% to 27% and the volume fraction of mobile phase B changed from 79% to 73%; from 1 to 8 min, the volume fraction of mobile phase A changed from 27% to 29% and the volume fraction of mobile phase B changed from 73% to 71%; from 8 to 11 min, the volume fraction of mobile phase A changed from 29% to 31% and the volume fraction of mobile phase B changed from 71% to 69%; from 11 to 14 min, the volume fraction of mobile phase A changed from 31% to 34% and the volume fraction of mobile phase B changed from 69% to 66%. From 14 to 16 min, the volume fraction of mobile phase A changed from 34% to 36%, and the volume fraction of mobile phase B changed from 66% to 64%. From 16 to 22 min, the volume fraction of mobile phase A was 36%, and the volume fraction of mobile phase B was 64%. From 22 to 25 min, the volume fraction of mobile phase A was 36% to 45%, and the volume fraction of mobile phase B was 64% to 55%. From 25 to 28 min, the volume fraction of mobile phase A was 45%, and the volume fraction of mobile phase B was 55%.

[0012] The extraction solvent in step (1) includes water or 30% to 100% methanol.

[0013] The extraction methods described in step (1) include ultrasonic treatment, heating and reflux, or shaking extraction.

[0014] The extraction time in step (1) is 15 to 60 minutes.

[0015] The extraction process in step (1) is performed 1 to 3 times.

[0016] The preparation of the standard decoction involves taking slices of Sophora japonica flowers (sophora buds), weighing them, soaking them in water, decocting them, filtering them while they are hot, concentrating the filtrate under reduced pressure, and freeze-drying them to obtain the standard decoction of Sophora japonica flowers (sophora buds).

[0017] This invention also provides a method for establishing a reference atlas of Sophora japonica buds, comprising the following steps:

[0018] (1) Prepare multiple batches of standard decoction test solutions of Sophora japonica buds;

[0019] (2) The test sample solution was tested according to the method for constructing the characteristic spectrum of Sophora japonica flower to obtain the characteristic spectrum of Sophora japonica flower standard decoction;

[0020] (3) Import the obtained feature chromatograms into the Chinese herbal chromatographic fingerprint chromatogram similarity evaluation system to establish a reference chromatogram for Sophora japonica standard decoction.

[0021] This invention also provides a method for distinguishing Sophora japonica buds from their adulterants, comprising the following steps:

[0022] (1) Preparation of test solution: Take the test sample, add water or 30% to 100% methanol for 15 to 60 minutes, cool, weigh again, replenish the lost weight with appropriate reagents, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in water, extract with water-saturated n-butanol by shaking 1 to 3 times, combine the n-butanol solutions, wash with ammonia test solution, take the n-butanol solution and evaporate to dryness, dissolve the residue in methanol, shake well, filter, take the filtrate to obtain the test solution;

[0023] (2) Preparation of reference solution: Take Sophora japonica buds reference material, decoct with water, take the supernatant, extract with water-saturated n-butanol, take the n-butanol layer, wash with ammonia solution, evaporate the solvent, make up to volume, shake well, filter, take the filtrate, and the reference solution of reference material is obtained; take soybean saponin Bb reference standard, dissolve in solvent, and the reference solution of reference standard is obtained.

[0024] (3) Ultra-high performance liquid chromatography detection: The test solution and the reference solution were injected into the ultra-high performance liquid chromatograph and detected by an electro-fogging detector to obtain characteristic chromatograms;

[0025] (4) Differentiation: When the retention times of the test sample solution correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material, and the peak corresponding to the soybean saponin Bb reference material is the S peak, calculate the relative retention times of peaks 1 to 10 and the S peak respectively. If the relative retention times are within ±10% of the specified value, and the relative peak area of ​​peak 7 is greater than 1.5, then the sample being tested is Sophora japonica buds; otherwise, the test sample is a counterfeit.

[0026] The extraction methods described in step (1) include ultrasonic treatment, heating and reflux, or shaking extraction.

[0027] The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) in step (3) are as follows: the column is HPH-C18, EC-C18, or SB-C18; acetonitrile is used as mobile phase A, and 0.01 mol / L ammonium formate aqueous solution is used as mobile phase B; the flow rate is 0.28–0.32 ml / min; the column temperature is 38–42 °C; the elution gradient is 0–1 min, with the volume fraction of mobile phase A changing from 21% to 27% and the volume fraction of mobile phase B changing from 79% to 73%; from 1 to 8 min, the volume fraction of mobile phase A changes from 27% to 29% and the volume fraction of mobile phase B changes from 73% to 71%; from 8 to 11 min, the volume fraction of mobile phase A changes from 29% to 31%. The volume fraction of mobile phase B changed from 71% to 69% at 11–14 min; from 14–16 min, the volume fraction of mobile phase A changed from 31% to 34% and the volume fraction of mobile phase B changed from 69% to 66%; from 16–22 min, the volume fraction of mobile phase A was 36% and the volume fraction of mobile phase B was 64%; from 22–25 min, the volume fraction of mobile phase A was 36% to 45% and the volume fraction of mobile phase B was 64% to 55%; from 25–28 min, the volume fraction of mobile phase A was 45% and the volume fraction of mobile phase B was 55%.

[0028] The counterfeit products include black locust flowers or mountain locust flowers.

[0029] The present invention also provides the application of the differentiation method in the quality testing of Sophora japonica standard decoction and formula granules.

[0030] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: 1. The method is simple, reproducible, accurate and reliable, easy to operate, takes less time, consumes less solvent, and causes less environmental pollution; 2. The present invention uses ultra-high performance liquid chromatography (UHPLC) to investigate the differences in components of Sophora japonica buds, Robinia pseudoacacia buds, and Sophora japonica buds, and rationally controls the chromatographic method. Acetonitrile is used as the mobile phase A, and 0.01 mol / L ammonium formate solution (adjusted to pH 4.5 with formic acid) is used as the mobile phase B for gradient elution, establishing 10 common peaks of Sophora japonica buds; a characteristic chromatogram of Sophora japonica bud standard decoction is constructed, and a comparative study is conducted on the adulterants (Robinia pseudoacacia buds and Sophora japonica buds), controlling the intrinsic quality of the entire Sophora japonica bud standard decoction. It can comprehensively reflect the characteristics of Sophora japonica bud standard decoction and adulterants (Robinia pseudoacacia buds and Sophora japonica buds), providing a new analytical means for the intrinsic quality control of Sophora japonica bud standard decoction and formula granules, and achieving the purpose of identifying adulterants (Robinia pseudoacacia buds and Sophora japonica buds) standard decoction and formula granules. Attached Figure Description

[0031] Figure 1 Characteristic chromatograms of Sophora japonica flower standard decoction under different chromatographic conditions;

[0032] Figure 2 Characteristic spectra of Sophora japonica flower standard decoction under different extraction solvents;

[0033] Figure 3 Characteristic spectra of standard Sophora japonica decoction prepared using different extraction methods;

[0034] Figure 4 Characteristic spectra of Sophora japonica flower standard decoction at different extraction times;

[0035] Figure 5 Characteristic spectra of Sophora japonica flower standard decoction after different extraction times;

[0036] Figure 6 Characteristic spectra of standard Sophora japonica decoction under different washing conditions;

[0037] Figure 7 Chromatograms of standard decoction samples and blank control samples of Sophora japonica flowers (Sophora japonica buds);

[0038] Figure 8 A comprehensive study of the characteristic spectra of standard decoctions containing Sophora japonica flowers (Sophora japonica buds);

[0039] Figure 9 Characteristic chromatograms of Sophora japonica flower standard decoction under different chromatographic columns;

[0040] Figure 10 Characteristic spectra of Sophora japonica flower standard decoction at different column temperatures;

[0041] Figure 11 Characteristic spectra of Sophora japonica flower standard decoction at different flow rates;

[0042] Figure 12 An overlay of characteristic spectra of 24 batches of Sophora japonica flower standard decoction;

[0043] Figure 13 A comparative chromatogram of the standard decoction of Sophora japonica flowers;

[0044] Figure 14 A control spectrum generated by overlaying characteristic spectra of standard decoction of Robinia pseudoacacia buds;

[0045] Figure 15 A control spectrum generated by overlaying characteristic spectra of standard Sophora japonica decoction;

[0046] Figure 16 To compare the standard decoction of Sophora japonica buds and counterfeit products;

[0047] Figure 17 Assignment of peak 5 in the characteristic chromatogram of the test sample;

[0048] Figure 18 Characteristic chromatograms of Sophora japonica bud reference medicinal materials (121270-201403). Detailed Implementation

[0049] Unless otherwise stated, the term “test sample” as used herein refers to an experimental sample used for testing or identification.

[0050] Unless otherwise stated, the term "reference material" as used herein refers to a standard substance used for identification, inspection, content determination and calibration of instrument performance.

[0051] Unless otherwise stated, the terms “precise weighing” as used herein mean that the weight should be accurate to one-thousandth of the weight taken, the term “weighing” means that the weight should be accurate to one-hundredth of the weight taken, the term “precise measurement” means that the volume should be measured accurately to one-thousandth of the volume taken, and the term “precise aspiration” means the operation of accurately measuring the sample using a microsyringe.

[0052] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0053] Instruments, reagents and samples:

[0054] Instruments: Thermo Vanquish UHPLC; Thermo Vanquish CAD detector; Chromeleam 7.2SR4 Chameleon workstation; Agilent Technologies 1290 Infinity UHPLC; 1290DAD diode array detector; 1290MCT column oven; 1290Vialsampler autosampler; 1290Fiexible pump quaternary pump; OpenLAB CDS2.3 chromatography workstation; ME204E / 02 electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); GKC114 temperature-controlled water bath (Nantong Huatai Experimental Instrument Co., Ltd.); KQ-250E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory); Acetonitrile (chromatographic grade, Thermo Fisher Scientific); Milli-Q pure water system (Millipore Scientific).

[0055] Reagents: Acetonitrile (chromatographic grade, Thermo Fisher Scientific); formic acid (chromatographic grade, Aladdin Scientific); ammonium formate (chromatographic grade, Aladdin Scientific); ultrapure water; all other reagents were of analytical grade.

[0056] Reference material: Soy saponin Bb (batch number: 145102) was purchased from Shanghai Shidander Biotechnology Co., Ltd.

[0057] The Sophora japonica bud reference material (batch number: 121270-201403) was purchased from the China National Institutes for Food and Drug Control.

[0058] Samples: 24 batches of Sophora japonica flower (flower bud) medicinal materials were collected; 2 batches of Robinia pseudoacacia flower bud medicinal materials and 3 batches of Sophora japonica flower bud medicinal materials were collected. All of the above Sophora japonica flower (flower bud) medicinal materials met the requirements of the "Sophora japonica flower" item in the 2020 edition of the Chinese Pharmacopoeia. Impurities and ash were removed from the above medicinal materials, and corresponding decoction pieces were prepared.

[0059] Table 1 Origin Information

[0060]

[0061] Preparation of standard decoction: Weigh 100g of Sophora japonica flower (Sophora japonica bud) slices, place them in a clay pot, add 1200ml of water and soak for 30min. Using a YMW mechanical split-type decoction pot, first bring to a boil over high heat, then simmer over low heat for 30min. Filter while hot using a 200-mesh standard sieve. For the second decoction, add 1000ml of water, bring to a boil over high heat, then simmer over low heat for 25min. Filter while hot using a 200-mesh standard sieve. Combine the two filtrates and concentrate under reduced pressure at 65℃. Concentrate to a relative density of 1.03~1.06 (65℃). Dispense the concentrate into vials and freeze dry in a freeze dryer to obtain the standard Sophora japonica flower (Sophora japonica bud) decoction.

[0062] Preparation of reference solution: Take 0.5g of Sophora japonica bud reference material, place it in a stoppered conical flask, add 25ml of water, seal tightly, heat under reflux for 60 minutes, cool, weigh again, replenish the lost weight with water, shake well, filter, take 20ml of the filtrate, extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol extracts, wash with ammonia solution until colorless, evaporate the n-butanol extract to dryness, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate as the reference solution for the reference material.

[0063] Take an appropriate amount of soybean saponin Bb reference standard, accurately weigh it, and add methanol to prepare a solution containing 20 μg of soybean saponin Bb per 1 ml, which will be used as the reference solution.

[0064] Example 1: Determination of chromatographic conditions

[0065] Chromatographic conditions 1: HPH-C18 (150 mm × 2.1 mm, 1.9 μm); acetonitrile as mobile phase A, 0.1% formic acid solution as mobile phase B, gradient elution according to the specifications in Table 2; flow rate 0.35 mL / min; column temperature 40 °C; detection using an electrospray detector, results are shown in Table 2. Figure 1 .

[0066] Table 2 Gradient Elution Table

[0067]

[0068] Chromatographic conditions 2: HPH-C18 (150 mm × 2.1 mm, 1.9 μm); acetonitrile as mobile phase A, and 10 mM ammonium formate aqueous solution (pH = 4.8) as mobile phase B, with gradient elution as specified in Table 3; flow rate 0.30 mL / min; column temperature 40 °C; evaporation tube temperature 70 °C. Results are shown in Table 3. Figure 1 .

[0069] Table 3 Gradient Elution Table

[0070]

[0071] Chromatographic conditions 3: HPH-C18 (150 mm × 2.1 mm, 1.9 μm); acetonitrile as mobile phase A, and 10 mM ammonium formate aqueous solution (pH = 4.5) as mobile phase B, with gradient elution as specified in Table 4; flow rate 0.30 mL / min; column temperature 40 °C; evaporation tube temperature 70 °C. Results are shown in Table 4. Figure 1 .

[0072] Table 4 Gradient Elution Table

[0073]

[0074] After optimization, acetonitrile-0.01 mol / L ammonium formate aqueous solution (adjusted to pH 4.5 with formic acid) was finally selected as the mobile phase. The final chromatographic conditions were as follows: HPH-C18 (150 mm × 2.1 mm, 1.9 μm); acetonitrile as mobile phase A, and 0.01 mol / L ammonium formate aqueous solution (adjusted to pH 4.5 with formic acid) as mobile phase B, with gradient elution as specified in Table 5; flow rate of 0.30 mL / min; column temperature of 40 °C; detection using an electro-fogging detector. The theoretical plate number, calculated based on the soybean saponin Bb peak, should be no less than 5000.

[0075] Table 5 Gradient Elution Table

[0076]

[0077] Example 2: Characteristic spectrum of saponins in Sophora japonica flowers (flower buds) and preparation of test solution

[0078] 1. Investigation of different extraction solvents

[0079] Take an appropriate amount of this product (batch number: HM-DG-1, a standard decoction prepared from Sophora japonica buds of batch number HM-YC-1), grind it into a fine powder, take 0.5g, accurately weigh it, place it in a stoppered conical flask, and accurately add 25ml each of water, 30% methanol, 50% methanol, 70% methanol, and methanol. Seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, weigh it again, replenish the lost weight with the appropriate reagents, shake well, filter, take 20ml of the filtrate, evaporate to dryness, redissolve the residue in 20ml of water, extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol solutions, wash 3 times with ammonia solution, evaporate the n-butanol solution to dryness, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the product. Accurately pipette 1 μl of each test solution and inject it into the ultra-high performance liquid chromatograph. Determine the chromatographic conditions as determined in Example 1. The results are shown in Table 6. Figure 2 .

[0080] Table 6 Comparison of different extraction solvents (peak area / sample weight)

[0081]

[0082] Conclusion: The extraction solvents were investigated using water, 30% methanol, 50% methanol, 70% methanol, and methanol respectively. It was found that 50% methanol had the highest extraction efficiency and better peak separation when used as the extraction solvent. Therefore, 50% methanol was selected as the extraction solvent.

[0083] 2. Examination of different extraction methods

[0084] Take an appropriate amount of this product (batch number: HM-DG-1), grind it into a fine powder, take 0.5g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, stopper tightly, and sonicate (power 250W, frequency 40kHz), heat under reflux, and shake to extract for 30 minutes. Cool, weigh it again, and make up the weight loss with 50% methanol. Shake well, filter, take 20ml of the filtrate, evaporate to dryness, redissolve the residue in 20ml of water, and extract twice with 20ml of water-saturated n-butanol each time. Combine the n-butanol extracts, wash three times with ammonia solution, evaporate the n-butanol extract to dryness, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate. Accurately pipette 1μl of each test solution and inject it into an ultra-high performance liquid chromatograph. Analyze according to the chromatographic conditions determined in Example 1. The results are shown in Table 7. Figure 3 .

[0085] Table 7 Comparison of different extraction methods (peak area / sample volume)

[0086]

[0087] Conclusion: Ultrasonic treatment has high extraction efficiency and is easy to operate, so ultrasonic treatment was ultimately chosen as the extraction method.

[0088] 3. Examination of different extraction times

[0089] Take an appropriate amount of this product (batch number: HM-DG-1), grind it into a fine powder, accurately weigh 0.5g, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, seal tightly, and sonicate (power 250W, frequency 40kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively. Cool, weigh again, and replenish the lost weight with 50% methanol. Shake well, filter, take 20ml of the filtrate, evaporate to dryness, redissolve the residue in 20ml of water, extract twice with water-saturated n-butanol (20ml each time), combine the n-butanol extracts, wash three times with ammonia solution, evaporate the n-butanol extract to dryness, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate. Accurately pipette 1μl of each test solution and inject it into an ultra-high performance liquid chromatograph. Determine the chromatographic conditions as determined in Example 1. The results are shown in Table 8. Figure 4 .

[0090] Table 8 Comparison of different extraction times

[0091]

[0092] Conclusion: The results show that the extraction efficiency is high after 30 minutes of ultrasonic treatment, indicating that the extraction is basically complete, and the extraction time is confirmed to be 30 minutes.

[0093] 4. Investigation of different extraction times

[0094] Take an appropriate amount of this product (batch number: HM-DG-1), grind it into a fine powder, accurately weigh 0.5g, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 50% methanol, shake well, filter, take 20ml of the filtrate, evaporate to dryness, redissolve the residue in 20ml of water, extract with water-saturated n-butanol 0, 1, 2, and 3 times, 20ml each time, combine the n-butanol extracts, wash 3 times with ammonia test solution, evaporate the n-butanol extract to dryness, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate. Accurately pipette 1μl of each test solution and inject it into an ultra-high performance liquid chromatograph, determine the chromatographic conditions determined in Example 1, and the results are shown in Table 9. Figure 5 .

[0095] Table 9 Comparison of different extraction times

[0096]

[0097]

[0098] Conclusion: The results show that there are more impurity peaks without extraction, while the peak shape is better, there are fewer impurity peaks, and the separation is better after extraction. Moreover, the extraction efficiency does not increase much after two extractions, indicating that the extraction is sufficient after two extractions. Considering all factors, the extraction number is determined to be 2 times.

[0099] 5. Investigation of different washing conditions

[0100] Take an appropriate amount of this product (batch number: HM-DG-1), grind it into a fine powder, take 0.5g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 50% methanol, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool, weigh it again, replenish the lost weight with 50% methanol, shake well, filter, take 20ml of the filtrate, evaporate to dryness, redissolve the residue in 20ml of water, extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol extracts, wash 3 times with water, 3 times with ammonia solution, and 20ml each time without adding any reagents, take the n-butanol extract, evaporate to dryness, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate. Accurately pipette 1μl of each test solution and inject it into an ultra-high performance liquid chromatograph, determine the chromatographic conditions determined in Example 1, and the results are shown in the figure. Figure 6 .

[0101] Conclusion: Ammonia washing resulted in fewer impurity peaks and better peak shape, thus ammonia washing was ultimately determined as the optimal washing condition.

[0102] Based on the above results, the method for preparing the test solution in the following embodiments of the present invention is as follows:

[0103] Take an appropriate amount of standard Sophora japonica flower (Sophora japonica bud) decoction, grind it into a fine powder, take 0.5g, place it in a stoppered conical flask, add 25ml of 50% methanol, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, shake well, filter, take 20ml of the filtrate, evaporate to dryness, redissolve the residue in 20ml of water, extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol solutions, wash three times with ammonia solution, 20ml each time, discard the ammonia solution, evaporate the n-butanol solution to dryness, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the final product.

[0104] Example 3: Methodological validation of the detection method of saponins in standard decoction of Sophora japonica flowers (Sophora japonica buds) using characteristic chromatograms.

[0105] 1. Specificity Examination

[0106] The standard decoction sample of Sophora japonica flowers (blooming buds) (batch number: HM-DG-1) and the blank control sample were tested according to the method described in Example 2. The results are shown in […]. Figure 7 .

[0107] Conclusion: The extraction solvent does not interfere with the determination of characteristic peaks, and the method used to determine the characteristic chromatograms of saponin components in the standard decoction of Sophora japonica flowers (Sophora japonica buds) is specific.

[0108] 2. Holistic assessment

[0109] Take a standard sample of Sophora japonica flower (flower bud) decoction (batch number: HM-DG-1), and under the chromatographic conditions determined in Example 1, change only the elution gradient: maintain the elution gradient with the highest acetonitrile ratio, double the elution time, and use the same preparation conditions for the test solution as in Example 2. Record the chromatogram, and the results are shown in [Figure 1]. Figure 8 .

[0110] Conclusion: No obvious chromatographic peaks eluted after the original gradient elution was completed, indicating that the chromatographic conditions basically met the requirement of maximizing information.

[0111] 3. Precision test

[0112] Take samples from the same batch (batch number: HM-DG-1), prepare test solutions according to the test solution preparation method in Example 2, inject 1 μl of the solution 6 times consecutively, and detect the samples under the chromatographic conditions determined in the example. Record the chromatograms. Using the peak corresponding to soybean saponins as the reference peak (peak S), calculate the relative retention time and relative peak area of ​​each peak. The results are shown in Tables 10 and 11.

[0113] Table 10 Precision Experiment Results - Relative Retention Time

[0114]

[0115] Table 11 Precision Experiment Results - Relative Peak Area

[0116]

[0117] The results show that the relative retention time RSD of each characteristic peak is less than 1%, and the relative peak area RSD is less than 2%, indicating good precision.

[0118] 4. Repeated examinations

[0119] Take an appropriate amount of standard decoction of Sophora japonica flowers (batch number: HM-DG-1), make 6 parallel portions, and prepare test solutions according to the test solution preparation method determined in Example 2. Inject 1 μl of each solution and determine the peak area value according to the chromatographic conditions determined in the example. The results are shown in Tables 12 and 13 below.

[0120] Table 12 Results of repeatability experiments (relative retention time)

[0121]

[0122] Table 13 Repeatability Experiment Results (Relative Peak Area)

[0123]

[0124] The results show that the relative retention time RSD of each characteristic peak is less than 1%, and the relative peak area RSD is less than 2%, indicating that the method has good repeatability.

[0125] 5. Stability Study

[0126] Samples from the same batch (batch number: HM-DG-1) were prepared into test solutions according to the method for preparing test solutions determined in Example 2. 1 μl of the solution was injected every 0, 4, 8, 12, 16, 20, and 24 hours, and the chromatograms were recorded under the chromatographic conditions determined in the example. The relative retention time and relative peak area of ​​each common peak were calculated using the peak corresponding to soybean saponins as the reference peak. The results are shown in Tables 14 and 15.

[0127] Table 14 Stability Experiment Results - Relative Retention Time

[0128]

[0129] Table 15 Stability Test Results - Relative Peak Area

[0130]

[0131] The results showed that the relative retention time (RSD) of each characteristic peak was less than 1%, and the relative peak area (RSD) of all peaks except for peaks 2 and 8 was less than 3%, indicating that the sample had good stability within 24 hours.

[0132] 6. Intermediate precision test

[0133] Three portions of the standard decoction of Sophora japonica flowers (bloom number: HM-DG-1) were prepared by two laboratory technicians according to the test solution preparation method determined in Example 2. 1 μl of each sample was injected at different times using different instruments, and the chromatograms were recorded according to the chromatographic conditions determined in the example. The relative retention times of each common peak were calculated using the peak corresponding to soybean saponins as the reference peak. The results are shown in Table 16.

[0134] Table 16 Intermediate Precision Experiment Results (Relative Retention Time)

[0135]

[0136] The results showed that the relative retention time (RSD) of each characteristic peak was less than 1%, indicating good intermediate precision of the sample.

[0137] 7. Durability Assessment

[0138] 7.1 Investigation of different chromatographic columns

[0139] This experiment investigated the effects of three different types of chromatographic columns on the characteristic chromatographic profile of Sophora japonica (Sophora japonica buds) standard decoction, with other chromatographic conditions the same as in Example 1 and the sample preparation method the same as in Example 2. The results are shown in [Figure 1]. Figure 9 .

[0140] The results showed that Sophora japonica flower (flower bud) samples could be well separated on the HPH C18 column, but the separation effect was poor on the EC-C18 and SB-C18 columns. Therefore, subsequent studies used an Infinity Poroshell HPH-C18 (Angilent, 2.1 mm × 150 mm, 1.9 μm) column.

[0141] 7.2 Investigation at different column temperatures

[0142] An Infinity Poroshell HPH-C18 (Angilent, 2.1 mm × 150 mm, 1.9 μm) column was used at a flow rate of 0.30 mL / min. The sample separation performance was investigated at column temperatures of 38 °C, 40 °C, and 42 °C. Other chromatographic conditions were the same as in Example 1, and the sample preparation method was the same as in Example 2. The results are shown in [Figure 2]. Figure 10 See Table 17.

[0143] Table 17 Effect of column temperature on separation performance (relative retention time)

[0144]

[0145] The results showed that small column temperature variations within the range of 38℃ to 42℃ could meet the system applicability requirements, and subsequent studies used a column temperature of 40℃.

[0146] 7.3 Investigation of different flow velocities

[0147] An Infinity Poroshell HPH-C18 (Angilent, 2.1 mm × 150 mm, 1.9 μm) column was used at a column temperature of 40 °C. The sample separation performance at flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min was investigated. Other chromatographic conditions were the same as in Example 1, and the sample preparation method was the same as in Example 2. The results are shown in [Figure 2]. Figure 11 Table 18.

[0148] Table 18 Effect of flow rate on separation performance (relative retention time)

[0149]

[0150] The results showed that small flow rate variations within the range of 0.28 ml / min to 0.32 ml / min could meet the system suitability requirements, and subsequent studies used a flow rate of 0.30 ml / min.

[0151] Example 4: Determination of the characteristic spectrum of saponin components in standard decoction of Sophora japonica flower (Sophora japonica buds)

[0152] 1. Chromatographic conditions: An HPH-C18 column (150 mm × 2.1 mm, 1.9 μm) was used; acetonitrile was used as mobile phase A, and 0.01 mol / L ammonium formate aqueous solution (adjusted to pH 4.5 with formic acid) was used as mobile phase B, with gradient elution according to the specifications in Table 5; the flow rate was 0.30 mL / min; the column temperature was 40 °C; and an electro-fogging detector was used for detection. The theoretical plate number, calculated based on the soybean saponin Bb peak, should be no less than 5000.

[0153] 2. Preparation of the reference solution: Take 0.5g of Sophora japonica bud reference material, place it in a stoppered conical flask, add 25ml of water, seal tightly, heat under reflux for 60 minutes, cool, weigh again, replenish the lost weight with water, shake well, filter, take 20ml of the filtrate, extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol extracts, wash with ammonia solution until colorless, evaporate the n-butanol extract to dryness, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate as the reference solution for the reference material. Separately, take an appropriate amount of soybean saponin Bb reference standard, accurately weigh it, add methanol to prepare a solution containing 20μg of soybean saponin Bb per 1ml, as the reference solution for the reference standard.

[0154] 3. Preparation of the test solution: Take an appropriate amount of standard Sophora japonica flower (Sophora japonica bud) decoction sample, grind it finely, take 0.5g, place it in a stoppered conical flask, add 25ml of 50% methanol, stopper tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, shake well, filter, take 20ml of the filtrate, evaporate to dryness, redissolve the residue in 20ml of water, extract twice with water-saturated n-butanol, 20ml each time, combine the n-butanol solutions, wash three times with ammonia solution, 20ml each time, discard the ammonia solution, evaporate the n-butanol solution to dryness, dissolve the residue in methanol, transfer to a 2ml volumetric flask, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0155] 4. Determination method: Accurately pipette 1 μl of the reference solution and the test solution into the ultra-high performance liquid chromatograph.

[0156] 5. Establishment of Feature Maps

[0157] The characteristic chromatograms of 24 batches of Sophora japonica flower (Huahua) standard decoction samples (prepared from Sophora japonica flower HM-YC-1 to 24) were superimposed using the "Similarity Evaluation System for Chromatographic Characteristic Chromatography of Traditional Chinese Medicine (Version 2012.0)" issued by the National Pharmacopoeia Commission. Common peaks with relatively stable relative retention time and relative peak area RSD of Sophora japonica flower (Huahua) were selected as characteristic peaks. See the superimposed graph below. Figure 12 See the generated comparison graph. Figure 13 The relative retention times and relative peak areas are shown in Tables 19 and 20.

[0158] Table 19 Sample Measurement Results (Relative Retention Time)

[0159]

[0160]

[0161] Table 19 Sample Measurement Results (Relative Peak Area)

[0162]

[0163] The chromatogram of the test sample should show 10 characteristic peaks, which should correspond to the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material. Among them, peak 5 should correspond to the retention time of the reference peak of soybean saponin Bb. The peak corresponding to the reference peak of soybean saponin Bb is the S peak. Calculate the relative retention times of the remaining characteristic peaks and the S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.47 (peak 1), 0.57 (peak 2), 0.84 (peak 3), 0.97 (peak 4), 1.05 (peak 6), 1.15 (peak 7), 1.21 (peak 8), 1.22 (peak 9), and 1.28 (peak 10).

[0164] 7. Comparison of Sophora japonica buds and counterfeit products

[0165] A control chromatogram was generated by overlaying the characteristic chromatograms of four batches (HM-DG-1 to 4) of the Sophora japonica flower standard decoction samples from the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine (Version 2012.0)" promulgated by the National Pharmacopoeia Commission. (See attached image.) Figure 14 The characteristic spectra of four batches of Sophora japonica flower bud standard decoction samples were overlaid to generate a control spectrum. Figure 15 A comparison of the standard decoction of Sophora japonica buds and counterfeit products can be found in [link to relevant documentation]. Figure 16 This indicates that there are significant differences in the chromatograms of Sophora japonica buds and their adulterants (Robinia pseudoacacia buds, Sophora japonica buds) in the standard decoction.

[0166] 8. Research on the material basis and identification of characteristic peaks of Sophora japonica buds

[0167] High-resolution mass spectrometry (HPLC) data were acquired using liquid chromatography-mass spectrometry (LC-MS) under the HPLC conditions specified for the characteristic chromatogram of Sophora japonica bud decoction. Isotope analysis was used to deduce the molecular formulas of the compounds in the Sophora japonica bud decoction. Common peaks were assigned based on comparisons with references and relevant standards. The retention time of peak 5 was consistent with that of the soybean saponin Bb reference standard; therefore, peak 5 in the characteristic chromatogram of the test sample was identified as soybean saponin Bb. (See attached image.) Figure 17 The retention times of the 10 characteristic peaks in the standard decoction of Sophora japonica buds were consistent with those in the reference herb Sophora japonica buds. (See...) Figure 18 .

[0168] 9. Determining the composition of Sophora japonica buds and counterfeit products (Robinia pseudoacacia buds, Sophora japonica buds).

[0169] Differential components were identified using mass spectrometry and reference standards. These components were then screened to differentiate between standard decoctions of Sophora japonica buds, Robinia pseudoacacia buds, and Sophora japonica buds. Peak 7 could distinguish Sophora japonica buds from adulterants (Robinia pseudoacacia buds and Sophora japonica buds). Due to the presence of adulteration in actual production, the relative peak area of ​​peak 7 was further used to differentiate adulterated samples. A minimum value of 1.80 was lowered by 15%, resulting in 1.53, as the specified value. Finally, soybean saponin Bb was designated as the reference peak. If the relative peak area of ​​peak 7 was greater than 1.50, the tested sample was considered Sophora japonica buds.

[0170] Table 2. Relative Peak Area Ranges of Sophora japonica buds and counterfeit products (Peak 7

[0171]

[0172] Summary:

[0173] a. If the retention times of the 10 characteristic peaks in the chromatogram of the tested sample correspond to those of the reference medicinal material, and the corresponding peak of the soybean saponin Bb reference standard is taken as the S peak, calculate the relative retention times of peaks 1 to 10 with the S peak. If the relative retention times are within ±10% of the specified value, and the relative peak area of ​​peak 7 is greater than 1.5, then the tested sample is Sophora japonica flower bud.

[0174] b. If the retention times of the 10 characteristic peaks in the chromatogram of the tested sample correspond to those of the reference drug, and the corresponding peak of the soybean saponin Bb reference standard is taken as the S peak, the relative retention times of peaks 1 to 10 and the S peak are calculated. The relative retention times should be within ±10% of the specified value, and the relative peak area of ​​peak 7 is less than 1.5. Then the tested sample is a counterfeit.

[0175] c. If the retention times of the 10 characteristic peaks in the chromatogram of the tested sample do not correspond to those of the reference drug, and the corresponding peak of the soybean saponin Bb reference standard is taken as the S peak, the relative retention times of peaks 1 to 10 and the S peak are calculated. If the relative retention times are not within ±10% of the specified value, and the relative peak area of ​​peak 7 is less than 1.5, then the tested sample is a counterfeit.

Claims

1. A method for constructing a feature map of Sophora japonica buds, characterized in that, Includes the following steps: (1) Preparation of test solution: Take Sophora japonica standard decoction, add extraction solvent for extraction, cool, weigh again, replenish the lost weight with appropriate reagents, shake well, filter, take the filtrate, evaporate to dryness, dissolve the residue in water, extract with water-saturated n-butanol, combine the n-butanol solutions, wash with ammonia test solution, take the n-butanol solution and evaporate to dryness, dissolve the residue in methanol, shake well, filter, take the filtrate, and the test solution is obtained; the extraction solvent is water or 30%~100% methanol; (2) Preparation of reference solution: Take soybean saponin Bb reference standard, add solvent to dissolve it, and the solution is obtained; (3) Ultra-high performance liquid chromatography (UHPLC) detection: The test solution and the reference solution were injected into the UHPLC and detected by an electro-fogging detector to obtain characteristic chromatograms. The characteristic chromatograms have 10 common peaks. The peak corresponding to the soybean saponin Bb reference is the S peak. The relative retention times of the remaining characteristic peaks and the S peak are calculated. The relative retention times are within ±10% of the specified values. The specified values ​​for peaks 1, 2, 3, 4, 6, 7, 8, 9, and 10 are 0, ... 0.47, 0.57, 0.84, 0.97, 1.05, 1.15, 1.21, 1.22, 1.28; the chromatographic conditions for the ultra-high performance liquid chromatography were as follows: the chromatographic column was HPH-C18, with dimensions of 2.1 mm × 150 mm and a diameter of 1.9 μm; acetonitrile was used as mobile phase A, and 0.01 mol / L ammonium formate aqueous solution was used as mobile phase B; the flow rate was 0.28~0.32 ml / min; the column temperature was 38~42℃; and the elution gradient was 0~1 min. The volume fraction of mobile phase A changed from 21% to 27%, and the volume fraction of mobile phase B changed from 79% to 73%. From 1 to 8 min, the volume fraction of mobile phase A changed from 27% to 29%, and the volume fraction of mobile phase B changed from 73% to 71%. From 8 to 11 min, the volume fraction of mobile phase A changed from 29% to 31%, and the volume fraction of mobile phase B changed from 71% to 69%. From 11 to 14 min, the volume fraction of mobile phase A changed from 31% to 34%, and the volume fraction of mobile phase B changed from 69% to 66%. %; 14~16 min, the volume fraction of mobile phase A changed from 34→36%, and the volume fraction of mobile phase B changed from 66→64%; 16~22 min, the volume fraction of mobile phase A was 36%, and the volume fraction of mobile phase B was 64%; 22~25 min, the volume fraction of mobile phase A was 36→45%, and the volume fraction of mobile phase B was 64→55%; 25~28 min, the volume fraction of mobile phase A was 45%, and the volume fraction of mobile phase B was 55%.

2. The method according to claim 1, characterized in that, The extraction methods described in step (1) include ultrasonic treatment, heating and reflux, or shaking extraction.

3. The method according to claim 1, characterized in that, The extraction time described in step (1) is 15 to 60 minutes.

4. The method according to claim 1, characterized in that, The extraction in step (1) is performed 1 to 3 times.

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