HPLC detection method for products in baicalin synthesis pathway

Abstract

The application discloses a kind of baicalin synthetic pathway product HPLC detection method, comprising the following steps: S1, baicalin bottom cell fermentation liquid sample is measured, add methanol solution, filter after ultrasonic treatment, take filtrate to obtain test solution;S2, the test solution and control sample solution are respectively injected into high performance liquid chromatograph, obtain the chromatogram of test solution and control sample solution after using HPLC method test;S3, the content of product in test solution is calculated according to the chromatogram of test solution and control sample solution by external standard method;Wherein, in step S2, the mobile phase A and mobile phase B used by the HPLC method are methanol and 0.08-0.12% phosphoric acid solution respectively.The detection method provided by the application is simple in operation, fast in detection speed, high in detection efficiency, small in error, good in reproducibility, strong in specificity, stable in test method, less in influenced factors and wide in applicability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine ingredient detection, and particularly relates to a HPLC detection method for products in a baicalin synthesis pathway. BACKGROUND

[0002] Scutellaria baicalensis Georgi is a dry root of the Labiatae plant Scutellaria baicalensis Georgi, which is recorded in the Chinese Pharmacopoeia 2010 edition (Part I), bitter in taste, cold in nature, and belongs to the lung, gallbladder, spleen, large intestine and small intestine channels; it has the effects of clearing heat and drying dampness, purging fire and detoxifying, stopping bleeding and preventing miscarriage; it is used for treating damp-warm, summer dampness, chest tightness and vomiting, damp-heat distention, dysentery, jaundice, lung heat cough, high fever and polydipsia, blood heat hematochezia, carbuncle and sore, and unstable fetus. The main components of Scutellaria baicalensis Georgi are chrysin, baicalein and baicalin, which have significant antioxidant, antiviral, antibacterial, anti-inflammatory, anti-allergic and anticancer effects.

[0003] Baicalin is mainly derived from Scutellaria baicalensis Georgi, but due to the overexploitation of environmental resources, high cost and many side reactions of chemical synthesis, etc., baicalin needs to be collected by a new method. In the baicalin biosynthesis pathway, phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin are the main components. Pinocembrin chalcone, as the most important intermediate product, is very expensive in terms of its irreplaceable role in the synthesis system and its commercial and medicinal value. The methods for producing this compound on the market all have many problems. The standard product price is about 1500 / 5mg. At present, almost all flavonoid synthesis pathways pass through the step of glycosylation to form chalcone, indicating the importance of chalcone in the flavonoid synthesis pathway.

[0004] However, the existing baicalin synthesis pathway does not effectively detect the products, which is not conducive to cost control and has low detection efficiency and inconvenient operation. SUMMARY

[0005] The main purpose of the present application is to provide a HPLC detection method for products in a baicalin synthesis pathway, which aims to solve the problems of ineffective detection of products in the existing baicalin synthesis pathway, low detection efficiency and inconvenient operation.

[0006] To achieve the above-mentioned purpose, the present application provides a HPLC detection method for products in a baicalin synthesis pathway, which comprises the following steps:

[0007] S1, a sample of Scutellaria baicalensis Georgi fermentation broth is measured, methanol solution is added, and after ultrasonic treatment, filtration is performed, and the filtrate is taken as a test solution;

[0008] S2, inject the test product solution and the control product solution into a high performance liquid chromatograph respectively, and obtain the chromatograms of the test product solution and the control product solution by testing with an HPLC method;

[0009] S3, calculate the content of the product in the test product solution by an external standard method according to the chromatograms of the test product solution and the control product solution;

[0010] In step S2, the mobile phase A and the mobile phase B used in the HPLC method are methanol and 0.08-0.12% phosphoric acid solution respectively.

[0011] Optionally, the product includes phenylalanine, cinnamic acid, pinobanksin chalcone, chrysin, baicalein and baicalin.

[0012] Optionally, in step S2, phenylalanine, cinnamic acid, pinobanksin chalcone, chrysin, baicalein and baicalin are separated by the HPLC method, and the content of phenylalanine, cinnamic acid, pinobanksin chalcone, chrysin, baicalein and baicalin is determined separately.

[0013] Optionally, step S1 includes:

[0014] The test product solution is obtained by taking the baicalin substrate cell fermentation liquor, centrifuging to collect the bacterial bodies and the supernatant, adding a methanol solution to the supernatant, concentrating the bacterial bodies, ultrasonic treatment, filtration, and taking the filtrate.

[0015] Optionally, the volume ratio of the supernatant to the methanol solution is (6-8):(2-4).

[0016] Optionally, the bacterial bodies are concentrated by using a phosphate buffered saline solution.

[0017] Optionally, step S2 includes:

[0018] The test product solution is obtained by taking the baicalin substrate cell fermentation liquor, centrifuging to collect the bacterial bodies and the supernatant, adding a methanol solution to the supernatant, concentrating the bacterial bodies, ultrasonic treatment, filtration, and taking the filtrate.

[0019] Optionally, in step S2, the detector of the high performance liquid chromatograph is an ultraviolet-visible spectrometric detector.

[0020] Optionally, in step S2, the gradient elution parameters used in the HPLC method are as follows:

[0021]

[0022]

[0023] Optionally, the conditions of the high performance liquid chromatograph are as follows:

[0024] The chromatographic column is a C18 chromatographic column; and / or,

[0025] The column temperature is 28-40 DEG C; and / or,

[0026] The flow rate is 0.6-1.2 ml / min.

[0027] In the technical scheme provided by the present application, each component generated in the biosynthesis pathway of baicalin is detected by using the HPLC method, the method is simple in operation, fast in detection speed, high in detection efficiency, small in error, good in reproducibility, strong in specificity, stable in detection method, less in affected factors, and wide in applicability; the specificity, stability, precision, and accuracy of the method all meet the methodological verification requirements, and the method has strong practicability; the components such as baicalin in Saccharomyces cerevisiae are detected by using the method, the content of the product generated in the synthesis pathway can be quickly and accurately detected, the detection method provided by the present application can be used for detecting multiple substances in the biosynthesis of baicalin, and is also suitable for component testing analysis and quality determination of traditional Chinese medicinal materials such as Scutellaria baicalensis and the like. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other related drawings can be obtained by those skilled in the art without creating any inventive labor.

[0029] Figure 1 The liquid chromatogram of the solvent in Example 2 of the present application;

[0030] Figure 2 The liquid chromatogram of the test solution in Example 2 of the present application;

[0031] Figure 3 The liquid chromatogram of the control solution in Example 2 of the present application;

[0032] Figure 4 The linear relationship diagram of the concentration x (mg / ml) of phenylalanine in the standard curve solution prepared in Example 3 of the present application and the peak area y;

[0033] Figure 5 The linear relationship diagram of the concentration x (mg / ml) of cinnamic acid in the standard curve solution prepared in Example 3 of the present application and the peak area y;

[0034] Figure 6 The linear relationship diagram of the concentration x (mg / ml) of pinocembrin chalcone in the standard curve solution prepared in Example 3 of the present application and the peak area y;

[0035] Figure 7 A linear relationship diagram of the concentration x (mg / ml) of apigenin in the standard curve solution prepared in Example 3 of the present application and the peak area y;

[0036] Figure 8 A linear relationship diagram of the concentration x (mg / ml) of baicalein in the standard curve solution prepared in Example 3 of the present application and the peak area y;

[0037] Figure 9 A linear relationship diagram of the concentration x (mg / ml) of baicalin in the standard curve solution prepared in Example 3 of the present application and the peak area y.

[0038] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not specified, they are all the conventional products which can be purchased in the market. In addition, the meaning of "and / or" appearing in the whole text includes three parallel solutions. Taking "A and / or B" as an example, it includes the solution of A, or the solution of B, or the solution of A and B satisfying simultaneously. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the premise that the ordinary skilled in the art can realize it. When the combination of the technical solutions appears contradictory or unachievable, it should be considered that the combination of the technical solutions does not exist and is not within the protection scope of the present application. Based on the embodiments in the present application, all the other embodiments obtained by the ordinary skilled in the art without making creative labor are within the protection scope of the present application.

[0040] Baicalin is mainly derived from Huangqi, but nowadays, especially the overexploitation of environmental resources, the high cost of chemical synthesis and the many side reactions, etc. make baicalin need new methods to collect. In the biosynthetic pathway of baicalin, phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin are the main components. Among them, pinocembrin chalcone as the most important intermediate product, whether it is irreplaceable to the synthesis system, or in its commercial value and medicinal value, is very expensive. The current market production method of this compound has many problems. The standard product price is about 1500 / 5mg. At present, almost all flavonoid synthesis pathways go through the step of glycosylation to form chalcone, indicating the importance of chalcone in the flavonoid synthesis pathway. Then in the existing baicalin synthesis pathway, the product is not effectively detected, which is not conducive to cost control, and the detection efficiency is low and the operation is not convenient.

[0041] In view of this, the present application provides a HPLC detection method for the product in the baicalin synthesis pathway, to solve the problems of complex operation and low detection efficiency of the existing detection method.

[0042] The present application provides a HPLC detection method for the product in the baicalin synthesis pathway, comprising the following steps:

[0043] S1, measure the Huangqi bottom cell fermentation liquid sample, add methanol solution, ultrasonic treatment and filter, take the filtrate to obtain the test sample solution;

[0044] S2, the test sample solution and the control sample solution are injected into the high performance liquid chromatograph respectively, and the chromatogram of the test sample solution and the control sample solution is obtained after HPLC test;

[0045] S3, the content of the product in the test sample solution is calculated by external standard method according to the chromatogram of the test sample solution and the control sample solution;

[0046] In step S2, the mobile phase A and the mobile phase B used in the HPLC method are methanol and 0.08-0.12% phosphoric acid solution respectively.

[0047] The technical scheme provided by the present application adopts HPLC to detect each component generated in the biosynthesis pathway of baicalin, and the method is simple in operation, fast in detection speed, high in detection efficiency, small in error, good in reproducibility, strong in specificity, stable in detection method, less in influencing factors, and wide in applicability; the specificity, stability, precision and accuracy of the method all meet the methodological verification requirements, and the method has strong practicability; the method can be used to detect baicalin and other components in Saccharomyces cerevisiae, and can quickly and accurately detect the content of the product generated in the synthesis pathway; the detection method provided by the present application can be used for detecting multiple substances in the biosynthesis of baicalin, and is also suitable for component testing and quality determination of traditional Chinese medicinal materials such as Scutellaria baicalensis and the like.

[0048] Further, the product includes phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin, and HPLC is used to separate phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin, and to separately determine the content of phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin. The HPLC method provided by the present application can detect the six important substances generated in the baicalin synthesis pathway, and the method also detects pinocembrin chalcone by using a UV detector for the first time. Traditional chalcone detection methods are mostly rough qualitative methods such as spectral analysis and thin layer chromatography. Compared with traditional chalcone detection methods, the method is simple, efficient, stable, and has a wide range of applications. At the same time, the detection of pinocembrin chalcone can more clearly reflect how high the conversion rate of the front-end and rear-end substances in the synthesis pathway is, and provide a better judgment method for finding out the problems existing in the flavonoid synthesis pathway.

[0049] The above step S1 is the preparation of the test sample solution: the baicalin chassis cell fermentation liquor is centrifuged to collect the bacteria and supernatant, methanol solution is added to the supernatant, the volume ratio of the supernatant and methanol solution is (6-8):(2-4), the bacteria are concentrated by PBS (phosphate buffered saline), for example, 10 ml of bacteria liquid is resuspended with 1 ml of 1M PBS, the supernatant treated with methanol and the concentrated bacteria are respectively subjected to ultrasonic treatment, and are respectively filtered, and the total filtrate is obtained as the test sample solution.

[0050] Before step S2, it also includes:

[0051] S20, preparation of the control sample solution: weigh the phenylalanine control sample, cinnamic acid control sample, pinocembrin chalcone control sample, chrysin control sample, baicalein control sample and baicalin control sample, and quantitatively prepare a mixed solution containing each control sample with methanol to obtain the control sample solution.

[0052] After the preparation of the test sample solution and the control sample solution, 20 μl of the test sample solution and the control sample solution are respectively injected into the high performance liquid chromatograph, and the chromatograms of the test sample solution and the control sample solution are obtained after the HPLC test. The detector of the high performance liquid chromatograph is an ultraviolet-visible spectrometric detector. In this step, the HPLC method is used to separate phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin, and the contents of phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin are determined separately. The HPLC method uses octadecylsilane-bonded silica gel as the filler, and the chromatographic column is a Waters symmetry C18 column with a size of 4.6*250 mm and a particle size of 5 μm or an equivalent column. The mobile phase is a mixture of methanol (A) and 0.1% phosphoric acid solution (D), and the gradient elution parameters are shown in Table 1. The flow rate is 0.8 ml / min, the detection wavelength is 210 nm, the column temperature is 30°C, and the injection amount is 20 μl.

[0053] Table 1 Gradient elution parameter table

[0054]

[0055] The technical solutions of the present application are further described in detail in combination with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present application and do not limit the present application.

[0056] Example 1 HPLC detection method of product components in baicalin synthesis pathway, comprising the following steps:

[0057] (1) Chromatographic conditions and system suitability test:

[0058] Octadecylsilane-bonded silica gel is used as the filler, and the chromatographic column is a Waters symmetry C18 column with a size of 4.6*250 mm and a particle size of 5 μm or an equivalent column. The mobile phase is a mixture of methanol (A) and 0.1% phosphoric acid solution (B), and the gradient elution parameters are shown in Table 2. The flow rate is 0.8 ml / min, the detection wavelength is 210 nm, the column temperature is 30°C, the injection amount is 20 μl, and the theoretical plate number calculated according to the chrysin peak should not be less than 5000.

[0059] Table 2 Gradient elution sequence

[0060]

[0061] (2) Solution preparation:

[0062] Preparation of test sample solution: 10 ml of Scutellaria baicalensis Georgi fermentation broth was centrifuged at 4000 rpm to collect the bacterial cells and supernatant. Methanol was added to the supernatant at a ratio of 7 / 3 (e.g., 0.7 ml of methanol was added to 0.3 ml of the supernatant). The bacterial cells were concentrated by 10 times (e.g., 10 ml of bacterial liquid was resuspended with 1 ml of 1M PBS), and PBS was added twice. The bacterial cells were ultrasonically treated (power 320 W, frequency 40 kHz) for 45 minutes, and then filtered with a 0.45 μm filter to obtain the test sample solution.

[0063] Preparation of control sample solution: 3 mg of phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin control samples were precisely weighed and placed in a 10 ml volumetric flask. 70% methanol was added to dissolve and dilute to the mark to obtain a mixed solution containing 0.3 mg of each control sample per 1 ml.

[0064] (3) Determination:

[0065] 20 μl of the test sample solution and the control sample solution were precisely measured and injected into the liquid chromatograph. The chromatogram was recorded by the above chromatographic conditions. The content of each component in the test sample solution was calculated by the external standard method using the control sample solution.

[0066] Example 2 Specificity test

[0067] The control sample solution, the test sample solution and the solvent were precisely measured at 20 μl, respectively, and determined by the above detection method. The chromatogram was recorded (see Figure 1 、 Figure 2 、 Figure 3 ).

[0068] As shown in the figure, in the chromatogram of the test sample solution, phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin can be baseline separated from the adjacent peaks, and the blank solvent does not interfere, indicating that the specificity of the method is good.

[0069] Example 3 Linear relationship test

[0070] 3 mg of phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin control samples were precisely weighed and placed in a 10 ml volumetric flask. 70% methanol was added to dissolve and dilute to the mark to obtain a mixed solution containing 0.3 mg of each control sample per 1 ml.

[0071] The results show that phenylalanine has a good linear relationship in the range of 5 mg / L-80 mg / L, the linear equation is y=149.14x+5.7427, and the correlation coefficient r is 1.0000 (see Figure 4 ), cinnamic acid has a good linear relationship in the range of 5 mg / L-80 mg / L, the linear equation is y=169.69x+1.4852, and the correlation coefficient r is 0.999 (see Figure 5 ), pinobanksin has a good linear relationship in the range of 5 mg / L-80 mg / L, the linear equation is y=108.24x-45.732, and the correlation coefficient r is 0.999 (see Figure 6 ), chrysin has a good linear relationship in the range of 5 mg / L-80 mg / L, the linear equation is y=85.607x+97.814, and the correlation coefficient r is 0.999 (see Figure 7 ), baicalein has a good linear relationship in the range of 5 mg / L-80 mg / L, the linear equation is y=115x-27.469, and the correlation coefficient r is 0.999 (see Figure 8 ), and baicalin has a good linear relationship in the range of 5 mg / L-80 mg / L, the linear equation is y=63.941x+13.343, and the correlation coefficient r is 0.999 (see Figure 9 ).

[0072] Example 4 Solution stability investigation

[0073] A batch of samples from Beijing Zhongke Zhi'an Biotechnology Co., Ltd. were prepared to prepare test solution, and the peak areas of phenylalanine, cinnamic acid, pinobanksin, chrysin, baicalein and baicalin were determined after 0, 8, 16, 20, 24, 40 and 60 hours of sample preparation.

[0074] The results show that the test solution is stable within 60 hours, the RSD of the peak area of phenylalanine is 0.60%, the RSD of the peak area of cinnamic acid is 0.70%, the RSD of the peak area of pinobanksin is 1.27%, the RSD of the peak area of chrysin is 0.95%, the RSD of the peak area of baicalein is 0.78%, and the RSD of the peak area of baicalin is 0.96%. The determination results are shown in Table 3 below.

[0075] Table 3 Solution stability experiment results statistics table

[0076]

[0077]

[0078] Example 5 Injection precision test

[0079] Take a batch of samples of Beijing Zhongke Quality Inspection Biotechnology Co., Ltd. according to the preparation method of the test solution in the foregoing detection method to prepare 1 part of the test solution, continuously sample 6 times, and determine the peak areas of phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin. The precision of the instrument is investigated.

[0080] The results show that the RSD of the peak area of phenylalanine is 0.78%, the RSD of the peak area of cinnamic acid is 1.10%, the RSD of the peak area of pinocembrin chalcone is 1.11%, the RSD of the peak area of chrysin is 1.08%, the RSD of the peak area of baicalein is 0.76%, and the RSD of the peak area of baicalin is 1.31%, indicating that the precision of the instrument is good. The determination results are shown in Table 4 below.

[0081] Table 4: Precision experiment results statistical table

[0082]

[0083] Example 6: Reproducibility test

[0084] Take a batch of samples of Beijing Zhongke Quality Inspection Biotechnology Co., Ltd. according to the preparation method of the test solution in the foregoing detection method to prepare 6 parts of the test solution, sample and determine the content and RSD of phenylalanine, cinnamic acid, pinocembrin chalcone, chrysin, baicalein and baicalin. The results are shown in Table 5 below.

[0085] The results show that the RSD of the peak area of phenylalanine is 0.34%, the RSD of the peak area of cinnamic acid is 0.54%, the RSD of the peak area of pinocembrin chalcone is 0.60%, the RSD of the peak area of chrysin is 0.64%, the RSD of the peak area of baicalein is 2.31%, and the RSD of the peak area of baicalin is 0.74%, indicating that the reproducibility of the instrument is good.

[0086] Table 5: Reproducibility experiment results statistical table

[0087]

[0088] Example 7: Sample recovery rate test

[0089] Precise pipetting control solution (solvent: 70% methanol): phenylalanine (concentration 3mg / ml) 1700ul, cinnamic acid (concentration 0.3mg / ml) 1000ul, pinocembrin chalcone (concentration 0.3mg / ml) 700ul, acerola (concentration 0.3mg / ml) 2000ul, baicalein (concentration 0.3mg / ml) 800ul, and baicalin (concentration 0.3mg / ml) 1200ul into the same flat-bottom flask, and 0.15g of sample with a known content is weighed into the flat-bottom flask, and 100% recovery solution is prepared according to the preparation method of the sample solution in the foregoing detection method, and 6 recovery solutions are prepared, and sample determination is performed, and the results are shown in Table 6 below. From the determination results, it can be seen that the HPLC detection method of the product components in the baicalin synthesis pathway of the present application is simple to operate, and has wide applicability.

[0090] Table 6: Recovery experiment result statistics table

[0091]

[0092]

[0093] In summary, the HPLC detection method of the product components in the baicalin synthesis pathway provided by the present application is simple to operate, has high detection efficiency, small error, good reproducibility, strong specificity, stable test method, fewer influencing factors, and wide applicability; the present method is the first time to analyze and qualify pinocembrin chalcone by using HPLC; the specificity, stability, precision, accuracy, etc. of the present method meet the methodological verification requirements, and have strong practicality.

[0094] The above is only the preferred embodiment of the present application, and does not limit the patent scope of the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the patent protection scope of the present application.

Claims

1. An HPLC method for detecting products in the baicalin synthesis pathway, characterized in that, The products include phenylalanine, cinnamic acid, chalcone, scutellarin, baicalin, and baicalin. The HPLC detection method for the products in the baicalin synthesis pathway includes the following steps: S1. Take a sample of baicalin chassis cell fermentation broth, add methanol solution, sonicate and filter, and take the filtrate to obtain the test solution. S2. Weigh out phenylalanine reference standard, cinnamic acid reference standard, chalcone reference standard, salicylic acid reference standard, baicalein reference standard, and baicalin reference standard, and quantitatively prepare a mixed solution containing each reference standard with methanol to obtain a reference solution; inject the test solution and the reference solution into a high-performance liquid chromatograph, respectively, and obtain the chromatograms of the test solution and the reference solution by HPLC; S3. Calculate the content of the product in the test solution using the external standard method based on the chromatograms of the test solution and the reference solution; In step S2, the mobile phase A and mobile phase B used in the HPLC method are methanol and 0.1% phosphoric acid solution, respectively. In step S2, the gradient elution parameters used in the HPLC method are as follows: ； The ultraviolet detection wavelength is 210 nm; The HPLC method uses the following column conditions: C18 column, 4.6 × 250 mm, 5 μm.

2. The HPLC detection method for the products in the baicalin synthesis pathway as described in claim 1, characterized in that, In step S2, phenylalanine, cinnamic acid, chalcone, succinate, baicalein and baicalin were separated by HPLC and their contents were determined individually.

3. The HPLC detection method for the products in the baicalin synthesis pathway as described in claim 1, characterized in that, Step S1 includes: Take the fermentation broth of baicalin chassis cells, centrifuge to collect the cells and supernatant, add methanol solution to the supernatant, concentrate the cells, sonicate, filter, and take the filtrate to obtain the test solution.

4. The HPLC detection method for the products in the baicalin synthesis pathway as described in claim 3, characterized in that, The volume ratio of the supernatant to the methanol solution is (6-8):(2-4).

5. The HPLC detection method for the products in the baicalin synthesis pathway as described in claim 3, characterized in that, Phosphate buffer solution was used for bacterial cell concentration.

6. The HPLC detection method for the products in the baicalin synthesis pathway as described in claim 1, characterized in that, In step S2, the detector of the high performance liquid chromatograph is an ultraviolet-visible spectrophotometer.

7. The HPLC detection method for the products in the baicalin synthesis pathway as described in claim 1, characterized in that, In step S2, the conditions of the high-performance liquid chromatograph are as follows: Column temperature is 28–40°C; and / or, The flow rate is 0.6–1.2 ml / min.

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