Quality evaluation method of cortex phellodendri medicinal material and application of quality evaluation method

Abstract

The invention relates to a quality evaluation method of a cortex phellodendri medicinal material and application thereof. Based on a multi-component alkaloid-phenolic acid-limonin synergistic effect mechanism of cortex phellodendri, berberine hydrochloride (alkaloid) and 3-O- / 5-O-feruloylquinic acid (phenolic acid) are introduced into a synchronous detection system for the first time; the defect that only two alkaloids, namely berberine hydrochloride and phellodendrine hydrochloride, are monitored in the existing pharmacopoeia, but key active components with remarkable anti-inflammatory and antioxidant activity, such as phenolic acids, are ignored, is overcome. The invention discloses a variable importance projection (VIP) algorithm based on a PLS-DA model. According to the method, 9 components are taken as the screening standard of symbolic components, core characteristic components including berberine hydrochloride and 3-O- / 5-O-feruloylquinic acid which contribute to the quality difference are intelligently screened from the 9 components, and the quality grades of different batches of cortex phellodendri medicinal materials are distinguished, so that the quality difference identification sensitivity is greatly improved; and a precise target spot is provided for producing area traceability and process regulation and control. By adopting the quality evaluation method provided by the invention, not only is the overall chemical substance basis of the medicinal materials comprehensively reflected, but also the quality grades of different batches of cortex phellodendri medicinal materials can be distinguished and the high-quality production area of cortex phellodendri can be locked through the VIP characteristic components.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicinal material quality evaluation, and more particularly to a quality evaluation method for Phellodendri Chinensis Cortex and application thereof. BACKGROUND

[0002] Phellodendri Chinensis Cortex is the dried bark of Phellodendron amurense Rupr. of Rutaceae, which is a commonly used bulk medicinal material in clinical practice of traditional Chinese medicine. It has the effects of clearing heat and drying dampness, purging fire and removing damp-heat, detoxifying and treating sores, and is often used for treating damp-heat diarrhea, jaundice and red urine, leucorrhea and vaginal itching, and eczema and sores. Modern pharmacological studies have shown that Phellodendri Chinensis Cortex contains rich active ingredients such as alkaloids (e.g., berberine hydrochloride, phellodendrine hydrochloride, jatrorrhizine), phenolic acids (e.g., chlorogenic acid, various feruloylquinic acids), limonoids (e.g., amurensitone, amurensin), and phenylpropanoids, and has significant anti-inflammatory, antibacterial, anticancer, and hypoglycemic effects. Therefore, Phellodendri Chinensis Cortex is not only directly used in clinical practice, but also is the core raw material of many important Chinese patent medicines such as Zhibai Dihuang Pill, Meizhu Mingmu Liquid, and Huanglian Shangqing Pill, and the market demand continues to grow.

[0003] However, the efficacy material basis and quality of Phellodendri Chinensis Cortex are highly dependent on factors such as production area, ecological environment, cultivation management mode, and processing technology. Differences in these factors directly lead to significant differences in the chemical composition and content of Phellodendri Chinensis Cortex from different sources and different batches, thereby seriously affecting the stability of its clinical efficacy and the product quality of Chinese patent medicines. At present, the quality control standards for Phellodendri Chinensis Cortex in the 2020 edition of the People's Republic of China Pharmacopoeia (hereinafter referred to as the Chinese Pharmacopoeia) have obvious limitations: only the contents of two single alkaloid components, berberine hydrochloride and phellodendrine hydrochloride, are specified. This evaluation method based on a single or a small number of index components cannot comprehensively and objectively reflect the overall chemical material basis and quality status of Phellodendri Chinensis Cortex, especially cannot effectively distinguish the internal quality fluctuations caused by differences in multiple active ingredients (such as phenolic acids and limonoids) between different batches. The single-sidedness and one-sidedness of the existing standards cannot meet the needs of modern Chinese medicine industry for fine and scientific quality control of Phellodendri Chinensis Cortex, and it is even more difficult to guarantee its effectiveness and safety in clinical application.

[0004] Therefore, it is urgent to establish an evaluation method that is more scientific, comprehensive, and can reflect the overall quality characteristics of Phellodendri Chinensis Cortex. SUMMARY

[0005] In view of the above technical problems, the present application provides a quality evaluation method for Phellodendri Chinensis Cortex and its application, so as to detect and monitor a plurality of key active ingredients contained in Phellodendri Chinensis Cortex, especially the ingredients with important pharmacological effects but not covered by the existing standards and pharmacopoeia, and reveal the quality difference rules among different origins and different batches of medicinal materials by using advanced chemometrics methods, so as to provide reliable scientific basis and technical support for the accurate grading of Phellodendri Chinensis Cortex, the screening of high-quality raw materials, the optimization of production process and the quality control of final products. At the same time, it is expected that the application of the quality evaluation method can make up for the shortcomings of the existing quality control method of Phellodendri Chinensis Cortex which is limited to two components of berberine hydrochloride and phellodendrine hydrochloride, so as to improve the quality level and clinical value of Phellodendri Chinensis Cortex and its related Chinese patent medicines by using a multi-index and overall quality evaluation system.

[0006] To achieve the above-mentioned purpose, in a first aspect, the present application provides a quality evaluation method for Phellodendri Chinensis Cortex, comprising the following steps:

[0007] S1: establishing the HPLC fingerprint of the Phellodendri Chinensis Cortex: gradient elution is carried out with acetonitrile-0.1% phosphoric acid water as the mobile phase, and a Diamonsil C18 column is used, and the detection wavelength is 215 nm; 18

[0008] S2: determining the content of the marker components in the Phellodendri Chinensis Cortex, wherein the marker components include berberine hydrochloride, 3-O-feruloyl quinic acid and 5-O-feruloyl quinic acid;

[0009] S3: based on the content of the marker components in the step S2, a quality evaluation model is constructed by a partial least squares discriminant analysis (PLS-DA) model, and the VIP value>1 is used as the screening standard of the marker components to distinguish the quality grades of different batches of Phellodendri Chinensis Cortex.

[0010] ​Different from the prior art, the quality evaluation method of the above technical scheme is based on the synergistic mechanism of the multi-component alkaloids-phenolic acids-citrin class of Cortex Phellodendri, and for the first time, berberine hydrochloride (alkaloid) and 3-O- / 5-O-feruloylquinic acid (phenolic acid) are included in the synchronous detection system, which makes up for the defect that the current pharmacopoeia only monitors two alkaloids of berberine hydrochloride and phellodendrine hydrochloride, and ignores the key active ingredients such as phenolic acids with significant anti-inflammatory and antioxidant activity. Based on the variable importance projection (VIP) algorithm of the PLS-DA model, the VIP value > 1 is used as the screening standard of the marker component, and the core characteristic components of berberine hydrochloride and 3-O- / 5-O-feruloylquinic acid with the largest contribution to the quality difference are intelligently screened from 9 components, and the quality grades of different batches of Cortex Phellodendri are distinguished based on this, which greatly improves the recognition sensitivity of the quality difference, and provides a precise target for provenance tracing and process control. By using the quality evaluation method provided by the present application, not only the overall chemical substance basis of the medicinal material can be comprehensively reflected, but also the quality grades of different batches of Cortex Phellodendri and the high-quality production area of Cortex Phellodendri can be distinguished and locked.

[0011] The use of acetonitrile-0.1% phosphoric acid water as the mobile phase can avoid the problem that phenolic acids such as 3-O- / 5-O-feruloylquinic acid are easily ionized in a pure acetonitrile-water system, resulting in peak tailing, improve the separation degree, and enhance the retention of alkaloids. The gradient elution program is based on the need for synchronous baseline separation of multiple categories of components, and under the condition of wavelength 215 nm, the response of multiple categories of components can be maximized, covering the characteristic absorption peaks of alkaloids, phenolic acids and citrin class. The use of Diamonsil C18 column can reduce the adsorption of phenolic acids through high-density silanol group tailing technology, improve the column recovery rate, and tolerate 0.1% phosphoric acid.

[0012] In some embodiments, the gradient elution conditions in step S1 are as follows: gradient elution: 0-6 min, 12% acetonitrile; 6-10 min, 12%-20% acetonitrile; 10-15 min, 20% acetonitrile; 15-25 min, 20%-25% acetonitrile; 25-35 min, 20%-40% acetonitrile; 35-45 min, 40%-55% acetonitrile; 45-50 min, 55%-60% acetonitrile; 50-55 min, 60-70% acetonitrile; flow rate: 1.0 mL / min; column temperature: 35°C; injection volume: 10 μL. In these embodiments, the targeted polarity span of the 8-stage gradient elution successfully achieves the simultaneous baseline separation of 9 types of components, for example, low acetonitrile concentration (12-20%) from 0 to 15 min to capture polar components such as chlorogenic acid and feruloylquinic acid, avoiding their being submerged by the solvent front, and high acetonitrile concentration (25-55%) from 25 to 45 min to desorb medium and low polarity components (phellodendrine, limonoids), thereby shortening the analysis time. The gradient elution program optimization covers more than 80% of the known active substances in Phellodendri Cortex.

[0013] In some preferred embodiments of the present application, the step S1 further comprises generating a control fingerprint by a similarity evaluation system, and marking 14 common peaks, and at least identifying 9 component peaks including chlorogenic acid, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, phellodendrine hydrochloride, magnoflorine, 5-O-feruloylquinic acid, jatrorrhizine hydrochloride, berberine hydrochloride, and phellodendrine. The fingerprint part clearly identifies 14 common peaks and 9 identified peaks. Both the integrity evaluation by the 14 peaks and the precise quality control focusing on the 9 key components are achieved. There is no report in the traditional methods and pharmacopoeia of simultaneously capturing components with large polarity differences such as alkaloids and phenolic acids. In particular, phellodendrine, a weak UV-absorbing substance, is easily missed by conventional methods. However, the use of Diamonsil C18 column with acetonitrile-phosphoric acid water system significantly improves the recovery rate of phellodendrine, filling the detection blind spot of this component in Phellodendri Cortex in the prior art.

[0014] In some preferred embodiments, the quality evaluation model in step S3 further combines cluster analysis HCA with the 9 component peaks as variables, adopts the intergroup connection method, and takes the squared Euclidean distance as the measurement standard to classify the different batches of Phellodendri Cortex into 3 categories. The classification results are consistent with those obtained by the partial least squares discriminant analysis PLS-DA model, which indicates that the component content difference in each group of samples is small, the quality is stable, and there is a certain difference in components between different groups, so as to analyze the differences of different batches of Phellodendri Cortex.

[0015] In some preferred embodiments of the present invention, the content range of berberine hydrochloride in the marker components is 66.34–125.54 mg / g; the content range of 3-O-feruloylquinic acid is 1.42–3.87 mg / g; and the content range of 5-O-feruloylquinic acid is 34.71–66.84 mg / g. The current pharmacopoeia only requires berberine ≥30 mg / g, which easily leads to defects such as high content of individual components but low overall quality. For example, a batch of berberine at 70.83 mg / g may be acceptable, but 5-O-feruloylquinic acid at only 37.79 mg / g—a near-low limit "high berberine - low phenolic acid"—may enter production. However, due to the large fluctuations in composition between different batches, it is necessary to increase the number of quality control components and set dual thresholds to intercept inferior raw materials, and to further implement quality grading standards for Phellodendron amurense, going beyond the original pharmacopoeia's distinction between acceptable and unacceptable quality.

[0016] More preferably, in the partial least squares discriminant analysis (PLS-DA) model, R² ≥ 0.975 and Q² ≥ 0.647. R² can also be expressed as "R²Y(cum)", a concept similar to... Figure 5 The R2X shown is similar, but it focuses on the model's cumulative explanatory power in the Y-axis direction, indicating how much information in the original data in the Y-axis direction is explained by the principal components. Similarly, "cum" represents the cumulative effect. Q2 can also be expressed as "Q2(cum)," which represents the model's cumulative predictive power. In some cases, Q2 is also used to describe the model's predictive performance on the dataset through principal component analysis. Here, "cum" also represents the cumulative effect, potentially involving the predictive power of multiple principal components. Preferably, the difference between R2 and Q2 is ≤0.3. A lower limit of 0.975 for R2 ensures a very strong fit, while a lower limit of 0.647 for Q2 ensures good accuracy and predictability. Furthermore, this invention requires a difference of ≤0.3 to effectively prevent overfitting, avoid high fit and low predictability, and ensure the model possesses high fit, high accuracy, and high reliability, providing a good industrial-grade model for the automatic grading of large-scale raw material batches.

[0017] In some embodiments, the sample pretreatment of the Cortex Phellodendri in step S1 comprises: taking 0.5 g of Cortex Phellodendri powder, adding 50 mL of 80% methanol-0.1% phosphoric acid aqueous solution, ultrasonic extraction for 30 min, and filtering to obtain a test solution. Phenolic acid components have poor stability in conventional methanol extraction, and the addition of 0.1% phosphoric acid aqueous solution can inhibit ionization and improve the recovery rate of 3-O- / 5-O-feruloylquinic acid. Therefore, such pretreatment can simultaneously extract phenolic acids and alkaloids from Cortex Phellodendri, while significantly improving the recovery rate. The solid-liquid ratio of Cortex Phellodendri powder to 80% methanol-0.1% phosphoric acid aqueous solution is 1:100 and the ultrasonic extraction time is 30 min, which can achieve an optimal balance between improving extraction efficiency and throughput, while taking into account the dissolution of polar and non-polar components in Cortex Phellodendri. This provides a faithful sample for fingerprinting and content determination of Cortex Phellodendri, and eliminates false negative / positive misjudgments.

[0018] The second aspect of the present application provides an application of the Cortex Phellodendri quality evaluation method according to the first aspect of the present application in the quality control of traditional Chinese medicine raw materials, comprising the following steps:

[0019] The contents of berberine hydrochloride, 3-O-feruloylquinic acid and 5-O-feruloylquinic acid in the Cortex Phellodendri are determined. If the Cortex Phellodendri meets the following conditions: the content of berberine hydrochloride is ≥30 mg / g; the content of 5-O-feruloylquinic acid is ≥35 mg / g; and the content of 3-O-feruloylquinic acid is ≥1.5 mg / g, it is determined as qualified raw material.

[0020] The above application creates a "double phenolic acid-alkaloid" synergistic quality control model, and uses a simplified detection process to not only reduce the misjudgment rate of inferior Cortex Phellodendri raw materials, but also accurately select high-quality batches to improve the clinical effectiveness of traditional Chinese medicines, and establish a raw material supply and management mechanism that automatically associates unqualified batches with production sites.

[0021] Further, in the preferred scheme of the application of the Cortex Phellodendri quality evaluation method according to the first aspect of the present application in the quality control of traditional Chinese medicine raw materials, the traditional Chinese medicine raw material is used for preparing Chinese patent medicines containing Cortex Phellodendri, and the Chinese patent medicines include Zhibai Dihuang Pill, Moshu Mingmu Liquid, Huanglian Shangqing Pill, Ermiao Pill, Simiao Pill, and Huangbai Capsule. Such Chinese patent medicines are not limited to the above-mentioned six kinds, but are divided into injection grade, oral grade and unqualified grade Cortex Phellodendri raw materials through the above-mentioned typical representatives, and are more reasonably applied to product development and adaptation.

[0022] The Cortex Phellodendri quality evaluation method of the present application can also be extended to the field of rapid quality control products. In this way, no matter how the preparation process of Chinese patent medicines containing Cortex Phellodendri is, as long as the quality control standards of the Cortex Phellodendri quality evaluation method of the present application are used, they belong to the scope of protection of the present application, and the detection service of Cortex Phellodendri is fully considered.

[0023] Based on the above, the third aspect of the present application provides a Phellodendri Chinensis Cortex quality evaluation kit using the Phellodendri Chinensis Cortex quality evaluation method of the first aspect of the present application, comprising hydrochloric acid berberine, 3-O-feruloyl quinic acid, 5-O-feruloyl quinic acid control and a mobile terminal pre-installed with PLS-DA analysis software, which is used to input the detection data of Phellodendri Chinensis Cortex and automatically output the instruction of acceptance or rejection. In such an embodiment, not only can the quality of Phellodendri Chinensis Cortex be quickly determined to solve the quality fluctuation problem caused by habitat differences, but also can promote the collaborative optimization and upgrading of the industry chain such as traceability, process control, raw material acceptance, Chinese medicine quality control and planting guidance.

[0024] The above summary of the invention is only a summary of the technical solutions of the present application. In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, and then can be implemented according to the content of the description and the drawings, and in order to enable the above and other purposes, characteristics and advantages of the present application to be more easily understood, the following will be described in combination with the specific embodiments of the present application and the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0025] The drawings are only used to show the principles, implementation manners, applications, characteristics and effects of the specific embodiments of the present application and other related contents, and cannot be considered as a limitation of the present application.

[0026] In the drawings of the specification:

[0027] Figure 1 The superimposed chromatogram of 10 batches of Phellodendri Chinensis Cortex test samples used in the specific embodiments;

[0028] Figure 2 HPLC chromatogram (A) of Phellodendri Chinensis Cortex test sample and HPLC chromatogram (B) of mixed control in the specific embodiments, wherein 1, chlorogenic acid; 2, 3-O-feruloyl quinic acid; 3, 4-O-feruloyl quinic acid; 4, hydrochloric acid berberine; 5, magnolia base; 6, 5-O-feruloyl quinic acid; 7, hydrochloric acid jatrorrhizine; 8, hydrochloric acid berberine; 9, phellodendron ketone;

[0029] Figure 3 Box plot of 9 kinds of chemical components of Phellodendri Chinensis Cortex from different producing areas in the specific embodiments, wherein 1, chlorogenic acid; 2, 3-O-feruloyl quinic acid; 3, 4-O-feruloyl quinic acid; 4, hydrochloric acid berberine; 5, magnolia base; 6, 5-O-feruloyl quinic acid; 7, hydrochloric acid jatrorrhizine; 8, hydrochloric acid berberine; 9, phellodendron ketone;

[0030] Figure 4 Cluster analysis tree diagram of different batches of Phellodendri Chinensis Cortex samples in the specific embodiments;

[0031] Figure 5For the PLS-DA score plot of different batches of Huangbai in the specific embodiment, the index R2X(cum) is used in the modeling of multivariate statistical analysis, which represents the cumulative explanatory ability of the model in the X-axis direction, that is, how much percentage of information in the original data is explained by the principal component, and "cum" here represents the cumulative, which means it may include the contribution of multiple principal components;

[0032] Figure 6 For the VIP score plot of different batches of Huangbai in the specific embodiment. DETAILED DESCRIPTION

[0033] To illustrate the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved, the following specific embodiments are combined with the accompanying drawings to explain in detail. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.

[0034] In this paper, "embodiment" means that the specific features, structures or characteristics described in combination with the embodiment can be included in at least one embodiment of the present application. The word "embodiment" appearing at various places in the specification does not necessarily refer to the same embodiment, and does not particularly limit its independence or association with other embodiments. In principle, in this application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0035] Unless otherwise defined, the meaning of the technical terms used herein is the same as that generally understood by those skilled in the art to which the present application belongs; the use of related terms in this paper is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0036] In the description of the present application, the word "and / or" is a description of the logical relationship between the objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this paper generally represents that the associated objects before and after are a "or" logical relationship.

[0037] In this application, such as "first" and "second" language is only used to distinguish one entity or operation from another entity or operation, and does not necessarily require or imply any actual quantity, primary or secondary or order relationship between the entities or operations.

[0038] In the present application, the terms "comprise", "contain", "include", or other similar phrases used in the statements mean to encompass the non-exclusive inclusion, and the terms do not exclude the presence of additional elements in the process, method or product comprising the stated elements, so that the process, method or product comprising a series of elements can not only include those defined elements, but also include other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0039] In the present application, the terms "greater than", "less than", "exceed" and the like are understood as not including the number itself, and the terms "above", "below", "within" and the like are understood as including the number itself. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly specified.

[0040] In the description of the embodiments of the present application, the spatial-related terms used, such as "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "perpendicular", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential", and the like, indicate the orientation or positional relationship based on the orientation or positional relationship shown in the specific embodiments or the drawings, and are only for the convenience of describing the specific embodiments of the present application or for the reader to understand, and do not indicate or imply that the indicated device or component must have a particular position, a particular orientation, or be constructed or operated in a particular orientation, and therefore cannot be understood as a limitation on the embodiments of the present application.

[0041] Unless otherwise explicitly specified or limited, in the description of the embodiments of the present application, the terms "mount", "connect", "connect", "fix", "set", and the like should be understood in a broad sense. For example, the "connection" can be a fixed connection, or a detachable connection, or an integral setting; it can be a mechanical connection, or an electrical connection, or a communication connection; it can be directly connected, or indirectly connected through an intermediate medium; it can be the internal communication of two elements or the interaction relationship between two elements. For those skilled in the art to which the present application belongs, the specific meaning of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0042] Phellodendron chinense Schneid. is a commonly used medicinal material in clinical practice, with the functions of clearing heat and drying dampness, and has a large market demand. Its pharmacological activities (such as anti-inflammatory, antibacterial, anticancer, and hypoglycemic) are derived from a complex system of chemical components, including alkaloids (such as berberine, phellodendrine, jatrorrhizine, and magnoflorine), phenolic acids (such as chlorogenic acid and various feruloylquinic acids), and limonoids (such as amurensin). However, the quality control of P. chinense is currently facing severe challenges: the current 2020 edition of Chinese Pharmacopoeia only uses two alkaloid components, berberine hydrochloride and phellodendrine hydrochloride, as quality control indicators, ignoring the effects of important active component groups such as phenolic acids (such as chlorogenic acid and feruloylquinic acid) and limonoids (such as amurensin), and cannot truly reflect the multi-component, multi-target synergistic pharmacodynamic material basis. This evaluation method based on a small number of indicator components is essentially a one-sided quality control strategy. It cannot capture the differences in the overall chemical profile of medicinal materials caused by factors such as origin, ecology, cultivation, and processing. Monitoring only two components, even if their contents meet the standards, cannot guarantee that the contents of other key pharmacologically active components are appropriate or that the quality is stable between batches, and cannot meet the current requirements of traditional Chinese medicine quality control.

[0043] Therefore, to break through the limitations of the current single component quality control, it is necessary to establish a scientific and comprehensive quality evaluation system that can systematically characterize the fingerprint of P. chinense, simultaneously precisely monitor multiple key active components, and use chemometrics to analyze the batch difference rules. The above work is of extreme importance and urgency for ensuring the safety, effectiveness, and uniformity and stability of P. chinense and its preparations.

[0044] Traditional Chinese medicine fingerprint technology is a quantifiable chromatographic identification method that can evaluate the quality of traditional Chinese medicinal materials from the overall properties of chemical components. In the embodiments of the present invention, 10 batches of P. chinense from different batches were selected for high performance liquid chromatography (HPLC) fingerprint analysis, and similarity analysis was performed to evaluate the stability and consistency of P. chinense from different origins. At the same time, based on the characteristics of traditional Chinese medicine with multiple targets, multiple pathways, and synergistic effects of multiple active components, the contents of 9 chemical components in different batches of P. chinense were detected by HPLC, and chemometric analysis methods such as hierarchical cluster analysis (HCA) and partial least squares discriminant analysis (PLS-DA) were used to analyze the differences between different batches of P. chinense. This three-dimensional evaluation mode of "overall characterization + multi-index quantification + pattern recognition" not only reveals the formation rules of the quality differences of medicinal materials from the overall chemical material basis level, but also conforms to the multi-target characteristics of traditional Chinese medicine, and has more comprehensive scientificity and applicability than the existing quality control methods.

[0045] Example 1, Quality Evaluation Method for P. chinense

[0046] 1 Instruments and materials used in the embodiments of the present invention

[0047] 1.1 Instruments

[0048] LC-20AT high performance liquid chromatograph (Shimadzu Corporation, Japan) online degasser, binary pump, autosampler, column oven, DAD detector, LCsolution workstation (Shimadzu Corporation, Japan); digital ultrasonic cleaner KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd.); AR224CN electronic analytical balance (Ohaus Instruments (Changzhou) Co., Ltd.); Milli-Q ultrapure water system (Millipore, USA).

[0049] 1.2 Materials

[0050] Phellodendrine hydrochloride (batch number: 104112-82-5, purity > 98%), magnoflorine (batch number: 2141-09-5, purity > 98%), 5-O-feruloylquinic acid (batch number: 40242-06-6, purity > 98%), jatrorrhizine hydrochloride (batch number: 6681-15-8, purity > 98%), 3-O-feruloylquinic acid (batch number: 1833-29-2, purity > 98%), berberine hydrochloride (batch number: 713-9404, purity > 98%), 4-O-feruloylquinic acid (batch number: 2613-86-7, purity > 98%), phellodendrofurone (batch number: 751-03-1, purity > 98%) were purchased from Chengdu Epha Biological Technology Co., Ltd.; chlorogenic acid (batch number: 0753-200111, purity > 98%) was purchased from Yuan Ye Biological Technology Co., Ltd. Methanol and acetonitrile were chromatographically pure, phosphoric acid was analytically pure, ultrapure water was prepared by a Milli-Q water purifier, and the rest of the reagents were analytically pure.

[0051] In the present application, unless otherwise specified, the other raw materials involved are commercially available products well known in the art.

[0052] The sources of the 10 batches of Phellodendri Chinensis Cortex used in the specific embodiments of the present application are shown in Table 1.

[0053] Table 1 Information of 10 batches of Phellodendri Chinensis Cortex samples

[0054] 2 Methods and results

[0055] 2.1 Chromatographic conditions

[0056] Column: Diamonsil C18 column (250 mm x 4.6 mm, 5 μm); mobile phase: 0.1% phosphoric acid water (A) - acetonitrile (B); gradient elution: 0-6 min, 12% B; 6-10 min, 12%-20% B; 10-15 min, 20% B; 15-25 min, 20%-25% B; 25-35 min, 20%-40% B; 35-45 min, 40%-55% B; 45-50 min, 55%-60% B; 50-55 min, 60-70% B. Flow rate: 1.0 mL•min; column temperature: 35 ℃; detection wavelength: 215 nm; injection volume: 10 μL.

[0057] 2.2 Preparation of test solution

[0058] About 0.5 g of Phellodendri chinensis Cortex powder (passed through a No. 4 sieve) was precisely weighed into a 100 mL stoppered conical flask, 50 mL of 80% methanol-0.1% phosphoric acid solution was added, weighed, and ultrasonically extracted (power 250 W, frequency 40 kHz) for 30 min, removed, allowed to cool, weighed, made up to weight, shaken well, filtered, and the filtrate was used as the test solution.

[0059] 2.3 Preparation of control solution

[0060] An appropriate amount of chlorogenic acid, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, berberine hydrochloride, magnoflorine, 5-O-feruloylquinic acid, jatrorrhizine hydrochloride, berberine hydrochloride, phellodendron ketone reference substances were weighed and precisely weighed to prepare a mixed control solution containing 90 μg / mL chlorogenic acid, 90 μg / mL 3-O-feruloylquinic acid, 100 μg / mL 4-O-feruloylquinic acid, 110 μg / mL berberine hydrochloride, 100 μg / mL magnoflorine, 110 μg / mL 5-O-feruloylquinic acid, 40 μg / mL jatrorrhizine hydrochloride, 370 μg / mL berberine hydrochloride, and 30 μg / mL phellodendron ketone. It should be noted that the concentrations of the above nine components in the mixed control solution are not fixed, but can be adjusted within a suitable concentration range.

[0061] 2.4 Fingerprint methodological investigation

[0062] 2.4.1 Precision test

[0063] Take the same test solution S1, under the chromatographic conditions of "2.1" continuous sample 6 times, with berberine hydrochloride (No. 12 peak) as the reference peak, the relative standard deviation (RSD) of the relative retention time of each common peak was calculated to be ≤2.62%, and the RSD of the relative peak area was ≤0.88%, indicating that the precision of the high performance liquid chromatograph was good.

[0064] 2.4.2 Reproducibility test

[0065] Take the same batch of Huangbai medicinal material powder S1, prepare 6 test solution according to the method of "2.2.1", and sample under the chromatographic conditions of "2.1", with berberine hydrochloride (No. 12 peak) as the reference peak, the RSD of the relative retention time of each common peak was calculated to be ≤0.89%, and the RSD of the relative peak area was ≤2.62%, indicating that the reproducibility of the method was good.

[0066] 2.4.3 Stability test

[0067] Take the same test solution S1, respectively, at room temperature for 0, 2, 4, 8, 12, 24 h, and determine according to the chromatographic conditions of "2.1", with berberine hydrochloride (No. 12 peak) as the reference peak, the RSD of the relative retention time of each common peak was calculated to be ≤1.09%, and the RSD of the relative peak area was ≤2.22%, indicating that the test solution S1 was stable at room temperature within 24 h.

[0068] 2.4.4 Establishment of fingerprint and similarity evaluation

[0069] According to the chromatographic conditions of "2.1", the test solution of 10 batches of Huangbai was analyzed, the data file was converted into "AIA" format, and was imported into "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version)". With the fingerprint of test solution S1 as the reference spectrum, the time window width was set to 0.1, the multi-point correction and Mark peak matching were carried out, and the control fingerprint was generated by median method. The HPLC fingerprint of 10 batches of Huangbai medicinal materials, the control spectrum and the test spectrum are shown in Figure 1 、 Figure 2 . The similarity of S1-S10 samples and the control fingerprint was calculated by computer software to be 0.997-1.000, indicating that the quality of Huangbai medicinal materials of different batches was stable.

[0070] 2.4.5 Identification of common peaks

[0071] The established fingerprint of 10 batches of Huangbai medicinal materials is shown in Figure 1, and 14 common peaks were determined. By comparing with the reference substances, 9 peaks were identified, in which peak 2 was chlorogenic acid, peak 3 was 3-O-feruloylquinic acid, peak 4 was 4-O-feruloylquinic acid, peak 5 was phellodendrine hydrochloride, peak 6 was magnoflorine, peak 7 was 5-O-feruloylquinic acid, peak 11 was jatrorrhizine hydrochloride, peak 12 was berberine hydrochloride, and peak 14 was phellodendrine.

[0072] 2.5 Determination of the contents of 9 components in Phellodendri Chinensis Cortex

[0073] 2.5.1 Linear range

[0074] The mixed reference substance solution of item 2.2.2 was precisely measured, and 1, 10, 20, 30, 40 and 50 μL were used as the sample volumes. The chromatographic conditions of item 2.1 were used for sample injection. The reference substance injection amount was used as the abscissa (X / μg), and the peak area was used as the ordinate (Y) for regression. The results are shown in Table 2, and it can be seen that each component has a good linear relationship in its respective range.

[0075] Table 2 Linear relationship of 9 components in Phellodendri Chinensis Cortex

[0076] 2.5.2 Precision test

[0077] The mixed reference substance solution was continuously injected 6 times under the chromatographic conditions of item 2.1, and the peak areas of each component were recorded. The results showed that the peak area RSDs of chlorogenic acid, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, phellodendrine hydrochloride, magnoflorine, 5-O-feruloylquinic acid, jatrorrhizine hydrochloride, berberine hydrochloride and phellodendrine were 0.69%, 1.18%, 1.09%, 0.92%, 1.17%, 0.63%, 0.69%, 0.93% and 0.65%, respectively, indicating that the precision of the instrument was good.

[0078] 2.5.3 Reproducibility test

[0079] Six test solutions were prepared in parallel according to the method described in section "2.2.1" using samples of Phellodendron bark. The average contents of chlorogenic acid, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, berberine hydrochloride, magnoflorine, 5-O-feruloylquinic acid, berberine hydrochloride, berberine hydrochloride, and berberine ketone were determined under the chromatographic conditions described in section "2.1". The results were 10.9801 mg / g, 2.771 mg / g, 7.514 mg / g, 7.770 mg / g, 7.8400 mg / g, 47.6565 mg / g, 2.2869 mg / g, 89.2814 mg / g, and 0.2768 mg / g, respectively. The RSDs (n=6) for mg / g were 0.22%, 1.10%, 1.39%, 1.31%, 1.62%, 1.26%, 0.96%, 1.22%, and 0.86%, respectively, indicating that the method has good repeatability.

[0080] 2.5.4 Stability Test

[0081] The Phellodendron amurense test solution was prepared and stored at room temperature in the dark for 0, 2, 4, 8, 12, and 24 days. Chromatographic analysis was performed under the conditions described in section "2.1", and the peak areas of each component were recorded. The results showed that the peak area RSDs (n=6) of chlorogenic acid, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, berberine hydrochloride, magnoflorine, 5-O-feruloylquinic acid, berberine hydrochloride, berberine hydrochloride, and berberine ketone were 1.12%, 1.50%, 1.18%, 1.46%, 1.08%, 1.01%, 1.11%, 1.05%, and 1.39%, respectively, indicating good instrument stability.

[0082] 2.5.5 Recovery Test

[0083] Six portions of Phellodendron bark powder S1 with known content of each component were accurately weighed (approximately 0.5 g each) and added to the mixed reference solution at approximately a 1:1 ratio. The solution was prepared according to the method described in section "2.2.1" and analyzed under the conditions described in section "2.1". The average recoveries (RSDs) of each component (n=6) were calculated to be 0.97%, 0.43%, 0.65%, 0.21%, 0.79%, 0.60%, 0.52%, 0.88%, and 0.49%, respectively (see Table 3 for details). The results indicate that the method is accurate and feasible.

[0084] Table 3. Recovery results of 9 components in Phellodendron bark (n=6)

[0085]

[0086] 2.5.6 Sample content determination

[0087] Take 10 batches of Phellodendri Chinensis Cortex medicinal materials to prepare test solution according to the method under item “2.2.1”, and determine the contents of chlorogenic acid, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, berberine hydrochloride, magnoflorine, 5-O-feruloylquinic acid, jatrorrhizine hydrochloride, berberine hydrochloride, and phellodendron ketone under the chromatographic conditions of item “2.1”, and the results are shown in Table Four. In order to more intuitively observe the differences of various chemical components in Phellodendri Chinensis Cortex from different producing areas, box plots are made, please refer to Figure 3 .

[0088] Table Four Determination results of 9 effective components in 10 batches of Phellodendri Chinensis Cortex (mg / g, n=3)

[0089] As can be seen from Table Four, the content determination results of the marker component berberine hydrochloride are 66.34-125.54 mg / g, the content determination results of 3-O-feruloylquinic acid are 1.42-3.87 mg / g, and the content determination results of 5-O-feruloylquinic acid are 34.71-66.84 mg / g. The content determination results of the other six components, chlorogenic acid, 4-O-feruloylquinic acid, berberine hydrochloride, magnoflorine, jatrorrhizine hydrochloride, and phellodendron ketone are 8.00-14.59 mg / g, 5.27-9.87 mg / g, 4.80-11.24 mg / g, 5.54-11.28 mg / g, 1.16-3.77 mg / g, and 0.2-0.65 mg / g, respectively.

[0090] 2.6 Multivariate statistical analysis

[0091] 2.6.1 Hierarchical cluster analysis (HCA)

[0092] Take the contents of 9 chemical components in different Phellodendri Chinensis Cortex samples as variables, import the data into SPSS 29.0 software, use group connection method, and take square Euclidean distance as the measurement standard to perform cluster analysis on 10 batches of Phellodendri Chinensis Cortex samples, and the cluster diagram is shown in Figure 4 . The results show that S1, S2, S5, S8, S9, and S10 are clustered into one class, S3, S4, and S7 are clustered into one class, and S6 is clustered into one class. The cluster analysis results show that the 10 batches of samples are divided into 3 classes by 9 chemical components.

[0093] 2.6.2 Partial least squares discriminant analysis (PLS-DA)

[0094] On the basis of HCA, import the contents of 9 chemical components in Phellodendri Chinensis Cortex samples from different producing areas into SIMCA 14.0 software for PLS-DA analysis, and the PLS-DA score plot is shown inFigure 5 , VIP score plot please refer to Figure 6 . Different batches of samples were divided into 3 categories, which was consistent with the results obtained by HCA. It showed that the content of each component in each group of samples was small, and the quality was stable. There was a certain difference between the components in different groups. In the PLS-DA partial least squares discriminant analysis score plot ( Figure 5 ): The horizontal coordinate (X axis): usually represents the first principal component (PC1), which is the direction of the maximum variability of the data. The value on this axis is the projection of the sample in this direction, reflecting the main source of variation. The vertical coordinate (Y axis): usually represents the second principal component (PC2), which is the direction of the second smallest variability of the data in the direction orthogonal to the first principal component. The value on this axis is the projection of the sample in this direction, reflecting the secondary variation source. R2X describes the fitting degree of the model, and Q2 describes the X prediction ability. Generally, Q2>0.5 indicates that the model has good reliability and predictability, and Q2>0.9 is very excellent, and the difference between R2Y and Q2 is preferably not greater than 0.3, that is, the numerical difference between R2Y and Q2 is ≤0.3. In this model, the statistical parameters of R2Y(cum) and Q2Y(cum) are 0.975 and 0.647, respectively. R2X represents the explanatory power of the model to the X variable (classification), R2X=0.975 close to 1 indicates high goodness of fit, Q2 represents the prediction ability of the model, Q2=0.647 greater than 0.5 indicates that the model has good reliability and predictability, indicating that the model has good repeatability and predictability. The difference between R2X and Q2 is 0.328, which is greater than 0.3, indicating that although the model has good predictability (Q2>0.5), the goodness of fit is very high (R2>0.97), which may indicate a slight risk of overfitting, which needs to be combined with other verifications. According to the PLS-DA model, further extract the variable importance projection (Variable Importance for the Projection, VIP) of 9 variables, and take VIP>1 as the standard to screen out chemical components with greater contribution to the classification results. The results show that berberine (1.734), 3-O-feruloyl quinic acid (1.624), and 5-O-feruloyl quinic acid (1.379) are the main marker components that cause the quality difference between different batches of Cortex Phellodendri samples.

[0095] Based on the above PLS-DA model and the three VIP>1 marker components (berberine hydrochloride, 3-O-feruloyl quinic acid, and 5-O-feruloyl quinic acid) screened out, a quality evaluation model for Cortex Phellodendri was constructed. Berberine hydrochloride, 3-O-feruloyl quinic acid, and 5-O-feruloyl quinic acid have the following pharmacological effects, respectively:

[0096] Berberine hydrochloride has:

[0097] 1. Antibacterial effect: Berberine hydrochloride can reduce the number of pili on the surface of bacteria, thereby inhibiting bacterial adhesion to the host mucosal epithelial cells and preventing bacterial colonization and reproduction in the body; it can also inhibit bacterial nucleic acid synthesis, affecting bacterial growth and metabolism. Clinically, it is often used to treat intestinal infections, diarrhea, and other diseases, such as acute enteritis and dysentery caused by Escherichia coli or Shigella.

[0098] 2. Anti-inflammatory effect: It can inhibit the release of inflammatory mediators, such as prostaglandins and leukotrienes, and reduce the inflammatory response; it can also regulate the production of cytokines and inhibit the activation of inflammatory signaling pathways, thereby exerting an anti-inflammatory effect.

[0099] 3. Blood glucose regulation: It can improve insulin sensitivity, promote glucose uptake and utilization, and lower blood glucose levels; it can also regulate the activity of enzymes related to glucose metabolism and improve glucose metabolism disorders. Clinically, it is used as an adjunct therapy for type 2 diabetes to help patients control blood glucose and improve insulin resistance.

[0100] 4. Lipid-regulating effect: It can lower the levels of cholesterol and triglycerides in the blood, reducing lipid deposition on the blood vessel walls; it can also promote lipid metabolism and excretion. Clinically, it is used to treat hyperlipidemia and can, to some extent, prevent the occurrence of cardiovascular diseases such as atherosclerosis.

[0101] 5. Antiarrhythmic effect: By affecting the ion channels of cardiomyocytes, regulating the electrophysiological activity of cardiomyocytes, and stabilizing the membrane potential of cardiomyocytes, it exerts an antiarrhythmic effect.

[0102] 3-O-feruloylquinic acid possesses:

[0103] 1. Antioxidant effect: It can eliminate excess free radicals in the body, reduce free radical damage to cells, and protect cells from oxidative stress. This helps maintain the normal structure and function of cells, and slows down the aging process of cells and tissues. For example, it can inhibit lipid peroxidation and reduce the production of oxidation products, thereby protecting the cardiovascular and nervous systems.

[0104] 2. Anti-inflammatory effects: It can regulate inflammation-related signaling pathways and inhibit the release of inflammatory mediators, such as tumor necrosis factor-α and interleukin-6. By inhibiting the inflammatory response, it can reduce tissue inflammatory damage and may have a certain therapeutic effect on inflammation-related diseases such as arthritis and enteritis.

[0105] 3. Antibacterial effect: It has an inhibitory effect on some bacteria and fungi. It can disrupt the cell membrane structure of pathogens, inhibit their growth and reproduction, thereby helping the body fight infection. For example, it has a certain inhibitory effect on common pathogens such as Escherichia coli and Staphylococcus aureus.

[0106] 4. Hypoglycemic effect: It can improve insulin resistance, promote glucose uptake and utilization, and regulate the activity of sugar metabolism-related enzymes, thereby reducing blood glucose levels. For diabetic patients, it may help control blood sugar and reduce the risk of diabetic complications.

[0107] 5. Neuroprotective effect: It can reduce nerve cell damage and improve nerve function. It may have a certain preventive and therapeutic effect on neurological diseases such as Alzheimer's disease and Parkinson's disease through mechanisms such as antioxidant and anti-inflammatory effects.

[0108] 5-O-feruloyl quinic acid has the following properties:

[0109] 1. Antioxidant effect: It can scavenge free radicals, inhibit lipid peroxidation, reduce oxidative stress damage to cells, and protect cell structure and function integrity, which may play a role in delaying tissue aging and protecting the cardiovascular system.

[0110] 2. Anti-inflammatory effect: By inhibiting the release of inflammatory factors (such as tumor necrosis factor and interleukin), regulating inflammatory signaling pathways, and reducing inflammation, it may have a certain effect on inflammation-related diseases such as respiratory tract inflammation and arthritis.

[0111] 3. Antibacterial and antiviral effects: It has certain inhibitory effects on some bacteria (such as Staphylococcus aureus) and viruses (such as influenza virus), which may exert anti-infective effects by damaging pathogen structures or inhibiting their proliferation.

[0112] 4. Cardiovascular protection: By regulating blood lipids, inhibiting platelet aggregation, and improving vascular endothelial function, it can help protect the cardiovascular system and reduce the potential risk of cardiovascular disease.

[0113] This model can effectively distinguish the quality differences of different batches of samples (clustering into different categories). By determining the content of these three marker components in a new batch of Cortex Phellodendri, inputting the PLS-DA model, it can predict the possible quality category (I, II, or III) or calculate the distance from the center of each category to evaluate its quality level (excellent, good, qualified, etc., which requires pre-defined category levels).

[0114] Traditional Chinese medicine fingerprint as a comprehensive and quantifiable chromatographic identification method can reflect the chemical composition and characteristics of traditional Chinese medicine and preparations, and further evaluate their quality stability and consistency. However, the evaluation results of similarity cannot well distinguish the differences between different batches of samples. Fingerprint combined with multi-component content determination and chemical pattern recognition analysis of differences between different batches of samples can more accurately reflect the quality of the traditional Chinese medicine.

[0115] The present application establishes the HPLC fingerprint of Phellodendri Chinensis Cortex, and 14 common peaks are calibrated, 9 of which are identified by comparison with reference substances. The similarity of the HPLC fingerprint of 10 batches of samples and the generated control spectrum is above 0.997, indicating that the Phellodendri Chinensis Cortex from different producing areas is similar in main components and cannot well distinguish the differences between different samples. Through multi-component content determination, it is found that the 9 chemical components in the same batch of medicinal materials are quite different, the content of berberine hydrochloride is the highest, the content of 5-O-feruloyl quinic acid is relatively high, the content of chlorogenic acid, 3-O-feruloyl quinic acid, 4-O-feruloyl quinic acid, berbamine hydrochloride and magnolia flower base is moderate, the content of jatrorrhizine and phellodendron ketone is relatively low. Further analysis by chemical pattern recognition method shows that HCA and PLS-DA can classify the Phellodendri Chinensis Cortex from 10 producing areas into 3 categories according to 9 chemical components. Through further analysis, berberine hydrochloride, 3-O-feruloyl quinic acid and 5-O-feruloyl quinic acid are the characteristic components of the chemical component differences between different batches, which have a greater impact on the quality of Phellodendri Chinensis Cortex. Among them, chlorogenic acid and 5-O-feruloyl quinic acid have moderate content and good separation degree in Phellodendri Chinensis Cortex, which can meet the requirements of content determination. It is suggested that berberine hydrochloride, 3-O-feruloyl quinic acid and 5-O-feruloyl quinic acid can be used as index detection components in the quality evaluation research of Phellodendri Chinensis Cortex, which can more comprehensively reflect the quality of Phellodendri Chinensis Cortex.

[0116] The three characteristic components screened by the present application not only contain the alkaloid berberine hydrochloride concerned by the pharmacopoeia, but also add phenolic acid components, which just make up for the blind area of the current standard. The box plot shows that the three components have the largest coefficient of variation in different producing area samples (the content of 5-O-feruloyl quinic acid in S6 batch is as high as 66.84 mg / g, while that in S5 is only 34.71 mg / g), which shows that they are indeed the key indicators for distinguishing quality. The design of gradient elution program (especially the steep gradient of 20%-40% acetonitrile at 25-35 min) successfully separates the feruloyl quinic acid isomers with similar polarity, which is the key to accurate quantification. The threshold of Q2>0.647 in the PLS-DA model is based on the cross-validation results of ten batches of samples, which has statistical significance.

[0117] Example two, application of the quality evaluation method of Phellodendri Chinensis Cortex in the quality control of traditional Chinese medicine raw materials

[0118] The quality of the purchased Phellodendri Chinensis Cortex raw materials is controlled to ensure that it meets the requirements for producing Phellodendri Chinensis Cortex-containing traditional Chinese medicines, such as but not limited to Zhibai Dihuang Pill, Moshu Mingmu Liquid, Huanglian Shangqing Pill, Ermiao Pill, Simiao Pill and Huangbai Capsule.

[0119] Sample pretreatment: prepare the test sample solution of the Phellodendri Chinensis Cortex raw material to be tested according to the method of step 1 of the embodiment.

[0120] Marker component content determination: according to the HPLC method and content determination method of step 3 of the embodiment, the content of the following 3 marker components in the raw material is specially determined: berberine hydrochloride, 3-O-feruloylquinic acid, 5-O-feruloylquinic acid;

[0121] Pass judgment: according to the result range determined in Example One and the actual quality requirements, the following minimum content limits are set as the judgment standard for the quality of the raw material: berberine hydrochloride content ≥ 30 mg / g, 5-O-feruloylquinic acid content ≥ 35 mg / g, 3-O-feruloylquinic acid content ≥ 1.5 mg / g;

[0122] Results and judgment: if the content of the above 3 components in the raw material to be tested meets or is higher than the set minimum limit (≥ 30 mg / g, ≥ 35 mg / g, ≥ 1.5 mg / g), the batch of raw material is determined to be qualified and can be used for the production of subsequent Chinese patent medicines such as, but not limited to, Zhibai Dihuang Pill, Moshe Mingmu Liquid, and Huanglian Shangqing Pill.

[0123] If the content of any component is lower than the corresponding minimum limit, the batch of raw material is determined to be unqualified and is rejected.

[0124] Compared with the 2020 edition of Chinese Pharmacopoeia which only detects two indicators of berberine hydrochloride and phellodendrine hydrochloride, the embodiment adds the key components 3-O-feruloylquinic acid and 5-O-feruloylquinic acid as control indicators to reflect the quality difference, which can more comprehensively evaluate the quality of Phellodendri Chinensis Cortex raw material and ensure the efficacy and stability of the produced Chinese patent medicines such as Zhibai Dihuang Pill, Moshe Mingmu Liquid, and Huanglian Shangqing Pill.

[0125] Example Three, Phellodendri Chinensis Cortex Quality Evaluation Kit

[0126] The Phellodendri Chinensis Cortex Quality Evaluation Kit of the embodiment is composed as follows:

[0127] Reference substance: berberine hydrochloride reference substance (purity > 98%), 3-O-feruloylquinic acid reference substance (purity > 98%), 5-O-feruloylquinic acid reference substance (purity > 98%), and other component reference substances such as chlorogenic acid and phellodendrine hydrochloride can also be included in other embodiments for fingerprint identification or total component determination to provide more accurate Phellodendri Chinensis Cortex quality grading and quality control results.

[0128] Mobile terminal pre-installed with analysis software:

[0129] Hardware: tablet computer or smart phone.

[0130] Software: pre-installed customized application App. The core functions of the App include: data input interface: for users to input the measured content values (unit: mg / g) of the three marker components berberine hydrochloride, 3-O-feruloylquinic acid, and 5-O-feruloylquinic acid in the sample to be tested.

[0131] Built-in PLS-DA quality evaluation model: This model is based on the model established in Example 1 (built using SIMCA 14.1, R2X(cum)=0.975, Q2(cum)=0.647). The model uses the content of the three marker components as X variables and can predict the quality category (I, II, III, or user-predefined levels such as excellent, good, and qualified) to which the sample belongs.

[0132] Decision rule database: stores the pre-set qualified determination criteria (such as in Example 2: berberine hydrochloride ≥ 30 mg / g, 5-O-feruloylquinic acid ≥ 35 mg / g, and 3-O-feruloylquinic acid ≥ 1.5 mg / g) and / or the content range corresponding to the quality level.

[0133] Analysis engine: input the content data input by the user into the built-in PLS-DA model for calculation to predict the quality category; at the same time, compare the input content values with the pre-set qualified determination criteria.

[0134] Result output interface: automatically displays the analysis results and gives clear instructions: displays the predicted quality category / level.

[0135] Based on the pre-set qualified determination criteria, output the instructions “accept” (qualified) or “reject” (unqualified).

[0136] In addition, the customized application App can also display the comparison of the measured values and standard values of each index. The specific use method is as follows:

[0137] The user performs pretreatment and HPLC analysis on the sample to be tested according to Example 1 or the standard method, and determines the content of the three marker components berberine hydrochloride, 3-O-feruloylquinic acid, and 5-O-feruloylquinic acid.

[0138] Open the mobile terminal App in the kit. In the data input interface of the App, input the measured content values of the three ingredients. Click the "analyze" or "determine" button. The App automatically performs the following operations: calls the built-in PLS-DA model, and predicts the sample quality category according to the input values (optional function). Compare the input three content values with the preset qualified standards (such as: ≥ 30 mg / g, ≥ 35 mg / g, ≥ 1.5 mg / g). The App clearly displays the final instruction in the result interface: "accept" (if all three indicators are ≥ the standard value) or "reject" (if any one indicator < the standard value). The kit provided in this embodiment integrates key control samples and intelligent analysis terminals. Users only need to input the measured content of three key indicators to quickly and conveniently obtain the determination result of whether the raw material is qualified, significantly improving the efficiency and standardization of the quality control of Cortex Phellodendri medicinal material raw materials on site or in the laboratory.

[0139] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited. Any technical solution obtained by replacing or modifying the equivalent structure or equivalent process based on the essential concept of the present application, using the content described in the specification and drawings of the present application, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc., are all included in the patent protection scope of the present application.

Claims

1. A method for quality evaluation of Phellodendron bark medicinal material, characterized in that, Includes the following steps: S1. Establish the HPLC fingerprint of the aforementioned Phellodendron bark medicinal material: gradient elution was performed using acetonitrile-0.1% phosphoric acid water as the mobile phase, and a Diamonsil C10 column was used. 18 Column, detection wavelength 215 nm; S2 determines the content of the marker components in the Phellodendron bark herb, which include berberine hydrochloride, 3-O-feruloylquinic acid, and 5-O-feruloylquinic acid; Based on the content of the marker components mentioned in step S2, S3 constructs a quality evaluation model using the partial least squares discriminant analysis (PLS-DA) model, and uses a VIP value > 1 as the screening criterion for marker components to distinguish the quality grades of different batches of Phellodendron amurense.

2. The method for quality evaluation of Phellodendron bark medicinal material according to claim 1, characterized in that, The conditions for gradient elution in step S1 are as follows: Gradient elution: 0–6 min, 12% acetonitrile; 6–10 min, 12%–20% acetonitrile; 10–15 min, 20% acetonitrile; 15–25 min, 20%–25% acetonitrile; 25–35 min, 20%–40% acetonitrile; 35–45 min, 40%–55% acetonitrile; 45–50 min, 55%–60% acetonitrile; 50–55 min, 60–70% acetonitrile. Flow rate: 1.0 mL•min; column temperature: 35℃; injection volume: 10 μL.

3. The method for quality evaluation of Phellodendron bark medicinal material according to claim 1, characterized in that, Step S1 further includes generating a reference fingerprint spectrum through a similarity evaluation system and identifying 14 common peaks, including at least 9 component peaks including chlorogenic acid, 3-O-feruloylquinic acid, 4-O-feruloylquinic acid, berberine hydrochloride, magnoflorine, 5-O-feruloylquinic acid, berberine hydrochloride, berberine hydrochloride, and berberine ketone.

4. The method for quality evaluation of Phellodendron bark medicinal material according to claim 3, characterized in that, In step S3, the quality evaluation model also incorporates cluster analysis (HCA) with the nine component peaks as variables, using the intergroup linkage method and the squared Euclidean distance as the metric to cluster different batches of Phellodendron amurense samples into three categories.

5. The method for quality evaluation of Phellodendron bark medicinal material according to claim 1, characterized in that, The content of the marker components is as follows: berberine hydrochloride ranges from 66.34 to 125.54 mg / g; 3-O-feruloylquinic acid ranges from 1.42 to 3.87 mg / g; and 5-O-feruloylquinic acid ranges from 34.71 to 66.84 mg / g.

6. The method for quality evaluation of Phellodendron bark medicinal material according to claim 1, characterized in that, In the partial least squares discriminant analysis (PLS-DA) model, R² ≥ 0.975, Q² ≥ 0.647, and preferably, the difference between R² and Q² is ≤ 0.

3.

7. The method for quality evaluation of Phellodendron bark medicinal material according to claim 1 or 3, characterized in that, The sample pretreatment of Phellodendron bark in step S1 includes: taking 0.5 g of Phellodendron bark powder, adding 50 mL of 80% methanol-0.1% phosphoric acid aqueous solution, ultrasonically extracting for 30 min, and filtering to obtain the test solution.

8. The application of the quality evaluation method for Phellodendron bark as described in any one of claims 1-7 in the quality control of raw materials for traditional Chinese medicine, characterized in that, Includes the following steps: The contents of berberine hydrochloride, 3-O-feruloylquinic acid, and 5-O-feruloylquinic acid in Phellodendron bark were determined. If the contents of berberine hydrochloride ≥30 mg / g, 5-O-feruloylquinic acid ≥35 mg / g, and 3-O-feruloylquinic acid ≥1.5 mg / g, the raw material was deemed qualified.

9. The application according to claim 8, characterized in that, The Chinese herbal raw materials are used to prepare traditional Chinese medicines containing Phellodendron bark, including Zhi Bai Di Huang Wan, She Zhu Ming Mu Ye, Huang Lian Shang Qing Wan, Er Miao Wan, Si Miao Wan, and Phellodendron bark capsules.

10. A quality evaluation kit for Phellodendron amurense using the quality evaluation method for Phellodendron amurense as described in any one of claims 1-7, characterized in that, The device includes berberine hydrochloride, 3-O-feruloylquinic acid, 5-O-feruloylquinic acid reference standards, and a mobile terminal pre-installed with PLS-DA analysis software. The mobile terminal is used to automatically output an acceptance or rejection instruction after inputting the test data of Phellodendron bark.

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