Quality evaluation method for suberect spatholobus stem decoction pieces based on'excellent shape and high quality '

Abstract

The invention discloses a suberect spatholobus stem decoction piece quality evaluation method based on a'excellent shape and high quality 'theory, and belongs to the technical field of traditional Chinese medicine quality evaluation. According to the method, key character indexes (the number of turns, the diameter and the delta E) are screened out by utilizing principal component analysis, correlation analysis and partial least squares discriminant analysis (OPLS-DA) by measuring the external character indexes (powder color delta E * > = 35, the diameter and the maximum number of turns of a tangent plane) and the internal quality indexes (catechin, epicatechin and the content of alcohol-soluble extracts) of the caulis spatholobi decoction pieces; and a quantitative correlation and prediction model of the content of the components and the content of the internal components is established. By setting a key character threshold (delta E * > = 35, the number of turns > = 5, and the diameter > = 5.0 cm) or using a prediction model, rapid and objective evaluation of the quality of the caulis spatholobi decoction pieces can be realized, traditional subjective identification and complex instrument detection are effectively replaced, and a reliable standard is provided for quality control and market circulation of medicinal materials.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality evaluation technology, specifically involving a quality evaluation method for chicken blood vine slices based on the theory of "superior shape and superior quality". This method enables objective and rapid evaluation of the quality of chicken blood vine slices, providing technical support for the quality control and standardized application of chicken blood vine slices. Background Technology

[0002] Chicken blood vine was first recorded in "Compendium of Materia Medica Supplement". It is the dried vine stem of Spatholobus suberectus Dunn, a legume. It has the effects of promoting blood circulation and nourishing blood, regulating menstruation and relieving pain, and relaxing muscles and tendons. Clinically, it is often used to treat blood deficiency and chlorosis, numbness of limbs, rheumatic pain and other symptoms. It is the core ingredient of traditional Chinese medicine such as "Wuji Baifeng Wan" and "Chicken Blood Vine Paste". It is also widely used in traditional Chinese medicine to regulate qi and blood.

[0003] Modern pharmacological studies have shown that the main active ingredients of chicken blood vine are flavonoids (such as catechins and epicatechins). These ingredients have antioxidant, anti-osteoporosis, and microcirculation-improving activities, and their content directly determines the clinical efficacy of chicken blood vine decoction pieces.

[0004] Historical herbal texts and modern pharmacopoeias have largely relied on subjective descriptions of the quality of chicken blood vine, such as "the more reddish-brown layers on the cut surface, the better" and "the better if the surface is reddish-brown or dark brown with longitudinal grooves." The lack of quantitative standards has led to significant differences in the judgments of different appraisers, resulting in inconsistent quality of processed chicken blood vine on the market.

[0005] Existing research at home and abroad mainly focuses on the isolation of chemical components, verification of pharmacological activities and identification by DNA barcoding of chicken blood vine. However, it has not systematically explored the quantitative correlation between appearance characteristics such as "number of rings, color and diameter" and active ingredients such as catechins and epicatechins, and it is impossible to quickly predict the internal quality through appearance.

[0006] Currently, the internal quality testing of *Spatholobus suberectus* mainly relies on large instruments such as high-performance liquid chromatography (HPLC), which requires complex sample pretreatment and has a long testing cycle, making it unsuitable for rapid quality screening in grassroots pharmacies and medicinal herb markets.

[0007] "Superior form and superior quality" is the core theory of traditional Chinese medicine quality evaluation, which has been verified in medicinal materials such as Danshen and Xuanshen.

[0008] In summary, by evaluating the quality of *Spatholobus suberectus* from multiple dimensions such as appearance, active ingredients, and yield, this study clarifies the scientific basis of "distinguishing appearance from quality" in the identification of *Spatholobus suberectus* slices, and provides a theoretical basis for constructing a rapid and non-destructive evaluation system for identifying the quality of *Spatholobus suberectus*. Summary of the Invention

[0009] To address the problems of subjective evaluation of chicken blood vine slices, unclear correlation between morphology and intrinsic quality, and complex detection methods in existing quality evaluation methods, this invention provides a chicken blood vine quality evaluation method based on morphological analysis.

[0010] This invention aims to utilize principal component analysis to jointly analyze the appearance, active ingredients, and yield of *Spatholobus suberectus*, constructing a multi-dimensional evaluation model. By rapidly predicting the content of active ingredients in *Spatholobus suberectus* based on its appearance characteristics, this invention achieves accurate quality evaluation and provides a theoretical basis for constructing a rapid and non-destructive quality assessment system for *Spatholobus suberectus*.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] A quality evaluation method for chicken blood vine slices based on "superior shape and high quality" is characterized by the following steps:

[0013] 1) Obtain data on 8 indicators of chicken blood vine.

[0014] To obtain the index data of chicken blood vine slices, we need to obtain the six appearance index data of chicken blood vine slices: powder color, slice diameter, and maximum number of slices.

[0015] Two intrinsic quality indicators of *Chicken Blood Vine* slices were obtained: catechin content and epicatechin content determined by high performance liquid chromatography (HPLC).

[0016] 2) Principal component analysis

[0017] A) Data Preprocessing and Modeling

[0018] Based on the data of the eight indicators obtained in step 1), a "trait-quality" sample database of chicken blood vine slices was constructed, and the data in the database were standardized and preprocessed. Pearson correlation analysis was used to clarify the direction and strength of the association between appearance traits and internal quality indicators. Partial least squares discriminant analysis (OPLS-DA) was used to screen the trait indicators that contribute significantly to quality grading. Multiple regression analysis was used to construct a predictive model of appearance traits on internal quality.

[0019] B) Screening of key trait indicators

[0020] Based on the OPLS-DA results (variable importance projection value VIP > 1) and the significance level of Pearson correlation (P < 0.05), 2-3 key appearance traits that can characterize the quality of chicken blood vine slices were selected.

[0021] 3) Quality Evaluation

[0022] The quality of chicken blood vine slices is evaluated based on the key appearance traits screened in step 2) or the prediction model constructed in step 2).

[0023] Further specifying, the method for determining the appearance characteristics in step 1) is as follows:

[0024] A) Measurement of powder color parameters

[0025] Take an appropriate amount of chicken blood vine powder, spread it evenly in a cuvette, and use a spectrophotometer (light source D65, aperture 8mm) to measure ΔL*, Δa*, and Δb*. Three parallel measurements were taken for each batch of samples, and six random measurements were taken for each sample. The average value was used to calculate the comprehensive color difference value ΔE*.

[0026] B) Measurement of the diameter of medicinal slices

[0027] The cross-sectional diameter of the chicken blood vine slices was measured using vernier calipers. Three slices were measured for each batch of samples, and the average value was taken.

[0028] C) Determination of the maximum number of rings in the medicinal slices:

[0029] Visually observe the cross-section of the chicken blood vine slices, count the maximum number of reddish-brown layers, and measure 3 slices from each batch of samples, taking the average value.

[0030] Further specifying that in step 1), the catechin content and epicatechin content are determined by HPLC, with the following specific conditions: chromatographic column: X-Peonyx column; mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B); gradient elution program: 0–10 min, 5%–15% A; 10–20 min, 15%–25% A; 20–30 min, 25%–35% A; 30–40 min, 35%–5% A; detection wavelength: 280 nm; flow rate: 1.0 mL / min. -1 The injection volume was 10 μL, and the column temperature was 30℃.

[0031] Further specifying, the preparation methods of the test solution and reference solution in the HPLC method are as follows:

[0032] A) Preparation of reference solution

[0033] Accurately weigh catechin and epicatechin standards, dissolve them in 45% methanol solution to prepare mixed reference solutions with concentrations of 1.36 mg / mL and 1.15 mg / mL, respectively. After filtration through a 0.22 μm filter membrane, store at 4°C for later use.

[0034] B) Preparation of test solution

[0035] Accurately weigh 1.0 g of chicken blood vine powder (passed through a No. 3 sieve), place it in a stoppered conical flask, add 60 mL of methanol, let stand for 30 min, then extract by ultrasonication for 45 min, filter and collect the filtrate; evaporate the filtrate to dryness in a water bath, dissolve the residue in 20 mL of distilled water, transfer to a separatory funnel, add 20 mL of ethyl acetate for extraction, repeat 4 times, combine the extracts and concentrate to dryness under reduced pressure; dissolve the residue in 45% methanol and make up to 10 mL in a volumetric flask, filter through a 0.22 μm filter membrane, and collect the filtrate.

[0036] Further specifying, in step 1), the method for determining the content of alcohol-soluble extract is as follows:

[0037] Accurately weigh approximately 2g of chicken blood vine powder (passed through a No. 3 sieve), place it in an Erlenmeyer flask, add 45mL of 95% ethanol, seal tightly and weigh. After standing for 1 hour, reflux and gently boil for 1 hour. After cooling, weigh again, replenish the lost weight with 95% ethanol, and filter. Take 25mL of the filtrate, evaporate to dryness in a water bath, dry in a 105℃ drying oven for 3 hours, cool for 30 minutes, and accurately weigh to calculate the alcohol-soluble extract content.

[0038] Further specifying that in step 2), the data preprocessing adopts the Z-score standardization method, and the data analysis tools are: Pearson correlation analysis and multiple regression analysis using SPSS 26.0 software, and OPLS-DA using SIMCA 14.1 software.

[0039] Further specifying, in step 4), the criteria for quality evaluation are:

[0040] When the key appearance characteristics meet the following criteria, such as ΔE*≥35, maximum number of rings≥5, and diameter≥2.0cm, the slices are judged to be of high quality. Alternatively, the predicted value of the intrinsic quality calculated by the prediction model is compared with the preset threshold values ​​for the content of high-quality ingredients (catechin ≥0.8mg / g, epicatechin ≥0.6mg / g, alcohol-soluble extract ≥8.0%). If the values ​​meet the threshold requirements, the slices are judged to be of high quality.

[0041] The beneficial effects of this invention are

[0042] 1. This invention is the first to transform the traditional subjective descriptions of chicken blood vine, such as "reddish-brown cut surface with many layers" and "reddish-brown color is better", into measurable quantitative indicators such as "number of layers" and "ΔE*", thereby eliminating human identification errors and making the evaluation results objective and consistent.

[0043] 2. This invention clarifies the direct correlation between appearance and active ingredients through multivariate statistical analysis (e.g., for every additional ring, the epicatechin content increases by an average of 0.12 mg / g), verifying the scientific validity of the "superior shape, superior quality" theory in *Spatholobus suberectus*.

[0044] 3. This invention does not rely on large instruments. It can complete the preliminary quality judgment by measuring the number of circles, diameter and ΔE* with a simple colorimeter. The detection time is shortened from 40 minutes of traditional HPLC to within 5 minutes, which is suitable for on-site screening in grassroots pharmacies and medicinal material markets.

[0045] 4. The quality trait thresholds (number of rings ≥ 5, ΔE* ≥ 35, diameter ≥ 2.0cm) specified in this invention can provide a unified standard for the procurement, production and supervision of chicken blood vine slices, reducing the circulation of inferior slices in the market. Attached Figure Description

[0046] Figure 1 Different grades of chicken blood vine slices according to the present invention Figure 2 HPLC chromatograms of mixed reference standard (A) and test sample (B), 1. catechins, 2. epicatechins Figure 3 OPLS-DA score chart (A) and VIP value chart (B) for three grades of chicken blood vine slices. Figure 4 Correlation heatmap of various indicators of chicken blood vine slices Detailed Implementation

[0047] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0048] The medicinal materials, reagents, and instruments used in this embodiment are as follows.

[0049] 1. Chicken Blood Vine (a type of medicinal herb)

[0050] The dried stems of *Spatholobus suberectus* Dunn., a legume, were identified by Professor Ma Wenfang of Guangxi University of Traditional Chinese Medicine from medicinal herb markets in Nanning and Yulin, Guangxi.

[0051] 2. Reagents

[0052] Catechins (110877-202306) and epicatechin (112534-202208) were purchased from Chengdu Mansite Biotechnology Co., Ltd.; acetonitrile, methanol, anhydrous ethanol, and ethyl acetate were purchased from Thermo Fisher Scientific (China) Co., Ltd.; 85% phosphoric acid was purchased from Tianjin Damao Chemical Reagent Factory; ultrapure water was prepared using a Milli-Q ultrapure water system.

[0053] Table 1. Information on the source of samples from *Spatholobus suberectus* (chicken blood vine).

[0054]

[0055] 3. Instruments

[0056] Waters e2695 liquid chromatograph (Waters Corporation).

[0057] Ultraviolet detector 2998 (Waters Corporation).

[0058] Analytical balance CPA225D (Sartorius Instrument Systems GmbH, Germany, 0.01%).

[0059] Ultrasonic cleaner KQ5200DE (Kunshan Ultrasonic Instrument Co., Ltd.)

[0060] Rotary evaporator N-1300 (Shanghai Ailang Instrument Co., Ltd.)

[0061] Milli-Q ultrapure water system (Guangxi Nanning Bomei Biotechnology Co., Ltd.)

[0062] Electric heating drying oven DHG-9240A (Shanghai Qixin Scientific Instruments Co., Ltd.).

[0063] PS2010 Spectroradiometer (Guangdong Sanenshi Intelligent Technology Co., Ltd.)

[0064] Taking the *Chicken Blood Vine* medicinal materials collected in Table 1 above as an example, the quality evaluation method of *Chicken Blood Vine* will be explained in detail.

[0065] I. Obtaining 8 key data points for *Chicken Blood Vine*

[0066] 1. Determination of pheasant blood vine slices

[0067] According to the "Specifications and Grades of Chinese Medicinal Herbs" standard published by the China Association of Traditional Chinese Medicine, *Spatholobus suberectus* slices are classified based on surface color, diameter, and number of rings. Representative types of *Spatholobus suberectus* slices are listed below. Figure 1 The results of the appearance characteristics analysis of 50 batches of *Spatholobus suberectus* (chicken blood vine) slices showed that the diameter of large slices (numbered S1-S17) ranged from 11 to 22 cm, with 10-18 coils; the diameter of medium slices (numbered S18-S32) ranged from 4 to 10.2 cm, with 4-10 coils; and the diameter of small slices (numbered S33-S50) ranged from 2 to 8.2 cm, with 1-3 coils. Specific results are shown in Table 2.

[0068] Table 2 Results of determination of the properties of chicken blood vine slices

[0069]

[0070] 2. Color determination of chicken blood vine powder

[0071] 2.1 Measurement conditions

[0072] Selectable light source: D65; Measuring aperture: 8mm; Light source: LED blue light excitation; Instrument error: ΔE*≤0.4. After calibrating the instrument with a black and white plate, sample measurements are performed. A white A4 sheet of paper is used as a standard sample for calibration (L*=96.49, a*=-0.21, b*=-8.09), and six parallel measurements are performed to calculate the average color value. The color values ​​L* (brightness), a* (red-green), and b* (yellow-blue) of the sample to be tested are compared with the colorimetric values ​​of the standard sample, and the differences are the colorimetric values ​​(ΔL*, Δa*, Δb*) of the sample. A certain amount of sample powder is taken and evenly spread in a petri dish, ensuring that there are no gaps between the powder particles and the surface is flat. Six locations are randomly selected on the surface of each sample for measurement, and three copies of each sample are prepared. The values ​​of ΔL*, Δa*, and Δb* are measured using a colorimeter, and the average value of each sample is calculated.

[0073] 2.2 Precision Experiment

[0074] Take an appropriate amount of S15 powder as a test sample and place it in a petri dish to cover the entire sample. Measure the powder 6 times according to the conditions in section "2.2.1" to obtain the color values ​​ΔL*, Δa*, and Δb*, respectively. The RSD values ​​are 0.07%, 0.12%, and 0.03%, respectively, indicating that the instrument has good precision.

[0075] 2.3 Stability Experiment

[0076] An appropriate amount of S15 powder sample was evenly spread in a petri dish. Following the powder color determination conditions in section "2.2.1", measurements were performed at fixed time intervals (14:00-15:00) on days 0, 1, 2, 3, 4, and 5. Each group was measured in parallel six times, and the mean value was used to calculate the RSD. Data analysis showed that the RSD values ​​of the three colorimetric parameters ΔL*, Δa*, and Δb* were 0.21%, 0.15%, and 0.31%, respectively, all below 3.0%, indicating good sample stability.

[0077] 2.4 Repeatability Experiment

[0078] Six parallel samples were prepared from S15 powder, each evenly spread in a petri dish. The powder color was measured simultaneously according to the powder color determination conditions described in section "2.2.1". Each sample was randomly measured six times. The average value and RSD of the three-color space parameters for the six samples were calculated. The results showed that the RSDs of the three-color space values ​​ΔL*, Δa*, and Δb* were 0.21%, 0.46%, and 0.70%, respectively, all less than 3.0%, indicating good color repeatability within the same batch of samples.

[0079] 2.5 Sample Color Measurement

[0080] Take an appropriate amount of chicken blood vine sample powder (passed through a No. 3 sieve), place it in a petri dish to cover it completely, and measure the color value of the sample according to the powder color determination conditions in section "2.2.1". Record the sample color indices ΔL*, Δa*, and Δb* respectively, and calculate the total color difference ΔE*=[(ΔL*)]. 2 +(Δa*) 2 +(Δb*) 2 ] 1 / 2 Three samples were selected from each batch and randomly measured six times. The average value was calculated and shown in Table 3.

[0081] Table 3. Color determination of Chicken Blood Vine Powder

[0082]

[0083] 3. Determination of effective components in chicken blood vine medicinal material

[0084] In this embodiment, high performance liquid chromatography was used to determine the content of active ingredients in chicken blood vine.

[0085] 3.1 Solvent Preparation

[0086] 3.1.1 Preparation of reference solvent

[0087] Accurately weigh epicatechin and catechin standards, dissolve them in 50% methanol solution, and prepare mixed reference solutions with concentrations of 1.15 mg / mL and 1.36 mg / mL, respectively. Filter the resulting solutions through a 0.22 μm pore size membrane and store them at 4°C for later use.

[0088] 3.1.2 Preparation of the test solution

[0089] Accurately weigh 1.0 g of *Spatholobus suberectus* powder, add 60 mL of methanol, let stand for 30 min, then extract ultrasonically for 50 min. Filter and collect the filtrate. Evaporate the filtrate to dryness under water bath conditions. Dissolve the residue in 20 mL of distilled water and transfer to a separatory funnel. Add 20 mL of ethyl acetate for extraction, repeating 4 times. Combine the ethyl acetate extracts and concentrate under reduced pressure until dry. Dissolve the residue in a small amount of 50% methanol, transfer to a 10 mL volumetric flask, and dilute to volume. Mix well and filter through a 0.22 μm microporous membrane to obtain the test solution.

[0090] 3.2 Column Conditions

[0091] X-Peonyx (250×4.6mm, 5μm) column; mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B); gradient elution: 0–15 min, 6%–9% A; 15–35 min, 9%–9.5% A; 35–45 min, 9.5%–13% A; 45–55 min, 13%–14% A; detection wavelength 280 nm, column temperature 30℃, flow rate 0.8 mL / min. Injection volume: 20 μL. The HPLC chromatograms of the mixed reference standard and the *Spatholobus suberectus* sample are shown below. Figure 2 .

[0092] 3.3 Examination of Linear Relationships

[0093] Accurately pipette 1, 5, 10, 20, 30, 40, and 50 μL of the mixed reference solution from section "2.3.2" and inject them into the high-performance liquid chromatograph (HPLC). Analyze under the chromatographic conditions described in section "2.3.1". Establish a standard curve with the injection volume (X) of the reference solution as the x-axis and the corresponding peak area (Y) as the y-axis. Linear regression analysis showed a good linear correlation between the peak area of ​​each target component and the injection volume. The specific regression equation and correlation coefficient are shown in Table 4.

[0094] Table 4 Linear Regression Equations

[0095]

[0096] 3.4 Precision Experiment

[0097] Accurately pipette the mixed reference solution and inject it six times consecutively under the chromatographic conditions specified in "2.3.1". The RSDs of the peak areas of catechin and epicatechin were 0.77% and 1.12%, respectively, indicating that the instrument has good precision.

[0098] 3.5 Stability Test

[0099] Accurately weigh 1.0 g of S15 sample powder and prepare the test solution according to method "2.3.1". Inject the test solution at room temperature at 0, 2, 4, 6, 8, 12 and 24 h. The RSDs of the peak areas of catechin and epicatechin were 0.45% and 1.33%, respectively, indicating that the test solution had good stability within 24 hours.

[0100] 3.6 Repeatability Experiment

[0101] Six 1.0g portions of S15 powder were accurately weighed and prepared into test solutions according to the method described in section "2.3.1". The solutions were then injected and analyzed. The results showed that the RSDs of the peak areas of catechin and epicatechin were 2.69% and 1.71%, respectively, indicating good repeatability. Six 1.0g portions of S15 sample were also accurately weighed and prepared in parallel according to the method described in "2.3.1", and then injected for analysis. The results showed that the RSDs of the peak areas of catechin and epicatechin were 2.69% and 1.71%, respectively, indicating good repeatability.

[0102] 3.7 Recovery Experiment

[0103] Six 1.0 g portions of S15 sample powder with known content were accurately weighed. Catechin and epicatechin reference standards were precisely added to each portion, and test solutions were prepared according to the method described in section "2.3.1". The recoveries were calculated. The results showed that the recovery rate of catechin was 99.35% with an RSD of 1.85%; the recovery rate of epicatechin was 101.40% with an RSD of 2.37%. Six 1.0 g portions of S15 sample with known content were accurately weighed. Catechin and epicatechin reference standards were quantitatively added to each portion, and test solutions were prepared according to the chromatographic conditions described in section "2.3.1". The results showed that the average recovery rate of catechin was 99.35% with an RSD of 1.85%, and the average recovery rate of epicatechin was 101.40% with an RSD of 2.37%. The recovery results met the requirements for method validation.

[0104] 3.8 Sample Content Determination

[0105] Accurately weigh 1.0 g of chicken blood vine powder, prepare the test solution according to the method in section "2.3.3", inject the sample for determination according to the chromatographic conditions in section "2.3.1", and calculate the content. Three samples are tested for each batch, and the average value is taken. See Table 5.

[0106] Table 5. Results of component content determination of *Chicken Blood Vine* slices (n=3, )

[0107]

[0108] 4. OPLS-DA Analysis

[0109] Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed using SIMCA14.1 on the relevant parameters (diameter, number of rings, ΔL*, Δa*, Δb*, ΔE*, catechins, and epicatechins) of the selected large, medium, and small slices of *Spatholobus suberectus* to examine the contribution of these parameters to the grading. The results are shown in [Figure number missing]. Figure 3 A. The results show that the samples at the three levels clustered into one class. VIP score chart ( Figure 3 B) The VIP values ​​of the four indicators, namely catechin, epicatechin, number of rings, and diameter, are greater than 1, indicating that these four indicators play an important role in differentiating different grades of chicken blood vine slices.

[0110] 5. Correlation analysis

[0111] Pearson correlation analysis was performed using SPSS 26.0 software to analyze the phenotypic characteristics (color, diameter, number of rings) and intrinsic quality indicators (catechins, epicatechins) of *Spatholobus suberectus*. The results are shown in Table 6. Figure 4 As shown in the figure. The analysis results showed that the color ΔL* value of chicken blood vine powder was significantly negatively correlated with the contents of catechin and epicatechin (P < 0.01); the color values ​​Δa*, ΔE*, diameter and number of rings were significantly positively correlated with catechin and epicatechin (P < 0.01); the Δb* value was not significantly correlated with the two components.

[0112] Table 6. Correlation analysis of the properties and content of *Spatholobus suberectus*

[0113] Note: *P < 0.05; **P < 0.01

[0114] 6. Regression Analysis

[0115] The contents of two quality indicators, catechin and epicatechin, flavonoids in *Spatholobus suberectus*, were used as independent variables, and the color values ​​ΔL*, Δa*, and Δb* were used as dependent variables. Regression analysis was performed using SPSS 26.0 software, and the results are shown in Tables 7-9.

[0116] Table 7 Summary of ΔL*, Δa*, Δb* models for *Spatholobus suberectus*

[0117]

[0118] Table 8. Analysis of Variance for Color Values

[0119]

[0120] Table 9 Regression Analysis of Color Values ​​and Component Content

[0121]

[0122] Table 10 Summary of Models for the Content of Effective Components in Chicken Blood Vine

[0123]

[0124] Table 11. Analysis of variance of the effective component content of *Spatholobus suberectus*

[0125]

[0126] Table 12 Regression analysis of the effective component content of *Spatholobus suberectus*

[0127]

[0128] Tables 8-12 show that the surface color of *Spatholobus suberectus* reflects the levels of catechins and epicatechins, which can help in quickly evaluating the quality of *Spatholobus suberectus* slices. When catechin and epicatechin contents are used as independent variables, the R² values ​​with respect to ΔL*, Δa*, and Δb* are 0.594, 0.593, and 0.058, respectively, indicating that the color value is affected by the effective components to a degree of 59.4%, 59.3%, and 5.8% (Table 7). Catechins and epicatechins are highly significantly correlated with ΔL* and Δa* values ​​(P<0.001), but not significantly correlated with Δb*. The regression equations are ΔL* = -31.7 + 0.738 × catechin - 1.722 × epicatechin, Δa* = 10.723 + 0.812 × catechin + 0.605 × epicatechin, and Δb* = 26.833 + 0.578 × catechin - 0.372 × epicatechin. The ΔL* and Δa* values ​​validated the significance of the regression equations; both p-values ​​were less than 0.05, indicating that the regression equations were statistically significant.

[0129] Tables 10-12 show that the regression equations between catechin and epicatechin content and color value are significant (P < 0.05). When catechin and epicatechin content are the dependent variables, the R² values ​​for catechin and epicatechin in *Spatholobus suberectus* with respect to ΔL*, Δa*, and Δb* are 0.505 and 0.721, respectively, indicating that the model can reflect the content of catechin and epicatechin in *Spatholobus suberectus* to a degree of 50.5% and 72.1% through phenotypic characteristics (color, diameter, number of rings), respectively. The regression equations between catechin and epicatechin content and color value are highly significant (P < 0.01). The regression equations are: catechin = 1.171 + 0.046 × ΔL* - 0.032 × Δa* + 0.112 × Δb*, epicatechin = -1.796 - 0.130 × ΔL* - 0.164 × Δa* + 0.164 × Δb*.

[0130] In summary, this study analyzed the correlation between the appearance and internal quality of *Spatholobus suberectus*, providing a scientific basis for the evaluation method of "identifying quality by appearance" and laying the foundation for the future development of rapid and non-destructive testing methods based on morphological characteristics. This is of great significance for the traditional identification and inheritance of experience in Chinese medicinal materials.

Claims

1. A quality evaluation method for *Spatholobus suberectus* slices based on "superior shape and high quality," characterized in that... Includes the following steps: 1) Obtain data on 8 indicators of chicken blood vine. To obtain the index data of chicken blood vine slices, we need to obtain the six appearance index data of chicken blood vine slices: powder color (ΔL*, Δa*, Δb*, ΔE*), slice diameter, and maximum number of slices. Two intrinsic quality indicators of chicken blood vine slices were obtained: the content of catechin and epicatechin determined by high performance liquid chromatography (HPLC), and the content of alcohol-soluble extract determined by referring to the pharmacopoeia method. 2) Principal component analysis A) Data Preprocessing and Modeling Based on the eight indicator data obtained in step 1), a "trait-quality" sample database of chicken blood vine slices was constructed, and the data in the database were standardized and preprocessed. Pearson correlation analysis was used to clarify the direction and strength of the association between appearance trait indicators and internal quality indicators. Partial least squares discriminant analysis (OPLS-DA) was used to screen trait indicators that significantly contribute to quality grading. Multiple regression analysis was used to construct a predictive model of appearance traits on internal quality. B) Screening of key trait indicators Based on the OPLS-DA results (variable importance projection value VIP>1) and the significance level of Pearson correlation (P<0.05), 2-3 key appearance traits that can characterize the quality of chicken blood vine slices were selected. 3) Quality Evaluation The predictive model constructed based on the key appearance traits screened in step 2) is used to evaluate the quality of chicken blood vine slices.

2. The method for evaluating the quality of *Spatholobus suberectus* slices based on "superior shape and high quality" according to claim 1, characterized in that, In step 1), the method for determining the appearance characteristics is as follows: A) Measurement of powder color parameters Take an appropriate amount of chicken blood vine powder, spread it evenly in a cuvette, and use a spectrophotometer (light source D65, aperture 8mm) to measure ΔL*, Δa*, and Δb*. Three parallel measurements were taken for each batch of samples, and six random measurements were taken for each sample. The average value was used to calculate the comprehensive color difference value ΔE*. B) Measurement of the diameter of medicinal slices The cross-sectional diameter of the chicken blood vine slices was measured using vernier calipers. Three slices were measured for each batch of samples, and the average value was taken. C) Determination of the maximum number of rings in the medicinal slices: Visually observe the cross-section of the chicken blood vine slices, count the maximum number of reddish-brown layers, and measure 3 slices from each batch of samples, taking the average value.

3. The method for evaluating the quality of *Spatholobus suberectus* slices based on "superior shape and high quality" according to claim 1, characterized in that... In step 1), the contents of catechin and epicatechin were determined by HPLC. Specific conditions were as follows: X-Peonyx column; mobile phase: acetonitrile (A) - 0.1% phosphoric acid aqueous solution (B); gradient elution program: 0–10 min, 5%–15% A; 10–20 min, 15%–25% A; 20–30 min, 25%–35% A; 30–40 min, 35%–5% A; detection wavelength: 280 nm; flow rate: 1.0 mL / min. -1 The injection volume was 10 μL, and the column temperature was 30℃.

4. The method for evaluating the quality of *Spatholobus suberectus* slices based on "superior shape and high quality" according to claim 3, characterized in that... The preparation methods for the test solution and reference solution in the HPLC method are as follows: A) Preparation of reference solution Accurately weigh catechin and epicatechin standards, dissolve them in 45% methanol solution to prepare mixed reference solutions with concentrations of 1.36 mg / mL and 1.15 mg / mL, respectively. After filtration through a 0.22 μm filter membrane, store at 4°C for later use. B) Preparation of test solution Accurately weigh 1.0 g of chicken blood vine powder (passed through a No. 3 sieve), place it in a stoppered conical flask, add 60 mL of methanol, let stand for 30 min, then extract by ultrasonication for 45 min, filter and collect the filtrate; after evaporating the filtrate to dryness in a water bath, dissolve the residue in 20 mL of distilled water, transfer it to a separatory funnel, add 20 mL of ethyl acetate for extraction, repeat 4 times, combine the extracts and concentrate under reduced pressure to dryness. The residue was dissolved in 45% methanol and diluted to a volumetric flask of 10 mL. The solution was then filtered through a 0.22 μm filter membrane, and the filtrate was collected.

5. The method for evaluating the quality of *Spatholobus suberectus* slices based on "superior shape and high quality" according to claim 1, characterized in that... In step 1), the method for determining the content of alcohol-soluble extract is as follows: Accurately weigh approximately 2g of chicken blood vine powder (passed through a No. 3 sieve), place it in an Erlenmeyer flask, add 45mL of 95% ethanol, seal tightly and weigh. After standing for 1 hour, reflux and gently boil for 1 hour. After cooling, weigh again, replenish the lost weight with 95% ethanol, and filter. Take 25mL of the filtrate, evaporate to dryness in a water bath, dry in a 105℃ drying oven for 3 hours, cool for 30 minutes, and accurately weigh to calculate the alcohol-soluble extract content.

6. The method for evaluating the quality of *Spatholobus suberectus* slices based on "superior shape and high quality" according to claim 1, characterized in that, In step 2), the data preprocessing adopts the Z-score standardization method, and the data analysis tools are: Pearson correlation analysis and multiple regression analysis are performed by SPSS 26.0 software, and OPLS-DA is performed by SIMCA 14.1 software.

7. The method for evaluating the quality of *Spatholobus suberectus* slices based on "superior shape and high quality" according to claim 1, characterized in that... In step 3), the criteria for quality evaluation are as follows: When the key appearance characteristics meet the following criteria, such as ΔE*≥35, maximum number of rings≥5, and diameter≥5.0cm, the slices are judged to be of high quality. Alternatively, the predicted value of the intrinsic quality calculated by the prediction model is compared with the preset threshold values ​​for the content of high-quality ingredients (catechin ≥0.8mg / g, epicatechin ≥0.6mg / g). If the values ​​meet the threshold requirements, the slices are judged to be of high quality.

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