Method for rapidly evaluating quality of andrographis paniculata medicinal material

Abstract

The invention discloses a method for rapidly evaluating the quality of an andrographis paniculata medicinal material, which adopts a near infrared spectrum technology, can be used for determination after a sample is crushed, does not need complex pretreatment operation on the sample, is simple, convenient and rapid, only needs several minutes for detection, does not need chemical reagents for dissolution, and can be used for rapidly evaluating the quality of the andrographis paniculata medicinal material. The contents of chlorogenic acid, andrographolide and dehydroandrographolide can be measured at the same time, and the content of limited components in the andrographis paniculata medicinal material is comprehensively evaluated.

Description

Technical Field

[0001] This invention relates to a method for rapidly evaluating the quality of Andrographis paniculata medicinal materials. Background Technology

[0002] Andrographis paniculata (Burm.F.) Nees is a tropical herb widely used medicinally in China, India, Thailand, and other countries and regions. Clinically, it possesses various pharmacological effects, including antidiarrheal, anti-inflammatory, and antiviral properties. The 2025 edition of the Chinese Pharmacopoeia primarily focuses on the content detection of andrographolide and dehydrated andrographolide in Andrographis paniculata. Currently, the quality evaluation method for Andrographis paniculata mainly relies on HPLC content determination of andrographolide compounds. This method is time-consuming; including pretreatment and detection time, the testing time for one sample is at least 4 hours. Furthermore, the detection process requires the use of large amounts of chemical reagents such as acetonitrile and methanol, which is environmentally unfriendly. Moreover, the current quality evaluation standards for Andrographis paniculata mainly focus on detecting the content of diterpenoid lactones, neglecting chlorogenic acid, another active ingredient. Studies have shown that chlorogenic acid in Andrographis paniculata is also an effective antioxidant and anti-inflammatory active ingredient. During the harvesting and initial processing of Andrographis paniculata, the key active components, andrographolide and dehydrated andrographolide, are significantly affected by environmental factors such as temperature and humidity, requiring rapid drying. Chlorogenic acid, an ester compound synthesized from caffeic acid and quinic acid, contains three unstable chemical groups in its molecular structure: an ester group, an unsaturated carbon-carbon double bond, and a polyphenolic hydroxyl group. This makes it highly sensitive to changes in light and temperature. Furthermore, in establishing an HPLC method for determining chlorogenic acid, we found that using reagents with high water content during pretreatment easily causes fluctuations in chlorogenic acid content, affecting the HPLC results. Therefore, rapid detection of moisture, andrographolide, dehydrated andrographolide, and chlorogenic acid in Andrographis paniculata during initial processing is crucial. Summary of the Invention

[0003] The purpose of this invention is to provide a method for rapidly evaluating the quality of Andrographis paniculata medicinal materials.

[0004] This invention is achieved through the following technical solutions:

[0005] A method for rapidly evaluating the quality of Andrographis paniculata medicinal materials includes the following steps:

[0006] 1) Collect calibration set samples and validation set samples from Andrographis paniculata medicinal material samples;

[0007] 2) Determine the content of andrographolide, dehydrated andrographolide, chlorogenic acid and moisture;

[0008] 3) Scan the Andrographis paniculata powder sample with a near-infrared spectrometer to obtain near-infrared spectral data;

[0009] 4) Import the near-infrared spectral data of the sample obtained in step 3) into the Brucker infrared spectrometer OPUS 7.2 software for spectral analysis. Vector normalization (SNV), minimum-maximum normalization (MMN), multivariate scattering correction (MSC), first derivative (FD), second derivative (SD), first derivative + vector normalization (FD + SNV), and first derivative + multivariate scattering correction (FD + MSC) are used, combined with partial least squares (PLS) to establish a near-infrared spectral model. A correction model is constructed to establish the relationship between the andrographolide, dehydrated andrographolide, chlorogenic acid, and moisture content of the *Andrographis paniculata* powder sample and the characteristic near-infrared spectral data. Specifically, scattering correction preprocessing methods are used for chlorogenic acid and moisture, and the spectral band range of 9403.6–5446.2 cm⁻¹ is iteratively optimized. -1 4424.1~4246.7 cm -1 and 6101.9~4597.6 cm -1 Andrographolide and dehydrated andrographolide were pretreated using a normalization-based method, and the spectral range of 9403.6–5446.2 cm⁻¹ was iteratively optimized. -1 and 4601.5~4246.7 cm -1 Based on the preprocessed characteristic spectral data, partial least squares method is used to independently establish the corresponding optimal model for each component; the iterative optimization of the spectral band range refers to intelligently and randomly selecting and combining multiple wavelength intervals to form a spectrum, and finally screening the optimal characteristic band interval for modeling.

[0010] 5) Collect near-infrared spectral data of the Andrographis paniculata powder sample to be tested according to the same spectral acquisition parameters as the calibration set sample. Based on the near-infrared spectral calibration model constructed in step 4), obtain the content of andrographolide, dehydrated andrographolide, chlorogenic acid and moisture in the Andrographis paniculata medicinal material to be tested.

[0011] Preferably, the chromatographic conditions for determining the contents of andrographolide, dehydrated andrographolide, and chlorogenic acid are as follows: the chromatographic column is an Agilent Eclipse Plus C18 (250 mm × 4.6 mm, 5 μm); gradient elution is performed using acetonitrile as mobile phase A and 0.2% formic acid as mobile phase B; the flow rate is 0.8 mL / min; the column temperature is 25℃; full wavelength scanning is performed, the detection wavelength is 250 nm, and the injection volume is 5 μL.

[0012] The beneficial effects of this invention are as follows: This invention uses near-infrared spectroscopy technology, and the sample can be measured immediately after pulverization. There is no need for complicated pretreatment of the sample, which is simple and fast. The detection time is only a few minutes. No chemical reagents are required for dissolution. Moreover, the contents of chlorogenic acid, andrographolide, and dehydrated andrographolide can be measured simultaneously, so as to comprehensively evaluate the contents of limited components in the medicinal material Andrographis paniculata. Attached Figure Description

[0013] Figure 1 This is the near-infrared spectrum of a mixed sample of Andrographis paniculata.

[0014] Figure 2 These are correlation coefficient graphs between the predicted and actual values ​​of each component; where (A): Correlation coefficient graph between predicted and actual values ​​of chlorogenic acid. (B): Correlation coefficient graph between predicted and actual values ​​of andrographolide. (C): Correlation coefficient graph between predicted and actual values ​​of dehydrated andrographolide. (D): Correlation coefficient graph between predicted and actual values ​​of moisture content.

[0015] Figure 3 This is an external validation of near-infrared calibration models for the content of three chemical components in Andrographis paniculata and their relationship to moisture content; specifically: (A): External validation of the near-infrared calibration model for chlorogenic acid content in Andrographis paniculata; (B): External validation of the near-infrared calibration model for andrographolide content in Andrographis paniculata; (C): External validation of the near-infrared calibration model for dehydrated andrographolide content in Andrographis paniculata; and (D): External validation of the near-infrared calibration model for moisture content in Andrographis paniculata. Detailed Implementation

[0016] The following is a further description of the invention, but not a limitation thereof.

[0017] Experimental materials:

[0018] Instruments and reagents:

[0019] Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, USA). Agilent Eclipse Plus C18 column (250 mm × 4.6 mm, 5 µm). Bruker MPA near-infrared spectrometer (BRUKER GmbH, Germany). JA3003 electronic analytical balance (Shanghai Balance Instrument Factory, China), SB25-12DTD ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd., China).

[0020] Andrographolide (batch number: 110797-202010), dehydrated andrographolide (batch number: 110854-202412), and chlorogenic acid (batch number: 110753-202119) were all purchased from the National Institutes for Food and Drug Control, China. Acetonitrile (chromatographic grade, Tedia, USA), formic acid (analytical grade, Guangzhou Chemical Reagent Factory, China), and distilled water were used in the experiments. All other reagents were of analytical grade.

[0021] Samples: 81 batches of Andrographis paniculata samples were collected in October 2024 from production areas such as Guangxi and Guangdong. They were identified by Dr. Zhang Huiye of Guangzhou Baiyunshan Hutchison Whampoa Chinese Medicine Co., Ltd. as the dried whole herb of Andrographis paniculata (Burm.F.Nees).

[0022] Example 1:

[0023] 1. 57 out of 81 batches of samples were set as calibration set samples for near-infrared spectroscopy detection, and 24 batches were set as validation set samples.

[0024] II. Determination of Flavonoids, Chlorogenic Acid, and Moisture Content in Andrographis paniculata

[0025] Preparation of mixed reference solution:

[0026] Take appropriate amounts of andrographolide reference standard, dehydrated andrographolide reference standard and chlorogenic acid reference standard respectively, weigh them accurately, put them in a 10 mL volumetric flask, add an appropriate amount of methanol to prepare a mixed reference standard solution with a final concentration of 0.269 mg / mL chlorogenic acid, 0.299 mg / mL andrographolide and 0.238 mg / mL dehydrated andrographolide.

[0027] Preparation of the test solution: Take about 1.0 g of Andrographis paniculata powder (passed through a No. 4 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 75wt% methanol, stopper tightly, weigh it, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 75wt% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0028] Chromatographic conditions: The column was an Agilent Eclipse Plus C18 (250 mm × 4.6 mm, 5 μm); acetonitrile was used as mobile phase A and 0.2% formic acid was used as mobile phase B, with gradient elution according to the gradient in Table 1; the flow rate was 0.8 mL / min; the column temperature was 25℃; full wavelength scanning was performed, the detection wavelength was 250 nm, and the injection volume was 5 μL.

[0029] Table 1

[0030] The contents of andrographolide, dehydrated andrographolide, and chlorogenic acid in Andrographis paniculata were determined by an external two-point method. The average contents of the three components in 57 batches of calibration set samples were 1.7795 mg / g, 15.2984 mg / g, and 4.5772 mg / g, respectively; the average contents of the three components in 24 batches of validation set samples were 1.9063 mg / g, 13.2370 mg / g, and 4.4315 mg / g, respectively.

[0031] The moisture content of each batch of Andrographis paniculata samples was determined using the following method: Approximately 5g of Andrographis paniculata powder was weighed and the moisture content of each sample was determined according to Method II (drying method) of "0832 Moisture Determination" in Part IV of the 2020 edition of the Chinese Pharmacopoeia. The average moisture content of the 57 calibration set samples was 8.0518%, and the average moisture content of the 24 validation set samples was 7.9968%.

[0032] Table 2. Chemical composition and moisture content of Andrographis paniculata calibration and validation set samples (n) 校正集 =57,n 验证集 =24)

[0033] III. Establishment of near-infrared calibration models for the content of andrographolide, dehydrated andrographolide, and chlorogenic acid in Andrographis paniculata

[0034] .1 Near-infrared spectral information acquisition

[0035] Near-infrared spectral information of *Andrographis paniculata* powder samples (passed through a No. 3 sieve) was acquired using a Bruker MPA near-infrared spectrometer. The measurement method was an integrating sphere diffuse reflectance system; resolution: 8 cm⁻¹. -1 64 scans; scanning wavelength range 3600~12500 cm⁻¹ -1 Relative humidity 25%~30%; room temperature (25±0.5)℃. Each sample underwent two spectral scans. Near-infrared spectra of 81 batches of samples are shown below. Figure 1 .

[0036] 2. Preprocessing and Modeling of Near-Infrared Spectral Information

[0037] The spectral information acquired by the near-infrared spectrometer is imported into the Brucker infrared spectrometer OPUS 7.2 software for analysis. Preprocessing methods include vector normalization (SNV), minimum-maximum normalization (MMN), multivariate scattering correction (MSC), first derivative (FD), second derivative (SD), first derivative + vector normalization (FD + SNV), and first derivative + multivariate scattering correction (FD + MSC). A near-infrared spectral model is then established using partial least squares (PLS).

[0038] The spectra and component contents of the Andrographis paniculata calibration set samples were imported into the Brucker infrared spectrometer OPUS 7.2 software for preprocessing and optimization. The seven spectral preprocessing methods were compared by using the obtained root mean square error of calibration set (RMSEC), calibration set correlation coefficient (Rc), root mean square error of cross-validation set (RMSECV), and cross-validation set correlation coefficient (Rv). The smaller the RMSECV and the closer the Rv is to 100%, the better the prediction effect of the model.

[0039] The near-infrared spectral preprocessing results for chlorogenic acid are shown in Table 3. The optimal spectral preprocessing method for the chlorogenic acid near-infrared correction model is multivariate scattering correction. The correction model established by this method has 7 principal components and a spectral range of 9403.6–5446.2 cm⁻¹ and 4424.1–4246.7 cm⁻¹. -1 The Rv was 98.8% and the RMSECV was 0.0882, indicating that the model has good predictive performance.

[0040] The near-infrared spectral preprocessing results for andrographolide are shown in Table 4. The optimal spectral preprocessing method for the andrographolide near-infrared calibration model is vector normalization. The calibration model established by this method has 9 principal components and a spectral range of 9403.6–5446.2 cm⁻¹ and 4601.5–4246.7 cm⁻¹. -1 The Rv is 99.29% and the RMSECV is 0.642, indicating that the model has good predictive performance.

[0041] The near-infrared spectral preprocessing results for dehydrated andrographolide are shown in Table 5. The optimal spectral preprocessing method for the near-infrared calibration model of dehydrated andrographolide is vector normalization. The calibration model established by this method has 10 principal components and a spectral range of 9403.6–5446.2 cm⁻¹ and 4601.5–4246.7 cm⁻¹. -1 The Rv was 97.27% and the RMSECV was 0.29, indicating that the model has good predictive performance.

[0042] The near-infrared spectral preprocessing results for moisture are shown in Table 6. The optimal spectral preprocessing method for the moisture near-infrared correction model is multivariate scattering correction. The correction model established by this method has 6 principal components and a spectral range of 6101.9–4597.6 cm⁻¹. -1 The Rv was 92.31% and the RMSECV was 0.19, indicating that the model has good predictive performance.

[0043] Table 3. Effects of different near-infrared spectral processing methods on chlorogenic acid correction model parameters

[0044] Table 4. Effects of different near-infrared spectral processing methods on the parameters of the andrographolide correction model.

[0045] Table 5. Effects of different near-infrared spectral processing methods on the parameters of the dehydrated andrographolide correction model.

[0046] Table 6. Effects of different near-infrared spectral processing methods on moisture correction model parameters

[0047] Simultaneously, the optimal model was determined using the main parameters of the NIR mathematical model, namely Rv and RMSECV. The models show that different preprocessing methods result in different spectral ranges across different wavelengths, but all exhibit good linear relationships. The correlation coefficients (Rv) for chlorogenic acid, andrographolide, dehydrated andrographolide, and water are all greater than 92%. Details of the optimal models for each component can be found in [link to relevant documentation]. Figure 2 .

[0048] IV. Validation and Evaluation of Near-Infrared Correction Models for Andrographolide, Dehydrated Andrographolide, Chlorogenic Acid, and Moisture Content in Andrographis paniculata

[0049] To verify the accuracy of the near-infrared calibration model, the near-infrared spectra of 24 samples from the Andrographis paniculata validation set were imported into the software, and the near-infrared calibration model established by the calibration set was used for prediction. The predicted values ​​were compared with the actual values ​​using correlation coefficient. The closer the correlation coefficient is to 1, the closer the predicted value is to the measured value.

[0050] In the validation set of 24 (n=2) Andrographis paniculata samples, the correlations between the predicted and measured values ​​of each component were as follows: chlorogenic acid R=0.9601, andrographolide R=0.9867, and dehydrated andrographolide R=0.9716. The correlations between the predicted and measured values ​​of each component were all greater than 0.96, indicating significant correlations. The correlation between the predicted and measured values ​​of moisture content in the same 24 (n=2) Andrographis paniculata samples was R=0.9331, which was greater than 0.93, also indicating significant correlations. These results demonstrate that the model established by this method has high prediction accuracy. The external validation results of the near-infrared prediction and correction model for the content of three chemical components and moisture content in Andrographis paniculata are shown below. Figure 3 As shown (i.e., the correlation results between predicted and measured values).

[0051] Table 7 Comparison of NIR predicted values ​​and HPLC measured values ​​of chlorogenic acid, andrographolide, and dehydrated andrographolide / mg*g -1

[0052] Table 8 Comparison of NIR predicted and measured values ​​of moisture / %

[0053] The predicted contents of chlorogenic acid, andrographolide, dehydrated andrographolide, and water in Andrographis paniculata are shown in Tables 7 and 8. The results show that the deviations between the actual and predicted values ​​are good, and the correlation model between the contents of the three chemical components in Andrographis paniculata and the water content established in this study has good predictive performance.

Claims

1. A method for rapidly evaluating the quality of Andrographis paniculata medicinal material, characterized in that, Includes the following steps: 1) Collect calibration set samples and validation set samples from Andrographis paniculata medicinal material samples; 2) Determine the content of andrographolide, dehydrated andrographolide, chlorogenic acid and moisture; 3) Scan the Andrographis paniculata powder sample with a near-infrared spectrometer to obtain near-infrared spectral data; 4) Import the near-infrared spectral data of the sample obtained in step 3) into the Brucker infrared spectrometer OPUS 7.2 software, analyze the spectral information, and construct a correction model for the relationship between the andrographolide, dehydrated andrographolide, chlorogenic acid, and moisture content of the Andrographis paniculata powder sample and the characteristic near-infrared spectral data using vector normalization, minimum-maximum normalization, multivariate scattering correction, first derivative, second derivative, first derivative + vector normalization, and first derivative + multivariate scattering correction. Specifically, scattering correction preprocessing methods were used for chlorogenic acid and moisture, and the spectral band range of 9403.6~5446.2 cm⁻¹ was iteratively optimized. -1 4424.1~4246.7 cm -1 and 6101.9~4597.6 cm -1 Andrographolide and dehydrated andrographolide were pretreated using a normalization-based method, and the spectral range of 9403.6–5446.2 cm⁻¹ was iteratively optimized. -1 and 4601.5~4246.7 cm -1 Based on the preprocessed characteristic spectral data, partial least squares method is used to independently establish the corresponding optimal model for each component; the iterative optimization of the spectral band range refers to intelligently and randomly selecting and combining multiple wavelength intervals to form a spectrum, and finally screening the optimal characteristic band interval for modeling. 5) Collect near-infrared spectral data of the Andrographis paniculata powder sample to be tested according to the same spectral acquisition parameters as the calibration set sample. Based on the near-infrared spectral calibration model constructed in step 4), obtain the content of andrographolide, dehydrated andrographolide, chlorogenic acid and moisture in the Andrographis paniculata medicinal material to be tested.

2. The method according to claim 1, characterized in that, The chromatographic conditions for determining the contents of andrographolide, dehydrated andrographolide, and chlorogenic acid were as follows: an Agilent Eclipse Plus C18 column; gradient elution with acetonitrile as mobile phase A and 0.2% formic acid as mobile phase B; flow rate of 0.8 mL / min; column temperature of 25℃; full wavelength scan with a detection wavelength of 250 nm and an injection volume of 5 μL.

3. The method according to claim 1, characterized in that, The preparation method of the mixed reference solution is as follows: Take appropriate amounts of andrographolide reference standard, dehydrated andrographolide reference standard and chlorogenic acid reference standard respectively, accurately weigh them, put them in a 10 mL volumetric flask, add an appropriate amount of methanol to prepare a mixed reference solution with a final concentration of 0.269 mg / mL chlorogenic acid, 0.299 mg / mL andrographolide and 0.238 mg / mL dehydrated andrographolide.

4. The method according to claim 1, characterized in that, The preparation method of the test solution is as follows: Take 1.0 g of Andrographis paniculata powder that has passed through a No. 4 sieve, weigh it accurately, place it in a stoppered conical flask, add 50 mL of 75wt% methanol accurately, stopper tightly, weigh it, sonicate for 30 minutes, cool it, weigh it again, make up the weight loss with 75wt% methanol, shake well, filter it, and take the filtrate.

5. The method according to claim 1, characterized in that, The number of samples in the calibration set is 57.

Images