Papaya pill adulteration identification method
By detecting the content ratio of benzoyl aconitine, benzoyl aconitine and benzoyl hypoconitine in papaya pills, an adulteration identification method was established using HPLC. This solved the problem of identifying genuine and counterfeit processed aconite and processed Sichuan aconite in papaya pills, and enabled accurate identification and quality control of counterfeit products.
Patent Information
- Application Number
- CN202511800446.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-02
- Publication Date
- 2026-03-03
AI Technical Summary
Existing quality standards make it difficult to accurately distinguish between genuine and counterfeit processed aconite and processed Sichuan aconite in papaya pills, leading to frequent cases of counterfeit products being used or under-used.
By detecting the content ratios of benzoylneoprothiolane, benzoylaconitine, and benzoylhypoconitine in samples of papaya pills, processed aconite, and processed Sichuan aconite, an adulteration identification method was established. The method was analyzed using HPLC, the ratios were calculated, and Cartesian product combinations were performed to determine the adulteration status.
This method enables the identification of genuine and counterfeit processed aconite and processed Sichuan aconite in papaya pills, effectively identifying the use of counterfeit ingredients and improving the quality control level of traditional Chinese medicine.
Smart Images
Figure CN121595745A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug testing and quality evaluation, and relates to a method for identifying adulteration in papaya pills. Background Technology
[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.
[0003] Plants of the genus *Aconitum* in the family Ranunculaceae are a very important group of medicinal plants, comprising approximately 350 species, of which about 167 are found in my country. Most are distributed in the high-altitude areas of western Sichuan, northern Yunnan, and eastern Tibet, with some also found in Northeast China and Inner Mongolia. Currently, about 40 species of *Aconitum* are commonly used medicinally, with the roots being the most frequently used part, making accurate differentiation based on their characteristics difficult. The 2020 edition of the *Chinese Pharmacopoeia* includes six medicinal varieties: *Aconitum carmichaelii* (dried tuberous root of *Aconitum carmichaelii*), *Aconitum carmichaelii* leaf (dried leaf of *Aconitum carmichaelii*), processed *Aconitum carmichaelii* (processed product of *Aconitum carmichaelii*), *Aconitum kusnezoffii* (dried mother root of *Aconitum carmichaelii*), *Aconitum kusnezoffii* (dried daughter root of *Aconitum carmichaelii*), and processed *Aconitum kusnezoffii* (processed product of *Aconitum kusnezoffii*).
[0004] Processed aconite is a processed product of aconite. Aconite is listed in the 2025 edition of the Chinese Pharmacopoeia, and its source is *Aconitum carmichaelii*, a plant in the Ranunculaceae family. Aconitum kusnezoffii The dried tuberous root of *Reichb.*. The main adulterant in processed *Aconitum carmichaelii* is *Aconitum japonicum*, which is similar in appearance to *Aconitum carmichaelii* and easily confused with it. In Xinjiang, there are instances of *Aconitum japonicum* tubers being harvested and sold as *Aconitum carmichaelii*. Because *Aconitum japonicum* is also a traditional medicinal material of the Kazakh and Hui ethnic groups, there have been clinical cases of *Aconitum japonicum* tubers being mistaken for *Aconitum carmichaelii*.
[0005] Processed Aconitum carmichaelii is a processed product of Aconitum carmichaelii; Aconitum carmichaelii is a plant of the Ranunculaceae family. Aconitum carmichaelii The dried mother root of *Aconitum carmichaelii*. The daughter roots of *Aconitum carmichaelii*, commonly known as "mud aconite," are generally processed into salted aconite, black aconite slices, and white aconite slices, depending on the size and uniformity of the root. Some smaller mud aconite roots, after processing, are often sold as processed *Aconitum carmichaelii*. Although processed *Aconitum carmichaelii* and *Aconitum carmichaelii* are different parts of the same plant, according to traditional Chinese medicine theory, they differ greatly in their medicinal properties and effects. *Aconitum carmichaelii* has the effects of restoring yang and rescuing from collapse, tonifying fire and assisting yang, dispelling cold and relieving pain; *Aconitum carmichaelii* has the effects of dispelling wind and dampness, warming the meridians and relieving pain. Therefore, the two should not be used interchangeably.
[0006] Prepared Aconitum kusnezoffii and Prepared Aconitum carmichaeli are the main medicinal flavors in the preparation of Mugua Pills, which have a strong effect of warming the channels and dispelling cold, can effectively expel wind-cold-damp pathogens, and relieve symptoms such as cold pain in joints and difficulty in flexion and extension. When combined with Chinese flowering quince, Clematis chinensis, etc., it enhances the effect of removing dampness and dredging collaterals, forming a complete action chain of "dispelling wind - dispersing cold - removing dampness". The current quality standard only controls the limit of toxic components of the two medicinal flavors, lacking the authenticity identification of the two medicinal flavors and the content control of active ingredients, resulting in the situation of using counterfeit products for feeding and less feeding. Therefore, it is urgent to develop and establish a method for evaluating the quality of Prepared Aconitum kusnezoffii and Prepared Aconitum carmichaeli in Mugua Pills. Summary of the Invention
[0007] In view of the problems of Mugua Pills and the like, the present invention provides a method for evaluating the quality of Mugua Pills. The quality of raw materials in Mugua Pills is determined by the content ratio of benzoyl neoline, benzoyl aconitine and benzoyl hypaconitine.
[0008] To achieve the above object, the present invention adopts the following technical solutions.
[0009] A method for identifying adulteration in Mugua Pills includes the following steps: (1) Detect the contents of benzoyl neoline and benzoyl hypaconitine in multiple samples of Prepared Aconitum carmichaeli and counterfeit Prepared Aconitum carmichaeli by HPLC method, and calculate the ratio of benzoyl neoline and benzoyl hypaconitine; (2) Detect the contents of benzoyl neoline and benzoyl aconitine in multiple samples of Prepared Aconitum kusnezoffii and counterfeit Prepared Aconitum kusnezoffii by HPLC method, and calculate the ratio of benzoyl neoline and benzoyl aconitine; (3) Combine the ratio data of the samples of Prepared Aconitum carmichaeli and counterfeits in step (1) and the samples of Prepared Aconitum kusnezoffii and counterfeits in step (2) in a Cartesian product combination according to a ratio of 1:1, and respectively obtain the lower limit value a of the ratio of benzoyl neoline and benzoyl hypaconitine for the combination containing the samples of Prepared Aconitum carmichaeli, and the lower limit value b of the ratio of benzoyl neoline and benzoyl aconitine for the combination containing the samples of Prepared Aconitum kusnezoffii; (4) Detect the contents of benzoyl neoline, benzoyl hypaconitine and benzoyl aconitine in Mugua Pills by HPLC method and calculate the ratio a1 of benzoyl neoline and benzoyl hypaconitine and the ratio b1 of benzoyl neoline and benzoyl aconitine; (5) Compare a1 with a. When a1 < a, it indicates that there is adulteration in the Prepared Aconitum carmichaeli in Mugua Pills; Compare b1 with b. When b1 < b, it indicates that there is adulteration in the Prepared Aconitum kusnezoffii in Mugua Pills; The adulteration of Prepared Aconitum carmichaeli is mud附子 or / and small附子, and the adulteration in Prepared Aconitum kusnezoffii is Aconitum soongaricum Stapf.
[0010] The HPLC methods in steps (1), (2), and (4) are the chromatographic conditions under the section on processed Aconitum carmichaelii in the 2020 edition of the Chinese Pharmacopoeia.
[0011] In step (4), the HPLC method also includes pretreatment before detection: For concentrated papaya pills: remove the coating, grind finely and pass through a No. 3 sieve, take 2.0g, accurately weigh, place in a stoppered conical flask, accurately add 25mL of a 1:100 v / v hydrochloric acid-methanol mixed solution, sonicate, filter, accurately measure 10mL of the filtrate, recover the solvent under reduced pressure below 40℃ until dry, add 10mL of 0.1mol / L hydrochloric acid solution to dissolve the residue, centrifuge, and take the supernatant; For the large honey pills of Papaya Pills: Crush and mix them, take 8.0g, accurately weigh them, place them in a stoppered conical flask, accurately add 50mL of a 1:100 v / v hydrochloric acid-methanol mixture, sonicate, filter, accurately measure 20mL of the filtrate, and recover the solvent under reduced pressure below 40℃ until dry. Add 10mL of 0.1mol / L hydrochloric acid solution to the residue to dissolve it, centrifuge for 20 minutes, and take the supernatant. The supernatant was then added to a solid-phase extraction column packed with a mixed cation exchange reversed-phase adsorbent. The column was eluted sequentially with 3 mL of water, 5 mL of ammonia solution, 5 mL of water, 5 mL of methanol, and 5 mL of acetonitrile. After the eluent was exhausted, the column was allowed to stand for 5 minutes. Then, it was eluted with 10 mL of a 90:10 v / v acetonitrile-concentrated ammonia solution mixture. The eluent was collected and the solvent was recovered under reduced pressure below 40°C until dry. The eluent was dissolved in an appropriate amount of mobile phase, transferred to a 2 mL volumetric flask, and diluted to the mark. The mixture was shaken well, and the filtrate was used as the HPLC sample. The ammonia solution is prepared by adding water to 100 mL of concentrated ammonia solution.
[0012] The present invention has the following advantages: The method provided by this invention can first identify adulteration of processed aconite and processed kusnezoffii as single herbs. Through multi-batch sample testing and modeling analysis, it can further identify adulteration of traditional Chinese medicine preparations containing processed aconite and processed kusnezoffii. Existing identification methods mainly rely on morphological characteristics to distinguish genuine and counterfeit processed aconite and processed kusnezoffii. When counterfeit products are added to traditional Chinese medicine preparations, traditional morphological identification becomes difficult to identify. Due to the similarity in chemical composition between processed aconite and processed kusnezoffii and counterfeit products, measuring a single chemical component cannot determine whether counterfeit products have been added. This invention, through extensive experimental accumulation, analyzes the regular differences in the ratios of benzoylneoprothiolane, benzoylneoprothiolane, and benzoylhypoaconitine in processed aconite, processed kusnezoffii, and related counterfeit products. Through modeling analysis, characteristic ratios that can be used to identify counterfeit products have been found, thereby determining whether counterfeit products have been added to traditional Chinese medicine preparations, solving the problem of identifying genuine and counterfeit processed aconite and processed kusnezoffii in papaya pill preparations. Attached Figure Description
[0013] Figure 1 The image shows the HPLC chromatogram of the mixed aconitine reference standard; the peaks after 15 min correspond to benzoylneoaconitine, benzoylneoaconitine, benzoylhypoaconitine, neoaconitine, hypoaconitine, and aconitine, respectively. Figure 2 The HPLC chromatogram of the prepared Aconitum carmichaelii sample (prepared Aconitum carmichaelii CW-1); Figure 3 The HPLC chromatogram of a sample of adulterated Aconitum carmichaelii (processed Aconitum carmichaelii 19); Figure 4 A scatter plot of the ratio of benzene / benzylene in Aconitum carmichaelii samples; Figure 5 A scatter plot of the ratio of benzene to benzene in Aconitum carmichaelii samples; Figure 6 A scatter plot of the ratio of benzene / benzene content in Aconitum carmichaelii samples; Figure 7 The HPLC chromatogram of processed Aconitum carmichaelii sample (processed Aconitum carmichaelii 8); Figure 8 The HPLC chromatogram of a sample of adulterated Aconitum carmichaelii (processed Aconitum carmichaelii 20); Figure 9 A scatter plot of the ratio of benzene / benzylene in the processed aconite sample; Figure 10 A scatter plot of the ratio of benzene to benzene in the processed aconite sample; Figure 11 A scatter plot of the ratio of benzene / benzylene in the processed aconite sample; Figure 12 Scatter plot of the benzene / benzene content ratio in the combined samples of processed aconite and processed cassia aconite; Figure 13 Box plot of the benzene / benzyl content ratio in the combined samples of processed aconite and processed aconite; Figure 14 Scatter plot of the benzene / benzene content ratio in the combined samples of processed Aconitum carmichaelii and Aconitum kusnezoffii; Figure 15 Box plot of the benzene / benzene content ratio in a combined sample of processed Aconitum carmichaelii and Aconitum kusnezoffii; Figure 16 This is the HPLC chromatogram of the papaya pill sample. Detailed Implementation
[0014] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.
[0015] Example 1: Establishment of a method for identifying adulteration in papaya pills 1. Identification of processed Aconitum carmichaelii Collect processed Aconitum carmichaelii samples (Table 1) and prepare test solutions according to the following methods: Accurately weigh approximately 2g of sample powder (passed through a No. 3 sieve) and place it in a stoppered conical flask. Add 3mL of ammonia test solution and accurately add 50mL of a 1:1 isopropanol-ethyl acetate mixture. Weigh the sample and sonicate it (300W power, 40kHz frequency; water temperature below 25℃) for 30 minutes. Cool the flask and weigh it again. Make up the lost weight with the 1:1 isopropanol-ethyl acetate mixture, shake well, and filter. Accurately measure 25mL of the filtrate and remove the solvent under reduced pressure below 40℃ until dry. Accurately dissolve the residue in 3mL of a 1:1 isopropanol-chloroform mixture, filter, and collect the filtrate.
[0016] Table 1 Information on Processed Aconitum carmichaelii Samples Preparation of the reference stock solution: Accurately weigh 15.70 mg of benzoyl neoaconitine reference standard, place it in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and set aside. (C) 苯甲酰新乌头原碱 =0.3055 mg / mL).
[0017] Accurately weigh 18.18 mg of benzoyl aconitine reference standard, place it in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and set aside. (C) 苯甲酰乌头原碱 =0.3563 mg / mL).
[0018] Accurately weigh 15.90 mg of benzoyl aconitine reference standard, place it in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and set aside. (C) 苯甲酰次乌头原碱 =0.3066 mg / mL).
[0019] Accurately weigh 28.89 mg of aconitine diester-type alkaloid reference extract (with the contents of neoaconitine, hypoaconitine, and aconitine already labeled), place it in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and set aside.
[0020] Preparation of mixed reference solutions: Accurately measure 4 mL of the above-mentioned benzoyl aconitine reference stock solution, 0.4 mL of benzoyl aconitine reference stock solution, 0.8 mL of benzoyl aconitine reference stock solution, and 3 mL of aconitine diester type alkaloid reference extract stock solution, and place them in the same 25 mL volumetric flask. Dilute to the mark with mobile phase (A:B=15:85v / v), and shake well to obtain the solution.
[0021] According to the chromatographic conditions for processed Aconitum carmichaelii in the 2020 edition of the Chinese Pharmacopoeia: octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-tetrahydrofuran (25:15 v / v) was used as mobile phase A, and 0.1 mol / L ammonium acetate solution (0.5 mL of glacial acetic acid was added per 1000 mL) was used as mobile phase B, with gradient elution performed as specified in Table 2; the detection wavelength was 235 nm.
[0022] Table 2 Gradient elution program Take 20 μL of the mixed reference solution and 5-10 μL of the prepared Aconitum carmichaelii test solution, inject them into the liquid chromatograph, and determine them.
[0023] Table 3. Determination results of processed Aconitum carmichaelii samples (unit: μg / g) Typical chromatograms of aconite alkaloid mixed reference standard, processed aconite sample, and processed aconite sample (pseudonym) are as follows: Figure 1-3 As shown. Based on the contents of benzoylneoprothizoline, benzoylaconitine, and benzoylhypoconitine measured in 25 batches of processed Aconitum carmichaelii, the analysis was performed using the ratios of benzoylneoprothizoline / benzoylaconitine (benzylneoprothizoline / benzylaconitine), benzoylneoprothizoline / benzoylhypoconitine (benzylneoprothizoline / benzylhypoconitine), and benzoylaconitine / benzoylhypoconitine (benzylaconitine / benzylhypoconitine).
[0024] The scatter plots of benzene-to-benzene / benzene-to-benzene, benzene-to-benzene-to-benzene, and benzene-to-benzene / benzene-to-benzene ratios in 25 batches of processed Aconitum carmichaelii are shown below. Figure 4-6 As shown, the 25 batches of samples can be divided into two categories: 19 batches of processed Aconitum carmichaelii and 6 batches of counterfeit products.
[0025] Table 4. Results of t-test analysis of prepared aconite root. p<0.05; p<0.01.
[0026] Since the sample sizes were all less than 50, the Shapiro-Wilk test was used to assess normality and analyze whether there were significant differences between the two groups. Table 4 shows that all three ratios exhibited normality. Further independent samples t-tests were used to analyze the differences in the three ratios between processed and counterfeit aconite, and the results showed that the p-values were all less than 0.01, indicating significant differences. The larger the absolute value of the t-value, the more significant the difference. The ratio of benzene to benzene, with the largest t-value, was selected as the indicator for distinguishing genuine from counterfeit products.
[0027] 2. Identification of processed aconite Collect processed Aconitum carmichaelii samples (Table 5) and prepare test solutions according to the following method: Accurately weigh approximately 2g of sample powder (passed through a No. 3 sieve) and place it in a stoppered conical flask. Add 3mL of ammonia test solution and accurately add 50mL of a 1:1 isopropanol-ethyl acetate mixture. Weigh the sample and sonicate it (300W power, 40kHz frequency; water temperature below 25℃) for 30 minutes. Cool the flask and weigh it again. Make up the lost weight with the 1:1 isopropanol-ethyl acetate mixture, shake well, and filter. Accurately measure 25mL of the filtrate and remove the solvent under reduced pressure below 40℃ until dry. Accurately dissolve the residue in 3mL of a 1:1 isopropanol-chloroform mixture, filter, and collect the filtrate.
[0028] Table 5 Information on Processed Aconitum carmichaelii Samples The preparation of reference standards and samples, and the chromatographic conditions and detection methods were the same as those for prepared Aconitum carmichaelii. The determination results are shown in Table 6.
[0029] Table 6. Determination results of processed Aconitum carmichaelii samples (unit: μg / g) Typical chromatograms of aconite alkaloid mixed reference standard, processed aconite sample, and processed aconite sample (pseudonym) are as follows: Figure 1 , 7 As shown in Figure -8. Based on the contents of benzoylneoprothizoline, benzoylaconitine, and benzoylhypoconitine measured in 24 batches of processed aconite, the analysis was performed using the ratios of benzoylneoprothizoline / benzoylaconitine (benzylneoprothizoline / benzylaconitine), benzoylneoprothizoline / benzoylhypoconitine (benzylneoprothizoline / benzylhypoconitine), and benzoylaconitine / benzoylhypoconitine (benzylaconitine / benzylhypoconitine).
[0030] The scatter plots of benzene / benzyl aconite, benzene / benzyl benzoate, and benzyl aconite / benzyl benzoate in 24 batches of processed aconite are shown below. Figure 9-11 As shown, the 24 batches of samples can be divided into two categories: 18 batches of processed aconite and 6 batches of counterfeit products.
[0031] Table 7 Results of t-test analysis of processed Aconitum carmichaelii p<0.05; p<0.01.
[0032] Since the sample sizes were all less than 50, the Shapiro-Wilk test was used to assess normality and analyze whether there were significant differences between the two groups. Table 7 shows that all three ratios exhibited normality. Further independent samples t-tests were used to analyze the differences in the three ratios between processed and counterfeit aconite. The results showed that the p-values for all three ratios were less than 0.01, indicating significant differences. The larger the absolute value of the t-value, the more significant the difference. The ratio of benzene / benzyl aconite with the largest t-value was selected as the indicator for distinguishing genuine from counterfeit products.
[0033] 3. Analysis of the results of the combination of processed aconite and processed Sichuan aconite The Papaya Pill formulation contains two herbs: processed Aconitum carmichaelii and processed Aconitum kusnezoffii. To determine whether the ratios of benzyl-methyl / benzyl-Aconitum carmichaelii and benzyl-methyl / benzyl-methyl can be used for adulteration analysis in the formulation, the significant differences in these ratios between processed Aconitum carmichaelii and processed Aconitum kusnezoffii were analyzed. Independent samples t-tests were used for the analysis.
[0034] Table 8. Results of t-test analysis of processed Aconitum carmichaelii and processed Aconitum kusnezoffii / Aconitum carmichaelii. p<0.05; p<0.01.
[0035] Table 9. Results of Benzene / Benzene-t Test Analysis of Processed Aconitum carmichaelii and Processed Aconitum kusnezoffii. p<0.05; p<0.01.
[0036] The differences in the ratios of benzene / benzene and benzene / benzene in processed aconite and processed cassia aconite are shown in Tables 8 and 9. The p values of both are greater than 0.05, indicating that processed aconite and processed cassia aconite show consistency and no significant difference. Therefore, the ratios of benzene / benzene and benzene / benzene can be used for adulteration analysis in preparations.
[0037] The ratio of processed aconite root to processed Sichuan aconite root in the Papaya Pill formulation is 1:1. 24 batches of processed aconite root and its adulterants and 25 batches of processed Sichuan aconite root and its adulterants were combined using Cartesian products in a 1:1 ratio to generate 600 sets of data on combinations of processed Sichuan aconite root and processed aconite root.
[0038] The scatter plot of the benzene / benzyl ratio in the combined samples is shown below. Figure 12 As shown, the box diagram is as follows Figure 13 As shown, combinations containing adulterated processed aconite and combinations containing genuine processed aconite can be clearly divided into two categories. The upper limit of the benzene / benzylene ratio for combinations containing adulterated processed aconite is 4.03, while the lower limit of the benzene / benzylene ratio for combinations containing genuine processed aconite is 3.49. There is a certain degree of overlap between the two groups. When the benzene / benzylene ratio in the sample is in the range of 3.49-4.03, it cannot be determined whether adulterated aconite has been added. When the benzene / benzylene ratio in the sample is less than 3.49, it indicates the presence of adulterated processed aconite.
[0039] The scatter plot of the benzene / benzene ratio in the combined samples is shown below. Figure 14 As shown, the box diagram is as follows Figure 15As shown, combinations containing adulterated processed aconite and combinations containing genuine processed aconite can be clearly divided into two categories. The upper limit of the benzene / benzene ratio for combinations containing adulterated processed aconite is 4.86, with one outlier at 5.38; the lower limit of the benzene / benzene ratio for combinations containing genuine processed aconite is 5.22. The two groups have no overlap except for one outlier. Tracing the outlier revealed that the total amount of monoester alkaloids in the processed aconite sample exceeded the standard limit, making it a non-compliant sample; this outlier was ignored. When the benzene / benzene ratio in a sample is less than 5.22, it indicates the presence of adulterated processed aconite.
[0040] In summary, the ratios of benzoylneoprothizoline / benzoylneoprothizoline (benzoylneoprothizoline / benzoylneoprothizoline) and benzoylneoprothizoline / benzoylneoprothizoline (benzoylneoprothizoline / benzoylneoprothizoline) can be used for adulteration analysis in papaya pill preparations; the specific standards are as follows: When the benzene / benzyl alcohol ratio in the sample is less than 3.49, it indicates that adulterated aconite has been added to the sample. When the ratio of benzene to benzene in the sample is less than 5.22, it indicates the presence of adulterated aconite products.
[0041] Application Example 1: Quality Testing of Papaya Pills Collect papaya pills from different manufacturers and batches, and prepare the test solution according to the following method: Papaya pills (concentrated water pills): Remove the coating, grind finely (through a No. 3 sieve), take 2.0g, accurately weigh, place in a stoppered conical flask, accurately add 25mL of hydrochloric acid-methanol (1:100) mixed solution, sonicate (power 500W, frequency 40kHz; temperature not exceeding 25℃) for 40 minutes, filter, accurately measure 10mL of the filtrate, recover the solvent under reduced pressure below 40℃ to dryness, add 10mL of 0.1mol / L hydrochloric acid solution to dissolve the residue, centrifuge (speed 8000 rpm) for 20 minutes, take the supernatant, add to a solid phase extraction column (using a mixed-type cation exchanger). Using a reverse-phase adsorbent as the packing material (500 mg, 6 mL, pre-washed sequentially with 6 mL each of methanol and water), the adsorbent is eluted sequentially with 3 mL of water, ammonia solution (5 mL concentrated ammonia diluted with water to 100 mL), water, methanol, and 5 mL each of acetonitrile. After the eluent has drained, the adsorbent is allowed to stand for 5 minutes, followed by elution with 10 mL of a mixed solution of acetonitrile and concentrated ammonia (90:10). The eluent is collected, and the solvent is recovered under reduced pressure below 40 °C until dry. An appropriate amount of mobile phase (A:B = 15:85 v / v) is added to dissolve the solvent, and the solution is transferred to a 2 mL volumetric flask, diluted to the mark, and shaken well. The filtrate is then collected.
[0042] Papaya pills (large honey pills): Cut into small pieces, mix well, take 8.0g, accurately weigh, place in a stoppered conical flask, accurately add 50mL of hydrochloric acid-methanol (1:100) mixed solution, sonicate (power 500W, frequency 40kHz; temperature not exceeding 25℃) for 40 minutes, filter, accurately measure 20mL of the filtrate, recover the solvent under reduced pressure below 40℃ until dry, add 10mL of 0.1mol / L hydrochloric acid solution to dissolve the residue, centrifuge (speed 8000 rpm) for 20 minutes, take the supernatant, and operate in the same way as above from "add to solid phase extraction column".
[0043] Preparation of the reference stock solution: Accurately weigh 15.70 mg of benzoyl neoaconitine reference standard, place it in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and set aside. (C) 苯甲酰新乌头原碱 =0.3055 mg / mL).
[0044] Accurately weigh 18.18 mg of benzoyl aconitine reference standard, place it in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and set aside. (C) 苯甲酰乌头原碱 =0.3563 mg / mL).
[0045] Accurately weigh 15.90 mg of benzoyl aconitine reference standard, place it in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and set aside. (C) 苯甲酰次乌头原碱 =0.3066 mg / mL).
[0046] Accurately weigh 28.89 mg of aconitine diester-type alkaloid reference extract (with the contents of neoaconitine, hypoaconitine, and aconitine already labeled), place it in a 50 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and set aside.
[0047] Preparation of mixed reference solutions: Accurately measure 4 mL of the above-mentioned benzoyl aconitine reference stock solution, 0.4 mL of benzoyl aconitine reference stock solution, 0.8 mL of benzoyl aconitine reference stock solution, and 3 mL of aconitine diester type alkaloid reference extract stock solution, and place them in the same 25 mL volumetric flask. Dilute to the mark with mobile phase (A:B=15:85v / v), and shake well to obtain the solution.
[0048] According to the chromatographic conditions for processed Aconitum carmichaelii in the 2020 edition of the Chinese Pharmacopoeia: octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-tetrahydrofuran (25:15 v / v) was used as mobile phase A, and 0.1 mol / L ammonium acetate solution (with 0.5 mL of glacial acetic acid added per 1000 mL) was used as mobile phase B, with gradient elution performed as specified in Table 2; the detection wavelength was 235 nm.
[0049] Accurately pipette 20 μl each of the mixed reference solution and the papaya pill test solution into the liquid chromatograph for determination.
[0050] Table 10. Results of Papaya Pill Sample Determination (Unit: μg / g) The spectrum of the reference solution is as follows Figure 1 As shown, the HPLC chromatogram of a typical papaya pill is as follows. Figure 16 As shown.
[0051] The ratios of benzyl-2-methyl / benzyl-2-methyl and benzyl-2-methyl / benzyl-2-methyl in 45 batches of papaya preparations were calculated, and the results are shown in Table 10. The benzyl-2-methyl / benzyl-2-methyl ratio in all 45 batches of papaya pills was greater than 3.49, indicating no adulteration with processed aconite. Since the proportions of the components in adulterated processed aconite differ significantly from those in genuine products, authenticity can be determined during the raw material inspection stage, minimizing the risk of subjective adulteration. Eighteen batches of papaya pills had a benzyl-2-methyl / benzyl-2-methyl ratio less than 5.22, suggesting the presence of adulterated processed aconite.
[0052] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.
Claims
1. A method for identifying adulteration in papaya pills, characterized in that, Includes the following steps: (1) The contents of benzoyl neoaconitine and benzoyl hypoaconitine in multiple processed Aconitum carmichaelii samples and adulterants were determined by HPLC, and the ratio of benzoyl neoaconitine to benzoyl hypoaconitine was calculated. (2) The contents of benzoylneoprothizoline and benzoylneoprothizoline in multiple processed aconite samples and adulterated processed aconite were detected by HPLC, and the ratio of benzoylneoprothizoline to benzoylneoprothizoline was calculated. (3) Combine the ratio data of processed Aconitum carmichaelii sample and counterfeit product in step (1) with processed Aconitum kusnezoffii sample and counterfeit product in step (2) in a 1:1 ratio using Cartesian product to obtain the lower limit value a of the ratio of benzoyl neoaconitine and benzoyl hypoaconitine in the combination containing processed Aconitum carmichaelii sample, and the lower limit value b of the ratio of benzoyl neoaconitine and benzoyl aconitine in the combination containing processed Aconitum kusnezoffii sample. (4) The contents of benzoyl neoaconitine, benzoyl hypoaconitine and benzoyl aconitine in papaya pills were determined by HPLC and the ratios a1 and b1 of benzoyl neoaconitine and benzoyl hypoaconitine were calculated. (5) Comparing a1 and a, when a1 < a, it indicates that the processed Aconitum carmichaelii in the Papaya Pill is adulterated; Comparing b1 and b, if b1 < b, it indicates that the processed aconite in the papaya pills is adulterated. Processed Aconitum carmichaelii is adulterated with processed Aconitum carmichaelii var. sarcodactylis and / or processed Aconitum kusnezoffii var. jungae. Processed Aconitum kusnezoffii var. jungae is adulterated with processed Aconitum kusnezoffii var. jungae.
2. The method for identifying adulteration of papaya pills according to claim 1, characterized in that, The HPLC methods in steps (1), (2), and (4) are the chromatographic conditions under the section on processed Aconitum carmichaelii in the 2020 edition of the Chinese Pharmacopoeia.
3. The method for identifying adulteration of papaya pills according to claim 1, characterized in that, In step (4), the HPLC method also includes pretreatment before detection: For concentrated papaya pills: remove the coating, grind finely and pass through a No. 3 sieve, take 2.0g, accurately weigh, place in a stoppered conical flask, accurately add 25mL of a 1:100 v / v hydrochloric acid-methanol mixed solution, sonicate, filter, accurately measure 10mL of the filtrate, recover the solvent under reduced pressure below 40℃ until dry, add 10mL of 0.1mol / L hydrochloric acid solution to dissolve the residue, centrifuge, and take the supernatant; For the large honey pills of Papaya Pills: Crush and mix them, take 8.0g, accurately weigh them, place them in a stoppered conical flask, accurately add 50mL of a 1:100 v / v hydrochloric acid-methanol mixture, sonicate, filter, accurately measure 20mL of the filtrate, and recover the solvent under reduced pressure below 40℃ until dry. Add 10mL of 0.1mol / L hydrochloric acid solution to the residue to dissolve it, centrifuge for 20 minutes, and take the supernatant. The supernatant was then added to a solid-phase extraction column packed with a mixed cation exchange reversed-phase adsorbent. The column was eluted sequentially with 3 mL of water, 5 mL of ammonia solution, 5 mL of water, 5 mL of methanol, and 5 mL of acetonitrile. After the eluent was exhausted, the column was allowed to stand for 5 minutes. Then, it was eluted with 10 mL of a 90:10 v / v acetonitrile-concentrated ammonia solution mixture. The eluent was collected and the solvent was recovered under reduced pressure below 40°C until dry. The eluent was dissolved in an appropriate amount of mobile phase, transferred to a 2 mL volumetric flask, and diluted to the mark. The mixture was shaken well, and the filtrate was used as the HPLC sample. The ammonia solution is prepared by adding water to 100 mL of concentrated ammonia solution.