Method for detecting contents and toxic quantities of various components in fructus psoraleae product and application of method

Abstract

The invention relates to a method for measuring the content of one or more components of fructus psoraleae in a fructus psoraleae product, which comprises the following steps: preparation of a test solution: extracting each component of the fructus psoraleae product by using an ultrasonic extraction method, and filtering to obtain a filtrate, namely the test solution; determining the content of one or more components of fructus psoraleae in the test solution by using a liquid chromatography; a chromatographic column is a C18 reversed-phase chromatographic column, and a mobile phase is an acetonitrile and phosphoric acid solution or an acetonitrile and formic acid solution; meanwhile, the invention provides a method for detecting the toxicity amount of a fructus psoraleae product. Through the application, a quantity-toxicity associated psoralea corylifolia quality control method can be established, the content of the index components and the toxicity quantity can be more intuitively reflected by using the visual color chart, and the method has important significance for improving the quality control of the psoralea corylifolia and the quality dynamic control in the production process of the psoralea corylifolia product.

Description

Technical Field

[0001] The invention belongs to the field of drug quality control, and in particular relates to a method for detecting the toxicity of a psoralea corylifolia product. Background Art

[0002] Psoraleae Fructus is the dried fruit of the leguminous plant Psoralea corylifolia L., which was first recorded in Leigong Paozhi Lun. Psoraleae has the effects of warming the kidney and supporting yang, absorbing qi and calming asthma, warming the spleen and stopping diarrhea. It is used for kidney yang deficiency, impotence and spermatorrhea, enuresis and frequent urination, cold pain in the waist and knees, asthma caused by kidney deficiency, and diarrhea at dawn; it is used externally to treat vitiligo and alopecia areata. Psoraleae is a traditional and commonly used Chinese medicinal material in my country. Psoraleae is widely used in clinical practice, and there are more than 200 Chinese patent medicine preparations containing Psoraleae. In recent years, the adverse reactions of liver damage in traditional Chinese medicines containing Psoraleae, such as Xianling Gubao oral preparations and Zhuanggu Joint Pills, have been reported, and the safety of Psoraleae has attracted attention. The chemical composition of Psoraleae is relatively complex. So far, more than 100 compounds have been isolated and identified from it, mainly including coumarins, monoterpenoids and flavonoids. Recent studies have shown that the main hepatotoxic components in Psoraleae are psoralen, isopsoralen chalcone, psoralen and bakuchiol [Huiying Shang, Xian Liu, Jinchao Pan, Hongbo Cheng, Zengchun Ma, Chengrong Xiao, Yue Gao. Exploring the mechanism and phytochemicals in Psoraleae Fructus-induced hepatotoxicity based on RNA-seq, in vitro screening and molecular docking, www.nature.com / scientificreports, https: / / doi.org / 10.1038 / s41598-023-50454-0, (2024) 14:1696].The quality control of Psoralea corylifolia is related to its safety. The 2020 edition of the Pharmacopoeia of the People's Republic of China only selected psoralen and isopsoralen as content determination indicator components to control the quality of Psoralea corylifolia medicinal materials. In addition to coumarins, recent studies have also detected flavonoids and monoterpene phenols. Wang D et al. used UHPLC-PDA to determine the contents of 9 components in Psoralea corylifolia (psoralen, isopsoralen, psoralen, isopsoralen, neopsoralen isoflavones, psoralenidine, psoralen B (isopsoralen chalcone), psoralen dihydroflavone methyl ether and bakuchiol) [Wang D, Jiaming G, Chai X, et al. Dynamic variations of bioactive compounds driven by enzymes in Psoralea corylifolia L. from growth to storage and processing. Arabian Journal of Chemistry[J].(2022)15,103461.】, Fan Ling et al. used HPLC to simultaneously determine the contents of 10 components in the traditional Chinese medicine Psoralea corylifolia (psoralen, isopsoralen, neopsoralen isoflavones, psoralen dihydroflavone, psoralenine, psoralenidine, isopsoralen chalcone, psoralen dihydroflavone methyl ether, Corylifol A and bakuchiol) [Fan Ling, Qiu Xinsong, Gao Yang, et al. Simultaneous determination of the contents of 10 components in the traditional Chinese medicine Psoralea corylifolia by HPLC[J]. Journal of Qiqihar Medical College, 2018, 39(16):1928-1931.]. Wang Juan et al. established the UPLC fingerprint of Psoralea corylifolia and determined the contents of 12 main components (psoralen, isopsoralen, isopsoralen dihydroflavonoids, neopsoralen isoflavones, psoralen A, imperatorin, psoralen, psoralen, 4,5-dehydroisopsoralen, psoralen B, isopsoralen chromone chalcone, bakuchiol). [Wang Juan, Zhou Zhixing, Yang Li, et al. Establishment of UPLC fingerprint of Psoralea corylifolia and determination of the contents of 12 main components [J]. Chinese Herbal Medicine, 2021, 52(02): 552-557.] It can be seen that the existing quality control methods of Psoralea corylifolia only reflect the contents of its different index components, but fail to simultaneously reflect the safety of its index components. Establishing a quality control method of Psoralea corylifolia with quantity-toxicity correlation and using a visual color card to more intuitively reflect the content and toxicity of the index components is of great significance to improving the quality control of Psoralea corylifolia. Summary of the invention

[0003] In order to solve the above problems in the prior art, that is, to establish a visual color chart that directly reflects the content of multiple components of Psoralea corylifolia and their toxicity, and directly reflects the quantity-toxicity relationship of multiple components of Psoralea corylifolia, the present application provides a method for detecting Psoralea corylifolia components.

[0004] The specific technical solutions of this application are as follows:

[0005] 1. A method for determining the content of one or more components of Psoralea corylifolia in a Psoralea corylifolia product, comprising:

[0006] Preparation of the test solution: using ultrasonic extraction to extract the various components of the psoralea corylifolia product, filtering, and obtaining the filtrate as the test solution; using liquid chromatography to determine the content of one or more components of psoralea corylifolia in the test solution; the chromatographic column is a C18 reverse phase chromatographic column; the mobile phase is acetonitrile and phosphoric acid solution, or acetonitrile and formic acid solution; the components are one or more of psoralen, 4'-O-methyl psoralea corylifolia chalcone, psoralen, Corylifol A, psoralea corylifolia dihydroflavone methyl ether, psoralen, isopsoralen, psoralen B, neopsoralen isoflavones, psoralen chalcone, psoralen A, bakuchiol, psoralen and isopsoralen.

[0007] 2. The method according to item 1 is characterized in that the use of ultrasonic extraction to extract the various components of the Psoralea corylifolia product includes taking 0.1-0.5g of Psoralea corylifolia powder, soaking the powder of the Psoralea corylifolia product in methanol, then adding water to the volume percentage of methanol to 80%, weighing the weight, then performing ultrasonic extraction, making up the weight with 80% volume percentage of methanol, and finally shaking well.

[0008] 3. The method according to item 2 is characterized in that the powder of the psoralea corylifolia product is added to methanol for immersion, the immersion time is 0-120 min, preferably 30-120 min, and the volume fraction of the methanol is 50-100%, preferably 80-100%.

[0009] 4. The method according to item 2 is characterized in that the mass of the Psoralea corylifolia powder is 0.1-0.25g.

[0010] 5. The method according to item 1 is characterized in that the time of ultrasonic extraction is 20-60 min, preferably 20-40 min.

[0011] 6. The method according to item 1 is characterized in that the concentration of the phosphoric acid solution is 0.05-1.0%, and the concentration of the formic acid solution is 0.05-1.0%.

[0012] 7. The method according to item 1 is characterized in that the chromatographic column is a ZORBAX Extend-C18 chromatographic column, and the column temperature of the chromatographic column is 25-35°C.

[0013] 8. The method according to item 1 is characterized in that the detection wavelength of the liquid chromatography is 242-325 nm, preferably 242-246 nm.

[0014] 9. A method for detecting the toxicity of psoralea corylifolia products, comprising

[0015] Measuring the content of each component in Psoralea corylifolia; preferably detecting by the method described in any one of claims 1 to 8; standardizing the content of each component to obtain a content standardized value; using biological LC 50 The value is used as an indicator, and the toxicity coefficient of each component is calculated with one component as the toxicity reference component; the toxicity of the component is calculated by the content and the toxicity coefficient, and the toxicity is standardized to obtain the toxicity standardized value; the quantity-toxicity relationship heat map is made by the content standardized value and the toxicity standardized value; the toxicity of the psoralea corylifolia product is detected by the quantity-toxicity relationship heat map;

[0016] Among them, the ingredients include one or more of psoralen, isopsoralen, psoralen, isopsoralen, psoralenidine, neopsoralen isoflavones, psoralen A, psoralen nin, psoralen chalcone, psoralen B, psoralen dihydroflavone methyl ether, Corylifol A, 4'-O-methylpsoralen chalcone, and bakuchiol.

[0017] 10. The method according to item 9, wherein the toxic reference ingredients are psoralen, isopsoralen, psoralen, psoralen, 4'-O-methylpsoralen chalcone, Corylifol A, and psoralen dihydroflavone methyl ether.

[0018] 11. The method according to item 9, wherein the organism is a zebrafish, a cell, a mouse, or a rat, preferably a zebrafish.

[0019] 12. Application of the method described in item 9 in the production process of Psoralea corylifolia products.

[0020] Beneficial Effects

[0021] The method described in the present application can be used to determine the content of one or more components of Psoralea corylifolia in Psoralea corylifolia products. Using the method of the present application, the content of each component in Psoralea corylifolia can be accurately determined, with high detection accuracy and separation.

[0022] The content-toxicity color chart and its preparation method provided in this application can intuitively reflect the relationship between the toxicity and content of each component in the psoralea corylifolia product. Specifically, when a more representative toxicity reference component is selected as the color chart, it can be applied to the industrial production of psoralea corylifolia products. According to the components to be played in the psoralea corylifolia products, the preparation method of the psoralea corylifolia products can be selected and optimized. It can also be used to detect the composition and toxicity changes in the production process of extraction and purification of traditional Chinese medicine containing psoralea corylifolia, which is conducive to the dynamic quality control of the production process of psoralea corylifolia products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the chromatogram of Psoralea corylifolia (Yunnan 9) with different mobile phases.

[0024] Figure 2 This is the chromatogram of Psoralea corylifolia (Yunnan 9) at different column temperatures.

[0025] Figure 3 This is the chromatogram of different elution gradients of Psoralea corylifolia (Yunnan 9).

[0026] Figure 4 These are the chromatograms of Psoralea corylifolia (Yunnan 9) at different detection wavelengths.

[0027] Figure 5 The results of different extraction methods for the test solution.

[0028] Figure 6 The figure shows the effect of different concentrations of methanol on the peak area of ​​the main components of Psoralea corylifolia.

[0029] Figure 7 This is the effect of extraction time on the peak area of ​​the main components of Psoralea corylifolia.

[0030] Figure 8 To investigate the effect of test solution preparation methods on the peak areas of the main components of Psoralea corylifolia.

[0031] Fig. 9 The chromatograms are representative of psoralea corylifolia and reference solution, where the peak numbers and their meanings are: 1. Psoralen; 2. Isopsoralen; 3. Psoralen; 4. Isopsoralen; 5. Neopsoralen isoflavones; 6. Psoralen A; 7. Psoralenine; 8. Psoralen chalcone; 9. Psoralenidine; 10. Psoralen B; 11. Psoralen dihydroflavone methyl ether; 12. Corylifol A; 13. 4'-O-methyl psoralen chalcone; 14. Bakuchiol; 15. Psoralen chromene chalcone.

[0032] Fig.10 To investigate the effects of psoralen coumarins, flavonoids and bakuchiol on zebrafish mortality.

[0033] Fig.11This is a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia (with psoralen as the toxicity reference component).

[0034] Fig.12 This is a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia (with isopsoralen as the toxicity reference component).

[0035] Fig.13 This is a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia (with Psoralea corylifolia as the toxicity reference component).

[0036] Fig.14 This is a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia (with Psoralea corylifolia as the toxicity reference component).

[0037] Fig.15 This is a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia (with 4'-O-methyl Psoralea corylifolia chalcone as the toxicity reference component).

[0038] Fig.16 This is a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia (with Corylifol A as the toxicity reference component).

[0039] Fig.17 This is a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia (with Psoralea corylifolia dihydroflavonoid methyl ether as the toxicity reference component).

[0040] Fig.18 This is a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia (with Bakuchiol as the toxicity reference component).

[0041] Fig.19 is the LC value of the toxic reference ingredient (assumed to be psoralen) 50 Visual color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia when the concentration (μg / mL) is 30.

[0042] Fig. 20 is the LC value of the toxic reference ingredient (assumed to be psoralen) 50 When the concentration (μg / mL) is 40, a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia is shown.

[0043] Fig.21 is the LC value of the toxic reference ingredient (assumed to be psoralen) 50 When the concentration (μg / mL) is 50, a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia.

[0044] Fig. 22 is the LC value of the toxic reference ingredient (assumed to be psoralen) 50When the concentration (μg / mL) is 60, a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia.

[0045] Fig.23 is the LC value of the toxic reference ingredient (assumed to be psoralen) 50 A visualized color card heat map showing the relationship between the content and toxicity of multiple components in Psoralea corylifolia when the concentration (μg / mL) is 500.

[0046] Fig.24 is the LC value of the toxic reference ingredient (assumed to be psoralen) 50 When the concentration (μg / mL) is 550, a visualized color card heat map of the content-toxicity relationship of multiple components in Psoralea corylifolia.

[0047] Fig.25 is the LC value of the toxic reference ingredient (assumed to be psoralen) 50 When the concentration (μg / mL) is 600, a color chart heat map showing the correlation between the content and toxicity of multiple components in Psoralea corylifolia is shown.

[0048] Fig.26 This is a visualized color card heat map of the content-toxicity relationship of multiple components in three processed products of Psoralea corylifolia (with Psoralea corylifolia as the toxicity reference component).

[0049] Fig. 27 This is a visualized color card heat map of the content-toxicity relationship of multiple components in raw Psoralea corylifolia, salt-roasted products and their decoctions (with Psoralea corylifolia as the toxicity reference component). DETAILED DESCRIPTION

[0050] The present invention is described in detail below. Although specific embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0051] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art will understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the present invention. The scope of protection of this application shall be determined by the attached claims.

[0052] The present application provides a method for determining the content of one or more components of Psoralea corylifolia in a Psoralea corylifolia product, which comprises: preparing a test solution: extracting multiple components of the Psoralea corylifolia product by ultrasonic extraction, filtering, and obtaining a filtrate as the test solution; determining the content of one or more components of Psoralea corylifolia in the test solution by liquid chromatography; the chromatographic column is a C18 reverse phase chromatographic column; the mobile phase is an acetonitrile and phosphoric acid solution, or an acetonitrile and formic acid solution;

[0053] The ingredients are one or more of psoralen, 4'-O-methyl psoralen chalcone, psoralen, Corylifol A, psoralen dihydroflavone methyl ether, psoralen glycosides, isopsoralen glycosides, psoralen B, neopsoralen isoflavones, psoralen chalcone, psoralen A, bakuchiol, psoralen and isopsoralen,

[0054] In one embodiment of the present application, it is characterized in that the use of ultrasonic extraction to extract the various components of the Psoralea corylifolia product includes taking 0.1-0.5g of Psoralea corylifolia powder, soaking the powder of the Psoralea corylifolia product in methanol, adding water to the volume percentage of methanol to 80%, weighing the weight, performing ultrasonic extraction at a power of 100-180W and a frequency of 40-60kHz, making up the weight with 80% volume percentage of methanol, and finally shaking well.

[0055] In one embodiment of the present application, the Soxhlet extraction method and / or the reflux extraction method can also be used to extract the various components of the Psoralea corylifolia product.

[0056] For example, in one embodiment of the present application, the mass of the extracted psoralea corylifolia powder can be 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, preferably 0.1-0.25 g. The power of the ultrasonic extraction can be 100 W, 110 W, 120 W, 130 W, 140 W, 150 W, 155 W, 160 W, 165 W, 170 W, 175 W, 180 W. The frequency of the ultrasonic extraction can be 40 kHz, 45 kHz, 50 kHz, 55 kHz, 60 kHz. The ultrasonic extraction time is 20-60 min, for example, it can be 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, preferably 20-40 min, for example, it can be 20 min, 25 min, 30 min, 35 min, 40 min.

[0057] In one embodiment of the present application, the powder of the psoralea corylifolia product is added to methanol for soaking, and the soaking time is 0-120 min. For example, the soaking time can be 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min, preferably 30-120 min, for example, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min. The volume fraction of the methanol is 50-100%, for example, the volume fraction can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and it should be understood that when the volume fraction of the methanol is 100%, the methanol is pure methanol.

[0058] In one embodiment of the present application, the powder of the Psoralea corylifolia product can also be soaked in pure methanol, and then water is added to the volume fraction of methanol to 50-100%. For example, the volume fraction can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0059] In one embodiment of the present application, the concentration of the phosphoric acid solution is 0.05-1.0%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%. The concentration of the formic acid solution is 0.05-1.0%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%.

[0060] In one embodiment of the present application, the column temperature of the chromatographic column is 25-35°C, for example, it can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C.

[0061] In one embodiment of the present invention, the detection wavelength of liquid chromatography is 242-325 nm, for example, the detection wavelength can be 242 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm. In one embodiment of the present invention, the detection wavelength can also be 242 nm, 246 nm, 254 nm, 270 nm, 280 nm, 310 nm, 325 nm, preferably 242-246 nm.

[0062] In one embodiment of the present application, an optimal elution gradient of the chromatographic column is also provided. When the mobile phase is acetonitrile-0.05% formic acid, liquid A is set to acetonitrile and liquid B is set to 0.05% formic acid solution. The elution gradient is 0 min (10% A: 90% B), 30 min (50% A: 50% B), 45 min (55% A: 45% B), 55 min (80% A: 20% B), 55-65 min (80% A: 20% B); in one case, the elution gradient is 0 min (10% A: 90% B) , 30min(50%A:50%B), 45min(60%A:40%B), 50min(70%A:30%B), 55min(80%A:20%B), 55-65min(80%A:20%B); in one case, the elution gradient is 0min(10%A:90%B), 30min(50%A:50%B), 45min(55%A:45%B), 55min(80%A:20%B), 55-65min(90%A:10%B).

[0063] In one embodiment of the present application, the chromatographic column is an Agilent Zorbax Extend-C18 chromatographic column.

[0064] In one embodiment of the present application, the preparation method of the test solution is to take about 0.1g of Psoralea corylifolia powder (passed through a No. 3 sieve), accurately weigh it, put it in a 25ml stoppered volumetric flask, add 25ml of 80% methanol (when measuring the raw Psoralea corylifolia, first add 20ml of methanol and soak it for 30min or more, and then add 5ml of water), weigh it, ultrasonically treat it (power 150W, frequency 40kHz) for 40 minutes, cool it, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0065] In one embodiment of the present application, the chromatographic conditions are preferably:

[0066] Chromatographic column: Agilent Zorbax Extend-C18 (4.6 mm × 250 mm, 5 μm); mobile phase: acetonitrile (A)-0.05% formic acid solution (B); column temperature: 25 ° C; wavelength: 246 nm; flow rate: 1.0 mL / min; elution gradient: 0 min (10% A: 90% B), 30 min (50% A: 50% B), 45 min (55% A: 45% B), 55 min (80% A: 20% B), 55-65 min (80% A: 20% B). In this article, "raw product" refers to pure natural medicinal materials that have not been processed or have been simply processed and purified.

[0067] The present application provides a method for detecting the toxicity of psoralea corylifolia products, comprising:

[0068] Measure the content of each component in Psoralea corylifolia; standardize the content of each component to obtain a standardized value; use biological LC 50 The value is used as an indicator to calculate the toxicity coefficient of the ingredient; the toxicity of each ingredient is calculated by content and toxicity coefficient, and standardized to obtain the standardized value of toxicity; a quantity-toxicity relationship heat map is made by the standardized value of content and the standardized value of toxicity; the toxicity of Psoralea corylifolia products is reflected by the quantity-toxicity relationship heat map.

[0069] It should be understood that in this application, the terms "dose-toxicity relationship heat map", "heat map", "color card map", "color map" and the like are all referred to in this document. Figures 11 to 19 These terms may be used interchangeably herein and are not intended to limit the scope of the present application.

[0070] Among them, the ingredients include one or more of psoralen, isopsoralen, psoralen, isopsoralen, psoralenidine, neopsoralen isoflavones, psoralen A, psoralen nin, psoralen chalcone, psoralen B, psoralen dihydroflavone methyl ether, Corylifol A, 4'-O-methylpsoralen chalcone, and bakuchiol.

[0071] In one embodiment of the present application, the toxicity reference component is preferably any one or more of psoralen, 4'-O-methylpsoralen chalcone, psoralen, Corylifol A, psoralen dihydroflavone methyl ether, psoralen, and isopsoralen.

[0072] Specifically, in the present application, a high performance liquid chromatograph is used to analyze the content of each component of Psoralea corylifolia. At the same time, according to the model of the high performance liquid chromatograph, the present application also discloses the specific conditions for the method for determining the content of each component of Psoralea corylifolia. Those skilled in the art may also use other known equipment to analyze the content of each component of Psoralea corylifolia.

[0073] After obtaining the content of each component of Psoralea corylifolia, the content values ​​of each component measured were further LOG 2 (M+1) standardization (Note: M is the content) is used as the standardized value of the content of each component.

[0074] Subsequently, zebrafish (2dfp-6dpf) were used to evaluate the toxicity of multiple components in Psoralea corylifolia. 50 The LC value of the compound on zebrafish was used as an indicator to evaluate the toxicity. 50 The value is negatively correlated with the toxicity. The more toxic the compound is, the lower the concentration LC that causes half of the fish to die. 50 The smaller the value, the less toxic it is. 50 In this application, "dfp" means "days post fertilization", which means "days after fertilization".

[0075] The contents of multiple components in Psoralea corylifolia were multiplied by the toxicity coefficient of each component, and the product was used as the toxicity value of each component in Psoralea corylifolia. 2 (M+1) standardization (note: M is the toxicity), that is, the toxicity standardization value associated with the content of each component in Psoralea corylifolia is obtained. In order to intuitively reflect and compare the toxicity, this application sets a compound as a toxicity reference component (such as psoralen), and its toxicity coefficient value is set to 1, and the toxicity coefficient values ​​of other components are the LC values ​​of the toxicity reference component (such as psoralen) 50 Divide the LC value of other components 50 Table 13 is prepared accordingly.

[0076] The standardized values ​​of the contents of multiple components in Psoralea corylifolia and the standardized values ​​of the corresponding toxicity were respectively made into heat maps, thus obtaining a quantity-toxicity color card that correlates the content of Psoralea corylifolia and the toxicity, so as to intuitively reflect the differences in the contents of each component and its corresponding toxicity by using color differences.

[0077] In one embodiment of the present application, the toxicity of multiple components in Psoralea corylifolia can be evaluated using zebrafish, cells, mice, or rats.

[0078] Therefore, the present application provides a quantity-toxicity relationship heat map (color card) in Psoralea corylifolia products, which can intuitively reflect the differences in the content of each component and its corresponding toxicity through color differences. The present application also provides the application of the quantity-toxicity relationship heat map in detecting the toxicity of Psoralea corylifolia products.

[0079] The present application also provides the application of any of the aforementioned methods in the production process of Psoralea corylifolia products.

[0080] Specifically, the content of the ingredients in the raw Psoralea corylifolia product is determined according to the aforementioned method, and a color chart is prepared according to the aforementioned method, that is, a corresponding color chart is prepared with each of the ingredients as a toxicity reference ingredient; after each processing step of preparing the raw Psoralea corylifolia product into the Psoralea corylifolia product, a corresponding color chart is prepared for each of the ingredients as a toxicity reference ingredient; a corresponding color chart is prepared for each of the ingredients in the Psoralea corylifolia product as a toxicity reference ingredient; and the obtained color charts are compared.

[0081] In one embodiment of the present application, the toxicity reference component is preferably any one or more of psoralen, 4'-O-methylpsoralen chalcone, psoralen, Corylifol A, psoralen dihydroflavone methyl ether, psoralen, and isopsoralen.

[0082] For example, raw Psoralea corylifolia requires washing and frying steps to obtain Psoralea corylifolia products. Therefore, those skilled in the art can obtain four color charts for a certain toxic reference component at four stages: raw Psoralea corylifolia product, before washing, before frying, and Psoralea corylifolia product. For another example, samples can be extracted at various stages of the Psoralea corylifolia production process to calculate the quantity-toxicity relationship of each component and prepare a color chart, and this process will not affect the overall production flow of the Psoralea corylifolia product.

[0083] It should be understood that a drug may have both positive and negative effects on the subject to which it is administered, or in short, may bring certain side effects in the process of treating a disease, which may be caused by different components in the drug, and those skilled in the art are capable of establishing the relationship between the content / toxicity of each component in the drug and the aforementioned positive and / or negative effects.

[0084] Therefore, when technicians in this field compare these four color charts together, they can intuitively see by color which ingredients have undergone significant changes in content during the production process of the Psoralea corylifolia product, and then infer that these ingredients may have positive and / or negative effects on the subjects. On the one hand, it can help technicians in this field to judge whether it is necessary to adjust the relationship between the dosage and the number of doses according to the changes in content / toxicity when administering the drug to the subject; on the other hand, technicians in this field can optimize the production process and production process based on the comparison results of the color charts, and more precisely and accurately control the content of each ingredient in Psoralea corylifolia, so that better effects can be obtained when the Psoralea corylifolia product is administered to the subject.

[0085] This application uses high performance liquid chromatography to determine the content of 14 components in Psoralea corylifolia, and further analyzes the content of each component. 2 (M+1) standardization was performed to obtain the content standardization value. In addition, in order to visually characterize the safety differences of these ingredients, zebrafish was used to evaluate the safety of each ingredient, and LC50 The value was used as the toxicity classification index and the toxicity coefficient of each component was calculated. The toxicity of the component in Psoralea corylifolia was obtained by multiplying the component content by the toxicity coefficient, and LOG 2 (M+1) standardization processing is performed to obtain the standardized value of toxicity. The standardized values ​​of the contents of multiple components in Psoralea corylifolia and the corresponding standardized values ​​of toxicity are made into a heat map (i.e., a color card with different colors), which is a color card of the correlation between the content and toxicity of Psoralea corylifolia, so as to intuitively reflect the difference in the content of multiple components in Psoralea corylifolia and their toxicity by color difference.

[0086] Moreover, the color chart prepared by the present application can be flexibly applied to the production process of psoralea corylifolia products (or psoralea corylifolia processed products). By comparing the quantity-toxicity color charts prepared in each link, the quantity-toxicity changes of each component of psoralea corylifolia can be intuitively reflected, and then the technical personnel in the field can optimize and adjust the production process of psoralea corylifolia and the use of psoralea corylifolia products. When the preferred reference components provided by the present application are used to prepare the color chart, the color contrast of the color chart is very obvious, the color chart effect is better, and there will be no difficulty in distinguishing colors. It is representative in reflecting the quantity-toxicity relationship of each component of psoralea corylifolia, that is, the above-mentioned preferred components can be better applied to the production process of psoralea corylifolia and the use of psoralea corylifolia.

[0087] Example

[0088] The specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0089] Raw materials and experimental conditions of Psoralea corylifolia:

[0090] Table 1 Information on Psoralea corylifolia from different origins

[0091]

[0092]

[0093] Table 2 Reference material information

[0094]

[0095]

[0096] Table 3 Information of reagents used in the experiment

[0097]

[0098] Chromatographic columns and related instruments:

[0099] Chromatographic column: Agilent Zorbax Extend-C18 (4.6×250mm, 5μm); Agilent 1260 high performance liquid chromatograph (Agilent Corporation, USA, G1311C quaternary pump, G1329B automatic sampler, G1316A column oven, G4212B detector); KQ2200V ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 1 / 100,000 analytical balance (METTLER TOLEDO MS105DU); 1 / 10,000 analytical balance (METTLER TOLEDO L304T); high-speed universal pulverizer (Tianjin Test Instrument Co., Ltd.).

[0100] It should be understood that in the embodiments, "raw product" refers to a pure natural medicinal material that has not been processed or has been simply processed and purified. Specifically, "raw product" refers to the directly used psoralea corylifolia product in Table 1, or the product after simple processing of the psoralea corylifolia product in Table 1. "Salt-fried product" refers to a clean psoralea corylifolia product (clean psoralea corylifolia) fried according to the salt-fried method (General Rule 0213 of the 2020 edition of the Chinese Pharmacopoeia) until it is slightly swollen.

[0101] Example 1. Determination of chromatographic conditions

[0102] 1. Determination of chromatographic mobile phase

[0103] Preparation of test solution: Take about 0.1 g of each Psoralea corylifolia product powder (passed through No. 3 sieve) shown in Table 1, accurately weigh, place in a 25 ml volumetric flask with a stopper, add 25 ml of 80% methanol, weigh the weight, ultrasonically treat (power 150 W, frequency 40 kHz) for 40 minutes, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain.

[0104] Take the test solution of Psoralea corylifolia (Yunnan 9), inject 10 μl for chromatographic analysis, and compare different mobile phases: (1) acetonitrile-water gradient elution; (2) acetonitrile-0.1% phosphoric acid solution gradient elution; (3) acetonitrile-0.1% formic acid solution gradient elution; (4) acetonitrile-0.05% formic acid solution gradient elution, and screen out the mobile phase with better peak shape and stable baseline.

[0105] Chromatogram Figure 1 The results showed that: under acetonitrile-water gradient elution, the chromatogram had few peaks and poor peak shape, among which psoralen (t R＝ 10.016) and isopsoralen (t R＝ 10.388) The chromatographic peak separation was poor and the peak area was small; the gradient elution chromatograms of acetonitrile-0.05% formic acid solution or acetonitrile-0.1% phosphoric acid or acetonitrile-0.1% formic acid solution had many peaks, good peak shape, stable baseline, and comparable effect. Psoralen (t R＝10.016) and isopsoralen (t R＝ 10.388) The chromatographic peak has a good shape and good separation. Therefore, the acetonitrile-0.1% phosphoric acid solution or acetonitrile-0.05% formic acid or 0.1% formic acid solution gradient elution system is selected, among which 0.05% formic acid has the advantage of less solvent consumption.

[0106] 2. Determination of column temperature

[0107] Take the test solution of Psoralea corylifolia (Yunnan 9) and compare different column temperatures: 25℃; 30℃; 35℃. Screen out the column temperature with better peak shape and separation.

[0108] Chromatogram Figure 2 The results showed that compared with the conditions of 30℃ and 35℃, the peaks in the chromatogram at 25℃ had better separation and peak shape, and Psoralea corylifolia (t R :37.279min)、Psoralea corylifolia chalcone (t R :38.274min), psoralen (t R :38.855min) The chromatographic peak separation is relatively good. Therefore, the preferred column temperature is 25℃.

[0109] 3. Determination of elution gradient

[0110] Take the test solution of Psoralea corylifolia (Yunnan 9), inject 10 μl for chromatographic analysis, compare different elution gradients of acetonitrile-0.05% formic acid (see Table 4), and screen out the elution gradient with better peak shape and separation. Among them, A represents acetonitrile and B represents 0.05% formic acid.

[0111] Table 4 Different elution gradient information

[0112]

[0113] Chromatogram Figure 3 The results showed that under the elution gradient 2 condition, the chromatogram had many peaks, good peak shape, high peak separation, and a stable baseline. R :37.279min)、Psoralea corylifolia chalcone (t R :38.274min), psoralen (t R :38.855min) chromatographic peak separation effect is better. Elution gradient 2 is better than the other two elution gradient conditions. At the same time, elution gradient 2 can be expressed as: 0min (10% A: 90% B), 30min (50% A: 50% B), 45min (55% A: 45% B), 55min (80% A: 20% B), 55-65min (80% A: 20% B).

[0114] 4. Determination of detection wavelength

[0115] Take the test solution of Psoralea corylifolia (Yunnan 9), inject 10 μl for chromatographic analysis, and use acetonitrile-0.05% formic acid gradient elution. Under the same chromatographic conditions, set different detection wavelengths: 325 nm, 310 nm, 280 nm, 270 nm, 254 nm, 246 nm, and 242 nm.

[0116] The optimal detection wavelength was determined by comparing the peak shapes and absorbance values ​​of the chromatographic peaks.

[0117] Chromatogram Figure 4 As shown, the results show that when the detection wavelength is 242nm and 246nm, the chromatographic peaks are the most and the separation is better, which can better take into account the sensitivity of various component peaks, among which 246nm has a better effect.

[0118] Through this embodiment, the preferred chromatographic conditions for determining the content of one or more components of Psoralea corylifolia in Psoralea corylifolia products are determined, and the preferred chromatographic conditions are as follows:

[0119] Chromatographic column: Agilent Zorbax Extend-C18 (4.6mm×250mm, 5μm); mobile phase: acetonitrile (A)-0.05% formic acid solution (B); column temperature: 25°C; wavelength: 246nm; flow rate: 1.0mL / min; elution gradient: 0min (10% A:90% B), 30min (50% A:50% B), 45min (55% A:45% B), 55min (80% A:20% B), 55-65min (80% A:20% B).

[0120] Example 2. Determination of the test solution preparation method

[0121] 1. Determination of the test solution extraction method

[0122] Soxhlet extraction: Take about 0.5g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve), weigh accurately, place in a Soxhlet extractor, add 50ml of methanol, heat and reflux to extract for 2 hours, cool, transfer to a 100ml volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate.

[0123] Reflux extraction: Take about 0.5g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve), weigh accurately, put it in a round-bottom flask, add 100ml of methanol, heat and reflux to extract for 2 hours, cool, transfer to a 100ml volumetric flask, add methanol to the scale, shake well, filter, and take the filtrate to obtain.

[0124] Ultrasonic extraction: Take about 0.1g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve), accurately weigh, place in a 25ml volumetric flask with a stopper, add 25ml of 80% methanol, weigh the weight, ultrasonically treat (power 150W, frequency 40kHz) for 40 minutes, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain.

[0125] 10 μL of the three test solutions were injected for chromatographic analysis according to the preferred chromatographic conditions in Example 1.

[0126] The results are as follows Figure 5 As shown, it shows that reflux extraction, Soxhlet extraction and ultrasound have equivalent effects, but the extraction time of reflux and Soxhlet extraction methods is long, which is 3 times of the ultrasound time in actual operation, and the ultrasonic extraction method has a high extraction rate for psoralen and isopsoralen, so the preferred method is the ultrasonic extraction method.

[0127] 2. Selection of extraction solvent concentration

[0128] Take three portions of about 0.1g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve), accurately weigh them, and place them in 25ml stoppered volumetric flasks respectively. Add 50% methanol, 80% methanol and 25ml of methanol, weigh the weight, and treat with ultrasound (power 150W, frequency 40kHz) for 40 minutes. Cool, weigh the weight again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain the product.

[0129] 10 μL of the above test solution was injected according to the preferred chromatographic conditions in Example 1 for chromatographic analysis.

[0130] The results are as follows Figure 6 As shown, the extraction efficiency of 50% methanol is low, and the extraction effects of 80% methanol and 100% methanol are equivalent. However, the extraction efficiency of 100% methanol for coumarin components such as psoralen and isopsoralen is low, so the 80% methanol extraction method is selected.

[0131] 3. Determination of extraction time

[0132] Take five portions of about 0.1g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve), accurately weigh, place in a 25ml volumetric flask with a stopper, add 25ml of 80% methanol, weigh the weight, and ultrasonically treat (power 150W, frequency 40kHz) for 20, 30, 40, 50, and 60 minutes respectively. Cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain.

[0133] 10 μL of the above test solution was injected according to the preferred chromatographic conditions in Example 1 for chromatographic analysis.

[0134] The results are as follows Figure 7As shown, the extraction effects of ultrasonic extraction for 30min, 40min, 50min, and 60min were equivalent to those of ultrasonic extraction for 20min, which were about 103%, 104%, 102%, and 98% of that for 20min. Therefore, the ultrasonic extraction time should not be less than 20min, and the optimal ultrasonic extraction time was 40min.

[0135] 4. Determination of the test solution configuration method (sampling volume, concentration)

[0136] Preparation method 1: Take about 0.1g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve), accurately weigh, put into a 25ml stoppered volumetric flask, accurately add 25ml of 80% methanol, weigh the weight, ultrasonically treat (power 150W, frequency 40kHz) for 40 minutes, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain.

[0137] Preparation method 2: Take about 0.25g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve), accurately weigh, put into a 50ml stoppered volumetric flask, accurately add 50ml of 80% methanol, weigh the weight, ultrasonically treat (power 150W, frequency 40kHz) for 40 minutes, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain.

[0138] Preparation method 3: Take about 0.5g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve), accurately weigh, put into a 100ml stoppered volumetric flask, accurately add 100ml of 80% methanol, weigh the weight, ultrasonically treat (power 150W, frequency 40kHz) for 40 minutes, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain.

[0139] 10 μL of the above test solution was injected according to the preferred chromatographic conditions in Example 1 for chromatographic analysis.

[0140] The results are as follows Figure 8 As shown, it can be seen that the peak areas of the 14 components measured at the same Psoralea corylifolia concentration (4 mg raw product / ml) are similar for different sampling amounts or different test solution concentrations. Among them, Preparation Method 1 has a smaller sampling amount and less solvent usage, and is better.

[0141] 5. Determination of the method of adding 25 ml of 80% methanol

[0142] β-glucosidase in Psoralea corylifolia can convert psoralen and isopsoralen into psoralen and isopsoralen. β-glucosidase in raw Psoralea corylifolia retains its activity and easily converts glycosides into aglycones in aqueous solvents. However, this conversion will not occur after frying of salt products, as β-glucosidase is inactivated. Therefore, β-glucosidase needs to be inactivated when measuring raw Psoralea corylifolia. The enzyme can be inactivated by immersion in methanol.

[0143] Method 1 (first add methanol to soak to kill the enzyme, then add water to 80% methanol): Take about 0.1g of Psoralea corylifolia (Yunnan 22, raw product) powder (pass through No. 3 sieve), accurately weigh, put it in a 25ml stoppered volumetric flask, accurately add 20ml of methanol, soak for 30min or 45min, 60min, 90min, 120min, then add 5ml of water, weigh the weight, ultrasonically treat (power 150W, frequency 40kHz) for 40 minutes, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate.

[0144] Method 2 (directly adding 80% methanol): Take about 0.1g of Psoralea corylifolia (Yunnan 22, raw product, salt-roasted product) powder (passed through No. 3 sieve), accurately weigh, put it in a 25ml volumetric flask with a stopper, accurately add 25ml of 80% methanol, weigh the weight, soak for 0min or 30min, 45min, 60min, 90min, 120min, ultrasonically treat (power 150W, frequency 40kHz) for 40 minutes, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate.

[0145] 10 μL of the above test solution was injected according to the preferred chromatographic conditions in Example 1 for chromatographic analysis.

[0146] The results are shown in Table 5. The β-glucosidase can be inactivated by first soaking in methanol. The chromatographic peak areas of psoralen, isopsoralen, psoralen and isopsoralen in the raw Psoralea corylifolia product test solution with different soaking times are equivalent, and the peak areas of the corresponding components of the salt-fried product are also equivalent; however, soaking in aqueous methanol (80% methanol) cannot inactivate the enzyme, and the peak areas of psoralen and isopsoralen are significantly reduced, while the peak areas of psoralen and isopsoralen are significantly increased, indicating that the glycosides are converted to aglycones, and the results of the measured glycoside and aglycone content are inaccurate. Therefore, when determining the raw Psoralea corylifolia product, it is necessary to first add methanol to soak to inactivate the β-glucosidase, and then add water to make the volume fraction of methanol reach 80%. However, the β-glucosidase in the salt-fried product is inactivated, and 80% methanol will not convert the glycosides into aglycones. Directly adding 80% methanol for ultrasonic treatment will not affect the content determination results of glycosides and aglycones.

[0147] Table 5 Effect of methanol immersion on the peak areas of psoralen, isopsoralen, psoralen and isopsoralen

[0148]

[0149]

[0150] Through this embodiment, the optimal preparation method of the test solution for determining the content of one or more components of Psoralea corylifolia in Psoralea corylifolia products is determined. The preferred preparation method of the test solution is as follows:

[0151] Take about 0.1 g of the Psoralea corylifolia powder (passed through a No. 3 sieve) in Table 1, accurately weigh it, put it in a 25 ml volumetric flask with a stopper, add 25 ml of 80% methanol (when measuring the raw Psoralea corylifolia, first add 20 ml of methanol and soak for 30 minutes or more, then add 5 ml of water), weigh it, treat it with ultrasound (power 150 W, frequency 40 kHz) for 40 minutes, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0152] Example 3. The psoralea corylifolia methods provided in Example 1 and Example 2 were tested.

[0153] 1. Investigation of linear relationship, detection limit and quantification limit

[0154] Accurately weigh appropriate amounts of reference substances (1) psoralen, (2) isopsoralen, (3) psoralen, and (4) isopsoralen, respectively, and add 80% methanol to a volumetric flask to dissolve and make up to volume to form reference substance stock solutions with concentrations of (1) psoralen (2.71 mg / ml), (2) isopsoralen (2.10 mg / ml), (3) psoralen (1.02 mg / ml), and (4) isopsoralen (1.11 mg / ml).

[0155] Accurately pipette appropriate amount of reference substance stock solution, place in 5 ml volumetric flask and mix (psoralen, isopsoralen, psoralen and isopsoralen are mixed as psoralen mixed standard), add 80% methanol to make up to volume, obtain initial concentration mixed reference substance psoralen mixed standard 1, and dilute in half to obtain reference substance mixed solutions of different concentrations. The concentration ranges are (1) 0.26-542.00 μg / ml, (2) 0.13-263.00 μg / ml, (3) 0.05-102.30 μg / ml, (4) 0.05-110.90 μg / ml.

[0156] Accurately weigh appropriate amounts of reference substances (5) new psoralen isoflavones, (6) psoralen A, (7) psoralen, (8) psoralen chalcone, (9) psoralen, (10) psoralen B, (11) psoralen dihydroflavonoid methyl ether, (12) Corylifol A (13) 4'-O-methyl psoralen chalcone, and (14) bakuchiol, and dissolve them in a volumetric flask with methanol and make up to volume to form a concentration of new psoralen isoflavones (1.36 mg / ml), psoralen A (1.01 mg / ml), psoralen (1.06 mg / ml), psoralen chalcone (0.78 mg / ml), psoralen (0.53 mg / ml), psoralen B (1.19 mg / ml), psoralen dihydroflavonoid methyl ether (1.06 mg / ml), Corylifol A (14) 4'-O-methyl psoralen chalcone, and bakuchiol. A (0.80 mg / ml), 4'-O-methylpsoralen chalcone (0.59 mg / ml), and bakuchiol (12.76 mg / ml) were used as control stock solutions.

[0157] Accurately pipette an appropriate amount of reference substance stock solution, place it in a 5 ml volumetric flask and mix it (new psoralen isoflavones, psoralen A, psoralen, psoralen chalcone, psoralen, psoralen B, psoralen dihydroflavone methyl ether, Corylifol A, 4'-O-methyl psoralen chalcone, and bakuchiol are mixed as the bakuchiol mixed standard), add 80% methanol to the scale to obtain the initial concentration mixed reference substance bakuchiol mixed standard 1, and dilute them in half to form reference substance mixed solutions of different concentrations. The concentration ranges are (5) 0.05-103.60 μg / ml, (6) 0.05-101.40 μg / ml, (7) 0.05-106.10 μg / ml, (8) 0.02-38.75 μg / ml, (9) 0.03-65.75 μg / ml, (10) 0.03-59.25 μg / ml, (11) 0.03-52.75 μg / ml, (12) 0.02-39.75 μg / ml, (13) 0.03-58.50 μg / ml, (14) 0.31-638 μg / ml.

[0158] According to the preferred chromatographic conditions of Example 1, linear regression was performed on the peak area versus the concentration to obtain a regression equation. As shown in Table 6, 14 compounds showed good linearity within the linear range (r≥0.9998).

[0159] Table 6 Linear relationship, detection limit and quantification limit results

[0160]

[0161] 2. Precision test

[0162] Take about 0.1g of Psoralea corylifolia (Yunnan 9) powder (pass through No. 3 sieve), weigh accurately, put it in a 25ml stoppered volumetric bottle, add 25ml of 80% methanol, weigh it, ultrasonically treat it (power 150W, frequency 40kHz) for 40 minutes, let it cool, weigh it again, make up the lost weight with 80% methanol, shake it well, filter it, and take the filtrate. Accurately draw 10μL into the liquid chromatograph, repeat the injection 6 times, record the chromatographic peak area of ​​each index component, and calculate the RSD. The experimental results are shown in Table 7.

[0163] Table 7 Precision test results

[0164]

[0165]

[0166] 3. Repeatability test

[0167] Take about 0.1g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve) in 6 portions, accurately weigh, place in a 25ml stoppered volumetric bottle, add 25ml of 80% methanol, weigh, ultrasonically treat (power 150W, frequency 40kHz) for 40 minutes, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate. Accurately draw 10μL into the liquid chromatograph, repeat the injection 6 times, calculate the content of each index component, and calculate the RSD. The experimental results are shown in Table 8.

[0168] Table 8 Repeatability test results (mg / g crude drug)

[0169]

[0170] 4. Stability test

[0171] The same Yunnan 9 test solution under the "precision test" was accurately drawn, and 10 μL was injected at 0, 2, 4, 6, 8, 12, 18, and 24 h respectively according to the preferred chromatographic conditions, and HPLC (high performance liquid chromatography) analysis was performed, and the chromatographic peak area of ​​each index component was recorded and the RSD was calculated. The experimental results are shown in Table 9.

[0172] Table 9 Stability test results

[0173]

[0174]

[0175] 5. Accuracy test

[0176] Take about 0.05g of Psoralea corylifolia (Yunnan 9) powder (passed through No. 3 sieve) with known content in 6 portions, add an equal amount of reference substance, put in a 25ml volumetric flask, add 80% methanol to the scale, weigh the weight, ultrasonically treat (power 150W, frequency 40kHz) for 40 minutes, let cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain. Analyze according to the preferred chromatographic conditions.

[0177] The experimental results are shown in Table 10. The average recoveries of the fourteen components in Yunnan 9 were 91.36% to 109.36%, and the RSDs were 1.96% to 5.35%.

[0178] Recovery rate = (total amount measured - known amount) / added amount * 100%

[0179] Table 10 Accuracy test results

[0180]

[0181] In this embodiment, by measuring the reference substances of each component in Psoralea corylifolia, it can be verified that the determination method provided by this application has very high precision, and its RSD can be as low as 0.27%. At the same time, this embodiment also conducts repeatability verification and accuracy tests on the determination method, and has excellent RSD at different times. The RSD of repeatability verification can be as low as 1%, and the RSD of accuracy test is mostly lower than 3%, and the stability test is around 2%. It further illustrates that the method of this application can fully and efficiently extract each component in the Psoralea corylifolia product, accurately determine the content of each component, and can fully recover each component with a recovery rate of more than 90%.

[0182] At the same time, it should be understood that in the "linear relationship, detection limit and quantification limit investigation", when the reference substance (i.e. the pure substance of each component) is used for chromatographic analysis, a standard curve of the concentration and peak area of ​​each component is obtained. Therefore, when the chromatographic analysis of each component in the Psoralea corylifolia sample is performed, a technician in this field can easily calculate the concentration based on the standard curve and the chromatographic area, and then calculate the specific content of each component.

[0183] Example 4. Determination of the contents of multiple components in psoralea corylifolia products and their standardization

[0184] To determine the contents of coumarins, flavonoids and monoterpene phenols in Psoralea corylifolia (such as psoralen, isopsoralen, psoralen, isopsoralen, neopsoralen isoflavones, psoralen A (psoralen dihydroflavone), psoralenin (psoralen isoflavones), psoralen chalcone, psoralenidine, psoralen B (isopsoralen chalcone), psoralen dihydroflavone methyl ether, Corylifol A, 4'-O-methylpsoralen chalcone or bakuchiol).

[0185] The determination was carried out using the preferred chromatographic conditions and test solution preparation method in Example 1.

[0186] Preparation of test solution: Take about 0.1g of Psoralea corylifolia powder (passed through No. 3 sieve), weigh accurately, put it in a 25ml volumetric flask with a stopper, add 25ml of 80% methanol (when measuring raw Psoralea corylifolia, first add 20ml of methanol and soak for 30min or more, then add 5ml of water), weigh the weight, ultrasonically treat (power 150W, frequency 40kHz) for 40 minutes, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, and take the filtrate to obtain.

[0187] Preparation of reference solution: Same as in “Example 3, investigation of linear relationship, detection limit and quantitation limit”.

[0188] Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine. The representative chromatogram of Psoralea corylifolia and reference solution is shown in Fig. 9 It can be seen that the assay method provided in the present application can well distinguish the 14 components in the Psoralea corylifolia product.

[0189] 1. Determination and standardization of the contents of multiple components in Psoralea corylifolia products

[0190] The method described in Example 1 and Example 2 was used to determine the content of one or more components of coumarins, flavonoids or monoterpene phenols in the 55 products in Table 1 (such as psoralen, isopsoralen, psoralen, isopsoralen, neopsoralen isoflavones, psoralen A (psoralen dihydroflavone), psoralen nin (psoralen isoflavones), psoralen chalcone, psoralen, psoralen B (isopsoralen chalcone), psoralen dihydroflavone methyl ether, Corylifol A, 4'-O-methylpsoralen chalcone or bakuchiol). The content values ​​of each component measured were further analyzed by LOG. 2 (M+1) standardization (Note: M is the content).

[0191]

[0192]

[0193] 2. Toxicity determination and standardization of multiple components in Psoralea corylifolia products

[0194] Raw materials and experimental conditions:

[0195] The fish used in the experiment were wild-type (offspring of the Tuebigen line) zebrafish, purchased from Nanjing Yaoshunyu Biotechnology Co., Ltd., about 6 months old. Adult zebrafish were allowed to mate freely in water at (28.0±0.5)℃ to reproduce eggs.

[0196] Reference substances shown in Table 2, sodium chloride (GENERAL-REAGNT, batch number: P1548228), potassium chloride (Nanjing Chemical Reagent Co., Ltd., batch number: 120913115884), magnesium sulfate heptahydrate (Shanghai Titan Technology Co., Ltd., batch number: P10434274), calcium chloride dihydrate (Guangdong Guanghua Technology Co., Ltd., batch number: 20100823), and Wahaha purified water.

[0197] Nikon SMZ800N stereo microscope (Nikon Corporation, Japan); biochemical incubator SPX-80 (Ningbo Haishu Saifu Experimental Instrument Factory); KQ3200B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); MS105DU analytical balance (Mettler Toledo Company, Switzerland).

[0198] (1) Toxicity determination and toxicity coefficient calculation of multiple components in Psoralea corylifolia

[0199] Zebrafish (2 dfp-6 dpf) were used to evaluate the toxicity of multiple components of psoralea corylifolia (representing coumarins, flavonoids and bakuchiol). The zebrafish median lethal concentration (LC50) was used to determine the toxicity of the components of psoralea corylifolia (representing coumarins, flavonoids and bakuchiol). 50 The LC value of the compound on zebrafish was used as an indicator to evaluate the toxicity. 50 The value is negatively correlated with the toxicity. The more toxic the compound is, the lower the concentration LC that causes half of the fish to die. 50 The smaller the value, the less toxic it is. 50 In order to intuitively reflect and compare the toxicity, this application intends to set a compound as the toxicity reference component (for example, psoralen), and its toxicity coefficient value is set to 1, and the toxicity coefficient value of other components is the LC value of the toxicity reference component (such as psoralen). 50 Divide the LC value of other components 50 value.

[0200] Take appropriate amount of (iso)psoralen, (iso)psoralen, neopsoralen isoflavones, psoralen A, psoralen, psoralen chalcone, psoralen, psoralen B, psoralen dihydroflavone methyl ether, Corylifol A, 4'-O-methylpsoralen chalcone, and bakuchiol reference substance, accurately weigh, add appropriate amount of DMSO to aid solubilization, and prepare with culture medium. The concentrations are shown in Table 12.

[0201] Table 12 Compound preparation concentration table

[0202] Compound Concentration (μmol / L) Psoralen 200、400、600、800、1000、1200、1500、2000 Isopsoralen 200、400、600、800、1000、1200、1500、2000 Psoralen 10、25、50、100、150、200 Isopsoralen 10、25、50、100、150、200 New Psoralea corylifolia Isoflavones 1、5、10、15、25、50 Psoralen 1、5、10、15、25、50 Psoralea corylifolia 100、200、300、400 Psoralea corylifolia Chalcone 5、7.5、12.5、25、50、100 Psoralen 100、200、400 Psoralen 1、5、10、15、25、50 Psoralea corylifolia dihydroflavonoid methyl ether 10、25、50、100、150 CorylifolA 10、25、50、100、150、300、600 4'-O-Methylpsoralen chalcone 10、25、50、100、150、300 Bakuchiol 1、2.5、5、7.5、10、15、25

[0203] Embryos were produced by natural mating of adult zebrafish, and the embryos were placed in culture medium and cultured in a 28.5°C incubator for 24 hours. Healthy zebrafish embryos 1 day after fertilization (1dpf) were taken and placed in a 24-well plate, with 10 embryos per well, 20 to 30 embryos per group, and exposed to different concentrations of each drug group, 2mL solution per well, and culture medium was used as a blank control. The eggs and fry were observed every day to see if they died and were removed in time, and the number of deaths was recorded, and the number of fish was recorded until 6dpf.

[0204] After the 14 components of Psoralea corylifolia were administered to zebrafish, the mortality rate of zebrafish varied with the administration time and concentration. The mortality results of zebrafish of 2dpf-6dpf for each component are shown in Fig. 9 .

[0205] The zebrafish LC of each component was calculated using the statistical software SPSS16.0 50 (6dpf) value. One of the above 14 components is set as the toxicity reference component, and its toxicity coefficient value is set to 1. The toxicity coefficient values ​​of the other components are the LC values ​​of the toxicity reference component. 50 Divide the LC value of other components 50 Taking psoralen as the toxicity reference component, its toxicity coefficient is 1, and the toxicity coefficient values ​​of other components are the LC 50 Divide the LC value of other components 50 Theoretically, any component can be selected as the toxicity reference component, see Table 13.

[0206]

[0207] The content of the measured components in Psoralea corylifolia was multiplied by their respective toxicity coefficients, and the product was used as the toxicity value of the measured components in Psoralea corylifolia. The toxicity value was further analyzed by LOG. 2 (M+1) standardization, that is, the toxicity standardization value associated with the measured component content in Psoralea corylifolia. Toxicity = content * toxicity coefficient; toxicity standardization value = LOG 2 (M+1) (Note: M is the toxic amount).

[0208] 3. Visualization of the content and toxicity of multiple components of Psoralea corylifolia

[0209] The standardized values ​​of the contents of multiple components in Psoralea corylifolia obtained in 1 and 2 of this embodiment and the standardized values ​​of the corresponding toxicity obtained are respectively made into heat maps, that is, the quantity-toxicity color card of the content of Psoralea corylifolia and the toxicity associated with the toxicity is obtained, so as to realize the intuitive reflection of the difference in the content of each component and its corresponding toxicity by color difference. Blue indicates a lower standardized value, and red indicates a higher standardized value.

[0210] For example, when psoralen is used as a reference toxicity reference, Fig.11On the left are the initials of the pinyin codes of the 55 psoralea products shown in Table 1 (the corresponding relationship is shown in Table 11), on the right are the corresponding relationships between the toxicity or standardized value and the color, and below are the contents and toxicity of the 14 components of psoralea products (for example, "psoralen-amount" means the standardized value of the content of psoralen, and "psoralen-toxicity" means the standardized value of the toxicity of psoralen).

[0211] According to the toxicity coefficients provided in Table 13, firstly, there are 14 ingredients in the psoralea corylifolia product, ranging from LC 50 Starting from the component with the largest value, each component of Psoralea corylifolia was set as a toxicity reference component, and the toxicity coefficient and toxicity of other components were calculated, and 14 color cards were made based on this. The applicant of this application found that LC 50 Too large or too small a value will result in unclear distinction of the colors in the color card, resulting in poor color card effect.

[0212] When using isopsoralen (LC 50 (μg / mL): 19.05) as the reference component, it can be seen that LC 50 Much greater than isopsoralen LC 50 The toxicity coefficient of the reference condition is very small, and the values ​​are very close and concentrated. In this case, the toxicity standardized values ​​calculated by the toxicity coefficient will also become very close, and the calculated toxicity standardized values ​​are also too small and concentrated. If it is made into a color card, it should be understood that the blue (the color with a lower standardized value) will become unclear (not shown in the figure). In addition, the content is low and the LC 50 The blue color of the toxicity color chart of relatively large components such as psoralen, psoralen, 4'-O-methyl psoralen chalcone, Corylifol A, and psoralen dihydroflavonoid methyl ether is not clearly distinguished, so it is further speculated that LC 50 Ingredients with values ​​lower than isopsoralen are not suitable as toxicity reference ingredients.

[0213] If the LC of other components in Psoralea corylifolia 50 The value is between 19.05 (isopsoralen) and 73.55 (psoralen), or LC 50 When the value (μg / mL) is greater than the maximum value 461.84 in the present application, the preferred toxicity reference component LC can be further determined based on the effect of the toxicity-quantity color chart. 50 (μg / mL) value is in the optimal range.

[0214] It is proposed to use a component in Psoralea corylifolia as an unknown toxicity reference component, and its LC 50The value is set in the range of 19.05 to 73.55, such as 30, 40, 50, 60 μg / mL and above the maximum value (461.84), such as 500, 550, 600 μg / mL. In this example, Psoralea corylifolia is assumed to be an unknown component X, and its LC 50 (μg / mL) is 30, it is named X30. Similarly, according to LC 50 The values ​​are changed and named X40, X50, X60, X500, X550 and X600 in sequence. The corresponding color charts ( Figure 19-25 ).

[0215] From the color chart, we can see that LC 50 (μg / mL) at 30, 40, 50, the toxicity color chart ( Fig.19 , Fig. 20 , Fig.21 ) and isopsoralen (LC 50 (μg / mL): 19.05) is not significantly improved compared with the color chart of the reference ingredient; at 60μg / mL, the effect ( Fig. 22 ) is close to psoralen (LC 50 (μg / mL): 73.55) is the color chart of the reference ingredient ( Fig.14 ); when LC 50 (μg / mL) equal to or greater than 500 ( Figure 23-25 ), the blue content area in the color card is very concentrated, and the difference is not obvious. At the same time, the toxicity area is collectively red, that is, some ingredients with low toxicity in essence will also be shown as having high toxicity or high toxicity in the color card. 50 When the concentration (μg / mL) is equal to or greater than 500, the performance in the color chart is poor.

[0216] And LC 50 When components with smaller values, such as isopsoralen and bakuchiol, are used as toxicity reference components, the color distinction of the heat map using the standardized toxicity value is not obvious ( Fig.12 , Fig.18 ). Therefore, the LC of the toxic reference component of Psoralea corylifolia can be determined 50 The optimal range of (μg / mL) value is 60-500. This also shows that LC 50 Ingredients with (μg / mL) values ​​between 60 and 500 can be used as reference ingredients for psoralen toxicity.

[0217] This application adopts LC 50The components with values ​​in the optimal range, such as psoralen, psoralen, psoralen, 4'-O-methyl psoralen chalcone, Corylifol A, and psoralen dihydroflavonoid methyl ether, were used as toxicity reference components. The toxicity coefficient of each component was calculated. The content of each component was multiplied by the toxicity coefficient to obtain the toxicity of each component in psoralen, and the toxicity was logarithmized. 2 (M+1) standardization, the content standardization value and the toxicity standardization value are made into a heat map (see Fig.11 , Figure 13 to Figure 17 ), the color distinction is more obvious.

[0218] In summary, psoralen, 4'-O-methylpsoralen chalcone, psoralenin and Corylifol A are the better toxicity reference ingredients; followed by psoralen dihydroflavone methyl ether, psoralen and isopsoralen; while psoralen B, neopsoralen isoflavones, psoralen chalcone, psoralen A, bakuchiol, psoralen and isopsoralen are not suitable as toxicity reference ingredients.

[0219] Example 5. Visual analysis of the content and toxicity of multiple components in different processed products of Psoralea corylifolia

[0220] 1. Preparation of different processed products of Psoralea corylifolia

[0221] Psoralea corylifolia (raw or purified, Yunnan 22): remove impurities.

[0222] Salted Psoralea corylifolia (Psoralea corylifolia salt-roasted product, Yunnan 22): Take clean Psoralea corylifolia and stir-fry it according to the salt-roasting method (General Rule 0213 of the 2020 edition of the Chinese Pharmacopoeia) until it is slightly swollen.

[0223] Stir-fried Psoralea corylifolia (Stir-fried Psoralea corylifolia, Yunnan 22): Take clean Psoralea corylifolia and stir-fry it according to the stir-frying method (General Rule 0213 of the 2020 edition of the Chinese Pharmacopoeia) until it slightly bulges, cracks and emits an aromatic aroma.

[0224] 2. Using the method described in Example 4, the contents of multiple components in three processed products of Psoralea corylifolia (Yunnan 22) were determined and standardized; the toxicity of multiple components in three processed products of Psoralea corylifolia was determined and standardized; the contents and toxicity of multiple components in three processed products of Psoralea corylifolia were visualized.

[0225] The standardized values ​​of the contents of multiple components in the three processed products of Psoralea corylifolia and the standardized values ​​of the corresponding toxicity were plotted as heat maps, thus obtaining the quantity-toxicity color chart associated with the contents of the three processed products and the toxicity ( Fig.26 ). The color chart shows that the content and toxicity of the three processed products are not much different, indicating that salt roasting and stir-frying have little effect on the content and toxicity of Psoralea corylifolia.

[0226] Example 6. Visual analysis of the content and toxicity of multiple components in the decoction of Psoralea corylifolia

[0227] 1. Preparation of Psoralea corylifolia decoction

[0228] Psoralea corylifolia decoction: Weigh 10g each of raw and salt-roasted Psoralea corylifolia (Yunnan 22) (not crushed), add 10 times the amount of water to each decoction twice, each time for half an hour, combine the decoctions, filter, add pure water to the filtrate to 200mL (50mg crude drug / mL), take an appropriate amount and add 80% methanol to prepare 10mg crude drug / ml for HPLC analysis.

[0229] 2. The method described in Example 4 was used to determine the standardized values ​​of the contents of multiple components in raw and salt-fried Psoralea corylifolia (Yunnan 22) and their decoctions, and a heat map was made with the standardized values ​​of the corresponding toxicity, thus obtaining a quantity-toxicity color chart ( Fig. 27 ).

[0230] It can be seen that the amount of flavonoids and bakuchiol in the water decoction of Psoralea corylifolia - the blue area of ​​the toxicity color card has obviously become darker or the red area has become lighter, indicating that the content of flavonoids and bakuchiol toxic components in the water decoction is low. Therefore, it shows that when flavonoids and bakuchiol are likely to have a huge impact on the subjects, those skilled in the art should use the water decoction to process Psoralea corylifolia, which is conducive to the safe use of Psoralea corylifolia.

[0231] Therefore, it can be understood that the amount-toxicity method of the present application and the color chart provided by the present application can synchronously and instantly react the relationship between the content and toxicity of each component in the psoralea corylifolia product, so that the observer can quickly identify the change in toxicity under different content levels, and then make a basic judgment on the toxicity of the product. When a certain component in the psoralea corylifolia Chinese medicine is regarded by those skilled in the art as being able to be used for treating or treating a certain disease or disease, those skilled in the art can fully use the above method and the above color chart to optimize the process of the psoralea corylifolia product, reduce the loss of the component in production, increase the proportion of the component in the medicine, and reduce the content or toxicity of other components at the same time, so as to achieve better effects when applied to the subject. The above is only a preferred embodiment of the present application, and is not intended to limit the present application in other forms. Any technician familiar with the profession may use the above-mentioned disclosed technical content to be changed or modified into an equivalent embodiment of equivalent changes. However, any simple modification, equivalent changes and modification made to the above embodiments according to the technical essence of the present application without departing from the technical solution content of the present application still belong to the protection scope of the technical solution of the present application.

Claims

1. A method for determining the content of one or more components of Psoralea corylifolia in a Psoralea corylifolia product, comprising: Preparation of the test solution: Use ultrasonic extraction to extract multiple components of the psoralea corylifolia product, filter, and the obtained filtrate is the test solution; Determine the content of one or more components of Psoralea corylifolia in the test solution by liquid chromatography; The chromatographic column is a C18 reverse phase column; The mobile phase is acetonitrile and phosphoric acid solution, or acetonitrile and formic acid solution; The ingredients are one or more of psoralen, 4'-O-methyl psoralen chalcone, psoralen, Corylifol A, psoralen dihydroflavone methyl ether, psoralen glycosides, isopsoralen glycosides, psoralen B, neopsoralen isoflavones, psoralen chalcone, psoralen A, bakuchiol, psoralen and isopsoralen.

2. The method according to claim 1, characterized in that: The ultrasonic extraction method for extracting the various components of the psoralea corylifolia product includes taking 0.1-0.5g of psoralea corylifolia powder, soaking the powder of the psoralea corylifolia product in methanol, then adding water to the volume percentage of methanol to 80%, weighing the weight, then performing ultrasonic extraction, supplementing the weight with 80% volume percentage of methanol, and finally shaking well.

3. The method according to claim 2, characterized in that The powder of the psoralea corylifolia product is added to methanol for soaking, the soaking time is 0-120 minutes, preferably 30-120 minutes, and the volume fraction of the methanol is 50-100%, preferably 80-100%.

4. The method according to claim 2, characterized in that: The mass of the psoralea corylifolia powder is 0.1-0.25g.

5. The method according to claim 1, characterized in that The ultrasonic extraction time is 20-60 min, preferably 20-40 min.

6. The method according to claim 1, characterized in that The concentration of the phosphoric acid solution is 0.05-1.0%, and the concentration of the formic acid solution is 0.05-1.0%.

7. The method according to claim 1, characterized in that The chromatographic column is a ZORBAX Extend-C18 chromatographic column, and the column temperature of the chromatographic column is 25-35°C.

8. The method according to claim 1, characterized in that: The detection wavelength of liquid chromatography is 242-325 nm, preferably 242-246 nm.

9. A method for detecting the toxicity of psoralea corylifolia products, comprising Measuring the content of each component in Psoralea corylifolia; preferably detecting by the method described in any one of claims 1 to 8; The content of each component is standardized to obtain a content standardized value; Using biological LC 50 The value is used as an indicator, one component is used as a toxicity reference component, and the toxicity coefficient of each component is calculated; The toxicity of the component is calculated by the content and the toxicity coefficient, and the toxicity is standardized to obtain a toxicity standardized value; The heat map of the relationship between quantity and toxicity was produced by using the standardized values ​​of content and toxicity; The toxicity of psoralea corylifolia products was detected by using the dose-toxicity relationship heat map; Among them, the ingredients include one or more of psoralen, isopsoralen, psoralen, isopsoralen, psoralenidine, neopsoralen isoflavones, psoralen A, psoralen nin, psoralen chalcone, psoralen B, psoralen dihydroflavone methyl ether, Corylifol A, 4'-O-methylpsoralen chalcone, and bakuchiol.

10. The method according to claim 9, wherein: The toxic reference components are psoralen, isopsoralen, psoralen, psoralen, 4'-O-methyl psoralen chalcone, Corylifol A, and psoralen dihydroflavone methyl ether.

11. The method according to claim 9, wherein: The organism is zebrafish, cell, mouse, rat, preferably zebrafish.

12. Use of the method according to claim 9 in the production process of psoralea corylifolia products.

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