Quality control method of aralia elata leaves

Abstract

The invention belongs to the technical field of quality control of traditional Chinese medicines, and particularly relates to a quality control method of aralia elata leaves. The quality control method of the aralia elata leaves comprises the following steps: (1) preparing a reference substance solution; (2) preparing a test solution; (3) a determination method: respectively and precisely absorbing the reference solution and the test solution, and taking octadecylsilane chemically bonded silica as a filler; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, the flow velocity is 0.7-0.9 mL / min, a gradient elution mode is adopted, and sample introduction, determination and chromatogram recording are carried out. The method has good technical effects in the aspects of the yield of the aralia elata leaves and the precision and the stability of the quality control method.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control technology, specifically relating to a quality control method for Aralia elata leaves. Background Technology

[0002] Aralia elata, also known as dragon tooth Aralia, thorny dragon tooth, and thorny bud, is a plant belonging to the genus Aralia in the family Araliaceae. It is a plant used for both medicinal and edible purposes. Its young shoots are a nutritious green food, while its roots and bark are used in traditional medicine to treat upper abdominal pain, insomnia, and arthritis. The leaves are used to treat edema and diarrhea, and the fruit is used to treat joint pain, dysmenorrhea, and bruises. Studies have shown that different parts of Aralia elata contain abundant triterpenoid saponins, which exhibit various pharmacological activities, including antioxidant, antitumor, anti-inflammatory, antiviral, anti-cardiovascular disease, hepatoprotective, anti-diabetic and anti-complication, and neuroprotective effects. Therefore, establishing quality evaluation and control methods that reflect the antitumor effects of Aralia elata leaves is of great significance for the effective utilization of its leaf resources.

[0003] Relevant patent documents retrieved: This document, published in China (CN101040884A) on September 26, 2007, discloses a quality control method for total saponins from the traditional Chinese medicine Aralia elata, belonging to the field of traditional Chinese medicine. It includes chromatographic conditions and system suitability testing, preparation of reference solutions, preparation of test solutions, and determination. The innovation of this invention lies in its high specificity, and the fact that the precision and accuracy of high-performance liquid chromatography (HPLC) are higher than those of gravimetric methods, ultraviolet spectrophotometry, and colorimetric methods. Therefore, it is more significant for determination. However, this invention only studies the quality control method for total saponins in Aralia elata, and its accuracy and precision still need to be improved.

[0004] Relevant non-patent literature retrieved: The journal or book title is "Traditional Chinese Medicines," and the article title is "A Study on Quality Evaluation of Aralia elata Leaves Based on Spectrum-Efficacy Relationship," 2023, 54(07):2172-2181. This article used 70% ethanol and water-saturated n-butanol to extract total saponins from each batch of Aralia elata leaves. HPLC-ELSD was used to analyze the saponins and establish a fingerprint spectrum. The MTT assay was used to test the inhibitory effects of total and monomeric saponins from Aralia elata leaves on human lung adenocarcinoma A549 cells, human liver cancer SMMC-7221 cells, and human colon cancer HT-29 cells. The grey relational analysis method was used to analyze the relationship between fingerprint peaks and antitumor effects. An HPLC-ELSD fingerprint spectrum of Aralia elata leaves containing 28 common peaks was established. The similarity between the spectrum of each batch and the common pattern spectrum was above 0.850, and five peak components were identified and assigned. However, this study also has the aforementioned problems. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention aims to provide a quality control method for Aralia elata leaves, so as to solve the technical problems such as low yield of Aralia elata leaves, low precision and poor stability of quality control methods, or a combination thereof.

[0006] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0007] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0008] The definition of standard chemical terms can be found in the reference "Administrative Measures for the Formulation and Revision of Standards for Traditional Chinese Medicine Decoction Pieces (Traditional Chinese Medicinal Materials) in Heilongjiang Province" (released in 2024).

[0009] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0010] In a first aspect, the present invention provides a method for quality control of Aralia elata leaves, comprising the following steps: (1) Preparation of reference solution; (2) Preparation of the test solution; (3) Determination method: Accurately pipette the reference solution and the test solution separately, using octadecylsilane-bonded silica gel as the packing material; use acetonitrile as mobile phase A and water as mobile phase B, with a flow rate of 0.7-0.9 mL / min, and adopt a gradient elution method. The gradient elution program is as follows:

[0011] Inject the sample, measure it, and record the chromatogram to obtain the result.

[0012] Preferably, the preparation method of the reference solution in step (1) includes the following steps: take an appropriate amount of Liaodong Aralia elata saponin V reference standard, accurately weigh it, add methanol to dissolve it, and the solution is obtained.

[0013] Preferably, the preparation method of the test solution in step (2) includes the following steps: accurately weigh the powder of this product, place it in a stoppered conical flask, add methanol, seal tightly, weigh, sonicate, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and obtain the product.

[0014] Preferably, the volume concentration of methanol is 50-75%. Other specific values ​​within the above range can be selected to achieve the technical effect of the present invention, including but not limited to 50, 55, 60, 65, 70, and 75%.

[0015] More preferably, the volume concentration of the methanol is 70%.

[0016] Preferably, the amount of methanol added is 20-30 mL. Other specific values ​​within the above range can be selected to achieve the technical effect of the present invention, including but not limited to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 mL.

[0017] More preferably, the amount of methanol added in step (3) is 25 mL.

[0018] Preferably, the concentration of the reference solution is 0.2-0.4 mg / mL. Other specific values ​​within the above range can be selected, all of which can achieve the technical effect of the present invention, including but not limited to 0.2 mg / mL, 0.3 mg / mL, and 0.4 mg / mL.

[0019] Preferably, the flow rate in step (3) is 0.8 mL / min.

[0020] Preferably, the temperature of the drift tube for sample injection in step (3) is 105-115℃. Other specific values ​​within the above range can be selected to achieve the technical effect of the present invention, including but not limited to 105℃, 110℃, and 115℃.

[0021] Preferably, the gas flow rate of the sample injection in step (3) is 3.0-3.4 L / min. Other specific values ​​within the above range can be selected, and all can achieve the technical effect of the present invention, including but not limited to 3.0 L / min, 3.1 L / min, 3.2 L / min, 3.3 L / min, and 3.4 L / min.

[0022] Preferably, the chromatographic column used in step (3) is a DIKMA Diamonsil C18, 250×4.6mm, 5μm or an Agilent ZORBAX SB-C18, 250×4.6mm, 5μm or an Agilent ZORBAX Eclipse XDB-C18, 250×4.6mm, 5μm.

[0023] Preferably, the instrument used for the determination in step (3) is a Thermo Fisher UltiMate 3000 liquid chromatograph-Aotai 6100 or a Thermo Fisher UltiMate 3000 liquid chromatograph-Aotai 6000.

[0024] Secondly, the present invention provides the application of the above-described quality control method in the testing of Aralia elata leaves and their corresponding products.

[0025] The present invention has at least the following beneficial effects: This invention has good technical effects in terms of the precision and stability of the method for controlling the yield and quality of Aralia elata leaves. Attached Figure Description

[0026] Figure 1 A graph for examining different extraction methods; Figure 2 The chromatograms are for different extraction solvents. Figure 3 Chromatograms of Aralia elata leaves obtained by ultraviolet and evaporative light detection; Figure 4 The chromatogram obtained by gradient elution 1; Figure 5 The chromatogram obtained by gradient 2 elution; Figure 6 The chromatogram obtained by gradient 3 elution; Figure 7 The chromatogram obtained by gradient 4 elution; Figure 8 The chromatogram obtained by gradient 5 elution; Figure 9 The chromatogram obtained by gradient 6 elution; Figure 10 Chromatogram of Aralia elata saponin V reference standard from Liaodong; Figure 11 Chromatogram of Aralia elata leaf sample; Figure 12 Overlay image of leaves from 15 batches of Aralia elata; Figure 13 Chromatograms were examined for different drift tube temperatures; Figure 14 To examine the chromatograms for different gas flow rates; Figure 15 Chromatograms were examined for different chromatographic columns; Figure 16 To examine chromatograms using different instruments; Figure 17 Chromatograms of saponin components from Aralia elata leaves obtained by ultraviolet and evaporative light detection; Figure 18 The graph shows the linear relationship of saponin V in Aralia elata. Detailed Implementation

[0027] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0028] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0029] Five batches of samples were collected from each of the three production areas: Changbai Mountain, Greater Khingan Mountains, and Lesser Khingan Mountains. Sample information is detailed in Table 1.

[0030] Table 1 Sample Information

[0031] Instruments: Thermo Fisher UltiMate 3000 liquid chromatograph, Aote ELSD6100 evaporative light detector, Tianjin Jinfen air generator (model: XWK-III), Mettler TOLEDO electronic balance (model: XSE205DU), Ningbo Xinzhi ultrasonic cleaner (model: SB25-12DTD), BOXUN constant temperature water bath (model: HH.S11-6).

[0032] Reagents: Acetonitrile (chromatographic grade, Honeywell Burdick & Jackson), methanol (chromatographic grade, Honeywell Burdick & Jackson), other reagents purchased from Sinopharm Chemical Reagent Co., Ltd., purified water prepared in-house. Reference standard: Aralia elata saponin V (Chengdu Desite Biotechnology Co., Ltd., batch number: DST230822-169, purity 93.94%) Preparation of reference solution: Take an appropriate amount of Liaodong Aralia elata saponin V reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 0.2 mg per 1 mL, which is used as the reference solution.

[0033] Example 1: Preparation of the test solution (1) Examination of different extraction methods The comparisons were made using the following methods respectively: Extraction: Weigh approximately 0.5g of Aralia elata leaf powder (passed through a No. 3 sieve), place it in a stoppered conical flask, add 25mL of 70% methanol, seal tightly, weigh, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, accurately measure 10mL of the filtrate and concentrate to dryness in an evaporating dish, dissolve the residue in 5mL of water, extract three times with 5mL of water-saturated n-butanol each time, combine the n-butanol extracts, evaporate to dryness, dissolve the residue in methanol, transfer to a 5mL volumetric flask, add methanol to the mark, shake well, and the product is obtained; Ultrasonic extraction: Accurately weigh approximately 1g of Aralia elata leaf powder (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 25mL of 70% methanol, seal tightly, weigh, and sonicate (600W power, 40kHz frequency) for 30 minutes. After cooling, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate. Results are shown below. Figure 1 In addition, based on the results of the determination of the leaf content of Aralia elata, and considering the ease of operation and sufficient extraction, ultrasonic treatment was selected as the sample extraction method.

[0034] (2) Investigation of different extraction solvents Accurately weigh approximately 1g of Aralia elata leaf powder (passed through a No. 3 sieve) and place it in a stoppered conical flask. Accurately add 25mL each of ethanol, methanol, 70% methanol, and 50% methanol, respectively. Seal the flask tightly and weigh. Sonicate the flask (600W, 40kHz) for 30 minutes, cool, and weigh again. Make up the lost weight with the corresponding extraction solvent, shake well, filter, and collect the filtrate. Results are shown below. Figure 2 In accordance with the results of the determination of the content of Aralia elata leaves, 70% methanol was selected as the extraction solvent.

[0035] Example 2 Selection of chromatographic conditions (1) Investigation of different detectors Aralia elata leaves contain abundant triterpenoid saponins, which exhibit various pharmacological activities including antioxidant, antitumor, anti-inflammatory, and antiviral effects. Therefore, this research focused on its saponin components. Two detection methods, ultraviolet (UV) detection and evaporative light scattering (EVS), were tested. It was found that saponins often show no UV absorption or only weak terminal UV absorption. Therefore, the response values ​​obtained using a UV detector for detecting saponins in Aralia elata leaves were low. In contrast, the EVS method showed better response and stronger specificity, demonstrating certain advantages over the UV method. Figure 3As shown in the figure. Therefore, further research will be conducted using HPLC-ELSD.

[0036] (2) Determination of chromatographic conditions Octadecylsilane-bonded silica gel was used as the packing material (based on literature reports, a DIKMA Diamonsil C18(2) 250mm×4.6mm, 5μm column was selected); acetonitrile was used as mobile phase A and water as mobile phase B, and gradient elution was performed according to the specifications in Tables 2-7; the flow rate was 0.8mL per minute; and the column temperature was 30℃. The obtained chromatograms are shown in [Table 7]. Figures 4-9 . Table 2 Gradient 1:

[0037] Table 3 Gradient 2:

[0038] Table 4 Gradient 3:

[0039] Table 5 Gradient 4:

[0040] Table 6 Gradient 5:

[0041] Table 7 Gradient 6:

[0042] By examining the elution time, resolution, and peak area of ​​the content determination index components of each chromatographic peak, and considering all factors, the final chromatographic conditions were determined as follows: octadecylsilane-bonded silica gel was used as the packing material (DIKMA Diamonsil C18 (2) 250mm × 4.6mm, 5μm column); acetonitrile was used as mobile phase A, and water was used as mobile phase B, with gradient elution performed according to Table 6; the flow rate was 0.8mL per minute; the column temperature was 30℃; and evaporative light scattering detector was used for detection. The theoretical plate number, calculated based on the V peak of Aralia elata saponins from Liaodong, should not be less than 5000. The resolution of each chromatographic peak was good, the method was feasible, and the results are shown in Table 6. Figures 10-11 .

[0043] Example 3: Establishment of Feature Map HPLC-ELSD chromatograms of 15 batches of Aralia elata leaf samples from different origins were determined. The results were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission. Common peaks were selected, and with Aralia elata saponin V (S peak) as a reference peak, the more stable common peaks were selected as characteristic peaks. (See attached table). Figure 12 .

[0044] Analysis of the relative retention time data of multiple batches of medicinal materials (see Table 8) revealed that the relative retention times of each characteristic peak were relatively stable and all within ±10% of the average value. Therefore, the average value of the relative retention time of each characteristic peak was used as the specified value.

[0045] Table 8. Relative retention times of characteristic peaks in the leaf characteristic spectrum of Aralia elata.

[0046] Based on the above method, the characteristic spectrum of Aralia elata leaves should show 6 characteristic peaks, among which the peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.46 (peak 1), 0.56 (peak 2), 0.62 (peak 3), 0.67 (peak 4), 0.97 (peak 5), and 1.00 (peak 6).

[0047] Example 4 Methodological Validation (1) Precision test The reference solution of Aralia elata saponin V was injected five times consecutively, 10 μL each time. The RSD of retention time and peak area were calculated respectively. The results are shown in Table 9.

[0048] Table 9 Results of Instrument Precision Testing

[0049] The results showed that the retention time and peak area RSD values ​​of the Liaodong Aralia elata saponin V reference standard were all less than 2.0%, indicating that the instrument precision was good.

[0050] (2) Stability test The same sample (Changbaishan medicinal material-1) was injected and analyzed at 0, 2, 4, 11, 14, 18, 20 and 24 hours, and the relative retention time of each characteristic peak and the S peak was recorded. The results are shown in Table 10.

[0051] Table 10 Stability test results

[0052] The results showed that the RSD of the relative retention time of each characteristic peak and the S peak was less than 2.0% within 0-24 hours, indicating that the method was relatively stable within 24 hours.

[0053] (3) Repeated examination Samples from the same batch (Changbai Mountain medicinal material - 1) were prepared into 6 test solutions according to the "Preparation Method of Test Solution". The solutions were injected and analyzed separately, and the relative retention times of each characteristic peak and the S peak were recorded. The results are shown in Table 11.

[0054] Table 11 Results of Repeatability Testing

[0055] The results showed that the RSD of the relative retention time of each characteristic peak and the S peak was less than 2.0%, indicating good repeatability.

[0056] (4) Durability test Investigation of drift tube temperature Samples from the same batch (Changbai Mountain medicinal material - 1) were taken and prepared into test solutions according to the "Preparation Method for Test Solution". Analysis was performed using three injection temperatures: 105℃, 110℃, and 115℃. The relative retention times of each characteristic peak and the S peak were recorded. The results are shown in Table 12. Figure 13 .

[0057] Table 12 Results of Temperature Investigation in Drift Tubes

[0058] The results showed that when the drift tube temperature was 105℃, 110℃, and 115℃, the relative retention times of each characteristic peak and the S peak were all within the specified range, indicating good durability.

[0059] Gas flow rate investigation Samples from the same batch (Changbai Mountain medicinal material - 1) were prepared into test solutions according to the "Preparation Method for Test Solution". Analysis was performed using three gas flow rates: 3.0 L / min, 3.2 L / min, and 3.4 L / min. The relative retention times of each characteristic peak and the S peak were recorded. The results are shown in Table 13. Figure 14 .

[0060] Table 13 Results of Gas Flow Rate Investigation

[0061] The results showed that when the gas flow rate was 3.0 L / min, 3.2 L / min, and 3.4 L / min, the relative retention time of each characteristic peak was within the specified range, indicating good durability.

[0062] Column analysis Samples from the same batch (Changbai Mountain medicinal material - 1) were prepared into test solutions according to the "Preparation Method for Test Solution". Three chromatographic columns were used: DIKMA Diamonsil C18(2) (250×4.6mm, 5μm), Agilent ZORBAX SB-C18 (250×4.6mm, 5μm), and Agilent ZORBAX Eclipse XDB-C18 (250×4.6mm, 5μm). The results are shown in Table 14. Figure 15 .

[0063] Table 14 Results of investigation using different chromatographic columns

[0064] The results showed that the separation effect of the three different types of chromatographic columns was good. The separation effect of the same brand (Agilent) was similar. However, the relative retention time of each characteristic peak was easily affected by the different brands of chromatographic columns. The relative retention time range specified in the standard could not be reproduced in the other two different types of chromatographic columns. In order to ensure the reproducibility of the method, it is recommended to fix the chromatographic column as DIKMA Diamonsil C18 (2) (250×4.6mm, 5μm).

[0065] Different instruments Samples from the same batch (Changbai Mountain medicinal material - 1) were taken and prepared into test solutions according to the "Preparation Method for Test Solution". Two HPLC systems, a Thermo Fisher UltiMate 3000-Aotei 6100 and a Thermo Fisher UltiMate 3000-Aotei 6000, were used for analysis. The results are shown in Table 15. Figure 16 .

[0066] Table 15 Results of investigation using different instruments

[0067] The results show that when the above instruments are used, the relative retention times of each characteristic peak and the S peak are all within the specified range, and the durability is good.

[0068] Example 5 Inspection (1) The heavy metal and harmful element tests on this product all met the relevant requirements.

[0069] Instruments and reagents Instruments: Mettler Toledo electronic balance (model: XSE205DU), microwave digester (model: MARS), Shanghai Broadcom constant temperature heating digester (model: BHW-09C), high performance liquid chromatography-inductively coupled plasma mass spectrometry (model: Perkin Elmer, USA; ICP-MS: Nexino350X; LC: A-10) Reagents: Purified water was prepared in-house; other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Reference standards: The single-element standard solutions of lead (Pb) (batch number: GBW08619-21032), cadmium (Cd) (batch number: GBW08612-21081), arsenic (As) (batch number: GBW08611-23052), and copper (Cu) (batch number: GBW08615-23061) used in the experiment were purchased from the National Institute of Metrology, China, with a mass concentration of 1000 μg / mL. The single-element standard solution of mercury (Hg) (1000 μg / mL, National Nonferrous Metals and Electronic Materials Analysis and Testing Center, batch number: GSB 04-1729-2004); Internal standards: germanium (Ge) (1000 μg / mL, batch number: GSB 04-1728-2004), indium (In) (1000 μg / mL, batch number: GSB 04-1728-2004), and indium (In) (1000 μg / mL, batch number: GSB 04-1729-2004). Bismuth (Bi) (1000 μg / mL, batch number: GSB 04-1731-2004) and gold (Au) single-element standard solution (1000 μg / mL, National Nonferrous Metals and Electronic Materials Analysis and Testing Center, batch number: GSB 04-1715-2004) were purchased from the National Nonferrous Metals and Electronic Materials Analysis and Testing Center. Heavy metals and harmful elements The determination of lead, cadmium, arsenic, mercury and copper in 15 batches of this product was carried out according to the method for determination of lead, cadmium, arsenic, mercury and copper (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 2321, Inductively Coupled Plasma Mass Spectrometry). The results are shown in Table 16.

[0070] Table 16. Results of heavy metal and harmful element determination in 15 batches of Aralia elata leaves.

[0071] According to the general provisions of the 2020 edition of the Chinese Pharmacopoeia for heavy metals and harmful elements in Chinese medicinal materials, lead shall not exceed 5 mg / kg; cadmium shall not exceed 1 mg / kg; arsenic shall not exceed 2 mg / kg; mercury shall not exceed 0.2 mg / kg; and copper shall not exceed 20 mg / kg. As shown in Table 16, none of the 15 batches of samples exceeded the standard limits.

[0072] (2) Leaching matter The extract of Aralia elata leaves was studied according to the method under General Chapter 2201 "Determination of Extractives" in Part IV of the 2020 edition of the Chinese Pharmacopoeia. Through investigation of different extraction solvents and extraction methods, a relatively reasonable method for extractive determination was identified, and reasonable limits were established: the extractives determined by the cold maceration method under General Chapter 2201 (Determination of Water-Soluble Extractives) should not be less than 30.0%.

[0073] Instruments and reagents Instruments: Mettler TOLEDO electronic balance (model: XSE205DU), BOXUN constant temperature water bath (model: HH.S11-6), IRM electric thermostatic drying oven (model: FD-230), EYELA condensate circulator (model: CA-1111), Guangming electronic temperature-controlled heating mantle (model: DZTW) Reagents: Purified water was prepared in-house; other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Moisture This product is the dried leaf of Aralia elata, a plant of the Araliaceae family. When conducting studies on extractives, it should be calculated on a dried basis. Therefore, the determination was performed according to Method II (drying method) under General Chapter 0832 of the 2020 edition of the Chinese Pharmacopoeia, Part IV. The specific procedure is as follows: Accurately weigh 2g of the test sample, open the cap and dry at 105℃ for 5 hours. Close the cap, transfer to a desiccator, cool for 30 minutes, and accurately weigh. Dry again at the same temperature for 1 hour, cool, and weigh until the difference between two consecutive weighings does not exceed 5mg. Calculate the water content (%) in the test sample based on the weight loss. See Table 17 for detailed results.

[0074] Table 17 Results of moisture determination by drying method

[0075] Calculation formula: Moisture (%) =

[0076] The moisture content determination results of 15 batches of samples can be used for the determination of extractives and content of this product.

[0077] Leachate determination method The extractives of Aralia elata leaves were studied according to the method described in Section 2201, "Determination of Extractives," of the 2020 edition of the Chinese Pharmacopoeia, Part IV. Water-soluble and alcohol-soluble extractives were explored during the study. By comparing the results of various methods, the appropriate method for determining the extractives of this product was determined. The specific research process is as follows: Determination of water-soluble extractives: Cold soaking method Accurately weigh approximately 4g of the test sample and place it in a 250-300mL Erlenmeyer flask. Accurately add 100mL of water, seal tightly, and allow to soak in cold water. Shake frequently for the first 6 hours, then let stand for 18 hours. Filter quickly using a dry filter. Accurately measure 20mL of the filtrate and place it in an evaporating dish that has been dried to constant weight. Evaporate to dryness on a water bath, then dry at 105℃ for 3 hours. Cool in a desiccator for 30 minutes and quickly and accurately weigh. Calculate the content (%) of water-soluble extractives in the test sample based on the dried product.

[0078] Hot soaking method Accurately weigh approximately 4g of the test sample and place it in a 100-250mL Erlenmeyer flask. Accurately add 100mL of water, seal tightly, and weigh. After standing for 1 hour, connect a reflux condenser and heat to boiling, maintaining a gentle boil for 1 hour. After cooling, remove the Erlenmeyer flask, seal tightly, and weigh again. Make up the lost weight with water, shake well, and filter through a dry filter. Accurately measure 25mL of the filtrate and place it in an evaporating dish that has been dried to constant weight. Evaporate to dryness on a water bath, then dry at 105℃ for 3 hours. Cool in a desiccator for 30 minutes and quickly and accurately weigh. Calculate the water-soluble extract content (%) in the test sample.

[0079] The content of water-soluble extracts in the test samples was calculated by cold leaching and hot leaching methods, respectively. The results are shown in Table 18.

[0080] Determination of alcohol-soluble extracts: This method uses 95% ethanol instead of water as the solvent, and the determination is carried out according to the cold leaching method and hot leaching method under the "Determination of Water-Soluble Extractives" section. The results are shown in Table 18.

[0081] Table 18 Comparison of extract content determination results between cold leaching and hot leaching methods

[0082] Calculation formula: Leachate content (%) =

[0083] (3) Determination of the method for determining leachate and sample testing The above results show that the content of alcohol-soluble extracts is lower than that of water-soluble extracts. For water-soluble extracts, the cold maceration method yields higher extracts than the hot maceration method. Furthermore, the hot maceration method using water as a solvent is highly sensitive to heating temperature and prone to boiling over, which is speculated to be related to the high content of saponins in Aralia elata leaves. Therefore, the cold maceration method (General Chapter 2201, Part IV, Chinese Pharmacopoeia 2020 Edition) under the water-soluble extract determination method was selected for testing 15 batches of samples. The test results for these 15 batches are shown in Table 19.

[0084] Table 19 Results of Leachate Content Determination in 15 Batches of Samples

[0085] Determining the limits Referring to the "Hong Kong Standards for Chinese Medicinal Herbs", statistical methods were applied to analyze the results of extract determination. Based on the extract determination data of 15 batches of samples, the limit formula for extract determination is as follows:

[0086] In the formula, x is the sample mean; α is the significance level (99% confidence level); t is the t-test value (one-tailed), with a reference standard set to 2.576; s is the standard deviation of the sample; n is the number of batches in the sample; n-1 is the degrees of freedom; MU is the uncertainty assessment, with a reference standard set to 5.35 based on the empirical value of expanded uncertainty of 0.14.

[0087] The calculated limits for leachate are shown in Table 20. Therefore, the proposed standard for the leachate test of this variety is: the content of leachate shall not be less than 30.0% as determined by the cold leaching method under the Water-Soluble Leachate Determination Method (General Chapter 2201, Part IV, Chinese Pharmacopoeia 2020 Edition). Upon testing, the leachate content of the above 15 batches of samples all met the requirements.

[0088] Table 20 Limit Calculation of Leachate

[0089] Example 6: Determination of Active Ingredient Content Saponins are the main active components of Aralia elata leaves. Liaodong saponin V was selected as the quality control indicator for Aralia elata leaves. An HPLC-ELSD method was established to determine the content of Liaodong saponin V in Aralia elata leaves. The method was verified to be accurate and reliable, and reasonable limits were established.

[0090] Instruments and reagents Instruments: Thermo Fisher UltiMate 3000 liquid chromatograph, Aote ELSD6100 evaporative light detector, Tianjin Jinfen air generator (model: XWK-III), Mettler TOLEDO electronic balance (model: XSE205DU), Ningbo Xinzhi ultrasonic cleaner (model: SB25-12DTD), BOXUN constant temperature water bath (model: HH.S11-6).

[0091] Reagents: Acetonitrile (chromatographic grade, Honeywell Burdick & Jackson), methanol (chromatographic grade, Honeywell Burdick & Jackson), other reagents purchased from Sinopharm Chemical Reagent Co., Ltd., purified water prepared in-house. Reference standard: Aralia elata saponin V (Chengdu Desite Biotechnology Co., Ltd., batch number: DST230822-169, purity 93.94%).

[0092] Among the saponin components of *Aralia elata* (Liaodong Aralia elata) IV, V, VI, VII, and TTP, the content of *Aralia elata* V showed a strong correlation with its antitumor effect and can be considered a quality control component for the antitumor activity of *Aralia elata* leaves. Therefore, *Aralia elata* V was selected as the content indicator for *Aralia elata* leaves.

[0093] 1. Selection of chromatographic conditions (1) Selection of different detectors Two detection methods, ultraviolet (UV) detection and evaporative light scattering (EVS), were tested. It was found that saponins often exhibit no UV absorption or only weak terminal UV absorption. Therefore, the response values ​​obtained by using a UV detector to detect saponins in Aralia elata leaves were low, while the EVS method showed better response and stronger specificity, demonstrating certain advantages over the UV method. Figure 17 As shown in the figure. Therefore, HPLC-ELSD was subsequently used for further research.

[0094] (2) Determination of chromatographic conditions The chromatographic column was packed with octadecylsilane-bonded silica gel; acetonitrile was used as mobile phase A and water as mobile phase B, and gradient elution was performed according to the specifications in Table 6 above; the flow rate was 0.8 mL per minute; the column temperature was 30 °C; and evaporative light scattering detector was used for detection. The chromatographic peaks showed good resolution, and the method was feasible.

[0095] 2. Examination of different extraction methods The extraction and ultrasonic extraction methods were the same as those in Example 1, and the results are shown in Table 21.

[0096] Table 21 Examination of Extraction Methods

[0097] Therefore, ultrasound was selected as the sample extraction method.

[0098] 3. Investigation of different extraction solvents Accurately weigh approximately 1g of Aralia elata leaf powder (passed through a No. 3 sieve) and place it in a stoppered conical flask. Accurately add 25mL each of ethanol, methanol, 70% methanol, and 50% methanol, respectively. Seal the flask tightly and weigh the contents. Sonicate the flask for 30 minutes, cool, and weigh again. Replenish the lost weight with the corresponding extraction solvent, shake well, filter, and collect the filtrate. The results are shown in Table 22; therefore, 70% methanol was chosen as the extraction solvent.

[0099] Table 22 Investigation of extraction solvents

[0100] (3) Sample size assessment For the samples, under the same other conditions, 0.5g, 1g and 2g samples were selected for comparison. The results are shown in Table 23. When the sample amount was 0.5g and 1g, the content of saponin V in Aralia elata showed a good linear relationship. When the sample amount was 2g, the content of saponin V in Aralia elata was relatively low. It is speculated that this is because the extraction was incomplete due to the large sample amount. Therefore, 1g was selected as the sample extraction amount.

[0101] Table 23 Sampling Quantity Examination

[0102] (4) Ultrasound time study For the samples, ultrasound treatment time was selected for 15 minutes, 30 minutes and 45 minutes under the same other conditions for comparison. The results are shown in Table 24. Therefore, the ultrasound treatment time was determined to be 30 minutes.

[0103] Table 24 Examination of ultrasound time

[0104] (5) Determination of the preparation method of the test solution Take about 1g of the powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 70% methanol, seal tightly, weigh it, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the product.

[0105] Example 7: Methodological Validation of Active Ingredients (1) Linear examination Accurately measure 0.5 mL, 0.75 mL, and 1.00 mL of the Liaodong Aralia elata saponin V reference solution (2.312 mg / mL) into 10 mL volumetric flasks. Then, accurately measure 1.00 mL and 2.00 mL of the Liaodong Aralia elata saponin V reference solution (2.312 mg / mL) into 5 mL volumetric flasks. Dissolve and dilute to the mark with 70% methanol in each flask and mix well. Accurately inject 10 μL of the reference standard into the liquid chromatograph. Plot a standard curve with the logarithm of the peak area integral as the ordinate and the logarithm of the injection volume (μg) as the abscissa, and obtain the regression equation. The results are shown in Table 25. Figure 18 .

[0106] Table 25 Injection Volume and Peak Area

[0107] (2) Precision test The reference solution of Aralia elata saponin V was injected five times consecutively, and the peak area was measured. The results are shown in Table 26.

[0108] Table 26 Results of Instrument Precision Testing

[0109] The results showed that the RSD values ​​of the peak areas of the five needles of Liaodong Aralia elata saponin V reference standard were all less than 2.0%, indicating that the instrument precision was good.

[0110] (3) Stability test Take the test solution of (Changbai Mountain-1 medicinal material) and inject it at 0h, 2h, 9h, 12h, 16h and 18h ​​respectively. Record the chromatographic peak area. The results are shown in Table 27.

[0111] Table 27 Stability test results

[0112] The results showed that the RSD of the peak area of ​​the test solution was less than 2.0% within 0-18 hours, indicating good stability.

[0113] (4) Repeated examination Take samples from the same batch (Changbai Mountain medicinal material-1), prepare 6 test solutions according to the "Preparation Method of Test Solution", inject and determine them separately, and the results are shown in Table 28.

[0114] Table 28 Results of Repeatability Testing

[0115] The results showed that when the same batch of samples was measured repeatedly 6 times, the RSD of saponin V content in Liaodong Aralia elata was less than 2.0%, indicating that the method had good repeatability.

[0116] (5) Recovery rate assessment Accurately weigh 0.5 g of sample (Changbai Mountain medicinal material - 1), add an appropriate amount of Liaodong Aralia elata saponin V reference standard, and prepare 6 test solutions according to the method under "Preparation of Test Solution". Inject the solutions and determine the recovery rate of Liaodong Aralia elata saponin V. The results are shown in Table 29.

[0117] Table 29 Recovery rate of saponin V from Aralia elata in Liaodong

[0118] The results showed that the average recovery rate of saponin V from Aralia elata in Liaodong was 100.12%, with an RSD of less than 2.0%, indicating a good recovery rate.

[0119] (6) Durability test Temperature investigation of drift tube Samples from the same batch (Changbaishan-1 medicinal material) were prepared according to the "Preparation of Test Solution". The test solution was analyzed at three temperatures: 105℃, 110℃, and 115℃. The content of saponin V in Liaodong Aralia elata was calculated. The results are shown in Table 30.

[0120] Table 30 Results of Temperature Investigation in Drift Tubes

[0121] The results showed that the drift tube exhibited good durability at temperatures of 105℃, 110℃, and 115℃.

[0122] Gas flow rate investigation Samples from the same batch (Changbaishan-1 medicinal material) were prepared into test solutions according to the "Preparation of Test Solution". The solutions were analyzed using three gas flow rates of 3.0 L / min, 3.2 L / min, and 3.4 L / min. The content of saponin V in Liaodong Aralia elata was calculated, and the results are shown in Table 31.

[0123] Table 31 Results of Gas Flow Rate Investigation

[0124] The results showed that the durability was good when the gas flow rate was 3.0 L / min, 3.2 L / min, and 3.4 L / min.

[0125] Column analysis Samples from the same batch (Changbai Mountain medicinal material - 1) were prepared into test solutions according to the "Preparation of test solution". Three chromatographic columns were used for analysis: DIKMA Diamonsil C18 (2) (250×4.6mm, 5μm), Agilent ZORBAX SB-C18 (250×4.6mm, 5μm), and Agilent ZORBAX Eclipse XDB-C18 (250×4.6mm, 5μm). The results are shown in Table 32.

[0126] Table 32 Results of Column Chromatography

[0127] The results showed that the column had good durability when it was DIKMA Diamonsil C18 (2), Agilent ZORBAX SB-C18, or Agilent ZORBAX Eclipse XDB-C18.

[0128] Different instruments Samples from the same batch (Changbaishan medicinal materials-1) were prepared into test solutions according to the "Preparation of test solution". The solutions were analyzed using two instruments: a Thermo Fisher UltiMate 3000 liquid chromatograph-Aotai 6100 and a Thermo Fisher UltiMate 3000 liquid chromatograph-Aotai 6000. The results are shown in Table 33.

[0129] Table 33 Results of investigation using different instruments

[0130] The results show that the above instruments have good durability.

[0131] Determination of sample content Take samples and prepare test solutions according to the "Preparation of Test Solution" procedure. Determine the content of saponin V in Aralia elata from Liaodong. The results are shown in Table 28.

[0132] Table 34. Saponin V Content of Aralia elata from Liaodong

[0133] The method for determining the content of Aralia elata leaves is a newly developed method, and this item is not included in the current quality standards; therefore, there is no reference limit for detection. The results of 15 batches of samples of Aralia elata from Liaodong ranged from 0.47% to 0.63%, with an average of 0.53%. Since Aralia elata leaves are mostly wild, the content varies greatly from place to place, exhibiting an unstable and uneven characteristic. Therefore, the content limit for this study is tentatively set at 60% of the average value, and the limit for Aralia elata from Liaodong is tentatively set at 0.32%.

[0134] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for quality control of Aralia elata leaves, characterized in that, Includes the following steps: (1) Preparation of reference solution; (2) Preparation of the test solution; (3) Determination method: Accurately pipette the reference solution and the test solution separately, using octadecylsilane-bonded silica gel as the packing material; use acetonitrile as mobile phase A and water as mobile phase B, with a flow rate of 0.7-0.9 mL / min, and adopt a gradient elution method. The gradient elution program is as follows: Inject the sample, measure it, and record the chromatogram to obtain the result.

2. The quality control method according to claim 1, characterized in that, The preparation method of the reference solution in step (1) includes the following steps: take an appropriate amount of Liaodong Aralia elata saponin V reference standard, accurately weigh it, add methanol to dissolve it, and the solution is obtained.

3. The quality control method according to claim 1, characterized in that, The preparation method of the test solution in step (2) includes the following steps: accurately weigh the powder of Aralia elata, place it in a stoppered conical flask, add methanol, seal tightly, weigh, sonicate, cool, weigh again, replenish the lost weight with methanol, shake well, filter, and the solution is obtained.

4. The quality control method according to claim 2 or 3, characterized in that, The volume concentration of methanol is 50-75%.

5. The quality control method according to claim 1, characterized in that, The concentration of the reference solution in step (1) is 0.2-0.4 mg / mL.

6. The quality control method according to claim 1, characterized in that, In step (3), the sample is injected using a drift tube at a temperature of 105-115℃.

7. The quality control method according to claim 1, characterized in that, The gas flow rate for the sample injection in step (3) is 3.0-3.4 L / min.

8. The quality control method according to claim 1, characterized in that, The determination described in step (3) was performed using high performance liquid chromatography; the chromatographic column was a DIKMA Diamonsil C18, 250×4.6mm, 5μm or an Agilent ZORBAX SB-C18, 250×4.6mm, 5μm or an Agilent ZORBAX Eclipse XDB-C18, 250×4.6mm, 5μm.

9. The quality control method according to claim 1, characterized in that, The determination in step (3) is performed using a Thermo Fisher UltiMate 3000 liquid chromatograph-Aotai 6100 or a Thermo Fisher UltiMate 3000 liquid chromatograph-Aotai 6000.

10. The application of the quality control method according to any one of claims 1-9 in the testing of Aralia elata leaves and their corresponding products.

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