A method for constructing a characteristic chromatogram for distinguishing between yinxiangmao traditional Chinese medicine formula granules and xianmao traditional Chinese medicine formula granules, and a preparation method for yinxiangmao traditional Chinese medicine formula granules and xianmao traditional Chinese medicine formula granules

Abstract

The application provides a method for constructing characteristic atlas of Yijiamao Chinese medicine formula granules and Xianmao Chinese medicine formula granules and a preparation method of Yijiamao Chinese medicine formula granules and Xianmao Chinese medicine formula granules, which comprises the following steps: (1) taking orsellin and curculigoside as control samples, respectively adding methanol to prepare solutions as control reference solution; (2) extracting a test sample solution by using 70% methanol and filtering; (3) respectively determining the control reference solution and the test sample solution by using high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography comprise the following steps: using octadecylsilane bonded silica gel as a filler, using acetonitrile as mobile phase A and 0.1wt% phosphoric acid aqueous solution as mobile phase B for gradient elution, and detecting at a wavelength of 270 nm. The method provided by the application has good stability, high precision and good reproducibility, can comprehensively reflect the quality condition of the formula granule preparation, and can effectively identify Yijiamao Chinese medicine formula granules and Xianmao Chinese medicine formula granules.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology of traditional Chinese medicine, and in particular to a method for constructing characteristic chromatograms for differentiating between wine-processed and curculigo-processed traditional Chinese medicine formula granules, as well as a method for preparing wine-processed and curculigo-processed traditional Chinese medicine formula granules. Background Technology

[0002] Curculigo orchioides has a long history of medicinal use, first recorded in the *Haiyao Bencao* (Sea Materia Medica). It is also mentioned in many other herbal texts. The medicinal form of Curculigo orchioides Gaertn. is currently the dried rhizome of this plant, belonging to the Amaryllidaceae family. Curculigo orchioides is a wild Chinese medicinal herb, and its rhizome has high medicinal value. It is warm in nature, bitter in taste, and slightly toxic. It has the effects of warming the kidneys and strengthening yang, dispelling cold and dampness, nourishing essence and blood, strengthening muscles and bones, and promoting blood circulation and reducing swelling. Wine-processed Curculigo orchioides is made by mixing clean Curculigo orchioides with rice wine, allowing it to soak thoroughly, then stir-frying until slightly dry, and finally drying it completely. The specific preparation conditions are: for every 100g of Curculigo orchioides slices, use 10g of rice wine, mix well, let it soak naturally for at least 60 minutes until thoroughly soaked, then stir-fry at 200-250℃ for 8-10 minutes. During the stir-frying process, the material's properties must be observed constantly. Once dry, remove the material and spread it out to cool to obtain wine-processed Curculigo orchioides slices. *Curculigo orchioides* (also known as *Curculigo pilosa* or *Curculigo pilosa*) has the effects of warming the kidneys and strengthening yang, dispelling cold and dampness, nourishing essence and blood, strengthening muscles and bones, and promoting blood circulation and reducing swelling. *Curculigo orchioides* and *Curculigo pilosa* (prepared with wine) can be distinguished by their specific appearance. However, *Curculigo orchioides* granules and *Curculigo pilosa* (prepared with wine) have different medicinal uses, making them difficult to distinguish by appearance alone. Therefore, providing an effective identification method for *Curculigo orchioides* granules and *Curculigo pilosa* (prepared with wine) is of great significance for quality control of these two granules. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum for distinguishing between wine-processed and curculigo-processed traditional Chinese medicine formula granules, and a method for preparing wine-processed and curculigo-processed traditional Chinese medicine formula granules. The method of the present invention is efficient and easy to operate, which is of great significance for the study of evaluating wine-processed and curculigo-processed traditional Chinese medicine formula granules, and ensures the uniformity and stability of sample quality.

[0004] The method of this invention can scientifically identify and study the traditional Chinese medicine formula granules of *Curculigo orchioides* and *Curculigo orchioides*. It has the characteristics of comprehensive detection and simple operation, can effectively identify the traditional Chinese medicine formula granules of *Curculigo orchioides* and *Curculigo orchioides*, and can ensure the uniformity and stability of the quality of the traditional Chinese medicine formula granules of *Curculigo orchioides*. In a first aspect, the present invention provides a method for constructing a characteristic spectrum for distinguishing between *Curculigo orchioides* herbal formula granules and *Curculigo orchioides* herbal formula granules, comprising: (1) Using styracin glucoside and curculigoside as reference standards, methanol was added to prepare solutions, which were used as reference solutions; (2) The test sample was extracted with 70% methanol and filtered to obtain the test sample solution; (3) The reference solution and the test solution were determined by high performance liquid chromatography. The chromatographic conditions of the high performance liquid chromatography included: the column was packed with octadecylsilane bonded silica gel, and gradient elution was performed with acetonitrile as mobile phase A and 0.1 wt% phosphoric acid aqueous solution as mobile phase B. The detection wavelength was 270 nm. Using the peak corresponding to the reference substance of styraxol glucoside as the S peak, the chromatogram of the herbal formula granules of the wine-processed curculigo root shows two identification characteristic peaks at relative retention times of 0.275 and 1.200 with the S peak. The chromatogram of the herbal formula granules of the curculigo root does not show the identification characteristic peaks.

[0005] According to the method for constructing characteristic chromatograms for distinguishing between *Curculigo orchioides* and *Curculigo orchioides* herbal formula granules provided by the present invention, the gradient elution is as follows: 0~12min, mobile phase A: 5%, mobile phase B: 95%; 12~25min, mobile phase A: 5→8%, mobile phase B: 95%→92%; 25~60min, mobile phase A: 8→15%, mobile phase B: 92→85%; Over 60-90 minutes, mobile phase A: 15% → 33%, mobile phase B: 85% → 67%.

[0006] Compared to other elution gradients, the above elution gradient, with an elution time of 90 minutes, resulted in complete elution of the chromatographic peaks, with a resolution greater than 1.5 for each peak, indicating good separation performance.

[0007] In some embodiments of the present invention, the theoretical plate number, calculated based on curculigoside, should be no less than 5000.

[0008] In some embodiments of the present invention, the concentration of the reference solution is 50-100 μg / mL.

[0009] In some embodiments of the present invention, in step (2), the mass ratio of the test sample to the volume ratio of the 70% methanol is 0.2 g: (10-50) ml. Those skilled in the art will understand that the test sample is a traditional Chinese medicine formula granule of Curculigo or Curculigo or Curculigo sylvestris.

[0010] According to the method for constructing characteristic chromatograms for differentiating *Curculigo orchioides* granules from traditional Chinese medicine formula provided by the present invention, in step (2), the extraction method includes ultrasonic treatment and / or reflux treatment, with ultrasonic treatment being preferred. Both ultrasonic treatment and / or reflux treatment can achieve better extraction, and the chromatograms of the two extraction methods have high similarity. Since ultrasonic treatment is simpler to operate, ultrasonic treatment is the preferred extraction method.

[0011] In some embodiments of the present invention, the ultrasonic treatment time is 20 min to 40 min.

[0012] In some embodiments of the present invention, the power of the ultrasonic treatment is 500-700W.

[0013] In some embodiments of the present invention, the frequency of the ultrasonic treatment is 30-50 kHz.

[0014] In some embodiments of the present invention, the flow rate of the mobile phase is 0.9-1.1 ml / min, preferably 1 ml / min. When the flow rate of the mobile phase is within the above range, the chromatographic peak shape is good and the resolution is moderate.

[0015] In some embodiments of the present invention, the column temperature of the chromatographic column is 25-35°C, preferably 30°C. When the column temperature is within the above range, the chromatographic peaks are symmetrical and the resolution is good.

[0016] In some embodiments of the present invention, the injection volume of the high-performance liquid chromatography is 5-15 μl, preferably 10 μl. Within this range, the chromatogram peak shape is good and the resolution is moderate.

[0017] Preferably, the chromatogram acquisition time for the high-performance liquid chromatography is not less than 90 minutes, and more preferably 90 minutes. After 90 minutes, the sample shows essentially no chromatographic peaks. According to the method for constructing characteristic chromatograms for distinguishing between *Curculigo orchioides* formula granules and *Curculigo orchioides* formula granules provided by the present invention, when the test sample is *Curculigo orchioides* formula granules, 12 characteristic peaks are presented in the chromatogram, among which the peak corresponding to the reference substance of styraxol glucoside 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: 0.275-peak 1, 0.320-peak 2, 0.491-peak 3, 0.783-peak 4, 0.929-peak 5, 1.000-peak 6, S, 1.068-peak 7, 1.200-peak 8, 1.789-peak 9, 2.660-peak 10, 2.824-peak 11, 3.428-peak 12.

[0018] According to the method for constructing characteristic chromatograms for distinguishing between *Curculigo orchioides* formula granules and *Curculigo orchioides* formula granules provided by the present invention, when the test sample is *Curculigo orchioides* formula granules, 10 characteristic peaks are presented in the chromatogram, among which the peak corresponding to the reference substance *Curculigo orchioides* glucoside 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: 0.320-peak 2, 0.491-peak 3, 0.783-peak 4, 0.929-peak 5, 1.000-peak 6, S, 1.068-peak 7, 1.789-peak 9, 2.660-peak 10, 2.824-peak 11, 3.428-peak 12.

[0019] The chromatogram of the Jiu Xianmao formula granules shows 12 characteristic peaks, which should correspond to the retention times of the two characteristic peaks in the chromatogram of the reference solution. The Jiu Xianmao and Xianmao formula granules have a total of 10 repeated chromatographic peaks, both of which contain two reference standards (styracin glucoside and curculigoside). Furthermore, the Jiu Xianmao and Xianmao formula granules can be distinguished by peaks 1 and 8.

[0020] The method provided by this invention has good stability, high precision, good reproducibility, convenience, and ease of mastery. It also comprehensively reflects the quality status of the formulated granule preparation, making its quality control more complete and adding another means to its quality standards. Under specific detection conditions, this invention can identify the characteristic chromatographic features of *Curculigo orchioides* granules by using the chromatographic peak of styraxol glucoside as a reference peak (S peak).

[0021] In a second aspect, the present invention provides a method for preparing the *Curculigo orchioides* herbal formula granules used in the above-mentioned method for constructing characteristic chromatograms to distinguish between *Curculigo orchioides* herbal formula granules, comprising: The medicinal slices of *Curculigo orchioides* were boiled in water, and the resulting aqueous extract was concentrated under reduced pressure to obtain a concentrated solution. The concentrated liquid was spray-dried to obtain spray-dried *Cistanche deserticola* powder. The spray-dried powder of *Cistanche deserticola* and maltodextrin are mixed and then dry granulated.

[0022] In some embodiments of the present invention, the decoction process includes a first decoction and a second decoction. The amount of water added in the first decoction is 8-12 times that of the *Curculigo orchioides* slices, and the first decoction time is 70-100 minutes. The amount of water added in the second decoction is 7-10 times that of the *Curculigo orchioides* slices, and the second decoction time is 50-60 minutes. The decoction temperature is 94-102°C.

[0023] In some embodiments of the present invention, the vacuum concentration temperature is 50-70°C, the vacuum degree is -0.050~-0.080 MPa, and the vacuum concentration is carried out to a density of 1.01~1.03.

[0024] In some embodiments of the present invention, the inlet air temperature of the spray dryer is 185-195°C and the outlet air temperature is 85-95°C.

[0025] In some embodiments of the present invention, the amount of maltodextrin added is 5-10%.

[0026] In a third aspect, the present invention provides a method for preparing the *Curculigo orchioides* herbal formula granules used in the above-mentioned method for constructing characteristic chromatograms to distinguish between *Curculigo orchioides* herbal formula granules, comprising: The sliced ​​Curculigo orchioides were boiled in water, and the resulting aqueous extract was concentrated under reduced pressure to obtain a concentrated solution. The concentrated solution was spray-dried to obtain spray-dried Curculigo orchioides powder. The spray-dried Curculigo orchioides powder and maltodextrin were mixed and then dry granulated.

[0027] In some embodiments of the present invention, the decoction process includes a first decoction and a second decoction. The amount of water added in the first decoction is 8-12 times that of the Curculigo orchioides slices, and the time for the first decoction is 70-100 minutes. The amount of water added in the second decoction is 7-10 times that of the Curculigo orchioides slices, and the time for the second decoction is 50-60 minutes. The temperature of the decoction is 94-102°C. In some embodiments of the present invention, the vacuum concentration temperature is 50-70°C, the vacuum degree is -0.050~-0.080 MPa, and the vacuum concentration is carried out to a density of 1.01~1.03. In some embodiments of the present invention, the inlet air temperature of the spray dryer is 185-195°C, and the outlet air temperature is 85-95°C. In some embodiments of the present invention, the amount of maltodextrin added is 5-10%. Attached Figure Description

[0028] Figure 1 This is a characteristic chromatogram of the Curculigo orchioides formula granules from Example 1 of the present invention; Figure 2 The characteristic spectrum of the traditional Chinese medicine formula granules of *Curculigo orchioides* from Example 1 of this invention; Figure 3 This is the chromatographic peak identification diagram of the traditional Chinese medicine formula granules of *Curculigo orchioides* from Example 1 of the present invention; Figure 4 This is a comparison of the characteristic spectra of the traditional Chinese medicine formula granules of *Curculigo orchioides* and *Curculigo orchioides* in Example 1 of the present invention; Figure 5 This is a chromatogram of the mobile phase investigation results in Example 2 of the present invention; Figure 6 This is a chromatogram of the elution gradient investigation results in Example 3 of the present invention; Figure 7 This is a chromatogram showing the detection wavelength of Example 4 of the present invention. Figure 8 This is a chromatographic result of the flow rate investigation in Example 5 of the present invention; Figure 9 This is a chromatographic result of column temperature investigation in Example 6 of the present invention; Figure 10 This is a chromatogram of the injection volume investigation results in Example 7 of the present invention; Figure 11 This is a chromatogram of the delayed performance test results from Example 8 of the present invention; Figure 12 The chromatogram shows the extraction method used in Example 9 of this invention. Figure 13 This is a chromatogram showing the extraction solvent investigation results of Example 10 of the present invention; Figure 14 This is a chromatogram showing the solvent addition amount investigation results of Example 11 of the present invention; Figure 15 This is a chromatogram of the extraction time investigation results in Example 12 of the present invention; Figure 16 These are the characteristic spectral verification images of 6 batches of *Curculigo orchioides* granules from Example 17 of this invention; Figure 17 These are the characteristic spectral verification images of six batches of Curculigo granules from Example 17 of this invention; Figure 18 This is a comparison diagram of the characteristic spectra of the wine-making granules and the characteristic spectra of the granules of *Curculigo orchioides* from Embodiment 17 of the present invention. Detailed Implementation

[0029] This invention provides a method for constructing characteristic chromatograms to distinguish between *Curculigo orchioides* and *Curculigo orchioides* herbal formula granules. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve this. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0030] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for constructing characteristic spectra for distinguishing between *Curculigo orchioides* and *Curculigo orchioides* herbal formula granules provided by the present invention.

[0031] Experimental instruments and materials High Performance Liquid Chromatograph (HPLC): Agilent 1260 HPLC system; Electronic balances: ME204E / 02, XPE26 (Mettler-Toledo Instruments Ltd.); Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.); Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.); Chromatographic column: YMC-Triart C18, 4.6 mm × 250 mm, 5 μm; Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade. Mossin glucoside reference standard (Sichuan Weikeqi Biotechnology Co., Ltd., batch number: WP23100914, content calculated as 99.63%). Curculigoside reference standard (China National Institutes for Food and Drug Control, batch number: 110771-201908, content calculated as 95.8%). Curculigo orchioides medicinal materials: XM-YC-01, XM-YC-02, XM-YC-03, XM-YC-04, XM-YC-05, XM-YC-06; Curculigo orchioides Chinese herbal formula granules: XM-KL-01, XM-KL-02, XM-KL-03, XM-KL-04, XM-KL-05, XM-KL-06; Jiu Xian Mao Chinese Medicine Formula Granules: JXM-KL-01, JXM-KL-02, JXM-KL-03, JXM-KL-04, JXM-KL-05, JXM-KL-06.

[0032] Preparation of Curculigo orchioides granules and wine-processed Curculigo orchioides granules according to embodiments of the present invention: (1) Processing Take the medicinal material of Curculigo orchioides, remove impurities, non-medicinal parts, insect-infested, moldy and other unqualified products, and cut it into slices of about 1cm after cleaning to obtain Curculigo orchioides slices.

[0033] Take 1000g of Curculigo orchioides slices, add 100g of rice wine, mix well, and let it sit naturally for more than 60 minutes until thoroughly moistened. Stir-fry at a temperature of 200-250℃ for 8-10 minutes. During the stir-frying process, observe the properties of the material at any time. Stir-fry until dry, remove the material, and spread it out to cool to obtain the Curculigo orchioides slices.

[0034] (2) Extraction and concentration Take 200g each of Curculigo orchioides slices and Curculigo orchioides wine slices. For the first decoction, add 10 times the amount of water and decoct for 90 minutes. For the second decoction, add 8 times the amount of water and decoct for 60 minutes. During the decoction process, the temperature is controlled at 94-102℃ to obtain the water extract.

[0035] The water extract was collected and concentrated under reduced pressure to a density of 1.01-1.03 at a temperature of 50-70℃ and a vacuum degree of -0.050--0.080 MPa to obtain concentrated Xianmao liquor and concentrated wine Xianmao liquor.

[0036] (3) Drying Take the concentrated liquid and spray dry it under the conditions of inlet air temperature of 190±5℃ and outlet air temperature of 90±5℃ to obtain spray-dried powder of Curculigo orchioides and spray-dried powder of Curculigo orchioides for wine.

[0037] (4) Particle preparation Take the spray-dried powder, add 4.5g of maltodextrin, mix well, perform dry granulation, and collect the particles between the 10-mesh and 40-mesh sieves to obtain Xianmao traditional Chinese medicine formula granules and wine Xianmao traditional Chinese medicine formula granules.

[0038] Example 1 (1) Preparation of reference solution Take appropriate amounts of styracil glucoside and curculigoside reference standards, accurately weigh them, and add methanol to prepare solutions containing 80 μg per 1 mL, which are used as reference solutions.

[0039] (2) Preparation of the test solution Take an appropriate amount (about 0.2g) of each of the two traditional Chinese medicine formula granules (Xianmao and Jiuxianmao) and place them in a stoppered conical flask. Add 25ml of 70% methanol, seal tightly, and sonicate (600W power, 40kHz frequency) for 30 minutes. Let cool, shake well, filter, and collect the filtrate to obtain the final product.

[0040] (3) High performance liquid chromatography determination Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine.

[0041] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A, and 0.1 wt% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1 at a flow rate of 1.0 mL / min; column temperature was 30 °C; detection wavelength was 270 nm. The theoretical plate number, calculated based on curculigoside, should be no less than 5000.

[0042] Table 1

[0043] See results Figure 1-4 , Figure 2 and Figure 3The chromatogram of the Zhongjiu Xianmao traditional Chinese medicine formula granules shows 12 characteristic peaks, which should correspond to the retention times of the two characteristic peaks in the chromatogram of the reference solution. The peak corresponding to the reference substance styrax glucoside is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated and should be within ±10% of the specified values. The specified values ​​are 0.275 (peak 1), 0.320 (peak 2), 0.491 (peak 3), 0.783 (peak 4), 0.929 (peak 5), 1.000 (peak 6, S), 1.068 (peak 7), 1.200 (peak 8), 1.789 (peak 9), 2.660 (peak 10), 2.824 (peak 11), and 3.428 (peak 12). Figure 1 The chromatogram of the Zhongxianmao traditional Chinese medicine formula granules showed 10 characteristic peaks, among which the peak corresponding to the reference substance styraxanthol glucoside was the S peak. The relative retention time of each characteristic peak and the S peak was calculated. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.320 (peak 2), 0.491 (peak 3), 0.783 (peak 4), 0.929 (peak 5), 1.000 (peak 6, S), 1.068 (peak 7), 1.789 (peak 9), 2.660 (peak 10), 2.824 (peak 11), and 3.428 (peak 12). Figure 4 In the study, the chromatographic peaks of both Jiu Xianmao granules and Xianmao granules were repeated 10 times, each containing two reference standards (codonopsis glycoside and cimenopausal glycoside), and Jiu Xianmao granules and Xianmao granules could be distinguished by peaks 1 and 8.

[0044] Example 2 (Mobile Phase Investigation) Example 2 used the same test solution and chromatographic conditions as Example 1, the difference being that Example 2 used acetonitrile-0.1% phosphoric acid, methanol-0.1% phosphoric acid, acetonitrile-0.1% glacial acetic acid, and acetonitrile-water for the investigation. Results are shown in […]. Figure 5 .

[0045] Figure 5 It can be seen that, compared with other mobile phases, the mobile phase of the present invention, which uses acetonitrile-0.1% phosphoric acid, has better peak separation effect and more stable baseline.

[0046] Example 3 (Eluting Gradient Investigation) Example 3 used the same test solution and chromatographic conditions as Example 1, employing acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, a flow rate of 1.0 ml / min, a column temperature of 30°C, and a detection wavelength of 270 nm. The difference was that Example 3 used three elution methods (gradient 1, gradient 2, and gradient 3) from Table 2.

[0047] Table 2

[0048] See results Figure 6 ,from Figure 6 It can be seen that when using an elution gradient of gradient 3 and an elution time of 90 minutes, the chromatographic peaks are completely eluted, the resolution of each chromatographic peak is greater than 1.5, and the separation effect is good.

[0049] Example 4 (Detection Wavelength) Example 4 uses the same test solution and chromatographic conditions as Example 1. The difference is that Example 4 uses a diode array detector to perform a full-band scan of the test solution and extracts chromatograms of the test solution at wavelengths of 230 nm, 270 nm, and 290 nm.

[0050] See results Figure 7 ,from Figure 7 It can be seen that the chromatographic peak shape is better when the detection wavelength is 270nm during the 0-90min period.

[0051] Example 5 (Flow Rate Investigation) Example 5 used the same test solution and chromatographic conditions as Example 1. The difference was that Example 5 investigated flow rates of 0.9 ml / min, 1 ml / min, and 1.1 ml / min.

[0052] See results Figure 8 ,from Figure 8 It can be seen that the chromatogram peak shape is the best and the resolution is moderate when the flow rate is 1.0 ml / min.

[0053] Example 6 (Column Temperature Investigation) Example 6 uses the same test solution and chromatographic conditions as Example 1. The difference is that Example 6 investigates column temperatures of 25°C, 30°C, and 35°C.

[0054] See results Figure 9 ,from Figure 9 It can be seen that when the column temperature is 30℃, the chromatogram peaks are symmetrical and the resolution is optimal.

[0055] Example 7 (Injection Volume Investigation) Example 7 used the same test solution and chromatographic conditions as Example 1. The difference is that Example 7 investigated the injection volumes of 5 μl, 10 μl, and 15 μl, respectively.

[0056] See results Figure 10 ,from Figure 10 It can be seen that the chromatogram peak shape is the best and the resolution is moderate when the injection volume is 10 μl.

[0057] Example 8 (Delayed Investigation) Using the test solution and chromatographic conditions of Example 1, the chromatogram acquisition time was extended by 90 min.

[0058] See results Figure 11 ,from Figure 11 It can be seen that the sample has almost no chromatographic peaks after 90 minutes, so the sample detection time can be set at 90 minutes.

[0059] Example 9 (Examination of Extraction Methods) Example 9 uses the same chromatographic conditions as Example 1, except that in step (2), the sample is extracted by reflux.

[0060] See results Figure 12 ,from Figure 12 It can be seen that the chromatograms of the two extraction methods are highly similar. Since ultrasonic extraction is simpler to operate, ultrasonic extraction is the preferred extraction method in this invention.

[0061] Example 10 (Extraction Solvent Investigation) Example 10 uses the same chromatographic conditions as Example 1, except that in step (2), water, 30% methanol, 70% methanol, and 25 ml of methanol are added respectively, sealed tightly, ultrasonically treated (power 600W, frequency 40kHz) for 30 minutes, cooled, shaken well, filtered, and the filtrate is collected.

[0062] See results Figure 13 ,from Figure 13 It is known that, compared with other extraction solvents, the extraction solvent used in this invention is 70% methanol, which yields a large amount of chromatographic peak information, which is beneficial for the identification of Curculigo orchioides granules and wine-processed Curculigo orchioides granules.

[0063] Example 11 (Investigation of Solvent Addition Amount) Example 11 uses the chromatographic conditions of Example 1, the only difference being in step (2), about 0.2g of the formulation particles are placed in a stoppered conical flask, 10ml, 25ml and 50ml of 70% methanol are added respectively, the flask is sealed, the weight is determined, the mixture is sonicated (power 600W, frequency 40kHz) for 30 minutes, shaken well, filtered, and the filtrate is collected.

[0064] See results Figure 14 ,from Figure 14 It can be seen that when the solvent addition amount is 25 ml, the chromatographic peak area of ​​the characteristic spectrum is moderate, so the preferred solvent addition amount for the test sample is 25 ml.

[0065] Example 12 (Examination of Extraction Time) Example 12 uses the chromatographic conditions of Example 1, the only difference being in step (2), about 0.2g of the formulation granules are placed in a stoppered conical flask, 20ml of 70% methanol is added, the flask is sealed tightly, and ultrasonic treatment is performed (power 600W, frequency 40kHz). The extraction time of the test sample is investigated at 20 minutes, 30 minutes and 40 minutes respectively. The sample is cooled, shaken well, filtered, and the filtrate is collected.

[0066] See results Figure 15 ,from Figure 15 It can be seen that when the extraction time is 30 minutes, the extraction is sufficient, so the optimal extraction time for the test sample is 30 minutes.

[0067] Example 13 (Precision Test) Take the sample solution of Jiuxianmao granules (batch number: JXM-KL-01) and inject it 6 times consecutively according to the experimental method of Example 1, 10 μl each time, and calculate the retention time and peak area of ​​each characteristic peak.

[0068] The results are shown in Tables 3 and 4.

[0069] Table 3 Retention Time

[0070] Table 4 Peak Area

[0071] As shown in Tables 3 and 4, the retention time RSD of each characteristic peak is 0.02%–0.09%, and the peak area RSD is 0.13%–2.08%, indicating that the method has good precision.

[0072] Example 14 (Reproducibility Study) Six portions of the *Curculigo orchioides* granules (batch number: JXM-KL-01) were accurately weighed and tested according to the experimental method in Example 1.

[0073] The results are shown in Tables 5 and 6.

[0074] Table 5 Relative Retention Time

[0075] Table 6 Relative Peak Area

[0076] As shown in Tables 5 and 6, the relative retention time RSD of each characteristic peak is 0–0.08% and the relative peak area RSD is 0–4.55%, indicating that the method has good repeatability.

[0077] Example 15 (Intermediate Precision Study) Using the experimental method of Example 1, two portions of each of the Jiu Xianmao granules (batch number: JXM-KL-01) were accurately weighed by different personnel (A, B) at different times (T1, T2) to prepare test samples for determination.

[0078] The results are shown in Tables 7 and 8.

[0079] Table 7 Relative Retention Time

[0080] Table 8 Relative Peak Area

[0081] As shown in Tables 7 and 8, the method has good stability when different personnel measure the same sample at different times.

[0082] Example 16 (Stability Study) The test solution from Example 1 was used to determine the chromatographic method of Example 1 at 0h, 3h, 6h, 9h, 12h, and 24h.

[0083] The results are shown in Tables 9 and 10.

[0084] Table 9 Retention Time

[0085] Table 10 Peak Area

[0086] As shown in Tables 9 and 10, the corresponding characteristic peak retention time RSD is 0–0.16%, peak area SD is 0–2.12%, and the sample solution is stable within 24 hours.

[0087] In summary, the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that this method is effective.

[0088] Example 17 (Detection of 6 batches of Curculigo orchioides granules and Curculigo orchioides granules) The characteristic spectra of six batches of Jiu Xianmao herbal formula granules and Xianmao herbal formula granules were determined using the method of Example 1. The relative retention time and relative peak area of ​​Jiu Xianmao herbal formula granules and Xianmao herbal formula granules were calculated respectively.

[0089] See results Figure 16-18 See Tables 11-14.

[0090] Table 11-6 shows the relative retention times of batches of *Curculigo orchioides* herbal formula granules.

[0091] Table 12-6 shows the relative peak area of ​​the herbal formula granules for the second batch of *Curculigo orchioides*.

[0092] Table 13-6 shows the relative retention times of batches of Curculigo orchioides granules.

[0093] Table 14-6 shows the relative peak area of ​​Xianmao traditional Chinese medicine granules.

[0094] According to Table 11-14 and Figure 16-17 Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak values, 12 peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 6 was designated as the S peak, the relative retention time (RSD) of the characteristic peaks in all six batches of *Curculigo orchioides* herbal formula granules was less than 2%.

[0095] from Figure 18 It can be seen that six batches of each of the traditional Chinese medicine formula granules of Jiuxianmao and Xianmao can be identified by peak 1 (retention time: 6.304 min) and peak 8 (retention time: 27.533 min), and the specified relative retention time values ​​with peak S (peak 6) are peak 1 (0.275) and peak 8 (1.200).

[0096] Setting limits for relative retention time Tables 15-16 summarize the investigation items and verification results based on Examples 13-16.

[0097] Table 15 Relative Retention Time

[0098] Table 16 Relative Peak Area

[0099] The relative retention time of each peak is specified to be ±10%. The RSD of the relative retention time of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The characteristic chromatogram of the test sample should show 12 characteristic peaks, among which the peak corresponding to the reference phenol glucoside is the S peak.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum for distinguishing between *Curculigo orchioides* (a type of Chinese herbal medicine) formula granules and *Curculigo orchioides* (another type of Chinese herbal medicine) formula granules, characterized in that, include: (1) Using styracin glucoside and curculigoside as reference standards, methanol was added to prepare solutions, which were used as reference solutions; The concentration of the reference solution is 50~100 μg / mL; (2) The test sample is extracted with 70% methanol and filtered to obtain a test sample solution; the mass ratio of the test sample to the volume ratio of the 70% methanol is 0.2g:(10-50)ml; (3) The reference solution and the test solution were determined by high performance liquid chromatography (HPLC). The HPLC conditions included: the column was packed with octadecylsilane-bonded silica gel, and gradient elution was performed using acetonitrile as mobile phase A and 0.1 wt% phosphoric acid aqueous solution as mobile phase B. The flow rate of the mobile phase was 0.9-1.1 ml / min. The detection wavelength was 270 nm. The theoretical plate number calculated based on cicroside should not be less than 5000. The injection volume for the high-performance liquid chromatography is 5-15 μl; The gradient elution is as follows: 0-12 min, mobile phase A: 5%, mobile phase B: 95%; 12-25 min, mobile phase A: 5→8%, mobile phase B: 95→92%; 25-60 min, mobile phase A: 8→15%, mobile phase B: 92→85%; 60-90 min, mobile phase A: 15→33%, mobile phase B: 85→67%. When the test sample is *Curculigo orchioides* granules, the chromatogram shows 12 characteristic peaks, among which the peak corresponding to the reference substance *Curculigo orchioides* glucoside is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified value is: 0.275 - Peak 1, 0.320 - Peak 2, 0.491 - Peak 3, 0.783 - Peak 4, 0.929 - Peak 5, 1.000 - Peak 6, 1.068 - Peak 7, 1.200 - Peak 8, 1.789 - Peak 9, 2.660 - Peak 10, 2.824 - Peak 11, 3.428 - Peak 12; When the test sample is a traditional Chinese medicine formula granule of Curculigo orchioides, 10 characteristic peaks are observed in the chromatogram. Among them, the peak corresponding to the reference substance of styraxanthol glucoside is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: 0.320-peak 2, 0.491-peak 3, 0.783-peak 4, 0.929-peak 5, 1.000-peak 6, S, 1.068-peak 7, 1.789-peak 9, 2.660-peak 10, 2.824-peak 11, 3.428-peak 12.

2. The method according to claim 1, characterized in that, The extraction methods include ultrasonic treatment and / or reflux treatment.

3. The method according to claim 2, characterized in that, The extraction method is ultrasonic treatment.

4. The method according to claim 3, characterized in that, The ultrasonic treatment time is 20-40 minutes; And / or, the power of the ultrasonic treatment is 500-700W; And / or, the frequency of the ultrasonic treatment is 30-50 kHz.

5. The method according to any one of claims 1-4, characterized in that, The column temperature of the chromatographic column is 25-35℃.

6. The method according to claim 1, characterized in that, The method includes: (1) Preparation of reference solution Take appropriate amounts of styracil glucoside and curculigoside reference standards, accurately weigh them, and add methanol to prepare solutions containing 80 μg per 1 mL, which are used as reference solutions. (2) Preparation of the test solution Take 0.2g of each of the following traditional Chinese medicine formulas: Curculigo orchioides granules and Curculigo orchioides granules (both made from wine), place them in a stoppered conical flask, add 25ml of 70% methanol, and seal tightly; sonicate for 30 minutes at a power of 600W and a frequency of 40kHz; cool, shake well, filter, and collect the filtrate to obtain the final product. (3) High performance liquid chromatography determination Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph and determine; Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material; the column length was 250 mm, the inner diameter was 4.6 mm, and the particle size was 5 μm; acetonitrile was used as mobile phase A, and 0.1 wt% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 1, with a flow rate of 1.0 mL per minute; the column temperature was 30 °C; the detection wavelength was 270 nm; the theoretical plate number, calculated based on curculigoside, should not be less than 5000. Table 1 。

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