Detection method and application of spikenard rhizome extract

Abstract

The present invention provides a detection method and use of a spikenard rhizome extract, the detection method comprising a fingerprint and / or a main component content determination method, the detection method can be used for quality detection and main component identification of a spikenard rhizome water extract having whitening activity, and has the characteristics of high precision, strong stability, good repeatability, and high sample recovery rate. Through fingerprint research, it was found that the main components of the spikenard rhizome water extract were deoxysappanol A and sappanone diol, and deoxysappanol A and sappanone diol were determined to be indicator components of the spikenard rhizome water extract. The component content of deoxysappanol A and sappanone diol was determined, and it was confirmed that deoxysappanol A and sappanone diol were the main active ingredients of the spikenard rhizome water extract that exerted whitening activity, and the spikenard rhizome water extract could be used as indicator components for the whitening activity of the spikenard rhizome water extract, and could be further applied to whitening-related industries.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a detection method and application of a spikenard rhizome extract. Background Art

[0002] Melanin is a biological pigment formed through a series of chemical reactions involving tyrosine or 3,4-dihydroxyphenylpropanamine. Melanocytes are typically located in the epidermis, and various skin diseases are caused by excessive epidermal pigment deposition. Tyrosinase inhibitors are important in skin whitening because they catalyze the biosynthesis of melanin. Consequently, tyrosinase inhibitors are becoming increasingly important in medical and cosmetic applications. However, many tyrosinase inhibitors suffer from limitations in their application, such as low activity and high cytotoxicity. Safety is a primary consideration for tyrosinase inhibitors, particularly those used in cosmetic and therapeutic products. Therefore, the search for non-cytotoxic drugs that inhibit melanin biosynthesis to achieve skin whitening effects is of great research significance.

[0003] Nardostachys jatamansi DC. is a perennial herbaceous plant of the genus Nardostachys in the family Caprifoliaceae. It has the effects of regulating qi and relieving pain, relieving depression and invigorating the spleen, and removing dampness and reducing swelling when applied externally. Currently, regarding product research on Nardostachys jatamansi, Chinese patent CN110652475A discloses the application of an alcohol extract of the root of Nardostachys jatamansi in improving skin repair activity and stabilizing skin structure. Chinese patent CN115634176A discloses a method for preparing an aqueous extract of the root of Nardostachys jatamansi obtained by plant tissue culture and its application in skin repair and whitening. Both disclose that different solvent extracts of the root of Nardostachys jatamansi have skin repair and whitening effects. It is well known that the quality and batch consistency of the root of Nardostachys jatamansi extract will vary due to differences in medicinal material processing, solvent extraction, storage methods, etc., which ultimately affect the skin repair and whitening activity of the root of Nardostachys jatamansi extract. However, how to control the quality of the water extract of Nardostachys chinensis rhizomes, identify the index components in the water extract of Nardostachys chinensis rhizomes, determine the content of the index components and evaluate the whitening activity are technical problems that have not yet been solved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a detection method for a spikenard rhizome extract.

[0005] Another technical problem to be solved by the present invention is to provide a use of the main components identified in the above-mentioned detection method of the Nardostachys rhizome extract.

[0006] The technical solution adopted in the present invention is:

[0007] A method for detecting a rhizome extract of Nardostachys chinensis, comprising a fingerprint and / or a main component content determination method, wherein the method can be used for quality detection and main component identification of a water extract of the rhizome of Nardostachys chinensis having whitening activity.

[0008] The fingerprint detection method of the water extract of Nardostachys chinensis rhizome is as follows:

[0009] (i) preparing an aqueous extract of Nardostachys pinnatifida rhizome as a test solution;

[0010] (ii) preparing a reference solution, wherein the reference substances are chlorogenic acid, caffeic acid, deoxygenated galbanol A, galbanone diol, isogamonic acid, galbanone B, derbyone, aristolochic acid, pentasinic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnic acid, galbanone A, and galbanone H standard substances;

[0011] (iii) Setting the chromatographic conditions: The chromatographic column is C 18 Mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid water, gradient elution program: 0-2 min, 2% A, 98% B; 2-4 min, 2%-10% A, 98%-90% B; 4-6 min, 10% A, 90% B; 6-9 min, 10%-15% A, 90%-85% B; 9-11 min, 15%-20% A, 85%-80% B; 11-15 min, 20% A, 80% B; 15-17 min, 20%-30% A, 80% ~70% B; 17~25 min, 30%~40% A, 70%~60% B; 25~29 min, 40%~50% A, 60%~50% B; 29~32 min, 50%~98% A, 50%~2% B; 32~34 min, 98% A, 2% B; 34~35 min, 98%~2% A, 2%~98% B; 35~39 min, 2% A, 98% B; column temperature 45℃, flow rate 0.3 mL / min, detection wavelength 254 nm;

[0012] (iv) establishing a fingerprint: taking the test sample solution prepared in step (i) and the mixed reference sample stock solution prepared in step (ii), measuring the sample using the chromatographic conditions in step (iii), injecting and analyzing the solution, and importing the data file into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" for similarity analysis;

[0013] The method for detecting the main components of the water extract of Nardostachys chinensis rhizome is as follows:

[0014] (1) Prepare an aqueous extract of Nardostachys chinensis rhizomes as a test solution;

[0015] (2) preparing a reference solution, wherein the reference solution comprises naringol and deoxynaringol A;

[0016] (3) Set the chromatographic conditions: the chromatographic column is C 18 , mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid in water, isocratic elution program: 0-20 min, 25% A, 75% B; column temperature: 40 ° C, flow rate: 0.3 mL / min, detection wavelength: 250 nm;

[0017] (4) Chromatographic detection: The test solution of step (1) and the reference solution of step (2) are measured according to the chromatographic conditions of step (3).

[0018] Preferably, in the above-mentioned detection method of the rhizome extract of Nardostachys chinensis, the specific preparation method of step (i) and step (1) is: weigh multiple batches of Nardostachys chinensis medicinal material powder, add pure water respectively, heat and reflux extract twice, obtain filtrate, centrifuge, and take supernatant.

[0019] Preferably, in the above-mentioned method for detecting the extract of Nardostachys rhizome, the material-liquid ratio used in the two extractions in step (i) and step (1) is 1:20-40 (g / mL).

[0020] Preferably, in the above-mentioned method for detecting the extract of Nardostachys rhizome, the single heating reflux time in step (i) and step (1) is 15 minutes.

[0021] Preferably, the detection method of the above-mentioned Nardostachys rhizome extract is prepared by the following method: adding 20 mL of pure water to every 1 g of Nardostachys rhizome powder and reflux extracting for 15 minutes, extracting twice, cooling, filtering, taking the filtrate, centrifuging at 14000 r / min for 10 minutes, and taking the supernatant.

[0022] Preferably, in the above-mentioned detection method of the extract of Nardostachys rhizome, the main components of the water extract of Nardostachys rhizome detected in the detection method are deoxynarbinol A and nardogenol, and the structural formula is as follows:

[0023]

[0024] Preferably, in the above-mentioned detection method of the rhizome extract of strychnine, in the step (ii), chlorogenic acid, caffeic acid, deoxystyracinol A, styracinone diol, isostyracinone, styracinone B, derbyone, aristolochic acid, pentastyracinic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine acid, styracinone A, and styracinone H standard products are respectively taken, dissolved in water, centrifuged, and the supernatant is taken to obtain.

[0025] Preferably, in the above-mentioned detection method of the rhizome extract of strychnine, the concentration of the mixed standard of chlorogenic acid, caffeic acid, deoxystrychnol A, strychnine diol, isostrychnine, strychnine B, derbyone, aristolochic acid, pentasinic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, strychnine A, and strychnine H in step (ii) is 0.1 mg / mL.

[0026] Preferably, in the above-mentioned method for detecting the extract of Nardostachys rhizome, the chromatographic column model in step (iii) is: Wasters ACQUITY UPLC BEH C-18, and the chromatographic column specifications are 2.1×50 mm and 1.7 μm.

[0027] Preferably, in the above-mentioned method for detecting the extract of Nardostachys rhizome, in step (iii), the chromatographic column temperature is 45° C., the flow rate is 0.3 mL / min, and the detection wavelength is 254 nm.

[0028] Preferably, the above-mentioned method for detecting the extract of Nardostachys rhizome adopts the chromatographic conditions of step (iii) to detect the test solution of the water extract of Nardostachys rhizome, with an injection volume of 3 μL, and compares the generated HPLC peak with the control fingerprint, and calculates the similarity between 0.93 and 1.00.

[0029] Preferably, the above-mentioned method for detecting the extract of the rhizome of Nardostachys rapa, and the specific method for establishing the fingerprint spectrum of step (iv) is: taking 38 batches of Nardostachys rapa medicinal materials of different origins and different storage years to prepare the test solution according to step (i), using the chromatographic conditions under step (iii) for determination, and recording the chromatogram; using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for data analysis, setting a reference spectrum, performing multi-point correction, automatic matching, using the average method, selecting a time window setting width of 0.1min, and generating a control spectrum; using the generated control fingerprint spectrum as a reference, measuring the similarity between the sample and the control fingerprint spectrum, and performing similarity evaluation of the sample pieces.

[0030] Preferably, in the above-mentioned method for detecting the extract of the rhizome of Nardostachys chinensis, the specific preparation method of step (2) is: weighing the reference substances of naringenone diol and deoxynaringenol A, adding pure water to the distilled volume, and shaking well to obtain the extract.

[0031] Preferably, in the above-mentioned method for detecting the extract of spikenard rhizome, the concentrations of the standards of naringenone diol and deoxynarbinol A in step (2) are both 1 mg / mL.

[0032] Preferably, in the above-mentioned method for detecting the extract of Nardostachys rhizome, an ACQUITY UPLC Hclass plus chromatograph is used in step (3), and the chromatographic column model is: ACQUITY UPLC BEH C18, and the chromatographic column specifications are 2.1 mm×100 mm and 1.7 μm.

[0033] Preferably, in the above-mentioned method for detecting the extract of Nardostachys rhizome, in step (3), the chromatographic column temperature is 40° C., the flow rate is 0.3 mL / min, the detection wavelength is 250 nm, and the injection volume is 2 μL.

[0034] Preferably, in the above-mentioned detection method of the Nardostachys chinensis rhizome extract, the Nardostachys chinensis rhizome extract has whitening activity.

[0035] Preferably, in the above-mentioned detection method of the spikenard rhizome extract, the spikenard rhizome extract is deoxyspandexol A and / or spikenardiol, and the whitening activity includes melanin inhibitory activity and / or tyrosinase inhibitory activity.

[0036] The application of naringenone diol detected by the above detection method as an indicator component of the whitening activity of the water extract of naringensis rhizome.

[0037] The application of the naringenone diol detected by the above detection method in the preparation of medicines, health products, cosmetics or skin care products with whitening activity.

[0038] Preferably, in the above application, the whitening activity refers to tyrosinase inhibitory activity and / or melanin inhibitory activity.

[0039] The deoxynarbinol A detected by the above detection method is used as an indicator component of the whitening activity of the water extract of the rhizome of Nardostachys juncea.

[0040] The deoxynarbinol A detected by the above detection method is used in the preparation of medicines, health products, cosmetics or skin care products with whitening activity.

[0041] Preferably, in the above application, the whitening activity refers to tyrosinase inhibitory activity and / or melanin inhibitory activity.

[0042] The beneficial effects of the present invention are:

[0043] The above-mentioned detection method for the extract of Nardostachys chinensis rhizome has the characteristics of high precision, strong stability, good repeatability, and high sample recovery rate. It is used in the quality standard detection of the aqueous extract of Nardostachys chinensis rhizome, and can effectively evaluate and ensure the quality of the aqueous extract of Nardostachys chinensis rhizome, thereby ensuring the whitening activity of the aqueous extract of Nardostachys chinensis rhizome. Specifically:

[0044] (1) UPLC fingerprints of 38 batches of water extracts of rhizomes of Nardostachys pinnatifida were established. After extensive experiments, the mobile phases were determined to be acetonitrile as mobile phase A and 0.1% formic acid water as mobile phase B; the gradient elution program was as follows: 0-2 min, 2% A, 98% B; 2-4 min, 2%-10% A, 98%-90% B; 4-6 min, 10% A, 90% B; 6-9 min, 10%-15% A, 90%-85% B; 9-11 min, 15%-20% A, 85%-80% B; 11-15 min, 20% A, 80% B; 15-17 min, 20% A, 80% B; %~30%A, 80%~70%B; 17~25min, 30%~40%A, 70%~60%B; 25~29min, 40%~50%A, 60%~50%B; 29~32min, 50%~98%A, 50%~2%B; 32~34min, 98%A, 2%B; 34~35min, 98%~2%A, 2%~98%B; 35~39min, 2%A, 98%B; the column temperature was 45℃, the flow rate was 0.3mL / min, and the detection wavelength was 254nm. According to the optimized experimental conditions, a total of 7 chromatographic peaks were identified. The results showed that the 38 batches of water extracts from Nardostachys chinensis rhizomes had good similarity, with similarity values ​​all greater than 0.939. The chemical composition differences between the batches were small and the similarity was good, indicating that the material benchmark of the fingerprint of water extracts from Nardostachys chinensis rhizomes established by the detection method described in the present invention has good stability, providing a reference basis for the systematic evaluation and quality control of water extracts from Nardostachys chinensis rhizomes products.

[0045] (2) At the same time, two index content detection methods for deoxygalbinol A and galbinol were established. The results of methodological content determination showed that: ① Precision test results: The RSD values ​​of the intra-day precision of deoxygalbinol A and galbinol were 0.27% and 0.60%, respectively; the RSD values ​​of the inter-day precision were both 0.43%. The intra-day precision and inter-day precision of the two index components showed that the method had good precision; ② Repeatability test results: The RSD values ​​of the peak areas of deoxygalbinol A and galbinol were 0.36% and 0.89%, respectively. The experimental results showed that the content detection method had good repeatability; ③ Stability test results: The relative deviations of the test sample solution of the water extract of galbinol rhizome were 0.19% and 0.32% within 24 hours, indicating that the test sample solution The liquid was measured within 24 hours, and the result remained stable; ④ the sample recovery test results showed that the sample recovery rates of deoxynarbinol A and nardinone diol were both between 95.11% and 99.66%, indicating that the content detection method of the present invention has a good sample recovery rate; ⑤ the content determination method of the main component content of the water extract of jacaranda rhizome was used to determine the contents of deoxynarbinol A and nardinone diol in 38 batches of water extracts of jacaranda rhizome. The content determination results of deoxynarbinol A in a total of 38 batches of water extracts of jacaranda rhizome were 0.2382% to 0.9051%, and the content determination results of nardinone diol were 0.0931% to 0.4218%. It can be seen that the content detection method established by the present invention is stable, accurate and reliable, and can be used in the quality inspection standard of water extracts of jacaranda rhizome to effectively ensure its intrinsic quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The UPLC fingerprint chromatograms of 38 batches of water extracts from rhizomes of Nardostachys pinnatifida were obtained.

[0047] Figure 2 Ultra-high performance liquid chromatograms of the test samples of the water extract of rhizome of jackfruit and the mixed standard of pentasinic acid, chlorogenic acid, caffeic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, deoxystyracinol A, styracinone diol, styracinone A, isostyracinone, aristolochic acid, derbyone, styracinone H and styracinone B: Peak 1 is pentasinic acid, peak 2 is chlorogenic acid, Peak 3 is caffeic acid, peak 4 is 8α-dihydrogeniposide, peak 5 is 7-deoxy-8-epi-strychnic acid, peak 6 is deoxygalbanol A, peak 7 is galbanone diol, peak 8 is galbanone A, peak 9 is isogamone, peak 10 is aristolochicone, peak 11 is derbyone, peak 12 is galbanone H, and peak 13 is aristolochicone B.

[0048] Figure 3The effects of different concentrations of water extract of Nardostachys pinnatifida rhizome, deoxynarbinol A, and nardogenol on the activity of B16F10 cells.

[0049] Figure 4 This is the effect of different concentrations of water extract of Nardostachys pinnatifida rhizome, deoxynarbinol A, and nardogenol on melanin content.

[0050] Figure 5 This is the effect of different concentrations of water extract of Nardostachys pinnatifida rhizome, deoxynarbinol A, and nardogenol on tyrosinase activity. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0052] (1) Optimization of test sample preparation conditions

[0053] The effects of different extraction factors on the components of the test solution were systematically investigated. The first was the investigation of the extraction method: ultrasonic extraction, heating reflux extraction and decoction were investigated. The results showed that the heating reflux extraction method had the highest extraction rate, so the heating reflux extraction method was selected to extract the rhizome of Nardostachys glutinosa; the second was the investigation of the extraction solvent: tap water, pure water and ultrapure water were investigated. The results showed that there was no significant difference in the extraction rate of the rhizome of Nardostachys glutinosa among the three solvents. The extraction rate of the main components, deoxygalbinol A and galbindinone diol, was higher in pure water solvent. The next step was to investigate the material-liquid ratio: the extraction system with three different material-liquid ratios of 1:10, 1:20 and 1:40 was investigated. The results showed that there was no significant difference in the extraction rate when the material-liquid ratio of 1:20 and 1:40 was used for extraction, while the material-liquid ratio of 1:20 was higher than that of the other extraction systems. During extraction, the extraction rate of the main components deoxynarbinol A and narbinone diol was higher, so a solid-liquid ratio of 1:20 can be selected for the extraction of narbinol rhizomes; investigation of extraction time: five different extraction times of 5min, 15min, 30min, 60min and 120min were investigated, and the results showed that there was no significant difference in the extraction rate and the content of the main components deoxynarbinol A and narbinone diol among the five extraction times, so an extraction time of 15min can be selected for the extraction of narbinol rhizomes; finally, the number of extractions was investigated: three extraction times, one, two and three times, were investigated, and the results showed that there was no significant difference in the extraction rate between extracting twice and extracting three times, so two extraction times, which saves time, can be selected for the extraction of narbinol rhizomes.

[0054] (2) Optimization of chromatographic conditions

[0055] The effects of five conditions, namely mobile phase pH, flow rate, injection volume, wavelength and column temperature, on the system adaptability of the main components of the water extract of jasmine rhizome, deoxy-jasmine alcohol A and jasmine ketone diol, were investigated. First, the mobile phases with different pH values ​​were investigated: acetonitrile-0.1% formic acid water and acetonitrile-pure water. The results showed that when the mobile phase was acetonitrile-0.1% formic acid water, the separation between the chromatographic peaks was large, and the target compounds were separated with good peak shape. Therefore, acetonitrile-0.1% formic acid water can be selected as the mobile phase. Then, three flow rates of 0.27mL / min, 0.3mL / min and 0.33mL / min were investigated. The results showed that at a flow rate of 0.3mL / min, the elution time of deoxy-jasmine alcohol A and jasmine ketone diol was earlier, and the separation between the chromatographic peaks was large. Therefore, 0.3mL / min can be determined as the optimal flow rate. Next, the three injections were tested. The injection volume of 1μL, 2μL and 3μL was investigated. The results showed that when the injection volume was 2μL, the separation of deoxygalbinol A and galbinone diol was good, so the injection volume of the water extract of galbindra rhizome was selected as 2μL; then, three detection wavelengths of 250nm, 252nm and 254nm were investigated. The results showed that at a wavelength of 250nm, the peak areas of deoxygalbinol A and galbinone diol were the largest and the separation was good, so the content detection was selected at 250nm; finally, three column temperatures of 35℃, 40℃ and 45℃ were investigated. The results showed that at 40℃, the separation of the two main components was good, so the column temperature of 40℃ was selected. In the fingerprint establishment method, in order to show more chromatographic peaks, the mobile phase ratio was continuously optimized and adjusted. Finally, after a lot of experimental exploration and ratio adjustment, the optimal mobile phase ratio was determined to be: 0-2 min, 2% A, 98% B; 2-4 min, 2%-10% A, 98%-90% B; 4-6 min, 10% A, 90% B; 6-9 min, 10%-15% A, 90%-85% B; 9-11 min, 15%-20% A, 85%-80% B; 11-15 min The elution time was 20 minutes, 20% A, 80% B; 15-17 minutes, 20%-30% A, 80%-70% B; 17-25 minutes, 30%-40% A, 70%-60% B; 25-29 minutes, 40%-50% A, 60%-50% B; 29-32 minutes, 50%-98% A, 50%-2% B; 32-34 minutes, 98% A, 2% B; 34-35 minutes, 98%-2% A, 2%-98% B; 35-39 minutes, 2% A, 98% B. To determine the content of the two main components, the chromatographic conditions were continuously optimized, and the final isocratic elution program was determined to be 0-20 minutes, 25% A, 75% B. Mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid in water.

[0056] Example 1

[0057] Establishment of fingerprint of aqueous extract of rhizome of Nardostachys pinnatifida

[0058] 1. Instruments and equipment

[0059] DZTW temperature-controlled electric heating mantle, Beijing Yongguangming Medical Instrument Co., Ltd.; ML 079 benchtop centrifuge, Eppendorf, Germany; Milli-Qacademic ultrapure water system, Millipore, USA; EYELA CCA-1111 rotary evaporator, EYELA, Japan; ACQUITY An H Class Plus ultra-high performance liquid chromatograph was used, purchased from Waters, USA; an Agilent 6550 UHPLC-QTOF-MS liquid chromatography-mass spectrometer was also used, purchased from Agilent, USA. Reference materials used in this example included quinoline, chlorogenic acid, caffeic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, deoxygalbanol A, galbanoldiol, galbanol A, isogamone, aristolochone, derbyone, galbanol H, and galbanol B, all of which were prepared by the research team in advance. All reference materials had a purity of ≥98%. Acetonitrile was chromatographically pure and purchased from Thermo Fisher Scientific; water was from Watsons.

[0060] 2. Experimental samples

[0061] A total of 38 batches of Nardostachys jatamansi medicinal materials from different origins and storage years were collected. Associate Researcher Zhang Peng of Tianjin University of Traditional Chinese Medicine identified them as dried roots and rhizomes of Nardostachys jatamansi DC., a species of the genus Nardostachys in the Caprifoliaceae family. Voucher specimens are stored at the State Key Laboratory of Component Banking at Tianjin University of Traditional Chinese Medicine. Detailed information is provided in Table 1.

[0062] Table 1 Source information of Nardostachys chinensis medicinal materials

[0063]

[0064]

[0065] 3. Chromatographic conditions

[0066] The chromatographic column was Wasters ACQUITY UPLC BEH C-18 (2.1×50 mm, 1.7 μm), and the gradient elution program was as follows: 0–2 min, 2% A, 98% B; 2–4 min, 2%–10% A, 98%–90% B; 4–6 min, 10% A, 90% B; 6–9 min, 10%–15% A, 90%–85% B; 9–11 min, 15%–20% A, 85%–80% B; 11–15 min, 20% A, 80% B; 15–17 min, 20%–30% A, 80%–70% B. ; 17-25 min, 30%-40% A, 70%-60% B; 25-29 min, 40%-50% A, 60%-50% B; 29-32 min, 50%-98% A, 50%-2% B; 32-34 min, 98% A, 2% B; 34-35 min, 98%-2% A, 2%-98% B; 35-39 min, 2% A, 98% B; the column temperature was 45°C, the flow rate was 0.3 mL / min, the detection wavelength was 254 nm, and the injection volume was 3 μL.

[0067] 4. Preparation of test solution:

[0068] Weigh 1.00 g of 38 batches of nard powder, place it in a 100 mL round-bottom flask, add 20 mL of pure water, reflux extract for 15 min, extract twice, cool, filter, take the filtrate, centrifuge at 14000 r / min for 10 min, and take the supernatant to obtain the product.

[0069] 5. Preparation of reference solution:

[0070] Take chlorogenic acid, caffeic acid, deoxygalbinol A, galbindone, isogamcinone, galbindone B, derbyone, aristolochic acid, pentasinic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnic acid, galbindone A, galbindone H and other standard substances, prepare them into 0.1 mg / mL mixed reference solution, centrifuge at 12000 r / min for 15 min, take the supernatant and place it in a sample vial.

[0071] 6. Establishment of fingerprint of aqueous extract of Nardostachys chinensis rhizome

[0072] 6.1 Precision test

[0073] Using the same sample solution of the water extract of Nardostachys rhizome, inject the sample six times continuously according to the chromatographic conditions in item "3." Record the UPLC chromatogram. Using peak 4, Nardostachysinone diol, as the reference peak, the RSDs of the relative peak areas and relative retention times of the common peaks were calculated to be 0.2% to 0.7% and 0.04% to 0.29%, respectively, indicating good instrument precision. The results are shown in Tables 2-1 and 2-2.

[0074] Table 2-1 Precision test results (relative peak area)

[0075]

[0076]

[0077] Table 2-2 Precision test results (relative retention time)

[0078]

[0079] 6.2 Stability test

[0080] The same sample solution of the aqueous extract of Nardostachys rhizome was injected at 0, 2, 4, 6, 8, 10, 12, and 24 hours using the chromatographic conditions under item "3." Using peak 4, Nardostachysinone diol, as the reference peak, the relative peak area RSDs of the common peaks ranged from 0.52% to 1.25%, and the relative retention time RSDs ranged from 0.07% to 0.36%, indicating that the sample solution was stable within 24 hours. The results are shown in Tables 3-1 and 3-2.

[0081] Table 3-1 Stability test results (relative peak area)

[0082]

[0083] Table 3-2 Stability test results (relative retention time)

[0084]

[0085] 6.3 Repeatability test

[0086] From the same aliquot of Nardostachys chinensis rhizome, six parallel aliquots of the test solution were prepared according to the test solution preparation method under "4." The samples were injected according to the chromatographic conditions under "3." UPLC chromatograms were recorded. Using peak 4, Nardostachys quinone diol, as the reference peak, the RSDs of the relative peak areas and relative retention times of the common peaks were calculated to be 0.86% to 1.32% and 0.03% to 0.36%, respectively, demonstrating good reproducibility. The results are shown in Tables 4-1 and 4-2.

[0087] Table 4-1 Repeatability test results (relative peak area)

[0088]

[0089] Table 4-2 Repeatability test results (relative retention time)

[0090]

[0091]

[0092] 7. Fingerprint establishment and similarity evaluation

[0093] Thirty-eight batches of Nardostachys rhizome samples were sampled and tested according to the test solution preparation method under "4" and the chromatographic conditions under "3," and the chromatograms were recorded. Data analysis was performed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)." A reference chromatogram was set up, and multi-point calibration, automatic matching, averaging, and a 0.1-minute time window were used to generate a control chromatogram. Using the generated control fingerprint as a reference, the similarity between the sample and the control fingerprint was measured and the sample similarity was assessed.

[0094] 38 batches of Nardostachys rhizome water extract samples were analyzed by fingerprint method, and the fingerprint of Nardostachys rhizome water extract and the reference fingerprint were generated by Chinese medicine fingerprint similarity software. Figure 1 A total of 7 chromatographic peaks were identified. The results showed that the 38 batches of water extracts from Nardostachys chinensis rhizomes were similar, with similarity values ​​greater than 0.939. The chemical composition differences between the batches were small and the similarity was good. This indicates that the established fingerprint material benchmark for water extracts from Nardostachys chinensis rhizomes is stable and can reflect its fingerprint characteristics. See Table 5 for details.

[0095] Table 5 Similarity of UPLC fingerprints of 38 batches of water extracts from rhizomes of Nardostachys pinnatifida

[0096]

[0097]

[0098] 8. Identification of characteristic peaks

[0099] Take chlorogenic acid, caffeic acid, deoxygalbinol A, galbinone diol, isogamcinol, galbinone B, derbyone, aristolochic acid, pentasinic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnic acid, galbinone A, galbinone H and other standard substances respectively, follow the preparation method of reference solution under item "5", and perform sample injection and detection under the chromatographic conditions under item "3", and record the chromatogram. By comparing with the chromatogram of the reference substance, the characteristic peaks were identified as quinoline (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), 8α-dihydrogeniposide (peak 4), 7-deoxy-8-epi-strychnine (peak 5), deoxygenated galbanol A (peak 6), galbanone diol (peak 7), galbanone A (peak 8), isogamone (peak 9), aristolochic acid (peak 10), derbyone (peak 11), galbanone H (peak 12), and aristolochic acid B (peak 13). The ultra-high performance liquid chromatograms of the test sample of the water extract of rhizome of Nardostachys pinnatifida and the mixed standard of pentastylic acid, chlorogenic acid, caffeic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, deoxystyrilol A, styrilone diol, styrilone A, isostyrilone, aristolochic acid, derbyone, styrilone H, and styrilone B are shown in Figure 4. Figure 2 shown.

[0100] Example 2

[0101] Determination of the main components of the aqueous extract of Nardostachys pinnatifida rhizome

[0102] 1. Chromatographic conditions

[0103] An ACQUITY UPLC H class plus chromatograph and an ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm) column were used. Mobile phase A was acetonitrile, mobile phase B was 0.1% formic acid in water, and the isocratic elution program was 0-20 min, 25% A, 75% B. The column temperature was 40°C, the flow rate was 0.3 mL / min, the detection wavelength was 250 nm, and the injection volume was 2 μL.

[0104] 2. Preparation of reference solution

[0105] Accurately weigh 10 mg of each of naringenone diol and deoxynaringenol A reference substances, place them in a 10 mL volumetric flask, add the corresponding solvent to the scale, and obtain a reference solution of 1 mg / mL. Dilute stepwise to obtain reference solutions of different concentrations.

[0106] 3. Preparation of test solution

[0107] Accurately weigh 1.00 g of 38 batches of nard powder, place it in a 100 mL round-bottom flask, add 40 mL of pure water, reflux extract for 15 min, extract twice, cool, filter, take the filtrate, centrifuge at 14000 r / min for 10 min, and take the supernatant to obtain the product.

[0108] 4. Linear relationship investigation

[0109] The mixed reference solution was diluted to different concentrations and injected into the liquid chromatograph. The peak area at the same retention time was calculated and a standard curve was plotted with concentration (X) as the abscissa and peak area (Y) as the ordinate. The results are shown in Table 6.

[0110] Table 6 Linear relationship investigation results

[0111]

[0112] 5. Methodology validation test

[0113] 5.1 Precision test

[0114] For intra-day precision, prepare a test solution according to the method in "3." Analyze the test solution using the chromatographic conditions in "1." Repeat the analysis for six consecutive injections. Record the peak areas of the two index components, deoxynarbinol A and naringone diol, and calculate the RSD. For inter-day precision, perform the same intra-day precision analysis, repeating the analysis for three consecutive days. Record the peak areas and RSDs of the two index components. Results are shown in Tables 7 and 8.

[0115] Table 7 Intra-day precision results

[0116]

[0117] The results in the table show that the intra-day relative deviations for deoxynarbinol A and naringenone diol were 0.27% and 0.60%, respectively; the inter-day relative deviations for both were 0.43%. The intra-day and inter-day precisions for these two index components indicate that the method has good precision. These experimental results demonstrate that the liquid chromatograph used has good precision.

[0118] 5.2 Stability test

[0119] The test solution was stored at room temperature. 2 μL of the sample was accurately aspirated at time intervals of 0, 2, 4, 6, 8, 10, 12, and 24 hours and injected into a liquid chromatograph to measure the peak area values ​​of deoxynarbinol A and naringone diol. The results are shown in Table 9.

[0120] Table 9 Stability test results

[0121]

[0122] The above experimental results show that the relative deviations of the test solution measured within 24 hours are 0.19% and 0.32%, respectively, indicating that the results of the test solution remain stable within 24 hours.

[0123] 5.3 Repeatability test

[0124] Six portions of the same batch of Nardostachys chinensis were used to prepare test solutions, and the peak areas were measured. The results are shown in Table 10.

[0125] Table 10 Repeatability test results

[0126]

[0127] The above experimental results show that the relative deviations of the two main components are 0.36% and 0.89%, respectively, indicating that the repeatability of the method is good.

[0128] 5.4 Sample recovery test

[0129] Six replicates of the same batch of nardostachys powder from the repeatability test were accurately weighed and added with appropriate amounts of deoxynardinol A and naringone diol. The sample recovery solutions were prepared according to the method. The results were measured and the recovery rate was calculated using the following formula. The results are shown in Table 11.

[0130] Sample recovery rate = (measured amount - original amount) / added amount × 100%.

[0131] Table 11 Sample recovery test results

[0132]

[0133] The experimental results showed that the recoveries of deoxynarbinol A and naringone diol in 6 samples were between 95.15% and 99.66% and 95.11% and 96.91%, respectively, and the recoveries were good.

[0134] 6. Sample measurement results

[0135] The contents of deoxynarbinol A and naringone diol in 38 batches of water extracts of rhizomes of Nardostachys pinnatifida were determined according to the method described above. The results are shown in Table 12.

[0136] Table 12 Content determination results of 38 batches of water extracts of Nardostachys chinensis rhizomes

[0137]

[0138]

[0139]

[0140] According to the above test results, the content of deoxynarbinol A in the water extracts of 38 batches of naringa rhizomes was determined to be 0.2382% to 0.9051%; the content of naringapinene diol was determined to be 0.0931% to 0.4218%.

[0141] Example 3

[0142] Effects of water extract of rhizome of naringa pinnatifida, deoxynaringaol A and naringapolone diol on the activity of B16F10 cells 1 Instruments and equipment

[0143] Materials: Glucose, sodium hydroxide (NaOH), and dimethyl sulfoxide (DMSO) were purchased from Beijing Solaibao Technology Co., Ltd.; 96-well plates were purchased from Koshida Electronic Materials Co., Ltd.; fetal bovine serum and phosphate-buffered saline were purchased from BI Biotechnology (Israel); lipopolysaccharide and Triton X-100 were purchased from Sigma (USA); penicillin-streptomycin (PS) and 0.25% trypsin-EDTA were purchased from Gibco (USA); melanocyte-stimulating hormone (α-MSH) and levodopa were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; kojic acid (KA) was purchased from Shanghai MacLean Reagent Co., Ltd.; 100 mm cell culture dishes were purchased from THERMO (USA); and 6-well cell culture plates were purchased from NEST (USA). Instruments: Automated microplate reader was purchased from Thermo Fisher Scientific.

[0144] 2 Preparation of complete culture medium

[0145] Prepared with 90% DMEM culture medium, 10% fetal bovine serum, and 1% penicillin-streptomycin; subculture when cells grow to 70-80% as observed under a microscope; discard the culture medium, wash twice with 1 mL PBS buffer, add 800 μL 0.25% trypsin-EDTA, and add 1 mL DMEM complete culture medium to terminate digestion after cells are observed to round and fall off under a microscope; transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 4 minutes at room temperature; discard the supernatant, add complete culture medium, blow evenly, and transfer to a culture flask; culture the culture flask in a 37°C, 5% CO2 cell culture incubator.

[0146] 3B16F10 cell counting and plate culture

[0147] Pipette 10 μL of evenly dispersed cell suspension into the space between the cell counting plate and the coverslip. Find the counting area under the microscope and calculate the cell density = total number of cells in the four squares / 4 × 10 4 × dilution factor; cells were seeded into corresponding culture dishes / plates at a certain density and cultured in a 37°C, 5% CO2 cell culture incubator.

[0148] 4 Solution preparation

[0149] Accurately weigh 30.7 mg of naringenan aqueous extract (AENJ) into a 1.5 mL centrifuge tube and add 0.614 mL of sterile water to completely dissolve it, resulting in a 50 mg / mL AENJ stock solution. Store in a 4°C refrigerator until ready for use. Subsequently, dilute with complete DMEM medium to obtain an AENJ sample solution of the corresponding concentration. Accurately weigh 5.1 mg of deoxynaringenanol A (DA) into a 1.5 mL centrifuge tube and add 265.5 μL of DMSO to completely dissolve it, resulting in a 100 mM DA stock solution. Store in a 4°C refrigerator until ready for use. Subsequently, dilute with complete DMEM medium to obtain a 100 mM deoxynaringenanol A sample solution. Accurately weigh 5.09 mg of naringenanone diol (ND) into a 1.5 mL centrifuge tube and add 201.8 μL of DMSO to completely dissolve it, resulting in a 100 mM ND stock solution. The solution was stored in a refrigerator at 4°C for future use and subsequently diluted with complete DMEM culture medium to obtain a ND sample solution of corresponding concentration.

[0150] 5 Operation process

[0151] 100 μg / mL, 10 μg / mL, 1 μg / mL of nardus serrata aqueous extract (DMEM as solvent), 10 μM, 1 μM, 10 -1 μM deoxynarbinol A (DMEM as solvent) and 100 μM, 10 μM, and 1 μM naringapine diol (DMEM as solvent) were added to different cell culture wells, 100 μL per well. A blank control group without drug was also set up. The blank control group and the drug-treated groups with different concentrations were all set up with 6 replicate wells, repeated twice, and cultured in an incubator for 24 hours; the supernatant was discarded with a pipette, washed twice with PBS buffer, and 1×CCK-8 working solution diluted in DMEM medium was added under light-proof conditions, 100 μL per well, incubated in a 37°C constant temperature incubator in the dark for 30 minutes, and the absorbance was measured at 450 nm with a microplate reader; the cell viability was calculated according to the instructions of the CCK-8 kit.

[0152] 6 Experimental results

[0153] Depend on Figure 3 It can be seen that there is no significant difference in the B16F10 cell viability between the sample solutions of different concentrations of nardostachys water extract, deoxynardostachys A and nardostim diol and the normal control group. This is a safe concentration for B16F10 cells and can be used for subsequent determination of melanin content and tyrosinase activity.

[0154] Example 4

[0155] Effects of aqueous extracts of rhizome of spikenard, deoxysappanol A, and spikenardone diol on melanin content

[0156] 1. Solution preparation

[0157] Accurately weigh 1 mg of α-MSH and dissolve it in 1 mL of sterile water to obtain a 600 μM α-MSH stock solution. Store at 4°C until needed. Dilute with complete culture medium to obtain a 100 nM α-MSH sample solution. Accurately weigh 10 mg of kojic acid and dissolve it in 1 mL of sterile water to obtain a 10 mg / mL kojic acid stock solution. Store at 4°C until needed. Prepare a 100 μg / mL sample solution by serial dilution with complete culture medium. Accurately weigh 4 g of NaOH, add purified water, stir to dissolve thoroughly, and dilute to 100 mL. Store at room temperature to obtain a 1 M NaOH solution.

[0158] 2 Operation process

[0159] 4*10 4 The cells were seeded at a density of 100 cells / mL in a 100 mm culture dish and cultured for 24 h. 600 μM α-MSH stock solution was diluted with DMEM complete medium to a 100 nM α-MSH sample solution, and the 100 nM α-MSH sample solution was diluted to 100, 10, and 1 μg / mL of nardus serrata aqueous extract, and the concentrations were 10, 1, and 10 μg / mL. -1 μM deoxynarbinol A, 100, 10, and 1 μM narathione diol, and 100 μg / mL kojic acid sample solutions; remove the culture dish, add the diluted sample solutions in sequence, and set up a blank control group with only DMEM complete medium, and culture in an incubator for 48 hours. Discard the supernatant, wash once with PBS, add about 700 μL of 0.25% trypsin-EDTA to each well, digest at 37℃ for 20s, and then add DMEM complete culture medium to stop digestion; transfer the cell suspension into a 1.5mL centrifuge tube, centrifuge at 1000rpm for 3min at room temperature to obtain the corresponding cell pellet; after discarding the supernatant, add 1mL PBS buffer to each tube, centrifuge at 13000rmp at room temperature for 10min to obtain cell melanin precipitate; after aspirating the supernatant as much as possible, add the corresponding volume of lysis solution (10% DMSO + 90% 1M NaOH) to each tube according to the number of cells, vortex mix, and place in an 80℃ metal bath and heat for 90min. If there is still precipitation, extend the time appropriately until the melanin is completely dissolved; take 100 μL of the mixed solution in each tube in the above (6) and add it to a 96-well plate, measure the absorbance value at 405nm on an enzyme reader, and use the lysis solution as a blank well. Melanin content calculation formula:

[0160] Melanin content / % = (sample well A 405 -Blank hole A 405 ) / (control well A 405 -Blank hole A 405 )×100%

[0161] 3 Statistical processing

[0162] All data were processed using SPSS26 statistical analysis software and expressed as mean ± standard deviation (x ± SD). One-way analysis of variance was used between groups, and P < 0.05 indicated that the difference was statistically significant.

[0163] 4 Experimental results

[0164] Depend on Figure 4 It can be seen that compared with the Control group, the melanin production of B16F10 cells in the α-MSH group was significantly increased; compared with the α-MSH group, the KA group significantly reduced the melanin content in B16F10 cells, and the 10 and 100 μg / mL nardus rapa aqueous extract groups significantly reduced the melanin content of B16F10 cells induced by α-MSH (P < 0.01, P < 0.001); 10 -1 , 1, and 10 μM deoxynarbinol group A significantly reduced the melanin content of B16F10 cells induced by α-MSH (P < 0.05, P < 0.01, P < 0.001); 1 μM naringazone diol group significantly reduced the melanin content of B16F10 cells induced by α-MSH (P < 0.05).

[0165] Example 5

[0166] Effects of aqueous extracts of rhizomes of Nardostachys pinnatifida, deoxynarbinol A and nardosinone diol on tyrosinase activity

[0167] 1. Solution preparation

[0168] Accurately weigh 1 mg of L-DOPA in 1 mL of pure water to obtain a 5 mM L-DOPA sample solution.

[0169] 2 Operation process

[0170] 4*10 4 The cells were seeded at a density of 100 cells / mL in 6-well plates and cultured for 24 h. The 600 μM α-MSH stock solution was diluted with DMEM complete medium to a concentration of 100, 10, and 1 μg / mL of nardus serrata aqueous extract. -1μM deoxynarbinol A, 100, 10, and 1 μM naringapolone diol, and 100 μg / mL kojic acid sample solutions were prepared; the 6-well plate was taken out and the diluted sample solutions were added in sequence, and a blank control group with only DMEM complete medium was set up and cultured in an incubator for 48 hours; the supernatant was discarded and 500 μL of Digest with 0.25% trypsin-EDTA at 37°C for 15 seconds, then immediately add complete DMEM to terminate digestion. Transfer the cell suspension to a 1.5mL centrifuge tube and centrifuge at 1000rpm for 3 minutes at room temperature. Discard the supernatant and add 1mL of 1× PBS buffer to each tube, mix thoroughly, and centrifuge at 1000rpm for 3 minutes at room temperature. Add the corresponding volume of 1× PBS solution containing 1% TritonX-100 to each tube according to the number of cells, and lyse at 4°C for 30 minutes. Re-vortex every 10 minutes to ensure complete lysis. Centrifuge at 12000rpm at 4°C for 10 minutes to obtain the supernatant of each sample. Transfer the supernatant to a new centrifuge tube, pipette, and take 50μL of each into a 96-well plate. Add an equal volume of 5mM L-DOPA solution to the supernatant, incubate at 37°C for 30 minutes, and measure the absorbance at 475nm on a microplate reader. Use the 5mM L-DOPA solution as a blank well. Tyrosinase activity is calculated using the following formula:

[0171] Tyrosinase activity / % = (sample well A 475 -Blank hole A 475 ) / (control well A 475 -Blank hole A 475 )×100%

[0172] 3 Statistical processing

[0173] All data were processed using SPSS26 statistical analysis software and expressed as mean ± standard deviation (x ± SD). One-way analysis of variance was used between groups, and P < 0.05 indicated that the difference was statistically significant.

[0174] 4 Experimental results

[0175] like Figure 5 As shown in the results, compared with the Control group, the tyrosinase activity of B16F10 cells in the α-MSH group was significantly increased; compared with the α-MSH group, the KA group significantly reduced the tyrosinase activity in B16F10 cells, and 10 μg / mL and 100 μg / mL of nardus rapa aqueous extracts significantly inhibited the tyrosinase activity of B16F106 cells induced by α-MSH (P < 0.01, P < 0.01); -1μM, 1μM, and 10μM deoxynarbinol A significantly inhibited the tyrosinase activity of B16F10 cells induced by α-MSH (P<0.05, P<0.05, P<0.01); 1μM and 10μM naringafoetida significantly inhibited the tyrosinase activity of B16F10 cells induced by α-MSH (P<0.001, P<0.01).

[0176] In summary, this study established the first ultra-high performance liquid chromatography fingerprint of the water extract of Nardostachys pinnatifida rhizome. Through fingerprint analysis, the main components of the water extract of Nardostachys pinnatifida rhizome were found to be deoxygalbinol A and galbinone diol. Deoxygalbinol A and galbinone diol were identified as indicator components of the water extract of Nardostachys pinnatifida rhizome, and the content of deoxygalbinol A and galbinone diol was determined. The tyrosinase inhibitory activity and melanin inhibitory activity of the water extract of Nardostachys pinnatifida rhizome and its main components, deoxygalbinol A and galbinone diol, were verified to be the main active components of the water extract of Nardostachys pinnatifida rhizome that exert whitening activity. In other words, deoxygalbinol A and galbinone diol can be used as indicator components of the water extract of Nardostachys pinnatifida rhizome to exert whitening activity, and deoxygalbinol A and galbinone diol can be further applied in whitening-related industries. The detection method of the present invention is efficient, simple, and has good repeatability, and provides a good reference basis for the medicinal material basis and comprehensive quality control of the water extract of Nardostachys chinensis rhizomes.

[0177] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements to the technical solutions of the present invention made by ordinary engineers and technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for detecting an extract of Nardostachys chinensis rhizome, characterized in that: The fingerprint detection method of the water extract of Nardostachys chinensis rhizome is adopted, and the specific steps are as follows: (i) preparing an aqueous extract of Nardostachys pinnatifida rhizome as a test solution; (ii) preparing a reference solution, wherein the reference solution is chlorogenic acid, caffeic acid, deoxygalbanol A, galbanol, isogamone, aristolochic acid B, derby ketone, aristolochic acid, 8 α -Dihydrogeniposide, 7-deoxy-8-epi-strychnine, galbanumone A and galbanumone H standards; (iii) Setting the chromatographic conditions: The chromatographic column is C 18 , mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid in water, gradient elution program: 0-2 min, 2% A, 98% B; 2-4 min, 2%-10% A, 98%-90% B; 4-6 min, 10% A, 90% B; 6-9 min, 10%-15% A, 90%-85% B; 9-11 min, 15%-20% A, 85%-80% B; 11-15 min, 20% A, 80% B; 15-17 min, 20%-30% A, 80%-70% B; 17-25 min, 30%-40% A, 70%-60% B; 25-29 min, 40%-50%A, 60%-50%B; 29-32 min, 50%-98%A, 50%-2%B; 32-34 min, 98%A, 2%B; 34-35 min, 98%-2%A, 2%-98%B; 35-39 min, 2%A, 98%B; column temperature 45°C, flow rate 0.3 mL / min, detection wavelength 254 nm; (iv) Establishing fingerprints: Take the test sample solution prepared in step (i) and the mixed reference stock solution prepared in step (ii), and perform determination using the chromatographic conditions of step (iii). Sampling and analyzing the solutions, import the data files into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" for similarity analysis.

2. The method for detecting the Nardostachys chinensis rhizome extract according to claim 1, wherein: The specific preparation method of step (i) is as follows: weighing multiple batches of nardostachys raphanus medicinal material powder, adding pure water respectively, heating and refluxing extraction twice, obtaining filtrate, centrifuging, and taking supernatant to obtain; the material-liquid ratio used in the two extractions in step (i) is 1:20-40 (g / mL); the single heating and reflux time in step (i) is 15 minutes.

3. The method for detecting the Nardostachys chinensis rhizome extract according to claim 1, wherein: The chromatographic column model in step (iii) is: Wasters ACQUITY UPLC BEH C-18, and the chromatographic column specifications are 2.1×50 mm, 1.7 μ m; column temperature was 45°C, injection volume was 3 μ L, the flow rate was 0.3 mL / min, and the detection wavelength was 254 nm.

4. The method for detecting the Nardostachys chinensis rhizome extract according to claim 1, wherein: The specific method for establishing the fingerprint spectrum of step (iv) is as follows: 38 batches of Nardostachys raphanidae medicinal materials of different origins and different storage years are taken to prepare test sample solutions according to step (i), and the chromatographic conditions under step (iii) are used for determination, and the chromatograms are recorded; the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" is used for data analysis, a reference spectrum is set, multi-point correction and automatic matching are performed, the average method is used, and a time window setting width of 0.1 min is selected to generate a control spectrum; the generated control fingerprint spectrum is used as a reference to measure the similarity between the sample and the control fingerprint spectrum, and perform similarity evaluation of the sample pieces.

5. The method for detecting the Nardostachys chinensis rhizome extract according to claim 1, wherein: The invention also includes a method for detecting the main components of the water extract of the rhizome of Nardostachys rapa, and the specific steps are as follows: (1) Prepare the water extract of Nardostachys chinensis rhizome as the test solution; (2) preparing a reference solution, wherein the reference solution is narcotone diol and deoxynarcotyl alcohol A; (3) Set the chromatographic conditions: the chromatographic column is C 18 , mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid in water, isocratic elution program: 0-20 min, 25% A, 75% B; column temperature: 40 °C, flow rate: 0.3 mL / min, detection wavelength: 250 nm; (4) Chromatographic detection: The test solution of step (1) and the reference solution of step (2) are measured according to the chromatographic conditions of step (3).

6. The method for detecting the extract of Nardostachys chinensis rhizome according to claim 5, wherein: The specific preparation method of step (1) is as follows: weigh multiple batches of nardus radiata powder, add pure water respectively, heat and reflux extract twice, obtain filtrate, centrifuge, and take supernatant to obtain; the material-liquid ratio used in the two extractions in step (1) is 1:20-40 (g / mL); the single heating and reflux time in step (1) is 15 minutes.

7. The method for detecting the extract of Nardostachys chinensis rhizome according to claim 5, wherein: In step (3), an ACQUITY UPLC H class plus chromatograph was used, and the chromatographic column model was ACQUITY UPLC BEH C18, with chromatographic column specifications of 2.1 mm × 100 mm, 1.7 μ m, column temperature was 40 °C, flow rate was 0.3 mL / min, detection wavelength was 250 nm, injection volume was 2 μ L.

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