Detection method and application of rhizoma nardostachyos rhizome extract

Abstract

The invention provides a detection method and application of a rhizoma nardostachyos rhizome extract, the detection method comprises a fingerprint spectrum and / or main component content determination method, the detection method can be applied to quality detection and main component identification of a rhizoma nardostachyos rhizome aqueous extract with whitening activity, and the detection method has the advantages of high precision, strong stability, good repeatability, high sensitivity and the like. And the sample adding recovery rate is high. According to the present invention, the main components of the rhizoma nardostachyos rhizome aqueous extract are the desoxynardostinol A and the nardostinone diol through the fingerprint spectrum research, the desoxynardostinol A and the nardostinone diol are determined as the index components of the rhizoma nardostachyos rhizome aqueous extract, and the component content determination of the desoxynardostinol A and the nardostinone diol is performed; it is proved that desoxynardosinol A and nardosinone diol are main active components of the rhizoma nardostachyos rhizome aqueous extract for playing the whitening activity, can be used as index components of the rhizoma nardostachyos rhizome aqueous extract for playing the whitening activity, and can be further applied to related whitening industries.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, in particular to a detection method and application of a Nardostachys chinensis rhizome extract. Background Art

[0002] Melanin is a biological pigment formed by a series of chemical reactions such as tyrosine or 3,4-dihydroxyphenylalanine. Melanocytes are usually located in the epidermis of the skin, and various skin diseases are caused by excessive deposition of epidermal pigments. Tyrosinase inhibitors are important in skin whitening because they catalyze the biosynthesis of melanin. Therefore, tyrosinase inhibitors are becoming increasingly important in medical and cosmetics. However, many tyrosinase inhibitors are limited in their application, such as low activity and high cytotoxicity. Among them, safety is the primary consideration for tyrosinase inhibitors, especially those used in cosmetics and therapeutic products. Therefore, it is of great research significance to find drugs that are non-cytotoxic and have an inhibitory effect on melanin biosynthesis to achieve the effect of skin whitening.

[0003] Nardostachys jatamansi DC. is a perennial herbaceous plant of the genus Nardostachys in the family Caprifoliaceae, which has the effects of regulating qi and relieving pain, relieving depression and invigorating the spleen, and removing dampness and swelling for external use. At present, for the product research of Nardostachys jatamansi, Chinese patent CN110652475A discloses the application of alcohol extract of Nardostachys jatamansi root in improving skin repair activity and stabilizing skin structure. Chinese patent CN115634176A discloses a preparation method of Nardostachys jatamansi root water extract obtained by plant tissue culture and its application in skin repair and whitening. Both disclose that different solvent extracts of Nardostachys jatamansi root have skin repair and whitening effects. It is well known that the quality and batch consistency of Nardostachys jatamansi root extract will vary due to differences in medicinal material processing, solvent extraction, storage methods, etc., which ultimately affects the skin repair and whitening activity of Nardostachys jatamansi root 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, and 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 method for detecting a Nardostachys chinensis rhizome extract.

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

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

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

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

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

[0010] (ii) preparing a reference solution, wherein the reference solution is chlorogenic acid, caffeic acid, deoxygenated galbanol A, galbanolone diol, isoglanol, galbanolone B, derbyone, aristolochic acid, pentasperglycine, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, galbanolone A, and galbanolone 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; 17~25min, 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;

[0012] (iv) Establishing fingerprint: taking the test sample solution of step (i) and the mixed reference sample stock solution of step (ii), measuring them under the chromatographic conditions of step (iii), injecting and analyzing the solution, and importing the data file into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" to perform similarity analysis;

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

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

[0015] (2) preparing a reference solution, wherein the reference solution is narcotone glycol and deoxynarcotyl alcohol A;

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

[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) to obtain the product.

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

[0019] Preferably, in the above-mentioned method for detecting the extract of Nardostachys chinensis rhizomes, the solid-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 grandiflora rhizomes, the single heating reflux time in step (i) and step (1) is 15 minutes.

[0021] Preferably, in the above-mentioned detection method of the Nardostachys affine rhizome extract, the Nardostachys affine rhizome water extract is prepared by the following method: every 1g of Nardostachys affine powder is added into 20mL of pure water and refluxed for 15min, extracted twice, cooled, filtered, the filtrate is taken, centrifuged at 14000r / min for 10min, and the supernatant is taken.

[0022] Preferably, in the above-mentioned detection method of the extract of rhizome of Nardostachys affine, the main components of the water extract of rhizome of Nardostachys affine 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 Nardostachys chinensis, in the step (ii), chlorogenic acid, caffeic acid, deoxygenated naringol A, naringone diol, isogranthene, aristolochic acid B, derbyone, aristolochic acid, pentasperglycine, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, naringone A, and naringone 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 nardostachys affine, the concentration of the mixed standard of chlorogenic acid, caffeic acid, deoxystyraxol A, styraxone diol, isostyraxone, styraxone B, derbyone, aristolochic acid, pentastyric acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, styraxone A, and styraxone H in step (ii) is 0.1 mg / mL.

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

[0027] Preferably, in the above-mentioned method for detecting the extract of Nardostachys grandiflora rhizomes, 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 chinensis rhizomes adopts the chromatographic conditions of step (iii) to detect the test solution of the water extract of Nardostachys chinensis rhizomes, with an injection volume of 3 μL, and compares the generated HPLC peak with the control fingerprint spectrum, and calculates the similarity between 0.93 and 1.00.

[0029] Preferably, the detection method of the above-mentioned Nardostachys rhizome extract, the specific method of establishing the fingerprint spectrum of step (iv) is: take 38 batches of Nardostachys rhizome extract from different origins and different storage years to prepare the test solution according to step (i), use the chromatographic conditions under step (iii) for determination, and record the chromatogram; use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" to perform data analysis, set a reference spectrum, perform multi-point correction, automatic matching, use the average method, select a time window setting width of 0.1min, and generate a control spectrum; use the generated control fingerprint spectrum as a reference, measure the similarity between the sample and the control fingerprint spectrum, and perform similarity evaluation of the sample pieces.

[0030] Preferably, in the above-mentioned method for detecting the extract of rhizomes of Nardostachys grandiflora, the specific preparation method of step (2) is: weigh the reference substances of naringenone diol and deoxynaringenol A, dilute to volume with pure water, and shake well to obtain.

[0031] Preferably, in the above-mentioned method for detecting the extract of Nardostachys chinensis rhizome, the concentrations of the standard substances of Nardostachys chinensis diol and deoxynardol A in step (2) are both 1 mg / mL.

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

[0033] Preferably, in the above-mentioned method for detecting the extract of Nardostachys affine rhizomes, 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 affine rhizome extract, the Nardostachys affine rhizome extract has whitening activity.

[0035] Preferably, in the above-mentioned detection method of the extract of Nardostachys affine rhizome, the extract of Nardostachys affine rhizome is deoxynarbinol A and / or nardostria diol, 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 rhizomes.

[0037] The application of 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 water extract of rhizomes of naringa pine to exert whitening activity.

[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 detection method of the extract of rhizome of Nardostachys affine 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 water extract of rhizome of Nardostachys affine, and can effectively evaluate and guarantee the quality of the water extract of rhizome of Nardostachys affine, thereby ensuring the whitening activity of the water extract of rhizome of Nardostachys affine. Specifically:

[0044] (1) UPLC fingerprints of 38 batches of water extracts of rhizomes of Nardostachys pinnatifida were established. After extensive experimental exploration, 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 %~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, the detection wavelength was 254nm, and according to the optimized experimental conditions, a total of 7 chromatographic peaks were identified. The results showed that 38 batches of water extracts from rhizomes of Nardostachys chinensis had good similarity, with similarity values ​​all greater than 0.939. The differences in chemical composition between batches were small and the similarity was good, indicating that the material benchmark of the fingerprint of water extracts from rhizomes of Nardostachys chinensis established by the detection method of the present invention had good stability, which provides a reference for the systematic evaluation and quality control of water extracts from rhizomes of Nardostachys chinensis.

[0045] (2) Two index content detection methods for deoxygalbinol A and galbinolone were established at the same time. The results of methodological content determination showed that: ① Precision test results: The RSD values ​​of the intra-day precision of deoxygalbinol A and galbinolone 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 galbinolone 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 determined within 24 hours and were 0.19% and 0.32%, respectively, 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 naringone 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 of deoxynarbinol A and naringone diol in 38 batches of naringone rhizome water extracts determined by the method for determining the main components of naringone rhizome water extracts, and the content determination results of deoxynarbinol A in 38 batches of naringone rhizome water extracts were 0.2382% to 0.9051%, and the content determination results of naringone 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 detection standard of naringone rhizome water extracts to effectively ensure its intrinsic quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The UPLC fingerprint chromatograms of 38 batches of water extracts of Nardostachys pinnatifida rhizomes are shown;

[0047] Figure 2 Ultra-high performance liquid chromatograms of the test samples of water extract of rhizome of Nardostachys pinnatifida and mixed standard of pentastylic acid, chlorogenic acid, caffeic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, deoxystyril alcohol A, pentastyrilone diol, pentastyrone A, isostastyrone, aristolochic acid, derbyone, pentastyrone H and pentastyrone B: chromatographic peak 1 is pentastylic acid, chromatographic peak 2 is chlorogenic acid, Chromatographic peak 3 is caffeic acid, chromatographic peak 4 is 8α-dihydrogeniposide, chromatographic peak 5 is 7-deoxy-8-epi-strychnic acid, chromatographic peak 6 is deoxygenated styracol A, chromatographic peak 7 is styracolone diol, chromatographic peak 8 is styracolone A, chromatographic peak 9 is isostyracolone, chromatographic peak 10 is aristolochicone, chromatographic peak 11 is derbyone, chromatographic peak 12 is styracolone H, and chromatographic peak 13 is styracolone B;

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

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

[0050] Figure 5 This paper shows the effects of different concentrations of water extract of Nardostachys chinensis rhizomes, deoxynarbinol A, and nardogenone diol 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 in conjunction with 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 rhizomes of Nardostachys grandiflora; 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 three solvents on the rhizomes of Nardostachys grandiflora, and the pure water solvent with a higher extraction rate of the main components, deoxygalbinol A and galbinolone diol, could be selected for extraction; the next step was the investigation of the solid-liquid ratio: three different solid-liquid ratio extraction systems of 1:10, 1:20 and 1:40 were investigated. The results showed that there was no significant difference in the extraction rate when using the 1:20 and 1:40 solid-liquid ratios, while the 1:20 solid-liquid ratio During extraction, the extraction rate of the main components deoxygalbinol A and galbinol diol was higher, so a solid-liquid ratio of 1:20 can be selected for extraction of galbinol 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 deoxygalbinol A and galbinol among the five extraction times, so an extraction time of 15min can be selected for extraction of galbinol rhizomes; finally, the number of extractions was investigated: three extraction times, one, two and three, 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 that save time can be selected to extract galbinol rhizomes.

[0054] (2) Optimization of chromatographic conditions

[0055] The effects of mobile phase pH, flow rate, injection volume, wavelength and column temperature on the system adaptability of the main components of the water extract of Nardostachys chinensis rhizomes, deoxynarbinol A and narbinone 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 a good peak shape. Therefore, acetonitrile-0.1% formic acid water can be selected as the mobile phase; then the 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 deoxynarbinol A and narbinone diol was earlier, and the separation between the chromatographic peaks was large, so 0.3mL / min was determined to be the optimal flow rate; next, the three injections were used. 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 galbinolone diol was good, so the injection volume of the water extract of galbinol 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 galbinolone diol were the largest, and the separation was better, so 250nm was selected for content detection; 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: 0-2min, 2% A, 98% B; 2-4min, 2%-10% A, 98%-90% B; 4-6min, 10% A, 90% B; 6-9min, 10%-15% A, 90%-85% B; 9-11min, 15%-20% A, 85%-80% B; 11-15min n, 20% A, 80% B; 15-17min, 20%-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. In order to determine the content of the two main components, the chromatographic conditions were continuously optimized, and the isocratic elution program was finally determined to be 0-20min, 25% A, 75% B. Mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid water.

[0056] Example 1

[0057] Establishment of fingerprint of water extract of rhizome of Nardostachys grandiflora

[0058] 1. Instruments and Equipment

[0059] DZTW temperature-controlled electric heating jacket, Beijing Yongguangming Medical Instrument Co., Ltd.; ML 079 desktop centrifuge, Eppendorf, Germany; Milli-Qacademic ultrapure water system, Millipore, USA; EYELA CCA-1111 rotary evaporator, EYELA, Japan; ACQUITY H class plus ultra-high performance liquid chromatograph, Waters, USA; UHPLC-QTOF-MS liquid mass spectrometer (Agilent 6550), Agilent, USA. In this example, the reference materials selected were pentaerythrin, chlorogenic acid, caffeic acid, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, deoxygalactoside A, galactoside diol, galactoside A, isogalactone, aristolochic acid, derbyone, galactoside H, and galactoside B, all of which were prepared by the research group in the early stage. The purity of all reference materials was ≥98%. Acetonitrile was chromatographically pure and purchased from Thermo Fisher Scientific; water was Watsons water.

[0060] 2. Experimental samples

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

[0062] Table 1 Source information of Nardostachys grandiflora

[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: 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 ; 17-25min, 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°C, the flow rate was 0.3mL / min, the detection wavelength was 254nm, and the injection volume was 3μL.

[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, take the supernatant, and obtain.

[0069] 5. Preparation of reference solution:

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

[0071] 6. Establishment of fingerprint of water extract of rhizome of Nardostachys grandiflora

[0072] 6.1 Precision test

[0073] Take the same sample solution of water extract of rhizome of Nardostachys pinnatifida, and inject it 6 times continuously according to the chromatographic conditions under item "3". Record the UPLC chart. Take peak 4, Nardostachysinone diol, as the reference peak. The RSD values ​​of relative peak area and relative retention time of each common peak are calculated to be 0.2% to 0.7% and 0.04% to 0.29%, respectively, indicating that the instrument has good precision. The results are shown in Table 2-1 and Table 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] Take the same sample solution of water extract of rhizome of Nardostachys pinnatifida, and inject the sample at 0, 2, 4, 6, 8, 10, 12 and 24 hours according to the chromatographic conditions under item "3", and take peak 4, Nardostachysinone diol, as the reference peak. The relative peak area RSD value of each common peak is between 0.52% and 1.25%, and the relative retention time RSD value is between 0.07% and 0.36%, indicating that the sample solution is stable within 24 hours. The results are shown in Table 3-1 and Table 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] Take the same portion of Nardostachys pine rhizome, prepare 6 portions of test solution in parallel according to the preparation method of test solution under item "4", inject according to the chromatographic conditions under item "3", record the UPLC chart, take peak 4 Nardostachys pine ketone diol as the reference peak, calculate the relative peak area and relative retention time of each common peak RSD values ​​of 0.86% to 1.32% and 0.03% to 0.36%, respectively, indicating that the method has good repeatability. The results are shown in Table 4-1 and Table 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] Take 38 batches of Nardostachys grandiflora samples respectively, and test them according to the preparation method of the test solution under "4" and the chromatographic conditions under "3", and record the chromatograms. Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" for data analysis, set the reference spectrum, perform multi-point correction, automatic matching, use the average method, select a time window setting width of 0.1min, and generate a control spectrum. Use the generated control fingerprint spectrum as a reference, measure the similarity between the sample and the control fingerprint spectrum, and perform the similarity evaluation of the sample.

[0094] Thirty-eight batches of Nardostachys chinensis rhizome water extract samples were analyzed by fingerprint method, and the fingerprint of Nardostachys chinensis rhizome water extract and the reference fingerprint were generated by the traditional Chinese medicine fingerprint similarity software. Figure 1 A total of 7 chromatographic peaks were identified, and the results showed that the 38 batches of water extracts of Nardostachys chinensis rhizomes had good similarity, with similarity values ​​greater than 0.939. The differences in chemical composition between the batches were small and the similarity was good, indicating that the material benchmark of the established fingerprint of water extracts of Nardostachys chinensis rhizomes was stable and could reflect its fingerprint characteristics, as shown in Table 5.

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

[0096]

[0097]

[0098] 8. Identification of characteristic peaks

[0099] Take chlorogenic acid, caffeic acid, deoxygalbinol A, galbindinone, isogamcinol, galbindinone B, derbyone, aristolochic acid, pentasperglycine, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnic acid, galbindinone A, galbindinone H and other standard substances respectively, follow the preparation method of the reference solution under "5", perform sampling and detection under the chromatographic conditions under "3", and record the chromatogram. By comparing with the chromatogram of the reference substance, the characteristic peaks were identified as aristolochic acid (peak 1), chlorogenic acid (peak 2), caffeic acid (peak 3), 8α-dihydrogeniposide (peak 4), 7-deoxy-8-epi-strychnic acid (peak 5), deoxygenated galactoside A (peak 6), galactoside diol (peak 7), galactoside A (peak 8), isogamone (peak 9), aristolochic acid (peak 10), derbyone (peak 11), galactoside H (peak 12), and galactoside 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-strychnic acid, deoxygenapol A, genapolone diol, genapolone A, isoganapolone, aristolochicone, derbyone, genapolone H, and genapolone B are shown in Figure 2 shown.

[0100] Example 2

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

[0102] 1. Chromatographic conditions

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

[0104] 2. Preparation of reference solution

[0105] Accurately weigh 10 mg of each of the reference substances of naringenone diol and deoxynaringenol A, place them in a 10 mL volumetric flask, add the corresponding solvent to the scale to obtain a reference solution of 1 mg / mL, and dilute them step by step 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, take the supernatant, and obtain.

[0108] 4. Linear relationship investigation

[0109] The mixed reference solution was diluted to different concentrations and injected into the liquid chromatograph respectively. The peak areas at the same retention time were calculated respectively, and the standard curve was drawn with the concentration X as the abscissa and the 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] Take an appropriate amount of this product, prepare the test solution according to the method under "3" for intra-day precision, and analyze the test solution according to the chromatographic conditions under "1", repeat the injection analysis for 6 consecutive times, record the peak areas of the two index components deoxygallinol A and gallinone diol, and calculate the RSD value. The inter-day precision is calculated according to the intra-day precision method, keep repeating the measurement for three days, record the peak areas and RSD values ​​of the two index components. The results are shown in Tables 7 and 8.

[0115] Table 7 Intra-day precision results

[0116]

[0117] From the results in the table, it can be seen that the relative deviations of intra-day precision of deoxygalbinol A and galbinol are 0.27% and 0.60% respectively; the relative deviations of inter-day precision are both 0.43%. The intra-day precision and inter-day precision of the two index components indicate that the method has good precision. The above experimental results show that the precision of the liquid chromatograph used is good.

[0118] 5.2 Stability test

[0119] Take the test solution, store it at room temperature, and accurately take 2 μL at time intervals of 0, 2, 4, 6, 8, 10, 12, and 24 hours, inject it into the liquid chromatograph, and determine the peak area values ​​of deoxygalbinol A and galbinol. 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 test solution is measured within 24 hours and the results remain stable.

[0123] 5.3 Repeatability test

[0124] Take 6 portions of the same batch of Nardostachys grandiflora medicinal materials, prepare the test solution according to the law, and measure the peak area respectively. 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] Take the same batch of naringa powder as the repeatability test, and weigh 6 portions in parallel, accurately add appropriate amounts of deoxynaringa alcohol A and naringa nediol to prepare the sample recovery solution according to the law, determine the results, and calculate the recovery rate with 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 show that the recoveries of deoxynarbinol A and naringone diol in the six samples are between 95.15% and 99.66% and 95.11% and 96.91%, respectively, and the recoveries are 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 rhizomes of Nardostachys chinensis

[0137]

[0138]

[0139]

[0140] According to the above determination results, the determination results of the content of deoxynarbinol A in the water extracts of 38 batches of naringa rhizomes were 0.2382% to 0.9051% respectively; the determination results of the content of naringapine ketone diol were 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), dimethyl sulfoxide (DMSO): Beijing Solaibao Technology Co., Ltd.; 96-well plate: Costar Electronic Materials Co., Ltd.; fetal bovine serum, phosphate buffer: BI, Israel; lipopolysaccharide, TritonX-100: Sigma, USA; penicillin-streptomycin (PS), 0.25% trypsin-EDTA: Gibco, USA; melanocyte stimulating hormone (α-MSH) and levodopa: Shanghai Yuanye Biotechnology Co., Ltd.; kojic acid (KA): Shanghai McLean Reagent Co., Ltd.; 100 mm cell culture dish: THERMO, USA; 6-well cell culture plate: NEST, USA. Instruments: Fully automatic microplate reader: Thermo Fisher Scientific.

[0144] 2. Preparation of complete culture medium

[0145] The preparation method is composed of 90% DMEM culture medium, 10% fetal bovine serum, and 1% penicillin-streptomycin; the cells are passaged when they grow to 70-80% under a microscope; the culture medium is discarded, and the cells are washed twice with 1mL PBS buffer, and 800μL 0.25% trypsin-EDTA is added. After the cells are observed to become round and fall off under a microscope, 1mL DMEM complete culture medium is added to terminate the digestion; the cell suspension is transferred into a 15mL centrifuge tube, and centrifuged at 1000rpm for 4min at room temperature; the supernatant is discarded, the complete culture medium is added, the tube is blown evenly, and the tube is transferred into a culture bottle; the culture bottle is placed in a 37°C, 5% CO2 cell culture incubator for culture.

[0146] 3B16F10 cell counting and plate seeding

[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 four squares / 4×10 4 × dilution multiple; the cells were seeded in the 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 naringenin aqueous extract (AENJ) in a 1.5 mL centrifuge tube, add 0.614 mL of sterile water to completely dissolve it, and obtain an AENJ mother solution with a concentration of 50 mg / mL. Store in a 4 ° C refrigerator for use, and then dilute with DMEM complete culture medium to obtain an AENJ sample solution of the corresponding concentration. Accurately weigh 5.1 mg of deoxynaringenin A (DA) in a 1.5 mL centrifuge tube, add 265.5 μL DMSO to completely dissolve it, and obtain a DA mother solution with a concentration of 100 mM. Store in a 4 ° C refrigerator for use, and then dilute with DMEM complete culture medium to obtain a deoxynaringenin A sample solution with a concentration of 100 mM. Accurately weigh 5.09 mg of naringenin diol (ND) in a 1.5 mL centrifuge tube, add 201.8 μL DMSO to completely dissolve it, and obtain an ND mother solution with a concentration of 100 mM. The solution was stored in a 4°C refrigerator for later use and subsequently diluted with DMEM complete 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 nard water extract (DMEM as solvent), 10 μM, 1 μM, 10 -1 μM deoxynarbinol A (DMEM as solvent) and 100 μM, 10 μM, and 1 μM naringenone diol (DMEM as solvent) were added to different cell culture wells, 100 μL for each well, and a blank control group without drug was set up. The blank control group and the drug-treated groups with different concentrations were set up with 6 duplicate 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 with DMEM culture medium was added under light-proof conditions, 100 μL for each well, incubated in a 37°C constant temperature incubator in light-proof for 30 minutes, and the absorbance was measured at 450 nm by an enzyme reader; the cell survival rate 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 different concentrations of naringa water extract, deoxynaringa alcohol A and naringa quinone diol sample solution groups 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 water extract of rhizome of Nardostachys pinnatifida, deoxynarbinol A and nardogenol on melanin content

[0156] 1. Solution preparation

[0157] Accurately weigh 1 mg of α-MSH in 1 mL of sterile water to obtain a 600 μM α-MSH stock solution, store it in a 4°C refrigerator for later use, and dilute it with complete culture medium to obtain a 100 nM α-MSH sample solution; accurately weigh 10 mg of kojic acid in 1 mL of sterile water to obtain a 10 mg / mL kojic acid stock solution, store it in a 4°C refrigerator for later use, and prepare a 100 μg / mL sample solution by stepwise dilution with complete culture medium. Accurately weigh 4 g of NaOH, add pure water, stir to fully dissolve it, and dilute to 100 mL. Store it at room temperature to obtain a 1M NaOH solution.

[0158] 2 Operation process

[0159] 4*10 4 The cells were seeded at a density of 100 / mL in a 100 mm culture dish and cultured for 24 h. The 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 nard water extract, and the concentrations were 10, 1, and 10. -1 μM deoxynarbinol A, 100, 10, 1 μM narcotrol and 100 μg / mL kojic acid sample solutions; take out the culture dish, add the diluted sample solutions in sequence, and set up a blank control group with only DMEM complete culture 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, then add DMEM complete culture medium to terminate digestion; transfer the cell suspension into a 1.5mL centrifuge tube, centrifuge at 1000rpm for 3min at room temperature to obtain the corresponding cell precipitate; after discarding the supernatant, add 1mL PBS buffer to each tube, centrifuge at 13000rmp for 10min at room temperature to obtain cell melanin precipitate; after absorbing 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 to 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 at 405nm on an ELISA reader, and use the lysis solution as a blank well. Melanin content calculation formula:

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

[0161] 3 Statistical analysis

[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 Nardostachys rapa aqueous extract groups significantly reduced the melanin content of B16F10 cells induced by α-MSH (P < 0.01, P < 0.001); -1 , 1, 10μM deoxynarbinol group A significantly reduced the melanin content of B16F10 cells induced by α-MSH (P<0.05, P<0.01, P<0.001); 1μM naringenonediol group significantly reduced the melanin content of B16F10 cells induced by α-MSH (P<0.05).

[0165] Example 5

[0166] Effects of water extracts from rhizomes of Nardostachys pinnatifida, deoxynarbinol A and naringone 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 a 6-well plate 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 nard water extract. -1μM deoxynarbinol A, 100, 10, 1 μM naringenone diol and 100 μg / mL kojic acid sample solution; take out the 6-well plate, add the diluted sample solution in turn, and set up a blank control group with only DMEM complete medium, and culture in the incubator for 48 hours; discard the supernatant, add 500 μL of 0.25% trypsin-EDTA, digest at 37℃ for 15s, then immediately add DMEM complete culture medium to terminate digestion; transfer the cell suspension into a 1.5mL centrifuge tube and centrifuge at 1000rpm for 3min at room temperature; discard the supernatant and add 1mL 1×PBS buffer to each tube, mix well and centrifuge at 1000rpm for 3min at room temperature; add the corresponding volume of 1×PBS solution containing 1% TritonX-100 to each tube according to the number of cells, lyse at 4℃ for 30min, and re-vortex every 10min to fully lyse; then centrifuge at 12000rpm, 4℃ for 10min to obtain the supernatant of different samples, transfer the supernatant to a new centrifuge tube, blow with a pipette and take 50μL in a 96-well plate, add an equal volume of 5mM L-DOPA solution to the supernatant, incubate at 37℃ for 30min, measure the absorbance at 475nm on an enzyme reader, and use 5mM L-DOPA solution as a blank well. Tyrosinase activity calculation formula:

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

[0172] 3 Statistical analysis

[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 figure, 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 nard water extract 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 naringenone diol significantly inhibited the tyrosinase activity of B16F10 cells induced by α-MSH (P<0.001, P<0.01).

[0176] In summary, this experiment established the ultra-high performance liquid chromatography fingerprint of the water extract of Nardostachys chinensis rhizome for the first time. Through the fingerprint study, it was found that the main components of the water extract of Nardostachys chinensis rhizome were deoxygalbinol A and galbinone diol. Deoxygalbinol A and galbinone diol were determined as the index components of the water extract of Nardostachys chinensis rhizome, and the content of deoxygalbinol A and galbinone diol was determined. By verifying the tyrosinase inhibitory activity and melanin inhibitory activity of the water extract of Nardostachys chinensis rhizome and its main components deoxygalbinol A and galbinone diol, it was confirmed that deoxygalbinol A and galbinone diol were the main active ingredients of the water extract of Nardostachys chinensis rhizome that exerted whitening activity, that is, deoxygalbinol A and galbinone diol can be used as the index components of the water extract of Nardostachys chinensis rhizome to exert whitening activity, and deoxygalbinol A and galbinone diol can be further applied to whitening related industries. The detection method of the 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 rhizomes of Nardostachys chinensis.

[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 design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for detecting a Nardostachys grandiflora rhizome extract, characterized in that: Including fingerprint and / or main component content determination method, wherein, The fingerprint detection method of the water extract of rhizome of Nardostachys rapa is as follows: (i) preparing an aqueous extract of rhizomes of Nardostachys grandiflora as a test solution; (ii) preparing a reference solution, wherein the reference solution is chlorogenic acid, caffeic acid, deoxygenated galactoside A, galactoside diol, isogalactone, galactoside B, derbyone, aristolochic acid, pentasperglycine, 8α-dihydrogeniposide, 7-deoxy-8-epi-strychnine, galactoside A, and galactoside H standard substances; (iii) Set 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; 17~25min, 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; (iv) Establishing fingerprint: taking the test sample solution of step (i) and the mixed reference sample stock solution of step (ii), measuring them under the chromatographic conditions of step (iii), injecting and analyzing the solutions, and importing the data files into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" for similarity analysis; The method for detecting the main components of the water extract of Nardostachys chinensis rhizome is as follows: (1) preparing an aqueous extract of rhizomes of Nardostachys grandiflora as a test solution; (2) preparing a reference solution, wherein the reference solution is narcotone glycol 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 water, isocratic elution program: 0-20min, 25% A, 75% B; column temperature: 40°C, flow rate: 0.3mL / min, detection wavelength: 250nm; (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) to obtain the product.

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

3. The method for detecting the extract of Nardostachys grandiflora rhizome according to claim 1 or 2, characterized in that: The main components of the water extract of the rhizome of Nardostachys affine detected in the detection method are deoxynarbinol A and nardogenol.

4. The method for detecting the extract of Nardostachys grandiflora rhizome according to claim 1, characterized in that: 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; the chromatographic column temperature is 45° C., the injection volume is 3 μL, the flow rate is 0.3 mL / min, and the detection wavelength is 254 nm.

5. The method for detecting the extract of Nardostachys grandiflora rhizome according to claim 1, characterized in that: The specific method for establishing the fingerprint spectrum of step (iv) is as follows: 38 batches of Nardostachys raphani medicinal materials of different origins and different storage years are taken to prepare the test solution according to step (i), and the chromatographic conditions under step (iii) are used for determination, and the chromatogram is recorded; the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" is used for data analysis, a reference spectrum is set, multi-point correction, automatic matching, 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, the similarity between the sample and the control fingerprint spectrum is measured, and the similarity of the sample pieces is evaluated.

6. The method for detecting the extract of Nardostachys grandiflora rhizome according to claim 1, characterized in that: In the step (3), an ACQUITY UPLC H class plus chromatograph is used, the chromatographic column model is: ACQUITY UPLC BEH C18, the chromatographic column specifications are 2.1 mm×100 mm, 1.7 μm, 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.

7. Use of naringenonediol detected by the detection method according to any one of claims 1 to 7 as an indicator component of the whitening activity of the water extract of naringensis rhizomes.

8. Use of naringenone diol detected by the detection method according to any one of claims 1 to 7 in the preparation of medicines, health products, cosmetics or skin care products with whitening activity; further preferably, the whitening activity refers to tyrosinase inhibitory activity and / or melanin inhibitory activity.

9. Use of deoxynarbinol A detected by the detection method according to any one of claims 1 to 7 as an indicator component of the whitening activity of the water extract of rhizomes of Nardostachys juncea.

10. Use of deoxygalbinol A detected by the detection method according to any one of claims 1 to 7 in the preparation of medicines, health products, cosmetics or skin care products with whitening activity; further preferably, the whitening activity refers to tyrosinase inhibitory activity and / or melanin inhibitory activity.

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