A method for identifying Yunnan small yellow ginger medicinal material

Abstract

The present invention belongs to the field of natural medicinal plant identification, and discloses a method for identifying Yunnan small yellow ginger medicinal materials. The present invention uses nuclear magnetic resonance carbon spectrum fingerprint to identify Yunnan small yellow ginger medicinal materials, and uses high performance liquid chromatography to refer to the Chinese Pharmacopoeia method to perform 6-gingerol quantitative analysis on Yunnan small yellow ginger medicinal materials, and measures 6-gingerol content of 0.15%-0.20%; those beyond this range are not Yunnan small yellow ginger varieties. The method can well reflect the categories of characteristic extracts in Yunnan small yellow ginger medicinal materials and the ratio between them, so as to achieve the purpose of identifying the varieties and quality of Yunnan small yellow ginger medicinal materials. The linear range is wide, the sensitivity is high, and the repeatability and feasibility are good. Not only can the intrinsic quality of Yunnan small yellow ginger medicinal materials be effectively controlled, but also the quality of Yunnan small yellow ginger can be objectively and effectively evaluated and controlled.

Description

Technical Field

[0001] The invention belongs to the field of natural medicinal plant identification, and in particular relates to an identification method for Yunnan small yellow ginger medicinal material. Background Art

[0002] Many areas in Yunnan belong to plateau basins, and the winter is cold. Small yellow ginger is one of the necessary foods for wintering. As early as 1958, small yellow ginger produced in Luoping area went abroad and became an important export commodity. Yunnan's unique climate makes the nutritional components of Yunnan small yellow ginger different from those in other places. Yunnan small yellow ginger mainly contains oily volatiles such as gingerol, gingerol terpenes, gingerene, phellandrene, citral, aromatic oils, as well as gingerol, vitamins, gingerol, resin, starch, fiber and a small amount of minerals. It has good detoxification, sterilization and medicinal effects. At present, the research on Yunnan small yellow ginger focuses on planting, cultivation, nutritional components and other aspects, but there is no report on the identification of Yunnan small yellow ginger by nuclear magnetic fingerprint. The use of nuclear magnetic fingerprint to identify Yunnan small yellow ginger can improve the quality of Yunnan small yellow ginger.

[0003] The quality of Yunnan small yellow ginger does not lie in a single ingredient, and its therapeutic effect is the result of the synergistic ratio of multiple ingredients. Therefore, it is imperative to establish a nuclear magnetic resonance carbon spectrum coupling fingerprint spectrum. The nuclear magnetic resonance carbon spectrum coupling fingerprint spectrum can not only control the active ingredients of Yunnan small yellow ginger, but also control the ratio between these active ingredients. The research and application of the nuclear magnetic resonance carbon spectrum coupling fingerprint spectrum of Yunnan small yellow ginger medicinal materials can not only solve the problem of identification and evaluation of Yunnan small yellow ginger medicinal materials in my country, but also provide a scientific guarantee for strengthening the systematization and standardization of the research on the intrinsic ingredients of Yunnan small yellow ginger medicinal materials, which is conducive to achieving international standards. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for identifying Yunnan small yellow ginger medicinal material using nuclear magnetic resonance carbon spectrum fingerprint technology.

[0005] In order to achieve the above object, the identification method of Yunnan small yellow ginger medicinal material of the present invention comprises the following two steps:

[0006] 1) The Yunnan small yellow ginger medicinal material is extracted by supercritical extraction to obtain characteristic extracts of Yunnan small yellow ginger gingerol and volatile oil; the characteristic extracts are measured and dissolved in organic solvents, and nuclear magnetic resonance carbon spectrum detection is performed respectively, and the characteristic spectrum should be consistent with the standard spectrum; the Yunnan small yellow ginger variety must meet the following conditions at the same time: the ratio of the integral of the δ211.2-211.6 absorption peak to the integral of the δ200.0-200.3 and δ199.7-199.9 absorption peaks is between 1.2-1.8, the ratio of the integral of the δ194.2-194.5 and the integral of the δ193.2-193.6 absorption peaks is between 1.0-1.8, and those beyond this range are not Yunnan small yellow ginger varieties, and there is no absorption peak between δ190-191.5.

[0007] 2) The 6-gingerol content of Yunnan Xiaohuangjiang was quantitatively analyzed by high performance liquid chromatography (HPLC) according to the Chinese Pharmacopoeia method, and the content of 6-gingerol was 0.15%-0.20%. The variety of Yunnan Xiaohuangjiang was not exceeded.

[0008] If steps 1 and 2 are met at the same time, it can be determined as Yunnan small yellow ginger medicinal material.

[0009] Among them, in step 1), the preparation method of the supercritical characteristic extract of Yunnan small yellow ginger includes: weighing the dried Yunnan small yellow ginger medicinal materials or decoction pieces, crushing them through 20 meshes, and controlling the extraction pressure to 15-30MPa, the extraction temperature to 35-50°C, and the extraction time to 1-2h during the extraction process.

[0010] Wherein, in step 1), the solvent for dissolving the characteristic extract of Yunnan small yellow turmeric gingerol and volatile oil is deuterated chloroform (CDCl3), deuterated methanol (CD3COCD3) or deuterated dimethyl sulfoxide (DMSO-d6), preferably deuterated methanol.

[0011] Wherein, in step 1), the characteristic peak of active ingredients in the characteristic extracts of Yunnan small yellow turmeric gingerol and volatile oil is an ester carbonyl absorption peak.

[0012] Wherein, in step 1), the peak intensity can be calculated by peak height method, area integration method or weight method.

[0013] Yunnan small yellow ginger slices can be identified using the above method.

[0014] The present invention uses a nuclear magnetic resonance carbon spectrum fingerprint to identify Yunnan small yellow ginger medicinal materials, which can well reflect the categories of characteristic extracts in Yunnan small yellow ginger medicinal materials and the ratios therebetween, thereby achieving the purpose of identifying the variety and quality of Yunnan small yellow ginger medicinal materials. The linear range is wide, the sensitivity is high, and the repeatability and feasibility are good. Not only can the intrinsic quality of Yunnan small yellow ginger medicinal materials be effectively controlled, but also the quality of Yunnan small yellow ginger can be objectively and effectively evaluated and controlled. Overall, it can not only solve the difficult problem of identifying and evaluating Yunnan small yellow ginger medicinal materials in my country, but also strengthen the systematization and standardization of the research on the intrinsic components of Yunnan small yellow ginger medicinal materials, providing a scientific guarantee for achieving international standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1-a This is the carbon nuclear magnetic resonance fingerprint of the characteristic extract of gingerol and volatile oil of Yunnan small yellow ginger medicinal material A in Example 1.

[0016] Figure 1-b This is a locally broadened and enlarged image of the characteristic peaks of the nuclear magnetic resonance carbon fingerprint of the characteristic extracts of gingerol and volatile oil of Yunnan small yellow ginger medicinal material A in Example 1.

[0017] Figure 2-a This is the carbon nuclear magnetic resonance fingerprint of the characteristic extracts of gingerol and volatile oil of Yunnan small yellow ginger medicinal material B in Example 2.

[0018] Figure 2-b This is a locally broadened and enlarged image of the characteristic peaks of the nuclear magnetic resonance carbon spectrum fingerprint of the characteristic extracts of gingerol and volatile oil of Yunnan small yellow ginger medicinal material B in Example 2.

[0019] Figure 3-a This is the carbon nuclear magnetic resonance fingerprint of the characteristic extracts of gingerol and volatile oil of commercially available medicinal material E in Example 3.

[0020] Figure 3-b This is a locally broadened and enlarged image of the characteristic peaks of the carbon nuclear magnetic resonance fingerprint of the characteristic extracts of gingerol and volatile oil of the commercially available medicinal material E in Example 3. DETAILED DESCRIPTION

[0021] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0022] Main instruments and equipment:

[0023] The NMR spectrometer was Bruker DPX 400.

[0024] Mass spectrometer: Q-Tof MicroTM model from Waters Micromass.

[0025] Semi-preparative high performance liquid chromatograph: Waters 600.

[0026] High performance liquid chromatograph: Agilent 1200.

[0027] 2000mL distillation flask, 5000mL distillation flask, spherical condenser, 2000mL separatory funnel.

[0028] DE-52AA Rotary Evaporator: Shanghai Yarong Biochemical Instrument Factory.

[0029] DEF-6020 Vacuum Drying Oven: Shanghai Jinghong Experimental Equipment Co., Ltd.

[0030] Column chromatography silica gel G and thin layer chromatography silica gel H: Qingdao Ocean Chemical Plant.

[0031] Silica gel chromatography column 6cm×70cm (diameter×height).

[0032] Yunnan small yellow ginger medicinal materials A, B, and C were collected from the geographical indication area of ​​Boai County. Yunnan small yellow ginger medicinal material D and commercial medicinal materials were purchased online; 6-gingerol, a chemical reference substance, was purchased from the National Institute for the Control of Pharmaceutical and Biological Products.

[0033] Reagents: chromatographically pure methanol (Tianjin Siyou Fine Chemicals Co., Ltd.) and analytically pure methanol (Tianjin Chemical Reagent Factory No. 1).

[0034] Example 1

[0035] 1. Identification study of Yunnan small yellow ginger medicinal material A

[0036] 1) Supercritical extraction method to obtain characteristic extracts of gingerol and volatile oil

[0037] Weigh Yunnan small yellow turmeric medicinal materials or pieces of decoction, grind them, pass through 20 meshes, extract Yunnan small yellow turmeric medicinal materials by supercritical extraction, control the extraction pressure to 15MPa, the extraction temperature to 45°C, and the extraction time to 1h during the extraction process, and obtain characteristic extracts of Yunnan small yellow turmeric gingerol and volatile oil;

[0038] 2) Detection of gingerol and volatile oil characteristic extracts from Yunnan small yellow ginger by C NMR fingerprint

[0039] 100-120 mg of the characteristic extracts of the Yunnan small yellow turmeric gingerol and volatile oil were respectively taken, dissolved in 0.5 mL of deuterated chloroform, and subjected to carbon NMR fingerprint detection to obtain carbon NMR fingerprints of the Yunnan small yellow turmeric gingerol and volatile oil.

[0040] 3) Analysis of the C NMR fingerprint of gingerol and volatile oil characteristic extracts from Yunnan small yellow turmeric

[0041] The characteristic spectrum of characteristic extracts of gingerol and volatile oil of Yunnan small yellow ginger should be consistent with the standard spectrum; Yunnan small yellow ginger A also meets the following conditions: the ratio of the integral of the δ211.43 absorption peak to the integral of the δ200.12 and δ199.90 absorption peaks is 1.56, between 1.2-1.8, the ratio of the integral of the δ194.30 and δ193.55 absorption peaks is 1.63, between 1.0-1.8; there is no absorption peak between δ190.0-191.5.

[0042] See the attached NMR carbon fingerprint Figure 1-a The local broadening and enlargement of the characteristic peak is shown in the attached Figure 1-b .

[0043] 2. Determination of gingerol content in Yunnan small yellow ginger by HPLC

[0044] i) Chromatographic conditions (refer to the 2020 edition of the Pharmacopoeia)

[0045] Mobile phase: acetonitrile: 0.4% phosphoric acid solution by mass = 13:87;

[0046] Chromatographic column: C18 (250*4.6mm, 5um);

[0047] Detection wavelength: 240nm.

[0048] The 6-gingerol content of Yunnan small yellow turmeric was quantitatively analyzed by high performance liquid chromatography, and the content of 6-gingerol was measured to be 0.9%-1.2%; those exceeding this range are not Yunnan small yellow turmeric varieties.

[0049] The determination result of Yunnan small yellow ginger medicinal material content was 0.18%.

[0050] If 1 and 2 are satisfied at the same time, it can be judged that it is Yunnan small yellow ginger medicinal material. Therefore, it can be judged that Example 1 is Yunnan small yellow ginger.

[0051] Example 2

[0052] 1. Identification study of Yunnan small yellow ginger medicinal material B

[0053] 1) Supercritical extraction method to obtain characteristic extracts of gingerol and volatile oil

[0054] Weigh Yunnan Xiaohuangjiang B medicinal material or slices, grind them, pass through 20 meshes, extract Yunnan Xiaohuangjiang medicinal material by supercritical extraction, control the extraction pressure to 20MPa, the extraction temperature to 40°C, and the extraction time to 1.5h during the extraction process, and obtain characteristic extracts of Yunnan Xiaohuangjiang gingerol and volatile oil;

[0055] 2) Detection of gingerol and volatile oil characteristic extracts from Yunnan small yellow ginger by C NMR fingerprint

[0056] 100-120 mg of characteristic extracts of gingerol and volatile oil of Yunnan small yellow turmeric were respectively taken, dissolved in 0.5 mL of deuterated chloroform, and subjected to carbon NMR fingerprint detection to obtain carbon NMR fingerprints of gingerol and volatile oil of Yunnan small yellow turmeric.

[0057] 3) Analysis of the C NMR fingerprint of gingerol and volatile oil characteristic extracts from Yunnan small yellow ginger

[0058] The characteristic spectrum of characteristic extracts of gingerol and volatile oil of Yunnan small yellow ginger should be consistent with the standard spectrum; Yunnan small yellow ginger B meets the following conditions at the same time: the ratio of the integral of the δ211.43 absorption peak to the integral of the δ200.12 and δ199.90 absorption peaks is 1.56, between 1.2-1.8, the ratio of the integral of the δ194.30 and δ193.55 absorption peaks is 1.63, between 1.0-1.8; there is no absorption peak between δ190.0-191.5.

[0059] See the attached NMR carbon fingerprint Figure 2-a The local broadening and enlargement of the characteristic peak is shown in the attached Figure 2-b .

[0060] 2. Determination of gingerol content in Yunnan small yellow ginger by HPLC

[0061] i) Chromatographic conditions (refer to the 2020 edition of the Pharmacopoeia)

[0062] Mobile phase: acetonitrile: 0.4% phosphoric acid solution by mass = 13:87;

[0063] Chromatographic column: C18 (250*4.6mm, 5um);

[0064] Detection wavelength: 240nm.

[0065] The 6-gingerol content of Yunnan small yellow turmeric was quantitatively analyzed by high performance liquid chromatography, and the content of 6-gingerol was measured to be 0.9%-1.2%; those exceeding this range are not Yunnan small yellow turmeric varieties.

[0066] The determination result of Yunnan small yellow ginger medicinal material content was 0.17%.

[0067] If both 1 and 2 are met, it can be judged as Yunnan small yellow ginger.

[0068] It can be judged from this that Example 2 is Yunnan small yellow ginger.

[0069] Example 3

[0070] 1. Identification study of commercially available turmeric medicinal material E

[0071] 1) Supercritical extraction method to obtain characteristic extracts of gingerol and volatile oil

[0072] Weigh the commercially available small yellow turmeric E medicinal material or the decoction pieces, grind them, pass through 20 meshes, and extract the Yunnan small yellow turmeric medicinal material by supercritical extraction. During the extraction process, the extraction pressure is controlled to be 20MPa, the extraction temperature is 40℃, and the extraction time is 1.5h to obtain the characteristic extracts of Yunnan small yellow turmeric gingerol and volatile oil;

[0073] 2) Detection of characteristic extracts of gingerol and volatile oil from commercially available turmeric by C NMR fingerprint

[0074] 100-120 mg of gingerol and volatile oil characteristic extracts of commercially available turmeric E were respectively measured and dissolved in 0.5 mL of deuterated chloroform, and carbon NMR fingerprint detection was performed to obtain the carbon NMR fingerprint of gingerol and volatile oil of Yunnan turmeric.

[0075] 3) Analysis of the C NMR fingerprint of gingerol and volatile oil characteristic extracts from commercially available turmeric

[0076] The characteristic spectra of gingerol and volatile oil characteristic extracts of commercially available small yellow turmeric should be consistent with the standard spectrum; Yunnan small yellow turmeric E simultaneously meets the following conditions: the ratio of the integral of the δ211.43 absorption peak to the integral of the δ200.12 and δ199.90 absorption peaks is 1.56, between 1.2-1.8, the ratio of the integral of the δ194.30 and δ193.55 absorption peaks is 1.63, between 1.0-1.8; there is no absorption peak between δ190.0-191.5.

[0077] See the attached NMR carbon fingerprint Figure 3-a The local broadening and enlargement of the characteristic peak is shown in the attached Figure 3-b .

[0078] 2. Determination of gingerol content in Yunnan small yellow ginger by HPLC

[0079] i) Chromatographic conditions (refer to the 2020 edition of the Pharmacopoeia)

[0080] Mobile phase: acetonitrile: 0.4% phosphoric acid solution by mass = 13:87;

[0081] Chromatographic column: C18 (250*4.6mm, 5um);

[0082] Detection wavelength: 240nm.

[0083] The 6-gingerol content of commercially available turmeric E was quantitatively analyzed by high performance liquid chromatography, and was found to be 0.12%.

[0084] If both 1 and 2 are met, it can be judged as Yunnan small yellow ginger.

[0085] It can be judged from this that Example 3 is not Yunnan small yellow ginger.

Claims

1. A method for identifying Yunnan small yellow ginger medicinal material, characterized in that: The following steps are involved: (1) The Yunnan small yellow turmeric medicinal material was extracted by supercritical extraction to obtain characteristic extracts of Yunnan small yellow turmeric gingerol and volatile oil; Measure the characteristic extract and use deuterated chloroform, deuterated methanol or deuterated dimethyl sulfoxide as solvent for nuclear magnetic resonance carbon spectrum detection. The characteristic spectrum should be consistent with the standard spectrum; Yunnan small yellow ginger varieties must meet the following conditions at the same time: the ratio of the integral of the δ211.2-211.6 absorption peak to the integral of the δ200.0-200.3 and δ199.7-199.9 absorption peaks is between 1.2-1.8, the ratio of the integral of the δ194.2-194.5 and δ193.2-193.6 absorption peaks is between 1.0-1.8, and those beyond this range are not Yunnan small yellow ginger varieties, and there is no absorption peak between δ190-191.5; (2) The 6-gingerol content of Yunnan small yellow turmeric was quantitatively analyzed by high performance liquid chromatography in accordance with the Chinese Pharmacopoeia method, and the content of 6-gingerol was measured to be 0.15%-0.20%. Those outside this range are not Yunnan small yellow turmeric varieties; If the conditions of steps (1) and (2) are met at the same time, it is judged to be Yunnan small yellow ginger medicinal material; In step (1), the preparation method of the Yunnan small yellow ginger supercritical characteristic extract comprises: weighing the dried Yunnan small yellow ginger medicinal materials or decoction pieces, crushing through 20 meshes, and controlling the extraction pressure to 15-30 MPa, the extraction temperature to 35-50 ° C, and the extraction time to 1-2 h during the extraction process.

2. The method for identifying the Yunnan small yellow ginger medicinal material as claimed in claim 1, characterized in that: In step (1), the solvent for dissolving the characteristic extract of Yunnan small yellow turmeric gingerol and volatile oil is deuterated methanol.

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