Method for extracting active ingredients in pekoe silver needle based on natural eutectic solvent

Abstract

The invention discloses a method for extracting active ingredients in pekoe silver needles based on a natural eutectic solvent, and belongs to the technical field of active substance extraction. According to the method for extracting the active ingredients in the pekoe needle through the natural deep-eutectic solvent, the natural deep-eutectic solvent is selected, the water content, the material-liquid ratio and the extraction conditions are optimized, and the problems that in the prior art, the extraction rate of the active ingredients of the pekoe needle is low and the like are solved. The proline glycerin (with the molar ratio of 1: 3) prepared by the invention is used as a natural eutectic solvent, and the extraction efficiency of total active substances (polyphenol, flavone and alkaloid) in the pekoe silver needle is 280mg / g of pekoe silver needle powder; the extraction rate of theanine reaches 21 mg / g of Baihao silver needle powder, and the extraction effect is far better than that of conventional water extraction and alcohol extraction.

Description

Technical Field

[0001] The invention relates to a method for extracting active ingredients in silver needle baihao based on a natural deep eutectic solvent, belonging to the technical field of active substance extraction. Background Art

[0002] Silver Needle Tea is a natural treasure trove of polyphenol compounds, rich in tea polyphenols, amino acids, flavonoids and other substances, which can help improve immunity, anti-oxidation, promote digestion, etc. Theanine (chemical name L-γ-glutamylethylamine) is a unique amino acid in tea, mainly found in green tea, black tea and white tea. The traditional water extraction method for extracting effective substances from tea has problems such as long extraction time, low extraction efficiency, and wastewater pollution, and a new extraction method is needed to replace it.

[0003] Deep eutectic solvent (DES) refers to a low melting point mixture formed by hydrogen bond acceptors and hydrogen bond donors in a certain molar ratio through hydrogen bonding. Compared with traditional solvents, DES is not only environmentally friendly, stable, low in volatility, easy to synthesize, with a wide range of polarity, but also designable.

[0004] At present, there are studies on the use of low eutectic solvents to extract effective substances. The document "Optimization of the process of extracting tea polyphenols from green tea with low eutectic solvents and analysis of the antioxidant activity of tea polyphenol extracts" discloses the use of choline chloride and lactic acid to prepare a low eutectic solvent to extract tea polyphenols from green tea, and the tea polyphenol extraction rate reaches 25.16%; the document "Extraction of total flavonoids from green tea with low eutectic solvents and their antioxidant activity" discloses the use of acetylcholine and lactic acid to prepare a low eutectic solvent to extract total flavonoids from green tea, and the total flavonoid extraction amount reaches 65.8 mg / mL. Both documents use the traditional heating extraction method, and the extraction efficiency is low; Patent CN108640966 A discloses the use of glycerol, proline and sucrose to prepare a low eutectic solvent to extract tea saponins from tea seed meal, and the tea saponin extraction rate is 25.84%; Patent CN 108992541 A discloses the use of glycerol, proline and citric acid to prepare a low eutectic solvent to extract tea polyphenols from tea seed meal, and the tea polyphenol extraction rate is 14.01%. In both patents, the preparation process of the low eutectic solvent uses freeze-drying technology, which increases the cost of plant extraction. There is a big difference between tea leaves and tea seeds, the content of active ingredients in them is different, and the processing methods of the extraction process are also different. At the same time, the above technology only stays in the extraction of polyphenols and flavonoids, and does not focus on the extraction effect of theanine.

[0005] Therefore, there is an urgent need to develop a method for efficiently extracting polyphenols, flavonoids, alkaloids and theanine from Silver Needle Needle to provide strong theoretical support and technical guidance for the extraction process of active substances, which has extremely high practical and economic value. Summary of the invention

[0006] In view of the defects existing in the prior art, the present invention provides a method for extracting active ingredients in Silver Needle with a natural deep eutectic solvent by selecting a natural deep eutectic solvent and optimizing the water content, the solid-liquid ratio and the extraction conditions, thereby solving the problems of low extraction rate of active ingredients in Silver Needle in the prior art.

[0007] The first object of the present invention is to provide a method for simultaneously improving the extraction effect of total phenols, flavonoids, alkaloids and theanine in silver needle baihao, using a natural deep eutectic solvent for extraction, and the extraction method comprises:

[0008] The natural deep eutectic solvent and water are mixed in a mass ratio of 3 to 19:1 to obtain a natural deep eutectic solvent aqueous solution; the silver needle white needle powder and the natural deep eutectic solvent aqueous solution are mixed in a material-liquid ratio of 1 g: 10 to 30 mL, heated by ultrasonication, and centrifuged to obtain a silver needle white needle extract;

[0009] The natural low eutectic solvent is prepared from two or more of proline, glycerol, betaine, citric acid, lactic acid, malic acid and levulinic acid.

[0010] In one embodiment, the preparation method of the natural deep eutectic solvent is:

[0011] Proline and glycerol are mixed in a molar ratio of 1:2.5-3.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-glycerol natural deep eutectic solvent; or,

[0012] Mix betaine and citric acid in a molar ratio of 1:1.5-2.5, and stir at 75-85°C for 2.5-3.5h to obtain a betaine-citric acid natural deep eutectic solvent; or,

[0013] Mixing betaine and lactic acid in a molar ratio of 1:1.5-2.5, stirring at 75-85°C for 2.5-3.5h to obtain a betaine-lactic acid natural deep eutectic solvent; or,

[0014] Mix betaine, malic acid and lactic acid in a molar ratio of 0.8-1.2:0.8-1.2:0.8-1.2, and stir at 75-85° C. for 2.5-3.5 hours to obtain a betaine-malic acid-lactic acid natural low eutectic solvent; or,

[0015] Mix betaine and glycerol in a molar ratio of 1:1.5-2.5, and stir at 75-85°C for 2.5-3.5h to obtain a betaine-glycerol natural deep eutectic solvent; or,

[0016] Proline and lactic acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-lactic acid natural deep eutectic solvent; or,

[0017] Proline and levulinic acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-levulinic acid natural low eutectic solvent.

[0018] In one embodiment, the heating ultrasound is performed at 20-80° C., 20-80 Hz for extraction for 20-120 min.

[0019] Optionally, the heating ultrasound is 40-50 Hz, and the extraction is performed at 55-65° C. for 50-70 min;

[0020] Optionally, the heating ultrasound is 15-25 Hz, and the extraction is carried out at 15-25° C. for 15-25 min.

[0021] In one embodiment, the centrifugation is performed at 8000-12000 rpm for 5-20 min.

[0022] Preferably, a method for simultaneously improving the extraction effect of total phenols, flavonoids, alkaloids and theanine in Baihao Yinzhen is to use a natural deep eutectic solvent for extraction, and the extraction method comprises:

[0023] Proline and glycerol are mixed in a molar ratio of 1:2.5-3.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-glycerol natural deep eutectic solvent;

[0024] The proline-glycerol natural low eutectic solvent and water are mixed at a mass ratio of 3.5-4.5:1 to obtain a proline-glycerol natural low eutectic solvent aqueous solution; the white needle silver needle and the proline-glycerol natural low eutectic solvent aqueous solution are mixed at a solid-liquid ratio of 1g:18-22mL, extracted at 40-50Hz, 55-65℃ for 50-70min, and centrifuged at 8000-12000rpm for 8-12min to obtain the white needle silver needle active ingredient extract.

[0025] Preferably, a method for simultaneously improving the extraction effect of total phenols, flavonoids, alkaloids and theanine in Baihao Yinzhen is to use a natural deep eutectic solvent for extraction, and the extraction method comprises:

[0026] Betaine and citric acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a betaine-citric acid natural deep eutectic solvent;

[0027] The betaine-citric acid natural low eutectic solvent and water are mixed in a mass ratio of 18-20:1 to obtain a proline-glycerol natural low eutectic solvent aqueous solution; the white needle silver needle and the proline-glycerol natural low eutectic solvent aqueous solution are mixed in a solid-liquid ratio of 1g:8-12mL, extracted at 15-25Hz, 15-25℃ for 15-25min, and centrifuged at 8000-12000rpm for 8-12min to obtain the white needle silver needle active ingredient extract.

[0028] The second object of the present invention is to provide the use of any of the above methods in extracting the effective ingredients of Silver Needle Needle.

[0029] The present invention also provides a method for extracting effective ingredients from silver needle white needles, using a natural deep eutectic solvent for extraction, the steps comprising:

[0030] The natural deep eutectic solvent and water are mixed in a mass ratio of 3 to 19:1 to obtain a natural deep eutectic solvent aqueous solution; the natural deep eutectic solvent aqueous solution and silver needle baihao powder are mixed in a solid-liquid ratio of 1g:10 to 30mL, heated with ultrasound, and centrifuged to obtain a silver needle baihao extract.

[0031] In one embodiment, the preparation method of the natural deep eutectic solvent is:

[0032] Proline and glycerol are mixed in a molar ratio of 1:2.5-3.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-glycerol natural deep eutectic solvent; or,

[0033] Mix betaine and citric acid in a molar ratio of 1:1.5-2.5, and stir at 75-85°C for 2.5-3.5h to obtain a betaine-citric acid natural deep eutectic solvent; or,

[0034] Mixing betaine and lactic acid in a molar ratio of 1:1.5-2.5, stirring at 75-85°C for 2.5-3.5h to obtain a betaine-lactic acid natural deep eutectic solvent; or,

[0035] Mix betaine, malic acid and lactic acid in a molar ratio of 0.8-1.2:0.8-1.2:0.8-1.2, and stir at 75-85° C. for 2.5-3.5 hours to obtain a betaine-malic acid-lactic acid natural low eutectic solvent; or,

[0036] Mix betaine and glycerol in a molar ratio of 1:1.5-2.5, and stir at 75-85°C for 2.5-3.5h to obtain a betaine-glycerol natural deep eutectic solvent; or,

[0037] Proline and lactic acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-lactic acid natural deep eutectic solvent; or,

[0038] Proline and levulinic acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-levulinic acid natural low eutectic solvent.

[0039] In one embodiment, the heating ultrasound is performed at 20-80° C., 20-80 Hz for extraction for 20-120 min.

[0040] In one embodiment, it is characterized in that the centrifugation is performed at 8000-12000 rpm for 5-20 min.

[0041] Preferably, a method for extracting active ingredients from Baihao Yinzhen is performed using a natural deep eutectic solvent, and the extraction method comprises:

[0042] Proline and glycerol are mixed in a molar ratio of 1:2.5-3.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-glycerol natural deep eutectic solvent;

[0043] The proline-glycerol natural low eutectic solvent and water are mixed at a mass ratio of 3.5-4.5:1 to obtain a proline-glycerol natural low eutectic solvent aqueous solution; the white needle silver needle and the proline-glycerol natural low eutectic solvent aqueous solution are mixed at a solid-liquid ratio of 1g:18-22mL, extracted at 40-50Hz, 55-65℃ for 50-70min, and centrifuged at 8000-12000rpm for 8-12min to obtain the white needle silver needle active ingredient extract.

[0044] Preferably, a method for extracting effective ingredients from Baihao Yinzhen is to use a natural deep eutectic solvent for extraction, and the extraction method comprises:

[0045] Betaine and citric acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a betaine-citric acid natural deep eutectic solvent;

[0046] The betaine-citric acid natural low eutectic solvent and water are mixed in a mass ratio of 18-20:1 to obtain a proline-glycerol natural low eutectic solvent aqueous solution; the white needle silver needle and the proline-glycerol natural low eutectic solvent aqueous solution are mixed in a solid-liquid ratio of 1g:8-12mL, extracted at 15-25Hz, 15-25℃ for 15-25min, and centrifuged at 8000-12000rpm for 8-12min to obtain the white needle silver needle active ingredient extract.

[0047] In one embodiment, the active ingredients include one or more of total phenols, flavonoids, alkaloids and theanine.

[0048] Beneficial Effects of the Invention

[0049] 1. The present invention uses a green biodegradable natural low eutectic solvent as an extractant, which is in line with the development concept of green extraction;

[0050] 2. The present invention adopts a natural low eutectic solvent as an extractant to improve the extraction efficiency of the active ingredients in Baihao Yinzhen. Among them, the extraction efficiency of the total active substances (polyphenols, flavonoids, alkaloids) in Baihao Yinzhen using proline glycerol (molar ratio of 1:3) as a natural low eutectic solvent is 280 mg / g Baihao Yinzhen powder; the extraction of theanine reaches 21 mg / g Baihao Yinzhen powder, and its extraction effect is far better than conventional water extraction and alcohol extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art will be briefly introduced below.

[0052] Figure 1 It is a comparison chart of the contents of the active ingredient extracts of Silver Needle prepared by Examples 1 to 7, Comparative Examples 1 to 7, water extraction and alcohol extraction;

[0053] Figure 2 This is a comparison chart of DPPH free radical scavenging rates of the active ingredient extracts of Silver Needle prepared by water extraction and alcohol extraction in Examples 1 to 7;

[0054] Figure 3 It is a comparison chart of ABTS free radical scavenging rates of the extracts of the active ingredients of Silver Needle prepared by water extraction and alcohol extraction in Examples 1 to 7.

[0055] Figure 4 It is a comparison chart of the cellular inflammatory factor levels of the silver needle active ingredient extracts prepared in Examples 2 to 5. DETAILED DESCRIPTION

[0056] The present invention uses a natural deep eutectic solvent to extract the active ingredients of Baihao Yinzhen. The present invention is innovatively established by selecting a natural deep eutectic solvent to extract the active ingredients of Baihao Yinzhen, and the content of the active ingredients obtained is nearly twice that of the traditional water extraction method, which solves the current problems of Baihao Yinzhen in the extraction of active ingredients that are not green enough and have a low yield.

[0057] In order to make the purpose, technical scheme and advantages of the present invention clearer and more specific, the present invention is further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with the accompanying drawings. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0058] raw material:

[0059] Silver needle white needle (powder, 800 mesh) was purchased from Fuding Tianjian Tea Co., Ltd.;

[0060] Proline was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 147-85-3;

[0061] Glycerol was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 56-81-5;

[0062] Betaine was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 107-43-7;

[0063] Lactic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No. 50-21-5;

[0064] Citric acid was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 77-92-9;

[0065] Malic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No. 6915-15-7;

[0066] Levulinic acid was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 123-76-2;

[0067] Sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd. with a CAS number of 57-50-1.

[0068] Extraction method:

[0069] Water extraction method:

[0070] Deionized water and Silver Needle Needle powder were mixed at a solid-liquid ratio of 1:20 g / ml, extracted at 60°C for 2 h, and extracted three times to obtain an extract.

[0071] Alcohol extraction method:

[0072] 75% ethanol and Silver Needle Needle powder were extracted at 60°C for 2 hours in a solid-liquid ratio of 1:20 g / ml, and the extraction was repeated three times to obtain an extract.

[0073] Detection method:

[0074] 1. Determination of flavonoids, total phenols and total alkaloids:

[0075] The flavonoid content in the extract of Baihao Yinzhen was determined by sodium nitrite-aluminum nitrate colorimetric method using rutin as the standard. The total phenol content in the extract of Baihao Yinzhen was determined by folin phenol colorimetric method using gallic acid as the standard. The total alkaloid content in the extract of Baihao Yinzhen was determined by sulfuric acid colorimetric method using berberine hydrochloride as the standard.

[0076] 2. Determination of theanine content:

[0077] The content of theanine in the extract of Silver Needle was determined by HPLC with theanine as the standard.

[0078] The chromatographic conditions are:

[0079] High performance liquid chromatography-ultraviolet detection (HPLC-UV) was performed using an Agilent 1100 series HPLC system (Agilent, San Jose, CA, USA) equipped with a diode array detector (DAD). The chromatographic column was a Diamonsil C18 reverse phase column (250 mm × 4.6 mm, 5 μm) with a flow rate of 1.0 mL min -1 , the column temperature was set to 35°C. The mobile phase was 0.1% (v / v) phosphoric acid aqueous solution (A) and acetonitrile (B). Gradient elution: 0-12min, 15-25% B; 12-15min, 25-45% B; 15-20min, 45-30% B; 20-30min, 30-20% B; 30-32min, 20-15% B. The detection wavelength was 507nm. The injection volume was 20μL and filtered through a 0.45μm membrane filter before use.

[0080] Example 1

[0081] A method for extracting the active ingredients of Baihao Yinzhen with a natural deep eutectic solvent, comprising the following steps:

[0082] (1) Preparation of natural deep eutectic solvent

[0083] Proline and glycerol were mixed in a round-bottom flask at a molar ratio of 1:3, and stirred at 80°C for 3 h to obtain a clear and transparent liquid, thereby obtaining a proline-glycerol low eutectic solvent;

[0084] (2) Extraction of active ingredients

[0085] The proline-glycerol low eutectic solvent (molar ratio 1:3) and water are mixed in a mass ratio of 4:1 to prepare a proline-glycerol low eutectic solvent aqueous solution (water content 20%), the proline-glycerol low eutectic solvent aqueous solution is placed in a conical flask, and the Silver Needle Needle powder is added and mixed, the solid-liquid ratio is 1g:20mL, 40Hz, 60℃ ultrasonic extraction for 60min, 10000rpm centrifugation for 10min, filtration, and the Silver Needle Needle active ingredient extract is obtained.

[0086] Example 2

[0087] A method for extracting the active ingredients of Baihao Yinzhen with a natural deep eutectic solvent, comprising the following steps:

[0088] (1) Preparation of natural deep eutectic solvent

[0089] Betaine and citric acid were mixed in a round-bottom flask at a molar ratio of 1:2, and stirred at 80°C for 3 h to obtain a clear liquid, thereby obtaining a betaine and citric acid eutectic solvent;

[0090] (2) Extraction of active ingredients

[0091] The betaine-citric acid low eutectic solvent (molar ratio 1:2) and water are mixed in a mass ratio of 19:1 to prepare a betaine and citric acid low eutectic solvent aqueous solution (water content 5%), the betaine and citric acid low eutectic solvent aqueous solution is placed in a conical flask, and the silver needle powder is added and mixed, the solid-liquid ratio is 1g:10mL, 20Hz, 20℃ ultrasonic extraction for 20min, 8000rpm centrifugation for 5min, filtration, and the silver needle active ingredient extract is obtained.

[0092] Example 3

[0093] A method for extracting the active ingredients of Baihao Yinzhen with a natural deep eutectic solvent, comprising the following steps:

[0094] (1) Preparation of natural deep eutectic solvent

[0095] Betaine and lactic acid were mixed in a round-bottom flask at a molar ratio of 1:2, and stirred at 80°C for 3 h to obtain a clear liquid, thereby obtaining a betaine-lactic acid deep eutectic solvent;

[0096] (2) Extraction of active ingredients

[0097] The betaine-lactic acid low eutectic solvent (molar ratio 1:2) and water are mixed in a mass ratio of 3:1 to prepare a betaine-lactic acid low eutectic solvent aqueous solution (water content 25%), the betaine-lactic acid low eutectic solvent aqueous solution is placed in a conical flask, and the silver needle peony root powder is added and mixed, the solid-liquid ratio is 1g:30mL, 80Hz, 80℃ ultrasonic extraction for 120min, 12000rpm centrifugation for 20min, filtration, and the silver needle peony root active ingredient extract is obtained.

[0098] Example 4

[0099] On the basis of Example 1, the natural low eutectic solvent in step (1) was changed to betaine, malic acid and lactic acid (molar ratio of 1:1:1), and the remaining steps were consistent with Example 1 to prepare an extract of the active ingredient of Baihao Yinzhen.

[0100] Example 5

[0101] On the basis of Example 1, the natural low eutectic solvent in step (1) was changed to betaine and glycerol (molar ratio 1:2), and the remaining steps were consistent with Example 1 to prepare an extract of the active ingredient of Baihao Yinzhen.

[0102] Example 6

[0103] On the basis of Example 1, the natural low eutectic solvent in step (1) was changed to proline and lactic acid (molar ratio 1:2), and the remaining steps were consistent with Example 1 to prepare an extract of the active ingredient of Baihao Yinzhen.

[0104] Example 7

[0105] On the basis of Example 1, the natural low eutectic solvent in step (1) was changed to proline and levulinic acid (molar ratio 1:2), and the remaining steps were consistent with Example 1 to prepare an extract of the active ingredient of Baihao Yinzhen.

[0106] Comparative Example 1

[0107] On the basis of Example 1, the frequency of ultrasound in step (1) was changed to 10 Hz, and the remaining steps were consistent with Example 1.

[0108] Comparative Example 2

[0109] On the basis of Example 1, the frequency of ultrasound in step (1) was changed to 100 Hz, and the remaining steps were consistent with Example 1.

[0110] Comparative Example 3

[0111] On the basis of Example 1, the temperature in step (1) was changed to 10° C., and the remaining steps were consistent with Example 1.

[0112] Comparative Example 4

[0113] On the basis of Example 1, the temperature in step (1) was changed to 100° C., and the remaining steps were consistent with Example 1.

[0114] Comparative Example 5

[0115] On the basis of Example 1, the low eutectic solvent in step (1) was changed to proline, glycerol and sucrose (molar ratio 3:4:0.5), and the remaining steps were consistent with Example 1.

[0116] Comparative Example 6

[0117] On the basis of Example 1, the low eutectic solvent in step (1) was changed to proline, glycerol and citric acid (molar ratio 1.5:4:0.1), and the remaining steps were consistent with Example 1.

[0118] Comparative Example 7

[0119] On the basis of Example 1, the molar ratio of proline to glycerol in step (1) was changed to 1:2, and the remaining steps were consistent with Example 1.

[0120] Example 8

[0121] 1. Take the extracts of active ingredients of Silver Needle obtained by water extraction and alcohol extraction in Examples 1 to 7, Comparative Examples 1 to 7, and test the extraction amounts of alkaloids, flavonoids, total phenols and theanine. The results are as follows: Figure 1 As shown in Table 1.

[0122] Table 1 Active ingredient content in the extract

[0123]

[0124]

[0125] 2. Antioxidant activity determination:

[0126] The extracts of Silver Needle Needle extracted with water and alcohol in Examples 1-7 were diluted with water to 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL for antioxidant activity testing.

[0127] (1) DPPH free radical scavenging ability

[0128] Weigh 8.0 mg of 1,1-diphenyl-2-picrylhydrazyl using a microbalance and weighing paper, pour directly into a 100 mL volumetric flask, dissolve and dilute with anhydrous ethanol, then sonicate for 5 minutes to prepare a 0.2 mmol / L DPPH solution (the solution should be used immediately after preparation and should be used up within 3.5 hours). Take 0.5 mL of different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL) of silver needle extract, add 0.5 mL of 0.2 mmol / L DPPH solution, vortex evenly, and react in the dark for 30 minutes. Use a 96-well plate and an enzyme reader to measure the OD517 nm absorbance value as A i The absorbance value of the mixture of DPPH solution and an equal volume of anhydrous ethanol is determined as A 0 , and the absorbance value of the mixture of Baihao Yinzhen extract and an equal volume of anhydrous ethanol is A j , the calculation formula is as follows:

[0129] Free radical scavenging rate (%) = (A 0 -A i +A j ) / A 0 ×100%.

[0130] The results are shown in Table 2 and Figure 2 As shown, the results show that the DPPH removal rates of Examples 1 to 7 are better than those of water extraction and alcohol extraction.

[0131] Table 2 DPPH test results

[0132] (mg / mL) 0.2 0.4 0.6 0.8 1.0 1.2 Example 1 36.72％ 57.81％ 60.12％ 84.12％ 91.59％ 95.41％ Example 2 33.1％ 53.96％ 57.41％ 81.55％ 87.69％ 93.71％ Example 3 24.33％ 49.33％ 58.94％ 79.26％ 85.23％ 88.3％ Example 4 30.16％ 41.75％ 55.74％ 70.22％ 81.02％ 85.47％ Example 5 27.82％ 51.32％ 56.43％ 73.24％ 79.33％ 81.62％ Example 6 26.63％ 40.37％ 49.91％ 67.81％ 76.24％ 79.14％ Example 7 29.94％ 42.79％ 51.32％ 65.41％ 78.35％ 80％ Water extraction 16.63％ 31.95％ 42.31％ 55.62％ 60.14％ 72.98％ Alcohol Extraction 17.2％ 37.41％ 47.71％ 57.04％ 68.51％ 76.32％

[0133] (2) ABTS+ free radical scavenging ability

[0134] Weigh 3 mg of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) in a 5 mL EP tube using a microbalance, add 0.735 mL of distilled water, and vortex to prepare a 7.4 mmol / mL ABTS solution. Then, weigh 1 mg of potassium persulfate in another 5 mL EP tube, add 1.43 mL of distilled water, and vortex to prepare a 2.6 mmol / L K 2 S 2 O 8 Solution. Take 0.735 mL of the prepared potassium persulfate stock solution into the EP tube containing the ABTS stock solution, vortex, and place it at room temperature in the dark for 12-16 hours to prepare the ABTS stock solution. Before use, dilute the ABTS stock solution with anhydrous ethanol to an OD of 734nm The ABTS working solution was obtained within 0.70±0.05. It should be noted that the solution should be prepared and used immediately to ensure its stability.

[0135] Take 0.2 mL of different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL) of silver needle extract, add 0.8 mL of ABTS working solution, vortex evenly, and place in the dark to react for 6 minutes. Use 96-well plate and microplate reader to measure OD 734nm , denoted as A i The absorbance value of the mixture of 0.8 mL ABTS working solution and 0.2 mL deionized water is A 0 , and the absorbance value of the mixture of 0.2mL of Baihao Yinzhen extract and 0.8mL of anhydrous ethanol is A j , the calculation formula is as follows:

[0136] Free radical scavenging rate (%) = (A 0 -A i +A j ) / A 0 ×100%.

[0137] The results are shown in Table 3 and Figure 3 As shown, the results show that the ABTS removal rates of Examples 1 to 7 are better than those of water extraction and alcohol extraction.

[0138] Table 3 ABTS test results

[0139] (mg / mL) 0.2 0.4 0.6 0.8 1.0 1.2 Example 1 48.25％ 72.87％ 82.98％ 91.09％ 96.27％ 97.2％ Example 2 42.08％ 67.31％ 78.64％ 89.77％ 92.24％ 94.36％ Example 3 36.37％ 66.35％ 76.12％ 86.93％ 90.88％ 95.71％ Example 4 38.62％ 61.72％ 68.9％ 89.37％ 91.37％ 94.65％ Example 5 31.7％ 65.57％ 71.39％ 85.12％ 90.24％ 91.72％ Example 6 29.14％ 57.38％ 69.99％ 87.62％ 92.97％ 93.07％ Example 7 26.64％ 50.14％ 72.06％ 84.91％ 88.67％ 90.48％ Water extraction 19.11％ 38.32％ 51.99％ 64.05％ 70.28％ 83.11％ Alcohol Extraction 20.32％ 41.33％ 56.2％ 69.67％ 73.54％ 85.42％

[0140] The above results show that the extract of active ingredients of Baihao Yinzhen exhibits excellent antioxidant capacity. Compared with traditional water extraction and alcohol extraction, the antioxidant capacity of DES extract is improved.

[0141] 3. Anti-inflammatory test of the extract of active ingredients of Baihao Yinzhen

[0142] Cell culture: RAW 264.7 mouse macrophages were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% double antibody in a culture environment of 37°C and 5% CO. 2 . Subculture or change medium according to the cell growth status.

[0143] Cell experiment: The active ingredient extracts of Silver Needle Needle in Examples 2 to 5 were diluted with DMEM culture medium to obtain an active ingredient extract of Silver Needle Needle with a concentration of 0.3125 μg / mL. The extracts were subjected to an MTT cell viability assay experiment:

[0144] Cytotoxicity assay: Digest cells in the logarithmic growth phase. Inoculate 1×10 4 Cells were cultured in 96-well cell culture plates. After 24 hours of culture, the supernatant was discarded. Sample solutions dissolved in culture medium at different concentrations were added to the sample wells. The blank control group was without extracts, and the group without cells and samples was zeroed. Multiple replicates were set for each well. After 24 hours of culture, the supernatant was discarded and washed with PBS. 100 μL of freshly prepared MTT solution of 0.5 mg / mL (dissolved in DMEM culture medium) was added to each well. After incubation for 4 hours, the supernatant was discarded, 100 μL of DMSO was added to each well to dissolve the crystals, and the cells were shaken at 37°C for 5 minutes. The absorbance value was read at 490 nm on an ELISA reader.

[0145] Determination of inflammatory factor levels: Macrophage suspension was prepared at a rate of 1×10 5 The cells were inoculated in a 24-well cell culture plate and incubated for 24 hours with 0.3125 μg / mL of the active ingredient extract of silver needle and 10 μg / ml LPS. The blank group was added with high-glucose medium, and the model group was added with 10 μg / mL LPS. During the test, the supernatant was taken to determine the IL-6 concentration in the culture medium according to the ELISA kit operating instructions. The results are shown in Tables 4, 5 and Figure 4 shown.

[0146] Table 4 Cell proliferation rate detection results

[0147] Group Cell proliferation rate Blank group 100％ Example 2 102.42％ Example 3 96.44％ Example 4 99.95％ Example 5 100.82％

[0148] Table 5 Cell experiment IL-6 inflammatory factor content detection results

[0149] Group IL-6 inflammatory factor content (pg / mL) Blank group 0 Model Group 3926.81493 Example 2 3189.55132 Example 3 3479.0647 Example 4 3476.0596 Example 5 3726.2086

[0150] The above results show that the extraction amounts of total phenols, flavonoids, alkaloids and theanine in Examples 1 to 7 are all higher than those in Comparative Examples 1 to 6 and water extraction and alcohol extraction, indicating that the extraction method of this patent has greatly improved the content of active ingredients in the extracts obtained compared with traditional water extraction and alcohol extraction. At the same time, by comparing Example 1 with Comparative Example 7, it can be seen that the molar ratio of proline-glycerol also has a great influence on the extraction effect of the active ingredients of Silver Needle.

[0151] On this basis, the DPPH and ABTS free radical scavenging rates of the active ingredient extracts of Examples 1 to 7 were higher than those of traditional water extraction and alcohol extraction. It can be seen that the antioxidant capacity of the extracts obtained by the extraction methods of the examples is greatly improved compared with traditional water extraction and pure extraction.

[0152] At the same time, the cytotoxicity test results of Examples 2 to 5 show that the active ingredient extracts of Silver Needle in the examples do not show obvious cytotoxicity, and can partially promote the proliferation of macrophages RAW264.7. The inhibition level of IL-6 inflammatory factors in Examples 2 to 5 is better than that in the model group, indicating that the active ingredient extracts of Silver Needle in Examples 2 to 5 have anti-inflammatory effects.

[0153] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A method for simultaneously improving the extraction effects of total phenols, flavonoids, alkaloids and theanine in Silver Needle, characterized in that: Extraction is performed using natural deep eutectic solvents. The extraction methods include: The natural deep eutectic solvent and water are mixed in a mass ratio of 3 to 19:1 to obtain a natural deep eutectic solvent aqueous solution; the silver needle white needle powder and the natural deep eutectic solvent aqueous solution are mixed in a material-liquid ratio of 1 g: 10 to 30 mL, heated by ultrasonication, and centrifuged to obtain a silver needle white needle extract; The natural low eutectic solvent is prepared from two or more of proline, glycerol, betaine, citric acid, lactic acid, malic acid and levulinic acid.

2. The method according to claim 1, characterized in that The preparation method of natural deep eutectic solvent is: Proline and glycerol are mixed in a molar ratio of 1:2.5-3.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-glycerol natural deep eutectic solvent; or, Mix betaine and citric acid in a molar ratio of 1:1.5-2.5, and stir at 75-85°C for 2.5-3.5h to obtain a betaine-citric acid natural deep eutectic solvent; or, Mixing betaine and lactic acid in a molar ratio of 1:1.5-2.5, stirring at 75-85°C for 2.5-3.5h to obtain a betaine-lactic acid natural deep eutectic solvent; or, Mix betaine, malic acid and lactic acid in a molar ratio of 0.8-1.2:0.8-1.2:0.8-1.2, and stir at 75-85° C. for 2.5-3.5 hours to obtain a betaine-malic acid-lactic acid natural low eutectic solvent; or, Mix betaine and glycerol in a molar ratio of 1:1.5-2.5, and stir at 75-85°C for 2.5-3.5h to obtain a betaine-glycerol natural deep eutectic solvent; or, Proline and lactic acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-lactic acid natural deep eutectic solvent; or, Proline and levulinic acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-levulinic acid natural low eutectic solvent.

3. The method according to claim 1, characterized in that The heating ultrasound is 20-80°C, 20-80Hz for extraction for 20-120min; the centrifugation is 8000-12000rpm for 5-20min.

4. The method according to claim 1, characterized in that Extraction is performed using natural deep eutectic solvents. The extraction methods include: Proline and glycerol are mixed in a molar ratio of 1:2.5-3.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-glycerol natural deep eutectic solvent; The proline-glycerol natural deep eutectic solvent and water are mixed at a mass ratio of 3.5 to 4.5:1 to obtain a proline-glycerol natural deep eutectic solvent aqueous solution; Silver needle baihao and proline-glycerol natural low eutectic solvent aqueous solution are mixed at a solid-liquid ratio of 1g:18-22mL, extracted at 40-50Hz and 55-65°C for 50-70min, and centrifuged at 8000-12000rpm for 8-12min to obtain an extract of the active ingredient of silver needle baihao.

5. Use of the method according to any one of claims 1 to 4 in extracting effective ingredients from Silver Needle Needle.

6. A method for extracting effective ingredients from Silver Needle Herb, characterized in that: Extraction using a natural deep eutectic solvent involves: The natural deep eutectic solvent and water are mixed in a mass ratio of 3 to 19:1 to obtain a natural deep eutectic solvent aqueous solution; the natural deep eutectic solvent aqueous solution and silver needle baihao powder are mixed in a solid-liquid ratio of 1g:10 to 30mL, heated with ultrasound, and centrifuged to obtain a silver needle baihao extract.

7. The method according to claim 6, characterized in that The preparation method of natural deep eutectic solvent is: Proline and glycerol are mixed in a molar ratio of 1:2.5-3.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-glycerol natural deep eutectic solvent; or, Mix betaine and citric acid in a molar ratio of 1:1.5-2.5, and stir at 75-85°C for 2.5-3.5h to obtain a betaine-citric acid natural deep eutectic solvent; or, Mixing betaine and lactic acid in a molar ratio of 1:1.5-2.5, stirring at 75-85°C for 2.5-3.5h to obtain a betaine-lactic acid natural deep eutectic solvent; or, Mix betaine, malic acid and lactic acid in a molar ratio of 0.8-1.2:0.8-1.2:0.8-1.2, and stir at 75-85° C. for 2.5-3.5 hours to obtain a betaine-malic acid-lactic acid natural low eutectic solvent; or, Mix betaine and glycerol in a molar ratio of 1:1.5-2.5, and stir at 75-85°C for 2.5-3.5h to obtain a betaine-glycerol natural deep eutectic solvent; or, Proline and lactic acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-lactic acid natural deep eutectic solvent; or, Proline and levulinic acid are mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 hours to obtain a proline-levulinic acid natural low eutectic solvent.

8. The method according to claim 6, characterized in that The heating ultrasound is 20-80°C, 20-80Hz and the extraction time is 20-120min.

9. The method according to claim 6, characterized in that Centrifugal: 8000-12000rpm for 5-20min.

10. The method according to claim 6, characterized in that The effective ingredients include one or more of total phenols, flavonoids, alkaloids and theanine.

Images