Method for extracting active ingredients in pollen typhae based on natural eutectic solvent

Abstract

The invention discloses a method for extracting active ingredients in pollen typhae based on a natural eutectic solvent, and belongs to the technical field of active substance extraction. By screening the natural eutectic solvent and optimizing preparation parameters, the invention provides the method for extracting the active components in the pollen typhae by using the natural eutectic solvent, and solves the problems of low extraction rate of the active components in the pollen typhae and the like in the prior art. According to the method, proline-lactic acid (the molar ratio is 1: 3) is used as a natural eutectic solvent, and the extraction efficiency of total active substances (polyphenol, flavone and alkaloid) in the pollen typhae reaches 167.9 mg / g of pollen typhae powder; the extraction of quercetin reaches 0.41 mg / g of pollen typhae 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 from pollen typhae based on a natural low eutectic solvent, belonging to the technical field of active substance extraction. Background Art

[0002] Typhae pollen is the dried pollen of Typha acuminatum, Typha orientalis or the same genus of Typhaceae, which can be used as medicine, and has the effects of astringent hemostasis, blood circulation and blood stasis, diuresis and stranguria, lipid-lowering and blood circulation. In recent years, quercetin has been found to have anti-inflammatory, antioxidant, lipid-lowering and other functions, and is also contained in a high content in Typhae pollen. However, the traditional water extraction method has problems such as long extraction time, low extraction efficiency, and wastewater environmental 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. Patent CN113354610A discloses a method for extracting flavonoid components in a prescription for nourishing yin, replenishing qi and promoting blood circulation using a low eutectic solvent prepared by proline and glycerol, but the material-liquid ratio used in the extraction is very large, and a lot of DES is required, which increases the cost; Patent CN117100789A discloses a method for extracting flavonoid components in lemon leaves using a low eutectic solvent prepared by proline and glycerol, but the final supernatant needs to be vacuum filtered, which increases the cost of extraction; The document "Green and efficient extraction of four bioactive flavonoids from Pollen Typhae by ultrasound-assisted deep eutectic solvent extraction" discloses a method for extracting flavonoids from Pollen Typhae using a low eutectic solvent prepared by choline chloride and 1,2-propylene glycol. In this method, acid hydrolysis is used, and the waste liquid after extraction is not environmentally friendly. At the same time, the above technical scheme is limited to extracting flavonoids from Pollen Typhae effective components, and does not focus on the extraction of other active substances and quercetin.

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

[0006] To solve the above problems, the present invention provides a method for extracting active ingredients from Pollen Typhae using a natural low eutectic solvent by screening a natural low eutectic solvent and optimizing the preparation parameters, thereby solving the problems of low extraction rate of active ingredients from Pollen Typhae 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 quercetin in Pollen Typhae, 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 natural deep eutectic solvent aqueous solution is mixed with Pollen Typhae powder in a solid-liquid ratio of 1 g: 10 to 30 mL, heated by ultrasonication, and centrifuged to obtain a Pollen Typhae extract;

[0009] The natural deep eutectic solvent is prepared from any two of proline, lactic acid, glycerol, levulinic acid, malic acid and urea.

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

[0011] 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,

[0012] 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,

[0013] 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 deep eutectic solvent; or,

[0014] Mix malic acid 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 malic acid-glycerol natural deep eutectic solvent; or,

[0015] Mix lactic acid 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 lactic acid-glycerol natural low eutectic solvent; or,

[0016] The urea and glycerol were mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 h to obtain a proline-lactic acid natural low eutectic solvent.

[0017] In one embodiment, the heating ultrasound is performed at 20-80° C., 20-80 Hz for extraction for 20-60 min.

[0018] In one embodiment, the centrifugation is performed at 6000-10000 rpm for 10-30 min.

[0019] Preferably, a method for simultaneously improving the extraction effect of total phenols, flavonoids, alkaloids and quercetin in Pollen Typhae uses a natural deep eutectic solvent for extraction, and the extraction method comprises:

[0020] Proline and lactic acid are mixed in a molar ratio of 1:1.5-2.5, stirred at 75-85°C for 2.5-3.5h to obtain a proline-lactic acid natural low eutectic solvent, the proline-lactic acid natural low eutectic solvent and water are mixed in a mass ratio of 8-10:1 to obtain a proline-lactic acid natural low eutectic solvent aqueous solution; the proline-lactic acid natural low eutectic solvent aqueous solution and Pollen Typhae are mixed in a solid-liquid ratio of 1g:15-25mL, ultrasonicated at 55-65Hz and 45-55°C for 25-35min, centrifuged at 8000-10000rpm for 15-20min, filtered, and an extract of the active component of Pollen Typhae is obtained.

[0021] Preferably, a method for simultaneously improving the extraction effect of total phenols, flavonoids, alkaloids and quercetin in Pollen Typhae uses a natural deep eutectic solvent for extraction, and the extraction method comprises:

[0022] Proline and glycerol are mixed at a molar ratio of 1:2.5-3.5, stirred at 75-85°C for 2.5-3.5 hours to obtain a proline-glycerol natural low eutectic solvent, and the proline-glycerol natural low eutectic solvent and water are mixed at a ratio of 18-20:1 to obtain a proline-glycerol natural low eutectic solvent aqueous solution; the proline-glycerol natural low eutectic solvent aqueous solution and Pollen Typhae are mixed at a solid-liquid ratio of 1 g:8-12 mL, ultrasonicated at 15-25 Hz and 15-25°C for 15-25 minutes, centrifuged at 6000-8000 rpm for 8-12 minutes, and filtered to obtain a Pollen Typhae active ingredient extract.

[0023] The second object of the present invention is to provide the application of any of the above methods in extracting effective components from Pollen Typhae.

[0024] The present invention also provides a method for extracting effective components from Pollen Typhae, using a natural deep eutectic solvent for extraction, the steps comprising:

[0025] The natural low eutectic solvent and water are mixed in a mass ratio of 3 to 19:1 to obtain a natural low eutectic solvent aqueous solution; the natural low eutectic solvent aqueous solution and Pollen Typhae powder are mixed in a solid-liquid ratio of 1 g: 10 to 30 mL, heated by ultrasound, and centrifuged to obtain a Pollen Typhae extract.

[0026] In one embodiment, the preparation method of the natural deep eutectic solvent is:

[0027] 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,

[0028] 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,

[0029] 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 deep eutectic solvent; or,

[0030] Mix malic acid 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 malic acid-glycerol natural deep eutectic solvent; or,

[0031] Mix lactic acid 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 lactic acid-glycerol natural low eutectic solvent; or,

[0032] The urea and glycerol were mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 h to obtain a proline-lactic acid natural low eutectic solvent.

[0033] In one embodiment, the heating ultrasound is performed at 20-80° C., 20-80 Hz for extraction for 20-60 min.

[0034] In one embodiment, the centrifugation is performed at 6000-10000 rpm for 10-30 min.

[0035] Preferably, a method for simultaneously improving the extraction effect of total phenols, flavonoids, alkaloids and quercetin in Pollen Typhae uses a natural deep eutectic solvent for extraction, and the extraction method comprises:

[0036] Proline and lactic acid are mixed in a molar ratio of 1:1.5-2.5, stirred at 75-85°C for 2.5-3.5h to obtain a proline-lactic acid natural low eutectic solvent, the proline-lactic acid natural low eutectic solvent and water are mixed in a mass ratio of 8-10:1 to obtain a proline-lactic acid natural low eutectic solvent aqueous solution; the proline-lactic acid natural low eutectic solvent aqueous solution and Pollen Typhae are mixed in a solid-liquid ratio of 1g:15-25mL, ultrasonicated at 55-65Hz and 45-55°C for 25-35min, centrifuged at 8000-10000rpm for 15-20min, filtered, and an extract of the active ingredient of Pollen Typhae is obtained.

[0037] Preferably, a method for simultaneously improving the extraction effect of total phenols, flavonoids, alkaloids and quercetin in Pollen Typhae uses a natural deep eutectic solvent for extraction, and the extraction method comprises:

[0038] Proline and glycerol are mixed at a molar ratio of 1:2.5-3.5, stirred at 75-85°C for 2.5-3.5 hours to obtain a proline-glycerol natural low eutectic solvent, and the proline-glycerol natural low eutectic solvent and water are mixed at a ratio of 18-20:1 to obtain a proline-glycerol natural low eutectic solvent aqueous solution; the proline-glycerol natural low eutectic solvent aqueous solution and Pollen Typhae are mixed at a solid-liquid ratio of 1 g:8-12 mL, ultrasonicated at 15-25 Hz and 15-25°C for 15-25 minutes, centrifuged at 6000-8000 rpm for 8-12 minutes, and filtered to obtain a Pollen Typhae active ingredient extract.

[0039] In one embodiment, the active ingredients include one or more of total phenols, flavonoids, alkaloids and quercetin.

[0040] Beneficial effects of the present invention:

[0041] 1. The present invention adopts green biodegradable natural low eutectic solvent as the extractant, which is in line with the development concept of green extraction.

[0042] 2. The present invention adopts a natural low eutectic solvent as an extractant to improve the extraction efficiency of the active ingredients in Puhuang. Among them, the extraction efficiency of the total active substances (polyphenols, flavonoids, alkaloids) in Puhuang using proline-lactic acid (molar ratio of 1:3) as a natural low eutectic solvent reaches 167.9 mg / g Puhuang powder; the extraction of quercetin reaches 0.41 mg / g Puhuang powder, and its extraction effect is far better than conventional water extraction and alcohol extraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] 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 are briefly introduced below.

[0044] Figure 1 It is a comparison chart of the active ingredient contents of the Typhae Pollen active ingredient extracts prepared in Examples 1 to 6 and Comparative Examples 1 to 9;

[0045] Figure 2 It is a comparison chart of DPPH free radical scavenging rates of the extracts of Typha Pollen active components prepared by water extraction and alcohol extraction in Examples 1 to 6;

[0046] Figure 3 It is a comparison chart of ABTS free radical scavenging rates of the extracts of Typha Pollen active components prepared by water extraction and alcohol extraction in Examples 1 to 6. DETAILED DESCRIPTION

[0047] The present invention uses a natural low eutectic solvent to extract the active ingredients of Pollen Typhae. The present invention is innovatively established by designing and synthesizing a natural low eutectic solvent to extract the active ingredients of Pollen Typhae, and the content of the active ingredients obtained is nearly twice that of the traditional water extraction method, which solves the current problem that the extraction of the active ingredients of Pollen Typhae is not green enough and has a low yield.

[0048] In order to make the purpose, technical solution and advantages of the present invention clearer and more specific, the present invention is further described in detail below 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 used to limit the present invention.

[0049] 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.

[0050] raw material:

[0051] Pollen Typhae (powder; 800 mesh) was purchased from Kangmei Chinese Medicine City, Haozhou City;

[0052] Proline was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 147-85-3;

[0053] Glycerol was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 56-81-5;

[0054] Lactic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No. 50-21-5;

[0055] Malic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., CAS No. 6915-15-7;

[0056] Levulinic acid was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 123-76-2;

[0057] Choline chloride was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 67-48-1;

[0058] 1,2-Propanediol was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., CAS No. 57-55-6;

[0059] Urea was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd. with a CAS number of 57-13-6.

[0060] Detection method:

[0061] 1. Determination of flavonoids, total phenols and total alkaloids:

[0062] The flavonoid content in the extract of Typhae Pollen was determined by sodium nitrite-aluminum nitrate colorimetric method using rutin as the standard. The total phenol content in the extract of Typhae Pollen was determined by folin phenol colorimetric method using gallic acid as the standard. The total alkaloid content in the extract of Typhae Pollen was determined by sulfuric acid colorimetric method using berberine hydrochloride as the standard.

[0063] 2. Determination of quercetin content:

[0064] The quercetin content in Pollen Typhae extract was determined by HPLC with quercetin as standard.

[0065] The chromatographic conditions are:

[0066] 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 0.05% (v / v) phosphoric acid aqueous solution (A) and acetonitrile (B). Gradient elution: 0-12min, 15-20% B; 12-15min, 20-40% B; 15-20min, 40-30% B; 20-30min, 30-20% B; 30-32min, 20-15% B. The detection wavelength was 370nm. The injection volume was 20μL and filtered through a 0.45μm membrane filter before use.

[0067] 3. Water extraction:

[0068] Deionized water and Typha acuminata were extracted at 60°C for 2 h in a solid-liquid ratio of 1:20 g / mL for three times to obtain an extract.

[0069] 4. Alcohol extraction method:

[0070] 75% ethanol and Typha latifolia were extracted at a solid-liquid ratio of 1:20 g / mL at 60°C for 2 h, and the extraction was repeated three times to obtain an extract.

[0071] Example 1

[0072] A method for extracting active ingredients of Pollen Typhae using a natural deep eutectic solvent, comprising the following steps:

[0073] (1) Preparation of natural deep eutectic solvent

[0074] Proline 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 and transparent liquid, i.e., a proline-lactic acid natural low eutectic solvent;

[0075] (2) Extraction of active ingredients

[0076] The proline-lactic acid natural low eutectic solvent (molar ratio 1:2) and water are mixed in a mass ratio of 9:1 to prepare a proline-lactic acid natural low eutectic solvent aqueous solution (water content 10%); the natural low eutectic solvent aqueous solution is placed in a conical flask, and Typha aquaphila powder is added and mixed, the solid-liquid ratio is 1g:20mL, 60Hz, 50℃ ultrasonic extraction for 30min, 8000rpm centrifugation for 20min, filtration, and Typha aquaphila active ingredient extract.

[0077] Example 2

[0078] A method for extracting active ingredients of Pollen Typhae using a natural deep eutectic solvent, comprising the following steps:

[0079] (1) Preparation of natural deep eutectic solvent

[0080] 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, i.e., a proline-glycerol natural deep eutectic solvent;

[0081] (2) Extraction of active ingredients

[0082] The proline-glycerol natural low eutectic solvent (molar ratio 1:3) and water are mixed at a mass ratio of 19:1 to prepare a proline-glycerol natural low eutectic solvent aqueous solution (water content 5%), the proline-glycerol natural low eutectic solvent aqueous solution is placed in a conical flask, and Typha aquaphila powder is added and mixed, the solid-liquid ratio is 1 g:10 mL, 20 Hz, 20°C ultrasonic extraction for 20 min, 6000 rpm centrifugation for 10 min, filtration, and Typha aquaphila active ingredient extract.

[0083] Example 3

[0084] A method for extracting active ingredients of Pollen Typhae using a natural deep eutectic solvent, comprising the following steps:

[0085] (1) Preparation of natural deep eutectic solvent

[0086] Proline and levulinic acid were mixed in a round-bottom flask at a molar ratio of 1:2, and stirred at 80°C for 3 hours to obtain a clear and transparent liquid, i.e., a proline-levulinic acid natural deep eutectic solvent;

[0087] (2) Extraction of active ingredients

[0088] The proline-levulinic acid natural low eutectic solvent (molar ratio 1:2) and water are mixed in a mass ratio of 3:1 to prepare a proline-levulinic acid natural low eutectic solvent aqueous solution (water content 25%), the proline-levulinic acid natural low eutectic solvent aqueous solution is placed in a conical flask, and Typha acuminata powder is added and mixed, the solid-liquid ratio is 1 g:30 mL, 80 Hz, 80°C ultrasonic extraction for 60 min, 10000 rpm centrifugation for 30 min, filtration, and Typha acuminata active ingredient extract.

[0089] Example 4

[0090] On the basis of Example 1, the natural low eutectic solvent in step (1) was changed to malic acid-glycerol (molar ratio 1:2), and the remaining steps were consistent with Example 1 to prepare the active ingredient extract of Typha acuminata.

[0091] Example 5

[0092] On the basis of Example 1, the natural low eutectic solvent in step (1) was changed to lactic acid-glycerol (molar ratio 1:2), and the remaining steps were consistent with Example 1 to prepare the active ingredient extract of Typha acuminata.

[0093] Example 6

[0094] On the basis of Example 1, the natural low eutectic solvent in step (1) was changed to urea-glycerol (molar ratio 1:2), and the remaining steps were consistent with Example 1 to prepare the active ingredient extract of Typha acuminata.

[0095] Comparative Example 1

[0096] On the basis of Example 2, the molar ratio of proline to glycerol in step (1) was changed to 1:2; the ultrasound in step (2) was performed at 35°C and 200W for 30 min, and the remaining steps were consistent with Example 2 to prepare a pollen active ingredient extract.

[0097] Comparative Example 2

[0098] On the basis of Example 1, the molar ratio of proline to lactic acid in step (1) was changed to 1:1, and the remaining steps were consistent with Example 1 to prepare a pollen pollen active ingredient extract.

[0099] Comparative Example 3

[0100] On the basis of Example 1, the molar ratio of proline to lactic acid in step (1) was changed to 1:3, and the remaining steps were consistent with Example 1 to prepare a pollen pollen active ingredient extract.

[0101] Comparative Example 4

[0102] On the basis of Example 1, the natural low eutectic solvent in step (1) was changed to proline and urea (molar ratio 1:1), and the remaining steps were consistent with Example 1 to prepare the active ingredient extract of Typha acuminata.

[0103] Comparative Example 5

[0104] On the basis of Example 1, the natural deep eutectic solvent in step (1) was changed to choline chloride and 1,2-propylene glycol (molar ratio of 1:4), and the choline chloride and 1,2-propylene glycol accounted for 30% in the deep eutectic solvent aqueous solution; the solid-liquid ratio was 1 mg:50 mL, the extraction was carried out for 35 min, and the remaining steps were consistent with Example 1 to prepare a Typha Pollen active ingredient extract.

[0105] Comparative Example 6

[0106] On the basis of Example 1, the temperature in step (2) was changed to 10° C., and the remaining steps were the same as in Example 1 to prepare a Puhuang active ingredient extract.

[0107] Comparative Example 7

[0108] On the basis of Example 1, the temperature in step (2) was changed to 100° C., and the remaining steps were the same as in Example 1 to prepare a Puhuang active ingredient extract.

[0109] Comparative Example 8

[0110] On the basis of Example 1, the ultrasonic frequency in step (2) was changed to 90 Hz (higher than that in Example 1), and the remaining steps were consistent with Example 1 to prepare a Puhuang active ingredient extract.

[0111] Comparative Example 9

[0112] On the basis of Example 1, the ultrasonic frequency in step (2) was changed to 10 Hz (lower than that in Example 1), and the remaining steps were consistent with Example 1 to prepare a Puhuang active ingredient extract.

[0113] Example 7

[0114] 1. The extracts of active ingredients of Pollen Typhae prepared by water extraction and alcohol extraction were taken from Examples 1 to 6, Comparative Examples 1 to 9, and the extraction rates of active ingredients (total phenols, flavonoids, alkaloids) and quercetin were tested. The results are as follows: Figure 1 And as shown in Table 1.

[0115] Table 1 Active ingredient content in the extract

[0116]

[0117] The results show that the extraction amounts of total phenols, flavonoids, alkaloids and quercetin in Examples 1-6 are all higher than those in Comparative Examples 1-9 and water extraction and alcohol extraction. It can be seen that the extraction method of the embodiment has been greatly improved compared with traditional water extraction and alcohol extraction.

[0118] 2. Antioxidant activity determination:

[0119] The Typha Pollen extracts of Examples 1-6 were diluted with water to 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL for antioxidant activity testing.

[0120] (1) DPPH free radical scavenging ability

[0121] 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 Puhuang extract of different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL) respectively, 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 ELISA reader to measure the OD517 nm absorbance value as A. i The absorbance of the mixture of DPPH solution and an equal volume of anhydrous ethanol is determined as A0, and the absorbance of the mixture of Typhae Pollen extract and an equal volume of anhydrous ethanol is determined as A j , the calculation formula is as follows:

[0122] Free radical scavenging rate (%) = (A0-A i +A j ) / A0×100%.

[0123] The results are shown in Table 2 and Figure 2 As shown, the results show that the DPPH removal rate of Examples 1-7 is better than that of water extraction and alcohol extraction.

[0124] Table 2 DPPH test results

[0125] (mg / mL) 0.2 0.4 0.6 0.8 1.0 1.2 Example 1 10.83％ 21.05％ 29.34％ 37.62％ 48.63％ 56.45％ Example 2 9.22％ 19.34％ 25.31％ 33.15％ 41.93％ 51.64％ Example 3 9.37％ 16.23％ 26.88％ 35.64％ 43.51％ 50.88％ Example 4 10.05％ 14.97％ 27.15％ 31.67％ 47.2％ 56.05％ Example 5 8.64％ 15.62％ 24.39％ 35.39％ 40.8％ 52.37％ Example 6 9.45％ 17.73％ 28.75％ 32.04％ 45.64％ 50.73％ Water extraction 3.77％ 8.91％ 16.82％ 19.67％ 24.83％ 26.31％ Alcohol Extraction 5.23％ 10.06％ 19.33％ 24.30％ 29.9％ 30.72％

[0126] (2) ABTS+ free radical scavenging ability

[0127] Use a microbalance to weigh 3 mg of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) into a 5 mL EP tube, 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 into another 5 mL EP tube, add 1.43 mL of distilled water, and vortex to prepare a 2.6 mmol / L K2S2O8 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 mother solution. Before use, dilute the ABTS mother solution with anhydrous ethanol so that its OD 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.

[0128] Take 0.2 mL of different concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mg / mL) of Typha lachryma-curcuma extract, add 0.8 mL of ABTS working solution, vortex evenly, and place in the dark to react for 6 min. Use 96-well plate and microplate reader to measure OD 734 nm , denoted as A i The absorbance of the mixture of 0.8 mL ABTS working solution and 0.2 mL deionized water is A0, and the absorbance of the mixture of 0.2 mL Typhae Pollen extract and 0.8 mL anhydrous ethanol is A j , the calculation formula is as follows:

[0129] Free radical scavenging rate (%) = (A0-A i +A j ) / A0×100%.

[0130] The results are shown in Table 3 and Figure 3 As shown, the results show that the ABTS removal rate of Examples 1-7 is better than that of water extraction and alcohol extraction.

[0131] Table 3 ABTS test results

[0132]

[0133]

[0134] The above results show that the extract of active ingredients from Pollen Typhae exhibits excellent antioxidant capacity, and the antioxidant capacity of DES extract is improved compared with traditional water extraction and alcohol extraction.

[0135] 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 effect of total phenols, flavonoids, alkaloids and quercetin in Pollen Typhae, characterized in that: Extraction is performed using natural deep eutectic solvents. The extraction methods include: The natural low eutectic solvent and water are mixed in a mass ratio of 3 to 19:1 to obtain a natural low eutectic solvent aqueous solution; the pollen typhae powder and the natural low eutectic solvent aqueous solution are mixed in a solid-liquid ratio of 1 g: 10 to 30 mL, heated by ultrasonication, and centrifuged to obtain a pollen typhae extract; The natural deep eutectic solvent is prepared from any two of proline, lactic acid, glycerol, levulinic acid, malic acid and urea.

2. The method according to claim 1, characterized in that: The preparation method of natural deep eutectic solvent is: 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 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, 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 deep eutectic solvent; or, Mix malic acid 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 malic acid-glycerol natural deep eutectic solvent; or, Lactic acid and glycerol 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 lactic acid-glycerol natural low eutectic solvent; or, The urea and glycerol were mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 h to obtain a proline-lactic 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 20-60min; the centrifugation is 6000-10000rpm for 10-30min.

4. The method according to claim 1, characterized in that: Proline and lactic acid are mixed at 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 low eutectic solvent, and the proline-lactic acid natural low eutectic solvent is mixed with water at a mass ratio of 8-10:1 to obtain a proline-lactic acid natural low eutectic solvent aqueous solution; The proline-lactic acid natural low eutectic solvent aqueous solution and Pollen Typhae are mixed at a solid-liquid ratio of 1 g: 15-25 mL, ultrasonicated at 55-65 Hz and 45-55° C. for 25-35 min, centrifuged at 8000-10000 rpm for 15-20 min, and filtered to obtain a Pollen Typhae active component extract.

5. Use of the method according to any one of claims 1 to 4 in extracting effective components from Pollen Typhae.

6. A method for extracting effective ingredients from Pollen Typhae, characterized in that: Extraction using a natural deep eutectic solvent involves: The natural low eutectic solvent and water are mixed in a mass ratio of 3 to 19:1 to obtain a natural low eutectic solvent aqueous solution; the pollen typhae powder and the natural low eutectic solvent aqueous solution are mixed in a material-liquid ratio of 1 g: 10 to 30 mL, heated by ultrasound, and centrifuged to obtain a pollen typhae extract.

7. The method according to claim 6, characterized in that The preparation method of natural deep eutectic solvent is: 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 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, 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 deep eutectic solvent; or, Mix malic acid 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 malic acid-glycerol natural deep eutectic solvent; or, Lactic acid and glycerol 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 lactic acid-glycerol natural low eutectic solvent; or, The urea and glycerol were mixed in a molar ratio of 1:1.5-2.5, and stirred at 75-85° C. for 2.5-3.5 h to obtain a proline-lactic acid natural low eutectic solvent.

8. The method according to claim 6, characterized in that The heating ultrasound is 20-80°C, 20-80Hz for extraction for 20-60min.

9. The method according to claim 6, characterized in that Centrifugation is performed at 6000-10000 rpm for 10-30 min.

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

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