Hybrid broussonetia papyrifera general flavone as well as extraction method, application and detection method thereof

Abstract

The invention provides hybrid broussonetia papyrifera total flavonoids as well as an extraction method, application and a detection method thereof, and relates to the technical field of plant active ingredient extraction. The invention discloses hybrid broussonetia papyrifera general flavone which contains 40 chemical components. The total flavonoids in the roots are extracted by adopting a microwave-assisted method, the total flavonoids in the stems are extracted by adopting an ultrasonic-assisted method, the total flavonoids in the leaves are extracted by adopting a soaking method, the differential extraction is performed on the total flavonoids in different parts of the roots, the stems and the leaves of the hybrid broussonetia papyrifera, and 40 flavonoid substances are separated from the roots, the stems and the leaves. The physicochemical properties such as pH values, conductivity values, oxidation resistance, scanning electron microscope microscopic characteristics and the like of the total flavonoids in the roots, stems and leaves obtained by adopting different extraction methods are different, so that the total flavone products of different parts of the hybrid broussonetia papyrifera can be comprehensively known; powerful physical and chemical data support and scientific basis are provided for development and utilization of the hybrid broussonetia papyrifera total flavonoids.

Description

Technical Field

[0001] The invention relates to the technical field of plant active ingredient extraction, and in particular to hybrid paper mulberry total flavonoids and an extraction method, application and detection method thereof. Background Art

[0002] Hybrid Broussonetia papyrifera is a new variety of plant bred by a research team from the Institute of Botany, Chinese Academy of Sciences, through space-borne and hybrid breeding techniques. Hybrid Broussonetia papyrifera is rich in crude protein, crude fat, amino acids, cellulose, mineral elements and other nutrients. The flavonoids in the leaves of Broussonetia papyrifera have obvious antioxidant and epidermal stem cell proliferation effects, and can effectively improve the excessive accumulation of fat in liver cells, and activate the intracellular oxidative stress defense system through the Nrf2 signaling pathway, thereby improving the body's ability to resist oxidative stress. At present, the chemical components of total flavonoids extracted from hybrid Broussonetia papyrifera are relatively small, which limits the application of total flavonoids from hybrid Broussonetia papyrifera. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a hybrid paper mulberry total flavonoids and its extraction method, application and detection method. The hybrid paper mulberry total flavonoids provided by the present invention contain 40 chemical components, which provides strong physical and chemical data support and scientific basis for understanding the chemical components of the total flavonoids in the hybrid paper mulberry and better developing and utilizing them.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The invention provides a hybrid paper mulberry total flavonoids, comprising one or more of the total flavonoids of hybrid paper mulberry root, hybrid paper mulberry stem and hybrid paper mulberry leaf; the chemical components of the hybrid paper mulberry total flavonoids include kaempferol, vitexin, apigenin, dihydroquercetin, dihydrokaempferol, quercetin, rutin, luteolin, naringenin, naringin chalcone, catechin, epicatechin, dihydromyricetin, isorhamnetin, kaempferol-3-O-glucose glucoside, quercetin-3-O-glucoside, trans-ferulic acid, 4-hydroxybenzoic acid, hydrocinnamic acid, trans-cinnamic acid, vanillic acid, vanillin, gallic acid, benzoic acid, salicylic acid, sinapic acid, syringic acid, syringaldehyde, protocatechuic acid, caffeic acid, p-coumaric acid, protocatechuic aldehyde, resveratrol, phthalic acid, isoliquiritigenin, daidzein, phloretin, 4',5,7-trigenin-7-glucoside, luteolin and gossypol.

[0006] The present invention also provides a method for extracting total flavonoids from hybrid paper mulberry as described in the above technical solution, including one or more of a method for extracting total flavonoids from the root of hybrid paper mulberry, a method for extracting total flavonoids from the stem of hybrid paper mulberry, and a method for extracting total flavonoids from the leaf of hybrid paper mulberry;

[0007] The method for extracting total flavonoids from the root of hybrid paper mulberry comprises the following steps: performing a first extraction on hybrid paper mulberry root powder using a first petroleum ether to obtain hybrid paper mulberry root defatted powder; performing microwave-assisted extraction on the hybrid paper mulberry root defatted powder using a first lower alcohol aqueous solution to obtain total flavonoids from the root of hybrid paper mulberry; the volume fraction of the lower alcohol in the first lower alcohol aqueous solution is 40-80%;

[0008] The method for extracting total flavonoids from hybrid paper mulberry stems comprises the following steps: performing a second extraction on hybrid paper mulberry stem powder using a second petroleum ether to obtain hybrid paper mulberry stem defatted powder; performing ultrasonic-assisted extraction on the hybrid paper mulberry stem defatted powder using a second lower alcohol aqueous solution to obtain total flavonoids from hybrid paper mulberry stems; the volume fraction of the lower alcohol in the second lower alcohol aqueous solution is 40-80%;

[0009] The method for extracting total flavonoids from the leaves of hybrid paper mulberry includes the following steps: performing a third extraction on hybrid paper mulberry leaf powder using a third petroleum ether to obtain hybrid paper mulberry leaf defatted powder; soaking and extracting the hybrid paper mulberry leaf defatted powder using a third lower alcohol aqueous solution to obtain total flavonoids from the leaves of hybrid paper mulberry; the volume fraction of the lower alcohol in the third lower alcohol aqueous solution is 65-85%.

[0010] Preferably, the ratio of the dry weight of the hybrid paper mulberry root defatted powder to the volume of the first lower alcohol aqueous solution is 1 g: 30-70 mL;

[0011] The lower alcohol in the first lower alcohol aqueous solution includes methanol and / or ethanol.

[0012] Preferably, the microwave power of the microwave-assisted extraction is 400-600 W, the temperature is 40-80° C., and the time is 5-25 min.

[0013] Preferably, the ratio of the dry weight of the hybrid paper mulberry stem defatted powder to the volume of the second lower alcohol aqueous solution is 1 g: 10-30 mL;

[0014] The lower alcohol in the second lower alcohol aqueous solution includes methanol and / or ethanol.

[0015] Preferably, the ultrasonic assisted extraction has an ultrasonic power of 300 to 1100 W, a temperature of 30 to 70° C., and a time of 1 to 3 h.

[0016] Preferably, the ratio of the dry weight of the defatted hybrid paper mulberry leaf powder to the volume of the third lower alcohol aqueous solution is 1 g: 5-25 mL;

[0017] The lower alcohol in the third lower alcohol aqueous solution includes methanol and / or ethanol.

[0018] Preferably, the soaking extraction is carried out at a temperature of 20 to 40° C. and for a time of 1 to 8 hours.

[0019] The present invention also provides the use of the hybrid paper mulberry total flavonoids described in the above technical solution or the hybrid paper mulberry total flavonoids obtained by the extraction method described in the above technical solution in cosmetics, health products or preparation of medicines.

[0020] The present invention also provides a method for detecting total flavonoids of hybrid paper mulberry, comprising the following steps:

[0021] The lower alcohol aqueous solution of total flavonoids of hybrid paper mulberry is subjected to high performance liquid phase tandem mass spectrometry detection to obtain the detection result of total flavonoids of hybrid paper mulberry;

[0022] The total flavonoids of hybrid paper mulberry include one or more of kaempferol, vitexin, apigenin, dihydroquercetin, dihydrokaempferol, quercetin, rutin, luteolin, naringenin, naringin chalcone, catechin, epicatechin, dihydromyricetin, isorhamnetin, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, trans-ferulic acid, 4-hydroxybenzoic acid, hydrocinnamic acid, trans-cinnamic acid, vanillic acid, vanillin, gallic acid, benzoic acid, salicylic acid, sinapinic acid, syringic acid, syringaldehyde, protocatechuic acid, caffeic acid, p-coumaric acid, protocatechuic aldehyde, resveratrol, phthalic acid, isoliquiritigenin, daidzein, phloretin, 4',5,7-trihydroxyisoflavone-7-glycoside, luteolin and gossypol;

[0023] The HPLC separation conditions for the HPLC tandem mass spectrometry detection include: the chromatographic column is a reverse phase chromatographic column, the mobile phase system includes mobile phase A and mobile phase B; the mobile phase A is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.1-0.5%; the mobile phase B is a formic acid acetonitrile solution, and the volume fraction of formic acid in the formic acid acetonitrile solution is 0.1-0.5%; the elution mode is gradient elution, and the gradient elution procedure is as follows:

[0024] 0-2min, the volume fraction of the mobile phase A is 90%;

[0025] 2-6 min, the volume fraction of the mobile phase A is reduced from 90% to 40%;

[0026] 6-9 min, the volume fraction of the mobile phase A is 40%;

[0027] 9-9.1 min, the volume fraction of the mobile phase A increases from 40% to 90%;

[0028] 9.1-12min, the volume fraction of the mobile phase A is 90%;

[0029] The mass spectrometry detection conditions of the high performance liquid phase tandem mass spectrometry detection include: the ion source is an electrospray ion source, the ion spray voltage is -2800V, the electrospray temperature is 350°C, and the ion transfer tube temperature is 320°C; the scanning mode is Fullscan-ddMS2, the scanning method is negative ion, and the primary mass spectrometry scanning range is 100-900m / z.

[0030] The invention discloses for the first time that the total flavonoids of hybrid paper mulberry contain kaempferol, vitexin, apigenin, dihydroquercetin, dihydrokaempferol, quercetin, rutin, luteolin, naringenin, naringin chalcone, catechin, epicatechin, dihydromyricetin, isorhamnetin, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, trans-ferulic acid, 4-hydroxybenzoic acid, hydrogenated cinnamic acid, trans-cinnamic acid, vanillic acid, vanillin, gallic acid The 40 chemical components of total flavonoids in hybrid paper mulberry, including benzoic acid, salicylic acid, sinapinic acid, syringic acid, syringaldehyde, protocatechuic acid, caffeic acid, p-coumaric acid, protocatechuic aldehyde, resveratrol, phthalic acid, isoliquiritigenin, daidzein, phloretin, 4',5,7-trihydroxyisoflavone-7-glycoside, luteolin and gossypol, provide strong physical and chemical data support and scientific basis for understanding the chemical composition of total flavonoids in hybrid paper mulberry and for better development and utilization of them.

[0031] The present invention adopts a microwave-assisted method to extract total flavonoids from the root, an ultrasound-assisted method to extract total flavonoids from the stem, and an immersion method to extract total flavonoids from the leaf, and performs the above-mentioned differentiated extraction on the total flavonoids in different parts of the root, stem and leaf of hybrid paper mulberry, and for the first time, 40 kinds of flavonoids are separated from the root, stem and leaf. The total flavonoids in the root, stem and leaf obtained by the present invention using different extraction methods have different physical and chemical properties such as pH value, conductivity value, antioxidant capacity and scanning electron microscope microscopic characteristics, which is helpful to fully understand the total flavonoid products in different parts of hybrid paper mulberry, and provides strong physical and chemical data support and scientific basis for the development and utilization of total flavonoids in hybrid paper mulberry.

[0032] As shown in the test results of the examples, the pH value order of the total flavonoid solutions in different parts of Broussonetia papyrifera is stem > root > leaf, and the pH values of all three parts satisfy 6.3 < pH < 7.0; the conductivity of the total flavonoid solutions in different parts of Broussonetia papyrifera all satisfies 966 μs / cm < conductivity < 1000 μs / cm; when the total flavonoid concentration is 0.1 mg / mL, the scavenging rates of the root and leaf parts on DPPH radicals are comparable and higher than that of the stem part, but the differences among the three are not significant (P > 0.05); when 0.2 mg / mL ≤ total flavonoid concentration ≤ 0.5 mg / mL, the scavenging rates of the total flavonoid solutions of the root, stem, and leaf parts on DPPH radicals are all extremely significantly (P < 0.01) higher than that of the leaf part under the same concentration condition, and follow the rule of stem ≥ root > leaf; the scavenging rates of the total flavonoid solutions in different parts of Broussonetia papyrifera on ABTS radicals generally show the result of root > stem ≥ leaf with extremely significant difference (P < 0.01). In addition, it is found that with the increase of the total flavonoid concentration, the scavenging rates of different parts on ABTS radicals all show an increasing trend, especially the stem and leaf parts are the most obvious, while the scavenging rate of the root part on ABTS radicals tends to be stable when the total flavonoid concentration ≥ 0.3 mg / mL; there are extremely obvious differences in the solid morphologies of the total flavonoids in different parts of Broussonetia papyrifera: the total flavonoids in the root part are mostly large lumps with multiple layers of superposition with a length of 6 μm and a width of more than 2.5 μm except for a very small number of small pieces; the total flavonoids in the stem part are sticky lumps with different lengths and widths and various shapes, and there are mostly small pieces; the total flavonoids in the leaf part are almost large plate-like similar to fish scale patterns; the block / size of the total flavonoids in different parts of Broussonetia papyrifera: leaf < stem < root. Obviously, this is related to the solid shape of flavonoid compounds; the looseness degree of the solid morphologies of the total flavonoids in different parts of Broussonetia papyrifera: root < leaf < stem, which may be related to the content of flavonoid glycosides in different parts and the adhesion states caused by the sugar molecule structures contained in flavonoid glycosides being different.

[0033] The present invention uses HPLC-MS / MS method to detect the total flavonoids of Broussonetia papyrifera, and can realize the qualitative and quantitative detection of 40 chemical components including kaempferol, vitexin, apigenin, dihydroquercetin, dihydrokaempferol, quercetin, rutin, luteolin, naringenin, naringin chalcone, catechin, epicatechin, dihydromyricetin, isorhamnetin, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, trans-ferulic acid, 4-hydroxybenzoic acid, hydrocinnamic acid, trans-cinnamic acid, vanillic acid, vanillin, gallic acid, benzoic acid, salicylic acid, sinapic acid, syringic acid, syringaldehyde, protocatechuic acid, caffeic acid, p-coumaric acid, protocatechualdehyde, resveratrol, phthalic acid, isoliquiritigenin, daidzein, phloretin, 4',5,7-trihydroxyisoflavone-7-glycoside, luteolin glycoside and gossypol. The detection method provided by the present invention has good stability and repeatability. Description of the Drawings

[0034] Figure 1It is a solution diagram of total flavonoids in different parts of hybrid paper mulberry, among which A is the total flavonoids in the root of hybrid paper mulberry, B is the total flavonoids in the stem of hybrid paper mulberry, and C is the total flavonoids in the leaf of hybrid paper mulberry;

[0035] Figure 2 This is the result diagram of pH value of total flavonoids solution in different parts of hybrid paper mulberry;

[0036] Figure 3 This is the result diagram of the conductivity value of total flavonoids solution in different parts of hybrid paper mulberry;

[0037] Figure 4 This is the result diagram of the scavenging rate of DPPH free radicals by total flavonoids solutions from different parts of hybrid paper mulberry;

[0038] Figure 5 This is the result diagram of the scavenging rate of ABTS free radicals by total flavonoids solutions from different parts of hybrid paper mulberry;

[0039] Figure 6 This is the SEM image of total flavonoids from the root of hybrid paper mulberry prepared in Example 7;

[0040] Figure 7 This is a SEM image of total flavonoids from the stem of hybrid paper mulberry prepared in Example 14;

[0041] Figure 8 This is a SEM image of total flavonoids from the leaves of the hybrid paper mulberry prepared in Example 17;

[0042] Fig. 9 is the total ion current of the standard;

[0043] Fig.10 This is the total ion current diagram of total flavonoids in the roots of hybrid paper mulberry;

[0044] Fig.11 This is the total ion current diagram of total flavonoids in the stem of hybrid paper mulberry;

[0045] Fig.12 This is the total ion current diagram of total flavonoids in the leaves of hybrid paper mulberry;

[0046] Fig.13 This is the standard curve of gallic acid and protocatechuic acid;

[0047] Fig.14 This is the standard curve of phthalic acid and protocatechuic aldehyde;

[0048] Fig.15 is the standard curve diagram of 4-hydroxybenzoic acid and caffeic acid;

[0049] Fig.16 This is the standard curve diagram of catechin and vanillic acid;

[0050] Fig.17 is the standard curve of dihydromyricetin and syringic acid;

[0051] Fig.18 is the standard curve of epicatechin and syringaldehyde;

[0052] Fig.19 is the standard curve of vanillin and p-coumaric acid;

[0053] Fig. 20 is the standard curve of trans-ferulic acid and rutin;

[0054] Fig.21 is the standard curve of vitexin and luteolin;

[0055] Fig. 22 This is the standard curve diagram of sinapinic acid and salicylic acid;

[0056] Fig.23 is the standard curve of dihydroquercetin and quercetin-3-O-glucoside;

[0057] Fig.24 It is the standard curve diagram of 4',5,7-genistein-7-glucoside and dihydrokaempferol;

[0058] Fig.25 is the standard curve of benzoic acid and kaempferol-3-O-glucoside;

[0059] Fig.26 is the standard curve of luteolin and resveratrol;

[0060] Fig. 27 is the standard curve of daidzein and trans-cinnamic acid;

[0061] Fig.28 is the standard curve of quercetin and hydrocinnamic acid;

[0062] Fig.29 is the standard curve of phloretin and naringin chalcone;

[0063] Fig.30 is the standard curve of naringenin and isorhamnetin;

[0064] Fig.31 This is the standard curve diagram of apigenin and kaempferol;

[0065] Fig.32 is the standard curve of gossypol and isoliquiritigenin;

[0066] Fig.33 This is the quality control chart of the quality control sample. DETAILED DESCRIPTION

[0067] The invention provides a hybrid paper mulberry total flavonoids, comprising one or more of the total flavonoids of hybrid paper mulberry root, hybrid paper mulberry stem and hybrid paper mulberry leaf; the chemical components of the hybrid paper mulberry total flavonoids include kaempferol, vitexin, apigenin, dihydroquercetin, dihydrokaempferol, quercetin, rutin, luteolin, naringenin, naringin chalcone, catechin, epicatechin, dihydromyricetin, isorhamnetin, kaempferol-3-O-glucose glucoside, quercetin-3-O-glucoside, trans-ferulic acid, 4-hydroxybenzoic acid, hydrocinnamic acid, trans-cinnamic acid, vanillic acid, vanillin, gallic acid, benzoic acid, salicylic acid, sinapic acid, syringic acid, syringaldehyde, protocatechuic acid, caffeic acid, p-coumaric acid, protocatechuic aldehyde, resveratrol, phthalic acid, isoliquiritigenin, daidzein, phloretin, 4',5,7-trigenin-7-glucoside, luteolin and gossypol.

[0068] The present invention provides a method for extracting total flavonoids from hybrid paper mulberry as described in the above technical scheme, including one or more of a method for extracting total flavonoids from hybrid paper mulberry roots, a method for extracting total flavonoids from hybrid paper mulberry stems and a method for extracting total flavonoids from hybrid paper mulberry leaves.

[0069] In the present invention, the method for extracting total flavonoids from the roots of hybrid paper mulberry includes the following steps: performing a first extraction on the hybrid paper mulberry root powder using a first petroleum ether to obtain a hybrid paper mulberry root defatted powder; performing microwave-assisted extraction on the hybrid paper mulberry root defatted powder using a first lower alcohol aqueous solution to obtain total flavonoids from the roots of hybrid paper mulberry; the volume fraction of the lower alcohol in the first lower alcohol aqueous solution is 40 to 80%.

[0070] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0071] The present invention utilizes the first petroleum ether to perform the first extraction on the hybrid paper mulberry root powder to obtain the hybrid paper mulberry root defatted powder.

[0072] In the present invention, the hybrid paper mulberry root powder is preferably obtained by dust removal, natural shade drying, processing, crushing and screening of paper mulberry roots. The screening is preferably through a 40-mesh sieve, and the part under the sieve is the hybrid paper mulberry root powder (that is, the particle size of the hybrid paper mulberry root powder is preferably ≥40 mesh).

[0073] In the present invention, the ratio of the dry weight of the hybrid paper mulberry root powder to the volume of petroleum ether (i.e., the solid-liquid ratio) is preferably 1g:20-60mL. In specific embodiments, it can be 1g:20mL, 1g:25mL, 1g:30mL, 1g:35mL, 1g:40mL, 1g:45mL, 1g:50mL, 1g:55mL or 1g:60mL.

[0074] In the present invention, the temperature of the first extraction is preferably room temperature, and the time of the first extraction is preferably 12 to 16 hours. In the present invention, the purpose of the first extraction is to defatting and removing pigments.

[0075] After the first extraction is completed, the present invention dries the solid components obtained by the first extraction to obtain hybrid paper mulberry root defatted powder. In the present invention, the drying temperature is preferably 65 to 75° C., and in a specific embodiment, it can be 65° C., 70° C. or 75° C. The present invention has no special limitation on the drying time, and the drying weight can be constant.

[0076] After obtaining hybrid paper mulberry root defatted powder, the present invention uses a first lower alcohol aqueous solution to perform microwave-assisted extraction on the hybrid paper mulberry root defatted powder to obtain total flavonoids from hybrid paper mulberry roots; the volume fraction of lower alcohol in the first lower alcohol aqueous solution is 40-80%.

[0077] In the present invention, the volume fraction of the lower alcohol in the first lower alcohol aqueous solution is 40-80%, and in specific embodiments can be 40%, 50%, 60%, 70% or 80%; the lower alcohol in the first lower alcohol aqueous solution preferably includes methanol and / or ethanol.

[0078] In the present invention, the ratio of the dry weight of the hybrid paper mulberry root defatted powder to the volume of the first low-alcohol aqueous solution (i.e., the solid-liquid ratio) is preferably 1g:30-70mL, and in specific embodiments it can be 1g:30mL, 1g:40mL, 1g:50mL, 1g:60mL or 1g:70mL.

[0079] In the present invention, the microwave power of the microwave-assisted extraction is preferably 400-600 W, and in a specific embodiment it can be 400 W, 450 W, 500 W, 550 W or 600 W; the temperature of the microwave-assisted extraction is preferably 40-80 ° C, and in a specific embodiment it can be 40 ° C, 50 ° C, 60 ° C, 70 ° C or 80 ° C; the time of the microwave-assisted extraction is preferably 5-25 min, and in a specific embodiment it can be 5 min, 10 min, 15 min, 20 min or 25 min.

[0080] After the microwave-assisted extraction is completed, the present invention sequentially filters the obtained microwave-assisted extraction system, centrifuges the obtained liquid components, and vacuum filters the obtained supernatant to obtain a total flavonoid solution from the roots of hybrid paper mulberry. In the present invention, the total flavonoids from the roots of hybrid paper mulberry are preferably stored in the form of a total flavonoid solution from the roots of hybrid paper mulberry, and the solvent in the total flavonoid solution from the roots of hybrid paper mulberry is removed before use.

[0081] In the present invention, the method for extracting total flavonoids from the stem of hybrid paper mulberry comprises the following steps: performing a second extraction on the hybrid paper mulberry stem powder using a second petroleum ether to obtain a hybrid paper mulberry stem defatted powder; performing ultrasonic-assisted extraction on the hybrid paper mulberry stem defatted powder using a second lower alcohol aqueous solution to obtain total flavonoids from the stem of hybrid paper mulberry; the volume fraction of the lower alcohol in the second lower alcohol aqueous solution is 40 to 80%.

[0082] In the present invention, the particle size of the hybrid paper mulberry stem defatted powder is preferably ≥ 60 mesh (i.e., passed through a 60-mesh sieve). In the present invention, other preparation conditions of the hybrid paper mulberry stem defatted powder are the same as those of the hybrid paper mulberry root defatted powder, which will not be repeated here.

[0083] In the present invention, the volume fraction of the lower alcohol in the second lower alcohol aqueous solution is 40-80%, and in specific embodiments can be 40%, 50%, 60%, 70% or 80%; the lower alcohol in the second lower alcohol aqueous solution preferably includes methanol and / or ethanol.

[0084] In the present invention, the ratio of the dry weight of the hybrid paper mulberry stem defatted powder to the volume of the second lower alcohol aqueous solution is preferably 1g:10-30mL, and in specific embodiments it can be 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL or 1g:30mL.

[0085] In the present invention, the ultrasonic power of the ultrasonic-assisted extraction is 300-1100 W, and in specific embodiments, it can be 300 W, 400 W, 500 W, 600 W, 700 W, 800 W, 900 W, 1000 W or 1100 W; the temperature of the ultrasonic-assisted extraction is 30-70°C, and in specific embodiments, it can be 30°C, 40°C, 50°C, 60°C or 70°C; the time of the ultrasonic-assisted extraction is 1-3 h, and in specific embodiments, it can be 1.0 h, 1.5 h, 2.0 h, 2.5 h or 3.0 h.

[0086] After completing the ultrasonic-assisted extraction, the present invention sequentially filters the obtained ultrasonic-assisted extraction system, centrifuges the obtained liquid components, and vacuum-filters the obtained supernatant to obtain a hybrid paper mulberry stem total flavonoid solution. In the present invention, the hybrid paper mulberry stem total flavonoid solution is preferably stored in the form of a hybrid paper mulberry stem total flavonoid solution, and the solvent in the hybrid paper mulberry stem total flavonoid solution is removed before use.

[0087] In the present invention, the method for extracting total flavonoids from the leaves of hybrid paper mulberry includes the following steps: using a third petroleum ether to perform a third extraction on the hybrid paper mulberry leaf powder to obtain a hybrid paper mulberry leaf defatted powder; using a third lower alcohol aqueous solution to soak and extract the hybrid paper mulberry leaf defatted powder to obtain total flavonoids from the leaves of hybrid paper mulberry; the volume fraction of the lower alcohol in the third lower alcohol aqueous solution is 65-85%.

[0088] In the present invention, the particle size of the hybrid paper mulberry leaf defatted powder is preferably ≥ 80 mesh (i.e., passed through an 80-mesh sieve). In the present invention, other preparation conditions of the hybrid paper mulberry leaf defatted powder are the same as those of the hybrid paper mulberry root defatted powder, which will not be repeated here.

[0089] In the present invention, the volume fraction of the lower alcohol in the third lower alcohol aqueous solution is 65-85%, and in specific embodiments it can be 65%, 70%, 75%, 80% or 85%; the lower alcohol in the third lower alcohol aqueous solution preferably includes methanol and / or ethanol.

[0090] In the present invention, the ratio of the dry weight of the defatted hybrid paper mulberry leaf powder to the volume of the third low-grade alcohol aqueous solution is 1g:5-25mL, and in specific embodiments it can be 1g:5mL, 1g:10mL, 1g:15mL, 1g:20mL or 1g:25mL.

[0091] In the present invention, the soaking extraction temperature is 20-40°C, and in specific embodiments it can be 20°C, 25°C, 30°C, 35°C or 40°C; the soaking extraction time is 1-8h, and in specific embodiments it can be 1h, 2h, 3h, 4h, 5h, 6h, 7h or 8h.

[0092] After the soaking and extraction is completed, the present invention sequentially filters the obtained soaking and extraction system, centrifuges the obtained liquid components, and vacuum filters the obtained supernatant to obtain a hybrid paper mulberry leaf total flavonoid solution. In the present invention, the hybrid paper mulberry leaf total flavonoid solution is preferably stored in the form of a hybrid paper mulberry leaf total flavonoid solution, and the solvent in the hybrid paper mulberry leaf total flavonoid solution is removed before use.

[0093] The present invention also provides the use of the hybrid paper mulberry total flavonoids described in the above technical solution or the hybrid paper mulberry total flavonoids obtained by the extraction method described in the above technical solution in cosmetics, health products or preparation of medicines. In the present invention, the medicine preferably includes one or more of antioxidant drugs, anti-inflammatory drugs, antibacterial drugs, hypoglycemic drugs, cardiovascular protective drugs, anticancer drugs, liver protective drugs and immunomodulatory drugs.

[0094] The invention provides a method for detecting total flavonoids of hybrid paper mulberry, comprising the following steps: subjecting a low-grade alcohol aqueous solution of total flavonoids of hybrid paper mulberry to high performance liquid phase tandem mass spectrometry detection to obtain a detection result of total flavonoids of hybrid paper mulberry.

[0095] In the present invention, the total flavonoids of hybrid paper mulberry include one or more of kaempferol, vitexin, apigenin, dihydroquercetin, dihydrokaempferol, quercetin, rutin, luteolin, naringenin, naringin chalcone, catechin, epicatechin, dihydromyricetin, isorhamnetin, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, trans-ferulic acid, 4-hydroxybenzoic acid, hydrogenated cinnamic acid, trans-cinnamic acid, vanillic acid, vanillin, gallic acid, benzoic acid, salicylic acid, sinapinic acid, syringic acid, syringaldehyde, protocatechuic acid, caffeic acid, p-coumaric acid, protocatechuic aldehyde, resveratrol, phthalic acid, isoliquiritigenin, daidzein, phloretin, 4',5,7-trihydroxyisoflavone-7-glycoside, luteolin and gossypol.

[0096] In the present invention, the preparation method of the lower alcohol aqueous solution of total flavonoids of hybrid paper mulberry preferably includes method 1 or method 2. In the present invention, the method 1 includes the following steps: centrifuging the total flavonoids solution of hybrid paper mulberry roots, the total flavonoids solution of hybrid paper mulberry stems or the total flavonoids solution of hybrid paper mulberry leaves obtained by the aforementioned extraction method to obtain a lower alcohol aqueous solution of total flavonoids of hybrid paper mulberry (sample solution to be tested). In the present invention, the method 2 preferably includes the following steps: dissolving the total flavonoids of hybrid paper mulberry in a lower alcohol aqueous solution and then centrifuging to obtain a lower alcohol aqueous solution of total flavonoids of hybrid paper mulberry. In the present invention, the type and volume fraction of the lower alcohol in the lower alcohol aqueous solution are consistent with the lower alcohol in the extraction of the total flavonoids of the roots of hybrid paper mulberry, the total flavonoids of the stems of hybrid paper mulberry or the total flavonoids of the leaves of hybrid paper mulberry, and will not be repeated here. In the present invention, the rotation speed of the centrifugal separation is preferably 9000-12000 r / min, more preferably 10000-12000 r / min; the time of the centrifugal separation is preferably 5-20 min, more preferably 10-15 min.

[0097] In the present invention, the HPLC separation conditions for HPLC tandem mass spectrometry detection include: the chromatographic column is a reverse phase chromatographic column, preferably a Waters HSS T3 chromatographic column; the mobile phase system includes a mobile phase A and a mobile phase B; the mobile phase A is a formic acid aqueous solution, the volume fraction of formic acid in the formic acid aqueous solution is preferably 0.1-0.5%, and in a specific embodiment, it can be 0.1%, 0.2%, 0.3%, 0.4% or 0.5%; the mobile phase B is a formic acid acetonitrile solution, the volume fraction of formic acid in the formic acid acetonitrile solution is preferably 0.1-0.5%, and in a specific embodiment, it can be 0.1%, 0.2%, 0.3%, 0.4% or 0.5%; the elution method is a gradient The gradient elution program is shown in Table 1; the mobile phase flow rate is preferably 0.1-0.5 mL / min, and in a specific embodiment it can be 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min or 0.5 mL / min; the column temperature is preferably 35-55°C, and in a specific embodiment it can be 35°C, 40°C, 45°C, 50°C or 55°C; the injection volume is preferably 1-5 μL, and in a specific embodiment it can be 1 μL, 2 μL, 3 μL, 4 μL or 5 μL.

[0098] Table 1 Gradient elution program

[0099] Time / min Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0 90 10 2 90 10 6 40 60 9 40 60 9.1 90 10 12 90 10

[0100] In the present invention, the mass spectrometry detection conditions of the high performance liquid phase tandem mass spectrometry detection include: the ion source is an electrospray ion source (ESI), the ion spray voltage is -2800V, the electrospray temperature is 350°C, and the ion transfer tube temperature is 320°C; the scanning mode is Full scan-ddMS2, the scanning mode is negative ion, and the primary mass spectrometry scanning range is 100-900m / z; the sheath gas pressure is preferably 40arb, and the auxiliary gas pressure is preferably 10arb.

[0101] In the present invention, the test results preferably include qualitative test results and / or quantitative test results.

[0102] In the present invention, the quantitative detection result is preferably obtained by using a standard curve method. In the present invention, the method for obtaining the quantitative detection result preferably comprises the following steps: obtaining the quantitative detection result according to the standard curve and the peak area obtained by the liquid chromatography tandem mass spectrometry detection; the standard curve uses the peak area of ​​the standard as the independent variable and the concentration of the standard as the dependent variable.

[0103] In the present invention, the method for drawing the standard curve preferably includes the following steps: detecting the mixed standard linear solution according to the conditions of the aforementioned liquid chromatography tandem mass spectrometry detection to obtain the peak area of ​​the mixed standard linear solution; drawing the standard curve of the standard with the peak area of ​​the standard as the independent variable and the concentration of the standard as the dependent variable.

[0104] In the present invention, the standards and concentrations in the mixed standard linear solution are shown in Table 2.

[0105] In the present invention, the preparation method of the mixed standard linear solution includes the following steps: adding the standard to a 80v / v% methanol-0.2wt% vitamin C-water mixed solution, vortex mixing and ultrasonic extraction, centrifugation to remove impurities, taking the supernatant and repeating the above steps once to ensure complete dissolution; combining the two supernatants to obtain a single standard mother solution with a concentration of 10mg / mL; taking each single standard mother solution and mixing it, using anhydrous ethanol as a diluent, preparing a mixed standard stock solution with a concentration of 1μg / mL and 10μg / mL, and storing it for standby use. Using anhydrous ethanol as a diluent, the mixed standard stock is diluted step by step to obtain a mixed standard linear solution. In the present invention, the ultrasonic extraction time is preferably 20 to 40min, more preferably 30min. In the present invention, the centrifugal speed is preferably 9000-12000 r / min, more preferably 10000-12000 r / min; the centrifugal time is preferably 5-20 min, more preferably 10-15 min.

[0106] Table 2 Mixed standard linear solution (ng / mL)

[0107]

[0108]

[0109] In order to further illustrate the present invention, the hybrid paper mulberry total flavonoids and their extraction method, application and detection method provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0110] In the following examples, the materials, reagents and consumables, instruments and testing methods used in the extraction of total flavonoids from different parts of hybrid paper mulberry are as follows.

[0111] Materials: The roots, stems and leaves of hybrid paper mulberry were collected from the Lankao Experimental Demonstration Base of the Hybrid Paper mulberry Industry Engineering Technology Research Center of Henan Academy of Sciences, Henan Academy of Sciences, and the hybrid paper mulberry Zhongke No. 101, which was 1-2 years old and 115-125 cm tall in May 2023. After dust removal, natural shade drying, processing and crushing, sieving (roots through 40 mesh sieve, stems through 60 mesh sieve), leaves through 80 mesh sieve), the sieved part is hybrid paper mulberry root powder, hybrid paper mulberry stem powder, and hybrid paper mulberry leaf powder.

[0112] Reagents and consumables: 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), rutin standard (B20771-100 mg, purity ≥98%, batch number T27F10Z81699), anhydrous ethanol, Wahaha purified water, sodium nitrite, aluminum nitrate, sodium hydroxide, potassium persulfate, medium-speed qualitative filter paper.

[0113] Instruments: TU-1810PC UV-visible spectrophotometer, Beijing Puxi; FW-100 high-speed universal pulverizer, Beijing Zhongxing; MDS-201 microwave extractor, Shanghai Precision Science; RE-5002 rotary evaporator, Wuxi Yiye; SHZ-D(III) circulating water multi-purpose vacuum pump, Gongyi Yuhua; HH-S6A digital display constant temperature water bath, Beijing Kewei; ME-204E analytical balance, Shanghai Toledo; KMH1-1100W0128-1-8 Goodyear ultrasonic GT SONIC, Guangdong Goodyear; vacuum pump glass filter, Sichuan Shuniu; TG16G Hetian centrifuge, Shanghai Hetian; DZF digital display blast drying oven, Shanghai Boxun; MX-S vortex mixing oscillator, Beijing Dalong Xingchuang; PHS-3E pH meter, Shanghai Yidian; DZS-706 conductivity meter, Shanghai Yidian; Quanta-200 scanning electron microscope, FEI, USA.

[0114] The extraction yield test method of total flavonoids from different parts of hybrid paper mulberry is as follows: the rutin standard is dissolved in anhydrous ethanol, and the volume is fixed to obtain rutin standard solutions of different concentrations, which are 0.207, 0.414, 0.621, 0.828, 1.034, 1.241 and 1.448 mg / mL, respectively. The absorbance values ​​of rutin standard solutions of different concentrations are tested respectively, which are 0.13, 0.25, 0.38, 0.51, 0.63, 0.76, and 0.89, respectively. The concentration of the rutin standard solution is used as the horizontal coordinate, and the absorbance value measured at the corresponding concentration of the rutin standard solution is used as the vertical coordinate to draw a standard curve (y=0.6131x-0.0003, correlation coefficient R 2=0.9999. ) Then, the extraction rate of total flavonoids in the total flavonoids solution of different parts of hybrid paper mulberry was determined by combining sodium nitrite-aluminum nitrate colorimetry and visible spectrophotometry. The test was repeated 5 times in parallel, and the extraction rate was the average value ± standard error.

[0115] Statistical analysis: The data were first summarized and sorted using WPS Office XLS, and then the Double Bar chart was drawn using IBM SPSS Statistics 20.0 software. The "mean ± standard error symbol" bar chart was used to represent the experimental data analysis results. P<0.05 (significant difference) and P<0.01 (extremely significant difference) were used as the criteria for judging the significance of the difference.

[0116] The experimental instruments, reagents and standards used in the qualitative and quantitative detection of the chemical components of total flavonoids in different parts of hybrid paper mulberry (see Table 3) are as follows.

[0117] Table 3 Standard product information

[0118]

[0119]

[0120] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (Vanquish, UPLC, Thermo, USA) and high-resolution mass spectrometer (Q Exactive, Thermo, USA); low-temperature high-speed centrifuge (Eppendorf5430R); nitrogen purge instrument (Thermo, Reacti-thermo); desktop rapid centrifugal concentrator and dryer (Hercy, ZLS-1); standard (Sigma); methanol / acetonitrile / formic acid (Anpu, chromatography grade).

[0121] Example 1

[0122] Microwave-assisted extraction of total flavonoids from the roots of hybrid papyrus—microwave-assisted extraction power

[0123] The hybrid papyrus root powder was extracted with petroleum ether for 12 to 16 hours, and the obtained solid component was dried at 65°C to obtain defatted hybrid papyrus root powder. The material-liquid ratio of hybrid papyrus root powder (dry weight) to petroleum ether was 1 g: 50 mL.

[0124] The defatted hybrid papyrus root powder was mixed with ethanol aqueous solution, and microwave-assisted extraction was performed. The insoluble impurities were filtered to remove them, and the liquid components were centrifuged at 3050r / min and 1370g for 20min. The obtained liquid components were vacuum filtered to obtain the total flavonoid solution of hybrid papyrus root. Among them, the solid-liquid ratio of defatted hybrid papyrus root powder to ethanol aqueous solution was 1g:30mL; the volume fraction of ethanol in the ethanol aqueous solution was 60%; the temperature of microwave-assisted extraction was 60℃, the time was 10min, the power was 400W, 450W, 500W, 550W and 600W respectively, and the total flavonoid extraction rates were 1.40±0.03%, 1.46±0.02%, 1.50±0.05%, 1.48±0.04% and 1.43±0.05% respectively. It can be seen that the optimal power of microwave-assisted extraction is 500W.

[0125] Example 2

[0126] Microwave-assisted extraction of total flavonoids from the roots of hybrid papyrus papyrifera—solid-liquid ratio

[0127] The only difference from Example 1 is that the microwave-assisted extraction power is 450 W, the solid-liquid ratio (g / mL) is 1 g:30 mL, 1 g:40 mL, 1 g:50 mL, 1 g:60 mL and 1 g:70 mL, and the total flavonoids extraction yields are 1.56±0.07%, 1.57±0.08%, 1.60±0.10%, 1.63±0.11% and 1.62±0.10%, respectively.

[0128] Example 3

[0129] Microwave-assisted extraction of total flavonoids from the roots of hybrid papyrus pachyphylla - ethanol volume fraction

[0130] The only difference from Example 1 is that the microwave-assisted extraction power is 450 W, the volume fraction of ethanol in the ethanol aqueous solution is 40%, 50%, 60%, 70% and 80%, respectively, and the extraction rates of total flavonoids are 1.59±0.05%, 1.60±0.06%, 1.63±0.07%, 1.64±0.08% and 1.61±0.07%, respectively.

[0131] Example 4

[0132] Microwave-assisted extraction of total flavonoids from the roots of hybrid papyrus—microwave-assisted extraction temperature

[0133] The only difference from Example 1 is that the microwave-assisted extraction power is 450 W, the microwave-assisted extraction temperatures are 40° C., 50° C., 60° C., 70° C. and 80° C., and the total flavonoid extraction yields are 1.56±0.04%, 1.58±0.03%, 1.62±0.04%, 1.60±0.04% and 1.61±0.05%, respectively.

[0134] Example 5

[0135] Microwave-assisted extraction of total flavonoids from the roots of hybrid papyrus—microwave-assisted extraction time

[0136] The only difference from Example 1 is that the microwave-assisted extraction power is 450 W, the microwave-assisted extraction time is 5 min, 10 min, 15 min, 20 min and 25 min, respectively, and the total flavonoid extraction rates are 1.58±0.05%, 1.59±0.07%, 1.62±0.07%, 1.64±0.08% and 1.63±0.06%, respectively.

[0137] Example 6

[0138] Microwave-assisted extraction of total flavonoids from the roots of hybrid papyrus

[0139] The total flavonoids from the roots of hybrid paper mulberry were extracted according to the method of Example 1. The extraction conditions were as follows: microwave-assisted extraction time was 20 min, temperature was 60° C., microwave power was 500 W, solid-liquid ratio was 1 g:50 mL, ethanol volume fraction was 60%, and the total flavonoid yield was 1.68±0.14%.

[0140] Example 7

[0141] Ultrasonic-assisted extraction of total flavonoids from the stems of hybrid papyrus—ultrasonic power

[0142] The hybrid papyrus stem powder was extracted with petroleum ether for 12 to 16 hours, and the obtained solid component was dried at 70°C to obtain defatted hybrid papyrus stem powder. The solid-liquid ratio of the hybrid papyrus stem powder to petroleum ether was 1 g:55 mL.

[0143] The defatted hybrid papyrus stem powder was mixed with ethanol aqueous solution, ultrasonic-assisted extraction was performed, insoluble impurities were filtered to remove, centrifuged at 3050r / min and 1370g for 20min, and the obtained liquid component was vacuum filtered to obtain a hybrid papyrus stem total flavonoids solution. Among them, the solid-liquid ratio of defatted hybrid papyrus stem powder to ethanol aqueous solution was 1g:20mL; the volume fraction of ethanol in the ethanol aqueous solution was 60%; the temperature of ultrasonic-assisted extraction was 50℃, the time was 2h, and the ultrasonic power was 300W, 500W, 700W, 900W and 1100W respectively, and the extraction rates of total flavonoids in hybrid papyrus stem were 0.33±0.03%, 0.34±0.02%, 0.36±0.03%, 0.38±0.05% and 0.39±0.05% respectively.

[0144] Example 8

[0145] Ultrasonic-assisted extraction of total flavonoids from the stems of hybrid papyrus pachyphylla—solid-liquid ratio

[0146] The only difference from Example 7 is that the ultrasonic power is 700 W, the solid-liquid ratio (g / mL) is 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL and 1g:30mL, and the yields of total flavonoids in the stems of hybrid paper mulberry are 0.41±0.06%, 0.42±0.06%, 0.43±0.06%, 0.46±0.06% and 0.46±0.05%, respectively.

[0147] Example 9

[0148] Ultrasonic-assisted extraction of total flavonoids from the stems of hybrid papyrus - ultrasonic time

[0149] The only difference from Example 7 is that the ultrasonic power is 700 W, the ultrasonic time is 1.0 h, 1.5 h, 2.0 h, 2.5 h and 3.0 h, respectively, and the extraction rates of total flavonoids in the stems of hybrid paper mulberry are 0.52±0.02%, 0.53±0.04%, 0.53±0.07%, 0.54±0.04% and 0.55±0.03%, respectively.

[0150] Example 10

[0151] Ultrasonic-assisted extraction of total flavonoids from the stems of hybrid papyrus - ultrasonic temperature

[0152] The only difference from Example 7 is that the ultrasonic power is 700 W, the ultrasonic temperatures are 30°C, 40°C, 50°C, 60°C and 70°C, and the extraction rates of total flavonoids in the stems of hybrid paper mulberry are 0.51±0.04%, 0.55±0.03%, 0.55±0.07%, 0.51±0.05% and 0.53±0.02%, respectively.

[0153] Embodiment 11

[0154] Ultrasonic-assisted extraction of total flavonoids from the stems of hybrid papyrus pachyphylla - ethanol volume fraction

[0155] The only difference from Example 7 is that the ultrasonic power is 700 W, the ethanol volume fractions are 40%, 50%, 60%, 70% and 80%, and the extraction rates of total flavonoids in the stem of hybrid paper mulberry are 0.53±0.02%, 0.56±0.02%, 0.54±0.01%, 0.56±0.05% and 0.56±0.03%, respectively.

[0156] Example 12

[0157] Ultrasonic-assisted extraction of total flavonoids from the stems of hybrid papyrus

[0158] The total flavonoids from the roots of hybrid paper mulberry were extracted according to the method of Example 7. The extraction conditions were as follows: ultrasonic time was 2.0 h, ultrasonic temperature was 50° C., ultrasonic power was 900 W, solid-liquid ratio was 1 g:25 mL, ethanol volume fraction was 70%, and the extraction rate of total flavonoids from the stems of hybrid paper mulberry was 0.59±0.06%.

[0159] Embodiment 13

[0160] Extraction of total flavonoids from hybrid papyrus leaves by room temperature immersion extraction method - extraction temperature

[0161] The hybrid paper mulberry leaf powder was extracted with petroleum ether for 12 to 16 hours, and the obtained solid component was dried at 60° C. to obtain defatted hybrid paper mulberry leaf powder. The solid-liquid ratio of the hybrid paper mulberry leaf powder to petroleum ether was 1 g: 60 mL.

[0162] The defatted hybrid paper mulberry leaf powder was mixed with an ethanol aqueous solution, soaked and extracted, filtered to remove insoluble impurities, centrifuged at 3050r / min and 1370g for 20min, and the obtained liquid component was vacuum filtered to obtain a hybrid paper mulberry leaf total flavonoid solution. Among them, the solid-liquid ratio of defatted hybrid paper mulberry leaf powder to ethanol aqueous solution was 1g:15mL; the volume fraction of ethanol in the ethanol aqueous solution was 75%; the extraction time was 4h, the extraction temperatures were 20℃, 25℃, 30℃, 35℃ and 40℃, and the extraction yields of total flavonoids in hybrid paper mulberry leaf were 5.16±0.03%, 5.19±0.08%, 5.16±0.04%, 5.13±0.06% and 5.17±0.02%, respectively.

[0163] Embodiment 14

[0164] Extraction of total flavonoids from hybrid papyrus leaves by room temperature immersion extraction method - extraction time

[0165] The only difference from Example 13 is that the extraction temperature is 30°C, the soaking extraction time is 1h, 2h, 4h, 6h and 8h, respectively, and the extraction yields of total flavonoids from the leaves of hybrid paper mulberry are 5.23±0.05%, 5.24±0.02%, 5.25±0.04%, 5.27±0.07% and 5.27±0.03%, respectively.

[0166] Embodiment 15

[0167] Extraction of total flavonoids from leaves of hybrid papyrus purpurogenus by room temperature immersion extraction - solid-liquid ratio

[0168] The only difference from Example 13 is that the extraction temperature is 30°C, the solid-liquid ratios are 1g:5mL, 1g:10mL, 1g:15mL, 1g:20mL and 1g:25mL, and the extraction yields of total flavonoids from the leaves of hybrid paper mulberry are 4.97±0.05%, 4.99±0.03%, 5.04±0.05%, 5.05±0.04% and 5.01±0.02%, respectively.

[0169] Example 16

[0170] Total flavonoids extracted from hybrid papyrus leaves by room temperature immersion extraction - ethanol volume fraction

[0171] The only difference from Example 13 is that the extraction temperature is 30°C, the volume fraction of ethanol is 65%, 70%, 75%, 80% and 85%, respectively, and the extraction yields of total flavonoids from the leaves of hybrid paper mulberry are 4.76±0.04%, 4.76±0.05%, 4.77±0.03%, 4.78±0.06% and 4.75±0.05%, respectively.

[0172] Embodiment 17

[0173] Extraction of total flavonoids from leaves of hybrid papyrus by soaking extraction at room temperature

[0174] The total flavonoids from the leaves of hybrid paper mulberry were extracted according to the method of Example 13. The extraction conditions were as follows: the volume fraction of ethanol was 75%, the solid-liquid ratio was 1 g:20 mL, the soaking extraction time was 6.0 h, the extraction temperature was 25°C, and the extraction yield of the total flavonoids from the leaves of hybrid paper mulberry was 5.22±0.27%.

[0175] Test Example 1

[0176] Testing of total flavonoid solutions from different parts of hybrid paper mulberry (the total flavonoid solutions from the roots of hybrid paper mulberry prepared in Examples 1 to 6, the total flavonoid solutions from the stems of hybrid paper mulberry prepared in Examples 7 to 12, and the flavonoid solutions from the stems and leaves of hybrid paper mulberry prepared in Examples 13 to 17).

[0177] 1. Observation of apparent characteristics

[0178] Take equal amounts of total flavonoids solution from the roots of hybrid paper mulberry, total flavonoids solution from the stems of hybrid paper mulberry, and flavonoids solution from the stems and leaves of hybrid paper mulberry, respectively, place them in 3 transparent glass ampoule bottles of the same size, and take photos using a high-definition digital camera under natural light.

[0179] Figure 1 The total flavonoids solutions of different parts of hybrid paper mulberry are shown in Figure 1, where A is the total flavonoids of hybrid paper mulberry roots, B is the total flavonoids of hybrid paper mulberry stems, and C is the total flavonoids of hybrid paper mulberry leaves. It can be seen that the total flavonoids solutions of the same volume of hybrid paper mulberry roots, stems, and leaves show a certain dark red and yellow color visible to the naked eye ( Figure 1Middle A), dark yellow with a green tint ( Figure 1 Middle B), dark green with a black tint ( Figure 1 In addition, after the sealed volumetric bottle was opened in a windless room, multiple people olfactory identification showed that the total flavonoid solutions of the hybrid paper mulberry roots, stems, and leaves all contained a more easily odorous volatile ethanol odor, and as the number of olfactory detections increased, the odor was somewhat similar to volatile methanol.

[0180] 2. pH value determination

[0181] After installing the instrument and electrodes, prepare pH = 4.00, pH = 6.86, pH = 9.18KH 2 PO 4 The electrode was calibrated with standard buffer solution, the protective bottle at the lower end of the electrode was removed, the rubber cover at the upper end of the electrode was pulled down to expose the small hole at the upper end, the electrode was cleaned with Wahaha purified water, and then the pH value of the total flavonoids solution in different parts of the hybrid paper mulberry was measured.

[0182] Figure 2 This is the result graph of the pH value of the total flavonoids solution in different parts of hybrid paper mulberry. It can be seen that the pH values ​​of the total flavonoids solution in different parts of hybrid paper mulberry are: stem>root>leaf, all greater than 6.3 and less than 7.0, but the difference is not significant (P>0.05).

[0183] 3. Conductivity value determination

[0184] After installing the instrument and electrodes, prepare 1408μS / cm KCl standard solution, put it in a 25℃ constant temperature water bath, control the solution temperature to 25.0℃, remove the protective bottle at the lower end of the electrode, clean the electrode with Wahaha purified water, and then measure the pH value of the total flavonoids solution from different parts of the hybrid paulownia.

[0185] Figure 3 This is the result graph of the conductivity values ​​of the total flavonoid solutions in different parts of hybrid paper mulberry. It can be seen that there is almost no significant difference in the conductivity values ​​of the total flavonoid solutions in different parts of hybrid paper mulberry (P>0.05), and they are all greater than 966μs / cm and less than 1000μs / cm.

[0186] 4. Determination of antioxidant index

[0187] 4.1 DPPH free radical scavenging activity

[0188] Dissolve DPPH powder in anhydrous ethanol to obtain a DPPH solution with a concentration of 0.1mmol / L. Sample group: Add 1.0mL of total flavonoid solutions from different parts of hybrid papyrus to 10mL brown volumetric flasks with concentrations of 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL and 0.5mg / mL, respectively, and then add 5mL of DPPH solution, and finally dilute to 10mL with anhydrous ethanol and shake to mix. Blank group: Replace the total flavonoid solutions from different parts of hybrid papyrus in the sample group with anhydrous ethanol. Let the sample group and the blank group stand in the dark for 30min. Determine its absorbance at an ultraviolet wavelength of 517nm. Calculate its DPPH free radical scavenging rate, and the calculation formula is as follows: DPPH free radical scavenging rate (%) = (A1-A0) ÷ A1 × 100%, where A0 is the absorbance of the sample group; A1 is the absorbance of the blank group. Each sample was tested in parallel 5 times.

[0189] Figure 4 The results of the scavenging rate of total flavonoids solutions in different parts of hybrid paper mulberry on DPPH free radicals show that the scavenging rate of total flavonoids solutions in different parts of hybrid paper mulberry on DPPH free radicals under the same and different concentration conditions has different degrees of difference. With the increase of concentration, the scavenging rate of total flavonoids solutions in roots and stems on DPPH free radicals showed a slight downward and increasing trend, respectively, while the leaves showed an extremely obvious downward trend. When the concentration was 0.1 mg / mL, the scavenging rate of DPPH free radicals in roots and leaves was equivalent and higher than that in stems, but the difference among the three was not significant (P>0.05); when 0.2 mg / mL≤total flavonoid concentration≤0.5 mg / mL, the scavenging rate of total flavonoids solutions in roots, stems and leaves on DPPH free radicals under the same concentration conditions was extremely significantly (P<0.01) higher than that in leaves, and followed the rule of "stem ≥root>leaf".

[0190] 4.2 ABTS free radical scavenging activity

[0191] Mix 50 mL of 7 mmol / L ABTS solution (solvent is anhydrous ethanol) and 2.5 mmol / L potassium persulfate aqueous solution evenly, and let stand at room temperature in the dark for 14 to 16 hours to obtain ABTS stock solution. Dilute the ABTS stock solution with distilled water until the absorbance at 734 nm is 0.7±0.02, and then obtain ABTS working solution. Sample group: Take 1.0 mL of total flavonoid solutions from different parts of hybrid papyrus (solvent is anhydrous ethanol) with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL and 0.5 mg / mL respectively in a brown volumetric flask, and then add 4.0 mL of ABTS working solution. Blank group: Replace the total flavonoid solutions from different parts of hybrid papyrus in the sample group with deionized water. After the sample group and the blank group were allowed to stand in a dark environment for 6 minutes, their absorbance at a wavelength of 734 nm was measured. The ABTS scavenging rate was calculated as follows: ABTS free radical scavenging rate (%) = (1-A0 / A1) × 100%, where A0 is the absorbance of the sample group and A1 is the absorbance of the blank group.

[0192] Figure 5 The figure shows the scavenging rate of ABTS free radicals by total flavonoids solutions from different parts of hybrid paper mulberry. It can be seen that under the same concentration conditions, the scavenging rate of ABTS free radicals by total flavonoids solutions from different parts of hybrid paper mulberry is generally very significant (P < 0.01), showing the result of roots > stems ≥ leaves. In addition, it was found that with the increase of total flavonoids concentration, the scavenging rate of ABTS free radicals in different parts showed an increasing trend, especially in stems and leaves, while the scavenging rate of ABTS free radicals tended to be stable when the total flavonoids concentration in the root was ≥ 0.3 mg / mL.

[0193] 5. Scanning electron microscope observation

[0194] The total flavonoids solution from different parts of hybrid paper mulberry was passed through a HPD600 macroporous resin chromatography column with a particle size of 0.3-0.5 mm, eluted with anhydrous ethanol, concentrated by rotary evaporation at 70-75°C, and freeze-dried in a vacuum at -40°C to constant weight, and then tested using a scanning electron microscope (accelerating voltage of 12 kV).

[0195] Figure 6 This is the SEM image of total flavonoids from the root of hybrid paper mulberry prepared in Example 7. Figure 7 This is the SEM image of total flavonoids in the stem of hybrid paper mulberry prepared in Example 14. Figure 8This is the SEM image of the total flavonoids in the leaves of hybrid paper mulberry prepared in Example 17. According to the SEM image, it can be intuitively and clearly seen that under the same scale field of view, the solid morphology of the total flavonoids in different parts of hybrid paper mulberry is extremely different: except for a very small number of small pieces, the total flavonoids in the roots are in the form of large lumps with a length of 6 μm and a width of more than 2.5 μm, which are superimposed on each other; the total flavonoids in the stems are in the form of sticky blocks of different lengths, widths and shapes, and most of them are small blocks; the total flavonoids in the leaves are almost in the form of large plates similar to fish scale patterns.

[0196] Scanning electron microscopes are often used to detect the microscopic surface structural characteristics of samples because of their advantages such as simple sample preparation, microscopic magnification that cannot be achieved by the naked eye, high resolution, and three-dimensional image perception. Most flavonoid compounds are crystalline solids, and a few (such as flavonoid glycosides) are amorphous powders. Analyzing the results of this study, it is not difficult to find that the total flavonoid block / particle size in different parts of hybrid paper mulberry is: leaf < stem < root. Obviously, this is related to the solid shape of flavonoid compounds. The looseness of the solid morphology of total flavonoids in different parts of hybrid paper mulberry is: root < leaf < stem, which may be related to the content of flavonoid glycosides in different parts, and the sugar molecular structure contained in flavonoid glycosides, resulting in different adhesion states.

[0197] The present invention uses the total flavonoids solution extracted from the roots, stems and leaves of hybrid paper mulberry as raw material, and systematically studies the physicochemical properties of the total flavonoids solution from the roots, stems and leaves of hybrid paper mulberry through a combination of physical and chemical methods, in order to provide strong physical and chemical data support and scientific basis for the further development and application of total flavonoids from hybrid paper mulberry in functional traditional Chinese medicine, beauty products and health care products.

[0198] Embodiment 18

[0199] Detection of total flavonoid solutions from different parts of hybrid paper mulberry prepared in Examples 6, 12 and 17.

[0200] Ultra-high performance liquid chromatography-tandem high-resolution orbitrap mass spectrometry (UHPLC-QE, Thermo, USA) was used to detect metabolites in the samples. The total flavonoids in different parts of hybrid paper mulberry were qualitatively and quantitatively analyzed by comparing the retention time and molecular mass (molecular mass error <10ppm) with those of the standard.

[0201] Preparation of standard solution: Add the standard to a 80v / v% methanol-0.2wt% vitamin C-water mixed solution, vortex mix and ultrasonically extract for 30 minutes, centrifuge at 12000r / min for 10 minutes to remove impurities, take the supernatant and repeat the above steps once to ensure complete dissolution; combine the two supernatants to obtain a single standard stock solution with a concentration of 10mg / mL. Take each single standard stock solution and mix it, use anhydrous ethanol as a diluent, and prepare a mixed standard stock solution with a concentration of 1μg / mL and 10μg / mL, and store it for later use. Use anhydrous ethanol as a diluent to dilute the mixed standard stock step by step to obtain a mixed standard linear solution. The concentration of the mixed standard linear solution is shown in Table 2.

[0202] Test sample solution: Accurately measure the total flavonoid solution samples from different parts of hybrid paper mulberry, centrifuge at 12000r / min for 10min, and the supernatant is the test sample solution.

[0203] The sample solution to be tested and the mixed standard linear solution were respectively subjected to high performance liquid chromatography-tandem mass spectrometry detection. Liquid chromatography parameters: chromatographic column: Waters HSS T3 (50×2.1mm, 1.8μm); mobile phase A is 0.1v / v% formic acid-ultrapure water, mobile phase B is 0.1v / v% formic acid-acetonitrile; mobile phase flow rate is 0.3mL / min; column temperature is 40℃; injection volume is 2μL; elution mode is gradient elution, and the gradient elution program is shown in Table 1; the samples are placed in the 4℃ automatic sampler during the entire analysis process. In order to avoid the influence of fluctuations in instrument detection signals, the continuous analysis of samples is carried out in random order. QC samples are evenly inserted into the sample analysis queue to monitor and evaluate the stability of the system and the reliability of experimental data. Mass spectrometry conditions: The data were collected using the QExactive high-resolution mass spectrometry detection system from Thermo Corporation of the United States; it was equipped with an electrospray ionization source (ESI), a sheath gas pressure of 40 arb, an auxiliary gas pressure of 10 arb, an ion spray voltage of -2800 V, an ion spray temperature of 350°C, and an ion transfer tube temperature of 320°C; the scan mode was Full scan-ddMS2, and the scan mode was negative ion; the primary mass spectrometry scan range (scan m / z range) was 100-900.

[0204] Qualitative results: The original spectrum obtained by UPLC-Q Exactive / MS analysis was pre-processed by using TraceFinder software for baseline filtering, peak identification, peak matching, retention time correction, peak alignment, etc., to obtain a data matrix containing retention time, mass-to-charge ratio and peak intensity. The total ion flow chromatogram (TIC) is a spectrum obtained by summing up the intensities of all ions in the mass spectrum at each time point and then continuously plotting them. The horizontal axis is the retention time (Time, min) and the vertical axis is the ion flow intensity (Intensity) of ion detection.

[0205] Fig. 9 is the total ion current of the standard product, Fig.10 This is the total ion current diagram of total flavonoids in the roots of hybrid paper mulberry. Fig.11 This is the total ion current diagram of total flavonoids in the stem of hybrid paper mulberry. Fig.12 The total ion current diagram of total flavonoids in the leaves of hybrid paper mulberry is Figures 9 to 12 It can be seen that the total flavonoids of the hybrid paper mulberry root, the total flavonoids of the hybrid paper mulberry stem and the total flavonoids of the hybrid paper mulberry leaf extracted by the present invention all contain kaempferol, vitexin, apigenin, dihydroquercetin, dihydrokaempferol, quercetin, rutin, luteolin, naringenin, naringin chalcone, catechin, epicatechin, dihydromyricetin, isorhamnetin, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, and quercetin-3-O-glucoside. glucoside, trans-ferulic acid, 4-hydroxybenzoic acid, hydrocinnamic acid, trans-cinnamic acid, vanillic acid, vanillin, gallic acid, benzoic acid, salicylic acid, sinapic acid, syringic acid, syringaldehyde, protocatechuic acid, caffeic acid, p-coumaric acid, protocatechuic aldehyde, resveratrol, phthalic acid, isoliquiritigenin, daidzein, phloretin, 4',5,7-trigenin-7-glucoside, luteolin and gossypol.

[0206] Quantitative results: The chromatograms of each mixed standard solution were fitted using TraceFinder software to obtain the standard curve, and then the compounds were absolutely quantified using the external standard method.

[0207] Fig.13 This is the standard curve of gallic acid and protocatechuic acid. Fig.14 is the standard curve of phthalic acid and protocatechuic aldehyde, Fig.15 is the standard curve of 4-hydroxybenzoic acid and caffeic acid, Fig.16 This is the standard curve of catechins and vanillic acid. Fig.17 is the standard curve of dihydromyricetin and syringic acid, Fig.18 This is the standard curve of epicatechin and syringaldehyde. Fig.19 This is the standard curve of vanillin and p-coumaric acid. Fig. 20 is the standard curve of trans-ferulic acid and rutin, Fig.21is the standard curve of vitexin and luteolin, Fig. 22 This is the standard curve of sinapinic acid and salicylic acid. Fig.23 is the standard curve of dihydroquercetin and quercetin-3-O-glucoside, Fig.24 This is the standard curve of 4',5,7-trigenisoflavone-7-glucoside and dihydrokaempferol. Fig.25 is the standard curve of benzoic acid and kaempferol-3-O-glucoside, Fig.26 is the standard curve of luteolin and resveratrol, Fig. 27 This is the standard curve of daidzein and trans-cinnamic acid. Fig.28 is the standard curve of quercetin and hydrocinnamic acid, Fig.29 This is the standard curve of phloretin and naringin chalcone. Fig.30 is the standard curve of naringenin and isorhamnetin, Fig.31 This is the standard curve of apigenin and kaempferol. Fig.32 is the standard curve of gossypol and isoliquiritigenin. Figure 13 to Figure 32 It can be seen that the linear relationship of each standard product is good.

[0208] The original data exported by the software were calculated to obtain the content of various chemical components in the total flavonoids solution of different parts of hybrid paper mulberry. The results are shown in Table 4.

[0209] Table 4 Quantitative detection results of various chemical components in total flavonoid solutions from different parts of hybrid paper mulberry (ng / mL)

[0210]

[0211]

[0212] Sample quality control: During the instrument analysis process, quality control samples (Quality Control, referred to as QC) are evenly inserted into the analysis sample sequence (QC is provided when the number of samples> 20), and the relative standard deviation (RSD) of the QC samples is calculated to determine the stability of the instrument during the detection process. Generally, when RSD<30%, it means that the detection system is relatively stable, which provides an important guarantee for the reliability and repeatability of the data. The sample quality control results are shown in Fig.33 and Table 5.

[0213] Table 5 Relative standard deviation of quality control samples

[0214]

[0215] Depend on Fig.33 As can be seen from Table 5, the detection method provided by the present invention is stable, and the detection data has high reliability and good repeatability.

[0216] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A hybrid paper mulberry total flavonoids, including one or more of the total flavonoids of hybrid paper mulberry root, hybrid paper mulberry stem and hybrid paper mulberry leaf; the chemical components of the hybrid paper mulberry total flavonoids include kaempferol, vitexin, apigenin, dihydroquercetin, dihydrokaempferol, quercetin, rutin, luteolin, naringenin, naringin chalcone, catechin, epicatechin, dihydromyricetin, isorhamnetin, kaempferol-3-O-glucose glycosides, quercetin-3-O-glucoside, trans-ferulic acid, 4-hydroxybenzoic acid, hydrocinnamic acid, trans-cinnamic acid, vanillic acid, vanillin, gallic acid, benzoic acid, salicylic acid, sinapic acid, syringic acid, syringaldehyde, protocatechuic acid, caffeic acid, p-coumaric acid, protocatechuic aldehyde, resveratrol, phthalic acid, isoliquiritigenin, daidzein, phloretin, 4',5,7-trigenin-7-glucoside, luteolin and gossypol.

2. The method for extracting total flavonoids from hybrid paper mulberry according to claim 1, comprising one or more of the methods for extracting total flavonoids from the roots of hybrid paper mulberry, the stems of hybrid paper mulberry, and the leaves of hybrid paper mulberry; The method for extracting total flavonoids from the roots of hybrid paper mulberry comprises the following steps: The hybrid paper mulberry root powder is first extracted with a first petroleum ether to obtain a hybrid paper mulberry root defatted powder; the hybrid paper mulberry root defatted powder is subjected to microwave-assisted extraction with a first lower alcohol aqueous solution to obtain total flavonoids from the hybrid paper mulberry root; the volume fraction of the lower alcohol in the first lower alcohol aqueous solution is 40 to 80%; The method for extracting total flavonoids from hybrid paper mulberry stems comprises the following steps: performing a second extraction on hybrid paper mulberry stem powder using a second petroleum ether to obtain hybrid paper mulberry stem defatted powder; performing ultrasonic-assisted extraction on the hybrid paper mulberry stem defatted powder using a second lower alcohol aqueous solution to obtain total flavonoids from hybrid paper mulberry stems; the volume fraction of the lower alcohol in the second lower alcohol aqueous solution is 40-80%; The method for extracting total flavonoids from the leaves of hybrid paper mulberry includes the following steps: performing a third extraction on hybrid paper mulberry leaf powder using a third petroleum ether to obtain hybrid paper mulberry leaf defatted powder; soaking and extracting the hybrid paper mulberry leaf defatted powder using a third lower alcohol aqueous solution to obtain total flavonoids from the leaves of hybrid paper mulberry; the volume fraction of the lower alcohol in the third lower alcohol aqueous solution is 65-85%.

3. The extraction method according to claim 2, characterized in that The ratio of the dry weight of the hybrid paper mulberry root defatted powder to the volume of the first lower alcohol aqueous solution is 1g:30-70mL; The lower alcohol in the first lower alcohol aqueous solution includes methanol and / or ethanol.

4. The extraction method according to claim 3 or 4, characterized in that The microwave power of the microwave-assisted extraction is 400-600W, the temperature is 40-80°C, and the time is 5-25min.

5. The extraction method according to claim 2, characterized in that The ratio of the dry weight of the hybrid paper mulberry stem defatted powder to the volume of the second lower alcohol aqueous solution is 1 g: 10-30 mL; The lower alcohol in the second lower alcohol aqueous solution includes methanol and / or ethanol.

6. The extraction method according to claim 2 or 5, characterized in that The ultrasonic assisted extraction has an ultrasonic power of 300 to 1100 W, a temperature of 30 to 70° C., and a time of 1 to 3 hours.

7. The extraction method according to claim 6, characterized in that The ratio of the dry weight of the defatted hybrid paper mulberry leaf powder to the volume of the third lower alcohol aqueous solution is 1 g: 5-25 mL; The lower alcohol in the third lower alcohol aqueous solution includes methanol and / or ethanol.

8. The extraction method according to claim 2 or 7, characterized in that The soaking extraction is carried out at a temperature of 20 to 40° C. and for a time of 1 to 8 hours.

9. Use of the hybrid paper mulberry total flavonoids according to claim 1 or the hybrid paper mulberry total flavonoids obtained by the extraction method according to any one of claims 2 to 8 in cosmetics, health products or preparation of medicines.

10. A method for detecting total flavonoids in hybrid paper mulberry, characterized in that: The following steps are involved: The lower alcohol aqueous solution of total flavonoids of hybrid paper mulberry is subjected to high performance liquid phase tandem mass spectrometry detection to obtain the detection result of total flavonoids of hybrid paper mulberry; The total flavonoids of hybrid paper mulberry include one or more of kaempferol, vitexin, apigenin, dihydroquercetin, dihydrokaempferol, quercetin, rutin, luteolin, naringenin, naringin chalcone, catechin, epicatechin, dihydromyricetin, isorhamnetin, kaempferol-3-O-glucoside, quercetin-3-O-glucoside, trans-ferulic acid, 4-hydroxybenzoic acid, hydrocinnamic acid, trans-cinnamic acid, vanillic acid, vanillin, gallic acid, benzoic acid, salicylic acid, sinapinic acid, syringic acid, syringaldehyde, protocatechuic acid, caffeic acid, p-coumaric acid, protocatechuic aldehyde, resveratrol, phthalic acid, isoliquiritigenin, daidzein, phloretin, 4',5,7-trihydroxyisoflavone-7-glycoside, luteolin and gossypol; The HPLC separation conditions for the HPLC tandem mass spectrometry detection include: the chromatographic column is a reverse phase chromatographic column, the mobile phase system includes mobile phase A and mobile phase B; the mobile phase A is a formic acid aqueous solution, and the volume fraction of formic acid in the formic acid aqueous solution is 0.1-0.5%; the mobile phase B is a formic acid acetonitrile solution, and the volume fraction of formic acid in the formic acid acetonitrile solution is 0.1-0.5%; the elution mode is gradient elution, and the gradient elution procedure is as follows: 0-2min, the volume fraction of the mobile phase A is 90%; 2-6 min, the volume fraction of the mobile phase A is reduced from 90% to 40%; 6-9 min, the volume fraction of the mobile phase A is 40%; 9-9.1 min, the volume fraction of the mobile phase A increases from 40% to 90%; 9.1-12min, the volume fraction of the mobile phase A is 90%; The mass spectrometry detection conditions of the high performance liquid phase tandem mass spectrometry detection include: the ion source is an electrospray ion source, the ion spray voltage is -2800V, the electrospray temperature is 350°C, and the ion transfer tube temperature is 320°C; the scanning mode is Full scan-ddMS2, the scanning method is negative ion, and the primary mass spectrometry scanning range is 100-900m / z.

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