Method for quantitatively detecting various active components in mulberry leaves

Abstract

The invention discloses a method for quantitatively detecting various active components in mulberry leaves, and particularly relates to the technical field of active component detection, the method comprises the following steps: (1) carrying out reflux extraction and concentration on mulberry leaves to obtain a pasty mulberry leaf extract; (2) extracting the pasty mulberry leaf extract obtained in the step (1) step by step, and respectively purifying to obtain a purified alkaloid extracting solution, a purified organic acid extracting solution and a purified flavone extracting solution in sequence; and (3) on the basis of UHPLC-ESI-Q-TOF / MS analysis, carrying out quantitative detection on the active components in the purified extracting solution obtained in the step (2). The method provided by the invention realizes gradient extraction of alkaloid, organic acid and flavonoid components in mulberry leaves and rapid detection of mulberry leaf components, and improves the sensitivity and accuracy of active component determination.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of active component detection, in particular to a method for quantitatively detecting multiple active components in mulberry leaves. BACKGROUND

[0002] Mulberry leaves are the leaves of Morus alba Linn, bitter, sweet, cold, lung, liver, and the effect of dispersing wind-heat, clearing liver and improving eyesight, first recorded in Shennong Bencao Jing. It has a bitter and sweet smell, and is slightly toxic. It is mainly used for cold and heat sweating. Mulberry leaves, as a medicinal and edible material, are included in the 2015 edition of Chinese Pharmacopoeia. Studies have found that mulberry leaves contain active substances such as alkaloids, flavonoids and organic acids. Among them, alkaloids have analgesic, anti-inflammatory and antibacterial effects, and can relieve symptoms such as cold and cough. Flavonoids have antioxidant, anti-aging and anti-inflammatory effects, and can improve skin problems and improve immunity. Organic acids have antioxidant, anti-inflammatory and antibacterial effects.

[0003] The detection of active components in mulberry leaves is of great significance to the fields of clarifying pharmacological mechanisms and functional verification, developing and producing medicinal or food products using mulberry leaves as raw materials, and optimizing mulberry extraction process. CN101806783A discloses a rapid determination method for active components in mulberry tea or mulberry leaves, which discloses a technical solution for determining active components in mulberry leaves by gas chromatography-mass spectrometry. However, in actual operation, due to the detection characteristics of gas chromatography, it is necessary to ensure that the sample to be detected remains stable at high temperature. However, active substances such as flavonoids will be damaged at high temperature, resulting in poor sensitivity of detection and affecting the reliability and accuracy of active component detection. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the above-mentioned defects existing in the prior art, and to provide a method for quantitatively detecting multiple active components in mulberry leaves, which realizes gradient extraction of alkaloids, organic acids and flavonoids in mulberry leaves, rapid detection of mulberry leaf components, and improves the sensitivity and accuracy of active component determination.

[0005] The technical solution adopted by the present application to solve its technical problem is as follows: a method for quantitatively detecting multiple active components in mulberry leaves, comprising the following steps: (1) refluxing and extracting mulberry leaves, concentrating the obtained mulberry leaf extract to obtain a paste-like mulberry leaf extract; (2) stepwise extracting the paste-like mulberry leaf extract obtained in step (1), and sequentially obtaining purified alkaloid extract, purified organic acid extract and purified flavonoid extract after purification treatment; (3) Based on UHPLC-ESI-Q-TOF / MS analysis, the active components in the purified extract obtained in step (2) are quantitatively detected; wherein, in the purified alkaloid extract, the alkaloid active components include Stizolobine, Noracohyoscine and 1-Deoxynojirimycin; in the purified organic acid extract, the organic acid active components include Chlorogenic acid, p-Coumaric acid, Caffeic acid and Quinic acid; in the purified flavonoid extract, the flavonoid active components include Rutin, Kaempferol, Quercetin, Isoquercitrin and Quercetin-3-triglucoside.

[0006] Preferably, in step (1), the specific method of reflux extraction is: after washing, drying and crushing the mulberry leaves, adding an alcohol solvent, performing reflux extraction, cooling to room temperature, centrifuging, and reserving the supernatant to obtain a mulberry leaf extract. The method of the present application repeatedly extracts the mulberry leaves with the alcohol solvent by reflux extraction to promote the dissolution of water-soluble and fat-soluble components such as flavonoids, organic acids and alkaloids, so as to achieve the effect of complete extraction of active components.

[0007] Preferably, the mass-volume ratio g / mL of the mulberry leaves to the alcohol solvent is 1:8-12.

[0008] Preferably, the temperature of the reflux extraction is 80-90℃, and the time is 2-5h. During the reflux extraction, the alcohol solvent is distilled out and then treated by condensation to return to the extraction liquid.

[0009] Preferably, the centrifugation speed is 2000-4000r / min, and the time is 2-4min.

[0010] Preferably, the alcohol solvent includes a methanol aqueous solution with a volume fraction of 50-90% and / or an ethanol aqueous solution with a volume fraction of 40-70%, etc. As the concentration increases, the extraction effect is better, thereby avoiding the case that when the concentration is too low, the solubility of active components is insufficient, leading to incomplete extraction, and also avoiding the case that when the concentration is too high, impurities are also extracted, reducing the purity and extraction rate of active components, and further affecting the subsequent detection results.

[0011] Preferably, in step (1), the mulberry leaf extract is concentrated by evaporation.

[0012] Preferably, in step (1), the concentration temperature is 60-80℃, and the concentration is stopped when the volume is 1 / 10 of the original volume. The method of the present application uses concentration treatment to remove the solvent and enrich the active components, and facilitates subsequent separation.

[0013] Preferably, in step (2), the specific method of stepwise extraction includes the following steps: a. Dissolve the paste-like mulberry leaf extract with dilute hydrochloric acid, then add chloroform with the same volume as the dilute hydrochloric acid, extract, centrifuge, and obtain a mixed extract; b. In the mixed extract obtained in step a, add ammonia water to adjust the pH value to alkaline, add chloroform with the same volume as the mixed extract obtained in step a, extract, shake up and down, then separate the layers to obtain a chloroform layer and a water layer, and the chloroform layer is marked as the alkaloid extract; c. In the water layer obtained in step b, add dilute hydrochloric acid with the same volume, then in the mixed solution after adding the dilute hydrochloric acid, add ethyl acetate with the same volume, extract, shake up and down, then separate the layers to obtain an ethyl acetate layer and a water layer, and the ethyl acetate layer is marked as the organic acid extract; d. In the water layer obtained in step c, add sodium hydroxide solution to adjust the pH value to alkaline, then centrifuge to obtain the flavone extract.

[0014] In step a, the purpose of treating with dilute hydrochloric acid is to convert the active substances in the mulberry leaf extract into salts for precipitation.

[0015] In step b, the purpose of adding ammonia water is to free the alkaloids.

[0016] In step c, the purpose of adding dilute hydrochloric acid is to promote the free precipitation of organic acids.

[0017] In step d, the purpose of adding sodium hydroxide is to promote the free precipitation of flavone components.

[0018] Preferably, in step a, the mass-volume ratio g / mL of the paste-like mulberry leaf extract to dilute hydrochloric acid is 1:1-3.

[0019] Preferably, in step a, the concentration of the dilute hydrochloric acid is 0.01-0.03 mol / L.

[0020] Preferably, in step a, the extraction time is 10-25 min.

[0021] Preferably, in step a, the centrifugation speed is 5000-6000 r / min, and the time is 2-3 min.

[0022] Preferably, in step b, the mass fraction of the ammonia water is 10-25%.

[0023] Preferably, in step b, the pH value is adjusted to 10-11.

[0024] Preferably, in step b, the extraction time is 10-25 min.

[0025] Preferably, in step b, the separation time is 20-40 min.

[0026] Preferably, in step c, the concentration of the dilute hydrochloric acid is 0.01-0.03 mol / L.

[0027] Preferably, in step c, the extraction time is 10-25 min.

[0028] Preferably, in step c, the standing and layering time is 20-40 min.

[0029] Preferably, in step d, the concentration of the sodium hydroxide solution is 0.1-1.0 mol / L.

[0030] Preferably, in step d, the pH value is adjusted to 8-9.

[0031] Preferably, in step (2), the purification treatment refers to membrane filtration with a filter membrane having a pore size of ≤0.22 μm. The solvent and macromolecular impurities in the extract are removed by membrane filtration.

[0032] Preferably, in step (3), the method for quantitatively detecting the active components in the purified extract is as follows: A standard curve is established: the active components are selected as standard products, and standard product solutions of different concentrations are prepared using methanol as a solvent; Under the same chromatographic and mass spectrometric conditions, each concentration standard product solution with a volume of V1 is injected into an ultra-high performance liquid chromatograph, and the chromatographic peaks of each active component are determined according to the characteristic ions of each active component by mass spectrometric detection, and the chromatographic peak areas of each active component are obtained; The standard curves of each active component are respectively established with the chromatographic peak areas of each active component as the ordinate and the concentrations of each active component as the abscissa; The chromatographic peak areas of each active component in the purified extract are obtained: under the same chromatographic and mass spectrometric conditions, the purified extract with a volume of V1 is injected into an ultra-high performance liquid chromatograph, and the chromatographic peaks of each active component are determined according to the characteristic ions of each active component by mass spectrometric detection, and the chromatographic peak areas of each active component are obtained; The content of each active component in the purified extract is determined: a standard curve of peak area Y versus concentration x (ng / mL) is plotted, and the content of the active component in the mulberry leaf is determined based on the linear equation obtained from the aforementioned standard curve.

[0033] Preferably, the standard product solutions for quantitatively detecting the alkaloid active components in the purified alkaloid extract are as follows: fagopyrin, noranisodrine and 1-deoxynojirimycin are selected as standard products, and fagopyrin standard product solutions with a concentration of 1-1000 ng / mL, noranisodrine standard product solutions with a concentration of 1-800 ng / mL and 1-deoxynojirimycin standard product solutions with a concentration of 0.2-2000 ng / mL are prepared using methanol as a solvent.

[0034] Preferably, the standard solution for quantitative detection of the active components of the purified organic acid extract is prepared by selecting chlorogenic acid, p-hydroxy cinnamic acid, caffeic acid and quinic acid as the standard, and preparing a chlorogenic acid standard solution with a concentration of 0.1-1000 ng / mL, a p-hydroxy cinnamic acid standard solution with a concentration of 2-700 ng / mL, a caffeic acid standard solution with a concentration of 1-600 ng / mL, and a quinic acid standard solution with a concentration of 10-2000 ng / mL, using methanol as the solvent.

[0035] Preferably, the standard solution for quantitative detection of the active components of the purified flavonoid extract is prepared by selecting rutin, kaempferol, quercetin, isoquercitrin and quercetin-3-triglucoside as the standard, and preparing a rutin standard solution with a concentration of 1-800 ng / mL, a kaempferol standard solution with a concentration of 1-600 ng / mL, a quercetin standard solution with a concentration of 2-500 ng / mL, an isoquercitrin standard solution with a concentration of 1-700 ng / mL, and a quercetin-3-triglucoside standard solution with a concentration of 10-1000 ng / mL, using methanol as the solvent.

[0036] Preferably, the detection wavelengths for the chromatography are 255 nm, 320 nm and 278 nm for the active components of the purified alkaloid extract, organic acid extract and flavonoid extract, respectively. Considering that alkaloids, organic acids and flavonoids have maximum absorption at 255 nm, 320 nm and 278 nm, respectively, the detection wavelengths of the UHPLC are set to 255 nm, 320 nm and 278 nm, respectively, for quantitative detection and analysis of the alkaloid extract, organic acid extract and flavonoid extract, to obtain more accurate and reliable detection results.

[0037] Preferably, the chromatography conditions are as follows: Agilent 1290 Infinity ultra-high performance liquid chromatograph; Waters ACQUITY UPLC BEH C18 chromatographic column; Column temperature 30°C, flow rate 0.3 mL / min, injection volume 1 μL; Detection wavelength 250-320 nm; The mobile phase is a water solution containing 0.1% formic acid as phase A and acetonitrile as phase B, and gradient elution is performed with the following program: elution time 0-40 min, phase A 95-5% and phase B 5-95%; The mass spectrometry conditions are as follows: Agilent 6530 quadrupole-time-of-flight tandem mass spectrometer; ESI ion source, data collected in positive and negative ion modes, data collection range m / z 100-1700; Ion source temperature 350℃, capillary voltage 3.5kV in positive ion mode, 4.0kV in negative ion mode; Atomization gas pressure 45Psi, drying gas flow rate 11L / min, sheath gas flow rate 11L / min, sheath gas temperature 350°C, fragment voltage 140V.

[0038] The method has the following beneficial effects: (1) The method is based on step-by-step extraction processing, and a continuous extraction and separation system is established for active ingredients in mulberry leaves, so that gradient extraction of alkaloids, organic acids and flavonoids in mulberry leaves is realized, the problem of long time consumption and inconvenient operation for detecting various active components in mulberry leaves in a single component extraction mode is solved, and the sensitivity and accuracy of active component determination are further improved by enriching the active ingredients during separation; (2) The method sets a standard product with a known concentration as a reference for quantitative analysis during detection, measures the response signals of the standard product and the sample, and compares them by using a standard curve to accurately calculate the concentration of each component in the sample, thereby ensuring the accuracy and reliability of the detection of each active component in mulberry leaves. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is the total ion chromatogram of the active components in the mulberry leaves of Example 1 of the present application; Figure 2 is the total ion chromatogram of the active components in the mulberry leaves of Example 2 of the present application; Figure 3 is the total ion chromatogram of the active components in the mulberry leaves of Example 3 of the present application. DETAILED DESCRIPTION

[0040] The present application will be further described below in conjunction with examples and drawings.

[0041] The mulberry leaves 1, 2 and 3 used in the examples of the present application are commercially available; the raw materials, equipment or chemical reagents used in the examples of the present application are obtained through conventional commercial channels, such as Agilent 1290 Infinity ultra-high performance liquid chromatography (UHPLC) and Agilent 6530 quadrupole-time-of-flight tandem mass spectrometer (Q-TOF), unless otherwise specified.

[0042] Example 1 (1) The mulberry leaves 1 were subjected to reflux extraction, and the obtained mulberry leaf extract was concentrated to 1 / 10 of the original volume at 70℃ by evaporation method, and then stopped, to obtain a paste-like mulberry leaf extract; the solvent was removed by concentration treatment, and the active ingredients were enriched, which facilitated subsequent separation; The specific method of the reflux extraction is as follows: after cleaning, drying and crushing the mulberry leaves, the mulberry leaves are added into 70% methanol aqueous solution with a mass-volume ratio of 1:8 g / mL, and then reflux extraction is carried out at 80°C for 2 hours. After cooling to room temperature, centrifugation is carried out at a rotation speed of 2500 r / min for 2 minutes, and the supernatant is reserved to obtain the mulberry leaf extract. During the reflux extraction, the alcohol solvent is condensed and returned to the extraction liquid. The alcohol solvent is repeatedly extracted by the reflux extraction to promote the dissolution of water-soluble and fat-soluble components such as flavonoids, organic acids and alkaloids, so as to achieve the effect of complete extraction of active ingredients. (2) The paste-like mulberry leaf extract obtained in step (1) is subjected to step-by-step extraction, and after purification treatment by using a filter membrane with a pore size of 0.22 μm, purified alkaloid extract, purified organic acid extract and purified flavonoid extract are obtained in sequence. The solvent and macromolecular impurities in the extract are removed by membrane filtration. The specific method of the step-by-step extraction includes the following steps: a. The paste-like mulberry leaf extract is dissolved in dilute hydrochloric acid with a mass-volume ratio of 1:1 g / mL, and then an equal volume of chloroform is added. After extraction for 20 minutes, centrifugation is carried out at 5000 r / min for 2 minutes to obtain a mixed extract. The purpose of treating with dilute hydrochloric acid is to convert the active substances in the mulberry leaf extract into salts for precipitation; b. In the mixed extract obtained in step a, 10% ammonia water is added to adjust the pH value to 10, and an equal volume of chloroform is added to the mixed extract obtained in step a. After extraction for 20 minutes, the mixture is shaken up and down and then left to separate into layers for 30 minutes to obtain a chloroform layer and a water layer. The chloroform layer is marked as the alkaloid extract. The purpose of adding ammonia water is to make the alkaloids free; c. In the water layer obtained in step b, an equal volume of dilute hydrochloric acid with a concentration of 0.01 mol / L is added, and an equal volume of ethyl acetate is added to the mixed solution after adding the dilute hydrochloric acid. After extraction for 20 minutes, the mixture is shaken up and down and then left to separate into layers for 30 minutes to obtain an ethyl acetate layer and a water layer. The ethyl acetate layer is marked as the organic acid extract. The purpose of adding dilute hydrochloric acid is to promote the free precipitation of organic acids; d. In the water layer obtained in step c, a sodium hydroxide solution with a concentration of 0.1 mol / L is added to adjust the pH value to 9, and then centrifugation is carried out to obtain a flavonoid extract. The purpose of adding sodium hydroxide is to promote the free precipitation of flavonoid components; In the embodiment of the present application, based on the step-by-step extraction process, a continuous extraction and separation system for active ingredients in mulberry leaves is established, gradient extraction of alkaloids, organic acids and flavonoids in mulberry leaves is realized, the problem of long time consumption and inconvenient operation for detecting various active components in mulberry leaves under single component extraction mode is solved, and the active components are enriched during separation, further improving the sensitivity and accuracy of active component determination. (3) Based on UHPLC-ESI-Q-TOF / MS analysis, the active components in the purified extract obtained in step (2) are quantitatively detected; wherein the alkaloid active components in the purified alkaloid extract include trachelanthine, noranisodrine and 1-deoxynojirimycin; the organic acid active components in the purified organic acid extract include chlorogenic acid, p-hydroxycinnamic acid, caffeic acid and quinic acid; the flavonoid active components in the purified flavonoid extract include rutin, kaempferol, quercetin, isoquercitrin and quercetin-3-triglucoside; The chromatographic conditions are as follows: Agilent 1290 Infinity ultra-high performance liquid chromatograph; Waters ACQUITY UPLC BEH C18 chromatographic column; Column temperature 30°C, flow rate 0.3 mL / min, injection volume 1 μL; Detection wavelength 250-320 nm; The mobile phase is 0.1% formic acid aqueous solution as A phase and acetonitrile as B phase for gradient elution, and the program is as follows: elution time 0-40 min, A phase 95-5%, B phase 5-95%; The mass spectrometry conditions are as follows: Agilent 6530 quadrupole-time-of-flight tandem mass spectrometer; ESI ion source, data acquisition range m / z 100-1700 under positive and negative ion modes respectively; Ion source temperature 350°C, capillary voltage 3.5 kV in positive ion mode and 4.0 kV in negative ion mode; Atomization gas pressure 45 Psi, drying gas flow rate 11 L / min, sheath gas flow rate 11 L / min, sheath gas temperature 350°C, fragment voltage 140 V.

[0043] The method for quantitatively detecting alkaloid active components in the purified alkaloid extract is as follows: Establishing standard curve: selecting Stizolobine, nor-Scopine and 1-Deoxynojirimycin as standard, preparing Stizolobine standard solution with concentration of 1-1000 ng / mL, nor-Scopine standard solution with concentration of 1-800 ng / mL and 1-Deoxynojirimycin standard solution with concentration of 0.2-2000 ng / mL by using methanol as solvent; Under the same chromatographic conditions and mass spectrometry conditions, injecting each concentration standard solution with volume of V1 into the ultra-high performance liquid chromatograph, detecting by mass spectrometry, determining the chromatographic peak of each active component according to the characteristic ion of each active component, and obtaining the chromatographic peak area of each active component, and the detection wavelength is 255 nm; Taking the peak area of the chromatographic peak of each active component as the ordinate and the concentration of each active component as the abscissa, the standard curve of each active component is established respectively; Obtaining the chromatographic peak area of each active component in the purified alkaloid extract: under the same chromatographic conditions and mass spectrometry conditions, injecting the purified alkaloid extract with volume of V1 into the ultra-high performance liquid chromatograph, detecting by mass spectrometry, determining the chromatographic peak of each active component according to the characteristic ion of each active component, and obtaining the chromatographic peak area of each active component; Determining the content of each active component in the purified alkaloid extract: drawing the standard curve of peak area Y vs. concentration x (ng / mL), and determining the content of alkaloid active components in mulberry leaves based on the linear equation obtained from the foregoing standard curve.

[0044] The method for quantitatively detecting the organic acid active components in the purified organic acid extract is the same as before, except that: Establishing standard curve: selecting chlorogenic acid, p-hydroxy cinnamic acid, caffeic acid and quinic acid as standard, preparing chlorogenic acid standard solution with concentration of 0.1-1000 ng / mL, p-hydroxy cinnamic acid standard solution with concentration of 2-700 ng / mL, caffeic acid standard solution with concentration of 1-600 ng / mL, and quinic acid standard solution with concentration of 10-2000 ng / mL by using methanol as solvent; The detection wavelength is 320 nm.

[0045] The method for quantitatively detecting the flavonoid active components in the purified flavonoid extract is the same as before, except that: Establishing standard curve: selecting rutin, kaempferol, quercetin, isoquercitrin and quercetin-3-triglucoside as standard, preparing rutin standard solution with concentration of 1-800 ng / mL, kaempferol standard solution with concentration of 1-600 ng / mL, quercetin standard solution with concentration of 2-500 ng / mL, isoquercitrin standard solution with concentration of 1-700 ng / mL, and quercetin-3-triglucoside standard solution with concentration of 10-1000 ng / mL by using methanol as solvent; The detection wavelength is 278nm.

[0046] In this embodiment of the invention, considering that alkaloids, organic acids, and flavonoids have maximum absorption at wavelengths of 255nm, 320nm, and 278nm, respectively, the detection wavelengths of UHPLC are set to 255nm, 320nm, and 278nm for quantitative detection and analysis of alkaloid extracts, organic acid extracts, and flavonoid extracts, in order to obtain more accurate and reliable detection results.

[0047] In this embodiment of the invention, during detection, a standard of known concentration is set as a reference for quantitative analysis. By measuring the response signals of the standard and the sample and comparing them using a standard curve, the concentration of each component in the sample can be accurately calculated, thereby ensuring the accuracy and reliability of the detection of each active component in mulberry leaves.

[0048] like Figure 1 As shown, the samples in the embodiments of the present invention have short peak elution times, all within 15 minutes, and good separation. The peak shapes of each compound are good, and the mass spectrometry response is high. By combining the retention times of standards such as 1-deoxynojirimycin, rutin, and chlorogenic acid with the MS mass spectrometry fragmentation rules for component identification, a variety of flavonoids, alkaloids, organic acids and other components can be accurately identified, resulting in accurate and clear detection results of active components.

[0049] Example 2 (1) Mulberry leaf 2 was refluxed and extracted. The mulberry leaf extract was concentrated at 75°C by evaporation to 1 / 10 of the original volume to obtain a paste-like mulberry leaf extract. The specific method of reflux extraction is as follows: After washing, drying and crushing the mulberry leaves, add a 60% ethanol aqueous solution at a mass-to-volume ratio of mulberry leaves to ethanol aqueous solution of 1:9 (g / mL). Reflux extraction is carried out at 85℃ for 4 hours. After cooling to room temperature, centrifuge at 3000 r / min for 4 minutes, and retain the supernatant to obtain the mulberry leaf extract. During the reflux extraction process, the alcohol solvent distilled off and then condensed back into the extract. By repeatedly extracting the mulberry leaves with the alcohol solvent through reflux extraction, the water-soluble and fat-soluble components such as flavonoids, organic acids and alkaloids are fully dissolved, thereby achieving the effect of complete extraction of active ingredients. (2) The paste-like mulberry leaf extract obtained in step (1) is extracted stepwise. After purification using a filter membrane with a pore size of 0.22 μm, purified alkaloid extract, purified organic acid extract and purified flavonoid extract are obtained in sequence. The solvent and macromolecular impurities in the extract are removed by membrane filtration. The specific method for step-by-step extraction includes the following steps: a. With a mass-to-volume ratio of mulberry leaf extract to dilute hydrochloric acid of 1:2 (g / mL), dissolve the mulberry leaf extract in dilute hydrochloric acid with a concentration of 0.02 mol / L, then add chloroform of the same volume as the dilute hydrochloric acid, extract for 25 min, and centrifuge at 5500 r / min for 3 min to obtain the mixed extract. b. In the mixed extract obtained in step a, add 15% ammonia water to adjust the pH value to 11, add chloroform of the same volume as the mixed extract obtained in step a, extract for 25 min, shake up and down and let stand for 30 min to separate the layers to obtain a chloroform layer and an aqueous layer. The chloroform layer is labeled as alkaloid extract. c. Add an equal volume of 0.02 mol / L dilute hydrochloric acid to the aqueous layer obtained in step b, and add an equal volume of ethyl acetate to the mixture after adding the dilute hydrochloric acid. Extract for 25 min, shake up and down, and let stand for 30 min to separate the layers, obtaining an ethyl acetate layer and an aqueous layer. The ethyl acetate layer is labeled as the organic acid extract. The purpose of adding dilute hydrochloric acid is to promote the free precipitation of organic acids. d. Add a 0.1 mol / L sodium hydroxide solution to the aqueous layer obtained in step c, adjust the pH to 8, centrifuge, and obtain the flavonoid extract; the purpose of adding sodium hydroxide is to promote the free precipitation of flavonoid components; (3) Same as step (3) in Example 1; (4) Same as step (4) in Example 1.

[0050] like Figure 2 As shown, the samples in the embodiments of the present invention have short peak elution times, all within 15 minutes, and good separation. The peak shapes of each compound are good, and the mass spectrometry response is high. By combining the retention times of standards such as 1-deoxynojirimycin, rutin, and chlorogenic acid with the MS mass spectrometry fragmentation rules for component identification, a variety of flavonoids, alkaloids, organic acids and other components can be accurately identified, resulting in accurate and clear detection results of active components.

[0051] Example 3 The only difference between this embodiment and Embodiment 1 is that, in step (1), the mulberry leaves 3 are subjected to reflux extraction. The rest is the same as in Embodiment 1.

[0052] like Figure 3 As shown, the samples in the embodiments of the present invention have short peak elution times, all within 15 minutes, and good separation. The peak shapes of each compound are good, and the mass spectrometry response is high. By combining the retention times of standards such as 1-deoxynojirimycin, rutin, and chlorogenic acid with the MS mass spectrometry fragmentation rules for component identification, a variety of flavonoids, alkaloids, organic acids and other components can be accurately identified, resulting in accurate and clear detection results of active components.

Claims

1. A method for quantitatively detecting multiple active components in mulberry leaves, characterized in that, Includes the following steps: (1) The mulberry leaves were refluxed and extracted, and the resulting mulberry leaf extract was concentrated to obtain a paste-like mulberry leaf extract; (2) The paste-like mulberry leaf extract obtained in step (1) is extracted stepwise and purified separately to obtain purified alkaloid extract, purified organic acid extract and purified flavonoid extract in sequence. (3) Based on UHPLC-ESI-Q-TOF / MS analysis, the active components in the purified extract obtained in step (2) were quantitatively detected; wherein, the alkaloid active components in the purified alkaloid extract include buckwheat alkaloid, norhyoscyamine and 1-deoxynojirimycin; the organic acid active components in the purified organic acid extract include chlorogenic acid, p-hydroxycinnamic acid, caffeic acid and quinic acid; the flavonoid active components in the purified flavonoid extract include rutin, kaempferol, quercetin, isoquercitrin and quercetin-3-triglucoside.

2. The method for quantitatively detecting multiple active components in mulberry leaves according to claim 1, characterized in that, In step (1), the specific method of reflux extraction is as follows: after washing, drying and crushing the mulberry leaves, add an alcohol solvent and perform reflux extraction. After cooling to room temperature, centrifuge and retain the supernatant to obtain mulberry leaf extract. The mass-to-volume ratio of mulberry leaves to alcohol solvent is 1:8-12 (g / mL). The reflux extraction temperature is 80-90℃ and the time is 2-5 hours. The centrifugation speed is 2000-4000 r / min and the time is 2-4 minutes. The alcohol solvent includes a methanol aqueous solution with a volume fraction of 50-90% and / or an ethanol aqueous solution with a volume fraction of 40-70%. The mulberry leaf extract is concentrated by evaporation. The concentration temperature is 60-80℃ and the concentration is stopped when the volume is reduced to 1 / 10 of the original volume.

3. The method for quantitatively detecting multiple active components in mulberry leaves according to claim 1 or 2, characterized in that, In step (2), the specific method for step-by-step extraction includes the following steps: a. Dissolve the paste-like mulberry leaf extract in dilute hydrochloric acid, then add an equal volume of chloroform to the dilute hydrochloric acid, extract, centrifuge, and obtain a mixed extract; b. In the mixed extract obtained in step a, ammonia water is added to adjust the pH value to alkaline, and chloroform of the same volume as the mixed extract obtained in step a is added. After extraction, the mixture is shaken up and down and allowed to stand to separate into layers, resulting in a chloroform layer and an aqueous layer. The chloroform layer is labeled as alkaloid extract. c. Add an equal volume of dilute hydrochloric acid to the aqueous layer obtained in step b, and add an equal volume of ethyl acetate to the mixture after adding dilute hydrochloric acid. Extract, shake up and down, and let stand to separate the layers to obtain an ethyl acetate layer and an aqueous layer. The ethyl acetate layer is labeled as organic acid extract. d. Add sodium hydroxide solution to the aqueous layer obtained in step c, adjust the pH to alkaline, centrifuge, and obtain flavonoid extract.

4. The method for quantitatively detecting multiple active components in mulberry leaves according to claim 3, characterized in that, In step a, the mass-to-volume ratio (g / mL) of the paste-like mulberry leaf extract to dilute hydrochloric acid is 1:1–3; the concentration of the dilute hydrochloric acid is 0.01–0.03 mol / L; the extraction time is 10–25 min; the centrifugation speed is 5000–6000 r / min, and the time is 2–3 min. In step b, the mass fraction of the ammonia water is 10–25%; the pH value is adjusted to 10–11; the extraction time is 10–25 min; the settling time is 20–40 min. In step c, the concentration of the dilute hydrochloric acid is 0.01–0.03 mol / L; the extraction time is 10–25 min; the settling time is 20–40 min. In step d, the concentration of the sodium hydroxide solution is 0.1–1.0 mol / L; the pH value is adjusted to 8–9.

5. The method for quantitatively detecting multiple active components in mulberry leaves according to claim 1 or 2, characterized in that, In step (2), the purification process refers to using a filter membrane with a pore size ≤ 0.22 μm.

6. The method for quantitatively detecting multiple active components in mulberry leaves according to claim 1 or 2, characterized in that, In step (3), the method for quantitatively detecting the active components in the purified extract is as follows: Establish a standard curve: Select the active component as a standard and prepare standard solutions of different concentrations using methanol as a solvent; Under the same chromatographic and mass spectrometric conditions, standard solutions of various concentrations with a volume of V1 were injected into an ultra-high performance liquid chromatograph. The chromatographic peaks of each active component were determined by mass spectrometry based on the characteristic ions of each active component, and the peak areas of each active component were obtained. Standard curves for each active component were established with the peak area of ​​the chromatographic peak of each active component as the ordinate and the concentration of each active component as the abscissa. To obtain the chromatographic peak area of ​​each active component in the purified extract: Under the same chromatographic and mass spectrometric conditions, a volume of V1 of the purified extract was injected into an ultra-high performance liquid chromatograph. The chromatographic peak of each active component was determined by mass spectrometry based on the characteristic ions of each active component, and the chromatographic peak area of ​​each active component was obtained. Determine the content of each active component in the purified extract: Plot a standard curve of peak area Y versus concentration x, and determine the content of active components in mulberry leaves based on the linear equation obtained from the aforementioned standard curve.

7. The method for quantitatively detecting multiple active components in mulberry leaves according to claim 6, characterized in that, The standard solutions for quantitatively detecting the alkaloid active components in the purified alkaloid extract are as follows: buckwheat alkaloid, norhyoscyamine, and 1-deoxynojirimycin are selected as standards, and standard solutions with concentrations of 1–1000 ng / mL, 1–800 ng / mL of norhyoscyamine, and 0.2–2000 ng / mL of 1-deoxynojirimycin are prepared using methanol as the solvent. The standard solutions for quantitatively detecting the organic acid active components in the purified organic acid extract are as follows: chlorogenic acid, p-hydroxycinnamic acid, caffeic acid, and quinic acid are selected as standards, and standard solutions with concentrations of 0.1–1000 ng / mL of chlorogenic acid, 2–700 ng / mL of 1-deoxynojirimycin, and 1-deoxynojirimycin are prepared using methanol as the solvent. The standard solutions for quantitative detection of flavonoid active components in the purified flavonoid extract were prepared using methanol as solvent. These solutions included 1-600 ng / mL of p-hydroxycinnamic acid standard solution, 1-600 ng / mL of caffeic acid standard solution, and 10-2000 ng / mL of quinic acid standard solution.

8. The method for quantitatively detecting multiple active components in mulberry leaves according to claim 6, characterized in that, The chromatographic detection wavelengths for quantitative determination of active components in purified alkaloid extract, organic acid extract, and flavonoid extract were 255 nm, 320 nm, and 278 nm, respectively.

9. The method for quantitatively detecting multiple active components in mulberry leaves according to claim 6, characterized in that, The chromatographic conditions are as follows: Agilent 1290 Infinity Ultra-High Performance Liquid Chromatography System; Waters ACQUITYUPLC BEH C18 column; Column temperature 30℃, flow rate 0.3 mL / min, injection volume 1 μL; Detection wavelength: 250–320 nm; The mobile phase consisted of an aqueous solution containing 0.1% formic acid as phase A and acetonitrile as phase B, with gradient elution performed as follows: elution time 0–40 min, phase A 95–5%, phase B 5–95%; The mass spectrometry conditions are as follows: Agilent 6530 quadrupole-time-of-flight tandem mass spectrometer; ESI ion source, data were collected in positive and negative ion modes respectively, with a data acquisition range of m / z 100~1700; The ion source temperature is 350℃, and the capillary voltage is 3.5kV in positive ion mode and 4.0kV in negative ion mode. The atomizing gas pressure is 45 Psi, the drying gas flow rate is 11 L / min, the sheath gas flow rate is 11 L / min, the sheath gas temperature is 350°C, and the fragment voltage is 140 V.

Images