CN110563774A

CN110563774A - three-stage separated matter of common sow thistle alkaloid and its separation process and application

Info

Publication number: CN110563774A
Application number: CN201910727780.2A
Authority: CN
Inventors: 李秀梅; 杨培龙; 潘方方; 石冬冬; 闻治国; 满晨
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Priority date: 2019-06-13
Filing date: 2019-08-07
Publication date: 2019-12-13
Anticipated expiration: 2039-08-07
Also published as: CN110563775B; CN110563774B; CN110256505A; CN110563775A; CN110477195B; CN110477195A; CN110256505B

Abstract

the invention belongs to the technical field of agricultural biology, and particularly relates to a three-stage separated matter of a common sowthistle herb alkaloid, a separation method and application thereof. The invention extracts alkaloid components from the common sow thistle by an ultrasonic method, and obtains a tertiary isolate with antioxidant capacity by utilizing preparative chromatographic separation, thereby having important practical significance for developing and utilizing alkaloid compounds in the common sow thistle.

Description

three-stage separated matter of common sow thistle alkaloid and its separation process and application

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a three-stage separated matter of a common sowthistle herb alkaloid, a separation method and application thereof.

Background

Sonchus brachyotus DC is an annual herb of Sonchus brachyotus of Compositae, also called herba Sonchi arvensis, herba Querci Acutissimae, herba Sonchi arvensis, and herba Ixeritis Sonchifoliae. The medicinal history of the long-crack common sow thistle is long, and according to records of the earliest medical monograph in China 'Shennong's herbal Jing and the 'herbal compendium' of Li Shizhen, the long-crack common sow thistle is bitter in taste and cold in nature, has the effects of clearing heat and removing toxicity, relieving swelling and expelling pus, cooling blood and removing stasis, clearing lung heat and relieving cough, benefiting liver and promoting urination, and helping digestion and harmonizing stomach, and is used for treating acute dysentery, enteritis, hemorrhoid and swelling and pain and other symptoms. The common sow thistle contains beta-sitosterol, luteolin, apigenin, quercetin and volatile oil, and has the functions of resisting bacteria, reducing blood pressure, reducing cholesterol, resisting tumor, treating hepatitis, resisting oxidation and the like. However, the effective antioxidant active ingredient in sonchus oleraceus has not been known so far.

Disclosure of Invention

The invention aims to provide a three-stage isolate of common sowthistle herb alkaloid.

The invention also aims to provide a separation method of the three-stage separator.

It is a further object of the present invention to provide the use of the above-mentioned tertiary isolate.

according to the specific embodiment of the invention, the three-stage separated substance of the common sow thistle alkaloid is prepared by the method comprising the following steps:

(1) Preparing extract of herba Sonchi arvensis;

(2) Preparing the common sow thistle extract obtained in the step (1) into a test solution of 100mg/mL, and separating by adopting a preparative chromatography, wherein the chromatographic conditions are as follows:

adopting a C18 silica gel column, taking 0.1% formic acid water solution as a mobile phase A, taking methanol as a mobile phase B, wherein the flow rate is 10mL/min, the column temperature is 27-33 ℃, the wavelength is 255-265nm, and the gradient elution procedure is as follows:

In the time range of 0-5min, the volume of the mobile phase A is changed from 95% to 60%, and the volume of the mobile phase B is changed from 5% to 40%;

in the time range of 5-15min, the volume of the mobile phase A is changed from 60% to 30%, and the volume of the mobile phase B is changed from 40% to 70%;

In the time range of 15-30min, the volume of the mobile phase A is changed from 30% to 20%, and the volume of the mobile phase B is changed from 70% to 80%;

the volume of mobile phase A is kept at 20% and the volume of mobile phase B is kept at 80% in the time range of 30-35 min;

collecting fractions with peak heights ranging from 420 mAU to 2000mAU to obtain a primary isolate;

(3) Concentrating and drying the primary isolate obtained in the step (2), preparing a test solution of 10mg/mL, and separating by using a preparative chromatography, wherein the chromatographic conditions are as follows:

collecting the fraction with peak height range of 0.2-1.8AU to obtain secondary isolate;

(4) Separating the secondary isolate obtained in step (3) by analytical chromatography under the following chromatographic conditions:

adopting a C18 silica gel column, taking 0.1% formic acid aqueous solution as a mobile phase A, taking methanol as a mobile phase B, and carrying out gradient elution at the flow rate of 1mL/min, the column temperature of 30 ℃ and the wavelength of 260nm, wherein the gradient elution procedure is as follows:

In the time range of 0-8min, the volume of the mobile phase A is changed from 95% to 75%, and the volume of the mobile phase B is changed from 5% to 25%;

in the time range of 8-10min, the volume of the mobile phase A is changed from 75% to 65%, and the volume of the mobile phase B is changed from 25% to 35%;

In the time range of 10-20min, the volume of the mobile phase A is changed from 65% to 40%, and the volume of the mobile phase B is changed from 35% to 60%;

In the time range of 20-24min, the volume of the mobile phase A is changed from 40% to 20%, and the volume of the mobile phase B is changed from 60% to 80%;

And in the time range of 24-27min, keeping the volume of the mobile phase A at 20% and the volume of the mobile phase B at 80%, and collecting the 16-18min fraction to obtain the final product.

in the reverse phase chromatographic column, the order of the common sow thistle alkaloid extract flowing out of the chromatographic column is that the components with stronger polarity are eluted out before the components with weaker polarity, namely the order of the common sow thistle alkaloid extract flowing out of the chromatographic column is influenced by the polarity of a mobile phase. Meanwhile, different mobile phase compositions, gradient programs, flow rates, column temperatures and the like can cause the polarity of the mobile phase to change, so that the compositions of effective components in fractions are different.

the method adopts the C18 chromatographic column to separate the alkaloid, can separate and obtain a chromatographic peak with good peak shape and no tailing under the condition of high pH, not only has extremely high sample loading amount, but also can keep high interface kinetic coefficient and obtain better column efficiency.

According to the third-stage separated matter of the long-split common sow thistle alkaloid, in the step (2), the column temperature is 30 ℃, and the wavelength is 260 nm.

according to the third-stage separated matter of the common sow thistle alkaloid of the embodiment of the invention, the common sow thistle extract is prepared by the following steps:

Using 75% ethanol as solvent, making herba Sonchi arvensis into extractive solution with liquid-to-material ratio of 30mL/g, performing ultrasonic extraction, standing, and collecting supernatant.

according to the three-stage separated matter of the common sow thistle alkaloid, the ultrasonic extraction power is 700W, the temperature is 55 ℃, and the ultrasonic time is 30 min.

According to the specific embodiment of the invention, the separation method of the common sow thistle alkaloid tertiary isolate comprises the following steps:

(1) Preparing extract of herba Sonchi arvensis;

According to the separation method of the three-stage separated matter of the common sow thistle alkaloid, which is the specific embodiment of the invention, in the step (2), the column temperature is 30 ℃, and the wavelength is 260 nm.

According to the separation method of the common sow thistle alkaloid tertiary isolate in the embodiment of the invention, the common sow thistle extract is prepared by the method comprising the following steps:

Using 75% ethanol as solvent, making herba Sonchi arvensis into extract solution with liquid-material ratio of 30mL/g, performing ultrasonic extraction, standing, collecting supernatant, performing rotary evaporation, cooling, and drying.

According to the separation method of the three-stage separated matter of the common sow thistle alkaloid, the power of ultrasonic extraction is 700W, the temperature is 55 ℃, and the ultrasonic time is 30 min.

The invention has the beneficial effects that:

the invention determines the extraction process of the common sow thistle alkaloid, and the extraction rate of the common sow thistle alkaloid can reach 20.30 percent. Multiple fractions are obtained by multi-stage preparative chromatography separation, the three-stage separated matter of the common sowthistle herb alkaloid is determined to have stronger oxidation resistance, and reliable theoretical and technical support is provided for developing and producing novel oxidation-resistant food or feed additives.

Drawings

FIG. 1 shows H₂O₂Effect on survival of Caco-2 cells;

FIG. 2 shows the pair of alkaloid extracts of Sonchus oleraceus H₂O₂the effect of impaired Caco-2 cell survival;

FIG. 3 shows the total antioxidant capacity of the extract of Gesneriana pauciflorus alkaloids;

FIG. 4 shows the scavenging ability of the alkaloid extract of Sonchus oleraceus to ABTS free radicals;

FIG. 5 shows the DPPH radical scavenging ability of the extract of Gesneriana longissima alkaloid;

FIG. 6 shows the scavenging ability of the extract of Gesneriana longissima alkaloid on hydroxyl radicals;

FIG. 7 shows superoxide anion scavenging ability of extract of Gesneriana longissima alkaloid;

FIG. 8 shows the total reducing power of the extract of Gesneriana pauciflorus alkaloids;

FIG. 9 shows the effect of Gesneriana lancifera alkaloid extract on ROS in the intestinal tract tissue of zebra fish;

FIG. 10 shows the effect of the extract of Gesneriana lancifera alkaloids on the SOD of intestinal tissues of zebra fish;

FIG. 11 shows the effect of the alkaloid extract from Gesneriana lancifera on the intestinal tissue MDA of zebra fish;

FIG. 12 shows the effect of the alkaloid extract from Gesneriana lancifera on the intestinal tissue CAT of zebra fish;

FIG. 13 shows preparative HPLC separation of Sonchus oleraceus extract;

FIG. 14 shows the alkaloid extract and fraction pair H of Sonchus oleraceus₂O₂effects of impaired Caco-2 cell survival;

FIG. 15 shows the total antioxidant capacity of the first fraction;

FIG. 16 shows the scavenging ability of the first fraction for ABTS free radicals;

FIG. 17 shows DPPH radical scavenging ability of the first fraction;

FIG. 18 shows the scavenging ability of the first fraction for hydroxyl radicals;

FIG. 19 shows superoxide anion scavenging ability of the first fraction;

FIG. 20 shows the total reducing power of the first fraction;

FIG. 21 shows the results of preparative HPLC separation of the primary isolate;

FIG. 22 shows the pair of secondary fractions H₂O₂the effect of impaired Caco-2 cell survival;

FIG. 23 shows the total antioxidant capacity of the secondary fraction;

FIG. 24 shows the scavenging ability of the secondary fraction for ABTS free radicals;

FIG. 25 shows the DPPH radical scavenging ability of the secondary fraction;

FIG. 26 shows the scavenging ability of the secondary fraction for hydroxyl radicals;

FIG. 27 shows superoxide anion scavenging ability of secondary fractions;

FIG. 28 shows the total reducing power of the secondary fraction;

FIG. 29 shows HPLC fractionation of the secondary isolate;

FIG. 30 shows the three-stage fraction vs. H₂O₂The effect of impaired Caco-2 cell survival;

FIG. 31 shows the total antioxidant capacity of the tertiary fraction;

FIG. 32 shows the scavenging ability of tertiary fractions for ABTS free radicals;

FIG. 33 shows DPPH radical scavenging ability of the tertiary fraction;

FIG. 34 shows the scavenging ability of the tertiary fraction for hydroxyl radicals;

FIG. 35 shows the superoxide anion scavenging capacity of the tertiary fraction;

fig. 36 shows the total reducing power of the tertiary fraction.

Detailed Description

example 1 preparation of alkaloid extract from Sonchus oleraceus

Cleaning herba Sonchi arvensis, oven drying at 40 deg.C to constant weight, pulverizing with miniature plant pulverizer, and sieving with 60 mesh analytical sieve. Weighing 10g of whole herb coarse powder of common sow thistle, placing the whole herb coarse powder into a 500mL ultrasonic cup, adding 75% ethanol to obtain an extraction solution with the liquid-material ratio of 30mL/g, adjusting the pH value of the extraction solvent to be 5, and carrying out ultrasonic extraction at the ultrasonic temperature of 55 ℃, the ultrasonic power of 700W and the ultrasonic time of 30 min.

Ultrasonic extracting, centrifuging at 5000rpm for 10min to obtain supernatant, rotary evaporating at 40 deg.C, and freeze drying to obtain herba Sonchi Oleracei alkaloid extract.

alkaloids are nitrogen-containing organic compounds, and alkaloid compounds have alkaline-like properties. The alkaloid has various parent nucleus structures, and examples thereof include pyrrole alkaloid, tropane alkaloid (solanaceae alkaloid), and tropane alkaloid (solanaceae alkaloid).

putting 0.1mL of extracting solution into a 1mL colorimetric tube, fixing the volume to a scale by using ethanol with a certain concentration, taking the corresponding ethanol concentration as a blank, measuring the light absorption value of the extracting solution at 410nm, and measuring the extraction rate of alkaloid in the sonchus oleraceus according to a standard curve, wherein the extraction rate is (the weight of the alkaloid in a sample/the weight of medicinal materials) multiplied by 100%.

the extraction rate of alkaloid in the invention is 20.30%.

example 2 verification of the antioxidant capacity of the Alkaloids extract of Sonchus oleraceus

2.1H₂O₂Establishing model for inducing Caco-2 cell oxidative damage

By investigating H₂O₂After the Caco-2 cells are acted for 24 hours at different concentrations (100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000 mu mol/L), the influence of the Caco-2 cells on the cell survival rate is observed, and H is determined₂O₂the Caco-2 cell oxidative damage model was induced, and the results are shown in FIG. 1.

As can be seen from FIG. 1, H₂O₂The cell survival rate is not significantly influenced when the concentration is 100-5000 mu mol/L, the cell survival rate is reduced by about 40% when the concentration is 7500 mu mol/L, and H is in high concentration₂O₂leads the survival rate of Caco-2 cells to be obviously reduced and the cell number to be obviously reduced by 10000 mu mol/L H₂O₂The survival rate of the treated cells decreased by about 60%, and the appropriate H was determined₂O₂H for inducing Caco-2 cell oxidative damage model₂O₂The concentration was 7500. mu. mol/L.

2.2 Gesneriana lansium alkaloid extract Pair H₂O₂Repair effect of induced Caco-2 cell oxidative damage model

the use concentration is as follows: extract pair H of 200 mug/mL common sow thistle alkaloid₂O₂The induced repairing effect of Caco-2 cell oxidative damage model is shown in FIG. 2.

The results showed that the cell viability was 71%, while H₂O₂after the Caco-2 cells are induced to be oxidized and damaged, the cell survival rate is56% thus, Sonchus oleraceus alkaloid extract Pair H₂O₂the model for inducing Caco-2 cell oxidative damage has a repairing effect.

2.3 anti-oxidant action of Sonchus oleraceus alkaloid extract

(1) total antioxidant capacity of Sonchus oleraceus alkaloid extract

27.8mg of FeSO are weighed out₄·7H₂O, dissolved and diluted to 1mL, and the concentration is 100 mM. Taking a proper amount of 100mM FeSO₄the solution was diluted to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM. The standard was prepared using distilled water or sample preparation solution.

a: to each assay well of a 96-well plate, 180. mu.L of FRAP working solution (TPTZ dilution 150. mu.L + TPTZ solution 15. mu.L + assay buffer 15. mu.L) was added.

b: adding 5 μ L of distilled water or PBS; adding 5 mu L of FeSO with various concentrations into the detection holes of the standard curve₄a standard solution; mu.L of each sample or 0.15-1.5mM Trolox was added to the sample wells as a positive control. Mix gently.

c: a593 was determined after incubation at 37 ℃ for 3-5 min. Measuring FeSO₄the standard curve is that y is 0.3119x +0.0526, R²＝0.999。

the Total Antioxidant Capacity (TAC) of the sample was calculated from the standard curve. As shown in FIG. 3, the total antioxidant capacity of the extract of sonchus oleraceus alkaloids at a concentration of 200. mu.g/mL was 0.89mmol/g, about 12 times that of the control, compared to the control at a concentration of 0.07 mmol/g, but the total antioxidant capacity of Trolox was about 7 times that of the extract of sonchus oleraceus alkaloids at a concentration of 6.66mmol/g positive control Trolox.

(2) ABTS free radical scavenging ability of common sow thistle alkaloid extract

7mmol/L ABTS (2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonie acid) diam-onium salt, ABTS) was mixed with 2.45mmol/L potassium persulfate at a ratio of 9:1 and allowed to stand at room temperature for 16 hours, using the octafold dilution before use as ABTS stock solution. Adding 2.5mL ABTS stock solutions into a series of samples with concentration ranges (1.6-1000. mu.g/mL, dissolved by absolute ethyl alcohol), mixing uniformly, reacting for 20min in a dark place, and measuring the light absorption value at the position of 734nm wavelength. The mixed solution of 1mL double distilled water and ABTS was used as a blank control, and BHT (butylated hydoxylutene) was used as a positive control.

ABTS clearance (%) - (a)₁-A₂)/A₁]X 100%, wherein A₁Denotes the absorbance of the blank, A₂And representing ABTS absorbance of the sample to be tested.

As can be seen from FIG. 4, the clearance rate was 20% when the concentration of the extract of the alkaloid from Sonchus oleraceus was 25. mu.g/mL, whereas the clearance rate was about 79.09% when the concentration was 200. mu.g/mL, which is about 4 times the clearance rate of the low concentration for ABTS free radicals.

(3) DPPH free radical scavenging ability of herba Sonchi arvensis alkaloid extract

samples with a certain concentration range (1.6-1000 mug/mL, dissolved by absolute ethyl alcohol) are respectively added with 0.3mL of 0.2mmol/L DPPH (1, 1-diphenyl-2-piperidinylhydrazine, DPPH) (dissolved by absolute ethyl alcohol) in each sample, the mixture is vortexed and mixed, the prepared samples are kept for 1h in a dark place, and then the absorbance of the prepared samples is measured at 517 nm. 1mL of absolute ethanol was used as a blank control instead of the extract, and BHT in the same concentration range was used as a positive control.

DPPH clearance (%) - (a)₁-A₂)/A₁]X 100%, wherein A₁Denotes the absorbance of the blank, A₂Indicates the DPPH absorbance of the sample to be tested.

As shown in FIG. 5, the clearance was 3.6% when the extract of Gesneriana longissima alkaloid was 25. mu.g/mL, whereas the clearance was approximately 32.6% when the extract was 200. mu.g/mL, which is approximately 9 times the clearance of DPPH free radicals at low concentrations.

(4) scavenging ability of herba Sonchi arvensis alkaloid extract on hydroxyl free radical

Using Fe³⁺the EDTA-ascorbic acid-hydrogen peroxide system generates hydroxyl radicals, the deoxyribose is attacked by the hydroxyl radicals and then cracked, and the deoxyribose reacts with the thiobarbituric acid under the acidic and heating conditions to generate a red compound, and the existence of the antioxidant can prevent the hydroxyl radicals from attacking the deoxyribose.

To the reaction system were added 400. mu.L of 2-deoxyribose (10mmol/L), 100. mu.L of ferric chloride (10mmol/L), 100. mu.L of EDTA-2Na (ethylene diamine acetic acid salt) (1mmol/L), 100. mu.L of 30% hydrogen peroxide (10mmol/L), 100. mu.L of a sample concentration (1.6-1000. mu.g/mL), 200. mu.L of ascorbic acid (1mmol/L) was added to initiate the reaction, the reaction was carried out at 37 ℃ for 1 hour, 1mL of a 0.5% TBA solution of sodium hydroxide (0.025mol/L) and 1mL of a 30% aqueous solution of TCA (trichloroacetic acid) were added, and the mixture was heated in a water bath at 80 ℃ for 30min and cooled. The absorbance was measured at 532nm, and 0.05mol/L PBS (phosphate buffered solution) (pH 7.4) was used in the reaction system instead of the sample as a blank control to calculate the clearance, and BHT in the same concentration range was used as a positive control.

hydroxyl radical scavenging rate (%) - (A)₁-A₂)/A₁]X 100%, wherein A₁Denotes the absorbance of the blank, A₂the absorbance of the sample to be measured is indicated.

As shown in FIG. 6, the clearance rate was 10.3% when the extract concentration of the common sowthistle herb alkaloid was 25. mu.g/mL, whereas the clearance rate was about 63.9% when the extract concentration was 200. mu.g/mL, which is about 6 times of the clearance rate of the hydroxyl radical at a low concentration.

(5) Scavenging ability of herba Sonchi arvensis alkaloid extract on superoxide anion

Superoxide anion is one of the most common free radicals in the body. Pyrogallic acid spontaneously generates superoxide anions in an alkaline environment. The rate of autoxidation is related to the concentration of superoxide anion. The test object can remove superoxide anion and slow down the autoxidation rate of pyrogallic acid.

adding 1mL of 200 mu g/mL sample to be tested into Tris-HCl buffer (pH value 8.2, 0.05mol/L) and 4.5mL of the sample to be tested, reacting at 25 ℃ for 10min, adding 600 mu L of 0.003mol/L pyrogallol (dissolved in 10mmol/L hydrochloric acid), immediately measuring the absorbance at the wavelength of 325nm after full reaction, measuring the absorbance once every 30s until the absorbance does not change obviously, replacing the sample with 10mmol/L hydrochloric acid as a blank control, and using BHT as a positive control. The rate of auto-oxidation of pyrogallol can be calculated as the slope from the absorbance-time curve.

As shown in fig. 7, the extract of the alkaloid from sonchus oleraceus has a scavenging ability for superoxide anions and its autoxidation rate is slowed down compared to BHT.

(6) Total reducing power of herba Sonchi arvensis alkaloid extract

determining total reducing power of alkaloid by iron reduction antioxidant power method, and measuring Fe in acidic environment³⁺-tripyridotriazine (Fe)³⁺TPTZ) can be reduced to a ferrous form by an antioxidant, presents a blue color and has a maximum absorption at 593nm, the greater the absorbance, the greater the reducing power.

preparing sample solution of 400 μ g/mL with sample 20mg, adding sample solution of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 μ L into 1mL with double distilled water, and adding PBS (pH 6.6) and 1% K of 0.2mol/L₃[Fe(CN)₆]2.5mL of each, reacted at 50 ℃ for 20min, then added with 2.5mL of 10% TCA, centrifuged at 3000r/min (r 3cm) for 10min, 2.5mL of the supernatant was added with 2.5mL of double distilled water, mixed with 0.5mL of 0.1% ferric chloride, and the absorbance A was measured at 700nm after 10min₂The light absorption value is measured by using the double distilled water method without adding the sample as a blank control, and the increased light absorption value indicates the increase of the reduction capacity.

As shown in FIG. 8, the increased absorbance was 0.006 when the concentration of the extract of sonchus oleraceus alkaloid was 20. mu.g/mL, whereas the increased absorbance was 0.046 at 200. mu.g/mL, which is about 7 times the total reducing power of the low concentration.

(7) In vivo antioxidant effect of herba Sonchi arvensis alkaloid extract

zebrafish intestinal injury was induced by injecting zebrafish with 0.2% Oxazolone (Oxazolone) for 1 day and fed with fish feed containing varying concentrations of sonchus oleraceus alkaloid extract for 5 days.

The results are shown in fig. 9-12, and the extract of herba Sonchi arvensis alkaloid can effectively reduce the content of ROS and MDA in intestinal tissue of zebra fish, and improve SOD and CAT activities.

In conclusion, the common sow thistle alkaloid extract has good antioxidation in vitro and in vivo.

Example 3 isolation of Primary alkaloid fraction from Sonchus oleraceus

3.1 Long-split Sonchus oleraceus alkaloid first-order fraction

Preparation of a test solution: dissolving herba Sonchi arvensis alkaloid extract with ultrapure water, preparing into test solution with concentration of 100mg/mL, and filtering with 0.22 μm filter membrane.

chromatographic conditions for separating the primary alkaloid fraction of sonchus oleraceus are as follows:

The extract of sonchus oleraceus alkaloids was purified by HPLC preparative chromatography on purified water from a sample obtained from a plant from sonchus longissimus in Durashell C18(L) (10 μm,30X 250mm) column, preparing chromatographic conditions for the first fraction of the alkaloid of common sowthistle herb: a chromatographic column: DurashellC18(L) (10 μm,30X 250mm), the mobile phase is a 0.1% formic acid-water (A) -methanol (B) system, the flow rate is 10mL/min, the column temperature is 30 ℃, and the wavelength is 260 nm. The gradient elution procedure is 0-5min, 5-40% B, 5-15min, 40-70% B, 15-30min, 70-80% B, 30-35min, 80-80% B, and the sample loading amount is 18 mL.

the extract of sonchus oleraceus alkaloid was separated into 6 fractions, SB1(s. brachyotus 1), SB2, SB3, SB4, SB5 and SB6, as shown in fig. 13, according to the chromatographic peak profile of sonchus oleraceus alkaloid extract in preparative chromatography, wherein the SB1 peak height ranged from 2 to 570mAU, the SB2 peak height ranged from 530 to 750mAU, the absorbance ranged, the SB3 peak height ranged from 420 to 2000mAU, the SB4 peak height ranged from 1200 to 2010mAU, the SB5 peak height ranged from 360 to 2010mAU, and the SB6 peak height ranged from 70 to 740 mAU.

3.2 Gesneriana oleracea alkaloid primary fraction Pair H₂O₂Repair effect of induced Caco-2 cell oxidative damage model

fractions SB1, SB2, SB3, SB4, SB5 and SB6 (concentration: 200. mu.g/mL) were investigated for H₂O₂And (3) the repair effect of the induced Caco-2 cell oxidative damage model.

The results are shown in FIG. 14The cell viability was 76%, 74%, 114%, 106%, 94%, 75%, respectively, thus SB3 for H₂O₂The induced Caco-2 cell oxidative damage model has obvious repairing effect.

3.3 Oxidation resistance of Primary Total alkaloid fraction of Sonchus oleraceus

(1) Total antioxidant capacity of primary alkaloid fraction of Sonchus oleraceus

The detection method was the same as in example 2.

From FIG. 15, it can be seen that the total antioxidant capacity of control, Trolox, extract, SB1-SB6 is 0.07, 6.66, 0.89, 0.09, 0.21, 1.98, 0.92, 0.89, 0.28mmol/g, respectively, and from these data, the total antioxidant capacity of SB3 is relatively high, and the total antioxidant capacity of SB3 is about 28, 0.3, 2, 22, 9, 2, 7 times that of control, Trolox, extract, SB1, SB2, SB4-SB6, and therefore, the antioxidant capacity of SB3 in the primary fraction of the alkaloids from Gesnezoinum gracile is relatively strong.

(2) ABTS free radical scavenging ability of primary alkaloid fraction of Sonchus oleraceus L

the detection method was the same as in example 2.

as shown in FIG. 16, when SB1-SB6 was at a concentration of 1.6. mu.g/mL, the removal rates for ABTS free radicals were 1.61%, 3.64%, 15.99%, 19.43%, 7.69%, 8.5%, respectively, and when it was 1000. mu.g/mL, the removal rates were 31.58%, 65.79%, 74.29%, 74.09%, 55.47%, 46.96%, respectively, which are about 20, 18, 5, 4, 7, 6 times the removal rates for ABTS free radicals at low concentrations, and since the first-order fraction drug concentrations were the same, the change in the removal rates for ABTS free radicals was relatively large for SB1 and SB2, and when the concentrations of SB3 and SB4 were the highest, the removal rates for ABTS free radicals were relatively high, while when the concentrations of SB4 were 40. mu.g/mL, the removal rates were higher than for BHT, while the other first-order fractions were all lower than for ABTS free radicals.

(3) scavenging ability of primary alkaloid fraction of herba Sonchi arvensis to DPPH free radical

The detection method was the same as in example 2.

As shown in FIG. 17, when SB1-SB6 was at a concentration of 1.6. mu.g/mL, the removal rates for DPPH radicals were 8.29%, 11.82%, 14.11%, 14.81%, 3.35%, 1.23%, respectively, and when it was 1000. mu.g/mL, the removal rates were 48.85%, 51.32%, 73.55%, 74.25%, 58.55%, 49.38%, respectively, which were about 5, 4, 5, 17, 40 times the removal rates for DPPH radicals at low concentrations, and the removal rates for DPPH radicals were relatively large for SB5 and SB6 due to the same drug concentration, relatively high when the concentrations of SB3 and SB4 were at their maximum, and lower than BHT when the concentrations of SB2-SB4 were 1.6. mu.g/mL, while the removal rates were higher than BHT when the first-order fractions were at other concentrations.

(4) scavenging ability of primary alkaloid fraction of herba Sonchi arvensis to hydroxyl free radical

the detection method was the same as in example 2.

As shown in FIG. 18, when SB1-SB6 was at a concentration of 1.6. mu.g/mL, the clearance of hydroxyl radicals was 6.85%, 10.37%, 27.77%, 21.44%, 20.39%, 18.1%, respectively, and when it was at a concentration of 1000. mu.g/mL, the clearance was 69.07%, 70.83%, 77.15%, 76.45%, 71.18%, 70.12%, respectively, which was about 10, 7, 3, 4 times the clearance of hydroxyl radicals at a low concentration, and the clearance of SB1-SB6 was less significantly changed due to the same drug concentration, when SB3 and SB4 were at a maximum concentration, the clearance of hydroxyl radicals was relatively high, SB4 (when the concentration was 1.6, 8, 40, 200. mu.g/mL), when SB5 was 1.6, 8. mu.g/mL), when SB 5. mu.4836. mu.6 g/mL and BHT were significantly higher than those at a concentration of 1.6. mu.6. mu.3876. mu.25 g/mL), while the other first fractions have lower clearance than BHT.

(5) scavenging ability of primary alkaloid fraction of herba Sonchi arvensis to superoxide anion

The detection method was the same as in example 2.

As shown in fig. 19, the primary alkaloid fractions from sonchus oleraceus, SB1-SB6, have a scavenging capacity for superoxide anions compared to BHT, and the autoxidation rates of SB2, SB3 and SB4 slowed down significantly.

(6) Total reducing power of primary alkaloid fraction of herba Sonchi Oleracei

As shown in fig. 20, when the concentration of SB1-SB6 was 20 μ g/mL, the absorbance values increased were 0, 0.014, 0.001, 0.022, and 0.019, respectively, and when the concentration was 200 μ g/mL, the absorbance values increased were 0.013, 0.014, 0.131, 0.075, 0.090, and 0.078, respectively, which are approximately 51, 54, 10, 100, 4, and 4 times the total reducing power of the low concentration, and since the concentrations of the drugs were the same, the total reducing powers of SB1, SB2, and SB4 were significantly changed, and the total reducing powers of SB3, SB5, and SB6 were not significantly changed, but SB3 was significantly higher than those of the other fractions, and the total reducing power was lower than that of BHT.

In conclusion, the detection result is consistent with the result of detecting the cell survival rate, so that the antioxidant activity of SB3 in the primary alkaloid fraction of the sonchus oleraceus is stronger.

Example 4 isolation of secondary alkaloid fraction from Sonchus oleraceus

4.1 Leptochloes Sonchi alkaloid secondary fraction

Rotary evaporating the effective active fraction SB3 at 40 deg.C, concentrating, and freeze drying. SB3 was dissolved in ultrapure water to prepare a sample solution having a concentration of 10mg/mL, which was then passed through a 0.22 μm filter. The crude product was purified by means of a preparative chromatography column Durashell C18(L) (10 μm,30X 250mm) was further fractionated.

The preparation chromatographic conditions are as follows: the mobile phase is a 0.1% formic acid-water (A) -methanol (B) system, the flow rate is 10mL/min, the column temperature is 30 ℃, and the wavelength is 260 nm. The gradient elution procedure is 0-5min, 5-40% B, 5-15min, 40-70% B, 15-30min, 70-80% B, 30-35min, 80-80% B, and the sample loading amount is 18 mL.

according to the chromatographic peaks of the fraction SB3 in the preparation chromatogram, the fraction SB3 is separated into 5 fractions, namely SB3-1, SB3-2, SB3-4 and SB3-5, wherein the peak height range of SB3-1 is 0-0.8AU, the peak height range of SB3-2 is 0.5-5.7AU, the peak height range of SB3-3 is 1.4-5.7AU, the peak height range of SB3-4 is 0.2-1.8AU and the peak height range of SB3-5 is 0.1-0.35AU, as shown in FIG. 21.

4.2 Gesneriana lansium alkaloid Secondary fraction Pair H₂O₂Induction of Caco-2Repair of oxidative damage model of cells

Fractions SB3-1, SB3-2, SB3-3, SB3-4, SB3-5 (concentration: 200. mu.g/mL) were investigated for H₂O₂Inducing the repair effect of the oxidative damage model of Caco-2 cells.

As shown in FIG. 22, the cell viability was 67%, 75%, 87%, 99%, 77%, respectively, and thus SB3-4 was exhibited for H₂O₂the repairing effect of the oxidative damage model of the Caco-2 cells is induced to be obvious.

4.3 Oxidation resistance of secondary alkaloid fraction of Sonchus oleraceus

(1) total antioxidant capacity of secondary alkaloid fraction of Sonchus oleraceus

The detection method was the same as in example 2.

as shown in FIG. 23, the total antioxidant capacity of control, Trolox, extract, SB3, SB3-1 to SB3-5 is 0.07, 6.66, 0.89, 1.98, 0.67, 2.31, 3.44, 5.52, 3.05mmol/g, respectively, so that the total antioxidant capacity of SB3-4 is relatively high, and the total antioxidant capacity of SB3-4 is about 78, 0.8, 6, 3, 8, 2 times as high as that of control, Trolox, extract, SB3, SB3-1, SB3-2, SB3-3, SB3-5, and thus the antioxidant capacity of SB3-4 in the secondary fraction of the alkaloids of Sonchus oleraceae is relatively high.

(2) ABTS free radical scavenging ability of long-crack common sow thistle alkaloid secondary fraction

The detection method was the same as in example 2.

as shown in FIG. 24, when the concentrations of SB3-1 to SB3-5 were 1.6. mu.g/mL, the respective clearance rates for ABTS free radicals were 0.61%, 2.85%, 2.24%, 11.18%, 4.47%, and when the concentration was 1000. mu.g/mL, the respective clearance rates were 75.61%, 75.81%, 76.21%, 76.63%, 76.02%, which were about 124, 27, 34, 7, 17 times the clearance rate for ABTS free radicals at a low concentration, and the clearance rates for ABTS free radicals were not significantly different from each other and were all lower than BHT when the concentrations of SB3-1, SB3-3, SB3-2, SB3-5, and SB3-4, and the secondary fractions of Sonchus oleraceus alkaloids were the greatest.

(3) DPPH free radical scavenging ability of long-crack common sow thistle alkaloid secondary fraction

The detection method was the same as in example 2.

As shown in FIG. 25, when the concentrations of SB3-1 to SB3-5 were 1.6. mu.g/mL, the respective clearance rates for DPPH free radicals were 5.04%, 2.14%, 8.45%, 3.78%, 3.53%, and when the concentrations were 1000. mu.g/mL, the clearance rates were 73.39%, 76.04%, 77.43%, 77.81%, 76.54%, respectively, which were approximately 15, 36, 9, 21, 22 times as high as the clearance rates for DPPH free radicals at low concentrations, and since the concentrations of the drugs were the same, the influence of the change in the clearance rates for DPPH free radicals was in the order of SB3-2, SB3-5, SB3-4, SB3-1, and SB3-3, and when the concentration of the second-stage fraction of the Sonchus oleraceae alkaloid was the greatest, the clearance rates for DPPH free radicals were not significantly different and were all lower than BHT.

(4) Scavenging ability of secondary alkaloid fraction of herba Sonchi arvensis to hydroxyl free radical

the detection method was the same as in example 2.

As shown in FIG. 26, when the concentrations of SB3-1 to SB3-5 were 1.6. mu.g/mL, the clearance rates for hydroxyl radicals were 8.94%, 12.94%, 16.71%, 20.47%, 6.59%, respectively, and when the concentrations were 1000. mu.g/mL, the clearance rates were 77.18%, 78%, 81.41%, 89.53%, 79.18%, respectively, which were about 9, 6, 5, 4, 12 times as high as the clearance rates for hydroxyl radicals at low concentrations, the clearance rates for hydroxyl radicals were all not significantly varied from SB3-1 to SB3-5 due to the same drug concentrations, and the clearance rates for hydroxyl radicals were relatively high when the concentrations of SB3-3 and SB3-4 were the greatest, and the clearance rates for hydroxyl radicals of the secondary fraction of the extended crack Sonchus alkaloids were all lower than that of BHT.

(5) scavenging ability of secondary alkaloid fraction of Sonchus oleraceus to superoxide anion

the detection method was the same as in example 2.

as shown in FIG. 27, the secondary fractions of the alkaloids from Sonchus oleraceus, SB3-1 to SB3-5, had a scavenging ability for superoxide anions and the autoxidation rates of SB3-3 and SB3-4 were significantly slowed down compared to BHT.

(6) Total reducing power of secondary alkaloid fraction of herba Sonchi arvensis

The detection method was the same as in example 2.

As shown in FIG. 28, when the concentrations of SB3-1 to SB3-5 were 20. mu.g/mL, the absorbance values increased were 0.018, 0.011, 0.023, 0.026, 0.016, respectively, and when the concentration was 200. mu.g/mL, the absorbance values increased were 0.140, 0.055, 0.253, 0.273, 0.150, respectively, which were about 8, 5, 11, 10.6, 9.7 times the total reducing power of the low concentrations, and since the concentrations of the drugs were the same, the total reducing power of the secondary fraction of Gesneriana lanigera alkaloid was changed in the order of SB3-3, SB3-4, SB3-5, SB3-1, and SB3-2, and the total reducing power was lower than that of BHT.

in conclusion, the detection result is consistent with the result of detecting the cell survival rate, so that the antioxidant activity of SB3-4 in the secondary alkaloid fraction of the sonchus oleraceus is stronger.

example 5 isolation of tertiary Alkaloids from Sonchus oleraceus

5.1 Tri-stage Total alkaloid fraction of Gesneriana lansium

Collecting SB3-4, rotary evaporating at 40 deg.C, concentrating, and freeze drying. The SB3-4 sample was dissolved in methanol to prepare a test solution with a concentration of 10mg/mL, and the solution was passed through a 0.22 μm filter. The active fraction SB3-4 obtained was further fractionated in analytical chromatography using a column Venusil C18 (250X 4.6mm,5 μm), analytical chromatographic conditions: the mobile phase is a 0.1% formic acid-water (A) -methanol (B) system, the flow rate is 1mL/min, the column temperature is 30 ℃, and the wavelength is 260 nm. Gradient elution procedure is 0-8min, 5% -25% B; 8-10min, 25% -35% B; 10-20min, 35% -60% B; 20-24min, 60% -80% B; 24-27min, 80% -80% B, and 20 mu L of sample loading amount.

the active ingredient was fractionated into 2 fragments, namely SB3-4-1 and SB3-4-2, based on the chromatographic peaks of the active ingredient in the analytical chromatogram, as shown in FIG. 29.

5.2 Gesneriana Sonchifolia alkaloid tertiary fraction Pair H₂O₂Inducing restoration effect of oxidative damage model of Caco-2 cells

Fractions SB3-4-1 and SB3-4-2 (200. mu.g/mL) were investigated for H₂O₂Repair of the induced oxidative damage model of Caco-2 cells.

The results are shown in FIG. 30, in which the cell viability was 105% and 86%, respectively, and SB3-4-1 was exhibited for H₂O₂The induced Caco-2 cell oxidative damage model has obvious repairing effect.

5.3 antioxidant Effect of the third level fraction of Alkaloids sonchifolia

(1) Total antioxidant capacity of tertiary alkaloid fraction of Sonchus oleraceus

the detection method was the same as in example 2.

As shown in FIG. 31, the total antioxidant capacity of control, Trolox, extract, SB3, SB3-4, SB3-4-1, SB3-4-2 is 0.065, 6.66, 0.89, 1.52, 9.45, 5.27mmol/g, respectively, and from these data, it is clear that the total antioxidant capacity of SB3-4-1 is relatively high, and the total antioxidant capacity of SB3-4-1 is about 145, 1, 11, 5, 2 times that of control, Trolox, extract, SB3, SB3-4, SB3-4-2, so that SB3-4-1 in the tertiary fraction of the alkaloids of Sonchus oleraceus L is stronger.

(2) ABTS free radical scavenging ability of long-crack common sow thistle alkaloid tertiary fraction

The detection method was the same as in example 2.

as shown in FIG. 32, when the concentration of SB3-4-1 and SB3-4-2 is 1.6 μ g/mL, the clearance rate of ABTS free radical is 50.2% and 44.29%, respectively, and when the concentration is 1000 μ g/mL, the clearance rate is 94.88% and 84.65%, respectively, which is about 2 and 2 times of the clearance rate of ABTS free radical at low concentration, therefore, the experiment shows that the tertiary fraction of the alkaloid of Sonchus oleraceus has the clearance ability to ABTS free radical and the clearance rate is higher than BHT.

(3) DPPH free radical scavenging ability of Sonchus oleraceus alkaloid tertiary fraction

The detection method was the same as in example 2.

As shown in FIG. 33, the removal rates of DPPH free radicals were 53.77% and 46.79% when SB3-4-1 and SB3-4-2 were 1.6. mu.g/mL, respectively, and 86.23% and 79.25% when SB 894-1 and SB3-4-2 were 1000. mu.g/mL, respectively, which are approximately 1.6 and 1.7 times the removal rate of DPPH free radicals at low concentrations, and therefore, experiments showed that the tertiary fraction of Sonchus oleraceus alkaloid had the ability to remove DPPH free radicals, whereas the removal rate of DPPH free radicals was lower than BHT and the remaining removal rate was higher than BHT when SB3-4-2 was 200. mu.g/mL.

(4) Hydroxyl radical scavenging capacity of sonchus oleraceus alkaloid tertiary fraction

The detection method was the same as in example 2.

as shown in FIG. 34, when the concentration of SB3-4-1 and SB3-4-2 was 1.6. mu.g/mL, the clearance of hydroxyl radicals was 45.95% and 23.22%, respectively, and when the concentration was 1000. mu.g/mL, the clearance was 88.23% and 78.41%, respectively, which are about 1.9 and 3.4 times of the clearance of hydroxyl radicals at low concentration, so that the experiment showed that the tertiary fraction of the alkaloid from Sonchus oleraceus had the clearance of hydroxyl radicals, whereas when the concentration of SB3-4-2 was 1000. mu.g/mL, the clearance of hydroxyl radicals was lower than that of BHT, and the clearance of the rest was higher than that of BHT.

(5) scavenging ability of tertiary alkaloid fraction of Sonchus oleraceus to superoxide anion

the detection method was the same as in example 2.

As shown in FIG. 35, the tertiary alkaloid fraction of Sonchus oleraceus has a scavenging ability for superoxide anions, and the autoxidation rates of SB3-4-1 and SB3-4-2 both have a reduced tendency, and the reduced tendency is lower than that of BHT.

(6) Total reducing power of tertiary alkaloid fraction of Sonchus oleraceus

the detection method was the same as in example 2.

as shown in FIG. 36, the absorbance increases of 0.024 and 0.016 for SB3-4-1 and SB3-4-2 at 20. mu.g/mL, respectively, and 0.293 and 0.233 for SB3-4-2 at 200. mu.g/mL, respectively, which is about 12 times and 15 times the total reducing power of the low concentration, and therefore, the experiment showed that the tertiary fraction of Gesnellenia elongata alkaloid has the total reducing power, whereas the total reducing power of SB3-4-1 is substantially higher than that of BHT, and the total reducing power of SB3-4-2 is lower than that of BHT.

In conclusion, the total antioxidant activity of the fraction SB3-4-1 is more remarkable, and the antioxidant effect and H of the tertiary fraction₂O₂Induced oxidation of Caco-2 cellsThe repairing effect of the damage model is consistent.

Claims

1. The three-stage separated substance of the common sow thistle alkaloid is characterized in that the three-stage separated substance is prepared by the method comprising the following steps:

(1) Preparing extract of herba Sonchi arvensis;

And in the time range of 24-27min, the volume of the mobile phase A is kept at 20%, the volume of the mobile phase B is kept at 80%, and fractions of 16-18min are collected to obtain a third-stage separated matter.

2. The tertiary isolate of extended crack common sow thistle alkaloid in claim 1, wherein in step (2), the column temperature is 30 ℃ and the wavelength is 260 nm.

3. the tertiary isolate of sonchus oleraceus alkaloids of claim 1, wherein sonchus oleraceus extract is prepared by a method comprising the steps of:

using 75% ethanol as solvent, making herba Sonchi arvensis into extractive solution with liquid-material ratio of 30mL/g, adjusting pH of the extractive solution to 5, performing ultrasonic extraction, standing, collecting supernatant, rotary evaporating, cooling, and drying.

4. The tertiary isolate of Sonchus oleraceus alkaloids of claim 3, wherein the power of ultrasonic extraction is 700W, the temperature is 55 ℃, and the ultrasonic time is 30 min.

5. The separation method of the three-stage separated substance of the common sow thistle alkaloid is characterized by comprising the following steps:

(1) preparing extract of herba Sonchi arvensis;

6. the method of claim 5, wherein in step (2), the column temperature is 30 deg.C and the wavelength is 260 nm.

7. the method of isolating tertiary alkali isolates of Sonchus oleraceus of claim 5, wherein the extract of Sonchus oleraceus is prepared by a method comprising the steps of:

Using 75% ethanol as solvent, making herba Sonchi arvensis into extract solution with liquid-material ratio of 30mL/g, performing ultrasonic extraction, standing, collecting supernatant, rotary evaporating, cooling, and drying.

8. the method of claim 5, wherein the power of ultrasonic extraction is 700W, the temperature is 55 deg.C, and the ultrasonic time is 30 min.

9. the use of a tertiary isolate of extended crack common sow thistle alkaloid according to claim 1.