CN110713506A

CN110713506A - Steady delphinidin-3-O-glucoside derivative and preparation method thereof

Info

Publication number: CN110713506A
Application number: CN201911156108.9A
Authority: CN
Inventors: 郑飞; 越皓; 陈长宝; 文连奎; 贺阳
Original assignee: Changchun University of Chinese Medicine
Current assignee: Changchun University of Chinese Medicine
Priority date: 2019-11-22
Filing date: 2019-11-22
Publication date: 2020-01-21

Abstract

The invention relates to a steady delphinidin-3-O-glucoside derivative and a preparation method thereof, belonging to the technical field of compounds and preparation thereof. The problem of the prior art that the stability of the anthocyanin is poor is solved. The structural formula of the derivative is shown as a formula I. The preparation method comprises the following steps: removing stalks of the amur grape, and pulping to obtain amur grape pulp; then extracting anthocyanin in the vitis amurensis serous fluid by adopting a non-thermal extraction technology to obtain an anthocyanin solution; centrifuging the anthocyanin solution, taking supernatant, concentrating under reduced pressure, eluting by macroporous resin, combining eluates, concentrating under reduced pressure, and freeze-drying to obtain an anthocyanin purified product; separating by preparative liquid chromatography to obtain delphinidin-3-O-glucoside; finally, caffeic acid and delphinidin-3-O-glucoside are subjected to reaction, dilution, ultrahigh pressure stabilization and other treatment, and the derivative is obtained. The derivative has better photo-thermal stability than delphinidin-3-O-glucoside, and has the advantages ofAntioxidant and antitumor effects.

Description

Steady delphinidin-3-O-glucoside derivative and preparation method thereof

Technical Field

The invention belongs to the technical field of compounds and preparation thereof, and particularly relates to a steady delphinidin-3-O-glucoside derivative and a preparation method thereof.

Background

Anthocyanins, also called anthocyanins, are widely present in plants, and fruits, vegetables, flowers and grains are rich in anthocyanins, such as blueberries, grapes, lonicera edulis, cowberries, mulberries, strawberries, purple sweet potatoes, purple corns, purple cabbages, black beans, black rice, roselle and the like. The anthocyanin has high content in grape, blueberry and indigo honeysuckle, and especially the vitis amurensis can be used as a good source of natural anthocyanin. Anthocyanins belong to the group of flavonoids, wherein the common anthocyanins include pelargonidin, cyanidin, delphinidin, peonidin, petunidin, malvidin, etc. The anthocyanin is used as a powerful natural antioxidant, is consistent with a mechanism of removing free radicals of flavonoid compounds, has special properties, is positively charged under an acidic condition, has high reaction activity due to the characteristic of natural electron deficiency, and has more advantages in the aspect of removing the free radicals; has wide space for treating human chronic diseases such as diabetes, hypertension and the like, has positive effects on reducing the risk rate of cardiovascular diseases and the incidence rate of obesity, and brings great help to human health.

The natural anthocyanin is bright in color, has great application potential in the fields of food processing and the like, and is widely applied to the fields of functional foods and health-care foods. However, natural anthocyanins have poor stability, are easily influenced by temperature, illumination, pH, oxygen, enzyme, metal ions and the like, are easily degraded in the processes of extraction, processing and storage, and have the phenomena of fading, discoloration, precipitation and the like, so that the application of the anthocyanins is limited. In the prior art, methods for improving the stability of anthocyanin comprise auxiliary color, microencapsulation, bioengineering technology and the like, but the methods can not meet the production requirement due to the limitation of various aspects such as technology, conditions, policy and the like; for example, the interaction color-assisting technology between organic acid and anthocyanin is an important way for improving the stability of anthocyanin, but the spontaneous color-assisting reaction is slow in speed and low in efficiency, and cannot meet the requirement of actual production.

The ultra-high pressure is a non-thermal processing technique for pressurizing a liquid or gas to 100MPa or more, and is mainly used for sterilization, component extraction, aging acceleration, and the like. In the prior art, the research on the application of the ultrahigh pressure technology to the improvement of the stability of the anthocyanin is few, and the stable treatment and mechanism of the ultrahigh pressure prodelphinidin-3-O-glucoside and caffeic acid are not reported.

Disclosure of Invention

In view of the above, the invention provides a steady delphinidin-3-O-glucoside derivative and a preparation method thereof, in order to solve the technical problem of poor stability of anthocyanin in the prior art.

The technical scheme adopted by the invention for solving the technical problems is as follows.

The invention provides a steady delphinidin-3-O-glucoside derivative, which has a structural formula shown as a formula I:

the invention also provides a preparation method of the stabilized delphinidin-3-O-glucoside derivative, which comprises the following steps:

removing stalks of the amur grape, and pulping to obtain amur grape pulp;

step two, extracting anthocyanin in the vitis amurensis serous fluid by adopting a non-thermal extraction technology to obtain an anthocyanin solution;

the non-thermal extraction technology is ultrasonic extraction or high-voltage pulse electric field extraction;

centrifuging the anthocyanin solution, taking supernatant, concentrating under reduced pressure, eluting by macroporous resin, combining eluent, concentrating under reduced pressure, and freeze-drying to obtain an anthocyanin purified product;

dissolving the anthocyanin purified product by using a methanol solution containing 0.5-1% (v/v) HCl, separating by using a preparative liquid chromatography, collecting an elution peak, and freeze-drying to obtain delphinidin-3-O-glucoside;

dissolving caffeic acid and delphinidin-3-O-glucoside in an organic solvent according to a mass ratio of 1 (3-5), uniformly mixing to obtain a mixed solution, and diluting the mixed solution by using a buffer solution with the pH of 3.0-4.0 until the concentration of the delphinidin-3-O-glucoside is 0.1-0.2 mg/ml; firstly, carrying out water bath at 50-60 ℃ for 30-60 min in the dark, then carrying out ultrahigh pressure stabilization treatment at the stable pressure of 160-500MPa for 1-15 min, purifying the obtained treatment liquid by using a macroporous resin column, carrying out reduced pressure concentration, and carrying out freeze drying to obtain the stabilized delphinidin-3-O-glucoside derivative.

Preferably, in the second step, the ultrasonic extraction process is as follows: the extraction solvent is 0.1% hydrochloric acid-75% ethanol solution, the liquid-material ratio is 7:1W/W, the power is 270W, and the time is 15 min.

Preferably, in the second step, the high-voltage pulsed electric field extraction process is as follows: the extraction solvent is 0.1% hydrochloric acid-75% ethanol solution, liquid-material ratio is 9:1w/w, electric field intensity is 15Kv/cm, and pulse number is 4.

Preferably, in the third step, the model of the macroporous resin is D101, and the elution process of the macroporous resin is as follows: and (3) loading and eluting at the rate of 1.5BV/h, standing for 30min after loading, washing with purified water with the volume of 5-8 times of the column volume to remove impurities, and eluting with 0.1% HCl-75% ethanol solution until colorless.

Preferably, in the third step, the supernatant is taken and concentrated under reduced pressure until the relative density is 1.1-1.2 and the temperature is 50 ℃; the combined eluates are decompressed and concentrated to the relative density of 1.2-1.3 at the temperature of 50 ℃.

Preferably, in the fourth step, the conditions for preparing the liquid chromatographic separation are as follows:

a chromatographic column: SunFere Prep C₁₈A column of dimensions 19mm x 50mm, 5 μm; the detection wavelength is 530 nm; the column temperature is 25 ℃; the flow rate is 5 mL/min; the sample injection amount is 150 mu L; mobile phase a-5% aqueous formic acid, B-methanol, gradient elution: 0-5 min, 15% B; 5-8 min, 15% -20% of B; 8-13 min, 10% -15% of B; 13-16 min, 25% -31% of B; 16-17 min, 31% -100% B; 17-19 min, 100% B; 19-20 min, 100% -15% of B; 20-23 min, 15% B.

Preferably, in the fifth step, the concentration of delphinidin-3-O-glucoside is 0.1 mg/ml.

Preferably, in the fifth step, the water bath temperature is 55 ℃ and the water bath time is 45 min.

Preferably, in the fifth step, the stable pressure is 200 to 400 MPa.

Preferably, in the fifth step, the mixture is concentrated under reduced pressure until the relative density is 1.2-1.3 and the temperature is 50 ℃.

Preferably, in the fifth step, the organic solvent is absolute ethyl alcohol.

Preferably, in the fifth step, the buffer is disodium hydrogen phosphate-citric acid buffer or acetic acid-sodium acetate buffer.

Compared with the prior art, the invention has the beneficial effects that:

the preparation method of the steady-state delphinidin-3-O-glucoside derivative adopts ultrasonic extraction or high-voltage pulse electric field extraction to extract anthocyanin in the vitis amurensis, so that the anthocyanin is protected from being damaged to the greatest extent; the prepared product is delphinidin-3-O- (6' -O-caffeoyl) -glucoside, and the ultra-high pressure stabilization treatment is adopted, so that compared with delphinidin-3-O-glucoside, the product has better photo-thermal stability and has the functions of oxidation resistance and tumor resistance. Through experimental detection, the hyperchromic rate of delphinidin-3-O- (6' -O-caffeoyl) -glucoside can reach more than 40%, and the R values of the preservation rates can reach more than 60% after heating for 2 hours at 100 ℃ and placing in the sun for 20 days at room temperature; after the HUVEC cells with oxidative damage are treated by the delphinidin-3-O- (6' -O-caffeoyl) -glucoside with different concentrations, the cell proliferation rate is remarkably increased, and the active oxygen is remarkably reduced; after the treatment of HepG-2 liver cancer cells and HL-60 acute promyelocytic leukemia cells, the cell inhibition rate is obviously increased.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

In fig. 1, a and B are HPLC profiles of delphinidin 3-O-glucoside and delphinidin-3-O-glucoside derivatives of example 1, respectively, and C is an HPLC profile of the product prepared in comparative example 1;

FIG. 2 is an MS/MS spectrum of the delphinidin-3-O-glucoside derivative of example 1.

Detailed Description

For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the detailed description, but it is to be understood that the description is intended to further illustrate the features and advantages of the invention and not to limit the claims to the invention.

The structural formula of the steady-state delphinidin-3-O-glucoside derivative is shown as the formula I:

the preparation method of the steady-state delphinidin-3-O-glucoside derivative comprises the following steps:

removing stalks of the amur grape, and pulping to obtain amur grape pulp;

dissolving caffeic acid and delphinidin-3-O-glucoside in an organic solvent according to a mass ratio of 1 (3-5), uniformly mixing to obtain a mixed solution, and diluting the mixed solution by using a buffer solution with the pH of 3.0-4.0 until the concentration of the delphinidin-3-O-glucoside is 0.1-0.2 mg/ml; firstly, carrying out water bath at 50-60 ℃ for 30-60 min in the dark, then carrying out ultrahigh pressure stabilization treatment at a stable pressure of 200-400MPa for 1-15 min, purifying the obtained treatment liquid by using a macroporous resin column, carrying out reduced pressure concentration, and carrying out freeze drying to obtain the stabilized delphinidin-3-O-glucoside derivative.

The principle of the invention is as follows: the forming mechanism of the stabilized 3-O-glucoside derivative is as follows: bonding delphinidin-3-O-glucoside with caffeic acid to remove one molecule of H₂O delphinidin-3-O- (6' -O-caffeoyl) -glucoside, the reaction process is as follows:

in the above technical scheme, in the step one, the vitis amurensis is not particularly limited, and can be obtained by a person skilled in the art according to a well-known manner. The higher the anthocyanin content, the better the vitis amurensis, preferably the vitis amurensis wild in Changbai mountain. The specific operations of destemming and beating the vitis amurensis are well known to those skilled in the art, and the process is not particularly limited.

In the second step, the non-thermal extraction technology is ultrasonic extraction or high-voltage pulse electric field extraction; the ultrasonic extraction process and the high-voltage pulse electric field extraction are non-thermal extraction techniques well known to those skilled in the art, and the invention researches that the effect is best when the following processes are adopted, so the preferred ultrasonic extraction process is as follows: extracting solvent 0.1% hydrochloric acid-75% ethanol solution at a liquid-material ratio of 7:1W/W and power of 270W for 15 min; the preferable high-voltage pulse electric field extraction process comprises the following steps: the extraction solvent is 0.1% hydrochloric acid-75% ethanol solution, the liquid-material ratio is 9:1w/w, the electric field intensity is 15Kv/cm, and the pulse number is 4.

In the above technical scheme, in the third step, the conditions of centrifugation, concentration under reduced pressure and freeze drying are not particularly limited. The preferred centrifugation conditions are: centrifuging at 4500r/min for 10 min; the supernatant fluid is obtained after centrifugation by either slowly pouring out the supernatant fluid or by filtration. Preferably, the supernatant is concentrated under reduced pressure until the relative density is 1.1-1.2 and the temperature is 50 ℃. Preferably, the combined eluates are concentrated under reduced pressure until the relative density is 1.2-1.3 and the temperature is 50 ℃. Preferred conditions for freeze-drying are: the pre-freezing temperature is-35 ℃, the heating temperature is 40 ℃, and the vacuum degree is 30 Pa. The model of the macroporous resin used for eluting the macroporous resin is preferably D101. The macroporous resin elution and the process of combining the eluents comprises the following steps: and (3) loading and eluting at the rate of 1.5BV/h, standing for 30min after loading, purifying and washing with water by 5-8 times of column volume to remove impurities, eluting with 0.1-75% HCl-75% ethanol until colorless, and mixing eluates.

In the technical scheme and the step four, the separation of the delphinidin-3-O-glucoside by using the preparative liquid chromatography is the prior art, and a person skilled in the art can separate the delphinidin-3-O-glucoside according to the retention time of the delphinidin-3-O-glucoside. The present invention provides a method for preparing a liquid chromatography separation, but is not limited thereto: a chromatographic column: SunFere Prep C₁₈A column of dimensions 19mm x 50mm, 5 μm; the detection wavelength is 530 nm; the column temperature is 25 ℃; the flow rate is 5 mL/min; the sample injection amount is 150 mu L; mobile phase a-5% aqueous formic acid, B-methanol, gradient elution: 0-5 min, 15% B; 5-8 min, 15% -20% of B; 8-13 min, 10% -15% of B; 13-16 min, 25% -31% of B; 16-17 min, 31% -100% B; 17-19 min, 100% B; 19-20 min, 100% -15% of B; 20-23 min, 15% B. The conditions for freeze-drying are not particularly limited, but prefreezing temperature-35 deg.C, heating temperature 40 deg.C, and vacuum degree 30Pa are preferred.

In the technical scheme, in the fifth step, the mass ratio of the caffeic acid to the delphinidin-3-O-glucoside is 1 (3-5), if the adding amount of the delphinidin-3-O-glucoside is less than 3 times of the mass of the caffeic acid, the product contains unbound delphinidin-3-O-glucoside, and if the adding amount of the delphinidin-3-O-glucoside is more than 5 times of the mass of the caffeic acid, the product also contains unbound delphinidin-3-O-glucoside. The organic solvent is preferably absolute ethanol, the dissolving sequence is not particularly limited, and caffeic acid can be dissolved firstly, and delphinidin-3-O-glucoside can be added. The mode of mixing uniformly is not particularly limited, and mechanical stirring and mixing can be adopted. The water bath temperature is 50-60 ℃, and if the water bath temperature is lower than 50 ℃, the hyperchromicity rate and the light and heat stability of the prepared derivative are reduced; the anthocyanin is easy to degrade, the higher the temperature is, the higher the degradation speed is, and if the water bath temperature is higher than 60 ℃, the anthocyanin is easy to degrade; preferably, the temperature of the water bath is 55 ℃ and the time of the water bath is 45 min. The stable pressure of the ultrahigh pressure stabilization treatment is 160-500MPa, the hyperchromicity rate and the light and heat stability of the prepared derivative are firstly increased and then reduced along with the increase of the stable pressure, if the stable pressure is less than 160MPa or more than 500MPa, the hyperchromicity rate and the light and heat stability of the prepared derivative are not ideal, and when the stable pressure is 200-400MPa, the hyperchromicity rate and the light and heat stability effects of the prepared derivative are better, so the stable pressure is preferably 200-400MPa, and when the stable pressure is 300MPa, the hyperchromicity rate and the light and heat stability effects of the prepared derivative are optimal, and the most preferable stable pressure is 300 MPa. The dwell time is preferably 3-7 min, and more preferably 5 min. The concentration of delphinidin-3-O-glucoside is preferably 0.1 mg/ml. The conditions of the concentration under reduced pressure and the freeze-drying are not particularly limited. Preferably, the mixture is concentrated under reduced pressure until the relative density is 1.2-1.3 and the temperature is 50 ℃. Preferably, the prefreezing temperature is-35 deg.C, the heating temperature is 40 deg.C, and the vacuum degree is 30 Pa. The buffer solution is preferably disodium hydrogen phosphate-citric acid buffer solution or acetic acid-sodium acetate buffer solution.

The invention is further illustrated by the following examples.

Example 1

Removing stalks of the amur grape, and pulping to obtain amur grape pulp;

step two, extracting anthocyanin in the vitis amurensis slurry by adopting a high-voltage pulse electric field to obtain anthocyanin solution; wherein the extraction solvent is 0.1% hydrochloric acid-75% ethanol solution, liquid-material ratio is 9:1w/w, electric field intensity is 15Kv/cm, pulse number is 4;

centrifuging the anthocyanin solution, taking supernatant, concentrating under reduced pressure (until the relative density is 1.1 and 50 ℃), eluting by macroporous resin, combining eluates, concentrating under reduced pressure (until the relative density is 1.2 and 50 ℃), and freeze-drying to obtain an anthocyanin purified product; wherein, the conditions for preparing the liquid phase chromatographic separation are as follows: a chromatographic column: SunFere Prep C₁₈A column of dimensions 19mm x 50mm, 5 μm; the detection wavelength is 530 nm; the column temperature is 25 ℃; the flow rate is 5 mL/min; the sample injection amount is 150 mu L; mobile phase a-5% aqueous formic acid, B-methanol, gradient elution: 0-5 min, 15% B; 5-8 min, 15% -20% of B; 8-13 min, 10% -15% of B; 13-16 min, 25% -31% of B; 16-17 min, 31% -100% B; 17-19 min, 100% B; 19E up to e20min，100％～15％B；20～23min，15％B；

dissolving caffeic acid with absolute ethyl alcohol, adding delphinidin-3-O-glucoside with the mass 3 times that of the caffeic acid, and uniformly mixing to obtain a mixed solution; diluting the mixed solution with disodium hydrogen phosphate-citric acid buffer solution with pH of 3.0 to make the concentration of delphinidin-3-O-glucoside be 0.1mg/ml, water-bathing at 50 deg.C for 30min in dark place, carrying out ultrahigh pressure stabilization treatment at stable pressure of 300MPa for 5min, purifying the obtained treatment solution with macroporous resin column, concentrating under reduced pressure (to relative density of 1.2, 50 deg.C), and freeze-drying to obtain stabilized delphinidin-3-O-glucoside derivative.

Example 2

Removing stalks of the amur grape, and pulping to obtain amur grape pulp;

step three, centrifuging the anthocyanin solution, taking supernatant, concentrating under reduced pressure (until the relative density is 1.2 and 50 ℃), eluting by macroporous resin, combining eluates, concentrating under reduced pressure (until the relative density is 1.3 and 50 ℃), and freeze-drying to obtain an anthocyanin purified product;

dissolving the anthocyanin purified product by using a methanol solution containing 0.5-1% (v/v) HCl, separating by using a preparative liquid chromatography, collecting an elution peak, and freeze-drying to obtain delphinidin-3-O-glucoside; wherein, the conditions for preparing the liquid phase chromatographic separation are as follows: a chromatographic column: SunFere Prep C₁₈A column of dimensions 19mm x 50mm, 5 μm; the detection wavelength is 530 nm; the column temperature is 25 ℃; the flow rate is 5 mL/min; the sample injection amount is 150 mu L; mobile phase a-5% aqueous formic acid, B-methanol, gradient elution: 0-5 min, 15% B; 5-8 min, 15% -20% of B; 8-13 min, 10% -15% of B; 13-16 min, 25% -31% of B; 16E17min，31％～100％B；17～19min，100％B；19～20min，100％～15％B；20～23min，15％B；

Dissolving caffeic acid with absolute ethyl alcohol, adding delphinidin-3-O-glucoside with the mass being 4 times that of the caffeic acid, and uniformly mixing to obtain a mixed solution; diluting the mixture with disodium hydrogen phosphate-citric acid buffer solution with pH of 3.0 to make the concentration of delphinidin-3-O-glucoside 0.1mg/ml, and water-bathing at 50 deg.C for 30min in dark place; performing ultrahigh pressure stabilization treatment at a stable pressure of 300MPa for 5min, purifying the obtained treatment solution with macroporous resin column, concentrating under reduced pressure (to relative density of 1.3, 50 deg.C), and freeze drying to obtain steady delphinidin-3-O-glucoside derivative.

Example 3

Removing stalks of the amur grape, and pulping to obtain amur grape pulp;

dissolving the anthocyanin purified product by using a methanol solution containing 0.5-1% (v/v) HCl, separating by using a preparative liquid chromatography, collecting an elution peak, and freeze-drying to obtain delphinidin-3-O-glucoside; wherein, the conditions for preparing the liquid phase chromatographic separation are as follows: a chromatographic column: SunFere Prep C₁₈A column of dimensions 19mm x 50mm, 5 μm; the detection wavelength is 530 nm; the column temperature is 25 ℃; the flow rate is 5 mL/min; the sample injection amount is 150 mu L; mobile phase a-5% aqueous formic acid, B-methanol, gradient elution: 0-5 min, 15% B; 5-8 min, 15% -20% of B; 8-13 min, 10% -15% of B; 13-16 min, 25% -31% of B; 16-17 min, 31% -100% B; 17-19 min, 100% B; 19-20 min, 100% -15% of B; 20-23 min, 15% B;

dissolving caffeic acid with absolute ethyl alcohol, adding delphinidin-3-O-glucoside with the mass 5 times that of the caffeic acid, and uniformly mixing to obtain a mixed solution; diluting the mixture with acetic acid-sodium acetate buffer solution with pH of 3.6 to make the concentration of delphinidin-3-O-glucoside 0.1mg/ml, and water-bathing at 50 deg.C for 30min in dark place; performing ultrahigh pressure stabilization treatment at a stable pressure of 300MPa for 5min, purifying the obtained treatment solution with macroporous resin column, concentrating under reduced pressure (relative density of 1.2 and 50 deg.C), and freeze drying to obtain steady delphinidin-3-O-glucoside derivative.

Example 4

Removing stalks of the amur grape, and pulping to obtain amur grape pulp;

secondly, extracting anthocyanin in the vitis amurensis serous fluid by adopting ultrasonic to obtain anthocyanin solution; wherein the extraction solvent is 0.1% hydrochloric acid-75% ethanol solution, the liquid-material ratio is 7:1W/W, the power is 270W, and the time is 15 min;

dissolving caffeic acid with absolute ethyl alcohol, adding delphinidin-3-O-glucoside with the mass being 3 times of that of the caffeic acid, and uniformly mixing to obtain a mixed solution; diluting the above mixed solution with acetic acid-sodium acetate buffer solution with pH of 4.0 to make the concentration of delphinidin-3-O-glucoside 0.2mg/ml, and water-bathing at 60 deg.C for 60min in dark place; performing ultrahigh pressure stabilization treatment at a stable pressure of 300MPa for 5min, purifying the obtained treatment solution with macroporous resin column, concentrating under reduced pressure (relative density of 1.2 and 50 deg.C), and freeze drying to obtain steady delphinidin-3-O-glucoside derivative.

Example 5

The stable pressure was replaced by 250MPa in step five of example 3, and the other conditions were the same as in example 3.

Example 6

The steady pressure was replaced with 350MPa in step five of example 3, and the other conditions were the same as in example 3.

Example 7

The pressure holding time in the fifth step of example 3 was replaced with 3min, and the other conditions were the same as in example 3.

Example 8

The pressure holding time in the fifth step of example 3 was replaced with 7min, and the other conditions were the same as in example 3.

Comparative example 1

The procedure of example 1 was repeated except that the amount of delphinidin-3-O-glucoside added in step five of example 1 was changed to 1 time the mass of caffeic acid, and the procedure was otherwise as in example 1.

Comparative example 2

The procedure of example 1 was repeated except that the amount of delphinidin-3-O-glucoside added in step five of example 1 was changed to 2 times the mass of caffeic acid, and the procedure was otherwise as in example 1.

Comparative example 3

The procedure of example 1 was repeated except that the amount of delphinidin-3-O-glucoside added in step five of example 1 was changed to 6 times the mass of caffeic acid, and the procedure was otherwise as in example 1.

Comparative example 4

The same procedure as in example 1 was repeated except that the steady pressure in step five of example 1 was changed to 150 MPa.

Comparative example 5

The steady pressure was replaced with 550MPa in step five of example 1, and the other conditions were the same as in example 1.

Comparative example 6

In the fifth step of example 1, the water bath temperature was replaced with 30 ℃ and the other conditions were the same as in example 1.

1.1 structural identification was carried out on the derivatives prepared in examples 1 to 8 and comparative examples 1 to 6.

Analytical chromatographic conditions were column: thermo Syncronis C₁₈Column (100 mm. times.3 mm, 1.7 μm), gradient elution, mobile phase: 0.1% aqueous formic acid (a) and acetonitrile (B); gradient elution: 0-5 min, 8-10% of B; 5-23 min, 10% B; 23-38 min, 10-15% of B; 38-48 min, 15-20% B; 48-53 min, 20-25% B; 53-66 min, 25-35% B; 66-71 min, 35-45% B; 71-76 min, 45-55% of B; 76-80 min, 55-80% B; the column temperature is 30 ℃; flow rate 0.2 mL/min^-1(ii) a The sample size was 5. mu.L. The mass spectrum condition adopts an electrospray positive ion scanning mode (ESI)⁺) The temperature of the dry gas is 350 ℃, the flow rate of the atomization gas is 35arb, the flow rate of the auxiliary gas is 10arb, and the mass scanning range m/z is 50-1000. The results are shown in FIGS. 1 and 2.

In FIG. 1, A and B are HPLC profiles of delphinidin 3-O-glucoside and delphinidin-3-O-glucoside derivatives, respectively, in example 1; FIG. 2 is an MS/MS spectrum of delphinidin-3-O-glucoside derivative of example 1. As can be seen from FIGS. 1 and 2, in the positive ion mode, the bonded derivative of delphinidin-3-O-glucoside and caffeic acid has an excimer ion peak m/z 627.13562, and fragment ions m/z 465.13562, 303.13525 and 141.13497 are visible in the secondary tandem mass spectrum. Wherein the fragment ion m/z 465.13562 is generated by loss of 1 caffeoyl group with molecular mass number 162 from molecular ion m/z 627.13562, and the fragment ion m/z 303.13525 is generated by continued loss of 1 glucose residue with molecular mass number 162 from fragment ion m/z 465.13562; m/z 141.13497 is generated by breaking C-C bond at 0/2 position of C ring of anthocyanin^0,2A^+·Radical cations. According to the report of the related literature and comprehensive analysis, the delphinidin-3-O- (6' -O-caffeoyl) -glucoside is identified. The analytical results of examples 2 to 8 were similar to those of example 1. The products prepared in the comparative examples 1-6 are structurally identified by an HPLC-MS/MS combined technology, wherein C in the figure 1 is an HPLC (high performance liquid chromatography) spectrum of the product prepared in the comparative example 1, and as can be seen from the figure 1, the product prepared in the comparative example 1In the prepared products, two compounds were detected, and both compounds contained unbound delphinidin-3-O-glucoside, as compared with example 1. The analysis results of the products prepared in comparative examples 2 to 6 were similar to that of comparative example 1.

1.2 evaluation of stability of the derivatives prepared in examples 1 to 8 and comparative examples 1 to 6.

And (3) determining an absorbance value at lambda 521nm by taking delphinidin which is not subjected to high-pressure bonding treatment as a reference, calculating a stabilized hyperchromicity ratio I (%) according to a formula 1-1, and evaluating the reaction effect.

I(％)＝[(A-A₀)/A₀]X 100 formula (1-1)

In the formula: a is the absorbance value of the steady-state delphinidin-3-O-glucoside solution; a. the₀The absorbance value of the untreated delphinidin-3-O-glucoside solution was obtained.

Using delphinidin without high pressure bonding treatment as a control, treating the sample at 100 ℃ for 2.0h and under sunlight for 20d, respectively, measuring absorbance values at lambda 521nm, and calculating the storage rate R (%) after stabilization according to the formula 1-2.

R(％)＝(A_tA) x 100 formula (1-2)

In the formula: a. the_tThe absorbance value of the steady-state delphinidin-3-O-glucoside solution after different treatment time of light and heat; a is the absorbance value of the steady delphinidin-3-O-glucoside solution before light and heat treatment.

The results are shown in tables 1 and 2. As can be seen from Table 1, the derivatives prepared in examples 1 to 3 had a hyperchromic ratio of 40% or more; the preservation rate R value can reach more than 60 percent after being heated for 2 hours at 100 ℃ and placed in the sunlight for 20 days at room temperature. As can be seen from Table 2, the derivatives prepared in comparative examples 1 to 6 have reduced color-increasing ratios and light stabilities as compared with the derivatives prepared in examples 1 to 8.

TABLE 1 evaluation results of stability of derivatives prepared in examples 1 to 8

TABLE 2 evaluation results of stability of derivatives prepared in comparative examples 1 to 6

1.3 the derivatives prepared in examples 1 to 3 were tested for antioxidant activity.

The assay used a cellular assay, culture of Human Umbilical Vein Endothelial Cells (HUVEC): taking out HUVEC cells from liquid nitrogen, quickly putting the HUVEC cells into a water bath kettle at 37 ℃, and slightly shaking a freezing tube to dissolve a freezing solution; transferring the cells into a centrifuge tube containing 5mL of culture medium, centrifuging at room temperature of 1000rpm/min for 5min, and removing the supernatant; suspending cells in cell culture medium containing 10% fetal calf serum, inoculating to culture dish, gently blowing, mixing, placing cells at 37 deg.C and 5% CO₂Culturing under saturated humidity condition. When the density of the cells reached 80%, the cells were passaged. Removing the culture medium, and washing with PBS buffer solution; adding 1-2 mL of 0.25% trypsin to digest cells, observing under a microscope, digesting for 1-2 min, and observing that the cells are separated from each other and become round, namely finishing digestion; finally, quickly removing pancreatin, adding complete culture medium, blowing to make cell, making into single cell suspension, passaging according to the ratio of 1:3, placing cell at 37 deg.C and 5% CO₂And (5) carrying out amplification culture under the saturated humidity condition. Selecting 4-9 generations of cells with good growth state for testing.

CCK-8 measures cell proliferation rate: taking HUVEC cells in logarithmic growth phase and good growth state, and performing cell growth treatment according to the ratio of 5 × 10³Inoculating each well in 96-well plate, adding 200 μ L culture solution into each well, collecting three parallel samples, standing at 37 deg.C and 5% CO₂Culturing for 24h in an incubator; blank group, model group, control group and example group are all treated for 24h, 20 microliter CCK-8 solution is added into each well, the mixture is cultured for 4h at 37 ℃, and the light absorption value OD 450 of each well is measured by a microplate reader.

Cell proliferation rate (%) ═ (OD)_{Sample set}/OD_{Control group}) X 100% formula (1-3)

Cell inhibition ratio (%) < 1- > (OD_{Sample set}/OD_{Control group}) X 100% formula (1-4)

Determination of intracellular ROS levels: taking HUVEC cells in logarithmic growth phase and good growth state, and performing cell growth treatment according to the ratio of 5 × 10³Inoculating into 96-well plate, adding culture medium 200 μ L per well, 37 deg.C, and 5% CO₂Culturing in an incubator for 24h, treating blank group, model group, control group and example group for 24h, and rinsing cells with PBS buffer solution for 1 time; then according to the operation instructions of the DCFH-DA cell ROS detection kit, diluting DCFH-DA by a serum-free culture solution according to a ratio of 1:1000 to enable the final concentration to be 10 mu moL L, adding the diluted DCFH into the cells by 200 mu L, incubating in an incubator at 37 ℃ for 20min, uniformly mixing every 3min, washing the cells for 2 times by using a serum-free culture medium, resuspending by using a PBS buffer solution, and carrying out fluorescence detection by using a microplate reader (the excitation wavelength is 480nm, and the emission wavelength is 525 nm).

Ratio (Ratio, R) ═ A_{Sample set}/A_{Control group}Formula (1-5)

In the formula: a. the_{Sample set}Is the absorbance value of the test group; a. the_{Control group}Absorbance values for the control group.

Establishing a HUVE cell oxidative damage model: investigation of H separately₂O₂After HUVEC cells were treated at concentrations of 100. mu. mol/L, 200. mu. mol/L, 300. mu. mol/L, 500. mu. mol/L, 750. mu. mol/L, and 1000. mu. mol/L for various periods of time (6 hours, 12 hours, 24 hours, and 48 hours), the cell inhibition rate was examined by the CCK-8 method. Each group is provided with 3 multiple holes, the results are averaged, and different concentrations H are calculated₂O₂Inhibition ratio (%) of cell proliferation, in terms of 50% (IC)₅₀) H of (A) to (B)₂O₂Concentration as H of subsequent experiment₂O₂Concentration, and detecting the level of intracellular ROS. Results are shown in Table 3, as treatment time increased, H₂O₂Half inhibitory concentration IC for inducing oxidative stress injury of HUVEC cells₅₀Gradually decreased, exhibiting concentration dependence; different concentrations of H₂O₂The results of intracellular ROS level measurements after different time treatments of HUVEC cells are shown in Table 4, while H₂O₂ROS ratio in cells at 300. mu. mol/L solution concentrationThere was a significant difference (p) from the beginning compared to the blank group<0.05), the ROS ratio in the cells was significantly different (p) compared with the blank group at 24h and 48h of culture<0.01) and the ROS ratio is 2.56 at 24h, the cell damage is obvious and stable, so the concentration of the selected induction model in the experiment is 300 mu mol/L H₂O₂HUVEC cells were induced in solution for 24 h.

TABLE 3H₂O₂Effect of inducing cells on half inhibitory concentration of cells at different times

TABLE 4 different concentrations H₂O₂Effect of different time of treating cells on intracellular ROS levels

Note: significant differences (p <0.05) and very significant differences (p <0.01) were indicated compared to the blank (0 μmol/L).

Examples 1 to 3 pairs of H₂O₂Effects of inducing oxidative damage of HUVEC cells: cells in logarithmic growth phase are taken for random grouping after passage for testing. Including the blank control group, H₂O₂An induction model group, a caffeic acid control group, a delphinidin-3-O-glucoside group (10. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L), an example 1 group (10. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L), an example 2 group (10. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L), and an example 3 group (10. mu. mol/L, 50. mu. mol/L, 100. mu. mol/L). Cell proliferation rate and intracellular ROS levels were determined separately as described above. Results are shown in tables 5 and 6, H₂O₂The cell proliferation rate of the induction model group is 0.52 times of that of the blank control group, and the induction model group has very significant difference (p)<0.01), indicating successful modeling with a significant increase in intracellular ROS release (p)<0.01). Caffeic acid control of different concentrationsGroup and H₂O₂Compared with an induction model group, the cell proliferation rate has no significant difference (p)>0.05), which indicates that caffeic acid with the concentration of 100 mu mol/L has no protective effect on oxidative damage cells when being treated for 24 hours; after the treatment of the delphinidin-3-O-glucoside group with different concentrations, the cell proliferation rate is remarkably increased, and the cell proliferation rate is increased with H at the concentrations of 50 mu mol/L and 100 mu mol/L₂O₂Significant difference (p) compared with the induction model group<0.05) and very significant difference (p)<0.01), the intracellular ROS release is significantly reduced (p)<0.01), which indicates the group of delphinidin-3-O-glucoside to H₂O₂The HUVEC cell oxidative damage induction has certain protection effect and is concentration-dependent. After treatment of the example groups at different concentrations, the cell proliferation rate was significantly increased, at concentrations of 50. mu. mol/L and 100. mu. mol/L, with H₂O₂The induction model group shows very significant difference (p)<0.01) and at the same time the intracellular ROS release is significantly reduced (p)<0.01), the cell proliferation rates were all very significantly different (p) compared to the delphinidin-3-O-glucoside group<0.01), no statistical difference in ROS levels (p)>0.05). The delphinidin-3-O- (6' -O-caffeoyl) -glucoside prepared in example was explained with reference to H as well₂O₂The HUVEC cell oxidative damage induction has a certain protection effect, and simultaneously, the antioxidant activity of the HUVEC cell oxidative damage induction is stronger than that of delphinidin-3-O-glucoside, and the HUVEC cell oxidative damage induction is concentration-dependent.

TABLE 5 Effect of different concentrations of the derivatives of the examples on the proliferation Rate of HUVEC cells

Note: significant differences (p <0.05) and very significant differences (p <0.01) compared to the blank group; compared with the model group, the delta represents significant difference (p <0.05), and the delta represents very significant difference (p < 0.01); compared with the group of non-stabilized anthocyanin, the tangle-solidup represents significant difference (p is less than 0.05), and the tangle-solidup represents very significant difference (p is less than 0.01).

TABLE 6 Effect of different concentrations of the derivatives of the examples on ROS levels in HUVEC cells

1.4 antitumor activity test was conducted on the derivatives prepared in examples 1 to 3.

The test adopts cell test, and the preparation of cell culture solution and buffer solution: solution 1: complete DMEM culture solution-10, transferring 50mL FBS, 5mL (10mmol/L) 100 XNEAA solution, 5mL (10,000U/mL penicillin and 10,000g/mL streptomycin) 100 Xdouble antibody, and adding incomplete DMEM culture solution to 500 mL; the pH was 7.35. Solution 2: complete RPMI medium-10, remove FBS100mL, 100 XNEAA solution 5mL (10mmol/L), 100 Xdouble antibody 5mL (10,000U/mL penicillin and 10,000g/mL streptomycin), add incomplete RPMI medium to 500 mL; the pH was 7.35. Culturing of tumor cells: HepG-2 liver cancer cells are cultured by using the solution 1, and HL-60 acute promyelocytic leukemia cells are cultured by using the solution 2. And respectively taking out the two cells from the liquid nitrogen, respectively recovering the two cells by using corresponding solutions, changing the solutions every day, and carrying out passage when the cell confluence rate is about 80 percent, wherein the passage ratio is 1: 3. And continuing to culture for 1-2 generations after the cells are stable.

Examples 1-3 examination of antitumor Activity:

screening the cells after subculture for 3-4 generations for anti-tumor activity, digesting the cells from the wall of the culture flask with 0.25% trypsin-EDTA when the cells grow to about 80%, centrifuging, adding corresponding solution to dilute the cells to 2 × 10⁵cells/mL, fully and uniformly blown.

The cell suspension was seeded in 96-well plates at 100. mu.L per well in CO₂Culturing at 37 deg.C for 24 h. Quickly sucking out the culture solution, adding 100 μ L culture medium containing different concentrations of medicine, and placing in CO again₂The incubator is used for 48 hours. Wherein the medicine isThe concentrations were 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, and 3.125. mu.M, respectively. Taking out 96-well plate after 48 hr, adding 10 μ L of CCK-8 solution into each well, slightly shaking to mix well, and placing in CO₂Culturing for 1-4 h at 37 ℃ in an incubator, and measuring the OD value after the result is stable, wherein the wavelength is 450 nm. Each 96-well plate was set with 6 blank wells as controls, and each compound was run in triplicate for each tumor cell, and all results were statistically analyzed. As shown in tables 7 and 8, the delphinidin-3-O- (6' -O-caffeoyl) -glucoside prepared in examples 1 to 3 inhibited the growth of HepG-2 cells by IC₅₀The concentrations are 63.25 μ M/L, 58.21 μ M/L and 60.38 μ M/L respectively, and IC for inhibiting HL-60 cell growth₅₀The concentrations are respectively 49.32 muM/L, 48.25 muM/L and 47.67 muM/L, and the antitumor activity is better.

TABLE 7 results of anti-HepG-2 activity of derivatives of the different examples

TABLE 8 results of anti-HL-60 activities of derivatives of different examples

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The stabilized delphinidin-3-O-glucoside derivative is characterized in that the structural formula is shown as a formula I:

2. the method for producing a stabilized delphinidin-3-O-glucoside derivative according to claim 1, comprising the steps of:

removing stalks of the amur grape, and pulping to obtain amur grape pulp;

3. The method for preparing the steady-state delphinidin-3-O-glucoside derivative of claim 1, wherein in the second step, the ultrasonic extraction process is: the extraction solvent is 0.1% hydrochloric acid-75% ethanol solution, the liquid-material ratio is 7:1W/W, the power is 270W, and the time is 15 min.

4. The method for producing a stabilized delphinidin-3-O-glucoside derivative according to claim 1, wherein in the second step, the high-voltage pulsed electric field extraction process comprises: the extraction solvent is 0.1% hydrochloric acid-75% ethanol solution, liquid-material ratio is 9:1w/w, electric field intensity is 15Kv/cm, and pulse number is 4.

5. The preparation method of the steady-state delphinidin-3-O-glucoside derivative of claim 1, wherein in the third step, the model of the macroporous resin is D101, and the elution process of the macroporous resin is as follows: the sampling and elution rate is 1.5BV/h, standing for 30min after sampling, washing with purified water with 5-8 times of column volume to remove impurities, and eluting with 0.1% HCl-75% ethanol solution until colorless;

in the third step, taking the supernatant, and concentrating the supernatant under reduced pressure until the relative density is 1.1-1.2 and the temperature is 50 ℃; the combined eluates are decompressed and concentrated to the relative density of 1.2-1.3 at the temperature of 50 ℃.

6. The method for producing a stabilized delphinidin-3-O-glucoside derivative according to claim 1, wherein in the fourth step, the conditions for the preparative liquid chromatography are as follows:

7. The method for preparing a stabilized delphinidin-3-O-glucoside derivative according to claim 1, wherein in the fifth step, the water bath temperature is 55 ℃ and the water bath time is 45 min. .

8. The method for producing a stabilized delphinidin-3-O-glucoside derivative according to claim 1, wherein in the fifth step, the stable pressure is 200-400 MPa.

9. The method for producing a stabilized delphinidin-3-O-glucoside derivative according to claim 1, wherein in the fifth step, the concentration is performed under reduced pressure until the relative density is 1.2 to 1.3 and the temperature is 50 ℃.

10. The method for producing a stabilized delphinidin-3-O-glucoside derivative according to claim 1, wherein in the fifth step, the organic solvent is absolute ethanol; the buffer solution is disodium hydrogen phosphate-citric acid buffer solution or acetic acid-sodium acetate buffer solution.