LU502858B1

LU502858B1 - Alkaloid antioxidant, preparation method and application thereof

Info

Publication number: LU502858B1
Application number: LU502858A
Authority: LU
Inventors: Ruitao Yu
Original assignee: Northwest Inst Plateau Bio Cas
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2023-03-31

Abstract

The invention discloses an alkaloid antioxidant, its preparation method and application thereof, and belong to the technical field of alkaloid compound extraction. The specific preparation method is as follows: Heating and refluxing the whole herb of Lepidium latifolium L. with ethanol to obtain crude extract; then, silica gel column chromatography, gel column chromatography, reversed-phase high-pressure liquid preparative chromatography separation and reversed-phase preparative column purification are used to obtain the alkaloid antioxidant. The alkaloid antioxidant separated and prepared by the invention has a remarkable total antioxidant capacity; furthermore, the raw material of the invention, Lepidium latifolium L., has a wide source, and large-scale operation can be realized by methods such as ethanol heating reflux extraction, and the purity of the product can be guaranteed to be more than 98% by high-pressure liquid chromatography.

Description

DESCRIPTION LU502858

ALKALOID ANTIOXIDANT, PREPARATION METHOD AND

APPLICATION THEREOF

TECHNICAL FIELD

The invention belongs to the technical field of alkaloid compound extraction, and in particular to an alkaloid antioxidant, its preparation method and application thereof.

BACKGROUND

Oxidative Stress (OS) refers to a state of imbalance between oxidation and antioxidation in the body, which tends to oxidize, leading to inflammatory infiltration of neutrophils, increased secretion of protease, and production of a large number of oxidation intermediates. Oxidative stress is a negative effect produced by free radicals in the body, and it is considered as an important factor leading to aging and diseases.

Lepidium latifolium L. is a plant of the genus Lepidium in the Cruciferae family (Brassicaceae), also known as horseradish and antidysenteric grass. Widely distributed and rich in resources, Lepidium latifolium L. is distributed in Gansu, Qinghai, Ningxia and other places, growing on the edge of fields, ridges, ditches and river valleys. It has certain value of development and utilization. As recorded in "Medicinal Plants in the Desert Regions of China",

Lepidium latifolium L. has the functions of clearing heat and eliminating dampness, and treating bacillary dysentery and enteritis. Modern pharmaceutical researches show that Lepidium latifolium L. has certain anti-oxidation and anti-aging effects. Therefore, it has important application value to isolate and identify antioxidants from Lepidium latifolium L. and apply them to antioxidant, anti-aging and related drug synthesis.

SUMMARY

In order to solve the problems in the prior art, the invention provides an alkaloid antioxidant, a preparation method and application thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

The first technical scheme of the invention: an alkaloid antioxidant, the molecular formula is CisH12N20,, and the structural formula is:

2 = LU502858 9 | + 9 5

Zz SS — ;

The second technical scheme of the invention: a preparation method of the alkaloid antioxidant, comprises the following steps: (1) reflux-extracting Lepidium latifolium L. with ethanol, and concentrating the extracting solution to obtain a crude extract: (2) extracting the crude extract obtained in step (1) with petroleum ether, ethyl acetate and n-butanol, and concentrating the extract to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction; (3) separating the n-butanol fraction obtained in step (2) by normal-phase silica gel column chromatography, performing gradient elution by using dichloromethane-methanol mixed mobile phase, concentrating and drying the eluate to obtain components Fr. 1-Fr. 4 in sequence; (4) separating the component Fr.4 obtained in the step (3) by using microporous resin open column chromatography, performing gradient elution by using a methanol-water mixed mobile phase, and concentrating the eluate to sequentially obtain the components Fr. 4-1 to Fr. 4-10; (5) separating the component Fr.4-2 obtained in step (4) by using a reversed-phase high-pressure liquid chromatographic column, eluting by using a formic acid solution-acetonitrile mixed mobile phase, detecting the eluate by an ultraviolet detector, collecting a chromatographic peak fraction, and drying to obtain the component Fr. 4-2-1; (6) purifying the component Fr.4-2-1 obtained in step (5) by using a reverse phase preparation column, eluting by using a formic acid solution-acetonitrile mixed mobile phase, detecting the eluent by an ultraviolet detector, collecting a chromatographic peak fraction, and drying to obtain the component Fr.4-2-1-2, namely the alkaloid antioxidant.

Further, in step (1), Lepidium latifolium L. is powder of Lepidium latifolium L., and the specific preparation method comprises the following steps: drying the whole plant of Lepidium latifolium L. in the shade, crushing and sieving by a 40-mesh sieve.

Further, in step (1), the volume fraction of the ethanol is 95%, and the solid-liquid ratio of the Lepidium latifolium L. to the ethanol is 1 kg: (5-50) L; the reflux extraction is carried out At 502858 70°C for 2-4 hours with 2-4 times; the vacuum degree of concentration 1s 0.07-0.09 MPa, and the temperature is 50-60 C.

Further, in step (2), the volume ratio of the crude extract to the petroleum ether, ethyl acetate or n-butanol is 1:1; the extraction times are 2-4 times, the vacuum degree of concentration is 0.07-0.09 MPa, and the temperature is 50-60°C.

Further, in step (3), before the normal-phase silica gel column chromatography separation, the n-butanol fraction needs to be mixed with 200-300 mesh silica gel, and the ratio of the n-butanol fraction to the silica gel is 1g: 1-3 mL; the volume ratio of dichloromethane to methanol in gradient elution of dichloromethane-methanol mixed mobile phase is 15:1, 10:1, 5:1 and 1:1 in turn; the vacuum degree of drying is 0.07-0.09 MPa, and the temperature is 50-60"C.

Further, in step (4), the stationary phase of the microporous resin open column chromatography is MCI microporous resin; before the separation by using microporous resin open column chromatography, the component Fr.4 needs to be dissolved in water and passed through a 0.45 micron microporous filter membrane, and the mass-volume ratio of Fr.4 to water is 1:1; the volume ratio of methanol in gradient elution of methanol-water mixed mobile phase is 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in turn; the vacuum degree of concentration is 0.07-0.09 MPa, and the temperature is 50-60°C.

Further, in step (5), the length of the reverse-phase high-pressure liquid chromatographic column is 250mm, the diameter is 20mm, the stationary phase is Sum Dubhe C18; the volume fraction of acetonitrile in the formic acid solution-acetonitrile mixed mobile phase is 14%, the volume fraction of formic acid in the formic acid solution is 0.2%; the elution time is 55min, and that elution flow rate is 19 mL/min; the wavelength of the ultraviolet detector is 254nm; the vacuum degree of the drying is 0.07-0.09 MPa and the temperature is 50-60°C.

Further, in step (6), the length of the reverse phase preparation column is 250mm, the diameter is 10mm, the stationary phase is Sum Unitary analytical column; the volume fraction of acetonitrile in the formic acid solution-acetonitrile mixed mobile phase is 5%, and the volume fraction of formic acid in the formic acid solution is 0.2%; the elution time is 60min, and the elution flow rate is 5.0mL/min; the wavelength of the ultraviolet detector is 254nm; the vacuum degree of drying is 0.07-0.09 MPa, and the temperature is 50-60°C.

The third technical scheme of the invention: an application of the alkaloid antioxidant in the preparation of antioxidant drugs. LU502858

Compared with the prior art, the invention has the following beneficial effects: (1) The alkaloid antioxidant separated and prepared by the invention has a remarkable total antioxidant capacity; (2) The purity of the alkaloid antioxidant separated and prepared by the invention can reach 98%; (3) The raw material of the invention is wide in source, and the method of extraction, silica gel column chromatography, gel column chromatography, high pressure liquid chromatography and the like can realize large-scale operation.

BRIEF DESCRIPTION OF THE FIGURES

In order to more clearly explain the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the embodiments. Obviously, the drawings in the following description is only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to these drawings without any creative effort.

FIG. 1 is a 'H-NMR spectrum of the alkaloid antioxidant prepared in embodiment 1 of the present invention;

FIG. 2 is a **C-NMR spectrum of the alkaloid antioxidant prepared in embodiment 1 of the present invention;

FIG. 3 is a control diagram of the antioxidant activities of the alkaloid antioxidant prepared in embodiment 1, vitamin C solution and quercetin solution.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention are now described in detail, which should not be construed as limiting the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In addition, for numerical ranges in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed.

Each smaller range between any stated value or stated range of intermediate values and any other stated value or intermediate value within the stated range is also included within the present invention. The upper and lower limits of these smaller ranges may independently be included PU502858 excluded from the range.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. While the present invention describes only the preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to said documents. In case of conflict with any incorporated literature, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations can be made to the specific embodiments of the present specification without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The specification and embodiment of that present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open ended terms that mean including, but not limited to.

Embodiment 1

Isolation and preparation of the alkaloid antioxidant from Lepidium latifolium L. (1) pretreatment of raw materials: drying the whole herb of Lepidium latifolium L. in the shade, crushing, and sieving with a 40-mesh sieve to obtain raw material powder; (2) extraction: reflux-extracting Skg of the raw material powder obtained in step (1) with

SOL 95vol.% ethanol at an extraction temperature of 70°C for 4 hours, repeating the extraction for 4 times, combining the four extractive solutions, and concentrating under reduced pressure at a vacuum degree of 0.09MPa and a temperature of 60°C to obtain a crude extract; (3) leaching: diluting the crude extract obtained in step (2) to 4L with water, sequentially extracting with 4L of petroleum ether, ethyl acetate and n-butanol, extracting each extractant for 4 times to obtain a petroleum ether fraction, an ethyl acetate fraction and a n-butanol fraction, and concentrating each fraction under reduced pressure under the conditions of vacuum degree of 0.09MPa and temperature of 60°C; (4) performing silica gel column chromatography coarse fraction: mixing 0.2L of the n-butanol fraction obtained in step (3) with 200g of 200-300 mesh silica gel, loading on a normal, 502858 phase silica gel column, performing gradient elution by using dichloromethane-methanol mixed mobile phase with the volume ratio of 15:1, 10:1, 5:1 and 1:1, collecting the eluent obtained by eluting by using the dichloromethane-methanol mixed mobile phase with the volume ratio of 1:1, concentrating the eluent under reduced pressure under the condition that the vacuum degree is 0.09MPa, the temperature is 60°C, and drying in an oven at 50°C to obtain a component Fr. 4; (5) performing gel column chromatography coarse fraction, namely dissolving 10g of the component Fr4 obtained in step (4) in 10mL of water, passing through a 0.45 micron microporous membrane, and performing separation by using microporous resin open column chromatography, the microporous resin open column chromatographic stationary phase is MCI microporous resin, the mobile phase A is water, and the mobile phase B is methanol, wherein the volume concentration of the methanol is 0, 10%, 20%, 30%, ..., linearly increasing 100%, performing gradient elution, and respectively segmenting to obtain 10 eluents, and respectively concentrating the 10 eluents under reduced pressure under the condition of vacuum degree of 0.09MPa and temperature of 60°C to obtain components Fr. 4-1 to Fr. 4-10; (6) high-pressure liquid chromatography separation: separating 2mL of the component

Fr.4-2 obtained in step (5) by reversed-phase high-pressure liquid chromatography, with a column length of 250mm, a diameter of 20mm and a stationary phase of Sum Dubhe C18, the mobile phase A is 0.2% formic acid-water and B is acetonitrile, where the volume fraction of acetonitrile is 14%, the injection amount is 0.5mL, the mobile phase flow rate is 19mL/min, and the elution time is 55min, after that, detecting the eluent by an ultraviolet detector with a detection wavelength of 254nm, and injecting the sample for 4 times under the same chromatographic conditions. Collecting and combining chromatographic peak fractions in the prepared chromatogram, and drying the fractions under reduced pressure under the conditions of vacuum degree of 0.09MPa and temperature of 60°C to obtain component Fr.4-2-1; (7) Reverse phase preparation column purification: dissolving the component Fr.4-2-1 obtained in step (6) in 1 mL of 10% methanol, purifying the dissolved Fr.4-2-1 by using the reverse preparation column, the length of the reverse phase preparation column is 250mm, the diameter is 10mm, the stationary phase is Sum Unitary analytical column; the mobile phase A is 0.2% formic acid-water solution, and the mobile phase B is acetonitrile solution, where the volume fraction of acetonitrile is 5%, the injection amount is 200uL, the mobile phase flow rate is 5.0mL/min, and the elution time is 60min, after that, detecting the eluent by an ultraviolet, 502858 detector with a detection wavelength of 254nm, and injecting the sample for 5 times under the same chromatographic conditions. Collecting and combining chromatographic peak fractions in the prepare chromatograms, and drying the fractions under reduced pressure under the condition of the vacuum degree being 0.09MPa and the temperature being 60°C to obtain a component

Fr.4-2-1-2, namely an alkaloid antioxidant in the Lepidium latifolium L.

The purity of the alkaloid antioxidant prepared in the embodiment is 98%.

Embodiment 2

Structural identification of alkaloid antioxidants

The alkaloid antioxidant prepared in embodiment 1 is subjected to structural identification using nuclear magnetic resonance (\H-NMR, C-NMR) and the results are shown in FIG. 1 and

FIG. 2. Where, FIG. 1 is a 'H-NMR spectrum of the alkaloid antioxidant prepared in embodiment 1 of the present invention; FIG. 2 is a "C-NMR spectrum of the alkaloid antioxidant prepared in embodiment 1 of the present invention, the specific physicochemical data of the alkaloid antioxidant are as follows:

Fr.4-2-1-2: yellow powder, 'H NMR (600 MHz, Methanol-d4) § 8.30 (1H, d, J = 5.2 Hz,

H-3), 8.20 (1H, d, J = 7.8 Hz, H-5), 8.04 (IH, d, J = 5.2 Hz, H-4), 7.72 (1H, d, J = 8.2 Hz, H-8), 7.60 (2H, t, J = 7.5 Hz, H-7), 7.30 (1H, t, J = 7.5 Hz, H-6), 7.24 (1H, d, J = 3.2 Hz, H-3"), 6.60 (1H, d, J = 3.2 Hz, H-4"), 4.77 (2H, s, H-6'). *C NMR (151 MHz, Methanol-d4) 8 157.34 (C-2'), 154.07 (C-5'), 143.15 (C-9b), 138.40 (C-3), 134.23 (C-1), 132.51 (C-9a), 132.31 (C-4b), 130.12 (C-4a), 122.65 (C-5), 122.12 (C-7), 121.28 (C-6), 115.04 (C-8), 113.30 (C-4), 111.18 (C-3"), 111.06 (C-4"), 57.58 (C-6").

The structural formula of the obtained alkaloid is as follows: 9

IAN

Lu 9

Zz > 1

Effect example:

Antioxidant activity experiment

For the alkaloid antioxidant prepared in embodiment 1, the total antioxidant capacity,502858 (T-AOC) determination kit 1s used to determine the total antioxidant capacity of the sample, and the specific steps are as follows: (1) Preparation of FeSO4-7H20 standard solution: Weighing 27.8mg of FeSO4-7H20 standard (provided by the total antioxidant capacity (T-AOC) determination kit), dissolving it in

ImL of 50% methanol, preparing a solution with a concentration of 100mM, and diluting it to standard solutions with concentrations of 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5mM respectively; (2) Preparation of positive control: Accurately weighing 3.6mg of vitamin C to prepare a vitamin C solution with a concentration of 20.43 mM, adding 50% methanol to dilute to 0.06mM, accurately weighing 6.1mg of quercetin to prepare a quercetin solution with a concentration of 20.18 mM, and adding 50% methanol to dilute to 0.22mM; preparation of sample solution:

Accurately weighing 5.3mg Fr.4-2-1-2(Perlolyrine), preparing a solution of perlolyrine with a concentration of 20 mM, and adding 50% methanol to dilute it to 0.28 mM. (3) In the 96-well plate, setting blank hole, standard hole and measuring hole, adding SuL of 50% methanol and 180 pL of frap working solution into the blank hole, adding FeSO4-7H20 standard solution into the standard hole, and adding 5 pL. of sample solution to be tested and 180 uL of frap working solution into the measuring hole, mixing well, incubating at 37°C for 3-5 minutes, measuring the absorbance at 593nm, and repeating three times for each sample; reading the OD value of each well with a microplate reader. (4) Standard curve: after subtracting the light absorption value of a blank hole from each hole, preparing a standard curve by taking the light absorption value of a standard substance as an abscissa and the concentration of the standard substance corresponding to each light absorption value as an ordinate, and preparing a curve formula by using EXCEL; (5) Calculation: substituting the absorbance value measured by the sample measuring tube (The blank absorption value needs to be subtracted.) into the standard curve formula, the obtained result is expressed as the concentration (mM) of FeSO4-7H,0 standard solution.

Taking vitamin C solution and quercetin solution as positive control, the results are shown in FIG. 3. It can be found that the total antioxidant capacity is: alkaloid antioxidant (Perlolyrine, 0.28mM) > quercetin (Quercitrin, 0.22mM) > vitamin C (Vitamin C, 0.06mM).

The above are only the preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. Any person familiar with the technical field who makes equivalent substitution or change according to the technical scheme and 502858 inventive concept of the present invention within the technical scope disclosed by the present invention should be covered in the scope of protection of the present invention.

Claims

CLAIMS LU502858

1. An alkaloid antioxidant, characterized in that, the molecular formula is Ci6H12N20,, and the structural formula is: Sa 95 9a A = qd

2. A preparation method of the alkaloid antioxidant according to claim 1, characterized by comprising the following steps: (1) reflux-extracting Lepidium latifolium L. with ethanol, and concentrating the extracting solution to obtain a crude extract: (2) extracting the crude extract obtained in step (1) with petroleum ether, ethyl acetate and n-butanol, and concentrating the extract to obtain petroleum ether fraction, ethyl acetate fraction and n-butanol fraction; (3) separating the n-butanol fraction obtained in step (2) by normal-phase silica gel column chromatography, performing gradient elution by using dichloromethane-methanol mixed mobile phase, concentrating and drying the eluate to obtain components Fr. 1-Fr. 4 in sequence; (4) separating the component Fr.4 obtained in the step (3) by using microporous resin open column chromatography, performing gradient elution by using a methanol-water mixed mobile phase, and concentrating the eluate to sequentially obtain the components Fr. 4-1 to Fr. 4-10; (5) separating the component Fr.4-2 obtained in step (4) by using a reversed-phase high-pressure liquid chromatographic column, eluting by using a formic acid solution-acetonitrile mixed mobile phase, detecting the eluate by an ultraviolet detector, collecting a chromatographic peak fraction, and drying to obtain the component Fr. 4-2-1; (6) purifying the component Fr.4-2-1 obtained in step (5) by using a reverse phase preparation column, eluting by using a formic acid solution-acetonitrile mixed mobile phase, detecting the eluent by an ultraviolet detector, collecting a chromatographic peak fraction, and drying to obtain the component Fr.4-2-1-2, namely the alkaloid antioxidant.

3. The preparation method according to claim 2, characterized in that, in step (1), Lepidium latifolium L. is powder of Lepidium latifolium L., and the specific preparation method comprises the following steps: drying the whole plant of Lepidium latifolium L. in the shade, crushing and 502858 sieving by a 40-mesh sieve.

4. The preparation method according to claim 2, characterized in that, in step (1), the volume fraction of the ethanol is 95%, and the solid-liquid ratio of the Lepidium latifolium L. to the ethanol is 1 kg: (5-50) L; the reflux extraction is carried out at 70°C for 2-4 hours with 2-4 times; the vacuum degree of concentration is 0.07-0.09 MPa, and the temperature is 50-60°C.

5. The preparation method according to claim 2, characterized in that, in step (2), the volume ratio of the crude extract to the petroleum ether, ethyl acetate or n-butanol is 1:1; the extraction times are 2-4 times; the vacuum degree of concentration is 0.07-0.09 MPa, and the temperature is 50-60°C.

6. The preparation method according to claim 2, characterized in that, in step (3), before the normal-phase silica gel column chromatography separation, the n-butanol fraction needs to be mixed with 200-300 mesh silica gel, and the ratio of the n-butanol fraction to the silica gel is 1g: 1-3 mL; the volume ratio of dichloromethane to methanol in gradient elution of dichloromethane-methanol mixed mobile phase is 15:1, 10:1, 5:1 and 1:1 in turn; the vacuum degree of drying is 0.07-0.09 MPa, and the temperature is 50-60 C.

7. The preparation method according to claim 2, characterized in that, in step (4), the stationary phase of the microporous resin open column chromatography is MCI microporous resin, before the separation by using microporous resin open column chromatography, the component Fr.4 needs to be dissolved in water and passed through a 0.45 micron microporous filter membrane, and the mass-volume ratio of Fr.4 to water is 1:1; the volume ratio of methanol in gradient elution of methanol-water mixed mobile phase is 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% in turn; the vacuum degree of concentration is 0.07-0.09 MPa, and the temperature is 50-60°C.

8. The preparation method according to claim 2, characterized in that, in step (5), the length of the reverse-phase high-pressure liquid chromatographic column is 250mm, the diameter is 20mm, the stationary phase is Sum Dubhe C18; the volume fraction of acetonitrile in the formic acid solution-acetonitrile mixed mobile phase is 14%, the volume fraction of formic acid in the formic acid solution is 0.2%; the elution time is 55min, and that elution flow rate is 19 mL/min; the wavelength of the ultraviolet detector is 254nm; the vacuum degree of the drying is 0.07-0.09 MPa and the temperature is 50-60°C.

9. The preparation method according to claim 2, characterized in that, in step (6), the length 02858 of the reverse phase preparation column is 250mm, the diameter is 10mm, the stationary phase is Sum Unitary analytical column; the volume fraction of acetonitrile in the formic acid solution-acetonitrile mixed mobile phase is 5%, and the volume fraction of formic acid in the formic acid solution is 0.2%; the elution time is 60min, and the elution flow rate is 5.0mL/min; the wavelength of the ultraviolet detector is 254nm; the vacuum degree of drying is 0.07-0.09 MPa, and the temperature is 50-60°C.

10. An application of the alkaloid antioxidant according to claim 1 in the preparation of antioxidant drugs.