CN112939737B - Compound extracted from burdock leaves and having effect of protecting alcoholic liver injury and preparation method and application thereof - Google Patents
Compound extracted from burdock leaves and having effect of protecting alcoholic liver injury and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a compound extracted from burdock leaves and having the function of protecting alcoholic liver damage, a preparation method and application thereof, which can effectively solve the problems of preparing the compound having the function of protecting alcoholic liver damage from burdock leaves and developing the new application of the burdock leaves in medicinal value, the method comprises the steps of soaking the burdock leaves in water, filtering, concentrating, extracting by ethyl acetate in sequence, concentrating the ethyl acetate extract liquid under reduced pressure, carrying out silica gel column chromatography, carrying out gradient elution by dichloromethane-methanol, recovering solvent under reduced pressure, carrying out MCI column chromatography again, carrying out gradient elution by methanol water solution, merging eluent, drying, recrystallizing in methanol to obtain the compound having the function of reducing blood fat and protecting alcoholic liver damage extracted from the burdock leaves, the preparation method has the advantages of easy operation, strong guidance, high separation speed and high product purity, and the compound can be effectively used for preparing the medicine for reducing blood fat and protecting alcoholic liver damage, develops the new application and medicinal value of the burdock leaves and has great economic and social benefits.
Description
Technical Field
The invention relates to a medicine, in particular to a compound extracted from burdock leaves and having the function of protecting alcoholic liver injury, a preparation method and application thereof.
Background
The burdock leaf is dry primary leaf of Arctiumlappa of Compositae, is widely distributed in China, has a long medicinal history in major production areas of Shandong and Xuzhou, and can prevent apoplexy and hand and foot paralysis in traditional herbal medicine. People still have the habit of using the burdock leaf tea to drink so as to prevent and treat hyperlipidemia.
The incidence of alcoholic liver disease is increasing worldwide, and the alcoholic liver disease is a disease which develops progressively and seriously harms the health and even the life in the later period. The modern medicine for treating alcoholic liver diseases has no specific treatment except for abstinence and symptomatic treatment. In recent years, traditional Chinese medicine accumulates abundant experience in preventing and treating alcoholic liver diseases.
However, the compounds separated from the burdock leaves for regulating blood fat and preventing and treating alcoholic liver injury and the application of the compounds in preparing the medicines for preventing and treating the alcoholic liver injury are not reported in a public way.
Disclosure of Invention
In view of the above situation, in order to overcome the defects of the prior art, the invention aims to provide a compound extracted from burdock leaves and having the effect of protecting alcoholic liver injury, and a preparation method and application thereof, which can effectively solve the problems of preparing the compound having the effect of protecting alcoholic liver injury from burdock leaves and exploiting the new application of burdock leaf medicinal value.
The invention solves the technical scheme that a compound which is extracted from burdock leaves and has the function of protecting alcoholic liver injury is a compound NBY-3 which is extracted from burdock leaves and has the function of protecting alcoholic liver injury, and the molecular structural formula is as follows:
the preparation method comprises the following steps:
(1) crushing 10kg of dried burdock leaves, adding water which is 6-15 times of the weight of the burdock leaves, soaking for 5-14 days at room temperature, filtering, and concentrating the filtrate to a concentrated solution (5-10L) which is equivalent to 0.1-3 g/mL of crude drug;
(2) sequentially extracting the concentrated solution with ethyl acetate for 4 times, wherein the dosage of each time is 1-5L, 0.5-3L and 0.5-3L, combining ethyl acetate extract, and concentrating under reduced pressure to obtain component Fr ETOAC (25-100 g); component Fr ETOAC Performing silica gel column chromatography with the inner diameter of 3-6 cm and the height of 8-30 cm, respectively performing gradient elution with dichloromethane-methanol at a gradient ratio of 100:3 and 3-10L, and a gradient ratio of 100:5 and 5-20L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2(20～30g)；
(3) Component Fr ETOAC -2, performing MCI column chromatography with the inner diameter of 1.5-4 cm and the height of 20-80 cm, performing gradient elution by using methanol water solutions with volume concentrations of 10%, 20% and 40%, wherein the dosage of each gradient is 0.5-5L, 0.5-5L and 2-10L respectively, collecting 40% methanol eluent, and recovering the solvent under reduced pressure to obtain a component Fr ETOAC -2-3(3～10g)；
(4) Component Fr ETOAC And (3) recrystallizing the-2-3 in methanol to obtain NBY-3 (30-80 mg) which is extracted from the burdock leaves and has the effects of reducing fat and protecting alcoholic liver injury.
The compound extracted from the burdock leaves is identified and named NBY-3, has the function of reducing blood fat and protecting alcoholic liver injury, can be effectively used for preparing the medicine for protecting the alcoholic liver injury, realizes the application in preparing the medicine for reducing the blood fat and protecting the alcoholic liver injury, has easy operation of the preparation method, strong guidance quality, high separation speed and high product purity, can be effectively used for preparing the medicine for reducing the blood fat and protecting the alcoholic liver injury, develops the new application and medicinal value of the burdock leaves, and has huge economic and social benefits.
Drawings
FIG. 1 is a molecular structural formula of compound NBY-3 of the present invention;
FIG. 2 is a graph relating the major HMBC and H-H COSY of compound NBY-3 of the present invention;
FIG. 3 is a NOESY correlation plot of compound NBY-3 of the present invention;
FIG. 4 is a photograph of compound NBY-3 of the present invention 1 H-NMR spectrum;
FIG. 5 is a photograph of compound NBY-3 of the present invention 13 A C-NMR spectrum;
FIG. 6 is an HSQC spectrum of compound NBY-3 of the present invention;
FIG. 7 is a HMBC spectrum of compound NBY-3 of the present invention;
FIG. 8 is a chart of the infrared spectrum of compound NBY-3 of the present invention;
FIG. 9 is a UV spectrum of compound NBY-3 of the present invention;
FIG. 10 is a mass spectrum of compound NBY-3 of the present invention;
FIG. 11 is a single crystal diffractogram of compound NBY-3 of the present invention;
FIG. 12 is a process flow diagram of compound NBY-3 of the present invention.
Detailed Description
The following examples are provided to explain the present invention in detail.
The invention may be embodied in the form of the following examples.
Example 1
In the specific implementation of the invention, the preparation method of the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury comprises the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 9 times of folium Arctii weight, soaking at room temperature for 8 days, filtering, and concentrating the filtrate to obtain concentrated solution (6L) equivalent to crude drug 2 g/mL;
(2) extracting the concentrated solution with ethyl acetate for 4 times (3L, 2L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (60g) (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 4cm and height of 20cm, respectively eluting with dichloromethane-methanol at gradient ratio of 100:3 and dosage of 6L, and dichloromethane-methanol at gradient ratio of 100:5 and dosage of 18L, collecting the 100:5 gradientEluting, recovering solvent under reduced pressure to obtain component Fr ETOAC -2(25g)；
(3) Component Fr ETOAC -2 performing MCI column chromatography with inner diameter of 3cm and height of 35cm, gradient eluting with 10%, 20% and 40% methanol water solution with each gradient dosage of 3L, 3L and 6L respectively, mixing eluates, and drying to obtain FrETOAC-2-3(3 g);
(4) component Fr ETOAC Recrystallizing the extract from folium Arctii with methanol to obtain NBY-3(50mg) with effects of reducing blood lipid and protecting alcoholic liver injury.
Example 2
In the specific implementation of the invention, the preparation method of the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury comprises the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 8 times of folium Arctii weight, soaking at room temperature for 11 days, filtering, and concentrating the filtrate to obtain concentrated solution equivalent to crude drug 3 g/mL;
(2) extracting the concentrated solution with ethyl acetate for 4 times (1.5L, 4.5L, 0.7L, and 2.8L each time), mixing the ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (20-40 g); component Fr ETOAC Performing silica gel column chromatography with inner diameter of 4cm and height of 28cm, respectively performing gradient elution with dichloromethane-methanol at gradient ratio of 100:3 and 4L, and gradient ratio of 100:5 and 18L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2(8～15g)；
(3) Component Fr ETOAC -2, performing MCI column chromatography with the inner diameter of 1.8cm and the height of 45cm, performing gradient elution by using 10%, 20% and 40% methanol aqueous solution with the volume concentration of 1L, 4L and 3L respectively, collecting 40% methanol eluent, and recovering the solvent under reduced pressure to obtain a component FrETOAC-2-3 (1.5-5 g);
(4) component Fr ETOAC -2-3 is recrystallized in methanol to obtain NBY-3 (15-35 mg) which is extracted from burdock leaves and has the effects of reducing fat and protecting alcoholic liver injury.
Example 3
In the specific implementation of the invention, the preparation method of the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury comprises the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 10 times of folium Arctii weight, soaking at room temperature for 6 days, filtering, and concentrating the filtrate to 5L concentrate corresponding to 0.5g/mL crude drug;
(2) extracting the concentrated solution with ethyl acetate for 4 times (4.5L, 1.5L, 2.8L, and 0.7L each time), mixing the ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (20-40 g); component Fr ETOAC Performing silica gel column chromatography with inner diameter of 5.5cm and height of 10cm, respectively eluting with dichloromethane-methanol at gradient ratio of 100:3 and 9L, and dichloromethane-methanol at gradient ratio of 100:5 and 6L, collecting 100:5 gradient eluate, recovering solvent under reduced pressure to obtain component Fr ETOAC -2(8～15g)；
(3) Component Fr ETOAC -2, carrying out MCI column chromatography with the inner diameter of 3.5cm and the height of 25cm, carrying out gradient elution by using 10%, 20% and 40% methanol aqueous solutions with the volume concentration of 4L, 1L and 9L respectively, collecting 40% methanol eluent, and recovering the solvent under reduced pressure to obtain a component FrETOAC-2-3 (1.5-5 g);
(4) component Fr ETOAC Recrystallizing the-2-3 in methanol to obtain NBY-3 (15-35 mg) which is extracted from the burdock leaves and has the effects of reducing fat and protecting alcoholic liver injury.
Example 4
In the specific implementation of the invention, the preparation method of the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury comprises the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 10 times of folium Arctii weight, soaking at room temperature for 5 days, filtering, and concentrating the filtrate to 1.5L corresponding to 0.4g/mL of crude drug concentrate;
(2) extracting the concentrated solution with ethyl acetate for 4 times (1.5L, 1L, 0.5L, and 0.5L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (23g) (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 3cm and height of 25cm, respectively, with dichloromethane-methanolGradient elution with dichloromethane-methanol gradient ratio of 100:3 and dosage of 3L and dichloromethane-methanol gradient ratio of 100:5 and dosage of 7L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2(9g)；
(3) Component Fr ETOAC -2 performing MCI column chromatography with inner diameter of 1.5cm and height of 45cm, performing gradient elution with 10%, 20% and 40% methanol aqueous solution, wherein the amount of each gradient is 1L, 1L and 4L, respectively, collecting 40% methanol eluate, and recovering solvent under reduced pressure to obtain a component FrETOAC-2-3(2.5 g);
(4) component Fr ETOAC Recrystallizing the extract from folium Arctii with methanol to obtain NBY-3(18mg) with effects of reducing blood lipid and protecting alcoholic liver injury.
Example 5
In the specific implementation of the invention, the preparation method of the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury comprises the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 9 times of folium Arctii weight, soaking at room temperature for 7 days, filtering, and concentrating the filtrate to 2.5L corresponding to 0.3g/mL of the concentrated solution;
(2) sequentially extracting the concentrated solution with ethyl acetate for 4 times (3L, 1.5L, 1L, and 0.5L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (28g) (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 4cm and height of 20cm, respectively performing gradient elution with dichloromethane-methanol at gradient ratio of 100:3 and 4L, and gradient ratio of 100:5 and 10L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2(11g)；
(3) Component Fr ETOAC -2 performing MCI column chromatography with an inner diameter of 2cm and a height of 40cm, performing gradient elution with 10%, 20% and 40% methanol aqueous solution, wherein the amount of each gradient is 2L, 2L and 5L respectively, collecting 40% methanol eluate, and recovering solvent under reduced pressure to obtain FrETOAC-2-3(3 g);
(4) component Fr ETOAC -2-3 is recrystallized in methanol to obtain extract from folium Arctii with effects of reducing blood lipid and protecting alcoholic liver damageCompound NBY-3(25mg) as a wound.
Example 6
In the specific implementation of the invention, the preparation method of the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury comprises the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 10 times of folium Arctii weight, soaking at room temperature for 9 days, filtering, and concentrating the filtrate to 4.5L corresponding to 0.2g/mL of the concentrated solution;
(2) extracting the concentrated solution with ethyl acetate for 4 times (4.5L, 3L, 2L, 0.5L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (35g) (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 6cm and height of 10cm, respectively performing gradient elution with dichloromethane-methanol at gradient ratio of 100:3 and dosage of 7L and dichloromethane-methanol at gradient ratio of 100:5 and dosage of 14L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2(13g)；
(3) Component Fr ETOAC -2 performing MCI column chromatography with an inner diameter of 3cm and a height of 30cm, performing gradient elution with 10%, 20% and 40% methanol aqueous solution by volume, wherein the amount of each gradient is 3.5L, 3.5L and 8L respectively, collecting 40% methanol eluate, and recovering solvent under reduced pressure to obtain a component FrETOAC-2-3(4 g);
(4) component Fr ETOAC Recrystallizing the extract from folium Arctii with methanol to obtain NBY-3(30mg) with effects of reducing blood lipid and protecting alcoholic liver injury.
Example 7
In the specific implementation of the invention, the preparation method of the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury comprises the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 9 times of folium Arctii weight, soaking at room temperature for 10 days, filtering, and concentrating the filtrate to 4.5L corresponding to 0.2g/mL of the concentrated solution;
(2) extracting the concentrated solution with ethyl acetate for 4 times (4.5L, 3L, 2L, 0.5L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (35g) (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 5.5cm and height of 15cm, respectively eluting with dichloromethane-methanol at gradient ratio of 100:3 and 9L, and dichloromethane-methanol at gradient ratio of 100:5 and 18L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2(15g)；
(3) Component Fr ETOAC -2 performing MCI column chromatography with an inner diameter of 4cm and a height of 20cm, performing gradient elution with 10%, 20% and 40% methanol aqueous solution by volume, wherein the amount of each gradient is 4.5L, 5L and 10L respectively, collecting 40% methanol eluate, and recovering solvent under reduced pressure to obtain FrETOAC-2-3(4.8 g);
(4) component Fr ETOAC Recrystallizing the extract from folium Arctii with methanol to obtain NBY-3(35mg) with effects of reducing blood lipid and protecting alcoholic liver injury.
It is noted that the above examples are only for illustrating the specific embodiments of the present invention, and the detailed description of the compound extracted from burdock leaves and the extraction method thereof, which has the activity of reducing blood lipid and protecting alcoholic liver injury, is illustrative and not intended to limit the scope of the present invention, and all changes and modifications that do not depart from the general concept of the present invention shall fall within the scope of the present invention.
The compounds prepared by the methods of examples 1-7 are identified to be the same compounds, the molecular structural formula of which is shown in figure 1, the compounds have the efficacy of reducing blood fat and protecting alcoholic liver injury, can be effectively used for preparing medicines for reducing blood fat and protecting alcoholic liver injury, and have very good beneficial technical effects through experiments, and the related data are as follows:
identification of compounds
The compound NBY-3 is a colorless crystal, and the adopted instruments and materials are as follows:
shimadzu double-beam 210A uv spectrometer (Shimadzu, Kyoto, japan);
LTQ orbitrap high resolution mass spectrometer (Thermo Fisher Scientific, Bremen, germany);
nuclear magnetic resonance apparatus: bruker AVANCE III 500-NMR spectrometer (Bruker, Billerica, German), TMS as internal standard;
thermo Fisher Scientific U3000 high performance liquid chromatograph;
BT25S precision balance (sidoris ltd, germany).
Column chromatography silica gel (100-; ODS (Beijing green Baicao science and technology development Co., Ltd.) MCI (Sparganium japonicum Co., Japan).
A deuterated reagent: DMSO-d 6 、CD 3 OD and CDCl 3 (Cambridge Isotope Laboratories, USA) chromatographic grade methanol (MeOH) and acetonitrile (MeCN) were purchased from TEDIA reagents Tiandi USA, and reagents such as petroleum ether, methanol, dichloromethane, ethyl acetate were analytically pure (Tianjin Fuyu Fine chemical Co., Ltd.).
DMEM high-glucose medium (Israel BI, lot # 0024419);
fetal bovine serum (Israel BI, lot number: 02-411-9);
lipopolysaccharide (LPS, Sigma-Aldrich, USA, batch number: L2994, adding physiological saline to prepare a stock solution);
OX-LDL (Shanghai-derived leaf Biotechnology Co., Ltd., batch No. S24897);
BCA protein quantification kit (Wuhan Boston biology, lot: AR 1110);
RIPA lysate (Roche Bio Inc., batch: AR 0102);
a biosafety cabinet (Saimer aircraft, USA, model 1384-A2);
carbon dioxide incubator (american type 3111, siemer fly);
enzyme-labeling instrument (Saimer Fei USA, model MULTISKAN FC).
Identification and analysis, Compound NBY-3 colorless crystals, [ alpha ]]2 D 523.411(c＝0.08g/100ml,CH 3 OH); by HR-ESI-MS [2M + H ]]Calculation of M/z 509.38315, [ M-OH [ ]] + The molecular formula C is presumed to be obtained by calculating m/z as 237.18468 15 H 26 O 3 The unsaturation degree is 3. Ultraviolet spectrum display (CH) 3 OH)λ max (log ε):201(0.668) nm; infrared spectrum shows the chemical changeThe composition has double bonds (1650, 963 cm) -1 ) Hydroxyl group (3311 cm) -1 ) The characteristic absorbs the signal. 1 H-NMR(CD 3 OD,500MHz) shows three methyl hydrogen signals [ delta ] H 1.27(3H,s,H-10)，1.22(3H,s,H-11)，0.75(3H,s,H-13)]One 1, 1-disubstituted double bond [ delta ] H 4.58(1H,t,J＝1.4Hz,H-12a)，4.94(1H,t,J＝1.4Hz,H-12b)]Four methylene hydrogen signals [ delta ] H 1.76(1H,m,H-5a)，1.23(1H,m,H-5b)，1.75(2H,m,H-6)，1.13(1H,t,J＝12.1Hz,H-1a)，1.58(1H,m,H-1b)，1.33(1H,t,J＝11.9Hz,H-4a)，1.76(1H,m,H-4b)]Two methine hydrogen signals [ delta ] H 2.40(1H,d,J＝12.1Hz,H-8a)，1.53(1H,m,H-2)]Two signals [ delta ] containing oxygen methine hydrogen H 4.21(1H,t,J＝2.2Hz,H-7)，3.97(1H,m,H-3)]。 13 C-NMR(CD 3 OD,125MHz) showed a 15 carbon signal comprising 3 methyl groups [ δ [ C 24.5(C-10)，29.7(C-11)，17.0(C-13)]4 sp 3 Hybrid methylene [ delta ] C 36.6(C-5)，30.4(C-6)，26.5(C-1)，50.9(C-4)]4 sp 3 Methine [ delta ] C 74.0(C-7)，44.6(C-8a)，55.2(C-2)，70.0(C-3)]Two sp 3 Hybrid quaternary carbons [ delta ] C 37.8(C-4a)，75.6(C-9)]One double bond carbon signal [ delta ] C 152.6(C-8)，109.1(C-12)]. The above information indicates that compound NBY-3 is likely to be a eudesmane-type sesquiterpene. The two-dimensional structure of compound NBY-3 was determined by correlation of HMBC and H-H COSY. H-5 and C-6/C-8a/C-4a/C-13 in HMBC spectra; h-7 and C-5/C-8/C-8 a; h-12 and C-7/C-8 a; h-1 and C-8a/C-2/C-4 a/C-9; h-2 and C-9; h-3 and C-1/C-2/C-4/C-9; h-4 and C-3/C-4 a/C-13; h 3 -10/H 3 -11 and C-9; h 3 13 is related to C-5/C-8a/C-4/C-4a, respectively. In the H-H COSY spectrum delta H 3.97(m, H-3) and δ H 1.53(m, H-2)/1.33(t, J ═ 11.9Hz, H-4a)/1.76(m, H-4b) correlation, δ H 4.21(t, J ═ 2.2Hz, H-7) and δ H 1.76(m,H-5a)/1.23(m,H-5b)/1.75(m,H 2 -6)/4.94(t, J ═ 1.4Hz, H-12a) correlation, δ H 2.40(d, J-12.1 Hz, H-8a) and δ H 4.94(t, J ═ 1.4Hz, H-12a)/4.58(t, J ═ 1.4Hz, H-12b)/1.13(t, J ═ 12.1Hz, H-1a)/1.58(m, H-1 b). The relative configuration of compound NBY-3 was determined using NOESY. H-8a has NOE correlation with H-2Signals indicating their same interface; at the same time, H 3 NOE-related signals for-13 and H-3, indicating H 3 -13 and H-3 on the other side; and H-7 and H-8a have no relevant signals, combined with three-dimensional modeling, it was determined that H-7 is on the equatorial bond and cannot be on the same side as H-8 a. Thus, H-7, H 3 -13 and H-3 are both on the other side. The absolute configuration was determined by CuK α X-single crystal diffraction to be 2S,3S,4aR,7R,8 aR.
According to data analysis and molecular structure, the molecular structural formula is as follows:
named compound NBY-3 extracted from folium Arctii and having effect in protecting alcoholic liver injury.
II, an activity experiment of the compound NBY-3 extracted from the burdock leaves and having the effect of protecting alcoholic liver injury.
Cell culture
Culturing RAW264.7 cells in DMEM complete medium (containing 10% newborn calf serum and 90% DMEM incomplete culture solution), standing at 37 deg.C and containing 5% CO 2 Culturing in an incubator. Cells in the logarithmic growth phase were selected for the experiments.
MTT method for detecting activity of RAW264.7 cells
Taking RAW264.7 cells in logarithmic growth phase, preparing into 1 × 10 cells with complete culture medium 5 The cell suspension was inoculated into a 96-well plate, 100. mu.l of the cell suspension was added to each well, and the plate was left at 37 ℃ with 5% CO 2 Culturing in an incubator. After 24h, the supernatant from the wells was discarded and 100. mu.l of complete medium containing compound NBY-3 at concentrations of 160, 80, 40, 20, 10, 0mM, respectively; the drug administration group and blank group are provided with 3 multiple wells at 37 deg.C and 5% CO 2 Culturing in an incubator. After 24h, add MTT10 μ l per well, continue culturing for 3h, discard supernatant, add 100 μ l DMSO per well, and put on bedAfter shaking for 10min, the purple crystals were fully dissolved and the OD of each well was measured at 490nm using a microplate reader. And cell viability was calculated as (a test/a space) × 100%.
TABLE 1 Effect of Compound NBY-3 on RAW264.7 cell Activity
Note: p <0.01, p < 0.001 compared to blank.
And (4) analyzing results:
effect of different concentrations of compound NBY-3 on RAW264.7 cell viability as shown in table 1: at a concentration of 160. mu.M of compound NBY-3, cell viability was less than 100%, and at other concentrations cell viability was greater than 100%. We chose compound NBY-3 at 20, 40. mu.M concentration administered to cells for further experimental study on cells.
In vitro lipid lowering experiment
RAW264.7 cells were cultured in a 12-well cell culture plate with DMEM medium at 37 ℃ for 12 hours. The cells were then grouped: blank control group, OX-LDL group (50 mug/mL), positive drug group (simvastatin 3 muM), drug group with different concentration (compound NBY-3), OX-LDL modeling is carried out for 24h, the culture medium is discarded, the culture medium is replaced by new culture medium, cells are collected after drug intervention is carried out for 24h, detection is carried out by a total cholesterol kit under the condition of 510nm of an enzyme labeling instrument, and the content of cholesterol is calculated by measuring OD value.
TABLE 2 determination of Compound NBY-3TC
Positive drugs: simvastatin was <0.05, P < 0.001 compared to model group
The effect of different concentrations of compound NBY-3 on TC in lipid accumulation model RAW264.7 cells caused by OX-LDL, as shown in Table 2, showed that different concentrations of compound NBY-3 decreased cholesterol levels caused by lipid accumulation caused by OX-LDL and were dose dependent.
Experiment for in vivo intervention of alcoholic liver injury
A medicine for treating alcoholic liver injury is prepared from the above compounds, and used for zebra fish experiment
1. Feeding zebra fish: all 3-4 month old wild type casper line adult zebra fishes are cultured in a zebra fish culture system of Beijing Aisheng company, the water temperature is 28 ℃, and the pH value is 7.0. Feeding twice daily, irradiating with fluorescent lamp for 14 hr, and dark environment for 10 hr. The remaining feeding and propagation conditions were referenced to Zebraphish Book.
Grouping and intervening: 75 healthy adult zebra fishes with uniform size are randomly divided into a normal group, a model group and a low-dosage and high-dosage group (the common feed is added with 0.4 percent and 0.2 percent of medicaments by mass ratio) of an administration group, and 15 fishes per jar. Except for the normal group, the alcohol concentration in the fish tank of each group is 1.0 percent, and the normal group is fed with the conventional fish water. The fish water is changed every day, each group of zebra fish regularly and quantitatively feeds feed for 3 times every day, 15mg of feed is fed for each fish every time, circulating water is closed before feeding, and the food intake condition of each group of fish is observed and recorded in the feeding process.
4. Index detection
Feeding adult zebra fish for 28 days continuously, after fasting for 24h, anaesthetizing the zebra fish, cutting aorta from the back and sucking blood, centrifuging, taking serum, mixing the serum of every 15 fish in the same treatment group as a sample, and measuring the levels of liver function indexes ALT, AST, TC and TG in the zebra fish serum according to the kit operation instructions.
TABLE 3 Effect of the prepared drugs on Biochemical indicators of liver function in serum of zebra fish with alcoholic liver disease
P <0.05, p <0.01 compared to normal group; comparison of # p <0.05, # p <0.01 with the high cholesterol group
Experiments show that the compound NBY-3 has an obvious lipid-lowering effect in vitro, and in vivo experiments can be effectively used for preparing medicines for preventing and treating alcoholic liver injury.
In conclusion, the invention has the advantages of rich raw materials, easy operation of the preparation method, strong guidance quality and high product purity of more than 98 percent, the compound has the efficacy of reducing blood fat and protecting alcoholic liver injury, can be effectively used for preparing the medicine for preventing and treating the alcoholic liver injury, develops new application and commercial value of burdock leaves, and has remarkable economic and social benefits.
Claims (9)
1. A compound extracted from folium Arctii for protecting alcoholic liver injury is compound NBY-3 extracted from folium Arctii for protecting alcoholic liver injury, and has a molecular structural formula:
2. the method for preparing the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury according to claim 1, which is characterized by comprising the following steps:
(1) crushing 10kg of dried burdock leaves, adding water which is 6-15 times of the weight of the burdock leaves, soaking for 5-14 days at room temperature, filtering, and concentrating the filtrate to a concentrated solution which is 0.1-3 g/mL of crude drug;
(2) sequentially extracting the concentrated solution with ethyl acetate for 4 times, wherein the dosage of each time is 1-5L, 0.5-3L and 0.5-3L, combining ethyl acetate extract, and concentrating under reduced pressure to obtain component Fr ETOAC (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 3-6 cm and height of 8-30 cm, and respectively performing gradient elution with dichloromethane-methanol and gradient elution with dichloromethane-methanolGradient ratio of 100:3 and dosage of 3-10L, gradient ratio of dichloromethane-methanol of 100:5 and dosage of 5-20L, collecting 100:5 gradient eluent, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2；
(3) Component Fr ETOAC -2, performing MCI column chromatography with the inner diameter of 1.5-4 cm and the height of 20-80 cm, performing gradient elution by using methanol water solutions with volume concentration of 10%, 20% and 40% respectively, wherein the dosage of each gradient is 0.5-5L, 0.5-5L and 2-10L, collecting 40% methanol eluent, and recovering the solvent under reduced pressure to obtain a component FrETOAC-2-3;
(4) component Fr ETOAC Recrystallizing the extract 2-3 in methanol to obtain a compound NBY-3 extracted from burdock leaves and having the effects of reducing blood fat and protecting alcoholic liver injury.
3. The method for preparing the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury according to claim 2, which is characterized by comprising the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 9 times of folium Arctii weight, soaking at room temperature for 8 days, filtering, and concentrating the filtrate to obtain concentrated solution equivalent to crude drug 2 g/mL;
(2) extracting the concentrated solution with ethyl acetate for 4 times (3L, 2L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 4cm and height of 20cm, respectively performing gradient elution with dichloromethane-methanol at gradient ratio of 100:3 and dosage of 6L and dichloromethane-methanol at gradient ratio of 100:5 and dosage of 18L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2；
(3) Component Fr ETOAC -2 performing MCI column chromatography with inner diameter of 3cm and height of 35cm, performing gradient elution with 10%, 20% and 40% methanol aqueous solution with each gradient dosage of 3L, 3L and 6L respectively, collecting 40% methanol eluate, and recovering solvent under reduced pressure to obtain FrETOAC-2-3;
(4) component Fr ETOAC Recrystallizing the extract 2-3 in methanol to obtain a compound NBY-3 extracted from burdock leaves and having the effects of reducing blood fat and protecting alcoholic liver injury.
4. The method for preparing the compound extracted from burdock leaves and having the effect of protecting alcoholic liver injury according to claim 2, characterized by comprising the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 10 times of folium Arctii weight, soaking at room temperature for 5 days, filtering, and concentrating the filtrate to 1.5L corresponding to 0.4g/mL of crude drug concentrate;
(2) extracting the concentrated solution with ethyl acetate for 4 times (1.5L, 1L, 0.5L, and 0.5L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 3cm and height of 25cm, respectively performing gradient elution with dichloromethane-methanol at gradient ratio of 100:3 and 3L, and gradient ratio of 100:5 and 7L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2；
(3) Component Fr ETOAC -2 performing MCI column chromatography with an inner diameter of 1.5cm and a height of 45cm, performing gradient elution with 10%, 20% and 40% methanol aqueous solution by volume, wherein the amount of each gradient is 1L, 1L and 4L respectively, collecting 40% methanol eluate, and recovering solvent under reduced pressure to obtain a component FrETOAC-2-3;
(4) component Fr ETOAC Recrystallizing the extract 2-3 in methanol to obtain a compound NBY-3 extracted from burdock leaves and having the effects of reducing blood fat and protecting alcoholic liver injury.
5. The method for preparing the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury according to claim 2, which is characterized by comprising the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 9 times of folium Arctii weight, soaking at room temperature for 7 days, filtering, and concentrating the filtrate to 2.5L corresponding to 0.3g/mL of the concentrated solution;
(2) extracting the concentrated solution with ethyl acetate for 4 times (3L, 1.5L, 1L, and 0.5L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (ii) a Component Fr ETOAC Go inPerforming silica gel column chromatography with diameter of 4cm and height of 20cm, respectively performing gradient elution with dichloromethane-methanol at gradient ratio of 100:3 and dosage of 4L, and dichloromethane-methanol at gradient ratio of 100:5 and dosage of 10L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2；
(3) Component Fr ETOAC -2 performing MCI column chromatography with an inner diameter of 2cm and a height of 40cm, performing gradient elution with 10%, 20% and 40% methanol aqueous solution by volume, wherein the amount of each gradient is 2L, 2L and 5L respectively, collecting 40% methanol eluate, and recovering solvent under reduced pressure to obtain a component FrETOAC-2-3;
(4) component Fr ETOAC -2-3 is recrystallized in methanol to obtain NBY-3 compound which is extracted from burdock leaves and has the functions of reducing fat and protecting alcoholic liver injury.
6. The method for preparing the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury according to claim 2, which is characterized by comprising the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 10 times of folium Arctii weight, soaking at room temperature for 9 days, filtering, and concentrating the filtrate to 4.5L corresponding to 0.2g/mL of the concentrated solution;
(2) extracting the concentrated solution with ethyl acetate for 4 times (4.5L, 3L, 2L, 0.5L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 6cm and height of 10cm, respectively performing gradient elution with dichloromethane-methanol at gradient ratio of 100:3 and dosage of 7L and dichloromethane-methanol at gradient ratio of 100:5 and dosage of 14L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2；
(3) Component Fr ETOAC -2 performing MCI column chromatography with an inner diameter of 3cm and a height of 30cm, performing gradient elution with 10%, 20% and 40% methanol aqueous solutions, wherein the amount of each gradient is 3.5L, 3.5L and 8L, respectively, collecting 40% methanol eluate, and recovering the solvent under reduced pressure to obtain a component FrETOAC-2-3;
(4) component Fr ETOAC Recrystallizing with methanol to obtain folium ArctiiThe compound NBY-3 extracted from the Chinese medicinal materials has the effects of reducing blood fat and protecting alcoholic liver injury.
7. The method for preparing the compound extracted from the burdock leaves and having the effect of protecting alcoholic liver injury according to claim 2, which is characterized by comprising the following steps:
(1) pulverizing dried folium Arctii 10kg, adding water 9 times of folium Arctii weight, soaking at room temperature for 10 days, filtering, and concentrating the filtrate to 4.5L corresponding to 0.2g/mL of the concentrated solution;
(2) extracting the concentrated solution with ethyl acetate for 4 times (4.5L, 3L, 2L, 0.5L each time), mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain component Fr ETOAC (ii) a Component Fr ETOAC Performing silica gel column chromatography with inner diameter of 5.5cm and height of 15cm, respectively eluting with dichloromethane-methanol at gradient ratio of 100:3 and 9L, and dichloromethane-methanol at gradient ratio of 100:5 and 18L, collecting 100:5 gradient eluate, and recovering solvent under reduced pressure to obtain component Fr ETOAC -2；
(3) Component Fr ETOAC -2 performing MCI column chromatography with an inner diameter of 4cm and a height of 20cm, performing gradient elution with 10%, 20% and 40% methanol aqueous solution by volume, wherein the amount of each gradient is 4.5L, 5L and 10L respectively, collecting 40% methanol eluate, and recovering solvent under reduced pressure to obtain a component FrETOAC-2-3;
(4) component Fr ETOAC Recrystallizing the extract 2-3 in methanol to obtain a compound NBY-3 extracted from burdock leaves and having the effects of reducing blood fat and protecting alcoholic liver injury.
8. The use of the compound extracted from burdock leaves and having the effect of protecting alcoholic liver injury as claimed in claim 1 in the preparation of a lipid-lowering and liver-protecting medicament.
9. The use of the compound extracted from burdock leaves and having the effect of protecting alcoholic liver injury as claimed in claim 1 for the preparation of a medicament for treating hyperlipidemia.
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| KR20020086372A (en) * | 2002-10-01 | 2002-11-18 | 주식회사 이롬라이프 | Composition for suppressing hyperlipidemia containing grains, fruits and vegetables, seeweeds and mushrooms |
| CN105616556A (en) * | 2016-02-03 | 2016-06-01 | 河南中医学院 | Preparation method of effective component for controlling alcoholic liver injury |
| CN105669797A (en) * | 2016-03-08 | 2016-06-15 | 重庆科瑞制药(集团)有限公司 | Method for separating burdock oil, arctiin, arctigenin, lappaol E and lappaol H from burdock |
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| KR20020086372A (en) * | 2002-10-01 | 2002-11-18 | 주식회사 이롬라이프 | Composition for suppressing hyperlipidemia containing grains, fruits and vegetables, seeweeds and mushrooms |
| CN105616556A (en) * | 2016-02-03 | 2016-06-01 | 河南中医学院 | Preparation method of effective component for controlling alcoholic liver injury |
| CN105669797A (en) * | 2016-03-08 | 2016-06-15 | 重庆科瑞制药(集团)有限公司 | Method for separating burdock oil, arctiin, arctigenin, lappaol E and lappaol H from burdock |
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