CN114716406A - Semen Scaphii Lychnophori extract with xanthine oxidase inhibiting activity, and its preparation method and application - Google Patents

Semen Scaphii Lychnophori extract with xanthine oxidase inhibiting activity, and its preparation method and application Download PDF

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CN114716406A
CN114716406A CN202210396244.0A CN202210396244A CN114716406A CN 114716406 A CN114716406 A CN 114716406A CN 202210396244 A CN202210396244 A CN 202210396244A CN 114716406 A CN114716406 A CN 114716406A
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compound
silica gel
gel column
extract
ethyl acetate
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CN114716406B (en
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刘超
孙金月
吕伸明
郭溆
陈莹莹
张梦启
王目旋
王青
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Shandong Academy of Agricultural Sciences
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Abstract

The invention belongs to the technical field of compound extraction, and particularly relates to a boat-fruited sterculia seed extract with xanthine oxidase activity inhibition function, and a preparation method and application thereof. The mechanism formula of the boat-fruited sterculia seed extract provided by the invention is specifically as follows:
Figure 896435DEST_PATH_IMAGE002
. The invention carries out deep and systematic research on chemical components of the boat-fruited sterculia seed, the xanthine oxidase resisting activity of the chemical components and the in vitro uric acid induced inflammation resisting activity of the chemical components, discovers a new compound, not only enriches the research scope of the compound, but also provides certain theoretical guidance for further application of the boat-fruited sterculia seed in the aspects of medicine and functional food, and lays a foundation for deep development of the plant.

Description

Semen Scaphii Lychnophori extract with xanthine oxidase inhibiting activity, and its preparation method and application
Technical Field
The invention belongs to the technical field of compound extraction, and particularly relates to a scaphium scaphigerum extract with xanthine oxidase activity inhibiting function, a preparation method and application thereof.
Background
Xanthine Oxidase (XO), which is a cytosolic enzyme present in various vertebrates including humans, belongs to the molybdenum flavoprotein family, and is widely present in milk, cow's milk, kidney, lung, heart and vascular endothelium. Xanthine oxidase is a key enzyme in the control of uric acid production in several species, including humans, and is the most active in the liver and gut. Excessive activity of xanthine oxidase can cause the increase of uric acid content in vivo, thereby causing diseases such as hyperuricemia and gout.
Sterculia Lychnophora (Sterculia Lychnophora Hance.)Sterculiae lychnophorae Semen, alias: semen Sterculiae Lychnophorae), which is mature and dry seed of Sterculia Lychnophora of Malaytea family, and is mainly distributed in Malaysia, Vietnam, India and China. Boat-fruited sterculia seed is a traditional Chinese medicine in China, is used for treating lung heat hoarseness, throat pain, dryness-heat constipation and headache and conjunctival congestion from ancient times, and is clinically used for treating acute tonsillitis. At present, few researches on the anti-hyperuricemia aspect of the crude extract of the scaphium scaphigerum are found, and the research is blank.
Disclosure of Invention
Aiming at the technical blank in the prior art, the invention provides a boat-fruited sterculia seed extract with xanthine oxidase activity inhibition function.
The invention also provides a preparation method of the boat-fruited sterculia seed extract.
The invention also provides an application of the boat-fruited sterculia seed extract.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a preparation method of a boat-fruited sterculia seed extract with xanthine oxidase activity inhibition, which is characterized by comprising the following steps:
(1) extracting semen Scaphii Lychnophori powder with 50% ethanol at room temperature, filtering the extractive solution, and concentrating under reduced pressure to obtain ethanol-free water extract; adding 95% ethanol into the water extract, separating polysaccharide from the water extract with 95% ethanol, centrifuging, pouring out, mixing the supernatant, and concentrating under reduced pressure to obtain ethanol extract; diluting the alcohol extract with distilled water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract part, ethyl acetate extract part and n-butanol extract part respectively;
(3) separating the ethyl acetate extract of semen Scaphii Lychnophori with silica gel column, and separating with CH2Cl2Stepwise elution with MeOH yields six components Fr 1-Fr 6;
(4) separating Fr2 on silica gel column with petroleum ether/ethyl acetate to obtain two components Fr 2a and Fr2 b; fr2b is separated by silica gel column and SephadexLH-20, and then is subjected to semi-preparative HPLC to obtain a compound 1, a compound 4 and a compound 5;
(5) fr 3 is separated by silica gel column by using petroleum ether/ethyl acetate to obtain four components Fr 3a-Fr 3 d; fraction Fr 3b on silica gel column and SephadexLH-20 (MeOH/CH)2Cl2 = 1/1) after separation, compound 6 and compound 7 are obtained by semi-preparative HPLC (); compound 3 crystallizes from Fr 3b in methanol;
(6) fr 4 is treated by a silica gel column and eluted by petroleum ether/ethyl acetate to obtain four components Fr 4a-Fr 4 c; passing the component Fr 4c through a silica gel column and Sephadex LH-20, and obtaining a compound 8 and a compound 9 on semi-preparative HPLC; and recrystallizing in petroleum ether/ethyl acetate solution at-20 ℃ to obtain a compound 2;
(7) eluting Fr 5 in silica gel column with petroleum ether/ethyl acetate to obtain four fractions Fr 5a-Fr 5 c; fr 5a is separated on a silica gel column and SephadexLH-20 to obtain a compound 10 and a compound 11, and then semi-preparative HPLC is carried out to obtain a compound 13; fr 5b is separated by a silica gel column and SephadexLH-20 to obtain a compound 12; fr 5c is separated on silica gel column and SephadexLH-20, and separated on semi-preparative HPLC to obtain compound 14 and compound 17; in addition, compound 15 and compound 16 crystallized from fr.5b.
Further, in the step (1), the specific process is as follows: extracting 10 kg semen Scaphii Lychnophori powder with 3 × 20L 50% ethanol at room temperature for 7 days, filtering the extractive solution, and concentrating under reduced pressure to obtain water extract without alcohol smell.
3. The method for preparing a compound according to claim 1 or 2, wherein in the step (2), the volume ratio of the water extract to the 95% ethanol is 1: 10, extracting twice; the centrifugation is 2000 r/min for 10 min; the concentration of the alcohol extract in distilled water is 0.25-0.30 kg/L.
Further, in the step (3), the silica gel column is 100-200 meshes; in the stepwise elution, CH2Cl2Volume ratio of/MeOH: 100/1 → 80/1 → 50/1 → 20/1 → 10/→ 8/10 → 6/1 → 4/1 → 2/1 → 1/1 → MA.
Further, in the step (4), the volume ratio of the petroleum ether to the ethyl acetate is 20/1-7/1; the silica gel column for separating Fr2 and Fr2b is 200-300 meshes; in the SephadexLH-20, MeOH/CH2Cl2 = 1/1; the conditions for the semi-preparative HPLC were: λ = 210 and 385nm, 2.5 mL/min; the mobile phase is as follows: compound 1 is MA H2O =65:32, 0.3% formic acid; compound 4 is MA: H2O = 63:37, 0.3% formic acid; compound 5 is MA H2O = 78: 22, 0.3% formic acid.
Further, in the step (5), the volume ratio of the petroleum ether to the ethyl acetate is 10/1-7/1; in the SephadexLH-20, MeOH/CH2Cl2 = 1/1; the silica gel column is 200-300 meshes; the conditions of the semi-preparative HPLC are lambda = 210-385nm, and 2.5 mL/min; the mobile phase is as follows: the compound 6 is MA: H2O = 32:68, 0.3% formic acid; compound 7 is MA: H2O = 38:62, 0.3% formic acid.
Further, in the step (6), the silica gel column is 200-300 mesh; the volume ratio of the petroleum ether to the ethyl acetate is 8/1-5/1; in the Sephadex LH-20, MeOH/CH2Cl2 = 1/1; the conditions of the semi-preparative HPLC are λ = 210-385nm, 2.5 mL/min, mobile phase: the compound 8 is MA: H2O = 55:45, 0.3% formic acid, compound 9 is MA: H2O = 50:50, 0.3% formic acid.
Further, in the step (7), the volume ratio of the petroleum ether to the ethyl acetate is 8/1-5/1; the SephadexLH-20 adopts MeOH; the conditions of the semi-preparative HPLC are lambda = 210-385nm, 2.5 mL/min, mobile phase: compound 13 is MA: H2O = 30:70, 0.3% formic acid; compound 14 is MA: H2O = 30:70, 0.3% formic acid; compound 17 is MA: H2O = 28:72, 0.3% formic acid.
The invention also provides a boat-fruited sterculia seed extract prepared by the preparation method, which has the following structural formula:
Figure DEST_PATH_IMAGE001
the invention also provides an application of the boat-fruited sterculia seed extract in preparing a medicament for inhibiting xanthine oxidase.
The boat-fruited sterculia seed ethyl acetate Extract (EF) extracted by the invention has good xanthine oxidase inhibition activity. The hyperuricemia cell model constructed by stimulating HK-2 cells by using uric acid as a stimulator shows that the stimulation of the uric acid can cause obvious inflammatory reaction, namely IL-6 and IL-1β、TNF-αIncreased production of isoinflammatory factors, intracellular IκBα、NF-κB. Phosphorylation of PI3K and AKT proteins was also significantly increased. This means that uric acid stimulation activates the PI3K/AKT signaling pathway and causes NF-κThe corresponding change in the B signaling pathway, thereby causing cellular inflammation. Three inflammatory factors, NF- κ B, I, were significantly reduced in HK-2 cells following EF treatmentκB. The phosphorylation levels of PI3K and AKT signaling pathway proteins were significantly down-regulated. Therefore, the ethyl acetate extract of scaphium scaphigerum shows excellent anti-inflammatory activity. The boat-fruited sterculia seed has great potential in resisting hyperuricemia.
The invention has the beneficial effects that:
(1) the invention extracts the effective components in the boat-fruited sterculia seed to the maximum extent by optimizing the reaction conditions, and the boat-fruited sterculia seed ethyl acetate extract obtained by extraction shows excellent anti-inflammatory activity and has great potential in the aspect of anti-hyperuricemia.
(2) The invention carries out deep and systematic research on chemical components of the boat-fruited sterculia seed, the xanthine oxidase resisting activity of the chemical components and the in vitro uric acid induced inflammation resisting activity of the chemical components, discovers a new compound, not only enriches the research scope of the compound, but also provides certain theoretical guidance for further application of the boat-fruited sterculia seed in the aspects of medicine and functional food, and lays a foundation for deep development of the plant.
Drawings
FIG. 1 is a schematic diagram showing the separation process of the ethyl acetate fraction from Sterculia Lychophora according to example 1.
FIG. 2 shows the results for compounds 1 and 21H-1Graph of the main correlation relationship in the H COSY and HMBC spectra.
Fig. 3 is a hydrogen spectrum and a carbon spectrum of compound 1.
Fig. 4 is a hydrogen spectrum and a carbon spectrum of compound 2.
Fig. 5 is a hydrogen spectrum and a carbon spectrum of compound 3.
Fig. 6 is a hydrogen spectrum and a carbon spectrum of compound 4.
FIG. 7 shows a hydrogen spectrum and a carbon spectrum of Compound 5.
Fig. 8 is a hydrogen spectrum and a carbon spectrum of compound 6.
Fig. 9 is a hydrogen spectrum and a carbon spectrum of compound 7.
FIG. 10 is a hydrogen spectrum and a carbon spectrum of Compound 8.
Fig. 11 is a hydrogen spectrum and a carbon spectrum of compound 9.
Fig. 12 is a hydrogen spectrum and a carbon spectrum of compound 10.
Fig. 13 is a hydrogen spectrum and a carbon spectrum of compound 11.
Fig. 14 is a hydrogen spectrum and a carbon spectrum of compound 12.
Fig. 15 is a hydrogen spectrum and a carbon spectrum of compound 13.
Fig. 16 is a hydrogen spectrum and a carbon spectrum of compound 14.
Fig. 17 is a hydrogen spectrum and a carbon spectrum of compound 15.
Fig. 18 is a hydrogen spectrum of compound 16.
Fig. 19 is a hydrogen spectrum and a carbon spectrum of compound 17.
FIG. 20 is a graph showing the effect of ethyl acetate extract of Sterculia Lychnophora on the viability of HK-2 cells.
FIG. 21 is a graph showing the effect of ethyl acetate extract of Sterculia Lychnophora on the morphology of HK-2 cells affected by uric acid.
FIG. 22 is a graph showing the effect of ethyl acetate extract from Sterculia Lychnophora on uric acid-induced production of inflammatory factors by HK-2 cells.
Detailed Description
The technical solution of the present invention is further explained and illustrated by the following specific examples.
Example 1
(1) Semen Scaphii Lychnophori (10 kg) powder was extracted with 50% ethanol (3X 20L) at room temperature for 7 days. Filtering the extractive solution, and concentrating under reduced pressure to obtain water extract with no alcohol smell of about 1L. Then (2 × 10L) 95% ethanol is used for separating polysaccharide in the water extract, centrifugation is carried out for 10 min at 2000 r/min, the supernatant is poured and combined, and the extract is concentrated under reduced pressure to obtain 1.30 kg. The extract was diluted with distilled water (4.5L) and extracted sequentially with petroleum ether (PE, 3X 4.5L), ethyl acetate (EA, 3X 4.5L) and n-butanol (n-Bu, 3X 4.5L) to obtain 5.6 g of a petroleum ether extract fraction (PF), 16.3 g of an ethyl acetate Extract Fraction (EF) and 105.2 g of an n-butanol extract fraction (BF), respectively. 2 mg of each of the three extracts was dissolved with a small amount of DMSO, and then diluted to 10 mL with a buffer to obtain 200µg·mL-1The test solution of (1). The three extracts were tested for xanthine oxidase inhibitory activity according to 3.2 experimental procedures.
(2) Separating the ethyl acetate extract of the sterculia scaphigera through a silica gel column (100-200 meshes) and using CH2Cl2Stepwise elution of/MeOH (100/1 → 80/1 → 50/1 → 20/1 → 10/→ 8/10 → 6/1 → 4/1 → 2/1 → 1/1 → MA) gives six components (Fr 1-Fr 6). The Fr 1 fraction was discarded because it was an oily substance, had too little polarity, hardly developed color under an ultraviolet lamp, and had little research value. Fr2 was separated on a silica gel column (200-300 mesh) using petroleum ether/ethyl acetate (20/1-7/1 v/v) to obtain two fractions (Fr 2a and Fr2 b). Fr2b was subjected to silica gel column (200-300 mesh) and SephadexLH-20 (MeOH/CH)2Cl2 = 1/1) separation, and reuse of semi-preparative HPLC (. lamda = 210-385nm, 2.5 mL/min) gave 1 (3 mg, 31.1 min, MA: H)2O =65:32, 0.3% formic acid) and 4 (6.7 mg, 24 min, MA: H)2O = 63:37, 0.3% formic acid) and 5 (32 mg, 24 min, MA: H)2O = 78: 22, 0.3% formic acid). Fr 3 was separated by means of a silica gel column (200-300 mesh) using petroleum ether/ethyl acetate (10/1-7/1 v/v) to give four fractions (Fr 3a-Fr 3 d). Fraction Fr 3b on silica gel column and SephadexLH-20 (MeOH/CH)2Cl2 = 1/1) separation by semi-preparative HPLC (. lamda = 210-385nm, 2.5 mL/min) to give 6 (2 mg, 38.1 min, MA: H)2O = 32:68, 0.3% formic acid) and 7 (3 mg, 17.2 min, MA: H)2O = 38:62, 0.3% formic acid). In addition, compound 3 (20 mg) crystallized from Fr 3b in methanol. Fr 4 was subjected to silica gel column (200-300 mesh) and eluted with petroleum ether/ethyl acetate (8/1 to 5/1 v/v) to obtain four fractions (Fr 4a-Fr 4 c). Passing the component Fr 4c through silica gel column and Sephadex LH-20 (MeOH/CH)2Cl2 = 1/1) gave 8 (2 mg, 18.2 min, MA: H) on semi-preparative HPLC (. lamda. = 210-385nm, 2.5 mL/min)2O = 55:45, 0.3% formic acid) and 9 (2.5 mg, 19.3 min, MA: H2O = 50:50, 0.3% formic acid). And recrystallized from a-20 deg.C petroleum ether/ethyl acetate solution to give 2 (4.1 mg). Fr 5 was eluted in a silica gel column using petroleum ether/ethyl acetate (8/1 to 5/1 v/v) to obtain four fractions (Fr 5a-Fr 5 c). Fr 5a was separated on silica gel column and SephadexLH-20 (MeOH) to give 10 (4.7 mg) and 11 (5.2 mg), followed by semi-preparative HPLC (. lamda = 210-385nm, 2.5 mL/min) to give 13 (5.5 mg, 22.3 min, MA: H)2O = 30:70, 0.3% formic acid). Fr 5b was isolated on a silica gel column and SephadexLH-20 (MeOH) to give 12 (6.4 mg). Fr 5c was also separated on silica gel column (200-300 mesh) and SephadexLH-20 (MeOH) to give 14 (40 mg, 19 min, MA: H: 2.5 mL/min) on semi-preparative HPLC (λ = 210-385nm, 2.5 mL/min)2O = 30:70, 0.3% formic acid) and 17 (8.1 mg, 24.3 min, MA: H2O = 28:72, 0.3% formic acid). Furthermore, compounds 15 (6.1 mg) and 16 (5.3 mg) crystallized from Fr.5b. (the specific separation scheme is shown in FIG. 1)
(3) Structure identification process of novel Compound 1
A colorless oil. Easily soluble in dichloroMethane, HR-ESI-MS M/z 209.0815 [ M + H ]]+Combining with nuclear magnetic carbon spectrum signal to deduce molecular formula C11H12O4Wherein the degree of unsaturation is 6. The hydrogen spectrum and carbon spectrum data of compound 1 are shown in table 1. DeltaH4.28/δC63.93/δC143.13 and deltaH4.24/δC64.48/δC147.74 are shown in FIG. 2. Compound 1 was identified as ethyl 2,3-dihydro-1, 4-benzodioxin-6-carboxylate. The hydrogen spectrum and carbon spectrum of compound 1 are shown in fig. 3.
(4) Structure identification process of novel Compound 2
A white solid. Easily dissolved in dichloromethane, HR-ESI-MS M/z 253.1067 [ M + H ]]+Combining the nuclear magnetic carbon spectrum signals (Table 1), the molecular formula C is deduced13H16O5Wherein the degree of unsaturation is 6. The hydrogen spectrum and carbon spectrum data of the compound 2 are shown in table 1, and the hydrogen spectrum and carbon spectrum are shown in fig. 4. DeltaH2.50/δH1.57/δH0.88 of1H-1HCOSY correlation and deltaH2.50/δC172.02 are shown in FIG. 2.
TABLE 1
Figure DEST_PATH_IMAGE003
(5) Process for identifying structure of compound 3-17
Compound 3: white solid, readily soluble in dichloromethane. EI-MS m/z: 367.16 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C30H52O, unsaturation degree is 5. Compound 3 isβ-sitosterol(s) ((s))β-sitosterol), the hydrogen and carbon spectra are shown in FIG. 5.
Compound 4: brown gum, EI-MS m/z: 2256.10 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C11H15NO4The unsaturation degree is 5. The compound 4 is 4- (2-formyl-5- (methoxymethyl) -1H-pyrrol-1-yl) butanoic acid, and the hydrogen spectrum and the carbon spectrum are shown as 6.
Compound 5:yellow oil, EI-MS m/z: 279.16 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C16H22O4The unsaturation degree was 6. Compound 5 was determined to be dibutyl phthalate (dibutyl phthalate), and the hydrogen and carbon spectra are shown in FIG. 7.
Compound 6: white solid, EI-MS m/z: 169.05 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C8H8O4The unsaturation degree is 5. Compound 6 is vanillic acid (vanillic acid) and the hydrogen and carbon spectra are shown in FIG. 8.
Compound 7: pale yellow solid, readily soluble in chloroform and methanol. EI-MS m/z: 193.05 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C10H8O4The unsaturation degree was 7. Compound 7 is scopoletin (scopoletin), and has a hydrogen spectrum and a carbon spectrum shown in figure 9.
Compound 8: white solid, readily soluble in diethyl ether, acetone, glacial acetic acid and chloroform. EI-MS m/z: 149.06 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C9H8O2The unsaturation degree was 6. Compound 8 is trans-cinnamic acid (cinnamic acid) and the hydrogen and carbon spectra are shown in figure 10.
Compound 9: white solid, soluble in hot water and methanol, insoluble in chloroform. EI-MSm/z: 183.06 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C9H0O4The unsaturation degree is 5. Compound 9 is ethyl 3, 4-dihydroxybenzoate, and its hydrogen spectrum and carbon spectrum are shown in FIG. 11.
Compound 10: white solid, readily soluble in methanol, poorly soluble in water, EI-MS m/z: 245.07 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C9H12N2O6The unsaturation degree was 5. Compound 10 is uridine and has a hydrogen and carbon spectrum as shown in FIG. 12.
Compound 11: white solid, readily soluble in methanol, poorly soluble in water, EI-MS m/z: 291.08 [M+H]+Determination of molecular formula in combination with nuclear magnetic carbon signalsIs C15H14O6The unsaturation degree was 9. Compound 11 is catechin, and its hydrogen and carbon spectra are shown in FIG. 13.
Compound 12: white solid, readily soluble in methanol, poorly soluble in water, EI-MS m/z: 282.28 [M+H]+Combined with nuclear magnetic carbon signals, the molecular formula is determined as C18H35NO, unsaturation 2. Compound 12 is oleamide and has a hydrogen spectrum and a carbon spectrum as shown in FIG. 14.
Compound 13: white solid, easily soluble in methanol, EI-MS m/z: 283.15 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C15H22O5The unsaturation degree is 5. Compound 13 is dihydrophaseolamic acid, and the hydrogen and carbon spectra are shown in FIG. 15.
Compound 14: white solid, soluble in hot water and methanol, insoluble in chloroform. EI-MS m/z: 155.03 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C7H6O4The unsaturation degree is 5. Compound 14 is 3, 4-dihydroxybenzoic acid and has a hydrogen spectrum and a carbon spectrum shown in FIG. 16.
Compound 15: white solid, dissolved in methanol and chloroform. EI-MS m/z: 345.26 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C19H36O5The unsaturation degree is 2. Compound 15 is methyl (9S,10R,11E,13R) -9,10,13-trihydroxyoctadec-11-enoate, and its hydrogen and carbon spectra are shown in FIG. 17.
Compound 16 was a white solid, dissolved in methanol and chloroform. EI-MS m/z: 331.24 [M+H]+Combined with nuclear magnetic carbon signals, the molecular formula is determined as C18H34O5The unsaturation degree is 2. Compound 16 is (9S,10R,13R, E) -9,10,13-trihydroxyoctadec-11-enoic acid, and its hydrogen and carbon spectra are shown in FIG. 18.
Compound 17: pale yellow solid, dissolved in methanol. EI-MS m/z: 181.05 [M+H]+Combined with nuclear magnetic carbon signal, molecular formula is determined as C9H8O4The unsaturation degree was 6. Compound 17 is caffeic acid and has a hydrogen spectrum and a carbon spectrum shown in FIG. 19.
Effect example 117 Compounds xanthine oxidase inhibitory Activity
The 14 compounds (1-17) separated and identified from the ethyl acetate extract of the scaphium scaphigerum are tested for xanthine oxidase inhibitory activity. The results of the experiment are shown in table 2.
TABLE 2
Figure DEST_PATH_IMAGE005
Effect example 2 detection of HK-2 cell Activity by Ethyl acetate extract of Sterculia Lychophora
For screening HK-2 cell culture concentrations, 0, 400, 600, 800, 1000, 1500 and 2000, respectively, were usedµg·mL-1The ethyl acetate extract solution of the boat-fruited sterculia seed acts on the cells for 24 hours, and then the CCK-8 reagent is used for detecting the cell viability. The results are shown in FIG. 20, when the cell concentration is 0-800µg·mL-1When the concentration is as high as 1000, the cell viability is above 80%µg·mL-1When the cells were viable, the cell viability dropped abruptly to below 80%. Thus, when the concentration of the ethyl acetate extract of the boat-fruited sterculia seed is between 0 and 800µg·mL-1Has no influence on the growth activity of the cells. Thus selecting 200, 400, 600µg·mL-1Three concentrations were used as dosing concentrations for the next cell experiment.
Effect example 3 protective Effect of ethyl acetate extract of Sterculia Lychnophora on HK-2 cells
Blank group: 2 mL of medium.
Model group: 4 mM uric acid 0.4 mL + 1.6 mL medium.
Positive control group: 4 mM uric acid 0.4 mL + 2 mM allopurinol 0.2 mL + 1.4 mL medium.
200 µg·mL-1Experimental groups: 4 mM uric acid 0.4 mL + 1 mg. mL-1Extract concentration 0.4 mL + 1.2 mL medium.
400 µg·mL-1Experimental groups: 4 mM uric acid 0.4 mL + 1 mg/mL-1Extract concentration 0.6 mL + 1.0 mL medium.
600 µg·mL-1Experimental groups: 4 mM uric acid 0.4 mL + 1 mg/mL-1Extract concentration 0.8 mL + 0.7 mL medium.
After 24 h of fine culture of HK-2 seeded on 6-well plates, the old medium was discarded and treated with the extracts and uric acid for 48 h according to pre-grouping, and the morphology of the cells in different groups was observed with an upright microscope.
HK-2 cells were treated with uric acid and extracts for 48 h in pre-groups and HK-2 cell morphology was observed with orthoscope. As shown in FIG. 21, the uric acid-treated HK-2 cells were remodeled into spindle shapes, rather than normal-morphology cobblestone shapes, as compared with the normally cultured HK-2 cells. However, the extract eases this process, the action of uric acid on the cells is reduced, and the number of cobblestone-like cells increases. Furthermore, these changes are very pronounced at higher concentrations, showing dose dependence. The results show that the stimulation of the extract may have a certain protective effect on the HK-2 cells treated by uric acid.
Effect example 4 inhibitory Effect of Ethyl acetate extract of Sterculia Lychnophora on uric acid-induced production of inflammatory factor by HK-2 cells
After 24 h of fine culture with HK-2 seeded in 6-well plates, the old medium was discarded, treated with the extracts and uric acid for 24 h according to pre-grouping, respectively, and the crushed cells were resuspended in commercial lysis buffer and incubated at 4 ℃ for 30 min. After low-temperature centrifugation (12000 r/min, 20 min), collecting supernatant for proinflammatory cytokines detection. TNF-αIL-6 and IL-1βThe concentration of (c) was determined using a commercial ELISA kit, and each group was replicated four times.
TNF-activated release from cells by external stimuliα、IL-1βAnd cytokines such as IL-6. To determine the inhibitory effect of the extract on uric acid-induced pro-inflammatory cytokine production, the present study investigated uric acid-induced IL-1 by enzyme immunoassay (ELISA)βIL-6 and TNF-αThe influence of (c). As shown in FIG. 22, IL-1 was observed after stimulation of HK-2 cells with uric acidβIL-6 and TNF-αThe expression of (A) is significantly increased. This indicates that hyperuricemia can indeed trigger cells to produce inflammation, and verifies the feasibility of cell models. In addition, treatment with EF was performed in a dose-dependent mannerReduced uric acid induced IL-1βIL-6 and TNF-αIs generated. This suggests that EF can reduce inflammation caused by uric acid.

Claims (10)

1. A preparation method of a boat-fruited sterculia seed extract with xanthine oxidase activity inhibition function is characterized by comprising the following steps:
(1) extracting semen Scaphii Lychnophori powder with 50% ethanol at room temperature, filtering the extractive solution, and concentrating under reduced pressure to obtain ethanol-free water extract; adding 95% ethanol into the water extract, separating polysaccharide from the water extract with 95% ethanol, centrifuging, pouring out, mixing the supernatant, and concentrating under reduced pressure to obtain ethanol extract; diluting the alcohol extract with distilled water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract part, ethyl acetate extract part and n-butanol extract part respectively;
(3) separating the ethyl acetate extract of semen Scaphii Lychnophori with silica gel column, and adding CH2Cl2Stepwise elution with MeOH yields six components Fr 1-Fr 6;
(4) separating Fr2 on silica gel column with petroleum ether/ethyl acetate to obtain two components Fr 2a and Fr2 b; fr2b is separated by silica gel column and SephadexLH-20, and then is subjected to semi-preparative HPLC to obtain a compound 1, a compound 4 and a compound 5;
(5) fr 3 is separated by silica gel column by using petroleum ether/ethyl acetate to obtain four components Fr 3a-Fr 3 d; fraction Fr 3b on silica gel column and SephadexLH-20 (MeOH/CH)2Cl2 = 1/1) after separation, compound 6 and compound 7 are obtained by semi-preparative HPLC (); compound 3 crystallized from Fr 3b in methanol;
(6) fr 4 is treated by a silica gel column and eluted by petroleum ether/ethyl acetate to obtain four components Fr 4a-Fr 4 c; passing the component Fr 4c through a silica gel column and Sephadex LH-20, and obtaining a compound 8 and a compound 9 on semi-preparative HPLC; and recrystallizing in petroleum ether/ethyl acetate solution at-20 ℃ to obtain a compound 2;
(7) eluting Fr 5 in silica gel column with petroleum ether/ethyl acetate to obtain four fractions Fr 5a-Fr 5 c; fr 5a is separated on a silica gel column and SephadexLH-20 to obtain a compound 10 and a compound 11, and then semi-preparative HPLC is carried out to obtain a compound 13; fr 5b is separated by a silica gel column and SephadexLH-20 to obtain a compound 12; fr 5c is separated on silica gel column and SephadexLH-20, and separated on semi-preparative HPLC to obtain compound 14 and compound 17; in addition, compound 15 and compound 16 crystallized from fr.5b.
2. The preparation method according to claim 1, wherein in the step (1), the specific process is as follows: extracting 10 kg semen Scaphii Lychnophori powder with 3 × 20L 50% ethanol at room temperature for 7 days, filtering the extractive solution, and concentrating under reduced pressure to obtain water extract without alcohol smell.
3. The method for preparing a compound according to claim 1 or 2, wherein in the step (2), the volume ratio of the water extract to the 95% ethanol is 1: 10, extracting twice; the centrifugation is 2000 r/min for 10 min; the concentration of the alcohol extract in distilled water is 0.25-0.30 kg/.
4. The preparation method according to claim 1 or 3, wherein in the step (3), the silica gel column is 100-200 mesh; in the stepwise elution, CH2Cl2Volume ratio of/MeOH: 100/1 → 80/1 → 50/1 → 20/1 → 10/→ 8/10 → 6/1 → 4/1 → 2/1 → 1/1 → MA.
5. The method according to claim 4, wherein in the step (4), the volume ratio of the petroleum ether to the ethyl acetate is 20/1 to 7/1; the silica gel column for separating Fr2 and Fr2b is 200-300 meshes; in the SephadexLH-20, MeOH/CH2Cl2 = 1/1; the conditions for the semi-preparative HPLC were: λ = 210 and 385nm, 2.5 mL/min; the mobile phase is as follows: compound 1 is MA H2O =65:32, 0.3% formic acid; compound 4 is MA: H2O = 63:37, 0.3% formic acid; compound 5 is MA: H2O = 78: 22, 0.3% formic acid.
6. According to claimThe preparation method of (4), wherein in the step (5), the volume ratio of the petroleum ether to the ethyl acetate is 10/1-7/1; in the SephadexLH-20, MeOH/CH2Cl2 = 1/1; the silica gel column is 200-300 meshes; the conditions of the semi-preparative HPLC are lambda = 210-385nm, and 2.5 mL/min; the mobile phase is as follows: the compound 6 is MA: H2O = 32:68, 0.3% formic acid; compound 7 is MA: H2O = 38:62, 0.3% formic acid.
7. The preparation method according to claim 4, wherein in the step (6), the silica gel column is 200-300 mesh; the volume ratio of the petroleum ether to the ethyl acetate is 8/1-5/1; in the Sephadex LH-20, MeOH/CH2Cl2 = 1/1; the conditions of the semi-preparative HPLC are λ = 210-385nm, 2.5 mL/min, mobile phase: the compound 8 is MA: H2O = 55:45, 0.3% formic acid, compound 9 is MA: H2O = 50:50, 0.3% formic acid.
8. The method according to claim 4, wherein in the step (7), the volume ratio of the petroleum ether/ethyl acetate is 8/1-5/1; the SephadexLH-20 adopts MeOH; the conditions of the semi-preparative HPLC are lambda = 210-385nm, 2.5 mL/min, mobile phase: compound 13 is MA: H2O = 30:70, 0.3% formic acid; compound 14 is MA: H2O = 30:70, 0.3% formic acid; compound 17 is MA: H2O = 28:72, 0.3% formic acid.
9. A sterculia scaphigera extract prepared by the method of any one of claims 1 to 8, wherein the formula is as follows:
Figure 74337DEST_PATH_IMAGE001
。
10. use of a compound according to claim 9 for the manufacture of a medicament for inhibiting xanthine oxidase.
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