阅读说明：本技术 米碎花中抗prrsv病毒成分的制备与应用 (Preparation and application of anti-PRRSV virus component in crushed rice flowers ) 是由 徐明行 韦珊珊 姚丽媛 杜逸琼 秦绪兵 林婷婷 于 2020-08-13 设计创作，主要内容包括：本发明设计医药技术领域,提供了一种米碎花的抗病毒应用,特别涉及米碎花中抗PRRSV成分的制备与应用。本发明该米碎花(学名：Eurya chinensis R.Br.)中提取分离的化合物Betulinic acid、α-amyrin、taxifolin、hydroxytyrosol,体外抗病毒实验首次发现具有抗PRRSV活性,为制备治疗猪蓝耳病毒提供了新的安全植物和天然化合物,为制备抗猪蓝耳病毒的药物或药物组合物提供有力的技术支持。米碎花具有良好的抗病毒活性,并且其具有抗病毒活性成分。鉴于其药食同源,又有很高的市场经济价值。(The invention relates to the technical field of medicines, provides an antiviral application of ground rice flower, and particularly relates to preparation and application of an anti-PRRSV component in ground rice flower. In vitro antiviral experiments show that the compounds Betulinic acid, alpha-amyrin, taxifolin and hydroxyytosol extracted and separated from the crushed rice flowers (the scientific name: Eurya chinensis R.Br.) have the PRRSV resistant activity for the first time, provide new safe plants and natural compounds for preparing and treating porcine reproductive and respiratory syndrome viruses and provide powerful technical support for preparing and treating the porcine reproductive and respiratory syndrome resistant medicines or pharmaceutical compositions. The crushed rice flower has good antiviral activity, and it has an antiviral active ingredient. Due to homology of medicine and food, the compound has high market economic value.)

1. The preparation and application of the anti-PRRSV virus component in the crushed rice flowers are characterized in that: the preparation of the anti-PRRSV component in the rice breaking flower can be used in the anti-PRRSV field;

2. the anti-PRRSV component of claim 1, wherein: the English names of the compounds are Betulinic acid, alpha-amyrin, taxifolin and hydroxypyrosol respectively, and the structures are shown as formula I;

3. the compound of claim 2, wherein: the compound can be obtained by preparation or synthesis.

4. The compound of claim 2, wherein: can be applied in the field of anti-PRRSV.

5. The use of a compound according to claim 4 in the field of combating PRRSV, characterized in that: the anti-PRRSV drug comprises effective amount of Betulinic acid, alpha-amyrin, taxifolin, hydroxyytosol and pharmaceutically acceptable carrier as active ingredients.

Technical Field

The invention relates to an antiviral application of crushed rice flowers, in particular to a preparation method and an application of an anti-PRRSV virus component in the crushed rice flowers.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious disease characterized by fever, anorexia, late abortion, premature birth, stillbirth, weak and mummy of infected pigs, Respiratory disorders in pigs of various ages, particularly piglets. The porcine reproductive and respiratory syndrome has high transmission speed, and is more likely to cause epidemic due to dense and frequent flow of pig herds especially in the countries with advanced technology and developed economy. After a severe epidemic period, the disease is often endemic, and damages pig production for a long time, thus causing huge economic loss to the pig industry. The disease was first reported in 1987 in the united states, followed by successive reports in germany, the netherlands, canada, etc., and then rapidly spread to asia and other regions, with enormous losses to the swine industry worldwide. PRRS is mainly caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The prevention and control of PRRSV is a difficult problem in China and even the world at present. PRRSV has a high infectivity, with 10 or fewer virus particles infecting pigs both through nasal and muscular routes, the infected pigs being detoxified from saliva, urine, semen and milk, and the virus infecting susceptible herds in a direct or indirect manner. PRRSV can be transmitted directly between herds at distances less than 30 m. The transmission mode between pig farms is mainly the transmission mode of infected pigs or semen with virus. Because of the great harm of the disease, the disease is classified as a B-type infectious disease by the International animal and epidemic department (OIE), and is classified as a second-type infectious disease in China. Scholars at home and abroad do a lot of work on the research of PRRSV prevention and control, and certain progress is made in the aspects of vaccine prevention and western medicine prevention and control. The PRRSV vaccine on the market at present mainly comprises inactivated vaccine and attenuated vaccine, but the protective effect of the inactivated vaccine is not ideal, the attenuated vaccine has the risk of virus dispersion, and the condition of strong toxicity may occur in the process of immunization. The common report in China shows that sow abortion and stillbirth are caused by inoculation of PRRSV vaccine. Therefore, research and development of natural and green medicaments for promoting the PRRSV vaccine effect or replacing the PRRSV vaccine status to achieve the aims of promoting the immune function and health condition of livestock and poultry and improving the resistance of animals to the blue-ear virus infection become one of the hot subjects in the field of animal infectious disease research.

Comminuted flower of Oryza sativa (scientific name: Eurya chinensis R.Br.), bush, root and whole plant of Theaceae can be used as medicine. Broken rice flowers are often used as tea drinks in Guangdong (especially Dongguan) areas, Guangdong people like drinking tea, the habit of drinking herbal tea in folk is long besides drinking conventional tea such as green tea, and broken rice flower tea is also used as one of the tea drinks. In addition, according to the records of classical book national Chinese herbal medicine assembly and Chinese materia medica: the nature, taste and meridian tropism of the rice flower: sweet, light, slightly astringent and cool. The functional indications are as follows: clear heat and remove toxicity, remove dampness and heal wound. For preventing influenza; it is used externally to treat burns, scalds and pustule sores. Biting by snake and insect; bleeding due to trauma. 'Mikuihua' alsoQuiltCan be used in Chinese medicinal composition for treating migraine, herpes zoster, pneumococcus, urethritis, and oral ulcer. Therefore, we speculate that the ground rice has good antiviral activity, and it has an antiviral active ingredient. Due to homology of medicine and food, the compound has high market economic value.

Disclosure of Invention

The invention aims to provide extraction, separation and identification of anti-PRRSV components of ground rice flowers and application thereof.

The extraction, separation and identification of the anti-PRRSV component of the ground rice flower are realized by the following technical scheme:

sun drying and pulverizing Mirabilis jalapa L.leaf, extracting with 95% ethanol, repeating the operation for three times, concentrating to obtain extract, suspending with pure water, and extracting with ethyl acetate to obtain ethyl acetate extract.

Decolorizing the ethyl acetate part with industrial macroporous resin D-101, eluting with 30% ethanol until the effluent is colorless, eluting with 90% ethanol until the effluent is colorless, collecting the effluent, concentrating under reduced pressure, and performing silica gel column chromatography to obtain 9 Er.1-Er.9 components.

Er.1 is chromatographed by a silica gel column (gradient elution of normal hexane and ethyl acetate system) to obtain Er.1-1, Er.1-2 and Er.1-3. Er.1-1 was chromatographed by HPLC (35% acetonitrile/water) to give the compound Betulinic acid.

Er.1-2 was chromatographed by HPLC (35% acetonitrile/water) to give the compound α -amyrin.

Er.3 was chromatographed on reverse-phase silica gel (80% methanol/water) and silica gel (chloroform: methanol system 25:1) with mobile phase to give the compound, hydroxyytosol.

Er.7 is chromatographed by a reverse silica gel column (30-100% methanol/water) to obtain Er.7-1, Er.7-2 and Er.7-3. Er.7-3 was chromatographed by HPLC (32% acetonitrile/water) to give the compound, taxifolin.

The structural formulas of the three compounds are respectively:

the four compounds with antiviral activity extracted, separated and identified from the rice flower are applied to resisting PRRSV. Compared with the prior art, the preparation and the application of the anti-PRRSV virus component in the ground rice flower have the remarkable advantages that:

the invention discovers for the first time that the rice breaking flower or the extract thereof can be used as a natural traditional Chinese medicine for resisting PRRSV, because the rice breaking flower or the extract thereof contains antiviral active ingredients, wherein Betulinic acid has high content in the crude extract of the rice breaking flower, and the content reaches 2 percent and is the main component in the rice breaking flower. The traditional Chinese medicine has unique superiority in preventing and controlling virus diseases. The traditional Chinese medicine antiviral agent not only can directly play an antiviral role in the aspects of preventing the adsorption, penetration, replication and the like of viruses, but also can regulate the immune function of an organism, participate in the immune response of the organism, play a role in immune surveillance and antiviral, is expected to become a novel medicine for preventing and treating the porcine reproductive and respiratory syndrome, and has a good application prospect in the aspect of preventing and treating the porcine reproductive and respiratory syndrome.

The PRRSV resistant compounds Betulinic acid, alpha-amyrin, taxifolin and hydroxypyrosol discovered by the invention can be obtained from the rice flower plant by extraction and separation, the extraction process is simple, and the effective components can be obtained quickly.

The PRRSV resistant compounds Betulinic acid, alpha-amyrin, taxifolin and hydroxypyrosol discovered by the invention have the advantages of simple synthesis process, low price, easily obtained raw materials, contribution to industrialization and wider application prospect.

According to the technical scheme, the invention proves that the ground rice has the compound capable of obviously inhibiting the infection and the replication of the PRRSV by utilizing an experimental method, has an obvious PRRSV virus resisting effect, is expected to become a novel medicament for preventing and treating the porcine reproductive and respiratory syndrome, and has a good application prospect in the prevention and treatment aspect of the porcine reproductive and respiratory syndrome.

Drawings

FIG. 1 is a structural formula of Betulinic acid;

FIG. 2 shows Betulinic acid¹A graph of H-NMR;

FIG. 3 shows Betulinic acid¹A graph of C-NMR;

FIG. 4 is a structural formula of α -amyrin;

FIG. 5 is a view of alpha-amyrin¹A graph of H-NMR;

FIG. 6 is of alpha-amyrin¹A graph of C-NMR;

FIG. 7 is a structural formula of taxifolin;

FIG. 8 is a schematic representation of taxifolin¹A graph of H-NMR;

FIG. 9 is a schematic representation of taxifolin¹A graph of C-NMR;

FIG. 10 is a structural formula of hydroxyytosol;

FIG. 11 is a schematic representation of hydroxynaphthols¹A graph of H-NMR;

FIG. 12 is a schematic representation of hydroxynaphthols¹A graph of C-NMR;

Detailed Description

The following detailed description will be given of the extraction, isolation and identification of the anti-PRRSV component from the ground rice flower according to the present invention, in conjunction with the preferred embodiments.

EXAMPLE 1 isolation preparation and structural characterization of the four Compounds

Drying and crushing 23Kg of crushed rice flower and leaf, leaching by 95% industrial alcohol, repeating the operation for three times, soaking for 96 hours for the first time, soaking for 72 hours for the second time and the third time, concentrating the three times of leachate under vacuum pressure by a large-scale rotary evaporator, and combining the concentrates to obtain a primary extract. Mixing the primary extract with pure water, stirring thoroughly to make it suspended, adding ethyl acetate of the same volume, stirring vigorously, layering in a 5000mL separating funnel, standing for thirty minutes, sequentially collecting the layered parts according to the layering sequence, concentrating the ethyl acetate part under reduced pressure and extracting the water part with ethyl acetate repeatedly for three times. The concentrated product in the ethyl acetate portion was combined and weighed to obtain 1.7Kg of ethyl acetate portion.

Decolorizing the ethyl acetate part with industrial macroporous resin D-101, eluting with 30% ethanol until the effluent is colorless, eluting with 90% ethanol until the effluent is colorless, collecting the effluent, and concentrating under reduced pressure to obtain 1.7Kg concentrate. The ethyl acetate fraction was subjected to column chromatography using 80-100 mesh silica gel, 90% ethanol fraction was subjected to gradient elution using n-hexane/ethyl acetate (v: v 100:1-1:100), and the column was washed with methanol, followed by thin layer chromatography to give 9 fractions, i.e., Er.1(32.5g), Er.2(61.3g), Er.3(56.8g), Er.4(85.4g), Er.5(74.1g), Er.6(141.2g), Er.7(64.2g), Er.8(92.1g), and Er.9(1092 g).

Er.1 is subjected to silica gel (200-mesh and 300-mesh) column chromatography, and the mobile phase is n-hexane: and (3) detecting an ethyl acetate system (100:1 to 1:1) by using a thin layer to obtain Er.1-1, Er.1-2 and Er.1-3. Er.1-1 was chromatographed by HPLC to give Betulinic acid as a compound with a mobile phase of 30% acetonitrile/water. The compound Betulinic acid has no ultraviolet ray, and the thin layer is blue in color. The Er.1-2 is chromatographed by HPLC to obtain the compound alpha-amyrin, and the mobile phase is a 35% acetonitrile/water system.

Er.3 is subjected to reverse silica gel column chromatography, and the mobile phase is methanol: water system (80% methanol), silica gel (200-: methanol system (v/v ═ 25:1) to give the compound Hydroxyytosol.

Er.7 is subjected to reverse silica gel column chromatography, and the mobile phase is methanol: and (3) detecting a water system (v/v is 30-100%) by a thin layer to obtain Er.7-1, Er.7-2, Er.7-3 and Er.7-4. Er.7-3 was chromatographed by HPLC with a mobile phase of 32% acetonitrile/water to give the compound, taxifolin.

Betulinic acid, white solid.¹H NMR(CD₃COCD₃,500MHz)δ_H:3.12(1H,dd,J＝11.5,5.0Hz,H-3),0.97(3H,s),0.75(3H,s),0.95(3H,s),0.85(3H,s),1.00(3H,s),4.70(1H,d,J＝2.1Hz,H-29b),4.58(1H,dd,J＝2.3,1.4Hz,H-29a),1.68(3H,s,H-30)；¹³C-NMR(CD₃COCD₃,126MHz)δ_C:40.1(C-1),28.2(C-2),79.8(C-3),40.2(C-4),57.0(C-5),16.9(C-6),35.8(C-7),42.1(C-8),52.2(C-9),38.3(C-10),22.2(C-11),27.0(C-12),39.8(C-13),43.7(C-14),31.0(C-15),33.5(C-16),50.6(C-19),152.2(C-20),31.9(C-21),38.3(C-22),28.8(C-23),16.8(C-24),16.3(C-25),19.6(C-26),15.2(C-27),110.2(C-29),19.7(C-30).

α -amyrin, white solid. Using nmr analysis, the data were as follows:¹H NMR(CD₃OD，500MHz)δ_H:0.91(3H,s),0.99(3H,s),1.03(3H,d,J＝6.5Hz),1.04(3H,s),1.06(3H,d,J＝6.5Hz),1.25(3H,s),1.27(3H,s),2.66(1H,d,J＝11.2Hz,18-H),3.48(1H,m,3-H),5.51(1H,brs,12-H)；¹³C-NMR(CD₃OD,126MHz)δ_C181.1(C-28),139.7(C-13),126.9(C-12),79.7(C-3),56.8(C-5),54.4(C-18),48.2(C-17,9),43.3(C-14),40.8(C-1),40.4(C-8),40.4(C-4),40.0(C-19),39.8(C-10),38.1(C-20),38.1(C-22),34.3(C-7),31.8(C-28),29.2(C-23),28.8(C-2),27.9(C-15),25.3(C-16),24.4(C-11),24.1(C-27),21.6(C-30),19.5(C-6),17.8(C-29),17.7(C-26), 25.4 (C-16), 24.1 (C-16). The Chinese name is alpha-amyrin.

Taxifolin, white crystals. Using nmr analysis, the data were as follows:¹H NMR(CD₃COCD₃,500MHz)δ_H:5.02(d,2-H),4.61(d,3-H),5.94(d,6-H),5.98(d,8-H),7.06(d,2'-H),6.86(d,5'-H),6.91(dd,6'-H),11.71(s,5-OH)；¹³C NMR(CD₃COCD₃,126MHz)δ_C198.2(C-4),167.9(C-7),165.1(C-5),164.2(C-8a),146.6(C-4'),145.8(C-3'),129.8(C-2'),120.8(C-6'),115.9(C-2'),115.8(C-5'),101.5(C-4a),97.1(C-6),96.0(C-8),84.5(C-2),73.2(C-3). Chinese name is taxifolin.

Hydroxyytosol, colorless oil. Using nuclear magnetic resonance spectroscopyThe data were analyzed as follows:¹H NMR(CD₃OD,500MHz)δ_H:6.67(H-5,d,J＝8.0),6.65(H-2,d,J＝1.9),6.51(H-6,dd,J＝8.0,J＝1.9),2.65(H-2',t,J＝7.2),2.65(H-1',t,J＝7.2)；¹³C-NMR(CD₃OD,126MHz)δ_C146.1(C-3),144.6(C-4),131.8(C-1),121.2(C-6),117.1(C-2),116.3(C-5),64.6(C-2'),39.7 (C-1'). The Chinese name is phenethyl alcohol.

Test example: in vitro anti-PRRSV activity of compounds alpha-amyrin, taxifolin and hydroxyytyrosol extracted from Michihua

Experimental materials: the Marc-145 cell line was a gift from the university of agriculture of south China. PRRSV was passed 2 times on Marc-145 cells, as a gift from southern China university of agriculture, and TCID was determined by Reed-Muench method₅₀Is 10^-1mL, the concentration for this test is 100 TCID₅₀

The newborn calf serum is purchased from Gibco company, inactivated in 56 deg.C water bath for 30 min, subpackaged, and stored at-20 deg.C for use. Penicillin and streptomycin are dissolved in triple distilled water to prepare solutions containing 1 ten thousand units of penicillin and streptomycin respectively, the solutions are filtered and sterilized by a 0.22 mu m microporous filter membrane, and the solutions are subpackaged in small bottles and stored at the temperature of minus 20 ℃ for later use.

DMEM culture solution is prepared from Gibco company, and is prepared by dissolving in distilled water, filtering to remove bacteria, packaging, and storing at 4 deg.C with serum and double antibody.

Phosphate Buffered Saline (PBS) was purchased from Shanghai Xinyu Biotech Ltd

The experimental method comprises the following steps:

the Marc-145 cells growing into a monolayer are digested and passaged in a conventional way, and the cell number is adjusted to 1 × 10⁶～1×10⁷Adding into 96-well culture plate at a concentration of 100 μ L/well, placing at 37 deg.C and 5% CO₂Culturing at constant temperature in incubator, sequentially adding 1 × 10 dilution after the cells grow into monolayer^-1～1×10^-11100 μ L/well of the virus solution of (1), each dilution of KLJHFGFDSAs'd ' f ' g ' h ' j ' k ' L

Calculating the TCID of the virus₅₀Is 10^-3Diluting to 10-100 TCID before use₅₀. And (5) standby.

Diluting 3 compounds with cell maintenance solution in multiple proportion for 8 concentration gradients in a safe concentration range, adding into Marc-145 cell culture plate with single layer growth, 4 μ L/well with 2 dilutions, allowing to act in 37 deg.C incubator for 72 hr, discarding medicinal liquid, and adding 100 TCID into each well₅₀The virus dilution (100 μ L) was set with 1 group of cell control and virus control, respectively, and the temperature was controlled at 37 ℃ with 5% CO₂Culturing in incubator at constant temperature, observing cytopathic condition every day, and measuring A by MTT method when the pathological change of virus control group reaches 70% -80%₅₇₀Obtaining the inhibition rate according to a formula, fitting a curve by using Excel, and calculating to obtain IC₅₀The value of (c). (inhibition rate ═ [ (administration-background)/(control-background)]×100％)

Determination of anti-PRRSV Activity of four Compounds

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.