Machilus robustus extract and preparation method and application thereof

Abstract

The present invention provides a Machilus robustus extract and a preparation method and application thereof, belonging to the technical field of Machilus robustus extract application. The present invention uses Machilus robustus as a raw material, extracts with ethanol aqueous solution, concentrates under reduced pressure, crystallizes at low temperature with vegetable oil and organic solvent, and dries to prepare Machilus robustus extract, and evaluates the effect of Machilus robustus extract on inflammatory response of RAW264.7 cells induced by lipopolysaccharide. The Machilus robustus extract can inhibit the production of inflammatory factors NO, TNFα and IL-6, downregulate the expression of inflammatory proteins iNOS, COX2 and NLRP3, reduce the mRNA levels of inflammatory genes iNOS, COX2, NLRP3, TNFα, IL-6 and IL-1β, inhibit the phosphorylation levels of ERK, P38, JNK and P65, and prevent the nuclear translocation of NF-κB P65, which is expected to be used in the preparation of drugs for inhibiting inflammatory response of RAW264.7 cells induced by lipopolysaccharide.

Description

Technical Field

[0001] The present invention relates to the technical field of Machilus rubra extract application, and in particular to Machilus rubra robustus extract and a preparation method and application thereof. Background Art

[0002] Machilus is a tree of the Lauraceae family, about 10 to 25 meters tall, with a breast diameter of up to 40 centimeters. The bark is brown, the branches are thick, cylindrical, the cortex is gray-brown, longitudinally fissured, and hairless. There are more than 100 species in the world, mainly distributed in tropical and subtropical regions in southeastern and eastern Asia. In my country, Machilus is produced in the southwest, central and southern parts of Taiwan Province, and in the north to Shandong, Hubei, Gansu and southern Shaanxi.

[0003] Currently, research on Machilus plants focuses on seed propagation and seedling growth, while there are few reports on the active ingredients of Machilus plants, and the related extraction process is complex, making it difficult to achieve industrialization. Summary of the invention

[0004] The object of the present invention is to provide a Machilus robustus extract and a preparation method and application thereof. The Machilus robustus extract provided by the present invention has good anti-inflammatory activity, and the preparation method is simple, fast and low-cost.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a Machilus robustus extract, comprising the following steps:

[0007] The Machilus robustus powder is soaked in an ethanol aqueous solution for extraction, and the solid-liquid separation is performed to obtain an extract;

[0008] The extract is concentrated to dryness under reduced pressure, the concentrate is mixed with vegetable oil and an organic solvent, low-temperature crystallization is performed, solid-liquid separation is performed, and the precipitate is retained and dried to obtain a Machilus robustus extract;

[0009] The temperature of the low-temperature crystallization is 2-8°C and the time is 18-32 hours; the low-temperature crystallization is carried out under static conditions.

[0010] Preferably, the temperature of the reduced pressure concentration is 40-60° C., the rotation speed is 40-60 rpm, and the vacuum degree is 40-100 bar.

[0011] Preferably, the particle size of the Machilus robusta powder is less than 100 mesh; the Machilus robusta comprises one or more of the branches, stems and leaves of Machilus robusta.

[0012] Preferably, the volume fraction of ethanol in the ethanol-water solution is 80-95%, and the dosage ratio of the Machilus robustus powder to the ethanol-water solution is 1 g: (20-30) mL.

[0013] Preferably, the soaking is performed 2 to 3 times, and the soaking time for each time is 18 to 24 hours.

[0014] Preferably, the soaking includes static soaking and stirring soaking, and the time of the stirring soaking is 8 to 14 hours.

[0015] Preferably, the vegetable oil is one or two of soybean oil, peanut oil, rapeseed oil, sesame oil and corn oil, and the mass ratio of the concentrate to the volume of the vegetable oil is 1 g: (4-6) mL.

[0016] Preferably, the organic solvent is one or two of n-hexane, petroleum ether, dichloromethane, ethyl acetate, n-butanol and acetone, and the volume ratio of the organic solvent to the vegetable oil is 1:1.

[0017] The present invention provides a Machilus robustus extract prepared by the preparation method described in the above scheme.

[0018] The present invention provides the use of the Machilus robustus extract described in the above scheme in the preparation of a drug or additive for inhibiting lipopolysaccharide-induced inflammatory response of RAW264.7 cells.

[0019] The invention provides a method for preparing Machilus robustus extract, comprising the following steps: soaking Machilus robustus powder in an ethanol aqueous solution for extraction, and separating the solid from the liquid to obtain an extract; concentrating the extract to dryness under reduced pressure, mixing the obtained concentrate with vegetable oil and an organic solvent, performing low-temperature crystallization, retaining the precipitate and drying to obtain the Machilus robustus extract; the low-temperature crystallization temperature is 2-8°C, and the time is 18-32 hours.

[0020] The invention uses Machilus robustus as a raw material, adopts the steps of ethanol aqueous solution extraction, reduced pressure concentration, low temperature crystallization of vegetable oil and organic solvent, drying and the like to prepare Machilus robustus extract, adopts Griess, ELISA, western blot and RT-PCR methods to evaluate the influence of Machilus robustus extract on inflammatory response of RAW264.7 cells induced by lipopolysaccharide, and the Machilus robustus extract can inhibit the production of inflammatory factors NO, TNFα and IL-6, down-regulate the expression of inflammatory proteins iNOS, COX2 and NLRP3, reduce the mRNA levels of inflammatory genes iNOS, COX2, NLRP3, TNFα, IL-6 and IL-1β, inhibit the phosphorylation levels of ERK, P38, JNK and P65, and prevent the nuclear translocation of NF-κB P65, and is expected to be applied in the preparation of drugs for inhibiting inflammatory response of RAW264.7 cells induced by lipopolysaccharide.

[0021] The Machilus robustus extract prepared by the invention has good anti-inflammatory activity and is used at a low concentration. When the concentration is as low as 12.5 μg / mL, it can still effectively inhibit the inflammatory response of RAW264.7 cells induced by lipopolysaccharide.

[0022] In addition, the preparation process of the present invention is simple and rapid, does not require column passing, and has low cost.

[0023] The Machilus robustus extract of the invention has a high yield of 4.3%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a data graph showing the effect of Machilus robustus extract on NO production and cell survival rate in Example 1 of the present invention;

[0025] Figure 2 This is a data diagram showing the effect of Machilus robustus extract on TNFα production in Example 1 of the present invention;

[0026] Figure 3 This is a data graph showing the effect of Machilus robustus extract on IL-6 production in Example 1 of the present invention;

[0027] Figure 4 This is a data graph showing the effect of Machilus robustus extract on the expression of iNOS, COX2 and NLRP3 proteins in Example 1 of the present invention;

[0028] Figure 5 This is a data graph showing that Machilus robustus extract affects the level of iNOS gene mRNA in Example 1 of the present invention;

[0029] Figure 6 This is a data graph showing the effect of Machilus robustus extract on the mRNA level of COX2 gene in Example 1 of the present invention;

[0030] Figure 7 This is a data graph showing that Machilus robustus extract affects NLRP3 gene mRNA levels in Example 1 of the present invention;

[0031] Figure 8 This is a data graph showing that Machilus robustus extract affects TNFα gene mRNA levels in Example 1 of the present invention;

[0032] Fig. 9 This is a data graph showing the effect of Machilus robustus extract on IL-6 gene mRNA level in Example 1 of the present invention;

[0033] Fig.10 This is a data graph showing the effect of Machilus robustus extract on IL-1β gene mRNA level in Example 1 of the present invention;

[0034] Fig.11This is a data graph showing the effect of Machilus robustus extract in Example 1 of the present invention on the phosphorylation levels of ERK, P38 and JNK;

[0035] Fig.12 This is a data graph showing the effect of Machilus robustus extract on the phosphorylation level of P65 in Example 1 of the present invention;

[0036] Fig.13 This is a data graph showing the effect of Machilus robustus extract on NF-κB P65 nuclear translocation in Example 1 of the present invention.

[0037] Fig.14 This is a data graph showing the effect of Machilus guangdongensis extract on NO production and cell survival rate in comparative example 1 of the present invention;

[0038] Fig.15 This is a data graph showing the effect of Machilus oleifera extract on NO production and cell survival rate in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0039] The present invention provides a method for preparing a Machilus robustus extract, comprising the following steps:

[0040] The Machilus robustus powder is soaked in an ethanol aqueous solution for extraction, and the solid-liquid separation is performed to obtain an extract;

[0041] The extract is concentrated to dryness under reduced pressure, the obtained concentrate is mixed with vegetable oil and an organic solvent, subjected to low-temperature crystallization, solid-liquid separation, and the precipitate is retained and dried to obtain the Machilus robustus extract.

[0042] In the present invention, unless otherwise specified, the raw materials used are commercially available products well known in the art.

[0043] The invention soaks Machilus robustus powder in an ethanol aqueous solution for extraction, and separates the solid and the liquid to obtain an extract.

[0044] In the present invention, the particle size of the Machilus robustus powder is preferably less than 100 mesh; the Machilus robustus preferably includes one or more of Machilus robustus branches, stems and leaves. In an embodiment of the present invention, the Machilus robustus powder is obtained by crushing Machilus robustus plants (including branches, stems and leaves) and then sieving.

[0045] In the present invention, the volume fraction of ethanol in the ethanol aqueous solution is preferably 80-95%, more preferably 85-90%, and in the embodiment of the present invention, specifically 95%. In the present invention, the dosage ratio of the Rhizoma Cyperi robusta powder to the ethanol aqueous solution is preferably 1g: (20-30) mL, more preferably 1g: (24-27) mL. In the embodiment of the present invention, specifically 1g: 20 mL.

[0046] In the present invention, the soaking preferably includes static soaking and stirring soaking; the time of the stirring soaking is preferably 8 to 14 hours, more preferably 10 to 12 hours. The present invention has no special requirements on the stirring rate of the stirring soaking, and the stirring rate well known in the art can be used. In the present invention, the number of soaking is preferably 2 to 3 times; the time of each soaking (including static soaking and stirring soaking) is preferably 18 to 24 hours, more preferably 20 to 22 hours.

[0047] The present invention has no special requirements for the solid-liquid separation method, and any solid-liquid separation method known in the art may be used, such as gauze filtration.

[0048] In the present invention, when multiple soakings are used (ie, multiple extractions are performed), the present invention preferably performs solid-liquid separation after each soaking, and finally combines the filtrate to serve as the extract.

[0049] After obtaining the extract, the present invention concentrates the extract to dryness under reduced pressure, mixes the obtained concentrate with vegetable oil and an organic solvent, performs low-temperature crystallization, separates the solid from the liquid, retains the precipitate and dries it to obtain the Machilus robustus extract;

[0050] The present invention preferably performs the reduced pressure concentration in a rotary evaporator. In the present invention, the reduced pressure concentration temperature is preferably 40 to 60°C, more preferably 45 to 55°C, and further preferably 50°C; the rotation speed is preferably 40 to 60 rpm, more preferably 45 to 55 rpm, and further preferably 50 rpm; the vacuum degree is preferably 40 to 100 bar, more preferably 50 to 80 bar, and further preferably 60 to 70 bar.

[0051] In the present invention, the vegetable oil is preferably one or two of soybean oil, peanut oil, rapeseed oil, sesame oil and corn oil; the volume ratio of the concentrate to the vegetable oil is preferably 1g:(4-6)mL, more preferably 1g:(4.5-5.5)mL, and further preferably 1g:5mL.

[0052] In the present invention, the organic solvent is preferably one or two of n-hexane, petroleum ether, dichloromethane, ethyl acetate, n-butanol and acetone. In the present invention, the volume ratio of the organic solvent to the vegetable oil is preferably 1:1.

[0053] In the present invention, the vegetable oil and the organic solvent are preferably mixed first, and then the mixed solution of the two is added to the concentrate.

[0054] In the present invention, the temperature of the low-temperature crystallization is 2-8°C, preferably 3-7°C, and more preferably 4-6°C; the time is 18-32h, preferably 20-30h, and more preferably 24h; the low-temperature crystallization is preferably carried out under static conditions.

[0055] In the low-temperature crystallization process of the present invention, the vegetable oil effectively wraps the impurity components in the alcohol extract and disperses them in the organic solvent, and the effective components are crystallized and precipitated under the action of low temperature.

[0056] The present invention provides a Machilus robustus extract prepared by the preparation method described in the above scheme.

[0057] The invention uses Machilus robustus plants as raw materials, adopts the steps of ethanol aqueous solution extraction, reduced pressure concentration, low temperature crystallization of vegetable oil and organic solvent, drying and the like to prepare Machilus robustus extract, adopts Griess, ELISA, western blot and RT-PCR methods to evaluate the influence of Machilus robustus extract on inflammatory response of RAW264.7 cells induced by lipopolysaccharide, and the Machilus robustus extract can inhibit the production of inflammatory factors NO, TNFα and IL-6, down-regulate the expression of inflammatory proteins iNOS, COX2 and NLRP3, reduce the mRNA levels of inflammatory genes iNOS, COX2, NLRP3, TNFα, IL-6 and IL-1β, inhibit the phosphorylation levels of ERK, P38, JNK and P65, and prevent the nuclear translocation of NF-κB P65, and is expected to be applied in the preparation of drugs for inhibiting inflammatory response of RAW264.7 cells induced by lipopolysaccharide.

[0058] The present invention provides the use of the Machilus robustus extract described in the above scheme in the preparation of a drug or additive for inhibiting lipopolysaccharide-induced inflammatory response of RAW264.7 cells.

[0059] The Machilus robustus extract provided by the present invention, its preparation method and application are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] The dried Machilus robustus plants were crushed and passed through a 100-mesh sieve; a certain weight of powder was weighed, and a 95% ethanol aqueous solution by volume was added according to 1g:20mL, and the mixture was soaked for 20 hours, during which time the mixture was continuously stirred for 12 hours, and the filtrate was filtered through gauze to retain the filtrate, and the filter residue was repeatedly extracted with the same volume of 95% ethanol aqueous solution for 3 times, the filtrate was filtered and combined, and the filtrate was concentrated to dryness under reduced pressure on a rotary evaporator, and the reduced pressure concentration was set at a temperature of 50°C, a rotation speed of 50rpm, and a vacuum degree of 40bar. A mixed solution of soybean oil and dichloromethane (the volume ratio of soybean oil to dichloromethane was 1:1) was added according to a ratio of the concentrate to soybean oil of 1g:4mL, and the mixture was fully mixed, and the mixture was allowed to stand at 4°C for 24 hours. The precipitate was collected by filtration and dried to obtain a Machilus robustus extract with a yield of 4.3%.

[0062] The Machilus robustus extract was dissolved in DMSO to 100 mg / mL, sterilized by UV for 30 min, and diluted to 400, 200, 100, and 50 μg / mL with serum-free DMEM high-glucose culture medium. LPS (lipopolysaccharide) was diluted to 400 ng / mL and used immediately. RAW264.7 cells were cultured to the logarithmic growth phase, and the cells were seeded in a 96-well plate and cultured overnight at a density of 5 × 10 4 / well, the volume was 100μL / well, the extract was added to pre-treat the cells for 2h, 50μL / well, and LPS was added for 24h, 50μL / well; the Control group was treated with culture medium (without drug) for 2h, 50μL / well, and culture medium (without LPS) was added for 24h, 50μL / well; the LPS group was treated with culture medium (without drug) for 2h, 50μL / well, and LPS was added for 24h, 50μL / well; the Griess method was used to detect the NO production in the cell supernatant, and the MTT method was used to detect the cell survival rate. The results are shown in Figure 1 As shown, Machilus robustus extract exhibited strong anti-inflammatory potential.

[0063] ELISA, western blot and RT-PCR were further used to evaluate the anti-inflammatory potential of Machilus robustus extract.

[0064] Specifically, RAW264.7 cells were cultured to the logarithmic growth phase and seeded in 6-well plates overnight at a density of 1 × 10 6 / well, with a volume of 3mL / well; the cells were pretreated with Machilus robustus extract for 2h, and LPS solution was added to treat the cells for 24h. The contents of PGE2, TNFα, IL-6 and IL-1β in the cell supernatant were detected by ELISA, the protein expressions of iNOS, COX2 and NLRP3 were detected by western blotting, and the gene expressions of iNOS, COX2, NLRP3, TNFα, IL-6 and IL-1β were detected by RT-PCR;

[0065] RAW264.7 cells were cultured to the logarithmic growth phase and seeded in 6-well plates overnight at a density of 1 × 10 6 / well, with a volume of 3mL / well; the cells were pretreated with Machilus robusta extract for 2h, and LPS solution was added to treat the cells for 6h. The expression and phosphorylation levels of ERK, P38, JNK and P65 proteins and the nuclear translocation of NF-κB P65 were detected by western blotting.

[0066] The results are as follows Figures 2 to 13As shown, Machilus robustus extract can inhibit the production of inflammatory factors NO, TNFα and IL-6, downregulate the expression of inflammatory proteins iNOS, COX2 and NLRP3, reduce the mRNA levels of inflammatory genes iNOS, COX2, NLRP3, TNFα, IL-6 and IL-1β, inhibit the phosphorylation levels of ERK, P38, JNK and P65, and prevent the nuclear translocation of NF-κB P65. It is expected to be used in the preparation of drugs for inhibiting lipopolysaccharide-induced inflammatory response in RAW264.7 cells.

[0067] Comparative Example 1

[0068] The dried Machilus guangdongensis plant was crushed and passed through a 100-mesh sieve; a certain weight of powder was weighed, and a volume of 95% ethanol aqueous solution was added according to 1g:20mL, and the mixture was soaked for 20h, during which time it was continuously stirred for 12h, and the filtrate was filtered through gauze to retain the filtrate, and the filter residue was repeatedly extracted with the same 95% ethanol aqueous solution for 3 times, and the filtrate was filtered and combined, and the filtrate was concentrated to dryness under reduced pressure on a rotary evaporator, and the reduced pressure concentration setting temperature was 50°C, the speed was 50rpm, and the vacuum degree was 40bar. A mixed solution of soybean oil and dichloromethane (the volume ratio of soybean oil and dichloromethane was 1:1) was added according to the ratio of the concentrate to soybean oil of 1g:4mL, and the mixture was fully mixed, and the mixture was allowed to stand at 4°C for 24h, and the precipitate was collected by filtration and dried to obtain the Machilus guangdongensis extract.

[0069] The extract of Machilus guangdongensis was dissolved in DMSO to 100 mg / mL, sterilized by UV for 30 min, and diluted to 400, 200, 100, and 50 μg / mL with serum-free DMEM high-glucose culture medium. LPS (lipopolysaccharide) was diluted to 400 ng / mL and used immediately. RAW264.7 cells were cultured to the logarithmic growth phase, and the cells were seeded in a 96-well plate and cultured overnight at a density of 5 × 10 4 / well, the volume was 100μL / well, the extract was added to pre-treat the cells for 2h, 50μL / well, and LPS was added for 24h, 50μL / well; the Control group was treated with culture medium (without drug) for 2h, 50μL / well, and culture medium (without LPS) was added for 24h, 50μL / well; the LPS group was treated with culture medium (without drug) for 2h, 50μL / well, and LPS was added for 24h, 50μL / well; the Griess method was used to detect the NO production in the cell supernatant, and the MTT method was used to detect the cell survival rate. The results are shown in Fig.14 As shown in the figure, the anti-inflammatory potential of Machilus guangdongensis extract is not as good as that of Machilus robustus extract.

[0070] Comparative Example 2

[0071] The dried Machilus willowii plants were crushed and passed through a 100-mesh sieve; a certain weight of powder was weighed, and a 95% ethanol aqueous solution was added by volume at a ratio of 1g:20mL, and the mixture was soaked for 20 hours, during which the mixture was continuously stirred for 12 hours, and the filtrate was filtered through gauze to retain the filtrate, and the residue was repeatedly extracted with the same 95% ethanol aqueous solution for 3 times, the filtrate was filtered and combined, and the filtrate was concentrated to dryness under reduced pressure on a rotary evaporator, and the reduced pressure concentration was set at a temperature of 50°C, a speed of 50rpm, and a vacuum degree of 40bar. A mixed solution of soybean oil and dichloromethane (the volume ratio of soybean oil to dichloromethane was 1:1) was added according to a ratio of the concentrate to soybean oil of 1g:4mL, and the mixture was fully mixed, and the mixture was allowed to stand at 4°C for 24 hours, and the precipitate was collected by filtration and dried to obtain a Machilus willowii extract.

[0072] The extract of Machilus willowii was dissolved in DMSO to 100 mg / mL, sterilized by UV for 30 min, and diluted to 400, 200, 100, and 50 μg / mL with serum-free DMEM high-glucose culture medium. LPS (lipopolysaccharide) was diluted to 400 ng / mL and used immediately. RAW264.7 cells were cultured to the logarithmic growth phase, and the cells were seeded in a 96-well plate and cultured overnight at a density of 5 × 10 4 / well, the volume was 100μL / well, the extract was added to pre-treat the cells for 2h, 50μL / well, and LPS was added for 24h, 50μL / well; the Control group was treated with culture medium (without drug) for 2h, 50μL / well, and culture medium (without LPS) was added for 24h, 50μL / well; the LPS group was treated with culture medium (without drug) for 2h, 50μL / well, and LPS was added for 24h, 50μL / well; the Griess method was used to detect the NO production in the cell supernatant, and the MTT method was used to detect the cell survival rate. The results are shown in Fig.15 As shown in the figure, the anti-inflammatory potential of Machilus willowii extract is not as good as that of Machilus robustus extract.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of Machilus robustus extract in the preparation of a drug or additive for inhibiting lipopolysaccharide-induced inflammatory response in RAW264.7 cells, characterized in that: The preparation method of the Machilus robustus extract comprises the following steps: The Machilus robustus powder is soaked in an ethanol aqueous solution for extraction, and the solid-liquid separation is performed to obtain an extract; the volume fraction of ethanol in the ethanol aqueous solution is 80-95%, and the dosage ratio of the Machilus robustus powder to the ethanol aqueous solution is 1 g: (20-30) mL; The extract is concentrated to dryness under reduced pressure, the concentrate is mixed with vegetable oil and an organic solvent, low-temperature crystallization is performed, solid-liquid separation is performed, and the precipitate is retained and dried to obtain a Machilus robustus extract; The temperature of the low temperature crystallization is 2-8°C and the time is 18-32h; The vegetable oil is soybean oil; the organic solvent is dichloromethane; and the volume ratio of the organic solvent to the vegetable oil is 1:

1.

2. The use according to claim 1, characterized in that: The temperature of the reduced pressure concentration is 40-60° C., the rotation speed is 40-60 rpm, and the vacuum degree is 40-100 bar.

3. The use according to claim 1, characterized in that: The particle size of the Machilus robusta powder is less than 100 meshes; the Machilus robusta includes one or more of the branches, stems and leaves of Machilus robusta.

4. The use according to claim 1 or 3, characterized in that: The soaking is performed 2 to 3 times, and the soaking time is 18 to 24 hours each time.

5. The use according to claim 4, characterized in that: The soaking includes static soaking and stirring soaking, and the time of the stirring soaking is 8 to 14 hours.

6. The use according to claim 1, characterized in that: The mass ratio of the concentrate to the volume of the vegetable oil is 1 g: (4-6) mL.

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