阅读说明：本技术 一种从莲房中提取抗炎成分的方法及应用 (Method for extracting anti-inflammatory component from lotus seed pot and application ) 是由 谢传奇 吴磊 熊伟 王书芬 于 2021-09-08 设计创作，主要内容包括：本发明属于天然产物化学领域,涉及一种从莲房中提取抗炎成分的方法,将干燥的莲房粉碎,用乙醇水溶液浸泡,碎渣过滤,滤液减压浓缩,并用超纯水分散,用乙酸乙酯萃取并浓缩,将浓缩液与硅胶混匀进行柱分离,用二氯甲烷和甲醇进行梯度洗脱,洗脱液进行薄层色谱分析,合并含有相同成分的洗脱液并浓缩,进行色谱鉴定,确定各成分的结构和纯度；对所得莲房单体成分进行抗炎作用分析,反油酸乙酯的抗炎能力最强,其可抑制炎症因子NO、PGE2和TNFα产生,降低iNOS和COX2的蛋白表达和mRNA水平,调节MAPKs和NF-κB信号活化水平；本发明制得的反油酸乙酯纯度高,可作为抗炎药物研发的物质基础。(The invention belongs to the field of natural product chemistry, and relates to a method for extracting anti-inflammatory components from lotus seedpod, which comprises the steps of crushing dried lotus seedpod, soaking the lotus seedpod in ethanol water solution, filtering broken slag, concentrating filtrate under reduced pressure, dispersing the filtrate with ultrapure water, extracting and concentrating the filtrate with ethyl acetate, uniformly mixing the concentrated solution and silica gel for column separation, performing gradient elution by using dichloromethane and methanol, performing thin-layer chromatography on eluent, combining the eluents containing the same components and concentrating the eluents, performing chromatographic identification, and determining the structure and purity of each component; the anti-inflammatory effect analysis of the obtained lotus seed pot monomer component shows that the ethyl elaeate has the strongest anti-inflammatory capability, can inhibit the production of inflammatory factors NO, PGE2 and TNF alpha, reduce the protein expression and mRNA level of iNOS and COX2, and regulate the activation level of MAPKs and NF-kB signals; the prepared ethyl elaeate is high in purity and can be used as a material basis for research and development of anti-inflammatory drugs.)

1. A method for extracting anti-inflammatory components from lotus seedpod is characterized in that the dried lotus seedpod is crushed, soaked in ethanol water solution, crushed slag is filtered, filtrate is concentrated under reduced pressure, dispersed by ultrapure water, extracted by ethyl acetate and concentrated, concentrated solution and silica gel are mixed uniformly for column separation, dichloromethane and methanol are used for gradient elution, thin-layer chromatography analysis is carried out on eluent, eluent containing the same components is combined and concentrated, and chromatography identification is carried out to determine the structure and purity of each component; and (4) analyzing the anti-inflammatory effect of the obtained lotus seed pot monomer component, and screening out the monomer component with the highest anti-inflammatory activity.

2. The method for extracting the anti-inflammatory component from the lotus seedpod as claimed in claim 1, wherein the mass volume ratio of the lotus seedpod dry matter to the ethanol water solution is 1: 15-20 percent, and the volume concentration of the ethanol water solution is 95 percent.

3. The method of claim 1, wherein the gradient ratio of dichloromethane to methanol is 100:1 and gradually adjusted to 1: 1.

4. The method of claim 1, wherein the chromatographic identification technique comprises Nuclear Magnetic Resonance (NMR) hydrogen spectrometry, mass spectrometry, and high performance liquid chromatography.

5. The method of claim 1, wherein the obtained 10 individual lotus seed pot components are isorhamnetin-3-O-beta-D-diglucoside, rutin, quercetin-3-O-beta-D-galactoside, kaempferol-3-O-rutinoside, quercetin, ethyl linoleate, ethyl elaeate, 7 beta-hydroxy betulinic acid, and balsamic alcohol; fully dissolving 10 lotus seed pot monomer components by using a DMSO solution to ensure that the concentration of mother liquor is 100 mg/mL; adjusting RAW264.7 cells to logarithmic phase, blowing off the cells with fresh culture solution, counting, adjusting cell concentration, and uniformly transferring the cells into a 96-hole sterile culture plate by using a multi-hole pipette with 100 mu L/hole; preparing 10 lotus seedpod monomer components into a solution to be treated with the concentration of 400 mug/mL by using a serum-free culture solution after 24h, wherein 3 multiple holes are formed in each treatment, 50 mug/L are formed in each hole, 6 drug-free multiple holes are formed in each treatment, 3 holes are used as LPS control, and 3 holes are used as blank control; preparing lipopolysaccharide solution with concentration of 4 μ g/mL with serum-free culture solution after 2h, wherein each well has a volume of 50 μ L, and the blank control well is replaced by serum-free culture solution; after 24h, detecting the content of NO in cell supernatant by a Griess reagent method, evaluating the anti-inflammatory capability of each component, and analyzing to obtain rutin, quercetin, ethyl linoleate, ethyl elaeate, 7 beta-hydroxy betulinic acid and balm alcohol with anti-inflammatory effect.

6. A method for extracting anti-inflammatory component ethyl elaeate from lotus seedpod comprises the steps of crushing dried lotus seedpod, soaking the lotus seedpod in an ethanol water solution, filtering broken slag, concentrating filtrate under reduced pressure, dispersing the filtrate with ultrapure water, extracting the filtrate with ethyl acetate and concentrating the filtrate, uniformly mixing the concentrated solution and silica gel to perform column separation, performing gradient elution by using dichloromethane and methanol, controlling the ratio of dichloromethane/methanol to 50/1 of eluent, performing thin-layer chromatography on the eluent, combining the eluents containing the same components and concentrating the eluent to obtain the ethyl elaeate.

7. The application of the anti-inflammatory component ethyl elaeate of lotus seedpod is characterized in that the ethyl elaeate is used as a basic substance for research and development of anti-inflammatory drugs.

8. The use of the anti-inflammatory component ethyl elaeate of nelumbo nucifera according to claim 7 for inhibiting the production of inflammatory factors NO, PGE2 and TNF α, reducing the protein expression and mRNA levels of iNOS and COX2, and modulating the level of activation of MAPKs and NF- κ B signaling.

Technical Field

The invention belongs to the field of natural product chemistry, and particularly relates to a method for extracting an anti-inflammatory component from lotus seed pots and application of the anti-inflammatory component.

Background

Inflammation is closely related to human health and is a key risk factor for chronic diseases such as autoimmune diseases, arthritis, obesity, diabetes, and cancer. According to the statistical data and reports of the world health organization, the global cancer incidence rate in 2018 is 1810 ten thousand, and the mortality rate is 960 ten thousand; over 6.5 billion adults were obese in 2016; in 2014 4.22 billion adults had diabetes. Therefore, the development of anti-inflammatory research is crucial to the life and health of people. Despite the enormous efforts of many scholars in anti-inflammatory research, the reality that many people in the world are suffering from inflammatory diseases shows that the continued intensive development of anti-inflammatory molecule discovery and its pharmacological action research still has important practical significance.

Lotus seedpod, also known as lotus seedpod, is a dried mature receptacle of Nelumbo nucifera Gaertn, a plant of the family Nymphaeaceae. According to the Chinese pharmacopoeia, the lotus seed pot is funnel-shaped or inverted cone-shaped in appearance, fine longitudinal lines and wrinkles are formed on the surface, a plurality of circular holes are formed in the top surface, and peduncle residues are formed at the base. Modern phytochemistry research shows that lotus seedpod contains rich natural active ingredients including alkaloids, flavones, terpenoids, organic acids, sterols, lipids, proteins, carbohydrates and the like. And pharmacological research shows that the lotus seed pot has various effects of enhancing memory, inhibiting bacteria, resisting inflammation, resisting oxidation, resisting cancer, preventing radiation, reducing blood fat and the like. However, the lotus seed pot is a non-edible raw material and is often discarded as a leftover in the processing process of lotus seeds, so that the health care and medicinal values of the lotus seed pot are not effectively developed and utilized.

Disclosure of Invention

The invention aims to provide a method for extracting anti-inflammatory components from lotus seedpod, which takes lotus seedpod as a raw material, adopts the steps of ethanol extraction, reduced pressure concentration, ethyl acetate extraction, silica gel column separation, dichloromethane/methanol elution, chromatographic identification and the like to prepare 10 monomer compounds, and analyzes the anti-inflammatory action of the obtained lotus seedpod monomer components, wherein the anti-inflammatory capability of ethyl elaeate (E9OAEE) is strongest, the production of inflammatory factors NO, PGE2 and TNF alpha can be inhibited, the protein expression and mRNA level of iNOS and COX2 can be reduced, and the signal activation level of MAPKs and NF-kappa B can be adjusted. The prepared ethyl elaeate is high in purity and can be used as a material basis for research and development of anti-inflammatory drugs.

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

a method for extracting antiinflammatory components from receptaculum Nelumbinis comprises pulverizing dried receptaculum Nelumbinis, soaking in ethanol water solution, filtering, concentrating the filtrate under reduced pressure, dispersing with ultrapure water, extracting with ethyl acetate and concentrating, mixing the concentrated solution with silica gel, separating with column, performing gradient elution with dichloromethane and methanol, performing thin layer chromatography, mixing eluates containing the same components, concentrating, and performing chromatographic identification to determine the structure and purity of each component; and (4) analyzing the anti-inflammatory effect of the obtained lotus seed pot monomer component, and screening out the monomer component with the highest anti-inflammatory activity.

Further preferably, the mass-volume ratio of the lotus seed pot dry matter to the ethanol water solution is 1: 15-20 percent, and the volume concentration of the ethanol water solution is 95 percent.

Further preferably, the gradient ratio of dichloromethane to methanol is gradually adjusted to 1:1 at 100: 1.

Further preferably, the chromatographic identification technology mainly comprises nuclear magnetic resonance hydrogen spectroscopy, mass spectrometry and high performance liquid chromatography.

Further preferably, the obtained 10 lotus seedpod monomer components are isorhamnetin-3-O-beta-D-diglucoside, rutin, quercetin-3-O-beta-D-galactoside, kaempferol-3-O-rutinoside, quercetin, ethyl linoleate, ethyl elaidic acid, 7 beta-hydroxy betulinic acid, betulinic acid and balsamic alcohol respectively; fully dissolving 10 lotus seed pot monomer components by using a DMSO solution to ensure that the concentration of mother liquor is 100 mg/mL; adjusting RAW264.7 cells to logarithmic phase, blowing off the cells with fresh culture solution, counting, adjusting cell concentration, and uniformly transferring the cells into a 96-hole sterile culture plate by using a multi-hole pipette with 100 mu L/hole; preparing 10 lotus seedpod monomer components into a solution to be treated with the concentration of 400 mug/mL by using a serum-free culture solution after 24h, wherein 3 multiple holes are formed in each treatment, 50 mug/L are formed in each hole, 6 drug-free multiple holes are formed in each treatment, 3 holes are used as LPS control, and 3 holes are used as blank control; preparing lipopolysaccharide solution with concentration of 4 μ g/mL with serum-free culture solution after 2h, wherein each well has a volume of 50 μ L, and the blank control well is replaced by serum-free culture solution; after 24h, detecting the content of NO in cell supernatant by a Griess reagent method, evaluating the anti-inflammatory capability of each component, and analyzing to obtain rutin, quercetin, ethyl linoleate, ethyl elaeate, 7 beta-hydroxy betulinic acid and balm alcohol with anti-inflammatory effect.

Further preferably, the cell concentration is 5x10^5/mL-10x10^ 5/mL.

More preferably, the final treatment concentration of each individual lotus seed pot monomer component is 100. mu.g/mL, and the final treatment concentration of LPS is 1. mu.g/mL.

Further preferably, of the 10 lotus seed pot monomer components, ethyl elaeate (E9OAEE) has the strongest anti-inflammatory capability, and the inhibition rate of NO generation reaches 90%.

More particularly, the invention provides a method for extracting anti-inflammatory component ethyl elaeate from lotus seedpod, which comprises the steps of crushing dried lotus seedpod, soaking the lotus seedpod in an ethanol water solution, filtering broken slag, concentrating filtrate under reduced pressure, dispersing the filtrate with ultrapure water, extracting and concentrating the filtrate with ethyl acetate, uniformly mixing the concentrated solution and silica gel for column separation, performing gradient elution by using dichloromethane and methanol, controlling the eluent ratio to be dichloromethane/methanol 50/1, performing thin-layer chromatography on the eluent, combining the eluents containing the same component and concentrating to obtain the ethyl elaeate.

The invention also provides application of the anti-inflammatory component of lotus seed pot-ethyl elaeate as a basic substance for research and development of anti-inflammatory drugs. Used for inhibiting the production of inflammatory factors NO, PGE2 and TNF alpha, reducing the protein expression and mRNA level of iNOS and COX2, and regulating the activation level of MAPKs and NF-kappa B signals.

The invention takes lotus seed pot as raw material, and adopts the steps of ethanol extraction, decompression concentration, ethyl acetate extraction, silica gel column separation, dichloromethane/methanol elution, chromatographic identification and the like to prepare 10 monomeric compounds. Through a lipopolysaccharide-induced RAW264.7 cell inflammation model, ethyl elaeate is screened to have the most remarkable anti-inflammatory activity, can inhibit the production of inflammatory factors NO, PGE2 and TNF alpha, reduce the protein expression and mRNA level of iNOS and COX2, and regulate the activation level of MAPKs and NF-kappa B signals. And spectrogram data shows that the prepared ethyl elaeate is high in purity and good in anti-inflammatory effect, and can be used as a basic substance for research and development of anti-inflammatory drugs.

Drawings

FIG. 1 is a graph showing the effect of 10 lotus seedpod monomer components on NO content in example 1 of the present invention;

FIG. 2 is a graph of the effect of a series of concentrations of ethyl elaeate on NO content (A) and cell viability (B) in example 1 of the present invention;

FIG. 3 is a graph of the effect of a series of concentrations of ethyl elaeate on PGE2(A) and TNF α (B) in example 1 of the present invention;

FIG. 4 is a graph showing the effect of ethyl elaeate on the expression of iNOS (A) and COX2(B) proteins at a series of concentrations in example 1 of the present invention;

FIG. 5 is a graph of the effect of a series of concentrations of ethyl elaeate on the mRNA levels of iNOS (A), COX2(B) and GAPDH (C) in example 1 of the present invention;

FIG. 6 is a graph plotting the effect of serial concentrations of ethyl elaeate on the phosphorylation levels of MAPKs signaling pathway proteins (ERK, P38 and JNK) in example 1 of the present invention;

FIG. 7 is a graph of the effect of a series of concentrations of ethyl elaeate on nuclear translocation of NF- κ B in example 1 of the invention.

Detailed Description

The invention is further illustrated by the following specific examples, which are intended to be illustrative, but not limiting, of the invention.

Example 1

A method for extracting anti-inflammatory components from receptaculum nelumbinis and application thereof are disclosed: weighing dry matter of receptaculum nelumbinis 2kg, pulverizing with pulverizer, soaking in 15 times volume of ethanol water solution for 3 days (72h), filtering the residue, concentrating the filtrate under reduced pressure to about 200mL with rotary evaporator, and repeating the process for 3 times; diluting and dispersing the concentrated solution by using ultrapure water with 3 times of volume, extracting the diluted solution by using ethyl acetate with the same volume, repeatedly extracting for 3 times until an extract phase is colorless and transparent, and concentrating the ethyl acetate extract phase under reduced pressure to form a paste, wherein the weight of the paste is 4.225 g; uniformly mixing the paste with 2 times of silica gel, performing column chromatography, eluting with pure dichloromethane as eluent, eluting with 100:1 dichloromethane/methanol as eluent, gradually increasing methanol ratio in the eluent from 100:1 to 1:1, performing thin layer chromatography on the obtained eluent, wherein the developer is sulfuric acid: methanol (10:1), developing solvent dichloromethane: methanol (10:1), combining the eluents containing the components with the same specific migration value, concentrating under reduced pressure, air drying at room temperature, and determining the structure and purity of the compound by using techniques such as nuclear magnetic resonance hydrogen spectrum, mass spectrum and high performance liquid chromatography. Finally obtaining 10 lotus seedpod monomer components which are respectively isorhamnetin-3-O-beta-D-diglucoside, rutin, quercetin-3-O-beta-D-galactoside, kaempferol-3-O-rutinoside, quercetin, ethyl linoleate, ethyl elaidic acid, 7 beta-hydroxy betulinic acid, betulinic acid and balsamic alcohol.

Wherein the extraction process of the ethyl elaeate comprises the following steps: mixing the paste with 2 times of silica gel, performing column chromatography, eluting with dichloromethane/methanol 50/1 eluent, and performing thin layer chromatography, wherein the developer is sulfuric acid: methanol (10:1), developing solvent dichloromethane: methanol (10:1), combining eluents containing components with the same specific migration value, concentrating under reduced pressure, drying at room temperature, identifying by using nuclear magnetic resonance hydrogen spectrum and mass spectrum technology to obtain a product of ethyl elaeate, and analyzing by using high performance liquid chromatography technology to obtain the ethyl elaeate with the purity of 98%.

Fully dissolving 10 lotus seed pot monomer components by using a DMSO solution to ensure that the concentration of mother liquor is 100 mg/mL; adjusting RAW264.7 cells to logarithmic phase, blowing off the cells by using fresh culture solution, counting to make the cell concentration be 5x10^5/mL, and uniformly transferring the cells into a 96-hole sterile culture plate by using a porous pipettor, wherein the volume of each hole is 100 mu L; preparing 10 lotus seedpod monomer components into a solution to be treated with the concentration of 400 mug/mL by using a serum-free culture solution after 24h, wherein 3 multiple holes are formed in each treatment, 50 mug/L are formed in each hole, 6 drug-free multiple holes are formed in each treatment, 3 of the multiple holes are used as LPS control, and 3 of the multiple holes are used as blank control; preparing Lipopolysaccharide (LPS) solution with concentration of 4 μ g/mL with serum-free culture solution after 2h, wherein each well has a volume of 50 μ L, and the blank control well is replaced by serum-free culture solution; after 24h, the content of NO in cell supernatant is detected by a Griess reagent method, and the anti-inflammatory capability of each component is evaluated. The results show that rutin, quercetin, ethyl linoleate, ethyl elaeate, 7 beta-hydroxybetulinic acid and vanillyl alcohol have anti-inflammatory effects, ethyl elaeate (E9OAEE) has the best effect of inhibiting NO production, and the inhibition rate reaches 90% (figure 1).

Preparing ethyl elaeate serial dilution concentrations of 25, 50, 100, 200 and 400 mu g/mL, detecting the influence of ethyl elaeate at each concentration on NO content by a Griess method, detecting the cytotoxicity of ethyl elaeate by an MTT method, detecting the influence of ethyl elaeate on RAW264.7 cell inflammatory factors PGE2 and TNF-alpha by an ELISA technology, and evaluating the influence of ethyl elaeate on the expression levels of inflammatory proteins iNOS and COX2 by a Western Blotting (WB) technology; the influence of elaidic acid ethyl ester on mRNA levels of inflammatory genes iNOS and COX2 is verified by using an RT-PCR technology, and the influence of elaidic acid ethyl ester on MAPKs and NF-kB signal pathways is confirmed by using a WB and immunofluorescence technology. The results show that ethyl elaeate at final concentrations of 6.25, 12.5, 25 and 50 μ g/mL can inhibit NO production and is less cytotoxic (fig. 2). Ethyl elaeate inhibited inflammatory factor PGE2 and TNF α production (fig. 3), decreased protein expression (fig. 4) and mRNA levels (fig. 5) of iNOS and COX2, modulated phosphorylation levels of MAPKs signaling pathway proteins ERK, P38 and JNK (fig. 6) and nuclear transport of NF- κ B transcription factors (fig. 7).

Example 2

A method for extracting anti-inflammatory components from receptaculum nelumbinis and application thereof are disclosed: weighing dry matter of receptaculum nelumbinis 2kg, pulverizing with pulverizer, soaking in 15 times volume of ethanol water solution for 3 days (72h), filtering the residue, concentrating the filtrate under reduced pressure to about 200mL with rotary evaporator, and repeating the process for 3 times; diluting and dispersing the concentrated solution by using ultrapure water with 3 times of volume, extracting the diluted solution by using ethyl acetate with the same volume, repeatedly extracting for 3 times until an extract phase is colorless and transparent, and concentrating the ethyl acetate extract phase under reduced pressure to form a paste, wherein the weight of the paste is 4.10 g; uniformly mixing the paste with 2 times of silica gel, performing column chromatography, eluting with pure dichloromethane as eluent, eluting with 100:1 dichloromethane/methanol as eluent, gradually increasing methanol ratio in the eluent from 100:1 to 1:1, performing thin layer chromatography on the obtained eluent, wherein the developer is sulfuric acid: methanol (10:1), developing solvent dichloromethane: methanol (10:1), combining the eluents containing the components with the same specific migration value, concentrating under reduced pressure, air drying at room temperature, and determining the structure and purity of the compound by using techniques such as nuclear magnetic resonance hydrogen spectrum, mass spectrum and high performance liquid chromatography. Finally obtaining 10 lotus seedpod monomer components which are respectively isorhamnetin-3-O-beta-D-diglucoside, rutin, quercetin-3-O-beta-D-galactoside, kaempferol-3-O-rutinoside, quercetin, ethyl linoleate, ethyl elaidic acid, 7 beta-hydroxy betulinic acid, betulinic acid and balsamic alcohol.

Fully dissolving 10 lotus seed pot monomer components by using a DMSO solution to ensure that the concentration of mother liquor is 100 mg/mL; adjusting RAW264.7 cells to logarithmic phase, blowing off the cells by using fresh culture solution, counting to make the cell concentration be 1x10^6/mL, and uniformly transferring the cells into a 96-hole sterile culture plate by using a porous pipettor, wherein the volume of each hole is 100 mu L; preparing 10 lotus seedpod monomer components into a solution to be treated with the concentration of 400 mug/mL by using a serum-free culture solution after 24h, wherein 3 multiple holes are formed in each treatment, 50 mug/L are formed in each hole, 6 drug-free multiple holes are formed in each treatment, 3 of the multiple holes are used as LPS control, and 3 of the multiple holes are used as blank control; preparing Lipopolysaccharide (LPS) solution with concentration of 4 μ g/mL with serum-free culture solution after 2h, wherein each well has a volume of 50 μ L, and the blank control well is replaced by serum-free culture solution; after 24h, the content of NO in cell supernatant is detected by a Griess reagent method, and the anti-inflammatory capability of each component is evaluated. The results showed that ethyl elaeate (E9OAEE) had the best inhibitory effect on NO production, with an inhibition rate of 90% (fig. 1).

Preparing ethyl elaeate serial dilution concentrations of 25, 50, 100, 200 and 400 mu g/mL, detecting the influence of ethyl elaeate at each concentration on NO content by a Griess method, detecting the cytotoxicity of ethyl elaeate by an MTT method, detecting the influence of ethyl elaeate on RAW264.7 cell inflammatory factors PGE2 and TNF-alpha by an ELISA technology, and evaluating the influence of ethyl elaeate on the expression levels of inflammatory proteins iNOS and COX2 by a Western Blotting (WB) technology; the influence of elaidic acid ethyl ester on mRNA levels of inflammatory genes iNOS and COX2 is verified by using an RT-PCR technology, and the influence of elaidic acid ethyl ester on MAPKs and NF-kB signal pathways is confirmed by using a WB and immunofluorescence technology. The results show that ethyl elaeate at final concentrations of 6.25, 12.5, 25 and 50 μ g/mL can inhibit NO production and is less cytotoxic (fig. 2). Ethyl elaeate inhibited inflammatory factor PGE2 and TNF α production (fig. 3), decreased protein expression (fig. 4) and mRNA levels (fig. 5) of iNOS and COX2, modulated phosphorylation levels of MAPKs signaling pathway proteins ERK, P38 and JNK (fig. 6) and nuclear transport of NF- κ B transcription factors (fig. 7).

Example 3

A method for extracting anti-inflammatory components from receptaculum nelumbinis and application thereof are disclosed: weighing dry matter of receptaculum Nelumbinis 2kg, pulverizing with pulverizer, soaking in 20 times volume of ethanol water solution for 3 days (72 hr), filtering the residue, concentrating the filtrate under reduced pressure to about 200mL with rotary evaporator, and repeating the process for 3 times; diluting and dispersing the concentrated solution by using ultrapure water with 3 times of volume, extracting the diluted solution by using ethyl acetate with the same volume, repeatedly extracting for 3 times until an extract phase is colorless and transparent, and concentrating the ethyl acetate extract phase under reduced pressure to form a paste, wherein the weight of the paste is 4.503 g; uniformly mixing the paste with 2 times of silica gel, performing column chromatography, eluting with pure dichloromethane as eluent, eluting with 100:1 dichloromethane/methanol as eluent, gradually increasing methanol ratio in the eluent from 100:1 to 1:1, performing thin layer chromatography on the obtained eluent, wherein the developer is sulfuric acid: methanol (10:1), developing solvent dichloromethane: methanol (10:1), combining the eluents containing the components with the same specific migration value, concentrating under reduced pressure, air drying at room temperature, and determining the structure and purity of the compound by using techniques such as nuclear magnetic resonance hydrogen spectrum, mass spectrum and high performance liquid chromatography. Finally obtaining 10 lotus seedpod monomer components which are respectively isorhamnetin-3-O-beta-D-diglucoside, rutin, quercetin-3-O-beta-D-galactoside, kaempferol-3-O-rutinoside, quercetin, ethyl linoleate, ethyl elaidic acid, 7 beta-hydroxy betulinic acid, betulinic acid and balsamic alcohol.

Fully dissolving 10 lotus seed pot monomer components by using a DMSO solution to ensure that the concentration of mother liquor is 100 mg/mL; adjusting RAW264.7 cells to logarithmic phase, blowing off the cells by using fresh culture solution, counting to make the cell concentration be 5x10^5/mL, and uniformly transferring the cells into a 96-hole sterile culture plate by using a porous pipettor, wherein the volume of each hole is 100 mu L; preparing 10 lotus seedpod monomer components into a solution to be treated with the concentration of 400 mug/mL by using a serum-free culture solution after 24h, wherein 3 multiple holes are formed in each treatment, 50 mug/L are formed in each hole, 6 drug-free multiple holes are formed in each treatment, 3 of the multiple holes are used as LPS control, and 3 of the multiple holes are used as blank control; preparing Lipopolysaccharide (LPS) solution with concentration of 4 μ g/mL with serum-free culture solution after 2h, wherein each well has a volume of 50 μ L, and the blank control well is replaced by serum-free culture solution; after 24h, the content of NO in cell supernatant is detected by a Griess reagent method, and the anti-inflammatory capability of each component is evaluated. The results showed that ethyl elaeate (E9OAEE) had the best inhibitory effect on NO production, with an inhibition rate of 90% (fig. 1).

Preparing ethyl elaeate serial dilution concentrations of 25, 50, 100, 200 and 400 mu g/mL, detecting the influence of ethyl elaeate at each concentration on NO content by a Griess method, detecting the cytotoxicity of ethyl elaeate by an MTT method, detecting the influence of ethyl elaeate on RAW264.7 cell inflammatory factors PGE2 and TNF-alpha by an ELISA technology, and evaluating the influence of ethyl elaeate on the expression levels of inflammatory proteins iNOS and COX2 by a Western Blotting (WB) technology; the influence of elaidic acid ethyl ester on mRNA levels of inflammatory genes iNOS and COX2 is verified by using an RT-PCR technology, and the influence of elaidic acid ethyl ester on MAPKs and NF-kB signal pathways is confirmed by using a WB and immunofluorescence technology. The results show that ethyl elaeate at final concentrations of 6.25, 12.5, 25 and 50 μ g/mL can inhibit NO production and is less cytotoxic (fig. 2). Ethyl elaeate inhibited inflammatory factor PGE2 and TNF α production (fig. 3), decreased protein expression (fig. 4) and mRNA levels (fig. 5) of iNOS and COX2, modulated phosphorylation levels of MAPKs signaling pathway proteins ERK, P38 and JNK (fig. 6) and nuclear transport of NF- κ B transcription factors (fig. 7).

Example 4

A method for extracting anti-inflammatory components from receptaculum nelumbinis and application thereof are disclosed: weighing dry matter of receptaculum Nelumbinis 2kg, pulverizing with pulverizer, soaking in 20 times volume of ethanol water solution for 3 days (72 hr), filtering the residue, concentrating the filtrate under reduced pressure to about 200mL with rotary evaporator, and repeating the process for 3 times; diluting and dispersing the concentrated solution by using ultrapure water with 3 times of volume, extracting the diluted solution by using ethyl acetate with the same volume, repeatedly extracting for 3 times until an extract phase is colorless and transparent, and concentrating the ethyl acetate extract phase under reduced pressure to paste, wherein the weight of the extract phase is 4.776 g; uniformly mixing the paste with 2 times of silica gel, performing column chromatography, eluting with pure dichloromethane as eluent, eluting with 100:1 dichloromethane/methanol as eluent, gradually increasing methanol ratio in the eluent from 100:1 to 1:1, performing thin layer chromatography on the obtained eluent, wherein the developer is sulfuric acid: methanol (10:1), developing solvent dichloromethane: methanol (10:1), combining the eluents containing the components with the same specific migration value, concentrating under reduced pressure, air drying at room temperature, and determining the structure and purity of the compound by using techniques such as nuclear magnetic resonance hydrogen spectrum, mass spectrum and high performance liquid chromatography. Finally obtaining 10 lotus seedpod monomer components which are respectively isorhamnetin-3-O-beta-D-diglucoside, rutin, quercetin-3-O-beta-D-galactoside, kaempferol-3-O-rutinoside, quercetin, ethyl linoleate, ethyl elaidic acid, 7 beta-hydroxy betulinic acid, betulinic acid and balsamic alcohol.

Fully dissolving 10 lotus seed pot monomer components by using a DMSO solution to ensure that the concentration of mother liquor is 100 mg/mL; adjusting RAW264.7 cells to logarithmic phase, blowing off the cells by using fresh culture solution, counting to make the cell concentration be 1x10^6/mL, and uniformly transferring the cells into a 96-hole sterile culture plate by using a porous pipettor, wherein the volume of each hole is 100 mu L; preparing 10 lotus seedpod monomer components into a solution to be treated with the concentration of 400 mug/mL by using a serum-free culture solution after 24h, wherein 3 multiple holes are formed in each treatment, 50 mug/L are formed in each hole, 6 drug-free multiple holes are formed in each treatment, 3 of the multiple holes are used as LPS control, and 3 of the multiple holes are used as blank control; preparing Lipopolysaccharide (LPS) solution with concentration of 4 μ g/mL with serum-free culture solution after 2h, wherein each well has a volume of 50 μ L, and the blank control well is replaced by serum-free culture solution; after 24h, the content of NO in cell supernatant is detected by a Griess reagent method, and the anti-inflammatory capability of each component is evaluated. The results showed that ethyl elaeate (E9OAEE) had the best inhibitory effect on NO production, with an inhibition rate of 90% (fig. 1).

Preparing ethyl elaeate serial dilution concentrations of 25, 50, 100, 200 and 400 mu g/mL, detecting the influence of ethyl elaeate at each concentration on NO content by a Griess method, detecting the cytotoxicity of ethyl elaeate by an MTT method, detecting the influence of ethyl elaeate on RAW264.7 cell inflammatory factors PGE2 and TNF-alpha by an ELISA technology, and evaluating the influence of ethyl elaeate on the expression levels of inflammatory proteins iNOS and COX2 by a Western Blotting (WB) technology; the influence of elaidic acid ethyl ester on mRNA levels of inflammatory genes iNOS and COX2 is verified by using an RT-PCR technology, and the influence of elaidic acid ethyl ester on MAPKs and NF-kB signal pathways is confirmed by using a WB and immunofluorescence technology. The results show that ethyl elaeate at final concentrations of 6.25, 12.5, 25 and 50 μ g/mL can inhibit NO production and is less cytotoxic (fig. 2). Ethyl elaeate inhibited inflammatory factor PGE2 and TNF α production (fig. 3), decreased protein expression (fig. 4) and mRNA levels (fig. 5) of iNOS and COX2, modulated phosphorylation levels of MAPKs signaling pathway proteins ERK, P38 and JNK (fig. 6) and nuclear transport of NF- κ B transcription factors (fig. 7).

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.