CN110742918B - Preparation method of nostoc extract with anti-inflammatory effect - Google Patents

Preparation method of nostoc extract with anti-inflammatory effect Download PDF

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CN110742918B
CN110742918B CN201911157260.9A CN201911157260A CN110742918B CN 110742918 B CN110742918 B CN 110742918B CN 201911157260 A CN201911157260 A CN 201911157260A CN 110742918 B CN110742918 B CN 110742918B
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赵杰
孙奕曦
潘冬梅
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Abstract

The invention provides a preparation method of a nostoc extract with anti-inflammatory effect, which comprises the following steps: firstly, 8 kilograms of roots and stems of the nostoc are taken, dried and crushed, soaked in ethanol for 2 hours, extracted by ultrasonic reflux for 3 times, the extracting solution is merged, filtered and decompressed and concentrated by a rotary evaporator to obtain an extract, the extract is uniformly mixed with a proper amount of diatomite and tightly packed by a filter paper cylinder and then placed into a Soxhlet extractor extracting cylinder, ethyl acetate is added into the dried Soxhlet extractor extracting cylinder, the Soxhlet extractor is installed and extracted in a constant-temperature electric heating jacket for 6 hours, the extracting solution is evaporated and concentrated by rotation to obtain the extract, the extract adopts a silica gel column for chromatography, and the eluent is evaporated by rotation to recover the solvent and then is frozen and spray dried to obtain extract dry powder.

Description

Preparation method of nostoc extract with anti-inflammatory effect
Technical Field
The invention relates to a preparation method of a nostoc extract with an anti-inflammatory effect, belonging to the technical field of traditional Chinese medicinal materials.
Background
The nostoc is dry overground part of nostoc of the nostoc genus of apocynaceae family, is a traditional folk herbal medicine, and is widely used in folk countries such as Fujian, Zhejiang and Guangdong. Bitter in taste, pungent and warm in nature, and slightly toxic. Has the effects of dispelling wind, removing dampness, promoting blood circulation, dredging collaterals, etc. The nostoc extract is used for treating symptoms such as rheumatic arthralgia, diarrhea, dermatophytosis, edema of the whole body, amenorrhea, traumatic injury and the like, the toxicity of the nostoc extract to RAW 264.7 cells for 24 hours at different concentrations is not clear, and a preparation method of the nostoc extract with an anti-inflammatory effect is urgently needed to solve the problems.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method of a nostoc extract with anti-inflammatory effect, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention is realized by the following technical scheme: a preparation method of a nostoc extract comprises the steps of firstly drying and crushing 8 kilograms of roots and stems of nostoc, soaking for 2 hours in 70% ethanol in an amount which is 6 times that of the roots and stems of the nostoc, carrying out ultrasonic reflux extraction for 3 times, respectively carrying out 30 minutes, 20 minutes and 20 minutes, combining extracting solutions, filtering, carrying out reduced pressure concentration by a rotary evaporator to obtain an extract, uniformly mixing the extract with a proper amount of diatomite, tightly packaging the extract by a filter paper tube, putting the packed extract into a Soxhlet extractor extraction cylinder, adding ethyl acetate in an amount which is 20 times that of the dried Soxhlet extractor extraction cylinder, installing the Soxhlet extractor, carrying out extraction for 6 hours in a constant-temperature electric heating jacket, carrying out rotary evaporation and concentration on the extracting solution to obtain an extract, carrying out silica gel column chromatography on the extract, eluting with trichloromethane-methanol according to a volume ratio of 6:4, carrying out rotary evaporation on the eluent, recovering a solvent, and carrying out freeze-spray drying to obtain an extract dry powder.
Further, an experimental study on the extraction of the effective parts of the nostoc flagelliforme and the anti-inflammatory effect of the nostoc flagelliforme comprises the following steps: preparing suspension with required concentration, capillary permeability experiment, rat pleurisy experiment caused by carrageenan, analysis of toxicity effect of nostoc extract on RAW 264.7 cells, and detection of IL-6 and TNF-alpha by enzyme-linked immunosorbent assay (ELISA);
(1) suspension formulated to the desired concentration: the animal experiment is ground by 1 percent CMC solution before use to prepare suspension with required concentration. The cell experiment is used after being dissolved in 0.1 percent DMSO solution before use;
(2) capillary permeability test: 40 mice were taken and randomly divided into four groups: model control group, indometacin group, nostoc extract high dose group, low dose group, 10 in each group, each half of male and female, the administration by intragastric administration according to the dosage of table 1, the model control group is given with 1% CMC solution with equal volume, 1 time/d, continuous administration for 5d, the mouse tail is given with intravenous injection of 0.6% Evans blue solution 10 mL/kg 30min after the last administration-1Then, 0.6 percent glacial acetic acid is injected into the abdominal cavity to form 10 mL/kg-130min later, dislocation and sacrifice, open the abdominal cavity, wash the abdominal cavity with 5mL of normal saline, suck the washing liquid out, combine, 3000 r.min-1Centrifuging for 15min, and collecting supernatant, and measuring absorbance A at 595nm wavelength;
(3) experiment of pleurisy in rats caused by carrageenan: 32 rats were randomly divided into 5 groups of 8 rats each, and the administration method was the same as (2), and 30min after administration at 5d, the rats were anesthetized with ether, the normal control group was injected with 0.2ml of sterile physiological saline to the right thoracic cavity, and the other groups were injected with 0.2ml of 1% carrageenan sterile solution. Killing a rat under anesthesia of chloral hydrate after 4 hours of inflammation, cutting the abdominal skin, exposing the diaphragm muscle, injecting 2ml of normal saline pleural lavage fluid (the pleural lavage fluid contains 5U/ml of heparin sodium and 10 mu g/ml of indometacin), gently shaking, uniformly mixing the intrathoracic liquids, taking 0.1ml as a white blood cell count, centrifuging the rest pleural effusion at 3000r/min for 15min, taking the supernatant, and measuring the contents of tumor necrosis factor-alpha and interleukin-6 by using an Elisa method;
(4) analysis of toxic effects of nostoc extracts on RAW 264.7 cells: taking RAW 264.7 cells in logarithmic growth phase at 5X 104Cell density of cells/ml was seeded in 96-well plates with 100. mu.l of cell suspension per well, with 8 replicate wells in each set. Setting blank group and different concentration extract administration group at 37 deg.C and 5% (v/v) CO2Culturing in the incubator for 24 hr, discarding the old culture medium, feeding the blank group to the fresh culture medium, respectively feeding the extract administration groups with different concentrations of the drugs, and further culturing in the incubator for 24 hr. After 24 hours, 30. mu.l of MTT (5mg/ml) was added to each well in the dark, the mixture was further cultured in an incubator at 37 ℃ for 4 hours, the cell supernatant was discarded, 100. mu.l of DMSO was added to each well to dissolve the reacted crystal, the mixture was shaken on a horizontal shaker for 5 minutes to mix well, and the OD value of each well was measured at a wavelength of 570nm using a microplate reader. Cell viability was calculated according to the following formula: the cell survival rate is equal to the average OD value of the experimental group/the average OD value of the control group multiplied by 100 percent;
(5) taking supernatant, and measuring the contents of tumor necrosis factor-alpha and interleukin-6 by an Elisa method: taking RAW 264.7 cells in logarithmic growth phase at 2X 105The cells/ml were seeded at a cell density in a 24-well plate, and 500. mu.l of the cell suspension was added to each well, and a blank group, an LPS (1. mu.g/ml) group, an extract at various concentrations, and an LPS (1. mu.g/ml) co-administration group were set. Culturing for 24 hr, discarding old culture medium, feeding blank group to fresh culture medium, feeding other groups according to setting, culturing for 24 hr, collecting cell supernatant, centrifuging at 3000rmp for 20min, collecting supernatantThe clear solution was assayed for IL-6 and TNF-. alpha.concentrations using an ELISA kit.
The invention has the beneficial effects that: according to the invention, the influence of the nostoc extract on the increase of permeability of mouse peritoneal capillary blood vessels caused by acetic acid, the influence of the nostoc extract on the number of white blood cells of thoracic cavity exudates and the influence of the nostoc extract on inflammatory media of the thoracic cavity exudates can be accurately and comprehensively analyzed through experimental research of extracting and observing the anti-inflammatory action of the effective parts of the nostoc, and the experimental analysis shows that the nostoc extract has no toxicity (the survival rate is more than 85%) on RAW 264.7 cells for 24 hours at the concentrations of 5, 20, 30 and 60 mu g/ml and has certain toxicity on the cells at the concentrations of 120 and 240 mu g/ml.
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Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a graph showing the comparison of the inhibition of LPS induced IL-6 and TNF- α production by RAW 264.7 cells by the extract of nostoc according to the preparation method of the extract of nostoc;
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
The invention provides a technical scheme that: a preparation method of a nostoc extract comprises the steps of firstly drying and crushing 8 kilograms of roots and stems of the nostoc, soaking the roots and stems of the nostoc for 2 hours by using 70% ethanol with 6 times of the weight, carrying out ultrasonic reflux extraction for 3 times with 30min, 20min and 20min respectively, combining extracting solutions, filtering, concentrating under reduced pressure by using a rotary evaporator to obtain an extract, uniformly mixing the extract with a proper amount of diatomite, tightly packaging the extract by using a filter paper tube, putting the packed extract into a Soxhlet extractor extraction cylinder, adding ethyl acetate with 20 times of the weight into the dried Soxhlet extractor extraction cylinder, installing the Soxhlet extractor, extracting the extract in a constant-temperature electric heating jacket for 6 hours, carrying out rotary evaporation and concentration on the extracting solution to obtain the extract, carrying out silica gel column chromatography on the extract, eluting chloroform-methanol according to the volume ratio of 6:4, carrying out rotary evaporation on the eluent to recover a solvent, carrying out freeze spray drying to obtain extract dry powder, and carrying out experimental study on the extraction and anti-inflammatory action of effective parts of the nostoc, the method comprises the following steps: preparing suspension with required concentration, capillary permeability experiment, rat pleurisy experiment caused by carrageenan, analysis of toxicity effect of nostoc extract on RAW 264.7 cells, and detection of IL-6 and TNF-alpha by enzyme-linked immunosorbent assay (ELISA);
(1) suspension formulated to the desired concentration: the animal experiment is ground by 1 percent CMC solution before use to prepare suspension with required concentration. The cell experiment is used after being dissolved in 0.1 percent DMSO solution before use;
(2) capillary permeability test: 40 mice were taken and randomly divided into four groups: model control group, indometacin group, nostoc extract high dose group, low dose group, 10 in each group, each half of male and female, the administration by intragastric administration according to the dosage of table 1, the model control group is given with 1% CMC solution with equal volume, 1 time/d, continuous administration for 5d, the mouse tail is given with intravenous injection of 0.6% Evans blue solution 10 mL/kg 30min after the last administration-1Then, 0.6 percent glacial acetic acid is injected into the abdominal cavity to form 10 mL/kg-130min later, dislocation and sacrifice, open the abdominal cavity, wash the abdominal cavity with 5mL of normal saline, suck the washing liquid out, combine, 3000 r.min-1Centrifuging for 15min, and collecting supernatant, and measuring absorbance A at 595nm wavelength;
(3) experiment of pleurisy in rats caused by carrageenan: 32 rats were randomly divided into 5 groups of 8 rats each, and the administration method was the same as (2), and 30min after administration at 5d, the rats were anesthetized with ether, the normal control group was injected with 0.2ml of sterile physiological saline to the right thoracic cavity, and the other groups were injected with 0.2ml of 1% carrageenan sterile solution. Killing a rat under anesthesia of chloral hydrate after 4 hours of inflammation, cutting the abdominal skin, exposing the diaphragm muscle, injecting 2ml of normal saline pleural lavage fluid (the pleural lavage fluid contains 5U/ml of heparin sodium and 10 mu g/ml of indometacin), gently shaking, uniformly mixing the intrathoracic liquids, taking 0.1ml as a white blood cell count, centrifuging the rest pleural effusion at 3000r/min for 15min, taking the supernatant, and measuring the contents of tumor necrosis factor-alpha and interleukin-6 by using an Elisa method;
(4) analysis of toxic effects of nostoc extracts on RAW 264.7 cells: taking RAW 264.7 cells in logarithmic growth phase at 5X 104Cells/mCell density of l was seeded in 96-well plates with 100. mu.l of cell suspension per well, with 8 replicate wells in each set. Setting blank group and different concentration extract administration group at 37 deg.C and 5% (v/v) CO2Culturing in the incubator for 24 hr, discarding the old culture medium, feeding the blank group to the fresh culture medium, respectively feeding the extract administration groups with different concentrations of the drugs, and further culturing in the incubator for 24 hr. After 24 hours, 30. mu.l of MTT (5mg/ml) was added to each well in the dark, the mixture was further cultured in an incubator at 37 ℃ for 4 hours, the cell supernatant was discarded, 100. mu.l of DMSO was added to each well to dissolve the reacted crystal, the mixture was shaken on a horizontal shaker for 5 minutes to mix well, and the OD value of each well was measured at a wavelength of 570nm using a microplate reader. Cell viability was calculated according to the following formula: the cell survival rate is equal to the average OD value of the experimental group/the average OD value of the control group multiplied by 100 percent;
(5) taking supernatant, and measuring the contents of tumor necrosis factor-alpha and interleukin-6 by an Elisa method: taking RAW 264.7 cells in logarithmic growth phase at 2X 105Cells/ml cell density was seeded in 24-well plates, 500. mu.l of cell suspension was added to each well, and a blank group, LPS (1. mu.g/ml) group, a common administration group of Candida extract and LPS (1. mu.g/ml) at different concentrations were set. After 24 hours of culture, the old culture medium is discarded, the blank group is given to the fresh culture medium, other groups are given according to the setting, after the culture is continued for 24 hours, cell supernatants of all groups are collected, the cells are centrifuged at 3000rmp for 20 minutes, the supernatants are taken, and the concentrations of IL-6 and TNF-alpha are detected by an ELISA kit.
Statistical analysis: data to
Figure GDA0003298030450000051
Mean comparisons were analyzed by One-way ANOVA. Statistical processing is done by SPSS 19.0 software. The difference is statistically significant when P is less than 0.05.
And (3) analyzing an experimental result: compared with the model control group, the effect of acetic acid on the increase of the capillary permeability of the mice is that the high-dose and low-dose groups of the nostoc extract can obviously inhibit the increase of the capillary permeability of the mice, and P is less than 0.05, which is shown in table 1.
TABLE 1 Permeability of Nostoc extracts to acetic acid-induced capillary vessels in mouse peritoneal cavity(iii) increased influence
Figure GDA0003298030450000061
n＝10)
Figure GDA0003298030450000062
P <0.05, P <0.01 compared to model control.
Compared with the blank control group, the number of white blood cells in the thoracic cavity exudate of the rat in the model control group is increased, and the tumor necrosis factor-alpha and the interleukin-6 are obviously increased. Compared with the model control group, the high dose of the nostoc extract can obviously reduce the number of leucocytes in exudate and reduce the content of tumor necrosis factor-alpha and interleukin-6 (P is less than 0.01); the low dosage of the extract can reduce the number of leucocytes in the exudate and reduce the content of tumor necrosis factor-alpha, and has no obvious influence on the content of interleukin-6, which is shown in a table 2-3.
TABLE 2 Effect of Candida extracts on the number of leukocytes in pleural effusion: (
Figure GDA0003298030450000063
n＝8)
Figure GDA0003298030450000064
Note: p <0.01 in comparison to model control group
TABLE 3 Effect of Candida extracts on inflammatory mediators of thoracic effusion: (
Figure GDA0003298030450000065
n＝8)
Figure GDA0003298030450000066
Figure GDA0003298030450000071
Note: compared with the model control group, P <0.05, P <0.01MTT results showed that the nostoc extracts were not toxic to RAW 264.7 cells for 24 hours at concentrations of 5, 20, 30, 60 μ g/ml (survival > 85%) and were somewhat toxic to cells at 120, 240 μ g/ml (survival < 85%) compared with the blank group. Therefore, we chose the concentration of non-toxic agent of 7.5, 15, 30 μ g/ml for the subsequent experiments on nostoc. The nostoc extract can inhibit LPS to induce RAW 264.7 cells to produce IL-6 and TNF-alpha.
As shown in fig. 1: LPS (1. mu.g/ml) stimulated the production of TNF-. alpha.and IL-6 by the cells compared to the blank control. The extract of Nostoc commune can inhibit LPS induced TNF-alpha and IL-6 production by RAW 264.7 cells.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (1)

1. A preparation method of a nostoc extract with anti-inflammatory effect is characterized in that: firstly, 8 kilograms of roots and stems of nostoc commune are taken, dried and crushed, soaked in 70 percent ethanol in an amount which is 6 times that of the roots and stems of the nostoc commune for 2 hours, extracted by ultrasonic reflux for 3 times, the time is respectively 30min, 20min and 20min, the extracting solutions are combined, filtered, decompressed and concentrated by a rotary evaporator to obtain an extract, the extract is uniformly mixed with a proper amount of diatomite, tightly packaged by a filter paper cylinder and then put into a Soxhlet extractor extracting cylinder, 20 times of ethyl acetate is added into the dried Soxhlet extractor extracting cylinder, the Soxhlet extractor is installed, the extracting is carried out in a constant-temperature electric heating jacket for 6 hours, the extracting solution is evaporated and concentrated by rotary evaporation to obtain the extract, the extract is subjected to silica gel column chromatography, chloroform-methanol is eluted according to the volume ratio of 6:4, and the eluent is evaporated and recovered in a rotary manner, and then is subjected to freeze spray drying to obtain the dry extract powder.
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