CN117695345A - Clerodendrum clerodendrum bungei extract and preparation method and application thereof - Google Patents
Clerodendrum clerodendrum bungei extract and preparation method and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a clerodendrum bungei extract, a preparation method and application thereof, and belongs to the technical field of natural medicines. Comprising the following steps: s1, cleaning, drying and crushing clerodendrum bungei medicinal materials, adding the clerodendrum bungei medicinal materials into water, heating, boiling, extracting, filtering, reserving filter residues, adding ethanol for precipitation, collecting solids, washing and drying to obtain clerodendrum bungei polysaccharide, and reserving filtrate; s2, adding the solid obtained in the step S1 into water, adding enzyme for enzymolysis, inoculating bacillus subtilis strain seed liquid, fermenting and culturing, sterilizing, inactivating enzyme, and filtering to obtain filtrate for use; s3, combining the filtrate in the step S1 and the filtrate in the step S2, adding an organic solvent for extraction, collecting an organic phase, removing the solvent under reduced pressure, washing, and drying to obtain the clerodendranthus spicatus organic extract; s4, uniformly mixing the clerodendranthus spicatus polysaccharide and the clerodendranthus spicatus organic extract to prepare the clerodendranthus spicatus extract, wherein the clerodendranthus spicatus extract has better anti-inflammatory and antioxidant properties, high activity, simple preparation method, wide original sources and wide application prospect.
Description
Technical Field
The invention relates to the technical field of natural medicines, in particular to a clerodendranthus spicatus extract and a preparation method and application thereof.
Background
And clematis is clematis filigree Clerodendrum japonicum (thunder.) sweet potato, also known as red dragon boat, red beard, etc. (Guangxi Zhuang nationality), produced from Guangxi, guizhou and Yunnan provinces, etc., and is a traditional common medicament among people. The tung mainly contains flavonoid, phenolic acid, tannin, volatile oil and other components, and the whole plant can be used as medicine, has sweet taste and cool nature, has pharmacological effects of anti-inflammatory, antioxidant, anti-tumor and the like, and is mainly used for treating migraine, traumatic stasis swelling, carbuncle swelling and sore; clinically, the traditional Chinese medicine composition is used for treating cough due to lung heat, heat stranguria and dysuria.
With the rapid development of medicine technology, many medicines for treating liver injury have been researched and reported, and mainly comprise Chinese herbal medicines, biological medicines, chemical medicines, trace elements and the like. However, some studies have shown that some drugs against liver injury also inevitably produce biotoxicity. It can be seen that the current anti-inflammatory treatment of traditional Chinese medicine is still a serious problem.
The traditional Chinese medicine is the treasure of the traditional Chinese medicine in China, and a plurality of traditional Chinese medicines and traditional Chinese medicine components have the effects of clearing heat and detoxicating, promoting diuresis and removing jaundice, promoting blood circulation and removing blood stasis, soothing liver and regulating qi, and the like, can directly treat the traditional Chinese medicine anti-inflammatory, and can not generate obvious toxic and side effects, so that the research and development of the medicines have important practical significance for treating the clinical traditional Chinese medicine anti-inflammatory.
Disclosure of Invention
The invention aims to provide a clerodendrum bungeana extract, a preparation method and application thereof, which have better anti-inflammatory and antioxidant properties, and the preparation method is simple, has wide original sources and wide application prospects by inhibiting the generation of NO, TNF-a, IL-12, IL-6 and IL-1 beta inflammatory factors to act.
The technical scheme of the invention is realized as follows:
the invention provides a preparation method of a clerodendranthus spicatus extract, which comprises the following steps:
s1, cleaning, drying and crushing clerodendrum bungei medicinal materials, adding the clerodendrum bungei medicinal materials into water, heating, boiling, extracting, filtering, reserving filter residues, adding ethanol for precipitation, collecting solids, washing and drying to obtain clerodendrum bungei polysaccharide, and reserving filtrate;
s2, adding the solid obtained in the step S1 into water, adding enzyme for enzymolysis, inoculating bacillus subtilis strain seed liquid, fermenting and culturing, sterilizing, inactivating enzyme, and filtering to obtain filtrate for use;
s3, combining the filtrate in the step S1 and the filtrate in the step S2, adding an organic solvent for extraction, collecting an organic phase, removing the solvent under reduced pressure, washing, and drying to obtain the clerodendranthus spicatus organic extract;
s4, uniformly mixing the clerodendranthus spicatus polysaccharide prepared in the step S1 and the clerodendranthus spicatus organic extract prepared in the step S3 to prepare the clerodendranthus spicatus extract.
As a further improvement of the invention, the solid-to-liquid ratio of the clerodendranthus spicatus medicinal material to water in the step S1 is 1:7-10g/mL.
As a further improvement of the invention, the heating and boiling extraction time in the step S1 is 2-4h.
As a further improvement of the invention, the ethanol is added in the step S1 until the ethanol content of the system is 80-90wt percent, and the precipitation time is 3-5h.
As a further improvement of the invention, the mass ratio of the solid, the enzyme and the water in the step S2 is 10-15:1-2:200.
As a further improvement of the invention, the enzyme in the step S2 comprises cellulase and pectase, the mass ratio is 5-7:1, the enzymolysis temperature is 40-45 ℃, and the enzymolysis time is 2-4h.
As a further improvement of the invention, the inoculation amount of the bacillus subtilis strain seed liquid in the step S2 is 2-4v/v%, and the bacterial content of the inoculated bacillus subtilis strain seed liquid is 10 7 -10 8 cfu/mL。
As a further improvement of the invention, the condition of the fermentation culture in the step S2 is 40-45 ℃,100-200r/min and 48-56h.
As a further improvement of the invention, the organic solvent in the step S3 is a mixed solution of n-butanol and cyclohexane, and the volume ratio is 3-5:7.
The invention further provides the clerodendranthus spicatus extract prepared by the preparation method.
The invention has the following beneficial effects: the invention prepares a clerodendrum bungeana extract, obtains clerodendrum bungeana polysaccharide with better antioxidant and anti-inflammatory activity through water extraction and alcohol precipitation, mixes water extract and fermentation products of various active substances obtained by breaking walls of plant cell walls after enzymolysis and fermentation, contains a large amount of active substances such as flavone, polyphenol, triterpene and the like through organic solvent extraction, has better anti-inflammatory and antioxidant properties, and plays roles in inhibiting the generation of NO, TNF-a, IL-12, IL-6 and IL-1 beta inflammatory factors, so that the prepared clerodendrum bungeana extract has high activity, simple preparation method, wide original sources and wide application prospect.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a preparation method of a clerodendrum cyrtophyllum extract, which comprises the following steps:
s1, taking 20 parts by weight of clerodendrum bungei medicinal material, cleaning, drying, crushing, adding the clerodendrum bungei medicinal material and water in a solid-to-liquid ratio of 1:7g/mL, heating, boiling and extracting for 2 hours, filtering, reserving filter residues, adding ethanol until the ethanol content of the system is 80wt%, precipitating for 3 hours, collecting solids, washing, drying to obtain clerodendrum bungei polysaccharide, and reserving filtrate;
s2, adding 10 parts by weight of the solid obtained in the step S1 into 200 parts by weight of water, adding 1 part by weight of enzyme, heating to 40 ℃, carrying out enzymolysis for 2 hours, inoculating bacillus subtilis strain seed liquid with the inoculum size of 2v/v%, at 40 ℃ and 100r/min, fermenting and culturing for 48 hours, sterilizing and inactivating enzyme, filtering, and reserving filtrate;
the enzyme comprises cellulase and pectase, and the mass ratio is 5:1;
the bacterial content of the seed liquid inoculated with the bacillus subtilis strain is 10 7 -10 8 cfu/mL；
S3, combining the filtrate in the step S1 and the filtrate in the step S2, adding an equal volume of organic solvent for extraction, collecting an organic phase, removing the solvent under reduced pressure, washing, and drying to obtain the clerodendranthus spicatus organic extract;
the organic solvent is a mixed solution of n-butanol and cyclohexane, and the volume ratio is 3:7;
s4, uniformly mixing the clerodendranthus spicatus polysaccharide prepared in the step S1 and the clerodendranthus spicatus organic extract prepared in the step S3 to prepare the clerodendranthus spicatus extract.
Example 2
The embodiment provides a preparation method of a clerodendrum cyrtophyllum extract, which comprises the following steps:
s1, taking 20 parts by weight of clerodendrum bungei medicinal material, cleaning, drying, crushing, adding the clerodendrum bungei medicinal material and water in a solid-to-liquid ratio of 1:10g/mL, heating, boiling and extracting for 4 hours, filtering, reserving filter residues, adding ethanol until the ethanol content of the system is 90wt%, precipitating for 5 hours, collecting solids, washing, drying to obtain clerodendrum bungei polysaccharide, and reserving filtrate;
s2, adding 15 parts by weight of the solid obtained in the step S1 into 200 parts by weight of water, adding 2 parts by weight of enzyme, heating to 45 ℃, carrying out enzymolysis for 4 hours, inoculating bacillus subtilis strain seed liquid with the inoculum size of 4v/v%,45 ℃, carrying out fermentation culture for 56 hours at 200r/min, sterilizing, inactivating enzyme, filtering, and reserving filtrate;
the enzyme comprises cellulase and pectase, and the mass ratio is 7:1;
the bacterial content of the seed liquid inoculated with the bacillus subtilis strain is 10 7 -10 8 cfu/mL；
S3, combining the filtrate in the step S1 and the filtrate in the step S2, adding an equal volume of organic solvent for extraction, collecting an organic phase, removing the solvent under reduced pressure, washing, and drying to obtain the clerodendranthus spicatus organic extract;
the organic solvent is a mixed solution of n-butanol and cyclohexane, and the volume ratio is 5:7;
s4, uniformly mixing the clerodendranthus spicatus polysaccharide prepared in the step S1 and the clerodendranthus spicatus organic extract prepared in the step S3 to prepare the clerodendranthus spicatus extract.
Example 3
The embodiment provides a preparation method of a clerodendrum cyrtophyllum extract, which comprises the following steps:
s1, taking 20 parts by weight of clerodendrum bungei medicinal material, cleaning, drying, crushing, adding the clerodendrum bungei medicinal material and water in a solid-to-liquid ratio of 1:8.5g/mL, heating, boiling and extracting for 3 hours, filtering, reserving filter residues, adding ethanol until the ethanol content of the system is 85wt%, precipitating for 4 hours, collecting solids, washing, drying to obtain clerodendrum bungei polysaccharide, and reserving filtrate;
s2, adding 12 parts by weight of the solid obtained in the step S1 into 200 parts by weight of water, adding 1.5 parts by weight of enzyme, heating to 42 ℃, carrying out enzymolysis for 3 hours, inoculating bacillus subtilis strain seed liquid with the inoculum size of 3v/v%, at 42 ℃ for 150r/min, fermenting and culturing for 52 hours, sterilizing, inactivating enzyme, filtering, and reserving filtrate;
the enzyme comprises cellulase and pectase, and the mass ratio is 6:1;
the bacterial content of the seed liquid inoculated with the bacillus subtilis strain is 10 7 -10 8 cfu/mL；
S3, combining the filtrate in the step S1 and the filtrate in the step S2, adding an equal volume of organic solvent for extraction, collecting an organic phase, removing the solvent under reduced pressure, washing, and drying to obtain the clerodendranthus spicatus organic extract;
the organic solvent is a mixed solution of n-butanol and cyclohexane, and the volume ratio is 4:7;
s4, uniformly mixing the clerodendranthus spicatus polysaccharide prepared in the step S1 and the clerodendranthus spicatus organic extract prepared in the step S3 to prepare the clerodendranthus spicatus extract.
Example 4
The difference compared to example 3 is that the enzyme is a single cellulase.
Example 5
The difference compared to example 3 is that the enzyme is a single pectase.
Comparative example 1
In comparison with example 3, the difference is that no enzyme is added in step S2.
Comparative example 2
The difference compared to example 3 is that in step S2, bacillus subtilis is not inoculated.
Comparative example 3
In comparison with example 3, the difference is that clerodendranthi polysaccharide was not added in step S4.
Comparative example 4
The difference from example 3 is that the organic extract of Clerodendranthus spicatus is not added in step S4.
Test example 1
Taking RAW264.7 cells in logarithmic growth phase, digesting with pancreatin, centrifuging, removing supernatant, and preparing into 7×10 with complete culture medium 4 Each mL of the cell suspension was inoculated into a 96-well plate, 100. Mu.L of the cell suspension was added to each well, and the mixture was placed at 37℃and 5% CO 2 After 24 hours of culture in the incubator, the supernatant in the wells was discarded, and 100. Mu.L of the complete medium containing the drug (final concentration of the extract of Clerodendranthus spicatus prepared in examples 1 to 5 and comparative examples 1 to 4: 0.25mg/mL; final concentration of LPS: 1. Mu.g/mL) was added, and the complete medium containing 1. Mu.g/mL of LPS and the complete medium containing no LPS, namely, the administration group, the model group and the blank group, were each provided with 4 duplicate wells, and placed at 37℃and containing 5% CO, were each added 2 Culturing in an incubator. The NO content secreted by the cells was determined by the kit. The results are shown in Table 1.
TABLE 1
| Group of | NO(μmol/L) |
| Blank group | 82.91±4.18 |
| Model group | 210.49±10.11 |
| Example 1 | 104.56±9.24 |
| Example 2 | 105.28±8.55 |
| Example 3 | 102.47±7.83 |
| Example 4 | 110.39±9.11 |
| Example 5 | 108.95±8.57 |
| Comparative example 1 | 125.48±7.82 |
| Comparative example 2 | 128.21±8.35 |
| Comparative example 3 | 135.19±10.52 |
| Comparative example 4 | 142.85±11.21 |
Annotation: * P <0.05 compared to the blank; # is P <0.05 compared to model group.
As can be seen from the above table, the clerodendrum cyrtophyllum extracts prepared in examples 1-3 of the present invention can significantly inhibit NO secretion.
Test example 2
The C57BL/6J mice were randomly divided into 11 groups of 6 mice each, which were control group, model group, examples 1-5 group, and comparative examples 1-4 group, respectively. After animals are adapted to the feeding environment, each administration group continuously irrigates the stomach according to the corresponding concentration to administer the corresponding group of the prepared clerodendranthus spicatus extract 7d (1 g/kg), and the control group and the model group are respectively administrated with physiological saline with the same volume in a manner of irrigating the stomach for 7d continuously. On day 7, the mice were weighed, and after 1 hour of administration, the model group and each of the administration groups were subjected to lipopolysaccharide tracheal instillation (5 mg/kg) to prepare an acute lung injury model, and the mice of the control group were instilled with the same volume of physiological saline.
After the end of the experiment, the lung tissue was removed and the weight (g): adding 9 times of physiological saline into the volume (mL) to homogenate, centrifuging, and taking the supernatant as a sample to be detected. The levels of TNF-alpha, IL-6, SOD, GSH were measured according to the kit method. The results are shown in Table 2.
TABLE 2
| Group of | TNF-α(pg/mL) | IL-6(pg/mL) | SOD(U/mg) | GSH(μmol/g) |
| Blank group | 108.2±7.5 | 32.2±2.2 | 125.2±30.1 | 8.2±0.5 |
| Model group | 240.3±55.2 | 129.5±6.9 | 60.4±11.4 | 4.8±0.9 |
| Example 1 | 125.2±26.8 | 59.6±4.5 | 105.5±12.5 | 7.5±0.7 |
| Example 2 | 124.8±22.8 | 60.5±3.8 | 106.3±11.4 | 7.4±0.5 |
| Example 3 | 127.4±21.9 | 62.2±5.6 | 108.3±11.2 | 7.7±0.6 |
| Example 4 | 132.5±19.5 | 68.9±6.2 | 100.1±10.3 | 6.9±0.5 |
| Example 5 | 134.2±20.1 | 69.4±6.1 | 98.6±9.6 | 7.0±0.4 |
| Comparative example 1 | 140.5±16.7 | 74.5±7.4 | 89.6±8.6 | 6.2±0.7 |
| Comparative example 2 | 144.2±12.5 | 76.7±6.5 | 84.5±8.1 | 5.8±0.9 |
| Comparative example 3 | 150.1±11.6 | 80.2±5.5 | 80.3±6.7 | 5.5±0.8 |
| Comparative example 4 | 158.9±13.5 | 86.9±4.7 | 72.5±5.8 | 5.0±0.9 |
Annotation: * P <0.05 compared to the blank; # is P <0.05 compared to model group.
As shown in the table above, the clerodendrum bungei extract prepared in the embodiments 1-3 of the invention can obviously reduce the inflammation index of mice and improve the oxidative stress index.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A method for preparing clerodendrum trichotomum extract, which is characterized by comprising the following steps:
s1, cleaning, drying and crushing clerodendrum bungei medicinal materials, adding the clerodendrum bungei medicinal materials into water, heating, boiling, extracting, filtering, reserving filter residues, adding ethanol for precipitation, collecting solids, washing and drying to obtain clerodendrum bungei polysaccharide, and reserving filtrate;
s2, adding the solid obtained in the step S1 into water, adding enzyme for enzymolysis, inoculating bacillus subtilis strain seed liquid, fermenting and culturing, sterilizing, inactivating enzyme, and filtering to obtain filtrate for use;
s3, combining the filtrate in the step S1 and the filtrate in the step S2, adding an organic solvent for extraction, collecting an organic phase, removing the solvent under reduced pressure, washing, and drying to obtain the clerodendranthus spicatus organic extract;
s4, uniformly mixing the clerodendranthus spicatus polysaccharide prepared in the step S1 and the clerodendranthus spicatus organic extract prepared in the step S3 to prepare the clerodendranthus spicatus extract.
2. The preparation method according to claim 1, wherein the solid-to-liquid ratio of the clerodendranthus spicatus medicinal material and water in the step S1 is 1:7-10g/mL.
3. The method according to claim 1, wherein the time of the heat boiling extraction in step S1 is 2 to 4 hours.
4. The method according to claim 1, wherein the ethanol is added in step S1 to a system ethanol content of 80-90wt%, and the precipitation time is 3-5 hours.
5. The method according to claim 1, wherein the mass ratio of solids, enzyme and water in step S2 is 10-15:1-2:200.
6. The preparation method according to claim 1, wherein the enzymes in the step S2 comprise cellulase and pectase in a mass ratio of 5-7:1, and the enzymolysis is performed at a temperature of 40-45 ℃ for 2-4h.
7. The method according to claim 1, wherein the seed solution of Bacillus subtilis strain in step S2 has an inoculum size of 2-4v/v%, and the seed solution of Bacillus subtilis strain has a inoculum size of 10 7 -10 8 cfu/mL。
8. The method according to claim 1, wherein the conditions for the fermentation culture in step S2 are 40-45℃and 100-200r/min, and the fermentation culture is performed for 48-56 hours.
9. The preparation method according to claim 1, wherein the organic solvent in the step S3 is a mixed solution of n-butanol and cyclohexane, and the volume ratio is 3-5:7.
10. A clerodendranthus spicatus extract prepared by the preparation method of any one of claims 1-9.
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