CN113648356A - Clerodendrum petasitum extract and application thereof in preparation of anti-inflammatory composition - Google Patents
Clerodendrum petasitum extract and application thereof in preparation of anti-inflammatory composition Download PDFInfo
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Abstract
The invention relates to the field of natural medicines, in particular to a clerodendrum japonicum extract and application thereof in preparing an anti-inflammatory composition. The Clerodendron petasitum extract is prepared by extracting Clerodendron petasitum with ethanol, sequentially extracting with petroleum ether and ethyl acetate, and eluting ethyl acetate with mixed solvent of weak polar solvent and strong polar solvent at certain ratio. The anti-inflammatory mechanism of ethyl clerodendrum japonicum acetate and its different gradient weak polar solvent-strong polar solvent part is effective in inhibiting the production of NO, TNF-a, IL-12, IL-6, and IL-1 beta inflammatory factors.
Description
Technical Field
The invention relates to the field of natural medicines, in particular to a clerodendrum japonicum extract and application thereof in preparing an anti-inflammatory composition.
Background
Clerodendron petasitum is Clerodendrendrum japonicum (Thunb.) Sweet, a Clerodendron quinquefolius, a Clerodendranthus spicatus, a Clinopodium androsaceus (Chunb.) A.C., a Clerodendrum spicatum, a Clinopodium japonicum (Chorda monster, Guangxi), produced in Guangxi, Guizhou, Yunnan, etc., and is a frequently used medicine in folk tradition. Clerodendron petasitum mainly contains flavonoids, phenolic acids, tannin, volatile oil, etc., and is sweet in taste and cool in nature, and has pharmacological effects of anti-inflammatory, anti-oxidation and anti-tumor, and can be used for treating migraine, traumatic blood stasis and swelling, carbuncle and skin sore; the traditional Chinese medicine composition is clinically used for treating cough with lung heat, stranguria with heat and difficult urination. In the previous research work, the Cheng tung ethyl acetate part is found to be the most effective part with the anti-inflammatory activity, however, the specific link and target point for exerting the anti-inflammatory effect and the main effective components are not clear.
Disclosure of Invention
The present invention aims at providing a clerodendrum japonicum extract and its use in the preparation of an anti-inflammatory composition.
The present invention provides a Clerodendron petersum extract obtained by sequentially extracting Clerodendron petersum with ethanol, and then eluting the ethyl acetate fraction with a mixed solvent of dichloromethane and methanol at a predetermined ratio.
The present invention also provides a method for producing the above-mentioned Clerodendron trichotomum extract, which comprises the steps of:
(1) reflux-extracting Clerodendron petasitum medicinal material with 50-95% of ethanol with the amount of 10-12 times of the weight of the medicinal material for 2-5 times, 1-3 h each time, mixing, and concentrating to obtain an ethanol extract;
(2) suspending the ethanol extract obtained in the step (1) with water according to the weight part of 1-1.5: 1 to obtain a suspension, adding petroleum ether into the suspension and the petroleum ether according to the volume ratio of 1: 1-1.5 for extraction for 2-4 times, and discarding the petroleum ether extract to obtain an extracted ethanol extract;
(3) adding ethyl acetate into the ethanol extract extracted in the step (2) and ethyl acetate according to the volume ratio of 1: 1-1.5 for extraction for 2-4 times, wherein the obtained ethyl acetate extract is an ethyl acetate part;
(4) and (3) loading the ethyl acetate part obtained in the step (3) into a silica gel column, sequentially carrying out gradient elution by using a weak polar solvent-a strong polar solvent according to a volume ratio of 10-70: 1, wherein the dosage of each gradient elution solvent is 8-10 times of the column volume, collecting each gradient elution part, concentrating and drying to obtain the ethyl acetate.
Preferably, in the step (1), the medicinal materials are soaked in 80% ethanol in an amount which is 12 times that of the medicinal materials for 24 hours, then are subjected to reflux extraction for 1.5 hours, then are filtered, and after 3 times of repeated extraction, the medicine residues are subjected to reflux extraction for 2 times by adding 60% ethanol in an amount which is 10 times that of the medicine residues, and each time lasts for 1.5 hours.
Preferably, in the step (2), the ethanol extract obtained in the step (1) is suspended with water according to the weight ratio of 1-1: 1.
Preferably, in the step (2), petroleum ether is added for extraction for 3 times according to the volume ratio of the suspension to the petroleum ether of 1: 1.
Preferably, in the step (3), the ethanol extract extracted in the step (2) and ethyl acetate are added with ethyl acetate for extraction for 3 times according to the volume ratio of 1: 1.
Preferably, in the step (4), the weak polar solvent is chloroform, and the strong polar solvent is methanol.
Preferably, in the step (4), the weak polar solvent is chloroform, and the strong polar solvent is acetone.
Preferably, in the step (4), the weak polar solvent is dichloromethane, and the strong polar solvent is methanol.
Preferably, in the step (4), collecting an elution part with a volume ratio of dichloromethane to methanol of 30-50: 1, concentrating and drying to obtain the methanol-methanol composite.
Preferably, in the step (4), the elution part with the volume ratio of dichloromethane to methanol being 50:1 is collected, concentrated and dried to obtain the compound.
The characteristics and advantages of the clerodendrum japonicum extract of the invention are:
1. the anti-inflammatory mechanism of ethyl clerodendrum japonicum acetate fraction and its different gradient dichloromethane-methanol elution fraction is effective in inhibiting the production of inflammatory factors such as NO, TNF-a, IL-12, IL-6 and IL-1. beta.
2. As the concentration of the drug in the ethyl acetate fraction of Clerodendron petorum and the fraction eluting with different polarity increases, the secretion of inflammatory factors decreases, and the anti-inflammatory activity is strongest at the elution fraction of Clerodendron petorum methylene chloride-methanol (50:1) and at the elution fraction of methylene chloride-methanol (30: 1).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1 preparation of Ethyl Clerodendron Clerodendrum acetate fraction
Medicinal materials
The medicinal materials are collected from Wuming district of southwestern, Guangxi, and identified as Clerodendrum japonicum (Thunb.) Sweet of Clerodendrum genus of Verbenaceae family by pharmacist of Assistant drug of Thymelah, department of pharmacy of first subsidiary hospital of Guangxi Chinese medicine university, and the medicinal parts are whole plants.
Second, preparation method
Soaking 11.5Kg of clerodendrum japonicum medicinal material in 80% ethanol in an amount which is 12 times that of the clerodendrum japonicum medicinal material for 24h, performing reflux extraction for 1.5h, filtering, repeatedly extracting for 3 times, adding 60% ethanol in an amount which is 10 times that of the clerodendrum japonicum medicinal material for reflux extraction for 2 times, performing reflux extraction for 1.5h each time, combining filtrates, concentrating until no ethanol smell exists to obtain 1178.75g of ethanol extract, adding 1000ml of pure water for suspension, adding petroleum ether with the volume ratio of 1:1 for extraction for 3 times, adding ethyl acetate with the volume ratio of 1:1 for extraction for 3 times, combining ethyl acetate extract, concentrating, freeze-drying to obtain 662.85g of clerodendrum japonicum ethyl acetate extract, and sealing and storing for later use.
Precisely weighing 0.15g of clerodendrum japonicum ethyl acetate extract, adding 0.1% DMSO for ultrasonic dissolution, diluting to 3mg/mL with RPMI1640 incomplete culture solution, filtering with sterile filter membrane, diluting with complete culture medium to desired concentration, and preparing.
Example 2 preparation of Clerodendron clerodendrum dichloromethane-methanol fraction
Dissolving 150g of ethyl acetate fraction of Clerodendron petiolatum in 350ml of methanol, mixing with 120g of column chromatography silica gel (200-300 meshes), drying, and loading onto column with diameter of 6cm and height of 60 cm. Respectively eluting with dichloromethane-methanol (70:1), dichloromethane-methanol (50:1), dichloromethane-methanol (30:1), dichloromethane-methanol (20:1), and dichloromethane-methanol (10:1) at gradient amount of 10 times of column volume, respectively collecting gradient elution parts, concentrating, and freeze drying. Obtaining dry paste of each part, sealing and storing for later use.
Weighing 0.15g of dry extract of different parts, adding 0.1% DMSO, dissolving with ultrasound, diluting with RPMI1640 incomplete culture solution to 3mg/mL, filtering with sterile filter membrane, diluting with complete culture medium to desired concentration, and mixing.
Example 3 preparation of Clerodendron petiolatum chloroform-methanol fraction
Dissolving 150g of ethyl acetate fraction of Clerodendron petiolatum in 350ml of methanol, mixing with 120g of column chromatography silica gel (200-300 meshes), drying, and loading onto column with diameter of 6cm and height of 60 cm. Performing gradient elution with chloroform-methanol (70:1), chloroform-methanol (50:1), chloroform-methanol (30:1), and chloroform-methanol (10:1) respectively, wherein the amount of each gradient is 10 times of the column volume, collecting gradient elution parts respectively, concentrating, and freeze drying. Obtaining dry paste of each part, sealing and storing for later use.
Weighing 0.15g of dry extract of different parts, adding 0.1% DMSO, dissolving with ultrasound, diluting with RPMI1640 incomplete culture solution to 3mg/mL, filtering with sterile filter membrane, diluting with complete culture medium to desired concentration, and mixing.
Example 4 preparation of Clerodendron petiolatum chloroform-acetone fraction
Dissolving 150g of ethyl acetate fraction of Clerodendron petiolatum in 350ml of methanol, mixing with 120g of column chromatography silica gel (200-300 meshes), drying, and loading onto column with diameter of 6cm and height of 60 cm. Gradient eluting with chloroform-acetone (70:1), chloroform-acetone (50:1), chloroform-acetone (30:1), and chloroform-acetone (10:1), respectively, the amount of each gradient is 10 times of column volume, and collecting gradient elution parts respectively. Obtaining dry paste of each part, sealing and storing for later use.
Weighing 0.15g of dry extract of different parts, adding 0.1% DMSO, dissolving with ultrasound, diluting with RPMI1640 incomplete culture solution to 3mg/mL, filtering with sterile filter membrane, diluting with complete culture medium to desired concentration, and mixing.
Example 5 Effect of Ethyl Clerodendron Clerodendrum acetate fraction and fraction thereof on LPS-induced NO secretion from RAW264.7 cells
First, experiment method
(1) Taking RAW264.7 cells in logarithmic growth phase, digesting with pancreatin, centrifuging at 1000rpm for 5min, discarding supernatant, and preparing into 5 × 10 with complete culture medium4The 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 left at 37 ℃ with 5% CO2Culturing in an incubator.
(2) After 24 hours, the supernatant in the well was discarded, and 100. mu.L of a drug-containing complete culture medium (wherein the ethyl clerodendrum japonicum acetate fraction, the methylene chloride-methanol (70:1) elution fraction, the methylene chloride-methanol (50:1) elution fraction, the methylene chloride-methanol (30:1) elution fraction, the methylene chloride-methanol (20:1) elution fraction, the methylene chloride-methanol (10:1) elution fraction, the chloroform-methanol (70:1) elution fraction, the chloroform-methanol (50:1) elution fraction, the chloroform-methanol (30:1) elution fraction, the chloroform-methanol (10:1) elution fraction, the chloroform-acetone (70:1) elution fraction, the chloroform-acetone (50:1) elution fraction, the chloroform-acetone (30:1) elution fraction, the chloroform-methanol (10:1) elution fraction, the chloroform-acetone (30:1) elution fraction, the chloroform-methanol fraction, and the chloroform-methanol fraction were added to be added to 100. mu.L of the total amount of each of the total amount of each of the total amount of each, Chloroform-acetone (10:1) elution sites, each elution site being administered at a final concentration of 0.25 mg/mL; final concentration of LPS 1. mu.g/mL), complete medium with LPS 1. mu.g/mL and complete medium without LPS, i.e., administration group, model group (M) and blank group (K), each group having 4 duplicate wells, placed at 37 ℃ and containing 5% CO2Culturing in an incubator.
(3) After 24 hours, the content of NO secreted by the cells was determined according to the kit.
Second, experimental results
Under the induction of LPS, the secretion of NO is increased significantly (P <0.05) in the model group compared with the control group, which is indicative of inflammation produced by macrophages. Addition of ethyl Clerodendron petiolate and the fraction thereof gave a significant decrease in NO secretion, compared with the model group: the ethyl acetate high-dose group (0.25mg/mL) and the medium-dose group (0.125mg/mL) can obviously reduce the secretion of the inflammatory factor NO (P <0.05), and the low-dose group has NO inhibition effect; the high, medium and low dose groups of clerodendrum japonicum methane-methanol (50:1) elution site can significantly inhibit (P < 0.05); the high and medium doses of clerodendrum japonicum methylene chloride-methanol (30:1) elution part and methylene chloride-methanol (20:1) elution part have inhibition effects (P <0.05), while the low dose group has inhibition tendency and has no significant difference; while the high dose group containing an elution site of Clerodendron petorum dichloromethane-methanol (70:1) and an elution site of Clerodendron petorum dichloromethane-methanol (10:1) was significantly inhibited (P <0.05), and neither the medium nor low dose groups showed any inhibition tendency. The results are shown in Table 1.
Example 6 Effect of Ethyl Clerodendron Clerodendrum acetate fraction and fraction thereof on the secretion of TNF-a, IL-12, IL-6 and IL-1. beta. by LPS-induced RAW264.7 cells
First, experiment method
(1) Taking RAW264.7 cells in logarithmic growth phase, digesting with pancreatin, centrifuging at 1000rpm for 5min, removing supernatant, and preparing into 5 × 10 with complete culture medium4The cell suspension was inoculated into a 96-well plate at one/mL, and 100. mu.L of the cell suspension was added to each well, and the plate was incubated at 37 ℃ in an incubator containing 5% CO 2.
(2) After 24 hours, the supernatant from the wells was discarded, and 100. mu.l of a drug-containing complete medium was added thereto, wherein the final concentrations of the drug-containing complete medium, ethyl clerodendrum japonicum, methylene chloride-methanol (70:1) and methylene chloride-methanol (50:1) and methylene chloride-methanol (30:1) were 0.25, 0.125 and 0.06mg/mL, respectively; final concentration of LPS 1. mu.g/mL), complete medium with LPS 1. mu.g/mL and complete medium without LPS,namely an administration group, a model group (M) and a blank group (K), each group being provided with 4 multiple wells and being placed at 37 ℃ and containing 5% CO2Culturing in an incubator.
(3) After 24 hours, the contents of inflammatory factors TNF-a, IL-12, IL-6 and IL-1 beta secreted by the cells were determined according to the kit.
(4) The experimental procedures of ELISA method are referred to the instruction manual.
Second, experimental results
Effect on TNF-a: the secretion of TNF-a was significantly increased in the model group under the induction of LPS compared to the control group (P < 0.05). The amount of TNF-a secreted by addition of ethyl clerodendrum japonicum and the fraction thereof was significantly reduced, as compared with the model group: the high dose group (0.25mg/mL) and the medium dose group (0.125mg/mL) of the ethyl clerodendrum japonicum acetate part can obviously reduce the secretion of the inflammatory factor TNF-a (P is less than 0.05), and the low dose has an inhibiting effect but has no obvious difference; the high, medium and low dose groups eluting Clerodendron clerodendrum dichloromethane-methanol (50:1) can remarkably inhibit the secretion of TNF-a (P < 0.05); the results are shown in Table 2.
Effect on the influence of IL-6: the secretion of IL-6 was significantly increased in the model group under the induction of LPS compared to the control group (P < 0.05). After the intervention of addition of ethyl acetate fraction of Clerodendron petorum and fraction thereof, the amount of IL-6 secretion was significantly reduced, as compared with the model group: the ethyl clerodendrum japonicum acetate high dose group (0.25mg/mL) and the medium dose group (0.125mg/mL) can remarkably reduce the secretion of the inflammatory factor IL-6 (P is less than 0.05), and the low dose has an inhibiting effect but has no remarkable difference; the high, medium and low dose groups eluting with Clerodendron clerodendrum dichloromethane-methanol (50:1) significantly inhibited IL-6 secretion (P < 0.05). The results are shown in Table 2.
Effect on IL-12: the secretion of IL-12 was significantly increased in the model group under the induction of LPS compared to the control group (P < 0.05). After addition of ethyl Clerodendron petiolate fraction and fraction thereof, the amount of IL-12 secretion was significantly reduced, as compared with the model group: the ethyl clerodendrum japonicum part high dose group (0.25mg/mL) and the medium dose group (0.125mg/mL) can remarkably reduce the secretion of the inflammatory factor IL-12 (P is less than 0.05), and the low dose has an inhibiting effect but has no remarkable difference; the high, medium and low dose groups eluting Clerodendron clerodendrum dichloromethane-methanol (50:1) can remarkably inhibit the secretion of IL-12 (P < 0.05); the results are shown in Table 2.
Effect on IL-1 β: the secretion of IL-1. beta. was significantly increased in the model group under the induction of LPS, compared to the control group (P < 0.05). Addition of ethyl Clerodendron petiolate fraction and fraction thereof reduced the secretion of the proinflammatory factor IL-1 β, compared to the model group: the high dose group (0.25mg/mL) and the medium dose group (0.125mg/mL) of the ethyl clerodendrum japonicum acetate part can obviously reduce the secretion of the inflammatory factor IL-1 beta (P is less than 0.05), and the low dose has an inhibiting effect but has no obvious difference; the high, medium and low dose groups eluting Clerodendron clerodendrum dichloromethane-methanol (50:1) can remarkably inhibit IL-1 beta secretion (P < 0.05); the results are shown in Table 2.
TABLE 1 influence of Ethyl Clerodendron Clerodendrum acetate fraction and fraction thereof on NO
n＝4)
| Group of | Concentration (mg/mL) | NO(μmol˙L-1) |
| Blank control group | -- | 83.62±4.30 |
| Model set | -- | 208.05±8.58Δ |
| Ethyl acetate moiety group | 0.25 | 187.04±4.96* |
| Methylene chloride-methanol (70:1) elution site | 0.25 | 180.74±8.30* |
| Methylene chloride-methanol (50:1) elution site | 0.25 | 158.01±5.08*# |
| Methylene chloride-methanol (30:1) elution site | 0.25 | 169.96±5.81*# |
| Methylene chloride-methanol (20:1) elution site | 0.25 | 179.06±7.10* |
| Methylene chloride-methanol (10:1) elution site | 0.25 | 183.86±10.31* |
| Chloroform-methanol (70:1) elution site | 0.25 | 185.65±5.26* |
| Chloroform-methanol (50:1) elution site | 0.25 | 194.42±3.85 |
| Chloroform-methanol (30:1) elution site | 0.25 | 198.17±4.36 |
| Chloroform-methanol (10:1) elution site | 0.25 | 202.24±5.18 |
| Chloroform-acetone (70:1) elution site | 0.25 | 197.42±8.13 |
| Chloroform-acetone (50:1) elution site | 0.25 | 194.82±6.30 |
| Chloroform-acetone (30:1) elution site | 0.25 | 190.36±5.62 |
| Chloroform-acetone (10:1) elution site | 0.25 | 181.42±3.68* |
Note: delta P<0.05, compared to a blank control group; p<0.05, compared with a model control group;#P<0.05, compared to the ethyl acetate fraction group.
TABLE 2 influence of Ethyl Clerodendron Clerodendrum acetate fraction on TNF-a
n＝4)
Note: Δ P <0.05, compared to placebo; p <0.05, compared to model control group; # P <0.05, compared to the ethyl acetate fraction group.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may be modified or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A clerodendrum japonicum extract, which is produced by a method comprising:
(1) reflux-extracting Clerodendron petiolatus medicinal material with 50-95% of ethanol in an amount of 10-12 times of the weight of the medicinal material for 2-5 times, each time for 1-3 hours, mixing, and concentrating to obtain an ethanol extract;
(2) suspending the ethanol extract obtained in the step (1) with water according to the weight part of 1-1.5: 1 to obtain a suspension, adding petroleum ether into the suspension and the petroleum ether according to the volume ratio of 1: 1-1.5 for extraction for 2-4 times, and discarding the petroleum ether extract to obtain an extracted ethanol extract;
(3) adding ethyl acetate into the ethanol extract extracted in the step (2) and ethyl acetate according to the volume ratio of 1: 1-1.5 for extraction for 2-4 times, wherein the obtained ethyl acetate extract is an ethyl acetate part;
(4) and (3) loading the ethyl acetate part obtained in the step (3) into a silica gel column, sequentially carrying out gradient elution by using a weak-polarity solvent and a strong-polarity solvent according to a volume ratio of 10-70: 1, wherein the dosage of each gradient elution solvent is 8-10 times of the column volume, collecting each gradient elution part, concentrating and drying to obtain the ethyl acetate gradient elution column.
2. The Clerodendron trichotomum Thunb extract according to claim 1, wherein in the step (1), the Clerodendron trichotomum Thunb extract is obtained by soaking in 12 times of 80% ethanol for 24 hours, reflux-extracting for 1.5 hours, filtering, repeating the extraction for 3 times, and reflux-extracting the residue with 10 times of 60% ethanol for 2 times, each time for 1.5 hours.
3. The Clerodendron trichotomum Thunb extract according to claim 1, wherein in the step (2), the ethanol extract obtained in the step (1) is suspended in water at a weight ratio of 1 to 1:1 to obtain a suspension, and petroleum ether is added to the suspension at a volume ratio of 1:1 to the petroleum ether to extract the suspension for 3 times.
4. The method of claim 1, wherein the ethanol extract obtained by the step (2) is extracted with ethyl acetate at a volume ratio of 1:1 with respect to ethyl acetate, and the extract is extracted 3 times.
5. The method of claim 1, wherein in the step (4), the weakly polar solvent is chloroform, and the strongly polar solvent is methanol.
6. The method of claim 1, wherein in the step (4), the weakly polar solvent is chloroform, and the strongly polar solvent is acetone.
7. The method of claim 1, wherein in the step (4), the weakly polar solvent is methylene chloride, and the strongly polar solvent is methanol.
8. The Clerodendron trichotomum extract according to claim 7, wherein the fraction eluted in step (4) is collected at a volume ratio of dichloromethane to methanol of 30 to 50:1, and the collected fraction is concentrated and dried.
9. The method of claim 8, wherein in the step (4), the eluted fraction having a volume ratio of dichloromethane to methanol of 50:1 is collected, concentrated and dried.
10. Use of a Clerodendron petersatumu extract for producing an anti-inflammatory composition, wherein the Clerodendron petersatumu extract is the Clerodendron petersatumu extract as defined in any one of claims 7 to 9.
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| CN (1) | CN113648356A (en) |
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| CN106620045A (en) * | 2016-12-30 | 2017-05-10 | 广西中医药大学 | Preparation method and application of 95% ethanol extract of clerodendrum japonicum roots |
| CN106728447A (en) * | 2016-12-30 | 2017-05-31 | 广西中医药大学 | The preparation method and application of Cheng forture paulownia root ethyl acetate extract |
| CN106822382A (en) * | 2016-12-30 | 2017-06-13 | 广西中医药大学 | The preparation method and application of Cheng forture paulownia root n-butanol extract |
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- 2021-09-14 CN CN202111072165.6A patent/CN113648356A/en active Pending
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| CN106620045A (en) * | 2016-12-30 | 2017-05-10 | 广西中医药大学 | Preparation method and application of 95% ethanol extract of clerodendrum japonicum roots |
| CN106728447A (en) * | 2016-12-30 | 2017-05-31 | 广西中医药大学 | The preparation method and application of Cheng forture paulownia root ethyl acetate extract |
| CN106822382A (en) * | 2016-12-30 | 2017-06-13 | 广西中医药大学 | The preparation method and application of Cheng forture paulownia root n-butanol extract |
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Application publication date: 20211116 |