CN109288826B - Application of aurantio-obtusin in preparing anti-inflammatory and anti-photoaging products - Google Patents
Application of aurantio-obtusin in preparing anti-inflammatory and anti-photoaging products Download PDFInfo
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- CN109288826B CN109288826B CN201811335671.8A CN201811335671A CN109288826B CN 109288826 B CN109288826 B CN 109288826B CN 201811335671 A CN201811335671 A CN 201811335671A CN 109288826 B CN109288826 B CN 109288826B
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention discloses application of aurantio-obtusin in preparing anti-inflammatory and photoaging prevention products, and the application proves that aurantio-obtusin has good anti-inflammatory effect by taking LPS (low-cholesterol) induced RAW264.7 cells to release NO and IL-6 as targets, and has good application prospect in preparing anti-inflammatory products; according to the invention, UVB induces HaCaT cells to secrete IL-6 and IL-1 alpha as target spots, so that the aurantio-obtusin has the effect of preventing photoaging, and the aurantio-obtusin is proved to have good application prospect in the aspect of preparing products for preventing photoaging.
Description
Technical Field
The invention relates to the field of aurantio-obtusin, in particular to application of aurantio-obtusin in preparing anti-inflammatory and anti-photoaging products.
Background
Cassia seed, originally recorded in Shennong Ben Cao Jing, is a dry mature seed of Cassia Obtusifolia L or Cassia tora L of Leguminosae, which is sweet, bitter, salty and slightly cold in nature, and has the effects of clearing heat, improving eyesight, moistening intestines and relaxing bowels.
Modern pharmacological research shows that the cassia seed has the effects of reducing blood fat, enhancing immunity, resisting oxidation, protecting liver, benefiting gallbladder, relieving asthma, inhibiting bacteria, resisting atherosclerosis and the like. The aurantio-obtusin is a special component in cassia seeds, has the character of yellow acicular anthraquinone compound, and has various biological activities such as blood sugar reduction, blood fat reduction, antiestrogen, collagen-induced platelet aggregation inhibition and the like.
At present, no report on the aspects of diminishing inflammation and preventing photoaging exists in the aurantio-obtusin.
Disclosure of Invention
The invention aims to provide application of aurantio-obtusin in preparing anti-inflammatory and anti-photoaging products so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
application of aurantio-obtusin in preparing antiinflammatory product is provided.
As a further scheme of the invention: the application amount of the aurantio-obtusin in preparing anti-inflammatory products is 5-20 mu mol/L.
As a further scheme of the invention: when the aurantio-obtusin is used for preparing an anti-inflammatory product, the dosage of the aurantio-obtusin is 5 mu mol/L.
As a further scheme of the invention: when the aurantio-obtusin is used for preparing an anti-inflammatory product, the dosage of the aurantio-obtusin is 20 mu mol/L.
Application of aurantio-obtusin in preparing products for preventing photoaging is provided.
As a further scheme of the invention: the dosage of the aurantio-obtusin is 10-20 mu mol/L when the aurantio-obtusin is used for preparing products for preventing photoaging.
As a further scheme of the invention: when the aurantio-obtusin is used for preparing a product for preventing photoaging, the dosage of the aurantio-obtusin is 10 mu mol/L.
As a further scheme of the invention: when the aurantio-obtusin is used for preparing a product for preventing photoaging, the dosage of the aurantio-obtusin is 20 mu mol/L.
Compared with the prior art, the invention has the beneficial effects that:
firstly, LPS induces RAW264.7 cells to release NO and IL-6 as target spots, so that the application proves that the aurantio-obtusin has good anti-inflammatory effect, and has good application prospect in the aspect of preparing anti-inflammatory products;
secondly, the invention verifies that the aurantio-obtusin has the effect of preventing photoaging by using UVB to induce HaCaT cells to secrete IL-6 and IL-1 alpha as target spots, and proves that the aurantio-obtusin has good application prospect in the aspect of preparing products for preventing photoaging.
Drawings
FIG. 1 is a graph of the effect of aurantio-obtusin on Raw264.7 cell viability.
FIG. 2 is a graph showing the effect of aurantio-obtusin on the secretion of NO by Raw264.7 cells induced by LPS.
FIG. 3 is a graph showing the effect of aurantio-obtusin on the secretion of IL-6 by Raw264.7 cells induced by LPS.
FIG. 4 is a graph of the effect of obtusin aurantiacum on IL-6 secretion from HaCaT cells.
FIG. 5 is a graph of the effect of obtusin aurantiacum on IL-1 α secretion by HaCaT cells.
Detailed Description
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
Lipopolysaccharide (LPS) -induced RAW264.7 mouse macrophages are a common model for studying inflammatory responses. Macrophages, stimulated by LPS, promote the secretion of inflammatory factors such as Nitric Oxide (NO) and interleukin-6 (interleukin 6). The invention takes LPS to induce RAW264.7 cells to release NO and IL-6 as targets, and examines the anti-inflammatory effect of the aurantio-obtusin.
Skin aging includes natural aging and photoaging, wherein photoaging may even cause various benign or malignant tumors to appear on the skin, besides causing changes in skin color, texture, elasticity, thickness, etc., which are serious effects. Skin photoaging is closely related to UV irradiation, and medium-wave ultraviolet UVB mainly acts on keratinocytes in the epidermis layer of the skin, so that the change of the skin appearance and the corresponding molecular biological change are caused, apoptosis of cells is caused, and an inflammatory factor IL-6 and the like are secreted. HaCaT is a commonly used human epidermal keratinocyte cell line, and the prevention photoaging effect of the aurantio-obtusin is examined by taking UVB (ultraviolet B) induced HaCaT cells to secrete IL-6 and IL-1 alpha as targets.
Materials and reagents: RAW264.7 cell line was purchased from Shanghai cell institute of Chinese academy of sciences; HaCaT was purchased from Shanghai cell institute of Chinese academy of sciences, aurantiamarin, dexamethasone and DMSO (dimethyl sulfoxide) were purchased from Sigma, DMEM high-sugar medium, fetal bovine serum FBS, pancreatin and the like were purchased from Gibco, CCK-8 was purchased from Shanghai dust Biotech limited; cytokine ELISA kits such as IL-6 were purchased from eBioscience, USA.
Preparing a liquid medicine: the aurantio-obtusin is prepared into 10mmol/L mother liquor by DMSO for standby, and is diluted into required concentration by DMEM culture medium when dosing.
Cell culture: RAW264.7 cells are cultured in DMEM high-glucose medium containing 10% FBS by mass fraction and placed in CO of 5% by mass fraction2And cultured in a cell culture box at 37 ℃ and saturated humidity. And (4) carrying out passage for 1 time after the cells grow to the logarithmic growth phase and carrying out passage for 2-3 d. Cells after at least 3 passages were taken for experiments.
Culturing HaCaT cells in DMEM high-sugar medium containing 10% FBS by mass fraction, and placing in CO with mass fraction of 5%237 ℃ and saturationCulturing in a humidity cell culture box. And (4) carrying out passage for 1 time after the cells grow to the logarithmic growth phase and carrying out passage for 2-3 d. Cells after at least 3 passages were taken for experiments.
Effect of drugs and compositions on growth of RAW264.7 cells: digesting RAW264.7 cells in logarithmic phase for 2min by using pancreatin with the mass fraction of 0.25% and EDTA with the mass fraction of 0.02%, discarding the pancreatin, neutralizing the pancreatin by using a DMEM high-sugar medium with the mass fraction of 10% FBS, gently blowing to form a single-cell suspension, centrifuging to discard a supernatant, resuspending the cells by using a complete medium, counting, and adjusting the cell suspension to 8 x 105/mL, 100. mu.L per well was inoculated into a 96-well plate at 37 ℃ with 5% CO by mass2Culturing for 24 hours under the saturation humidity condition, completely sucking supernatant of each hole, adding 100 mu L of DMEM high-sugar culture medium into each hole, and randomly dividing into a negative control group (the DMEM high-sugar culture medium containing DMSO with the mass fraction of 0.1%), a toxicity control group (the DMEM high-sugar culture medium containing DMSO with the mass fraction of 5%) and a drug group; after the corresponding medicine is added, each group is provided with 4 compound holes with the mass fraction of 5 percent CO2After culturing for 24h at 37 ℃ under the saturated humidity condition, adding CCK-8 according to 10 mu L per well in advance for 4h, and detecting the absorbance value (A450) at 450nm by an enzyme-labeling instrument after 4 h. As can be seen in fig. 1, compared to the blank control group, the different concentrations of aurantio-obtusin had no significant effect on the growth of RAW264.7 cells, i.e., aurantio-obtusin had no cytotoxic effect at the concentration of 5-20 μmol/L.
And (3) detecting inflammatory factors: digesting RAW264.7 cells in logarithmic phase for 2min by using pancreatin with the mass fraction of 0.25% and EDTA with the mass fraction of 0.02%, discarding the pancreatin, neutralizing the pancreatin by using a high-sugar medium of 10% FBS DMEM, gently blowing to form a single-cell suspension, centrifuging to discard the supernatant, resuspending the cells by using the complete medium, counting, and adjusting the cell suspension to 8 x 105/mL, 100. mu.L per well was inoculated into a 96-well plate at 37 ℃ with 5% CO by mass2Culturing for about 8h under saturated humidity condition to allow cells to adhere to the wall, completely sucking supernatant in each hole, adding 100 muL serum-free DMEM high-sugar medium into each hole, randomly dividing into blank control group, LPS (1 mug/mL) group, LPS + positive control group (5 mumol/L Dex), LPS (1 mug/mL) + drug group, adding corresponding drugs, and arranging 4 multiple holes in each group with mass fraction of5％C02Culturing at 37 deg.C under saturated humidity for 24 hr, and collecting supernatant for detecting inflammatory factors such as NO and interleukin-6 (IL-6). As can be seen in fig. 2, different concentrations of aurantio-obtusin significantly inhibited NO secretion from RAW264.7 cells induced by bacterial Lipopolysaccharide (LPS) and exhibited a dose-dependent effect. As can be seen in fig. 3, different concentrations of aurantio-obtusin can significantly inhibit bacterial Lipopolysaccharide (LPS) -induced IL-6 secretion from RAW264.7 cells, with 20 μ M concentration of aurantio-obtusin having the best anti-inflammatory effect.
UVB photoaging inflammatory factor detection: digesting Hacat cells in logarithmic growth phase for 2min by using pancreatin with the mass fraction of 0.25% and EDTA with the mass fraction of 0.02%, discarding the pancreatin, neutralizing the pancreatin by using a high-sugar medium containing FBS DMEM with the mass fraction of 10%, gently blowing to form a single-cell suspension, centrifuging to discard a supernatant, re-suspending the cells by using a complete medium, counting, and adjusting the cell suspension to 8 x 104/mL, 400. mu.L per well was inoculated into 24-well plates with a mass fraction of 5% CO at 37 ℃2Culturing for 24h under saturated humidity condition, sucking up supernatant of each well, washing with 500 μ L PBS twice, covering cells with a small amount of PBS, and irradiating cells with UVB irradiator (irradiation intensity of 8 mJ/cm)2) After irradiation, PBS was used for washing once, 400. mu.L of the culture medium was added to the blank group and the UVB model group, and the culture medium containing aurantio-obtusin at different concentrations was added to the administration group. After further incubation for 24 hours, the supernatant was aspirated for detection of IL-6 and IL-1 α. As can be seen in FIG. 4, different concentrations of aurantio-obtusin significantly inhibited the secretion of IL-6 by Hacat cells by UVB irradiation with a dose-dependent effect. As can be seen in FIG. 5, 20. mu. mol/L of aurantio-obtusin can significantly inhibit the secretion of IL-1. alpha. by Hacat cells caused by UVB irradiation, and the effective inhibition rate reaches 22.4%.
In summary, we found that aurantio-obtusin has a good inhibitory effect on inflammatory factors using LPS-induced RAW264.7 mouse macrophage cell line. Secondly, on the basis of an anti-inflammatory result, the protection effect of aurantio-obtusin on UVB-induced human epidermal keratinocyte cell line HaCaT photoaging is detected, and researches show that aurantio-obtusin can inhibit the secretion of aging-related inflammatory factors caused by UVB.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes 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. Any reference sign in a claim should not be construed as limiting the claim concerned.
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 (3)
1. Application of aurantio-obtusin in preparing products for preventing photoaging is provided.
2. Use of aurantio-obtusin in the preparation of a product for preventing photo-aging according to claim 1, wherein the aurantio-obtusin is used in an amount of 10-20 μmol/L.
3. Use of aurantio-obtusin in the preparation of a product for the prevention of photoaging according to claim 2, wherein said aurantio-obtusin is used in an amount of 20 μmol/L.
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| CN201811335671.8A CN109288826B (en) | 2018-11-11 | 2018-11-11 | Application of aurantio-obtusin in preparing anti-inflammatory and anti-photoaging products |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101077341A (en) * | 2006-05-23 | 2007-11-28 | 成都中医药大学 | Application of aurantio-obtusifolin or its derivates in preparing hypolipidemic drug |
| CN101967090A (en) * | 2010-06-28 | 2011-02-09 | 南京泽朗农业发展有限公司 | Technology for extracting aurantio-obtusin |
| CN106581431A (en) * | 2016-12-10 | 2017-04-26 | 济南昊雨青田医药技术有限公司 | Medicine composition for preventing and treating amygdalitis |
| CN106727859A (en) * | 2016-12-10 | 2017-05-31 | 济南昊雨青田医药技术有限公司 | It is a kind of to treat pharmaceutical composition of pharyngitis and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101077341A (en) * | 2006-05-23 | 2007-11-28 | 成都中医药大学 | Application of aurantio-obtusifolin or its derivates in preparing hypolipidemic drug |
| CN101967090A (en) * | 2010-06-28 | 2011-02-09 | 南京泽朗农业发展有限公司 | Technology for extracting aurantio-obtusin |
| CN106581431A (en) * | 2016-12-10 | 2017-04-26 | 济南昊雨青田医药技术有限公司 | Medicine composition for preventing and treating amygdalitis |
| CN106727859A (en) * | 2016-12-10 | 2017-05-31 | 济南昊雨青田医药技术有限公司 | It is a kind of to treat pharmaceutical composition of pharyngitis and preparation method thereof |
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