CN111840348A - Application of eclipta alba extract in preparation of pharmaceutical preparation for inhibiting differentiation of MSCs (mesenchymal stem cells) to adipocytes - Google Patents
Application of eclipta alba extract in preparation of pharmaceutical preparation for inhibiting differentiation of MSCs (mesenchymal stem cells) to adipocytes Download PDFInfo
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- CN111840348A CN111840348A CN202010784041.XA CN202010784041A CN111840348A CN 111840348 A CN111840348 A CN 111840348A CN 202010784041 A CN202010784041 A CN 202010784041A CN 111840348 A CN111840348 A CN 111840348A
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- eclipta alba
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- alba extract
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Abstract
The invention relates to the technical field of traditional Chinese medicine extraction and extracts, in particular to application of an eclipta alba extract in preparation of a medicinal preparation for inhibiting differentiation of MSCs (mesenchymal stem cells) to adipocytes. The eclipta alba extract can inhibit the differentiation of MSCs to fat cells, and the molecular mechanism is that the eclipta alba extract containing the eclipta alba glycoside IV can inhibit the expression of PPAR gamma protein, so that the eclipta alba extract can be applied to the preparation of the anti-obesity pharmaceutical preparation.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine extraction and extracts, in particular to application of an eclipta alba extract in preparation of a medicinal preparation for inhibiting differentiation of MSCs (mesenchymal stem cells) to adipocytes.
Background
Differentiation of adipocytes begins with Mesenchymal Stem Cells (MSCs) and ends with mature adipocytes. Excessive differentiation of mesenchymal stem cells into adipocytes causes obesity. Therefore, the differentiation of MSCs into adipocytes is inhibited, and the occurrence of obesity can be suppressed to some extent.
Eclipta alba is the overground part of Eclipta prostrata L. of Eclipta prostrata of Compositae, and has the effects of enriching blood, cooling blood to stop bleeding, clearing heat and detoxicating, and nourishing yin and tonifying liver and kidney. Modern pharmacological research shows that the eclipta alba has pharmacological effects of resisting tumor, regulating immunity, resisting inflammation and the like, and is clinically used for treating coronary heart disease, hemorrhagic diseases and the like.
Disclosure of Invention
The inventors have found that ecliptin iv and an eclipta alba extract containing ecliptin iv can inhibit the differentiation of mesenchymal stem cells into adipocytes based on extensive studies on an eclipta alba extract using mouse bone marrow-derived mesenchymal stem cells (BMMSCs) as a model, and have completed the present invention.
The invention provides an application of an eclipta alba extract in preparing a pharmaceutical preparation for inhibiting differentiation of MSCs (mesenchymal stem cells) to adipocytes, wherein the eclipta alba extract contains ecliptin IV.
In a second aspect, the invention provides an application of an eclipta alba extract in preparing a pharmaceutical preparation for inhibiting the expression of PPAR gamma protein, wherein the eclipta alba extract contains ecliptin IV.
In a third aspect, the invention provides application of an eclipta alba extract in preparing a pharmaceutical preparation for resisting obesity, wherein the eclipta alba extract contains ecliptin IV.
In a fourth aspect, the present invention provides an anti-obesity pharmaceutical composition.
The pharmaceutical composition comprises the eclipta alba extract and a pharmaceutically acceptable carrier or excipient.
Alternatively, the pharmaceutical composition comprises the yerbadetajoside IV and a pharmaceutically acceptable carrier or excipient.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows morphological observations of mouse BMMSCs primary cells; wherein, A, B, C shows the photograph results of the primary culture for 3 days, 5 days and 7 days by inverted phase contrast microscope;
FIG. 2 shows the results of flow cytometry to identify the expression of surface markers of normal mouse BMMSCs; A. unlabeled nude cells set P1 gate as target cell population at sscvs. fsc, i.e. mononuclear cell population; B1-B4 is to respectively carry out PE-CD90, PE-sca-1, PE-CD29 and PE-Cy7-CD140 alpha gating P2 on the mononuclear cell group and screen MSCs positive expression cells; B5-B6 is to carry out Horizon V450-CD34 and APC-CD45 gating P2 on the mononuclear cell group, and to screen negative cells which do not express hematopoietic cell markers;
FIG. 3 shows the results of oil-red-O staining of primary cultured mouse BMMSCs differentiated into adipocytes induced by adipocyte-inducing agent;
FIG. 4 shows that rhoifolin IV inhibits differentiation of primary cultured mouse BMMSCs into adipocytes;
FIG. 5 shows that ecliptin IV reduces the expression level of PPAR gamma proteins in primary culture mouse BMMSCs;
FIG. 6 shows that Ecliptae herba extract reduces the expression level of PPAR γ protein in mouse bone marrow;
FIG. 7 shows that Ecliptae herba extract inhibits mouse BMMSCs from differentiating into adipocytes.
Detailed Description
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 only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Application of eclipta alba extract in preparing a pharmaceutical preparation for inhibiting differentiation of MSCs (mesenchymal stem cells) to adipocytes, wherein the eclipta alba extract contains ecliptin IV.
Meanwhile, the invention also provides:
application of eclipta alba extract in preparation of pharmaceutical preparations for inhibiting expression of PPAR gamma protein, wherein the eclipta alba extract contains ecliptin IV.
Through research, the eclipta alba extract can inhibit the expression of PPAR gamma protein in the induction process of MSCs cells to fat cells.
As known to those skilled in the art, PPAR γ is a key downstream signaling factor of the Wnt/β -catenin signaling pathway. PPAR γ is responsible for promoting differentiation of MSCs into adipocytes.
Further, the present invention provides:
application of eclipta alba extract in preparing pharmaceutical preparations for resisting obesity, wherein the eclipta alba extract contains ecliptin IV.
The eclipta alba extract can inhibit differentiation of MSCs cells to fat cells, and a large number of researches of the inventor show that the molecular mechanism is that the ecliptin IV and the eclipta alba extract containing the ecliptin IV inhibit expression of PPAR gamma protein related to differentiation of mesenchymal stem cells to fat cells, so that the eclipta alba extract can be applied to preparation of pharmaceutical preparations for resisting obesity.
In the application, the eclipta alba extract is preferably an alcohol extract of eclipta alba.
The application of the invention is preferably that the eclipta alba extract is prepared by the following method:
1) extracting Ecliptae herba with ethanol water solution; concentrating to obtain crude extract;
2) dissolving the obtained crude extract in water, and extracting with petroleum ether and ethyl acetate to obtain petroleum ether extract, ethyl acetate extract and water layer extract;
3) combining the extracts obtained in the step 2) and drying to obtain the finished product.
More preferably, the eclipta alba extract is prepared by the following method:
1) extracting Ecliptae herba with ethanol water solution; concentrating until no alcohol exists to obtain a crude extract; wherein,
the ethanol water solution is preferably 50 to 90 percent by volume, more preferably 60 to 80 percent by volume, and most preferably 70 percent by volume;
and/or the amount of the ethanol aqueous solution is preferably 2 to 10 times, more preferably 4 to 7 times, and most preferably 5 times that of the eclipta alba.
And/or, the extraction time is preferably 1 to 4 hours, more preferably 2 to 3 hours;
and/or, the number of extractions is preferably 1-6, more preferably 2-4, most preferably 3;
2) dissolving the obtained crude extract in water (3-20 times of water, preferably 6-15 times of water, and most preferably 10 times of water. ) Then petroleum ether and ethyl acetate are used for extraction, and after the solvent is removed, petroleum ether extraction part extract, ethyl acetate extraction part extract and water layer extract are respectively obtained;
wherein, petroleum ether and ethyl acetate are preferably used for extraction for 1 to 6 times respectively, more preferably 2 to 4 times, and most preferably 3 times;
the solvent removal can be carried out by a method commonly used in the art such as distillation under reduced pressure, and the present invention is not limited thereto.
3) Combining the extracts obtained in the step 2) and drying to obtain the eclipta alba extract.
Drying may be carried out in a manner commonly used in the art, and the present invention is not limited thereto.
More preferably, the eclipta alba extract at least contains the active ingredient of the eclipta alba glycoside IV. Therefore, the invention further provides an anti-obesity pharmaceutical composition.
The pharmaceutical composition comprises the eclipta alba extract and a pharmaceutically acceptable carrier or excipient.
Alternatively, the pharmaceutical composition comprises the yerbadetajoside IV and a pharmaceutically acceptable carrier or excipient.
The pharmaceutical composition according to the present invention is preferably in the form of one of tablets, capsules, powders, granules, lozenges, pills, solutions, suspensions, emulsions, syrups, powders, fine granules, pellets, elixirs or injections.
The pharmaceutical composition according to the present invention, preferably, the acceptable carrier or excipient is selected from at least one of a solvent, a diluent, a dispersant, a suspending agent, a surfactant, an isotonic agent, a thickener, an emulsifier, a preservative, a binder, a lubricant, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetener, an ion exchanger, a mold release agent, a coating agent, a flavoring agent, and an antioxidant.
As used herein, "pharmaceutically acceptable" means having no substantial toxic effect when used in the usual dosage amounts, and thus being approved by the government or equivalent international organization or approved for use in animals, more particularly in humans, or registered in the pharmacopoeia.
As used herein, the term "pharmaceutical composition" has its ordinary meaning. The pharmaceutical composition of the present invention can be prepared by obtaining the active ingredients of the raw materials of the pharmaceutical composition of the present invention by extraction, separation and purification means commonly used in pharmaceutical manufacturing, optionally mixing with one or more pharmaceutically acceptable carriers or excipients, and then forming a desired dosage form, using conventional techniques in the pharmaceutical field, particularly in the field of formulation. The pharmaceutical composition according to the present invention is a pharmaceutical formulation which may be suitable for oral, parenteral or topical, topical administration. The pharmaceutical composition can be prepared into various forms such as tablets, powder, granules, capsules, oral liquid and the like. The medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.
Dosage forms for oral administration may include, for example, tablets, pills, hard or soft capsules, solutions, suspensions, emulsions, syrups, powders, fine granules, pellets, elixirs and the like, without limitation. In addition to the active ingredient, these preparations may contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silica, talc, stearic acid or its magnesium salt, calcium salt, and polyethylene glycol). Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone. If necessary, it may further contain pharmaceutically acceptable additives such as disintegrating agents (e.g., starch, agar, alginic acid or sodium salt thereof), absorbents, coloring agents, flavoring agents, sweetening agents, and the like. Tablets may be prepared according to conventional mixing, granulating or coating methods.
Dosage forms for parenteral administration may include, for example, injections, drops for medical use, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches and the like, without being limited thereto.
The pharmaceutical compositions according to the present disclosure may be administered orally or parenterally, for example rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, or subcutaneously.
As used herein, the term "about" generally refers to a range of error permitted in the art, such as ± 10%, e.g., ± 5%, e.g., ± 2%.
Example 1 preparation of eclipta alba extract
20kg of Ecliptae herba (purchased from san shan Yao Co., Ltd., Anguo, lot number: mhl0001, origin: Hebei) was extracted with 100L 70% ethanol under reflux for 2 hr for 3 times. Distilling at 45 deg.C under reduced pressure, concentrating until no alcohol exists, and estimating the mass of the extract to be about 1 kg.
Dissolving the crude extract in 10L water, and extracting with petroleum ether and ethyl acetate for 3 times to obtain 255g (named as EPA-P) of petroleum ether extract, 150g (named as EPA-E) of ethyl acetate extract and 600g (named as EPA-W) of water layer extract.
Mixing the above extracts, and drying to obtain Ecliptae herba extract. Drying may be carried out in a manner commonly used in the art, and the present invention is not limited thereto.
Example 2 separation and identification of chemical Components in Ecliptae herba extract
255g of petroleum ether extraction part extract (named EPA-P), 150g of ethyl acetate extraction part extract (named EPA-E) and 600g of water layer extract (named EPA-W) are obtained by the method of example 1; wherein 600g of water layer (EPA-W) is adsorbed and separated by macroporous resin D101, and is eluted by pure water, 30% ethanol, 70% ethanol and pure ethanol, and 30% ethanol eluate is EPA-W-D3.
The EPA-W-D3 fraction (25 g) is mixed with silica gel (50 g), and the mixture is loaded on a silica gel column (250 g) and subjected to silica gel column chromatography (600 ml). Performing gradient elution with dichloromethane-methanol to obtain 16 fractions; TLC (thin layer chromatography) detection was followed by pooling, and pooled fractions 1-4 were recorded as: EPA-W-D3-1; the combined fractions 5-6 were recorded as: EPA-W-D3-5; the combined fractions 7-8 were recorded as: EPA-W-D3-7; the combined fractions 9-10 were recorded as: EPA-W-D3-9; the combined fractions 11-13 were recorded as: EPA-W-D3-11; fraction 14 was recorded as: EPA-W-D3-14. Wherein EPA-W-D3-11 was prepared by preparative liquid chromatography to give compounds 6(348mg) and 7(83 mg).
150g of ethyl acetate fraction (EPA-E) was stirred with 200g of silica gel and loaded on a column of 1.6Kg of silica gel, and silica gel column chromatography was carried out in a column volume of 4L. The elution was performed with a gradient of dichloromethane-methanol (dichloromethane-methanol volume ratio) of 1:0, 100:1, 50:1, 25:1, 12.5:1, 7:1, 4:1, 1:1, 0:1, two column volumes were collected for each gradient. The fractions were checked by TLC analysis for a total of 15 fractions.
The 8 th and 9 th fractions were combined and designated EPA-E8, and separated by Sephadex LH-20 gel column chromatography (methanol elution) and preparative liquid chromatography to give compound 14(32 mg). The 11 th fraction is named EPA-E11, and is eluted by Sephadex LH-20 gel and methanol, and subjected to Flash medium pressure chromatography (ODS-C)18) Gradient eluting with 40% methanol → 100% methanol, separating by preparative liquid chromatography, eluting with methanol water of series proportion, detecting each eluate fraction by TLC and HPLC, mixing the same fractions to obtain compound 10(130mg), Compound 17(1290 mg). The 13 th, 14 th and 15 th fractions were combined and designated EPA-E13, and separated by SephadexLH-20 gel preparative liquid chromatography to give Compound 1(95mg), Compound 4(25mg) and Compound 5(210 mg). Combining the 5 th and 6 th fractions, named EPA-E5, and performing silica gel column chromatography, Sephadex LH-20 gel, and Flash medium pressure chromatography (ODS-C)18) And preparative liquid chromatography gave compound 2(49mg), compound 11(114mg), and compound 16(43 mg).
255g of petroleum ether fraction (EPA-P) are stirred with 500g of silica gel, and the mixture is subjected to silica gel column chromatography and gradient elution by using petroleum ether-ethyl acetate (volume ratio is 1:0 → 0: 1) to obtain 17 fractions. Analyzing and detecting each component by TLC, combining to obtain 8 components, and naming the fraction 1 as EPA-P-1; fraction 2-4 is EPA-P-2; fraction 5-7 is EPA-P-5; fraction 8-10 is EPA-P-8; fraction 11-14 is EPA-P-11; fraction 15 is EPA-P-15; fraction 16 was EPA-P-16; fraction 17 was EPA-P-17.
The component EPA-P-1 was prepared by preparative liquid chromatography to give compound 8(88 mg). The EPA-P-5 component is processed by Flash medium pressure chromatography (ODS-C)18) EPA-P-5-F6 was isolated and prepared by preparative liquid chromatography to give compounds 9(98mg) and 13(45 mg). The EPA-P-8 component is subjected to Flash medium pressure chromatography (ODS-C)18) EPA-P-8-F3 and EPA-P-8-F7 were isolated and prepared by preparative liquid chromatography to give Compound 3(43 mg). The EPA-P-11 fraction was subjected to repeated Sephadex LH-20 gel separation, preparative liquid chromatography and recrystallization to give compounds 15(61mg) and 12(149 mg). The percentages in this example are volume percentages; the fraction designations are all named in the order of elution.
By passing1H、13C-NMR is carried out, and the structures of the compounds 1-17 are determined; the structures of identified compounds 1-17 are shown in table 1.
The model number of the NMR instrument is: BRUKER AVANCE iii 500 superconducting nuclear magnetic resonance spectrometer (TMS as internal standard, BRUKER, switzerland);
specifically, the method comprises the following steps:
TABLE 1 list of compounds
Example 3 Helianoside IV inhibits differentiation of BMMSCs into adipocytes
Mesenchymal Stem Cells (MSCs) are multipotent differentiated stem cells and are a common source of adipocytes and osteoblasts. The canonical Wnt/beta-catenin signal pathway plays an important role in the differentiation process of the MSCs, and one of the downstream key signal factors, namely PPAR gamma, is responsible for promoting the differentiation of the MSCs to adipocytes. The inventor finds that the ecliptin IV can inhibit the differentiation of the MSCs to the adipocytes by inhibiting the expression of the PPAR gamma protein.
Subject:
female C57BL/6J mice at 6-8 weeks, SPF grade, purchased from beijing wakakon biotechnology, inc, license number: SCXK (Jing) 2014-. The animals are raised in the animal house of Tianjin Chinese medicine university at room temperature of 22 + -2 deg.C and relative humidity of 58-65%.
Main reagents and experimental materials:
α mem (thermo); fetal bovine serum (FBS, Thermo); double antibody (penicillin, streptomycin) (Hyclone); oil red O powder (sigma); tropaellaroside IV (purified by the method of example 2 of the present application);
the main apparatus is as follows: CO 22Incubator (formula 3111, THERMO usa); laser confocal microscopy (LSM, zeiss, germany); a bench top high speed centrifuge (ALLEGRA X-15R, Beckman, USA); a low temperature refrigerator at-40 ℃ (MDF-U5410, Sanyo corporation, Japan); one in ten thousand balance (BS110S, Sartorius, germany).
1. In vitro culture and induced differentiation of mouse mesenchymal stem cells
The experimental method comprises the following steps:
primary culture of BMMSCs
1) C57/BL6J female mice are killed by breaking the neck and soaked in 75% alcohol after 6-8 weeks;
2) separating the long bones on both lower sides of the mouse in D-PBS by using sterile scissors and tweezers;
3) separating the skin muscle tissue of the long bone in D-PBS using sterile scissors forceps;
4) the long bone was placed in a full culture of α MEM + 10% FBS + 1% double antibody, and the bone marrow was blown out with a 1ml syringe, followed by filtering the single cells with a 70 μm cell sieve;
5) at 2X 106cells/mL in 25cm2In a culture bottle;
6) after 72 hours, the cells were passaged by changing the medium 2-3 times per week until the cells were confluent at 90%.
Passage of BMMSCs
1) Cells were washed 2 times with D-PBS;
2) adding 1mL of trypsin-0.25% EDTA, placing in an incubator at 37 ℃ for 2min, and repeatedly blowing and beating cells by using a 1mL pipette;
3) adding 1mL of full culture medium to terminate digestion, blowing the cells by using a 1mL liquid shifter again until 90% of the cells are detached from the wall, and collecting cell suspension to a 15mL centrifuge tube;
4) centrifuging at 20 deg.C for 5min at 300 g;
5) passages were performed at 1:2 or 1: 3.
Mouse BMMSCs flow identification
1) Digesting BMMSCs third-generation cells, and centrifuging at 300g and 4 deg.C for 5 min.
2) Add 1mL of precooled PBS and adjust cells to 3 x 107cells/mL。
3) And (3) incubating the fluorescence labeled antibody, and respectively setting a negative cell group (without the antibody) and an antibody group: respectively adding the fluorescence labeled monoclonal antibodies, uniformly mixing by vortex, and incubating for 30min on ice in a dark place;
4) adding 1mL PBS, mixing, centrifuging at 1400rpm and 4 deg.C for 5 min;
5) the supernatant was discarded, 500. mu.L of 1% paraformaldehyde was added to resuspend the cells, and the cells were loaded within 24 h.
Mouse BMMSCs induction culture
1) Digesting BMMSCs third generation cells, adjusting cell number to 1 × 105cells/mL；
2) Planting 100 μ L/well in 96-well plate, and setting blank control group and fat induction group respectively;
3) when the cell fusion rate is 95%, adding adipocyte inducer (MSCgo) into the adipocyte inducing groupTMAdoptive Differentiation Medium, BI, 053301B, performed as described), BMMSCs complete Medium (α MEM with 5% FBS) was added to the blank control group;
4) changing the liquid every 3 days, and not carrying out passage during the induction period;
5) oil red O staining was performed after 10 days (mesenchymal stem cell adipogenic differentiation staining kit, BI, C37a00150, operating according to the instructions).
The experimental results are as follows:
growth and morphological changes of primary BMMSCs in mice
Under an inverted phase contrast microscope, after 3 days of primary BMMSCs cell culture, liquid is changed for the first time, so that adherent growth of cells is seen, and the cells are circular or long fusiform (figure 1A); after 5 days, the adherent cells gradually increased, and a large number of cell colonies appeared, exhibiting vortex-like or cartwheel-like growth (fig. 1, panel B); after 7 days of culture, the cells covered the area of the bottom of the flask to about 80% (FIG. 1, panel C).
Identification of BMMSCs in normal mice
At present, no established molecular marker specifically expressed is accepted as a gold standard for identification of MSCs, but MSCs can still be identified to some extent by relying on co-expression of multiple molecules. The MSCs derived from the mouse bone marrow mainly express stem cell molecules such as sca-1, CD44, CD105, CD140a and CD 29; does not express hematopoietic cell molecular markers, such as CD45 and CD 34. The expression rate of the surface molecules of mouse BMMSCs separated by the experiment is as follows: sca-1 was 86%, CD29 was 99%, CD140a was 88.7%, and CD90 was 63.7%. Whereas CD45 was 14% and CD34 was 1%, the streaming results are shown in FIG. 2.
Mouse BMMSCs in vitro induction differentiation culture
Mouse BMMSCs after 10 days of adipocyte inducer treatment were stained with oil-Red-O, and the results are shown in FIG. 3, where the head cut indicates adipocytes stained with oil-Red-O.
Tropaeolumoside IV inhibits the formation of fat cells in the induction process of mouse BMMSCs
Respectively setting BMMSCs of a blank control group and mice of a fat induction group (induction group), wherein the induction group cells comprise only adding an adipocyte inducer, simultaneously adding the adipocyte inducer and having a final concentration of 10-5M Ecliptae glycoside IV (DMSO dissolved, wherein the final concentration of DMSO is not more than 1 ‰, and the culture medium of other groups of cells contains equal amount of DMSO), and after 10 days of culture, the result of oil red O staining is shown in FIG. 4.
As can be seen, oil O staining (arrow) after induction, whereas 10 was added-5After M drought lotus glycoside IV, the cells are not stained by oil red O, which shows that drought lotus glycoside IV can obviously inhibit BMMSCs from differentiating to fat cells.
2. Tropaeolumoside IV inhibits PPAR gamma protein expression of mouse BMMSCs cells
The inventor finds in research that the mouse BMMSCs have the tendency that the PPAR gamma protein expression in cells is increased after the adipocyte inducer is added into the cells, and the adipocyte inducer is added by 10 percent compared with the adipocyte inducer-5M-Helianoside IV inhibits protein expression of PPAR gamma (P)<0.05)。
Blank control cells, induction cells and different combinations of RIPA: mixing protease inhibitor PMSF according to a ratio of 100:1 for extracting holoprotein, carrying out SDS-PAGE gel electrophoresis on the extracted holoprotein, carrying out membrane transfer, incubating and developing by a PPAR gamma primary antibody (1:1000, Affinity Biosciences, AF6284) and a horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:10000, China fir gold bridge, ZB-2301), and taking beta-actin protein as an internal reference.
As shown in fig. 5, it can be seen that the expression level of PPAR γ protein decreased with the increase of the concentration of rosellin iv treatment (n is 3, P is less than 0.05, compared with the non-drug-added group).
The experimental results prove that the ecliptin IV can inhibit the expression of PPAR gamma protein so as to inhibit the differentiation of BMMSCs to fat cells.
Example 4 ethanol extract of eclipta alba inhibits the formation of adipocytes
The differentiation direction of the MSCs is influenced by various factors, such as exogenous chemical drugs including dexamethasone, xanthine, indomethacin and the like, so that the MSCs are differentiated to fat cells; aging causes increase of ROS and oxygen pressure in the body, so that differentiation capability of MSCs to fat cells is enhanced. In SAMP6 mice, a number of MSCs differentiated into adipocytes. This example isolates MSCs in the bone marrow of SAMP6 mice three months after administration of ethanol extract of eclipta alba, and detects PPAR γ protein expression in cells; the MSCs in the bone marrow of the SAMP6 mouse were cultured for adipocyte induction, and adipocyte formation was observed.
1. Laboratory animal
The 4-month-old male rapid aging mouse P6(SAMP6) and the 4-month-old male anti-rapid aging mouse R1(SAMR1), SPF grade, were provided by the department of medicine of Beijing university (department of Experimental zoology), and the animals were housed in the institute of Radiology of Tianjin, at room temperature of 22. + -. 2 ℃ and relative humidity of 58-65%.
2. Test drug
2.1 drug dose
2.2 modes of administration
The extract of eclipta alba prepared in example 1 was dissolved in water to prepare an extract solution having a concentration of 48mg/mL for administration in an amount of 0.1mL/10g based on the body weight of the mouse.
The preparation is administered by regular drenching every day, and the control group and the model group are administered with purified water of the same volume for 12 weeks.
3. Animal tissue treatment
3.1 SAMP6 mouse bone marrow tissue extraction method
1) Mice were sacrificed by cervical dislocation;
2) the hind limbs of the mice were cut off, taking care to maintain the integrity of the bones;
3) the skin musculature on the leg bones was separated with a surgical instrument and the right femur was placed in a 1.5mL EP tube and stored at-20 ℃ and the left femur was placed in an EP tube containing 4% paraformaldehyde and stored at 4 ℃.
4) The left cervical bone and the remaining bone marrow were each blown with a 1mL syringe until the bone became white, and the bone marrow tissue was collected in a 15mL centrifuge tube for bone marrow protein extraction.
3.2 SAMP6 Total protein extraction from mouse bone marrow
1) Centrifuging at 1400rpm for 5 min;
2) discarding the supernatant, adding 1mL PBS, mixing the cells uniformly, adding 4mL erythrocyte lysate, oscillating, and incubating on ice for 10 min;
3) centrifuging at 1400rpm for 10 min;
4) the supernatant was aspirated to completion as much as possible, 150. mu.L of RIPA was added, transferred to a precooled 1.5mL EP tube, and lysed on ice for 30 min;
5) centrifuging: 14000RPM, 15min, 4C;
6) collecting supernatant, and quantifying BCA to obtain total bone marrow protein.
The expression level of PPAR γ in total bone marrow was measured by western blotting in the same manner as in example 2, and the results are shown in fig. 6. It can be seen that the amount of PPAR γ in the bone marrow of the administered group was significantly reduced (n ═ 8, P <0.05, compared to the model group).
3.3 mouse BMMSCs Induction
BMMSCs of mice of a control group, a model group and an administration group are respectively obtained by the same method as example 2, and oil red O staining observation is carried out after 10 days of induction by adding an adipocyte inducer, and the result is shown in figure 7.
From the above results, it can be seen that ecliptin iv in the eclipta alba extract can reduce the expression of PPAR γ protein associated with adipocyte formation in mouse BMMSCs cultured in vitro, and can inhibit the differentiation of BMMSCs into adipocytes; furthermore, the eclipta alba extract can reduce the content of PPAR gamma protein in the bone marrow of a mouse and inhibit the differentiation of BMMSCs to fat cells, and on the basis, the ecliptin IV and the eclipta alba extract containing the ecliptin IV can be used for preparing a pharmaceutical preparation for inhibiting the differentiation of MSCs to fat cells and further preparing an anti-obesity pharmaceutical composition.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. Application of eclipta alba extract in preparing a pharmaceutical preparation for inhibiting differentiation of MSCs (mesenchymal stem cells) to adipocytes, wherein the eclipta alba extract contains ecliptin IV.
2. Application of eclipta alba extract in preparation of pharmaceutical preparations for inhibiting expression of PPAR gamma protein, wherein the eclipta alba extract contains ecliptin IV.
3. Application of eclipta alba extract in preparing pharmaceutical preparations for resisting obesity, wherein the eclipta alba extract contains ecliptin IV.
4. The use according to any one of claims 1 to 3, wherein the Ecliptae herba extract is an alcohol extract of Ecliptae herba.
5. The use of claim 4, wherein the eclipta alba extract is prepared by the following method:
1) extracting Ecliptae herba with ethanol water solution; concentrating to obtain crude extract;
2) dissolving the obtained crude extract in water, and extracting with petroleum ether and ethyl acetate to obtain petroleum ether extract, ethyl acetate extract and water layer extract;
3) combining the extracts obtained in the step 2) and drying to obtain the finished product.
6. An anti-obesity pharmaceutical composition comprising drought lotus glycoside IV and pharmaceutically acceptable carrier or excipient.
7. An anti-obesity pharmaceutical composition comprising an extract of eclipta alba containing eclipta alba glycoside IV and a pharmaceutically acceptable carrier or excipient.
8. The pharmaceutical composition according to claim 6 or 7, wherein the rosemariside IV is used as the only active ingredient of the pharmaceutical composition.
9. The pharmaceutical composition of any one of claims 6-8, wherein the pharmaceutical composition is in the form of one of a tablet, a capsule, a powder, a granule, a lozenge, a pill, a solution, a suspension, an emulsion, a syrup, a powder, a fine granule, a pellet, an elixir, or an injection.
10. The pharmaceutical composition according to any one of claims 6 to 8, wherein the acceptable carrier or excipient is selected from at least one of solvents, diluents, dispersing agents, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, preservatives, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavors, sweeteners, ion exchangers, mold release agents, coating agents, flavors, and antioxidants.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105530944A (en) * | 2013-07-03 | 2016-04-27 | 庆熙大学校产学协力团 | Pharmaceutical composition for preventing or treating cognitive impairment or concentration impairment disorders, comprising eclalbasaponin or derivative thereof |
| CN105748546A (en) * | 2015-05-14 | 2016-07-13 | 大连医科大学 | Application of Herba Ecliptae extract to preparation of anti-osteoporosis multi-target medicines or healthcare products |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105530944A (en) * | 2013-07-03 | 2016-04-27 | 庆熙大学校产学协力团 | Pharmaceutical composition for preventing or treating cognitive impairment or concentration impairment disorders, comprising eclalbasaponin or derivative thereof |
| CN105748546A (en) * | 2015-05-14 | 2016-07-13 | 大连医科大学 | Application of Herba Ecliptae extract to preparation of anti-osteoporosis multi-target medicines or healthcare products |
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| ABHISHEK GUPTA 等: "Ethyl acetate fraction of Eclipta alba: a potential phytopharmaceutical targeting adipocyte differentiation", 《BIOMEDICINE & PHARMACOTHERAPY》 * |
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