CN102048851B - Loquat leaf cell extract for protecting liver and/or reducing fat and preparation method and application thereof - Google Patents
Loquat leaf cell extract for protecting liver and/or reducing fat and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a loquat leaf cell extract for protecting liver and/or reducing fat and a preparation method and application thereof. The extract and the medicinal composition thereof contain a compound of a formula (1); and the loquat leaf cell extract can be used for manufacturing medicaments.
Description
Technical field
The invention relates to the extract of Folium Eriobotryae cell in the application of the liver protecting and/or reducing fat, and the preparation method of this extract.
Background technology
The formal name used at school of Folium Eriobotryae is Eriobotrya japonica, and the ancient reed Fructus Citri tangerinae by name of Chinese has another name called golden ball or reed branch, and it is a genus of the Maloideae in the Rosaceae, is evergreen dungarunga.Studies show that at present Folium Eriobotryae has the curative effect that reduces body fat, anti-inflammatory, antitumor, blood sugar lowering and improve abnormal metabolism, related description can be with reference to No. the 2006104182nd, Japanese Patent Publication, its content and in herein for your guidance.
In addition, document is confirmed already, and the triterpenic acid in the Folium Eriobotryae (triterpene acid) composition has anti-inflammatory and antineoplastic active (please refer to Biol.Pharm.Bull.28 (10), 1995-1999 (2005)); In US Patent No. 6,908, among the 632B1, the Corosolic acid (corosolic acid is a kind of triterpenic acid) that discloses in the Folium Eriobotryae extract has the function of regulating and controlling blood sugar.By above-mentioned document as can be known, the triterpenic acid composition in the Folium Eriobotryae all has curative effect for numerous disease.
Through finding, except Folium Eriobotryae, triterpenic acid (such as termentic acid (tormentic acid), hyptadienic acid (hyptadienic acid), Crataegolic acid (maslinic acid) and Corosolic acid etc.) also can obtain in other plant, and other biological activity of tool.For instance, in US Patent No. 6,784, among the 206B2, the Corosolic acid in teaching Flos Caryophylli Lagerstroemia indica L. (Lagerstroemia speciosa) or Ba Naba (Banaba) leaf can be applicable to fat-reducing and regulating and controlling blood sugar; Termentic acid composition in the Japan visitor Ochnaceae (Vochysia divergens) can in order to slow down nerve or inflammatory pain (please refer to European Journal of Pharmacology, 453 (2002), 203-208); In addition, Crataegolic acid then has hypoglycemic effectiveness (please refer to Biol.Pharm.Bull.30 (11) 2075-2078 (2007)).
Although triterpenic acid can extract in different plants and get, but its content in natural plants is extremely low, if want in natural plants, to make triterpenic acid, with in response to the market demand, then must plant a large amount of plants, and need complicated purification step and use a large amount of solvents, activated carbon and purification resin, thereby increase manufacturing cost.
Industry is is actively researched and developed the method for preparing triterpenic acid, for example utilizes the mode (please refer to Phytochemistry, 59,315-323 (2002)) of tissue culture's Folium Eriobotryae cell, but its effect that promotes triterpenic acid content is limited.In WO 2007/004827A1, a kind of method for preparing Corosolic acid of teaching then, it is the mode of cultivating Folium Eriobotryae or Flos Caryophylli Lagerstroemia indica L. leaf cell with the two-stage, obtains the Corosolic acid of high-load.Yet the method must be screened first the cell strain with high production capacity, and adds derivant in culture fluid, and must be through the incubation step of two-stage, not only technique very complex and manufacturing cost are high, and only can promote the content of Corosolic acid, and are unprofitable to the lifting of other triterpenic acid content.Therefore, a kind of conveniently and the method for producing in large quantities triterpenic acid in the urgent need to.
The present invention be directed to the research that the demand is done, the present application people adds the mode of Folium Eriobotryae cell raffinate by in the culture fluid of Folium Eriobotryae cell, can significantly promote the content of the triterpenic acid of cultivating gained Folium Eriobotryae cell.In addition, the present application people finds that in addition the extract of Folium Eriobotryae cell has liver-protective effect, and the termentic acid in the extract and hyptadienic acid active component also can be used for reducing fat.
Summary of the invention
The first purpose of the present invention is to provide a kind of extract of the Folium Eriobotryae cell for the protection of liver, and it comprises following formula (1) chemical compound:
Wherein, R
1And R
2Independently to be separately hydrogen or methyl; R
3And R
4Independently to be separately hydrogen, methyl or hydroxyl; R
5To be connected with the carbon atom of No. 1 and No. 2 institute's marker location and to form the have following formula pentacyclic structure of hexatomic ring or following formula (3) of (2):
The second purpose of the present invention is to provide a kind of method for preparing the extract of above-mentioned Folium Eriobotryae cell.
The 3rd purpose of the present invention is to provide a kind of medical composition, comprises the above-mentioned extract of effective dose.
The 4th purpose of the present invention is to provide a kind of extract of above-mentioned Folium Eriobotryae cell that uses in the application of making liver-protective medicament.
It is a kind of for reducing fat and/or liver-protective medical composition that the 5th purpose of the present invention is to provide, and it comprises the chemical compound with following formula (4) and/or formula (5) of effective dose:
The 6th purpose of the present invention is to provide a kind of extract of the Folium Eriobotryae cell for reducing fat, and it comprises the chemical compound with above-mentioned formula (4) and/or formula (5).
The 7th purpose of the present invention is to provide a kind of method for preparing the chemical compound with above-mentioned formula (4) and/or formula (5).
The chemical compound that the 8th purpose of the present invention is to provide a kind of use to have above-mentioned formula (4) and/or formula (5) or its medical acceptable salt or ester are in the application of making reducing fat and/or liver-protective medicament.
The 9th purpose of the present invention is to provide a kind of extract that uses the Folium Eriobotryae cell in the application of the medicament of making reducing fat, and wherein this extract comprises the chemical compound with above-mentioned formula (4) and/or formula (5).
Detailed technology of the present invention and better enforcement aspect will be described in the following content, know usually that for field tool under the present invention the knowledgeable understands feature of the present invention according to this.
Description of drawings
Figure 1 shows that the flow chart of the extract of the preparation Folium Eriobotryae cell in one embodiment of the invention;
Figure 2 shows that the extract of Folium Eriobotryae cell is thrown the H.E. colored graph that gives its hepatic tissue cell behind the mice of Carbon Tetrachloride Induced hepatic fibrosis;
Figure 3 shows that the extract of Folium Eriobotryae cell is thrown the Sirius Red colored graph that gives its hepatic tissue cell behind the mice of Carbon Tetrachloride Induced hepatic fibrosis;
Figure 4 shows that the immunostaining figure that the extract of Folium Eriobotryae cell is thrown the CD14 that gives its no cell in storehouse behind the mice of Carbon Tetrachloride Induced hepatic fibrosis;
Figure 5 shows that the extract of Folium Eriobotryae cell is thrown the microscope figure that gives its adipose cell behind the mice; And
Figure 6 shows that Folium Eriobotryae cell extraction thing, termentic acid or hyptadienic acid thrown and give that its behind the adipose cell is divided into the colored graph of adipose cell before the 3T3-L1.
The specific embodiment
The present application people is carrying out finding that the extract of Folium Eriobotryae cell has liver-protective effect behind the sv cell experiment and intravital zoopery many times, and its active component is following formula (1) chemical compound:
Wherein, R1 and R2 independently are hydrogen or methyl separately; R3 and R4 independently are hydrogen, methyl or hydroxyl separately; R5 is connected with the carbon atom of No. 1 and No. 2 institute's marker location and forms the have following formula pentacyclic structure of hexatomic ring or following formula (3) of (2):
Particular words it, the extract of Folium Eriobotryae cell of the present invention is to comprise one or more following triterpenic acid chemical compounds:
Wherein, formula (4) to the chemical compound of formula (7) is respectively hyptadienic acid, termentic acid, Crataegolic acid and Corosolic acid.
More particular words it, the extract of Folium Eriobotryae cell of the present invention is the chemical compound that comprises formula (4) and/or formula (5).
In herein, " the liver protecting " word is to contain protection by the caused hepatic injury of chemical substance.For example, extract of the present invention can be used for protection by as the caused hepatic injury of chemical substance such as carbon tetrachloride, ethanol and thiacetamides (thioacetamide) or inhibition by the caused hepatic fibrosis of hepatic injury.
The present invention also provides a kind of method for preparing the extract of Folium Eriobotryae cell, and it comprises: a) cultivate a Folium Eriobotryae cell in a culture fluid, wherein this culture fluid comprises a Folium Eriobotryae cell raffinate and this Folium Eriobotryae cell; And b) the Folium Eriobotryae cell a) cultivated of extraction step.
Wherein, the Folium Eriobotryae cell of step in a) is to obtain through following steps: 1) get one and have the blade of the Loquat Seeds Seedling of wound, and treat that wound grows callus (callus tissue) cell; And 2) taking out this callus cell is also cultivated.In abovementioned steps 1) in, can make in any way the blade of Loquat Seeds Seedling produce wound, for example, can cut the mode of drawing, tearing or shearing, obtain the Loquat Seeds seedling leaf with wound, better then is to utilize the mode of drawing of cutting; Afterwards, can utilize the known induction mode of any technical staff, make the wound of the blade of Loquat Seeds Seedling grow callus, for example utilize a solid medium, and add suitable nitrogenous source, carbon source, plant growth regulator, cell division is regulated hormone (6-benzyladenine (BA) for example, fragmentation element (kinetin), or zeatin (zeatin) etc.) or cell cycle regulation hormone (α-naphthaleneacetic acid (NAA) for example, 2,4-dichlorphenoxyacetic acid (2,4-D) or diindyl Yin acetic acid (IAA)) etc., cultivate about 1 month time, this can be referring to Avanzados et al., Plant Cell Culture Protocols, Methods in Molecular Biology, vol.318, p.59-70, document content and in herein for your guidance.
General knowledge knows that callus cell is the cell mass by the plant tissue differentiation, has to be regenerated as the potentiality that have the new plant of identical character and inherited character with original plant.If callus cell is incubated on the suitable culture medium, then can form root or bud by adventitious organogenesis, through further cultivating, can grow up to new Loquat seedling eventually again; Cultivate callus cell if utilize the cell suspension cultures mode, then it can divide how new Folium Eriobotryae cell, this can be referring to Ma et al., Leaf callus induction and suspension cultureestablishment in lychee (Litchi chinensis Sonn.) cv.Huaizhi, Acta PhysiologiaePlantarum, vol.31, p.401-405, document content and in herein for your guidance.Implementing in the aspect in the part of the inventive method, namely is to utilize the cell suspension cultures mode to make the Folium Eriobotryae cell.
Being used for step Folium Eriobotryae cell raffinate a) of the inventive method, is to make through following steps: I) get a Folium Eriobotryae cell, and working concentration is that about 80 volume % are to this Folium Eriobotryae cell of alcohol extraction of about 100 volume %; II) product through extraction concentration step I) is to obtain one first concentrate; III) working concentration is extremely these first concentrate of dissolve with methanol of about 60 volume % of about 40 volume %, collects one first and does not dissolve part; And IV) working concentration is about 70 volume % this first does not dissolve part to the dissolve with methanol of about 95 volume %, collects one second and does not dissolve part, to make Folium Eriobotryae cell raffinate.
Wherein, the Folium Eriobotryae cell above-mentioned steps I) also can make by following steps: 1) get one and have the blade of the Loquat Seeds Seedling of wound, and treat that wound grows callus cell; And 2) taking out this callus cell is also cultivated.Correlative detail as mentioned in about described in the incubation step of the Folium Eriobotryae cell of step in a).
Moreover, step I) concentration of alcohol be preferably about 90 volume % to about 98 volume %; Step II I) methanol concentration is preferably about 45 volume % to about 55 volume %; And methanol concentration step IV) is preferably about 80 volume % to about 90 volume %.Implement in the aspect in the part of the inventive method, when in Step II I) with step IV) in when distinguishing the methanol of the about 50 volume % of working concentration and about 85 volume %, can obtain the Folium Eriobotryae cell raffinate of satisfactory especially concentration; In other words, through the prepared Folium Eriobotryae cell of the step of this specific part aspect raffinate, it does not comprise in fact about 50 volume % methanol solubilized part and about 85 volume % methanol solubilized parts.
In the total amount of culture fluid, the content of the Folium Eriobotryae cell raffinate of step in a) for about 0.5ppm to about 20ppm, be preferably extremely about 5ppm of about 1ppm, the best is about 1ppm.If the content of Folium Eriobotryae cell raffinate is lower than 0.5ppm, then stimulate/induce the triterpenic acid produce an effect limited; And if the content of Folium Eriobotryae cell raffinate is higher than 20ppm, then can suppresses the growth of Folium Eriobotryae cell, thereby affect the productive rate of triterpenic acid.
According to the method for preparing Folium Eriobotryae cell extraction thing of the present invention, after completing steps cultivation program a), then carry out step b) extraction procedures.Furtherly, step b) comprise in fact following steps: b1) working concentration is the Folium Eriobotryae cell that about 80 volume % a) cultivate to the alcohol extraction step of about 100 volume %; B2) product through extraction concentration step b1) is to obtain one second concentrate; B3) working concentration is extremely these second concentrate of dissolve with methanol of about 60 volume % of about 40 volume %, collects one the 3rd and does not dissolve part; B4) to be about 70 volume % dissolve part and is filtered collection filtrate to the dissolve with methanol the 3rd of about 95 volume % working concentration; And b5) optionally concentrates this filtrate.
Implement in the aspect above-mentioned steps b1 in the part of the inventive method) concentration of alcohol be preferably about 90 volume % to about 98 volume %; Step b3) methanol concentration is preferably about 45 volume % to about 55 volume %; And methanol concentration step b4) is preferably about 80 volume % to about 90 volume %.In addition, can optionally repeat repeatedly step b) in extraction and/or dissolving step, the effective ingredient in the Folium Eriobotryae cell is separated, and as far as possible with the extraction of whole effective ingredient and go out with invalid components.
Generally speaking, according to the application form of the extract of Folium Eriobotryae cell, can be optionally further with step b) the extract bone dry of the Folium Eriobotryae cell that obtains, conveniently to transport, to store or to utilize.For example, can utilize a drying steps (for example concentrating under reduced pressure and/or pass into gas) to remove the organic solvent that residues in the operation in the extract.Implementing in the aspect in of the present invention one, is to utilize concentrating under reduced pressure to carry out drying, with the extract of the Folium Eriobotryae cell that obtains white powder.
Implement in the aspect in of the present invention one, the step b that comprises following program) to obtain the extract of Folium Eriobotryae cell of the present invention: use the Folium Eriobotryae cell of about 95 volume % alcohol extractions through cultivating, with filter paper filtering, collect filtrate and concentrating under reduced pressure, to obtain about 50 these concentrate of volume % dissolve with methanol of a concentrate → use, with filter paper filtering, collect not dissolving part → about 85 volume % dissolve with methanol of use and do not dissolve part, with filter paper filtering, collection filtrate → make filtrate carry out concentrating under reduced pressure obtains the extract of Folium Eriobotryae cell.Aforesaid operations is as shown in Figure 1.Implement the extract of the prepared Folium Eriobotryae cell of aspect by this, it does not comprise in fact about 50 volume % methanol solubilized compositions and about 85 volume % methanol can not solvent components, and contains the triterpenic acid active component of satisfactory especially content.
Specifically, method of the present invention is by adding Folium Eriobotryae cell raffinate to culture fluid, stimulate/induce the Folium Eriobotryae cell to produce the triterpenic acid of high-load, needn't cultivating or screen the cell strain with high production capacity by the two-stage.Compared to prior art, method of the present invention can be economical and be made in a large number easily triterpenic acid.Generally speaking, with the dry weight basis of the extract of Folium Eriobotryae cell, the content of triterpenic acid composition is at least about 60 % by weight, and is better for 75 % by weight, better for 80 % by weight, best at least about 85 % by weight.
The present invention is also about a kind of medical composition for the protection of liver, and it comprises the extract of the Folium Eriobotryae cell of effective dose, and this extract can be obtained by the inventive method.In the medical composition for the protection of liver of the present invention, the content of the extract of Folium Eriobotryae cell is not absolute key point, as long as can provide institute's wish liver-protective benefit.
Can any suitable mode use according to medical composition of the present invention, for example, but not as limit, can be oral, subcutaneous or the dosing mode such as intravenous use.In addition, medical composition of the present invention can use separately or with medical adjuvant, and in fact can be used in veterinary and the mankind pharmaceutically.
In addition, the present invention again about a kind of extract that uses the Folium Eriobotryae cell or by the extract of the prepared Folium Eriobotryae cell of the inventive method in the application of making medicament, it can be in order to prepare the medicament for the protection of liver of any suitable form of tool.For example; when the extract that uses the Folium Eriobotryae cell during in the medicament made for the protection of liver; usually for each person every day consumption be per kilogram of body weight about 25 to the chemical compound of about 90 milligrams formula (4) and/or formula (5), be preferably per kilogram of body weight about 30 to the chemical compound of about 75 milligrams formula (4) and/or formula (5).
Wherein, be suitable for the medicament of oral administration medicine supplying in the preparation tool, can be by the extract of Folium Eriobotryae cell of the present invention be mixed with the adjuvant that is fit to this project and can sharp affects the extract activity, such as: solvent, oil-based solvent, diluent, tranquilizer, absorption delay agent, collapse powder, emulsifying agent, adhesive, lubricant, hygroscopic agent etc.For example, solvent can be selected from water and sucrose solution, diluent can be selected from lactose, starch and microcrystalline Cellulose, the absorption delay agent can be selected from spherical chitosan and the poly-candy of Fructus Vitis viniferae amido, lubricant can be selected from magnesium carbonate, oil-based solvent can be selected from plant or animal oils, such as olive oil, sunflower oil and cod-liver oil etc.In this, can utilize prior art method to make suitable oral administration medicine supplying form, for example: lozenge, capsule, granule, powder, fluid extract, solution, syrup, suspension agent, Emulsion, and tincture etc.
Be suitable for medicament subcutaneous or the intravenous types of administration as for tool, be with Folium Eriobotryae cell extraction thing with optionally be generally used for this purpose material, mixed such as solubilizing agent, emulsifying agent or other adjuvant etc., to make such as venoclysis liquid, Emulsion venoclysis liquid, injection, dry powder injection, suspension injection, to reach dry powder suspension injection etc.The solvent that may adopt such as: water, physiological salt solution, alcohols (such as ethanol, propanol or glycerol etc.), sugar juice (for example: glucose or mannitol solution) or aforesaid combination.
Optionally, except the adjuvant composition of above-mentioned available pharmaceutical preparation, can add the additives such as flavoring agent, toner, coloring agent in addition, the suitable sense of mouth and visual experience when taking to improve the gained medicament; Can add in addition the preservative agent, antiseptic, antibacterial, antifungal of reasonable volume etc., to improve the storage characteristics of gained medicament.
Moreover, can and contain one or more other active component in medical composition of the present invention (or medicament), further strengthen the effect of medical composition of the present invention (or medicament) or increase the utilization motility and allotment degree of pharmaceutical formulation, needing only this other active component does not have adverse influence to the benefit of the extract of Folium Eriobotryae cell.
On the other hand; shown in hereinafter embodiment; the present application people's will is found outward; have the hyptadienic acid of following formula (4) and the termentic acid chemical compound of formula (5) in the extract of Folium Eriobotryae cell and have reducing fat and/or liver-protective effect; for example reduce the body fat accumulation or reduce triglyceride content in the blood; so can be particularly useful in treatment hypertriglyceridema or fat-reducing, comprise reduction body weight, control body weight, avoid obesity etc.In lower without being limited by theory, salty letter hyptadienic acid or termentic acid can by suppressing the mechanism of adipose cell formation, be reached the effect of reducing fat.In herein, " reducing fat " word is to contain the body fat that reduces in the tissue and/or the blood fat in the blood.
Therefore, the present invention also about a kind of for reducing fat and/or liver-protective medical composition, it comprises the chemical compound with following formula (4) and/or formula (5) of effective dose:
Or its medical acceptable salt or ester; And a kind of extract of the Folium Eriobotryae cell for reducing fat, it also comprises the chemical compound with formula (4) and/or formula (5).
In natural Folium Eriobotryae, the content of hyptadienic acid (chemical compound that namely has formula (4)) is extremely low, even can't record, and the content that can promote in prior art is also limited.Yet, according to the method for the extract of preparation Folium Eriobotryae cell of the present invention, can make the extract of high-load hyptadienic acid, such as among the embodiment hereinafter confirmation.According to the present invention, the extract of Folium Eriobotryae cell contains usually at least about 3 % by weight, and is better for 4 % by weight, and better hyptadienic acid at least about 5 % by weight is with the dry weight basis of extract.In addition, the extract of Folium Eriobotryae cell of the present invention contains usually at least about 20 % by weight, and is better for 30 % by weight, and better termentic acid at least about 40 % by weight is with the dry weight basis of extract.
Wherein, behind the extract that makes the Folium Eriobotryae cell, can utilize a purification step obtaining hyptadienic acid or termentic acid, such as distributing by liquid phase or the means such as tubing string chromatography.In an enforcement aspect of the present invention, be to utilize high-effect liquid chromatography (LC) mode to carry out purification, to obtain target chemical compound hyptadienic acid and termentic acid.
For reducing fat and/or liver-protective medical composition, the content with chemical compound of formula (4) and/or formula (5) is not absolute key point in of the present invention, as long as desired reducing fat and/or liver-protective benefit can be provided.
In addition; the present invention also has the extract of Folium Eriobotryae cell of chemical compound that the chemical compound of formula (4) and/or formula (5) or its medical acceptable salt or ester or use comprise have formula (4) and/or formula (5) in the application of making medicament about a kind of use, and wherein this medicament is for reducing fat and/or the liver protecting.
Similar to above-mentioned medicament for the protection of liver (or medical composition); the medicament that is used for reducing fat also can be prepared into any suitable form; and can mix with adjuvant or other active component of the activity of the chemical compound that can sharp impact has formula (4) (being hyptadienic acid) and/or formula (5) (being termentic acid), with the application of increase medicament.
Generally speaking, when use comprises the extract of Folium Eriobotryae cell of chemical compound of have formula (4) and/or formula (5) when making the medicament that is used for reducing fat, usually for each person every day consumption be per kilogram of body weight about 75 to the chemical compound of about 180 milligrams formula (4) and/or formula (5), be preferably per kilogram of body weight about 90 to the chemical compound of about 150 milligrams formula (4) and/or formula (5).
Hereby with following implementation aspect further to illustrate the present invention.Wherein those are implemented aspect and only are provided as explanation, but not in order to limit category of the present invention.
[embodiment]
The extract of [embodiment 1] preparation Folium Eriobotryae cell
(1) sets up aseptic seedling
In the orchard of a Taichung Bian Keng, gather ripe Folium Eriobotryae (Eriobotrya japonica Lindl.) seed, and with water flushing 30 minutes.Reach about 1 minute with 70% soak with ethanol seed, change again and be soaked in 1% liquor natrii hypochloritis of containing 0.01%Tween 20, and place the ultrasonic vibrating device, carry out surface sterilization after about 15 minutes, move to again aseptic operating platform.Clean seed three to four times with aquesterilisa, in the MS culture medium that contains 30 g of/liter sucrose (Murashige-Skoog medium), cultivate seed.After two to three weeks, seed begins to germinate, and collects the blade of rear two months seed Seedling that germinates, with as test material.
(2) callus induction (callus tissue)
Cut about 0.3 centimetre of blade drawing the seed Seedling, and with its immigration solid medium (6-benzyladenine (BA that comprises 2.5ppm in the MS basic recipe, 6-benzyladenine), the α-naphthaleneacetic acid of 1ppm (NAA, α-naphthaleneacetic acid), 3% (30 g/liter) sucrose and 0.3% (3 g/liter) quartzy Eucheuma gelatinosum (gelrite)) the middle cultivation.After one month, hang oneself and cut the wound of drawing and grow light yellow callus.
(3) cultivate the Folium Eriobotryae cell
Get about 60 g above-mentioned light yellow callus (can be divided into newborn Folium Eriobotryae cell), and utilize 590 microns screen cloth to sieve, after making callus cell be dispersed into a certain size, its immigration is contained 4,000 milliliter of culture fluid (BA that comprises 2.5ppm in the MS basic recipe, the NAA of 1ppm and 3% (30 g/liter) sucrose) bioreactor (BioFlo110 Bioreactor, New Brunswick Scientific, the U.S.) (condition of culture: ventilation is the 0.3v.v.m. (volumetric flow of gas of per minute per unit liquid volume in cultivation in, gasvolume flow per unit of liquid volume per minute), mixing speed is 40 rev/mins, and temperature is 24 to 26 ℃).Per ten days new culture fluid of subculture, and when each subculture, stay 500 milliliters of old suspension cell liquid (being culture fluid), add again 4,500 milliliters of new culture fluid.With other 4,500 milliliters of old suspension cell liquid (containing about 320 g of cell fresh weight) move into and contain in the bioreactor (volume is 30 liters stainless steel cylinder) of 25.5 liters of culture fluid (NAA and 3% (30 g/liter) sucrose that comprises BA, the 1ppm of 2.5ppm in the MS basic recipe), and the Folium Eriobotryae cell raffinate that adds 1ppm is to culture fluid, to continue to cultivate the Folium Eriobotryae cell through differentiation.After 18 days, stop cultivating.
(4) preparation Folium Eriobotryae cell raffinate
In one independently cultivates batch, obtain the Folium Eriobotryae cell through cultivating, be dried (dry precontract 5,400 g, dry after about 320 g), and after carrying out reflux extraction three times with the ethanol of 95 volume %, with filter paper filtering, collect filtrate and carry out concentrating under reduced pressure.With 50 volume % dissolve with methanol concentrate, behind the dissolving secondary, with filter paper filtering, get and do not dissolve part.Afterwards, this does not dissolve part with 85 volume % dissolve with methanol again, dissolves three times, with filter paper filtering, collects and does not dissolve part, namely obtains Folium Eriobotryae cell raffinate.
(5) preparation Folium Eriobotryae cell extraction thing
In one independently cultivates batch, obtain the Folium Eriobotryae cell through cultivating, under 60 ℃, be dried (dry precontract 5,400 g, dry after about 320 g), and after carrying out reflux extraction three times with 10 liters 95 volume % ethanol, with filter paper filtering, collect filtrate and carry out concentrating under reduced pressure.With 5 liters 50 volume % dissolve with methanol concentrate, behind the dissolving secondary, with filter paper filtering, get and do not dissolve part.Afterwards, this does not dissolve part with 10 liters 85 volume % dissolve with methanol again, dissolves three times, with filter paper filtering, collects filtrate, and carries out concentrating under reduced pressure, can obtain the Folium Eriobotryae cell extraction thing of about 53 g of white powderies.The Folium Eriobotryae cell extraction thing that makes through above-mentioned steps does not comprise in fact 50 volume % methanol solubilized compositions and 85 volume % methanol can not solvent components.
(6) purification triterpenic acid chemical compound
The extract of about 1 g white powdery Folium Eriobotryae cell is placed silica gel (LiChroprep RP-18, E.Merck, 40 to 63 microns) on, take methanol the ratio of water is distributed as 8: 2 to 10: 0 concentration, the high-effect liquid chromatography (LC) instrument of recycling preparation type (HPLC, Pu: the Shimadzu LC-8A (Japan, capital of a country) of side; Mobile phase: 85 volume % methanol; Flow velocity: 3 ml/min; Tubing string: YMC, J ' Sphere series ODS-H80 tubing string (internal diameter is 10 millimeters, and length is 250 millimeters, and particle size is 5 microns) purification can obtain respectively two kinds of triterpenic acid chemical compounds.
Utilize mass spectrum (Jeol GCmate, Japan, Tokyo) and NMR (
1H,
13C, DEPT, COSY, HMQC, HMBC, Jeol 500MHz, Japan, Tokyo) the purified chemical compound of analysis, wherein, main component is through confirming as hyptadienic acid (hyptadienic acid has the chemical compound of formula (4)) and termentic acid (tormenticacid has the chemical compound of formula (5)).
Hyptadienic acid (7.2 milligrams):
1H-NMR (pyridine-d
5): δ 0.98,1.08, and 1.15,1.17,1.42,1.70 (each 3H, s, H-23to H-29), (1.10 3H, d, J=6.6Hz, H-30), (3.04 1H, s, H-18), 4.44, (4.56 each 1H, d, J=14.9Hz, H-1), (5.60 1H, t, J=3.4Hz, H-12).
13C-NMR (pyridine-d5): δ 16.7 (C-30), 17.6 (C-6), 18.8 (C-26), 18.9 (C-25), 21.7 (C-24), 25.3 (C-27), 26.9 (C-21), 27.0 (C-11), 27.1 (C-16), 27.1 (C-29), 29.6 (C-15), 30.1 (C-23), 34.5 (C-7), 38.4 (C-22), 41.9 (C-4), (42.3 2C, C-8, C-14), 42.4 (C-9), 43.6 (C-20), 48.2 (C-17), 50.9 (C-10), 54.7 (C-18), 60.8 (C-1), 63.6 (C-5), 72.6 (C-19), 128.1 (C-12), 133.6 (C-3), 140.2 (C-13), 156.7 (C-2), 180.6 (C-28).
Termentic acid (25.3 milligrams):
1H-NMR (pyridine-d
5): δ 0.92 (3H, s, H-24), δ 1.00 (3H, s, H-25), δ 1.12 (3H, s, H-26), δ 1.12 (3H, s, H-30), δ 1.28 (3H, s, H-23), δ 1.43 (3H, s, H-29), δ 1.65 (3H, s, H-27), 1.74 (1H, t, J=12.7Hz, H-1), 1.90 (1H, dd, J=12.0,3.8Hz, H-1), δ 2.34 (1H, td, J=13.2,4.1Hz, H-15), δ 3.05 (3H, s, H-18), δ 3.14 (1H, td, J=13.1,4.1Hz, H-16), δ 3.77 (3H, s, H-3), δ 4.31 (1H, dt, J=10,2.6Hz, H-2), δ 5.59 (1H, s, H-12).
13C-NMR (pyridine-d
5): δ 17.1 (C-30), 17.2 (C-25), 19.1 (C-6), 22.8 (C-24), 17.7 (C-26), 24.5 (C-11), 25.1 (C-27), 26.9 (C-16), 27.4 (C-21), 27.6 (C-29), 29.7 (2C, C-15, C-23), 34.0 (C-7), 39.0 (C-22), 39.1 (C-10), 39.3 (C-4), 41.1 (C-8), 42.6 (C-1), 42.8 (C-20), 43.3 (C-14), 48.1 (C-9), 48.7 (C-17), 49.2 (C-5), 55.1 (C-18), 66.6 (C-2), 73.2 (C-19), 79.8 (C-3), 128.5 (C-12), 140.4 (C-13), 181.2 (C-28).
(7) the triterpenic acid content of the extract of mensuration Folium Eriobotryae cell
Measure the triterpenic acid content of Folium Eriobotryae cell extraction thing with HPLC.The used condition of HPLC is as follows: pump is Shimadzu LC-10ATvp; RI-detector is Shimadzu RID-10A; Tubing string is HyPURITYC-18 tubing string (internal diameter is 4.6 millimeters, and length is 250 millimeters, and particle size is 5 microns); Solvent system is methanol/0.15 volume % water-containing acetic acid, methanol: 0.15 volume % water-containing acetic acid=85: 15 volume ratios, and flow velocity is 0.5 ml/min, temperature is 35 ℃.As shown in table 1, with the dry weight basis of the extract of Folium Eriobotryae cell, the content of hyptadienic acid is about 5 % by weight, and the content of termentic acid is about 39 % by weight.
Table 1
| Cultivate batch | The Folium Eriobotryae dry cell weight (g) | Triterpenic acid content (milligram/dry cell weight) | The extract dry weight of Folium Eriobotryae cell (g) | Hyptadienic acid content (%) in the Folium Eriobotryae cell extraction thing | Herba Potentillae Chinensis acid content (%) in the Folium Eriobotryae cell extraction thing | Folium Eriobotryae cell extraction thing total triterpene acid content (%) |
| Control group (is not added Folium Eriobotryae | 269.9 | 66.8 | 29.35 | 2.52 | 18.08 | 66.9 |
| The cell raffinate) | ||||||
| 1 | 323.4 | 192.6 | 53.7 | 5.46 | 39.02 | 80.8 |
| 2 | 346.1 | 162.86 | 55.3 | 4.19 | 40.57 | 83.21 |
| 3 | 310.5 | 165.24 | 41.9 | 5.56 | 37.73 | 75.57 |
Table 2
As shown in table 2, the content of triterpenic acid composition in former plant is not high, and the content that utilizes method for tissue culture to promote is also limited, so be uneconomic with Folium Eriobotryae as the source of triterpenic acid.Yet, compared to prior art, utilize the method for the extract of preparation Folium Eriobotryae cell of the present invention, can obtain four kinds of triterpenic acid compositions of high-load, its total content is more up to 173.9 milligrams/g (in dry cell weights).
In addition, it should be noted that the disclosure according to WO2007/004827A1, the Folium Eriobotryae cell only can be cultivated in 250 milliliters container, and preparation method of the present invention can apply to the container of the scale more than 150 liters in fact.
The liver of the extract protection mice of [embodiment 2] Folium Eriobotryae cell
The extract of experiment A, Folium Eriobotryae cell suppresses liver inflammation and hepatic fibrosis and alleviates hepatic injury
36 ICR mices (available from Le Sikesheng skill company) are divided into four groups, and one group is control group, and in addition three groups then with hepatic injury and the hepatic fibrosis of Carbon Tetrachloride Induced mice.Throw respectively carboxymethyl cellulose (CMC) solvent or the Folium Eriobotryae cell extraction thing (70 or 200 mg/kgs of body weight) of mice 0.5 % by weight of giving experimental group.
On the other hand, carry out inducing of hepatic fibrosis in mice.Throw weekly and give twice carbon tetrachloride of ICR mice (10 volume % are dissolved in (0.1 milliliter/10 g body weight) in the olive oil), by a definite date eight weeks.Between induction period, throw every day and give the mice extract.Induce end,, in the situation of anesthesia (with carbon dioxide narcosis), taken a blood sample by the abdominal vein of mice, for the mensuration of Plasma Biochemical value in mice.In addition, promptly take off the liver of mice, and clean with the ice normal saline solution.Afterwards, liver is divided into four parts, respectively it is dipped in the 10 volume % neutral formalin, use for pathological section.With 100 ℃ temperature oven dry a copy of it, for the mensuration of hepatic fibrosis degree, and other portion is stored under-80 ℃, for the mensuration of bran amido sulfur (glutathione).
In addition, after obtaining mouse blood, with 4,700 rev/mins rotating speeds with its centrifugal 15 minutes, and use automatic biochemical analyzer (Cobas Mira, Roche, Rotkreuz, Switzerland) and commercial reagent (Roche Diagnostics, Mannheim, Germany) bran propylhomoserin the third amino transaminase (alanine transaminase, ALT) and bran propylhomoserin oxalacetic acid transaminase's (aspartate transaminase, AST) activity in the mensuration mice plasma.Wherein, bran propylhomoserin the third amino transaminase and bran propylhomoserin oxalacetic acid transaminase are the pointers of liver inflammation, and measurement result is as shown in table 3.
Protein measuring is to carry out according to the method for Lowry in the hepatic tissue, and (this can be referring to Lowry et al.Protein measurement with the folin phenol reagent.J.Biol.Chem.1951 as standard substance with Ox blood plasma albumen; 193:262-275, document content and in herein for your guidance), the result is as shown in table 4.
The mensuration of the bran amido sulfur in the hepatic tissue is the method according to Hissin, and this can be referring to Hissin et al.Afluorometric method for determination of oxidized and reduced glutathione in tissues.Anal.Biochem.1976; 74:214-226, document content and in herein for your guidance.Wherein, weigh 0.5 g liver organization, and add 5 milliliters 1.15 % by weight Klorvess Liquids, after homogenizing with homogenizer, get 1 milliliter homogenate, add again 1 milliliter 10 % by weight trichloroacetic acids, behind the mix homogeneously, with the centrifugal force of 3000g centrifugal 15 minutes.Then, get 0.01 milliliter supernatant, and add 0.18 milliliter of phosphoric acid-stretch ethylenediaminetetraacetic acid (phosphate-EDTA) buffer and 0.01 milliliter of σ-o-phthalaldehyde(OPA) (σ-phthaldehyde, 1 mg/ml methanol) solution, behind the mix homogeneously, detect with fluorescent, excitation wavelength 350 nanometers of the wavelength of 420 nanometers.The content of bran amido sulfur is that the result is as shown in table 4 with " micromole/g tissue " expression.
Chronic hepatitis or hepatic injury meeting cause hepatic fibrosis, and mainly being the hypertrophy of extracellular matrix collagen protein, causes hepatic fibrosis, wherein, hydroxyl proline (hydroxyproline) is amino acid special in the collagen protein, so measure the degree that the content of hydroxyl proline in the liver can estimate hepatic fibrosis.The mensuration of the collagen protein in the liver organization (or hydroxyl proline) content is the method with reference to Neuman, and this can be referring to Neuman et al.Thedetermination ofhydroxyproline.J.Biol.Chem.1950; 184:299-306, document content and in herein for your guidance.Wherein, after the liver organization hydrolysis, with hydrogen peroxide oxidation, with p-dimethyl amine benzaldehyde (p-dimethylaminobenzoaldehyde) colour generation, under the wavelength of 540 nanometers, measure light absorption value again.The content of hydroxyl proline is that the result is as shown in table 4 with " microgram/g tissue " expression.
With formalin fixedly behind the liver organization, carry out paraffin embedding and microsection manufacture, and use two kinds of stainings to dye, a kind of is general H.E. staining (hematoxylin and Yihong dyeing, hematoxylin and eosinstain), another kind of then be the special staining of collagen protein, i.e. Sirius Red dyeing.The mensuration of hepatic fibrosis then is to utilize image analysis system (Image-Pro Plus version 5.1, Media Cybernetics, the Maryland State, the U.S.) to analyze the ratio of hepatic fibrosis.Above-mentioned result of the test is shown in Fig. 2, Fig. 3 and table 5.
Table 3
All numerical value are all mean+/-standard error (hits=9).Compared to control group,
###P＜0.001;
Add the CMC group compared to carbon tetrachloride,
*P＜0.05,
*P＜0.01.
Table 4
All numerical value are all mean+/-standard error (hits=9).Compared to control group,
###P＜0.001;
Add the CMC group compared to carbon tetrachloride,
*P＜0.05,
*P＜0.01,
* *P＜0.001.
Table 5
All numerical value are all mean+/-standard error (hits=9).Compared to control group,
###P＜0.001; Add the CMC group compared to carbon tetrachloride,
*P＜0.01,
* *P＜0.001.
As shown in Table 3, the extract of Folium Eriobotryae cell can suppress the rising by the caused mice plasma ALT of carbon tetrachloride and AST activity, and this result shows that the extract of Folium Eriobotryae cell can alleviate the liver Tibetan inflammation that carbon tetrachloride causes.
In addition, can find out from table 4, carbon tetrachloride causes that the hepatic injury meeting of mice causes the minimizing of the content of protein in liver, Gai Yinqi suppresses the ability of liver synthetic protein, so cause the protein in liver content decrease, and the caused oxidative pressure of carbon tetrachloride or injury can consume and reduce the content of bran amido sulfur in the liver, and table 4 shows that the extract of Folium Eriobotryae cell of the present invention can promote the content of protein and bran amido sulfur, shows that it can alleviate hepar damnification.Moreover as shown in Figure 2, pathological section also shows that the extract of Folium Eriobotryae cell can improve the serious downright bad situation of the caused hepatocyte of carbon tetrachloride.
The result of table 4 also illustrates, the extract of Folium Eriobotryae cell can reduce the lifting of hydroxyl Proline content in the liver that carbon tetrachloride causes, and therefore, the extract of Folium Eriobotryae cell can suppress hepatic fibrosis.From Fig. 3 and table 5 as can be known, the result of pathological section also shows that the extract of Folium Eriobotryae cell can suppress hepatic fibrosis.
The extract of experiment B, Folium Eriobotryae cell suppresses the performance of the no cell surface receptor CD14 in storehouse
The extract of Folium Eriobotryae cell may alleviate hepatic fibrosis by suppressing inflammation, this mechanism can be referring to Salminea et al., Terpenoids:natural inhibitors of NF-κ B signaling withanti-inflammatory and anticancer potential.Cell Mol.Life Sci 2008; 65:2979-2999, document content and in herein for your guidance.Carbon tetrachloride can cause the lipopolysaccharide (lipopolysaacharide in the blood, LPS) increase, lipopolysaccharide enter behind the liver can with liver macrophage (macrophage, be the no cell in storehouse (Kupffercell)) surface receptor CD14 effect, to activate the no cell in storehouse and to promote cytohormone to produce, cause inflammatory response, and then cause hepatic injury, this mechanism can be referring to Su, 2002.Lipopolysaccharides in liver injury:molecular mechanisms of Kupffer cell activation.Am.J.Physiol.283, G256-G265, document content and in herein for your guidance.Therefore, below carry out the immunostaining of surface receptor CD14, understand Folium Eriobotryae cell extraction thing by the performance situation of observing CD14 and whether be used for alleviating hepatic fibrosis by suppressing inflammation.
At first, the pathological section among the above-mentioned experiment A is carried out with 3 volume % hydrogen peroxide removal intrinsic oversxidases, adding 5 % by weight Lac Bovis seu Bubali again, and reacting 30 minutes, to block non-specific combination after Tuo La and dehydration of alcohol process.Then, add CD14 antibody, after cultivating 2 hours under the room temperature, clean with phosphate buffered solution (PBS) again, add secondary antibody, and under room temperature, cultivated 30 minutes.Utilize immune detection cover group (available from BioGenex, San Ramon, Canada) and benzidine amine (diaminobenzidine, DAB) to carry out colour generation, again with behind the brazilwood extract dyeing, dehydration and mounting, the result is as shown in Figure 4.
As seen from Figure 4, carbon tetrachloride can promote the performance of CD14, and the extract of Folium Eriobotryae cell then can suppress the performance of CD14, and therefore, the extract of Folium Eriobotryae cell can suppress the activation of the no cell in storehouse or suppress the inflammatory response of liver.
Consolidated statement 3 to table 5, and the result of Fig. 2, Fig. 3 and Fig. 4 as can be known, the extract of Folium Eriobotryae cell can suppress the liver inflammation, and then suppresses hepatic fibrosis.
The extract of [embodiment 3] Folium Eriobotryae cell, termentic acid and hyptadienic acid suppress the activation of the no cell in liver storehouse
Owing to can produce nitric oxide behind the no cell activation in storehouse, so can observe the activation situation of the no cell in storehouse or the degree of liver inflammation by measuring nitric oxide production concentration.
Isolate the no cell in liver storehouse of rat (available from Le Sikesheng skill company) according to following mode: at first, with rat anesthesia, re-use and be dissolved in HBSS (Hanks ' balanced salt solution)) in 0.05 % by weight the 4th collagen type catabolic enzyme (type IV collagenase), carry out perfusion (totally 200 milliliters) with the flow velocity of 20 milliliters of per minutes.Afterwards, take out rat liver and shredded, digest with 0.5 g/liter the 4th collagen type catabolic enzyme again and reach 10 minutes.Then, filter liver samples with the nylon wire (strile nylon guaze) through sterilization, after the wash filtrate, carry out centrifugal with 50%/25% volume percoll solution gradient.After the centrifugal end, collect the Cell sap of 25%percoll and 50%percoll, again in addition centrifugal, and with isolated cell culture in containing DMEM culture medium (Dulbecco ' s modified Eagle ' s medium, Hyclone, the U.S., contain penicillin, 100 ug/ml of 10 volume % hyclones, 100 active unit/litres streptomycin, and 2 mMs/liter L-bran amic acid) 96 porose discs (5 * 10
5Cells/well) in.Cultivate after 15 minutes, remove medium supernatant, add again the DMEM culture medium, cultivate after 24 hours, replaced medium, and Folium Eriobotryae cell extraction thing (10,50 or 100 ug/ml), termentic acid (5,25 or 50 ug/ml) or the hyptadienic acid (10,25 or 50 ug/ml) of interpolation variable concentrations, cultivated again 60 minutes.Afterwards, add the lipopolysaccharide of 0.1 micro-molar concentration to culture medium, after 24 hours, collect the supernatant of culture medium, and use leather sharp scholar (Griess) reagent (available from Sigma) to measure nitric oxide production content, in addition, (3-(4 with MTS, 5-di-methylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, available from Promega, Madison, Wisconsin State, the U.S.) the measurement cell survival rate.Wherein, the action principle of MTS is: because the activity of living cells tool dehydrogenase, so MTS can be reduced into the water miscible product of reddish violet, and it has the highest light absorption value under the wavelength of 490 nanometers, therefore, can judge that by the light absorption value ratio survival rate of cell, cell survival rate are to be calculated by following formula.The as a result series of above-mentioned test is in table 6.
The light absorption value of the light absorption value/control group of survival rate=experimental group * 100%
Table 6
All numerical value are all mean+/-standard error (hits=3).Compared to control group,
###P＜0.001;
Add the dimethyl sulfoxide group compared to lipopolysaccharide,
*P＜0.05,
*P＜0.01.
As seen from Table 6, the lipopolysaccharide of 0.1 micro-molar concentration can not make the no cell death in storehouse, and under this condition, Folium Eriobotryae cell extraction thing, termentic acid and hyptadienic acid all can suppress the nitric oxide production generation brought out by lipopolysaccharide, and have the concentration interdependence.Therefore, the above results proves that the extract of Folium Eriobotryae cell, termentic acid and hyptadienic acid all can pass through to suppress activation or the liver inflammatory response of the no cell in storehouse, and reach the effect that suppresses hepatic fibrosis.
The extract of [embodiment 4] Folium Eriobotryae cell suppresses high lipid food and brings out the mice obesity
The extract of experiment A, Folium Eriobotryae cell suppresses Mouse Weight and fat weight
(available from Taibei Le Sikesheng skill company) tests with male C57BL/6 mice.24 mices are divided into four groups, one group of normal feedstuff of feeding (control group), three groups of feeding high lipid food 58Y1 (available from TestDiet) and give respectively the carboxymethyl cellulose solvent, 200 mg/kgs of 0.5 % by weight or the extract of 400 mg/kgs Folium Eriobotryae cell then in addition, and record weekly average food ration and the body weight change of every mice, around scheduling to last, the result is shown in following table 7 and table 8.
Behind the mice after testing all around so that carbon dioxide narcosis is above-mentioned, the fat of Qi Fu Testis both sides is also taken out in blood sampling, after weighing, a part of fat is soaked in the 10 volume % neutral formalin.
As shown in table 7, the food intake of the mice of feeding high lipid food is lower than the mice of the normal feedstuff of feeding, and give in the mice of extract of Folium Eriobotryae cell through throwing, dispensing dosage is 400 mg/kgs the only food ration reduction in the first week of mice, and second to then not obviously impact of food ration all around.
As shown in table 8, all around, with the body weight of the mice of high lipid food feeding apparently higher than control group, and compared to the mice that gives CMC through throwing, absolute weight and the relative weight (with respect to the percentage ratio of body weight) of final body weight, secondary Testis fat of mice of extract of giving the Folium Eriobotryae cell through throwing is all lower.
Table 7
All numerical value are all mean+/-standard error (hits=6).Compared to control group,
###P＜0.001;
Add the CMC group compared to high lipid food,
*P＜0.05.
Table 8
T-CHOL in the extract inhibition mice plasma of experiment B, Folium Eriobotryae cell and the concentration of triglyceride
Utilize automatic biochemical analyzer (Cobas Mira, Roche, Rotkreuz, Switzerland) and commercial reagent (RocheDiagnostics, Mannheim, Germany) to measure the concentration of T-CHOL and triglyceride in the mice plasma, the result is as shown in table 9.
Table 9
All numerical value are all mean+/-standard error (hits=6).Compared to control group,
###P＜0.001;
Add the CMC group compared to high lipid food,
*P＜0.05.
As shown in table 9, give the final total plasma cholesterol of mice of extract of Folium Eriobotryae cell and the concentration of triglyceride through throwing and all be lower than the mice that gives CMC through throwing.
The extract of experiment C, Folium Eriobotryae cell suppresses the growth of mice adipose cell
H.E. dyeing is carried out in the fat section that experiment obtains among the A, and analyzed the diameter of adipose cell with image analysis system, the result is such as table 10 and shown in Figure 5.
Table 10
All numerical value are all mean+/-standard error (hits=6).Compared to control group,
###P＜0.001;
Add the CMC group compared to high lipid food,
* *P＜0.01.
The result that the adipose cell of table 10 and Fig. 5 is analyzed shows, compared to the mice that gives CMC through throwing, the adipose cell diameter of mice of extract of Folium Eriobotryae cell that gives high dose through throwing is less.Therefore, the extract of Folium Eriobotryae cell of the present invention has the effect that suppresses the adipose cell growth.
Table 7 shows clearly that to table 10 and the 5th figure the extract of Folium Eriobotryae cell of the present invention can effectively reduce formation and the content of body fat, also has the effect of blood fat reducing simultaneously.
The extract of [embodiment 5] Folium Eriobotryae cell, termentic acid and hyptadienic acid suppress Adipocyte Differentiation
Be to contain in the incubator that 5% carbon dioxide and temperature be 37 ℃, in the DMEM culture fluid (Hyclone, the U.S.) that contains 10 volume % hyclones (Hyclone, the U.S.), with 1 * 10 in saturated humidity
4The density of individual cells/square cm is cultivated the front adipose cell (preadipocyte) of 3T3-L1.
In 24 porose discs, cultivated the 3T3-L1 cell two days, after making cell present the full up state of monolayer (monolayerconfluents) and tool contact inhibition, throwing is given differentiation agent and (is comprised 5 ug/ml insulins, 0.5 mM/liter IBMX (3-isobutyl-1-methylxanthine, 3-isobutyl-1-methylxanthine) and 1 micromole/litre DEX (dexamethason)), and dimethyl sulfoxide (Dimethyl sulfoxide, DMSO) or variable concentrations (0.5, the extract of Folium Eriobotryae cell 5 or 50 ug/ml), termentic acid, or hyptadienic acid, and cultivated.Two days later, upgrade culture fluid (the DMEM culture fluid that contains 5 ug/ml insulins and 10% volume FBS), and changed a culture fluid in per two days, be continued until the 8th day that adds behind the differentiation agent.
In incubation, under microscopical observation, the 3T3-L1 cell becomes spherical by the spindle shape and increases, and can find oil droplet densely distributed in Cytoplasm, and at this moment, the 3T3-L1 cell has been divided into adipose cell (adipocyte).With 10 volume % neutral formalin fixed fat cells one hour, under room temperature, after Oilred O (0.1 mg/ml) dyeing two hours, oil droplet can be dyed to redness, again with behind the washed with de-ionized water cell three times, with microscopic examination and take a picture, subsequently with quantitative isopropyl alcohol with the oil droplet stripping.At last, measure light absorption value with the wavelength of 492 nanometers, and calculate extract, termentic acid or the hyptadienic acid of Folium Eriobotryae cell to the suppression ratio of Adipocyte Differentiation.The suppression ratio of cell differentiation is to calculate with following formula.
Differentiation suppression ratio (%)=[(differentiation agent+dimethyl sulfoxide) light absorption value-control group light absorption value-(differentiation agent+substances) light absorption value]/[(differentiation agent+dimethyl sulfoxide) light absorption value-control group light absorption value] * 100%
In addition, under differentiation agent existed, with the impact of MTS viewing test material (being extract, termentic acid and the hyptadienic acid of Folium Eriobotryae cell) on the 3T3-L1 cell survival rate, series was in table 11 as a result.
The light absorption value of the light absorption value/control group of survival rate=experimental group * 100%
Table 11
All numerical value are all mean+/-standard error (number of samples=3).Compared to control group,
#P＜0.05,
###P＜0.001;
Add the dimethyl sulfoxide group compared to differentiation agent,
*P＜0.05,
*P＜0.01,
* *P＜0.001.
Can clearly be observed by Fig. 6 and table 11, the extract of Folium Eriobotryae cell, termentic acid and hyptadienic acid all can suppress the differentiation of adipose cell.Therefore, in this embodiment, prove that first termentic acid and hyptadienic acid have the effect of reducing fat.
Above-described embodiment only is to illustrate principle of the present invention and effect, but not is used for restriction the present invention.Any ripe personage in technique all can be in the situation of know-why of the present invention and spirit, and above-described embodiment is made amendment and changed.Therefore, the scope of the present invention the application's claim limited range as described later.
Claims (17)
1. method for the preparation of the extract of liver-protective Folium Eriobotryae cell, described extract comprises following formula (4) and/or formula (5) chemical compound:
Described method comprises:
A) in a culture fluid, cultivate a Folium Eriobotryae cell, wherein this culture fluid comprises a Folium Eriobotryae cell raffinate and this Folium Eriobotryae cell, and in the total amount of this culture fluid, the consumption of this Folium Eriobotryae cell raffinate is 0.5ppm to 20ppm, and is to make through following steps:
I) get a Folium Eriobotryae cell, and working concentration is this Folium Eriobotryae cell of alcohol extraction of 80 volume % to 100 volume %;
II) product through extraction concentration step I) is to obtain one first concentrate;
III) working concentration is this first concentrate of dissolve with methanol of 40 volume % to 60 volume %, collects one first and does not dissolve part; And
IV) working concentration be 70 volume % to 95 volume % dissolve with methanol this first do not dissolve part, collect one second and do not dissolve part, to make this Folium Eriobotryae cell raffinate; And
B) the Folium Eriobotryae cell a) cultivated of extraction step comprises following steps:
B1) working concentration is the Folium Eriobotryae cell that the alcohol extraction step of 80 volume % to 100 volume % a) is cultivated;
B2) product through extraction concentration step b1) is to obtain one second concentrate;
B3) working concentration is this second concentrate of dissolve with methanol of 40 volume % to 60 volume %, collects one the 3rd and does not dissolve part;
B4) working concentration is that the dissolve with methanol the 3rd of 70 volume % to 95 volume % does not dissolve part and filtered, and collects filtrate; And
B5) optionally concentrate this filtrate.
2. method according to claim 1, wherein the Folium Eriobotryae cell of step in a) is to obtain through following steps:
1) gets one and have the blade of the Loquat Seeds Seedling of wound, and treat that wound grows callus cell; And
2) taking out this callus cell is also cultivated.
3. method according to claim 1 is characterized in that, in the total amount of this culture fluid, the consumption of the Folium Eriobotryae cell raffinate of step in a) is 1ppm to 5ppm.
4. method according to claim 1 is characterized in that, step I) in the Folium Eriobotryae cell be to make through following steps:
1) gets one and have the blade of the Loquat Seeds Seedling of wound, and treat that wound grows callus cell; And
2) taking out this callus cell is also cultivated.
5. method according to claim 1, the concentration of ethanol this step I wherein) is 90 volume % to 98 volume %, the concentration of methanol this Step II I) is 45 volume % to 55 volume %, and this step IV) the concentration of methanol be 80 volume % to 90 volume %.
6. method according to claim 1, it is characterized in that, the concentration of ethanol this this step b1) is 90 volume % to 98 volume %, this step b3) the concentration of methanol be 45 volume % to 55 volume %, and this step b4) the concentration of methanol be 80 volume % to 90 volume %.
7. medical composition for the protection of liver comprises the according to claim 1 prepared extract of each described method in 6 of an effective dose.
8. medical composition according to claim 7 is characterized in that, it is for the protection of by the caused hepatic injury of chemical substance or suppress hepatic fibrosis.
9. medical composition according to claim 8 is characterized in that, this chemical substance is carbon tetrachloride, ethanol or thiacetamides.
A use according to claim 1 in 6 the prepared extract of each described method it is characterized in that in the application of making medicament this medicament is for the protection of liver.
11. application according to claim 10 is characterized in that, this medicament is for the protection of by the caused hepatic injury of chemical substance or suppress hepatic fibrosis.
12. application according to claim 11, wherein this chemical substance is carbon tetrachloride, ethanol or thiacetamides.
13. method for preparing the chemical compound of have following formula (4) and/or formula (5), it is characterized in that, comprise utilization according to claim 1 in 6 each described method and carry out a purification step to obtain the chemical compound of formula (4) and/or formula (5) preparing the extract of a Folium Eriobotryae cell:
14. method according to claim 13 is characterized in that, this purification step comprises and carries out a high-effect liquid chromatography (LC).
15. a use has the chemical compound of following formula (4) and/or formula (5) or its medical acceptable salt or ester in the application of making medicament:
Wherein this medicament is for reducing fat and/or the liver protecting.
16. application according to claim 15 is characterized in that, this medicament is by the caused hepatic injury of chemical substance or inhibition hepatic fibrosis for fat-reducing, treatment hypertriglyceridema, protection.
17. application according to claim 16 is characterized in that, this chemical substance is carbon tetrachloride, ethanol or thiacetamides.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910206764 CN102048851B (en) | 2009-11-09 | 2009-11-09 | Loquat leaf cell extract for protecting liver and/or reducing fat and preparation method and application thereof |
Applications Claiming Priority (1)
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| CN1439648A (en) * | 2003-03-28 | 2003-09-03 | 中国医学科学院药用植物研究所 | Loquat leaf triterpene acid composition and its uses in preparation of medicine against inflammation and antitussive |
| CN1640885A (en) * | 2004-01-08 | 2005-07-20 | 中国科学院上海药物研究所 | Loguat leaf total dehydrocamphenilic acid and its preparation method and use |
| CN101224246A (en) * | 2008-01-25 | 2008-07-23 | 江苏省中国科学院植物研究所 | Preparing method of loquat leaf total triterpenic acid and antidiabetic use thereof |
| CN101428077A (en) * | 2008-12-19 | 2009-05-13 | 中国医药集团总公司四川抗菌素工业研究所 | Traditional Chinese medicine composition for treating diabetes |
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| CN1439648A (en) * | 2003-03-28 | 2003-09-03 | 中国医学科学院药用植物研究所 | Loquat leaf triterpene acid composition and its uses in preparation of medicine against inflammation and antitussive |
| CN1640885A (en) * | 2004-01-08 | 2005-07-20 | 中国科学院上海药物研究所 | Loguat leaf total dehydrocamphenilic acid and its preparation method and use |
| CN101224246A (en) * | 2008-01-25 | 2008-07-23 | 江苏省中国科学院植物研究所 | Preparing method of loquat leaf total triterpenic acid and antidiabetic use thereof |
| CN101428077A (en) * | 2008-12-19 | 2009-05-13 | 中国医药集团总公司四川抗菌素工业研究所 | Traditional Chinese medicine composition for treating diabetes |
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| 陈剑等.中药枇杷叶的研究进展.《药用植物研究与中药现代化——第四届全国药用植物学与植物药学术研讨会论文集》.2004,355-361. * |
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