CN102824391B - Application of kalimeris in preparation of medicament for reducing blood fat or functional foods - Google Patents
Application of kalimeris in preparation of medicament for reducing blood fat or functional foods Download PDFInfo
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- CN102824391B CN102824391B CN201210336242.9A CN201210336242A CN102824391B CN 102824391 B CN102824391 B CN 102824391B CN 201210336242 A CN201210336242 A CN 201210336242A CN 102824391 B CN102824391 B CN 102824391B
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Abstract
The invention relates to a preparation method for triterpenoid compounds in kalimeris and a function for reducing blood fat of the triterpenoid compounds. The invention provides a method for extracting and preparing the triterpenoid compounds from the kalimeris as well as a blood-fat reducing function and application of the triterpenoid compounds obtained by the method. The method for preparing the triterpenoid compounds in the kalimeris comprises the following steps of: with a full kalimeris plant as a raw material, carrying out reflux extraction or maceration extraction or percolation extraction with 80 percent ethanol; filtering and carrying out vacuum concentration on filtrate; sequentially extracting with petroleum ether and ethyl acetate; enabling a concentrated solution of the ethyl acetate extraction part to pass through a macroporous absorption resin chromatograph column; sequentially carrying out water washing and eluting with 20 percent ethanol for removing impurities; then eluting with 40-60 percent ethanol solution; collecting an ethanol elution solution; and carrying out vacuum concentration, freezing and drying on the ethanol elution solution to obtain an extract of the triterpenoid compounds in the kalimeris. The total content of the triterpenoid compounds as the extract of the kalimeris is not lower than 50 percent; and the triterpenoid compounds comprise main components of suberone, epifriedelanol, sitosterol, stigmasterol, spinach sterol, spinach sterone and daucosterol and have remarkable efficacy of reducing the blood fat.
Description
Technical field
Patent of the present invention relates to functional food and field of phytochemistry, the separation preparation of the natural activity factor and the application of functional food.
Background technology
Cardio-cerebralvascular diseases has become the first killer of current harm humans life and health, and the death causing due to cardio-cerebralvascular diseases is in the world in first.In blood plasma cholesterol concentration too high be an Etiological that causes cardio-cerebralvascular diseases.At present, blood fat reducing class medicine all has toxic and side effects in various degree, as gastrointestinal reaction, liver, injury of kidney etc., most of blood fat reducing Western medicine can make blood fat more concentrate on liver metabolism, make lipid more be deposited in liver and increase the weight of hepatic cell fattydegeneration and liver function injury, making patient's lowering fat and protecting liver difficulty in treatment satisfactory to both parties.In addition, most chemical synthetic drugs are because of the mechanism of action of its single target spot, and the effect being difficult to reach satisfied aspect the hyperlipemia for the treatment of cause of disease complexity, especially not yet needs the sub-health population of Drug therapy in hyperlipidemia edge for those.And natural function food is because of its many target spots and multicomponent synergism, safe, the feature having no side effect, has extensively caused people's concern, especially suitable those Hyperlipidemias but not yet reach the colony of medical treatment degree.Therefore, property active blood fat reducing component in exploitation Natural Food source becomes day by day urgent study hotspot.
Modern medicine thinks, Herba Kalimeridis is cool in nature, acrid in the mouth, has the effects such as removing heat from blood, heat clearing away, dampness removing, removing toxic substances.Have at present the separation of bibliographical information Herba Kalimeridis triterpenoid compound exquisite, but the literary composition of its hypolipidemic activity and extraction thereof there is not yet report.About the report of Herba Kalimeridis, be and select chromatography carrier not of the same race, by repeatedly repeatedly column chromatography identify and the functional activity of gained monomeric compound and part material comprise antibacterial, protection CCl
4the hepatic injury of induction etc.
For example: 2006, woods material reported that in Herba Kalimeridis, separation obtains triterpenoid compound.Adopting the dry herb of Herba Kalimeridis, is 95% ethanol merceration 3 times by volume fraction, each 2 d, and merging filtrate, respectively with petroleum ether, ethyl acetate, n-butanol extraction concentrated.Petroleum ether part is through silica gel column chromatography, and petroleum ether-ethyl acetate system gradient elution, obtains 6 monomeric compounds.
2010, Wang Gang adopted 80% ethanol extraction Herba Kalimeridis, used respectively petroleum ether, water saturated n-butanol extraction, and petroleum ether portion adopts respectively silica gel column chromatography and gel column chromatography Sephadex LH-20 to carry out separation and purification, Structural Identification 9 compounds.Experiment shows that compound chrysophanol and vanillin are to CCl
4the hepatic injury of induction has certain protective effect.
2010, Liu Jingsong adopted 80% ethanol extraction Herba Kalimeridis, used respectively petroleum ether, water saturated n-butanol extraction, and petroleum ether portion adopts silica gel column chromatography and gel column chromatography Sephadex LH-20 to carry out purification, has identified 5 triterpenoid compound.Activity shows that compound V is to CCl
4the hepatic injury of induction has certain protective effect.
2010, permitted the clear 80% industrial alcohol reflux, extract, that adopts of literary composition, with petroleum ether, ethyl acetate, n-butyl alcohol, extract respectively.Petroleum ether portion is carried out to silica gel column chromatography separation and obtain 14 fats compounds.
2008, permitted the clear 80% industrial alcohol reflux, extract, Herba Kalimeridis that adopts of literary composition, with ethyl acetate, n-butyl alcohol, extract respectively.Ethyl acetate extraction part is carried out to silica gel column chromatography separation, to n-butanol portion, first use silica gel column chromatography separated, use Sephadex LH-20 pillar separated, final evaluation obtains 8 compound Screening of Antibacterial Activities and shows: succinic acid (1) and syringic acid (5) have inhibitory action to bacillus subtilis again.
2008, the refined employing 80% ethanol extraction Herba Kalimeridis of woods, used respectively petroleum ether, water saturated n-butanol extraction, n-butanol portion was carried out to silica gel column chromatography separated with Sephadex LH-20 chromatographic column, obtained cupreol.
2010, little 80% ethanol that appears in the newspapers of Gong extracted respectively 3 times, extract respectively, and Dichlorodiphenyl Acetate ethyl ester extracts, and partly carrying out column chromatography separation separation from Herba Kalimeridis obtained 11 compounds with other petroleum ether, ethyl acetate and n-butyl alcohol.
Above extracting method is the refining means of compound, the reactive compound existing in the distinct Herba Kalimeridis of object.And these procedures are too loaded down with trivial details, sample loss is large (cause output too low) also; A large amount of solvent elutions also can increase substantially cost in addition; And silica gel column chromatography and the separation and purification of Sephadex LH-20 chromatographic column are difficult to suitability for industrialized production.
Summary of the invention
In order effectively to utilize bioactive triterpene constituents in Herba Kalimeridis, the invention provides the new purposes of Herba Kalimeridis, determined that its fat biological is active.
The invention discloses the application of Herba Kalimeridis in preparing blood lipid-lowering medicine or functional food.
Particularly, described Herba Kalimeridis specifically refers to the extract obtaining by following method: Herba Kalimeridis Herb is dry, pulverizing, crosses 20 mesh sieves and divides, 80% ethanol extraction, solid-liquid ratio 1g:12mL, 60 ℃ of water-baths, stir and extract 2 times, filter, residue in kind repeats to extract 1 time, and the extracting solution concentrating under reduced pressure of merging is removed to ethanol, uses successively petroleum ether and the ethyl acetate extracting twice of 3 times of volumes, combined ethyl acetate extraction part, concentrates to obtain paste; Paste carries out separation with column chromatography method, and column chromatography be take macroporous adsorbent resin as immobile phase, and impurity is removed in first water and 10% ethanol drip washing, rear employing 20% and 40-60% ethanol elution, collect ethanol elution thing concentrating under reduced pressure, vacuum drying, and obtain Herba Kalimeridis extract.
By animal model contrast test, measure, in the present invention, blood fat reducing Herba Kalimeridis extract has on average declined more than 20% to the serum total cholesterol content of hyperlipidemia mice compared with matched group, serum triglycerides content declines and is up to 56%, hdl concentration raises and is up to 68%, the reduction of low density lipoprotein, LDL content is up to 65%, to sum up shows to have comparatively significant blood fat reducing function.
By animal model contrast test, measure, blood fat reducing Herba Kalimeridis extract of the present invention can Effective Regulation plasma triglyceride (TG) two kinds of enzymes, the activity of lipoprotein lipase (LPL) and hepatic lipase (HL), thus effectively reduce the content of Triglycerides in Serum.
By animal model contrast test, measure, blood fat reducing Herba Kalimeridis extract of the present invention can Effective Regulation blood plasma in the activity of key enzyme (HMG-CoA, CYP7a1, ACAT) of cholesterol metabolism, reduce the autologous synthetic of cholesterol, stop cholesterol esterification simultaneously, and can impel more cholesterol to be converted into bile acid, thereby reach the object that reduces cholesterolemia.
By animal model contrast test, measure, blood fat reducing Herba Kalimeridis extract of the present invention can Effective Regulation Mice Body in (serum and liver) MDA, SOD, the isocyatic variation of GSH-PX, reduce the accumulation of active oxygen in hyperlipidemia Mice Body, can decompose rapidly lipid peroxide, defence effect of free radicals damage.
Above result of the test shows, the extract of Herba Kalimeridis of the present invention can pass through number of ways, reaches blood fat reducing function.
Beneficial effect of the present invention is mainly reflected in: provide a kind of and take Herba Kalimeridis and prepare as raw material, wherein in macroporous adsorbent resin 20% component, the triterpenoid compound gross mass content in flavone compound and macroporous adsorbent resin 60% component, all more than 50%, and all has the plant extract of good blood fat reducing function.This extract and the comparison of domestic existing product Xuezhikang, better to demand prevention hyperlipidemia and recovery normal lipid effect.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the present invention is further described.
Fig. 1 is the impact of Herba Kalimeridis component on hyperlipemia in mice serum TC, TG, HDLC, LDLC.
Fig. 2 is the impact of Herba Kalimeridis component on mice HL.
Fig. 3 is the impact of Herba Kalimeridis component on mice LPL.
Fig. 4 is the impact of Herba Kalimeridis component on sterol in feces and Determination of Bile Acids.
Fig. 5 is the impact of Herba Kalimeridis component on mice MDA.
Fig. 6 is the impact of Herba Kalimeridis component on SOD in Mice.
Fig. 7 is the impact of Herba Kalimeridis component on mice GSH-PX.
the specific embodiment
In the present invention, the concentration of ethanol is concentration expressed in percentage by volume.
Embodiment 1: Herba Kalimeridis Herb is dry, pulverizing, crosses 20 mesh sieves and divides, and takes 500g in reactor, add 80% ethanol 7000ml, 60 ℃ of heating in water bath, stir and extract (reflux, extract,, or dipping extraction, or percolation extracts) 60min, filter, collect extracting solution, under residue similarity condition, extract once again, merge extracted twice liquid, concentrating under reduced pressure is removed ethanol.Use successively petroleum ether and the ethyl acetate extracting twice of 3 times of volumes, combined ethyl acetate extraction part, concentrates to obtain paste; Paste adopts macroporous adsorptive resins separated, and water and 20% ethanol drip washing, remove impurity first successively, and rear employing 40%-60% alcoholic solution eluting is collected ethanol elution thing, and concentrating under reduced pressure, lyophilization, and obtain Herba Kalimeridis extract T.
Extractive content is measured: take oleanolic acid as standard substance, adopt vanillin-glacial acetic acid-perchloric acid coloration method to measure, product triterpenes content must not be lower than 50%.
It is 52.5% that Herba Kalimeridis triterpene extracts T measures total triterpenes compounds content, mainly contains suberone, epifriedelinol, sitosterol, stigmasterol, spinasterol, the materials such as Herba Spinaciae sterone.
Embodiment 2: Herba Kalimeridis Herb is dry, pulverizing, crosses 20 mesh sieves and divides, and takes 500g in reactor, add 80% ethanol 7000ml, 60 ℃ of heating in water bath, stir and extract (reflux, extract,, or dipping extraction, or percolation extracts) 60min, filter, collect extracting solution, under residue similarity condition, extract once again, merge extracted twice liquid, concentrating under reduced pressure is removed ethanol.Use successively petroleum ether and the ethyl acetate extracting twice of 3 times of volumes, combined ethyl acetate extraction part, concentrates to obtain paste; Paste adopts macroporous adsorptive resins separated, and ethanol elution thing is collected in water and 20% ethanol drip washing first successively, and concentrating under reduced pressure, lyophilization, and obtain Herba Kalimeridis extract F.
Extractive content is measured: take oleanolic acid as standard substance, adopt vanillin-glacial acetic acid-perchloric acid coloration method to measure, product triterpenes content 20.6%; Take rutin as standard substance, adopt coloration method or ultraviolet method to measure, product flavonoid content 62.3%.
Embodiment 3: embodiment 3: blood fat reducing function test:
3.1 experimental animals and feed formula
3.1.1 experimental animal
Healthy clean level male mice (CD-1), surrounding age, body weight provides for 18～22g ，Gong 72Zhi，You Nanjing Medical University's experimental animal center, the quality certification number: SYXK(Soviet Union) 2008-2007, mice is raised in the aseptic air conditioning chamber of Nanjing Medical University's animal center, 19 ~ 21 ℃ of room temperatures, relative humidity 50 ~ 60%, auto-control is each 12h round the clock, freely drinks water.
3.1.2 feed formula
Normal feedstuff meets national animal feeding standard ，You Nanjing Medical University's experimental animal center to be provided, and its main nutrient composition is as table 1, and high lipid food is configured voluntarily by test chamber, and formula is as table 1.
3.2 Herba Kalimeridis component animal model in vivo tests
Herba Kalimeridis crude extract is first by petroleum ether, ethyl acetate extraction, by ethyl acetate part through macroporous resin initial gross separation, using the sample sets of the component part blood fat reducing test in body obtaining, thereby further verify the hypolipemic function of triterpenoid compound, mice is divided into 9 groups at random, the same time of every day is carried out gavage, raises 4 weeks, and mice group and dosage are concrete as table 2.
Table 2 mice group and dosage
| Mice group | Number of elements | Dosage is selected |
| |
8 | Normal feedstuff+50mg/Kg/d |
| |
8 | High lipid food+50mg/Kg/d |
| |
8 | High lipid food+150mg/Kg/d |
| F component |
8 | High lipid food+50mg/Kg/d |
| F component |
8 | High lipid food+100mg/Kg/d |
| T component |
8 | High lipid food+50mg/Kg/d |
| T component |
8 | High lipid food+100mg/Kg/d |
Note: 20%, 60% ethanol elution component: use respectively F group, T organizes expression
Experimental result shows: with normal group comparison, mice serum TC, TG, HDL-C, LDL-C index and tremulous pulse medicated porridge hardenability value (AI) are as shown in Figure 1, Herba Kalimeridis extract all has reduction (P < 0.05) in various degree to T-CHOL (TC), triglyceride (TG), low density lipoprotein, LDL (LDLC) concentration, and high density lipoprotein (HDL-C) concentration raises to some extent.
On the impact of mice lipoprotein lipase liver esterase (HL), lipoprotein lipase (LPL) as shown in Figure 2 and Figure 3, Herba Kalimeridis extract can be by exciting enzyme means of subsistence footpath to promote lipoprotein metabolism to hyperlipidemia mice, thereby reduction triglyceride, and the rate-limiting enzyme of triglyceride is had to certain optimum effect.
The impact of the key enzyme vigor that Mice Body inner cholesterol is formed is as shown in table 3 and Fig. 4, and Herba Kalimeridis extract all suppresses the activity of HMG-CoA reductase and ACAT, reduces the autologous synthetic of cholesterol, stops cholesterol esterification simultaneously.Meanwhile, the changes of contents by sterol and bile acid in the active of mensuration CYP7a1 and mensuration stool in mice, found that, Herba Kalimeridis extract all can strengthen the activity of CYP7a1, impels more cholesterol to be converted into bile acid, reaches the object that reduces cholesterolemia.
The impact of table 3 Herba Kalimeridis component on mice HMG-CoA, CYP7a1, ACAT
| Group | HMG-CoA（U/L） | CYP7a1（U/L） | ACAT（U/L） |
| Normal group | 180.1 scholars 9.7 a,b | 13.87 scholar 1.4 a,b | 80.13 scholar 5.6 b,c |
| Model group | 185.1 scholars 8.6 a | 10.56 scholar 1.3 c | 84.22 scholar 8.6 a,b |
| Positive controls | 178.2 scholars 11.3 a,b | 12.47 scholar 1.8 b,c | 75.58 scholar 10.7 b,c |
| Low dosage F group | 181.5 scholars 6.8 a,b | 10.72 scholar 1.4 c | 68.31 scholar 8.5 c |
| High dose F group | 183.7 scholars 10.5 a,b | 14.49 scholar 1.4 a,b | 87.88 scholar 8.7 a |
| Low dosage T group | 176.6 scholars 14.4 a,b | 13.62 scholar 1.7 a,b | 80.85 scholar 11.0 a,b,c |
| High dose T group | 149.49 scholars 8.2 e | 14.32 scholar 1.0 a,b | 72.60 scholar 6.8 c |
On the impact of hyperlipemia in mice MDA, SOD, GSH-PX enzyme activity, as shown in Fig. 5, Fig. 6, Fig. 7, Herba Kalimeridis extract all can reduce the accumulation of active oxygen and decompose rapidly lipid peroxide, has important defence effect of free radicals damage.
Claims (2)
1. the application of Herba Kalimeridis extract in preparing blood lipid-lowering medicine or functional food, it is characterized in that being prepared as of described Herba Kalimeridis extract: get indian kalimeris leaf or Herba Kalimeridis stem or Herba Kalimeridis whole herb with root dry, pulverize, with the alcoholic solution of 80% volumetric concentration, extract, solid-liquid ratio is 1g:12mL, in 60 ℃ of stirring in water bath, extracting 60min filters, residue in kind repeats to extract 1 time, the extracting solution concentrating under reduced pressure merging is removed to ethanol, use successively petroleum ether and the ethyl acetate extracting twice of 3 times of volumes, combined ethyl acetate extraction part, concentrates to obtain paste; Macroporous adsorptive resins on paste, first uses distilled water drip washing, then, with the alcoholic solution eluting of 10% volumetric concentration, removes after impurity, adopts 20% and 40-60% alcoholic solution eluting, by eluate concentrating under reduced pressure, vacuum drying.
2. application according to claim 1, is characterized in that adopting 20% and 40-60% alcoholic solution eluting macroporous resin, elution volume be 4-6 doubly, flow velocity is 2-4 mL/min.
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