CN1115671A - Active component of cordyceps sinensis and its separation method - Google Patents
Active component of cordyceps sinensis and its separation method Download PDFInfo
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Abstract
The present invention includes the structure of specific active component in Cordyceps sinensis, the relevant separation process to obtain the specific active component, and the application of the said specific active component in the inhibition of active nephrosis cells.
Description
The present invention relates to a kind of separation method of Cordyceps active component, can suppress activatory human kidney diaphragm cell, make and improve gA nephritis, avoid the course of disease to proceed to uremic active component (Ha-A) to extract.
Pharmaceutical research confirms in recent years, a kind of larva that parasitizes lepidopteran insects, and grow the Clavicipitaceae Cordyceps fungus of Stroma, and its active and pharmacological action are multiaspect, it comprises:
(1) immune system: the Cordyceps leachate can improve animal macrophage phagocytic index, stimulate that spleen Thy-1 hyperplasia is secreted, brought out to first Jie's white matter (IL-1), immune stimulatory globulin M (IgM) is synthetic and secretion, T lymphopoiesis and more second Jie's white matter (IL-2) receivers of bone-marrow-derived lymphocyte performance.Its water Extract also can improve ordinary person and leukaemic's natural killer cytoactive.
(2) renal function aspect: Cordyceps can alleviate injury of renal tubular, guarantees Na on its cell membrane
+-K
+-ATPase enzyme, its effect may be relevant with the peroxidating of minimizing cytolipin; The hematuria that also can lower chronic renal insufficiency and improve rat with delay the kreatinin value and rise.
(3) cardiovascular system: the Cordyceps mycelium extract can increase the laboratory animal coronary artery blood flow, reduces tremulous pulse, brain and peripheral resistance and blood pressure lowering.Its water solublity master composition adenosine has the activity of loose vascular smooth muscle and blood vessel dilating, and Cordyceps also can be caught into platelet generation, and Cordyceps also has the effect of anti-hypoxia, inhibition brain monoamine oxidase, MAO (MAOB) in addition.Yet above-mentioned pharmacological action is carried out because of the Cordyceps active ingredient that does not adopt complete purification as yet, and the relevant histology who improves the nephropathy change changes and the research of clinical symptoms had not also had the pattern of employing immunoglobulin A (IgA) nephritis to carry out.
IgA nephritis is clinically with paroxysmal hematuria and/or albuminuria performance, often show with chronic disease, people's about 20% its course of disease after suffering from this disease can proceed to uremia gradually, still there be not the carrying out that the treatment agent that confirms to have any structure can stop disease so far, so extract and the searching curative is very heavy problem with additive method.
The mechanism of causing a disease of IgA nephritis is owing to cause the kidney diaphragm that nephropathy IgA immunocomplex is deposited on the kidney pompon, make the kidney diaphragm cell activation that is the attitude of stopping originally, activatory kidney diaphragm cell can be emitted first medium (IL-1), the 6th Jie's white matter (IL-6), tumor necrosis factor (TNF α), make the hyperplasia of kidney diaphragm, the kidney diaphragm cell of hypertrophy can disengage IL-1, IL-6, PDGF (PDGF), growth factor-beta transition (TGF-β) etc., the effect of these cytohormones and somatomedin is as from secretin (autocoids), can make the hypertrophy of kidney diaphragm be vicious cycle, and can impel kidney diaphragm cell to emit O
2 -, H
2O
2, platelet activating factor (PAF), prostaglandin E
2(PGE
2), thromboxan B
2(TXB
2), and neutral proteinase etc., therefore cause kidney diaphragm hypertrophy, pompon sclerosis, cause the injury of kidney basement membrane.
Set up the stripped pattern of emitting cytohormone behind the cultural method of the human kidney diaphragm cell that exsomatizes, the human kidney diaphragm cell activation and can injure the chemical substance of kidney basement membrane in the past already, and set up the zootype of IgA nephritis.People utilize the activation kidney diaphragm cell that suppresses stripped, and the zootype of acute toxicity test and IgA nephritis is found out specific composition and a kind of active ingredient that can treat IgA nephritis.
The objective of the invention is:
The specific composition (fractions) and an active ingredient that (1) will be produced in Cordyceps mycelium are used for (a) and suppress the nephridial tissue pathology that activatory kidney diaphragm cell proliferation, (b) improve zootype IgA nephritis animal and change and hematuria/albuminuria.And
(2) obtain this " active constituent " and specific " active ingredient " has separation method.
Above-mentioned purpose of the present invention realizes in the following manner:
A kind of Cordyceps is active to be formed and separation method, it is characterized in that: adopt Cordyceps (Stroma partly) in 45-50 ℃ of dark place oven dry, pulverizing the back soaked 24 hours with 20 times of methanol (w/v), Extract is through concentrating under reduced pressure, thick extract separates with silica gel column chromatography, collect dashing of 1: 1 (v/v) extremely clean ethyl acetate scope of n-hexane/ethyl acetate ratio and put forward part (F-2 layer), in fairly large separation journey, normal hexane-ethyl acetate the mixed liquor that also methanol extract (or F-2 layer) can be carried out cutting than segmentation dashes to be carried, carry out in the cumulative mode of ethyl acetate, the ratio of collecting is go out partly (C-11) of 1: 2 (v/v), F-2 all can still possess above-mentioned activity in 4 ℃ of preservations at least three months with tangible " can suppress human kidney diaphragm cell activation " biological activity of C-11 layer of equal tool, these two groups " active compositions "; Desire when obtaining " active ingredient " in " the active composition ", can remove some impurity again with the preparation silicon thin-layer chromatography with C-11 layer, its developping solution n-hexane/ethyl acetate (1: 1v/v), and expansion secondary, reclaim Rf=0.5-0.7 scope (T-4 district band), be beneficial to half follow-up preparation scale anti-phase formula high performance liquid chroma-tography and carry out purification to T-4 layer again, wherein, anti-phase formula chromatography is a methanol towards extract, and the tubing string immobile phase is C
18(8 * 250-mm, 5-μ m) type, eluent flow rate is 2ml/min, the UV-detector wavelength set is in 254nm, " the activity index composition (HA-A) " can be by complete purification.
It is characterized in that, Cordyceps strain numbering (CS-1) is inoculated in: in the liquid phase medium of glucose 2% peptone 0.5% malt extract 2% Rhizoma Solani tuber osi dextrose bouillon culture fluid 24g/l composition, under 26 ± 1.0 ℃, leave standstill cultivation (culture period 30 days), collect its mycelium, in 45-50 ℃ of oven dry, the dry weight thing that obtains grind the back and extract 24 hours with the methanol of 20 times of volumes, thick extraction quality testing pressure is concentrated, and (VGH-CS-1-MX), VGH-CS-1-MX employing anti-phase formula high performance liquid chroma-tography analyzes " active composition " and the method for " active ingredient " that contains H1-A activity index composition.
It is characterized in that isolated from Cordyceps " active form " F-2 can calculate the GI of active natural goods H1-A composition purified from F-2
50(50% propagation suppresses) is 40 little not ears (μ M), extrapolates H1-A treatment dosage thus and is about 40 milligrams.
It is characterized in that wherein the SPECTRAL DATA of active ingredient and structure are:
(1) molecular formula: C
28H
42O
2
(2)
1H-NMR: referring to Fig. 6
(3) proton fragment (EIMS) (m/z): 410 molecular ions, base peak, 395,367,349,325,285,267,253,241,213,191,173,125.
(4)
13C-NMR(CDCl
3)(δ)：
11.95(q)，17.65(q)，19.66(q)，19.97(q)21.12(q)，23.73(q)，24.59(t)，27.74(t)，29.47(t)，30.69(t)，33.11(d)，34.67(t)，35.57(t)，40.31(d)，41.81(d)，41.89(t)，42.35(s)，42.88(d)，48.43(d)，53.35(d)，72.05(d)，126.81(d)，132.15(d)，134.05(s)，135.49(d)，160.96(s)，161.60(s)，186.65(s)，
Because IgA nephritis is clinically with paroxysmal hematuria (broad perspectives or microcosmic) and/or albuminuria performance, IgA nephritis is often with the chronic disease performance, and it is carrying out to be accompanied by the asymptomatic course of disease in the middle of the acute exacerbation.According to reports, France, 20 years about 20 to 30% its courses of disease of patient in Italian and Hispanic data show morbidity back can proceed to renal failure.Therefore can estimate approximately and proceed to renal failure gradually by diagnosing out this to count annual about 1-2% patient after being ill.Very unfortunate, as not have confirmation to stop this disease to be carried out fully so far Therapeutic Method.Why can form IgA nephritis, not quite clear so far, yet gone up by the section of the kidney of IgA nephritis and can see a large amount of IgA and the 3rd complement (C3) is deposited on kidney diaphragm portion, expression is caused kidney to injure through reducing road activity complement by the IgA immunocomplex is deposited on kidney diaphragm portion probably again.
These cause nephropathy IgA immunocomplex and how to cause the injury of kidney pompon, what sketch then is: the kidney diaphragm cell that is dormant state originally, via after causing nephropathy and exempting from negative fit stimulation, can make kidney diaphragm cell activation, activatory kidney diaphragm cell can be emitted first Jie's white matter (IL-1), the 6th Jie's white matter (IL-6), tumor necrosis factor (TNF α), make kidney diaphragm cell proliferation, differentiation, comprise PDGF and emit various somatomedin, growth factor beta transition (TGF-β) etc., the effect of these cytohormones and somatomedin is as from secretin, can make the hypertrophy of kidney diaphragm be vicious cycle, and can impel kidney diaphragm cell to emit O
2, H
2O
2, platelet activating factor, prostaglandin, Thromboxan B
2(TXB
2), Neutral proteinaxe etc., therefore cause interstitial proliferation, pompon sclerosis, cause the injury of kidney basement membrane.
Treating people as can be known by the above wishes by stoping the synthetic or IgA immunocomplex place of IgA to be set about, yet the technology of immunology is to accomplish in today, old friends have to take the second best, hope is with special separation method, in the natural thing of commonly seeing, extract and to suppress activatory kidney diaphragm cell, make it no longer to breed and emit on the medicines structure of cytohormone and growth factor and set about; Breed when activatory kidney diaphragm cell is suppressed no longer, the step of backward disengaging cytohormone, somatomedin promptly is blocked, so we seek the in-vitro screening method of curative to suppress activatory kidney diaphragm cell proliferation for us.Laboratory at us can be with kidney diaphragm cell activation, with (
3H)-and the bonded method of thymidine can be used as the synthetic index of DNA, and old friends add IL-1 and IL-6 and make it activation with the above-mentioned cultivation mankind's kidney diaphragm cell, reduce to add it " active ingredient " of Cordyceps (
3H)-thymidine is in conjunction with the medicine pattern that suppresses the kidney diaphragm cell proliferation of activation as in vitro Screening.
Intravital screening system: zootype must possess specificity, can cause the nephridial tissue damage to reach and the closely similar characteristic of corresponding human diseases, based on above-mentioned consideration, it is to adopt the IgA nephritis pattern that the people set up such as Rifai A that the present invention tests, employed antigen is R36A, and it is by purified a kind of C one polysaccharide that comes out of a kind of streptococcus pneumoniae.The antibody that uses is the IgA monoclonal antibody of antagonism R36A, and this kind house mouse myeloma cell strain: the strain of TEPC-15 hybridoma cell can be produced the IgA monoclonal antibody of (secretion) anti-phosphorylcholine (PC), and this monoclonal antibody can combine with PC very single-mindedly.When above-mentioned antigen and antibodies are in the same place, promptly form the IgA immunocomplex, experimentally be from house mouse lumbar injection antigen (R36A), by tail vein injection antibody (the IgA monoclonal antibody of anti-R36A), in the house mouse blood vessel, can form the IgA immunocomplex, be circulated to kidney and be deposited on kidney diaphragm portion through blood, cause hematuria/albuminuria.The kidney of mouse is taken out the histological examination of doing the renal tissue section, and as seen reaching with the human identical variation of IgA nephritis with ematoxyline-Eosin (HE) dyeing is kidney diaphragm cell proliferation one by one, kidney diaphragm hypertrophy.Multiple do with frozen section that fluorescence staining can be seen and the mankind's the identical variation of IgA nephritis is IgA one by one and is deposited on kidney diaphragm portion.
Toxicity:
Cordyceps is to the house mouse lumbar injection, its LD
50Value is the LD of 21.7 ± 2.6g/kg intravenous injection
50Be 24.5 ± 2.2g/kg.Oral maximum tolerated dose is 252.5-300g/kg, illustrate no matter adopt intravenous injection, lumbar injection or irritate stomach, its toxicity is all very low, and the anxious toxotest that case of the present invention is done, its method is: to contain after " active form " feedstuff feeding ICR house mouse of 2% 5 days it sacrifice, have or not anxious toxicity to inspect.
So, Cordyceps is that a pharmacological action is extensive, and the medicine that toxicity is very little, research in the past known " IgA nephritis " is a kind of by nephritis that immunologic mechanism caused, and not having specific drugs at present can treat, based on this reason, causing people is object of study with Cordyceps, and reach " improve and bring out mice generation IgA nephritis in the body " with " suppress In vitro culture activatory human kidney diaphragm hyperplasia " be screening technique, seek in the Cordyceps and can suppress the hyperplasia of activatory kidney diaphragm exsomatizing, effectively " activation is formed " that can stop IgA nephritis to take place in the body and/or worsen is with " active ingredient is in the hope of being used for the treatment of IgA nephritis.
And will be described in further detail by drawings and Examples about obtaining " the active composition " and the explanation of " active component ".
Fig. 1 extracts active the composition and the active ingredient (flow process of H1-A) for the present invention from the Cordyceps Stroma.
Fig. 2 forms the anti-phase liquid chromatography (LC) spectrum of T-4 (numbering CS-F2-C11-T4) for the present invention is active.
Fig. 3 is the reverse-phase chromatography spectrum of the active ingredient H1-A of the complete purification of the present invention.
Fig. 4 is the reverse-phase chromatography spectrum that Cordyceps liquid phase of the present invention is cultivated mycelium methanol extract.
Fig. 5 is an active ingredient H1-A structural diagrams of the present invention.
Fig. 6 for the hydrogen nuclear magnetic resonance of active ingredient H1-A of the present invention (
1H-NMR) spectrum.
Referring to Fig. 1, extract the method for Active components and active ingredient (H1-A) from the Cordyceps sinensis stroma for the present invention, at first the Cordyceps sinensis finished product is adopted baking box oven dry (35-60 ℃) or air-dryly all can, because Cordyceps sinensis finished product water content is very high, take more polar substances out of when dry processing can be avoided follow-up extraction, thereby affect the performance of silica gel column chromatography purifying. Dry product must grind with grinder or pulverizer, to improve the efficient that extracts.
Because the contained active ingredient of Cordyceps sinensis (refer to the described kidney diaphragm cell that can suppress activation of the present patent application specification and improve renal function), the scope that belongs to low polarity extracts and all can extract fully with methyl alcohol (or alcohols of other low carbon number), acetone, ether, ethyl acetate, chloroform, carrene. Consider in the lump if active ingredient is reclaimed the factors such as high and the non polar impurities that is drawn in the lump be less, then to use methyl alcohol and ethyl acetate the most suitable, then use methyl alcohol among the embodiment, the program of its extraction is then such as person as described in the flow process among the figure.
And chromatography method of the present invention is asked attached ginseng the 2-3 figure, and it can be divided into two kinds of scopes:
(a) in a Cordyceps sinensis corpse or other object for laboratory examination and chemical testing on a small scale, predict the method for its contained biologically active and a kind of active ingredient.
(b) in fairly large Cordyceps sinensis, set up the chromatography flow process according to activity index, to obtain " Active components " and a kind of " active ingredient ".
With regard to a item (small-scale chromatography):
A little Cordyceps sinensis methyl alcohol Extract is passed through the little tubing string of anti-phase formula (reversed phasecartridgecolumn, annotate, this tubing string is activation in advance), and continue to carry with the methyl alcohol punching, Active components and active ingredient are not adsorbed, but can remove the much impurity of very low polarity. Rush that extract can carry out screening or with anti-phase formula high performance liquid chromatography (HPLC), active ingredient (H1-A) is carried out quantitatively (chromatography condition sees that embodiment is described), concentrate also can carry out active primary dcreening operation.
If use ethyl acetate extraction Cordyceps sinensis, after then will extracting liquid and suitably concentrating, the n-hexane (or benzinum) that adds equal proportion, mixed liquor filters in modes such as mineral wools in advance, then by a small-sized silicagel column, tubing string continues that (1: 1V/ V) punching is carried, and rushes extract and can carry out high performance liquid chroma-tography or active detection as aforementioned after concentrating with ethyl acetate/n-hexane.
With regard to the b item (fairly large chromatography):
Because the host of Cordyceps sinensis is the larval phase insect, so finished product is piled up extremely many low polar metabolites, cause the more low very big interference of polar impurity that belongs to, so in the purifying flow process include in two-period form silica gel column chromatography (namely repeating secondary (positive) or silica gel column chromatography) for the first time chromatography be at the graduation Active components and other are in a large number than the lower a group impurity of the polarity of active ingredient, the mode of roughly latter being mixed low ratio ethyl acetate with n-hexane is rushed proposition (such as n-hexane: ethyl acetate=4: 1v/v), immediately draw high then mobile phase polarity, " Active components " punching that contains not only a kind of active ingredient is put forward. This step first can amputation the impurity at height two ends, the usefulness that provides immediately the very high Active components of active amplification can carry out the pathology zoopery, in addition person thereby significantly reduce for the second time scale of silica gel column chromatography. According to Cordyceps sinensis composition characteristic, front its chromatography can be considered the big graduation of polarity, and the latter draws by can be considered the segmentation that each given activity is formed with active ingredient, and needn't be considered as repetition. If intend carrying out chromatographic purifying from the mycelium Extract, the process that then only must carry out aforementioned second stage gets final product, and the low polar impurity contained because of mycelium significantly is low than the stroma finished product.
Preferred embodiment of the present invention:
1. adopt Cordyceps (Stroma partly) in 45-50 ℃ of dark place oven dry, pulverizing the back soaked 24 hours with 20 times of methanol (w/v), Extract is through concentrating under reduced pressure, thick extract separates with silica gel column chromatography, collects dashing of 1: 1 (v/v) extremely clean ethyl acetate scope of n-hexane/ethyl acetate ratio and puies forward part (F-2 layer).In fairly large separation process, the normal hexane-ethyl acetate mixed liquor that also methanol extract (or F-2 layer) can be carried out finer partition dashes to be carried, and carries out in the cumulative mode of ethyl acetate, and the ratio of collecting is go out partly (C-11) of 1: 2 (v/v).F-2 and C-11 layer of all tangible " can suppress human kidney diaphragm cell activation " biological activity, these two groups " active compositions " all can still possess above-mentioned activity in 4 ℃ of preservations at least three months.
Desire when obtaining " active ingredient " in " the active composition ", can remove some impurity again with the preparation silicon thin-layer chromatography with C-11 layer, its developping solution n-hexane/ethyl acetate (1: 1v/v), and launch secondary, reclaim Rf=0.5-0.7 scope (T-4 district band).Though this process is identical with the separation mechanism of aforementioned silica gel column chromatography, but still can remove some impurity of C-11 layer again, carry out purification to T-4 layer again to help half follow-up preparation scale anti-phase formula high performance liquid chroma-tography.Wherein, anti-phase formula chromatography is a methanol towards extract, and the tubing string immobile phase is C
18(8 * 250-mm, 5-μ m) type is 2ml/min towards the extract flow velocity, and the UV-detector wavelength set is in 254nm, and " activity index composition (H1-A) " can be by complete purification.
" active form " and purification active ingredient are obtained in summary, and (flow and method of H1-A) sees also accompanying drawing 1.The anti-phase liquid chromatography (LC) of the active T-4 of composition is composed and the H1-A purity of complete purification sees also accompanying drawing 2,3.
Mycelium is cultivated and is identified that " activity index composition " method of production is as described below.
Cordyceps strain numbering (CS-1) is inoculated in the liquid phase medium that contains following composition: glucose 2% peptone 0.5% malt extract 2% Rhizoma Solani tuber osi-dextrose bouillon culture fluid 24g/l
Under 26 ± 1.0 ℃, leave standstill cultivation (culture period 30 days), collect its mycelium, in 45-50 ℃ of oven dry.Methanol with 20 times of volumes after institute's dry weight thing that obtains grinds extracts 24 hours, slightly extracts the quality testing pressure out and concentrates (VCH-CS-1-MX), and VGH-CS-1-MX adopts the analysis of anti-phase formula high performance liquid chroma-tography, confirms to contain H1-A activity index composition (seeing shown in Figure 4).
Total concluding a research item one content contains the method for obtaining and identify " the active composition " and " active ingredient " in partly artificial certainly Cordyceps and the liquid phase mycelia culture.
2. " active form " and " active ingredient (H1-A) "
A. " active form ": to the separation process that is purified into active ingredient (H1-A), isolated activity shows powerhouse in the composition of each chromatography, comprises F-2, C-11, T-4 after referring to be extracted out by methanol.
B. " active ingredient " refer to H1-A (SPECTRAL DATA and structure and hydrogen nuclear magnetic resonance (
1H-NMR) spectrum is consulted shown in the accompanying drawing 5,6).In addition, the application potential of above-mentioned in order to confirm " the active composition ", avoiding it is an obvious poisonous or mutagenic matter, once carries out white mice acute toxicity test and Ames test (Ames Test) in the F-2 graduation stage, all shows no overt toxicity and mutagenicity.
3. the detection mode that adopted of the present patent application case has two (exsomatize with live body detection mode each one).One is pattern for utilizing the activatory human kidney diaphragm cell of inhibition that exsomatizes, measuring Cordyceps " the active composition " or " active ingredient " handles down, to the downtrod degree of activation that caused by first Jie's white matter and the 6th Jie's white matter is activity index, kidney diaphragm cell activation degree then indicates it with isotope
3The efficient that the H-thymidine changes into DNA is foundation (consulting subordinate list 1-1 to 1-5).
4. acute toxic test: to contain and not contain each 6 of 2%CS-F2 feedstuff feeding ICR house mouses after 5 days, with it sacrifice, its result is shown in subordinate list 2, the matched group that brings out IgA nephritis with the IgA immunocomplex can cause can obviously improve the swollen phenomenon of liver (average liver heavy/body weight ratio is 6.75 ± 0.09) behind hepatomegaly phenomenon (average liver heavy/body weight ratio is 7.12 ± 0.12) the feeding 2%F-2, all the other liver functions then two groups all in normal range, (consult shown in the subordinate list 2).
5. the living animal pattern adopts then respectively that (mode of α-R36A-IgAmAb) makes to produce class IgA nephritis disease, and animal has hematuria and albuminuria phenomenon, and pathological examination also can show IgA nephritis pathological changes from lumbar injection house mouse antigen (R36A) and tail vein injection antibody.Cordyceps " active form " (as F-2) then with 0.5% and 1% mixed in feedstuff, to test its effect of improving aforementioned symptom and pathological manifestations (consulting shown in the subordinate list 3-1 to 3-4), also support Cordyceps effectively to truly have " the active composition " effect of improvement experimental " IgA nephritis ".
In sum, the present invention's isolating from Cordyceps " active composition " F-2, in the external kidney diaphragm hyperplasia that suppresses activation, cause the carrying out of the step of kidney injury with blocking-up backward, can stop IgA nephritis to worsen in vivo, and improve hematuria/albuminuria, and F2 can't produce acute toxicity, even can improve the hepatomegaly phenomenon of being brought out because of the IgA immunocomplex, calculate the GI of the purified active natural goods H1-A composition that comes out from F2
50(50% propagation suppresses) is 40 little not ears (μ M), calculate to be about H1-A40 milligram thus, promptly effective in cure in treatment human IgA nephritis, and this kind Cordyceps of the present invention activity is formed and separation method is not seen on any open publication, meets three property requirements of application for a patent for invention.
The above person is that the present invention extracts the preferable specific embodiment of one of special construction active ingredient with the particular separation method; if the change of being done under this invention's idea; it is included when spiritual that its function does not exceed description and diagram yet, all should be in protection scope of the present invention.
Table 1-1: the graduation F-2 of the methanol extract of Cordyceps is exsomatizing to activatory people
Class kidney diaphragm cell inhibiting effect 0 50 micrograms/microlitre 100 mcg/ml F-2 (n=3) 0 51.0 ± 6.3%
*94.0 ± 11.3% matched group (n=3) 000
* suppress percentage ratio
Matched group: for not adding the difference of Cordyceps
Table 1-2: the graduation C-11 of the methanol extract of Cordyceps is exsomatizing to activatory people
The effect of class kidney diaphragm cell inhibiting
0 50 micrograms/microlitre 100 mcg/ml C-11 (n=3) 0 88.4 ± 11.6%
*90.4 ± 11.8%F-2 (n=3) 0 23.9 ± 2.3%
*84.4 ± 10.5% matched group (n=3) 000
* suppress percentage ratio
Matched group: for not adding the difference of Cordyceps
Table 1-3: the composition T-4 of the methanol extract of Cordyceps is exsomatizing to activatory people
The effect of class kidney diaphragm cell inhibiting
0 50 micrograms/microlitre 100 mcg/ml C-11 (n=3) 0 69.0 ± 9.7%
*99.0 ± 10.5%T-4 (n=3) 0 85.0 ± 5.0% 98.0 ± 0.7% matched group (n=3) 000
* suppress percentage ratio
Matched group: for not adding the difference of Cordyceps
Table 1-4: the composition H1-A of the methanol extract of Cordyceps
Exsomatizing to the effect of activatory human kidney diaphragm cell inhibiting
0 10 mcg/ml, 20 mg/ml, 40 mcg/ml, 50 mg/ml T-4 (n=3) 0 42.2 ± 9.9%
*66.3 ± 18.5% 71.5 ± 2.5% 97.9 ± 3.5%H1-A (n=3) 0 23.0 ± 9.6% 51.2 ± 11.9% 66.3 ± 6.8% 82.7 ± 11.5% matched group (n=3) 0000
* suppress percentage ratio
Matched group: for not adding the difference of Cordyceps
Table 1-5: the composition H1-A of the methanol extract of Cordyceps and positive control (Lovastatin)
Exsomatizing to the effect of activatory human kidney diaphragm cell inhibiting
20 little not ear 50 little not ear 75 little not ear 1000 little not ear H1-A (n=3) 30.8 ± 4.9% 61.6 ± 7.5% 69.5 ± 5.6% 83.2 ± 3.5%Lovastatin (n=3) 19.8 ± 2.0% 37.1 ± 4.3% 60.9 ± 3.8% 73.0 ± 4.0% matched groups (n=3) 0000
Table 2: the F-2 that raises with normal feedstuff and 2% ratio is mixed in feedstuff
Acute toxic test to IgA nephritis house mouse
| Matched group (6) | 1%F2 (6) | |
| Heavy (gram) body weight (gram) liver of liver weight/body weight ratio (100 grams/gram) liver function: alanine pyruvic acid transaminase (ALT) (units per liter) bran wheat acid oxalic acid transaminase (AST) (units per liter) cholesterol (milligram/gram liver) | ????1.03±0.03 ????14.5±0.34 ????7.12±0.12 ????83.3±10.3 ????315±36.9 ????1.76±0.18 | ?1.00±0.04 ?14.8±0.40 ?6.75±0.09 ?77.5±3.59 ?346±24.2 ?1.46±0.02 |
Table 3-1: raise with normal feedstuff and be mixed in feedstuff with the F-2 of 0.5% and 1% ratio respectively
Histopathology influence to IgA nephritis house mouse
No.1????No.2????No.3????No.4????No.5????No.6????Total
(No. 1) (No. 2) (No. 3) (No. 4) (No. 5) (No. 6) (sum total) 1%F-2 (n=6) 2+ 2+ 2+ 2+ 2+ 2+ 12+
*0.5%F-2 (n=6) 4+ 4+ 4+ 3+ 4+ 3+ 22+ matched group (n=6) 4+ 4+ 4+ 4+ 4+ 4+ 24+
Grade: 0,1+, 2+, 3+, 4+ (5 grades)
H﹠amp; E dyeing; 1%F-2, v.s. matched group: P＜0.05
Feeding 1%F-2 group has the improvement on the significant histopathology
Table 3-2: raise with normal feedstuff and be mixed in feedstuff with the F-2 of 0.5% and 1% ratio respectively
Kidney immunopathology result to IgA nephritis house mouse
IgA immunocomplex deposition
No.1??????No.2??????No.3????No.4?????No.5????No.6????Total
(No. 1) (No. 2) (No. 3) (No. 4) (No. 5) (No. 6) (sum total) 1%F-2 (n=6) 0 1+ 1+ 0 1+ 1+ 4+
*0.5%F-2 (n=6) 1+ 1+ 1+ 1+ 1 1+ 6+ matched group (n=6) 2+ 2+ 2+ 2+ 2+ 1+ 11+
Grade: 1,1+, 2+, 3+ (4 grades)
1%F-2, v.s. matched group: P＜0.05
Feeding 1%F-2 group has the IgA immunocomplex deposition of remarkable minimizing kidney diaphragm portion
Table 3-3: raise with normal feedstuff and be mixed in feedstuff with the F-2 of 0.5% and 1% ratio respectively
Kidney immunofluorescence check result to IgA nephritis house mouse
The 3rd complement deposit
No.1?????No.2????No.3????No.4????No.5????No.6????Total
(No. 1) (No. 2) (No. 3) (No. 4) (No. 5) (No. 6) (sum total) 1%F-2 (n=6) 1+ 0 1+ 0 1+ 0 3+
*0.5%F-2 (n=6) 2+ 1+ 2+ 0 3+ 1+ 9+ matched group (n=6) 2+ 2+ 2+ 2+ 3+ 3+ 14+
Grade: 0,1+, 2+, 3+ (4 grades)
1%F-2, v.s. matched group: P＜0.05
Feeding 1%F-2 group has the 3rd complement deposit of remarkable minimizing kidney diaphragm portion
Table 3-4: raise with normal feedstuff and be mixed in the F-2 of 0.5% and 1% ratio respectively
Feedstuff is to the uroscopy result of IgA nephritis house mouse
Hematuria and/or albuminuria (＞+/ 3+)
Matched group (n=6) 0.5%F-2 (n=6) 1%F-2 (n=6) the 5th day 2/6 (33.2%) 2/6 (33.2%) 2/6 (33.3%) the 6th day 2/6 (33.3%) 2/6 (33.2%) 2/6 (33.3%) the 7th day 3/6 (50.0%) 1/6 (16.6%)
*1/6 (16.6%)
*
*P＜0.05
By the 7th day, the house mouse of feeding 0.5% and 1%F-2 group had remarkable minimizing hematuria and albuminuria
Claims (4)
1. the active separation method of forming of a Cordyceps, it is characterized in that: adopt Cordyceps (Stroma partly) in 45-50 ℃ of dark place oven dry, pulverizing the back soaked 24 hours with 20 times of methanol (w/v), Extract is through concentrating under reduced pressure, thick extract separates with silica gel column chromatography, collect 1: 1 (v/v) can be put in the large-scale separation journey of n-hexane/ethyl acetate ratio to clean ethyl acetate scope towards putting forward part (F-2 layer), also methanol extract (or F-2 layer) can be segmented other normal hexane-ethyl hexanoate mixed liquor towards carrying, with the cumulative mode row of ethyl acetate it, the ratio of collecting is the elution part (C-11) of 1: 2 (v/v), F-2 all can still possess above-mentioned activity in 4 ℃ of preservations at least three months with tangible " can suppress human kidney diaphragm cell activation " biological activity of C-11 layer of equal tool, these two groups " active compositions "; Desire when obtaining " active ingredient " in " the active composition ", can remove some impurity again with the preparation silicon thin-layer chromatography with C-11 layer, its developping solution n-hexane/ethyl acetate (1: 1v/v), and expansion secondary, reclaim Rf=0.5-0.7 scope (T-4 district band), be beneficial to half follow-up preparation scale anti-phase formula high performance liquid chroma-tography and carry out purification to T-4 layer again, wherein anti-phase formula chromatography eluant is a methanol, and the tubing string immobile phase is C
18(8 * 250-mm, 5-μ m) type, eluent flow rate is 2ml/min, and the UV-detector wavelength set is in 254nm, and " activity index composition (H1-A) " can be by purification person fully.
2. as claim 1 an active separation method of forming of described a kind of Cordyceps, it is characterized in that, Cordyceps strain numbering (CS-1) is inoculated in: in the liquid phase medium of glucose 2% peptone 0.5% malt extract 2% Rhizoma Solani tuber osi dextrose bouillon culture fluid 24g/l composition, under 26 ± 1.0 ℃, leave standstill cultivation (culture period 30 days), collect its mycelium, in 45-50 ℃ of oven dry, the dry weight thing that obtains grind the back and extract 24 hours with the methanol of 20 times of volumes, (VGH-CS-1-MX), VGH-CS-1-MX adopt anti-phase formula high performance liquid chroma-tography and analyze " the active composition " and the method person of " active ingredient " of containing H1-A activity index composition thick extract concentrating under reduced pressure.
3. as claim 1 an active separation method of forming of described a kind of Cordyceps, it is characterized in that isolated from Cordyceps " the active composition " F-2, the GI of purified active natural goods H1-A composition from F-2
50(50% propagation suppresses) is 40 little not ears (μ M), and H1-A treats dosage and is about 40 milligrams.
4. as claim 1 an active separation method of forming of described a kind of Cordyceps, it is characterized in that the structure of active ingredient is:
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| Application Number | Priority Date | Filing Date | Title |
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| CN94108215A CN1076623C (en) | 1994-07-25 | 1994-07-25 | Active component of cordyceps sinensis and its separation method |
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| CN94108215A CN1076623C (en) | 1994-07-25 | 1994-07-25 | Active component of cordyceps sinensis and its separation method |
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| CN1115671A true CN1115671A (en) | 1996-01-31 |
| CN1076623C CN1076623C (en) | 2001-12-26 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103751224A (en) * | 2014-01-27 | 2014-04-30 | 正源堂（天津）生物科技有限公司 | Method for extracting antitumor active components from cordyceps militaris and application thereof |
| CN103784481A (en) * | 2014-01-27 | 2014-05-14 | 正源堂（天津）生物科技有限公司 | Method for extracting anti-tumor active component from Cordyceps militaris and application of active component |
| CN106032547A (en) * | 2015-03-10 | 2016-10-19 | 上海医药工业研究院 | Preparation method and purification method of ergosterol compounds |
-
1994
- 1994-07-25 CN CN94108215A patent/CN1076623C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103751224A (en) * | 2014-01-27 | 2014-04-30 | 正源堂（天津）生物科技有限公司 | Method for extracting antitumor active components from cordyceps militaris and application thereof |
| CN103784481A (en) * | 2014-01-27 | 2014-05-14 | 正源堂（天津）生物科技有限公司 | Method for extracting anti-tumor active component from Cordyceps militaris and application of active component |
| CN103784481B (en) * | 2014-01-27 | 2016-06-01 | 正源堂（天津）生物科技有限公司 | A kind of method and application thereof extracting antitumor component from Cordyceps militaris (L.) Link. |
| CN106032547A (en) * | 2015-03-10 | 2016-10-19 | 上海医药工业研究院 | Preparation method and purification method of ergosterol compounds |
| CN106032547B (en) * | 2015-03-10 | 2020-05-19 | 上海医药工业研究院 | Preparation method and purification method of ergosterol compound |
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| CN1076623C (en) | 2001-12-26 |
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