CN102697822B - Application of sargentg loryvine stem active fraction to preparation of anti-inflammatory-immune and anti-ageing medicaments - Google Patents

Application of sargentg loryvine stem active fraction to preparation of anti-inflammatory-immune and anti-ageing medicaments Download PDF

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CN102697822B
CN102697822B CN201210180781.8A CN201210180781A CN102697822B CN 102697822 B CN102697822 B CN 102697822B CN 201210180781 A CN201210180781 A CN 201210180781A CN 102697822 B CN102697822 B CN 102697822B
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caulis sargentodoxae
water
preparation
fraction
ethyl acetate
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汤建
马瑞丽
欧阳臻
张蓉
徐秀泉
于小凤
闻崇炜
赵神洲
许辉
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Jiangsu University
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Abstract

The invention discloses an application of a sargentg loryvine stem active fraction to preparation of anti-inflammatory-immune and anti-ageing medicaments, which belongs to the technical field of medicine. A method for applying the sargentg loryvine stem active fraction comprises the following steps of: performing reflux extraction on sargentg loryvine stem with water, and precipitating with ethanol to obtain sargentg loryvine stem polysaccharides and water-soluble fractions; and performing reflux extraction on sargentg loryvine stem with 50-80 percent ethanol, gradually leaching with petroleum ether, methylene dichloride and ethyl acetate in sequence to obtain a petroleum ether fraction, a methylene dichloride fraction and an ethyl acetate fraction, and treating a residual water phase with a macroporous adsorption resin HP-20 to obtain a water-soluble fraction. As proved by pharmacodynamics experimental research, the four fractions have remarkable inhibiting effects on xylene-caused mouse ear edema and carrageenan-caused rat paw edema, and the contents of MDA (Methane Dicarboxylic Aldehyde) and PGE2 (Phenyl Glycidyl Ether 2) in voix pedis inflammation tissues can be lowered. The four fractions have high in-vitro antioxidant activity, the content of MDA in blood serum and liver tissues can be lowered effectively, and the SOD (Super Oxygen Dehydrogenises) activity is increased. The sargentg loryvine stem active fraction can be applied to preparation of anti-inflammatory-immune medicaments and anti-ageing medicaments.

Description

Caulis Sargentodoxae active site, the application in preparing anti inflammatory immunity and antiaging agent
Technical field
The invention belongs to medical technical field, be specifically related to from Chinese crude drug Caulis Sargentodoxae the position that preparation has the active and activity of fighting against senium of anti inflammatory immunity, and in the application of preparing aspect anti inflammatory immunity medicine and antiaging agent.
Background technology
Caulis Sargentodoxae is the rattan of Sargentodoxaceae plant Caulis Sargentodoxae Sargentodoxa cuneata (Oliv.) Rehd.et Wils..There is the effects such as removing toxicity for eliminating carbuncles, promoting blood circulation and stopping pain, expelling wind and removing dampness, clinically separately or prescription in order to treat the diseases such as traumatic injury, rheumatic arthritis.Recent study discovery, Caulis Sargentodoxae mainly contains the chemical compositions such as phenols, organic acid, lignanoid, triterpene saponin, flavone, anthraquinone.Modern pharmacology research shows that Caulis Sargentodoxae extract or compound have the biological activitys such as protection cardiovascular system, antiviral, antitumor, antibacterial, antioxidation.
Caulis Sargentodoxae water decoction shows immunosuppressive action in mouse model, can reduce phagocytic index, IgM growing amount, periphery blood T lymphocyte number and T, the bone-marrow-derived lymphocyte conversion ratio of mice, to the equal inhibited (Li Li of the humoral immunization of the cellular immunization of the T cell mediated of mice and bone-marrow-derived lymphocyte mediation, Deng. Chinese Journal of Immunology .2009,25 (3), 223-224).Caulis Sargentodoxae decocting liquid obviously reduce lymphocyte of adjurant arthritis rat matrix metalloproteinase MMP-2 and MMP-9 expression (pay treasure, etc. Guizhou medicine .2009,33 (12), 1097-1099).Two kinds of phenol glucosides in Caulis Sargentodoxae have the activity of obvious inhibition sheep gonad prostaglandin synthetase, and have obvious anti-inflammatory activity (JP 06199885. for Sakakibara I, et al.).
Caulis Sargentodoxae is as a kind of conventional Chinese medicine, and without obvious toxic-side effects, clinical use safety, is suitable for doing further research and development.The at present research of this medical material aspect anti inflammatory immunity be take medical material water decoction and individual compound as main, there is no that Caulis Sargentodoxae extract is further refined to active site and for the preparation of the report of anti inflammatory immunity medicine.Research and the application aspect defying age of Caulis Sargentodoxae extract and active site also has no report.
In order to test and assess the anti inflammatory immunity activity of Caulis Sargentodoxae active site of the present invention and the application potential of defying age aspect, used the biological activity at method test well-known to those skilled in the art each position of medical material.These known method of testings comprise that mice caused by dimethylbenzene xylene ear swelling method of testing, carrageenin cause rat paw edema method of testing, and inside and outside antioxidation model etc., it is active that result shows that selected Caulis Sargentodoxae active site has good anti inflammatory immunity, in antioxidation model, there is better activity in vivo and in vitro, show good defying age application potential.
Summary of the invention
The object of this invention is to provide the effective site that Caulis Sargentodoxae has anti inflammatory immunity activity and defying age application potential.
Caulis Sargentodoxae polysaccharide, water soluble part W, through anti-inflammatory activity screening widely, are confirmed in each position of Caulis Sargentodoxae extract 1ethyl acetate fraction and water soluble part W with Caulis Sargentodoxae 50%-80% ethanol extraction 2there is remarkable inhibition mice caused by dimethylbenzene xylene ear swelling and carrageenin and cause rat paw edema effect, demonstrate good anti inflammatory immunity active, and show good antioxidation activity in vitro in removing DPPH free radical and reducing power experiment, can significantly reduce malonaldehyde in serum and liver organization (MDA) content, and increase superoxide dismutase (SOD) activity, the extract that shows Caulis Sargentodoxae has good inside and outside antioxidation, has antidotal potentiality.
Therefore, first aspect of the present invention relates to provides four kinds of active sites of Caulis Sargentodoxae and preparation method thereof:
Caulis Sargentodoxae polysaccharide (HT pS) and water soluble part W 1(HT w1) preparation method, according to following step, carry out: dry Caulis Sargentodoxae medical material, be crushed to 20 order coarse powder, powder is water reflux, extract, twice at 100 ℃, each 3 hours, after extracting solution concentrating under reduced pressure, adds dehydrated alcohol, the precipitation of spending the night at 4 ℃-20 ℃, preferred 5-15 ℃; The centrifugal 10-30min of 3000r/min, preferably 15-20min; Drying precipitated part obtains Caulis Sargentodoxae polysaccharide (HT pS); Concentrated supernatant, to the 1/30-1/5 of original volume, is preferably concentrated into the 1/20-1/10 of original volume; Cross HP-20 macroporous adsorbent resin, first wash with water to closely colourless, then use 2BV-10BV 80% (v/v) ethanol elution, preferably 4BV-6BV; Flow velocity is 10-60mL/min, preferably 20-30mL/min; Collect 80% ethanol (v/v) eluent, concentrated, lyophilization obtains water soluble part W 1(HT w1).
Caulis Sargentodoxae ethyl acetate extract (HT aE) and water soluble part W 2(HT w2) preparation method, according to following step, carry out: in Caulis Sargentodoxae medicinal powder, add 8-12 50%-80% alcohol reflux doubly, and filtered and recycled ethanol.The aqueous dispersion of Caulis Sargentodoxae ethanol extraction, uses petroleum ether, dichloromethane, ethyl acetate fractional extraction successively, and concentrating under reduced pressure reclaims solvent, obtains respectively petroleum ether part, dichloromethane position, ethyl acetate extract (HT aE), water position.Water position is concentrated into the 1/30-1/10 of original volume, is preferably concentrated into the 1/20-1/15 of original volume; Cross HP-20 macroporous adsorbent resin, first wash with water to closely colourless, then use 2BV-10BV 95% (v/v) ethanol elution, preferably 4BV-6BV; Flow velocity is 10-60mL/min, preferably 20-30mL/min; Collect 95% ethanol elution, concentrate to obtain water soluble part W 2(HT w2).
Second aspect of the present invention relates to the purposes for the preparation of anti inflammatory immunity and antiaging agent to described four positions.Observe respectively sample xylol under various dose and cause that mice ear, carrageenin cause rat paw edema degree, carrageenin causes prostaglandin (PGE in rat paw inflammatory tissue 2) and the impact of malonaldehyde (MDA) content.It is active that result shows that four active sites of Caulis Sargentodoxae all have significant anti inflammatory immunity, can be used for preparation treatment inflammatory disease as the medicine of the diseases such as rheumatism or rheumatoid arthritis.Simultaneously in model, there is significant antioxidant activity in vitro, and can significantly reduce MDA content in serum and liver organization, and it is active to increase SOD, show that Caulis Sargentodoxae active site has good potentiality preparing aspect antiaging agent.
The pharmaceutical composition that provides Caulis Sargentodoxae active site and pharmaceutically acceptable adjuvant to form is provided third aspect of the present invention.
Caulis Sargentodoxae active site can independent or several part combinations, more further with auxiliary material combination, dosage form comprises: tablet, capsule, unguentum, patch, injection etc.
Carrier of the present invention or excipient comprise carrier and the excipient of the conventional application of pharmaceutics, such as filler, binding agent, antiseptic, correctives, diluent, coloring agent etc.
The following examples and biological activity test are to further description of the present invention, below cited embodiment be construed as limiting never in any form.
The specific embodiment
Embodiment 1
Caulis Sargentodoxae polysaccharide (HT pS) and water soluble part W 1(HT w1) preparation:
Get the dry Caulis Sargentodoxae stem that 70g is ground into 20 about orders, by 8 times of amount pure water reflux, extract, twice, each 3 hours, merge extractive liquid, was evaporated to 200mL, added dehydrated alcohol to 1000mL, put 4 ℃ of refrigerator overnight precipitations, centrifugal at 100 ℃, drying precipitated must HT pS2.8g; Concentrated supernatant, crosses HP-20 macroporous adsorbent resin, first washes with water to without glucide, then is washed till clarification with 80% ethanol, collects 80% ethanol elution and concentrates, and lyophilization obtains HT w13.4g.
Embodiment 2
Ethyl acetate extract (HT aE) and water soluble part W 2(HT w2) preparation:
Get the dry Caulis Sargentodoxae stem that 80g is ground into 20 order left and right, 10 times of amount 80% ethanol water-bath reflux, extract, twice at 85 ℃, each 2h, merge extractive liquid,, decompression recycling ethanol.The aqueous dispersion of Caulis Sargentodoxae ethanol extraction, with petroleum ether, dichloromethane, ethyl acetate extraction, concentrating under reduced pressure reclaims solvent, obtains respectively petroleum ether part, dichloromethane position, ethyl acetate extract, water position successively.Water position is concentrated first to be washed with water to colourless by HP-20 macroporous adsorbent resin, then uses 95% ethanol elution to colourless, collects 95% ethanol elution and concentrates to obtain water soluble part W 2, lyophilization obtains HT aE2.62g, HT w25.96g.
Embodiment 3
HT pS, HT w1, HT aEand HT w2xylol causes the impact of mice ear reaction
Choose 80 of 20 ± 2g Kunming mouses, be divided at random 10 groups, male and female half and half, 8 every group.The 1st group: 1% tween 80, blank group; The 2nd group: 25mg/kg indomethacin, positive controls; 3rd, 4 groups: HT pShigh low dose group (200mg/kg, 100mg/kg); 5th, 6 groups: HT w1high low dose group (200mg/kg, 100mg/kg); 7th, 8 groups: HT aEhigh low dose group (200mg/kg, 100mg/kg); 9th, 10 groups: HT w2high low dose group (200mg/kg, 100mg/kg).Each organize mice with 20mL/kg gastric infusion 1h after, mouse right ear is coated with to 50 μ L caused by dimethylbenzene xylene scorching, after half an hour, the dislocation of mice cervical vertebra is put to death, the card punch that is 7mm with diameter is laid round auricle at the same position of left and right ear, analytical balance is weighed, the difference of auris dextra and left ear weight is swelling, calculates inhibitory rate of intumesce, auris dextra weight-left ear weight after swelling (mg)=cause is scorching.Inhibitory rate of intumesce=(the average swelling of the average swelling-administration of matched group group) average swelling * 100% of/matched group, experimental result is in Table 1.Data show, HT w2it is the strongest that position suppresses mice ear activity.
The impact of table 1 mice caused by dimethylbenzene xylene ear swelling (
Figure BDA00001720785000041
n=8)
No. Animal group Dosage (mg/kg) Average swelling (mg) Suppression ratio (%)
1 Blank group - 10.9±4.9 -
2 Indomethacin group 25 2.2±1.4 30.92
3 HT PS 200 4.1±2.9 ** 62.59
4 HT PS 100 7.5±3.6 30.92
5 HT W1 200 6.0±3.0 * 50.33
6 HT W1 100 6.6±2.5 * 44.95
7 HT AE 200 4.1±2.8 * 61.79
8 HT AE 100 8.3±3.2 25.24
9 HT W2 200 3.2±3.1 ** 70.53
10 HT W2 100 8.7±4.0 19.47
**p＜0.01， *p＜0.05
Embodiment 4
HT pS, HT w1, HT aEand HT w2on Carrageenan causes the impact of normal rat paw swelling reaction and chooses 60 of 180-200g male SD rats, is divided at random 10 groups, 6 every group.The 1st group: 1% tween 80, blank group; The 2nd group: 10mg/kg indomethacin, positive controls; 3rd, 4 groups: HT pShigh low dose group (100mg/kg, 50mg/kg); 5th, 6 groups: HT w1high low dose group (100mg/kg, 50mg/kg); 7th, 8 groups: HT aEhigh low dose group (100mg/kg, 50mg/kg); 9th, 10 groups: HT w2high low dose group (100mg/kg, 50mg/kg).With 20mL/ (kgd) isometric(al) gastric infusion 1 time, 4d continuously.After last administration, 1h measures the sufficient pawl volume (mL) of mark under every Rat Right metapedes pawl ankle joint upper end shank-feathering, tie-in 2 times, average as causing the initial volume before inflammation, synchronously give every rat right hind leg vola mind-set ankle joint direction inserting needle subcutaneous injection 1% carrageenin 0.1mL and cause inflammation, measure respectively the sufficient pawl volume (mL) that causes mark under inflammation rear 1.0,2.0,3.0 and 4.0h foot pawl ankle joint upper end shank-feathering, the results are shown in Table 2.Data show, except HT aEposition, the inhibitory rate of intumesce of low dose group is more remarkable, wherein HT w1it is the strongest that position suppresses swelling performance.
Swelling (ml)=to scorching metapedes volume-to scorching front foot volume
Inhibitory rate of intumesce (%)=(the average swelling of the average swelling-administration of matched group group) average swelling * 100% of/matched group
Table 2 carrageenin cause rat paw edema degree reaction impact (
Figure BDA00001720785000051
n=6)
Figure BDA00001720785000052
**p＜0.0l， *p＜0.05
Embodiment 5
HT pS, HT wl, HT aEand HT w2on Carrageenan causes MDA and PGE in rat swelling foot sole of the foot inflammation tissue 2content influence
1) assay method of MDA in scorching foot tissue:
According to embodiment 3, cause after scorching 4h, put to death rat, on articulatio talocruralis, 0.5cm cuts at place inflammatory swelling foot, weigh, after peeling, shred, the normal saline (NS) that is placed in 5mL soaks after 1h, the inflammation foot that soaked NS is immersed in 0.45mL0.5% trichloroacetic acid (TCA), after 4h, abandon scorching foot, the hydrochloric acid 1.0mL that adds 0.1mol/L, add again after 0.55mL0.67% thiobarbituricacidα-, boiling water bath 20min, add 2mLNS dilution, with the centrifugal 10min of 1000r/min, getting supernatant is that 532nm place measures its light absorption value in wavelength, with this, represent the content of MDA, experimental result is in Table 3.In each position high dose group of data declaration, inflammation tissue is subject to radical damage degree lower, wherein HT w1it is the strongest that the protection inflammation tissue at position is avoided radical damage ability.
2) prostaglandin (PGE in scorching foot tissue 2) assay method:
The sufficient normal saline soak of above-mentioned inflammation is centrifugal, draw supernatant 0.3mL, add 0.5mol/KOH-methanol solution 2mL, at 50 ℃, isomerization 20min, is diluted to 5mL with methanol, in wavelength, is that 278nm place measures its absorbance.With every gram of suitable absorbance of inflammatory tissue, represent PGE 2content, experimental result is in Table 3.From data, the inhibition PGE of low dose group 2the effect producing is stronger, wherein HT wlposition suppression ratio is the most remarkable.
The results are shown in Table 3.
Table 3 carrageenin causes MDA and PGE in rat swelling foot inflammation tissue 2content (
Figure BDA00001720785000061
n=6)
No. Group Dosage (mg/kg) MDA PGE 2
1 Blank group 0.574±0.011 0.356±0.019
2 Positive group 10 0.123±0.029 ** 0.186±0.016 **
3 HT PS 100 0.199±0.014 ** 0.280±0.015 **
4 HT PS 50 0.263±0.044 ** 0.232±0.015 **
5 HT W1 100 0.194±0.006 ** 0.244±0.028 **
6 HT W1 50 0.214±0.021 ** 0.197±0.013 **
7 HT AE 100 0.206±0.025 ** 0.318±0.012 *
8 HT AE 50 0.264±0.041 ** 0.233±0.016 **
9 HT W2 100 0.197±0.046 ** 0.251±0.014 **
10 HT W2 50 0.25±0.028 ** 0.209±0.017 **
**p<0.01， *p<0.05
Embodiment 6
HT pS, HT w1, HT aEand HT w2antioxidation activity in vitro is measured
1) mensuration of DPPH radical scavenging activity:
In the Caulis Sargentodoxae extract solution of 1mL variable concentrations, add the alcoholic solution of 1.0mL 200 μ mol/DPPH, then add mix homogeneously after 2.0mL 80% ethanol, place after 30min with ultraviolet spectrophotometer at the mensuration light absorption value A of 517nm place at dark place i, measure the alcoholic solution of 1.0mL DPPH and the light absorption value A of 3.0mL alcoholic solution mixed liquor simultaneously olight absorption value A with 3mL ethanol and 1.0mL sample mix liquid j, clearance rate formula: clearance rate (%)=[1-(A i-A j)/A 0] * 100%.Data show, HT w2and HT aEit is the strongest that DPPH ability is removed at position.
2) mensuration of reducing power:
The Caulis Sargentodoxae extract solution of 1.0mL variable concentrations, adding 2.5mLpH is 6.6 phosphate buffer and 2.5mL 1% potassium ferricyanide, after mix homogeneously, in 50 ℃ of water-baths, be incubated 20min, adding 10% trichloroacetic acid 2.5mL mixes again, with 3000r/min centrifugalize 10min, pipette supernatant 2.5mL in test tube, adding distil water 2.5mL and 0.1%FeCl 3aqueous solution 0.5mL, measures light absorption value in 700nm place after normal-temperature reaction 10min, light absorption value is higher, illustrates that the reproducibility of this reactant mixture is stronger.With Vc, contrast, with distilled water, prepare before use.Data show, HT w2and HT aEposition reducing power is stronger, but is obviously weaker than Vc.
Four kinds of active site scavenging ability of DPPH free radical experiments of table 4
Figure BDA00001720785000071
No tests; *: the concentration of Vc is respectively 10,25,50 and 100 μ g/mL
Four kinds of active site reducing power experiments of table 5
Figure BDA00001720785000072
Embodiment 7
HT pS, HT w1, HT aEand HT w2in body, antioxidation and activity of fighting against senium are measured
According to embodiment 3, cause after scorching 4h, after anesthetized rat, femoral artery is got blood, disconnected cervical vertebra is got liver after putting to death rat, by serum and liver homogenate tissue be stored in-20 ℃ to be measured, in serum and liver organization, MDA content and SOD activity data show in Table 6 results, HT w1in position removing free radical, protection body inflammatory process, avoid radical damage ability the strongest.
Embodiment 8
Caulis Sargentodoxae ethyl acetate extract (HT aE) preparation of capsule preparations
6g Caulis Sargentodoxae ethyl acetate extract (HT aE) freeze-dried powder, add 3g soluble starch and 9mL70% ethanol, mix, 60 ℃ of oven dry, pulverize.By every 0.45g, make capsule.
Embodiment 9
Caulis Sargentodoxae water soluble part W 2(HT w2) preparation of injection
1.6g Caulis Sargentodoxae water soluble part W 2(HT w2) freeze-dried powder, add 40mL distilled water, be heated to 80 ℃, add 2% (v/v) benzyl alcohol, after mix homogeneously, cross 0.22 μ m filter membrane, the clear and bright liquid embedding obtaining in 2mL ampoule, steam sterilization 30min, standby.
Table 6 carrageenin causes MDA, SOD in foot swelling rat blood serum and liver organization, GSH-Px and CAT activity influence
Figure BDA00001720785000081
**p<0.01， *p<0.05。

Claims (2)

1. the preparation method of Caulis Sargentodoxae polysaccharide and water soluble part, is characterized in that carrying out according to following step: dry Caulis Sargentodoxae medical material, is crushed to 20 order coarse powder, powder is water reflux, extract, twice at 100 ℃, and each 3 hours, after extracting solution concentrating under reduced pressure, add dehydrated alcohol, the precipitation of spending the night at 5-15 ℃; The centrifugal 15-20 min of 3000 r/min; Drying precipitated part obtains Caulis Sargentodoxae polysaccharide; Concentrated supernatant is to the 1/20-1/10 of original volume; Cross HP-20 macroporous adsorbent resin, first wash with water to closely colourless, then use 4BV-6BV volume ratio 80% ethanol elution, flow velocity is 20-30 mL/min; Collected volume is than 80% ethanol elution, and concentrated, lyophilization obtains water soluble part.
2. the preparation method of Caulis Sargentodoxae ethyl acetate extract and water soluble part, is characterized in that carrying out according to following step: in Caulis Sargentodoxae medicinal powder, add 8-12 volume ratio 50%-80% alcohol reflux doubly, and filtered and recycled ethanol; The aqueous dispersion of Caulis Sargentodoxae ethanol extraction, uses petroleum ether, dichloromethane, ethyl acetate fractional extraction successively, and concentrating under reduced pressure reclaims solvent, obtains respectively petroleum ether part, dichloromethane position, ethyl acetate extract, water position; Water position is concentrated into the 1/20-1/15 of original volume; Cross HP-20 macroporous adsorbent resin, with 4BV-6BV volume ratio 95% ethanol elution, flow velocity is 20-30 mL/min.
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