CN101966221B

CN101966221B - Application of prickly pear in preparation of medicaments or health-care products

Info

Publication number: CN101966221B
Application number: CN2010105050013A
Authority: CN
Inventors: 刘华钢
Original assignee: Individual
Current assignee: Guangxi Kang Yu Biological Ltd By Share Ltd
Priority date: 2008-09-09
Filing date: 2008-09-09
Publication date: 2011-10-26
Anticipated expiration: 2028-09-09
Also published as: CN101966221A

Abstract

The invention discloses application of prickly pear in preparation of medicaments or health-care products for reducing blood pressure, blood fat and blood sugar. Because prickly pear polysaccharides contained in the prickly pear can control the happening and development of high blood pressure, have positive significance for controlling the happening and development of hyperlipemia and fatty degeneration of liver, have good potential application value in preventing and treating diabetes and complications thereof and have a certain anti-tumor effect, so that the application of the prickly pear lays a foundation for developing the medicaments or health-care products for preventing and treating high blood pressure, hyperlipemia and diabetes and effectively promotes full use of prickly pear resources. In addition, because the prickly pear belongs to natural plant resources, has good medicinal effect and heath-care effect, has the effect of regulating and protecting a plurality of organs and systems of human bodies, has relatively low cost and side effect, and treatment based on syndrome differentiation, is flexibly increased and reduced in dosage, and is very suitable for treatment of chronic diseases, the prickly pear has good application prospect in preparation of the medicaments or health-care products for reducing blood pressure, blood fat and blood sugar.

Description

The application of fruit of Radix et Caulis Opuntiae Dillenii in preparation medicine or health product

The application is dividing an application of " application in preparation medicine or health product of fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides ", original application day is JIUYUE in 2008 9 days, application number is 200810073775.6, and denomination of invention is fruit of Radix et Caulis Opuntiae Dillenii and the application of polysaccharides in preparation medicine or health product.

(1) technical field:

The present invention relates to fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides in preparation blood pressure lowering, blood fat reducing, blood sugar lowering, the medicine of antitumor action or the application in the health product, belong to the technical field of utilizing of natural plant resource.

(2) background technology:

Weizhou Island, the North Sea, Guangxi is the extinct volcano island of China's maximum, and growth provides competent nutrient to the lava on the island to Radix et Caulis Opuntiae Dillenii, and Shi Yi tropical climate has formed wild Radix et Caulis Opuntiae Dillenii in blocks in addition.And the Radix et Caulis Opuntiae Dillenii four seasons on the island can bear a considerable number of fruit of Radix et Caulis Opuntiae Dillenii.The Weizhou Island is produced fruit of Radix et Caulis Opuntiae Dillenii and is had distinctive kermes, wherein rich in mineral substances (as selenium) and sulfacid.But up to now, the fruit of Radix et Caulis Opuntiae Dillenii that the Weizhou Island abounds with except minute quantity by local resident or traveller edible, the overwhelming majority all withered mashed in the ground, or by bird, Serpentis etc. as the meal in the abdomen, cause the significant wastage of resource, had a strong impact on the abundant development and use of fruit of Radix et Caulis Opuntiae Dillenii.

The medicinal function of Chinese herbal medicine and active component thereof has the characteristics of many target spots, multipath, too many levels, becomes the modern medicines hot of research and development, and therefore natural plants causes people's extensive concern and attention in the effect aspect disease prevention and the treatment in recent years.Radix et Caulis Opuntiae Dillenii has good ornamental value, edibility, health care value and medical value as a kind of natural plants, and the medicine source is abundant, nontoxic free from extraneous odour, development safe ready.In recent years, zoopery and human trial prove, Radix et Caulis Opuntiae Dillenii has pharmacological actions such as human body immunity improving power, blood sugar lowering, blood fat reducing, antitumor, antibiotic, antiinflammatory.Fruit of Radix et Caulis Opuntiae Dillenii (Opuntia ficus-indica) is the fruit of Opuntia (Opuntia) plant, and sarcocarp contains abundant trace element, protein, aminoacid, vitamin, polysaccharide, flavonoid and pectin etc.American Indian and Mexican have history in several thousand with it as food and drug use.The Radix et Caulis Opuntiae Dillenii rhizome is had effects such as blood fat reducing and blood sugar lowering both at home and abroad report is all arranged, but fruit of Radix et Caulis Opuntiae Dillenii whether also has blood fat reducing and the blood sugar lowering isoreactivity does not appear in the newspapers.In view of fruit of Radix et Caulis Opuntiae Dillenii resource abundance, it is bright-colored that fruit of Radix et Caulis Opuntiae Dillenii is produced in Weizhou Island, the North Sea, Guangxi, taste and sweet mouthfeel, the content height of vitamin and polysaccharide, so the applicant has carried out relevant pharmacological testing research and explores to fruit of Radix et Caulis Opuntiae Dillenii and from isolated effective site polysaccharides wherein.

(3) summary of the invention:

Technical problem to be solved by this invention is: according to the contained composition of fruit of Radix et Caulis Opuntiae Dillenii, be used for preparing the medicine or the health product of blood pressure lowering, blood fat reducing, blood sugar lowering, antitumor action with fruit of Radix et Caulis Opuntiae Dillenii and from the polysaccharides that extraction separation wherein goes out.

The present invention constitutes like this: fruit of Radix et Caulis Opuntiae Dillenii (Opuntia ficus-indica) is in preparation blood pressure lowering, blood fat reducing, blood sugar lowering, the medicine of antitumor action or the application in the health product.

Polysaccharides is in preparation blood pressure lowering, blood fat reducing, blood sugar lowering, the medicine of antitumor action or the application in the health product.

Described polysaccharides is the polysaccharide composition that extracts from fruit of Radix et Caulis Opuntiae Dillenii, and its total sugar content is 70%～80%.

The preparation method of concrete polysaccharides is: get fruit of Radix et Caulis Opuntiae Dillenii, the water supersound extraction 0.5～2.5 hour that adds 4～10 times of weight, extracting solution concentrates, the ethanol of adding 40～95%, concentration of alcohol to 40～90% of regulating concentrated solution precipitates, centrifugal 2～10min under 1000～10000rpm rotating speed then, centrifugal sediment is dissolved in water, with volume ratio is 1～5: 2～10 dehydrated alcohol and the extraction of ethyl acetate mixed liquor 1～4 time, water layer is removed albumen with Sevage+ papain method, concentrate, drying, promptly.

Wherein the detailed process of Sevage+ papain method is: with Sevage solution (volume ratio: chloroform: n-butyl alcohol=4～10: 1～5) behind the extraction water solution 1～3 time, water intaking layer liquid, transfer pH to 5～7, the papain that in aqueous solution, adds 1～4u/100ml, 45～65 ℃ of insulation 20～28h, centrifugal 2～the 10min of 1000～10000rpm removes the bottom precipitation, gets polysaccharides deproteinization liquid.

Specifically, the application process of fruit of Radix et Caulis Opuntiae Dillenii, polysaccharides is: get fruit of Radix et Caulis Opuntiae Dillenii or polysaccharides, add or do not add medicinal adjuvant, be prepared into pharmaceutical preparation according to conventional method.

Perhaps with fruit of Radix et Caulis Opuntiae Dillenii or polysaccharides and the combination of other medicinal ingredient, and add or do not add medicinal adjuvant, be prepared into pharmaceutical preparation according to conventional method.

Can also get fruit of Radix et Caulis Opuntiae Dillenii or polysaccharides, add or do not add adjuvant, be prepared into health product according to conventional method.

Perhaps with fruit of Radix et Caulis Opuntiae Dillenii or polysaccharides and other combinations of substances, add or do not add adjuvant, be prepared into health product according to conventional method.

In order to verify effect of the present invention, preparation that below will be by polysaccharides and the test of pesticide effectiveness research that fruit of Radix et Caulis Opuntiae Dillenii natural juice and polysaccharides carry out further set forth the present invention.

One, the extraction purification of polysaccharides

Get fruit of Radix et Caulis Opuntiae Dillenii, the water supersound extraction 0.5～2.5 hour that adds 4～10 times of weight, extracting solution concentrates, and adds 40～95% ethanol, concentration of alcohol to 40～90% of regulating concentrated solution precipitates, centrifugal 2～10min under 1000～10000rpm rotating speed then, centrifugal sediment is dissolved in water, and is 1～5: 2～10 dehydrated alcohol and the extraction of ethyl acetate mixed liquor 1～4 time with volume ratio, water layer is removed albumen with Sevage+ papain method, concentrate, drying promptly gets polysaccharides.

Sevage+ papain method: with Sevage solution (volume ratio: chloroform: n-butyl alcohol=4～10: 1～5) behind the extraction water solution 1～3 time, water intaking layer liquid, transfer pH to 5～7, the papain that in aqueous solution, adds 1～4u/100ml, 45～65 ℃ of insulation 20～28h, centrifugal 2～the 10min of 1000～10000rpm removes the bottom precipitation, gets polysaccharides deproteinization liquid.

After measured, polyoses content reaches 60～80% in the polysaccharides that is extracted.

Two, the experimental study of fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides (CPFP) hypotensive activity

1. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides single and continuous 9 all administrations are to the systolic pressure and heart rate (HR) influence of original hypertensive rat (SHR)

1.1 laboratory animal

Select (w) age in 16 weeks male SHR60 only, systolic pressure (SBP) 〉=23.94kPa (1kPa=7.5mmHg), body weight (250 ± 50) g is provided by Shanghai Slac Experimental Animal Co., Ltd., the quality certification number: SCXK (Shanghai) 2003-0003; Male Wistar rat of the same age is provided by Guangxi Medical University's Experimental Animal Center, the quality certification number: osmanthus is moving is betrothed to 2000 No. 001.Raising is in being equipped with the Animal House of air-conditioning, and room temperature (25 ± 2) ℃ is freely drunk water.

1.2 experimental technique

1.2.1 grouping and administration

The parallel adaptability pressure measurement training of domestication 2w waits to conform before the experiment, is divided into 6 groups at random: i.e. model group (NC): give distilled water; CPFP high dose group (HD), middle dosage group (MD), low dose group (LD): give 3.06gkg respectively ^-1D ^-1, 1.58gkg ^-1D ^-1, 0.79gkg ^-1D ^-1CPFP; Fruit of Radix et Caulis Opuntiae Dillenii natural juice group (NJ): give 3.15gkg ^-1D ^-1Fruit of Radix et Caulis Opuntiae Dillenii natural juice (CPNJ); Captopril group (PC): give 0.01gkg ^-1D ^-1Captopril; Normal control group (Wt): the Wistar rat is given distilled water.Every group 10, route of administration is for irritating stomach.

1.2.2 pressure testing method

Aiming at rat tail veutro with the small electrical hair-dryer warms, it is little when red to treat that rat tail skin becomes, with the pressure arteries and veins condom of sphygomanometer to rat tails proximal part place, high quick transducer places last 1/3 place of tail, transducer face is aimed at the afterbody veutro, fixing, degree of tightness is motionless for well with hand push with transducer.Treat after the rat calmness, open recording system, as seen there is pulse wave to occur, make the pressure of pressing in the arteries and veins cover be elevated to the pulse wave complete obiteration rising 3.99kPa that pressurizes again with gas cell, falling low-tension pulse cover internal pressure gradually by the slow venting of gas cell valve then, is zero generally holding time 5～6 seconds from beginning to be deflated to manifold pressure.Examine pulse wave and occur first ripple once more from being blocked, pairing pressure was SBP when this first ripple occurred.The meansigma methods of getting continuous 3 pressure measurement values be as surveying SBP value, measure the medication first time respectively before, SBP and the HR of SHR weekly during 2 hours (h), 4h, 6h, 8h, 12h, 24h and the successive administration 9w after the medication.And then extrapolate HR indirectly according to pulse wave.

Carry out statistical procedures 1.2.3 statistical procedures is used SPSS11.0 software, measurement data all adopts Expression, measurement data relatively adopts the t check between two groups, organizes mean more and relatively adopts variance analysis.

1.3 experimental result

1.3.1 single-dose is to SBP and the HR influence of SHR

2h promptly had tangible pressure reduction effect after single gave CPFP, and the 4h pressure reduction effect peaks.Compared significant difference (P＜0.05) before HD and the treatment during 4h, MD and LD pressure reduction effect than HD slightly a little less than, with relatively there are no significant before treatment difference (P＞0.05).After PC gave captopril, the 2h blood pressure drops peaked, and with NC with before treating significant difference (P＜0.05) was arranged relatively, and blood pressure lowering amplitude maximum is 0.80 ± 8.03kPa, is better than HD blood pressure lowering amplitude maximum (0.68 ± 8.02kPa).From the time that efficacy of antihypertensive treatment is kept, SBP returns to the preceding level of treatment behind the PC administration 8h, returns to the preceding level of treatment behind each administration group administration 12h of CPFP, and its under voltage stability is better than PC; Present tangible dose-effect and time-effect relationship from blood pressure lowering amplitude CPFP blood pressure lowering.Above-mentionedly organize respectively after the administration that HR does not have significant change (P＞0.05) in the 24h.

1.3.2 the 9w administration is to SBP and the HR influence of SHR continuously

Each administration group SBP fall behind medication 2w begins obviously, slows down to the 8w SBP range of decrease, and is basicly stable a lower level.PC, HD and MD respectively 4w, 5w and 6w begin with treatment before compared highly significant meaning (P＜0.01), before and after LD and the NJ self more variant (P＜0.05).NC is tending towards raising at the experimental session blood pressure, and the SBP 1.00kPa that on average raise behind the 9w and has compared significant differences (P＜0.01) before the treatment.Contrast PC, HD, MD, LD and NJ between group and all significantly be better than NC (P＜0.01) at 3w, 4w, 5w, 6w and 5w antihypertensive effect respectively, the maximum average of its blood pressure lowering is respectively 2.16 ± 0.18,1.59 ± 1.44,1.42 ± 0.16,0.87 ± 0.18 and 0.82 ± 0.19kPa, be better than CPFP administration group from the efficacy of antihypertensive treatment of blood pressure lowering amplitude PC, but there was no significant difference (P＞0.05) between them.In addition, also present tangible dose-effect and time-effect relationship from blood pressure lowering amplitude CPFP successive administration 9w blood pressure lowering.Each group of medication is less to the influence of HR, medication anteroposterior diameter statistical procedures there was no significant difference (P＞0.05).

2. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of original hypertensive rat (SHR) plasma renin (RA) and Angiotensin II (AngII)

2.1 laboratory animal is with experiment 1.

2.2 experimental technique

2.2.1 grouping and administration are with experiment 1, administration time is 9w.

2.2.2 experimental procedure

Successive administration 9w, fasting after the last administration (can't help water) 24h is with 20% urethanes intraperitoneal anesthesia (5mlkg ^-1), contain ethylenediaminetetraacetic acid (EDTA), dimercaptopropanol, BAL and oxine test tube from what each 2ml of abdominal aortic blood placed pre-cooling respectively, mixing, 4 ℃, 3000 rev/mins of (rpm) centrifugal 10min, separated plasma ,-20 ℃ of sealings are preserved to be measured.

The active mensuration of blood plasma RA is promptly measured the speed that the nervous plain I of blood plasma medium vessels (AngI) produces.Get double blood plasma, portion allows it directly and antibody response, surveys the concentration of its AngI, in contrast pipe; Another part allows itself and antibody response again behind 37 ℃ of incubation 30min, the concentration of surveying its AngI is for measuring pipe.The concentration of mensuration pipe AngI deducts the concentration of control tube AngI and divided by the incubation time, then is the generation speed of AngI in the unit interval, claims the RA activity.

The measurement operation step of AngII level is carried out in strict accordance with analyzing the medicine box operation instructions, treat mixing behind the application of sample EO, room temperature is placed 15min, the centrifugal 15min of 3500rpm, suct clear liquid mensuration and respectively manage sedimentary radiocounting (cpm), and directly provided related parameter, standard curve and sample concentration by the program that the r enumerator is worked out in advance.

2.2.3 statistical procedures is with experiment 1.

2.3 experimental result

HD and PC have the effect that reduces plasma renin activity, and comparing with NC all has significant difference (P＜0.05), but there was no significant difference (P＞0.05) between HD and the PC.Reducing aspect the plasma A ngII, PC is the strongest, and HD takes second place, and with NC significant differences (P＜0.01) is arranged more all, but compares not statistically significant (P＞0.05) between HD and the PC; LD is relative with NJ relatively poor, with NC than there was no significant difference (P＞0.05).Thereby prompting HD has the mechanism of action that is similar to angiotensin converting enzyme inhibitor.

3. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of original hypertensive rat (SHR) endothelin level (ET) and nitric oxide (NO)

3.1 laboratory animal is with experiment 1.

3.2 experimental technique

3.2.1 grouping and administration are with experiment 1, administration time is 9w.

3.2.2 experimental procedure

Successive administration 9w, fasting after the last administration (can't help water) 24h is with 20% urethanes intraperitoneal anesthesia (5mlkg ^-1), respectively get blood 2ml from ventral aorta and place and contain 10%EDTA30 μ l test tube, mixing.Behind 4 ℃ of about 2h of placement, the centrifugal 10min separated plasma of 3000rpm ,-20 ℃ of sealings are preserved to be measured.Multiple melting before the test analyzed the medicine box description according to ET, NO and carries out the strictness operation.

3.2.3 statistical procedures is with experiment 1.

3.3 experimental result

Compare with NC, HD and MD have the effect (P＜0.05) of obvious reduction SHR blood plasma ET, but compare there was no significant difference (P＞0.05) with PC; PC, HD and MD can significantly increase NO content (P＜0.01 or P＜0.05), but the difference (P＞0.05) of comparing with PC that there are no significant.

4. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of original hypertensive rat (SHR) blood plasma NA and epinephrine (Adr)

4.1 laboratory animal is with experiment 1.

4.2 experimental technique

4.2.1 grouping and administration are with experiment 1, administration time is 9w.

4.2.2 chromatographic condition

Chromatographic column is C ₁₈Chromatographic column (250mm * 4.0mm, 5 μ m); Column temperature is a room temperature; Mobile phase is methanol-0.15molL ^-1Potassium phosphate buffer (1: 9, pH6.0); Flow velocity is 1mlmin ^-1The detection wavelength is 280nm.Be not less than 3000 with the NA and the Adr theory of computation number of plates respectively under this condition.

4.2.3 experimental procedure

Successive administration 9w, fasting after the last administration (can't help water) 24h is with 20% urethanes intraperitoneal anesthesia (5mlkg ^-1), place from abdominal aortic blood 1.5ml to contain the heparin test tube, mixing, the centrifugal 15min of 2000rpm under 4 ℃ of conditions, separated plasma; Get blood plasma 100 μ l, add 0.05molL ^-1Perchloric acid 200 μ l, the centrifugal 2min of 10000rpm pours Millipore Ultra-free-Mc centrifuge tube into, and 10000rpm is centrifugal, and 3min postposition-20 ℃ refrigerator is to be measured.

4.2.4 statistical procedures is with experiment 1.

4.3 experimental result

The mensuration of plasma sample: get " 4.2.3 " blood sample ultrafiltrate of handling well by above-mentioned chromatographic condition analysis, HD and PC and NC compare behind the administration 9w, and NA significantly reduces (P＜0.05).

5. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to original hypertensive rat (SHR) cardiac weight and exponential influence thereof

5.1 laboratory animal is with experiment 1.

5.2 experimental technique

5.2.1 grouping and administration are with experiment 1, administration time is 9w.

5.2.2 experimental procedure

Successive administration 9w, fasting after the last administration (can't help water) 24h is with 20% urethanes intraperitoneal anesthesia (5mlkg ^-1), behind abdominal aortic blood, core fast, put in the pre-cold saline and clean, cut off remnant tissue, blot surface moisture with filter paper, the separation left and right ventricles is also weighed, and interventricular septum is returned left chamber.Take by weighing left ventricular mass, calculate the Left ventricular mass index (the heavy and weight ratio of left ventricle) of expression left ventricular hypertrophy degree.

5.2.3 statistical procedures is with experiment 1.

5.3 experimental result

Compare with NC, the left ventricular mass of each administration group, left ventricular mass/right ventricle's weight and ventricular weight/body weight have significant difference (P＜0.05 or P＜0.01); CPFP administration group and PC relatively left ventricular mass do not have significant difference (P＞.05).

6. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of original hypertensive rat (SHR) aorta and renal artery tectology

6.1 laboratory animal is with experiment 1.

6.2 experimental technique

6.2.1 grouping and administration are with experiment 1, administration time is 9w.

6.2.2 experimental procedure

6.2.2.1 aorta HE dyeing pathomorphism is observed

Successive administration 9w, fasting after the last administration (can't help water) 24h is with 20% urethanes intraperitoneal anesthesia (5mlkg ^-1), behind abdominal aortic blood, win thoracic aorta rapidly, use formalin fixed, paraffin embedding, HE dyeing.With observation by light microscope and take pictures.

6.2.2.2 the SABC of aorta and renal artery bFGF, Ki-67 detects

Successive administration 9w, fasting after the last administration (can't help water) 24 hours is with 20% urethanes intraperitoneal anesthesia (5mlkg ^-1), behind abdominal aortic blood, win thoracic aorta and renal artery rapidly, formalin fixed, dehydration, transparent, waxdip, embedding, section.Tissue slice dewaxes through dimethylbenzene, after the gradient ethanol aquation, is soaked in the water stand-by.

The acid reparation: 800～1500ml is in pressure cooker to get the citrate buffer solution (pH=6.0 ± 0.1) that configures, and electric furnace is heated to boiling.The tissue slice of dewaxing after the aquation places on the high-temperature resistance plastice staining rack, puts into ebullient buffer, covers pot cover, buckles pressure valve, continues to be heated to jet, clocks from jet the beginning, and pressure cooker leaves thermal source behind the 1.5min, is cooled to room temperature.Take out slide, with after the tap water flushing, be placed in ovum and educate in the box earlier.PBS (NaCl 9.0g+Na ₂HPO ₄12H ₂O 6.0g+NaH ₂PO ₄2H ₂O 0.4g+ distilled water 1000ml) flushing is three times, and each 3min at interval removes PBS liquid.Every section adds 50 μ l, 3% hydrogen peroxide, hatches 10min under the room temperature, with the activity of blocking-up endogenous peroxydase.PBS flushing three times, each 3min at interval removes PBS liquid.

Every section adds bFGF-2 (sc-79) or Ki-67 (cloneSP6) the rabbit Chinese People's Anti-Japanese Military and Political College Mus polyclonal antibody (all by dilution in 1: 50) of 50 μ l, 4 ℃ of following overnight incubation.PBS flushing three times, each 3min at interval removes PBS liquid, and every section adds 50 μ l instant two step method (abiotic element) PV6001 rabbit two step method detection kit, hatches 15min under the room temperature.PBS flushing three times, each 3min at interval removes PBS liquid.

The DAB colour developing: A, B, each 50 μ l mixing of C reagent in 850 μ l distilled water and the DAB reagent are mixed with 1mlDAB colour developing liquid.Every section adds the freshly prepared DAB solution of 50 μ l, the tap water flushing, and haematoxylin was redyed 30 seconds, and indigo plant is returned in the tap water flushing.Section is through gradient alcohol dehydration drying, neutral gum sealing.

The result judges: 1. the bFGF coloration result is judged: with reference to the Mattern method, mark from following two aspects: the positive staining degree of depth (does not see that dyeing is 0, slight dyeing is 1, moderate dyeing is 2, degree of depth dyeing is 3) and the percentage ratio of positive cell (not seeing dyes is designated as 0, and staining cell＜25% is 1, and 25-50% is 2,＞50% is 3), with this two aspects score addition.Under the condition of unknown pathological diagnosis, under high power lens, observe 10 visuals field respectively after, average, record.Score 0-2 is divided into negative the expression, (3-6) positive expression above 2 minutes, and wherein 3-4 is divided into the weak positive, and 5-6 is divided into strong positive.

2. the Ki-67 label index is judged: reference literature, dye the pale brown color of homogeneous with karyon, endochylema and after birth are not colored as positive cell, count 1000 cells of 5 above high power fields at random, and positive staining cell check figure is Ki-67 index (Ki-67I) in average per 100 cells.

6.2.3 statistical procedures is with experiment 1.

6.3 experimental result

6.3.1 influence to SHR histopathology form

6.3.1.1 each group of histological examination changes not obvious.

6.3.1.2 light microscopic is checked down

Wt: aorta is most of near normal, indivedual endotheliocytic swellings, and middle film does not see obviously and thickens (6-8 layer) that the adventitia connective tissue is not seen deposit.NC: the aorta artery wall thickness differs, neointimal hyperplasia, present little height fluctuating shape, the visible a small amount of deposit of SES, endotheliocyte is imperfect, the attached wall of intimal surface visible red cell, film smooth muscle cell proliferation in the part, film obviously thickens (reaching the 11-13 layer) in the tube wall, and the adventitia connective tissue is not seen deposit.HD: the slight hypertrophy of aortic tunica intima, part thickens, endotheliocytic swelling, film slightly thickens (8-10 layer) in the part, and the adventitia connective tissue is not seen deposit.MD: the slight hypertrophy of aortic tunica intima, visible little projection.Endotheliocyte is imperfect, and film thickens (10-11 layer) in the part, and the adventitia connective tissue is not seen deposit.LD: the slight hypertrophy of aortic tunica intima presents little height fluctuating shape, the visible a small amount of deposit of SES, endotheliocyte is imperfect, the attached wall of intimal surface visible red cell, film smooth muscle cell proliferation in the part, middle film obviously thickens (10-12 layer), and the adventitia connective tissue is not seen deposit.NJ: the slight hypertrophy of aortic tunica intima, visible little projection.Endotheliocyte is imperfect, and film thickens (9-11 layer) in the part, and the adventitia connective tissue is not seen deposit.PC: the slight hypertrophy of aortic tunica intima, part thickens, endotheliocytic swelling, film slightly thickens (8-10 layer) in the part, and the adventitia connective tissue is not seen deposit.

6.3.2 influence to SHR aorta and renal artery smooth muscle cell (VSMC) bFGF and Ki-67

6.3.2.1 influence to each group aorta and renal artery VSMC bFGF content

The main diffusivity of bFGF positive staining is distributed in the tunica media of artery VSMC endochylema, observes to be graininess under high power field; Nucleus is painted hardly.Compare with NC, Wt aorta and renal artery VSMCbFGF content obviously reduce (P＜0.01), and HD and MD aorta VSMC bFGF content significantly descend (P＜0.01 or P＜0.05), and the content of the renal artery VSMC bFGF of HD is obviously decline (P＜0.05) also; Compare the equal no significant difference of VSMC bFGF content (P＞0.05) of HD, MD and NJ with PC.

6.3.2.2 influence to each group aorta and renal artery VSMC Ki-67I

The Ki-67 positive reaction all is distributed in the nucleus of aortic tunica media VSMC, the endochylema dye-free.Compare with NC, the aorta of Wt, HD and PC and renal artery VSMC Ki-67I obviously reduce (P＜0.01 or P＜0.05), and the renal artery VSMC Ki-67I of NJ also obviously reduces (P＜0.05); Compare with PC, and the equal no significant difference of VSMC Ki-67I of HD, MD and NJ (P＞.05).

The blood pressure lowering experimentation shows: 2h promptly had tangible pressure reduction effect after single gave polysaccharides, and the 4h pressure reduction effect peaks, and blood pressure lowering amplitude maximum is 0.68 ± 8.02kPa.During 4h HD with the treatment before compared significant difference (P＜0.05).From the time that efficacy of antihypertensive treatment is kept, SBP returns to the preceding level of treatment behind each administration group administration 12h of polysaccharides.Present tangible dose-effect and time-effect relationship from the blood pressure lowering of blood pressure lowering amplitude polysaccharides.In continuous 9w administration process, polysaccharides shows the hypotensive effect that continues to SHR, presents dose-effect and timeliness between each dosage group and relies on trend, and its effect characteristics are gently, stablize.Compared significant difference (P＜0.01) before HD and MD and the treatment with NC; LD compares with NC with NJ also significant difference (P＜0.01), but more is not very obvious before and after self, but also variant (P＜0.05).The maximum average of HD, MD, LD and NJ blood pressure lowering is respectively 2.16 ± 0.18,1.59 ± 1.44,1.42 ± 0.16,0.87 ± 0.18 and 0.82 ± 0.19kPa.Illustrate that thus polysaccharides has the effect of good blood pressure lowering.

Therefore, polysaccharides can obviously reduce the systolic pressure of SHR, and is tangible timeliness and dose-effect relationship; Can obviously reduce the content of SHR blood plasma RA, AngII, ET, norepinephrine (NE) and elevation of NO; Protection SHR aortic endothelial cell reverses smooth muscle cell proliferation, has the effect that reverses the SHR target organ damage to a certain extent.

Three, the experimental study of fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides (CPFP) effect for reducing blood fat

1. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to hyperlipemia (HLP) rat fat and the morphologic influence of liver

1.1 laboratory animal

60 of SD rats, male, body weight 160 ± 10g is provided by Guangxi Medical University's Experimental Animal Center, and the quality certification number is: SCXK osmanthus 2003-0003; Animal feed is produced (standard feed) by Guangxi Medical University's Experimental Animal Center.The experimental session rat freely ingests, drinks water, ventilation and natural lighting.

1.2 experimental technique

1.2.1 modelling and experiment grouping

Get 60 of rats, be divided into 10 of blank groups (KB) and high fat at random and make 50 of model group (ZM).After the conventional raising 1 week (w), water 12 hours (h) is can't help in fasting, and blood sampling detects basic blood fat.The reference literature method prepares high lipid food, and is promptly formulated by 87.3% normal feedstuff, 10% Adeps Sus domestica, 2% cholesterol, 0.5% Fel Sus domestica salt, 0.2% propylthiouracil.The ZM rat with high lipid food feed 3w after, fasting (can't help water) 12h, blood sampling detects blood fat.Whether build up with definite HLP model with the blood fat comparison before the modeling.After the HLP model causes, the HLP rat is divided into 5 groups by serum total cholesterol (TC) level: CPFP high dose group (HD), CPFP low dose group (LD), fruit of Radix et Caulis Opuntiae Dillenii natural juice group (NJ), positive controls (LF) and HLP model group (HL), 10 every group.

1.2.2 medication

The rat modeling finishes each group and gives relative medicine filling stomach respectively.HD and LD give 3.06gkg respectively ^-1D ^-1And 0.79gkg ^-1d ^-1CPFP, NJ gives 3.15gkg ^-1D ^-1Fruit of Radix et Caulis Opuntiae Dillenii natural juice; LF gives 3.0mgkg ^-1D ^-1Lovastatin, blank group (KB) and HL are with 10mlkg ^-1Normal saline is irritated stomach.Every day 1 time, 3w continuously.Each group is all freely drunk water.

1.2.3 food ration and body weight

Same time sheet of every day food-intake, every w organizes rat body weight same time sheet, so that adjust dosage.

1.2.4TC, the mensuration of triglyceride (TG), highdensity lipoprotein-cholesterol (HDL-C), LDL-C (LDL-C), apolipoprotein AI (apoAI) and apolipoprotein B (apoB)

Administration 3w, fasting 12h before the last administration, behind the administration 1h, get blood, pack in anticoagulant or the not anticoagulant test tube, at 4 ℃ of following 3000 rev/mins of (rpm) centrifugal 10min, get serum or blood plasma respectively, on automatic clinical chemistry analyzer, measure the content of TC, TG, HDL-C, LDL-C, apoAI and apoB respectively.

1.2.5LDL-C/HDL-C, the calculating of TG/HDL-C ratio and atherogenic index (AI)

Lipid determination result (TC, TG, HDL-C and LDL-C content) according to individual event calculates blood fat aggregative index (LDL-C/HDL-C, TG/HDL-C ratio and AI) respectively, and wherein AI calculates according to the Frieldwald formula: AI=(TC-HDL-C)/HDL-C.

1.2.6 liver is heavy and the liver coefficient

After each is organized rat and puts to death, win liver rapidly, blot surface moisture with pre-cold saline rinsing reuse filter paper after, weigh.According to liver re-computation liver coefficient: liver coefficient=liver weight (g)/body weight (g) * 100%.

1.2.7 liver histopathology is observed

Get hepatic tissue, be cut into 4-5mm ³Fritter, 10% neutral formalin is fixed, the gradient ethanol dehydration: 75% ethanol 90min～95% ethanol 90min～100% ethanol 90min * 3 time～100% dimethylbenzene 60min * 2 time, 62 ℃ of paraffin 2h * 2 time, section 6 μ m are affixed on the common microscope slide after the embedding.Dye through HE, observe under the optical microscope and take pictures.

Carry out statistical procedures 1.2.8 statistical procedures is used SPSS11.0 software, measurement data all adopts

Expression, measurement data relatively adopts the t check between two groups, organizes mean more and relatively adopts variance analysis.

1.3 experimental result

1.3.1 rat food ration and body weight change during the modeling

Average day food ration of rat and body weight all have increase in various degree during the modeling.The average day food ration of ZM rat all is lower than KB, and wherein 1w has significant difference (P＜0.05), may be because just food high lipid food incompatibility is relevant; 2nd, 3w there are no significant difference (P＞0.05); The ZM rat body weight all is higher than KB, but there was no significant difference (P＞0.05).

1.3.2 the foundation of rat HLP model

Survey blood fat behind the ZM rat 3w of feed high lipid food.With compare before the modeling, serum TC, TG, LDL-C level significantly raise (P＜0.01), HDL-C level obviously descend (P＜0.05).Illustrate that the ZM rat exists metabolism disorder of blood lipid, the success of HLP model copy.

1.3.3 influence to rat food ration and body weight

During the administration, each organizes the rat well-grown, and is movable normal.Respectively organize rat food ration and body weight before the administration and all do not have significant difference (P＞0.05).Behind the administration 3w, average day food ration of rat and body weight all increase steadily, compare with KB, HL, and average day food ration of each administration group rat and body weight there was no significant difference (P＞0.05) illustrate that CPFP has no adverse effects to rat food ration and body weight change.

1.3.4 influence to HLP rat TC, TG and LDL-C

Before the administration, the serum TC of rat, TG and LDL-C level compare there was no significant difference (P＞0.05) mutually between each HLP model group, but all apparently higher than KB (P＜0.01).

Behind the administration 3w, each group is relatively preceding with administration, and KB Serum TC, TG and LDL-C raise 1.84%, 6.06% and 7.14% respectively, difference that there are no significant (P＞0.05); HL Serum TC, TG and LDL-C raise 23.84%, 27.27% and 31.82% respectively, and significant difference (P＜0.05) is all arranged; HD, LD, NJ and LF blood fat reduce respectively: TC (23.10%, 18.29%, 19.60% and 31.64%), TG (34.37%, 27.27%, 24.64% and 35.48%) and LDL-C (26.70%, 24.76%, 19.29% and 38.42%), each organizes TC, TG and LDL-C all significantly reduces (P＜0.01 or P＜0.05).

Compare with HL, each administration group Serum TC, TG and LDL-C level be very obviously decline (P＜0.01) all; With LF relatively, HD Serum TC, TG and LDL-C level there are no significant difference (P＞0.05) illustrates that CPFP can significantly reduce HLP Serum TC, TG and LDL-C, HD and LF effect are all better.

1.3.5 influence to HLP rat HDL-C

Before the administration, rat blood serum HDL-C level compares there was no significant difference (P＞0.05) mutually between each HLP model group, but all is starkly lower than KB (P＜0.01).

Behind the administration 3w, with comparison before the administration, KB rat blood serum HDL-C level raises 0.85%, but there was no significant difference (P＞.05); HL reduces by 8.70%, there was no significant difference (P＞0.05); HD, LD, NJ and LF raise 19.78%, 8.51%, 8.60% and 13.83% respectively, and HD rat blood serum HDL-C raises significant difference (P＜0.05), all the other administration groups there are no significant difference (P＞0.05).

With HL relatively, each administration group rat blood serum HDL-C level all raises to some extent, HD and LF have significant difference (P＜0.05), and the CPFP HLP rat blood serum HDL-C that can raise is described.

1.3.6 influence to HLP rat apoAI and apoB

Behind the administration 3w, with HL relatively, HD, LD, NJ and LF serum apoAI raise 33.49%, 5.58%, 3.49% and 21.16% respectively, but there are no significant difference (P＞0.05); HD, LD, NJ and LF serum apoB reduce by 49.26%, 40.16%, 47.18% and 50.15% respectively, and significant differences (P＜0.01) is all arranged.ApoAI and apoB level there are no significant difference (P＞0.05) between each administration group.

1.3.7 influence to HLP rat fat aggregative index (LDL-C/HDL-C, TG/HDL-C ratio and AI)

Behind the administration 3w, compare with HL, HD, LD, NJ and LF blood fat aggregative index reduce respectively: LDL-C/HDL-C (57.68%, 52.66%, 49.84% and 62.38%), TG/HDL-C (63.89%, 57.41%, 52.78% and 66.67%), AI (62.23%, 53.68%, 51.54% and 65.80%) all have significant differences (P＜0.01).Compare with LF, HD there was no significant difference (P＞0.05), LD and NJ rat blood serum LDL-C/HDL-C and AI ratio have significant difference (P＜0.01 or P＜0.05).

1.3.8 the influence of and liver coefficient heavy to the HLP rats'liver

Behind the administration 3w, with the HL comparison, liver weight of HD, LD, NJ and LF rat and liver coefficient all have reduction in various degree, and liver is heavy to reduce by 15.16%, 10.15%, 11.25% and 16.99% successively, and HD and LF reduce significant difference (P＜0.05); The liver coefficient reduces by 12.10%, 9.80%, 10.09% and 13.26% successively, and HD, LD, NJ and LF reduce significant difference (P＜0.01 or P＜0.05).With LF relatively, the heavy and liver coefficient of each administration group liver there are no significant difference (P＞0.05).

1.3.9 influence to HLP rat liver steatosis

1.3.9.1 histological examination

KB: the no abnormal variation of liver, liver surface is smooth, and is glossy, do not see phenomenons such as hyperemia, edema; HL: liver volume increases, the peplos anxiety, and the edge circle is blunt, and tangent plane is greasy, and tarnish is milk yellow, and the visible big fine particle of syringe needle belongs to typical hepatic cell fattydegeneration; Each administration group: liver color and luster, quality, volume all have clear improvement.

1.3.9.2 om observation

KB: the morphology of hepatic tissue is acted normally, the lobules of liver clear in structure, hepatocyte is arranged and is streak, with the central vein is the radial traveling in center, do not see that hepatocyte cloudy swelling, fat become and necrosis, little blood vessel and bile capillary between the visible lobule in portal area are not seen changes such as cell infiltration and the formation of silt gallbladder, and 10 rat hepatocytes there is no steatosis (0/10);

HL: the lobules of liver structure owes clear, the hepatic cell fattydegeneration (10/10) of moderate to severe all appearred in 10 rats, the fat vacuole that differs in size appears in the endochylema, the hepatocyte volume generally becomes big change circle, nucleus is squeezed on one side, sinus hepaticus obviously narrows down or disappears, the painted normal cell of hepatocyte is shallow, endochylema is light to be dyed, see point-like or the necrosis of kitchen range shape in the acinus more, the portal area with gently-the moderate inflammation cellular infiltration, do not see obvious hepatic fibrosis, most of central veins of hepatic lobules Zhou Xianxian cholesterol crystal;

Each administration group: hepatic tissue all has improvement in various degree, and especially HD and LF hepatic tissue have improvement largely.

2. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of hyperlipemia (HLP) hemorheology of rat

2.1 laboratory animal is with experiment 1.

2.2 experimental technique

2.2.1 hemorheological mensuration

Administration 3w, fasting 12h before the last administration behind the administration 1h, gets blood 3.5ml, washes rotary viscosimeter with serum, surveys whole blood viscosity (high, medium and low cutting), hemorheology indexs such as plasma viscosity and packed cell volume value.

2.2.2 statistical procedures is with experiment 1.

2.3 experimental result

Influence to the HLP hemorheology of rat

Behind the administration 3w, with HL relatively, HD, LD, NJ and LF whole blood viscosity (height cuts, in cut, low cutting), plasma viscosity, erythrocyte (RBC) hematocrit value all have significance to reduce (P＜0.01 or P＜0.05); Compare with LF, the equal significance of the RBC hematocrit value of HD, whole blood viscosity (height is cut) and plasma viscosity reduces (P＜0.01 or P＜0.05), and effect is better than LF; The RBC hematocrit value of LD and NJ also significantly reduces (P＜0.05), illustrates that CPFP has obvious improvement effect unusually to the hemorheology of HLP rat.

3. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of hyperlipemia (HLP) rat anti Oxidation

3.1 laboratory animal is with experiment 1.

3.2 experimental technique

3.2.1 sample process

Administration 3w, fasting 12h before the last administration behind the administration 1h, gets blood, in the not anticoagulant test tube of packing into, at 4 ℃ of centrifugal 10min of following 3000rpm, gets serum, packing ,-20 ℃ of preservations.

3.2.2 the active mensuration of serum superoxide dismutases (SOD)

Measuring principle: measure with xanthine oxidase, produce ultra-oxygen anion free radical by xanthine and xanthine oxidase response system, latter's oxidation azanol forms nitrite, is aubergine under the effect of developer.In strict accordance with the operation of SOD testing cassete description, measure the absorbance (OD) of SOD at the 550nm place, calculate activity of SOD in serum according to following formula:

Extension rate before activity of SOD in serum (U/ml)=(control tube OD value-mensuration pipe OD value) ÷ control tube OD value ÷ 50% * sample test.Annotate: to reach 50% o'clock pairing SOD amount be a SOD unit of activity (U) to the SOD suppression ratio in every 1ml reactant liquor.

3.2.3 serum malonaldehyde (MDA) Determination on content

Measuring principle: adopt thiobarbituricacid (TBA) method.MDA in the lipid peroxide catabolite can with the TBA condensation, form red product.In strict accordance with the operation of SOD testing cassete description, measure the OD value of MDA at the 532nm place, calculate Content of MDA according to following formula:

Extension rate before MDA content (nmol/ml)=(measuring the pipe OD value-blank pipe of mensuration OD value) ÷ (the blank pipe of standard pipe OD value-standard OD value) * standard substance concentration * sample test.

3.2.4 statistical procedures is with experiment 1.

3.3 experimental result

Influence to HLP rat anti Oxidation

Behind the administration 3w, HD, LD, NJ and LF activity of SOD in serum raise 50.06%, 40.64%, 37.24% and 34.33% respectively; Compare with HL, HD, LD and NJ have significant differences (P＜0.01), and LF has significant difference (P＜0.05).Content of MDA reduces by 36.33%, 32.72%, 27.78% and 28.66% respectively, and each administration group all has significant differences (P＜0.01).In addition, SOD and MDA there are no significant difference (P＞0.05) between each administration group group.

Experiment conclusion: 1. polysaccharides has the effect of blood fat reducing and adjusting lipoprotein metabolism: can reduce TC, TG, LDL-C and apoB level in the hyperlipidemia rats blood, rising HDL-C and apoAI level; And the change of the liver protecting cellularity function, strengthen its effect to lipid metabolism.

2. polysaccharides can improve blood viscosity and rheological characteristic, directly improves blood constituent: can reduce whole blood viscosity (high, neutralization low), plasma viscosity, the RBC hematocrit of hyperlipidemia rats, and improve hemorheological property.By improving dense, sticking, the poly-state of blood, promote the metabolism of lipid material.

3. polysaccharides can be removed free radical, reduce the generation of lipid peroxide: can effectively reduce MDA content in the hyperlipidemia rats serum, SOD activity in the rising serum, the prompting polysaccharides may be by improving the activity of free radical scavenging enzyme, safeguard interior free yl stable state and balance, increase the oxidation resistance of LDL, reduce the generation of lipid peroxide, thereby performance prevents and treats hyperlipemia and atherosclerotic effect.

Therefore, polysaccharides has blood fat reducing and regulates the effect of lipoprotein metabolism, can improve blood viscosity and rheological characteristic, directly improve blood constituent, remove free radical, reduce the generation of lipid peroxide, thereby performance prevents and treats hyperlipemia and atherosclerotic effect.

Four, the experimental study of fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides (CPFP) hypoglycemic activity

1. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of diabetes (DM) rat carbohydrate metabolism and general signs

1.1 laboratory animal

The SD rat, male and female half and half, body weight (200 ± 10) g is provided by Guangxi Medical University's Experimental Animal Center, and the quality certification number is: SCXK osmanthus 2003-0003; Animal feed is produced (standard feed) by Guangxi Medical University's Experimental Animal Center.The DM model mouse is freely ingested, is drunk water, and ventilates natural lighting.

1.2 experimental technique

1.2.1DM rat model is set up and index determining

1.2.1.1DM rat model is set up and grouping

SD rat fasting 12 hours (h) is with 60mgkg ^-1(STZ is dissolved in 0.1molL to streptozotocin (STZ) lumbar injection ^-1, pH4.2 citric acid/sodium citrate buffer solution in, now with the current, place on ice).The above-mentioned buffer of normal control group (Sd) rats by intraperitoneal injection equivalent.(fasting 12h before the rat blood sampling) measures fasting glucose (FBG) value, all FBG 〉=13mmolL with blood glucose (BG) analyzer behind the 72h ^-1Be considered as the DM rat model.

To become mould DM rat to sort from high to low and carry out the physics numbering, at random the DM rat will be divided into dosage group (MD) and CPFP high dose group (HD) among DM model control group (NC), insulin group (IN), natural juice group (NJ), the CPFP by BG concentration.

1.2.1.2 dosage and method

IN gives 10U.kg ^-1D ^-1Insulin (Ins), NJ gives fruit of Radix et Caulis Opuntiae Dillenii natural juice 3.15gkg ^-1D ^-1, MD and HD give 1.58gkg respectively ^-1D ^-1, 3.06gkg ^-1D ^-1Every morning empty stomach administration 1 time, 8 weeks of continuous irrigation stomach (w).

1.2.1.3 general signs is observed

Observe the general situation such as ergasia, hair change, amount of drinking water, food-intake and defecation of rat, and record body weight change situation.

1.2.1.4 the mensuration of blood glucose

Administration 4w and 8w respectively organize FBG with BG analyzer mensuration, and calculating and statistical analysis medicine are to the influence of DM rat BG level.

1.2.1.5 the mensuration of glycolated hemoglobin (GHB)

Administration 8w end is pulled out rat eye blood sampling 2-4ml and is placed centrifuge tube, and with 500～1000 rev/mins (rpm), centrifugal 5～10 minutes (min) abandons supernatant and stay sedimentary erythrocyte, then with normal saline washing 2～3 times.Measure kit measurement GHB content with GHB, concrete operations see the test kit description for details, and calculating and statistical analysis medicine are to the influence of DM rat GHB level.Computing formula: GHB gram number * 2 * 10g in every 10g GHB absorbance (OD)=mensuration pipe OD/2ml blood

Carry out statistical procedures 1.2.2 statistical procedures is used SPSS11.0 software, measurement data all adopts

1.3 experimental result

1.3.1 influence to the general situation of DM rat due to the STZ

1.3.1.1 general state is observed

The Sd rat is in high spirits, and movable frequent, hair is pure white glossy, the bedding and padding drying; NC rat lethargy, mobility is low, and tail is clammy, withered and yellow, the no color and luster of hair, comes off, the perpendicular hair back of a bow, bedding and padding are tide extremely, and hypogastric region and external genital hair soak, and it is rare quiet to defecate, and the symptom of " three-many-one-little " occurs; After giving CPFP and Ins, DM rat spirit takes a turn for the better gradually, and activity increases, and fur gloss is gradually good, and bedding and padding are relatively drier, and the sx of " three-many-one-little " is reduced on rare pool of defecating.

1.3.1.2 influence to the DM rat body weight

Sd and IN rat body weight are sustainable growth trend in experiment; The NC rat body weight progressively descends, and illustrates that generation is compensatory in the DM rat body, metabolism disorder; After giving CPFP, be significantly higher than the NC body weight same period (P＜0.05) during DM rat body weight 8w.

1.3.2 medicine is to the influence of MD rat FBG due to the STZ

Behind administration 4w and the 8w, each organizes FBG all in various degree reduction.NC comparison HD, MD and NJ all have remarkable hypoglycemic activity (P＜0.01) with the same period, and hypoglycemic effect presents certain dose-effect and time-effect relationship.All there is not significant difference (P＞0.05) between HD, MD and the NJ.

1.3.3 medicine is to the influence of DM rat GHB due to the STZ

The result shows: compare with NC, the GHB content of HD, MD and NJ significantly reduces (P＜0.01 or P＜0.05), but does not have significant difference (P＞0.05) between HD, MD and the NJ.Prompting HD, MD, NJ be blood sugar lowering more stably all.

2. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the lipometabolic influence of diabetes (DM) rat

2.1 laboratory animal is with experiment 1.

2.2 experimental technique

2.2.1 the mensuration of serum total cholesterol (TC), triglyceride (TG), L DL-C and highdensity lipoprotein-cholesterol (HDL-C)

Blood is got at administration 8w end, and 4 ℃, the centrifugal 10min of 3500rpm measures serum TC, TG, LDL-C and HDL-C content on automatic clinical chemistry analyzer, and calculates atherogenic index (AI): AI=(TC-HDL-C)/HDL-C according to the Frieldwald formula.

2.2.2 statistical procedures is with experiment 1.

2.3 experimental result

Compare with Sd, NC rat TC, TG and LDL-C significantly raise (P＜0.01), HDL-C content significantly descend (P＜0.05); Behind the administration 8w, compare with NC, the TC of HD and IN and TG content significantly descend (P＜0.05), and LDL-C content also reduces to some extent, and wherein the LDL-C content of HD reduces effect significantly (P＜0.05); Simultaneously, the HDL-C content of HD and IN significantly raise (P＜0.05).

3. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of diabetes (DM) pancreas in rat tectology

3.1 laboratory animal is with experiment 1.

3.2 experimental technique

The morphologic observation 3.2.1 pancreatic tissue HE dyes

Administration 8w end, get blood after, get pancreas rapidly, use formalin fixed, paraffin embedding, HE dyeing.Light microscopic is observed the myocardial pathology variation down and is taken pictures.

3.2.2 the SABC of pancreatic tissue α cell and β cell detects

Cut open the belly and get pancreas in administration 8w end, formalin fixed, dehydration, transparent, waxdip, embedding, section.Tissue slice dewaxes through dimethylbenzene, after the gradient ethanol aquation, is soaked in the water stand-by.

The acid reparation: 800-1500ml is in pressure cooker to get the citrate buffer solution (pH=6.0 ± 0.1) that configures, and electric furnace is heated to boiling.The tissue slice of dewaxing after the aquation places on the high-temperature resistance plastice staining rack, puts into ebullient buffer, covers pot cover, buckles pressure valve, continues to be heated to jet, clocks from jet the beginning, and pressure cooker leaves thermal source behind the 1.5min, is cooled to room temperature.Take out slide, with after the tap water flushing, be placed in ovum and educate in the box earlier.PBS (NaCl 9.0g+Na ₂HPO ₄12H ₂O 6.0g+NaH ₂PO ₄2H ₂O 0.4g+ distilled water 1000ml) flushing is three times, and each 3min at interval removes PBS liquid.

Every section adds 50 μ l, 3% hydrogen peroxide, hatches 10min under the room temperature, with the activity of blocking-up endogenous peroxydase.PBS flushing three times, each 3min at interval removes PBS liquid.Every section adds Glucagon Ab-1 or the Insulin Ab-5 (all by dilution in 1: 50) of 50 μ l, 4 ℃ of following overnight incubation.PBS flushing three times, each 3min at interval removes PBS liquid, and every section adds the quick SABC Maxvision of 50 μ l instants ^TMAnti-rabbit-HRP detection kit hatches 15min under the room temperature.PBS flushing three times, each 3min at interval removes PBS liquid.

The DAB colour developing: A, B, each 50 μ l mixing of C reagent in 850 μ l distilled water and the DAB reagent are mixed with 1mlDAB colour developing liquid.Every section adds the freshly prepared DAB solution of 50 μ l.The tap water flushing, haematoxylin was redyed 30 seconds, and indigo plant is returned in the tap water flushing.Section is through gradient alcohol dehydration drying, neutral gum sealing.

Observation is respectively organized the SABC sheet and is carried out comprehensive grading under the mirror.1, express area: 1. the positive expression cell number is 0 to count 0 fen.2. positive expression cell number≤25% is counted 1 fen; 3. the positive expression cell number is 26～50% to count 2 fens; 4. the positive expression cell number accounts for 51～75% and counts 3 fens; 5. positive expression cell number＞75% is counted 4 fens.2. expression intensity: 1. positive signal is strong, is sepia, and little bulk is counted 3 fens, and 2. positive signal is medium, is brown, and the coarse granule shape is counted 2 fens; 3. positive signal a little less than, be light brown, fine particulate is counted 1 fen; 4. do not have positive signal, count 0 fen.Two additions, 0 minute negative (-), 2-3 is divided into the weak positive (+), and 4-5 is divided into moderate positive (++), and 6-7 is divided into strong positive (+++).Counting adopts double-blind method.

3.3 experimental result

3.3.1 influence to DM rat tissue pathomorphism

Islets of langerhans quantity is more in the Sd pancreas, and volume is bigger, the rounded or oval-shaped cell mass of islets of langerhans, and sharpness of border, islets of langerhans inner cell quantity is more, marshalling, endochylema is plentiful, and it is circular that nuclear mostly is; NC islets of langerhans quantity obviously reduces, structural deterioration, and profile disappears, and islets of langerhans inner cell quantity obviously reduces, the cell irregular arrangement, obscure boundary, cavity appears in part cellular swelling or shrinkage necrosis in the islets of langerhans; IN islets of langerhans quantity showed increased, profile is complete, distribution rule, cell boundaries is clear; It is lighter that HD and MD and NC compare pancreatic disorders, and the islets of langerhans profile is complete, and islets of langerhans inner cell quantity increases, distribution rule, and endochylema is loose, light dying, cell boundaries is more clear.

3.3.2 medicine is to the influence of MD rat Langerhans islet β cell due to the STZ and α cell

3.3.2.1 beta Cell of islet Ins expression

The result shows: compare with NC, each administration group Ins The positive expression rate significantly raises (P＜0.05), shows that each administration group has significant protective effect to beta Cell of islet damage due to the STZ.Immunohistochemical staining shows each group expression Ins degree: NC islets of langerhans middle body coloring degree obviously shoals than normal group, and area dwindles; Painted intensification of beta Cell of islet and area increase in each administration group.

3.3.2.2 alpha Cell of islet glucagon expression

The result shows: compares with NC, the no significant difference of each administration group glucagon expression (P＞.05).Each group of immunohistochemical staining demonstration is expressed glucagon degree: compares with NC, Sd and the no significant difference of each administration group α cell tryptase glucagon expression (P＞.05).

4. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of diabetes (DM) rat anti Oxidation

4.1 laboratory animal is with experiment 1.

4.2 experimental technique

4.2.1 malonaldehyde in the cardiac muscle (MDA) and superoxide dismutase (SOD) are measured

Administration 8w end, get blood after, it is dirty to core, and cleans with the ice normal saline, filter paper blots, and after weighing, is made into 10% myocardium homogenate with normal saline, in the centrifugal 15min of refrigerated centrifuger 3000rpm, get supernatant, be diluted to normal saline on request and be used for myocardium MDA after the certain multiple and SOD measures.

4.2.1.1 the mensuration of Content of MDA

Measuring principle: adopt thiobarbituricacid (TBA) method.MDA in the lipid peroxide catabolite can with the TBA condensation, form red product, at the 532nm place absorption maximum is arranged.In strict accordance with the operation of SOD testing cassete description, measure the OD value of MDA at the 532nm place, calculate Content of MDA according to following formula:

4.2.1.2 the mensuration of activity of SOD in serum

4.3 experimental result

4.3.1 medicine is to the influence of DM rat heart muscle MDA and SOD

NC rat heart muscle MDA content has significantly than Sd and increases due to the streptozotocin (STZ), and the SOD activity then significantly reduces, and shows that DM rat heart muscle oxidation resistance descends.Behind the medication 8w, with NC relatively, HD, MD and all significantly reductions (P＜0.01) of NJ cardiac muscle MDA value, corresponding SOD activity obviously increases (P＜0.01), all there were significant differences (P＜0.01) but compare with IN.

5. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the Ultrastructural influence of diabetes (DM) rat heart muscle

5.1 laboratory animal is with experiment 1.

5.2 experimental technique

Behind the administration 8w, after getting blood, win heart, putting into 2.5% glutaraldehyde of pre-cooling rapidly (prepares with phosphate buffer, pH7.2) fixing, use PBS (pH7.2) rinsing 10min three times afterwards, put into 1% osmic acid (Osmic acid .), fixing 2h, PBS rinsing 10min three times, 50%～90% ethanol dewaters step by step, the acetone dehydration, the epoxy resin embedding, 38 ℃, 45 ℃, 60 ℃ of polyase 13 6h successively, the section of LKB-V ultramicrotome, about 70nm, the two dyeing of acetic acid uranium-lead citrate are observed Ultrastructural variation such as myocardial mitochondria and cardiac muscle fiber and are taken pictures under H-500 type transmission electron microscope.

5.3 experimental result

Observe under the Electronic Speculum: Sd cardiac muscle fiber no abnormality seen, myocardium muscle segment para-position is neat, no sarcostyle dissolving, structure of mitochondria is complete, and the ridge queueing discipline is high-visible.The visible myocardium muscle segment para-position entanglement of NC is excess shrinkage, part sarcostyle dissolving, and mitochondrial swelling, the mitochondrion obscure boundary, part ridge fracture dissolving forms little cavity in the mitochondrion, shows that the DM rat heart muscle has certain damage due to the streptozotocin STZ.Each administration group rat heart muscle muscle segment para-position is neat substantially, rarely seen small part dislocation, and the sarcostyle dissolving is less, and mitochondrion is seen swelling and cavity slightly.

6. fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides are to the influence of diabetes (DM) rats'liver and skeletal muscle Insulin receptor INSR (InsR) and skeletal muscle glucose transporter 4 (GLuT4) mRNA expression

6.1 laboratory animal is with experiment 1.

6.2 experimental technique

6.2.1RNA extracting

6.2.2RNA purity and integrity analysis

6.2.3 total RNA reverse transcription becomes cDNA

6.2.4 reverse transcription-polymerase chain amplified reaction (RT-PCR)

6.3 experimental result

6.3.1 medicine is to the influence of DM rat liver due to the streptozotocin (STZ) and skeletal muscle InsR

The result shows: the quantity of each administration group DM rat liver and skeletal muscle InsR all has increase (P＜0.01) than NC.

6.3.2 medicine is to the influence of DM rat skeletal muscle GLuT4 due to the STZ

The result shows: the quantity of each administration group rat skeletal muscle GLuT4 all has increase (P＜0.01) than NC.

Experiment conclusion: by observing the influence of polysaccharides, and studied the influence of polysaccharides to pancreas and diabetic cardiomyopathy from the histology to diabetes rat carbohydrate metabolism, lipid metabolism and antioxidation due to the STZ.Inquired into the possibility mechanism of polysaccharides blood sugar reducing function then from gene level.Prompting:

1. 60mgkg is adopted in this research ^-1Once abdominal cavity injection STZ sets up diabetes rat model, irritate stomach polysaccharides 8w with optimal dose to diabetes rat after, the result shows that polysaccharides can improve the symptom of diabetes " three-many-one-little ", significantly reduces BG and GHB.Simultaneously, also reduce TC, TG, LDL-C and the AI of diabetes rat, the content of rising HDL-C, this shows that polysaccharides has promoted the utilization of sugar and fat, alleviated the damage of sugared toxicity and fat toxicity, helped the recovery of diabetes and prevent the generation of complication body.

2. tectology is observed and shown: polysaccharides is in blood sugar lowering and blood fat reducing, improved the degree of injury of diabetes rat pancreas due to the STZ, increase beta Cell of islet and secretion of insulin, therefore promote the beta Cell of islet reparation, increasing insulin secretion may be one of polysaccharides mechanism of action.

3. diabetes have reduced the rat anti oxidative function, and the MDA level is raise, and SOD is active to be reduced.Give to have reduced the MDA level behind the polysaccharides, the increased SOD activity, this shows that polysaccharides can improve the anti-oxidation function of body, alleviates peroxide injury.

4. Electron microscope showed: polysaccharides has in various degree improvement to diabetes rat cardiac muscle muscle segment para-position entanglement due to the STZ, sarcostyle dissolving and mitochondrial swelling, shows that it can alleviate the cardiac damage that diabetes cause.

5. by RT-PCR liver and skeletal muscle InsR, skeletal muscle GLuT4 are analyzed as can be known, polysaccharides can improve the content of InsR and GLuT4, illustrates that its blood sugar lowering mechanism may produce blood sugar reducing function by increasing InsR and GLuT4 content.

Therefore, polysaccharides energy blood sugar lowering, glycolated hemoglobin and blood fat, improve carbohydrate metabolism and disorders of lipid metabolism, improve the oxidation resistance of body, delay developing of complication, improve the situation of body, this with the impaired beta Cell of islet of its reparation, promote insulin secretion, to increase InR relevant with GLuT4 content.

Five, the experimental study of fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides (CPFP) antitumor action

(1) research of extracorporeal anti-tumor function

1. experiment material

1.1 cell strain: human hepatoma cell strain HEPG-2, human oophoroma cell line SKOV3, human stomach cancer cell line SGC7901 are provided by medical science experimental center Cytology Lab; Human hepatoma cell strain HCC-LM3, people's normal cell lines of human liver strain L02 etc. are provided by the The 2nd Army Medical College tumor.

2. experimental technique

2.1 the cultivation of cell, frozen and recovery

People's hepatocarcinoma, gastric cancer, 4 kinds of cell strains of ovarian cancer are selected in this experiment for use, in 37 ℃, 5%CO ₂Incubator in, cultivate in the RPMI-1640 of 10% new-born calf serum and each 100U/ml of mycillin.Inverted microscope observation of cell growing state, 0.25% trypsinization goes down to posterity, and the trophophase cell of taking the logarithm is used for experiment.

When frozen, the trophophase cell of taking the logarithm, digestion, centrifugal, remove supernatant.Add cryopreserving liquid, make cell density be about 1 * 10 ⁷/ ml is sub-packed in the frozen pipe, place earlier 4 ℃ 60 minutes, place again-20 ℃ 60 minutes, put again in-80 ℃ of ultra cold storage freezers and preserve.

During recovery, the frozen pipe of the tumor cell in the ultra cold storage freezer is changed over to rapidly in 37 ℃ of water, keep frozen pipe spout part to be positioned on the water surface, stir and quicken to thaw, after treating to thaw fully, with the frozen tube wall of 75% cotton ball soaked in alcohol wiping.To thaw the back cell transfer in centrifuge tube, add centrifugal 5 minutes of the culture medium 1000rpm of about 6 times of frozen liquid measures, abandon supernatant, repeat this step once.Add culture fluid again, the mixing cell goes in the culture bottle and cultivates.

2.2 tetrazolium bromide (MTT) method is determined the inhibitory action of polysaccharides extract to tumor cell

This experimental selection gastric cancer 7901, hepatocarcinoma HEPG-2, LM3 ovarian cancer SKOV3, people's normal liver cell LO2 cell strain adopt mtt assay to study the anticancer effect of polysaccharides.The take the logarithm tumor cell line of trophophase, concentration is 5 * 10 ⁴Individual/ml, join 96 orifice plates, every hole adds celliferous culture fluid 100 μ l, puts 37 ℃, 5%CO ₂After incubator is hatched 48h, add the polysaccharides of variable concentrations respectively, adjusting every porocyte cumulative volume with PBS solution is 200 μ l, adds 20 μ lMTT (2mg/ml), 37 ℃, 5%CO behind the 48h ₂After incubator is hatched 4h, abandon supernatant, every hole adds DMSO 200 μ l, puts into microplate reader, and the middling speed vibration was measured absorbance (OD value), each concentration parallel assay 3 hole after 5 minutes in 490nm wavelength place.With PBS solution is blank, obtains growth inhibition ratio (IR) by following formula.

2.3Annexin V-FITC dyeing observation of cell apoptosis

(1) the LM3 cell is grown in 6 orifice plates, be in adding CPFP (2.500mg/ml) cell death inducing in the exponential phase, and set up negative control group;

(2) use twice of PBS washed cell;

(3) in the Binding Buffer of 500 μ l, add 2 μ l Annexin V-FITC, 5 μ lPropidium Iodide, mixing;

(4) above-mentioned drips of solution is added on the coverslip surface, makes the long evenly covering of coverslip surface that cell is arranged;

(5) lucifuge, room temperature reaction are 5 minutes.Coverslip is inverted on the microscope slide, and under fluorescence microscope, dichroic filter (FITC and rhodamine) observes, it is green that Annexin V-FITC fluorescence signal is, and the PI fluorescence signal takes on a red color.

2.4 flow cytometry detection by quantitative apoptosis rate

The take the logarithm tumor cell of trophophase, after each concentration C PFP effect, collecting cell, regulating cell density with binding buffer liquid is 1 * 10 ⁶/ ml gets 100 μ l suspensions, adds 5 μ l Annexin-v/FITC and 10 μ l iodate, the third ingot solution, and the room temperature lucifuge was hatched 15 minutes, added 400 μ l PBS mixings, flow cytometry analysis.

2.5RT-PCR detect the expression of P53 gene mRNA

2.6 Western blotting detects the P53 correlative protein expression

2.7 statistical analysis

Use SPSS 13.0 softwares and carry out statistical procedures, measurement data all adopts

Expression, measurement data relatively adopts t check, the relatively employing X of two groups of rates between two groups ²Check is organized mean more and is relatively adopted variance analysis.

3. experimental result

3.1CPFP inhibitory action to tumor cell

The result shows that the common dose of CPFP various dose and 5-FU has different inhibitory action to the propagation of human body ovarian cancer SKOV-3, gastric cancer 7901, hepatocarcinoma HEPG-2, hepatocarcinoma LM3 cell.A little less than CPFP and the 5-FU comparison, inhibition ability.Under the same concentrations, CPFP is obvious than other strain cells to the effect of LM3 cell inhibiting, but considers that drug level is bigger, and the doses polysaccharides is little to the inhibitory action of tumor cell on the whole.To under with the close condition of a kind of inhibition rate of tumor cell, CPFP is very little and positive control medicine 5-FU is obvious to people's normal liver cell LO2 inhibitory action to the inhibitory action of people's normal liver cell LO2 relatively.

3.2CPFP the influence of pair cell apoptosis

CPFP (2.500mg/ml) effect LM3 cell is after 48 hours, and Annexin V-FITC dyeing can be observed the apoptotic cell under the fluorescence microscope.Flow cytometry CPFP effect is descended the LM3 apoptosis rate as can be known, and the apoptosis rate of NS group is 9.76%; CPFP (0.625mg/ml) group apoptosis rate is 19.77%; CPFP (2.500mg/ml) group apoptosis rate is 34.12%, infers that thus CPFP may have apoptosis-induced effect.

3.3CPFP influence to the P53 expression

The result shows, increases with CPFP dosage, and the expression of P53 gene mRNA has certain down regulation trend in the LM3 cell.The result of protein detection also finds same trend.

4. conclusion:

(1) polysaccharides is to the inhibitory action of tumor cell: this experimentation shows that the polysaccharides various dose is very little to the inhibitory action of people's normal liver cell LO2, but the common dose of positive control antitumor drug 5-FU is obvious to the inhibitory action of LO2.The common dose of polysaccharides various dose and positive control antitumor drug 5-FU all has different inhibitory action to the propagation of human body ovarian cancer SKOV-3, gastric cancer 7901, hepatocarcinoma HEPG-2, hepatocarcinoma LM3 cell.A little less than the inhibitory action of polysaccharides and the 5-FU comparison, inhibition ability, and bigger with concentration.The cytotoxicity that is polysaccharides is less, the ability of direct killing cell a little less than.Infer that polysaccharides may play a role by other approach to the inhibition of tumor.

(2) polysaccharides pair cell effect of apoptosis: this experimentation showed polysaccharides effect LM3 cell after 48 hours, and Annexin V-FITC dyeing can be observed the tumor cell and the drug-induced following tumor cell of apoptosis of the active growth under the fluorescence microscope.Can observe normal LM3 cell, its clear-cut; Apoptotic cell shrinkage wherein, color are turned white, and the more peripheral normal cell of shape all changes to some extent, is green fluorescence.According to Flow cytometry LM3 natural death of cerebral cells rate as can be known, the apoptosis rate of NS group is 9.76%; Polysaccharides (0.625mg/ml) group apoptosis rate is 19.77%; Polysaccharides (2.500mg/ml) group apoptosis rate is 34.12%, can infer that thus polysaccharides may have certain apoptosis-promoting effect.

(3) expression analysis of the P53 under the polysaccharides effect: this experiment RT-PCR outcome research shows, the variation of the expression intensity of LM3 cell P53 gene mRNA has certain trend under the effect of various dose polysaccharides, with the increase of polysaccharides dosage, the down-regulated expression of P53 gene mRNA.According to Western blot result as can be seen, the expression intensity difference of LM3 cell P53 gene protein under the effect of various dose polysaccharides, its variation has certain trend, and with the increase of polysaccharides dosage, the expression of P53 gene protein weakens to some extent.Infer that thus it may be one of its antitumor mechanism in the expression of molecule and protein level that polysaccharides reduces saltant P53.

(2) research of anti-tumor in vivo effect

1. experiment material:

1.1 laboratory animal: Kunming mouse, male and female half and half, 18～20g provides (experimental animal production licence: SCXKG osmanthus 2006-0003, experimental animal occupancy permit: SYKG osmanthus 2003-0005) by Guangxi Medical University's Experimental Animal Center.S180 tumor-bearing mice provenance is provided by the Shanghai cell.

2. experimental technique

2.1 animal acute toxicity test

Because of being subjected to the influence of polysaccharides (CPFP) concentration and volume, single administration can't be measured its LD50, so according to the dose of the tolerant Cmax of animal, maximum volume, every 12h administration 1 time, administration is 2 times altogether, mensuration CPFP maximum dosage-feeding.Irritate stomach by the grouping of acute toxicity test conventional method divided dose.

2.2S180 the inhibition of tumor-bearing mice oxter transplanted tumor experiment

2.2.1S180 the strain of tumor-bearing mice tumor is protected and is planted, goes down to posterity and the preparation of transplanted tumor model

(1) guarantor's kind of S180 tumor strain:

Get 4～5 of the kunming mices of body weight 20g, the strain of abdominal cavity inoculation S180 tumor passed a generation in regular 7～9 days, and the abdominal cavity is protected kind.

(2) foundation of S180 oxter transplanted tumor model

Get that went down to posterity 7 days in the abdominal cavity and well-grown S180 sarcoma mice, taking off cervical vertebra puts to death, 75% ethanol disinfection animal skin, aseptic condition is operation down, carefully cut off mouse peritoneal, draw well-grown animal ascites, note observing the ascites color, ascites be the milky person that do not have the flocky precipitate for good, bloody ascites is then unavailable.Trypan blue dyeing proof viable count＞98%, ascites by 1: 2 dilution proportion, is blown and beaten mixing with normal saline, the cell counting count board counting, adjusting cell number is 2 * 106 living cells/ml.Get 50 of Kunming mouses, armpit skin conventional alcohol disinfecting in right side is held the 1ml syringe and is given mice oxter subcutaneous injection 0.2ml tumor cell suspension, makes focal tumor model.Note in the operating process being close to subcutaneous inserting needle, tumor cell suspension is not spilt.After modeling the 4th day with observed S180 oxter transplanted tumor model on the 6th day become the tumor situation.

2.2.2 laboratory animal grouping and administration:

Behind the inoculation 24h, mice is divided into five groups at random by sex, body weight, and 10 every group, successive administration 10 days: normal saline group (NS): normal saline 0.2ml10g-1d-1, po; CTX group: CTX 0.02gkg-1d-1, ip; CPFP senior middle school low dose group: 1.97gkg-1d-1,0.99gkg-1d-1,0.49gkg-1d-1, po.

The matter assessment of bids is accurate cancels for having a following experimental data:

(1) the heavy tumor less than 1g or 20% mice of the average tumor of normal saline group mouse tumor is heavy less than 0.4g, and the expression tumor growth is bad.

(2) administration group dead mouse surpasses 20%, or average weight descends above 15% after going tumor, the untoward reaction of expression medicine.

2.2.3 calculating tumour inhibiting rate

Weighed in the 11st day and put to death animal, cut off the right oxter of mice subcutaneous tissue, peel off tumor, thymus, spleen, weigh, calculate tumour inhibiting rate, thymus index, spleen index by following formula.

2.3CPFP to S180 tumor-bearing mice Immune Effects

2.3.1S180 the mensuration of tumor-bearing mice thymus index, spleen index

Thymus index=thymic weight (mg)/body weight (g)

Spleen index=spleen weight (mg)/body weight (g)

2.3.2 the splenocyte transformation function is measured

(1) experimental technique: the inductive mice spleen lymphocytes proliferation experiment of phytohaemagglutinin (mtt assay)

(2) principle: after the T cell is subjected to phytohaemagglutinin (PHA), canavaline (Con A) etc. to cause mitogen or specific antigen stimulation, blast transformation takes place, living cells particularly proliferative cell is decomposed into bluish violet formazan crystallization by the mitochondrion hydrolytic enzyme with MTT (tetramethyl azo azoles salt) and develops the color, and its optical density value can reflect the propagation situation of cell.

(3) preparation of splenocyte suspension: take off cervical vertebra after medication finishes and put to death mice, aseptic taking-up spleen in super-clean bench, shred repeatedly with eye scissors, the PBS flushing to centrifuge tube, adds lymphocyte separation medium 1ml along the centrifuge tube sidewall through 200 order nylon net filters, centrifugal 1500 rev/mins, 10 minutes, draw intermediate layer cell, adjusting cell concentration with the RPMI-1640 culture medium that contains 10% calf serum is the single splenocyte suspension of 5 * 106/ml.

The mensuration of (4) lymphocyte transformation function: the mouse boosting cell suspension for preparing is added in 96 well culture plates 100 μ l cell suspension/holes.Every Mus is established control wells: 3 multiple holes, and every hole adds 100 μ l RPMI-1640s; Con A stimulates the hole: 3 multiple holes, every hole adds 100 μ l Con A (10mg/L) solution.After in 37 ℃, 5%CO2 incubator, hatching 40h, take out every hole and add 10 μ l MTT (5mg/ml) solution, continue to hatch 4h.After the taking-up, the careful suction removed supernatant, and every hole adds DMSO 150 μ l, vibrates 10 minutes, and crystal is fully dissolved, and selects the wavelength at 570nm place on microplate reader, surveys the OD value, calculates stimulation index (SI).

SI=stimulates hole (adding Con A hole) OD value/control wells OD

2.4CPFP effect to S180 tumor-bearing mice serum free radical

The modeling of S180 tumor-bearing mice, grouping, administration are after 10 days, and the 11st day eyeball got blood and put to death mice.Blood plasma 2000r/ minute centrifugal 10 minutes, collect the upper strata and do not have hemolytic serum in the EP pipe, standby in-20 ℃ of refrigerators, measure superoxide dismutase (SOD), malonaldehyde (MDA), nitric oxide (NO) in the week.

2.5 transmission electron microscope observing CPFP is to the Ultrastructural influence of S180 tumor-bearing mice tumor tissue

2.6CPFP influence to S180 tumor-bearing mice tumor tissue pathology form and P53, Bax, vegf protein expression

The modeling of S180 tumor-bearing mice, grouping, administration are after 10 days, and eyeball was got blood in the 11st day, take off cervical vertebra and put to death mice, peel off tumor tissue, and 10% neutral formalin is fixed, and it is standby to make wax stone in the 24h.

2.6.1 observe tumor tissue pathology form

The result judges: (* 100) observe necrosis area under the low power lens, are judged as kitchen range shape necrosis (+) by the necrosis area size fractionation, strip necrosis (++), big lamellar necrosis (+++).

2.6.2 the SABC method detects P53, Bax, vegf protein is expressed

The result judges: the P53 positive staining is positioned neoplastic cell nuclei.Pale brown color or the positive express cell of brown granular appear in nucleus.The Bax positive staining is positioned tumor cell matter.Pale brown color or the positive express cell of brown granular appear in Cytoplasm.The VEGF positive staining is positioned tumor cell matter.Pale brown color or the positive express cell of brown granular appear in Cytoplasm.5 visuals field of random observation under every section high power lens, the counting positive percentage or the positive are organized relative gray value.

2.7 date processing

Use SPSS13.0 software and carry out statistical procedures, measurement data all adopts

Expression, measurement data relatively adopts the t check between two groups, and the relatively employing X2 check of two groups of rates is organized mean more and is relatively adopted variance analysis.

3. experimental result

3.1 animal acute toxicity test

Observe in test, the mouse gavaging medicine is in 2 weeks, each dosage group of CPFP and the no abnormal difference of normal saline control animals, and 14d behind the medicine puts to death mice, dissects the perusal heart, liver, spleen, lung, kidney, brain, small intestinal, all no abnormal discovery.Result of the test shows: irritate stomach in the mice 24h altogether and give CPFP4.746gkg-1, maximum tolerated dose reaches 4.746gkg-1.

3.2CPFP inhibition experiment to S180 tumor-bearing mice oxter transplanted tumor

3.2.1 tumor modeling situation is observed and each experimental group tumor piece compares

In the mouse inoculation 2 days, outward appearance does not have significant change, and is movable, diet is normal; Behind the inoculation oncocyte the 3rd～4 day, the part mice can touch subcutaneous grain of rice size lump; The 4th～6 day lump in inoculation back is obvious gradually, and each is formed ratio of outflow and all is about 80%.High, medium and low dosage group of polysaccharides and CTX group tumor growth are slow than the normal saline group.But the minimizing of CTX group mice diet loses weight obviously, the fur tarnish, and perpendicular hair phenomenon is obvious, and activity obviously reduces, normal crowding together; And the high, medium and low dosage group of polysaccharides mice is obviously active than CTX group and normal saline group mice, and it is not obvious to lose weight, and fur is glossy, and perpendicular hair phenomenon is not obvious.Administration finishes back execution and respectively organizes mice, get the tumor block analysis relatively. in normal saline group and the polysaccharides, low dose group tumor block size all is about (0.8--1.1cm) * (0.8--1.0cm), polysaccharides high dose group tumor block size is about 0.4cm * 0.6cm, the about 0.3cm-0.45cm of CTX group tumor block size.

3.2.2CPFP to S180 mice oxter transplanted tumor tumor-inhibiting action

The tumor of polysaccharides height, middle dosage, CTX group heavily is starkly lower than the normal saline group, and its tumor of polysaccharides low dose group is heavy then close with the normal saline group.Show that the height of polysaccharides, middle dosage group and CTX group all have the obvious suppression rate to tumor, wherein best with the effect of high dose group and CTX group.

3.3CPFP to S180 tumor-bearing mice Immune Effects

3.3.1CPFP influence to thymus index and spleen index

Compare with the normal saline group, each dosage group thymus index of CPFP increases to some extent than the normal saline group, and wherein high, middle dosage has significant difference (P＜0.05), index and spleen index there was no significant difference (P＞0.05); Cyclophosphamide group thymus index obviously reduces, and compares with the normal saline group that there were significant differences (P＜0.05), index and spleen index no significant difference (P＞0.05).Prompting CPFP can impel thymus development, improves the caused thymus inhibitory action of tumor-bearing mice, and the growth of spleen is not had obvious facilitation; CTX can significantly suppress the thymus of tumor-bearing mice, and spleen is not had obvious inhibitory action.

3.3.2CPFP influence to the splenocyte transformation function

Compare with the normal saline group, the CPFP height, all there were significant differences for middle dosage group (P＜0.01), CTX group splenocyte conversion capability is starkly lower than normal saline group (P＜0.01), prompting CPFP can obviously improve the splenocyte transformation function of tumor-bearing mice, and the splenocyte transformation function of cyclophosphamide group tumor-bearing mice is starkly lower than the normal saline group.

3.4 polysaccharides is to S ₁₈₀The influence of tumor-bearing mice SOD, MDA, NO

Compare with the normal saline group, polysaccharides height, middle dosage group all can improve SOD content (P＜0.05 or P＜0.01), and the high, medium and low dosage group of polysaccharides all can reduce MDA, NO content (P＜0.05 or P＜0.01).

3.5 polysaccharides is to the Ultrastructural influence of tumor tissue

The normal saline group: the oncocyte form is irregular, and iuntercellular has connection, and fine hair is abundant.The organelle structure is more clear: nuclear morphology is irregular, and the caryoplasm ratio is big, and nuclear heteromorphism is strong, and the interior heterochromatin of nuclear is many, and kernel is obvious.Polysaccharides 1.97gkg ^-1Group: visible apoptotic cell, the iuntercellular gap increases, chromatin margination in the nucleus, kernel concentrates or is cracked, and visible similar round apoptotic body, mitochondrial vacuolar degeneration in the kytoplasm, mitochondrial crista merges, and is the early apoptosis sign.The CTX group: as seen nuclear atypia is arranged, and large stretch of cavity district appears in cytoplasm, dilatation of rough endoplasmic reticula, and ribosome comes off, and nuclear membrane disappears, and karyorrhexis is apoptosis sign in late period.

3.6S ₁₈₀Protein expression levels such as tumor-bearing mice tumor tissue pathology form and Bax are observed

3.6.1CPFP for S ₁₈₀The influence that tumor tissue pathology form changes

Low power lens is observed down, normal saline group and the visible growth of tumour cell of CPFP low dose group are active, the partial necrosis kitchen range is arranged, CPFP high dose, middle dosage group and CTX positive controls to the destructive power of tumor greater than normal saline group and CPFP low dosage, observe big lamellar necrosis is arranged in the visible tumor, it is comparatively serious that necrosis area reaches degree more greatly, and through rank test, each organizes downright bad no significant difference.

3.6.2CPFP influence for tumor tissue Bax protein expression

Compare with the normal saline group, Bax protein expression numerical value all increases in the CPFP group tumor tissue, compares high, middle dosage group P＜0.05 with the normal saline group.

3.6.3CPFP influence to tumor tissue P53 protein expression

With the normal saline group relatively, the P53 protein expression obviously reduces in the CPFP group tumor tissue, high dose group P＜0.01, CTX, in, low dose group P＜0.05.

3.6.4CPFP influence for the expression of tumor tissue vegf protein

Compare with the normal saline group, vegf protein is expressed all and is reduced high dose group P＜0.01, middle dosage group P＜0.05, low dosage nonsignificance in the CPFP group tumor tissue.

4. conclusion:

(1) polysaccharides is to S ₁₈₀The tumor-inhibiting action of tumor-bearing mice

Experimental result shows, compares with the normal saline group, and polysaccharides height, middle group of inhibition tumor have significant difference (P＜0.01 or P＜0.05), and CTX group tumour inhibiting rate suppresses tumor significant difference (P＜0.01).CTX is the antitumor drug of extensive use clinically, is the chemical compound of chlormethine and phosphinylidyne amido be combined into, can directly destroy DNA and stop it to duplicate.CTX at body through liver cell pigment P ₄₅₀Oxidation, driffractive ring generate intermediate product aldehyde phosphoamide, and decomposite strong effectively phosphamide chlorine mustard in tumor cell, can alkanisation take place with DNA, form cross link, suppress the growth and breeding of tumor cell, thereby the performance antitumaous effect is cell cycle nonspecific agent (CCNSA), determined curative effect.In this experiment, CTX group inhibitory rate is more than 60%, to S ₁₈₀Sarcoma has good inhibition effect.Polysaccharides high dose, middle dosage group tumour inhibiting rate are higher than 30%, and experiment is multiple to be weighed three times, as a result basically identical.Tumour inhibiting rate that it is generally acknowledged Chinese medicine surpasses 30%, the experiment triplicate, and continuous stable curative effect for several times, and statistical significance is arranged, can evaluate medicine has certain curative effect.This experimental result shows: polysaccharides is to S ₁₈₀Mice oxter transplanted tumor has the obvious suppression effect.

(2) polysaccharides is to S ₁₈₀The tumor-bearing mice antitumor immune function

This result of study shows; polysaccharides can improve thymus index; index and spleen index there is not obvious influence; illustrate that it mainly promotes the thymocyte proliferation differentiation; promote cellular immune function; improve tumor-bearing mice immune organ situation to a certain extent, the function of immune system that damages because of the lotus tumor is had certain protection and promotes restitution.In this experimentation, the polysaccharides group is to S ₁₈₀Tumor-bearing mice T lymphocyte proliferation activity has raising largely, and the prompting polysaccharides can strengthen the lymphocytic proliferation activity of T and improve the immunne response ability, is one of mechanism of its performance antitumor action.

(3) polysaccharides is to S ₁₈₀The effect of tumor-bearing mice interior free yl

Remove free radical and be one effective anticancer, press down the cancer means.The vigor of SOD has reflected the ability of body removing oxygen-derived free radicals.S has mainly been measured in this experiment ₁₈₀Tumor-bearing mice blood plasma MDA and SOD content show that polysaccharides can reduce S ₁₈₀Tumor-bearing mice blood plasma MDA, increased SOD content.Polysaccharides high dose, middle dosage and normal saline group relatively have significant difference (P＜0.05 or P＜0.01).Prompting polysaccharides antitumor mechanism may have certain relation with the removing free radical.

(4) polysaccharides is to S ₁₈₀The influence of tumor-bearing mice tumor tissue pathology form

Low power lens is observed necrosis area in each experimental group tumor down, normal saline group and the visible growth of tumour cell of polysaccharides low dose group are active, the partial necrosis kitchen range is arranged, may be because tumor tissue growth is too fast, the little necrosis region that ischemia forms, polysaccharides high dose, middle dosage group tumor body have big lamellar necrosis generally less than the normal saline group in the visible tumor, and it is comparatively serious that necrosis area reaches degree more greatly.No significant difference through between each group of rank test, may organize tumor all has downright bad relevant with each.

(5) immunohistochemical method and polysaccharides are to S ₁₈₀The influence of tumor-bearing mice Bax, P53 protein expression

Bax and P53 are two kinds of common oncogenes or antioncogene, and its protein expression can be used as a kind of prognosis sign in the research of various treating malignant tumors.In the experimental result: compare with the normal saline group, Bax protein expression numerical value all increases in the polysaccharides group tumor tissue, high, middle dosage group P＜0.05, low dose group nonsignificance.With the normal saline group relatively, the P53 protein expression obviously reduces in the polysaccharides group tumor tissue, high dose group P＜0.01, CTX, in, low dose group P＜0.05.Show that polysaccharides can improve the Bax expression of gene protein; Reduce the sudden change of P53 gene, this may be one of its antineoplastic important mechanism.

(6) polysaccharides is to S ₁₈₀The influence of expression of tumor-bearing mice vegf protein and NO content

Originally studies show that vegf protein is expressed and the reduction of NO content in the polysaccharides group tumor tissue, it might also be one of polysaccharides antineoplastic mechanism that prompting suppresses tumor vascular growth.

Therefore, polysaccharides has certain inhibitory action to tumor cell, and is not obvious to normal cell inhibiting effect; Effect with certain inhibition tumor growth; Can improve thymus index, spleen index, improve the lymphopoiesis function, the enhancing body cellular immunization.

The present invention studies by experiment and learns: polysaccharides contained in the fruit of Radix et Caulis Opuntiae Dillenii can be by regulating endothelial function, inhibition renin-angiotensin system and reversing smooth muscle cell proliferation three aspects and play hypertensive development of control; Tangible blood fat reducing, antioxidation are arranged, improve hemorheology and liver fat Denaturation, positive effect is arranged preventing and treating hyperlipemia and adipohepatic generation development; Can improve carbohydrate metabolism and lipid metabolism, increase beta Cell of islet, Insulin receptor INSR InsR and Glu4, improve oxidation resistance, the reparation damaged myocardium cell of body, good potential using value be arranged preventing and treating diabetes and complication thereof; And has certain antitumor action.Therefore the present invention for the exploitation Nantural non-toxic prevent and treat hypertension, hyperlipidemia, diabetes and anti-tumor drug or health product are laid a good foundation, effectively promoted making full use of of fruit of Radix et Caulis Opuntiae Dillenii resource.

Because natural drug is in the therapeutic process of hypertension, a plurality of internal organs such as the heart, brain, kidney and system had regulate and protective effect, can improve patient's quality of life simultaneously preferably; Can be during the treatment hyperglycemia from integrally-regulated, strengthening the body resistance improves the function of regulating beta Cell of islet and receptor, reduces the antagonism of antibody, action temperature and lasting; In blood pressure lowering and blood sugar lowering, can reach blood fat reducing; Natural drug and active component antitumor thereof have the characteristics of many target spots, multipath, too many levels; And Chinese herbal medicine has relative low price, and side effect is less relatively, determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs, and plus-minus is suitable for the treatment of chronic disease very much flexibly; Therefore fruit of Radix et Caulis Opuntiae Dillenii and polysaccharides will have a good application prospect aspect medicine for preparing blood pressure lowering, blood fat reducing, blood sugar lowering, antitumor action or the health product.

(4) specific embodiment:

Embodiments of the invention 1: the preparation of polysaccharides:

Get fruit of Radix et Caulis Opuntiae Dillenii, the water supersound extraction 1.5 hours that adds 10 times of weight, extracting solution concentrates, and adds 95% ethanol, the concentration of alcohol to 70% of regulating concentrated solution precipitates, centrifugal 10min under the 5000rpm rotating speed then, centrifugal sediment is dissolved in water, and is 1: 2 dehydrated alcohol and the extraction of ethyl acetate mixed liquor 2 times with volume ratio, water layer is removed albumen with Sevage+ papain method, concentrate, drying promptly gets polysaccharides.

Wherein the detailed process of Sevage+ papain method is: with Sevage solution (volume ratio: chloroform: n-butyl alcohol=4: 1) behind the extraction water solution 2 times, water intaking layer liquid, transfer pH to 5～7, the papain that in aqueous solution, adds 2u/100ml, 65 ℃ of insulation 26h, the centrifugal 5min of 10000rpm removes the bottom precipitation, gets polysaccharides deproteinization liquid.

After measured, polyoses content reaches 71.1% in the polysaccharides that is extracted.

Embodiments of the invention 2: get fruit of Radix et Caulis Opuntiae Dillenii making beating (one-tenth pasty state), filter (removing slag, go seed), water to the 500～1000ml that adds 1～20 times of weight adds the Mel or the xylitol of the commercially available bean jelly of 0～20% weight or ocean glue, 0～20% weight again, stirs, put into pot and boil the antiseptic that the back adds allowed band, after filtration, tinning is sealed, sterilization is placed cooling and is condensed; Injection molding after perhaps stirring, oven dry (keeping 10～50% moisture content), depanning gets semi-finished product, and packing promptly gets fruit of Radix et Caulis Opuntiae Dillenii cream.This unguentum is oral, each 250 grams, every day 1-2 time; Can be used for anti-curing oncoma.

Embodiments of the invention 3: with fresh fruit of Radix et Caulis Opuntiae Dillenii or Radix et Caulis Opuntiae Dillenii fruit juice is raw material, through all or part of fermentation or blend the solution of brewing alcoholic strength 〉=7% (V/V) into, filters, and tinning is sealed, and promptly gets Radix et Caulis Opuntiae Dillenii fruit wine.This medicated wine is oral, each 10ml, every day 1-2 time; Can be used for anti-curing oncoma.

Embodiments of the invention 4: get fruit of Radix et Caulis Opuntiae Dillenii making beating (one-tenth pasty state), filter (removing slag, go seed), sugaring stirs evenly, injection molding, and oven dry (keeping 10～60% moisture content), depanning gets semi-finished product, and packing promptly gets the fruit of Radix et Caulis Opuntiae Dillenii GUODANPI.Said preparation is oral, each 1-5 gram, every day 1-2 time; Can be used for anti-curing oncoma.

Embodiments of the invention 5: after fruit of Radix et Caulis Opuntiae Dillenii decocts with water 0.5～2 hour, add the milk of 1～30% volume and the tea of 1～30% volume, add sweet taste flavoring agent again, stir evenly, tinning is sealed, and sterilization promptly gets fruit of Radix et Caulis Opuntiae Dillenii milk tea, this medicinal tea is oral, each 10-25ml, every day 1-2 time; Can be used for anti-curing oncoma.

Embodiments of the invention 6: get the polysaccharides or the fruit of Radix et Caulis Opuntiae Dillenii of embodiment 1 preparation, add correctives, control suitable relative density, packing, sterilization make polysaccharides oral liquid or Radix et Caulis Opuntiae Dillenii fruit juice.Contain polysaccharides 0.1 gram or fruit of Radix et Caulis Opuntiae Dillenii 1 gram in every 1ml oral liquid, take 10ml at every turn, every day 2-3 time, be used for hypertensive treatment.

Embodiments of the invention 7: the polysaccharides or the fruit of Radix et Caulis Opuntiae Dillenii of getting embodiment 1 preparation; add solvent, mixing; leave standstill, filter; reconcentration to relative density is 1.30～1.35 (50～60 ℃); add that excipient is granulated or with the concentrated solution spray-drying process; packing, sterilization make polysaccharides granule or fruit of Radix et Caulis Opuntiae Dillenii granule.Every bag of 1-5 gram is taken 1 bag at every turn, every day 2-3 time, is used for the treatment of hyperlipidemia.

Embodiments of the invention 8: get the polysaccharides or the fruit of Radix et Caulis Opuntiae Dillenii of embodiment 1 preparation, add suitable excipient, make polysaccharides sheet or Radix et Caulis Opuntiae Dillenii chankings with the lift-over granulation.Every contains 0.1-0.5 gram polysaccharides or 1-5 gram fruit of Radix et Caulis Opuntiae Dillenii, takes the 1-5 sheet at every turn, every day 2-3 time, is used for the treatment of hyperglycemia.

Embodiments of the invention 9: get the polysaccharides or the fruit of Radix et Caulis Opuntiae Dillenii of embodiment 1 preparation, add suitable excipient, directly use the packing of fully-automatic capsule racking machine, make polysaccharides capsule or fruit of Radix et Caulis Opuntiae Dillenii capsule.Every contains 0.1-0.5 gram polysaccharides or 1-5 gram fruit of Radix et Caulis Opuntiae Dillenii, takes the 1-5 grain at every turn, every day 2-3 time, is used for the treatment of hyperglycemia.

Embodiments of the invention 10: get the polysaccharides or the fruit of Radix et Caulis Opuntiae Dillenii of embodiment 1 preparation, add suitable excipient, make drop pill.Every ball contains 0.1-0.5 gram polysaccharides or 1-5 gram fruit of Radix et Caulis Opuntiae Dillenii, takes the 1-5 ball at every turn, every day 2-3 time, is used for hypertensive treatment.

Claims

1. fruit of Radix et Caulis Opuntiae Dillenii is in the medicine of preparation hypotensive activity or the application in the health product.

2. according to the application of the described fruit of Radix et Caulis Opuntiae Dillenii of claim 1, it is characterized in that: get fruit of Radix et Caulis Opuntiae Dillenii, add or do not add medicinal adjuvant, be prepared into pharmaceutical preparation according to conventional method.

3. according to the application of the described fruit of Radix et Caulis Opuntiae Dillenii of claim 1, it is characterized in that: with the combination of fruit of Radix et Caulis Opuntiae Dillenii and other medicinal ingredient, and add or do not add medicinal adjuvant, be prepared into pharmaceutical preparation according to conventional method.

4. according to the application of the described fruit of Radix et Caulis Opuntiae Dillenii of claim 1, it is characterized in that: get fruit of Radix et Caulis Opuntiae Dillenii, add or do not add adjuvant, be prepared into health product according to conventional method.

5. according to the application of the described fruit of Radix et Caulis Opuntiae Dillenii of claim 1, it is characterized in that: with fruit of Radix et Caulis Opuntiae Dillenii and other combinations of substances, add or do not add adjuvant, be prepared into health product according to conventional method.