CN103142774A - Application of total saponin extract of lobedfruit schizocapsarhizome in treatment of liver cancer and nasopharyngeal carcinoma - Google Patents

Abstract

The invention belongs to the field of medicaments and provides an application of natural plant lobedfruit schizocapsarhizome and total saponin extract of the lobedfruit schizocapsarhizome, wherein the natural plant extract is total saponins of the lobedfruit schizocapsarhizome (the Latin name is Schizocapsa plantaginea Hance). The invention provides an extract technology of the total saponin extract of the lobedfruit schizocapsarhizome and the application of the total saponin extract of the lobedfruit schizocapsarhizome in treatment of liver cancer and nasopharyngeal carcinoma. The extract has strong antineoplastic activity and certain targeting property. Serum pharmacology tests prove that the extract still has strong pharmaceutical activity after be absorbed and metabolized, and the saponin extract can inhibit the expression of tumor cells VEGF and CD34 so as to inhibit the growth of capillaries of a tumor tissue and then inhibit the growth of a tumor. A plant source of the extract is a unique plant species, which has great development and utilization value, is planted in a certain scale and is cheap and easily obtained, in Guangxi province. The invention provides an antitumor extract with a wide prospect, so that a novel medicament for treating tumors is provided.

Description

The application of dehiscent fruit potato total saponin extracts in anti-hepatocarcinoma and nasopharyngeal carcinoma

Technical field

The invention belongs to medicine and genetic engineering field, relate to the application of dehiscent fruit potato and total saponin extracts thereof.

Background technology

The dehiscent fruit potato, Liliopsida, Liliidae, Liliales Rhizoma amorphophalli potato section, dehiscent fruit potato platymiscium, the Latin formal name used at school: Schizocapsa plantaginea Hance, another name: Semen Capparis, water dog be young, long must fruit, winter leaf seven, Hei Dongye, arrow root potato, objective mother company, objective mother seven, horse old man, Mi Hewa, a song chicken, water Semen Arecae, water bird son, summer radish, Rhizoma Schizocapsae Plantagineae, Rhizoma Schizocapsae Plantagineae, water shrimp public affairs, Cipangopaludina chinensis seven, Rhizoma Bolbostematis, Herba Senecionis Chrysanthemoidis, little Cipangopaludina chinensis seven.Be born on limit, ditch, ridge meadow more.Be distributed in the provinces and regions such as Jiangxi, Hunan, Guangdong, Guangxi and Guizhou.Be used as medicine with tuber, cure mainly laryngopharynx swelling and pain, acute gastroenteritis, urinary tract infection, toothache, chronic gastritis, Stomach duodenum ulcer, rheumatic arthritis, menoxenia, malaria, traumatic injury; Sore swollen toxin is controlled in external, traumatic hemorrhage.The chemical composition of dehiscent fruit potato has had preliminary Separation Research at present, therefrom isolated and surpassed 40 kinds of monomeric substances, its chemical composition comprises: rhizome contains aminoacid, sapogenin luxuriant sapogenin (yamogenin) according to appointment, steroid saponins (A-D) comprises and dehiscent fruit potato saponin (lieguonin or taccaoside), the highly oxidized steroidal of multiple steroid bitter principle arrow root potato ketone lactone (taccalonolide) A-M(), flavone and alkaloid.And its chemical composition activity has been carried out preliminary experiment.The spectromatic analysis that extracts from the dehiscent fruit potato (taccalonolide A) has cytotoxicity to the P388 tumor cell, and the Mus plasmodium there is killing action, nearest research shows that also spectromatic analysis and E (taccalonolideAand E) firstly have the natural steroid that is similar to the taxol effect to microtubule, and it has been carried out certain structure of modification.C27 steroidal saponin taccaoside C and taccaoside D have inhibitory action to human leukemia CCRF-CEM tumor cell line, Eca 1 (human esophagus cancerous cell), SPC-A-1 (human lung adenocarcinoma cell), BGC 1 (the low differentiation of mankind gastric adenocarcinoma cells, AGS (mankind's gastric adenocarcinoma cells), six tumor cells such as K562 (the former leukaemia of the chronic marrow of the mankind) all have inhibitory action (IC50=1.90; 6.6; 6.5; 5.1; 8.9; 7.9 μ g/m1).As seen antitumor research at present mainly concentrates on the multiple steroid bitter principle arrow root potato ketone lactone taccalonolides of non-saponins, the rarely seen taccaoside C of saponins, approximately luxuriant sapogenin (yamogenin) and dehiscent fruit potato saponin), ((lieguonin or taccaoside A-B) has no document and mentions dehiscent fruit potato saponin first, second.These researchs still mainly rest on the non-specific antitumor action of external preliminary observation dehiscent fruit potato active component, have not yet to see report with specific, targeting for tumor hepatocarcinoma and nasopharyngeal carcinoma research.

Summary of the invention

The objective of the invention is to utilize the total saponins that extracts from the tuber of Guangxi Special plant dehiscent fruit potato that tumor is treated.

The invention provides a kind of extract of new inhibition tumor cell, this extract is dehiscent fruit potato Latin formal name used at school: the total saponins composition that extracts in Schizocapsa plantaginea Hance and each saponin monomer compound composition that comprises.

The application of dehiscent fruit potato total saponin extracts in anti-hepatocarcinoma, anti-nasopharyngeal carcinoma.

In the present invention, tumor can be people's hepatocarcinoma, nasopharyngeal carcinoma.

The present invention utilizes dehiscent fruit potato saponin as Hepatoma therapy first, the medicine of nasopharyngeal carcinoma, add described dehiscent fruit potato total saponin extracts (should comprise: luxuriant sapogenin (yamogenin) in tumor cell, steroid saponins (A-D)), this saponin can obviously suppress the propagation of hepatoma carcinoma cell or nasopharyngeal carcinoma cell, and lower to the normal cell restraint.

The application of dehiscent fruit potato total saponin extracts in anti-hepatocarcinoma, anti-nasopharyngeal carcinoma is add in tumor cell and give described dehiscent fruit potato total saponin extracts in people's tumor mouse transplantation model.

In the present invention, dehiscent fruit potato total saponins is used for tumor cell (for example Liver cancer cell SMMC-7721, BEL-7404 and human nasopharyngeal epithelioma 1 CNE-1), result shows that dehiscent fruit potato total saponins has obvious inhibitory action to tumor cell in experiment in vitro.Through dehiscent fruit potato total saponins is used for normal liver cell HL-7702, contrast proof dehiscent fruit potato total saponins has obvious selectivity to interstitial laser photocoagulation.Indicating that dehiscent fruit potato total saponins has wide Research Prospects at anti-tumor aspect.

In the present invention, dehiscent fruit potato total saponins is used for Serologic test.Make dehiscent fruit potato saponin the medicinal liquid gavage rat of rat tolerance dose concentration every day, and after continuous seven days, abdominal aortic cannulation is got blood, and centrifuging and taking gets serum.Utilize Contained Serum culture of tumor cell (Liver cancer cell SMMC-7721 for example, BEL-7404 and human nasopharyngeal epithelioma 1 CNE-1), result shows that Contained Serum has obvious inhibitory action to tumor cell, proves that dehiscent fruit potato total saponins still has antitumor action through after Absorption And Metabolism.Prompting dehiscent fruit potato total saponins can be taken orally.

In swollen neoplastic process, the formation of tiny blood vessels has very important facilitation to the growth of tumor tissues with diffusion.Tumor cell can promote to organize the expression of interior VEGF (VEGF), and angiogenic growth is accelerated, and tiny blood vessels increases, and further promotes the growth of tumor body.

In the present invention, certain density dehiscent fruit potato total saponins is added drop-wise on the aseptic acetate fiber filter paper that is placed on chick chorioallantoic membrane, continue to hatch and get chick chorioallantoic membrane after three days and be placed in microscopically and observe, find that on the Embryo Gallus domesticus chick chorioallantoic membrane, Angiogenesis obviously weakens, and be the concentration dependence, prompting dehiscent fruit potato saponin can reduce the generation of tiny blood vessels, and the tiny blood vessels perfusion that reduces in interstitial laser photocoagulation is had certain effect.

ELISA measures tyrosine protein kinase (TPK) content, dehiscent fruit potato saponin also has inhibitory action to tyrosine kinase content, but be weaker than BAY 43-9006, illustrate that dehiscent fruit potato saponin can by suppressing the above-mentioned effect of tyrosine kinase performance of modulate tumor cell and angiogenic growth, reach the targeting anti-tumor effect.

In the present invention, dehiscent fruit potato total saponins is used for nude mice suppresses the tumor model, result shows that size, the weight of nude mice tumor reduce along with the increase of dosage.It is downright bad that the HE coloration result finds that dehiscent fruit potato saponin can cause the tumor cell small pieces, and focal necrosis and little focal necrosis are collapsed with blood vessel in various degree.Showed by immune group result, dehiscent fruit potato total saponins can make tiny blood vessels minimizing in tumor.The expression dyeing demonstration of VEGF, dehiscent fruit potato total saponins can make tumor tissues vegf expression amount reduce.

Generally speaking, dehiscent fruit potato total saponins also can reduce the angiogenesis in tumor tissues except can directly suppressing people's hepatocarcinoma and Growth of Nasopharyngeal Carcinoma, reduces the hemoperfusion of tumor tissues, play the effect of inhibition tumor cell and angiogenic growth, and can reduce the bloody path transfer of tumor cell.

The invention provides a kind of new Hepatoma therapy and the medicine of nasopharyngeal carcinoma, this medicine is the total saponins composition of dehiscent fruit potato.This medicine is applied to laryngopharynx swelling and pain originally, gastroenteritis, and traumatic injury is studied its mechanism and application at hepatocarcinoma and treatment of nasopharyngeal carcinoma in the present invention first, for treatment of cancer provides new medicine storage.This medicine has pair tumor group to be woven with certain selectivity, and can suppress the generation of tumor tiny blood vessels, is a kind of powerful antitumor drug that acts on.

This extract anti-tumor activity is strong and have certain targeting, through the serum pharmacological checking, this extract still has very strong pharmaceutically active through after Absorption And Metabolism, but and the Angiogenesis of the expression of this saponin extract inhibition tumor cell VEGF, CD34 and TPK content and then inhibition tumor tissues, suppress tumor growth.The plant origin of this extract is the distinctive plant variety of Guangxi local, and larger value of exploiting and utilizing is arranged, and now existing certain planting scale, cheap and easy to get.The invention provides a kind of antineoplastic extract that bright prospects are arranged, for the treatment of tumor has increased new medicine.

Description of drawings

Fig. 1 is for measuring the cellular morphology figure (100 *) of Contained Serum extracorporeal anti-tumor function.Fig. 1-1 is 30% blank serum cytological map, and as can be seen from the figure, cell is normal adherent growth state, overlapping growth phenomenon is arranged, be thought of as serum to due to the promotion growth of cell, dead cell (floating cell) is less, cell outline is clear, and is bright, in endochylema without granule; Fig. 1-2, Fig. 1-3, be respectively 30% BAY 43-9006 Contained Serum group, 30% dehiscent fruit potato saponin Contained Serum group cytological map.Compare with Fig. 1-1, Fig. 1-2, Fig. 1-3 attached cells tail off, and Growth of Cells is slow, and dead cell increases, and cell breakage has obvious fragment to be suspended in the middle of culture medium, illustrate that containing dehiscent fruit potato saponin metabolite serum has inhibitory action to tumor cell.

To be medicine compare with negative control group the ， ﹡ p＜0.01(that affects of transplanted tumor in nude mice weight Fig. 2).

To be medicine compare with negative control group the ， ﹡ p＜0.01(that affects of transplanted tumor in nude mice volume Fig. 3).

Fig. 4 is that drug effect is by the cell microphotograph of HE dyeing.After HE dyeing, nucleus is blue, similar round, and color is more shallow, and the obvious shrinkage of necrotic zone visible cell core diminishes, and color is darker.Three or five neoplasm necrosis cell aggregationes are called little focal necrosis, and fritter neoplasm necrosis cell aggregation is called focal necrosis, can divide the strip necrosis according to the big or small degree of necrotic zone, and large lamellar is downright bad, and a loose tumor cell is only arranged in large lamellar necrotic zone.The HE coloration result is found the substantially acellular downright bad phenomenon of negative control group tumor cell, rich blood vessel, and quantity is more.(seeing Fig. 4-1).As seen the BAY 43-9006 group has large lamellar downright bad, the downright bad and focal necrosis in various degree of strip, and blood vessel obviously subsides, atrophy, number tail off (seeing Fig. 4-2); The visible small pieces of dehiscent fruit potato saponin high dose group are downright bad, focal necrosis and little focal necrosis (seeing Fig. 4-3); The visible focal necrosis of dosage group and little focal necrosis (seeing Fig. 4-4) in dehiscent fruit potato saponin; The downright bad part of dehiscent fruit potato saponin low dose group is less, as seen in various degree little focal necrosis (seeing Fig. 4-5).Dehiscent fruit potato saponin senior middle school low dose group all has in various degree the blood vessel phenomenon of collapsing.Fig. 4-6 are the BAY 43-9006 group downright bad figure of large lamellar (40 *).

To be the different pharmaceutical treatment group compare) ， ﹡ ﹡ p＜0.05(and compare with negative control group the ， ﹡ p＜0.01(that affects of MVD in people's hepatocarcinoma SMMC-7721 transplanted tumor Fig. 5 with negative control group).

Fig. 6 is that different pharmaceutical is on the expression impact of people's hepatocarcinoma SMMC-7721 transplanted tumor nude mice CD34.1: negative control group; 2: the BAY 43-9006 group; 3: dehiscent fruit potato saponin high dose group; 4: dosage group in dehiscent fruit potato saponin; 5: dehiscent fruit potato saponin low dose group.

Fig. 7 is different pharmaceutical treatment group vegf expression situation.

Fig. 8 is the expression (100 *) of VEGF in tumor tissues, 1: negative control group; 2: the BAY 43-9006 group; 3: dehiscent fruit potato saponin high dose group; 4: dosage group in dehiscent fruit potato saponin; 5: dehiscent fruit potato saponin low dose group.The positive expression dyeing of VEGF is brown color, mainly fills the air after birth and endochylema in tumor cell, and it is blue that nucleus is, without positive dyeing.In negative control group, VEGF is high expressed state (+++), can see large regional brown color dyeing by figure (8-1).There is the individual cells dyeing that is positive in low express (+) of BAY 43-9006 group, dehiscent fruit potato saponin high dose VEGF in tumor region, compare significant difference with negative control group.

Fig. 9 is the variant medicine group of microscopically (40 *) Embryo Gallus domesticus blood vessel audio-visual picture.1: negative control group; 2: the BAY 43-9006 group; 3: dehiscent fruit potato saponin low dose group; 4: dosage group in dehiscent fruit potato saponin; 5: dehiscent fruit potato saponin high dose group.

The specific embodiment

The separation of dehiscent fruit potato total saponins composition

1.1 alcohol extraction

With bubble after pulverizing medicinal materials in the alcoholic solution of about 3 times of volumes 75%, reflux, extract, 2h.Sucking filtration obtains filtrate.Medicinal residues are put back to add same concentration in round-bottomed flask, volume ethanol repeats once.Merge medicinal liquid twice, filter, be concentrated into extractum, move in clean bottle standby.Every 100g is dry, and medical material can extract approximately 10g of extractum, and productive rate is 10%.

1.2 polarity section extraction

With above-mentioned gained extractum with the proper silica gel mixed grinding to becoming powdered, be placed in sand core funnel, be installed on bottle,suction, add the petroleum ether sucking filtration colourless to Extract, collect sucking filtration gained petroleum ether solution rotary evaporation and obtain the petroleum ether extraction component, be placed in 60 ℃ of baking oven dry for standby.Add successively afterwards ethyl acetate, n-butyl alcohol to repeat above step.Obtain altogether petroleum ether, ethyl acetate, three components of n-butyl alcohol.Three component productive rates are: petroleum ether group section 20%; Ethyl acetate group section 15%; N-butyl alcohol group section 50%.

1.3 macroporous adsorbent resin is processed

3 ~ 6g n-butyl alcohol component is dissolved in 50% ethanol of 50 times of volumes, the slow D101 macroporous adsorptive resins by (approximately 1 ~ 2 second one) 150ml, until liquid level during near the resin column top 50%, 60%, 100% ethanol with 150 times of volumes rinse successively.Collect flushing liquor, rotary evaporation is concentrated respectively to press variable concentrations, is placed in 60 ℃ of baking oven dry for standby.Each eluate productive rate is: 50% ethanol elution thing 73%; 60% ethanol elution thing 19%; 100% ethanol elution thing 7%.

1.4 the macroporous adsorbent resin separator evaluation of pesticide effectiveness

Choose human liver cancer cell pearl SMMC-7721, use mtt assay to measure its IC50, result is as follows:

This shows, this separation method can be good at the anti-tumor active ingredient in the enrichment drug extract.Extract solvent for use low toxicity and recoverable, detached dowel can repeat to separate and reproducible utilization, economical and efficient.

1.5 the chemical identification of elution fraction

By discrimination test as seen, this separation method gained saponin constituent purity is high, and good enrichment is arranged.

Therefore, the D101 macroporous adsorbent resin 100% separating obtained product of ethanol elution is the described total saponin extracts of this experiment.

Measure dehiscent fruit potato total saponins to the lethal effect of human liver cancer cell SMMC-7721, BEL-7404, KB cell CNE-1 and Human normal hepatocyte HL-7702

2.1 the concentration of medicine configuration

Dehiscent fruit potato saponin is made into 0.6mg/ml, as stock solution, half-and-half dilutes 4 times, obtain altogether the medicinal liquid of five concentration.0.22 μ m filtering with microporous membrane in safety cabinet is stored in-4 ℃.

2.2 mtt assay detects medicine to be measured

2.2.1 the cultivation of human hepatoma cell strain SMMC-7721, BEL-7404

Choose human liver cancer cell pearl SMMC-7721, BEL-7404 and be placed in respectively culture bottle, cultivate in 37 ℃, the incubator of 5%CO2 with the DMEM culture fluid that contains 10% hyclone.Observe every day, changes according to circumstances liquid.After cell covers with, use 0.25% trypsinization, go down to posterity.When the cell growing way is better, gets a part and use cryopreserving liquid frozen in-80 ℃ of cryogenic refrigerators.Get freeze-stored cell dissolving fast in 37 ℃ of water-baths after one week, recovery, the survival condition of observation of cell.

2.2.2 the cultivation of Human normal hepatocyte HL-7702

Choose Human normal hepatocyte HL-7702 and be placed in culture bottle, cultivate in 37 ℃, the incubator of 5%CO2 with the PRMI-1640 culture fluid that contains 10% hyclone, 1 μ g/mL insulin.Observe every day, changes according to circumstances liquid.After cell covers with, use 0.25% trypsinization, go down to posterity.When the cell growing way is better, gets a part and use cryopreserving liquid frozen in-80 ℃ of cryogenic refrigerators.Get freeze-stored cell dissolving fast in 37 ℃ of water-baths after one week, recovery, the survival condition of observation of cell.

2.2.3 the cultivation of KB cell CNE-1

Choose KB cell CNE-1 and be placed in culture bottle, cultivate in 37 ℃, the incubator of 5%CO2 with the PRMI-1640 culture fluid that contains 10% hyclone.Observe every day, changes according to circumstances liquid.After cell covers with, use 0.25% trypsinization, go down to posterity.When the cell growing way is better, gets a part and use cryopreserving liquid frozen in-80 ℃ of cryogenic refrigerators.Get freeze-stored cell dissolving fast in 37 ℃ of water-baths after one week, recovery, the survival condition of observation of cell.

2.2.4 the external tumor-inhibiting action of mtt assay detection of drugs

The trophophase cell of taking the logarithm, after 0.25% trypsinization, suction pipe is blown and beaten into cell suspending liquid, microscopically blood cell counting plate counting cells.1 * 104/ hole is inoculated in 96 orifice plates, every hole 190 μ L.Cultivate in incubator make cell adherent continuously more than 12h after, every hole adds the medicinal liquid of the variable concentrations of 10 μ L said extracted, each liquor strength is done 4 parallel holes.Continue to cultivate 24h, every hole adds the MTT solution of 20 μ L5mg/mL, continues to cultivate 4h.Suck supernatant, every hole adds 150 μ LDMSO, uses microplate reader in 490nm wavelength place's survey OD value in 15 minutes.Replication three times.Calculate cell proliferation inhibition rate.Suppression ratio (%)=(matched group OD value-experimental group OD value)/matched group OD value * 100%.

2.3 experimental result

Dehiscent fruit potato saponin is to BEL-7404, and the IC50 concentration of SMMC-7721, CNE-1, HL-7702 cell is respectively 7.16 μ g/ml, 2.83 μ g/ml, 5.68 μ g/ml, 42.38 μ g/ml.Dehiscent fruit potato saponin is to BEL-7404, and SMMC-7721, CNE-1 cell inhibition are remarkable, and normal cell is had certain differentiation effect, point out this extract to have good antitumor application prospect.

The preparation and the extracorporeal anti-tumor function that contain dehiscent fruit potato total saponins metabolite serum detect

3.1 dehiscent fruit potato total saponins maximum tolerated dose experiment

Get 18 of body weight 18 ~ 22g mices, female, hero half and half is divided into 3 groups, 6 every group at random.Be respectively blank group and administration group, the administration group is divided into again high dose group and low dose group.Mice fasting 12h before experiment can't help water.The blank group gavages normal saline, and the administration group gavages dehiscent fruit potato total saponins.Dehiscent fruit potato total saponins high dose group is 300mg/kg, and low dose group is 100mg/kg.Each dosage gavages by 1mL/1g, and 3 times/d, interval 8h.14d observes dead mouse, poisoning and active situation continuously, and records the body weight change of mice.With the dosage that just the there is no dead mouse maximum tolerated dose as this tested medicine.

3.2 determining of rat oral gavage dosage

According to dehiscent fruit potato saponin maximum tolerated dose experiment, with reference to animal per kg body weight folding coefficient table, the dosage of rat is defined as 300mg/kg/d.BAY 43-9006 determines that according to clinical reference dose conversion the gavage amount is 360mg/kg/d.Medicine is dissolved in distilled water, is made into 20mg/mL.BAY 43-9006 is suspended in 0.5% sodium carboxymethyl cellulose and makes 24mg/mL.

3.3 preparation Contained Serum

Get 30 of 200 ± 20g rats, male and female half and half are divided into 3 groups at random, 10 every group.Be respectively the blank group, BAY 43-9006 group, dehiscent fruit potato saponin group.According to the above drug dose that configures, gavage by 1.5mL/200g, 2 times/d, interval 12h, gavage is 7 days continuously.After last gavage 1h, 10% urethane anesthesia, abdominal aortic blood.Standing 1h in obtained blood room temperature, the centrifugal 5min of 5000rpm collects serum.Serum mixing on the same group, 0.22 μ m filtering with microporous membrane.56 ℃, the 30min deactivation.Be stored in-20 ℃.

3.4 mtt assay detects the anti tumor activity in vitro of Contained Serum

Take the logarithm trophophase SMMC-7721, CNE-1 cell is inoculated in 96 orifice plates, and inoculum density is 1 * 104/ hole, cell attachment fully after, suck supernatant, add the culture medium of serum-free, suck supernatant after serum starvation 24h, (volume ratio of Contained Serum in cultivating system is respectively 5%, 10%, 15% to add the Contained Serum of variable concentrations, 25%, 30%) culture medium 200 μ L establish 3 multiple holes for every group, after continuing to cultivate 48h, every hole adds the MTT solution of 20 μ L 5mg/mL, continues to cultivate 4h.Suck supernatant, every hole adds 150 μ LDMSO, uses microplate reader in 490nm wavelength place's survey OD value in 15 minutes.Duplicate detection three times.Calculate cell proliferation inhibition rate.Suppression ratio (%)=(blank serum group OD value-Contained Serum group OD value)/blank serum group OD value * 100%.

3.5 experimental result

3.5.1, Contained Serum antitumor activity in vitro result

BAY 43-9006 group, dehiscent fruit potato saponin group all have the effect of extracorporeal suppression tumor cell in various degree.Wherein, 15%, the suppression ratio of SMMC-7721 is respectively 15.49%, 24.65%, 32.26%, 10%, 15%, 25%, 30% dehiscent fruit potato saponin group Contained Serum group is 11.15%, 17.12%, 25.31%, 27.30% to 25%, 30% BAY 43-9006 group Contained Serum.

15%, the suppression ratio of CNE-1 is respectively 13.12%, 22.67%, 34.88%, 10%, 15%, 25%, 30% dehiscent fruit potato saponin group Contained Serum group is 13.81%, 18.59%, 24.33%, 30.18% to 25%, 30% BAY 43-9006 group Contained Serum.

Compare with blank serum group, group difference is remarkable, has statistical significance (p＜0.01).10% dehiscent fruit potato saponin Contained Serum group is compared with 10% BAY 43-9006 Contained Serum group, and its external tumor-inhibiting action is (p＜0.05) significantly.

Table 1 Contained Serum external to the SMMC-7721 function influence (

± s, n=6)

Table1?The?In?vitro?antitumor?effect?of?medicated?serum(

±s,?n=6)

Table 2 Contained Serum external to the CNE-1 function influence (

± s, n=6)

Table2?The?In?vitro?antitumor?effect?of?medicated?serum(

±s,?n=6)

This result proves after dehiscent fruit potato saponin is through oral absorption metabolism still have the effect that suppresses tumor, illustrates that it has effect and possible oral administration in body.

Immunohistochemical Method is measured dehiscent fruit potato total saponins to the impact of CD34, vegf expression in people's Liver Cancer Bearing Nude Mice model tumor tissues

4.1 the foundation of people's Liver Cancer Bearing Nude Mice model

Choose 3-4 age in week, 32 of the nude mices of body weight 16 ~ 18g, male and female half and half.The take the logarithm hepatocarcinoma SMMC-7721 of trophophase uses 0.25% trypsinization, and collecting cell is made cell suspending liquid with PBS, is modulated into 5 * 107ml-1, and is subcutaneous at the side axillary fossa of nude mice, inoculates respectively the 0.2ml cell suspending liquid.15d after inoculation selects the successful nude mice of inoculation, is divided at random 5 groups, and 6 every group, male and female half and half are respectively the blank group, BAY 43-9006 group, the high, medium and low dosage group of dehiscent fruit potato saponin.Dehiscent fruit potato saponin is dissolved in sterilization is configured to variable concentrations in distilled water, dehiscent fruit potato saponin group gavages respectively 40mg/kg/d, 20mg/kg/d, 5mg/kg/d, and the BAY 43-9006 group gives 30mg/kg/d.0.1ml/10g, 1 time/d, continuous gavage 21d.The blank group is left intact.From the administration first day, every 1 day weighing nude mice body weight, the major diameter (A) of vernier caliper measurement tumor and perpendicular path length (B) by formula calculated gross tumor volume (V).The cervical vertebra dislocation is put to death, and gets tumor tissue, claims tumor heavy, dissects nude mice, observes the neoplasm metastasis situation, calculates tumour inhibiting rate, tumor relative volume (RTV).Get tumor tissues, immerse 10% formaldehyde and fix, paraffin embedding, serial section, HE dyeing and immunohistochemical staining.

 

4.2 Immunohistochemical Method detects the expression of VEGF, CD34 in tumor tissues

All establish negative positive control dyeing for every group in experiment, positive control is respectively hepatocarcinoma, and negative control is cut into slices with homologue and replaced property the 1st antibody with PBS.

Roasting sheet: paraffin section is positioned in 60 ℃ of calorstats and toasts 20min.Dewaxing, aquation: conventional dimethylbenzene dewaxing, gradient ethanol aquation, dimethylbenzene I 20min--dimethylbenzene II 20min-100% ethanol 10min-95%5min-80%5min-70%5min.0.01MPBS liquid (pH7.4) is washed three times, each 5min.Antigen retrieval: VEGF (alkalescence is repaired): tissue slice is placed in edta buffer liquid (PH8.0 ± 0.1) beaker that boiling is housed, after boiling 20min, is placed in room temperature cooling.CD34(is acid to be repaired): 1500ml 0.01M citrate buffer (pH6.0) is placed in the rustless steel pressure cooker, puts into section after boiling, continue to be heated to jet, jet beginning timing is rinsed with tap water after 1.5min and is cooled to room temperature.Take out section and rinse three times with PBS, each 5min removes PBS liquid.Every section adds 1 3%H2O2, hatches 10min under room temperature.PBS rinses 3 times, each 5min.Drip primary antibodie 50ul, 4 ℃ of lower refrigerator overnight incubation.PBS rinses 3 times, each 3min.Drip the anti-reagent C of 50ul two, hatch 10min for 37 ℃.PBS rinses 3 times, each 5min.Drip two anti-reagent D 50 μ L, hatch 10min for 37 ℃.PBS washes 3 times, each 5min.Drip the freshly prepared DAB solution reaction dyeing of 50ul, microscopically is observed.Tap water rinses, and haematoxylin is redyed, and tap water rinses and returns indigo plant, and is transparent, drying, neutral gum mounting.

VEGF is tumor cell endochylema antigen, judges after section statining tumor cell and the endotheliocyte endochylema positive cell of person that the brown yellow granule occurs.Every section is chosen at random 5 high power fields (* 100) and is carried out the result judgement.Comprehensively score according to the percentage ratio that the intensity that dyes and positive cell account for the tumor cell sum.Score by dye levels: be unstained 0 minute, light color 1 minute, moderate dyeing 2 minutes, strong positive dyeing is 3 minutes.Positive percentage: staining cell number＜5% is 0 minute, and 6%～25% is 1 minute, and 26%～50% is 2 minutes, and＞51% is 3 minutes.Be its final score after addition separately with the dye levels of every section and staining cell percentage rate.Counted feminine gender (-) in 0 minute, counted in 1～2 minute (+), counted in 3～4 minutes (++), counted in 5～6 minutes (+++).

CD34 is positioned at after birth and endochylema, and it is positive for expressing that cell membrane and endochylema are brown color.Method with reference to Weidner, (* 100) select blood capillary intensive district under low power lens, be hot-spots, then (* 400) select 5 visual field counting blood capillary numbers at random under high power lens, get 5 visual field meansigma methodss as MVD value (individual blood capillary/* 400).Microvascular statistical standard: in tumor region, any cell membrane dyeing is the single or multiple endotheliocytes of brown color and with Tumors cell and Interstitial cell, clearly boundary person is arranged, and is counted as 1 blood capillary; The blood vessel that the flesh layer is thicker is not counted; The blood vessel that tube chamber surpasses 8 red blood cell diameters is not counted; Get rid of fibrosis and edge response district, hemorrhage zone etc.

4.3 experimental result

4.3.1, ordinary circumstance and the growth characteristics of people's hepatocellular carcinoma in nude mice

In whole processing procedure, predrug period, drug treating group, the nude mice of control group mental status are good, and obvious uneasiness is not arranged, how moving or refusing to eat phenomenon.After nude inoculation approximately Subcutaneous tumor in 5 ~ 6 days begin growth, the visible tumor body of naked eyes is the limitation growth of expanding, finger presses can be touched enclosed mass, and peplos is arranged.32 none routine repeated inoculations of nude mice, wherein 30 nude mices become tumor, tumor formation rate 93.75%.To the experiment end, all nude mices survivals.It is spherical that the tumor body is class, and the nodosity shape is partially hard, slidably.Near tumor and the tumor piece as seen enrich blood vessel, with muscle, the adhesion of skin phase, as seen in various degree infiltration of basilar part, part can be to muscle and rib.

4.3.2, dehiscent fruit potato saponin, BAY 43-9006 be on the impact of people's hepatocarcinoma SMMC-7721 transplanted tumor in nude mice

Result shows, the growth of the inhibition human liver cancer cell transplanted tumor that variant medication therapy groups and positive control BAY 43-9006 group all can be in various degree.After gastric infusion, in BAY 43-9006 group, dehiscent fruit potato saponin high dose group, dehiscent fruit potato saponin, the tumor tumor of dosage group is heavy compares obvious decline with negative control group, and its difference has statistical significance (p＜0.01).The tumor relative volume (RTV) of BAY 43-9006 group, dehiscent fruit potato saponin high dose group, middle dosage group is for being 3.56 ± 0.59,3.83 ± 0.62,5.21 ± 0.77, compares with the RTV7.27 of negative control group ± 0.55, all significantly reduces (p＜0.01).In each treatment group, BAY 43-9006 and dehiscent fruit potato saponin high dose group are the most obvious to the suppression ratio of tumor, for not being 51.75%, 47.71%.See Table 3.

The impact of the different treatment groups of table 3 on people's hepatocarcinoma SMMC-7721 transplanted tumor in nude mice

Table?3?Effect?of?different?drug?treatment?group?of?human?liver?SMMS-7721?transplant?tumors

﹡ p＜0.01(compares with negative control group)

4.3.3, the different pharmaceutical group is on the impact of tumor growth and blood vessel situation thereof

Normal tumor cell nucleus after HE dyeing is blue, similar round, and color is more shallow, and the obvious shrinkage of necrotic zone visible cell core diminishes, and color is darker.Three or five neoplasm necrosis cell aggregationes are called little focal necrosis, and fritter neoplasm necrosis cell aggregation is called focal necrosis, can divide the strip necrosis according to the big or small degree of necrotic zone, and large lamellar is downright bad, and a loose tumor cell is only arranged in large lamellar necrotic zone.The HE coloration result is found the substantially acellular downright bad phenomenon of negative control group tumor cell, rich blood vessel, and quantity is more.As seen the BAY 43-9006 group has large lamellar downright bad, the downright bad focal necrosis that reaches in various degree of strip, and blood vessel obviously subsides, atrophy, number tails off; The visible small pieces of dehiscent fruit potato saponin high dose group are downright bad, focal necrosis and little focal necrosis; The visible focal necrosis of dosage group and little focal necrosis in dehiscent fruit potato saponin; The downright bad part of dehiscent fruit potato saponin low dose group is less, as seen in various degree little focal necrosis.Dehiscent fruit potato saponin senior middle school low dose group all has in various degree the blood vessel phenomenon of collapsing.

4.3.4, the different pharmaceutical group is on the impact of MVD in people's hepatocellular carcinoma in nude mice

Showed by immune group result, blood capillary can present single endotheliocyte or endotheliocyte bunch, and its form and size have than notable difference, and the part can form streak or ring-type.The MVD of matched group is 46.40 ± 1.30, and with BAY 43-9006 group 19.03 ± 1.63, dehiscent fruit potato saponin high dose group 26.40 ± 1.25 is compared significant difference (p＜0.01).In dehiscent fruit potato saponin, the MVD of dosage group is 43.80 ± 1.25, compares significant difference (p＜0.05) with matched group.Dehiscent fruit potato saponin low dose group and negative control group no significant difference.See Table 4.

Table 4 different pharmaceutical treatment group on the impact of MVD in people's hepatocarcinoma SMMC-7721 transplanted tumor (

± s, n=6)

Table?4?Effects?of?different?drug?treatment?groupon?MVDof?SMCC-7721xenografted?tumor(

±s,n=6)

﹡ compares p＜0.01 ， ﹡ ﹡ and compares p＜0.05 with negative control group with negative control group

4.3.5, the different pharmaceutical treatment group is on the impact of vegf expression in people's hepatocellular carcinoma in nude mice

In tumor tissues, the dyeing of the positive expression of VEGF is brown color, mainly fills the air after birth and endochylema in tumor cell, and it is blue that nucleus is, without positive dyeing.In negative control group, VEGF is high expressed state (+++), can see large regional brown color dyeing.There is the individual cells dyeing that is positive in low express (+) of BAY 43-9006 group, dehiscent fruit potato saponin high dose VEGF in tumor region, compare significant difference (p＜0.01) with negative control group.In dehiscent fruit potato saponin, dosage vegf expression situation is ++, compare significant difference (p＜0.01) with negative control group.See Table 5.

Table 5 different pharmaceutical treatment group vegf expression situation (

± s, n=6)

Table?5?The?expression?of?VEGF?in?different?drug?group(

±s,n=6)

﹡ compares p＜0.01 with negative control group

This presentation of results dehiscent fruit potato saponin has very strong inhibitory action to transplanted tumor, also embodies anti-tumor activity in integral animal test, and its effect may be by suppressing the transplanted tumor cell and slow down microvascular growth in the tumor body, reduces that the tumors of nutrients supply realizes.

Dehiscent fruit potato total saponins is to chick chorioallantoic membrane (CAM) model vessel growth

5.1 the preparation of false air chamber and dosing

Get the similar Embryo Gallus domesticus on the 7th of volume and be divided at random administration group and blank group, 10 pieces every group.Warm water washing is clean, and 75% cotton ball soaked in alcohol is cleaned twice sterilization, the egg air chamber upwards, long holder is 45 ℃ with the egg holder, puts into temperature (37.0 ± 0.5) ℃, hatches in the CO2 incubator of relative humidity 60%.The 2nd day, on super quiet workbench, the eggshell place cracks eggshell above air chamber, open 1.5cm * 1.5cm window with ophthalmic tweezers, expose the chorion inner membrance of below, the sterilization qualitative filter paper disk that is cut in advance diameter 5mm is placed on the upper less blood vessel of CAM place, and experimental group adds the medicine of 100 μ L variable concentrations, and the blank group adds 100 μ L sodium chloride injections.Then the hinge with sterilization seals the chorion opening, and Embryo Gallus domesticus is carried out packet marking, continues to put into incubator and hatches.

The administration group is set as BAY 43-9006 group 12.5mg/L, and the high, normal, basic dosage group of dehiscent fruit potato saponin is respectively 5mg/mL, 2.5mg/mL, 1.25mg/mL.

5.2 the making of CAM specimen

After 48h is hatched in dosing, add methanol: the fixative 1.5mL of acetone (1: 1), fix 15 min under room temperature, after the blood vessel of CAM solidifies fully, remove chorion and membrana putaminis more than the CAM plane with ophthalmic tweezers, and with eye scissors centered by the filter paper carrier, the complete CAM that cuts, put in the dish ware that fills distilled water, unfold CAM with ophthalmic tweezers, it is tiled on microscope slide.Dry in the shade.Observe the angiogenic growth situation at microscopically (* 40), and take pictures with digital camera.Vascular counts records centered by carrier, the blood vessel number in radius 5mm.Observe out the thickness of large, medium and small blood vessel with inverted microscope, counting.According to blood vessel diameter, blood vessel is divided three classes: 1.＞0.2mm is trunk; 2.＞0.05mm＜0.2mm is medium vessels; 3.＜0.05mm is little blood vessel.

5.3 experimental result

Observe variant administration group chick chorioallantoic membrane blood vessel, compare with the blank group, see from table 6, compare with the blank group, in dehiscent fruit potato saponin, low dose group has obvious inhibitory action (p＜0.01) to three grades of blood vessels, and high dose group all shows inhibitory action (p＜0.01) to one, two, three blood vessel.Dehiscent fruit potato saponin high dose group is to secondary, three grades of blood vessel inhibitory action obvious (comparing p＜0.01 with the blank group).

Table 6 different pharmaceutical group on the impact of Embryo Gallus domesticus CAM angiogenesis ( ± s, n=10)

Table6?The?effect?of?different?drug?group?on?angiogenesisin?chorioallantoic?membrane(CAM)?(

±s,?n=10)

* with the blank group than p＜0.01

This presentation of results dehiscent fruit potato saponin can suppress tiny blood vessels and generate, and reduces the hemoperfusion of tumor, has a positive effect to suppressing tumor growth.

5.4 tyrosine kinase assay result

Can be seen by table 7, drug effect 24h, BAY 43-9006 administration group, dehiscent fruit potato saponin high dose group are compared with the blank group, and there were significant differences for its tyrosine kinase content (p＜0.01).Drug effect 48h, the different dosing group is compared with the blank group, and its tyrosine kinase has notable difference (p＜0.05), wherein high, the middle dosage group (p＜0.01) of BAY 43-9006 group, dehiscent fruit potato saponin.The tyrosine kinase changes of contents of different time various dose sees Table 7.

 

The tyrosine kinase content results of the different timeliness of table 7, dose-effect

Table?3-6?The?results?of?tyrosine?kinase?content?on?different?ageing,?concentration-response?relationship

﹡ compares p＜0.05 than p＜0.05 ， ﹡ ﹡ with the BAY 43-9006 low dose group with the blank group than p＜0.01 ， ﹟ with the blank group

In sum, dehiscent fruit potato total saponins is clear and definite to the inhibitory action of tumor cell, respond well, and in zoopery, transplanted tumor is had good effect, is a kind of research direction of good novel anti-tumor medicine.

The present invention's experiment material used and reagent

1.1 cell strain

People's hepatocarcinoma SMMC-7721, BEL-7404, HL-7702, CNE-1 be available from Chinese Academy of Sciences's cell bank, by the cultivation of going down to posterity of this laboratory.

1.2 animal origin and raising

The Wistar mice, the SPF level, male and female half and half are provided by Guangxi Medical University's Experimental Animal Center.

The Wistar rat, the SPF level, male and female half and half are provided by Guangxi Medical University's Experimental Animal Center.

Embryo Gallus domesticus: available from Nanning City good phoenix agriculture and animal husbandry Co., Ltd.

BALB/C nude mouse (4～5 week), body weight (15～18) g, male and female half and half available from Guangxi Medical University's Experimental Animal Center, are raised in the Experimental Animal Center SPF of Guangxi Medical University level Animal House.

1.3 key instrument and equipment

CO2 incubator (U.S. Thermo company), MK3 microplate reader (U.S. Thermo company), inverted phase contrast microscope (Japanese OLYMPUS company), Hfsafe 1200 safety cabinet Hong Kong heating power Development Co., Ltds), TDL-80-2B low speed desk centrifuge (Anting Scientific Instrument Factory, Shanghai), the vertical autoclave of LDZX-50FBS (Shenan Medical Appliances Factory, Shanghai) is hatched box (Foochow step new), 3K15 High speed refrigerated centrifuge (German sigma company)

1.4 main agents

people's spleen tryrosinase ELISA detection kit (source, Shanghai leaf bio tech ltd), 1640 culture medium (U.S. Gibco company), DMEM high glucose medium (U.S. Gibco company), DMSO(U.S. Sigma company), hyclone (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.), trypsin U.S. Sigma company), MTT(U.S. AMRESCO company), sodium carboxymethyl cellulose (the Chengdu Long Huagongshijichang of section), strong biochemical corp in PBS(Nanning), D101 macroporous adsorbent resin (Tianjin recovery fine chemistry industry institute), ethanol, petroleum ether, ethyl acetate, n-butyl alcohol (the Chengdu Long Huagongshijichang of section), Leupeptin(import packing), Aprotinin(import packing), the green skies of PMSF(biotechnology research institute), RIPA lysate (green skies biotechnology research institute), DMR+Q550 pathological image analyser (German Lycra), calorstat (Foochow steps new), plastics are high temperature resistant section frame (Foochow steps new), slide gauge (Shanghai metering products factory), chemical plant, Gansu Province, west, EDTA(Guangdong), the digital medical image analysis of Motic Med 6.0 system

1.5 the preparation of main agents

The preparation of medicine: drug extract is dissolved in appropriate tri-distilled water and is made into desired concn, 0.22 μ m filtering with microporous membrane degerming in super-clean bench.The medicine that is insoluble to tri-distilled water is dissolved in appropriate DMSO and is made into finite concentration, makes the DMSO content that adds in the effect system be not more than 0.1%.

The preparation of PRMI-1640 culture medium: the PRMI-1640 powder fully is dissolved in 1000mL, add 2.0g NaHCO3, the final concentration that adds penicillin, streptomycin to make to contain penicillin, streptomycin in system is 100UmL-1, filtration sterilization and packing in super-clean bench, 20 ℃ of refrigerators of Bao Cun Yu –, add hyclone before use, be mixed with the PRMI-1640 culture medium of 10% hyclone.

The preparation of PBS: get a bag PBS powder, be dissolved in fully in the 2L tri-distilled water, autoclaving after packing, room temperature is preserved.

The preparation of MTT solution: get 250mgMTT, be made into 5mg/mL with 50mLPBS, fully after the dissolving in safety cabinet with 0.22 μ m filtering with microporous membrane.Be sub-packed in the EP pipe according to operating position, every pipe 1mL, frozen in-20 ℃.All adopt masking foil to cover lucifuge in preparing and packaging and frozen process.

The preparation of 0.25% pancreatin: accurately take pancreatin 0.25g, dissolved in 100mLPBS, in safety cabinet with 0.22 μ m filtering with microporous membrane, and packing, every bottle of 10mL.The complete rear employing sealing compound sealing of packing.-20 ℃ of preservations are put before use and are thawed, and are placed in 37 ℃ of water-bath balances.

The preparation of cells frozen storing liquid: RPMI-164070%, new-born calf serum 20%, DMSO10%.

The preparation of Contained Serum: Contained Serum is placed 56 ℃ of water-bath 30min deactivation, 0.22 μ m filtering with microporous membrane degerming in super-clean bench, and being deployed into final concentration with serum-free PRMI-1640 culture medium is 5%, 10%, 15%, 25%, 30%.

Claims (3)

1. the application of dehiscent fruit potato total saponin extracts in anti-hepatocarcinoma, anti-nasopharyngeal carcinoma.

2. dehiscent fruit potato total saponin extracts as claimed in claim 1, be dehiscent fruit potato Latin formal name used at school: the total saponins composition that extracts in Schizocapsa plantaginea Hance and each saponin monomer compound composition that comprises.

3. the application of dehiscent fruit potato total saponin extracts as claimed in claim 1 in anti-hepatocarcinoma, anti-nasopharyngeal carcinoma is characterized in that: add in tumor cell and give described dehiscent fruit potato total saponin extracts in people's tumor mouse transplantation model.

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