CN106668042A - Application of Chonglou saponin VII to preparation of anti-lung-cancer medicament - Google Patents

Abstract

The invention relates to medical preparations containing organic active ingredients, and particularly relates to application of Chonglou saponin VII to preparation of an anti-lung-cancer medicament. The Chonglou saponin VII disclosed by the invention has a good inhibiting effect on growth of lung cancer cells and animal transplantation tumors, and the effect is specifically shown as follows: (1) the Chonglou saponin VII has a very high growth inhibiting effect on A549 and NCI-H1299 lung cancer cells in vitro; (2) the Chonglou saponin VII can induce apoptosis and cell cycle arrest, and has obvious influence on proteins related to apoptosis and a cell cycle; (3) the Chonglou saponin VII has an obvious inhibiting effect on in-vivo transplantation tumors.

Description

Applications of the Rhizoma Paridis saponin VII in anti-lung-cancer medicament is prepared

Technical field：

The present invention relates to contain the compound of the glycosyl being connected with non-saccharide compound by glycosidic inkage, with Rhizoma Paridis saponin VII is related to, the compound can be used to prepare the medicine of anti-lung cancer.

Background technology：

Pulmonary carcinoma is global modal malignant tumor, and its M ＆ M is occupy first of all kinds of malignant tumor, it has also become one of disease of serious harm human health and life.In recent years, affected by various factors, the M ＆ M of world community pulmonary carcinoma all substantially rises, and China is the most country of patients with lung cancer quantity in the world.Therefore, seek new effective lung cancer therapy medicine, further improve clinical efficacy, oneself becomes current in the urgent need to the problem studied.There is advantageous advantage in China as the aggregation ground of Chinese herbal medicine, therefore, find from Chinese herbal medicine have efficiently, the antitumor drug of low toxicity feature be one of means for being particularly suitable for.

Rhizoma Paridis (Rhizoma Paridis) are derived from the dry rhizome of liliaceous plant Rhizoma Paridiss or Rhizoma Paridis, and history of the treatment with more than one thousand years in Chinese medicine anti-cancer formulation, is the conventional medical herbs in Chinese medicine medicine for preventing formula.Modern study proves that the main active substances of Rhizoma Paridis are steroidal saponins, mainly includes dioscin and the big class compound of pennogenin two, and compound Rhizoma Paridis saponin VII is the one kind in above-mentioned steroidal saponin, shown in the following formula I of its chemical structural formula.

Have now been found that Rhizoma Paridis saponin VII has the multiple biological activities such as antitumor, antiinflammatory, antibacterial, hemostasis, but there is not yet the report of the compounds for treating pulmonary carcinoma.

The content of the invention：

The technical problem to be solved in the present invention is to provide the new application of Rhizoma Paridis saponin VII, i.e., the new opplication in pharmacy.

Above-mentioned new application is actually, applications of the Rhizoma Paridis saponin VII in anti-lung-cancer medicament is prepared.

In above-mentioned application, the medicine is made up of Rhizoma Paridis saponin VII and medically acceptable adjuvant, wherein, weight/mass percentage compositions of the Rhizoma Paridis saponin VII in medicine is 10%～50%.The medicine can be common oral formulations, such as granule, tablet or capsule.

Growths of the Rhizoma Paridis saponin VII of the present invention to lung carcinoma cell and animal-transplanted tumor has good inhibitory action, is in particular in：(1) to A549 and NCI-H1299 lung carcinoma cells there is very strong growth inhibition effect in vitro；(2) can inducing cell apoptosis and cell cycle arrest, and the albumen related to cell cycle to apoptosis has a significant effect；(3) there is obvious inhibitory action to internal transplanted tumor.

Description of the drawings

Fig. 1 is statistical analysiss figures of the Rhizoma Paridis saponin VII to the retardance situation in lung carcinoma cell cycle, and the PVVII of figure represents Rhizoma Paridis saponin VII.

Fig. 2 is that Rhizoma Paridis saponin VII induces lung carcinoma cell that the statistical analysiss figure of apoptosis occurs, and the PVVII of figure represents Rhizoma Paridis saponin VII.

Fig. 3 is the statistical analysiss figure of impacts of the Rhizoma Paridis saponin VII to apoptosis and G2/M checkpoints associated protein, and the PVVII of figure represents Rhizoma Paridis saponin VII.

Specific embodiment

Rhizoma Paridis saponin VII used by following embodiments 1～5 is purchased from Chengdu Man Site bio tech ltd.

Embodiment 1：Inhibitory action of the Rhizoma Paridis saponin VII to external lung carcinoma cell

Take the logarithm the tumor cell of trophophase, be inoculated in 96 well culture plates with 3000/ hole, overnight incubation, after cell growth state is good, original culture medium is discarded, then according to the drug level of design：With 0,0.78,1.56,3.12,6.25,12.5 μM acts on A549 and NCI-H1299 cells 24 to Rhizoma Paridis saponin VII, and culture is stopped after 48,72h；Be subsequently adding the MTT solution of 0.5mg/ml, after processing 4h, abandon supernatant, be eventually adding 150 μ l DMSO vibrations and mix, be placed in microplate reader and determine its absorbance (measure wavelength is 570nm).As a result as shown in table 1, Rhizoma Paridis saponin VII can substantially suppress the propagation of lung cell A549 and NCI-H1299, be a kind of anti-tumor medicine (IC with cytotoxicity₅₀It is shown in Table 1).

Half suppression ratio of the Rhizoma Paridis saponin VII of table 1 to A549 and NCI-H1299 cells

Embodiment 2：Impact situations of the flow cytometry analysis Rhizoma Paridis saponin VII to the lung carcinoma cell cycle

Take the logarithm trophophase A549 and NCI-H1299 cell, with 2 × 10⁵/ hole is inoculated in six well culture plates, overnight incubation, discards culture medium, adds Rhizoma Paridis saponin VII (0,1,2,4 μM), after processing cell 24h；With pancreatin digestion, cell, centrifugation, PBS 2 times are collected, then be 75% ethanol solution suspension cell with the volume fraction of pre-cooling, 4 DEG C of refrigerator overnights are placed in after sealing preserve.Next day, centrifugation, PBS washes 1 time, then 500 μ l PBS solutions are added (containing 25 μ lPI (1mg/ml) and 0.5 μ l RNases (10mg/ml) in every solencyte, it is vortexed and mixes, then cell is placed in into 37 DEG C of lucifuge incubation 30min, after fully mixing, using the distribution situation of flow cytomery cell cycle, sample is detected in l h and finished.As a result as shown in figure 1, lung carcinoma cell is Jing after Rhizoma Paridis saponin VII effects, the distribution of the DNA of cell shows as the increase of G2/M phases cells ratio.

Embodiment 3：The apoptosis situation of flow cytomery Rhizoma Paridis saponin VII

Take the logarithm trophophase A549 and NCI-H1299 cell, with 2 × 10⁵/ hole is inoculated in six well culture plates, overnight incubation, discards culture medium, adds (0,1,2,4 μM) of Rhizoma Paridis saponin VII to process after cell 48h, digestion, collects cell, centrifugation, PBS 2 times, outwells supernatant, is filtered dry with filter paper back-off；Again with the μ l/Tube suspension cells of l × Binding Buffer 100 in apoptosis test kit, each 5 μ l/Tube of Annexin-FITC and PI are subsequently adding, lucifuge, room temperature places 15min；Last 1 × Binding the Buffer for being separately added into 400 μ l/Tube again, after fully mixing, using the apoptosis situation of flow cytomery cell, sample is detected in 1h and finished.As a result as shown in Fig. 2 lung carcinoma cell is Jing after Rhizoma Paridis saponin VII effects, the apoptosis rate of cell is presented dose dependent to be increased.

Embodiment 4：Western blotting methods detect apoptosis and G2/M checkpoints associated protein

Rhizoma Paridis saponin VII (0,1,2,4 μM) I (0,0.5,1,2 μM) is incubated after 48h respectively jointly with A549 and NCI-H1299 cells, pancreatin digestion, collection cell, and with RIPA lysates total protein is extracted, and then carries out protein quantification.Cell death related protein Fas, DR3, DR5 are detected using western blotting, DcR3, PARP, Cleaved PARP, Cleaved Caspase-3, p53 and phosphor-p53 and G2/M checkpoint associated protein Cyclin B1, the expression of p21Waf1/Cip1.As a result as shown in figure 3, Rhizoma Paridis saponin VII can significance raise A549 cells in p53 and p-p53 protein expression, lowered the protein expression level of Cyclin B1 and p21Waf1/Cip1；And Rhizoma Paridis saponin VII energy significances raise p21Waf1/Cip1 in NCI-H1299 cells, Cyclin B1 are lowered.In addition, Rhizoma Paridis saponin VII raises Fas, DR3, the DR5 in A549 and NCI-H1299 cells, the protein expression level of PARP, Cleaved PARP and Cleaved Caspase-3 reduces the protein expression level of DcR3.

Embodiment 5：Set up impacts of the Nude Mouse Model research Rhizoma Paridis saponin VII to growth of xenografted

Set up Non-small cell lung carcinoma A549 cell Nude Mouse Models：From the female BAl BIc/c nu/nu mices in 4 to 6 weeks, tumor cell is made into suspension, then according to cell number is 2 × 10⁶/ be only inoculated under the right side axillary fossa of nude mice.2～3 body weight and the size with 2～3 tumors of vernier caliper measurement are weighed weekly.Then according to below equation calculates gross tumor volume：Tumor Volume (TV)=1/2 × (L × W²), wherein L is the length of the tumor longitudinal axis, and W is the length of tumor transverse axis, and draws the growth curve of nude mouse tumor.When the average external volume of tumor reaches about 100mm³When, nude mice is randomly divided into into 8 groups, 8 per group.According to following administration：Blank control group (0.9% normal saline)；The high, medium and low dosage group (4,3,2mg/kg) of Rhizoma Paridis saponin VII.Nude mice intraperitoneal injection 5 times weekly, are administered continuously 4 weeks.After administration terminates, nude mice is put to death, then take out tumor tissues frozen in -80 DEG C.Partial tumors tissue extracts total protein with the cracking of RIPA lysates, then carries out protein quantification.Cell death related protein DR3, DR5, p53 and G2/M checkpoint associated protein Cyclin B1, the expression of p21Waf1/Cip1 are detected using western blotting methods.As a result as shown in table 2 below and Fig. 3, Rhizoma Paridis saponin VII is capable of the growth of significance suppression A549 transplanted tumor in nude mice, with anti-tumor in vivo activity.

The Rhizoma Paridis saponin VII of table 2 process 4 weeks after each group nude mice body weight change, tumor volume growth curve, tumor weight and tumour inhibiting rate (Mean ± SD)

Claims (3)

1. applications of the Rhizoma Paridis saponin VII in anti-lung-cancer medicament is prepared.

2. application according to claim 1, it is characterised in that the medicine is by Rhizoma Paridis saponin VII and medically acceptable Adjuvant composition, wherein, weight/mass percentage compositions of the Rhizoma Paridis saponin VII in medicine be 10%～50%.

3. application according to claim 1 and 2, it is characterised in that the medicine is granule, tablet or capsule.