CN1923212A - Application of paris polyphyla var. yunnanensis stem and leaf monomer saponin in pharmaceutical industry - Google Patents

Abstract

The invention relates to a method for using yunnannogenin haulm saponin to produce medicine. Wherein, it uses the haulm of yunnannogenin as material, while yunnannogenin saponin I-IV can restrain the increment of leukemia cell K562 and HL60, while their IC50 value e lower than 10ug/ml; the saponin II can induce K562 and HL60 cell to dry externally, relative to the decrease of Bc1-2/Bax ratio, which is the molecule target of leukocythemia resistance.

Description

The application of Rhizoma Paridis stem and leaf monomer saponin in pharmacy

Technical field

The present invention relates to the purposes of Rhizoma Paridis stem and leaf monomer saponin, relate in particular to the purposes in pharmaceutical field.

Background technology

Leukemia (leukeamia) betides malignant tumor of hematopoiesis system.China's leukemia sickness rate is 2.76/10 ten thousand, and in the mortality of malignant tumors, leukemia occupies the 6th (male) and the 8th (women), is in the 1st (Ye Rengao, Lu Zaiying chief editor among the adult the child and below 35 years old.Internal medicine.The 6th edition.Beijing: People's Health Publisher, in June, 2004,625).In recent years progressively deeply, aspect leukemic treatment, because the application of effective combined chemotherapy (chemotherapy) and the progress of bone marrow transplantation have many patients disease free survival for a long time to leukemic research.But the leukemia chemotherapy medicament toxicity is bigger, and the patient finally produces drug resistance and causes chemotherapy failure.Bone marrow transplantation exists bone marrow to select donor difficulty and the expensive problem that is fit to, and it is higher relatively to transplant the back related mortality.Scholars constantly explore the leukemic effective way of treatment, and as Biotherapeutics, molecular targeted treatment etc., but traditional chemotherapy still is considered to the most basic treatment means (what open-birth, Chen Mingqing, Wang Xicai chief editor.Modern clinical tumor practical therapeutics.Yunnan science and technology publishing house, in October, 2004,179).Therefore, searching can be eliminated the leukemia clone and to the less medicine of normal hematopoietic cell damage, become control leukemia perfect method and approach.

Rhizoma Paridis (Paris Polyphylla Var.Yunnanensis L) is Trilliaceae (Trillaceae) paris plant, have another name called Herba Typhonii gigantei, Rhizoma Paridis, three layers of grass, Pharmacopoeia of the People's Republic of China nineteen ninety-five version, version in 2000 and version in 2005 are recorded dry rhizome (Chinese Pharmacopoeia Commission that this medicine is Rhizoma Paridis (Rhizoma Paridis) or Rhizoma Paridis.People's Republic of China's distiller (one one).Beijing: Chemical Industry Press 2005:214) is medicinal raw material.

The Rhizoma Paridis root is the primary raw material of multiple Chinese patent medicine and compatibility drug, and employing amount among the people is very big, and after the mid-90 in last century, only the consumption in Yunnan Province just reaches hundreds of tons every year, and national requirement reaches about 1000 tons.And its speed of growth is extremely slow, therefore annual consumption head and shoulders above the increment of wild Rhizoma Paridis, make the Rhizoma Paridis resource exhausted day by day.The Rhizoma Paridis stem and leaf is then grown rapidly, and annual its increment is equivalent to 7～8 times of root growth amount in the same year, is not exploited as yet so far.According to relevant department's statistics, with the amount calculating of medicinal Rhizoma Paridis root in recent years, be dropped with regard to the stem and leaf that at least 3500 tons of Rhizoma Paridis are arranged every year, causes the huge waste of resource.So, to the research of Rhizoma Paridis aerial parts, be not only the making full use of of resource, also be protection to nature.Up to now, both at home and abroad all unused land top is medicinal, does not more have the research report that monomer saponin in the Rhizoma Paridis stem and leaf is used for the leukemia aspect.

Summary of the invention

The object of the present invention is to provide the new purposes of Rhizoma Paridis stem and leaf monomer saponin, i.e. new application in pharmacy.

In fact, the present invention relates to Rhizoma Paridis stem and leaf monomer saponin improves or treats application in the leukemic medicine in preparation.

The preparation method of the Rhizoma Paridis stem and leaf monomer saponin that the present invention relates to comprises the steps:

(1) stem and leaf with the Rhizoma Paridis aerial parts is a raw material, obtains extract with organic solvent extraction;

(2) extract is through the saponin resin column, and 70% eluting partly is a total saponins;

(3) total saponins is through silica gel column chromatography, and eluting gets several portions, merges same area, respectively again through reversed phase column chromatography, after eluting obtains identical saponin, respectively again recrystallization to obtain purity be Rhizoma Paridis stem and leaf monomer saponin I, II, III, IV more than 96%.

Wherein, the described organic solvent of step (1) is selected from a kind of in ethanol, methanol, acetone, chloroform or the ethyl acetate.

When Rhizoma Paridis stem and leaf monomer saponin of the present invention is used on the medicine, can directly use, perhaps use with the form of pharmaceutical composition.This pharmaceutical composition contains 0.1%～99%, is preferably 0.5%～90% Rhizoma Paridis stem and leaf monomer saponin of the present invention, and all the other are acceptable on the materia medica, to nontoxic and inert pharmaceutically suitable carrier of humans and animals and/or excipient.

In order to understand essence of the present invention better, will its application in field of medicaments be described with the medicine biological activity test result of Rhizoma Paridis stem and leaf monomer saponin of the present invention below.

One, material and method

1 experiment material

1.1 sample, cell strain and reagent

(1) sample: Rhizoma Paridis saponin I～IV, purity is 96% bioactive extracts, its structural formula is:

Wherein,

	R	Molecular formula	Molecular weight
				Rhizoma Paridis saponin I Rhizoma Paridis saponin I I Rhizoma Paridis saponin I II Rhizoma Paridis saponin I V	glc. 2rha. | 4 ara. glc. 2rha. | 4 rha. 4rha glc. 2rha. | 3 glc. glc. 2rha.	C 44H 70O 16 C 51H 82O 20 C 45H 72O 17 C 39H 62O 12	854 1014 884 722

glc.py＝β-D-glucopyranosyl

rha.py＝α-L-rhamnopyranosyl

ara.fur＝α-L-arabinofuranosyl

Above-mentioned Rhizoma Paridis saponin I～IV is colourless acicular crystal in methanol solution, 216 ℃～218 ℃ of fusing points separate purification in Kunming Inst. of Botany, Chinese Academy of Sciences plant research department.

(2) cell strain: K562 (human red blood cell leukemia cell line), HL60 (strain of people's acute promyelocytic leukemia cell) are all available from Chinese Academy of Sciences's Shanghai cell biological institute.

(3) main agents:

A.RPMI-1640 culture medium (Gibco company, lot number: 31800-022);

B. calf serum (Hangzhou Sijiqing Biological Engineering Material Co., Ltd., lot number: 040623);

C.Hoechst33258 (solarbio of sigma company, B2883);

D. propidium iodide (PI, Sigma company);

E.RNA enzyme (RnaseA, Huamei Bio-Engrg Co.);

F. penicillin (middle promise pharmaceutcal corporation, Ltd product, lot number: 04101029);

G. streptomycin (Shangdong Ruiyang Pharmaceutical Co., Ltd's product, lot number: 03120207);

H. dimethyl sulfoxide (DMSO, AMRESCO company product);

I. symphysis albumen-V (Annexin-VFITC is available from IMMUNOTECH company);

J. Mus monoclonal antibody Bcl-2 (production code member: MAB-0014), exempt from multi-resistance Bax (production code member: RAB-0092), rabbit survivin polyclonal antibody (production code member: RAB-0536), all step neoplasm technology company limited available from Foochow; Anti-Fas antibody is a sigma company product;

K.Immuno-Bridge+ reagent (Bioisystech Co., Ltd of China fir Golden Bridge in Beijing);

L. (VP-16, Pudong Pharmaceutical Factory, Yadong Pharmaceutical Co., Shanghai Pharmaceutica produce etoposide, lot number: 028003);

M. cisplatin (DDP, the biological Pharma Inc. in Geju City, Yunnan produces lot number: 20020802);

N. (MTT, AMRESCO produce dimethyl tetrazole indigo plant, lot number: 0752B18);

O. sodium lauryl sulphate (SDS, Shanghai biological engineering company limited).

1.2 experimental instrument and equipment

(1) CO2 incubator: Japanese SANYO company, MCO-15AC type;

(2) medical clean work station: SuZhou Antai Air Tech Co., Ltd., SW-CJ-IFD type;

(3) low speed autobalancing centrifuge: Beijing Medical Centrifugal Machine Factory, LD25-2 type;

(4) inverted microscope: Japanese OLYMPUS TOKYO company, CK type;

(5) inverted phase contrast microscope: optical instrument factory, Chongqing, AC220VIN;

(6) fluorescence microscope: LEICA DMIRB type;

(7) filter: the new Asia, Shanghai purifies device factory;

(8) Constant Temp. Oven: the Shanghai medical apparatus and instruments factory of making a leapleap forward, GZX-DH-300-S-II type;

(9) portable autoclave sterilizer: Shanghai Huaxian Medical Nuclear Instruments Co., Ltd., YXQ.SG41.280 type;

(10) flow cytometer: U.S. BACKMAN-COULTER company, EPICS XL type;

(11) electronic balance: the two outstanding test instrunment in Changshu City factory, JJ200 type;

(12) ELISA Plate automatic reading instrument: U.S. Bioteck company, RIDMODEI-3550 type;

(13) disposable 96 orifice plates: U.S. NUNC company;

1.3 reagent configuration

1.3.1RPMI-1640 the configuration of complete culture solution

(1) one bag of RPMI-1640 culture medium is dissolved in the 1L tri-distilled water;

(2) add 2.2 gram sodium bicarbonate, fully mixings;

(3) filter (filter membrane is diameter 0.22 μ m+0.45 μ m) filters;

(4) add 10% calf serum, 100U/ml penicillin and 100 μ g/ml streptomycins, transferring pH value is 7.2, is the RPMI-1640 complete culture solution.

1.3.2PBS balanced salt solution configuration

(1) takes by weighing sodium dihydrogen phosphate 2.96g, sodium hydrogen phosphate 29.01g, sodium chloride 9.0g;

(2) it is dissolved in the 600ml distilled water, transfers between pH value to 7.2～7.4;

(3) be settled to 1L with distilled water at last, being concentration is the PBS solution (needs according to variable concentrations during use dilute) of 0.1mmol/l.

1.3.3 fixative configuration

Get methanol 30ml, acetic acid 10ml (3: 1), mixing keeps in Dark Place standby.

2 experimental techniques

2.1 cell culture

K562, HL60 cell are placed RPMI 1640 complete culture solutions that contain 10% calf serum, at 37 ℃, 5%CO ²The conventional cultivation in the incubator.The take the logarithm cell of trophophase, calculating the cell motility rate with 4g/L trypan blue dyeing back be＞98%, the adjustment cell density is 5 * 10 ⁴/ ml is standby.

2.2 the configuration of sample

The Rhizoma Paridis stem leaf saponin is established 5 concentration, is dissolved as 10mg/ml with DMSO earlier, and reuse RPMI RPMI-1640 dilutes respectively, and the ultrasound wave mixing is standby.

2.3 improvement mtt assay

(1) gets standby K562, HL60 cell suspension inoculation in 96 bundle culture plates, every hole 90ul;

(2) add the given the test agent of variable concentrations, every hole 10ul makes that its final concentration is respectively 25,5,1,0.1,0.01ug/ml, and each concentration is all established 3 multiple holes;

(3) the positive contrast of DDP, VP-16, the negative contrast of equal-volume cell suspension, 1%DMSO is the solvent contrast, equal-volume RPMI-1640 culture fluid is a blank;

(4) cell is put 37 ℃ after the dosing, 5%CO ₂Cultivated 48 hours in the incubator;

(5) every hole adds MTT (5mg/ml) 10ul, continues to cultivate 4 hours;

(6) every Kong Jiasan connection liquid [10%SDS+5% isobutanol+0.012mol/L HCL (W/V/V)] 100ul left standstill 12 hours;

(7) establish 570nm for measuring wavelength, 630nm is a reference wavelength, measures the OD value in each hole with microplate reader.Calculate cell proliferation inhibition rate by following formula:

Suppression ratio (%)=[1-(dosing holes OD value-blank well OD value)/(negative hole OD value-blank well OD value)] * 100%

Adopt LOGIT method calculation of half inhibitory concentration IC ₅₀

2.4 morphological observation

Get standby K562, HL60 cell suspension is gone in the culture bottle, adds the saponin I I (making its final concentration is 5ug/ml) after the dilution, places 37 ℃, 5%CO ²The conventional cultivation 24 hours in the incubator, then:

A. the cell sample after centrifugal collection saponin I I handles adds the 0.5ml fixative in centrifuge tube, slowly hanged cell, fixes 10 minutes;

B. the centrifugal fixative that goes washes twice with the PBS solution of 0.01mmol/L, each 3 minutes (manually rocking during the washing);

C. last centrifugal back is inhaled and is gone most of liquid to keep 50ul liquid, has slowly hanged cell again, drops on the microscope slide, makes cell distribution even as far as possible;

D. dry slightly, cell is attached to is difficult on the microscope slide with liquid flow;

E. evenly drip and go up 0.5ml Hoechst33258 dyeing liquor, dyeed 5 minutes.Remove liquid, little drying with absorbent paper from the edge suction;

F. wash twice with 0.01mmol/L PBS, each 3 minutes.Remove liquid, little drying with absorbent paper from the edge suction;

G. place and to detect the nucleus (excitation wavelength is about 350nm, and emission wavelength is about 460nm) that is blue under the fluorescence microscope.

2.5 Flow cytometry dna content

A. cell culture: described with test method 2.1.

B. sample preparation: the K562 of the trophophase of taking the logarithm, HL60 cell, adjusting cell density is 2 * 10 ⁵/ ml is divided into 3 groups at random, and every group of cell suspension is 10ml.First group is sample sets (add saponin I I, making its final concentration is 5ug/ml), second group of positive matched group (add VP-16, making its final concentration is 5ug/ml), the 3rd group of negative matched group (not adding any medicine).

C. centrifugal collecting cell behind three component other places reasons 24h, 48h, the 72h;

D.0.01mmol/L PBS liquid washed twice adds 70% cold ethanol (dehydrated alcohol+0.01mmol/L PBS liquid preparation) and makes single cell suspension, and 4 ℃ of preservations are spent the night;

It is E. centrifugal that (centrifugal control is residual liquid to the greatest extent for 1000g * 10min), abandon supernatant, PBS liquid washing 1 time;

F. each 200ul of RnaseA that adds Triton X-100 and 200ug/ml, put 37 ℃ 30 minutes;

G. add the PI dyestuff 1ml of 100ug/ml, the room temperature lucifuge left standstill 15 minutes;

H. detect (excitation wavelength 488nm, emission wavelength 670nm) on the flow cytometer, every duplicate samples is measured 10000 cells, and accompanying software detects dna content and calculates corresponding apoptosis rate.

2.6 Phosphatidylserine (PS) transposition detects

A. cell culture: described with test method 2.1.

B. sample preparation: the K562 cell of the trophophase of taking the logarithm, adjusting cell density is 2 * 10 ⁵/ ml is divided into 3 groups at random, and every group of cell suspension is 10ml.First group is sample sets (add saponin I I, making its final concentration is 5ug/ml), second group of positive matched group (add VP-16, making its final concentration is 5ug/ml), the 3rd group of negative matched group (not adding any medicine); Each group was all handled 24 hours.

C. centrifugal collecting cell, PBS liquid washed twice, low-speed centrifugal is abandoned supernatant;

D. cell is resuspended in Diluted binding buffer liquid, cell is transferred to 1 * 10 ⁶/ ml;

E. get 100ul cell suspension and be added in 12 * 75mm test tube, add 5ul Annexin VFITC marking fluid and 2.5ul PI, mixing;

F. test tube is placed the ice bath lucifuge to hatch 10 minutes, add the cold Diluted bindingbuffer of 10ul, the up flow type cell instrument detects (excitation wavelength 488nm, emission wavelength 670nm) immediately, every duplicate samples is measured 10000 cells, accompanying software analysis result.

2.7 immunohistochemical staining

2.6.1Bcl-2 the SABC of expressing detects

The immunohistochemical staining operation is undertaken by the test kit description, and concrete steps are as follows:

A. cell culture: described with test method 2.1.

B. sample preparation: the K562 cell of the trophophase of taking the logarithm, adjusting cell density is 2 * 10 ⁵/ ml is divided into 3 groups at random, and every group of cell suspension is 10ml.First group is sample sets (add saponin I I, making its final concentration is 5ug/ml), second group of positive matched group (add VP-16, making its final concentration is 5ug/ml), the 3rd group of negative matched group (not adding any medicine); Each group was all handled 24 hours.

C. centrifugal collecting cell, 0.01mmol/L PBS washes 3 times, the centrifugal supernatant of abandoning;

D. add 4% paraformaldehyde, 1～2ml mixing, room temperature is fixed 15 minutes;

E. the centrifugal supernatant of abandoning, PBS are washed 5 minutes * 3 times, the centrifugal supernatant of abandoning; Cell is dripped on the slide little drying;

F. drip 3%H ₂O ₂, room temperature lucifuge 30 minutes is with the blocking-up endogenous peroxidase activity;

G.PBS washes 3 minutes * 3 times, dries slightly;

H. Dropwise 5 % sheep blood serum sealing was hatched 30 minutes for 37 ℃, inclined, and did not wash;

I. drip working solution Bcl-2 (is anti-), hatched 1 hour for 37 ℃, PBS flushing 2 minutes * 3 times;

J. drip reagent 1, incubated at room 20 minutes, PBS flushing 2 minutes * 3 times;

K. drip reagent 2, incubated at room 20 minutes, PBS flushing 2 minutes * 3 times;

The colour developing of L.DAB solution;

M. tap water fully wash, redye, dehydration, transparent, mounting.

2.6.2Bax the SABC of expressing detects

A. cell culture: the same;

B. sample preparation: the K562 cell of the trophophase of taking the logarithm, adjusting cell density is 2 * 10 ⁵/ ml is divided into 3 groups at random, and every group of cell suspension is 10ml.First group is sample sets (add saponin I I, making its final concentration is 5ug/ml), second group of positive matched group (add VP-16, making its final concentration is 5ug/ml), the 3rd group of negative matched group (not adding any medicine); Each group was all handled 24 hours.

C. centrifugal collecting cell, 0.01mmol/L PBS washes 3 times, the centrifugal supernatant of abandoning;

D. add 4% paraformaldehyde, 1～2ml mixing, room temperature is fixed 15 minutes;

E. the centrifugal supernatant of abandoning, PBS are washed 5 minutes * 3 times, the centrifugal supernatant of abandoning; Cell is dripped on the slide little drying;

F. drip 3%H ₂O ₂, room temperature lucifuge 30 minutes is with the blocking-up endogenous peroxidase activity;

G.PBS washes 3 minutes * 3 times, dries slightly;

H. Dropwise 5 % sheep blood serum sealing was hatched 30 minutes for 37 ℃, inclined, and did not wash;

I. drip working solution Bax (is anti-), hatched 1 hour for 37 ℃, PBS flushing 2 minutes * 3 times;

J. drip reagent 1, incubated at room 20 minutes, PBS flushing 2 minutes * 3 times;

K. drip reagent 2, incubated at room 20 minutes, PBS flushing 2 minutes * 3 times;

The colour developing of L.DAB solution;

M. tap water fully wash, redye, dehydration, transparent, mounting.

2.6.3Fas the SABC of expressing detects

Similar with said method, one anti-is Fas in the I step.More than 3 kinds of groupizations all establish with PBS and replace an anti-negative control group of doing.

2.8 the fluorescent immune method flow cytometer detects apoptosis protein

2.8.1Bcl-2 Protein Detection

A. cell culture is the same;

B. sample preparation: the HL60 cell of the trophophase of taking the logarithm, adjusting cell density is 2 * 10 ⁵/ ml is divided into 3 groups at random, and every group of cell suspension is 10ml.First group is sample sets (add saponin I I, making its final concentration is 5ug/ml), second group of positive matched group (add VP-16, making its final concentration is 5ug/ml), the 3rd group of negative matched group (not adding any medicine).

C. with cell harvesting to the 10ml centrifuge tube, the centrifugal 5min of 300 * g abandons supernatant; Add 4mlPBS with the centrifugal 5min of 300 * g, abandon supernatant, cell is transferred to 1 * 10 with PBS ⁶/ ml.

D. respectively at adding 100ul cell suspension and 100ulIntraPrep Reagent 1 in Bcl-2 and the homotype control tube, mixing is hatched 15min in room temperature;

E. add the 4mlPBS washing, the centrifugal 5min of 300 * g abandons supernatant;

F. every pipe adds 100ul IntraPrep Reagent 2, and mixing is hatched 5min under the room temperature gently;

Add 20ul Bcl-2FITC in the G.Bcl-2 pipe, add IgG in the homotype control tube ₁-FITC 20ul, mixing, the room temperature lucifuge is hatched 15min;

H. add 4mlPBS, the centrifugal 5min of 300 * g abandons supernatant; Add and contain upward machine mensuration of 10g/L paraformaldehyde PBS liquid 0.5ml.

2.8.2Bax Protein Detection

A～F step and said method are similar;

G. add 5.0ul Bax mixing, the room temperature lucifuge is hatched 30min;

H. add 4mlPBS, the centrifugal 5min of 300 * g abandons supernatant;

I. add 2.0ul sheep anti mouse FITC2, lucifuge is hatched 30min;

J. add 4mlPBS, the centrifugal 5min of 300 * g abandons supernatant, adds the 0.5mlPBS mixing.

Last machine testing: with Flow-Chek calibration light path and stream, with the more accurate PMT voltage of Flow-Set, adjust feminine gender with the homotype contrast, flow cytometer detects the percentage rate of Bcl-2 albumen and Bax protein expression respectively.

2.9 statistical procedures

Adopt the SPSS10.0 statistical analysis software to carry out data analysis and handle, experimental result is with (mean ± standard deviation) expression, and each group difference adopts one factor analysis of variance, if whole variant, then relatively checks with LSD in twos.The significance of difference between two sample means is checked with t.

Two, experimental result

1. Rhizoma Paridis saponin I～IV is to the half-inhibition concentration (IC of K562 and HL-60 cell ₅₀) be respectively 0.81 ± 0.8,0.08 ± 0.04,0.45 ± 0.26,0.18 ± 0.15ug/ml and 2.13 ± 1.03,0.05 ± 0.03,0.01 ± 0.006,0.09 ± 0.01ug/ml, all less than 10ug/ml, show docs-effect dependence preferably.

2. when handling K562, HL60 cell 24h with 5ug/ml saponin I I, differing in size appears in cell, shape irregularity, karyon division pyknosis, chromatin morphological characteristic such as concentrate.

3. the hypodiploid peak all occurs when handling K562, HL60 cell 24h, 48h and 72h respectively with saponin I I, and increase with the prolongation of action time.G appears in the K562 cell ₂/ M the phase blocks, and the HL60 cell S phase occurs and blocks.

4. when handling K562 cell 24h with saponin I I, the PS transposition appears in 11.2% cell, positive controls only 3.55%.Immunohistochemical staining shows the low Bcl-2 of expression of processed group K562 cell albumen, and Bax albumen does not have significant change, and Bcl-2/Bax ratio descends; The Fas protein expression does not have obvious rising than matched group.(5) saponin I I handles HL60 cell different time, and the fluorescent immune method flow cytometer detects finds that Bcl-2 albumen downward modulation (4.8% → 1.6%), Bax albumen raise (12% → 63.5%), the more preceding obvious decline of Bcl-2/Bax ratio.

Compared with prior art, the present invention has following outstanding advantage:

1. up to now, still do not have in the prior art Rhizoma Paridis stem and leaf monomer saponin as effective ingredient at the report of treatment aspect the leukemia, also this composition does not have the report of anti-leukocythemia liveness.Seeking from traditional Chinese medicine and plant resources and can effectively treat leukemic medicine, is the field of Showed Very Brisk in the domestic and international new drug research.The present invention as preparing and preventing leukemic medicine to further investigate, has obtained challenging pharmacological tests to described Rhizoma Paridis stem and leaf monomer saponin.

2. Rhizoma Paridis saponin I～IV can suppress the propagation of human leukemia cell line K562, HL60, its IC ₅₀Value has stronger external anti-leukocythemia liveness all less than 10ug/ml.External K562, the HL60 apoptosis of inducing of saponin I I is along with the prolongation apoptosis rate of action time increases.It is relevant that saponin I I induces K562, HL60 apoptosis and Bcl-2/Bax ratio to descend, and analyzing Bcl-2 and Bax should be the antileukemie molecular target of Rhizoma Paridis stem leaf saponin II, has crucial meaning, is indicating good prospect in medicine.

3. to make full use of the stem and leaf of the existing Rhizoma Paridis that is abandoned in vain be raw material in the present invention, turns waste into wealth, and makes full use of plant resources, with low cost, preparation technology is simple, and can be made into peroral dosage form or injection type, provides a kind of economical and practical medicine for the mankind finally capture leukemia.

Description of drawings

Fig. 1 is the concentration-suppression ratio curve chart of Rhizoma Paridis stem leaf saponin to the K562 cell;

Fig. 2 is the concentration-suppression ratio curve chart of Rhizoma Paridis stem leaf saponin to the HL60 cell;

Fig. 3 observes 5ug/ml saponin I I to K562,24 hours influence of HL60 cytosis (* 200) for inverted phase contrast microscope, wherein, and A:HL60 cell matched group; B:HL60 cell processed group; C:K562 cell matched group; D:K562 cell processed group;

Fig. 4 be fluorescence microscope 5ug/ml saponin I I to K562,24 hours influence of HL60 cytosis (* 400), wherein, A:K562 cell matched group; B:K562 cell processed group; C:HL60 cell matched group; D:HL60 cell processed group;

Fig. 5 is that K562 cell cell cycle behind 5ug/ml saponin I I effect different time changes;

Fig. 6 is a 5ug/ml saponin I I effect K562 cell different time cell death inducing;

Fig. 7 is that HL60 cell cell cycle behind 5ug/ml saponin I I effect different time changes;

Fig. 8 is a 5ug/ml saponin I I effect HL60 cell different time cell death inducing;

Fig. 9 is the PS transposition behind 5ug/ml saponin effect 24h of K562 cell, wherein, and A: matched group; B: processed group; C: positive controls;

The expression (* 200) of Bcl-2 when Figure 10 detects 5ug/ml saponin I I effect K562 cell 24h for SABC, wherein, A: matched group; B: processed group;

The expression (* 200) of Bax when Figure 11 detects 5ug/ml saponin I I effect K562 cell 24h for SABC, wherein, A: matched group; B: processed group;

When Figure 12 detects 5ug/ml saponin I I effect K562 cell 48h for SABC the expression (* 200) of Fas wherein, A: matched group; B: processed group.

The specific embodiment

Can further be well understood to the present invention by specific embodiment given below.But they are not the qualifications to content of the present invention.

Embodiment 1

---the preparation of Rhizoma Paridis stem and leaf monomer saponin

Stem and leaf with the Rhizoma Paridis aerial parts is a raw material, pulverizes, and uses ethanol extraction; The hydrotrope behind the recovery ethanol, last saponin resin column is water respectively, 70% ethanol, 95% ethanol elution, collecting the partially recycled ethanol of 70% ethanol elution is total saponins; Total saponins is through silica gel column chromatography, and (8: 2v/v) eluting gets ten parts, and respectively with the second, four, six or nine parts are steamed separately except that organic solvent, obtains four fractions with organic solvent system; Each fraction is again through reversed phase column chromatography, and (8: 2--9: 1v/v) eluting gets saponin I, II, III, IV, individual composition with organic solvent system; The difference recrystallization, obtaining purity separately is Rhizoma Paridis stem and leaf monomer saponin I, II, III, the IV of 96%-99%.

Embodiment 2

The Rhizoma Paridis stem leaf saponin is to the influence of K562, HL60 cell proliferation

Rhizoma Paridis stem leaf saponin I～IV can obviously suppress the propagation of K562, HL60 cell, and its effect is dose-effect relationship preferably.As calculated, Rhizoma Paridis stem leaf saponin I～IV is to the IC of K562 cell ₅₀Be respectively 0.81 ± 0.8,0.08 ± 0.04,0.45 ± 0.26,0.18 ± 0.15ug/ml, to the IC of HL60 ₅₀Be respectively 2.13 ± 1.03,0.05 ± 0.03,0.01 ± 0.006,0.09 ± 0.01ug/ml, all less than 10ug/ml.The results are shown in Table 1, its concentration-suppression ratio curve is seen Fig. 1, Fig. 2.

Experiment repeats 3 times.The effect that Rhizoma Paridis stem leaf saponin I～IV suppresses the K562 cell proliferation sees Table 1-4, and the effect that VP-16 (positive controls) suppresses the K562 cell proliferation sees Table 5; The effect that Rhizoma Paridis stem leaf saponin I～IV suppresses the HL60 cell proliferation sees Table 6-9, and the effect that DDP (positive controls) suppresses the K562 cell proliferation sees Table 10.

Table 1. Rhizoma Paridis stem leaf saponin I suppresses the effect of K562 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5	72 66	81 76	76 69					76.33±4.50** 70.33±5.12**

1 0.1 0.01	60 12 0	71 30 17	73 35 23	68.00±7.00** 25.67±12.09* 13.33±11.93
					IC 50	1.74	0.38	0.32	0.81±0.80
Solvent	8.5	5.36	6.18	6.68±1.62

Annotate: compare * P＜0.05, * * P＜0.01 with the molten matched group of matchmaker.

Table 2. Rhizoma Paridis stem leaf saponin II suppresses the effect of K562 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5 1 0.1 0.01	89 90 92 34 18	96 97 99 48 26	88 84 82 50 36					91.00±4.35** 90.33±6.05** 91.00±8.54** 44.00±8.71** 26.67±9.01**
IC 50					0.14	0.06	0.05	0.08±0.04
	Solvent	8.5	5.36	6.18					6.68±1.62

Annotate: compare * P＜0.05, * * P＜0.01 with the molten matched group of matchmaker.

Table 3 Rhizoma Paridis stem leaf saponin III suppresses the effect of K562 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5 1 0.1 0.01	100 48 60 36 19	100 45 40 47 18	100 58 54 50 34					100.00±0** 50.33±6.80** 51.33±10.26** 44.33±7.37** 23.67±8.96*
IC 50					0.5	0.69	0.17	0.45±0.26
	Solvent	8.5	5.36	6.18					6.68±1.62

Annotate: compare * P＜0.05, * * P＜0.01 with the molten matched group of matchmaker.

Table 4 Rhizoma Paridis stem leaf saponin IV suppresses the effect of K562 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5 1 0.1 0.01	100 59 46 48 22	100 70 59 60 30	100 70 70 56 32					100.00±0** 66.33±6.35** 58.33±12.01** 54.67±6.11** 28.00±5.29**
IC 50					0.36	0.10	0.08	0.18±0.15
	Solvent	8.5	5.36	6.18					6.68±1.62

Annotate: compare * P＜0.05, * * P＜0.01 with the molten matched group of matchmaker.

Table 5 VP-16 is to the inhibitory action of K562 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5 1	99 71 19	93 56 5	100 50 23					97.33±3.79 59.00±10.81 15.67±9.45

Table 6 Rhizoma Paridis stem leaf saponin I suppresses the effect of HL60 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5 1 0.1 0.01	55 61 47 23 20	69 74 58 16 6	59 63 56 34 6					61.00±7.21** 66.00±7.00** 53.60±5.85** 24.33±9.07* 10.67±8.08
IC 50					3.30	1.34	1.75	2.13±1.033
	Solvent	9.18	8.35	9.61					9.046±0.52

Annotate: compare * P＜0.05, * * P＜0.01 with the molten matched group of matchmaker.

Table 7 Rhizoma Paridis stem leaf saponin II suppresses the effect of HL60 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5 1 0.1 0.01	82 86 92 87 50	100 100 100 89 60	93 88 86 86 44					91.67±9.07** 91.33±7.57** 92.67±7.02** 87.33±1.52** 51.30±8.00**
IC 50					0.01	0.069	0.069	0.049±0.034
	Solvent	9.18	8.35	9.61					9.046±0.52

Annotate: compare * P＜0.05, * * P＜0.01 with the molten matched group of matchmaker.

Table 8 Rhizoma Paridis stem leaf saponin III suppresses the effect of HL60 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5 1 0.1 0.01	100 100 99 99 48	100 100 100 90 40	100 100 92 81 72					100±0.00** 100±0.00** 97.00±4.35** 90.00±9.00** 53.33±16.65**
IC 50					0.01	0.014	0.002	0.009±0.006
	Solvent	9.18	8.35	9.61					9.046±0.52

Annotate: compare * P＜0.05, * * P＜0.01 with the molten matched group of matchmaker.

Table 9 Rhizoma Paridis stem leaf saponin IV suppresses the effect of HL60 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5 1 0.1 0.01	100 72 36 43 51	100 100 50 61 31	100 100 50 51 37					100±0.00** 90.67±16.16** 45.33±8.08** 51.67±9.01** 39.67±10.26**

IC 50	0.11	0.08	0.08	0.09±0.0173
					Solvent	9.18	8.35	9.61	9.046±0.52

Annotate: compare * P＜0.05, * * P＜0.01 with the molten matched group of matchmaker.

Table 10 DDP is to the inhibitory action of HL60 cell proliferation

Dosage ug/ml	Suppression ratio (%)
					Experiment for the first time	Experiment for the second time	Experiment for the third time	Mean value S
	25 5 1	100 70 18	100 73 34	100 74 26					100.00±0.00 72.33±2.08 26.00±8.00

Embodiment 3

---morphological observation

K562, HL60 cell, are observed under inverted phase contrast microscope after 24 hours through the saponin I I of 5ug/ml effect respectively, find that cell is not of uniform size, shape irregularity, karyon division pyknosis.After Hoechst33258 dyeing, under fluorescence microscope, observe, find that cell volume diminishes, karyopycnosis, visible fine and close graininess hyperfluorescence in nucleus or the cytoplasm, the cell blebbing forms apoptotic body when serious.And normal cell is and fills the air even fluorescence, and fluorescence than processed group a little less than (seeing Fig. 3,4).

Embodiment 4

---FCM analyzes the result of apoptotic cell

Form apoptotic body behind the apoptosis, cause cell DNA to be lost, after 5ug/ml saponin I I handles K562 cell 24h, 48h, 72h, in the dna content figure that flow cytometer detects, at G ₁Occurred the hypodiploid peak before phase, apoptosis rate is respectively 2.1% (24h), 5.1% (48h), 7.9% (72h), is certain time-effect relationship (see figure 3).Compare with matched group, difference has statistical significance (P＜0.05).We also observe G behind the drug effect ₀/ G ₁The phase cell slightly rises, G ₂/ M phase cell obviously descends, and cell is arrested in G ₂/ M the phase (seeing Fig. 5,6).

After 5ug/ml saponin I I handles HL60 cell 24h, 48h, 72h, in the dna content figure that flow cytometer detects, G ₁Occurred the hypodiploid peak before phase, apoptosis rate is respectively 8.3% (24h), 15.6% (48h), 27.1% (72h), is certain time-effect relationship (see figure 5).Compare with matched group, difference has significant statistical significance (P＜0.01).After also observing drug effect 24h in this experiment, S phase cytosis, G ₂/ M phase cell reduces.Behind effect 48h～72h, S phase cell reduces, G ₂/ M phase cell increases gradually.Cell is arrested in the S phase (seeing Fig. 7,8).

Embodiment 5

---FCM detects cell PS transposition result

After Annecin VFITC and the PI double labelling, FCM detects and finds 5ug/ml saponin I I effect K562 cell 24 hours, and V appears in 11.2% cell ⁺PI ^-(early apoptosis), V appears in 1.37% cell ⁺PI ⁺(apoptosis in late period).And positive controls is respectively 3.55%, 0.78% (see figure 9).

Embodiment 6

---Bcl-2 immunohistochemical staining result

Bcl-2 albumen borrows its terminal hydrophobicity carboxyl terminal of being made up of 19 aminoacid to link to each other with film, is positioned on cell membrane, endoplasmic reticulum, mitochondrial membrane and the nuclear membrane.5ug/ml saponin I I effect K562 cell 24 hours carries out SABC with the Bcl-2 monoclonal antibody and detects, and it is higher that the result shows that blank group Bcl-2 expresses, and is strong positive reaction in the cell, and cell is dark-brown.And experimental group cell color obviously shoals, and shows that the Bcl-2 expression obviously reduces (see figure 10).

Embodiment 7

---the immunohistochemical staining result

Bax albumen mainly is positioned Cytoplasm, and the K562 cell is after saponin I I handles, and Bax protein staining intensity is not seen obvious increase (seeing Figure 11).

Embodiment 8

---Fas immunohistochemical staining result

(Apo-1, CD95) receptor is an I type transmembrane protein to Fas, is the directly cell surface antigen of cell death inducing of a class.5ug/ml saponin I I effect K562 cell 48 hours carries out SABC with the Fas monoclonal antibody and detects, and K562 dyes not have and obviously strengthens (seeing Figure 12) after finding to handle.

Embodiment 9

---indirect fluorescent method flow cytometer detects Bcl-2 albumen and Bax protein expression

Bcl-2 albumen and Bax protein expression situation see Table 11 behind 5ug/ml saponin I I processing HL60 cell 24h, the 48h.

Table 11. Rhizoma Paridis stem leaf saponin II induces HL60 apoptosis protein expression rate

	Bax(％)		Bcl-2(％)
					24h	48h		24h		48h
	Saponin I I VP-16	63.50 4.20	53.4 4.20	1.60 3.10							4.50 4.80
Negative control					12.00		4.80

Research Rhizoma Paridis stem leaf saponin monomer I, II, III, IV vitro inhibition Leukemia Cell Proliferation are screened its type and concentration, find that saponin monomer II activity is stable, cytotoxicity is stronger.Choose the effect that saponin I I further studies its induced apoptosis in leukemia cell lines, inquire into the mechanism of its leukemia resisting action.For being developed to the leukemic medicine of new targeted therapy, Rhizoma Paridis stem leaf saponin II provides reliable experimental evidence and new approaches.

Test data shows, Rhizoma Paridis stem leaf saponin II can reduce the Bcl-2 protein expression level, destroy the balance between Bcl-2 and the Bax, logical Rhizoma Paridis stem leaf saponin II provides certain theoretical basis to the inhibited proliferation of other tumor cells, provides certain theoretical foundation for Rhizoma Paridis stem leaf saponin II is developed to new targeting anti-leukemia medicine.

Claims (2)

1. Rhizoma Paridis stem and leaf monomer saponin improves or treats application in the leukemic medicine in preparation.

2. Rhizoma Paridis stem leaf saponin II improves or treats application in the leukemic medicine in preparation.

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