CN107698664A

CN107698664A - The obtained polypeptide of antitumor activity is extracted in garlic

Info

Publication number: CN107698664A
Application number: CN201610685428.3A
Authority: CN
Inventors: 马恒标
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-09-05
Filing date: 2014-09-05
Publication date: 2018-02-16
Also published as: CN106146619A; CN104327158A; CN106317202A; CN104327158B; CN106188237A; CN107021997A; CN106220711A

Abstract

The present invention relates to isolated active peptides from garlic, and its amino acid sequence is as shown in SEQ ID NO.1 7.The polypeptide has the effect of preferably treatment breast cancer, while to normal cytotoxic side effect.

Description

The obtained polypeptide of antitumor activity is extracted in garlic

Technical field

The invention belongs to biological field, and breast cancer can be treated more particularly to what a kind of extraction from garlic obtained The polypeptide of antitumor activity.

Background technology

Cancer, also known as malignant tumour, it is the disease as caused by control growth and proliferation of cell mechanism is not normal.Cancer cell except Grow it is out of control outer, can also locally invade arround normal structure even via body-internal-circulation system or lymphatic system be transferred to body its His part.

Cancer is the general designation of a major class malignant tumour.The characteristics of cancer cell is unrestricted, hyperplasia without end, makes patient's body Interior nutriment is largely consumed；Cancer cell discharges a variety of toxin, human body is produced a series of symptoms；Cancer cell can also turn Move on to whole body growth and breeding everywhere, cause human body to be become thin, be powerless, anaemia, poor appetite, heating and serious organ function by Damage etc..On the other side have a benign tumour, and benign tumour is then easily removed clean, does not shift typically, does not recur, to organ, Tissue only extruding and blocking action, but cancer（Malignant tumour）The 26S Proteasome Structure and Function of tissue, organ can be also destroyed, causes necrosis Bleeding concurrent infection, patient are dead finally due to organ failure.Today of science high speed development is arrived, we have reason It is not incurable disease to believe cancer.The medical experts for being directed to Natural medicine research study discovery：Anion is thin to suppressing cancer Dysuria with lower abdominal colic, which is moved, encouraging effect, it is important that cancer patient is treated to body without any damage using natural factor anion Evil, without any toxic side effect.

In the treatment of cancer of routine, mainly there are drug therapy, operative treatment, radiotherapy and chemotherapy.At present Though the antineoplastic used has certain curative effect to most of tumours, there are still treated effect is low, poor selectivity, poison Side effect is obvious, is also easy to produce the problems such as cells resistance.Therefore, efficient, low toxicity, the clear and definite antineoplastic of action target spot are found It is still biology, the study hotspot in medical research field.

Garlic is Liliaceae allium garlic, and vegetable formal name is referred to as AliurnSatlrum and AlliumCepa.Perennial grass Wood, there is strong smelly pungent, be artificial cultivation.Standby save in China north and south is produced.Garlic is used as medicine in China to be had Long history, modern medicine study find that garlic has the effect such as antibacterial, antitumor, reducing blood lipid, platelet aggregation-against.And And a variety of protein with cancer suppression effect, lipid, polysaccharide etc. have been isolated to it.But reality is gone back in the prior art There is no a kind of especially limited garlic polypeptide for cancer with preferable inhibitory activity.

The content of the invention

The technical problems to be solved of the present invention are to provide a kind of garlic series polypeptide of anti-breast cancer activity material.This is more Peptide is to be obtained from just unearthed garlic garlic clove by extraction.

Present invention also offers a kind of method for extracting active peptides in garlic, this method includes, will be fresh unearthed big Garlic garlic clove refrigerates, and into powder, water extraction, ultrafiltration membrance filter, bidimensional electrophoretic separation, mass spectrum, separation, is dried to obtain.

The amino acid sequence that the present invention also provides the specific peptide is：

NB-DS-5：RCPSIGICLTT

NB-DS-8：LRMKSPPGGSRTT

NB-DS-9：SRLLMKGRRSPPMTT

NB-DS-10：GSPYLRMKGVGGSMRT

NB-DS-13：MYVSRLGSSGIGL

NB-DS-14：CPSRGSSAMARL

NB-DS-16：MARTSPYGEQSRP

A technical scheme provided by the invention is：

A kind of polypeptide, its sequence are SEQ ID NO：1 to 7 any shown polypeptide with more than 30% homology.

A kind of polypeptide, its sequence are SEQ ID NO：1 to 7 is any shown.

A kind of pharmaceutical composition, include the SEQ ID NO of therapeutically effective amount：1 to 7 it is any shown in polypeptide and its pharmaceutically Acceptable salt.

A kind of pharmaceutical composition, include the SEQ ID NO of therapeutically effective amount：1 to 7 it is any shown in polypeptide and pharmaceutically acceptable Carrier or diluent.

Application of the pharmaceutical composition of the present invention in the medicine for treating cancer is prepared.

The pharmaceutical composition of the present invention is preparing the application in being used to treat the medicine of breast cancer.

Advantages of the present invention：

A series of active peptides extracted from garlic of the present invention have good inhibition to various cancers, are expected into one Step is prepared into the medicine for the treatment of cancer.

Embodiment

The extraction of the garlic polypeptide of embodiment 1

Take just unearthed fresh garlic（Within 2 ages in days）, peeling, into garlic clove, liquid nitrogen frozen 1h.Grind into powder, add PH7.0 PBS is dissolved, and is filtered.Supernatant uses ultra-filtration and separation, and milipore filter uses 0.3KDa, 5KDa, 50kDa ultrafiltration Film is filtered, and obtains molecular weight 0.3-5kDa and 5-50kDa different component, detects the antitumor activity of each component, knot Fruit shows that molecular weight 0.3-5kDa component antitumor activity is optimal.

There is optimal antitumor component by what ultrafiltration obtained, cross DEAE FF anion-exchange chromatography prepacked columns, on Sample buffer solution is 4mmol/L pH8.5 Tris-HCl buffer solutions, and each applied sample amount is 10mL, and eluent is containing 1mol/ L NaCl 4mmol/L pH8.5 Tris-HCl, eluent is first successively with 3% (v/v), 6% (v/v), 9% (v/v), 12% (v/v), 24% (v/v), 100% (v/v) concentration progress gradient elution, flow velocity 5mL/min, Detection wavelength 280nm, Collection of illustrative plates is chromatographed, each peak is collected, detects the antitumor activity of each peak component, the results showed that peak 5,8,9,10,13,14,16 groups Dividing has optimal antitumor activity, and vacuum freeze drying after corresponding peak component desalination is preserved.As required by the application The anti-tumour active polypeptide of protection.

The anti-breast cancer activity detection of polypeptide described in embodiment 2

Described each peak component antitumor activity analysis is with MDA-MB-231 cells, and antitumor activity is followed the trail of using mtt assay Component.Using PBS as control.

Mtt assay：Take the logarithm the MDA-MB-231 cells in growth period, after being digested with pancreatin, with 4 × 10⁴Individual/hole it is close Degree is inoculated in 96 orifice plates, per the μ L of hole 180, at 37 DEG C, cultivates 24h.Then it is loaded, sample solution should first pass through micropore filter Membrane filtration is degerming, and sample is diluted into 5 concentration gradients with nutrient solution, and 20 μ L are added per hole, 4 parallel holes, are put Continue to cultivate 48h in cell culture incubator.Then, MTT is added（5mg/mL）20 μ L, put CO₂37 DEG C of cultures of incubator 4h.Nutrient solution in reject hole, DMSO 150 μ L, 37 DEG C of constant temperature oscillation 30min is then added per hole fully to dissolve first a ceremonial jade-ladle, used in libation knot It is brilliant.ELIASA detects each hole absorbance (A) value at 570nm wavelength, and experiment is at least repeated 3 times.Cell inhibitory rate=［ (A pairs According to-A samples)/A controls ］ × 100%.Using Excel analysis software calculation of half inhibitory concentration IC50.As a result such as the institute of table 1 Show：

Polypeptide	IC50 （μmol/L）
		NB-DS-5	3.27
NB-DS-8	3.56
		NB-DS-9	3.94
NB-DS-10	4.02
		NB-DS-13	3.88
NB-DS-14	4.02
		NB-DS-16	3.99
PBS control	Unrestraint effect

Polypeptide is to different tumour cells and the cell toxicant of normal cell described in embodiment 6.

Respectively with human liver cancer cell BEL-7402, human glioma cells U251, Non-small cell lung carcinoma cell A549 etc. Tumour cell is target cell, and control is used as using normal human lung cancer cell A549 HFL1.Using mtt assay, the more of the application are detected Peptide has inhibited proliferation to various tumour cells, wherein, the IC50 values to BEL-7402 are 5.96 μm of ol/L, right HFL1 cytotoxicity does not almost have.Therefore, polypeptide of the invention can be in treatment human liver cancer, glioma, lung cancer, mammary gland Applied in the medicine of cancer.As a result it is as shown in table 2：

Polypeptide	IC50 （μmol/L）BEL-7402	IC50（μmol/L）U251	IC50 （μmol/L）A549	IC50（μmol/L）Normal epidermis cell
					NB-DS-5	32.51	29.87	31.85	Without obvious inhibition
NB-DS-8	37.48	28.56	32.27	Without obvious inhibition
					NB-DS-9	32.56	27.66	33.76	Without obvious inhibition
NB-DS-10	33.57	29.31	37.46	Without obvious inhibition
					NB-DS-13	35.96	32.53	40.05	Without obvious inhibition
NB-DS-14	36.45	31.48	29.17	Without obvious inhibition
					NB-DS-16	34.99	34.28	33.48	Without obvious inhibition
PBS control	Without obvious inhibition	Without obvious inhibition	Without obvious inhibition	Without obvious inhibition

Sequence table

The ＞ Ma Hengbiao of ＜ 110

The obtained polypeptide of antitumor activity is extracted in the ＞ garlics of ＜ 120

〈160〉7

〈210〉1

〈211〉11

〈212〉PRT

The ＞ of ＜ 213 are artificial synthesized

〈400〉1

RCPSIGICLTT

〈210〉2

〈211〉13

〈212〉PRT

The ＞ of ＜ 213 are artificial synthesized

〈400〉2

LRMKSPPGGSRTT

〈210〉3

〈211〉15

〈212〉PRT

The ＞ of ＜ 213 are artificial synthesized

〈400〉3

SRLLMKGRRSPPMTT

〈210〉4

〈211〉16

〈212〉PRT

The ＞ of ＜ 213 are artificial synthesized

〈400〉4

GSPYLRMKGVGGSMRT

〈210〉5

〈211〉13

〈212〉PRT

The ＞ of ＜ 213 are artificial synthesized

〈400〉5

MYVSRLGSSGIGL

〈210〉6

〈211〉12

〈212〉PRT

The ＞ of ＜ 213 are artificial synthesized

〈400〉6

CPSRGSSAMARL

〈210〉7

〈211〉13

〈212〉PRT

The ＞ of ＜ 213 are artificial synthesized

〈400〉7

MARTSPYGEQSRP

Claims

1. a kind of polypeptide, its sequence is SEQ ID NO：The polypeptide with more than 30% homology shown in 6.

2. a kind of polypeptide, its sequence is SEQ ID NO：Shown in 6.

3. any described polypeptide pharmaceutically may be used with it in a kind of pharmaceutical composition, including the claim 1-2 of therapeutically effective amount The salt of receiving.

4. any described polypeptide and it can pharmaceutically be connect in a kind of pharmaceutical composition, including the claim 1-2 of therapeutically effective amount The carrier or diluent received.

5. application of the pharmaceutical composition in the medicine for treating cancer is prepared described in claim any one of 3-4.

6. the pharmaceutical composition described in claim any one of 3-4 is preparing the application in being used to treat the medicine of breast cancer.