CN102784140B

CN102784140B - Application of indirubin in reduction of arsenic content in body blood

Info

Publication number: CN102784140B
Application number: CN201110132422.0A
Authority: CN
Inventors: 许羚; 王鲲; 何迎春; 陈君超; 郑青山; 石劲敏; 李禄金; 盛玉成; 吕映华; 杨娟; 刘红霞
Original assignee: Shanghai University of Traditional Chinese Medicine
Current assignee: Shanghai University of Traditional Chinese Medicine
Priority date: 2011-05-20
Filing date: 2011-05-20
Publication date: 2014-04-09
Anticipated expiration: 2031-05-20
Also published as: CN102784140A

Abstract

The invention discloses a new application of indirubin. Indirubin is a main component contained in indigo naturalis, and has a chemical structure of a bi-indole compound. Indirubin has inhibition effect on various transplantable animal tumors, and can destroy leukemia cells; indirubin can also enhance the phagocytosis effect of mononuclear phagocyte systems of animals. According to the invention, indirubin can reduce the arsenic content in body blood. Arsenic is a toxic substance with severe harm to human body. Research shows that when compared with a blank control group, rats administrated with indirubin have obviously reduced arsenic concentration in blood.

Description

Indirubin is the purposes in arsenic content in reducing body blood

Technical field:

The invention belongs to medical technical field, be specifically related to indirubin effect in arsenic content in reducing body blood.

Background technology:

Arsenic is present in nature widely, in industrial and agricultural production application quite wide, be a kind of toxicant that body is had to serious harm.Arsenic is protoplasm poisonous substance, its with histiocyte endoenzyme be easily in sulfydryl be combined, thereby the activity of inhibitory enzyme, cause cellular metabolism obstacle, the toxicity of formation to central nervous system, cardiovascular system, gastronintestinal system, causes that oral cavity, esophagus, gastric mucosal erosion, swelling are hemorrhage, toxic hepatitis, subacute severe hepatitis, heart fat infiltration, central nervous system's anoxia, glomerular injury etc.In recent years, endemic arsenic poisoning is more serious both at home and abroad, in China's animal husbandry, be still widely used organo-arsenic as feed additive (growth promoter), cause common food (animality and vegetalitas) all can detect arsenic, especially with marine product for the highest, as the mankind of food chain terminal, unavoidably there is in vivo certain arsenic especially and accumulate.At present, about the Therapeutic Method of arseniasis, mainly contain selenium supplement treatment, with sulfydryl, drive arsenic treatment and utilize treatment by Chinese herbs etc., but up to now, without any medicine, any therapy, obtain satisfied row's arsenic curative effect.Therefore, finding effectively medicine or the therapy of row's arsenic is a urgent task.

Summary of the invention:

Indirubin is main composition contained in Indigo Naturalis, and its chemical constitution is a bisindole compound.Molecular formula is C ₁₆h ₁₀n ₂o ₂.Indirubin has inhibitory action to multiple transplanted animal tumor, can destroy leukaemia; Indirubin can also strengthen the phagocytic activity of the mononuclear phagocyte system of animal.But indirubin is only as cancer therapy drug in medical research before, and document shows that indirubin is evident in efficacy to chronic grain leukemia in the past.

In the present invention, will for indirubin, provide a new purposes, while using separately, have the effect of obvious reduction body blood arsenic content.

Accompanying drawing explanation: Fig. 1 is blank group and the average whole blood arsenic concentration-time plot of indirubin suspension oral gavage group rat (n=12)

The specific embodiment:

-materials and methods

1.1 laboratory animal

Laboratory animal adopts male Sprague-Dawley rat, and male, body weight 320g～370g, is provided by Shanghai Slac Experimental Animal Co., Ltd., and production licence number is SCXK (Shanghai) 2007-0005.

Feeding environment is regular grade experimental animal room, temperature: 22 ℃～23 ℃, and relative humidity 40%～70%, occupancy permit number is SCXK (Shanghai) 2006-0003.

1.2 medicines and reagent

(1) As standard inventory solution 1gL ^-1(As standard stock solution, 1gL ^-1): Merck company provides, lot number HC 961324;

(2) nitric acid (Nitric acid): HPLC level, Merck company, lot number K40748356004;

(3) hydrochloric acid (Hydrochloride acid): HPLC level, Merck company, lot number K39343417841;

(4) Fluohydric acid. (Hydrofluoric acid): top grade is pure, and Chemical Reagent Co., Ltd., Sinopharm Group provides, lot number 20100520;

(5) high-purity helium (99.999%): Shanghai success gas company limited provides;

(6) indirubin raw material: purity 95%, lot number MRHGG 100313, Shaanxi Sen Fu Bioisystech Co., Ltd provides;

(7) sodium carboxymethyl cellulose (Carboxymethylcellulose sodium, CMC): Chemical Reagent Co., Ltd., Sinopharm Group provides, lot number 20100520;

(8) ultra-pure water (purified water): Millipore A10 ultra-pure water instrument preparation;

1.3 reagent preparations

(1) preparation of arsenic series standard solution

ICP-AES arsenic standard solution: precision measures As standard inventory solution (1gL ^-1) 0.25mL, add in 50mL volumetric flask, with deionized water, be assigned to scale, obtain 5mgL ^-1arsenic standard solution; Precision measures As standard inventory solution 0.05mL, adds in 50mL volumetric flask, with deionized water, is assigned to scale, obtains 1mgL ^-1arsenic standard solution; Precision measures 5mgL ^-1arsenic standard solution 5mL, add in 50mL volumetric flask, with deionized water, be assigned to scale, obtain 0.5mgL ^-1arsenic standard solution; Precision measures 1mgL ^-1arsenic standard solution 5mL, add in 50mL volumetric flask, with deionized water, be assigned to scale, obtain 0.1mgL ^-1(100 μ gL ^-1) arsenic standard solution.

Precision measures above-mentioned 100 μ gL ^-1arsenic standard solution 25mL, add in 50mL volumetric flask, with deionized water, be assigned to scale, obtain 50 μ gL ^-1arsenic standard solution; Precision measures 50 μ gL ^-1arsenic standard solution 20mL, add in 50mL volumetric flask, with deionized water, be assigned to scale, obtain 20 μ gL ^-1arsenic standard solution; Precision measures 20 μ gL ^-1arsenic standard solution 25mL, add in 50mL volumetric flask, with deionized water, be assigned to scale, obtain 10 μ gL ^-1arsenic standard solution; Precision measures 10 μ gL ^-1arsenic standard solution 25mL, add in 50mL volumetric flask, with deionized water, be assigned to scale, obtain 5 μ gL ^-1arsenic standard solution; Precision measures 5 μ gL ^-1arsenic standard solution 10mL, add in 50mL volumetric flask, with deionized water, be assigned to scale, obtain 1 μ gL ^-1arsenic standard solution.

The preparation of (2) 0.5% carboxymethylcellulose sodium solutions

Accurately take the sodium carboxymethyl cellulose powder art of 5.00g, put in 800ml beaker, add 500ml distilled water, 60 ℃ of magnetic agitation 4h, are transferred in 1000ml volumetric flask, are settled to scale.4 ℃ store for future use.

(3) preparation of medicine suspension

Indirubin suspension: precision takes indirubin 52.6mg, grinds to form fine powder, slowly adds a small amount of 0.5%CMC solution, continue to be ground to evenly, be transferred in 25mL tool plug graduated cylinder, repeatedly shift and merge, add to 25mL, be mixed with the suspension that concentration is 2gL-1.Face and join existing use.

1.4 instrument

(1) U.S. Perkin Elmer OPTIMA-DV of company 5300 type inductance couplings and plasma emission spectrometer (ICP-AES);

(2) 7500Ce of U.S. Agilent company inductance coupling and plasma mass spectrograph (ICP-MS);

(3) one of German Saturious BT2202S percentage analytical balance (taking blood sample);

(4) the rich fast experiment SSW of the company limited type electric heating constant temperature tank in Shanghai;

(5) Switzerland Mettler Toledo AL204-IC precision balance (taking raw material);

(6) Switzerland Mettler Toledo XP-205DU 100,000/precision balance (taking reference substance)

1.5 instrument settings

(1) ICP-AES instrumental method arranges

Instrument working parameter: RF power 1300w, nebulizer flow 0.8Lmin ^-1, assisted gas flow 0.2Lmin ^-1, plasma gas flow rate 15Lmin ^-1, peristaltic pump flow velocity 15mLmin ^-1, the time of integration 1～20s, analytical wavelengths 118.979nm.

(2) ICP-MS instrumental method arranges

Instrument working parameter: radio-frequency power 1500w; Sampling depth 8.0mm; Carrier gas flux 0.7Lmin ^-1; Assisted gas flow 0.2Lmin ^-1; Plasma gas flow rate 15Lmin ^-1; Reaction gas helium; 2 ℃ of S/C temperature; Sample hoisting velocity 0.5rps; The high salt type of nebulizer: Babington; Sampling spiroid type and diameter: nickel cone, 0.8mm; Intercepting cone type and diameter: nickel cone, 0.4mm; Drainage pattern: Spectrum; Count/quality: 3; Number of repetition: 3; Detection mode: automatically; Analytical wavelengths 118.979nm.

1.6 EXPERIMENTAL DESIGN

Choose 12 rats, non-fasting, body weight is (357.8 ± 10.7) g, be divided at random 2 groups, every group before administration and after administration 0.25,0.5,0.75,1,2,3,4,6,8,10,12,24,32,48,56,72,80,96,104,120,144h rat eye socket venous plexus gather anticoagulated whole blood 0.5mL, preserve until analyze for-40 ℃.

The administration arrangement of the different groups of table 1-1

1.7 containing arsenic sample pretreatment

Precision takes rat whole blood 0.5g to be measured in brand-new gas phase head space bottle, adds 4mL nitric acid, 1mL hydrochloric acid and 0.5mL Fluohydric acid., seals.Be positioned in 50 ℃ of water-baths and be incubated overnight.Second day is uncapped sample in head space bottle is transferred in the brand-new Falcon pipe of 15mL, and repeatedly rinse merges all liq and adds water to 15g.The centrifugal 10min of 4000rpm, gets supernatant 6mL sample introduction ICP-AES or ICP-MS and analyzes.

1.8 date processing and statistics

AUC _0-t(area under 0-144h blood arsenic-time graph) adopts statistical moment to calculate.AUC between two groups _0-t, difference adopts SNK check (q check), by SAS 9.1.3, realized.

Two results

1. arsenic standard curve

ICP-AES: standard curve was former point curve, concentration is respectively 0,0.1,0.5,1,5mgL ^-1, regression equation is that the range of linearity is 0～5mgL ^-1.

ICP-MS: standard curve was former point curve, concentration is respectively 0,1,2,5,10,20,50,100 μ gL ^-1, regression equation is Y=7.035e-3*X-2.813e-4, the range of linearity is 0～100 μ gL ^-1.

2. Pharmacokinetics in Rat

Blank group rat whole blood arsenic concentration is in Table 2-1, and the whole blood arsenic concentration after gavage drug suspension is in Table 2-2, two groups of AUC _0-tbetween value and group, comparative result is in Table 2-3.Use indirubin can make the AUC of blood arsenic _0-tsignificantly decline.

Total arsenic concentration (the mgL of whole blood of the blank group of table 2-1 rat ^-1)

Table 2-2 rat oral gavage indirubin (50mgkg ^-1) after the total arsenic concentration (mgL of whole blood ^-1)

The blood arsenic AUC of the different groups of table 2-3 _0-t

Note: " * " and blank group be P < 0.05 relatively

Claims

1. the application of indirubin in the medicine of the content for the preparation of heavy metal arsenic in reducing blood.