CN103961338A - Application of hydroxytyrosol in DOX (doxorubicin) cardiotoxicity resistance as well as pharmaceutical composition taking hydroxytyrosol as main active ingredient - Google Patents

Abstract

The invention discloses an application of hydroxytyrosol in DOX (doxorubicin) cardiotoxicity resistance as well as pharmaceutical composition taking hydroxytyrosol as a main active ingredient. According to the invention, a cell membrane integrity fluorescent probe and a cell nucleus fluorescent probe are adopted to label myocardial cells simultaneously, the myocardial cell protection effect of a to-be-screened substance is surveyed from different action mechanisms, a novel application of hydroxytyrosol in DOX cardiotoxicity resistance is discovered, hydroxytyrosol has a higher protective effect on both myocardial cell membranes and cell nucleuses, and the myocardial cell protection rates of 100 mu M-hydroxytyrosol are 18.7% and 24.8% respectively under FDA (fluorescein diacetate) and hoechst33342 fluorescence labeling.

Description

The pharmaceutical composition that the application of hydroxytyrosol in adriamycin myocardial toxicity and the hydroxytyrosol of take are main active

Technical field

The invention belongs to drug screening and evaluation methodology field, be specifically related to the pharmaceutical composition that the application of hydroxytyrosol in adriamycin myocardial toxicity and the hydroxytyrosol of take are main active.

Background technology

Hydroxytyrosol is the effective ingredient in Fructus Canarii albi, occupies critical role in Mediterranean diet.In research in early days, it is believed that it is that the middle oleuropein of olive oil plays a significant role, through the research that deepens continuously, find, the hydrolyzate hydroxytyrosol of oleuropein has stronger physiologically active.It is simple in structure, and molecular weight is little, has good water solublity and fat-soluble, can prevent that radiation, oxygen-derived free radicals and other factors from causing damage to retina, has good treatment and health care, is subject to the extensive concern of domestic and international researcher.

There are some researches show, hydroxytyrosol has two enzyme phase systems of Detoxication in can inducing cell, can prevent the damage that in medicated cigarette, toxic component acrylic aldehyde causes retina, regulate mitochondrial function, vision is had to protective effect, the ocular disease such as macula lutea degeneration are had to improvement effect (Zhu Lu, LiuZhong bo, Feng Zhihui, et al.Hydroxytyrosol protects against oxidativedamage by simultaneous activation of mitochondrial biogenesis and phase IIdetoxifying enzyme systems in retinal pigment epithelial cells[J] .J NutrBiochem, 2010, 21 (11): 1089-1098.).

Publication number is that the Chinese patent literature of CN101674817B discloses hydroxytyrosol in induction or strengthened the purposes in repair of cartilage or regenerating bone or cartilage, this application people finds, the propagation of chondrocyte can effectively be induced or strengthen to hydroxytyrosol, stimulate chondrocyte proliferation and improve the ability that they produce extracellular matrix components, and then promote repair of cartilage and regeneration.

Application number is that 201210203464.3 Chinese patent literature discloses the purposes of hydroxytyrosol for improvement of mitochondrial function, this application people's discovery, hydroxytyrosol improves mitochondrial function by activation and the biogenous raising of mitochondrion of mitochondrial respiratory chain cpd.

Although reported in prior art that hydroxytyrosol has multiple biological activity, also do not reported that up to now perhydroxyl radical butyl alcohol can alleviate the myocardial toxicity that amycin brings out.

Amycin (Doxorubicin, DOX) is one of broad-spectrum anti-cancer drug of commonly using clinically, is widely used in the oncotherapys such as solid tumor, leukemia, lymphoma, breast carcinoma.But amycin can produce the myocardial toxicity of cumulative bad, dose dependent, and be further developed into irreversibility myocardial damage, finally cause congestive heart failure, therefore seriously suppressed its clinical practice.Therefore the active substance of finding acquisition adriamycin myocardial toxicity is significant to the clinical practice of amycin.

Summary of the invention

The invention provides the application of hydroxytyrosol in adriamycin myocardial toxicity, applicant studies discovery, and hydroxytyrosol has remarkable resistance to the myocardial toxicity because using amycin to bring out, and myocardial cell is had to stronger protective effect.

The new purposes of hydroxytyrosol of the present invention is found by the following method:

(1) ZHENQI FUZHENG KELI is dissolved to centrifuging and taking supernatant with pure water;

(2) with macroporous resin by described supernatant successively water, 40% ethanol, 95% ethanol elution, collect respectively each elution fraction, be evaporated to dry;

(3) elution fraction after concentrated is carried out to separation by preparative liquid chromatography, collect each fraction, as material to be screened, standby after being dried;

(4) myocyte that cores, adds material to be screened to protect in advance;

Described myocardial cell is preferably exponential phase cell, can select common H9c2 myocardial cell; It is 100～10000/hole for 96 orifice plates that cell density is preferably 1～100/μ L(), more preferably 20～40/μ L, most preferably is 40/μ L, fluorescence intensity comparing after suitable cell density is conducive to dye.

The final concentration of material to be screened can first carry out preliminary experiment to be determined, can myocardial cell not produced to toxicity, is advisable, and pre-guard time is preferably 30～180min, more preferably 30min.Guard time is long in advance may affect cell viability, affects screening and evaluation result.

As do not make specified otherwise, final concentration of the present invention all refer to material join in system after concentration in total system.

(5) after having protected in advance, add amycin to damage;

As preferably, the damage concentration of amycin is 0.3～0.6 μ M, and the damage time is 6～24h.Preferred damage concentration is 0.3 μ M, and the damage time is 24h, causes the process of chronic myocardial toxicity to simulate amycin low dosage in practical clinical.Meanwhile, suitable damage concentration and damage time can avoid myocardial cell excessively impaired, affect screening and evaluation result.

(6) to add in the myocardial cell after damage simultaneously, there is difference and excite cell membrane integrity fluorescent probe and the nucleus fluorescent probe with emission wavelength, carry out fluorescent labeling;

As preferably, described cell membrane integrity fluorescent probe is diacetic acid fluorescein (FDA), and it excites with emission wavelength and is respectively 488nm and 530nm.FDA is a kind of nonpolar ester type compound, and itself there is no fluorescence, but can be by the enzyme hydrolysis in living cells after it is by cell membrane and the fluorescent material (green-emitting fluorescence) of polarization; Therefore this fluorescent material can not pass through cell membrane, can in the complete cell of cell membrane, retain, in the incomplete cell of cell membrane, scatter and disappear very fast, thus can be according to the film integrality of its fluorescence intensity judgement myocardial cell.

The maximum excitation of hoechst33342 and emission wavelength are respectively 346nm and 460nm.Hoechst33342(sends out blue-fluorescence) can be combined by the base specific on nucleus double-stranded DNA, when DNA is impaired, the fluorescent probe being combined on DNA just reduces, and fluorescence intensity is corresponding weakening also.After being combined with double-stranded DNA, the maximum excitation of hoechst33342 and emission wavelength are respectively 350nm and 461nm.

FDA and hoechst33342 excite and emission wavelength non-overlapping copies, do not produce interference while guaranteeing to gather fluoroscopic image.

As preferably, the fluorescent labeling concentration of FDA and hoechst33342 is 8～12 μ g/mL, the fluorescent labeling time is 10～20min, preferred fluorescent labeling concentration is 8～12 μ g/mL, the fluorescent labeling time is 10min, and high concentration and for a long time labelling dyeing meeting cause certain damage to cell.

Amycin causes that the damage mechanism of myocardial toxicity mainly comprises that the cell viability that destroys the integrity of cell membrane and cause declines, and changes mitochondrial membrane potential in anoxic and cell death inducing etc.Based on this, the present invention has selected two kinds of fluorescent probes, from different mechanism of action, investigates the protection effect of material to be screened to myocardial cell.

(7) dyeed rear collection, analysis of cells fluoroscopic image, calculate respectively material to be screened to myocardial cell cell membrane and nuclear protective rate;

The computing formula of protective rate is:

Wherein, f is the fluorescence intensity of dosing protection group, and Fm is the fluorescence intensity of damage group, and Fc is the fluorescence intensity of normal cell group.

After having dyeed, discard culture fluid, after washing one time with PBS, blot, on fluorescence inverted microscope platform, read fluoroscopic image, fluorescence acquisition parameters is: 2.5 times of object lens magnifications, time of exposure 1000ms, first use green fluorescence optical filter (exciting light 440-520nm, utilizing emitted light 505nm) take, then use blue-fluorescence optical filter (exciting light 320-400nm, utilizing emitted light 400nm) to take; 6 width images are taken in every hole, and gained fluoroscopic image is added up through FluoinsightCell work station image mosaic, image recognition and data data output system, calculates protective rate.

The Chinese patent literature that described FluoinsightCell work station image mosaic, image recognition and data data output system have been CN101788480 at publication number is open.

(8) from material to be screened, filter out the cell membrane of myocardial cell and nuclear protective rate are all greater than to 15% active substance.

Pass through said method; the present invention finds from the extracting solution of ZHENQI FUZHENG KELI; it is active that hydroxytyrosol has stronger adriamycin myocardial toxicity, and under FDA and hoechst33342 fluorescent labeling, the hydroxytyrosol of 100 μ M is respectively 18.7%, 24.8% to the protective rate of myocardial cell.

The present invention also provides a kind of pharmaceutical composition that hydroxytyrosol is main active of take.

As preferably, in described pharmaceutical composition, the percentage by weight of hydroxytyrosol is 0.01～99.9%; More preferably 30～99%, more preferably 50～99%.

The dosage form of described pharmaceutical composition can be selected injection, injectable sterile powder, tablet, capsule, spirit, powder, granule, syrup, solution, tincture, aerosol, powder spray or suppository; Be preferably injection, injectable sterile powder, tablet, capsule or syrup; More preferably injection, tablet or capsule.

Compared with prior art, beneficial effect of the present invention is:

The present invention adopts cell membrane integrity fluorescent probe and nucleus fluorescent probe with tense marker myocardial cell; from different mechanism of action, investigate the protection effect of material to be screened to myocardial cell; found the new purposes of hydroxytyrosol in adriamycin myocardial toxicity; show as hydroxytyrosol myocardial cell cell membrane and nucleus are all had to stronger protective effect; under FDA and hoechst33342 fluorescent labeling, the hydroxytyrosol of 100 μ M is respectively 18.7%, 24.8% to the protective rate of myocardial cell.

Accompanying drawing explanation

Fig. 1 is that the fluorescence intensity of FDA labeled cell is with the change curve of cell density;

Fig. 2 is that the fluorescence intensity of Hoechst labeled cell is with the change curve of cell density;

In Fig. 1, Fig. 2, Fluorescence intensity represents fluorescence intensity, and Log Cell number represents the logarithm value of cell quantity;

Fig. 3 is that the survival rate of Hoechst labeled cell is with the change curve of DOX concentration;

Fig. 4 is that the survival rate of FDA labeled cell is with the change curve of DOX concentration;

In Fig. 3, Fig. 4, Cell Survival(%) represent cell survival rate (%), Log DOX(μ M) represent the logarithm value of doxorubicin concentration (μ M), Cell number represents cell quantity (/ hole);

Fig. 5 is monochromatic fluorescent labeling and the impact of Two Colour Fluorescence labelling on cell survival rate;

Wherein, Cell Survival(%) represent cell survival rate (%), Log DOX(μ M) represent the logarithm value of doxorubicin concentration (μ M), mono fluorescence Hoechst represents the monochromatic fluorescent labeling of Hoechst, mono fluorescence FDA represents the monochromatic fluorescent labeling of FDA, dualfluorescence Hoechst represents the Hoechst fluorescent labeling in Two Colour Fluorescence labelling, and dualfluorescence FDA represents the FDA fluorescent labeling in Two Colour Fluorescence labelling;

Fig. 6 be under Two Colour Fluorescence labelling rutin to the protection design sketch of myocardial cell (10 times of Microscopic observations);

Wherein, control represents normal cell, and DOX represents to add 0.3 μ M amycin and processes the cell after 24h, and DOX+rutin represents to add 100 μ M rutin and protects in advance and add 0.3 μ M amycin after 30min and process the cell after 24h; Merge represents the fluorescence microscopy picture of FDA and Hoechst to merge the Two Colour Fluorescence displaing micro picture obtaining, and by Two Colour Fluorescence displaing micro picture, is merged and can clearly be seen myocardial cell configuration and nucleus position;

Fig. 7 is the dose-effect figure of rutin to myocardial cell protection effect;

Wherein, Rutin represents rutin, Protective Percent(%) represent protective rate (%), Log Concentration(μ M) logarithm value of indicated concentration (μ M);

Fig. 8 a is that preparation component Myocardial prolection composition the selection result and the LC-MS of 40% ethanol extraction analyzes total ion current figure under negative ion mode;

Fig. 8 b is that preparation component Myocardial prolection composition the selection result and the LC-MS of 95% ethanol extraction analyzes total ion current figure under negative ion mode;

In Fig. 8 a, 8b, Protective Percent(%) represent protective rate (%), relative abundance represents relative abundance, Time(min) express peak time (minute);

Fig. 9 is the dose-effect figure of hydroxytyrosol to myocardial cell protection effect;

Wherein, Hydroxytyrosol represents hydroxytyrosol, Protective Percent(%) represent protective rate (%), Log Concentration(μ M) logarithm value of indicated concentration (μ M).

The specific embodiment

(1) FDA and Hoechst labeled cell density are linear investigates

The take the logarithm H9c2 cell of trophophase, with after the Digestive system digestion containing 0.25% pancreatin and 0.02%EDTA, with different cell density (0,10,100,500,1000,2000,4000,6000,8000,10000/hole) plant in middle 60 holes of 96 orifice plates, periphery replaces avoiding edge effect with PBS, be placed in cell culture incubator and cultivate respectively 24h, 48h, 72h; Discard the culture fluid in every hole, lucifuge, by concentration, be that the FDA of 10 μ g/mL and the Hoechst of 10 μ g/mL carry out monochromatic fluorescent labeling to the cell of different incubation times respectively, in cell culture incubator, hatch 10min, discard culture fluid, PBS washes one time, blots, and on fluorescence inverted microscope platform, reads fluoroscopic image.

Fluorescence acquisition parameters is: 2.5 times of object lens magnifications, time of exposure 1000ms, green fluorescence optical filter (exciting light 440-520nm, utilizing emitted light 505nm), blue-fluorescence optical filter (exciting light 320-400nm, utilizing emitted light 400nm).6 width images are taken in every hole, and through the splicing of FluoinsightCell work station fluoroscopic image, image recognition and data output system, result are added up, and statistical result as shown in Figure 1, 2.

From Fig. 1,2, when cell density is 0 to 10000/hole, the fluorescence intensity of FDA and Hoechst labeled cell increases with the increase of cell density, and fluorescence intensity increases with the increase of cell culture time, and is good linear relationship (R2>0.9).The fluorescence intensity that these two kinds of fluorescent dyes are described within the scope of the required cell number of conventional cell experiment, all can reflect preferably 96 orifice plates in the actual number of cell, can be used for accurate evaluation Injury by Adriamycin degree and medicament protection effect.

(2) damage of the doxorubicin concentration of FDA and the different cell densities of Hoechst labelling is linear investigates

The take the logarithm H9c2 cell of trophophase, with after the Digestive system digestion containing 0.25% pancreatin and 0.02%EDTA, plants in the middle of 96 orifice plates in 60 holes, periphery replaces avoiding edge effect with PBS, and the cell density of each plate is respectively 100,500,1000,2000,4000,6000,8000,10000/hole, be placed in cell culture incubator and cultivate 24h, discard culture fluid; The amycin (Concentraton gradient is 0.001,0.01,0.1,0.5,1,2,6,10,100 μ M) that every plate adds variable concentrations damages, and the damage time is 24h; Every plate arranges normal cell group and does not add amycin group; After having damaged, discard the culture fluid in every hole, lucifuge, adding respectively 100 μ L concentration is that the FDA of 10 μ g/mL and the Hoechst of 10 μ g/mL carry out monochromatic fluorescent labeling to the cell of different incubation times, in cell culture incubator, hatch 10min, discard culture fluid, PBS washes one time, blots, and on fluorescence inverted microscope platform, reads fluoroscopic image.

Fluorescence acquisition parameters and Treatment Analysis method are with (1), and statistical result as shown in Figure 3,4.

As seen from Figure 3, when DOX concentration is 0 to 2 μ M, the survival rate of Hoechst labeled cell increases and reduces with DOX concentration, and during 2 μ M, cell survival rate is close to 1%.

As seen from Figure 4, when DOX is 0.001 to 0.5 μ M, the survival rate of FDA labeled cell increases and to reduce with DOX concentration, when DOX is 0.5 to 10 μ M, has occurred survival rate plateau, and when DOX is higher concentration 100 μ M, cell survival rate just reduces.

Infer thus, Hoechst can present amycin comparatively sensitively to certain organelle degree of injury, thus the nucleus damage situations of reflecting myocardium cell.

(3) the dose-effect relationship comparison of toxicity of adriamycin under monochromatic fluorescence and Two Colour Fluorescence labelling H9c2 cell

The take the logarithm H9c2 cell of trophophase, with after the Digestive system digestion containing 0.25% pancreatin and 0.02%EDTA, with the density kind in 4000/hole, in the middle of 96 orifice plates in 60 holes, periphery replaces avoiding edge effect with PBS, is placed in cell culture incubator and cultivates respectively 24h; Discard culture fluid, add doxorubicin hydrochloride (DOX) (0.01,0.1,0.3,0.5,1,2 μ M) the damage 24h of variable concentrations; Normal cell group is set and does not add amycin group; One plate FDA labelling, plate Hoechst labelling.Dyeing condition and cell fluorescent images obtain with processing method with (1).

One plate is used FDA and Hoechst labelling simultaneously, in culture fluid, add FDA and Hoechst simultaneously, make both final concentrations be 10 μ g/mL, every hole adds 100 μ L, and in cell culture incubator, incubated cell 10min, discards culture fluid, PBS rinsing one time, first use green fluorescence optical filter (exciting light 440-520nm, utilizing emitted light 505nm), take one time, then use blue-fluorescence optical filter (exciting light 320-400nm, utilizing emitted light 400nm) to clap one time.Fluoroscopic image obtains and processing method same (1).

Under more monochromatic fluorescent labeling and Two Colour Fluorescence labelling, the dose-effect relationship of H9c2 cell survival rate and doxorubicin concentration, comparative result is shown in Fig. 5.

As seen from Figure 5, when doxorubicin concentration is 0.01-2 μ M, the cell survival rate of monochromatic fluorescent labeling and Two Colour Fluorescence labelling gained does not have obvious difference, and while showing Two Colour Fluorescence labelling, two fluorescent probes can not affect each other.Because Hoechst labelling is more responsive, so the concentration (0.3 μ M) of amycin is damage concentration while selecting Hoechst labeled cell survival rate to be about 50%.

(4) for evaluating the adriamycin myocardial toxicity of positive compound rutin

The take the logarithm H9c2 cell of trophophase, with after the Digestive system digestion containing 0.25% pancreatin and 0.02%EDTA, is inoculated in 60 holes in the middle of 96 orifice plates with the density in 4000/hole, and periphery replaces avoiding edge effect with PBS, is placed in 37 ℃ of cell culture incubators and hatches 24h; Adding respectively concentration is the known myocardial preservation compound rutin of 10,20,40,60,80,100 μ mol/L, pre-protection 30min, do not abandon culture fluid, add amycin, making its final concentration is 0.3 μ M, normal cell group (only add culture fluid and do not do other processing) and model group (only add amycin, do not add rutin) are set, in cell culture incubator, hatch 24h; Discard the culture fluid in every hole, and wash one time with PBS, lucifuge adds 100 μ L containing the culture fluid of 10 μ g/mL FDA and 10 μ g/mL Hoechst, hatches 10min in cell culture incubator; Discard culture fluid, PBS washes one time, blots, and on fluorescence inverted microscope platform, reads fluoroscopic image; Two Colour Fluorescence image obtain with processing method with (3) (Fig. 6).By formula, calculate protective rate and draw protective rate to the amount effect curve of concentration (Fig. 7) and calculate the EC50 value of rutin.The computing formula of protective rate is:

Wherein f is the fluorescence intensity of dosing protection group, and Fm is the fluorescence intensity of the i.e. damage group of model group, and Fc is the fluorescence intensity of normal cell group.

As seen from Figure 6, at high power Microscopic observation, FDA labeled cell, when cell is during in normal condition (control, normal cell group), form is fusiformis, and cellular morphology is complete, and green fluorescence is brighter; After Injury by Adriamycin (model group), it is irregular that cellular morphology becomes, and some cells are free to be bubbled out now, and green fluorescence intensity reduces simultaneously; After the pre-protection of rutin (dosing protection group), cellular morphology is more many than model group rule, and what have is even fusiformis, and cavity reduces, and green fluorescence intensity increases than model group.Hoechst labeled cell, can significantly see that nucleus that model group is labeled is than the minimizing of normal cell group, and the nucleus that dosing protection group is labeled increases than model group.Confirmed that method of the present invention can be preferably for evaluating the myocardium protecting action of medicine.

Simultaneously; as shown in Figure 7; rutin has good protective effect to myocardial cell; according to FDA and Hoechst fluorescence intensity, calculate; in 10-100 μ M model; rutin is concentration dependent to the protective effect of myocardial cell, and when concentration is 100 μ M, protective rate is about respectively 53.6%(FDA dyeing) and 38.9%(Hoechst dyeing).

(5) the inventive method is for screening the myocardial preservation composition of body resistance-strengthening Zhenqi

Utilize the inventive method to there being the active component in the ZHENQI FUZHENG KELI (be purchased from revise Pharmaceutical) of myocardium protecting action to screen, specific as follows:

1) ZHENQI FUZHENG KELI is dissolved to centrifuging and taking supernatant with pure water;

2) with macroporous resin by described supernatant successively water, 40% ethanol, 95% ethanol elution, collect respectively each elution fraction, be evaporated to dry;

3) elution fraction after concentrated is carried out to separation by preparative liquid chromatography, collects each fraction, after lyophilization as component to be screened, for screening experiment;

4) inoculation H9c2 cell is on 96 orifice plates, cell density is 4000/hole, cultivate after 24h, add the culture fluid (having the concentration of component of cytotoxicity to reduce to 25 μ g/mL or 10 μ g/mL during 50 μ g/mL) that contains 50 μ g/mL component to be screened, after pre-protection 30min, the DOX damage 24h that adds 0.3 μ M, and Normal group (not adding DOX) and model control group (DOX of 0.3 μ M) be set, method according to the myocardial preservation material of setting up in (4) part, use FDA and Hoechst to carry out fluorescent labeling, fluorescent labeling completes by FluoinsightCell work station and takes and process, calculate the protective rate of each Components On Myocytes to be screened,

Preparation component Myocardial prolection composition the selection result and LC-MS that Fig. 8 a, 8b are respectively 40%, 95% ethanol extraction analyze total ion current figure under negative ion mode.Screening obtains B5, B6, B19, B20, five active components of B22.

5) by the analysis of liquid matter, obtain containing hydroxytyrosol in B component 6;

6) inoculation H9c2 cell is on 96 orifice plates, cell density is 4000/hole, cultivate after 24h, add and contain variable concentrations (10, 20, 40, 60, 80, 100 μ M) culture fluid of hydroxytyrosol, after pre-protection 30min, the DOX damage 24h that adds 0.3 μ M, and Normal group (not adding DOX) and model control group (DOX of 0.3 μ M) be set, according to the 4th) screening technique of the myocardial preservation material set up in part, use FDA and Hoechst to carry out fluorescent labeling, fluorescent labeling completes by FluoinsightCell work station and takes and process, obtain the dose-effect figure (Fig. 9) of hydroxytyrosol to myocardial cell protection effect.

As seen from Figure 9, hydroxytyrosol is all proportionate with concentration to the prolection of myocardial cell.During FDA labelling, when the protective rate of 100 μ M hydroxytyrosols is 18.7%, Hoechst labelling, the protective rate of 100 μ M hydroxytyrosols is 24.8%.While showing Hoechst labelling, have higher protective rate during than FDA labelling, prompting hydroxytyrosol may have better anti-apoptotic effect.

Claims (8)

1. the application of hydroxytyrosol in adriamycin myocardial toxicity.

2. take the pharmaceutical composition that hydroxytyrosol is main active for one kind.

3. pharmaceutical composition as claimed in claim 2, is characterized in that, in described pharmaceutical composition, the percentage by weight of hydroxytyrosol is 0.01～99.9%.

4. pharmaceutical composition as claimed in claim 3, is characterized in that, in described pharmaceutical composition, the percentage by weight of hydroxytyrosol is 30～99%.

5. pharmaceutical composition as claimed in claim 4, is characterized in that, in described pharmaceutical composition, the percentage by weight of hydroxytyrosol is 50～99%.

6. pharmaceutical composition as claimed in claim 2, it is characterized in that, the dosage form of described pharmaceutical composition is injection, injectable sterile powder, tablet, capsule, spirit, powder, granule, syrup, solution, tincture, aerosol, powder spray or suppository.

7. pharmaceutical composition as claimed in claim 6, is characterized in that, the dosage form of described pharmaceutical composition is injection, injectable sterile powder, tablet, capsule or syrup.

8. pharmaceutical composition as claimed in claim 7, is characterized in that, the dosage form of described pharmaceutical composition is injection, tablet or capsule.