Technical field

The present invention relates to the medicinal application field, relate in particular to the antibacterial applications of arteannuin and derivant thereof.

Background technology

Infectious disease is owing in the pathogenic microorganism intrusive body, breed caused disease in vivo in large quantities, is one of major reason of clinical patient death.Cause the reason first antibacterial of death to breed the damage that the release toxin causes histoorgan in a large number; It two is that the antibacterial composition passes through immune cell activated, induce proinflammatory cytokines such as TNF-α, IL-1, IL-6, NO to discharge, cause the formed water fall effect of multiple inflammatory mediator, inflammatory reaction is enlarged even out of hand, finally causing with cell self property destruction is that the systemic inflammatory of feature is pyemic generation.Bacterial infection once was human first cause of the death, and antibiotic invention has brought human light of hope.But antibioticly generally make increasing antibacterial produce drug resistance,, wait for that human will be dark next time if do not solve the problem of antibiotic abuse.

Herba Artemisiae Annuae extract is used for treating the relevant heating of malaria the history in more than 1,000 year, and except that traditional malaria effect, Herba Artemisiae Annuae class material also has the effect of others, as relieving asthma, anticancer, schistosomicide and to immune adjusting etc.The research of relevant arteannuin and derivant thereof both at home and abroad is a lot, but research range mainly concentrates on malaria, anticancer, antischistosomal effect and the mechanism thereof.

Artesunate (Artesunate), chemistry dihydroartemisinine-1 by name, 2-α-monomester succinate, molecular formula is C19O8 H28, molecular weight 384 is the derivants with antimalarial arteannuin of sesquiterpene structure.Artesunate is novel antimalarial, and than arteannuin, artesunate can be mixed with the dosage form of any routine, comprises solid and liquid form, and administration is very convenient.Artesunate is as antimalarial, the height of not only tiring, and be difficult for producing toleration.

To the existing report of the antiinflammatory action of arteannuin and derivant thereof, discover that arteannuin can suppress the activation of the synthetic and NF-κ B of LPS/TNF-α inductivity NO synthase; Artesunate to LPS and merge that interferon stimulates Turnover of Mouse Peritoneal Macrophages NO synthetic the obvious suppression effect arranged; Artesunate also has similar protective effect to the mouse macrophage RAW264.7 that LPS stimulates; Tan Yuqing etc. find that also Herba Artemisiae Annuae extract, arteannuin can reduce endotoxin shock mice LPO, ACP, endotoxin, TNF-α, P450 concentration, the increased SOD activity, reduce mouse death rate, prolong the mean survival time of mice, Mouse Liver, lung tissue form are also had the certain protection effect; Discovery arteannuin such as Wang Jun can suppress CpGODN, LPS, heat-inactivated EC inducing cell discharges inflammatory factor, and the sepsis model mice is had remarkable protective effect.But whether artesunate has antibacterial action, whether can improve the drug resistance of bacterial antibiotic, strengthen antibiotic antibacterial efficacy and infection that antibacterial is caused whether effective, announcement does not appear in the newspapers.China is the main grown place of arteannuin and its derivant, because this curative effect of medication height, toxicity are low, WHO has classified arteannuin as the main medicine of world's treatment malaria, China is enlarging Herba Artemisiae Annuae plantation and arteannuin production, therefore widen the indication of artemisinin derivatives, be applied to the control of bacterial infective diseases, undoubtedly to the utilization that improves artemisinin derivatives, increase its economic worth and improve the drug resistance phenomenon that the antibiotic abuse caused significant.

Summary of the invention

The objective of the invention is to arteannuin and derivant thereof, particularly artesunate is applied to antibiotic field, not only widen the purposes of arteannuin and derivant thereof, particularly artesunate, and can alleviate the abuse of existing antibacterials and the drug resistance phenomenon that causes thereof.

Based on above-mentioned purpose, the technical scheme that the present invention uses is with a kind of use in conjunction of carrying out in a kind of and antibacterials in arteannuin and the derivant thereof, this application is not the application as the treatment of diseases method, but the application in the medicine that is equipped with treatment disease (antibiotic), wherein above-mentioned two kinds of components in proportions are 1: 1～4096: 1, last two kinds of compositions can be prepared into compositions, also can be two kinds of independent medicaments.Above-mentioned two kinds of compositions can produce Synergistic antimicrobial and render a service, and can reduce the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of antibacterials.

Above-mentioned bacterium comprises gram positive bacteria and gram negative bacteria; Said derivative comprises dihydroartemisinine, Artemether, arteether and artesunate; Above-mentioned antibacterials comprise the medicine of beta-lactam antibiotic (penicillins, cephalosporins, atypia beta-lactam antibiotic), aminoglycoside, clindamycin class, quinolones, in a preferred embodiment, above-mentioned antibacterials are benzylpenicillin, ampicillin, cefuroxime, cefpiramide, gentamycin, clindamycin, lomefloxacin, Gatifloxacin, ampicillin sodium-sulbactam sodium and safe energy, are preferably gentamycin, ampicillin sodium-sulbactam sodium, cefpiramide and Gatifloxacin.

Above-mentioned application is preferably the use in conjunction of artesunate and antibacterials.

The independent use of artesunate can suppress the growth of gram-negative bacteria and gram positive bacteria, with the antibacterial efficacy that can obviously strengthen antibacterials after the antibacterials use in conjunction, reduces the using dosage of antibacterials; Experiment shows that arteannuin and artesunate and antibacterials use in conjunction can obviously reduce antibacterial (comprising Grain-negative, positive bacteria) and attack mouse death rate, significantly suppresses mice serum LPS content and cytokine TNF-α and discharges.

This finder studies the antibiotic and antiinflammatory action of artesunate inside and outside.

At first, this research worker has been carried out pharmacology's analysis to the vitro antibacterial activity of this artesunate, and with the antibiotic use in conjunction after find that the independent use of artesunate can suppress the growth of gram-negative bacteria and gram positive bacteria, with can obviously strengthen antibiotic antibacterial efficacy after the antibiotic use in conjunction, reduce antibiotic using dosage; Secondly, artesunate and antibiotic use in conjunction and can obviously reduce antibacterial (comprising Grain-negative, positive bacteria) and attack mouse death rate significantly suppress mice serum LPS content and cytokine TNF-α release.

Therefore the present invention has widened the purposes of arteannuin and derivant thereof, particularly artesunate, has improved the antibacterial efficacy of antibacterials, the drug resistance phenomenon that has alleviated the antibacterials abuse and caused.

Description of drawings

Fig. 1 represents that artesunate works in coordination with gentamycin, cefpiramide, ampicillin sodium-sulbactam sodium to the antibacterial curve of the associating of escherichia coli ATCC35218;

Fig. 2 represents that artesunate works in coordination with ampicillin, Gatifloxacin, the ampicillin sodium-sulbactam sodium antibacterial curve of associating to the golden bacterium ATCC25923 of Portugal;

Fig. 3 represents that artesunate works in coordination with gentamycin, Gatifloxacin, ampicillin sodium-sulbactam sodium to the antibacterial curve of the associating of escherichia coli clinical separation strain.

The specific embodiment

The present invention is further illustrated below in conjunction with embodiment, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.

Artesunate (AS) and antibacterials can produce Synergistic antimicrobial and render a service according to the proportioning in table 1, the table 2, reduce the minimum inhibitory concentration and the minimum bactericidal concentration of antibacterials.

The MIC (μ g/ml) of antibacterials when table 1 variable concentrations artesunate (AS) uses with antibacterials are collaborative
									Antibacterials	Escherichia coli ATCC35218				The escherichia coli clinical separation strain
AS0	AS32	AS128	AS256	AS0	AS32	AS128	AS256
								Gentamycin		4	0.25	0.125	0.0625	＞256	＞256	＞256	＞256
Streptomycin	＞256	＞256	＞256	＞256	＞256	＞256	＞256		＞256
								Ampicillin sodium-sulbactam sodium		64	64	32	8	128	128	128	128
Cefpiramide	64	32	8	4	＞256	＞256	＞256		＞256
								Gatifloxacin		0.5	0.5	0.5	0.5	64	64	64	32

Table 2 artesunate (AS) can produce the minimum mass ratio of antibacterial action when using with different antibacterials are collaborative
							Antibacterials	Escherichia coli ATCC35218			The escherichia coli clinical separation strain
AS32	AS128	AS256	AS32	AS128	AS256

Gentamycin	128∶1	1024∶1	4069∶1	＞1∶8	＞1∶2	＞1∶1
							Streptomycin	＞1∶8	＞1∶2	＞1∶1	＞1∶8	＞1∶2	＞1∶1
Ampicillin sodium-sulbactam sodium	1∶2	4∶1	32∶1	1∶4	1∶1	2∶1
							Cefpiramide	1∶1	16∶1	64∶1	＞1∶8	＞1∶2	＞1∶1
Gatifloxacin	64∶1	256∶1	512∶1	1∶2	1∶1	2∶1

Experimental example 1

This experimental example is to study artesunate and ten kinds of antibacterials MIC (minimum inhibitory concentration) and the MBC (minimum bactericidal concentration) to different bacterium

Adopt the micropore dilution method, adjusting bacterial concentration is 10 ⁵CFU/ml is inoculated in the 96 hole aseptic culture plates, and 10 kinds of antibacterials such as artesunate and penicillin sodium, ampicillin etc. are 5.14mg/ml with the normal saline dilution respectively.Add various medicines to containing in the antibacterial culturing hole, doubling dilution successively, the ultimate density of the 1st～10 hole medicine is followed successively by 256,128,64,32,16,8,4,2,1,0.5ug/ml.Put 37 ℃ of incubators and hatch 24h and 48h, read the positive and negative control hole, negative control hole is limpid, positive control hole muddiness.Medicine is the lowest concentration of drug that suppresses antibacterial naked eyes visible growth behind the 24h to the MIC of antibacterial, and medicine is the lowest concentration of drug that suppresses antibacterial naked eyes visible growth behind the 48h to the MBC of antibacterial.This time experimental result shows the independent use of artesunate bacteria growing inhibiting fully, and has observed the inhibitory effect of different antibacterials to these five kinds of antibacterials.(seeing Table 3).

During independent use of table 310 kind of antibacterials and AS to the MIC and the MBC of different bacterium

		Penicillin G sodium	The ampicillin	Cefuroxime	Cefpiramide	Gentamycin	Clindamycin	Lomefloxacin	Gatifloxacin	Ampicillin sodium-sulbactam sodium	Safe energy	Artesunate
													35218	MIC	＞256	＞256	2	64	4	256	＜0.5	＜0.5	64	＜0.5	＞256
MBC	＞256	＞256	2	64	4	256	＜0.5	＜0.5	64	＜0.5	＞256
												Staphylococcus aureus		MIC	64	128	＞256	＞256	＞256	＞256	256	128	＞256	64	＞256
MBC	＞256	＞256	＞256	＞256	＞256	＞256	＞256	256	＞256	64	＞256
													Escherichia coli	MIC	＞256	＞256	＞256	＞256	64	＞256	＞256	＞256	64	＜0.5	＞256
MBC	＞256	＞256	＞256	＞256	128	＞256	＞256	＞256	128	1	＞256
												Klebsiella Pneumoniae		MIC	64	64	2	2	2	＞256	2	1	8	＜0.5	＞256
MBC	64	128	2	2	2	＞256	2	1	16	2	＞256
													Enterococcus	MIC	＞256	＞256	＞256	＞256	4	256	64	2	32	1	＞256
MBC	＞256	＞256	＞256	＞256	128	＞256	256	32	32	1	＞256
												Pseudomonas aeruginosa		MIC	＞256	＞256	＞256	256	＞256	＞256	32	8	＞256	32	＞256
MBC	＞256	＞256	＞256	256	＞256	＞256	32	16	＞256	32	＞256

Experimental example 2

This experimental example is to study after artesunate and the antibacterials use in conjunction influence to escherichia coli MIC and MBC.

Adopt checkerboard type micropore dilution method, adjusting bacterial concentration is 10 ⁵CFU/ml is inoculated in the 96 hole aseptic culture plates, will be to the antibacterials of five kinds of medium sensitivities of antibacterial dosage and the variable concentrations artesunate difference compatibility that is lower than MIC, and the medicine behind the observation compatibility is to the MIC and the MBC of escherichia coli.Though this time experimental result shows that artesunate uses bacteria growing inhibiting fully separately,, illustrate that there is collaborative inhibitory effect the collaborative back of artesunate and antibacterials to antibacterial with the MIC and the MBC that can obviously reduce antibacterials after the antibacterials use in conjunction.(seeing Table 4).

Minimum inhibitory concentration after each antibacterials of table 4 and artesunate are collaborative

	Escherichia coli ATCC35218	The escherichia coli clinical separation strain
			Gentamycin+AS	1/16MIC+32μg/ml AS	No effect
Streptomycin+AS	No effect	No effect
			Ampicillin sodium-sulbactam sodium+AS	1/8MIC+256μg/ml AS	No effect
Cefpiramide+AS	1/8MIC+256μg/ml AS	No effect
			Gatifloxacin+AS	No effect	1/2MIC+256μg/mlAS

Experimental example 3

This experimental example is to study artesunate and the inhibition strength of the collaborative back of the different antibacterials of 1/2MIC concentration to the escherichia coli growth.

Adjusting bacterial concentration is 10 ⁶CFU/ml, measuring bacterium liquid OD600 is 0.002 (1OD=5 * 10 ⁸CFU/ml), with reference to experimental example 1 result, adding final concentration respectively separately is the antibacterials of 256 μ g/ml artesunate or 1/2MIC concentration, and after adding the antibacterials of 256 μ g/ml artesunate and 1/2MIC concentration simultaneously, as for 37 ℃ of constant temperature shaking table 150rpm jolting, measure 1,3,5,7,9,12,16 respectively, the OD value of bacterium liquid during 24h, calculate the amount of bacteria of each time point.Result of study shows that artesunate 256 μ g/ml can make escherichia coli ATCC35218, the staphylococcus aureus ATCC25923 and the escherichia coli clinical separation strain speed of growth obviously slow down, compare with independent use antibacterials, united behind the artesunate obviously bacteria growing inhibiting, the inhibition degree is than using antibacterials or artesunate all strong (Fig. 1,2,3) separately.

Experimental example 4

This experimental example is to study artesunate use in conjunction antibacterials are attacked mice to gram-negative bacteria protective effect

Cleaning level 70 of the kind white mice in Kunming (Medical University Of Chongqing's Experimental Animal Center provides), body weight 19.9 ± 0.5g/ only, male and female half and half, be divided into matched group, artesunate group, escherichia coli group at random, escherichia coli+gentamycin group, artesunate (1.5,5,15mg/kg)+gentamycin+escherichia coli group.Every group of 10 animals.Contrast does not give any reagent; The artesunate of arteannuin group intramuscular injection 15mg/kg; The escherichia coli group gives 0.8 * 10 ⁶The escherichia coli ATCC35218 that/kg lives; Escherichia coli+gentamycin group gives the escherichia coli ATCC35218 that lives after the gentamycin 0.5mg/kg intramuscular injection; Gentamycin+artesunate+escherichia coli group after giving artesunate, gives gentamycin and escherichia coli immediately.Give normal diet and drinking-water after administration finishes, observe mice ordinary circumstance and mortality rate (table 5) in 7 days.The result can obviously reduce mortality of mice after showing artesunate Synergistic antimicrobial medicine, shows the coordinating protection effect occurring after artesunate and the antibacterials associating.

The collaborative gentamycin of table 5 artesunate is attacked mice to escherichia coli ATCC35218 protective effect

Processed group	Sum	Death toll	Mortality rate (%)	P
					Contrast	10	0	0	-
E.coli	10	10	100.0	-
					AS(15mg/kg)	10	10	100.0	-
Gentamycin (0.5mg/kg)	10	9	90.0	-
					AS (1.5mg/kg)+celebrating is big+E.coli	10	6	60.0	0.024 *
AS (5mg/kg)+celebrating is big+E.coli	10	0	0	0.001 **
					AS (15mg/kg)+celebrating is big+E.coli	10	0	0	0.001 **

Experimental example 5

This experimental example is to study artesunate use in conjunction antibacterials are attacked mice to gram-positive bacteria protective effect

Cleaning level 70 of the kind white mice in Kunming (Medical University Of Chongqing's Experimental Animal Center provides), body weight 19.9 ± 0.5g/ only, male and female half and half, be divided into matched group, artesunate group, golden Portugal bacterium group at random, gold Portugal bacterium+Gatifloxacin group, artesunate (5,15,45mg/kg)+Gatifloxacin+golden Portugal bacterium group.Every group of 10 animals.Contrast does not give any reagent; The artesunate of arteannuin group intramuscular injection 45mg/kg; Gold Portugal bacterium group gives 1.0 * 10 ⁷The golden bacterium ATCC25923 of Portugal that/kg lives; Gold Portugal bacterium+Gatifloxacin group gives the golden bacterium ATCC25923 of Portugal that lives after the Gatifloxacin 0.5mg/kg intramuscular injection; Gatifloxacin+artesunate+golden Portugal bacterium group after giving artesunate, gives Gatifloxacin and golden Portugal bacterium immediately.Give normal diet and drinking-water after administration finishes, observe mice ordinary circumstance and mortality rate (table 6) in 7 days.The result can obviously reduce mortality of mice after showing artesunate Synergistic antimicrobial medicine, shows the coordinating protection effect occurring after artesunate and the antibacterials associating.

The collaborative Gatifloxacin of table 6 artesunate is attacked mice to the golden bacterium ATCC25923 of Portugal protective effect

Processed group	Sum	Death toll	Mortality rate (%)	P
					Contrast	10	0	0	-
SA	10	10	100.0	-
					AS(45mg/kg)	10	10	100.0	-
Gatifloxacin (0.5mg/kg)	10	9	90.0	-
					AS (5mg/kg)+Gatifloxacin+SA	10	7	70.0	0.060
AS (15mg/kg)+Gatifloxacin+SA	10	5	50	0.009 **
					AS (45mg/kg)+Gatifloxacin+SA	10	2	20	0.000 **

Experimental example 6

This experimental example is to study artesunate is attacked mice to the deactivation escherichia coli protective effect.

Cleaning level 60 of the kind white mice in Kunming (Medical University Of Chongqing's Experimental Animal Center provides), body weight 19.9 ± 0.5g/ only, male and female half and half are divided into matched group, artesunate group, deactivation escherichia coli group at random, artesunate (5,15,45mg/ml)+deactivation escherichia coli group.Every group of 8 animals.Contrast does not give any reagent; The intramuscular injection of artesunate group gives the artesunate of 45mg/kg, 4h, and 24h, repeat administration is once behind the 48h; Deactivation escherichia coli group gives 1.25 * 10 ¹¹The deactivation escherichia coli ATCC35218 of/kg; Artesunate+deactivation escherichia coli group after intramuscular injection gives artesunate, gives deactivation escherichia coli ATCC35218 immediately, repeats administered intramuscular artesunate 1 time behind 4h, 24h, the 48h.Give normal diet and drinking-water after administration finishes, observe mice ordinary circumstance and mortality rate (table 7) in 7 days.The result shows that artesunate can reduce mortality of mice, shows that the mice that pro-inflammatory cytokine is attacked has protective effect.

Table 7 artesunate is attacked the protective effect of mice to the deactivation escherichia coli

Processed group

Sum

Death toll

Mortality rate (%)

P

Contrast	8	0	0	-
					AS(45mg/kg)	8	0	0	-
E.coli	8	8	100.0	-
					AS(5mg/kg)+E.coli	8	8	100.0	-
AS(15mg/kg)+E.coli	8	6	25.0	0.048 *
					AS(45mg/kg)+E.coli	8	4	50.0	0.011 *

^*p＜0.5； ^**p＜0.01vsE.coli

Experimental example 7

This experimental example is to study artesunate the deactivation escherichia coli is attacked the influence of mice serum release of cytokines

Cleaning level 40 of the kind white mice in Kunming (Medical University Of Chongqing's Experimental Animal Center provides), body weight 19.9 ± 0.5g/ only, male and female half and half, be divided into matched group, artesunate group, deactivation escherichia coli group, artesunate (5,15,45mg/kg)+deactivation escherichia coli group at random, 3 of every treated animals.Contrast does not give any reagent.The intramuscular injection of artesunate group gives the artesunate of 45mg/kg; Artesunate (5,15,45mg/kg)+deactivation escherichia coli group gives to give deactivation escherichia coli ATCC35218 immediately behind the artesunate; Administration finishes and plucks eyeball behind the 4h and get the centrifugal immediately indwelling supernatant of blood and be stored in-20 ℃, cytokine TNF-α to be determined (table 8).The result shows that artesunate can significantly reduce the deactivation escherichia coli and attack the mice serum release of cytokines.

Table 8 artesunate reduction deactivation escherichia coli ATCC35218 attack mice serum cytokine TNF-α release (n=6, )

Processed group	TNF-α(pg/ml)	P
			Contrast	58.10±15.31
AS(45mg/kg)	44.57±27.79
			E.coli	1399.06±185.63
AS(5mg/kg)+E.coli	864.71±321.11	0.092
			AS(15mg/kg)+E.coli	914.18±141.22	0.023 *
AS(45mg/kg)+E.coli	680.13±103.30	0.004 **

^*p＜0.5； ^**p＜0.01vsE.coli

The present invention has improved antibiotic antibacterial efficacy with the use in conjunction of artesunate and antibacterials, and artesunate and antibiotic use in conjunction can also be prevented, treat bacterial infection.

Those skilled in the art can be according to content of the present invention, and the associating of arteannuin and derivant and antibacterials can be reached effect of the present invention equally.