Patent Application: US-47253706-A

Abstract:
the present application provides a natural aqueous extract obtainable from a cinnamon bark which has antiviral activity against enveloped viruses including influenza a , parainfluenza virus and hsv - 1 viruses , as well as in vivo activity in inhibition of influenza a and parainfluenza viruses . the present application also concerns a method for the extraction of said cinnamon extract and applications thereof .

Description:
the active material was isolated by three steps as follows : a ) the bark was purchased in the market and was ground into powder before it was stirred in aqueous phosphate buffer 0 . 01m - 0 . 02m , ph 7 . 0 , overnight . the supernatant was separated by centrifugation and was used as the crude neutralizing extract ; b ) the active material in the crude extract was precipitated by kcl 0 . 15m or 0 . 08m mgcl2 , and the precipitate was dissolved in water or 0 . 01m phosphate buffer , ph 7 . 0 ( ce ppt . ); c ) this solution was submitted onto column of sepharose 4b followed by a stepwise elution with phosphate buffer and various concentrations of galactose . the active antiviral material was eluted from the column by 0 . 15m galactose ( fig1 a , b , c , fraction b or ii ). hemagglutinating unit ( hau ) was determined by using 0 . 4 % washed human red blood cells . viral hemolytic activity was tested in vitro in two successive steps : 1 ) attachment of the free virus onto 1 ml of 4 % washed human erythrocytes for 15 minutes at room temperature ; 2 ) incubation of the infected cells in 37 ° c . for 3 hours followed by centrifugation . the hemolytic activity of the viruses was determined by measuring the absorbance of the supernatant at 540 nm . 60 ml of crude extract were precipitated by mgcl 2 0 . 08m or kcl 0 . 15m . the precipitate was dissolved in water or in 0 . 01m phosphate buffer and was submitted on 10 ml column of sepharose 4b pre - washed with phosphate buffer 0 . 01m , ph 7 . 0 . after submission , the column was washed with the buffer followed by stepwise elution of galactose 0 . 15m , 0 . 3m , and various concentrations of acetonitrile , as shown in fig1 a , b , c . the antiviral material was found in fraction b eluted from the column by 0 . 15m galactose ( fig1 ( c )) or fraction ii in fig1 a , b . in vitro inhibition of hemolytic activity by influenza a by crude extract of the invention various amounts of crude extract were incubated with 256 hau samples of influenza a pr8 virus to test the inhibitory effect on the hemolytic activity of the virus , as described in the experimental procedure . virus alone or the crude extract alone was used as controls . the results are shown in fig1 ( a ). the hemolytic activity of the virus was totally inhibited by 250 μg of the crude extract . in vitro inhibition of hemolytic activity of sendai virus by the extract of the invention various amounts of crude extract were incubated with 256 hau samples of sendai virus to test the inhibitory effect on the hemolytic activity of the virus , as described in the experimental procedure . virus alone or the crude extract alone was used as controls . the results are shown in fig1 ( b ). the hemolytic activity of the virus was totally inhibited by 250 μg of the crude extract . the cinnamon extracts ( ce ) or autoclaved ce was kept at room temperature or in the refrigerator for 4 years before testing their ability to inhibit the hemolytic activity of sendai virus ( s . v .). 200 μg of ce were mixed with 256 hau of the virus and hemolysis was tested as described in the experimental procedures . the results are shown in fig2 . as can be seen , the antiviral activity of ce was maintained after all treatments although it lost some activity after autoclaving at 134 ° c . inhibition of influenza a pr8 by various fractions of the extract of the invention , treated with various reagents autoclaved ce fractions were incubated with 256 hau of influenza a pr8 virus at room temperature for 15 minutes . after application on human erythrocytes , the mixture was transferred to 37 ° c . for 3 hours . the results are shown in fig3 . 50 - 100 μg of each ce fractions was sufficient to inhibit the viral hemolytic activity . ce ppt ( isolated fraction obtained by salting out with kcl 0 . 15m ) expressed the strongest antiviral activity . ce ppt was incubated with 0 . 01m or 0 . 1m hcl and h 2 so 4 at room temperature for 3 hours followed by neutralization to ph 7 before examining its ability to neutralize the virus , as described in fig3 . the results after this treatment are shown in fig4 ( a ). ce ppt was incubated with 0 . 01m or 0 . 1m naoh at room temperature for 3 hours , followed by neutralization to ph 7 , before examining its ability to inhibit the hemolytic activity of the virus , as described in fig3 . the results are shown in fig4 ( b ). the treated material remained partially active . ce ppt is the precipitated fraction obtained by salting out with kcl 0 . 15m . ce fractions were dialyzed against water before examining the antiviral activity as described in fig3 . the results are shown in fig5 . the active material in the ce fractions has a molecular weight greater than 10 kda ( the dialysis bag cut - off ). inhibition of influenza a pr8 by the extract of the invention after incubation for various time periods 50 - 200 μg samples of the ce ppt fraction were incubated with the virus for 1 - 30 minutes at room temperature , before adding the erythrocytes . hemolytic activity of the virus was determined as described in fig3 . the results are shown in fig6 ( a ). short incubation ( one minute ) was sufficient to neutralize the virus . 50 - 200 μg samples of the ce ppt fraction were incubated with the virus for 1 min . or 20 minutes at room temperature before adding the erythrocytes . hemolytic activity of the virus was determined as described in fig3 . the results are shown in fig6 ( b ). short incubation ( one minute ) was sufficient to neutralize the virus . 256 hau of influenza a pr8 virus were absorbed to human erythrocytes at room temperature before application of various ce fractions , and incubation at 37 ° c . as described in methods . the results are shown in fig7 . each of the ce fractions inhibited the hemolytic activity of the virus , although this required at least two - fold amount of each fraction compared to the direct interaction between the free virus and the ce fractions . two ce fractions were absorbed onto human erythrocytes , and the excess was washed twice with pbs before application of 256 hau of influenza a pr8 virus at room temperature and incubation at 37 ° c . as described in methods . the results are shown in fig8 . both the refrigerated crude extract and the isolated fraction ce ppt protected the erythrocytes from the hemolytic activity of the virus , but ce ppt was more effective . the amount needed for the protections was 4 - 10 times higher than the amount that inhibited the virus by direct interaction . in vivo effect of treatment of the extract of the invention on influenza a infected mice 3 . 5 week old mice were injected i . v . ( caudal vein ) with 250 μl of pbs containing 128 hau of influenza a virus alone or influenza a mixed with 250 μg of the crude extract or the crude extract alone . the mice were weighed at 2 - 3 day intervals . the results are shown in fig9 . while the mice infected with the virus alone lost weight and most of them died within 7 - 10 days , the mice injected with a mixture of the virus and the crude extract continued to gain weight almost on a level with those injected with the crude extract alone . each group included 10 mice . 3 . 5 week old mice were allowed to inhale 50 μl of water containing 64 hau of sendai virus alone , or virus mixed with 125 μg of the crude extract , or the crude extract alone . the mice were weighed at 2 - 3 day intervals . the results are shown in fig1 . while the mice infected with the virus alone lost weight and most of them died within 7 - 10 days , the mice treated internasally with a mixture of the virus and the crude extract recovered and gained weight . each group included 10 mice . in vivo effect of the extract of the invention on influenza a pr8 infection 3 . 5 weeks old mice were injected into the caudal vein with 128 hau of influenza a pr8 pre - incubated with 250 μg of the ce inhibitor for 30 minutes at room temperature . the mice were weighed every 2 - 3 days for 3 weeks . the results are shown in fig1 ( a ) and 11 ( b ). weight loss of over 2 gr . was marked as a weight loss event . no deaths occurred among the mice infected with the virus pre - incubated with the inhibitor . each group included 10 mice . 100 pfu aliquots of hsv1 were mixed with 50 μg ( b ) of autoclaved ce ppt in 100 μl medium m - 199 and immediately submitted on vero cells in 24 wells plate . after 2 hours incubation at 37 ° c ., 5 % co 2 , each well was overlaid with additional one ml medium and the incubation continued 3 days . the cells were washed twice with pbs before fixation with methanol and staining with giemsa . the results are shown in fig1 ( a ). as can be seen in lane ( a ), cells with hsv alone were detached and washed from plate . against this , cells with hsv mixed with 50 μg ce ppt were not affected , indicating that the extracts of the invention protected the vero cells from hsv - 1 infection . 50 μg fixed aliquots of ce ppt were incubated with samples containing 10 2 - 10 6 pfu of hsv1 for 1 hour in 100 μl of medium m - 199 . each sample was applied on confluent vero cell monolayer growth in 24 wells plate and the plate was incubated at 37 ° c ., 5 % co 2 for 2 hours . one ml medium was added to each well and incubation continued 3 days . the cells were washed twice with pbs before fixation with methanol and staining with giemsa . results are shown in fig1 ( b ). the lanes were as follows : a — 10 2 pfu , b — 10 3 pfu , c — 10 4 pfu ( a - c — virus was totally inhibited ); d — 10 5 pfu — virus was partially inhibited ; e — 10 6 pfu — virus was hardly inhibited ; f — 10 2 pfu of virus without inhibitor , cells were detached and washed from wells . aliquots containing 10 6 pfu of hsv1 were mixed with 50 μg - 400 μg of ce ppt in 100 μl medium m - 199 . each mixture immediately submitted on confluent vero cell monolayers in 24 cells plate . after 1 hour incubation at 37 ° c ., 5 % co 2 , the cells from each well were overlaid with one ml m - 199 and the incubation continued 3 days . the cells were washed twice with pbs before fixation with methanol and staining with giemsa . the results are shown in fig1 ( c ). the lanes were as follows : a — 10 6 pfu of virus without inhibitor , cells were detached and washed from wells ; b — f : 10 6 pfu of virus with various amounts of ce ppt as follows : b — 50 μg , c — 100 μg , d — 200 μg , e — 300 μg , f — 400 μg . there is direct correlation between inhibition and increasing amounts of the ce ppt . as can be seen from all these results the extract of the invention was able to protect vero cells from the damaging effects caused by hsv - 1 infection . effects of the extract of the invention on the weight loss of mice infected with virus three and a half week old mice were infected with 32hau of sendai virus which was pre - incubated for 20 minutes with 125 μg of the ce ppt inhibitor or treated with the ce ppt immediately after infection with the virus . the mice were then weighed every 2 - 3 days during a 3 - week period . as fig1 a shows , the two groups of mice which had been treated with the inhibitor started to gain weight 8 days post infection ( p = 0 . 017 ). the control group which had not been treated with the inhibitor continued losing weight . in a different experiment , 3 . 5 - week old mice were infected internasally with 32 hau of sendai virus and immediately thereafter treated with 125 μg of the ce ppt inhibitor . a second group of mice was treated with the ce ppt inhibitor one hour post infection . the mice were weighed every 2 - 3 days for a period of 2 . 5 weeks . as fig1 b shows , mice which had been treated with the ce ppt inhibitor continued to gain weight whereas mice in the control group lost weight significantly ( p =& lt ; 0 . 001 ). in another set of experiments , immunization with the ce ppt inhibitor was tested . 3 . 5 week old mice were immunized intranasally ( i . n ). with 32 hau of sendai virus mixed with 125 μg of the ce ppt . the controlled group received only water . three weeks post immunization both groups of mice were infected with 64 hau of the naïve virus alone . the mice were weighed every 2 - 3 days over a period of 40 days . as fig1 c shows , the immunized mice were not affected by the subsequent virus infection and kept gaining weight ( p = 0 . 013 ). similarly , two groups of mice were immunized 3 times by the sendai virus mixed with the ce ppt inhibitor via two different routes of administration : oral and subcutaneously ( s . c ) as shown in fig1 d . two weeks after the third administration of the virus plus the ce ppt , the mice of both groups were infected with 80 hau of the naïve virus , as were the mice of the control group . the immunized mice were not affected by the subsequent virus infection and continued gaining weight . basically , no difference was observed between the mice to which the virus plus the ce ppt were administered orally or the mice which were administered s . c ( p =& lt ; 0 . 001 ). hiv - 1 activity was tested on mt2 cells ( cd4 + t - cells ) using the model of syncytia formation in cell culture . 20 - 120 μl aliquots of the vnf ( ceppt ) fraction , 0 . 5 mg / ml , were incubated with 50 μl virus for 5 minutes in a final volume of 200 μl rpmi medium at room temperature . 90 μl of each mixture were added onto the cells in duplicates . after 3 days of incubation at 37 ° c . in a 5 % co 2 humidified incubator , the infectivity was determined by monitoring syncytia formation . syncytia were observed in 95 - 100 % of the control wells without ceppt and served as the 100 % infectivity to which other wells were compared . as shown in fig1 a and b , 8 - 10 μg of ceppt in 8 - 10 μl was sufficient to neutralize the virus completely . the inhibition of avian influenza h9n2 by vnf was tested by the in vitro hemolysis assay as done previously ( borkow and ovadia , 1994 , 1999 ). the hemolytic activity of the influenza virus ( release of hemoglobin from red blood cells ) was examined on human erythrocytes as follows : human blood was obtained from the blood bank and was used fresh . prior to use , erythrocytes were washed 5 times with phosphate buffered saline ( pbs ), ph 7 and diluted to a concentration of 4 %, with the same buffer . the washed diluted erythrocytes were mixed with the virus alone or with a virus preincubated with the viral neutralizing fraction ( vnf ) for 20 minutes at room temperature . after the attachment , excess virus was removed by washing with pbs before adding 200 μl of 0 . 1 m sodium citrate buffer at ph 4 . 6 for three min ., in order to achieve fusion of the virus with the erythrocytes . the mixture was then washed in pbs , centrifuged and incubated in 0 . 8 ml pbs at 37 ° c . for 3 hours . intact erythrocytes were removed by centriftigation and 300 μl aliquot was withdrawn from the supernatant of each sample into wells of an elisa plate for measurement of the absorbance in an elisa plate reader at 540 nm . release of hemoglobin into the measured supernatant indicates viral hemolytic activity . as fig1 shows , the hemolytic activity of the virus was neutralized by the vnf ( ceppt ) in a dose dependent manner . influenza h9n2 virus was absorbed onto human erythrocytes at room temperature before application of vnf ( ceppt ) on the infected cells . the cells were then incubated at 37 ° c . and the hemolytic activity was determined as described in a previous figure . as fig1 shows , ceppt inhibited the hemolytic activity of the avian influenza virus after it was attached on the infected cells as it did to the free virus . hemagglutinating activity of the newcastle disease virus ( ndv ) was tested by mixing a drop of chicken blood with a drop of the virus suspended in pbs on a microscope slide ( left side of the picture ). as shown in fig1 , right hand - side picture , preincubation of the virus ( 10 8 eid 50 ) with 10 mg of vnf ( ceppt ) resulted in hemagglutination inhibition . no such hi was observed in the absence of the nvf ( left hand - side picture ). one ml containing 4 . 5 mg of vnf ( ceppt ) and 10 7 eid 50 of influenza h9n2 were incubated for 20 minutes at room temperature before preparing 10 fold dilutions from this mixture . 0 . 1 ml of each dilution was injected into each allantoic cavity of 10 embryonated chicken spf eggs , 11 days old . same dilutions of the virus alone or vnf alone were used as controls ( 10 eggs in each group ). the eggs were observed during the following week for vitality and viral hemagglutinating activity . as fig1 a and b demonstrate , vnf decreased the viral infectivity by 5 logs ( fig1 b ) and increased the survival of the embryos at the similar rate ( fig1 a ). this experiment is similar to the previous one carried out with the avian influenza h9n2 . one ml containing 5 mg of vnf ( ceppt ) and 10 8 eid 50 of newcastle disease virus were incubated for 20 minutes at room temperature before preparing 10 fold dilutions from this mixture . 0 . 1 ml of each dilution was injected into each allantoic cavity of 10 chicken spf eggs ( 11 days old ). same dilutions of the virus alone or vnf alone were used as controls ( 10 eggs in each group ). the eggs were observed during the following week for vitality and viral hemagglutinating activity . as fig2 a and b demonstrate , vnf decreased the viral infectivity by 5 logs and increased the survival of the embryos at the similar rate . 0 . 5 ml containing 2 . 5 mg of vnf ( ceppt ) were absorbed onto 250 mg of each three filtering materials ( names on the graph ) and dried overnight at room temperature . 1 ml of human influenza h1n1 virus containing 1280 hau was filtered through each one , and the passing fluid was tested for hemolytic activity on washed human erythrocytes as described above . as fig2 demonstrates , the lab filter paper absorbed with the ceppt was most efficient in absorbing the vnf and reduced the hemolytic activity of the filtered virus significantly . two different approaches of vaccination were used : vaccination in - ovo was compared with the customary intraocular vaccination of 1 - 2 day old chicks . in - ovo vaccination of the first group was carried out by injecting 0 . 1 ml of pbs containing 10 5 . 3 eid 50 of ndv preincubated with 1 mg of vnf into spf chicken eggs at day 18 of the embryonic development . second group was vaccinated 1 - 2 days after hatching by dripping the same dose into the eyes of the chicks ( the customaryintraocular vaccination ). non - vaccinated chicks were used as controls . blood samples were withdrawn from each chick at days 7 , 14 , 24 post - vaccination and the serum titer was determined by hemagglutination inhibition assay of serial dilutions of each serum . the serum titer after in - ovo vaccination was as good as the tedious customary intraocular vaccination of 1 - 2 day old chicks . in - ovo vaccination was much more comfortable and safe . table serum titer of chicks following vaccination with ndv + ceppt average serum titer ( hemagglutination inhibition ) group day 7 day 17 day 24 in - ovo ( d18 ) 7 . 6 ± 0 . 5 9 . 2 ± 0 . 8 9 . 0 ± 0 . 1 ndv + ceppt intraocular 4 . 0 ± 1 . 0 8 . 1 ± 1 . 0 8 . 7 ± 0 . 7 ( d2 ) ndv only non - vaccinated 2 . 0 ± 0 . 1 1 . 9 ± 0 . 2 3 . 1 ± 0 . 2 ± hernandez et al ., ( 2004 ). influence of two plant extracts on broilers performance , digestibility , and digestive organ size , poult . sci ., 83 ( 2 ): 169 - 74 . kalemba and kunicka , ( 2003 ). antibacterial and antifungal properties of essential oils , curr . med . chem ., 10 ( 10 ): 813 - 29 . khan et al , ( 2003 ). cinnamon improves glucose and lipids of people with type 2 diabetes , diabetes care , 26 : 3215 - 3218 . lay nd roy , ( 2004 ). antimicrobial and chemo - preventive properties of herbs and spices , curr . med . chem ., 11 ( 11 ): 1451 - 60 . mau , j . l ., et al ., ( 2001 ). antimicrobial effect of extracts from chineese chive , cinnamon , and corni fructus . j . agric . food chem . 49 : 183 - 188 . murcia et al , ( 2004 ). antioxidant evaluation in dessert spices compared with common food additives , influence of irradiation procedure , j . agric . food chem ., 52 : 1872 - 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