sodium nitrate ( nano2 ) is one of important environmental toxicants and poses important health risks .
most countries use nano2 as food additive as a source of color and flavor preservation in meat products and this chemical can also be found in water resources .
consumption of nano2 at low levels for long time caused histopathologic changes , nitrosative tissue damage , and lipid peroxidation in liver and kidney , as well as inducing chromosomal aberrations , decreased immune system , increased cancer colon risk , hypoxia , enlargement of the carotid body , and a vasodilation due to the activity with haemoglobin in the blood to form methaemoglobin , which has a much higher ( up to 20 times ) affinity for oxygen [ 4 , 5 ] .
acute intraperitoneal treatment of wistar rats with nano2 ( at the dose of 50  mg / kg bw ) influences the blood rheological properties and erythrocyte hematometric indices . as very little data are available on the effort to cure the intoxication of nano2 ,
the aim of this work was to evaluate the curative effect of chlorophyll from sauropus androgynus ( l ) merr as antioxidant to cure its toxic effect in inducing oxidative stress .
dietary chlorophyll can be found in fresh fruits and vegetables as chlorophyll a and chlorophyll b , thermally processed fruits and vegetables as metal - free pheophytins and pyropheophytins , and thermally processed green vegetables as zn - pheophytins and zn - pyropheophytins .
chlorophyll in form of underutilized greens in fresh vegetables , supplements , liquid solutions , extracts , or tablets can be used effectively as healthy and beneficial nutrient supplement .
antioxidant activity is one of the beneficial effects of chlorophylls to prevent oxidative dna damage and lipid peroxidation both by reducing reactive oxygen species ( ros ) and chelating metal ions [ 811 ] .
chlorophylls can act as a hydrogen donor to break the chain reaction , due to the porphyrin in its chemical structure . 

sauropus androgynus ( l ) merr was identified as potentially rich sources of chlorophyll [ 13 , 14 ] .
the antioxidant activities of the dark green leaves of s. androgynous were reported to have biologically nutritive value . among others , they have antidiabetic activity in diabetic mice induced by alloxan , reduce fever , increase breastmilk production , and prevent hoarse voice ; have antidyslipidemic activity and prevent the cardiovascular disorder in wistar male rats induced with fat - rich diet ; affect the growth performances , resist diseases , and enhance nonspecific immune responses in grouper diets .
the previous study reported that antioxidant activity of chlorophyll from s. androgynous leaves is able to decrease schistocytes percentage and malondialdehyde ( mda ) level and also increase the level of haemoglobin ( hb ) and ferritin in female mice induced by nano2 .
this work may provide new information for toxicological testing to validate the safety and traditional uses of these plants .
cu - chlorophyllin reported has a higher antioxidant activity than that of natural chlorophylls due to the presence of the chelated metal in the porphyrin ring .
the antioxidant activities of the cu - chelated compounds were found to be much higher than those of natural chlorophylls and of mg - free derivatives .
this study also compared the curative effect of natural chlorophyll in s. androgynus leaves compared to cu - chlorophyllin antioxidant activity in female rats induced subacute sodium nitrite ( nano2 ) .
haematological blood assays and the level of mda , ferritin , and transferrin in blood serum were analysed as curative effect indicator of chlorophyll , while the histopathologic view of liver and kidney tissues was used to evaluate its toxicity .
fresh leaves of s. androgynus ( l ) merr were collected from the inhabitant park in penggaron lor village , genuk , semarang , central java , indonesia .
cu - chlorophyllin from k - liquid was obtained from a drug store in semarang , central java , indonesia .
a given amount ( 200  g ) of fresh leaves was cleaned and was thereafter blended with acetone  :  methanol ( 7  :  3 , v / v ) using an electric blender until the pigment was removed .
the organic phase material that resulted was added steadily with na2so4 anhydrate to ensure no water in the extract . the solvent removal and drying process of chlorophyll used rotary evaporator and n2 gas .
a suspension of 0.016  mg of chlorophyll from s. androgynus ( l ) merr leaf extract ( csa ) was dissolved in 1  ml distilled water , used as a dose 1 of csa , while for making 1/2 dose csa 0.008  mg / ml was diluted from 1 dose csa .
thirty female wistar albino rats ( 150200  g ) were obtained from the faculty of pharmacy , universitas gadjah mada , yogyakarta , indonesia .
rats were maintained in the animal house of faculty of medicine , universitas islam sultan agung in standard hard bottom polypropylene cages at 23c  2c , 12  :  12  h light / dark cycle and free access to laboratory chow and tap water throughout the study .
ethical clearance was obtained from the ethics committee of the faculty of medicine , universitas islam sultan agung , indonesia , with document number 150/v/2015/komisi bioetika .
nano2 was administered 50  mg / kg body weight ( dissolved in 1  ml distilled water ) , which refers to rate of ld50 on rats .
nano2 as much as 1  ml / day was given intraperitoneally to the rats for 10 days for treated animals ( groups ii , iii , iv , v , and vi ) .
the control rats ( group i ) were treated with the same volume of distilled water . during the following 14 days
, the rats from groups iii and iv were given orally dose 1/2 ( 0.008  mg / ml ) and dose 1 ( 0.016  mg / ml ) of csa , respectively , whereas the rats from groups v and vi were given cu - chlorophyllin from k - liquid with dose 1/2 ( 0.008  mg / ml ) and dose 1 ( 0.016  mg / ml ) , respectively .
cu - chlorophyllin was dissolved in distilled water based on the instruction in the packing .
the solution of chlorophyll and cu - chlorophyllin , those given to rat , were 3  ml / head / day according to the conversion of adult man doses .
blood samples were collected from the animals via the periorbital sinus 24  h after the last treatment . about 3  ml of the blood
the clotted blood samples were centrifuged at 3000  rpm for 10  min to obtain the serum , which was used for biochemical analyses .
another 2  ml of the blood samples was collected into heparinized tubes that were used for haematological assays . the packed cell volume ( pcv )
red blood cell ( rbc ) counts were determined using the new improved neubauer hemocytometer .
schistocytes are detected in the peripheral blood smear stained using wedge procedures and observed by microscopy in 100x every 1000 erythrocytes .
commercially available kits were used according to the respective manufacturer 's protocol for the measurement of mda , ferritin , and transferrin .
mda level was measured using thio barbituric acid reactive substance ( tbars ) test with 532  nm wavelength spectrophotometer , whilst ferritin level was measured using enzyme linked immunosorbent assay ( elisa ) method .
all the animals were then sacrificed by anesthetizing with diethyl ether 24  h after the last treatment .
the organ tissues were processed and embedded in paraffin wax and sections were made of about 46  m . after staining with haematoxylin and eosin , slides were examined under the microscope ( olympus , japan ) for histopathological changes and photographed .
the histopathologic parameters for liver were scored as follows : ( 1 ) showing no changes or normal , ( 2 ) parenchymatous degeneration , ( 3 ) hydropic degeneration , and ( 4 ) necrosis , while the scoring for proximal tubule epithelium was as follows : ( 1 ) lesion less than 25% indicating no change , ( 2 ) lesion 25%<50% , ( 3 ) lesion 50%<75% , and ( 4 ) lesion more than 75% showing severe changes .
results are reported as mean values  sem and statistically analysed by one - way anova test with 95% significance level .
if the data characteristics did not allow for the one - way anova test to be conducted , then the kruskal - wallis test became the alternative .
the hb in induced nano2 rats group was significantly lower ( p < 0.05 ) than that of rats in control groups .
it indicated that the subacute administration of nano2 can cause anaemia which was indicated by the decrease of hb level , pcv percentage , increase the rbc concentration , and schistocytes percentage .
changes in blood parameters and immune response are the direct toxic effects due to high dose administration of nano2 .
pvc indicates the proportion of whole blood occupied by the rbc and depends on the hb level in rbc .
the percentages of schistocytes in nano2-induced rats group were significantly higher than control group which proved that nano2 is able to damage the resulting fragmented rbcs called as schistocytes .
the schistocytes may have different forms such as triangular , helmet , or comma shaped of broken or fragmented erythrocytes .
nano2 affects the haematological and hemorheological parameters in mature rats [ 5 , 24 ] .
the group that was treated with one - dose chlorophyll from s. androgynous showed significant difference in hb level , rbc concentration when compared with the nano2 group ( p < 0.05 ) , but no significant difference with the cu - chlorophyllin in the same dose and control group .
this indicated that the natural chlorophyll from s. androgynous leave is as effective as cu - chlorophyllin to cure the oxidative stress caused by nano2 induction .
the haematological parameters as pcv , hb , and schistocytes in in the lower dose group of s. androgynous chlorophyll and cu - chlorophyllin were not significantly different ( p > 0.05 ) compared to those in the nano2 group , which indicated that lower dose of chlorophyll was not more effective at ameliorating the anaemia symptoms induced by nano2 .
nano2 is one of methaemoglobin - forming drugs that may exacerbate oxidative toxicity under certain chronic or acute hemolytic settings .
the increasing of hb level showed that nano2 induced erythrocyte methemoglobinemia by increasing reactive oxygen species ( ros ) .
the fusion of the heme moiety of hemoglobin released from red blood cells into endothelium could provide catalytically active iron to the vasculature .
ferritin as a cytoprotection against free radicals in vitro will be increased in the increasing of ros . 

the mda level increased significantly in nano2 induction group , whereas transferrin and ferritin level were reduced significantly with exposure to nano2 when compared with control .
nitrate is reduced to nitrite   and nitrite is converted to nitrosonium ions which in turn reacts with amines and amides to form nitrosamines and nitrosamines , respectively .
n - nitrosamines have the ability to induce rapid oxidative stress   and then cause lipid peroxidation ; therefore it disturbs the cellular homoeostasis .
the serum mda level is one of the molecules used as indicator of lipid peroxidation to estimate oxidative stress .
present study showed that rats who received nano2 for 10 days showed increasing of the mda serum significantly compared to control group .
this result is in accordance with the previous studies that elevated mda level in nano2-given mice   and elevated levels of serum mda in rats given nano2 .
results from this study revealed that treatment with chlorophyll of s. androgynus restored mda levels after nano2 treatments to normal values . the mda level in group
ii which was not treated by the chlorophyll remained high ( see table 2 ) .
the decreasing of mda levels in group iii till group vi after the nano2 treatments is the results of the antioxidant activity of chlorophyll of s. androgynus and cu - chlorophyllin .
the same effects were observed for serum ferritin and transferrin . decreased serum transferrin and ferritin levels
could be a result of damage , particularly protein oxidation by reactive oxygen species generated by nano2 toxicity .
there was a decrease ( p < 0.05 ) in serum transferrin and ferritin in the nano2-induced rat followed by therapy with s. androgynus chlorophyll and cu - chlorophyllin group compared with the control group .
the histopathologic parameters for liver and proximal tubule epithelium of kidney were not significantly different among groups ( table 3 ) , although there were increasing score of hepatocyte and proximal tubule epithelium damage in nano2-induced group .
histopathology of hepatocyte of rat liver in control group showed normal or no change with score 1 , while the hepatocyte in nano2 induced group showed hydropic degeneration ( figure 1 ) .
the hepatocyte and proximal tubule kidney in group treated with dose 1 cu - chlorophyllin showed the highest score of hepatocyte histopathologic view even not significantly different from the control .
there was founded necrosis cell in proximal tubule kidney of rats treated by cu - chlorophyllin ( figure 2 ) .
the histology of kidney in the group cured with dose 1 of chlorophyll showed that the lesion cell was less than 50% . in the present study , intake of nano2 for 10 days
this result was in accordance with roth and kate smith   that rats given nano2 via drinking water at 13 
g / l during pregnancy and lactation showed decreasing of erythropoietic development , changing in some histopathological such as cytoplasmic vacuolization of centrilobular hepatocytes , and decreased hematopoiesis in bone marrow and spleen .
have detected hydropic cellular degeneration in liver and tubular degeneration in kidney in 20  mg / kg / day nano2-induced mice for 8 months .
the histopathological changes are mediated by oxidative stress produced by the action of the metabolic pathways generated against the toxic compounds .
( 2005 ) reported that cu - chlorophyllin tested by -carotene bleaching method and the stable radical 2,2-diphenyl-1-picrylhydrazyl ( dpph ) scavenging assay methods presented a higher antioxidant activity than that of natural chlorophylls , showing the importance of the nature of the chelated metal in the porphyrin ring .
the mechanism of antioxidant activity displayed by the natural chlorophyll derivatives does not seem to be based on the ability to donate hydrogen but maybe , the protection of linoleic acid against oxidation , and/or preventing decomposition of hydroperoxides .
this is suggestive of hepatocytes protection from antioxidant activity from chlorophyll of s. androgynus against nano2-induced damages .
the histopathologic parameters for liver showed nano2-induced toxicity and ameliorative potentials of chlorophyll of s. androgynus on nano2-induced oxidative stress .
chlorophyllin copper ( cu(ii)-chlorophyllin ) is one of derivatives of chlorophyll that reported increased peripheral leukocyte count and improving symptoms of dizziness and fatigue in individuals with leukopenia .
chlorophyll is converted to mg - free pheophytin derivatives during digestion and then transported to peripheral tissues .
chlorophylls degraded rapidly to pheophytins in response to the high acidity of the gastric phase .
in the past , chlorophyll has been used to treat gastrointestinal problems , anaemia , and cancer .
fahey et al .   reported that chlorophyll can improve the function of essential detoxification pathways .
chlorophyllin was 410-fold more potent as a phase 2 enzyme inducer than chlorophyll , since it has other detoxification properties because it is much more water - soluble than chlorophyll .
.   showed that sasa senanensis rehder leaf extract containing fe(ii)-chlorophyllin demonstrated superoxide anion and hydroxyl radical - scavenging activity five times higher than a similar product containing cu(ii)-chlorophyllin and comparable to a product containing cu(ii)-chlorophyllin , ginseng , and pine leaf extracts .
therefore , the data obtained from this study further proved the usefulness of chlorophyll of s. androgynus as a food supplement that may be recommended to cure humans and animals from nano2 toxicity .
the present study has demonstrated the curative effect of chlorophyll solution from s. androgynus leaves on haematological , serum biochemical , and histological parameters , altered by sodium nitrate exposure in female wistar rats .
this amelioration may be partly due to the antioxidant activity of chlorophyll from s. androgynus leaves that possess remarkable potential to cure the oxidative stress caused by sodium nitrate .
further assessment of molecular evaluations of biological activities of chlorophyll from s. androgynus needs to be studied .
 sodium nitrate ( nano2 ) widely used as food additive for coloring and preserving meat has been reported to induce oxidative stress and cause histopathologic changes , nitrosative tissue damage , and lipid peroxidation in liver and kidney . 
 therefore , the present study compared the curative effect of chlorophyll from sauropus androgynus ( l ) merr and cu - chlorophyllin as antioxidant in nano2-induced female wistar rats based on haematological , serum biochemical , and histological evaluation . 
 thirty male wistar rats were randomly assigned into six groups of five rats each . 
 nano2 were given at a subacute dose of 50  mg / kg bw intraperitoneally for 10 days . 
 chlorophyll from s. androgynus and cu - chlorophyllin from k - liquid were given in the following 14 days at the two doses : 0,016  mg / ml and 0.008  mg / ml . nano2 exposure resulted in significant reductions ( p < 0.05 ) in values of packed cell volume ( pcv ) , haemoglobin ( hb ) concentration and red blood cell ( rbc ) count , transferrin , and ferritin and elevation in malondialdehyde ( mda ) level and schistocytes percentage with insignificant reductions in serum albumin and transferrin levels . 
 histology of kidney and liver were changed insignificantly ( p > 0.05 ) to normal values . 
 chlorophyll from s. androgynus and cu - chlorophyllin possess antioxidant potentials to protect against toxicities induced by sodium nitrate . 
1. Introduction
2. Material and Methods
3. Results and Discussion
4. Conclusion