Anti-anemia Effect of Chlorophyll from Katuk ( Sauropus androgynus ) Leaves on Female Mice Induced Sodium Nitrite

Context: Sodium nitrite (NaNO2) in blood is highly reactive with haemoglobin (Hb), thus affecting hematopoiesis and induction of methemoglobinemia. Aim: This study was conducted to determine the effect of chlorophyll from katuk (Sauropus androgynus) leaves on the level of Hb, Malondialdehyde (MDA), ferritin, and schistocytes percentage in female mice induced NaNO2. Settings and Design: Experimental research was conducted using 24 female mice strain Balb-c. Methods and Material: NaNO2 0.3 ml/head/ day given during 18 days, while the chlorophyll or Cu-chlorophyllin as much as 0.7 ml/head/day given the following day for 14 days. Statistical analysis used: Results are reported as mean values ± SD and statistically analyzed by One Way Anova test with 95% significance level. Results: The Hb levels of blood plasma in the control group, NaNO2 induction, induction NaNO2 and chlorophyll of katuk leaves (NaNO2+katuk), induction of NaNO2 and Cu-chlorophyllin from K-LiquidTM (NaNO2+Cu-chlorophyllin) in sequence is 13.29 g/dl; 11.83 g/dl; 14.54 g/dl; 13.99 g/dl, whilst the MDA levels in each group is 2.10 ± 0.11 mol/L, 3.44 ± 0.38 mol/L, 2.31 ± 0.18 mol/L, 2.31 ± 0.13 mol/L, and the ferritin levels is 62.71 ± 6.42 ng/ml; 63.22 ± 7.59 ng/ml; 67.45 ± 8.03 ng/ml, and 64.74 ± 7.80 ng/ml, respectively. The fragment schistocytes percentage’s in each group is 0%, 0.11%, 0.01%, 0.03%. The ferritin levels tend to increase in NaNO2+katuk. Mann Whitney test results obtained no significant difference in Hb, MDA level and schistocytes percentage between the groups of mice that received NaNO2+katuk NaNO2+Cu-chlorophyllin (p>0.05). This indicates that chlorophyll from S. androgynus leaves as effective as Cu-chlorophyllin in decrease the MDA levels after NaNO2 treatment, and although not significant, it can increase ferritin levels. Conclusion: The antioxidant activity of chlorophyll from katuk leaves are able to decrease schistocytes percentage’s and MDA level. The increasing of Hb and ferritin level indicates its potential in the treatment of haemolityc anaemia. Further studies aimed at the mechanisms of action of this chlorophyll are needed.


INTRODUCTION
Sodium nitrite (NaNO 2 ) is an inorganic salt used as a common preservative and color fixative in cured meat products, fish and some types of cheese and occurs naturally in many foods, particularly vegetables.Recent studies about toxicology of NaNO 2 show that sodium nitrite in blood is highly reactive with hemoglobin, thus affecting hematopoiesis and induction of methemoglobinemia-a condition in which there is a reduction in hemoglobin's ability to transport oxygen. 1 The adduction of NaNO 2 in acute doses significantly decrease the number of erythrocyte causing a state of anaemia in rat. 2 Therefore, it important to prevent the anemia due to the negative effects of NaNO 2 .The rate of anaemia occurrence in Indonesia is considerably high.According to annual report of Dinkes Prov.Jateng (2013), the rate was 40.1% in pregnant mother in 2013.The percentage of anaemia prevalence in women is higher than that of in men, that was 23.9%. 3 The condition of anaemia that is not treated immediately will lead to complications such as cardiomegaly since erythrocytes and blood viscosity decreased causes an increase in cardiac output and blood flow as a result of tissue hypoxia. 4Prevention of anaemia has been undertaken by the government through the provision of iron supplementation. 5However, the use of the supplementation emerge side effects such as less comfortable circumstances in the pit of the stomach, nausea, vomiting, and constipation.Therefore, it is needed an anaemia therapy with natural ingredients, one of which is using chlorophyll of plants.In Indonesia, Katuk (Sauropus androgynus) leaves are often consumed as a breastmilk booster.The leaves contain plenteous chlorophyll.Nurdin   et al. (2009) notified that 8% of the leaves dry weight contain chlorophyll-a and chlorophyll-b as much as 1136.6 mg/kg and 372.5 mg/kg of the substances, respectively. 6Porphirin structure of the chlorophyll can reduce diphenilpycrylhydazil (DPPH) free radical form diphenilpycrylhydrazine that is non radical. 7Heme structure of the chlorophyll can bind a Magnesium (Mg) ion acting as an iron ion in human and animals. 8owever, a role of chlorophyll as antioxidant with parameter hemoglobin (Hb), Malondialdehide (MDA) and ferritin level, also schistocytes percentages in experimental animals induced by NaNO 2 as pathological treatment for anaemia have not been investigated.MDA is an end product of lipid peroxidation. 9State of increasing oxidative stress in the body induces the increase of MDA level as well.Oxidative stress which is uncompensated in normal erythrocyte will bring in hemoglobin oxidation become methemoglobin and membrane ravage in the form of uplift of membrane permeability to cell lysis. 10This condition causes the level of iron (Fe) stock in the body decrease progressively, reflected by deflation of ferritin concentration in serum. 11Examination of routine ferritin level is carried out to determine diagnosis of iron deficiency, because it is proven that ferritin level as an earlier indicator declines in the condition where anaemia of iron deficiency occur. 12Schistocytes are fragments of red blood cells (RBCs) produced by extrinsic mechanical damage within the circulation.The detection of schistocytes is an important morphological clue to the diagnosis of thrombotic microangiopathic anemia (TMA). 13his study aimed to examine the effect of chlorophyll in anaemic female mice induced NaNO 2 during 18 days.Antianemia effect of chlorophyll is observed after Katuk leaves treatment for 14 days by measuring Pharmacognosy Journal, Vol 8, Issue 4, Jul-Aug, 2016 hemoglobin, MDA and ferritin levels of blood plasma, also the percentage of schistocytes in blood smear.The result of this research is expected to encourage the use of Katuk leaves chlorophyll as anaemia drug.

Material
Fresh leaves of katuk (Sauropus androgynus) (Figure 1) were obtained from the inhabitant park in Penggaron Lor Village, Genuk, Semarang, Indonesia.All chemicals were of analytical grade.Cu-chlorophyllin from K-Liquid TM was obtained from a drug store in Semarang.Ethical clearance was obtained from the Ethics Committee of the Faculty of Medicine Universitas Islam Sultan Agung, Indonesia.

Chlorophyll Extraction
Chlorophyll crude extract was prepared from S. androgynus leaves by extraction with acetone: methanol (7:3, v/v). 13A given amount of leaves (200 g) was mixed with cetone: methanol (7:3, v/v) and magnetically stirred until the pigment was removed.Ascorbic acid was added to the solution to avoid degradation of the pigment.The extract was filtered to remove insoluble material.The filtrate obtained was then partitioned with diethyl ether three times sequentially.The organic phase material resulted were added with Na 2 SO 4 anhydrate as far as water contained was bound maximum.The solvent removal and drying process of chlorophyll used rotary evaporator and N 2 gas.

Experimental design
The experiments were carried out on two-month-old female Balb-C mice.The animals were divided into three sodium nitrite-treated groups (n=6 in each group) and an age-matched control group (n=6).Mice were maintained in the animal house of Faculty of Medicine Universitas Islam Sultan Agung in standard hard bottom polypropylene cages at 23ºC ± 2ºC, 12:12 h light/dark cycle and free access to laboratory chow and tap water throughout the study.NaNO 2 was administered orally by gavage at 3.75 mg/kg body weight (dissolved in 1 ml aquades), refers to rate of LD 50 on mice. 14NaNO 2 as much as 0.3 ml/g of body weight were given to the mice for 18 days for treated animals (group II, III and IV). 15 The control rats were injected with the same volume of distilled water.The following days after day 18 until day 32, the mice from group III were given with chlorophyll solution from S. androgynus leaves, whereas the mice from group IV were given with Cu-chlorophyllin from K-Liquid.TM The doses of chlorophyll and Cu-chlorophyllin those were given to mice were 0.7 ml/head/day according to the conversion of adult man doses. 16ood collection and plasma analysis Blood samples from the mice were taken at the day 33 to measure Hb, MDA and ferritin level in the blood plasma.Whole blood samples were obtained in heparinized tubes, centrifuged and plasma was stored at -20ºC until further analysis.Hb level was measured by cyanmethemoglobin methods using spectrophotometer 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.Schistocytes are detected in the peripheral blood smear stained using wedge procedures and observed by microscopy in 100X every 1000 erytrocyte.

Statistical analysis
Results are reported as mean values ± SD and statistically analyzed 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.A post hoc test was conducted where needed.Cu-chlorophyllin from K-Liquid TM 13,99 ± 0,81 a 2.31 ± 0.13 a 64.74 ± 7.80 a 0,03 ± 0,05 a NB: Supercripts in different letter showed the significantly different at α 5%.

RESULTS
The hemoglobin (Hb) decreased after NaNO 2 induction treatment group for 18 days.In contrast, mean of, MDA, ferritin level, and schistocytes percentage's in mice which were induced with NaNO 2 induction increased draw on the control group level (Table 1).Control group showed normal erythrocytes, whereas in the group induced NaNO 2 found schistocytes (Figure 2).Katuk chlorophyll and Cu-chlorophyllin group showed the lower schistocytes percentage compared with the group with the induction of NaNO 2 .Adduction of Katuk leaves chlorophyll and Cu-chlorophyllin proven to increase Hb level in mice.Although it has been induced by NaNO 2 (Figure 3).Adduction of Katuk leaves chlorophyll and Cu-chlorophyllin proven to reduce MDA level in mice.Although it has been induced by NaNO 2 , the levels were approaching the level of MDA in the control group after treatment, even Mann Whitney statistical test shown that MDA level of Katuk leaves treatment was not different from the level of control group (p>0.05).Moreover, MDA level of Katuk leaves was equal to that of Cu-chlorophyllin from K-Liquid, with the statistical test result shown that there was no differences between them (p>0.05, Figure 4), proving that chlorophyll from Katuk leaves as effective as chlorophyll from market products.One Way Anova test resulted that there were no differences in ferritin levels between all groups.However, the level in Katuk leaves (67.45 ± 8.03) tend to be higher than those of control group and other groups, followed by Cu-chlorophyllin from K-Liquid (64.74 ± 7.80) (Figure 5).

DISCUSSION
The present study demonstrates the effect of acute NaNO 2 treatment caused a significantly reduced decrease in hemoglobin level, as well as increased MDA, ferritin level and schistocytes percentages.It is suggested that the decrease in hemoglobin level may be attributed to microcytic and/or hypochromic anemia possibly as consequence of the toxic effect of NaNO 2 .Natrium nitrite (NaNO 2 ) can trigger oxidative stress condition which is indirectly caused haemolytic anaemia.It was can be seen from the mean of Malondiadehide (MDA) level in control group compare to those of the other groups that were induced by NaNO 2 .Adduction of NaNO 2 leads free radical formation from nitrite ion, and then the reaction continues to form lipid peroxidase and produce one of some products in the form of MDA.Natrium nitrite is one of substances that are classified as moderately toxic based on LD 50 .Toxic effect from NaNO 2 in this research was demonstrated by death of 4 mice which experiencing skins discoloration, feathers loss and the eyes excreting ichor. 17The decrease of erythrocyte is caused by lysis of membrane structure of the erythrocyte by dint of nitrite ion and nitrite metabolism and as a product of lipid peroxidase which reacted with sulfhydryl group from fat layer and protein components of erythrocyte membrane.Nitrite triggers the formation of free radicals due to its ability to stimulate iron ion oxidation on the oxyhaemoglobin forming methemoglobin as ROS. 18,19Therefore, anaemia that comes up is not iron deficiency anaemia, but rather haemolytic anaemia.In case of haemolytic anaemia, receptor transferrin serum increase when bone marrow erythropoiesis activities increase as well and found no functional iron depletion, it means the level of serum ferritin is normal or increase.Ferritin is an acute-phase protein, so that the value increases in inflammation condition, in pre-latent phase it is familiar known as iron depletion or storage iron deficiency.At this state, iron stock decrease, but the iron in plasma and erythrocyte is still normal. 20xcessive oxidant compounds induce a chain reaction of free radicals or lipid peroxidase which through several processes to form MDA. Oxidant compounds which successfully suppressed will not cause a continued reaction or lipid peroxidase, so that the MDA level will decline.Oxidative stress circumstances of NaNO 2 as anaemia pathologic treatment in female mice can be mitigated by antioxidant activities of Katuk leaves chlorophyll which MDA level of Katuk leaves chlorophyll treatment group is identical to that of the control group.Katuk leaves chlorophyll treatment can increase ferritin level in mice, although statistically there was no significant difference.Among other treatment groups, Katuk leaves treatment group gave the highest level of ferritin compare to the control group.The increase equal to the level of Cu-chlorophyllin from K-Liquid, however there was no significant difference between them.Katuk leaves chlorophyll potentially can be used as antioxidant, because it is able to prevent the formation of oxidant compound excessively. 9In the state of infection and inflammation, it occur disruption of iron release from reticuloendothelial cells, consequently, the level of ferritin intracellular and serum increase. 21Copper (Cu) of Cu-chlorophyllin is a micro essential substance which serves as part of the enzymes in the body and plays a role in iron absorption.Copper of seruloplasmin (a glycoprotein synthesized in liver) contribute in Fe oxidase process produce other copper femetaloproteins i.e. cytochrome oxydase, tironase, monoamin oxidase, superoxide dismutase (SOD) and lysyl oxidase before transported into plasma. 22his is in accordance with Togatorop (2013) revealed that chlorophyll of Katuk leaves can relieve toxic chemicals caused by cigarette smoke including free radicals in the body.Antioxidative effect of chlorophyll coming from porphyrin structure that is tetrapirol shaped and conjugated polyenes which can capture oxygen singlet 23 along with the existence of Mg ion that makes free radicals tend to give electrons to the Mg, thus neutralize free radicals. 24As a metaloenzim, SOD activities depend on the presence of Cu, Zn, and Mn metals.Copper in erythrocyte have the form of dismutase superoxide metaloenzyme that act as antioxidants. 25reatment of Katuk leaves chlorophyll can decrease Malondialdehide (MDA) level.The decrease of MDA level in Katuk leaves treatment was very effective and significant draw on negative control group.The decline is also almost the same as that of the Cu-chlorophyllin treatment group as a positive control group.According to this, chlorophyll of Katuk leaves potentially can be used as an alternative for healing anaemia caused by oxidative stress.The point must also be considered for future research is necessary to assess the level of oxidative stress that is caused by NaNO 2 induction.Besides, it is needed to measure quantitatively the level of chlorophyll from Katuk leaves extract and Cu-chlorophyllin from K-Liquid TM along with assessment on ferritin level in latent phase of anaemia.

CONCLUSION
The results of this study demonstrate the potential of chlorophyll from katuk (Sauropus androgynus) leaves as antioxidant caused by oxidative stress due to NaNO 2 induction as indicated by increasing of MDA level.Furthermore, our studies also indicate that the katuk chlorophyll may also be useful in the treatment of haemolytic anaemia, indicated by its ability to decrease Schistocytes percentages, also increase Hb and ferritin level.Further studies aimed at the mechanisms of action of this chlorophyll are needed.

ACKNOWLEDGEMENT
Financial support for this work was provided by the research grant from Faculty of Medicine Universitas Islam Sultan Agung, Semarang, Indonesia with contract number 22/P-KEL/UPR-FK/XI/2013, fiscal year 2013/2014.Suparmi would like to thank to the Indonesian Directorate General of Higher Education (DIKTI) for granting her a supporting fee to present this abstract in International Anatomical and Biomedical

Figure 2 :
Figure 2: Typical shapes for specific identification of schistocytes showed a polychromatophilic erythrocyte in the white arrow.

Figure 3 :
Figure 3: Hb levels in four treatment groups after 18 days NaNO 2 treatment followed by 14 days chlorophyll from treatment.S. androgynus leaves or Cu-chlorophyllin of K-Liquid TM.

Figure 4 :
Figure 4: MDA levels in four treatment groups after 18 days NaNO 2 treatment followed by 14 days chlorophyll from treatment.S. androgynus leaves or Cu-chlorophyllin of K-Liquid TM.

Figure 5 :
Figure 5: Ferritin levels in four treatment groups.