Evidence for the Presence of Iron Chelator Constituent In The Wheatgrass for the Treatment of Patients With Thalassemia Major

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1 Research Article ISSN: Available online through Evidence for the Presence of Iron Chelator Constituent In The Wheatgrass for the Treatment of Patients With Thalassemia Major Prakash I Shah 1, Ramesh K.Goyal 2, C.B.Tripathi 3, Mehul Gosai 3 1 Shantilal Shah Pharmacy College, Bhavnagar University, Bhavnagar-3642, Gujarat, India 2 Shobhanaben Pratapbhai Patel school of Pharmacy and Technology Management, NMIMS, Mumbai, 3 Govt. Medical College, Bhavnagar,Gujarat, India Received on: ; Revised on: ; Accepted on: ABSTRACT Triticum aestivum, (Poaceae), the wheatgrass/juice and its formulation has been shown to produce beneficial effects in patients with thalassemia major. In the present investigation we have studied the effect of wheatgrass juice and capsules and the mechanism of action in patients with thalassemia major. It was prospective, open labeled, non-randomized, parallel design and no controlled clinical study in 28 patients with thalassemia major. Treatment with wheatgrass juice and capsules produced significant elevation in mean blood transfusion interval. There was no change in the mean haemoglobin levels but there was reduction in HbA 2 level and induction of foetal haemoglobin (HbF). Treatment of the patients also produced decrease in serum ferritin, serum iron, and total iron binding capacity (TIBC). There was normal excretion of urobilinogen in the urine of patients with thalassemia. Bilirubin, bile salts, leukocytes, nitrites, protein, and glucose were absent in the urine sample of thalassemia major. Universal CAS assay also showed the presence of phytosiderophore in wheatgrass capsules and juice. The FTIR spectroscopy also confirmed the presence of various functional groups in the chemical structure of phytosiderophore. In conclusion our data provides sufficient evidence that wheatgrass can produce decrease in iron over load possibly by virtue of phytosiderophore present in wheatgrass/juice. KEY WORDS: Thalassemia major patient, Total iron binding capacity, Chromo Azurol S, phytosiderophore, HbF- Foetal haemoglobin, Iron chelation. INTRODUCTION Thalassemia is characterized by impaired production of one or more polypeptide chains of globin 1. It may involve any of the four polypeptides (α, β, γ, δ,) that occur in normal haemoglobin synthesis. The most prevalent thalassemia syndromes are those that involve diminished or absent synthesis of the α 2 β 2 globin chains of HbA1. Wheatgrass is reported to produce beneficial effect on haemoglobin level and increases mean blood transfusion interval in thalasemics 2,3, but mechanism of action is still not known. Electrophoretic pattern of haemoglobin comprises of different haemoglobin fractions HbA, HbA2 and HbF. Generally foetal haemoglobin (HbF) level is elevated in thalassemia major patients. To the best of our knowledge,effects of wheatgrass on thalassemia major patients on different fractions of haemoglobin has not been reported. The management of beta thalassemia major patients requires life long regimen of regular blood transfusion coupled with iron chelation therapy 4. Blood transfusion produces on long term serious and unavoidable side effects because of deposition of iron in the heart, liver, pancreas, glands and vital organs of the body. This may lead to heart failure, cirrhosis of liver, diabetes mellitus and malfunctioning of other glands. Iron overload in thalassemia major patients may be treated with chelating agents like desferrioxamine 5,6. Earlier data revealed that treatment with wheatgrass tablet for 9 months period significantly reduces serum ferritin in beta thalassemia patients. This suggests that wheatgrass may possess iron chelating property 7. To date this is the only published clinical trial addressing that wheatgrass reduces iron overload in beta thalassemia patients. The measurement of serum ferritin is the most commonly used indirect estimate of body iron *Corresponding author. Dr. Prakash I Shah Senior Lecturer, Shantilal Shah Pharmacy College, Sardar Vallabhbhai patel campus, Bhavnagar University, Bhavnagar, Gujarat,India. stores8. In the present study we have carried out effect of wheatgrass capsules and juice on serum iron, serum ferritin and total iron binding capacity to substantiate the possibility of wheatgrass. It can be assumed that iron may be excreted in any form in the urine sample of thalassemia major patients treated with wheatgrass. Based on our hypothesis, we carried out the estimation of urobilinogen, bile salts, bilirubin, ketone bodies, protein, blood, leukocytes, nitrites and glucose in the urine sample of thalassemics. If iron is not excreted in urine sample of thalassemics treated with wheatgrass tablet, and iron over load is reduced in thalassemics; it is possible that there may be a substance present in wheatgrass which might have iron chelating property. We also carried out FTIR spectroscopy of acetonic extract of wheatgrass dry powder and juice to confirm the various functional groups present in the chemical structure of Phytosiderophore. The in-vitro experiments of iodometric titration and colorimeter assay were also confirmed the iron complexation property of wheatgrass capsules and juice. In the light of above the precise objective of the present investigations was to investigate the mechanism of action of wheatgrass as iron chelator in thalassemics. MATERIALS AND METHODS Patient Selection, Inclusion and Exclusion Criteria The study was conducted at Sir.Takhtasinhji hospital, Bhavnagar. The necessary permission of institutional review board Government medical college, Bhavnagar was obtained prior to the commencement of clinical study. It was prospective, open labeled, non-randomized and parallel design clinical trial on thalassemia major patients. The protocol was approved by the Ethics Committee of the Institutional Review Board. The informed consent form approved by the ethics committee in vernacular language was taken from all the patients/parents or their relatives. The study included a total of 28 thalassemia major patients age range from 3 to years registered indoor patients for the blood transfusion at Sir.T

2 Table 1: Profile of patients in the present clinical study hospital, Bhavnagar and Patients with major illness, major secondary complications like diabetes, thyroid, or cardio vascular complications or patients with sickle cell anemia were excluded from the study. These patients were divided into 2 groups. Group-I (n=13) were the thalassemia major patients, given commercially available (Nutrila) capsules each containing 3 mg. of wheatgrass (Triticum aestivum) dry powder received from Green Era foods and Neutraceuticals, Bhavnagar. Group-II (n=9) were the thalassemia major patients given traditionally prepared ml fresh wheatgrass juice received at our center. Wheatgrass juice was prepared by taking 4 gm. wheatgrass leaves with water in a household mixture. The patients were given counseling on day, every month as and when they come for blood transfusion, on 6 months and on completion of treatment of 9 months.the blood samples were collected by venipuncture on day, 6 months and after 9 months for the bio chemical estimation. Out of twenty eight (n=28) thalassemia major patients 6 patients discontinued the study before six months and 4 patients discontinued the study after six months of study period due to indiscipline in taking wheatgrass juice or capsules. Analysis of blood and urine sample of Thalassemics The blood samples collected in plain tubes were analyzed for complete blood count (CBC) using three part differential cell counter automated haematology analyzer Sysmex KX-21, Japan. Haemoglobin electrophoresis was also performed by using fully automated Genio S Electrophoresis machine make by Interlab srl, Italy. The haemolysate was prepared from the collected blood sample. It was loaded in sample tray in Genio S Electrophoresis machine and Electrophoresis was carried out. Serum was separated from the blood collected in EDTA containing tube and serum ferritin was measured by the kit DPC (Germany) on fully automated immulyte by Chemilluminescence method. Serum iron and TIBC were estimated spectrophotometrically using standard kit obtained from Raichem Laboratory, USA on fully automated random access Kone lab- biochemistry analyzer. Urine sample of thalassemia major patients were collected in bottle and urobilinogen, bilirubin, leukocytes, nitrites, ketones, bile pigment, protein, glucose and blood were analyzed using Diruhi H- automated urine chemistry analyzer. Chemical and spectroscopical analysis of wheatgrass Wheatgrass capsules and juice were analyzed for the presence of phytosiderophore by Chrome Azurol S (CAS) assay as described by Schwyn etal, The FTIR spectroscopy was also carried out to characterize phytosiderophore. In vitro assay by iodometric titration and identification tests for ions were performed by standard methods. Statistical analysis Results are presented as Mean ± SEM. Statistical differences between the means of the various groups were evaluated using student s paired t test. Data were considered as statistically significant at P value of 5% (P.5). RESULTS Profile of thalassemia major patients included in the study The age of thalassemia major patients included in the study was 3 to years and out of 22 thalassemia major patients patients were male, while 12 patients were female (Table-1). Total number of thalassemia major patients 22 Age range of thalassemia patients 3 to years Number of male patients Number of female patients 12 Patients above years age 7 Patients below years age Patients with serum ferritin below 2 Patients with serum ferritin above Effect of wheatgrass on different haemoglobin fractions Wheatgrass capsules when given for a period of 6 months to the patients there was an increase in HbA 2 level, but it was decreased after 9 months treatment period as compared to initial level. There was also an increase in the total haemoglobin level after 9 months treatment with wheatgrass capsules as compared to the initial level. There were no significant changes found in the level of HbA and HbF after treatment with wheatgrass capsules as compare to initial level in thalassemia major patients (Table-2). Table-2: Effect of Wheatgrass capsule (n=13) or juice (n=9) in Patients with thalassemia major on different haemoglobin fractions Parameter Mean Value After Mean Value After Treatment with capsules Treatment with juice Mean Value After 6 After 9 Mean Value After 6 After 9 Before Months Months Before Months Months Treatment (n=13) (n=12) Treatment (n=9) (n=6) with capsules with juice (n=13) (n=9) HbA (%) 88. ± ± ± ±.54* 88.2 ± 1.18* 91.3 ± 1.1 HbA 2 (%) 2.7 ±.11* 3.5 ±.2* 2.49 ± ±.17* 3.98 ±.43* 2.5 ±.18 HbF (%) 8.98 ± ± ± ±.59* 7.81 ±.82* 6.6 ±.67 TotalHb 6.51 ± ± ± ± ± ±.19 (gm%) * Indicates significantly different from initial value (P <.5) Effects of wheatgrass on iron over load in thalassemics Treatment with wheatgrass capsules (Table-3) as well as juice (Table-4) for the period of 6 and 9 months produced decrease in serum transferrin and serum TIBC levels as compared to initial level. There was also decrease in serum ferritin level after 6 and 9 months treatment period with wheatgrass capsules as compared to initial levels; but it was statistically significant for the period of 6 months. Table-3: Effect of Wheatgrass Capsule on Patients with thalassemia major with respect to changes in Iron status (n=13) Parameter Normal Mean Value Mean Value After Treatment Value Before After After Treatment 6 Months 9 Months (n=13) (n=13) (n=12) Serum Transferrin 6- gm/dl ± ± ±13.8 (Serum Iron) Serum Ferritin - ng/dl 6516±57 ±743.6* 4749 ±98 Total iron binding ± ± ±27.99 capacity (TIBC) microgm/dl Table-4: Effect of Wheatgrass Juice on Patients with thalassemia major with respect to changes in Iron status (n=9) Parameter Normal Mean Value Mean Value After Treatment Value Before After After Treatment 6 Months 9 Months (n=13) (n=13) (n=12) Serum Transferrin 6- gm/dl ± ± ± 23.6 (Serum Iron) Serum Ferritin - ng/dl 5 ± ± ± Total iron binding ± ± ±33.5 capacity (TIBC) microgm/dl

3 Effect of wheatgrass capsule and juice on mean blood transfusion (B.T.) interval in thalassemia major patients Treatment with wheatgrass capsules (Fig. 1) or juice (Fig. 2) for the period of 9 months produced significant elevation in mean blood transfusion interval as compared to initial level. Effect of Wheatgrass Capsule on Mean Blood Transfusion Interval Mean Blood transfusion Interval * Initial 9 Months Wheatgrass capsule treatment period Fig.-1: Effect of Wheatgrass Capsule on Patients with thalassemia major with respect to changes in mean blood transfusion interval (n=13) Mean Blood transfusion interval Effect of Wheatgrass Juice on Blood Transfusion Interval * Initial 9 months Wheatgrass juice treatment Period Fig.-3: FTIR spectrum of Acetonic extract of wheatgrass juice extract at the group frequencies were 1739 cm -1 and 16 cm -1. A broad band of NH 2 / -OH group was also appeared around 34 cm -1. The FTIR Spectrum of aqueous wheatgrass juice + CAS solution (Complex) as showed in figure-4. When Iron (Fe) was induced in the juice extract under the similar condition iron was Co-ordinated via COOH group and hence a single peak around 16 cm -1 was observed, which indicates that metal Co-ordinated with C=O (Carbonyl) group of phytosiderophore namely Mugienic acid. The FTIR Spectrum of Wheatgrass dry powder in acetone extract as showed in figure-5 clearly indicated the presence of NH 2 / -OH group in the structure of Phytosiderophore by depicting group frequency around 3 cm -1 and 36 cm -1. Asymmetric stretching was also appearing around 294 cm -1, while a sharp peak around 17 cm -1 indicates presence of COOH group in plain and out of plane bending was also appeared for CH 2 group Fig.-2: Effect of Wheatgrass Juice on Patients with thalassemia major with respect to changes in blood transfusion interval (n=9) Chemical analysis of wheatgrass The results of acetonic extract containing dry wheatgrass powder and wheatgrass juice showed the presence of phytosiderophore. The iodometric titration also supported the presence of phytosiderophore by showing complexation produced by wheatgrass capsules as well as juice. In vitro studies by colorimeter assay with ferric ammonium sulphate (FAS) with potassium thiocynate (KCNS) showed decrease in absorbance as compared to initial values. This indicates that there is decrease in free iron when treated with wheatgrass capsules as well as juice. FTIR spectroscopy of wheatgrass The FTIR Spectrum of aqueous wheatgrass juice extract (Fig-3) showed presence of 2 types of C=O (Carbonyl) group in the wheatgrass juice Fig.- 4: FTIR spectrum of wheatgrass juice with CAS solution The FTIR Spectrum of dry powder in acetone extract + CAS solution (Complex) as showed in figure-6. The group frequencies were also appeared in accordance to complex of phytosiderophore with metal ion. A broad

4 Fig.-5: FTIR spectrum of wheatgrass dry powder of blood transfusions. There was elevation in mean blood transfusion interval in thalassemia major patients as compared to initial levels. Probably, this action may be attributed to similarity of structure between chlorophyll and haemoglobin. The foetal haemoglobin (HbF) is synthesised by the formation of tetramer of a 2 b 2 -globin chain.it is reported that the drugs like hydroxyurea, 5-AzaC and butyrate compounds have been utilised to elevate foetal hemoglobin (HbF) in the treatment of thalassemics. HbF has substantially higher affinity for oxygen than other adult haemoglobin fractions 11. The mechanism of induction of HbF by butyrate is that; it Inhibits histone deacetylases and there by increasing histone acetylation, which opens up the chromatin structure and makes DNA more accessible to transcription factors. This results in increased transcription of the gamma-globin gene by making its DNA more accessible to transcription factors 13. Early studies demonstrated that treatment of tissue culture cells with the cytidine analog 5- azac lead to cellular differentiation and DNA hypomethylation. Incorporation of 5- azac into baboon DNA resulted in a 4- to 7-fold increase in gammaglobin gene expression in the adult, and to hypomethylation of the gamma -globin promoter 12,13. It is also reported that, DNA hypomethylation and histone deacetylation seem to be the chief mechanism of action of the agents that induce HbF synthesis 14. These suggest that inhibitors of DNA methyl transferase and histone deacetylase may activate transcription of foetal haemoglobin (HbF) by synthesising formation of a 2 b 2 -globin chains tetramer. Earlier studies reported that In-vitro experiment of wheatgrass aqueous extract increases production of foetal haemoglobin (HbF) level. In our investigations also treatment with wheatgrass juice produced significant elevation in the foetal haemoglobin (HbF) level in thalassemia major patients. The probable mechanism of action of foetal haemoglobin (HbF) induction by wheatgrass juice may be attributed to inhibition of histone deacetylases or inhibition of DNA methyl transferase Fig.-6: FTIR spectrum of wheatgrass dry powder with CAS solution peak around 3 cm -1 indicates presence of NH 2 / -OH group. The original frequencies of -C=O (Carbonyl) group around 1737 cm -1 was now appeared around 16 cm -1 clearly indicating that metal is co-ordinated with carboxyl group. DISCUSSION Treatment with wheatgrass tablets for 9 months period produced significant reduction in serum ferritin level in beta thalassemia patients. Chlorophyll present in the wheat grass has structure similar to haemoglobin. In bone marrow at the cellular level the cells get heme for the production of haemoglobin. Since heme production occurs faster, globin chains combine with it and form less of insoluble inclusion bodies. The destruction of RBCs is les and so life span of RBCs is prolonged. We found that administration of wheatgrass produced significant decrease in the frequency It is well known that HbA 2 is synthesised by the formation of tetramer of alpha (2), delta (2) chains. Thalassemics have elevated HbA 2 level. In the present study treatment with wheatgrass capsules and wheatgrass juice for 9 months period; there were decrease in the HbA 2 level. However, there was significant elevation in HbA 2 level after 6 months treatment period as compare to initial levels. Thus, we suggest that both wheatgrass capsules as well as juice produced reduction in HbA 2 level after long treatments of 9 months; probably by virtue of decrease in synthesis of tetramer of alpha (2), delta (2) chains and hence, improves conditions in thalassemics. Another observation in the present study was that treatment with wheatgrass capsules and juice for the period of 6 and 9 months produced decrease in serum ferritin level in patients with thalassemia major. Treatment with both wheatgrass capsules as well as juice produced decrease in serum transferrin and total iron binding capacity (TIBC) levels as compared to initial levels. These results indicate that wheatgrass capsules or wheatgrass juice appears to reduce iron over load in thalassemia major patients. It is well known that bilirubin is formed from the breakdown of haemoglobin in the reticulo-endothelial system (mainly spleen, liver, and bone marrow). The indirect bilirubin (water insoluble) then bind to serum albumin and transported through the blood to the liver. The unconjugated bilirubin being insoluble in water can not be filtered through the glomerulus. In the liver, bilirubin is removed by parenchymal cells. It is then converted to, water soluble (conjugated) bilirubin by the action of enzyme glucuronyl transferase, by conjugation with glucuronic acid. This conjugated bilirubin is excreted by the liver through the bile duct and into the duodenum. Since conjugated bilirubin is easily filtered through the glomerular and excreted in urine. In the intestine, bilirubin is converted by bacterial enzymes to several related compounds, collectively called as urobilinogen. Most of the urobilinogen is

5 lost in feces as stercobilinogen and about to % of urobilinogen is reabsorbed into the blood stream and returns to liver. It is re excreted by the kidneys into the urine. In thalassemia major patients, the red blood cells are affected, there is an increase in unconjugated bilirubin, while conjugated bilirubin is normal and hence no bilirubin will be excreted in the urine because unconjugated bilirubin can not be filtered at the glomerular, due to the increased formation of bilirubin, the rate of formation of urobilinogen increases considerably and it is excreted in urine in high proportion. Urine analysis of pre and post treated thalassemia major patients treated with wheatgrass capsules and juice revealed that excretion of urobilinogen in normal proportion and other substances such as bilirubin, bile salts, leukocytes, nitrites, protein, and glucose were also absent. These results indicate that, Urobilinogens or bile salts were not excreted in abnormal proportion in urine sample through kidney in any of the thalassemia major patients either pre or post treated with wheatgrass capsules and juice. Now, Iron has no any other routes for excretion so there must be constituent present in wheatgrass which may responsible for chelation of iron. It is possible that there may be a substance present in wheatgrass which might possess iron chelating property. Barley and rice are grasses, which secrete mugienic acid a phytosiderophore which possess acidification, chelation and reduction processes in iron uptake by plants 16. The Mossbauer spectroscopic in vivo studies on siderophore uptake in fungi, reported that siderophores can also function as intracellular iron storage compounds 17. In the present study also acetonic extract of dry wheatgrass powder as well as juice confirmed the presence of phytosiderophore by the chromo azurol S (CAS) assay 9. It can be presumed that phytosiderophore present in wheatgrass can function as intracellular storage for iron and act as iron chelator. The structure of phytosiderophore (mugienic acid) as 3-hydroxymugienic acid, 2 -deoxymugienic acid, avenic acid,and distichonic acid 18. In the present investigations the FTIR Spectroscopy further confirmed the presence of Carbonyl group (-C=O), Carboxylic acid group ( COOH), and NH 2 / -OH groups in supporting the chemical structure of phytosiderophore (mugienic acid). Thus, our studies suggest that wheatgrass capsules as well as juice contain chemical substance Phytosiderophore belong to Mugienic acid. In conclusion our data suggest both wheatgrass capsules and juice produces beneficial effects in patients with thalasemia major. The mechanisms may not be induction of foetal haemoglobin or the presence of chlorophyll. Although at this stage we cannot conclude on the mechanism of action but our study provides sufficient evidence for the iron chelating property of wheatgrass that may be attributed to presence of phytosiderophore. Source of support: Nil, Conflict of interest: None Declared REFERENCES 1) Weatherall, D. J. The thalassemias. B.M.J. 1997, 314: ) R.K. Marwah, Deepak Bansal, Siftinder Kaur and Amita Trehan, In: Indian pediatrics 4, Volume 41 July. 3) Karnail singh. MS Pannu, Palwinder Singh and Jaswinder Singh January. Effect of Wheatgrass Tablets on the frequency of Blood Transfusions in Thalassemia Major,Indian journal of paediatrics,, volume-77:9. 4) Steinberg, M. H. Thalassemia: molecular pathology and management. Am. J. Med. Sci. 1988, 296: ) Cooper, C. E., Lynagh, G. R., Hoyes, K. P., Hider, R. C., Cammack, R., Porter, J. B. The relationship of intracellular iron chelation to the inhibition and regeneration of human ribonucleotide reductase. J. Biol. Chem 1996,. 271: ) Hoyes, K. 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