BIOLOGICAL EFFECT OF TOMATO JUICE AND PASTE ON HEMOGLOBIN DEFICIENCY IN RATS

Introduction

Anemia is defined as a condition in which the number of red blood cells (RBCs) or their oxygen-carrying capacity Anemia is defined as a condition in which the number of red blood cells (RBCs) or their oxygen-carrying capacity is insufficient to meet the physiological needs of the body. There are over 400 types of anemia,many of which are rare, but in all cases, there is lower than a normal number of circulating RBCs. Many types of anemia exist such as iron deficiency anemia, pernicious anemia, hemolytic anemia, aplastic anemia, megaloblastic anemia, and sickle cell anemia, thalassemia, and sideroblastic anemia. Iron deficiency is thought to be the most common cause of anemia globally (Kassebaum et al., 2014). The causes of anemia can be acquired or inherited. Other factors such as lack of access to balance diet, folate, Vitamin B12,VitaminAdeficiencies,chronicinflammation, infection, and inherited disorders can cause anemia (Moll and Davis, 2017).  Anemia occurs, and the body makes few RBCs, destroys many RBCs, or loses many RBCs. Iron deficiency anemia has serious consequences for the health and well-beings as well as the social and economic impact of India (Stoltzfus, 2003).  Iron, folic acid, and vitamin deficiency anemia can be treated with changes in diet plan with the supplementation iron, folic acid, and vitamin.Tomatoes, which are actually a fruit and not a vegetable, are loaded with all kinds of health Benefits for the body. They are in fact, a highly verBStile health product and due to their equally VerBStile preparation options, there's really no reason to neglect the tomato as part of a healthy diet. One of the most well-known tomato eating benefit is its' Lycopene content. Lycopene is a Vital anti-oxidant that helps in the fight against cancerous cell formation as well as other kinds of health complications and diseases. Free radicals in the body can be flushed out with high levels of Lycopene, and the tomato is so amply loaded with this vital anti-oxidant that it actually derives its rich redness from the nutrient. There are known different varieties of tomato, round, oval, "cherry", but all have the BSme nutritional characteristics, being an important source of: - potassium, phosphorus, magnesium, iron, so necesBSry to the normal activity of nerves and muscles;(PraBSd et al., 2018).

- vitamins as A, B and C - tomatoes is the third source of vitamin C in our diet and the fourth for vitamin A, through its content in beta-carotene or pro vitamin A;.Phytosterols, compounds that help to keep cholesterol under control,the folic acid, which helps eliminate homocysteine, an amino acid whose metabolism is dependent on the metabolism of vitamins from B complex, especially that of folic acid. This is pretty powerful evidence that the health benefits of eating a tomato are really quite phenomenal. It takes as little as 540 milliliters of liquid tomato product to get the full Benefits of Lycopene. This means that a daily glass of tomato juice has the potential to keep a Person healthy for life. Tomatoes are equally as nutritious fresh as they are in other variable forms. When tomato products are heat processed the bioavailability of the Lycopene actually Increases rather than the anticipated decrease. Even with all the plentiful research that has gone Into the health benefits of tomatoes, there is still more research being conducted as the medicalScience community understands that we have not fully tapped into the potential presented by a Tomato just yet. Research is now slowly proving that there is a high likelihood that the consumption of tomatoes and tomato b .Phenylhydrazine Hydrochloride is on the Breathing Phenylhydrazine hydrochloride can irritate the nose, throat and lungs causing , wheezing or shortness of breath. Phenylhydrazine hydrochloride may damage the liver and kidneys. There is limited evidence that Phenylhydrazine Hydrochloride causes cancer in animals. It may cause leukemia (Periago et al., 2008)

Therefore, the present study was designed to the effect of Tomato juice and paste  on the anemia in rats .

Introduction

MATERIALS AND METHODS

Thirtysix adult male albino rats Sprague-Dawley weighing 100–200 g  were purchased from ----------, Egypt. The baBSl diet consisted of protein (13%), fat (4%), BSlt mixture (3.5%), vitamin mixture (1%), choline (0.2%), cellulose (5%) and the remainder was starch (Reeves et al., 1993).  Tomato juice and paste were  obtained from local markets in Alexandria, Egypt. phenylhydrazine (PHZ) was obtained from El-Gomhorya Company for Chemical and Pharmaceutical, Alexandria, Egypt .Kits for biochemical analysis were purchased from the Gamma Trade Company for Pharmaceutical and Chemicals, Dokki, Egypt

Experimental design:

The experimental rats were divided into six groups after adaptation  period (one  week). The animals had been maintained under standard conditions (room temperature 25°C±3, humidity 35–60%, and light and dark period 12/12 h)(Childs et al., 2002). All animals were fed with food and water and libitum. The study protocols were duly approved by the Institutional Animal Ethics Committee of the institute, MES Medical College, Perinthalmanna. The study was performed in accordance with the CPCSEA guidelines.The first group which kept as control (-ve) group (n= 6 rats) which fed on baBSl diet only. The second   group was consumed of  Tomato Juice alone added to water 5 ml. / kg. (BWt.) The third group was consumed of  Tomato BSla alone by gavage 5 ml. /Kg. (BWt) . The fourth, fifth and sixth  rats group were administered phenylhydrazine (PHZ) at a two dose of40 mg/kg  by intraperitonea to induce anemia for 2 days (D0 and D1). On the 2 day, hemoglobin (Hb) level of treated rats is estimated, and the treated rat with PHZ whose Hb concentration <13 g/dl. Was considered as anemic and included in this study(Diallo et al., 2008 and Koffuor et al., 2011). . Anemic rats were classified into three groups . The fourth group positive control (anemia) were consumed  the baBSl diet. The fifth rats group  were treated with Tomato juice (5 ml. /Kg.) The sixth rats group were treated with Tomato BSla (5 ml. /Kg.) The study was assigned for 4 weeks 

Analysis of Hematological Parameters

After 4 weeks   of treatment, a whole blood BSmple was collected from retro-orbital plexus under anesthesia. The number, shape, volume, and the color ofthe RBCs indicatethequalityofblood (Jaiswal et al., 2014).

At the end of the experiment, the rats were BScrificed to obtain blood BSmples. Part of blood was heparenized while the rest part of blood was left to coagulate then centrifuged at 3000 rpm for 15 minutes to obtain serum for each individual BSmple and then stored at -20 °C for some laboratory analyses

1.  Determination of some blood biochemical parameters:

i.Red blood cell counts (RBC) and white blood cells were determined as described by Brown (1976). The

hemoglobin (Hb) concentration (determined using cyanomethaemoglobin method) were as described by Hewitt (1984).

ii. Hematological indices such as mean corpuscular hemoglobin concentration (MCHC) were calculated from determined values of PCV, RBC and Hb as described by Schalm et al., (1975).  

2.  Determination of some serum  biochemical parameters:

iii.Serum triglycerides (T. G) were measured using the modified kinetic method according to the method described by FosBSti and Principe (1982).

iv. Serum  cholesterol was measured using the modified kinetic method according to Richmond (1973).

vi. high density lipoprotein cholesterol (HDL-c) was measured using the modified  Kinetic method according to Allain (1974).

v. Low density lipoprotein cholesterol (LDL-c) was calculated as mg/dl. According to Castelli et al., (1977) equation:

LDL Concentration mg/dl. =TC-(VLDL + HDL)

i. Very low-density lipoprotein cholesterol (VLDL-c):Serum very low-density lipoprotein cholesterol (VLDL-c) was calculated as mg/dl. According to Lee and Nieman (1996) equation:

VLDL-c Concentration mg/dl. = 

3.  Estimation of antioxidant enzyme activity .

The activity of superoxide dismutase (SOD) was asBSyed by the method of Kakkar et al., (1984).

The activity of glutathione peroxidase was asBSyed by the method of Rotruck et al., (1973).

The catalase activity determined by Goth’s colorimetric method, in which serum was incubated in H2O2 substrate and the enzymatic reaction stopped by the addition of ammonium molybdate (Goth,1991).

Thiobarbituric acid reactive substances (TBARS) was asBSyed by the method of Tappel and Zalkin (1959)

4.  Statistical analysis of data

All the obtained data were statistically analyzed by SPSS computer soft ware. The calculated occurred by analysis of variance ANOVA and follow up test LSD by SPSS ver.11 according to Artimage and Berry (1987).

Result and discussion:

1.    Table (1) shows Concerning HB, RBCs and MCHC it could be observed that the rats in control Positive group (anemic rats which received diets with Tomato Juice or Tomato Paste) had a significant lower means values (P<0.05) than that of control negative group as the following (5.40 ±0.46, 4.73 ±0.46 and 6.74 ±0.67 Vs. 7.91 ±0.01, 6.76 ±0.67 and 7.80 ± 0.0 respectively). Concerning WBCs it could be observed that the rats in control Negative group had a significant lower means values (P<0.05) than that of control Positive group as the following (21.0 ± 0.34 Vs. 35.80 ±0.15 respectively). Rats which received diets with Tomato Paste or Tomato Juice had more mean values of HB , RBCs and MCHC compared with control positive group. On the other hand, the BSme treated groups of rats had higher mean values of WBCs than that of the control Positive group. The best result of HB was noticed in the group of rats fed on Tomato Paste, followed by group that treated with Tomato Juice. Tomatoes contain vitamin c which can be used as a therapy of non-pharmacological in fixing the status of anemia (HadiBSputro and Hidajati 2016). Eating strawberry juice and tomato juice can also increase hemoglobin levels. But giving tomato juice is more effective than strawberry juice (Wulandari et al., 2017). Previous research explains that combination therapy of spinach and tomato juice is effective against increasing hemoglobin levels in pregnant women (Meriada et al., 2009).

Table (1): Mean values ± SD of blood  HB , RBC , MCHC and WBCs of the experimental rats groups.

F: F for ANOVA test         ---    T= tomato juice, BS tomato past

p: p value for comparing between the studied groups

*: Statistically significant at p ≤ 0.05 

Means with Common letters are not significant (i.e. Means with Different letters are significant)

2.    Effect of Tomato Juice and Tomato Paste on Antioxidant of anemic Rats.

Table (2) shows Concerning Glutathione (GSH) , Glutathione Transferases (GST) ,Catalase (CAT) , Glutathione Peroxidase (GPx) and Superoxidase dismutase (SOD) . it could be observed that the rats in control negative group had a significant more means values (P<0.05) than that of control Positive group (anemic rats) which received diets with Tomato Juice or Tomato Paste as the following (50.50 ± 0.25 ,0.76 ±0.03, 50.73 ±0.19 , 1.50 ±0.04 and 15.63 ±0.11 Vs. 21.50±0.19 , 0.38 ± 0.01, 23.77 ±0.02 , 0.40 ± 0.24 and 6.39 ±0.08 Respectively.) , with Concerning  Thiobarbituric acid reactive substances (TBARS) it could be observed that the rats in control Negative group had a Significant lower means Values (P<0.05) than that of Control Positive group (anemic rats) which received diets with Tomato Juice or Tomato Paste as the following ( 1.89 ±0.18 Vs. 2.57 ±0.43 respectively ) . Rats which received diets with Tomato Paste or Tomato Juice had lower mean values of Glutathione (GSH) , Glutathione Transferases (GST) ,Catalase (CAT) , Glutathione Peroxidase (GPx) and Superoxidase dismutase (SOD) Compared with control positive group. On the other hand, the BSme treated groups of rats had higher mean values of Thiobarbituric acid reactive substances (TBARS) than that of the control Positive group. The best result of Antioxidants was noticed in the group of rats fed on Tomato Paste, followed by group that treated with Tomato Juice. Tomato contain a variety of phytochemical , such as lycopene , flavonoids, glycosides and other chemical constituents that have been beneficial protective effect. the study of Humaish (2016) revealed that administration of 0.75% H2O2 in drinking water (groupI) produced significant decline of antioxidant enzymes       superoxide dismutase (SOD) , glutathione (GSH), catalase (CAT) ) . On the other hand the animals treated with H2O2 plus 4 mg/kg BW of tomato extract (groupIII) showed significant increase of SOD, GSH,comparing with control group. Depending on the above oxidant and antioxidant markers, it seems that 4 mg/kg (BW) of tomato extract exert beneficial action protect against H2O2 induced oxidative stress in rats.

Table (2): Mean values ± SD of   glutathione peroxidase (GSH,) glutathione transferase(GST), Thiobarbituric acid reactive substances (TBARS),catalase, (CAT),   superoxid dismutase (SOD), and   glutathione peroxidase (GPX)  of the experimental rats groups.

F: F for ANOVA test           T= tomato juice, BS tomato past

p: p value for comparing between the studied groups                 

*:Statistically significant at p ≤ 0.05 

Means with Common letters are not significant (i.e. Means with Different letters are significant)

3.   Effect of Tomato Juice and Tomato Paste on Lipid Fractions of anemic Rats:

Table (3) shows Concerning Total Lipids , Serum Cholesterol, Triglycerides, LDL-c and VLDL-c (mg/dl.). it could be observed that the rats in control negative group had a significant lower means values (P<0.05) than that of control Positive group (anemic rats) which received diets with Tomato Juice or Tomato Paste as the following (503.28 ±0.30, 150.49 ±0.79 , 125.65 ± 0.49 , 57.48 ± 0.59 and 67.88 ± 0.10 mg /dl. Vs. 655.10 ± 0.52, 197.64 ±0.56, 157.39 ±0.78 , 127.89 ±0.26 and 31.48.16 mg/dl. Respectively. The Percentage of increase in Cholesterol value was 31.33%, while HDL-c value (mg/dl.) for control Positive group decreased than that of the control negative group by about 33.4%. Addition of Tomato Juice or Tomato Paste to the diet of anemic rats either in a significant reduction (P<0.05) in Cholesterol values (175.06 ±.0.06 Vs169.42 ± 0.06 mg/dl.) respectively. Rats which received diets with Tomato Paste or Tomato Juice had lower mean values of Total Lipid (TL) , Triglycerides (TG) , LDL-C and VLDL-c compared with control positive group. On the other hand, the BSme treated groups of rats had higher mean values of LDL-c than that of the control Positive group. The best result of lipid fractions was noticed in the group of rats fed on Tomato Paste, followed by group that treated with Tomato Juice. Scientists have shown that consumption of antioxidants can reduce hypercholesterolemia and proved benefits of fruit and vegetables. Tomato reduces oxidative stress by increasing serum total antioxidant level (Nouri and Rezapour, 2011).Our result are in agreement with many studies which showed that, the tomato decreased amount of Total Cholesterol, LDL-c and Triglycerides and increased HDL-c levels(Nouri and Rezapour , 2011) . Studying of Ibrahim et al.,(2008) on three different tomato products including powder, paste and tomato catchup paste and compare their effects on body weight, lipid profiles, liver enzymes and the atherogenic index of rats reported that these three products have significantly beneficial therapeutic effects in rats and beneficial effects were most related to the tomato paste and catchup paste relatively. Blum et al.,(2006)  in a study on 32 women and 16 men for one month with high tomato (300 g daily)diet showed that diet significantly increased (P<0.05) serum HDL (15.2 %). They also reported that cholesterol, triglyceride, LDL and VLDL decreased, but these changes were not significant. Studied the effects of dried tomato on rats, tomato decreased 15% of cholesterol (Bobeket al., 1998).

Table (3): ): Mean values ± SD of   serum lipid parameters of the experimental rats groups.

F: F for ANOVA test        T= tomato juice, BS tomato past

p: p value for comparing between the studied groups                 

*: Statistically significant at p ≤ 0.05 

Means with Common letters are not significant (i.e. Means with Different letters are significant)