Ameliorative effect of Hesperidin on Hematological changes in Methionine induced Hyperhomocysteinemia in Rats

Presentation By Anjali.CH, Saisantosh.CH, Saisree.D, Ramesh.D, Amrutha.D

Cardiovascular disease is the most common cause of death in industrialized countries such as the

US, and is on the rise in developing countries too.

The National Heart, Lung, and Blood Institute of the National Institutes of Health has identified

many risk factors for cardiovascular disease, including an elevated LDL-cholesterol level, high

blood pressure, a low HDL-cholesterol level, obesity and diabetes.

In recent years, researchers have identified another risk factor for cardiovascular disease, a

elevated homocysteine level.

Homocysteine is an amino acid normally found in blood, but elevated levels have been linked with

coronary heart disease and stroke.

Introduction

Elevated homocysteine levels may impair endothelial vasomotor function, which determines

how easily blood flows through blood vessels.

Hyperhomocysteinemia induces vascular damage because of the highly reactive thiol group in

homocysteine that it is readily oxidized leading to the formation of homocysteine.

Homocysteine mixed disulfides and homocysteine thiolactone.

During these oxidative processes, several reactive species are generated.

The oxidation of homocysteine promotes oxidation of low density lipoprotein cholesterol,

which causes injury to vascular endothelial cells and leads to endothelial dysfunction.

Methionine is the only dietary source of homocysteine, which disrupts and interfere

endothelial integrity.

Flavonoids, which are polyphenolic compounds, can directly quench free radicals and inhibit enzymes of

oxygen reduction pathways.

Hesperidin, a bioflavonoid richly found in oranges and lemons, has been reported to be an effective

supplement in the treatment of several neurodegenerative diseases.

Above all, hesperidin has been reported to contribute to the intracellular antioxidant defense systems as a

powerful agent against superoxide, singlet oxygen, and hydroxyl radicals.

But there are no reports on hesperidin role on hematological changes induced by Hyperhomocysteinemia

1.HYPERHOMOCYSTEINEMIA

Hyperhomocysteinemia is associated with cardiovascular, renal, and

neurodegenerative diseases .

Total plasma Hcy values of approximately 10 μmol/L for men and 8

μmol/L for women constitute the normal range .

However, even a small increase in total plasma Hcy is associated

with an increased risk of coronary artery disease both for men and

women .

Moreover, a number of studies have demonstrated that smoking, ex-

cessive alcohol consumption/alcohol abuse, obesity, type II diabetes,

and an unhealthydiet contribute to mild Hyperhomocysteinemia.

RISKS OF HYPERHOMOCYSTEINEMIA

In patients with profound Hyperhomocysteinemia leading to homo-cystinuria.

Skeletal and ocular (eye) problems .

Mental retardation/developmental delay.

Thrombotic complications.

TREATMENT OF HYPERHOMOCYSTEINEMIA

Unlike many other thrombophilic disorders, hyperhomocysteinemia can be treated directly with vitamin supplementation.

The primary vitamin used to lower homocysteine levels is folate and studies have clearly shown a decrease in homocysteine levels with fo-late.

Taking folate and vitamin B12 can lead to an additional decrease in homocysteine levels above that seen with folate alone

HESPERIDIN

Hesperidin is a flavanone glycoside named after the term 'Hesper-idium', referring to citrus fruits which are the main source of hesperidin.

Hesperidin has antioxidant, anti-inflammatory, hypolipidemic, vasoprotective and anticarcinogenic and cholesterol lowering actions.

Hesperidin can inhibit following enzymes: phospholipase A2, lipoxy-genase, HMG-CoA reductase and cyclo-oxygenase. Hesperidin im-proves the health of capillaries by reducing the capillary permeabil-ity.

MATERIALS AND METHODS

This study was approved by Lalitha college of pharmacy, Anurag group of Institution's, Hyderabad, Telangana. Animals were pro-duced from Teena labs, kukatpally, Hyderabad, which is re-gistered with committee for the purpose of control and supervision of experiments on animals(CPCSEA).

Albina rats of either sex(150-200gms) produced from the central an-imal house facility in the college and acclimatized under standard laboratory conditions at 25 ±2°c, 50±15RH and normal pho-toperiod for 7days, were used for the experiment.

Approval was taken from Institutional Animal Ethical Committee in School of Pharmacy, Anurag group of Institutions, Ghatkesar, Hy-derabad, prior to the experiments

Drugs and chemicalsHesperidin was a purchased from Otto chemicals Hyderabad. L-Methion-ine, RBC diluting fluid, WBC diluting fluid and Cedar wood oil were pur-chased from SD-fine chemicals. All other chemicals used were of analytical grade.

Experimental protocolThe animals were weighed and divided into three groups of five animals each.

Group I: ControlThe healthy rats were selected and treated as control and they received normal saline orally.

Group II: L-MethionineThe healthy rats were selected and treated with L-Methionine(1gm/kg/day) for 30 days orally.

Group III: HesperidinThe healthy rats were selected and treated with Hesperidin (25mg/kg/day) for 30 days orally.

Sample CollectionAt the end of the 30th day, blood samples were collected into hep-arinized tubes by cardiac puncture from all animals, under light ether anesthesia.

Hematological AssaysHemoglobin ,Complete Blood Count (CBC) includes packed cell volume (PCV), red blood cell (RBC) count, white blood cell (WBC) count were performed according to manual visual methods and differential count was measured using an automatic hematological assay ana-lyzer, Advia 60® Hematology system (Bayer Diagnostics Europe Ltd, Ireland) according to manufacturer’s instructions at Likitha Di-agnostic labs Hyderabad.

  

1. Estimation of HaemoglobinThe haemoglobin concentration of blood was estimated by the method us-ing Shali’s haemoglobinometer (Samuel, 1986). The level of the fluid in the haemoglobin tube was read and expressed in gram percentage.

2. Red Blood Cell (RBC) Count

Erythrocytes / Red Blood Cells were counted by the method of Samuel (1986) using haemocytometer. The method involves an accurate dilu-tion of a measured quantity of blood with a fluid which was isotonic with blood and which will prevent its coagulation. A dilution of 1:2000 was usually necessary. The dilute blood was placed in a Neubauer counting chamber and the number of cells in a circumscribed volume was enumerated under a microscope.

3.WHITE BLOOD CELL (WBC) COUNTLeukocytes / White Blood Cells were counted by the method of Samuel, (1986) using haemocytometer. Leukocytes were less numerous, a dilu-tion of 1 in 20 was used and the diluent was usually the one which destroys the red blood corpuscles. The cells in the four corner blocks were counted.Number of cells counted x Blood dilution x Chamber depthNumber of leukocytes = Area of chamber countedDilution : 20Area counted : 4sq.mmDepth : 0.1mm

4.Differential CountDifferential count is to estimate the percentage distribution of white blood corpuscles in a blood smear (Merck Veterinary Manual, 1979).

5. Packed Cell Volume (PCV) (Merck Veterinary Manual, 1979)The packed cell volume of blood is obtained by centrifuging a tube of blood and measuring the percentage of packed red blood cells (RBC’s) in the total sample.  

Statistical Analysis: Data were expressed as mean ±SEM and subjected to one-way analysis of variance (Annova) followed by Tukey’s multiple comparison test using Graph Pad Prism version 5.0. The P values of <0.05 were considered statistically significant for differences in mean using the least of signif-icance difference.

The mean platelets counts, packed cell volume were significantly increased with significant decrease in blood haemoglobin levels and RBC in me-thionine-treated group as compared to the control . While methionine treatment caused a significant change in total WBC count, Eosino-phil, monocyte and neutrophil counts as compared to the control rats. Fur-ther, treatment with Hesperidin to methionine-treated rats significantly de-creased the elevated platelets, PCV, Total WBC, Eosinophil, Monocyte and Neutrophil counts as compared to the L-Methionine treated rats and significant changes were also observed in RBC count and blood haemo-globin levels

Fig- 8 Effect of Hesperidin on Hemoglobin levels

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Fig-9 Effect of Hesperidin on RBC count

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Fig-10 Effect of Hesperidin on PCV

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Fig-11 Effect of Hesperidin on Platelet count

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Fig- 8 Effect of Hesperidin on Hemoglobin levels

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Fig-9 Effect of Hesperidin on RBC count

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Fig-10 Effect of Hesperidin on PCV

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Fig-11 Effect of Hesperidin on Platelet count

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Fig-15 Effect of Hesperidin on monocyte count

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CONCLUSION

Recent epidemiological data have shown that hyperhomocysteine-

mia can be detected in 20 and 40% of patients with coronary artery

disease and cerebrovascular disease, respectively.

Numerous studies have demonstrated that hyperhomocysteinemia

produces endothelial damage and dysfunction increasing the

risk of thermogenesis and thrombogenesis through oxidative

mechanisms.

An imbalance in dietary methionine may contribute to the develop-

ment of atherosclerosis by increasing homocysteine levels.

Methionine administration for a period of 30 days induced a

hematological changes in rats (P<0.01)

The mean platelets counts, packed cell volume were significantly in-creased with significant decrease in blood haemoglobin levels and RBC in methionine-treated group as compared to the control.

While methionine treatment caused a significant change in total WBC count, Eosinophil, monocyte and neutrophil counts as compared to the control rats.

Further, treatment with Hesperidin to methionine-treated rats signific-antly decreased the elevated platelets, PCV, Total WBC, Eosinophil, Monocyte and Neutrophil counts as compared to the L-Methionine treated rats and significant changes were also observed in RBC count and blood haemoglobin levels.

Our study results demonstrate that oral treatment with Hesperidin at 25 mg /kg /day, p.o. reversed many of hematological changes induced by methionine.

Hesperidin may, thus, be a new treatment option as a plant derived supplement along with other drugs as an adjuvant for patients with hyperhomocysteinemia, as homocysteine emerges as a risk factor for cardiovascular diseases.

But still further study is required to know the proper mechanism be-hind the effects of Hesperidin as our study is preliminary and ba-sic one.

The reversal of hematological changes by Hesperidin may be due to its potent antioxidant property.

 

Thank You

Presentation By Anjali.CH, Saisantosh.CH, Saisree.D, Ramesh.D, Amrutha.D