The Haematological Profiles of High Fat Diet Mice Model with Moringa oleifera Leaves Ethanol Extract Treatment

1Study Program of Magister of Anti Aging and Aesthetic Medicine, Faculty of Medicine, Universitas Padjadjaran, Indonesia, 45363. 2Department of Biomedical Sciences, Faculty of Medicine, Universitas Padjadjaran, Indonesia, 45363. 3Central Laboratory, Universitas Padjadjaran, Indonesia, 45363. 4Study Program of Magister of Biotechnology, Postgraduate School, Universitas Padjadjaran, Indonesia, 40132. 5Medical Laboratory Technologist, Bakti Asih School of Analyst, Bandung, Indonesia, 40192. *Corresponding Author E-mail: n.atik@unpad.ac.id

Obesity is known as an imbalance between energy intake and energy expenditure. 1 The morphological hallmark of obesity is an increase in adipocyte mass and number, but the characteristics of obesity that need to be concerned are dysfunction of adipose tissue and chronic low-grade inflammation. 2 These conditions have negative impacts, such as alteration in iron metabolism, immune system, and platelet activity and further can change the haematological parameter.
People with obesity have a higher risk to undergo iron deficiency anaemia compare to lean people. 3 Anaemia is a decrease in the total amount of haemoglobin or the number of RBC. Iron deficiency anaemia is commonly known due to the lack of sufficient iron to form normal RBC, caused by inadequate intake of iron, chronic blood loss, or a combination of both. 4 In overweight/obesity, iron deficiency anaemia can be caused by dilutional hypoferremia, inadequate dietary iron intake, increased iron requirements, and/or impaired iron absorption. 3 The characteristic of obesity as a systemic low-grade inflammation makes iron deficiency is linked to inflammatory markers. Adipose-derived cytokines such as IL-6 and IL-1 act as potent inducers of hepcidin expression in adipose tissue. 2 This elevation can decrease iron absorption and impair the effectiveness of iron fortification 3,5 Adipose tissue is composed of mature adipocytes and stromal vascular fraction, including macrophages. Macrophages residing in adipose tissue are called adipose tissue macrophages (ATMs). 6,7 In lean phenotype, ATMs display the anti-inflammatory macrophage (M2). In obesity, there is a phenotype switch in macrophage from M2 to M1, a classical pro-inflammatory macrophage. 7,8 Many studies indicate that increased accumulation of ATMs is associated with obesityrelated conditions such as type 2 diabetes, cardiovascular disease, and fatty liver. 7 Obesity alters the lymphoid tissues, pro-inflammatory leucocyte distribution and population, and the amount of WBC, lymphocyte, neutrophil and monocyte. 9,10 The WBC count will increase when the inflammation and leucocyte activation occurred. 10 Moringa tree grows in Asia and Africa. It has 13 species, and the most often cultivated is Moringa oleifera (MO). 11 Traditionally, people use all parts of the tree for medication, but the leaves are useful for treating malaria, typhoid fever, joint pain, skin disease and to enhance breast milk. 12 The leaves are known rich of vitamin A, B1, B2, B3, carotenoid, polyphenol, phenolic acid, flavonoid, alkaloid, glucosinolates, isothiocyanates, tannin and saponin. 11,12 The leave part is rich of minerals such as calcium, potassium, zinc, magnesium, iron and copper. 13 It also contains iron 25 times higher than spinach. 13 The vitamin C and carotenoid contents in the leave part can enhance iron absorption. 14 Many studies were performed to explore MO leaves potencies for health conditions, such as hypercholesterolemia, hypertension, hyperglycaemia, type 2 diabetes, insulin resistance, non-alcoholic fatty liver disease, anticancer, and antiinflammation. 11 Some studies have shown the effect of MO leave on blood profiles. In animals experiment, MO leave is more effective as supplementation for iron deficiency anaemia than synthetic supplementation. 14 In anaemic rats, MO leaves ethanol extract (MOLE) could increase haemoglobin, RBC, haematocrit, and total iron concentration, and decrease WBC count. 15,16 A study involving women with iron deficiency anaemia showed that MOLE supplementation for three weeks increased haematocrit, Mean Corpuscular Haemoglobin(MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), and decreased platelet concentration significantly as compared to the control group. 17 Based on the background, this study aimed to investigate the haematological parameter of high fat diet mice in parallel with MOLE oral administration.

Material and Methods animals
Considering the Declaration of Helsinki for the welfare of animals used for research,the Research Ethics Committee Universitas Padjadjaran Bandung has approved the experimental animal protocol (Approval No. 107/UN6.KEP/EC/2019). Forty male DDY mice with 5 weeks of age were obtained from PT. Biofarma Bandung, Indonesia. Mice were acclimatized for 2 weeks in the Animal Laboratory Faculty of Medicine, Padjadjaran University, Bandung, Indonesia. Mice were placed in room controlled-temperature, 12 light and 12 dark cycle and had free access to diet and water.

extract and drug Preparation
MO leaf powder is produced by PT. Moringa Organik Indonesia, from fresh leaves. School of Life Sciences and Technology, Bandung Institute of Technology accomplished the identification of the plant. The leaves powder was extracted by maceration method with 95% ethanol. The ethanol extract was concentrated with freeze-dried, then kept in the refrigerator until used in the treatment. Two doses were selected from the previous study. 18 Simvastatin was purchased from PT.Kimia Farma Pharmacy, Indonesia, with dose as previously described. 18

high Fat diet and Mole treatment
Mice were divided into 5 groups (n= 8 mice per group). The SD group: mice were given standard diet as negative control group; HFD group: high fat diet treatment; HFD+S group: high fat diet+ simvastatin 0,8 mg/20gBW/day treatment as positive control group; HFD+MOLE1: high fat diet+MOLE 5,6 mg/20gBW/day treatment; and HFD+MOLE2 : high fat diet + MOLE 11,2 mg/20gBW/day treatment. Simvastatin and MOLE were given by intragastric tube every day. SD and HFD were given ad libitum and the treatment was performed for 7 weeks.

sample Collection
At the end of experimental day, mice were fasted overnight. Mice were euthanized after anesthetized, blood samples were collected via cardiac puncture and drawn into EDTA tubes (Becton Dickinson, New Jersey, USA). The fresh blood in the EDTA tubes was then processed for haematology examination by used haematologyanalyser (The Samsung LABGEO HC10, Suwon, Korea) to measure 18 whole blood indices.

statistical analysis
All of the data were submitted to the Saphiro-Wilk test of normality. Values are shown as Mean ± SD for variables that met the criteria of normality, and non-parametric variables are shown as median (min-max) values. Statistical analysis was performed using Graphpad Prism 7.00 program (GraphPad Software, Inc. San Diego, CA) results and disCussion haematological analysis for rBC andrelated parameters Red Blood Cells (RBC) and RBC-related parameters are presented in figure 1. The amount of RBC was tending to lower in the negative control group compared with the normal control group [5,805(4,12-9,07) vs 7,535(6,13-9,32)]. After treatment with MOLE dose 1, the amount of RBC was tend to increase as compared with the negative control group [8,265(5,57-11,61) vs 5,805 (4,12-9,07), p=0,6589].
The examination of RBC-related parameters, such as the concentration of haemoglobin (HB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) andred cell distribution width (RDWC), showed that there was no significant alteration found among groups in all RBC-related parameters.

the haematology analysis of WBC and related parameters
The amount of WBCs was tend to increase in negative control group as compared with normal control group [(3,59±1,974) vs (2,419±0,8047), p=0,5471]. This increase of WBC in treatment groups could be due to the reaction in bone marrow because of inflammation. 17 There were no significant differences among all groups (p=0,3856), indicating that MOLE did not affect the alteration of WBC count (Figure 2).

the haematology analysis of platelet and related parameters
We further examined the platelet count, plateletcrit (PCT), mean platelet volume (MPV) and platelet distribution width (PDWC). There was no significant difference found among groups in all parameters (Figure 3). Although there was a rise of platelet count with a higher dose of MOLE, but it did not show any significant differences among groups (p=0,3856), indicating that MOLE did not affect the platelet count. It was similar to findings of Adepapo et al 21 . However, we found that the MPV value tended to increase in the negative control group as compared with the control group [4,2(2,6-6,7) vs 3,5(3,2-4,2), p>0,9999]. MPV is elevated in some conditions, such as hypercholesterolemia, diabetes mellitus and hypertension, that also lifethreatening conditions for people with obesity. 22 MOLE might improve these conditions since we noticed that MPV values in the treatment group 1 and 2tended to decrease as compared with the negative control group [3,6(3,2-4,1) and 3,55(2,6-6,8) vs 4,2(2,6-6,7)].

ConClusion
In summary, the study showed that MOLE might have potencies to prevent the worsening of hematological parameters in the sub-chronic high fat dietby slight increased of haemoglobin, slight decreased of WBC, decreased the granulocyte percentage, and slight decreased of MPV.

aCknoWledgeMent
We would like to thank to Neni Anggraeni, Suhendra Praptama and Asep Saepuloh for excellent assistance.