Effects of Oral Intake of Olive Leaf Extract on Hematological Parameters: A Double-Blinded, Randomized, Placebo-Controlled Trial in Progress
Authors: Ferdousi Farhana , Alam Masud , Araki Risa , Suidasari Sofya , Yokozawa Miki , Yamauchi Ken , Hashimoto Koichi , Haque Rashidul , Isoda Hiroko 
Published: November 2021

Abstract
Background:
Anemia is one of the major public health problems that affected 2.36 billion individuals worldwide in 2015. Half of all causes of anemia are iron or other nutrient deficiency anemia (WHO, 2015). Currently available treatment options for anemia include iron or other nutrient supplementation, nutritional interventions in the form of food fortification, blood transfusion, and erythropoietic agent infusion. However, conventional treatments are not without adverse effects. In this context, medicinal plants with bioactive components have been receiving considerable attention from researchers as natural hematinic agents complemented with or alternatives to conventional treatment.
Olive leaf (Olea europaea L. Oleaceae) extract (OLE) is rich in bioactive compounds and have been reported to have several health benefits (Gorzynik-Debicka et al., 2018). Previously we showed that apigetrin and apigenin, components of OLE, induced erythroid differentiation in K562 cells (Tsolmon et al., 2011 and Isoda et al., 2014). Also, OLE and its phenolic compounds induced erythroid differentiation in hHSCs (Kondo et al., 2021, Samet et al., 2015).
Additionally, we have reported that long-term consumption of OLE tea increased RBC count, Hb%, and hematocrit (Ht) in female volunteers aged between 40 - 70 years old (Ferdousi et al., 2019). However, none of the participants were anemic at the baseline. Therefore, in the present study, we aimed to investigate the effects of oral intake of OLE on hematological parameters in healthy to mildly anemic young adults.
Study Design and Methods
A double-blinded, randomized, placebo-controlled study will be conducted in Bangladesh. Total 165 apparently healthy adults with Hb level at least 10g/dl for females (nonpregnant, non-breastfeeding) and 11g/dl for males, and aged between 18 to 59 years will be enrolled from the local community. Major exclusion criteria will include currently pregnant women or breastfeeding mothers, moderate to severe anemia (Hb concentration &lt; 10 g / dL for women and &lt; 11 g / dL for men), any medical condition, such as hematological diseases, malignant tumors, aplastic anemia, uncontrolled hypertension, chronic kidney disease, irritable bowel syndrome, and cardiovascular diseases, and currently taking medicine for diabetes or hypertension and taking regular iron or other supplements, etc.
After providing informed written consent, study participants will be randomly assigned to placebo, 250 mg OLE and 500 mg OLE groups. Participants will take OLE or placebo capsules in two divided doses every day for 12 weeks. Hematological parameters, Hb concentration, RBC count, WBC count, Ht, MCV, MCH, MCHC, serum Fe and ferritin levels will be investigated at baseline and after 6 and 12 weeks of intervention. Liver and renal function tests will be conducted for safety assessment. Additionally, lipid profile will be investigated to understand how lipid parameters will be regulated if anemia condition is improved by OLE intervention (Araki et al., 2019). Given that fatigue is one of the most common symptoms of anemia, we will also evaluate the effect of OLE on the score of each item of the Fatigue Assessment Scale (FAS) questionnaire during the intervention.
The analysis will be carried out on an intention-to-treat basis involving all the participants who will consume the test samples at least once. We will replace the missing data from dropouts using the baseline observation carried forward method. ANOVA with Bonferroni's post hoc test will be used for normally distributed data; otherwise, the Mann-Whitney U test or Kruskal-Wallis H(K) test will be used. Categorical variables will be compared by the Chi-square or Fisher's exact test. A one-way repeated-measures ANOVA with Bonferroni's post hoc test (Within-group) and ANOVA followed by Bonferroni's post hoc test (between-group) will be performed. Significance is at p &lt; 0.05.
This protocol is registered with UMIN-CTR Clinical Trial (UMIN000039023), and has been approved by University of Tsukuba Hospital Clinical Research Ethics Review Committee (R01-292) and the Ethical Review Committee of icddr,b (PR-21017). This study is funded by the Japan Society for the Promotion of Science (19K20106) and partly by the Japan Science and Technology Agency-SATREPS (JPMJSA1506).

Disclosures
Suidasari: Nutrition Act Co. Ltd.: Current Employment. Yokozawa: Nutrition Act Co. Ltd.: Current Employment. Yamauchi: Nutrition Act Co. Ltd.: Current Employment.