Catha edulis Forsk and Ascorbic Acid Effects on Hematological Indices in Rat

Introduction

Catha Edulis Forsk (CEF) is one of the stimulants chewed by the people of Ethiopia.¹ It is extensively chewed regardless of its adverse effects. Anemia, cancer, schizophrenia, anxiety, depression, diabetes mellitus, and inflammation are more common among CEF chewers.² Hematological changes are observed in these diseases.³ It also affects the therapeutic effects of drugs.^4,5 Certain disease conditions are aggravated by CEF.^6,7 Cathinone, Cathine, tannins, ascorbic acid (AA), and electrolytes are some of the composites in it.^8,9 The compounds found in this psychostimulant are expected to affect hematological indices.

Its subacute effects on hematologic indices have been evaluated in an animal model. Most of the findings are controversial and incomplete.¹⁰ AA has been taken as a protective agent against different diseases effects.^11–14 The purpose of taking AA is to regulate hematological and other changes in disease conditions. Acute administration of AA reduces oxidative stress and increases blood cell counts.¹⁵ Yet, no studies have been conducted on subchronic hematological effects of CEF and AA.

Investigate the subchronic effects of CEF and AA on hematological indices in rats is the aim of the current study.

Materials and Methods

Diethyl ether, chloroform (Sigma-Aldrich, Germany), Tween 80, AA, and 70% ethanol were chemicals used in this study. These chemicals were purchased from local suppliers in Addis Ababa, Ethiopia.

Plant Materials Collection

Bundles of fresh CEF leaves (9kg) were collected from Aweday, Eastern Ethiopia. The plant specimen was identified and a voucher number (October 16, 2018, AA002) was given. The leaves have been deposited at the National Herbarium of Ethiopia, Addis Ababa University.

Plant Material Extraction

After the edible parts of the leaves were separated and washed with tap water, the leaves were freeze-dried at −20^OC¹⁶ for 2 days and crushed using mortar and pestle. Two hundred grams of freeze-dried crushed leaves were placed into a conical flask wrapped with aluminum foil. A total of 400 mL organic solvents, ie, 300 mL diethyl ether and 100 mL chloroform (3:1v/v ratio) were added into the flask. The mixture was shaken under dark conditions for 48hrs using a rotary shaker (New Brunswick Scientific Co, USA) at 120 rpm and 20^OC. It was then filtered initially using cotton gauze followed with grade I Whatman filter paper (Cat No 1001 150). The organic solvents were then removed through evaporation using Rota-vapor under a controlled temperature of 36°C, rotation of 120 rev/min, and 240 Pascal negative pressure. The water in the extract was removed through lyophilization and the dry residue was weighed using an analytical balance and stored in a desiccator till used. The CEF juice (ChJ) was prepared from 12g/kg b.w of fresh leaves using 2% tween 80 in distilled water (v/v). In this study, 12 grams of fresh CEF leaves for a rat weighing 1 kilogram was taken as a selected dose. The leaf extract for each rat was calculated according to the weight of each rat (calculated dose = Weight of the rat (g) * the selected dose (g)/1000 (g)). The fresh leaves with tween 80 in distilled water (T80W) were crushed using a blender machine. The juice was then squeezed and filtered using the gauze and grade I Whatman filter paper. The T80W used to extract the given weight of the leaves was determined based on the total weight of each rat and vehicle volume used (2.5 mL/kg b.w).

Animal

A total of 36 adult wild-type male white albino rats aged between 7 and 8 weeks were used. Their weight was ranged between 213 and 229g. They were purchased from Laboratory Animal Breeding Section of the Ethiopian Public Health Institution. Three rats per plastic cage under natural light and dark (12:12hrs) cycles at room temperature were housed. Pellet and water were available ad libitum throughout the experimental period. Rats were weighed twice a week to ensure appropriate dosing based on body weight changes. Department of Medical Physiology and Institutional Review Board (IRB) Committee of the College of Health Sciences approved the study. Rules in animal care and use¹⁷ have been used during animal handling.

Grouping and Dosing

Rats were randomly assigned into six groups (n= 6/group) and received T80W, CEF extract (Ce) 100 milligram/kilogram, 200 milligram/kilogram and 300 milligrams/kilogram, AA (200 milligram/kilogram), and CEF juice (ChJ 2.5 mL/kg). They were administered for twelve weeks. T80W was used as a vehicle and the quantity for CEF extract and AA were selected based on previous reports.^18,19

Test Substances and Volume Determination

Fresh CEF extract, AA, ChJ, and vehicle were prepared every day. CEF extract was dissolved in T80W. AA was powdered and dissolved in T80W to make a stock solution of 80 mg/mL. Dose of the extract administered in each rat was calculated based on the total body weight (b.w) of each rat. Appropriate standard vehicle volume (2.5 mL/kg b.w) was used to determine how much volume was used to dissolve the calculated dose of CEF extract and AA. Each rat in its respective group received a single daily oral vehicle, CEF extract, ChJ, and AA. 1 mL was the final volume and all these were administered orally.

Blood Collection

The procedure used by Ketema et al²⁰ was used during blood withdrawal. Three milliliters of whole blood were collected from each rat through cardiac puncture after they were anesthetized using sodium pentobarbital (1mL/kg b.w). Blood was collected at 9:am and put into a tube containing ethylenediaminetetraacetic acid (EDTA) 24 hours after the last administration of test substances. Total leukocytes, Neutrocytes (NEUT), lymphocytes (LYMPH), monocytes (MONO), Eosinophils (EO), basophils (BASO), Hemoglobin (Hg), Hematocrit (HCT), Red blood cell size (RBCs), and red blood cell size variability (RsV) were analyzed by CBC machine (Sysmex KX-21). Hemoglobin concentration per red blood cell (HgpRBC), Hemoglobin concentration per volume of red blood cells (HgpvRBCs), platelet, Platelet size variability (PsV), plateletcrit (PCT), and platelet-large cell ratio (P-LCR) were also analyzed. NEUT to LYMPH ratio (NLR) was determined from quantified cells.

Data Analysis

The statistical analysis was done using SPSS version 21.0 and graphs were plotted using Microsoft Excel. Mean ±S.E.M have been used to express the values. Differences in hematological indices between three and more groups were analyzed using one-way ANOVA followed by Tukey’s post hoc analysis. An independent t-test was also used in this study to compare values obtained from only two groups such as the test versus control groups.

Results

Significant differences in total leukocyte count (F(_{5, 30)} =1.61, p > 0.05), MONO (F (_{5, 30)} = 1.37, p > 0.05), EO (F (_{5, 30)} = 1.64, p > 0.05) and BASO (f (_{5, 30)} = 1.39, p > 0.05) were not observed between groups. Significant difference in NEUT (F (_{5, 30)} = 6.42, P < 0.001), LYMPH (F (_{5, 30)} = 3.27, p < 0.05) and NLR (F (_{5, 30)} = 6.97, p < 0.001) was observed between groups.

NEUT and NLR in rats receiving CEF extract were significantly higher when compared with the T80W (t _(26.07) =−3.72, p < 0.01, 95% CI [−11.69,-3.37] and t _(24.72) = −3.22, p < 0.01, 95% CI [−20.28,-4.44]) and AA (t _(7.44) = −2.34, p < 0.05; 95% CI [−9.25,-0.11] and t _(27.99) = −2.06, p < 0.05; 95% CI [−17.06,-0.06], respectively) as shown in Figure 1.

Figure 1 Effects of Catha edulis Forsk on HgpRBC, NEUT, and NLR in Rats. Each bar represents the mean ± SEM of these indices in rats received AA, T80W, and khat. **Statistical difference at p < 0.01 and *Statistical difference at p < 0.05 when rats received CEF, AA and T80W were compared to each other. Abbreviations: CEF, Catha edulis Forsk; AA, ascorbic acid; HgpRBC, hemoglobin concentration per red blood cell; NEUT, neutrocyte; NLR, neutrocyte to lymphocyte ratio.

Rats received Ce100 milligram/kilogram had significantly higher NEUT (p < 0.001, 95% CI [7.09, 29.21]) and NLR (p < 0.001, 95% CI [13.46, 54.49]) compared with rats received T80W (Table 1). NEUT and NLR in rats received Ce100 milligram/kilogram were also significantly higher when compared with rats received AA (p < 0.01, 95% CI [4.25, 26.37] and p < 0.01, 95% CI [9.65, 50.69], respectively). However, LYMP was significantly less in this group of rats when compared with rats received T80W (p < 0.05, 95% CI [−31.12,-0.89]) (Table 1).

Table 1 Effect of Catha edulis Forsk and Ascorbic Acid on Hematological Indices

Significant differences in RBC count (F _{(5, 30)} = 2.75, p < 0.05), HGB (F _{(5, 30)} = 3.69, p-value less than 0.05), HCT (F _{(5, 30)} = 3.60, p < 0.05) and RBCS (F _{(5, 30)} = 4.07, P value less than 0.01) were observed between groups. Post hoc analysis results indicated no significant differences in RBC count, Hg concentration, and HCT (Table 1).

HgpRBC in rats received the Ce was significantly less than in those received T80W (t _(24.27) = 3.57, p < 0.05; 95% CI [0.41, 1.52]) as shown in Figure 1. Rats received Ce 100 milligram/kilogram (p < 0.05, 95% CI [0.07, 11.63]), Ce 200 milligram/kilogram (p value less than 0.05, 95% CI [−11.97,-0.41]) and Ce 300 milligram/kilogram (p < 0.05, 95% CI [−12.73,-1.17]) had significantly less RBCS than received AA 200 milligram/kilogram. RsV in rats received Ce300 milligram/kilogram (p < 0.05, 95% CI [0.01, 4.57]) and AA (p < 0.05, 95% CI [0.10, 4.66]) was significantly higher when compared with rats received T80W (Table 1).

Significant differences were observed in aPs (F _{(5, 30)} = 3.82, p < 0.01), PCT (F (_{5, 30)} = 5.51, P-value less than 0.01), PsV(F (_{5, 30)} = 7.14, p < 0.001) and P-LCR (F (_{5, 30)} = 8.95, p < 0.001) between groups. But, no significant difference in platelet count F _{(5, 30)} =0.79, p > 0.05) among the groups was observed. PCT was higher in rats received Ce100 milligram/kilogram and Ce300 milligram/kilogram compared to rats received T80W (P-value less than 0.05, 95 CI [0.05, 0.57] and p < 0.05, 95% CI [0.05, 0.57], respectively). PCT in these groups was also significantly higher when compared to rats received AA (p < 0.05, 95% CI [0.01, 0.53] and p < 0.05, 95% CI [0.01, 0.53], respectively). PsV in rats received Ce300 milligram/kilogram was significantly greater than in received T80W (p < 0 0.01, 95% CI [0.31, 2.80]). Rats received Ce100 milligram/kilogram (p < 0.01, 95% CI [−9.43, −1.57]), Ce200 milligram/kilogram (p-value less than 0.001, 95% CI [−10.23, −2.37]) and Ce300 milligram/kilogram (p < 0.01, 95% CI [−9.25, −1.39]) had significantly less P-LCR than rats received ascorbic acid. As shown in the table, rats receiving ChJ 2.5 mL/kg had higher P-LCR than in rats received T80W (p- vale was less 0.05, 95% CI [0.60, 8.46].

Discussion

In this study, total leukocytes, monocytes, basophils, and eosinophils were not affected by crude CEF extract and juice (Table 1). Alele et al²¹ also revealed that total leukocyte was not affected by this extract. However, Bin-Jaliah et al¹⁰ and Ketema et al²⁰ revealed that total leukocyte was reduced by CEF extract indicating that this extract suppressed immunity. Disagreement between these findings might be because of variation in extraction solvent, duration of administration, and animal species used to see the effects.

Even though the statistical difference in the total leukocytes did not observe between rats receiving the vehicle and AA in this study, previous studies indicated that white cell count was significantly increased by vitamin C.^19,22 In the previous study, Vitamin C (30 milligrams/kilogram and 70 milligrams/kilogram) was given for four weeks twice per day. However, it was administered (200 milligrams/kilogram) for twelve weeks per day in the current evaluation. Discrepancy between these findings might be attributed to the dose and duration of administration. These findings showed that vitamin C administered for a short period with a minimum dose increases white blood cells. In our study, neutrocytes and NLR were increased significantly while lymphocytes count was reduced, particularly by the lower dose. The tolerance effects of CEF might be less at the lower dose. Although much has not been done on the tolerance effects of CEF, a previous study showed that CEF responses are gradually reduced, indicating that it has a tolerance effect.²³

An increase in the Neutrocytes count by extract in this study might be because of its inflammatory,^24,25 oxidative stress,^26,27 adrenocortical function, and sleep physiology²⁸ effects. CEF increases adrenocortical function and cortisol secretion.^6,29 In turn, cortisol increased neutrophil count.³⁰ On the other hand, stress increases neutrophil count³¹ and cathinone in CEF causes oxidative stress.^26,32 CEF-induced sleep restriction increases neutrophil count.^33,34 Like the previous study,¹⁰ the lower dose of CEF extract reduced lymphocyte count in our study. However, another study revealed that lymphocyte count was increased in schizophrenic patients.³⁵ CEF increased dopamine levels in healthy and schizophrenic patients.⁷ Bogale et al³⁶ reported that schizophrenic-like symptoms were observed in mice received CEF extract. In this way, CEF should have increased lymphocyte count, but opposite result was obtained in our study that might be because of variation in the duration of administration.

Reduction in lymphocyte count observed in the current study might be through ECF hematopoietic effects. A previous study indicated that the destruction of dopaminergic cells affected blood cell synthesis.^37,38 On the other hand, this stimulant affects bioavailability of dopamine in body fluid.⁷ Adrenalin level that affects hematopoiesis could be affected by khat. Another study indicated that sympathetic fibers innervating bone marrow released adrenalin and dopamine affect stem cell activities.²⁸ This indicates that dysregulation of adrenergic and dopaminergic fibers might be associated with hematopoietic disturbances.

The higher neutrophil-to-lymphocytes ratio observed in this study could be associated with depression effects of the CEF.^2,26 This ratio is high in patients with severe depression disorder.³ Psychiatric disorders associated with subchronic inflammation increased NLR and CEF showed inflammatory responses.^3,24,25 CEF extract and juice did not affect red blood cell count, hemoglobin concentration, size of red blood cells (sRBCs), and Hematocrit (HCT) in this study (Table 1). However, when the comparison was made between rats received CEF and AA, sRBCs were less in rats at all doses of the extract (Table 1). Nevertheless, Owu et al³⁹ showed that AA (vitamin C, 200 milligrams/kilogram) administered in rats for 28 days reduced sRBCs indicating that the duration of vitamin C administration could contribute to the differences.

Rats received higher dose of the extract and AA had greater RsV compared with rats received vehicle. Catha edulis Forsk juice also reduced MCHC when compared with vehicle (Table 1). However, in the study conducted before, the PCV and hemoglobin concentration were significantly reduced, while RBCS was increased.¹⁰ The discrepancy could be attributed to the dose of the extract, duration of administration, and solvent used to extract the plant material. The dose of the Catha edulis Forsk extract used by Bin-Jaliah et al¹⁰ was 500 milligram/kilogram and administered for only one week, while the higher dose in our study was 300 milligrams/kilogram administered for twelve weeks. The solvent used in this previous study was hydro-ethanol while diethyl/chloroform (v/v; 3:1) was used in our study. Like in our study, Bin-Jaliah et al¹⁰ also revealed that RsV was increased by the crude extract of CEF.

In this study, the increase in RsV in rats received CEF might be the result of the adverse effects of Catha edulis Forsk on the liver. Previous studies indicated that RsV significantly increased in patients with progressive liver fibrosis and inflammation.^40,41 On the other hand, CEF chewing was associated with acute liver injury, subchronic inflammation and cirrhosis.^24,42

In this study, CEF juice reduced HgpvRBC (Table 1). Another study also showed CEF reduced HgpvRBCs.²⁴ However, Ketema et al²⁰ showed that statistical difference in HgpvRBCs was not observed between mice received the extract and vehicle.

The solvent, animal model and duration of administration difference used by the previous and current study might be the reason for variation in the results. Ketema et al²⁰ used methanol to extract CEF and was administered for four weeks in mice while we used diethyl ether and chloroform to extract the leaves and administered for twelve weeks in rats.

Hemoglobin reduction in each RBC observed in this study might be resulted from its effect on iron absorption problems across the gastrointestinal tract. Previous studies indicated that tannins reduced absorption of iron and vitamins; involved in the synthesis of red blood cells.^43,44 One of the components found in the leaves of CEF is tannin,^8,9 suggesting that CEF affects iron and vitamin absorption. Another study also showed that CEF reduced level of iron and vitamin B12 in the serum.⁴⁵ Gastrointestinal problems such as esophagitis, gastritis, delaying gastric empty and impaired intestinal absorption, some of the effects of the leaves.² Liver problems induced by CEF²⁴ could also be attributed to bleeding problems and iron deficiency among people who are chewing the leaves of CEF.⁴⁶

The average HgpRBC reduction in rats received CEF (Figure 1) in our study might be because of its effect on the renal system. Erythropoietin is one of the proteins involved in the synthesis of RBCs and this could be affected by CEF extract. However, Ketema et al²⁰ and Bin-Jaliah et al¹⁰ also could not see any significant differences in HgpRBC in mice and rats, respectively.

Statistical difference in the total number of platelet has not been seen in our study (Table 1). Alsalahi et al²⁴ and Alele et al²¹ also found analogous outcomes. But Ketema et al²⁰ reported that its methanolic extract (100 and 200 milligrams/kilogram) administered for four weeks reduced platelet number in mice. Contrarily, a study conducted by Bin-Jaliah et al¹⁰ revealed that its hydro-ethanolic extract (500 milligrams/kilogram) administered for one week increased platelet count in the rat model. The dissimilarity of these findings might be because of administration duration and extraction solvent variation.

In this study, lower and higher doses of CEF extract increased PCT, while the higher dose increased PsV and aPs. Its juice significantly increased the P-LCR. These findings indicated that CEF increases platelet activities. Strong platelet activity is represented by higher PCT, PsV, and P-LCR.⁴⁷ platelet activities are increased when there is organ inflammation,⁴⁸ anxiety and depression disorders.⁴⁹ On the other hand, an increase in platelet activities and oxidative stress are among the risk factors for thrombosis.^50,51 This, in turn, indicated that CEF might aggravate organ inflammation, anxiety disorders, and thrombosis. Previous studies indicated that organ inflammation is more prevalent among people who are using CEF.^25,52

AA also increased P-LCR and PsV. Studies indicated that a higher dose of ascorbic acid contributed to oxidative stress,⁵³ the release of thromboxane A₂ and prostaglandin E₂ in subjects with depression and thrombosis.⁵⁴ Platelet activities were higher in patients with depression, schizophrenia and other psychiatric problems and contributed to the secretion of serotonin.⁵⁵

In conclusion, CEF extract, CEF juice, and AA altered some of the hematological indices. AA and CEF increased platelet activities and size variability of red blood cells. The mechanisms of action for these changes need to be studied.