AMERICAN JOURNAL OF PHYSIOLOGY, BIOCHEMISTRY AND PHARMACOLOGY, 2018 VOL 7, NO. 2, PAGE 75–85 eJManager 10.5455/ajpbp.20180616012821

ORIGINAL RESEARCH Open Access Acute and sub-chronic oral toxicity studies of the leaves aqueous extract of umbellatum Poir. on mice Hermine Boukeng Jatsa1,4, Joseph Bertin Kadji Fassi1,4, Mérimé Christian Kenfack1,4, Nestor Gipwe Feussom1,4, Mireille Poumeni Kameni1, Nadège Distele Simo1,4, Emilienne Tienga Nkondo1,4, Alain Bertrand Dongmo2, Louis-Albert Tchuem Tchuente3,4 1Laboratory of Animal Physiology, Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé I, Yaoundé, Cameroon 2Laboratory of Animal Physiology, Department of Animal Biology, Faculty of Science, University of Douala, Douala, Cameroon 3Laboratory of Biology, Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé I, Yaoundé, Cameroon 4Centre for Schistosomiasis and Parasitology, Yaoundé, Cameroon

ABSTRACT ARTICLE HISTORY Aim: The schistosomicidal activity of Clerodendrum umbellatum leaves aqueous extract Received 16 June 2018 (CuAE) has been previously demonstrated. The present study was performed to estab- Accepted 02 July 2018 lish the acute and sub-chronic oral toxicity profile of CuAE in mice. Published 06 July 2018 Methods: For acute oral toxicity study, CuAE was administered per os to female mice KEYWORDS at a single dose of 2,000 mg/kg. Animals were observed for 14 days in order to identify the signs of toxicity or death. In the repeated dose 28-day oral toxicity study, the extract Clerodendrum umbellatum; acute oral toxicity; repeated was administered daily and per os to female and male mice at doses of 200, 400, and dose 28-day oral toxicity; 800 mg/kg for 28 consecutive days. A satellite group was also set up. Body weight was NOAEL; mice measured weekly. Hematological, biochemical, and histopathological parameters were analyzed. Results: CuAE at 2,000 mg/kg produced no sign of toxicity or mortality. In the sub-chronic toxicity study, no mortality, no significant change in the body weight, the relative organ weights, and the hematological parameters were recorded in all treated mice. CuAE at 400 mg/kg significantly increased transaminases activities in male mice. Except for -cre atinine and high-density lipoprotein-cholesterol in which concentrations are increased after administration of CuAE at 800 mg/kg, the levels of others biochemical markers didn’t change. Histopathological abnormalities found in the kidneys were reversible.

Conclusion: These results indicated that the LD50 of CuAE is greater than 2,000 mg/kg and CuAE, therefore, belongs to the category five of relatively non-toxic substances. From the sub-chronic toxicity study, the no-observed-adverse-effect level of CuAE for both male and female mice was considered to be 200 mg/kg/day.

Introduction in Africa, especially in Cameroon, Ethiopia, Soudan, It has been considered that if a drug is effective, it Congo, and Senegal [2,3]. In Cameroon, the leaves will have side effects. However, herbal medicines are are used for the treatment of epilepsy, headache, generally considered to be safe and effective agents. intestinal helminthiasis, irregular menstruation, Although they are widely used and assumed to be infective dermatitis, asthma, and metaphysical safe, medicinal can potentially be toxic [1]. powers. The tops are used for treating whitlow and Clerodendrum umbellatum Poir. is a belong- vulvovaginitis. To treat intestinal helminthiasis, ing to the family or Labiatae (formally traditional healers recommend to crush some of ). It is a woody climbing shrub found the fresh leaves in a little water and drink one part

Contact Hermine Boukeng Jatsa [email protected] Laboratory of Animal Physiology, Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé I, Yaoundé, Cameroon.

© EJManager. This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, noncommercial use, distribution and reproduction in any medium, provided the work is properly cited. H. B. Jatsa, J. B. Kadji Fassi, M. C. Kenfack, N. G. Feussom, M. P. Kameni, N. D. Simo, E. T. Nkondo, A. B. Dongmo, L. T. Tchuente of the extract. The rest of the extract in which has guidelines (EEC Directive of 1986; 86/609/EEC) and Capsicum frutescens fruits is used were approved by the Animal Ethical Committee of to purge the patient [2]. Pharmacological studies the Laboratory of Animal Physiology of the Faculty revealedbeen added that five C. umbellatum leaves aqueous extract of Sciences, University of Yaoundé I—Cameroon. (CuAE) displayed schistosomicidal activity [4,5]. Acute oral toxicity study Moreover, its phytochemical analysis revealed the - The acute oral toxicity—acute toxic class method sides, tannins, and triterpenes [4]. In a murine was conducted in compliance with the Organization modelpresence of Schistosomaof alkaloids, mansoniflavonoids, infection, saponins, the sapono metha- for Economic Cooperation and Development nolic fraction from CuAE exhibited schistosomicidal, (OECD) Guideline for the Testing of Chemicals No. hepatoprotective, and antioxidant properties [6,7]. 423 [8]. To achieve this test, nine female mice of Despite the wide use of C. umbellatum leaves in 9 weeks of age, weighing 21–23 g were randomly traditional medicine and its pharmacological activi- divided into three groups of three mice each. Mice ties, there is a paucity of data related to the evaluation in the control group received distilled water. The of the toxicity of this plant. Herein, an acute oral tox- dose of CuAE used as the starting dose was 2,000 icity and a repeated dose 28-day oral toxicity studies mg/kg body weight. Prior to the treatment, food but were performed to evaluate the safety of CuAE. not water was withheld for 3 hours. CuAE dissolved in distilled water was administered in a single dose Material and Methods of 2,000 mg/kg by gavage in a volume of 1 ml/100 g body weight, using a suitable intubation cannula Preparation of the plant extract to a group of three mice (test group). CuAE adminis- The leaves of Clerodendrum umbellatum Poir. were tration was repeated to another group of three mice collected in April 2012 in the locality of Mekok, - near Sangmelima, in the South region of Cameroon. mals were fasted for 2 hours and then observed. Botanical authentication was done at the National (confirmationAnimals were group). observed Following for thegeneral treatment, behavior ani Herbarium of Yaoundé, Cameroon by comparison to a changes continuously for 30 minutes, every hour specimen registered under the voucher number 7405 Service Recherche Forestière Cameroun (SRF)/Cam. for 14 days after administration of the extract. Leaves were dried in the shade, powdered, and Observationsduring the first were 24 focused hours andon parameters at least once such daily as mixed with water (100 g/l) for 24 hours of macera- piloerection, sensitivity to sound and touch, loco- motion, aggressiveness, and appearance of feces. The number of survivors was noted after 24 hours. Medicaltion at room Research temperature. and Study The of mixture Medicinal was Plants filtered, to Animals were weighed on day 0, and then on days givefrozen Cu atAE −20°C, with a and recovery lyophilized rate of at 9%. the TheInstitute extract of 7 and 14. At the end of the study, all surviving ani-

as lungs, liver, kidneys, spleen, stomach, intestine, Experimental animals was kept in a freezer at 4°C until the use. andmals ovaries were sacrificed were removed and some and internal weighed. organs A gross such The male and female BALB/c mice bred in the ani- pathological examination of these organs was also mal facility of the Centre for Schistosomiasis and performed. The LD50 was evaluated and CuAE clas- Parasitology of Yaoundé—Cameroon were used in this study. They were housed in polypropyl- ene cages in a bioterium under natural 12 hours sified according to the Globally Harmonized System Repeated dose 28-day oral toxicity study light/12 hours dark cycles and the temperature was (GHS) for the classification of chemicals [9]. This study was conducted in compliance with fed with rodents’ diet and water ad libitum. Before the OECD Guideline for the Testing of Chemicals startingmaintained the between experiments, 22°C andanimals 25°C. were The micerandomly were No. 407: Repeated dose 28-day oral toxicity study selected, marked on the tail to permit individual in rodents [10]. Study design cage) for 5 days to allow for acclimatization to the laboratoryidentification, conditions. and kept Females in their cageswere nulliparous (five mice/ Both the male and female mice of 7–8 weeks of age and non-pregnant. All procedures were conducted weighing 20–26 g for female and 25–30 g for male in accordance with the principles of laboratory were used in this study. Sixty mice were randomly animal use and care of the European Community

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Table 1. Body weight and relative weight of vital organs after administration of a unique dose of 2,000 mg/kg of CuAE to mice. Body weight (g) Relative weight of organs (g/100 g body weight) Groups Initial Final Ovaries Heart Stomach Liver Intestine Lungs Spleen Kidneys weight weight (×10−3) 21.87 ± 24.12 ± 0.027 ± Control 0.41 ± 0.01 1.75 ± 0.17 5.68 ± 0.31 8.58 ± 0.70 1.31 ± 0.06 0.79 ± 0.09 1.23 ± 0.04 0.47 0.43 0.006 21.90 ± 24.10 ± 0.024 ± Test group 0.41 ± 0.06 2.09 ± 0.27 5.63 ± 0.40 8.16 ± 0.56 1.35 ± 0.10 0.84 ± 0.02 1.11 ± 0.06 0.45 0.33 0.005 Results are expressed as a mean ± SEM. n = 3.

- 15 minutes. The serum was collected and stored at prised a control group receiving distilled water as the vehiclefive females and three each. testThe groups first batch receiving of four Cu groupsAE at doses com total proteins by the Biuret method [11]. Alanine of 200, 400, and 800 mg/kg, respectively. The second aminotransferase−20°C until analyzed. (ALT) The and serum aspartate was aminotrans assayed for- batch of two groups comprised the control satellite ferase (AST) were analyzed according to the method group receiving distilled water and the test satellite of Reitman and Frankel [12] using commercial kits group receiving CuAE at the highest dose of 800 mg/ (BIOCLIN, Brazil). Total cholesterol and high-density kg. CuAE was administered daily and per os to each lipoprotein (HDL) were evaluated using a colorimet- mouse in a volume of 1 ml/100 g body weight by a ric method by commercial kits (INMESCO, Germany). suitable intubation cannula for 28 consecutive days. Creatinine was analyzed using a kinetic method [13]. Animals were observed twice daily for morbidity and Histopathological study - - litemortality. groups On were day kept 29, thefor observationfirst batch of of animals reversibility, (con in 10% neutral buffered formalin. The organs were persistence,trol group and or test delayed groups) occurrence was sacrificed. of toxic The effects, satel The liver and the kidneys were removed and fixed (sections of 5 µm) and staining with hematoxylin andembedded eosin. inPreparations paraffin blocks were followed examined by sectioning under a Sample collection for 14 days’ post-treatment and then sacrificed. light microscope and any alteration compared to During the experimental period, body weight, food, the normal structure was registered. and water intakes were recorded once a week. Statistical analysis collected in the retro-orbital sinus into ethylene Results were expressed as a mean ± standard error diamineBefore the tetraacetic sacrifice, acid blood (EDTA) from tubes each for animal hemato was- of the mean (SEM). Statistical analysis was per- logical analysis and into dry tubes for the assess- formed by one-way analysis of variance followed ment of biochemical markers. Animals were then between groups. P < 0.05 was considered statisti- were removed and weighed: liver, kidneys, lungs, by Dunnett test to evaluate significant differences- spleen,sacrificed ovaries, by decapitation testicles, heart, and the stomach, following and organs intes- ried out using GraphPad Prism version 4.00 for tine. The relative weight of each organ (gram of Windowscally significant. (GraphPad All statisticalSoftware, San analyses Diego, were CA). car organ/100 g of body weight) was calculated. Results Hematological analysis Acute oral toxicity Hematological analysis was performed using an automated hematology analyzer to examine red Oral administration of CuAE at the single dose of blood cell (RBC), hemoglobin (HGB), hematocrit - (HCT), mean corpuscular hemoglobin concentration mal change in behavioral properties of mice during (MCHC), mean corpuscular volume (MCV), mean cor- 142,000 days mg/kg of observation was followed and by no no mortality. significant In abnor addi- puscular hemoglobin (MCH), total white blood cells tion, the body weight and the weight of organs (WBCs), and differential leukocytes and platelets. were statistically similar in both control and test groups (Table 1). All internal organs examined at Biochemical analysis necropsy were free from any gross pathological Blood collected in dry tubes was allowed for complete changes. Regarding these results, the LD50 of CuAE clottingwww.ajpbp.com and then centrifuged at 3,500 rpm at 4°C for 77 H. B. Jatsa, J. B. Kadji Fassi, M. C. Kenfack, N. G. Feussom, M. P. Kameni, N. D. Simo, E. T. Nkondo, A. B. Dongmo, L. T. Tchuente

Table 2. Food and water intakes after administration of CuAE to mice for 28 consecutive days. Test groups Satellite groups CuAE CuAE CuAE CuAE Control Control (200 mg/kg) (400 mg/kg) (800 mg/kg) (800 mg/kg) Male W1 38.68 ± 0.007 32.21 ± 0.002 40.51 ± 0.003 35.72 ± 0.004 30.48 ± 0.007 30.05 ± 0.009 W2 38.65 ± 0.006 36.69 ± 0.004 35.17 ± 0.004 38.96 ± 0.003 30.32 ± 0.004 26.05 ± 0.006 W3 30.72 ± 0.008 34.21 ± 0.007 30.25 ± 0.003 28.55 ± 0.006 23.05 ± 0.007 23.17 ± 0.002 W4 32.21 ± 0.002 38.02 ± 0.001 32.18 ± 0.004 24.05 ± 0.007### 19.69 ± 0.006 19.83 ± 0.002 W5 17.27 ± 0.008 21.38 ± 0.008 Food intake W6 25.89 ± 0.002 28.86 ± 0.002 (g/mouse/ Female week) W1 31.78 ± 0.001 20.05 ± 0.007### 29.60 ± 0.005 25.52 ± 0.007## 30.45 ± 0.005 30.09 ± 0.007 W2 33.23 ± 0.002 31.10 ± 0.006 30.25 ± 0.002 27.04 ± 0.004## 30.30 ± 0.003 26.03 ± 0.003 W3 22.55 ± 0.002 23.38 ± 0.005 19.11 ± 0.007# 23.37 ± 0.006 23.03 ± 0.007 23.17 ± 0.006 W4 23.53 ± 0.003 20.98 ± 0.006 22.20 ± 0.004 24.19 ± 0.007 19.69 ± 0.002 19.83 ± 0.002 W5 17.25 ± 0.008 17.09 ± 0.006 W6 25.89 ± 0.004 23.10 ± 0.005 Male W1 22 ± 0.004 24 ± 0.007 18 ± 0.005 20 ± 0.007 25 ± 0.004 20 ± 0.006# W2 21 ± 0.007 20 ± 0.006 18 ± 0.005 22 ± 0.005 20 ± 0.006 23 ± 0.007 W3 26 ± 0.008 27 ± 0.007 21 ± 0.004 29 ± 0.006 22 ± 0.007 25 ± 0.007 W4 28 ± 0.006 30 ± 0.005 30 ± 0.007 31 ± 0.006 30 ± 0.006 26 ± 0.008# W5 32 ± 0.005 34 ± 0.006 Water intake W6 32 ± 0.005 35 ± 0.004 (ml/mouse/ Female week) W1 14 ± 0.007 15 ± 0.005 15 ± 0.006 14 ± 0.005 17 ± 0.007 14 ± 0.006 W2 15 ± 0.005 19 ± 0.006# 17 ± 0.008 15 ± 0.006 17 ± 0.007 18 ± 0.005 W3 17 ± 0.007 20 ± 0.006 15 ± 0.006 22 ± 0.007## 18 ± 0.006 18 ± 0.005 W4 20 ± 0.006 24 ± 0.007# 20 ± 0.005 22 ± 0.008 20 ± 0.006 20 ± 0.006 W5 23 ± 0.007 26 ± 0.007# W6 26 ± 0.008 28 ± 0.007 Results are expressed as a mean ± SEM. n = 5. W = week. #p < 0.05; ##p < 0.01; ###p < 0.001: values are significantly different from controls at the same week. exceeds 2,000 mg/kg and according to the GHS for the food consumption was recorded in both sexes. CuAE belongs to - sumption was recorded in male mice (p < 0.05), substances.the classification of chemicals, whileHowever, an increase a significant in water reduction consumption in water was noted con category five, the category of relatively non-toxic in female mice (p < 0.05). Sub-chronic oral toxicity Effects ofClerodendrum umbellatum leaves aqueous Effects ofClerodendrum umbellatum leaves aqueous extract on clinical mortality signs, body, and organ extract on food and water intakes weights Table 2 summarizes the effects of CuAE on food The sub-chronic oral toxicity study in mice was and water intakes during sub-chronic treatment. performed for 28 days and none of the CuAE- Daily administration of CuAE to male mice for 28 treated animals died or exhibited abnormal clinical signs at any of the tested doses. The variation of the of food and water intakes. Comparatively, to con- consecutive days resulted in a significant change body weight of mice is shown in Figure 1. During the experimentation, the body weight variation 36.91%, 15.25%, and 19.70% was, respectively, of the mice of both sexes in all test and satellite recordedtrol mice, aafter significant administration reduction ofof foodCuAE intake at 200, by 400, or 800 mg/kg. This reduction was 25.33% to that of their controls. However, a body weight in male mice treated with 800 mg/kg of CuAE. On groups was statistically not significant compared- tion of CuAE at 200 or 800 mg/kg to male mice. The by 26.67% and 29.41% in female mice treated bodyloss was weight noted of female in the micefirst treatedweek after with administra CuAE at all withthe contrary, CuAE at water200 or intake 800 mg/kg,significantly respectively. increased In doses decreased after administration of the extract

78the satellite groups, no significant modification of A J Physiol Biochem Pharmacol • 2018 • Vol 7 • Issue 2 Toxicity of Clerodendrum umbellatum leaves aqueous extract

Figure 1. Body weight variation of mice after administration of CuAE to mice for 28 consecutive days. W0: the begin- ning of the treatment. Results are expressed as a mean ± SEM. n = 5. and remained under the normal range during the Effects ofClerodendrum umbellatum leaves aqueous experimentation. The relative weight of the organs extract on hematological parameters

The effects of CuAE on hematological parameters of CuAE to mice of the test groups (200, 400, and 800 both male and female mice after 28 days of an oral mg/kg)did not as vary well significantly as to the mice after of administration the test satellite of administration are summarized in Table 4. No sig- group (Table 3).

nificant difference was observed from the results www.ajpbp.com of the hematological profile of mice of both sexes79 H. B. Jatsa, J. B. Kadji Fassi, M. C. Kenfack, N. G. Feussom, M. P. Kameni, N. D. Simo, E. T. Nkondo, A. B. Dongmo, L. T. Tchuente

Table 3. The relative weight of vital organs after administration of CuAE to mice for 28 consecutive days. Relative weight of organs (g/100 g body weight) Test groups Satellite groups CuAE CuAE CuAE CuAE Organs Control Control (200 mg/kg) (400 mg/kg) (800 mg/kg) (800 mg/kg) Male Heart 0.44 ± 0.01 0.50 ± 0.03 0.45 ± 0.04 0.48 ± 0.04 0.54 ± 0.02 0.57 ± 0.07 Stomach 1.09 ± 0.16 1.26 ± 0.04 1.02 ± 0.09 1.13 ± 0.07 1.46 ± 0.07 1.13 ± 0.13 Liver 4.40 ± 0.28 4.74 ± 0.26 4.85 ± 0.05 4.90 ± 0.49 4.39 ± 0.11 4.51 ± 0.20 Intestine 8.51 ± 0.11 8.13 ± 0.60 9.85 ± 0.26 8.82 ± 0.16 7.84 ± 0.28 8.07 ± 0.20 Lungs 0.67 ± 0.06 0.76 ± 0.06 0.72 ± 0.05 0.65 ± 0.09 0.71 ± 0.04 0.74 ± 0.03 Spleen 0.50 ± 0.11 0.42 ± 0.02 0.56 ± 0.04 0.52 ± 0.12 0.43 ± 0.02 0.49 ± 0.05 Kidneys 1.45 ± 0.14 1.54 ± 0.05 1.54 ± 0.05 1.59 ± 0.16 1.55 ± 0.07 1.65 ± 0.08 Testicles 0.73 ± 0.05 0.78 ± 0.06 0.83 ± 0.04 0.78 ± 0.06 0.67 ± 0.03 0.74 ± 0.08 Female Heart 0.42 ± 0.02 0.49 ± 0.04 0.47 ± 0.01 0.46 ± 0.02 0.48 ± 0.03 0.48 ± 0.03 Stomach 1.32 ± 0.06 1.72 ± 0.10 1.75 ± 0.10 1.27 ± 0.10 1.49 ± 0.08 1.22 ± 0.09 Liver 4.67 ± 0.10 4.56 ± 0.21 5.28 ± 0.64 5.01 ± 0.10 4.78 ± 0.09 4.71 ± 0.09 Intestine 10.04 ± 0.25 9.52 ± 0.48 11.18 ± 0.34 9.90 ± 0.32 9.92 ± 0.18 9.14 ± 0.46 Lungs 0.77 ± 0.02 0.71 ± 0.05 0.64 ± 0.03 0.85 ± 0.06 0.87 ± 0.05 0.81 ± 0.05 Spleen 0.53 ± 0.03 0.54 ± 0.06 0.55 ± 0.05 0.53 ± 0.03 0.62 ± 0.08 0.51 ± 0.05 Kidneys 1.14 ± 0.42 1.28 ± 0.09 1.08 ± 0.05 1.45 ± 0.71 1.07 ± 0.04 1.32 ± 0.04 Ovaries (×10−3) 0.037 ± 0.004 0.068 ± 0.017 0.044 ± 0.008 0.065 ± 0.015 0.042 ± 0.004 0.050 ± 0.006 Results are expressed as a mean ± SEM. n = 5.

Table 4. Hematological parameters of mice after administration of CuAE for 28 consecutive days. Test groups Satellites groups CuAE CuAE CuAE CuAE Parameters Control Control (200 mg/kg) (400 mg/kg) (800 mg/kg) (800 mg/kg) Male RBC (106/µl) 8.22 ± 0.33 8.92 ± 1.18 8.04 ± 1.61 7.28 ± 1.67 8.84 ± 0.28 7.32 ± 0.44 HGB (g/dl) 12.22 ± 0.94 12.84 ± 2.09 12.38 ± 1.56 10.35 ± 2.73 14.60 ± 0.27 12.30 ± 0.72 HCT (%) 32.22 ± 0.65 37.82 ± 4.96 33.42 ± 5.00 28.80 ± 6.84 36.72 ± 1.00 30.85 ± 2.04 MCHC (g/dl) 36.70 ± 2.54 33.46 ± 2.23 37.56 ± 1.40 36.02 ± 0.29 39.78 ± 0.56 40.05 ± 0.70 MCH (pg) 14.74 ± 0.68 14.20 ± 0.86 16.80 ± 2.03 14.00 ± 0.24 17.04 ± 0.35 16.82 ± 0.28 MCV (fl) 40.60 ± 1.20 42.60 ± 1.43 45.00 ± 5.80 38.72 ± 0.92 43.00 ± 0.70 42.25 ± 0.99 WBC (103/µl) 20.64 ±11.81 13.14 ± 2.22 16.90 ± 6.02 12.80 ± 4.47 11.28 ± 0.95 4.65 ± 0.77 Lym (%) 74.06 ± 4.55 71.15 ± 4.72 69.32 ± 6.65 71.57 ± 5.18 70.02 ± 1.21 71.75 ± 4.02 Mono (%) 14.34 ± 1.90 16.02 ± 1.51 19.28 ± 2.51 15.17 ± 2.27 19.84 ± 1.36 16.87 ± 2.35 Gran (%) 11.62 ± 3.59 12.84 ± 2.85 11.36 ± 4.72 13.27 ± 3.29 10.18 ± 1.96 11.37 ± 3.76 Plat (103/µl) 279.4 ± 14.82 261.4 ± 38.88 214.8 ± 55.16 262.5 ± 70.47 327.2 ± 44.53 282.7 ± 14.82 Female RBC (106/µl) 7.39 ± 1.77 7.15 ± 1.95 7.93 ± 2.16 7.78 ± 1.12 7.78 ± 1.62 6.82 ± 2.87 HGB (g/dl) 11.70 ± 1.12 11.18 ± 1.47 11.70 ± 1.83 11.67 ± 1.10 12.82 ± 1.09 12.08 ± 2.44 HCT (%) 31.78 ± 6.28 28.84 ± 3.52 32.96 ± 10.14 29.69 ± 2.92 32.08 ± 7.52 27.00 ± 10.24 MCHC (g/dl) 36.90 ± 5.19 32.60 ± 7.53 35.14 ± 1.65 38.97 ± 2.21 40.50 ± 1.34 35.30 ± 5.19 MCH (pg) 16.00 ± 0.71 13.12 ± 3.02 14.48 ± 0.98 14.87 ± 0.67 16.60 ± 0.52 14.00 ± 1.86 MCV (fL) 43.40 ± 1.60 40.40 ± 0.24 41.20 ± 2.00 38.50 ± 1.39 41.20 ± 1.39 40.60 ± 1.60 WBC (103/µl) 8.97 ± 2.55 6.12 ± 1.51 5.40 ± 0.97 8.50 ± 1.47 6.56 ± 0.96 6.08 ± 1.39 Lym (%) 59.30 ± 5.97 72.14 ± 1.56 71.40 ± 2.54 68.40 ± 7.57 74.66 ± 2.01 75.06 ± 4.26 Mono (%) 19.06 ± 1.31 16.14 ± 1.69 14.78 ± 0.57 19.32 ± 1.64 15.20 ± 0.65 11.82 ± 1.04 Gran (%) 20.26 ± 5.81 11.74 ± 1.63 13.82 ± 2.08 12.32 ± 5.68 10.08 ± 1.87 13.06 ± 3.48 Plat (103/µl) 230.8 ± 29.48 229.4 ± 21.98 154.6 ± 36.92 237.2 ± 43.54 232.6 ± 43.87 240.4 ± 46.30 Results are expressed as a mean ± SEM. n = 5. Lym = lymphocytes; Mono = monocytes; Gran = granulocytes; Plat = platelets.

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Table 5. Biochemical parameters of mice after administration of CuAE for 28 consecutive days. Test groups Satellite groups CuAE CuAE CuAE CuAE Parameters Control Control (200 mg/ kg) (400 mg/ kg) (800 mg/kg) (800 mg/kg) Male ALT (U/l) 81.30 ± 3.31 86.03 ± 8.65 127.40 ± 8.75## 72.12 ± 5.55 96.50 ± 7.36 114.37 ± 11.95 AST (U/l) 170.80 ± 8.12 169.60 ± 7.18 252.60 ± 26.23# 152.25 ± 11.09 201.00 ±14.73 236.75 ± 23.90 Total proteins 9.98 ± 0.83 9.42 ± 0.86 10.73 ± 0.21 9.60 ± 0.95 11.46 ± 0.67 10.65 ± 0.63 (mg/dl) Total cholesterol 123.34 ± 6.43 103.84 ± 4.21 164.56 ± 6.81 132.99 ± 7.10 138.04 ± 6.92 142.33 ± 10.78 (mg/dl) HDL-cholesterol 13.15 ± 1.07 16.78 ± 1.76 16.77 ± 1.83 16.83 ± 2.09 20.11 ± 0.91 19.16 ± 2.35 (mg/dl) Creatinine 0.42 ± 0.11 0.48 ± 0.08 0.42 ± 0.02 0.78 ± 0.16# 0.42 ± 0.10 0.19 ± 0.04 (mg/dl) Female ALT (U/l) 77.50 ± 5.54 103.90 ± 6.82 90.70 ± 15.94 110.50 ± 6.10 132.70 ± 2.35 136.30 ± 11.94 AST (U/l) 168.20 ± 12.76 208.00 ± 19.18 198.40 ± 33.86 249.75 ± 29.78# 260.60 ± 11.32 262.00 ± 16.38 Total proteins 9.18 ± 0.41 8.65 ± 1.34 10.56 ± 0.53 10.38 ± 0.23 12.65 ± 1.1 14.57 ± 0.51 (mg/dl) Total cholesterol 103.17 ± 6.60 122.78 ± 11.1 106.85 ± 11.41 124.78 ± 5.65 149.19 ± 9.02 152.72 ± 1.06 (mg/dl) HDL-cholesterol 15.98 ± 0.92 18.87 ± 1.13 20.04 ± 1.93 21.6 ± 1.30# 20.08 ± 0.97 21.45 ± 0.89 (mg/dl) Creatinine 0.73 ± 0.04 0.65 ± 0.09 0.68 ± 0.08 0.51 ± 0.05 0.39 ± 0.05 0.35 ± 0.05 (mg/dl) Results are expressed as a mean ± SEM. n = 5. #p < 0.05; ##p < 0.01: values are significantly different from controls. of the test and satellite groups were compared to consecutive days is presented in Figure 2. Liver those of control and control satellite groups. tissue displayed normal hepatocytes, sinusoidal vein, centrolobular vein, and portal vein in all Effects ofClerodendrum umbellatum leaves aqueous test and satellite groups of female mice. Similarly, extract on biochemical parameters kidney sections revealed the normal architecture The effects of repeated administration of CuAE of glomerulus and Bowman’s capsules with dis- for 28 days on serum biochemical parameters are tinct Bowman’s space, renal corpuscle, and renal summarized in Table 5. This treatment induced a tubules (Fig. 3). In the test groups, the liver tissue p < 0.01) and AST activ- of male mice treated with CuAE at 400 mg/kg or ities in male mice receiving CuAE at 400 mg/kg (p < those belonging to the test satellite group showed 0.05)significant and in increase female ofmice ALT treated ( with CuAE at 800 mg/kg (p < 0.05) compared to the control group. CuAE at 800 mg/kg sig- CuvascularAE at 800 congestion mg/kg, kidneyand inflammatory sections showed cells atro(ICs)- p < 0.05) the HDL-cholesterol infiltration (Fig. 2). In male mice treated with concentrationRegarding the lipidin female profile, mice. At this same dose, These alterations of the kidneys were reversible Cunificantly increased ( - asphy observed of glomeruli in the and test tubular satellite clarifications group (Fig. 3).(TCs). tration in male mice compared to control mice. AE significantly increased creatinine concen Discussion the test groups were reversible. The total protein Clerodendrum umbellatum is widely used as a andModifications total cholesterol of these levels biochemical were unaffected parameters by the in medicinal plant for the treatment of epilepsy, repeated administration of CuAE at all doses (200, headache, intestinal helminthiasis, irregular men- 400, and 800 mg/kg) to mice of both sexes. struation, infective dermatitis, asthma, whitlow, Effects ofClerodendrum umbellatum leaves aqueous vulvovaginitis, and metaphysical disorders [2]. extract on the histology of the liver and the kidneys The schistosomicidal activity of its leaves aque- ous extract has been demonstrated in Schistosoma Histology of the liver sections of male and female mansoni-infected mice [4]. In this regard, it was of mice who received CuAE at different doses for 28 great importance to investigate the toxicological www.ajpbp.com 81 H. B. Jatsa, J. B. Kadji Fassi, M. C. Kenfack, N. G. Feussom, M. P. Kameni, N. D. Simo, E. T. Nkondo, A. B. Dongmo, L. T. Tchuente

Figure 2. Histology of the liver of mice after administration of CuAE to mice for 28 consecutive days (hematoxy- lin-eosin (HE), ×400). H = hepatocytes; CV = centrolobular vein; Vc = vascular congestion; PV = portal vein.

Figure 3. Histology of the kidney of mice after administration of CuAE to mice for 28 consecutive days (HE, ×400). G = glomerulus; RT = renal tubules; aG = atrophy of glomerulus.

effects of this plant extract and ensure its safety. LD50 is greater than 2,000 mg/kg. This resulted in The acute oral toxicity and sub-chronic oral toxicity classifying Cu studies of CuAE were then carried out on male and non-toxic substances according to the GHS for female mice. AE in the category five of relatively

In the acute toxicity study, CuAE at the single dose between the oral LD50 of CuAE and that of others of 2,000 mg/kg did not alter the behavioral pat- speciesthe classification of Clerodendrum of chemicals as C. infortunatum [9]. A comparison leaves terns of the mice and induced no mortality during methanolic extract (>2,000 mg/kg) [14], C. inerme 14 days of study. It was therefore stated that its oral leaves ethyl acetate, chloroform, ethanolic, and

82 A J Physiol Biochem Pharmacol • 2018 • Vol 7 • Issue 2 Toxicity of Clerodendrum umbellatum leaves aqueous extract aqueous extracts (>3,000 mg/kg) [15], C. capitatum oral administration of CuAE to male and female leaves hydroethanolic extract (>5,000 mg/kg) [16], mice for 28 consecutive days did not affect neither and C. infortunatum leaves and roots alcoholic RBCs count nor HGB, nor HCT, suggesting that this extracts (>3,000 mg/kg) [17] demonstrated that extract has no oxygenation and anemia risk [22]. some species of this genus are relatively non-toxic Total WBCs and differential leukocytes of mice of both sexes of the test groups and the test satellite

50 of - and safe, and are therefore classified in category CuAE was greater than the LD50 of C. myricoides root trol groups. This result indicates that CuAE did not aqueousfive according extract to the(1,134 GHS. mg/kg), However, which the oralbelongs LD to interferegroup showed with the no immune significant system. difference A normal with hema con- category four of low toxic substances [18]. In a repeated dose 28-day oral toxicity study, C. inerme leaves ethyl acetate, chloroform, ethano- no clinical sign of toxicity and no mortality was lic,tological and aqueous profile of extracts animals of treated C. infortunatum chronically leaves with recorded in both male and female mice treated and roots alcoholic extracts was also reported in with CuAE at different doses (200, 400, and 800 the literature [14,15,17]. mg/kg). These results indicate that the mice were The levels of the liver-associated enzymes are not affected by repeated exposure to CuAE. The widely used to evaluate toxic effects to the liver. - Transaminases (ALT and AST) are important ity in animals and it is an important indicator of enzymes in assessing the liver function. ALT is spe- thevariation adverse of effect body weightof drugs is and the firstchemicals sign of[19]. toxic In the current study, the body weight of mice of the and heart. These enzymes are mainly found in the - cytoplasmcific to the liverof animal while cells. AST isAn associated increase inwith the the level liver of cantly compared to that of their controls. However, ALT and AST in the serum indicates the altered integ- thetest bodyas well weight as satellite of female groups mice did treated not vary with signifi CuAE rity of cell membrane as well as cell lysis or death. decreased during the experimentation and could be This results in their secretion into the bloodstream linked to the reduction of food intake also recorded [16,23]. The increase of ALT and AST activities in in this study. Similar results were obtained by the serum of male mice after administration of CuAE Hayelom et al. [18] after administration of C. myri- at 400 or 800 mg/kg indicates liver tissue damage. coides root aqueous extract to mice. Male mice were - less susceptible than female mice to the inhibiting topathological study of the liver, which revealed effect of the extract on food intake and body weight These findings were further confirmed by the his gain. This could be due to the anabolizing potential mice. Except for this abnormality, the liver structure of androgens. This inhibiting effect of CuAE could be wasICs infiltrationclosed to the on normal the liver structure. preparations The variation of these explained by the probable presence of anorexigenic of transaminases activities has been reported in compounds in the extract [18]. In fact, an increase subchronic oral toxicity studies of some species of of food intake, as well as a body weight gain, was Clerodendrum [16–18]. Synthesized by the liver, the recorded after stopping the administration of CuAE to mice. Organ-to-body weight is an import- Results from the present investigation revealed that ant index to diagnose whether an organ has been CutotalAE proteindid not reflectsalter the liver total functional protein concentration.capacity [24]. exposed to injury. Hence, if mice are exposed to It can, therefore, be suggested that CuAE does not toxic substances, the weight of the damaged organ promote severe hepatic alterations since the syn- will either increase or decrease as will the organ- thesis function of the liver remains intact and the to-body weight ratio [16,20]. Our results demon- alteration in ALT liver enzyme demonstrates that strated that repeated administration of CuAE did the damage may be reversible. of vital organs. Our results are in accordance with was observed in the levels of total cholesterol in thosenot induce of many significant authors who side found effects that on the the chronic weight Regarding the lipid profile, no significant change administration of C. infortunatum leaves methano- increase in HDL-cholesterol levels in the female lic extract as well as C. capitatum did not affect the miceall test treated and satellite with Cu AEgroups. at 800 However, mg/kg was a significant recorded. weight of vital organs [14,16]. When LDL-cholesterol circulates in the blood, it can The hematopoietic system is one of the most slowly build up in the inner walls of the arteries, sensitive targets for toxic compounds and an inducing the formation of plaques leading to ath- important index of physiological and pathologi- erosclerosis. In contrast, HDL-cholesterol tends cal status in man and animal [21]. In this study, an to carry cholesterol away from the arteries to the www.ajpbp.com 83 H. B. Jatsa, J. B. Kadji Fassi, M. C. Kenfack, N. G. Feussom, M. P. Kameni, N. D. Simo, E. T. Nkondo, A. B. Dongmo, L. T. Tchuente liver. Thus, an increase in HDL-cholesterol pro- [5] Khan SA, Shahid S, Ahmad W, Ullah S. tects against cardiovascular diseases [25,26]. This Pharmacological importance of Clerodendrum result showed that Cu - genus: a current review. Int J Pharmaceut Sci Res cial effects by reducing cardiovascular risk factors. 2017; 2:22–30. Creatinine is a biomarkerAE couldof the haverenal some function. benefi Its [6] Jatsa HB, Kenfack MC, Simo ND, Feussom NG, Nkondo ET, Tchuem Tchuente L-A, et al. Schistosomicidal, level is increased in the serum when the cortex hepatoprotective and antioxidant activities of the and/or the glomeruli are damaged. Thus, higher methanolic fraction from Clerodendrum umbella- than normal levels of serum creatinine are an indi- tum Poir leaves aqueous extract in infection in mice. BMC Complement Altern - Med 2015; 15:248–56. cantlycation increasedof a deficiency in the in male renal mice function treated [16,22]. with the In [7] Wang J-H, Luan F, He X-D, Wang Y, Li M-X. extractthe current at the study,highest serum dose of creatinine 800 mg/kg. level This signifi result Traditional uses and pharmacological properties of Clerodendrum phytochemicals. J Trad Complement fact, glomeruli atrophy and TCs were observed on Med 2018; 8:24–38. kidneywas associated sections withof these histopathological mice. Nevertheless, findings. these In [8] OECD. OECD guidelines for testing of chemicals. Test guidelines 423: Acute oral toxicity—acute toxic alterations of the kidney structure were reversed 14 days after stopping treatment. This result indicates Development, Paris, France, 2001. that kidney damage caused by CuAE is reversible. class method. Office of Economic and Community system for human health and environmental effects Conclusion [9] ofOECD. chemical Harmonized substances. integrated 28th Jointhazard Meeting classification of the Chemicals Committee and the Working Party on From the present study, it can be concluded that the Chemicals, Part 2. Paris, France, 1998. acute and sub-chronic (28 days) oral administra- [10] OECD. OECD guidelines for testing of chemicals. tions of CuAE did not produce any clinical signs of Test guidelines 407: Repeated dose 28-day oral toxicity or mortality in the male and female mice. Community Development, Paris, France, 2008. The LD50 value of CuAE was higher than 2,000 mg/ kg, and the no-observed-adverse-effect level of the [11] Gornaltoxicity AG, study Bardwil in rodents. GS, David Office MM. ofDetermination Economic and of extract is considered to be 200 mg/kg/day for both serum proteins by the means of Biuret reactions. J the male and female mice. Biol Chem 1949; 177:751–66. [12] Reitman S, Frankel S. A colorimetric method for the Acknowledgments determination of serum oxaloacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; Authors are grateful to the International Foundation 28:56–63. for Science, Grant F/3622 2F, to Hermine Boukeng [13] Bartels H, Bohmer M, Heierli C. Serum creatinine Jatsa. We also thank Mr. Jean Pierre Takala for his without protein precipitation. Clin Chim Acta 1972; 37:193–7. technical support in histology study. [14] Das S, Bhattacharya S, Biswas M, Kar B, Kumar RBS, Pramanik G, et al. Acute and sub-chronic toxicity References study of Clerodendron infortunatum leaf in adult [1] Nasri H, Shirzad H. Toxicity and safety of medicinal male albino mice. Am Eur J Sci Res 2011; 6:188–91. plants. J Herb Med Pharmacol 2013; 2:21–2. [15] Bhushan B, Sardana S, Bansal G. Acute and sub- [2] Adjanohoum JE, Aboubakar N, Dramane K, Ebot ME, acute toxicity study of Clerodendrum inerme, Ekpere JA, Enow-Orock EG, et al. Traditional medi- Jasminum mesnyi Hance and Callistemon citrinus. J cine and pharmacopeia: contribution to ethnobo- Acute Dis 2014; 3:324–7. [16] Idoh K, Agbonon A, Potchoo Y, Gbeassor M. CSTR/OUA, Porto Novo, Benin, 1996. Toxicological assessment of the hydroethanolic [3] Cheektanical M,and Pollar floristic BJ, Darbyshire studies in I,Cameroon. Onana JM, CNPMS, Wild C. leaf extract of Clerodendrum capitatum in Wistar The plants of Kupe Mwanenguba and the Bakossi rats. Pan Afr Med J 2016; 24:66; doi:10.11604/ mountains, Cameroon. Royal Botanic Gardens, Kew pamj.2016.24.66.8771 National Herbarium of Cameroon, 2004. [17] Nandi S, Ukil B, Lyndem LM. Acute and sub-acute [4] Jatsa HB, Ngo Sock ET, Tchuem Tchuente L-A, toxicological evaluation of the alcoholic leaf and Kamtchouing P. Evaluation of the in vivo activity of root extracts of Clerodendrum infortunatum L. 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