Atsafack et al. BMC Complementary and Alternative Medicine (2015) 15:44 DOI 10.1186/s12906-015-0581-z RESEARCH ARTICLE Open Access Toxicological studies of stem bark extract from Schefflera barteri Harms (Araliaceae) Serge Secco Atsafack1, Jules-Roger Kuiate1, Raymond Simplice Mouokeu2, Martin Luther Koanga Mogtomo3, Alembert Tiabou Tchinda4, Tamokou Jean De Dieu1, Huguette Magnifouet Nana1, Rébecca Madeleine Ebelle Etame3, Lucie Biyiti5 and Rosalie Annie Ngono Ngane3* Abstract Background: The use of herbal medicines as complements or alternatives to orthodox medicines has been on the increase. There has been the erroneous belief that these medicines are free from adverse effects. Schefflera barteri is popularly used in the West region of Cameroon for the treatment of various diseases such as diarrhea, spasm, pneumonia and animals bite. Considering the ethnopharmacological relevance of this plant, this study was designed to investigate the possible toxic effects of the stem bark extract of S. barteri. Methods: The extract was prepared by maceration of stem bark dry powder in methylene chloride/methanol mixture. Phytochemical analysis was performed by chemical reaction method. Oral acute toxicity study was carried out by administering single geometric increasing doses (2 to 16 g/kg body weight) of plant extract to Swiss albino mice. For sub-acute toxicity study, repeated doses (100, 200, 400 and 800 mg/kg bw) of plant extract were given to Wistar albino rats for 28 consecutive days by oral route. At the end of the treatment period, hematological and biochemical parameters were assessed, as well as histopathological studies. Results: Phytochemical analysis of stem bark extract of S. barteri revealed the presence of anthocyanins, anthraquinons and saponins. Acute toxicity results showed that the LD50 was greater than 16000 mg/kg. Sub-acute treatment significantly (P < 0.05) increased the level of serum transaminase, proteins and HDL cholesterol. On the other hand, the extract significantly (P < 0.05) reduced the level of leucocytes as well as neutrophils, basophils and monocytes in female. No significant variation of serum creatinine, LDL cholesterol, serum triglycerides as well as liver, spleen, testicles and ovaries proteins was noted. Histopathological analysis of organs showed vascular congestion, inflammation of peri-portal and vacuolization of hepatocytes at the level of the liver. Leucocytes infiltration of peri-portal veins were noticed on lungs and liver cells as well as inflammatory peri-bronchial and basal membranes seminar tube merely joined on lungs and testis respectively. Conclusion: The results suggest that acute administration of the stem bark extract of S. barteri is associated with signs of toxicity, administration over a long duration provokes hepatotoxicity, testes and lungs toxicities. Keywords: Schefflera barteri, Acute toxicity, Sub-acute toxicity, Histopathological analysis * Correspondence: angono@yahoo.com 3 Laboratory of Biochemistry, University of Douala, P.O. Box 24157, Douala, Cameroon Full list of author information is available at the end of the article © 2015 Atsafack et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Atsafack et al. BMC Complementary and Alternative Medicine (2015) 15:44 Background In recent times, focus on plant research has increased all over the world and a large body of evidence has collected to show immense potentials of medicinal plants used in various traditional systems [1-3]. The World Health Organization (WHO) estimates that 70 to 80% of the people in developing countries use traditional medicine as a major source of health care. However, many people underestimate the toxicity of natural products and do not realize that these agents could be as toxic or more than synthetic drugs. So far, many plants have been reported to be toxic to both human and animals [1,4]. It should therefore, be emphasized that the traditional use of any plant for medicinal purposes, by no means, warrants the safety of such plant. Plants in folk medicine should therefore, be evaluated for safety or toxicity and necessary recommendations made on their use. Schefflera barteri locally called “Dehethe” in Dschang (Cameroon), “Rukiganame” or “Omwamira” in Uganda, is a shrub belonging to the Araliaceae family. It is distributed throughout Africa’s mountainous forests gallery (Guinea, Sierra Leone, Niger, Uganda…) [5]. In the highlands of the West Region of Cameroon, S. barteri is well known for medicinal purpose [6]. The stem bark is widely used for peg fence [7]. From ethnopharmacological data, the leaves or the stem bark are also used to treat diarrhea, spasm, pneumonia and bite from animals. In Uganda, S. barteri is reported to reduce dog insensitivity, tiredness and aggressiveness [8]. In spite of the use of S. barteri in traditional medicine, scientific data on the plant is limited. Also, systematic evaluation of its toxic effects is lacking. Therefore, this study was designed to investigate the acute and sub-acute toxicity of S. barteri stem bark extract. Methods Plant material The stem bark of S. barteri was collected in Baleveng, Menoua Division, West Region of Cameroon, in March 2010. Identification of the plant was done at the National Herbarium, in Yaounde-Cameroon, using a voucher specimen registered under the reference HNC N° 26155/RSF-Cam. Page 2 of 9 the freezer at 4°C for further studies. Phytochemical analysis of this extract was performed by standard chemical reaction methods [9]. Experimental animals Fifty Swiss albino mice (25 males and 25 females, 8 - 10 weeks old) weighing 18-24 g, and 50 Wistar albino rats (25 males and 25 females, 8 - 10 weeks old), weighing 120-185 g were used for acute and sub-acute toxicity studies respectively. These animals were bred in the animal house of the University of Dschang and housed in plastic cages under normal laboratory conditions (12 hr light/dark cycle: 23 ± 2°C). They were fed with standard diet. Food and water were given ad libitum to all animals used for the experiments. They were handled according to standard protocols for the use of laboratory animals. The studies were conducted according to the ethical guidelines of the Committee for Control and Supervision of Experiments on Animals (Registration no. 173/CPCSEA, dated 28 January, 2000), Government of India, on the use of animals for scientific research. Toxicological investigations Acute toxicity study Fifty mice were randomly allocated into five groups of ten animals each (5 females and 5 males. Group I (Control) was administered orally with vehicle (2.5% (v/v) DMSO/tween 80). Remaining groups (II, III, IV and V)) were administered with geometric increased doses of 2000, 4000, 8000 and 16000 mg/kg body weight of S. barteri extract respectively via gastric intubation. Those doses were chosen after several screenings on mice. They were prepared using 2.5% (v/v) DMSO/tween 80 and the administered volume was not more than 1 ml as a unique administration. The experimental animals were deprived of food for 18 hr prior to extract administration. They were observed continuously for 3 hr thereafter for activity (locomotion), reaction to noise, reaction to pinch, state of excrements and mortality. After this period, the animals were given food and water ad libitum. Dead animals in each group were noticed within 48 hr following the administration of the extract. The surviving animals were monitored daily for 14 days for changes in body weight, food and water consumptions [10]. Preparation of plant extract S. barteri stem bark were air-dried at room temperature (23 ± 2°C) and milled to coarse particles. A 100 g sample of the powdered material was macerated three times at room temperature in 500 ml of a mixture of methylene chloride/methanol (1:1) for 48 hr, and then filtrated. The filtrate was concentrated using a rotary evaporator (Büchi R200) and the obtained volume was later dried at 50°C to yield 10.05 g of extract. The extract was kept in Sub-acute toxicity Fifty albino rats of both sexes were used. They were grouped into five groups of ten animals each (5 males and 5 females). The control group (Group 1) received orally throughout the experiment a solution of 2.5% (v/ v) DMSO/tween 80. The test group (2, 3, 4 and 5) received the plant extract at 100, 200, 400 and 800 mg/kg body weight. The administration of various doses of the Atsafack et al. BMC Complementary and Alternative Medicine (2015) 15:44 Page 3 of 9 extract was done by gastric intubation once a day, for 28 consecutive days [11]. (Olympus CH02). Any alterations compared to the normal structures were registered [14]. Food intake and weight gain estimation Statistical analysis Food intake and weight gain was recorded every two days during the experimental time. Results were expressed as mean value ± standard deviation (S.E.M.). Within group, comparisons were performed by the analysis of variance using ANOVA test. Significant difference between control and experimental groups were assessed by Waller Duncan-test. Sample collection Rats were fasted overnight on the 28th day and urine was collected from individual metabolic cages, centrifuged and store at +4°C for 24 hours. Upon fasting, the blood samples were collected by cardiac puncture into heparinized and non-heparinized tubes from chloroform anaesthetized rats. Animals were further sacrificed and used for gross pathological examinations and relative organ indices determination. Results Qualitative phytochemical screening Phytochemical screening of the stem bark methanol/ methylene chloride extract of S. barteri revealed the presence of saponins, anthraquinons and anthocyanins while alkaloids, phenols, sterols and triterpenes were not detected. Haematological analysis The heparinized blood was used for hematological analysis (hematocrit, total red cell (RBCs), total white blood cell (WBCs), lymphocytes, neutrophils, monocytes, eosinophils and basophils) [12]. Biochemical analysis The non-heparinized blood was allowed for complete clotting and then centrifuged at 3000 × g for 5 min. The supernatants (serum samples) were aspired and frozen at -15°C. The serum was assayed for creatinine, aspartate amino transferase (AST), alanine amino transferase (ALT), total cholesterol, high density lipoprotein (HDL), triglycerides and total protein using commercial kits (IMNESCO GmbH, Germany). Urine was assayed for total protein and creatinine using the same commercial kits. Acute oral toxicity Mice behavior was affected in both sexes by acute treatment with S. barteri extract. From 4 000 mg/kg, a reduction of locomotion, reaction to noise and reaction to pinch were noticed. No death were recorded within 48 hours after administration of the extract in animals of both sexes at doses less than or equal to 16 000 mg/kg of body weight. Food consumption recorded during the periods of observation following the administration of extract is presented in Table 1. A significant (P < 0.05) reduction of food consumption was noticed for all treated mice (i.e. from 2000 mg/kg). The decrease was more pronounced as the doses increased. The weights of the experimental animals recorded during the two weeks of observation are presented in Tissues proteins analysis Immediately after blood collection, the liver, lungs, heart, kidneys, spleen, testis and ovaries were isolated, freed of blood, and weighed using an electronic balance (Mettler PE 160, France). A section of each organ was used for estimation of protein concentration. For this purpose, the homogenate of each organ was prepared in 0.9% NaCl solution at 10% (i.e. 10 g organ in 100 ml of solution). The protein concentrations were determined by the Biuret method [13]. Table 1 Effect of daily intake of the methanol/methylene chloride stem bark extract of S. barteri on food consumption in mice according to sex and dose Sexes Food consumption (g) Week 1 Male Histopathological study Immediately after collecting the blood samples, vascular perfusion was performed for the organ mentioned above and tissue section were further performed (5-micron thickness). These tissues were further fixed in 10% formalin and then, embedded in paraffin for histopathological analysis. They were routinely stained with haematoxylin and eosin (H & E), and examined under a light microscope Dose (g/kg) Female Week 2 a 5.38 ± 0.31a 2 b 4.07 ± 0.13 4.76 ± 0.23b 4 4.08 ± 0.22b 4.64 ± 0.12b 8 c 2.83 ± 0.49 3.58 ± 0.18c 16 2.54 ± 0.38c 3.30 ± 0.08c 0 a 5.41 ± 0.26 6.32 ± 0.13a 2 4.50 ± 0.33b 5.21 ± 0.44b 4 c 3.94 ± 0.46 4.70 ± 0.09c 8 3.75 ± 0.47c 4.26 ± 0.15d c 4.00 ± 0.88d 0 16 4.88 ± 0.16 3.71 ± 0.52 Data are expressed as mean ± S.E.M. n = 5. Values for a given group in a line followed by same letter as superscript are not significantly different according to Waller Duncan’s multiple comparison test (P < 0.05). Atsafack et al. BMC Complementary and Alternative Medicine (2015) 15:44 Figure 1. Mice that received the extract at doses of 8000 and 16000 mg/kg showed a reduction in body weight all over the experimentation time in both sexes, with the reduction being more visible during the first week of experimentation. Sub-acute toxicity General signs No death or significant change in general behavior or other physiological activities were observed during the treatment period either in the controls group or in the extract treated groups. Food intake, weight gain and organ indices The extract did not affect food consumption of male rats but the females were negatively affected during the second week of treatment (Table 2). Throughout the experiment, weight gain decreased in female rats from 400 mg/kg but the males were not affected (Figure 2). The results of the effects of S. barteri extract on relative organ indices of both male and female rats are summarized in Table 3. There were no significant changes in the lung, kidney, and ovaries or testis to body weight ratios in both groups. However, the extract significantly increased heart and spleen to body weight in both male and female. The significant increase in liver to body weight was observed in male. Hematological parameters Hematological analysis indicated that hematocrit, red blood cells count (RBCs), lymphocytes, eosinophils, basophils and monocytes were not affected in males (Table 4). However, total WBCs significantly decreased in both groups with females being more affected. Page 4 of 9 Similarly, neutrophils, basophils and monocytes significantly decreased in female from 200 mg/kg b.w. Biochemical parameters Biochemical values of rats treated with the methylene chloride/methanol extract from S. barteri are shown in Table 5. This extract did not affected serum creatinine of animal of both sexes, although, a decrease in the urine creatinine level was noted. Total cholesterol and urinary proteins was not affected in both males and females. Triglycerides decreased significantly in females. HDL-cholesterol increased significantly only in females while LDL-cholesterol decreased significantly in males and females. ALT and AST levels significantly increased in both sexes. Serum proteins increased significantly while hepatic proteins, spleen proteins and testis/ovaries proteins decreased significantly. Histopathology analysis Histopathological analysis of organs portions after treatment with S. barteri stem bark extract revealed varying effect (Figure 3). At the level of the liver, vascular congestion, leucocytes infiltration, periportal inflammation and vacuolization of hepatocytes were noted in both sexes. Leucocytes infiltration and inflammatory peribronchial were noticed on the lungs. Inflammatory peribronchial and merely joined basal membrane seminar tube were observed on lungs and testis respectively. At the level of the kidney, a congestion of glomeruli and a widening of the urinary space in the 800 mg/kg treated rats as compared to the control group were observed. Discussion Although significant advances have been made in the development and application of in vitro toxicity assays, Figure 1 Body weight evolution of mice in acute toxicity of the methylene/chloride extract of S. barteri. Atsafack et al. BMC Complementary and Alternative Medicine (2015) 15:44 Page 5 of 9 Table 2 Effect of daily intake of the methylene chloride /methanol extract of S. barteri on food consumption in rats according to sex and dose Sexes Male Female Doses (mg/kg) Food consumption (g) Week 1 Week 2 Week 3 Week 4 0 23.28 ± 2.22a 28.99 ± 1.82a 28.65 ± 3.54a 21.97 ± 1.94a 100 22.27 ± 2.21a 30.64 ± 0.84a 28.39 ± 0.67a 24.35 ± 2.57a 200 a 23.16 ± 1.98 a 31.40 ± 1.33 a 28.44 ± 2.17 22.05 ± 2.82a 400 21.61 ± 1.99a 30.07 ± 1.10a 26.93 ± 2.18a 22.24 ± 2.50a 800 a 24.18 ± 1.85 a 28.09 ± 2.40 a 26.58 ± 3.18 23.39 ± 2.93a 0 20.85 ± 0.58a 26.31 ± 1.41ab 23.92 ± 1.18a 21.97 ± 2.41a a a a 100 20.24 ± 1.25 27.59 ± 1.82 23.62 ± 1.85 23.35 ± 1.39a 200 21.35 ± 1.13a 27.64 ± 1.71a 23.32 ± 1.61a 22.05 ± 2.39a 400 a 21.89 ± 1.20 b 24.49 ± 2.35 a 22.57 ± 3.56 22.24 ± 2.70a 800 21.82 ± 2.07a 24.13 ± 1.42b 22.15 ± 2.33a 23.39 ± 2.75a Data are expressed as mean ± S.E.M. n = 5. Values for a given group in a line followed by different letter as superscript are significantly different according to Waller Duncan’s multiple comparison test (P < 0.05). in vivo safety evaluation remains the most useful tool for identifying target organ toxicity [15]. The rat has been the species of choice for the vast majority of preclinical toxicology studies performed in the evaluation of pharmaceutical candidates. Recent finding revealed that mouse is a suitable model for very early safety assessment since earlier identification of preclinical toxicities are generally predictive of human toxicity and could save time, money, and effort spent [16]. The acute toxicity study showed no mortality at a dose limit of 16000 mg/kg b.w. by oral administration. The extract S. barteri is therefore relatively harmless based on Hodge and Sterner Scale [17]. However, the reduction in mice activity and reaction to noise may be due to depressant and sedative effect on the central nervous system [18]. The reduction of reaction to pinch and reactivity may be due to its inhibitory action on nocireptors or inhibition of the production of algogenic substances (prostaglandins, histamines), or inhibition of the pain signal transmission at the central level [19]. Phytochemical studies of stem bark of S. barteri revealed the presence of saponins and such substances may provoke anorexia and weight loss in animals [20]. Their presence in this plant could justify the decrease of both food consumption and weight loss observed during acute toxicity study in mice. Improvement of weight gain noted in the second week Figure 2 Body weight evolution of rats in sub-acute toxicity of the methylene/chloride extract of S. barteri. Atsafack et al. BMC Complementary and Alternative Medicine (2015) 15:44 Page 6 of 9 Table 3 Relative organ weights indices (g/100g) of rats in sub-acute toxicity of the methylene Chloride/methanol extract of S. barteri Male Female Doses (mg/kg) Liver Spleen Kidneys Lungs Heart Testis/ovaries Control 3.68 ± 0.25b 0.32 ± 0.06b 0.82 ± 0.04a 0.67 ± 0.03a 0.34 ± 0.03c 1.21 ± 0.06a 100 3.84 ± 0.27b 0.37 ± 0.03a 0.79 ± 0.04a 0.68 ± 0.07a 0.37 ± 0.04b 1.17 ± 0.07a 200 a 4.10 ± 0.65 a 0.37 ± 0.03 a 0.79 ± 0.08 a 0.68 ± 0.03 b 0.38 ± 0.02 1.16 ± 0.03a 400 4.11 ± 0.39a 0.37 ± 0.05a 0.79 ± 0.04a 0.69 ± 0.13a 0.40 ± 0.05a 1.16 ± 0.07a 800 a 4.63 ± 1.35 a 0.37 ± 0.06 a 0.79 ± 0.26 a 0.70 ± 0.17 a 0.40 ± 0.06 1.16 ± 0.35a Control 4.19 ± 0.36a 0.28 ± 0.08c 0.89 ± 0.08a 0.69 ± 0.14a 0.34 ± 0.03c 0.07 ± 0.02a a c a a b 100 4.20 ± 0.98 0.28 ± 0.06 0.89 ± 0.02 0.68 ± 0.14 0.37 ± 0.04 0.07 ± 0.02a 200 4.32 ± 0.33a 0.31 ± 0.04b 0.90 ± 0.07a 0.68 ± 0.10a 0.40 ± 0.02a 0.07 ± 0.01a 400 a 4.30 ± 0.75 b 0.31 ± 0.02 a 0.90 ± 0.08 a 0.69 ± 0.04 a 0.41 ± 0.03 0.06 ± 0.01a 800 4.31 ± 0.50a 0.34 ± 0.07a 0.91 ± 0.06a 0.69 ± 0.06a 0.42 ± 0.05a 0.06 ± 0.02a Data are expressed as mean ± S.E.M. n = 5. Values for a given group in a line followed by different letter as superscript are significantly different according to Waller Duncan’s multiple comparison test (P < 0.05). may be justified by the biotransformation and elimination of the responsible compounds contained in absorbed extract. The daily oral administration of the CH2Cl2/MeOH extract of S. barteri stem bark for 28 days did not affect red blood cells, suggesting that oral administration of this extract has no oxygenation and anaemia risk [21,22]. However the decrease in white blood cells indicates that the 28- day’s administration of this extract resulted in the weakening of the immune system [20]. The decrease of neutrophils, basophils and monocytes thus observed may be related to leucocyte infiltrations in the liver and lung revealed by histopathological analysis of these organs. A significant decrease of hepatic proteins levels was noted, moreover, liver relative weight also increased. These parameters are indicators of hepatic toxicity. Furthermore, a significant increase of AST and ALT in serum was also observed. It is well known that many toxic compounds accumulate in the liver where they are detoxified [23]. Liver damage and its recovery are usually assessed by measuring the level of serum transaminases, particularly ALT. Indeed, changes in their serum level are biological markers of liver dysfunctioning and/or Table 4 Hematological parameters of rats in sub-acute toxicity of the methylene chloride/methanol extract of S. barteri Sexes Parameters studied 6 Control 3 a Total RBC (x 10 /mm ) 3 Male 3 3 200 mg/kg 400 mg/kg a a a 3.84 ± 0.56 a 3.80 ± 0.37 3.73 ± 0.47a 3.78 ± 0.55 226.00 ± 19.50 222.00 ± 16.10 218.00 ± 12.07 214.00 ± 10.25 194.00 ± 16.46b Hematocrit (%) 45.40 ± 5.63a 50.00 ± 2.73a 48.40 ± 1.81a 49.20 ± 3.96a 49.20 ± 3.56a a a 800 mg/kg Total WBC (x 10 /mm ) 1&110 /mm ) a a 0.80 ± 0.44 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.44a Neutrophils (%) 26.60 ± 4.77a 27.00 ± 5.00a 27.20 ± 4.14a 27.00 ± 7.00a 27.60 ± 3.03a a Basophils (%) 0.40 ± 0.54 Monocytes (%) 6.20 ± 1.30a 3 a 3 61.00 ± 2.34a 61.80 ± 8.13 3.35 ± 0.50a 3.37 ± 0.36a 248.00 ± 13.82 246.00 ± 20.74 244.00 ± 11.40 168.00 ± 8.34 150.00 ± 20.00b Hematocrit (%) 46.40 ± 2.79a 48.60 ± 5.32a 48.00 ± 5.43a 49.40 ± 3.71a 49.80 ± 2.59a 1.40 ± 0.54 1.20 ± 0.44 1.20 ± 0.44 1.200 ± 0.00 1.17 ± 0.00a Neutrophils (%) 30.00 ± 5.33a 29.20 ± 2.50a 25.80 ± 1.10b 24.60 ± 2.61b 23.60 ± 3.03b bc bc a Basophils (%) 1.60 ± 0.54 Monocytes (%) 6.20 ± 0.77a a b Eosinophils (%) Lymphocytes (%) a a 3.30 ± 0.40a Total WBC (x 10 /mm ) a a 5.40 ± 0.19a a 64.80 ± 4.55 3.37 ± 0.24a a 5.40 ± 0.07a a 65.60 ± 2.96 0.40 ± 0.30a 0.40 ± 0.44 5.60 ± 0.55a a 3.39 ± 0.42a a 0.41 ± 0.44 6.20 ± 1.30a 66.00 ± 5.00 a a 0.40 ± 0.54 a Total RBC (x 106/mm3) a a Eosinophils (%) Lymphocytes (%) Female 3.90 ± 0.22 3 100 mg/kg 1.20 ± 0.44 5.20 ± 0.83ab a 61.80 ± 5.40 a 64.00 ± 1.58 a 1.00 ± 0.70 4.60 ± 1.51b c 0.60 ± 0.54d 0.80 ± 0.44 4.20 ± 1.30b a 65.40 ± 3.58 4.60 ± 1.51b a 65.20 ± 2.58 65.20 ± 2.64a Data are expressed as mean ± S.E.M. n = 5. Values for a given group in a line followed by same letter as superscript are not significantly different according to Waller Duncan’s multiple comparison test (P < 0.05).WBCs = white blood cells, RBCs = red blood cells. Atsafack et al. BMC Complementary and Alternative Medicine (2015) 15:44 Page 7 of 9 Table 5 Biochemical parameters of rats in sub-acute toxicity of the methylene/chloride extract of S. barteri Male Female Parameters studied control 100 mg/kg 200 mg/kg 400 mg/kg 800 mg/kg Urinary creatinine (mg/dl) 38.64 ± 3.80a 37.76 ± 3.28a 37.44 ± 3.14a 30.96 ± 1.07b 28.96 ± 3.40b Serum creatinine (mg/dl) 21.04 ± 1.02a 24.60 ± 2.47a a 24.80 ± 4.01a a 24.80 ± 4.01a 24.84 ± 3.20a Total cholesterol (mg/dl) 122.30 ± 3.37 122.80 ± 3.47 121.92 ± 5.14 121.84 ± 2.65 121.73 ± 1.78a HDL cholesterol (mg/dl) 90.42 ± 7.31a 90.73 ± 7.18a 90.95 ± 6.51a 92.86 ± 4.39a 93.73 ± 5.12a a a a LDL cholesterol (mg/dl) 4.15 ± 0.60 Triglycerides (mg/dl) 141.62 ± 5.38a ab 4.31 ± 1.56 b 3.45 ± 1.23 138.80 ± 8.20a c a 137.60 ± 3.10a 137.82 ± 0.35a 45.88 ± 1.56 52.00 ± 2.19 54.43 ± 7.36 66.07 ± 5.78 66.24 ± 5.29a AST (U/L) 7.60 ± 1.10b 9.65 ± 0.95ab 9.77 ± 0.73a 9.79 ± 2.50a 9.91 ± 0.63a Serum protein (mg/dl) 8.35 ± 0.32 Urinary protein (mg/dl) 3.48 ± 0.81a b 133.00 ± 3.15a ALT(U/L) c b 1.40 ± 0.17b 1.41 ± 0.29 c b 8.68 ± 1.22 a 11.37 ± 1.83 3.42 ± 0.29a a a 3.35 ± 0.45a 3.09 ± 0.56a 562.12 ± 71.22 436.16 ± 39.54 348.67 ± 24.80 273.41 ± 33.30 282.62 ± 32.76d Spleen protein (mg/g) 332.00 ± 31.87a 291.01 ± 27.49ab 290.00 ± 32.62ab 280.00 ± 26.53b 280.00 ± 29.60b a c 3.06 ± 0.83a Hepatic protein (mg/g) a b 14.66 ± 1.76a 13.73 ± 0.79 b d b 54.69 ± 8.33 53.49 ± 4.25b Testis protein (mg/g) 87.10 ± 11.07 84.48 ± 5.43 63.74 ± 13.74 Urinary creatinine (mg/dl) 64.32 ± 6.16a 63.24 ± 5.06a 54.52 ± 2.30b 48.68 ± 3.30c 48.40 ± 0.35c a a a a Serum creatinine (mg/dl) 31.08 ± 3.00 Total cholesterol (mg/dl) 119.60 ± 2.58a 31.44 ± 2.79 31.04 ± 2.10 31.68 ± 1.24a 121.73 ± 5.15a 121.73 ± 5.15a 122.34 ± 3.01a 122.59 ± 4.99a 73.26 ± 1.18 77.41 ± 7.43 96.44 ± 2.72 94.44 ± 7.77a 26.98 ± 5.44a 30.98 ± 5.10a 28.71 ± 4.39a 13.05 ± 2.57b 12.16 ± 2.48b a a a b HDL cholesterol (mg/dl) 71.61 ± 3.35 LDL cholesterol (mg/dl) b 31.44 ± 1.20 b b a Triglycerides (mg/dl) 96.60 ± 8.90 87.40 ± 7.67 87.00 ± 9.78 71.80 ± 8.08 68.81 ± 4.00b ALT(U/L) 42.58 ± 9.10b 49.57 ± 2.82a 49.60 ± 5.37a 49.65 ± 8.94a 49.91 ± 4.38a bc b a c AST (U/L) 9.10 ± 0.80 Serum protein (mg/dl) 7.13 ± 0.61b a 9.58 ± 0.76 10.38 ± 1.36 11.96 ± 0.99 12.61 ± 0.99a 8.10 ± 0.20b 12.56 ± 0.97a 12.79 ± 1.0 a 13.34 ± 0.83a a 3.34 ± 0.56a a a Urinary protein (mg/dl) 3.30 ± 10.59 3.23 ± 0.37 3.33 ± 0.14 3.35- ± 0.17 Hepatic protein (mg/g) 645.02 ± 19.55a 527.62 ± 36.79b 380.26 ± 26.11c 359.04 ± 43.43c 350.88 ± 27.47c a b b b Spleen protein (mg/g) 335.20 ± 34.45 284.00 ± 26.08 288.00 ± 27.27 272.40 ± 37.64 268.00 ± 17.44b Ovaries protein (mg/g) 546.00 ± 39.77a 480.00 ± 84.85ab 451.20 ± 31.29 b 449.30 ± 44.18b 416.68 ± 24.99b Data are expressed as mean ± S.E.M. n = 5. Values for a given group in a line followed by different letter as superscript are significantly different according to Waller Duncan’s multiple comparison test (P < 0.05). ALT = alanine transaminase; AST = Aspartate transaminase; HDL = high density lipoprotein; LDL = low density lipoprotein. damage [24]. Thus, S. barteri extract may be associated with hepatotoxicity. These findings were further confirmed by the histopathological studies on the liver which revealed marked necrosis, vascular congestions, peri- portal inflammations and cell vacuolizations. Urine creatinine decreased while serum creatinine was not affected. Creatinine is a marker of kidney toxicity, its levels increased in the serum when the cortex and/ or the glomerula are damaged [25]. Glomerula damage is also indicated by the increase of the urine protein levels [26]. No variation of serum creatinine and urine protein levels indicates that the kidneys are normal as shown by histopathological study. A significant increase in HDL-cholesterol levels in the treated females and reduction in LDL-cholesterol and triglycerides levels in some treated animals were observed. This showed that the extract had some beneficial effects by reducing cardiovascular risk factors, which contribute to death of diabetic patient [27]. Histopathological examination revealed many abnormalities. Vascular congestions on the liver section could be due to the inflammation, blockage or vasoconstriction action of the S. barteri extract on the walls of blood vessels. This extract could contain some substances capable of acting like non steroidal anti-inflammatory drugs that provoke hypersensibility reaction which led the lung and liver inflammations observed [28]. The presence of the empty vacuole-like spaces in the hepatocytes could be due to abnormal infiltration of extracellular substances into the hepatocytes or to malfunctioning of the latter [20]. The joined basal membrane of the seminar tube could be due to cellular retraction with reduction of cytoplasmic compounds or cells loss caused by apoptosis [29]. It may also be due to the osmotic gradient modification through the cytoplasmic membrane [30]. The congestion of glomeruli and widening of the urinary space was observed. Drug concentration in the blood is affected by capillary constriction, leading to a decrease Atsafack et al. BMC Complementary and Alternative Medicine (2015) 15:44 Page 8 of 9 Figure 3 Histology of organs of the control rats and those exposed of Schefflera barteri for 28 days. The organs of the rats exposed S. barteri extract showed: Light vacuole-like space (l) and congestions (c) in liver, inflammations (i), peri-portal /peri-bronchial and leucocytes infiltration (if) in both the liver and lungs, fusion (fu) of basal membranes of seminar tubes (m) in testis; congestion of glomeruli and widening of the urinary space, normal spleen and ovary. Histological analysis of the organs of the control rats showed normal structure: hepatocytes (h), alveols (a), bronchi (b), glomeruli (g), membrane, follicle (f). in glomerular filtration of that drug which minimizes its effect and protects the tubular cells [31]. This may affect the shrinkage and atrophy of the glomeruli. At the same time, the mesangial cell processes may be retracted due to the contraction of their filaments, which may be stimulated by angiotensin II present in these cells. Conclusion S. barteri extract is relatively harmless by acute oral administration. Although sub-acute administration is associated with side effects on the central nervous system, immune system, liver and testis. Therefore, for a S. barteri extract based treatment, the dose, frequency and duration of the treatment should be carefully defined to avoid adverse effect of the plant extract. Competing interests The authors declare that they have no competing interests. Authors’ contributions SSA is the field investigator. RANN and JRK designated the study and supervised the work. RSM is co-field investigator and conceived the manuscript. ATT prepared the plant extract. JDT contributed to the phytochemical studies. HNM Atsafack et al. BMC Complementary and Alternative Medicine (2015) 15:44 and MLKM contributed in manuscript writing and editing. RMEE and LB revised the manuscript. All authors read and approved the final manuscript. Acknowledgements This works was supported by AIRES-Sud (Appuis Intégrés pour le Renforcement des Equipes Scientifiques du Sud), a program of the French Ministry of Foreign and European Affair implemented by the “Institut de Recherche pour le Developpement” (IRD-DSF). We are grateful to Dr Désiré Dzeufiet for histopathological analysis, Pr. Antoine Mvondo Ze, and Pr. Telesphore Nguelefack for their technical assistance. Author details Laboratory of Microbiology and Antimicrobial Substances, University of Dschang, P.O. 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