Effect of aqueous leaves extract of Costus afer Ker Gawl (Zingiberaceae) on the liver and kidney of male albino Wistar rat.

BACKGROUND: The use of medicinal plants in Nigeria has significantly increased over recent years as it is easily accessible, cheap and the strong belief that herbal remedies are natural and therefore non toxic. AIMS: This study aims to investigate the sub-chronic toxicity (28-day) of the aqueous extract of Costus afer Ker Gawl leaves on the liver and kidney of male albino Wistar rats.
MATERIALS AND METHODS: A total of 20 male albino Wistar rats (113-205 g) divided into four groups of five weight-matched animals each, were used for the study. Group 1 received standard feed and water ad libitium and served as the control. Group 2, 3 and 4 received 375, 750 and 1125 mg/kg of aqueous extract of C. afer leaves respectively. The animals were sacrificed under ether anesthesia and the organs were harvested, weighed and histopathological studies carried out. The effect of C. afer on the hepatic biomarkers aspartate aminotransferase (AST), alanine aminotransferase (ALT); alkaline phosphatase (ALP); triglyceride (TG); total bilirubin (TB); conjugated bilirubin (CB); albumin (ALB) and kidney biomarkers urea, creatinine, sodium, potassium and bicarbonate were investigated. STATISTICAL ANALYSIS: Data were evaluated using Mann Whitney. If P ≤ 0.05 groups were considered to be significantly different.
RESULTS: C. afer contained alkaloids, saponins, flavonoids, anthraquinones, cardiac glycosides, terpenoids, phenolic compounds and tannins. The average body, organ, relative weights, feed and fluid intake showed no significant changes (P > 0.05) when compared to the control. The liver function tests (ALT, ALP, AST, CB, TB and ALB) showed significant differences (P < 0.05) in the test groups when compared with the control while TG showed no statistical difference (P > 0.05). The kidney function tests (urea, creatinine, sodium, potassium and bicarbonate) showed no significant differences (P > 0.05) in the test groups when compared to the control.
CONCLUSION: Costus afer may be hepatotoxic but non-toxic to the kidney.

INTRODUCTION

The plant Costus afer Ker-Gawl (Costaceae) is among 150 species of stout, perennial and rhizomatous herbs of the genus Costus.[1] It can be found in the forest belt of Senegal, South Africa, Guinea, Niger, Sierra Leone, Ghana, Cameroon and Nigeria.[1] The plant is commonly known as ginger lily or bush cane. It is known as “Okpete” or “Okpoto” in Igboland, “Kakizawa” in Hausa “tete-egun” in Yoruba and “Mbritem” in Efik all in Nigeria.[2] Anglophone Cameroon calls it “Monkey sugar cane”. It bears white and yellow flowers. The stem, seeds, leaves and rhizomes are harvested from the wild plant and they contain several bioactive metabolites. This plant is used as a remedy for cough, inflammation, arthritis, as laxative, aparient, purgative, diuretic, in rheumatism and treatment of several other diseases.[3] In Nigeria, the plant extract is used as fodder to treat goats with retained placenta. The decoction of the stem or powdered fruits is used as cough medicine. Whole its boiled root is applied to cuts and sores.[3] Soothing formulation for rheumatic pains is prepared with boiled leaves.[3] It has been shown to reduce carageenan-induced edema in the rat paw, and check diarrhoea caused by arachidonic acid and castor oil.[4] It ameliorated all signs associated with adjuvant-induced polyarthritis in rats and a dose of 100-300 mg/kg daily is well-tolerated in humans.[4] In Ohafia, Abia State, Nigeria and in some parts of Anglophone Cameroon, the decoction of the plant is administered to diabetic patients to alleviate the clinical signs.[5] An infusion of the inflorescence is taken to treat tachycardia. The same infusion or a rhizome infusion is taken to treat stomach complaints. A stem decoction, mashed stem or the pounded fruit, sometimes mixed with sugar cane juice, are taken to treat cough, respiratory problems and a sore throat.[4]

In Nigeria, the debarked stem is chewed to treat nausea and to quench thirst. A cold water extract of the stem is taken to treat small epileptic attacks. Rhizome pulp is applied to abscesses and ulcers to heal them, applied to teeth to cure toothache and mixed with water to treat diarrhea and amoebic dysentery. A rhizome decoction or the raw rhizome is taken to treat leprosy and venereal diseases. Leaf sap is used as eye drops and as nose drops to treat headache with vertigo. It is also used to treat edema and fever. Leaf sap or a rhizome decoction is taken to treat malaria. Stem sap is applied to treat urethral discharges, venereal diseases, jaundice and to prevent miscarriage. Stem sap is acid and rubefacient and burns on open wounds, but it is also anodyne and healing and is applied to different skin ailments. The stem decoction is widely taken to treat rheumatoid arthritis. An infusion of the dried aerial parts is taken to treat hypertension. The powdered stems is used as an enema to treat worms and hemorrhoids. The pulped stem taken in water is strongly diuretic.[4] Other species of the genus also have medicinal effects, e.g. Costus lucanusianus is used in the Ivory Coast for the treatment of impending abortion. It also exhibits uterine relaxant activity. Costus schlechteri and C. afer are used in the treatment of diabetes mellitus.[4]

The liver holds a unique position in the human body due to its gastrointestinal connections and varied functions. Liver receives a large amount of nutrients and noxious compounds entering the body through the digestive tract and portal vein.[5] As a result of its continuous involvement, it is susceptible to toxic injuries caused by certain agents and hence any damage to hepatic cells will disturb body metabolism.[6] In spite of the tremendous advancement in modern medicine, there is hardly any drug that can stimulate liver function, offer protection to the liver from damage or help in regeneration of hepatic cells.[7] There are reports of liver damage due to natural and drug treatments forcing discontinuation of treatment and the urgency of re-evaluating the pharmacokinetics and pharmacodynamics of these compounds.[89]

Nephrotoxicity is a major complication characterized by morphological destruction of intracellular organelles and cellular necrosis, followed by functional alterations including the depletion of the antioxidant defense system and mitochondrial damage.[10] Oxidative damage is thought to be one of the main mechanisms involved in nearly all chronic renal pathologies.[11] Kim-1 is a more sensitive biomarker, which out performs traditional biomarkers of kidney injury.[12] Kim-1 is expected to represent an important genomic marker for the potential screening of nephrotoxicants.

Considering the complexity of herbal medicines, it is now necessary to evaluate their safety before the clinical use. In spite of various studies conducted on multiple species of C. afer plant, to the best of our knowledge, information available regarding the safety of C. afer Ker Gawl following repeated exposure of aqueous extracts in experimental animals is scarce. The present study was designed to investigate the hepato and nephrotoxic effects of aqueous extracts of leaves of C. afer Ker Gawl. Histopathological examinations as well as biochemical parameters were used to assess toxicity.

MATERIALS AND METHODS

Sample collection

C. afer leaves were collected from a farmland in Choba campus of the University of Port Harcourt Rivers State, Nigeria.

Sample identification

The plant was identified and authenticated by A.O. Ozioko, International Centre for Ethnomedicine and Drug Development (INTERCEDD), University of Nigeria Nsukka, Enugu State and the Voucher Number is INTERCEDD/033 and Professor M.E. Bassey of Botany department, University of Uyo Akwa Ibom State, Nigeria.

Phytochemical screening

Phytochemical screening of freshly prepared sample of C. afer Ker Gawl was carried out in the Department of Pharmacognosy, University of Port Harcourt, Nigeria employing the of Sofowora.[13]

Animal care and handling

The animals were used for the study were twenty male albino rats (150-200 g) obtained from the Department of Pharmacology Animal House, Nnamdi Azikiwe University, Awka, Nigeria. The male albino Wistar rats were left to acclimatize for 14 days. They were housed in standard cages at room temperature and average humidity. The study was cleared by the University of Port Harcourt's Ethics Committee. The bedding of the cages (sawdust) was changed daily and the cage also washed and disinfected weekly. Feed (Top Feeds Premier Feeds Flour Mills Nig. Plc., Lagos State) and water were made available to them ad libitum.

Acute toxicity

The acute toxicity study of aqueous leaf extract C. afer Ker Gawl was conducted following Lorkes method.[14] The male albino Wistar rats were divided into six groups of five weight-matched animals. The different groups received 100, 200, 400, 800, 1600 mg/kg of aqueous leaf extract C. afer Ker Gawl. Body weight, signs of toxicity and mortality were observed for 24 h and daily for 14 consecutive days.

Subchronic toxicity

Subchronic toxicity testing was conducted according to World Health Organization and Organization for Economic Co-operation and Development guidelines.[15] Rats were divided into four groups of 5 weight-matched animals each. In the present study, the doses of aqueous extract of C. afer Ker Gawl leaves used were 375, 750 and 1125 mg/kg/day which represents 25%, 50% and 75% of LD50 (1500 mg/kg). The aqueous extracts were administered orally to each group of rats daily for 28 days, while the control group received water. The animals were weighed every week and the feed and fluid consumption was measured daily. On the 28th night, the rats were fasted and on the 29th day they were sacrificed under ether anesthesia and their livers and kidneys were harvested. The organ weights were taken. Whole blood was obtained from ocular orbit vein from each of the rats, collected into heparinized bottles and the blood samples (plasma) were analyzed for the activity of the biochemical parameters of the kidney (renal function tests) and liver (liver function tests).

Histopathological analysis

The liver and kidney were fixed in 10% of formaldehyde. After 72 h, the organs were dehydrated in graded alcohol, cleared in xylene and embedded in paraffin. Tissue processing was carried out by an autotechnician and the prepared 5 μm thick sections were mounted on slides and stained with hematoxylin and eosin. Stained sections were microscopically evaluated and the pictures of the slides were taken for comparison.

Statistical analysis

Analysis using the Mann-Whitney test was carried out to examine the statistical significance of differences in the mean levels of the various parameters and biomarkers between the control and test groups using Graph Pad Prism 5.03. P < 0.05 was considered to be statistically significant.

RESULTS

Phytochemical tests

Phytochemical studies of C. afer (leaves) revealed the presence of alkaloids, terpenoids, phenolic compounds, cardiac glycosides, saponins, anthraquinones, tannins and flavonoids.

No death was recorded at all the doses. The results of the in vivo acute toxicity study indicated that the LD50 of the aqueous leaf extract of C. afer is more than 2000 mg/kg.

Sub chronic toxicity

The effect of C. afer Ker Gawl leaves on the absolute and relative organ weight, feed and fluid intake are shown in Table 1. The extract was shown to increase the absolute and relative weight of the liver but had no significant change the absolute and relative weight of the kidney. The feed and fluid intake increased in a dose dependent manner. The extract produced decrease in body weight [Figure 1].

Table 1

Effect of Costus afer Ker Gawl leaves on the absolute and relative organ weight, feed and fluid intake

Figure 1

Effect on the aqueous leave extract of Costus aferon percentage weight again

The extract affected all the liver enzymes measured in a dose dependent manner as can be seen in Table 2. The alanine aminotransferase (ALT), alkaline phosphatase (ALP), conjugated bilirubin (CB), total bilirubin and aspartate aminotransferase (AST) were significantly increased (P < 0.05) while the alb showed a decrease compared with the control. The extract did not affect the kidney parameters measured as all the values were almost the same with the control as shown in Table 3.

Table 2

Effect of aqueous leaf extract of Costus afer Ker Gawl on hepatic biomarkers

Table 3

Effect of different doses of Costus afer Ker Gawl leaves renal function tests

The photomicrographs show that the extract caused histopathological changes in the liver. The degree of inflammation observed was dose dependent. In Figure 2a (control) there was no inflammation or distortion of tissues showing central vein, hepatic sinusoids and hepatocytes were clearly seen. The 375 mg/kg extract treated group Figure 2b show mild inflammation typified by collection of inflammatory cells while Figure 2c i.e. 750 mg/kg extract treated group showed inflammatory cell aggregates. The histopathological changes were most pronounced in Figure 2d (1125 mg/kg of the extract) with areas of perivascular hepatocytic architectural distortion and vacuolar change.

Figure 2

Photomicrograph of liver sections of albino rats stained with Hematoxylin and Eosin (H and E), magnification ×400 for all the groups. (2a) section of the liver shows normal histology for the control group; (2b) section of the liver of the 375 mg/kg treated group showing a collection of inflammatory cells; (2c) section of the liver of the 750 mg/kg treated group showing inflammatory cell aggregates. (2d) A section of the liver of the 1125mg/kg treated group showing perivascular hepatocytes architectural distortion, inflammation and vacuolar change. CV = central vein; HS= Hepatic sinusoid; HC = hepatocytes; CIA = Collection of inflammatory cells; ICA = Inflammatory cells aggregates; PHAD/VC = perivascular hepatocytes architectural distortion and vacuolar change

The kidney of the control group was normal showing numerous renal tubules and interstitial tissues [Figure 3a]. Figure 3b-d show the histology of rat kidney, which received 325, 750 and 1125 mg/kg respectively. Treatment with extract did affect the renal architecture.

Figure 3

Photomicrograph of Kidney sections of albino rats All panels were stained with H and E and magnification ×400; (3a) A section of the kidney of the control group showing normal histology with numerous glomeruli, renal tubules and blood vessels. The interstitial space appears unremarkable. (3b) A section of the kidney of the 375 mg/kg group showing normal histology; (3c) A section of the kidney of the 750 mg/kg group showing normal histology (3d) A section of the kidney of the 1125 mg/kg group showing normal histology. RT = Renal tubules; IS = interstitium

DISCUSSION

The present study has investigated the sub-chronic hepatic and nephrotic effects of aqueous leaf extract of C. afer in male Wistar albino rats. There was a reduction in mean percentage body weight gain of rats that received aqueous leaf extract of C. afer. The alteration in overall body weight or organ-body weight ratio is an indication of impairment in the normal functioning of the organs. Organ weights are widely accepted for the evaluation of test article-associated toxicities.[16] Since there was an increase in relative and absolute organ weight of the liver accompanied by significant change in other biochemical parameters in the treated animals at all tested doses, we may conclude that the extract is hepatotoxic,[1718] but this was not the case with kidney where no significant changes were observed both in the relative and absolute kidney weights and even renal function biomarkers - urea, creatinine, electrolyte estimated in all the doses., This is an indication that C. afer leaves may be non-toxic to the kidney of Wistar albino rats.[1719]

Liver function tests conducted through blood assays give information about the state of the liver, describing its functionality (albumin [ALB]), cellular integrity (transaminases) and its link with the biliary tract (ALP).[8] ALT is the enzyme produced within the cells of the liver and the serum levels of this enzyme increase after liver damage due to increased membrane permeability or liver cell necrosis and cytosol leakage into the serum.[20]

Assessment of liver and kidney function is very important in toxicity evaluation of drugs and plant extracts as they are both necessary for the survival of an organism. AST and ALT activities are commonly measured to monitor liver damage. A mild or higher activity of AST indicates liver injury or myocardial infarction[2122] and the ratio of AST/ALT may be employed in disease diagnosis. An AST/ALT ratio greater than 1 suggests myocardial infarction, while a ratio <1 may be due to the release of ALT from the affected liver.[23] An AST/ALT ratio of more than 2 is indicative of alcoholic hepatitis or cirrhosis.[24] The ratio of AST/ALT ranges from 1.2 to 1.4, an indication of the abnormal functioning of the liver. In the present study, the extract induced hepatic damage to the liver, whereas the kidney was not affected as revealed by the results of blood chemistry analysis. The significant alterations in the indicators of liver damage (ALT, AST, ALP, total and CB, ALB and triglycerides [TG]), suggests that sub-chronic administration of aqueous leaf extract of C. afer Ker Gawl affects hepatocyte function in albino rats. Reduction in ALB and increase in TG are indications of diminished synthetic function of the liver or might be due to impaired hepatocellular function.[2526] The change in serum concentration of ALB and TG in the treated and control groups further suggest that the extract impaired hepatocellular or secretory functions of the liver at all the doses tested an interpretation that is confirmed by the histological analysis, which showed lesions.

The absolute and relative weight of the kidney was not affected when compared to the control.[27] Kidney function was evaluated by means of serum urea, creatinine and blood electrolyte concentrations. Increased blood creatinine is a good indicator of compromised kidney function. In the present study, serum creatinine, urea and electrolytes such as sodium, potassium and bicarbonates were not affected by the extract treatment. However, a slight but not significant increase was observed in the levels of urea, sodium and potassium when compared with the control. These results suggest that the extract does not alter the kidney function, this agrees with the photomicrographs of the kidney with no lesion or inflammation seen. Blood urea nitrogen (BUN) and creatinine are considered as the traditional indicators of kidney damage, however, no change was observed in BUN and creatinine levels in our study.

The functional studies in toxicology should be coupled with the appropriate histological studies, because appropriate morphological studies are useful for the anatomical localization of action of toxin.[28] The photomicrographs show that the extract caused histopathological changes in the liver. The degree of inflammation was observed to be dose dependent. The control group (Group 1) was normal as there was no inflammation or distortion of tissues. The central vein, hepatic sinusoids and hepatocytes were clearly seen. However 375 mg/kg of the extract produced mild inflammation as a collection of inflammatory cells were seen, whereas 750 mg/kg of the extract presented with inflammatory cell aggregates and the highest dose 1125 mg/kg showed the most pronounced histopathological changes as they presented with areas of perivascular hepatocytic architectural disarray/distortion and vacuolar change. The toxic effect of aqueous extract of C. afer on liver may be due to any one or more of the phytochemicals present in the extract. It might also be due to their synergistic effect.

The kidney of the control group was normal showing numerous renal tubules and interstitial tissues. The different doses of the extracts 375 mg/kg, 750 mg/kg and 1125 mg/kg showed normal histological features. Though aqueous leaf extract of C. afer Ker Gawl is of pure natural origin and is easily accessible and may contain biologically active principles, incessant consumption of aqueous leaf extract of C. afer Ker Gawl should be discouraged. Going by the result obtained in this present study, there is an indication of C. afer being potentially hepatotoxic in all doses used and especially under a prolonged usage. The no observed adverse effect level (NOAEL) of C. afer in the liver is below 375 mg/kg while NOAEL in the kidney is above 1125 mg/kg. These values are considered as rather very high and may have serious toxicological implications.