Timothy Auta

1Department of Biological Sciences,Federal University Dutsin-Ma,Katsina State,Nigeria.

Abstract Considering the possible health hazards associated with consumption of aqueous wood-ash extracts,this study investigated the sub-chronic toxicity of aqueous wood-ash extract of Parkia biglobosa on Mus musculus.Parkia biglobosa (5 kg) were collected and aqueous extracts of wood-ash were prepared by percolation method.Doses of 0(control),5,50 and 100 mg/kg,once a day,were administered to mice (n = 10/treatment) 90 days.Blood was collected by retro-orbital phlebotomy and assayed for serum creatinine,aspartate aminotransferase and alanine aminotransferase,and the kidney and liver were excised for histopathology using standard methods.Data were analysed using descriptive statistics and ANOVA at α0.05. Serum creatinine (mg/dL),aspartate aminotransferase (UI/L)and ALT (UI/L) increased with dosage and peaked at 100 mg/kg:2.1 ± 0.2,128.8 ± 8.0 and 58.0 ± 2.9 compared to the control:0.8 ± 0.1,89.8 ± 5.1 and 28.5 ± 1.4.Tubular degeneration and necrosis in the kidney,hepatocyte atrophy and dissociation of cords in the liver were observed at the 100 mg/kg dose level,while control organs had normal tissue architecture.Aqueous wood-ash extracts of P.biglobosa showed sub-chronic toxicity in Mus musculus.

Key words:Wood-ash extract,Parkia biglobosa,Pathology,Biochemistry,Necrosis

Background

Plant extracts have been used for thousands of years for medical,pharmaceutical,sanitary purposes,aromatherapy,phototherapy,perfumery and cosmetic applications besides food and ‘beverage flavoring [1-3].Methods and solvents used in indigenous extraction of plant parts and components primarily depend on the purpose for which the extract is used [4,5],which can be performed by various extraction procedures.Water decoction has been reported to be the most common preparation used by the traditional people in Africa [6-9].Native Americans consume wood ash as an additive for mineral and as bread leaver,while the Chinese mostly consume wood ash for religious ceremonies and for curing incurable diseases [10].In Nigeria,wood-ash extracts were used as food additives or for medicinal purposes (as decoction),which have been a continuous practice among different ethnic groups since the ancestral period [11].Wood-ashes are normally collected from household fireplaces and processed to obtain the extracts,using tap,well,stream or any other source of water.This practice is common among the Gbagyi,Nupe,Ebira and other ethnic groups in North-Central Nigeria/Middle-Belt regions,where wood-ash is used for preparing foods like “Tuwo” (meal prepared using maize/corn flour) and as medicine for stomach ache [11].Wood-ash extracts have been reported to have toxic effects on both male and female reproductive activities as reported in 2016 [12,13].

Several authors have published studies on the use and toxicity of various extracts ofParkiabiglobosa(P.biglobosa).Though in 2008 [14],it was reported the use ofP.biglobosaas herbal medicaments in African countries and its non-toxicity to humans,which differs from literature in 2004 [15] that reported that histopathological changes in the liver and gill of fish exposed toP.biglobosaextracts,indicating subtle cellular damages like necrosis,lesions,oedema and hepatocytes.The seeds extracts have also been reported to have seemingly toxic effects on rats [16].In 2018 [17],it was reported that aqueous wood-ash extract ofP.biglobosaimpaired spermatogenesis in male albino mice.It is apparent from the foregoing that limited information exists on the sub-chronic toxicity of aqueous wood-ash extract ofP.biglobosato mammals.These bring to the fore the need to provide important scientific information on the possible adverse health effects of the aqueous wood-ash extract ofP.biglobosaon mammals.Considering the possible health hazards associated with consumption of aqueous wood-ash extracts,this study was aimed at investigating the subchronic toxicity of aqueous wood-ash extract ofP.biglobosaonMus musculus.

Methods

Collection and processing of the wood-ash extract of P.biglobosa

P.biglobosa(African locust bean) (Figure1) woods were collected as described in 2015 [11],they were properly identified and authenticated at the Herbarium of the Department of Biological Sciences,Ahmadu Bello University,Zaria; where voucher specimenP.biglobosa(PB-324) was deposited.The woods were further processed into ash and extracted using a method described in 2015 [11].

Procurement and acclimatization of Mus musculus for the study

Ethical approval,with reference number UIACUREC/App/2014/006 was obtained from the University of Ibadan Animal Care and Use for Research Ethical Committee.A total of 190 healthy adultMus musculuswere purchased from the animal house of Institute for Medical Advance Research and Training(IMRAT),College of Medicine,University of Ibadan,Oyo State for the range finding and acute toxicity studies.They were housed under standardized(temperature:28-31℃,photoperiod:approximately 12 hours natural light per day) animal house conditions and fed with standard mice feed and water ad libitum.The animals were randomly selected and kept in their cages to acclimatise for 2 weeks before commencement of each experiment,according to Organization for Economic Cooperation and Development guidelines[18].

Range finding test

After the acclimatization period,a preliminary range finding test was carried out to assess the toxicological

Figure1 An African locust bean (Parkia biglobosa)tree

effects of aqueous wood-ash extract ofP.biglobosafollowing the single dose administration regime according to methods described by OECD [19].A total of 9 groups (n = 10/treatment) were used for the range finding test.Mus musculusin group 1 served as the control,while groups 2,3,4,5,6,7,8 and 9 were administered single doses ofP.biglobosaaqueous wood-ash extract of 5,50,100,300,500,1000,1500 and 2000 mg/kg,respectively.The administration was carried out by gavage,using a suitable intubation cannula.Animals were observed individually after dosing during the first 30 mins,and at intervals of 2 hrs during the first 24 hrs,with special attention given during the first 4 hrs and daily thereafter,for forty eight(48) hrs,except where they were found dead.After 48 hrs,the number of deceased mice was counted in each group and percentage mortality calculated [18].

Acute toxicity test

Acute toxicity (14 days) tests to determine the median lethal dose (LD50) were carried out for aqueous woodash extractsP.biglobosa,using OECD guidelines [18].Mice were randomly divided into 6 groups (n =10/treatment),based on their weight (with a weight difference of not more than 20% in each group as recommended by OECD [18].Group 1 served as control while mice in groups 2,3,4,5 and 6 were orally administeredP.biglobosaaqueous wood-ash extract at same 300,500,1000,1500 and 2000 mg/kg dose levels once.They were observed twice daily (9.00 h and 16.00 h) for the first week and once daily subsequently,for general toxic effects such as overt signs of toxicity(salivation,diarrhoea,lachrymation,tremors,ataxia,yellowing of hair,loss of hair,postural abnormalities or behavioural changes),stress (fur erection or exophthalmia),aversive behaviour (biting paw and penis,intense grooming behaviours,scratching behaviour,licking of tail or vocalization) and mortality for 14 days [18].Mortalities were recorded and the median lethal dose (LD50) was computed using probit analysis [20].From the result of a median lethal dose,1/79,1/8and ¼ fractions ofP.biglobosaextract i.e 5,50 and 100mg/kg doses were selected for the lethal studies.

Sub-chronic toxicity test

Sub-chronic toxicity test was conducted for 90 days in accordance with OECD guideline for the testing of chemicals [21],with slight modification where n =10/treatment group,instead of the recommended n =5/treatment group was adopted to increase the chances of mice survival due to a lack of literature on the toxicity of aqueous wood-ash extracts ofP.biglobosa.

A total of 40 mice were used for the sub-chronic toxicity tests and individuals were divided into 4 groups(n = 10/treatment),based on their weight (with weight difference of not more than 20% in each group as recommended by OECD [18].Mice in groups 1 served as control,received distilled water while those in 2,3 and 4 were administered daily oral doses ofP.biglobosaaqueous wood-ash extract at 5,50 and 100 mg/kg respectively for 90 days.

Blood collection and biochemical analysis

At the end of the 90-day exposure periods,blood samples were collected by retro-orbital phlebotomy,using capillary tubes from individual mice into plain Eppendorf tubes for biochemical analysis.Blood samples for biochemical analysis were collected in a plain Eppendorf bottle and allowed to clot (25-30 min)at room temperature (28-30℃) and centrifuged at 3,200 rpm for 15 min.Serum obtained were diluted by a factor of distilled water to the required quantity for the serum biochemistry analysis,depending on the quantity of blood obtained from each mouse.The serum obtained was stored at -20℃ until when analysed.

Aspartate aminotransferase (AST) assay.AST activity was measured using Randox Diagnostics Kit(Cat.No.AS 101) according to manufacturer’s protocol with Jenway Spectrophotometer (6305 model) at Hg 546 nM wavelength.AST activity (U/L) = (Δ OD/min)× 1,745.

Alanine aminotransferase (ALT) assay.ALT activity was measured using Randox Diagnostic Kit (Cat.No.AL 100),according to manufacturer’s protocol with Jenway Spectrophotometer (6305 model) at Hg 546 nM wavelength.

Alkaline phosphatase (ALP) assay.ALP assay was carried out using Randox Diagnostics Kit (Cat.No.AP 542),according to manufacturer’s protocol at 25℃ and wavelength of 405 nm using a 1 cm light path cuvette with a Jenway Spectrophotometer (6305 model).

Creatinine and urea assay.Using Randox kit,levels of creatinine and urea were measured at 37℃ and 546 nM wavelength,according to the manufacturer’s protocol using Jenway Spectrophotometer (6305 model).

Histopathology of kidney and liver.Mice were sacrificed by cervical dislocation and samples of kidney and liver were excised and rinsed in normal saline to remove blood.Samples were fixed in Bouin’s fluid for 24 hrs to enable tissue hardening.They were then transferred into 10% phosphate-buffered formalin,plastic bottles.The tissues were placed into cassettes(caskets) and dehydrated in a graded series of ethanol dilutions and embedded in paraffin wax.Sections of 5 μm were cut and stained with Haemoxylin and Eosin(H&E) and examined using a light microscope at magnifications of 40 and 100 objectives,respectively.

Statistical analysis

Data were expressed as mean ± standard error of the mean.Probit analysis was used to determine the LD50ofP.biglobosaaqueous wood-ash extracts.One-way Analysis of Variance (ANOVA) and Duncan Multiple Range Test (DMRT) were used for analyses of data.The difference between the groups were considered significant atP＜ 0.05.All statistical analyses were conducted with Microsoft Office Excel 2007 and SPSS-16 for Windows.

Results

Mortalities range of 10%-70% was recorded in among mice in the 300-2,000 mg/kg dose of aqueous wood-ash extracts ofP.biglobosa.At the end of the range-finding test,no mortality was recorded in groups administered 0-100 mg/kg of the aqueous wood-ash extract ofP.biglobosaextract.Highest mortality of 70% was recorded in group administered 2,000 mg/kg dose concentrations ofP.biglobosaaqueous wood-ash extract (Table1).Lowest mortality of 10% was recorded in the 300 mg/kg dose concentration group ofP.biglobosa.

Mortalities ranging from 30%-100% were recorded in the 300-2,000 mg/kg dose concentrations (Group 1-6).100% mortalities were recorded in the 1,500 and 2,000 mg/kg dose concentrations,respectively.A 14-day LD50of 396.80 mg/kg was obtained (Table2).

Mortalities ofMusmusculusduring the 90-day subchronic administration of aqueous wood-ash extracts ranged between 10%-50%,with the control group having the lowest mortality and the 100 mg/kg dose group having the highest (Table3).

As presented in Table4,only mice in the 50 mg/kg group (4.0 ± 0.35) showed significant (P＜ 0.05)decrease in hepatosomatic index compared to the control group (4.91 ± 0.21).The relative weight of the kidney was significantly (P＞ 0.05) higher in 5 mg/kg dose group (1.72 ± 0.17) compared to the control group(1.53 ± 0.09).Similarly,mean activities of AST,ALT and ALP,and levels of urea,creatinine,total cholesterol,triglyceride and low density lipoprotein in serum significantly (P＜ 0.05) increased from control group to the highest dose concentration (100mg/kg) at 90 days(Table5).

Significant (P＜ 0.05) increase from control to the highest (100 mg/kg) concentration were:AST ((95.92 ±14.47) U/L to (214.55 ± 16.97) U/L,ALT ((52.33 ± 5.49)U/L to (209.67 ± 23.04) U/L),ALP ((63.83 ± 8.71) U/L to (345.83 ± 19.83) U/L),urea ((43.5 ± 2.59) mg/L to(77.17 ± 6.03) mg/dL) and creatinine ((0.88 ± 0.19)mg/dL to (2.70 ± 0.25) mg/dL).

Table1 Mortalities of Mus musculus administered oral doses of Parkia biglobosa aqueous wood-ash extracts after 48 hrs

Table2 Mortalities among Mus musculus orally exposed to varying concentrations of Parkia biglobosa aqueous wood-ash extracts for 14 days (n=10/treatment)

Table3 Mortalities of Mus musculus exposed to varying concentration of oral doses of the aqueous wood-ash extracts of Parkia biglobosa for 90 days (n=10/treatment)

Table4 Organosomatic indices of Mus musculus administered Parkia biglobosa aqueous wood-ash extract during the sub-chronic study

Table5 Serum biochemical profile of Mus musculus after oral administration of aqueous wood-ash extracts of Parkia biglobosa for 90 days

Several pathological alterations were observed in the kidney and liver of mice administered oral doses,once a day,ofP.biglobosaaqueous wood-ash extract for 90 days.The kidney of the control and the 5 mg/kg dose group,showed well-distended tubules with numerous glomeruli at the end of 90 days.Multiple foci of moderate aggregates of inflammatory cells in the renal interstitium (suggestive of nephritis) and sloughing off of tubular epithelium were observed at the 50 and 100 mg/kg doses,respectively (Figure2).

The histopathological examination of the liver of unexposed mice revealed hepatocytes arranged in cords around the perioral region,with no observed pathological changes.Varying degrees of pathological degeneration ranging from mononuclear cellular infiltration (5 mg/kg) to necrosis (100 mg/kg) (Figure3).

Discussion

In this study,the effects of sub-chronic oral administration with different doses of aqueous woodash extracts ofP.biglobosawere recorded in the mice,as there were toxic effects observed by direct visual observation of the mice throughout the experiments,which were dose and time-dependent.Death and apparent behavioural changes (such as the reduction in water and food in-take and self-isolation) recorded during the course of the experiment,mostly in groups treated with 50 and 100 mg/kg of extracts,as compared to the control group could be associated with the high levels of polycyclic aromatic hydrocarbons and heavy metals [11] present in the extracts.The varying clinical and behavioural changes in mice exposed to the extract in this study included salivation,ataxia,splayed gait and hyper-excitability to tremors,convulsions and choreoathetosis,which are all indicators of a disruption of the central nervous system.These observations are like the results of the studies conducted in 2010 [22] and 2015 [23].While oral dose ofP.biglobosais 50 mg/kg BW,for 90 days induced intermittent diarrhoea,decreased feed intake,and thick eye discharge,paralysis of the hind limb whereas,a lower dose of 5 mg/kg displayed mild to moderate toxicosis with diarrhoea,decreased feed intake,loss of body weight,dyspnoea,ataxia,eye discharge,salivation and few deaths,after displaying lack of coordination and signs of tremors.This is similar to what was reported in 2010 [24] and 2015 [23].

The high mortality recorded in the groups exposed toP.biglobosaextract implicated the extract to be highly toxic.The different stressful conditions observed in this study could be due to the toxic effects of the aqueous wood-ash extract on the general physiology of theMusmusculus.Alterations in liver weight may suggest treatment-related changes including hepatocellular hypertrophy [25-27].The change in the relative weight of the liver could be attributed to the presence of alkaloids in the extracts,which have been reported to have hepatotoxic activity [18],indicating that the extracts might have toxic potential on the liver ofMus musculuswith increasing dose.This was corroborated by the biochemical findings that indicated high levels of ALT,AST and ALP among mice administered the aqueous wood-ash extracts.Also,histological study provided more information regarding the hepatotoxicity of the extracts.Increased heart weight may be the only evidence of myocardial hypertrophy that is often macroscopically and microscopically difficult to recognize [25,28].Changes in kidney weight may reflect renal toxicity,tubular hypertrophy or chronic progressive nephropathy [25].The activities or levels of some enzymes like AST,ALT,ALP,creatinine and urea also indicate the toxic effects of substances in the environment.These enzymes are normally found within the cells of liver,heart,gills and kidneys but their significant increase in plasma or serum indicates the tissue injury or organ dysfunction [29].

Figure2 Photomicrographs of mice kidney after oral administration of aqueous wood-ash extract of Parkia biglobosa for 90 days.(A) Control:normal architecture of kidney tissues; showing no visible sign of lesion.(B) 50 mg/kg of Parkia biglobosa:multiple foci (box) of moderate aggregates of inflammatory cells are revealed in the renal interstitium (suggestive of nephritis).(C) 100 mg/kg of Parkia biglobosa:moderate sloughing-off of tubular epithelium (arrow).H&E.A&C 100X and B 400X.

Figure3 Photomicrographs of mice liver after orally administered aqueous wood-ash extract of Parkia biglobosa at 90 days,once a day.(A) Control:normal hepatocytes at the periportal region and the hepatic portalvein are shown.(B) 5 mg/kg:no visible lesion of hepatocytes are evident.There are a few foci of mild aggregates of mononuclear cells.(C) 50 mg/kg:mild multifocal thinning of cords with dilated sinusoids are shown (arrow).(D)100 mg/kg:widespread moderate thinning of cords and moderate Kupffer cell hyperplasia.A&B 400X C&D 100X.

It was observed that the aqueous wood-ash extracts ofP.biglobosainduced renal failure in the mice in the sub-chronic test at doses of 50 and 100 mg/kg body weight.This was evident from the renal function tests as serum concentrations of creatinine and urea significantly increased in the treated group,suggesting impairment of renal function [30].Urea concentration has been reported to increase in acute and chronic intrinsic renal disease [31].The observed significant increase in creatinine may be attributed to pathological changes in the kidneys probably associated with intrinsic renal lesions,decreased perfusion of the kidney,or obstruction of lower urinary tract by the extracts,that is,damage of nephron structural integrity [32,33].The abnormal cytoarchitecture of the kidney found in histological examination confirmed these.

The histopathological changes in the selected organs reflected some significant effects with variable intensities.The kidney is an organ sensitive to external factors,which might induce histopathological change as well as functional deficit [34].It is well known that the kidney plays a pivotal role in the regulation of various chemicals [35].Anatomically,the cortex of the kidney has more functional structures than the medulla and mainly nephrons.Most chemicals and toxins carried in the blood stream will have a great chance to influence cortical rather than medullary function [3].Pathological changes in the liver of mice exposed to the aqueous wood-ash extract implicated this plant extract as toxicant because these organs are important for the diffusion of oxygen and detoxification of xenobiotics[36].

Conclusion

Sub-chronic administration of aqueous wood-ash extract ofP.biglobosamay lead to a reduction in body and organ weight,soft faeces,piloerection,paralysis of hind limbs,frequent diarrhoea and eventually death.The present findings,therefore,imply that the extract is hazardous,which can affect the well-being of the human users who are liable to get wood ash extract toxicosis,following its continual oral exposure,leading to hepatic and renal failure.Further study using other wood species as sources of ash and for a longer period of time will be undertaken to ascertain the chronic toxicity of wood ash or the possible reversal of its effects on the organs.There is a need for this extract to be characterised,to ascertain the bioactive components of the extracts responsible for the activities observed.Further studies are needed to explore the mechanism(s)underlying the role of the major metabolite(s) of the extract.The use of other solvents in the extraction of the wood ashes,to ascertain the chemical constituents of the ashes is highly recommended,as not all components might have been soluble in aqueous solvents.Supplementary experiments are underway to determine the possible reversal or worsening toxicity on exposure to the wood ash for a longer period of time.