Hepato-protective potentials of aqueous, chloroform and methanol leaf extracts of Whitfieldia lateritia on 2, 4-dinitrophenylhydrazine-induced anaemia in rats

This study aimed at investigating the hepato-protective potentials of the aqueous, chloroform and methanol leaf extracts of Whitfieldia lateritia on 2, 4-dinitrophenylhydrazine (2,4-DNPH)-induced anaemia in rats. The toxicity study, quantitative phytochemical screening, total and direct bilirubin concentrations, mean protein, albumin and globulin concentrations, as well as mean liver marker enzymes activities (ALT, AST and ALP) were carried out using standard procedures. Thirty-six wistar rats were grouped into six (n =6). Group I: normal control; Group II: negative control; Group III: administered 0.6 ml/kg body weight (b.w) of Astifer (standard Haematinic); Group IV to VI were administered 400 mg/kg b. w. of the aqueous, chloroform and methanol leaf extracts, respectively. Induction of anaemia was achieved in the test groups (II-VI) by administration of 2, 4-dinitrophenylhydrazine (20 mg/kg b.w.) once daily for seven days. Administration of extracts commenced subsequently and lasted for 21 days. Animals were sacrif iced on the 22nd day and blood collected for laboratory analysis. ALT, AST and ALP activities of group II anaemic rats showed signif icant (P < 0.05) reduction compared with normal control rats. Group III rats showed signif icant (P < 0.05) increase in ALT, AST and ALP activities compared with group II anaemic rats. Group IV rats showed signif icant (P < 0.05) increase in ALT and AST activity compared with group III rats. The total bilirubin concentration of group II rats was non-signif icantly (P > 0.05) higher compared with the normal control rats. Groups IV and VI rats showed non-signif icant (P > 0.05) reduction in total bilirubin concentration compared with group V rats. In conclusion, W. lateritia leaf has benef icial hepato-protective properties in Wistar rats at therapeutic dose that supports its use in the treatment of hepatic diseases.


INTRODUCTION
Plants are primary sources of medicine, f ood, shelter, f iber and other necessities used every day by humans. The roots, stem, f lowers, leaves, seeds and f ruits provide f ood f or animals and human beings (Edeogaet al., 2005). The consumption of plants is not just f or its nutrient value but also f or its medicinal ef f ects or purposes and even in modern times plants are being used in many pharmaceutical industries. Plants provide a vast array of secondary metabolites against environmental stress or other f actors like nest attacks, wounds, blood boosting and injuries. The phytochemical properties produced by plants are said to have various therapeutic uses f rom time immemorial. Plants serve as an essential aspect of human diet supplying the body with minerals, vitamins and salts, certain hormone precursors and of course energy and protein (Onyenuga and Fef uga, 2018). Plant leaves have nutrient and medicinal benef its which make them highly needed in diets (Odoemelam, 2015). Among these are the leaves of W.lateritia, popularly known as blood plant.
Morphologically, W.lateritia is a f lowering plant belonging to the f amily of Acanthaceae that grows wild in the evergreen plantation of East Af rica (Bumbariet al., 2009). The leaves have high economic and medicinal use. (Okaf or and Okigbo, 2012). The leaves are used in f olkloric medicine af ter boiling and other processes for boosting of blood, treatment of anaemia, liver damage and inf lammation (Aja et al., 2016). W.lateritia is predominantly f ound in Sierra Leone but recently has been observed in several parts of the world like Nigeria. Here in Ebonyi state it can easily be obtained in a large number in places like Ivo, Ikwo, Unwana and Izzi Local Government Areas. W.lateritia is called "Ogwuobara" in Igbo, "Ogu n' eje" in Yoruba, and "Magnijini" in Hausa language. The rise in the treatment of diseases with herbal medicine is almost universal among developed and underdeveloped countries and of ten more af f ordable than purchasing synthetic drugs. Despite the rise of W. lateritia leaves f or the treatment of various diseases, there is paucity of documented data available. It is theref ore very important f or us to augment the available inf ormation on W. lateritia leaf research (Aja et al., 2016). Many of the modern medicine in the early history contain vitamins that have a lasting importance to health when consumed by humans and is usef ul in the ef f ective treatment of human diseases (Aja et al., 2016). Medicinal plants are not dif ferent in terms of how they work, herbal plants are becoming more of mainstream as improvement in analysis and quality central along with advantages in clinical research have shown the treatment and prevention of diseases. Herbal plants have been studied and used as alternative treatment f or diseases caused by microorganisms, but the f ull potential of plants remain under exploited. The leaves of W.lateritia are purportedly used f or blood boosting, and also f or treating dif f erent ailments in f olkloric medicine. The medicinal and nutritional potential available in the plant depends on its chemical composition. The general public is becoming very interested in the plant because of its medicinal and nutritional importance; however, there are little or no documented reports to this regard, it is on this basis that this work was designed. Hence it is pertinent to investigate its anti-anaemic potentials and of course the saf e dose. Theref ore, inf ormation on its phytochemicals, proximate, vitamin and mineral compositions might be of benef its to scientists in drug design. Determination of possible phytochemicals (quantitatively) that may be present in the leaf extracts of W.lateritia, LD50 determination, determination of liver f unction parameters (AST, ALP and ALT), and determination of total protein, albumin and globulin in anaemic albino rats administered with aqueous, chlorof orm and methanol leaf extracts of W. lateritia. Next is the determination of total bilirubin and direct bilirubin of the anaemic albino rats.

Ethical approval
The National Institute of Health (NIH) approved guideline f or the care and use of laboratory animals was adopted f or this study.

Plant materials
Fresh leaves of Whitfieldia lateritia used f or this study were collected f rom Unwana community, Af ikpo-North Local Government of Ebonyi State, Nigeria. The leaf was authenticated by Dr Garuba Omosun of the Plant Science and Biotechnology Department of Michael Okpara University of Agriculture; some of the leaves were deposited in the herbarium with authentication number W0045, f or ref erence purposes.

Sample preparation
The leaves were destalked, washed with water and shade dried f or two weeks at ambient temperature with constant turning to avert f ungal growth. The dried leaves were milled to obtained the vegetable leaf meals (VLMs) using an electric blender and was stored at 4 o C temperature in ref rigerator in well labeled airtight containers f or analysis.

Extraction
The 10% methanol, chlorof orm and aqueous extracts of the leaves were carried out according to the methods describe by Cowan, (1999). Exactly 400g of powdered leaves was macerated in a stoppered container with the solvents and allowed to stand at room temperature f or a period of at least 3 days with f requent agitation until the soluble matter dissolved. The mixture was then strained, the marc (the damp solid material) was pressed, and the combined liquids were clarif ied by f iltration or decantation af ter standing. Theref ore aqueous, chlorof orm and methanol, are the solvents used f or extraction and were chosen on the basis that; These solvents will maximize the yield of the active compounds and minimize extraction of unwanted compounds in the crude extract. Secondly the active compounds will be soluble in the solvents. Again the nature of polarity of therapeutic values will maintain its potency in the solvent. Finally, it is relatively saf e to use these solvents even when subjected to heat. Exactly 400g of the powder was macerated in 4 litres of aqueous solution for 24hrs. The extract was then sieved with cheese cloth and later f iltered using buckner f unnel and Whatman No1 f ilter paper. The f iltrate was allowed to evaporate to dryness using rotary evaporator and then stored in an air tight sterile container in the ref rigerator at 4℃ until required.

Acute Toxicity and Lethal Dose (LD50) test
The acute toxicity study of all extracts of W. lateritia leaves was carried out using the modified method of Lorke, (1983). The test was divided into two phases. In the f irst phase, total of nine randomly selected adult mice were divided into three groups (n=3) and received 10, 100 and 1000 mg/kg b. w. of the extracts, and there were no signs of toxicity and death recorded af ter 24 hours of observation. The doses f or phase two were determined based on the outcome of the phase one. There was no death recorded, a f resh batch of animals were used f ollowing the same procedure with higher doses of 1600, 2900, and 5000 mg/kg body weight of the extract. The animals were observed f or 24 hours f or signs of toxicity and death. The LD50 was calculated as the geometric mean of the high nonlethal dose and lowest lethal dose (Lorke, 1983).

Induction of Haemolytic Anaemia with 2,4 DNPH and collection of blood samples.
A modif ied method described by Berger (1980), was used in this study. The animals of Groups II to VI received 2,4-dinitrophenylhydrazine (20 mg/kg body weight) once daily f or seven days. On the eighth day, their blood samples were collected by tail snip of each rat into heparinized capillary tubes f or haematological analysis. The tails were f irst sterilized by swabbing with 70% ethanol and then the tip of the tails pierced. Bleeding was enhanced by gently milking the tail f rom the body towards the tip. Blood of approximately 2 ml was drawn into heparinized capillary tubes containing anticoagulant for haematological parameters analysis. Rats with packed cell volume (PCV) less or equal to 30 (≤30) were considered anaemic and selected for the experimental groups. On the twenty-f irst day the animals were euthanized by use of chloroform and blood was collected through cardiac puncture f or f urther laboratory experiments.

Experimental Design
A total of 36 healthy rats were used f or the experimental study, they were randomly allotted into six (6) groups (I to VI) with 6 animals per group (n=6). Appropriate solvent (10% tween 80) vehicle was used to dissolve the extracts. All test substances were administered once daily f or 21 consecutive days by oral-f eeding cannula. All tested substances were prepared f resh bef ore administration through oral gavage according to design below: Group I: Normal control: non-anaemic rats administered 7 ml/kg b. w. of 10% tween 80. Group II: Negative control: 2,4dinitrophenylhydrazine-induced anemic rats administered 7 ml/kg b. w of 10% tween 80.

Group
IV: Test Group I: 2,4dinitrophenylhydrazine-induced anemic rats administered 400 mg/kg b. w. of aqueous leaf extract of W.lateritia.

Phytochemical analysis
Selected phytochemical determination was carried out on extracted leaf samples using standard methods of AOAC (2010) except for f lavonoid and alkaloid which were determined as described by Harborne (1993).

Biochemical Analysis
The f ollowing parameters were determined in blood samples collected by ocular puncture: Serum globulin using the method described by Ochie and Kolhatkar (2000), serum Albumin using the method described by Trease and Evans (1989) and total protein using the method described by Ochie and Kolhatkar (2000).

Assay of serum alanine aminotransferase (ALT) activity
The ALT substrate and phosphate buf f er, 0.5ml each were pipetted into two sets of test tubes labelled B (Sample blank) and T (sample test) respectively. The serum (0.1ml) sample was added to the sample test (T) only and mixed properly: then incubated f or exactly 30 min in a water bath at 37 0 C. A volume, 0.5ml each of 2,4dinitrophenyl hydrazine was added to both the test tubes labelled T (sample test) and B (Sample blank) immediately af ter the incubation. Also, 0.1ml of serum sample was added to the (B sample blank) only. The entire medium was mixed thoroughly and allowed to stand f or exactly 20min at 25 0 C. Af ter that, 5.0 ml each of sodium hydroxide (NaOH) solution was added to both test tubes and also mixed thoroug hly. Absorbance of the sample (Asample) against the sample blank was read at a wavelength of 456nm af ter 5 min.

Assay of serum aspartate aminotransferase (AST) activity.
The method of Reitman and Frankel (1957) was used. The AST substrate and phosphate buf f er, 0.5ml each, were pipette into the sample blank (B) and sample test (T) test tubes respectively. The serum sample, 0.1ml was added to the sample test (T) only and mixed immediately. It was then incubated in a water bath f or exactly 30min at 37 o C. A volume, 0.5ml of 2,4dinitrophenyl hydrazine was added to both sample blank (B) and sample test (T) test tubes immediately af ter incubation. Also, 0.1ml of the sample was added to the sample blank (B) only. The medium was mixed and allowed to stand for exactly 20min at 25 0 C. Finally, 5.0ml of sodium hydroxide (NaOH) was added to both the sample blank (B) and sample (T) test tubes and mixed thoroughly. The absorbance of sample (Asample) was read at a wavelength of 546nm against the sample blank af ter 5mins.

Determination of total and direct bilirubin of the colourimetric method
To determine total or unconjugated bilirubin (Tb), two separate cuvettes labelled SB (sample blank) and S (sample) were set up. A volume of 0.2ml of reagent 1 was pipette into the sample blank and the test sample cuvettes respectively. A drop of reagent 2 was added into the test sample (S) cuvette. Also, 1ml of reagent 3 was added to the two cuvettes labelled SB and S. the sample (0.20ml) was added to the two cuvettes which were mixed thoroughly and allowed to stand for 10 minutes at 25 0 C then the absorbance of the sample against the sample blank was read at a wavelength of 560nm.

Procedure for direct or conjugated bilirubin (Db)
Two separate cuvettes labelled SB (sample blank) and S (sample) were set up. A volume of 0.20ml reagent 1 was pipette into the sample blank and the test sample cuvettes respectively. A drop of reagent 2 was added into the test sample(S) cuvette. Also, 2ml, of sodium chloride (9g/L) each was added to the two cuvettes labelled SB and S. the sample 0.20ml each was added to the two cuvettes which were mixed thoroughly and allowed to stand f or 10 minutes at 25 0 C then the absorbance of the sample against the sample blank (ASB) was read at a wavelength of 650nm.

Determination of serum alkaline phosphatase (ALP)
The procedure of Plummer (1971) was used. The serum sample (0.5ml) was pipetted into the sample test tubes (T) and 0.5ml of the standard was pipette into the sample blank (B) respectively. One ML (1ml) of distilled water was added to the sample test tubes (T) and sample blank (B).
A drop of phenolphthalein monophosphatase was also added to both sample blank (B) and sample test (T) and mixed immediately; then incubated into a water bath f or exactly 20 minutes at 25 0 C. A volume of 0.5ml of 2-amino-2 methyl-1propanol was added to both sample blank (B) and sample test tubes (T) immediately af ter incubation. Then the absorbance of the (Asample) was read at a wavelength of 550nm against the sample blank af ter 5min.

Statistical analysis
The data obtained f rom the laboratory test were subjected to one way analysis of variance (ANOVA). Dif f erences between means at P < 0.05 were accepted as signif icant. The results were expressed as mean standard deviation (SEM). This analysis was estimated using Statistical Package f or Social Science (SPSS), version 17 and represented with appropriate charts.

Result of acute toxicity
Acute toxicity tests on W. lateritia in albino rats established a high LD50, which suggests that the aqueous, chlorof orm and methanol extracts of the leaf W. lateritia may be generally regarded as saf e with a remote risk of acute intoxication and sedation at high dose above 5000mg/kg b.w. The result is as shown in Table 4.

Mean protein, albumin and globulin concentrations of 2, 4-dinitrophenylhydrazine induced anaemic rats treated with W. lateritia leaf extracts
Figure 2 revealed that the total protein, albumin, and globulin concentration of 2, 4dinitrophenylhydrazin induced anaemic rats administered 10% tween 80 were signif icantly (P < 0.05) lower compared with the normal control rats. The anaemic rats treated with 0.6 ml/kg b. w. of astif er showed signif icant (P< 0.05) increase in total protein, albumin and globulin concentration compared with the anaemic rats administered 10% tween 80. Anaemia induced rats treated with 400 mg/kg b. w. of aqueous and methanol extracts of W. lateritia leaf showed signif icant (P < 0.05) reduction in total protein and albumin concentration compared with anaemic rats treated with 0.6 ml/ kg b. w. of astif er, while the chlorof orm extract treated anaemic rats showed non-signif icant (P > 0.05) reduction compared with astif er treated anaemic rats. Anaemic rats treated with 400 mg/kg b. w. of chlorof orm and methanol extracts showed signif icant (P < 0.05) increase in total protein, albumin and globulin concentration compared with the anaemic rats treated with 400 mg/kg b. w. of aqueous extract of the leaf . Group I: normal control; Group II: negative control; Group III: administered 0.6 ml/kg body weight (b.w) of Astif er (standard Haematinic); Group IV to VI were administered 400 mg/kg b. w. of the aqueous, chlorof orm and methanol leaf extracts, respectively.

Mean liver marker enzyme activities of 2, 4dinitrophenylhydrazine induced anaemic rats treated with W. lateritia leaf extracts
The alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities of anaemic rats administered 10% tween 80 showed signif icant (P < 0.05) reduction compared with normal control rats. The rats treated with 0.6 ml/kg b. w. of astif er af ter 2, 4-dinitrophenylhydrazine anaemia induction showed signif icant (P < 0.05) increase in ALT, AST and ALP activities compared with anaemic rats administered 10% tween 80. Anaemic rats treated with 400 mg/kg b. w. of aqueous extract of W. lateritia leaf showed signif icant (P < 0.05) increase in ALT and AST activity compared with astif er treated anaemic rats. The rats treated with 400 mg/kg b. w. of chlorof orm and methanol extracts of W. lateritia leaf af ter anaemia induction showed non-signif icant (P > 0.05) reduction in AST, ALT and ALP activities compared with anaemic rats treated with 400 mg/kg b. w. of aqueous extract of the leaf as shown in Figure 3.

Mean Bilirubin Concentration of 2, 4dinitrophenylhydrazine induced anaemic rats treated with W. Lateritia leaf Extracts
The total bilirubin concentration of 2, 4dinitrophenylhydrazine induced rats administered 10% tween 80 was non-signif icantly (P > 0.05) higher compared with the normal control rats. Astif er treated anaemic rats at 0.6 ml/kg b. w showed non-signif icant (P > 0.05) reduction in total bilirubin concentration compared with anaemic rats administered 10% tween 80. Anaemia induced rats treated with 400 mg/kg b.w. of aqueous and methanol extracts of W. lateritia leaf showed non-signif icant (P > 0.05) reduction in total bilirubin concentration compared with anaemic rats treated with chlorof orm extract of the leaf . The direct bilirubin concentration of anaemia induced rats administered 10% tween 80 showed signif icant (P < 0.05) reduction compared with normal control rats. Anaemic rats treated with 0.6 ml/kg b. w of astif er and 400 mg/kg b. w. of extracts of W. lateritia leaf showed non-signif icant (P > 0.05) increase in direct bilirubin concentration compared with anaemic rats administered 10% tween 80. The anaemic rats treated with aqueous and methanol extracts of W. lateritialeaf showed non-signif icant (P > 0.05) increase in direct bilirubin concentration compared with astif er treated anaemic rats as shown in Figure 4.

DISCUSSION
Anaemia is a well-known lif e-threatening condition. It may be caused by excessive blood loss, haemolysis, and def iciency associated with RBC synthesis (due to iron def iciency). The synthetic drugs available f or its management/cure are not without their side ef f ects, they may also be inaccessible and costly to some rural suf f erers. All these limitations of the synthetic drugs have necessitated researches , f ocusing on less toxic herbal therapies known for their anti-anaemic ef f icacies as claimed by ethno medicinal practitioners. Phytochemicals are plant  Table 3. It is obvious f rom the result that the leaves are rich in phenols, tannins, f lavonoids, reducing sugars, and alkaloids. The presence of these biologically active compounds suggest that plants could serve as potential sources of drugs and their secondary metabolites could exert some biological activities when taken by animals (Edeoga et al., 2005). Epidemiological studies have shown that high consumption of vegetables is associated with a lowered incidence of anaemia, liver disease, heart disease, inf lammation, arthritis, neurodegenerativ e diseases and other immune related diseases (Krishna et al., 2014). Flavonoids have been shown to augment humoral response by stimulating the macrophage and B-lymphocytes involved in antibody synthesis. Reports indicate that several types of f lavonols stimulate human peripheral blood leucocyte prolif eration (Sharif if ar et al., 2009) and possess antibacterial, antiinf lammatory, anti-allergic, anti-viral, hepatoprotective and antineoplastic activities.
Flavonoids also f unction as reducing agents, f ree radical scavengers and quenchers of singlet oxygen f ormation (Kayode and Kayode, 2011), thus very usef ul in counteracting the f ree radicals generated by immune depressants. The alkaloid content or composition was appreciably high. Alkaloids being a class of secondary metabolites and with their wide range of pharmacological activities including antimalaria, anticancer, antibacterial and antihyperglyceric activities have been of essence in drug design (Edeoga et al., 2005). Tannins have been shown to play major roles as anti-diarrheal and anti-hemorrhagic agent (Edeoga et al., 2005). The terpenoids have also been shown to decrease blood sugar levels in animal studies. Glycosides may be crucial in the transduction of intracellular signals mediated by neurotransmitters, hormones and neuromodulators receptors. When activated, these molecules can act on several intracellular targets (De-Weerdet al., 2007). The immune response of both human and animals may be inf luenced by several essential nutrients which modif y the immune system f unction. It is generally assumed that many important inf ections of human and animals have been associated with a nutritional def iciency which generates a suppression of immune response Group 4 Group 5 Group 6 (Chandra, 1990). Tannin, a class of astringent, is a phenolic biomolecule that binds to and precipitates proteins and various other organic compounds including amino acids and alkaloids (Edeoga et al., 2005). Hence the leaf extracts of W. lateritia could be of great importance to human health. Acute toxicity tests on W. lateritia in albino rats using the method of Lorke (1983) established a high LD50, which suggests that the aqueous, chlorof orm and methanol extracts of the leaf W. lateritia may be generally regarded as saf e with a remote risk of acute intoxication and sedation at high dose above 5000mg/kgb. w. Signs of acute toxicity include decreased locomotor activity, decreased f eed intake, tremor, change of hair colour, prostration and death (Barbosa-Ferreira et al., 2005). None of these signs was noticed in the experimental mice given the extracts. The degree of saf ety is also consistent with its popular use locally. Thus, since W. lateritia is believed to have hepato-protective potentials by many traditional healers, the experimental determination of this good saf ety margin would justif y the plant as relatively saf e at the dose level (400mg/kg b. w) used in this study.
The result f or the serum total protein in Figure 2 shows that serum protein was elevated in the control group 1 when compared to the group induced with anaemia and treated with the vehicle tween 80 and leaf extracts. This suggests that induction of anaemia also led to decreased serum total protein as seen in group 2. The treatment however boosted the serum total protein signif icantly (P<0.05) in the group treated with the standard astif er multivitamin, aqueous, chlorof orm and methanol leaf extracts. This suggests that the chlorof orm extract had comparative ef f ect with that of the standard drug (astif er). The decrease in the total serum protein in the group induced with anaemia could be as a result of the toxicity induced by 2, 4 DNPH and diminished synthesis of protein by liver (Naomet al., 2008), while the increase in the groups treated with astif er multivitamin, aqueous, chlorof orm and methanol extract is due to the presence of phytochemical in the plant extract which are well known to have direct inf luence on the liver and boost protein synthesis as conf irmed by Aja et al., (2016). This is in line with the study of (Sarkiyayi and Alduirasheed, 2013), who's preliminary investigation on anti-anaemia in W. lateritia leaf extracts buttress the assertion.
Protein is important f or structure f ormation and repairs of tissue building (Thompson et al., 2013).
Liver f unction tests (LFTs or Lf s) are groups of blood tests that give inf ormation about the state of the liver. The liver is a vital organ that f unctions in detoxif ication, storage, and other biochemical metabolisms necessary f or the body (Mbuh et al., 2003). This study equally examined the changes in aspartate aminotransf erase (AST), alanine aminotransf erase (ALT), alkaline phosphate (ALP), total bilirubin and direct bilirubin activities in induced anaemic albino rats. The change in the mean serum of ALT, AST and ALP are presented in Figure 3 in the result, it shows that ALT was not signif icant (P>0.05) in group treated with 400mg/kg b.w of chloroform when compared with the control group. The result also shows that AST concentration in aqueous treated was signif icant (P<0.05) when compared with the control. The result shows that ALP was signif icantly (P> 0.05) decreased in all the groups when compared to the control except in the astif er treated group. This agrees with a similar study by (Kayode and Kayode, 2011) on f luted pumpkin (another popular blood boosting plant). ALT is purely cytoplasmic catalyzing the transamination reaction (Mauro et al., 2006). The decrease in ALT concentration may be as a result of the administration of the astif er and extracts o f W. lateritia. Any type of liver cell injury can reasonably increase ALT levels. The changes in the mean serum total bilirubin and direct bilirubin concentration are presented in Figure 4. The result shows that there was a signif icant increase (P<0.05) in mean serum total bilirubin concentration in groups treated with 20mg/kg b. w of tween 80 (vehicle), 0.6 ml/kg b.w of Astif er and 400mg/kgb. w of chlorof orm extract of W. lateritia when compared to the control group, while the result f or the mean serum direct bilirubin concentration shows that there was a signif icant decrease (P>0.05) in mean serum direct bilirubin concentration when compared with control group. Unconjugated bilirubin is a breakdown product of heme (a part of haemoglobin red blood cells). This agrees with the f indings of Shivaraji et al., , whose work showed that when total bilirubin level exceeds 17µmol/L, it indicates liver disease, when total bilirubin level exceeds 40µmol/ L, bilirubin deposition at the sclera, skin and mucous colour occur (Shivaraji et al., 2016).

CONCLUSION
All the data obtained f rom this study showed strong preliminary evidence that W. lateritia leaf extracts have hepato-protective potentials as proven by several biochemical, and liver marker enzyme activities.Accordingly, the extract can be used as an ef f ective herbal product f or the prevention of hepatic diseases and liver related issues. It is believed to be due to its phytochemicals like glycosides, f lavonoids etc. that contributed to its ef f iciency.

Conflict of interest
Authors have no conf lict of interest to declare.

AUTHOR CONTRIBUTIONS
ESI designed the study, wrote the protocol, and supervised the work. AOA perf ormed all the laboratory work. MOJ and APO perf ormed the statistical analysis. UEN managed the analyses of the study. AOA and OCE wrote the f irst draft of the manuscript. AOA, MOJ and APO perf ormed the literature search. All authors read and approved the f inal draf t of the manuscript.