Phytochemistry and Toxicity Studies of Telfairia Occidentalis Aqueous Leaves Extract on Liver Biochemical Indices in Wistar Rats

This study assessed the effect of oral administration of aqueous extract of Telfairiaoccidentalis (TO) leaves on liver biochemical indices in Wistar albino rats. Fifty rats weighing 150-200g were div ided into 2 groups A and B, to test for acute (10 days) and sub-acute (4 weeks) effect respectively. Each group consisted of 25 rats, which were fu rther div ided into 5 sub-groups 1, 2, 3, 4 and 5. Sub-group 1 served as control and was orally gavaged with standard animal feed only, while sub-groups 2, 3, 4 and 5 were the test groups. In addition to the standard animal feed, 500mg/kg, 1000mg/kg, 2000mg/kg and 4000mg/kg of the extract was orally gavaged to the test groups 2, 3, 4 and 5 respectively. The standard animal feed g iven to the control and test groups contained the same amount of daily calories and protein. The acute and sub-acute administration of the extract caused significant differences in values of Aspartate amino Transferase(AST), Aspartate to Alanine amino Transferase(AST/ALT) rat io, Total protein, A lbumin, Globulin, Albumin/Globulin ratio, Total body weight, Total and Conjugated bilirubin (P < 0.001) between test and control groups. Serum levels of other parameters were statistically non-significant. There was no obvious evidence of hepato-toxicity detected in this study.


Introduction
As the acceptance of herbal remedy is gradually increasing worldwide [1], there is a growing concern about the hepatotoxicity of several hundreds of dietary herbs/vegetables which have remained either un-investigated or poorly investigated, and are increasingly used by patients with liver diseases [2]. Parado xically, so me herbal remedies used in the treatment of liver d iseases may be hepatotoxic themselves [3], and at times the specific hepatotoxic component is not known because of the difficulty in proper analysis of the product or plant material. It has been difficu lt diagnosing herbal hepatotoxicity especially if unsuspected, because consumers generally consider herbal remed ies to be safe and more effective and view them as natural alternatives to orthodox med icine. [3]. A recent report has shown that only 40% of people who use herbal medicine info rmed their primary care physicians [4]. Apart fro m the safety and the acclaimed therapeutic efficacy, other reasons for the recent surge in the consumption of herbal remed ies include easy accessibility, more acceptability fro m cu ltural and spiritual perspectives [5], non-involvement of expert consultation and of in jury may range fro m acute hepatitis, sub acute hepatic necrosis, cholestatic hepatitis, cirrhosis and acute liver failure [3,9].Several mechanis ms have been postulated as being the possible processes through which these herbal remedies cause liver injury or dysfunctions. These include inhibit ion of cyclo-o xygenase and/or cytochrome p450 activity or an immune mediated liver in jury [10].
However, the hepato-protective effects of some botanicals have also been confirmed by clinical research and their use in prevention and treatment of liver diseases supported. A typical examp le is the Silybummarianum, wh ich is currently the most well researched plant in the treat ment of liver disease in many countries including Germany. The hepato-protective effects of Sily marin in hu man after mushroom poisoning (Aman ita spp) have been repeatedly demonstrated and confirmed [11]. In the light of these, continuous research to reappraise the acclaimed hepato-protective/toxic effects in some of the extensively consumed herbs/vegetables is recommended. Prior studies have shown that these dietary vegetables/herbs are the common sources of drug induced liver in jury. Thus, this research work evaluates the acute and sub-acute effect of TO leaves extract on rat liver b iochemical indices since it is such a widely consumed vegetable in Nigeria, most sub Saharan African countries, and worldwide.
We hope the results of this research will p rovide additional and useful informat ion about the effect of this widely consumed vegetable on the liver bio-chemical indices.

Materials and Methods
Collection and i dentification of pl ant materials Fresh leaves of TO were obtained fro m the agricu ltural farm in Uyo Local Govern ment Area in AkwaIbom State, Nigeria. A Taxono mist in the Department of Botany, University of Uyo, Nigeria, identified the leaves.

Preparation of Telfairiaoccidentalis aqueous leaves extract
The fresh leaves were rinsed to remove sand and other debris, oven dried at 40 0 c and pulverized into powder using electric blender to give a gram weight of 865g. This was soaked in 2 liters of distilled water and allowed overnight for about 12 hours and stirred at intervals. The mixture was sieved with a clean white cloth. The filtrate was dried by heating in water bath at 40 o c to obtain a solid extract. The solid extract was weighed with an electronic weighing balance (OHA US) before the stock solution was prepared. The stock solution was prepared by dissolving 15g of extract in 10ml of water to give concentration of 1500mg/ ml. The solution was well labeled and preserved in a refrigerator at 4 o c until required for use.

Phytochemical screening of Telfairiaoccidentalis leaves extracts
One hundred grams (100g) of the blended leaves was soaked in 500ml of 95% ethanol in a sterile conical flask and stored at room temperature and thereafter filtered using a Whatman No 2 filter paper. The filtrate was evaporated to dryness and preserved in clean bottles at room temperature until required for used.
The phytochemical analysis of the extract was carried out to determine the presence of saponins, phenolics, alkaloids, tannin, flavonoids, glycosides, steroids, deoxysugar, terpens and anthraquinone using standard procedures according to Trease and Evans [22] and Sofowora [23].
Determinati on of the presence of anti-nutrients One hundred grams (100g ) of the extract TO leaves was used to determine the presence of some anti-nutrients such as tannin, oxalate, cyanide and phytate.
The presence of tannic acid (a po lyphenol) was determined using the method described by Makkar and Goodchild [24]. The hydrogen cyanide was determined using the method described by Fasuyi et al [13]. The presence of phytate was determined as described by Young and Greaves [25]. The presence of oxalate was determined by the titrimetric method of Moir [26] as modified by Ranjhan and Krishna [27].
Collection and maintenance of ani mals Fifty adult male Wistar albino rats weighing 150-200g were obtained fro m the animal house of the Faculty of Basic Medical Sciences, Un iversity of Uyo, Nigeria. Their init ial weights noted and they were div ided into two groups A and B. Each group consisting of twenty-five rats of 5 sub-group: 1, 2, 3, 4 and 5 with five rats per subgroup. Subgroup 1 in each group served as the control.
They were housed in a standard wooden cage with wooden shavings as their beddings, kept and maintained in the animal house of Faculty of Basic Medical Sciences, University of Uyo for one week prior to the study to allow for acclimatization. They had free access to water ad lib itum and good light source and maintained at roo m temperature. Determinati on of LD 50 Lethality studies to determine the LD 50 was performed according to the procedure described by Lorke [28]. Twenty rats weighing 150-200g were rando mly divided into 5 groups with each group having 4 rats. They were treated with 1000, 2000, 3000, 4000 and 5000mg/kg respectively with no mortality recorded after 24 hours. In the second phase, doses of 5500, 5750 and 6000mg/kg p roduce negative behavioral changes followed by 100% mo rtality at 5500mg/ kg concentration of the extract. The median lethal dose (LD 50 ) was then calculated as follows: LD 50 = √D o x D 100 D o = Ma ximu m dose that produce 0% mortality D 100 = Minimu m dose that produce 100% mo rtality LD 50 = √5000×5500 = 5244.04mg/ kg Fro m here, convenient doses were chosen to preclude the lethal range.

Administration of extract
Animals in subgroup 1 (control) in both groups were orally gavaged with standard animal feed (Bendel Feed and Flour Mill Ltd. Benin) only, while those in subgroups 2, 3, 4 and 5 (test groups) in addition to the standard animal feed, were orally gavaged with 500mg/kg, 1000mg/kg, 2000mg/kg, and 4000mg/kg body weight of the extract respectively. The different doses were chosen based on the LD 50 (5244.04mg/kg) to preclude the lethal dose.The standard animal feed given to the control and test groups contained the same amount of daily calories and protein.

Blood sample collection
After 10 days of extract ad min istration, all animals in-group A were weighed again and the sacrificed after being anaesthsized. Dissection to expose the heart was performed and blood was obtained through cardiac puncture into specimen bottles with no anticoagulant. The samples were analyzed in the Chemical Pathology Unit o f the University of Uyo Teaching Hospital to test for the acute effect of the extract on liver biochemical indices. The liver of each rat was removed and the weight noted.
Similar procedures were repeated for the experimental animals in group B after four weeks of extract administration and tested for sub-acute effect. The research protocols were carried out in the University of Uyo, according to the rules in Nigeria (Revised Helsin ki Declaration, 2008) governing the use of laboratory animals as acceptable internationally.

Assay of bi ochemical parameters
Determination of serum AST, A LT and A LP: serum levels of these enzymes were determined using appropriate test kits (Rando x Laboratories U.K).
Seru m assay for AST and ALT: were determined according to the procedure described by Segal and Matsuzawa [29] The princip le of the method for measuring ALT involved monitoring the concentration of pyruvate hydrazone formed with 2, 4, d initrophenyl hydrazine, wh ile for AST, it involved monitoring the concentrations of oxaloacetate hydrazone formed with 2, 4 dinit rophenyl hydrazine.
Alkaline phosphatase: (A LP) was determined by the method of Ahamed and King [30].
Seru m total protein : was estimated using the Biu ret method based on the principle that protein react with copper (II) to produce a blue-vio let color co mpound in alkaline med iu m and that the color intensity is proportional to the concentration of the total protein in the sample.
Seru m albu min concentration was estimated using the albumin-bro mocresol green reaction. (BCG) as described by Doumas et al [31]. The princip le is based on the albu min quantitatively binding to the indicator 3, 3'5, 5' tetra-bromo-m-cresol sulphonaphthalein (bro mocresol green) which absorb maximally at 578n m. Seru m total and conjugated bilirub in was estimated using colorimet ric method.
Statistical Anal ysis Descriptive statistics was computed for each of the liver function indices and reported as means ± standard deviation. Furthermore, one-way analysis of variances (ANOVA) was used to compare differences in liver function indices of rats following the administration of different concentration of TO leaves ext ract. Post analysis to determine the groups that differs significantly fro m each other was performed using the Least Significance DifferenceTest (LSD).
All statistical analysis were enhanced using the statistical package for social sciences version 17 (SPSS 17.0) and P < 0.05 was considered to be statistically significant. Table 1 shows the phytochemical constituents of TO leaves extract to include: saponin, tannin, flavoniods and phenolics.  Table 2 shows the anti-nutrient content to include tannin, hydrogen cyanide, phytic acid and oxalate. Table 3 shows that acute administration of TO leaves extract on experimental animals caused a non-significant differences in A LP (Fcal = 2.51, P = 0.074), A LT (Fcal = 2.68, P = 0.061), A lbu min (Fcal = 2.40, P = 0.084), Liver weight (Fcal = 0.83, P = 0.524) and ratio of liver to body weight (Fcal = 1.04, P = 0.414). However, significant differences in levels of ALT (Fcal = 33.02, P<0.001), Total protein (Fcal = 329.53, P < 0.001), Globulin (Fcal = 13.54, P < 0.001), Total bilirubine (Fcal = 47.65, P < 0.001), Conjugated bilirub in (Fcal = 34.25, P < 0.001) and Albumin/globulin rat io (Fcal = 32.85, P < 0.001) were obtained between test and control groups. Group 5 had the highest level of AST, total protein, globulin and total bilirubin, wh ile group 3 had the highest body weight. Appropriate post analysis to determine which groups were different fro m each other in the effect of the extract on serum liver b iochemical indices follo wing the 10 days administration showed non-significant differences in serum levels of AST between groups 2 and 3, 2 and 4, 3 and 4 and 3 and 5 with P = 0.069, 0.221, 0.519 and 0.144 respectively. However, significant differences were detected in other groups. Significant serum levels of total protein were also detected in all studied groups, whereas ALB/ G ratio differs significantly among groups with the exception of between group 1 and 2 (P=0.576) and 2 and 3 (P=0.627).
Pair wise co mparison of the effectsfollowing 4 weeks administration of the extract showed that, the serum levels of AST was significantly d ifferent between all paired groups except groups 3 and 5 (P = 0.578). Similarly, total serum protein and albu min fractions were significantly d ifferent in all groups with (P < 0.05). Significant levels of globulin was detected in all paired groups with exception of group 1 and 3, 2 and 3 and 4 and 5 with P = 0.093, 0.262 and 0.164 respectively. ALB/ Gratio also showed significant differences between the paired groups except group 1 and 2 (P = 0.805) and group 2 and 5 (P = 0.925). Significant levels in total bilirubin was detected in other paired groups except groups 1 and 4 (P = 0.199) and 3 and 5 (P = 0.653). In addition, significant differences in body weight was detected in other paired g roups at (P < 0.005) except groups 1 and 2 (P = 0.237), 1 and 3 (P = 0.999), 1 and 5 (P = 0.341) and 1 and 4 (P = 0.341). Similarly, Liver/Body weight ratio was significant in other groups excerpt 1 and 2 and 2 and 5 with P = 0.205 and 0.761 respectively.

Discussion
Findings of this study showed that there were significant differences in values of AST, AST/ALT ratio, Total protein, Albumin, Globulin, ALB/ Grat io, Total body weight, Total and Conjugated bilirubin between control and test groups in both phases of the study. Serum levels of other parameters were statistically non-significant even though the experimental g roups recorded higher values than control groups. The significant changes in the above-mentioned parameters are unlikely to be due to the to xic effect o f the extract on liver cells. This is because, of the two major enzy mes (AST & ALT) usually used as surrogate markers for hepatic injury [32][33][34], only AST was significantly elevated.
A significant h igh level of AST and ALT is common in most acute and sub-acute hepatocellular disorders with ALT being higher than or equal to AST. In addit ion, prior studies have empirically shown that these transaminase enzymes are widely distributed in other cells of the body and the serum levels could be elevated in injury affect ing these cells such as muscle in jury due to strenuous exercise, rhabdomyolysis and myositis [35]. Nevertheless, the activities of ALT outside the liver cells are very low and therefore, ALT is considered more specific for hepatocellular damage than AST [36]. Isolated significant increase in AST may have been extrahepatic as this enzyme is also found in other tissue such as kidneys, brain, pancreas, lungs, leukocytes, erythrocytes, cardiac and skeletal muscles [37].
Again, preponderance of emp irical evidences suggest that dietary protein levels could affect the activities of liver enzy mes. Koutsos et al [38] demonstrated the effect of various concentrations of dietary protein level on the activity of the liver en zy mes. In this study, it was shown that levels of AST and A LT were increased fo llo wing a very high concentration of crude protein (70%) meal. Up-regulat ion of these liver enzy mes (AST and A LT) involved in amino acid catabolism is the probably mechanis m [38]. Th is also explains the reason for the highest level of the enzy mes (AST and ALT) in animals in-g roup five fed with the highest concentration of the ext ract. It could have also correlated with the extra-hepatic activit ies of these enzymes.
Raised serum A LP level usually reflects impaired excretion and bile flow as in the obstruction that affects biliary system. Increase serum level may reflect increase synthesis in the phase of inadequate excret ion as in conditions leading to increase biliary p ressure. In this study, such insignificant increase observed could not have indicated hepatobiliary obstruction; rather extra hepatic sources (e.g. bone) may be implicated [35].
In addition, significant raised levels of serum total protein, albumin, albumin/globulin rat io in this study could further buttressed the non-toxic effect of the extract to the liver. The liver synthesizes protein and low levels could reflect impaired synthesis. Normal level reflects normal synthesis while high level is co mmon in high protein d iet. It could therefore be correct to assert that, the significant high levels of seru m protein and protein fract ions observed in the experimental groups was due to the high protein content of TO leaves extract ad min istered at high concentrations to the tested animals. The p lausibility of this assertion lends support from several previous studies, which convincingly demonstrated the presence of rich amino acid and protein content of TO leaves extract.
Akoroda [39], in h is study showed that TO leaf extract is highly nutritive and is very rich in protein (21%-37%). Kayode et al [40] made similar observation. In other studies the extract of this leaf was emp irically shown to constitute a rich source of an array of amino acids such as alanine, aspartate, glycine, glutamate, histidine, lysine, methionine, tryptophan, cysteine and leucineetc [12,41], wh ich could provide a readily availab le precursors for protein synthesis. High amino acid or protein diet is associated with high hepatic synthesis and hence high serum protein [42] as recorded in this study.
In contradistinction to previous studies [13,43], there was a significant reduction in body weight of the experimental animals in both phases of the study. Prior studies have shown that the health benefits or adverse health effect obtained fro m consumption of plant food is rooted in the concentration of phytochemicals and nutritional constituents, which are determine by the quantity consumed [44]. Telfiariaoccidetalis leaves meal has severally demonstrated growth stimulating effect with associated weight increase at an optimal threshed concentration of the extract beyond which loss in weight ensued.
Fasuyi et al [13] observed that birds kept on 15% TO leaves meal d ietary inclusion level had the highest weight gained with subsequent weight reduction at doses greater than 15% ext ract concentration. Iweala and Obidoa [43] showed that, if the dose of the extract is kept low at a tolerable level, it could be ad min istered or consumed for a longer period without weight loss. Similarly, Saalu et al [44] demonstrated evidence of testicular protective effect in rats treated with dose of TO extract < 400mg/ kg body weight and testicular to xic effect at concentrations > 400mg/kg body weight. It could be correct to infer that the weight loss in this study was associated with the h igh doses of the extract ad min istered to the experimental an imals.
Harper et al [45] demonstrated that, an excess level of amino acids is associated with decreased food intake, muscle deposition and loss in body weight in ch icken and rats. Water intake was also significantly greater in b irds fed with very high protein (CP of 70%) d iet signify ing the possibility of hyper-osmolar diuresis leading to dehydration and subsequent polydipsia. All these could have contributed to the weight loss associated with high protein diet, wh ich was more significant (P < 0.001) in-g roup fed with the highest concentration of the ext ract in both phases of the study. The plausibility of previous findings obtains validity fro m the present study.
Again, some o f b iochemical changes noted in this study could have been due to the effect of some of the phytochemical constituents detected. These include saponin, tannin, flavanoids, alkalo ids and phenolics. Also present were, hydrogen cyanide, phytate and oxalate. These bioactive constituents have empirically, been shown to possess varied biological effects probably due to their structural differences and concentrations found in different plant species. For examp le, saponins are steroids and are present in a number of p lants and plants products. Prior studies have shown that saponin-containing vegetables have a characteristic bitter taste and foaming properties. They have anti-nutrient properties, are associated with hemolysis, and retard growth because; they primarily cause a reduction in feed intake [46]. Sharma et al [47] observed that 4-7 weeks of adlibitu m feeding of saponin containing feed resulted in some toxic man ifestations such as listlessness, anorexia, weight loss and gastroenteritis in sheep.
Thus, the presence of saponin compounds as a major constituent in the extract of TO as detected in this study could have contributed to loss in weight, which was mo re significant (P < 0.001) in-group, fed with the highest concentration of the extract in both phases of the study. Additionally, the significant levels of total and conjugated bilirubin, which signifies hemolytic d isorders, could be associated with the effect of saponin as it's known to cause hemolysis of red blood cells especially when used at high concentrations [46].
Tannin another bioactive constituent is a water-soluble polyphenol with anti-in flammatory and anti-microbial effect. It has both bacteriocidal as well as bacteriostatic effect against staph. aureus and other micro-organis ms. Prior studies have shown that the presence of tannins in the extract provides the effect for its use as purgative [21], anti-asthmatic, antitussives and anti-hay fever [19].
Tannin also has some anti-nutrient effect achieved by forming complexes with protein. It binds and inhibits protein such as digestive enzymes [47]. However, the to xicity of tannin depends on the form with wh ich it occurs. Generally, the hydrolysable form is more to xic than the condense form which is more d igestible and reducing in effect [47]. The absence of a significant toxic effect in this study could imp ly that the extract contained predominantly the condense form of tannin. However, the plausibility of this assertion is an area for further research.
Hydrogen cyanide, detected as an anti-nutrient has been shown to be a potentially to xic substance. However, its effect on the liver is very rare. It is absorbed as soon as released and is rapidly deto xified in the liver by the liver en zy me rhodanese which convert cyanide to thiocyanate by catalyzing the t ransfer of sulfur of thiosulfate to the cyanide to form thiocyanate (a less toxic fo rm) which is excreted in the urine [48,49]. The presence of the extract of TO with its cyanide content is noted to trigger on the activit ies of rhodanese. The present of the extract also provides the sulfur ions containing amino acid needed to enhance this process. This could explain the low to xicity of cyanide to the liver despite its high concentration in the extract.
Phytic acid structurally represented as C 6 H 6 [OPO(OH) 2 ] is another anti-nutrient detected in TO leaves extract. It is an hexaphosphoric acid ester of inositol. It is naturally stored in plant tissues as calcium and/or magnesium salt [50]. It represents the majo r storage form of phosphorus and is a strong chelator due to the presence of reactive phosphate groups. Several studies have shown that phytic acid is hepatoprotective in action. Abd-El-Rahaman et al [51] showed that animals treated with phytic acid added meal had a significant reduction in levels of some liver en zy mes (AST & A LT) (ev idence of hepatoprotective effect) against significant increases levels in those treated with Afloto xin B (evidence of hepatotoxicity). There were also significant increases in serum total biliribin, protein, albu min, caspase-3-transferase and glutathione-S-transferase levels.
Shibata [50] showed that there were no significant differences in levels of total protein, albumin to globulin ratio, AST, A LT between broilers treated with phytic acid added meal than control. However, abdominal fat weight reduction was noted in experimental than control groups. This reduction was proportional to the quantity of phytic acid added.
The present study results are congruent with the above mentioned and go further to support the fact that most of the phytochemical constituents of Telfiariaoccidentalisare not hepatotoxic, but could enhance some other physiological changes e.g. weight loss, raised protein levels, mild hemolysisetc as detected in this study.
The hepatoprotective effect as observed in this study stand in clear support to previous studies with similar documentations.

Conclusions
The result of this study showed that the extract of Telfairiaoccidentalis is not toxic to the liver even at very high doses.