Hepatoprotective action of various partitions of methanol extract of Bauhinia purpurea leaves against paracetamol-induced liver toxicity: involvement of the antioxidant mechanisms

PCM is safe when consumed in therapeutic doses; interaction with other drugs and side effects are rarely observed [29]. However, overdose of PCM is known to result in liver injury, with the potential for liver failure progression [30, 31]. Given its clinical relevance, PCM is a widely preferred toxicity model for the evaluation of hepatoprotective agents [29]. Via the glucuronidation process, more than 90 % of PCM that has been consumed in therapeutic doses is conjugated by the liver into inactive components, non-toxic sulphate, and glucuronide prior to their excretion in the urine. Meanwhile, less than 5 % of PCM is metabolized to a highly reactive toxic metabolite: N-acetyl-p-benzoquinoneimine (NAPQI), via CYP450 [32]. NAPQI is commonly reduced by glutathione (GSH) to a non-toxic mercaptate conjugate. GSH has been highlighted as being responsible for the antioxidant defense in humans by scavenging the free radicals produced through the metabolism processes within the liver [33] in order to prevent subsequent cell damage. Excessive intake of PCM causes the metabolic pathways, particularly the sulphation and glucuronidation pathways, to become saturated, resulting in the shunting of more PCM down the CYP450 pathway to generate unnecessary high amounts of NAPQI. Excessive production of NAPQI depletes the GSH stores in the body [34]. GSH concentrations dropped to low levels in the centrilobular cells, subsequently affecting the ability of GSH peroxidase, the major peroxide detoxification enzyme, to function efficiently under conditions of GSH deficiency. In the situation when the GSH supply decreases to less than 30 % of the normal, the unbound NAPQI will bind nonspecifically to tissue macromolecules, including intracellular proteins, particularly those with sulfhydryl groups, consequently causing cell dysfunction and death [32, 35].

Despite the advancement in the field of modern medicine with the development of various types of new drugs to treat various types of ailments, their effectiveness is sometimes surpassed by their adverse side effects. Therefore, researches are being carried out throughout the world to find alternative drugs with less, or, possibly, no side effects to help treat those ailments. In the case of liver toxicity, limited number of drugs has been developed to help fight this disease, but their uses are usually accompanied by unwanted side effects. In an attempt to contribute to the search for potent and safe liver-protecting agent, the hepatoprotective potential of B. purpurea was investigated. The reason for choosing B. purpurea was based on the findings that the plant possesses remarkable antioxidant and anti-inflammatory activities [7, 8, 9, 12, 13], which have been generally known to play part in the mechanisms of hepatoprotection.

In the present study, three types of partitions, namely PEBP, EABP and AQBP, were prepared from MEBP and subjected to the PCM-induced liver toxicity in rats’ model. From the results of serum biochemical analysis, EABP was found to be the most effective partition followed by the AQBP with PEBP shows no effect on the liver function enzymes level. Only EABP, at all doses tested,  were able to reduce the serum enzymes’ level, which was earlier increase by the overdose PCM. This finding was supported by the microscopic observations and histopathological scoring. Overall, these observations suggested that the hepatoprotective activity of B. purpurea could be due to the synergistic action of a mixture of polar and non-polar compounds with the intermediate polarity compounds being the main hepatoprotective compounds in B. purpurea. Moreover, the suggested synergistic action of compounds present in EABP was further supported by the phytochemical analysis of EABP that demonstrated the presence of flavonoids, tannins and saponins. These classes of compounds have been reported to exert antioxidant [36, 37, 38] and anti-inflammatory [39
40, 41] activities.

TPC values were influenced by the non-polar to polar solvent extractions. In this case, the value decreased in the following order: EABP??AQBP??PEBP. Based on the solvent polarity chart, ethyl acetate is classified as an intermediate solvent, hence is able to extract intermediate-polarity phenolic compounds, while aqueous and petroleum ether extract polar and non-polar compounds, respectively. The present findings were further supported by Azlim Almey et al. [42], who suggested that higher extraction yields of total extractable polyphenols and total soluble solids are collected as the solvent polarity increases. In the present study, although EABP was considered to have the highest TPC value (271 mg GAE/100 g extract) in comparison to the other partitions (PEBP?=?105 mg GAE/100 g extract; AEBP?=?133 mg GAE/100 g extract), the value was lower than the standard value (1000 mg GAE/100 g extract) for any substances to be considered as having a high TPC [19]. It is important to highlight that MEBP did possess a high TPC value of approximately 1218 mg GAE/100 g extract [14]. Usually, it is anticipated that extracts with a high amount of polyphenolic content should also exhibit high antioxidant activity [43]. However, there is also report on the presence of extract with low TPC value exhibiting high antioxidant activity [44], which supported the presence finding as demonstrated by EABP. Reihani and Azhar [45], while discussing on their finding that the curry leaf demonstrated a comparatively low antioxidant activity despite showing the highest TPC value, cited that the difference could be related to poor specificity of the TPC assay [46, 47]. Moreover, Singleton et al. [47] also suggested that the phenolic compounds, depending on the number of phenolic groups they have, react differently to the Folin–Ciocalteu reagent.

In discussing the relationship between TPC value and antioxidant activity, it is worth to discuss on phenolic compounds in general. Phenolic compounds, broadly distributed in the plant kingdom, are the most abundant secondary metabolites of plants. They are compounds having one or more aromatic rings with one or more hydroxyl groups. Currently, more than 8000 phenolic structures have been identified, ranging from simple molecules (i.e. phenolic acids) to incredibly polymerized substances (i.e. tannins). The most common plant phenolics include phenolics acids, flavonoids, tannins, with flavonoids being the most plentiful polyphenols in our diets. Flavonoids are themselves classified into several subclasses depending on the oxidation state of the central C ring. The five well-known subclasses of flavonoids are flavonols, flavones, flavanones, flavanonols, and dihydroflavonols. Their structural dissimilarity in each subgroup is in part attributable to the degree and pattern of hydroxylation, methoxylation, prenylation, or glycosylation [48]. Tannins, another major group of polyphenols, are usually subdivided into two classes, namely hydrolysable tannins and condensed tannins. Hydrolysable tannins are compounds containing a central core of glucose or another polyol esterified with gallic acid (known as gallotannins), or with hexahydroxydiphenic acid (known as ellagitannins). The high diversity in the structure of these compounds is attributable to the many possibilities in forming oxidative linkage [49]. On the other hand, condensed tannins are oligomers or polymers of flavan-3-ol connected through an interflavan carbon bond. The structure diversity of this class of tannins can be due to various factors such as the discrepancy in hydroxylation pattern, stereochemistry at the three chiral centers, and the location and type of interflavan linkage, as well as the degree and pattern of methoxylation, glycosylation and galloylation [50]. The fact that there are various types of polyphenolic compounds in EABP might support the claim made by Singleton et al. [47] that those polyphenolic compounds react differently to the Foin-Ciocalteu reagent resulting in the low TPC value obtained.

Polyphenolics, as antioxidants, are believed to have the capability to donate hydrogen to free radicals, consequently breaking the chain reaction of lipid peroxidation at the initiation stage [51]. The fact that EABP exerted high antioxidant activity when assess using the superoxide radical scavenging and ORAC assays seems to suggest the presence of high polyphenolic compounds in EABP. However, this suggestion seems to be contradicted by EABP’s low TPC value indicating that the antioxidant activity did not correlate with the TPC value. According to Wong et al. [52], since TPC value is obtained following the Folin–Ciocalteu reaction, which is based on redox reactions, the assay detects not only polyphenolic compounds, but also other biological substances that are reactive towards the Folin–Ciocalteu reagents (i.e. amino acids, carbohydrates and ascorbic acid) [46, 47]. It is plausible to suggest that some of the polyphenolic compounds present in EABP did not work via redox reaction against the Folin-Ciocalteu reagents leading to low TPC value. In addition, the low to moderate antioxidant activity observed in the DPPH radical scavenging assay could be due to the presence of some polyphenolic compounds that may not scavenge DPPH radicals (DPPH?) due to steric resistance [52]. Following the DPPH radical scavenging assay, EABP was also found to exert the highest antioxidant activity further suggesting the parallel relationship between total phenolic value and antioxidant activity. In the SOD scavenging assay, EABP again demonstrated potential free radical scavenging activity. The ability of EABP to effectively attenuate both assays suggests its potential to work via the mechanism of reduction. Interestingly, EABP and AQBP were also able to exert remarkable antioxidant activity when measured using the ORAC assay, which is based on the peroxyl radical absorbance capacity of an extract [53]. Based on all of the above analysis, EABP is believed to have promising antioxidant activity followed by AQBP and PEBP.

Following subjection to the in vitro LOX and XO assays, all partitions of MEBP were found to exert very low anti-inflammatory activity. LOXs are enzymes that have a major role in manipulating the biosynthesis of leukotrienes, which have been ascribed to the pathophysiology of several allergic and inflammatory diseases [54]. Meanwhile, XO is important for catalyzing the sequential oxidation of hypoxanthine to xanthine, and to the production of hydrogen peroxide and uric acid [55]. The enzymatic activity via the LOX and XO pathways essentially involve modulation of the inflammation mechanism [56, 57]. Although the present anti-inflammatory findings might be in contrast to previous anti-inflammatory report on B. purpurea [15], several factors are to be considered. Firstly, the type of inflammatory models used to evaluate the anti-inflammatory potential of extract and partitions were significantly different, wherein assessment of the earlier was made using in vivo models whereas assessment of the latter was made using the in vitro models. Moreover, the in vivo models used were more related to the cyclo-oxygenase (COX)-dependent model [15]. COX plays a major role in catalyzing the production of prostaglandin [58]. The role of COX and prostaglandin in the modulation of B. purpurea pharmacological action could be justified by the ability of MEBP to exert antiulcer activity. As discussed by Zakaria et al. [9] and Abdul Hisam et al. [59], the aqueous (AEBP) or chloroform (CEBP) extracts of B. purpurea exert antiulcer activity via a local and non-specific mechanism known as cytoprotection. It is claimed that cytoprotection take place as a result of the ability of certain compounds to trigger the synthesis of prostaglandin, which consecutively lead to the synthesis of mucus and bicarbonate. Thus, from the results obtained it is plausible to suggest that the anti-inflammatory property of B. purpurea that may contribute to the hepatoprotective activity of EABP or AEBP occur via the non-LOX, non-XO-, but COX-mediated pathway.

The presence of hepatoprotective activity in B. purpurea, particularly its methanol extract (MEBP) and partitions (EABP and AQBP), could be attributed to the presence of various phytoconstituents in the extract and its partitions. Flavonoids, tannins, saponins and triterpenes have been reported elsewhere to exert various pharmacological activities including hepatoprotective activity [40, 60, 61, 62]. Although the presence of other classes of compounds should not be ignored, the presence of flavonoids in the partition, particularly, will be highlighted in this discussion based on the HPLC findings. The presence of flavonoids in B. purpurea has been reported earlier by Yadav and Bhadoria [63], supported the presence of flavonoids in MEBP or its partitions, namely EABP and PEBP, as demonstrated by the phytochemical screening data. This claimed was further strengthen by the recent HPLC findings, which show that the detected peaks fall within the UV-Vis spectra (?_max) of flavonoid-type compounds [64]. According to Tsimogiannis et al. [64], flavonoids are categorized into five major subgroups: flavonols, flavones, flavanones, flavanonols, and dihydroflavonols. There are 2 absorbance bands on the UV-Vis spectra for determination of flavonoid types wherein for flavonols or flavones the spectra that fall in the respective region of 350–385 nm or 310–350 nm was labeled band A while for band B the spectra lies in the region of 250–290 nm. EABP had 3 major peaks detected from different wavelengths with ?_max at 256–353 nm (Fig. 3), and the 4 major peaks of AQBP were detected  at 251–354 nm (Fig. 4). Referring to the chromatogram of the extracts, the major peaks that fell in the region mentioned by Tsimogiannis et al. [64] are apparently flavonoid-based compounds. The most outstanding properties of almost all groups of flavonoids is how they act as antioxidant [65, 66] and anti-inflammatory [67, 68] agents. The ability of EABP to exert the most effective hepatoprotective activity could be linked partly to the presence of flavonoid-based compounds, which might act as antioxidant and COX-modulated anti-inflammatory agents as discussed above. Other than the types of polyphenolic compounds discussed above, it is also important to highlight on the presence of complex polyphenolic compounds such as those that form dimeric flavonoids or those that form a complex with carbohydrate or protein [69]. Focusing on the leaves of B. purpurea, our literature search demonstrated that the bioactive compounds identified from the leaves were not single pure polyphenolic compounds but rather dimeric flavonoids of which one of them were associated with carbohydrates, namely bis [3’,4’-dihydroxy-6-methoxy-7,8-furano-5’,6’-mono methylalloxy]-5-C-5-biflavonyl and (4’-hydroxy-7-methyl 3-C-?-L-rhamnopyranosyl)-5-C-5-(4’-hydroxy-7-methyl-3-C-?-D-glucopyranosyl) bioflavonoid [70]. The presence of a complex polyphenolic compounds might explained why there is no match of HPLC peaks when comparison were made between the HPLC chromatogram of MEBP or EABP against several pure flavonoids (i.e. fisetin, quercetin, rutin, quercitrin, naringenin, genistein, pinostrobin, hesperetin, dihydroquercetin, flavanone, 4’.5’7’-trihydroxy flavanone) (data not shown), thus, suggesting that the flavonoids present in MEBP and EABP are not pure polyphenolic compound(s) but rather a complex polyphenolic compounds as described above.