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The Hepatoprotective Effect of Cochlospermum Religiosum Bark Extract and its Isolated Constituent Through Modulation of FABP1 Protein.

Introduction

The liver plays several critical functions in our body along with detoxification process. During the detoxification process, the exogenous chemicals might induce the free radicals and might harm the liver and its functions [1]. Also, these toxins are favourable to cause the liver disease like cirrhosis, fatty liver, hepatitis, fibrosis and hepatocellular carcinoma, etc [2].

Paracetamol is widely used as an antipyretic and analgesic drug which is procured by the patients Over the Counter (OTC) in India, highly responsible to produce acute hepatic damage when consumed an overdose. Its reactive metabolite causes the free radical stress and glutathione (GSH) depletion leading to liver necrosis [3]. To treat the hepatic (acute and chronic) disorders, modern medicine faces the challenges and utilization of these drugs on long duration might also lead to the various side effects as the list above. Hence the alternative medicine, enriched with the secondary metabolites like phenolic compounds like flavonoids, anthocyanins, flavonols, flavones, isoflavonoids, flavan-3-ols, and proanthocyanidins, (a) which possess the potential to terminate the free radicals/protection against the Reactive Oxygen Species(ROS), (b) improve the level of antioxidant enzymes, (c) modulate gene expression [4], (d) regenerate the hepatic cells and (e) protects the liver against damage [5].

Cochlospermum religiosum (L) belonging to the family Cochlospermaceae is commonly called as the silk-cotton tree, buttercup tree, Kattupparutti (Tamil) and Galgal (Hindi). The tree produces a gum called as katira. It is used to treat fistula, diarrhoea, cough, pharyngitis, dysentery, syphilis, trachoma and gonorrhoea. The dried flowers and leaf are used as antipyretic, stimulants, sedative and laxative [6]. It is also used as an anti-inflammatory agent in Siddha Drug 'KalnarParpam' [7]. The leaves are traditionally used to treat liver diseases and jaundice [8].

The roots of C. religiosum have profound hepatoprotective activity [9]. Traditionally the bark possesses the broad spectrum of activity and hence it is used to treat for microbial infections and also its extract is used to treat jaundice [10]. Owing to the protect the liver from various damages and regeneration of liver cells, an attempt has been made to isolate the Butyl heptadec-9-enoate by bioassay-guided fractions of the ethanolic extract of C. religiosum and researched for it's in vivo hepatoprotective activity against paracetamol-induced Wistar rats. Also, it was investigated for in vitro cytotoxicity against HepG2 cell lines. Furthermore the mechanism action of the isolated compound was explored by molecular docking analysis.

Materials and Methods

All the solvents were purchased from Sigma Aldrich, USA. The procured solvents were used for the experimental procedures of extraction of the bark samples. The bark of Cochlospermum religiosum Linn, was collected during the month of June, from the Tirunelveli region, Tamil Nadu, India. The plant material was authenticated by Dr.P. E. Rajashekar Principal scientist, Division of Plant Genetic Resources, (DPGR), Indian Institute Horticultural Research- ICAR, Bengaluru. The collected fresh samples were washed with double distilled water in order to remove the external sand and debris. It was shade dried to restore the volatile organic compounds, after drying the herbarium specimen was stored at Krupanidhi College of Pharmacy, Bangalore, and the specimen no. is CR-319/KCP/ 2017-18. It was combined together into fine powder in a mechanical blender and sieved with mesh no.20 to get fine powder and further it was subjected to extraction.

All analysis was performed on by analytical RP-HPLC (Shimadzhu LC- Prominence 20AT). The analytical column used was column (C18, 250 mm x 4.6 mm, 5u particle) with a mobile phase consisting of HPLC grade Methanol:Water (50:50) and employing gradient elution at a flow rate of flow rate 1ml/min.. The temperature of the column was maintained at 25[degrees]C and the detection wavelength was set at 254 nm. The volume of the sample injection was 10ul.

Extraction of bark of C. religiosum and preliminary phytochemical investigations

The stem bark of C. religiosum was shade dried. Ethanol (250 x 3) ml was used as the solvent to extract 200 g of dried powder of C. religiosum using hot continuous soxhlet extractor for 24 h. The extracted solvent was further evaporated under reduced pressure to obtain dried extract of EECR (Ethanolic extract of C.religiosum). EECR was subjected to preliminary chemical tests to identify different phytoconstituents [11].

Determination of total phenolic content

The total phenolic of the extract EECR was determined by a reported method [12]. About 50 mg of the EECR extract was dissolved in 50 ml of methanol. Serial dilution was performed to get the lower concentration of samples. For the minimal concentration serial dilution methodology was followed. 200 [micro]l of the extract (1 mg/ml) was taken in a test tube and it was mixed with 1 ml of Folin-Ciocalteu reagent and 800 [micro]l of sodium carbonate. After shaking, it was kept for under dark for 2 h. The absorbance was measured at 750 nm. The total phenol content of the extract was determined using the calibration curve and the inference was expressed as gallic acid equivalent in mg/g of the extract.

Determination of total flavonoid content

The total flavonoid of the extract EECR was measured by Swain and Hill's assay method [13]. About 50 mg of EECR extract were dissolved in 50 ml of methanol. For the minimal concentration serial dilution methodology was followed. To 0.2 ml of the EECR extract, 2 ml of double distilled water and 4 ml of vanillin reagent was added rapidly in a test tube. The mixture is allowed for incubation for 15 min and measured for its absorbance at 500 nm. Double distilled water is used as blank. The total flavonoid content of the extract was determined using the calibration curve and the observation was expressed as phloroglycinol acid equivalent in mg/ g of the extract.

Isolation of bioassay guided fraction by column chromatography

The extract (EECR, 2g) was re-suspended in 10ml ethanol and was used for liquid liquid partition chromatography. Chloroform, methanol and hexane soluble fractions such as C.religiosum bark liquid-liquid chloroform fraction (CRBLCF), C.religiosum bark liquidliquid methanol fraction (CRBLMF) and C.religiosum bark liquidliquid hexane fraction (CRBLHF) were separated using separating funnel. There was no yield for CRBLHF. CRBLCF and CRBLMF were analyzed by TLC and HPLC to check the purity. Since there was more yield with CRBLMF (Cochlospermum religiosum bark liquid-liquid methanol fraction), it was further subjected to column fractionation.

A 1 g sample of C.religiosum bark liquid-liquid methanol fraction (CRBLMF) was fractionated into four fractions CRB1, CRB2, CRB3 and CRB4 of chloroform:methanol in the following ratios: 1:19, 1:4, 1:1 and 0:1 (v/v) using column chromatography using silica gel (mesh 60-120) as stationary phase and gradient elution technique was implemented [14]. The fractions were analyzed by TLC to check for purity and further analyzed by HPLC to check percent purity. Fraction CRB4 was showing 82.9% purity more than other fractions (CRB1, CRB2 and CRB3); it was taken up for further analysis such as LCMS, NMR and CHN analysis.

Characterization of compound CRB4

The isolated compound was characterized by LCMS/MS, CHN and NMR analysis. For LCMS/MS analysis the samples was dissolved in methanol, 5 [micro]l of the sample was injected which will give both LC and MS information with run conditions such as column of Cl8, 4.6*100mm, 5 [micro]m, with mobile phase of methanol and water at 90:10 ratio, with rate of flow of 0.2 ml/min was injected at a volume of 5 [micro]l and detection was done as 254 nm.

Paracetamol induced in vivo hepatoprotective activity in Wistar rats

Male Wistar albino rats between 150 and 200 g were procured from Radiant Research Pvt Ltd, Bangalore with permission from Institutional Animal Ethical committee (CPCSEA Reg No. RRPL/ 06/CPCSEA). The animals have been housed in well-known polypropylene cages and maintained under good laboratory conditions (12:12hr light & dark; 35-60% relative humidity and at 25 [+ or -] 50 C ambient temperature). The animals had been fed with water and rat pellet diet ad libitum.

Acute toxicity study of C.religiosum for dose determination

The oral acute toxicity was conducted consonantly OECD guiding principle 423, which stipulates the usage of three animals which were fasted overnight (12 h) and weight were recorded [15]. Test dose of ethanol extract of C.religiosum (EECR) have been calculated with regard to the weight of fasted animal and administered orally about 2000mg/kg. b.w. The animals had been regularly observed of behavioural modifications and signs of toxicity for the first 24h after dosing. Observation was continued each day for a complete of 14 days [16].

In vivo paracetamol induced hepatotoxicity of C. religiosum

The experiment was carried in accordance the technique of Soliman et al., 2014 [17]. Six groups were taken for the study, each group consist of six Wistar rats. Group I served as Normal Control received 0.5% carboxy methyl cellulose (CMC) and Group II served as toxic control and were administered with Paracetamol i.e 400 mg/kg.b.w. [18] for seven days. Group III rats received standard drug (Liv-52, 5ml/kg, b.W) [19-20] for seven days and concurrently administered paracetamol (400 mg/kg). Group IV, V and VI were acquired test drug EECR (100 mg/kg, 200 mg/kg and 400mg/kg) each day up to seven days respectively and concurrently administered Paracetamol (400 mg/kg). On the eighth day the blood was collected from retro-orbital plexus of rats.

Determination of key liver function biochemical markers and oxidative stress markers

The collected blood from Wistar rats was centrifuged at 2500rpm for 15min at 30[degrees]C and serum was used for the estimation of diverse biochemical parameters specifically aspartate amino transferase (AST) [21], alanine aminotransferase (ALT) [21]. alkaline phosphatise (ALP) [22], total protein (TP)[23] and total bilirubin [24-28]. Animals have been sacrificed and livers have been excised and washed in 50 mM ice cold potassium phosphate buffer of pH 7.4. 10% w/v liver homogenates have been prepared in Tris-Hcl buffer (0.15 M) and the suspension was centrifuged for 15 min at 3000 rpm and supernatant was accrued and analyzed for superoxide dismutase (SOD), Catalase (CAT), lipid peroxidation (LPO), reduced glutathione (GSH), glutathione peroxidise (GPx), gamma glutamyl-transferase (GGT), glutathione S-transferase (GST) [26,2731].

Morphological assessment of rat liver

Liver have been taken out followed by washing with saline (0.9%). Small parts of liver tissues are stored in 10% formaldehyde for histopathological examination. These tissues have been impacted in paraffin wax. Sections of 5-6 [micro]m in thickness have been cut and stained with hematoxylin-eosin stain. The level of damage was observed using microscope.

In vitro cytotoxicity assay of extract (EECR) and isolated compound (CRB4) in HepG2 Cell line

HepG2 cells which are human liver hepatoma cell line were procured from National centre for cell sciences, Pune, India were seeded (1x 105cells/ T25 Flask) and cultured in Dulbecco's modified Eagle medium (DMEM) containing 1% Streptomycin and10% Fetal Bovine serum (FBS) 37[degrees]C with 5% C[O.sub.2] (Binder) for 24h. Cells have been sub-cultured each third day by trypsinisation with 0.25% Trypsin-EDTA solution. 50,000 cells/well cell density was seeded to 96 well plate (Tarson India pvt Ltd). After 24h, 100 [micro]l of different concentrations (10, 20, 40, 80, 160, 320 [micro]g/ml) of EECR (Crude) and CRB4 (isolated compound) were added to the well. Once monolayer was formed after 24hr, the supernatant had been discarded and washed with medium followed by addition of different concentrations of drugs. The plates have been then incubated for 24 h at 37[degrees]C with 5% C[O.sub.2] atmosphere. After incubation the supernatant was discarded and 100 [micro]l of 0.6mg/ml MTT was added to every well. The plates have been incubated for 4hr at 37[degrees]C with 5% C[O.sub.2] The supernatant was discarded and 100 DMSO was added. The OD was measured at 590 nm using plate reader (Tecan, SpectraFluor Plus) and percentage inhibition was calculated [32].

In vitro hepatoprotective activity study in HepG2 cells against Paracetamol induced hepatotoxicity

Two sets of 5.0*[10.sup.4] cells were seeded to 96 well plate with culture media containing 100 [micro]M PCM in presence of EECR (Crude) and CRB4 (isolated compound) at the concentrations of20, 40 and 80 [micro]g/ml for 24 h.

For first set, 100 [micro]l 0.6 mg/ml MTT was added to every well. The plates have been incubated for 4hr at 37[degrees]C. The supernatant had been discarded and 100 DMSO was added. The OD was measured at 590 nm using plate reader (Tecan, SpectraFluor Plus) and percentage hepatoprotective activity was calculated.

Statistical analysis

Statistical analysis had been computed by ANOVA (Analysis of Variance) with Tukey test where P<0.05 was significant in all test. All data reported as mean [+ or -] SEM. [IC.sub.50] value was calculated using version 5.0 Graph Pad Prism.

Molecular docking analysis

The complexation of the isolated compound butyl oleate with Liver fatty acid binding protein (L-FABP) was assessed by molecular docking analysis further to understand the mechanism of action.

To identify the possible interactions of butyl oleate, Glide module of Schrodinger Suite was used. The chemical structure of the isolated compound was drawn in 2D and optimized using Ligprep [33] module of Schrodinger software using default parameters. The 3D structures of L-FABP in complex with oleate (PDB ID: 2LKK), Iodide derivative of human LFABP (PDB ID: 3B2I) were retrieved from Protein Data Bank (PDB). Cocrystallized structure of L-FABP with ligand was minimized by OPLS-2005 force field and grid was generated at the active site of L-FABP with the ligand. The ligand was docked on to grid file by using Glide-SP (Standard Precision) methodology [34]. The hydrogen bonding and hydrophobic interactions of the isolated compound butyl oleate was observed for best docked poses and compared with active compounds. All the molecular docking studies were carried on a laptop running windows 7, 64 bit operating system with configuration of 3.00 GB RAM, Intel core i3 CPU M350 @ 2.27 GHz.

Results and Discussion

It is well known that traditional system of medicine presents tremendous hope as far as the treatment of liver diseases is concerned. C.religiosum bark is traditionally used to treat jaundice. The present study was designed to establish the scientific evidences for the usage C.religiosum plant in hepatic conditions. The percentage yield from powdered C. religiosum was found to be 4.2% (W/W) of a green coloured powder of EECR. When EECR was subjected to the preliminary qualitative phytochemical analysis by standard procedures it revealed the presence of carbohydrates, flavonoids, alkaloids, phenols, tannins and terpenoids. Studies have shown the stem bark of Cochlospermum religiosum contains many primary and secondary metabolites including phenolic compounds and flavonoids [35]. The hepatoprotective potential of EECR can be explained based on the respective phytoconstituents detected in the extract. For example flavonoids have been reported to exert antioxidant [36] and hepatoprotective [37] activities.

Determination of total phenolic and flavonoid content in EECR

The estimation of total phenolic and flavonoid content has also revealed that is contains considerable amount is comparison to standand gallic acid and phloroglucinol.

Folin-Ciocalteu method was used to measure total phenolic content and is reported as gallic acid equivalents (GAE) and is found to be 375.89 [+ or -] 4.35 mg GAE/g extract. The total flavonoid content in EECR is expressed in phloroglycinol (PGE) which is found to be 71.9 [+ or -] 0.9 mg/g extract.

The plant polyphenols known as flavonoids that represent the most common and extensively distributed group of phyto-constituents serve as strong antioxidants, exhibit their protection against oxidative stress-induced cellular damage through various mechanisms such as free radical scavenging, metal ion chelation and anti-lipid peroxidation [38]. And therefore these antioxidant chemicals, particularly polyphenols, could contribute to its antioxidant and hepatoprotective activities [39]. Based on all of the reports, the hepatoprotective activity is C.religiosum can be suggested possibly with involvement of synergistic actions of flavonoids, phenols and terpenoids.

HPLC profiling of extract, fraction and isolated compound

The compounds (crude, fraction and isolated compound) were isolated and identified based on the retention times, UV-visible spectra and mass spectra. The absorption spectra was obtained at 254 nm wavelength showing the Rf value of 8.377 with 72.4 %, 2.81 with 32.7% and 2.62 with 82.9% for crude, fractionated compound and isolated compound respectively. The HPLC chromatogram for the isolated compound CRB4 was obtained as shown in the figure 1.

Characterization of Compound CRB4

LCMS/MS analysis: Total ion chromatogram in positive mode and negative mode are shown in figures 2(a) and (b) respectively. The positive mode mass spectrum of compounds show abundant [(M + H).sup.+1] mass of 733 and 363 (Retention time-0.339 min). The negative mode mass spectra of compounds show abundant(M H)-1mass of 362, 296, and 177 (Retention time 0.334min).

The compound CRB4 confirmed a pseudo molecular ion in its mass spectrum with peak at m/z 363 for [[M+K].sup.+] ion and suggests a molecular formulae [C.sub.21][H.sub.40][O.sub.2] .

NMR spectral analysis

[sup.1]H-NMR pectra: In the [sup.1]H-NMR spectra (figure 3(a),(b); figure 4(a),(b)) the triplet signal at [delta] 0.87 is due a methyl group, the strong singlet at [delta] 1.27 suggests the presence of long chain methylene groups, the multiplet signal at [delta] 1.60 is due to methylene groups attached to unsaturated systems and the signal at [delta] 2.05 is due to a methylene group adjacent to a carbonyl group. The bunch of signals at [delta] 4.13 is due to the protons under oxygen function i.e. a methylene group under oxygen function (-C[H.sub.2]O).

[sup.13]C-NMR pectra: In the [sup.13]C-NMR spectra (figure 5(a), (b), (c)) the signals at [delta] 13.73 and 18.65 are attributed to methyl carbon, the signal at [delta] 65.57 suggests the presence of a -C[H.sub.2]OH group, the signals at [delta] 128.72 and 128.84 suggests the presence of a -CH = CH- group, the signal at 167.72 is due to a carbonyl group. The rest of the signals at [delta] 19.16, 19.18, 19.75, 22.34, 28.10, 29.70, 30.57, and 38.06 are due to the presence of a long chain of methylene groups.

CHN analysis

The structure of the isolated compound CRB4 was elucidated from the mass spectrophotometric, NMR and CHN analysis. The signals at a 128.72 and 130.84 suggests the presence of a -CH = CHgroup. It matches with the molecular formulae, by the a pseudo molecular ion peak at m/z 363 for [[M+K].sup.+] ion. A search on the literature suggests that a fatty acid with one C=C is oleic acid (the position of the double bond will be at C-9. Taking this into consideration the structure of the compound has been identified as Butyl oleate (figure 6(a) (b)).

Butyl oleate is a fatty acid ester lipid molecule [40]. Studies have suggested that fatty acid methyl esters present in various plants possess antioxidant properties [41-42]. Oleic acid is found in plant extract has shown to possess potent antioxidant and anti-inflammatory property and also it has been shown that it may inhibit the progression of adreno leukodystrophy (ALD), a life threatening disease that affects the brain and adrenal glands [43]. Oleic acid present in olive oil has been reported to be responsible for its hypotensive (blood pressure reducing) effects [44]. And there are also reports that show that phenolic acid ester derivatives possess potent anti-oxidant properties, may have useful applications as antioxidants [45].

Paracetamol induced in vivo hepatoprotective activity in Wistar rats

Acute toxicity study of C.religiosum for the dose determination

Oral administration of ethanolic extract of C.religiosum (EECR) did not show any sign of toxicity and mortality up to 2000mg/kg body weight dose. Therefore 1/5, 1/10 and 1/20th of 2000mg/kg was selected for the in vivo studies.

Determination of key liver function biochemical markers and oxidative stress markers

The major organ of the body concerned with regulation of internal chemical environment is liver. Therefore, damage to the liver imposed by a hepatotoxic agent is of grave outcome. Acetaminophen, a popular pain killer drug is also called as paracetamol or APAP (N-acetyl-p-aminophenol), even though considered as safe, an overdose of APAP can result in severe hepatic injury due to the formation of its , a toxic metabolic intermediate NAPQI, mainly via the cytochrome P4502E1 (CYP2E1) enzyme pathway [46]. NAPQI is a highly reactive free radical which can readily oxidize lipids; consequently, it initiates the process of lipid peroxidation to cause the damage in liver cells [47]. PCM induced liver toxicity is a well known experimental model to determine the effectiveness of hepatoprotective agents [48]. The mechanism of paracetamol induced liver injury is the covalent binding of its toxic metabolite, n-acetyl-p-benzoquinone-amine to the protein sulfhydryl groups and causing severe lipid peroxidation and cell death [49]. There is an increase in the serum enzyme markers due to leakage of serum enzymes resulted due to impaired transport mechanism of hepatocytes caused by paracetamol overdose [50] thus causing an increase in serum enzyme levels.

Liver serum biomarkers such as AST, ALT, ALP, TB and TP, till today remain as the standard methods for the assessment of liver injury. The present study reports the prospective hepatoprotective effects of EECR against liver injury produced by paracetamol in wistar rats. Liver injuries are often associated with a rise in serum AST, ALT, ALP and TB levels and lowering of TP levels. The EECR had been subjected for in vivo hepatoprotective and antioxidant studies at three different dose levels (100mg/kg, 200mg/kg, 400mg/kg) using Paracetamol induced male wistar strain of albino rats.

A decrease level of AST, ALT, ALP and total bilirubin and increase levels of total proteins had been observed in the treated group compared to paracetamol induced rats. The EECR at concentration 100, 200 and 400mg/kg extensively (p<0.05) recovered the biochemical parameters near to the normal. Liv 52 (5ml/kg) and EECR at 400mg/kg showed recovering property of all biochemical parameters near to normal (table 1).

Increased lipid peroxidation causes tissue injury and collapse of antioxidant defence mechanism to avoid excessive free radical formation in PCM induced toxicity. In liver homogenate, there has been significant decrease in CAT, SOD, GST, GPx and GSH similarly increase in GGT and LPO levels was detected in treated group compared to paracetamol induced group. Group treated with Liv 52 (40mg/kg) and EECR 400mg/kg showed increase level of CAT, SOD, GST, GPx and GSH also decrease level of GGT and LPO significantly (P<0.05). No significant change in above parameters was observed in EECR dose at 100 and 200 mg/kg (table 2 & 3).

EECR treatment to the rats significantly reduced the raised levels of LPO in dose dependant manner. Decline is SOD is one of the sensitive indices for assessing liver cell injury [51]. It is one of the essential enzymes in the enzymatic antioxidant defence system. It converts superoxide anion reduces into hydrogen peroxide and reduces the toxic effect. EECR causes a significant elevation in liver SOD activity and thus lessens free radical induced oxidative injury to liver. Catalase defends the tissues from highly reactive hydroxyl radicals by decomposing hydrogen peroxide. Therefore a decline in catalase activity may cause various harmful effects due to hydrogen peroxide and superoxide radical assimilation. The standard drug Liv-52 and the higher dose of EECR i.e., 400mg/kg increase the catalase activity level. Restoration of catalase activity and thereby the decrease in hepatic damage might reflect the antioxidant activity of the phytoconstituents present in the plant used in this experiment.

GSH, a non enzymatic antioxidant is one of the plentiful tripeptide found in the liver. It gets rid of the reactive oxygen species for instance superoxide radicals, hydrogen peroxide and preserves protein thiols of membrane. In PCM intoxicated rats, the reduced levels of GSH are linked with lipid peroxidation. EECR administration significantly restored the levels of GSH, GPx, GST and GGT in a dose dependant manner. The presence of phenolic compounds and flavonoids as disclosed in preliminary phytochemical assessment of EECR. The observed hepatoprotective and antioxidant activities of EECR are may be due to the presence of these phytoconstituents.

Morphological assessment of rat liver

The protective effect EECR on liver toxicity was further established by morphological studies of the liver, which essentially assisted the results from the serum assays. Morphological assessment of the liver showed macro and microvesicular steatosis and necrosis in hepatocytes in paracetamol treated rats (figure 7b). Liv-52 (figure 7c) and EECR, at various dose levels (100, 200 and 400 mg/kg, b.w) treated groups (figure 7d,e,f) showed protection which shows arrangement of normal hepatocytes in lobular pattern around the central veins and very minimal fatty change seen compared to paracetamol treated groups. The maximum protection of EECR against liver damage was achieved at a dose of400 mg/kg, b.w than other doses (100 and 200 mg/kg, b.w), which showed a more or less normal architecture of the liver having reversed to a large extent, the hepatic lesions produced by paracetamol, almost comparable to the normal control groups (figure 7a) which may be due to presence higher ratios of phytoconstituents compared to other doses (100 and 200 mg/kg, b.w).

Hepatoprotective effect of EECR and CRB4 in paracetamol induced HepG2 Cell line

In recent times in vitro cytotoxicity and hepatoprotective activity of plant extract and bioassay guided fractions has gained importance in screening at the primary level [52]. HepG2 cell line is a popular and an effective in vitro model for assessing hepatoprotective potential of phyto compounds and bioassay guided fractions due to its functional similarity to an intact liver [53-54]. Further to confirm the hepatoprotective activity, both crude extract EECR and the isolated compound CRB4 were subjected to in vitro studies using HEPG2 cell line and study discloses the hepatoprotective effect of EECR and CRB4 against PCM induced toxicity in HepG2 cells which was well in-coordination with the results of in vivo hepatoprotective studies of EECR.

Both EECR and CRB4 were found to show 75.56 and 30.78% toxicity at 320[micro]g/ml. For hepatoprotective activity 80 [micro]g/ml concentrations were considered as highest due to less than 50 percent cytotoxicity. The extracts EECR and CRB4 were found to show 41.2 and 22.53% cytoxicity at 80 [micro]g/ml compared to Paracetamol control which was showing 100% cytotoxicity (table 4).

The extracts EECR and CRB4 were found to show 26.98 and 44.44% hepatoprotectivity at 80 [micro]g/ml compared to untreated control and the standard drug Liv 52 which was showing a protection of 54.25% (table 5).

The mechanism of C.religiosum on PCM induced toxicity could be possibly due to the antioxidant potential of the phytoconstituents present such as of fatty acid esters, total phenolic and flavonoid components as illustrated in the (figure 9)

Molecular docking analysis

In order to investigate the binding capacity of the isolated compound butyl oleate from Cochlospermum religiosum on the proteins related to liver toxicity in humans, we docked the compound to the target proteins by doing computational analysis, glide docking. Both glide standard (SP) and extra precision (XP) mode had been introduced, where extra precision mode used for cross validation purpose. The high binding affinity of the ligand with the protein was explained clearly by interation analysis (figure 8). Previous studies have demonstrated that Human liver fatty acid binding protein (L-FABP) plays an important protective function in PCM induced toxicity [55]. In our study, docking of butyl oleate with 2LKK protein showed binding energy of -6.964 Kcal/mol showed a glide emodel score of -49.690 (table 7), the binding interation of the keto group of butyl oleate was established with hydrogen bond with ARG 122 which is the major interaction of butyl oleate with 2LKK creating a hydrophobic region with LEU 91,PHE 95, ILE 98, PHE 50, ILE 52, ALA 54, PHE 15, PHE 18, MET 19, ILE 22 (figure 8(a)).

In the docking analysis of butyl oleate with the derivative of human L-FABP (PDB ID: 3B2I) showed a energy of -7.232 Kcal/ mol showed a glide emodel score of -51.076 [table 7], the binding interation of the keto group of butyl oleate was established with hydrogen bond with ARG 122 which is the major interaction of butyl oleate with 3B2I creating a hydrophobic region with PHE 120, MET 113, ILE 109, ILE 41, PHE 50, ILA 52, ALA 54, ILE 35, LEU 28 (figure 8(b)).

On the basis of the docking scores of our ligand butyl oleate with the selected proteins, it can be inferred that the ligand-protein complex is more stable with 2LKK protein than with 3B2I protein showing a binding energy of -6.964 Kcal/mol.

Mechanism of C.religiosum in PCM induced liver toxicity

Paracetamol induced liver toxicity is one of the well known and commonly used animal models for studying the hepatoprotective property of plants. The nonsteroidal antiinflammatory drug Paracetamol (PCM) also called as Acetaminophen is often regarded as a safe, eventhough, excessive intake of this drug lead to acute liver failure and hepatic cell damage [56]. The mechanism of paracetamol overdosage on the liver is due to depletion of GSH content where the toxic metabolite, n-acetyl-p-benzoquinone-amine (NAPQI), covalently binds to the cellular mitochondrial proteins reduces the oxidation of mitochondrial fatty acids and this results in massive necrosis and apoptosis of hepatic cells [57-58]. Due to hepatic cellular damage caused by paracetamol overdose, a heavy disturbance is caused in the transport functions of hepatocytes and these results in the leakage of cellular enzymes to the plasma.

The loss of performance capability of cell membrane in liver is related with liver cell damage and it is indicated by high levels of AST, ALT and ALP [59]. Serum proteins are mainly derived from liver. The elevation of TB in blood stream can be attributed to over production, increased haemolysis, decreased conjugation or impaired bilirubin transport and hence its estimation is an important parameter to determine the normal functioning of the liver [60]. Antioxidant enzymes such as SOD, CAT, GPx, and GST are very important to encounter with oxygen species and offering protection to organisms. SOD is a protective enzyme, which converts superoxide radicals to hydrogen peroxide. The peroxisomes of eukaryotic cells contain a hemeprotein called Catalase which catalyses the conversion of hydrogen peroxide to water and oxygen. The chemically induced oxidative destruction of lipid and proteins leads to acute oxidative stress and to prevent this, maintaining the redox status is critical which is taken care by the protection offered by GPx in animals. Lipid peroxidation has been assumed to be the cause for deleterious process in liver injury due to overdosage of paracetamol. Excessive lipid peroxidation leads to tissue damage and failure of protection given by antioxidant mechanisms in preventing formation of excessive free radicals which is indicated with increase in LPO and GGT level of liver [61]. The toxic effects of reactive oxygen species produced by toxicants is indicated by a reduction of GSH, GPx, GST, SOD and CAT enzyme activity (figure 9). The mechanism of C.religiosum on PCM induced toxicity could be possibly due to the antioxidant potential of the phytoconstituents present such as of fatty acid esters, total phenolic and flavonoid components. Previous studies have shown the leaves of Cochlospermum reliosum show good antibacterial activity due to it's their antioxidant activity [62]. Myricetin is a naturally occurring flavonol was isolated from the leaves of Cochlospermum reliosum which showed anti-carcinogenic property due to its potent antioxidant potential [63]. This suggests that EECR and CRB4 can reduce reactive oxygen species that may decrease the oxidative damage to the liver and boost the activities of the liver antioxidant enzymes, thus protecting the liver from the damage induced by PCM. Also, the possible mechanism could be by the restoration of hepatic regeneration through a stimulated synthesis of protein or increase speed of detoxification and excretion.

The fatty acid-binding proteins (FABPs) are a family of highly expressed intracellular lipid-binding proteins (iLBPs) that serves to bind these free ligands with high affinity. FABPs are found expressed throughout tissues that are highly active in FA metabolism and consists several isoforms. To date, nine FABP protein-coding genes have been identified in the human genome. These include liver- (L-FABP), intestine-(I-FABP), heart- (H-FABP), adipocyte(A-FABP), epidermal- (E-FABP), ileal-(Il-FABP), brain- (B-FABP), myelin- (M-FABP) and testis-FABP (T-FABP) [64].

L-FABP or FABP1 is a liver fatty acid binding protein (FABP), is a soluble protein abundantly found in the cytoplasm of hepatocytes, and to a lesser extent in the nucleus [65-66] and outer mitochondrial membrane [65,67]. Unlike other members in the FABP family, each molecule of FABP1 is capable of binding two molecules of long-chain fatty acids [68]. The inactivation of free radicals by the smethionine and cysteine amino acids of FABP1 is thought to be the mechanism of FABP1's antioxidant activity [69]. FABP1 is present at high concentrations (approximately 0.4-0.8 mM) in hepatocytes and also binds many lipid peroxidation products [7071]. For these reasons FABP1 might serve as an endogenous cellular protectant [72-73]. Wang and colleagues had earlier suggested that FABP1 levels could be targeted through appropriate pharmacological treatment to minimize cellular damage [73-74]. Thus, FABP1 may be a new therapeutic strategy to suppres s ROS levels occurring in the liver [75] during chemotherapy [76-77] or drug-induced liver injury [78-79]. In summary, there is considerable evidence that FABP1 exerts cytoprotection in liver and kidney and that FABP1 is an effective endogenous antioxidant. The mechanism of C.religiosum on PCM induced toxicity could be possibly due to the antioxidant potential of the phytoconstituents present such as of fatty acid esters acting through the modulation of FABP1 protein, total phenolic and flavonoid components as illustrated in the (figure 9).

Conclusion

Overall, it could be concluded that EECR and CRB4 protects liver against oxidative stress induced by PCM both in vivo and in vitro and it is also effective in enhancing the activities of hepatic enzymes implicated in attacking ROS. The hepatoprotective action may be mainly mediated by the enhancement of hepatic glutathione regeneration potential and decreased level of lipid peroxidation attributed to the main chemical constituents of EECR such as total phenols, flavonoids and the isolated compound butyl heptadec-9-enoate or butyl oleate found to be a fatty acid ester.

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Jyothi Yachamaneni (1), Dhanaraj Sangeetha *, (2) Sai Saraswathi Vijayaraghavalu (2)

(1) Krupanidhi College of Pharmacy, Bangalore 560035. Karnataka, India

(2) Department of Chemistry, School of Advanced Sciences, Vellore Institute of Technology, Vellore 632014, Tamil Nadu, India

Received: 5 July 2020

Accepted: 1 November 2020

Published online: 3 August 2021

* Coresponding author

E-mail address: dsangeetha@vit.ac.in (Prof. D. Sangeetha, Asst. Professor (Sr), Department of Chemistry, School of Advanced Sciences, Vellore Institute of Technology, Vellore 632 014, India)

Caption: Figure 1: HPLC chromatogram of isolated compound CRB4

Caption: Figure 2: (a) & (b), LCMS chromatogram of CRB4 fraction. (Positive mode). No peaks were found in negative mode

Caption: Figure 3: (a),(b) NMR Chromatographic profile of CRB4 (isolated compound)

Caption: Figure 4: (a),(b) NMR Chromatographic profile of CRB4 (isolated compound)

Caption: Figure 5: (a),(b) NMR Chromatographic profile of CRB4 (isolated compound); (c) Structure of isolated compound

Caption: Figure 6: (a) CHN chromatogram of CRB4 (isolated compound); (b) The structure of the probable isolated CRB4 compound may be butyl heptadec-9-enoate or butyl oleate from stem bark of C. religiosum

Caption: Figure 7: Photomicrographs of sections of the liver of rats in control and treated groups of EECR in Paracetamol induced liver toxicity (HXE=10)

Caption: Figure 8: (a) 2D docked pose of Butyl oleate with 2LKK protein; (b) 2D docked pose of Butyl oleate with 3B2I protein

Caption: Figure 9: A schematic diagram showing the mechanism of protective effect of C.religiosam in PCM induced toxicity. PCM causes excessive oxidative stress and decreases the activity of antioxidant enzymes, such as GSH, GPx, GST, SOD and CAT, and increase in, LPO and GGT, possibly resulting in the elevation of [H.sub.2][O.sub.2]. The disturbance of oxidant/antioxidant balance caused oxidative stress of the cellular systems and results in liver toxicity, specifically thee elevation of the ALT, AST, ALP, TB and reduced TP levels. The Administration of C.religiosam (EECR and CRB4) was found to maintain the oxidant/antioxidant balance during PCM treatment, resulting in the prevention of the cell damage
Table 1: Effect of EECR on key liver function biochemical markers
on paracetamol Induced hepatotoxicity in rats
                                            ALT
Groups          Treatment                 (IU/L)
I          Normal Control 0.5%       38.83 [+ or -] 3.34
               Sodium CMC
II                 PCM             88.34 [+ or -] 4.88 (c)
         (400mg/kg of b.w, i.p.)
III              Liv-52            45.01 [+ or -] 5.62 ***
              (5ml/kg, p.o)
IV                EECR              66.39 [+ or -] 3.33 *
          (100mg/kg, b.w, p.o)
V                 EECR             57.96 [+ or -] 3.42 ***
          (200mg/kg, b.w, p.o)
VI                EECR             50.64 [+ or -] 3.01 ***
          (400mg/kg, b.w, p.o)
                  AST                      ALP
Groups           (IU/L)                  (IU/L)
I          110.3 [+ or -] 10.08       70.03 [+ or -] 6.00
II       209.9 [+ or -] 42.91 (c)   178.3 [+ or -] 7.90 (c)
III        121.5 [+ or -] 16.83     89.82 [+ or -] 3.40 ***
IV          153 [+ or -] 7.73        140.1  [+ or -] 9.23
V          142.4 [+ or -] 9.72      129.5 [+ or -] 6.96 **
VI         133.9 [+ or -] 5.13      116.6 [+ or -] 7.73 ***
                  TB                     TP
Groups         (mg/dl)                 (g/dl)
I          2.90 [+ or -] 0.06       7.497 [+ or -] 0.32
II       12.8 [+ or -] 0.22 (c)   3.478 [+ or -] 0.11 (c)
III      4.83 [+ or -] 0.13 ***   6.92 [+ or -] 0.19 ***
IV       8.05 [+ or -] 0.31 ***   5.05 [+ or -] 0.20 ***
V        5.77 [+ or -] 0.22 ***   5.893 [+ or -] 0.15 ***
VI       4.94 [+ or -] 0.17 ***   6.195 [+ or -] 0.14 ***
Values are expressed as Mean [+ or -] SEM. (n=6); * p<0.05, ** p<0.01,
*** p<0.001 compared to group III. (a) p<0.05, (b) p<0.01, (c) p<0.001
compared to group I. AST-Aspartate aminotransferase; ALT--Alanine
aminotransferase; ALP-Alkaline phosphatase.TB-Total bilirubin,
TP-Total protein
Table 2: Effect of EECR on oxidative stress markers on
paracetamol induced hepatotoxicityin rats
Groups               Treatment              SOD (U/mg of protein)
I         Normal Control 0 5% Sodium CMC     4.41 [+ or -] 0.48
II          PCM (400mg/kg of b.w, Lp.)         1.40 [+ or -] 0.19 (c)
III            Liv-52 (5ml/kg, p.o)           2.84 [+ or -] 0.21
IV           EECR (10Cmg/kg, b.w, p.o)        2 43 [+ or -] 0.43
V           EECR (200 mg/kg, b.w, p.o)        2.57 [+ or -] 0.36
VI           EECR (400mg/kg, b.w, p.o)       2.88 [+ or -] 0.28
                                          LPO
Groups     CAT (U/mg protein)     ([micro]M/mg protein)
I          42 29 [+ or -] 1.15      0.34 [+ or -] 0.01
II        25.32 [+ or -] 0.99 (c)   1.55 [+ or -] 0.06 (c)
III       35 48 [+ or -] 1.13 ***   0.55  [+ or -] 0.02 ***
IV          29.07 [+ or -] 1.51     1.31  [+ or -] 0.05 **
V           30.17 [+ or -] 1.71     0.74  [+ or -] 04 ***
VI         32.76 [+ or -] 1.63 *    0.59 [+ or -] 0.02 ***
                  GSH
Groups    ([micro]M/mg Protein)
I           2.69 [+ or -] 0.21
II            0 93 [+ or -] 0 06 (c)
III          1.89 [+ or -] 0.14 ***
IV          1.01 [+ or -] 0.06
V           0.24 [+ or -] 0.06
VI         1.44 [+ or -] 0.12
Values are expressed as Mean [+ or -] SEM. (n=6); * p<0.05, ** p<0.01,
*** p<0.001 compared to group III. (a) p<0.05, (b) p<0.01, (c) p<0.001
compared to group I. SOD-Super oxide dismutase, CAT-Catalase, LPO-Lipid
peroxidase, GSH-Reduced glutathione
Table 3: Effect of EECR on antioxidant parameters on
paracetamol induced hepatotoxicity in rats
Groups                Treatment
I          Normal Control 0.5% Sodium CMC
II           PCM (400mg/kg of b.w, i.p.)
III             Liv-52 (5ml/kg, p.o)
IV            EECR (100mg/kg, b.w, p.o)
V             EECR (200mg/kg, b.w, p.o)
VI            EECR (400mg/kg, b.w, p.o)
Groups             GPx (nmol/min)                   GGT (IU/L)
I             3512 [+ or -] 9.02             16.33 [+ or -] 1.38
II           113.3 [+ or -] 2.57 (c)           27 [+ or -] 1.50 (c)
III          292.7 [+ or -] 6.93 ***          15.5 [+ or -] 1.17 **
IV             159 [+ or -] 7.52             24.33 [+ or -] 1.17
V            215.7 [+ or -] 14.95 ***        23.33 [+ or -] 1.28
VI           244.7 [+ or -] 11.67 ***        21.33 [+ or -] 1.68
Groups            GST (nmol/min)
I           109 8 [+ or -] 2.46
II          34.17 [+ or -] 3.60 (c)
III         94.33 [+ or -] 2.02 ***
IV          50 83 [+ or -] 5 72
V           63.33 [+ or -] 3.77 ***
VI          80.83 [[+ or -] 5.77 ***
Table 4: Cytotoxicity effect of EECR and CRB4 in HepG2 Cell line
Compound name      Cone, ug/ml   Cell viability %
Control                 D              100
                       10              4.56
                       20             10 88
EECR                   40             23.89
                       80              41.2
                       320             7550
                       10              3.42
                       20             12.81
CRB4                   40             16.07
                       80              2253
                       160            27.56
                       320             3078
Liv52                  100            45.75
Table 5: Hepatoprotective effect of EECR and CRB4 in paracetamol
induced HepG2 cell line
                         Cone
Compound name         [micro]g/ml   % Hepatoprotectivity
Paracetamol (PCM)         30                 0
                          20                6.12
EECR                      40               12.47
                          80               26.98
                          20               28 09
CRB-4                     40               31.05
                          80               44.44
Liv-52                    100              54 25
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Title Annotation:Original Article
Author:Yachamaneni, Jyothi; Sangeetha, Dhanaraj; Vijayaraghavalu, Sai Saraswathi
Publication:Trends in Biomaterials and Artificial Organs
Geographic Code:9INDI
Date:Jul 1, 2021
Words:8893
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