Hepatoprotective and antioxidant activity of ethanolic extract of Artabotrys odoratissimus stem on rifampicin-induced hepatotoxicity in rats

Hindustan Abdul Ahad,Chinthaginjala Haranath,Bhupalam Pradeepkumar,Beludari Mohammed Ishaq

1Department of Industrial Pharmacy,Raghavendra Institute of Pharmaceutical Education and Research(RIPER)-Autonomous,KR Palli Cross,Cheyyedu-515721,Ananthapuramu,Andhra Pradesh,India;2Department of Pharmacology,Raghavendra Institute of Pharmaceutical Education &Research (RIPER)-Autonomous,KR Palli Cross,Cheyyedu-515721,Andhra Pradesh,India;3Department of Pharmaceutical Analysis,Santhiram College of Pharmacy,Nandyal-518501,Kurnool Dist.Andhra Pradesh,India.

Keywords:Artabotrys odoratissimus,Rifampicin,Liver protection,Histopathology

Background

The Liver fills in as the mainline of defense and forestalls harmful impacts by detoxifying poisonous substances,which keeps up homeostasis.Different poisonous synthetic substances,alcohols,medications,diseases and immune system problems induced hepatotoxicity.Investigations have shown that some natural concentrates and their substance ingredients can hinder these previously mentioned and secure liver tissue from the etiologies of persistent hepatic grievance [1].Presently many medicaments are advised to treat liver issues.

As the tremendous limits in manufactured drug items,Items from the nature are emerged and an urge to discover lead components from them.One such plant that is presently under scrutiny for its possible hepatoprotective (HPA) and cell reinforcement action in our research facility isArtabotrys odoratissimus(A.odoratissimus) (family:Annonaceae) [2],commonly known as Nag champa[3].

Earlier many researchers explored the medicinal values of herbs grow in ndhra Pradesh,India [4–6].A.odoratissimusis one such plant that is utilized in fables medication to treat liver sicknesses.A.odoratissimushas been proved for various pharmacological activities such as antimicrobial activity [7],anti-fertility activity[8],antifungal and anti-fumonisin[9].it also is utilized for treating Jaundice[10].Very little work done on the plant that work against malignant growth and hostile to microbial,against oxidant,hostile to diabetic exercises.Moreover,we additionally investigated the phytochemical elements ofA.odoratissimusand demonstrated the existence of flavonoids,tannins and triterpenes.The polyphenolic flavonoids present inA.odoratissimus,demonstrated to show different pharmacological applications including HPA[11,12].

However,no efforts have been made to this date to consider the HPA ofA.odoratissimusstem.Along these lines,this examination was done to get bits of knowledge into the utility of an ethanolic concentrate ofA.odoratissimusstem in rifampicin (RCN) incited liver harm in rats,as the creature model to build up a palatable HPA of medication.

Materials and methods

Animals

Wister albino rats (either sex) were used for the study.Institution Animal Ethics Committee has approved the project (RIPA/col/IAEC/20/3R) and the CPCSEA consent number is 878/ac/05/CPCSEA/004/2015.The animals were kept at 27 ± 2 °C,RH of 50 ± 6% and light and dark cycles of 10 and 14 h,for 7 days before and throughout the trials.Animals were given a standard diet(Varna Enterprises,Bengaluru,India)and the food was stopped 18 h before the start of the experiment and water ad libitum.All the try-outs were done in the morning rendering to the ethical strategies[13,14]for the exploration of experimental pain in cognizant animals.

Plant authentication and extraction

The stems ofA.odoratissimuswere collected from Ananthapuramu,Andhra Pradesh,India.And authenticated in the Department of Botany,Sri Krishnadevaraya University,Ananthapuramu,Andhra Pradesh.The herbarium specimen was procured(Voucher specimen no-20/H47/B-07)and preserved.

Extraction and isolation

A.odoratissimusstems were shade dried.Later powdered and a quantity of 60 mg was defatted with petroleum ether,later extracted with 300 mL of 95%ethanol using soxhlet apparatus for 50 h and finally vacuum dried to get dark brown residue (150 g,10%)[15,16].Later the powdered extract was subjected for infra-red spectra.The extraction process was repeated until a constant spectrum produced(Figure 1).

Phytochemical studies

All the extracts were exposed for phytochemical studies [2,17,18].Various chemical tests were performed on the extract to identify the phytochemical components.

Test for carbohydrates.Molish test.Take 2–3 mL of test sample add few drops of alpha α-naphtol solution in alcohol and conc.H2SO4from the walls of the test tube.Violet ring formation specifies the existence of carbohydrates.

Reducing sugars.Fehling test:to the sample equal quantity of Fehling A and B solutions were added.Later warmed in a water bath for 5–10 min.Yellow brick red precipitate formation designates the existence of reducing sugars.Benedicts test:to the sample Benedict’s reagent was added.Later warmed in a water bath for 5 min.Green;yellow to red colour designates the manifestation of reducing sugars.

Test for non-reducing sugars.A) Negative for Fehling’s and Benedict’s test.B)Tannic acid test:upon inclusion of 20%tannic acid to test solution precipitate will be formed indicating the existence of starch.

Test for proteins.A)Biuret test.Test solution plus 4%NaOH and 1% CuSO4,violet or pink colour formation signposts the occurrence of proteins.B)Millon’s test.5 mL of Millon’s reagent plus 3 mL of test solution a white precipitate was shaped which cracks to brick-red precipitate designates the attendance of proteins.C)Xanthoprotein test.Up on the inclusion of 1 mL of concentrated H2SO4to 3 mL of test solution a white precipitate is acquired specifying tyrosine/tryptophan.D) Test for proteins holding sulphur.To 5 mL of test solution,add 2 mL of 40% NaOH and few drops of 10%lead acetate solution was appended and simmered.Black or brownish colour signposts the existence of sulphur comprising proteins.E) Precipitation test.Test solution gives a precipitate with the reagents (absolute alcohol,5%mercuric chloride)signposts the existence of proteins.

Figure 1 Infra-red spectra of Artabotrys odoratissimus stem extract

Test for steroids.A) Salkowaski reaction.2 mL of chloroform plus 2 mL of concentrated H2SO4to 2 mL of test solution and shaked well.Red coloration of chloroform layer and greenish-yellow fluorescence of acid layer designates the existence of steroids.B)Liberman Buchard reaction.To 2 mL of test solution,with chloroform,2 mL of acetic anhydride plus 2 drops of concentrated H2SO4from the walls of the test tube.A fine play of colours from red to blue and lastly to green will seem designates the incidence of steroids.C) Libermanns test.To 3 mL of test solution,add 3 mL of acetic anhydride.After warming,cooled and few drops of concentrated H2SO4acid were included.Blue colour creation signposts the attendance of steroids.

Test for aminoacids.A) Ninhydrin test.To 3 mL of test solution add 3 drops of 5%ninhydrin solution heat in boiling water bath for 10 min.purple or bluish colour designates the manifestation of amino acids.B)Test for tyrosine.To 3 mL of test solution add 3 drops of Million’s reagent.Heat the solution red colour designates the incidence of tyrosine.C) Test for tryptophan.To 3 mL of test solution,plus glycoxalic acid and concentrated H2SO4,makes a violet ring at the junction of 2 layers signposts the existence of tryptophan.

Test for glycosides.To 2–3 mL of test solution appended with dilute H2SO4and heat on water bath for 1–2 min.After neutralisation with 10% NaOH add Fehlings A and B reagents.Creation of brick red precipitate designates the existence of glycosides.

Test for alkaloids.The presence of alkaloids can be established by Dragendroffs reagent (orange brown precipitate).Mayer’s reagent (precipitate),Hager’s reagent (yellow precipitate),Wagner’s test (reddish brown precipitate).

Test for tannins.To the sample addition of the following reagents confirms the existence of tannins.Five percent ferric chloride(blue to black colour),lead acetate (white precipitate),gelatine (white precipitate),bromine water(discolouration).

Test for flavonoids.A) Shindo test.To the sample 5 mL ethanol (95%),few drops of concentrated H2SO4and few magnesium turnings.Spinning of the solution into pink colour specifies the attendance of flavonoids.B) Ferric chloride test.To the sample few drops of ferric chloride penetrating green colour creation endorses the existence of flavonoids.

Acute toxicity studies

Overnight fasted Albino rats maintained at standard conditions were administrated with the extract in the growing dose and espied safe up to a dose of 2 g/kg for all extracts[19,20].

The acute toxicity study was made in adult female albino rats by“fixed dose”method of organization for economic co-operation and development rules No.420.Fixed-dose method in annex 2 d:test procedure starting with a dose of 2,000 mg/kg bd.wt.was embraced.The animals were not fed overnight.Individual and polyherbal extracts were given orally at a dose level of 2,000 mg/kg.Then the animals were observed for 3 h to identify any changes in general behavioural,nervous,autonomic contours and for every 30 min of next 3 h and finally for fatality after 24 h for 14 days.

Experimental animal and design

The experiment was steered according to the revised measures designated hitherto[21,22].RCN(50 mg/kg)was dissolved in 0.5 % carboxy methyl cellulose for oral administration.Six groups of rats 6 in each(R1–R6) were intoxicated by RCN for 7 days.Group(R1) i.e.,normal control receives pure water (oral) for 7 days,and then intraperitoneally given with 10 mL/kg/body weight (0.9% w/v NaCl).Group R2(hepatotoxicity control) receives pure water for 7 days and then orally intoxicated.Group R3 (standard)receives standard drug (Silymarin 25 mg/kg) (oral).Ethanol extract ofA.odoratissimusstem(100,200 and 400 mg/mL/kg/body weight were given to group 4,5 and 6.

After 48 h of intoxication,the rats were euthanized by ether and then sacrificed.The blood was collected by cardiac puncture in heparinized tubes for the valuation of dissimilar enzyme levels of serum glutamic oxaloacetic transaminase (SGOT),serum glutamate pyruvate transaminase (SGPT),alkaline phosphatase (ALP) and bilirubin with standard kits.The liver was instantly appropriated and splashed with ice-cold saline for histological observation.

Biochemical constituents

The biochemical constraints (serum enzymes) viz.,aspartate aminotransferase [23],SGPT [24,25],serum ALP [24]and total bilirubin [26,27]were assayed using assay kits(Eagle Biosciences).

SGOT and SGPT.Principle:the function of SGOT is to convert L-Aspartate and α-ketoglutarte to oxaloacetate and glutamate.Dinitro phenyl hydrazine reacts with oxaloacetate which yields hydrazone imitative (brown coloured complex).The function of SGPT is to convert the L-Alanine and α-ketoglutarte to pyruvate and glutamate.Dinitro phenyl hydrazine reacts with pyruvate which yields hydrazone derivative(brown coloured complex).The reaction does not obey Beer’s lamberts law,so a calibration graph is strategized using pyruvate as a standard.The activity of SGOT and SGPT can be calculated from the calibration curve.

The kit contains,L1-substrate reagent,L2-DNPH reagent,L3-sodium hydroxide reagent in 1:10 dilution(4N) and S-Pyruvate standard (2mM).Procedure:five test tubes took and to tube 1,0.5 mL of L1,0.1 mL of distilled water and 0.5 mL of L2 were appended and placed aside for 20 min then appended 5 mL of L3 mixed well and confess to stand for 10 min (blank).In test tube 2,0.45 mL of L1,0.05 mL of S,0.1 mL of distilled water and 0.5 mL of L2 were appended and kept aside for 20 min and appended 5 mL of L3 confess for 10 min.The absorbance was appropriated at 505 nm which showed enzyme activity of 24.In test tube 3,appended 0.40 mL of L1,0.1 mL of S,0.1 mL of distilled water and 0.5 mL of L2 were appended and left for 20 min,and then 5 mL of L3 was appended and confessed for 10 min.The absorbance was appropriated at 505 nm,which showed enzyme activity of 61.In test tube 4,0.35 mL of L1,0.15 mL of S,0.1 mL of distilled water and 0.5 mL of L2 were appropriated and kept aside for 20 min,appended 5 mL of L3 and confessed for 10 min.The absorbance appropriated at 505 nm,which showed enzyme activity of 114.In test tube 5,0.3 mL of L1,0.2 mL of S,0.1 mL of distilled water and 0.5 mL of L2 were appropriated and left aside for 20 min,appended 5 mL of L3 and confessed for 10 min.The absorbance was appropriated at 505 nm,which showed enzyme activity of 190.Assay:for a blank test tube,0.5 mL of L1 was appropriated and kept aside for 60 min at 37oC.0.5 mL of L2 was appended,mixed well and confessed to the stand for 20 min,to this appended 0.1 mL of distilled water and 5 mL of L3.Into the sample test tube,0.5 mL of L1,0.5 mL of sample were appropriated and set for 60 min at 37oC.To this appended 0.5 mL of L2 and kept aside for 20 min,5 mL of L3 was appended and the absorbance was checked at 505 nm.The activity can be estimated from the calibration plot.

Alkaline phosphatase.Principle:alkaline phosphatase hydrolyses di-sodium phenyl phosphate to form phenol at alakaline pH.The formed phenol reacts with 4 amino anti pyrine and forms a red colour complex in the attendance of oxidising agents like potassium ferricyanide.Procedure:the quantitative estimation of colour indicates the activity of ALP.L1-buffered substrate (pH 10).L2-chromogen reagent.L3-phenol standard (10%).Working solution-one vial of L1 mixed with 2.2 mL of purified water.To the blank appended 0.5 mL of working solution,1.5 mL of water and set at 37oC for 3 min,append 0.05 mL of water,mixed well and set at 37oC for 15 min add 1 mL of L2 and absorbance was taken at 510 nm.To the standard append 0.5 mL of working solution,1.5 mL of water and set at 37oC for 3 min,appended 0.05 mL of L3,mixed well and set at 37oC for 15 min add 1 mL of L2 and the absorbance was taken at 510 nm.To the control append 0.5 mL of working solution,1.5 mL of water and set at 37oC for 3 min mixed well and set at 37oC for 15 min,append 0.05 mL of serum,1 mL of L2 and the absorbance was taken at 510 nm.To the test,append 0.5 mL of working solution,1.5 mL of water and set at 37oC for 3 min,append 0.05 mL of serum mixed well set at 37oC for 15 min,append 1 mL of L2 and the absorbance was taken at 510 nm.Total activity of ALP in KA units=(AT-AC)/(AS-AB)*10.

Bilurubin.Principle:unconjugated bilirubin in the incidence of caffeine benzoate accelerator reacts with diazotised sulphanilic acid and forms coloured azo bilirubin complex.Procedure:for unconjugated bilirubin.Two blank test tubes append 1 mL direct reagent(L1)and 0.1 mL of sample and mix well.After set at 37oC for 5 min,the absorbance was measured at 546 nm.To the sample test tube,1 mL of L1 was appended,one drop of nitrite reagent was appended and 0.1 mL of sample.Total bilirubin.Two blank test tubes were taken and append 1 mL total bilirubin reagent (L1) and 0.1 mL of sample and mixed well after set at 37oC for 10 min.Spot the absorbance at 546 nm.To the sample test tube 1 mL of L1 was appended and one drop of total nitrite reagent and 0.1 mL of sample were appended.Total or direct bilirubin=AT/AS*10

In vitro anti-oxidant activity

1,1-diphenyl 2-picryl hydrazyl-scavenging activity.The free radical scavenging activity of the extract was restrained in terms of hydrogen giving or radical hunting ability using the stable radical 1,1-diphenyl 2-picryl hydrazyl (DPPH) [28–30].The solution of 0.1 mm in ethanol was made and 1.0 mL of this solution was appended to 3.0 mL of all the extracts solution in water at various proportions (10–100 μg/mL).The absorbance was restrained at 519 nm after 1 h of standing [31].The lower absorbance of the mixture designates developed free radical-scavenging activity.Ascorbic acid was utilized as a standard drug.

Scavenging of hydrogen peroxide (H2O2).A solution of H2O2(20mM)in pH 7.4 phosphate buffer saline was prepared.To 2 mL of this various levels of extract or standard in methanol (1 mL) were appended.Fifteen min later the absorbance was measured at 232 nm[32–34].

Histopathological studies

The liver tissue was divided out and fixed in 10%formalin,dehydrated in gradual ethanol (50–100%),clean in xylene,and entrenched in paraffin.Segments were made,stained with hematoxylin and eosin dye for photomicroscopic remarks viz.,cell necrosis,fatty change,hyaline regeneration,ballooning degeneration[35–38].

Statistical analysis

The data are expressed as average ± standard error of the mean.The data were assessed with one-way analysis of variance[39,40],and the changes amid the groups were dogged with the Dunnett post hoc test as given by the graph pad PRISM-V7 software.

Results and discussion

Results

Phytochemical study.All extracts were studied for phytochemical observation to find the qualitative presence of alkaloids,glycosides,amino acids,proteins,flavonoids and phenolic compounds.

Acute toxicity studies.Ethanolic and aqueous extracts did not show any toxicity,and the half lethal dose up to 2 g/kg dose.After 24 h,7 days and 14 days treatment,the body weight of rats in ethanolic extracts were 98.1±0.8,103.3±0.7,105.4±0.5 g,and these were 97.5±0.6,103.0 ± 0.3,104.2 ± 0.4 g in the control group.There was no statistical difference in weight gain in rats among the 2 groups.There were also found that no rats in treatment group died because of drug toxicity(no mortality observed).

In vitro antioxidant study.The study was executed by using DPPH free radical and H2O2scavenging(Table 1).

Effect of the A.odoratissimus on biochemical

parameters.The ethanolic extract ofA.odoratissimusunveiled significant (P＜0.05) HPA in RCN induced models by taming liver function,as specified by the lessening in levels of the liver enzymes likened with the control group.

Histopathological studies of the liver (pre-treated with 10% dimethyl sulfoxide) discovered significant(P＜0.05) damage to the building of the liver,with severe necrosis of liver cells (Table 2).The untreated(normal) shows the normal lobular construction with undisturbed cytoplasm and well-defined sinusoids line and nucleus around the perivenular area (Figure 2(a)).Normal histological structures were experiential in the liver of rats given with silymarin (25g m/kg) (Figure 2(b)).Liver cells disarrangement,necrosis and inflammation were observed in RCN rats(Figure 2(c)),(Figure 2(d)) and (Figure 2(e)) with varying doses.(Figure 2(e))is a normal cell intoxicated with RCN.

Table 1 In vitro antioxidant activity of Artabotrys odoratissimus

Table 2 Effect of the ethanolic extract of Artabotrys odoratissimus stem on biochemical parameters in rifampicin induced hepatotoxicity

Discussion

The liver has a requisite role for its artistic metabolic and detoxification aptitude.As it is visible to several drugs and xenobiotics,a myriad of transitional and products is produced and leads to hepatocellular death and constitutes the principal causes of liver disease.

Figure 2 Histopathological cell sections of the liver cells.(a)Normal;(b)section of liver tissue of 100 g/kg RCN treated group showing massive coagulative necrosis,hemorrhage and inflammation;(c) section of 25 g/kg of silymarin liver tissue pre-treated on the liver followed by RCN showing preservation of normal hepatocytes;(d)section 100 mg/kg Artabotrys odoratissimus liver tissue intoxicated with RCN showing tissue necrosis and inflammation;(e) section 200 mg/kg Artabotrys odoratissimus liver tissue intoxicated with RCN showing tissue necrosis and inflammation;(f) section 400 mg/kg Artabotrys odoratissimus liver tissue intoxicated with RCN showing normal histology.(400x)CV,centrilobular.CN,coagulative necrosis.I,inflammation.H,haemorrhage.

To ensure the existence of an individual and maintain the function of the liver,the conventional treatment focuses on sign management and liver transplantation in severe cases of liver disease[41,42].However,there are no drugs currently utilized to increase the detoxification power of the organ.Therefore,testing and the use of botanical HPA plants are noticeably growing.Therefore,it would be highly authoritative to validate the success of the plant extracts in the existence of chemical-induced hepatotoxicity.

RCN is applied to treat tuberculosis.All these agents were known to induce hepatotoxicity.Therefore,the same hepato toxins were chosen to induce hepatotoxicity in rats and evaluate the HPA ofA.odoratissimus.Several studies indicate that RCN can produce hemorrhagic hepatic necrosis.Thus,in this study,rats directed with RCN resulted in the increased liver weight of rats via the development of penetration,vacuolization,and inflammation in the liver(Figure 2).RCN induces hepatotoxicity releases reactive free radicals,which leads to cellular damage by membrane lipids peroxidation and covalently bind with other macromolecules within hepatocytes.In the present work,ethanolic extract showed a depletion of these in a dose-dependent manner unlike other plants showed hepato-protective activity[43,44].

Past examinations indicated that alkaloids and flavonoids have cancer limiting activity.Preliminary phytochemical investigations on the ethanolic concentrates revealed the presence of metabolites that produces such activity.It is sensible to propose that the phytochemicals present in the plant may act separately or synergistically to create the noticed HPA ofA.odoratissimus.Perhaps,flavonoids and alkaloids present in the unrefined stem remove applied HPA impact by their free extremist rummaging action,avoidance of lipid peroxidation and harm to cells as such an activity has been proposed for some different plants.Besides,alkaloids and flavonoids show cancer limiting actions by their free radical hunting movement.

As the liver is ceaselessly presented to oxidative pressure,the arrival of free radicals is the primary hepatotoxicity system of poisons.Oxidative pressure harms cell segments,for example,proteins,lipids and nucleic acids.To affirm the cancer stoppage assets of the plant,in vitro DPPH and H2O2screening were done.In these tests an ethanolic concentrate ofA.odoratissimuswas seen to repress with the most extreme estimation at the convergence of 100 μg/mL.The rough ethanolic concentrate ofA.odoratissimushad a determined IC50estimation is almost like the determined IC50estimation of the known cancer preclusion agent,ascorbic injury.As it is clarified for different plants ethanolic concentrate may act through their free extremist rummaging,the balance of free revolutionaries and restraint of rot by means of a few pathways.The studies were in preliminary stage so,the data that was obtained was only included.Still a detailed work is under process.

To summarize,this investigation gave additional proof that the ethanolic extract,had an equivalent HPA with that of the standard drug.Results acquired uncovered that there was a portion subordinate decrease in all biomarkers of liver injury in pre-and post-treatment.Thusly,this information appears to show that the HPA of the plant is dispersed to polar bioactive standards contained in the ethanolic portion.Despite the fact that the HPA component of the plant extricate is yet not explained,the noticed cell reinforcement action is one of the foreseen systems.Most importantly,the ethanolic concentrate of the stem ofA.odoratissimuswould be remunerated as protected dependent on the aftereffects of an intense oral harmfulness study.Besides,separation and portrayal of novel cell reinforcements will be done in future examinations by utilizing HPLC/LC-MS/MS methods.

Conclusion

TheA.odoratissimusstem owns HPA activity against RCN persuaded liver toxicity.This activity may be due to its flavonoids and other components of the plant.However,harmonizing in vitro and in vivo exams will be essential to approve the mechanism accountable for this HPA outcome.