INTRODUCTION:

The liver disports a symbolic role in metabolism and abrupt withdrawal of exogenous toxins and curative agents¹. A number of natural and synthetic executors act as hepatotoxins and produce a variety of liver diseases. There are number of herbal extracts, which are revealed to have antihepatotoxic activity².

Aponogeton natans (Linn.) Engl. and Krause.belongs to Aponogetonaceae family. Aponogeton natans (Linn.) Engl. and Krause occurs in plains and marshy places in Asia, Australia, India and Srilanka. Traditionally leaf pastes are applied with hot water to cure cuts and wounds³.

Fresh tuber paste is scalded with 200 ml of coconut oil and used on hair before bath for three days to cure fungal infection⁴. Aponogeton natans (Linn.) Engl. and Krause is an ingredient in preparation of an important Ayurvedic preparation Useerasava. This asava is useful in haemothermia, anaemia, contaminant of blood and diabetes⁵. A perusal of existing reports affirms that the no detailed hepatoprotective study had been done earlier. The research has been outlined to investigate the hepatoprotective screening of various extracts of Aponogeton natans (Linn.) Engl. and Krause. leaf with leafstalks against CCl₄ induced liver toxicity in experimental animals.

MATERIALS AND METHODS:

Plant material:

Fresh parts of Aponogeton natans (Linn.) Engl. and Krause.were collected from Salipur, Cuttack, Odisha, India which was identified and authenticated by Prof. P. Jayaraman, PARC, Chennai. The voucher specimen was given the No. PARC/2009/398.

Preparation of extract:

The air dried powdered leaves with leafstalks was loaded into soxhlet apparatus and was subjected to extraction for about 72 hours with petroleum ether (60-80ºC), benzene, chloroform and methanol successively. The solvents were distilled off after extraction and the extracts were concentrated under reduced pressure. The extracts were kept in refrigerator until tested⁶. The four extracts were then subjected to phytochemical analysis. For the pharmacological tests, the extracts were dissolved in 1% Tween-80 in normal saline solution to prepare 200 mg/kg concentrations.

In vitro Antioxidant assessment:

Superoxide scavenging activity:

Superoxide scavenging activity of the plant extract was determined by McCord and Fridovich method, 1969, which depends on light induced superoxide generation by riboflavin and the corresponding reduction of nitroblue tetrazolium. The plant extracts of 0.1 ml of different concentrations and 0.1 ml of 6 μM ethylenediamine tetraacetic acid consist of NaCN, 0.1 ml of 50 μM nitroblue tetrazolium, 0.05 ml of 2 μM riboflavin were transferred to a test tube, and final volume was made up to 3 ml using phosphate buffer. Then the assay tubes were uniformly illuminated with an incandescent light (40 Watts) for 15 minutes and thereafter the optical densities were measured at 560 nm. A control was prepared using 0.1 ml of respective vehicle in the place of plant extract/ascorbic acid. The percentage inhibition of superoxide production was evaluated by comparing the absorbance values of control and experimental tubes^{7, 8.}

Percentage inhibition was determined by using following equation:

% Inhibition= [(A0– A1) / A0 × 100]

Where; A0 is absorbance of control and A1 is absorbance of test sample. IC₅₀value was calculated from the values obtained for % Inhibition.

Hydroxyl radical scavenging activity:

Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extracts for hydroxyl radicals generated from the Fe2+/EDTA/H2O2 system (Fenton reaction). The hydroxyl radical attacks deoxyribose, which eventually results in the formation of thiobarbituric acid reacting substances (TBARS) (Elizabeth and Rao, 1990). Fenton reaction mixture consisting of 200 µl of 10 mM ferrous sulphate (FeSO4. 7H2O), 200 µl of 10 mM EDTA and 200 µl of 10 mM 2- deoxyribose and was blended with 1.2 ml of 0.1 M phosphate buffer (pH 7.4) and 200 µl of plant extracts. Thereafter, 200 µl of 10 mM H₂O₂ was added before the incubation at 37^oC for 4 hours. Then, 1 ml of this Fenton reaction blend was employed with 0.2 ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 0.8% thiobarbituric acid and 1.5 ml of 20 % acetic acid. The total volume was then made to 5 ml by adding distilled water and kept in an oil bath at 100⁰ C for 1 hour. After the mixture had been cooled, 5 ml of 15:1 v/v butanol-pyridine blend was added. Following vigorous shaking, the tubes were centrifuged at 4000 rpm for 10 min and the absorbance of the organic layer containing the thiobarbituric acid reactive substances was measured at 532 nm. A control was assembled using 0.1 ml of vehicle in the place of plant extract/ascorbic acid. The percentage inhibition of hydroxyl radicals by the extract/compound was determined by comparing the absorbance values of the control and the experimental tubes as calculated for hydroxyl radical assay^9,
10.

DPPH radical scavenging activity:

Free radical scavenging action was predispose by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with slight alteration. An aliquot of 3 ml of 0.004% DPPH solution in ethanol and 0.1 ml of plant extracts at various concentrations were blended. The blend was shaken energetically and allowed to reach a constant state at room temperature for 30 min. Decolorization of DPPH was resolute by measuring the absorbance at 517 nm. A control was arranged by using 0.1 ml of respective vehicle in the place of plant extract/ascorbic acid^{11, 12}.

The percentage inhibition action was figured as [(A0-A1)/A0]×100, where A0 was the absorbance of the control, and A1 was the absorbance of the plant extract/ ascorbic acid. IC50 values (i.e. concentration of a sample, which is required to scavenge exact 50% of free radicals) were also calculated from the data obtained.

Experimental animals:

Wistar albino rats of both the sex weighing 140-160 grams were used for the hepatoprotective studies. The animals were kept in standard conditions of day and night cycles at 22ºC in polypropylene cages. The animals were given on standard pellet water ad libitum. The rats were acclimatized to laboratory conditions by housing them in propylene cages prior to the experiments for one week. The experiment was conducted in Institute of Pharmacy and Technology, Salipur, Cuttack and CPCSEA recognized local ethical committee approved the protocol bearing No. 19/IAEC-IPT/13.

Chemicals:

All the laboratory reagents and kits used in the assays were of analytical grade, and were procured from Qualigens Fine Chem Pvt Ltd, Sulab laboratory and Himedia Lab Pvt Ltd.

Evaluation of hepatoprotective activity:

Diclofenac sodium induced hepatotoxicity model:

The screening for hepatoprotective activity against diclofenac sodium induced hepatotoxicity was followed as per standard procedure. The rats troops were divided into seven groups with six rats in each. Group I served as normal control and received the vehicle alone (2 ml, p.o.) for 5 days. Group II served as toxic control animals received diclofenac sodium (2 ml/kg, in olive oil, i.p.) (on the 3rd and 4th day. Group III, IV, V, VI received 200 mg/kg of pet ether, benzene, chloroform, methanol extracts respectively and Group VII was treated with silymarin (10 mg/kg) for 5 days and on the 3^rd and 4^th day diclofenac sodium (2 ml/kg, in olive oil, i.p.) was administered 1 hour after the treatment of the extracts. Group VII (Standard) was treated with standard drug silymarin (100 mg/kg p.o.) for 5 days and on the 3^rd and 4^th day diclofenac sodium (50 mg/kg i.p.) was administered 1 hour after the treatment of the drug. The animals were sacrificed 48 hour after the last injection of diclofenac sodium under mild ether anesthesia. The blood was collected and allowed to stand for 30 min at 37°C and centrifuged to separate the serum to estimate various biochemical parameters^{13, 14}.

In the above hepatoprotective model, various in vivo antioxidant parameters were estimated.

Preparation of liver homogenate:

The liver was quickly removed and perfused immediately with ice-cold saline (0.9% NaCl). A portion of the liver was homogenized in chilled Tris-HCl buffer (0.025 M, pH 7.4) utilising a homogenizer. The homogenate obtained was centrifuged at 5,000 rpm for 10 minutes, the supernatant was collected for analysis¹⁵.

Biochemical parameters:

The biochemical parameters were estimated as per the standard procedure prescribed by the manufacturer’s instruction manual provided in the kit (Coral Clinical Systems, Verna Goa, India).

Invivo Anti-oxidant parameters:

Liver tissue were homogenized in KCl [10 mM] phosphate buffer (1.15%) with EDTA (pH 7.4) and centrifuged at 12000 rpm for 60 min. The supernatant was used for assay of the marker enzymes (GPx, SOD, CAT, and LPO) and protein estimation.

Histopathological study of liver:

The histopathological study was carried out for liver tissue. The liver was transferred to 4% formalin solution and processed for histopathological studies following the standard procedure described by. The microtome sections were taken and stained with hematoxylin and eosin. The section thus obtained was scanned in Carl-Zeiss microscope with photographic facility and photomicrographs were taken^16,17.

Statistical Analysis:

All experiments were arranged in triplicates and data obtained were processed for statistical analysis. Data in respect to various parameters of Aponogeton natans Linn. extracts were processed statistically using one way ANNOVA followed by Tukey’s multiple comparision test.

RESULT AND DISCUSSION:

In vitro antioxidant activity:

Superoxide anion plays an important role in the formation of more reactive species such as hydrogen peroxide, hydroxyl radical and singlet oxygen, which induce oxidative damage in lipids, proteins and DNA^18,19. Therefore, studying the scavenging activity of plant extracts/compounds on superoxide radical is one of the most important ways of clarifying the mechanism of antioxidant activity.

In the present study, the petroleum ether, benzene, chloroform and methanol extracts of Aponogeton natans were found to possess concentration dependent scavenging activity on superoxide generated by photoreduction of riboflavin and the results were given in Fig. 1. The mean IC50 values for superoxide radical of petroleum ether, benzene, chloroform and methanol extracts of Aponogeton natans were found to be 521.629μg, 443.098μg, 376.111μg and 310.81μg respectively. The mean IC50 value of ascorbic acid was found to be 255.6 μg.

The petroleum ether, benzene, chloroform and methanol extracts of Aponogeton natans were found to possess concentration dependent scavenging activity on hydroxyl radicals and the results were given in Fig. 2. The mean IC50 values for hydroxyl radical of petroleum ether, benzene, chloroform and methanol extracts of Aponogeton natans were found to be 486.379μg, 394.032μg, 342.727μg and 260.322μg respectively. The mean IC50 value of ascorbic acid was found to be 185.384μg.

The petroleum ether, benzene, chloroform and methanol extracts of Aponogeton natans were found to possess concentration dependent scavenging activity on DPPH radicals and the results were given in Fig. 3. The mean IC50 values for DPPH radical of petroleum ether, benzene, chloroform and methanol extracts of Aponogeton natans were found to be 344.193μg, 305.396μg, 189.726μg and 127.00μg respectively. The mean IC50 value of ascorbic acid was found to be 27.68μg.

Among the four types of Aponogeton natans Linn. extracts, the methanol extract showed better scavenging activity The order of scavenging activity was in the following manner: ascorbic acid > methanolic extract > chloroform > benzene > petroleum ether.

Fig. 1:

ANPE: Aponogeton natans pet. ether extract; ANBE: Aponogeton natans benzene extract; ANCE: Aponogeton natans chloroform extract; ANME: Aponogeton natans methanol extract

Fig. 2

ANPE: Aponogeton natans pet. ether extract; ANBE: Aponogeton natans benzene extract; ANCE: Aponogeton natans chloroform extract; ANME: Aponogeton natans methanol extract

Fig. 3

ANPE: Aponogeton natans pet. ether extract; ANBE: Aponogeton natans benzeneextract;

ANCE: Aponogeton natans chloroform extract; ANME: Aponogeton natans methanol extract

Biochemical parameters of diclofenac sodium induced hepatotoxicity model:

The results observed from serum biochemical parameters in pretreated of Aponogeton natans Linn.extracts with respect to induction of hepatotoxicity using Diclofenac sodium are given in (Fig. 4). A marked reduction in total protein levels was observed in the group treated with diclofenac sodium which were significantly decreased to 3.54±0.612 when set side by side or compared with the normal control group. The SGOT, SGPT, ALP, Total bilirubin levels increased in the group treated with diclofenac sodium to a significant level. Rats treated with diclofenac sodium (toxin control) developed significant liver damage and it was well indicated by elevated levels of hepato specific enzymes like SGOT 442.13±2.544), SGPT 490.95±2.589, ALP 242.28±2.547 and Total bilirubin 3.62±0.6854 in serum when compared to control group.

The groups received the pre-treatment of Aponogeton natans Linn. extracts methanol extract (ANME) and chloroform extract (ANCE) at dose levels of 200 mg/kg body weight significantly controlled the change in the biochemical parameters. The extract ANME and ANCE at dose levels of 200 mg/kg exhibited significant increase 7.51±1.085, 7.14±0.443 respectively in the serum total protein level as compared to toxic control group and the effect was comparable with the standard group administered with silymarin 8.41±0.561. The SGOT, SGPT, ALP and Total bilirubin level decreased in drug treated groups to significant level. The SGOT 176.93±2.561, 163.15±2.561, SGPT 383.71±3.874,187.11±2.679, ALP 158.25±2.141, 111.56±3.018 and Total bilirubin 1.13±0.2720, 0.89±0.1266 levels decreased significantly in methanol extract (ANME ) and standard silymarin group as compared to toxic control group. The SGOT 425.98±3.775, SGPT 478.41±1.454, ALP 225.23±3.610 and Total bilirubin 1.55±0.3305 levels significantly decreased in ANCE as compared to toxin control group. Administration of pet. ether (ANPE) and benzene (ANBE) extracts did not display effect of increase in the serum enzyme levels as compared to toxic control group .

Fig. 4

ANPE: Aponogeton natans pet. ether extract; ANBE: Aponogeton natans benzene extract; ANCE: Aponogeton natans chloroform extract; ANME: Aponogeton natans methanol extract

In vivo anti-oxidant parameters:

SODs, Catalase and GPx is an extremely efficient intracellular buffer for oxidative stress and SODs, Catalase and GPx acts as a antioxidant that reduces free radicals²⁰. The level of SODs, Catalase and GPx depleted when animals were injected with diclofenac sodium. The depleted level of SODs, Catalase and GPx rose with the pretreatment of methanol extract (ANME). The results are given in (Fig. 5).

The groups received the pre-treatment of Aponogeton natans Linn. extracts methanol extract (ANME) and chloroform extract (ANCE) at dose levels of 200 mg/kg body weight significantly controlled the change in the antioxidant enzyme levels. The methanol extract ANME and chloroform extract ANCE at dose levels of 200 mg/kg exhibited significant increase SOD, catalase and GPx to 30.1±1.266, 41.58±1.248, 33.3±0.11 and 26.48±1.619, 38.69±0.862, 28.92±0.17 respectively as compared to toxic control group. The LPO level in methanol extract (ANME) and chloroform extract (ANCE) at dose levels of 200 mg/kg body weight decreases significantly to 13.32±1.014 and 18.17±1.111 respectively as compared to toxic control group. Administration of pet. ether (ANPE) and benzene (ANBE) extracts did not display any significant effect in the antioxidant levels as compared to toxic control group.

Fig. 5

ANPE: Aponogeton natans pet. ether extract; ANBE: Aponogeton natans benzene extract; ANCE: Aponogeton natans chloroform extract; ANME: Aponogeton natans methanol extract

Histopathological study of liver in diclofenac sodium induced hepatotoxic rats

In normal control animal liver, histological sections showed hepatocytes that were well-preserved, uniform cytoplasm and sinusoidal spaces. When analysed and compared with the normal control group, liver tissue in the rats treated with diclofenac sodium revealed extensive liver injuries, characterized by severe hepatocellular degeneration, necrosis and inflammatory cell infiltration (Fig. 6a).

Photomicrograph of sections taken from the liver of rats treated with diclofenac sodium slowed extensive necrosis and poptosis around central vein (Fig. 6b). Histology appearance of rat liver treated with A.natans pet ether extract (ANPE) and diclofenac sodium. Representative sections of liver tissue of rats treated with diclofenac sodiumand Aponogeton natans showing necrosis, inflammation around central veins and microvesicular steatosis . The liver did not show any marked recovery (Fig. 6c).

Photomicrographs of liver sections taken from rats treated with Aponogeton natans benzene extract (ANBE) and diclofenac sodium showed necrosis, inflammation around centralveins with bridginging necrosis and microvesicular steosis (Fig. 6d).

Histological presentation of rat liver administered with Aponogeton natans chloroform extract (ANCE) and diclofenac sodium treated rat liver, showed mild portal inflammation, focal lobular inflammation and rare apoptosis around central veins (indicating marked improvement over diclofenacsodium group (Fig. 6e).

Photomicrogrphy sections in liver tissue of rats treated with Aponogeton natans methanol extract (ANME) and diclofenac sodium showed good recovery with the absence of necrosis and fatty deposition. The central vein has nominal portal inflammation. It showed superior recovery as compared with other groups (Fig. 6f).

Histological display of rat liver treated with silymyrin and diclofenac sodium showed that Silymyrin administered rats showed significant recovery with disappearance of fatty deposition and necrosis. The portal tracts appeared almost clear indicating a potent hepatoprotective activity (Figure 6g).

Fig. 6a: Histopathological appearance of normal rat liver.

Fig. 6b: Histopathological appearance of rat liver treated with diclofenac sodium.

Fig. 6c: Histopathological appearance of rat liver treated with A.natans pet ether extract and diclofenac sodium.

Fig. 6d: Histopathological appearance of rats treated with A. natans benzene extract and diclofenac sodium.

Fig. 6e: Histopathological appearance of rats treated with A. natans chloroform extract and diclofenac sodium.

Fig. 6f: Histopathological appearance of rats treated with A. natans methanol extract and diclofenac sodium.

Fig. 6g: Histopathological appearance of rats treated with silymarin and diclofenac sodium.

DISCUSSION:

The invitro antioxidant results of the present study suggested that the methanol extracts (ANME) have potent antioxidant activity or free radical scavenging activity. The antioxidant activity of methanol extracts may be due to the presence of phenols and poly phenolic compounds²³. Hepatocellular injury from metabolic inhibition, oxygen radical toxicity, immunologically mediated damage, or some other mechanism results in predominant elevations of aminotransferase and alkaline phosphatase¹⁸. The ALT, AST and ALP levels were significantly elevated when rats were administered with diclofenac sodium indicating hepatocellular damage. The increased levels of these enzymes were significantly decreased by pretreatment with methanol extract (ANME) in dose dependent manner. This is the indication of stabilization of plasma membrane as well as repair of hepatic tissue damages caused by diclofenac sodium.

The significant increased liver weight of diclofenac sodium exposed animals seems to be due to toxic potential of diclofenac sodium. The significant increase in weight of liver was, however, found to be associated with concomitant increase of serum AST and ALT enzyme levels. It is important to note that the elevated activity of serum AST and ALT recorded in this study may be due to loss of enzymes of liver tissue. Pretreatment of methanol extract (ANME) decreased the liver weight significantly indicating recovery of liver tissue from damage. Significant decrease in total protein of the liver contents is a reflection of hepatic toxicity. The significant reductions of protein in diclofenac intoxicated group indicate depletion in the protein reserve and thus suggest hepatic toxicity. Methanol extract (ANME) administration increased the total protein content leading to normalization.

The SODs, catalase and GPx are enzymatic antioxidants widely distributed in all animal tissues that decomposes hydrogen peroxide and protects the tissue from highly reactive hydroxyl radicals^21,
22. Therefore, the reduction in the activity of these two enzymes may result in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxide. In the present study, methanol extract (ANME) significantly restored the hepatic catalase and GPx activity, which indicated that methanol extract (ANME) could scavenge reactive free radicals that eventually lessen the oxidative damage to the tissues and subsequently improved the activities of these antioxidant enzymes. The preventive effect of methanol extract was also confirmed by the results of histopathological study, as evidenced by a dose related decrease in the incidence and severity of histopathological hepatic lesions.

Particularly Aponogeton natans Linn. methanol extract pretreatment prevented the reduction in the antioxidant enzyme activities and consequent oxidative damage to the liver. In fact, the pretreatment of methanol extract (ANME) alone significantly boosted the antioxidant enzyme activities. Also reported good hepatoprotective activity in their studies; and suggested that the hepatoprotective activity of plant extract could be a result of boosting the antioxidant capacity of the liver. It was further confirmed by the histopathological assessment of the liver tissue. Hence in the present investigation the hepatoprotective activity of Aponogeton natans Linn. methanol extract may be attributed to chiefly polyphenolics, flavonoids, tannins and triterpenoids. The results of this investigation provide pharmacologic evidence on the folkore use of Aponogeton natans Linn. leaf with leafstalks for hepatoprotective activity.

Photomicrography shows that methanol, chloroform extracts and Silymarin have a protective effect in plasma membrane of hepatocytes and possesses multiple mechanism of actions against hepatotoxic agents.

CONCLUSION:

The results obtained from diclofenac sodium induced hepatotoxic model in rats indicated that after treatment of toxicants there was significant rise in SGOT, SGPT, ALP and Total bilirubin and Total protein levels. Treatment with Aponogeton natans Linn. methanol extract increased the level of SGOT, SGPT, ALP, total bilirubin and total protein activities when compared to toxic control. In case of antioxidant enzyme, Aponogeton natans Linn. methanol extract was found to significantly increase the level of SOD, CAT and GPX. The level of LPO which was elevated due to hepato toxicant was brought to also come to normal. The above results were further supported by histopathological evidences. It is event from the literature that the plant of Aponogeton natans (Linn.) Engl. and Krause having therapeutic potential to act as hepatoprotective and antioxidant agent. Further study can be extended to establish the plant stable dosage form to exploit it commercially to be utilized as alternative remedies to the available synthetic therapeutic agents.

ACKNOWLEDGEMENT:

The authors are thankful to Prof. P.Jayaraman, Director, Plant Anatomy Research Centre, Tambaram, Chennai, India for authenticating the plant material.

CONFLICT OF INTEREST:

Nil

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Received on 06.05.2018 Modified on 18.06.2018

Research J. Pharm. and Tech 2018; 11(10): 4431-4438.

DOI: 10.5958/0974-360X.2018.00811.9