Hepatoprotective potency of Lagenandra toxicaria and Ariopsis peltata against CCl 4 induced liver fibrosis in Wistar rats

Lagenandra toxicaria (LT) and Ariopsis peltata (AP) belongs to the family Araceae. LT is being traditionally used to cure bilious symptoms and wound healing. AP leaves are edible and the rhizome has medicinal value. With such a wide range of medicinal applications, it's essential to scientifically authenticate traditional usage and find the plant's bioactive components. Here, we have examined the anti - inflammatory and hepatoprotective potency of these plant extracts. The in - vivo liver fibrosis was induced in Wistar male rats using CCl 4 and was treated with various concentrations of the methanol and water extracts of the plant rhizomes. In the in - vitro anti inflammatory assay, LT methanol extract showed 42 % protection at 600 mg/ml which is higher than the other extracts. Among the AP and LT extracts, the 600 mg/kg methanol extract of LT treated rats showed a decreased (p<0.05) serum alanine/aspartate aminotransferase and total bilirubin levels. The plant extract brought about an amelioration of CCl 4 induced pathological effects and a significant reduction in the severity of inflammation, fatty metamorphosis, necrosis and liver fibrosis. Overall, the results suggest that LT rhizome could be effective in the treatment of liver fibrosis. It is possible that this is related to the presence of anti - inflammatory and antioxidant chemicals such as 9 - octadecenamide, flavonoids and phenols in the extract.


Introduction
Liver is a major organ that regulates various physiological functions and about 500 essential tasks are attributed to the liver (1). Hence, damage or loss of any liver function can have serious effects on our health. According to recent statistics, liver illnesses kill over two million people each year throughout the world. Cirrhosis complications account for almost one million deaths (2). Repeated liver injury by toxicants, synthetic drugs, or hepatic viruses cause sustained inflammation resulting in the accumulation of excess extracellular matrix (ECM) which leads to the fibrosis of the liver tissue. The accumulated ECM proteins damage the liver by forming a fibrous scar leading to liver cirrhosis and cancer (3,4). The management of inflammation, oxidative damage, apoptosis and related tissue responses are the essential therapeutic strategies for preventing liver failure (5,6). Herbal medications have recently acquired attention due to their anti-fibrotic qualities as well as other benefits such as low side effects, low toxicity and low cost. For the treatment of liver disorders, 65 % of patients in Europe and the United States rely on herbal remedies (6). Many phytochemicals such as silymarin, armepavine, salvianolic acid, plumbagin, rhein, glycyrrhetinic acid, epigallocatechin-3-gallate, osthole and curcumin have been isolated, recorded, and thoroughly researched for their efficacy in treating liver fibrosis (4). The plant constituents have anti-inflammatory and antioxidant properties that can have multiple effects in counteracting liver fibrosis and recovering the damaged cells (7). Among the Araceae members, several plants were reported for their hepatoprotective activity. According to one report (8) Colocasia esculenta corm ethanolic extract showed significant hepatoprotective activity against CCl4 and paracetamolinduced liver damage compared to silymarin. Similarly, the ethanolic and methanolic extracts of Amorphophallus campanulatus corm could restore the CCl4 induced hepatic damage and reduce ALT, AST and ALP (7). Quercetin, extracted from the ethyl acetate fraction of A. paeoniifolius corm was reported to have a hepatoprotective effect against CCl4 induced hepatotoxicity (9). Study showed that Alocacia macrorrhiza, exhibits anti-inflammatory potency against carrageenan-induced paw oedema in rats and further it also shows hepatoprotective and anti-inflammatory activity (10). It is crucial that ethnomedicinally beneficial plant's efficacy toward liver therapy has to be scientifically evaluated. The present study examined the antioxidant, anti-inflammatory and hepatoprotective activities of the rhizome extracts of two plants of Araceae viz. Lagenandra toxicaria and Ariopsis peltata. Lagenandra toxicaria is traditionally used for treating bilious complaints and wound healing (11). Ariopsis peltata leaves are edible and rhizomes are used indigenously to treat bilious complaints (12).

Reagents
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) and Total bilirubin (TBL) analysis kits were purchased from Agappe diagnostics Pvt. Kerala, India. Hydrogen peroxide and CCl4 were purchased from NICE, Kerala, India. All other reagents used were of analytical quality.

Plant collection
Lagenandra toxicaria and Ariopsis peltata rhizomes were collected from Markanja village, Sullia taluk (12⁰34'38.7" N, 75⁰.29'54.8" E) and Charmadi ghat in Belthangadi taluk of Dakshina Kannada District (13⁰04'35" N, 75⁰27'06" E), Karnataka, India. Plants were identified and the voucher specimens were deposited in the herbarium of the Department of Applied Botany (AK08 & AK09), Mangalore University. The plant rhizomes were washed in running tap water and air-dried, powdered and stored for further study.

Preparation of extract
Using the soxhlet apparatus and a water bath, 25 g of dried rhizome powder was extracted in 250 ml of methanol and water. The extracts were vacuum dried at 45 °C and stored at 4 °C for further use (13). The percentage yield was meas-ured using the formula (Eqn 1).

Analysis of total phenolic and flavonoid content
The total phenol and flavonoid content of the extract was analyzed using Gallic acid and Quercetin standards respectively by spectrophotometry method (14).

Antioxidant analysis
Antioxidant properties were analyzed using Ferric reducing antioxidant power (FRAP) assay and DPPH assay. DPPH solution was prepared using methanol. One ml of different concentrations of plant extracts (0.1 to 1 mg/ml) was added to the DPPH solution. The reaction mixture was vortexed and kept for incubation in dark, at room temperature and the absorbance was measured at 517 nm. The IC50 of the plant extract was determined (15) using the formula (Eqn. 2).
1 ml of the extract (0.1 to 1 mg/ml) was mixed with 2.5 ml of phosphate buffer (0.2 mol, pH 6.6) and 2.5 ml of 1 % potassium ferricyanide and incubated at 50 °C for 20 min. Then added 2.5 ml of 10 % trichloroacetic acid and centrifuged for 10 min at 3000 rpm. From each tube, 2.5 ml of supernatant was collected and diluted twice with distilled water, 0.5 ml of freshly prepared FeCl3 solution (0.1%) was added to the diluted mixture, the absorbance was measured at 700 nm (15).

In-vitro Anti-inflammatory analysis using human blood
The anti-inflammatory activity of the extracts was examined by HRBC (Human red blood cells) membrane stabilization method. Five millilitres of blood was obtained from a healthy human volunteer in a sterile tube and used for the experiment, following the standard protocol (16). Diclofenac was used as the standard drug and the control was without the extracts. At 560 nm, the absorbance of the supernatant was measured using a spectrophotometer. The % of hemolysis was calculated by assuming that 100 % hemolysis occurred in the control. The following formula (Eqn. 3) was used to determine the percentage of hemolysis.

Animals and experiment
NUCARE (Nitte University Centre for Animal Research and Experimentation), Deralakatte, Mangalore, provided female Mus musculus mice weighing 25-30 g and Wistar male rats weighing 180-200 g. The animals were maintained at a constant temperature of 22 °C, with 12 hr of darkness and 12 hr of light. The animals were fed with conventional laboratory food and had access to clean water. The animal experiments were approved by the Nitte Institute's animal ethical committee, with the protocol number (NGSMIPS/ IAEC/MARCH-2019/124).

Acute toxicity study
Test No. 423 (OECD, 2001; Acute oral toxicity -acute toxic class technique) was used to conduct an acute oral toxicity study following OECD rules for chemical testing. The study involved 5 animal groups, each group containing 6 animals that are randomly assigned. The animals were fasted for 3-4 hr and the plant extract was administered with an initial dose of 2000 mg/kg body weight (B. W). The treated animals were observed for 14 d and if two out of 3 animals died then the dose was considered toxic. If one out of 3 animals is dead then the same dose was repeated for its toxicity confirmation and if the mortality is observed again then the experiment was repeated with lower doses of the extract.

Induction of liver fibrosis
The male Wistar rats weighing 180-210 g were procured and housed in a suitable environment following the OECD guidelines. All animal experiments were carried out following the procedure of the Institutional Animal Care and Use Committee of Nitte Deemed to be University for the care and use of laboratory animals. The experimental design involved 12 groups containing n = 8 animals in each group. CCl4 was given twice a week at an interval of 3 d. The standard drug silymarin and the plant extracts were administered every day for 8 weeks (17). After eight weeks of treatment, the animals were sacrificed by cervical dislocation. The blood samples were collected and used for serum enzyme analysis. The liver tissues were isolated and a portion was stored in formalin for histopathological studies. The remaining tissues were homogenized (10%, w/v) in 50 mM phosphate buffer (pH. 7.0) and stored at -80 0 C for testing.

Liver index calculation
The body weight and liver weight of each animal was recorded and the liver index of the rats was calculated using the formula (Eqn. 4) followed by standard procedure (18).

Examination of serum ALT, AST and Total bilirubin activities
The serum was isolated from collected blood samples and the levels of serum AST, ALT and total bilirubin activities were examined by Agappe kits (India) using semi autoanalyzer (STAR 21) following the maker's guidelines.

Liver histology examination
In a 10 % buffered formalin solution, a piece of the liver tissue was fixed, dehydrated in a 50-100 % ethanol series, cleaned in xylene and paraffin-embedded. The paraffinembedded tissues were sectioned (5 mm) and stained with hematoxylin-eosin (H&E) for histological observation of liver injury and Masson's trichrome for observation of collagen deposition in the liver (19). 0 -3 scoring system (grade 0 -absent, grade 1 -present in one-third of the lobule, grade 2 -present in two-third of the lobules, grade 3present in all the lobules) was used to indicate the level of necrosis, inflammation and fatty metamorphosis. For fibrosis, collagen was graded (20) grade 0 -normal, grade 1fibrosis present (collagen fiber present as small septa) grade 2 -mild fibrosis (collagen fiber extended as septa from portal tract to central vein forming complete septa); grade 3 -moderate fibrosis (collagen fibers formed thin complete septa) and grade 4 -severe fibrosis (collagen fibers formed thick septa). All histological assessments were carried out by a skilled operator who was blinded to the study.

Examination of lipid peroxidation and antioxidant enzyme
Isolated liver tissues were washed with chilled saline solution to remove the blood cells. A 10 % tissue homogenate was prepared and the supernatant was collected to determine the malondialdehyde (MDA) levels and catalase activity of the liver was calculated using hydrogen peroxide as a standard following standard methods (21).

Bioactive compound examination using GC-MS
Lagenandra toxicaria rhizome methanol extract was analyzed using GC-MS instrument, Shimadzu QP-2010 ultra. The oven temperature was retained at 60 to 280 0 C at a speed of 10 0 C per min. The injector volume was 1 µl and the split ratio was 10. The bioactive compounds were identified using the Willey/NIST mass spectral library of the GC-MS data system. The % composition of the different components of the sample was calculated from the peak area integrated by the analysis program.

Statistical analysis
The data are presented as mean with ± standard deviation. To investigate and compare the data, one-way ANOVA was performed, followed by Newman-Keuls multiple comparison tests (PRISM GraphPad, version 5.0; GraphPad Software Inc., San Diego, CA).

Yield of extracts
The Lagenandra toxicaria and Ariopsis peltata showed yield of 12.1 % and 12.6 % in methanol and 14.8 % and 17.2 % in water respectively.

Phenolic and flavonoid content analysis
The phenolic and flavonoid content of the plant extracts is given in Table 1. The methanol extract of both the plants had higher phenol and flavonoid content. Both the methanol and water extract of L. toxicaria was rich in phenolics and flavonoids. Water extract of A. peltata had lower content.

Antioxidant assay
L. toxicaria methanol extract had an antioxidant activity of 477.57 ± 3.75 µg/ml which is higher compared to that of the water extract, ie, 851.53 ± 7.69 µg/ml. The antioxidant activity in A. peltata rhizome methanol and water extract was 1216.62 ± 6.61 µg/ml and 1512.82 ± 10.62 µg/ml (Fig.  1a). The reducing power of all the extracts increased with increase in concentration as evidenced by the increased absorbance at 700 nm. The L. toxicaria methanol extract had the highest reducing power, with a higher absorbance at 1000 µg/ml than the other extracts. A. peltata water extracts showed lower absorbance compared to the other extracts (Fig. 1B).

Anti-inflammatory activity
Inhibition of hemolysis by the plant extracts was analyzed using the human red blood cells. Both the plant extracts showed stabilization of HRBC at different concentrations (200 mg/ml, 400 mg/ml, 600 mg/ml and 800 mg/ml). The L. toxicaria methanol extract showed more protection compared to all other extracts. At 400 mg/ml it showed 31.19 ± 6.09 % of protection and at 600 mg/ml, it showed 41.99 ± 3.32 % of protection against hemolysis. At higher concentrations, the protection was decreased. The results are represented in Table 2.

Acute toxicity study
The methanol and water extracts of both the plants at 2000 mg/kg dose did not show any behavioural changes or mortality in treated animals. Hence, the extracts were considered to be nontoxic.

Body weight and liver index
In the positive and negative control groups the body weight decreased significantly (p<0.05) compared to the normal control group. A mortality of two animals each were seen in these 2 groups. The extract and silymarin treated group did not show any mortality except for the 400 mg/kg A. peltata water extract where one animal was dead. A significant increase in body weight was seen in the L. toxicaria extract treated groups The liver weight and liver index increased significantly in the positive control group compared to the normal control group. The liver weight and index decreased significantly in the standard control and in all the extract treated groups Table 3.

Serum markers of CCl4 induced liver fibrosis in animals
The concentrations of AST, ALT and TBL are good indicators of liver health An increase in these enzymes is suggestive of abnormal liver function. In this study, higher levels of AST, ALT and TBL were observed in the positive control groups. The concentration of AST, ALT and TBL levels in different treatment groups is given in Fig. 2. Among the extract-treated groups, the 600 mg/kg L. toxicaria methanol extract showed the concentrations comparable to normal control group.

Liver histology observation
The H&E staining (Fig. 3a) showed prominent hepatic steatosis, necrosis, infiltration of inflammatory cells, hepato       cyte disorganization and sinusoidal architectural changes confirming the successful induction of liver fibrosis. The pathological effects of CCl4 treatment were seen to be ameliorated in both the plant extract treated groups. The 600 mg/kg L. toxicaria methanol extract showed a significant reduction of the liver necrosis and inflammation among all the extracts. Histological scoring of extract treated groups indicates a significant reduction in severity of inflammation, fatty metamorphosis and necrosis (Fig. 4). Further, the Massonson trichrome (Fig. 3b) shows severe fibrosis in the positive control group. The collagen deposition score (Fig. 4) was significantly reduced in the extract-treated groups. Lagenandra toxicaria methanol and water extract treated groups showed very low severity scoring comparable to the standard drug-treated group.

Liver antioxidant enzyme and oxidative stress marker
The liver homogenate of the positive control group showed decreased catalase activity and a higher MDA levels. In contrast, the plant extract treated groups showed significantly lower levels of MDA and significantly increased levels of catalase activity. The 600 mg/kg metha-nol extract of L. toxicaria showed results comparable to the standard drug (Table 4).

GC-MS analysis
The chromatogram revealed the presence of 21 compounds. The retention time of these compounds are represented in Table 5 and Fig. 5. The compounds were identified by comparing their peak retention time, % peak area and mass spectral fragmentation patterns to those of the Willy and NIST libraries' recognised compounds. The chromatogram showed a higher amount of 9-

Discussion
Liver fibrosis is stimulated by oxidative stress, inflammation and cellular apoptosis from viral infection or other toxicants (5,22). Inflammatory cells have been linked to the induction of neutrophils and lymphocytes, which in turn promote fibrosis (23). The oxidative stress activates the NF-kB which stimulates the hepatic stellate cells. It also induces production of cytokines like TNF-α, TGF-β, IL-1β, IL-6, Cox-2 that promotes inflammation and fibrosis. NF-kB regulates the inflammatory pathway genes (24,25). It was opined that the antioxidant and anti-inflammatory compounds play a major role in the treatment of liver fibrosis (5). Here, both the plant extracts showed good antioxidant activity. In the DPPH and FRAP assays, L. toxicaria methanol extract showed higher antioxidant potency than A. peltata.
The stabilization of the lysosomal membrane is critical for limiting the inflammatory response. Erythrocyte membrane is similar to the lysosomal membrane (16,26). The stabilization of the erythrocyte membrane by HRBC method confirms the anti-inflammatory activity of the extracts. The In-vitro anti-inflammatory study clearly showed that the percentage of protection increased in a concentration dependent manner up to 600 mg/ml in all the extracts and the protection decreased at 800 mg/ml concentration. Among the other extracts, the L. toxicaria methanol extract showed higher anti-inflammatory activity at 600 mg/ml. The CCl4 induced liver fibrosis model is widely used to study different aspects of liver fibrosis and healing (27). Repeated administration of CCl4 induces lipid peroxidation and the injured hepatocytes release the cellular components. These induce the inflammatory response leading to fibrosis (28,29). At present, liver transplantation is the  most effective strategy to address liver fibrosis. It has many limitations, like the shortage of organ donors, immunological rejection, surgical complications and its high cost (5). In recent years, various herbal remedies have been demonstrated to possess anti-fibrotic activities and is being used to treat fibrosis (7). According to one study (30) bile duct ligation induced hepatic fibrosis in rat model was treated with armepavine showed antifibrotic activity through the anti NF-κB activation pathway. Salvianolic acid B is a water-soluble, bioactive compound isolated from Salvia miltiorrhiza. It also significantly lowers the NF-κB, and IκBα levels and reduces the CCl4 induced liver fibrosis (31). The biochemical analysis and histological studies showed that the CCl4 induced damages were successfully reversed by the plant extracts. The L. toxicaria methanol extract treated rats showed higher recovery than the other extracts. The severity scoring of the pathological changes have significantly decreased after the treatment.
The innate antioxidant enzyme defence system is a natural guard of organs against oxidative stress. The enzyme catalase protects the cells by breaking the toxic hydrogen peroxide into water and oxygen (32). CCl4 inhibits the natural antioxidants in the liver tissue and increases the MDA (33). Our study showed that both the plant extracts co-treatment in CCl4 treated animals showed increased levels of antioxidant enzymes. Also, a reduction in the lipid peroxidation was seen in all the extract treated groups. The 600 mg/kg methanol extract of L. toxicaria showed very low lipid peroxidation compared to the other extract treated groups ( Table 4).
The GC-MS analysis of the L. toxicaria methanol extract showed some major compounds like Cyclopentanol, Alpha-methyl-sorboside, Palmitic acid, 13 -Hexyloxacyclotridec -10 -en -2 -one, 9-octadecenamide and stigmasterol. Studies have shown that octadecenamide, palmitic acid and Stigmasta-5, 20 (22)-dien-3-ol-have antiinflammatory ativity (34). The GC-MS findings indicate a higher percentage of 9-octadecenamide. The 9-octade cenamide which is an oleamide was reported to have antioxidant and anti-inflammatory activity (35,36). The therapeutic effects of oleamide are being widely studied. Oleamide extracted from the green algae Codium fragile reduces the production of IL-1, IL-6 and translocation of NF-κB. The effects of oleamide in the carrageenan-induced rat inflammatory model were comparable to the non-steroidal, antiinflammatory medication, diclofenac (37). A decreased histamine and -hexosaminidase secretion and reduced interleukin-4 (IL-4) and TNF-α production were seen in the oleamide derived from Arctium lappa extract which is proof of its anti-allergic potential (38). Oleamides are also linked to certain neuroprotective properties including anti -inflammatory responses in a rat model of Alzheimer's disease (39). Several liver illnesses, such as hepatitis, liver fibrosis, cirrhosis and hepatocellular carcinoma, are linked to the NF-κB pathway. As a result, the NF-κB pathway is a possible target for hepatoprotective drug development (40). Studies have shown that oleamide has a very good affinity towards the NF-kB and would inhibit the activation of inflammatory pathways (37,41). Therefore, the 9octadecenamide offered anti-inflammatory and antioxi-dant properties of L. toxicaria methanol extract could be the mechanism that leads to extracts' anti-fibrotic action against CCl4 induced liver fibrosis.

Conclusion
Good amounts of bioactive compounds, including phenols and flavonoids were present in the water and methanol extracts of Lagenandra toxicaria and Ariopsis peltata. The methanol extract of L. toxicaria exhibits a greater antioxidant capacity than the other extracts. This can be attributed to the higher concentration of phenols and flavonoids. Both the plant extracts showed resistance to CCl4-induced liver fibrosis. The methanol extract of L. toxicaria 600 mg/ kg showed higher amelioration of CCl4 induced collagen accumulation and inflammation. The GC-MS analysis of L. toxicaria methanol extract revealed the presence of bioactive compounds such as octadecenamide, palmitic acid and stigma steroids, which have anti-inflammatory and antifibrotic activities. Therefore, the L. toxicaria plant extract can be effectively used to treat liver fibrosis