Introduction

Liver provides protection to an organism against foreign particles by eliminating them, through the process of excretion [1]. Liver damage caused by various hazardous chemicals such as carbon tetrachloride (CCl4), thioacetamide, chronic alcohol consumption, and even some drugs used to treat various diseases, may accumulate in the liver tissue and cause alteration in the normal physiological function of the liver by generating free radicals, which is the major reason for the cause of heart related problems like stroke, autoimmune diseases like rheumatoid arthritis, diabetes, and cancer [2]. Patients with liver injury reported to have oxidative stress. The imbalance in the level of pro-oxidants and antioxidants leads to oxidative stress leads to potential damage for the organism [3]. Currently available synthetic drugs used for the treatment of liver diseases are inadequate and known for various side effects. Earlier scientific studies reported that, the use of plant phenolics, reduces the risk of liver diseases by acting as antioxidants [4]. Recently, interests in consuming edible plants that are rich in antioxidants and health-promoting phytochemicals have been increased worldwide. CCl4 is frequently used chemical for inducing liver damage in experimental animals [5, 6]. CCl4 is a potent hepatotoxin, when administrated leads to necrosis in liver tissue, ultimately leads to liver injury. CCl4 induced liver injury depends on metabolism of toxic substance by the liver NAPDH-cytochrome system results in the formation of highly reactive intermediate trichloromethyl free radicals, which binds to cell protein covalently which leads to membrane lipid peroxidation and finally cell necrosis.

India is the land of medicinal plants and it is important to register the data by scientific researches on such plants could be of clinical importance. Treating various health issues like cancer, arthritis, diabetes has been documented in Ayurvedic medical system practiced mainly in the Asian continents for more than 5000 years [7]. Ipomoea staphylina Linn. (Convolvulaceae) is widely found in Asian countries including Sri Lanka, India, and China. In Asian traditional medicine system, I. staphylina is an extensive climber, commonly used for the treatment of liver diseases, purgation, stomach disorders, pain, inflammation, and in rheumatism [8]. Plants of the same genus such as Ipomoea hederacea [9] and Ipomoea obscura [10] have been reported for their hepatoprotective activity. Recently, antimicrobial activity of aqueous extract of Ipomoea reniformis has been reported [11]. The antioxidant and hepatoprotective activity of of I. staphylina has not been evaluated scientifically till now. Therefore, identification of phytocompounds from I. staphylina aquous leaves extract (AIS) and evaluate its antioxidant and hepatoprotective activity against CCl4 induced oxidative damage in Wistar rats was the main aim of this research.

Methodology

Chemicals

All the chemicals used were of great purity and sensible grade. High quality chemicals used in the research were bought from Sigma Chemicals Co. (St. Louis, MO, USA) like CCl4, Folin‘s reagent, Na2CO3, AlCl3, NaCl, KCl, K2S2O8, Na2HPO4, KH2PO4, H2O2,gallic acid, sodium nitrite, rutin, C2HCl3O2, DPPH, ABTS, C6N6FeK3, BHT and FeCl3.

Plant collection and extraction

Plants sample collection and extraction preperations were done by standard protocol followed in Plant metabolite lab, Department of Environmental Biotechnology, Bharathidasan University, Trichirappalli. I. staphylina plants samples were collected from local areas near Bharathidasan University, Tiruchirappalli district, Tamilnadu, India and authenticated in the Department of Plant Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India (Collection number 1002, Centre for Research and development of Siddha-Ayurveda Medicines, Bharathidasan University). Leaves were picked, washed, shade dried and ground to powder. Ten grams of the powder was extracted with 250 mL of distilled water (in the ratio 1:25), by proper shaking for 2 days. Whatmann filter paper No.1 was used to filter the extract and followed by lyophilization using a vacuum freeze dryer (Christ alpha 1–2/LD plus, Osterode am Harz,Germany) to produce a yield of 10% of dry extract. The dried extract was stored at − 20 °C in deep freezer for further study. The extract was dissolved in distilled water of known concentrations and used for experimental studies [11].

Determination of total phenolic content

Total phenolic content was determined by the standardized protocol [12]. To 100 μL of the AIS (aquous extract of I. staphylina), 1.5 mL of distilled water and 300 μL of Folin-Ciocalteu reagent was added and incubated for 6 min at room temperature. Then, 300 μL of sodium carbonate (2 g/10 mL) was added and allowed to stand for 30 min at 40 °C for colour development. Absorbance was read at 765 nm using the spectrophotometer (Shimadzu UV-1601, Columbia, MD, USA). Commercial gallic acid was used as the standard. Total phenolic content was expressed as mg of GAE/g of extract.

Determination of total flavonoids content

Total flavonoids content was estimated using the method of Ordon ez et al. [13]. The above mentioned method was standardized in our laboratory to calculate the amount of total flavonoids present in the sample. To 0.5 mL of AIS, 0.5 ml of 2% aluminium chloride solution was added. After 1 h at room temperature, the absorbance was read at 420 nm using a UV-spectrophotometer, and expressed as mg of RE/g of extract. Estimation of total flavonoid in the extracts was carried out in triplicates. Commercial rutin standard was used this assay.

ABTS free radical scavenging activity

For ABTS assay, the method of Re et al. [14] was adopted with some modifications. The stock solutions included 7 mM ABTS solution and 2.4 mM potassium persulfate solution was prepared first. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark condition. The solution was then diluted by mixing 1 mL ABTS solution with 40 mL of methanol to obtain an absorbance of 0.706 ± 0.002 units at 734 nm using a spectrophotometer. AIS (1 mL) of various concentrations ranging from 20 to 100 μg were allowed to react with 1 mL of the ABTS solution after 7 min the absorbance was read at 734 nm using the spectrophotometer. ABTS scavenging capacity of the extract was compared with that of BHT standard and percentage of inhibition calculated as:

$$ \mathrm{ABTS}\ \mathrm{radical}\ \mathrm{scavenging}\ \mathrm{activity}\ \left(\%\right)=\left[\left(\mathrm{Abs}\ \mathrm{control}\hbox{-} \mathrm{Abs}\ \mathrm{sample}\right)\right]/\left(\mathrm{Abs}\ \mathrm{control}\right)\Big]\times 100 $$

Where Abs control is the absorbance of ABTS radical + methanol; Abs sample is the absorbance of ABTS radical + sample extract/standard. The sample concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage against sample concentration. All determinations were carried out in triplicates. ABTS radical-scavenging activity of BHT was also assayed for comparison.

DPPH free radical scavenging activity

Effect of extract on DPPH radical was estimated using the method of Liyana-Pathiranan and Shahidi [15]. A solution of 0.135 mM DPPH in methanol was prepared and 1 mL of this solution was mixed with AIS (1 mL) of various concentrations ranging from 20 to 100 μg. The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. Absorbance of the mixture was read spectrophotometrically at 517 nm. BHT was used as reference standard. The ability to scavenge DPPH radical was calculated by the following equation

$$ \mathrm{DPPH}\ \mathrm{radical}\ \mathrm{scavenging}\ \mathrm{activity}\ \left(\%\right)=\left[\left(\mathrm{Abs}\ \mathrm{control}\hbox{-} \mathrm{Abs}\ \mathrm{sample}\right)\right]/\left(\mathrm{Abs}\ \mathrm{control}\right)\Big]\times 100 $$

where Abs control is the absorbance of DPPH radical + methanol; Abs sample is the absorbance of DPPH radical + sample extract/standard. All determinations were carried out in triplicates.

Determination of reducing power activity

Reducing power of the AIS was determined according to the method of Oyaizu [16]. One mL of AIS of various concentrations (20–100 μg) mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. Then 2.5 mL of 10% trichloroacetic acid was added to the mixture, and centrifuged at 3000 rpm for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL distilled water and 0.5 mL of 0.1% ferric chloride. Absorbance was measured at 700 nm using a spectrophotometer. Increased absorbance of the reaction mixture indicated increased reducing power.

Polyphenolic compounds identification using HPLC analysis

Identification of polyphenolic compounds from AIS using RP-HPLC with C18 column (4.6 × 250 mm, 5 μm particle size). HPLC system was coupled with a PDA detector (PDA-2998), Waters, USA. Mobile phase consist of HPLC water with 0.1% formic acid (Solvent A) and 100% methanol (HPLC grade) (Solvent B). Gradient elution program followed; Solvent A and B was 0–10% (5 min), 10–15% (5 min), 15–20% (5 min), 20–30% (5 min), 30–40% (5 min); Flow rate was 1 mL/min and maximum pump pressure 4000 psi was maintained. Polyphenolics were identified at wavelength of 280 nm [17]. Compounds were identified and confirmed by comparing its tR and λ max compared with commercial standard.

In vivo studies

Acute toxicity test

To evaluate the acute toxicity of AIS, a single oral dose of AIS of various concentrations 0.5, 1, 1.5, 2, 2.5 and 3.0 g/kg b.w. was given to Wistar rats then allowed free access to food and water. The animals were observed for signs of toxicity such as, urination, salivation, asthenia and defaecation for 72 h after the oral administration for the acute oral toxicity. AIS was safe up to the dose of 2 g/kg b.w. p.o. for Wistar rats and did not show any acute toxicity signs. We chose a wide concentration range for AIS in our preliminary study and their inhibitory effects against CCl4 induced hepatotoxicity were evaluated (data not shown). By avoiding non effective and toxic concentrations, the 250 and 500 mg/kg b.w. concentrations were selected. Experimental protocols were approved by the CPCSEA, Chennai, Tamil Nadu, India (BDU/IAEC/2012/29).

Treatment

Animals were randomly divided into five groups of 6 rats per group. Group I served as normal control and was given olive oil (1 mL/kg of b.w.) daily for a period of 8 weeks. For inducing hepatotoxicity (in vivo), animals of Groups II-V were administered orally 1 mL/kg of b.w. of CCl4 (1:1, v/v, CCl4 in olive oil) twice a week for 8 weeks. Group II served as CCl4-treated model group (disease control). Group III received silymarin (at 50 mg/kg of b.w.) daily for a period of 8 weeks, which served as positive control. Groups IV and V were administered orally with AIS at 250 and 500 mg/kg of b.w. daily for 8 weeks [18]. At the end of the experiment, all animals were sacrificed. Serum samples were collected into heparinized tubes (50 U/mL). Liver samples were dissected out and washed immediately with ice cold saline to remove as much blood as possible. One part of the liver samples was immediately stored at −20 °C until analysis, another part was excised and fixed in 10% formalin solution for histopathological analysis.

Determination of serum biochemical parameters

Serum biochemical parameters such as SGOT, SGPT, ALP and bilirubin were estimated in the serum of experimental animals using commercial reagent kits and were performed according to the manufacturer’s (Nice Chemicals [P] Ltd. Cochin, Kerala, India) instructions.

Antioxidant enzymes activities

Liver homogenates (10% w/v) prepared in phosphate-buffered saline (PBS containing 137 mM sodium chloride, 2.68 mM potassium chloride, 10.14 mM sodium phosphate dibasic and 1.76 mM potassium phosphate monobasic in 1000 mL distilled water pH 7.2) were used for antioxidant studies. Catalase activity was determined by the method of Sinha [19] in which dichromatic acetic acid is reduced to chromic acetate when heated in the presence of hydrogen peroxide, with the formation of perchloric acid as an unstable intermediate. The green colour developed was read at 590 nm against blank on a spectrophotometer. CAT activity was expressed as units/mg protein (mmoL of hydrogen peroxide consumed/min/mg protein). SOD activity was determined by the method of Marklund and Marklund [20]. The degree of inhibition of pyrogallol auto-oxidation by the supernatant of the tissue homogenate or haemolysate was measured. The change in absorbance was read at 470 nm against blank every minute for 3 min using a spectrophotometer. Enzyme activity was expressed as U/mg of protein. GSH content was estimated by the method of Moron et al. [21]. To the supernatant of the tissue homogenate, 0.5 mL of 10% trichloroacetic acid was added followed by centrifugation. To the resulting protein-free supernatant, 4 mL of 0.3 M sodium phosphate dibasic (pH 8.0) and 0.5 mL of 0.04% (w/v) 5, 5-dithiobis-2-nitrobenzoic acid were added. Absorbance of the resulting yellow colour was read spectrophotometrically at 412 nm. Results were expressed as μg/mg protein.

Determination of lipid peroxidation level

The mean concentration of MDA, a measure of the intensity of lipid peroxidation, was assayed in the form of thiobarbituric acid reacting substances (TBARS) by the method of Ohkawa et al. [22]. Briefly, to 0.2 mL of 8.1% sodium dodecyl sulphate, 1.5 mL of 20% acetic acid (pH 3.5) and 1.5 mL of 0.81% thiobarbituric acid aqueous solution were added in succession. To this reaction, 0.2 mL of tissue homogenate was added separately. This mixture was then heated in a boiling water bath for 60 min. After cooling to room temperature, 5 mL of butanol:pyridine (15:1, v/v) solution was added. The mixture was then centrifuged at 2000 x g for 15 min. Upper organic layer was separated and the intensity of the resulting pink color was read at 532 nm. Tetramethoxypropane was used as an external standard. The level of lipid peroxides was expressed as nanomoles of MDA formed/mg protein.

Histological studies

Liver samples were fixed in Bouin’s fixative and processed to obtain 5 μm thick paraffin sections and stained with hematoxylin and eosine (H&E) for histological observations.

Statistical analysis

Statistical analysis was performed using the statistical package SPSS (Statistical Package for Social Science, SPSS Inc., Chicago, IL, USA). Analysis of variance was performed by ANOVA procedures and significance of each group was verified with one-way analysis of variance followed by Duncan post hoc test (P ≤ 0.05). Values obtained are means of six replicate determinations ± standard deviation.

Results

Preliminary phytochemical analysis

Preliminary phytochemical analysis [18] of AIS revealed the presence of phenolics, flavonoids, steroids, tannins, saponins and terpenoids. Total phenolics and flavonoid content of AIS were 20.78 ± 0.86 mg of GAE/g of the extract and 17.78 ± 0.45 mg of RE/g of extract respectively.

Free radical scavenging activity

Inhibition range of ABTS (Fig. 1a) and DPPH (Fig. 1b) free radicals by AIS are shown in Figures. The quenching effect of AIS against ABTS and DPPH radicals in a dose dependent manner is clearly depicted in the result obtained. IC50 values of AIS to quench ABTS and DPPH radicals are 32.08 ± 0. 12 and 45.24 ± 0.65 μg/mL, respectively.

Fig. 1
figure 1

a Antioxidant activity of AIS. a ABTS.+ radical scavenging activity of AIS compared with BHT. Each value is expressed as the mean ± standard deviation (n = 3). b DPPH radical scavenging activity of AIS compared BHT. Each value is expressed as the mean ± standard deviation (n = 3)

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Reducing power activity

The reducing potential of the AIS was shown in Fig. 2 in comparison with a standard BHT at 700 nm. In the reducing power assay, the principle is the ability of the antioxidants present in the sample to reduce Fe3+ to Fe2+ by donating an electron and results in the formation of perl’s blue coloured solution. The intensity of the colour formed can be measured at 700 nm. The reducing effect of the extract can be predicted by increase in absorbance in dose dependent manner. The reducing capacity of AIS increases proportionally with increase in the concentration of the extract used. The results shows that the reducing at100 μg/mL concentration had an optical density of 0.4274, which was comparatively similar to that of the standard BHT with an optical density of 0.4766.

Fig. 2
figure 2

Reducing power of AIS compared with BHT. Each value is expressed as the mean ± standard deviation (n = 3)

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HPLC identification of polyphenolic compounds

A typical HPLC profile of the phenolic and flavonoids constituents of AIS was presented in Fig. 3. HPLC analysis of AIS showed that phenolic compounds such as protocatechuic acid, chlorogenic acid, caffeic acid, vanillin and p-Coumaric acid and also flavonoids such as naringinin, qurecetin, rutin and flavon were detected (Fig. 3c). Flavonoids are the most abundant phytocompounds present in the AIS as compared with phenolic compounds. Chromatograms revealed some unidentified peaks that may correspond to unknown phenolic compounds.

Fig. 3
figure 3

HPLC profiling of polyphenolic compounds identified in AIS

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In vivo studies

Serum biochemical parameters

Mean serum activities of SGOT, SGPT, ALP, and bilirubin were found to be higher in samples from Group II rats (Disease control) when compared to values in samples from Group I (Normal control) rats. However, the mean serum activities of above mentioned parameters were significantly (P ≤ 0.05) lower in groups treated with AIS (500 mg/kg b.w) and silymarin (Table 1).

Table 1 Protective effect of AIS on CCL4 induced elevation in hepatic enzymes, Bilirubin, TBARS content, and depletion of enzymatic and non enzymatic enzymes in Wistar rats
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Antioxidant enzyme activities

CCl4 treatment caused a significant decrease in the level of tissue antioxidant enzymes such as SOD, CAT, and the cellular antioxidant molecule GSH in liver [23]. AIS administration has up regulated the activities of these endogenous antioxidant enzymes (Table 1). The levels of liver SOD, CAT, and GSH in the CCl4-treated group were less compared with the normal control group. The result obtained indicates the tremendous increase (P ≤ 0.05) in SOD, CAT and GSH activity in the AIS treated groups (at 500 mg/kg b.w.). The activity of AIS at the dose of 500 mg/kg was comparable to that of the positive control, silymarin. Reduced glutathione is presumed to be an important endogenous defense against peroxidative destruction of cellular membranes. In the present study, CCL4 administrated animals shows decline in the presence of reduced glutathione level. Treatment with AIS was very effective in restoring the glutathione content which had been substantially decreased in CCl4 group (Table 1). Significantly (P ≤ 0.05) higher MDA level was noted in tissue samples obtained from CCL4 administrated (Group II) rats than in those from normal (Group I) rats, whereas the mean levels of MDA in samples from AIS-treated groups (Group IV and Group V) were significantly (P ≤ 0.05) lower than the levels in samples from (Group II) rats given in Table 1.

Histopathological examination

The report of histopathological studies supports the results of the biochemical studies and it demonstrates that the hepatic damage caused by CCl4 during intoxication is reduced in the liver sample of animals treated with AIS. Histological examination of the liver sections of control animals revealed the presence of normal hepatocytes with well-preserved cytoplasm, a prominent nucleus and distinct sinusoidal spaces (Fig. 4a). CCl4 treated groups liver tissue cells show severe damage to liver tissue by cell necrosis (Fig. 4b). Interestingly, the administration of AIS against this liver damage shows protective capacity of AIS against liver necrosis. AIS at a dose of 500 mg/kg b.w. was more effective when compared to the other dose (250 mg/kg bw) (Fig. 4c)and also showed regaining of normal histological appearance (Fig. 4d), which clearly indicates the protective and curative effect of AIS against CCl4-induced hepatic damage. The standard, silymarin-treated rats liver section showed almost normal liver with no sign of necrosis (Fig. 4e).

Fig. 4
figure 4

Histopathological analysis of liver tissue of male Wistar rats administrated with CCl4 and treated with AIS. a Control group shown normal lobular architecture with clear central vein (X-40). b CCl4 administered group shown severe hepatocyte necrosis, fatty degeneration and vacuolation (X-40). c AIS treated group at 250 mg/kg shown mild improvement in chord arrangement but perinuclear vacuolation was visible (X-40). d AIS treated group at 500 mg/kg shown normal cellular architecture with distinct hepatic cells sinusoidal spaces (X-40). e Silymarin (positive drug) treated group exhibited almost normal histology (X-40); Hepatocytes (HC), Central vein (CV) and Cellular inflammation (IN)

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Discussion

Phenolic compounds acting as antioxidants present in the plant products are gaining attention among different people due to their health benefits. In this study, antioxidant assays like ABTS, DPPH and reducing power assays have been carried to explore the antioxidant activity of AIS. These assays are the mostly used common assays by researchers to evaluate the antioxidant capacity of samples from plants. In the present study, scavenging activity of AIS on DPPH and ABTS radicals were observed in a concentration-dependent pattern. The antioxidant ability of the secondary metabolites of plants is also related to its reducing capacity [24]. As already discussed above, the reducing capacity of a substance is based on the reduction of the complex compound ferric tripyridyl triazine (Fe3+-TPTZ) to ferrous tripyridyl triazine (Fe2+-TPTZ), which results in the formation of coloured solution. Reducing power efficacy of the AIS increases in a concentration dependant manner. Due to the chemical complexity of plant extract, the isolation and quantification of polyphenolic compounds are very difficult. HPLC is an important analytical method in identification of bioactive compounds from plant sources. In this study we identified important bioactive compounds using HPLC. These phytochemicals have effective antioxidant potential to treat various human diseases [25]. Antioxidant properties of bioactive compounds (phenolics and flavonoids) are responsible for the hepatoprotection by plant extracts. These compounds effectively reduce the oxidative stress and other inflammatory responses [26, 27].

Protective effect of AIS was further evaluated in CCl4- induced oxidative stress in the livers of Wistar rats by observing the histological and biochemical changes in rats. The liver injury caused by CCl4 is mainly by the release of free radicals, which are generated during the metabolism of CCL4 by the cytochrome P450 (CYP) system. The content of CYP in liver is more abundant than that in other organs such as lung, kidney, and intestine. By the activation of liver CYP, by CCl4 intoxication produces hepatotoxic metabolite trichloromethyl free radicals (•CCl3 or CCl3OO•), which immediately propagate a chain of lipid peroxidation events and ultimately leads to the breakdown of membrane structure and result in the leakage of hepatic cell marker enzymes into the blood. In our study, significant increase (P ≤ 0.05) in the levels of hepatic enzymes such as SGOT, SGPT, ALP and bilirubin were observed in serum sample of experimental animals after administration of CCl4. The increased levels of liver enzymes in blood of rats administrated with CCL4were reduced by the treatment with AIS. The degree of liver cell membrane damage is known clinically by the increased activity of SGOT, SGPT, and ALP enzyme in serum. The hepatic TBARS level is also commonly used as an indicator of liver tissue damage which involves a series of chain reactions [28]. In our results, rats treated with CCl4 showed an increase in TBARS which was restored towards normal by the treatment with AIS.

Antioxidant enzyme such as SOD converts superoxide anion into H2O2 and O2, whereas CAT reduces H2O2 to H2O, resulting in the quenching of free radicals. SOD and CAT plays an important role in the elimination of ROS derived from the redox process in liver tissues and considered to be most important enzymes [29]. Intoxication with CCl4 to Wistar rats reduced the antioxidant capacity of rat liver by inhibiting the enzymatic activity of SOD and CAT. However, treatment with AIS improved the levels of these enzymes significantly in a dose dependent manner (250 and 500 mg/kg b.w). The activity of AIS was directly proportional to the increased concentration, suggesting that it could offer protection against hepatic damage caused by toxic substances. CCl4 administration reduces the level of GSH in liver tissue of rats. Administration of AIS as a drug to treat hepatic damage significantly increased the GSH content in liver tissue (P ≤ 0.05). Similarly hydroalcoholic extract of I. staphylina leaves significantly decreased the serum urea, creatinine, uric acid and protein levels in treated groups, which strongly supports our data [30]. In support with the results of biochemical parameters, treatment with AIS reduced the histological alteration induced by CCl4.

Conclusion

Our study demonstrated that the AIS leaves showed favorable potency to protect the liver damage induced by CCl4, which suggested that I. staphylina can be used as a potential hepatoprotective drug. The hepatoprotecive activity of AIS against severe damage caused by CCL4 intoxication was clearly proved by the results obtained. This is mainly achieved by its ability to neutralize the free radicals. Simultaneously inflammatory responses were suppressed and improved activity of drug-metabolizing enzyme is noted well from the results obtained. HPLC results revealed that I. staphylina extract have potential bioactive secondary metabolites. Future studies are in progress to better understand the mechanism of action of bioactive compounds in AIS responsible for its hepatoprotective and antioxidant effects.