Determination of phytoconstituents of n-hexane extract of leaves of Morus nigra and evaluation of their effects on biochemical and histopathological parameters in paracetamol intoxicated mice liver

*Correspondence: T. H. Mallhi. Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Government College University Faisalabad, Faisalabad, Pakistan. E-mail: tauqeer.hussain.mallhi@hotmail.com) Determination of phytoconstituents of n-hexane extract of leaves of Morus nigra and evaluation of their effects on biochemical and histopathological parameters in paracetamol intoxicated mice liver


INTRODUCTION
Conventional drug therapy for liver ailments poses less efficacy and life-threatening adverse events.On the other hand, the use of traditional or herbal remedies portends less adverse outcomes with significant benefits (Bachar, Mahmud, Qais, 2012).Therefore, it is imperative to screen complementary and alternative medicine (CAM), particularly herbal drugs, for the treatment of liver diseases in order to get more efficacious and safer agents (Mallhi et al., 2014a;2014c).The beneficial effects of plants in several diseases could be attributed to the presence of phytochemicals (biologically active and non-essential chemical compounds).The phytochemicals modulate physiological functions and biotransformation reactions involved in the detoxification process, thus providing protection against cytotoxic, genotoxic, and metabolic actions of environmental toxicants (Saha, Das, 2003).

Collection of plant
The leaves of Morus nigra were collected in April-May 2012 from Allied Hospital Faisalabad, University of Agriculture University Faisalabad, local Nursery farms in Faisalabad, Pakistan.The plant was identified by Dr. Mansoor Hameed, Associate Professor at Department of Botany, University of Agriculture Faisalabad, Pakistan and kept in department herbarium for reference (AUF-PI/2012/MN3).

Preparation of plant extract
Leaves of the plant were washed and dried, and were finally grounded to the powder form (3.2 kg) by using commercial grinder.The powder was soaked in n-hexane (8 L) for 7-10 days with occasional shaking.Muslin cloth was served to filter the solution and marc was pressed to get all filtrate.Rotary evaporator (70 °C) was used to evaporate filtrate that resulted in a jelly like paste (dark brown color).The paste was stored in amber colored glass bottle at room temperature.

Phytochemical screening
The preliminary phytochemical screening was performed by using methods as described by Farhan et al. (2012) (Table I).

Determination of phytoconstituents by HPLC analysis
Active constituents of the n-hexan extract of Morus nigra leaves were qualitatively determined by using isocratic HPLC.A small amount of extract was added in distilled water (5 mL).After adding methanol (12 mL), the mixture was kept for 5 minutes.Subsequently, 6 mL distilled water was added to the mixture and kept for more 5 minutes.After adding 10 mL of 15 M HCl, the mixture was placed in an oven for 2 hours.Syringe filter

Saponins
Extract in test tube was shacked for 5 min using a vortex Appearance of big foamy Alkaloids 0.2 g powder of the plant was dissolved in 10 mL of HCl 1% and they were transferred to a water bath for few minutes. 1 mL of the filtrated extract was treated with 2-4 drops of Dragendorff's reagent

Phenols
In beakers, 5 mL of each extract was taken and 1 mL of FeCl 3 1% and 1 mL of K 3 (Fe(CN) 6 ) 1% were added Fresh radish blue color Terpenoids 1 mL of acetic anhydride and 2 mL of concentrated sulphuric acid were added to beakers containing 1 mL of extract

Reddish brown on the interface
Volatile Oils l0 mL of extract was filtered by filter paper till saturation and then exposed for UV light Appearance of a bright pinkish color

Flavonoids
Two solutions A and B from plant extract were prepared.The solution A contains 5 mL of ethanolic extract.The solution B consists of 5 mL of ethanolic solvent added to 5 mL of KOH 50%.Then the two solutions A and B were mixed together.

Appearance of yellow color
was used for filtration of final solution.Phenolics were separated using a shim-pack CLC-ODS (C-18) column, 25 cm × 4.6 mm, 5 µm.The mobile phase used was an isocratic (ACN:Dichloromethane:Methanol -60:20:20) that was run at a flow rate of 1 mL/min.Samples were analyzed using UV-Visible detector at 280 nm at room temperature while kaempferol was detected at 248 nm wavelength.Methanol solution was used to wash the HPLC system for about one hour before use.Retention times (RT) were compared with standards in order to identify flavonoid contents in the extract (Sultana et al., 2008).

Animals
Twenty-five Swiss albino mice of the both sex, weighing about 22-35 g were kept in the departmental animal house.The mice were housed in standard laboratory conditions at a temperature of 25 ± 2 °C, relative humidity (50-55%) and a 12 h light/dark cycle (five mice per cage).The acclimatization period was of ten days (Iwalokun et al., 2006).All the animals fed standardized nutritionally balanced diet and drinking water ad libitum.All the experiments were carried out in accordance with the European Community guidelines (EEC Directive of 1986; 86/609/EEC).The plan of the study was specifically approved by the institutional animal research committee (Reference: ARC/GCUF/2013/11M).

Experimental protocol
All the animals were divided into five groups with five mice per group.

1.
Group I served as normal control receiving distilled water only, for seven days 2.
Group III (silymarin control) had daily administration of silymarin 50 mg/kg (reference drug) for 7 days with subsequent administration of paracetamol 3 h after silymarin 4.
Group IV received n-hexane extract of Morus nigra (NHMN) at doses 250 mg/kg p.o. for 7 days and received paracetamol 250 mg/kg 3 h after the extract dose.
At the end of experiment, mice were fasted for 12 h and anesthetized with light chloroform.Animals were sacrificed through cervical decapitation on the 8th day (at the same time of last dose).Eppendorf tubes were used to collect blood samples.Serum was separated by centrifugation (7000 rpm for 15 min at 4 °C).A substantial elevation of liver transaminases and total bilirubin (TBR) was considered as hepatotoxicity throughout the experiment (Mallhi et al., 2014b).

Biochemical Investigation
Serum level of total bilirubin was determined by using the technique as described by Ehrlich (1883).Reitman and Frankel`s methods were used to determine the concentration of ALT and AST in serum (Reitman, Frankel, 1957) while values of ALP were determined by using Kind and King`s method (1954).

Statistical Analysis
Results were presented as Mean ± standard error (SE).All the continuous data were subjected to Student t-test by using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA).P values were calculated by comparing group III (silymarin), group IV (NHMN 250 mg/kg) and group V (NHMN 500 mg/kg) with group III (PCM) (Table III).Treatment groups (NHMN) were also compared with silymarin group and respective p values are mentioned in text where comparison was stated.P values at ≤0.05 were considered as statistically significant.

RESULTS
Phytochemical screening of n-hexane extract of Morus nigra leaves revealed the presence of phenols, flavonoids, alkaloids and terpenoids.Flavonoids were moderately present, while small fractions of phenols, alkaloids and terpenoids were also found as shown in Table II.
The HPLC chromatogram was compared with the standard retention times (RT) that showed the presence of qurecetin (2.577 min), oleanolic acid (5.213 min), luteolin (8.431 min), apigenin (13.843 min), vitamin C (24.131 min) and kuwanon C (29.453 min) in the extract (Figure 1).HPLC analysis at the wavelength of 248 nm for the detection of kaempferol showed no peak, demonstrating the absence of kaempferol in the extract.Chemical structures of identified compounds are shown in Figure 2.

Serum ALT activity
The mean value of ALT in paracetamol intoxicated mice was significantly (p<0.001) higher when compared to normal control receiving distilled water (112.1 ± 4.18 IU/L vs 32.80 ± 2.08 IU/L respectively).Pretreatment with 250 and 500 mg/kg n-hexane extract of Morus nigra decreased the serum ALT concentration significantly and percentages of reduction were 54.3% and 62.9%, respectively.The reduction in serum ALT with NHMN was comparable to reduction caused by silymarin (p = 0.432).Mean values of ALT in NHMN (250 mg/kg) and NHMN (500 mg/ kg) were significantly differ (p<0.001) from normal and paracetamol controls (Table III).

Serum AST activity
The mean value of AST was 36.5 ± 3.61 IU/L in normal control that was significantly (p<0.001)lower than in the paracetamol control group (101.2 ± 9.17 IU/L).Treatment with NHMN prior to the paracetamol administration reduced mean levels of AST to 45.40 ± 7.13 IU/L and 43.8 ± 6.61 IU/L with doses of 250 and 500 mg/kg of NHMN respectively.These reduced values are comparable to that of silymarin control (39.80 ± 7.80 IU/L, P=0.189).Percentage reduction in mean levels of AST was 55.1% and 56.7% with 250 and 500 mg/kg of extract dose, respectively.A significant difference (p<0.01) was observed in mean values of serum AST in NHMN groups when compared with paracetamol control (Table III).

Serum ALP activity
Similarly mean values of ALP were significantly higher (413.40 ± 21.76 IU/L) in the paracetamol intoxicated group as compared to normal control (p<0.01).The groups treated with NHMN 250 mg/kg and 500 mg/kg significantly (p<0.01)reduced mean serum ALP levels by 28.5% and 33.6% as compared to PCM group and this reduction was comparable with silymarin (p = 0.221) control shown in Table III.

Total bilirubin
Paracetamol intoxication caused significant (p<0.05)elevation in total bilirubin concentration (2.01 ± 0.22 g/dL) as compared to normal control (0.89 ± 0.03 g/dL).While the administration of 250 mg/kg and 500 mg/kg NHMN subjected to significant reduction (p<0.01) in TBR count by 56.4% and 54.5% respectively when compared to paracetamol control.The reduction caused by NHMN was comparable (p = 0.512) to reduction caused by silymarin (55.1%) (Table III).

Histopathology
The biochemical investigations were confirmed by histopathological examinations as shown in Figure 3.The group I receiving distilled water showed normal liver architecture and healthy hepatocytes (histology score = 0 for all domains).Paracetamol administration caused inflammation (score = 3), microvesicular steatosis (score = 1), necrosis, ballooning (score = 3) and sinusoidal constriction of hepatocytes while treatment with silymarin and NHMN provided improvement in inflammation and necrosis accompanied by sinusoidal dilatation.Examination of mice liver treated with silymarin showed to great extent improved sections represented by healthy hepatocytes and nucleated cells.Histology scoring system showed mild steatosis (score = 1), negative to microvesicular steatosis, mild inflammation (score = 1)   resulting in the reduced levels of GSH in blood and liver.Depletion of GSH can result in mitochondrial dysfunction, an increase of lipid peroxidation, and development of acute hepatic necrosis.Hepatocellular necrosis releases enzymes such as AST and ALT into the circulation that can be measured in the serum as a sign of liver injury (Yahya et al., 2013).
Morus nigra is a popular and widely distributed species of the Moraceae family that has been included in Modern Chinese Materia Medica due to its long history of medicinal use (Xiao et al., 2002).Traditionally, Morus nigra has been used as a medicinal agent to nourish the yin and blood, benefit the kidneys, and treat weakness, fatigue, anemia, and premature graying of hairs.It is also utilized to treat urinary incontinence, tinnitus, dizziness, and constipation in the elderly and the anemic (Xiao et al., 2002).In current study, hepatoprotective activity of leaves of Morus nigra was evaluated by using mice as experimental model.Morus nigra n-Hexane extract with 250 mg/kg reduced elevated ALT by 54.3% (p<0.001),AST by 55.1% (p<0.01),ALP by 28.5% (p<0.01) and TBR by 56.4% (p<0.01) as compared to paracetamol control.The dose of 500 mg/kg reduced mean values of ALT by 62.9% (p<0.001),AST by 56.7% (p<0.01),ALP by 33.6% (p<0.01) and TBR by 54.5% (p<0.01) as compared to paracetamol control in present study.The reduction of mean values of liver enzymes and total bilirubin was comparable (p>0.05) with silymarin in the current study.A high dose (500 mg/kg) of NHMN demonstrated a better reduction of liver enzymes and total bilirubin as compared to low dose (250 mg/kg) but the difference was statistically insignificant (p>0.05).Similarly, histopathological improvements were more profound with 500 mg/kg and these improvements were also comparable to silymarin.Improvement in histology score was also observed with extract doses and was comparable to silymarin.
The hepatoprotective activity of Morus nigra could be attributed to the presence of chemical compounds that are biologically active.Phytochemical screening demonstrated that plant contains hepatoprotective phenols, flavonoids, alkaloids and terpenoids (Fernando, Soysa, 2014).Qualitative analysis by isocratic flow HPLC showed the presence of six phytoconstituents in n-hexane extract of leaves of Morus nigra and these findings are consistent with previous investigations (Wang, Wang, Chen, 2007).Quercetin (Janbaz, Saeed, Gilani, 2004), oleanolic acid (Chen et al., 2005), luteolin (Domitrović et al., 2009), apigenin (Zheng et al., 2005;Mallhi et al., 2014c) and vitamin C (Uboh et al., 2012); all are well known to possess liver protective potential and desired pharmacological activity in our study might be attributed to the presence of these compounds in Morus nigra n-hexane extract.Kuwanon C in our extract is also present in closely related species, Morus alba (Family: Moraceae) and has been found to possess antimicrobial activity against several bacteria and molds (Butt et al., 2008).
The possible mechanism of action by which n-hexane extract of Morus nigra provided hepato-protection might be related to the free radical scavenger and antioxidant activities of the identified compounds (Saeed, Khan, Shabbir, 2012).A number of reactive oxygen species (ROS) like singlet oxygen (1O2), superoxide anion radical (O2 −• ), hydroxyl radical (OH • ), nitric oxide radical (NO • ), and alkyl peroxyl (ROO • ) are produced regularly in the human body as a result of oxidation processes.Protective mechanisms exist to mediate their harmful effects but sometimes ROS overrides the defense capabilities of the body resulting in oxidative damage to molecules and membranes, as happened in PCM group.In such cases, there is a need to strengthen this mechanism by antioxidant supplementation (Butt et al., 2012).Morus nigra is important in respect to antioxidant compounds and quercetin is most important for antioxidant potential of mulberry plant.It has been previously reported that leaves of mulberry contain higher amount of quercetin and support for possible application of mulberry leaves extract or functional components to reduce in vivo and in vitro oxidation process (Chen, Li, 2007;Enkhmaa et al., 2005;Katsube et al., 2006).In addition, fruits of Morus alba have also been reported to possess antioxidant activity (Ercisli, Orhan, 2007) that appeals the need to investigate various parts of Morus nigra for its antioxidant and hepatoprotective potential.In present study, qualitative determination of active compounds of extract was carried out by using HPLC analysis.Comparison of chromatogram with retention times showed presence of some potential antioxidant compounds including quercetin, oleanolic acid, luteolin and apigenin.Quercetin by multiple mechanisms interplay, demonstrates hepatoprotective effect on liver injury, by increasing antioxidant system activities against oxidative stress and lowering the expressions of proinflammatory cytokines (Chen, 2010).Oleanolic acid (OLA) causes enhancement of glutathione regeneration capacity and reduction of lipid peroxidation.OLA has also been documented to possess dose dependent hepatoprotection (Bai et al., 2007) and it might contribute to liver protective activity in present study.Extract of Morus nigra also showed presence of luteolin, a flavone type flavonoid.In presented study, PCM administration causes GSH depletion with an association of increased lipid peroxidation, which led to tissue injury and liver damage.Presence of luteolin in plant extract results in reversal of these changes and elevation of GSH levels.Furthermore, luteolin also inhibits lipid peroxidation, emphasizing its antioxidant effect (Bigoniya, 2013).Hepatoprotection of NHMN might be attributed to these mechanisms of luteolin.Furthermore, due to apigenin-related anti-inflammatory and antioxidant properties, NHMN could counteract the pro-oxidant effect of PCM or NAQPI through scavenging superoxide radicals, consequently declining hepatic glutathione-Stransferase (GST) and decreasing release of γ-glutamyl transpeptidase in serum (Asadi-Samani et al., 2015); as a result, NHMN could be assumed as a potent plant against experimentally induced hepatocarcinogenesis in mice.All these mechanisms might contribute to the hepatoprotective activity of n-hexane extract of Morus nigra in present study and phytoconstituents with antioxidant activities enhance the chances of Morus nigra extract supplementation, especially in liver ailments.However, future studies are required to investigate the pharmacological activities of these compounds

CONCLUSIONS
The results obtained from current study indicate the presence of antioxidant compounds, i.e. quercetin, oleanolic acid, luteolin, apigenin and ascorbic acid and their potential hepatoprotective activity against paracetamol induced liver injury in mice.

FIGURE 2 -
FIGURE 2 -Chemical structure of identified compounds in n-hexane extract of leaves of Morus nigra.

TABLE I -
Protocols for phytochemical screening of n-hexan extract of the leaves of Morus nigra Morus nigra

TABLE II -
Phytochemical screening of n-hexan extract of the leaves of Morus nigra

Active Compounds n-Hexan extract of the leaves of Morus nigra
(+++) high amount after added of reagent immediately; (++) moderate amount after 5 min of reagent added; (+) low amount after 10 min of reagent added and (-) absent of active compound after 20 min FIGURE 1 -HPLC chromatogram of n-hexane extract of Morus nigra showing presence of qurecetin, oleanolic acid, luteolin, apigenin, vitamin C and kuwanon C at 280 nm wavelength.

TABLE III -
Effect of n-hexane extract of Morus nigra (NHMN) on liver enzymes and total bilirubin Results are presented as Mean ± SE and compared with Student t test.p values are served as statistically significant difference when compared to PCM control.*p < 0.01, **p<0.001