Leaves of Schinus polygamous ( Cav . ) Cabrera ( Anacardiaceae ) are a potential source of hepatoprotective and antioxidant phytochemicals

Phytochemical investigation of the crude ethanol extract of the leaves of Schinus polygamus (Cav.) Cabrera (Anacardiaceae) results in isolation of eight compounds identified as triterpenoids, 3-Oacetyllupeol (I), βeta-sitosterol (II), lupeol (III). In addition to the polyphenols, gallic acid (IV), methyl gallate (V), quercetin-3-α-O-rhamnoside (VI), kaempferol (VII) and quercetin (VIII). In Egypt, degenerative diseases in general and toxic hepatitis in particular remain a serious public health problem. The hepatoprotective, curative and anti-oxidant properties of the major phytochemicals, compounds III and IV isolated from leaves of S. polygamus were investigated. Liver damage was induced by CCl4 (1 mlkg -

antioxidant defense system of the body (Hopps et al., 2010;Santiago and Mayor, 2014).However, at certain conditions, oxidative stress is triggered due to the imbalance between the production of ROS and the antioxidant systems of the body (Pepe et al., 2009).High risk health conditions such as neurodegenerative diseases, hepatitis, diabetes, cancer and inflammation are usually related to oxidative stress (Fearon and Faux, 2009;Fang et al., 2002).Based on growing interest in free radical biology and lack of effective therapies for most chronic diseases, the usefulness of antioxidants in protection against these diseases is warranted (Huang et al., 2010).In this regard, phytochemicals from various natural products had become the subject of most drug development researches (Santiago and Mayor, 2014).Plants are the most commonly known sources of natural antioxidants which includes polyphenolic and terpenoids compounds (Grassmann, 2005;Dimitrios, 2006).
Family Anacardiaceae (Bailey, 1953) has long reputation in folk medicine for its nutritional value of edible fruits and seeds, and for variable ailments such as treatment of bowel complain, chronic wounds, jaundice, hepatitis and relieving inflammatory conditions (Abbasia et al., 2010).There is an extensive body of literature addressing the escalated distribution of hepatic diseases among the people in Egypt (Shabana et al., 2011;Lehman, 2008;Strickland et al., 2002).Hepatoprotective herbal drugs can offer help by blocking absorption of toxins into liver cells and the formation of inflammatory substances that contribute to liver degeneration (Shabana etal., 2011).
As part of on-going study of medicinal plants belongs to family Anacardiaceae, the closely related genera, Harpephyllum caffrum Bernh, Rhus coriaria L and Schinus polygamus Cav. were subjected to biological testing in order to confirm the claimed herbal benefits of these drugs by different communities (Van Wyk et al., 2009;Rayne and Mazza, 2007;Shabana et al., 2008Shabana et al., , 2011Shabana et al., , 2013)).
Schinus polygamus (Cav.)Cabrera, family Anacardiaceae is a tree cultivated for ornamental purpose.Traditionally the leaves have been used for wounds cleansing, while the bark produces a balsamic resin, is used to treat arthritic and chronic bronchitis (Munoz et al., 1981).The presence of well-known flavonoids was reported in S. polygamus, namely, kaempferol, quercetin and quercetin-3-O-galactoside (Mandich et al., 1984).The n-hexane extract of S. polygamus were reported to contain β-sitosterol to which analgesic, anti-inflammatory and antipyretic activities of the Argentine and the Chilean species were attributed (Erazo et al., 2006;Gonzalez et al., 2004).The essential oil obtained from S. polygamus leaves exhibited antimicrobial activities (Gonzalez et al., 2004).
Nowadays, the current research on plant based medications focus on isolation of biologically active substances from potent plants (Kingston, 2011).
However, nothing could be traced on phytochemicals of Egyptian S. polygamus and its biological potentiality.
Considering the health benefits one can derive from S. polygamus (Mandich et al., 1984;Munoz et al., 1981), its phytochemical, biochemical and pharmacological studies were scarce.Moreover, chemical profiling of the antioxidative components present in the plant is not yet been fully elucidated.Therefore this study was undertaken to back up its ethnobotanical uses while intensively evaluating the antioxidative components of S. polygamus as well as assessing its potential role as a powerful antioxidant and hepatoprotective

Plant
The leaves of S. polygamus (Cav.)Cabrera were obtained from trees growing in El Orman botanical garden, Giza in April 2013.The plant was kindly authenticated by Dr. Mohamed El-Gebaly, Botany Specialist.Voucher specimen 1842013 is kept in the herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.

Preparation of the extracts
Two kilograms of air-dried powdered leaves of S. polygamus were extracted by cold percolation with 90% ethanol (5 × 1 L).The ethanol extract was evaporated under reduced pressure to give 350 g green semi-solid residue.250 g of the crude extract were successively partitioned between n-hexane, chloroform, ethyl acetate and n-butanol saturated with water.The solvent in each case was completely evaporated under reduced pressure to yield 30, 10, 150 and 3 g, respectively.

Apparatus for phytochemical study
Ultraviolet lamp (λmax=254 and 330 nm), Shimadzu, a product of Hanovia lamps for localization of spots on chromatograms.EI-MS were recorded with a Varian Mat 711, Finnigan mass SSQ 7000 Mass spectrometer, 70 eV.IR spectra were observed as KBr discs on Shimadzu IR-435, PU-9712 infrared spectrophotometer. 1 HNMR (300 MHz) and 13 CNMR (75 MHz) spectra were recorded on Jeol EX-300MHz and Bruker AC-300 spectrometer operating at 300 ( 1 H) and 75 ( 13 C) MHz in CDOD3 and CDCl3 as a solvent and chemical shifts were given in δ (ppm).

Phytochemical investigation of the n-hexane soluble fraction
Thirty grams of the n-hexane soluble fraction were chromatographed on a VLC column, 210 g silica gel (12.5 × 7 cm) using n-hexane, n-hexane-chloroform and chloroform-ethyl acetate mixtures as elutes.Fractions, 200 ml each, were collected and monitored by TLC.Similar fractions were pooled together to obtain three major fractions (A-C).
Fraction A (4.0 g), eluted with n-hexane, was rechromatographed on silica gel sub-column using n-hexane and1% chloroform in nhexane as solvent systems to obtain compound I (0.02 g).
Fraction B (2.5 g), eluted with 15% chloroform in n-hexane, was purified on silica gel column using n-hexane-chloroform mixtures (10% chloroform in n-hexane) as an eluent to obtain compound II (0.03 g).

Phytochemical investigation of the ethyl acetate soluble fraction
Ethyl acetate soluble fraction (150 g) was purified on column chromatography (300 g polyamide, 50 × 3 cm) using 100% water and water-methanol mixtures in order of decreasing polarity.Fractions, 100 ml each, were collected and monitored by TLC.Similar fractions were pooled together to obtain two major fractions (a,b).
Fraction a (2 g), eluted with 15 and 25% methanol in water revealed the presence of three major spots.Further rechromatography using sephadex LH 20 and water-methanol in decreasing polarity as eluent led to the isolation of two phenolic compounds IV and V (0.5 and 0.08 g), respectively.
Fraction b (5.37 g), eluted with 30 to 50% methanol in water, was further subjected to rechromatography on RP silica column using water-methanol as elute and resulted in separation of three compounds VI (0.03 g), VII (0.05 g) and VIII (0.03 g).

Material for biological study
The tested solutions were prepared by dissolving them in distilled water containing few drops of Tween 80 to yield a concentration of 5% w/v.

Animals
Adult Sprague-Dawely rats weighing 160 ± 10 g were provided from the animal-breeding unit of National Research Center, El-Dokki, Giza, Egypt.They were kept under standard conditions with temperature at 23 ± 2°C and a 12/12 h light/dark cycle and allowed free access to food and water throughout the experiment.This study was conducted in accordance with the standard guidelines used in handling of the experimental animals and approved by the Institutional Animal Care and Use Committee (IACUC) (No. 9-031),

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College of Pharmacy, Cairo University.

Experimental design
For testing the hepatoprotectiveeffect on the liver, 36 adult male albino rats were used in the experiment and divided into six groups (each of 6).Group served as control, received saline (1 ml kg -1 /day) and received CCl4 1 mlkg -1 (1:1 CCl4 in liquid paraffin orally) once daily for 7 days.Group received CCl4 1 mlkg -1 and silymarin 25 mgkg -1 orally for 7 days as a reference drug.Four groups were administered the tested compounds at a daily dose of 50 and 100 mgkg-1 b.wt.(Paget and Barnes, 1964).The compounds, as well as, the reference drug, were administered to the rats for another seven days after liver damage.
For testing the antioxidant effect on liver, five groups of animals (6 animals each) were divided.One group was kept as a control, while for the other groups, group with liver injury was kept nontreated, another group received daily the reference drug (Vitamin E) and the other groups received the tested isolates of S. polygamus daily in the given doses.Blood samples were taken after 7 days.

Measurement of AST, ALT and ALP serum levels
The animals were scarified and blood sample was collected from the retro-orbital sinus plexus under mild ether anesthesia.Collected blood was allowed to clot and serum was separated at 3500 rpm for 15 min for carrying out biochemical investigation serum levels of aspartate aminotransferase (AST) (Thewfeld, 1974), alanine aminotransferase (ALT) (Thewfeld, 1974), and alkaline phosphatase (ALP) (Kind and King, 1954;Klassan and Plaa, 1969) enzymes were measured in each group at zero time, after 7 days of receiving the tested drug, 72 h after induction of liver damage and after 7 days of treatment with the tested samples.

Estimation of antioxidant activity
The antioxidant activity was calculated by the determination of glutathione in blood of CCl4-induced liver injury rats adopting the methods of (Beutler, 1975;Beutler et al., 1963) using vitamin E as a reference drug.Blood samples were taken after 7 days for the determination of glutathione.

Statistical analysis
All data were expressed as mean ± standard error (SE).The statistical comparison of difference between the control group and the treated groups were carried out using two-way analysis of variance (ANOVA) followed by Duncan's multiple range test.

Phytochemical investigation
Column chromatographic fractionation of the n-hexane soluble fraction of the crude ethanol extract of leaves of S. polygamus led to the isolation of three compounds (I to III), whereas, the ethyl acetate soluble fraction afforded five compounds (IV to VIII) which were characterized through their physicochemical and spectral data.

Compound I
Lupeol acetate (0.04 g, crystallize from methanol as white needle crystals, m.p. (218°C), was suggested to be a triterpenoid since it responded positively to libermann-Burchard's test.The presence of m/z 249 ion indicates the presence of CH 3 CO at C-3 position.Skeletal mass peaks are represented by m/z 218 and 203 and the base peak at 189 all in accordance with lupene skeleton of triterpenoid.The 1 HNMR spectrum (300 MHz in CDCl 3 ) exhibited four tertiary methyl signals of the lupene skeleton at 0.80, 0.86, 0.95, and 1.05 as singlets.The C-30 vinylic methyl at δ1.75 ppm (s) acetyl methyl resonate at δ1.97 ppm (s), where α-proton at C-3 appear at δ4.22 ppm dd (J=3, 6 Hz).The C-29 olefinic protons H a , H b appeared at δ5.71 and 5.60 ppm as broad singlets.
13 CNMR (75 MHz, in CDCl 3 chemical shift assignments for compound I is revealed the presence of oxycarbon signal at δ83.0 assigned to (C-3) with downfield shift about (+4.0 ppm) compared with that of lupeol, which confirm the substitution with acetyl group (Chatterjee et al., 2000).Two signals assigned for olefinic carbons (C-20 and C-29) at δ=145.09 and 109.3 ppm, respectively and showed the presence of a carbonyl at δ172.8.All the aforementioned resonances are in accordance with compound I proposed structure and comparable to lupeol acetate (Jamal et al., 2008).As far as the available literature is concerned, this is the first report on isolation of lupeol acetate (C 32 H 52 O 2 ) from S. polygamus.

Compound II
Beta-sitosterol white powder (0.03 g); m.p.:134 to 135°C; MS (m/z): exhibited molecular ion 414 (M + ), and other fragmentation pattern as 396, 339, 325, 310, 298, 257, 140, 139, 125, 97, 71, and 57.Its mass spectral data suggested the molecular formula as C 29 H 50 O.Compound II also showed positive Liebermann-Burchard reaction indicated its sterol nature.The 1 HNMR spectra (CDCl 3, 300 MHz) showed the presence of six methyl signals that appeared as two methyl singlet at δ0.68 and 1.01; three methyl doublets that appeared as δ0.81, 0.83, and 0.93; and a methyl triplet at δ0.84.Olefinic proton was presented at δ5.37 suggested the presence of tri substituted double bond at C-5/C-6 in its structure.The 1 HNMR spectra showed a proton corresponding to the proton connected to the C-3 hydroxy group which appeared as a triplet of doublet of doublets at δ3.53.The 13 CNMR showed twenty nine carbon signal including six methyls, eleven methylenes,ten methane together with three quaternary carbons.Thus, the structure of II was assigned as β-sitosterol that was consistent to the reported literature values (Jamal et al., 2008).

Compound III
Lupeol (0.25 g, crystallize from methanol as white needle crystals, m.

In vivo hepatoprotective activity
The control group (received saline) served as a baseline for all the biochemical parameters.A significant increase in the activity of the serum enzymes AST, ALT and ALP (P<0.05) was observed upon treatment with CCl 4 .Positive control (Silymarin group) showed highly significant decrease (P<0.05) in AST, ALT and ALP as compared to negative control group.The treatment of intoxicated rats with compounds III and IV at a dose of (50 and 100 mg kg -1 ) produced a significant hepatoprotective effects.Hepatic protection was evidenced by the ability of gallic acid to normalize the  high enzyme parameters in a dose-dependent manner (50 and 100 mgkg -1 ) by 47.9 and 102.9% for AST comparable to silymarin.Gallic acid (100 mgkg -1 ) exhibited a highly significant reduction (p<0.05) in AST, ALT and ALP better than lupeol.Results presented in Table 1 showed that low dose, 50 mgkg -1 of lupeol exhibited normalization by 93%, for AST comparable to silymarin.

In vivo antioxidant activity
Concerning the antioxidant activity, the phytochemicals of the leaves of S. polygamus exhibited significance antioxidant activity.Results (Table 2) revealed that lupeol and gallic acid in dose 100 mgkg -1 exerted a remarkable antioxidant activity (91.4 and 96.4%), respectively.The reduced level of glutathione in intoxicated rats was greatly restored by lupeol and gallicacid relative to vitamin E (potency 100%).These results clearly elucidated the significant in vivo antioxidant activity provided by both lupeol and gallic acid, so they could be considered as powerful antioxidant agents.Gallic acid showed highly significant rise (p<0.05) in glutathione better than lupeol.

DISCUSSION
Free radicals and reactive oxygen species (ROS) play a role in liver diseases pathology and progression, dietary antioxidants have been proposed as therapeutic agents to counteract liver damage (Higuchi and Gores, 2003).Natural antioxidant may act as protectors but, more importantly, may exert modulator effects in cells through actions in antioxidant, drug metabolizing and repairing enzymes as well as working as signaling molecules in important cascades for cell survival (Singh et al., 2008).
Terpenoids are useful phytochemicals isolated from plants (Firn, 2010).They can regulate the activity of nuclear hormone receptors, which are involved in maintain the energy homeostasis in the body and manage obesity induced metabolic disorders including type 2 diabetes, hyperlipidemia, insulin resistance and cardiovascular disease (Goto et al., 2010).
In the present study, the in vivo antioxidant and hepatoprotective activities of major isolated compounds (lupeol, gallic acid) from the leaves of S. polygamus on experimental rats were evaluated.The hepatoprotection showed that the tendency of these molecules to preserve liver enzymes was particularly pronounced with gallic acid.The results of the present study are in accordance with the previously reported reviews, which demonstrated that lupeol ameliorates aflatoxin B1-induced peroxidative hepatic damage in rats (Preetha et al., 2006).
Hepatoprotective effect of lupeol on tissue defense system in cadmium-induced hepatotoxicity in rats was known (Sunitha et al., 2001).In relation to that, it was observed that lupeol can substantially normalized degenerative alterations in the hepatocytes with granular cytoplasm (Akçam et al., 2013).Additionally, lupeol induced growth inhibition and apoptosis in hepatocellular carcinoma SMMC7721 cells by down-regulation of the death receptor expression (Zhang et al., 2009).Moreover, have some effect on lipid peroxidation and antioxidant status in rat kidney after chronic cadmium exposure (Nagaraj et al., 2000).
Henceforth, Lupeol has several biological activities, anti-inflammatory, and anti-tumor activities wherein according to some review (Wal et al., 2011;Gallo and Sarachine, 2009;Ragasa et al., 2009), was previously identified in family Anacardiaceae (Shabana et al., 2011).Lupeol and its ester derivative, lupeollinoleate were investigated for their possible hepatoprotective effect against cadmium-induced toxicity in rats (Sunitha et al., 2001).The mechanism of cadmium-mediated acute hepatotoxicity involves two pathways, one for the initial injury produced by direct effects of cadmium and the other for the subsequent injury produced by inflammation.Primary injury appears to be caused by the binding of Cd 2+ to sulfhydryl groups on critical molecules in mitochondria.Thiol group inactivation causes oxidative stress, the mitochondrial permeability transition, and mitochondrial dysfunction (Rikans and Yamano, 2000).The fact that antioxidant agents inhibit carbon tetrachloride-induced liver damage (Wu et al., 2009) prompt to study the antioxidant effect of the isolated compounds.
Gallic acid with the lowest hydrophobicity than methylgallate and alpha-tocopherol was found to be the most active antiradical agent (Asnaashari et al., 2014).The low hydrophobicity of gallic acid can be treated by using gallic acid-lecithin complex (Liu et al., 2014).The hepatoprotective and antioxidant effects of gallic acid were evaluated against paracetamol-induced hepatotoxicity in mice (Rasool et al., 2010).Gallic acid showed significant protection against paracetamol induced liver injury in rats by improving the antioxidant defense condition, lessening lipid peroxidation and conserving the pathological changes of the liver.Hepatotoxicity, results not from paracetamol itself, but from one of its metabolites N-acetyl-Pbenzoquinoneimine (NAPQI), which depletes the liver's natural antioxidant glutathione and directly damages cells in the liver, leading to liver failure (Rasool et al., 2010).
Carbon tetrachloride is a well-known hepatotoxic agent and the preventive action of drugs on liver damage by CCl 4 has been widely used as an indicator of their liver protective activity.Changes associated with CCl 4 -induced liver damage are similar to that of acute viral hepatitis (Huang et al., 2010).Carbontertachloride treatment initiated lipid peroxidation, caused leakage of enzymes like alanine tranaminase and lactate dehydrogenase (Singh et al., 2008;Krithika et al., 2009) levels.CCl4induced lipid peroxidation as well as covalent binding of CCl 4 metabolites to cell components, and also restored lipoprotein metabolism (Boll et al., 2001).The antioxidant vitamin E (alpha-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited.The covalent binding of the CCl 3* radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl 3-OO*, initiates lipid peroxidation.The former process may result in adduct formation and, ultimately, cancer initiation, whereas the latter results in loss of calcium homeostasis and, ultimately, apoptosis and cell death (Boll et al., 2001).
In CCl 4 -induced liver injury model, oxidative stress could be provoked, which prompts lipid peroxidation that injure hepatocellular membrane, followed by substantial release of pro-inflammatory chemokines and cytokines, which in consequence of liver damage (Feng et al., 2011).A large amount of plants, especially medicinal plants, has been investigated to eliminate the hepatic damage stimulated by CCl 4 .For example, Coptidis rhizome, a traditional Chinese medicinal plant used to clear heat and scavenge toxins, belongs to liver meridian in Chinese medicinal practice (Feng et al., 2010(Feng et al., , 2012)).
Anti-oxidative therapy, mainly using natural antioxidants, represents a reasonable therapeutic approach for the prevention and/or treatment of liver diseases due to the role of oxidative stress in initiation and progression of hepatic damage (Li et al., 2015).However, although concept of anti-oxidative therapy has been improved nowadays, there is a long way to go for the application of antioxidants in liver disease.Therefore, translational research is of great importance for antioxidative therapy.Considering ROS and oxidative stress act positively in certain circumstances and the difference between animals and humans, the effective dose and safe dose, duration of treatment, absorption and bioavailability of antioxidants require thorough investigation.Furthermore, in the future, large-scale samples and appropriate duration of anti-oxidative treatment for liver diseases should be performed (Li et al., 2015).

Conclusion
The current study provides a scientific basis for the claims that S. polygamus is effective against certain liver-related diseases (Shabana et al., 2013).Taken together, our findings provide evidence that the major isolated compounds (lupeol and gallic acid) from S. polygamus exhibit hepatoprotective and antioxidant activities.These compounds might be useful for the prevention of toxic-induced and free radical-mediated liver diseases since it has been suggested that antioxidant compounds may be used as prophylactic agent.
Moreover, this study demonstrates that S. polygamus have potential hepatoprotective activity which is mainly attributed to the antioxidant potential, which might occur by reduction of lipid peroxidation and cellular damage.Previous reports were prevailed the isolation of lupeol and gallic acid from different genera belongs to Family Anacardiaceae (Shabana et al., 2008(Shabana et al., , 2011)).As a part of the ongoing investigation of plants belongs to family Anacardiaecae (Shabana et al., 2008(Shabana et al., , 2011(Shabana et al., , 2013)), it can be concluded that gallic acid and lupeol could be consider as potent hepatoprotective agents and chemotaxonomic markers for plants which belongs to family Anacardiaecae.

*
Statistically significant different from zero time group at p< 0.01.•Statistically significant different from 72h group at p< 0.01.a Pretreated with tested sample.b After induction of liver damage.*Phytochemicals, L=lupeol; G=gallic acid were tested at (100, 50 mg/kg b wt).**Silymarinat dose level (25 mg/kg b wt).C %change (change in liver enzymes after induction of liver disease from zero time).D %change (change in liver enzymes after 7 days from induction of liver disease).
It gives characteristic violet color with ρanisaldehyde/H 2 SO 4 spray reagent.The IR√ Max + -CH 3 COO] suggesting the presence of acetyl group in the structure.

Table 1 .
Effects of the of the isolated phytochemicals of S. polygamus *and silymarin** drug in male albino rats(n=6)on serum AST, ALT, and ALP levels.

Table 2 .
Antioxidant activity of the isolated phytochemicals of S. polygamus in male albino rats (n=6).Statistically significant different from control group at p < 0.01.b % of change from control= (Mc -Mt) × 100 / Mc; Mc is the mean change in control animals; Mt is the mean change in drug-treated animals; c % Potency calculated as regard the standard drug. *