Phytoconstituents and Hepatoprotective Effect of Suaeda monoica Forssk and Suaeda pruinosa Lange. (2024)

A mangrove and mangrove associate plants are proved to have rich ofhigh value secondary metabolites viz, saponins, alkaloids, polyphenolswhich possess antibacterial, antifungal, antiplasmodial andhepatoprotective activities (1).

Suaeda (Chenopodiaceae) is a genus of plants also known as seepweeds, includes annual and perennial herbs or rarely small trees. Thereare about 110 species in the Suaeda genus (S.) (2). They are distributedon coasts, deserts, lakeside and saline and alkaline land all over theworld.

A number of species have been found to be valuable feed forlivestock in arid area like Suaeda pruinosa Lange, S. vera and S.vermiculata (3), while others have been utilized to desalinize irrigatedfarmlands like Suaeda maritima (4). In Mexico, some species such asSuaeda pulvinata are cooked in traditional dishes known as romeritos.Suaeda species are commonly used in folk medicine (5-6). Some speciespossess hypoglycemic, anti-inflammatory, hypolipidemic, cardiotonic,antioxidant, antimicrobial and anticancer activity (7-8).

In Saudi Arabia it is represented by 9 species according to Boulos(9). Five species were found in Al Jouf area represented by;S.aegyptica, S. vera and S. vermiculata, S. fruiticosa and S. mollis(10).

Suaeda vera subspecies pruinosa (Lange), are facultative halophytesthat tolerate moderate salt soils, dry soils, nitrified and saline(halonitrofilos) in very sunny places arid climate during May to October(11). The specific epithet " pruinosa " refers to the presenceof pruina, which is kind of thin waxy coating on stems and leaves, whichoffers a glaucous appearance. In the area south of Alicante has beenused to make the " stone soda " and was said to be as good asthat made with fine barrilla (Halogetumsativus). The nutritive value offorage species (crude protein ranges) in Suaeda pruinosa Lange is27.17%.

Suaeda monoica Forssk. ex J. F. Gmel is a salt marsh mangrove herbgrowing in hypersaline soils. It is smaller in size, simple leaves whichare edible. Traditionally, the leaf from S. monoica is known to use as amedicine for hepatitis (12) and scientifically it is reported to be usedas ointment for wounds (13) and possess antiviral activity (7) becauseof the presence of triterpenoids and sterols (5).

Methanolic extracts of S. monoica, exhibits moderate sun protectiveactivity and a safe, effective and affordable sun screen formulation(14). Endophytic fungi were isolated from the leaves and stems of S.monoica, the isolation was according to the method described bySuryanarayanan et al (15). Phytochemical screening of methanolicextracts from S. monoica, showed the presence of flavonoids, tannins,phenols, polyphenols, steroids and Alkaloids (16).

A polysaccharide extracted from the leaves of S. monoica showedpositive activity against human immune deficiency viruses (7,13).

Therefore, the present attempt has been made to identify thephytoconstituent of S. monoica and Suaeda pruinosa extracts and to findout the anti- inflammatory effect of each extract on the rat liverintoxicated by paracetamol overdose.

MATERIALS AND METHODS

Plant material and preparation of extract

Suaeda sp. were collected during months of June, July, 2016-2017from El Doubia at El Riyadh- El Dallamroad. Air-dried and powderedaerial parts of Suaeda monoica and Suaeda pruinosa were extracted withpetroleum ether. The solvent was evaporated under reduced pressure thenextracted with methyl alcohol in [H.sub.2]O(80%), the solvent wasstripped off under reduced pressure gave 50 g. The petroleum etherextract 10 g was subjected to subsequent investigation.

Extract of amino acids

Lyophilized tissues were ground in a mortar and extracted with MCW(methanol: chloroform:water 12:5:3 v/v/v), following the proceduredescribed by Marur et al.(1994) (17). A sample of ground material (100mg) was extracted with 10 mL of MCW for 3 days in a closed tube in arefrigerator. After centrifugation at 1200 rpm for 5 min on a benchcentrifuge, the supernatant was collected and the residue extracted witha further 6 mL of MCW. After centrifugation the supernatants werecombined (16mL) and shaken with chloroform (4mL) followed by water (6mL). On standing, the resulting separation of phases enabled the removalof chlorophyll and the aqueous phase was taken to dryness under a streamof [N.sub.2] gas. This material was resuspended in water and used foramino acid determination.

Preparation of fatty acid methyl esters

This method is a modified Folch procedure (18), utilizing 2:1 (v/v)chloroform/ methanol as the organic solvent.

Chemicals and reagents

Amino acids analysis

Methanol (HPLC grade), Chloroform (HPLC grade) chloroform/methanol(HPLC grade)2:1 (v/v), 0.88% (8.8 g/liter) KCl in distilled [H.sub.2]O,Nitrogen gas: high purity (<5 mg [O.sub.2]/kg), Centrifuge tube withspout or 50-ml glass tube, Polytron PT-3100 hom*ogenizer equipped with aPT-DA 3012/2EC aggregate (probe) or equivalent, Whatman no. 40 filterpaper, 15- and 50-ml test tubes with Teflon-lined screw cap, Test tubeshaker, Tabletop centrifuge, Vacuum aspirator, N-EVAP model 112 nitrogenevaporator (Organomation) with moisture trap in line from N2 source,Glass pipets, Glass beakers.

Organic solvent for preparation of lipid sample

Nitrogen gas: high purity (<5 mg [O.sub.2]/ kg), 0.5 N NaOH: 2 gNaOH diluted to 100 ml in methanol; store up to 6 months at 2[degrees]to 8[degrees] C10% to 14% boron trifluoride (BF3) in methanol (Sigma orSupelco; also see recipe).

Isooctane or hexane, Anhydrous sodium sulfate: store at100[degrees]C N-EVAP model 112 nitrogen evaporator (Organomation) withmoisture trap in line from N2 source (Fig. D1.2.1), 15-ml test tubeswith Teflon-lined screw caps 1000C heating block, Test tube shaker,Tabletop centrifuge, GC sample vials with Teflon caps.

METHODS

Separation of the amino acids was carried out on a Superpac(Pharmacia) ODS-2 column coupled to an LKB dual pump HPLC system (model2150), controlled by a gradient generator, model 2152. Knowing theconcentration of amino acids in the sample, an appropriate volume wastaken for the preparation of o-phthaldialdehyde (OPA) (19-20) and9-fluorenylmethyl-chloroformate (FMOC-Cl) derivatives (21) prior toseparation and analysis by reverse-phase HPLC. The analysis of prolinewas carried out by a second analysis using FMOC-Cl derivatives on eachsample. FMOC derivates were prepared as described by (22).

Extracted lipid sample

Fatty acid compositional analysis of lipids carried out bygas-liquid chromatography (GLC) modified Folch procedure (18).

GC method

Column: J+W DB-23 (50% cyanopropyl) methylpolysiloxane FID: Temp:270 [degrees]C, Injection: --mode: split ratio 30:1,--temperature: 270[degrees]C, injection volume: 1 [micro]L

Oven:--intial temp: 140 [degrees]C,--temp ramp: 10 [degrees]C /min,--final temp: 260[degrees], Hold at final temperature for 3 minutes.For sequences run prior to 03/30/07, the final temperature was held for5 minutes.

HPLC conditions for phenolic compounds

HPLC analysis was carried out using an Agilent 1260 series. Theseparation was carried out using C18 column (4.6 mm x 250 mm i.d., 5[micro]m). The mobile phase consisted of water (A) and 0.02%tri-floro-acetic acid in acetonitrile (B) at a flow rate 1 ml/min. Themobile phase was programmed consecutively in a linear gradient asfollows: 0 min (80% A); 0-5 min (80% A); 5-8 min (40% A); 8-12 min (50%A); 12-14 min (80% A) and 14-16 min (80% A). The multi-wavelengthdetector was monitored at 280 nm. The injection volume was 10 [micro]lfor each of the sample solutions. The column temperature was maintainedat 35 [degrees]C.

Biochemical studies Materials and Methods

Drugs and Chemicals Paracetamol was purchased from AlexandriaCompany for Pharmaceuticals and Chemical Industries. Kits fordetermination of aspartate aminotransferase (AST), alanineaminotransferase (ALT), alkaline phosphatase (ALP),bilirubin and ureawere obtained from Biomed Diagnostics, Egypt. Theinterleukin-1[beta](IL-1[beta]) and tumor necrosis factor alpha(TNF-[alpha]) were carried out using an ELISA reagent kits obtained fromBiosource, USA.

Animals

Male rats weighing 100-120 g were obtained from the Animal BreedingLab of National Research Centre, Cairo, Egypt. The animals were keptunder constant temperature conditions (22 [+ or -] 2 [degrees]C),relative humidity (50-60%), and lighting (12 h light/dark cycle). Foodand water were accessible ad libitum. The study was carried outaccording to the guidelines of the Ethics Committee of the NationalResearch Centre.

Experimental design

Six groups each of six male albino rats were selected. Group1:served as negative control. Group 2: rats received a single dose ofparacetamol (positive control). Group 3 & 4: rats received Suaedamonoica and Suaeda pruinose before paracetamol induction. Group 5 &6: rats received the Suaeda monoica and Suaeda pruinose extract afterparacetamol induction. Twenty-four hour after treatments, the rats ofall groups were anesthetized and blood samples were collected directlyfrom retro-orbital plexus. The blood samples were allowed to clot for20-30 min. Serum was separated by centrifugation at 37[degrees]C andused for estimation of various biochemical parameters. Animals weresacrificed by decapitation. Livers were rapidly isolated; a part of eachwas hom*ogenized using cold saline to prepare a 10% hom*ogenate that wasused for estimation of malondialdehyde (MDA). The second part of theliver was preserved in 10% formalin for histopathological andhistochemical examinations.

Biochemical analyses

The activities of AST and ALT were determined according to themethod of Reitman and Frankel (23). Alkaline phosphatase was determinedusing the method described by Demetriou et al (24). Bilirubin wascarried out by the method described by Young (25). Creatinine wasmeasured by the method of Bartels and Bohmer (26). Urea was calculatedby the method of Tabacco et al (27). Arylesterase activity ofparaoxonase 1 (POX-1) was measured spectrophotometrically usingphenylacetate as a substrate (28-29). IL-1[alpha] was carried out usingan ELISA reagent kit and expressed as pg/ml. TNF-[alpha] was determinedby ELISA using kit (Biosource International, USA) and microtiter platereader (Fisher Biotech, Germany). Lipid peroxidation was assayed bymeasuring the level of malondialdehyde (MDA) in the tissue hom*ogenatesusing the method of Ruiz-Larrea et al (30).

Histopathological and Histochemical studies

After the experimental period, animals were sacrificed, liverremoved immediately, sliced and washed in saline. Liver pieces werepreserved in 10% formalin for histopathological studies. Sections weretaken and stained with hematoxylin and eosin (H&E) (31) and thenthey were examined for histopathological changes and photographed. Thehistochemical study was performed by periodic acid-Schiff method (32)for visualization of the polysaccharide in the liver. These materialswere demonstrated in sections with 5 [micro]m thickness. statisticalanalysis

The protection percent is calculated by 1-(T-V/C-V)x100. Data wereexpressed as mean [+ or -] SD. The data were analyzed by one-way ANOVAfollowed by Duncanas multiple tests, using SPSS software (SAS InstituteInc., Cary,NC, USA). A probability value of less than 0.05 wasconsidered statistically significant.

RESULTS AND DISCUSSION

Preliminary phytochemical analysis

Plant materials were screened using the methods previouslydescribed (33). Phytochemical analysis gave positive test for alkaloids,terpenoids, steroids, tannins, saponins, flavonoids and phenols. Theidentification of major chemical groups was carried out by thin layerchromatography (TLC) on silica gel. TLC was developed in chloroform/methanol/water (26:14:3), spots were visualized with sulfuric acid 20%in methanol solution under 254 and 356 nm gave yellow spots forFlavonoids, and purple color suggested the existence of triterpene, pinkcolor for sterols and brown color for monoterpenes. Data shown that themain compounds were observed in S. monoica and S. pruinosa phenolic andterpenes /or sterols.

Amino acids analysis

Separation of the amino acids for methanolic extracts of each plantwas carried out on HPLC system revealed that, the highest percentage ofMethionine in S. pruinosa (0.999) while S. monoica (0.889) and Histidinewas (0.216) in S. monoica and (0.167) in S.pruinosa extracts. Valine,Isoleucine and Phenylalanine were absent from the extracts of S.monoicaand S.pruinosa (table1).

GC of fatty acid methyl esters in the petroleum ether extract

Fatty acid analysis of lipids carried out by gas-liquidchromatography (GC)34 showed high percentage of long chain fatty acids,the percentage of unsaturated fatty acids was (polyunsaturated fattyacids linolenic acid was (38.96%) followed by linoleic acid (59.84%) andmonounsaturated fatty acid, oleic was 71.69% and the percentage ofsaturated fatty acids 65.26% for S.pruinosa, while the high percentageof linolenic acid (33.72%) followed by linoleic acid (52.42%) and oleicacid (40.71%) and 77.80 saturated fatty acids for S. monoica (table2).

HPLC of phenolic compounds

HPLC of phenolic compounds in Methanolic extract from the aerialparts of S.monoica and S. pruinosa revealed that, the identifiedcompounds in methanolic extract of S. pruinosa were nine whilemethanolic extract of S. monoica showed eight compounds. The extract ofeach plant contained Gallic Acid, Catechin, Coffeic Acid, Syringic Acid,Rutin, Coumaric Acid, Vanillin, Quercetin but cinnamic acid found inmethanolic extract of S. pruinosa only (table 3). Histopathologicalstudies

At the end of the experiment, and after blood samples were obtainedliver samples were taken, washed with normal saline and processed forhistopathological study. Initially the materials were fixed in 10%buffered neutral formalin and paraffin sections were taken at 5 [micro]mthickness processed in alcohol-xylene series and was stained with alumhematoxylin and eosin, then were examined for histopathological changes.

The microscopic examination of liver of control rats shows thenormal hepatic lobules and portal areas structure. The central vein issurrounded by the hepatocytes with eosinophilic cytoplasm and distinctnuclei. The hepatic sinusoids are shown between the hepatocytes (Fig.1.A, B).

Histopathological investigations of liver of rat administered asingle dose of paracetamol (3 g/kg/b.w) showed widespread swelling andballooning degeneration and focal necrosis that associated withinflammatory infiltration in the hepatocytes (Fig. 1.C). In some ratsdilated and congested portal tracts (Fig. 1.D).

In rats given Sum extract and paracetamol, examination showed thehepatic lobules that appeared more or less like control (Fig. 2.A). Insome rats, hydrobic degeneration and congested portal tracts were shown(Fig. 2.B). Histopathological investigation of liver of rats given Supextract and paracetamol showed the hepatic lobules that appeared more orless like control (Fig. 2.C). In some rats hydrobic degeneration andcongested portal tracts were noticed (Fig. 2.D).

Sections of liver of rats given paracetamol and Sum, and Supextracts showed the hepatic lobules that appeared more or less likecontrol (Fig. 3A, B). But in rats given paracetamol and Sup extract fewfoci of necrosis in the hepatocytes were showed (Fig. 3.B).

Examination of control liver sections stained by PAS reaction showsthe abundance of glycogen in the cell of hepatic lobule. The glycogenparticles appear accumulated at one side of the cytoplasm leaving theother side almost devoid of such materials (Fig. 4.A). Histochemicalinvestigations of liver of rats administered a single dose ofparacetamol dose (3 g/kg body weight) and were sacrificed after 24 hshowed marked diminution in glycogen distribution in the hepatocytes ascompared with control group(Fig. 4.B). Examination of sections of liverthat stained with PAS in rat orally given paracetamol after 7 days ofSum and Sup plants extracts administration (prophylactic groups, 100mg/kg b.w) indicated an increase in glycogen contents as compared withparacetamol only. The distribution of this inclusion was more or lesslike normal control (Fig. 4C, D). On the other hand, in case oftherapeutic groups in which paracetamol was conducted once before Sumand Sup plant extracts administration (100 mg/kg b.w) daily for 7 daysshowed heterogeneous distribution of glycogen, where the healthy zonesexhibited normal distribution and the injured zones exhibited a decreasein this inclusion (Fig.4E, F).

Biochemical studies

Liver regulates many important metabolic functions and contains ahost of enzymes. Any injury in this organ causes distortion of thesemetabolic functions. The present work aimed to compare the possiblehepatoprotective impact of Suaeda Sp. administered for one week prior toand after a single acetaminophen toxic dose (3g/ kg body weight) in malerats.

The present study showed damage of liver due to paracetamol overdosage which is confirmed by elevated levels of all biochemicalparameters measured (SGPT, SGOT, ALP, TB, urea, creatinine, IL1, TNF andMDA). The study also showed decrease in POX enzyme. These elevatedenzymatic activities in serum, are indicators of cellular leakage andloss of functional integrity of cell membrane in liver (35). Aqueousextract of S. monoica and S. peruinosa contain alkaloids, terpenoids,steroids, tannins, quinones, saponins, flavonoids and phenols. They canscavenge off free radicals which are the principles for thehepatoprotective activity for protection or therapeutic effect.Decreased serum parameters level following extract treatment indicatedthe effectiveness of the extract in restoring normal functional statusof the liver.

Suaeda had successfully reversed the PCM-induced hepatotoxic effectby its ability to reduce the elevated levels of ALT, AST, and ALPsuggesting

that these biochemical restorations could be due to the extractability to inhibit the cytochrome P450 or/and ability to promote the PCMglucuronidation (36-37). Furthermore, the ability to lower the enzymeslevel can be associated with the ability of Suaeda to prevent lipidperoxidation of endoplasmic reticulum that is rich in polyunsaturatedfatty acid by disrupting the binding of activated radicals to themacromolecules. This process can possibly be achieved via theantioxidant activity of Suaeda extracts due to the presence of highamount of a linolenic and linoleic fatty acid (38). Besides, mechanismsof protection that can take place include activation of liverregeneration by enhancing the protein and glycoprotein synthesis oraccelerated detoxification and excretion (39), prevention of lipidperoxidation process, and stabilization of hepatocellular membrane (38).The two Suaeda extracts contain methionine and cysteine amino acidswhich act as -SH donner in synthesis of GSH and prevent APAP metaboliteto interact with macromolecules. The results are confirmed with DiPierro and Rosson, (40) who found that a mixture of L-cysteine,L-methionine, and L-serine were effective at a lower dose than N- acetylcysteine (NAC) in APAP over dose and improved mouse survival rates.Moreover, the mixture significantly reduced ALT level and inducedhepatic GSH synthesis and decrease MDA accumulation (40).

Increase in serum level of ALP is due to increased synthesis in thepresence of increasing biliary pressure 41 and reflects the pathologicalalteration in biliary flow (42).

Bilirubin, a metabolic product of hemoglobin, undergoes conjugationwith glucuronic acid in hepatocytes to increase its water solubility.Determination of serum bilirubin represents an index for assessment ofhepatic function, and any abnormal increase in the levels of serumbilirubin indicates hepatobiliary diseases and severe disturbance ofhepatocellular function (43). Decreased serum bilirubin level followingextract treatment indicated the effectiveness of the extract inrestoring normal functional status of the liver.

Paracetamol-induced toxicity in rats may have altered membranestructure and function as well as lipids metabolism in the liver.

Therefore, it is possible to propose that the extract/compoundsexerting an anti-inflammatory activity might also demonstratehepatoprotective activity. Interestingly, this is supported by the studyof Gupta et al (44) who suggested that the combination ofhepatoprotective effect and antioxidant activity synergisticallyprevents the process of initiation and progression of hepatocellularinjury in prophylactic and therapeutic treatments (44).

Vanillin, a phenolic aldehyde has been reported to possessantioxidant and free radical scavenging ability which could possiblyaccount for the hepatoprotective property of this plant. The activitiesof antioxidant counteract the redox state precipitated intracellularlyand hence ensure hepatoprotection against paracetamol-induced liverinjury (45-47). The antioxidant activity of this extract may alsoexplain the mechanism of the hepatoprotective activity of Suaeda sp. Thefindings of this study corroborate the effect that was reported forHomalium zeylanicum (48).

Lipid peroxidation (LPO) is one of the characteristic features ofoxidative stress in PCM hepatotoxicity. According to Luqman and Rizvi(49), LPO is known to injure the cells by inactivation of membraneenzymes, decrease fluidity of the membrane and release, into cytotoxic,aldehydes such as MDA (49). The elevated level of hepatic MDA has beenregarded as an indicator of cellular damage and indicates the inabilityof the antioxidant defense system to protect against the production ofexcessive free radicals. In this study, MDA increased and POX decreasedas a result of paracetamol over dose. The enhanced antioxidant defensein addition to reduced LPO product in the liver is indicative for theantioxidant effect of the tested extracts.

The biochemical results were supported by the histopathologicalfindings. The results demonstrated that rats exposed to PCM alone showedsevere cellular damage that might be due to the production of freeradicals by PCM and subsequent LPO. These histopathologic findings wereameliorated significantly in the groups of rat wither were pre-treatedor treated with either S. monoica or S. peruisma extracts, indicatingpronounced protection of hepatocytes against PCM induced hepatic damage.It can be referred to the antioxidant effect of the tested extracts thatmarkedly decreased the oxidative stress and thereby reduced thehistopathological alterations of the liver.

The present study found that two Suaeda contain cysteine andMethionine with excessive amount which considered as -SH donner for GSHsynthesis. Di Pierro and Rossoni (40) found that DDM-GSH (amixture ofL-cysteine, L-methionine, and L-serine in a weight ratio of 2:1:1) waseffective at a lower dose thanN-acetylcysteine (NAC) in reducingAPAP-induced hepatotoxicity; improving mouse survival rates, decreasingsALT and MDA accumulation and increase hepatic GSH synthesis in themouse model (40).

Suaeda sp. may inhibit the activity of certain cytochrome P450enzymes whose inhibition could safeguard against APAP hepatotoxicity(37). With regard to the potential hepatoprotective effects of Suaeda,it was demonstrated that rats which were chronically supplemented withSuaeda extract (100 mg/kg) and APAP (3 g/kg) exhibited an improvement inAPAP-induced liver necrosis and increased serum ALT and AST levels 50.In the present study, paracetamol hepatotoxicity was associated with anincrease in serum level of IL1a. Similar results were reported by otherinvestigators (51-52).

Signaling through the IL-1 receptor (IL-1R) was recently shown toplay an important role in paracetamol-induced hepatotoxicity (53).Activation of NF-[kappa]B/Rel transcription factor leads to theincreased production of various inflammatory mediators, including IL1[alpha] and TNF-[alpha]. (54).

Activation of Toll-like receptors (TLRs) by pathogen-associatedmolecular patern results in upregulation of pro- IL1 a via NF-[kappa]Bpathway. This is followed by a second signal that results in a caspase 1mediated cleavage of pro- IL1[alpha] to release the active molecule(55).

Pretreatment with studied extracts decreased serum IL1 a andTNF-[alpha] in rats subjected to paracetamol hepatotoxicity. It wassuggested that the two extracts have inhibitory effect on NF-[kappa]B/Rel activity in rat serum (56).

In summary, our present study demonstrated that S. monoica and S.peruisma possessed a protective action against paracetamol-induced liverinjury. The underlying mechanisms of their hepatoprotective activitymight be attributed to the presence of the amino acids, unsaturatedfatty acids and phenolic compounds. These encouraging findings would behelpful for developing potential hepatoprotective agents for remedyingAPAP-induced liver injury.

http://dx.doi.org/10.13005/bpj/1620

(Received: 27 January 2019; accepted: 26 February 2019)

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F.S. Elsharabasy [1], N.S. Metwally [2], A.H. Mahmoud [2], M.S.Soliman [2], E.R. Youness [3], A.H. Farrag [4] and Sherifa Arafa [5].

(1) Department of Chemistry, Prince Sattam Bin Abdul-AzizUniversity, Saudi Arabia and Chemistry of Natural and Microbial ProductsDepartment, National Research Center, Dokki, Egypt.

(2) Therapeutic Chemistry Department, Pharmaceutical IndustriesResearch Division, National Research Center, Dokki, Egypt.

(3) Medical Biochemistry Department, Medical Research division,National Research Center, Dokki, Egypt.

(4) Pathology Department, National Research Center, Dokki, Egypt.

(5) Department of Biology, Prince Sattam Bin Abdulaziz University,Saudi Arabia and Department of Botany, Faculty of Science, CairoUniversity, Egypt.

Caption: Fig. 2. Sections of liver of A, B) control rat liver showsnormal structure of hepatic lobule and portal tract, respectively, C)rat administered a single dose of paracetamol (2 g/kg/b.w) showswidespread swelling and ballooning degeneration and focal necrosis thatassociated with inflammatory infiltration in the hepatocytes, D) ratadministered a single dose of paracetamol shows the portal andperiportal area with dilated and congested veins (long arrow) (H & Estain, Scale bar: 20 [micro]m)

Caption: Fig. 3. Sections of liver of A)rat given Sum extract andparacetamol show the hepatic lobule that appear more or less likecontrol, B) rat given Sum extract and paracetamol show hydrobicdegeneration and congested portal tract, C)rat given Sup extract andparacetamol show the hepatic lobule that appear more or less likecontrol, D) rat given Sup extract and paracetamol show hydrobicdegeneration and congested portal tract (H & E stain, Scale bar: 20[micro]m)

Caption: Fig. 4. Sections of liver of A) rat given paracetamol andSum extract show the hepatic lobule that appear more or less likecontrol, and B) rat given paracetamol and Sup extract show the hepaticlobule that appear more or less like control. Notice few foci ofnecrosis in the hepatocytes (H & E stain, Scale bar: 20 [micro]m)

Caption: Fig. 5. Liver sections of A) normal rat showing normaldistribution of glycogen in the hepatocytes, B) rat administered onedose of paracetamol (3 g/kg/b.w) showing a marked diminution in theglycogen contents as compared with control group, C, D): rats given Sumand Supextracts for 7 consecutive days before a single dose paracetamolshowing glycogen contents in the hepatic lobule appeared more or lesslike control, E, F) rats given Sum or Supextracts for successive daysbefore paracetamol showing heterogeneous distribution of glycogen. Thehealthy zones exhibite normal distribution and the injured zonesexhibite a decrease in this inclusion (PAS stain, Scale bar: 20[micro]m)

Table 1. Amino acids identified in the methanolextract from the aerial parts of each S. monoicaand S. pruinosa [micro]mol/mg dry wtIdentified amino acids S. monica S. pruinosaHistidine 0.216 0.167Arginine 0.137 0.130Threonine 0.121 0.115Alanine 0.091 0.084Proline 0.082 0.077Tyrosine 0.105 0.081Valine ND NDMethionine 0.889 0.999Cysteine 0.151 0.143Isoleucine ND NDLeucine 0.122 0.116Phenylalanine ND NDlysine 0.312 0.297Aspartic acid 0.111 0.086Glutamic acid 0.035 0.027Glycine 0.128 0.122Table 2. GC of fatty acid methyl esters in thepetroleum ether extract from the aerial parts of S.monoica and S.pruinosa Plant samplesIdentified fatty acids(mg/100 g D.W) S. monica S. pruinosa Area% Area%Lauric acid (C12:0) 15.78 10.49Myristic acid(C14:0) ND NDMyristoleic (C14:1) 7.55 8.27Pentadecylic acid (C15:0) ND NDPalmitic acid (C16:0) 39.41 33.39Palmitoleic acid (C16:1) 20.65 25.17Stearic acid (C18:0) 22.61 21.38Oleic acid (C18:1) 40.71 38.25Linoleic acid (C18:2) 52.42 59.84Linolenic acid (C18:3) 33.72 38.96Table 3. HPLC of phenolic compounds in Methanol extractfrom aerial parts of each S. monoica and S. pruinosa S. monoicaCompounds Area Conc. ([micro]g/ml) Conc. ([micro]g/g)Gallic Acid 250.08 13.43 268.60Catechin 132.56 25.58 511.61Coffeic Acid 165.36 6.13 122.50Syringic Acid 362.51 18.86 377.23Rutin 474.19 65.43 1308.50Coumaric Acid 83.54 1.86 37.20Vanillin 209.57 6.37 127.32Quercetin 89.57 8.45 168.95Cinnamic Acid 0.00 0.00 0.00 S. pruinosaCompounds Area Conc. ([micro]g/ml) Conc. ([micro]g/g)Gallic Acid 160.71 8.63 172.61Catechin 155.22 29.95 599.08Coffeic Acid 159.21 5.90 117.95Syringic Acid 285.90 14.88 297.51Rutin 374.03 51.61 1032.12Coumaric Acid 97.57 2.17 43.45Vanillin 253.38 7.70 153.94Quercetin 12.56 1.18 23.70Cinnamic Acid 17.27 0.16 3.28Table 4. Effect of Suaeda. Sp. extract on rat liver and kidneytreated with over dose paracetamolParameter ALT ASTGroup name1-Clean 12.13[+ or -] 1.17 24.74[+ or -] 2.23control (2,3,4,5,6) (2,3,4)42-Positive 60.11[+ or -] 2.41 92.56[+ or -] 2.78control (1,3,4,5,6) (1,3,4,5,6)43-S. monoica 41.21[+ or -] 3.44 45.99[+ or -] 2.39Pre-treated (1,2,4,5,6) (1,2,5,6)4-S. pruinosa 49.07[+ or -] 2.93 46.64[+ or -] 2.83Pre-treated (1,2,3,5,6) (1,2,5,6)5-S. monoica 30.59[+ or -] 2.03 28.44[+ or -] 1.58Post-treated (1,2,3,4) (1,2,3,4)6-S. pruinosa 31.93[+ or -] 2.17 27.87[+ or -] 2.25Post-treated (1,2,3,4) (1,2,3,4)Parameter ALP Total bilirubinGroup name1-Clean 92.61[+ or -] 2.93 0.92[+ or -] 0.15control (2,3,4,5,6) (2,3,4,5,6)42-Positive 261.98[+ or -] 4.07 1.65[+ or -] 0.18control (1,3,4,5,6) (1,3,4,5,6)43-S. monoica 226.39[+ or -] 1.99 1.46[+ or -] 0.03Pre-treated (1,2,5,6) (1,2,5,6)4-S. pruinosa 228.89[+ or -] 3.70 1.49[+ or -] 0.03Pre-treated (1,2,5,6) (1,2,5,6)5-S. monoica 179.73[+ or -] 2.72 1.37[+ or -] 0.03Post-treated (1,2,3,4) (1,2,3,4)6-S. pruinosa 166.62[+ or -] 2.42 1.38[+ or -] 0.05Post-treated (1,2,3,4) (1,2,3,4)Parameter urea CreatinineGroup name1-Clean 28.97[+ or -] 1.95 1.15[+ or -] 0.19control (2,3,4,5,6) (2,3,4,5,6)42-Positive 66.49[+ or -] 2.57 4.20[+ or -] 0.59control (1,3,4,5,6) (1,3,4,5,6)43-S. monoica 52.95[+ or -] 1.94 3.48[+ or -] 0.27Pre-treated (1,2,5,6) (1,2,5,6)4-S. pruinosa 55.98[+ or -] 1.13 3.48[+ or -] 0.38Pre-treated (1,2,5,6) (1,2,5,6)5-S. monoica 39.54[+ or -] 2.06 2.29[+ or -]0.20Post-treated (1,2,3,4) (1,2,3,4)6-S. pruinosa 39.87[+ or -] 2.097 2.54[+ or -] 0.23Post-treated (1,2,3,4) (1,2,3,4)Values represent mean of six animals [+ or -] SESignificant change at P < 0.05. LSD: Least significance differenceTable 5. Effect SU. Sp. extract on antioxident of rat treated withover dose paracetamolParameter 1-Clean 2- PositiveGroup name control controlMDA 19.91[+ or -] 1.65 59.59[+ or -] 2.43 (2,3,4,5,6) (1,3,4,5,6)POX-1 26.61[+ or -] 1.70 13.18[+ or -] 1.14 (2,3,4,5,6) (1,3,4,5,6)Parameter 3- S. monica 4- S. pruinosaGroup name Pre- treated Pre -treatedMDA 48.46[+ or -] 1.86 49.36[+ or -] 1.72 (1,2,5,6) (1,2,5,6)POX-1 18.11[+ or -] 1.84 18.54[+ or -]1.29 (1,2) (1,2)Parameter 5--S. monica 6- S. pruinosaGroup name Post-treated Post-treatedMDA 37.09[+ or -] 1.75 38.02[+ or -] 2.09 (1,2,3,4) (1,2,3,4)POX-1 18.54[+ or -]1.29 18.94[+ or -] 1.63 (1,2) (1,2)Values represent mean of six animals [+ or -] SESignificant change at P < 0.05. LSD: Least significance differenceTable 6. Effect SuaedaSp. extract on immune response in rattreated with overParameter 1-Clean 2- PositiveGroup name control controlIL-1P (Pg/ml) 29.00[+ or -] 1.58 93.46[+ or -] 2.78 (2,3,4,5,6) (1,3,4,5,6)TNF (Pg/ml) 17.79[+ or -] 1.18 45.46[+ or -] 1.63 (2,3,4,5,6) (1,3,4,5,6)Parameter 3- S. monica 4- S. pruinosaGroup name Pre- treated Pre -treatedIL-1P (Pg/ml) 77.17[+ or -] 1.74 71.55[+ or -] 2.49 (1,2,5,6) (1,2,5,6)TNF (Pg/ml) 38.251[+ or -] 1.77 39.53[+ or -] 2.08 (1,2,5,6) (1,2,5,6)Parameter 5- S. monica 6- S. pruinosaGroup name Post-treated Post-treatedIL-1P (Pg/ml) 54.65[+ or -] 2.24 54.89[+ or -] 1.92 (1,2,3,4) (1,2,3,4)TNF (Pg/ml) 30.94[+ or -] 1.59 30.44[+ or -] 2.38 (1,2,3,4) (1,2,3,4)Values represent mean of six animals [+ or -] SESignificant change at P < 0.05. LSD: Least significance differenceFig. 1. Chemical Structures of compounds identified in methanolicExtract from the aerial parts of S.monoica and S.pruinosaCinnamic acid derivatives R1 R2 R3 R4Caffeic acid OH OH H HCoumaric Acid H OH H HCinnamic Acid H H H HBenzoic acid derivatives R1 R2 R3 R4Gallic acid H OH OH OHSyringic Acid OC[H.sub.3] OH OC[H.sub.3] H