Phytochemical and pharmacological study of Ficus palmata growing in Saudi Arabia

Abstract

Phytochemical study of the aerial parts of Ficus palmata utilizing liquid–liquid fractionation and different chromatographic techniques resulted in the isolation of a new isomer of psoralenoside namely, trans-psoralenoside (5) in addition to, one triterpene: germanicol acetate (1), two furanocoumarins: psoralene (2), bergapten (3), one aromatic acid vanillic acid (4) and the flavone glycoside rutin (6). Structures of the isolated compounds were established through physical, 1D- and 2D-NMR and MS data. The total extract and fractions of the plant were examined in vivo for its possible effects as hepatoprotective, nephroprotective, antiulcer and anticoagulant activities in comparison with standard drugs. Hepatoprotective activity was assessed via serum biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and total bilirubin. Tissue parameters such as non-protein sulfhydryl groups (NP-SH), malonaldehyde (MDA) and total protein (TP) were also measured. In addition to tissue parameters, nephroprotective effect was evaluated by measuring the serum levels of sodium, potassium, creatinine and urea. Histopathological study for both liver and kidney cells was also conducted. Antiulcer activity was explored by observing stomach lesions after treatment with ethanol. Whole blood clotting time (CT) was taken as a measure for the anticoagulant activity of the extract. Antioxidant activity of the total extract and fractions of the plant was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and ascorbic acid as standard.

1. Introduction

Ficus is the genus of the family Moraceae that comprises about 800 species (Harrison, 2005). Most of the members of the family are very high trees, shrubs and rarely herbs often with milky juice (Hutchinson et al., 1958). There are five species of Ficus growing in Saudi Arabia; Ficus vasta, Ficus carica, Ficus salicifolia, Ficus palmata and Ficus glumosa (Migahed, 1996). A number of Ficus species are used in folk medicine as anti-tumor, anti-inflammatory and tonic medicament (Lansky et al., 2008, Kitajima et al., 1999) Microbial diseases such as epilepsy and jaundice (Noumi and Fozi, 2003, Betti, 2004), bronchitis, influenza whooping cough, tonsillitis, toothache, bacillary dysentery, enteritis and bruises are also reported to be treated by Ficus extracts. Antioxidant activities were also reported for Ficus extracts. (Abdel-Hameed, 2009, Çalişkan and Polat, 2011). The chemical review on genus Ficus, reveals the presence of sterols and/or terpenes (Kuo and Li, 1997, Kuo and Chaiang, 1999), coumarins (Chunyan et al., 2009), furanocoumarin glycosides (Chang et al., 2005), isoflavones (Li and Kuo, 1997), lignans (Li and Kuo, 2000) and chromone (Basudan et al., 2005).

2. Materials and methods

2.1. Plant materials

The plants of F. palmate Forsk. were collected in March, 2008 from the Agabat Tanoma in the kingdom of Saudi Arabia. The plant was identified by Dr. Mohammed Yusuf, Taxonomist of the Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. A voucher specimen (# 15362) has been deposited at the herbarium of the department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.

2.2. General experimental procedures

Melting points were determined in open capillary tubes using Thermosystem FP800 Mettler FP80 central processor supplied with FP81 MBC cell apparatus, and were uncorrected. Ultraviolet absorption spectra were obtained in methanol and with different shift reagents on a Unicum Heyios α UV–Visible spectrophotometer. ¹H and ¹³C NMR spectra were recorded on a UltraShield Plus 500 MHz (Bruker) (NMR Unite at the College of Pharmacy, Salman Bin Abdulaziz University) spectrometer operating at 500 MHz for proton and 125 MHz for carbon, respectively. The chemical shift values are reported in δ (ppm) relative to the internal standard TMS or residual solvent peak, the coupling constants (J) are reported in Hertz (Hz). 2D-NMR experiments (COSY, HSQC, HMBC and NOESY) were obtained using standard Bruker program. MS were obtained using Liquid Chromatography/Mass Spectrometer (Quattro micro API) equipped with a Z-spray electrospray ion source (Micromass^®, Quattro micro™, WATERS). Silica gel 60/230–400 mesh (EM Science) and RP C-18 silica gel 40–63/230–400 mesh (Fluka) were used for column chromatography, while silica gel 60 F254 (Merck) was used for TLC.

2.3. Extraction, fractionation and purification

Dry leaves of Ficus palmata (1900 gm) were extracted with 95% ethanol to exhaustion and the solvent was evaporated under reduced pressure using rotary vacuum evaporator to obtain viscose extract. Equal volume of water was added and the resulted extract successively partitioned with petroleum ether (60–80 °C) (3 × 500), chloroform (3 × 500), ethyl acetate (3 × 400) and butanol (2 × 300). The solvents were evaporated to obtain 36.9, 9.7, 8.0 and 27.5 gm of petroleum ether, chloroform, ethyl acetate and butanol, respectively. The left aqueous layer was dried to give 33.4 gm.

A portion of petroleum ether layer (17.2 gm) was and chromatographed over silica gel column (400 gm, 5 cm i.d.). Elution the column with petroleum ether; petroleum ether: CHCl₃ (90:10–0:100) in a gradient system. On the bases of TLC behavior, similar fractions were pooled together affording three main fractions (A–C). Fraction A (2.6 gm) was further purified on silica gel column (50 gm, 2 cm i.d.) eluted with petroleum ether: CHCl₃ (90:10) followed by crystallization from MeOH afforded 45 mg of 1. A similar treatment of fraction B (3.2 gm) afforded 140 mg of β-sitosterol. Fraction C (2.7 gm) was rechromatographed over silica gel column eluted with petroleum ether: EtOAc mixtures in a gradient system resulted in the isolation of 75 mg of 2 and 14 mg of 3 after crystallization from MeOH.

Part of the chloroform layer (5 gm) was chromatographed over silica gel column (200 gm, 3 cm i.d.) eluted with petroleum ether: CHCl₃ (90:10) and polarity was gradually increased with CHCl₃ till 100% then MeOH was used in an increasing ratio. Fractions eluted with petroleum ether: CHCl₃ (85:15) (700 mg) afforded 40 mg of 2 on crystallization from MeOH. Fraction D eluted from the column with CHCl₃:MeOH (75:25) was subjected to reversed phase RP-18 PTLC using MeOH:H₂O (70:30) as a developing system to yield 4 (5.5 mg).

The EtOAc layer (8 gm) was chromatographed over silica gel column. The column eluted with CHCl₃:MeOH:H₂O (100:0:0–40:60:6) and then with MeOH. Fraction E eluted from the column with CHCl₃:MeOH:H₂O (75:25:2.5) afforded 15 mg of 5 after crystallization from MeOH.

A portion of ButOH layer (2.5 gm) was chromatographed on medium pressure RP-18 silica gel column started with 5% ACN in H₂O, increasing polarity with ACN afforded 6.5 mg of compound 6.

2.4. Animals

Wistar albino rats (150–200 g) roughly the same age (8–10 weeks), obtained from the Experimental Animal Care Center, College of Pharmacy, King Saud University, Riyadh were used. The animals were housed under constant temperature (22 ± 2 °C), humidity (55%) and light/dark conditions (12/12 h). They were provided with Purina chow and free access to drinking water ad libitum (Abdel-Kader et al., 2010). The experiments and procedures used in this study were approved by the Ethics Committee of the College of Pharmacy, King Saud University.

2.5. Chemicals

Silymarin, rutin, warfarin and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma Chemical Co., St Loui, MO, USA. Ascorbic acid was purchased from E. Merck (Germany).

2.6. Hepatoprotective & nephroprotective activity

Male Wistar rats were divided into fifteen groups of five animals each. Group I received normal saline and was kept as a control group. Groups II–XV received single dose of CCl₄ (1.25 ml/kg body weight). Group II received only CCl₄ treatment. Group III was administered silymarin (Sil) at a dose of 10 mg/kg p.o. (20.7 μmole/kg) Groups IV–XIII were treated with 100 and 200 mg/kg of the different fractions, while the last two groups received 200 and 400 mg/kg of the total extract respectively. Treatment started 6 days prior to CCl₄ and continued till day seven. After 24 h, following CCl₄ administration in day 7 the animals were sacrificed using ether anesthesia. Blood samples were collected by heart puncture and the serum was separated for evaluating the biochemical parameters.

2.7. Determination of AST, ALT, GGT, ALP, bilirubin, creatinine, urea, potassium and sodium levels

The biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and total bilirubin were estimated by reported methods (Edwards and Bouchier, 1991). The enzyme activities were measured using diagnostic strips (Reflotron^®, ROCHE) and were read on a Reflotron^® Plus instrument (ROCHE). Serum creatinine and blood urea were assayed using Randox Diagnostic kits (Randox Laboratories Ltd., Crumlin, U.K.) by the reported method (Varley and Alan,1984). Potassium level was measured using diagnostic strips (Reflotron^®, ROCHE) while photometric determination of sodium level was done using Mg-uranylacetate method (Henry et al., 1974).

2.8. Determination of non-protein sulfhydryl groups (NP-SH), malonaldehyde (MDA) and total protein (TP)

The liver and kidney samples were separately cooled in a beaker immersed in an ice bath. The tissues were homogenized in 0.02 M ethylenediaminetetraacetic acid (EDTA) in a Potter–Elvehjem type C homogenizer. Homogenate equivalents of 100 mg tissues were used for the measurements. Non-protein sulfhydryl groups (NP-SH) were quantified by mixing homogenated with 4 ml of distilled water and 1 ml of 50% trichloroacetic acid (TCA) in 15 ml test tubes. The tubes were shaken intermittently for 10–15 min and centrifuged for 15 min at approximately 3000 rpm to precipitate the protein. 2 ml of the supernatant was mixed with 4 ml of 0.4 M Tris buffer, pH 8.9 and 0.1 ml of 0.01 M DTNB [5,5′-dithio-bis-(2-nitrobenzoic acid)] and the samples were shaken. The absorbance was measured spectrophotometrically within 5 min of addition of DTNB at 412 nm against a reagent blank with no homogenate (Sedlak and Lindsay, 1968).

For the level of MDA aliquots of homogenate were incubated at 37 °C for 3 h in a metabolic shaker. Then 1 ml of 10% aqueous TCA was added and mixed. The mixture was then centrifuged at 800 rpm for 10 min. One milliliter of the supernatant was removed and mixed with 1 ml of 0.67% w-thiobarbituric acid in water and placed in a boiling water bath for 10 min. The mixture was cooled and diluted with 1 ml distilled water. The absorbance of the solution was then measured at 535 nm. The content of MDA (nmol/g wet tissue)(index of the magnitude of lipid peroxidation) was then calculated, by reference to a standard curve of MDA solution (Utley et al., 1967).

For TP determination parts of the homogenate were treated with 0.7 ml of Lowry’s solution, mixed and incubated for 20 min in dark at room temperature. Diluted Folin’s reagent (01 ml) was then added and samples were incubated at room temperature in dark for 30 min. The absorbance of the resulted solutions was then measured at 750 nm (Lowry et al., 1951).

2.9. Histopathology

The fixed liver and kidney samples were placed in cassettes and loaded into tissue baskets. They were subjected to dehydration, clearing and infiltration by immersion in different concentrations of ethanol (70–100%), xylene (3 times, 1 h each) and finally paraffin wax (4 times, 1 h each). The tissues were then transferred into molds filled with paraffin wax. After orienting, the tissues by hot forceps the molds were chilled on cold plates and excess wax were trimmed off using a knife. The rotary microtome (Leitz 1512) was used for making thin sections (3 μm). The sections were placed onto clean slides that were drained vertically for several minutes before placing them onto a warming table at 37–40 °C (Prophet et al., 1994). The slides were then deparaffinized, hydrated and stained in Mayer’s hematoxylin solution for 15 min. The slides were then washed in lukewarm running tap water for 15 min and placed in distilled water. After they were immersed in 80% ethyl alcohol for one to two minutes they were counterstained in eosin-phloxine solution for 2 min. The slides were then dehydrated and cleared through two changes each of 95% ethyl alcohol, absolute ethyl alcohol, and xylene (2 min each) and finally mounting with resinous medium.

2.10. Antiulcer activity

Rats were divided into thirteen groups of 5 animals each. All animals received 1 ml 80% ethanol. Group I served as positive control. Groups II–XI were treated with 100 and 200 mg/kg of different fractions, while groups XII and XIII received 200 and 400 mg/kg of the total extract 30 min before the administration of ethanol. One hour after the administration of ethanol, rats were scarified and examined for stomach lesions. Patchy lesions of the stomach induced by ethanol were scored according to the method described by Robert et al. (1983).

2.11. Determination of whole blood clotting time (CT)

Clotting time was determined according to the reported method (Dacie and Lewis, 1968). Rats were divided into thirteen groups of 5 animals each. Group I received 10 mg/kg of warfarin and served as positive control. Groups II–XI were treated with 100 and 200 mg/kg of different fractions, while groups XII and XIII received 200 and 400 mg/kg of the total extract. 0.4 ml of blood samples were collected at 0, 30, 60, 120 min from the retrorbital sinus venous channels. The blood samples were dispensed separately into prewarmed test tubes fixed in a rack previously placed in a water bath maintained at 37 °C. Tubes were tilted to check for the sign of blood clot every 30 s. Using a stopwatch, the time interval between blood collection and the time the clot appearing in each test tube was recorded in min.

2.12. In vitro antioxidant activity using DPPH radical scavenging assay

The method was carried out as described by Brand et al. (1995). Various concentrations (10, 50, 100, 500 and 1000 μg/ml) of the crude extract and fractions were used. The assay mixtures contained in total volume of 1 ml, 500 μL of the extract, 125 μL prepared DPPH and 375 μL solvent. Ascorbic acid was used as the positive control. After 30 min incubation at 25 °C, the decrease in absorbance was measured at λ = 517 nm. The radical scavenging activity was calculated from the equation:

$$\%\text{radical scavenging activity}=((\text{A control}-\text{A sample})/\text{A control})\times 100$$

2.13. Statistical analyses

For each set of experiments where two or more than two groups were compared, an analysis of variance (ANOVA) test was used to determine the significance of the differences. Differences between the control and CCl₄-treated group were compared for significance using Dunnette test for non paired samples (Woolson and Clarke, 2002). All the values shown are the mean ± S.E.

3. Results

Phytochemical study of the aerial parts of F. palmata resulted in the isolation of two furanocoumarin derivatives, rutin, germanicol acetate, vanillic acid and psoralenoside methyl ether. The structures of the compounds are presented in Fig. 1. The total extract and different fractions were tested for hepatoprotective and nephroprotective effect against CCl₄ induced toxicity. The results are presented in Table 1, Table 2, Table 3. Antiulcer potential of the extract and fractions was also explored against ethanol-induced lesions. Results are presented in Table 4. The results of anticoagulant effect in comparison with warfarin based on whole blood clotting time (CT) are presented in Table 5. Total extract and fractions were tested for their antioxidant activity using DPPH radical scavenging assay. Results of this study are presented in Table 6.

Fig. 1.

Histopathological appearance of Liver cells; (L-1) normal cells; (L-2) liver cells of rats treated with CCl₄ showed Kupffer cells activation, cytoplasmic vacuolization of hepatocytee and focal hepatic necrosis associated with inflammatory cells infiltration; (L-3) liver cells of rats treated with CCl₄ and Sil small focal hepatic necroses with few inflammatory cells infiltration and slight activation of kupffer cells; (L-4) liver cells of rats treated with CCl₄ and 400 mg/kg total extract showing excellent recovery with absences of histopathological changes; (L-5) liver cells of rats treated with CCl₄ and 200 mg/kg total extract showing slight hydropic degeneration of some hepatocytes; (L-6) (L-7) liver cells treated with 100 and 200 mg/kg of the petroleum ether fraction resulted in complete protection and absence of any histopathological changes; (L-8) liver cells treated with 200 mg/kg chloroform fraction showing moderate protection of hepatocytes with only slight hydropic degeneration.

Table 1.

Effect of F. palmata total extract and fractions on serum biochemical parameters.

Treatment (n = 5)	Dose (mg/kg)	Biochemical parameters
		AST (units/l)		ALT (units/l)		GGT (units/l)		ALP (units/l)		Bilirubin (mg/dl)
		Mean ± S.E.	% Decrease	Mean ± S.E.	% Decrease	(Mean ± S.E.)	% Decrease	Mean ± S.E.	% Decrease	Mean ± S.E.	% Decrease
Normal		94.33 ± 3.52		34.63 ± 4.01		3.73 ± 0.24		313.33 ± 14.52		0.56 ± 0.02
CCl4	1.25 ml/kg	193.66 ± 4.66⁎⁎⁎		165.00 ± 5.03⁎⁎⁎		9.53 ± 0.37⁎⁎⁎		526.66 ± 20.27⁎⁎⁎		2.78 ± 0.08⁎⁎⁎
Sily. + CCl4	10	111.73 ± 10.35⁎⁎⁎	42.30	53.16 ± 5.80⁎⁎⁎	67.77	4.40 ± 0.36⁎⁎⁎	53.84	357.66 ± 9.20⁎⁎⁎	32.08	0.98 ± 0.11⁎⁎⁎	64.55
Total	200	149.66 ± 7.51⁎⁎	22.71	113.16 ± 7.99⁎⁎	31.41	6.76 ± 0.35⁎⁎	29.02	442.66 ± 22.45⁎	15.94	1.66 ± 0.13⁎⁎⁎	40.11
Total	400	140.66 ± 2.60⁎⁎⁎	27.36	99.23 ± 4.80⁎⁎⁎	39.85	6.70 ± 0.47⁎⁎	29.72	417.66 ± 10.92⁎⁎	20.69	1.34 ± 0.13⁎⁎⁎	51.61
Pet. ether	100	166.33 ± 3.71⁎⁎	14.11	115.66 ± 7.05⁎⁎⁎	29.89	7.50 ± 1.02	21.32	493.33 ± 11.66	6.32	1.61 ± 0.12⁎⁎⁎	41.91
Pet. ether	200	147.66 ± 5.92⁎⁎⁎	23.75	95.06 ± 4.78⁎⁎⁎	42.38	5.96 ± 0.47⁎⁎	37.41	487.33 ± 11.78	7.46	1.45 ± 0.07⁎⁎⁎	47.90
CHCl3	100	168.66 ± 7.21⁎	12.90	151.33 ± 4.97	8.28	7.60 ± 0.40⁎	20.27	459.66 ± 13.04⁎	12.72	2.68 ± 0.03	3.59
CHCl3	200	144.00 ± 3.60⁎⁎⁎	25.64	135.66 ± 3.75⁎⁎	17.77	6.26 ± 0.23⁎⁎⁎	34.26	418.33 ± 10.47⁎⁎	20.56	2.20 ± 0.15⁎	20.95
EtOAc	100	189.33 ± 2.60	2.23	172.66 ± 4.63		8.60 ± 0.32	9.78	528.33 ± 19.05		2.60 ± 0.13	6.47
EtOAc	200	182.66 ± 6.69	5.67	153.66 ± 6.64	6.86	8.53 ± 0.40	10.48	557.33 ± 22.51		2.27 ± 0.14⁎	18.32
n-Butanol	100	186.33 ± 6.38	3.78	155.33 ± 4.91	5.85	8.76 ± 0.21	8.04	553.00 ± 5.68		2.62 ± 0.14	5.86
n-Butanol	200	148.66 ± 9.52⁎⁎	23.23	154.33 ± 9.26	6.46	9.10 ± 0.20	4.54	525.33 ± 13.77		2.60 ± 0.10	6.58
Water	100	173.66 ± 10.47	10.32	146.33 ± 7.53	11.31	8.23 ± 0.20⁎	13.63	482.66 ± 10.71	8.35	2.74 ± 0.13
Water	200	153.00 ± 5.68⁎⁎	20.99	133.33 ± 5.04⁎⁎	19.19	7.66 ± 0.23⁎⁎	19.58	481.00 ± 30.51	8.67	2.47 ± 0.09⁎	11.13

^⁎p < 0.05.

^⁎⁎p < 0.01.

^⁎⁎⁎p < 0.001.

Table 2.

Effect of F. palmata total extract and fractions on kidney function.

Treatment (n = 5)	Dose (mg/kg)	Biochemical parameters
		Sodium (mmol/l)		Potassium (mmol/l)		Creatnine (mg/dl)		Urea (mg/dl)
		Mean ± S.E.	% Change	Mean ± S.E.	% Change	(Mean ± S.E.)	% Change	Mean ± S.E.	% Change
Normal		56.56 ± 4.42		2.83 ± 0.17		78.06 ± 6.13		6.03 ± 0.35
CCl4	1.25 ml/kg	192.33 ± 11.05⁎⁎⁎		11.80 ± 0.60⁎⁎⁎		177.33 ± 8.00⁎⁎⁎		17.66 ± 1.20⁎⁎⁎
Sily. +CCl4	10	96.60 ± 7.84⁎⁎⁎	49.77	3.86 ± 0.14⁎⁎⁎	67.23	131.66 ± 7.05⁎⁎	25.75	11.60 ± 0.89⁎⁎	34.33
Total	200	143.33 ± 5.78⁎⁎	25.47	7.63 ± 0.24⁎⁎⁎	35.31	134.33 ± 4.63⁎⁎	24.24	10.46 ± 0.53⁎⁎	40.75
Total	400	136.66 ± 6.69⁎⁎	28.94	6.46 ± 0.37⁎⁎⁎	45.19	116.53 ± 9.19⁎⁎	34.28	8.70 ± 0.61⁎⁎⁎	50.56
Pet. ether	100	138.33 ± 14.26⁎	28.07	7.40 ± 0.70⁎⁎	37.28	127.66 ± 6.96⁎⁎	28.00	9.86 ± 0.23⁎⁎⁎	44.15
Pet. ether	200	119.33 ± 3.75⁎⁎⁎	37.95	5.60 ± 0.16⁎⁎⁎	52.54	126.66 ± 10.39⁎⁎	28.57	6.90 ± 0.51⁎⁎⁎	60.94
CHCl3	100	151.66 ± 10.47⁎	21.14	8.30 ± 0.64⁎⁎	29.66	125.66 ± 30.47⁎⁎	15.22	10.20 ± 0.75⁎⁎	42.26
CHCl3	200	134.00 ± 5.68⁎⁎	30.32	7.40 ± 0.36⁎⁎⁎	37.28	150.33 ± 11.46	29.13	9.36 ± 0.40⁎⁎⁎	46.98
EtOAc	100	176.66 ± 10.65	8.14	10.63 ± 0.26	9.88	173.00 ± 4.35	2.44	14.20 ± 0.45⁎	19.62
EtOAc	200	169.66 ± 11.25	11.78	10.36 ± 0.35	12.14	166.66 ± 3.48	6.01	13.06 ± 0.76⁎	26.03
n-Butanol	100	166.66 ± 13.54	13.34	10.73 ± 0.63	9.03	157.00 ± 4.16	11.46	15.13 ± 1.40	14.33
n-Butanol	200	169.66 ± 7.31	11.78	10.40 ± 0.58	11.86	154.66 ± 6.96	12.78	14.56 ± 0.52⁎	17.54
Water	100	180.66 ± 8.37	6.06	10.23 ± 0.47	13.27	159.33 ± 8.83	10.15	15.00 ± 0.73	15.09
Water	200	158.33 ± 4.80⁎	17.67	9.46 ± 0.42⁎	19.77	161.66 ± 12.23	8.83	13.50 ± 0.51⁎	23.58

^⁎p < 0.05.

^⁎⁎p < 0.01.

^⁎⁎⁎p < 0.001.

Table 3.

Effect of F. palmata total extract and fractions on NP-SH, MDA and TP in rat liver and kidney.

Treatment (n = 5)	Dose (mg/kg)	NP-SH (nmol/l)		MDA (nmol/l)		Protein (g/l)
		Liver	Kidney	Liver	Kidney	Liver	Kidney
Normal		5.55 ± 0.33	11.86 ± 0.86	1.07 ± 0.08	1.60 ± 0.08	129.03 ± 6.05	81.93 ± 3.22
CCl4	1.25 ml/kg	1.18 ± 0.17∗∗∗	3.48 ± 0.58⁎⁎⁎	4.60 ± 0.47⁎⁎⁎	6.15 ± 0.25⁎⁎⁎	57.41 ± 3.22⁎⁎⁎	35.48 ± 1.93⁎⁎⁎
Sily. +CCl4	10	4.34 ± 0.34⁎⁎⁎	6.65 ± 0.43⁎⁎	2.87 ± 0.43⁎	3.44 ± 0.20⁎⁎⁎	74.83 ± 8.09	57.41 ± 3.22⁎⁎
Total	200	1.96 ± 0.12⁎	7.22 ± 0.78⁎	2.54 ± 0.22⁎⁎	3.74 ± 0.23⁎⁎⁎	80.00 ± 4.82⁎⁎	51.61 ± 4.08⁎
Total	400	2.46 ± 0.22⁎⁎	8.23 ± 0.69⁎⁎	1.99 ± 0.12⁎⁎	2.56 ± 0.26⁎⁎⁎	96.12 ± 6.44⁎⁎	62.58 ± 4.51⁎⁎
Pet. ether	100	4.50 ± 0.46⁎⁎⁎	6.95 ± 0.50⁎⁎	2.02 ± 0.25⁎⁎	2.94 ± 0.23⁎⁎⁎	90.96 ± 8.04⁎⁎	59.35 ± 4.08⁎⁎
Pet. ether	200	4.55 ± 0.49⁎⁎⁎	8.96 ± 0.35⁎⁎⁎	1.49 ± 0.10⁎⁎⁎	2.08 ± 0.16⁎⁎⁎	110.96 ± 4.08⁎⁎⁎	72.25 ± 2.78⁎⁎⁎
CHCl3	100	2.94 ± 0.40⁎⁎	7.48 ± 0.94⁎	2.41 ± 0.10⁎⁎	3.42 ± 0.46⁎⁎	79.35 ± 3.99⁎⁎	52.25 ± 5.20⁎
CHCl3	200	3.24 ± 0.34⁎⁎	9.29 ± 0.21⁎⁎⁎	1.95 ± 0.18⁎⁎	2.60 ± 0.23⁎⁎	90.96 ± 6.35⁎⁎	67.09 ± 4.08⁎⁎⁎
EtOAc	100	1.64 ± 0.13	7.38 ± 0.465⁎⁎	4.74 ± 0.31	5.69 ± 0.51	52.90 ± 3.41	36.77 ± 2.86
EtOAc	200	1.57 ± 0.10	7.60 ± 0.83⁎	4.52 ± 0.34	4.88 ± 0.15⁎⁎	56.12 ± 6.77	40.00 ± 2.68
n-Butanol	100	1.31 ± 0.28	3.07 ± 0.39	3.91 ± 0.32	3.82 ± 0.26⁎⁎⁎	65.16 ± 4.26	52.25 ± 2.86⁎⁎
n-Butanol	200	1.27 ± 0.17	2.41 ± 0.32	4.00 ± 0.37	3.27 ± 0.06⁎⁎⁎	62.58 ± 6.61	60.64 ± 3.07⁎⁎⁎
Water	100	1.03 ± 0.06	3.70 ± 0.94	3.14 ± 0.14⁎	4.83 ± 0.41⁎	74.83 ± 2.78⁎⁎	45.80 ± 2.86⁎
Water	200	1.38 ± 0.13	2.89 ± 0.35	2.81 ± 0.22⁎	4.38 ± 0.41=	79.35 ± 5.20⁎	49.67 ± 3.85⁎

^⁎p < 0.05.

^⁎⁎p < 0.01.

^⁎⁎⁎p < 0.001.

Table 4.

Effect of F. palmata total extract and fractions on the gastric lesion induced by 80% ethanol.

Treatments	Dose (mg/kg)	Ulcer index (Mean ± S.E.)
Ethanol 80%	1 ml	8.00 ± 0.57
Total ext. + ethanol 80%	200	4.00 ± 0.57⁎⁎
Total ext. + ethanol 80%	400	2.00 ± 0.57⁎⁎⁎
Pet. ether + ethanol 80%	100	5.00 ± 0.57⁎⁎
Pet. ether + ethanol 80%	200	3.33 ± 0.33⁎⁎
CHCl3 + ethanol 80%	100	6.33 ± 0.66
CHCl3 + ethanol 80%	200	4.00 ± 0.57⁎⁎
EtoAC + ethanol 80%	100	7.66 ± 0.33
EtoAC + ethanol 80%	200	6.66 ± 0.66
n-Butanol + ethanol 80%	100	8.00 ± 0.57
n-Butanol + ethanol 80%	200	7.33 ± 0.66
Water + ethanol 80%	100	7.66 ± 0.88
Water + ethanol 80%	200	6.00 ± 0.57⁎

^⁎p < 0.05.

^⁎⁎p < 0.01.

^⁎⁎⁎p < 0.001.

Table 5.

Effect of F. palmata total extract and fractions on clotting time.

Treatment (n = 5)	Dose (mg/kg)	0 min	30 min	60 min	120 min
Norma saline	1 ml	2.46 ± 0.14	2.8 ± 0.05	2.46 ± 0.08	2.63 ± 0.08
Warfarin	10	2.60 ± 0.05	5.70 ± 0.32⁎⁎⁎	7.83 ± 0.63⁎⁎⁎	8.23 ± 0.67⁎⁎⁎
Total	200	2.33 ± 0.17	4.23 ± 0.37⁎⁎	5.46 ± 0.38⁎⁎⁎	6.16 ± 0.24⁎⁎⁎
Total	400	2.83 ± 0.17	5.20 ± 0.26⁎⁎⁎	6.00 ± 0.43⁎⁎	6.70 ± 0.26⁎⁎⁎
Pet. ether	100	2.66 ± 0.12	2.80 ± 0.05	4.06 ± 0.31⁎⁎	5.70 ± 0.32⁎⁎⁎
Pet. ether	200	2.83 ± 0.17	4.53 ± 0.38⁎⁎	5.63 ± 0.56⁎⁎	5.70 ± 0.32⁎⁎⁎
CHCl3	100	2.83 ± 0.17	5.10 ± 0.34⁎⁎	4.93 ± 0.20⁎⁎⁎	5.03 ± 0.24⁎⁎⁎
CHCl3	200	2.36 ± 0.24	3.10 ± 0.34	4.60 ± 0.26⁎⁎	5.10 ± 0.25⁎⁎⁎
EtOAc	100	3.06 ± 0.08	3.23 ± 0.43	3.13 ± 0.17	3.30 ± 0.25
EtOAc	200	2.86 ± 0.20	3.36 ± 0.35	2.83 ± 0.17	3.50 ± 0.37
n-Butanol	100	2.56 ± 0.35	2.96 ± 0.32	3.23 ± 0.20	3.60 ± 0.26
n-Butanol	200	2.56 ± 0.40	3.60 ± 0.47	4.33 ± 0.40⁎	4.60 ± 0.26⁎⁎
Water	100	3.03 ± 0.27	5.03 ± 0.17⁎⁎	5.23 ± 0.23⁎⁎	4.93 ± 0.58⁎
Water	200	3.03 ± 0.24	4.00 ± 0.36	5.03 ± 0.17⁎⁎	5.70 ± 0.32⁎⁎

^⁎p < 0.05.

^⁎⁎p < 0.01.

^⁎⁎⁎p < 0.001.

Table 6.

Free radical-scavenging activity (DPPH-assay).

Treatment	Radical scavenging activity (%)
	10	50	100	500	1000
Total ext.	26.1	27.0	40.9	93.8	94.5
Pet. ether	2.7	7.8	11.7	22.8	47.8
CHCl3	6.0	22.1	48.1	92.3	92.1
EtOAc	30.2	80.9	97.3	97.0	96.5
n-butanol	8.9	11.4	37.0	83.0	92.5
Water	11.1	18.8	23.2	28.3	50.8
Ascorbic acid (STD)	41.0	86.4	95.5	98.1	98.3

3.1. The physical and spectral data of compounds 1–6

3.1.1. Germanicol acetate (1)

C₃₂H₅₂O₂; White crystals; mp 279–280 °C; ¹H NMR (500 MHz, CDCl₃): δ_H 0.73 (3H,s, H-27), 0.84 (3H,s, H-24), 0.85 (3H,s, H-23), 0.91 (3H,s, H-26), 0.93 (3H,s, H-29), 0.94 (3H,s, H-30), 1.02 (3H,s, H-28), 1.08 (3H,s, H-25), 2.05 (3H, s, CH₃-CO), 4.49 (1H, dd, J = 11.5, 6.2 Hz, H-3α), 4.80 (1H, s, H-19); ¹³C NMR (125 MHz, CDCl₃): δ_C 14.6 (C-27), 16.1 (C-25), 16.5 (C-24), 16.8 (C-26), 18.2 (C-6), 21.1 (C-11), 21.3 (CO-CH₃), 23.7 (C-2), 25.3 (C-28), 26.2 (C-12), 27.5 (C-15), 27.9 (C-23), 29.2 (C-30), 31.4 (C-29), 32.4 (C-20), 33.3 (C-21), 34.4 (C-17), 34.5 (C-7), 37.2 (C-22), 37.4 (C-16), 37.8 (C10), 38.4 (C-4), 38.5 (C-1), 38.6 (C-13), 40.8 (C-8), 43.3 (C-14), 51.1 (C-9), 55.6 (C-5), 81.0 (C-3), 129.8 (C-19), 142.7 (C-18), 171.1 (CO-CH₃); ESIMS m/z 491 (19, [M + Na]⁺), 469 (100, [M + H]⁺).

3.1.2. Psoralene (2)

C₁₁H₆O₃; Colorless crystals; mp 162–163 °C; UV λmax (MeOH): 240, 245, 292, 327; ¹H NMR (500 MHz, CDCl₃): δ_H 6.40 (d, J = 9.5 Hz, H-3), 7.49 (s, H-8), 7.50 (d, J = 1.5 Hz, H-3′), 7.71 (s, H-5), 7.72 (d, J = 1.5 Hz, H-2′), 7.82 (d, J = 9.5 Hz, H-4); ¹³C NMR (125 MHz, CDCl₃): δ_C 99.88 (C-8), 106.39 (C-3′), 114.66 (C-3), 115.42 (C-10), 119.85 (C-5), 124.88 (C-6), 144.10 (C-4), 146.92 (C-2′), 152.04 (C-9), 156.42 (C-7), 161.04 (C-2); ESIMS m/z 209 (33, [M + Na]⁺), 187 (100, [M + H]⁺).

3.1.3. Bergapten (5-Methoxypsoralen) (3)

C₁₂H₈O₄; White powder; mp191–192 °C; UV λmax (MeOH): 221, 248, 260, 269, 311; ¹H NMR (500 MHz, CDCl₃): δ_H 6.29 (d, J = 9.5 Hz, H-3), 7.15 (s, H-8), 7.04 (d, J = 1.5 Hz, H-3′), 7.61 (d, J = 1.5 Hz, H-2′), 8.17 (d, J = 9.5 Hz, H-4); ¹³C NMR (125 MHz, CDCl₃): δ_C 60.09 (OCH₃), 93.82 (C-8), 105.06 (C-3′), 105.06 (C-10), 112.53 (C-3), 112.66 (C-6), 139.29 (C-4), 144.80 (C-2′), 149.58 (C-9), 152.70 (C-5), 158.38 (C-7), 161.26 (C-2); ESIMS m/z 239 (24, [M + Na]⁺), 217 (100, [M + H]⁺).

3.1.4. Vanillic acid (4)

C₈H₈O₄; White powder; mp 210–211 °C; UV λmax (MeOH): 258, 292; ¹H NMR (500 MHz, DMSO-d₆): δ_H 3.81 (3H, s, OCH₃), 6.85 (1H, d, J = 8 Hz, H-5), 7.47 (1H, d, J = 2 Hz, H-2), 7.52 (1H, dd, J = 8, 2 Hz, H-6); ¹³C NMR (125 MHz, DMSO-d₆): δ_C 55.42 (OCH₃), 112.64 (C-2), 114.99 (C-5), 121.76 (C-1), 123.47 (C-6), 147.14 (C-3), 150.99 (C-4), 167.37 (C O); ESIMS m/z 191 (42, [M + Na]⁺), 169 (100, [M + H]⁺).

3.1.5. Psoralenoside (5)

C₁₇H1₈O₉; White powder; mp 253 °C; UV λmax (MeOH): 244, 285, 324; ¹H NMR (500 MHz, CD₃OD): δ_H 3.45 (m, H-4″), 3.53 (m, H-3″), 3.53 (m, H-5″), 3.61 (t, J = 8 Hz, H-2″), 3.68 (dd, J = 5.5, 10.5 Hz, H-6″), 3.95 (bd, J = 10.5 Hz, H-6″), 5.03 (d, J = 8.25 Hz, H-1″), 6.57 (d, J = 16.5 Hz, H-3), 6.84 (d, J = 2 Hz, H-3’), 7.46 (s, H-8), 7.74 (d, J = 2 Hz, H-2′), 7.93 (s, H-5), 8.20 (d, J = 16.5 Hz, H-4); ¹³C NMR (125 MHz, CD₃OD): δ_C 61.02 (C-6″), 69.81 (C-4″), 73.38 (C-2″), 76.59 (C-3″), 76.81 (C-5″), 98.76 (C-8), 101.78 (C-1″), 106.01 (C-3′), 117.27 (C-10), 119.27 (C-3), 120.58 (C-6), 122.45 (C-5), 140.37 (C-4), 145.64 (C-2′), 154.27 (C-9), 156.75 (C-7), 176.50 (C-2); ESIMS m/z 389 (18, [M + Na]⁺), 367 (100, [M + H]⁺).

4. Discussion

Phytochemical investigation of the aerial parts of Ficus palmata resulted in the isolation of 6 compounds germanicol acetate (1) (Gonzàlez et al., 1981, Dat et al., 2002), psoralene (2) (O’Neil, 2001; Abu-Mustafa and Fayez, 1967), bergapten (5-methoxypsoralen) (3), vanillic acid (4) (Pouchert and Behnke, 1992) and the flavone glycoside rutin (6) (Bilia et al., 1996) which were identified by a comparison of their physical and spectral data with literature as well as direct comparison with authentic material whenever available.

¹³C NMR showed signals for 6 oxygenated carbons from δ_C 61.02 to 76.81 and 101.78 ppm and their correlated protons from δ_H 3.45 to 5.03 ppm in the ¹H NMR assigned for β-d-glucopyranoside (experimental) indicating the glycosidic nature of 5. The aglycone part showed 11 carbon signals and 6 protons. The signals were comparable to those of 2 with some significant differences. As 2 have no free OH for glycosilation one should expect a ring opening to obtain 5. C-2 in 5 showed signal at δ_C 176.50 while the same signal in 2 appeared at δ_C 161.04 ppm (experimental) indicating a ring opening of the α-pyrone ring in 5 and conversion of C-2 into carboxylic group. The resulting hydroxyl at C-9 consequently is the site of connection of glucose. This fact was proved undoubtedly from the results of an HMBC experiment where the H-1″ proton signal of glucose at δ_H 5.03 ppm showed correlation with the C-9 carbon at δ_C 154.27 ppm. A closely related compound was isolated from Psoralea cotylifolia and named psoralenoside (Qiao et al., 2006). The major difference between psoralenoside and 5 is the orientation around C-3, C-4 double bond. In case psoralenoside the J_3,4 = 12.4 Hz (Qiao et al., 2006) indicates a cis-orientation of the two protons, however, that value in 5 is 16.5 Hz (experimental) clearly indicates a trans-orientation between H-3 and H-4. From the above discussion 5 was identified as a new isomer of psoralenoside and the name trans- psoralenoside was proposed to differentiate from the known cis isomer.

4.1. Serum parameters related to hepatoprotective activity

Hepatic toxicity following CCl₄ administration is reflected by increase in the biochemical parameter levels such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and total bilirubin (Table 1) (Edwards and Bouchier, 1991). Pretreatment of rats with Sil, significantly (p < 0.001) decreased the raised levels of AST, ALT, GGT, ALP and bilirubin induced by CCl₄ (42.30, 67.77, 53.84, 32.08 and 64.55% respectively) (Table 1) indicating a good recovery from the hepatotoxic agent. Treatment with F. palmata total extract showed dose dependent reduction in the levels of all the measured parameters. Animal treated with 400 mg/kg body weight of F. palmata showed a significant (p < 0.01–p < 0.001) reduction in the levels of AST, ALT, GGT, ALP and bilirubin (27.36, 39.85, 29.72, 20.69, and 51.61%) indicating good protection against liver damage induced by CCl₄. Treatment with 200 mg/kg body weight resulted in less improvement in the parameters, However, all the results were highly significant (p < 0.01–p < 0.001) except reduction in ALP (15.94%, p < 0.05) (Table 1).

All fractions were tested for hepatoprotective effect at 100 and 200 mg/kg body weight. The best protective effect was obtained with petroleum ether and chloroform fractions. 200 mg/kg body weight of the petroleum ether showed better results than 200 mg/kg body weight of the total extract in reducing the elevated levels of ALT, GGT, ALP and bilirubin. The higher dose of the chloroform fractions was better than the total extract in lowering the levels of AST, GGT and ALP (Table 1). The water layer at 200 mg/kg body weight showed some activity while the ethyl acetate and butanol were almost inactive (Table 1).

4.2. Serum parameters related to nephroprotective activity

The Kidney regulates plasma ionic composition including sodium, potassium, calcium, magnesium, chloride. It is also concerned with the removal of nitrogenous metabolic waste products such as urea, creatinine and uric acid (Pocock and Richards, 2006). Elevations of serum electrolytes, urea and creatinine are considered reliable parameters for investigating drug-induced nephrotoxicity in animals and man (Adelman et al., 1981). CCl₄ exhibits a significant rise in the biochemical markers of kidney functions like serum urea, serum creatinine, sodium and potassium levels (Table 2). Pretreatment with Sil (10 mg/kg p.o) decreased the raised levels of serum urea, serum creatinine, percentage of sodium and potassium (34.33, 25.75, 49.77 and 67.23%) induced by CCl₄ (Table 2). Dose dependent reduction in the elevated parameters resulted from the treatment with F. palmata total extract. Animals treated with 400 mg/kg body weight of F. palmata showed highly significant (p < 0.01–p < 0.001) reduction in the levels of serum urea, serum creatinine, sodium and potassium levels (50.56, 34.28, 28.94 and 45.19%) indicating a good protection against CCl₄ induced nephrotoxicity. Reduction in the levels of serum urea and serum creatinine was more than that resulted from the treatment with the standard drug Sil. Treatment with 200 mg/kg body weight resulted in less protection than the higher dose and significance ranged from p < 0.01–p < 0.01. Reduction in the levels of serum urea was more than that resulted from treatment with the standard drug Sil (Table 2).

At 100 mg/kg dose the petroleum ether fraction improved all the measured parameters with values slightly better than those resulted from pretreatment with total extract at 200 mg/kg body weight. Similarly, the chloroform fraction at the higher dose improved the levels of urea, creatinin, sodium and potassium (30.32, 37.28, 29.13 and 46.98% respectively). However, the 100 mg/kg body weight was less effective than the lower dose of the total extract except in the level of potassium that was reduced by 42.26%. The effect of the other fractions was very weak and statistically insignificant (Table 2).

4.3. Tissue parameters related to both hepatoprotective and nephroprotective

Glutathione-S-transferases (GSTs) catalyze the transfer of reduced glutathione (GSH) to reactive electrophiles, a function that serves to protect cellular macromolecules from interaction with electrophiles that contain electrophilic heteroatoms (–O, –N, and –S) and in turn protects the cellular environment from damage. Severe reduction in GSH content can predispose cells to oxidative damage, a state that is linked to a number of human health issues (Brunton et al., 2006).

Treatment of animals with CCl₄ at a dose of 400 mg/kg decreased the hepatic and renal NP-SH from 5.55 ± 0.33, 11.86 ± 0.86 to 1.18 ± 0.17, 3.48 ± 0.58 μmol/gm wet weight tissue, respectively (Table 3). Pretreatment of the animals with Sil at a dose of 10 mg/kg significantly increased the reduced NP-SH content (p < 0.01, P < 0.001) to 4.34 ± 0.34 and 6.65 ± 0.43 μmol/gm wet weight tissue in hepatic and renal tissues respectively. Animals that received the total extract of F. palmata showed a significant dose dependent recovery of the NP-SH contents. Pre-treatment with 400 mg/kg F. palmata total extract significantly increased the reduced content of NP-SH in the liver and kidney tissues to 2.46 ± 0.22 and 8.23 ± 0.69 μmol/gm wet weight tissues respectively (p < 0.01). The 200 mg/kg dose was less effective but statistically significant (p < 0.05). The NP-SH contents in the liver and kidney tissues were increased to 1.96 ± 0.12 and 7.22 ± 0.78 μmol/gm wet weight tissues respectively. Restoring the NP-SH content in the kidney tissue exceeded that caused by Sil (Table 3). Treatment with the petroleum ether fraction at 200 and 100 mg/kg resulted in a highly significant improvement in the liver contents of NP-SH (4.55 ± 0.49 and 4.50 ± 0.46 μmol/gm wet weight tissue, respectively) comparable to that caused by Sil. Regarding the kidney contents of NP-SH the higher dose of the petroleum ether fraction restored the NP-SH contents to 8.96 ± 0.35 μmol/gm wet weight tissue exceeding the improvement resulted from treatment of the total extract and Sil The results were highly significant. The chloroform fraction at 200 mg/kg resulted in a highly significant best improvement in the NP-SH contents in this study (9.29 ± 0.21 μmol/gm wet weight tissue). The effect of the ethyl acetate fraction at the two doses used was comparable to that of the lower dose of the extract (Table 3).

Malonaldehyde (MDA) is the main end-product of polyunsaturated fatty acid peroxidation (PUFA) following Reactive oxygen species (ROS) insult (Esterbauer et al., 1991). MDA is a reactive aldehyde and is one of many reactive electrophile species that cause toxic stress in cells and form covalent protein adducts (Farmer and Davoine, 2007). The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism (Del Rio et al., 2005). Normal control group showed 1.07 ± 0.08 and 1.60 ± 0.08 nmol/l concentrations of MDA in their healthy liver and kidney tissues respectively. The levels of MDA greatly increased after CCl₄ treatment to 4.060 ± 0.47 and 6.15 ± 0.25 nmol/l in liver and kidney tissues respectively. The standard drug Sil was effective in reducing these elevated levels to 2.87 ± 0.43 and 3.44 ± 0.20 nmol/l in liver and kidney tissues respectively. F. palmata total extract resulted in a dose dependent reduction in the MDA levels. Treatment of the animals with 400 mg/kg body weight decreased the level of MDA to 2.54 ± 0.22 nmol/l (p < 0.01) in liver tissues and 3.74 ± 0.23 nmol/l (p < 0.001) in kidney tissues reflecting the level of protection similar to that of Sil (Table 3). Both petroleum ether and chloroform fractions at the 200 mg/kg resulted in a highly significant (p < 0.001 and p < 0.01, respectively) decrease in the level of MDA in liver tissues to 1.49 ± 0.10 and 1.95 ± 0.18 nmol/l, respectively. Similar improvement by the two fractions was observed in the kidney tissues where the MDA contents were reduced to 2.08 ± 0.16 and 2.60 ± 0.23 nmol/l. The resulted improvement exceeded that resulted from pretreatment with the standard drug Sil in the liver and kidney tissues (2.87 ± 0.43 and 3.44 ± 0.20 nmol/l, respectively) (Table 3).

One of the most important liver functions is protein synthesis. Liver damage causes disruption and disassociation of polyribosomes on the endoplasmic reticulum thereby reducing the biosynthesis of protein. The TP levels depressed in hepatotoxic conditions due to defective protein biosynthesis. Restoring the normal levels of TP is an important parameter for liver recovery (Navarro and Senior, 2006). Treatment of rats with CCl₄ resulted in a more than 50% reduction in liver and kidney tissue protein contents (Table 3). Treatment with Sil increased the levels of TP in both liver and kidney tissues to 74.83 ± 8.09 g/l and 57.41 ± 3.22 g/l respectively. Highly significant results (p < 0.01) resulted from the treatment with 400 mg/kg body weight F. palmata total extract. The TP levels increased in liver tissues to 96.12 ± 6.44 g/l and in kidney tissues to 62.58 ± 4.51 g/l respectively. These results indicated that the extract has more protection at this dose than Sil. Traetment with 200 mg/kg body weight F. palmata total extract increased levels of proteins to 80.00 ± 4.82 and 51.61 ± 4.08 in liver and kidney tissues respectively. Both petroleum ether and chloroform fractions at the 200 mg/kg resulted in a highly significant (p < 0.001 and p < 0.01) increase in the TP levels in liver tissues (110.96 ± 4.08 and 90.96 ± 6.35 g/l) and kidney tissues (72.25 ± 2.78 and 67.09 ± 4.08 g/l) respectively. These results are better than the improvement caused by Sil and total extract (Table 3).

4.4. Histopathological study

The histological appearance of the liver and kidney cells reflects their conditions (Prophet et al., 1994). The histopathology of the normal hepatic cells is presented in Fig. 1, L-1. Treatment with CCl₄ resulted in Kupffer cells’ activation, cytoplasmic vacuolization of hepatocytee and focal hepatic necrosis associated with inflammatory cells’ infiltration. (Fig. 1, L-2). Liver samples of rats treated with Sil prior CCl₄ administration showed small focal hepatic necroses with few inflammatory cells’ infiltration and slight activation of kupffer cells (Fig. 1, L-3). Treatment with 400 mg/kg body weight F. palmata total extract showed excellent recovery with absences of histopathological changes (Fig. 1, L-4). The liver cells of rats treated with the lower dose of the total extract (200 mg/kg body weight) showed slight hydropic degeneration of some hepatocytes (Fig. 1, L-5). Pretreatment with 100 and 200 mg/kg body weight of the petroleum ether fraction resulted in complete protection and absence of any histopathological changes (Fig. 1, L-6 and L-7). The chloroform fraction at doses of 200 mg/kg body weight showed moderate protection of hepatocytes with only slight hydropic degeneration (Fig. 1, L-8).

Histopathological study revealed the normal renal architecture in control group (Fig. 2, K-1). Kidney cells of rats treated with CCl₄ showed dramatic histopathological changes appearing as cytoplasmic vaculation, vacular degeneration of epithelial lining renal tubules, focal inflammatory cells infiltration and preivascular edema (Fig. 2, K-2). Pretreatment with Sil resulted in good protection indicated by slight vacuolation of epithelial lining renal tubules and endothelial lining glomerular tuft (Fig. 2, K-3). Renal cells of rats treated with CCl₄ and 400 or 200 mg/kg body weight F. palmata total extract showed complete recovery of renal cells with no histopathological changes (Fig. 2, K-4 and K-5). kidney cells of rats treated with CCl₄ and 200 mg/kg of the petroleum ether fraction showed complete recovery as no histopathological changes were observed (Fig. 2, K-6), while cells of rats treated with 100 mg/kg of the petroleum ether fraction showed slight vaculations of epithelial lining of some renal tubules (Fig. 2, K-7). Treatment with CCl₄ and 200 mg/kg of the chloroform fraction showed complete recovery as no histopathological changes were observed (Fig. 2, K-8).

Fig. 2.

Histopathological appearance of kidney cells; (K-1) normal cells; (K-2) kidney cells of rats treated with CCl₄ cytoplasmic vaculation, vacular degeneration of epithelial lining renal tubules, focal inflammatory cells infiltration and preivascular edema; (K-3) kidney cells of rats treated with CCl₄ and Sil showing slight vacuolation of epithelial lining renal tubules and endothelial lining glomerular tuft; (K-4) (K-5) kidney cells of rats treated with CCl₄ and 400 or 200 mg/kg total extract showing complete recovery of renal cells with no histopathological changes; (K-6) kidney cells of rats treated with CCl₄ and 200 mg/kg of the petroleum ether fraction showing no histopathological changes; (K-7) kidney cells of rats treated with CCl₄ and 100 mg/kg of the petroleum ether fraction showing slight vaculations of epithelial lining of some renal tubules; (K-8) kidney cells of rats treated with CCl₄ and 200 mg/kg of the chloroform fraction showing no histopathological changes.

4.5. Antiulcer activity

Peptic ulcer is one of the most common, chronic gastrointestinal disorders. It has become a common global health problem affecting a large number of people worldwide and still a major cause of morbidity and mortality (Chan and Leung, 2002). Inflamed lesions or excavations of the mucosa and tissue that protect the gastrointestinal tract characterize peptic ulcer. Damage of mucus membrane that normally protects the esophagus, stomach and duodenum from gastric acid and pepsin causes peptic ulcer (Brenner and Stevens, 2006). Many plants showed good antiulcer activity such as Jasminum grandiflorum, Anogeissus latifolia, Solanum nigrum, Azadirachta indica and Ocimum sanctum (Sen et al., 2009; Kumar et al., 2011). F. palmata total extract was tested at 200 and 400 mg/kg body weight for possible antiulcer effect against 80% ethanol induced lesions. All the fractions were tested at two doses of 100 and 200 of mg/kg body weight. The ulcer index of the group treated with 80% ethanol was 8.00 ± 0.57 (Table 4). Protection against ulcer by F. palmata total extract was dose dependent and highly statistically significant (p < 0.01–p < 0.001). The best protection was observed with the higher dose of the total extract where ulcer index is 2.00 ± 0.57 (Table 4). Good protection also resulted from treatment with petroleum ether and chloroform fraction with ulcer indexes of 3.33 ± 0.33 and 4.00 ± 0.57 respectively (Table 4).

4.6. Determination of whole blood clotting time (CT)

Coumarins and coumarin derivatives are well known for their anticoagulant effect (Murray et al., 1982; Penning-van Beest et al., 2005). F. palmata total extract and all fractions were subjected to CT assay using warfarin as standard. The increase in the CT resulted from treatment with total extract, petroleum ether and chloroform fractions was statistically significant, time dependent and dose dependent. The total extract at a dose of 400 mg/kg body weight resulting in an increase in CT reached 6.70 ± 0.26 while warfarin time was 8.23 ± 0.67 after 120 min. The effect of the 200 mg/kg body weight was very close to that of the high dose after 120 min although it was less at the early stages of the experiment. The petroleum ether fraction ended up with the same efficacy after 120 min; however, the higher dose (200 mg/kg) was more effective after 30 and 60 min. The chloroform fraction was the second effective among the fractions. At the 200 mg/kg dose the chloroform fraction increased the CT to 5.10 ± 0.25 after 120 min.

4.7. Antioxidant activity

As demonstrated in Table 6, the ethyl acetate fraction was able to reduce the stable free radical DPPH, to yellow- colored DPPH at low concentrations (50 and 100 μg/ml). The effect was almost similar to that of the standard ascorbic acid. The higher concentrations (500 and 1000 μg/ml) of the crude extract and chloroform fraction were able to reduce the DPPH although the lower concentrations showed only weak activity.