Hepatoprotective activity of Cyperus alternifolius on carbon tetrachloride–induced hepatotoxicity in rats

Abstract

Objective: The present work explored the potential hepatoprotective activity of total ethanol and successive extracts of Cyperus alternifolius L (Cyperaceae) against carbon tetrachloride (CCl₄)–induced hepatotoxicity in rats and to isolate their bioactive constituents.

Methods: For isolation and identification of the compounds, column chromatography and spectroscopic analysis were used, a model of hepatotoxicity by CCl₄ in rats was used to evaluate the total ethanol extract and its successive fractions.

Results: Phytochemical screening of C. alternifolius revealed the presence of different phytochemical groups. The plant proved to be safe for human use because it did not induce any signs of toxicity or mortality in mice when administered orally at doses up to 5000 mg kg⁻¹. The total alcoholic extract in doses of 100 and 200 mg kg⁻¹ and the successive extracts (ether, chloroform and ethyl acetate) in a dose of 10 mg kg⁻¹ exhibited a significant (p ≤ 0.05) protective effect by lowering the elevated serum levels of aspartate aminotransferase: 230.4, 218.8, 224.6, 227.4 and 231.6 U L⁻¹, respectively, compared with 111.6 U L⁻¹ for silymarin (25 mg kg⁻¹). Serum levels of alanine aminotransferase were also reduced: 77.4, 72.7, 79.7, 76.0 and 79.7 U L⁻¹ compared to 63.7 U L⁻¹ for silymarin. Alkaline phosphatase: 164.6, 158.0, 163.6, 154.7 and 166.4 U L⁻¹ compared to 138.2 U L⁻¹ for silymarin. Total bilirubin: 0.50, 0.46, 0.55, 0.52 and 0.57 mg dl⁻¹ compared to 0.42 mg dl⁻¹ for silymarin. Cholesterol: 213.1, 200.0, 192.7, 193.6 and 197.1 mg dl⁻¹ compared to 180.3 mg dl⁻¹ for silymarin. Triglycerides: 237.3, 222.4, 209.5, 206.8 and 210.2 mg dl⁻¹ compared to 196.8 mg dl⁻¹ for silymarin. Eight phenolic compounds were isolated from C. alternifolius for the first time and identified as esculetin 1, umbelliferon 2, imperatorin 3, psoralen 4, xanthotoxin 5, quercetin 6, quercetin-3-O-rutinoside 7 and gallic acid 8.

Conclusions: The results concluded that C. alternifolius possesses significant protective effect against hepatotoxicity induced by CCl₄.

Keywords::

• LD₅₀
• liver damage
• marker enzymes
• total bilirubin
• cholesterol
• triglycerides

Introduction

The genus Cyperus is a large genus of the sedge family, Cyperaceae. Members of Cyperaceae are monocotyledon, grass-like, flowering plants, commonly found in wet areas and known as sedges (Gregory & Cutler, 1960). The Cyperaceae is rich in secondary metabolites including tannins, phenolic acids, flavonoids (Trease & Evans, 1976), coumarins (Sharma, 1993) and sesquiterpenoids (Morimoto et al., 1999). The genus Cyperus comprises about 550 species which are widely distributed all over the world (Vare & Kukkonen, 2005). The most common species includes Cyperus alternifolius L (umbrella sedge). Phytochemical study on the aerial parts of Cyperus species led to isolation of flavonoids and coumarins (Sayed et al., 2008) as the most important constituents (Yu et al., 2007).

Liver ailments remain a serious health problem (Baranisrinivasan et al., 2009). Various xenobiotics are known to cause hepatotoxicity, one among them is carbon tetrachloride (CCl₄) (Kodavanti et al., 1989), which has proved to be highly useful as an experimental agent for the induction of acute liver injury. Conventional and synthetic drugs used in the treatment of liver diseases are sometimes inadequate and can have serious adverse effects. It is therefore necessary to search for alternatives to replace currently used drugs of doubtful efficacy and safety (Arhoghro et al., 2009). Natural remedies from medicinal plants are considered to be effective and safe alternative treatments for hepatotoxicity (Awaad et al., 2006; Awaad & Al-Jaber, 2010). The current study was undertaken to evaluate the potential protective effect of C. alternifolius on CCl₄-induced hepatotoxicity in rats.

Materials and methods

Plant material

The aerial parts of C. alternifolius were collected during the flowering stage in March 2005 from Orman Garden, Egypt. The sample was kindly identified by Dr. M. Gebali, botanist, and by comparison with the published plant description (El-Gohary, 2004). A voucher specimen has been deposited in the herbarium of the Desert Research Center. Plant material was air-dried in shade, reduced to fine powder, packed in tightly closed containers and stored at room temperature for phytochemical and biological studies.

Extraction

The air-dried powder of C. alternifolius aerial parts (1 kg) was extracted by percolation in 90% ethanol (Awaad et al., 2006) at room temperature for 2 days. The ethanol extract was filtered and the residues were re-percolated for four times. The total ethanol extract was concentrated under reduced pressure at a temperature not exceeding 35°C to yield a dry extract of 190 g.

The total ethanol extract (50 g) was dispersed in 1000 ml of distilled water and extracted successively with diethyl ether, chloroform, ethyl acetate and n-butanol, respectively. Each extract was dried over anhydrous sodium sulfate and concentrated under reduced pressure at a temperature not exceeding 35°C to yield 8.5, 10.6, 15.6 and 19.0 g dry extracts, respectively.

Preparation of the extracts for biological studies

The total ethanol and successive extracts of C. alternifolius and the standard silymarin were suspended in distilled water using Tween 80 (3% v/v).

Experimental animals

Both sex of adult albino mice (25–30 g body weight) and albino rats (180–200 g body weight) were used. They were housed randomly in groups in polypropylene cages, fed with standard rodent diet and water ad libitum. Animals were allowed to acclimatize to the work area environment for 2 weeks before use. Each animal was used for one experiment only. The care and handling of the animals were in accordance with the internationally accepted standard guidelines and was approved by an institutional review board.

Determination of median lethal dose (LD₅₀)

LD₅₀ of the total ethanol extract of C. alternifolius plant was estimated in mice by using the method of Lorke (1983). In a preliminary test, animals in groups of three, received one of 10, 100 or 1000 mg kg⁻¹ of the tested extract suspended in the vehicle (3% v/v Tween 80). Animals were observed for 24 h for signs of toxicity and number of deaths. Depending on the results of the preliminary test, doses of 1250, 2500 and 5000 mg kg⁻¹ of the tested extract were administered to fresh groups, each of six mice. Control animals were received the vehicle and kept under the same conditions. Signs of toxicity and number of deaths per dose were recorded within 24 h and the LD₅₀ was calculated as the geometric mean of the dose that resulted in 100% mortality and that which caused no lethality at all.

Experimental induction of hepatic damage

CCl₄ was dissolved in corn oil in the ratio 1:1 v/v. Liver damage was induced in rats following subcutaneous (SC) injection of CCl₄ in the lower abdomen at a dose of 3 ml kg⁻¹ (Théophilea et al., 2006).

Hepatoprotective activity

Sixty adult albino rats were randomly divided into 10 groups of six animals, each. Rats of the 1st (normal control) and 2nd (CCl₄-intoxicated control) groups received the vehicle in a dose of 1 ml kg⁻¹. Animals of the 3rd group (reference) received silymarin at a dose of 25 mg kg⁻¹. The 4th, 5th and 6th groups were treated with the total ethanol extract of C. alternifolius plant at the dose levels of 50, 100 and 200 mg kg⁻¹, respectively. The 7th, 8th, 9th and 10th groups were administered 10 mg kg⁻¹ of the successive extracts (ether, chloroform, ethyl acetate and n-butanol, respectively). All medications were administered orally by gastric intubation for 8 consecutive days. Two hours after the last dose, normal control rats were given a single dose of corn oil (3 ml kg⁻¹, SC), whereas animals of the 2nd to 10th groups were received a single SC dose of CCl₄ (3 ml kg⁻¹).

Blood sampling

After 24 h of corn oil and CCl₄ injections, blood samples (2 ml) were collected from all rats by puncturing their retro-orbital plexus of veins. Serum was separated by centrifugation at 2500 rpm for 15 min at 4°C and used for the biochemical assay.

Biochemical analysis

The serum activity of liver marker enzymes (AST and ALT) was assayed following the method of Reitman and Frankel (1957), whereas the activity of alkaline phosphatase (ALP) was estimated by the method of Babson et al. (1966). The levels of total protein (Henary et al., 1974) and albumin (Doumas et al., 1971) were estimated in serum. The serum concentrations of total bilirubin (Walter & Gerarade, 1970), cholesterol (Zoppi & Fenili, 1976) and triglycerides (Fossati & Prencipe, 1982) were assayed.

Statistical analysis

Values are expressed in mean ± SEM for six animals in each group. p value was calculated using analysis of variance followed by Dunnett’s test for multiple comparisons. Values of p < 0.05 were considered significant in all cases.

Isolation of the phenolic compounds

TLC examination of the different extractives (Stahl, 1969) using two different solvent systems (chloroform-methanol (a) 98:2 and ethyl acetate-methanol-water (b) 60:5:4) and spray reagents revealed that both ether and chloroform extracts are identical (same numbers and color of spots); in addition both extracts exhibit the higher activity as hepatoprotective. Therefore, both extracts were collected together (15 g), fractionated on column packed with silica gel (450 g) and eluted gradually with benzene-ethyl acetate, 120 fractions were collected (60 ml each) and reduced to 3 main fractions, these sub-fractions were reapplied on preparative thin layer chromatography using system a. For final purifications, each compound was reapplied on column packed with Sephadex LH20 and eluted with methanol. The fractions were concentrated under reduced pressure to produce compounds 1–5. Hepatoprotection was achieved in rats following medication of ethyl acetate and butanol extracts but was less significant, accordingly they were subjected to fractionation as follows.

The ethyl acetate and butanol were found to be identical adopting solvent systems (ethyl acetate-methanol-water 30:5:4) (d), visualized by UV before and after spraying with aluminum chloride, accordingly. Both of them were collected together (20 g) and fractionated on column packed with silica gel (600 g) and eluted with ethyl acetate-methanol-water (120:5:4), 100 fractions were collected (50 ml each) and reduced to 3 main fractions in different yields 3.4, 2.7 and 1.6, respectively (according to the number, color and R_f values of the spots), these sub-fractions were reapplied on preparative paper chromatography using system acetic acid-water (85:15). For final purifications, each compound was reapplied on column backed with Sephadex LH20 and eluted with methanol. The fractions were concentrated under reduced pressure to produce compounds 6-8.

Esculetin 1

Needle crystals (20 mg), R_f = 0.12 (system a), m.p. 272–274°C. UV λ_max (MeOH): 226, 257, 293 and 346 nm. EI-MS m/z (% rel. int.): M⁺ 179 (100), 150 (20), 132 (3), 122 (22), 121 (18), 94 (20) and 69 (10). ¹H NMR (DMSO-d₆): δ 7.9 (1H, d, J = 9.5 Hz, H-4); δ 7.1 (1H, S, H-5); δ 6.79 (1H, S, H-8) and δ 6.2 (1H, d, J = 9.5 Hz, H-3). ¹³C NMR (DMSO): 161.28 (C-2′), 110.77 (C-3′), 144.60 (C-4′), 112.25 (C-5′), 148.40 (C-6′), 150.33 (C-7′), 102.53 (C-8′), 142.84 (C-9′), 110.77 (C-10′).

Umbelliferone 2

White crystals (25 mg), R_f = 0.75 (system a), m.p. 272–274°C. UV λ_max (MeOH): (nm), 244, 257, 320 and 324. EI-MS m/z (% rel. int.): 162 (M⁺) (100), 134 (15), 106 (10), 78 (9), 77 (10) and 51 (5). ¹H NMR (DMSO-d₆): δ 7.79 (1H, d, J = 9.5 Hz, H-4); δ 7.42 (1H, d, J = 8.4 Hz, H-5); δ 6.77 (1H, dd, J = 8.4, J = 2.2 Hz, H-6); δ 6.71 (1H, d, J = 2.2 Hz, H-8) and δ 6.1 (1H, d, J = 9.5 Hz, H-3). ¹³C NMR (DMSO): 161.101 (C-2′), 121.40 (C-3′), 145.10 (C-4′), 129.78 (C-5′), 113.42 (C-6′), 162.11 (C-7′), 103.10 (C-8′), 146.5 (C-9′), 112.34 (C-10′).

Imperatorin 3

Obtained as needle crystal from methanol (30 mg). R_f = 0.65 (system a), m.p. 102–103°C. λ_max (EtOH) 302, 265 and 250 nm. EI-MS m/z (% rel. int.): 270 (100), 201 (50), 215 (10), 255 (30) and 174 (80). ¹H NMR (CDCl₃): δ 6.35 (1H, d, J = 9.5 Hz, H-3); δ 7.73 (1H, d, J = 10 Hz, H-4); δ 7.29 (1H, S, H-5); δ 6.79 (1H, d, J = 2.1 Hz, H-4′); δ 7.69 (1H, d, J = 2.6 Hz, H-5′); δ 5.08 (2H, d, OCH₃, H-8) and δ 1.68 (6H, S, 2CH₃). ¹³C NMR (DMSO): 161.46 (C-2), 113.58 (C-3), 145.43 (C-4), 113.80 (C-5), 126.41 (C-6), 148.73 (C-7), 131.06 (C-8), 143.75 (C-9), 116.62 (C-10), 147.15 (C-2′), 106.59 (C-3′), 69.51 (C-1′′), 119.53 (C-2′′), 139.70 (C-3′′), 16.67 (C-4′′), 24.53 (C-5′′).

Xanthotoxin 4

Obtained as needle crystals from methanol (50 mg). R_f = 0.54 (system a), m.p. 144–146°C. λ_max (EtOH) 202, 186, 249 and 300 nm. MS showed M⁺ at m/e 216 (100), and fragment ions at m/e 202 (20), 186 (18), 185 (23), 173 (21), 145 (16) and 71 (12). IR (KBr) V_max cm⁻¹: 1736, 1628, 1579, 1510, 1214 and 1008. ¹H NMR (CDCl₃): δ 6.33 (1H, d, J = 9.5 Hz, H-3); δ 7.8 (1H, d, J = 9.5 Hz, H-4); δ 7.35 (1H, S, H-5), δ 7.1 (1H, d, J = 2.5 Hz, H-4′); 7.62 (1H, d, J = 2.5 Hz, H-5′).¹H NMR (CDCl₃): 161.36 (C-2), 113.59 (C-3), 142.80 (C-4), 116.65 (C-5), 126.66 (C-6), 147.73 (C-7), 60.41 (C-8), 147.17 (C-2′), 106.60 (C-3′), 145.30 (C-4′), 113.70 (C-5′).

Psoralen 5

Obtained as white needle crystals, soluble in methanol, m.p. 102–103°C, R_f value: 0.46 (system a). UV showing λ_max at 250, 265 and 302 nm. EI-MS showed m/z: 200 (100%), 270 (70%), 202 (50%), 253 (25%) and 238 (35%). IR (KBr) V_max cm⁻¹: 1721, 1628, 1579, 1510, 1214 and 1008. ¹H NMR (CDCl₃): δδ 6.36 (1H, d, J = 9.6 Hz, H-3), δ 8.00 (1H, d, J = 9.6 Hz, H-4), δ 7.54 (1H, s, H-5), δ 7.86 (1H, d, J = 2.3 Hz, H-2′), δ 6.92 (1H, d, J = 2.3 Hz,H-3′), δ 4.95 (1H, d, J = 7.2 Hz, H-1′′), δ 5.53 (1H, t, J = 7.2 Hz, H-2′′), δ 1.68 (3H, S, H-4′′), δ 1.64 (3H, S, H-5′′). ¹³C NMR (methanol-d₃): 161.46 (C-2), 113.58 (C-3), 145.43 (C-4), 113.80 (C-5), 126.41 (C-6), 148.73 (C-7), 131.06 (C-8), 143.75 (C-9), 116.62 (C-10), 147.15 (C-2′), 106.59 (C-3′), 69.51 (C-1′′), 119.53 (C-2′′), 139.70 (C-3′′), 16.67 (C-4′′), 24.53 (C-5′′).

Quercetin 6

Yellow needle crystals (20 mg) R_f = 0.7 (system b), m.p. 313–315°C. UV λ_max (MeOH): (nm) 255, 269 (sh), 370; (AlCl₃): 270, 290 (sh), 455; (AlCl₃/HCl): 270, 357, 426; (NaOAc): 274 (Dec.), 325; (NaOAc/H₃BO₃): 261, 385; (NaOMe): 246, 330, 398. ¹H NMR (DMSO-d₆): δ 8.16 (1H, d, J = 2 Hz, H-6); δ 7.67 (1H, d, J = 2 Hz, H2′); δ 7.6 (1H, dd, J_6′,2′ = 8 Hz, H-6′); δ 6.80 (1H, d, J = 8 Hz, H-5′) and δ 6.3 (1H, d, J = 2 Hz, H-8).

Quercetin-3-O-rutinoside 7

Yellow crystals (600 mg) R_f = 0.5 (system b), m.p. 190°C. UV λ_max (MeOH): (nm) 256, 265 (sh), 290, 355; (AlCl₃): 274, 302 (sh), 330 (sh), 432; (AlCl₃/HCl): 270, 298, 359, 399; (NaOAc): 272, 324, 398; (NaOAc/H₃BO₃): 263, 292 (sh), 368; (NaOMe): 272, 310, 410. ¹H NMR (DMSO-d₆): δ 8.10 (1H, d, J = 2 Hz, H2′); δ 7.86 (1H, d, J = 8 Hz, H-6′); δ 6.89 (1H, d, J = 8 Hz, H-5′); δ 6.65 (1H, d, J = 2 Hz, H-8); δ 6.5 (1H, d, J = 2 Hz H-6); δ5.13 (1H, d, J = 7.50 Hz H1′′); δ 4.55 (1H, d, J = 1.3 Hz, H1′′′); δ 3.82 (1H, dd, J = 10 Hz, J = 2 Hz H-6′′); δ 3.65 (1H, dd, J = 3.5, H2′′′); δ 3.47–3.87 (6H, m, sugar protons) and δ 1.23 (3H, d, J = 6 CH₃).

Gallic acid 8

Obtained as white crystals, soluble in methanol. R_f value = 0.74 (system b). m.p. 252°C. ¹H NMR (methanol-D₃): δ (ppm) 7.0 (2H, s). ¹³C NMR (methanol-d₃): δ.120.7 C-1, 108.9 C-2, 145.5 C-3, 138.2 C-4, 145.0 C-5, 108.9 C-6, 169.1 C-7 (carbonyl).

Results and discussion

Isolation of phenolic compounds

Eight phenolic compounds were isolated from C. alternifolius for the first time (Figure 1) and identified as esculetin, umbelliferon, imperatorin, psoralen, xanthotoxin, quercetin, rutin and gallic acid using different spectroscopic analysis such as ¹H NMR, ¹³C NMR DEPT, COSY, HMBC, HMQC and mass spectrometry.

Figure 1.  The isolated compounds of C. alternifolius L.

Compounds 1–5 were isolated from ether and chloroform extracts. They were identified as esculetin, umbelliferon, imperatorin, psoralen and xanthotoxin by comparing their TLC chromatograms, UV spectrum in methanol and with different shift reagents, EI-MS, ¹H NMR and ¹³C NMR spectra with authentic samples and published data (Murray et al., 1982). Compound 6 migrated with quercetin. Its spectral data were in agreement with those published for this compound (Mabry et al., 1970).

Acid hydrolysis (Harborne et al., 1975) of compound 7 revealed the sugar rhamnose glucose which identified by PC and TLC (systems e and f) and an aglycone which was found to be identical with compound 6 when compared with its TLC and UV shift reagents. There are substituted at position 3 as indicated by their UV spectra upon addition of diagnostic shift reagent. Accordingly, based on the results obtained and some of the published data (Geissman, 1962; Mabry et al., 1970; Harborne et al., 1975), this compound was identified as quercetin-3-O-rutinoside (rutin).

Compound 8 was isolated from ethyl acetate and butanol extracts. It was obtained as a white crystal after crystallization from methanol and identified as gallic acid.

Determination of median lethal dose (LD₅₀)

The total ethanol extract of C. alternifolius did not produce any behavioral changes and mortality in mice in doses up to 5000 mg kg⁻¹. Accordingly, it suggested that oral LD₅₀ of the total extract was higher than 5000 mg kg⁻¹. Therefore, the tested plant can be categorized as highly safe because substances possessing LD₅₀ higher than 50 mg kg⁻¹ are non-toxic (Buck et al., 1976).

Hepatoprotective activity

Hepatotoxicity induced by CCl₄ is the most commonly used model system for the screening of hepatoprotective activity of plant extracts. Acute exposure to CCl₄ produces rapid cellular injury due to its reductive dehalogenation in the endoplasmic reticulum of hepatocytes to generate an unstable highly reactive complex CCl₃• or trichloroperoxyl radical (CCl₃O₃). These radicals attack microsomal lipids and proteins causing lipid peroxidation. Products of lipid peroxidation may cause damage to the biological membranes leading to serious cellular injury and leakage of serum marker enzymes like AST, ALT and ALP (Cotran et al., 1994) and finally cell death (Wei, 1998). The prevention of this phenomenon can be considered as hepatoprotective activity (Poon et al., 2003).

Estimations of serum transaminases and ALP are the most sensitive tests for diagnosis of liver diseases (Mahendale et al., 1985). In this study, SC injection of CCl₄ to rats leads to a marked elevation in the levels of serum AST, ALT and ALP after 24 h of exposure compared to normal control rats, indicating acute hepatocellular damage (Table 1). This might be due to the release of these enzymes from the cytoplasm, into the blood circulation rapidly after rupture of the plasma membrane and cellular damage (Sallie et al., 1991). Pretreatment with the total ethanol extract of C. alternifolius plant (100 and 200 mg kg⁻¹) and the ethereal, chloroform and ethyl acetate successive extracts (10 mg kg⁻¹) significantly reversed the levels of hepatic enzymes when compared to CCl₄-treated animals (Table 1). In the group where silymarin was given, the levels of hepatic enzymes were significantly lower (p < 0.05) than in the CCl4-treated group. The protective effect of plant extracts against CCl₄ may be attributed to the presence of flavonoids (Gilani & Janbaz, 1995) or coumarins (Park et al., 2004) among the plant constituents.

Table 1.  Effect of the total ethanolic and successive extracts of C. alternifolius on the activity of AST, ALT and ALP enzymes and levels of total proteins and albumin in serum of rats with CCl₄-induced hepatotoxicity (n = 6).

Groups	AST (U L^−1)	ALT (U L^−1)	ALP (U L^−1)	Total proteins (g dl^−1)	Albumin (g dl^−1)
Normal control	87.3 ± 4.71^b,c	44.6 ± 2.95^b,c	85.6 ± 4.9^b,c	6.16 ± 0.25^b	4.02 ± 0.16^b
CCl4-intoxicated control	265.7 ± 10.16^a	91.4 ± 5.08^a	190.5 ± 8.3^a	5.10 ± 0.15^a	3.38 ± 0.10^a
Silymarin (25 mg kg^−1) + CCl4	111.6 ± 6.80^a,b	63.7 ± 4.11^a,b	138.2 ± 5.8^a,b	5.82 ± 0.17^b	3.87 ± 0.11^b
Total alcohol extract (50 mg kg^−1) + CCl4	241.4 ± 8.11^a,c	82.6 ± 4.45^a,c	173.4 ± 6.2^a,c	5.48 ± 0.18^a	3.43 ± 0.11^a,c
Total alcohol extract (100 mg kg^−1) + CCl4	230.4 ± 8.32^a–c	77.4 ± 4.16^a–c	164.6 ± 6.1^a–c	5.65 ± 0.16^b	3.61 ± 0.06^a,b
Total alcohol extract (200 mg kg^−1) + CCl4	218.8 ± 7.33^a–c	72.7 ± 4.12^a,b	158.0 ± 6.0^a–c	5.74 ± 0.16^b	3.68 ± 0.07^b
Etherial extract (10 mg kg^−1) + CCl4	224.6 ± 7.10^a–c	79.7 ± 4.11^a–c	163.6 ± 8.8^a–c	5.84 ± 0.14^b	3.79 ± 0.11^b
Chloroformic extract (10 mg kg^−1) + CCl4	227.4 ± 6.27^a–c	76.0 ± 4.00^a–c	154.7 ± 7.6^a,b	5.89 ± 0.15^b	3.86 ± 0.10^b
Ethyl acetate extract (10 mg kg^−1) + CCl4	231.6 ± 7.14^a–c	79.7 ± 5.16^a–c	166.4 ± 9.4^a–c	5.46 ± 0.16^a	3.43 ± 0.12^a,c
Butanol extract (10 mg kg^−1) + CCl4	236.1 ± 6.11^a–c	80.2 ± 4.64^a,c	170.3 ± 9.0^a,c	5.47 ± 0.16^a	3.50 ± 0.19

Values represent the mean ± SE of six rats in each group.

^ap < 0.05: Statistically significant from the normal control group.

^bp < 0.05: Statistically significant from CCl₄-intoxicated group.

^cp < 0.05: Statistically significant from the reference (silymarin) group.

The administration of CCl₄ alone adversely interferes with protein metabolism probably by inhibiting the synthesis of proteins in the liver such as albumin. In the present study, the levels of total protein (5.10 ± 0.15 g dl⁻¹) and albumin (3.38 ± 0.10 g dl⁻¹) were significantly (p < 0.05) reduced after intoxication with CCl₄ in comparison with normal control rats (6.16 ± 0.25 and 4.02 ± 0.16 g dl⁻¹, respectively). The lowered levels of total protein and albumin in the serum reveal the intensity of hepatopathy (Aniya et al., 2005). Moreover, defects in protein metabolism, evidenced by changes in total protein and/or albumin level, are used to indicate the severity of the hepatic diseases (Henry, 1984). Pretreatment with the total ethanol extract of C. alternifolius plant (100 and 200 mg kg⁻¹) and the ethereal, chloroform and ethyl acetate successive extracts (10 mg kg⁻¹) to CCl₄-intoxicated rats showed a significant reversal of these parameters toward the normal (Table 1). The effects of these extracts were comparable to that of standard silymarin activity. This assures the hepatoprotective activity of these extracts against damage by CCl₄.

Further, animals receiving CCl₄ had higher serum levels of bilirubin, cholesterol and triglyceride, as compared to the intact rats (Table 2). The rise in the level of serum bilirubin is the most sensitive parameter that confirms the intensity of jaundice (Drotman & Lawhorn, 1978). The ability of the total ethanol extract (100 and 200 mg kg⁻¹) and the ethereal and chloroform successive extracts (10 mg kg⁻¹) to reduce the level of total bilirubin in the serum of intoxicated rats (Table 2) suggests their potential in clearing bilirubin from the serum when its level is elevated.

Table 2.  Effect of the total ethanolic and successive extracts of C. alternifolius L plant on levels of total bilirubin, cholesterol and triglycerides in serum of rats with CCl₄-induced hepatotoxicity (n = 6).

Groups	Total bilirubin (mg dl^−1)	Cholesterol (mg dl^−1)	Triglycerides (mg dl^−1)
Normal control	0.26 ± 0.01^b	112.4 ± 6.58^b	141.3 ± 7.36^b
CCl4-intoxicated control	0.63 ± 0.03^a	244.9 ± 8.80^a	252.8 ± 7.13^a
Silymarin (25 mg kg^−1) + CCl4	0.42 ± 0.02^a,b	180.3 ± 7.47^a,b	196.8 ± 6.20^a,b
Total alcohol extract (50 mg kg^−1) + CCl4	0.55 ± 0.03^a,c	232.1 ± 9.86^a,c	242.6 ± 8.85^a,c
Total alcohol extract (100 mg kg^−1) + CCl4	0.50 ± 0.02^a–c	213.1 ± 8.90^a–c	237.3 ± 7.85^a–c
Total alcohol extract (200 mg kg^−1) + CCl4	0.46 ± 0.02^a,b	200.0 ± 8.65^a,b	222.4 ± 6.41^a–c
Etherial extract (10 mg kg^−1) + CCl4	0.55 ± 0.03^a–c	192.7 ± 7.85^a,b	209.5 ± 7.73^a,b
Chloroformic extract (10 mg kg^−1) + CCl4	0.52 ± 0.03^a–c	193.6 ± 7.90^a,b	206.8 ± 7.23^a,b
Ethyl acetate extract (10 mg kg^−1) + CCl4	0.57 ± 0.03^a,c	197.1 ± 8.73^a–c	210.2 ± 8.70^a–c
Butanol extract (10 mg kg^−1) + CCl4	0.62 ± 0.02^a,c	240.5 ± 8.50^a,c	229.7 ± 8.11^a,c

Values represent the mean ± S.E. of six rats in each group.

^ap < 0.05: Statistically significant from the normal control group.

^bp < 0.05: Statistically significant from CCl₄-intoxicated group.

^cp < 0.05: Statistically significant from the reference (silymarin) group.

Administration of the total ethanol extract (50 mg kg⁻¹) and butanol successive extract (10 mg kg⁻¹) once daily for 8 days before CCl₄, did not elicit any significant effect on the altered serum levels of ALT, ALP, total protein, albumin, total bilirubin, cholesterol and triglycerides, when compared with CCl₄-intoxicated group.

Conclusions

In the present study, the total ethanol extracts (100 and 200 mg kg⁻¹) showed significant hepatoprotective activity against acute hepatotoxicity induced by CCl₄ in rats, which was comparable with the standard drug silymarin. The bioactive principles of the ether and chloroform successive extracts that have been identified could be responsible for the observed hepatoprotective effect. Further studies with the individual isolated compounds are underway which will enable us to understand the exact mechanism of hepatoprotective activity of C. alternifolius.

Acknowledgment

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for the work through the research group project NO RGP-VPP-060.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.