A new cytotoxic ceramide from Heteroxenia ghardaqensis and protective effect of chloroform extract against cadmium toxicity in rats

2.5.1 2S,3R-4E,8E-2-(hexadecanoylamino)-docosa-4,8-diene 1,3-diol (1)

Yellow powder, mp 80.0–82.0 °C, [α]_D²⁵ – 7.6° (c 0.01, CHCl₃); IR ν_max (KBr) cm⁻¹: 3416, 2956, 2919, 2850, 1642, 1618, 1049, 964, 720, 621. HRESI-MS (M + Na)⁺; at m/z 615.5245 (M + Na, 100%), 593.4753 (M + 1, 20%), ESI-MS: 593 (M + 1, 100%), 522 (35%), 507 (15%), 481 (10%), 423 (20%), 395 (80%), 368 (60%), 328 (25%), 298 (20%), 255 (15%), 223 (25%), 198 (70%). ¹H NMR (CDCl₃, 600 MHz): 3.68 (dd, J = 11.7, 3.8, H-1a, 1H), 3.88 (dd, J = 11.7, 3.5, H-1b, 1H), 3.90 (m, H-2, 1H), 4.29 (m, H-3, 1H), 5.36 (dd, J = 15.8, 6.18, H-4, 1H), 5.76 (dt, J = 15.8, 6.18, H-5, 1H), 2.06 (m, H-6, 2H), 2.10 (m, H-7, 2H), 5.40 (dt, J = 15.2, 6.54, H-8, 1H), 5.52 (dt, J = 15.2, 6.5, H-9, 1H), 1.94 (m, H-10, 2H), 2.22 (t, J = 7.5, H-2′, 2H), 1.62 (m, H-3′, 2H), 1.28 (br s, n CH₂), 0.86 (t, J = 6.8, H-22 and 16′, 6H), 6.27 (d, J = 7.5, NH, 1H)), 2.93 (br s, OH-1, 3). ¹³C NMR (CDCl₃, 150 MHz): 62.5 (C-1), 54.5 (C-2), 76.8 (C-3), 129.0 (C-4), 133.6 (C-5), 32.0 (C-6), 32.2 (C-7), 131.4 (C-8), 129.2 (C-9), 32.7 (C-10), 174.1 (C-1‘), 36.9 (C-2‘), 25.8 (C-3′), 32.4 (C14′, C-20), 22.7 (C-15′, C-21), 29.3–29.8 (n CH₂), 14.2 (C-22, 16′). GC–MS of fatty acid methyl ester 1B: MS m/z: 270, 251, 227, 185, 158, 143, 130, 101, 87, 74, 69, and 55.

2.5.2 2S,3R-4E-2-(octadecanoylamino)-octadec-4-ene-1-ol (3)

White amorphous solid, mp 78.0–79.0 °C. IR ν_max (KBr) cm⁻¹; 3445, 2922, 2853, 1642, 723. Positive ESI-MS (M + H)⁺; at m/z 550 (M⁺, 100%,), 507 (45%), 481 (35%), 381 (25%), 325 (55%), 283 (80%), 269 (75%), 225 (60%), 168 (30%). ¹H NMR (CDCl₃, 600 MHz): 3.64 (d, J = 5.5, 3.8, H-1a, 1H), 3.85 (m, H-1b, 1H), 4.02 (m, H-2, 1H), 2.25 (m, H-3, 2H), 5.30 (m, H-4, 1H), 5.35 (m, H-5, 1H), 1.98 (m, H-6, 2H), 2.28 (t, J = 7.5, H-2′, 2H), 1.58 (m, H-3′, 2H), 1.26 (brs, n CH₂), 0.86 (t, J = 7.5, H-18 and 18′, 6H), 5.39 (d, J = 7.0, NH, 1H). ¹³C NMR (CDCl₃, 150 MHz): 64.5 (C-1), 50.8 (C-2), 34.8 (C-3), 129.8 (C-4), 130.1 (C-5), 32.0 (C-6), 174.1 (C-1′), 36.6 (C-2′), 25.8 (C-3′), 26.0 (C-3′), 29.3–29.8 (n CH₂), 32.4 (C-16 and 16′), 22.7 (C-17, 17′), 14.2 (C-18, 18′).

2.6.1 Cell culture

Human hepatocarcinoma cell lines (Hep-G2), which were purchased from ATCC, USA, were used to evaluate the cytotoxic effect of the tested compounds. Cells were routinely cultured in Eagle’s Minimum Essential Medium which was supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine, containing 100 units/ml penicillin G sodium, 100 units/ml streptomycin sulphate, and 250 μg/ml amphotericin B. Cells were maintained at sub-confluence at 37 °C in humidified air containing 5% CO₂. For sub-culturing, monolayer cells were harvested after trypsin/EDTA treatment at 37 °C. Cells were used when confluence had reached 75%. Tested ceramides were weighed, dissolved in dimethyl sulphoxide (DMSO), and diluted thousand times in the assay to begin with the intended concentration. All cell culture materials were obtained from Cambrex BioScience (Copenhagen, Denmark). All chemicals were purchased from Sigma/Aldrich, USA. All experiments were repeated three times.

2.6.2 Reagents preparation

MTT solution: 5 mg/ml of MTT in 0.9% NaCl. Acidified isopropanol: 0.04 N HCl in absolute isopropanol.

2.6.3 Calculation

Percentage of relative viability (V) was calculated using the following equation: $$V\%=A_{\text{treated}}/A_{\text{cont}}\times 100$$ where V%: Percentage of relative viability, A_treated: Absorbance of treated cells, A_cont: Absorbance of control cells.

The half maximal inhibitory concentration (IC₅₀) was calculated from the equation of the dose response curve.

2.6.4 Anti-tumour activity

Cytotoxicity of tested ceramides (1 and 2) against HepG2 was measured using the MTT cell viability assay. MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay is based on the ability of active mitochondrial dehydrogenase enzyme of living cells to cleave the tetrazolium rings of the yellow MTT and forms a dark blue insoluble formazan crystals which is largely impermeable to cell membranes, resulting in its accumulation within healthy cells. Solubilization of the cells results in the liberation of crystals, which are then solubilized. The number of viable cells is directly proportional to the level of soluble formazan dark blue colour. The extent of the reduction of MTT was quantified by measuring the absorbance at 570 nm (Hansen et al., 1989).

Cells (0.5 × 105 cells/well), in serum-free media, were plated in a flat bottom 96-well microplate, and treated with 20 μl of serial dilutions of the tested compounds for 48 h at 37 °C, in a humidified 5% CO₂ atmosphere. After incubation, media were removed and 40 μl MTT solution/well was added and incubated for an additional 4 h. MTT crystals were solubilized by adding 180 μl of acidified isopropanol/well and plate was shaken at room temperature, followed by photometric determination of the absorbance at 570 nm using microplate ELISA reader. Triplicate repeats were performed for each concentration and the average was calculated. Data were expressed as the percentage of relative viability compared with the untreated cells compared with the vehicle control for the solid sample only, with cytotoxicity indicated by <100% relative viability.

2.7.1 Experimental animals

Thirty male rats were used and divided into 6 groups (5 rats/group). Group I: the control group, group II: rats given one dose of chloroform extract (60 mg/kg b.w.) for 10 days, group III: rats given one dose of cadmium chloride daily equivalent to 1/10 LD₅₀ (8.8 mg/kg b.w.) (Koriem et al., 2009) for 10 days, group IV: rats given one dose of cadmium chloride daily equivalent to 1/10 LD₅₀ (8.8 mg/kg b.w.) and chloroform extract of coral (20 mg/kg b.w.) for 10 days, group V: rats given one dose of cadmium chloride daily equivalent to 1/10 LD₅₀ (8.8 mg/kg b.w.) and chloroform extract of coral (40 mg/kg b.w.) for 10 days, group VI: rats given one dose of cadmium chloride daily equivalent to 1/10 LD₅₀ (8.8 mg/kg b.w.) and chloroform extract of coral (60 mg/kg b.w.) for 10 days.

2.7.2 Experimental design

Thirty male rats were used and divided into 5 groups (6 rats/group). Group I: the control group, group II: rats given one dose of cadmium chloride daily equivalent to 1/10 LD₅₀ (8.8 mg/kg b.w.) (Koriem et al., 2009) for 10 days, group III: rats given one dose of cadmium chloride daily equivalent to 1/10 LD₅₀ (8.8 mg/kg b.w.) and chloroform extract (20 mg/kg b.w.) for 10 days, group IV: rats given one dose of cadmium chloride daily equivalent to 1/10 LD₅₀ (8.8 mg/kg b.w.) and chloroform extract (40 mg/kg b.w.) for 10 days, group V: rats given one dose of cadmium chloride daily equivalent to 1/10 LD₅₀ (8.8 mg/kg b.w.) and chloroform extract (60 mg/kg b.w.) for 10 days.

2.7.3 Histopathological study of adrenal glands

Adrenal glands were dissected out and fixed instantaneously in 10% formal saline for 24 h. The specimens were washed in tap water, dehydrated in ascending grades of ethanol, cleared in xylene, embedded in paraffin wax (m.p 55–60 °C). Paraffin (m.p. 55–60 °C) sections of 6 μm thicknesses were prepared and stained with haematoxylin and eosin (Koriem et al., 2009). In this method the paraffin sections were stained in haematoxylin for 5 min. Sections were washed in running water for bluing and then stained in 1% watery eosin for 2 min, washed in water, dehydrated, cleared and mounted in Canada balsam. The cytoplasm stained shades of pink and red and the nuclei gave blue colour.

3.2.1 Histopathological results

Microscopic examination of the adrenal glands of control and rats that administered with chloroform extract of coral (60 mg/kg/bw) showed the normal structure, outer capsule, zona glomerulosa, sudanophobe layer, zona fasciculate, zona reticularis of the cortex and medulla (Fig. 5A and B). Examination of sections of adrenal glands of rats treated with CdCl₂ (8.8 mg/kg b.w.) showed hydropic degeneration, focal necrosis in the adrenal cortex and disturbance of the medulla (Fig. 5C), congestion and haemorrhagic area in the medulla (Fig. 5D). Sections of adrenal glands of rats treated with of CdCl₂ and chloroform extract of coral (20 and 40 mg/kg/b.w.) showed mild hydropic degeneration in the cortex (Fig. 5E and F, respectively). Sections of adrenal gland of rats treated with of CdCl₂ and chloroform extract of coral (60 mg/kg/bw) showed mild hydropic degeneration in the cortex and normal medulla (Fig. 5G).

Close

Figure 5

Sections of adrenal glands of (A) control rat shows normal structure. Notice 1 – outer capsule of the adrenal gland, 2 – zona glomerulosa of the cortex, 3 – sudanophobe layer of the cortex, 4 – zona fasciculata of the cortex, 5 – zona reticularis of the cortex and 6 – medulla, (B) rat treated with chloroform extract of coral (60 mg/kg/bw) shows normal of the cortex and normal medulla, (C) rat treated with CdCl₂ shows hydropic degeneration (arrow) and focal necrosis (arrowhead) in the adrenal cortex and disturbance of the medulla (thin arrow). (D) Rat treated with CdCl₂ shows congestion (arrow) and haemorrhagic area (arrowhead) in the medulla. (E) Rat treated with of CdCl₂ and chloroform extract of coral (20 mg/kg/bw) shows mild hydropic degeneration (arrow) in the cortex and normal structure of medulla. (F) Rat treated with of CdCl₂ and chloroform extract of coral (40 mg/kg/bw) shows mild hydropic degeneration (arrow) in the cortex. (G) Rat treated with of CdCl₂ and chloroform extract of coral (60 mg/kg/bw) shows mild hydropic degeneration in the cortex and normal medulla (H & E stain, bar: 20 μm).

Export to PPT

The histopathological study showed that distraction of cadmium chloride for ten days caused hydropic degeneration, focal necrosis in the adrenal cortex as well as congestion and haemorrhagic area in the medulla. These results were in accordance with (Mithal, 1981; Abdel-Aziem et al., 2001; Jin et al., 2013). Cadmium intoxication caused congested blood vessels, haemorrhage just adjacent to the medulla and circumscribed haemorrhage in cortex. Also it was reported that cadmium has degenerative effect on the endothelium of blood vessels. On the other hand, the same author suggested that haemorrhage has occurred due to the effect of cadmium (Mithal, 1981).

The histopathological investigation showed that the toxic effects of cadmium in rats treated with chloroform extract of coral (20, 40, and 60 mg/kg/bw) were found to be reduced as presented by decrease the hydropic degeneration in the cortex and normal medulla. The decreasing of the cadmium chloride toxicity can be attributed to the presence of gorgostane sterols (Elshamy et al., 2013), cholestane sterols, ceramides, and fatty acid esters (Inagaki et al., 2004; Djeridane et al., 2010; Mohamed et al., 2012).

The sterol content plays a basic role in the biological activity of the chloroform extract because of its high antioxidant activity (Djeridane et al., 2010). It was reported that the alcoholic extract of H. fuscescens has moderate antioxidant activity that related to the isolated sterols, especially gorgostane derivatives, ceramides and long chains (Elshamy et al., 2013; Mohamed et al., 2012).

Huang et al. (2010) stated that the high ceramide content increases antioxidant activity. Also, significant attenuation against the Cd-induced hepatotoxicity was found due to its ceramides content (Prabu et al., 2012; Smalinskiene et al., 2009). The presence of saturated fatty acid esters plays an important role as antioxidant agents because they are rich in fatty acids and their esters (De La Cruz et al., 1999).