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The effects of carbenoxolone, a semisynthetic derivative of glycyrrhizinic acid, on peripheral and central ischemia-reperfusion injuries in the skeletal muscle and hippocampus of rats.


As carbenoxolone, a semisynthetic derivative of glycyrrhizinic acid, has a free radical scavenging property, thus the effects of carbenoxolone during ischemia-reperfusion was evaluated on an animal model of ischemia-reperfusion injury in the rat hind limb and hippocampus. Peripheral and central ischemia were induced by free-flap surgery in skeletal muscle and four-vessel-occulation (4VO) of rat, respectively. Carbenoxolone (50-200 mg/kg) and normal saline (10 ml/kg) were administered intraperitoneally. In peripherlal ischemia, during preischemia, ischemia and reperfusion conditions the electromyographic (EMG) potentials in the muscles were recorded. The malondialdehyde (MDA) was measured by the thiobarbituric acid (TBA) test after reperfusion in peripheral and central ischemia. In peripheral ischemia, the average peak-to-peak amplitude during ischemic-reperfusion was found to be significantly larger in carbenoxolone group (100-200 mg/kg) in comparison to control group. The MDA levels were recovered significantly upon carbenoxolone (100-200 mg/kg) therapy in the skeletal muscle and hippocampus of ischemic rats. These results suggest that carbenoxolone can salvage the skeletal muscle and hippocampus from acute ischemia-reperfusion injury.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Carbenoxolone; Glycyrrhizinic acid; Four-vessel-occlusion; Lipid peroxidation; Malondialdehyde ischemia-reperfusion; Skeletal muscle


Licorice is a perennial herb native to the Mediterranean region, central to southern Russia, and Asia Minor to Iran, now widely cultivated throughout Europe, the Middle East, and Asia (Blumenthal et al., 2000). The traditional medicinal properties and pharmacological activities of Glycyrrhiza include the treatment of peptic ulcers, asthma, pharyngitis and abdominal pain (Leung, 1980). It has been shown expectorant, antitussive, antidote (Moon and Kim, 1997), antiinflammatory (Tangri et al., 1965), antioxidant activity (Kiso et al., 1984) and hepatoprotective properties (Haraguchi et al., 1998). Bioactive components of licorice root are the triterpene glycoside, glycyrrhizic acid (GL) and its aglycone, 18b-glycyrrhetinic acid (GLA) (Blumenthal et al., 2000). GL is a noteworthy drug among phythotherapeutics and its chemical modification is the perspective route to design new bioactive compounds for medicine. Carbenoxolone, the succinyl ester of GLA (Davidson and Baumgarte, 1988), is now used in clinical treatment of ulcer diseases (Turpie and Thomson, 1965). It has some pharmacological properties such as the inhibition of gap junctional intercellular communication (Davidson and Baumgarte, 1988) and recently it was shown that carbenoxolone has anticonvulsant, sedative and muscle relaxant activities in mice (Hosseinzadeh and Nassiri Asl, 2003).

Recent evidence supports this subject using an in vitro ischemic model which carbenoxolone (as a gap junction blocker) significantly has been decreased the spread of cell death during ischemic episode and impairment of intrinsic neuronal activities using electrophysiological recording (Frantseva et al., 2002). Thus, gap junction channels appear to be a novel target to reduce brain damages during the cerebral ischemia.

During ischemic-reperfusion, membrane structures are damaged (Weglicki et al., 1984). Also the activation of phospholipases in reperfusion catalyses the lipolysis and peroxidation of polyunsaturated fatty acids. Thiobarbutiric acid reactive substances (TBARS) are the end products of lipid peroxidation. These products are extremely toxic and reactive (Horakova et al., 1991). The extension of lipid peroxidation by these end products causes dysfunctions in the cell membrane of the lipid-rich brain. The tissue damages could be determined using the measurement of these end products (Halliwell and Gutteridge, 1984). Limb ischemia is often produced during surgical procedures, which provide a bloodless operative field (Feller et al., 1989). Furthermore, in vascular impairment including vascular trauma, acute arterial thrombosis and arterial embolism this phenomenon is occurred. Ischemia-reperfusion may produce tissue edema, compartment syndromes, fibrotic contractures and contractile dysfunction (Tuncel et al., 1997). Prolong ischemia causes dysfunction and eventually tissue necrosis through loss of cellular energy charge and accumulation of potentially toxic products of cellular metabolism. Although blood flow is essential to salvage ischemic skeletal muscle, several evidences indicate that highly reactive metabolites of molecular oxygen are formed during reperfusion (Carden and Korthuis, 1989). Reactive oxygen metabolites (ROM), namely superoxide anion, are generated by activated neutrophils, monocytes and macrophages as well as other cell types and can be augmented in the setting of excess oxygen (Tzeng and Billiar, 1994).

The aim of this study was to examine the effects of carbenoxolone, the synthetic constituent of licorice, during ischemia-reperfusion. In our peripheral ischemia model, a muscle flap was isolated on its pedicle and muscles activities and lipid peroxidation respectively assessed using measuring electromyographic (EMG) potentials and the tissue malondialdehyde (MDA) level in muscle flap. In central ischemia, the lipid peroxidation was assessed using measuring the tissue MDA level following the ischemic-reperfusion in the rat hippocampus.

Materials and methods


Male rats, 200-250 g were obtained from the animal house of Pharmaceutical Research Center, Bu-Ali Research Institute of Mashhad University of Medical Sciences. The animals were housed in colony rooms with 12/12 h light/dark cycle at 21[+ or -]2[degrees]C and had free access to food and water.

All animal experiments were carried out in accordance with Mashhad University of Medical Sciences, Ethical Committee Acts.


Carbenoxolone was obtained from Sigma. Thiobarbituric acid (TBA), n-butanol, phosphoric acid, potassium chloride and tetramethoxypropane (TMP) were purchased from Merck. Xylazine and ketamine were obtained from Loughrea, Co. (Galway, Ireland) and Rotexmedica (GmbH, Germany), respectively. Phenytoin (as a positive control) in the form of ampoule (200 mg/5 ml) was obtained from IPDIC, Iran.

All chemical were dissolved in distilled water. Carbenoxolone was dissolved in physiological saline solution.

Surgical procedure

Peripheral ischemia

An incision in the inner side of the hind leg from the inguinal ligament to the tendon calcaneus insertion was made. Then it was divided up and the triceps surae was dissected as a muscle flap and that insertion to femur was cut, then the muscle isolated was on its vascular pedicle. Immediately after, having previously dissected the femoral vessels, the artery and vein were clamped with a single clamp of microsurgery. The absence of bleeding was verified in the muscle flap. Then the incision was closed to prevent desiccation. For reperfusion periods, the clamp of the femoral vessels of animals was taken off and then the bleeding of the muscle flap was verified (Fernandez et al., 2000).

Five groups of animals were used: Group 1 including sham-operated animals, Group 2 served as ischemic control to which saline (10 ml/kg) was injected i.p.. To the three groups of rats, carbenoxolone (50,100,200 mg/kg; i.p.) were injected. All drugs were administrated 1 h before reperfusion.

Except Group 1, other groups underwent 2 h ischemia and 1 h reperfusion. During preischemic, ischemic, reperfusion conditions the EMG activity in the muscles were measured for all groups and at end of the reperfusion time MDA level was determined by the TBA test.

Central ischemia (Global ischemia model)

The cerebral ischemia was induced using four-vessel-occlusion (4VO) (Pulsinelli and Brierley, 1979) in male NMRI rats (200-250 g). Animals were anesthetized with a mixture of ketamine (60 mg/kg) and xylazine (6 mg/kg), which administered i.p. The vertebral arteries were exposed and closed permanently by cauterization through the alar foramenae. The next day, anesthesia was induced with ketamine/xylazine. The carotid arteries were exposed and occluded for 20 min, using microvascular clamps. Subsequently, both clamps were removed and both arteries inspected for immediate reperfusion. Sham-operated animals underwent both surgical procedures without arterial occlusions. Six groups of animals were used: Group 1 including sham-operated animals, Group 2 served as ischemic control to which saline (10 ml/kg) was injected i.p. To another group of animals phenytoin (50 mg/kg) was administered (i.p.) as a positive control and to the three groups of rats, carbenoxolone (50, 100, 200 mg/kg; i.p.) were injected. All drugs were administrated immediately at the onset of reperfusion. Carbenoxolone, phenytoin and saline were also given every 24h for 2 consecutive days (before the day of decapitation). After 72 h, animals were decapitated and the hippocampus was removed for the TBA test.

Malondialdehyde assay

The tissue MDA level was determined by the TBA test. At the end of the treatments, the tissues were obtained to determine MDA. Muscle or hippocampus proportion was homogenized with cold 1.5% KCL to make a 10% homogenate. To 0.5 ml of 10% homogenate pipetted into a 10-ml centrifuge tube were added 3 ml of 1% phosphoric acid and 1 ml of 6% TBA aqueous solution. The mixture was heated for 45 min in a boiling water bath. After cooling, 4 ml of n-butanol was added and mixed vigorously (Yavuz et al., 1997). After centrifugation at 60,000g for 10 min (muscle tissue) or 20,000g for 20 min (hippocampal tissue), the color of butanol layer was measured at 535 nm. 1, 1, 3, 3-tetramethoxypropane was used as a standard of MDA.

Electromyography data collection

To determine the muscles activities during ischemia-reperfusion, intramuscular EMG signals were recorded with PowerLab data acquisition systems. Two pairs of pin electrodes in terminating alligator clips were inserted into the triceps surae (muscle flap) in hind leg, and adductor muscles. The distance between the two electrodes of a pair in each muscle was 5 mm. A grounding electrode was gently attached to the rat's tail (Ossowska et al., 1996). The EMG signals were collected with sampling frequency of 12 ppm (MacLab/4SP). Duration for each stimulation was 20ms. The raw EMG signals were low-pass filtered at 50 Hz and EMG signal is expressed as average peak-to-peak amplitude for a 10 min recording periods.

Statistical analysis

The data were expressed as mean values[+ or -]SEM and tested with analysis of variance followed by the multiple comparison test of Tukey-Kramer.


Peripheral ischemia

The average peak-to-peak amplitudes in the control group and sham were (1.68[+ or -]0.063, 2.18[+ or -]0.068 V) and (1.59[+ or -]0.104, 2.20[+ or -]0.074 V), respectively during ischemia and reperfusion. Carbenoxolone at the doses of (100-200 mg kg) had significantly greater amplitude (2.01[+ or -]0.026, 1.98[+ or -]0.025 V) (2.03[+ or -]0.023, 1.95[+ or -]0.028 V) than controls, respectively, during ischemia and reperfusion, respectively. The difference between the effect of carbenoxolone 100 mg/kg and 200 mg/kg was not significant. Carbenoxolone in a dose of 50 mg/kg could significantly increase the average peak-to-peak amplitudes only in the reperfusion phase (Table 1).

The MDA levels of muscle portion in the sham and saline groups were found 7.06[+ or -]1.92 and 30.27[+ or -]3.84 nmol/g, respectively. Carbenoxolone reduced the MDA level dose dependently. At the doses of 100 and 200 mg/kg, MDA levels were reduced up to 66.60% and 95.93%, respectively by carbenoxolone treatment. Carbenoxolone in a dose of 50 mg/kg could not significantly reduce MDA level (Table 2).

Central ischemia

The MDA levels of hippocampus portion in the sham and saline were found to be 167.0[+ or -]2.8 and 286.0[+ or -]2.5 nmol/g, respectively. In phenytoin group, the MDA level was reduced up to 58.04% compared to the saline group (Table 3).

Carbenoxolone reduced the MDA level dose dependently. At the doses of 100 and 200 mg/kg, MDA levels were reduced up to 41.61% and 62.94%, respectively by carbenoxolone treatment. At a dose of 50 mg/kg, carbenoxolone did not reduce the MDA level significantly (Table 3).


In the present study, carbenoxolone significantly decreased the MDA level following ischemic-reperfusion in the rat skeletal muscle and hippocampus. This effect was dose dependent. A significant decrease in average peak-to-peak amplitude of EMG signal was seen in both ischemia and reperfusion phases in control group as compared to the sham group. Furthermore carbenoxolone in doses of (100-200 mg/kg) induced the increased in EMG activity during ischemia-reperfusion which was associated with a reduction in MDA level as compared to control.

Peroxidation of membrane lipids may also contribute to myocyte dysfunction as a consequence of alterations in membrane fluidity and porosity, second messenger function, cellular compartmentation, cell volume regulation, the expression and/or mobility of membrane receptors and other proteins and ultimately, cell lysis (Girotti, 1985). Reperfusion-induced contractile dysfunction may result as a consequence of oxidant-mediated damage to structural, transport and contractile proteins and inactivation of receptors and essential enzymes (Akimitsu et al., 1994).

In recent study, carbenoxolone (as a gap junction blocker), in the in vitro ischemic model, significantly decreased the spread of cell death during ischemic episode and impairment of intrinsic neuronal activities using electrophysiological recording (Frantseva et al., 2002). Experimental data indicate that the gap junction communication is affected after an ischemic period. The increased immunoreactivity for connexin-43 was seen 2 days after mild ischemic injury in the rat hippocampus and striatum (Hossain et al., 1994).

Astrocytes, which exhibited a high degree of coupling through gap junctions, composed mainly of connexin-43 (Dermietzel, 1998). Also, recent studies support a role for interastrocytic gap junctions in the propagation and amplification of neuronal injury. These channels remain functional in postischemic brain (Lin et al., 1998; Rami et al., 2001) reported that the inhibition of gap junction communications by octanol (a gap junction channels blocker) in a rodent model of global transient cerebral ischemia reduces the extension of ischemic injury. They also found that redistribution of connexin-43 immunoreactivity after ischemia correlates well with the phenomenon of selective vulnerability. Although, Naus et al. (2001) found that Cx43 heterozygote null mice exhibited a significantly larger infarct volume compared to wild-type following focal ischemia. But the contribution of gap junctions to cell injury is a complicated phenomenon that depends on the specific insult and network in which it operates (Perez Velazquez et al., 2003).

ROM, namely superoxide anion, are generated by activated neutrophils, monocytes and macrophages, can be augmented in the setting of excess oxygen following the ischemia reperfusion (Tzeng and Billiar, 1994). Compounds with steroidal structures were shown to be potent [O.sub.2.sup.-] scavengers and to be active in the protection neuron against peroxide injury (Hall et al., 1987). Carbenoxolone in micromolar concentration has been shown to suppress the generation of superoxide anions and hydrogen peroxide in macrophages (Suzuki et al., 1983). On the other hand, superoxide anion can cause lipid peroxidation, which disrupts cell and mitochondrial membranes as well as impairing calcium transport (Braunwald and Kloner, 1985).

Thus, in addition of previous mechanism which we mentioned above, these two properties of carbenoxolone may also contribute to the protection against increased lipid peroxidation in brain following ischemic-reperfusion.

Accumulation of intracellular free calcium [C[a.sup.2+]i] may play an essential role in the ischemia-reperfusion injury of skeletal muscle. Intracellular calcium over-loading activates intracellular calcium-dependent proteases, phosphatases, and phospholipases, leading to cell injury and more generation of ROM (Akimitsu et al., 1994). Although it has been shown that [C[a.sup.2+]i] levels significantly increase during ischemia-reperfusion in 1 or 2 h of ischemia and these changes probably depending on the muscle fiber type exposed (Ivanics et al., 2000). In global ischemia, neuronal glutamate release will evoke calcium waves in syncytia of astrocytes (Verkhratsky and Kettenmann, 1996) that propagate through gap junctions and, in turn, will activate neurons at a distance. During the acute phase of stroke, spontaneous waves of spreading depression are evoked within the ischemic peri-infarct zone (Nedergaard and Astrup, 1986).

We suggest carbenoxolone probably by closing gap junction channels decreased intercellular C[a.sup.2+] waves and propagation of transcellular signals which excerbate cell injury as calcium overload, oxidative stress and metabolic inhibition result this phenomena (Lin et al., 1998). It remains to be investigated to what extent of the protective effects of carbenoxolone during ischemia-reperfusion is related to inhibition of gap junction channels in rat skeletal muscle.

The most important point of this study is that carbenoxolone increases the EMG activity and decreases MDA level of skeletal muscle and hippocampus during ischemia-reperfusion. This showed that carbenoxolone prevented lipid peroxidation during acute ischemia-reperfusion model. As carbenoxolone in micromolar concentration has been shown to suppress the generation of superoxide anions and hydrogen peroxide (Suzuki et al., 1983), it may act via radical scavenging properties. Another mechanism can be attributed to closing gap junctions channels and reduction of intercellular C[a.sup.2+] waves, leading to decreased lipid peroxidation.

Phenytoin also significantly reduced the MDA level in our experiment. In cerebral ischemia animal models, phenytoin has been shown protective effects in different hippocampal regions dentate granular cells (Vartanian et al., 1996). Thus, in our experiments, we applied phenytoin as a positive control which has protective effects during the ischemic-reperfusion.

In conclusion, carbenoxolone, the synthetic constituent of licorice, can probably protect tissues against lipid peroxidation in peripheral and central ischemia-reperfusion damages.


The authors are thankful to the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support.


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H. Hosseinzadeh (a,*), M. Nassiri Asl (b), S. Parvardeh (b)

(a) Pharmaceutical Research Center, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, IR Iran

(b) Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Science, Mashhad, IR Iran

Received 29 June 2004; accepted 6 July 2004

*Corresponding author. Tel.: +98 511 8823252; fax: +98 511 8823251.

E-mail address: (H. Hosseinzadeh).
Table 1. Effects of carbenoxolone (50-200 mg/kg) on average peak-to-peak
amplitudes of EMG signals, before, during and after ischemia-reperfusion
in rat skeletal muscle

Group (n = 10) Preischemia (V) Ischemia (V) Reperfusion (V)

Control (10 ml/kg) 2.21 [+ or -] 1.68 [+ or -] 1.59 [+ or -]
 0.077 0.063## 0.104###
Sham 2.02 [+ or -] 2.18 [+ or -] 2.20 [+ or -]
 0.034 0.068 0.074
Carbenoxolone 50 mg/kg 2.21 [+ or -] 1.85 [+ or -] 1.86 [+ or -]
 0.072 0.023 0.077*
Carbenoxolone 100 mg/kg 2.16 [+ or -] 2.01 [+ or -] 2.03 [+ or -]
 0.068 0.026*** 0.023***
Carbenoxolone 200 mg/kg 2.07 [+ or -] 1.98 [+ or -] 1.95[+ or -]
 0.038 0.025*** 0.028***

Values are mean[+ or -]SEM. #p<0.05, ##p<0.01 and ###p<0.001 when
compared to intra-group, *p<0.05 and ***p<0.001 when compared with
corresponding between groups with Tukey-Kramer.

Table 2. The effect of carbenoxolone on MDA levels in skeletal muscle of
rat after reperfusion

Group (n = 10) MDA (nmol/g)

Saline (10 ml/kg) 30.27 [+ or -] 3.84
Sham 7.06 [+ or -] 1.92***
Carbenoxolone (50 mg/kg) 29.91 [+ or -] 0.74
Carbenoxolone (100 mg/kg) 10.11 [+ or -] 0.164**
Carbenoxolone (200 mg/kg) 1.23 [+ or -] 0.061***

Results are expressed as mean[+ or -]SEM. **p<0.01, ***p<0.001.
Significance calculated between saline group and sham and carbenoxolone
groups with Tukey-Kramer.

Table 3. The effect of carbenoxolone on MDA levels after the global
ischemic-reperfusion in rats

Treatment MDA levels (nmol/g)

Saline (10 ml/kg) 286 [+ or -] 2.5
Sham 167 [+ or -] 2.8***
Phenytoin (50 mg/kg) 120 [+ or -] 1.82***
Carbenoxolone (50 mg/kg) 240 [+ or -] 0.24
Carbenoxolone (100 mg/kg) 167 [+ or -] 0.97***
Carbenoxolone (200 mg/kg) 106 [+ or -] 1.5***

Results are expressed as mean[+ or -]SEM. ***p<0.001.
Compared to saline group, Tukey-Kramer test, n = 10.
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Author:Hosseinzadeh, H.; Nassiri Asl, M.; Parvardeh, S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Sep 1, 2005
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