Heart and kidney oxidative stress status in septic rats treated with caraway extracts

Abstract

Context: Carum carvi L., (Umbelliferae) known as caraway, is a famous traditional herbal plant supposed to contain active components with pharmacological properties.

Objective: In this study, the effects of caraway extracts on preventing sepsis induced by oxidative tissue injuries have been investigated by measuring heart and kidney oxidative stress parameters.

Materials and methods: Sepsis was induced in rats (n = 6) by experimental cecal ligation and puncture (CLP) model. Then, either hydroalcoholic extract or essential oils (50 and 100 mg/kg body weight) were injected intraperitonially immediately after CLP operation. Twenty-four hours after CLP, the rats were anesthetized when kidney and heart tissues were removed to analyze the tissue oxidative stress parameters, that is, glutathione (GSH) and lipid peroxidation (LP).

Results: Sepsis induction caused a significant increase in kidney but not heart LP, indicating that kidney was more affected by sepsis induction than heart. Kidney LP and plasma urea/creatinine ratio levels were readily reversed in rats treated with essential oils but not in those treated with hydroalcoholic extract. Unlike LP, the heart and kidney GSH levels were not affected in all treated groups.

Discussion and conclusion: Our data imply that caraway oils probably have a protective role in kidney tissue against oxidative injury in advanced stages of sepsis.

Key words::

• Tissue injury
• sepsis
• antioxidant

Introduction

Septic shock is a severe form of sepsis associated with the development of progressive damage to multiple organs in the late stage. It is also an important cause of mortality in intensive care units (Hubbard et al., 2005). During sepsis, reductions of enzymatic and nonenzymatic antioxidants [e.g. glutathione (GSH)] and also over-production of oxygen-free radicals [reactive oxygen species (ROS)] ultimately lead to oxidative stress (Peralta et al., 1993; Gutteridge, 1995). Oxidative stress plays an important role in the pathogenesis of sepsis and its complications. Experimental and clinical studies have shown that any harmful tissue events, such as infection, trauma, or anoxia, are perceived by macrophages and monocytes, which in turn secrete cytokines such as interleukin (IL)-1 and tumor necrosis factor-α. These cytokines activate inflammatory cells, such as neutrophils, macrophages or monocytes, platelets, and mastocytes, releasing large amounts of ROS leading to cellular injury via several mechanisms including the peroxidation of membrane lipids and the oxidative damage of proteins and DNA (Hubbard et al., 2005; Andrades et al., 2005). These events can eventually cause secondary damages to organs including heart and kidney, which may contribute to organ dysfunction and failure (Singer & Brealey, 1999).

The frequent question for the treatment of sepsis nowadays favors the investigation of oxidative stress and the role of antioxidative defense systems to develop new pharmacological approaches to this life-threatening disease (Victor et al., 2004). There is substantial evidence indicating that natural antioxidant and herbal drugs exert their antioxidant properties by scavenging oxygen-free radicals (Huh et al., 1994; Gómez-Zubeldia et al., 2000). Silymarine (a mixture of bioactive flavonoliganans isolated from Asteraceae silybum) can protect APAP (N-acetyl-para-aminophenol OR acetaminophen) -induced damage in rat brain by preventing lipid peroxidation (LP) and replenishing the GSH levels (Nencini et al., 2007). A membrane radical scavenger, that is Tempol-attenuated organ dysfunction and injury by reducing the plasma concentration of IL1β and NO (nitric oxide) and decreasing the neutrophil infiltration in the liver and lung tissues (Liaw et al., 2005).

Carum carvi L. (Umbelliferae), known as caraway, is a famous traditional herbal plant and is normally found in northern and central Europe, Siberia, Turkey, Iran, India, and North Africa. In Iran, the seeds of caraway, commonly known as “Zireh Siah,” have been used extensively in food. The seeds have been traditionally used as a medicinal plant in traditional medicine for treating aliments such as flatulence, colic pain, and bronchitis. In addition, caraway has been widely used as stomachic, antispasmodic, antibacterial, antiulcerogenic, and antiproliferative agent (Eddouks et al., 2004).

In vitro and in vivo studies indicated that essential oils and aqueous extract of caraway have various physiological and pharmacological properties (De Carvalho & Da Fonseca, 2006; Lemhadri et al., 2006; Mazaki et al., 2006). One previous study showed the suppressive effects of caraway extracts on cytochrome P-4501A1, a phase-I xenobiotic metabolizing enzyme, which was over-expressed with dioxane in hepatoma cell line (Naderi-Kalali et al., 2005). More freshly, we proved the in vitro antioxidant and antibacterial properties of caraway oil preparation (Fatemi et al., in press). In addition, we showed that caraway essential oils modulate the parameters related to oxidative liver and lung injuries, that is, myeloperoxidase (MPO) activity, thiobarbituric acid reactive substances (TBARS), and GSH levels in cecal ligation and puncture (CLP) rat model (Fatemi et al., 2010a,b).

In regard to our previous studies (Fatemi et al., 2010a,b, in press) and also the importance of free radicals in the mortality of septic patients, we decided to consider the effects of caraway oils and hydroalcoholic extract on preventing oxidative heart and kidney injuries in sepsis for the first time. Thus, in this study, after analyzing the composition of caraway extracts and their antioxidant capacities, we considered the effects of these fundamental constitutes on parameters related to oxidative kidney and heart injuries in septic rats.

Materials and methods

Chemicals

Fresh caraway seeds were purchased from a local market in Tehran, Iran. The plants were collected in June 2008 from the Varzaneh town in Isfahan province, Iran. A scientist of botany, Mrs. Bagherzadeh (M.Sc., Institute for Research in Forests and Rangelands, Isfahan, Iran) authenticated the plant materials. Aluminum trichloride (AlCl₃), methanol, trichloroacetic acid, ethylenediaminetetraacetic acid, dimethyl sulfoxide (DMSO), and Tris-base were obtained from Merck Co., Germany. Quercetin, 2,2-diphenylpicrylhydrazyl (DPPH), Trolox, thiobarbituric acid (TBA), dithionitrobenzene, and GSH were from Sigma Chemical Co., USA. Serum urea, creatinine, and creatinine kinase-MB (CK-MB) detection kits were from Pars Azmoon Co., Tehran, Iran.

Oil extraction and analysis

Caraway seeds were subjected to oil extraction using a Clevenger-type apparatus. The extraction was carried out for 2 h and the oil was stored in dark glass bottles in a freezer (−20°C) until further use. Gas chromatographic (GC) analysis was performed using GC (9-A-Shimadzu; Japan) equipped with a flame ionization detector. Quantitation was performed using Euro Chrom 2000 software (KNAUER Company, Germany) by the area normalization method. The analysis was carried out using a DB-5 fused-silica column (30 m × 0.25 mm; film thickness, 0.25 µm) using a temperature program of 40–250°C at a rate of 4°C/min, injector temperature of 250°C, detector temperature of 265°C. The carrier gas was helium (99.99%). The GC/MS unit consisted of a Varian-3400 GC coupled to a Saturn II ion trap detector. The type of the column and the conditions used for analysis was similar to those of GC analysis. The constituents were identified by comparing their mass spectra with those of authentic standards. The peaks were further identified by comparing their retention indices with those of authentic standards.

Preparation of hydroalcoholic extract and estimation of total flavonoids

Powdered caraway seeds (30 g) were mixed with distilled water (50 ml) and methanol (50 ml) at 70–80°C and maintained at 60°C for 24 h. Then, the hydroalcoholic extract was filtered through a Whatman filter (#4, with pore size 20–25 µm) to remove particulate matters. The filtrate was then freeze-dried for further use.

Total flavonoid content of the hydroalcoholic extract was determined using Dowd method as adapted by Arvouet-Grand et al. (1994). Briefly, 5 ml of the extract solution (0.1 mg/ml) was mixed with an equal volume of 2% aluminum trichloride (AlCl₃) prepared in methanol. The absorption was recorded at 415 nm using Shimadzu UV-3100 spectrophotometer after 10 min against a blank sample. The blank sample was prepared by mixing 5 ml hydroalcoholic extract with 5 ml methanol alone. Total flavonoids were determined using a standard curve prepared with quercetin (0–100 mg/l) as the standard. The mean of three readings was used and expressed as milligram quercetin equivalents (QE)/gram of hydroalcoholic extract.

Radical-scavenging capacity (DPPH assay) of the extracts

This spectrophotometric assay uses the stable radical DPPH as a reagent (Burits & Bucar, 2000). The essential oils in methanol (50 µl of 1:5 dilution) were added to 5 ml of DPPH solution (0.004% DPPH in methanol). After 30 min incubation at room temperature, the absorbance was recorded against the blank at 517 nm. Trolox, (1 mM) a stable antioxidant, was used as a synthetic reference. The inhibition of free radical (DPPH) was calculated as percentage (I%) by the following formula:

where A_blank is the absorbance of the control reagent (containing all reagents except the test compound) and A_sample is the absorbance of the test compound. All the assays were carried out in triplicate.

The radical scavenging activity of the hydroalcoholic extract was also measured spectrophotometrically using the DPPH radical (Blois et al., 1958). The caraway hydroalcoholic extract (50 µg/ml) was added to 2 ml DPPH solution (125 μM DPPH in methanol) and the final volume was adjusted to 4 ml with water. The mixture was mixed and incubated at 37°C in dark for 30 min. Changes in the absorbance of DPPH were recorded at 517 nm. A parallel experiment was performed in which caraway extract was replaced with vitamin C (5 µg/ml) and considered as a positive control. Percent inhibition was calculated by comparing the absorbance values of the blank and the sample as discussed above.

CLP model

Male albino rats of Wistar strain (150–170 g) were purchased from the Pasteur Institute of Iran. Animal studies were approved by the Medical Ethics Committee of Tarbiat Modares University.

Polymicrobial sepsis in rats was induced by CLP according to the method of Hubbard et al. (2005). Briefly, rats were anesthetized by a single injection [intraperitoneal (i.p.)] of ketamine (90 mg/kg) and xylozine (10 mg/kg) mixture. A small midabdominal incision (2–3 cm) was made and the cecum was exposed. A distended portion of the cecum just distal to the ileocecal valve was isolated, filled with fecal content, and tied with a 3-0 silk suture in a manner not to disrupt bowel continuity. The ligated portion of the cecum was punctured twice with a 20-gauge needle. The cecum was then placed in its original position within the abdomen, the abdomen was then closed with a 3-0 suture in two layers, and the animals were allowed to recover. In the sham-operated rat, the cecum was exposed, manipulated, and returned to the peritoneal cavity without being ligated and punctured. After surgery, normal saline [3 ml/100 g body weight (b.w.)] was given subcutaneously to all rats to prevent dehydration.

Animal treatments and tissue processing

As shown in Table 1, animals were divided into seven groups. The caraway essential oils, hydroalcoholic extracts (50 and 100 mg/kg b.w.) and also indomethacin (10 mg/kg b.w.) were dissolved in 0.5 ml DMSO and injected immediately after CLP operation. Twenty-four hours after CLP, blood samples were collected and kidney and heart tissues were removed, washed, and processed for biochemical analysis.

Table 1.  Treatment groups.

Groups	DMSO	EO (mg/kg b.w.)		HE (mg/kg b.w.)		Indomethacin (10 mg/kg b.w.)
		50	100	50	100
SOP	+	−	−	−	−	−
CLP	+	−	−	−	−	−
CLP+E.O50	−	+	−	−	−	−
CLP+E.O100	−	−	+	−	−	−
CLP+H.E50	−	−	−	+	−	−
CLP+H.E100	−	−	−	−	+	−
CLP+Ind10	−	−	−	−	−	+

In sham-operated rats (SOP), laparotomy was done and animals received DMSO as vehicles; in CLP rats, animals received vehicles only; in CLP + EO groups, essential oils (50 or 100 mg/kg) were injected (i.p.) immediately after CLP operation, In CLP + H.E groups, hydroalcoholic extracts (50 or 100 mg/kg) were injected (i.p.) immediately after CLP operation. A group of CLP rats treated with indomethacin (10 mg/kg b.w.) as positive control.

Measurement of LP products in liver

A weighed portion of tissues was homogenized in phosphate buffer (100 mM, pH 7.0) and used to measure the levels of TBARS as indices for LP. The concentration of TBARS was measured spectrophotometrically using TBA reagent based on the procedure described by Buege and Aust (1987).

GSH estimation

GSH was estimated in tissue homogenates according to the procedure of Seldak and Lindsay (1986).

Plasma biomarkers

To confirm the kidney and heart injury, serum urea, creatinine and creatine kinase-MB were determined respectively, using commercial kits on an autoanalyzer (Technicon RA-1000).

Statistical analysis

Data are presented as means ± standard error. The results were subjected to one-way analysis of variance followed by Tukey’s honestly significant differences using SPSS 13.0 software. The significance was considered as P < 0.05.

Results

Analysis of caraway extracts

Based on GC and GC/MS analyses, 17 known compounds were identified in the essential oil samples extracted from Iranian caraway seeds (Figure 1 and Table 2). The peaks identified as cumin aldehyde (20.90%), γ-terpinene (16.78%), γ-terpinene-7-al (16.69%), and p-cymene (7.23%) constitute the major compounds of the essential oils (Table 2).

Table 2.  Essential oil analysis prepared from caraway seeds.

Peak no	Name	Percentage	RI
1	α-Pinene	0.50	979.40
2	β-Pinene	4.51	989.60
3	Myrceme	0.38	1002.29
4	p-Cymene	7.23	1031.57
5	Limonene	1.28	1040.95
6	γ-Terpinene	16.78	1086.57
7	p-Cymen-8-ol	0.80	1089.44
8	Cumin aldehyde	20.90	1119.08
9	α-Terpinen-7-al	2.90	1137.72
10	Bornyl acetate	1.20	1245.58
11	γ-Terpinen-7-al	16.69	1308.77
12	Cuminyl acetate	4.38	1341.03
13	Myristicine	2.70	1489.74
14	Elemicine	3.55	1531.56
15	Germacrene B	1.58	1576.74
16	Dill apoil	1.02	1628.19
17	Carvone	0.65	1234.46

The essential oils extracted from seeds using a Clevenger-type apparatus. Analysis was performed on a GC equipped with a flame ionization detector. The peaks were identified by comparing their mass spectra with those in the computer library as well as authentic standards. RI: Retention Index.

Figure 1.  MS chromatogram plot. The constituents were identified by comparing their mass spectra with those of authentic standards. The peaks were further identified by comparing their retention indices with those of authentic standards.

The total flavonoid content of hydroalcoholic extract of caraway seeds was found to be 18.5 mg of QE/g hydroalcoholic extract calculated from a standard curve generated by quercetin.

DPPH radical-scavenging activity of caraway extracts

The antioxidant capacities of the essential oils and hydroalcoholic extract prepared from caraway seeds were determined by in vitro DPPH radical-scavenging activity (Figure 2A and 2B). The essential oils notably reduced the concentration of DPPH free radical, with an efficacy higher than that of Trolox (Figure 2A). Also, the hydroalcoholic extract reduced the concentration of DPPH free radical, with an efficacy lower than that of vitamin C (Figure 2B).

Figure 2.  (A) Free radical-scavenging activity of the caraway seed extracts. The radical scavenging activity of the oil extract was measured spectrophotometrically using the DPPH free radical. Trolox (1 mM) was used as positive control. (B) The radical scavenging activity of the hydroalcoholic extract was measured spectrophotometrically using the DPPH free radical. Vitamin C (5 µg/ml) was used as positive control. Results are mean ± SEM of three analyses carried out on essential oil and hydroalcoholic extract derived from caraway seeds.

The effects of caraway extracts on parameters related to oxidative kidney and heart injuries in CLP rats

Administration (i.p.) of caraway oils (50 and 100 mg/kg b.w.) to septic rats resulted in a significant reduction in kidney MDA levels (Table 3). CLP operation in rats without further treatments resulted in increased TBARS level, an index of LP. Treatment of rats with either caraway oil doses (50 and 100 mg/kg b.w.) could reverse the MDA level to normal level (laparatomy group) while hydroalcoholic extracts with similar concentrations failed to alter the TBARS level. The caraway preparations at a dose of 50 or 100 mg/kg b.w. given to rats were comparable with the effects of indomethacin (10 mg/kg b.w.), which is considered as a positive control. As shown in Table 3, kidney GSH was not changed in all treated animals (P > 0.05).

Table 3.  Effect of caraway extracts on kidney and heart oxidative parameters in sepsis rats.

	Groups
	Tissues	SOP (DMSO)	CLP (DMSO)	EO (50 mg/kg b.w.)	EO (100 mg/kg b.w.)	HE (50 mg/kg b.w.)	HE (100 mg/kg b.w.)	Indomethacin (10 mg/kg b,w.)
TBARS (nmol/g tissue)	Kidney	7.77 ± 0.19	9.77 ± 0.84*	7.5 ± 0.06**	7.33 ± 0.3**	8.6 ± 1.3	8.48 ± 1.4	7.2 ± 0.24**
	Heart	3.5 ± 0.23	4.01 ± 0.15	4 ± 0.07	4.06 ± 0.06	3.9 ± 0.06	3.8 ± 0.2	3.7 ± 0.06
GSH (µmol/g tissue)	Kidney	30 ± 0.7	29.68 ± 0.56	32 ± 0.6	33.09 ± 5.5	30.5 ± 0.9	26.53 ± 0.84	31 ± 2.5
	Heart	18.18 ± 0.36	20.73 ± 0.2	18.4 ± 0.5	22.6 ± 0.2	21.56 ± 0.9	24.9 ± 1.6	19.6 ± 0.3

In sham-operated rats (SOP), laparotomy was done and animals received DMSO as vehicles; in CLP rats, animals received vehicles only; in CLP + EO groups, essential oils (50 or 100 mg/kg) were injected (i.p.) immediately after CLP operation, In CLP + HE groups, hydroalcoholic extracts (50 or 100 mg/kg) were injected (i.p.) immediately after CLP operation. A group of CLP rats treated with indomethacin (10 mg/kg b.w.) as positive control. All the animals were scarified 24 h after treatments. Values are mean ± SEM carried out in duplicate. ^*P < 0.05 is considered significantly different from laparatomy group within each parameter. ^**P < 0.05 is considered significantly different from CLP group within each parameter.

Under similar conditions of treatments, the levels of heart TBARS and GSH levels were not affected by CLP operation (Table 3). Administration of both caraway extracts also caused no significant changes in heart LP and GSH levels (P > 0.05).

Cardiac and renal biomarkers in plasma

The data presented in Table 4 are consistent with results in Table 3, which shows that the ratio of urea/creatinine, the indicator of renal function, was approximately greater than in septic rats as compared with that in control group. Similar to indomethacin, caraway essential oils (50 and 100 mg/kg b.w.) influenced this ratio. In this connection, a marked increase in urea/creatinine ratio due to CLP operation was significantly suppressed in CLP rats treated with caraway oils and also indomethacin (P < 0.05). Nevertheless, caraway hydroalcoholic extracts failed to alter this parameter.

Table 4.  Effect of caraway extracts on renal and cardiac biomarkers in plasma of sepsis rats.

Groups	SOP (DMSO)	CLP (DMSO)	E.O (50 mg/kg b.w.)	E.O (100 mg/kg b.w.)	H.E (50 mg/kg b.w.)	H.E (100 mg/kg b.w.)	Indomethacin (10 mg/kg b,w)
Urea/creatinine (mg/dl)	84.85 ± 12.5	462.47 ± 81*	53.38 ± 15**	79.16 ± 21**	474.28 ± 23	477.33 ± 14	36.84 ± 11**
CK-MB (U/L)	475.2 ± 33	480.75 ± 34	391.33 ± 38	508.33 ± 85	397 ± 90	649 ± 9	423.33 ± 66

In sham-operated rats (SOP), laparotomy was done and animals received DMSO as vehicles; In CLP rats, animals received vehicles only; In CLP + EO groups, essential oils (50 or 100 mg/kg) were injected (i.p.) immediately after CLP operation, In CLP + H.E groups, hydroalcoholic extracts (50 or 100 mg/kg) were injected (i.p.) immediately after CLP operation. A group of CLP rats treated with indomethacin (10 mg/kg b.w.) as positive control. All the animals were scarified 24 h after treatments. Values are mean ± SEM carried out in duplicate. ^*P < 0.05 is considered significantly different from laparatomy group within each parameter. ^**P < 0.05 is considered significantly different from CLP group within each parameter.

Plasma CK-MB (as the indicator of heart injury) was also unaffected in all treated animals (P > 0.05) (Table 4).

Discussion

Previously, we reported the suppressive effects of caraway essential oils on colon premalignant lesions induced by dimethylhydrazine (Dadkhah et al., in press). More recently, we proved the in vitro antioxidant and antibacterial properties of caraway oil preparation (Fatemi et al., in press). To continue with this, after indicating the influence of caraway essential oils and hydroalcoholic extract on oxidative liver and lung injury parameters (MPO activity, TBARS, and GSH levels) in CLP rat model (Fatemi et al., 2010a,b), attempts were made to find out the mechanism by which the caraway extracts reduce the possible kidney and heart damages that may occur in septic rats.

In the first step, we evaluated the antioxidant strength of caraway efficient extracts in vitro. As shown in this study (Figure 2A), the radical scavenging capacity of caraway seed essential oils (DPPH assay) containing cumin aldehyde (20.90%), γ-terpinene (16.78%), γ-terpinene-7-al (16.69%), and p-cymene (7.23%) (Table 2) implied that caraway oil is a potent free-radical scavenger and antioxidant. As shown in Figure 2B, the hydroalcoholic extract of caraway seeds containing 18 mg/g quercetin equivalent of flavonoids is also an effective antioxidant.

Our in vivo results (Table 3) clearly showed that sepsis causes partial oxidative damage in the kidney as demonstrated by the increased LP. These data, together with the biochemical findings (urea/creatinine ratio) confirmed the fractional kidney damage. Other studies also indicated that the oxidative stress in sepsis has a direct relationship with organ injuries in CLP model. Releasing large amounts of ROS in sepsis can cause cellular injury via several mechanisms including the peroxidation of membrane lipids and the oxidative damages to proteins and DNA (Gutteridge, 1995; Hubbard et al., 2005).

Treatment of septic rats with caraway oils and also indomethacin could significantly reverse the increased LP product (P < 0.05) but conversely, caraway hydroalcoholic extract did not modulate the kidney LP level (P > 0.05). The protective role of caraway oils in kidney was further confirmed by showing that the urea/creatinine ratio increased in septic rats (the indicator of renal function) is reversed in caraway oil and indomethacin-treated groups (Table 3). On the other hand, GSH, which is an important constituent of the intracellular protective mechanism against various noxious stimuli (Villa et al., 2002), is not affected in kidney after CLP operation indicating that the injury was not serious enough to cause GSH depletion. So, the GSH level was not changed after treatment of septic rats with both caraway preparations. Quite the opposite of this result, our findings indicate that heart was not severely injured under CLP operation. The oxidative parameters of heart injury, that is GSH, LP, and also plasma CK-MB were not affected in all treated animals indicating that sepsis due to CLP operation caused no damage to heart (Table 3).

Our previous results also indicated that hydroalcoholic extract of caraway seeds has no effects in modulating the oxidative stress parameters in lung tissue (Fatemi et al., 2010b). In this regard, there are different reasons that can justify the superiority of essential oils over hydroalcoholic fractions in modulating the partially injured kidney. First, the effectiveness of the essential oils in remedy of sepsis is probably due to pharmacokinetic properties and the disposition of the caraway monoterpenes in the body. Considering the physicochemical properties of the terpenes, such as lipophilicity and molecular mass and size, penetration into and permeation through the skin as well as subsequent systemic effects can be expected (Crowell et al., 1992; Phillips et al., 1995; Stott et al., 1998; Hongratanaworakit & Buchbauer, 2006). The presence of aromatic compound glucosides, alkyl glycosides, glucides, nucleoside, and flavonoids in hydroalcoholic fraction of caraway seeds (Matsumura et al., 2002) offer relatively poor bioavailability to this fraction.

The bioavailability of dietary polyphenols is limited not only by the physiochemical properties of the molecule but also by enzyme and microbial-mediated biotransformation and active efflux (Scalbert et al., 2002; Lambert & Yang, 2003; Manach et al., 2004, 2005; Yang et al., 2008), which directly undergo phase II metabolism (Li et al., 2001; Lambert & Yang, 2003; Lu et al., 2003a,b; Hu et al., 2003). On the other hand, the more protective role of essential oils, rather than hydroalcoholic extracts in septic rats can partly be due to antimicrobial properties of caraway essential oils. In this connection, our data indicate that caraway essential oils but not hydroalcoholic extracts obtained from caraway seeds possessed antimicrobial activity against some Gram-negative and Gram-positive bacteria (Fatemi et al., in press; data not shown). Other studies also implied the severe antimicrobial activity of essential oils (Rasooli et al., 2006, 2008; Costa et al., 2008).

On the other hand, despite the fact that polyphenols have antioxidant activities, there are different reports indicating the prooxidant properties of the flavonoids (Galati & O’Brien, 2004; Murzakhmetova et al., 2008; Babich et al., 2008; Panemangalore & Bebe, 2009). Robaszkiewicz et al. (2007) demonstrated concentration-dependent effects of quercetin on A549 cells in vitro, including augmentation of cell proliferation and increase in total antioxidant capacity (TAC) of the cells at low quercetin concentrations and a decrease in cell survival and viability, thiol content, TAC and activities of superoxide dismutase, catalase and GSH S-transferase at higher concentrations of the flavonoid (>50 mM) (Robaszkiewicz et al., 2007). In addition, in the presence of H₂O₂, catechol B ring of flavonoids is oxidized by MPO to produce phenoxyl radicals responsible for oxidative damage to macromolecules (Galati & O’Brien, 2004). Intracellular accumulations of ROS and protein carbonyls were detected in the HL-60 cells treated with apigenin (a flavonoid) in a dose-dependent manner. When these cells were treated with MPO inhibitors, the ROS level enhanced by apigenin was significantly reduced. The gathered data suggested that oxidation of apigenin by MPO and production of B-ring phenoxyl radicals might be responsible for the prooxidant effects (Miyoshi et al., 2007).

One study also indicated the prooxidant activity of polyphenols arising from their interactions with metals such as iron and copper leading to the production of hydroxyl radicals via fenton-like reaction (Perron & Brumaghim, 2009). Considering this information, we believe that polyphenols have antioxidant activities especially in reduced forms. When these compounds are changed to oxidized types such as phenoxyl or quinone forms, they can act as prooxidant agents. So, in regard to the increased levels of ROS and MPO in sepsis (data not shown; Fatemi et al., 2010a), it is assumed that polyphenols and flavonoids present in hydroalcoholic extract are changed to oxidized form and act as the prooxidant resulting from the lack of hydroalcoholic extract effects in improving tissue injury induced by sepsis. Alternatively, it should be considered further to assess the protective effect of caraway hydroalcoholic extract in sepsis.

Our results indicated ineffectiveness of i.p. injection of caraway hydroalcoholic extract. In this connection, using other models of sepsis induction and also evaluating other routes of the administration, that is, oral, intravenous, and subcutaneous, should be considered. In addition, different time of injections, for example, pre-treatments or post-treatments in different time intervals should be examined entirely.

Conclusion

In conclusion, our data implied that i.p. administration of caraway essential oil (but not hydroalcoholic extract) modulated sepsis-induced limited kidney damage in the advanced stages of sepsis. Its protective role is justified by its antioxidant properties, which involve the inhibition of LP and decreasing the plasma urea/creatinine ratio. Of course further in vivo studies are needed to consider the exact mechanism of caraway extract effects in sepsis-induced organ failure.

Declaration of interest

The authors declare no conflict of interest.