Antimicrobial, anti-inflammatory and antioxidant activity of Nigella sativa in clinically endometritic cows

ABSTRACT

The aim of this study was to investigate the response to intrauterine infusion of Nigella sativa (NS) and ceftiofur and to evaluate the possible antimicrobial, antioxidant and anti-inflammatory effects of NS in clinically endometritic cows. A total of 30 clinically endometritic cows (10 cows in each group) were randomly divided into 3 treatment groups: an i.u. infusion of 100 mL of methanol extract of NS was given once a day during 3 days in group NS; an i.u. infusion of a 20-mL suspension (Excenel®, Pfizer) containing 1 g of ceftiofur as ceftiofur hydrochloride was given once a day during three days in group CH; cows with no treatment belonged to group C. After the treatment, there were slight vaginal discharges in 3/10 animals in group NS and 2/10 animals in group CH. GSH-Px and superoxide dismutase levels increased and malondialdehyde concentration decreased significantly (P < .05) in groups NS and CH after treatment when compared to pretreatment ones. Hp and SAA levels of groups NS and CH decreased significantly (P < .05) after treatment when compared to group C. NS had antimicrobial, antioxidant and antiinflamatory effects in clinical endometric cows. Although the antimicrobial effect of NS was less than that of CH, this study indicates a therapeutic value of NS in clinically endometritic cows. NS might be a promising therapy for cows with clinical endometritis in the future.

KEYWORDS:

• Nigella sativa
• antimicrobial
• antioxidant
• antiinflamatory
• endometritic cows

1. Introduction

Endometritis is defined as inflammation of the glandular layer of uterus and is associated with delayed uterine involution and poor reproductive performance (Agarwa et al. 2013). Treatment of endometritis has been routinely carried out with intrauterine infusion of antibiotics (Singh et al. 2014). Indiscriminate use of antibiotics for treatment of uterine infections has led to emergence of resistance strains. As a result of this, the attention is now moving towards the herbal formulations (Hemiaiswarya et al. 2008).

The seeds of NS have been used traditionally for centuries in the Middle East, Northern Africa and Asia for the treatment of various diseases such as asthma, cough, bronchitis, headache, rheumatism, dysentery, infections, fever, influenza and eczema (Burits & Bucar 2000; Gilani et al. 2004). The extracts also showed in vitro and in vivo antimicrobial effects (Mashhadian & Rakhshandeh 2005). Many studies have reported antioxidant activity of NS extract and its active constituent (thymoquinone) against biologically hazardous reactive oxygen species (Houghton et al. 1995).

The most sensitive acute phase proteins (APPs) are haptoglobin (Hp) and serum amyloid A (SAA) in cattle, and APPs are blood proteins that can be used to assess the innate immune system's systemic response to infection, inflammation, trauma and stress (Piccione et al. 2012).

Although several research reporting a long list of examples claiming the usefulness of NS in medicine exists, very few studies have reported its influence on the reproductive system and almost all of them have focused on the male reproductive system (Agarwal et al. 1990; Gilani et al. 2004). Moreover, the effect of NS seeds on female reproductive functions remains largely unknown (Kabir et al. 2001).

The aim of this study was to investigate the response to administration efficiency of intrauterine infusion of NS and ceftiofur and to evaluate the possible antimicrobial, antioxidant and anti-inflammatory effects of NS in clinically endometritic cows.

2. Material and methods

This study was carried out on 30 Holstein cows (age: 3–7 years) maintained at a dairy farm in Elazig province of Turkey. All the animals were kept under similar feeding and managemental conditions during the experiment. All the cows were considered for experimentation on the basis of the presence of white flakes in cervicovaginal mucous, pH and colour reaction of cervicovaginal mucous to white side test (Popov 1969).

Totally 30 clinically endometritic cows (allocating 10 cows in each group) were randomly divided into 3 treatment groups: an i.u. infusion of 100 mL of methanol extract of NS was given once a day during three days in group NS; an i.u. infusion of a 20-mL suspension (Excenel®, Pfizer) containing 1 g of ceftiofur as ceftiofur hydrochloride was given once a day during three days in group CH; cows with no treatment belonged to group C. The intrauterine infusions were performed using individually wrapped, single-use infusion pipettes (Continental Plastic, Delavan, WI).

2.1. Nigella sativa seeds and reflux extraction

The seeds were collected from local herbal drugs shops in Elazig province of Turkey. Blackseed powder (100 g) was used with 600 mL of methanol with an extraction period of 10–12 h. The extract was filtered using filter paper and the solvent was evaporated using rotary distillation apparatus. In order to obtain a completely dry extract, the resultant extract was transferred to glass dishes and was left in a 50°C oven for 24 h. Then, it was stored at 4°C until assessment of its antimicrobial activity.

2.2. Blood samples and biochemical assays

Blood samples were taken from the jugular vein with 10 mL heparinized test tube and silicone (for serum) vacutainer tubes before treatment and one week after treatment. Samples were centrifuged at 3.000 × g at 4°C for 10 min to separate the plasma from the erythrocytes. To obtain packed erythrocytes, the remaining erythrocytes were washed twice with an isotonic solution of sodium chloride. To obtain erythrocyte hemolysates, 500 μL of packed erythrocytes were destroyed by adding four volumes of cold redistilled water. The resulting suspension was centrifuged twice, firstly for 10 min in the tube centrifuge at 1.500 × g at 4°C and then in an Eppendorf centrifuge at 5.000 × g for 5 min at 4°C. Clear supernatant was obtained as hemolysates to determine glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). The lysate was frozen at −20°C until the time of analysis.

Thiobarbituricacid reactive substances, measured as malondialdehyde (MDA), were determined in plasma. In the study, plasma MDA, which is the last product of lipid peroxidation, was determined spectrophotometrically (Yagi 1984). Plasma MDA was expressed in mmol/L.

Erythrocyte GSH-Px enzyme activity was determined using the method previously described in Paglia and Valentine (1967).

Erythrocyte SOD activity was measured at 560 nm and was expressed as U/gHb (Sun et al. 1988).

Hp measurement was based on prevention of the peroxidase activity of haemoglobin, which is directly proportional to the amount of Hp. The analytical sensitivity of this test in serum has been determined as 0.0156 mg/mL for Hp by the manufacturer (Tridelta Development Plc, Ireland).

SAA was measured by a solid-phase sandwich-ELISA. The analytical sensitivity of this test in serum has been determined as 0.3 μg/mL for SAA by the manufacturer (Tridelta Development Plc, Ireland).

2.3. Sampling of vaginal swap and bacteriologic examination

After restraining the animal and securing its tail, the perennial region was washed with soap and water. Sampling for bacteriological examination was performed immediately after diagnosis of clinically endometritis and on day 14 after the treatment. Vaginal aspirates were collected about 5–10 mL from vaginal fornix using sterilized vaginal speculum and cotton swab sticks. The aspirates were poured in sterile test tubes containing sterile Stuart media as transport media and were kept in icebox and transported to the laboratory within 3 h after collection. The samples were cultivated in the blood agar media plates. The inoculated blood agar plates were incubated for 24 h at 37°C. The bacterial isolates grown in the blood agar media plates were identified by employing Cowan & Steel's (1973) method.

2.4. Statistical analysis

All results were expressed as mean ± standard deviation (SD). One-way repeated measure analysis of variance using SPSS/PC was used to determine statistical differences between mean values of the studied parameters among the groups. Differences were considered as significant at P < .05.

3. Results

A purulent (40%) or a mucopurulent (60%) vaginal discharge was recorded in clinically endometritic cows. After treatment, there were slight vaginal discharge in 3/10 animals in group NS and 2/10 animals in group CH.

All vaginal swab samples taken from clinical endometritic cows (NS, CH and C) before treatment had positive bacteriology. Isolates from vaginal swab samples were identified; Staphylococcus aureus and E. coli were the most predominant isolates identified among all groups. In addition, A. pyogenes, Streptococcus spp., Pasteurella spp., Pseudomonas spp., Corynebacterium spp., Acinetobacter spp. and Bacillus spp. were occasionally isolated. E. coli found mostly in samples taken from uncured animals in group NS on day 14. The swab samples of group CH were negative and yielded no bacterial growth on day 14 after treatment.

Serum oxidative stress parameters and APP concentrations before and after the treatment in NS, CH and C groups are showed in Table 1.

Table 1. The mean levels and standard deviations (±SD) of MDA, GSH-Px, SOD, Hp and SAA in clinically endometritic cows.

Parameters		Group C n = 10	Group NS n = 10	Group CH n = 10
MDA (nmol/mL)	BT AT	3.06 ± 0.08 2.88 ± 0.06^a	3.43 ± 0.10^A 1.85 ± 0.08^Bb	3.20 ± 0.09^A 2.06 ± 0.10^Bb
SOD (U/gHb)	BT AT	676.25 ± 27.5 698.86 ± 24.6^a	632.41 ± 32.4^A 966.14 ± 28.3^Bb	672.24 ± 26.6^A 884.67 ± 26.3^Bb
GSH-Px (U/gHb)	BT AT	38.36 ± 2.45 44.55 ± 3.48^a	34.66 ± 3.12^A 73.16 ± 5.20^Bb	36.20 ± 3.38^A 64.62 ± 5.67^Bb
SAA (mg/mL)	BT AT	86.25 ± 7.43 68.14 ± 5.59^a	78.86 ± 8.84^A 8.65 ± 0.52^Bb	82.25 ± 9.25^A 15.24 ± 2.84^Bb
Hp (mg/mL)	BT AT	0.724 ± 0.07 0.556  ± 0.06^a	0.866 ± 0.08^A 0.048 ± 0.01^Bb	0.808 ± 0.09^A 0.114 ± 0.03^Bb

^a,bValues with different superscript letters within a row differ significantly at P < .05.

^A,BValues with different superscript letters within a column differ significantly at P < .05.

BT: pretreatment; AT: posttreatment.

Mean erythrocyte GSH-Px and SOD levels of groups NS and CH increased significantly (P < .05) after treatment in comparison with group C. The same parameters increased insignificantly (P > .05) in group NS compared to group CH.

MDA levels of groups NS and CH decreased significantly (P < .05) after treatment when compared to group C. However same parameter decreased insignificantly (P > .05) in group NS compared to group CH.

Mean erythrocyte GSH-Px and SOD levels increased and MDA concentration decreased significantly (P < .05) in groups NS and CH after treatment compared to pretreatment ones.

Hp and SAA levels decreased significantly (P < .05) in groups NS and CH after treatment compared to pretreatment ones.

Hp and SAA levels of groups NS and CH decreased significantly (P < .05) after treatment when compared with group C. However same parameter decreased insignificantly (P > .05) in group NS compared to group CH.

5. Discussion

Cephapirin is a frequently prescribed antibiotic to treat cows with clinical endometritis (LeBlanc et al. 2002). Ceftiofur sodium (Drillich et al. 2001), ceftiofur hydrochloride (Kasimanickam et al. 2010) and ceftiofur crystalline free acid (Brick et al. 2012) have been shown to be effective (resulting in clinically recovery) for treatment of clinical endometritis. In the recent decades, the need of new anti-infective agents because of the emergence of multiple antibiotic resistances has led to the search of new sources of potential antimicrobials (Hemiaiswarya et al. 2008). Among them, plants offer a wide range of biodiversity of great value for the pharmaceutical industry.

Antimicrobial activities have been reported for the extracts of NS seed in vitro and in vivo (Hanafy & Hatem 1991, Morsi 2000). The results of this study was well correlated with an earlier reported work (Singh et al. 2005; Harzallah et al. 2012), where NS seed oil has been shown to be effective against a wide spectrum of organisms (bacteria such as B. cereus, B. subtilis, S. aureus, S. epidermis, E. coli, and P. aeruginosa). In the present study, S. aureus, E. coli, A. pyogenes, Streptococcus spp., Pasteurella spp., Pseudomonas spp., Corynebacterium spp., Acinetobacter spp. and Bacillus spp. were identified from vaginal swab samples in all cows before treatment and E. coli was identified only from two cows in NS group after treatment on day 14. High counts of the bacterial types were noticed in group C in contrast to the rapid reduction of bacterial counts in groups NS and CH after treatment on day 14. However, swab samples taken from group CH were negative and yielded no bacterial growth. Previous studies have shown the antimicrobial activity of methanol chloroform and diethyl ether extracts of NS seeds on standard and hospital microorganisms such as E. coli, C. albicans, S. aureus and P. aeroginosa (Hanafy & Hatem 1991; Mashhadian & Rakhshandeh 2005). Thymoquinone, pcymene (monoterpene), longifolene (sesquiterpene) and thymohydroquinone were the compounds responsible for the strong antimicrobial activity of NS (Bourgou et al. 2010). In addition, Thymoquinone or thymohydroquinone are active components of the seed that have different pharmacological activities such as anti-inflammatory and antioxidant effects (Khan 1999). Antioxidant and anti-inflammatory activity of the NS may enhance phagocytic activity by protecting the phagocytic cell (Salem 2005); so it can play therapeutic roles in reducing microorganisms. Furthermore, microbial elimination may be related to the uterotrophic activity of NS on the uterus (Saadat et al. 2011). Gram-negative bacteria have an effective permeability barrier composed of the outer membrane, which restricts the penetration of amphiphatic compounds and multidrug resistance pumps that extrude toxins across this barrier (Nagi et al. 2008). It is possible that the apparent ineffectiveness of the plant antimicrobial activity for E. coli is largely due to this permeability barrier (Hasan et al. 2013).

Many studies have indicated the significance of Hp and SAA as a clinically useful parameter for measuring the occurrence and severity of inflammatory responses in cattle with endometritis (Ceron et al. 2012; Chan et al. 2010; Sahoo & Mohanty 2014). In the present study, serum Hp and SAA levels in endometritic cows were found to be higher than reference values of healthy cows. Serum APPs have been proposed as predictors of uterine infection (Huzzey et al. 2009; Chan et al. 2010; Dubuc et al. 2010).

The anti-inflammatory effect of NS was confirmed by the study of Mutabagani and El-Mahdy (1997) in rats. It has been shown that the ethanolic extract of NS possessed ability to reduce the serum levels of acute inflammatory biomarker C-reactive protein to be APPs highly significantly in albino rats (Bashir et al. 2014). In present study, Hp and SAA levels decreased significantly (P < .05) in groups NS and CH after treatment compared to pretreatment ones. Hp and SAA levels of groups NS and CH decreased significantly (P < .05) after treatment when compared to group C. However, same parameter decreased insignificantly (P > .05) in group NS compared to group CH. This may be from the anti-inflammatory effect of NS. This effect may be consistent with the inhibitory effects of thymoquinone and other components of NS on many inflammatory mediators (Khan 1999; Bourgou et al. 2010). Both the crude fixed oil of NS and purethymoquinone inhibited cyclooxygenase and 5-lipoxygenase pathways of arachidonate metabolism in rat peritoneal leukocytes (Houghton et al. 1995). The effect was demonstrated via the dose-dependent inhibition of the formation of thromboxane B₂ and leukotriene B₄. These proteins provide an insight to know the degree of recovery after medication, as well as indication of inflammation (Hirvonen et al. 1999). Thus, the decline in Hp and SAA levels after treatment can be considered as a sign of recovering of clinically endometritis.

Inflammation induces oxidative stress by producing oxidants and intracellular antioxidant mechanisms against these inflammatory stresses involve antioxidant enzymes, including GSH-Px and SOD. Recently, it has been shown that faulty cellular antioxidant systems cause organisms to develop a series of inflammatory diseases (Liao et al. 2012). It was previously reported that plasma MDA concentration was found to be increased in the cows with puerperal metritis compared to the control group, while decreases of GSH-Px and CAT activities were observed (Kizil et al. 2010). In the present study, plasma MDA concentration increased and SOD and GSH-Px activities decreased in endometritic cows. These changes attributed to the increase of MDA concentrations confirmed the occurrence of an oxidative stress during endometritis because SOD and GSH-Px are involved in the conversion of radicals into less effective metabolites.

NS was reported to have a preventive effect on oxidative stress by causing to rise of antioxidant enzymes (Khattab & Nagi 2007; Yaman & Balikci 2010). The antioxidant activity of NS seeds and oil was basically dependent on its active constituent thymoquinone and carvacrol (Thippeswamy & Naidu 2005). It has been shown that thymoquinone could inhibit iron-dependent microsomal lipid peroxidation efficiently in rats with doxorubicin-induced hyperlipidemic nephropathy (Badary et al. 2000). NS oil prevented lipid peroxidation and increased the antioxidant defense system in diabetic rabbits (Meral et al. 2001). Kanter et al. (2006) was reported that treatment with NS oil reduced tissue MDA and protein carbonyl levels while preventing the inhibition of GSH-Px and SOD enzymes. It was reported that MDA and NO levels decrease and SOD and GSH-Px activities increase with Nigella sativa plus gentamicin application (Yaman & Balikci 2010). In the present study, there were statistically significant (P < .05) increases for GSH-Px and SOD levels and significantly (P < .05) decreases for MDA levels in groups NS and CH when compared to group C after treatment. However, insignificant (P > .05) increases in GSH-Px and SOD levels and insignificant (P > .05) decreases in MDA level were observed for group NS compared to group CH, which may be due to the antioxidant property of NS (Yaman & Balikci 2010).

6. Conclusion

The development of effective alternative therapies to antibiotics for the treatment of clinic endometritis is needed due to the emergence of multidrug-resistant bacteria associated with uterine infections in cows. NS was found to have antimicrobial, antioxidant and antiinflamatory effects for clinically endometric cows. Although the antimicrobial effect of NS was less than CH, this study indicates therapeutic value of NS for clinically endometritic cows. NS might be a promising therapy for cows with clinically endometritis in the future. Therefore, the optimal dose for use of intrauterine NS and the underlying mechanisms by which the infection is controlled warrant further investigation.

Disclosure statement

No potential conflict of interest was reported by the authors.