Inhibition of Benzoyl Peroxide–Induced Cutaneous Oxidative Stress, Toxicity, and Ear Edema in Mice by Nardostachys jatamansi.

Abstract

Recent years have shown considerable efforts to identify new chemopreventive agents that could be of clinical value. In the current study, modulatory effect of Nardostachys jatamansi. (Jones) DC on benzoyl peroxide–induced oxidative stress, toxicity, and ear edema is investigated. Pretreatment with jatamansi at doses of 2.5 and 5 mg/kg body weight in acetone prior to the application of benzoyl peroxide (20 mg/animal per 0.2 ml acetone) resulted in significant inhibition of benzoyl peroxide–induced cutaneous oxidative stress, toxicity, and ear edema in a dose-dependent manner. The cutaneous microsomal membrane lipid peroxidation and xanthine oxidase activities were significantly reduced (p < 0.05). Moreover, the depleted levels of phase II metabolizing enzymes and glutathione were recovered significantly (p < 0.05). Our findings suggest that N. jatamansi. is an effective chemopreventive agent in mouse skin with potential of ameliorating benzoyl peroxide–induced cutaneous oxidative stress, toxicity, and ear edema.

• KEYWORD
• Antioxidants
• carcinogenesis
• chemoprevention
• herb
• Nardostachys jatamansi
• promoter

Introduction

The increased generation and decreased degradation of reactive oxygen species (ROS) may result in oxidative stress, which in turn is implicated in a large number of diseases such as myocardial ischemia, carcinogenesis, inflammatory disease, cataract, aging, Alzheimer disease, diabetes mellitus, rheumatoid arthritis, and muscular dystrophy (Ames, 1983). The role of ROS in carcinogenesis is well documented, as superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (⁻OH), and singlet oxygen can act as initiator and/or promoter (Slaga, 1981), cause DNA damage (Sultana, 1995), activate procarcinogen (Marnett, 1987), and alter the cellular antioxidant defense system and phase II metabolizing enzymes (Reiners, 1991). The antioxidant machinery of cellular defense system comprises antioxidant and phase II metabolizing enzymes such as glutathione-S-transferase and quinone reductase and small molecular antioxidants such as glutathione that protect against free radical mediated tissue injury (Perchellet, 1989). Reiners et al. (1991) reported the depleted levels of antioxidant enzymes in a chemically induced skin tumorigenesis model. Moreover, ROS may also induce chromosomal aberration and sister chromatid exchange (Emerit, 1982) leading to mutagenesis, cellular transformation, and lipid peroxidation that converts the endogenous lipid to genotoxic intermediates (Leob, 1988).

Benzoyl peroxide (BPO) is a free radical generating compound. In the pharmaceutical industry it is used as a cosmetic additive, particularly in antiacne preparations (Karasz, 1977). The hyperplastic and morphological effects of BPO on murine skin is well documented (Slaga, 1981). There is thus some concern regarding the frequent human contact with BPO. However, because of its wider applicability and unavoidable exposure to men, there is a need to define compounds that may be effective in abrogating BPO-induced oxidative stress and toxicity.

Nardostachys jatamansi. (Jones) DC (Valerianaceae) is indigenous to the alpine Himalayan regions of India. In Ayurveda, (rhizomes of N. jatamansi. are used as a bitter tonic, stimulant, antispasmodic, and to treat epilepsy, hysteria, and convulsions (Bagchi, 1991). The decoction of the drug is also used in neurological disorders, insomnia, and disorders of cardiovascular system (Uniyal, 1969). The commercially available drug comprises the rhizome with or without some taproot attached. In the Unani (ancient Greco-Arab) system of medicine, jatamansi has been reported as hepatotonic, cardiotonic, diuretic, and analgesic (Turner, 1568). Shakir et al. (2000) reported jatamansi to have hepatoprotective activity against thioacetamide-induced liver injury. Because it is effective in various disorders, especially those where oxidative/free radical mediated injury is implicated, we speculated that N. jatamansi. might ameliorate BPO-induced cutaneous oxidative stress and toxicity. We prophylactically treated mice with acetonic extract of N. jatamansi. and observed that it inhibited BPO-induced cutaneous oxidative stress, epidermal inflammation (ear edema), and toxicity.

Materials and Methods

Animals

Eight-week-old adult male Swiss albino mice (20–25 g) were obtained from the Central Animal House facility of Hamdard University and were housed in a ventilated room at 30°C under a 12-h light-dark cycle. The mice were allowed to acclimatize for 1 week before the study and had free access to standard laboratory feed (Hindustan Lever Ltd, Mumbai, India) and water. The dorsal skin of the mice was shaved with an electric clipper (Oster A2) followed by the application of hair removing cream (Anne French, Geoffrey Manners, Mumbai, India) at least 2 days before treatment. Only mice showing no signs of hair regrowth were used in the experiments. Excess cream was washed with lukewarm water. The project was undertaken with prior approval from the University Animals Ethics Committee. Utmost care was taken to ensure that animals were treated in the most humane and ethically acceptable manner.

Chemicals

Reduced glutathione, oxidized glutathione, glutathione reductase, reduced nicotinamide adenine dinucleotide phosphate, bovine serum albumin (BSA), 1,2-dithio-bis-nitrobenzoic acid, 1-chloro-2,4-dinitrobenzene, flavin adenine dinucleotide, Tween-20, 2,6-dichloro indophenol, thiobarbituric acid, and benzoyl peroxide were obtained from Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals were obtained from S.D. Finechem (Surat, India).

Plant material

N. jatamansi. whole plant was obtained from a local market (New Delhi) and was identified and authenticated by Prof. M. Iqbal, (Head) Medicinal Plant Division, Department of Environmental Botany, Hamdard University, New Delhi, India. A voucher specimen was deposited in Department of Medical Elementology and Toxicology, Faculty of Science, Hamdard University, New Delhi, India. The plant material was crushed manually with a mortar and pestle was used subsequently for extract preparation. Extreme precautions were considered in order to prevent the contamination of both plant material and extract.

Preparation of extract

Briefly, crushed plant material (200 g) was repeatedly extracted with 2000 ml solvents of increasing polarity starting with petroleum ether, benzene, ethyl acetate, acetone, methanol, and double-distilled water. The reflux time for each solvent was 4 h. The extracts were cooled at room temperature, filtered, and evaporated to dryness under reduced pressure in a rotary evaporator (Buchi Rotavapor). The yield obtained for each solvent was 3, 4.2, 6.3, 10, 25, and 11 g, respectively. The residues were stored at 4°C. The acetone fraction was used for further study.

Experimental protocol

Thirty mice were randomly allocated to five groups of six animals each. Group I served as normal control and received a dermal application of 0.2 ml acetone. Group II served as BPO treated control. Groups III and IV received a single topical application of N. jatamansi. at a dose of 2.5 and 5 mg/kg, respectively. Group V received a single oral dose of 5 mg/kg of the plant extract.

After 12 h, groups II, III, and IV received a topical application of BPO at a dose of 20 mg/kg per 0.2 ml acetone. After 16 h of BPO application, mice were killed by cervical dislocation and processed for subcellular fractionation.

Tissue preparation

Skin tissue from the mice in each group was excised and washed with ice-cold saline. The tissue was homogenized in cold phosphate buffer (0.1 M, pH 7.4) by a polytron homogenizer (Kinematica Polytron, Switzerland), and subcellular fractions were prepared as reported elsewhere (Raza et al., 1995).

Biochemical estimations

The glutathione (GSH) in skin was determined by the method of Jollow et al. (1974). Activity of enzyme was measured by the earlier reported method (Mohandas et al., 1984). Glutathione reductase activity was measured by the method of Carlberg and Mannervik (1975). Catalase activity was measured by according to the method of Clairborne (1985). Glutathione-S.-transferase was measured by the method of Athar and Iqbal (1998). The method of Zaheer et al. (1965) was used to assay the activity of glucose-6-phosphate dehydrogenase activity. The assay for microsomal lipid peroxidation was carried out according to the method of Iqbal et al. (1996). Xanthine oxidase was estimated by the method of Athar et al. (1996). Quinone reductase activity in the cytosolic fraction of skin was estimated by the method of Iqbal et al. (1999). The protein concentration in all samples was determined by the earlier reported method (Lowry, 1951).

Measurement of ear edema

Ear edema was measured according to the earlier published report (Huang et al., 1991) with slight modification. Briefly, female albino mice were treated on the right ear with 25 µl acetone (control) or test compound (i.e., 2.5 and 5.0 mg/kg of N. jatamansi.) before the application of inflammatory agent (BPO 20 mg/mouse) in acetone. The mice were sacrificed after 16 h of BPO treatment, and ear punches (6 mm diameter) were weighed. The increased weight of the ear punch is the measure of inflammation.

Results

Treatment with BPO resulted in the depletion of cutaneous glutathione by 54% and the activity of antioxidant enzymes, viz., glutathione reductase, catalase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase, to a level of 36%, 45%, 42%, and 25%, respectively, of the acetone-treated control group. Prophylactic treatment with N. jatamansi. resulted in significant recovery (p < 0.05) of these enzymes as shown in Table 1.

Table 1 Effect of prophylactic treatment of animals with N. jatamansi. on benzoyl peroxide–mediated depletion in the level of cutaneous glutathione and on the activities of various antioxidant enzymes.

Treatment groups	Reduced glutathione (nmol GSH/g tissue)	Glutathione reductase (nmol NADPH oxidized/min per mg protein)	Catalase (nmol H2O2 consumed/min per mg protein)	Glutathione peroxidase (nmol NADPH oxidized/min per mg protein)	Glucose-6-phosphate dehydrogenase (nmol NADP reduced/min per mg protein)
Acetone-treated control	0.028 ± 0.002	79.5 ± 6.0	170.0 ± 9.0	55.0 ± 1.5	354.0 ± 9.0
N. jatamansi. (5 mg/kg)	0.03 ± 0.004	83.0 ± 8.1	185.0 ± 14.0	61.0 ± 1.8	370.0 ± 11.0
Benzoyl peroxide	0.013 ± 0.003**	50.5 ± 3.5**	94.0 ± 10.0***	32.0 ± 2.5***	264.0 ± 12.0***
N. jatamansi. (2.5 mg/kg) + benzoyl peroxide	0.02 ± 0.002^#	62.2 ± 3.0^#	130.0 ± 12.0^#	42.0 ± 2.0^#	295.0 ± 7.0^#
N. jatamansi. (5 mg/kg) + benzoyl peroxide	0.031 ± 0.003^###	66.0 ± 4.6^#	165.0 ± 16.0^##	59.4 ± 1.2^###	335.0 ± 9.5^###

Each value represents mean ± SE. Benzoyl peroxide applied in the dose of 20 mg/0.2 ml acetone per animal. Values marked with asterisks differ significantly from the corresponding value for the acetone-treated control (**p < 0.001, ***p < 0.001). Values marked with number symbols differ significantly from the corresponding value for mice treated with benzoyl peroxide alone (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).

The BPO treatment decreased the levels of cutaneous glutathione-S.-transferase and quinone reductase to 57% and 17% respectively, when compared with control group. Pretreatment of animals with N. jatamansi. extract showed a significant recovery (p < 0.01) of the depleted levels of enzymes as shown in Table 2.

Table 2 Effect of prophylactic treatment of animals with N. jatamansi. on benzoyl peroxide–mediated depletion in the activities of phase II metabolizing enzymes, viz., glutathione-S.-transferase and quinone reductase.

Treatment groups	Gutathione-S.-transferase (nmol CDNB conjugate formed/min per mg protein)	Quinone reductase (nmol dichloroindophenol reduced/min per mg protein)
Acetone-treated control	152.0 ± 4.0	312.0 ± 7.0
N. jatamansi. (5 mg/kg)	156.0 ± 2.5	324.0 ± 9.5
Benzoyl peroxide	65.8 ± 4.5***	259.0 ± 8.0***
N. jatamansi. (2.5 mg/kg) + benzoyl peroxide	85.0 ± 3.0^##	294.0 ± 11.0^#
N. jatamansi. (5 mg/kg) + benzoyl peroxide	112 ± 7.0^###	306.0 ± 9.0^##

Each value represents mean ± SE. Benzoyl peroxide applied in the dose of 20 mg/0.2 ml acetone per animal. Values marked with asterisks differ significantly from the corresponding value for the acetone-treated control (***p < 0.001). Values marked with number symbols differ significantly from the corresponding value for mice treated with benzoyl peroxide alone (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).

BPO treatment enhanced the susceptibility of cutaneous microsomal membrane for ironascorbate–induced lipid peroxidation to 85% and xanthine oxidase to 84% when compared with control group. However, N. jatamansi., exhibited significant protection (p < 0.001) against BPO-mediated lipid peroxidation and induced xanthine oxidase (Table 3).

Table 3 Effect of prophylactic treatment of animals with N. jatamansi. on benzoyl peroxide–mediated enhancement in the susceptibility of cutaneous microsomal membrane for iron ascorbate–induced lipid peroxidation and xanthine oxidase.

Treatment groups	Xanthine oxidase (µg uric acid formed/min per mg protein)	Lipid peroxidation (nmol malondialdehyde formed/h per g tissue)
Acetone-treated control	55.0 ± 3.5	3.4 ± 0.12
N. jatamansi. (5 mg/kg)	52.0 ± 0.63	3.3 ± 0.14
Benzoyl peroxide	102.0 ± 2.6***	6.3 ± 0.32***
N. jatamansi. (2.5 mg/kg) ± benzoyl peroxide	80.5 ± 3.3^###	5.3 ± 0.28^#
N. jatamansi. (5 mg/kg) ± benzoyl peroxide	67.0 ± 6.9^###	4.1 ± 0.22^###

Each value represents mean ± SE. Benzoyl peroxide applied in the dose of 20 mg/0.2 ml acetone per animal. Values marked with asterisks differ significantly from the corresponding value for the acetone-treated control (***p < 0.001). Values marked with number symbols differ significantly from the corresponding value for mice treated with benzoyl peroxide alone (^#p < 0.05, ^###p < 0.001).

The effect of prophylactic treatment of animals with N. jatamansi. on BPO-induced ear edema is shown in Table 4. N. jatamansi. dose-dependently inhibited BPO-induced ear edema to a significant level (p < 0.001).

Table 4 Effect of N. jatamansi. on benzoyl peroxide–induced ear edema of mouse ears.

Treatment groups	Weight/punch (mg)	Inhibition (%)
Acetone-treated control	13.9 ± 0.29
N. jatamansi. (5 mg/kg)	14.3 ± 1.03
Benzoyl peroxide	32.1 ± 1.06***
N. jatamansi. (2.5 mg/kg) + benzoyl peroxide	22.4 ± 1.44^###	30%
N. jatamansi. (5 mg/kg) + benzoyl peroxide	15.1 ± 0.56^###	52%

Each value represents mean ± SE. Benzoyl peroxide applied in the dose of 20 mg/0.2 ml acetone per animal. Values marked with asterisks differ significantly from the corresponding value for the acetone-treated control (***p < 0.001). Values marked with number symbols differ significantly from the corresponding value for mice treated with benzoyl peroxide alone (^###p < 0.001).

Discussion

The results indicate that pretreatment with the extract of N. jatamansi. protects the animals from the cutaneous oxidative stress induced by BPO. Because the N. jatamansi. treatment increased the antioxidant enzymes, it seems the extract induced the antioxidant enzymes present in the body, which neutralize the reactive oxygen free radicals. The higher level of phase II metabolizing enzymes in the N. jatamansi.–treated animals supports this. As the level of depletion of these enzymes is directly related to the level of reactive oxygen free radicals present, the higher level of phase II metabolizing enzymes in N. jatamansi.–treated animals indicates that the level of reactive oxygen free radicals was considerably less. The decreased levels of xanthine oxidase and of lipid peroxidation in N. jatamansi.–treated animals reveal the status of oxidative stress, which is controlled well by the N. jatamansi.. All these findings suggest that the N. jatamansi. extract seems to either neutralize the generated free radicals or it induces the antioxidant enzymes present in the body that neutralizes the free radicals. But the N. jatamansi. treatment per se. did not increase the levels of antioxidant enzymes. Thus, there are chances that N. jatamansi. may also possess antioxidant molecules. These findings are consistent with previous studies (Sanz, 1994). The decrease in ear edema in N. jatamansi.–treated rats can be explained by the antioxidant nature of the extract, as the reactive oxygen free radicals cause cellular injury and inflammation in tissues (Ames, 1983).

BPO has been reported as a cutaneous tumor promoter (Slaga, 1981). The role of reactive oxygen free radicals in carcinogenesis is well established (Floyd, 1990). Earlier cancer chemopreventive studies show that after the treatment with chemopreventive agents, the levels of antioxidant enzymes were elevated in the organs of the experimental animals (Sun, 1990). The significant enhancement in the activity of antioxidant enzymes such as catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase in the skin by prophylactic treatment with N. jatamansi. on BPO-induced cutaneous oxidative stress suggests that it may contribute to the chemopreventive effect. These findings are consistent with earlier reports where chemopreventive action was attributed to antioxidant activity of such agents (Sanz, 1994).

Thus, it can be suggested that the chemoprevention of carcinogenesis due to reactive oxygen free radicals may be possible with N. jatamansi.. The plant is reported to contain coumarins, lignans, neolignans, and sesquiterpenes (Shanbagh, 1964; Bagchi, 1991). Coumarins (Morris, 1992), terpenes (Suh, 1998), and lignans (Owen, 2000) from different sources have been reported to be chemopreventive in carcinogenesis. Thus, the antioxidant and the possible chemopreventive activity of N. jatamansi. may be attributed to the presence of coumarins, lignans, neolignans, and sesquiterpenes. Chemoprevention of carcinogenesis by N. jatamansi. needs to be investigated in detail. Further studies are necessary to find out the phytoconstituents responsible for the activity of N. jatamansi..

In conclusion, it can be suggested that N. jatamansi. is effective in protecting the experimental animals from the cutaneous oxidative stress and inflammation induced by the BPO and may also have a possible role in preventing ROS-induced carcinogenesis.

Acknowledgments

The authors are thankful to ICMR, New Delhi, for providing the necessary funds to carry out the research. Authors are thankful to Mr. Siraj Hussain (Vice Chancellor, Jamia Hamdard) for providing adequate facilities for research work.