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Modulation of radiation-induced biochemical alterations in mice by rosemary (Rosemarinus officinalis) extract
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Phytomedicine 14 (2007) 701–705 www.elsevier.de/phymed
SHORT COMMUNICATION
Modulation of radiation-induced biochemical alterations in mice by rosemary (Rosemarinus officinalis) extract Dhanraj Soyal, Archana Jindal, Inder Singh, P.K. Goyal Radiation and Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur 302004, India Received 2 June 2006; accepted 24 October 2006
Abstract Radioprotective effect of leaves extract of Rosemarinus officinalis (ROE) has been studied against 6 Gy g-radiations in the liver of Swiss albino mice at various post-irradiation intervals between 12 h and 30 days. In control animals (without ROE treated irradiated), an elevation in glycogen, protein, acid and alkaline contents was found till day 5th, but thereafter decreased at successive intervals without returning to normal. Cholesterol level was found to be lower than normal till 10th day, then increased up to 20th day but later declined without restoring normal level. A similar trend of variation in these biochemical parameters was observed in experimental group (ROE pretreated irradiated) also but to a lower extent. ROE significantly delayed and inhibited the rise in these biochemical parameters. Almost normal values of such constituents were regained by day 30th in experimental animals; whereas in control animals, normal values were not ever attained. In control animals, there was an elevation in lipid peroxidation (LPx) and a decrease in glutathione (GSH) in blood and liver; whereas in experimental group, decline in LPx accompanied by an increase in GSH concentration was observed. r 2006 Elsevier GmbH. All rights reserved. Keywords: g-irradiation; Biochemical parameters; Lipid peroxidation; Glutathione; Swiss albino mice; Rosemarinus officinalis
Introduction Since the pioneering work of Patt et al. (1949) demonstrating that cysteine protects mice and rats against radiation-induced sickness and mortality, several compounds have been tested for their radio protective capacity (Weiss Joseph and Landauer Michael, 2003). The research findings have been affirmed that pretreatment of certain chemical compounds like 2-MPG (Sugahara et al., 1970), WR-2721 (Yuhas et al., 1980) and diltiazem (Nunia and Goyal, 2004) protect mammals against deleterious effects of ionizing radiation. The Corresponding author.
E-mail addresses: [email protected], [email protected] (P.K. Goyal). 0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.12.011
major drawback of synthetic compounds has been that they are highly toxic at the optimum protective dose. Therefore, it is desirable to use other materials, which are less toxic and offer high protection. Rosemarinus officinalis, belonging to family Labiatae, commonly called as rosemary, is widely found along the north and south coasts of the Mediterranean Sea and also in the sub-Himalayan areas (Kotb, 1985). Rosemary is an evergreen branched and bushy shrub. It has been used as an antispasmodic in renal colic and dysmenorrhea and in relieving respiratory disorders. It has also been used as an analgesic, anti-rheumatic, carminative, cholagogue, diuretic, expectorant, anti-epileptic, for effects on human fertility, hepatoprotective and antimutagenic but its radioprotective effects are not known (EI-Gadi and Bshina, 1989; Al-sereiti et al., 1999).
ARTICLE IN PRESS 702
D. Soyal et al. / Phytomedicine 14 (2007) 701–705
Liver is the most versatile organ in the body with an amazing capacity to perform diverse biochemical functions. In fact, a large number of different functions of the liver have been identified. The plant, Rosemarinus officinalis, has not yet been assessed for its possible radio-modulatory potential. Therefore, the present study has been targeted on liver to evaluate radiationinduced biochemical lesions and the radio protective potential of this plant in g-irradiated mice.
behavioral changes, mortality, morbidity, sickness, food and water consumption till their sacrifice or survival. Liver was collected from autopsied animals at 12, 24 h 3rd, 5th, 10th, 20th and 30th day post-treatment. Homogenate of liver was prepared, and glycogen, protein and cholesterol contents were measured using Montgomery (1957), Lowry et al. (1951) and Lieberman (1951) methods, respectively. Activity of acid and alkaline phosphatase was also assayed by using commercially available kits. Spectrophotometer (Systronics UV-VIS-108) was used to measure the optical densities.
Materials and methods
Glutathione (GSH) assay
Preparations of Rosemarinus officinalis extract (ROE)
Liver and blood glutathione (GSH) levels were estimated, after 1 h of DDW/ROE/radiation treatment, according to the methods of Moron et al. (1979) and Beutler et al. (1963), respectively. The absorbance was read at 412 nm using a Systronics UV-VIS-108 Spectrophotometer.
Rosemarinus officinalis, Linn was identified by a competent botanist from Department of Botany, University of Rajasthan, Jaipur. Fresh leaves of this plant were collected during October–December of the year. These leaves were air dried, powdered and extracted with double-distilled water (DDW) by refluxing for 36 h (12 h 3). The ROE thus obtained was vacuum evaporated so as to get in powder form. For the experiment, ROE was redissolved in the sterile doubledistilled water and the dose of required concentration was prepared.
Modification of radiation response Adult male Swiss albino mice (6–8 weeks old) weighing 2572 gm from an inbred colony were used for the present study. Animal care and handling were performed according to guidelines issued by World Health Organization (Geneva, Switzerland) and the Indian National Science Academy (New Delhi, India). Dose selection of ROE was done in a separate experiment based on drug tolerance study (Jindal et al., 2006). Mice selected for this study, were divided into four groups. Animals in Group-I were administered orally with DDW (volume equal to ROE) to serve as normal (vehicle treated) while animals in Group-II were given ROE orally at a dose of 1000 mg/kg b.wt. once in a day for 5 consecutive days. Animals of Group-III received an equal volume of DDW (as in Group-I) while animals in Group-IV (experimental) were given ROE (as in Group-II). After 30 min of DDW or ROE administration on 5th day, animals of Group III and IV were whole-body irradiated at Cobalt Teletherapy Unit (ATC-C9) at Cancer Treatment Centre, Department of Radiotherapy, SMS Medical College and Hospital, Jaipur (at the dose rate of 0.85 Gy/min). Animals of all the groups were monitored for weight change,
Lipid peroxidation (LPx) assay The lipid peroxidation (LPx) level in liver and serum was measured by the assay of thiobarbituric acid reactive substances (TBARS) using the method of Ohkhawa et al. (1979) in which the absorbance was read at 532 nm with a Systronics UV-VIS-108 spectrophotometer.
Statistical analysis The statistical significance of the differences between normal and DDW+irradiated (control) as well as control and ROE+irradiated (experimental) was evaluated by using the Student’s ‘t’ test.
Results The animals of DDW+irradiation group (control) showed signs of radiation sickness within 3–5 days after exposure to 6 Gy of g-irradiation. The pretreatment of mice with ROE (experimental group) delayed the onset of radiation-induced mortality and symptoms of sickness as compared to control group. In irradiated control animals (Group-III), an increase in glycogen and protein contents was noticed as early as at 12 h. Interval and raised further until day 5th, but later decreased from day 10th reaching to near normal by day 30th post-treatment. On contrary, cholesterol level was found to be significantly lower than normal at early intervals. However, it increased later at consecutive intervals without restoring the normal value. An increase over normal in the acid and alkaline
Table 1. Variations (mean7S.E.) in different biochemical parameters in liver of mice after exposure to 6 Gy g-radiation in the presence (experimental) or absence (control) of Rosemarinus officinalis extract (ROE) Biochemical parameter
Treatment Post-treatment autopsy interval 12 h
24 h
3rd day
5th day
10th day
20th day
30th day
Protein 150.0174.031 mg/gma
Group-II 152.0273.639 153.0173.344 154.4573.714 152.8974.827 150.9174.166 150.7173.863 148.3173.783 Group-III 205.5173.434b 208.4771.565b 224.8871.783b 244.4270.662b 234.5172.197b 175.4772.303b 155.6471.073 Group-IV 167.3671.443b 175.6370.900b 190.6671.282b 195.7271.326b 165.1773.409b 154.8073.551b 151.2570.811d
Cholesterol 4.3970.065 mg/gma
Group-II Group-III Group-IV
4.3670.04 4.0870.242 4.2970.204
4.3870.06 3.8370.169 4.1370.145
4.3870.04 3.4270.043 3.9870.111
4.3870.03 4.3570.06 4.4170.05 3.7370.387b 3.9770.075b 4.6570.111b 4.1070.088 4.14 0.192b 4.35 0.100b
4.4170.11 4.4870.244 4.4170.257
Acid phosphatase 1.9870.041 KAUa
Group-II Group-III Group-IV
1.9170.06 2.4570.023b 2.4070.029
1.8570.086 3.0170.334c 2.5170.391
1.8170.051 3.1270.801 2.8970.891
1.7970.055 3.4970.201b 2.9270212d
1.7870.079 3.0870.156b 2.4670.290
1.8870.063 2.7670.107b 2.2370.190d
1.9370.068 2.5070.079d 1.9970.175d
Alkaline Phosphatase 2.5670.093 KAUa Group-II Group-III Group-IV
2.5970.06 3.6170.078b 3.1370.105c
2.6170.104 4.3970.063b 3.4170.172b
2.6470.098 5.0170.081b 3.3970.212b
2.6370.081 5.7870.209b 3.4570.313b
2.6270.094 4.0570.378c 3.2070.407
2.5870.085 3.5870.300c 2.6970.205d
2.5470.047 3.1570.061b 2.5970.177c
4.2970.15 6.1170.351b 5.8770.878
4.3970.243 7.0570.132b 6.5870.519
4.3770.14 7.9670.381b 6.8170.710
4.3670.267 8.6570.302b 7.8370.317d
4.2670.137 7.8270.263b 6.2670.221b
4.2770.281 4.9270.086c 4.6470.348
4.3170.718 4.4670.078 4.2870.127
Group-II: ROE treated. Group-III: DDW+radiation. Group-IV: ROE+radiation. Statistical comparison between normal v/s control and control v/s experimental. a Group-I DDW treated. b po 0.001. c po 0.01. d po 0.05.
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Group-II Group-III Group-IV
D. Soyal et al. / Phytomedicine 14 (2007) 701–705
Glycogen 4.2170.234 mg/gma
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Table 2. Glutathione (GSH) and Lipid peroxidation (LPx) levels in Swiss albino mice after exposure to gamma radiation in the presence (experimental) or absence (control) of Rosemarinus officinalis extract (ROE) Treatment
GSH level (mean7S.E.)
LPx level (mean7S.E.)
Blood (mg/ml) Liver (mmol/gm) Serum TBARS (MDA)nmol/ml Liver TBARS (MDA)nmol/mg Normal ROE alone DDW+6 Gy (control) ROE+6 Gy (experimental)
3.5670.16 3.6770.17 2.6170.10b 2.9570.12a
64.4971.63 64.9273.12 40.6571.72b 54.3571.81b
1.1570.08 1.0370.13 3.6270.18b 2.4670.20b
2.6570.12 2.2070.19a 6.7070.29b 3.5970.22b
Stastistical comparision between normal v/s control; control v/s experimental. a po 0.05. b po 0.001.
phosphatase activities was noticed at early intervals and found maximum on day 5th but later started to decrease from day 10th, without returning to normal level even till the end of study (Table 1). In the experimental animals (ROE pretreated irradiated), elevated values of glycogen and protein were noted at the similar intervals like control, but such increase was significantly lesser. The normal level of glycogen and protein attained by day 30th and 20th post-irradiation, respectively in this group. Cholesterol level declined up to day 3rd however, it increased later and returned to near normal level on day 30th. A significant increase over normal in acid and alkaline phosphatase was recorded until day 5th, but at all the intervals such increase was significantly lesser than the control, and normal value regained on day 30th postirradiation (Table 1). ROE treated irradiated animals showed a significant increase in GSH content (blood and liver) with respect to control, but the values remained below normal. Exposure of animals to g-radiation increased LPx in Groups III and IV, whereas ROE pretreatment significantly reduced LPx induction in the ROE+irradiation group, thereby protecting liver and blood against radiation-induced LPx (Table 2).
Discussion An elevation in liver glycogen and total protein concentration over normal were observed after irradiation in control and experimental groups. It is because more substrate is made available as a result of tissue break down. Moreover, the persistently higher level of hepatic glycogen after irradiation could be due to the stimulation of the pituitary adrenal system. Similar results were observed with Emblica officinalis also (Sharma and Goyal, 2005). Mukarjee and Goldfeder (1974) suggested that such rise in protein may be due to an increased transport of amino acids through plasma
membrane as a consequence of permeability change in irradiated cell membranes in the experimental group. Liver cholesterol showed a decrease up to day 3rd after irradiation which confirms the observations of Sharma and Goyal, (2005). The reduction in cholesterol during early intervals might be due to the stress response caused by irradiation, which stimulates the synthesis of steroid hormones via hypothalamic–pituitary system. An increase in the liver phosphatase activities of mice due to irradiation was observed in the present study. Similar increase in such enzymes was observed earlier (Sharma and Goyal, 2005; Bharatiya and Khan, 1983; Khan et al., 1984) after irradiation with various doses. Radiation-induced cell death may be a possible reason for increased activity of ACP and ALP. Aqueous extract of the young sprouts of rosemary has an anti-lipoperoxidant activity, as it reduced the formation of malonaldehyde significantly in a dosedependent manner, and significantly decreased the release of lactic dehydrogenase (LDH) and aspartate aminotransferase (ASAT), thus confirming the antihepatotoxic action of ROE (Joyeux et al., 1990). The active constituents of Rosemarinus officinalis like carnosol, carnosic acid, caffeic acid, rosmarinic acid, ursolic acid, different diterpenes, phenols and flavonoids are reported to have antioxidant, antimutagenic, radioprotective properties (Fluck et al., 1976; Leung and Foster 1996; Del Bano et al., 2003, 2006). Ionizing radiation induces lipid peroxidation (LPx) which can lead to DNA damage and cell death (Noda et al., 1993). An increase in the glutathione (GSH) level by ROE may be responsible for the scavenging of radiation-induced free-radicals including LPx and thereby protecting against radiation-induced mortality. It has been reported that LPx starts to increase as soon as the endogenous GSH is exhausted, and the addition of GSH promptly stops further peroxidation (Kilic et al., 2000). My present study shows that Rosemarinus officinalis leaves extract provides a significant protection against radiation-induced biochemical alterations in liver.
ARTICLE IN PRESS D. Soyal et al. / Phytomedicine 14 (2007) 701–705
References Al-Sereiti, M.R., Abu-Amer, K.M., Sen, P., 1999. Pharmacology of rosemary Rosemarinus officinalis Linn. and its therapeutic potentials. Indian J. Exp. Biol. 37, 124–130. Beutler, E., Duron, O., Kelly, B.M., 1963. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 61, 582–888. Bharatiya, H.C., Khan, A.S., 1983. Modification of acid phosphatase activity in the liver of Swiss albino mice by 2mercaptopropionylglycine against gamma irradiation. Radiobiol. Radiother. 24, 235–238. Del Bano, M.J., Lorente, J., Castillo, J., Garcia, O.B., Del Rio, J.A., Ortuno, A., Quirin, K.W., Gerard, D., 2003. Phenolic diterpenes, flavones, and rosmarinic acid distribution during the development of leaves, flowers, stems, and roots of Rosemarinus officinalis. Antioxidant activity. J. Agric. Food Chem. 51, 4247–4253. Del Bano, M.J., Castillo, J., Garcia, O.B., Lorente, J., MartinGil, R., Acevado, C., Alcaraz, M., 2006. Radioprotective-antimutagenic effects of rosemary phenolics against chromosomal damage induced in human lymphocytesby gamma-rays. J Agric. Food Chem. 54 (6), 2064–2068. EI- Gadi, A., Bshina, S.M., 1989. Usage of some plants in Libyan folk medicine. Part I Dar Alkutob Alwatania. Benghazi. Fluck, H., Jasperson-Schib, R., Rawson, J.M., 1976. Medicinal Plants and their Uses. W., Foulsham & Co., Slough. Jindal, A., Sancheti, G., Soyal, D., Goyal, P.K., 2006. Radioprotective potential of Rosemarinus officinalis against lethal effects of radiation, a preliminary study. J. Environ. Pathol. Toxicol. Onkol., Communicated. Joyeux, M., Rolland, A., Fleurentin, J., Mortier, F., Dorfman, P., 1990. Planta Med. 56, 171. Khan, A.S., Sharma, J., Pareek, B.P., Bhartiya, H.C., 1984. Radioprotective effect of 2-mercaptopropionylglycine 2MPG on the alkaline phosphatase activity in the liver of Swiss albino mice against gamma irradiation. Radiobiol. Radiother. 25, 579–583. Kilic, D., Sayan, H., Gonal, B., Egcham, I., 2000. The effects of granulocyte macrophase colony stimulating factor on glutathione and lipid peroxidation in a rat model. Eur. J. Surg. Oncol. 26, 701–704.
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Kotb, D.F., 1985. Medicinal plants in Libya. Arab Encyclopedia House, Tripoli, 720p. Lieberman, B., 1959. Microanalysis. In: Wolten, E.J.I. (Ed.), Medical Biochemistry King. Churchill Pub, London, pp. 42–57. Leung, A.Y., Foster, S., 1996. Encyclopedia of Common Natural Ingredients Used in Foods, Drugs and Cosmetics, second ed. Wiley, New York, pp. 446–448. Lowry, G.H., Rosenbrough, N., Farr, A., Randall, R., 1951. Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193, 265–275. Montgomery, R., 1957. Determination of glycogen. Arch. Biochem. Biophys. 67, 378–382. Moron, M.S., Depierre, J.W., Mannervik, B., 1979. Levels of GSH GR and GST activities in rat lung and liver. Biochem. Biphys. 512, 67–78. Mukarjee, H., Goldfeder, A., 1974. Protein biosynthesis in the liver of X-irradiated mice. Int. J. Radiat. Biol. 25, 445–450. Noda, Y., McGeer, P.L., McGeer, E.G., Comporti, M., 1993. Lipid peroxidation, biopathological significance. Mol. Aspects Med. 14, 199–207. Nunia, V., Goyal, P.K., 2004. Prevention of gamma radiation induced anemia in mice by diltiazem. Radiat. Res. 45, 11–17. Ohkhawa, A., Oshishi, N., Yogi, K., 1979. Assay for lipid peroxidation in animals tissue by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Patt, H.M., Tyree, E.B., Straube, R.L., Smith, D.E., 1949. Cysteine protection against X-irradiation. Science 110, 213–214. Sharma, A., Goyal, P.K., 2005. Radioprotective capacity of Emblica officinalis against radiation induced biochemical alterations in liver of Swiss albino mice. J. Cell. Tissue. Res. 51, 341–344. Sugahara, T., Tanaka, Y., Nagata, N., Kano, E., 1970. Radiation protection by 2-MPG. In: Proceedings of International Symposium on Thiola, Osaka, Japan, pp. 267. Weiss Joseph, F., Landauer Michael, R., 2003. Protection against ionizing radiation by antioxidant nutrients and phytochemicals. Toxicology 189, 1–20. Yuhas, J.M., Spellman, J.M., Cullo, F., 1980. The role of WR2721 in radiotherapy and/or chemotherapy. Cancer Clin. Trials 3, 221.
Phytomedicine 14 (2007) 701–705 www.elsevier.de/phymed
SHORT COMMUNICATION
Modulation of radiation-induced biochemical alterations in mice by rosemary (Rosemarinus officinalis) extract Dhanraj Soyal, Archana Jindal, Inder Singh, P.K. Goyal Radiation and Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur 302004, India Received 2 June 2006; accepted 24 October 2006
Abstract Radioprotective effect of leaves extract of Rosemarinus officinalis (ROE) has been studied against 6 Gy g-radiations in the liver of Swiss albino mice at various post-irradiation intervals between 12 h and 30 days. In control animals (without ROE treated irradiated), an elevation in glycogen, protein, acid and alkaline contents was found till day 5th, but thereafter decreased at successive intervals without returning to normal. Cholesterol level was found to be lower than normal till 10th day, then increased up to 20th day but later declined without restoring normal level. A similar trend of variation in these biochemical parameters was observed in experimental group (ROE pretreated irradiated) also but to a lower extent. ROE significantly delayed and inhibited the rise in these biochemical parameters. Almost normal values of such constituents were regained by day 30th in experimental animals; whereas in control animals, normal values were not ever attained. In control animals, there was an elevation in lipid peroxidation (LPx) and a decrease in glutathione (GSH) in blood and liver; whereas in experimental group, decline in LPx accompanied by an increase in GSH concentration was observed. r 2006 Elsevier GmbH. All rights reserved. Keywords: g-irradiation; Biochemical parameters; Lipid peroxidation; Glutathione; Swiss albino mice; Rosemarinus officinalis
Introduction Since the pioneering work of Patt et al. (1949) demonstrating that cysteine protects mice and rats against radiation-induced sickness and mortality, several compounds have been tested for their radio protective capacity (Weiss Joseph and Landauer Michael, 2003). The research findings have been affirmed that pretreatment of certain chemical compounds like 2-MPG (Sugahara et al., 1970), WR-2721 (Yuhas et al., 1980) and diltiazem (Nunia and Goyal, 2004) protect mammals against deleterious effects of ionizing radiation. The Corresponding author.
E-mail addresses: [email protected], [email protected] (P.K. Goyal). 0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.12.011
major drawback of synthetic compounds has been that they are highly toxic at the optimum protective dose. Therefore, it is desirable to use other materials, which are less toxic and offer high protection. Rosemarinus officinalis, belonging to family Labiatae, commonly called as rosemary, is widely found along the north and south coasts of the Mediterranean Sea and also in the sub-Himalayan areas (Kotb, 1985). Rosemary is an evergreen branched and bushy shrub. It has been used as an antispasmodic in renal colic and dysmenorrhea and in relieving respiratory disorders. It has also been used as an analgesic, anti-rheumatic, carminative, cholagogue, diuretic, expectorant, anti-epileptic, for effects on human fertility, hepatoprotective and antimutagenic but its radioprotective effects are not known (EI-Gadi and Bshina, 1989; Al-sereiti et al., 1999).
ARTICLE IN PRESS 702
D. Soyal et al. / Phytomedicine 14 (2007) 701–705
Liver is the most versatile organ in the body with an amazing capacity to perform diverse biochemical functions. In fact, a large number of different functions of the liver have been identified. The plant, Rosemarinus officinalis, has not yet been assessed for its possible radio-modulatory potential. Therefore, the present study has been targeted on liver to evaluate radiationinduced biochemical lesions and the radio protective potential of this plant in g-irradiated mice.
behavioral changes, mortality, morbidity, sickness, food and water consumption till their sacrifice or survival. Liver was collected from autopsied animals at 12, 24 h 3rd, 5th, 10th, 20th and 30th day post-treatment. Homogenate of liver was prepared, and glycogen, protein and cholesterol contents were measured using Montgomery (1957), Lowry et al. (1951) and Lieberman (1951) methods, respectively. Activity of acid and alkaline phosphatase was also assayed by using commercially available kits. Spectrophotometer (Systronics UV-VIS-108) was used to measure the optical densities.
Materials and methods
Glutathione (GSH) assay
Preparations of Rosemarinus officinalis extract (ROE)
Liver and blood glutathione (GSH) levels were estimated, after 1 h of DDW/ROE/radiation treatment, according to the methods of Moron et al. (1979) and Beutler et al. (1963), respectively. The absorbance was read at 412 nm using a Systronics UV-VIS-108 Spectrophotometer.
Rosemarinus officinalis, Linn was identified by a competent botanist from Department of Botany, University of Rajasthan, Jaipur. Fresh leaves of this plant were collected during October–December of the year. These leaves were air dried, powdered and extracted with double-distilled water (DDW) by refluxing for 36 h (12 h 3). The ROE thus obtained was vacuum evaporated so as to get in powder form. For the experiment, ROE was redissolved in the sterile doubledistilled water and the dose of required concentration was prepared.
Modification of radiation response Adult male Swiss albino mice (6–8 weeks old) weighing 2572 gm from an inbred colony were used for the present study. Animal care and handling were performed according to guidelines issued by World Health Organization (Geneva, Switzerland) and the Indian National Science Academy (New Delhi, India). Dose selection of ROE was done in a separate experiment based on drug tolerance study (Jindal et al., 2006). Mice selected for this study, were divided into four groups. Animals in Group-I were administered orally with DDW (volume equal to ROE) to serve as normal (vehicle treated) while animals in Group-II were given ROE orally at a dose of 1000 mg/kg b.wt. once in a day for 5 consecutive days. Animals of Group-III received an equal volume of DDW (as in Group-I) while animals in Group-IV (experimental) were given ROE (as in Group-II). After 30 min of DDW or ROE administration on 5th day, animals of Group III and IV were whole-body irradiated at Cobalt Teletherapy Unit (ATC-C9) at Cancer Treatment Centre, Department of Radiotherapy, SMS Medical College and Hospital, Jaipur (at the dose rate of 0.85 Gy/min). Animals of all the groups were monitored for weight change,
Lipid peroxidation (LPx) assay The lipid peroxidation (LPx) level in liver and serum was measured by the assay of thiobarbituric acid reactive substances (TBARS) using the method of Ohkhawa et al. (1979) in which the absorbance was read at 532 nm with a Systronics UV-VIS-108 spectrophotometer.
Statistical analysis The statistical significance of the differences between normal and DDW+irradiated (control) as well as control and ROE+irradiated (experimental) was evaluated by using the Student’s ‘t’ test.
Results The animals of DDW+irradiation group (control) showed signs of radiation sickness within 3–5 days after exposure to 6 Gy of g-irradiation. The pretreatment of mice with ROE (experimental group) delayed the onset of radiation-induced mortality and symptoms of sickness as compared to control group. In irradiated control animals (Group-III), an increase in glycogen and protein contents was noticed as early as at 12 h. Interval and raised further until day 5th, but later decreased from day 10th reaching to near normal by day 30th post-treatment. On contrary, cholesterol level was found to be significantly lower than normal at early intervals. However, it increased later at consecutive intervals without restoring the normal value. An increase over normal in the acid and alkaline
Table 1. Variations (mean7S.E.) in different biochemical parameters in liver of mice after exposure to 6 Gy g-radiation in the presence (experimental) or absence (control) of Rosemarinus officinalis extract (ROE) Biochemical parameter
Treatment Post-treatment autopsy interval 12 h
24 h
3rd day
5th day
10th day
20th day
30th day
Protein 150.0174.031 mg/gma
Group-II 152.0273.639 153.0173.344 154.4573.714 152.8974.827 150.9174.166 150.7173.863 148.3173.783 Group-III 205.5173.434b 208.4771.565b 224.8871.783b 244.4270.662b 234.5172.197b 175.4772.303b 155.6471.073 Group-IV 167.3671.443b 175.6370.900b 190.6671.282b 195.7271.326b 165.1773.409b 154.8073.551b 151.2570.811d
Cholesterol 4.3970.065 mg/gma
Group-II Group-III Group-IV
4.3670.04 4.0870.242 4.2970.204
4.3870.06 3.8370.169 4.1370.145
4.3870.04 3.4270.043 3.9870.111
4.3870.03 4.3570.06 4.4170.05 3.7370.387b 3.9770.075b 4.6570.111b 4.1070.088 4.14 0.192b 4.35 0.100b
4.4170.11 4.4870.244 4.4170.257
Acid phosphatase 1.9870.041 KAUa
Group-II Group-III Group-IV
1.9170.06 2.4570.023b 2.4070.029
1.8570.086 3.0170.334c 2.5170.391
1.8170.051 3.1270.801 2.8970.891
1.7970.055 3.4970.201b 2.9270212d
1.7870.079 3.0870.156b 2.4670.290
1.8870.063 2.7670.107b 2.2370.190d
1.9370.068 2.5070.079d 1.9970.175d
Alkaline Phosphatase 2.5670.093 KAUa Group-II Group-III Group-IV
2.5970.06 3.6170.078b 3.1370.105c
2.6170.104 4.3970.063b 3.4170.172b
2.6470.098 5.0170.081b 3.3970.212b
2.6370.081 5.7870.209b 3.4570.313b
2.6270.094 4.0570.378c 3.2070.407
2.5870.085 3.5870.300c 2.6970.205d
2.5470.047 3.1570.061b 2.5970.177c
4.2970.15 6.1170.351b 5.8770.878
4.3970.243 7.0570.132b 6.5870.519
4.3770.14 7.9670.381b 6.8170.710
4.3670.267 8.6570.302b 7.8370.317d
4.2670.137 7.8270.263b 6.2670.221b
4.2770.281 4.9270.086c 4.6470.348
4.3170.718 4.4670.078 4.2870.127
Group-II: ROE treated. Group-III: DDW+radiation. Group-IV: ROE+radiation. Statistical comparison between normal v/s control and control v/s experimental. a Group-I DDW treated. b po 0.001. c po 0.01. d po 0.05.
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Group-II Group-III Group-IV
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Glycogen 4.2170.234 mg/gma
703
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Table 2. Glutathione (GSH) and Lipid peroxidation (LPx) levels in Swiss albino mice after exposure to gamma radiation in the presence (experimental) or absence (control) of Rosemarinus officinalis extract (ROE) Treatment
GSH level (mean7S.E.)
LPx level (mean7S.E.)
Blood (mg/ml) Liver (mmol/gm) Serum TBARS (MDA)nmol/ml Liver TBARS (MDA)nmol/mg Normal ROE alone DDW+6 Gy (control) ROE+6 Gy (experimental)
3.5670.16 3.6770.17 2.6170.10b 2.9570.12a
64.4971.63 64.9273.12 40.6571.72b 54.3571.81b
1.1570.08 1.0370.13 3.6270.18b 2.4670.20b
2.6570.12 2.2070.19a 6.7070.29b 3.5970.22b
Stastistical comparision between normal v/s control; control v/s experimental. a po 0.05. b po 0.001.
phosphatase activities was noticed at early intervals and found maximum on day 5th but later started to decrease from day 10th, without returning to normal level even till the end of study (Table 1). In the experimental animals (ROE pretreated irradiated), elevated values of glycogen and protein were noted at the similar intervals like control, but such increase was significantly lesser. The normal level of glycogen and protein attained by day 30th and 20th post-irradiation, respectively in this group. Cholesterol level declined up to day 3rd however, it increased later and returned to near normal level on day 30th. A significant increase over normal in acid and alkaline phosphatase was recorded until day 5th, but at all the intervals such increase was significantly lesser than the control, and normal value regained on day 30th postirradiation (Table 1). ROE treated irradiated animals showed a significant increase in GSH content (blood and liver) with respect to control, but the values remained below normal. Exposure of animals to g-radiation increased LPx in Groups III and IV, whereas ROE pretreatment significantly reduced LPx induction in the ROE+irradiation group, thereby protecting liver and blood against radiation-induced LPx (Table 2).
Discussion An elevation in liver glycogen and total protein concentration over normal were observed after irradiation in control and experimental groups. It is because more substrate is made available as a result of tissue break down. Moreover, the persistently higher level of hepatic glycogen after irradiation could be due to the stimulation of the pituitary adrenal system. Similar results were observed with Emblica officinalis also (Sharma and Goyal, 2005). Mukarjee and Goldfeder (1974) suggested that such rise in protein may be due to an increased transport of amino acids through plasma
membrane as a consequence of permeability change in irradiated cell membranes in the experimental group. Liver cholesterol showed a decrease up to day 3rd after irradiation which confirms the observations of Sharma and Goyal, (2005). The reduction in cholesterol during early intervals might be due to the stress response caused by irradiation, which stimulates the synthesis of steroid hormones via hypothalamic–pituitary system. An increase in the liver phosphatase activities of mice due to irradiation was observed in the present study. Similar increase in such enzymes was observed earlier (Sharma and Goyal, 2005; Bharatiya and Khan, 1983; Khan et al., 1984) after irradiation with various doses. Radiation-induced cell death may be a possible reason for increased activity of ACP and ALP. Aqueous extract of the young sprouts of rosemary has an anti-lipoperoxidant activity, as it reduced the formation of malonaldehyde significantly in a dosedependent manner, and significantly decreased the release of lactic dehydrogenase (LDH) and aspartate aminotransferase (ASAT), thus confirming the antihepatotoxic action of ROE (Joyeux et al., 1990). The active constituents of Rosemarinus officinalis like carnosol, carnosic acid, caffeic acid, rosmarinic acid, ursolic acid, different diterpenes, phenols and flavonoids are reported to have antioxidant, antimutagenic, radioprotective properties (Fluck et al., 1976; Leung and Foster 1996; Del Bano et al., 2003, 2006). Ionizing radiation induces lipid peroxidation (LPx) which can lead to DNA damage and cell death (Noda et al., 1993). An increase in the glutathione (GSH) level by ROE may be responsible for the scavenging of radiation-induced free-radicals including LPx and thereby protecting against radiation-induced mortality. It has been reported that LPx starts to increase as soon as the endogenous GSH is exhausted, and the addition of GSH promptly stops further peroxidation (Kilic et al., 2000). My present study shows that Rosemarinus officinalis leaves extract provides a significant protection against radiation-induced biochemical alterations in liver.
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