Role of Rosemary Leaf Extract Against Various Doses of Gamma Radiation

Transcription

1 Trees for Life Journal a forum on benefiial trees and plants Open aess, freely available online Researh Artiles Role of Rosemary Leaf Extrat Against Various Doses of Gamma Radiation Garima Sanheti and P. K. Goyal* Radiation & Caner Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur (India) *Corresponding author Abstrat The present investigation reports the radiomodulatory effet of Rosmarinus offiinalis (rosemary) leaf extrat against radiation indued hematologial alterations in Swiss albino mie at various post autopsy intervals (i.e., between 24 hours to day 30). Treatment of animals with rosemary extrat (1000 mg/ kg body wt) prior to irradiation was found to delay the onset of mortality and redued the symptoms of radiation sikness suh as ruffled hairs, lethargy, anorexia and diarrhea in omparison to radiation alone treated animals. Rosemary treated experimental groups exhibited a dose dependent rise (9 < 6 < 3 Gy) in the number of leuoytes (i.e., lymphoytes, monoytes, basophils, eosinophils and neutrophils) by the 30th day post autopsy interval in omparison to the ontrol. resulted in a signifiant inrease in lipid peroxidation levels (p< 0.01, p< 0.001) and a redution in glutathione levels (p<0.05, p<0.001) in blood as observed in radiation alone treated animals. Conversely, treatment of mie with rosemary extrat exhibited a signifiant derease (p< 0.01, p< 0.001) in lipid peroxidation level and an inrease (p< 0.05, p<0.001) in glutathione ontent. Introdution Radiation protetion is at a ross road after radiation inidents and unaeptable tragedies suh as those at Chernobyl and Three Mile Island. Radiation indued damage to the normal tissues an be partially redued by the use of radioprotetors that redue the damaging effets of radiation, inluding radiationindued lethality (4,22,38). Various workers have investigated the potential appliation of radioprotetive hemials in the event of planned and unplanned exposure (i.e., linial onology, radiation site leanup, military senarios, radiologial terrorism, radiation aidents, et.) (18,25,40). A substantial amount of researh has been arried out in the field of hemial radioprotetion during the last few deades; however, no safe and ideal syntheti radioprotetors are available to date. Reently, interest has generated in developing the potential drugs of plant origin for the amelioration of radiation effets. Plants and their produts are well known to have an advantage over the syntheti ompounds in terms of their potential low/no toxiity at the effetive dose with minimum or no side effets (3,9,33,34,41). However, the use of mediinal plants suffers from lak of robust sientifi evidene to support their use. Therefore, studies supporting or rejeting their role in the treatment of various health disorders is of great need (3). Rosemary (Rosmarinus offiinalis), belonging to the family Lamiaeae, is a ommon mediinal and aromati plant grown in many parts of the world. It is indigenous to Southern Europe, partiularly on the dry roky hills of the Mediterranean region. Rosemary is used as a u linary herb, a beverage drink, as well as in osmetis; in folk mediine it is used as a toni and stimulant, analgesi, anti rheumati, arminative, diureti, expetorant, antiepilepti, antispasmodi in renal oli, dysmenorrhoea, for relief of respiratory disorders, effets on Copyright: 2007 Garima Sanheti et al. This is an Open Aess artile distributed under the terms of the Creative Commons Attribution Liense whih permits unrestrited use, distribution, and reprodution in any medium, provided the original work is properly ited. Contat: P. K. Goyal pkgoyal2002@rediffmail.om Reeived: July 29, 2006 Aepted: January 28, 2007 Published: August 1, 2007 The eletroni version of this artile is the omplete one and an be found online at: Trees for Life Journal 2007, 2:2 PEER VIEWED

2 human fertility, and the stimulation of hair growth (1). Rosemary has been shown to be safe in toxiity studies in animal models when added as an antioxidant to food (35). Sine time immemorial, the plant has been used traditionally by people for uring various health disorders around the world. Caribs of Guatemala use rosemary to ure various human diseases (12). Rosemary has been desribed as a wonder drug in literature and in various medieval drug monographs as well (36,44). Thus, wide aeptability and diverse pharmaologial and anti oxidative properties of the plant stimulated us to evaluate the radio modulatory effet of Rosmarinus offiinalis in Swiss albino mie exposed to various doses of gamma radiation. Materials and Methods Animal are and handling Male Swiss albino mie (Mus musulus), 6 8 weeks old, weighing g., from an inbred olony were used for the present study. The animals were provided standard mie feed (proured from Hindustan Lever Ltd., India) and water ad libitum and were maintained under ontrolled onditions of temperature and light (Light: dark, 10 hrs: 14 hrs.). Four animals were housed in a polypropylene age with loally proured paddy husk (Oryza sativa) as bedding throughout the experiment. Tetrayline ontaining water (0.13 mg/ml) was provided one a fortnight and was given as a preventive measure against infetions. Animal are and handling were performed aording to the guidelines set by the World Health Organization (WHO), Geneva, Switzerland and the INSA (Indian National Siene Aademy), New Delhi, India. The Departmental Animal Ethial Committee approved the present study. The obalt teletherapy unit (Co 60) at the Caner Treatment Centre, Radiotherapy Department, SMS Medial College & Hospital, Jaipur, India, was used for irradiation. Unanaesthetized animals were restrained in well ventilated Perspex boxes and exposed to various doses of gamma radiation (i.e., 3, 6 and 9 Gy) at a distane (SSD) of 80 m from the soure at a dose rate of 0.85 Gy/min. Taxonomi desription of the plant Rosemary is an evergreen shrub growing to 1.5 m by 1.5 m at a medium rate. The leaves of rosemary are about 1 inh long, linear, revolute, dark green above and paler and glandular beneath, with amphoraeous aromati odour. The sented hermaphrodite flowers are small and pale blue. Muh of the ative volatile priniple resides in their alyes. There are various other varieties of the plant, but the green leaved variety is the kind used mediinally (13). Preparation of plant extrat The identifiation of the plant Rosmarinus offiinalis (family: Lamiaeae) was done by a botanist, Dr. Deepak Aharya, (Vouher Speimen No: DDC/2001/DEPTBT/ ACHARYA2430) of the Department of Botany, Danielson College, Chhindwara, Madhya Pradesh (India). The non infeted leaves of the plant were olleted, arefully leaned, shade dried and powdered in a grinder. The plant material was prepared by extrating 200 gm of leaf powder with double distilled water by refluxing for 36 hrs (12 hrs. x 3) at 55 ± 5 C. Pellets of the extrat were obtained by evaporation of its liquid ontents in the inubator. An approximate yield of 22 % extrat (w/w) was obtained. The required dose for treatment was prepared by dissolving the drug pellets in double distilled water and administered by oral gavage with a miropipette (100 μl/ animal) at a dose of 1000 mg/ kg body wt./animal (1000 mg of 22% of original plant weight). Heneforth, rosemary leaf extrat will be alled. Design Optimum dose determination A dose seletion of Rosmarinus offiinalis () was done on the basis of a drug tolerane study. For this purpose, various doses of extrat (100, 200, 400, 800, 1000, 1500 and 2000 mg/kg body wt.) were tested for their tolerane (one in a day for 5 onseutive days) in Swiss albino mie. One hour after the last administration of, mie were exposed to 8 Gy gamma irradiation. All these animals were then observed for 30 days for soring signs of radiation sikness or mortality. Thus, the optimum tolerated dose of (1000 mg/ kg b. wt.) was determined and used for further detailed experimentation (Fig 1). The LD50/30 and Dose Redution Fator The effiay of any protetive agent is evaluated by the determination of its dose redution fator (DRF). The DRF of R. offiinalis extrat () based on LD50/30 survival experiment was alulated after irradiating a large number of Swiss albino mie to different doses of gamma rays in the presene (experimental) or absene (ontrol) of. The perentage of mie surviving at eah radiation dose till 30 days following suh exposures was used to onstrut survival dose response urves. Regression analysis was done to obtain LD50/30, and dose redution fator was omputed as: DRF = LD 50/30 of Animals LD 50/30 of Animals

3 The LD50/30 values for ontrol and experimental animals obtained from the survival data are 6.85 and respetively. The dose redution fator of R. offiinalis against radiation treatment was alulated on the basis of the survival experiment and was measured as Modifiation of radiation response A total of 48 animals used for the experiment were assorted into 4 groups. Mie of group 1 (sham irradiated) were orally administered double distilled water (DDW) at a dose of 1000 mg/ kg body weight, volume equal to. Animals belonging to group 2 ( alone) were given daily rosemary extrat at a dose of 1000 mg/ kg/ animal for 5 onseutive days, one hour before irradiation. Animals of group 3 (radiation ontrol) were exposed to various doses of gamma rays alone (i.e., 3, 6 and 9 Gy) one hour after DDW treatment on day 5. Group 4 ( experimental) reeived (1000 mg/ kg body wt./ animal) as in group 2. One hour after last administration of, mie were exposed to various doses of gamma rays, (i.e., 3, 6 and 9 Gy), respetively. These animals were observed daily for any sign of sikness, morbidity, behavioral toxiity and mortality. A minimum of 6 animals from eah group were neropsied on days 1, 3, 5, 10, 20 and 30 post treatment intervals to study hematologial and biohemial parameters. Hematologial study For the study, blood was olleted from the orbital sinus of animals from eah group in a vial ontaining 0.5 M EDTA. Differential leuoyte ounts (lymphoytes, monoytes, basophils, eosinophils and neutrophils) were determined by adopting standard proedures. The number of eah type of leuoytes (lymphoytes, monoytes, basophils, eosinophils and neutrophils) and the total number of leuoytes ounted were reorded. The perentage of eah type of leuoyte was alulated by the formula: Number of type of leuoyte Total number of leuoytes ounted 100 Biohemial determinants Biohemial alterations were studied in animals of all the groups at one hour post exposure to gamma radiation. The level of glutathione (GSH) was determined in blood by the method of Beutler et al. (5). The lipid peroxidation (LPx) level in the serum was measured by the assay of thiobarbituri aid reative substanes (TBARS) aording to the method of Okhawa et al. (30). Statistial analysis The result for all the groups at various neropsy intervals were expressed as mean ± standard error of the mean (S. E. M.) to evaluate whether the mean of the sample drawn from experimental ( experimental) deviated signifiantly from respetive ontrol ( ontrol). Student s t test was used by the method of Bourke et al. (7). The signifiane level was set at different levels as p<0.05, p<0.01 and p< Results Data are presented in Tables 1 3 and Figures 1 4. The radioprotetive effet of rosemary leaf extrat () was studied in mie treated with 1000 mg/ kg body wt. before exposure to 3, 6 and 9 Gy of gamma radiation. No notieable signs of behavioral hange, sikness or mortality were observed in Sham irradiated/ treated group. Animals exposed to 3 and 6 Gy gamma rays alone did not show mortality throughout the experimental period, but slight laziness was observed in some animals. Animals exposed to 9 Gy gamma rays exhibited epilation, ruffled hair, watering of eyes, weight loss and beame lethargi. No animal ould survive in the 9 Gy irradiated alone group beyond day 10. Animals pretreated with did not exhibit mortality or any symptoms of radiation sikness. General health, ativeness, food and water intake were found to be normal in pretreated irradiated animals. Survival perentage Body weight (gm) Fig 1: Days survival of mie pretreated with different doses of and exposed to 8 Gy gamma radiation Post-irradiation time (days) Fig 2: Variation in the body w eight of m ie at post-irradiation intervals treated w ith (experimental) or without (Radiation ontrol) mg 200mg 400mg 800mg 1000mg 1500mg 2000mg ontrol Shamirradiat ed alone 3 Gy + 3 Gy 6 Gy + 6 Gy 9 Gy Post-irradiation time (days)

4 Fig 3: Variations in the glutathione (GSH) and lipid peroxidation (LPx) level in peripheral blood of mie w ith/w ithout Rosemary extrat () treatment GSH (µg/ ml) LPx (n mole/ ml) Sham irradiated alone 3 Gy + 3 Gy 6 Gy + 6 Gy 9 Gy + 9 Gy Signifiane level (determined by Student's 't' test for statistial omparison between the groups at): a p<0.05 b p<0.01 p<0.001 Survival Perentage a a Fig 4: Survival dose-response urve for determination of LD 50/30 (Survival data olleted for treatment of radiation doses and alulated by regression analysis) b 3 Gy 6 Gy 9 Gy b After whole body exposure to different doses of gamma radiation (i.e., 3, 6 and 9 Gy), lymphoyte perentages remained signifiantly lower than normal, and ould not regain a normal value even by the last day of autopsy interval (day 30). No signifiant hanges in monoytes, eosinophils and basophil ounts were registered in any of the groups. However, monoytes followed a pattern similar to lymphoytes. Following irradiation, a signifiant inrease above normal was observed in neutrophil ounts. A normal value ould not be restored in any of the irradiated groups till day 30 post exposure. Daily administration of 1000 mg/ kg of for 5 onseutive days rendered reovery in the different types of leuoytes (i.e., lymphoytes, monoytes, eosinophils, basophils and neutrophils) in omparison to irradiated alone groups, and values lose to normal were registered in a dose dependent manner (3> 6> 9 Gy) by post treatment day 30. Biohemial determinants There was no signifiant differene observed in the levels of glutathione (GSH) and lipid peroxidation (LPx) in the blood ontent of sham irradiated (group 1) or alone treated animals (group 2). In onomitant treatment of and radiation (group 4), GSH was found to be further lowered than the radiation treated group. A signifiant elevation in the values of blood GSH as ompared to group 3 was estimated in experimental animals. An inrease in LPx levels above normal was evident in serum of irradiated mie, while a signifiant derease in suh values was evident in the pretreated irradiated group. Disussion Radiation injuries are manifested as a result of enhaned prodution of free radials due to oxidative stress. Exposure to radiation auses ionization of moleules in the ells, whih sets off potentially damaging reations via free radial prodution (23). Free radial mediated proesses and oxidative stress have been impliated in the pathogenesis of the aging proess and various diseases suh as atheroslerosis, liver damage, arthritis, aner, and neurodegenerative disorders (2). The prevailing view is that intake of antioxidant nutrients an redue the risk of free radialrelated health problems and may prove to be protetive against ionizing irradiation. The present study revealed that the number of lymphoytes delined in a dose dependent manner after exposure to 3, 6 and 9 Gy gamma irradiation. A rapid depression was observed at early intervals whih may be attributed to diret destrution of suh ells in peripheral blood of mie (31). No signifiant hanges in monoytes, eosinophils and basophils were observed after whole body exposure to different doses of gamma radiation. It may be attributed to the fat that mature granuloytes are radioresistant whereas lymphoytes are extremely sensitive to radiation (8). Furthermore, neutrophil granules altered inversely as ompared to lymphoytes. These ells exhibited an early rise while lymphoytes and monoytes delined soon after exposure thus showing an opposite behavior. This an be explained by an abortive rise phenomenon as desribed earlier by workers (26,39). Aording to Hall (14), by the time the number of irulating ells in the blood reahes minimum value as the mature irulating ells begin to die off, the supply of new ells from the depleted preursor population beomes inadequate to replae these, thereby making radiation effets beome apparent. Also, this abrupt inrease may have appeared due to an abortive rise phenomenon (6,26) or an be interpreted as stimulation effet (11). Hastening the maturation of granuloyte preursors in bone marrow and their release into general irulation an be attributed to a rise in neutrophil ounts (29,42). It is evident from the present study that administration of redued radiation sikness and

5 mortality, and provided protetion to differential leuoytes ounts (i.e., lymphoytes, monoytes, basophils, eosinophils and neutrophils) in the peripheral blood of mie from the damaging gamma radiation. It has been observed that rosmarini aid (found in rosemary) is effetive in relation to blood irulation and to improve hemodynamis in olusive arterial diseases (1). Rosemary has been found to ontain ertain antioxidative (32) and free radial savenging ativity (45) in its ative ompounds like affei aid, arnosoli aid, hlorogeni aid, rosmanol, rosmarini aid, arnosol, different diterpenes (16,43), rosmari diphenol, rosmariquinone (17) and other natural antioxidants suh as ursoli aid, alkaloid rosmariine and gluooli aid (21). In a reent study, arnosi aid was found to render protetion to UVA irradiated human skin fibroblasts (29). The basi effet of radiation on ellular membranes is believed to indue lipid peroxidation (LPx) by the prodution of free radials that have the potential to damage DNA and ause ell death (24,27). The level of radiation indued LPx inreased onsiderably in a dose dependent manner in the entire group 3 irradiated animals, whereas a derease in the values was observed in treated group 4. The inhibition observed in the LPx level in blood of administered animals may have been responsible for the observed radioprotetion by plant extrat. This view is supported by the investigation of an anti lipoperoxidant ativity of young sprouts of Rosmarinus offiinalis that signifiantly redued the formation of malondialdehyde in rat hepatoytes1 (9). Sotelo Felix et al. proposed that arnosol ould savenge free radials indued by arbon tetrahloride, onsequently avoiding the propagation of lipid peroxides in the liver of mie (37). Studies onduted by Haraguhi et al. report an inhibition of superoxide and lipid peroxidation by 4 diterpenoids from rosemary, i.e. arnosi aid, arnosol, rosmanol and epirosmanol (15). Del Bano et al. investigated the effiay of arnosi aid, arnosol and rosmarini aid (ative onstituents of rosemary) and found these to be radioprotetive against hromosomal damage indued by γ rays (10). The exat mehanism of ation of rosemary is yet to be eluidated; however, it may at as a protetive by savenging free radials triggered by radiation. Glutathione (GSH) is one of the antioxidant enzymes that at as the first line of defense against prooxidant stress, thus performing as a free radial savenger. Oral administration of DDW or did not signifiantly influene the endogenous GSH level in blood. In the present study, GSH levels were found to be lower in the blood of irradiated alone animals than that observed in the pre treated mie. The levels of GSH were found to be elevated in the blood of mie after administration. One of the mehanisms of protetion against radiation an be an elevation in the glutathione level that is mediated through the modulation of ellular antioxidant level. Rosmarini aid has been experimentally found to have a signifiant antioxidant role through free radial savenging ativity (1). Kili et al. observed that lipid peroxidation starts as soon as the endogenous GSH gets exhausted, and the addition of GSH stops further peroxidation promptly (20). Inrease in the GSH onentration, towards normal, ould have resulted in redued levels of LPx, thereby proteting against damage aused by radiation in the pretreated irradiated group. The mehanism of the radioprotetive ation of Rosmarinus offiinalis leaf extrat in this animal model may thus be its free radial savenging ativity and its ability to thus protet ellular moleules from oxidative damage. Furthermore, it inhibited lipid peroxidation and modulated GSH levels in blood of these Swiss albino mie. The ativity of rosemary may also be attributed to stimulating or proteting hematopoiesis in bone marrow and a subsequent inrease of hematologial onstituents in the peripheral blood. Sine signifiant protetion was obtained at a non toxi low dose, may have an advantage over the known radioprotetors. Further investigations are in progress to study the exat mehanism of ation and linial appliability of R. offiinalis in radioprotetion.

6 Table 1: Variation in differential leuoyte ounts (DLC) of 3 Gy irradiated Swiss albino mie at various autopsy intervals Post- Interval Treatment Group Lymphoytes Monoytes Eosinophils Basophils Neutrophils Day 1 Day 3 Day 5 Day 10 Day 20 Day 30 Shamirradiated 48.4± ±0.50 b 1.4± ± ± ± ± ± ± ± ± ± ± ± ± ±1.28 b 2.4± ± ± ± ±1.38 b 1.6±0.92 b 2.4± ± ± ± ± ± ± ± ±1.50 b 1.8±0.40 b 1.8± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.42 a 1.8± ± ± ± ± ± ± ± ± ± ± ± ± ±0.52 alone 67.4± ± ± ± ±0.45 a indiates signifiant (p< 0.05) differene with respet to Sham-irradiated v/s ontrol, as well as ontrol v/s experimental b indiates signifiant (p<0.01) differene with respet to Sham-irradiated v/s ontrol, as well as ontrol v/s experimental indiates signifiant (p<0.001) differene with respet to Sham-irradiated v/s ontrol, as well as ontrol v/s experimental

7 Table 2: Variation in differential leuoyte ounts (DLC) of 6 Gy irradiated Swiss albino mie at various autopsy intervals Post- Interval Treatment Group Lymphoytes Monoytes Eosinophils Basophils Neutrophils Day 1 Day 3 Day 5 Day 10 Day 20 Day 30 Shamirradiated 51.8± ± ±0.35 a 0.2± ± ± ± ± ± ±1.80 b 53.8±2.77 b 2.4±0.17 a 1.8± ±0.21 a 41.6± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.78 a 2.2±0.33 a 0.6± ± ± ± ±0.56 a 0.4± ±0.93 b 54.6± ±0.48 b 1.8±0.45 a 0.2± ± ± ±0.46 a 2.0± ± ± ± ±0.60 a 2.0± ± ± ± ± ± ± ± ± ± ± ± ±0.52 alone 67.4± ± ± ± ±0.45 a indiates signifiant (p< 0.05) differene with respet to Sham-irradiated v/s ontrol, as well as ontrol v/s experimental b indiates signifiant (p<0.01) differene with respet to Sham-irradiated v/s ontrol, as well as ontrol v/s experimental indiates signifiant (p<0.001) differene with respet to Sham-irradiated v/s ontrol, as well as ontrol v/s experimental

8 Table 3: Variation in differential leuoyte ounts (DLC) of 9 Gy irradiated Swiss albino mie at various autopsy intervals Post- Interval Treatment Group Lymphoytes Monoytes Eosinophils Basophils Neutrophils Day 1 Day 3 Day 5 Day 10 Day 20 Day 30 Shamirradiated 52.0± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.68 N.S. N.S. N.S. N.S. N.S. 65.6± ± ± ± ±3.68 N.S. N.S. N.S. N.S. N.S. 64.2± ± ± ± ± ± ± ± ± ±0.52 alone 67.4± ± ± ± ±0.45 N.S. = No Survival a indiates signifiant (p< 0.05) differene with respet to Sham-irradiated v/s ontrol, as well as ontrol v/s experimental b indiates signifiant (p<0.01) differene with respet to Sham-irradiated v/s ontrol, as well as ontrol v/s experimental indiates signifiant (p<0.001) differene with respet to Sham-irradiated v/s ontrol, as well as ontrol v/s experimental

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