Jagetia, G. C., Baliga, M. S., Venkatesh, P. and Ulloor, J. N. Influence of Ginger Rhizome (Zingiber officinale Rosc) on Survival, Glutathione and Lipid Peroxidation in Mice after Whole-Body Exposure to Gamma Radiation. Radiat. Res. 160, 584–592 (2003).
The radioprotective effect of the hydroalcoholic extract of ginger rhizome, Zingiber officinale (ZOE), was studied. Mice were given 10 mg/kg ZOE intraperitoneally once daily for five consecutive days before exposure to 6–12 Gy of γ radiation and were monitored daily up to 30 days postirradiation for the development of symptoms of radiation sickness and mortality. Pretreatment of mice with ZOE reduced the severity of radiation sickness and the mortality at all doses. The ZOE treatment protected mice from GI syndrome as well as bone marrow syndrome. The dose reduction factor for ZOE was found to be 1.15. The optimum protective dose of 10 mg/kg ZOE was 150 of the LD50 (500 mg/kg). Irradiation of the animals resulted in a dose-dependent elevation in the lipid peroxidation and depletion of GSH on day 31 postirradiation; both effects were lessened by pretreatment with ZOE. ZOE also had a dose-dependent antimicrobial activity against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and Candida albicans.
INTRODUCTION
The first report of the use of chemicals to protect mammals against radiation-induced damage appeared in 1949, when Patt et al. (1) reported that cysteine protected mice and rats against radiation-induced sickness and mortality. Since then, several chemical compounds and their analogues have been screened for their radioprotective effects. However, the practical applicability of the majority of these synthetic compounds has been limited because of toxicity at radioprotective doses (2). Thus it was considered important to explore alternatives to the synthetic compounds that would be radioprotective at nontoxic doses. Plants have been used to treat various ailments in humans since time immemorial, and herbal preparations have usually been considered safer and less toxic than synthetic compounds. Therefore, it is natural that the choices of alternative radioprotectors would include plants and plant products.
The rhizome of Zingiber officinale, commonly known as ginger, is consumed worldwide as a spice and flavoring agent. The rhizome of ginger has been reported to possess diverse medicinal properties in the traditional Indian system of medicine, the Ayurveda, and it is widely used in several medicinal preparations (3–5). Depending on the season, climate, geographic region, and processing, ginger has been reported to possess terpenes like ar-curcumene, farnesene, β-bisabolene, γ-selinene, β-elemene, β-sesquiphellandrene, phellandrene, camphene, α-pinene, β-pinene, cumene, myrecene, limonene, cymene, cineole, citral, zingiberene and basabolene. Ginger also contains the oxygenated monoterpenes like 2-heptanol, 2-nonanol, n-nonanol, n-decanol, methyl heptenone, 1,8-cineole, borneol, bornyl acetate, linalool, geraniol and neral and oxymethyl phenols like gingerol, shogaol, zingerone and paradol (4, 6). Most of these compounds have been reported to possess antioxidative, free radical scavenging activities (7, 8), to increase antioxidant enzymes (9), and to decrease lipid peroxidation (10).
2-Mercaptopropionylglycine (MPG), a synthetic thiol compound, has been reported to be a radioprotector and has been used in clinics as a detoxifying agent (11). Ocimum sanctum has also been reported to be useful in a number of therapeutic applications, including antimicrobial and radioprotective activities (12–14). Earlier studies have shown that an aqueous extract of Ocimum sanctum was most effective at a nontoxic dose of 10 mg/kg once daily for 5 consecutive days and protected mice against radiation-induced mortality, chromosomal damage, and stem cell damage (13, 14).
The common use, wide acceptability by humans, and diverse medicinal and antioxidative properties attributed to ginger rhizome stimulated us to examine the radioprotective effect of the hydroalcoholic extract of ginger (Zingiber officinale) in mice after whole-body irradiation with different doses of γ radiation, using Ocimum sanctum and 2-mercaptopropioonyl glycine (MPG) as positive controls.
MATERIALS AND METHODS
The animal care and handling were carried out according to the guidelines issued by the World Health Organization, Geneva, and the INSA (Indian National Science Academy, New Delhi). The study was approved by the Institutional Animal Ethical Committee. Ten- to 12-week-old male Swiss albino mice weighing 30 to 36 g were selected from an inbred colony maintained under controlled conditions of temperature (23 ± 2°C), humidity (50 ± 5%) and light (14 and 10 h of light and dark, respectively). The animals had free access to the sterile water and food. Four animals were housed per polypropylene cage containing sterile paddy husk (procured locally) as bedding.
Preparation of the Extract
The ginger rhizomes (small local variety) were collected during April and the leaves of Ocimum santum in September 2001. The plants were identified by Dr. G. K. Bhat, Department of Botany, Poorna Prajna College, Udupi, India. The ginger rhizomes were separated, cleaned, freed from scales, shade-dried and powdered. The extract of ginger powder was prepared as described earlier (15). Briefly, 100 g of the ginger powder was extracted with 50% ethanol at 50 to 60°C in a Soxhlet apparatus for 72 h. The cooled liquid extract was concentrated by evaporation. The aqueous extract of Ocimum sanctum was prepared as described earlier (14). The extracts were stored at −70°C until use. The extract of ginger was characterized by HPLC. The extract of Zingiber officianale and Ocimum sanctum will be referred to as ZOE and OSE, respectively.
Preparation of the Drug Solution and Mode of Administration
2-Mercaptopropionylglycine (MPG; a kind gift from Santen Pharmaceuticals Limited, Osaka, Japan) was dissolved in the sterile double-distilled water at 2 mg/ml immediately before use. The pH of the solution was adjusted to 6.4 by the addition of 0.1 N sodium hydroxide before administration. OSE and ZOE extracts were dissolved at a concentration of 1 mg/ml in double-distilled water. The animals were given double-distilled water or drugs intraperitoneally unless stated otherwise and then were divided into the following groups:
Double-distilled water + radiation
The animals in this group received 0.01 ml double-distilled water/g body weight before exposure to γ radiation.
MPG + radiation
The animals in this group were given 20 mg MPG/kg before exposure to 10 Gy γ radiation (11).
OSE +radiation
The animals in this group were injected with 10 mg OSE/kg once daily for 5 consecutive days before irradiation with 10 Gy (14).
ZOE + radiation
This group of animals was injected with different doses or 10 mg ZOE/kg once daily for 5 consecutive days before exposure to radiation (16).
Determination of Acute Toxicity of ZOE
The acute toxicity of ZOE was determined according to Prieur et al. (17) and Ghosh (18). Briefly, the animals were fasted by withdrawing their food and water for 18 h and were divided into groups of 10 mice/group. Groups were injected with various doses of freshly prepared ZOE (125, 200, 250, 300, 400, 500, 600, 700, 800 and 1000 mg/kg body weight). The animals were provided with food and water immediately after administration of the drug. Mortality was observed for 14 days after drug treatment.
Selection of Optimum ZOE Dose
The optimum radioprotective dose of ginger was selected by giving the animals 5, 10, 20, 40 or 80 mg ZOE/kg once daily for 5 consecutive days. One hour after the last administration, the animals were exposed to 10 Gy of γ radiation (16). A minimum of 25 animals were used for each drug dose.
Selection of Route of ZOE Administration
The most effective route of administration for radioprotection was selected by giving the animals 10 mg ZOE/kg orally or intraperitoneally once daily for 5 consecutive days before exposure to 10 Gy of γ radiation. A minimum of 25 animals were used for each group.
Selection of Dosing Schedule for ZOE
To select the best schedule for radioprotection, mice were given ZOE as a single dose of 50 mg, 25 mg once daily for 2 days, or 10 mg once daily for 5 consecutive days before exposure to 10 Gy of γ radiation. A minimum of 25 animals each were used for each schedule.
Radioprotective Effect of ZOE
To ascertain the radioprotective ability of ZOE, the animals were divided into two groups as described above. The animals in the ZOE + radiation group received 10 mg/kg ZOE once daily for 5 consecutive days before exposure to 6, 7, 8, 9, 10, 11 or 12 Gy of γ radiation. A minimum of 25 animals were used for each radiation dose in each concurrent group.
Irradiation
One hour after the last administration of double-distilled water, ZOE or OSE and 30 min after the administration of MPG, the immobilized animals were exposed whole-body to 60Co γ radiation (16) in a specially designed well-ventilated acrylic box. Ten animals were irradiated each time at a dose rate of 1.66 Gy/min at a source-to-animal distance (midpoint) of 70 cm.
Unless otherwise started, the animals were monitored daily for the development of symptoms of radiation sickness and for mortality for 30 days postirradiation. The dose reduction factor (DRF) was calculated by the method of Miller and Tainter (19):
Estimation of Glutathione and Lipid Peroxidation
The livers of the surviving animals in the ZOE + radiation and double-distilled water + radiation groups were studied on day 31. Mice were killed and the livers were perfused with ice-cold saline transcardially. Each whole liver was removed, blotted dry, and weighed, and a 10% homogenate was prepared with an ice-cold 0.2 M sodium phosphate buffer, pH 8.0, using a homogenizer (Yamato LSG LH-21, Japan). A minimum of 4 animals were used for each radiation dose in each group.
Total protein, was estimated by the method of Lowry et al. (20) using bovine serum albumin as standard, while the GSH contents and lipid peroxidation were estimated as described earlier (21, 22).
Antimicrobial Activity
The antimicrobial activity of ZOE was evaluated on the enteric pathogens Escherichia coli, Klebsiella, Salmonella typhimurium, Shigella dysenteriae, Vibrio cholera o1, Pseudomonas aeruginosa and Staphylococcus aureus. The antifungal activity of the ginger extract was studied using Candida albicans, an agent that causes diarrhea in immunocompromised hosts.
The antimicrobial activity was estimated by the agar diffusion method (23). Briefly, the bacterial and Candida colonies were inoculated into peptone water and nutrient broth and incubated at 37°C for 4–6 h. The turbidity was compared using McFarland's turbidity standard tube no. 5. Bacteria or fungus were inoculated in sterile petri dishes containing Muller-Hinton agar (MHA) and Sabourauds dextrose agar (SDA) by the lawn culture method. Wells of 6 mm in diameter were punched into the agar medium using a sterile borer. These wells were inoculated with 15, 22.5, 30, 37.5 or 45 mg of ZOE. The double-distilled water and solvent controls were run simultaneously. The plates were incubated at 37°C overnight, and the diameters of the zones of inhibition were measured in millimeters.
The statistical significance of differences between the treatments was determined using the Z test for the survival studies, while Student's t test was used for biochemical and antimicrobial activities. All the results are expressed as means ± SEM (standard errors of the mean).
RESULTS
HPLC Fingerprinting
ZOE was analyzed using HPLC. The chromatogram shows a total of nine peaks; peak 4 corresponds to gingerol, while the other eight peaks represent unknown compounds (Fig. 1).
Determination of Acute Toxicity of ZOE
Administration of ZOE at doses of 125, 200, 250 and 300 mg/kg did not induce mortality during the whole observation period. A dose of 400 mg/kg body weight resulted in a 20% reduction in the survival of mice, while a 50% reduction in survival was observed for 500 mg/kg body weight, 600 mg/kg resulted in 80% mortality, and 100% mortality was observed at doses of 700 mg/kg and higher.
Selection of Optimum Dose of ZOE
The optimum dose of ZOE for radiation protection was selected by giving mice 0, 2.5, 5, 10, 20 or 40 mg /kg ZOE before whole-body exposure to 10 Gy γ radiation. The administration of these doses of ZOE for 5 consecutive days did not induce mortality. The first mortality in the double-distilled water + 10 Gy radiation group was observed on day 3, and all the animals died by 16 days postirradiation (Fig. 2). Irradiation of mice with 10 Gy induced symptoms of radiation sickness including reduction in food and water intake, irritability, watering of eyes, epilation, weight loss, emaciation, lethargy, diarrhea and facial edema. The pretreatment of mice with ZOE delayed or reduced the severity of those symptoms. The onset of radiation-induced mortality was also delayed in the ZOE + radiation group compared to the double-distilled water + 10 Gy radiation group. The longest delay was observed for 10 and 20 mg/kg ZOE, where the first mortality occurred by day 8 postirradiation, while the shortest delay was observed for 2.5 mg/kg, where the first mortality occurred on day 5 postirradiation (Fig. 3).
The majority of the animals (67%) in the double-distilled water + 10 Gy radiation group died within 10 days after irradiation. The treatment of mice with ZOE ameliorated gastrointestinal tract (GI) injury as shown by an increase in their 10-day survival. The lowest mortality was observed in the animals treated with 20 mg/kg (12%) followed by 10 mg/kg ZOE (25%). A significant elevation in the 10-day survival was observed for treatment with 10 and 20 mg/kg ZOE (P < 0.001) treatment (Fig. 3).
Treatment of mice with ZOE resulted in a drug dose-dependent increase in the survival of irradiated animals at 30 days up to a dose of 10 mg/kg (P < 0.01), where the highest survival (33%) was observed (Fig. 3). An increase in the drug dose to 20 and 40 mg resulted in a 13% (P < 0.002) and a 21% reduction in the survival compared to 10 mg/kg ZOE (Fig. 2).
Selection of Route of Administration
Greater survival was seen for intraperitoneal than oral administration (Figs. 4, 5). Therefore, intraperitoneal administration was used for all subsequent studies.
Selection of Dosing Schedule of ZOE
The daily administration of 10 mg/kg ZOE for 5 consecutive days (cumulative dose of 50 mg/kg) was found to be better than a single administration of 50 mg or 25 mg daily for 2 consecutive days since it afforded greater protection against both the GI and hemopoietic syndromes (Figs. 6, 7). Further studies were carried out using this regimen.
Radioprotective Effect of ZOE
The radioprotective action of ZOE was evaluated by using the optimum dose of 10 mg/kg before exposure of the mice to 6, 7, 8, 9, 10, 11 or 12 Gy of γ radiation. Irradiation of the mice with different doses of γ radiation resulted in the development of symptoms of radiation sickness within 2–4 days after exposure. Higher radiation doses resulted in the early onset of the symptoms. Facial edema was observed after 9 Gy or more. Some animals exposed to higher radiation doses exhibited difficulty in locomotion during the second week after exposure. The severity of the symptoms increased with increasing radiation dose.
Whole-body irradiation of mice with 6 Gy did not induce mortality. However, with increasing dose of radiation, survival decreased in a dose-dependent manner until a nadir was reached at 10 Gy, where no survivors were observed beyond 16 days postirradiation (Fig. 8). As the dose increased, the time of mortality advanced (Fig. 8). The LD50/30 was 8.2 Gy for this group. Pretreatment with 10 mg/kg ZOE delayed or reduced the severity of radiation sickness and delayed the onset of mortality by 4–5 days compared to the concurrent double-distilled water + radiation group. Deaths from the GI syndrome were fewer compared to the double-distilled water + radiation group for all doses (Fig. 9a). The number of survivors increased significantly in the ZOE + radiation group at 30 days postirradiation (P < 0.05 for 9 Gy and P < 0.01 for 10 Gy) compared to the concurrent double-distilled water + radiation group (Fig. 9b). The LD50/30 was 9.4 Gy, an increase of 1.4 Gy, giving a dose reduction factor of 1.1 (Fig. 9b).
Glutathione
The administration of ZOE alone before sham irradiation did not alter the glutathione content. Irradiation resulted in a significant and dose-dependent decline in the GSH contents in the double-distilled water + radiation group. ZOE treatment before irradiation elevated the GSH content significantly (P < 0.01, 0.005) compared to the concurrent double-distilled water + radiation group.
Lipid Peroxidation
Administration of ZOE before sham irradiation did not increase lipid peroxidation. Irradiation increased the lipid peroxidation in a dose-dependent manner in both the double-distilled water + radiation and ZOE + radiation groups. Pretreatment with ZOE significantly (P < 0.02, 0.001) reduced the induction of lipid peroxidation, thereby protecting against radiation-induced lipid peroxidation at all the doses studied. In spite of the reduction in the lipid peroxidation by ZOE, the lipid peroxidation levels were above control levels at day 31 postirradiation (Fig. 10b).
Antimicrobial Activity
The zones of inhibition for the alcoholic extract of ginger extract are shown in Table 1. The ginger extract showed low to moderate inhibitory activity on enteric bacteria and Candida albicans.
DISCUSSION
Dietary ingredients may be very useful if they protect against the deleterious effects of ionizing radiation. Ginger is a dietary ingredient that possesses several medicinal properties (3–5). The aim of the present study was to evaluate the radioprotective effect of ginger in mice exposed whole-body to different doses of γ radiation.
The exposure of animals to γ radiation resulted in radiation-induced sickness and mortality; the higher doses killed all the animals within 10 days, in agreement with earlier reports (14, 16, 24). In mice, death within 10 days postirradiation is due to gastrointestinal damage (14, 16, 25). The bone marrow stem cells are more sensitive to radiation damage than the intestinal crypt cells, but the peripheral blood cells have a longer transit time than the intestinal cells. Hence the gastrointestinal syndrome appears earlier than the bone marrow syndrome. In mice, death from 11 to 30 days postirradiation is due to the hemopoietic damage (14, 25).
The radioprotective effect of ZOE increased in a dose-dependent manner up to 10 mg/kg (once daily for 5 consecutive days); above that dose, the radioprotective effect declined. Earlier studies on radioprotection showed similar effects for other agents (11, 14, 16, 26). The reason may be that above a particular concentration, a compound may start manifesting its toxic effects (11, 14, 16, 26). Oral administration at an equimolar dose did not afford significant protection. This may be due to the faster absorption of ZOE through the i.p. route. A higher oral dose of ZOE may be required to produce blood levels required for radioprotection, since a significant amount may be lost during the actions of various enzymes in the digestive tract. A similar observation has also been reported for Ocimum sanctum (14).
Treatment with ZOE once daily for consecutive 5 days was superior to the single- or two-dose administration of 25 mg for radioprotection, which is in good agreement with the earlier findings for Moringa oleifera (27). The increased survival of mice after 10 mg/kg ZOE after various radiation doses indicates the effectiveness of ginger in arresting deaths from both the GI and bone marrow syndromes. This reduction in death from the GI syndrome may be due to the protection of the intestinal epithelium. Ginger has been reported to ameliorate chemotherapy-induced nausea and vomiting and to protect against gastrointestinal hemorrhage in humans (28, 29). Ginger and its active constituents β-sesquiphellendrene, β-bisabolene, ar-curcumene, 6-shogaol, zingiberene, terpenoid, 6-gingerol and 6-gingesulfonic acid have been reported to inhibit experimentally induced gastric ulcerations in rats (30–34). The activities of ginger may have helped to protect the intestinal epithelium, allowing better absorption of nutrients, and resulting in the prolongation of life.
The hemopoietic syndrome is induced at low doses of radiation and is manifested by hemopoietic stem cell depletion and ultimately by the depletion of mature hemopoietic and immune cells. The reduction in deaths from bone marrow syndrome by ZOE may be the result of protection of the stem cell compartment of the bone marrow. The LD50 was increased to 9.4 Gy, with a DRF of 1.15.
As far as we are aware, this is the first report of a radioprotective effect of ginger. However, other plants including Panax ginseng (35), Ocimum sanctum (14) and Mentha arvensis (16) have been reported to protect mice against radiation-induced mortality. A direct comparison of the present findings with polyherbal preparations like triphala, cystone and abana (36–38) is not possible because of the presence of several plant ingredients, including ginger, in these drugs. The present findings are in good agreement with our earlier findings on mint and Ocimum sanctum, where optimum radioprotection was observed with a similar dose schedule and route of administration (14, 16).
The observed antimicrobial activity of ZOE is in good agreement with the earlier reports on ginger (5, 39). The antimicrobial activity of ZOE may be due to the presence of terpenes in it, which have also been reported to be antibacterial (40, 41). The inhibition of protein, DNA and RNA synthesis and cell wall synthesis and disruption of plasma membrane are the principal mechanisms of microbial killing (42). The antimicrobial activity of ZOE may be due to one or all of these pathways. Other plants such as Ocimum sanctum and mint that show antimicrobial activity (5, 12, 16) have also been reported to be radioprotective.
The mechanism of action of ZOE is not known. Free radical scavenging is a common mechanism of radioprotection. Ginger and its volatile oil have been reported to scavenge free radicals like superoxide anion and H2O2 in vitro (43, 44). The aqueous and non-aqueous extracts of ginger along with curcuminoids and cassumunin A and B that are present in it have been reported to be antioxidants (45, 46). Unlike most antioxidants and free radical scavengers, ginger has been found to be anti-inflammatory (47).
One of the peaks in the chromatogram of ZOE corresponds to gingerol, which may have contributed to the observed radioprotective action. This possibility is supported by the observation that gingerol is an antioxidant, an anti-inflammatory agent, and a scavenger of superoxide anion (7, 48, 49). Ginger has also been reported to contain sulfonated compounds like 4-gingesulfinic and shagosulfonic acids (50), which may also be responsible for observed radioprotection.
Ionizing radiation induces lipid peroxidation, which can lead to DNA damage and cell death (51–53). Therefore, an agent that protects against such damage can provide protection against radiation damage. The administration of ZOE significantly reduced the amount of lipid peroxidation compared to the concurrent control groups. This inhibition of lipid peroxidation by ginger extract may also have been responsible for the observed radioprotection. Ginger and its constituent compound 6-dehydrogingerdione have been found to inhibit the induction of lipid peroxidation in vitro (54–58).
The significant elevation in the GSH level by ZOE at all radiation doses may be responsible for the scavenging of radiation-induced free radicals, including lipid peroxidation, and thereby protecting against radiation-induced mortality. Ginger has been reported to elevate GSH levels in mice and rats and to reduce lipid peroxidation (55). It has also been found to increase the acid-soluble sulfhydryl, glutathione-S-transferase and aryl hydrocarbon hydroxylase, superoxide dismutase, catalase and glutathione peroxidase in experimental animals (55, 57). Several investigators have reported that lipid peroxidation starts as soon as the endogenous GSH is exhausted, and that the addition of GSH promptly stops further peroxidation (58).
In conclusion, ginger provided protection against radiation-induced mortality in mice by protecting against the GI and bone marrow syndromes. The free radical scavenging, elevation in antioxidant status, and reduction in lipid peroxidation appear to be the important mechanisms of radioprotective action by ginger.
Acknowledgments
We thank Prof. M. S. Vidyasagar, and Dr. J. Velumurugan, Department of Radiotherapy and Oncology, Kasturba Medical College, Manipal, India, for providing the necessary irradiation facilities and radiation dosimetry. We also thank Prof. P. G. Shivananda and Dr. Mamatha Ballal, Department of Microbiology, for their assistance and help in carrying out antimicrobial studies. We thank Prof. N. Udupa, Dean, and Mr. Subramanyam, College of Pharmaceutical Sciences, Manipal, for analyzing the ginger extract with HPLC. Thanks are also due to Prof. Gopalkrishna Bhat Poorna Prajna College Udupi, Karnataka, for identifying the plants. Financial support from the Indian Council for Medical Research, Govt. of India, New Delhi, is gratefully acknowledged.
REFERENCES
FIG. 2.
Kaplan-Meier survival curves for mice treated with various doses of ginger, 10 mg/kg Ocimum extract, or 20 mg/kg MPG before exposure to 10 Gy γ radiation
FIG. 3.
Effect of ZOE, OSE (10 mg/kg) or MPG (20 mg/kg) on the survival of mice exposed to 10 Gy of γ radiation. Solid bars, 10-day survival; mottled bars, 30-day survival. IR = irradiation, SIR = sham irradiation. Error bars are 95% confidence limits
FIG. 4.
Effect of route of administration on the Kaplan-Meier survival curves of mice treated with 10-mg/kg ZOE before exposure to 10 Gy γ radiation
FIG. 5.
Effect of route of administration on the survival of mice exposed to 10 Gy of γ radiation. Solid bars: 10-day survival: mottled bars: 30-day survival. IR = irradiation, SIR = sham irradiation. Error bars are 95% confidence limits
FIG. 6.
Effect of different dosing schedules on the Kaplan-Meier survival curves for mice treated with 10 mg/kg ZOE before exposure to 10 Gy γ radiation. IR = irradiation, SIR = sham irradiation
FIG. 7.
Effect of drug dose schedule of 50 mg/kg ZOE on the survival of mice exposed to 10 Gy of γ radiation. Solid bars: 10-day survival; mottled bars: 30-day survival. IR = irradiation, SIR = sham irradiation. Error bars are 95% confidence limits
FIG. 8.
Kaplan-Meier estimate of survival of mice treated with 10 mg/kg ZOE before exposure to various doses of radiation. Panel a: 7 Gy; panel b: 8 Gy; panel c: 9 Gy, panel d: 10 Gy, panel e: 11 Gy, panel f: 12 Gy. Double-distilled water + radiation (squares); ZOE + radiation (circles)
