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British Journal of Radiology (2004) 77, 1027-1035
© 2004 British Institute of Radiology
doi: 10.1259/bjr/83720350
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Full Paper

Radiprotective effect of abana, a polyherbal drug following total body irradiation

M S Baliga1, G C Jagetia1, P Venkatesh1, R Reddy2 and J N Ulloor3

1 Department of Radiobiology, Kasturba Medical College, Manipal-576 104, 2 College of Pharmaceutical Sciences, Manipal-576 104, Karnataka, India and 3 Department of Psychiatry and Program in Behavioral Neuroscience, Boston University School of Medicine, Boston, MA, USA

Correspondence: Dr Ganesh Chandra Jagetia, Professor and Head of the Department of Radiobiology, Kasturba Medical College, Manipal-576 104, Karnataka, India


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effects of 20 mg/kg body weight of abana (ABE) on radiation-induced sickness and mortality in mice exposed to 7 Gy to 12 Gy of gamma irradiation were studied. Treatment of mice with abana 1 h before irradiation delayed the onset of mortality and reduced the symptoms of radiation sickness when compared with the non-drug treated irradiated controls (double distilled water, DDW). Abana provided protection against both the gastrointestinal and haemopoietic deaths. However, animals of both the ABE+irradiation and DDW+irradiation groups did not survive up to 30 days post-irradiation beyond 11 Gy irradiation. The LD50/30 was found to be 8.5 Gy for the DDW+irradiation group and 10.3 Gy for ABE+irradiation group. The administration of abana resulted in an increase in radiation tolerance by 1.8 Gy, and the dose modification factor (DMF) was found to be 1.2. The irradiation of animals resulted in a dose dependent elevation in lipid peroxidation, and a reduction in glutathione (GSH) concentration on day 31 post-irradiation. Treatment of mice with abana before irradiation caused a significant depletion in lipid peroxidation followed by a significant elevation in GSH concentration in the liver of mice at day 31 post-irradiation. Abana scavenged •OH, DPPH, ABTS•+ and NO• in a concentration dependent manner in vitro. Our results indicate that the radioprotective activity of abana may be due to free radical scavenging and increased GSH level in irradiated mice.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The observation by Patt et al [1] that pre-treatment of rats with cysteine protected them against X-ray-induced mortality opened a new field of research in radiation biology. Subsequently, several chemical compounds were synthesized and tested for their radioprotective properties and the sulphydryl compounds were found to be good radioprotectors. However, the practical applicability of the of majority of thiol compounds remained limited, due to their high systemic toxicity at the optimum protective doses [2]. The high toxicity of thiol compounds necessitated a search for alternative compounds, which could be less toxic and highly effective at non-toxic doses.

The traditional Indian system of medicine, the Ayurveda, gives a detailed account of several diseases and their treatments. The majority of drugs and/or drug formulations used in Ayurveda are principally derived from herbs and plants. Studies carried out in the past decade and a half have shown that herbal preparations like Liv. 52, protected mice against radiation-induced sickness, mortality, dermatitis, spleen injury, liver damage, decrease in peripheral blood cell counts, pre-natal development, lipid peroxidation and radiation-induced chromosome damage [3–6]. The other Ayurvedic preparations like brahmarasayana, narasimharasayana, ashwagandharasayana and amrithaprasham, a group of herbal preparations used to improve the general health, have also been reported to reduce radiation-induced leucopenia and lipid peroxidation in mouse liver [7]. Recently, some other herbal drugs like triphala, cystone, mentat and chyavanaprasha have been reported to protect mice against radiation-induced lethality [8–11].

Abana, is a herbal formulation, which is used clinically in India as a cardioprotective agent. It has been reported to reduce hypertension [12–14] and other cardiovascular diseases in man [15–18]. It has also been reported to inhibit platelet aggregation [19]. Earlier studies have shown that abana protected mouse bone marrow cells against radiation-induced micronuclei formation and reduced radiation-induced mortality [20, 21]. However, information on the mechanism of action by which abana protects and the dose modification factor (DMF) is lacking. The DMF is an important aspect in radiobiology as it clearly gives an indication of the quantitative capacity of the drug to enhance the tolerance of tissues to radiation and its ability to mitigate radiation-induced sickness and mortality. Therefore, the present study was undertaken to obtain an insight into the mechanism of action and also to evaluate the effects of abana on radiation-induced mortality in mice whole body exposed to different doses of gamma-radiation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals
Dimethyl sulphoxide (DMSO), deoxyribose, ethylene diamine trichloroacetic acid (EDTA), ascorbic acid, nitoblue tetrazolium (NBT), 1,1-diphenyl-2-picryl hydrazyl (DPPH), sodium nitroprusside, Greiss reagent, 2,2-azinobis (3-ethyl benzothiazoline-6-sulphonic acid) diammonium salt (ABTS), trichloro-acetic acid (TCA), thiobarbituric acid (TBA), 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) were procured from Sigma Chemical Co., St. Louis, USA. Ferric chloride, sodium bicarbonate, hydrochloric acid (HCl), sodium chloride, potassium hydrogen phosphate, disodium hydrogen phosphate, potassium chloride and hydrogen peroxide were procured from Ranbaxy Fine Chemicals, New Delhi, India.

Composition of abana
The abana (Himalaya Drug Co., Bangalore) is a mixture of several medicinal plants in defined proportions. The abana consist' of Terminalia arjuna (Roxb. Ex DC) Wight & Arn., Combretaceae (30 mg); Withania somnifera (Linn.) Dunal, Solanaceae (20 mg); Nepata hindostana (Roth.) Haines, Labiatae (20 mg); Asparagus racemosus Willd, Liliaceae (10 mg); Tinospora cordifolia (Willd.) Miers ex Hook, f & Thoms, Menispermaceae (10 mg); Centella asiatica (Linn.) Urban, Apiaceae (10 mg); Glycyrrhiza glabra Linn., Fabaceae (10 mg); Terminalia chebula Retz, Combretaceae (10 mg); Phyllanthus embelica (Linn.) Gaertn, Euphorbiaceae (10 mg); Boerhaavia diffusa Linn., Nyctaginaceae (10 mg); Convolvulus pluricaulis Choisy, Convolvulaceae (10 mg); Ocimum sanctum Linn., Labiatae (10 mg); Nardostachys jatamansi D C., Valerianaceae (10 mg); Piper longum Linn., Piperaceae (10 mg); Carum copticum Hiern, Apiaceae (10 mg); Zingiber officinale Rosc., Zingiberaceae (10 mg); Eclipta alba (Linn.) Hassak, Asteraceae (10 mg); Cyperus rotundus Linn., Cyperaceae (5 mg); Acorus calamus Linn., Araceae (5 mg); Embelia ribes Burm. F., Myrsinaceae (5 mg); Syzygium aromaticum (Linn.) Merrill and Perry, Myrtaceae (5 mg); Celastrus paniculatus Linn., Celastraceae (5 mg); Santalum album Linn., Santalacae (5 mg); Elettaria cardamomum Maton, Zingerberaceae (5 mg); Foeniculum vulgare Mill, Apiaceae (5 mg); Rosa damascena Mill. Rosaceae (5 mg); Cinnamomum cassia Blume, Lauraceae (5 mg); Crocus sativus Linn., Iridaceae (2 mg); Aloe vera (Linn.) Burm., Liliaceae (1 mg); Daucus carota Linn., Apiaceae (1 mg); Nelumbium speciosum Gaertn. Nymphaeaceae (1 mg); Punica granatum Linn. Punicaceae (1 mg) and Pyrus malus Linn. Rosaceae (1 mg).

Preparation of the extract
The extract of abana (ABE) was prepared by extracting 100 g of abana powder in 50% ethanol (l l) at 50–60°C in a Soxhlet apparatus for 72 h as described earlier [20, 21]. The cooled liquid extract was concentrated by evaporating its liquid contents in vacuo. An approximate 25% yield of the extract was obtained.

Radioprotective effect
Animal care and handling
The animal care and handling were done according to the guidelines set by the World Health Organization (WHO), Geneva, Switzerland and the INSA (Indian National Science Academy, New Delhi). 8 to 10 week old male Swiss albino mice weighing 30 g to 36 g were selected from an inbred colony maintained under controlled conditions of temperature (23±2°C), humidity (50±5%) and light (10 h and 14 h of light and dark, respectively). The animals were provided with sterile food and water ad libitum. Five to six animals were housed in a polypropylene cage containing sterile paddy husk (procured locally) as bedding throughout the experiment. The animals were euthenized on a daily basis to avoid unnecessary suffering during the examination period according to WHO guidelines.

Preparation of drug and mode of administration
The required amount of abana was dissolved in double distilled water (DDW) and administered intraperitoneally once daily for 5 consecutive days [20, 21]. The animals were divided into the following groups:

DDW+irradiation
The animals of this group were administered with 0.01 ml/g body weight of sterile DDW intraperitoneally.

ABE+irradiation
The animals of this group were injected intraperitoneally with 10 mg/kg body weight of abana once daily consecutively for 5 days [20, 21].

Irradiation
1 h after the last administration of DDW or ABE on the fifth day, the prostrate and immobilized animals (achieved by inserting cotton plugs in the restrainer) were whole-body exposed to 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy and 12 Gy of 60Co gamma radiation (Theraton, Atomic Energy Agency, Ontario, Canada) in a specially designed well ventilated acrylic box. A batch of 10 animals was irradiated each time at a dose rate of 1.33 Gy min–1 at a source to animal distance (midpoint) of 102 cm. Immediately after irradiation, the animals were sorted into individual polypropylene cages. The animals of both groups were monitored daily for the development of symptoms of radiation sickness and mortality. A total of 18 animals were used for each dose of radiation in each concurrent group. The dose reduction factor (DRF) was calculated by the method of Miller and Tainter [22]. Go


{771027E001}

Biochemical estimations
The animals that survived up to 30 days after exposure were euthenized on day 31 post-irradiation for estimation of glutathione (GSH) and lipid peroxidation (LPx). After removal of the spleen, the animals were perfused with ice cold saline transcardially. The whole liver from each surviving animal was removed, blot dried, weighed and a 10% homogenate was prepared in ice-cold 0.2 M sodium phosphate buffer pH 8.0 using a homogenizer (Yamato LSG LH-21, Japan). Four survivors from each concurrent group were used for the estimation of GSH and LPx at each irradiation dose.

Glutathione (GSH)
GSH content was measured by the method of Moron et al [23]. Briefly, proteins were precipitated by 25% TCA, centrifuged and the supernatant was collected. The supernatant was mixed with 0.2 M sodium phosphate buffer pH 8.0 mM and 0.06 mM DTNB and incubated for 10 min at room temperature. The absorbance of the sample/s was read against the blank at 412 nm in an ultraviolet-visible light (UV-Vis) double beam spectrophotometer (UV-260; Shimadzu Corporation, Tokyo, Japan) and the GSH concentration was calculated from the standard curve.

Estimation of protein
Total proteins, were estimated by the method of Lowry et al [24] using bovine serum albumin as standard.

Lipid peroxidation (LPx)
LPx was measured by the method of Beuege and Aust [25]. Briefly, tissue homogenate was mixed with TCA-TBA-HCl and was heated for 15 min in a boiling water bath. After centrifugation the absorbance was recorded at 535 nm using a UV-Vis double beam spectrophotometer. The LPx has been expressed as MDA in nM per mg protein.

Free radical scavenging
Separate experiments were carried out to evaluate the free radical scavenging activity of abana in vitro.

Hydroxyl radical scavenging activity
The scavenging of the hydroxyl free radical (•OH) was estimated by the method of Halliwell et al [26]. Briefly, the reaction mixture contained deoxyribose (2.8 mM), KH2PO4–NaOH buffer, pH 7.4 (0.05 M), FeCl3 (0.1 mM), EDTA (0.1 mM), H2O2 (1 mM), ascorbate (0.1 mM) and ABE (10–500 µg ml–1) in a final volume of 2 ml. This was incubated for 30 min at ambient temperature followed by the addition of 2 ml of trichloroacetic acid (2.8% W/V) and thiobarbituric acid. The reaction mixture was kept in a boiling water bath for 30 min, cooled and the absorbance was read at 532 nm in a UV-Vis double beam spectrophotometer.

DPPH scavenging activity
The principle for the reduction in DPPH free radicals is that, the antioxidant reacts with stable free radical, DPPH and converts it to 1,1-diphenyl-2-picryl hydrazine. The ability to scavenge the stable free radical DPPH is measured by decrease in the absorbance at 517 nm [27]. To the ethanolic solution of DPPH (0.05 mM), an equal volume of ABE (10–500 µg ml–1) dissolved in water, was added to a final volume of 1.0 ml. An equal amount of methanol was added to the control. After 20 min, absorbance was recorded at 517 nm in a UV-Vis double beam spectrophotometer.

Total antioxidant activity assay
Total antioxidant potential was determined by ABTS (2,2-azinobis (3-ethyl benzothiazoline-6-sulphonic acid) diammonium salt) assay described earlier [28]. This technique measures the relative ability of antioxidant substances to scavenge the ABTS•+radical cation generated in the aqueous phase. The reaction mixture contained ABTS (0.00017 M), ABE (10–500 µg ml–1), and buffer in a total volume of 3.5 ml. The absorbance was measured at 734 nm UV-Vis double beam spectrophotometer.

Nitric oxide scavenging activity
Nitric oxide was generated from sodium nitroprusside and measured by the Greiss reaction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide [29, 30], which interacts with oxygen to produce nitrite ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide [29, 31]. Sodium nitroprusside (5 mM) in phosphate-buffered saline was mixed with different concentrations of ABE (20–400 µg ml–1) and incubated at 25°C for 150 min. The samples from the above were allowed to react with Greiss reagent (1% sulphanilamide, 2% H3PO4 and 0.1% napthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with napthylethylenediammine was read at 546 nm and referred to the absorbance of standard solutions of potassium nitrite treated in the same way with Griess reagent.

Statistical analysis
All the data are expressed as mean±SEM (standard error of the mean). The Student's t-test was used for the free radical scavenging and for the biochemical assay (GSH and LPx). The "Z" test [32] was used for survival studies using the following formula: Go


{771027E002}

where {771027E003}


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Radioprotective effect
The animals of DDW+irradiation group exhibited signs of radiation sickness within 2–4 days after exposure to different doses of gamma-radiation depending on the irradiation dose. Exposure of mice to higher radiation doses resulted in an early appearance of the signs. They mainly included reduction in the food and water intake, irritability, epilation, weight loss, emaciation, lethargy, diarrhoea and ruffling of hairs. A few animals also exhibited facial oedema between 1 and 2 weeks after exposure to doses above 10 Gy. Some of the animals exhibited paralysis and difficulty in locomotion during the second week after exposure to doses above 9 Gy. The severity of the symptoms increased and advanced with increase in radiation dose (Figure 1Go).



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  		Figure 1. Kaplan-Meir's estimate of survival of mice treated with 20 mg kg–1 body weight of abana before exposure to various doses of gamma-radiation. (a) 8 Gy, (b) 9 Gy, (c) 10 Gy, (d) 11 Gy and (e) 12 Gy of radiation. {blacksquare}: double distilled water+irradiation; {circ}: abana+irradiation.

 
The results are expressed as percent survival after exposure to various doses of gamma radiation. The whole body irradiation of mice to 7 Gy did not induce mortality in both the groups (Figure 2Go). However, with a further increase in exposure dose, the survival declined in a dose dependent manner until a nadir was reached at 11 Gy and thereafter, where no survivors were reported beyond 19 days post-irradiation. The increase in exposure dose resulted in an advancement in the onset of mortality due to gastrointestinal (GI) deaths (Figures 1 and 2GoGo). The survival was plotted against exposure dose on a semi-log plot and the LD50/30 was found to be 8.5 Gy for the DDW+irradiation group (Figure 3Go).



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  		Figure 2. Effect of 20 mg kg–1 of abana on the radiation-induced mortality on day 10 post-irradiation in the mice exposed to different doses of gamma-radiation. {blacksquare}: double distilled water+irradiation and •: abana+irradiation (95% confidence limits).

 


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  		Figure 3. Effect of 20 mg kg–1 of abana on the radiation-induced mortality on day 30 post-irradiation in the mice exposed to different doses of gamma-radiation. {blacksquare}: double distilled water+irradiation and •: abana+irradiation (95% confidence limits).

 
Treatment of mice with 20 mg kg–1 ABE before irradiation delayed or reduced the severity of radiation sickness and also decreased radiation-induced mortality when compared with the concurrent DDW+irradiation group. ABE treatment protected mice against both GI and haemopoietic deaths as evidenced by the greater number of survivors at 10 days and 30 days post-irradiation when compared with the concurrent DDW+irradiation group. ABE treatment increased 10 day survival by 1.3-fold, 3.3-fold, and 5.7-fold for 9 Gy, 10 Gy and 11 Gy, respectively, when compared with DDW+irradiation. The administration of ABE increased the survival for 12 Gy irradiation, where approximately 8% survivors were observed, up to 10 day post-irradiation as against no survivors in DDW+irradiation group (Figure 2Go).

Similarly, ABE treatment increased the 30 day survival in mice exposed to different doses of gamma-radiation. This increase in survival was statistically significant for 9 Gy (p<0.0001) and 10 Gy (p<0.002) exposure when compared with the concurrent DDW+irradiation group. ABE pre-treatment increased animal survival by approximately 17% after exposure to 11 Gy, while no survivors were observed by 30 days post-irradiation in the concurrent DDW+irradiation group. The LD50/30 was found to be 10.3 Gy, resulting in an increase of 1.8 Gy when compared with the DDW+irradiation. The DRF was found to be 1.2 (Figure 3Go).

Glutathione
The results are expressed as GSH µmol g–1 tissue (Table 1Go). GSH concentration remained unaltered in the DDW+sham-irradiation group. Similarly, the administration of ABE alone before sham-irradiation did not alter the GSH concentration. The exposure of animals to different doses of gamma-radiation caused a significant and dose dependent decline in GSH concentration in the DDW+irradiation group, while ABE pre-treatment elevated GSH concentration significantly when compared with the concurrent DDW+irradiation group. In spite of the significant elevation in GSH concentration by ABE, the levels were below normal in both the DDW+irradiation and ABE+ irradiation groups (Figure 4aGo).


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  		Table 1. Effect of treatment with abana on the glutathione and lipid peroxidation levels in the live of the mice exposure to different doses of gamma-radiation

 


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  		Figure 4. Alteration in the (a) glutathione and (b) lipid peroxidation levels in the liver of mice treated with 20 mg kg–1 abana before exposure to different doses of gamma-radiation.

 
Lipid peroxidation
Lipid peroxidation is expressed in terms of nmol MDA/mg protein (Table 1Go). LPx remained unaltered in DDW+sham-irradiation group. Treatment of mice with ABE alone did not alter the LPx in the DDW+sham-irradiation group (Table 1Go). The irradiation of mice to various doses of gamma-radiation induced LPx in dose dependent manner in the DDW+irradiation group and a peak level was observed at 10 Gy irradiation. ABE pre-treatment significantly reduced the LPx in the ABE+irradiation group thereby protecting against radiation-induced lipid peroxidation at all the exposure doses studied. In spite of the decline in the LPx by abana, the LPx was higher than the DDW+sham-irradiated controls (Figure 4bGo).

Free radical scavenging
Scavenging of •OH radical
ABE inhibited the generation of •OH radicals in a concentration dependent manner. The lowest concentration, i.e. 10 µg ml–1, was a poor scavenger of •OH radicals, while with increasing ABE concentration, the radical scavenging also increased and approximately 53% scavenging of •OH was observed at 200 µg ml–1 ABE. The highest scavenging of 77.7% was observed for a dose of 500 µg ml–1 ABE, the highest concentration employed (Figure 5aGo).



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  		Figure 5. Influence of various doses of abana (ABE) on the scavenging of different free radicals in vitro. (a) Hydroxyl, (b) DPPH, (c) ABTS and (d) NO.

 
DPPH scavenging
ABE treatment inhibited the generation of DPPH in a dose dependent manner by ABE treatment. The DPPH scavenging activity increased gradually but steadily with increasing ABE concentration and 100 µg ml–1 ABE scavenged more than 50% of DPPH radicals (Figure 5bGo). A further increase in ABE concentration resulted in an elevation in DPPH scavenging activity and maximum scavenging was observed at 400 µg ml–1. A further increase in ABE concentration did not significantly increase the scavenging of DPPH radical (Figure 5bGo).

The total antioxidant activity
The total antioxidant activity was measured by ABTS assay. ABE caused a concentration dependent inhibition of ABTS•+ radicals. An almost 50% inhibition of ABTS radicals was observed at a concentration of 80 µg ml–1 ABE (Figure 5cGo). Further increase in ABE concentration resulted in a gradual increase in the antioxidant activity and an almost saturation levels were observed at 300 µg ml–1 with a marginal increase thereafter (Figure 5cGo).

Nitric oxide scavenging
Abana treatment caused a concentration dependent inhibition of NO generation in vitro. This inhibition of NO was gradual and 61% NO generation was inhibited at a concentration of 200 µg ml–1. A further increase in the abana concentration inhibited NO generation with increasing concentration up to 400 µg ml–1, thereafter a marginal increase was seen (Figure 5dGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
There is a continued interest in and need for the identification and development of non-toxic and effective radioprotective compounds that can reduce the effect of radiation. Such compounds could potentially protect humans against the genetic damage, mutation, alteration in the immune system and teratogenic effects of toxic agents including radiation, which act through the generation of free radicals.

A single whole-body exposure of mammals to ionizing radiation results in a complex set of syndromes whose onset, nature and severity are a function of both total radiation dose and radiation quality. At the cellular level, ionizing radiation can induce damage in biologically important macromolecules such as DNA, proteins, lipids and carbohydrates in various organs. While some damage may be expressed early, the other may be expressed over a period of time depending upon cell kinetics and radiation tolerance of the tissues. Proliferating cells are highly sensitive to irradiation, therefore, the effect of whole body irradiation is mainly felt by the highly proliferating germinal epithelium, gastrointestinal epithelium and the bone marrow progenitor cells. Of these the germinal epithelium does not have a life supporting function to the exposed individual, while the gastrointestinal epithelium and the bone marrow progenitor cells are crucial for the sustenance of life and any damage to these cells will impair the normal physiological processes drastically, causing an adverse impact on survival. The gastrointestinal epithelium is less sensitive than the bone marrow progenitor cells, but as the cell transit time is quick, it is expressed earlier than the haemopoietic syndrome. In mice death within 10 days post-irradiation is due to gastrointestinal damage, while death between 11 days and 30 days post-irradiation is due to haemopoietic damage inflicted by radiation [8–11, 33, 34].

Treatment of mice with ABE before irradiation to different doses of gamma-radiation reduced the symptoms of radiation sickness and mortality. This clearly indicates the effectiveness of abana in arresting GI death, where the number of survivors for all the treatment groups was higher than that of the DDW+irradiation group. The administration of 20 mg kg–1 ABE resulted in the protection of mice and this reduction in GI death may also be due to the protection of intestinal epithelium, which would have allowed proper absorption of the nutrients. Terminalia chebula and Glycyrrhiza glabra (part of ABE formulation) have been reported to reduce cysteamine-induced duodenal ulcers in rats by increasing the beta-glucuronidase activity in the Brunner's glands [35]. Terminalia chebula and Glycyrrhiza glabra have also been reported to protect epithelial cells against the cytopathic effects of influenza A virus and indomethacin-induced gastric ulcers in rats [36, 37]. The antimicrobial and antiviral activity of ABE and its constituent plants may have played some role in protecting mice against the radiation-induced GI deaths [38, 39].

Treatment of mice with abana significantly reduced bone marrow deaths in the ABE+irradiation group. The bone marrow cells have been reported to be protected against radiation-induced damage by various other plants [3–5, 7–10, 20]. The constituent plant extracts of Ocimum sanctum and Tinospora cordifolia have also been reported to ameliorate the radiation or cyclophosphamide-induced damage in mice [40, 41]. The other plant constituents of ABE like Embelica officinales, Withania somnifera, Boerhaavia diffusa and Rubia cordifolia, have been reported to be rejuvenating, stress reducers, immunomodulator and are widely used to treat general debility [42–46]. Some other radioprotective compounds like ginosan, melatonin and 16,16-dimethyl prostaglandin E2 have been reported to be immunomodulators and this property of ABE would have been also helped to protect mice against the radiation-induced mortality.

The exact mechanism by which ABE protects against radiation-induced damage is not fully understood. Since ABE scavenged free radicals like •OH, DPPH and NO• in a concentration dependent manner in vitro therefore free radicals scavenging seems to be one of the important mechanism of radioprotection by ABE. Another mechanism may be elevation of antioxidant status by ABE, since it has inhibited the generation of the ABTS•+ radical in vitro. The raised GSH may have been responsible for the protection against radiation-induced mortality.

Depletion of intracellular GSH has been implicated as one of the causes of radiation-induced damage, while increased levels of intracellular GSH are responsible for the radioprotective action. ABE pre-treatment helped to restore GSH levels when compared with the concurrent DDW+irradiation group. This resulted in the inhibition of radiation-induced lipid peroxidation, thereby protecting against radiation damage. Ionizing radiation induces lipid peroxidation, which can lead to DNA damage and cell death [47, 48]. Some of the plants, which are part of abana formulations like Santalum album [49], Embelia ribes [50], Piper longum, Zinger officinale, Syzygium aromaticum, Elettaria cardamum [51], Rubia cordifolia [52] and Ocimum sanctum [53] have been reported to increase GSH. While the other plants used in abana like Asparagus racemosus, Glycyrrhiza glabra, Phyllanthus embelica, Boerrhaavia diffusa, Ocimum sanctum, Eclipta alba have been found to possess antioxidant properties [54].

Therefore an agent that reduces lipid peroxidation can provide protection against radiation-induced damage. ABE reduced mortality. Ginger and bael extracts have been found to protect against radiation-induced lipid peroxidation [34, 55]. The other herbal drugs like the Liv. 52, brahmarasayana, narasimharasayana, ashwagandharasayana and amrithaprasham, have also been found to protect against radiation-induced damage by inhibiting the radiation-induced lipid peroxidation [6, 7].

In conclusion abana pre-treatment reduced radiation-induced sickness and mortality in mice by protecting against the radiation-induced GI and bone marrow damage. Free radical scavenging, elevation in antioxidant status and GSH and reduction in lipid peroxidation appear to be important mechanisms of radioprotection.


   Acknowledgments
 
The authors are grateful to Himalaya Drug Co., Bangalore, India for providing the abana powder as a free gift to carry out the study. We also 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 help in radiation dosimetry, respectively.

Received for publication October 2, 2003. Revision received May 18, 2004. Accepted for publication July 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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