Printer Friendly
The Free Library
22,728,043 articles and books

Radioprotection by a herbal preparation of Hippophae rhamnoides, RH-3, against whole body lethal irradiation in mice.



Summary

Purpose: Hippophae rhamnoides L. has been well documented to have anti-oxidative, immunostimulative and regenerative properties and therefore a herbal preparation of H. rhamnoides coded as RH-3 was investigated for its radioprotective action.

Materials and methods: RH-3 was administered intraperitonially (i.p.) to mice 30 minutes before whole body irradiation and whole body survival, spleen Colony forming units (CFU CFU

see colony-forming units.
) and haematological Adj. 1. haematological - of or relating to or involved in hematology
hematologic, hematological
 parameters were studied. To investigate free radical scavenging and antioxidant potential, Fenton reaction, radiation mediated OH radical scavenging and chemically generated superoxide anions scavenging were studied in vitro while inhibition of lipid peroxidation was studied in liver homogenate homogenate /ho·mog·e·nate/ (ho-moj´in-at) material obtained by homogenization.

homogenate

material obtained by homogenization.
 of mice.

Results: A dose of 30 mg/kg body weight of RH-3 rendered 82% survival as compared to no survival in irradiated control. The endogenous CFU counts in mouse spleen on 10th post-irradiation day with and without RH-3 demonstrated radioprotective effect. Various hematological hematological, hematologic

pertaining to or emanating from blood cells.


hematological tests
total and differential white cell counts, hematocrit estimation, erythrocyte count.
 parameters also corroborated the radioprotective effect of RH-3. In a dose dependent manner, RH-3 inhibited Fenton reaction and radiation mediated generation of hydroxyl radicals in vitro, superoxide anion mediated Nitroblue tetrazolium nitroblue tetrazolium

a yellow dye converted to a blue color on reduction.


nitroblue tetrazolium test
used to measure the phagocytic activity of polymorphonuclear leukocytes by the amount of color change in the dye.
 (NBT) reduction and Fe[SO.sub.4] mediated lipid peroxidation in liver.

Conclusion: Free radical scavenging, acceleration of stem cell proliferation and immunostimulation are the radioprotective attributes, which require further investigations.

Key words: Radioprotection ra·di·o·pro·tec·tion
n.
Protection against the harmful effects of radiation.



radi·o·pro·tec
, Hippophae rhamnoides, lethal irradiation, herbal radioprotector, antioxidant

* Introduction

Development of effective and non-toxic radioprotective agents is of considerable interest for radiation medicine, space flights, nuclear industries and emergencies. A large number of chemical and biological agents have been screened and reviewed in this connection (Weiss et al. 1990, Maisin 1992). Several molecular drugs of synthetic and natural origin are being tried in experimental models and human clinical trials to mitigate radiation injury caused by whole body gamma radiation exposure ranging from sub-lethal to supra-lethal doses. Among molecular radioprotectors, WR-2721 (Weiss et al. 1990) and related compounds have been the most promising so far. However, severe side effects such as nausea, vomiting, hypotension and neurotoxicity neurotoxicity /neu·ro·tox·ic·i·ty/ (noor?o-tok-sis´it-e) the quality of exerting a destructive or poisonous effect upon nerve tissue.  (Landauer et al. 1987) associated with most of the radioprotective agents tried at therapeutic levels, have restrained their use. In fact no radioprotective agent available today meets all the requisites of an ideal radioprotector (Maisin 1998). In view of this, search for new er more effective agents is inevitably continuing. Recently a number of plant products have been evaluated for radioprotective action (Shimoi et al. 1994, 1996, Goel et al. 1998, Umadevi et al. 1999). Our hypothesis has been that plant extracts eliciting radioprotective efficacy contain a large number of active constituents like antioxidants, immunostimulants, cell proliferation promoters cytokines etc. Some of them may individually as well as combinely render protection against radiation induced pathology. In this process some toxic effects could be generated which could be countered by several other types of molecules present in the whole extract. Immense interest is, therefore generating in herbal drugs globally.

H. rhamnoides L. (F. Elaeagnaceae), commonly known as sea buckthorn buckthorn, common name for some members of the Rhamnaceae, a family of woody shrubs, small trees, and climbing vines widely distributed throughout the world.  and native to Europe & Asia, is a deciduous shrub, 2-4 m in height, very hardy and salt tolerant. It can withstand -43 to 40 [degrees]C grows on acid alkaline soils with pH of 5.8-8.3 (optimal pH 6-7) and even nutritionally poor soils like river bank steep slopes and is a nitrogen fixing plant. In Tibetean and Indian systems of medicine, for centuries, H. rhamnoides has been exploited for treatment of sluggish digestion, stomach malfunctioning (Nikitin et al. 1989, Xiao et al. 1992), circulatory disorders, ischemic heart disease Ischemic heart disease
Insufficient blood supply to the heart muscle (myocardium).

Mentioned in: Myocarditis

ischemic heart disease 
 (Liu et al. 1988, Zhang 1987), burn and wound healing (Ianev et al. 1995, Nikulin et al. 1992), hepatic injury (Cheng 1992, Cheng et al. 1994) and neoplasia (Nikitin et al. 1989, Luginov et al. 1983). H. rhamnoides contains a large number of constituents like flavonoids flavonoids,
n.pl common plant pigment compounds that act as antioxidants, enhance the effects of vitamin C, and strengthen connective tissue around capillaries.
 (Wu et al. 1994, Zhang 1987, Liu et al. 1988), vitamin A, C, E, and K, tannins (Ianev et al, 1995, Spirodonov et al. 1997) sugars, fats and various t race elements like Se, Zn, Cu, and S (Ianev et al. 1995). Due to these molecules, it can act as strong antioxidant (Wang et al. 1992) and inhibitor of succinate succinate /suc·ci·nate/ (suk´si-nat) any salt or ester of succinic acid.

succinate semialdehyde  ?.


suc·ci·nate
n.
 oxidation (Spirodonov et al. 1997). Radiation exposure induces lipid peroxidation (Von Sonntag 1987) and subsequently disturbs ion homeostasis especially the [Ca.sup.++] influx (Trump et al. 1992). Dilitiazam, a known [Ca.sup.++] channel blocker, could therefore render significant radioprotection (Goel et al. 1996). H. rhamnoides has also been reported to control [Ca.sup.++] influx from extracellular to intracellular component (Wu et al. 1994) and therefore it may also inhibit radiation induced calcium mediated cytotoxicity.

For radioprotection, stimulation of stem cell proliferation, free radical scavenging, antioxidant, calcium channel blocking, immunostimulation and DNA repair enhancement are considered essential. This plant since has been demonstrated to have such properties, RH-3, a preparation of H. rhamnoides, was therefore investigated for its radioprotective efficacy in experimental animals exposed to whole body lethal gamma irradiation. Free radical scavenging properties were also studied under in vitro conditions.

* Materials and methods

RH-3 preparation

Fresh whole berries were collected from Lahaulspiti (an altitude of about 3800 meters) in Himachal Pradesh, India and the plant was confirmed as Hippophae rhamnoides by comparison with the Voucher specimen (IHBT IHBT I Have Been Trolled  No. 1047) kept in herbarium herbarium, collection of dried and mounted plant specimens used in systematic botany. To preserve their form and color, plants collected in the field are spread flat in sheets of newsprint and dried, usually in a plant press, between blotters or absorbent paper.  of Biodiversity Centre at Institute of Himalayan Biosource Technology, Palampur, Himachal Pradesh, India. The berries were washed and shade dried. Known quantity of the dried material was extracted using absolute alcohol and tripled distilled water (50:50, v/v; three changes). Finally the extract was lyophilized, weighed and stored at 4 [degrees]C. RH-3 was a code name of this product prepared by Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India and was studied for its madioprotective properties in our Laboratory at Delhi. The preparation was diluted in triple-distilled water for desired concentration and the dose expressed in mg refers to weight of dried RH-3. HPLC-fingerprint analysis of the 50% alcoholic extract shows several peaks of flavo noids which ranged between 7 to 18 minutes retention time (see Figure HPLC HPLC high-performance liquid chromatography.

HPLC

high performance liquid chromatography.

HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed
).

Treatment

Desired doses of RH-3 were administered i.p. 30 minutes before whole body gamma radiation.

Animals

Male Swiss albino Strain 'A' inbred mice (12 weeks old, weighing 25 [+ or -] 2 g) were maintained on standard food pellets (Lipton India) and water ad libitum. Animal experiments were conducted according to 'INSA -- Ethical guidelines for use of animals in Scientific Research published by Central Drug Research Institute, Lucknow, India.

Maximum tolerated dose

Acute toxicity of single dose administration of RH-3 was studied in terms of survival, changes in behavior, neuromuscular co-ordination and respiratory disorder etc. Period of observation for acute toxicity was 2 days for a single dose regimen.

Irradiation

Whole body irradiation was given through [Co.sub.60] gamma cell (Model 220 -- Atomic Energy of Canada) at an absorbed dose rate of 0.66 Gy/min and fresh air was continuously circulated in the irradiation chamber to avoid generation of hypoxic conditions. Mice were kept in perforated plastic bottles for irradiation individually. For studying free radical scavenging in vitro, different solutions were exposed to various desired doses of gamma rays delivered by Gamma chamber 5000 (BRIT, India), having a dose rate of 1.78 Gy/sec during the course of experiment.

Survival studies

Animals were observed for survival daily up to 30 post-irradiation days. Data were presented as % survival after the expiry of 30 post-irradiation days. The body weight was also recorded every alternate day.

Haemopoietic Stem Cell assay

For endogenous colony forming unit assay (Till and McCulloch 1961), mice were sacrificed by cervical dislocation 10 days after various treatments. Spleen was removed and fixed in Bouin's solution for 24 h. Macroscopic colonies (CFU) visible per spleen were scored in each mouse.

Haematological studies

Hemoglobin, Total leukocyte count and Differential lymphocyte counts were studied in blood samples drawn from heart of mice sacrificed by cervical dislocation.

Free radical scavenging and antioxidant studies

These studies were undertaken to unravel the mode of action of RH-3. All spectrophotometric measurements were done with the help of chemito UV-Vis spectrophotometer spectrophotometer, instrument for measuring and comparing the intensities of common spectral lines in the spectra of two different sources of light. See photometry; spectroscope; spectrum. . All the experiments were done in triplicate.

* Protein: Total proteins were estimated in 10% liver homogenate (Lowry et al. 1952).

* Scavenging of hydroxyl radicals: The radiation and [FeSO.sub.4] generated OH radicals quenched by RH-3 were estimated using 2-deoxyribose as the marker substrate (Gutteridge 1981). Each tube contained 0.5 ml of 2-deoxyribose and 0.5 ml of phosphate buffered saline Phosphate buffer saline (abbreviated PBS) is a buffer solution commonly used in biochemistry. It is a salty solution containing sodium chloride, sodium phosphate and potassium phosphate. The buffer helps to maintain a constant pH.  (pH 7.4). In one tube 0.1 ml of 100 [mu]M [FeSO.sub.4] was added and mixed thoroughly. Other tubes were exposed to desired doses of gamma radiation in the presence or absence of plant extract. Thereafter 0.5 ml of (1% Thiobarbituric acid, TBA) was added and vortexed. 0.5 ml of 10% Trichloro acetic acid (TCA TCA

1. trichloroacetic acid.

2. tricarboxylic acid cycle (Krebs cycle).

TCA Tricyclic antidepressant, see there
) was added and vortexed again thoroughly and the whole solution was incubated at 100 [degrees]C for 15 minutes; Each tube was cooled and measured for the intensity of chromogen chromogen /chro·mo·gen/ (kro´mah-jen) any substance giving origin to a coloring matter.

chro·mo·gen
n.
1. A substance that lacks definite color but may be transformed into a pigment.
 by recording at 532 nm.

* Scavenging of superoxide anions: The superoxide quenching ability of RH-3 was estimated using nitrobluetetrazolium (NBT) as the marker substrate (Kakkar et al. 1984). In each tube 1.2 ml of sodium pyrophosphate buffer (0.052 M, pH 8.3), desired concentration of plant extract and 0.1 ml of 186 [mu]M phenazine phen·a·zine   also phen·a·zin
n.
A yellow crystalline compound, C6H4N2C6H4, used in the manufacture of dyes.



phenazine  

1.
 methosulfate were taken followed by addition of 300 [mu]l of 300 [mu]M nitrobluetetrazolium and total volume was adjusted to 3 ml. Thereafter reaction was initiated by adding 200 1 of NADH NADH the reduced form of NAD.

NADH
n.
The reduced form of NAD.


NADH,
n.pr a coenzyme that incorporates niacin and involved in the Krebs cycle.
 (780 [mu]M) and the solution was incubated at 37 [degrees]C for 90 seconds. The reaction was stopped by adding 1 ml of glacial acetic acid glacial acetic acid
n.
Acetic acid that is at least 99.8 percent pure.
. The resultant mixture was shaken with 4 ml of n-butanol and allowed to stand for 10 minutes at room temperature. By centrifugation butanol bu·ta·nol  
n.
Either of two butyl alcohols derived from butane and used as solvents and in organic synthesis.



[butan(e) + -ol1.
 layer was separated and color intensity of chromogen in the butanol was measured at 560 nm against butanol. The percentage of inhibition by extract was calculated by considering the optical density of the chromogen in the absence o f extract as 0% inhibition of NBT reduction.

* Lipid peroxidation: Randomly selected 6-8 weeks strain 'A' mice were sacrificed by cervical dislocation, dissected and abdominal cavity were perfused with 0.9% saline; whole liver was taken out and visible blood clots were carefully and maximally removed and weighed amount of liver was processed to get a 10% homogenate in cold buffered saline (pH 7.4); using potter elevejam homogenizer and filtered through a mesh to get a clear homogenate. 2 ml of 10% liver homogenate was taken in a series of 35-mm petri dishes to which desired amount of RH-3 was added and mixed gently to form a homogeneous solution. Lipid peroxidation was initiated by adding 100 [mu]1 of ferrous ammonium sulfate (0.5 mM) and thereafter petri dishes were incubated at 37 [degrees]C for 30 minutes. 100 [mu]1 of homogenate was pipetted out for estimating lipid peroxidation levels in terms of Thiobarbituric Acid Reactive Substances Thiobarbiturate reactive substances (TBARS) are the low-molecular-weight end products, whose main component is malondialdehyde, that are formed during the decomposition of lipid peroxidation products.  (TBARS TBARS Thiobarbituric Acid Reactive Substances
TBARS Tiberium-Based Armor Reinforcement Substance
) following method of Beuge and Aust (1978).

Analysis of data & Statistical analysis

All experiments were repeated thrice and the data were analyzed statistically and expressed as mean [+ or -] MSE; student-t-test was applied for significance and P value <0.05 was considered significant.

* Results

Maximum tolerated dose

Single doses of RH-3 up to 40 mg/kg b.w. was tolerated by mice without any apparent adverse manifestation, except being little drowsy for 3 to 5 minutes. However, RH-3 beyond 45 mg/kg b.w. manifested mortality within 48 h in a dose dependent manner (Table 1).

Survival studies

Survival of mice up to 30 post-irradiation days has been shown in (Figure la and lb). All irradiated animals without RH-3 treatment were dead with in 15 days. On 30th post-irradiation clay maximum survival (81.7%) was achieved by administration of 30 mg/kg b.w. of RH-3, 30 minutes before 10 Gy irradiation. Doses of RH-3, less than 25 mg or more than 35 mg/kg b.w. were less effective for 30 days post-irradiation survival. Effect of time interval between administration of RH-3 and irradiation was also studied on 30 days post-irradiation survival and it was observed that 30 minutes interval was most optimal for radioprotective effect.

Haemopoietic stem cells

Effect of various radiation doses on colony forming capacity and its modulation by pre-irradiation administration of RH-3 (30 mg/kg b.w., --30 min.) has been depicted in Table 2. CFU counts in spleen decreased with increasing radiation dose in the absence of RH-3. RH-3 administration to unirradiated rats did not influence CFU counts. Pre-irradiation treatment with RH-3 rendered protection against all radiation doses used here and was found to be significantly higher (p <0.05) than the corresponding irradiated groups without RH-3 treatment. The CFU counts however declined with increasing radiation doses even in presence of RH-3.

Haematological studies

* Haemoglobin: Changes in the amount of haemoglobin (Hb) in mice in different treatment groups have been shown at different post-irradiation intervals (Figure 2). RH-3 alone decreased the haemoglobin level to 12.8% [+ or -] 3.7 up to 10th post-treatment day and thereafter on 1 day it was observed to be equivalent to untreated control (15 g%). In irradiated group (10 Gy) haemoglobin decreased continuously attaining the value of 10.8 [+ or -] 1.14 g% by 10th day as compared to 15 g% in untreated control; data for 15th day could not be collected since all the animals in this group were dead by that time. In RH-3 + 10 Gy group the haemoglobin level remained low (11.86 [+ or -] 0.49) up to 7 days but thereafter it started recovering with time and increased to 12.4 +/- 0.29 g% in 15 days.

* Total leucocyte counts (TLC TLC total lung capacity; thin-layer chromatography.

TLC
abbr.
1. thin-layer chromatography

2.
): Treatment with any single dose of RH-3 alone increased TLC up to 7 days and thereafter it reverted to control value (Figure 3). In irradiated group TLC decreased sharply up to 10 days and data for 15 days could not be collected since by that time all untreated irradiated animals were dead. RH-3 treatment 30 minutes before irradiation did not exhibit any protection over untreated irradiated group up to 7 days but the recovery became evident thereafter and was quite steep during successive three days. Within 15 days TLC became very close to unirradiated control.

* Differential leucocyte counts (DLC): The effect of RH-3 in protecting lymphocyte, polymorphs and monocytes against radiation damage has been depicted in Figures 4a, b and c. In the group treated with RH-3 alone the number of lymphocytes, polymorphs and monocytes increased upto 7 days but reverted to normal value within 10 post-treatment days. Lymphocytes and monocytes however again showed an upward trend as seen on 15th post-treatment day. In irradiated group (10 Gy), the number of lymphocyte, polymorphs and monocytes decreased sharply up to 7 days as compared to control values. There was no recovery and all animals died with in 15 post-irradiation days. On 7th and 10th post irradiation days the TLC was so low that DLC could not be studied. In RH-3 + 10 Gy group, the lymphocyte and monocytes decreased sharply up to 7 days as happened to irradiated untreated animals. There was no sign of significant recovery up to 10 days. However on 15th day lymphocytes, monocytes and polymorphs were seen to have significa nt recovery.

Effect of RH-3 on free radical scavenging

These studies were conducted under in Vitro conditions.

* OH radical scavenging:

-- Fenton reaction mediated OH radical generation: The effect of varied concentrations of RH-3 on scavenging of Fenton reaction mediated OH radical as determined by inhibition of 2-deoxyribose degradation has been depicted in Figure 5. RH-3 rendered inhibition of 2-deoxyribose degradation in a dose dependent manner; maximum effect (75.71%) was achieved at a concentration of 2 mg/mi (P < 0.05).

-- Fixed radiation induced OH radical generation and scavenging by varied herbal concentrations: The effect of different concentrations of RH-3 on inhibition of 2-deoxyribose degradation by a radiation dose (200 Gy) has been depicted in Figure 6. RH-3 inhibited 2-deoxyribose degradation in a dose dependent manner. At a concentration of 0.2 mg/ml of RH-3 about 90% inhibition was achieved, though maximum inhibition (97.09 %) was achieved at a concentration of 2 mg/ml (P < 0.05).

* Superoxide anion scavenging: Figure 7 shows that increasing dose of RH-3 inhibited superoxide anions in a proportionate manner. A concentration of 2 mg/ml rendered maximal inhibition (85.66%; P < 0.05) of superoxide anion mediated formazan production.

* Inhibition of lipid peroxidation: Inhibition of Fenton reaction mediated lipid peroxidation by RH-3 as expressed by inhibition of TBARS formation has been depicted in Figure 8. RH-3 elicited a dose dependent inhibition of lipid peroxidation, maximal inhibition (68.92 %) being achieved at a concentration of 2 mg/ml (P < 0.05).

* Discussion

The present studies revealed that pre-irradiation administration of single dose of RH-3 (30 mg/kg b.w.) rendered 81.7% survival in mice against 10 Gy whole body irradiation. Untreated irradiated mice suffered 100% mortality within 10-15 days (Goel et al. 1998). The maximum protective effect was achieved at subtoxic level of RH-3. Infact, most of the molecular and herbal radioprotective agents often render maximum radioprotective effect at subtoxic level approaching maximum tolerated dose level (Riklis et al, 1990, Schuchter et al. 1993, Goel et al. 1996, Umadevi et al. 1999).

Radiation protection by chemicals at cellular and subcellular sub·cel·lu·lar  
adj.
1. Situated or occurring within a cell: subcellular organelles.

2. Smaller in size than ordinary cells: subcellular organisms.

3.
 level likely reflects both wholesome effect of scavenging of radiation-induced free radicals and the repair of damaged targets and molecules. However, repair of damage and recruitment of cells to substitute apoptotic and necrotic cells are other very important manifestations of an ideal radioprotector. The recruitment of cells may contribute towards recovery of a number of tissues like bone marrow, intestine and skin etc. This could be possible due to proliferative stimulation rendered to the stem cells of the tissues/systems by certain molecules like cytokines, growth factors etc present in the herbal preparation. Administration of such chemicals has directly been shown to render significant radioprotection (Neta et al. 1994). RH-3 is a herbal preparation of H. rhamnoides and contains a plethora of molecules of diversified nature ranging from antioxidant to proliferative and immunostimulatory nature. Therefore, it became imperative to investigate the effect of RH-3 through various parameters for understanding the important facets of mode of action of a natural mixture of molecules available in RH-3.

H. rhamnoides has been well reported to contain several antioxidant molecules like vitamin A, C, E and K, tannins and flavonoids. It also contains certain trace elements like Se, Zn, Cu and S which are part of metallo-enzymes and some of which are known to manifest antioxidant activity and radioprotection (Ianev et al. 1995). The antioxidant potential was therefore monitored in vitro by estimating OH radicals generated by irradiation or Fenton reaction. RH-3 inhibited OH radical by scavenging them, thus confirming its antioxidant capabilities. The extraction was since done with polar solvents it is possible that antioxidants like vitamin A & E might not be there though vitamin C, tannins and other antioxidant molecules may be present. Therefore the antioxidant capabilities of this extract under present study could be a partial manifestation only.

The unirradiated mice spleen did not have any shortage of stem cells due to the absence of a cytocidal agent like radiation. Treatment with RH-3 possibly did not induce any proliferative stimulation in spleen and therefore CFU counts remained comparable to the untreated control. The enhanced CFU counts in spleen of RH-3 treated irradiated mice in comparison to untreated irradiated control, evidently indicate its radioprotective manifestation. The presence of RH-3 constituent molecules in cellular milieu at the time of irradiation and induction of damage could offer radioprotection by scavenging of free radicals. However some of the constituent molecules of RH-3 may not be getting immediately metabolized and persist even after irradiation. Such molecules could have influenced repair process at least the fast repair.

The reduction in the quantity of hemoglobin is a reflection of bone marrow stem cells activity; its depression, stimulation and recovery could be seen in the later part of second post-irradiation week. Administration of RH-3 before lethal irradiation ensured faster recovery in TLC in general and lymphocytes, polymorphs and monocytes in particular. Increase in TLC by RH-3 alone (Fig. 4a, b, c) is an interesting observation and could be an attribute responsible for immunostimulation. Some molecules enhanced bone marrow proliferation in a specific direction elevating TLC and down regulating RBC RBC red blood cell.

RBC or rbc
abbr.
red blood cell


RBC,
n See red blood cell count.


RBC

red blood cells; red blood (cell) count (see blood count).
 production resulting in the decrease of haemoglobin (Figure 3). Further, iron chelation Chelation
The process by which a molecule encircles and binds to a metal and removes it from tissue.

Mentioned in: Heavy Metal Poisoning

chelation
 studies done in our laboratory (unpublished data) could also be responsible for decreased haemoglobin.

The radioprotective effect generated by RH-3 at molecular level in terms of free radical scavenging as studied through in-vitro studies (Figure 5-8) could explain the cellular survival, proliferation enhancement, immunostimulation and ultimately the whole body survival. However, it is not revealed by this study whether enhancement of repair of DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 damage is also contributed by any component of RH-3 and it needs to be investigated further.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]
Table 1

Estimation of maximum tolerated dose: Survival of strain 'A' mice was
observed up to 48 hours after administration of single dose of RH-3.

(* n5 for each dose of RH-3)

Dose in mg/kg b.w.  * % Survival

 25                 100
 35                 100
 40                 100
 45                  80
 55                  60
 60                  40
100                   0
Table 2

CFU counts observed on 10th post-irradiation day in spleen of animals
given different doses of radiation or 30 mg/kg b.w. i.p. of RH-3 or
both. Time interval between drug administration and irradiation was 30
minutes.

Treatment  Radiation  Mice No  Mean CFU
RH-3       (Gy)                counts/Spleen *

-           5          6       13.58 [+ or -] 0.74
+           5          6       18.33 [+ or -] 0.96
-           7.5       12        2.25 [+ or -] 0.82
+           7.5        6        14.0 [+ or -] 1.17
-          10         12        0.37 [+ or -] 0.09
+          10          6         6.0 [+ or -] 0.88

* MSE - Mean standard error.


Acknowledgements

Thanks are due to Dr. T. Lazar Mathew, Director, INMAS INMAS Institute of Nuclear Medicine and Allied Sciences (Delhi, India) , for his support during the course of this study, Prof. C. K. Gupta, Consultant in Biostatistics for his help in statistical analysis of the data and Shri Ashok Kumar Sharma, AA'B' for providing Computer support.

* References

Beuge, J. A. and Aust, S. D.: Microsomal microsomal

pertaining to or emanating from microsome.
 lipid peroxidation. Methods in Enzymology Methods in Enzymology is a series of scientific publications on the topics of biochemistry by the Academic Press, now part of Elsevier. Each volume is centered on a specific topic of biochemistry, such as DNA repair, yeast genetics, or biology of the nitric oxide.  52: 302-310, 1978.

Blumberg, A. L., Nelson, D. F., Gramkowski, M., Glover, D., Glick, J. H., Yuhas, J. M. and Kligerman, M. M.: Clinical trials of WR-2721 with radiation therapy. International Journal of Radiation Oncology, Biology Physics 8: 561-563, 1982.

Cheng, T. J.: Protective action of seed oil of Hippophae rhamnoides L. (HR) against experimental liver injury in mice. Chung Hua Yu Fang I Hsueh Tsa Chih 4: 227-229, 1992.

Cheng, T. J., Pu, J. K., Wu, L. W., Ma, Z. R., Cao, Z. and Li, T. J.: A preliminary study on hepato-protective action of seed oil on Hippophae rhamnoides L. (HR) and mechanism of the action. Chung Kuo Chung Yao Tsa Chih 19: 367-370-384, 1994.

Goel, H. C., Ganguly, S. K., Prasad, J. and Jain, V.: Radioprotective effects of diltiazem on cytogenetic cytogenetic /cy·to·ge·net·ic/ (-je-net´ik)
1. pertaining to chromosomes.

2. pertaining to cytogenetics.


cytogenetic

pertaining to or originating from the origin and development of the cell.
 damage and survival in gamma ray exposed mice. Indian Journal of Experimental Biology 34: 1194-1200, 1996.

Goel, H. C., Prasad, J., Sharma, A. and Singh, B.: Anti-tumor and radioprotective action of P. hexandrum. Indian Journal of Experimental Biology 36: 583-587, 1988.

Gutteridge, J. M. C.: Thiobarbituric acid-reactivity following iron-dependent free-radical damage to amino acid and carbohydrates. FEBS FEBS Federation of European Biochemical Societies  Letters 128: 343-346, 1981.

Ianev, E., Radev, S., Balutsov, M., Klouchek, E. and popov, A.: The effect of an extract of sea buckthorn (Hippophae rhamnoides L.) on the healing of experimental skin wounds in rats. Khiruginia (Sofiia) 48: 30-33, 1995.

Kakkar, P., Das, B. and Viswanathan, P. N.: A modified spectrophotometric assay of superoxide dismutase. Indian Journal of Biophysics biophysics, application of various methods and principles of physical science to the study of biological problems. In physiological biophysics physical mechanisms have been used to explain such biological processes as the transmission of nerve impulses, the muscle  and Biochemistry 21: 130-133, 1984.

Landauer, M. R., Davis, H. D., Dominitz, J. A. and Weiss, J. F.: Dose and time relationships of the radioprotector WR-2721 on locomotor activity in mice. Pharmacology Biochemical Behaviour 27: 573-576, 1987.

Liu, F. M., Li, Z. X. and Shi, S.: Effect of total flavones of Hippophae rhamnoides L. on cultured rat heart cells and on cAMP level and adenylate cyclase in myocardium myocardium /myo·car·di·um/ (-kahr´de-um) the middle and thickest layer of the heart wall, composed of cardiac muscle.

hibernating myocardium  see myocardial hibernation, under
. Chung-kuo-yao-li-Hsueh-Pao 9: 539-542, 1988.

Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurement with Folin-Phenol reagent. Journal of Biological Chemistry The Journal of Biological Chemistry (often abbreviated JBC) is a scientific journal founded in 1905 and published since 1925 by the American Society for Biochemistry and Molecular Biology.  193: 265-274, 1952.

Luginov, A. S., Mironov, V. A., Amirov, N. S. H., Aruin, L. I. and Vasilev, G. S.: Effect of preparation from Hippophae berries on the healing of experimental stomach ulcers. patol-Fiziol-Eksp-Ter 6: 67-70, 1983.

Maisin, J. R.: Perspectives in chemical radiation protection. Proceedings International conference on low dose irradiation and Biological Defence mechanisms. Kyoto, Japan 12-16 July 1992. International Congress Series (Excerpta Medica medica (māˑ·dē·k ) 1013: 135-142, 1992.

Maisin, J. R.: Chemical radioprotection: past, present and future prospects. International Journal of Radiation Biology 73: 443-450, 1998.

Neta, R., Steifel, S. M. and Finkelman, F.: Interleukin-12 protects bone marrow from and sensitizes intestinal tract to ionizing radiation. Journal Immunology 153: 4230-4237, 1994.

Nikitin, V.A., Chistiakow, A.A. and Bugaewa, V.: Therapeutic endoscopy in combined therapy of gastroduodenal gas·tro·du·o·de·nal
adj.
Relating to the stomach and the duodenum.



gastroduodenal

pertaining to the stomach and duodenum.
 ulcers. Khiruginia-mosk 4:33-35, 1989.

Nikulin, A.A., Iakusheva, E.N. and Zakharova, N.M.: A comparative pharmacological evaluation of sea buckthorn, rose and plantain plantain (plăn`tĭn), any plant of the genus Plantago, chiefly annual or perennial weeds of wide distribution. Many species are lawn pests and the pollen is often a hay fever irritant. P.  oil in experimental eye burn. Eksp Klin Farmakol 55:64-66, 1992.

Riklis, E., Kol, R. and Marko, R.: Trends and developments in radioprotection: the effect of nicotinamide nicotinamide (nĭk'ətĭn`əmīd): see vitamin.  on DNA repair. International Journal of Radiation Biology 57:699-708, 1990.

Schuchter, L.M. and Glick, J.H.: The current status of WR-2721 (Amifostine): a chemotherapy and radiation therapy protector. Biological therapy cancer updates 3:1-10, 1993.

Shimoi, K., Masuda, S., Furgior, M., Esaki, S. and Kinae, N.: Radioprotective effect of antioxidative plant flavonoids in [gamma]-irradiated mice. Carcinogenesis 15:2669-2672, 1994.

Shimoi, K., Masuda, S., Shen, B., Furugori, M. and Kinae, N.: Radioprotective effects of antioxidative plant flavonoids in mice. Mutation Research 350:153-161, 1996.

Spirodonov, N.A., Arkhipov, V.V., Foigel, A.G., Tolkachev, O.N., Sasov, S.A., Syrkin, A.B. and Tolkachev, V.N.: The cytotoxicity of Chamaenerium angustifoliwn (L.) Scop and Hippophae rhamnoides L., tannins and their effect on mitochondrial mitochondrial

pertaining to mitochondria.


mitochondrial RNAs
a unique set of tRNAs, mRNAs, rRNAs, transcribed from mitochondrial DNA by a mitochondrial-specific RNA polymerase, that account for about 4% of the total cell RNA that
 respiration. Eksp Klin Farmacol 60:60-63, 1997.

Till, J.E. and McCulloch, E.A.: A direct measurement of radiation sensitivity of normal bone marrow. Radiation Research 14:213-222, 1961.

Uma Devi, P., Ganasoundari, A., Rao, B.S.S. and Srinivasan, K.K.: In vivo Radioprotection by Ocimum flavonoids: survival of mice. Radiation Research 151:74-78, 1999.

Von Sonatag C, The chemical basis of radiation biology, (Taylor and Francis, London) 1987.

Wang, Y., Lu, Y., Liu, X., Gou, Z. and Hu, J.: The protective effect of Hippophae rhamnoides L. on hyperlipidemic serum cultured smooth muscles cells in vitro. Chung Kuo Chung Yao Tsa Chih 17:624-6, 601, 1992.

Weiss, J.F., Kumar, K.S., Walden, T.L., Neta, R., Landaver, M.R. and Clark, E.P.: Advances in radioprotection through use of combined agent regimens. International Journal of Radiation Biology 57:709, 1990.

Wu, J., Yu, X.J., Ma, X., Li, X.G. and Liu, D.: Electrophysiologic effect of total flavones Hippophae rhamnoides L on guinea pig papillary papillary /pap·il·lary/ (pap´i-lar?e) pertaining to or resembling a papilla, or nipple.
papillary,
adj similar to a small, nipple-shaped elevation or projection.
 muscles and cultured rat myocardial myocardial /myo·car·di·al/ (-kahr´de-al) pertaining to the muscular tissue of the heart.

myocardial

pertaining to the muscular tissue of the heart (the myocardium).
 cells. Chung Kuo Yao Li Hsueh Pao 15:341-343, 1994.

Xiao, M., Yang, Z., Jiu, M., You, J. and Xiao, R.: The antigastroulcerative action of [beta]-cytosterol-D-Glucoside and it's aglycan in rats. Hua-His-I-Ko-ta-Hsueh-Hsueh-Pao 23:98-101, 1992.

Zhang, M.S.: A control trial of flavanoids Hippophae rhamnoides L. in treating ischemic heart disease. Chung-Hua-Hsin-Hsueh-Kuan-Ping-Tsa-Chih 15:97-99, 1987.

A.K. Sinha (1)

(1.) Institute of Himalayan Bioresource Technology, Palampur, India

* Address

H.C. Goel, Head, Radiation Biology division, Institute of Nuclear Medicine and Allied sciences, Lucknow Marg, Delhi-110 054, India

Tel: ++91-011-3970081; Fax: ++91-011-3919509; e-mail: radbio@nda.vsnl.net.in
COPYRIGHT 2002 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2002 Gale, Cengage Learning. All rights reserved.

 Reader Opinion

Title:

Comment:



 

Article Details
Printer friendly Cite/link Email Feedback
Author:Goel, H.C.; Prasad, J.; Singh, S.; Sagar, R.K.; Prem Kumar, I.; Sinha, A.K.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:9INDI
Date:Jan 1, 2002
Words:4635
Previous Article:Stevioside induces antihyperglycaemic, insulinotropic and glucagonostatic effects in vivo: studies in the diabetic Goto-Kakizaki (GK) rats.
Next Article:Antimutagenic and anticarcinogenic effects of phyllanthus amarus.



Related Articles
Effects of Podophyllum hexandrum on radiation induced delay of postnatal appearance of reflexes and physiological markers in rats irradiated in utero.
The evaluation of the radioprotective effect of Triphala (an ayurvedic rejuvenating drug) in the mice exposed to [gamma]-radiation.
Novel microencapsulation from Tagra Biotechnologies.
Radioprotection by mangiferin in DBAx[C.sub.57]BL mice: a preliminary study.
Modulatory influence of Adhatoda vasica Nees leaf extract against gamma irradiation in Swiss albino mice.

Terms of use | Copyright © 2014 Farlex, Inc. | Feedback | For webmasters