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Radionprotective effect from poliphenols of Bloody Cranesbill

RADIONPROTECTIVE EFFECT OF POLIPHENOLS FROM BLOODY CRANESBILL /GERANIUM SANGUINEUM - L/

Georgi Velev, St. Ivancheva 1, R. Lazarov 2

Botanical Institute Bulgarian Academy of Sciences, Department of Radiology of NOC.
Antiradiation effect of poliphenols from Geranium sanguineum - L. G. Velev, St. Ivancheva R. Lazarov

Botanical Institute Bulgarian Academy of Sciences, Department of Radiology of NOC.
Use of biologically active natural substances from natural products gives new opportunities of modern pharmacology. In particular, to create alternative to synthetic preparations, which usually are very toxic. Known are synthetic radiation protectors like WR- 2721, ОК-78 /5/. Not occasionally in the pharmacies of USA over 70% of the offered preparations are of natural origin. Protection of human and animal organisms from ionization radiations has interested scientists even since their discovery. Radio-modifying effect of a number of natural products has been subject of studies by many authors. /1,2,3,4/. These preparations have been involved in mass prophylactics of people working in environment with ionization radiations.

GSе lyophilized substrate of extract from the rootage of Bloody Cranesbill /Geranium Sanguineum- L/. It is a complex of poliphenolic compounds with the following percentage ratio between the components: tanins /20-30%/, flavonoids /0.03-0.08%/, catehins and protocyanidines /0.15-0.25%/, poliphenolic acids /0.01-0.02%/ and ballast substances/7/. Extract is obtained after roots are dried well to 12 % moisture, after grinding to dimension of the particle 0.5 mm and then they are extracted through lyophilization with ethyl alcohol. Because the biologically active constituents are thermally unstable, drying is carried out through lyophilization at temperature 35-40°С. The obtained substance is powder like of brown-red color and dissolves well in water and alcohol.  

The aim of the present study is assessment of radiation protective capacities of poliphenols, contained in the standard preparation GS, obtained from the roots of Bloody Cranesbill. In achieving this task standard radiobiological methodology is used for survival of rodents, test of leukocytes, granulocytes, lymphocytes and platelets in the peripheral blood of rats, phagocytal activity of neutrophyles, examination of basic cell populations in cancer affected patients, undergoing radiation therapy.  

MATERIALS AND METHODS

1. Experimental animals
The trials for survival, endogenous spleen colonies / Е - КОЕ g/ and phagocytal activity of neutrophyles have been carried out on white no breed mice of male gender of body mass 20-25 g. The examination of the peripheral blood indicators has been carried out on male rats of Vistar breed of body mass 150 -170 g.
Clinical study  of GS has been carried out in the Scientific Centre on cancer affected patients, undergoing radiation therapy. The group consisted in 12 people, and the control one in 11.

2. Application of the preparation
On the test mice water solution of lyophilizate was introduced through metal probe in dosages 30 mg/kg body mass 5 consecutive days before radiation. It was administered to the rats also through metal probe but in dosages 5 mg/kg body mass. The cancer patients took in peros in the form of capsules at empty stomach 150 mg daily /~2.5 mg/kg/, starting 10 days before the onset of radiations and going on through the whole course of the treatment.

Radiation. The mice were exposed to radiation with 10 Gyв gamma - emitter "Ray - 1300" with four sources, 60Co and power 1.3 u/min. The Vistar rats were subjected to 4 Gyв with the same gamma - emitter.
With the cancer patients, a treatment course was carried out with ionizing radiation in conventional dosages and volume.

EXPERIMENTS AND EXPERIMENTAL GROUPS

In tracing the survival of mice, exposed sharply with 10 Gy gamma – rays we used four groups of 20 experimental animals.
The first group was given solution of GS in dosage 30mg/kg with metal probe five consecutive days before radiation. Prophylactic scheme of application.

The second group took in the same dosage five consecutive days but after exposure to radiation. Therapeutic scheme of application.
The two control groups we gave distilled water – to the one before and to the other after subjection to radiation – in parallel with the trial groups.
Tracing of blood indicators in dynamics we carried out in two groups of Vitastar rats while one of them we gave solution from the lyophilizate  GS five consecutive days before radiation with 4 Gy gamma rays in dosages 5 mg/kg body mass, and the control – with distilled water. We traced the leukocytes, granulocytes and lymphocytes. We also traced the in dynamics the platelets.

The cancer patients in which we traced the basic leukocyte populations, administering to them radiation and taking in as a radiation protector GS were 12. Their average age was 47.3 - 6 men and 6 women. According to nozoological units they were distributed as follows: four patients underwent treatment of Hodgkin’s disease, four – on occasion of uterus tumors, one – of rectal tumor, one – of lung tumor, one – of nasal pharynx, one – of the brain. In the controlled group four of the patients were with of Hodgkin’s disease, two – with non-of Hodgkin’s disease lymphomas, two – with tumor of the tongue, one of the nasal pharynx, one – with uterus tumor, one – with breast tumor. Or, in total the controlled group was of eleven patients, subjected only to radiology.

METHODS
* Survival
We traced the survival rate of mice on 3-™ , 70-™ 16-™ , 23-™ , 30-™ day after subjection to radiation with 10 Gy gamma - rays, at prophylactic and therapeutic  scheme of application of GS.
   

* Endogenous spleen colonies / Е - КОЕ g/ we determined by the method of J. Till, Е. Мс. Kulloch, L. Siminovitch. We used five groups of mice, each with 20 animals. We applied preventively 5 consecutive days GS in dosages, as follows: I group - 30 mg/kg body mass , II group - 20 mg/kg, III group - 10 mg/kg, IV group - 5 mg/kg and V – control one, which we gave distilled water. On the fifth day they were subjected to 10 Gy gamma - rays, and on the eight day after the radiation we removed the spleens on which we counted endogenous spleen colonies.

 * Tracing of hematological indicators from peripheral blood: leukocytes, granulocytes, lymphocytes, platelets in male Vistar rats with body mass 150-170 g. GS was administered for five consecutive days prophylactically in dosages of 5 mg/kg body mass, then they were subjected to 4 Gy from the working and the controlled group. We monitored hematological indicators in dynamics.

The phagocytes activity was determined and marked by 14 С 20-hour’s culture of Echerichiacoli and cultivated in monolayer /8/ 14 С Echenchiacoli  was obtained after incubation of bacterial culture in the nutrition media of Тimakov, containing radioactive markers 14 С timin and 14 С acenin. Isolation of neutrophyles through NH4Cl- isotopic solution was done, after triple gradient centrifugation and washing of the cell suspension with solution of Henk's. The obtained neutrophyles are of retained vitality. The incubation was done for 1 hour at 37° С in ratio neutrophyles / Echerichtacoli 1: 10, where after the non-included in the monolayer activity was removed through quadruple rinsing with physiological solution. The lysis of the cells is carried out 0.15 % on sodium lauril  sulphate, scintillation cocktail is added and the ?-activity of liquid scintillation counter is measured. The phagocytosis is calculated in percentages from the output activity for 1.106 cells.

RESULTS AND DISCUSSION
The working and the controlled groups were of 20 trial animals each. We watered the working group with GS 30 mg/kg body mass for five consecutive days before and after radiation with 3 Gy, and the controlled one we watered with distilled water.

I. Effect of GS on the survival rate of mice after sharp radiation
The results of the monitoring of the survival rate of mice indicate statistically reliable difference in the prophylactic ally treated group on 16"™, 23"™ и 30" and one day after radiation. This most probably is due to three factors: stimulation of the hemopoiesis /all cell rows/,availability of powerful antioxidants like flavonoids, antocianes, poliphenol acids and etc. and increased phagocytic activity of the neutrophyles /the thirtieth day survived prophylactically treated animals are 63% ±5.1%, and for the control 33%  ± 4. 5% /.
With the therapeutically treated group, statistically reliable difference we obtained on the I6"™1 day. The survived ones were 55% ±5.3% for the trial group and 38% ±4.6% for the control one.

II. Tracing of endogenous spleen colonies /Е-КОЕd/
In group one whereto we applied GS in dosages 40 mg/kg body mass we obtained average number Е-КОЕd 7. 17 ±1. 34, II 'nd group 30 mg/kg- 6.57 ±0.83, ПГ group 20 mg/kg- 3.29 ±0.41, IV'™ group 10 mg/kg- 1. 80 ± 0. 33. In the controlled group we did not find spleen colonies. The preparation probably helps the greater retention of stem cells after radiation and speeds up their proliferation or modifies both processes which leads to faster recovery of blood formation after radiation./1/

III. Effect of GS on hemopoeisis of Vitastar breed rats
We traced in dynamics the blood indicators, in particular leukocytes, granulocytes, lymphocytes and platelets. We watered the experimental group of animals of 5 mg/kg body mass with GS, where after they were subjected to gamma-emitter with 4 sources 60Со in dosages 4 Gy/ the extrapolation coefficient of rats/ mice was 0.157 /.  During the first 10 days after radiation the leukocytesте of the experimental group were higher than the ones of the control, and on the 23'rd day the difference was statistically reliable. The platelets of the treated group were of higher values than the ones of the control, except for the 5th day, here we did not find trustworthy difference.

STUDY OF BLOODY CRANESBILL
FIRST GROUP - LEU. OR, L.Y,

IV. Effect of GS on the phagocytic activity of the neutrophyles.
The neutrophylic granulocytes are some of the most sensitive to ionizing radiation blood cells. The applied radio-isotopic method gives us integral assessment for the functional state of the phagocytic neutrophyles.
GS demonstrated, applied in prophylactically –therapeutic scheme, statistically trustworthy difference. The data for the control are 7.21% ±1.53%, experimental group, treated with GS10.67% ±1.61%.
The obtained result gives us grounds to consider that GS has stimulating effect on the phagocytic neutrophyles.

V. Effect of GS  as regards to Т-lymphocytes in cancer patients, subjected to radiation.
The application of GS stimulated selectively the Т-lymphocytic population, expressed in statistically trustworthy limits in comparison with the control group. The monitored changes in the percentage part of the total lymphocytes did not indicate correlation with dynamics in certain Т-lymphocytic sub-population. The ratio between Т-helpers/inducers and Т-cytotoxic/suppressors remained constant. The analysis of the remaining lymphocytic sub-populations – general:  В-lymphocytes, NK-cells did not indicate statistically significant differences. On this stage of the study, the effect of the application of GS is considered as positive. The retention of the Т-lymphocytic population, responsible for the cells immune response in cancer patients is of particular significance.

CONCLUSIONS
   1. GR significantly increases the survival of mice, radiated with lethal dosages;
   2. GR impacts positively the hemopoeisis in rats, helps the rapid recovery of the indicators in peripheral blood;   3. GR significantly impacts the quantity of endogenous spleen colonies /Е- КОЕd/;
   4. GR impacts positively the function of the neutrophilic population. FА of the treated group with GS is statistically trustworthy higher than the one of the control group.
   5. The application of GS in cancer patients, subjected to radiation indicated retention of the Т-lymphocytic population, which determines the benefit of its simultaneous application in this case. These results determine also the possibility of GS to be applied prophylactically in contingents, working in environment with ionizing radiation.


SUMMARY
The development of standardized preparations from natural products, having radiation protective effect, acquire still greater significance in prophylaxis of contingents, receiving greater dosages of ionization radiation or likely possibility of getting such ones.

In the first group we have in mind:
1. Cancer patients undergoing radiation therapy.
2. Groups of people, feeding the reactors in NPP with fuel.
3. Military troops, located in "hot" spots on the planet where some kind of nuclear military ammunitions are used.

The second group is:
4.   Employees in NPP and people, living nearby.

The idea of this study is obtaining of standard preparation of radiation protective effect, of low or practically absent toxicity which can be taken for a long period. The used in our country synthetic radiation protectors / WR- 2721, ОК - 78 /, are highly toxic and this does not allow their long application. The standard product , obtained after gradual extraction with ethyl alcohol and following sublimation drying, called Gerisan /GS/, we studied mostly at prophylactic scheme of application in mice, subjected to radiation with 10 Gy gamma-rays. We traced survival endogenous spleen colonies and phagocytic activity. Prophylactic – therapeutic scheme we applied to rats, subjected to 4 Gy gamma rays, whereof we studied blood indicators.
We administered GS also to cancer patients, undergoing radiation therapy. GS, administered preventatively, increases post-radiation survival of mice, supports post-radiation survival of hemopoeisis in rats and impacts the rapid recovery of Т-lymphocytes in cancer patients, undergoing radiation therapy.


Key words: ionization radiation, radiation protection, GeraniumSanguineum, hemopoeisis, phagocytic  activity.