RADIOPROTECTIVE EFFECT OF BLOOD GERANIUM POLYPHENOLS /GERANIUM SANGUINEUM - L/

Georgi Velev, St. Ivancheva ¹, R Lazarov ²

Botanical Institute at BAS, Clinic of Radiology at NOC. Antiradiation effect of polyphenols from Geranium sanguineum - L. G. Velev, St. Ivancheva R. Lazarov ².

Botanical Institute Bulgarien Academy of Sciences, Department of radiology of NOC.

The use of biologically active substances from natural products gives new opportunities to modern pharmacology. Namely, to create an alternative to synthetic preparations, which are usually very toxic. Synthetic radioprotectors such as WR- 2721, OK-78 /5/ are known. It is no coincidence that over 70% of the preparations available in US pharmacies are of natural origin. The protection of human and animal organisms from ionizing radiation has interested scientists since their discovery. The radio-modifying effect of a number of natural products has been the subject of research by many authors /1,2,3,4/. These preparations come into consideration for mass prophylaxis of people working in ionizing radiation environments.

GSis a lyophilic substrate of the extract of the rhizomes of Blood Geranium /GeraniumSanguineum- L/. It is a complex of polyphenolic compounds with the following percentage ratio between the components: tannins /20-30%/, flavonoids /0.03-0.08%/, catechins and prontocyanidins /0.15-0.25%/, polyphenolic acids /0.01-0.02%/ and ballast substances/7/.The extract is obtained after the roots are thoroughly dried to 12 % moisture, finely ground to a particle size of 0.5 mm and extraction with ethyl alcohol is carried out. Due to the fact that biologically active constituents are thermolabile, drying is done by lyophilization at 35-40°C. The resulting substance is powdery with a brownish-reddish colour and dissolves well in water and alcohol.

The aim of the present study was to evaluate the radioprotective potential of polyphenols contained in the standard GS preparation derived from blood geranium roots. To achieve the aim, standard radiobiological methodology was used for rodent survival, leukocyte, granulocyte lymphocyte and platelet studies in peripheral blood of rats, phagocytic activity of neutrophils, study of major cell populations in cancer patients undergoing radiotherapy.

MATERIALS AND METHODS

1. Experienced animals

Survival experiments, endogenous splenic colonies / E - KOE g/ and phagocytic activity of neutrophils were performed with white inbred male mice with a body mass of 20-25 g. The study regarding peripheral blood indices was performed with male Wistar rats of 150 -170 g body mass.

A clinical trial of GSSwas conducted at the NSC in cancer patients undergoing radiotherapy. The group consisted of 12 people and the control of 11.

1. Application of the preparation.

Experimental mice were administered an aqueous solution of the lyophilizate via a metal probe at doses of 30 mg/kg body weight for 5 consecutive days before irradiation. The rats were also administered by metal probe, but in doses of 5 mg/kg bw. Cancer patients took it in the form of capsules on an empty stomach 150 mg/day /~2.5 mg/kg/ starting 10 days before the start of irradiations and continuing throughout the course of treatment.

Irradiation. The mice were irradiated with a 10-Gu gamma-irradiator "Beam -

1300″ with four sources, 60Coand power 1.3 u/min. The Wistar rats were irradiated with 4Gy of the same gamma-irradiator.

Cancer patients were treated with ionizing radiation in conventional doses and volume.

EXPERIMENTS AND EXPERIMENTAL GROUPS

We used four groups of 20 experimental animals in the follow-up of survival of mice acutely irradiated with 10 Gugama-rays.

Group 1 was watered with GS solution at a dose of 30 mg/kg using a metal probe for five consecutive days before irradiation. Prophylactic administration schedule.

A second group took the same dose for five consecutive days, but after irradiation. Therapeutic administration schedule.

The two control groups were watered with distilled water - one before, the other after irradiation - in parallel with the experimental groups.

Dynamic monitoring of blood parameters was performed in two groups of Wistar rats, one of which was watered with a solution of the lyophilizate GSS for five consecutive days before irradiation with 4 Gugama rays at doses of 5 mg/kg body weight, and the control with distilled water. We monitored leukocytes, granulocytes and lymphocytes. We also traced platelets in dynamics.

The cancer patients in whom we followed the major leukocyte populations, administering radiotherapy and taking GSS as radioprotector were 12. Their mean age was 47.3 years - 6 men and 6 women. The patients were divided according to the following nosological entities: four patients were treated for Hodgkin's disease, four for uterine tumours, one for rectal tumours, one for lung tumours, one for nasopharyngeal tumours and one for brain tumours. In the control group, four patients had Hodgkin's disease, two had non-Hodgkin's lymphomas, two had tongue tumors, one had a nasopharyngeal tumor, one had a uterine tumor, and one had a breast tumor. Or, the control group was eleven patients in total who underwent radiotherapy alone.

METHODS

• Survivability

We tracked survival in mice at 3-^™ , 70-^™ 16-™ , 23-™ , 30-™ days after irradiation with 10 Gugama - rays, in prophylactic and therapeutic administration schedule of GS.

• Endogenous splenic colonies /E - COE g/ were determined by the method of J. Till, E. Mc. Kulloch, L. Siminovitch. We used five groups of mice, in each of which there were 20 animals. We administered preventively for 5 consecutive days GSin doses as follows: group I - 30 mg/kg body weight, group II - 20 mg/kg, group III - 10 mg/kg, group IV - 5 mg/kgand group V - control, which we watered with distilled water. On the fifth day we irradiated with 10 Gugama - rays and on the eighth day after irradiation we removed the spleens on which we counted endogenous splenic colonies.
• Monitoring of peripheral blood haematological parameters: leucocytes, granulocytes, lymphocytes, platelets in male Wistar rats with a body mass of 150-170 g. GSbe administered prophylactically for five consecutive days at 5 mg/kgbody mass, after which we irradiated the treatment and control groups with 4 GS. We monitored hematological parameters in dynamics. Phagocytic activity was determined in labeled by ¹⁴ With a 20-hour culture of Escherichia coli cultured in a monolayer /8/ ¹⁴ C Enchiasi was obtained after incubation of a bacterial culture in Timakov's medium containing as radioactive markers ¹⁴ With thymine and ¹⁴ S acenin. Isolation of neutrophils by NH4Cl-isotopic solution was performed, followed by threefold gradient centrifugation and washing of the cell suspension with Henk's solution. The resulting neutrophils had preserved vitality. Incubation was carried out for 1 hour at 37°C in a neutrophil/Echerichtacoli1:10 ratio, after which the activity not included in the monolayer was removed by washing four times with saline. Lysis of the cells was performed with 0.15 % of sodium lauryl sulfate, scintillation cocktail was added, and the ?-activity was measured on a liquid scintillation counter. Phagocytosis is calculated as a percentage of baseline activity for 1.10⁶ cells.

RESULTS AND DISCUSSION

Working and control groups of 20 experimental animals each. We watered the working group with GS30 mg/kgbody weight for five consecutive days before and after 3 Gy irradiation, and we watered the control with distilled water.

I. Effect of GSon survival of mice after acute irradiation.

The results of the mouse survival follow-up showed a statistically significant difference in the prophylactic treatment group at 16″™, 23″™ and 30″^1и day after irradiation. This is most likely due to three factors: stimulation of hematopoiesis /all cell lines/, the presence of potent antioxidants such as flavonoids, anthocyanins, polyphenolic acids, etc. and increased phagocytic activity of neutrophils /30″^You Day 1 survivors of prophylactically treated animals were 63% ±5.1% and control 33% ±4.5%/.
In the treatment group, we obtained a statistically significant difference at I6″™¹ day. Survivors were 55% ±5.3% for the experimental group and 38% ±4.6% for the control.

II. Tracking of endogenous splenic colonies /E-CoEd/

At D^you group, to which we administered GSat doses of 40 mg/kg body weight, we obtained a mean number of E-CoEd 7.17 ±1.34, II '^ra group 30 mg/kg- 6.57 ±0.83, PG^ta Group 20 mg/kg- 3.29±0.41, Group IV'™ 10 mg/kg- 1.80±0.33. In control group we did not find splenic colonies. The preparation probably either promotes greater preservation of stem cells after irradiation or accelerates their proliferation or modifies both processes resulting in faster ?recovery of hematopoiesis after irradiation./1/

III. Effect of GSon the haemopoiesis of Wistar rats.

We monitored blood parameters, namely leukocytes, granulocytes, lymphocytes and platelets. We irradiated the experimental group of animals with 5 mg/kgbody weight with GS, then irradiated in a gamma irradiator with 4 sources ⁶⁰Co at doses of 4 Gy/the extrapolation coefficient of rat/mouse was 0.157/ In the first 10 days after irradiation, the leukocytes of the working group were higher than those of the control, and at 23′^thia day the difference is statistically significant. Platelets in the treatment group had higher values than those of the control, except at 5″^til day, here we find no statistically significant difference.

'BLOOD GERANIUM' STUDY'

FIRST GROUP - LEU. OR, L.Y,

IV. Effect of GSon the phagocytic activity of neutrophils.

Neutrophil granulocytes are among the most sensitive blood cells to ionizing radiation. The applied radioisotope method gives us an inferential assessment of the functional state of phagocytosing neutrophils.

GShowed, administered in a prophylactic-therapeutic scheme, a statistically significant difference. The data for the control was 7.21% ±1.53%, the working group treated with GS10.67% ±1.61%.

These results give us reason to believe that GShas a definite stimulatory effect on phagocytizing neutrophils.

V. Effect of GSon T lymphocytes in cancer patients undergoing radiotherapy.

The administration of GS stimulated selectively the T-lymphocyte population, expressed in statistically significant ranges when compared with the control group. Observed changes in the percentage of total lymphocytes showed no correlation with dynamics in any particular T-lymphocyte subpopulation. The ratios of T helper/inducers and T cytotoxic/suppressors remained constant. The analysis of other lymphocyte subpopulations - total: B-lymphocytes, NK-cells did not show statistically significant differences. At this stage of the study, the effect of the administration of GSis reported as positive. Preservation of the T-lymphocyte population, which is responsible for the cellular immune response in ocular patients, is of particular importance.

FINDINGS

1. GRsignificantly increased survival in mice irradiated with lethal doses;
2. GRpositively affects hematopoiesis in rats, helps to restore peripheral blood indicators faster;
3. GR significantly affects the amount of endogenous splenic colonies /E-CoEd/;
4. GRpositively affects neutrophil population function. The FA of the GS-treated groupwas statistically significantly higher compared with the control group.
5. The administration of GS in cancer patients undergoing radiotherapy showed preservation of the T-lymphocyte population, which determines the usefulness of its concomitant administration in this case. These results also determine the possibility of G50C↩Sto be administered prophylactically in contingents working in environments with ionizing radiation.