Revaz G.Melkadze

Georgian technical university, Georgia

RADIOPROTECTIVE PROPERTIES OF BIOACTIVE ADDITIVE "GRAIL”

Due to the expansion of the areas of human activities in which ionizing radiation is applied, especially in case of emergencies at nuclear power facilities, also in the case of using nuclear weapons in wars and armed conflicts and the use of radioactive substances for terrorist and subversive purposes, the issue of anti-radiation protection is gaining increasing diligence.

In order to be protected from radiation, apart from technical and engineering solutions (protection in time, by distance and screening), pharmacological agents (medical anti-radiation protection) are also widely used.

The current system of medical anti-radiation protection is based on carrying out a set of therapeutic- preventive measures aiming at preserving the lives and health of the people exposed to ionizing radiation [1-3]. In this respect, a special role belongs to radio protective means, including the drugs or formulations, which, if applied for preventive purposes, can have a protective effect manifested in preserving the life of an irradiated organism or weakening the severity rate of radiation damage, with a prolonged active capacity and life span.
Out of the great number of radio protective remedies (synthetic, microbiological, organic etc.) available today, due to their low-toxic nature and identity with the chemical substances generated within a human organism, preferences are made for natural herbal medicines [4-7].

The remedy alleged as radio protective in this article, is an aqueous of biologically active additive “Grail” (BG) of ecologically clean medicinal-food plants, products of apiculture and viniculture, created within the framework of a transnational program for the elimination of the consequences of the Chernobyl nuclear power plant disaster [8,9].

1. Radioprotective effect of BG a dose of an irradiation 750 sGr

The majority of the rats developed an acute radiation disease of the 2^nd and 3^rd grade 3 days after being exposed to radiation. The rats showed off signs of general oppression, immobility, weak reaction to external irritants. Their hair turned dull, fragile, easy to pick out. The consumption of food and water also decreased.

The defecation of the half of the rats during the first week was frequent and liquid, with some spots of blood observed in the intestinal contents of some of them. By the end of the 1^st week, 60% of the rats were observed to have serous- blood-tinged discharge.

The animals started dying by the end of the first week. Upon dissection of the killed rats a typical picture of lesions characteristic to the acute radiation disease was observed: hemorrhage into different organs and tissues, spleen atrophy, fatty degeneration of liver, necrotic changes in gastral and intestinal walls.

A week after exposing to radiation, the animals were examined on hematological indicators: the content of leucocytes, erythrocytes and platelets in the blood of rats was identified. The count of blood corpuscles was done according to standard clinical methods.

Findings of investigation showed that the BG helps to increase the survival rate of the animals by 41 % and also prolongs the average lifetime of the killed rats by 90%.

The comparison of the survival and average lifetime of the irradiated animals with cytological indicators of blood detected a certain correlation. Preventive administration of the BG decreases leucopenia and thrombocytopenia, while keeps the number of erythrocytes unchanged in comparison with the examined indicators. Consequently, the decrease of the survival rate and average lifetime of the rats in control experiments is identified by enhancing leucopenia and thrombocytopenia.

Table 1. Survival and average lifetime of the irradiated rats during control experiments and of those with pre-administration of the BG

Group of animals	Irradiation dose, sGr	Number of rats	Average weight of animals, g	% of the survived animals	Average lifetime of the killed rats
Control	750	30	187,9±6,6	16,0	8,6±1,2
Experiment	750	30	189,2±5,0	56,7	16,2±2,2^*

Table 2. The effect of the BG on cytological indicators of blood of rats

Group of animals	Number of the examined animals	Leucocytes, thousand.	Erythrocytes, mln.	Thrombocytes, thousand
	In 1 micro liter of blood
Without exposing to radiation	12	10,1±0,2	9,3±0,3	790±72
Control	12	0,5±0,07	7,3±0,2	350±22
Experiment	12	0,9±0,15^*	8,2±0,8	560±53^*

^* the shifts credible in comparison with control data

2. Radio protective effect of BG on a bacterial- endotocsemical component of an acute radiation sickness Bone marrow form of an acute radiation sickness

The first series of experiments with the irradiated mice under a dose of 800 sGr showed that intragastric administration of the BG promotes a certain increase of a survival rate of the totally irradiated animals (table 3). Thus, in the experimental groups, administered the BG in a dose of 10 and 3 ml/kg, during 12 through 30 days survived the animals with acute radiation sickness from 66% to 33%, while the survival rate in the control group amounted from 33% (12 days) to 7% (30 days). Under the BG effect, the lethality peak shifted to a more delayed (later) data in developing acute radiation sickness, reflected on a tendency of increasing average lifetime of the exposed animals, with a mortality histogram shifted to the right.

Table 3. The effect of the BG on the survival dynamics of rats with a bone marrow for of acute radiation disease

Groups	Dosage ml/kg	n	Survival rate (%)						Average lifetime, days
			Days, Acute Radiation Sickness
			6	9	12	15	18	30
Control	Placebo	15	100	53	33	20	13	7	8.2
BG	10.0	15	100	93	66*	33	33	20	10.7
The same	3.0	15	100	86	46	40	33	33*	11.2^U
„	1.0	15	100	66	40	33	27	13	8.8

* - Reliable distinction with a control after TMF at Р£0.05; ^u – after the same U criterion

The evaluation of the body weight dynamics also demonstrated that the administration of the BG in all the tested doses exerted a positive impact on the course of acute radiation sickness, weakening the fall of the animals’ body weight to some extent at the height of a bone marrow syndrome (the impact of the preparation on the weight decrement is reliable during a variance analysis of a set of indicators for a period from the 6^th through the 12^th day, Р ≤ 0.05).

3. Mixed form of acute radiation sickness

During the 2^nd series of experiments, under a radiation dose- 950 sGr with the purpose of enhancing intestinal components under radiation damage, a part of control mice (13%) developed a typical intestinal form of acute radiation syndrome, resulted into lethality on the 4^th-5^th days, while the remaining animals were dead within up to 13 days, with an average lifetime of 7.6 ± 0.5 days (table 4).

The administration of the concentrate at the given model of acute radiation sickness also increased the survival of animals at the height of bone marrow death (from the 6^th through the 12^th day), caused a peak shift up to 12 days and somewhat increased an average lifetime. By the lethality level on the 9^th-12^th days, the BG therapeutic effect is statistically reliable by a cumulative action in a dose of 10 and 3 ml/kg. After a 30-day survival criterion, a reliable therapeutic effect of the BG on the given model of acute radiation disease was absent; however, a few animals in the experimental groups managed to survive (13% and 7%) under 100% death of the control animals.

The BG therapeutic effect is also proved by a slight fall of the body weight of test mice at the height of acute radiation sickness.

Table 4. The BG effect on the survival dynamics of mice (CВА х C₅₇BL₆) F₁ at a mixed form of acute radiation sickness (950 sGr)

Groups	Doses, ml/kg	n	Survival rate (%)										Average lifetime, days
			Days, Acute radiation sickness
			6		9	12		15	18	30
Control	Placebo	15	67		33	7		0	0	0			7,6
BG	10,0	15	73	53			46*	33*	20		13	8,2
The same	3,0	15	87	53			33	20	7		7	8,7
“-“	1,0	15	53	53			33	20	13		0	7,7
* - Reliable distinction with a control after TMF at Р£0.05

The results of this research demonstrate that the intragastric administration of the BG from the 1^st day of acute radiation sickness promotes a small increase of a survival rate of the animals with a bone marrow form of acute radiation sickness (approximately by 25%) at a 93% death rate among the control animals.

The BG therapeutic effect is also manifested by a significant peak shift of the death of animals at a more delayed (late) period and a lesser fall of the body weight at the height of acute radiation disease.

At a more acute form of acute radiation sickness with a typical intestinal syndrome, the BG reliably reduces the number of the animals killed at an earlier period after being exposed to radiation and decreases the fall of the body weight at the height of acute radiation sickness; however, it does not have a reliable effect after a 30-day survival criterion.

By the nature of the impact on the course of bone marrow and mixed forms of acute radiation sickness, the BG resembles of the effect provided by an a microbic gnotobiotic state of animals, for which a peak shift of an early lethality to the right at a time scale and a slight increase of a survival rate of animals under a total dose of 900-1000 sGr is also characteristic.

Thus, the obtained results allow us to conclude that the BG can exert a small therapeutic effect at an experimental acute radiation sickness, probably, by reducing bacterial-toxic components of radiation damage.

4. Corrective effect of BG at the intestine’s colonization resistance disorders

It has been identified that a 3-day fasting decreases the intestine’s colonization resistance of rats. Thus, with the rats fed on fodder, indicator microbes did not colonize the intestine, while the microbes were observed with the fasting rats a day after the administration in the amount of 10⁴-10⁵ m.t./1 g of a blind gut content. The total number of colon bacilli also increased among the fasting animals.

2 out of the 6 rats, administered the concentrate during a 3-day fasting, as well as the control animals, were not colonized by indicator microbes, while in the remaining cases the amount of colonization was <10² m.t./g. Within this group of rats was also observed least of all the presence of colon bacilli. The balm did not have a substantial effect on the state of colonization resistance of intact animals.

The results of the research demonstrate that the balm significantly weakens the resistance disability effect of fasting on the intestine’s colonization resistance and could be used an effective remedy for preventing and correcting dysbiotic disorders provoked by a feeding schedule disorder and possibly, other influences too.

5. Anti-diarrhea effect

5.1. Castor diarrhea

The results of the experiment are provided in table 5.

The administration of castor oil in a dose of 0.3 ml to the control mice caused an intensive diarrhea in 100% of the cases, developed after 23 min. on the average (from the 12^th to the 37^th min.). It lasted from 9 to 28 minutes (21 ±2 min. on the average).

During this period, the animals had 1 through 15 acts of defecation (7± 1.3 on the average) with a liquid discharge mixed with a large amount of mucus.

Table 5. The impact of one-time gastral administration of the BG on castor diarrhea among mice

№	Groups	Dose, ml/kg	n	Diarrhea frequency and intensity indicators
				Frequency of occurrences %	Number of acts, М±m	Latent period, min.	Duration, min.
1.	Control	Placebo	10	100	7.0±1.3	23.2+2.1	21.0±2.0
2.	BG	3.0	10	60	5.3±0.9	31.3±5.0	16.7±1.8
3.	the same	10.0	10	40^х^,+	4.2±0.8	45.7±4.1*^1	13.4±1.6*^1^,^.2
4.	Loperamide (mg/kg)	1.0	10	70	3.4±0.6	55.6±3.8*^1^,^.2	14.5±4.8*^1^,^.2
5.	The same	2.0	10	20^х	1-2	50-70	1-3

Notes: the BG and placebo were administered in the amount of 0.1 ml/10 g. 30 minutes before the intragastral administration of castor oil (0.3 ml). The figures in the brackets indicate a median of indicators; X- a reliable distinction with control after TMF.

* - the same after Student t criterion; + - the same after Manna-Witney criterion at R<0.05; The indexes 1,2 at reliability marks indicate the groups, within which the distinction was identified.

Preliminary administration of the BG in a dose of 10ml/kg prevented completely the development of diarrhea with 60% of mice (R≤0.05 after TMF), while significantly delayed it and reduced its duration. At the same time, the number of defecation decreased slightly among the animals with developed diarrhea, even though visually the volume of diarrhea reduced. Upon administering the BG in a dose of 3 ml/kg diarrhea was prevented in 40% of cases. At the given selection, this is a reliable effect (R<0.05), against the background of an unchanged intensity of diarrhea.

As demonstrated by comparative tests of anti-diarrhea preparation loperamide on the given model, castor diarrhea is rather hard to be dealt with by a corrective therapy. As can be seen from Table 5, loperamide in a dose of 1 mg/kg under the given experimental conditions did not have a substantial inhibiting impact on castor diarrhea after an alternative criterion; it had a preventive effect against developing diarrhea with 80% of mice when administered in a dose of 2 mg/kg.

Thus, by its anti-diarrhea efficiency on the given model, the BG in a dose of 10 ml/kg can be compared to loperamide, administered in a comparatively higher dose for this remedy, taken by a human in a dose of 1-2 mg.

5.2. Early post-radiation diarrhea

The results of the research are provided in Table 6 and Table 7.

During the experiments with mice it was identified that the BG in a dose of 3 and 10 ml/kg significantly inhibits early post-radiation diarrhea developed within 1 hour after being exposed to radiation. The anti-diarrhea effect of the preparation after an alternative criterion amounts to 30 through 50% (table 6). Among the other animals (with developed diarrhea) the BG in a dose of 10 ml/kg significantly increases a latent period of developing disorder, and reduces the number of defecations.

Loperamide, a highly effective remedy on the given model, exerts a protective effect nearly of the same strength as the BG in a dose of 10 ml/kg (table 7).

Table 6. The BG impact in comparison with loperamide at an early post-radiation diarrhea of mice

Groups	Dose, ml/kg	n	Diarrhea frequency and intensity indicators
			frequency of occurrences %	Number of acts, М±m	Latent period, min.	Duration, min.
Control	Placebo	15	100	8.9±0.8	14.6±1.3	37.1±3.9
BG	3.0	10	70^х	5.8±1.2	21.3±1.8	28.0±4.7
the same	10.0	10	50^х	2.7±0.6*	26.5±2.7*	20.9±3.4*
Loperamide (mg/kg)	0.3	10	20^х	2	31.0	15.3

Notes: x- a reliable distinction with control after TMF; * - the same after Student t criterion at R≤0.05.;

During the experiments with rats on an early post-radiation diarrhea model (300 Gr), the BG in a dose of 3 and 1 ml/kg prevented the development of diarrhea, respectively, with 40% (R<0.05 after TMF) and 30% (R>0.05) of the animals under 100% development of diarrhea among the control animals. In both cases, the administration of the BG led to reducing the intensity of diarrhea, the delay of its development, and decreasing its duration. In its effectiveness, the BG in a dose of 3 ml/kg on the given model stands closer to loperamide in a dose of approximately 0.5 mg/kg (table 7).

The research shows that the BG possesses a distinct anti-diarrhea activity on the models of diarrhea of various genesis a driving member of these models is an enhanced secretion of water and electrolytes in entoblast canal lumen. As known, similar mechanism is the main component in developing diarrhea during many infections and toxic infectious diseases, food poisoning, intoxications by chemical factors. An uncontrolled secretion of water is the base for diarrhea also during cholera.

Table 7. The BG impact in comparison with loperamide at an early post-radiation diarrhea of rats

Groups	Dose, ml/kg	n	Diarrhea frequency and intensity indicators
			frequency of occurrences %	frequency of occurrences %	frequency of occurrences %	frequency of occurrences %
Control	Placebo	10	100	3.3±0.6	28.5±3.2	11.7±2.6
BG	1.0	10	70	1.5±0.5*	34.3±4.6	8.2±2.1
the same	3.0	10	60*	1.4±0.2*	38.2±5.1	4.6±2.7*
Loperamide (mg/kg)	0.5	10	50*	1.5±0.4*	30.1±3.2	13.0±3.6
The same	1.0	10	10*	2	7	6

Notes: x- a reliable distinction with control after TMF (R£0.05); * - the same after Student t criterion.

The BG anti-diarrhea effect is realized during the intragastral administration of the medication a short time before exposing intestinal secretion inductors i.e. the time needed only for passing of this remedy through intestinal canal. Under the given conditions, the BG therapeutic effect could be induced mainly by a direct activity of its chemical compounds on intestinal wall, leading to reducing the mucus responsiveness and decreasing water secretion.

The obtained results and their analysis enables us to conclude that the BG could be applied as an anti-diarrhea remedy, with moderately manifested efficiency, useful for treating enterocolitis and diarrhea syndromes of various aetiology.

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