C. Plamadeala et al.

/ Journal of Advanced Research in Physics 4(1), 011306 (2013)

1

Abstract Cytogenetic study was accomplished in order to
investigate the radiation impact on the embryos of Triticum
aestivum L. (bread wheat). VARIAN CLINAC 2100SC
radiotherapy device was used to irradiate wheat caryopses with
X-ray doses of 22.4 Gy, 33.6 Gy, 44.8 Gy, 56 Gy, 67.2 Gy.
Before the irradiation, wheat caryopses were let to germinate
in INCUCELL room in controlled environmental conditions.
Radioprotective action of ethanol extracts from Hypericum
perforatum L. and Viola tricolor L., known for their
pharmaceutical use was tested. Ethanolic plant extracts were
prepared in concentration of 0.5% by Plantavorel laboratory
in Piatra Neamt. During the qualitative cytogenetic studies
some many types of chromosomal aberrations were identified
micronuclei, delayed and expelled chromosomes, chromatin
simple or multiple bridges as well as combined complex
aberrations. The main results are consistent with different
decrements of the mitotic index revealed in Triticum aestivum
embryos treated with ethanolic extracts of Hypericum
perforatum L. and Viola tricolor L. simultaneously with almost
identical diminution of chromosomal aberration level.

Keywords cell division, X-rays, chromosomal aberrations,
radioprotector extract.
I. INTRODUCTION

Various experimental studies proved since decades ago
that ionizing radiations have the capacity to break the DNA
chain and induce chromosomal aberrations [1-3]. It is well
known that ionizing radiations (like X-rays in our study) are
characterized by genotoxic potential, meaning ionizing
radiations have the ability to affect the molecules of DNA,
the chromosomes and the genetic activity, causing
hereditary modifications. Cytogenetic study carried out on
chromosomal aberrations induced by radiation in plant
meristemes is recognized as an useful complementary tool
since much similarity with mammal chromosome damages
induced in irradiated cell cultures was noticed. Additional
advantage of low cost and higher availability of plant
meristem investigation as well as the total preservation of
result accuracy are also important.
In order to decrease the toxic effect of the ionizing
radiations on the DNA molecules radioprotector molecules
can be useful. A radioprotector substance may be
administrated before or during the irradiation its effects
being given by the ability of increasing the radioresistance,
thus diminishing the probability of appearance of
Manuscript received October 28, 2013.
*
cristinaplamadeala@rocketmail.com
chromosomal aberrations or further genetic mutations.
Herbal extracts consisting in mixtures of multiple substances
are supposed to influence and monitor radiation injuries by
free radical scavenging [4] when radiation impact result in
molecular breakings and reactive molecular fragment
releasing.
Indirect findings also emphasized the role of multiple
substance extracts on the enzyme activity in the irradiated
tissue. Karchuli et al. (2009) studied the radioprotective
effect of hydroalcoholic extract of seeds of Bixa orellana in
bone marrow cells of mice exposed to 4.0 Gy radiation dose
and found that diminished number of aberrant metaphases
resulted due to the radioprotector administration [5]. The
radioprotective action of mint leaf extract was evidenced
being supposed to occur through the decreased frequency of
chromosomal aberrations induced in bone marrow of mice
exposed to 8 Gy gamma radiations [6]. Hannequin et al. [7]
showed that intravenous administration of an ethanol extract
of Gingko biloba leaves is effective in patients after brain
irradiation as it was in the recovery of Chernobyl workers
after the nuclear accident in 1986. The radioprotective
action of Panax ginseng has been also reported [8], related
to recovery of thrombocyte and erythrocyte counts in blood
after irradiation.

II. MATERIALS AND METHODS

Cytogenetic investigations were carried out on the root
meristems developed after seed germination. The studied
biological materials were caryopses of Triticum aestivum L.
(bread wheat) with a uniform gene pool, supplied by the
Biology Faculty of Alexandru Ioan Cuza University, Iasi,
Romania. We used three groups of samples, each of them
containing fifty seeds. The first group was used as the
control group; the second group was treated with an
ethanolic extract of Hypericum perforatum L. (goatweed)
and the third with ethanolic extract of Viola tricolor L.
(heartseas). Ethanolic extracts of goatweed and heartseas
were provided by the Plantavorel laboratory from Piatra
Neamt to be tested as radioprotector. Both ethanolic extracts
were used in concentration of 0.5%.
The seeds from the control group were soaked in distilled
water for four hours, while the seeds from the other two
groups were soaked in the radio protector substance extracts,
also for four hours. After soaking, all the samples were let in
the INCUCELL room to germinate for 12 hours, in
Comparative cytogenetic analysis of
radioprotector effect of two vegetal extracts
Cristina Plamadeala
1*
, Alina Aparaschivei
1
, Iulia Bara
2
, Ramona Focea
1, 3
, Dorina Creanga
1

1
Faculty of Physics, Alexandru Ioan Cuza University, 11 Blvd. Carol I, Iasi, 700506, Romania
2
Faculty of Biology, Alexandru Ioan Cuza University, 11 Blvd. Carol I, Iasi, 700506, Romania
3
University Hospital Sf. Spiridon, Iai, Romania

2 C. Plamadeala et al. / Journal of Advanced Research in Physics 4(1), 011306 (2013)

conditions of darkness and constant temperature of
21.00.1
o
C.
After germination, the caryopses were irradiated with an
X-ray photon beam generated by the linear particle
accelerator VARIAN CLINAC 2100SC of University
Hospital Sf. Spiridon, Iasi, with a dose rate of 2.24
Gy/min. The samples were exposed for 10, 15, 20, 25 and
30 minutes, thus obtaining the calculated radiation doses of
22.4, 33.6, 44.8, 56 and 67.2 Gy, respectively [9].
After irradiation, the germinated caryopses were put again
in the INCUCELL device being kept into it for 12-24 hours,
until the roots of the seeds were no longer than 1 cm. Then
the cell chromosomes were colored using Fuelgen rapid
staining method [10]. After coloring, the top of the roots
were cut and crushed through the squash method onto
microscope slides. For cell visualization an optic
microscope NYKON Y-FL eclipse e600 was used with
attached NIKON e950 photo camera.
Qualitative studies aimed to evidence the capacity of X-
ray ionizing radiations to cause different types of
chromosomal aberrations during the mitotic cycle.
Quantitative studies included the calculation of two
cytogenetic parameters mitotic index (M.I.) equal to the
ratio of the dividing cell number to the total number of
observed cells and the aberration index (A.I.) equal to the
ration of aberrant mitosis cell number and the total observed
cells in the investigated tissue sample.
Five microscopic slides were prepared from each
experimental variant (combinations of radiation
dose/radioprotector type) and from each slide ten
observation fields were analyzed; average values and
standard deviations were calculated for the graphical
representation. Statistic t-test was applied to see the
statistical significance of the differences between the
radioprotector supplied samples and the control ones
(related to the criterion of p<0.05).

III. RESULTS AND DISCUSSION

Qualitative analyses provided information about the main
types of chromosomal aberrations micronuclei (Fig. 1a),
chromatin bridges, delayed and expelled chromosomes (Fig.
1b). Most chromosomal aberrations were detected in the
irradiated samples at high doses of X-rays, while small
number of aberrations was recorded in the seeds that were
not exposed to radiations. We consider these aberrations are
the result of environmental factor gradients.


Fig. 1. a) Cell in prophase with a micronucleus (1); b) cell in ana-telophase
with chromatin bridges (2) and expelled (3 and 4) and delayed (5)
chromosomes

In Fig. 2 the percentages of cells in each of the mitosis
phases (prophase, metaphase, anaphase and telophase) for
the control group are shown.
For the control samples (radiation dose of 0 Gy) we can
see a normal development most of the cells (about 18%
from all dividing cell percentage) being in the first mitosis
phase (prophase). It can be noticed that although the
prophase cell percentage decreased in irradiated samples
under the control level (between 8% and 15%), however, the
percentage of the cells in metaphase, anaphase and telophase
increased with the dose. More, linear increase with radiation
dose of metaphase cell percentage (from 2% in 0 Gy control
sample to about 10% in 67.2 Gy sample) could be
evidenced.

Fig. 2. The influence of X-rays on the percentages of cells in different
mitosis phases for the control group


Fig. 3. The influence of X-rays on the percentages of cells in different
mitosis phases for the samples treated with ethanolic extract of Hypericum
perforatum L. 0.5%

In Fig. 3 one can see the effect of vegetal extract from H.
perforatum plants on the wheat mitosis phases. It is evident
that for relatively higher doses, the total percentage of
prophase cells has decreased (ranging from 4 to about 10%)
comparatively with control group, except for 67.2 Gy, where
a significant positive variation with about 2% was noticed
C. Plamadeala et al. / Journal of Advanced Research in Physics 4(1), 011306 (2013)
3
(from 15% in Fig. 2 to 17%, in Fig. 3; p<0.05). The other
mitosis phase cells (metaphase, anaphase and telophase)
appeared to be non-significantly changed compared to the 0
Gy sample except for the dose of 67.2 Gy. For example
metaphase cell percentage was significantly increased
(p<0.05) from 2% in 0 Gy sample (Fig. 3) only up to 4% in
67.2 Gy exposed one and supplied with radioprotector while
about 10% level was assessed in the lack of the
radioprotector for the same 67.2 Gy dose (Fig. 2).
Consequently it resulted that the radioprotector plant extract
had a general inhibitory action on the whole mitosis
phenomenon. Deeper investigation is needed in the next
study stage in order to clarify the causes of the atypical
response for the 67.2 Gy dose.

Fig. 4. The influence of X-rays on the percentages of cells in different
mitosis phases for the samples treated with ethanolic extract of Viola
tricolor L. 0.5%

In Fig. 4 one can see that compared with the control
group, the inhibitory effect of the ethanolic extract of Viola
tricolor L. is lower than that of the extract of Hypericum
perforatum. L.
Fig. 5. The influence of radiation and radioprotector plant extracts on the
mitotic index (M.I.)

Indeed, the prophase cell percentage is diminished but
only to 11-16% (compared to the 0 Gy sample characterized
by 18% prophase cells) while in general no remarkable
changes could be noticed for the percentages of cells in the
other mitosis phases; exception was recorded also for the
67.2 Gy sample where metaphase cell percentage was
significantly increased to about 5% (Fig. 4).
As seen in Fig. 5, mitotic index (M.I.) decreases under the
influence of both of the radioprotector extracts in all
irradiated samples. Higher influence was obtained for H.
perforatum extract compared to the ethanolic extract of V.
tricolor as in H. perforatum samples lower division rate was
evidence. Otherwise the radiation exposure seems to have
stimulatory effect on cell mitosis in the radioprotector lack
(control group) as suggested also in the above graph
discussion.

Fig. 6. The influence of radiation and radioprotector substance on the
aberration index (A.I.)

Simultaneously with the diminution of the mitotic index
(M.I.) in radioprotector treated seedlings compared to the
control group, the reduction of the aberration index (A.I.)
was also evidenced (Fig. 6). There is no significant
difference between the radioprotector effect of the
Hypericum perforatum and Viola tricolor, except for the
radiation dose of 67.2 Gy; in this case one may conclude
that for high doses of radiation, the ethanolic extract of
Hypericum perforatum has a better radioprotective action on
the caryopses of Triticum aestivum.
As for the mechanisms of radioprotective action of
vegetal extracts used in this study the main evident assertion
may be formulated from the comparison of mitotic index
and aberration index graphs.
It appears that simultaneously with the decreasing of cell
division rate given by the mitotic index graph (Fig. 5), the
embryonic tissue radiosensitivity has diminished too as
emphasized by aberration index graph (Fig. 6) which is in
concordance with Bergonier-Tribondeau law. Indeed,
according to the systematic observations underlying the
radiosensitivity scale for tissues and species, a tissue
radiosensitivity is higher when mitotic activity,
differentiability capacity, oxygen and water contents are
higher. The radioprotector vegetal extracts had certainly
inhibitory effects on cell proliferation ability that ensured
also the radiosensitivity diminution as shown by lower
percentage of chromosomal aberrations. The biochemical
issues are related to the nature of molecules present in the
vegetal extracts able to determine such action mechanisms.
4 C. Plamadeala et al. / Journal of Advanced Research in Physics 4(1), 011306 (2013)

Other author reports dedicated to radioprotective action of
vegetal extracts explained the radioprotection molecular
mechanism by indirect action on the enzymes controlling
toxic products of lipid peroxidation caused by radiations
which could also happen in the present case.
For example, Kumar et al. (2003) [8] focused on lipid
peroxidation caused by radiation and consequently on the
effect of radioprotector (ginseng extract) administration on
the related enzyme activity (acid and alkaline phosphatases).
Samarth &Kumar (2003) has reported the mint extract
radioprotective effect [11]. They found that radiation
decreased the activity of peroxidase enzyme glutathione S-
transferase (able to keep down the lipid peroxidation
phenomenon) while mint extract administration has increase
the peroxidase activity and consequently reduced the toxic
effect of lipid destruction by peroxidation. But no relation
between peroxidase enzyme activity and the cell division
ability was mentioned.
The next stage of our experimental study will also take
into consideration the biochemical aspects aiming to shape
the complex frame of molecular changes induced by vegetal
extracts in the studied vegetal embryos exposed to ionizing
radiation. For the present moment we underline that the
main qualitative difference between the actions of the two
vegetal extracts from the comparison of Fig. 5 and Fig. 6
can be seen. While in Fig. 5 distinct graphical tracks of the
two radioprotectors can be seen, in Fig. 6 almost identical
patterns were obtained. This could mean that even if both
radioprotectors act according to Bergonier Tribondeau
radiosensitivity law (both radioprotectors having the same
influence action on chromosome duplication during
mitosis), different mechanisms underline the radioprotective
influence on the mitotic activity.

IV. CONCLUSION

The analysis of the cytogenetic investigation result carried
out in this work allows to formulate two principal
conclusions. First, the ethanolic extracts of both
radioprotective plants (H. perforatum L. and V. tricolor L.)
have inhibitory effects on cellular division rate, with higher
amplitude in the case of H. perforatum. Second, that the
protective actions on the cell chromosomes are basically
identical which caused almost equal decreasing effects on
the occurrence of chromosomal aberrations. Further studies
are needed to enable us to discriminate among the
peculiarities of each plant extract composition and
specificity of action.
REFERENCES
[1] C. Savakan, M.C. Toker, The effects of various doses of
gamma irradiation on the seed germination and root tip
chromosomes of rye (Secale cereale L.), in Turkish J. Bot., 1991.
[2]. L. Cheng, H. Yang, B. Lin, Y. Wang, F. Zhang, Effect of
gamma-ray radiation on physiological, morphological characters
and chromosome aberrations of minitubers in Solanum tuberosum
L., Int. J. Radiat. Biol., 2010. 86(9):791-799
[3]. H. Okamoto and A. Tatara, Effects of low-dose radiation on
the cell cycle duration of barley roots, in Environ. Exper. Bot.,
1995.35(3):379-388
[4].G. C. J agetia, Radioprotective Potential of Plants and Herbs
against the Effects of Ionizing Radiation, in J. Clin. Biochem.
Nutr., 2007. 40(2): 7481.
[5] M.S. KARCHULI, GANESH N, PROTECTIVE EFFECT OF BIXA
ORELLANA L. AGAINST RADIATION INDUCED CHROMOSOMAL
ABERRATION IN SWISS ALBINO MICE, IN INTERNATIONAL JOURNAL OF
PHYTOMEDICINE, 2009. 1: 18-21
[6] Samarth RM, Kumar A.Mentha piperita (Linn.) leaf extract
provides protection against radiation induced chromosomal
damage in bone marrow of mice, in Indian. J. Exp. Biol., 2003.
;41(3):229-327
[7]. Hannequin D., Thibert A., Vaschalde Y., Development of a
model to study the anti-oedema properties of Ginkgo biloba
extract, in Presse Med., 1986.15: 1575-1576
[8]. Kumar M., Sharma M.K., Saxena P.S., Kumar A.,
Radioprotective effect of Panax ginseng on the phosphatases and
lipid peroxidation level in testes of Swiss albino mice, in Biol.
Pharm. Bull., 2003. ;26(3):308-312.
[9]. R. Focea, G. Capraru, M. Racuciu, D. Creanga, T. Luchian,
Aberrant cell divisions in root meristeme of maize following
exposure to X-rays low doses compared to similar effects of 50 Hz
electromagnetic exposure, in EPJ Web of Conferences, 2012, 24.,
06004.
[10]. M. M. Campeanu, M. Maniu, I. C. Surugiu, Genetica -
metode de studiu (Genotoxic effects of ELF fields in plants), in
Ed. Corson, Iasi, 2002.
[11] Samarth RM, Kumar A.Mentha piperita (Linn.) leaf extract
provides protection against radiation induced chromosomal
damage in bone marrow of mice, in Indian. J. Exp. Biol., 2003.
41, 229237