the effect of biologically active substances of aralia mandshurica

Annals of RSCB
Vol. XV, Issue 2
THE EFFECT OF BIOLOGICALLY ACTIVE SUBSTANCES OF
ARALIA MANDSHURICA AND DEUTERIUM DEPLETED WATER ON
THE STRUCTURE OF SPLEEN IN MUS MUSCULLUS
Cornelia Editoiu¹, Carmen Popescu², Greta Ispas³, G. C. Corneanu ,
M. Zagnat , I. Stefanescu
¹“VICTOR BABES” CLINIC HOSPITAL OF INFECTIOUS DISEASES AND
PNEUMOPHTISIOLOGY, CRAIOVA; ² EMERGENCY HOSPITAL, CRAIOVA; ³
MEDICAL CLINIC, CRAIOVA; ”VASILE GOLDIS” WEST UNIVERSITY, ARAD;
“GRIGORE TRAIAN POPA” UNIVERSITY OF MEDICINE AND PHARMACY, IASI;
N.R.-D.I. FOR CRYOGENICS AND ISOTOPES SEPARATION, RAMNICU VALCEA
Summary
In this experiment it was analyzed the effect of some biologically active substances
extracted from the cortex of the Aralia mandshurica plants (Chisinau population) on
laboratory mice (Mus muscullus), compared with a stress factor (X- ray radiation). The
dilutions were made in distilled water and in deuterium depleted water. The combined
administration of Aralia mandshurica plants cortex extracts presented a protective effect
against X-ray radiation but also an immunostimulating effect, especially if the dilution was
carried out with DDW.
Key words: Aralia mandshurica, volatil oil, polyphenols, Mus muscullus, radioprotective
effect
corniedi@yahoo.co.uk
Introduction
Aralia mandshurica (Araliaceae
Fam.,
Dicotyledonatae
Class,
Angiospermatophyta
Phylum)
(Sârbu,
1999) is a shrub of 3 to 6 meters high,
originating from Primorie region in Siberia
(Russia), grows spontaneously in the Far
East and is inserted in culture, including in
Moldavia. As a vegetable product, the plant
roots are used because of the therapeutic
properties (immunostimulating - Corneanu
et al, 2003, stimulating and repairing Zagnat, 2004). The plant root contains
triterpenoid saponoside - aralozide (4.7%),
volatil oil (0.12%), flavonozide, resins and
the aralin alkaloid (Nistreanu, 2000;
Stanescu et al, 2004). In the Oriental
traditional medicine are used extracts from
the roots of species particularly in cases of
physical and mental strain, convalescence,
asthenic states, the beneficial effects being
attributed to aralozides which have the
property to increase the ability of
adaptability of the organism in conditions
of overload (Stanescu et al, 2002).
The deuterium depleted water / ultra
light water (DDW) is low-deuterium water,
30-120 ppm deuterium; its physical aspect
looks like colorless liquid, flavored water,
with a low content in ion and hydrogendeuterium isotope. The effects of this kind
of water are great: increased vascular
reactivity, nonspecific immune stimulation,
increased animal resistance to sublethal and
lethal doses of radiation, remission of tumor
cell lines, etc. (Titescu, 1999). DDW affects
both the normal cells proliferation and the
pathological ones, being involved in the
processes of apoptosis and mechanisms anti
carcinogens (Corneanu et al, 2005).
In this experiment we analyzed the effect of
administration of some biologically active
substances in dilutions made with normal
water or ultra light water, to the action of a
stress factor, the X-rays.
The experiment is part of a doctoral
thesis done under the direction of Professor
Doctor Corneanu Gabriel.
212
Annals of RSCB
Vol. XV, Issue 2
Materials and methods
In this case, there were used Mus
muscullus healthy young animals, with
about 25 g per specimen. Following the
terms of professional bioethics, the version
number of animals was of 3 individuals.
The biologically active substances
used (extraction of volatil oil and extract of
polyphenols with rest of flavones) were
obtained from the cortex of the Aralia
mandshurica plants, Chisinau population,
and the dilutions were made in distilled
water and in deuterium depleted water, with
30 ppm deuterium.
The stress factor was the X-rays
released by a RAP-150/300 device (ex
USSR), in a single dose, acute to the
parameters: 250 kV, 5mA, d.f. = 500 mm, 1
mm Al. filter, the total dose rate 52.8 R /
min, the total acute dose 528 R (5.28 Gy).
The bioactive substances were
administered intraperitoneally, 0.5 ml
solution, of 0,01% concentration, an
injection at two days. Three injections were
administered before the X-irradiation
(which took place a day after the third
injection), followed by two shots after Xirradiation. The slaughter was made one
day after the last injection and spleen was
taken to analyze its structure. The
experimental variations are shown in Tables
1 and 2.
Table 1. Mus muscullus experimental variants
treated with bioactive substances diluted with distilled water
Variations
non- irradiated
C (control)
V1
V3
Treatment bioactive substances
diluted with distilled water
Untreated
Volatil oil A. mandshurica
Polyphenols A. mandshurica
Variations irradiated
CX
V1-X
V3-X
Table 2. Mus muscullus experimental variants
treated with bioactive substances diluted with deuterium depleted water
Variations
Treatment bioactive substances diluted with
deuterium depleted water
non- irradiated
C-DDW (control)
Untreated
V2
Volatil oil A. mandshurica
V4
Polyphenols A. mandshurica
Spleen is the largest lymphoid
organ of the body, with an important role in
immune function and serves as a filter for
the abnormal or senescent cells. The White
Pulp is the spleen lymphoid component
and includes B and T lymphocytes
compartments. In terms of histology it
consists of: periartehale pehlimfatice
sheaths and splenic Malpighi corpuscles.
The Periarteholare lymphatic sheaths
(PALS) represent lymphoid tissue sleeves
(timodependent areas) which contain
Variations irradiated
C-DDW-X
V2-X
V4-X
effector and regulatory T lymphocytes,
interdigitating
dendritic
cells
and
macrophages. Red Pulp surrounds the white
pulp and consists of splenic tissue cords
(the Bilroth cords) situated among the
venous sinuses. The Bilroth cords are
composed of a three-dimensional network
of reticular cells and reticular fibers,
forming a functional unit with sinuses
adventice. In this network, free spleen cells,
with
phagocytic
function,
move
(macrophages). The core of these
213
Annals of RSCB
Vol. XV, Issue 2
macrophages also shows nuclear appendix
(Corneanu et al, 2007). At the Bilroth
Cords level, the traffic has an “open”
nature, because blood comes in direct
contact with the spleen cells (Cesta, 2006,
Raica et al, 2004). The process of apoptosis
plays an important role in the development
of spleen, especially the lymphoid
compartment, predominantly in the germ
centers of B cells (Corneanu et al, 2005).
Results and discussion
numerous lymphocytes also at the red pulp
level; rare megakaryocytes and connective
thin fibrous septa are observed in the
interstitial spaces and
into capillaries
sinusoids.
Irradiated control variant (C-DDW-X): the
hypertrophy and hyperplasia of white pulp, the
presence of large splenic follicles, some with
germ centers and numerous lymphocytes at
the red pulp level, too.
V2 (Volatil oil): numerous cells with sub
capsular changes, the deleting of white pulp,
diffusely lymph-plasma infiltrated, and the red
pulp hyperplasia with the proliferation of
megakaryocytes.
V2 (Volatil oil)-X: hyperplasia of splenic
follicles, the red pulp expansion by sinusoids
dilated capillaries and by fibrous connective
septa hypertrophy and moderate increases in
the number of megakaryocytes.
V4 (polyphenols): the white pulp
hypertrophy, rare cells with nuclear atypia,
numerous follicles with germ centers.
V4 (polyphenols)-X: marked atrophy of white
pulp evidenced by the presence of some small
and rare splenic follicles; the red pulp
hypertrophy marked by intense dilation of
capillary sinusoids with this many
megakaryocytes. At the interstitial level, the
fibrous connective septa appear uneven, some
of them hypertrophied and others normal.
Corneanu et al, 2007 have shown that the
action of a stress factor (DDW or X-rays), the
macrophages in the spleen were in great
number; also, for the lymphocytes from the
Malpighi lymphatic glomerule in the spleen
was emphasized the presence of many
megakaryocytes with numerous residual
corpuscles, as a result of the phagocytic
activity.
Mus muscullus experimental variants treated
with bioactive substances diluted with distilled
water:
The Control Variant (C) Spleen: histological
appearance characteristic of the species, with
the presence of the white and red pulps: the
white pulp is composed of lymphatic follicles
consisting of small and medium lymphocytes
and centered by a splenic arteriola. Red pulp
appears among the lymphoid follicles,
diffusely distributed and consisted of capillary
sinusoids filled with blood and other blood
figurative elements (polymorpho-nuclear,
lymphocytes) and also with the highlighting of
rare megakaryocytes.
The Irradiated Control Variant(CX):
hypertrophy and hyperplasia of lymphatic
follicles, nuclei with a tendency to dark (like
defensive reaction to the stress factor), rare
megakaryocytes and thin fibro conjunctive
septa.
Vl (Volatil oil) cells with nuclear atypia,
limfoplasmatic inflammatory infiltrate follicles
arranged in the form of germ centers.
Vl (Volatil oil)-X: reticular cell hypertrophy,
the red pulp hypertrophy with the proliferation
of hypotrophy megakaryocytes and the white
pulp hypotrophy.
V3 (polyphenols): white pulp hypertrophy,
with
some
confluent
follicle;
rare lymphoid follicles with germ centers,
many cells with nuclear atypia.
V3 (polyphenols)-X: Rare cells with nuclear
atypia, numerous follicles with germ centers.
Mus musculus experimental variants treated
with bioactive substances diluted with
deuterium depleted water:
The Control Variant (C-DDW): Spleen: the
white pulp hypertrophy and marked
hyperplasia, with the presence of large splenic
follicles, some with germ centers and
214
Annals of RSCB
Vol. XV, Issue 2
Conclusions
X-radiations induce structural changes
- Malpighi lymphatic follicle hypertrophy and
hyperplasia, many lymphocytes present also in
the red pulp and thin fibrous connective septa.
The administration of DDW, only,
produces structural alterations in the white
pulp, with the presence, in the red pulp of
lymphocytes whose cell membrane is broken;
the lymphocyte alteration induced by DDW
being stronger than the one induced by X-rays.
The administration of essential oil
from the cortex of Aralia mandshurica, on
non-irradiated variants produces reduced
inflammatory infiltrates, probably due to the
monoterpenes hydrocarbons contained in the
essential oil which, diluted in DDW, has an
antiseptic,
anti-inflammatory
and
immunomodulating effect. Similar issues were
reported in other studies, too (Istudor, 2001).
Acknowledgements
These researches are part of the
grant 33.062/2004, sponsored by CNCSISBucharest. The authors address sincere
thanks to Mr. Dr. Marin Popa, head of Flaw
Laboratory from SC Electroputere SA
In irradiated variants, administration of
the essential oil diluted with DDW shows a
stronger protective effect on spleen structure
than in distilled water, observed in splenic
follicles hyperplasia, red pulp expansion and
immunostimulative by increasing the number
of megakaryocytes.
In case of using the polyphenols
extract from the cortex of Aralia mandshurica
diluted with DDW has a protective effect on
the structure of spleen, observed in the marked
hypertrophy of the red pulp by intense dilation
of capillary sinusoids and with the presence of
numerous megakaryocytes.
The flavones derivatives develop antiinflammatory, anti-allergic, antimicrobials,
antioxidant and capillary protective effects
(Stanescu et al, 2002).
Craiova, who performed X-irradiation of
animals. Also thank Dr. Mr. Marin Zagnat,
UMF Iasi, which used to extract bioactive
substances (volatile oil and polyphenols)
from the cortex of Aralia mandshurica.
References
Cesta MF., 2006, Normal structure, function, and
histology of the spleen. Toxicol. Pathol. 34:455465.
Corneanu G., Craciun C., Ciupina V., Prodan G.,
Corneanu M., Atyim P., Stefanescu I., Iacob M.,
Corneanu L-M., 2005, The efect of Nigella sativa
L. alkaloids and of deuterium-depleted water on
Mus musculus L. spleen, Revue de Cytologie et
Biologie vegetales-Le Botaniste. 28 (s.i.). 84-91.
Corneanu G., Corneanu M., Atyim P., Craciun C.,
2007, The COX-2 gene implication in the
immune response (experiments on animals, Mus
musculus L.), Analele Universitatii din Craiova,
Agricultura-Montanologie-Cadastru.
vol.
XXXVII/A. 525-535.
Istudor V., 2001, Farmacognozie. Fitochimie si
fitoterapie, Ed. Medicala, Bucuresti.
Nistreanu A., 2000, Farmacognozie. USMF
..Nicolae Testemitanu", Chisinau.
Raica M., Mederle O., 2004, Histologie teoretica si
practica, Ed. Brumar, Timisoara: 287-301, 137140.
Sarbu A., 1999, Biologie vegetala, Note de curs,
Editura Universitatii Bucuresti.
Stanescu U., Miron A., Hancianu M., Aprotosoaie
C., 2002, Bazele farmaceutice, farmacologice si
clinice ale fitoterapiei, (vol. II). Ed. « Gr. T.
Popa », Iasi, 76-145.
Stanescu U., Miron A., Hancianu M., Aprostosoaie
C., Plante medicinale de la A la Z, monografii
ale plantelor de interes terapeutic, 2004, (vol.
II). Ed. « Gr. T. Popa », UMF Iasi, 613.
10. Titescu G., Iliescu R., Nastasa V., Bild W.,
Stefanescu I., Haulica I., Lupusoru C., 1999, Apa
echilibrata in deuteriu. Research concerning the
radioprotective and immunostimulating efects of
deuterium-depleted water, Rom. J. Physiol. 36
(3-4): 18-205.
215
Annals of RSCB
Vol. XV, Issue 2
Annex with Figures
Figura 1. Lymphatic follicles of mouse
spleen (oc. 10 x ob. 10) C
Figura 3. Numerous lymphocytes at the
red pulp level (oc. 10 x ob. 10) C-DDW
Figura 5. Hyperplasia red pulp level
and rare megakaryocytes of mouse
spleen (oc. 10 x ob. 10) V2
Figura 7. Numerous follicles with germ
centers of mouse spleen
(oc. 10 x ob. 20) V4
Figura 2. Thin fibro conjunctive septa
of mouse spleen (oc. 10 x ob. 20) C-X
Figura 4. Big splenic follicles of mouse
spleen (oc. 10 x ob. 10) C-DDW-X
Figura 6. Dilatation of red pulp capillary
and hypertrophy of fibro conjunctive
septa of mouse spleen (oc. 10 x ob. 40) V2-X
Figura 8. Atrophy of white pulp of mouse
spleen (oc. 10 x ob. 10) V4-X
216