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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 hydrogen-deuterium 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.

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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 X-irradiation. 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

Treatment bioactive substances diluted with distilled water

Variations irradiated

C (control) Untreated CX V1 Volatil oil A. mandshurica V1-X V3 Polyphenols A. mandshurica V3-X

Table 2. Mus muscullus experimental variants

treated with bioactive substances diluted with deuterium depleted water

Variations non- irradiated

Treatment bioactive substances diluted with deuterium depleted water

Variations irradiated

C-DDW (control) Untreated C-DDW-X V2 Volatil oil A. mandshurica V2-X V4 Polyphenols A. mandshurica V4-X

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

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

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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

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

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.

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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).

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 anti-inflammatory, anti-allergic, antimicrobials, antioxidant and capillary protective effects (Stanescu et al, 2002).

Acknowledgements These researches are part of the

grant 33.062/2004, sponsored by CNCSIS-Bucharest. The authors address sincere thanks to Mr. Dr. Marin Popa, head of Flaw Laboratory from SC Electroputere SA

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. 34:455-465.

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, 137-140.

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.

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Annex with Figures

Figura 1. Lymphatic follicles of mouse Figura 2. Thin fibro conjunctive septa spleen (oc. 10 x ob. 10) C of mouse spleen (oc. 10 x ob. 20) C-X

Figura 3. Numerous lymphocytes at the Figura 4. Big splenic follicles of mouse red pulp level (oc. 10 x ob. 10) C-DDW spleen (oc. 10 x ob. 10) C-DDW-X

Figura 5. Hyperplasia red pulp level Figura 6. Dilatation of red pulp capillary and rare megakaryocytes of mouse and hypertrophy of fibro conjunctive spleen (oc. 10 x ob. 10) V2 septa of mouse spleen (oc. 10 x ob. 40) V2-X

Figura 7. Numerous follicles with germ Figura 8. Atrophy of white pulp of mouse

centers of mouse spleen spleen (oc. 10 x ob. 10) V4-X (oc. 10 x ob. 20) V4

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