SOCIETATEA ROMANA DE ONCOLOGIE COMPARATA

1 ROMANIAN SOCIETY OF COMPARATIVE ONCOLOGY

SOCIETATEA ROMANA DE ONCOLOGIE COMPARATA
ROMANIAN SOCIETY OF COMPARATIVE ONCOLOGY

REVISTA ROMAN^
DE ONCOLOGIE COMPARATA

ROMANIAN JOURNAL
OF COMPARATIVE ONCOLOGY

EDITURA CERES I
CERES PUBLISHING HOUSE
 TESTAREA EFICAGIT~TII UNOR EXTRACTE DIN PLANTE
ASUPRA EVOLUnEl HEPATOMULUI RS1, IN PERSPECTIVA

THE TEST OF EFFICACY OF SOME PLANT EXTRACTS

IN THE EVOLUTION OF THE RS1 HEPATOMA,
IN VIEW TO USE -m
AS ADJUVANTS IN CHEMOTHERAPY
EUGENIA TIRIFON', ZENAIDA STOENESCU2, M. VASILESCU~, IULIANA GRUIA',
MONICA NISTOROIU', MARIETA PANAIT', MADALINA CHE$NOIU', ANA-MARIA BURDU$EL',
IOLANDA DUMITRESCU', ANTONELA BU$CX1, DANIELA F M T I ~ ' ,
CRlNA TIRIFON2, N. MANOLESCU', S. CINCX'
lInstitutu1 Oncotogic ,,Pro$ DI:Al. 7kestioreanu" Bucure~ti; 2Universitatea de Medicid ~i Famacie
,,Carol Davila" B u c u ~ t i ;3Ministerul Agriculturii ji Dezvoltirii Rurale, Bucure~ti / 'Institute of Oncology
Pro$ DI: Alex. Trestioreanu Bucharest, Romania; 2University of Medicine and Pharmucy ,,Carol Davila"
Bucharest, Romania; 3Ministry of Agriculrure and Rural Development, Bucharest, Romania

REZUMAT
fn acest experiment aujost testate efectele unor extracte
din rridricini de plante medicinale administrate la ~obo-
h i Rtar cu hepatom RSI, in varianfele-preventiv,tera-
peutic gi tardiv terapeutic, tn scopul indenf@cdriiunor
posibile ejecte imunostimulutoare pilsau antitumorale.
LuGnd fn considerare udtoarele rezultate: nivel cres-
cut de supraviepire, scriderea sintezei de AND pard la
limite nonnale, diminuarea markerilor de stres oxidativ,
valorile concentrafiilorde citocromi P450 ,vi a activitdfii
GST, sa poate stabili ejicacitatea produselor testate.
Nivelul scdzut a1 toxicitafii ~i a1 ejectelor secundare su-
gereazd cd extractele testate ar putea fi utilizale ca posi-
bili adjuvanti in chimioterapia antitumoral6 la anima-
lele de laborator.
Cuvinte cheie: citostatice, metastaze, chimioterapie, siner-
gism, imunostimulutor
ABSTRACT
To identifi possible new immunostimulating a d o r anti-
tumor egects, some extracts from medicinal plant roots
were tested in preventive, therapeutic and tardive thera-
peuric mode on 140 Wistar rats with RS1 Hepatoma.
T&g into acount the following results: a higher sur-
vival rate, decreases the DNA synthesis lo the normal
limit, diminution the oxidative stress markers, the values
of cytocromes P45O concs and GST activities, revealed
the eflciency of studied extracts. The lack of toxicity aiul
of secondary eaects, suggerates that the investigated
extracts could represent some possible adjuvants to anti-
tumoral chemotherapy in laboratory animals.
Key words: cytostatics, metastares, chemotherapy, syner-
gism, immune-stimulating

- Complica@e chimioterapiei limiteazti utilizarea

citostaticelor, deoarece organismul animalelor devine sen-
sibil la infectii, iar sdderea imunitg$i favorized evo-
lutia cancem1ui $i apariga metastazelor. Restabilirea ca-
pacitgfii do a p h a organismului este o problemti ma-
jori cu care se confruntii specYialigtiiin timpul fi dupg te-
rapia citostaticg. (4)
- fn scopul prevenirii complica@lor chimiokrapiei,
s-a testat actiunea imunostimulatoare gilsau antiturnoralti
prin administrarea preventivg $i terapeutid a unei aso-
cieri cu efect sinergic de extracte din rgdicini de plank
(Acanthopanax senticosus / Eleutherococcus senticosus,
- The complications of chemotherapy are limiting
the use of cytostatics because the animal body becomes
sensible to infections and the lower of immunity favors
cancer evolution and the metastasis. (4)
- For prevention of complications of chemotherapy,
the irnmunestimulating andlor antitumoral action by pre-
ventive and therapeutic administering of an association
of some plant roots extracts with synergic effect (Acan-
t h o p m senticosus / Eleutherococcus senticosus, Ca-
lendula oficinalis, Chelidonium rnajus), to Wistar rats
with RS 1 hepatoma.
 REVISTA R O DE
~ONCOLOGIE
U COMPARAT~I ROMANIAN JOURNAL OF COMPARATIVE ONCOLOGY
Calendula oficinalis, Chelidonium mujus), la ~obolani
Wistar cu hepatom RS 1.
Experimentul s-a efectuat pe 140 qobolani Wistar
inoculati cu hepatom RS1 (indus prin administrarea sub-
cutanat2 a 2.500.000 - 4.000. 000 celule tumorale/0,5 ml
solutie 0,9% NaCI).
Extractele din plante testate, au fost administrate in
citeva variante, astfel:
Loturile experimentale:
- Lot I (preventiv) - extractele administrate preven-
tiv, cu 2 luni inainte de inocularea tumorii;
- Lot II (terapeutic) - extractele administrate con-
comitent (in acelqi timp) cu inocularea tumorali;
- Lot III (terapeutic tardiv) - extractele administrate
la 25 de zile dupi inocularea tumorali;
- Lot IV - martor inoculat cu tumorg $i netratat;
- Lot V - martor netratat, neinoculat tumoral;
- Lot VI - martor tratat $i neinoculat cu tumora;
- La toate animalele, extractele au fost administrate
at& in hrani, c k $i prin aplicafii periturnorale.
S-au determinat urmitorii paramehi: spor de crevtere
in greutate, evaluarea tumorali (volum, greutate .yi aspect
macroscopic), rata de supraviewire $i mortalitate, concen-
tratia dialdehidei malonice, activitatea ceruloplasminei,
concentrapa de tioli, concentraoa citocromilor P450, ac-
tivitatea GST, fracoa de celule aflate ?nsintei5, indicele de
proliferare $i tndicele ADN, indice apoptotic. (1,2)
REZULTATE $I DISCUW
.
1. Morfometria gi rata de supraviepire
Examinarea macroscopici a evolutiei tumorale re-
levi c i in cazurile administrkii preventive a extractelor,
se produce reducerea evolufiei tumorale.
in cazul adrninistrZrii terapeutice, se produce liza
tumorali.
Fig. 1 - Lot I (preventiv) / (preventive)
Extractele din plante administrate cu 2 luni inainte
de inocularea tumorali; aspect macroscopic la 25 zile
dupi inoculare-/ Extracts administered previously
to tumor innoculation (2 months before); macroscopic
aspect at 25 days afer innoculation
NO. 1512008
MATERIAL AND METHOD
The experiment was made on 140 Wistar rats with
RS1 Hepatoma (induced by administration, subcuta-
neous of 2,500,000 - 4,000,000 tumor cells 10.5 ml0,9%
NaCl) and tested extracts were administrated in several
modes.
Experimental lots:
- Lot I (preventive) - extracts administered previ-
ously to tumor innoculation (2 months before);
- Lot II (therapeutic) - extracts administered con-
comitantly (at the same time) with tumour innoculation
- Lot ILI (tardy therapeutic) - extracts administered
at 25 days after tumor inoculation;
- Lot IV - ,,control" without treatment and inoculat-
ed with tumor;
- Lot V - ,,control" without treatment and tumor;
- Lot VI - ,,controlutreatment and without tumor;
- To all animals, extracts were administered both in
food and in peritumoral applications.
There were surveyed: whole body weight, tumor
volums, survival rates, and after sacrifications: tumor
weight, macroscopic aspects, as well as some molecular
markers: malondialdehyde, thiols, P450 cytochromes
concs, ceruloplasrnin and GST activities, S-phase cell
fractions, prolipherative index. (1,2)
$ESULTS AND DISCUSSIONS
1. Morphometry and survival
The Macroscopic Exam of the tumor evolution re-
veals, for the cases under previously or therapeutic treat-
ment, a reduced tumor evolution.
In therapeutic cases appear the tumor lysis.
1.a. Survival Mean Time
The extracts administration increases survival rates:
Fig. 2 - Lot 11(terapeutic)/(therapeutic)
Extractele administrate concomitent cu inocularea
tumoral5i; aspect macroscopic la 25 zile dupg
inocularea tumorali / Extracts administered concomi-
tently with tumour innoculation; macroscopic
aspect at 25 days after innoculation
 REVISTA ROW
DE ONCOLOGIE COMPARAT~I RONlANlAN JOURNAL OF COMPARATIVE ONCOLOGY No. 1512008

Fig. 4 - Lot IV - Martor - inoculat cu tumora $i

netratat / ,,Control1'(with tumor and without treatment)
Aspect macroscopic a1 evolufiei tumorale la 25 zile
dupi inocularea tumorali / Macroscopic aspect of
Fig. 3 - Lot I1 (terapeutic)/(therapeutic)
tumor evolution at 25 days after the tumor innoculation
Aspect macroscopic a1 tumorii izolate la 25 zile
dupi inocularea tumorali /Macroscopic aspect
of isolated t u m r at 25 days afrer innoculation

1.a. Timpul mediu de supraviefuire

Administrarea extractelor din plante favorizeazi
cre$terea ratei de supravietuire, astfel: 42 zile - varianta
preventivi, 38 zile - terapeutic $i 37 zile - terapeutic tar-
div, comparativ cu martorul- 31 zile (graficul 1).

1.b. Greutate/volum tumoral

Valorile greut2~ii/volurnuluitumoral sunt semnifica-
tiv reduse, attit fn varianta preventivi - 1,39 cm3 / 1,48 g,
c3t $i fn cea terapeutici - 3,83 cm3 / 4,64 g, comparativ
cu administrarea tardivi - 12,55 cm3 / 14,27 g, sau cu
cele ale martorului - 23,05 cm3 / 24,31 g (graficul2).
2. Analiza fazelor ciclului celular yi a indicilor de
proliferare celularii prin metoda citometriei in flux
Tumorile RS1 sunt agresive (S = 19,93%, $i neardi-
ploide (indice AND = 1,22).
Sensibilitatea celulelor tumorale la extractele din
plante, diferi in func@ede momentul administririi pro-
duselor testate.
Fig. 5 - Lot IV - Martor - inoculat cu tumora $i
netratat / ,,Controlu (with tumor and without treatment)
Aspect macroscopic a1 evolutiei tumorale la 25 zile
dupi inocularea tumorali / Macroscopic aspect of
tumor evolution at 25 days afrer the tumor innoculation
42 days for preventive, 38 d for therapeutic, and 37 days
for tardive therapeutic, comparing to 3 1 d for control lot.
(graphic 1).

LOT 1 -2 months before tumor

innoculation

I LOT I I - conncomitently with

tumor innoculation

I LOT I l l - 25 days after tumor

innoculation

d LOT IV - "control"without
treatment and inoculated with
tumor

Graficul 1 / Graphic 1 - Tmpul mediu de supraviefuire (zile) /Mean time survivaWdays

 REVISTA ROMBN~~ 1 ROMANIAN JOURNAL OF COMPARATIVE ONCOLOGY NO. 1512008
DE ONCOLOGIE COMPARAT~~

I LOT 1 -2months before tumor

innoculation

I LOT I I - conncomitently with

tumor innoculation

I LOT I l l - 25 days after tumor

innoculation
' ' ^T
IV - "control" without
3tment and inoculated with
tumor
---- -- -

hic 2 - Media volumului tumoral /lotlcm3/Mean volume

Channels (FU-A)
Fig. 6 - Lot I - preventiv /Lot I - preventive Fig. 7 - Mot IV - r ioculat cu tumori
$i f81-5tratament / Lot IV - ,,controla without
J treatnzent and innoculated with tumor

1.b. Tumor weight/volume

The weight/volurne tumor values are signific
duced in both the preventive - 1,39 cm3 / 1,48 g
concomitant phase - 3,83 cm3/ 4,64 g, if com
the late administration - 12,55 cm3 / 14,27
ones of the control lot - 23,05 cm31 24,3 1 g

2. The analysis of cell cycle phases "*r

o The sensitivity of tumor cells to the plant extracts
- - -
Fig. 8 Lot I terapeutic /Lot 1 therapeutic lot
La tumorile analizate, efectul antitumoral atinge ni-
vele maxime, in varianta administriirii preventive a extrac-
proliferative indices, determined by the fhW@ume-
tric method
RS 1 tumors are aggressive (S = 19,93%), and near-
diploid (DNA index = 1,22)
treatment varies depending on the moment when the tested
product is administrated.
At the analyzed tumors, the antitumor effect reaches
the maximum level when the plant extracts are previously
administrated, with decreasing of DNA synthesis to the

1002
 REVISTA R O MDE~ONCOLOGIE COMPARAT~I ROMANIAN JOURNAL OF COMPARATIVE ONCOLOBY NO. 1512008

i ILOT 1 -2 months before tumor

innoculation

LOT II - conncomitently with

tumor innoculation

L O T Ill - 25 days after tumor

innoculation

1 w LOT IV - "control. without

treatment and inoculated with
tumor
.-- .. .- - . . .. -

Graficul 3 /Graphic 3 - Valorile Indicelui de Stres Oxidativ / I S 0 Value

telor din plante, prin scriderea sintezei ADN pin3 la limite
normale (S = 4%) ~i acumularea celulelor in faza GI.
Distribuirea cantitativg a AND-ului in fazele ciclu-
lui celular pentru lotul preventiv.
Distribuirea cantitativi a AND-ului in fazele ciclu-
lui celular pentru lotul martor.
Distribuirea cantitativg a AND-ului in fazele ciclu-
lui celular pentru lotul I1 (extractele administrate con-
comitent cu inocularea turnoralg).
normal limit (S = 4%) and accumulation of cells in G1
phase.
DNA quantitative distribution in the cell cycle phas-
es for the preventive lot
The DNA quantitative distribution in cell cycle
phases for the control lot
DNA quantitative distribution in cell cycle phases
for lot I1 (extracts administered concomitently with tu-
mour innoculation)

3. Valorile Indidui de Sh.es Oxidativ (ISO) 13. The oxidative stress index (IS0 Value)
Produsele testate prezinti un efect protector asupra The products have a oxidative stress protection
stresului oxidativ, prin scgderea valorilor indicelui (ISO), effect, decreasing the oxidative stress index, especially in
in special ?n varianta administr8rii preventive (graficul3). the prevention phase (graphic 3).

4. Markerii biologici ai sistemului de detoxifiere
P450 prezintil cea mai sernnificativg descrevtere
(cca 40%) Fn cazul asocierii sinergice dintre evolutia tu-
moral3 ~i administrarea preventivl a extractelor din
plante (lot I), deoarece acestea actioneazi in primele faze
ale evolutiei tumorale (graficul4).
4. Bilogical markers for the detoxification system
P450 - the most signifficant degrease appears (cca
40%) in the case of the synergic association of the tumor
evolution, with the preventive administration of extracts
(lot I), because these act in the very early stages of tumor
evolution (graphic 4).

0.45
0.401
/ W LOT 1 -2 months before tumor
innoculation
0,4 I
I W LOT II - conncomitently with
0,35 ' I tumor innoculation

W LOT I11 - 25 days after tumor

ILOT IV - "control" without

: treatment and inoculated with
; tumor
I W LOT V - "control" without
I treatment and tumor

,
.I LOT Vl - "control" treatment and
without tumor

Graficul4 / Graphic 4 - Activitatea citocromilor P450 / The activity of P450 c y t o c h m m ~

 REVISTA ROW
DE ONCOLOGIE COMPARAT~I ROMANIAN JOURNAL OF COMPARATIVE ONCOLOGY NO.1512008
Graficul5 /Graphic no. 5 - Activitatea glutationtransferazei (GST) /
GST activities
GST - activitatea enzimaticri cre$te cu aproximativ
50% la animalele shitoase qi tratate (Lot IV) datorid efec-
tului inductiv al extractelor asupra GST-ului, comparativ cu
animalele shitoase $i netratate (Lot V) (graficul5). Nive-
lul concentratiei P450 scade, in timp ce nivelul activititii
GST crqte, ca rezultat a1 actiunii enzimatice protectoare a1
extractelor din plante aspura organismului animal.
CONCLUZII
1. Abinistrarea extractelor din plante produc creqterea
ratei de supraviepire: 42 zile pentru varianta preven-
tivi, 38 zile pentru cea terapeuticg $i 37 zile pentru
varianta terapeutic tardiv, comparativ cu 3 1 zile pentru
lotul martor.
2. Indiferent de varianta administririi, la loturile tratate,
s-a observat o descrqtere semnificativi a volumului
tumoral (peste 50%), comparativ cu lotul martor.
3. Dirninuarea valorilor markerilor oxidativi, indicg efec-
tul protector a1 produselor asupra stresului oxidativ, in
special in varianta adrninistriirii preventive.
4. La tumorile analizate prin citometrie in flux, afectul
antitumoral atinge nivele maxime, in varianta admin-
istriirii preventive a produselor testate, respectiv, prin
sciderea sintezei de ADN, pihi la limita normali
(S=4) $i acurnularea celulelor in faza GI.
5. Valorile enzimelor implicate in sistemele de detoxi-
fiere, respectiv, concentratia citocromi P450 $i activi-
tatea GST, relevti eficienfa produselor testate. S-a ob-
servat o creftere semnificativi a activitioi GST, at& la
animalele shitoase, clt $i la cele tratate, in special in
varianta preventivri $i terapeutici.
... A
.
I
LOT I - 2 months before tumor
innoculation
LOT I I - conncomitently with tumor
innoculation
LOT I l l - 25 days after tumor
innoculation
LOT IV - "control"without
treatment and inoculated with
tumor
LOT V - "control"without treatment
and tumor
I LOT VI- "control" treatment and
without tumor
GST - the enzyme activity aproximatively grows
with 50% at the healthy animals (VI) due to the inductive
effect of plants extract on GST, compared with the healthy
and nontreated ones (lot V) (graphic no 5).
P450 concentration degreases, while GST activity is
increased as a result of the plants protective enzymes
action.
CONCLUSIONS
1. The extracts administration increases survival rates: 42
days for preventive, 38 d for therapeutic, and 37 d for
tardive therapeutic, comparing to 3 1 d for control lot.
2. A significant tumor volume decrease in extracts cases,
(more than 50%) was observed, comparing to tumor
case.
3. Diminution of the oxidative markers indicated a pro-
tective effect over oxidative stress, mainly in 'preven-
tive' administration.
4. At the analyzed tumors by flow-citometry, the antitu-
mor effect reaches the maximum level when the plant
extracts are previously administrated, with decreasing
of DNA synthesis to the normal limit (S = 4%) and
accumulation of cells in G 1 phase. *
5. Values of cytocromes P450 concs and GST activities -
enzymes involved in detoxication pathways - revealed
the efficiency of studied extracts. Significant increas-
es of GST activities were noticed, both in healthy ani-
mals and in tumor cases, especially in preventive and
therapeutic cases.
 REVISTA R O MDE~ONCOLOGIE
~ COMPARAT~ I ROMANIAN JOURNAL
.- OF COMPARATIVE ONCOLOGY No. 1512008

6. Lulnd in considerare rezultatele mentionate anterior,
respectiv: nivel crescut de supraviepire, greutate cor-
poralI crescutg, sciderea indicelui de proliferare tu-
moralii, efectul protector asupra stresului oxidativ si a1
sistemului de detoxifiere, se poate considera c l produ-
sele testate prezintSi efect imunostimulator. Extractele
din plante testate pot fi utilizate ca posibili adjuvanti in
chimioterapia la animalele de laborator, datoriti nive-
lelor reduse privind toxicitatea qi efectele secundare.
6. Taking into acount the previous mentioned results: a
higher survival rate, a higher weight, decreases of pro-
liferative index, effects on enzymes and systems invol-
ved in oxidative stress or detoxication, we assume that
plant extracts exhibit an irnmunostirnulatory effect.
We shall mention the lack of toxicity and also of the
secondary effects, suggesting so that the investigated
extracts may represent some possible adjuvants for
chemotherapy in laboratory animals.

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(22). 7 130-9

2. Omllra T, Sato R - The carbon monoxide - binding pigment of liver micro-

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somes. Solubilization, purijcation, properties. J.Biol.Chem 1964, 239, (7),

2379-85.

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3. Voinea-Fanica Ene Ghid de terapie nauristc, editia a IV-a revizuid, Editura
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4. Voinea-Fanica Ene - Imunoterapia cancerulzri cu mijloace naturale, extras
P

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