Indian Journal of Experimental Biology Vol. 43, March 2005, pp. 264-267

Efficacy of Terminalia arjuna (Roxb.) on N-nitrosodiethylamine induced hepatocellular carcinoma in rats

S Si valokanathan , M Ilayaraja & M P Balasubramanian*

Department of Pharmacology and En viron mental Toxicology, Dr. ALM Post-Graduate Institute of Bas ic Medi ca l Sciences. Uni versity of Madras, Taramani , Chennai 600 11 3, India.

Received 12 April 2004; revised 19 November 2004

The effec t of ethanoli c extract of Tenninalia (// j una bark on carbohydrate metaboli zing enzy mes of N­nitrosocli ethylamine induced hepatocellular carcinoma in Wistar albino rats were stucliecl. The plasma and li ver glyco lytic enzy mes such as hexokinase, phosphogluco isomerase, aldolase were significantl y increased in cancer induced animals while glyconeogenic enzyme, glucose-6-phosphatase was decreased. These enzy mes were reverted signifi cantly to near normal range in treated animal s after oral admini stration of T. (//jliiW for 28 clays. The modulation of the enzy mes co nstitute the depletion of energy metaboli sm leads to inhibition of cancer growth. This inh ibitory ac ti vity may be clue to the anti cancer acti vity of constituents present in the ethanolic extract ofT. cujuna.

Keywords: Terminalia arjuna , Hepatocellular carcinoma. Carbohydrate metabolizing enzy mes

IPC Code: lnt Cl 7 A61 P

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver and among the most common cancers worldwide 1

• The attributable ri sk factors for HCC are viral infection, aflatoxin exposure in diets, cigarette smoking, alcohol consumption and oral contracepti ves. For many years cancer chemotherapy has been dominated by potent drugs that e ither intetTupt the synthesis of DNA or destroy its structure once it has formed. Unfortunately, their toxicity is not limited to cancer ce ll s and normal ce ll s are also harmed. Efforts to develop less toxic drugs that affect only malignant cells and mechanism based approach are necessary in cancer chemotherapy. According to WHO estimates, more than 80% people in developing countries depend on traditional medicine for their primary heaith needs2

. Numerous medicinal plants and their formulation s are used for liver di sorders in ethnomedical practices as well as traditional system of medicine in lndia3 A wide variety of chemical agents have been shown to possess chemopreventive properties against a broad spectrum of cancet.4 . A recent survey that more than 60% of cancer patients use vitamins or herbs at some point in their therap/ .

*Corres pondent auth or Phone: 91-44-2492531 7, 24925548 Fax: 91 -44-4926709 Emai l: mpbpet@rediffmail.com

Terminalia arjuna Roxburgh a tropical woody tree growing throughout Indi a and locally known as kumbuk ; Arjun a in English , is a member of the Combretaceae family, which includes 250 species of Terminalia. A few randomi zed c linical trials of unstandardi sed concotion of the tree bark have been performed in coronary heart disease (CHD) pati ent in lndiac'. Bes ides, th e plant exhibits vari ety of medici nat properti es such as fun g icida l, antibacteri a l, antifertility and antihuman immunodeficiency viru s induced di sease. The plant was found to be usefu l in the treatment o f can cer also7

. Kapoor8 reported that the powdered bark is tradition ally used as diuretic and genera l to nic in cases of c irrhos is of th e liv er. In thi s contex t Pettit~ is of the opinion than th a t a compoun ds ofT. arjuna possess cancer cell growth inhibitory activity again st various cell lines s uch as P388

, OVC AR-3, SF-295 , A498, NCI-4460, KM20L2 and S K-MEL-5 . Thus there is a paucity of information regarding the anticancer activity ofT. arjuna against liver cance r. Hence, the present study has been undertaken to evaluate the anticancer activity of e thanol ic extract of T. arjuna on carbohydrate metabol izing enzymes such as hexokinase (HEX), phosphoglucoisomerase (PGI), aldolase (ALD) and glucose-6-phosphatase (G6P) in N- nitrosodiethylamine induced liver cancer in rats.

SIVALOKANATHAN et al.: EFFICACY OF TERMINAL/A ARJUNA ON HEPATOCELLULAR CARCINOMA 265

Materials and Methods Plant material-The fresh bark of Terminalia

arjuna was collected during September 2002 in Chennai, Tamil Nadu, India. The plant was authenticated by botanist of Captain Srinivasa Murti Drug Research Centre for Ayurveda, Chennai, Tamil Nadu, Indi a. A voucher specimen (No. 064) has been deposited in the herbarium of the same department.

Preparation of plant extract-The shade dried T.wjuna bark was coarsely powdered (1 kg) and soaked in I 000 ml of ethanol for 10 days at room temperature. The extract was filtered and concentrated to obtain the solid residue and the final weight was noted. The yield of the total ethanolic extract was 8.5 %. Primary phytochemical screening of the ethanol ic extract of T. arjuna bark revealed the presence of triterpenoids, phenol , flavonoids, tannin and saponins.

Animals- Healthy male Wi star albino rats (60-80 g) used in the present study were obtained from Tamil Nadu Veterinary College and University, Chennai , Tamil Nadu , India. The animals were maintained at

25° ± 2° C and were provided with standard commercially ava ilable pell et diet (M/s Hindustan Lever Limited, Mumbai, Indi a) and water ad libitum.

Experimental design- The rats were divided into fo ur groups of 6 animals each. Group I animals were g iven drinking water, Groups II and III animals were ad mini stered with sing le i.p 111Jection of N­nitrosodi ethylamine (DEN, S igma Chemical Company, USA) at a dose of 200 mg/kg body weight in saline to induce li ver cancer. Two weeks after ad mini strati on of DEN, phenobarbita l (PB, Sigma) at a concentration of 0 .05% was incorporated into rat chaw for up to 14 success ive weeks to promote the cancer. After the induction period , group III animals were treated with ethano li c ex tract of T. arjuna tree bark orally at a concentration of 400mg/kg body

weight for 28 days . Group IV an imals served as plant extract control.

Collection of plasma and liver homogenate- After the experimental period all the animals were sacrificed by decapitation. Blood was collected in

tubes containing EDT A and centrifuged at 2000 x g for l 5 min and plasma was collected. Liver was

perfused in situ with cold 0.15 M NaCI at 3iC and homogenized in ice-cold U.l M Tri s-HCI buffer (pH 7.4).

Bichemical assays-Hexokinase, phosphoglucoi o­merase, aldolase and glucose-6-phosphatase were assayed according to the method of Branstrup 10

,

Horrocks 11, King 12 and Gancedo 13

, respective ly. Statistical analysis- Stati stical differences were

calcul ated by independent sample- ' t' test by using

SPSS 7.5 student version . The mean ± SE was compared in each group. Comparisons were made between group I and II, group I and III, group I and IV and group II and III.

Results and Discussion The results of the efficacy of T. arjuna on plasma

carbohydrate metabolizing enzymes of DEN induced hepatocell ular carcinoma was presented in. Table I. The levels of HEX, PGI an-d ALD in plasma of group II animals were elevated significantly where as G6P were decreased . In group III T. wjuna treated animals these enzymes were returned significantly to near normal range. Correspondingly the ac tiviti es of HEX, PGI , ALD were increased significantly in liver ti ssue of group II animals when compare with group I animals (Fig.l). On the other hand, the level s o f G6P in li ver tissues of group II animals were decreased. These enzymes levels were normalized in T. wj111w treated animals. However, there were no signi ficant changes observed in group Ill and group IV animal s when compared with group I control animals.

Tab le !- Plasma carbohydrate metaboli zing enzy mes of control and experimental animals [Values are mea n ± SE from 6 animals in each group]

Parameters Group I Group II Group Ill Group IV

HEX 11.11 ± 0.43 19.15±0.4la .

12.16 ± 0.40 aNS b' 9.95 ± 0.34 aNS PGI 12.66 ± 0.31 18. 12 ± 0.38 a

. 13.07 ± 0.46 aNS b' 12.00 ± 0.51 aNS

ALD 12.52 ± 0.44 15.15 ± 0.36 a** J3.08 ± 0.35 aNS b** 13. !6 ± 0.44 aNS G6P 3 1.05 ± 0.49 21.1 3 ± 0.48 a

. 29.02 ± 0.44 at b' 32.08 ± 0.51 aNS

Units- HEX : n moles of glucose-6-phosphate liberated/mg protein/min ; PGI: n moles of fructose liberated/mg protein/min ; ALD: nmoles of glyceraldehyde liberated/mg protein/min, and G6P: n moles of inorganic phosphorus liberated/mg protein/min. a: as compared with group I; b: as compared with group II. P values: * <0.001 ; **<0.0 1; t<0.05 and NS =Not significant

266 INDIAN J EXP BIOL, MARCH 2005

30

a'

25

20

15

10

HEX PGI

DC antral 1'2lDEN 0 DEN+ T .arjuna IIDT.arjuna

a'

ALD

aNS

G6P

Fig.l-Effccts of ethano lic extract of T. wjuna on li ver ca~bohydrate metaboli sing enzy mes in rats [Values are mean ± SE from 6 animals in each group! a: as compared with group I; b: as compared with group II. Units-HEX: nmoles of glucose-6-phosphate liberatecl/mg protein/m in; PGI: nmoles of fructose li berated/mg protein/min ; ALD: nmol es o f glycerolclehycle li beratcd/mg protein/min , and G6P: nmoles of inorgan ic phosphorus liberatecl/mg protein/min. P va lues: *<0.00 I;

''<0.0 I; 1<0.05; Ns = Not significant.

The most common biochemical phenotype of highly malignant, rapidly growing tumors is the ir ability to. utilize glucose at high rates. Numerous studi es have demonstrated that highly malignant tumors, i.e . those that are poorly differentiated and grow rapidly, exhibit the capacity to metabolize glucose to lactate at much higher rates than normal cells 14•

16. The growth rate of hepatomas and their

glycolytic enzymes activities are s ignificantly correlated 17. In the present study increased activities of hexokinase, phophoglucoisomerase and aldolase in cancer bearing animals may be due to the elevated rate of glycolysis in determining the glycolytic capacity of cancer cells.

It is well known that the high rate of glucose metabolism is dependent on elevated levels of hexokinase 18 which catalyses the first step in the glycolytic pathway. In hepatoma cells, type II hexokinase activity is over expressed and binds to the outer mitochondrial membrane 19'20 . In the present studies the group II cancer bearing animals also show elevated level of hexokinase exceeding that of the normal rats. This increased level of hexokinase in cancer tissues is due to the large amount of glucose metabolism21 . Alteration in the activity of phosphoglucoisomerase may be expected to influence the proportion of glucose-6-phosphate metabolized via

the glycolytic pathway. This enzyme is an indicator of metabolic growth and increases in cancer condition22

.

The group II cancer bearing animals show increased level of phosphoglucoisomerase in liver and plasma, may be due to the higher rate of glycolysis in liver and subsequent leakage of this enzyme into the blood. Aldolase, is a key enzyme in glycolytic pathway and elevated levels of this enzyme were reported in metastatic conditions of breast tissues23

. In group II cancer bearing animals the acti vi ties of aldolase was increased. Aldolase was less freq uently raised than phosphoglucoisomerase.

The reduction in gluconeogenesis is manifested rapidly in arow ina tumors in which the levels of

0 0 ~

glucose-6-phosphatase were decreased. Schamhart reported decreased activities of glucose-6-phosphatase in hepatoma cells, revealing the progressive failure of glucogenesis in liver tumor. In the present investigation , glucose-6-phosphatase was inhibited in group-II cancer bearing animals. This is in accordance with the findings of Graham25 On the contrary, a sign ificant decrease of glycolytic enzymes and the increase of gluconeogenic enzymes level in drug treated group III animals indicate the regression of cancer. This can be attributed to the anticancer potency ofT. wjuna bark extract.

Generally large number of recognized inhibitors of mutagens/carcinogens are basically of plant origin and

. hl d. h . I 26.27 K t /28 . of hig y 1verse c emtca nature . aur e a , usmg Ames assay reported that tannin fraction of T.mjww possess antimutagenic activity against 4-nitro­phenylenediamine (NPD) in TA 98, tester strain of Salmonella typhimurium. According to them anticarcinogenic and antimutagenic activity of plant phenols is due to an interaction of the compound with target tissue DNA. Teel29 also suggested that ellagic acid of T. arjuna masks the sites of DNA. Therefore, the anticancer activity of T. cnjuna on DEN induced liver cancer may be attributed to the possible binding of one of the active compound with DNA and interruption of the macromolecule synthesis. This may leads to depletion of energy metabolism in cancer tissues and normalize the abnormal cells behaviour. This anticancer activity of T. arjuna on regulation of carbohydrate metabolism can provide new imp01tant information and potentially new approach to cancer chemotherapy.

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