THERAPEUTIC EFFECTS OF ZANTHOXYLUM TETRASPERMUM …

www.wjpps.com Vol 3, Issue 5, 2014.

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Narayanasamy et al. World Journal of Pharmacy and Pharmaceutical Sciences

THERAPEUTIC EFFECTS OF ZANTHOXYLUM TETRASPERMUM

W.A. STEM BARK ON CARBOHYDRATE METABOLIZING

ENZYMES IN MAMMARY CARCINOMA MICE

Mr.Narayanasamy K.1* and Dr.Ragavan B2

1*Dept of Biochemistry, Sree Narayana Guru College, Coimbatore, Tamil Nadu, India.

2 Dept of Biochemistry, P.S.G.College of Arts & Science, Coimbatore, Tamil Nadu, India.

ABSTRACT

Mammary carcinoma is a heterogeneous disease that appears to

progress from an in situ tumor to invasive cancer. Little is known

about the molecular events driving this progression. The genus

Zanthoxylum is known as “Timoor” that is used as mouth fresh, tooth

care and spice. Zanthoxylum tetraspermum (Wight & Arn.) belongs to

the family“Rutaceae” possesses some biological activities. The present

study evaluated the effect of Z.tetraspermum stem bark extract (300

and 600mg / kg body weight) in liver and kidney of N-methyl-N-

nitrosourea (MNU) induced mammary carcinoma mice on

carbohydrate metabolizing enzymes and pentose phosphate pathway

enzyme. Mammary carcinoma-bearing mice showed a significant (P<0.05) rise in glycolytic

enzymes like hexokinase, Phosphoglucoisomerase, aldolase and pentose phosphate pathway

enzyme glucose-6-phosphate dehydrogenase. Mammary carcinoma-bearing mice also causes

a simultaneous fall in gluconeogenic enzymes like glucose-6-phosphatase and fructose 1, 6-

diphosphatase.The activities of mitochondrial enzymes like succinate dehydrogenase and

malate dehydrogenase were significantly (P<0.05) lowered in mammary carcinoma-bearing

mice. Z.tetraspermum stem bark extract administration to tumor-induced mice significantly

(P<0.05) reversed the activities of glycolytic enzymes, pentose phosphate pathway enzyme,

gluconeogenic enzymes and the mitochondrial enzymes which indicated the antitumor

activity of the plant extract. The effect of oral Z.tetraspermum at the dose of 600mg / kg body

weight was more than the 300 mg / kg body weight. Comparison of normal mice, mice

administered only with plant stem bark extract and mice administered with 5-Fluoro Uracil

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Article Received on 25 March 2014, Revised on 20 April 2014, Accepted on 13 May 2014

*Correspondence for Author

Mr.Narayanasamy K.

Dept of Biochemistry, Sree

Narayana Guru College,

Coimbatore, Tamil Nadu,

India.


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(5-FU) as positive drug control group showed no significant variations in enzyme activities.

The results obtained in the present study indicate the therapeutic effect of the stem bark

extract and validates the traditional use of this plant in mammary carcinoma animals.

Key words: Z. tetraspermum, MNU, Mammary carcinoma, metabolic enzymes.

INTRODUCTION

Breast cancer is the most prevalent cancer in women worldwide, excluding nonmelanoma

skin cancer, and is the second leading cause of cancer deaths in women1. Once metastasis has

occurred, the survival rate is drastically reduced to a median of 2–3 years; therapy is then

aimed at controlling symptoms, prolonging survival and improving quality of life2.This is a

complex disease thought to occur via a multistep process and the use of adjuvant therapy,

continues to be fatal in many patients. Metastatic disease is the most common cause of breast

cancer death3 and is preceded by a sequence of events leading to the transformation of normal

breast epithelium. Histologically, progression may proceed through stages of atypical ductal

hyperplasia; ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) 4. The first

critical step in this process is invasion, which requires the loss of cellular adhesion and gain

of motility.

Traditional medicines have been the starting point for the discovery of many important

modern drugs. This has led to chemical and pharmacological investigations and general

biological screening of medicinal plants all over the world. Approximately 80% of the

world’s population relies on the use of traditional medicine, which is predominantly based on

plant materials. In recent years there has been considerable emphasis on the identification of

plant products as possible anticarcinogens with antioxidant properties5.

Zanthoxylum has been studied for several types of biological activities such as larvicidal,

anti-inflammatory, analgesic, antinociceptive, antioxidant, antibiotic, hepatoprotective,

antiplasmodial, cytotoxic, antiproliferative, anthelminthic, antiviral, anticonvulsant and

antifungal6-17. Zanthoxylum tetraspermum is a potent unidentified medicinal plant. It is

vernacularly called “Tooth ache tree” and belonging to the family of “Rutaceae”. It is an

aromatic, spiny, thorny, stout, deciduous climbing shrub or small tree, with brown bark and

alternate branches are armed with strong brown prickles. The wood is yellowish and soft18.

The plant is found in the Western Ghats in the Nilgiris, Aanaimalai hills, Kolli hills at

attitudes of 1,200 to 1,800m and in Kerala and Karnataka. The plant is credited in Srilanka


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with stimulant, astringent and digestive properties and is prescribed in dyspepsia and

diarrheas19-21

Z.tetraspermum is used for treating microbial infections, antifungal activities, tumors and

tooth ache.The phytochemical investigations of Z.tetraspermum stem bark have revealed the

presence of two benzophenanthrene alkaloids such as 8-acetonyl dihydronitidine, 8-acetonyl

dihydro avicine22 and decrine from Z. tetraspermum, Z. caudatum and Zanthoxylum

limonella23.The presence of the alkaloids such as Liriodenine, sesamin, lichexanthone and

piperitol gamma-gamma-diethyl ether from the Z. tetraspermum has been reported and they

have shown significant anti-bacterial and anti-fungal activity24. The presence of an alkaloid

Norsanguinarine, a polyhydroxy and a phenolic compound cyclohexanetetrol,

methoxyphenol, Gallopamil in the aqueous extract and a phenolic compound 2-methoxy-4-

vinylphenol in the ethanolic extract, from the Z. tetraspermum has also been reported25.

Toothpaste containing Z. nitidum extract decreased the incidence of dental plaque and

enhanced gingival health26. An alkaloidal extract of the stem barks of Z.chiloperone

exhibited antifungal activity against Candida albicans, Aspergillus fumigatus and

Trichophyton mentagrophytes27. Bafi-Yeboa et.al.28 investigated Z. americanum leaf, fruit,

stem, bark and root for antifungal activity with 11 strains of fungi. All extracts exhibited a

broad spectrum of antifungal activity. Alkamides isolated from the leaves of Z. syncarpum

showed moderate antiplasmodial activity, with IC50 values of 4.2 and 6.1 mM against

plasmodium falciparum D6 and W2 clone29. Ethanolic extracts of the trunk bark of Z. fagara,

Z.elephantiasis and Z. martinicense showed antifungal activity30. The petroleum ether,

chloroform and methanol extracts of the leaves and barks of Z. budrunga have been evaluated

for their antibacterial, antifungal and cytotoxic properties31. Benzophenanthrene alkaloids, 8-

acetonyldihydronitidine and 8-acetonyldihydroavicine were isolated from Z. tetraspermum

stem bark which showed significant antibacterial activity32.

Cytotoxic activity of essential oil of Z. rhoifolium was evaluated against HeLa (human

cervical carcinoma), A-549 (human lung carcinoma), HT-29 (human colon adenocarcinoma),

and Vero (monkey kidney) cell lines and mice macrophages by Da Silva et.al. 33. They

observed that the essential oil is cytotoxic against tumor cells (CD50 = 82.3, 90.7 and 113.6

µg/ml for A-549, HeLa, HT-29 cell lines, respectively). The fruit essential oils of Z. leprieurii

and Z. xanthoxyloides could be used as food supplements to protect against emergent diseases

such as cardiovascular problems, cancer and diabetes34.


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Carbohydrate metabolism plays a characteristic role in cancerous conditions35. One of the

most common and profound phenotypes of highly growing malignant tissues is that their

capacity to utilize and catabolize glucose at high rates. The high glycolytic rate is important

for rapidly proliferating cancers not only as major energy source but also to provide such

cells with precursors for nucleotide and lipid biosynthesis. Mitochondria are the intracellular

organelles responsible for ATP synthesis through the coupling of oxidative phosphorylation

to respiration in human and animal cells. The principal mitochondrial substrate is pyruvate

formed by glycolysis that enters the tricarboxylic acid cycle (TCA cycle) and the respiratory

chain to promote the generation of ATP. Mitochondria are the major intracellular source

during oxidative phosphorylation and are the primary target of reactive oxygen species

(ROS), which are generated under normal conditions as by-products of aerobic metabolism in

animal and human cells36. The defects in the respiratory chain lead to enhanced production of

ROS and free radicals in mitochondria, resulting in mitochondrial DNA mutations which

indirectly impair glucose sensing by reducing intracellular concentrations of ATP, an

important metabolic fuel37.

Since the therapeutic efficacy of Zanthoxylum tetraspermum was not yet carried out, the

objective of this study is focused on the metabolic enzymes associated with the carbohydrate

metabolizing and mitochondrial TCA cycle enzymes and cytotoxic activity of the plant

extract.

MATERIALS AND METHODS

Plant Material and Extraction

The whole plant material of Zanthoxylum tetraspermum. Wight & Arn. [Syn.Fagara

tetrasperma] 38 was collected from the silent valley ever green forest of Western Ghats,

Palakkad district, Kerala, South India. The plant was identified with the help of Institute of

Forest Genetics and Tree Breeding, Coimbatore, Tamil Nadu, South India. The stem bark of

the plant was shade dried at room temperature for 15 days. Then, they were powdered using

mixer grinder and subjected to extraction. The coarse powder (500gm) was extracted with

mixture of ethanol and water (1:1 ratio) for 72 hrs.The extract was then concentrated in vacuo

until the solvent was completely removed. The yield of the extract was found to be 12.6

grams.


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Animals

Female albino Sprague-Dawley mice between 40-50 days old were used for the experiments.

The animals were housed in individual, well ventilated cages (12-h light: dark cycle) and

were fed a commercial pelleted diet (M/s. Hindustan Lever foods, Mumbai, India) with water

freely available. This project received approval from the Animal Ethics Committee by

CPCSEA96/IAEC No.158/99/10.

Induction of mammary carcinoma

Mammary carcinoma was induced in female mice by a single intraperitoneal dose of N-

methyl-N-nitrosourea (MNU) injected into each of 30 female albino Sprague–Dawley mice

(aged 50 days). At day 50, all mice received a single dose of MNU 50 mg/kg

intraperitoneally. (MNU, reagent grade, was obtained from Sigma, USA, dissolved in 0.9%

saline). Two weeks after MNU treatment, a time by which the animals had recovered from

MNU-induced toxicity, the mice were divided into groups. The tumor was allowed to grow

for three months and the mice were palpated regularly to determine the appearance of

mammary tumor. After three months, mammary carcinoma was confirmed by histological

examination.

Experimental design

The animals were divided into eight groups of six animals each. The groups were formed as

follows:

Group – I = Normal healthy Mice.

Group – II =Control Mice (MNU Induced, 50mg MNU/ kg; ip)

Group –III =Extract treated Mice (MNU + 300mg extract/kg; oral; daily) for 4 weeks.

Group –IV =Extract treated Mice (MNU + 600mg extract/kg; oral; daily) for 4 weeks.

Group –V =Drug treated Mice (MNU + 5-Fluoro Uracil 300mg/kg; oral; daily) for 4 weeks.

Group –VI =Drug treated Mice (MNU + 5-Fluoro Uracil 600mg/kg; oral; daily) for 4 weeks.

Group –VII =Plant extract only (Plant extract 300mg/kg; oral; daily) for 4 weeks.

Group –VIII =Plant extract only (Plant extract 600mg/kg; oral; daily) for 4 weeks.

Groups III to VI were induced with mammary carcinoma and after three months, treatment

began with plant extract, 5-FU administered orally for four weeks as indicated above. Groups

VII and VIII animals were administered with Z.tetraspermum only on the same dosage as

Groups III and IV animals and by a similar route.


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Biochemical analysis

At the end of the experimental period, animals were fasted overnight and then killed by

cervical decapitation. The liver and kidneys from all the animals were removed, washed in

ice-cold isotonic saline and blotted individually on ash-free filter paper. The tissues were

homogenized in 0.1M Tris HCl buffer (pH 7.4) and used for estimation of carbohydrate

metabolizing and mitochondrial enzymes.

Carbohydrate metabolizing enzyme assays

Hexokinase activity was measured with respect to the amount of glucose utilized after the

addition of ATP39. Aldolase activity was assayed according to the method of King (1965) 40

with fructose-1, 6-diphosphatase as substrate and dinitrophenyl hydrazine as coloring reagent.

Phosphoglucoisomerase was measured using 2, 6-dichlorophenol indophenols dye according

to the method of Gracy and Tilley (1975) 41. The activity of glucose-6-phosphate

dehydrogenase was assayed by the method of Ells and Kirkman (1961) 42 with respect to the

amount of inorganic phosphorus liberated after the addition of the substrate glucose-6-

phosphate. The activities of glucose-6-phosphatase and fructose-1, 6-diphosphatase were

assayed43 with respect to the amount of inorganic phosphorus liberated after the addition of

their respective substrates glucose-6-phosphate or fructose-1, 6-diphosphate.

Mitochondrial TCA cycle enzyme assays

The purity of mitochondria was assessed by estimating succinate dehydrogenase activity by

the method of Slater and Bonner (1952) 44 in which the rate of reduction of potassium

ferricyanide is assessed. The activity of malate dehydrogenase was estimated by the method

of Mehler et.al., (1948)45.

STATISTICAL ANALYSIS

The statistical evaluation was done using one-way analysis of variance (ANOVA). Individual

differences between treatments were examined using Tukey’s HSD test. In all cases P< 0.05

denoted significance.

RESULTS

Glycolytic enzymes

The activities of glycolytic enzymes such as hexokinase, aldolase and

phosphoglucoisomerase were significantly (P<0.05) increased in the liver and kidney of

cancerous group II animals. Administration of two different doses (300mg and 600mg / kg


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body weight) of plant extract for 4 weeks in mammary carcinoma-bearing mice (group III

and IV) showed significantly (P<0.05) reduced activity of hexokinase, aldolase and

phosphoglucoisomerase when compared with cancerous group II. Similarly, administration of

same doses of 5-FU for 4 weeks in mammary carcinoma-bearing mice (group V and VI) also

showed the significantly (P<0.05) reduced activity of glycolytic enzymes. Group IV mice had

showed significantly (P<0.05) effective activity than the group III animals. However, the

plant extract alone treated animals (groups VII and VIII) did not show any significant

changes when compared with normal mice (group I). No significant variation is shown when

the group III and IV mice compared with Group V and VI respectively (Tables-1&2,

Figures.1-3, 5-7).

Pentose phosphate pathway enzyme

The activity of pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase was

significantly (P<0.05) increased in the liver and kidney of group II mice. Administration of

the doses 300mg and 600mg / kg body weight of plant extract in mammary carcinoma-

bearing mice (group III and IV) showed significantly (P<0.05) reduced activity of glucose-6-

phosphate dehydrogenase when compared with cancerous group II mice. The administration

of same doses of 5-FU for 4 weeks in mammary carcinoma-bearing mice (group V and VI)

also showed the significantly (P<0.05) reduced activity of glucose-6-phosphate

dehydrogenase enzyme. Group IV mice had showed significantly (P<0.05) effective activity

than the group III animals. And the plant extract alone treated animals (groups VII and VIII)

did not show any significant variation when compared with normal (group I) mice. No

significant variation is shown when the group III and IV animals compared with Group V and

VI respectively (Tables-1&2, Figures.4 & 8).

Gluconeogenic enzymes

The activities of gluconeogenic enzymes like glucose-6-phosphatase and fructose-1, 6-

diphosphatase in the liver and kidney of cancerous group II animals were significantly

(P<0.05) decreased when compared with normal mice (group I). Administration of two

different doses of plant extract (300mg and 600mg / kg body weight) in group III and IV

mice showed significantly (P<0.05) increased activity of gluconeogenic enzymes when

compared with cancerous group II. Administration of 5-FU (300mg and 600mg / kg body

weight) for 4 weeks in mammary carcinoma-bearing group V and VI mice also showed the

significantly (P<0.05) increased activity of gluconeogenic enzymes. The oral dose of the


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extract with 600mg / kg body weight (group IV mice) was more than the 300 mg / kg body

weight (group III mice). The plant extract alone treated animals (groups VII and VIII) did not

show any significance when compared with normal mice (group I). No significant variation is

shown when the group III and IV mice compared with Group V and VI respectively (Tables-

3& 4, Figures.9-12).

Mitochondrial TCA cycle enzymes

The activities of TCA cycle enzymes like succinate dehydrogenase and malate

dehydrogenase in the liver and kidney mitochondria of normal and experimental animals are

shown in Tables-5 & 6 respectively (Figures.13-16). The activity of mitochondrial enzymes

succinate dehydrogenase and malate dehydrogenase were significantly decreased in the liver

and kidney of cancerous group II mice (P<0.05) when compared with the normal (group I)

animals. Mammary carcinoma-bearing mice (group III and IV) treated with two different

doses (300mg, 600mg) of plant extract showed a significant increase in mitochondrial

enzymes activity when compared with group II animals (P<0.05). Mammary carcinoma-

bearing mice (group V and VI) treated with two different doses (300mg, 600mg) of 5-FU also

showed a significant increase in mitochondrial enzymes activity when compared with group

II mice (P<0.05). No significant variation is shown when the group III and IV compared with

Group V and VI respectively. Group IV mice treated with a dose of 600mg has significantly

showed an effective activity when compared with the group III animals treated with a dose

of 300mg (P<0.05). Groups VII and VIII treated with the plant extract alone (2 different

doses) showed no significant variation when compared with normal mice (group I).

Table-1: Liver Hexokinase, Aldolase, Phosphoglucoisomerase and Glucose-6-phosphate

dehydrogenase activity in control and experimental mice

Treatment / Groups

Hexokinase (µg of glucose -6-

phosphate /min / mg protein)

Aldolase (µmoles of

glyceraldehyde formed /min / mg protein at 37°C)

Phospho glucoisomerase

(Fructose / min / mg protein)

Glucose-6-phosphate

dehydrogenase (units / minute / mg

protein) I- Normal 4.3417 0.56380 0.2134 0.04393 0.2500 0.03742 0.3305 0.05546 II- Tumor induced 8.8950 0.70222 a * 0.5933 0.04844 a * 0.3833 0.03327 a * 1.0983 0.05419 a

* III- MNU + 300mg extract 5.0083 0.55003 b,f * 0.3700 0.04336 b,f * 0.2400 0.02366 b,f * 0.4917 0.06047 b,f

* IV- MNU + 600mg extract 4.2883 0.40216 b,f * 0.3350 0.04231 b,f * 0.2050 0.05431 b,f * 0.3550 0.02074 b,f

*


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Values are expressed as Mean S.D (n = 6); Statistical comparison by Tukey’s HSD: a –

Group II is compared with Group I; b – Group III, IV is compared with Group II; c – Group

III is compared with Group V; d – Group IV is compared with Group VI; e – Group VII, VIII

is compared with Group I; f – Group III is compared with Group IV. NS – Non-

significant. *P < 0.05.

Table-2: Kidney Hexokinase, Aldolase, Phosphoglucoisomerase and Glucose-6-

phosphate dehydrogenase activity in control and experimental mice

Values are expressed as Mean S.D (n = 6); Statistical comparison by Tukey’s HSD: a –

Group II is compared with Group I; b – Group III, IV is compared with Group II; c – Group

III is compared with Group V; d – Group IV is compared with Group VI; e – Group VII, VIII

is compared with Group I; f – Group III is compared with Group IV. NS – Non-

significant. *P < 0.05.

V- MNU + 300 mg 5FU

4.0083 0.52396 c NS

0.3600 0.03162 c NS

0.2033 0.04633 c NS

0.3350 0.01225 c NS

VI- MNU + 600 mg 5FU

4.2367 0.07174 d NS

0.3250 0.00548 d NS

0.2167 0.00816 d NS

0.3183 0.03764 d NS

VII- 300 mg extract

3.9750 0.26075 e NS

0.2417 0.02483 e NS

0.1833 0.04926 e NS

0.2744 0.04410 e NS

VIII- 600 mg extract

4.3805 0.44793 e NS

0.2467 0.03266 e NS

0.1850 0.04324 e NS

0.3150 0.03209 e NS

Treatment / Groups

Hexokinase (µg of glucose -6-

phosphate /min / mg protein)

Aldolase (µmoles of

glyceraldehyde formed /min / mg protein at 37°C)

Phospho glucoisomerase

(Fructose / min / mg protein)

Glucose-6-phosphate

dehydrogenase (units / minute / mg

protein) I- Normal 2.1532 1.27034 0.1221 0.01025 0.1230 0.07438 0.0991 0.04543 II- Tumor induced 3.7872 0.31740 a * 0.3450 0.02739 a * 0.2467 0.02733 a * 0.2050 0.03564 a *

III- MNU + 300mg extract 2.8117 0.05981 b,f * 0.0758 0.03393 b,f * 0.1700 0.02683 b,f * 0.1407 0.02823 b,f *

IV- MNU + 600mg extract 2.1217 0.08565 b,f * 0.1281 0.00369 b,f * 0.1385 0.01813 b,f * 0.1315 0.02071 b,f *

V- MNU + 300 mg 5FU

2.3219 0.26548 c

NS 0.1056 0.02188 c

NS 0.1417 0.02704 c

NS 0.1450 0.02258 c

NS VI- MNU + 600 mg 5FU

2.0350 0.24566 d

NS 0.1288 0.00674 d

NS 0.1542 0.04124 d

NS 0.1417 0.01722 d

NS VII- 300 mg extract

2.4883 0.27795 e NS

0.1104 0.01760 e NS

0.1483 0.02483 e NS

0.1400 0.02366 e NS

VIII- 600 mg extract

2.2267 0.24736 e NS

0.0997 0.02406 e NS

0.1533 0.02875 e NS

0.1383 0.02714 e NS


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Table- 3: Liver Glucose-6-phosphatase and Fructose-1, 6- diphosphatase activity in

control and experimental mice

Values are expressed as Mean S.D (n = 6); Statistical comparison by Tukey’s HSD: a – Group II is compared with Group I; b – Group III, IV is compared with Group II; c – Group III is compared with Group V; d – Group IV is compared with Group VI; e – Group VII, VIII is compared with Group I; f – Group III is compared with Group IV. NS – Non-significant. *P < 0.05. Table – 4: Kidney Glucose-6-phosphatase and Fructose-1, 6- diphosphatase activity in


Values are expressed as Mean S.D (n = 6); Statistical comparison by Tukey’s HSD: a – Group II is compared with Group I; b – Group III, IV is compared with Group II; c – Group III is compared with Group V; d – Group IV is compared with Group VI; e – Group VII,

Treatment / Groups Glucose-6-phosphatase

(Nano moles of Pi liberated /min / mg protein)

Fructose-1,6 – Diphosphatase (µmoles of Pi liberated /min /

mg protein) I- Normal 1.9283 1.32388 1.1017 0.07250

II- Tumor induced 0.2714 0.06628 a * 0.3067 0.04926 a *

III- MNU + 300mg extract 1.5167 0.38051 b,f * 0.7300 0.00894 b,f *

IV- MNU + 600mg extract 1.5883 0.09988 b,f * 0.9583 0.02229 b,f *

V- MNU + 300 mg 5FU 1.6767 0.12111 c NS 0.7950 0.03886 c NS

VI- MNU + 600 mg 5FU 1.7017 0.08864 d NS 0.9883 0.03312 d NS

VII- 300 mg extract 1.6567 0.06250 e NS 1.0783 0.03869 e NS

VIII- 600 mg extract 1.8270 1.13960 e NS 1.0800 0.06197 e NS

Treatment / Groups Glucose-6-phosphatase

(Nano moles of Pi liberated /min / mg protein)

Fructose-1,6 – Diphosphatase (µmoles of Pi liberated /min /

mg protein) I- Normal 2.7438 1.20828 1.4433 0.02422

II- Tumor induced 1.1700 0.04899 a * 0.4450 0.02429 a *

III- MNU + 300mg extract 2.3050 0.09772 b,f * 1.1550 0.02881 b,f *

IV- MNU + 600mg extract 2.5083 0.19590 b,f * 1.2483 0.01722 b,f *

V- MNU + 300 mg 5FU 2.6017 0.21018 c NS 1.1533 0.04844 c NS

VI- MNU + 600 mg 5FU 2.6633 0.03983 d NS 1.3233 0.01506 d NS

VII- 300 mg extract 2.2411 0.90671 e NS 1.4033 0.01966 e NS

VIII- 600 mg extract 2.4088 0.41996 e NS 1.4117 0.01472 e NS


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VIII is compared with Group I; f – Group III is compared with Group IV. NS – Non-significant. *P < 0.05. Table- 5: Liver Succinate dehydrogenase and Malate dehydrogenase activity in control

and experimental mice

Values are expressed as Mean S.D (n = 6); Statistical comparison by Tukey’s HSD: a – Group II is compared with Group I; b – Group III, IV is compared with Group II; c – Group III is compared with Group V; d – Group IV is compared with Group VI; e – Group VII, VIII is compared with Group I; f – Group III is compared with Group IV. NS – Non-significant. *P < 0.05. Table- 6: Kidney Succinate dehydrogenase and Malate dehydrogenase activity in


Values are expressed as Mean S.D (n = 6); Statistical comparison by Tukey’s HSD: a – Group II is compared with Group I; b – Group III, IV is compared with Group II; c – Group III is compared with Group V; d – Group IV is compared with Group VI; e – Group VII, VIII is compared with Group I; f – Group III is compared with Group IV. NS – Non-significant. *P < 0.05.

Treatment / Groups Succinate dehydrogenase

(SDH) (µmoles / min / mg protein)

Malate dehydrogenase (MDH)

(µmoles of NADH oxidized / min / mg protein)

I- Normal 0.41517 0.027795 0.39167 0.005354 II- Tumor induced 0.20017 0.003189 a * 0.15533 0.003077 a * III- MNU + 300mg extract 0.29533 0.004179 b,f * 0.22533 0.003933 b,f * IV- MNU + 600mg extract 0.35467 0.004274 b,f * 0.26917 0.002927 b,f * V- MNU + 300 mg 5FU 0.31133 0.012533 c NS 0.22800 0.002366 c NS VI- MNU + 600 mg 5FU 0.35933 0.008042 d NS 0.27167 0.002160 d NS VII- 300 mg extract 0.39983 0.002137 e NS 0.38317 0.003488 e NS VIII- 600 mg extract 0.41133 0.006408 e NS 0.38383 0.008472 e NS

Treatment / Groups

Succinate dehydrogenase (SDH)

(µmoles / min / mg protein)

Malate dehydrogenase (MDH)

(µmoles of NADH oxidized / min / mg protein)

I- Normal 0.31017 0.010108 0.23767 0.030303 II- Tumor induced 0.15550 0.004231 a * 0.10083 0.005115 a * III- MNU + 300mg extract 0.22433 0.002582 b,f * 0.14083 0.006274 b,f * IV- MNU + 600mg extract 0.25333 0.002805 b,f * 0.14967 0.003502 b,f * V- MNU + 300 mg 5FU 0.23083 0.003764 c NS 0.15750 0.001517 c NS VI- MNU + 600 mg 5FU 0.26667 0.003615 d NS 0.17083 0.004262 d NS

VII- 300 mg extract 0.30150 0.004637 e NS 0.21583 0.015355 e NS VIII- 600 mg extract 0.30517 0.004446 e NS 0.22817 0.005845 e NS


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I I I I I I I V V VI VI I VI I I0

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Liver hexokinase activity

Normal Tumor Induced Extract(300mg)Extract(600mg) 5FU(300mg) 5FU(600mg)Plant only(300mg) Plant only(600mg)

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Normal Tumor Induced Extract(300mg) Extract(600mg)

5FU(300mg) 5FU(600mg) Plant only(300mg) Plant only(600mg)

Fig .1: Liver Hexokinase activity in control Fig.2: Liver Aldolase activity in control and and experimental mice. experimental mice.

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/mg

prot

ein

Liver phosphoglucoisomerase activity



III III IV V VI VII VIII

0

0.2

0.4

0.6

0.8

1

1.2Ac

tivit

y /m

in/m

g pr

otei

n

Liver glucose.6.phosphate dehydrogenase activity


Fig.3: Liver Phosphoglucoisomerase activity Fig.4: Liver Glc.6.P.DHase activity in control and experimental mice. in control and experimental mice.

II I I I I I V V VI VI I VI I I

00.5

11.5

22.5

3

3.54

Act

ivit

y /m

in/m

g pr

otei

n

Kidney hexokinase activity


I I II I I I V V VI VI I VI I I

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Kidney aldolase act ivity


Fig.5: Kidney Hexokinase activity in control Fig.6: Kidney Aldolase activity in control and

and experimental mice. experimental mice.


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II I

I I I I V V VI VI I VI I I

0

0.05

0.1

0.15

0.2

0.25

Kidney phosphoglucoisomerase act ivity


II I I I I I V V VI VI I VI I I

0

0.05

0.1

0.15

0.2

0.25

Kidney glucose.6.phosphate dehydrogenase act ivity


Fig.7: Kidney Phosphoglucoisomerase activity Fig.8: Kidney Glc.6.P.DHase in control and experimental mice. activity in control and experimental mice


0.5

1

1.5

2

Act

ivity

/min

/mg

prot

ein

Liver glucose.6.phosphatase activity




0.2

0.4

0.6

0.8

1

1.2A

ctiv

ity /m

in/m

g pr

otei

n

Liver fructose.1,6.diphosphatase activity



Fig.9: Liver Glc.6.phosphatase activity in control Fig.10: Liver Fruc.1, 6. diphosphatase

and experimental mice. activity in control and experimental mice.


0

0.5

1

1.5

2

2.5

3

Kidney glucose.6.phosphatase activity


I I I I I I I V V VI VI I VI I I

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Act

ivit

y /m

in/m

g pr

otei

n

Kidney fructose.1,6.diphosphatase act ivity


Fig.11: Kidney Glc.6.phosphatase activity Fig.12: Kidney Fruc.1,6.diphosphatase

in control and experimental mice. activity in control and experimental mice


1105


III

III IV V VI VII VIII00.05

0.10.15

0.20.25

0.30.35

0.40.45

Act

ivity

/min

/mg

prot

ein

Liver succinate dehydrogenase activity




0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Act

ivity

/min

/mg

prot

ein

Liver malate dehydrogenase activity


Fig.13: Liver SDH activity in control Fig.14: Liver MDH activity in control and

and experimental mice experimental mice


0.05

0.1

0.15

0.2

0.25

0.3

0.35

Act

ivity

/min

/mg

prot

ein

Kidney succinate dehydrogenase activity




0.05

0.1

0.15

0.2

0.25A

ctiv

ity /m

in/m

g pr

otei

n

Kidney malate dehydrogenase activity



Fig.15: Kidney SDH activity in control Fig.16: Kidney MDH activity in control and experimental mice and experimental mice. DISCUSSION

Malignant cells have a diminished respiratory rate, coupled with an excessive rate of aerobic

glycolysis. Mammary carcinoma-bearing mice have showed an increase in the activity of

glycolytic enzymes hexokinase, aldolase and phosphoglucoisomerase. This allowed us to

infer the elevated rate of glycolysis in tumor conditions since tumor cell proliferation is

dependent on glucose availability; these cells acquire the major part of their energy from the

glycolytic pathway46. The degree of elevation of these enzymes is directly related to the

extent of morphological differentiation and growth rate of hepatomas47. Hexokinase plays a

critical role in initiating and maintaining the high glucose catabolic rates of rapidly growing

tumors48 and accomplishes the entry of glucose into the glycolytic pathway by

phosphorylation to glucose-6-phosphate.The proliferating cells undergo a shift from oxidative


1106


to glycolytic metabolism, where the energy requirements of the rapidly dividing cells are

provided by the ATP from glycolysis49.

The mitochondrial porin-bound hexokinase II is increased under the control of mutated

p53.This would direct mitochondrial ATP preferentially to form glucose-6-phosphate and

hence is expected to increase the biosynthetic pentose phosphate pathway. Hence, the

glycolytic capacity of cancer cells depends totally on hexokinase activity for its metabolic

fuel50. In this study, the observed increase in the activity of hexokinase in mammary

carcinoma animals might have been due to the increased metabolic need of energy fuel for

proliferating tumor cells and simultaneously increased activity of hexokinase51.

Administration of two different doses (300mg, 600mg) of plant extract and similar doses of

5-FU for 4 weeks to mammary carcinoma-bearing mice significantly (P<0.05) reduced the

enzyme activity and this may have been due to the antitumor activity of the alkaloids,

phenolic compounds and other phytochemicals present in the hydroethanolic stem bark

extract of Z.tetraspermum.Similar type of results on hexokinase were reported in mammary

carcinoma rats by Arathi and Sachdanandam (2003) 51.

Aldolase, another key enzyme in the glycolytic pathway, was increased in diethylnitrisamine-

induced tumor conditions. Aldolase was found to be elevated in tumor-bearing animals and in

breast cancer52. The elevated activity of phosphoglucoisomerase and aldolase may be due

to cell impairment and necrosis. In experimental carcinogenesis the cells are subjected to

carcinogen-induced damage, and very often exhibit glycolysis after a period of increased

oxygen uptake. The present result on Aldolase activity is in accordance with the study of

Semecarpus anacardium Linn. nut milk extract on carbohydrate metabolizing enzymes in

mammary carcinoma rats51.

Phosphoglucoisomerase serves as a good index of tumor growth and is significantly elevated

in cancerous cells. In agreement with this study, Campbell & King, (1962)53 reported that

phosphoglucoisomerase was an indicator of metastatic growth and was elevated in patients

with neoplasms, especially after metastasis. Alterations in the activity of

phosphoglucoisomerase might be expected to influence the proportion of glucose-6-

phosphate metabolized via the glycolytic pathway54. The high glycolytic rate of most tumors

can be adopted as a major source of energy in the deranged cell. Increase in the activity of

glycolysis results from rise of the tumor growth rate and is accompanied by a decrease in the

activity of the pentose phosphate pathway and respiratory chain55.


1107


The plant extract treated animals showed a significant drop in the activity of glycolytic

enzymes and concomitant elevation in the activity of gluconeogenic enzymes. This

modulation may be due to the antitumor activity of the plant stem bark extract either by

inhibiting the glycolytic enzyme activity or by suppression of tumor progression56.The

activity may be attributed to the presence of alkaloids, polyphenolic content and other

aromatic phytochemical constituents in the plant stem bark extract, which has an effective

role over aerobic glycolysis in a dose dependant manner57.Comparison of group I, with

groups VII and VIII showed no significant variations in enzyme activity. The present

results are in agreement with the study reported by Sujatha and Sachdanandam (2002) 58.

The activity of gluconeogenic enzymes such as glucose-6-phosphatase and fructose-1, 6-

diphosphatase were inhibited significantly in tumor-bearing animals.Lactate production from

glucose rises and concomitantly glucose production from pyruvate decreases during the

progression of tumor growth. The observed reduction in the activity of these enzymes in

tumor-bearing animals may be due to the higher lactate production of neoplastic tissues, and

it has been proved that tumor utilizes a large proportion of lactate for glycolysis and protein

synthesis. A crucial point in regulation of aerobic glycolysis and of energy metabolism in

general is represented by the transport of metabolites across the mitochondrial membrane

from the cytosol to the matrix space of mitochondria. The present study is coinciding with the

findings on the therapeutic effect of Semecarpus anacardium Linn. nut milk extract on

carbohydrate metabolizing enzymes in mammary carcinoma rats51.

The mammary carcinoma-bearing mice showed decreased activity of mitochondrial TCA

cycle enzymes when compared with normal mice. Decreased activity of these enzymes might

be due to the alteration in the morphology and ultrastructure of cancer cells and the ability of

mitochondria to undergo metabolic changes when compared with normal cells, and also the

number of mitochondria was drastically reduced in tumor cells. The decrease in the

mitochondrial content might be due to the marked deficiency in one or electron transport

chain compounds59.We may speculate that tumor cells may be able to produce compounds

capable of being transported to normal host cells and act as uncoupling agents, thus lowering

ATP production in the normal tissues and contributing to tumor-induced cachexia60.

Enhanced mitochondrial lipid peroxidation has been reported to inactivate succinate oxidase,

succinate dehydrogenase and the components of the respiratory chain61.Further supporting

our observation, the decreased activity of succinate dehydrogenase and malate dehydrogenase


1108


in kidney has been reported in gentamicin-induced nephrotoxicity62. Enhanced free radical

generation and the loss of mitochondrial respiration have been observed in Parkinson’s

disease, Alzheimer’s disease and cardiac ischemia/reperfusion injury51.

CONCLUSION

The results obtained in the present study indicate that deranged energy metabolism in N-

methyl-N-nitrosourea (MNU) induced mammary carcinoma in mice was rectified and

favorable restoration of glycolysis, HMP shunt enzyme and TCA cycle enzymes was

achieved by administration of Z.tetraspermum stem bark extract.

ACKNOWLEDGEMENT

The authors are thankful to Dr.R.Rajendran, Principal, P.S.G.College of Arts and Science,

Coimbatore-14, Dr.K.V.Surendran, Principal, Sree Narayana Guru College, Coimbatore,

Dr.V.P.Prabhakaran, Secretary, Sree Narayana Guru Educational Trust, Coimbatore for

giving permission to carry out this work.

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THERAPEUTIC EFFECTS OF ZANTHOXYLUM TETRASPERMUM …