Cancer accounts for high morbidity and high mortality rate throughout the world. Cancer of breast is common in women in developed countries and more than 40% of all breast cancer cases are found in developing countries.1)
Can-cer is based on genetic and epigenetic alterations that affect the regulation and function of genes. These pathologic changes are selected during progression to yield cells that multiply aggressively beyond the limits normally set by their intrinsic proliferative capacity, neighboring cells, limited mi-togens, and blood supply. It is expected that the cataloging of specific changes in gene expression will provide diagnostic markers for any given type. Furthermore, proteins encoded by specific genes could potentially serve as targets for thera-peutic intervention.2)
In addition to the therapeutic strategies already known, some recent reports indicate new areas for the development of target selective drugs for the treatment of metastatic breast cancer.3) The earliest adjuvant chemotherapy, with single-agent alkylating and anti-metabolic drugs, was soon replaced by combination therapy, as it was demonstrated that simulta-neous combination of two or more agents provided better re-sults.4,5)
Kalpaamruthaa is a Siddha formulation, which has been formulated in our laboratory. They contain Semecarpus
anacardium LINN. (SA), Emblica officinalis and honey with different ratio. Kalpaamruthaa (KA) is a Siddha preparation which has been formulated in our laboratory, the components studies of this drug reveals the presence of flavonoids, ascor-bic acid, polyphenols, tannins, sugars and sterols. Flavonoids are seemingly more widely dispersed in the food supply and are of more varied composition than are isoflavones. For these reason, their absorption and metabolism have been less completely characterized. Cameron and Pauling6) believed
that ascorbic acid combats cancer by promoting collagen synthesis and thus prevents tumors from invading other
tis-sues. Flavonoids are polyphenols widely distributed and known to possess biological and pharmacological activities, including anti-inflammatory action against free radicals.7) Isoflavonoids are members of the broad class of plant polyphenols that have been shown in vivo to have benefit in the treatment of a wide variety of chronic diseases, including cancer. Another important consideration in vivo is the local-ized metabolism of isoflavones that may occur in the vicinity of tumor cells. Many experiments to verify the association between isoflavone intake and disease prevention have been reported.8)
The biochemical evaluation of glycoprotein components, marker enzymes and lysosomal enzymes of liver, kidney and blood of control and experimental rats was carried out to study the therapeutic efficacy of KA on 7,12-dimethylbenz-(a)anthracene (DMBA) induced mammary carcinoma bear-ing rats (Sprague–Dawley strain). This study was undertaken to arrive at the effective dose of KA in mammary carcinoma condition in rats.
MATERIALS AND METHODS
Materials Mammary carcinoma was induced in 8-week
old rats by gastric intubation of 7,12-dimethylbenz(a)an-thracene (Sigma, St. Louis, MO, U.S.A.; 25 mg/ml).9)
Animals Female albino Sprague–Dawley rats, 50—55 d,
were obtained from the National Institute of Nutrition, Hy-derabad, and maintained at 30 °C (approx.) on a 121 h day–night rhythm (well-ventilated spacious cages). The rats were fed commercial rat feed supplied by Hindustan Lever, Ltd., Mumbai with the name Gold Mohur rat feed. Food and water were given ad libitum.
The rats were divided into seven groups with six animals in each group and were given the dose regimen as given below.
Anticancer Effect of Kalpaamruthaa on Mammary Carcinoma in Rats
with Reference to Glycoprotein Components, Lysosomal and Marker
Enzymes
Krishnamurthy VEENA,aPalanivelu SHANTHI,band Panchanatham SACHDANANDAM*,a
aDepartment of Medical Biochemistry, Dr. A. L. Mudaliar Post-Graduate Institute of Basic Medical Sciences, University of
Madras; and bDepartment of Pathology, Dr. A. L. Mudaliar Post-Graduate Institute of Basic Medical Sciences, University
of Madras; Taramani Campus, Chennai-600 113, India. Received September 15, 2005; accepted November 16, 2005
A promising approach to reduce the occurrence of cancer is its treatment, specifically by chemical interven-tion through minor dietary constituents. Epidemiological studies suggest that specific pharmacologically active agents present in the diet might reduce cancer. A remarkable surge of interest in chemoprevention research has, thus, lead to the identification of many phytochemicals of dietary origin as effective potential chemotherapeutic agents. In the present investigation, attempt has been made to study the potency of Kalpaamruthaa (KA), a herbal preparation, against cancer. The changes in level of glycoprotein components, marker enzymes and lyso-somal enzymes were carried out in 7,12-dimethylbenz(a)anthracene (DMBA) induced Sprague–Dawley rats. The changes in the body weights and volume were also determined. KA was administered at the dosage level of 100, 200, 300, 400 and 500 mg/kg body weight (BW) in olive oil orally for 14 d, after the induction period is completed (90 d). On administration of KA, the levels of the above enzymes and the changes in the body weights and volume were significantly normalized in a dose dependant manner. The present study shows that KA is effective at the dosage level of 300 mg/kg body weight in mammary carcinoma bearing rats.
Key words breast cancer; flavonoid; ascorbic acid; glycoprotein; Semecarpus anacadium
© 2006 Pharmaceutical Society of Japan ∗ To whom correspondence should be addressed. e-mail: psachdanandam2000@yahoo.co.in
Group I: Control animals
Group II: Breast cancer was induced in overnight fasted animals by a single dose of DMBA in olive oil (25 mg/kg body weight) by gastric intubation.
Group III: Breast cancer induced animals (as in Group II) were treated with the drug, Kalpaam-ruthaa (100 mg/kg body weight/d) in olive oil orally by gastric intubation for 14 d. Group IV: Breast cancer induced animals (as in Group
II) were treated with the drug, Kalpaam-ruthaa (200 mg/kg body weight/d) in olive oil orally by gastric intubation for 14 d. Group V: Breast cancer induced animals (as in Group
II) were treated with the drug, Kalpaam-ruthaa (300 mg/kg body weight/d) in olive oil orally by gastric intubation for 14 d. Group VI: Breast cancer induced animals (as in Group
II) were treated with the drug, Kalpaam-ruthaa (400 mg/kg body weight/d) in olive oil orally by gastric intubation for 14 d. Group VII: Breast cancer induced animals (as in Group
II) were treated with the drug, Kalpaam-ruthaa (500 mg/kg body weight/d) in olive oil orally by gastric intubation for 14 d. Animals were weighed, explored by inspection and palpa-tion and the two major and perpendicular diameters of each were measured with a caliper.
Volume: Total volume was measured using the formula n4/3·p(d1/2) · (d2/2)2, where d1and d2are the two diameter
of the (d1d2). At sacrifice, the volume of each was calcu-lated using its three diameters: n4/3·p(d1/2) · (d2/2)(d3/2).
After the experimental period, the animals were sacrificed by cervical decapitation. Liver and kidney were immediately excised from the animals and the organs were weighed accu-rately and washed well with ice-cold saline and homogenized in Tris–HCl buffer (0.1M, pH 7.4). Blood was also collected for analyses. Body weight and weight were recorded. Protein was estimated by the method of Lowry et al.10) Hexose and
hexosamine in the aliquots were determined by the method of Niebes11) and Wagner,12) respectively. The level of sialic acid was measured by the method of Warren.13)The levels of
lactate dehydrogenase (LDH) and 5 nucleotidase (5 ND) was estimated by the method of King (a) and Luly et al.,14,15) respectively. Acid phosphatase and Cathepsin-D was meas-ured by the method of King (b)16) and Sapolsky,17)
respec-tively.
Statistical Analysis Values are given as the meansS.D.
of six rats. The results were statistically evaluated using
Stu-dent’s t-test and ANOVA. RESULTS
Table 1 indicates the changes in the body weight, organ weights and volume during cancer and drug treated condi-tion. The body weight and organ weights such as liver and kidney were significantly decreased (p0.001) whereas the volume was found to be enormously elevated (p0.001) in mammary carcinoma bearing animals. On treatment with KA, the body weight, organ weights and the volume were significantly recouped back to near normal conditions in a dose dependent manner. The dosage level of 300 mg/kg body weight was found to be the effective dose with a significant (p0.001) effect.
Table 2 shows the glycoprotein levels in plasma, liver and kidney of control and experimental animals. In mammary carcinoma bearing rats, the protein bound carbohydrate com-ponents such as hexose, hexosamine and sialic acid levels were significantly increased (p0.001) when compared to control animals. After treatment, the glycoprotein levels were significantly decreased in drug treated animals in a dose de-pendant manner when compared to mammary carcinoma bearing rats. The drug treatment at the concentration of 300, 400 and 500 mg/kg showed a highly significant effect (p0.001) when compared with cancer induced animals.
Table 3 depicts the activities of lysosomal enzymes namely, acid phosphatase, cathepsin-D in liver and kidney of control and experimental animals. The activities of lysosomal enzymes were found to be significantly increased (p0.001) in carcinoma induced animals than in control animals. On drug treatment, the levels were significantly decreased in a dose dependent manner showing a favorable change in groups treated with 300 mg/kg.
The levels of marker enzymes (LDH, 5ND) in plasma, liver and kidney of control and experimental animals were depicted in Table 4. Activities of marker enzymes were found to be significantly increased in mammary carcinoma bearing animals. There was a significant decrease in the levels of these enzymes in KA treated group animals with a higher significance in 300, 400 and 500 mg/kg treatment.
DISCUSSION
Interest in the use of herbal products has grown dramati-cally in the Western world. With the narrow therapeutic range associated with most anticancer drugs, there is an in-creasing need for understanding possible adverse drug inter-actions in medical oncology.18)KA is non-toxic and has
sev-566 Vol. 29, No. 3
Table 1. Effect of KA on the Changes in the Body Weights, Organ Weights and Volume of Cancer and Drug Treated Animals
Parameters Group I Group II Group III Group IV Group V Group VI Group VII
Body weight (g) 282.8321.01 241.5720.99a,# 249.9121.27b,NS252.0123.17b,NS271.2524.90b,@ 272.2225.16@ 271.1725.95b,@ Liver weight 3.80.31 2.30.18a,# 2.40.18b,NS 2.60.19b,* 3.30.23b,# 3.40.24b,# 3.40.24b,# (g/100 g body weight) Kidney weight 0.870.07 0.610.05a,# 0.630.05b,NS 0.640.05b,* 0.810.07b,# 0.810.07b,# 0.820.07b,# (g/100 g body weight) Volume (cm3) — 12610.6 1169.8b,NS 1119.5b,@ 897.8b,# 877.5b,# 877.5b,#
Values are expressed as meanS.D. of six animals. Initial weights of the animals are more or less and similar. aWhen compared with Group I; bWhen compared with Group
II. Statistical significance: ∗ p0.05, @p0.01, #p0.001 and NS, non-significant. Group I Control; Group II Mammary carcinoma induced; Groups III, IV, V, VI and VII
eral activities that are capable of inhibiting the initiation, de-velopment and progression of cancer.
In mammary carcinoma bearing animals, there was a sharp drop in their body weight. This may be due to the cancer cachexia. Cancer cachexia results in progressive loss of body weight, which is mainly accounted by wasting of host body compartments such as skeletal muscle and adipose tissue. Weight loss and tissue wasting are observed in cancer pa-tients.19)The weight loss implies poor prognosis and shorter
survival time for cancer patients.20) On drug treatment, the gradual increase in body weight indicates the counteractive property of the drug. Food rich in polyphenols, flavonoids, catechins could mediate favourable changes due to their an-tioxidant, immunopotentiating and anti-inflammatory proper-ties.21)This inhibiting property may be due to the presence of
flavonoids, as they have been reported to impart antiprolifer-ative action on several cancer cells.22)
Flavonoids in the KA might influence the studied carcino-genic parameters and might be the main inhibitory represen-tative in the drug. Chakraborty et al.,23) have showed that
flavonoid acts as inducers of apoptosis in cells and thus acts as a potent anti-proliferative agent and this strategy suggest their potential use in cancer control. The flavonoid may in-hibit growth and cause regression of s via modulating the protein kinases activity and through induction of apoptosis.24) The mechanism of carcinogen inactivation, antiproliferation, cell cycle arrest, induction of apoptosis on differentiation, in-hibition of angiogenesis and antioxidation property of flavonoids may be responsible for growth inhibition.25)
Glycoproteins play a vital role in cell-to-cell recognition,
Table 2. Effect of KA on Glycoprotein Components in Mammary Cancer and Drug Treated Animals
Parameters (mg/dl) Group I (Control) Group II Group III Group IV Group V Group VI Group VII Plasma
Hexose 132.018.17 192.2511.64a,# 189.2813.51b,* 187.3113.96b,@ 148.2111.73b,# 150.3312.58b,# 151.2710.85b,# Hexosamine 31.412.92 48.314.12a,# 45.014.07b,@ 44.913.90b,@ 40.803.56b,# 40.033.15b,# 39.223.02b,# Sialic acid 72.226.97 134.5712.31a,# 130.8411.57b,@ 127.979.14b,@ 99.648.36b,# 99.028.91b,# 98.619.04b,# Liver
Hexose 3.420.25 8.237.65a,# 5.020.48b,@ 4.970.41b,@ 3.950.30b,# 4.060.39b,# 3.920.31b,# Hexosamine 3.270.3 9.580.92a,# 8.810.82b,NS 5.870.49b,* 4.040.37b,# 4.100.35b,# 4.070.38b,# Sialic acid 2.770.22 5.160.47a,# 5.240.49b,NS 4.920.34b,* 3.610.26b,# 3.420.29b,# 3.150.30b,# Kidney
Hexose 2.820.21 4.220.38a,# 4.150.34b,NS 4.120.32b,NS 3.040.27b,# 3.070.24b,# 3.050.28b,# Hexosamine 2.150.17 4.270.36a,# 4.120.33b,NS 4.010.30b,* 2.710.18b,# 2.650.2b,# 2.630.22b,# Sialic acid 2.450.21 3.950.30a,# 3.830.32b,NS 3.790.30b,NS 2.630.19b,# 2.540.15b,# 2.490.21b,# Values are expressed as meanS.D. of six animals. Comparisons are made between: a
Groups I with II; b
Groups III, IV, V, VI and VII with Group II. Statistical significance are expressed as: #p0.001; @p0.01; ∗ p0.05; and NS, non-significant. Group I Control; Group II Mammary carcinoma induced; Groups III, IV, V, VI and VII CancerKA
treated animals at a concentrations of 100, 200, 300, 400 and 500 mg/kg body weights respectively.
Table 3. Effect of KA on Lysosomal Enzymes in Mammary Carcinoma and Drug Treated Animals
Parameters Group I Group II Group III Group IV Group V Group VI Group VII
Liver Cathepsin D 32.753.00 64.595.86a,# 62.415.14b,NS 61.415.72b,@ 37.913.18b,# 37.083.04b,# 36.833.12b,# Acidphosphatase 7.040.62 19.861.84a,# 18.211.73b,NS 14.761.33b,@ 9.610.88b,# 10.010.91b,# 9.570.87b,# Kidney Cathepsin D 48.384.15 76.307.11a,# 74.856.96b,* 73.816.54b,@ 53.494.86b,# 52.404.67b,# 52.874.92b,# Acidphosphatase 2.700.13 5.630.41a,# 5.320.47b,@ 5.270.46b,@ 3.020.27b,# 3.050.29b,# 2.940.22b,# Values are expressed as meanS.D. of six animals. Values of ACP are expressed as mmol of phenol formed/min/mg protein. Cathepsin D is expressed mmol of tyrosine liber-ated/h/mg protein. Comparisons are made between: a
Groups I with II; b
Groups III, IV, V, VI and VII with Group II. Statistical significance are expressed as: #p0.001; @ p0.01;
∗ p0.05; and NS, non-significant. Group I Control; Group II Mammary carcinoma induced; Groups III, IV, V, VI and VII CancerKA treated animals at a concentrations of 100, 200, 300, 400 and 500 mg/kg body weights respectively.
Table 4. Effect of KA on Marker Enzymes in Mammary Carcinoma and Drug Treated Animals
Parameters Group I Group II Group III Group IV Group V Group VI Group VII
Plasma LDH 1.070.09 2.010.15a,# 1.850.14b,NS 1.820.11b,* 1.150.09b,# 1.130.10b,# 1.130.09b,# 5-Nucleotidase 2.940.23 5.610.52a,# 5.580.51b,NS 5.450.48b,NS 3.010.22b,# 3.020.28b,# 3.010.26b,# Liver LDH 1.360.11 0.520.04a,# 0.650.05b,@ 0.660.06b,@ 1.040.09b,# 1.070.08b,# 1.330.10b,# 5-Nucleotidase 2.550.20 5.910.51a,# 5.630.47b,NS 4.740.42b,NS 3.010.29b,# 3.100.28b,# 2.980.22b,# Kidney LDH 1.990.12 1.360.12a,# 1.430.09b,NS 1.640.11b,* 1.850.13b,# 1.880.14b,# 1.900.13b,# 5-Nucleotidase 1.720.11 3.140.24a,# 3.010.22b,NS 2.980.22b,NS 2.020.18b,# 2.000.14b,# 2.050.17b,# Lactate dehydrogenase is expressed as mmol of pyruvate liberated/min/mg protein. 5-Nucleotidase is expressed as nmol of Pi liberated/min/mg protein. Values are expressed as meanS.D. of six animals. a
When compared with Group I; bWhen compared with Group II. Statistical significance: ∗ p0.05, @
p0.01, #p0.001 and NS, non-significant.
Group I Control; Group II Mammary carcinoma induced; Groups III, IV, V, VI and VII Cancer + KA treated animals at a concentrations of 100, 200, 300, 400 and 500 mg/kg body weights respectively.
intracellular processing of proteins, cell activation and ability of cancer cells to metastasize.26)It is used for detecting
ma-lignant diseases at an early stage and for evaluating various therapeutic mechanisms. Alterations in the glycoprotein me-tabolism have been exploited for the diagnosis of growth.27) The elevated levels of hexose; hexosamine and sialic acid in cancerous conditions are useful indicators of carcinogenic process, and these changes alter the structure, rigidity and function of cell membranes.28)In plasma, the increased levels of glycoprotein components in cancer condition may be due to the leakage of the disturbed membrane components from either disintegrating or dying neoplastic cells or as a conse-quent shedding of plasma membrane.29)
On drug treatment, glycoprotein component levels were reverted back to near normal levels. This could be due to the cytostabilising property of the drug and flavonoids possess inhibitory action against carcinogenesis.30)The alkaloids and other components of drug may alter the expression of glyco-syltransferases and interfere with cell membrane glycopro-tein synthesis and structure, indicating its potent anti prop-erty. This reduction in the levels of glycoprotein components indicates that the KA has ability to suppress malignancy by modulating cell transformation, decreasing the degree of metastasis, inhibiting the progression of growth, controlling the cancer cell proliferation and differentiation by causing ef-fective favourable changes in the structure of cell mem-branes. The fruits have been reported to contain constituents with variable biological activity and possess antioxidant,31) adaptogenic,32) and hepato-protective33)and anti-activities.34) Chrysin is a natural, biologically active compound extracted from many plants, honey. It possesses potent anti-inflamma-tion, anti-cancer and anti-oxidation properties.35)
LDH is a tetrameric enzymes recognized as a marker with potential use in assessing the progression of the proliferating malignant cells. In the present study, the activities of LDH increase in-bearing animals, possibly as a result of over pro-duction by cells.36)LDH is a fairly sensitive marker for solid neoplasm.37)Numerous reports reveal elevated LDH activity
in various types of s.38)This may be due to the higher
glycol-ysis in the cancerous condition, which is the only energy-pro-ducing pathway for the uncontrolled proliferating malignant cells. The results of the current study show that 5-ND activ-ity was elevated in cancerous animals. Dao et al. (1980)39)
have reported that the increased activity of 5-nucleotidase seems to have originated from the proliferating breast cells. This elevation of the marker enzyme may be correlated with the progression of the malignancy. Walia et al. (1995)40)have
reported higher activities of 5-ND in breast cancer patients. 5-ND activity was found to be increased in cancerous breast tissue of patients.41)
The abnormal variation in the marker enzymes reflects the overall change in metabolism that occurs during malig-nancy.42)This elevation indicates the severity of the disease, or may be due to the release of enzyme from normal tissue invaded by elements.43) The altered activities in the drug
treated animals can be attributed to the presence of flavonoids. The drug SA contains a number of minerals and vitamins. Analysis of SA revealed the presence of calcium, iron, copper, sodium and aluminium44) and it’s also proved
the antiproliferative effects on several cancer cells.22)Other constituents of the drug namely bhilawanols are mainly
lo-calized maximally in cell membrane fraction suggesting their effect through alterations in membrane fractions. KA would change the permeability of the membrane, affecting cellular growth.45)
Lysosomes are a group of cytoplasmic organelles present in numerous animal tissues, characterized by their content of acid hydrolases. These cytoplasmic vesicles contain hy-drolytic enzymes that are capable of digesting the macromol-ecules like polysaccharides, nucleic acids and lipids.46) The invasion in the cancerous conditions may lead to the elevated activity of lysosomal enzymes. This increase may be due to the abnormal fragility of the lysosomes in the cancerous con-dition, which depends on the damage of cell membrane due to enormous production of free radicals in the cancerous con-dition.47) Cathepsin-D is a useful marker for identifying
breast cancer patients with increased risk of recurrent dis-ease.48) The activity of cathepsD of malignant tissues in-creases approximately 3-folds and 2-folds when compared with normal and benign breast disease tissue, respectively.49)
An increased expression of cathepsin-D in malignant breast tissue when compared to normal breast tissue observed in our investigation is in line with previous reports.50)
Reversion of their levels in drug treated animals may be due to the stabilizing property of the drug on lysosomal membrane which could have been imparted by the flavonoids, as it is well established that flavonoids have in-hibiting property on lysosomal membranes. The drug may modify the lysosomal membrane in such a way that it is ca-pable of fusing with the plasma membrane and thereby pre-venting the discharge of acid hydrolases or by inhibiting the release of lysosomal enzymes.51)Numerous reports have
ap-peared on the inhibition of acid hydrolases by flavonoids.52) Studies of cancer treatment in experimental animals have assessed the impact of a wide variety of flavonoids and a se-lected few isoflavones for their efficacy in inhibiting cancer in a number of animal models. Citrus flavonoids were the focus of studies by So et al. (1996).53) The presence of flavonoids has been shown to impart inhibitory action against carcinogenesis.30)The active principles or extracts of P. em-blica have been shown to possess several pharmacologic
ac-tions, e.g., antitumor, and cytotoxic activities.54) Phytogens are phenolic nonsteroidal plant compounds with estro-gen-like biological activity. Most flavonoids are nonestro-genic or weakly estrononestro-genic; however, the isoflavones such as genistein, other flavonoids such as apigenin and kaempferol, and the polyphenolic stilbenes such as resveratrol act through estrogen receptor-mediated mechanisms and also have antie-strogenic effects.55) Polyphenols, which is present in our drug, existing ubiquitously in nature. They exhibit a broad spectrum of biological activities including anti-inflammatory, antiviral, antiatherogenic, antibacterial, as well as anticancer effects.56,57)
The results of the present of study indicate that Kalpaam-ruthaa (modified Siddha preparation) possesses strong anti-cancer effects. This drug exerted a strong antianti-cancer effect in a dose dependent manner and is pronounced at the levels of 300 mg/kg body weight.
REFERENCES
1) Sharma B. K., Ray A., IJCB, 15, 110—117 (2000).
2) Hermeking H., Curr. Opin. Oncol., 15, 44—49 (2003). 3) Bange J., Zwick E., Ullrich A., Nat. Med., 7, 548—552 (2001). 4) Carter S. K., Cancer, 30, 1543—1555 (1972).
5) Fossati R., J. Clin. Oncol., 16, 3439—3460 (1998). 6) Cameron E., Pauling L., Oncology, 27, 181—192 (1973). 7) Lahouel M., Fillastre J. P., Haema., 7, 313—320 (2004). 8) Barnes S., Proc. Soc. Exp. Biol. Med., 217, 386—392 (1998). 9) Sujatha V., Muthumanickam V., Rani G., Sachdanandam P., J. Pharm.
Pharmacol., 51, 241 (1999).
10) Lowry O. H., Rosenbrough N. J., Farr A. L., Randall A. J., J. Biol. Chem., 193, 265—275 (1951).
11) Niebes P., Clin. Chim. Acta, 42, 399—408 (1972). 12) Wagner W. D., Anal. Biochem., 94, 394—397 (1979). 13) Warren L., J. Biol. Chem., 234, 1971—1975 (1959).
14) King E. J., “Practical Clinical Enzymology,” ed. by Van D., Nostrand Company Ltd., London, 1965, pp. 83—93.
15) Luly P., Barnabei O., Tria E., Biochem. Biophys. Acta, 282, 447—452 (1972).
16) King E. J., “Practical Clinical Enzymology,” ed. by Van D., Nostrand Company Ltd., London, 1965, pp. 191—208.
17) Sapolsky A. I., Atlman R. D., Il D. S., Fed. Proc., 32, 1489—1493 (1973).
18) Sparreboom A., Cox M. C., Acharya M. R., Figg W. D., J. Clin. Oncol., 22, 2489—2503 (2004).
19) Argiles J. M., Aczon-Bieto J., Mol. Cell. Biochem., 81, 3—17 (1988). 20) Khan S., Tisdale M. J., Int. J. Cancer, 80, 444—447 (1999).
21) Barber M. D., Fearon K. C., Tisdale M. J., McMillan D. C., Ross J. A., Nutr. Cancer, 40, 110—124 (2001).
22) Birt D. F., Hendrich S., Wang W., Pharmacol. Ther., 90, 157—177 (2001).
23) Chakraborty S., Roy M., Taraphdar A. K., Bhattacharya R. K., Phy-tother. Res., 18, 595—600 (2004).
24) Choi Y. J., Kang J. S., Park J. H. Y., Lee Y. J., Choi J. S., Kang Y. H., J. Nutr., 133, 985—991 (2003).
25) Ren W., Oiao Z., Wang H., Zhu L., Zhang L., Med. Res. Rev., 23, 519—534 (2003).
26) Sell S., Human Path., 21, 1003—1019 (1990).
27) O’Kennedy R., Berno G., Moran E., Smyth H., Carroll K., Thornes R. D., Obrien A., Fennelly J., Butler M., Cancer Lett., 58, 91—100 (1991).
28) Selvam S., Nagini S., Ind. J. Physiol. Pharmacol., 39, 252—254 (1995).
29) Bosman H. B., Spataro H. C., Myres M. W., Res. Commun. Chem. Pathol. Pharmacol., 12, 499—512 (1975).
30) Elangovan V., Nalini R., Balasubramanian S., Sekar N., Govindasamy S., Ind. J. Pharmacol., 26, 266—269 (1994).
31) Rotermund H., Schreiber G., Maeno H., Weinssen U., Weigand K.,
Cancer Res., 30, 2139—2146 (1970).
32) Landel A. M., Hammond W. G., Meguid M. M., Cancer, 55, 230— 237 (1985).
33) Bhattacharya A., Chatterjee A., Ghosal S., Bhattacharya S. K., Ind. J. Exp. Biol., 37, 676—680 (1999).
34) Jeena K. J., Joy K. L., Kuttan R., Cancer Lett., 136, 11—160 (1999). 35) Jose J. K., Kuttan R., J. Ethnopharmacol., 75, 65—69 (2001). 36) Helmes M. H., Modia A., El-Moneim A., Moustafe M. S., EI-Balc A.,
Safinoz M. E. L., Med. Sci. Res., 26, 613—617 (1998).
37) Lippert M., Papadopoulos N., Javadpour N. R., Urology, 18, 50—53 (1981).
38) Engan T., Hannisdal E., Acta Oncol., 29, 151—154 (1990).
39) Dao J. L., Bock F. G., Crouch S., Proc. Soc. Exp. Biol. Med., 102, 635—638 (1959).
40) Walia M., Mahajan M., Singh K., Ind. J. Med. Res., 101, 247—249 (1995).
41) Canbolat O., Duark F., Cetin R., Kavutcu H., Demirci S., Ozturk S., Breast Cancer Research and Treatment, 37, 189—193 (1996). 42) Stefanini M., Cancer, 55, 1931—1936 (1985).
43) Schwartz M. A., West M., Walsch W. S., Zimmerman H. J., Cancer,
15, 346—353 (1962).
44) Premalatha B., Sachdanandam P., Phytother. Res., 14, 352—357 (2000).
45) Patwardhan B., Ghoo R. B., David S. B., Ind. J. Pharm. Sci., 50, 130— 132 (1988).
46) Novikoff A. B., Hers H. G., Van Hoff F., (eds.), “Lysosomes and Storage Diseases,” Academic Press, New York, 5. (1973).
47) Geetha A., Ind. J. Exp. Biol., 31, 288—290 (1993). 48) Barret A. J., Ann. N.Y. Acad. Sci., 674, 1—5 (1992).
49) Schultz D. C., Bazel S., Wright L. M., Cancer Res., 54, 48—54 (1994).
50) Lah T. T., Calaf G., Kalman E., Shinde B. G., Russo J., Jarosz D., Zabrecky J., Somers R., Daskal I., Biol. Chem. Hoppe. Seyler., 376, 357—363 (1995).
51) Carevic O., Djokic S., Agents and Action, 25, 124—131 (1988). 52) Havsteen B., Biochem. Pharmacol., 32, 1141—1148 (1983).
53) So F. V., Guthrie N., Chambers A. F., Moussa M., Carroll K., Nutr. Cancer, 26, 167—181 (1996).
54) Mohamed S. M., Mackeen M. M., Lajis N. H., Rahma A. A., Al A. M., Nat. Prod. Sci., 5, 172—176 (1999).
55) Cos P., De Bruyne T., Apers S., Vanden Berghe D., Pieters L., Vliet-inck A. J., Planta Med., 69, 589—599 (2003).
56) Cos P., Calomme M., Pieters M., Vietinck A. J., Vanden Berghe D., “Studies in Natural Products Chemistry,” Vol. 22, ed. by Atta-Ur-Rah-man, Elsevier Science, Amsterdam, 2000, pp. 307—341.
57) Middleton E., Jr., Kandaswami C., Theoharides T. C., Pharm. Rev., 52, 673—751 (2000).
