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Protective role of Trianthema portulacastrum against diethylnitrosoamine-induced experimental hepatocarcinogenesis
Cancer Letters 129 (1998) 7–13
Protective role of Trianthema portulacastrum against diethylnitrosoamine-induced experimental hepatocarcinogenesis Sukanya Bhattacharya*, Malay Chatterjee Division of Biochemistry, Department of Pharmaceutical Technology, Jadavpur University, Calcutta, India Received 26 August 1997; received in revised form 5 January 1998; accepted 5 February 1998
Abstract The chemopreventive efficacy of the Indian medicinal plant Trianthema portulacastrum L. Aizoaceae was tested in a chemical rat hepatocarcinogenesis model in male Sprague–Dawley rats. Hepatocarcinogenesis was induced by the potent carcinogen diethylnitrosoamine (DENA). Treatment of the rats to the basal medium with aqueous, ethanolic and chloroform fractions of the plant extract at a dose of 100 mg/kg body weight once daily reduced the incidence, numerical preponderance, multiplicity and size distribution of visible neoplastic nodules. Morphometric evaluation of focal lesions showed a reduction of altered liver cell foci/cm2 and a reduction of the average focal area. A decrease in the percentage of liver parenchyma occupied by foci seems to suggest the anticarcinogenic potential of the plant extract in DENA-induced hepatocarcinogenesis. 1998 Published by Elsevier Science Ireland Ltd. All rights reserved Keywords: Trianthema portulacastrum L. Aizoaceae; Diethylnitrosoamine; Hepatocarcinogenesis
1. Introduction The plant Trianthema portulacastrum L., family Aizoaceae, is an exotic weed and a native of tropical America. It is now naturalized throughout India in cultivated fields, river beds, waste ground, etc. [6]. The plant is a diffuse prostrate glabrous succulent herb with many angular branched stems [6,11]. Its flowers are pink or white, solitary and apical in position. The plant has a long history of folklore medicine in India, Bangladesh, The Philippines and other Asian countries [7,11]. It has been used in traditional folk
* Corresponding author. c/o The Principal, Vidyasagar College, Calcutta 39, Sankar Ghosh Lane, Calcutta 700 006, India.
medicine for therapy of a broad spectrum of ailments [7,11]. In India the plant is consumed as a vegetable and is thought to be efficaceous in the treatment of several diseases [5]. The root is cathartic and abortifacient with irritant properties. An infusion of the roots is administered in jaundice, stranguary and dropsy. Leaves have been reported to be diuretic and therefore useful in the treatment of oedema and ascites. A decoction of the herb is a vermifuge, which is useful in the treatment of rheumatism. It is said to be antidotal to alcoholic poison [5,6]. Recently, our laboratory has reported the hepatoprotective activity of the plant in the case of CCL4induced hepatotoxicity in mice [4]. Further work from this laboratory has also demonstrated a clear protective effect of the extract in reversing lipid peroxidation, glutathione status and activities of related
0304-3835/98/$19.00 1998 Published by Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (98 )0 0085-8
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S. Bhattacharya, M. Chatterjee / Cancer Letters 129 (1998) 7–13
antioxidant enzymes in CCL4-induced chronic liver damage in mice. In view of the above, the present study was designed to evaluate the anticarcinogenic potential of the aqueous ethanolic and chloroform fractions of the plant against a defined model of experimental hepatocarcinogenesis.
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats weighing 110–120 g were obtained from the Indian Institute of Chemical Biology (C.S.I.R.), Calcutta, India. These animals were used for the entire study and were housed 10– 12 rats per cage in galvanized metallic cages. The animals were acclimatized to standard laboratory conditions (temperature 25 ± 10°C, relative humidity 60 ± 5% and a 0600 to 1800 h photoperiod) 1 week prior to the actual commencement of the experiment. The animals were supplied with standard pellet diet (Hindustan Lever, Bombay, India) and double distilled demineralized drinking water ad libitum. 2.2. Plant material Dried overground parts of the plant T. portulacastrum L. were supplied by United Chemicals and Allied Products (Calcutta, India). The plant was authenticated by the Botanical Survey of India and a voucher specimen number (JU/Pharm/Biochem/ SPO3) has been deposited in the same herbarium. 2.3. Extraction Air-dried overground parts of T. portulacastrum L. were crushed in a Waring blender and the fine pulverized material was subjected to exhaustive extraction in a Soxhlet’s extractor with petroleum ether (b.p. 60– 80°C). The petroleum ether extract was discarded. The residue was then successively extracted with benzene, chloroform and acetone. Finally, the residue was extracted with ethanol (95% w/v) [20]. The liquid concentrates obtained in the cases of chloroform extract and ethanolic extract were separately subjected to distillation in a distilling flask
using a glass condenser. Each distillation took about 60 min. The extract in each case was taken in a roundbottomed flask placed within a heating mantle. The distillate was collected in another flat-bottomed flask. The flask containing the extract and the flask containing the final distilled product were connected to a condenser which was connected to a water source by rubber tubing. The heating mantle was connected to an electric source. The distillate observed in each case was eventually reduced to solid residue by rotary vacuum evaporation below 45°C to yield 4.6 and 4.8% w/w of chloroform and alcohol extracts, respectively. This was stored at 4°C until use. The chloroform fraction and the ethanolic fraction were separately suspended in deionized water prior to anticarcinogenic evaluation. Aqueous extract of the plant was prepared by dissolving the fine pulverized material in double distilled water. Then, 0.2 ml of 0.05% Tween 80 was added to the sticky chloroform fraction in distilled water using a Potter– Elvehjhum homogenizer. Tween 80 was added as an emulsifying agent to enhance the solubility of the fraction in water. 2.4. Experimental regimen Rats were divided at random into different groups with 10–12 rats per group. Group I′ animals were the Trianthema controls and received crude Trianthema extract and Tween 80. Group I animals belonged to the normal (untreated) control group. Group II animals belonged to the DENA control group (i.e. animals receiving carcinogen and no drug). Group III animals belonged to the DENA + aqueous extracttreated group. Group IV animals belonged to the DENA + Et-OH extract-treated group. Group V animals belonged to the DENA + CHCL3 extract-treated group. The initiation of hepatocarcinogenesis was performed by a single intraperitoneal injection of diethylnitrosoamine (DENA) (Sigma, St. Louis, MO) at a dose of 200 mg/kg body weight in 0.9% NaCl solution [14,18]. Plant drug administration at a dose of 100 mg/kg body weight to the basal medium of each animal once daily was started from the day of carcinogen administration in groups III–V and continued throughout the course of the study. Group I′ animals (Trianthema controls) received the plant drug at the same dose as the Et-OH control group
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different planes under a photomicroscope for specific hepatocellular lesions [1]. Altered hepatocyte foci were counted (number per cm2) and their number and size in mm2 were calculated in the different groups of rats according to published criteria [3,6,18]. The exact percentage area of liver parenchyma occupied by focal lesions was also taken into consideration (Table 4).
and CHCL3 control animals (data not shown) also received the extract (100 mg/kg body weight) throughout the course of the study. After 22 weeks on this regimen, animals were sacrificed and morphometric evaluations were made. 2.5. Morphometry The rats were killed under light ether anaesthesia and fasted overnight prior to sacrifice. After sacrifice, the livers were excised, blotted dry, weighed and examined on the surface for visible macroscopic liver lesions (neoplastic nodules). The greyish white lesions were easily recognized and distinguished from the surrounding non-nodular reddish brown liver parenchyma. The nodules were spherical in shape and were measured in two perpendicular directions to the nearest millimetre to obtain the average diameter of each nodule. Nodules were divided into three categories in accordance with their respective size and the total area of liver parenchyma occupied (≤1, .1–3 and ≥3 mm) as described by Moreno et al. [3,13]. The nodule incidence percentage, the total number of nodules and the nodule multiplicity were calculated (Table 2). From the measured diameters, the individual nodule volume and the nodule volume as a percentage of the total liver volume were calculated (Table 3). For histology, thin sections of excised liver slices from right, left and caudate lobes of each liver were dissected out and quickly immersed in 10% neutral buffered formalin. Embedded liver tissues were microtomed (5 mm thick) and stained with haematoxylin and eosin. Histological slides were scanned in
2.6. Statistical analysis The statistical analysis for the hepatocyte lesions in different groups was performed by Fisher’s exact probability test. All other data were analyzed by Student’s t-test.
3. Results 3.1. Food and water intake There was no treatment-related change in the average daily food and water intake among the different experimental animals. 3.2. Mortality All animals survived during the entire course of the experiment 3.3. Body and liver weights Table 1 depicts the final body and liver weights of animals in grams and the relative liver weights of the
Table 1 Body and liver weights of rats belonging to different groups Group
No. of rats
Final body weight (g)
(I′) Trianthema control (I) Normal (II) DENA control (III) Aqueous extract + DENA (IV) Et-OH extract + DENA (V) Chloroform extract + DENA
10 10 10 12 11 12
313.2 314.1 301.2 311.9 305.1 312.7
± ± ± ± ± ±
29.1 24.6 21.1 37.0 28.6 32.8
Values represents the mean ± SE. a P , 0.02 compared to the normal group by Student’s t-test. b P , 0.05 compared to the DENA control group by Student’s t-test.
Liver weight (g) 10.4 10.5 14.0 10.1 12.2 10.3
± ± ± ± ± ±
2.4 2.2 3.4 2.0 2.6 2.2
Relative liver weight (g liver/100 g body weight) 3.32 3.34 4.64 3.23 3.99 3.29
± ± ± ± ± ±
0.32 0.15 0.47a 0.32b 0.40 0.35b
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Table 2 Effect of T. portulacastrum on the development of DENA-induced hepatic nodules in rats Group
No. of rats with nodules/total no. of rats
Nodule incidence (%)
Total no. of nodules
Average no. of nodules/ nodule-bearing liver (nodule multiplicity) (mean ± SE)
(I′) Trianthema control (I) Normal (II) DENA control (III) Aqueous extract + DENA (IV) Et-OH extract + DENA (V) Chloroform extract + DENA
– – 10/10 6/12 6/11 3/12
– – 100 50a 54.5c 25a
– – 335 119 131 22
– – 33.5 19.8 21.8 7.3
a
± ± ± ±
3.2 2.1b 2.8d 0.9e
P , 0.01, cP , 0.05 compared to the DENA control group by Fisher’s exact probability test. P , 0.01, dP , 0.02, eP , 0.001 compared to the DENA control group by Student’s t-test.
b
experimental animals sacrificed in the 22nd week following DENA administration. There was a slight decrease in the final body weight of rats subjected to DENA (carcinogen) alone as compared to the normal control group. The result is statistically significant. In groups III–V, animals maintained nearly normal body weights after treatment indicating clearly that the aqueous, Et-OH and CHCL3 extracts of T. portulacastrum had no toxic effects on the growth responses of the rats and were fairly well tolerated. The relative liver weights did not vary significantly between the groups, but in groups III and V the variation was significant (P , 0.05) as compared to the DENA control group by Student’s t-test. In the DENA control group the change in liver weight was significant (P , 0.02) as compared to the normal control (group I). Control animals displayed body weights, liver weights and relative liver weights close to the normal untreated group (group I). 3.4. Hepatic neoplastic nodules The administration of aqueous, ethanolic and chloroform extracts of T. portulacastrum at a dose of 100 mg/kg body weight to the basal diet of each animal once daily brought about a significant reduction in the nodule incidence in DENA-induced hepatocarcinogenesis in rats (Table 2). In the DENA group, receiving the carcinogen alone, the nodule incidence was 100%. It dropped to 54.5% in the EtOH extract-treated group (group IV) and to as little as 50% in the aqueous extract-treated group. It was, however, remarkably low in group V following treat-
ment with the chloroform extract of T. portulacastrum. In this case the nodule incidence was 25%. All results are statistically significant. In groups III and V, a P-value of ,0.01 was observed and in group IV, a P-value of ,0.02 was observed compared to the DENA control group by Fisher’s exact probability test. The average number of nodules per nodule-bearing animal (nodule multiplicity) was also considered in our study. In group IV, a P-value of ,0.02 was observed, in group III, a P-value of ,0.01 was observed and in group V, a P-value of ,0.001 was observed compared to the DENA control group by Student’s t-test. Table 3 depicts the size distribution and growth of DENA-induced hepatic nodules in the different groups of rats. The mean nodular volume and the nodule volume as a percentage of the liver volume are shown after 22 weeks of plant drug supplementation. A notable observation in our study was the reduction in the appearance of nodules greater than 3 mm in the treated groups (i.e. groups III–V), compared to the DENA control group. The mean nodular volume showed a decrease from 1.61 cm3 in the DENA control group (group II) to 0.81 cm3 in group III and 0.93 cm3 in group IV. The extract-treated groups revealed a reduced nodular volume and liver volume percentage. In groups III and IV, a P-value of ,0.05 was observed compared to the DENA control group by Student’s t-test. The mean nodular volume was 0.75 cm3 in group V. In group V, a P-value of ,0.001 was observed compared to the DENA control group by Student’s t-test.
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S. Bhattacharya, M. Chatterjee / Cancer Letters 129 (1998) 7–13 Table 3 Effect of T. portulacastrum on the size distribution and growth of DENA-induced hepatic nodules in rats Group
(I′) Trianthema control (I) Normal (II) DENA control (III) Aqueous extract + DENA (IV) Et-OH extract + DENA (V) Chloroform extract + DENA a
No. of rats
– – 10 6 6 3
Size of nodules (mm)
≤1
>1–3
≥3
– – 85 40 39 11
– – 130 46 50 8
– – 120 33 42 3
Mean nodular volume (cm3) (mean ± SE)a
Nodular volume/ liver volume (%) (mean ± SE)b
– – 1.61 0.81 0.93 0.75
– – 67.3 48.7 50.3 31.9
± ± ± ±
0.27c 0.09e 0.14d 0.06f
± ± ± ±
5.9 4.5d 4.6d 3.2g
Individual nodule volume was calculated from the perpendicular diameters measured on each nodule. One gram of liver was assumed to occupy 1 cm3. Value represents the mean ± SE; dP , 0.05; eP , 0.02; fP , 0.01 and gP , 0.001 compared to DENA control group by Student’s t-test.
b c
3.5. Altered hepatocyte foci The effect of T. portulacastrum on the development and growth of altered liver cell foci induced by DENA in rats is given in Table 4. There was a remarkable decrease in the foci incidence from 100% in the carcinogen-fed group to 72.7% in the aqueous extract + DENA-treated group, 75% in the DENA + Et-OH extract-treated groups and 58.3% in the DENA + CHCL3 extract-treated group. There was also a reduction in the number of foci/cm2 in the extract-treated groups. A P-value of ,0.05 was observed in groups III and IV compared to group II. The percentage area of liver parenchyma occupied by foci was also significantly reduced in all the plant drug-treated groups. In group III, a P-value of ,0.01 was observed compared to group II and in group IV, a P-value of ,0.02 was observed compared to group II by Student’s t-test. In
group V, a P-value of ,0.001 was observed compared to the DENA control group by Fisher’s exact probability test.
4. Discussion The present study clearly demonstrates that the anticarcinogenic property of Trianthema portulacastrum L. Aizoaceae is very promising. Although the findings are of a preliminary nature, the present investigations have very clearly demonstrated that the administration of the chloroform extract of T. portulacastrum could bring about a marked reduction of DENA-induced hepatocarcinogenesis. The chloroform extract was remarkable in that it was able to suppress nodule development/hepatocellular lesion formation in rats to such a degree that in most rats
Table 4 Effect of T. portulacastrum on the development and growth of altered liver cell foci induced by DENA in rats Group
No. of rats with foci/total no. of rats
Foci incidence (%)
No. of foci/cm2 (mean ± SE)
Mean focal area (cm2) (mean ± SE)
Area of liver parenchyma occupied by foci (%) (mean ± SE)
(I′) Trianthema control (I) Normal (II) DENA control (III) Aqueous extract + DENA (IV) Et-OH extract + DENA (V) Chloroform extract + DENA
– – 10/10 8/11 9/12 7/12
– – 100 72.7 75.0 58.3d
– – 20.1 12.71 13.11 11.15
– – 0.38 0.20 0.22 0.15
– – 2.91 2.10 2.25 1.62
a
± ± ± ±
2.45 1.85a 1.75a 1.52b
± ± ± ±
0.06 0.05a 0.04a 0.02b
P , 0.05, bP , 0.01, cP , 0.02, eP , 0.001 compared to the DENA control group by Student’s t-test. P , 0.05 compared to the DENA control group by Fisher’s exact probability test.
d
± ± ± ±
0.19 0.16b 0.13c 0.06e
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of group V, nodule development was practically nil or if nodules developed, they were very feeble and very much reduced in size (Figs. 1 and 2). From our study, it could be clearly visualized that following plant drug treatment in DENA-treated animals, a lesser number of rats developed nodules. Furthermore, the nodules decreased in size as compared to the group treated with the carcinogen alone. There was a general reduction of the nodular volume and also of the nodular volume as a percentage of the liver volume. Nodules greater than 3 mm were found to be far less in number in the group treated with the chloroform extract of the plant. The body weights of the rats may be considered to be more or less uniform. The lowering of the nodule incidence appears to be unconnected to the nutritional status of the experimental animals. The animals had normal food and water intake and the plant drug did not affect this intake of food and water, showing that the plant extract was apparently non-toxic and therefore well tolerated by the animals. Furthermore, the extract could confer a great degree of protection against chemical hepatocarcinogenesis, particularly that initiated by DENA. There is ample evidence to show that hepatic preneoplastic lesions are putative precursors of hepatocellular carcinoma in rats and perhaps also in humans [10,15,17,21]. The existing data suggest that liver-altered foci represent neoplasia development and provide information regarding the anticarcinogenic potential of an agent in an experimental model system due to the consistent manner
Fig. 1. Rat liver treated with DENA (22 weeks after i.p. injection of DENA) showing greyish white nodules.
Fig. 2. Rat liver returning to normality after treatment with chloroform extract of Trianthema portulacastrum L. after DENA administration (22-week study).
in which foci appear during post-initiation events of hepatocarcinogenesis [9,16,17]. Since there is a strong correlation between foci and hepatocarcinogenesis, our observed data prove beyond doubt that the plant extract administration can remarkably affect post-initiation stages of hepatocarcinogenesis and may perhaps affect the power with which DENA can induce foci to appear. Most of the antineoplastic agents available in the market produce many toxic side-effects, such as nephrotoxicity, cirrhosis, etc. [8,19], but since the control as well as the experimental animals indulged in normal food and water intake, the extract may be taken to be non-toxic. The fundamental question of how the plant extract exerts its influence on DENA-induced hepatocarcinogenesis comes to mind. It has been reported that DENA confers carcinogenic action by its metabolic conversion to a reactive ethylating agent, which can alkylate cellular DNA [2,12]. The inhibitory effect of the plant extract on DENA-induced hepatocarcinogenesis is likely to be related to the detoxification of the carcinogen through inhibition or by induction of enzymes. Alternatively, the extract may have a role in interfering with carcinogen DNA-binding to elicit its antitumorigenic response. Otherwise the plant extract could play a role in repairing genetic lesions or by reinforcing immune surveillance mechanisms. The fact remains that the chloroform extract of T. portulacastrum has proved significant in reducing nodule incidence by as much as 25% as compared
S. Bhattacharya, M. Chatterjee / Cancer Letters 129 (1998) 7–13
to 100% in the DENA-treated group (a tremendous reduction in the percentage of nodule incidence is thus observed). The chloroform fraction of T. portulacastrum has emerged as the most active fraction inhibiting chemically-induced rat hepatocarcinogenesis.
Acknowledgements This work was supported by a research grant (No. 9/96 (264) 95/EMR-1) from the Council of Scientific and Industrial Research (C.S.I.R.) of the Government of India. Financial assistance to Sukanya Bhattacharya S.R.F. (NET) is gratefully acknowledged. The authors are indebted to Dr Anupam Bishayee for his technical assistance. References [1] P.L. Bannasch, Cytology and cytogenesis of neoplastic (hyperplastic) hepatic nodules, Cancer Res. 36 (1976) 2555–2562. [2] H. Bartsch, E. Hictanen, C. Malavelle, Carcinogenic nitrosoamines: free radical aspects of their action, Free Rad. Biol. Med. 71 (1989) 637–643. [3] A. Bishayee, M. Chatterjee, Inhibition of attenuated liver cell foci and persistent nodule growth by vanadium during diethylnitrosoamine-induced hepatocarcinogenesis in rats, Anticancer Res. 15 (1995) 455–462. [4] A. Bishayee, A. Mandal, M. Chatterjee, Signs of early hepatotoxicity in mice by Trianthema portulacastrum L., Phytomedicine 3 (1996) 155–161. [5] Y.R. Chadham, in: Wealth of India – Raw Materials, Vol. X, C.S.I.R., New Delhi, 1976, p. 281. [6] A. Chatterjee, S. Pakrashi, in: The Treatise of Indian Medicinal Plants, Vol. 1, Publication and Information Directorate, New Delhi, 1994, p. 77. [7] R.N. Chopra, I.C. Chopra, K.L. Handa, in: Indigenous Drugs of India, U.N. Dhur, Calcutta, 1958, p. 528. [8] M.J. Cleave, P.C. Hydes, in: H. Sigel (Ed.), Antitumor Properties of Metal Complexes, Vol. II, Marcel Decker, New York, 1980, pp. 1–62.
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[9] E. Farber, The pathology of experimental liver cancer, in: H.M. Cameron, D.A. Linsell, G.P. Warwick (Eds.), Liver Cell Cancer, Elsevier/North Holland, Amsterdam, 1976, pp. 234–247. [10] E. Farber, R. Cameron, The sequential analysis cancer development, Adv. Cancer Res. 35 (1980) 125–226. [11] K.R. Kirtikar, B.D. Basu, in: E. Blatter, J.F. Qius, K.S. Mhaskar (Eds.), Indian Medicinal Plants, Vol. II, Lalit Mohen Basu, Allabahad, 1975, p. 1180. [12] P.N. Magee, R. Montesano, R. Preussmann, in: C.E. Searle (Ed.), N-Nitrosocompounds and Related Carcinogens, Chemical Carcinogenesis, ACS Monograph Series, No. 173, American Chemical Society, Washington, DC, 1976, pp. 491–625. [13] F.S. Moreno, M.B.S.L. Rizzi, M.L.Z. Dagli, M.V.C. Peateado, Inhibitory effect of B-corotence on preneoplastic lesions induced in Wister rats by the resistant hepatocyte model, Carcinogenesis 12 (1991) 1817–1822. [14] H.C. Pitot, L. Barness, T. Goldsworthy, T. Kitagawa, Biochemical characterization of stages of hepatocarcinogenesis after a single dose of diethylnitrosoamine, Nature 271 (1978) 456–458. [15] H.C. Pitot, A.E. Sirica, The stages of initiation and promotion in hepatocarcinogenesis, Biochim. Biophys. Acta 605 (1980) 191–215. [16] H.M. Raes, P. Scholze, B. Jantsch, Growth kinetics diethylnitrosoamine-induced enzyme deficient preneoplastic liver cell populations in vivo and in vitro, Cancer Res. 32 (1972) 2577–2586. [17] E. Scherer, P. Emmelot, Kinetics of induction and growth of precancerous liver cell foci and liver tumor formation by diethylnitrosoamine in rats, Eur. J. Cancer 11 (1975) 689– 696. [18] H.L. Stewart, G.M. Williams, C.H. Keyesser, L.S. Lombard, R.J. Montali, Histological typing of liver tumors of rat, J. Natl. Cancer Inst. 64 (1980) 177–207. [19] H.J. Thompson, L.D. Meeker, S. Kokosha, Effect of inorganic and organic form of dietary selenium on the promotional stage of mammary carcinogenesis in rats, Cancer Res. 44 (1984) 2803–2806. [20] B. Vohra, S.A. Shah, S.A.H. Nagvi, S. Ahmed, M.S.Y. Khan, Studies on Trianthema portulacastrum, Planta Med. 47 (1983) 106–108. [21] G.M. Williams, The pathogenesis of rat liver cancer caused by chemical carcinogenesis, Biochim. Biophys. Acta 605 (1980) 194–210.
Protective role of Trianthema portulacastrum against diethylnitrosoamine-induced experimental hepatocarcinogenesis Sukanya Bhattacharya*, Malay Chatterjee Division of Biochemistry, Department of Pharmaceutical Technology, Jadavpur University, Calcutta, India Received 26 August 1997; received in revised form 5 January 1998; accepted 5 February 1998
Abstract The chemopreventive efficacy of the Indian medicinal plant Trianthema portulacastrum L. Aizoaceae was tested in a chemical rat hepatocarcinogenesis model in male Sprague–Dawley rats. Hepatocarcinogenesis was induced by the potent carcinogen diethylnitrosoamine (DENA). Treatment of the rats to the basal medium with aqueous, ethanolic and chloroform fractions of the plant extract at a dose of 100 mg/kg body weight once daily reduced the incidence, numerical preponderance, multiplicity and size distribution of visible neoplastic nodules. Morphometric evaluation of focal lesions showed a reduction of altered liver cell foci/cm2 and a reduction of the average focal area. A decrease in the percentage of liver parenchyma occupied by foci seems to suggest the anticarcinogenic potential of the plant extract in DENA-induced hepatocarcinogenesis. 1998 Published by Elsevier Science Ireland Ltd. All rights reserved Keywords: Trianthema portulacastrum L. Aizoaceae; Diethylnitrosoamine; Hepatocarcinogenesis
1. Introduction The plant Trianthema portulacastrum L., family Aizoaceae, is an exotic weed and a native of tropical America. It is now naturalized throughout India in cultivated fields, river beds, waste ground, etc. [6]. The plant is a diffuse prostrate glabrous succulent herb with many angular branched stems [6,11]. Its flowers are pink or white, solitary and apical in position. The plant has a long history of folklore medicine in India, Bangladesh, The Philippines and other Asian countries [7,11]. It has been used in traditional folk
* Corresponding author. c/o The Principal, Vidyasagar College, Calcutta 39, Sankar Ghosh Lane, Calcutta 700 006, India.
medicine for therapy of a broad spectrum of ailments [7,11]. In India the plant is consumed as a vegetable and is thought to be efficaceous in the treatment of several diseases [5]. The root is cathartic and abortifacient with irritant properties. An infusion of the roots is administered in jaundice, stranguary and dropsy. Leaves have been reported to be diuretic and therefore useful in the treatment of oedema and ascites. A decoction of the herb is a vermifuge, which is useful in the treatment of rheumatism. It is said to be antidotal to alcoholic poison [5,6]. Recently, our laboratory has reported the hepatoprotective activity of the plant in the case of CCL4induced hepatotoxicity in mice [4]. Further work from this laboratory has also demonstrated a clear protective effect of the extract in reversing lipid peroxidation, glutathione status and activities of related
0304-3835/98/$19.00 1998 Published by Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (98 )0 0085-8
8
S. Bhattacharya, M. Chatterjee / Cancer Letters 129 (1998) 7–13
antioxidant enzymes in CCL4-induced chronic liver damage in mice. In view of the above, the present study was designed to evaluate the anticarcinogenic potential of the aqueous ethanolic and chloroform fractions of the plant against a defined model of experimental hepatocarcinogenesis.
2. Materials and methods 2.1. Animals Male Sprague–Dawley rats weighing 110–120 g were obtained from the Indian Institute of Chemical Biology (C.S.I.R.), Calcutta, India. These animals were used for the entire study and were housed 10– 12 rats per cage in galvanized metallic cages. The animals were acclimatized to standard laboratory conditions (temperature 25 ± 10°C, relative humidity 60 ± 5% and a 0600 to 1800 h photoperiod) 1 week prior to the actual commencement of the experiment. The animals were supplied with standard pellet diet (Hindustan Lever, Bombay, India) and double distilled demineralized drinking water ad libitum. 2.2. Plant material Dried overground parts of the plant T. portulacastrum L. were supplied by United Chemicals and Allied Products (Calcutta, India). The plant was authenticated by the Botanical Survey of India and a voucher specimen number (JU/Pharm/Biochem/ SPO3) has been deposited in the same herbarium. 2.3. Extraction Air-dried overground parts of T. portulacastrum L. were crushed in a Waring blender and the fine pulverized material was subjected to exhaustive extraction in a Soxhlet’s extractor with petroleum ether (b.p. 60– 80°C). The petroleum ether extract was discarded. The residue was then successively extracted with benzene, chloroform and acetone. Finally, the residue was extracted with ethanol (95% w/v) [20]. The liquid concentrates obtained in the cases of chloroform extract and ethanolic extract were separately subjected to distillation in a distilling flask
using a glass condenser. Each distillation took about 60 min. The extract in each case was taken in a roundbottomed flask placed within a heating mantle. The distillate was collected in another flat-bottomed flask. The flask containing the extract and the flask containing the final distilled product were connected to a condenser which was connected to a water source by rubber tubing. The heating mantle was connected to an electric source. The distillate observed in each case was eventually reduced to solid residue by rotary vacuum evaporation below 45°C to yield 4.6 and 4.8% w/w of chloroform and alcohol extracts, respectively. This was stored at 4°C until use. The chloroform fraction and the ethanolic fraction were separately suspended in deionized water prior to anticarcinogenic evaluation. Aqueous extract of the plant was prepared by dissolving the fine pulverized material in double distilled water. Then, 0.2 ml of 0.05% Tween 80 was added to the sticky chloroform fraction in distilled water using a Potter– Elvehjhum homogenizer. Tween 80 was added as an emulsifying agent to enhance the solubility of the fraction in water. 2.4. Experimental regimen Rats were divided at random into different groups with 10–12 rats per group. Group I′ animals were the Trianthema controls and received crude Trianthema extract and Tween 80. Group I animals belonged to the normal (untreated) control group. Group II animals belonged to the DENA control group (i.e. animals receiving carcinogen and no drug). Group III animals belonged to the DENA + aqueous extracttreated group. Group IV animals belonged to the DENA + Et-OH extract-treated group. Group V animals belonged to the DENA + CHCL3 extract-treated group. The initiation of hepatocarcinogenesis was performed by a single intraperitoneal injection of diethylnitrosoamine (DENA) (Sigma, St. Louis, MO) at a dose of 200 mg/kg body weight in 0.9% NaCl solution [14,18]. Plant drug administration at a dose of 100 mg/kg body weight to the basal medium of each animal once daily was started from the day of carcinogen administration in groups III–V and continued throughout the course of the study. Group I′ animals (Trianthema controls) received the plant drug at the same dose as the Et-OH control group
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different planes under a photomicroscope for specific hepatocellular lesions [1]. Altered hepatocyte foci were counted (number per cm2) and their number and size in mm2 were calculated in the different groups of rats according to published criteria [3,6,18]. The exact percentage area of liver parenchyma occupied by focal lesions was also taken into consideration (Table 4).
and CHCL3 control animals (data not shown) also received the extract (100 mg/kg body weight) throughout the course of the study. After 22 weeks on this regimen, animals were sacrificed and morphometric evaluations were made. 2.5. Morphometry The rats were killed under light ether anaesthesia and fasted overnight prior to sacrifice. After sacrifice, the livers were excised, blotted dry, weighed and examined on the surface for visible macroscopic liver lesions (neoplastic nodules). The greyish white lesions were easily recognized and distinguished from the surrounding non-nodular reddish brown liver parenchyma. The nodules were spherical in shape and were measured in two perpendicular directions to the nearest millimetre to obtain the average diameter of each nodule. Nodules were divided into three categories in accordance with their respective size and the total area of liver parenchyma occupied (≤1, .1–3 and ≥3 mm) as described by Moreno et al. [3,13]. The nodule incidence percentage, the total number of nodules and the nodule multiplicity were calculated (Table 2). From the measured diameters, the individual nodule volume and the nodule volume as a percentage of the total liver volume were calculated (Table 3). For histology, thin sections of excised liver slices from right, left and caudate lobes of each liver were dissected out and quickly immersed in 10% neutral buffered formalin. Embedded liver tissues were microtomed (5 mm thick) and stained with haematoxylin and eosin. Histological slides were scanned in
2.6. Statistical analysis The statistical analysis for the hepatocyte lesions in different groups was performed by Fisher’s exact probability test. All other data were analyzed by Student’s t-test.
3. Results 3.1. Food and water intake There was no treatment-related change in the average daily food and water intake among the different experimental animals. 3.2. Mortality All animals survived during the entire course of the experiment 3.3. Body and liver weights Table 1 depicts the final body and liver weights of animals in grams and the relative liver weights of the
Table 1 Body and liver weights of rats belonging to different groups Group
No. of rats
Final body weight (g)
(I′) Trianthema control (I) Normal (II) DENA control (III) Aqueous extract + DENA (IV) Et-OH extract + DENA (V) Chloroform extract + DENA
10 10 10 12 11 12
313.2 314.1 301.2 311.9 305.1 312.7
± ± ± ± ± ±
29.1 24.6 21.1 37.0 28.6 32.8
Values represents the mean ± SE. a P , 0.02 compared to the normal group by Student’s t-test. b P , 0.05 compared to the DENA control group by Student’s t-test.
Liver weight (g) 10.4 10.5 14.0 10.1 12.2 10.3
± ± ± ± ± ±
2.4 2.2 3.4 2.0 2.6 2.2
Relative liver weight (g liver/100 g body weight) 3.32 3.34 4.64 3.23 3.99 3.29
± ± ± ± ± ±
0.32 0.15 0.47a 0.32b 0.40 0.35b
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Table 2 Effect of T. portulacastrum on the development of DENA-induced hepatic nodules in rats Group
No. of rats with nodules/total no. of rats
Nodule incidence (%)
Total no. of nodules
Average no. of nodules/ nodule-bearing liver (nodule multiplicity) (mean ± SE)
(I′) Trianthema control (I) Normal (II) DENA control (III) Aqueous extract + DENA (IV) Et-OH extract + DENA (V) Chloroform extract + DENA
– – 10/10 6/12 6/11 3/12
– – 100 50a 54.5c 25a
– – 335 119 131 22
– – 33.5 19.8 21.8 7.3
a
± ± ± ±
3.2 2.1b 2.8d 0.9e
P , 0.01, cP , 0.05 compared to the DENA control group by Fisher’s exact probability test. P , 0.01, dP , 0.02, eP , 0.001 compared to the DENA control group by Student’s t-test.
b
experimental animals sacrificed in the 22nd week following DENA administration. There was a slight decrease in the final body weight of rats subjected to DENA (carcinogen) alone as compared to the normal control group. The result is statistically significant. In groups III–V, animals maintained nearly normal body weights after treatment indicating clearly that the aqueous, Et-OH and CHCL3 extracts of T. portulacastrum had no toxic effects on the growth responses of the rats and were fairly well tolerated. The relative liver weights did not vary significantly between the groups, but in groups III and V the variation was significant (P , 0.05) as compared to the DENA control group by Student’s t-test. In the DENA control group the change in liver weight was significant (P , 0.02) as compared to the normal control (group I). Control animals displayed body weights, liver weights and relative liver weights close to the normal untreated group (group I). 3.4. Hepatic neoplastic nodules The administration of aqueous, ethanolic and chloroform extracts of T. portulacastrum at a dose of 100 mg/kg body weight to the basal diet of each animal once daily brought about a significant reduction in the nodule incidence in DENA-induced hepatocarcinogenesis in rats (Table 2). In the DENA group, receiving the carcinogen alone, the nodule incidence was 100%. It dropped to 54.5% in the EtOH extract-treated group (group IV) and to as little as 50% in the aqueous extract-treated group. It was, however, remarkably low in group V following treat-
ment with the chloroform extract of T. portulacastrum. In this case the nodule incidence was 25%. All results are statistically significant. In groups III and V, a P-value of ,0.01 was observed and in group IV, a P-value of ,0.02 was observed compared to the DENA control group by Fisher’s exact probability test. The average number of nodules per nodule-bearing animal (nodule multiplicity) was also considered in our study. In group IV, a P-value of ,0.02 was observed, in group III, a P-value of ,0.01 was observed and in group V, a P-value of ,0.001 was observed compared to the DENA control group by Student’s t-test. Table 3 depicts the size distribution and growth of DENA-induced hepatic nodules in the different groups of rats. The mean nodular volume and the nodule volume as a percentage of the liver volume are shown after 22 weeks of plant drug supplementation. A notable observation in our study was the reduction in the appearance of nodules greater than 3 mm in the treated groups (i.e. groups III–V), compared to the DENA control group. The mean nodular volume showed a decrease from 1.61 cm3 in the DENA control group (group II) to 0.81 cm3 in group III and 0.93 cm3 in group IV. The extract-treated groups revealed a reduced nodular volume and liver volume percentage. In groups III and IV, a P-value of ,0.05 was observed compared to the DENA control group by Student’s t-test. The mean nodular volume was 0.75 cm3 in group V. In group V, a P-value of ,0.001 was observed compared to the DENA control group by Student’s t-test.
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S. Bhattacharya, M. Chatterjee / Cancer Letters 129 (1998) 7–13 Table 3 Effect of T. portulacastrum on the size distribution and growth of DENA-induced hepatic nodules in rats Group
(I′) Trianthema control (I) Normal (II) DENA control (III) Aqueous extract + DENA (IV) Et-OH extract + DENA (V) Chloroform extract + DENA a
No. of rats
– – 10 6 6 3
Size of nodules (mm)
≤1
>1–3
≥3
– – 85 40 39 11
– – 130 46 50 8
– – 120 33 42 3
Mean nodular volume (cm3) (mean ± SE)a
Nodular volume/ liver volume (%) (mean ± SE)b
– – 1.61 0.81 0.93 0.75
– – 67.3 48.7 50.3 31.9
± ± ± ±
0.27c 0.09e 0.14d 0.06f
± ± ± ±
5.9 4.5d 4.6d 3.2g
Individual nodule volume was calculated from the perpendicular diameters measured on each nodule. One gram of liver was assumed to occupy 1 cm3. Value represents the mean ± SE; dP , 0.05; eP , 0.02; fP , 0.01 and gP , 0.001 compared to DENA control group by Student’s t-test.
b c
3.5. Altered hepatocyte foci The effect of T. portulacastrum on the development and growth of altered liver cell foci induced by DENA in rats is given in Table 4. There was a remarkable decrease in the foci incidence from 100% in the carcinogen-fed group to 72.7% in the aqueous extract + DENA-treated group, 75% in the DENA + Et-OH extract-treated groups and 58.3% in the DENA + CHCL3 extract-treated group. There was also a reduction in the number of foci/cm2 in the extract-treated groups. A P-value of ,0.05 was observed in groups III and IV compared to group II. The percentage area of liver parenchyma occupied by foci was also significantly reduced in all the plant drug-treated groups. In group III, a P-value of ,0.01 was observed compared to group II and in group IV, a P-value of ,0.02 was observed compared to group II by Student’s t-test. In
group V, a P-value of ,0.001 was observed compared to the DENA control group by Fisher’s exact probability test.
4. Discussion The present study clearly demonstrates that the anticarcinogenic property of Trianthema portulacastrum L. Aizoaceae is very promising. Although the findings are of a preliminary nature, the present investigations have very clearly demonstrated that the administration of the chloroform extract of T. portulacastrum could bring about a marked reduction of DENA-induced hepatocarcinogenesis. The chloroform extract was remarkable in that it was able to suppress nodule development/hepatocellular lesion formation in rats to such a degree that in most rats
Table 4 Effect of T. portulacastrum on the development and growth of altered liver cell foci induced by DENA in rats Group
No. of rats with foci/total no. of rats
Foci incidence (%)
No. of foci/cm2 (mean ± SE)
Mean focal area (cm2) (mean ± SE)
Area of liver parenchyma occupied by foci (%) (mean ± SE)
(I′) Trianthema control (I) Normal (II) DENA control (III) Aqueous extract + DENA (IV) Et-OH extract + DENA (V) Chloroform extract + DENA
– – 10/10 8/11 9/12 7/12
– – 100 72.7 75.0 58.3d
– – 20.1 12.71 13.11 11.15
– – 0.38 0.20 0.22 0.15
– – 2.91 2.10 2.25 1.62
a
± ± ± ±
2.45 1.85a 1.75a 1.52b
± ± ± ±
0.06 0.05a 0.04a 0.02b
P , 0.05, bP , 0.01, cP , 0.02, eP , 0.001 compared to the DENA control group by Student’s t-test. P , 0.05 compared to the DENA control group by Fisher’s exact probability test.
d
± ± ± ±
0.19 0.16b 0.13c 0.06e
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of group V, nodule development was practically nil or if nodules developed, they were very feeble and very much reduced in size (Figs. 1 and 2). From our study, it could be clearly visualized that following plant drug treatment in DENA-treated animals, a lesser number of rats developed nodules. Furthermore, the nodules decreased in size as compared to the group treated with the carcinogen alone. There was a general reduction of the nodular volume and also of the nodular volume as a percentage of the liver volume. Nodules greater than 3 mm were found to be far less in number in the group treated with the chloroform extract of the plant. The body weights of the rats may be considered to be more or less uniform. The lowering of the nodule incidence appears to be unconnected to the nutritional status of the experimental animals. The animals had normal food and water intake and the plant drug did not affect this intake of food and water, showing that the plant extract was apparently non-toxic and therefore well tolerated by the animals. Furthermore, the extract could confer a great degree of protection against chemical hepatocarcinogenesis, particularly that initiated by DENA. There is ample evidence to show that hepatic preneoplastic lesions are putative precursors of hepatocellular carcinoma in rats and perhaps also in humans [10,15,17,21]. The existing data suggest that liver-altered foci represent neoplasia development and provide information regarding the anticarcinogenic potential of an agent in an experimental model system due to the consistent manner
Fig. 1. Rat liver treated with DENA (22 weeks after i.p. injection of DENA) showing greyish white nodules.
Fig. 2. Rat liver returning to normality after treatment with chloroform extract of Trianthema portulacastrum L. after DENA administration (22-week study).
in which foci appear during post-initiation events of hepatocarcinogenesis [9,16,17]. Since there is a strong correlation between foci and hepatocarcinogenesis, our observed data prove beyond doubt that the plant extract administration can remarkably affect post-initiation stages of hepatocarcinogenesis and may perhaps affect the power with which DENA can induce foci to appear. Most of the antineoplastic agents available in the market produce many toxic side-effects, such as nephrotoxicity, cirrhosis, etc. [8,19], but since the control as well as the experimental animals indulged in normal food and water intake, the extract may be taken to be non-toxic. The fundamental question of how the plant extract exerts its influence on DENA-induced hepatocarcinogenesis comes to mind. It has been reported that DENA confers carcinogenic action by its metabolic conversion to a reactive ethylating agent, which can alkylate cellular DNA [2,12]. The inhibitory effect of the plant extract on DENA-induced hepatocarcinogenesis is likely to be related to the detoxification of the carcinogen through inhibition or by induction of enzymes. Alternatively, the extract may have a role in interfering with carcinogen DNA-binding to elicit its antitumorigenic response. Otherwise the plant extract could play a role in repairing genetic lesions or by reinforcing immune surveillance mechanisms. The fact remains that the chloroform extract of T. portulacastrum has proved significant in reducing nodule incidence by as much as 25% as compared
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to 100% in the DENA-treated group (a tremendous reduction in the percentage of nodule incidence is thus observed). The chloroform fraction of T. portulacastrum has emerged as the most active fraction inhibiting chemically-induced rat hepatocarcinogenesis.
Acknowledgements This work was supported by a research grant (No. 9/96 (264) 95/EMR-1) from the Council of Scientific and Industrial Research (C.S.I.R.) of the Government of India. Financial assistance to Sukanya Bhattacharya S.R.F. (NET) is gratefully acknowledged. The authors are indebted to Dr Anupam Bishayee for his technical assistance. References [1] P.L. Bannasch, Cytology and cytogenesis of neoplastic (hyperplastic) hepatic nodules, Cancer Res. 36 (1976) 2555–2562. [2] H. Bartsch, E. Hictanen, C. Malavelle, Carcinogenic nitrosoamines: free radical aspects of their action, Free Rad. Biol. Med. 71 (1989) 637–643. [3] A. Bishayee, M. Chatterjee, Inhibition of attenuated liver cell foci and persistent nodule growth by vanadium during diethylnitrosoamine-induced hepatocarcinogenesis in rats, Anticancer Res. 15 (1995) 455–462. [4] A. Bishayee, A. Mandal, M. Chatterjee, Signs of early hepatotoxicity in mice by Trianthema portulacastrum L., Phytomedicine 3 (1996) 155–161. [5] Y.R. Chadham, in: Wealth of India – Raw Materials, Vol. X, C.S.I.R., New Delhi, 1976, p. 281. [6] A. Chatterjee, S. Pakrashi, in: The Treatise of Indian Medicinal Plants, Vol. 1, Publication and Information Directorate, New Delhi, 1994, p. 77. [7] R.N. Chopra, I.C. Chopra, K.L. Handa, in: Indigenous Drugs of India, U.N. Dhur, Calcutta, 1958, p. 528. [8] M.J. Cleave, P.C. Hydes, in: H. Sigel (Ed.), Antitumor Properties of Metal Complexes, Vol. II, Marcel Decker, New York, 1980, pp. 1–62.
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