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Cancer chemopreventive activity of Achyranthes aspera leaves on Epstein–Barr virus activation and two-stage mouse skin carcinogenesis
Cancer Letters 177 (2002) 1–5 www.elsevier.com/locate/canlet
Cancer chemopreventive activity of Achyranthes aspera leaves on Epstein–Barr virus activation and two-stage mouse skin carcinogenesis Asima Chakraborty a, Adelheid Brantner a,*, Teruo Mukainaka b, Yoshitoshi Nobukuni b, Masashi Kuchide b, Takao Konoshima c, Harukuni Tokuda b, Hoyoku Nishino b b
a Institute of Pharmacognosy, University of Graz, Universitaetsplatz 4/I, A-8010 Graz, Austria Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan c Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8414, Japan
Received 30 April 2001; received in revised form 5 September 2001; accepted 6 September 2001
Abstract Achyranthes aspera leaves have been assessed for chemopreventive activity. The MeOH extract, alkaloid, non-alkaloid and saponin fractions exhibited significant inhibitory effects (concentration 100 mg) on the Epstein–Barr virus early antigen activation induced by the tumor promotor 12-O-tetradecanoylphorbol-13-acetate in Raji cells. In this in vitro assay the nonalkaloid fraction containing mainly non-polar compounds showed the most significant inhibitory activity (96.9%; 60% viability). In the in vivo two-stage mouse skin carcinogenesis test the total methanolic extract possessed a pronounced anticarcinogenic effect (76%). The present study suggests that A. aspera leaf extract and the non-alkaloid fraction are valuable antitumor promotors in carcinogenesis. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Achyranthes aspera; Antitumor promotor; Epstein–Barr virus activation; Two-stage carcinogenesis
1. Introduction Achyranthes aspera L. (Amaranthaceae) is an indigenous medicinal plant of Asia, South America, and Africa that is commonly used by traditional healers for the treatment of fever, especially malarial fever, dysentery, asthma, hypertension and diabetes [1–3]. The root extract is well reputed for its pronounced insect molting hormonal activity. A decoction of the whole plant is described to have diuretic properties and the aqueous * Corresponding author. Tel.: 143-316-380-5528; fax: 143-316380-9860. E-mail address: [email protected] (A. Brantner).
extract is given for pneumonia. The dried herb is used to treat children for colic and also as an astringent in gonorrhea treatment [4]. The roots of A. aspera are reported to have application in infantile diarrhea and cold [5] while dry leaves are employed against asthma [6]. The seeds are regarded as having emetic and hydrophobic properties [7]. Leaf extracts are reported to possess hypoglycemic, thyroid-stimulating and antiperoxidative activities [8,9]. Two oleanolic acid-based saponins [10], amino acids and oleanolic acid [5] have been isolated from the unripe fruits and seeds. The occurrence of ecdysterone and oleanolic acid has been reported from the roots. Leaves and stems also contain ecdysterone. Alkaloids of the betaine type or betalaine were identified in the leaves and roots [3].
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00766-2
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A. Chakraborty et al. / Cancer Letters 177 (2002) 1–5
From the shoots of A. aspera an aliphatic dihydroxyketone (36,47-dihydroxyhenpentacontan-4-one) has been isolated and characterized [4]. A careful survey of the literature revealed that no study of chemopreventive activity has so far been conducted on this plant. The present paper deals with the evaluation of A. aspera leaves as a cancer chemopreventor. The methanolic extract, and the alkaloid, non-alkaloid, and saponin fractions were investigated by the Epstein–Barr virus early antigen (EBV-EA) activation assay in vitro and the two-stage mouse skin carcinogenesis test in vivo. The EBV is used for an in vitro model because its infection is implicated in various kinds of human neoplasms including certain types of cutaneous T or natural killer cell proliferative disorders. EBV encoded gene (B 958 EBV) has already been mapped for promotor. EBV harboring non-producer Raji cells were activated to express EBV-EA by the use of 12-O-tetradecanoylphorbol-13-acetate (TPA). EBV-EA induction by TPA is related to the activation of protein kinase C and phospholipase A2 [11]. 2. Materials and methods 2.1. Chemicals TPA and 7,12-dimethylbenz[a]anthracene (DMBA) were obtained from Sigma (USA).
30 h, 668C). The drug/solvent ratio was 1:6. The crude methanolic extracts were concentrated under reduced pressure to yield 100 g of a green gummy residue. An equivalent (1.5 g) was dissolved in 5% aqueous HCl and extracted with CHCl3 to separate the non-alkaloid part (100 mg). The acidic fraction was basified with aqueous NH4OH (pH 9) and extracted with CHCl3. The organic solvent was evaporated to furnish the crude alkaloid extract (14 mg). All fractions were evaporated to dryness. For the determination of the alkaloid fraction the chloroform extracts obtained after acidification and basification were tested with Dragendorff’s and Mayer’s reagent. The chloroform part obtained after basification gave a positive result in both assays. In the other chloroform fraction no alkaloids could be proved. Saponins were separated from the aqueous fraction. The aqueous extract (250 ml) was successively extracted with ether and nBuOH. The saponin mixture was extracted from the syrupy n-BuOH fraction by MeOH. A large volume of ether was added to the methanolic part. The brown precipitate of the crude saponins was filtered and purified twice by redissolving in MeOH and precipitating with ether. The purification gave a light brown powdery mixture of crude saponins (40 mg). The aqueous non-saponin fraction is under investigation. The MeOH extract (Me), non-alkaloid fraction (Nal), alkaloid fraction and isolated saponin fraction were used for subsequent experiments.
2.2. Animals
2.4. In vitro EBV-EA activation assay
Pathogen-free female ICR mice (6 weeks old) were purchased from Nippon SLC Co. Ltd. (Shizuoka, Japan). They were kept in groups of five animals per polycarbonated cage in a temperature-controlled room at 24 ^ 28C. Animals were fed a MP solid diet (Oriental yeast Ltd., Chiba, Japan) and water ad libitum.
The inhibition of EBV-EA activation was assayed using Raji cells (EBV genome-carrying human lymphoblastoid cells; EBV non-producer type; the Raji cells were obtained from Professor G. Klein, Department of Microbiology and Tumor Biology Center, Karolinska Institute, Sweden) cultivated in 10% FBS RPMI-1640 medium (Nakalai Tesque, Kyoto, Japan). The indicator cells (1 £ 10 6 Raji cells/ml) were incubated in 1 ml of medium containing 4 mM n-butyric acid as an inducer, 32 pmol of TPA (20 ng/ml DMSO) and test samples (1, 10, 100 mg/ml TPA) at 378C in a CO2 incubator. After 48 h the cell suspensions were centrifuged at 1000 rev./min and the supernatant was removed. The pellet was resuspended in 0.1 ml of phosphate buffer solution and the cell suspension was smeared. The activated
2.3. Preparation of extracts and fractions The leaves of A. aspera were collected at Calcutta, India in December 1999. Authentication of plant material was carried out by Dr Sitesh Das at the Ayurvedic Research Institute, Calcutta, where the herbarium voucher has been kept. Air-dried (30 ^ 28C) powdered leaves (250 g) were extracted in a soxhlet apparatus with MeOH (250 g,
A. Chakraborty et al. / Cancer Letters 177 (2002) 1–5
cells were stained with high titer EBV-EA-positive sera from nasopharyngeal carcinoma patients and the conventional indirect immunofluorescence technique was employed for detection. In each assay at least 500 cells were counted. The experiments were repeated three times. The average extent of EA induction was determined and compared with the positive control experiments in which the cells were treated with n-butyric acid plus TPA. The EA induction was found to be more than 30%. 2.5. In vivo two-stage carcinogenesis test on mouse skin papillomas promoted by TPA The mice were divided into two groups. Each group was composed of 15 mice housed in bunches of five per cage. Hair was clipped in the dorsal region with surgical clippers with proper care before the first day of initiation. The mice were initiated with DMBA (100 mg/0.1 ml acetone) [12]. One week after initiation, papilloma formation was promoted twice a week by application of TPA (1 mg/0.1 ml acetone). The positive controls were given 1 mg TPA/0.1 ml acetone alone topically to the shaved dorsal skin. The other group of mice was treated with the test samples (methanolic extract or non-alkaloid fraction; 50 mg/ 0.1 ml acetone each) 1 h before each TPA treatment. Animals were examined weekly for the development of tumors on the skin for 20 weeks. The tumor incidence was statistically analyzed by Student’s t-test in treated mice and controls. 3. Results and discussion The total methanolic extract of A. aspera and the
3
alkaloid, non-alkaloid and saponin fractions were tested for their antitumor activity using a short-term in vitro assay of TPA-induced EBV-EA activation in Raji cells. These inhibitory effects and the viabilities of Raji cells are shown in Table 1. All tested substances revealed inhibitory effects on EBV-EA activation by TPA. The total methanolic extract and alkaloid fraction exhibited strong inhibitory activities (93.3 and 93.2% at 100 mg/ml and 51.8 and 52.7% at 10 mg/ml, respectively). The extract and alkaloid fractions preserved high viabilities (70% at 100 mg/ml). The non-alkaloid fraction showed the most significant inhibitory effect on EBV-EA activation among all fractions tested (96.9% inhibition at 100 mg/ml) and more than 60% inhibition at 10 mg/ ml with weak cytotoxicity (60% viability at 100 mg/ ml). This inhibitory activity is higher than retinoic acid (27.7% at 100 mg/ml and 0% at 10 mg/ml, respectively). The saponin fraction showed moderate inhibitory activity (89.5% at 100 mg/ml and 48% at 10 mg/ ml) with high viabilities (70% at 100 mg/ml). The results of this in vitro experiment suggest the nonalkaloid fraction to be a valuable antitumor promotor. On the basis of these in vitro results the non-alkaloid fraction and the total methanolic extract were selected for the in vivo two-stage carcinogenesis assay on mouse skin papillomas using DMBA as an initiator. The tumor initiating property as well as the tumor-promoting potential of the samples were evaluated. Fig. 1A indicates the time course of tumor formation. The first tumors were observed in the group treated with DMBA 1 TPA at 6 weeks. The tumor initiating activity of the Nal group was higher (7 weeks) in comparison to the animals treated with
Table 1 Relative ratio of A. aspera extract and fractions on EBV-EA activation with respect to positive control a TPA (20 ng/ml)
MeOH extract Non-alkaloid fraction Alkaloid fraction Saponin fraction Retinoic acid a
Sample concentration (mg/ml TPA) (% to control (% viability)) 100
10
1
6.7 ^ 2.9 (70) 3.1 ^ 2.5 (60) 6.8 ^ 2.6 (70) 10.5 ^ 2.3 (70) 72.3 ^ 4.2 (60)
48.2 ^ 3.8 39.7 ^ 2.7 52.7 ^ 3.1 52.0 ^ 2.8 100.0 ^ 0
94.5 ^ 0.8 91.7 ^ 1.7 94.0 ^ 0.6 100.0 ^ 0 100.0 ^ 0
At a concentration of 100 mg ratio/TPA, all samples were statistically different from control (TPA 20 ng/ml, 100%) and showed a significant inhibitory effect against EBV-EA activation.
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A. Chakraborty et al. / Cancer Letters 177 (2002) 1–5
Fig. 1. Inhibitory effects of methanol extract (Me) and non-alkaloid fraction (Nal) on mouse skin carcinogenesis induced by DMBA-TPA. (A) Percentage of mice with papillomas. (B) Average number of papillomas per mouse. X, DMBA (100 mg) 1 TPA (1 mg) alone; K, DMBA (100 mg) 1 TPA (1 mg) 1 Nal (50 mg); A, DMBA (100 mg) 1 TPA (1 mg) 1 Me (50 mg). Results were statistically different from the control group (*P . 0:01, **P . 0:05).
Me (8 weeks). After 10 weeks the percentage of tumor-bearing mice treated with DMBA 1 TPA was 100% whereas 42% animals of the Nal group and 14% of the Me group showed the appearance of tumors. During the total experimental period of 20 weeks 100% (DMBA 1 TPA), 100% (Nal) and 64% (Me) of mice had developed tumors (Fig. 1A). The effect of the Nal treatment is comparable to that of retinoic acid (45% after 10 weeks, 95.5% after 20 weeks; concentration 50 mg), which is a known tumor inhibitor. Fig. 1B shows the average number of tumors per mouse. After 20 weeks of promotion it was observed that 9.5 ^ 2.0 tumors/mouse (8.3 ^ 1.8 tumors/mouse after 15 weeks) were found in the control group (DMBA 1 TPA). At the same evaluation period only 2.3 ^ 0.3 tumors/mouse (1.8 ^ 0.5 tumors/ mouse after 15 weeks) in the Me group and 5.6 ^ 1.3 tumors/mouse (4.2 ^ 1.4 tumors/mouse after 15 weeks) in the Nal group were formed. Treatment with Me caused a 76% reduction in the average number of tumors/mouse after 20 weeks of promotion. After treatment with Nal a reduction of 41% was observed. Retinoic acid in comparison caused 4.4 ^ 1.4 tumors/mouse causing a 53.6% reduction of tumors after 20 weeks of promotion.
The results of the in vitro primary screening EBVEA activation test indicate that the non-alkaloid fraction containing mainly non-polar compounds (ecdysterone, dihydroxyketone) showed the most significant inhibitory activity (96.9% at 100 mg/ml) among all tested fractions. The effect is stronger than retinoic acid, a well known chemopreventor as well as an anticarcinogenic agent [13] working on the suppression of the epigenetic tumor promotion [14]. The topical exposure on mouse skin led to a strong anticarcinogenic effect of the methanolic extract at the dose level tested. The anticarcinogenic effect was proved firstly by a delayed tumor formation and secondly by a smaller number of papillomas per mouse in comparison to the other test samples. As a mechanism of action the direct free radical scavenging activity of the plant extract [9] or the inhibition of ornithine decarboxylase activity by oleanolic acid [15] can be suggested. The total methanolic extract of A. aspera leaves contains ecdysterone and dihydroxyketone as nonpolar compounds and alkaloids, oleanolic acid, saponins, flavonoids (total content of hydrolyzed glycosides and free aglycons; mean 3.02 ^ 0.15%; N ¼ 3) [16] and tannins (total content, mean 0.44 ^ 0.03%; N ¼ 3) [17].
A. Chakraborty et al. / Cancer Letters 177 (2002) 1–5
The present study suggests that A. aspera leaf extract and the non-alkaloid fraction are valuable antitumor promotors in carcinogenesis. Further investigations are in progress to clarify the influence of the individual compounds on the cancer chemopreventive activity of A. aspera leaves and to specify their possible mechanism of action. Acknowledgements This study was supported partly by Grants-in-Aid from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan. References [1] R.D. Girach, A.S.A. Khan, Ethnomedicinal uses of Achyranthes aspera leaves in Orissa (India), Int. J. Pharmacogn. 30 (1992) 113–115. [2] W. Tang, G. Eisenbrand, Achyranthes bidentata BI, in: Chinese Drugs of Plant Origin, Springer-Verlag, Berlin, 1992, pp. 13–17. [3] K.H. Bhom R. Liersch, Achyranthes, in: R. Haensel, K. Keller, H. Rimpler, G. Schneider (Eds.), Hagers Handbuch der Pharmazeutischen Praxis, V, Springer-Verlag, Berlin, 1992, pp. 54–59. [4] T.N. Misra, R.S. Singh, H.S. Pandey, C. Prasad, An aliphatic dihydroxyketone from Achyranthes aspera, Phytochemistry 30 (1991) 2076–2078. [5] S.K. Borthakur, N. Goswami, Herbal remedies from Dimoria of kamrup district of Assam in northeastern India, Fitoterapia 66 (1995) 333–340.
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[6] V. Singh, Traditional remedies to treat asthma in the North West and Trans-Himalayan region in J. & K. state, Fitoterapia 66 (1995) 507–509. [7] A.K. Batta, S. Rangaswami, Crystalline chemical components of some vegetable drugs, Phytochemistry 12 (1973) 214–216. [8] M.S. Akhtar, J. Iqbal, Evaluation of the hypoglycemic effect of Achyranthes aspera in normal and alloxan diabetic rabbits, J. Ethnopharmacol. 31 (1991) 49–57. [9] P. Tahiliani, A. Kar, Achyranthes aspera elevates thyroid hormone level and decreases hepatic lipid peroxidation in male rats, J. Ethnopharmacol. 71 (2000) 527–532. [10] V. Seshadri, A.K. Batta, S. Rangaswami, Structure of two new saponins from Achyranthes aspera, Ind. J. Chem. 20B (1981) 773–775. [11] K. Ohmori, K. Miyazaki, M. Umeda, Detection of tumor promotors by early antigen expression of EB virus in Raji cells using a fluorescence microplate-reader, Cancer Lett. 132 (1998) 51–59. [12] M. Takasaki, T. Konoshima, H. Etoh, I.P. Singh, H. Tokuda, H. Nishino, Cancer chemopreventive activity of euglobal-G1 from leaves of Eucalyptus grandis, Cancer Lett. 155 (2000) 61–65. [13] K. Shudo, From cancer prevention to cancer treatment, Yakugaku Zasshi 120 (2000) 987–995. [14] T. Yamamoto, Chemoprevention and FAR therapy regimen comprised of 5-fluorouracil, vitamin A and radiation, Gan To Kagaku Ryoho 28 (2001) 454–460. [15] T. Oguro, J. Liu, C.D. Klaassen, T. Yoshida, Inhibitory effect of oleanolic acid on 12-O-tetradecanoylphorbol-13-acetateinduced gene expression in mouse skin, Toxicol. Sci. 45 (1998) 88–93. [16] European Pharmacopoeia, 3rd Edition, Suppl., Council of Europe, Strasbourg Cedex, 1998, p. 203. [17] European Pharmacopoeia, 3rd Edition, Suppl., Council of Europe, Strasbourg Cedex, 2000, p. 83.
Cancer chemopreventive activity of Achyranthes aspera leaves on Epstein–Barr virus activation and two-stage mouse skin carcinogenesis Asima Chakraborty a, Adelheid Brantner a,*, Teruo Mukainaka b, Yoshitoshi Nobukuni b, Masashi Kuchide b, Takao Konoshima c, Harukuni Tokuda b, Hoyoku Nishino b b
a Institute of Pharmacognosy, University of Graz, Universitaetsplatz 4/I, A-8010 Graz, Austria Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan c Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8414, Japan
Received 30 April 2001; received in revised form 5 September 2001; accepted 6 September 2001
Abstract Achyranthes aspera leaves have been assessed for chemopreventive activity. The MeOH extract, alkaloid, non-alkaloid and saponin fractions exhibited significant inhibitory effects (concentration 100 mg) on the Epstein–Barr virus early antigen activation induced by the tumor promotor 12-O-tetradecanoylphorbol-13-acetate in Raji cells. In this in vitro assay the nonalkaloid fraction containing mainly non-polar compounds showed the most significant inhibitory activity (96.9%; 60% viability). In the in vivo two-stage mouse skin carcinogenesis test the total methanolic extract possessed a pronounced anticarcinogenic effect (76%). The present study suggests that A. aspera leaf extract and the non-alkaloid fraction are valuable antitumor promotors in carcinogenesis. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Achyranthes aspera; Antitumor promotor; Epstein–Barr virus activation; Two-stage carcinogenesis
1. Introduction Achyranthes aspera L. (Amaranthaceae) is an indigenous medicinal plant of Asia, South America, and Africa that is commonly used by traditional healers for the treatment of fever, especially malarial fever, dysentery, asthma, hypertension and diabetes [1–3]. The root extract is well reputed for its pronounced insect molting hormonal activity. A decoction of the whole plant is described to have diuretic properties and the aqueous * Corresponding author. Tel.: 143-316-380-5528; fax: 143-316380-9860. E-mail address: [email protected] (A. Brantner).
extract is given for pneumonia. The dried herb is used to treat children for colic and also as an astringent in gonorrhea treatment [4]. The roots of A. aspera are reported to have application in infantile diarrhea and cold [5] while dry leaves are employed against asthma [6]. The seeds are regarded as having emetic and hydrophobic properties [7]. Leaf extracts are reported to possess hypoglycemic, thyroid-stimulating and antiperoxidative activities [8,9]. Two oleanolic acid-based saponins [10], amino acids and oleanolic acid [5] have been isolated from the unripe fruits and seeds. The occurrence of ecdysterone and oleanolic acid has been reported from the roots. Leaves and stems also contain ecdysterone. Alkaloids of the betaine type or betalaine were identified in the leaves and roots [3].
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00766-2
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A. Chakraborty et al. / Cancer Letters 177 (2002) 1–5
From the shoots of A. aspera an aliphatic dihydroxyketone (36,47-dihydroxyhenpentacontan-4-one) has been isolated and characterized [4]. A careful survey of the literature revealed that no study of chemopreventive activity has so far been conducted on this plant. The present paper deals with the evaluation of A. aspera leaves as a cancer chemopreventor. The methanolic extract, and the alkaloid, non-alkaloid, and saponin fractions were investigated by the Epstein–Barr virus early antigen (EBV-EA) activation assay in vitro and the two-stage mouse skin carcinogenesis test in vivo. The EBV is used for an in vitro model because its infection is implicated in various kinds of human neoplasms including certain types of cutaneous T or natural killer cell proliferative disorders. EBV encoded gene (B 958 EBV) has already been mapped for promotor. EBV harboring non-producer Raji cells were activated to express EBV-EA by the use of 12-O-tetradecanoylphorbol-13-acetate (TPA). EBV-EA induction by TPA is related to the activation of protein kinase C and phospholipase A2 [11]. 2. Materials and methods 2.1. Chemicals TPA and 7,12-dimethylbenz[a]anthracene (DMBA) were obtained from Sigma (USA).
30 h, 668C). The drug/solvent ratio was 1:6. The crude methanolic extracts were concentrated under reduced pressure to yield 100 g of a green gummy residue. An equivalent (1.5 g) was dissolved in 5% aqueous HCl and extracted with CHCl3 to separate the non-alkaloid part (100 mg). The acidic fraction was basified with aqueous NH4OH (pH 9) and extracted with CHCl3. The organic solvent was evaporated to furnish the crude alkaloid extract (14 mg). All fractions were evaporated to dryness. For the determination of the alkaloid fraction the chloroform extracts obtained after acidification and basification were tested with Dragendorff’s and Mayer’s reagent. The chloroform part obtained after basification gave a positive result in both assays. In the other chloroform fraction no alkaloids could be proved. Saponins were separated from the aqueous fraction. The aqueous extract (250 ml) was successively extracted with ether and nBuOH. The saponin mixture was extracted from the syrupy n-BuOH fraction by MeOH. A large volume of ether was added to the methanolic part. The brown precipitate of the crude saponins was filtered and purified twice by redissolving in MeOH and precipitating with ether. The purification gave a light brown powdery mixture of crude saponins (40 mg). The aqueous non-saponin fraction is under investigation. The MeOH extract (Me), non-alkaloid fraction (Nal), alkaloid fraction and isolated saponin fraction were used for subsequent experiments.
2.2. Animals
2.4. In vitro EBV-EA activation assay
Pathogen-free female ICR mice (6 weeks old) were purchased from Nippon SLC Co. Ltd. (Shizuoka, Japan). They were kept in groups of five animals per polycarbonated cage in a temperature-controlled room at 24 ^ 28C. Animals were fed a MP solid diet (Oriental yeast Ltd., Chiba, Japan) and water ad libitum.
The inhibition of EBV-EA activation was assayed using Raji cells (EBV genome-carrying human lymphoblastoid cells; EBV non-producer type; the Raji cells were obtained from Professor G. Klein, Department of Microbiology and Tumor Biology Center, Karolinska Institute, Sweden) cultivated in 10% FBS RPMI-1640 medium (Nakalai Tesque, Kyoto, Japan). The indicator cells (1 £ 10 6 Raji cells/ml) were incubated in 1 ml of medium containing 4 mM n-butyric acid as an inducer, 32 pmol of TPA (20 ng/ml DMSO) and test samples (1, 10, 100 mg/ml TPA) at 378C in a CO2 incubator. After 48 h the cell suspensions were centrifuged at 1000 rev./min and the supernatant was removed. The pellet was resuspended in 0.1 ml of phosphate buffer solution and the cell suspension was smeared. The activated
2.3. Preparation of extracts and fractions The leaves of A. aspera were collected at Calcutta, India in December 1999. Authentication of plant material was carried out by Dr Sitesh Das at the Ayurvedic Research Institute, Calcutta, where the herbarium voucher has been kept. Air-dried (30 ^ 28C) powdered leaves (250 g) were extracted in a soxhlet apparatus with MeOH (250 g,
A. Chakraborty et al. / Cancer Letters 177 (2002) 1–5
cells were stained with high titer EBV-EA-positive sera from nasopharyngeal carcinoma patients and the conventional indirect immunofluorescence technique was employed for detection. In each assay at least 500 cells were counted. The experiments were repeated three times. The average extent of EA induction was determined and compared with the positive control experiments in which the cells were treated with n-butyric acid plus TPA. The EA induction was found to be more than 30%. 2.5. In vivo two-stage carcinogenesis test on mouse skin papillomas promoted by TPA The mice were divided into two groups. Each group was composed of 15 mice housed in bunches of five per cage. Hair was clipped in the dorsal region with surgical clippers with proper care before the first day of initiation. The mice were initiated with DMBA (100 mg/0.1 ml acetone) [12]. One week after initiation, papilloma formation was promoted twice a week by application of TPA (1 mg/0.1 ml acetone). The positive controls were given 1 mg TPA/0.1 ml acetone alone topically to the shaved dorsal skin. The other group of mice was treated with the test samples (methanolic extract or non-alkaloid fraction; 50 mg/ 0.1 ml acetone each) 1 h before each TPA treatment. Animals were examined weekly for the development of tumors on the skin for 20 weeks. The tumor incidence was statistically analyzed by Student’s t-test in treated mice and controls. 3. Results and discussion The total methanolic extract of A. aspera and the
3
alkaloid, non-alkaloid and saponin fractions were tested for their antitumor activity using a short-term in vitro assay of TPA-induced EBV-EA activation in Raji cells. These inhibitory effects and the viabilities of Raji cells are shown in Table 1. All tested substances revealed inhibitory effects on EBV-EA activation by TPA. The total methanolic extract and alkaloid fraction exhibited strong inhibitory activities (93.3 and 93.2% at 100 mg/ml and 51.8 and 52.7% at 10 mg/ml, respectively). The extract and alkaloid fractions preserved high viabilities (70% at 100 mg/ml). The non-alkaloid fraction showed the most significant inhibitory effect on EBV-EA activation among all fractions tested (96.9% inhibition at 100 mg/ml) and more than 60% inhibition at 10 mg/ ml with weak cytotoxicity (60% viability at 100 mg/ ml). This inhibitory activity is higher than retinoic acid (27.7% at 100 mg/ml and 0% at 10 mg/ml, respectively). The saponin fraction showed moderate inhibitory activity (89.5% at 100 mg/ml and 48% at 10 mg/ ml) with high viabilities (70% at 100 mg/ml). The results of this in vitro experiment suggest the nonalkaloid fraction to be a valuable antitumor promotor. On the basis of these in vitro results the non-alkaloid fraction and the total methanolic extract were selected for the in vivo two-stage carcinogenesis assay on mouse skin papillomas using DMBA as an initiator. The tumor initiating property as well as the tumor-promoting potential of the samples were evaluated. Fig. 1A indicates the time course of tumor formation. The first tumors were observed in the group treated with DMBA 1 TPA at 6 weeks. The tumor initiating activity of the Nal group was higher (7 weeks) in comparison to the animals treated with
Table 1 Relative ratio of A. aspera extract and fractions on EBV-EA activation with respect to positive control a TPA (20 ng/ml)
MeOH extract Non-alkaloid fraction Alkaloid fraction Saponin fraction Retinoic acid a
Sample concentration (mg/ml TPA) (% to control (% viability)) 100
10
1
6.7 ^ 2.9 (70) 3.1 ^ 2.5 (60) 6.8 ^ 2.6 (70) 10.5 ^ 2.3 (70) 72.3 ^ 4.2 (60)
48.2 ^ 3.8 39.7 ^ 2.7 52.7 ^ 3.1 52.0 ^ 2.8 100.0 ^ 0
94.5 ^ 0.8 91.7 ^ 1.7 94.0 ^ 0.6 100.0 ^ 0 100.0 ^ 0
At a concentration of 100 mg ratio/TPA, all samples were statistically different from control (TPA 20 ng/ml, 100%) and showed a significant inhibitory effect against EBV-EA activation.
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A. Chakraborty et al. / Cancer Letters 177 (2002) 1–5
Fig. 1. Inhibitory effects of methanol extract (Me) and non-alkaloid fraction (Nal) on mouse skin carcinogenesis induced by DMBA-TPA. (A) Percentage of mice with papillomas. (B) Average number of papillomas per mouse. X, DMBA (100 mg) 1 TPA (1 mg) alone; K, DMBA (100 mg) 1 TPA (1 mg) 1 Nal (50 mg); A, DMBA (100 mg) 1 TPA (1 mg) 1 Me (50 mg). Results were statistically different from the control group (*P . 0:01, **P . 0:05).
Me (8 weeks). After 10 weeks the percentage of tumor-bearing mice treated with DMBA 1 TPA was 100% whereas 42% animals of the Nal group and 14% of the Me group showed the appearance of tumors. During the total experimental period of 20 weeks 100% (DMBA 1 TPA), 100% (Nal) and 64% (Me) of mice had developed tumors (Fig. 1A). The effect of the Nal treatment is comparable to that of retinoic acid (45% after 10 weeks, 95.5% after 20 weeks; concentration 50 mg), which is a known tumor inhibitor. Fig. 1B shows the average number of tumors per mouse. After 20 weeks of promotion it was observed that 9.5 ^ 2.0 tumors/mouse (8.3 ^ 1.8 tumors/mouse after 15 weeks) were found in the control group (DMBA 1 TPA). At the same evaluation period only 2.3 ^ 0.3 tumors/mouse (1.8 ^ 0.5 tumors/ mouse after 15 weeks) in the Me group and 5.6 ^ 1.3 tumors/mouse (4.2 ^ 1.4 tumors/mouse after 15 weeks) in the Nal group were formed. Treatment with Me caused a 76% reduction in the average number of tumors/mouse after 20 weeks of promotion. After treatment with Nal a reduction of 41% was observed. Retinoic acid in comparison caused 4.4 ^ 1.4 tumors/mouse causing a 53.6% reduction of tumors after 20 weeks of promotion.
The results of the in vitro primary screening EBVEA activation test indicate that the non-alkaloid fraction containing mainly non-polar compounds (ecdysterone, dihydroxyketone) showed the most significant inhibitory activity (96.9% at 100 mg/ml) among all tested fractions. The effect is stronger than retinoic acid, a well known chemopreventor as well as an anticarcinogenic agent [13] working on the suppression of the epigenetic tumor promotion [14]. The topical exposure on mouse skin led to a strong anticarcinogenic effect of the methanolic extract at the dose level tested. The anticarcinogenic effect was proved firstly by a delayed tumor formation and secondly by a smaller number of papillomas per mouse in comparison to the other test samples. As a mechanism of action the direct free radical scavenging activity of the plant extract [9] or the inhibition of ornithine decarboxylase activity by oleanolic acid [15] can be suggested. The total methanolic extract of A. aspera leaves contains ecdysterone and dihydroxyketone as nonpolar compounds and alkaloids, oleanolic acid, saponins, flavonoids (total content of hydrolyzed glycosides and free aglycons; mean 3.02 ^ 0.15%; N ¼ 3) [16] and tannins (total content, mean 0.44 ^ 0.03%; N ¼ 3) [17].
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The present study suggests that A. aspera leaf extract and the non-alkaloid fraction are valuable antitumor promotors in carcinogenesis. Further investigations are in progress to clarify the influence of the individual compounds on the cancer chemopreventive activity of A. aspera leaves and to specify their possible mechanism of action. Acknowledgements This study was supported partly by Grants-in-Aid from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan. References [1] R.D. Girach, A.S.A. Khan, Ethnomedicinal uses of Achyranthes aspera leaves in Orissa (India), Int. J. Pharmacogn. 30 (1992) 113–115. [2] W. Tang, G. Eisenbrand, Achyranthes bidentata BI, in: Chinese Drugs of Plant Origin, Springer-Verlag, Berlin, 1992, pp. 13–17. [3] K.H. Bhom R. Liersch, Achyranthes, in: R. Haensel, K. Keller, H. Rimpler, G. Schneider (Eds.), Hagers Handbuch der Pharmazeutischen Praxis, V, Springer-Verlag, Berlin, 1992, pp. 54–59. [4] T.N. Misra, R.S. Singh, H.S. Pandey, C. Prasad, An aliphatic dihydroxyketone from Achyranthes aspera, Phytochemistry 30 (1991) 2076–2078. [5] S.K. Borthakur, N. Goswami, Herbal remedies from Dimoria of kamrup district of Assam in northeastern India, Fitoterapia 66 (1995) 333–340.
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