Global Journal of Pharmacology, 3 (2): 99-106, 2009 ISSN 1992-0075 © IDOSI Publications, 2009

Antitumor and Phytotoxic Activities of Leaf Methanol Extract of Oldenlandia diffusa (Willd.) Roxb
M. Soriful Islam, Most. Mauluda Akhtar, M. Mostafizur Rahman, M. Atikur Rahman, Kanak Kanti Sarker and M. Firoz Alam
Biotechnology and Microbiology laboratory, Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh Abstract: Oldenlandia diffusa (Willd.) Roxb., a well known medicinal plant in Bangladesh has been identified for antitumor properties through Agrobacterium tumefaciens infection using potato disc bioassay and phytotoxic effects on radish root and seed. Significant tumor inhibition was observed at 100ppm and 1000ppm of leaf methanol extract. Maximum tumor inhibition 40.98, 41.93 and 41.89% were observed at 1000ppm for the accessions of the Agrobacterium tumefaciens AtTa0112, AtAc0114 and AtSl0105, respectively. Phytotoxicity assay shows significant root length inhibition by the extract at the concentrations of 1000ppm and 10000ppm. Similarly, seed germinations were also significantly inhibited at the concentrations 1000ppm and 7500ppm extracts. Overall results supported that Oldenlandia diffusa might be a potential source of antitumor agent that could be used for further drug development for tumor treatment in human. Key words: Oldenlandia diffusa % Potato disc bioassay % Antitumor INTRODUCTION Cancer is one of the most life-threatening diseases and serious public health problems in both developed and developing countries. It is a group of diseases characterized by the disregulate proliferation of abnormal cells that invade and disrupt surrounding tissues [1]. Due to the toxic and adverse side effects of synthetic drugs as well as conventional treatments are being failed to fulfill their objectives (tumor control), for these consequence herbal medicine has made a comeback to improve the fulfillment of our present and future health needs [2]. The development of efficient anticancer agents, such as vinblastine and vincristine isolated from the Catharanthus roseus (L.) G. Don, provided convincing evidence that plants could be a source of novel cancer chemotherapeutic agents [3]. Oldenlandia diffusa (Rubiaceae) is well known medicinal plant for its tremendous use all over the world including Bangladesh. It has long been use in China for the treatment of hepatitis, tonsillitis, sore throat, appendicitis, urethral infection and malignant tumors of the liver, lung and stomach [4]. Isolated chemical constituents i.e. Oleanolic acid and ursolic acid from this plant species are effective as antibiosis, anticancer, liver protection and transaminase degrading agents [5-7]. Pharmacological studies have demonstrated that O. diffusa has antitumor, immunomodulatory [8-10], anti-inflammatory, hepatoprotective [11], anti-oxidative [12, 13] and neuroprotective [14] activities. The aqueous extract of this plant is effective in inhibiting the growth of eight different cancer cell lines and inducing apoptosis reported by Gupta, et al. [15]. Different bioassays offer vast advantages for screening of medicinal plant extracts for different purposes i.e. antitumor, antibacterial, antioxidant, phytotoxic properties. Potato disc bioassay is one of them that are developed based on Agrobacterium tumefaciens infection on potato disc is useful for checking antitumor properties of plant extract. The rationale for the use of the bioassay is that the tumorogenic mechanism initiated in plant tissues by Agrobacterium tumefaciens is in many ways similar to that of animals [16]. According to Kempf, et al. [17], Bartonella henselae, a tumor causing bacteria in human shares a similar pathogenicity strategy with plant pathogens A. tumefaciens. Several scientists have used this method over the past 15 years and they appear to be adaptable to the purpose of standardization or quality control of bioactive compounds in such heterogeneous botanicals [18]. According to leading

Corresponding Author: Dr. M. Firoz Alam, Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh

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where the tumors cells lack starch [28] and each experiment was carried out in triplicate. 3 and 5 days of interval. Sterilized Whatman No. 20µl of bacterial suspension (1. Bangladesh. Osaka-Japan). Following design was used for preparing inoculums: 600µl test extract+150µl Double Distilled Water (DDW)+750µl A. Bangladesh using standard procedure [19-22]} were used during antitumor and antibacterial assay. 1 filter paper kept on Petri dish and 5 ml extracts (1000ppm and 10000ppm) were added separately. Usually. After inoculation. University of Rajshahi. [23].0×109cfu) was used for preparing seeded LB agar plates and negative controls were prepared using solvent only. AtAc0114 and AtSl0105 {isolated from crown gall sample and identified in the Biotechnology and Microbiology Laboratory. radish seed phytotoxicity assay was performed [26]. USA). Solanaceae) were cut into 5mm×8mm in size from the center of potato tissue by sterilize cork borer.10 (Russel. the present study was under taken to assess specifically leaf methanol extracts for antitumor and phytotoxic properties. Six to seven loops of bacterial suspensions (1. diffusa was evaluated for antitumor properties using potato disc bioassay described by Turker and Camper [26] and Hussain. Pharmacol. Camptothecin (30ppm) was used as positive control replacing test extract.0×109 cfu) were transferred into sterilize phosphate buffer saline (PBS) and this was used during antitumor assay as inoculums. 5 ml DDW was added and then 20 radish seeds were placed on Petri dishes followed by tightly sealed and incubation at 23±2°C. Antitumor Potato Disc Assay: Methanol extract of O. Filter paper was dried at room temperature for reducing extra solvent. Extract Preparation: Collected plant materials directly crushed into small pieces and sun drying until reducing water content followed to pulverize using electric blender (Nokia. Department of Botany.. =20% inhibition of tumor is considered as a significant value for plant extracts [29]. et al. MATERIALS AND METHODS Plant Material: Fresh leaves of Oldenlandia diffusa were collected from Rajshahi University campus. 1 filter paper. A. 3 (2): 99-106. et al. Each assay was carried out in three times. Bacterial Culture Preparation: Agrobacterium strains were cultured on Luria-Bertani (LB) agar medium which was prepared using the following compositions: 10g of yeast extract+10g of bacto peptone+5g of NaCl+20g of agar dissolved in 1 liter of water according to Devi. Percentage of tumor inhibition was calculated using standard formula described by Hussain. antibacterial assay of leaf methanol extract of O. Tumors were observed on potato discs after 21 days under stereo microscope followed by staining with Lugol’s iodine (10% KI and 5% I2) after 30 minutes. the antibacterial activity of O. Filtration was done through Teton cloth and Whatman No. Radish Seed Phytotoxicity Assay: To evaluate phytotoxic properties of leaf methanol extract of O. Agrobacterium Strains: Three A. Freed. Cefotaxime (30µg/ml) followed by air dried and placed on seeded LB agar plates and incubated at 37°C for 24 hours. et al. China). Michigan State University. This plant species was taxonomically identified by Dr.. diffusa was performed to check viability of Agrobacterium strains using agar 100 disc diffusion assay [24. Root length was measured after 1. Thermostatic water bath. Petri dishes were sealed by parafilm and incubated at 27-30°C for 3 weeks. Fifty gram powder was dipped into 250ml methanol into a conical flask with rubber corks and left for two days with occasional shaking. Mahbubur Rahman (Taxonomist). Single colony was transferred into LB broth and incubated at 30°C for 48 h. Department of Botany. After incubation. Sterilized potatoes (Solanum tuberosum L. Rajshahi University.M. Assistant Professor. Antibacterial Assay: Before antitumor study. 2009 background of antitumor potential of O. tumefaciens in PBS. [27]. Only DDW containing Petri dish was used as control. Two types of determination were done for this purpose:For Root Length Determination: Whatman No. 7500ppm and 10000ppm). 1 filter paper discs (6mm in diameter) were impregnated with 10µl of tested extract (250mg/ml) as well as antibiotics (Kanamycin (30µg/ml). 25]. 100ppm and 1000ppm) and relevant study (antibacterial assay250mg/ml and Phytotoxicity assay-1000ppm. [27]. D. respectively. diffusa was determined by measuring zone of inhibition against all the studied Agrobacterium strains. Filtrates were collected in a beaker and dried up to semisolid using Water bath (4 holes analogue. diffusa. Semi solid extracts were then dissolved into solvent and prepared particular concentration for antitumor (10ppm. diffusa. . tumefaciens strains namely AtTa0112. Each potato disc was overlaid with 50µl of appropriate inoculums.H.Global J. Data were statistically analyzed using MSTAT software version 2.

Plant-based compounds have been playing an important role in the development of several clinically useful anticancer agents i..28 and 13. vinblas-tine. Whereas. Antibacterial Assay: This portion of this study was done to justify whether plant extract is lethal for gene transformation system of Agrobacterium tumefaciens or has no effect because genetic transformation system is required for finally induction tumor.92 74. diffusa inhibit tumor growth in highly significant way in a concentration dependent manner across the strains (Table 1 and Table 2).16 0. of tumor 101 % tumor inhibition .27 Mean Square 9.71 1. 2009 For Seed Germination Determination: This part of the determination is similar to that of earlier determination except for the extract concentrations and number of seeds. 3 (2): 99-106. Other studies [31. diffusa on potato disc at different concentrations (10ppm. Pharmacol. Mean no. 1: Figure shows percentage of tumor inhibition by the leaf methanol extract of O.e.66.81 14. Maximum 40.71 35. including taxol. C=1000ppm. AtAc0114 and AtSl0105 Agrobacterium strains. Here two different concentrations (1000ppm and 7500ppm) and 100 radish seeds were used. diffusa and tumor induction by A. based on the previous antitumor [810].0000 Mean no. B=100ppm.01 9. inhibition zone was recorded for only studied antibiotic kanamycin and Cefotaxime (positive control). negative control did not show any visible zone of inhibition (Figure not shown) as expected. A=10ppm. but not at 10ppm. Germinated seeds were counted after every day up to 5 days. tumefaciens strains on potato discs Source of variation Strains (S) Concentration (C) S×C Error Total Degree of Freedom 2 3 6 24 35 Sum of squares 19. Significant tumor inhibition was observed at 100ppm and 1000ppm concentrations. diffusa has been studied using different bioassay for this purpose.98.93 and 41. RESULTS AND DISCUSSION Drug discovery is the key attempt of our age to overcome many life-threatening diseases like cancer. Results of this assay confirmed extract has no effect on the viability of Agrobacterium strains because no zone of inhibition zone was recorded against all the studied A. vincristine.62 43. 14. 41. Specifically leaf methanol extract of O.43% tumor inhibition were recorded for AtTa0112.57 0. topotecan and irinotecan and etoposide derived from epipodophyllotoxin [30]. the camptothecin derivatives. 32] also represented that Agrobacterium is susceptible to kanamycin and Cefotaxime. Oldenlandia diffusa is the valuable medicinal plant for its multipurpose uses. as well as anticancer [15] reports.40 Prob 0.0000 0. tumefaciens strains. 100ppm and 1000ppm) Table 1: Statistical analysis of tumor inhibition by the leaf methanol extract of O. of % tumor tumor inhibition 10 9 8 7 6 5 4 3 2 1 0 A B C D A B C D A B C D AtTa011 AtAc01 AtSl010 45 40 35 30 25 20 15 10 5 0 Fig.Global J. Antitumor Potato Disc Assay: Results showed that leaf methanol extract of O. Each experiment was carried out in three times.22 0.89% and minimum 13.41 F Value 23. The present study has been undertaken to identify this plant as a source of phytotoxic and antitumor agent.

1). of Gall Means followed by different letter (S) down the column are significantly different among the concentration as well as days at p<0. It was also observed that A.52 B 4. 3 (2): 99-106. 2009 Plate 1: Photographs show gradual tumor inhibition by the leaf methanol extract of O.Global J. Camptothecin served as a positive control and 100% tumor inhibition was observed. Data values are means of three replicates respectively (Fig.91 B 0. tumefaciens AtSl0105 strains was more prominent for producing tumor (8. tumefaciens strains. diffusa on potato discs in a concentration as well as strain dependent manner. 102 . diffusa Variables Strains AtSl0105 AtAc0114 AtTa0112 LSD value Concentration Negative control 10ppm 100ppm 1000ppm LSD value 7.25 AB 5. Data were compared with control.80 AB 4. [27] have also shown that tumor inhibition rate on potato discs are dependent on concentration of plant extract and also tumor producing A.23 C 1. In the present investigation. A3 B3 and C3 as 1000ppm plant extract and A4 B4 and C4 as 30ppm Camptothecin (positive control) Table 2: Analysis of mean data of antitumor activity of leaf methanol extract of O. Similar phenomenon was observed by Turker and Camper [26]. 1 and Plate 1).084 6. This result may be attributed due to its DNA damaging activities. et al.1 ± 0. Camptothecin is a cytotoxic quinoline alkaloid which inhibits the DNA enzyme topoisomerase I (topo I) isolated from the bark and stem of Camptotheca acuminata (Camptotheca.2 ± 0. Pharmacol.32) than other strains AtAc0114 (7. A1 B1 and C1 as negative control and A2 B2 and C2 as 10ppm.72 A 5.05.4±0. Happy tree) [33]. Hussain.9390 Mean no.20) and AtTa0112 (6..25) suggests their differential sensitivity (Fig.25 A 6.

67 C 49.973 % seed germination Fig.000ppm Table 3: Analysis of mean data of root length inhibition by leaf methanol extract of O. It is well documented that alcohols (ethanol. diffusa Variables Concentration Control 1..711 C 1. These findings may be attributed to the nature of biological active compounds and their strong solubility with appropriate solvent. diffusa. inhibition by leaf methanol extract of O. 2009 Control Average root length (mm) 40 30 20 10 0 Day 1 Day 3 Day 5 1.131 B 21.000ppm Means followed by different letter (S) down the column are significantly different among the concentration as well as days at p<0.000ppm 10. for instance. the major secondary metabolites (terpenoids.30 A 8.33 B 54.05. Table 4: Analysis of mean data of seed germination. 38]. of Means followed by different letter (S) down the column are significantly different among the concentration as well as days at p<0. Different studies have shown many secondary metabolites as a source of bioactive compounds with allelochemical potential have great chemical diversity and are involved in many metabolic and ecological processes [34-36].000ppm 10. seed germinations were also significantly inhibited by the both concentrations 1000ppm and 7500ppm (Fig.Global J. inhibitors of enzymes and antitumor.00 A 30. The mentioned structure diversity is reflected in a variety of biological activities as. In the present investigation. Data values are means of three replicates.547 17.000ppm 7. Above mention discussions have provided knowledge that O.547 67. methanol) used as a solvent for plant extract 103 . Data compared with control. diffusa.000ppm 10. 3 (2): 99-106. immunosuppressive and antiparasitic agents [37. 2: Histogram shows regular root length inhibition by the leaf methanol extract at two different concentrations (1000ppm and 10000ppm) of O.500ppm LSD value Days Day 1 Day 2 Day 3 Day 4 Day 5 LSD value 14. phenolics and alkaloids) are of potential Mean no. 3: Graph shows phytotoxicity assay on radish seed germination percentage at two different concentrations (1000ppm and 7500ppm) of leaf methanol extract of O.07 C 1. 3 and Table 4).05. 2 and Table 3).722 B 5. Data values are means of three replicates medicinal interest. Radish Seed Phytotoxicity Assay: Statistical analysis shows that root length were significantly inhibited by the extracts at both the concentrations 1000ppm and 10000ppm (Fig.00 E 26.00 B 15. In drug discovery.589 C 8. plant extract was prepared using methanol as a solvent. These observations may be accomplished due to the presence of active biological compounds. In another cases.56 A 2. These results were analyzed compared with control. diffusa Variables Concentration Control 1.22 D 42.000ppm LSD value Days Day 1 Day 3 Day 5 LSD value 2. Control 120 germinated seeds 100 80 60 40 20 0 Day 1 Day 2 Day 3 Days Day 4 Day 5 1.973 Root length (mm) Fig.01 A 2. Pharmacol. Data compared with control. diffusa might be potential source of antitumor and phytotoxic properties.

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