Electronic Journal of Pharmacology and Therapy Vol. 4, 9-13 (2011) ISSN: 0973- 9890 (Available online at www.tcrjournals.

com)

Original Article

Indexed in: ProQuest database Abstract, USA (ProQuest Science Journals, Health and Medical complete), EBSCO databases (USA), Indian Science Abstract, Medical and Aromatic Plant Abstract, New Delhi

PILOT OBSERVATION ON POSSIBLE AMELIORATIVE EFFECTS OF AQUEOUS EXTRACTS FROM BARK OF ALSTONIA SCHOLARIS ON BLEOMYCIN INDUCED CHROMOSOMAL DAMAGE IN IN VITRO CULTURED HUMAN LYMPHOCYTES
CHAKRABORTY, S.1, MISTRY, D.2 AND PITHAWALA, M.2
1

Department of Zoology, S. S. R. College of Arts Commerce and Science, Sayli Silvassa, 396 230; 2 C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Gopal Vidyanagar, Bardoli Mahua Road, Tarsadi, 394350. E. mail: Received: May 22, 2011; Accepted: June 18, 2011 Abstract: In present study possible ameliorative efficacy of aqueous extract of bark of Alstonia scholaris (ASE) in cultured human lymphocytes against Bleomycin-induced cytogenetic alterations has been reported. Cultures were treated either alone with Alstonia scholaris aqueous bark extract (50g/ml) or in combination with radiomimetic drug Bleomycin (15g/ml). Positive implications about radioprotective effects by Alstonia scholaris bark extract on Bleomycin induced cytogenetic alteration in the form of chromosomal aberrations in in vitro human lymphocyte culture was recorded. Key words: Alstonia scholaris, Bleomycin, Human cytogenetics

INTRODUCTION Alstonia scholaris Linn, (Family: Apocynaceae) is properly known as the Saptaparni or Devils tree in India. It is a medium to large sized tree, of about 40 m height with a somewhat tessellated corky grey to grey-white bark. The outer blaze is cream to yellowish in color with abundant, milky latex that flows rapidly when cut. The bark is about 1/2 inch thick. The Alstonia species are rich in alkaloids, steroids and triterpenoids and these substances may cause toxicity to animals [1]. Bleomycin sulfate is a mixture of glycopeptide [2] antibiotic containing approximately primarily Bleomycin A2 (70%) and B2 (30%). It is isolated from Streptomyces verticillus. The drug binds to 9

DNA, inhibits DNA synthesis, and causes single strand scission of DNA in vivo and in vitro at specific base sequences [2]. Bleomycin has been shown to cause cell cycle arrest in G2 and in mitosis.When irradiation occurs during the G0/ G 1 phase of the cell cycle, large-scale rear rangements appear as exchange-type chromosome aberrations at the next mitosis. In vitro treatment of bleomycin also produces such chromosomal aberrations hence is often referred to as well as used as radiomimetic drug. With respect to radiation damage to human, it is important to protect humans from adverse effects induced by ionizing radiation. The use of radioprotectors represents an obvious strategy to improve the therapeutic index in radiotherapy. Although synthetic radioprotectors such as the

E. J. Pharmacol. Therapy aminothiols have yielded the highest protective factors, typically they are more toxic [3] than naturally occurring protectors [4]. In general, the best radioprotective agents are reported to result in the highest behavioral toxicity in mice [5,6]. Hence, the search for alternative sources, including bioactive principles of plant origin, has been an ongoing task worldwide. The radioprotective efficacy of a hydro-alcoholic extracted material from the bark of Alstonia scholaris (ASE) in mice against radiationinduced hematological and biochemical alterations has been recently reported [7]. Swiss albino mice were administered with ASE (100 mg/kg body weight/d for 5 consecutive day) orally prior to whole-body gamma irradiation (7.5 Gy). Pretreatment with ASE caused a significant increase in glutathione levels in serum as well as in liver in comparison to irradiated animals [7]. Their study showed that ASE protects against radiation-induced biochemical alterations in Swiss albino mice. In another study, Jahan and Goyal [8] demonstrated that Alstonia scholaris bark extract (ASE) pretreatments in mice provides protection against radiation induced cytogenetic damage in the form of chromosomal aberrations and micronuclei induction in bone marrow of mice. The aim of the present study was to know whether ASE could also show such radioprotective activity in humans. Human lymphocytes can easily be cultured in laboratory and forms an excellent in vitro culture system for early and rapid detection of activities of various compounds. Any change induced in chromosome compliments after addition of compounds reflects the genotoxic potentials of those compounds. We, therefore, analyzed the effects of aqueous Alstonia scholaris bark extracts on bleomycin induced chromosomal aberrations from in vitro cultured human lymphocytes. MATERIALS AND METHODS Plant material: Authentic samples of Alstonia scholaris Linn. R.Br. (Apocynaceae) stem bark were locally collected. 10 Treatment protocols: Four separate culture vials were set up from each of the twelve blood samples collected. First culture vial was kept untreated so as to act as control. After 24 hours Extraction (Aqueous) method: Standard protocol [7] with some modification was followed. Approximately 50gm of Alstonia scholaris bark was collected and dried for 2-3 days at room temper ature. After dr ying completely, it was ground crumbly in a mixture and the extract was prepared by refluxing with double distilled water (DDW) in Soxhlet extractor for 36 hrs at 40C. The liquid extract was allowed to cool and concentrated by evaporating its liquid contents in vacuo and freeze dried. The extracts were stored at low temperature until further use. Extracts were dissolved in sterile pyrogen free DDW prior to use. Lymphocyte culture: About 5ml of RPMI 1640 (Hikaryo, ready mix) culture media was taken. To this was added 50l Heparin and 0.6ml of blood. The incubation temperature was 37C for duration of 72 hrs. At 69th hr, 100l of colchicine was added. After 2 hrs, cultures were terminated. A treatment of pre-warmed hypotonic potassium chloride (0.075M) solution at 37C for 25min was given followed by fixative (3:1 methanol: Acetic acid) washes so that clear white lymphocyte pellet was obtained. This was suspended in about half ml of fixative for final preparation of slides. On chilled sterile slides, about 4-5 drops of cell suspension was dropped from convenient height with the help of pasture pipette. Slides were air dried and immediately coded or labeled. Air- dried slides were stained in 2 % Giemsa staining solution prepared in Sorensons buffer. Optimum staining time varied between slides and the stain batches, however, was generally of the order of about 57 minutes. For counting chromosomal aberrations one hundred well spread metaphase plates, each containing not less than 44 chromosomes were considered. Both chromosomes as well as chromatid type of aberrations were considered. For statistical analysis, Students t-test was employed.

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Table 1: Chromosomal aberrations induced in in vitro cultured human lymphocytes after addition of Alstonia scholaris bark extract alone as well as with Bleomycin. (Figures indicate total aberrations per 1200 cells). # Significantly greater than the control at (P< 0.001), * Significantly less than only bleomycin treatment at (P< 0.01), *Significantly greater than the control at (P< 0.05)
Groups Normal Bark Extract Bleomycin Bleomycin +Bark extract 50 mg/ml 15ug/ml 50 ug/ml + 15ug/ml (Control) Chomatid Gaps 12 16 32 22 Chromatid Break 7 6 17 10 Chromosome Gap 3 2 9 3 Chromosome Break 2 1 6 4 Dicentric 1 0 14 5 Acentric Fragment 1 0 2 2 Ring 0 0 1 0 Hypodiploid 10 13 20 11 Hyperdiploid 1 2 2 1 Prematurely separated centromeres 10 12 4 7 Total aberrations - excluding chromatid gaps 35 36 75 # 43* Aberration

Sr No 1 2 3 4 5 6 7 8 9 10 11

of initiation, second culture vial was treated with Alstonia scholaris aqueous bark extract (ASE) at the concentration of 50g/ml. The third culture vial was treated with 15g/ml of Bleomycin (BLM) and fourth vial received combined dose of ASE and Bleomycin at the concentration of 50g/ml+15g/ml. RESULTS The present study was initiated to rule out the possibility of ameliorative effects of Alstonia scholaris aqueous bark extr act against Bleomycin induced damage on in vitro cultured human lymphocyte chromosomes. There was statistically significant reduction (P<0.01) in total number of chromosomal aberrations (Table 1) after addition of Alstonia scholaris aqueous bark extract in the final concentration of 50g/ ml along with bleomycin (15ug/ml) as compared with alone treatment of bleomycin in the same concentration. However, for this combined treatments total chromosomal aberrations still remained significantly higher than control at (P<0.05). Alone addition of Alstonia scholaris aqueous bark extracts did not induce any significant aberrations. Both chromosome type as well as chromatid type of aberrations were statistically high at (P< 0.001) after addition of Bleomycin as compared to control as well as alone addition of A. scholaris bark extracts. 11

DISCUSSION Ionizing radiation produce deleterious effects in the living organisms and the rapid technological advancement has increased human exposure to ionizing radiation enormously. High levels of irradiation can induce mortality in mammals. There is therefore a need to protect mammals against such effects of ionizing radiation. With respect to radiation damage to humans, it is important to protect biological systems from radiation-induced genotoxicity or lethality. Numerous attempts have been made to investigate different means for controlling and protecting from radiation hazards using chemical, physical and biological means. The main radioprotective class is thiol synthetic compounds such as amifostine and aminothiols. Amifostine is a powerful radioprotective agent compared with other agents [9-11], but this drug is limited in use for clinical practice due to side effects and toxicity. It was established during the last two decades that products derived from natural sources could be used as non-toxic radioprotectors. Plants and natural products have a number of advantages: they are not toxic at applied concentrations, are relatively cheap, can be applied orally and many of them are used in traditional medicine. A

E. J. Pharmacol. Therapy number of plants and herbs, applied in vitro [12, 13] and in vivo [14,15] before or after irradiation, possess radioprotective properties, which prolong life [16] improve haematological parameters [17-19], and reduce the negative effects of radiation on chromosome and DNA level [20,21]. The search for less-toxic radiation protectors has spurred interest in the development of natural products. Recently, Jahan and Goyal [8], demonstrated the radioprotective effects of Alstonia scholaris bark extract (ASE) on cytogenetic alterations in the for m of chromosomal aberrations and micronuclei induction in bone marrow of male Swiss albino mice. In a similar study another group of researchers showed that Alstonia scholaris bark extract pretreatments render protection against radiation-induced biochemical alterations in mice [7]. The present study was carried out on in vitro cultured human lymphocytes. The cultures were treated either alone with Alstonia scholaris aqueous bark extract (ASE) as well as in combination with radiomimetic drug Bleomycin. Positive implications about radioprotective effects by Alstonia scholaris aqueous bark extract on Bleomycin induced cytogenetic alteration in the form of chromosomal aberrations in in vitro human lymphocyte culture was recorded. So far in the reported literature there seems plethora of investigations which have proved the potentials of plant extracts as radioprotective. Several plants viz., Gingko biloba [14], Centella asiatica [22], Hippophae rhamnoides [23], Ocimum sanctum [24], Panax ginseng [25], Podophyllum hexandrum [26], Tinospora cordifolia [27], Emblica officinalis [28], Phyllanthus amarus [29], Amaranthus paniculatus [30], Piper longum [31], Syzigium cumini [32], Mentha arvensis [33], Mentha piperita [34], Zingiber officinale [12], Ageratum conyzoides [35], Aegle marmelos [36] and Aphanamixis polystachya [21] show potential radioprotective effects. Radio-protective effect of Lycopersicon esculentum extract against 12 radiation induced chromosomal aberration in Swiss albino mice has been reported very recently [37]. A cascade of events leading to DNA damage like single or double fragments, base changes, DNADNA or DNA-protein adducts are triggered by reactive oxygen species (ROS) in the form of and , as well as peroxide radicals induced by ionizing radiation/s [38]. Following irradiation, double-chain fragments are the primary cause of cellular death. The probable mechanisms allowing certain plants and other natural substances to exhibit radioprotective properties are various. Most of the plants contain polyphenols, which neutralize free radicals, and the increase in cellular antioxidants in irradiated system is likely the primary mechanism of radioprotection [39]. Polyphenols in plants can regulate the mRNA expression of antioxidant enzymes, such as catalase, glutathione transferase, glutathione per-oxidase, and superoxide dismutase, thus countering oxidative stress caused by ionizing radiation. Plants and herbs can suppress the activation of the protein kinase C, mitogen-activated protein kinase, cytochrome P-450, as well as genes which are probably responsible for damage induced by ionizing radiation. In conclusion, the evidence provided in the current study give reason to believe that the aqueous extracts from bark of plant Alstonia scholaris possess a radioprotective potential. However to further justify, additional in vitro and in vivo studies need to be performed so as to confirm these data and to discover the mechanisms of the possible protective effect. REFERENCES

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