Isolation of Cannabinoids from the plant Cannabis sativa and its potential anticancer activity

Tariq. A. L1* and Reyaz. A.L2
  1. Department of Biotechnology KRS Autonomous College of Arts and Science Tiruchengode-637215
  2. Department of Biotechnology Periyar University Salem-636011
Corresponding Author:Tariq. A. L Email: [email protected]
Received:23 January 2012 Accepted: 04 February 2012
Citation: Tariq. A. L1* and Reyaz. A.L2 “Isolation of Cannabinoids from the plant Cannabis sativa and its potential anticancer activity”, Int. J. Drug Dev. & Res., Jan-March 2012, 4(1): 241-246. doi: doi number
Copyright: © 2010 IJDDR, Tariq. A. L et al. This is an open access paper distributed under the copyright agreement with Serials Publication, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

The plant leaves were identified as Cannabis sativa L. The cannabniods were extracted by aqueous extract found a total yield of 3.8g while as acetone extract 4.8g. The protein content in crude extract of Cannabis sativa L for aqeous extract found 112μg/ml and for acetone extract 160μg/ml. The molecular weight of protein by SDS PGAGE found to be 70KDa. The HPLC intension percentage for aqueous was 11 while for acetone extract it found 25. The actone extract exhibited more anticancer activity against HT29, MCF7 and SF-26 Cells

Introduction

The applications of cannabinoids have been known to exert palliative effects in cancer patients [1]. Cannabinoids are proposed as potential antitumoral agents on the basis of experiments performed both in cultured cells and in animal models of cancer [2]. It is broadly uses as medicine in China, India, Southeast Asia, South Africa, South America for malaria, rheumatic pains, constipation and childbirth [3]. Cannabinoids have anti-inflammatory and antinociceptive properties that involve receptor dependent and independent mechanisms [4]. Cannabis sativa (Bhang) is a major constituent of the Jatiphaladya commonly grown herb of high medicinal importance [5]. The proapoptotic and antineoplastic properties of cannabinoids shows emphasis effects of N-acylethanolamines [6, 7,8]. Cannabinoids present an interesting therapeutic potential such as antiemetics, appetite stimulants in debilitating diseases, analgesics, treatment of multiple sclerosis, spinal cord injuries, Tourette’s syndrome, epilepsy and glaucoma [9]. The dronabinol has an anti-depressive potential that can readily be used in medical practice [10]. A simple and reliable thin layer chromatographic method is used in a micellar solution of sodium dodecyl sulfate as mobile phase for the identification of all the four herbal drugs with preliminary separation on silica gel ‘G’ TLC plate [11, 12].

MATERIALS AND METHOD

Collection and preservation of specimen
The plant leaves of Cannabis sativa were collected from Kolli hills, Tamil Nadu and were taken to the laboratory; subsequently they were preserved in 10% neutralized formalin at low temperature
Extraction of crude toxin:
The aqueous extract of sample was prepared by [2] squeezing the sand free specimens in triple distilled water. The resultant solution was filtered and dialyzed by using sigma dialysis membrane-500 (Av Flat width-24.26 mm, Av. Diameter-14.3mm and capacity approx-1.61ml/cm) against D-glucose to remove the excess water. The supernatant so obtained stored at 4oC and then used as crude aqueous extract.
Acetone extraction
The sample was dried in air for 2 days and after complete drying, crushed in the form of fine powder then 10g sample was added into 200ml of acetone, covered and allowed to stand for 5 hours [13]. The solvent was then removed after squeezing the sample and filtered through Whatman filter paper No1. The solvent was evaporated at low pressure by using a Buchi Rotavapor R-200 at 40C and stored in refrigerator to use as crude acetone extracts.
Purification of Crude Protein
Purification of the crude extract was carried out [13] by using DEAE cellulose anion exchange chromatography equilibrated with 10mM Tris-HCl buffer pH 8.3. The 15ml of crude extract was eluted with 10mM Tris-HCl buffer pH 8.3 at the flow rate of 20ml/h. A linear gradient consisting of 50ml of 10mM Tris-HCl buffer pH 8.3 and 50ml of the same buffer with 0.3M NaCl. The 5ml of fractions elute was collected and absorbance measure at 280nm.
Protein Estimation
Protein estimation of Cannabis sativa L extracts was done as described by [14] by using Bovine serum albumin standard and absorbance was read at 650nm under spectrophotometer.
Characterization of protein:
Sodium Dodecyl Sulphate–PAGE
One dimensions of sodium dodecyl sulphate polyacrylamide gel electrophoresis was carried out following the modified method of [15]. The molecular weight of the protein determined by staining the protein with 10% methanol, 7% acetic acid and 0.2% coomassie brilliant blue for 4 hours and destained with 10% methanol, 25% acetic acid solution for 12 hours.
HPLC Chromatography
The column used here by following [16] “Normal Phase” having Cartridge 8 x 100 mm Bondapak C18 and Solvent contains 0.5% phosphoric acid in 40% aqueous acetone. In HPLC, narrow columns with internal diameters 2-80 mm was used. The solvents was filtered through fine membranes (0.4-0.5 micron) and all solutions were injected into the Water’s instrument comprises of three main components: the injector (located on the top righthand corner of the console), the solvent delivery system (right-hand side) and the UV detector (lefthand side).
Source and Maintenance of Cell Lines
Cells lines representing the most common human cancers (WHO, 2006) were obtained from the National Cancer Institute (NCI). The cell lines include the breast adenocarcinoma (MCF-7), the glioblastoma (SF-268) and the colon adenocarcinoma (HT-29). The three cell lines [17] were cultured in 200 ml flasks (NunclonTM) with the complete culture medium (RPMI-1640 or DMEM). The cells were maintained at 37 ºC in a 5% CO2 humidified incubator. The culture medium (20 ml) was replaced three times weekly and the cells were trypsinised when nearly confluent.
Preparation of cell suspension
All the cells were detached from the culture flasks by addition of 1 ml of 0.25% trypsin-0.1% EDTA [17]. Trypsin was then inactivated by the addition of 10 ml of experimental medium (RPMI-1640 or DMEM). The cells were separated into a single cell suspension by a gentle pipetting action and 1 ml of the cell suspension was returned to the culture flask containing 20 ml of culture medium. The remaining cell suspension was centrifuged at 1000 rpm (Sorvall® T6000D) for 3 minutes and the supernatant discarded. Experimental medium (20 ml) was added to the cells which were then repeatedly pipetted to allow cells to disperse in to the medium. An aliquot of the trypsinised cell suspension was stained with 2 mg/ml trypan blue (1:1 ratio) in order to obtain a more quantitative analysis of the cell suspension. Trypan blue is a water-soluble dye, which is insoluble in the cell membrane lipids. It will thus only cross cell membranes of dead/non-viable cells. On visualization with the light microscope, viable cells appear transparent, while nonviable cells are stained dark blue. The haemocytometer was used to determine the total number of viable and non-viable cells by counting cells in the 25 squares (each square is subdivided into 16 smaller squares of 0.1 mm2) at the top and bottom of the haemocytometer and the average number of cells per unit volume (millimeter, ml) of medium calculated. The cell suspension was adjusted with experimental medium to approximately 1.5 x 105 cells/ml. The experiment was carried out only when there was at least 95% cell viability.
Percentage cell viability was calculated as follows
% Cell viability = Abs test sample–Abs background sample–Mean Abs blank x100 (Eq. 10.1) Mean Abs control– Mean Abs blank Where: Abs: absorbance at 492 nm

Result

Identification of Leaves
The present study deals with to study the anticancer activities of herbal plant Cannabis sativa L. The sample collected from Kolli hills, Tamil Nadu, was identified based on the taxonomical characters (Figure 1). Kingdom-Plantae, Class-Magnoliopsida, Order-Rosales, Family-Cannabaceae, Genus- Cannabis, Species-Sativa L
Extraction of Protein
Acetone extract of plant sample Cannabis sativa L yielded a total amount of 3.2g of crude extract from 500g of sample. Similarly from aqueous extract at a total amount of 4. 8g of crude extract was yielded.
Protein Content
The optical density values of aqueous extract obtained 0.530nm which was corresponds to 0.6mgg/ml. While in acetone extract the optical density found 0.676nm which corresponds to 1.6mg/ml (Table 1).
DEAE Chromatography
The optical density value of cannabinoid of aqueous extract at 280nm was 1.530nm and for acetone extract the optical density of cannabinoid was 1.745nm.
Molecular weight of Protein
Sodium dodecyly sulphate polyacrylamide gel electrphrosesis on 12% gel, the crude protein toxins yielded 3 bands in aqueous extract and 5 bands in acetone extract of Cannabis sativa L with one well defined band of 70 kDa (Figure 2).
HPLC chromatography
Under the high performance liquid chromatography it was analyzed that in the acetone extract of retention time of 3.27min the percentage intension was found to be 11 for aqueous extract and 25 for acetone extract. In the aqueous extract the retention time of 4.75min, the percentage intension was found to be 32 for aqueous and 30 for acetone extract.
Anticancer activity
The aqueous extract exhibited anticancer activity against HT-29, MCF-7 and SF-268 cell lines. It showed potential anticancer activity as the concentration was increased which lead the reduction of Trypan blue that means inhibited the proliferation of cancer cells and the background appeared transparent. In acetone extract which showed more anticancer activity against activity against HT-29, MCF-7 and SF-268 cell lines. It showed potential anticancer activity as the concentration was increased which lead the reduction of Trypan blue that means inhibited the proliferation of cancer cells and the background appeared transparent (Figure 3).

Discussion

It is noteworthy that most of the CB1 receptor antagonists developed to date, including rimonabant, has inverse agonist properties, so their effects do not necessarily reflect reversal of the tonic action of the endocannabinoids [18]. Rimonabant counteracts the anti-tumour effects of anandamide-related compounds and other cannabinoid agonists in thyroid, breast and prostate cancers. Unexpectedly, rimonabant per se showed a potential anti-tumour action in thyroid, mantle-cell lymphoma and breast tumours both in vitro and in vivo [1]. Cannabinoids in cancer therapy as palliative agents the potential application of cannabinoid agonists as anticancer agents is still at the preclinical level. Meanwhile the cannabinoids are emerging as valuable adjunctive agents for optimizing the management of multiple symptoms of cancer and the treatment of therapyrelated side-effects [19,20]. Regarding effectiveness, cannabinoids exert notable antitumour activity in animal models of cancer, but their possible antitumour effect in humans has not been established. Regarding toxicity, cannabinoids not only show a good safety profile but also have palliative effects in patients with cancer, indicating that clinical trials with cannabinoids in cancer therapy are feasible (17, 21]. As cannabinoids are relatively safe compounds, it would be desirable that clinical trials using cannabinoids as a single drug or in combined anticancer therapies could accompany these laboratory studies to allow us to use these compounds in the treatment of cancer [12].
1. Where A1, A2 are blank controls and A3 to A9 are various aqueous concentration of cannabinoids in microtitre wells containing of HT-29 cell. Where B1, B2 are blank controls and B3 to B9 are various aqueous concentration of cannabinoids in microtitre wells containing of HT-29 cell. Where C1, C2 are blank controls and A3 to A9 are various aqueous concentration of cannabinoids in microtitre wells containing of HT-29 cell.
2. Where D1, D2 are blank controls and D3 to D9 are various acetone concentration of cannabinoids in microtitre wells containing of HT-29 cell. Where E1, E2 are blank controls and E3 to E9 are various acetone concentration of cannabinoids in microtitre wells containing of HT-29 cell. Where F1, F2 are blank controls and F3 to F9 are various acetone concentration of cannabinoids in microtitre wells containing of HT-29 cell.

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Table 1
 

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Figure 1 Figure 2 Figure 3
 

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