Received date: 28/07/2014; Revised date: 09/08/2014; Accepted date: 19/08/2014
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The present paper deals with study of In-vitro cytotoxicity effect of isolated gossypol from Bt and Non-Bt cotton seeds on HeLa cell lines. Gossypol is a polyphenolic binaphthyl diadehyde natural yellow colored pigment. It is not only resistance substance for cotton plant’s self –defense system against insect pests and possibly some diseases but also an important phytochemical compound of immense interest due to its several biological properties including anti-cancer,anti-HIV,anti-oxidation, antimicrobial and as male contraceptive. During this study gossypol exhibited broad spectrum of anti-cancer activity against the HeLa cell lines. The cytotoxicity effect of gossypol was detemined by MTT (3-(4-,5-dimethylthiazolyl-2)-2,5-dipheniltetrazolium bromide) assay. Gossypol from Bt and Non-Bt cotton seeds has shown dose dependent cytotoxicity effect against HeLa cell lines. In-vitro screening of the gossypol showed potential cytotoxic activity against HeLa cell lines. Mortality rate of 11.5884% and 22.6058% with 3μg/1μl concentration of isolated gossypol from Bt and Non-Bt cotton seed extracts was observed on HeLa cell lines respectively. Hence the inhibitory concentration at 50% (IC50) was fixed at 3μg/1μl of gossypol for HeLa cells. The standard anti-cancer drug Doxorubicin (1mg/ml) was also used in this study to confirm anti-cancer activity of gossypol isolated from Bt and Non-Bt cotton seed with 1.7828% cell viability. The present study confirms the mild toxic effect of gossypol on HeLa cell lines and can preferably be used as anti-cancerous drug in combination with other natural similar compounds to replace the synthetic chemical drugs for fewer side effects.
Gossypol, HeLa Cells, MTTssay
Plantsre the basic source of knowledge of modern medicine.lmostll the parts of the plant, viz, bark, root, stemnd seedsre known to have various medicinal properties . The trend of using natural products has increasednd thective plant extractsre frequently screened for new drug discoveriesnd for the presence ofntimicrobials.ccording to the world health organization (WHO), Medicinal plants would be the best source to obtain variety of drugs.bout 80% of individual medicines have compounds derived from medicinal plants. Therefore, such plants should be investigated to better understand their properties, safetynd efficiency.The plant selected for this study is Cotton, the most important textile fiber cropnd is the second most important oil crop in world. Cotton belongs to the family Malvaceae, genus Gossypiumnd comprises of 50 different species, of these only four speciesre cultivated in India.
Gossypiumrboreumnd Gossypium harbaceum belongs to the old world diploid group, wheres new world tetraploid cultivated speciesre Gossypium hirsutumnd Gossypium barbdance.
Inndhra Pradesh cotton is cultivated inll the three differentgro climatic regions viz., Coastal region, Telangana regionnd Rayalaseema region. Cotton seed which remainsfter cotton ginned is used to produce cotton seed oil. The plant has been found to possess several ethno medicinal uses. Cotton seedsnd leaves feature in traditional medicine in various formsndre taken internallyndpplied externally to treat skin problemsnd injuries. For headaches, drink is made from powdered cotton seedsnd mixed with milk. Dysentery islso treated withn infusion of seedsnd leaves. Spotsnd other skin conditionsre treated using cotton seed or extracts from the leaves. In Western medicine, cotton is put to use in the form of dressings, bandages, swabsnd cotton wool. Scientific investigations have shown that cotton rootsnd seeds contain certain compounds that may be beneficial to the health, potentially for treating Cancernd HIV. In India, Cervical Cancer is the most common cancer in women, even more common than breast cancer. Every year, in India 132,000 new casesre diagnosednd 74,000 women die due to this cancer .Cervical Cancer is caused by the Human Papilloma virus (HPV).(Source: WHO/ICO Information Centre on Human Papilloma Virus (HPV)nd Cervical Cancer).
Cotton contain gossypol; polyphenolic compound that isn integral part of the cotton plant’s self-defence systemgainst insect pestsnd possibly some diseases . Somemount of gossypol tends to react with many natural substances in cottonseednd forms the bound gossypol that is non-harmful. However the unreacted gossypol knowns “free gossypol” is toxic. Thus free gossypol isnnti-nutritional factorndnti-cancergent that limits the use of cottonseednd its products . Gossypol [2, 2N-Bi (8-formyl-1, 6,7, trihydroxy5-isopropyl-3-methyl naphthalene)] is crystalline compound. The molecular formula of gossypol is C30H30O8.The inclusion compound formation by gossypol has been studiedt different thermodynamic conditions. Most of the investigated molecules form more than one inclusion compound with gossypol. Polymorphism exhibited by gossypol inclusion compounds is dimorphismnd trimorphism.Traditionally, the determination of cell growth is done by counting viable cellsfter staining with vital dye.Trypan blue staining is simple way to evaluate cell membrane integrity (and thusssume cell proliferation or death) but the method is not sensitivend cannot bedapted for high throughput screening. Measuring the uptake of radioactive substances, usually tritium-labeled thymidine, isccurate but it islso time-consumingnd involves handling of radioactive substances. Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, tetrazole) is reduced to purple formazan in the mitochondria of living cells.
Thebsorbance of this colored solution can be quantified by measuringt certain wavelength (usually between 500nd 600 nm) by spectrophotometer. Thebsorption max is dependent on the solvent employed. This reduction takes place only when mitochondrial reductase enzymesrective,nd therefore conversion can be directly related to the number of viable (living) cells. When themount of purple formazan produced by cells treated withngent is compared with themount of formazan produced by untreated control cells, the effectiveness of thegent in causing death of cells can be deduced, through the production of dose-response curve . Solutions of MTT solubilized in tissue culture media or balanced salt solutions, without phenol red,re yellowish in color. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple MTT formazan crystals whichre insoluble inqueous solutions. The crystals can be dissolved incidified isopropanol. The resulting purple solution is spectrophotometrically measured.n increase in cell number results inn increase in themount of MTT formazan formedndn increase inbsorbance. The use of the MTT method does have limitations influenced by: (1) the physiological state of cellsnd (2) variance in mitochondrial dehydrogenasectivity in different cell types. Nevertheless, the MTT method of cell determination is useful in the measurement of cell growth in response to mitogens,ntigenic stimuli, growth factorsnd other cell growth promoting reagents, cytotoxicity studies,nd in the derivation of cell growth curves [1-20].
Isolationnd Detection of Gossypol
For extraction of gossypol three grams of Btnd Non-Bt cotton seed kernels obtained manually were crushednd extracted with diethyl ether (5 x 20 ml) . The solvent was evaporatedt low temperature tilln oily material containing gossypol was obtained. This was stored for further use. Gossypol was extracted withqueouscetone with the same method. The residual leftfter the extraction of free gossypol withqueouscetone was soaked in 2M HCl solution (75 ml) for 10 minnd then refluxed for 30 min.fter cooling, the solution was filtered. The residue was washed withbsolute ethanol (15ml).
Then chloroform was evaporated from the extractt low temperature tilln oily material containing gossypol was obtained. Specific chemical tests were performed for detection of gossypol in samples of seed extracts. For this purpose 5 ml of seed extract crude was dissolved in small volume of ethanol in 25 ml conical flasknd final volume was made up to the mark bydding more ethanol. Two ml of each sample solution was taken in the test tube separatelynd equalmount of solidntimony chloride wasdded in each test tubend mixed thoroughly. Similar types of tests were performed with stannic chloridend leadcetate.
Evaluation of cytotoxicctivity:
Cell viabilitynd cytotoxicityssay were used for drug screeningnd cytotoxicity tests of chemicals. HeLa (Human cervixdeno carcinoma), cell lines were obtained from NCCS Pune. (HeLa cells were provided by Prof.Satyanarayana Singh Coordinator, DBT-ISLARE, CFRD Osmania University, Hyderabad).
MTTssay is the best known method for determining mitochondrial dehydrogenasectivity in the living cells. In this method, MTT is reduced to purple formazan by NADH. However, MTT formazan is insoluble in water,nd it forms purple needle-shaped crystals in the cells. Therefore prior to measuring thebsorbance,n organic solvent is required to solubilize the crystals.dditionally, the cytotoxicity of MTT formazan makes it difficult to remove cell culture media from the plate wells due to floating cells with MTT formazan needles, giving significant well-to-well error (Alley,M.C.,etl 1988). HeLa cells were cultured in RPMI 1640 medium with 10% FBS, 100 units/ml penicillin,nd 100 mg/ml streptomycin. HeLa cell lines were sub culturednd were maintainedt 370Ct 5% CO2 in CO2 incubator. Cultures were continuously monitored every 24 hr. observed undern inverted microscope tossess the degree of confluencynd to confirm thebsence ofny microorganism contaminants. In-vitro study of cytotoxicity effect of Btnd Non-Bt cotton seeds isolated Gossypol wasssessed by MTT (3- (4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)ssay. Cell lines were sub culturednd 250μl of media (containing 10000cells) were transferred into 96 well platesnd incubated for 24 hr. The cells were subcultured with 100μl of fresh medium. Isolated Gossypol wasddedt different concentration (1-3μg/μl)nd then final volume was made to 200μl with the mediand incubated for 4 hr.fter incubation media containing drug was removed. 20μl of MTT reagent (6mg/ml in PBS) wasdded to each well containing mediand incubated for 3 hrt 37 °C underntmosphere of 5% CO2 until purple precipitate was observed. Media was removed without disturbing cellsnd 200μl DMSO (MTT solvent) wasdded to dissolve the purple precipitate.bsorbance was readt 570 nm with reference filter of 630 nm. Percentage cytotoxicity was calculatednd used for finding the IC50 value of the concentration required for 50% cell death by Gossypol, isolated from Btnd Non-Bt cotton seed.
For detection of gossypol in the samples of Btnd Non-Bt cottonseed extracts three specific chemical conformation tests with SbCl3, Pb(CH3COO)2nd SnCl3 were performed. Turbid reddish complexppeared in case of SbCl3fter 15 min. While reddish precipitateppearedfter 10 minutes with Stannic chloride. Test with leadcetate gave yellowish precipitate thatppearedfter 20 min confirming presence of gossypol in the samples. In this study we have employed dose dependentpproach to evaluate the toxicity of the isolated gossypol from both Btnd non-Bt cotton seeds on HeLa cell linest different concentration (1μg/1μl -3μg/1μl).
Results revealed that there is significance cytotoxicity observed with dose dependent concentration of Btnd non-Bt cotton seed isolated gossypol like 1μg/1μl,2μg/1μlnd 3μg/1μl respectively on HeLa cell lines (Table 1 and Fig 1 ). Doxorubicin is used for standardnti cancerous drug. From the result it is confirmed that DMSO has no effect on HeLa cellsnd is useds dissolving solvent for gossypol.t 1μg/1μl of non-Btnd Bt gossypol dose the viabilitynd cytotoxicity percentage of HeLa cell lines is 81.21%, 93.37%, 18.78%nd 3.87% respectively. Similarlyt 2μg/1μl of Non-Btnd Bt gossypol dose the viabilitynd cytotoxicity percentage of HeLa cell lines is 95.57%, 93.37%, 19.6%nd 9.58% respectively.t 3μg/1μl of Non-Btnd Bt gossypol dose the viabilitynd cytotoxicity percentage of HeLa cell lines is 97.2%, 92.5%, 22.6%nd 11.5%.ccording to our previous reports the levels of gossypol is more in non-Bt cotton seeds correspondingly the cytotoxicity was found to be more for non-Bt cotton seed isolated gossypol with 3μg/1μl dose dependent concentration.
Cytoxicityctivity of gossypol on Human Breast cancer cells was reported by Jerzy w.Jarsozewski etll;(1990), Gossypol inhibited growthndpoptosis of Human Headnd neck Squamous cell Carcinoma in vivo was reported by Keith G. wolter etl . Increase in the percentage ofpoptotic cells was observed in treated tumors versus control gossypol inducedpoptosis in ovarian cancer cells through oxidative stress were reported by Romano etl . In comparison tobove three references, in this studylsonticancerousctivity of isolated gossypol from Btnd non Bt showed mild cytotoxicity on HeLa cancer cell lines. The in vitro screening of the Gossypol showed potential cytotoxicctivitygainst the HeLa cancer cell linesnd mortality rate of 11.5884nd 22.6058 % was observed in 3μg/1μl concentration of isolated gossypol from Btnd non Bt respectively. Hence the inhibitory concentrationt 50% (IC50) was fixedt 3μg/1μl (Graphnd table 2) of gossypol for HeLa cells [21-37].
Presentnalysis revealed moremount of gossypol in Non-Bt cotton seeds. Gossypols natural compound showed more percentage of cell viability compared to the standardnti-cancer drug Doxorubicin. Cytotoxicity for viable cells was maximum for non-Bt cotton seed gossypol indicating the effective control of HeLa cells over period of time. The present study confirms the mild toxic effect of gossypol on HeLa cell linesnd can preferably be usedsnti-cancerous drug in combination with other natural similar compounds to replace the synthetic chemical drugs for fewer side effects.
• uthorsre highlycknowledged to UGC Govt. Of India (RFSMS-FELLOWSHIP) for funding this project.
• uthors express their sincere thanks to Y.Rama Reddy, Principal scientist (Plant Breeding), REGIONALGRICULRAL RESEARCH STATION, NANDYAL, Kurnul Dist.518503,ndhra Pradesh, India, for timely providing seed materials.
• uthorsre highlycknowledged to Central Facilities for Researchnd Development (CFRD) Osmania University, Hyderabad, for support of instrumentation to complete this work.
• uthors express their sincere thanks to. Kishorend Jalapathi.Pochampalli (Assistant professor) Department of Chemistry, University College of science, Saifabad, Osmania University Hyderabad,ndhra Pradesh, India-5000004.