Pharmacognosy Journal |Year 2017 | Volume 9 | Issue 4 |Page 499-503
Mukti Ram Paudel1*, Mukesh B Chand1, Basant Pant2, Bijaya Pant1
1Central Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, NEPAL.
2Annapurna Research Center, Maitighar, Kathmandu, NEPAL.
Mukti Ram Paudel
Central Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, NEPAL.
Submission Date: 21-11-2016;
Review completed: 05-01-2017;
Accepted Date: 02-02-2017.
DOI : 10.5530/pj.2017.4.81
Article Available online
© 2016 Phcog.Net. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Context: Dendrobium longicornu is a traditional medicinal plant widely used in Asia. It has many bioactive compounds like bibenzyl, phenanthrenes, phenolic compounds. There has been little research in the cytotoxic and antioxidant effects of D. longicornu. Aims: The aim of this study was to investigate the cytotoxic and antioxidant activities of this plant. Settings and Design: Antioxidant and cytotoxic activity of Dendrobium longicornu extracts. Methods and Material: The plant extracts were prepared by soxhlet’s extractor in organic solvents, acetone and ethanol. The total polyphenol content (TPC) in the extracts was determined spectrophotometrically by the Folin-Ciocalteu method and the total flavonoid content (TFC) by aluminium chloride method. The antioxidant activity was determined using DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The cytotoxic activity was evaluated against human brain tumor cells (U251) and cervical cancer cells (HeLa) using MTT assay. Statistical analysis used: Regression analysis was done for calculation of IC50. Duncan multiple range test and Dunnett test were done to compare the data. Results: The Dendrobium longicornu acetonic extract (DLA) showed significantly highest TPC and TFC than Dendrobium longicornu ethanolic extract (DLE). The antioxidant activity was also significantly higher in DLA followed by DLE. Highest cytotoxicity (i.e., lowest IC50 value) was found for the DLA on U251 cells and DLE on HeLa cells. Conclusions: This result concluded that D. longicornu is a potential source of antioxidant and cytotoxic agents.
Key words: Dendrobium longicornu, DPPH, flavonoid, MTT, Polyphenol.
Dendrobium longicornu is an important medicinal orchid used to treat fever and coughs.1 Variety of its chemical constituents such as bibenzyls, phenanthrenes, lignin glycoside and phenolic compounds have been reported.2,3 Besides these, biological activities of this plant have not been reported yet. Reactive oxygen species and reactive nitrogen species, including singlet oxygen, hydrogen peroxide, superoxide anion, hydroxyl radical and nitrous oxide are often generated in the living organisms as byproducts of biological reaction, are known to play a vital role in oxidative cellular damaged. Oxidative stress resulting from these free-radicals plays an important role in manifesting various disorders, including ageing, diabetes, cancer and cardiovascular diseases.4 Recently, the natural antioxidant study has become popular by DPPH radical scavenging assay because of its simplicity and high sensitivity. This assay is based on the theory that a hydrogen donor is an antioxidant. The antioxidant effect is proportional to the disappearance of DPPH radical in test samples.5 Biologically active compounds, include the phenolic compounds, phenanthrenes, bibenzyls, alkaloids, are widely used in medicine.6,7 The phytochemicals screened for biological activity, bioassay that appears capable of bioactivity present in the crude extracts is the MTT assay. This method provides a front-line screen that can be backed up more specific bioassay. It appears that MTT assay is predictive of cytotoxicity of any phytochemicals.8
Dendrobium longicornu produce natural antioxidants, they are potent source of useful new compounds with biological activities. The primary objectives of the present study are to estimate the total polyphenol and flavonoid contents in the crude extracts of this plant, and to evaluate the antioxidant and cytotoxic activities of the crude extracts.
SUBJECTS AND METHODS
The stems of Dendrobium longicornu were collected from Chitlang, Makawanpur district, Central Nepal. The collected stems were air-dried in shade at room temperature (20°C), grinded to fine powder and stored in tight-seal dark container until used.
Preparation of plant extracts
Plant extracts were prepared using soxhlet extractor. Grinded stem powder was extracted with two solvents, acetone and ethanol in the ratio of 1:10 (w/v).9 Solvent evaporated under room temperature to obtain dry extract. Each extract was dissolved in 99% ethanol (analytical grade) to make a concentration of 1 mg/ml stock solution and stored at 4°C.
Estimation of total polyphenol content
Total polyphenol content (TPC) in the plant extracts was determined using Folin-Ciocalteu’s reagent method.10 Stock solution of each extract (1 mg/ml) was used to estimate total phenolic content. The reaction mixture was prepared by mixing 0.5 ml of stock solution of extract, 2.5 ml of 10% Folin-Ciocalteu’s reagent and 2.5 ml 7.5% NaHCO3. Blank was concomitantly prepared, containing 0.5 ml ethanol, 2.5 ml 10% Folin-Ciocalteu’s reagent and 2.5 ml of 7.5% of NaHCO3. The reaction mixtures were thereafter incubated in room temperature for 45 min. The absorbance was measured using Genesys UV-visible spectrophotometer at 765 nm. The estimation of polyphenol content was carried out in triplicate. The same procedure was repeated for the standard solution of gallic acid. The total polyphenol content of extract was expressed as micrograms of gallic acid equivalent (GAE) per milligrams of dry extract.
Estimation of total flavonoid content
Total flavonoid content (TFC) in the plant extracts was determined using Aluminium chloride method.10 Stock solution of each extract (1 mg/ ml) was used to estimate total flavonoid content. The reaction mixture was prepared by mixing 1 ml extract and 1 ml of 2% AlCl3 solution (dissolved in ethanol). The reaction mixtures were incubated for an hour at room temperature. The absorbance was measured using spectrophotometer at 415 nm. This experiment was carried out in triplicate. The same procedure was repeated for the standard solution of quercetin hydrate. The total flavonoid content of extract was expressed as micrograms of quercetin equivalent (QE) per milligrams of dry extract.
Antioxidant activity assay
In vitro antioxidant activity was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay according to the method described earlier.11 Stock solution of each extract at concentration of 1 mg/ml diluted to prepare the series concentrations (50, 100, 200, 400 and 800 µg/ml) for antioxidant assay. An aliquot of 1.5 ml of 0.25 mM DPPH solution in ethanol and 1.5 ml of extract at various concentrations were mixed. The mixture was shaken vigorously and allowed to reach a steady state at room temperature for 30 min. Decolourization of DPPH was determined by measuring the absorbance at 517 nm using Genesys UV-visible spectrophotometer. Antioxidant assay was carried out in triplicate for series of concentrations. Antioxidant activity of extracts was expressed as 50% inhibitory concentration of extract (IC50 µg/ml of extract). IC50 of extracts was calculated using polynomial regression equation where abscissa represents the series concentrations of extracts and ordinate the triplicate percentage antioxidant activity.
Cytotoxic activity assay
Cytotoxic activity of crude plant extracts of Dendrobium longicornu was determined by using MTT assay.8,12 Human brain tumor cells (U251) and cervical cancer cells (HeLa) were cultured in RPMI 1640 medium and incubated under 5% CO2 at 37°C for 48 hours to reach 80% confluence. The cells were harvested by gentle scraping with a cell scraper and resuspended in the medium. From the suspension, 5x103 cells in 100 µl medium was dispensed into each well of 96-well microtiter cell culture plate and incubated under the same conditions of cell culture for 48 hours to allow adherence and growth of cells. The supernatants were gently aspirated, and 100 µl of D. longicornu extracts were added over a range of four cytotoxic concentrations (100 - 800 µg/ml) prepared in medium and incubated for 24 hours. Ten µl of 5 mg/ml MTT was added to every well and the plate was reincubated for further 4 hours. The formazan crystals formed were dissolved in 100 µl DMSO. The plate was then read on a microplate reader (iMarkTM, Bio-Rad) at 595 nm. The number of dead cells per well was calculated as a percentage of the control so measuring cell death after drug exposure. A dose-response curve was plotted for each drug to calculate the inhibition of cell growth by 50%. IC50 of extracts was calculated from the regression equation where abscissa represents the series of concentrations of extracts and ordinate the triplicate percentage inhibition of cell growth.
Data are expressed as mean of three analyses. Equations for standard gallic acid and quercetin were obtained from linear regression model for calculation of total phenolic and flavonoid contents respectively. IC50 of extracts for antioxidant and cytotoxic activities was calculated by using the appropriate linear/non-linear regression equation with F-statistic at p ≤ 0.05. All the analysis was done using R.13
Total polyphenol and total flavonoid contents
Total polyphenol content (TPC) and total flavonoid content (TFC) of Dendrobium longicornu were shown in Figure 1. Linear regression equation of gallic acid (y = 0.0154 x − 0.3285) in the range 25 - 100 µg/ml with R2 value of 0.989 was constructed and linear equation of quercetin hydrate (y = 0.0242 x − 0.1845) was constructed with R2 value of 0.976 for estimation of TPC and TFC respectively. According to result, the highest TPC and TFC were found in Dendrobium longicornu acetonic extract
(DLA) showed significant differences with Dendrobium longicornu ethanolic extract (DLE) at p ≤ 0.05.
The antioxidant activity of the extracts has strong relationship with the solvent employed, mainly due to different antioxidant potential of crude extracts with different polarities. Mean percentage DPPH radical scavenging activity of extracts at their different concentrations was shown in Figure 4. Dendrobium longicornu acetonic extract (DLA) showed higher DPPH radical scavenging activity. IC50 for each extract was calculated using the regression equation from percentage radical scavenging activity. Acetonic extract (DLA) had lowest IC50 than ethanolic extract (DLE) means DLA showed highest antioxidant activity (Figure 2). All these presented values were found to be significant different with that of standard ascorbic acid (AA) at p ≤ 0.05.
The cytotoxic effect of the Dendrobium longicornu extracts against the human cancer cells evaluated using the MTT assay (Figure 5 and 6). The ethanolic extract (DLE) showed the highest percentage inhibitory effect on growth of HeLa cells (Figure 4a) and acetonic extract (DLA) showed on U251 cells (Figure 4b) at the concentrations ranging from 100 - 800 µg/ml. Concentration of extract required for fifty percent cell growth inhibition (IC50) was calculated from the percentage cell growth inhibition at different concentration using appropriate regression equation by F-statistics at p ≤ 0.05. Lowest IC50 showed by DLA on U251 cells is 620.56 µg/ml (y = 18.060 ln(x) − 66.137; F = 28.797; p = 0.002) and by DLE on HeLa cells is 294.70 µg/ml (y = 40.257 ln(x) − 178.899; F = 80.935; p ≤ 0.001). However, acetonic extract showed relatively weak inhibitory effects on the HeLa cells and ethanolic extract on U251 cells.
The quantitative analysis of acetonic and ethanolic extracts of Dendrobium longicornu for TPC and TFC, revealed the high content of these components, approve and complement other previous works.14–17 These phytoconstituents play a significant role in the medicinal properties. The polyphenol and flavonoid compounds are mainly found in acetonic extract could be attributable to the observed high antioxidant properties.18-20 This is manifested by strong free radical scavenging activity; acetonic extract (DLA) superior with lowest IC50 as similar with the standard ascorbic acid (AA).21–23
Figure 1: Total polyphenol and flavonoid contents of crude extracts.
Figure 2: Percentage free radical scavenging activity of crude extracts.
Figure 3: IC50 of crude extracts.
Figure 4 (a): Percentage inhibition of growth of human cervical cancer cells (HeLa) by crude extracts.
Figure 4 (b) Percentage inhibition of growth of human brain tumor cells (U251) by crude extracts.
Figure 5 (a): Living human cervical cancer cells (HeLa) before addition of crude ethanol extract.
Figure 5 (b): Dead human cervical cancer cells (HeLa) after addition of crude ethanol extract.
Figure 6 (a): Living human brain tumor cells (U251) before addition of crude acetone extract.
Figure 6 (b): Dead human brain tumor cells (U251) after addition of crude acetone extract.
The role of antioxidants is their interaction depends on the oxidative free radicals. The summary of DPPH method is that antioxidants react with the stable free radical, 2,2-diphenyl-1-picrylhydrazyl (deep violet colour) and convert it to 2,2-diphenyl-1-picrylhydrazine with discolouration. The discolouration indicates the scavenging potential of the extract with high polyphenol and flavonoid contents.5,24
The result of cytotoxicity of the acetonic extract of D. longicornu showed significant cell growth inhibition against U251 cell lines, whereas ethanolic had no significant cytotoxic activity against the U251 cells. The cytotoxicity of acetonic extract may be due to the presence of high content of flavonoid compounds. Similarly, cytotoxic activity of the ethanolic extract of D. longicornu showed signicant result against the HeLa cell lines due to presence of high amount of flavonoid compounds. The flavonoids have reported for their cytotoxic activity by earlier researchers.25-27
The extracts of Dendrobium longicornu have been shown to exert antiproliferative effects on different human cancer cell lines. The ethanolic extract showed cytotoxic actitvity on HeLa cells whereas acetonic extract on U251 cells. The conventional cancer treatments like chemotherapy and radiotherapy are costly and also cause adverse effects. Hence, it is necessary to identify another anticancer drugs, which are more potent, selective and less toxic than conventional treatment. This research on plant based compounds has been efficacious in the field of anticancer drugs discovery.
Authors kindly acknowledge to International Foundation for Science (IFS), Sweden and University Grant Commission (UGC), Nepal for financial support. We also thankful to Department of Neurosurgery of Kagoshima University, Japan for providing U251 cell lines and Everest Biotech Pvt. Ltd., Nepal for providing HeLa cell lines.
CONFLICT OF INTEREST
AA: Ascorbic acid; DLA: Dendrobium longicornu acetone extract; DLE: Dendrobium longicornu ethanol extract; DPPH: 2,2-diphenyl-1-picrylhydrazyl; GAE: Gallic acid equivalent; HeLa: Human cervical cancer cells; IC50: 50% inhibitory concentration; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; QE: Quercetin equivalent; TFC: Total flavonoid content; TPC: Total polyphenol content; U251: Human brain tumor cells.
|1.||Manandhar NP. A survey of medicinal plants of Jajarkot district, Nepal. J Ethnopharmacol. 1995;48(1):1-6. doi:10.1016/0378-8741(95)01269-J. https://doi. org/10.1016/0378-8741(95)01269-J.|
|2.||Hu JM, Chen JJ, Yu H, Zhao YX, Zhou J. Five new compounds from Dendrobium longicornu. Planta Med. 2008;74(05):1-5. doi:10.1055/s-2008-1074492. https://doi.org/10.1055/s-2008-1074492.|
|3.||Hu JM, Fan W, Zhou L, Zhao Y, Zhou J. A new phenolic compound from Dendrobium longicornu. Bull Korean Chem Soc. 2010;31(10):3025-6. doi:10.5012/bkcs.2010.31.10.3025. https://doi.org/10.5012/bkcs.2010.31.10.3025.|
|4.||Salman KA, Ashraf S. Reactive Oxygen Species: A link between chronic inflammation and cancer. Asia-Pacific J Mol Biol Biotechnol. 2013;21(2):41-9.|
|5.||Mensor LL, Menezes FS, Leitao GG, et al. Screening of Brazilian plant extracts for antioxidant activity by the use of DPPH free radical method. Phyther Res. 2001;15(2):127-30. doi:10.1002/ptr.687. https://doi.org/10.1002/ptr.687.|
|6.||Xu J, Han Q Bin, Li SL, et al. Chemistry, bioactivity and quality control of Dendrobium, a commonly used tonic herb in traditional Chinese medicine. Phytochem Rev. 2013;12(2):341-67. doi:10.1007/s11101-013-9310-8. https://doi. org/10.1007/s11101-013-9310-8.|
|7.||Ng TB, Liu J, Wong JH, et al. Review of research on Dendrobium, a prized folk medicine. Appl Microbiol Biotechnol. 2012;93(5):1795-803. doi:10.1007/s00253-011-3829-7. https://doi.org/10.1007/s00253-011-3829-7.|
|8.||Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63. doi:10.1016/0022-1759(83)90303-4. https://doi.org/10.1016/0022-1759(83)90303-4.|
|9.||Mukherjee S, Pathak D, Parikh J, Jagtap S, Shaikh S, Tupe R. Antiglycation and antioxidant activity of a rare medicinal orchid Dendrobium aqueum Lindl. Med Chem Drug Discov. 2012;2(2):17-29.|
|10.||Stankovic MS. Total phenolic content, flavonoid concentration and antioxidant activity of Marrubium peregrinum L extracts. Kragujev J Sci. 2011;33(2011):63-72.|
|11.||Zhao GR, Xiang ZJ, Ye TX, Yuan YJ, Guo ZX. Antioxidant activities of Salvia miltiorrhiza and Panax notoginseng. Food Chem. 2006;99(4):767-74. doi:10.1016/j.foodchem.2005.09.002. https://doi.org/10.1016/j.foodchem.2005.09.002.|
|12.||Sargent JM, Taylor CG. Appraisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukaemia. Br J Cancer. 1989;60(2):206-10. https://doi. org/10.1038/bjc.1989.252|
|13.||R core team R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria; 2014.http://www.r-project.org/.|
|14.||Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complement Altern Med. 2012;12(1):221. https://doi.org/10.1186/1472-6882-12-221.|
|15.||Cai Y, Luo Q, Sun M, Corke H. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci. 2004;74(17):2157-84. https://doi.org/10.1016/j.lfs.2003.09.047.|
|16.||Alhakmani F, Kumar S, Khan SA. Estimation of total phenolic content, in-vitro antioxidant and anti-inflammatory activity of flowers of Moringa oleifera. Asian Pac J Trop Biomed. 2013;3(8):623-7. https://doi.org/10.1016/S2221-1691(13)60126-4.|
|17.||Eddebbagh M, Massoudi M, Abourriche A, et al. Correlation of the Cytotoxic and Antioxidant Activities of Moroccan Pomegranate (Punica Granatum) with Phenolic and Flavonoid Contents. J Pharm Pharmacol. 2016;4(9):511-9. https:// doi.org/10.17265/2328-2150/2016.09.006.|
|18.||Katalinic V, Milos M, Kulisic T, Jukic M. Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols. Food Chem. 2006;94(4):550-7. https://doi.org/10.1016/j.foodchem.2004.12.004.|
|19.||Prior RL, Wu X, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem. 2005;53(10):4290-302. https://doi.org/10.1021/jf0502698.|
|20.||Yang D, Liu LY, Cheng ZQ, et al. Five new phenolic compounds from Dendrobium aphyllum. Fitoterapia. 2015;100:11-8. https://doi.org/10.1016/j.fitote.2014.11.004.|
|21.||Moretti M, Cossignani L, Messina F, et al. Antigenotoxic effect, composition and antioxidant activity of Dendrobium speciosum. Food Chem. 2013;140(4):660-5. https://doi.org/10.1016/j.foodchem.2012.10.022.|
|22.||Moukette BM, Pieme CA, Njimou JR, Biapa CPN, Marco B, Ngogang JY. In vitro antioxidant properties, free radicals scavenging activities of extracts and polyphenol composition of a non-timber forest product used as spice: Monodora myristica. Biol Res. 2015;48(1):15. https://doi.org/10.1186/s40659-015-0003-1.|
|23.||Peteros NP, Uy MM. Antioxidant and cytotoxic activities and phytochemical screening of four Philippine medicinal plants. J Med Plants Res. 2010;4(5):407-14.|
|24.||Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci Technol. 1995;28:25-30. https://doi. org/10.1016/S0023-6438(95)80008-5 25.|
|25.||Patel RM, Patel SK. Cytotoxic activity of methanolic extract of Artocarpus heterophyllus against A549, HeLa and MCF-7 cell lines. J Appl Pharm Sci. 2011;1(7):167-171.|
|26.||Awah FM, Uzoegwu PN, Ifeonu P, et al. Free radical scavenging activity, phenolic contents and cytotoxicity of selected Nigerian medicinal plants. Food Chem. 2012;131:1279-1286. doi:10.1016/j.foodchem.2011.09.118.|
|27.||Jeune MAL, Kumi-Diaka J, Brown J. Anticancer activities of pomegranate extracts and genistein in human breast cancer cells. J Med Food. 2005;8(4):469-475. doi:10.1089/jmf.2005.8.469.|
Dendrobium longicornu is widely used traditional medicinal orchids, had many valuable secondary metabolites. The biological activities of this orchid had not been explored. However, the present study explores the biological activities such as antioxidant, cytotoxicity of the crude extracts of this orchid. This study proved baseline for the pharmacological uses of this orchid.
Mukti Ram Paudel: He is PhD scholar of the Tribhuvan University at Central Department of Botany, Kathmandu, Nepal. He has been doing the research on bioactivity of secondary metabolites isolated from the different Dendrobium species. He has published more than half dozen of papers in national and international peer-review academic journals and has presented his papers in national and international conferences.
Mukesh Babu Chand: He is free-lancer researcher of plant science. He did research on antioxidant activities of some wild orchids of Nepal. He has published his research in national and international peer-review journals and has presented his papers in national conferences.
Basant Pant: He is Professor of neurosurgery and Chairman of Annapurna Research Center. He has published various papers in brain tumors in reputed peer-reviewed academic journals. Notably has worked on angiotensin-II induced hypertensive chemotherapy.
Bijaya Pant: She is Professor of Tribhuvan University at Central Department of Botany, Kathmandu, Nepal. She has been doing research on tissue culture and bioactivity of some medicinal orchids of Nepal. She has published more than hundred papers on tissue culture and bioactivity of orchids in national and international peer-review academic journals and has presented her papers in national and international conferences.