M. Srilatha1*, K. Lavanya2, Subha T.1
1Department of Biotechnology, Sona College of Arts and Science, Salem - 636 005, Tamilnadu, India
2Department of Biochemistry, PSG College of Arts & Science, Coimbatore- 641014 Tamil Nadu India
*Address for Corresponding Authors
Department of Biotechnology, Sona College of Arts and Science, Salem - 636 005, Tamilnadu, India
Background: The rising burden of cancer worldwide calls for an alternative treatment solution. Herbal medicine provides a very feasible alternative to western medicine against cancer. Objectives: The present study assessed the in vitro antioxidative potential and cytotoxicity of Tabernaemontana divaricata leaves protein fraction. Materials and Methods: The antioxidant activity of protein fraction of T. divaricata was performed by several antioxidative assays including 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging assay, ABTS, superoxide radical (SO) scavenging assay and Hydrogen peroxide (H2O2) with standard protocol, followed by Cytotoxicity test with tryphan blue assay. Results: From the results, T.divaricata has been found to have the significant antioxidant activity in a dose-dependent manner and IC50 value was 59.1μg/ml for DPPH, 48 µg/ml for ABTS, 60 µg/ml for SO and 38.5 µg/ml for H2O2. Further, the cytotoxicity analysis was determined against Dalton Lymphoma Ascites (DLA) cell line and the IC50 value was found to be 62μg/ml for protein fraction of T.divaricata. Conclusion: Hence, the current study attests that T. divaricata is a fine source of natural antioxidants with anticancer agents and can be used in pharmaceutical preparations for the treatment of diseases induced by oxidative stress.
Keywords: T. divaricata, Antioxidants, Dalton Lymphoma ascites, Trypan blue assay
Medicinal plants are considered as potential source for drug development and many novel products have reached clinical trials. Scientists are investigating properties of medicinal plants in order to develop novel drugs against disease like cancer, from natural products. Medicinal herbs have profound scope and have been used to find potential anticancer compounds in them (Riaz et al., 2016) Oxidative stress can damage cells as well as tissues, which therefore leads to various dreadful degenerative diseases like cancer (Wolf and Dean, 1987). About 127 lakhs of new cancer cases was estimated according to the International Agency for Research on Cancer in 2008. The global burden is may rise to 21.4 million by 2030 (Jacques et al., 2010). Hence, supplementation of herbal antioxidants is necessary to suppress the oxidative stress in a healthier way. Use of man-made antioxidants like butylated hydroxy toluene (BHT) and butylated hydroxy anisole (BHA) are restricted due to their side effects (Madhavi and Salunkhe, 1995). Recently, various antioxidants are acquired from naturally available plants that have the capacity to scavenge free radical or active oxygen (Knekt et al., 1996). Free radicals are a class of highly reactive, chemical species which contains one or more unpaired electrons due to which they are highly unstable and cause damage to other molecules by extracting electrons from them in order to attain stability. Reactive oxygen species (ROS) and free radicals, such as superoxide anion, DPPH and hydroxyl radical, are constantly formed in the human body by normal metabolic action. Their action is opposed by a balanced system of antioxidant defenses, including antioxidant compounds and enzymes. Excess production of these free radicals and reactive oxygen species leads to a number of oxidative degenerative disorders in the body. In order to neutralize the free radical damage, the biological system acts synergistically to deactivate the free radicals before they attack the cell. Recently, much attention has been given to naturally occurring antioxidants, which may play an important role in inhibiting both free radicals and oxidative chain reactions. Ascetic fluid is the direct nutritional source for tumor cells, and therefore, a rapid increase in ascetic fluid with tumor growth would be a means to meet the nutritional requirement of tumor cells (Prasad and Giri, 1994); (Haldar et al., 2010).Therefore, searching for safer and effective antioxidants from natural plants is of great interest among researchers. Based on that phytomolecule from plant sources have been widely reported to possess antioxidant activity (Srivastava et al., 2013). In addition, plant proteins have also been demonstrated to be a good source of antioxidants for instance, active peptides from walnuts (Chen et al., 2012) pulses (Lopez-Girona et al., 2012) and corn gluten meal have been assessed for their antioxidant capacities (Zhuang et al., 2013).
Materials and Methods
Collection and authentication of Plant
The fresh leaves of Tabernaemontana divaricata (Apocynacea) were collected from Coimbatore district, India. Taxonomic authentication was done by Dr.G.V.S.Murthy Taxonomist, TNAU, Coimbatore and Tamilnadu. India and the authentication number BSI/SRC/5/23/2015/Tech/2083.
Preparation of Protein extract of Tabernaemontana divaricata leaf (TdPf)
Protein was extracted by recrystallization of ammonium sulphate. Fresh leaves of Tabernaemontana divaricata leaves 20% were taken and homogenized with PBS buffer pH 7.2 and were centrifuged for 5000 rpm for 10minutes. Pellets were discarded and supernatant were saved. To the supernatant add known volume of ammonium sulphate 10-100% and was centrifuged at 10,000 rpm, 4oC for 10minutes. The supernatant were discarded and the pellet was suspended with Dialysis membrane for salting out. The crude extract was kept at -20oC (Kiba et al., 2003).
The chemicals and solvents used in the study were of highest purity and analytical reagents grade. The Chemicals were purchased from SD Fine Chem., Himedia and Sigma, India.
In vitro Antioxidants assays
DPPH (Diphenyl- 1-Picryl-Hydrazyl) Radical Scavenging Activity of TdPf
The ability of the leaf protein fraction to scavenge the DPPH radical was quantified using spectrophotometric assay. The scavenging ability of leaves extract towards the stable free radicals was determined through DPPH assay by the method of Pathiranan and Shahidi (2005). Ascorbic acid was used as standard. The reactants were vortexed and incubated in dark at room temperature for half an hour. The absorbance was measured at 518 nm in a spectrophotometer (Pathiranan and Shahidi, 2005).. The assay was carried out in triplicates. DPPH radical scavenging activity was calculated as a percentage using the formula:
ABTS radical scavenging activity assay was performed according to (Arnao et al., 2001) with some modifications. The ABTS stock solution was prepared by mixing ABTS solution (7 mM, 3 mL) and ammonium per sulphate (2.45 mM, 15 mL) in distilled water. The mixture was left in the dark at room temperature for 16 h before use. Fresh ABTS working solution was prepared by mixing ABTS stock solution in 0.2 M sodium phosphate buffered saline (pH 7.4) to an absorbance of 0.7 ± 0.02 at 734 nm. Then 50 μL of the sample (0.3-1.5 μM) was added to 5 mL of fresh ABTS working solution. The reaction mixture was kept in the dark for 6 min and absorbance was monitored at 734 nm (Long and Halliwell, 2001). BHA was used as a standard. The control was conducted in the same manner, except that distilled water was used instead of sample. The percentage inhibition was calculated using the formula:
% Inhibition = [(A0 − A)/A0] × 100
Where A0 = absorbance of control; A = absorbance of sample.
The superoxide radical generated from the photo reduction of riboflavin was detected by NBT (Nitro blue tetrazolium) reduction by the method of McCord and Fridovich, 1969).The reaction mixture contained EDTA (0.1 M), 0.0015% NaCN, riboflavin (0.12 mM), NBT (1.5 mM) and protein fraction of T.divaricata at various concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/ml)and phosphate buffer (67 mM, pH 7.8) in a total volume of 3 ml. The tubes were uniformly illuminated for 15 minutes and the optical density was measured at 530 nm before and after the illumination (McCord and Fridovich, 1969). The percent scavenged was calculated by the following equation.
The IC50 value was calculated, which is the effective concentration at which the antioxidant activity is 50%.
Hydrogen Peroxide Assay
The ability of the plant extract to scavenge H2O2 was determined according to the method of (Ruch et al., 1989). A solution of H2O2 (4mM) was prepared in phosphate buffer (pH 7.2). H2O2 concentration was determined spectrophotometrically from its absorption at 230nm was determined after 10minutes against a blank solution containing phosphate buffer without H2O2. The scavenging activity of H2O2 by plant extract and the standard compounds was calculated using the formula
In vitro cytotoxicity analysis
Maintenance of DLA tumor cells Cell line
Dalton’s Lymphoma Ascites (DLA) tumor cells were procured from Amala Cancer Research Centre, Thirussur, Kerala and the cells were propagated by intraperitoneal transplantation of 1x106 cells in 100μl of PBS. After 10-15 days, the cells were drawn from the intraperitoneal cavity and used for the in vitro.
Trypan Blue exclusion assay
The cytotoxic effect of protein fraction was evaluated by using DLA tumor cells by intra peritoneal propagated DLA cells. In vitro cytotoxicity studies were carried out to find out the 50 per cent effective concentration (EC50) of TdPf by trypan blue exclusion method17. The Dalton’s Lymphoma Ascites cells were propagated in the peritoneal cavity of mice were taken and washed with saline thrice by centrifuging at low speed. 0.1 ml of PBS containing 1x106 cells was used for the in vitro assay. Various concentrations (10 to 100 g/0.1ml of PBS) of protein fraction containing 1x106 DLA tumor cells were incubated at 370 C for three hours. At the end of the incubation period 0.1 ml of trypan blue was added and layered the cells on the haemocytometer for counting. The dead cells were blue in color and counted to calculate the percentage of dead cells (per cent cytotoxicity) using the formula:
Antioxidants have the capacity to protect the body from oxidative stress damage. Epidemiological studies indicate that intake of fruits; vegetables as well as indigenous herbal products have the capacity to prevent the free radicals in the human body. In this study, the antioxidant properties and cytotoxic effect of tumor cells of the protein fraction of T. divaricata was assessed.
In vitro Antioxidants assays
The results showed the antioxidant activity of the protein fraction by scavenging the DPPH free radicals when compared to the scavenging the standard ascorbic acid. The IC50 concentration of Ascorbic acid (49μg/ml) was found to be less than that of TdPf (59μg/ml). It means that the protein extract of plant at higher concentration captured more DPPH radicals resulting into decrease in absorbance and increase in IC50 value are shown in the Figure 1.
The ABTS is a stable blue colour radical reduced by antioxidants to green coloured ABTS radical. ABTS has the ability to donate hydrogen atoms to free radicals slowing the process of lipid peroxidation (Desire et al., 2016). The IC50 concentration of Ascorbic acid (35μg/ml) was found to be less than that of TdPf (48μg/ml). The scavenging activities of Phyto protein fraction of Td and the standard ascorbic acid are shown in the Figure 2.
Protein fractions showed free radical scavenging effect on Superoxide in a concentration dependent manner. The results clearly indicated that proteins isolated from T. divaricata leaves have higher superoxide scavenging potential activity with IC50 value 60 µg/ml. It was also observed that Low molecular weight proteins exhibited stronger superoxide activity as compared with the standard Ascorbic acid as shown in the Figure 3.
Hydrogen Peroxide Assay
The T. divaricata protein fraction was capable of scavenging H2O2 in a concentration dependent manner (Figure 4). Scavenging of H2O2 T. divaricata might be due to the donation of electrons to H2O2 thus neutralizing it to water. The IC50 concentration of Ascorbic acid (42μg/ml) was found to be less than that of TdPf (38.5μg/ml).This dose dependent trend was also observed in the aqueous extract of P. boissieriana and showed moderate nitric oxide and H2O2 scavenging activity (Ebrahimzageh et al., 2009).
Figure 1. Observation of different assay: (a) DPPH, (b) ABTS, (c) SO, (d) H2O2
Effect of T.divaricata Protein fraction on antitumorigenic activity to DLA tumor Cells
In vitro antitumorigenic effect of TdPf was assessed by cytotoxic studies against intraperitoneally propagated DLA tumor cells using trypan blue exclusion method. Figure 5 and 6 shows the antitumorigenic effect and dose dependent in vitro cytotoxic effect of TdPf to DLA tumor cells.
Figure 2. Antitumorigenic effect of T. divaricata to DLA tumor cells: (a) DLA tumor cells without T.divaricata Pf, (b) DLA tumor cells with T.divaricata Pf. A-Live cell B-Dead cell
Fifty percent effective concentration (EC50) was found to be 62 μg/ml of T.divaricata Protein fraction. This cytotoxic effect showed the antiproliferative role of T.divaricata against DLA tumor cells.
Figure 3. In vitro cytotoxic effect of T. divaricata protein fraction to DLA tumor cells in trypan blue assay
Free radicals, especially ROS can react with substances in the body resulting in cellular damage and human disorders. According to this, the elimination of ROS and free radicals is considering as one of the most important defense mechanisms of a living body against different disorders. Antioxidant abilities of the peptides are thought to be due to their amino acid composition and hydrophobicity (Sarmadi and Ismail, 2010). Some amino acids, such as Tyr, Lys, Arg, Gly, Leu and His had been reported to exhibit antioxidant ability (Xie et al., 2008). Antioxidant compounds can inhibit oxidative reactions using both free radicals scavenging and interrupting the radical chain reaction of lipid peroxidation (Kim et al., 2007). The DPPH free radical scavenging assay was frequently used to evaluate the antioxidative role of plant extracts. The scavenging of DPPH by the protein fraction was found to be increased sharply with increasing concentration of the protein fraction when compared to standard ascorbic acid reflected its antioxidative potential. The free radical scavenging activity of the protein extract was examined in in vitro using DPPH radical from the leaves of Leucas linifolia (Ramakrishna et al., 2012). The formation of the ABTS radical cation takes place almost instantaneously after adding ammonium per sulphate to an ABTS solution. The scavenging ability of peroxides against ABTS radicals was concentration dependent. A more appropriate format for the assay is decolourization technique in that the radical is generated directly in stable form prior to reaction with putative antioxidants (Ilhami Gulcin, 2006). Similar to our results cacao protein fractions also showed dose dependent ABTS scavenging effect which was comparable to that of standard antioxidant ascorbic acid (Huang et al., 2005) Bean seed protein (Comfort et al., 2011). ), low molecular weight fraction of chickpea protein hydrolysate (Li et al., 2008), also reported strong superoxide radical scavenging activity. The protein fraction from the leaves of Cynodon dactylon showed a dose dependent radical scavenging activity for hydrogen peroxide which was found to be comparable with standard ascorbic acid (Santhi and Annapoorani, 2009). The results obtained in the present study revealed that TdPf can be effectively scavenged H2O2 and prevented the inhibition of enzymes and oxidation of SH thiol groups. The dose dependent cytotoxic activity of the leaf extract of Hymenodictyon against different cell lines were reported (Kamuhabwa et al., 2010). The antitumorigenic effect of Solanum nigrum against HeLa cell line and Vero cell line (Patel et al., 2009).
Similar observations was showed maximum cytotoxicity to DLA tumor cells by four ayurvedic herbs such as Curcuma longa L., Ocimum sanctum L., Tinospora cordifolia (Wild) and Zizypus mauritiana (Adhvaryu et al, 2010). The potent antitumorigenic effect of aqueous extract of Areca catechu was also reported (Chetan et al., 2010). The EC50 of Dendrobium formosum on DLA cells was found to be 350μg/ml (Prasad and Koch, 2014). Similar results were also reported by Gopika showed significant in vitro cytotoxicity activity against DLA cell lines (Gopika et al., 2015).
The authors are thankful to the Avinashilingam University for providing necessary laboratory facilities to carry out the work successfully.
Conflicts of interest
We declare that we have no conflicts of interests
Not necessary for this work.
Source of funding
This work did not received fund from any funding agency
Adhvaryu MR, Reddy N, Parabia MH 2010. Antitumor activity of four ayurvedic herbs in Dalton Lymphoma Ascites bearing mice and their short-term in vitro cytotoxicity on DLA cell line. African Journal of Traditional Complementary and Alternative Medicine, 5(4): 409-418.
Arnao MB, Cano A, Acosta M. 2001. The hydrophilic and lipophiolic contribution to total antioxidant activity. Food Chemistry, 73:239-44.
Chen N, Yang H, Sun Y, Niu J, Liu S. 2012. Purification and identification of antioxidant peptides from walnut (Juglansregia L.) protein hydrolysates. Peptides, 38:344-349.
Chetan CA, Rajesh MP, Sanjay LD, Jitesh KJ. 2010. In vitro cytotoxicity study of Agave americana, Strychnos nuxvomica and Areca catechu extracts using MCF-7 cell line. Journal of Advanced Pharmaceutical Technology and Research, 1(2): 245-252.
Comfort FA, Joseph BF, Tayo NF, Rotimi EA. 2011 Effect of peptide size on antioxidant properties of African yam bean seed (Sphenostylis stenocarpa) protein hydrolysate fractions. International Journal of Molecular Science, 12: 6685-6702.
Desire AA, Ascension NM, Florentine NFC, Steve VO, Christine FM, Francois-Xavier E 2016. Antibacterial and antioxidant activities of ethanolic leaves extracts of Dissotis multiflora Triana (Melastomataceae).International Journal of Pharmaceutical Sciences and Drug Research, 8(1):50-56.
Ebrahimzadeh MA, Nabavi SM, Nabavi SF, Eslami B. 2009 Antioxidant activity of aqueous extract of Pyrus boissieriana fruit. Pharmacology online, 1: 1318-1323.
Gopika G, Sujesh M, Babu TD. 2015. Evaluation of Cytotoxic and Antitumor activity of Phyllanthu acidus (L.) Skeels leaf extracts, International Journal of Novel Research in Life Sciences, 2(2): 19-26.
Haldar PK, Kar B, Bala A, Bhattacharya S, Mazumder UK. 2010. Antitumor activity of Sansevieria roxburghiana rhizome against Ehrlich ascites carcinoma in mice. Pharmaceutical Biology, 48:1337-43.
Huang D, Ou B, Prior RL. 2005. The chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53(6):1841-1856.
Ilhami Gulcin. 2006. Antioxidant activity of caffeic acid. Toxicology, 217(2): 213-220.
Jacques F, Hai-Rim S, Freddie B, David F, Colin M, Donald MP. 2010. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. International Journal of Cancer, 127(12):2893-2917.
Kamuhabwa A, Nshimo C, Witte PD. 2010. Cytotoxicity of some medicinal plant extracts used in Tanzanian traditional medicine. Journal of Ethno Pharmacology, 70:143-149.
Kiba A, Saitoh H, Nishihara M, Omiya K, Yamamura S. 2003 C-Terminal domain of a hevein-like protein from Wasabia japonica has potent antimicrobial activity. Plant and Cell Physiology, 44:296-303.
Kim SY, Je JY, Kim SK. 2007. Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion. Journal of Nutritional Biochemistry, 18: 31-38.
Knekt P, Jarvinen R, Reunanen A, Maatela J. 1996. Flavonoid intake and coronary mortality in Finland: A cohort study. British Medical Journal, 312(7029):478-81.
Li Y, Jiang B, Zhang T, Mu W, Liu J. 2008. Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH). Food Chemistry, 106: 444-450.
Long LH, Halliwell B. 2001 .Antioxidant and prooxidant abilities of foods and beverages. Methods in Enzymology, 335:181-190.
Lopez-Girona A, Mendy D, Ito T, Miller K, Gandhi AK, Kang J, Karasawa S, Carmel G, Jackson P, Abbasian M, Mahmoudi A, Cathers B, Rychak E, Gaidarova S, Chen R, Schafer PH, Handa H, Daniel TO, Evans JF, Chopra R. 2012. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalide. Leukemia, 26: 2326-2335.
Madhavi DL, Salunkhe DK. Toxicological aspects of food antioxidants. 1995. In: Madavi DL, Deshpande SS, Salunkhe DK, (Eds.). Food Antioxidants. Dekker, New York, 267.
McCord JM, Fridovich I. 1969 Superoxide dismutase an enzymic function for erythrocuprein (hemocuprein). Journal of Biological Chemistry, 244:6049-6055.
Patel S, Gheewala N, Suthar A, Shah A. 2009 In vitro cytotoxicity activity of
Solanum Nigrum extract against Hela cell line and Vero cell line. International Journal of
Pharmaceutical and Pharmacy Science, 1: 38-46.
Pathiranan CM, Shahidi F. 2005. Antioxidant activity of commercial soft and hard wheat (Triticum aestivum L) as affected by gastric pH conditions. Journal of Agricultural and Food Chemistry, 53:2433-2440.
Prasad R, Koch B. 2014. Antitumor activity of ethanolic extract of Dendrobium formosum in cell lymphoma: an in vitro and in vivo study. Biomed Research International, 751-53.
Prasad SB, Giri A. 1994. Antitumor effect of cisplatin against murine ascites Dalton's lymphoma. Indian Journal of Experimental Biology,; 32:155-62.
Ramakrishna H, Murthy SS, Divya R, Manatharani DR, Panduranga Murthy G. 2012.Hydroxyl radical and DPPH scavenging activity of crude protein extract of
Leucas linifolia: A folk medicinal plant. Asian Journal of Plant Science and Research,
Riaz M, Zia-Ul-Haq M, Saad B. 2016. Anthocyanins and Human Health: Biomolecular and Therapeutic Aspects. Springer Int Publishing, pp 125-138.
Ruch RJ, Cheng SJ, Klaunig JE. 1989. Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea, Carcinogen, 10:1003-1008.
Salomi MJ, Panikkar KR. 1989. Cytotoxic action of Nigella sativa seeds. Amala research bulletin, 9:17-21.
Santhi R, Annapoorani S. 2009 Antioxidative role of Terminalia catappa leaf protein against ELA induced mice. International Journal of Drug Development and Research, 1(1):81-84.
Sarmadi BH, Ismail A. 2010. Antioxidative peptides from food proteins- A review, Peptides, 31(10): 1949-1956.
Srivastava P, Singh VK, Singh BD, Srivastava G, Misra BB, Tripathi V. 2013. Screening and Identification of salicin compound from Desmodium gangeticum and its in vivo anticancer activity and docking studies with cyclooxygenase (COX) proteins from Mus musculus. Proteomics and Bioinformatics, 6(5): 109-124.
Wolf SP, Dean RT. 1987. Glucose auto-oxidation and protein modification. The potential role of autoxidative glycosylation in diabetes. Biochemical Journal, 245(1):243-50.
Xie ZJ, Huang JR, Xu XM, Jin ZY. 2008. Anti-oxidant activity of peptides isolated from alfalfa leaf protein hydrolysate. Food Chemistry, 111: 370-376.
Zhuang H, Tang N, Yuan Y. 2013. Purification and identification of antioxidant peptides from corn gluten meal. Journal of Functional Foods, 5: 1810-1821.