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Research Article
 

Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines



Sridevi I. Puranik, Murigendra B. Hiremath, Rajendra B. Nerli and Shridhar C. Ghagane
 
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ABSTRACT

Background and Objectives: Tabernaemontana divaricata belongs to family Apocynaceae is an ornamental, flowering, evergreen shrub that is used in traditional medicine for the treatment of urinary disorders, abdominal tumours and dysentery. The aim of the study was to investigate in vitro antitoxicity and anticancer activities from leaf extracts of Tabernaemontana divaricata. Materials and Methods: The present study involves at screening of preliminary phytochemicals, determination of total phenolic contents, evaluation of methanol, ethanol and aqueous leaf extracts of T. divaricata using in vitro 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) assay, ferric ion reducing antioxidant power (FRAP) assay, Phosphomolybdenum (PM) method and evaluation of in vitro anticancer activity on T-24 human bladder cancer cell line. Results: The preliminary screening of phytochemicals revealed a broad spectrum of secondary metabolites present in all five different solvent extracts. Aqueous extract showed high presence of phenols (62.54±0.18 mg g1 GAE, r2 = 0.9882) among methanol and ethanol extracts in comparison to Gallic acid (80.28±0.14 mg g1 GAE, r2 = 0.9954). Ethanol extracts exhibited potential antioxidant activity at a concentration of 100 mg mL1 (62.41±0.67%). The IC50 value was 188.9±07 µg mL1 for T-24 bladder cancer cell line whiles the standard drug Doxorubicin showed an IC50 value of 0.23 µM mL1. Morphological variations was observed in cancerous cells undergoing cell shrinkage and membrane blebbing in human bladder cancer cell line T-24 while the extract showed no cytotoxicity towards normal cells MEF-L929. Conclusion: Based on these results it can be concluded that the tested extracts holds significant antioxidant and anticancer properties. However further investigation with lead compounds of T. divaricata on in vivo trials will enable its therapeutic use.

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Sridevi I. Puranik, Murigendra B. Hiremath, Rajendra B. Nerli and Shridhar C. Ghagane, 2018. Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines. International Journal of Cancer Research, 14: 100-108.

DOI: 10.3923/ijcr.2018.100.108

URL: https://scialert.net/abstract/?doi=ijcr.2018.100.108
 
Received: June 27, 2018; Accepted: September 25, 2018; Published: November 09, 2018



INTRODUCTION

Bladder cancer (BC), the fourth most common cancer in men and 7th most common in women, the average age at diagnosis is 60 years with male to female ratio of 2.6:1 that represents an important health problem1. Bladder carcinogenesis is thought to develop from the interaction of environmental exposures and genetic susceptibility2. The early treatment for BC is surgical, radiotherapy and chemotherapy, but post-treatment metastasis occurs in 60% patients, 20% patients are progressed to the middle and advanced stage; moreover, the 5 year survival rate is less3 than 50%. There is an urgent need for research on herbal treatment using herbal sources4. The secondary metabolites may be useful adjuncts in the prevention of cancer recurrence, interrupting carcinogenesis via cell-to-cell interactions or anti-oxidation pathways5. The understanding of the role of herbal remedies in the development and course of bladder cancer is expanding rapidly. As experimental design becomes more sophisticated, research will shift its focus from particular synthetic drug to herbal synergistic compounds.

Plants are well known as a major source of modern medicines. From ancient times, humans have utilized plants for the treatment and prevention of diseases, leading to the dawn of traditional medicine6. Tabernaemontana divaricata belongs to the Apocynaceae family, growing evidence suggests that this plant has medicinal benefits and is used to treat various diseases such as diarrhoea, urinary disorders, abdominal tumours, epilepsy, eye infections, inflammation, leprosy, paralysis, rabies, rheumatic pain, skin diseases, ulceration and vomiting7. The leaf extracts could possibly be used as pharmacological interventions in many different ailments because of the antioxidants and potential secondary metabolites present in the leaves. The present study was thus carried out to investigate the preliminary phytochemical screening, determination of total phenolic contents, in vitro antioxidant properties and in vitro anticancer activities of T. divaricata leaf extracts against T-24 human bladder cancer cell line and MEF-L929 normal cell lines.

MATERIALS AND METHODS

Materials
Collection of plant material and authentication: The plant Tabernaemontana divaricata was collected from different regions of Western Ghats, Karnataka, during the year 2017. It was authenticated by taxonomist Dr. Kotresh. The voucher specimen was deposited in Department of Botany Karnatak Science College, Dharwad. The plant material was washed thoroughly, checked for any microbial contamination. The leaves were separated, shade dried and powdered. The powder was then stored in airtight container at -20°C until used.

Procurement of chemicals and cell line
Chemicals: All chemicals used for the experiments were of analytical grade. Gallic acid and ascorbic acid were procured from Sigma Aldrich (Mumbai, India). Folin-Ciocalteu reagent (FCR), Molybdate reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and other chemicals used during the experiments were procured from Hi media Laboratories, Pvt. Ltd. (Mumbai, India) and Merck Specialties Pvt. Ltd. (Mumbai, India). The sub-culturing, Dulbecco Modified Eagle Medium (DMEM) was obtained from Sigma-Aldrich (St. Louis, MO, USA) which was supplemented with 10% fetal bovine serum, (FBS, from Sigma-Aldrich), 1% penicillin streptomycin and 1% nonessential amino acids from (Abbott Healthcare Pvt. Ltd, Mumbai, India).

Cell lines: The T-24 bladder cancer and MEF-L929 normal mouse embryo fibroblast cell lines were procured by the National Centre for Cell Science (NCCS) Pune, India.

Methods
Extraction: The powdered leaf material was subjected for soxhlet extraction by following the method of Jenson8 with slight modification in the temperature gradient. The five solvents namely chloroform, ethyl acetate, methanol, ethanol and aqueous were selected for extraction. The extracts were then vacuum dried in rotary vacuum evaporator under the reduced pressure at 40°C.

Preliminary phytochemical analysis: All the five extracts were qualitatively screened for the presence of phytoconstituents namely alkaloids, flavonoids, glycosides, phenols, lignins, saponins, sterols, tannins, anthraquinone and reducing sugar by following the methods of Ghagane et al.9.

Total phenolic content (TPC): The total phenolic content of each of T. divaricata leaf extracts were carried by Folin-Ciocalteu method with slight modification10. About 1 mg mL1 of extracts were prepared in their respective solvents and were quantified by using spectrophotometer at 760 nm using gallic acid as reference. The results were compared to the standard curves and total phenolic content was expressed as mg g1 gallic acid equivalent (GAE) per gram dry powder for the samples.

In vitro antioxidant assays
2,2-Diphenyl-1-picrylhydrazyl(DPPH) free radical scavenging assay: The free radical scavenging effect of T. divaricata (L.) extract was assessed with the stable scavenger DPPH with slight modifications of the method described by Brand-Williams et al.11. Different concentrations of extracts were prepared in ethanol. Positive control was ascorbic acid. The DPPH solution (0.004%) was prepared in ethanol and 5 mL of this solution was mixed with the same volume of extract and standard solution separately. These solutions were kept in dark for 30 min to read absorbance at 517 nm spectrophotometric by using ethanol as blank. The scavenging activity against DPPH was calculated using the equation:

Image for - Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines

where, A is the absorbance of the control reaction (1 mL of ethanol with 1 mL of DPPH solution) and B is the absorbance of the test sample. The results were analyzed in triplicate. The IC50 value is the concentration of sample required to inhibit 50% of the DPPH free radical.

Ferric ion reducing antioxidant power (FRAP) assay: The FRAP assay was performed by following the method of Oyaizu12 with slight modification. About 0.1% extracts were prepared with different dilutions. About 1 mL of extract was mixed thoroughly with 2.5 mL of 0.2 mM phosphate buffer (pH 7.4) and equal volume of K3Fe(CN)6 (1% w/v). The mixture was incubated at 50°C for 20 min, followed by the addition of 2.5 mL of TCA (10% w/v). It is then centrifuged at 3000 rpm for 10 min. The upper layer of the solution is collected and mixed with 2.5 mL of distilled water and 0.5 mL of ferrous chloride (0.1% w/v). Finally the absorbance is measured at 700 nm against blank. Ascorbic acid was used as positive reference standard. Increased absorbance of the reaction mixture indicates higher reducing power of the plant extract.

Phosphomolybdenum (PM) assay: The PM assay was determined by the method described by Prieto et al.13. About 0.1% of T. divaricata (L.) (methanol, ethanol and aqueous extracts) leaf extracts was added to each test tube individually containing 3 mL of distilled water and 1 mL of molybdate reagent solution. These tubes were kept incubated at 95°C for 90 min. After incubation the absorbance was measured at 695 nm. Ascorbic acid was used as reference standard.

Cell line assay
Culturing of cell lines: The cells were sub-cultured in Dulbecco Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% non-essential amino acids in tissue culture flasks and incubated in a CO2 incubator in a 5% CO2 and 95% humidity atmosphere. After trypsinization the cells were counted and the cell viability was tested by trypan blue using haemocytometer. A known number of cells (2×103 cell/well in 100 μL of medium) were seeded into 96-well plates, respectively, to execute the MTT assay.

Treatment groups: Tabernaemontana divaricata (L.) (5 mg mL1) of leaf methanol, ethanol and aqueous extract were prepared in 0.1% di-methyl sulfoxide (DMSO) for treating T-24 bladder cancer cell line and the normal cell line (MEF-L929). The reactant mixtures were diluted with media and cells were treated with different concentrations of the extract (3.125-200 μg mL1) and incubated for 72 h, respectively. The following treatment groups are setup of the study. Negative control: cells alone. Positive control: cells+Doxorubicin (Standard drug). Test groups: cells+methanol extract of T. divaricata (L.) leaves cells+ethanol extract of T. divaricata (L.) leaves and cells+aqueous extract of T. divaricata (L.) leaves. A similar treatment regime was followed for the normal mouse embryo fibroblast (MEF-L929) cell line.

MTT cell viability assays: The MTT cell viability assay was performed by following the methods described14-16. After 72 h the media of the treated cells (100 μL) were removed and the cell cultures were incubated with 25 μL of MTT at 37°C for 4 h. After incubation, the formazan produced was solubilized by addition of 100 μL DMSO. The suspension was placed on a micro-vibrator for 5 min and the absorbance was recorded at 540 nm by an ELISA reader and the results were analyzed in triplicate and percentage of inhibition was calculated.

Statistical analysis: All results were expressed as Mean±SD for three replications. Statistical analysis of data was performed by analysis of variance (ANOVA) and level of statistical significance between groups using GraphPad Prism version 7.0 for Windows (GraphPad Software, San Diego, CA, USA). At least three independent analyses were carried out per sample.

RESULTS

Total yield of crude extract: Leaf powder of 100 g of T. divaricata gave total yield of 13.88 g in chloroform, 196.84 g in ethyl acetate, 17.92 g in methanol, 14.44 g in ethanol and 11.72 g in aqueous solvents, respectively.

Table 1: Preliminary phytochemical of T. divaricata extract
Image for - Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines

Table 2: Total phenolic content of T. divaricata leaf methanol, ethanol and aqueous extracts
Image for - Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines

Table 3:
Determination of percentage inhibition of 2, 2-diphenyl-1-picrylhydrazyl radical scavenging activity of T. divaricata (%)
Image for - Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines

Phytochemical analysis: The different leaf extracts of T. divaricata subjected for preliminary phytochemical screening with solvents chloroform, ethyl acetate, methanol, ethanol and aqueous, showed a range of secondary metabolites (Table 1). Alkaloids were detected in chloroform, ethanol and aqueous extract, flavonoids were moderately present in ethyl acetate, methanol and ethanol extracts, glycosides were present in all five solvents, phenols were detected in methanol, ethanol and aqueous extracts, lignin’s were present in all extracts except aqueous, saponins were present in chloroform, ethyl acetate, ethanol and aqueous extracts, except ethanol sterols showed its presence in all four extracts except ethanol, tannins were present in methanol, ethanol and aqueous extracts. Anthraquinone were absent, Phlobatannins in aqueous, reducing sugar in chloroform and volatile oil showed its presence in ethanol extract.

Total phenol content: Total phenolic contents vary considerably among extracts of T. divaricata methanol, ethanol and aqueous, presented in Table 2. The amount of total phenolic content were calculated using the standard curve of Gallic acid (R2 = 0.9954) value from the equation:

y = mx+c

where, y is absorbance of unknown, m is the value from the graph, x is concentration, a is value from the graph and the results were expressed as Gallic acid equivalent per gram.

Antioxidant activities
DPPH assay: The antioxidant activities of T. divaricata leaf extracts with different concentrations of methanol, ethanol and aqueous were investigate using DPPH free radical scavenging activity. The results were presented in Table 3. The antioxidant capacity of the extract was compared with ascorbic acid as standard antioxidant.

FRAP and PM assay: In the present study, the presence of antioxidants in the ethanol, methanol and aqueous extracts were subjected to FRAP and PM assay along with standard ascorbic acid. The results are presented in triplicates with Mean±SD (Fig. 1a, b).

Image for - Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines
Fig. 1(a-b): (a) FRAP assay for extracts of T. divaricata and (b) PM assay for extracts T. divaricata
  Data is presented as mean SEM (n = 3). *Significant value (p<0.05)

Table 4: IC50 values of cell proliferation inhibition of T. divaricata leaf extracts (μg mL1)
Image for - Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines

Anticancer assay: Anticancer activity of T. divaricata leaf methanol, ethanol and aqueous extracts on T-24 bladder cancer cells lines. The effect of methanol, ethanol and aqueous extracts of T. divaricata leaves on cell viability were evaluated individually by MTT assay and the IC50 values were calculated from dose-dependent response studies assessed 72 h post-treatment. Tabernaemontana divaricata leaf extracts inhibited the growth of T-24 bladder cancer cells in a dose-dependent manner. The cytotoxicity for normal mice embryo fibroblast (MEF-L929) cell line was also evaluated and there was no inhibition observed on normal cell lines. The cell viability of the T. divaricata leaf extracts was compared to standard chemotherapeutic drug doxorubicin which is commonly used in the treatment of bladder cancer (Fig. 2a, b). The IC50 values were calculated in a dose-dependent response and are presented in Table 4. The combination of the extracts along with the standard drug also induced a higher cytotoxicity in cancer cells. These results revealed morphological changes and shrinkage of cells leading to cell apoptosis induced by the extracts in the human bladder cancer cell lines as depicted in Fig. 3a-c.

Image for - Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines
Fig. 2(a-b):
(a-b) Viability inhibition (%) of T. divaricata methanol, ethanol, aqueous leaf extracts and standard drug (doxorubicin) on T-24 human bladder cancer cell lines
  Data is presented as mean SEM (n = 3). *Significant value (p<0.05)

DISCUSSION

This study states the importance of herbal medicine in the use of bladder cancer treatment. This study is the ancient practice of herbal medicinal treatment for various diseases. Medicinal plants contain very strong secondary metabolites that bear the antioxidant, radical scavenging agents that help in prevention of cancers and autoimmune disorders17. The lack of literature on the use of herbal remedies on the treatment of bladder cancer gave a pavement to evaluate the antioxidant and anticancer activity on bladder cancer cell line7. The plant was collected from Western Ghats of Uttar Kannada district and identified according to their taxonomical features as T. divaricata and screened for the presences of phytoconstituents with five different solvent extracts. Phytochemical analysis of T. divaricata leaf revealed the presences of rich secondary metabolites in the selected leaf extracts. These secondary metabolites are found to have various biological and therapeutic properties18.

Image for - Evaluation of in vitro Antioxidant and Anticancer Activity of Tabernaemontana divaricata Leaf Extracts Against T-24 Human Bladder Cancer Cell Lines
Fig. 3(a-b):
(a) Morphological changes of T-24 human bladder cancer cell lines and (b) MEF-L929 normal cell line for 72 h. Morphological changes of standard drug (Doxorubicin). CS: Cellular shrinkage, BL: Membrane blebbing (Magnification for T-24 was 20X and MEF-L929 was 20X

In the present study, alkaloids were noticed in chloroform, ethanol and aqueous extracts, flavonoids were moderately present in ethyl acetate, methanol and ethanol extracts, glycosides were present in all five solvents, phenols were detected in methanol, ethanol and aqueous extracts, lignin’s were present in all extracts except aqueous, saponins were present in chloroform, ethyl acetate, ethanol and aqueous extracts, except ethanol sterols showed its presence in all four extracts, tannins were present in methanol, ethanol and aqueous extracts, anthraquinone was absent, phlobatannins in aqueous, reducing sugar in chloroform and volatile oil showed its presence in ethanol extract (Table 1). Tabernaemontana divaricata, garden plant in tropical countries is a rich source of alkaloids with various pharmacological properties, the results of phytochemicals revealed the presence of various secondary metabolites. The phenolic compound of methanol, ethanol and aqueous extracts were further quantified using FCR method, the results showed that aqueous extract had higher quantity of phenolic compound present in the leaf sample (62.54±0.18 mg g1 GAE) than methanol (51.71±0.90 mg g1 GAE) and ethanol extracts (56.86±0.37 mg g1 GAE) but slightly lower than the standard Gallic acid (80.28±0.14 mg g1 GAE) (Table 2). The presence of phenolic compound in the leaf extracts of T. divaricata is concordance with the properties of the plants that are used in the folk medicine for anti-infection, anti-inflammation, analgesic, anti-tumour, anti-oxidative effect and the effect in neuronal activity4.

Tabernaemontana divaricata has aroused interest due to the important biologic activity of its extracts, particularly, antioxidant activity, that has been associated with the secondary metabolites present7. Radical scavenging activities are very critical due to the lethal properties of free radical ions present in biological systems through reactive oxygen species (ROS) which ultimately impacts many human diseases such as most cancers17. Free radical scavenging activity is one of the mechanisms for anti-oxidation6. Antioxidant activity of T. divaricata leaves extracts were studied by screening its ability to decolorize the stable DPPH radical, FRAP and PM assays.

The results for 100 mg mL1 leaf extracts concentration are presented in Table 3. Ethanol leaf extract proved more potent with (62.41±0.67%) radical scavenging activity for minimum concentration of 100 mg mL1 as compared to methanol (48.58±0.38%) and aqueous extracts (53.87±0.19%). But however the radical scavenging effect of aqueous extract was slightly lower in degree as compared to the standard ascorbic acid (65.25±0.72%). As like DPPH radical scavenging activity assay, FRAP and PM assay also exhibited maximum potent to ethanol leaf extract and significant as compared to that of methanol and aqueous extracts. The existence of antioxidant, in the various extract have been shown to be an impart antioxidant activity by breaking the free radical chain11. This is because of the secondary metabolites like phenols, alkaloids and flavonoids present in the extracts due to which the antioxidants are the compounds capable of donating electron or hydrogen molecule for reduction19,20. Higher concentration resolute was unswervingly relative to greater antioxidant potential of the leaf extract. Among the methods tested for antioxidant capacity, DPPH (pink to yellow), FRAP (colourless to green) methods had end point from coloured to colourless whereas, in PM assay it was green to blue (Fig. 1a, b).

The evaluation of anticancer activity of T. divaricata leaf methanol, ethanol and aqueous extracts on (T-24) human bladder cancer cell line and (MEF-L929) normal cell line was evaluated for progression and morphology of cells, respectively. The MTT assay an in vitro model system to determine the cell viability inhibition was used in screening the extracts that influence basic cellular functions and morphology. As a result significant growth inhibition was observed in bladder cancer cell line (T-24), while there was no adverse effect on the growth of normal cells (MEF-L929). The impact of leaf extracts were showed the concentration (3.125, 6.25, 12.5, 25, 50, 100, 200 μg mL1) and incubation time period 72 h dependent, where in the inhibition (%) was calculated in triplicates diluted serially. Among the different concentrations gradients 200 μg mL1 of ethanol and aqueous extracts were the most efficient in hampering the progression of cells. The methanol extract showed less effect throughout the range of experimented concentrations and was not statistically significant on (T-24) human bladder cancer cell lines for a single time point of 72 h. The results were compared with the standard Doxorubicin drug21,22 used in the treatment of bladder cancer showed significant inhibition than the extracts on cancer cell lines (Fig. 2a, b). However, the results showed significant inhibition of ethanol extract was also potent extract with IC50 value (188.9±07.05 μg mL1) for T-24, methanol extract was 426.6±12.07 μg mL1 and aqueous extract showed 566.9±15.11 μg mL1. Variations in IC50 value was observed among methanol and aqueous extracts (Table 4) while the standard drug Doxorubicin exhibited IC50 value (0.23 μM mL1). The results revealed changes at structural and morphological level where the cancerous cells underwent shrinkage and membrane blebbing leading to cell death occurring apoptosis in (T-24) human bladder cancer cell line while the extract showed no cytotoxicity towards normal cell lines (Fig. 3a-c).

CONCLUSION

In the light of these findings, it is evident that T. divaricata leaves may be exploited as an important source of antioxidants. This study has gathered experimental evidence that T. divaricata leafs are more potent as natural antioxidant for its capacity to protect cells from oxidative DNA damage. It observed that ethanol leaf extracts of T. divaricata had significant anticancer activity on T-24 human bladder cancer cell line using in vitro model. At in vitro model system all the parameters tested T. divaricata stood first. Presence of polyphenols in leaves may therefore be responsible for their overall antioxidant potential. Thus T. divaricata could serve as an ideal candidate for bio-pharmaceutical therapeutic products and nutraceutical. Further study on various other cancerous cell lines should be carried out to identify the predominant phyto-molecules responsible for the antioxidant and anticancer activity to validate its therapeutic application at large scale.

SIGNIFICANCE STATEMENT

The study provides a platform for implementation of herbal preparations on in vivo animal models.

The current conclusions of the study paves the path for development of less toxic molecules in the treatment of cancer. The findings of the research work are unique in the treatment of bladder cancer as there are very less studies around the globe which have used herbal drug.

ACKNOWLEDGMENT

The authors are grateful to Dr. Kotresha K, Department of Botany, Karnataka Science College, Dharwad, Karnataka, for identifying the plant. The authors acknowledge Dr. G. U. Hulepanavarmath, Head, Dept. of English, Rani Chennamma University, Belagavi, for rendering the English Editing of the manuscript.

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