Evaluation of Anti-cancer, Antioxidant and Antimicrobial Activities of Alstonia scholaris L

 

Ravula Nanditha, Saravanan J, Praveen T K*, Deepa S, Emdormi Rymbai

Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Ooty, The Nilgiris 643 001.

 

ABSTRACT:

The objective of the current study is to investigate the anticancer, antioxidant and antimicrobial activities of the bark extracts of Alstonia scholaris L.  The barks were extracted with n-hexane, chloroform, ethyl acetate and methanol using soxhlet apparatus. The antioxidant potential of all the extracts was estimated by DPPH method. The antimicrobial property of the plant extracts was determined by disc diffusion method using gram positive, gram negative and fungal strains. The anticancer property was determined by MTT assay on HepG2 and HT-29 cells. All extracts exhibited significant antoxidant potential with fraction 1 of ethyl acetate extract showing the highest antioxidant potential. All extracts displayed antibacterial activity against all the selected gram-positive strains and some gram negative strains. All the extracts were found to have antifungal activity against Saccharomyces cerevisiae and Candida albicans at a concentration of 2mg but only methanolic extract was found to have activity against Aspergillus niger and Aspergillus flavus. The maximum cytotoxic effect was exhibited by ethyl acetate and methanolic extract on HepG2 and HT-29 respectively.

 

 

INTRODUCTION:

Alstonia scholaris, also known as devil’s tree or dita bark tree, belongs to the family Apocynaceae and is widely distributed in India, Ceylon, tropical Africa and east Australia¹. The chief phytoconstituents present in Alstonia scholaris are alkaloids, irridoids, coumarins, flavonoids, leucoanthocyanines, reducing sugars, simple phenolics, steroids, saponins, and tannins². The important alkaloids reported are echitamine, tubotaiwine, akuammicine, echitamidine, picrinine, ditamine. Non alkaloid constituents of the flowers are n-hexacosane, lupeol beta-amyrin, ursolic and palmitic acids. Among the other phytoconstituents, isookanine-7-o-alpha irhamnpyranoside, a new flavone glycoside and alstonoside, a secoiridoid glycoside have been reported. It is also found to have steroids.

 

Alstonia scholaris is reported for its anticancer activity on skin carcinogenesis in mice³.  It is reported to possess antimalarial property which is used in the marketed ayurveda preparation Ayush-64, NRDC, India. The alkaloid fraction of the leaves showed anti-tussive, anti-asthmatic and expectorant activities and is proved to be a valuable lead material for respiratory diseases drug development⁴. (Nidhi Saxena et al., 2013).

 

Cancer is the second most prevalent disease after cardiovascular diseases causing maximum number of deaths. Cancer is predicted to cause approximately 12 million deaths in 2020. The currently available chemotherapeutic drugs have certain limitations like; low selectivity leading to high toxicity, poor pharmacokinetic properties and only palliative. This study aims to study the potential anticancer, antioxidant and antimicrobial properties of Alstonia scholaris L.

 

MATERIALS AND METHODS:

Plant Material:

The bark of Alstonia scholaris L. was collected in the month of August from Ooty, Tamilnadu, India. The collected plant material was authenticated by Prof. P Jayaraman, Director, Plant anatomy research centre, National Institute of herbal science, Chennai, Tamilnadu.

Extraction and separation:

The barks of Alstonia scholaris L. were shade dried, milled to a coarse powder. About 200g of the powder was extracted with n-hexane, chloroform, ethyl acetate and methanol separately for 18 h at 40°C by continuous hot percolation using soxhlet apparatus. The extracts were filtered and concentrated to a dry mass by using rotary evaporator.

 

3.5g of the extract was packed into silica gel column by wet packing method. The column was eluted in a gradient manner using n-hexane: ethyl acetate (50: 50, 40: 60, 30: 70, and 20: 80). 50ml fractions were collected and based on the TLC profiles the fractions were combined to get a total of 42 fractions.

 

In vitro antioxidant activity by DPPH method:

DPPH solution (0.2mg/ml) was freshly prepared using methanol as solvent; 0.5mL of this solution was mixed with 0.5mL of different dilutions (10, 50, 100, 150 and 200𝜇g/mL) of the test sample. The volume of the solution was adjusted to 5mL with methanol. After incubation in the dark for 30 minutes at room temperature, the absorbance was measured at 517nm. Butylated.

 

Hydroxy toluene was used as standard. The antioxidant activity was evaluated by comparing the absorbance with the negative control (0.5mL of DPPH solution and 4.5mL of methanol) ⁵.

 

Percentage inhibition =100 – [A sample /A control X 100]

 

ANTIBACTERIAL ACTIVITY:

Bacterial strains:

The antibacterial activity of Alstonia scholaris L was evaluated using 3 Gram-positive bacteria: Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis and 3 Gram-negative bacteria: Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae.

 

Antibacterial Activity by disc diffusion method:

The antibacterial activity of Alstonia scholaris L was evaluated by disc diffusion method. 100μl of bacterial suspension was applied uniformly on the surface of Mueller-Hinton agar plate. The dried plant extracts were dissolved separately in 5% dimethylsulphoxide (DMSO) to a final concentration of 100mg/ml. Sterilized discs (3 mm in diameter) impregnated with 20μl of extract (100 mg/ml) were arranged on the surface of inoculated plates and incubated at 37°C for 24 hours. Penicillin G disc (10μg) was used as positive control whereas the solvent DMSO (5%) was used as negative control. After incubation, the zone of inhibition was measured with HiMedia zone scale. The study was performed in triplicate^6,7,8,9,10.

 

ANTIFUNGAL ACTIVITY:

Fungal Strains:

The antifungal activity of Alstonia scholaris L was evaluated using 3 fungal cultures: Saccharomyces cerevisiae, Candida albicans, Aspergillus niger and Aspergillus flavus.

 

Antifungal activity by disc diffusion method:

The antifungal activity of Alstonia scholaris L was evaluated by disc diffusion method. 100μl of fungal suspension was applied uniformly on the surface of Mueller- Hinton agar plate. The dried plant extracts were dissolved separately in 5% dimethylsulphoxide (DMSO) to reach a final concentration of 100mg/ml. Sterilized discs were impregnated with 20μl of extract (100mg/ml) and arranged on the surface of inoculated plates and incubated at 37°C for 24 hours. Amphotericin B disc (10μg) served as positive control and the solvent DMSO (5%) as negative control. After incubation, the zone of inhibition was measured with HiMedia zone scale. The study was performed in triplicate^{11,12,13,14,15}.

 

ANTICANCER ACTIVITY:

Invitro anticancer activity by MTT assay:

HepG2 and HT-29 cells were trypsinised, washed and seeded in plates. The plates were then incubated at 37°C and 5% CO₂ for 24 h in a CO₂ incubator. The test extracts were added at a concentration of 40, 80, 160 and 320 ppm. 100μl MTT at a concentration of 5μg/ml was then added to each well and incubated for 48 h at 37°C and 5% CO2 in a CO2 incubator. 100μl of DMSO was added to each well and incubated at room temperature for 15 minutes and the OD was read at 570nm. A well with cell lines and MTT but without extract was used as control. The experiment was done in triplicate and average value of % inhibition was taken for calculation of IC50 value^{16,17,18,19,20}.

 

% Inhibition=100-[(OD of the well with drug/OD of the control) x 100]

 

Genotoxicity Study:

The genotoxic effect of ethyl acetate extract of Alstonia scholaris L. was studied on mitomycin induced chromosomal aberrations on in vitro cultured liver cells. About 5ml fresh cultures were taken and incubated at a temperature of 37°C for duration of 72 hrs. At 69^th hr, 100ml of colchicine was added. After 2 hrs, cultures were terminated. A treatment of pre-warmed hypotonic potassium chloride solution at 37°C for 25min was given followed by fixative (3:1 methanol: Acetic acid) washes so that clear white lymphocyte pellet was obtained. This was suspended in about 0.5ml of fixative for final preparation of slides. Air- dried slides were stained in 2 % Giemsa stain prepared in Sorenson’s buffer. Chromosomal aberrations were counted in 100 well spread metaphase chromosomes²¹.

 

RESULTS:

Extraction and separation:

The yields of the n-hexane, chloroform, ethyl acetate and methanol extracts are given in Table-I. The highest yield (5.08gm) was obtained with ethyl acetate. The ethyl acetate extract on separation by column chromatography gave a total of 42 fractions. The list of fractions is given in Table-2. The qualitative phytochemical analysis revealed the presence of alkaloids, flavonoids and phenolic compounds in all the four extracts. Saponins were found in all the extracts except in the chloroform extract whereas glycosides were present only in ethyl acetate and methanol extracts. Terpenoids were found only in methanol extract.

 

Table I: Soxhlet extraction of bark of Alstonia scholaris L

 

Antioxidant activity:

The antioxidant activity of A. scholaris L was evaluated by DPPH method. The antioxidant activity increased proportionately with increasing concentrations of all the four test extracts. The maximum antioxidant activity was exhibited by ethyl acetate extract at a concentration of 300µg.

 

Table II: Antioxidant activity of Alstonia scholaris L. (IC50 in μg/ml)

 

Antibacterial Activity:

All four extracts of A. scholaris stem bark were tested for in vitro antibacterial activity against selected gram positive and gram negative bacterial strains. All the extracts at a concentration of 2mg were found to possess significant antibacterial activity against all the selected gram positive strains: Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis and gram negative, Klebsiella aerogenes. Only ethyl acetate and methanolic extracts of A. scholaris were active on E.coli and Pseudomonas aeruginosa.

 

Antifungal activity:

The four extracts of A. scholaris stem bark were tested for in vitro antifungal activity against selected fungal strains. All the extracts were found to have antifungal activity against Saccharomyces cerevisiae and Candida albicans at a concentration of 2mg but only methanolic extract was found to have activity against Aspergillus niger and Aspergillus flavus.

Table III: Antibacterial activity of Alstonia scholaris L. on Gram positive strains

Values are expressed as zone of inhibition (mm) and an average of triplicate

 

Table IV: Antibacterial activity of Alstonia scholaris L. on Gram negative strains

Values are expressed as zone of inhibition (mm) and an average of triplicate

 

Table V: Antifungal activity of Alstonia scholaris L.

 

In vitro anticancer activity:

All the extracts of Alstonia scholaris L except the extract of hexane (ASHE) showed a dose dependent decrease in the cell proliferation on both HepG2 cell lines and HT-29 cell lines. The maximum cytotoxic effect (IC50: 225.8±0.36) on HepG2 cell lines was exhibited by ethyl acetate extract of Alstonia scholaris L (ASEE) followed by ASME (IC50: 399.1±1.12) and ASCE (IC50: 416.7±1.32). The maximum cytotoxic effect (IC50: 239.4±3.51) on HT-29 cell lines was exhibited by methanolic extract of Alstonia scholaris L (ASME) followed by ASEE (IC50: 264.1±1.98) and ASCE (IC50: 403.3±0.79).

 

Table VI: Cytotoxic activity of Alstonia scholaris L. on HepG2 cell lines and HT-29 cell lines

Values are expressed as Mean ± SD, n=3

Genotoxicity Study:

Genotoxicity studies of ethyl acetate extract of Alstonia scholaris at concentrations of 80ppm, 160ppm, and 320ppm on THLE3 cell lines showed a dose dependent increase in the number of chromosomal aberrations.

Table VII: Chromosomal aberration studies of ASEE on THLE3 cell lines

 

 

Fig I: Dicentric chromosome and chromosome breaks in THLE3 cell lines

 

 

Fig II: Minute in THLE3 cell lines (ASEE-320ppm)

 

 

Fig III: Fragment in THLE3 cell lines (Mitomycin-2 μg/ml)

 

 

Fig IV: Minute in THLE3 cell lines (ASEE-160ppm)

 

DISCUSSION:

The phytoconstituents are one of the major sources in the discovery of new drugs. Plants have been used as medicine in the treatment of various diseases by humans from time immemorial. In this study, the antioxidant, antimicrobial and anticancer property of the plant, Alstonia scholaris was evaluated.

 

The bark of Alstonia scholaris was extracted by soxhlet apparatus using four different solvents: n-hexane, chloroform, ethyl acetate and methanol. The maximum yield was obtained with ethyl acetate followed by methanol, chloroform and n-hexane respectively. Phytochemical analysis revealed the presence of phenols and flavonoids in all the four extracts. The antioxidant potential of the extracts was determined by using in vitro DPPH method. The ethyl acetate extract of Alstonia scholaris L showed the maximum antioxidant activity with an IC 50 Value of 436.6µg/ml. It was therefore decided to fractionate the ethyl acetate extract using silica gel column. The fractionation of ethyl acetate extract of Alstonia scholaris L yielded a total of 42 fractions. All the fractions were subjected to in-vitro antioxidant study by DPPH method and among all the 42 fractions, fraction I showed maximum antioxidant effect with an IC₅₀ value of 32.1 µg/ml, which was very close to that of the standard BHT (20.30 µg/ml). The phenols present in the food and plants possess strong antioxidant properties and thus are being increasingly investigated. Phenol compounds widely distributed in the plant tissues include flavonoids, tannins, hydroxyl cinnammate esters and lignin²². The phytochemical anlaysis of the barks of Alstonia scholaris has shown the presence of phenols, flavanoids and terpenoids. These phytoconstituents may be responsible for the antioxidant activity exhibited by Alstonia scholaris.

 

The antimicrobial property of all the extracts was studied against gram-positive organisms, gram-negative organisms and fungal strains. Among the four extracts, ethyl acetate extract of Alstonia scholaris L displayed maximum antibacterial activity followed by methanolic extract. The zone of inhibition shown by ethyl acetate extract of Alstonia scholaris L was very close to that of standard drug against both gram positive and gram-negative organisms. The chloroform and n-hexane extracts did not show any significant antimicrobial activity against E. coli and P. aeruginosa. The antifungal activity of all the four extracts was studied against four fungal strains. The methanolic extract of Alstonia scholaris L showed significant antifungal activity against all the four selected fungal strains wheras ethyl acetate extract of Alstonia scholaris L showed antifungal activity close to the standard against S. cerevisiae and C. albicans but no activity against A. niger and A. flavus. The chloroform and n-hexane extract showed moderate antifungal activity against S. cerevisiae and C. albicans but no activity against A. niger and A. flavus.

 

The in vitro anticancer activity of all the four extracts was studied by MTT assay on HepG2 and HT-29 cell lines. The n-hexane extract did not show any significant cytotoxic effect. The maximum cytotoxic effect was displayed by ethyl acetate extract with an IC₅₀ of 225.8 µg/ml followed by methanolic (399.1 µg/ml) and chloroform extract (416.7 µg/ml) on HepG2 cell line. The maximum cytotoxic effect on HT-29 cell lines was displayed by methanolic extract with an IC₅₀ of 239.4 µg/ml followed by ethyl acetate extract (264.1 µg/ml) and chloroform extract (403.3 µg/ml) on HepG2 cell line. The genotoxicity study of ethylacetate extract showed a dose dependent increase in the number of chromosomal aberrations. The free radicals play a major role in pathophysiology of various diseases including cancer. These free radicals cause cancer by causing damage to the DNA of the cell^{23, 24}. The presence of phenols and flavanoids in all the extracts might have contributed to the antioxidant property. The antioxidant property of Alstonia scholaris might have played a significant role in the anticancer property.

 

CONCLUSION:

Among the four extracts, the ethyl acetate extract of Alstonia scholaris showed good to moderate antimicrobial, anticancer and antioxidant activities. However, further studies related to isolation and identification of the lead compounds from Alstonia scholaris is required to prove the possible anticancer and antimicrobial property.

 

ACKNOWLEDGEMENTS:

The authors would like to thank the Department of Science and Technology – Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions (DST-FIST), New Delhi for their infrastructure support to our department. The authors would also like to express their special thanks to management of JSS College of Pharmacy, Ooty and JSS Academy of Higher Education and Research, Mysuru.

 

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Accepted on 30.01.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4153-4157.