Cytotoxic activity of aqueous extracts of Anogeissus leiocarpus and Terminalia avicennioides root barks against Ehrlich ascites carcinoma cells.

OBJECTIVES: Folkloric claims on the use of a mixture of Anogeissus leiocarpus and Terminalia avicennioides root barks in tumor management exist without scientific evidence. This study aimed at investigating the phytochemical constituents and in vitro antiproliferative activity of these plants and their mixture.
MATERIALS AND METHODS: Phytochemical screening was carried out on the aqueous extracts after which various concentrations (0 to 1 000 μg/ml) were incubated with Ehrlich ascites carcinoma cell lines for 3 and 24 hours.
RESULTS: The extracts contained alkaloids, tannins, flavonoids, phenolics, saponins, phlobatannins, and terpenes. The separate extracts and their 1:1 mixture significantly (P<0.05) decreased the computed percentage viability of the cell lines in a dose- and time-dependent manner.
CONCLUSIONS: The antiproliferative activity may be due to the presence of the bioactive compounds in the extracts and has a potential in the management of tumor.

Introduction

Cancer is the ultimate end stage of carcinogenesis characterized by abnormal cell and tissue proliferation, which eventually leads to invasion and metastasis. It affects one-third of persons and is a major cause of deaths in the developed world during the year 2000.[1] Available management options such as surgery, radiation therapy, immunotherapy, and chemotherapy are either toxic, expensive, or both. This led to the search for alternative therapies in botanicals with anticancer activity. Two such plants acclaimed to be used in managing tumor by traditional herb sellers in Osogbo, south-western Nigeria are Anogeissus leiocarpus and Terminalia avicennioides.

A. leiocarpus (DC) (family: Combretaceae) (Guill and Perr) is known as chewing stick tree (English) and Ayin (Yoruba, south western Nigeria). It has been claimed to be used to treat diarrhea, dysentery, malaria, bacterial infections and used as chewing sticks. The stem bark has been reported to contain flavonoids, tannins, phenols, and alkaloids.[2]

T. avicennioides (family: Combretaceae) (Guill and Perr) is known as Baushe (Hausa, northern Nigeria), Idi (Yoruba), and Edo (Igbo, southeast Nigeria).[34] Its roots are used as chewing sticks and the plant has been reported to exhibit gastroprotective effect against HCl-induced gastric damage in rats.[5] The roots contain glycosides, phenols, tannins, ellagic acid, saponins, alkaloids, and steroids.[34] A mixture of aqueous extracts of both plant root barks have been claimed to be used traditionally in the management of tumor in the south western part of Nigeria.

There has not been any scientific verification of the anticancer activity of these plants and their mixture. This study aims at lending scientific credence to this claim by determining the phytochemical constituents and also in vitro anticancer activity of aqueous extracts of A. leiocarpus and T. avicennioides root barks, as well as their mixture against Ehrlich ascites carcinoma (EAC) cell lines.

Materials and Methods

Plant materials

Fresh roots of A. leiocarpus and T. avicennioides were obtained from a farmland in Offa, Nigeria and authenticated at the Department of Botany, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria, with voucher numbers 13775 and 15428, respectively.

Cell lines

EAC cell lines were obtained from Molecular Biology Laboratory, Department of Biology, Faculty of Arts and Sciences, Gaziantep University, Gaziantep, Turkey.

Preparation of extracts

Fresh roots of both plants were separately oven-dried at 40°C for 3 weeks and pulverized with blender (AKIRA BL-1531, Indonesia). A known amount (300 g) of each powder was extracted in 5 L of distilled water and placed on orbital shaker maintained at 300 rpm for 24 hours. This was then filtered with Whatman No. 1 filter paper and the filtrate concentrated on water bath to yield 30 g of T. avicennioides (10%) and 28.5 g of A. leiocarpus (9.5%). A 1:1 mixture of both extracts was also prepared for use in the present study.

Phytochemical screening

Qualitative screening

The aqueous extracts of the root barks of A. leiocarpus and T. avicennioides were screened for their phytochemical constituents according to the standard methods[67] as follows:

Alkaloids: To 1 mL of 1% HCl, 3 mL of the extract was added and heated for 20 minutes, cooled, and filtered. A few drops of Wagner's reagent (2 g of iodine and 6 g of KI in 100 mL of distilled water) were then added to 1 mL of the filtrate. A reddish brown precipitate indicated the presence of alkaloids.

Tannins: To 1 mL of freshly prepared 10% KOH, 1 mL of the aqueous extract was added. A dirty white precipitate indicated the presence of tannins.

Phenolics: To 2 drops of 5% FeCl3, 1 mL of the extract was added. A greenish precipitate indicated the presence of phenolics.

Glycosides: To 10 mL of 50% H2SO4, 1 mL of the extract was added, heated in boiling water for 15 minutes, and 10 mL of Fehling's solution was added and boiled again. The absence of a brick-red precipitate indicated that glycosides were not present.

Saponins: To 5 drops of olive oil, 3 mL of the aqueous extract was added in a test tube and shaken vigorously. A stable emulsion indicated the presence of saponins.

Flavonoids: To 1 mL of 10% NaOH, 3 mL of the extract was added. A yellow coloration indicated the presence of flavonoids.

Steroids: To 5 drops of concentrated H2SO4, 1 mL of the extract was added. The absence of a red coloration indicated that steroids were not present.

Phlobatannins: A few drops of 1% HCl were added to 3 mL of the extract. A red precipitate indicated the presence of phlobatannins.

Terpenes: To 1 mL of the extract, 5 drops of acetic anhydride was added, followed by a drop of concentrated H2SO4. The mixture was steamed for 1 hour and neutralized with NaOH, followed by chloroform. The presence of a blue-green color indicated the presence of terpenes.

Anthraquinones: A known volume (3 mL) of the extract was shaken with 10 mL of benzene, filtered, and 5 mL of 10% NH3 solution was added to the filtrate. The absence of pink or violet color in the ammoniacal (lower) phase indicated that hydroxyl anthraquinones were not present.

Quantitative determinations

The detected phytochemicals were quantified as described:

Alkaloids: This was done by the alkaline precipitation gravimetric method described by Harborne.[7] Briefly, an amount of sample (0.5 g) was dispersed in 10% acetic acid solution in ethanol in a ratio of 1:10 (10%). The mixture was left undisturbed for 4 hours at 28°C and filtered with Whatman No. 1 filter paper. The filtrate was concentrated to one quarter of its original volume by evaporation and treated with 3 drops of conc. NH4OH. The alkaloid precipitate was received in a weighed filter paper, washed with 1% ammonia solution, and oven dried at 80°C. Alkaloid content, expressed as a percentage of the weight of sample analyzed and converted to mg/mL. This was done in triplicate.

Flavonoids: This was assayed using the procedure described by Jagadish et al.[8] Briefly, the extract (1.5 mL) was added to 1.5 mL of 2% methanolic AlCl3 solution. The mixture was vigorously shaken on orbital shaker for 5 minutes at 200 rpm and the absorbance was read at 367 nm after 10 minutes of incubation. Quercetin was used as a standard for the calibration curve. The assay was carried out in triplicate.

Phenolics: This was determined using Folin-Ciocalteu method, as described by Olajire and Azeez.[9] The extract (0.5 mL) was added to 10 mL deionized distilled water and 2.5 mL of 0.2 N Folin-Ciocalteu phenol reagent. The mixture was left undisturbed at room temperature for 5 minutes and then 2 mL of 2% sodium carbonate was added. The absorbance of the resulting solution was read at 780 nm and repeated three times. Quercetin was used as a standard for calibration curve. This was done in triplicate.

Tannins: Tannin content was determined by Folin-Denis colorimetric method described by Kirk and Sawyer,[10] with slight modification. 0.5 g of sample was dispersed in 50 mL of distilled water and shaken. The mixture was left undisturbed for 30 minutes at 28°C and filtered through Whatman No. 1 filter paper. The filtrate (2 mL) was dispersed into a 50-mL volumetric flask and 2.5 mL of 10% Na2 CO3 solution was added. The content of each flask was made up to 50 mL with distilled water and incubated at 28°C for 90 minutes. Absorbance was read at 260 nm using the reagent blank. Tannic acid was used for the calibration curve. The procedure was repeated three times.

Saponins: This was done using the procedure described by Brunner.[11] A known mass (1 g) of finely ground sample was weighed into a 250-mL beaker and 100 mL of isobutyl alcohol was added. The mixture was shaken for 5 hours to ensure uniform mixing and filtered through Whatman No. 1 filter paper into a 100-mL beaker, after which 20 mL of 40% magnesium carbonate solution was added. The resulting mixture was again filtered through Whatman No. 1 filter paper to obtain a clear colorless solution. A known volume (I mL) of the colorless solution was pipetted into a 50-mL volumetric flask and 2 mL of 5% FeCl3 solution was added and made up to the marked level with distilled water. It was left undisturbed for 30 minutes for blood red color to develop. The absorbance was read after color development at a wavelength of 380 nm. Standard saponin was used for calibration curve.

Phlobatannins

This was done as recommended by AMC-RSC.[12] The extract (0.5 g) was weighed into a 50-mL beaker and 20 mL of 50% methanol was added, covered with paraffin and placed in a water bath set at 80°C for 1 hour. The mixture was properly shaken to ensure uniform mixing after which it was filtered through a Whatman No. 1 filter paper into a 50-mL volumetric flask, rinsed with aqueous methanol and then made up to the marked level with distilled water. The extract (1 mL) was pipetted into a 50 mL volumetric flask, 20 mL of distilled water, 2.5 mL of Folin-Dennis reagent, and 10 mL of 17% sodium carbonate were added to the solution in the 50-mL flask. This mixture was homogenized thoroughly for 20 minutes and absorbance read at a wavelength of 550 nm.

Terpenes

This was quantified according to the recommendation of AMC-RSC.[12] A sample of extract (0.5 g) was weighed into a 50-mL conical flask, 20 mL of 2:1 Chloroform-Methanol mixture was added, shaken thoroughly, and left undisturbed for 15 minutes. The mixture was later centrifuged for another 15 minutes. Supernatant obtained was discarded, and the precipitate was re-washed with another 20 mL chloroform-methanol mixture for re-centrifugation. The resultant precipitate was dissolved in 40 mL of 10% sodium deodecyl sulfate solution. 0.01 M FeCl3 solution (1 mL) was added to the mixture at 30s interval, shaken, and left undisturbed for 30 minutes. The absorbance was read at 510 nm.

Cell viability assay

In vitro cytotoxic activity was determined using Trypan Blue cytotoxicity assay.[13] Briefly, aqueous extracts were dissolved in water to a final concentration of 200 μg/mL and made up to 800 μL with Phosphate buffered saline (PBS). EAC (100 μL) with concentration of about 106 cells/mL was added to the tubes. This was then incubated at 37°C for 3 hours and 24 hours followed by addition of 100 μL (0.4% in PBS) of trypan blue dye (Sigma-Aldrich, St. Louis, USA) to all the test tubes. The control consisted of PBS in place of the extracts. The cells that did not take up the dye and those that took up the dye were declared viable and non-viable, respectively. The number of cells was thereafter counted using the Cedex counter. % viability was expressed as:

Statistical analysis

Data were expressed as mean ± S.D for each analysis and significant differences were determined by Analysis of Variance (ANOVA) and Tukey's Post Hoc test for multiple comparisons at 95% confidence level using SPSS software (SPSS Inc., Chicago, IL, USA).

Results

Table 1 shows the phytochemical constituents of aqueous extracts of A. leiocarpus and T. avicennioides root barks. The extracts contained alkaloids, tannins, flavonoids, phenolics, saponins, phlobatannins, and terpenes. Anthraquinones, glycosides, and steroids were not detected.

Table 1

Phytochemical composition of aqueous extracts of A. leiocarpus and T. avicennioides root barks

The extracts and their mixture significantly (P<0.05) reduced the viability of EAC cells after incubation for 3 hours [Figure 1] and 24 hours [Figure 2] in a manner that was directly related to the concentrations (0 to 1 000 μg/mL) of the aqueous extracts of A. leiocarpus, T. avicennioides, and their mixture.

Figure 1

EAC cell viability following 3 hours of incubation with various concentrations (μg/mL) of aqueous extracts of A. leiocarpus, T. avicennioides, and their mixture. Bars carrying different letters are significantly different (P<0.05)

Figure 2

EAC cell viability following 24 hours of incubation with various concentrations (μg/mL) of aqueous extracts of A. leiocarpus, T. avicennioides, and their mixture. Bars carrying different letters are significantly different (P<0.05)

Discussion

Various natural products have been shown to have anticancer activities and also act as cancer preventive agents. Dietary chemopreventive agents are predominantly present in fruits, vegetables, grains, spices, and herbs like Jatropha curcas[1415] and have diverse chemical structures.[16] These natural products have been known to affect metabolic activation of chemical carcinogens. For example isothiocyanates in cruciferous vegetables like cauliflower, broccoli, and cabbage are known to interfere with nitrosamine metabolism.[17] Others like sulforaphane, zerumbone, curcumin, and chalcone (an α, β-unsaturated flavonoid) also affect various stages of carcinogenesis.[16]

This study has shown that aqueous extracts of A. leiocarpus and T. avicennioides root barks contain alkaloids [Table 1] which have been reported to possess anesthetic, stimulant, and anticancer properties.[18] Tannins, flavonoids, and phenolics have antioxidant and antimicrobial properties.[18] The presence of phenolics, flavonoids, and saponins have been shown to correlate well with free radical and nitric oxide scavenging activity.[1419] Flavonoids have strong anticancer properties and saponins have cholesterol-lowering and cytotoxic properties.[20] Polyphenols can inhibit tumor formation and inactivate carcinogens and mutagens.[2122] Triterpenes have demonstrated antibacterial activities.[23] The presence of these phytochemicals in the aqueous extracts of A. leiocarpus and T. avicennioides root barks might confer many of the mentioned pharmacological properties on the extracts.

One of the methods that have been used for investigating anticancer activity is cytotoxicity assay or cell viability assay using the EAC cell lines in vitro.[1324] The significant (P<0.05) decrease in the in vitro cancer cell viability with increasing dose and time [Figures 1 and 2] indicates that the extracts are cytotoxic against the EAC cell lines. This supports the notion that plants can provide potent anticancer principles if exploited.

The presence of tannins, flavonoids, phenolics, and saponins, among others in the extracts used in the present study, may be responsible for the antiproliferative activities on the EAC cell lines. This may, therefore, explain the rationale behind the use of the aqueous extracts of A. leiocarpus and T. avicennioides root barks and their mixture in the management of tumor in folk medicine. Further studies are needed to isolate and characterize the active principles of anti-cancer in the extracts.