In this study, we evaluated the anti-cancer property of five bark extracts and the isolates from chloroform and ethyl acetate of Annickia chlorantha by the tetrazolium salt method (MTT method). The anti-cancer activity was performed on human prostate cancer cell lines PC-3 and hormone-dependent breast cancer cell lines MCF-7. Results indicated that the two isolates displayed interesting cytotoxicity towards MCF-7 cell lines with CC 50 of 3.84 CC 50/mL and 4.87 CC 50/mL for chloroform and ethyl acetate respectively; while the total bark extracts showed CC 50 of 24.33 CC 50/mL, 36.49 CC 50/ mL and 73.52 CC 50/mL for chloroform, ethyl acetate and methanol extracts respectively. By the other hand on PC-3, the CC 50 of the isolates were higher than the one on MCF-7, more than 10 CC 50/mL for both chloroform and ethyl acetate isolates and 49.14 CC 50/mL, 77.33 CC 50/mL, 89.38 CC 50/mL and 92.37 CC 50/mL, respectively for chloroform, ethyl acetate and methanol soluble extracts. From this study, we identified that the two isolates had anti-cancer properties against MCF-7 cell lines.
Cancer is considered as an important and large family of diseases, in which abnormal cells grow and form a subset of neoplasm, with the potential to spread to other parts of the body. This abnormal growth of cells can many times lead to death. This disease remains a major public health burden in both developing and developed countries and constitutes the second leading cause of death worldwide [
In this paper, we are reporting for the first time the study based on anticancer properties of five crude extracts of Annickia chlorantha, from Democratic Republic of the Congo, which has been used in African traditional medicine as anti-malarial, anti-fever and for dyeing and back ache properties [
Even this plant has shown an interesting in vitro activity on plasmodium strains, it is however cytotoxic and can be oriented to the treatment of cancer [
The tested plant material (stem barks) used in the present study was collected in Abumombazi (Nord Ubangi Province, Democratic Republic of the Congo) during a field work in March-April 2014 by Professor Koto- te-Nyiwa Ngbolua and was authenticated by Mr B.L. Nlandu of the INERA (Institut National d’Etudes et Recherches Agronomiques). Voucher specimen N0 Ngb020EQ is on deposit at the Bioprospection Laboratory of the Department of Biology (Faculty of Sciences, University of Kinshasa).
The plant material of Annickia chlorantha (Oliv.) Setten & Maas stem (Annonaceae) was shade dried at ±27˚C and 900 g of powder were extracted by maceration, sequentially with petroleum ether 60˚C - 80˚C, chloroform, ethyl acetate, methanol and methanol 80%. The extraction was optimised by repeating the maceration twice and each solvent extract was concentrated under reduced pressure and allowed to dry at room temperature and weighed to give petroleum ether 60˚C - 80˚C, dichloromethane, ethyl acetate, methanol and aqueous methanol-solubles, respectively.
Each soluble was weighed, coded and subjected to column chromatography over silica gel 60 (0.040 - 0.63 mm [230 - 400 mesh]). The column was eluted with the mixture of petroleum ether 60˚C-80˚C-ethyl acetate and collected fractions were monitored on thin layer chromatography (TLC) (silica gel 60 F254 aluminum barking from Merck, Germany).
Chromatographic techniques (thin layer and column chromatographies) were used to perform the separation.
Anticancer activities were assayed by the standard MTT colorimetric procedure against MCF-7 and PC-3 cell lines. MCF-7 and PC-3 cell lines were obtained from National Centre for Cell Science (NCCS), Pune. It was maintained in Roswell Park Memorial Institute (RPMI 1640) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, amphotericin (3 μg/mL), gentamycin (400 μg/mL), streptomycin (250 μg/mL), penicillin (250 units/mL) and 1 mg/ml insulin in the incubator at 5% CO2, 37˚C. All extracts were dissolved in dimethylsulfoxyde (DMSO) to give a stock concentration of 100 μg/μL. The stock solution was then serially diluted with culture medium. The test concentrations for the extracts were 0.5, 1, 5, 10, 50 and 100 μg/mL. The test concentrations for isolates were 0.1, 1 and 10 μg/mL. The concentration of DMSO never exceeded 1% in any of the 96 wells.
The cells were trypsinized at sub-conflency and 2 × 104 cells/well were seeded in 96 well plates using culture medium, the viability was tested using Trypan blue dye with help of haemocytometer and 95% of viability was confirmed. After 24 hrs, the fresh medium with the extracts were added at respective wells and kept incubated for 72 hrs. After incubation the following assays were performed. The fresh medium was changed again for all groups and 10 μl of MTT (5 mg/mL stock solution) was added and the plates were incubated for an additional 4 h. The medium was discarded and the formazan blue, which was formed in the cells, was dissolved with 100 μl of DMSO. The optical density was measured at 570 nm. The percentage toxicity was calculated as following.
where: At = Absorbance value of test compound,
Ab = Absorbance value of blank and Ac=Absorbance value of control.
The graph pad prism software was used to calculate CC50 of the extracts/isolates.
The phytochemical screening was performed on the isolates in order to identify their chemical groups.
The different extracts were concentrated under reduced pressure and yielded respectively (1.67 g, 0.19%), (2.17 g, 0.24%), (4.02 g, 0.45%), (7.55 g, 0.84%) and (1.81 g, 0.20%) of solvent-soluble, for petroleum-ether 60˚C - 80˚C, chloroform, ethyl acetate, methanol and methanol 80%.
For the separation of the chloroform extract, 2.10 g of the crude extract were eluted on the column and a total of 220 fractions of 10 mL volumes each were collected. Similar fractions according to the TLC analysis were pooled to give four (C1, C2, C3 and C4) combined fractions. The combined fraction C1 eluted with petroleum ether 60˚C-80˚C-ethyl acetate (8:2) afforded one spot, white powder, of the major compound. Combined fractions C2, C3 and C4 eluted with petroleum ether 60˚C-80˚C-ethyl acetate (7:3), (6:4), (1:1) and (3:7), respectively afforded a mixture of spots each, in small quantities.
For the ethyl acetate extract, 3.71 g of the crude extract were eluted on the column and a total of 304 fractions of 10 mL volumes each were collected. Similar fractions according to the TLC analysis were pooled to give five (E1, E2, E3, E4 and E5) combined fractions. The combined fractions E1 and E2 eluted with petroleum ether 60˚C - 80˚C-ethyl acetate (9:1) and (7:3), respectively afforded one spot each of white powder. The fraction E2 was in faint. Combined fractions E3, E4, E5 eluted with petroleum ether 60˚C - 80˚C-ethyl acetate (6:4), (1:1), (4:6) and (2:8), respectively afforded a mixture of spots each, in small quantities.
The chemical screening performed on the isolates showed positive reaction only with Mayer and Dragendorff tests. This allowed concluding that the isolates could be alkaloids. This information is correlating with the literature review [
By the one hand, from the analysis, as shown in
However, the ethyl acetate and methanol 80% extracts showed middle IC50 of 36.49 CC50/mL and 73.52 CC50/mL, respectively and more than 100 CC50/mL for methanol and petroleum ether 60˚C - 80˚C extracts. By the other hand on human prostate cancer cells (PC-3), the isolates’ CC50 were higher than the one on MCF-7, more than 10 CC50/mL for both chloroform and ethyl acetate isolates, and 49.14 CC50/mL, 77.33 CC50/mL, 89.38 CC50/mL and 92.37 CC50/mL respectively for chloroform ethyl acetate, methanol 80% and methanol and more than 100 CC50/mL for petroleum ether 60˚C - 80˚C extract.
Further work is in progress to elucidate the chemical structures of constituents isolated in chloroform and ethyl acetate extracts. Indeed, Wall et al. [
As shown in
Extract/Isolate | CC50 (CC50/mL) | |
---|---|---|
MCF-7 | PC-3 | |
Petroleum-ether 60˚C - 80˚C | >100 | >100 |
Chloroform | 24.33 | 49.14 |
Ethyl acetate | 36.49 | 77.33 |
Methanol | >100 | 92.37 |
Methanol 80% | 73.52 | 89.38 |
Chloroform isolate | 3.84 | >10.00 |
Ethyl acetate isolate | 4.87 | >10.00 |
Paclitaxel | 27.73 | 22.34 |
Dose (CC50/mL) | Chloroform isolate | Ethyl acetate isolate |
---|---|---|
0.1 | 10.58 | 0.00 |
1.0 | 57.82 | 34.73 |
10.0 | 96.95 | 92.69 |
in
In
However in
It can then be confirmed that the positive control-drug Paclitaxel has anti-proliferative effect on PC-3 cell lines. However the two isolates and the negative control didn’t show effective effect on the PC-3 cell lines.
The Control of cell proliferation is considered to be a potentially effective strategy for the control of tumor growth [
The aim of this work was to evaluate the anticancer properties of five crude extracts of Annickia chlorantha, and two isolates obtained from the chloroform and ethyl acetate extracts of this plant species. We have shown that the isolates have an inhibitory effect on the MCF-7 cell lines and only the positive control-drug Paclitaxel itself has anti-proliferative effect on the damaged cells PC-3; however the two isolates don’t have any effect on the PC-3 cell lines. It can be suggested that Annickia chlorantha can be considered as a possible therapeutic agent against human breast cancer.
The authors are deeply thankful to FICCI, for the award of the prestigious CV Raman International Fellowship for African Researchers, program 2013, under Visiting Fellowship (Research grant: INT/NAI/CVRF/2014), accomplished at PSG College of Pharmacy (Peelamedu, Coimbatore/India) by Mr. Damien Sha-Tshibey Tshibangu. Thanks to all the authorities of PSG College of Pharmacy.
The authors are also indebted to PAH ARES/UNIKIN for the research grants awarded to Mr. Damien Sha- Tshibey Tshibangu, Pius T. Mpiana and Koto-te-Nyiwa Ngbolua in the Project: “Renforcement des Capacités d’Expérimentation et de Travail de Terrain/2015”.
Damien S. Tshibangu,Selvaraj Divakar,Muthiah Ramanathan,Govindarajan Syamala,Koto-te-Nyiwa Ngbolua,Jean Chrysostome V. Mudogo,Dinangayi D. Tshilanda,Nicole M. Misengabu,Pius T. Mpiana, (2016) In Vitro Anticancer Assessment of Annickia chlorantha (Oliv.) Setten & Maas Stem (Annonaceae) Bark from Democratic Republic of Congo. Journal of Biosciences and Medicines,04,23-29. doi: 10.4236/jbm.2016.44004