PRELIMINARY ASSESSMENT OF THE ANTIPROLIFERATIVE POTENTIAL OF THE HERBAL FORMULA EKPO AGANWO

{ DOWNLOAD AS PDF }

About Authors:
Enegide Chinedu*, David Arome, Solomon Fidelis Ameh
Department of Science Laboratory Technology (Physiology & Pharmacology Technology),
University of Jos, Jos Nigeria
chinex.snow@gmail.com

Abstract
Background: Cancer is a major global public health problem and has metamorphosed to a leading cause of death globally. Latest predictions by the Word Health Organization suggest that cancer death rate may escalate to about 15 million deaths by 2030. Though various anticancer agents have been discovered and are in use currently, they have not been able to tame the disease effectively. This has left the scientific community with the task of discovering/developing better agents with high efficacy and minimal adverse effect. There have been increased interests by both developed countries and the scientific community towards the importance of herbs as a fountainhead in the development of newer drugs, as many classes of drugs now in use contain models from natual product. Ekpo aganwo (EA) is an herbal formulation prepared from the stem bark of Khaya senegalensis and is its main constituent.
Aim: This study was design as a preliminary assessment of the antiproliferative potential of EA.
Materials and Method: The modified Ayinde et al., bench top assay method was employed for the study.
Result: Methotrexate compared with control, caused a significant (P<0.05) decrease in radicle lenght throughout the study. The percentage inhibition was however highest after 72 hours, with a rate of 86.67%. EA 10 mg/ml did not produce any significant decrease in radicle growth after 48 hours, but however showed a significant (P<0.05) decrease in radicle growth after 72 hours of experiment. The percentage inhibition after 72 hours was 24.56%. EA doses of 20 - 100 mg/ml produced significant (P<0.05) decrease in radicle growth throughout the experiment. The percentage inhbition of EA doses of 20 - 100 mg/ml was 28.07 - 59.65 % after 72 hours.
Conclusion: The results obtained have favoured the postulation of EA having an anticancer potential, and this have been supported by the significant antiproliferative activity it produced during the experiment.

REFERENCE ID: PHARMATUTOR-ART-2096

INTRODUCTION
Cancer is a major global public health problem and has metamorphosed to a leading cause of death globally. The World Health Organization have reported that the recorded cancer death rate for the year 2004 was about 7.4 million, while that of the year 2008 was about 7.6 million. However, latest predictions by Word Health Organization also suggests that cancer death rate may escalate to about 15 million deaths by 2030. ^[1] Cancer evolves from series of molecular events that fundamentally changes cells normal property. The hallmarks of cancer however are uncontrollable and sustained proliferation, others being evasion of aptosis, angiogenesis and metastasis. ^[2] Though various anticancer agents have been discovered and are in use currently, they have not been able to tame the disease effectively. Most of these agents are known to elicit dangerous adverse effects, expensive and are not even readily available in most rural areas. This has left the scientific community with the task of discovering/developing better agents with high efficacy and minimal adverse effect. However, history have shown that herbal medicine have played a major role in the development of modern medicine. Herbal medicine has evolved to a common and widely used therapy for various ailments by a large proportion of the world's population. ^[3] It has been stated by the World Health Organisation (WHO) that about 80% of developing countries population and 75% of the world population solely employ traditional medicines in the treatment/management of health issues. ^[4] There have been increased interests by both developed countries and the scientific community towards the importance of herbs as a fountainhead in the development of newer drugs, as many classes of drugs now in use contain models from natual product. ^[5] There have been claims by herbal practitioners of successful use of herbal preparations in the treatment of certain ailments, but these are with minute scientific evidences. ^[6] Therefore, the scientific assessment of such herbal preparations with the aim of affirming their forkloric useage should be an issue of top priority to the scientific community.

Ekpo aganwo (EA) is an herbal formulation prepared from the stem bark of Khaya senegalensis which is its main constituent. EA is used traditionally in the treatment of several ailments such as hypertension, diarrhea, stomach upset, back pain and fever. While there is also a believe that this formula can also be a useful anticancer agent. In the light of this, it is therefore important to screen this formula scientifically so as to ascertain if it is having an anticancer property. This study therefore, was design as a  preliminary assessment of the antiproliferative potential of EA, using bench top assay method, involving the antiproliferative effect of EA on guinea corn (Sorghum bicolor) seed radicle.

MATERIALS AND METHODS
Material
Methotrexate injection (Korea United Pharm. Inc) was purchased from a pharmacy outlet in Jos. The herbal formula "ekpo aganwo" was supplied by an herbal practitioner "Iya Abdul" who is based and practices in Jos, Nigeria. The herbal formula was supplied in powdered form and was reconstituted with distilled water before use.

Experimental Plant (Sorghum bicolor)
The experimental plant guinea corn (Sorghum bicolor) was purchased from Angwan-rukuba market, Jos. A viability test was carried-out on the seeds. This was done by pouring them into a bowl of water and observed for floating. The floating ones were discarded, while the submerged ones were dried and cleansed with absolute alcohol for usage.

Antiproliferative Evaluation
"The modified Ayinde et al., ^[7] bench top assay method was adopted for this study. Methotrexate was prepared to a concentration of 0.05 mg/ml. The powdered herbal formulation was reconstituted with distilled water and different concentrations (10, 20, 40, 60, 80 and 100 mg/ml) of the herbal formulation were prepared. Eight (8) sterile petri-dishes were layered with cotton-wool and filter paper (Whatman No. 1). Twenty (20) seeds of guinea corn (Sorghum bicolor) were placed into each layered petri-dish. The control seeds were treated with 10 ml of distilled water, while the methotrexate group was treated with 10 ml of the prepared methotrexate solution. The test group seeds were treated with different concentrations of EA. The first group of test seeds was treated with 10 ml of 10 mg/ml concentration, the second group of test seeds was treated with 10 ml of 20 mg/ml  concentration and the third group was treated with 10 ml of 40 mg/ml. The fourth was treated with 10 ml of 60 mg/ml and another group of test seeds was treated with 10 ml of  80 mg/ml concentration. The last group was treated with 10 ml of 100 mg/ml. All seeds in the various groups were incubated in a dark room and observed for growth after 24 hours. The mean lenght (mm) of the radicles emerging from the seeds in each group were however taken after 48 and 72 hours respectively.  The percentage growth was calculated using the formula:

 % Growth = mean radicle length treated × 100

                  mean radicle length control

While the percentage inhibition was calculated using the formula:

% Inhibition = 100 - % Growth

Statistical analysis
Two way ANOVA (Analysis of Variance) and Bonferroni post-test were used to test for significance. P<0.05 were considered significant.

RESULT

Table 1: Inhibitory effect of the herbal formula “ekpoaganwo”on the percentage growth of guinea corn (Sorghum bicolor) seeds radicle

* = P<0.05, MTX= Methotrexate, EA=ekpo aganwo

DISCUSSION
The search for new anticancer agents that are more effective and elicit less toxic effects has kindled great research interest in herbal medicine. This is because herbal medicine have grown to become the bed-rock of modern medicine development. ^[8]Herbal medicine have longed been employed in the treatment of various ailments, especially in developing countries and even developed countries. This have been accompanied with some advantages which includes easy accessability, less toxicity and even cheaper. ^[9] There have been increased claims of success in the use of herbal medicine. However, this have been greeted with increased scientific investigation on these herbs, so as to validate these claims. Scientific screening of herbs for their likely anticancer effects require a lot of materials as well as efforts and time. Even in most times, the result obtained are not really promising and doesn't in anyway reflect the said claims. These challenges have led to the establishment of various bench top procedures which have been reportedly employed by researchers as pointers of potentially promising anticancer herbs. In thesame vain, McLaughlin et al., ^[10] David et al., ^[11] Shogbaike et al., ^[12] and Ayindeet al., ^[13] have used germinating seeds radicles as parameters in preliminary screening of suspected potential anticancer agents. This method has terriffic advantages which includes reproducibility of result, simplicity of process, rapid, time and cost effective. It can be a very useful alternative in laboratories where suitable human cell lines are not readily available. It is also a priceless method that can be used to screen and eliminate several herbs that may be claimed to be anticancers, which however doesn't possess such property. Cancerous cells are known to show rapid proliferation and this is experienced also in meristematic cells of seeds (includingSorghum bicolor) under favourable conditions. This shows why this method was employed in this study.

The experimental result obtained from the study shows a high unhindered proliferation of the control seeds radicle throughout the 72 hours of study. This affirms the ability of Sorghum bicolor seeds to proliferate rapidly under favourable conditions. Methotrexate compared with control as shown in Fig. 1, caused a significant (P<0.05) decrease in radicle lenght throughout the study. The percentage inhibition was however highest after 72 hours, with a rate of 86.67%. This justifies it's current use as an anticancer agent. EA 10 mg/ml did not produce any significant decrease in radicle growth after 48 hours, but however showed a significant (P<0.05) decrease in radicle growth after 72 hours of experiment. The percentage inhibition after 72 hours was 24.56%. EA doses of 20 - 100 mg/ml produced significant (P<0.05) decrease in radicle growth throughout the experiment. The percentage inhbition ofEA doses of 20 - 100 mg/ml as shown in Table 1, was 28.07 - 59.65 % after 72 hours. It's effect was however dose dependent, as higher doses gave better effects (i.e effect produced by EA increased with dose increase). This result shows a remarkable antiproliferative activity by the herbal formula "ekpo aganwo, thereby favouring the postulation of it having an anticancer potential.

CONCLUSION
The results obtained have favoured the postulation of EA having an anticancer potential, and this have been supported by the significant antiproliferative activity it produced during the experiment. The authors are therefore considering furthering to anotber stage of this study on animal models and human cell lines.

REFERENCES
1. World Health Organization. NMH Fact sheet No297 2013. who.int/mediacentre/factsheets/
2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70.
3. Farnsworth, N. R. in Biodiversity Wislson, E. O Ed., National Academy Press Washington,  DC 1988, pp. 83 -97.
4. World Health Organization. NMH Fact sheet No134 2008. who.int/mediacentre/factsheets/
5. Chinedu, E., Arome, D and Solomon F.A. Herbal Plants a Reliable Source for Drug Discovery and Development. PHARMATUTOR-ART-2017. Available online at https://www.pharmatutor.org/articles/herbal-plants-reliable-source-drug-discovery-development. Accessed on December 7th, 2013.
6. Chindo, B. A., Anuka J. A. and Gamaniel K. S. Toxicity Screenings of Ficus platyphylla Stem Bark in Rats. Pharmacologia, 2012; 3: 499-505
7. Ayinde, B.A, Omogbai, E.K.I and Ikpefan E.O. Comparative Cytotoxic and Antiproliferative Effects Of Persea americana mill (Lauraceae) Leaf, Stem And Root Barks. Nigerian Journal of Pharmaceutical Sciences, 2011;Vol. 10, No.1, , ISSN: 0189-823.
8. Pezzuto, J. M. Biochemical Pharmacology  1997, 53, 121-133
9. Allison, B., Fontaine, R., Heshka, S., Mentore, L. and Heymsfield, B. Alternative treatments for weight loss: A critical review. Critical Review of Food Science and Nutrition 2001; 41:1–28.
10. McLaughlin, J.L., Chang, C and Smith D.I. Bench-top bioassays for the discovery of bioactive natural products: an update. In: Atta-ur- Rahman, editor. Studies in Natural Products Chemistry. Amsterdam: Elsevier Science Publishers B.V, 1991; 9:383-409.
11. David, A., Chinedu. E and Ameh, F.S. Evaluation of the Anti-proliferative Activities of Aqueous Leaf and Root Extracts of Cymbopogon citratus. J Pharm BiolSci, 2013; 1 (5): 56-60.
12. Shogbaike, D.A., Ogundaini, A.O and Adesanya S.A.  The effects of some synthesized stilbene analogues on Artemia salina  naupalii and germination of Sorghum bicolor seeds. Nig. J. Nat. Prod. and Med., 2002;6: 19-25.
13. Ayinde, B.A and Agbakwuru, U. Cytotoxic and growth inhibitory effects of the methanol extract Struchium sparganophora Ktze (Asteraceae) leaves. Phcog  Mag. ,2010; 6 (24): 293297.

NOW YOU CAN ALSO PUBLISH YOUR ARTICLE ONLINE.

SUBMIT YOUR ARTICLE/PROJECT AT articles@pharmatutor.org

Subscribe to Pharmatutor Alerts by Email

FIND OUT MORE ARTICLES AT OUR DATABASE