Evaluation of Selected Nigerian Medicinal Plants for Antioxidant, Antimicrobial and Cytotoxic activities

Background: Increased exposure to pathogens and free radicals contributed to the high incidence and mortality rate of various cancers in Nigeria and globally. Promotion of scientic research on medicinal plants in collaboration with traditional health practitioners to validate claims made on safety, ecacy and quality of traditional medicinal plants in the treatment of cancer is imperative. This study aims at screening extracts of selected Nigerian medicinal plants used traditionally for cancer treatment for antioxidant, antimicrobial and cytotoxic activities towards identication of potential source of new anticancer agents. Methods: Twenty-one extracts from sixteen medicinal plants species were screened for their cytotoxicity on RD, HeLa and Hep-2 cancer cell lines using MTT assay. The DPPH radical scavenging activity of the extracts as well as their Total Phenolic Content (TPC) were evaluated. The extracts were also evaluated for their antimicrobial activity using spectrophotometric growth inhibition method. Results: Extracts of Tetrapleura tetrapleura and Xylopia aethiopica showed high DPPH inhibitory activity and phenolic content. Extracts of X. aethiopica and Anchomanes difformis showed broad spectrum of antibacterial activities while root extracts of Crotolaria retusa and T. tetraptera exhibited antifungal activities comparable (P<0.05) to Ketoconazole. Extracts of Capcicum frutescens, Aspilia africana, X. aethiopica, T. tetraptera and C. retusa showed broad spectrum of cytotoxic activities. Conclusions: Extracts of T. tetraptera and X. aethiopica demonstrated satisfactory activities in all the biological tests which could be linked to their high phenolic contents. The ndings support the ethnomedicinal uses of most of the tested medicinal plants.


Background
Cancer remains one of the leading cause of death worldwide with an estimate of 18.1 million new cancer cases and 9.6 million cancer deaths (1). Several factors have been found to be associated with the development of cancer but notable are the actions of free radicals and pathogenic microorganisms (2,3).
Studies have shown that reactive free radicals interact with macromolecules such as DNA in the cell, leading to damages to cell structure and functions (4). In addition pathogenic microbes have been found to be associated with some kind of cancer (5).
Cancer treatment faces challenges of resistance, toxicities to normal cells and ineffectiveness of some anticancer agents (6)(7)(8)(9). This has necessitated the search for new anticancer agents from medicinal plants. Studies have shown that plants are rich in phenols which are able to attenuate the actions of free radicals (10) and are able to elicit antimicrobial activities (11,12). In addition, plants are also veritable source of anticancer agents (13)(14)(15).
The dependence of vast majority of African populace on medicinal plants for healthcare needs (16) formed the basis for the inquiry into plants used traditionally in the treatment of cancer in South Western Nigeria. Retrieving information on the pharmacological relevance of medicinal plants is often achieved through the conduction of ethnomedicinal surveys (17). An ethnomedicinal survey was carried out in Ile-Ife, Osun state, Nigeria between June to December 2017. From the survey, twenty-one extracts from sixteen plants species were selected for this study. This study aims at screening the selected plant extracts for antioxidant, antimicrobial and cytotoxic activities towards identi cation of potential source of new anticancer agents.

Plant material
The plant parts used for this study (Table 1) were collected and identi ed at the botanical garden of the University of Ibadan, Ibadan, Nigeria. Authentication of the plant material was done at the herbarium of the Forest Research Institute of Nigeria, Ibadan Nigeria, where voucher specimens were also deposited.
The plant materials were dried at room temperature and pulverized into powder. Freshly crushed endosperm of Cocos nucifera was used in this study.

Extraction
Each plant material (200 g) was macerated in 80% methanol for 78 h at room temperature. Crushed endosperm of Cocos nucifera (200 g) was extracted in n-hexane. Extracts were ltered through lter paper (Whatman No. 1) and concentrated to dryness in vacuo.

DPPH radical scavenging assay
The free radical scavenging activities of the extracts against 2,2-diphenyl-1, picrylhydazyl (DPPH) was evaluated following the method of (18) with slight modi cation. Extracts and ascorbic acid were made into various concentrations (1.56, 3.13, 6.25, 12.5, 25, 50 and 100 µg/mL) in methanol while a 0.004% (w/v) solution of DPPH in methanol was freshly prepared. Each concentration of the extract (1 mL) was mixed with 3 ml of the DPPH solution and incubated in the dark for 30 min at 27± 2 C. In the control experiment, 1 mL of methanol was used in the place of the extracts. Absorption of the reaction was measured at 517 nm. The experiment was repeated three times. The concentration at which there is 50% inhibition of the DPPH • (IC 50 ) was determined using Graph pad prism (5.0) while the percentage inhibition of the extracts was calculated using the formula; % Inhibition = Absorbance of control -Absorbance of test sample x 100 Absorbance of control Total phenolic content (TPC) assay The total phenolic content of the extracts was determined using Folin -Ciocalteu (FC) reagent following the method of (19) with slight modi cation. The extracts were made into 100 µg/mL while 10% FC (v/v) in methanol was freshly prepared. The FC reagent (25 µL) was added to 50 µL of the extracts in 96well plates and allowed to stand for 3 min. For the blank, methanol was used in place of the extracts. A solution of 7.5% Na 2 CO 3 (125 µL) was added into each well and afterwards incubated in the dark for 2 h at 25 ± 2 C. The absorbance was recorded with Thermo Fisher Scienti c microplate reader at 758 nm. The experiment was carried out in triplicates. The total phenolic content was therefore expressed as Gallic acid equivalents (GAE) (18).
In vitro antimicrobial assay

Test organisms
Reference bacterial and fungi strains were obtained from the Department of Medical Microbiology and Parasitology, University College Hospital, Ibadan Nigeria. The bacterial strains include: Escherichia coli ATCC 25923, Pseudomonas aeruginosa ATCC 10145, and Salmonella typhi ATCC 24683 while the fungi strain used was Candida albicans ATCC 24433. Nutrients broth and sabouraud dextrose broth was used for the maintenance of the bacterial and fungal strains respectively at 4 C.

Preparation of inoculums
A small piece of colony from a day-old cultures of each test organisms was adjusted to cell density of 1x10 8 CFU/mL in sterile distilled water using McFarland Standard No. 0.5.

Spectrophotometric growth inhibition method
The method of (20) was adopted with some modi cations. Extracts and standard drugs (streptomycin and ketoconazole) were made into concentrations 1000, 500, 250, 125, 62.50, 31.25 and 15.63 µg/mL in freshly prepared nutrient or sabouraud dextrose broth. An aliquot of 75 µL of each test concentration was gently mixed together with 75 µL of the inoculum in wells of 96 -well plate. Sterile distilled water was used in the control experiment. The absorbance at 540 nm was taken before and after 24 h of incubation at 37 C. Differences in optical densities were taken as microbial growth index. The experiment was carried out in triplicates. The concentration at which there is 50% microbial inhibition (IC 50 ) was CC 50 values of all test samples were determined with nonlinear regression plot of log (cytotoxic concentration) against normalized percentage cytotoxicity. One-way at P < 0.05 followed by Tukey's test was used test for the signi cant difference between the extracts and the standard drugs.

Results
The antioxidant activities of the extracts were assessed by evaluating their inhibitory activities against DPPH. Except for bark extract of T. tetrapleura with an IC 50 of 4.59 µg/mL and root extract of X.
aethiopica with an IC 50 of 6.01 µg/mL, the inhibitory activities of other extracts are signi cantly (P<0.05) different from ascorbic acid ( Table 2).
Phenols are class of compounds with proven antioxidant activities, therefore samples with high phenol contents often exhibit antioxidant activities. Among all tested extracts, leaf extracts of Nicotiana tabacum and P. osun, bark extract of T. tetraptera and root extract of X. aethiopica had the highest phenolic contents ( The antimicrobial study showed that the extracts exhibit inhibitory activity against tested bacterial and fungal strains ( Table 3). The root and bark extracts of X. aethiopica and leaf extracts of A. difformis, Morinda lucida and Pterocarpus osun displayed comparable (P<0.05) antibacterial inhibitory activity against Salmonella typhi when compared to Streptomycin. All tested extracts were active against Pseudomonas aeruginosa, however, none was comparable (P<0.05) to the activity of Streptomycin with IC 50 of 0.9 µg mL -1 . Similarly, A. difformis, bark extract of X. aethiopica elicit comparable activities against E. coli. Root extracts of X. aethiopica and leaf extract of A. difformis showed broad spectrum of antibacterial activities. Antifungal studies against Candida albians showed that root extracts of Crotolaria retusa, Tetrepleura tetraptera and Ketoconazole exhibited comparable (P<0.05) antifungal activities.
Extracts were evaluated for their cytotoxicity effects on human larynx epithelioma (Hep 2), Human Rhabdomyosarcoma (RD) and cervical adenocarcinoma (HeLa) cell lines. MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay was used to monitor viable cells after treatment with extracts. Most extracts were active against Hep 2, yet, extracts of Aspilia africana, T. tetraptera and N. tabacum were the most cytotoxic with IC 50 of 1.3, 1.7 and 2.9 µg/mL respectively which were comparable (P<0.05) to the standard drug (Table 4). On RD cell line, the cytotoxicity of the standard drug was comparable to that of Culcasia scanden, Capsicum frutescens and root extract of X. aethiopica with IC 50 of 0.9, 1.6 and 1.6 µg/mL, respectively. Only the bark extract of X. aethiopica demonstrated comparable (P<0.05) cytotoxic activity against HeLa cell line when compared with standard drug. Extracts of C. frutescens, A. africana,X. aethiopica,T. tetraptera and Crotolaria retusa showed broad spectrum of cytotoxic activities against the tested cell lines.

Discussions
Medicinal plants are very important in health care delivery in developing nations of the world (16). Rising prevalence and mortality of cancer and related diseases necessitated sourcing for treatment alternatives from medicinal plants (22). Bioactive compounds in extracts of medicinal plants are responsible for the diverse pharmacological activities demonstrated by these plants (23,24). Identifying potential medicinal plants for cancer treatment is often achieved through information retrieved from traditional health practitioner (TMPs) (17). This present study seeks to scienti cally justify the ethnomedicinal use of sixteen medicinal plant species used in the treatment of cancer and microbial infections.
Free radicals are proven contributor to the development and progression of most disease conditions via their damaging effects on macromolecules including proteins, DNA and RNA (25). Antioxidants on the other hand inhibits the actions of free radicals and therefore could potentially prevent the onset and progression of these diseases (26). Previous reports showed that Nigerian medicinal plants elicit antioxidant activities (18,26). In our study, the bark extract of T. tetraptera had the highest inhibitory activity against the DPPH radical as well as very high total phenolic content. Phenolic compounds have been widely studied for their biological activities and are known to contribute to the antioxidant activity of extracts (10,18,19,27). It could be safely posited that phenolic compounds of T. tetraptera are partly responsible for its antioxidant activity as well as other reported biological activities such as hypoglycemic, antimicrobial, anti-in ammatory and antilipidemic activities (28). Other extracts including bark and root extracts of X. aethiopica and root extract of T. tetraptera showed high total phenolic contents which also correspond to their high inhibitory activities against DPPH radical.
In addition to free radicals, there are various evidences linking chronic microbial infections with cancer. Salmonella typhi for example has been linked to the development of cancer of the gall bladder (29) while Helicobacter pylori has been linked to gastric cancer (5). Several reports demonstrated the antimicrobial potentials of medicinal plants. Phenolic compounds in extracts of medicinal plants contribute to their antimicrobial activities going by their damaging effect on the cell membrane and disruption of metabolism and synthesis of nucleic acids (30).
Results showed that the study plants elicited antimicrobial activities against S. typhi, P. aeroginosa, E. coli and C. albicans. Root extracts of X. aethiopica and leaf extract of A. difformis elicit broad spectrum of antibacterial activities. Fruit extract of X. aethiopica and derivative of its major constituent had earlier been demonstrated to elicit antimicrobial activities (31,32). Our ndings demonstrated that the root and bark extracts of X. aethiopica equally hold antimicrobial properties, and considering their high phenolic content, phenolic compounds might be responsible for the observed activities.
As with the reports of (22), (26) and (21), extracts of this study showed varying cytotoxic activities against tested cell lines. According to National Cancer Institute (NCI) on screening of medicinal plants for cytotoxic activities, plant extracts with CC 50 < 30 µg/mL are considered active (22). Based on the NCI standard, nineteen extract were active against at least one cancer cell line, while ten extracts were active against at least two cancer cell lines. Only six extracts were cytotoxic to all the cancer cell lines.
Our study also showed that bark and root extracts of T. tetraptera demonstrated cytotoxicity against all tested cancer cell lines. (22) reported the cytotoxicity of extract of T. tetrapleura against breast (BT-549) cancer cell line. Similarly, in vitro and in vivo models of (37) also demonstrated the cytotoxicity of fruit extract of T. tetraptera against Ehrlich Ascites tumour cells. Bioactive coumarin, saponins, terpenes and some phenolics have been isolated from extracts of this plants (28). The high phenolic content of T. tetraptera extract as shown in this study might contribute to its cytotoxicity, however, further works will aim at identifying the active cytotoxic compounds.
Stem extract of Crotolaria retusa was found to be more cytotoxic than leaf, seed, pod and ower extracts of the plant although in an un-selective manner (38). We report the cytotoxicity of its root extract to Hep 2, RD and HeLa cell lines. Similarly, we found leaf extract of Aspilia africana to be cytotoxic against the three tested cell lines. (39) reported the cytotoxicity of leaf extracts obtained using various green methods of extraction against AGS, A549, and HeLa cell lines. Gallic acid, chlorogenic acid, syringic acid, ferruric acid, and quercetin were detected in the most active extract of A. africana.
Capsicum frutescens elicited broad spectrum of cytotoxicity against all tested cell lines. Its fruits, commonly used in African cuisine is widely studied for its nutritional and health bene ts (40). Although evidences suggest that capsaicin, a constituent of the fruits is a human carcinogen (41), however the same compound has been reported to demonstrate cytotoxic and cancer prevention potentials (40). A more recent report demonstrated that capsaicin and piperine from Piper nigrum could reverse resistance of cancer cells to doxorubicin (42). Other notable cytotoxic extracts are the bark extracts of Pterocarpus osun, leaf extract of Culcasia scandens and Pativera alliaceae which were cytotoxic to at least two cancer cell lines.

Conclusions
In this study, we evaluated the antimicrobial, antioxidant and cytotoxicity of extracts of medicinal plants used traditionally in the treatment of cancer in South Western Nigeria. Results from the study justi es the traditional use of these extracts in the treatment of cancer and accompanying conditions. Extracts of T. tetraptera and X. aethiopica demonstrated satisfactory activities in all the biological tests which could be linked to their high phenolic contents. Further puri cation might potentiate their pharmacological effects which will favour their pharmaceutical application in the development of anticancer and antimicrobial therapeutics. Our current endeavour is devoted towards unraveling mechanism of action as well as identifying the active constituents present in the extracts of these plants.

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Ethics approval and consent to participate: Not applicable Consent for publication: All Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.