In vitro antiproliferative activities of some Ghanaian medicinal plants

Background Cancer continues to pose a significant threat to human well-being due to the overwhelming rate of morbidity and mortality associated with it. Hence, the quest for newer, effective and safer anticancer agents has become more crucial. Over the years, some medicinal plants have been used to treat abnormal tissue growths (tumours) in Ghana. Even though sufficient literature points out that people found some relief in their use, there is limited scientific evidence of their antiproliferative activities. Method Ethanolic extracts of nine medicinal plant materials from seven plant species, including the stem bark of Terminalia superba , Talbotiella gentii and Ceiba pentandra and the leaves of Morinda lucida , Dracaena arborea , Dioscorea dumetorum , Thaumatococcus danielli , Ceiba pentandra and Talbotiella gentii , were evaluated for antiproliferative activities against four human cancer cell lines (hepatocellular carcinoma, colorectal adenocarcinoma, cervical carcinoma, and mammary adenocarcinoma) using an MTT-based assay. Results The extract of C. pentandra leaves, exhibited generally higher antiproliferative activity, which was particularly substantial against human hepatocellular carcinoma (HepG2) cells (IC 50 = 16.3 µg/mL) and human colorectal adenocarcinoma (RKO) cells (IC 50 = 18.7 µg/mL). All the other plant materials demonstrated weak (IC 50 : 201–500 µg/ mL) to moderate (IC 50 : 21–200 µg/mL) antiproliferative activities against the four cancer cell lines. Conclusion The extracts of the plant materials demonstrated varied antiproliferative activities. Extract of C. pentandra leaves exhibited the highest antiproliferative activity. The IC 50 values of C. pentandra leaves met the benchmark to be considered effective against HepG2 and RKO cancer cell lines in particular. Therefore, there is the need to further undertake fractionation work on C. pentandra leaves. The antiproliferative effect of extract of C. pentandra leaves against other cancer cell lines and normal cell line could also be explored in the future to ascertain the anticancer potential of this plant material. Generally, findings from this work support the indigenous use of these plant materials in treating abnormal tissue growth in Ghana.


Introduction
Cancer is a broad term describing a group of diseases characterised by abnormal cell growth and progression, usually beyond their natural boundaries, that can invade nearby body parts or spread to other tissues [1].Cancer is a significant health concern and one of the leading causes of mortality.It is the second leading cause of disease and mortality globally [2,3].In 2022, there were an estimated 20 million new cases and nearly 10 million deaths attributed to cancers [4].Although several drugs have been developed over the years to combat this deadly disease [5][6][7], the development of resistance and the reported adverse drug reactions associated with their use [8][9][10][11] have hindered treatment success, which has led to the continuous search for new agents.The frightening projection of a 57% surge in cancer-related morbidity and mortality by the year 2032 has also amplified the interest in newer effective, and safer antitumour agents [12].
Ghana is endowed with numerous plants of medicinal value [24,25].Several herbalists and traditional healers use these plants to treat or manage various diseases, including abnormal tissue growth [24,25].Plants such as Morinda lucida, Talbotiella gentii, Dracaena arborea, Dioscorea dumetorum, Thaumatococcus danielli, Ceiba pentandra, and Terminalia superba have been cited in various studies to be used in folkloric management of tumours in Ghana [26].
Previous studies on the antiproliferative properties of some of these plant species have pointed to their anticancer potential.Morinda lucida, for instance, has been reported by Appiah-Opong et al. [27] to induce apoptosis in human promyelocytic (leukemic) HL-60 cell line.Young et al. [28] also reported the ability of methanol extract of Dracaena arborea to decrease the cell viability of metastatic human breast cancer cells (MDA-MB-231).The cytotoxic potential of bark extracts of Ceiba pentandra has also been reported against Ehrlich ascites carcinoma (EAC), and human mammary adenocarcinoma (MCF-7) cells [29].Despite these encouraging findings, significant gaps still exist as most of these studies have focused on one cell line.Additionally, there is a dearth of scientific work on several of these plant materials regarding their antiproliferative activities.Therefore, this study seeks to evaluate the aforementioned plant species for their antiproliferative activities on a panel of human cancer cells.This evaluation will help to support their traditional use for treating abnormal tissue growth by traditional healers or herbalists.

Cell lines
The cell lines included the human cancer cell lines: HepG2 (human hepatocellular carcinoma), RKO (human colorectal adenocarcinoma), Hela (human cervical carcinoma) and MCF-7 (human mammary adenocarcinoma).All the cell lines were generously donated by Prof. Mohamed Mutocheluh of the Virus Research and Molecular Biology Laboratory of the Clinical Microbiology Department of the School of Medical Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Ghana.

Cell culture
A working medium was prepared from DMEM reconstituted by adding 1% NEAA, 1% Penicillin/Streptomycin and 10% FBS.The cancer cell lines (HepG2, RKO, Hela and MCF-7) were cultured in the working medium.The cells were incubated in an incubator maintained at 37 ºC and 5% CO 2 under relative humidity of 95%.The continuous logarithmic growth of the cells was ensured by subculturing the cells at least twice a week.The cells at about 80% confluence were washed with PBS, detached using trypsin and collected for centrifugation.After centrifuging at 200 x g for 5 min, the cells were resuspended in the working medium.The enumeration of cells was achieved using a haemocytometer, and the viability of cells was determined using trypan blue exclusion.Eppendorf 96-well TC-treated cell culture plates were used to seed cells at 4 × 10 4 cells/100µL per well.

Plant materials collection and authentication
Nine plant materials (viz., C. pentandra bark, C. pentandra leaves, D. dumetorum leaves, D. arborea leaves, M. lucida leaves, T. gentii bark, T. gentii leaves, T. superba bark and T. danielli leaves) were collected from different parts of Ghana (Akosombo, Kwahu Bepong, and Mampong-Akwapem).Prof. Gustav Komlaga from the Department of Pharmacognosy, and Dr. George Henry Sam from the Department of Herbal Medicine, both of the Faculty of Pharmacy and Pharmaceutical Sciences, KNUST, authenticated the plants.Herbarium specimens with voucher numbers (Table 1) were deposited in the Herbarium of the same Faculty.

Preparation of plant extracts
The plant materials were rinsed with tap water and airdried at room temperature for three weeks.The dried plant materials were then ground into coarse powder.Each powdered material (2000 g) was placed in a glass jar, and 80% ethanol was added.The mixture was left for 72 h, and occasionally gently stirred.It was subsequently filtered using Whatman No. 1 filter paper.The process was performed thrice to ensure that all constituents had been extracted.The filtrate was then concentrated using rotary evaporator (R-114, Buchi, Switzerland).Drawell freeze drier (DW-10 N) was used to concentrate the sample further by removing the water.The dried extract was kept in a well-closed glass bottle and put in the refrigerator at 4ºC until required.The extract of C. pentandra leaves which exhibited the highest antiproliferative properties was further fractionated.The extract was first suspended in water and then extracted with petroleum ether (PE).The resultant water fraction was further extracted sequentially with ethyl acetate (EtOAc) and n-butanol (n-BuOH).The four different fractions obtained (PE, EtOAc, n-BuOH, and water fraction) from the fractionation process were dried, weighed and kept in a wellclosed glass bottle and put in the refrigerator at 4 ºC until required.

Preparation of test solutions
Stock test solutions of crude extracts (1 mg/mL) were prepared in 1% dimethylsulfoxide (DMSO).The positive control, 5-fluorouracil (5FU) solution, was also prepared in a similar way.The negative control was prepared from 2 µl of DMSO and 1998 µl of working medium (reconstituted DMEM).Each treatment stock solution of the crude extracts was diluted with the working medium to give the different concentrations (200 µg/mL, 100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL and 1 µg/mL).Following the confirmation of C. pentandra leaves as significantly bioactive, a time-dependent study was conducted on the extract by assessing its antiproliferative activities after 24 h, 48 h and 72 h of treatment at concentrations between 3.125 and 50 µg/mL.Using the working medium, the 1 mg/mL stock solution was diluted to give a concentration of 50 µg/mL.The serial dilution method was then used to prepare the other required concentrations (that is, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, and 3.125 µg/mL).Similar approach was employed in the determinations of the antiproliferative activity of the four fractions of C. pentandra leaves obtained during the fractionation process.In all cases, the solutions (that is, test solutions, positive and negative control) contained ≤ 0.1% of DMSO which has been demonstrated to have no effect on the growth of cells [30].

Antiproliferative study
Cell viability was determined using MTT-based assay [31].Cells seeded in 96-well plates were incubated at 37 °C and 5% CO 2 under humid conditions for 24 h for the cells to attach.The old medium was discarded, and cells were incubated in 100 mL of fresh medium containing test crude extracts at a concentration of 1 µg/ mL to 200 µg/mL for all the plants' crude extracts and 3.125 µg/mL to 50 µg/mL for the C. pentandra fractions.All tests were performed in triplicate.The positive (5FU) and negative (DMEM) controls were also done in triplicate.This was then incubated for 72 h.To determine the time-dependent effect of extract of the active plant material (that is, C. pentandra leaves), the treated cells were incubated for 24 h, 48 h and 72 h.After incubation, 20 µl of MTT reagent was added to each well, and the plate was incubated for another 3 h.The supernatant was then aspirated from each well, and 120 µl of isopropanol was added, with three of the wells serving as blank and sterility control.The plate was kept in the incubator for 30 min for the formazan crystals to dissolve.The absorbance was read on iMark Microplate Reader (Bio-Rad, USA) with the measurement filter set at 595 nm.The procedure was repeated two more times.

Data analysis
The data were analysed using GraphPad Prism (version 8.0.2).Cell viability was determined by the ratio of cells cultured in the presence of extracts or fractions to cells cultured in the absence of extracts or fractions (cells were mock-treated with DMSO).Comparisons of IC 50 values to 30 µg/mL were done using a one-sample t-test.All other comparisons between treatments were conducted using the one-way ANOVA and Tukey's test for post-hoc analysis.In all comparisons, p < 0.05 was considered as statistically significant.

Results
Extracts from the nine plant materials used by traditional healers in Ghana to treat abnormal cell growth were evaluated for their antiproliferative activities using an MTT-based assay.The plant samples demonstrated a wide range of activities on cell viability, as shown in Fig. 1A and B. All the plant extracts inhibited cell growth in a dose-dependent manner with significant differences as shown in Fig. 1A and B and Supplementary Data S1.
As summarised in Table 2, only one plant material, C. pentandra leaves demonstrated appreciable antiproliferative properties of IC 50 between 16 µg/mL and 42 µg/mL.The remaining plant samples produced moderate to weak antiproliferative activities.As shown in Fig. 1A and B, the effects of the various extracts on cell viability were concentration dependent.Extracts from C. pentandra bark, D. arborea leaves, M. lucida leaves, and T. danielli leaves showed moderate antiproliferative properties against the cell lines with IC 50 values ranging from 76 µg/mL to 193 µg/mL.Extracts from D. dumetorum leaves, T. gentii leaves and bark, and T. superba bark, on the other hand, showed moderate antiproliferative properties against most of the cell lines.However, T. superba bark, T. gentii leaves, and D. dumetorum leaves displayed weak toxicity (IC 50 > 200 µg/mL) against Hela, HepG2, and MCF-7 cell lines respectively.
Extracts of C. pentandra leaves demonstrated the highest activity against all the cell lines with IC 50 values of 16.3 µg/mL, 18.7 µg/mL, 41.6 µg/mL, 30.2 µg/mL against HepG2, RKO, Hela and MCF-7, respectively.The IC 50 values were found to be significantly lower against HepG2 (p < 0.001) and RKO (p < 0.01) when compared to the American National Cancer Institute (NCI) threshold of < 30 µg/mL.
The cell viability of the extract of C. pentandra leaves was determined after 24 h, 48 h and 72 h of treatment.As shown in Fig. 2, the test materials demonstrated good inhibitory effect after 24 h, 48 h and 72 h of treatment against the HepG2 cell line.Results showed a time-dependent antiproliferative effect which was not statistically significant (p = 0.3430).Table 3 provides the IC 50 values obtained for the test.The test material demonstrated IC 50 values ranging from 35.6 ± 1.6 to 71.4 ± 3.8 after 24 h of treatment, 21.5 ± 0.9 to 57.4 ± 4.5 after 48 h of treatment and 16.1 ± 0.6 to 40.9 ± 3.1 after 72 h of treatment.
Figure 3A and B provide the percentage inhibition of the various fractions against HepG2 and Hela cell lines.Inhibition of cell viability by the different fractions of C. pentandra leaves showed varied results.All four fractions inhibited cell growth in a dose-dependent fashion with significant differences as shown in Fig. 3A and B and Supplementary Data S2.Ethyl acetate fraction demonstrated the highest inhibition of cell viability against both cell lines.As shown in Table 4, ethyl acetate fraction had the lowest IC 50 values (that is, exerted the highest inhibitory effect), with HepG2 being the most susceptible.The IC 50 values were significantly lower against HepG2 (p < 0.01) and Hela (p < 0.05) when compared to NCI threshold of < 30 µg/mL.

Discussion
The level of antiproliferative activity has been classified as high, moderate, weak or no toxicity.This was based on protocols established by the American National Cancer Institute (NCI) and the Geran Protocol [32,33], stated as follows: No effect = IC 50 ≥ 501 µg/mL; Weak effect = IC 50 201-500 µg/mL; Moderate effect = IC 50 21-200 µg/mL; and High effect = IC 50 ≤ 20 µg/mL.With the exception of extract of C. pentandra leaves, which demonstrated high antiproliferative activity (IC 50 ≤ 20 µg/mL) against two cell lines (Table 2), all other plant extracts exhibited weak or moderate antiproliferative activity against the four established cancer cell lines used.
The IC 50 values of M. lucida leaves extract were within the range of 100 to 185 µg/mL for the different cancer cells (HepG2, RKO, Hela and MCF-7), which indicated that the extract was moderately antiproliferative.The results contradicted reports on other Morinda species with substantial antiproliferative activities.The leaves of another species, M. tinctoria, have been shown to demonstrate marked activity [34].Similarly, Noor and coworkers reported a higher antiproliferative effect (IC 50 39.9 µg/mL) of M. citrifolia leave extract on MCF-7 [35].
Other previous studies have also demonstrated that four monoterpenes isolated from M. morindoides leaves exhibited marked antiproliferative activity (<10 µg/mL) against MT-4 (human T-cell) cells [36], while damnacanthal and nordamnacanthal isolated from the roots of M. elliptica have been shown to possess marked antiproliferative activity against HL-60 and Wehi-3B (myelomonocytic leukaemia) cell lines [37].The extract of T. gentii leaves produced IC 50 values ranging from 129.9 to 201.5 µg/mL against the cancer cells, while the bark produced IC 50 values between 105 and 300 µg/mL.To the Dracaena arborea showed moderate antiproliferative activity against all four cancer cell lines with IC 50 values of 89.9 to 163.4 µg/mL.Methanol extract of D. cinnabari has been reported to display high antiproliferative activity against oral squamous cell carcinoma (H103) cell lines [38].A homoisoflavonoid isolated from another Dracaena species (D. cambodiana) has also been reported to have substantial activity (IC 50 = 1.4 µg/mL and 2.9 µg/ mL) against K562 (chronic myelogenous leukemia) and SGC-7901 (human gastric cancer) cells, respectively [39].The results of the effect of leave extract of D. arborea on HepG2 and Hela cells offered some evidence for the traditional healing practices associated with this plant material.D. dumetorum leaf extract also showed moderate antiproliferative activity (IC 50 of 76.3 to 132.5 µg/mL) against RKO, MCF-7 and HepG2 cells while displaying weak toxicity (IC 50 = 245.1 µg/mL) against Hela cells.The   results were, however, at variance with similar studies on other Dioscorea species, which reported high antiproliferative activity (IC 50 = 19.77µg/mL) for D. bulbifera against CCRF-CEM (acute lymphoblastic leukemia) cell line [40].
Thaumatococcus danielli leaves exhibited moderate antiproliferative activities (IC 50 of 129.1 to 193.0 µg/mL) against the selected panel of cancer cell lines.Although significant antioxidant activity has been reported for the seeds [41] and the leaves [42,43] of T. danielli, there is a dearth of published work on the antiproliferative activity of any part of this plant.Similarly, published reports on the other species of Thaumatococcus (T.flavus) concerning its antiproliferative properties are scanty.T. superba stem bark was another medicinal plant material evaluated in the present study that demonstrated weak to moderate antiproliferative effects (IC 50 between 117.5 µg/ mL and 206.4 µg/mL) against the cancer cells.Our finding is similar to other researchers, such as Fyhrquist et al., who reported a wide range of antiproliferative effect (IC 50 range of 18.0 µg/mL to 250.0 µg/mL) for other species of Terminalia [44].The findings of Spiegler and coworkers also revealed that the extract of T. superba stem bark had either no activity or weak activity [45].
Although the bark of C. pentandra demonstrated moderate antiproliferative activity (IC 50 = 108.8± 1.8 to 162.9 ± 2.7), its leaves exhibited the highest antiproliferative activities (IC 50 of 16.3 to 41.6 µg/mL) against the four established cancer cell lines with HepG2 (IC 50 = 16.3 µg/ mL) and RKO (IC 50 = 18.7 µg/mL) cells being the most susceptible.These findings were consistent with earlier studies in which the stem bark of C. pentandra revealed antiproliferation potential against EAC (Ehrlich ascites carcinoma), MCF-7 and B16F10 (murine melanoma) cells [29].The aerial part of C. pentandra had also been reported to show substantial antiproliferative activity against HepG2 and MCF-7 cell lines with IC 50 of 14.895 µg/mL and 18.859 µg/mL, respectively [46].The antiproliferative potency of the extract of C. pentandra leaves is further demonstrated by its ability to substantially inhibit cell growth after 48 h and 24 h of treatment.
The results of C. pentandra leaves extract were within the criteria established by the American National Cancer Institute, which states that, for a crude extract to be considered promising for further work, it must show IC 50 < 30 µg/mL.Therefore, the C. pentandra leaves could be considered as a good candidate for further work since it demonstrated significantly lower bioactivity against HepG2 (IC 50 = 16.3 µg/mL, p < 0.001) and RKO (IC 50 = 18.7 µg/mL, p < 0.01) when compared to 30 µg/ mL.The findings also give credence to its use by traditional healers and herbalists for the treatment of cancerlike diseases.
The extract of C. pentandra leaves was further fractionated, yielding four fractions which were assessed for their antiproliferative activity.The PE fraction demonstrated the least effectiveness against all four cell lines with IC 50 values greater than 50 µg/mL.The most active fraction was the ethyl acetate fraction with appreciable effectiveness (IC 50 between 10.7 ± 1.1 µg/mL and 68.62 µg/ mL).This is consistent with the work of Orabi and his co-workers, who demonstrated the effectiveness of ethyl acetate extract of C. pentandra against diethylnitrosamine-induced hepatocellular carcinoma in rats [47].One of the most important biological events in the development of malignancies is oxidative stress [48,49].It has been shown that the ethyl acetate fraction of C. pentandra possesses a very high level of antioxidant activity and can significantly reduce oxidative stress [50].Therefore, the tremendous antiproliferative effect of the ethyl acetate fraction found in this study may have resulted from its capacity to decrease oxidative stress.

Conclusion
The study concluded that C. pentandra leaf extract exhibit high antiproliferative effect with HepG2 and RKO cells being the most susceptible.Extracts of M. lucida, C. pentandra bark, T. danielli leaves and D. arborea leaves demonstrated moderate antiproliferative activity while the other plant materials displayed varied antiproliferative activities.The findings of the present study therefore support the indigenous use of these medicinal plants in treating abnormal tumour growth in the various communities in Ghana.Furthermore, the ethyl acetate fraction of C. pentandra leaves was found to demonstrate a high antiproliferative effect against HepG2 and Hela cancer cells.It is therefore essential to undertake isolation work on the ethyl acetate fraction in order to further identify the bioactive compounds that are responsible for the antiproliferative activity observed in C. pentandra leaves.Identification of bioactive compounds and the determination of their mechanisms of action will help to better understand the therapeutic potential of C. pentandra leaves.

Fig. 1 A
Fig. 1 A representative graph of the inhibitory effects of plant extracts on HepG2 (A) and RKO (B) cells.The cells were treated with plant extracts (1-200 µg/mL), and the cell viability was assessed at 72 h using an MTT-based assay.Data are presented as the means, with the standard deviations as error bars, from three different experiments, each conducted in triplicate.a p < 0.05, b p < 0.01, c p < 0.001, and d p < 0.0001 correspond to significant difference between the untreated and treated cells as calculated by one-way ANOVA and Tukey's test, ns: difference not significant.The comparisons within the treatment are indicated in the supplementary data.Data for positive control (5-fluorouracil) are presented for comparison

Fig. 2 AFig. 3 A
Fig. 2 A representative graph of time-dependent inhibitory effects of ethanolic extracts of C. pentandra leaves on HepG2 cells.The cells were treated with the ethanolic extract (3.125-50 µg/mL), and the cell viability was assessed at 24, 48 and 72 h using an MTT-based assay.Data are presented as the means, with the standard deviations as error bars, from three different experiments, each conducted in triplicate.There was no statistical difference in the viability at the various time intervals (p = 0.3430), as calculated by one-way ANOVA

Table 1
Medicinal plants screened for antiproliferative activities

Table 2
IC 50 values of different plant extracts on the various cancer cell lines -IC 50 values are presented as means and standard deviations of three independent experiments, with each experiment conducted in triplicate.Using a one-sample t-test, IC 50 values were compared to 30 µg/mL.*p < 0.05, **p < 0.01 and ***p < 0.001 correspond to significant differences a

Table 3
IC 50 values of C. Pentandra leaves extract on cancer cell lines at different times

Table 4
IC 50 values of C. Pentandra leave fractions on the various cancer cell lines Aqueous -IC 50 values are presented as means and standard deviations of three independent experiments, with each experiment conducted in triplicate.Using a one-sample t-test, IC 50 values were compared to 30 µg/mL.*p < 0.05 and **p < 0.01 correspond to significant differences a