Ethiopian Medicinal Plants Traditionally Used for the Treatment of Cancer, Part 2: A Review on Cytotoxic, Antiproliferative, and Antitumor Phytochemicals, and Future Perspective

This review provides an overview on the active phytochemical constituents of medicinal plants that are traditionally used to manage cancer in Ethiopia. A total of 119 articles published between 1968 and 2020 have been reviewed, using scientific search engines such as ScienceDirect, PubMed, and Google Scholar. Twenty-seven medicinal plant species that belong to eighteen families are documented along with their botanical sources, potential active constituents, and in vitro and in vivo activities against various cancer cells. The review is compiled and discusses the potential anticancer, antiproliferative, and cytotoxic agents based on the types of secondary metabolites, such as terpenoids, phenolic compounds, alkaloids, steroids, and lignans. Among the anticancer secondary metabolites reported in this review, only few have been isolated from plants that are originated and collected in Ethiopia, and the majority of compounds are reported from plants belonging to different areas of the world. Thus, based on the available bioactivity reports, extensive and more elaborate ethnopharmacology-based bioassay-guided studies have to be conducted on selected traditionally claimed Ethiopian anticancer plants, which inherited from a unique and diverse landscape, with the aim of opening a way forward to conduct anticancer drug discovery program.


Introduction
Cancer is a major global health challenge that affects millions of people annually across the world. Recent estimates showed about 18.1 million new cases of cancer and 9.6 million cancer-related deaths worldwide [1]. Moreover, due to population growth, aging, and increased prevalence of key risk factors, this figure is expected to rise in the coming years. According to the same report, different from other parts of the world, cancer death (7.3%) is higher than cancer incidence (5.2%) in Africa. This is mainly attributed to lack of adequate health care facilities as well as professionals, lack of early cancer detection system, and poor access to chemotherapeutic treatments. Due to these and other factors, including socio-economic conditions, the majority of the population of Africa has relied on traditionally used medicinal herbs and/or plants as a monotherapy or in combination with clinically approved anticancer drugs.
Medicinal plants have been a rich source of clinically effective anticancer agents for the past few decades. Over 60% of the currently used anticancer drugs are either directly derived from plants or inspired by their novel phytochemicals [2] and/or unique ligands as secondary metabolites. In spite of such success, the importance of medicinal plants as a source of leads for anticancer drug discovery was marginalized in comparison with other advanced approaches. This could be due to issues associated with intellectual property rights and securing not enough amounts of plant material which results in the slowness of working with natural products [3]. However, despite these drawbacks, medicinal plant-based drug discovery and development has made a comeback to find potent and affordable natural products with a new mechanism of action and better toxicological profile due to structural diversity of natural product small molecules (NPSM). For instance, among small molecules approved for cancer treatment between 1940 and 2014, 49% are derived and/or originated from natural products [4].
Ethiopia inherited a unique array of fascinating flora from its diverse landscape. Due to the geographical location and diversity, which favors the existence of different habitat and vegetation zones, Ethiopia is home to a variety of plant species. The Ethiopian flora is estimated to contain 6027 species of higher plants of which more than 10% are estimated to be endemic [5]. Different authors have compiled ethnobotanical and ethnopharmacological profiles and reviews of Ethiopian traditionally used medicinal plants [6,7]. However, published reports regarding isolated bioactive compounds of traditionally used Ethiopean medicinal plants, especially those with cytotoxic properties are scant. However, investigations conducted on plants with cytotoxic properties out side Ethiopia, include the study on Catha edulis Forsk [8,9], Artemisia annua L., Rumex abyssinicus Jacq. [9]., Carissa spinarum L., Dodonaea angustifolia L.f., Jasminum abyssinicum Hochst. ex DC., Rumex nepalensis Spreng., Rubus steudneri Schweinf. and Verbascum sinaiticum Benth. [10], Viola abyssinica Steud. ex Oliv. [11], Xanthium strumarium L. [12], Senna singueana (Del). Lock [13], Glinus lotoides L. [14], Kniphofia foliosa Hochst [15], Sideroxylon oxyacanthum Baill., Clematis simensis Fresen, and Dovyalis abyssinica (A. Rich) Warburg [16]. Thus, for further evaluation, identification, or modification of anticancer leads, thorough review of the chemistry and pharmacology of medicinal plants from relatively uncovered traditional medical systems is crucial. Therefore, in continuation of our previous mini-review [17], in which we documented both ethnobotanical and ethnopharmacological evidence of Ethiopian anticancer plants involving mostly the cytotoxic and antioxidant activities of crude extracts, here, in this review, we comprehensively document the cytotoxic and antiproliferative constituents from anticancer plants those traditionally used in Ethiopia. The secondary metabolites reported from each medicinal plant species are categorized based on the class of natural products they belong to.

Phytochemistry of Ethiopian Anticancer Plants
The present review reports secondary metabolites isolated from 27 plants that are traditionally used to treat different types of cancer in Ethiopia. Phytochemical investigations of traditionally used Ethiopian anticancer plants have led to the isolation of compounds that belong to different classes of natural products [10,57]. In this review, we have not included plants those displayed compounds with very low cytotoxic/antiproliferative activity (i.e., IC 50 (Concentration that inhibited cell proliferation by 50%)/ED 50 (Effective dose for 50% of the population) > 50 µg/mL or > 100 µM, in most cases, except few where compounds tested against a panel of cell lines) or plants from which no anticancer compounds were isolated/reported. This review compiled and discussed the potential anticancer/antiproliferative agents based on the types of secondary metabolites, such as terpenoids, phenolic compounds, alkaloids, steroids, and lignans.

Terpenoids
Terpenoids are classified according to the number of their isoprene unit as hemi-, mono-, di-, tri-, tetra-, and polyterpenes [58]. Various studies reported that the anticancer activity of terpenoids is due to the inhibition of inflammation, cancer cell proliferation, angiogenesis and metastasis, and induction of programmed cell death [59]. Triterpenoids are one important class of terpenoids, which contain isopentenyl pyrophosphate oligomers [60]. They are biosynthesized by plants through cyclization of 30-carbon intermediate squalene and include various structural subclasses [61]. Several triterpenoids have been shown to have anticancer activity.
Several terpenoids have been isolated from Ethiopian plants that have claims of having anticancer activity, although these plants may have been collected from other sources. For example, sonhafouonic acid (8) from Zehneria scabra, collected from Cameroon, demonstrated potent cytotoxicity against brine shrimp assay [66], while Lin et al. [67] showed the antiproliferative activity of euphol (9), isolated from Euphorbia tirucalli from Taiwan against human gastric cancer cells. Euphol selectively promotes apoptosis by mitochondrial-dependent caspase-3 activation and growth arrest through induction of p27kip1 and inhibition of cyclin B1 in human gastric CS12 cancer cells. It also showed a selective and strong cytotoxicity against other groups of human cancer cell lines such as glioblastoma (the most frequent and aggressive type of brain tumor) [67,68]. The molecular mechanism of action of another anticancer triterpenoid, maslinic acid (10), isolated from the leaves of Olea europaea has been studied, which induced apoptosis in HT29 human colon cancer cells by directly inhibiting the expression of Bcl-2, increasing that of Bax, releasing cytochrome-C from the mitochondria and activating caspase-9 and then caspase-3 [69]. Similarly, the leaf extract of Ricinus communis collected from Malta was also reported for its cytotoxicity against several human tumor cells and induction of apoptosis against human breast tumors, SK-MEL-28. The monoterpenoids 1,8-cineole, camphor and α-pinene, and the sesquiterpenoid β-caryophyllene, isolated from R. communis, also showed cytotoxicity against similar cell lines in a dose-dependent manner [70].

Alkaloids
Vinblastine (30) and vincristine (31) (Figure 4) are one of the most effective bis-indole vinca alkaloids as anticancer drugs, isolated from the leaves of Catharanthus roseus [84]. This is one of the most precious anticancer plants indigenous to Madagascar. Previously, approximately 30 bis-indole alkaloids and over 60 monomeric indole alkaloids have been isolated from the aerial parts and roots of C. roseus [85,86]. Wang et al. [87] isolated three new cytotoxic dimeric indole alkaloids (32)(33)(34) along with other five known compounds from the whole plant of C. roseus collected from China (Table 4). Among the isolated compounds, leurosine (36) showed the most potent cytotoxic activity with IC50 value of 0.73 ± 0.06 µM. Furthermore, the isolated three new compounds (32-34) also showed potent cytotoxicity against triple-negative breast cancer (MDA-MB-231) cell line with IC50 values ranging from 0.97 ± 0.07 µM to 7.93 ± 0.42 µM. Another alkaloid, cathachunine (40), also showed a promising cytotoxic activity against HL-60 by inducing an intrinsic apoptotic pathway

Steroids and Lignans
Steroids and lignans, in addition to other phytochemicals, are common secondary metabolites reported from Ethiopian plants. Evidence and epidemiological studies suggest that phytosterols and lignans are protective against a wide range of diseases and possess anticancer activity [93]. Withanolides are cytotoxic steroidal lactones, reported from various plants of the family Solanaceae [94], of which withaferine-A (44) and

Preclinical, In Vivo, and Clinical Studies on Ethiopian Anticancer Plants
Preclinical studies generate data on the efficacy, safety, and pharmacokinetic properties of lead compounds, which will later be used to select better molecules for clinical trials. Assessment of the findings of preclinical in vivo animal studies supports the traditional use of plants to manage cancer in Ethiopia (Table 6). Despite the preclinical efficacy data, there are no clinically significant anticancer agents isolated from traditionally used Ethiopian plants. Moreover, there are also no clinical trials conducted on anticancer plants that are collected from Ethiopia. Among reviewed phytochemicals only ursolic acid, secoisolariciresinol (52), and colchicines (41), isolated from plants collected elsewhere, were considered further for clinical trial.

Preclinical, In Vivo, and Clinical Studies on Ethiopian Anticancer Plants
Preclinical studies generate data on the efficacy, safety, and pharmacokinetic properties of lead compounds, which will later be used to select better molecules for clinical trials. Assessment of the findings of preclinical in vivo animal studies supports the traditional use of plants to manage cancer in Ethiopia (Table 6). Despite the preclinical efficacy data, there are no clinically significant anticancer agents isolated from traditionally used Ethiopian plants. Moreover, there are also no clinical trials conducted on anticancer plants that are collected from Ethiopia. Among reviewed phytochemicals only ursolic acid, secoisolariciresinol (52), and colchicines (41), isolated from plants collected elsewhere, were considered further for clinical trial.  Ricinus communis Fruit extract Significantly reduced tumor volume in 4T1 syngeneic mouse model [113] --

Vernonia amygdalina
Aqueous crude extract Increase efficacies and optimizes treatment outcomes when given with paclitaxel in athymic mice [116] _ -

Conclusions
Despite the traditional use of various Ethiopian plants for the treatment of cancer by herbal medicine practitioners for many decades, only a few active anticancer crude extracts, herbal preparations, and pure compounds were tested and so far no clinical trial was conducted on them. In this review, an attempt has been made to document antiproliferative, antitumor, and cytotoxic natural products small molecules isolated from medicinal plants that are traditionally used to treat cancer in Ethiopia. However, among the reported active compounds, only few have been isolated from plants that are originated and collected from Ethiopian geographic location, despite their wider presence and traditional claim at home. The majority of compounds reported in this review are isolated from plants (corresponding to Ethiopian species) that were collected from different regions of the world. However, the comprehensive list of active compounds (IC 50 and ED 50 values) provided in this review will help to identify the most potent source(s) of these compounds, as bioactive marker(s), of local flora. Based on the higher frequency of citation Croton macrostachyus, Jatropha curcas, Plumbago zeylanica, and Vernonia hymenolepsis are potential candidates for follow-up bioassay guided investigations. Furthermore, plants with reported antiproliferative compounds such as Podocarpus falcatus, Linum usitatissimum, and Zehneria scabra should also be examined for additional cytotoxic compounds and evaluated against a battery of cancer cell lines.
Generally, the ecological variation has a huge impact on the biosynthesis, yield of active constituent and biological potency of secondary metabolites produced by plants of similar species from different geographical regions. Thus, Ethiopian anticancer plants might have novel active constituents to fight cancer, based on traditional medical use, than those collected from other regions due to their unique geographical location and inherent climatic condition of the diverse landscape. Unfortunately, these valuable plant resources are disappearing rapidly due to climate change, rapid urbanization, agricultural land expansion, and artificial deforestation; therefore, Ethiopian flora is facing a great challenge, and thus it is high time to examine the anticancer plants systematically with the aim to carry out chemical and biological invesigations, as well as clinical trials on promising anticancer plant extracts based on ethnopharmacological knowledge.