Orchidaceae-Derived Anticancer Agents: A Review

Simple Summary Orchids are commonly used in folk medicine for the treatment of infections and tumors but little is known about the actual chemical composition of these plants and their anticancer properties. In this paper, the most recent literature on orchid-derived bioactive substances with anticancer properties is reviewed. According to the published data, numerous species of orchids contain potential antitumor chemicals. Still, a relatively insignificant number of species of orchids have been tested for their bioactive properties and most of those studies were on Asian taxa. Broader research, ’including American and African species, as well as the correct identification of samples, is essential for evaluating the usefulness of orchids as a plant family with huge anticancer potential. Abstract Species of orchids, which belong to the largest family of flowering plants, are commonly used in folk medicine for the treatment of infections and tumors. However, little is known about the actual chemical composition of these plants and their anticancer properties. In this paper, the most recent literature on orchid-derived bioactive substances with anticancer properties is reviewed. For the assessment, previous papers on the anticancer activity of Orchidaceae published since 2015 were considered. The papers were found by exploring electronic databases. According to the available data, many species of orchids contain potential antitumor chemicals. The bioactive substances in a relatively insignificant number of orchids are identified, and most studies are on Asian taxa. Broader research on American and African species and the correct identification of samples included in the experiments are essential for evaluating the usefulness of orchids as a plant family with vast anticancer potential.


Introduction
According to the World Health Organization (WHO) "Guidelines on Safety Monitoring of Herbal Medicines in Pharmacovigilance Systems", up to 80% of the world's population rely on herbal medicines as a primary source of healthcare. As summarized by Ekor [1], the use of herbal medicines is increasing also in developed countries [2,3]. It is not surprising that the utilization of plants in medicine is as old as mankind itself and even modern pharmacotherapy includes numerous herb-derived drugs [4,5]. Noteworthy, about 10% of known vascular plants are currently used as therapeutics [6]. In 2019 alone, almost 2000 new species of plants were discovered according to the "State of the World's Plants and Fungi 2020" report, published by the Royal Botanic Gardens Kew. These plants could be potential sources of new phytochemicals that can be used in medicine [7].

Orchidaceae
Orchidaceae is one of the largest families of flowering plants with more than 27,000 accepted species [21] and more than 31,000-35,000 species are estimated to exist in total [22,23]. This is a cosmopolitan group growing in almost every habitat except deserts and glaciers. The plants are found above the Arctic Circle, in Patagonia, and even on Macquarie Island [24,25]. However, the greatest diversity of orchids is recorded in tropical regions, especially in mountainous areas [26].
Orchids can grow as epiphytes, lithophytes, or terrestrial perennial herbaceous plants that lack any permanent woody structures. Adult plants are mostly able to acquire carbon through photosynthesis, but some taxa are mycoheterotrophic [27]. Orchids are extremely diverse and their growth can be sympodial or monopodial. Many species produce storage organs like bulbs or pseudobulbs. Their flat or pleated leaves may be variously arranged on the stem (alternate, opposite, arranged spirally), or may grow only at the base of the plant. Orchid flowers are extremely diverse, usually zygomorphic, and most often containing both male and female reproductive organs. The outer whorl has three sepals and the inner whorl has three petals; however, one petal (lip) is usually modified and differs in appearance from the other two. A central flower structure called the column comprises both the male (anther) and female (stigma) parts of the flower. The ovary is composed of three carpels.
Orchids are often called "masters of deception" due to the diversity of deceptive mechanisms for attracting pollinators, e.g., generalized food deception, food-deceptive floral mimicry, brood-site imitation, shelter imitation, pseudo antagonism, rendezvous attraction, and sexual deception [28,29]. Many nectar-less orchids mimic other pollinatorrewarding plants [30] or produce various pseudo pollen or pollen-like papillae to lure insects [31]. A large group of species is able even to produce chemicals similar to insect sex pheromones [32][33][34][35] and this means of pollination, called pseudo copulation, is found only in orchids.
Orchids became one of the most popular ornamental plants in the Victorian era and currently, the official global orchid trade is estimated to ca. 72 million specimens per year [45]. These plants are widely used as medicines, food, and as herbs with other cultural values [46,47]. Currently vanilla together with salep and chikanda are globally and regionally important food products [45]. Orchids were first used in Chinese traditional medicine [48,49], but they are also popular in Ayurvedic therapies [50] and are commonly used by native tribes in tropical America as well as in Africa [51,52].

Importance of Symbiosis
As mentioned before, all orchids are associated with specific mycobiota and different fungi species are found in various plant parts [40,[70][71][72][73]. Preliminary studies already proved that some of these microorganisms are characterized by antimicrobial activities [74,75] and that interactions of symbiotic fungi with plants contribute to secondary metabolites production .
Unfortunately, the comparative studies on compounds extracted from fungi-infected and in vitro cultivated, fungi-free orchids were not conducted so far. Considering the enormous number of orchid species, their symbionts remain poorly recognized. Noteworthy, most of the experiments on orchid endophytic fungi included only root tissue, [76,77] while in traditional medicine, stems, and leaves are organs usually used for therapies [43,48,49,[78][79][80][81]. The importance of recognition of orchid endophytic fungi for secondary metabolites synthesis and their potential application in medicine were summarized by Sarsaiya et al. [39] and Pant et al. [82].
Interestingly, some of the bioactive compounds were found in an invasive orchid species, Arundina graminifolia, which is an Asian native herb. It would be important to study also the populations of this species which are currently invading Central and South America [83] in the context of the differences in symbiotic mycobiota of non-native plants as well as the similarity of secondary metabolites produced by native and invasive populations. Similarly, the compounds produced by Liparis nervosa which grows in Asia, Africa, and America should be compared with plants collected in various geographical regions.

Importance of Taxonomy and Plant Material Preservation
In this study, as experts in orchid taxonomy [84][85][86][87], it is crucial to emphasize the fundamental role of the correct identification of plants for further studies on the usefulness of phytochemicals in cancer therapy [88][89][90]. The diversity of orchids and superficial similarity of related species often leads to erroneous identification of taxa [91,92]. The detailed studies on various orchids revealed that numerous commonly recognized species are actually species-complexes that include several distinct species [93][94][95].
Most of the reports reviewed in this paper were on Dendrobium, which is one of the most complicated taxa in terms of species nomenclature and classification [96]. Currently, there are more than 1000 species in this genus, and new species and varieties are described frequently from tropical Asia [97,98]. Diagnostic characters which allow to identify particular Dendrobium species are related to flower morphology and therefore plants cannot be correctly classified in the vegetative stage [99]. To further investigate orchids used for treating cancer, it is vitally important that they are correctly identified. Initial identification of a plant should not only be confirmed by expert taxonomists but also voucher material further verified and preserved in the form of dried herbarium specimens [100] and preferably complemented with DNA barcodes [101][102][103]. The molecular identification without properly preserved plant material can be doubtful [104,105]. Unfortunately, the good practices summarized by Bussmann [91] are rarely applied in studies on orchid secondary metabolites, therefore it is not possible to confirm the identification of examined species.

Secondary Metabolites of Orchids
The basic knowledge on the diversity of orchid secondary metabolites was summarized by Sut et al. [71], Teoh [106], and Pant et al. [82] but the authors of these papers did not present data on the action mechanism of particular secondary metabolites, the importance of symbiotic fungi or other issues related with using orchid-derived biocompounds. Experiments on alkaloids, terpenes, stilbenoids, bibenzyls, phenanthrenes, flavonoids, and polysaccharides isolated from Orchidaceae indicated their potential medical usefulness [106].
Noteworthy, some of the bioactive compounds can be actually produced by the symbiotic microbes associated with orchids [121].
It should be emphasized that our team is also currently working on the identification of phytochemicals in the orchid species described for the first time and on the determination of their biological properties, including anticancer activity.

Biotechnological Methods for Orchidaceae Family
As shown, plants of the Orchidaceae family can be a source of many valuable, biologically active compounds that can be widely used as a basis or a supplement to the modern forms of oncological therapy. Plants growing in natural habitats are often the only source of these valuable compounds. Due to the fact that these plants usually do not synthesize large amounts of these compounds, it is very difficult to meet the constantly growing demand for these metabolites. What is more, many species capable of their synthesis are under strict protection. The solution to this problem is the use of biotechnological methods allowing constant access to valuable biomass from in vitro cultivation and, in many cases, increasing the level of their synthesis and accumulation. For this purpose, efficient in vitro propagation protocols have already been developed for many medically valuable orchid species. Such an approach often involves the induction of callus tissue which can then be stimulated to differentiate to give rise to new shoots, or in the case of embryogenic callus, it may be the start of somatic embryos. Pujari et al. described three simple, fast, and economical in vitro tissue culture protocols for Dendrobium ovatum that can be used to develop the right amount of material for biological research in an endangered orchid. Additionally, the authors also demonstrated the enhancement of moscatilin production in the in vitro cultures of this valuable plant [122]. Another type of culture that has found application for the Orchidaceae is the protoplast or thin cell layer (TCL) culture. Vudala et al. developed an effective micropropagation protocol for Hadrolaelia grandis with thin cell layer culture systems that can be the starting point for in vitro plant breeding, even on a large scale [123]. Additionally, Brattacharyya et al. developed a protocol for the regeneration of Dendrobium aphyllum, an important therapeutic orchid by the t-TCL method. For this purpose, Murashige and Skooga (MS) medium was supplemented with 15 µM meta-topoline along with 10 µM TDZ and 10 µM AgNO 3 . This combination was found to be the most optimal for shoot proliferation [124]. In addition, an adventitious shoot can also be a valuable strategy, which in a relatively short time, using appropriate growth regulators, allows to multiply valuable plant material. As presented by Mahendran et al. who developed a protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant of Malaxis densiflora. In these in vitro studies, seed-derived protocorm explants were cultured on 1/2 Murashige and Skoog medium with 2,4-D, Picloram, and Dicamba alone or in combination with BAP, TDZ, and Kn. It was shown that the best results were obtained on 1/2 MS with 3.39 µM of 2,4-D and 6.80 µM of TDZ. This protocol is another example of work on the possibility of efficient in vitro culture of human-important members of the Orchidaceae family [125]. Another strategy worth considering, among the sources of extremely valuable compounds, is the cultivation of various tissue and cell cultures in special bioreactors [126,127]. These devices, which allow for the maintenance of plant material in sterile conditions in vitro, often allow the optimization of the entire breeding process, which is extremely important from a technological and economic point of view. Bioreactors ensuring optimal conditions for growth and development by strict control of many key parameters have long been used even on an industrial scale in many other plant families. In addition, the possibility of stimulating production with various physical and chemical factors, combined with genetic modifications in the future, will certainly allow the development of efficient and comprehensive solutions allowing the use of the Orchidaceae family as a kind of mini-factories producing compounds desired in many areas of life.

The Anticancer Activity of Plant Extracts from Orchidaceae
Extracts of many species of orchids have anticancer properties. Isolates from various plant parts exhibit cytotoxic activity against leukemia and melanoma, as well as against brain, breast, cervical, gastric, liver, and lung cancer cells.
Extracts of several species of Dendrobium ( Figure 1) have a cytotoxic effect and inhibit the growth of cervical cancer and glioblastoma brain tumor cells [128][129][130][131][132]. It is hypothesized that polyphenol compounds found in orchid extracts inhibit cancer cells by xenobiotic-metabolizing enzymes altering the metabolic activation of potential carcinogens [133]. On the other hand, flavonoids can modify hormone production and prevent the growth of cancer cells [133]. In contrast, phenolics can interrupt cellular division during the telophase stage of mitosis. These chemicals also affect cell proliferation by reducing the amount of cellular protein, the mitotic index, and colony formation [131]. The ethanolic extract of Dendrobium chrysanthum perturbs cell cycle progression and results in a delay in the growth of cells. It also exerts anticancer activity [129]. A similar situation for extracts of D. venustum in which phoyunnanin E triggered apoptosis of lung cancer cells by suppression of survivin [134]. Another Dendrobium species, D. crepidatum, is significantly cytotoxic against both cervical cancer (HeLa) and glioblastoma brain tumor (U251) cell lines [128].
The volatile oil of Anoectochilus roxburghii induces apoptosis in tumor cells and triggers an enzyme cascade resulting in the apoptosis of lung cancer cells (NCI-H446) [138]. The ethyl acetate extract of Anoectochilus formosanus induces apoptosis in human breast cancer cells (MCF-7) and the aqueous extract effectively inhibits the growth of colon cancer cells in mice [138].
Some studies on Pleione by Liu et al. [139] indicate that an ethyl acetate extract of Pleione bulbocodiodes inhibits the growth of mice cancer cells LA795 (lung adenocarcinoma). Wang et al. [140] also indicate that some components of the extract of Pleione yunnanensis strongly inhibit the growth of lung adenocarcinoma cells. Other compounds obtained from this species are very cytotoxic against colon cancer cells (HepG2), liver cancer cells (BGC-823), and breast cancer cells (MCF-7).
Other orchid extracts that are effective against breast cancer (MCF-7) are those from Eulophia nuda tubers [141], leaves of Aerides odorata [142], and leaves of Vanilla [143]. It is hypothesized that the cytotoxic activity is related to the synergistic action of the phytoconstituents present in these species [141]. Other studies are presented in Table 1.

The Anticancer Effect-Potential Mechanism of Action and Activation of Signalling Pathways of Pure Compounds from Orchids
Several classes of phytoconstituents of great chemical diversity have been isolated from therapeutically-used orchids [71].
Another group of orchid metabolites that have antitumor activities are phenanthropyrans and phenanthrenes [160][161][162][163][164][165][166]. Nudol isolated from Dendrobium nobile arrests the cell cycle of osteosarcoma (U2OS) cells, induces cell apoptosis via the caspase-dependent pathway and suppresses the migration of these cells [161]. Cypripedin isolated from Dendrobium densiflorum is effective against lung cancer by activating caspase-3 and downregulating the antiapoptotic proteins Bcl-2 and Bcl-xL in cells [167]. Denbinobin also isolated from Dendrobium and Ephemerantha also promotes caspase-3 activity in lung adenocarcinoma cells [168,169] and a polysaccharide extracted from Anoectochilus roxburghii inhibits in this way the growth and proliferation of human prostate cancer (PC-3) cells [138].
One of the most extensively studied orchid compounds is dendrobine and its derivatives [174,175]. This chemical induces apoptotic cell death via a mitochondrial-mediated pathway in lung cancer cells (A549). The combination of dendrobine with cisplatin enhances their cytotoxicity by stimulating JNK/p38 stress signalling pathways and, consequently, inducing apoptosis involving the pro-apoptotic proteins Bax and Bim [176].
[167] Cytotoxic effect (>50 µM). Concentration-dependent cell apoptosis. At non-toxic concentrations inhibition of anchorage-independent growth of the cancer cells, as indicated by the decreased colony size and number. Ephemeranthol A also had an inhibitory effect on migration. We further found that ephemeranthol A exerts its antimetastatic effects via inhibition of EMT, as indicated by the marked decrease in N-cadherin, vimentin, and Slug.
Furthermore, this compound suppressed the activation of focal adhesion kinase (FAK) and protein kinase B (Akt) proteins, which are key regulators of cell migration. As for the anticancer activity, ephemeranthol A induced apoptosis by decreasing Bcl-2 followed by the activation of caspase 3 and caspase 9. [169] Dendrobium officinale Kimura et. Migo Leaves ViceninII A549 and H1299 Cytotoxic effect effect (>10 µM). ViceninII targets the TGF-_/Smad and PI3K/Akt/mTOR pathways and inhibit TGF-1-induced EMT phenotypes in lung adenocarcinoma A549 and H1299 cells. [177]

In Vivo Studies of Extracts and Pure Compounds from the Orchidaceae Family
In vivo studies are the next important step after in vitro and involve testing compounds and assessing the safety of their efficacy on living organisms such as animals, plants or whole cells. The Orchidaceae family is a valuable source of secondary metabolites (selected presented on Figure 2), and despite the limited number of studies meeting our criteria, this is also applicable to the in vivo studies which are presented below. In the in vivo studies, Su et al. [214] evaluated the antitumour effects of moscatilin, a natural compound isolated from the orchid Dendrobium moscatum in the mouse xenograft model. MDA-MB-231 cells were axillary injected into nude mice to establish the mouse model of breast cancer. These data suggested that moscatilin suppresses breast cancer growth and progression in vivo, and therefore can be used as a potential therapeutic agent for the treatment of breast cancer [214]. Sun et al. investigated the possibility of erianin (a natural compound derived from Dendrobium candidum), as a potential therapy in colorectal cancer (CRC). The authors tested the function of erianin on tumor growth in a mouse model by injection of SW480 cells into NOD/SCID mice. These data indicated that erianin inhibited tumor growth via β-catenin in vivo [215]. On the other hand, Zhang et al. investigated the inhibitory effect of Dendrobium officinale polysaccharide (DOPA) on human gastric cancer cell SGC-7901 xenografts in nude mice, where the nude mice with SGC-7901 xenografts were randomly divided into model, 5-fluorouracil (5-Fu), low-dose DOPA, middle-dose DOPA, and high-dose DOPA group. DOPA inhibited the growth of SGC-7901 cell xenografts in nude mice. The authors suspect that the mechanism may be related to its increase of serum TNF-α and IL-2 levels, up-regulation of Bax protein expression, and down-regulation of Bcl-2 protein expression [216]. Zhao et al. tested Dendrobium officinale extracts (4.8 and 2.4 g/kg) which were administered orally to rats from the gastric carcinogenesis model. Compared to the cancer model group, the high-dose of Dendrobium officinale extracts significantly inhibited the rate of carcinogenesis. Further analysis showed that Dendrobium officinale extracts regulated DNA damage, oxidative stress, and carcinogenesis-related cytokines, and induced cell apoptosis to prevent gastric cancer [217]. Song et al. noted that dendrobine (an alkaloid isolated from Dendrobium nobile) enhanced the chemotoxicity of cisplatin against A549 xenograft tumor female BALB/c mice. Treatment with dendrobine or cisplatin resulted in an obvious reduction of tumour size, whereas combination treatment dramatically decreased the tumor size. Additionally, the authors showed that dendrobine chemo-sensitized A549 cells to cisplatin induced apoptosis through the JNK/p38 pathway in vivo [176]. In turn, Fang et al. investigated if polysaccharides isolated from Rhizoma pleionis (PRP) suppress H22 tumor growth in vivo in a model of malignant ascites in BALB/c mice. H22 cells were transplanted into the left abdominal cavity of mice, and then animals were treated either with PRP in saline at various doses (75,150, and 300 mg/kg) or with cyclophosphamide (CTX) (20 mg/kg) or cyclophosphamide (CTX) (20 mg/kg). The authors revealed that on the tenth day after tumor cell inoculation, the mouse abdominal perimeter and weight in the PRP treatment group were significantly smaller than those in the control group. Collectively, these results demonstrated that PRP has significant antitumour properties in the H22 tumor model [218]. Other studies in xenograft analysis showed that chrysotoxene (phenanthrene derivative that was first isolated from Dendrobium chrysotoxum) (20 mg/kg) indicated that it significantly (p < 0.01) the inhibited growth of HepG2 cell-induced tumors by regulating the aforementioned apoptotic proteins (Smac, Cytochrome c, Survivin, Bcl-2, Bax, Apaf-1, c-caspase-9, and c-caspase-3), compared with the control group. Finally, the authors suggested that chrysotoxene may be a potential candidate drug for treating patients with hepatoblastoma [219]. Biswas et al. showed that Bulbophyllum sterile petroleum ether fraction ameliorates tumour progression in Ehrlich ascites carcinoma model in vivo. The authors revealed that the petroleum fraction of bulbs (PFB) and petroleum fraction of roots (PFR) at the dose of 200 mg/kg reduced the body weight compared to control. Cisplatin, which served as control, was injected on the first day and reduced the increase in body weight as compared to control. Additionally, the results suggested that the active fractions of bulbs and roots possess anticancer activity, likely by inducing apoptosis through the phospho-p53 dependent pathway [137]. A similar antitumour effect in an in vivo model was also shown by Jia et al. These results of antitumour activity demonstrated that the tumor weight of mice in three different dosage groups was significantly lower than that of the model group (p < 0.05, p < 0.01). Moreover, the authors exhibited that the polysaccharide from the fibrous root of Bletilla striata had a significant inhibitory effect on the tumor growth on S 180 tumor bearing mice. For this reason, the authors suggest that the mechanism of antitumour might be that it could enhance the immune function by regulating the levels of TNF and IL-2 in serum [220]. Kim et al. showed that dendrobine inhibited γ-irradiation-induced migration and invasion of A549 cells by suppressing sulfatase2 (SULF2) expression, thus inhibiting IR-induced signalling. To investigate the inhibitory effects of dendrobine in vivo, a mouse model of IR-induced metastasis, by injecting BALB/c nude mice with γ-irradiated A549 cells via the tail vein, has been established. These results noted that the number of pulmonary metastatic nodules in mice significantly reduced with dendrobine treatment (2 Gy/Dendrobine, 10.87 ± 0.71), by prevention of IR-induced signalling. For this reason, the authors report that this compound may serve as a therapeutic enhancer in non-small cell lung cancer (NSCLC) patients [221].
The studies presented above confirm the enormous anticancer potential of the compounds contained in this family, which makes them potential candidates for future anticancer therapies.

Conclusions
The review of the literature revealed that orchids have not been equally well studied throughout the world. The largest number of studies refers to Asian orchids, and little is known about the chemical constituents of American and African plants, except the pantropical Vanilla.
The literature reports that both extracts and pure compounds extracted from orchids have a strong cytotoxic effect on various cancer cell lines by inducing intrinsic and extrinsic apoptotic pathways. In addition, in vivo studies have shown that pure compounds or extracts can be used as a potential therapeutic agent in anti-cancer therapies. Considering the very low percentage of orchids examined in terms of their secondary metabolites, further analyses are very likely to reveal the existence of numerous new substances suitable for anticancer therapy.