Recent Research Progress on Natural Stilbenes in Dendrobium Species

Dendrobium is the second biggest genus in the Orchidaceae family, and many of them have been utilized as a traditional Chinese medicine (TCM) for thousands of years in China. In the last few decades, constituents with great chemical diversity were isolated from Dendrobium, and a wide range of biological activities were detected, either for crude extracts or for pure compounds. Stilbene compound is one of the primary active constituents in the genus Dendrobium. At present, 267 stilbene compounds with clarified molecular structures have been extracted and isolated from 52 species of Dendrobium, including 124 phenanthrenes and 143 bibenzyls. At the same time, activity studies have indicated that 157 compounds have pharmaceutical activity. Among them, most of the compounds showed antitumor activity, followed by antioxidant, anti-inflammatory and anti-α-glucosidase inhibitory activities. Additionally, 54 compounds have multiple pharmacological activities, such as confusarin (14), 2,4,7-trihydroxy-9,10-dihydro-phenanthrene (43), moscatilin (148), gigantol (150) and batatasin III (151). This review summarizes current knowledge about the chemical composition of stilbene, bioactivities and pharmacologic effects in 52 species of Dendrobium. We also expect to provide a reference for further research, development and utilization of stilbene constituents in the Dendrobium genus.


Introduction
The genus Dendrobium, which belongs to the family Orchidaceae, includes more than 1500 species widely distributed throughout Asia, Europe and Australia [1]. According to the literature, there are almost 80 species of this genus in China; many of them are used in traditional or folk medicine [2]. Dendrobium serves numerous functions in traditional Chinese medicine, including as an antipyretic, as an anti-inflammatory agent, for benefit of the eyes and digestive system, and regulating blood sugar level [3]. Recent scientific research on the genus led to the isolation of a series of compounds, such as stilbenes, polysaccharides, alkaloids, amino acids, sesquiterpenes, fluorenones, flavonoids, phenolic acids, phenylpropanoids, lignans, amides, alkaloids and steroids [4]. Pharmacological studies have disclosed that some of them display wide bioactive activities.
The stilbene components mainly include phenanthrenes and bibenzyls in Dendrobium species. The variety of the substituents on the benzene ring endows stilbene compounds with numerous biological activities. In recent years, more studies have focused on the stilbene components of Dendrobium and multiple pharmacological effects have been discovered. Preclinical studies have found that the phenanthrene compounds in Dendrobium species showed good antioxidant, anti-inflammatory and antitumor activity [5]. Meanwhile, bibenzyls, another class of stilbene compounds in Dendrobium species, have attracted Table 1. Phenanthrenes isolated in Dendrobium species.

Simple Phenanthrene
The structural characteristics of simple phenanthrene are generally only hydroxy and methoxy substituents on the aromatic ring. Their great structural diversity stems from the number and position of their oxygen functions. The hydroxy and methoxy moieties numbers are between 3 and 6, and can usually be found on C-2, C-3, C-4, C-5, C-6 or C-7. At present, 31 simple phenanthrene compounds, 1-31, have been extracted and isolated from 44 species of Dendrobium. Among these species, moscatin (1) is the most widely distributed simple phenanthrene, with an amount of 14, followed by nudol (15), with an amount of 8. In addition, substitutions at position C-1, C-8, C-9 and C-10 are quite rare: compounds 23, 24 and 25 substituted with a methoxy group at C-1; compounds 9, 14 and 17 substituted with a methoxy group at C-8; compound 26 substituted with a methoxy group at C-9; compound 27 substituted with a hydroxy group at C-9; and compound 28 substituted with a methoxy group at C-9 and C-10.

Dihydrophenanthrene
The main difference between dihydrophenanthrene and simple phenanthrene is that the C-9 and C-10 sites are linked by single bonds. Dihydrophenanthrene substituents are also hydroxyl and methoxy, generally located at positions 2, 3, 4, 5 and 7, which are basically consistent with phenanthrene compounds. Currently, 36 dihydrophenanthrene compounds, 32-67, have been extracted and isolated from 44 species of Dendrobium. Meanwhile, substitutions at position C-1, C-6, C-8 and C-9 are quite rare: compound 65

Simple Phenanthrene
The structural characteristics of simple phenanthrene are generally only hydroxy and methoxy substituents on the aromatic ring. Their great structural diversity stems from the number and position of their oxygen functions. The hydroxy and methoxy moieties numbers are between 3 and 6, and can usually be found on C-2, C-3, C-4, C-5, C-6 or C-7. At present, 31 simple phenanthrene compounds, 1-31, have been extracted and isolated from 44 species of Dendrobium. Among these species, moscatin (1) is the most widely distributed simple phenanthrene, with an amount of 14, followed by nudol (15), with an amount of 8. In addition, substitutions at position C-1, C-8, C-9 and C-10 are quite rare: compounds 23, 24 and 25 substituted with a methoxy group at C-1; compounds 9, 14 and 17 substituted with a methoxy group at C-8; compound 26 substituted with a methoxy group at C-9; compound 27 substituted with a hydroxy group at C-9; and compound 28 substituted with a methoxy group at C-9 and C-10.

Dihydrophenanthrene
The main difference between dihydrophenanthrene and simple phenanthrene is that the C-9 and C-10 sites are linked by single bonds. Dihydrophenanthrene substituents are also hydroxyl and methoxy, generally located at positions 2, 3, 4, 5 and 7, which are basically consistent with phenanthrene compounds. Currently, 36 dihydrophenanthrene compounds, 32-67, have been extracted and isolated from 44 species of Dendrobium. Meanwhile, substitutions at position C-1, C-6, C-8 and C-9 are quite rare: compound 65 substituted with a methoxymethyl group at C-1; compounds 66 and 67 substituted with a hydroxy group at C-1; compounds 34, 57 and 60 substituted with a methoxy group at C-6; compound 62 substituted with a methoxy group at C-8; and seven compounds 35-41 substituted with a methoxy group at C-9. Among the 44 species of Dendrobium, hircinol (53) is the most widely distributed dihydrophenanthrene, with an amount of 15, followed by the coelonin (55), with an amount of 9.

Other Phenanthrenes
A total of 16 phenanthraquinone (68-83) were isolated from 19 species of Dendrobium. Among these species, D. nobile has the maximum number, containing seven monomer compounds, followed by D. wardianum, containing three monomer components, and other species containing minor monomer compounds with a number ranging from 1 to 2. In addition, phenanthrene-1,4-quinones are the most commonly occurring phenanthrenequinones, densiflorol B (68) is the most widely distributed phenanthraquinone in our analysis of the Dendrobium species, with an amount of 6. A total of 14 diphenanthrene (84-97)

Bibenzyls
Bibenzyls are another class of stilbene chemical ingredients that have been widely reported in the genus Dendrobium. Bibenzyl compounds refer to compounds formed by two benzyl units linked by a single methyl C-C bond. The character of bibenzyls is that the C3, C5 and C-4 positions are usually hydroxyl or methoxy on the core structure, and the C2 or/and C4 often have a p-hydroxyl or phenyl substitution. Although the structure of the parent nucleus of bibenzyl compounds is simple, the position and number of methoxy and hydroxyl groups on the aromatic ring, the bridge chain substituents connecting the aromatic ring and the change of the aromatic ring leads to various structural types and, thus, to various biologic activities. Bibenzyls can be divided into simple bibenzyl, bridged carbon bibenzyl and bibenzyl derivatives. In our analysis of 52 species of Dendrobium, only D. hainanense did not discover bibenzyl; the other species all had different amounts of bibenzyl. Currently, 143 bibenzyl compounds (125-267) have been discovered and isolated from 51 species of Dendrobium. Among these species, D. nobile has the maximum number of bibenzyl, containing 32 monomer components, followed by D. candidum and D. gratiosissimum, containing 29 and 21 monomer components. The distribution and structure of these compounds in Dendrobium are shown in Table 2 and Figure 2.      (148), chrysotoxine (149), gigantol (150), batatasin III (151)

Simple Bibenzyl
Currently, 68 simple bibenzyl  have been discovered and separated from 51 species of Dendrobium. The structural characteristics of simple bibenzyl are generally only hydroxy and methoxy substituents on the aromatic ring. Regardless of the simplicity of the skeletons of simple bibenzyl compounds, they have various structural types due to the position and number of methoxy and hydroxyl groups on the aromatic ring. The hydroxy and methoxy substituents numbers are between 3 and 6, and can usually be found on C-3, C-4, C-5, C-3′, C-4′ or C-5′. In addition, substitution at position C-2 is quite rare: compound 181 substituted with a hydroxy group at C-2 and compound 190 substituted with a methoxy group at C-2. Moscatilin (148) and gigantol (150) were found in most Dendrobium, with the species amount of 35 and 30, respectively. Batatasin III (151), tristin (130) and 4,4′-dihydroxy-3,5-dimethoxybibenzyl (157) were also found in Dendrobium, with the species amounts of 23, 16 and 10, respectively.

Bridged Carbon Bibenzyl
The main difference between bridged carbon bibenzyl and simple bibenzyl lies in the change of the aromatic ring and the bridge chain substituents connecting the aromatic ring. Similarly, bridged carbon bibenzyl has various structural types due to the position and number of methoxy and hydroxyl groups on the aromatic ring. In addition, it also exhibits various structural types depending on the substituents of the bridge chain. The number of hydroxyl and methoxy substituents on the aromatic rings ranges from 4 to 5, and can normally be found on C-3, C-4, C-5, C-3′, C-4′ or C-5′. Up to now,

Simple Bibenzyl
Currently, 68 simple bibenzyl  have been discovered and separated from 51 species of Dendrobium. The structural characteristics of simple bibenzyl are generally only hydroxy and methoxy substituents on the aromatic ring. Regardless of the simplicity of the skeletons of simple bibenzyl compounds, they have various structural types due to the position and number of methoxy and hydroxyl groups on the aromatic ring. The hydroxy and methoxy substituents numbers are between 3 and 6, and can usually be found on C-3, C-4, C-5, C-3 , C-4 or C-5 . In addition, substitution at position C-2 is quite rare: compound 181 substituted with a hydroxy group at C-2 and compound 190 substituted with a methoxy group at C-2. Moscatilin (148) and gigantol (150) were found in most Dendrobium, with the species amount of 35 and 30, respectively. Batatasin III (151), tristin (130) and 4,4 -dihydroxy-3,5-dimethoxybibenzyl (157) were also found in Dendrobium, with the species amounts of 23, 16 and 10, respectively.

Bridged Carbon Bibenzyl
The main difference between bridged carbon bibenzyl and simple bibenzyl lies in the change of the aromatic ring and the bridge chain substituents connecting the aromatic ring. Similarly, bridged carbon bibenzyl has various structural types due to the position and number of methoxy and hydroxyl groups on the aromatic ring. In addition, it also exhibits various structural types depending on the substituents of the bridge chain. The number of hydroxyl and methoxy substituents on the aromatic rings ranges from 4 to 5, and can normally be found on C-3, C-4, C-5, C-3 , C-4 or C-5 . Up to now,

Bibenzyl Derivatives
The genus Dendrobium is one of the primary sources of bibenzyl derivatives. Although, the structure complexity of bibenzyl derivatives means that multiple structural types are also due to the position and number of methoxy and hydroxyl groups on the aromatic ring, and changes in the aromatic ring. At present, 53 bibenzyl derivatives (215-267) have been identified and isolated from 27 species of Dendrobium. Among these species, D. candidum has the maximum number of bibenzyl derivatives, containing fourteen monomer components, followed by D. fimbriatum, containing nine monomer components and other species containing minor monomer components, with amounts ranging from 1 to 7.

Pharmacological Activity of Stilbenes in Dendrobium Species
Of the 267 isolated stilbene constituents, 157 compounds were found to have pharmacological activities, 103 monomer compounds showed single pharmacological activities and 54 monomer compounds had multiple pharmacological activities. In the following, stilbene constituents that were isolated from the different Dendrobium species are described according to different pharmacological effects, such as anti-oxidant, anti-inflammatory, antitumor, α-Glucosidase and pancreatic lipase inhibitory activities, such as antimicrobial, neuroprotective and anti-platelet aggregation effects.

Anti-Oxidant Activity
Oxidative stress is believed to be implicated in aging, neurodegenerative disease, diabetes mellitus, cardiovascular disease and tumors. Antioxidants can be profitable in combating these processes and diseases. The DPPH and ABTS free radical have been widely used to evaluate the antioxidant capacity of fractions and pure compounds to obtain the more effective and nontoxic compounds for the prevention and/or treatment of such diseases [77]. There are 58 stilbene constituents in the genus Dendrobium, which showed antioxidant activity by DPPH and ABTS free radicals scavenging assays (Table 3). While positive results have been obtained for the in vitro anti-oxidant activity of most stilbene components, further human studies are needed to substantiate the beneficial and negative effects of these compounds.
Many compounds have been discovered to have excellent in vitro antioxidant activity; however, their in vivo activities and mechanisms of action have rarely been explored. In 2021, su, et al. [131] assessed the protective effect of lusianthridin (30) on hemin-induced low-density lipoprotein oxidation (he-oxLDL). The research demonstrated that compound 30 protected LDL oxidation induced by hemin, and had the potential protective effect in foam cell formation. In addition, compound 30 could inhibit the formation of TBARs, reduce REM, reduce oxidized lipid products, as well as preserve the level of cholesterol arachidonate and cholesterol linoleate (Figure 3).

Anti-Infammatory Activity
Macrophages play a major role in inflammation and host defense mechanisms against bacterial and viral infections. The NO radical, synthesized by the oxidation of Larginine catalyzed by nitric oxide synthase, is involved in a number of physiological and pathological processes in mammals. Meanwhile, excessive production of NO by iNOS in macrophages is involved in various acute and chronic inflammatory diseases. Therefore, inhibitors of NO production in macrophages are an important target in the treatment of certain inflammatory diseases [59]. Due to some phenolic compounds from different Dendrobium species showing significant inhibitory effects on NO production, discovering new phenolic compounds and evaluating their prospective anti-inflammatory activities have become of great interest to many research groups worldwide. At present, 45 stilbene constituents in the genus Dendrobium have shown anti-inflammatory activity in LPS-

α-. Glucosidase and Pancreatic Lipase Inhibitory Activities
α-Glucosidase is a membrane bound enzyme in the small intestine, which is responsible for digesting starch and disaccharide into glucose. Inhibition of this enzyme can delay carbohydrate digestion, which can prevent excess glucose absorption. Pancreatic lipase is the key enzyme responsible for lipid digestion. Inhibition of pancreatic lipase enzyme can reduce lipid absorption and may help to protect pancreas β-cells [47]. In recent years, the number of reports on natural compounds with α-glucosidase and lipase inhibitory activities have continuously increased. Significant research has been focused on the search for alternative α-glucosidase inhibitors with non-sugar core structure, particularly the polyphenols, due to their abundant availability in nature and their promising biological activities [41]. At present, 29 stilbene constituents in the genus Dendrobium showed α-glucosidase and pancreatic lipase inhibitory activities (Table 5). The research found that the inhibition of carbohydrate and lipid hydrolyzing enzymes by compound 73 may decrease the rate of their cleavage, component release and absorption in the small intestine, and consequently, suppress postprandial hyperglycemia and hyperlipidemia. Additionally, lusianthridin (32) and moscatilin (148) at non-toxic concentrations may find a role in helping to regulate glucose metabolism by stimulating glucose uptake in skeletal muscle, the largest site of glucose disposal, leading to the prevention of MetS and type II diabetes [41]. In addition, dendrofalconerol B (263), a bisbibenzyl compound, exhibited strong pancreatic lipase and α-glucosidase effects. This compound also exhibited anti-adipogenic activity through the suppression of PPARγ and C/EBPα expression (Figure 3) [111].

Antitumor Activity
There are 70 stilbene compounds in the genus Dendrobium that have exhibited antitumor activity. Among them, 19 compounds were active against single cancer cells and 51 compounds were active against multiple cancer cells (Tables 6 and 7). Moscatilin (148) is the most common stilbene compound of the genus Dendrobium, which also has anticancer activity against a variety of cancer cells. Moscatilin was shown to induce apoptosis in human colorectal cancer cells through tubulin depolymerization, DNA damage and c-Jun N-terminal kinase (JNK) activation [134]; apoptosis of human pancreatic cancer cells via reactive oxygen species and the JNK/stress-activated protein kinases (SAPK) pathway [135]; and apoptosis and mitotic catastrophe in human esophageal cancer cells by early promotion of the M phase cell cycle blockade and the regulation of mitotic catastrophe-associated proteins [136]. Moscatilin induces apoptosis of human pharyngeal squamous carcinoma cells by activating caspases through the JNK signaling pathway [137]. Moscatilin impedes HCC invasion and uPA expression through the Akt/NF-κB signaling pathway [138]. Moscatilin was also reported to inhibit the migration and metastasis of human breast cancer cells by inhibiting Akt and the Twist signaling pathway [139]. In addition, moscatilin suppressed tumor angiogenesis and growth in human umbilical vein endothelial cells, blocking ERK1/2, Akt and the eNOS pathway [140].    Studies on the anti-cancer mechanism of other stilbenes have found that nudol (15) caused cell cycle arrest at G2/M phase in U2OS cells, and it also induced cell apoptosis through the caspase-dependent pathway and inhibited migration of OS cells [141], and chrysotoxene (23)-induced apoptosis of HepG2 cells in vitro and in vivo via activation of the mitochondria-mediated apoptotic signaling pathway [142]. Lusianthridin (32) suppresses lung cancer stem cells via the inhibition of Src-STAT3-c-Myc; thereby, regulating the stemness of the cells [143]. The research disclosed that cypripedin (69) possesses cytotoxic activity against NSCLC and synergies cisplatin-induced cell death. The underlying mechanism is believed to be through the downregulation of the anti-apoptotic protein Bcl-xL, which results in the imbalance of apoptosis regulatory proteins, causing the loss of mitochondrial membrane integrity and the release of cytochrome c [144]. Denbinobin (71) has been reported to inhibit A549 cells and human glioblastoma multiforme cell apoptosis through caspase and apoptosis signal-regulating kinase 1 activation. In addition, compound 71 inhibits CXCL12-induced PC3 cell migration by inhibiting Rac1 activity [145]. phoyunnanin E (94) inhibits migration and growth in an anchorage-independent manner with detailed mechanisms of action covering EMT suppression, reduction of migratoryassociated integrins αv and β3 and suppression of FAK/Akt signals which consequently suppressed downstream migratory proteins in lung cancer [146]. In addition, Phoyunnanin-E-mediated apoptosis via a p53-dependent pathway by increasing the accumulation of cellular p53 protein [147]. Chrysotoxine (149) markedly suppressed 6-OHDA-induced apoptosis in human neuroblastoma SH-SY5Y cells, presumably through direct scavenging of intracellular ROS induced by 6-OHDA, thus blocking ROS-mediated downstream signaling pathways [148]. In addition, compound 149 was shown to suppress active Src/Akt signal and in turn depleted Sox2-mediated cancer stem cells [149]. Batatasin III (151) has promising anti-cancer properties by inhibiting cell proliferation, migration and invasion by suppressing EMT and FAK/AKT/CDC42 pathways [150]. Chrysotobibenzyl (172) inhibited lung cancer cell migration via Cav-1, integrins β1, β3 and αν, and EMT suppressions [151]. Alteration to down-stream signaling of p53 including activation of pro-apoptosis protein, reduction of anti-apoptosis and suppression on protein kinase B (Akt) survival pathway were notified in 4,5,4 -trihydroxy-3,3 -dimethoxybibenzyl (161)treated lung cancer cells [152]. Erianin (184) down-regulates the expression of inflammation factors through the regulation of IDO-induced tumor cells angiogenesis mimicry and endothelial cell-dependent angiogenesis by targeting the JAK2/STAT3 pathway and its down-stream genes MMP-2/MMP-9, and thereby inhibits the angiogenesis of lung cancer cells (Figure 3) [153].
After nearly two decades of research, significant progress has been made in isolation and structure identification, and a series of naturally occurring stilbenes with various structures have been isolated. However, activity investigations on most compounds are limited to in vitro screening, and few systematic pharmacological studies are available in the literature due to the scarcity of samples. The synthesis of active stilbenes must be developed to obtain sufficient samples for systematic pharmacological studies. In addition, due to their rapid metabolism and excretion in mammalian bodies, improvements in de-livery systems, stability and solubility are required.
While there is still a long way to go, Dendrobium species are undeniably one of the most promising sources of biologically active stilbenes. Meanwhile, hopefully even more research groups will deal with the phytochemistry, pharmacology, bioavailability and potential utilization of Dendrobium.

Conclusions
Dendrobium has been used as a medicinal herb in China for thousands of years. Nonetheless, due to the high degree of personalization of TCM diagnosis and treatment of TCM, its clinical efficacy cannot be comprehensively evaluated by evidence-based medicine. The fruitful achievements in its chemical constituent investigations in recent years have laid a foundation for the study of its therapeutically material basis and action mechanism, which will help clarify its diverse pharmacologic effects and develop into a modern pharmaceutical preparation.
Stilbenes are rich sources of lead compounds in the search for new drugs and medicines. Dendrobium species are promising sources of biologically active stilbenes. At present, 267 stilbene compounds with clarified molecular structures have been extracted and isolated from 52 species of Dendrobium, that includes 31 simple phenanthrene, 36 dihydrophenanthrene, 16 phenanthraquinone, 14 diphenanthrene, 27 phenanthrene derivatives, 68 simple bibenzyl, 22 bridged carbon bibenzyl and 53 bibenzyl derivatives. Meanwhile, activity studies have shown that 157 stilbene compounds have pharmaceutical activity. Among them, 62 compounds showed antioxidant activity; 51 compounds showed anti-inflammatory activity; 31 compounds showed anti-α-glucosidase and pancreatic lipase inhibitory activities; 73 compounds exhibited antitumor activity; 7 compounds showed antimicrobial activity; 11 compounds exhibited neuroprotective effects; 7 compounds showed antiplatelet aggregation effects; 7 compounds showed gastrointestinal motility activities and 7 compounds showed tyrosinase inhibitory and anti-aging activities. In addition, 19 phenanthrenes and 35 bibenzyls have multiple pharmacological activities, such as confusarin (14), 2,4,7-trihydroxy-9,10-dihydrophenanthrene (43), tristin (130), 3,3 ,5-trihydroxybibenzyl (133), 3,4'-dihydroxy-5-methoxybibenzyl (135), moscatilin (148), gigantol (150), batatasin III (151), 4,4 -dihydroxy-3,5-dimethoxybibenzyl (157) and dendrocandin U (231). These compounds showed a variety of pharmacological activities, mainly including antioxidant, anti-inflammatory, antitumor, anti-α-glucosidase inhibitory activities. For many compounds, however, despite having been discovered to have good inhibitory activity in vitro results, their in vivo activities had rarely been determined. It is hoped that the pharmacological mechanism of these compounds can be further studied in the future. In addition to their activity, the pharmacological toxicity and physicochemical properties of the compounds also deserve attention. Here we briefly review the published studies of the last 20 years that were related to stilbene chemical constituents and pharmacologic activities in 52 species of Dendrobium. This can not only provide significant pharmacological effect and chemotaxonomic knowledge of the genus Dendrobium, but also provide a scientific basis for developing new medicines utilizing this interesting plant and stilbene constituents.