Identification, Biological Activities and Biosynthetic Pathway of Dendrobium Alkaloids

Dendrobium is a genus of flowering plants belonging to the Orchidaceae family with more than 1,400 species. Many Dendrobium species have been used as medicinal plants in several Asian countries for thousands of years. Alkaloids were reported as the major biological markers due to their complex chemical compositions and various types. In this review, we summarized the structural types of alkaloids, their pharmacological activities, as well as the mechanisms of biological activities. More than sixty alkaloids were isolated and identified from the Dendrobium genus. Moreover, the pharmacological effects of Dendrobium alkaloids as hepatic lipid and gluconeogenesis regulation, as neuroprotection, and as anti-tumor, anti-inflammatory, anti-diabetes, and anti-virus factors were described. Besides, the total chemical synthesis of dendrobine is provided, while the biosynthetic pathway of dendrobine has been proposed based on the functions of associated genes. For applications of these invaluable herbs, more researches on the extraction of biological markers from compounds are needed. Further confirmation of the proposed biosynthetic pathways is anticipated as well.


INTRODUCTION
Apart from Asteraceae, the orchid family is the second-largest flowering family, which has 28,000 species distributed in about 736 genera (Chase et al., 2015), among which Dendrobium is one of the largest genera. It contains more than 1,500 species (www.theplantlist.org), most of which are epiphytic or lithophytic, and it is widespread in South, East, and Southeast Asia, like China, Japan, Philippines, Vietnam, India, and Indonesia. Some species are also found in New Guinea, Australia, and the islands of the Pacific (Zhu et al., 2009). The plants of Dendrobium species have been used as traditional or folk medicine in many Asian countries for thousands of years. For instance, there are 96 Dendrobium species in China, and about 30 species, known as shí hú (石斛) or shí hú lán (石斛 兰), have been widely used as ethnic medicine for tonifying the stomach, nourishing Yin (to enhance the production of body fluids, such as blood, saliva, tears, etc.), and clearing heat and toxic matter (Yang et al., 2006;Flora of China, 2020). The earliest written record of the medicinal usage of Dendrobium was found in the ancient text "Shen Nong's Herbal Classic" 2000 years ago, in which it was considered to be a "superior grade" herbal medicine. Hundreds of years later, Dendrobium was documented in detail in "Compendium of Materia Medica" in Ming Dynasty (1590 AD). Nowadays, Dendrobium nobile Lindl., Dendrobium chrysotoxum Lindl., Dendrobium fimbriatum Hook., Dendrobium officinale Kimura et Migo, and Dendrobium huoshanense Z. Z. Tang et S. J. Cheng are included in Chinese Pharmacopoeia (2020 edition). Among these five species, Dendrobium nobile Lindl. is one of the 50 fundamental herbs used in traditional Chinese medicine (TCM). Generally, the fibrous stems of Dendrobium are employed as the officinal parts in ethnopharmacology, persevered by dry processing (Chinese Pharmacopoeia Commission, 2020). These stems are usually used either alone or mixed with other tonic Chinese herbs, like x ı yáng sh en (American Ginseng) and gǒu qǐ zǐ (Barbary Wolferry Fruit) (Lin et al., 2018). Aside from China, Dendrobium species have also been used as ethnomedicines in Japan, Indian, and Thailand (Table 1). Given their high medicinal value and wide ethno-applications, the Dendrobium genus was recognized as a prized folk medicine (Ng et al., 2012).
Due to the important pharmacological activities and economical value of Dendrobium genus, up to now, many phytochemical and pharmacological researches have been implemented. The active constituents in Dendrobium are polysaccharides, alkaloids, flavonoids, amino acids, bibenzyls, and several trace elements (He et al., 2020). The polysaccharides from Dendrobium exhibit immunomodulatory and hepatoprotective activities; and the alkaloids are antioxidant, anticancer, and neuroprotective, while other compounds display anti-angiogenesis, anticytotoxicity, and anti-mutagenesis effects (Ng et al., 2012;Xu et al., 2013). Alkaloids are the earliest identified category of compounds in Dendrobium (Chen and Chen, 1935). More importantly, Dendrobium alkaloids are the key constituents that responsible for their pharmacological activities, making them potential candidates for new drugs. Therefore, some important bioactive markers such as dendrobine (20) have attracted many scientists to investigate their chemical, pharmaceutical, and biological mechanisms, as well as biogenetic pathways (Li Q. et al., 2017).
Dendrobium alkaloids with complex chemical structures consist of pyrrole, indolizidine, terpenoid alkaloids, organic amine alkaloids, indole, quinazoline, and others . In accordance with other genera of Orchidaceae plants, indolizidine alkaloids and organic amine alkaloids are the major constituents of this genus (Lam et al., 2015). These chemicals are considered as active ingredients for effects like antiinflammatory, cytotoxic, antitumor, cytoprotection, gluconeogenesis regulation, and preventing neuronal apoptosis (Ng et al., 2012). For instance, Dendrobium nobile Lindl. is a famous TCM recorded in Chinese Pharmacopoeia (2020 edition). The alkaloids of Dendrobium nobile Lindl. (DNLA) are considered to have beneficial effects on liver metabolism, hepatic lipid homeostasis, neuronal activity, and resistance effects on tumors, cancers, and virus based on previous studies ( Table 2). Dendrobine (20), a sesquiterpene alkaloid, makes up 92.6% of the DNLA (Xu et al., 2017). Dendrobine (20) is the first identified active alkaloid of Dendrobium nobile Lindl. (Chen and Chen, 1935), and is regarded as the standard agent for qualitative and quantitative evaluation of Dendrobium nobile Lindl. (Li R. et al., 2017).
In this review, we aim to summarize the structural types, pharmacological activities, and the mechanisms of biological activities of Dendrobium alkaloids. Additionally, the proposed biogenetic pathways of dendrobine (20) are also included.

STRUCTURAL IDENTIFICATION OF DENDROBIUM ALKALOIDS
Alkaloids are representatives of the first category of compounds extracted from Dendrobium . Dendrobium alkaloids were isolated by the traditional alkaloid extraction method given their basic chemical structure. Dried powders of Dendrobium spp. were liquid-liquid extracted with various solvents, such as ethanol, methanol, or chloroform, then fractionated successively with water, petroleum ether, ethyl acetate, n-butyl alcohol, etc. . Subsequently, these fractions were purified on different silica gel column chromatography systems with various polarity ranges of solvents (Morita et al., 2000). Moreover, high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) coupled with mass spectrometry were developed to discover new compounds of Dendrobium (Xu et al., 2020). Up to now, more than sixty alkaloids (Figures 1-5 (Inubushi et al., 1964;Lüning et al., 1965;Elander et al., 1969;Ekevag et al., 1973;Begum et al., 2010;Lam et al., 2015;Hu et al., 2016;Hu et al., 2016;Xu et al., 2019).

Amine Alkaloids
Amine alkaloids are a class of widely spread natural amines with basic nitrogen but cannot form a ring in the skeleton. Most Dendrobium devonianum Paxton (Zhang et al., 2013). Pierardine (61) was isolated from Dendrobium pierardii R. Br. (Elander et al., 1969).  (63) are another type of alkaloids isolated from two species of Dendrobium, whose structures were confirmed by X-ray crystallography (Hemscheidt and Spenser, 1993).

Metabolic Analysis of Dendrobium
At present, metabolomics has been widely utilized in the field of medicinal plants, such as bioactive components identification, drug metabolism, toxicology, and investigation on metabolic pathways, etc. (Liang et al., 2009). Alkaloids are regarded as chemical markers in quantitative analysis of Dendrobium. Generally, the metabolic profiling of Dendrobium alkaloid compounds was established by liquid chromatography coupled to single (LC-MS) and tandem (LC-MS/MS) mass spectrometry, in combination with multivariate data analyses, where secondary metabolites can be accurately quantified based on their fingerprint chromatograms. For example, the comparative metabolite analysis of Dendrobium officinale Kimura et Migo and Dendrobium huoshanense Z. Z. Tang et S. J. Cheng showed that the accumulation of alkaloids was species-specific (Song et al., 2020). Ten potential anti-inflammatory alkaloid components were detected from the extraction of Dendrobium aphyllum (Roxb.) C. E. C. Fisch by UPLC-MS , while eight water-soluble metabolites containing rare imidazolium alkaloids and anosmines (4) were identified by the screening of Dendrobium nobile Lindl., Dendrobium officinale Kimura et Migo, and Dendrobium loddigesii Rolfe, using chromatography along with spectroscopic techniques (Chen et al., 2018). Besides, DNLA was reported to improve hepatic lipid homeostasis based on the results of UPLC-MS of 48 kinds of hepatic bile acids in the livers of high fat diet (HFD)-fed mice . Furthermore, the combination of

PHARMACOLOGICAL ACTIVITIES
Dendrobium alkaloids are active components with antiinflammatory, antitumor, and anti-viral effects, which can also regulate hepatic lipid and gluconeogenesis, and protect from hyperglycemia. For a better understanding of the bioactivities of Dendrobium alkaloids, previous studies on pharmacological efficacy are summarized.

Anti-inflammatory Activity
Inflammation induced by endotoxin such as lipopolysaccharide (LPS), is an immune defense response of organisms to tissue injury and microbial agents (Guha and Mackman, 2001). Most anti-inflammatory activities were tested with the LPS-induced RAW264.7 model by evaluating the indices of nitric oxide (NO) production and the expression of inducible NO synthase (Chen et al., 2018). The anti-inflammatory activities of Dendrobium alkaloids have been reported. For example, anosmines (63) that were presented in four Dendrobium species exhibited inhibitory activity against NO production and inflammation in LPSactivated RAW264.7 cells without cytotoxic activity (Chen et al., 2018). Besides (+)-homocrepidine A [(+)-13] isolated from Dendrobium crepidatum Lindl. ex Paxton was evaluated for its anti-inflammatory activity (NO inhibition) with LPSinduced RAW 264.7 macrophages, and the half maximal inhibitory concentration (IC 50 ) value was 3.6 µM. However, the other enantiomeric isomer (-)-homocrepodine A [(-)-13], displayed an IC 50 value of 22.8 µM, which was almost 7 times less active than [(+)-13]. Besides, their racemic mixtures (±)-homocrepodine A [(±)-13], showed a moderate inhibitory effect (IC 50 5.0 µM). Similar pharmacological activities were observed in (±)-dendrocrepidamine A [(±)-14] (Hu et al., 2020). Compared with the enantiomers of racemic indolizidine and their racemic mixtures, homocrepidine B (15) also displayed moderate anti-inflammatory activity with the IC 50 value of 27.6 µM (Hu et al., 2016). Furthermore, the total alkaloids, mainly consisted of six indolizine-type compounds from the same Dendrobium species, showed protective effects against the LPS-induced acute lung injury in mice by the down-regulation of the TLR4-mediated MyD88/MAPK signaling pathway . Taken together, the pharmacological investigations of Dendrobium alkaloids on anti-inflammatory shed light on scientific guidance for the source of this genus.

Improved Regulation of Hepatic Lipid Homeostasis and Gluconeogenesis
The liver is quite essential to the regulation of lipid and glucose homeostasis. On the other hand, the disruption of homeostasis will result in metabolic disorders of the liver, including fatty liver and diabetes, which are the most common chronic liver disease all over the world (Rinella, 2015). DNLA was found to impact the regulation of liver glucose and the expressions of lipid metabolism genes in mice livers by increasing the expressions of PGC1a, Glut2, Cpt1a, Acox, ATGL/Pnpla2, and FoxO1 genes, and decreasing the mRNA transcription from the Srebp1 gene (Xu et al., 2017). Moreover, excessive accumulation of hepatic lipids is responsible for liver metabolic dysfunction. Modulation of bile acids has been reported as an effective intervention strategy for maintaining hepatic lipid homeostasis (Chiang, 2013). DNLA exerted protective effects on hepatic lipid homeostasis by enhancing taurine-conjugated bile acids and decreasing the cholic acid/chenodeoxycholic acid ratio . To be specific, DNLA decreased four types of bile acids and increased five types of bile acids among 48 kinds of hepatic bile acids in the livers of high-fat diet (HFD)-fed mice . On the other hand, DNLA regulated hepatic gluconeogenesis by mediating the hepatic antioxidant components through hepatic metallothionein and the gene expression of the nuclear factor erythroid 2-related factor 2 antioxidant pathway, which plays critical roles in host defense against abnormal gluconeogenesis (Xu et al., 2017). The mechanisms were further elucidated that DNLA improved mitochondrial function and inhibited mitochondrial apoptotic cell death . Overall, considering the beneficial effects of Dendrobium alkaloids on liver metabolism, Dendrobium alkaloids could be used as natural compounds in the development of new treatments for hyperlipidemia and hyperglycaemia.

Anti-tumor Activity
It was reported that Dendrobium alkaloids could inhibit tumor cell growth and mediate apoptosis. Song et al., 2019;Wang et al., 2014). Specifically, the alkaloid extracts of Dendrobium candidum Wall. ex Lindl. were reported to significantly inhibit the growth of transplanted Lewis tumors, meanwhile, the mixed alkaloids could improve the spleen index and regulate the expressions of TNF-α and IL-2 (Wang et al., 2014). Moreover, the fat-soluble alkaloids extracted from Dendrobium nobile Lindl. were found to induce the apoptosis of human colorectal cancer HT-29 cells with an IC 50 value of 0.72 mg/ml at 48 h, where the cell cycle was arrested in G2 phase. Besides, the extraction decreased the mitochondrial membrane potential (ΔΨm) and induced ROS accumulation by increasing expression levels of apoptotic proteins, such as Caspase-9, Caspase-3, and intracellular cytochrome C , which may be related to the mitochondria-mediated apoptotic pathway. The combined treatment using sesquiterpene alkaloids, dendrobine (20) and cisplatin, was also effective for inhibiting the non-small cell lung cancer cells (NSCLC) in vitro and in vivo, where the cytotoxicity was induced by the simulation of c-jun NH 2 -terminal kinase (JNK)/p38 stress signaling pathways, and the expression change of pro-apoptotic proteins Bax and Bim further led to the apoptosis (Figure 6, revised from song et al., 2019, created with BioRender.com). Besides, dendrobine (20) also mediated apoptotic cell death by the mitochondrial-mediated pathway (Song et al., 2019). On the whole, due to the distinct association with cell death signaling pathways, dendrobine (20) can be regarded as a potential agent for the development of novel anti-NSCLS strategies especially when combined with cisplatin. (Song et al., 2019).

Renal Protective and Anti-Diabetic Effects
In China, the dried stems of some Dendrobium species including Dendrobium huoshanense Z. Z. Tang et S. J. Cheng, Dendrobium officinale Kimura et Migo, and Dendrobium nobile Lindl. have been used to nourish kidney and improve the symptoms of diabetes (Cakova et al., 2020). For instance, shihunidine (2) and shihunine (1) isolated from Dendrobium loddigesii Rolfe displayed inhibitory effects on Na + /K + -ATPase of the rat kidney (Li et al., 1991). Li et al. (2019) recently reported that DNLA showed vital hypoglycemic effects in diabetic rats. The shihunine (1) extracts from Dendrobium loddigesii Rolfe at the dose of 50 mg/kg decreased the triglycerides level by 43.7%, compared with the non-treated db/db mice, and inhibited the expression of cleaved cysteine aspartic acid-specific protease 3. The result of western blot analysis also verified the agonistic effects of shihunine (1) extracts on the expressions of adenosine monophosphate-activated protein kinase phosphorylation and glucose transporter four in the liver or adipose tissues. Moreover, in clinical application, Dendrobium combined with other herbs, such as Astragalus spp. and Schisandra Michx., was applied for the therapy of diabetes (Cakova et al., 2020).

CHEMICAL SYNTHESIS AND BIOSYNTHETIC PATHWAY OF DENDROBINE
Dendrobine (20) is the first identified sesquiterpene alkaloid from Dendrobium nobile Lindl., which is recommended as the exclusive chemical marker for the quality control of this species by Chinese Pharmacopoeia (2015 and 2020 edition). The rule suggested that the mass fraction of dendrobine (20) should be greater than 0.4% in the medicinal Dendrobium nobile Lindl..

Chemical Synthesis of Dendrobine
Dendrobine (20) with a complicated tetracyclic ring system and seven contiguous stereocenters displayed remarkable bioactivities. Up to now, several cases are available on the total chemical synthesis of dendrobine (20). Connolly and Heathcock (1985) first synthesized dendrobine (20) in 1985. Several decades later, Kreis and Carreira (2012) achieved the total chemical synthesis based on 18 cascaded reactions with a key amine group, and the main synthesis pathway is summarized in Figure 7 ( Kreis and Carreira, 2012). Other three dendrobinealkaloids (-)-dendrobine (20) (-)-mubironine B (27), and (-)-dendroxine (24) were also obtained by total synthesis (Guo et al., 2018). Despite the advances of these total synthesis methods, it remains challenging to overcome the compound yield after a series of reactions (Li Q. et al., 2017).

Biosynthesis of Dendrobine
Dendrobine (20) belongs to the class of terpenoid indole alkaloids (TIAs) . The biogenetic pathway of TIAs is conservative among alkaloid-producing plants (Li Q. et al., 2017). Based on the results of transcriptome and metabolomic analysis, the putative dendrobine (20) biosynthetic pathway was proposed, and a series of key metabolic genes were labeled in Figure 8 ( Guo et al., 2013;Li Q. et al., 2017;Chen et al., 2019). Three core stages were involved in the biogenetic pathway, including the formation of isopentenyl diphosphate (IPP), the construction of sesquiterpene skeleton, and the process of post-modification. Firstly, the mevalonate (MVA) and 2-Cmethyl-D-erythritol 4-phosphate (MEP) pathways were considered as the upstream of dendrobine (20) biosynthetic pathway, mainly for the synthesis of IPP . Three key enzyme-coding genes involved in the MVA pathway, acetyl-CoA C-acetyltransferase (AACT) gene, phosphomevalonate kinase (PMK) gene, and diphosphomevalonate decarboxylase (MVD) gene, were observed to be positively associated with dendrobine (20) accumulation in Dendrobium nobile Lindl. through large-scale transcriptome sequencing, and then validated through qRT-PCR analysis (Li Q. et al., 2017). In contrast, hydroxymethylglutaryl-CoA synthase (HMGS) gene and 3hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene were found to be less effective in dendrobine (20) biosynthesis in the same species (Li Q. et al., 2017), though HMGS and HMGR both played significant roles in alkaloid biosynthesis in Dendrobium officinale Kimura et Migo . The result shows that HMGS and HMGR may differently contribute to the production of dendrobine (20) in Dendrobium spp.. In the MEP pathway, rate-determining genes 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) isolated from protocorms of Dendrobium officinale Kimura et Migo were largely up-regulated by the methyl jasmonate (MeJA) treatment, suggesting their significant roles in the sesquiterpene biosynthesis based on the analysis of KEGG enrichment and relative expression (Fan et al., 2016;Chen et al., 2019). The crucial impacts of DXS and DXR in Dendrobium officinale Kimura et Migo were later confirmed by the high correlations between total alkaloid contents and their transcripts , furthermore, DXS was a leaf-specific expression gene accounting for high alkaloids content in leaves (Shen et al., 2017). IPP is an important downstream product of MVA and MEP pathways, which is the precursor for the construction of synthetic terpenes. IPP formed the skeleton of muurolenetype sesquiterpene initially catalyzed by TPS21 enzyme (Li Q. et al., 2017), then this sesquiterpene was further oxidized by monooxygenases and/or dioxygenase to produce picrotoxanelactone. Cytochromes P450s (CYP450s) is a complex superfamily of monooxygenase, and they are vital for the formation of sesquiterpene alkaloids (dendrobine). At present, some CYP450s have been discovered in a few Dendrobium species (Coon, 2005;Guo et al., 2013;Li Q. et al., 2017;Yuan et al., 2018;Chen et al., 2019). For instance, 59 full-length CYP450s candidate genes involved in the dendrobine (20) biosynthesis were identified and characterized in Dendrobium officinale Kimura et Migo through tissue-specific transcriptomic analysis, phylogenetic analysis, and further gene expression pattern induced by MeJA treatment . In Dendrobium huoshanense Z. Z. Tang et S. J. Cheng, 229 genes were identified as putative CYP450s, 7.8% of which were CYP71 family members  Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 605994 11 associated with hydroxylation steps of alkaloid biosynthesis (Yuan et al., 2018). However, the family members and expression patterns of CYP450s remain unclear in most Dendrobium plants. It is worth mentioning that all other 25 dendrobine-type alkaloids (20-42, 51-52) identified from Dendrobium were believed to share similar biosynthesis pathways due to the mutual sesquiterpene backbone of these alkaloids (Guo et al., 2013;Chen et al., 2019).
Although the common biosynthesis pathway for most TIAs through the construction of strictosidine backbone exists in many plants , no enzyme involved in strictosidine formation has been verified in dendrobine (20) biosynthesis. However, due to the complex dendrobine (20) metabolism, accurate identification of genetic networks from a large number of candidate genes is needed in the future.
The metabolism of dendrobine (20) was affected by abiotic and biotic stresses. For example, light intensity was reported to influence the content of dendrobine (20) . MeJA, a signaling molecule in the biosynthesis of alkaloids, could induce the accumulation of Dendrobium alkaloids by an active precursor supply . Besides, symbiosis with mycorrhizal fungus could stimulate the biosynthesis of dendrobine (20) by regulating the expressions of genes involved in the MVA pathway (Li Q. et al., 2017). Other relevant factors need to be further elucidated.

CONCLUSION
In this paper, we summarized the structural types, pharmacological activities, and mechanisms of Dendrobium alkaloids, as well as the suggested biogenetic pathway of dendrobine (20), which is an important type of sesquiterpene alkaloids. Despite the advances of the investigation on alkaloids, more emphasis should be laid on the discovery of more novel skeletons in Dendrobium genus based on abundant alkaloid metabolites, and the improvement of isolation methods. Moreover, many current studies on Dendrobium were only focused on their crude extracts, or the activity of mixtures, which necessitates the need for figuring out the typical pharmacological activity of pure Dendrobium alkaloids. Additionally, further investigation on novel pharmacological activities of these alkaloids should be implemented. Meanwhile, in-depth researches on the biological mechanisms of these activities are also desired. Finally, although the biosynthetic pathway of dendrobine (20) has been proposed, further confirmation is anticipated.

AUTHOR CONTRIBUTIONS
DZ and SC contributed to the conception of the review. YZ and EK helped collect and perform the chemical analysis of compounds. FY helped to analyze the pharmacological activities of alkaloids. ZM and YS contributed to the mechanisms and biosynthetic pathways of alkaloids and wrote the manuscript.

FUNDING
This work was supported financially by the National Natural Science Foundation of China (No. 31960082;No. 81560622).