Traditional Uses, Phytochemistry, Pharmacology, and Quality Control of Dendrobium officinale Kimura et. Migo

Dendrobium officinale, a well-known plant used as a medicinal and food homologous product, has been reported to contain various bioactive components, such as polysaccharides, bibenzyls, phenanthrenes, and flavonoids. It is also widely used as a traditional medicine to strengthen “Yin”, nourish heart, tonify five viscera, remove arthralgia, relieve fatigue, thicken stomach, lighten body, and prolong life span. These traditional applications are in consistent with modern pharmacological studies, which have demonstrated that D. officinale exhibits various biological functions, such as cardioprotective, anti-tumor, gastrointestinal protective, anti-diabetes, immunomodulatory, anti-aging, and anti-osteoporosis effects. In this review, we summarize the research progress of D. officinale from November 2016 to May 2021 and aim to better understand the botany, traditional use, phytochemistry, and pharmacology of D. officinale, as well as its quality control and safety. This work presents the development status of D. officinale, analyzes gaps in the current research on D. officinale, and raises the corresponding solutions to provide references and potential directions for further studies of D. officinale.


INTRODUCTION
The genus Dendrobium (Orchidaceae) includes more than 1,000 species (Teixeira and Ng, 2017). Among them, Dendrobium officinale Kimura et. Migo is a precious medicinal plant recorded in Chinese Pharmacopoeia, which is widely used as a traditional Chinese medicine (Editorial Board of China Pharmacopoeia Committee, 2020). Moreover, there is a synonym of "Dendrobium catenatum Lindl." in the plant list (http://ipni.org/urn:lsid:ipni.org:names:628140-1). It is well known that D. officinale is distributed in several countries around the world, such as the United States, Japan, and Australia. In particular, D. officinale exhibits a broader distribution in the different regions of China, including Anhui, Zhejiang, Hunan, Fujian, Guangxi, Sichuan, and Yunnan Provinces . Due to the overexploitation and depletion of its wild plant resources, it has been considered as a secondary endangered plant in the "China Plant Red Data Book" (Fu, 1992). On the other hand, the artificial cultivation technology of D. officinale has made a significant breakthrough (Cheng Y et al., 2019). However, the existing cultivation resources of D. officinale are mixed, which results in the uneven product quality and the unsound evaluation system, greatly affecting the practical and reasonable development and utilization of D. officinale.

Bibenzyls
Bibenzyl components in D. officinale have attracted a lot of attention due to their promising anti-tumor properties . To date, 19 bibenzyls have been isolated, which were identified predominantly from the stems and leaves of D. officinale. The typical parent nucleus structure of bibenzyls presented in the stems of D. officinale is two benzene rings, which can be replaced by different substituents. Some bibenzyls (4, 5, and 7) were found to have significant anti-tumor activity (Tian et al., 2019;Zhao et al., 2020), which attracted further research and development. The chemical structures of these bibenzyls are shown in Figure 2.

Flavonoids
It has been reported that flavonoids belong to a large group of secondary metabolites in D. officinale, which possess anti-tumor activity (Xing et al., 2018). Frontiers in Pharmacology | www.frontiersin.org August 2021 | Volume 12 | Article 726528 7 the red color stems of D. officinale . In addition, the transcriptome and component metabolism analyses have become the typical approaches to investigate flavonoid biosynthesis mechanisms in D. officinale . The chemical structures of these flavonoids are shown in Figures 5-7.

Alkaloids
Alkaloids are a class of nitrogenous organic compounds possessing various biological activities. They are also active constituents of Dendrobium plants. It is worth noting that the regulation of genes related to alkaloid biosynthetic pathways through comparative transcriptomic analysis has gradually become a research interest (Jiao et al., 2018). The chemical structures of these alkaloids are shown in Figure 8.

Organic Acids
Organic acids are a kind of acid organic compounds containing a carboxyl group. Eight types of organic acids have been identified in D. officinale. The chemical structures of these organic acids are displayed in Figure 9.

Cardioprotective Activity
Cardiovascular diseases are the major causes of mortality globally, and the main cause of more than 10 million people death. Among cardiovascular diseases risk factors, cardiomyopathy and high blood pressure are the most common ones. Oral administration of D. officinale fine powders at the doses of 0.09, 0.18, and a very high dose of 1.1 g/kg for 30 days protected isoproterenol (ISO)induced cardiac hypertrophy, indicated by the decreased myocardial collagen synthesis, increased myocardial fibrosis and ventricular remodeling, and significantly reduced levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and cardiac troponin I (cTN-I) in plasma relative to the model group (ISO 5 mg/kg) (Xiao et al., 2018). In addition, it was well known that diabetic cardiomyopathy was a typical cardiovascular complication mediated via hyperglycemia. One study indicated that D. officinale water-soluble extracts prevented diabetic cardiomyopathy and might be a candidate for therapeutic use . D. officinale water extracts (75, 150, and 300 mg/kg) intragastrically once daily for 2 weeks could protect left anterior descending coronary artery (LAD)-induced myocardial ischemia through decreasing creatine kinase (CK)-MB, lactate dehydrogenase (LDH), malondialdehyde (MDA), and increasing superoxide dismutase (SOD) and Meis 1 levels . Treatment with D. officinale stems polysaccharide DOP-GY at the doses of 6.25, 12.5, and 25 µg/ ml exhibited protective effects on hydrogen peroxide (H 2 O 2 )induced H9c2 cardiomyocyte apoptosis via phosphatidylinositol/ protein kinase B (PI3K)/Akt and mitogen-activited protein kinase (MAPK) signaling pathways, as demonstrated by the decreased levels of LDH, lipid peroxidation damage (LPD), reactive oxygen species (ROS), and pro-apoptosis protein (Zhang et al., 2017a). Another study demonstrated that D. officinale polysaccharides could decrease malondialdehyde levels, increase SOD activities, and inhibit the generation of intracellular ROS in H9c2 cardiomyocytes (Zhao et al., 2017). Besides, Yue et al. (2017) obtained a novel homogeneous heteroxylan from alkali-extracted D. officinale crude polysaccharide (S32), which possessed significant anti-angiogenic effects (S32, 13.51 μM) on human microvascular endothelial cells (HMEC-1) by inhibiting their migration and disruption of tube formation in a dosedependent manner, compared with a vehicle group (Yue et al., 2017). However, some of these experimental doses were too high and there was a lack of positive controls.
It has been suggested that D. officinale has cardioprotective activity by treating hypertension. It was reported that blood pressure was significantly reduced after treating with D. officinale (10 g/d) (Wu et al., 2018b). The treatment of D. officinale ultrafine powder (DOFP) at very high doses of 200 and 400 mg/kg for 20 weeks exhibited anti-hypertensive activity on overeating greasy-induced metabolic hypertension in rats by inhibiting the activation of lipopolysaccharide/toll-like receptor 4 (LPS/TLR4) signal pathway, as demonstrated by the decreased levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-c), LPS, C-reactive protein (CRP), interleukin 6 (IL-6), TLR4, myeloid differentiation factor (MyD88), IL-1β, and tumor necrosis factor alpha (TNF-α) and the increased levels of high-density lipoprotein cholesterol (HDL-c) and nitric oxide (NO) relative to valsartan (8 mg/kg)-treated positive control (Su et al., 2021). Moreover, Yan et al. (2019a) reported that the effective component of alcohol extract of D. officinale in the treatment of metabolic hypertension was apigenin flavonoid glycosides (Yan et al., 2019a). In contrast, more investigations are still needed to reveal the underlying mechanism of the antihypertensive effect of apigenin flavonoid glycosides. The results showed that treatment with D. officinale flowers at a very high dose of 3.1 g/kg for 6 weeks could significantly improve vascular diastolic function by reducing systolic blood pressure and mean arterial pressure in high glucose and fat compound alcohol-induced hypertensive rats, inhibiting the thickening of thoracic aorta and the loss of endothelial cells, reducing plasma content of endothelin 1 (ET-1) and thromboxane B2 (TXB2), and increasing the content of prostacycline (PGI2) and NO, compared with model control group and valsartan (5.7 mg/kg) positive control group (Liang K. L et al., 2018). Treating with D. officinale ultrafine powder DOFP could improve the intestinal flora and increase the production, transportation, and utilization of short-chain fatty acid (SCFA), activate the intestinal-vascular axis SCFA-GPCR43/41 pathway, increase vascular endothelial function, and finally decrease the blood pressure in alcohol, and high sugar and fat diets (ACHSFD)-induced metabolic hypertension model rats . These results suggested that D. officinale may have a potential clinical application in the treatment of hypertension. In this study, however, the optimal dose, constituents, and side effects of D. officinale are not assessed. Moreover, further detailed clinical trials should be employed to assess the value of D. officinale as a drug for the treatment of hypertension. In addition, this evidence is still tenuous; no double-blind trials involving D. officinale have been performed, and more evidence from randomized controlled trials is required to elucidate other mechanisms that may be responsible for anti-hypertension effects. Although D. officinale possesses a potential therapeutic effect on cardiovascular diseases, especially cardiomyopathy and hypertension (Figure 12), more in-depth investigations on its effective monomer compounds, molecular mechanism, and clinical trials are warranted to identify effective cardioprotective agents with minimized side effects.

Anti-Tumor Activity
As a type of traditional medicine and ordinary food, D. officinale has benefits on human health supported by its effectiveness in the prevention and treatment of cancer diseases . Several studies have reported all crude extracts, polysaccharides, and other pure compounds isolated from D. officinale exhibited anti-tumor activities Guo et al., 2019;Zhao et al., 2020). Administration of D. officinale methanol extracts at a dose of 0.25, 0.5, and 1 mg/ml could inhibit the growth of SMMC-7721, BEL-7404 cells, and primary liver cancer cells, and promote their apoptosis via activating mitochondria apoptosis pathway and suppressing the Wnt/β-catenin pathway . Similarly, another study demonstrated that D. officinale polysaccharide (DOPA-1) induced HepG-2 cell apoptosis by influencing mitochondrial function, ROS production, and apoptosis-related protein expression . Importantly, a rat study also confirmed the anti-tumor effects of D. officinale in vivo. The 2-weeks administration of D. officinale polysaccharide DOP at very doses of 2.4, 4.8, and 9.6 g/kg suppressed 1-methyl-2-nitro-1-nitrosoguanidine-(MNNG)induced (150 µg/ml) precancerous lesions of gastric cancer in rats via modulating Wnt/β-catenin pathway and altering endogenous serum metabolites . In addition to crude extracts and polysaccharides, it is more evident that gigantol (4), moscatilin (5), erianin (7), orchinol (25), and isoviolanthin (71) isolated from D. officinale are also responsible for the anticancer activity of D. officinale (Xing et al., 2018;Liu et al., 2019;Lee et al., 2020;Zhao et al., 2020). Gigantol (4) was found to repress invasiveness and growth of SW780, 5,637, and T24 human bladder cancer cells by inhibiting the Wnt/epithelial-mesenchymal transition (EMT) signaling . Likewise, moscatilin (5) was demonstrated to induce apoptosis in FaDu human head and neck squamous carcinoma cells (HNSCC) via c-Jun N-terminal kinase (JNK) signaling pathway (Lee et al., 2020). The anti-tumor function of erianin (7) was investigated by two independent studies. The results suggest that erianin (7) induced cell apoptosis through the   Frontiers in Pharmacology | www.frontiersin.org August 2021 | Volume 12 | Article 726528 13 cell proliferation, repressing tumor cell migration and invasion, and improving body immunity ( Figure 13). The studies described above suggest that the compounds identified in D. officinale demonstrate significant inhibitory effects on different types of tumor cells, such as SMMC-7721, BEL-7404, HepG-2, SW780, and HCC cell lines. Further investigations may explore the structural modification and the structure-activity relationship (SAR) studies for these bioactive compounds of D. officinale, thereby facilitating the further discovery and development of new anti-tumor candidates, along with the characterization of the key regulatory genes, metabolic pathways, and heterologous biosynthesis pathways of active ingredients from D. officinale. The currently published studies on the anti-tumor effects of the compounds from D. officinale are mainly focused on in vitro and in vivo experiments, while clinical studies have yet to be conducted and the exact molecular mechanisms remain elusive.

Gastrointestinal Protective Effect
D. officinale is traditionally used to nourish "Yin" and thicken stomach. D. officinale extracts have thus been frequently applied to treat gastrointestinal diseases as a traditional Chinese medicine. It was reported that polyphenols from fermentation liquid of D. officinale improved intestinal health via the regulation of intestinal microbiota and their metabolites, thereby relieving oxazolone-induced intestinal inflammation in Zebrafish (Gong et al., 2020). Moreover, D. officinale is often used in combination with other traditional Chinese medicines to achieve its better therapeutic effects. It was found that the mixture of D. officinale and American ginseng at a dose of (0.32 × the dog's weight × 6 g)/ 12 kg) could function as a prebiotic agent to enhance SCFAproducing genera and reverse gut dysbiosis . Likewise, the combination of D. officinale and other Chinese herbal medicine, such as Acanthopanax senticosus, Panaxnotoginseng, Didymocarpus hancei and Valeriana officinalis can also alleviate gastric mucosal injury (Guo, 2020b).
The 14-days treatment of D. officinale glucomannans at 0.16 g/kg produced more SCFAs (mainly acetate and butyrate) in cecum and colon (Shi et al., 2020). It should be noted that polysaccharides LDOP-1 isolated from D. officinale could protect ethanol-induced gastric mucosal injury in vitro (250, 125, and 62.5 mg/ml for 2 h) and in vivo (100 and a very high dose of 400 mg/kg for 30 days) by regulating AMP-activated protein kinase (AMPK)/mTOR signaling pathway demonstrated by the increased levels of p-AMPK, light chain 3β (LC-3β), heme oxygenase-1 (HO-1) and Beclin-1, the decreased levels of p-mTOR and p62, and the reversed levels of caspase3, Bax, and Bcl-2 detected both in vitro and in vivo (Ke et al., 2020). Furthermore, it was suggested that D. officinale polysaccharide (200 mg/kg/d) exhibited protective effects against DSS-induced colitis by inhibiting pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the colonic mucosa, modulating the abundance of gut microbiota, and promoting the production and utilization of SCFAs in the colon . However, positive control or dose-dependent effect analysis was not performed, which might require further validation. In addition, the structure of the correlation between polysaccharide administration and health outcomes, as well as the functional role of the polysaccharides themselves has not been examined in depth.

Anti-Diabetes Activity
Diabetes is a common disease with glucose metabolism disorder, which seriously affects human health around the world . Numerous ethnomedicinal studies supported the traditional use of D. officinale for clearing heat, nourishing "Yin", benefiting the stomach, and promoting body fluid, thereby leading D. officinale to become an essential medicine to treat "Xiao Ke" disease (Diabetes) for thousands of years (Yan et al., 2019b).
Over the past few years, the hypoglycemic effect of D. officinale has become an attractive research field. One study has confirmed that the administration of water extracts from the stems of D. officinale at the doses of 75, 150, and a very high dose of 300 mg/kg for 12 weeks decreased serum insulin, TC, TG, LDL-c, and increase HDL-c in high-fat diet/streptozotocin (HFD/STZ) (30 mg/kg)-induced diabetic mice in a dosedependent manner (Zeng et al., 2020). The possible mechanism of D. officinale water extracts can be associated with improving lipid transport and suppressing insulin resistance and fibrosis via EMT. Another study similarly found that a 4-weeks treatment with D. officinale water extracts at very high doses of 350 and 700 mg/kg elevated the liver glycogen synthesis, energy, and amino acid metabolism as well as taurine-mediated defense against oxidative stress in STZinduced diabetic mice (Zheng et al., 2017). It was found that the   in STZ-induced (60 mg/kg) rats via suppressing vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT-1), and connective tissue growth factor (CTGF) relative to the irbesartan (17.5 mg/kg)-treated positive control (Chang et al., 2019). Additionally, a previous study also revealed that the hypoglycemic effect of the 3-months combined treatment of D. officinale (10 g/d) and metformin (0.5 g/d) was superior to the single-use . However, these studies have important limitations-only one or two doses of D. officinale are used in these studies. In addition, the evaluation of D. officinale doses that are much higher, and it has no translational value from a therapeutic viewpoint.

Immunomodulatory Activity
A large number of experiments have provided evidence for the immunomodulatory activity of D. officinale. Polysaccharide is the main active component for immunoregulation. Oral administration of 0.25% D. officinale polysaccharide DOW-5B (w/v) for 25 days displayed significant immunomodulatory effects via increasing the content of butyrate, immunoglobulin M (IgM), IL-10, and TNF-α in vivo (Li et al., 2020a). Another study also demonstrated that D. officinale polysaccharide DOP-1-1 stimulated immunity by enhancing the level of nuclear factor kappa-B (NF-κB) while inhibiting the level of IκBα through TLR4 signaling . Moreover, it was found that D. officinale polysaccharides (FDP-1) treatments, ranging from 12.5 to 200 µg/ml, exhibited immunomodulatory activity through increasing cell proliferation and NO and IL-1β production in a dose-dependent manner (Tian et al., 2019). Neither positive control nor dose-effect analysis in vivo was assessed in these studies. In addition, there is a general lack of systematic research on the relationship between immune activity and structure of D. officinale polysaccharides.

Anti-Aging Activity
The oral administration with a very high dose of 1 g/kg of D. officinale juice and a very high dose of 0.32 g/kg of D. officinale polysaccharide for 9 weeks exhibited an anti-aging effect in D-galactose-induced (0.125 g/kg) aging mice, supported by the significantly increased contents of SOD, glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) in serum, as well as the enhanced SOD level in heart, liver, kidney, and cerebrum (Liang et al., 2017). Similar to this study, the treatment with D. officinale polysaccharide (DOP) at the doses of 50 and 100 mg/kg for 4 weeks exhibited more potent antifatigue activity than Rhodiola rosea extract as a positive control in BALB/c mice, as revealed by the increased TG (or fat) mobilization and the decreased lipid oxidation (LOD) and cell variability of T and B lymphocytes in the weight-loaded swimming test (Wei et al., 2017). However, the underlying mechanisms by which D. officinale's anti-aging effects remain unclear. Additionally, the current pharmacological research lacks component analysis and clinical pharmacological experiments.

Anti-Osteoporosis Activity
A previous study revealed that the administration of D. officinale water extracts at very high doses of 150, 300, and 600 mg/kg for 13 weeks prevented ovariectomy (OVX)-induced bone loss in Wistar rats by decreasing the levels of TG, alkaline phosphatase (ALP), bone glucose protein (BGP) while increasing acid phosphatase (ACP) and bone mineral density (BMD) in comparison with Xian-Ling-Gu-Bao capsule (240 mg/kg) as a positive control . Meanwhile, D. officinale water extract treatments (10, 40, 80 μg/ml) were found to suppress receptor activator expression of the nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells . In addition, D. officinale polysaccharide DOP treatments at very high doses of 200 or 400 µg/ml exhibited an antiosteoporosis effect through the activation of NRF2 signaling, thereby attenuating adipogenic differentiation and promoting osteogenic differentiation in BMSCs (Peng et al., 2019). Notably, no reports about the anti-osteoporosis effect of small molecule compounds from D. officinale have been documented so far.

Protective Effects on Liver and Kidney
One study has demonstrated that the treatment with D. officinale ethanol extracts at the doses of 4.375 and 17.5 mg/kg for 9 weeks prevented liver and kidney damage in hyperuricemic rats by suppressing the protein levels of NF-κB and TLR4 compared with the model group (0.15% adenine, 10% yeast extract, and 89.85% standard diet) (Lou et al., 2020). Likewise, another study revealed that D. officinale flower water extracts (50, 100, 150, and 200 mg/kg) showed protective effects on alcohol-induced (10 ml/kg) liver injury by its anti-steatosis, anti-oxidative, and anti-inflammatory effects . However, additional evidence from randomized controlled trials is required to identify other regulatory mechanisms that may be responsible for the protective effects on liver and kidney. The bioactive constituents of these extracts also remain unknown.

Other Activities
In addition to the bioactivities described above, D. officinale was also found to have other therapeutic effects, such as neuroprotective effect, anti-photoaging effect, and pulmonary protective function. For instance, in hypoxic-ischemic brain damage (HIBD) neonatal rat model (vehicle group, normal saline 10 ml/kg), the administration of aqueous extracts of D. officinale at the doses of 75, 150, and 300 mg/kg for 14 days suppressed the neuronal apoptosis by reducing cleaved caspase-3 and Bax, increasing Bcl-2, enhancing the expression of neurotrophic factors and K + -Cl-cotransporter 2 (KCC2), and decreasing the expression of hypoxia-inducible factor-1α (HIF-1α) and histone deacetylase 1 (HDAC1), leading to neuroprotective effects in neonatal rats against HIBD (Li and Hong, 2020b). D. officinale protocorm treatments at the doses of 10, 25, and 50 mg/ml exerted an anti-photoaging effect through decreasing erythema and protected skin from dryness by increasing CAT, SOD, and GSH-Px expression levels and decreasing thiobarbituric acid reactive substances (TBARS) and MMPs levels relative to the model group (UV irradiation) and positive control group (UV irradiation and a formulation of matrixyl) (Mai et al., 2019). In addition, D. officinale polysaccharides prevented lung injury by ameliorating cigarette smoke-induced mucus hypersecretion and viscosity by decreasing the expression of mucin-5AC (MUC5AC) mRNA and secretory protein in vitro and in vivo .

QUALITY CONTROL
The wild resources of D. officinale have gradually decreased, while the supply of artificially cultivated D. officinale has increased correspondingly. Consequently, wild resource collection has progressively become a non-mainstream, and the artificial cultivation mode occupies a dominant position in D. officinale industry (Ni et al., 2018). According to the description in the Editorial Board of Chinese Pharmacopoeia (2020) edition, the peak area ratio of mannose to glucose, moisture content, total ash content, ethanol extract, and polysaccharide content should reach 2.4-8.0%, less than 12.0%, more than 6.0, 6.5, and 25.0% of D. officinale stem, respectively (China Pharmacopoeia Committee, 2020). However, the quality of D. officinale may be affected by regions, tissues, harvest time, cultivation techniques, growth years, endophytes, and others which may disturb the long growth cycle (Cheng J et al., 2019). For instance, Li et al. (2017a) found that the contents of polysaccharides in D. officinale from Yunnan, Fujian, Jiangsu, and Zhejiang were variable (Li et al., 2017b). Among them, the highest content of polysaccharides was 54.42% in Zhejiang, followed by 43.26% in Fujian (Li et al., 2017a). Previous studies have revealed that the contents of polysaccharides in stems, leaves, and flowers of D. officinale are 34.61%, 23.51%, and 13.47%, respectively . It was also found that the polysaccharide content of D. officinale is gradually increased during the entire flower-opening process from buds to full bloom, in which the polysaccharide content is the highest in the full bloom stage (13.75%), followed by the micro bloom stage (11.52%). In contrast, the lowest content appears in the bud stage (9.50%) . In addition, D. officinale is usually planted on trees (fixed to the trunk with fine twine). Therefore, the polysaccharide content of D. officinale is significantly affected by different auxiliary tree species. Those planted on the evergreen tree Phoebe zhennan as the accessory hosts exhibit the highest polysaccharide content (37.8%) substantially different from those grown on the Michelia ilsonii, Davidia nvolucrate and Taxus chinensis var. mairei (Gu and Xie, 2021). Moreover, the polysaccharide content of 5-yearold D. officinale stems is the highest, followed by 3-year-old, 4year-old, 2-year-old, and 1-year-old ones (Qin et al., 2017). Besides, endophytes play an important role in promoting the accumulation of polysaccharides. For example, DO14 (Pestalotiopsis sp.) isolated from D. officinale treated with 240 mg/L protein-polysaccharide fractions (PPF) can promote the accumulation of polysaccharides. This endophytic fungus could be used as biological fertilizer to improve the yield and quality of D. officinale . In addition, exploiting suitable artificial-sheltered cultivation mode, screening the best cultivation substrate, and developing aseptic germination technology could be utilized to enhance the quality production of D. officinale in current agronomical practices (Cheng Y et al., 2019;Zuo et al., 2020).
It is well known that the polysaccharides' bioactivities vary from different sources, production regions, and cultivation conditions of D. officinale. Moreover, it has been reported that the bioactivities of D. officinale polysaccharides are related to their chemical characteristics and advanced structures. However, D. officinale polysaccharides are macromolecular compounds with large molecular weight and complex structure, thereby generating a great challenge to implement analytical technologies (Ma et al., 2018). Therefore, it is necessary to establish a safe and effective quality assessment method for the quality control and clinical application of D. officinale. Herein, the qualitative and quantitative analytic methods of D. officinale polysaccharides are summarized and discussed.
There are several rapid and accurate methods for quantitative estimation of natural polysaccharides and their different fractions in D. officinale (Wu et al., 2018a). Generally, the large D. officinale polysaccharides are acid hydrolyzed into oligosaccharides, and then LC-MS is used to separate and characterize the products efficiently. The previous results indicate that the variations in the mass values of different peaks present structural differences of various metabolized products. Moreover, MS can be used to explore the oligosaccharide hydrolysates of D. officinale polysaccharides in detail from the aspects of identity, structure, and properties (Ma et al., 2018). An oligosaccharidemarker approach by labeling them with fluorescence reagent paminobenzoic acid ethyl ester (ABEE) (Te-Man-ABEE and Pen-Man-ABEE) was recently applied for quality assessment of D. officinale polysaccharides using UHPLC-QTOF-MS (Wong et al., 2019). The results revealed that the two oligosaccharide markers exhibited a satisfactory linearity relationship with D. officinale polysaccharides (R 2 ≥ 0.997) in the range of 0.68-16.02 µg. These markers also revealed satisfactory precision (relative standard deviation, RSD <7.0%) and recovery (91.41-118.30%) in unknown sample determination. It is speculated that the oligosaccharide-marker method is a simple, rapid, and reliable approach for the qualitative and quantitative determination of specific polysaccharides from D. officinale and other herb formulas (Wong et al., 2019).
Although single-component quality assessment can be used to control the quality of natural Chinese herbal medicine (Wei et al., 2020), the chemical compositions of D. officinale are complex. D. officinale contains a lot of effective nutritional compositions, including flavonoids, crude fiber, amino acids, proteins, and fat. These active components of D. officinale are affected by tissues and harvest times. A previous study found that the contents of flavonoids in the stems, leaves, and flowers of D. officinale were 0.052%, 0.251%, and 1.835%, respectively (Li et al., 2019a). Another research reported that the content of total flavonoids in the flowers of D. officinale is the highest at the full flowering stage (1.66%) followed by the bract stage (1.52%) and micro flowering stage (1.41%) . It should be noted that the contents of five representative flavonoid glucosides from D. officinale in 25 batches with different sources were determined by UHPLC-ESI-MS/MS, where principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied . The content of crude fiber in the autumn stem is 59.7% higher than that in the spring stem, and the fiber in the autumn leaf is 122.9% higher than that in the spring leaf (Li et al., 2017a). The total protein content of D. officinale stems in autumn is 10.9% higher than that in spring, while that of the leaves in autumn is 9.3% higher than that in spring. The results also revealed that the total protein content in the leaves is higher than that in the stems, and the ratio of total protein in the autumn leaves is 67.03 mg/g, which is 31.4% higher than that in the autumn stems (51.02 mg/g). The content of fat in the stems and leaves of D. officinale ranges from 10.0 to 15.0 mg/g. The average content of fat in the leaves (13.90 mg/g) is slightly higher than that in the stems (11.50 mg/g) . In particular, Wu and Feng (2019) revealed that the content of polysaccharides is the highest in those samples collected from October to the following March (Wu and Feng, 2019). Yu et al. (2014) found that it is more reasonable to harvest D. officinale at biennials pre-bloom than at specific harvesting months according to the content of polysaccharides . According to the Editorial Board of Chinese Pharmacopoeia (2020) edition, however, D. officinale should be harvested from November to the following March (China Pharmacopoeia Committee, 2020).
Additionally, there are some noteworthy scientific gaps, which can be resolved from the following aspects. First, the genuine, defective, and counterfeit varieties of D. officinale are mixed together, so there is an urgent need to breed varieties with better agronomic characters, high yield, high quality, and intense stress resistance. . Second, it is also an effective way to ensure the quality of D. officinale by controlling pesticide residues and using endophytes to control diseases and pests (Yu et al., 2014;Zhou et al., 2018). Third, at present, chemical methods are primarily used to evaluate the quality of D. officinale, while the quality evaluation of traditional Chinese medicine is closely related to its biological activity. Therefore, the evaluation methods are suggested to be improved by combining chemical approaches and bioassays, providing a foundation for the industrial production and clinic use of D. officinale (Wei et al., 2020).

SAFETY
The stems and leaves of D. officinale were approved by National Health and Family Planning Commission (NHFPC) of People's Republic (PR) of China to be utilized as a novel food material on Jan 15, 2013 and Jan 15, 2017 (National Health and Family Planning Commission of the People's Republic of China, 2013; National Health and Family Planning Commission of the People's Republic of China, 2017), respectively. Therefore, several safety studies of D. officinale have been performed. It has been reported that acute toxicity test (12.0 g/kg), genetic toxicity tests (Ames test, micronucleus test of bone marrow, and sperm shape abnormality test in mice) (1,000, 2000, and 4,000 mg/kg), and 90-days feeding test (1.08, 1.67, and 5.00 g/kg) in rats were employed to assess the safety of the stems from D. officinale. These results indicated that D. officinale was a type of health food product without noticeable toxicity, genetic toxicity, and mutagenicity within the range of the test doses . Moreover, oral administration with D. officinale stems at the doses of 25, 1,250, and 2,500 mg/kg did not exhibit any apparent effect on pregnant rats or deformity effect on fetal rats (Qin et al., 2019). Likewise, oral administration with the leaves and flowers of D. officinale at the doses of 0, 2.0, 4.0, and 6.4 g/kg for 90 days did not exhibit apparent adverse effects on sperm quality and testicular tissue morphology in parent and offspring rats (Fu et al., 2017;Fu et al., 2020a). Besides, D. officinale flowers (0, 2.0, 6.4 g/kg) had no apparent adverse effects on pregnant and offspring rats before birth (Fu et al., 2020b).
D. officinale is considered to have edible and medicinal values. However, due to its thermal tonic property, it is prohibited for patients with wind-heat cold, dampness, and allergies, teenagers, and pregnant women. According to the Pharmacopoeia of the People's Republic of China (China Pharmacopoeia Committee, 2020), the dosage of 6-12 g/d of D. officinale is appropriate, and intake of trace elements recommended by the Food and Drug Administration of the United States will not induce poisonous effects.

CONCLUSION AND FUTURE PROSPECTS
D. officinale, as a medicinal or food homologous product, plays a crucial role in healthcare. This study summarizes and updates the botany, traditional uses, bioactive components, pharmacology, quality control, and safety of D. officinale. Available data indicate that over 120 compounds have been isolated and identified from D. officinale, including polysaccharides, bibenzyls, flavonoids, alkaloids, phenanthrenes, etc. D. officinale is associated with multiple beneficial pharmacological properties, such as cardioprotective, anti-tumor, gastrointestinal protective, anti-diabetes, anti-aging, and anti-osteoporosis activities. Furthermore, it is evident that D. officinale is a non-toxic, which can be listed as a toxicologically safe functional food. However, its clinical applications has been rarely described, and critical improvements are still required for its industrial applications.
Firstly, phytochemical studies have demonstrated that D. officinale mainly contains polysaccharides, bibenzyls, phenanthrenes, and flavonoids while little is known about the analysis and function of organic compounds such as protein and fatty acid. Growing evidence has shown that structure-based drug design plays a vital role in developing novel drugs, and a series of strategies can be adopted to obtain effective therapeutic drugs. Further studies need to be conducted to isolate and identify more compounds from D. officinale with novel structures, emphasizing on bioactivity-guided, structurally modified, and chemically synthesized molecules. Besides, most of the D. officinale containing health products are mainly derived from its stems rich in chemical compounds, while non-medicinal parts are rarely exploited. Therefore, it may be interesting to extend the research to the non-medicinal parts of the inexpensive flowers, leaves, and roots of D. officinale to ensure the fully utilization of its edible and medicinal values (Zhang et al., 2017a).
Second, D. officinale is traditionally used to relieve fatigue, nourish "Yin", heart, and stomach, and expel evil heat, which is closely related to its preventive effects on cardiac, gastrointestinal and diabetes diseases according to modern pharmacology. Pharmacological research of the cardioprotective, gastrointestinal tract and anti-diabetes protective effects of D. officinale mainly concentrates on its crude extracts and polysaccharides. However, the optimal dose, constituents, and side effects of D. officinale are not assessed. Besides, there is a lack of in-depth study on the mechanism of action of D. officinale monomeric compounds or the specific mechanism of D. officinale in animals is not comprehensive enough. Therefore, the highquality and well-designed in vivo, in vitro, and clinical studies are encouraged to be carried out and to explore the molecular mechanisms and relationship between active chemical constituents and potential cardioprotective, gastrointestinal tract regulatory and anti-diabetes effects.
Third, the dosage of D. officinale used in previous studies were different (ranging from 4.375 mg/kg to 20 g/kg). It is not possible to define an exact upper cut-off dose, and the test dose needs to be pharmacologically relevant. In many cases, 100-200 mg/kg extracts for in vivo studies should be assumed as the upper limit for meaningful pharmacological studies. For pure compounds, a much lower dose range (e.g., 30-50 μM) should be considered for in vitro studies (Heinrich et al., 2020). In some cases, the pharmacological activity of D. officinale is present only with doses that might be too high for clinical use. According to the Chinese Pharmacopoeia, 6-12 g/day administration of D. officinale extract is common for human, but any clinical application at such doses should be accompanied with conservative safety insurance. Besides, the acute toxicity and sub-toxicity assessments of D. officinale were mostly carried out based on animal experiments. Furthermore, comprehensive placebo-controlled and double-blind clinical trials are necessary to provide sufficient evidence ensuring drug efficacy and patient safety.
Finally, D. officinale possesses various biological activities, which has been applied as health care medicine, health food, and health tea. In addition, with the development of analytical techniques and quality control methods, such as the improvement and update in chromatography techniques and molecular identification methods, new quality markers and quality control measures are likely to be adopted for better quality assessment of Chinese herbal medicine in the future (Leong et al., 2020).
In conclusion, D. officinale is one of the most popular medicinal and food homologous products in China. Modern pharmacology investigations have revealed its cardioprotective, gastrointestinal protective, anti-diabetes, and antiaging effects, which robustly support its traditional application in nourishing "Yin", heart, and stomach, expelling evil heat, and relieving fatigue. This paper provides a full-scale review about the progress of botany, traditional uses, phytochemistry, pharmacology, quality control, and toxicology of D. officinale. The information summarized in this work can provide a foundation for further applying the medicinal and edible value of D. officinale in the future.

AUTHOR CONTRIBUTION
WC collated documents and wrote the manuscript; JL and JZ helped to perform the arrangement of tables and pictures; JW and LY helped to check chemical structure formula; LQ and BZ contributed significantly to analysis and manuscript preparation. All authors have read and approved the final version of the manuscript.

FUNDING
This work was supported by the National Natural Science Foundation of China (82003896).