Echinacoside, an Inestimable Natural Product in Treatment of Neurological and other Disorders

Echinacoside (ECH), a natural phenylethanoid glycoside, was first isolated from Echinacea angustifolia DC. (Compositae) sixty years ago. It was found to possess numerous pharmacologically beneficial activities for human health, especially the neuroprotective and cardiovascular effects. Although ECH showed promising potential for treatment of Parkinson’s and Alzheimer’s diseases, some important issues arose. These included the identification of active metabolites as having poor bioavailability in prototype form, the definite molecular signal pathways or targets of ECH with the above effects, and limited reliable clinical trials. Thus, it remains unresolved as to whether scientific research can reasonably make use of this natural compound. A systematic summary and knowledge of future prospects are necessary to facilitate further studies for this natural product. The present review generalizes and analyzes the current knowledge on ECH, including its broad distribution, different preparation technologies, poor pharmacokinetics and kinds of therapeutic uses, and the future perspectives of its potential application.


Introduction-Treasure from the Garden: The Discovery and Distribution of ECH
Phenylethanoid glycosides (PhGs) are naturally occurring water-soluble compounds that are widely distributed in the plant kingdom, and most of which are isolated from garden plants and medicinal herbs. Structurally, these compounds are characterized by cinnamic acid and hydroxyl phenyl ethyl moieties that are attached to a β-glucopyranose (apiose, galactose, rhamnose, xylose, etc.) via a glycosidic bond. In recent years, interest has been growing in using PhGs [1] as their potential in the prevention and treatment of various human diseases and disorders.
Echinacoside (ECH, Figure 1), a natural PhG, was first isolated from Echinacea angustifolia DC. (Compositae), a garden plant sixty years ago, [2] and subsequently prepared from the species of Cistanches [3] as well as the aerial part of landscape herb, Penstemon crandallii A. Nels. (Scrophulariaceae) [4], whole plants of Pedicularis striata Pall. [5] and now successively found in 40 plant species [6][7][8][9][10][11][12] belonging to 18 genus and 10 families (Figure 2). To date, the species of genus Cistanches (Orobanchaceae) and Echinacea (Asteraceae) were the main natural plant sources for the preparation of ECH. ECH was found in both underground and aboveground parts of medicinal herbs but with widely varying levels of content ( Figure 2), including different stages of plant growth [8,13], different parts of the same plant [14], and vice versa, the same parts of different plants [15,16]. Until now, the highest content of ECH was found in haustorium phloem of Cistanches tubulosa which was reached almost 15.5% [17], thus could be a good resource for the isolation of pure ECH.   ECH was found in both underground and aboveground parts of medicinal herbs but with widely varying levels of content (Figure 2), including different stages of plant growth [8,13], different parts of the same plant [14], and vice versa, the same parts of different plants [15,16]. Until now, the highest content of ECH was found in haustorium phloem of Cistanches tubulosa which was reached almost 15.5% [17], thus could be a good resource for the isolation of pure ECH.  ECH was found in both underground and aboveground parts of medicinal herbs but with widely varying levels of content (Figure 2), including different stages of plant growth [8,13], different parts of the same plant [14], and vice versa, the same parts of different plants [15,16]. Until now, the highest content of ECH was found in haustorium phloem of Cistanches tubulosa which was reached almost 15.5% [17], thus could be a good resource for the isolation of pure ECH.

Preparation of ECH
As PhG compounds exhibited significant activities in the prevention and treatment of various human diseases and disorders, it was important to develop sustainable methods to produce sufficient quantities of ECH for pharmaceutical applications. Firstly and also usually, like other natural bioactive compounds, high purity of ECH was obtained from medicinal plants by using classic isolation methods and semi-preparative liquid chromatography (LC) [18] or high-speed counter-current chromatography methods [14] as Figure 3 shown, and the yield of ECH was usually between 0.2%~0.4%. An efficient ultrasound-assisted aqueous two-phase extraction process for preparation of ECH from Cistanche deserticola enhanced the content of ECH in the extracts (27.56 mg/g) which was 2.46-fold higher than the amounts obtained in ultrasound-assisted extraction [19]. Interestingly, it was found the content of ECH in medicinal herbs was significantly influenced by the factors of preparation processing [20], including the slice thickness, drying temperature, and the time for inactivation of the enzyme [21]. Therefore, it was worth noting that ECH was demonstrated to be highly susceptible to "enzymic" degradation and oxidation in hydroalcoholic solutions during the extraction process, and ECH in biosamples was susceptible to degradation at a higher temperature during the whole process, thus the operation must be carried out carefully at a lower temperature. Secondly, besides the above classic isolation method, plant cell/tissue culture, called "green cell factories", has become increasingly attractive as a cost-effective alternative to classical approaches for the sustainable mass production of plant-derived molecules [22]. Several published data demonstrated an increased accumulation of ECH in both plant tissue culture [23] and cell suspension culture [24] of Cistanche deserticola, and some revulsant including tyrosine, phenylalanine, cladosporium fulvum, methyl jasmonate and salicylic acid were found could promote the accumulation of ECH [25][26][27]. Thirdly, as an inestimable natural product which possesses a broad spectrum of beneficial activities, the chemical synthesis of ECH is needed to satisfy its comprehensive application. A group from National Taiwan University has completed the total synthesis of ECH from building blocks over 7 steps with yield was 4.5% [28] which was showed in Figure 3.

Preparation of ECH
As PhG compounds exhibited significant activities in the prevention and treatment of various human diseases and disorders, it was important to develop sustainable methods to produce sufficient quantities of ECH for pharmaceutical applications. Firstly and also usually, like other natural bioactive compounds, high purity of ECH was obtained from medicinal plants by using classic isolation methods and semi-preparative liquid chromatography (LC) [18] or high-speed countercurrent chromatography methods [14] as Figure 3 shown, and the yield of ECH was usually between 0.2%~0.4%. An efficient ultrasound-assisted aqueous two-phase extraction process for preparation of ECH from Cistanche deserticola enhanced the content of ECH in the extracts (27.56 mg/g) which was 2.46-fold higher than the amounts obtained in ultrasound-assisted extraction [19]. Interestingly, it was found the content of ECH in medicinal herbs was significantly influenced by the factors of preparation processing [20], including the slice thickness, drying temperature, and the time for inactivation of the enzyme [21]. Therefore, it was worth noting that ECH was demonstrated to be highly susceptible to "enzymic" degradation and oxidation in hydroalcoholic solutions during the extraction process, and ECH in biosamples was susceptible to degradation at a higher temperature during the whole process, thus the operation must be carried out carefully at a lower temperature. Secondly, besides the above classic isolation method, plant cell/tissue culture, called "green cell factories", has become increasingly attractive as a cost-effective alternative to classical approaches for the sustainable mass production of plant-derived molecules [22]. Several published data demonstrated an increased accumulation of ECH in both plant tissue culture [23] and cell suspension culture [24] of Cistanche deserticola, and some revulsant including tyrosine, phenylalanine, cladosporium fulvum, methyl jasmonate and salicylic acid were found could promote the accumulation of ECH [25][26][27]. Thirdly, as an inestimable natural product which possesses a broad spectrum of beneficial activities, the chemical synthesis of ECH is needed to satisfy its comprehensive application. A group from National Taiwan University has completed the total synthesis of ECH from building blocks over 7 steps with yield was 4.5% [28] which was showed in Figure 3.

Pharmacokinetics and Strategy
Generally, the systemic effects of natural PhGs mainly depend on their bioavailabilities through the gastrointestinal barrier. However, both in vitro and in vivo experimental data appeared to reflect their pitiful fates in the gut, including relatively poor bioavailabilities and rapid rates of excretion [29]. Dozens of studies have shown that the content of PhGs in plant within μg, following ingestion, they appeared in the circulation as phase II metabolites, and their plasma levels rarely exceed nM concentrations [30]. Animal study confirmed that the absolute bioavailability of ECH was only 0.83% [31], as Table 1 shown, the absorption and elimination of ECH was extremely fast in rats and the serum concentration was very low, and ECH could not be identified in any human plasma sample at any time after ECH tablet ingestion [32]. The serum concentration-time curves for intragastric and intravenous administration were fitted to one-compartment model and two-compartment model, respectively. The metabolites of ECH in rat feces were identified as acteoside, decaffeoylacteoside, lugrandosie and 3,4-dihydrophenyl ethanol after oral administration [33], and a portion of ECH was transformed into acteoside [34]. Now, to predict the absorption of orally administrated drugs, Caco-2 monolayer is widely used as a model of the human intestinal mucosa. It was shown that ECH permeated poorly through the Caco-2 monolayers although one potential metabolite, cinnamic acid, diffused readily with an apparent permeability of 1 × 10 −4 cm/s. The data implied ECH was not likely to cross the normal intestinal barrier [35][36][37], but it can through the blood-brain barrier in permanent

Pharmacokinetics and Strategy
Generally, the systemic effects of natural PhGs mainly depend on their bioavailabilities through the gastrointestinal barrier. However, both in vitro and in vivo experimental data appeared to reflect their pitiful fates in the gut, including relatively poor bioavailabilities and rapid rates of excretion [29]. Dozens of studies have shown that the content of PhGs in plant within µg, following ingestion, they appeared in the circulation as phase II metabolites, and their plasma levels rarely exceed nM concentrations [30]. Animal study confirmed that the absolute bioavailability of ECH was only 0.83% [31], as Table 1 shown, the absorption and elimination of ECH was extremely fast in rats and the serum concentration was very low, and ECH could not be identified in any human plasma sample at any time after ECH tablet ingestion [32]. The serum concentration-time curves for intragastric and intravenous administration were fitted to one-compartment model and two-compartment model, respectively. The metabolites of ECH in rat feces were identified as acteoside, decaffeoylacteoside, lugrandosie and 3,4-dihydrophenyl ethanol after oral administration [33], and a portion of ECH was transformed into acteoside [34]. Now, to predict the absorption of orally administrated drugs, Caco-2 monolayer is widely used as a model of the human intestinal mucosa. It was shown that ECH permeated poorly through the Caco-2 monolayers although one potential metabolite, cinnamic acid, diffused readily with an apparent permeability of 1 × 10 −4 cm/s. The data implied ECH was not likely to cross the normal intestinal barrier [35][36][37], but it can through the blood-brain barrier in permanent middle cerebral artery occlusion (MCAO) rats [38]. Furthermore, a recent study estimated the dynamic pharmacokinetic of ECH between Parkinson's disease rat and normal rats showed that the plasma concentrations of ECH in Parkinson's disease rats were higher than that in the normal rats after oral administration. The reasons why the elimination rate of ECH slowed down in Parkinson's disease rats may be as follows: ECH is partially hydrolyzed to aglycone in the body and in the state of pathophysiology, a low activity of certain enzymes induced by 6-hydroxydopamine (6-OHDA) damage might lead to the decreased clearance rate and increased retention time of ECH. Even if ECH was mainly excreted in the urine, the decreased blood circulation of kidney induced by 6-OHDA damage might play an important role in the decreased elimination rate and increased retention time of ECH [39]. 50 mg/kg p.o./MCAO rats C 5min = 29.83 ng/mL, C 15min = 31.28 ng/mL, C 30min = 40.21 ng/mL, C 45min = 26.49 ng/mL, C 60min = 21.20 ng/mL, C 90min = 14.04 ng/mL, C 120min blow LOD. [38] 8.4 ± 1.6 µg/mL/Caco-2 monolayers Permeated poorly, 0% was uptake at 90 min, and the apparent permeability was zero. [35,36] 200 µM/Caco-2 monolayers Passive diffusion, apparent permeability was nearly 10 −7 cm/s. [37] MCAO, middle cerebral artery occlusion; LOD, limit of detection; p.o., intragastric administration; i.p., intraperitoneal injection.

Pharmacological Properties and Underlying Mechanisms
ECH was proved possessing kinds of pharmacological activities since it was found sixty years ago, the data of this review were mainly gathered by consulting the database of PubMed, Springer, Elsevier, and Scholar in the last 30 years. Among the broad range of therapeutic applications of ECH, its neuroprotective bioactive has attracted the more attention of pharmaceutical scientists than the others. Dozens of reports have discovered that ECH was effective in Parkinson's and Alzheimer's diseases by using both animal experiments and cell lines tests, which were shown in Table 2. Besides the neuroprotective action, the cardioactive property, anti-inflammatory activity, antioxidant and anti-osteoporotic activities as well as other pharmacological potentials of ECH were presented in Tables 3-7, respectively. The data of Table 2 and Figure 4 showed that ECH could prevent the progress of neurodegeneration in Parkinson's and Alzheimer's diseases. Several Parkinson's or Alzheimer's animal models induced by 6-OHDA, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), D-galactose and β-amyloid Aβ- (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) as well as cerebral ischemia rats were used to estimate the neuroprotective effects of ECH. PC12 and neuroblastoma SH-SY5Y cell lines were employed to discover the related mechanisms, which were related to the mitogen-activated protein kinase (MAPK), NF-kappa B, caspase 3 and 8 as well as reactive oxygen species (ROS)/activating transcription factor 3 (ATF3)/C/EBP-homologous protein (CHOP) pathways, as Figure 4 showed. However, most of the above data were obtained by using cells or animals, the reliable clinical trials were limited, large-scale evidence-based human clinical trials with specific neuroprotective therapeutic settings are necessary. The same problems have been found by applying other evaluation methods of the different pharmacological properties. anti-osteoporotic activities as well as other pharmacological potentials of ECH were presented in Tables 3-7, respectively. The data of Table 2 and Figure 4 showed that ECH could prevent the progress of neurodegeneration in Parkinson's and Alzheimer's diseases. Several Parkinson's or Alzheimer's animal models induced by 6-OHDA, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), D-galactose and β-amyloid Aβ-(25-35) as well as cerebral ischemia rats were used to estimate the neuroprotective effects of ECH. PC12 and neuroblastoma SH-SY5Y cell lines were employed to discover the related mechanisms, which were related to the mitogen-activated protein kinase (MAPK), NF-kappa B, caspase 3 and 8 as well as reactive oxygen species (ROS)/activating transcription factor 3 (ATF3)/C/EBP-homologous protein (CHOP) pathways, as Figure 4 showed. However, most of the above data were obtained by using cells or animals, the reliable clinical trials were limited, large-scale evidence-based human clinical trials with specific neuroprotective therapeutic settings are necessary. The same problems have been found by applying other evaluation methods of the different pharmacological properties.   6-OHDA-induced neurotoxicity in rats 3, 5 and 7 mg/kg for 7 days, i.p.
Prevented the decreased of the extracellular levels of DA, DOPAC and HVA, elevated the concentrations of DA, DOPAC and HVA in the right striatum of awake, freely-moving rats. [43] 6-OHDA-induced neurotoxicity in PC12 cells 0.1, 1 and 10 µM Significantly enhanced cell viability, oxidation-reduction activity and mitochondrial membrane potential, reduced ROS production, as well as inhibited mitochondria-mediated apoptosis. [44] MPTP-induced neurotoxicity in mice 30 mg/kg for 14 days, p.o.
Suppressed the reduction of nigral dopaminergic neurons, striatal fibers, DA and DA transporter, prevented the apoptosis cells and Bax/Bcl-2 ratio of mRNA and protein, increased the expression level of GDNF and BDNF mRNA and protein, and improved the gait disorder. [45] MPTP-induced Parkinson's disease in C57BL/6 mice 20 mg/kg for 14 days, p.o.
Protected the C57BL/6 mice against MPTP-induced behavioral default, increased the number of spontaneous movement and latent period of mice on the rotating rod., and decreased the level of protein biliverdin reductase B. [46] MPTP-induced Parkinson's disease in C57BL/6 mice 30 mg/kg for 14 days, p.o.
Suppressed the dopaminergic neuron loss, maintained dopamine and dopamine metabolite content, inhibited the activation of microglia and astrocytes in the substantia nigra; downregulated the level of p38MAPK and the NF-kappaB p52 subunit. [47] MPTP-induced Parkinson's disease in mice 5 and 20 mg/kg for 15 days, p.o.
Reduced behavioral deficits and cell death, increased striatal DA, DA metabolite levels and tyrosine hydroxylase expression; reduced caspase-3 and caspase-8 activation in MPP-induced apoptosis in cerebellar granule neurons. [48] MPTP-induced neurotoxicity in SH-SY5Y cells 10, 20 and 40 µg/mL Improved cell survival, suppressed the generation of ROS and the expression of apoptotic genes (ATF3, CHOP, and SCNA), and decreased the caspase-3 activity in a dose-dependent manner; restored the GDNF expression, improved dopaminergic neuron survival and protected these neurons against apoptosis; protected apoptosis through ROS/ATF3/CHOP pathway.
[59]  Rotenone-induced Parkinson's disease in rats 20, 40 and 80 mg/kg for 4 weeks, i.p. Suppressed the neurological disability and the loss of dopaminergic neurons in substantia nigra, increased DA concentrations in striatum, no effect on liver and kidney damage. [73] Rotenone-induced injury in SHSY5Y, Hela and HEK293T cells 5, 10 and 20 µg/mL Protected cells over-expressed with TrkA or TrkB against rotenone injury, elevated the pERK levels and inhibited cytochrome c release and caspase-3 activation. [74] Permanent MCAO-induced neurotoxicity in rats 10, 20 and 40 mg/kg for 4 weeks, i.p. Increased the content of GSH and activity of GSH-Px, decreased the activity of NOS; arranged the rat tissue structure of hippocampal CAI area in order. [75] D-galactose induced subacute aging in mice 50 mg/kg for 6 weeks, p.o.

30-300 µM
Relaxed the endothelium-intact rings, enhanced the cyclic guanosine monophosphate production in aortic rings through NO-cyclic guanosine monophosphate pathway. [86] Noradrenaline induced contractions in isolated rat aortic strip
One of the traditional uses of Cistanche deserticola was for treatment of irritable bowel syndrome disease, and ECH was the main bioactive ingredients in this herbal responsible for the activity. To date, dozens of in vivo studies demonstrated the anti-inflammatory property of ECH, the data of Table 4 showed that ECH could suppress the acute colitis in mice induced by dextran sulphate sodium [88], attenuate acute hepatotoxicity in rats induced by D-galactosamine/lipopolysaccharide [89] and carbon tetrachloride (CCl 4 ) [90], increase hyaluronan levels and decrease wound contraction for wound healing, modulate inflammatory markers including transforming growth factor (TGF)-β1, NO, alanine aminotransferase (ALT), myeloperoxidase, inflammatory cytokines, and etc.; however, the molecular mechanisms of the anti-inflammatory of ECH were limited, which were only related to the expressions of TGF-β1, capase-3 and TNF-α.  Suppressed the development of acute colitis, prevented colonic damage, protected intestinal epithelium from inflammatory injury, up-regulated the expression of TGF-β1, and increased the number of Ki67 + proliferating cells. [88] SD rats were abraded to generate erythema and cicatrization 0.4 mg/mL, topical Decreased the edematous process, increased hyaluronan levels and less wound contraction. [93] Removed vocal fold lamina propria to generate injury in pigs 3-12 mg/mL for 15 days, topical Improved the phonation threshold pressure and the vocal economy, maintained a stable hyaluronan and collagen content. [94] D-galactosamine/lipopolysaccharideinduced acute liver injury in mice and primary cultured mouse hepatocytes 25-100 mg/kg, p.o.
It was worth mentioning that, to date, numerous in vitro and in vivo studies have demonstrated the strong antioxidant property of ECH (Table 5). In DPPH assay, the EC 50 of ECH was 6.6 µM which was 9.5-fold than Trolox; on xanthine/xanthine oxidase generated superoxide anion radical test, the IC 50 of ECH was 2.74 µM than tocopherol, and etc. [98][99][100][101]. In vivo experiments, ECH could prompt the ability of anti-oxication, anti-fatigue and anti-stress in vascular dementia rats or subacute aging mice model, and the indirect antioxidant activities of ECH due to the induction or/and activation of major endogenous antioxidant enzymes and inactivation of pro-oxidant enzymes. In addition, the molecular mechanisms of this activity showed that ECH reduced nuclear protein levels of transcription regulator protein BACH1, enhanced heme oxygenase 1 mRNA levels, down-regulated expression of p53, up-regulated the SIRT1 [102]. Furthermore, the structure-activity relationship of antioxidant property of ECH was also estimated. It was believed that the inhibitory oxidative hemolysis activity of ECH was related to the number of phenolic hydroxy groups. ECH, possessing four phenolic hydroxy groups, exhibited stronger antioxidant activities than cistanoside D possessing only two phenolic hydroxy groups, and compound permethylacteoside with no phenolic hydroxy group inhibited oxidative hemolysis weakly [103,104]. Table 5. Anti-oxidative property of ECH in selected models.
Cistanche deserticola is a traditional Chinese medicine (TCM) called "Desert ginseng" in China owing to its excellent medical functions and nourishing effect. According to the theory of TCM, Cistanche deserticola can supplement the kidney, and kidney stores essence and the essence can transform into bone marrow to nourish the bones, which means Cistanche deserticola could promote the formation of the bone [112]. As ECH is the main constituent of Cistanche deserticola, thus maybe possesses anti-osteoporotic property. And in the anti-osteoporotic tests as Table 6 shown, ECH exhibited anti-osteoporotic effect on the promotion of bone formation and suppression of bone resorption [113], and the molecular targets of ECH were also discovered that it could increase the osteoprotegerin (OPG) level and decrease the receptor activator for nuclear factor-κB Ligand (RANKL) level [114] as well as promoted the phosphorylation of ERK1/2 to activate MAPK/ERK pathway [115,116]; however, the dosage of ECH were so high that even reached at 270 mg/kg body weight/day, which made some difficult in the future clinical trials and enhanced the medicinal costs. Since the results of the report showed the dosage of 30 mg/kg body weight/day of ECH was also effective in ovariectomized (OVX) rats, a proper dosage of ECH in future treatment of osteoporosis disease should be selected with more tests. Completely corrected the increased urine concentration of calcium, inorganic phosphorus, and hydroxyproline; enhanced bone quality, improved total bone mineral density and biomechanical strength of tibia, promoted the bone formation and suppressed the bone resorption. [113] OVX rat model of osteoporosis 30, 90 and 270 mg/kg/day for 12 weeks, p.o.
Inhibited the rat lens aldose reductase with IC 50 was 3.1 µM; inhibited the increase in postprandial blood glucose levels, improved glucose tolerance without producing significant changes in body weight or food intake. [87] Antiviral activity Mouse macrophage model/100-1000 µg/mL Possessed high antiviral activities with different antiviral profile and limited immune activation properties. [118] Anti-hepatic fibrosis effect Hepatic stellate cell lines/125, 250 and 500 µg/mL Inhibited hepatic stellate cell activation with IC 50 was 520.3 µg/mL, suppressed the conduction of the signaling pathways in transforming growth factor-beta1/smad, including increasing the mRNA level and protein expression of smad7, and decreased both the mRNA and protein levels of smad2 and smad3 in hepatic stellate cell. [119] Anti-tumor activity Pancreatic adenocarcinoma cell lines/20, 50, 100 µM Inhibited the proliferation of pancreatic adenocarcinoma cells by inducing the production of reactive oxygen species and the perturbation of mitochondrial membrane potential and thus triggering apoptosis, and this activity was main through modulating MAPK activity. [120] Testis and sperm injury protect activity Testicular and sperm toxicity induced by BPA/ 6 mg/kg for 6 weeks, p.o.
Besides the above significant bioactive, ECH also proved to possess additional antidiabetic effect, antiviral activity, anti-hepatic fibrosis effect, anti-tumor property, testis and sperm injury protect activities as Table 7 shown.

Discussion
ECH, a natural PhGs compound has been isolated from dozens of medicinal or horticultural plants, exhibited highly positive activities in treatment of nervous, cardiovascular and bone disorders, especially for the prevention and treatment of a variety of nervous system disorders including Parkinson's and Alzheimer's diseases. Given the above potential in pharmaceutical applications, the preparations of ECH including the classic isolation from herbs, plant cell/tissue culture and even the chemical synthesis have attracted the interests of plenty pharmaceutical scientists. This review has presented the discovery of ECH including its distribution in the plant kingdom, and the preparation of ECH including the methods of classic isolation from medicinal plants, "green cell factories" of plant cell/tissue culture and chemical synthesis, and the pharmacokinetics data of ECH was also posted for further medicinal uses. Then, the most important section of this paper, the remarkable pharmacological properties of ECH were elucidated, including neuroprotective activity, cardioactive property, anti-osteoporotic effect, anti-inflammatory and antioxidant activities. However, there were four important questions should be pay attention before ECH was used for clinical applications: first, both in vivo and in vitro experiments of ECH reflected its dissatisfied pharmacokinetic property. Concerning the in vivo experiments, ECH exhibited a pitiful fates in the gut, including relatively poor bioavailability [31] (the absolute bioavailability was only 0.83%) and rapid rates of metabolism and excretion, following ingestion, ECH appeared in the circulation as phase II metabolites, and its plasma levels rarely exceed nM concentrations [30]; and in vitro tests showed that ECH permeated poorly through the Caco-2 monolayers, which implied that ECH was not likely to cross the intestinal and blood brain barriers, thus appeared failed to explain its neuroprotective activity and other bioactive [35]. However, it was reported that in neuronal cells and non-neuronal cells which were exposed to rotenone, ECH was able to cross the blood-brain barrier freely [74]. Second, it should be noted that several of the activities especially the anti-osteoporotic one occurred at relatively high concentrations of ECH (30-270 mg/kg/day, orally for 12 weeks) [113], and in the antidiabetic experiments [87], the dose of ECH even reached at 500 mg/kg p.o. And others occurred at normal concentrations about 5 to 50 mg/kg. Third, the underlying molecular mechanisms of ECH with neuroprotective and cardiovascular properties, anti-osteoporotic, and anti-inflammatory activities have not been elucidated in detail. Although some common molecular signal pathways and several distinct targets have been disclosed, the responses of molecular targets to ECH with the above effects remain unclear. Forth, sixty years after the discovery of ECH, to date, very little efforts are done for its clinical trials and the safety and toxicity tests, and reliable clinical data describing the health effects of ECH are limited. Therefore, the in vivo animal studies of ECH should be considered with caution and more clinical trial on its efficacy and safety should be performed.

Conclusions
In summary, ECH was an inestimable natural product that exhibited highly positive activities in nervous and cardiovascular system disorders as well as bone disease from the wealth of laboratory data, and thus was believed have a promising potential in the treatment of Parkinson's and Alzheimer's diseases, atherosclerosis, osteoporosis, acute colitis, wound injury, and hepatitis. The excellent antioxidant property, which was 9.5-fold greater than Trolox, also implied an ideal application of ECH in the future clinical trials. However, at present, despite the wealth of experimental data that was available describing the potent pharmacological effects of ECH, many issues remain unresolved with respect to effective clinical applications. First, the low bioavailability and extremely fast metabolism of ECH in animals reflected its dissatisfied pharmacokinetic property in the future clinical application. The rapid rates of metabolism and excretion of ECH was due to the multiple metabolic pathways that were involved to eliminate plant-derived toxins, thus further intensive studies are required to confirm the clinical potential of ECH, thereby enabling its acceptance as a therapeutic agent. Another important issue was that, although the present review findings provided a sound basis to confirm that ECH is a potential candidate for intervention in neurodegenerative diseases such as Alzheimer's and Pakinson's disorders, the molecular signal pathways, especially the molecular targets to ECH with the above effects, remain unclear. Thus, further studies are needed to interpret the directed molecular mechanisms. The last issue is also the most important problem, implying that large-scale evidence-based human studies with specific therapeutic settings are necessary. Although plenty of laboratory data shed light on the protection of ECH against dozens of diseases, the reliable clinical data are limited. More clinical trials on the safety and drugability of ECH are needed.
Funding: This work was supported by the grants from the National Natural Science Foundation of China (No.81560684) and Ningxia key research and invention program of science and technology cooperation of the East and the West (No. 2017BY084).

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in this manuscript: