Insights into Recent Studies on Biotransformation and Pharmacological Activities of Ginsenoside Rd

It is well known that ginsenosides—major bioactive constituents of Panax ginseng—are attracting more attention due to their beneficial pharmacological activities. Ginsenoside Rd, belonging to protopanaxadiol (PPD)-type ginsenosides, exhibits diverse and powerful pharmacological activities. In recent decades, nearly 300 studies on the pharmacological activities of Rd—as a potential treatment for a variety of diseases—have been published. However, no specific, comprehensive reviews have been documented to date. The present review not only summarizes the in vitro and in vivo studies on the health benefits of Rd, including anti-cancer, anti-diabetic, anti-inflammatory, neuroprotective, cardioprotective, ischemic stroke, immunoregulation, and other pharmacological effects, it also delves into the inclusion of potential molecular mechanisms, providing an overview of future prospects for the use of Rd in the treatment of chronic metabolic diseases and neurodegenerative disorders. Although biotransformation, pharmacokinetics, and clinical studies of Rd have also been reviewed, clinical trial data of Rd are limited; the only data available are for its treatment of acute ischemic stroke. Therefore, clinical evidence of Rd should be considered in future studies.


Biotransformation
The content of ginsenoside Rd differs from different wild ginseng and parts of ginseng, and ranges from 0.02% to 1.66% [4]. Moreover, it is difficult and costly to isolate Rd from natural products; thus, a microbial enzymatic transformation has become the predominant conversion modality of ginsenoside Rd due to its distinct selectivity, mild reactive conditions, and environmental compatibility ( Figure 1).

Enzymatic Transformation
Ginsenoside Rd-characterized by tetracyclic, dammarane-type triterpenes with three sugar moieties-is structurally similar to Rb1, Rb2, and Rc, but lacks one outer glycoside moiety at the C-20 position. The preparation of ginsenosides via hydrolysis of glycosidic bonds using enzymatic transformation methods has exceptional advantages, e.g., high selectivity, mild reactive conditions, and being environmentally-friendly. Therefore, it is viable to obtain ginsenoside Rd from Rb1, Rb2, and Rc by hydrolyzing the monosaccharide residue using a specific glycosidase, such as α-L-arabinofuranosidase, α-L-arabinopyranosidase, β-glucosidase, or pectinase (Table 1). " " means convert to, "/" means not mentioned.

Arabinofuranosidase
Ginsenoside Rc, one of the major components of ginseng, comprising 7-22% of total ginsenoside, has an arabinofuranosyl moiety and three glucopyranosyl moieties; thus, arabinofuranosidase and glucosidase could convert Rc to a deglycosylated ginsenoside [5]. In order to improve the biotransformation rate of ginsenoside Rd and optimize the enzymatic properties, studies about arabinofuranosidase have emerged in recent years. An et al. [5] identified a recombinant α-L-arabinofuranosidase AbfA, which was cloned from a soil bacterium; Rhodanobacter ginsenosidimutans Gsoil 3054T could biotransform ginsenoside Rc to ginsenoside Rd. Recombinant AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it only hydrolyzed arabinofuranoside moieties from ginsenoside Rc and derivatives, not other sugar groups from ginsenosides Rb1 or Rb2. The following year, the same research team reported a novel recombinant α-L-arabinofuranosidase (Abf22-3) from the ginsenoside converting Leuconostoc sp. 22-3 isolated from the Korean fermented food kimchi, which could biotransform ginsenoside Rc into Rd [6]. Results showed that over 99.5% of Rc was converted to Rd after 24 h under optimal conditions of pH 6.0 and 30 • C. In another study, a molar yield of ginsenoside Rd was nearly 100% using a thermostable recombinant α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus at a pH 5.5 and at 80 • C [7]. Later, Xie et al. [8] cloned and overexpressed the novel thermostable α-L-arabinofuranosidase (Tt-Afs) from T. thermarum DSM5069, which showed a high conversion and productivity of Rd. In addition, the ginsenoside Rc-hydrolyzing α-L-arabinofuranosidase gene, BsAbfA, was cloned from Bacillus subtilis and optimized. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA were important in effectively recognizing ginsenoside Rc, providing an effective biotransformation pathway of ginsenoside Rc into Rd [9].
Among the major ginsenosides, ginsenoside Rb2 accounts for 1-22% of the total ginsenosides in ginseng root [32] and could also be used for converting into Rd. Kim et al. [10] reported a recombinant enzyme α-L-arabinopyranosidase (AbpBs), which could efficiently catalyze the conversion of ginsenoside Rb2 to Rd by selectively hydrolyzing the outer arabinopyranoside moiety at the C-20 position.

β-glucosidase
β-glucosidases, a heterogeneous group of enzymes, are capable of cleaving the βglycosidic linkages of aryl and alkyl β-glucosides, β-linked oligoglucosides, and several other oligosaccharides. Some recombinant enzymes, especially β-glucosidases with different substrate specificities, have been widely applied to produce the rare ginsenosides. To date, considerable attention has been placed on the transformation of ginsenoside Rb1 into Rd with the use of β-glucosidases. The thermostable β-glucosidase Tt-BGL from extremophile Thermotoga thermarum DSM5069 selectively converts ginsenoside Rb1 into ginsenoside Rd, with high productivity [11]. Additionally, ginsenoside Rd has been used as an intermediate for the transformation of other rare ginsenosides. Quan et al. [12] reported that the recombinant β-glucosidase Bgp3 from Microbacterium esteraromaticum isolated from the ginseng field could catalyze the conversion of ginsenoside Rb1 to the more pharmacologically active major ginsenoside Rd and ginsenoside CK. Subsequently, they isolated a novel recombinant glycosidase Bgp2 from Microbacterium esteraromaticum, which belonged to the glycosyl hydrolase family 2 protein and could hydrolyze the ginsenoside Rb2 along the following pathway: Rb2 Rd 20(S)-Rg3 through the selective hydrolysis of the arabinopyranose and glucose moieties [13]. The ginsenoside-hydrolyzing β-glucosidase gene Bgy2, a member of the glycosyl hydrolase family 3 protein, was cloned and identified from Lactobacillus brevis [14]. Under the optimal conditions (pH 7.0, 30 • C), 1.0 mg/mL ginsenoside Rb1 was converted into 0.59 mg/mL ginsenoside Rd, with molar conversion productivities of 69%. Moreover, Rb1-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11,239 was studied (and optimized) by Chang et al. [15]. The enzyme hydrolyzed β-(1 6)-glucoside at the C-20 position of ginsenoside Rb1 to generate Rd and Rg3, and hydrolyzed β-(1 2)-glucoside at the C-3 position to generate F2.

Pectinase
Pectinase specifically hydrolyzes protopanaxadiol (PPD)-type ginsenosides and is a selective enzyme that converts ginsenoside Rb1 to Rd. Fang et al. [16] explored one-pot production process of ginsenoside Rd by coupling enzyme-assisted extraction with selective enzymolysis, and provided a higher yield at 52.5 • C and pH 6.0, suggesting that pectinase could be used as an efficient enzyme for producing ginsenoside Rd.

Microbial Transformation
Microbial transformation is also a major production method of Rd. The mechanism of enzymatic transformation involves hydrolyzing ginsenosides using the catalytic activity of the enzyme, which has the advantages of a short reaction cycle, low pollution, and high product purity; however, the reaction conditions are difficult to control, the enzyme is easy to inactivate, and the separation and purification processes of the enzymes are complicated. In contrast, microbial transformation is characterized by low costs, few byproducts, and wide applications, but the drawbacks of a long conversion time and a low biotransformation rate are inevitable. Therefore, the enzymatic-microbial transformation of ginsenosides have their own characteristics and complement each other in the actual production process. The production of Rd could be achieved through microbial methods, including fungus, bacteria, gut microbiota, and food microorganisms (Table 1).

Fungal System
A mutant filamentous fungus Paecilomyces bainier 229-7 that transformed ginsenoside Rb1 to Rd with high selectivity and substrate tolerance was obtained (and identified) by Feng et al. [17]. The highly substrate-tolerant mutant produced ginsenoside Rd from Rb1 with a bioconversion rate as high as 94.9% under optimized culture conditions in shake flasks, along with an 89% bioconversion rate in 10 L fermenter, with a chromatographic purity of 92.6% purified by macroporous resin, which rendered it a promising strain for the preparation of Rd in the pharmaceutical industry. Later, the same team reported on the effects of external calcium treatments on the biotransformation of ginsenoside Rb1 to ginsenoside Rd by Paecilomyces bainier 229-7. Results suggested that both Ca 2+ channels and calmodulin (CaM) were involved in ginsenoside Rd biotransformation via regulation of β-glucosidase activity [18]. Additionally, ginsenoside Rb1-converting fungus Aspergillus versicolor LFJ1403 was isolated and identified from the ginseng field soil and the biotransformation of ginsenoside Rb1 to Rd using an extracellular enzyme directly from the fungus spore production phase was investigated. The results of HPLC showed that Rd was the only product in this process, and the conversion rate was increased to 96% in shake flasks, indicating that the spore suspension biotransformation system had potential in the industrial production of Rd [19]. A novel ginsenoside Rd transformation fungus, Aspergillus niger TH-10a obtained from screening the survival library of LiCl and UV irradiation, could efficiently convert ginsenoside Rd from Rb1, and achieve the highest transformation rate of about 86% at 32 • C and pH 5.0 [20].

Bacteria System
Some studies have focused on the discovery and identification of ginsenoside-transforming bacteria. To identify a microorganism that was capable of converting Rb1 into other ginsenosides, 12 Microbacterium spp. were screened by Hansoo et al. [21], and M. trichothecenolyticum was identified to convert Rb1 into Rd and then into Rh2 based on TLC and HPLC analyses of reaction products. Then, Akter et al. [22] isolated a gram-positive, aerobic, motile, rod-shaped bacterial strain (MAH-16T) from a soil sample of a vegetable garden and identified it as a member of the genus Paenibacillus barengoltzii SAFN-016T according to the 16S rRNA gene sequence comparisons, which might be responsible for the biosynthesis of ginsenoside Rd from major ginsenoside Rb1. Later, they also isolated a novel, gram-positive, and ginsenoside-converting bacterium (MAHUQ-46T) from forest soil, which was closely related to Paenibacillus pinihumi S23T (97.3% similarity) [23]. Furthermore, a Gram-negative, strictly aerobic, non-spore-forming, and rod-shaped bacterial strain (FW-6T) was isolated from a freshwater sample and displayed β-glucosidase activity that could transform ginsenoside Rb1 to Rd [24].
There are various microorganisms in the ecological environment of plants; some are attached to the surfaces of plants, while others live in the plants. Endophytes are fungi or bacteria commonly found in higher plants that live in the tissues and organs of healthy plants for some (or all) of their stages. Previous research focused on microorganisms that attached to plant surfaces and the rhizosphere, but the study of endophytes in plants was fledgling. An endophytic bacterium, G9y, with the ability to specifically convert ginsenoside Rc to Rd, was isolated from Panax quinquefolius; the transformation mechanism might be related to the production of α-L-arabinofuranosidase, which specifically hydrolyzes the terminal arabinofuranosyl moieties at the C-20 position of ginsenoside Rc [25]. Ginsenoside Rc was completely converted to Rd by bacterium G9y within 25 h after inoculation under the optimized conditions of pH 7.0 and 45 • C.

Pharmacological Activity
Ginsenoside Rd is known for its beneficial pharmacological activities. To date, extensive studies of in vitro cell biology and in vivo animal models have demonstrated that ginsenoside Rd offers potential anti-cancer, anti-diabetic, anti-inflammatory, neuroprotective, cardioprotective, ischemic stroke, immunological, and other pharmacological activities. In this section, we summarize recent studies on various health-promoting activities of ginsenoside Rd to provide a systematic summary and analysis of the pharmacological effects and the potential molecular mechanisms.

Anti-Cancer
The promising anti-cancer activity of ginsenoside Rd has been identified in various types of cell lines and animal models, including gastric cancer, colorectal cancer, lung cancer, breast cancer, glioblastoma, etc. ( Table 2). The underlying anti-cancer mechanisms of ginsenoside Rd are shown in Figure 2. Ginsenoside Rd significantly inhibits cell proliferation and induces cell cycle arrest and cell apoptosis by increasing the expression of caspase-3, caspase-9, and the ratio of Bax/Bcl-2 in human gastric cancer [33], cervical cancer Hela cells [34], and human glioma U251 cells [35]. The possible mechanisms of Rd inhibiting glioma cells might be related to inhibition of telomerase activity by downregulating human telomerase catalytic subunit (hTERT) expressions at both mRNA and protein levels [35]. Moreover, it was reported that Rd reduced the proliferation and migration of glioblastoma cells by upregulating the tumor suppressor miR-144-5p and downregulating its target toll-like receptor 2 [36]. Lee et al. [37] identified fourteen proteins contributing to cell growth inhibition after ginsenoside Rd treatment in HT29 through two-dimensional gel electrophoreses, MALDI-TOF and TOF-MS, including proteins associated with mitosis (such as stathmin 1, microtubule-associated protein RP/EB family, stratifin) and associated with apoptosis (Rho GDP dissociation inhibitor, tropomyosin 1, annexin 5). The combination of combretastatin A4 phosphate (CA4P), a vascular disrupting agent, and Rd, had synergistic anti-tumor effects in hepatocellular carcinoma, which the mechanism might be related to the inhibition of HIF-1α via PI3K/AKT/mTOR signaling pathway [38]. The divalent cation-selective channel transient receptor potential melastatin 7 (TRPM7) channel was shown to affect the proliferation of some types of cancer cells. Several studies reported that ginsenoside Rd inhibited the proliferation and survival of gastric and breast cancer cells by inhibiting TRPM7 channel activity [39,40]. Moreover, ginsenoside Rd significantly inhibited metastasis in the human hepatocellular carcinoma, colorectal cancer, and breast cancer [41][42][43]. Research by Wang et al. [43] showed that Rd treatment attenuated breast cancer metastasis in part through derepressing miR-18a-mediated Smad2 expression regulation. A blockade of angiogenesis was an important approach for cancer treatment and prevention; thus, some studies investigated the effects of ginsenoside Rd on angiogenesis, in vitro and in vivo. Results demonstrated that Rd inhibited VEGF-induced migration, tube formation, and proliferation of primary cultured human umbilical vascular endothelial cells (HUVECs) dose-dependently [44]. Furthermore, Rd normalized the structure of tumor vessels, and improved the anti-tumor effect of 5-FU in xenograft mice [45]. Clinical drug resistance to chemotherapy is always considered a major obstacle in the successful treatment of cancer. Notably, ginsenoside Rd was reported to reverse doxorubicin resistance in MCF-7/ADR cells through downregulating the multidrug resistance 1 (MDR1) protein [46]. In addition, Rd could overcome cisplatin resistance in NSCLC by downregulating the nuclear factor erythroid 2-related factor 2 (NRF2) pathway [47].
Manipulation of gut microbiota composition through the treatment of prebiotics could be a novel preventive measure against cancer development. Interestingly, Rd exerted anti-cancer effects by holistically reinstating mucosal architecture, improving mucosal immunity, promoting beneficial bacteria, and downregulating cancer-cachexia associated bacteria [48]. a major obstacle in the successful treatment of cancer. Notably, ginsenoside Rd was reported to reverse doxorubicin resistance in MCF-7/ADR cells through downregulating the multidrug resistance 1 (MDR1) protein [46]. In addition, Rd could overcome cisplatin resistance in NSCLC by downregulating the nuclear factor erythroid 2-related factor 2 (NRF2) pathway [47]. Manipulation of gut microbiota composition through the treatment of prebiotics could be a novel preventive measure against cancer development. Interestingly, Rd exerted anti-cancer effects by holistically reinstating mucosal architecture, improving mucosal immunity, promoting beneficial bacteria, and downregulating cancer-cachexia as- Figure 2. Anti-cancer mechanism of ginsenoside Rd. "↓" means downregulation, "↑" means upregulation.  "/" means not mentioned, "↑" means upregulation, "↓" means downregulation.

Anti-Diabetic
Diabetes mellitus is characterized by chronic hyperglycemia, which also results in the abnormal accumulation of methylglyoxal (MG, one of the most reactive advanced glycation end-product precursors) and induces neuronal cell death in the central nervous system. Ginsenoside Rd and Rh2 were shown to ameliorate the cell viability of MG-treated astrocytes and improve insulin signaling, indicating that Rd and Rh2 might have therapeutic potential in treating diabetes-induced neurodegeneration [49]. Kaviani et al. [50] evaluated the effects of ginsenoside Rd on the apoptosis-associated cell death in human pancreatic islets, and results showed that Rd inhibited the progress of death of cultured human pancreatic islets by diminishing the apoptosis of the islet cells. Moreover, Jung et al. [51] developed a new pectin lyase-modified ginseng (GS-E3D), with enhanced ginsenoside Rd content, which had a potent protective role in diabetes-induced renal dysfunction through antioxidative and antiapoptotic activities. Diabetic retinopathy (DR) is a complex complication of diabetes that can lead to blindness. A recent report demonstrated that ginsenoside Rd ameliorated diabetes-driven vascular damage through enhancement of AMPK/SIRT1 interaction, which supported the potential vascular protective evidence of Rd for early DR [52] (Table 2).

Anti-Inflammatory and Antioxidative
Inflammatory response is a complex network composed of multiple mediators, cells, and pathways, which is involved in the occurrence and development of various diseases, such as cancer, atherosclerosis, and neurodegenerative diseases. Ginsenoside Rd exhibited significant anti-inflammatory activities against many inflammatory diseases, such as chronic hepatitis [53], neuroinflammation [54], osteoarthritis [55], and gastritis [56], through the downregulation of inducible nitric-oxide synthase (iNOS) and COX-2 by inhibiting NF-κB, furthering the inhibition of the production of NO and PGE2 [57,58] (Table 3). However, recent studies have strengthened the understanding of the mechanistic implications at molecular and cellular levels. The anti-inflammatory mechanism of ginsenoside Rd is shown in Figure 3.  Ginsenoside Rd ameliorated colitis by inducing p62-driven mitophagy-mediated NLRP3 inflammasome inactivation and upregulating of AMPK/ULK1 signaling pathway in DSS-induced murine colitis model [59]. Moreover, ginsenoside Rd attenuated the inflammatory response in rats with TNBS-induced relapsing colitis and recurrent ulcerative colitis via modulating p38 and JNK signaling pathways, inhibiting neutrophil infiltration   "/" means not mentioned, "↑" means upregulation, "↓" means downregulation.
Ginsenoside Rd ameliorated colitis by inducing p62-driven mitophagy-mediated NLRP3 inflammasome inactivation and upregulating of AMPK/ULK1 signaling pathway in DSS-induced murine colitis model [59]. Moreover, ginsenoside Rd attenuated the inflammatory response in rats with TNBS-induced relapsing colitis and recurrent ulcerative colitis via modulating p38 and JNK signaling pathways, inhibiting neutrophil infiltration and promoting the antioxidant capacity [60,61]. A recent study evaluated the utility of Rd in gastrointestinal mucosal regeneration and clarified that Rd could stimulate the proliferation and differentiation of endogenous intestinal stem cells and improve recovery of intestinal function in a rat model of inflammatory bowel disease (IBD) by increasing the expression levels of Bmi, CDX-2, and Msi-1 [62]. In addition, ginsenoside Rd and bifidobacterialfermented ethanol-extracted red ginseng could alleviate allergic rhinitis by suppressing IgE, IL-4, IL-5, and IL-13 expression and restoring the composition of gut microbiota [63]. Zhang et al. [64,65] reported that ginsenoside Rd significantly inhibited the production of pro-inflammatory cytokines and mediators in carrageenan-induced rat paw edema; the detailed mechanisms might be related to reducing the inflammatory cell infiltration into inflammatory sites, inhibiting the tissue lipid peroxidation and increasing the antioxidant enzyme activities through downregulation of NF-κB activation.
Additionally, oxidative stress-induced cell damage has been implicated in a variety of disease, such as aging, neurodegenerative disorders and certain chronic diseases. Ginsenoside Rd could be considered a potential antioxidant agent for prolonging the lifespan in senescence-accelerated mice and C. elegans [66,67]. Moreover, ginsenoside Rd was reported to have an anti-oxidative effect by enhancing glutathione levels in H4IIE cells via NF-κBdependent γ-glutamylcysteine ligase induction [69]. Ye and co-workers investigated the protective role of ginsenoside Rd against the cytotoxicity in PC12 cell lines induced by exposure to hydrogen peroxide, indicating the potential neuroprotective effects [68].

Cognition and Neuroprotection
Various ginseng species and ginsenosides have been documented to possess therapeutic effects in many central nervous system (CNS) ailments, for instance, Alzheimer's disease, Parkinson's disease, spinal cord injury, depression, and other cognitive impairment. The protective effects could be ascribed to reducing neuroinflammation, improving oxidative stress, regulating neurotransmitter release, and promoting nerve regeneration. Recent studies have shown that ginsenoside Rd could be a promising natural neuroprotective agent [4]. The current review summarizes the recent progress in neuroprotective effects of ginsenoside Rd in detail (Table 4, Figure 4).
Alzheimer's disease (AD), is a neurodegenerative disease characterized by the sophisticated and unknown pathogenesis. Currently, the main popular hypotheses include neuronal dysfunction triggered by deposition of amyloid β (Aβ) proteins, neurofibrillary tangles triggered by hyperphosphorylation of tau protein, and cholinergic nerve degeneration [70]. Several studies examined the neuroprotective effects of Rd against neuronal insults in Aβ [25][26][27][28][29][30][31][32][33][34][35] or Aβ 1-40 induced AD rat models by ameliorating oxidative stress, alleviating the inflammation and reducing neuronal apoptosis [71,72]. Rd could also improve learning and memory ability in Aβ-protein precursor (APP) transgenic mice through inhibiting the transcription activity of NF-κB [73]. Furthermore, ginsenoside Rd attenuated Aβ-induced pathological tau phosphorylation by altering the functional balance of GSK-3β and PP-2A in Aβ-treated cortical neurons and in Aβ 1-40 induced rat model and APP transgenic mice model [74]. A deficiency of the neurotransmitter acetylcholine (ACh) is also the major characteristic of Alzheimer's disease. Results by Kim revealed that Re and Rd effectively induced the expression of cholinergic markers ChAT/VAChT genes and elevated ACh in Neuro-2a cells, as well as played an important role in neuronal differentiation and the nerve growth factor (NGF)-TrkA signaling pathway [75]. Ginsenoside Rd reduced OA-induced neurotoxicity and tau hyperphosphorylation in OA induced rat model (10 mg/kg) or in cultured cortical neurons (2.5 or 5 µM for 12 h) by enhancing the activities of protein phosphatase 2A (PP-2A) indicating that Rd might be a potential preventive drug candidate for AD and other tau pathology-related neuronal degenerative diseases [76]. Another recent research documented the protective effects of Rd against ovariectomy rat model. Rd enhanced learning and memory function of ovariectomy rats by increasing levels of sAPPα in the hippocampi, reducing extracellular Aβ and activating estrogen-like activity [77].  Recent findings highlighted the efficacy of ginsenoside Rd as neuroprotective compounds for Parkinson's disease (PD) prevention and treatment through reducing oxidative stress, improving mitochondrial integrity and functions, and inhibiting apoptosis [78][79][80]. As a potential neuroprotective agent, ginsenoside Rd exhibited anti-neurotoxicity effect on various neurotoxic injury responses induced by Pb, trimethyltin (TMT), or kainic acid (KA) [81][82][83]. Cong et al. [84] evaluated the neuroprotective effects of ginsenoside Rd in a rat model of spinal cord injury (SCI), and the results demonstrated that Rd (25 and 50 mg/kg) significantly improved the locomotor function of rats after SCI through reversing the redox-state imbalance, inhibiting the inflammatory response and apoptosis in the spinal cord tissue. Another study investigated the protective effects of Rd on spinal cord mitochondrial dysfunction by regulating mitochondrial permeability transition pore formation and cytochrome c release [85].
Additionally, several recent studies focused on the effects of stress-related disorders of Rd and other protopanaxatriol-type ginsenosides. Sustained stress has been considered a risk factor for human ailments, including depression, anxiety, and cognitive dysfunction. Brain-derived neurotrophic factor (BDNF), a neurotrophin, is crucial to the survival, growth, and maintenance of neurons involved in emotional and cognitive function in brain. Results of Han et al. [86] showed that Rd mitigated anxiety/depression, colitis and gut dysbiosis by regulating NF-κB-mediated BDNF expression. Moreover, Rd improved cognitive impairment in chronic restraint stress mice by mitigating oxidative stress and inflammation, while upregulating the hippocampal BDNF-mediated cAMP-reflecting element binding (CREB) protein signaling pathway [87]. Moreover, Rd ameliorated impairment of learning and memory behaviors in chronic cerebral hypoperfusion (CCH) mice through regulation of BDNF by reestablishing the balance between Ac-H3 and HDAC2 [88]. The occurrence of metabolic and psychiatric disorders may be caused by higher levels of glucocorticoids. Ginsenoside Rd could inhibit adrenocorticotrophic hormone (ACTH)-induced corticosterone production through blockading the MC2R-cAMP/PKA/CREB pathway in adrenocortical cells, which might represent an important therapeutic option for the treatment of stress-related disorders [89]. Ginsenoside Rd also exerted neuroprotective effects after noise-induced auditory system damage through a mechanism involving the SIRT1/PGC-1α signaling pathway, which could be an attractive pharmacological target for the development of novel drugs for noise-induced hearing loss treatment [90].
The effect of ginsenoside Rd on inducing neural stem cells differentiation remains to be obscure. One study showed that ginsenoside Rd enhanced the proliferation but did not affect the differentiation of neural stem cells in adult rats and cultured neural stem cells [91], however, another study illustrated that Rd promoted the differentiation of neurospheres into astrocytes in a dose-dependent manner [92]. PC12 cells respond to nerve growth factor (NGF) and could be serve as a model for neuronal cells. Wu et al. [93] provided the first evidence that Rd promoted the neurite outgrowth of PC12 cells by upregulating GAP-43 expression via ERK-and ARK-dependent signaling pathways. Furthermore, Rd inhibited glutamate-induced Ca 2+ entry in a concentration-dependent manner and prevented glutamate-induced apoptosis in rat cortical neurons, which provided potential evidence of Rd as a new neuroprotective drug for the prevention of neuronal apoptosis and death induced by cerebral ischemia [94].

Ischemic Stroke
In previous articles, the promising role of ginsenoside Rd on ischemic stroke has been described [3,95], the underlying mechanisms include the suppression of oxidative stress and inflammation, activation of PI3K/AKT pathway, suppression of the NF-κB as well as reduction of cytochrome c-releasing and apoptosis-inducing factor and so on. Thus, current review summarized the recent progress of Rd on ischemic stroke from 2015 to 2020 and focused on the molecular mechanisms underlying the beneficial role of ginsenoside Rd on ischemic stroke (Table 5).
There are multiple molecular mechanisms of ischemic stroke, of which oxidative DNA damage can trigger dysfunction and death of brain neurons and eventually lead to poor outcomes. The endonuclease VIII-like (NEIL) proteins NEIL1, NEIL2, and NEIL3, are major DNA glycosylases that remove oxidative base lesions. Yang et al. [96] investigated the effect of Rd on the expression of NEILs in the MCAO rat model and found that Rd significantly upregulated NEIL1 and NEIL3 expressions in both mRNA and protein levels.  "/" means not mentioned, "↑"means upregulation, "↓"means downregulation.  "↑"means upregulation, "↓"means downregulation.
The NMDA receptor (NMDAR) is a major excitatory neurotransmitter in central nervous system, which is involved in the pathological process of central nervous system diseases such as cerebral infarction, cerebral hemorrhage, ischemic stroke, brain trauma, etc. A recent study found that Rd could exert an inhibitory effect on NMDAR-triggered currents and sequential excitotoxicity through mitigation of DAPK1-mediated NR2B phosphorylation by attenuating calcineurin activity [97]. Rd protected ischemia-reperfusion injury (IRI) models rats and cultured neurons via inhibiting the hyperphosphorylation of NMDAR 2B subunit (NR2B subunit) and decreasing its expression levels in cell membrane [98].
It was well known that inflammation played an important role in the pathogenesis of ischemic stroke; however, the detailed mechanism of inflammatory modulation after ischemic stroke remained elusive. Microglia, the main immune cells in brain, are activated and subsequently release proinflammatory cytokines and other inflammatory mediators, worsening the neurologic outcome for stroke patients. Zhang et al. [99] demonstrated that Rd could safely improve the outcome of patients with ischemic stroke and revealed that administration of Rd in middle cerebral artery occlusion rat models could significantly inhibit ischemia-induced microglial activation and proteasome activity in microglia. Then, in 2020, the same research team further illuminated the downstream mechanisms that Rd was efficient for attenuating the pathogenesis of cerebral ischemia-induced bloodbrain barrier damage by suppressing proteasome-mediated inflammation and sequentially suppressing NF-κB/MMP-9 pathway [100]. Moreover, nod-like receptor protein 3 (NLRP3) inflammasome plays a key role in mediating inflammatory response in the process of cardiovascular disorder, diabetes and ischemic stroke. It was reported that the combination of Panax ginseng and Angelica sinensis treatment attenuated cerebral injury via inhibition of NLRP3 inflammasomes activation and microglial pyroptosis after stroke, along with Drp1-mediated mitochondrial fission [101].

Cardiovascular Protection
A previous study has shown that ginsenoside Rd blocked Ca 2+ influx through receptorand store-operated Ca 2+ channels in vascular smooth muscle cells, which might contribute to the cerebrovascular benefits [102] (Table 5). Nowadays, there is growing evidence that cerebrovascular remodeling is the common pathological basis of hypertension target organ damage, and circulatory dysfunction. Thus, effective cerebrovascular remodeling reversal therapy is an important measure to improve the prognosis of patients with hypertension, atherosclerosis, etc. Guan et al. [103] examined the effects of ginsenoside Rd on blood pressure, cerebrovascular remodeling and Ca 2+ entry in freshly isolated basilar arterial vascular smooth muscle cells (BAVSMCs). Results showed that the attenuation of hypertensive cerebrovascular remodeling after Rd treatment, which the underlying mechanism might be associated with inhibition of voltage-independent Ca 2+ entry and basilar artery smooth muscle cells (BASMCs) proliferation. Later, they investigated whether Rd influenced H 2 O 2 -induced apoptosis in BAVSMC [104]. The data strongly provided evidence that Rd potentiated H 2 O 2 -induced apoptosis of BASMCs through the mitochondria-dependent pathway. Then, ginsenoside Rd, as a voltage-independent Ca 2+ channels blocker, reduced ox-LDL uptake and cholesterol accumulation in macrophages via inhibition of scavenger receptor A activity and expression, suggesting that Rd prevented the development of atherosclerosis [105]. Additionally, Lu et al. [106] focused on the effects of Rd on L-type calcium channel current in isolated rat ventricular myocytes and its potential mechanism and drew a conclusion that Rd might exert its protective effects via blocking of Ca 2+ channel in cardiomyocytes.
Cardiac hypertrophy, the gradual compensatory function of chronic stress load, eventually leads to myocardial ischemia and chronic heart failure. Results from Zhang et al. [107] revealed that ginsenoside Rd improved cardiac dysfunction and remodeling induced by pressure overload, which was related to the inhibition of protein levels of AKT, calcineurin A, ERK1/2 and TGF-β1. Myocardial ischemia-reperfusion (MI/R) injury refers to the structural and functional damage of myocardial cells caused by ischemic myocardium after resuming blood reperfusion; the mechanisms are still diverse and unknown. Rd-mediated cardioprotective effects against myocardial ischemia/reperfusion were found by both reducing intracellular reactive oxygen species, inhibiting mitochondria-mediated apoptosis and Ca 2+ influx. Evidence suggested that Rd attenuated myocardial ischemia/reperfusion injury via inhibition of AKT/GSK-3β signaling and mitochondria-dependent apoptotic pathway in MI/R injury rat model and an in vitro neonatal rat cardiomyocyte (NRC) model [108]. Another report showed that Rd protected against MI/R injury as evidenced by improving cardiac function, decreasing infarct size and levels of serum creatine kinase, LDH and cTnI via Nrf2/HO-1 signaling, which played a key role in attenuating oxidative stress [109]. A recent study investigated the potential protective efficacy of Rd against nicotine-induced vascular endothelial cell injury by preserving normal vascular endothelial NO signaling, suppressing platelet aggregation and vasoconstriction, and by preventing endothelial cell-monocyte adhesion [110].

Immunological Activities
Recently, more attention has been paid to the effect of ginsenoside Rd on immune regulation in many immune-mediated diseases (Table 5). Multiple sclerosis (MS), one of the most common central nerve demyelinating diseases, is an autoimmune inflammatory disease affecting the central nervous system of the body. Ginsenoside Rd effectively ameliorated the clinical severity in EAE mice, providing a potential for amelioration of neuroimmune dysfunction diseases [111]. While the underlying mechanism of Rd in inhibiting the clinical course of EAE remains unclear. Furthermore, recent study investigated the potential mechanisms underlying the efficacy of Rd in alleviating the injury of EAE by modulating inflammation and autoimmunity via the downregulation of related proinflammatory cytokines IL-6 and IL-17, upregulation of inhibitory cytokines TGF-β and IL-10, and modulation of Treg/Th17 imbalance [112]. Similarly, Guillain-Barré syndrome (GBS) is also one of the most common immune-mediated neuropathies, characterized by demyelination and axonal damage, mainly peripheral nerve demyelination. A recent study by Ren et al. [113] provided the evidence of preventive effect of Rd on GBS by modulating monocyte subsets conversion and elevating the transcription factors, such as Nr4a1, through the in vivo experimental autoimmune neuritis mice model and in vitro mouse bone marrow stem cells.
Organ transplant rejection, a manifestation of the body's immune response, seriously affects the prognosis of patients. It was reported that ginsenoside Rd could effectively antagonize transplant rejection via regulating the balance of Th1/Th2 type cytokines secretion, as well as reducing the percentages of CD4+ T cells and CD8+ T cells in the peripheral blood of rat recipients [114]. In parallel, ginsenoside Rd from Panax ginseng could enhance Th1 immunity, which might qualify Rd as an immunoadjuvant to induce surface mannan extract to produce a protective antibody [115,116]. However, it was noted that ginsenoside Rd and 20(S)-Rg3 isolated from red ginseng were identified as potential allergens that induced the release of mediators associated with anaphylactoid reactions [117].

Others
In addition, ginsenoside Rd was reported to have renal protection, lung protection, promotion of wound healing and bone differentiation, weight loss and other pharmacological activities (Table 6). Yokozawa and coworkers [118,119] evaluated the protecting effects of Rd against cisplatin-induced renal injury, a process in which apoptosis played a central role. Another study demonstrated that Rd possessed a protective function against renal ischemia/reperfusion injury (IRI) via downregulating M1 macrophage polarization [120]. Likewise, the protective effect of Rd on lipopolysaccharide (LPS)-induced acute lung injury (ALI) was recently investigated to explore the improvement of survival in endotoxemic mice by inhibiting the PI3K-AKT signaling pathway [121]. "↑"means upregulation, "↓"means downregulation.
Ginsenoside Rd also had beneficial effects on weight loss, skin whitening and hair growth. Ginsenoside Rb1 and Rd were reported as representative compounds for improving the accelerated movement of the small intestine [122]. The beneficial effects of ginsenoside Rd on obesity and insulin resistance were found by Yao and co-workers in 2020, and its mechanisms were related to upregulation of thermogenesis in a cAMP-dependent manner [123]. Moderate melanogenesis inhibition activity of Rd at 20 µM purified from Panax ginseng berry in in melan-a cells, while floralginsenoside A (FGA) was observed to display the most potent inhibitory effect, which the potential whitening mechanism might be related to inhibition of melanin content and tyrosinase activity [124]. Moreover, ginsenoside Rb and Rd were reported to promote cell proliferation in HFs through p63 induction in follicular keratinocytes, which might be the therapeutic agent for the prevention of hair loss [125]. Kim et al. [126] identified ginsenoside Rd as the most active anti-osteoporotic agents via inducing the differentiation and mineralization of MC3T3-E1 cells through the activation of the AMPK/BMP-2/Smad signaling pathways. Moreover, ginsenoside Rd was screened through a Duchenne muscular dystrophy (DMD) hiPSC-derived myoblast screening platform and identified to significantly ameliorate some of the skeletal muscle phenotypes caused by dystrophin deficiency [127]. Later, the wound-healing effect of the ginsenoside Rd isolated from ginseng leaves was tested through in vitro the keratinocyte progenitor cells (KPCs), human dermal fibroblasts (HDFs) and animal wound models [128]. Ginsenoside Rd was also reported to prevent and rescue rat intestinal epithelial cells from irradiation-induced apoptosis [129].

Pharmacokinetics and Clinical Studies
Researchers have focused on ginsenoside Rd for its bioactivities; however, little is known about its pharmacokinetic behavior, solubility, bioavailability, safety, and clinical efficacies. A systematic and comprehensive understanding of Rd is not only necessary to further study its pharmacological actions and protective mechanisms, but to also provide a scientific basis for the clinical application of Rd and the development of new dosage forms. This review provides a profile of the pharmacokinetics, metabolism, safety, tolerance, and clinical efficacy of Rd (Table 7).

Preclinical Studies
Previous literature reviewed the validity of Rd as a neuroprotective agent for acute ischemic stroke, including the pharmacokinetics, pharmacodynamics, clinical efficacy, safety, and putative therapeutic mechanisms of Rd [95]. In fact, in 2007, Wang et al. [130] first reported on the pharmacokinetic studies of ginsenoside Rd in dog plasma by liquid chromatography-mass spectrometry after solid-phase extraction. Sun et al. [131] analyzed the pharmacokinetic, tissue distribution, and excretion of ginsenoside Rd in rodents performed by HPLC and radioactive tracer assays. Results showed that intravascular administration with 20, 50, or 150 mg/kg Rd was rapidly distributed to various tissues; the dynamic changes were consistent with a two-compartment mode. Then, pharmacokinetic characteristics of eight ginsenosides, including Rd, Rh1, Rh2, Rg1, Rg2, Rg3, and so on, were investigated after an oral administration of GTSSL at a single dose of 400 mg/kg to rats based on the LC-ESI-MS/MS method [132]. Jeon et al. [133] investigated and compared ginsenoside pharmacokinetics in mice and rats following the repeated oral administration of red ginseng extract (RGE); results showed the pharmacokinetics and metabolic pathways of ginsenosides exhibited species differences. In mouse plasma, seven PPD-type ginsenosides (20(S)-Rb1, Rb2, Rc, Rd, Rg3, CK, and 20(S)-PPD) and one protopanaxatriol (PPT)-type 20(S)-Re were detected, whereas 20(S)-Rb1, Rb2, Rc, Rd, 20(S)-PPD, and 20(S)-PPT were detected in SD rat plasma. In addition, the T max and T 1/2 of 20(S)-PPD and 20(S)-PPT in rats were greater than those in mice, suggesting the species-dependent difference in the gut metabolism and absorption of ginsenosides. Recent study examined the pharmacokinetic profiles of ginsenosides Rd and Rg3 in mice orally gavaged with red ginseng (RG). Results showed that Rd absorbed was substantially high in fermented RG extract (fRGe)-treated mice, which suggested that oral administration of RG extracts could modify gut microbiome and consequently affect the bioavailability of RG ginsenosides [134]. Shenqi Jiangtang granule (SJG) is a traditional Chinese medicine prescription; Zhang et al. investigated the plasma pharmacokinetics during absorption of SJG after oral administration in rats [135]. The results showed that in vivo absorption and exposure of gomisin D and ginsenoside Rd were better than other analytes. It has been proven that some PPD-type ginsenosides, including G-Rb1, G-Rd, and partial PPT-type ginsenosides, have antidepressant and neuroregulatory effects. Recent literature reviewed the absorption and metabolism of Rd between normal and depressed rats [136]. As shown in Table 7, AUC values and Cmax values of Rd in the depression model group were increased and CL/F was decreased as compared with the normal group, suggesting the bioavailability of ginsenosides in the depression model could be improved.

Clinical Studies
In vivo pharmacokinetics and metabolism data of ginsenoside Rd may also be valuable for better understanding its pharmacologic activities and clinical application. In early 2007, Liu et al. Identified the metabolites of Rd in a rat and pharmacokinetic study in healthy volunteers [137]. Seven metabolites of Rd, mainly including oxygenation, glycosylation deglycosylation, were detected from rat urine collected from 0 to 24 h after oral and intravenous administration. The average half-life time of Rd in human plasma was detected as 19.29 h, indicating that the ginsenoside Rd may be metabolized slowly after intravenous administration. Later, the pharmacokinetics and safety of Rd were assessed in healthy Chinese participants through a phase I, randomized, open-label, single, and multiple-dose study [138]. Data showed that Rd was well tolerated with no dose-related adverse events, and had a good pharmacokinetics and safety profile, allowing it to be explored in future clinical studies in patients with acute ischemic stroke. Liu et al. [139,140] documented the improvement effects of Rd against acute ischemic stroke through a phase Π randomized, double-blind, placebo-controlled trial. In patients with acute ischemic stroke, Rd had favorable safety and tolerability, and improved the clinical symptoms of acute ischemic stroke. Two recent clinical trials showed that Rd had fewer side effects than glucocorticoid and could improve the outcome of patients with ischemic stroke by suppressing microglial proteasome activity and sequential inflammation [99].
Moreover, gut microbiota is not only involved in the biotransformation of ginsenosides, but also in the pharmacokinetics of ginsenosides in humans. However, few studies focused on the roles of the gut microbiota on the pharmacokinetics of ginsenosides in humans, and the effects had not yet been fully elucidated. A recent study determined the serum concentrations of the ginsenosides in humans after the administration of RG extracts (RG and fRG) and researchers analyzed their correlations with the fecal ginsenosidemetabolizing activities [141]. The gut bacteria seemed to exert their metabolic activity mainly on the biotransformation into ginsenoside CK via Rd rather than Rg3, suggesting that the profile and composition of the gut microbiota might affect the bioavailability and the pharmacological effects of ginsenosides. Overall, clinical studies on ginsenoside Rd are still limited, and mainly focused on phase 1 and phase 2 clinical studies of acute ischemic stroke, indicating that further studies on the potential efficacy of natural products in experimental animal models and randomized clinical trials are essential.

Concluding Remarks
Collectively, this review systematically summarized recent advances on the biotransformation, pharmacological, pharmacokinetic, and clinical studies of ginsenoside Rd. Most pharmacological activities of Rd, including anti-cancer, anti-inflammatory, antioxidative, cardiovascular protection, and immunoregulation effects were exhibited and summarized. Other health-beneficial activities that have previously received less attention, such as kidney protection, lung protection, promotion of wound healing and bone differentiation, and anti-obesity, were also included. Moreover, the limited number of pharmacokinetic and clinical studies on ginsenoside Rd have also been documented.
Overall, Rd is a very promising candidate agent for the treatment of diverse diseases, while the following issues require greater attention in the future: (i) the exact mechanisms and targets that contribute toward the pharmacological activity of ginsenoside Rd require further detailed investigation; (ii) experimental animal model studies and randomized clinical trials should be performed to evaluate the therapeutic efficacy of ginsenoside Rd; (iii) the effects of ginsenoside Rd combined with chemotherapy, target therapy, or immunotherapy need to be determined.