Biotransformation, Pharmacokinetics, and Pharmacological Activities of Ginsenoside Rd Against Multiple Diseases

Panax ginseng C.A. Mey. has a history of more than 4000 years and is widely used in Asian countries. Modern pharmacological studies have proved that ginsenosides and their compounds have a variety of significant biological activities on specific diseases, including neurodegenerative diseases, certain types of cancer, gastrointestinal disease, and metabolic diseases, in which most of the interest has focused on ginsenoside Rd. The evidentiary basis showed that ginsenoside Rd ameliorates ischemic stroke, nerve injury, cancer, and other diseases involved in apoptosis, inflammation, oxidative stress, mitochondrial damage, and autophagy. In this review, we summarized available reports on the molecular biological mechanisms of ginsenoside Rd in neurological diseases, cancer, metabolic diseases, and other diseases. We also discussed the main biotransformation pathways of ginsenoside Rd obtained by fermentation.

INTRODUCTION Panax ginseng C.A. Mey. is a well-known herbal medicine widely used in China, Korea, Japan, and other East Asian countries. At present, the ginseng root and its extract are the most widely used herbal medicine. Modern pharmacological studies have proved that ginsenosides are the main active ingredient of ginseng and have a wide range of pharmacological effects, such as anti-inflammatory Yi, 2021), anticancer (Zhang et al., 2021a), and anti-viral (Kang et al., 2021), regulate immunity (Kang et al., 2021), metabolism (Wang et al., 2021a), and improve cardiovascular system (Wang et al., 2021b;Sarhene et al., 2021) and nervous system (Brioschi Guevara et al., 2021) function, whereas most attention has been focused on the ginsenoside Rd.
Ginsenoside Rd, a natural compound extracted from the root of Panax ginseng C.A. Mey., is one of the protopanaxadiol (PPD)type ginsenosides, while the proportion of ginsenoside Rd in ginseng is very low (Liu et al., 2020a). Interestingly, the promising effects of the pretreatment and treatment of ginsenoside Rd on neurological diseases, cancer, gastrointestinal disease, and metabolic diseases have been studied extensively in in vivo and in vitro models Chen et al., 2022;Zhou et al., 2022).
Existing studies related to ginsenoside Rd have shown that various ginsenosides, such as Rb1, Rb2, and Rc, can be transformed into ginsenoside Rd after absorption and metabolism in vivo (Park et al., 2010;Shin and Oh, 2016). In addition, Rd can be prepared in a variety of ways based on the indepth study of biotransformation and the development of modern fermentation technology (He et al., 2019). Based on the above results, we summarized the biotransformation process of other ginsenosides into Rd, thereby hoping to play a positive role in the large-scale industrial production of Rd. In this study, the biotransformation sources, pharmacokinetics, pharmacological effects, and molecular mechanisms of ginsenoside Rd on various systemic diseases in recent years were reviewed, and their therapeutic potential was discussed.
β-glucosidase cleaves the glycoside at the C-3 position of ginsenoside Rd and produces the ginsenoside compound CK (Renchinkhand et al., 2020). Ginsenoside M1 is formed by the hydrolysis of the C-3 glucose group in ginsenoside Rd by snailase (Renchinkhand et al., 2017).

PHARMACOKINETICS
Intestinal flora can promote the metabolic transformation of ginseng extract and Rb1 into ginsenoside Rd in rats and can enter the blood for absorption in rats (Kim et al., 2014a). Ginsenoside Rd is distributed in various organs, with the highest content in the lungs, followed by the liver, kidney, heart, and intestine, and the lowest content in the brain (Sun et al., 2012). After taking urine 0-24 h after oral administration and intravenous administration, liquid chromatography-mass spectrometry (LC-MS) is used to confirm that oxidation and  glycosylation (Yang et al., 2006a;Yang et al., 2007a) are the main metabolic pathways of ginsenoside Rd in rats. The absolute bioavailability of Rd in dogs is 0.26% (Wang et al., 2007). As in clinical trials, ginsenoside Rd shows linear pharmacokinetics, is well tolerated in the dose range of 10-75 mg after an intravenous administration, and is slowly cleared from plasma, and the elimination rate does not change after repeated administration (Zeng et al., 2010).

Ischemic Stroke
In ischemic stroke, ginsenoside Rd plays a neuroprotective role by restoring mitochondrial function, reducing neuronal apoptosis, and eliminating neuroinflammation ( Figure 2). As for the therapeutic window study, ginsenoside Rd shows an obvious neuroprotective effect in the middle cerebral artery occlusion (MCAO) model (Ye et al., 2011a). Importantly, the results of a clinical trial showed that ginsenoside Rd has a positive effect on the prognosis of acute ischemic stroke . In Ca 2+ influx and mitochondrial dysfunction, ginsenoside Rd, a potential Ca 2+ channel blocker (Li et al., 2010), significantly reduces the burst of glutamate by increasing the expression of glutamate transporter-1 (GLT-1) and inhibits the channels of Ca 2+ influx (Zhang et al., 2013a) to protect the rat hippocampal neurons (Zhang et al., 2012a). Similar to a calcineurin inhibitor, ginsenoside Rd exerts a neuroprotective effect by inhibiting the elevation of N-methyl-D-aspartate (NMDA) receptors and the hyperphosphorylation of the N-methyl-D-aspartate receptor 2B (NR2B) subunit in the MCAO model and oxygen-glucose deprivation (OGD) cultured neurons (Xie et al., 2016b;Zhang et al., 2020a). Ginsenoside Rd pretreatment exerts neuroprotective effects by inhibiting the Ca 2+ overload and specificity attenuated the expression of transient receptor potential melastatin (TRPM) 7 and acid-sensing ion channel (ASIC) 1a while promoting ASIC2a expression following focal ischemia (Zhang et al., 2012b). Remarkably, the results of a clinical trial based on Ca 2+ disorder and subsequent neurotoxicity induced by acuteischemic stroke, ginsenoside Rd can be considered a calcium channel antagonist and a neuroprotectant (Liu et al., 2009). As for mitochondrial dysfunction, ginsenoside Rd markedly protects the mitochondria, as indicated by regulating enzyme activity, reducing mitochondrial hydrogen peroxide production and depolarizing mitochondrial membrane potential (MMP), decreasing reactive oxygen species (ROS) production in isolated mitochondria from Sprague-Dawley (SD) rats (Ye et al., 2011b), and reducing the mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage and cell apoptosis in MCAOinduced ischemic stroke model Yang et al., 2016). These findings are also confirmed in primary cultured hippocampal neuron cells (Ye et al., 2009). In addition, in elderly stroke mice, ginsenoside Rd can play an equivalent neuroprotective role in elderly transient focal ischemic mice by regulating lipid peroxide accumulation, mitochondrial complex activity, and MMP (Ye et al., 2011c).

Other Nervous System Diseases
Ginsenoside Rd has a significant neuroprotective effect on a variety of neurological diseases, which may be related to its promotion of stem cell proliferation (Shi et al., 2005) and differentiation into astrocytes (Lin et al., 2012). Ginsenoside Rd may promote neurite outgrowth by upregulating growthassociated protein of 43 kDa (GAP-43) expression via ERK-and ARK-dependent signaling pathways in NGF-induced PC12 cells .
In H 2 O 2 -induced PC12 cells, ginsenoside Rd, as a neuroprotective agent, has neuroprotective effects on neurodegenerative diseases (Ye et al., 2008). In the converting monocyte phenotype and macrophages of the Guillain-Barre syndrome (GBS) mouse model, ginsenoside Rd attenuates experimental autoimmune neuritis (Ren et al., 2021). Ginsenoside Rd can regulate MMP by decreasing intracellular ROS and enhancing the activity of antioxidant enzymes and mitochondrial complex, thereby increasing intracellular ATP levels and ultimately reducing 1-methyl-4-phenylpyridinium (MPP + )-induced cell death in Parkinson's disease (PD) (Liu et al., 2015b). Meanwhile, in the Aβ 25-35 -induced neuronal damage model, apoptosis and oxidative stress are ameliorated by ginsenoside Rd by regulating antioxidant capacity and the production of apoptotic proteins (Liu et al., 2015c). Learning and memory abilities can be improved in ginsenoside Rd-pretreated APP transgenic mice by significantly suppressing the NF-κB pathway to reduce the generation of proinflammatory factors (Liu et al., 2015d). Ginsenoside Rd-mediated neuroprotective effects against Alzheimer's disease (AD) progression play a significant role in Neuro2a cells (Kim et al., 2014b). Ginsenoside Rd pretreatment can inhibit tau protein phosphorylation by maintaining a balance of GSK-3β, cyclindependent kinase 5 (CDK5/P25), and protein phosphatase 2A (PP-2A)  to inhibit tau phosphorylation of tau protein at Ser199/202, Ser396, or Ser404 in okadaic acid-induced rats, APP transgenic mice, and cortical neurons to increase PP-2A activity for protection against AD (Li et al., 2011a;Li et al., 2021), respectively. Moreover, ginsenoside Rd increases the soluble amyloid-β precursor protein α (sAPPα) level and reduces extracellular Aβ to enhance the cognitive and memory functions of ovariectomy rats (Yan et al., 2017).
In experimental autoimmune encephalomyelitis, ginsenoside Rd exerts a neuroprotective role by regulating the immune response and inflammatory reaction via a signal pathway of IFN-g/IL-4, BDNF/ NGF , and Foxp3/RORγt/JAK2/STAT3 (Jin et al., 2020a). In spinal cord injury (SCI) models, ginsenoside Rd shows anti-inflammatory effects consistent with dexamethasone that could significantly decrease the biomarkers of apoptosis, inflammation, oxidative damage factor, and repaired damaged mitochondria; particularly, there is no obvious difference in terms of dexamethasone in anti-inflammatory Cong and Chen, 2016), and these effects depended on the ASK1/JNK pathway . In the pathology of noise-induced hearing loss (NIHL), ginsenoside Rd could alleviate the apoptosis and oxidative stress damage on neuron cells by activating the SIRT1/ PGC-1α signaling pathway (Chen et al., 2020). In addition, ginsenoside Rd treatment effectively eliminates the oxidative injury and the production of proinflammatory factors and peroxides in the chronic restraint stress (CRS) paradigm . Ginsenoside Rd pretreatment may be neuroprotective in old rats following acute Pb exposure through limited microglial activation and maintained neural stem cell proliferation (Wang et al., 2013a).
To summarize, ginsenoside Rd can play a significant role in neuron damage by inhibiting the production of excitatory amino acids, reducing the intracellular Ca 2+ influx mediated by the NMDA pathway, changing the neurotoxicity of Ca 2+ to mitochondrial function damage, and regulating apoptosisinducing and neuroinflammatory factors ( Table 2). Table 3 and Figure 3, ginsenoside Rd can inhibit the proliferation of various cancer cells by participating in the apoptotic pathway. As a potential therapeutic and specific 26S proteasome inhibitor, ginsenoside Rd plays an important role in anticancer therapy by targeting 26S proteasome (Chang et al., 2008). Ginsenoside Rd can appreciably inhibit the proliferation of gastric cancer cells and can stimulate apoptosis by downregulating cyclin D1, thereby inducing cell cycle arrest in the G0/G1 phase and enhancing the expression of caspase-3 and caspase-9 and the ratio of Bax/Bcl-2 (Tian et al., 2020). After heat processing, the anticancer activity of deglycosylated Rd could be improved via the apoptotic pathway for AGS cells (Kim et al., 2013b).

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In non-small-cell lung cancer (NSCLC), ginsenoside Rd, as a therapeutic drug, inhibits the nuclear factor erythroid 2associated factor 2 (NRF2) pathway, and the synergistic effect of ginsenoside Rd in A549 and cisplatin (DDP)-resistant A549 cell lines (A549/DDP) can be weakened by knocking out NRF2 (Chian et al., 2019). As for glioblastoma, ginsenoside Rd decreases the proliferation of human glioma U251 cells and promotes apoptosis by downregulating the expression of hTERT and Bcl-2, upregulating the expression of the caspase-3 level, and inhibiting the telomerase activity of U251 cells (Gu et al., 2019). Ginsenoside Rd inhibits the proliferation and migration of glioblastoma cells by decreasing the expression of tumorsuppressor Mir-144-5p and promoting the expression of the target of Mir-144-5p toll-like receptor 2 (Liu et al., 2020b). In colorectal cancer cells, ginsenoside Rd, a therapeutic agent, targets epidermal growth factor receptor (EGFR)/SOX2 signaling (Phi et al., 2019).
Finally, in HepG2 cells and the HepG2 cell-injected nude mice-induced hepatocellular carcinoma model, the combination of CA4P and ginsenoside Rd has synergistic antitumor effects via the PI3K/AKT/mTOR signaling pathway-related inhibition of HIF-1α (Yang et al., 2021a). HepG2 cells treated with ginsenoside Rd noticeably promoted matrix metalloproteinases' (MMPs) activation, and MAPK signaling pathways were involved in cancer cell migration, thereby suggesting that ginsenoside Rd inhibits the activity of HepG2 cells in a dose-dependent and timedependent manner (Yoon et al., 2012).

Metabolic Diseases
Laboratory data of ginsenoside Rd suggest that it has effects on multiple metabolic diseases. The browning of white adipose tissue induced by cold stress and cAMP levels are increased by ginsenoside Rd. In particular, Rd alleviates obesity and insulin resistance by upregulating thermogenesis through the cAMP/ protein kinase A (PKA) signaling pathway (Yao et al., 2020). In fast-food diet-induced non-alcoholic fatty liver disease (NAFLD), fermented ginsenoside Rd with Cordyceps militaris regulates lipid metabolism and the inflammatory response via mTORC1 signaling (Choi et al., 2019). Ginsenoside Rd inhibits the progress of the death of islet transplantation by decreasing the apoptosis of the islet cells (Kaviani et al., 2019). In the atherosclerosis process, ginsenoside Rd decreases oxidized lowdensity lipoprotein (Ox-LDL) and cholesterol by inhibiting Ca 2+ influx (Li et al., 2011b). In diabetic db/db mice and mesangial cells, pectin-lyase-modified ginsenoside Rd relieves diabetic nephropathy via alleviated ROS production (Jung et al., 2021).

Other Diseases
Ginsenoside Rd has positive effects on skin injury, osteoporosis, kidney injury, vessel injury, heart injury, lung injury, aging, and inflammation. In animal wound models, ginsenoside Rd significantly increases wound healing by promoting the proliferation and migration level of keratinocyte progenitor cells (KPCs) and human dermal fibroblasts (HDFs) (Kim et al., 2013c). Ginsenoside Rd also has a positive effect on rejection caused by a transplant skin allograft (Wang et al., 2012a). Beyond that, ginsenoside Rd, as an antiosteoporotic agent, promotes differentiation and mineralization in osteoblastic MC3T3-E1 cells . In animal models of renal I/R injury and cultured proximal tubule cells, ginsenoside Rd has a protective effect by inhibiting inflammation and regulating biochemical indexes of renal function (Yokozawa et al., 1998;Ren et al., 2016). In addition, ginsenoside Rd downregulates NF-κB and the expression of iNOS and COX-2 in lipopolysaccharide (LPS)-induced Institute of Cancer Research (ICR) mice, and RAW264.7 cells were suppressed (Kim et al., 2013d). In the nicotine-induced vascular endothelial injury model, ginsenoside Rd plays an important role in the prevention of cardiovascular diseases via participation in NO signaling and regulates platelet and vascular function (Zhang et al., 2020c). Ginsenoside Rd upregulates Cyto C release and caspase-9/caspase-3 activation and decreases the MMP and the ratio of Bcl-2/Bax via the mitochondriadependent pathway in H 2 O 2 -induced apoptosis in basilar artery smooth muscle cells (BASMCs) . Furthermore, ginsenoside Rd could relieve the cisplatin-induced kidney injury (Yokozawa and Liu, 2000;Yokozawa and Dong, 2001) and kidney proximal tubules cephaloridine injury under cephaloridine treatment (Yokozawa and Dong, 2001). In an adrenocorticotrophic hormone (ACTH)-induced corticosterone secretion cell model, ginsenoside Rd inhibits ACTH-induced corticosterone production by inhibiting the MC2R-cAMP/PKA/cyclic AMP response element binding (CREB) pathway in adrenocortical cells (Jin et al., 2020b). In myocardial I/R-induced rats and simulated I/R-induced primary neonatal rat cardiomyocyte models, ginsenoside Rd promotes cardioprotection via the activation of AKT/GSK-3b signaling (Wang et al., 2013b). In addition, ginsenoside Rd can protect against LPS-induced acute lung injury by inhibiting the PI3K/AKT signaling pathway (Yang et al., 2021b). Other studies have indicated that ginsenoside Rd can significantly enhance the survival time of Caenorhabditis elegans via lipid metabolism and the activation of the stress response signaling pathway  and can alleviate the oxidative damage caused by aging in senescence-accelerated mice (Yokozawa et al., 2004). Finally, the anti-inflammatory activity of ginsenoside Rd is well documented, is considered to be associated with its antioxidant effects (Kim et al., 2007;Zhang et al., 2013b), and selectively produces prostaglandin E2 (PGE2) by activating the CCAAT/ enhancer binding protein (C/EBP) and CREB to express COX-2 (Jeong et al., 2007). Ginsenoside Rd exerts anti-inflammation effects in carrageenan-induced inflammation rats via the inhibition of the NF-κB signaling pathway (Wang et al., 2012b) and in ovalbumin-induced allergic rhinitis mice by regulating multiple inflammatory factors  and elicits a Th1 and Th2 immune responses (Yang et al., 2007b). Ginsenoside Rd enhances the Th1 response to surface mannan extract in mice, which protects mice from disseminated candida infection by stimulating higher titers of Th1 antibodies and a Th1-dominated immune response (Han and Rhew, 2013).

CONCLUSION AND PERSPECTIVE
As a widely used herbal medicine, ginseng appears in the form of dietary supplements nowadays. Available evidence suggests that the antiapoptotic, antioxidant, and anti-inflammatory activities, which suppress the calcium influx of ginsenoside Rd, may have an important role in the neuroprotective and anticancer effects. Ginsenoside Rd play a crucial role in neuroprotective, anticancer effects, metabolism, and other diseases by regulating PI3K/AKT, inhibiting Cyto C released and caspase activation, and regulating the release of inflammatory factors, which play a crucial role in neuroprotective, anticancer effects, metabolism, and other diseases.
In addition, ginsenoside Rd has potential therapeutic effects on regulating metabolism and in multiorgan protection. However, attributable to the shortage of clinical studies on ginsenoside Rd, it is difficult to make a clear decision. In addition to exploring its various activities, it is suggested to verify existing activities in a deeper mechanism, design clinical trials to prove its safety and effectiveness, and obtain a more extensive clinical application.

AUTHOR CONTRIBUTIONS
JnL, QH, and YY collected, analyzed, and reviewed the literature and wrote the main manuscript; PJ, JC, ME, ZZ, HQ, JaL, and ZC added/checked references and assembled figures/tables; DZ and LZ revised the manuscript; and XL and LZ designed and supervised the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.