Diosgenin: An Updated Pharmacological Review and Therapeutic Perspectives

Plants including Rhizoma polgonati, Smilax china, and Trigonella foenum-graecum contain a lot of diosgenin, a steroidal sapogenin. This bioactive phytochemical has shown high potential and interest in the treatment of various disorders such as cancer, diabetes, arthritis, asthma, and cardiovascular disease, in addition to being an important starting material for the preparation of several steroidal drugs in the pharmaceutical industry. This review aims to provide an overview of the in vitro, in vivo, and clinical studies reporting the diosgenin's pharmacological effects and to discuss the safety issues. Preclinical studies have shown promising effects on cancer, neuroprotection, atherosclerosis, asthma, bone health, and other pathologies. Clinical investigations have demonstrated diosgenin's nontoxic nature and promising benefits on cognitive function and menopause. However, further well-designed clinical trials are needed to address the other effects seen in preclinical studies, as well as a better knowledge of the diosgenin's safety profile.


Introduction
Currently, innovative approaches are being adopted to combat the onset and progression of human health problems [1][2][3]. One strategy involves using compounds derived from edible dietary plants, seeds and traditional medicinal herbs that have an impact on not only treating but also preventing cancer, either alone or along with existing treatment approaches [4][5][6]. Natural medicines which are derived from medicinal herbs or different vegetables or plants and fruits with promising health benefits are proved to be effective in treating and also preventing a wide range of human ailments, including obesity, neurological disorders, cancer, metabolic syndromes, cardiovascular disease, and diabetes [7][8][9][10]. A recent review by Newman and Cragg [11] reported the various natural products as a source of safe drugs for the treatment of various ailments in humans [11]. Over the decades, natural compounds derived from Chinese herbs have been used to a large extent in traditional practices of medicine. There are natural compounds that can be taken from plants, such as the anticancer medicine paclitaxel from Taxus brevifolia quercetin found in numerous vegetables and fruits [12,13] and the antimalarial medication artemisinin from Artemisia apiacea, which have a wide spectrum of pharmacological effects [14].
Sapogenins are a group of chemicals that are found in a variety of natural products in glycoside form and enhance overall health. The most effective bioactive chemicals derived from natural product sources are steroidal sapogenins (otherwise known as spirostans) [15,16]. The majority of steroidal sapogenins show pharmacological action in vitro and in preclinical animal models [15,17]. Sapogenins have been the subject of several clinical investigations, which are either finished or ongoing [18,19]. Diosgenin is a steroidal sapogenin that can be found in the Rhamnaceae, Liliaceae, Scrophulariaceae, Dioscoreaceae, Amaryllidaceae, Solanaceae, Leguminosae, and Agavaceae families [20][21][22][23]. It is plentiful in Rhizoma polgonati, Smilax china, Dioscorea villosa, Trigonella foenum-graecum, and Dioscorea rhizome, among other plants with medicinal values. In the pharmaceutical sector, diosgenin is considered to be a fundamental material for starting the steroidal medicines to synthesize. One of the leading causes of death in the whole world is considered to be chronic diseases, and despite recent advancements in the procedures of treatment, synthetic medications still exhibit grating side effects along with chemoresistance, restricting the usage of these medications.
Phytochemicals have gained popularity due to their low risk of negative effects [8,24]. Diosgenin is a phytochemical that has acquired prominence due to its usefulness in the treatment of lethal diseases such as nervous system disorders, diabetes, cancer, arthritis, asthma, cardiovascular disease, and others [25]. Scientists have used it in the treatment of inflammation, malignancies, hyperlipidemia, and infections as it has a wide spectrum of therapeutic attributes and pharmacological actions [26]. Diosgenin has been investigated extensively for the management and treatment of cancer [17], skin illnesses [27], cardiovascular illnesses [28], diabetes mellitus [29], atherosclerosis [30], and osteoporosis [31].
Diosgenin has been largely talked of and written about for its huge potential in pharmacological studies, as well as the intriguing rudimentary means of action, validating and expanding the comprehensive knowledge gained through its conventional use. Several mechanistic and preclinical studies have been conducted in this context, primarily during the last two decades, to better apprehend the true advantages and significance of diosgenin against manifold illnesses [32,33]. Overall, the findings of multiple investigations suggest that diosgenin could be used as a novel multi-target-based therapeutic or chemopreventive drug for a variety of chronic diseases. As a result, it has become an agenda of great interest to develop effective ways to derive diosgenin from various natural resources, furthermore to establish medication dosage forms to grant its administration [34,35]. Recently, the role of diosgenin in diabetic and cardiac diseases has been reviewed, but a holistic review of various biological activities based on preclinical and clinical studies is still missing in the literature to the best of our literature review [36,37]. The fundamental objective of this review is to summarize the in-depth pharmacological activity moving from preclinical to clinical evidence and safety issues, to upcoming strategies to overcome present limitations.

Review Methodology
To conduct this comprehensive study, the most relevant studies on the pharmacological properties of diosgenin were analyzed. We searched for scientific publications published in journals electronic databases such as PubMed/MEDLINE, DOAJ, Scopus, Web of Science, and SciFinder, using the next MeSH terms: "Diosgenin/analogs & derivatives," "Diosgenin/pharmacology," "Diosgenin/ therapeutic use," "Humans," Anti-Asthmatic Agents/pharmacology" "Chronic Disease/prevention & control," "Cardiovascular Diseases/drug therapy," "Inflammation/drug therapy," "Neoplasms/drug therapy," "Nervous System Diseases/drug therapy" "Saponins/pharmacology," "Saponins/ therapeutic use," "Saponins/toxicity," "steroids," and "Signal Transduction/drug effects." Studies that explained the molecular mechanisms, signaling pathways, molecular targets, and well-defined doses of pharmacological experiments were included. Duplicates, experimental pharmacological studies involving other test substances, and homeopathic preparations were excluded. The most relevant information was summarized in tables and images. The taxonomy of plants has been validated according to The Plant List [38,39] and the chemical formulas verified with the PubChem database [40].

Sources
The compound diosgenin was discovered by Fuji and Matsukawa in 1936 [41], and its application in the synthesis of cortisone and other drugs was reported by Marker and coworkers [42]. The Dioscorea genus is the main source of diosgenin, and~137 species of this genus contain diosgenin 2 Oxidative Medicine and Cellular Longevity [26] including Dioscorea nipponica [43], Dioscorea zingiberensis [44], Dioscorea composita [45], and Dioscorea deltoidea [46,47]. It is also isolated from other botanicals including Trigonella foenum-graecum, Costus speciosus, Tribulus terrestris L., Rhizoma polgonati, and Paris polyphylla ( Table 1). The compound diosgenin is mostly produced from the hydrolysis process of steroidal saponins in the presence of strong acid, base, or enzyme catalyst, while microbial transformation technics are gaining much attention due to their highly specific nature, low cost, and environmentally friendly conditions [48].

Biosynthesis
Microwave-assisted extraction (MAE) and ultrasoundassisted extraction (UAE) methods were used for a better yield of diosgenin by using different solvent systems including acetone, ethanol, hexane, and petroleum ether with different concentrations ( [49]. In the industry of pharmaceutical sector, most hormonal medicines have diosgenin as their precursor. This makes diosgenin a classic precursor which is generated mostly by Dioscorea species. The mechanisms underlying the emergence and evolution or progression of biosynthesis of diosgenin in plants are unknown. The geneses of the dios-genin biosynthesis pathway, as well as its evolution, in yam, were validated in recent experimental research. The authors used a variety of diosgenin biosynthesis routes in this study [55] (Figure 1). Metabolic engineering has also been used to produce diosgenin from yeast. Because cholesterol biosynthesis is more efficient in animal cells than in plant cells, researchers have created a strain with yeast chassis that produces cholesterol by integrating animal genes [56]. As a common precursor for triterpenoid biosynthesis, 2,3-oxidosqualene could be produced in significant amounts from it; Saccharomyces cerevisiae strain BY-T3 which was previously modified was chosen as the starting strain [57]. First, Cheng and coauthors discovered that positive selection-driven P450 gene neofunctionalization and duplication were significant in the diosgenin biosynthesis pathway formation. They discovered that CpG islands, which developed to maintain the balance of carbon flux between the production of diosgenin and starch and modulate or control gene expression in the diosgenin pathway, were responsible for diosgenin enrichment in the yam lineage. Finally, we heterologously produced diosgenin to 10 mg/L in yeast that was genetically modified by combining genes from plants, mammals, and yeast [55].

Pharmacological Properties of Diosgenin:
Underlying Molecular Mechanisms and Signaling Pathways 5.1. Neuroprotective. The oxidative stress caused by the reactive oxygen stress leads to various ailments such as neurological disorders, cardiovascular diseases, and cancer [58][59][60]. Health benefits of diosgenin administration are most commonly determined as a neuroprotective agent in terms of  3 Oxidative Medicine and Cellular Longevity cognitive effects [61][62][63], neuroinflammation [51,[64][65][66][67], multiple sclerosis [68,69], spinal cord injury [70,71], stroke and thrombosis [72], and neuropathic pain [43,[73][74][75]. Few recent investigations based on diosgenin and related aspects have been studied through in silico, in vitro, and in vivo approaches and discussed here. Regarding this, a new structure of the neuronal network has been developed to calculate the solubility of the diosgenin compound. The ordinary neural network modeling was used to improve the calculation accuracy and estimate the solubility of diosgenin in the nalkanols with more carbon atoms due to its rational design concept [76].
In another study, diosgenin was evaluated against cerebral ischemia-reperfusion injury through in silico (proteome dynamic approach), in vitro, and in vivo methods. The authors investigated 5043 regulatory proteins from the brain samples and different signaling pathways [77]. Screening of small molecules for targeting the fungal virulence factors without any effect on viability was conducted by Aaron and the group [78]. They demonstrated that Cryptococcus neoformans (Cn) was prohibited from crossing the bloodbrain barrier via suppressing the proteolytic activity of Mpr 1 with maintaining Cn viability. In another word, out of 240 compounds, diosgenin with two other compounds significantly inhibited Mpr 1 proteolytic activity (IC 50 : <10 μM) without any cell toxicity and blocked Cn crossing the BBB [78].
Cheng et al. evaluated the antiapoptotic effects of diosgenin in D-galactose-induced ageing brain (cerebral cortical apoptosis) [79]. A total of 36 male (12-week-old) Wistar rats were recruited and divided into four groups (control: 1 mg/ kg/day of saline, i.p.; DD0: 150 mg/kg/day of D-galactose, i.  The overall results of this study showed the suppressing (D-galactose-induced neuronal Fas-dependent and mitochondria-dependent apoptotic pathways) and enhancing (Bcl-2 family-associated prosurvival and IGF-1-PI3K-AKT survival pathways) effects of diosgenin, which may trigger neuroprotective effects against D-galactoseinduced ageing brain [79]. The neuroprotective effects of diosgenin in the diabetic mice model were also evaluated by Leng and the group [80]. All mice (male C57) were fed for up to 8 weeks (high fat diet) and intraperitoneally injected with streptozotocin (dose: 100 mg/kg for 2 days). Eligible mice were dived into four groups including control (n = 6), diabetic group (n = 6), low-dose diosgenin group (n = 6, 50 mg/ kg), and high-dose diosgenin group (n = 6, 100 mg/kg). The main outcome of this study indicates that diosgenin significantly reduced the level of blood glucose and increased the body weight of diabetic mice. Diosgenin attenuated the level of MDA (in dose-dependent manner) but increased the activity of antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)) and expression of signaling pathways (nuclear factor-erythroid factor 2related factor (Nrf2), heme oxygenase (HO), and NAD(P)H dehydrogenase [quinone] (NQO)-1) in diabetic mice. These signaling pathways were involved in its neuroprotective activities [80].
Oyelaja-Akinsipo et al. investigated the neuroprotective and glucose-lowering ability of compound diosgenin in hyperglycemia-mediated cerebral ischemic brain injury using the zebra-fish model of type II diabetes mellitus [81]. Diabetes was developed in an experimental model by using streptozotocin (20 mg/kg b.w.) for 28 days, and two different doses (20 and 40 mg/kg b.w) of diosgenin were used. The results of this study indicated that diosgenin significantly decreased the concentration of glucose from 175.87 to 105.68 mg/d/L and 82.06 mg/d/L in both doses. The compound treatment increased the body weight and growth in diabetic zebra fishes (p < 0:05 and p < 0:001) and also enhanced the catalytic activity. Its activity also protects the brain from the possibility of hyperglycemic-mediated brain injury and apoptotic brain cell death [81].
The most representative studies on neuroprotective activities of diosgenin have been presented in Table 2.

Anticancer.
Cancer is the most dangerous disease that affects people around the globe, with one in every six deaths due to cancer [92][93][94]. Different therapeutic tactics, including radiotherapy, chemotherapy, and laser-based therapy, are currently under practice [59,95,96]. A series of experiments on cytotoxicity aspects of diosgenin was conducted by several researchers worldwide. In a recent study, cytotoxic effects of standardized extracts, fraction and their compounds (Trigonella foenum-graecum) were evaluated against human cancer cells (SKOV-3, HeLa, and MOLT-4 cells).     MOLT-4)). The fraction significantly increased reactive oxygen species production and caspases activity in the cells [97]. A total of 28 diosgenin amino acid ester derivatives (3a-3g and 7a-7g) were designed and synthesized by Ma et al. and evaluated for their cytotoxicity against six human cancer cells including K562, T24, MNK45, HepG2, A549, and MCF-7 [98]. The majority of derivatives displayed cytotoxic potential against these six tumor cells. Out of 28 derivatives,  7 Oxidative Medicine and Cellular Longevity upregulation of Bax and downregulation of Bcl-2 and BIRC5 pre-mRNA transcripts of genes [54].

Oxidative Medicine and Cellular Longevity
Yin and coworkers developed and synthesized thirty-two new diosgenin derivatives and evaluated their cytotoxic activity against three human cancer cells (A549, MCF-7, and HepG2). Among them, compounds 8, 18, 26, and 30 were more potent compared to diosgenin. Compound 8 showed strong and low cytotoxic activity against HepG2 cells (IC 50 : 1.9 μM) and low L02 cells (IC 50 : 18.6 μM), respectively. Additionally, compound 8 induces G 0 /G 1 cell cycle arrest and apoptosis in HepG2 cells. A molecular docking study also suggested that p38α-MAPK is a suitable target for compound 8 and fits well its active site [99]. Two new azasteroids were synthesized from diosgenin through the modification in the A and B rings and evaluated for their antiproliferative activity [100].
Diosgenin significantly inhibited the cell viability and motility of breast cancer cells and stimulate apoptosis via suppression of S-phase kinase-associated protein Skp-2 in breast cancer cells [101]. Although NF-κB promotes cancer initiation and development, but some reports demonstrate its role in tumor suppression [102]. Part of this several studies has been conducted to investigate the role of diosgenin, and its derivatives against different types of cancers are presented in Figure 2 and Table 3.

Antiatherosclerosis.
Atherosclerosis is a disease of the middle and large arteries characterized by the formation in the inner tunic and middle deposits of atheromatous plaques, which contain accumulations of LDL-cholesterol, lipophage, and sometimes calcifications on former lesions that prevent normal blood flow through the vessel. It is a disease in which plaque accumulates inside the arteries and can trigger serious problems including heart failure, stroke, or death [132][133][134]. The pathogenic mechanisms are com-plex, involving lipid peroxidation, oxidative stress, inflammation, or altered immune response, causing aging and degenerative brain damage [135][136][137].
The antiatherosclerosis potential of diosgenin and its derivatives were studied by few researchers. In this context, the curative effects of diosgenin on macrophage cholesterol metabolism and its mechanism were investigated by Lv and the group [30]. The diosgenin treatment significantly enhanced the expression of ATP-binding cassette transporter A1 (ABCA1) protein without any effect on liver X receptor α levels. Additionally, diosgenin treatment also inhibits aortic atherosclerosis progression via downregulation of miR-19b proteins in THP-1 macrophages/MPM-derived foam cells ( Figure 3). The in vivo study of the diosgenin compound and its impact on Wistar rats treated with an atherogenic diet were performed by Binesh and coworkers. In this study, the atherogenic diet triggered the inflammatory mediators in the heart, liver, and brain via upregulation of COX-2, TNFα, and NFkBp65 levels of the rats, while diosgenin treatment downregulated the level of these inflammatory markers and inhibit the development of atherosclerosis (Figure 3) [64]. The same research group reported the downregulation of NF-κB expression and polarization of macrophages by diosgenin treatment [138]. In another study, compound dioscin was evaluated for its inhibitory activity against atherosclerosis and postmenopausal atherosclerosis in ovariectomized LDLR-/-mice [139,140].

Antiasthmatic.
Asthma is a long-term condition which results in inflammation of the lower respiratory tract and affects children and adults. Junchao and coworkers evaluated the molecular mechanism of anti-trachea inflammatory effects produced by diosgenin via interactions with glucocorticoid receptor alpha [141]. They used ovalbumin-induced asthmatic mice and primary tracheal epithelial cells as

5.5.
Hepatoprotective. Liver disease can have genetic causes or it can be caused by a variety of factors that affect the liver, such as viruses or alcohol consumption [142][143][144]. Obesity is also associated with liver disease. Over time, liver damage results in scarring (cirrhosis), which can lead to liver failure, a life-threatening condition [145,146]. Under this topic, Xie et al. [147] reported on the impact of diosgenin on transforming growth factor (TGF)-β1-induced hepatic stellate cells and its mechanism of action for antifibrotic effects.
The results of this study showed that diosgenin significantly inhibited the proliferation of TGF-β1-induced hepatic stellate cells and reduced the expression of collagen I and alpha-smooth muscle action as well as the expression of TGF-β receptors I and II. Additionally, diosgenin also downregulated the expression of TGF-β1-induced phosphorylation of Smad3 in hepatic stellate cells and displayed potential effects to treat liver fibrosis. In another study, Zhang and coworkers described the dioscin and its effect on alcoholic liver fibrosis (in vitro and in vivo). The results demonstrated that dioscin improved the condition of alcoholic liver fibrosis via modulation of toll-like receptor 4/ myeloid differentiation primary response 88/NF-κB signaling pathway [148]. Other important studies related to diosgenin and its derivatives have been presented in Table 4 and shown in Figure 4.

Clinical Studies
A clinical pilot study was conducted to investigate the efficacy and safety profile of diallyl thiosulfinate associated with nuciferine and diosgenin in the cure of primary and secondary erectile dysfunction. A total of 143 candidates (age 18-39 Y) were selected and treated orally with 9 Oxidative Medicine and Cellular Longevity nuciferine and diosgenin for up to three months (single dose, alternative days). After three months of treatment, each candidate was screened in terms of the international index of erectile-5, premature ejaculation diagnostic tool, and male sexual health questionnaire. A significant enhancement was observed in terms of the international index of erectile-5 (8.7 vs. 14.01; p < 0:001) when compared with baseline and follow-up visits. The overall observation of the study indicates that these three compounds are capable to enhance the control of ejaculation in candidates suffering from premature and erectile dysfunction without any side effects [157].
In another study, Tohda et al. evaluated the impact of diosgenin-rich yam extract on synaptic loss and memory dysfunction using a transgenic mouse model of Alzheimer's. A placebo-controlled, randomized, double-blind, cross-over study was performed on 28 healthy volunteers (age: 20-81 years) and randomly treated with yam extract or placebo (12-weeks intake and 6-week washout period). The Japanese version of the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) test and the adverse effects were evaluated. The diosgenin-rich yam extract consumption (12 weeks) significantly increased the synaptic fluency or enhance cognitive function without any side effects [158].
The standardized multinutrient supplement including folic acid, selenium, vitamin E, catechins, glycyrrhizin, dios-genin, damiana, and omega-3-fatty acids showed beneficial effects on in vitro fertilization/intracytoplasmic sperm injection in terms of embryo quality [159]. The curative effects of Dioscorea villosa extract on menopausal systems, lipids, and sex hormones were evaluated [160].
A placebo-controlled, randomized, double-blind, crossover study was conducted on 23 healthy women suffering from symptoms of menopause. All candidates were treated with wild yam cream or placebo for up to three months, and no significant adverse effects were observed in both the treatments. Additionally, no changes were also recorded in weight, systolic or diastolic blood pressure, total serum cholesterol, triglyceride, high-density lipoprotein cholesterol, glucose, estradiol, or serum [160].

Toxicological Profile: Safety and Side Effects
Only a few articles have acknowledged diosgenin's toxicity, even though there has been substantial research on its part and maneuver in the treatment along with prevention of cancer and further different chronic disorders. A study was published in 2009 on breast cancer cells by Srinivasan and coworkers where it was shown that diosgenin modulates AKT to regulate breast cancer cell survival and that this drug has no effect on normal breast epithelial cells (MCF-10A) except for its selective toxicity to cancer cells [108]. Steroidal saponins of different doses from D. zingiberensis were given to mice that were experimental in another in vitro investigation. Diosgenin was the metabolite and the main component of these saponins. There were no harmful effects up to a level of 562.5 mg/kg, according to the findings. However, steroidal saponins, which include diosgenin, displayed deleterious consequences and even death in a dosedependent manner at dosages of 1125 mg/kg and higher. Interestingly, the steroidal saponins dosage, which is traditional, is 510 mg/kg/day, implying that steroidal saponins, together with diosgenin, have no significant toxicity at this dosage [161]. A recent study also discovered that diosgenin derivatives had antithrombotic properties. In vivo experiments revealed that they appeared to be protective and comparable to aspirin, with a decreased risk of bleeding and less stomach mucosal injury [162]. Furthermore, investigations have shown that diosgenin has a modest inhibitory impact on cytochrome P450 enzymes (CYPs), suggesting diosgenin fused with any other medicine would be safe to consider that they would have no toxicity [163]. These investigations demonstrated that diosgenin and its derivatives are nontoxic and have underlined their utility in the medicaments of chronic disorders including cancer.

Therapeutic Perspectives and Limitations
Diosgenin is a natural compound mostly found in Dioscorea species and starting material for the commercial synthesis of different steroids including cortisone, pregnenolone, and progesterone. The natural antioxidant compound diosgenin possesses different biological activities such as anticancer [164,165], antidiabetic [28,36,152,[166][167][168][169], multiple sclerosis [68,69], and spinal cord injury [70,71] and helps in the management of these diseases. Therefore, diosgenin could be functional and helpful in the treatment and prevention of a variety of disorders. The identification of diosgenin-specific targets, on the other hand, is critical for further validating its use in the treatment and elimination of diseases.
The main therapeutic limitations of diosgenin are represented by its low bioavailability; therefore, a special emphasis is being paid to the production of nanoformulations or conjugate complexes to improve the compound's bioavailability and pharmacokinetic features to develop it into a possible medicine. The potential of this chemical, its analogs, or combinations of this molecule with others has previously been demonstrated; however, carrier systems such as nanoparticles must be developed to govern diosgenin to the location where it works, boosting effectiveness and lowering adverse impacts. For example, encapsulated diosgenin PCL [poly(caprolactone)]-pluronic nanoparticles (PCL-F68-D-NPs) were developed by the nanoprecipitation method to improve performance in brain cancer therapy [170]. The developed nanoparticles (PCL-F68-D-NPs) displayed significant cytotoxicity against U87-MG cells compared to free diosgenin. Additionally, developed nanoparticles demonstrated suitable properties in terms of size distribution, stability, morphology, chemical and mechanical properties, encapsulation, and loading efficiency [170].

Overall Conclusions
A steroidal saponin, diosgenin, and its chemical and structural variants are useful in the treatment of a great variety of chronic conditions, including cardiovascular disease, several forms of lethal malignancies, nervous system problems, and autoimmune diseases. They have gotten a lot of interest from researchers all across the world. Diosgenin and its derivatives have been shown to have pharmacological benefits against cancer, diabetes, osteoporosis, Alzheimer's disease, and stroke in several investigations. Diosgenin has been shown to act on several molecular targets that are essential players in the occurrence and incidence of many serious disorders. Its multitargeting capability allows it to influence multiple molecular targets and signaling pathways at the same time. Diosgenin has an edge over the most commercial medicinal medicines available today because of this property. Furthermore, investigations demonstrating its nontoxic nature significantly promote the inclusion of this medicine in additional clinical studies or trials in the forthcoming days. Overall, diosgenin demonstrated great promise in the treatment and prevention of a variety of chronic diseases; nevertheless, additional clinical research is needed in the nearing days to come to confirm the preclinical findings and demonstrate the effective and secure usage of these purely natural compounds.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest. C., P.S., W.C.C., and A.R. and contributed to the final revision and major edits on the manuscript. All the authors read and approved the final manuscript.