Therapeutic properties and structural characterization of steroidal saponins: a review

Medicinal plants are sources of bioactive substances that can act to maintain human health. Among the compounds widely distributed in medicinal plants, there are steroidal saponins, an important class of secondary metabolites that are characterized as the active principle of these natural products. The structure of steroidal saponins is composed of a steroidal aglycone covalently linked to portions of carbohydrates and due to the complexity of its structure, the structural characterization processes are laborious. Steroidal saponins have been investigated over the years, due to their potent therapeutic properties such as antimicrobial, anti-inflammatory and cytotoxic. In this work were summarized the studies found in the scientific literature in the last two decades, about the investigation of the therapeutic properties and structural characterization of the steroidal saponins. Furthermore, recent studies have suggested that some saponins like candidates for the treatment of patients with Coronavirus disease (COVID-19). Studies on steroidal saponins are of great importance, as they can be potent therapeutic agents.


Introduction
Saponins are a group of bioactive glycosides, widely distributed in the plants. They can be classified into two groups based on the nature of their aglycone skeleton: steroidal saponins which are present mainly in the monocotyledonous angiosperms and triterpenoid saponins which occur mainly in the dicotyledonous angiosperms [1] .
Steroidal saponins are a group of natural compounds that consist of a steroidal aglycone, designated sapogenin, covalently linked to sugar moieties [1] . Because of its amphipathic nature, these substances have the capacity to form a foam when in contact with water and they possess pharmacological and medicinal properties [2] , such as antifungal, anti-inflammatory, anticancer and antiulcerogenic activities [1] . There are distributed in various plant species and are found in abundance in the families Agavaceae, Alliaceae, Asparagaceae, Dioscoreaceae, Liliaceae, Melanthiaceae, Solanaceae, Trilliaceae and Zygophillaceae.
Many industrial and commercial applications are reports to saponins, they are found in beverages, cosmetics and pharmaceutical products. Furthermore, they are used as raw materials for the production of steroid Revista Fitos. Rio  hormones, as food additives and due its therapeutic properties, have been investigated over the years, toward the development of new natural medicines [2] .
Steroidal saponins possess 27 carbon atoms in the aglycone and can be divided into spirostane and furostane based on the nature of their aglycone skeleton. The type spirostane possess a skeleton hexacycle ABCDEF-ring system and sugar moieties commonly linked at C-3. Already, the furostane type presents a pentacyclic ABCDE-ring system with the sixth open F ring and sugar moieties commonly linked at C-3 and C-26 (FIGURE 1) [1,3] . The image was adapted of reference [1] .
Steroidal saponins can also have functional groups in the aglycone [4] where the most common are -OH and -OCH3. Furthermore, can also contain insaturations in ring. The sugars usually found are glucose, galactose, arabinose, xylose and rhamnose [5] . According to the literature, the furostane steroidal saponin isolated from Yucca gloriosa L. rhizomes (Agavaceae) showed the functional group OCH3 linked at C-22 and sugar moieties of galactose and glucose (FIGURE 2A) [6] . Already, the furostane steroidal saponin from Allium sativum L. var. Voghiera (Alliaceae) showed the group OH linked at C-5 and at C-22 [7] (FIGURE 2B). It's also reported that the spirostane steroidal saponin from Asparagus filicinus (Asparagaceae) presented the group OH linked at C-17 and showed moieties of the glucose, arabinose and xylose [8] (FIGURE 2C) and the spirostane steroidal saponin from Smilacina japonica (Liliaceae) showed a insaturation on C-ring, the functional group OH linked at the C-17 and C-24 and presented the sugars galactose, glucose and xylose [9] (FIGURE 2D). This large structural diversity is responsible for the various biologic activities of saponins and because they are complex substances become labor intensive the processes of structural characterization.  [6] . (B) Furostane steroidal saponin from Allium sativum L. var. Voghiera [7] . (C) Spirostane steroidal saponin from Asparagus filicinus [8] (D) Spirostane steroidal saponin from Smilacina japonica [9] .

Results and Discussion
The search in Science Direct, Capes periodicals portal and Google academic using the terms "steroidal saponins" resulted in 11,922; 5,514 and 34,900 articles respectively. This large amount of articles show that research on steroidal saponins is of great relevance arousing the interest primarily of researchers in the field of chemistry and health.
The following topics will address the two biosynthetic pathways described in the literature (FIGURE 3 and

Biosynthesis
Steroidal saponins are derived of the C5 isoprene units, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) and is described that such isoprene units can be formed by two biosynthetic pathways, the mevalonate pathway and an alternative pathway known as deoxyxylulose phosphate [10] .
In the mevalonate pathway occurs the formation of the mevalonic acid as precursor of the reaction. Initially, two molecules of acetyl-coenzyme A are connected for Claisen condensation to give acetoacetyl-CoA.
Subsequently, another molecule of acetyl coenzyme A is incorporated via a stereospecific aldol addition giving the ester β-hydroxy-β-methylglutaryl-CoA (HMG-CoA). Subsequently occurs a hydrolysis and enzymatic reduction giving the mevalonic acid. Then, the mevalonic acid is transformed in the isoprene unit isopentenyl diphosphate (IPP), through the successive phosphorylation of the hydroxyl groups, followed by decarboxylation and elimination of a group pyrophosphate. Subsequently, an isomerase removes a proton at C-2 of IPP giving the dimethylallyl diphosphate (DMAPP) (FIGURE 3) [10] .
Deoxyxylulose phosphate pathway was posteriorly discovered and probably is more widely utilized in nature than is the mevalonate pathway. The compound 1-Deoxy-D-xylulose 5-phosphate is the precursor of the reaction and is formed from two products of glycolysis, pyruvic acid and D-glyceraldehyde 3-phosphate.
In this biosynthetic pathway, initially the pyruvic acid reacts with the thiamine diphosphate (TPP) that mediates the decarboxylation of pyruvate producing an acetaldehyde equivalent bound in the form of an enamine. This, reacts as a nucleophile, in an addition reaction with the D-glyceraldehyde 3-phosphate.
Saponins have as precursor the oxidosqualene, that is formed from the isoprene units IPP and DMAPP.
Initially a unit C5 of the IPP is condensed with a unit C5 of DMAPP resulting in the molecule C10 of the geranyl diphosphate (GPP). This is linked with a unit of IPP resulting in a unit C15 of farnesyl diphosphate (FPP). The union of two molecules of FPP originates the squalene, that by action of squalene monooxygenase form oxidosqualene. In sequently, occur a series of reactions of the cyclization, rearrangement, migration of hydride, methyl, formation of carbocation, forming a great diversity of steroid skeletons [2] (FIGURE 5) [10] . It is reported that oxidosqualene cyclization can proceed via the "chair-chairchair" or via the 'chair-boat-chair' conformation. Triterpenes saponins originate from the 'chair-chair-chair' conformation, while steroids saponins arise from the 'chair-boat-chair conformation [11] .
Spirostane saponins are formed by enzymatic hydrolysis of furostane saponins, such hydrolysis occurs by action of the enzyme β-glucosidase, that is an enzyme specific to cleave the glucose unit linked to C-26 allowing that the oxygen to be free and make an intramolecular bond with the carbon in C-22, leading to the closure of the F ring, but this mechanism isn't fully elucidated [12] .
The biogenetic relationship between the furostane and spirostane derivatives also is still contestable [3] . It's reported that furostanolic saponins to be usually contained in fresh plants and it are gradually converted into spirostanol saponins during the drying process. Moreover, there are reports that usually furostane saponins showed low toxicity, while spirostanes are highly toxic [3] .

Structural characterization
Steroidal saponins are complex substances, so the structural characterization process takes a lot of work,  The image was adapted of reference [1] .
According to the literature, through the difference of the chemical shift between the diastereotopic hydrogens H26a and H26b, it is possible to identify the configuration at the C-25. The stereochemistry is defined S if ≠ a,b ≥ 0.57 ppm and defined R if ≠ a,b≤ 0,48 ppm [6,18] . Furostane steroidal saponin from Yucca gloriosa L.
rhizomes showed C-25 configuration was deduced to be R based on the difference of chemical shifts (FIGURE 2A) [6] .
The complete assignments of the overall structure of the aglycone skeleton are achieved by a combination of 1 H, 13  3) [9] . In addition, were found HMBC correlations of the olefinic hydrogen H-11 with C-10, C-12, C-13 and C-14 [9] .
The acid hydrolysis with HCl 2M of the sugars of the saponin from Smilacina japonica (FIGURE 2D) generated glucose, galactose and xylose, which were identified by thin layer chromatography [9] . The

Antifungal activity
The antifungal activity is very reported in the Alliaceae [7,19,20] and Dioscoreaceae [21,22] families. The aginoside, a spirostane steroidal saponin from Allium nigrum L. was evaluated against phytopathogens showed a significant antifungal activity [20] . Furostane and spirostane steroidal saponins from Persian leek were evaluated against various fungal pathogens (Penicillium italicum, Aspergillus niger, Trichoderma harzianum and Botrytis cinerea) and Persicosides A and B, two spirostane steroidal saponins showed the highest activity on the tested fungi than the other tested saponins based on furostanol [20] . A spirostane saponins from Dioscorea villosa (discoreae) presented antifungal activity against Candida albicans, Candida glabrata and Candida tropicalis [21] . These results show the relationship of the spirostane skeleton on the activity, indicating that a spirostane aglycon is a structural feature increasing the antifungal activity of saponin compounds [20] .

Antibacterial activity
Antibacterial activity is also reported for spirostane saponins. Seven spirostanol saponins from P. polyphylla var. yunnanensis showed significant antimicrobial activity against P. acnes [23] . One spirostanol saponin from Cordyline fruticosa (L.) A. Chev. also showed a moderate antibacterial activity against the Gram-positive Enterococcus faecalis [24] . The authors point out that saponins deserve more attention as antibacterials, since this property is often assumed as less important over the antifungal activity. They believe that saponins might represent new and effective antibacterial agents [24] .
Sun et al. [26] isolated four steroidal furostanol saponins from the rhizomes of Aspidistra elatior Blume (Asparagaceae), their structures were determined based on chemical methods and spectral data and the isolated compounds, named aspidsaponins E-H were tested in vitro assay for inhibitory activities against LPS-induced nitric oxide production in RAW264.7 macrophages. Among them, compounds aspidsaponins G and aspidsaponins H showed excellent anti-inflammatory activities with IC50 values 82.1 and 65.9 μM, respectively [26] .
Three steroidal saponin from Allium flavum (Amaryllidaceae) exhibited moderate cytotoxicity against human colorectal cancer cell line (SW480) [31] . Five steroidal saponins from Ophiopogon japonicus (Liliaceae) showed cytotoxicity activity against five human cancer cell lines (HepG2, HLE, BEL7402, BEL7403 and Hela) [32] . A phytochemical study on T. Tschonoskii rhizomes, result in the isolated of twenty-four steroidal saponins. The citotoxic activity was analyzed against HepG2 cells and the results showed that two compounds that possess aglycone of pennogenin exhibited a remarkable cytotoxic activity, which presumed that the aglycone of pennogenin is critical for the cytotoxic activity. The authors believe that the structural changes on pennogenin due to substituents or the configuration difference could result in the activity disappeared [33] . Seven steroidal saponins from Dioscorea zingiberensis Wright (Dioscoreaceae) inhibited the proliferation of a panel of established human and murine cancer cell lines in vitro, where the zingiberensis saponin had more cytotoxic effect than the other saponins, demonstrated that this saponin is an effective natural agent for cancer therapy [34] .

Gastroprotective activity
It is reported that four steroid saponins from Paris polyphylla var. yunnanensis (Liliaceae) strongly inhibited gastric lesions induced by ethanol and indomethacin [43] . Preliminary biological investigations made with a furostane saponin isolated from Agave angustifolia var. marginata, indicated a significant protective effect against induced gastric ulcers using in vivo experimental models and demonstrated negligible toxicity on membrane integrity in the in vitro assays [44] .
He et al. [51] investigated therapeutic potentials of Chinese Herbal Medicine (CHM) to combat renal injury in COVID-19 patients. In this study, were selected active ingredients from CHM, contends mainly flavonoids and saponins which generally have the effects of anti-inflammation and anti-tumor. The diosgenin, a sapogenin that can reduce apoptosis by regulating PI3K/Akt, ERK and JNK signaling pathways was a the top listed one [51,52] . Furthermore, the authors believe that the stigmasterol and sitogluside sapogenins may play the role of preventing renal injury by acting on multiple targets in oxidativestress, inflammation, or apoptotic pathways. The authors suggest that CHM are promising to protect the kidney through the mechanisms of anti-oxidation, inhibition of inflammation and apoptosis pathways [51] .