Inhibiting Angiogenesis by Anti-Cancer Saponins: From Phytochemistry to Cellular Signaling Pathways

Saponins are one of the broadest classes of high-molecular-weight natural compounds, consisting mainly of a non-polar moiety with 27 to 30 carbons and a polar moiety containing sugars attached to the sapogenin structure. Saponins are found in more than 100 plant families as well as found in marine organisms. Saponins have several therapeutic effects, including their administration in the treatment of various cancers. These compounds also reveal noteworthy anti-angiogenesis effects as one of the critical strategies for inhibiting cancer growth and metastasis. In this study, a comprehensive review is performed on electronic databases, including PubMed, Scopus, ScienceDirect, and ProQuest. Accordingly, the structural characteristics of triterpenoid/steroid saponins and their anti-cancer effects were highlighted, focusing on their anti-angiogenic effects and related mechanisms. Consequently, the anti-angiogenic effects of saponins, inhibiting the expression of genes related to vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1-α (HIF-1α) are two main anti-angiogenic mechanisms of triterpenoid and steroidal saponins. The inhibition of inflammatory signaling pathways that stimulate angiogenesis, such as pro-inflammatory cytokines, mitogen-activated protein kinase (MAPKs), and phosphoinositide 3-kinases/protein kinase B (PI3K/Akt), are other anti-angiogenic mechanisms of saponins. Furthermore, the anti-angiogenic and anti-cancer activity of saponins was closely related to the binding site of the sugar moiety, the type and number of their monosaccharide units, as well as the presence of some functional groups in their aglycone structure. Therefore, saponins are suitable candidates for cancer treatment by inhibiting angiogenesis, for which extensive pre-clinical and comprehensive clinical trial studies are recommended.


Introduction
Saponins are one of the most widespread secondary metabolites presents in the plant kingdom and marine organisms, generating stable foam in aqueous solutions, such as soap [1]. Humans have long used saponin-rich plants in their diets to maintain health and therapeutic benefits, including some Fabaceae plants [2], such as pea (Pisum sativum), beans (Phaseolus spp.), fava bean (Vicia faba), soybean (Glycine max), mung bean (Vigna radiate), and licorice (Glycyrrhiza glabra). Additionally, Allium genus plants [3] are also rich in saponins, such as garlic (Allium sativum), onion (Allium cepa), and chives (Allium schoenoprasum). Moreover, herbs, including tea (Camellia sinensis), banana (Musa spp.), and ginseng (Panax ginseng), are found as saponin sources [4]. Saponins are composed of two parts, including aglycone (sapogenin) as non-polar moiety and glycoside (saccharide chain) as polar moiety. Triterpenoid saponins and steroidal saponins with, respectively, 30 and 27 carbon atoms, are the 2 main chemical structures of sapogenin core of saponins isolated from natural sources [5]. Furthermore, farnesyl diphosphate synthesized from the mevalonate biosynthetic pathway is the main precursor of the sapogenin [6,7]. Saponins have a high structural diversity due to the presence of functional groups on the sapogenin core, the number and type of sugars in the structure, and the way sugars bind to each other and to the aglycone part. Such structural diversity causes various biological and pharmacological effects [8]. Accordingly, several effects have been reported for saponins, such as anti-microbial, anti-leishmaniasis, anti-oxidant, anti-cholesterol, anti-lipidemia, analgesic, anti-inflammation, anti-diabetic, and anti-coagulant effects. [1,9]. Saponins also revealed the prominent anti-cancer and cytotoxic activities via regulating multiple signal pathways of cellular apoptosis, cell cycle arrest, proliferation, autophagy, and inflammation [10].
Angiogenesis, as the formation of new blood vessels, is one of the most important strategies to provide the energy needed for the survival and proliferation of cancer cells. Today, the inhibition of angiogenesis is used as a new and practical idea to control the growth and metastasis of tumors [11]. Since the discovery of the significant role of angiogenesis in the growth and progression of tumors by Folkman in 1971 [12], several drugs with anti-angiogenic effects have been introduced to the global pharmaceutical market, including bevacizumab, lenalidomide, axitinib, sorafenib, and everolimus. These drugs treat cancer by inhibiting the angiogenesis triggers, including the vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1-α (HIF-1α) release [13]. On the other hand, plants and their secondary metabolites have always been considered by researchers to treat cancer. According to investigations, plant secondary metabolites have also had a significant effect on inhibiting the growth and progression of tumors through blocking angiogenesis stimulation cellular signaling pathways [14]. Vinca alkaloids and paclitaxel, which have been isolated from Catharanthus roseus (Apocynaceae) and Taxus brevifolia (Taxaceae), respectively, are among anti-angiogenesis natural products found in pharmaceutical markets [15].
Sobolewska et al. reviewed the cytotoxic effects and anti-cancer mechanisms of saponins emphasizing the structure-activity relationship and their natural sources [16,17]. In this present review, the dramatic effects of saponins have been described on inhibiting the growth and metastasis of cancer via inhibiting the gene expression of angiogenesis mediators, including VEGF/VEGFR2, HIF-1α, fibroblast growth factor 2 (FGF2), and phosphoinositide 3-kinases/protein kinase B (PI3K/Akt). The anti-cancer effects of saponin was also shown to be applied through blocking inflammatory cellular signaling pathways, including pro-inflammatory cytokines, mitogen-activated protein kinase (MAPKs) pathways, and nuclear factor κ light chain enhancer of activated B cells (NF-κB). In this article, we aimed to review the chemical structure of saponins and their natural sources. Additionally, saponins with anti-angiogenic effects and their mechanisms are highlighted as promising candidates for cancer treatment.

Study Design
A comprehensive review was performed using electronic databases, including PubMed, Scopus, ScienceDirect, and ProQuest. The keywords (("angiogenesis" OR "Neovascularization" OR "cancer" [title/abstract/keywords]) AND ("saponin" OR "steroidal saponin" OR "triterpenoid" [all fields]) were searched until December 2022. Only English language reports were included. After excluding duplicates, two independent researchers checked the primary retrieved results based on the title and abstract. Selected articles were then screened based on their full text. The whole in vitro, in vivo, and clinical trial studies in which the anti-angiogenesis effects and mechanisms of saponins were included. To complete the search strategy, the reference lists and citation hand search with the authors' expertise in saponins as major therapeutic agents in angiogenesis were also used. Those reports included in the final article are summarized.

Chemistry, Biosynthesis, and Natural Sources
Saponins are a large group of secondary metabolites that are abundant in natural sources, including plants and marine organisms. These compounds have a high molecular weight, containing 27-30 carbons (C). In their structure, C3 is bound to the hydroxyl group in all aglycone saponins [52]. Saponins possess a polar/non-polar structure. In their polar sections, saponins have sugar chains (glycosylated forms), while the sapogenin structure makes the nonpolar part. Such chemical properties cause soap-like foams behaviors in aqueous solutions for saponins [53].
Squalene and 2,3-oxidosqualene are critical precursors in the biosynthesis of saponins. Accordingly, two units of farnesyl pyrophosphate formed in the mevalonate biosynthetic pathway and converted to squalene in the presence of the enzyme squalene synthase. The produced squalene is then converted to 2,3-oxidosqualene by the squalene epoxidase enzyme. Consequently, squalene and 2,3-oxidosqualene are converted to various aglycone saponins by different enzymes, cyclase and cytochrome P450. Finally, aglycone saponins are glycosylated by glycosyltransferase enzymes [6,7,54] (Figure 1). Saponins are typically divided into two main categories, including triterpenoid saponins (C30, Figure 2A) and steroidal saponins (C27, Figure 2B). However, alkaloidal saponins ( Figure 2C), which contain nitrogen atoms, are also classified in this category [5,55]. Consequently, steroidal saponins are divided into three categories, including spirostanes, furostanes, and cholestanes or open-chain steroidal saponins ( Figure 2B). The spirostanes Saponins are typically divided into two main categories, including triterpenoid saponins (C30, Figure 2A) and steroidal saponins (C27, Figure 2B). However, alkaloidal saponins ( Figure 2C), which contain nitrogen atoms, are also classified in this category [5,55]. Consequently, steroidal saponins are divided into three categories, including spirostanes, furostanes, and cholestanes or open-chain steroidal saponins ( Figure 2B). The spirostanes have a common spiro-carbon at the junction of two heterocyclic rings of furan and pyran in their structure and A and B rings are mainly trans isomer. The A and B rings of furostanes are found in both the cis and trans isomeric position and mainly consist of four hydrocarbon rings attached to a furan ring. Consistently, cholestane steroids do not have O-heterocyclic rings, and there is an opened ring in this group of saponins [52,56].  Oleananes and dammaranes ( Figure 2A), with, respectively, five and four hydrocarbon rings, are two main groups of triterpenoid saponins isolated from natural sources, especially the plant kingdom. The terpenoid saponins rings are in an all-trans isomeric position. Additionally, their aglycone mostly contains functional groups including COOH, OH, and CH 2 OH at C28, C3, and C24, respectively [5,53,57].
Natural sources-isolated saponins are in glycosylated form and contain various saccharide chains. These saccharide chains are mainly oligosaccharides and composed of Dextro (D) or Leavo (L) isomers of sugars, such as glucose, galactose, rhamnose, arabinose, xylose, and glucuronic acid. However, sugars, such as quinovose, apiose, and fucose, are also found at a lesser frequency [53,[58][59][60][61]. According to the number of saccharide chains attached to the saponin structure, these compounds are divided into two main categories: monodsmosidic and bidesmosidic, although in limited cases, tridesmoside saponins are also seen [62]. Although steroid saponins are generally monodsmosidic and are usually attached to the sugar chain at the C3 position, terpenoid saponins are bidesmosidic. Furthermore, tridesmoside, and the sugar chains, in addition to C3, are attached to the functional groups in C28 and C24 carbon. However, in some cases, the saccharide chain may bind to other carbons in the saponins structure [63][64][65].
In summary, due to their unique chemical structure and structural diversity, saponins have shown a bright future among natural compounds.

Pharmacological and Biological Activities
The structural diversity of saponins have led to their various biological and pharmacological effects. In addition to their biological effects, such as cytotoxicity [17], antibacterial [80,81], anti-viral [82], anti-fungal [83,84], anti-leishmania [85,86], anti-inflammation [87,88], and anti-oxidant [89,90] mechanisms, saponins have shown prominent pharmacological effects, such as cardioprotective [91], neuroprotective [92], anti-cancer [93,94], hepatoprotective [95], wound healing [96,97], analgesic [98,99], anti-rheumatoid [100,101] anti-convulsant [102] and immunomodulatory [103,104] activities. More recently, saponins have also been used as adjuvants in vaccines [105,106]. QS-21, isolated from Quillaja saponaria bark (Quillajaceae), is one of the saponins that has passed clinical trials in different phases for use as an adjuvant in vaccination [107]. Accordingly, studies showed the beneficial therapeutic effects of saponins on various diseases, including metabolic diseases, such as obesity [108,109], diabetes [110,111], hypercholesterolemia [112], hypertension [113,114], and osteoporosis [115]. Dutta et al. showed that rasmuside A, as a steroidal saponin isolated from Asparagus racemosus fruits, had an anti-leishmaniasis effect on the Leishmania donovani strain at 1.31 ug/mL [116]. The saponin-rich fraction of green tea seeds (Camellia sinensis, Theaceae) showed significant anti-bacterial effects against Escherichia coli, Staphylococcus aureus and Salmonella spp. via the penetration and destruction of the bacterial cell membrane/wall [117]. Another study showed anti-oxidant and anti-inflammation activities for Camellia sinensis root saponins [118]. Aginoside, as a spirostane saponin isolated from Allium nigrum (Liliaceae) bulbs, showed strong anti-fungal activities at 400 ppm against wild fungi, such as Fusarium spp., that invade plants [119]. Moreover, Li et al. showed that the saponin-rich extract of Panax ginseng steamed root ameliorated the ischemic injuries at 200 mg/kg and 400 mg/kg, orally via modifying the hemodynamic parameters, such as the left ventricular systolic pressure and heart rate, as well as a decreasing of the intracellular calcium ion overload [120]. Additionally, a clinical study has been designed on the healing effects of Panax notoginseng saponins on patients with hypertensive intracerebral hemorrhage (NCT02999048). Onjisaponin derivatives, as the triterpenoid saponins isolated from Polygala tenuifolia root (Polygalaceae), showed prominent neuroprotective effects and increased the survival of PC21 cell lines against glutamate-induced neurotoxicity [121]. Additionally, Meng et al. showed in vitro (0.5, 1, and 2.5 µM) and in vivo (2.5 mg/kg, 5 mg/kg, and 10 mg/kg, intragastrically, rats) potentials of Paris saponin VII as a steroidal saponin separated and purified from the rhizome of Trillium tschonoskii Maxim (Melanthiaceae). In their study, Paris saponin VII ameliorated rheumatoid arthritis by inhibiting the expression of inflammatory signaling pathways, such as mitogen-activated protein kinase, and reducing inflammatory cytokines [100]. On the other hand, the saponin-rich extract of sea cucumber (Pearsonothuria graeff ) revealed marked anti-obesity and anti-hyperlipidemia effects in the rat fed with 0.08% saponin-rich extract and a high-fat diet for eight weeks [122]. Macrophyllosaponin B and astragaloside VII, as two terpenoid saponins found in Astragalus spp., showed regulatory effects on the immune system at 60 µg/albino mice intraperitoneal (i.p.) via blocking the activities of interlukin-4 and stimulating the expression of interlukin-2 and interferon-γ [123]. The saponin-rich fraction of Clerodendrum infortunatum leaves (Lamiaceae) showed anti-convulsant effects at 45 mg/kg, i.p., and relieved the central and peripheral pain at 35 and 40 mg/kg, i.p., respectively, in mice [124]. In addition, several clinical trials have been conducted on the beneficial therapeutic effects of saponins and plants rich in saponins, such as onion (Allium cepa, Araliaceae) and fenugreek (Trigonella foenum-graecum, Fabaceae) [110,[125][126][127][128]. So far, several clinical trials have been reported on the therapeutic effects of ginsenoside saponins on various diseases, including cardiovascular disorders, diabetes, blood lipids, high blood pressure, and obesity [129,130].
Furthermore, saponins have shown significant cytotoxic and anti-cancer activity through various mechanisms in in vivo and in vitro studies, which we will discuss in more detail in the next section. Overall, saponins have high therapeutic potential and could be introduced as promising candidates for the treatment of complex diseases, such as metabolic disorders and cancer.

An Overview on the Anti-Cancer Mechanisms of Saponins
According to the World Health Organization, cancer is rapidly spreading worldwide and has been the main cause of death in more than 60% of countries (112 out of 183 countries studied) in the world [131]. Therefore, researchers in the field of cancer are trying to find effective treatments for it. Due to their low side effects, multi-therapeutic targeting, and in most cases, their low-cost phytochemicals have been promising candi-dates for cancer treatment. Among the phytochemicals, saponins have been prominent anti-cancer candidates in vitro and in vitro [10,94].
PI3K/Akt/mTOR is a critical signaling pathway in controlling cell division, survival, differentiation, and metabolism. The dysregulation of this pathway in several cancer types occurs and causes cancer cell growth, angiogenesis, metastasis, and cell migration. On the other hand, enhancing the gene expression of PI3K/Akt/mTOR mediators increases cancer cells' resistance to chemotherapy. Therefore, the inhibition of such pathways could be one of the pivotal strategies for cancer treatment [132,133]. Timosaponin AIII, as a glucosylated spirostane saponin of Anemarrhena asphodeloides (Asparagaceae) rhizomes, combated taxol-resistant lung cancer cells in vitro (2-8 µM) and in vivo (2.5 and 5 mg/Kg) via reducing PI3K/Akt/mTOR gene expressions [134]. As a steroidal saponin isolated from Aspidistra letreae (Asparagaceae), aspiletrein A showed cytotoxic effects on human lung cancer, including H460, H23, and A549 at 6.25 µM and 12.5 µM through inhibiting the expression of Akt [135]. Diosgenin as a spirostanol saponin found in Trigonella foenum graecum (Fabaceae) showed prominent anti-cancer activities with different mechanisms, including downregulating PI3K/Akt/mTOR gene expression [136]. DG-8d, as a synthetic derivative of diosgenin, obstructed PI3K/Akt signaling pathway at 20 µM, thereby inhibiting the growth of non-small cell lung cancer cell lines [137]. In another study, A-24 (2-8 µM) as a spirostanol saponin of Allium chinense (Liliaceae) bulbs revealed antiproliferation effects through blocking the PI3K/Akt/mTOR cellular signaling pathway on SGC-7901 and AGS, as human gastric adenocarcinoma cell lines [138].
The Bcl-2 family plays a critical role in apoptosis. Bax and Bcl-2 are two vital members of the Bcl-2 family. In this line, Bax induces apoptosis, and Bcl-2 prevents cell death by blocking the Bax activities. Therefore, the Bax/Bcl-2 ratio is a critical factor for the determination of anti-apoptosis activities of anti-cancer agents in vitro and in vivo studies [139,140]. Zingiberensis, as a glycosylated steroidal saponin isolated from Dioscorea zingiberensis rhizomes (Dioscoreaceae), showed cytotoxic activities on ovarian (SK-OV-3) and lung cancer (C26) cell lines at 1.51 and 0.81 µM, respectively. They found that reducing the Bcl-2 gene expression was the main anti-cancer mechanism of zingiberensis [141]. Raju et al. showed that feeding with 1% (w/w) and 0.05% (w/w) of diosgenin significantly inhibited the growth of colon cancer at 42% and 24%, respectively, in F344 rats. The anti-cancer effect was applied by reducing Bcl-2 gene expression and enhancing caspase-3 gene expression as two signaling pathways of apoptosis induction [142].
Glycolysis and oxidative phosphorylation are pivotal sources of energy for cancer cells and thereby play a critical role in energy production for cancer cells and their survival [143]. Thus, employing the inhibitors of such metabolic pathways could be introduced as potential anti-cancer compounds [144]. Gracillin (5 µM), as a spirostanol saponin isolated from Dioscorea collettiivar rhizomes (Dioscoreaceae), inhibited the growth of breast cancer tumor via blocking the oxidative phosphorylation and glycolysis [145].
Overgeneration of ROS induces cell cycle arrest, apoptosis, and autophagy in cancer cells. Therefore, stimulation of ROS production is one of the important strategies in chemotherapy and the discovery of anti-cancer agents [146,147]. Spirostane saponins, SAP-1016 and SAP-884, and furostane saponins, KE-1046 and KE-1064, showed cytotoxic activities against colon and breast cancer cell lines. SAP-1016 at 0.05 µM revealed more potential anti-cancer activities via enhancing ROS production [148]. Macrostemonoside (100 µM), found in Allium macrostemon (Liliaceae) bulbs, is another spirostane saponin with anti-colorectal cancer activities by increasing ROS production [149].
MAPK signaling pathways are complex and made of kinases, which associated regulation plays a major role in cell death, cell migration and invasion, angiogenesis, and progressing cancer cells. In fact, the downregulation of MAPK pathways and its downstream is one of the main mechanisms of anti-cancer agents [160,161]. Peng et al. showed that the total saponins of Conyza blini (Asteraceae) significantly inhibited the growth of cervical carcinoma tumor via downregulating the MAPKs/transforming growth factor beta (TGF-β)/nuclear factor erythroid 2-related factor 2 (Nrf2) as a vital cellular signaling pathway of inflammation and cell proliferation at 15 µg/mL and 20 µg/mL. The liquid chromatography-mass spectroscopy analysis of Conyza blini saponins confirmed the presence of triterpenoid saponins, such as 2β, 16α, 23-trihydroxyoleanolic acid, and 2β, 23-dihydroxyoleanolic acid [162]. Triterpenoid saponins isolated from Glycyrrhiza glabra root (Fabaceae) showed anti-cancer effects on breast, cervical, liver, pancreatic, colorectal, lung, gastric, and other types of malignancies via downregulating the PI3K/Akt, MAPKs signaling pathways [163].
Inhibiting Wnt/β-catenin cellular signaling pathways is another anti-cancer mechanism of saponins. This signaling pathway plays a critical role in cell proliferation, migration, and apoptosis. 1C (25 mM) as a ginsenoside saponin found in Panax ginseng (Araliaceae) revealed anti-prostate cancer activities via inhibiting the Wnt/β-catenin signaling pathway [164]. Zafar et al. reported that tubeimoside-1, a triterpenoid saponin found in Bolbostemma paniculatum (Cucurbitaceae), has broad anti-cancer effects on several cancer cell lines, such as the pancreas, esophageal, gastric, neuroblastoma, and bladder via regulating the gene expression of different signaling pathways involved in cell cycle arrest, apoptosis, and inflammatory cytokine generation [165]. Furthermore, tubeimoside-1 showed anti-colon cancer effects at 10, 25, and 50 µg/mL through inhibiting Wnt/β-catenin [166].
Additionally, increasing reactive oxygen species (ROS) generation is another 1C anticancer mechanism [164]. Moreover, Lin et al. reported that glycyrrhizic acid as one the main triterpenoid saponins of Glycyrrhiza glabra root inhibited breast cancer via enhancing ROS production [167].
In summary, saponins exhibit significant anti-cancer activities against various malignancies by regulating apoptotic and inflammation signaling pathways and inducing cell cycle arrest (Figure 3). Therefore, they have always been considered promising candidates for cancer treatment. On the other hand, the inhibition of angiogenesis in tumors as another main mechanism of saponins anti-cancers has attracted the attention of many researchers.

Anti-Angiogenic Potentials
The inhibition of angiogenesis and direct killing of cancer cells are two main strategies in the treatment of cancer [172]. Ever since Folkman proposed the theory of angiogenesis inhibition for the treatment of cancer in 1971, many efforts have been made to find molecules with anti-angiogenic effects [12]. These investigations led to the introduction of some drugs, such as sorafenib, bevacizumab, sunitinib, everolimus, and axitinib, with anti-angiogenic effects in the treatment of various cancers [13]. Natural sources, especially phytochemicals, have been among the main candidates for the discovery of molecules with anti-angiogenic effects [173]. Alkaloid-based drugs (e.g., vincristine, vinblastine, and camptothecin) [174], terpenoid-based drugs (e.g., paclitaxel) [175], flavonoid-based drugs

Anti-Angiogenic Potentials
The inhibition of angiogenesis and direct killing of cancer cells are two main strategies in the treatment of cancer [172]. Ever since Folkman proposed the theory of angiogenesis inhibition for the treatment of cancer in 1971, many efforts have been made to find molecules with anti-angiogenic effects [12]. These investigations led to the introduction of some drugs, such as sorafenib, bevacizumab, sunitinib, everolimus, and axitinib, with anti-angiogenic effects in the treatment of various cancers [13]. Natural sources, especially phytochemicals, have been among the main candidates for the discovery of molecules with anti-angiogenic effects [173]. Alkaloid-based drugs (e.g., vincristine, vinblastine, and camptothecin) [174], terpenoid-based drugs (e.g., paclitaxel) [175], flavonoid-based drugs (e.g., naringenin, kaempferol, and hesperidin) [176], and coumarin-based drugs (e.g., galbanic acid, esculetin, and daphnetin) [19] are among the major phytochemicals reported to inhibit angiogenesis. Additionally, Saponins, as one of the largest groups of natural compounds, have shown notable anti-angiogenic effects in the treatment of various cancers, in vitro and in vivo.

Anti-Angiogenic Potentials of Steroidal Saponins
The inhibition of angiogenesis is one of the critical anti-cancer mechanisms of steroidal saponins. Diosgenin (5,15, and 25 µM), as a steroidal saponin found in Dioscorea spp. root (Dioscoreaceae) and Trigonella foenum-graecum (Fabaceae) seeds, reduced the level of two main mediators of angiogenesis (VEGF and FGF2) in glioblastoma cell lines [177]. In addition, dioscin and deltonin (Figure 4), as two glycosylated form of diosgenin found in Dioscorea spp. (Dioscoreaceae), inhibited angiogenesis induced by VEGF (50 ng/mL) in HUVEC via down-regulating the VEGFR2 downstream pathways, including MAPK/Akt pathways at 1 and 4 µM [178,179]. The sugar moiety of dioscin consists of two rhamnose units and one glucose unit, while the sugar chain of deltonin consists of two glucose units and one rhamnose unit. Investigations showed that the presence of more rhamnose units in the sugar moiety of steroidal saponins enhanced their anti-cancer activities [17]. Additionally, a steroidal saponin of Tribulus terrestris (Zygophyllaceae), terrestrosin D (Figure 4), showed significant anti-angiogenesis activities at 50 mg/kg in male Balb/c mice with prostate cancer. In their study, contrary to expectations, terrestrosin D increased the level of VEGF in HUVEC, so direct endothelial cell destruction by this saponin was suggested as a possible mechanism of its anti-angiogenic effect [180]. Steroidal saponins from the rhizome of Polyphylla paris (Melanthiaceae), including Paris saponin I, II, VI, and VII, blocked induced VEGF angiogenesis in HUVEC vascular endothelial cells at 2 and 4 µM. Furthermore, polyphyllin D, as another Polyphylla paris steroidal saponin, prevented angiogenesis in zebrafish embryos model at 0.313 µM and 0.156 µM. These saponins inhibited the critical downstream pathways of angiogenesis, including PI3K/Akt/mTOR/ribosomal protein S6 kinase beta-1 (S6K1), SRC/endothelial nitric oxide synthase (eNOS), phospholipase Cγ (PLCγ)/ERK, Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3), and VEGFR2 [181,182]. Moreover, Paris saponin II (Figure 4) revealed anti-cancer activities against ovarian cancer via blocking NF-κB as one of the signaling pathways of inflammation and VEGF stimulant generation, thereby inhibiting angiogenesis in in vitro (2.5 µM) and in vivo (25 mg/kg, i.p., female Balb/c mice) [183,184]. The gene expression of MMPs as one of the critical triggers of angiogenesis was suppressed by ACS (Figure 4), a steroidal saponin isolated from Ophiopogon japonicas roots (Asparagaceae) at 1.25, 2.5 and 5 µM in HUVECs and C57/BL mice [185]. ERK1/2, HIF-1α and Akt are stimulated under hypoxia conditions and caused the release of VEGF, thereby initiating angiogenesis. These signaling pathways were inhibited in HUVEC by DT-13 as another steroidal saponin of Ophiopogon japonicas roots (Asparagaceae) at 0.01, 0.1, and 1 µM [186]. In addition, the steroidal saponin of Convallaria majalis rhizome (Asparagaceae), convallamaroside, reduced neovascularization in mice with kidney cancer at 5-100 µg/day [187]. In summary, the steroidal saponin revealed prominent anti-angiogenesis activities by inhibiting the angiogenesis cellular signaling pathways, including PI3K/Akt/mTOR, MAPKs, and JAK/STAT, that were stimulated with VEGF. The studies confirmed sugar moiety attached to C-3 increased the steroidal saponins anti-cancer and anti-angiogenesis activities.

Anti-Angiogenic Potentials of Triterpenoid Saponins
Triterpenoid saponins have shown remarkable anti-angiogenic activities by modulating different cellular signaling pathways, which are discussed below. AG36, as a triterpenoid saponin isolated from Ardisia gigantifolia (Primulaceae) rhizomes, inhibited breast tumor growth in female BALB/c mice at 0.75 and 1.5 mg/kg/day through antiangiogenic effects. In their study, AG36 (20 5, 10, and 20 µM) inhibited VEGF and its receptor, as well as focal adhesion kinase (FAK)/PI3K/Akt cascade, as stimulators of VEGF production. According to a docking study, the presence of a four-sugar chain was necessary for strong interaction between AG36 and VEGFR2 [188]. Arenaroside derivatives ( Figure 5), including Arenaroside C, D, E, and G, as the polyhydroxylated triterpenoid saponins isolated from Polycarpaea arenaria (Caryophyllaceae), showed anti-angiogenic effects at doses less than 5 µM. In their study, it was found that the presence of the acetyl group at C-22 of polyhydroxylated triterpenoid saponins led to more anti-angiogenic effects [189]. Furthermore, ginsenosides are the dammarane type of triterpenoid saponins that are found in the organs of the Panax spp. (Araliaceae), especially Panax ginseng roots. The ginsenosides revealed significant anti-angiogenesis activities in several reports [190,191]. In this line, ginsenoside-Rb1 ( Figure 5) inhibited angiogenesis in HUVEC at 100-500 nM by enhancing the gene expression of pigment epithelium-derived factor (PEDF), as the most abundant endogenous anti-angiogenesis glycoproteins [192]. Additionally, Lu et al. showed that ginsenoside-Rb1 upregulated the PEDF gene expression via blocking the microRNA-33a (miR-33a) as one of the main miRNAs of angiogenesis activator. In their study, it was found that ginsenoside-Rb1 inhibited miR-33a by increasing the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and PEDF. Since ginsenosides have steroid-like rings in their chemical structure, in their study, the role of steroid receptors was confirmed in the regulation of miR-33a and PEDF [193]. Ginsenoside-Rb2 prevented angiogenesis in C57BL/6 mice with melanoma at 100 µg/mouse, intravenously [194]. Of other ginsenosides, ginsenoside K is one of the active deglycosylated metabolites of ginsenoside Rb1, which is metabolized by the intestinal bacterial flora after the oral ingestion of Panax ginseng roots [195]. Ginsenoside K abrogated the activities of sphingosine kinase 1 (SPHK1), as an angiogenesis mediator enzyme, in HUVEC at 10 µg/mL. The higher lipophilicity of ginsenoside K is related to its aglycone structure, and this is probably the reason for its strong affinity for SPHK1 [196]. Ginsenoside Rg3 (20 mg/kg), alone and in combination with gemcitabine, inhibited angiogenesis in female C57BL/6 mice with lung cancer by reducing VEGF gene expression [197]. Moreover, senegin III (0.1, 1, and 10 µM), as a triterpenoid saponin of Polygala senega roots (Polygalaceae), inhibited VEGF-induced angiogenesis by increasing PEDF. This triterpenoid saponin obstructed the angiogenesis induced by bFGF (200 ng/mL, subcutaneously) at 1 and 2 mg/Kg (i.p.; 10 days) in mice. The results confirmed that the presence of the C-28 sugar chain was necessary for its anti-angiogenesis activities [198]. Oldhamianoside II (5, 10, and 20 mg/kg), a bidesmosidic oleanolic acid derivative saponin of Gypsophila oldhamiana roots (Caryophyllaceae), markedly revealed anti-cancer activities against sarcoma and ovarian cancers via blocking the multi-targets of angiogenesis pathways, including VEGF and VEGFR2, COX-2, bFGF, and pro-inflammatory cytokines, such as TNF-α and IL-6 [199,200]. A triterpenoid saponin isolated from Camellia sinensis seeds (Theaceae), theasaponin E1 (2 µM and 10 µg/mL), showed anti-angiogenesis activities by decreasing the gene expression of VEGF, Akt, and HIF-1α. Theasaponin E1 ( Figure 5) contains a four-sugar chain attached to C-3, and studies indicated that this chain is necessary for anti-angiogenesis activities [201,202]. Chiisanoside, a triterpenoid saponin isolated from Acanthopanax sessiliflorus (Araliaceae) leaves, reduced the VEGF plasma level in (H22) tumor-bearing mice at 60, 120, and 240 mg/kg, and in this way, it suppresses angiogenesis and tumor growth [203]. Additionally, Pulsatilla saponin D (Figure 5), as a triterpenoid saponin of Pulsatilla koreana (Ranunculaceae) roots, inhibited gene expression of the promotion angiogenesis mediators, including VEGF and HIF-1α at 10 µM in human liver, pancreatic, gastric, and colon cancer cell lines. The structureactivity relationship studies of Pulsatilla saponins showed that the presence of the carboxyl group at the C-28 position was necessary for the anti-cancer activities of these saponins, although it increased their hemolytic effects [204][205][206][207][208]. Platycodin D ( Figure 5) [209,210], julibroside J8 [211], maesasaponins (I-VII.1) [212], astragaloside IV [213], capilliposide B ( Figure 5) [214], raddeanin A ( Figure 5) [215,216], and gleditsiosides B [217] are other triterpenoid saponins with anti-angiogenesis activities described in Table 1. Triterpenoid saponins showed anti-angiogenesis activities via blocking the pro-angiogenesis mediators, including VEGF and HIF-1α, as well as suppressing the PI3K/Akt, MAPKs, and NF-κB as stimulation angiogenesis cellular signaling pathways. Furthermore, these saponins increased the PEDF as an endogenous anti-angiogenesis protein. Investigations indicated the presence of sugar moiety in C-3 and C-28, and the carboxyl and acetyl functional groups at C-28 and C-22, respectively, enhances the triterpenoid saponin anti-angiogenesis and anti-cancer activities.

Anti-Angiogenic Potentials of Total Saponin Extraction
The total saponin extraction of plants showed prominent anti-angiogenesis activities. Ahmad et al. showed that the total saponin extract of Rumex hastatus (Polygonaceae) aerial parts suppressed angiogenesis at IC50 64.9 µg/mL in the chorioallantoic membrane (CAM) angiogenesis model [224]. The crude Astragalus saponins extracted from Astragalus membranaceus (Fabaceae) exhibited anti-cancer effects against colon and gastric cancer at 80 and 50 µg/mL, respectively, via inhibiting angiogenesis-inducing signaling pathways, including VEGF, bFGF, HIF-1α, and PI3K/mTOR/Akt [225,226]. Additionally, the crude saponins extract from Actinidia valvata (Actinidiaceae) roots inhibited the gene expression of VEGF and bFGF, thereby revealing anti-angiogenesis activities in hepatoma H22 mice at 1 g/kg/d [227]. In another study, Albiziasaponins of Albizia lebbeck (Fabaceae) showed anti-angiogenesis activities at 0.5, 0.1, and 1 µg/mL in the CAM model [228]. Moreover, the total saponins of sea cucumber, Holothuria leucospilota, suppressed the VEGF gene expression at 3, 6, or 12 µg/mL in breast cancer cell lines [223]. Regarding the excellent anti-angiogenic effects of extracts rich in saponins, it can be concluded that the presence of several saponins with different anti-angiogenic cellular mechanism targets has made the synergy of their anti-angiogenic effects.
In summary, saponins showed notable anti-angiogenesis activities via several mechanisms, including suppressed gene expression involved in the release and activation of VEGF, such as PI3K/mTOR/Akt, HIF-1α, bFGF, JAK/STAT, VEGFR2, and MAPK pathways. Additionally, inhibiting the gene expressions of pro-inflammatory cytokines including COX-2, TNF-α, and IL-6, enhancing the PEDF gene expression, and direct inhibitory effects on vein endothelial cell proliferation are other anti-angiogenesis mechanisms of saponins. Furthermore, studies showed the direct relationship between chemical structure and saponin anti-angiogenesis activities. Finally, due to the prominent anti-angiogenic activities of triterpenoid and spirostanol saponins (Figures 4 and 5), these compounds can be introduced as promising anti-cancer candidates.

Conclusions
Saponins are natural compounds with a unique and diverse chemical structure, which has led to numerous pharmacological and biological effects from these compounds. These compounds have noteworthy anti-cancer effects on different types of cancer. The inhibition of angiogenesis as one of the main approaches for cancer treatment has always been considered by researchers. Accordingly, saponins showed prominent anti-angiogenic effects by various mechanisms (Figure 6). The inhibition of VEGF and HIF-1α are among the most critical anti-angiogenesis mechanisms of saponins. These compounds exerted anti-angiogenic effects via inhibiting the signaling pathways, including MAPKs, FGF2, and PI3K/Akt/mTOR. Despite the fact that humans and animals widely consume plants containing saponins, these compounds have side effects, such as bleeding, digestive complications, hypertension, genotoxicity, kidney/liver necrosis, and hormonal interference in high doses [163,[229][230][231][232]. Furthermore, due to the complexity of the chemical structure of saponins, there is a possibility of their interference with chemotherapy regimens, so more research is needed surrounding their use in cancer treatment [93]. Recently, extensive bioavailability studies (NCT04335435) on saponins have been designed in order to prescribe the appropriate dose and reduce their side effects and drug interactions. On the other hand, the isolation and purification of saponins from natural sources is complex and requires multiple techniques and time-consuming steps, so it is necessary to conduct more studies to find new ways to isolate and purify these compounds. Therefore, triterpenoids and steroidal saponins can be introduced as promising natural compounds for the treatment of cancer. For this purpose, extensive experimental and clinical trial studies are recommended.

Conflicts of Interest:
Authors declare there is no conflict of interest.