Biosynthesis of glyceollins

Soyabeans contain abundant isoflavones, a varied group of polycyclic compounds, which have been reported to have numerous biological activities including suppression of sex-hormone dependent cancers⁽ Reference Wood, Clarkson and Appt ^{16 –} Reference Hwang, Kim and Jee ^{19 )} and amelioration of post-menopausal complications, including osteoporosis and hot flashes⁽ Reference Messina ^{20 )}. Isoflavonoids could be classified as phytoanticipins because they are stored in plant cells in anticipation of pathogenic attack⁽ Reference Dakora and Phillips ^{21 )}. All soyabeans and their products contain significant amounts of the isoflavones daidzein and genistein, either as the aglycone or as different types of glycoside conjugates. These include 6′-O-malonylglucosides, 6′-O-acetylglucosides and the β-glucosides of daidzein and genistein⁽ Reference Farmakalidis and Murphy ^{22 ,} Reference Kudou, Fleury and Welt ^{23 )}, all of which can be separated by reversed-phase HPLC. Smaller amounts of glycitein conjugates are often found in soyabean or soya protein, whereas conjugates of glycitein are abundantly present hypocotyledon or germ⁽ Reference Kudou, Fleury and Welt ^{23 )}. The malonyl and acetyl glycosides are susceptible to heat and readily convert to the more stable β-glycoside⁽ Reference Barnes, Kirk and Coward ^{24 )}. Therefore, the relative proportions of these conjugates can vary considerably among different soya foods depending on the extent of processing of the soyabean⁽ Reference Setchell ^{25 )}.

Glyceollins are de novo synthesised from daidzein in the soyabean in response to environmental stresses such as fungal infection. Although the glyceollins are detected at high concentrations in soyabeans during stress, they have also been detected at trace levels in non-elicitor-treated soyabean seeds⁽ Reference Kraus, Spiteller and Mithöfer ^{26 )}.

The compounds possess pterocarpanoid skeletons with cyclic ether decoration originating from a C5 prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterised during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned. However, some genes need to be identified for later biosynthetic steps⁽ Reference Akashi, Sasaki and Aoki ^{27 )}. According to a recent study by Akashi et al., dimethylallyl diphosphate: (6αS, 11αS)-3,9,6α-trihydroxypterocarpan ((2)-glycinol) 4-dimethylallyltransferase appears to be involved in yielding the direct precursor of glyceollin I⁽ Reference Akashi, Sasaki and Aoki ^{27 )}.

Glyceollins are derived from the precursor molecule, daidzein through several intermediate structures including glycinol⁽ Reference Tilghman, Boué and Burow ^{28 )}. Glycinol, which was previously shown to be oestrogenic, is derived from daidzein via a pterocarpan by cyclisation and 6α-hydroxylation⁽ Reference Boue, Tilghman and Elliott ^{29 )}. For the biosynthesis of the glyceollins, glycinol is prenylated to produce glyceollidin I and II, followed by cyclisation into the corresponding glyceollins⁽ Reference Banks and Dewick ^{30 )}.

The most well-known elicitors that effectively stimulate biosynthesis of glyceollins include some fungal strains such as Pseudomonas glycinea, Meloidogyne incognita, Heterodera glycines, Aspergillus sojae, Aspergillus awamori, Aspergillus oryzae and Rhizopus oligosporous ⁽ Reference Dakora and Phillips ^{21 ,} Reference Lee, Kim and Chun ^{31 )}.

Whether an elicitor is a fragment of a fungal cell or a defined chemical molecule such as a β-glucan, host plants must recognise these phytoalexin-inducing factors. Such molecules from both pathogens and mutualists are presumably recognised by receptors located on the cell wall or membrane. Typical biotic elicitors causing glyceollin production in soyabean include β-1,3-1,6-oligoglucoside, β-1,3-glucan and cyclic β-1,6-1,3-glucan, which are cell wall components of pathogenic or mutualistic symbiotic micro-organisms⁽ Reference Smith and Banks ^{32 )}. For instance, cyclic β-1,6-1,3-glucans synthesised by both free-living cells and bacteroids of B. japonicum are active elicitors of glyceollins in soyabean⁽ Reference Gore and Miller ^{33 ,} Reference Miller, Hadley and Gustine ^{34 )}.

Glyceollin synthesis is also induced by abiotic elicitors including iodoacetate, UV, Tx-100, and metal ions such as Fe, Cu, Hg and Ag⁽ Reference Dakora and Phillips ^{21 )} as well as during fungal infection. The mechanisms by which biotic and abiotic factors affect isoflavonoid phytoalexin formation in plants are unclear. Because elicitors have diverse structural features, they may act simply by injuring plant cells, which then stimulates the phytoalexin biosynthetic pathway. Or they may cause the host plant to release a constitutive elicitor that triggers phytoalexin formation⁽ Reference Dakora and Phillips ^{21 )}.

Antifungal activity

According to our previous study glyceollins were shown to inhibit several lines of fungal species. More specifically, the glyceollins (200 and 600 μg/disk) revealed a remarkable antifungal effect against Phytophthora capsici and Sclerotinia sclerotiorum, and to a lesser degree Fusarium oxysporum and Botrytis cinerea, within the growth inhibition range of 10·9–61·0%, along with their respective minimum inhibitory concentration values ranging from 25 to 750 μg/ml. The glyceollins also had a strong suppressive effect on spore germination of all tested plant pathogens along with concentration- and time-dependent kinetic inhibition of P. capsici, which is responsible for pepper disease⁽ Reference Kim, Suh and Lee ^{35 )}. Phytoalexins appear to exert their antifungal activity by altering membrane permeability although the presence of other mechanisms could not be excluded⁽ Reference Keen and Bruegger ^{36 )}.

Antioxidant activity

In healthy aerobes, the production of reactive oxygen species (ROS) is approximately balanced with the antioxidant defence systems⁽ Reference Halliwell ^{37 )}. Oxidative stress can be defined as a disturbance in the pro-oxidant–antioxidant balance in favour of the former, leading to potential damage⁽ Reference Sies ^{38 )}, or a serious imbalance between ROS production and antioxidant defences⁽ Reference Halliwell ^{37 )}. Although ROS might be harmful at the high concentration, the generation of ROS, within certain boundaries, is essential to maintain homoeostasis. For instance, macrophages utilise ROS to combat infective agents. Likewise, cytosolic ROS may be involved in the regulation of some important cellular events such as proliferation, gene expression and signal transmission. However, it is generally agreed that an excessive production of ROS is associated with ageing, cancer development, atherosclerosis and neurodegenerative disorders⁽ Reference Markesbery ^{39 ,} Reference Finkel and Holbrook ^{40 )}.

Glyceollins isolated from soyabean that sprouted in the presence of A. sojae showed strong antioxidant activity and ROS scavenging potential when assessed by an in vitro model⁽ Reference Kim, Suh and Kim ^{13 )}. The antioxidant activities of glyceollins were confirmed by measuring ferric reducing antioxidant power, 2,2-diphenyl-1-picrylhydrazyl radical scavenging, singlet oxygen quenching, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging, hydroxyl radical scavenging activity and lipid peroxidation inhibition. In addition, the antioxidant potential of glyceollins were measured by a fluorescent probe, 2,7-dichlorofluorescin diacetate, and dihydroethidium in mouse hepatoma hepa1c1c7 cells in which they were insulted with H₂O₂ to generate ROS. The compound showed a strong reducing power and inhibited lipid peroxidation and significant scavenging activities of radicals including singlet oxygen superoxide anion, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl⁽ Reference Kim, Suh and Kim ^{13 )}. It was also found that glyceollins significantly suppressed H₂O₂-induced LDL oxidation production (Fig. 2), suggesting their potential as natural antioxidants and nutraceuticals.

Fig. 2.

(Colour online) Inhibition of LDL oxidation by glyceollins. The isolated LDL was subjected to an oxidation in the presence of H₂O₂ and the inhibitory activities of glyceollins and soya extracts containing glyceollins against the oxidation were assayed by the classical method described elsewhere. Both the methanolic (80%) extracts of the soya sprout elicited with fungal infection (inoculated) and the soya sprout not treated with fungus (non-inoculated) were compared with glyceollins for the preventive activity against LDL oxidation (W-K Kim and J-S Lim, unpublished results).

Antioxidant enzyme induction

Many natural antioxidants have been found to induce phase 2/antioxidant enzymes such as NAD(P)H:quinone oxidoreductase 1, haem oxygenase 1, glutathione reductase, glutathione S-transferase A1 (also known as glutathione S-transferase Ya in mouse), and glutamate-cysteine ligase via the nuclear factor-erythroid 2 p45 subunit-related factor 2 (Nrf2)-mediated pathway⁽ Reference Kensler, Wakabayashi and Biswal ^{41 –} Reference Jeong, Jun and Kong ^{44 )}. Our recent study indicated that glyceollins have the potential to induce antioxidant enzymes and phase 2 detoxifying enzymes through the Nrf2-Kelch-like enoyl-CoA hydratase-associated protein 1 (Keap1) pathway⁽ Reference Kim, di Luccio and Kong ^{11 )}, although the mechanism of action how the compounds activate the Nrf2 signalling pathway needs to be defined (Fig. 3).

Under unstressed conditions, Nrf2 is present in cytosol in the form of a heterodimer with Keap1, and it is rapidly degraded by the proteosomal pathway. Once activated by oxidative stress or electrophiles, it migrates into the nucleus and binds to the antioxidant response element of specific genes, enhancing their transcription. Although the whole mechanism by which some natural compounds cause liberation of Nrf2 from Keap1 remains unclear, the conformational change of Keap1 by direct interaction with the compounds or an indirect signal generated from the binding of the compounds to cellular membrane seems to be responsible for the nuclear translocation of Nrf2 following the transcriptional activation of antioxidant enzymes.

Fig. 3.

(Colour online) Possible mechanism of antioxidant and phase 2 enzyme induction by glyceollins. Enzymes involved in removal of reactive oxygen species and phase II detoxification of xenobiotics to reduce cellular stress include glutathione S-transferases (GST), quinone: NAD(P) oxidoreductase (NQO1), epoxide hydrolase, haem oxygenase 1 (HO-1), UDP-glucuronosyl transferases and γ-glutamylcysteine ligase (γ-GCL). The transcription of these enzymes is coordinately regulated through the antioxidant response elements (ARE). The nuclear factor–erythroid 2 p45 subunit-related factor 2 (Nrf2) is a transcription factor that binds to ARE and activate these genes. Glyceollins appear to induce antioxidant/phase 2 detoxifying enzymes by promoting the nuclear translocation of Nrf2 which is, in turn, facilitated by the release of Nrf2 from Nrf2–Kelch-like enoyl–CoA hydratase-associated protein 1(Keap1) complex and the phosphorylation of Nrf2 by phosphoinositide 3-kinase (PI3K) signalling pathway⁽ Reference Surh ^{87 )}.

In particular, cysteine residues abundantly present in Keap1 could be modified by exogenous oxidants or antioxidants, facilitating Keap1 separation from Nrf2⁽ Reference Kobayashi, Li and Iwamoto ^{45 ,} Reference Ogura, Tong and Mio ^{46 )}. It is also speculated that the Nrf2–Keap1 complex is separated from each other by phosphorylation of either Nrf2 or Keap1 by certain signalling pathways including mitogen-activated protein kinase or phosphoinositide 3-kinase, which causes a conformational change⁽ Reference Sun, Huang and Zhang ^{47 –} Reference Lim, Kim and Kim ^{49 )}.

The importance of Nrf2 in cytoprotection has been proven from the result of when the knockout of Keap1 in mice led to juvenile lethality due to hyperkeratosis of the oesophagus⁽ Reference Wakabayashi, Itoh and Wakabayashi ^{50 )}. Hepatocyte-specific knockout of the Keap1 gene consistently elevates the accumulation of Nrf2 in the nucleus and protects hepatocytes against acute drug toxicity and inflammatory liver injury⁽ Reference Okawa, Motohashi and Kobayashi ^{51 ,} Reference Osburn, Yates and Dolan ^{52 )}.

Halliwell pointed out that antioxidants would only significantly influence a disease process if free radicals or other reactive species caused or significantly contributed to the progression of the disease⁽ Reference Halliwell ^{37 ,} Reference Halliwell ^{53 )}. Evidence supports the view that increased free radical formation is usually a consequence of tissue damage by a disease or toxin⁽ Reference Gutteridge and Halliwell ^{54 )}.

Nrf2-mediated phase 2 enzyme induction has been widely accepted as a promising approach for cancer chemoprevention as well as protection against oxidative stress⁽ Reference Jeong, Jun and Kong ^{44 ,} Reference Yu and Kensler ^{55 –} Reference Seo, Park and Kim ^{58 )}. Considering Nrf2-mediated antioxidant enzyme induction, glyceollins have the potential to prevent chemically induced carcinogenesis, neurodegenerative diseases such as Alzheimer's disease and atherosclerosis.

Anti-inflammatory activity

Inflammation is a process that includes multiple steps, which are regulated by activating inflammatory or immune cells⁽ Reference Lundberg ^{59 )}. It is widely accepted that inflammation plays a key role in coronary artery disease and in the progression of atherosclerosis⁽ Reference Libby, Ridker and Maseri ^{60 )}. Similarly, inflammation has long been considered a major precursor for the development of cancer in both infectious and non-infectious conditions. Non-infectious chronic inflammatory disorders such as inflammatory bowel disease, which are typically associated with chemokine signalling, increased cell proliferation, reduced cell cycles, accumulation of mutations, and inadequate DNA repair are well-known harbingers of malignancy⁽ Reference Xavier and Podolsky ^{61 )}. Thus, chronic inflammation is considered to be common cause of atherosclerosis and may contribute to the incidence and/or promotion of certain types of cancer⁽ Reference Ross, Stagliano and Donovan ^{62 )}.

Macrophages play a central role in the regulation of the inflammatory response by releasing pro-inflammatory cytokines, chemokines and other mediators that induce migration of additional cells to inflamed tissue⁽ Reference Bosca, Zeini and Traves ^{63 )}. The activated macrophages secrete inflammatory cytokines such as TNFα, IL-6, and induce ROS and PGE₂ ⁽ Reference Kaplanski, Marin and Montero-Julian ^{64 )}.

Phyto-oestrogen is known to have antioxidative, and anti-inflammatory properties by the inhibition of inducible NO synthase gene expression and NO production, as well as inhibition of the expression of inflammatory cytokines⁽ Reference Labinskyy, Csiszar and Veress ^{65 )}. The molecular mechanisms are implicated in the oestrogen receptor (ER)-dependent pathway or by the activation of the inflammatory NF-кB transcription factor through mitogen-activated protein kinases⁽ Reference Park, Lee and Heo ^{66 ,} Reference Fan, Gao and Wang ^{67 )}.

Our recent study showed that glyceollins markedly suppressed the inflammatory response in lipopolysaccharide (LPS)-activated murine macrophages and the skin in vivo ⁽ Reference Kim, Sung and Kim ^{68 )}. Interestingly, glyceollins suppressed LPS-dependent secretion of IL-6, NO and expression of cyclo-oxygenase 2 (COX-2) from RAW264.7 cells. NO is a molecule produced by inducible NO synthase in a reaction that converts arginine and O₂ into citrulline and NO⁽ Reference Kanwar, Kanwar and Burrow ^{69 )}. It plays an important role in inflammation⁽ Reference Kanwar, Kanwar and Burrow ^{69 )}. Upregulation of COX-2 expression by the transcriptional activation of the COX-2 gene, results in the increased production of PGE₂ which is a critical factor in the inflammatory condition⁽ Reference Lee, Soyoola and Chanmugam ^{70 )}.

It seems that the anti-inflammatory activity of glyceollins is associated with the suppression of LPS-dependent phosphorylation and the induction of NF-κB⁽ Reference Kim, Sung and Kim ^{68 )}. It is known that inactive NF-κB normally stays bound to inhibitory κB in the cytosol. NF-κB can be activated by LPS and pro-inflammatory cytokines, which induce increased protein phosphorylation and proteolysis of inhibitory κB protein⁽ Reference Verma, Stevenson and Schwarz ^{71 )}. The activated NF-κB is, in turn, translocated into the nucleus, binding to NF-κB-binding sites in the promoter regions of target genes and inducing the transcription of pro-inflammatory mediators such as inducible NO synthase, COX-2, TNFα, IL-1β, IL-6 and IL-8⁽ Reference Baeuerle and Baltimore ^{72 )}. Activation of macrophages plays an important role in the initiation and propagation of inflammatory responses by the production of cytokines and mediators, such as IL-1β, TNFα, NO and COX-2⁽ Reference Reddy and Reddanna ^{73 ,} Reference Hamalainen, Nieminen and Vuorela ^{74 )}.

Glyceollins also suppress LPS-dependent phosphorylation of extracellular-signal-regulated kinase 1 and 2 and p38, suggesting their ability to inhibit the essential targets responsible for the inflammation process⁽ Reference Kim, Sung and Kim ^{68 )}. Thus, glyceollins appear to exert anti-inflammatory activity via regulating the NF-κB signalling pathway (Fig. 4).

Fig. 4.

(Colour online) Hypothetical anti-inflammatory mechanism of glyceollins. Pro-inflammatory agents such as lipopolysaccharides (LPS) interact with Toll-like receptors (TLR) and relay signals to activate NF-κB, which acts as transcriptional activator to induce several pro-inflammatory mediators. Glyceollins seem to inhibit both expression and nuclear translocation of NF-κB, and thereby suppress LPS-induced inflammation. ARE, antioxidant response elements; ERK, extracellular-regulated protein kinase; IKK, inhibitor of NF-κB; MCP, monocyte chemoattractant protein-1.

Anti-cancer and oestrogen-modulating activities

Oestrogen antagonists have been exploited to prevent or treat oestrogen-positive breast cancer. Soya isoflavones have been extensively studied for their potential to prevent and/or treat sex-hormone-dependent tumours⁽ Reference Burow, Boue and Collins-Burow ^{14 ,} Reference Dong and Qin ^{17 –} Reference Messina ^{20 )}. This key finding led to the proposal that a diet containing soyabean may be beneficial in the prevention or treatment of hormone-dependent diseases because of the presence of these bioactive non-nutrients⁽ Reference Dong and Qin ^{17 –} Reference Messina ^{20 ,} Reference Setchell, Borriello and Hulme ^{75 )}. Soyabeans and their products have long been copiously consumed by the people of East Asian countries including Korea, China and Japan⁽ Reference Kim and Kwon ^{76 –} Reference Yamamoto, Sobue and Sasaki ^{78 )}, countries where the incidence of breast cancer is relatively low⁽ Reference Messina ^{79 ,} Reference Wu, Ziegler and Nomura ^{80 )}. Therefore, it is quite plausible that isoflavones could prevent breast cancer by antagonising oestrogen action. Salvo et al. reported that treatment with glyceollin suppressed E2-stimulated tumour growth of MCF-7 cells (−53·4%) and BG-1 cells (−73·1%) in ovariectomized athymic mice⁽ Reference Salvo, Boue and Fonseca ^{15 )}. These tumour inhibitory effects corresponded with significantly lower E2-induced progesterone receptor expression in the tumours, suggesting that the glyceollin mixture may be functioning as selective ER modulators, selectively antagonising ER function in a tissue type-specific manner⁽ Reference Burow, Boue and Collins-Burow ^{14 ,} Reference Salvo, Boue and Fonseca ^{15 )}. In contrast to these reports, our data consistently suggested the oestrogenic activity of glyceollins, as shown in the E-screen assay and in the enhanced proliferation of MCF-7 cells carrying ER, and the increased expression of pS2, a typical biomarker for oestrogenic activity⁽ Reference Kim, di Luccio and Kong ^{11 )}. Therefore, we speculate that like isoflavones, glyceollins appear to exert both oestrogen-like and anti-oestrogenic activities depending on the physiological condition. That is, glyceollins could act as oestrogen agonists under conditions that are lacking oestrogen, as could be seen in post-menopausal women. Conversely, these compounds could act as anti-oestrogens in the presence of endogenous oestrogen, and may also compete with oestradiol for binding to ER.

Meanwhile, glyceollins were found to inhibit the proliferation and cause the apoptosis of murine hepatoma cells (W-K Kim and J-S Lim, unpublished results). This has nothing to do with the oestrogenic activity of the compounds, but it may be associated with their inhibitory activity on the cell signalling pathway related to apoptosis. In particular, apoptotic activity of the compounds is believed to be associated with their stimulation of the c-Jun N-terminal kinase signalling pathway because cytochrome c release, increased by glyceollins, was suppressed by c-Jun N-terminal kinase inhibitor (W-K Kim and J-S Lim, unpublished results). Cytochrome c release from the mitochondria is known to be essential for the activation of caspase 3, one of the terminator enzymes executing the apoptotic process.

Anti-atherosclerosis

Due to their antioxidant properties⁽ Reference Kim, Suh and Kim ^{12 )}, glyceollins may have an effect on preventing and/or treating atherosclerosis. In addition, glyceollins inhibited LPS-induced inflammation in macrophage cells and suppressed the proliferation and migration of human aortic smooth muscle cells, suggesting that the compounds had potential to retard the atherosclerotic process⁽ Reference Kim, Sung and Kim ^{68 ,} Reference Kim, Cha and Choi ^{81 )}. However, currently there is no direct evidence supporting anti-atherosclerotic effect in human subjects.

Other beneficial effects