Functional Analysis of Natural Polyphenols and Saponins as Alternative Medicines

Hiroshi Sakagami1, Tatsuya Kushida2, Toru Makino3, Tsutomu Hatano4, Yoshiaki Shirataki5, Tomohiko Matsuta1, Yukiko Matsuo6 and Yoshihiro Mimaki6 1Meikai University School of Dentistry, Saitama, 2Non-Profit Organization Bio-Knowledge Bank, Tokyo, 3HumaLabo Co., Ltd., Tokyo, 4Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, 5Faculty of Pharmaceutical Sciences, Josai University, Saitama, 6Tokyo University of Pharmacy and Life Sciences, School of Pharmacy, Tokyo, Japan


Introduction
Hot water extracts of plants have been utilized to treat various diseases without severe side effects. The presence of numerous kinds of components in the extract made it difficult to identify the causative substances. The hot water extracts contain relatively higher amounts of high-molecular weight polysaccharides and lignin-carbohydrate complexes (LCCs) and relatively lower amounts of low-molecular weight tannins, flavonoids, terpenes and saponins.
Polysaccharides and LCCs are easily extractable by hot-water or alkaline solution and thus be expected to be present in higher amounts in the extract, but the complete determinations of the chemical structures have never been achieved due to the structural complexity. On the other hand, tannins, flavonoids, terpenes and some saponins are mostly difficult to be dissolved in water, but more easily extractable with methanol, and the structures of thousands of compounds have been identified. These low molecular weight compounds are thus expected to be present in lower amounts in the extract. Therefore, the information of how much these lower molecular materials are present in hot-water extract is limited, albeit important.

Yield and putative amounts in methanol and hot water extracts
Usually, LCCs are extracted by alkaline extract, and concentrated by acid-precipitation. The yield of LCCs are 0.04 to 7.9% of the plant materials   (Table 2). Gel filtration chromatography of the alkaline extract of Sasa senanencis Rehder revealed that LCC represents major parts of the extract (Matsuta et al, 2011). The major part of polyphenols in LCCs from LEM is composed of lignin precursors such as p-coumaric acid, vanillic acid, syringic acid and ferulic acid, but not tannin nor flavonoids ( Pretreatment of mice with LCC derived from pine cone extract increased the survival time of tumor-bearing mice (Sakagami et al., 1987) and bacterial-inoculated mice, possibly due to the accumulation of polymorphonuclear cells and monocytes/macrophages that produce active oxygens (Harada et al., 1988).
Pretreatment of mice with LCC induced antimicrobial activity against various microorganisms (Staphylococcus aureus SH10, Escherichia coli GN2411, Pseudomonas aeruginosa H7, Klebsiella pneumoniae ST101, Candida albicans YA2), but not Salmonella enteritidis 116-54 (Harada et al., 1988;Oh-hara et al., 1990). When the sugar moiety of LCC was destroyed by www.intechopen.com A Compendium of Essays on Alternative Therapy 272 treatment with sulfuric acid or trifluoroacetic acid, the antimicrobial activity was significantly reduced. Furthermore, dehydrogenation polymers of phenylpropenoids, which do not contain sugar, showed much lower antimicrobial activity. These data suggest the importance of sugar moiety of LCC for the induction of antimicrobial activity.
Pretreatment with LCCs protected infant mice from Hymenoleptis nana (Cestoda) infection. Subcutaneous administration of LCCs (10 mg/kg) to 1 week old mice evoked strong protective immunity against oral infection by Hymenolepis nana eggs. Significant antiparasite effects were also induced in 4 week old mice by intraperitoneal or oral administration of LCCs. Injection of LCCs by any root (s.c, i.p., p.o.) induced higher antiparasite activity than other natural antitumor polysaccharides [pine cone polysaccharide (Frs. I, II), PSK (proteinbound polysaccharide), Schizophyllan (water-soluble glucan), TAK (water-insoluble glucan), carboxymethyl TAK] (Abe et al., 1989). These data suggest that the conjugation of sugar and polyphenols is essential for the expression of in vivo biological activity.

Cytokine production
a. In vivo study.
Endogenously produced TNF have been reported to induce resistance against microbial infection (Nakane et al., 1988). When mice were intravenously treated with eliciting agents such as OK-432 (Picibanil) (Sakagami et al., 1990a) or Lactobacillus casei , TNF was induced in the blood, reaching a maximum level after 2-3 hours, and thereafter rapidly declined. Priming the mice with LCC, much higher level of endogenous TNF was induced, accompanied by hepatic accumulation of Kupffer cells. The priming effect of LCC may be involved in the induction of antitumor, antimicrobial and antiparasite activities. The endogenous TNF production primed by LCC decreased during the aging of mice and upon the tumor implantation into the mice (Hanaoka et al., 1989), suggesting the possibility that the resistance of the hosts against microbial infection may decline with aging.
b. In vitro study.
In contract, contamination of lipopolysaccharide (LPS) at more than 0.0001% of sample significantly affected cytokine determination in vitro. When the biological activity derived from contaminating LPS was subtracted, both cacao mass and husk LCCs lost the stimulation activity of nitric oxide (NO) and cytokine (TNF-, IL-1 , IFN-) production in mouse macrophage-like cell lines (RAW264.7, J774.1). However, cacao mass LCC enhanced LPS-induced iNOS expression in RAW264.7 cells. These data demonstrated several new biological activities of LCC distinct from LPS and further confirmed the promising antiviral and immunomodulating activities of LCCs .

Anti-viral activity
a. Anti-HIV activity: Anti-human immunodeficiency virus (HIV) activity was assessed quantitatively by a selectivity index (SI=CC 50 /EC 50 , where CC 50 is the 50% cytotoxic concentration against mockinfected MT-4 cells, and EC 50 is the 50% effective concentration against HIV-infected cells).
b. Anti-influenza virus activity: LCCs inhibited the plaque formation and RNA polymerase (enzyme engaged in viral replication) activity of influenza virus (Nagata et al., 1990). Limited digestion demonstrated that phenylpropenoid polymer, but not sugar moiety, is important for anti-influenza virus activity (Harada et al., 1991). This was confirmed by our finding that dehydrogenation polymers of phenylpropenoids without carbohydrate inhibited both the plaque formation and RNA polymerase more effectively than LCCs (Sakagami et al., 1990b).
LCC instantly adsorbed to the influenza virus when mixed in vitro, as demonstrated by sucrose gradient centrifugation, and abrogated the infectivity of the influenza virus in mouse infection model .
Recently, carboxylated lignins, synthesized using 4-hydroxy cinnamic acid scaffold by enzymatic oxidative coupling, have been reported to inhibit the entry of HSV-1 entry into the cells (Thakkar et al., 2010). Sulfated LCC (PPS-2b) (MW8500) also showed anti-HSV activity possibly by inhibiting the viral binding and penetration into host cells. Prunella cream formulated with a semi-purified fraction significantly reduced the skin lesion and mortality induced by HSV-1 infection in Guinea pigs (Zhang et al., 2007) The anti-HSV activity of sulfated lignins depended on their molecular weight, with the maximum at 39.4 kDa (Raghuraman et al., 2007).
d. Clinical application.
A clinical pilot study was carried out to evaluate anti-HSV-1 activity of a pine cone LCC and ascorbic acid treatment, with forty eight healthy patients of both genders between 4 and 61 years old (mean: 31 years), with active lesions of HSV-1. According to the HSV-1 stage at the presentation the patients were classified into the prodromic, erythema, papule edema, vesicle/pustule and ulcer stages. One mg of LCC-ascorbic acid tablet or solution was orally administered three times daily for a month. The patients who began the LCC-ascorbic acid treatment within the first 48 hours did not develop HSV-1 characteristic lesions, whereas those patients who began the treatment later experienced a shorter duration of cold sore lesions and a decrease in the symptoms. The majority of the patients reported the reduction in the severity of symptoms and the reduction in the recurrence episodes after the LCCascorbic acid treatment compared with previous episodes, suggesting its possible applicability for the prevention and treatment of HSV-1 infection (López et al., 2009).

Synergistic action with vitamin C
Vitamin C exhibited either antioxidant or prooxidant activity, depending on the concentration (Sakagami et al., 2000b). We have reported that ascorbate derivatives that produced the doublet signal of ascorbate radical (sodium-L-ascorbate, L-ascorbic acid, Disoascorbic acid, 6--D-galactosyl-L-ascorbate, sodium 5,6-benzylidene-L-ascorbate) induced apoptosis (characterized by internucleosomal DNA fragmentation and an increase in the intracellular Ca 2+ concentration) in HL-60 cells. On the other hand, ascorbate derivatives that did not produce radicals (L-ascorbic acid-2-phosphate magnesium salt, L-ascorbic acid 2-sulfate and dehydroascorbic acid) did not induce apoptosis (Sakagami et al., 1996a(Sakagami et al., , 1996b. This suggests the possible involvement of the ascorbate radical in apoptosis-induction by ascorbic acid-related compounds. We accidentally found that LCCs from the pine cone of Pinus parviflola Sieb et Zucc, pine cone of Pinus elliottii var. Elliotti, leaf of Ceriops decandra (Griff.) Ding Hou and, thorn apple of Crataegu Cuneata Sieb. et Zucc modulated the radical intensity of ascorbate bi-phasically, depending on the concentrations. At higher concentration, LCCs strongly enhanced the radical intensity of sodium ascorbate, which rapidly decayed, possibly due to the breakdown of ascorbic acid or to the consumption of ascorbyl radical. LCCs, not only from pine cones (Fr. VI), but also from Catuaba bark, pine seed shell, A. nikoense Maxim. and C. Cuneata Sieb. et Zucc. enhanced the radical intensity and cytotoxic activity of sodium ascorbate (Sakagami et al., 2005). On the other hand, tannins such as gallic acid, EGCG, and tannic acid counteracted the radical intensity and cytotoxic activity of sodium ascorbate (Satoh et al, 1999).
Sodium ascorbate rapidly reduced the oxygen concentration in the culture medium, possibly due to oxygen consumption via its pro-oxidation action. Simultaneous addition of LCCs further enhanced the ascorbate-stimulated consumption of oxygen (Sakagami et al., 1997). These data suggest that the synergistic enhancement of the cytotoxic activity of LCCs and ascorbate might be due at least in part to the stimulated induction of hypoxia.

State in hot-water extract: Possible binding to other compounds
Solvent fractionation of Alkaline Extract of the Leaves of Sasa senanensis Rehder (SE) demonstrated that (i) chlorophyllin in SE was recovered from the water layer, that contains majority of compounds (more than 81%) and inhibited the NO production by macrophages more potently than other n-hexane, diethyl ether and ethylacetate layers . Three-dimensional HPLC analysis demonstrated that the majority of SE components are recovered from one major peak. Furthermore, LCC isolated from SE showed the unique greenish color of chlorophyllin (absorption maximum = 452 nm) . These data strongly suggest the possible association of chlorophyllin with LCC in the native state or during extraction with alkaline solutions.

Site of action, signaling pathway, and receptor identification
A receptor for LCC, and the related signal transduction pathways had been largely unknown. On the other hand, -glucans and -mannans, which constitute the cell wall of fungi and plants, are recognized by dectin-1 and dectin-2, respectively, which are members of C-type lectins, and prominently expressed in the cell membrane of dendritic cells and macrophages as the transmembrane receptors (Brown & Gordon, 2001;McGreal et al., 2006;Vautier et al., 2010). It is also known that dectin-1 and 2 are essential for host defense against fungi, such as, Pneumocystis carinii, and Candia albicans (Saijo et al., 2007;. Furthermore, genetic and immunological studies have demonstrated the molecular mechanism regarding the immunostimulation and phylaxis recently. It became evident that, in the dendritic cells and macrophages, activated dectin-1 and 2 by -glucans and -mannans, respectively, can induce the activation of NFкb thorough signal transduction molecules such as syk and Card9-Bcl10-Malt1 (Gross et al., 2006;Saijo et al., 2010;Vautier et al., 2010).

Future directions
We have previously reported that tumor-specificity of oligomeric tannins (both hydrolysable and condensed types) is higher than that of monomeric tannins (Sakagami et al., 2000a), although the tumor-specificity of tannins was much lower than that of chemotherapeutic drugs . This suggests the importance of determining the optimal molecular weight of LCC for the expression of the highest biological activity. However, with the increase in the molecular weight, the water-solubility of LCC may decline, and make it difficult to be sterilized by millipore filteration.
There is a possibility that bacterial endotoxin (LPS) in the soil and air may contaminate LCC during the isolation step, since LPS is similarly extracted with alkaline solution and precipitated with acid. Most of previous studies have not paid attention to such LPS contamination in the LCC preparations. Alkaline extraction step that is necessary for the preparation of LCC has both merit and demerit. Merit is the chemical inactivation of LPS. Demerit is the degradation of LCC into its smaller size. Therefore, the conditions for alkaline extraction should be optimized to maximize the LPS inactivation and minimize the loss of biological activity.
It is generally accepted that lignin is linked by various amounts of polysaccharides (polymers of glucose or other sugar), yielding LCCs having broad ranges of molecular weights. Phenylpropenoid polymer portion of LCC have potent anti-vial activity, whereas carbohydrate portion have immunopotentiation activity, possibly via activation of TLR pathway and chemokine expression (Fig. 3). After extraction with alkaline solution, the structure of LCC may be either unmodified or modified. When LCC is prepared from the leaves, chlorophyllin may be associated during extraction step with alkaline solution. This association may enhance anti-inflammation activity and inhibition of cytochrome P-450 (CYP). When washing procedure is incomplete, LPS from bacteria in the soil and air may associate with LCC, considering that both LPS and LCC are precipitated by acid. This association may trigger the back-ground level of immune response-related genes expression. The associations with chlorophyllin, LPS and others may produce diverse biological activities, depending on the plant species and preparative procedures.

NO TNF
? D e c t i n -１

Lignin carbohydrate complex (LCC)
Mannan receptor G lu c a n r e c e p t o r

Anti-viral activity Immunopotentiation
Cell membrane

NO TNF
? D e c t i n -１

Lignin carbohydrate complex (LCC)
Mannan receptor G lu c a n r e c e p t o r

Phenylpropenoid polymers Polysaccharides
Anti-viral activity Immunopotentiation Cell membrane

Yield and putative amounts in methanol and hot water extracts
Tannins are defined to be naturally occurring polyphenols with potent binding activity with proteins and other biomolecules such as polysaccharides and lipids. The binding of tannins with those molecules is attributed to the hydrogen bonding and/or hydrophobic interaction, in addition to electrostatic interaction. In some cases covalent bonding also attributes to the interaction with proteins. Tannins are structurally classified into two major groups. Those belonging to one group are called hydrolysable tannins: i.e., esters of gallic acid and its oxidative derivatives with glucose or related sugars. The other ones are belonging to condensed tannins: i.e., flavan oligomers or polymers where their constituent monomeric flavans are connected mainly by C-4 -C-8 or C-4 -C-6 linkages. Some other types of polyphenolic compounds such as those containing several caffeic acid units (caffeetannins), and phloroglucinol oligomers or polymers (phlorotannins) are also regarded to be tannins. The binding properties of tannins, and therefore their biological and/or pharmacological properties attributed to the binding, vary depending on their structures. Since tannins bind strongly with cellulose and lignin of plant cells with hydrogen bonding and hydrophobic interaction, combinations of acetone and water are used as the solvents for extraction of tannins to release them from the plant cells. Some examples for the extractability of tannins from plant materials are shown below.
Geranium thunbergii is a plant species belonging to the family Geraniaceae, and its aerial part has been used as a folkmedicine for treatments of intestinal disorders including diarrhoea in Japan. Geraniin, the major constituent of this medicinal herb, is the representative hydrolysable tannin composed of galloyl, hexahydroxydiphenoyl (HHDP), and dehydrohexahydroxydiphenoyl (DHHDP) groups, in addition to a glucopyranose core. Several other tannins with related structures, including didehydrogeraniin, furosin, furosinin, elaeocarpusin (=ascorgeraniin), and geraniinic acids B and C, were also isolated from the same source plant species. Quantitative analysis of this medicinal herb using methylene blue for the estimation of total tannin indicated that the tannin contents vary 0.9 % -2.4 % of the aerial part, depending on the collected month. The quantitative experiments applied to several Geranium species indicated that the extractability for tannins with methanol is lower than that with the mixture of acetone and water (ca. 30-50% lower). The quantitative analysis of geraniin, the major tannin of G. thunbergii, by high-performance liquid chromatography (HPLC) of the aqueous acetone extracts indicated that the contents were 0.6 % -1.8 % of the aerial parts of the plant species (Okuda et al., 1980).
Most of the medicinal herbs have been extracted with hot water used in the traditional medicine, although some types of constituents are decomposed in hot water to give various products. Geraniin is also easily hydrolyzed in water to give several lower molecular weight polyphenols, such as corilagin, gallic acid, ellagic acid, and brevifolin (Fig. 4). Thus, the behaviour of geraniin during the extraction of the constituents from this medicinal herb with hot water was investigated. The first increment of the geraniin concentration was then followed by its decrease accompanied by the increase of corilagin, a hydrolysis product from geraniin. The highest concentration of geraniin during the decoction was observed at the time just after the boiling.

Fig. 4. Structural changes of geraniin in hot water
Analogous quantitative analysis was also performed for the extraction of rosmarinic acid (Fig. 5), called Labiatae-tannin, from dried leaves of Perilla frutescens. As a result, the concentration of this polyphenolic compound increased until the time of the boiling. The concentration did not decrease during the continuous heating of the aqueous solution, while rosmarinic acid was quite unstable when preparing the dried herb by heated air (Okuda et al., 1986).  Clinical isolates of MRSA found in Okayama University Hospital, which acquired antibiotic resistance against -lactams, aminoglycosides, macrolides, and some other antibiotics, were used as target bacteria. The addition of (-)-epicatechin gallate (25 mg/L), isolated from green tea leaves, decreased the minimum inhibitory concentration (MIC) of oxacillin, 128-512 mg/L for four MRSA strains, to 0.5-1 mg/L (Shiota et al., 1999). An analogous decrease of MIC of oxacillin was found for the addition of tellimagrandin I, a hydrolysable tannin isolated from rose petals. The addition of tellimagrandin I also brought the decrease of the www.intechopen.com

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MIC of tetracycline for two MRSA strains, from 2 to 0.25, and 8 to 1 mg/L, respectively (Shiota et al., 2000). Corilagin, isolated from leaves of Arctostaphyllos uva-ursi, also caused noticeable decreases of the MIC values of several -lactam antibiotics. The mechanisms of the effects of the hydrolysable tannins were also examined, and the suppression of the function of penicillin-binding protein 2a (PBP-2a), and also the inhibitory effects onlactamase have been suggested (Shimizu et al., 2001).
Further investigation revealed that a product obtained by incubation of (-)-epigallocatechin gallate (EGCG) in solution showed lowering effects on MIC of -lactam antibiotics. Since EGCG is unstable even in neutral solution, we investigated the changes in the structure of EGCG, and found formation of dimeric products. Among the products, theasinensin A (Fig.  7) caused marked decrease of MIC of oxacillin, and related -lactam antibiotics. MIC of streptomycin for the MRSA strains and for a methicillin-sensitive Staphylococcus strain decreased upon the addition of theasinensin A (Hatano et al., 2003a). Condensed proanthocyanidin obtained from pericarpus of Japanese pepper (Zanthoxylum piperitum) also decreased MIC of -lactam antibiotics (Kusuda et al., 2006).

State in hot-water extract: Possible binding to other compounds
Although some hydrolysable tannins such as geraniin were unstable in water as mentioned above, the stability of hydrolysable tannins in water is dependent on their structures. Structural changes of different oligomeric hydrolysable tannins during the hot water treatment were investigated, and differences in the instability of those tannins have also been shown. For example, isorugosin D, which is a regio-isomer of rugosin D concerning the orientation of valoneoyl group, was more stable, relative to rugosin D, against the hot-water treatment. Oenothein B, which has a macrocyclic structure with two valoneoyl groups in the molecule, was much more stable relative to those dimers (Fig. 8) (Yoshida et al., 1992).
Investigation on the metabolic profiles after oral administration of galloylglucoses revealed formation of 4-O-methylgallic acid. The HHDP group in ellagitannins is metabolised into urolithins A and B, and some compounds structurally related to them (Ito et al., 2008). On the other hand, metabolism of catechins and related oligomeric flavanoids gives protocatechuic acid, and its methyl derivatives (Fig. 9). Our investigation of the binding of EGCG and those low-molecular-weight metabolites revealed that they affects the bindings of site-I-and site-II-binding drugs to human serum albumin, depending on their structures (Nozaki et al., 2009).

Site of action, signaling pathway, and receptor identification
Pentagalloyglucose has been reported to induce autophagic cell death in prostate cancer cells (Hu et al., 2009) and inhibit its bone metastasis by transcriptionally repressing EGFinduced metalloproteinase secrtion (Kuo et al., 2009). On the other hand, proanthocyanidines induce apoptotic cell death in human non-small cell lung cancer cells, at least in part due to inhibition of COX-2, PGE 2 and PGE 2 receptor expression (Shama et al., 2010).

Future directions
Many researchers have reported various pharmacological and biological activity of polyphenolic compounds including tannins. However, some of those compounds, especially several tannins and condensed proanthocyanidins or catechins are unstable in water and/or on heating. Therefore, the actual pharmacologically active compounds may be different from the administered compounds. The changing procedures should be clarified. Our study on the structural changes of catechins will give a solution of the problem (Taniguchi et al., 2008).
Binding of polyphenolic compounds, especially the binding of tannins, to biomolecules should be further investigated, too, since their pharmacological activity starts with the association or complex formation of the polyphenols and biomolecules. Our study on the formation of complex from tannins and related polyphenols with serum albumin revealed that the formed supermolecule has the "molecular weight" of 9.5 x 10 5 for a combination (Hatano et al., 2003b). The structural details of those supermolecules are still in an enigma.

Flavonoids
Flavonoids are important secondary metabolites i n h i g h e r p l a n t s , and classified into flavones, isoflavones, flavonols, flavanones, catechins and anthocyanidins, based on the skeletons of aglycone moieties (Fig. 10). Backbone structures of all flavonoids are derived from two metabolites, malonyl-CoA and p-coumaroyl-CoA, and the biosynthetic pathway of flavonoids are the condensation of three molecules of malonyl-CoA with one molecule of pcoumaroyl-CoA (Martens & Mithöfer, 2005).

Yield and putative amounts in methanol and hot water extracts
Flavonoids of yellow pigment are usually extracted by methanol, ethanol or alcohol-water mixtures, and fractionated by diethyl ether and ethyl acetate. Anthocyanines of red pigment are usually extracted by methanol and hydrochloric acid or trifluoroacetic acid mixtures (with yield of about 1-5%). The use of mineral acid can lead to the loss of attached acyl group.

Anti-osteoporosis activity
Osteoporosis is considered to be one of the hormonal deficiency diseases observed in the menopausal women and elderly persons. When estrogen is reduced in the body, the production of pro-inflammatory cytokines such as IL-1 and IL-6 are elevated, and the osteoclastogenesis enhanced, leading to the bone resorption. Glucosidic isoflavones of PIII (Isocal, glucosidic isoflavones extracted from Sophorae fructus) stimulated the osteoblastic proliferation by suppressing the IL-1 and IL-6 production and elevating the NO level. In bone marrow primary culture, PIII effectively suppressed the osteoclastogenesis. These effects of PIII was slightly lower than 17-estradiol, but higher than two soybean isoflavones [daidzin (1), genistin (2)]. PIII may preferentially induce anti-osteoporosis response by attenuating their osteoclastic differentiation and by upregulating the NO (Fig.11) (Joo et al., 2003).
Eleven isoflavonoids were examined for their possible antimicrobial property against twelve known Gram-positive and Gram-negative sensitive bacteria. Daidzein (3) and calycosin failed to show antimicrobial activity and formononetin, genistein (4), biochanin A, irisolidone, 7,4'-dihydroxy-3'-methoxyisoflavone, licoisoflavone A and licoisoflavone B had moderate antimicrobial action. Sophoraisoflavone A and 6,8-diprenylgenistein showed higher antimicrobial activity in vitro against 214 strains of bacteria. Their MIC ranged from 25 to 200 mg/L in most strains. At the concentrations of 30 and 60 g/mouse, sophoraisoflavone A and 6,8-diprenylgenistein significantly protected the mice from 50 median lethal dose (MLD) of a virulent strain of Salmonella Typhimurium (Dastidar SG et al., 2004).

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The inhibition of macrophage NO production by these isoflavanones might, at least in part, be explained by their radical scavenging or reduction activity .

Enzyme inhibitory activity
Sophoflavescenol with a C-8 prenylated flavonol inhibited cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), more efficiently than PDE3 and PDE4. Among twelve prenylated flavonoids with the resorcinol moiety, kuraridin (10), sophoraflavanone G (5), kurarinone (9) and norkurarinol inhibited the tyrosinase activity more effectively than kojic acid, a tyrosinase inhibitor. The substitution of a lavandulyl or hydroxylavandulyl group at the C-8 position and a methoxy or hydroxy group at the C-5 position (Fig. 13) may be required for their inhibitory effect    Fig. 13. Sophora flavescens that inhibit tyrosinase activity

State in hot-water extract: Possible binding to other compounds
Neutral aqueous solutions in anthocyanins are unstable and quickly decolorized. The copigmentation reaction, first reported by Robinson, is a colour-stabilizing mechanism (Oszmianski et al., 2004) and regarded as significant factors for stabilizing the structure of anthocyanins (Marcovic et al., 2000). The copigmentation reaction occurs, when planar molecule interaction between anthocyanin and other pigments occurs to form the complex. The common copigment compounds are flavonoids such as rutin and quercetin (Bakowska et al., 2003), other polyphenols, amino acids and organic acids.

Site of action, signaling pathway, and receptor identification
It has been recently reported that baicalin, a flavone, induced the osteoblastic differentiation via the activation of Wnt/ -catenin signalling pathway (Guo et al, 2011). Apigenin induced cell growth retardation via leptin/leptin receptor pathway in human A549 adenocarcinoma cell line (Bruno et al., 2011).

Future direction
Flavonoids are distributed into virtually all parts of the plant, the root, heartwood, sapwood, bark, leaf, fruit and flower. Although various pharmacological and biological activities of flavonoids are reported, the reason why biosynthesis of prenylflavonoids is restricted in Leguminosae, Moraceae and a few species of plants is unclear. Further chemical and physiological investigations are needed to answer this question.

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Saponins
Saponins are naturally occurring glycosides with the ability of forming a soapy lather when shaken with water, and classified as triterpene saponins or steroidal saponins on the basis of the structural features of the aglycone moieties. Triterpene saponins are found in a wide variety of dicotyledonous plants, but are rare in monocotyledons (Dinda et al., 2010). On the other hand, steroidal saponins are mainly distributed among a limited species of monocotyledonous plants (Mahato et al., 1982). Some marine organisms are known to produce saponins with unique chemical structures and biological properties (Minale et al., 1996).
Several important Chinese herbal medicines such as Astragalus Root, Licorice Root, Polygala Root, Ginseng, Bupleurum Root, Anemarrhena Rhizome and Ophiopogon Tuber contain saponins as the main secondary metabolites, which may be responsible for their specific pharmacological activities. Depending on the fundamental skeletons of the aglycone moieties, triterpene saponins have been classified into more than 10 types, oleanane-type, ursane-type, lupan-type, dammarane-type, and so on. As for steroidal saponins, they have been classified as being spirostan-type, furostan-type, or cholestane-type. Cardiac glycosides and pregnane glycosides are parts of steroidal glycosides, but are not usually included in the saponin category.

Yield and putative amounts in methanol and hot water extracts
Saponins can to be extracted with hot water. However, since saponins are structurally composed of a hydrophobic aglycone unit and a hydrophilic sugar moiety, methanol and a mixture of ethanol and water (4:1, v/v) allow saponins, including cardiac and pregnane glycosides with deoxysugar residues, to be exhaustively extracted from crude materials. OSW-1 was quantitatively extracted from a plant material using methanol. Pure ethanol fails to extract saponins with high polar properties such as glycyrrhizinic acid whose sugar moiety is composed of two glucuronic acid units (Kuroda et al., 2010).

Cytotoxic and antitumor activities of OSW-1
Isolation and identification of OSW-1: OSW-1 was isolated from the methanol extract of the bulbs of Ornithogalum saundersiae, a Liliaceae plant native to South Africa. The structure of OSW-1 was determined by conventional spectroscopic analysis and hydrolysis, and was revealed to be a cholestane-type saponin with an acylated sugar unit at the C-16 hydroxy group of the cholestane skeleton (Kubo et al., 1992).
Cytotoxic activities: OSW-1 exhibited significantly potent cytotoxic activities against various cultured malignant tumor cells such as mouse mastocarcinoma, human pulmonary adenocarcinoma, human pulmonary large cell carcinoma and human pulmonary squamous cell carcinoma, including adriamycin (ADM)-resistant P388 leukemia and camptothecin (CPT)-resistant P388; the activities are around 10-100 times more potent than those of the clinically used anticancer drugs, mitomycin C (MMC), ADM, cisplatin (CDDP), CPT and taxol (TAX). To the contrary, normal human pulmonary fibroblasts are little sensitive to OSW-1 .
Panel screening: Evaluation of OSW-1 in the Japanese Foundation for Cancer Research 38 cell-line assay (Yamori et al., 1999) showed that the mean concentration required to achieve GI 50 against the panel cells tested was 5.6 nM. OSW-1 displayed differential cytotoxicities, with breast cancer, CNS cancer and lung cancer subpanel cell lines showing particular sensitivity, but with colon cancer, ovarian cancer, stomach cancer and prostate cancer subpanel cell lines being relatively resistant to it. The pattern of the differential cytotoxicities of OSW-1 was evaluated using the Compare Program and was revealed not to correlate with those shown by any of the other compounds, including currently used anticancer drugs; correlation coefficient values were less than 0.5. This indicates that OSW-1 must have a unique mode of action and the potentiality as the lead of a new anticancer agent.
In vivo evaluation: OSW-1 was not haemolytic in human erythrocytes at 100 g/mL. In in vivo evaluations, OSW-1 was effective versus mouse P388 with an increased life span of 59% by one-time administration (i.p.) of 0.01 mg/kg. As for the response of Hep134 rat xenograft to OSW-1, administration of OSW-1 on the second day and the fourth day (each 0.04 mg/kg, i.p.) significantly reduced tumor growth compared to the non-treated group (unpublished data).
OSW-1 analogues: O. saundersiae yielded several OSW-1-related compounds, which were slightly different from OSW-1 in the structures of the aromatic acid moieties. A few more polar analogues having a glucosyl unit at C-4 of the terminal xylosyl moiety were also isolated and identified. Phytochemical studies of Ornithogalum thyrsoides and Galtonia candicans, which are taxonomically related to O. saundersiae, resulted in the isolation of a series of OSW-1 derivatives, possessing a glucosyl, diglucosyl, or triglucosyl unit at the C-3 hydroxy group of the aglycone moiety without exceptions (Mimaki, 2006). These OSW-1 analogues, together with those partially modified by hydrolysis and reduction, were useful for elucidating the structure-activity relationship (SAR) of OSW-1.
Total synthesis of OSW-1: Since the potentiality of OSW-1 as a new lead compound for a new anticancer agent was evident, OSW-1 has been an attractive synthetic target for organic www.intechopen.com A Compendium of Essays on Alternative Therapy 290 chemists. Furthermore, a number of OSW-1 analogues with modified acylated diglycosides, side-chains and/or steroidal nuclei have been obtained by means of chemical synthesis for SAR studies. Guo and Fuchs reported the first synthesis of the protected aglycone of OSW-1 (Guo & Fuchs, 1999). By employing the same approach, Deng and his co-workers established the first total synthesis of OSW-1 in 1999 (Deng et al., 1999). Yu and Jin reported a total synthesis of OSW-1 based on their own new strategy (Yu & Jin, 2001). Most recently, Xue and his co-workers have succeeded in the gram scale total synthesis of OSW-1 in 10 linear steps with an overall yield of 6.4%, starting from (+)-dehydroisoandrosterone (Xue et al., 2008).  SAR of OSW-1: The aglycone of OSW-1 is a cholesterol (cholest-5-en-3-ol) derivative with a -hydroxy group at C-16, an -hydroxy group at C-17 and a carbonyl group at C-22. The sugar moiety attached to C-16 of the aglycone was assigned as O-(2-O-p-methoxybenzoyl--D-xylopyranosyl)-(1→3)-2-O-acetyl--L-arabinopyranosyl. The acylated sugar moiety is essential for the exhibition of the potent cytotoxic activities of OSW-1 (Morzycki et al., 2004;Tang et al., 2007). Slight differences in the aromatic acid structure gave no effects on the activities , but the following modifications, 1) deacylation , 2) glucosylation at C-4 of the xylosyl unit (Kuroda et al., 2001), 3) removal of the C-4 hydroxy group of the arabinosyl unit (Tschamber et al., 2007), 4) change in the linkage position of the xylosyl unit from C-3 of the arabinosyl moiety to its C-4 (Zheng et al., 2010, significantly reduced its cytotoxicities. Although replacement of the aglycone by disparate steroids led to inactive compounds (Ma et al., 2000(Ma et al., , 2001a(Ma et al., , 2001b, the steroidal aglycone moiety tolerate certain modifications without a significant loss in cytotoxic potency. The C-17 hydroxy group was shown to be of little importance for the cytotoxicities (Zheng et al., 2010. The C-22 carbonyl group was reported to be a pharmacophore requirement (Wojtkielewicz et al., 2007); however, a current SAR study concluded that it is not necessary for the cytotoxic activities . A number of the side chain modified analogues of OSW-1 have been synthesized, and some C-23-oxa analogues have been found to be significantly cytotoxic (Deng et al., 2004;Shi et al., 2005). The steroidal A-and B-rings can be generally modified without a loss of cytotoxic potency, as was evident when the 3-O-glucopyranosyl- (Mimaki, 2006), 3-Obiotinyl- (Kang et al., 2009) and 5,6-dihydro-  OSW-1 derivatives were as active as the parent. However, the activity of a derivative with an aromatic A-ring was exceptionally much less than that of OSW-1 (Tschamber et al., 2007). The linkage of the acylated diglycoside to the C-16 hydroxy group of the aglycone is essential for the potent cytotoxicity (Ma et al., 2001b), except in the case of a few synthetic analogues Zheng et al., 2011).

Other biological activities of OSW-1
At first, OSW-1 was isolated from a cyclic AMP phosphodiesterase inhibitor from O. saundersiae bulbs (Kubo et al., 1992). Tamura and his co-workers found that OSW-1 inhibited ovarian E 2 secretion. The decrease in the levels of ovarian steroid induced by OSW-1 was shown to be due to its direct inhibitory action on the gene expression of the steroidal enzyme and the proliferation of granulose cells in the ovary (Tamura et al., 1997).

Other cholestane-type saponins with cytotoxic activities
O. saundersiae bulbs produced a novel 24(23→22)abeo-cholestane glycoside named saundersioside B, which has a six-membered hemiacetal ring between C-16 and C-23, and a five-membered acetal ring between C-18 and C-20. Saundersioside B possesses an aromatic acid ester group at the sugar moiety attached to C-3 of the aglycone and inhibited HL-60 cell growth through induction of apoptosis .
Candicanoside A, isolated from G. candicans, has two epoxy functional groups between C-16 and C-23, and between C-18 and C-23 in its rearranged cholestane skeleton (Mimaki et al., 2000). A novel polyoxygenated 5-cholestane glycoside, designated as galtonioside A, has also been isolated from G. candicans . These cholestane-type saponins also display potent cytotoxic activities against cultured tumor cells with differential cytotoxicities in panel screenings. The obtained cytotoxic profiles are related to that of OSW-1.

State in hot-water extract: Possible binding to other compounds
Although some spirostan-type saponins such as digitonin are characterized by the formation of complex with cholesterol, it has not been reported that cholestane-type saponins interact with other compounds. When OSW-1 is dissolved in water, methanol, or other alcohols and the solution is allowed to stand for a few days, OSW-1 gradually decomposes to afford deacyl derivatives.

Site of action, signaling pathway, and receptor identification
The action mechanism of OSW-1 has been recently disclosed to damage the mitochondrial membrane and cristae in human leukemia and pancreatic cancer cells, leading to losses of transmembrane potential, increases in cytosolic calcium contents, and activation of calciumdependent pathways. No anticancer compounds reported to date have this mechanism (Zhou et al., 2005). Furthermore, OSW-1 has been shown to induce apoptosis in mammalian cells through the mitochondrial pathway, involving the caspase-8-depending cleavage of Bcl-2 (Zhu et al., 2005).

Future directions
A number of crude drugs, including those used in Kampo prescriptions, are known to contain saponins. However, the pharmacological roles of saponins in Kampo prescriptions remain to be elucidated, except for glycyrrhizinic acid in Licorice Root. Manifestation of the specific pharmacological effects of each saponin will lead to providing evidence on the efficacy of Kampo medicines.
It is notable that O. saundersiae, an ornamental plant without a folkloric medicinal background, produces OSW-1, a cholestane-type steroidal saponin with high apparent potential for effectively treating some cancers that are resistant to available medicines. Thus, OSW-1 clearly warrants further chemical and biological investigations for its clinical applications. www.intechopen.com

Conclusion
This review summarizes unique biological properties of LCCs, tannins, flavonoids and saponins, suggesting their functionality as alternative medicines. LCCs showed broad and potent anti-viral activity and synergism with vitamin C. Since virus is one of major risk factor of oral cavity cancer (Sakagami; 2010a), anti-viral action of LCC may reduce the incidence of virus-triggered diseases such as cancer. The use of a genetic algorithm-kernel partial least squares algorithm combined with an artificial neural network (Jalai-Heravi, 2008) may be useful to predict the optimal structure of LCCs for the expression of biological activities. Identification of dectin-2 as LCC receptor awaits further confirmation with siRNA and gene overexpression experiments. Tannins are found to be relatively unstable in hot water, and easily decomposed and associated with serum albumin. Biological significance of such degradation products and associates remains to be investigated. Saponins with high polarity can to be extracted with hot water, and therefore should be present in many herbal extracts and Kampo medicines. The antitumor potentital of OSW-1 should be further pursued. .