**Phytochemicals of the Chinese Herbal Medicine** *Tacca chantrieri* **Rhizomes**

Akihito Yokosuka and Yoshihiro Mimaki

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53668

## **1. Introduction**

[24] Okuda T, Hatano T, Agata I, Nishibe S. The Components of Tannic Activities in Labi‐ atae Plants. I. Rosmarinic acid from Labiatae Plants in Japan. Yakugaku Zasshi 1986;

[25] Kimura Y, Okuda H, Okuda T, Hatano T, Arichi S. Studies on the Activities of Tan‐ nins and Related Compounds, X. Effects of Caffeetannins and Related Compounds on Arachidonate Metabolism in Human Polymorphonuclear Leukocytes. Journal of

[26] Kimura Y, Okuda H, Okuda T, Hatano T, Agata I, Arichi S. Studies on the Activities of Tannins and Related Compounds from Medicinal Plants and Drugs. VI. Inhibitory Effects of Caffeoylquinic Acids on Histamine Release from Rat Peritoneal Mast Cells.

[27] Shibata S, Saito T. Flavonoid Compounds in Licorice Root. Journal of Indian Chemi‐

[28] Hatano T, Yasuhara T, Fukuda T, Noro T, Okuda T. Phenolic Constituents of Lico‐ rice. II. Structures of Licopyranocoumarin, Licoarylcoumarin and Glisoflavone, and Inhibitory Effects of Licorice Phenolics on Xanthine Oxidase. Chemical and Pharma‐

[29] Hatano T, Fukuda T, Miyase T, Noro T, Okuda T. Phenolic Constituents of Licorice. III. Structures of Glicoricone and Licofuranone, and Inhibitory Effects of Licorice Constituents of Monoamine Oxidase. Chemical and Pharmaceutical Bulletin 1991; 39

[30] Hatano T, Yasuhata T, Miyamoto K, Okuda T. Anti-human Immunodeficiency Virus Phenolics from Licorice. Chemical and Pharmaceutical Bulletin 1988; 36 2286-2288.

[31] Uchiumi F, Hatano T, Ito H, Yoshida T, Tanuma S. Transcriptional Suppression of the HIV Promoter by Natural Compounds. Antiviral Research 2003; 58 89-98.

[32] Hatano T, Shintani Y, Aga Y, Shiota S, Tsuchiya T, Yoshida T. Phenolic Constituents of Licorice. VIII. Structures of Glicophenone and Glicoisoflavanone, and Effects of Licorice Phenolics on Methicillin-Resistant *Staphylococcus aureus*. Chemical and Phar‐

[33] Sun XL, Ito H, Masuoka T, Kamei C, Hatano T. Effect of *Polygala tenuifolia* Root Ex‐ tract on Scopolamine-Induced Impairment of Rat Spatial Cognition in an Eight-Arm

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tion. PhD thesis. Okayama University; 2008 (in Japanese).

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66 Alternative Medicine

Natural Products 1987; 50 392-399.

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ceutical Bulletin 1989; 37 3005-3009.

maceutical Bulletin 2000; 48 1286-1292.

1238-1243.

Chemical and Pharmaceutical Bulletin 1985; 33 690-696.

The family Taccaceae is composed of two genera, *Tacca* and *Schizocapsa*, and about 10 spe‐ cies, with most distributed in tropical regions of Asia, the Pacific Islands, and Australia [1]. *Tacca chantrieri* André is a perennial plant that occurs in the southeast region of mainland China, and its rhizomes have been used for the treatment of gastric ulcers, enteritis, and hepatitis in Chinese folk medicine. According to a Chinese herbal dictionary, *T. plantaginea* has also been used for the same purposes as *T. chantrieri* [2]. The chemical constituents of *T. plantaginea* have been extensively examined and a series of highly oxygenated pentacyclic steroids named taccalonolids, which have a γ-enol lactone, have been isolated as characteris‐ tic components of the herb [3], but there has been only one report of the secondary metabo‐ lites of *T. chantrieri*, in which a few trivial sterols such as stigmasterol and daucusterol, and a diosgenin glycoside were found [4]. Therefore, we focused our attention on the constituents of *T. chantrieri* rhizomes, and a detailed phytochemical investigation of this herbal medicine has been carried out.

In this chapter, we describe the phytochemicals isolated from *T. chantrieri* rhizomes and their biological activities with a focus on cytotoxicity against human cancer cells.

## **2. Isolation and structural determination**

*T. chantrieri* specimens were collected in Yunnan Province, People's Republic of China. The rhizomes of *T. chantrieri* (fresh weight, 7.3 kg) were extracted with hot MeOH (3 L × 2). The MeOH extract was concentrated under reduced pressure, and the extract was passed through a polystyrene resin (Diaion HP-20) column eluted with MeOH/H2O gradients,

EtOH, and EtOAc. The 50% MeOH and MeOH eluate portion was subjected to silica gel and octadecylsilanized silica gel column chromatography to afford a total of 41 compounds, clas‐ sified into diarylheptanoids (**1** and **2**), diarylheptanoid glucosides (**3**–**9**), ergostane gluco‐ sides (**10**–**21**), withanolide glucosides (**22** and **23**), spirostan glycosides (**24**–**28**), furostan glycosides (**29**–**32**), pseudofurostan glycosides (**33**–**37**), pregnane glycosides (**38**–**40**), and a phenolic glucoside (**41**) (Fig.1). Their structures were determined through extensive spectro‐ scopic studies and through chemical transformations followed by chromatographic and spectroscopic analysis.

**3. Diarylheptanoids and diarylheptanoid glucosides**

R2 R4

 R2 OH OMe OH OMe OH OH OH OH OMe

 R3 H H OH OMe H OH OMe H H

phenyl)-7-(4-hydroxyphenyl)heptane by analysis of the 1D (1

 R4 OH OMe OH OMe OH OH OH OH OMe

OH OH R1 R3 3 5

sides (Fig. 2) [5].

Figure 2

[M]<sup>+</sup>

**Figure 2.** Structures of **1–9** and their derivatives

**1 1a 2 2a 5a 7a 8a 9a 9b**

 R1 OH OMe OH OMe OMe OMe OMe H H

Diarylheptanoids consist of two phenyl groups linked by a linear seven-carbon aliphatic chain. Compounds **1** and **2** are diarylheptanoids and **3**–**9** are diarylheptanoid monogluco‐

HO OH

HO OH

**4**

H and 13C) and 2D (1

3 5

O

OH

 R1 OH OMe OH OMe OMe H

OH R1 R2 3 5

> R2 H H OH OH OMe H

O

Phytochemicals of the Chinese Herbal Medicine *Tacca chantrieri* Rhizomes

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69

> O OH

O OH

HO HO HO

HO

Compound **1** was isolated as a viscous syrup, [α]D +1.7˚ (MeOH). HREIMS of **1** showed an

SY, HMQC, and HMBC) spectra. The absolute configuration of the 3,5-dihydroxy moieties of the new diarylheptanoids were determined by applying the CD exciton chirality method to acyclic 1,3-dibenzoates [6]. The trimethyl derivative (**1a**) was converted to the correspond‐ ing 3,5-bis(*p*-bromobenzoate) (**1b**) and its CD spectrum exhibited positive (237.4 nm, Δε +29.9) and negative (253.3 nm, Δε –20.0) Cotton effects, which were consistent with a nega‐ tive chirality. Thus, the absolute configurations were determined as 3*R* and 5*R* (Fig. 3). The

 peak at *m/z* 332.1623, corresponding the empirical molecular formula of C19H24O5, which was also deduced by analysis of its 13C NMR and DEPT spectral data. The IR spec‐ trum suggested the presence of hydroxy groups (3347 cm-1) and aromatic rings (1611 and 1515 cm-1). The UV spectrum showed an absorption maximum due to substituted aromatic rings (281.4 nm). The planar structure of **1** was assigned as 3,5-dihydroxy-1-(3,4-dihydroxy‐

HO HO HO

5

H-<sup>1</sup>

H CO‐

**Figure 1.** Extraction, partition, and purification procedures

## **3. Diarylheptanoids and diarylheptanoid glucosides**

EtOH, and EtOAc. The 50% MeOH and MeOH eluate portion was subjected to silica gel and octadecylsilanized silica gel column chromatography to afford a total of 41 compounds, clas‐ sified into diarylheptanoids (**1** and **2**), diarylheptanoid glucosides (**3**–**9**), ergostane gluco‐ sides (**10**–**21**), withanolide glucosides (**22** and **23**), spirostan glycosides (**24**–**28**), furostan glycosides (**29**–**32**), pseudofurostan glycosides (**33**–**37**), pregnane glycosides (**38**–**40**), and a phenolic glucoside (**41**) (Fig.1). Their structures were determined through extensive spectro‐ scopic studies and through chemical transformations followed by chromatographic and

spectroscopic analysis.

68 Alternative Medicine

**Figure 1.** Extraction, partition, and purification procedures

Diarylheptanoids consist of two phenyl groups linked by a linear seven-carbon aliphatic chain. Compounds **1** and **2** are diarylheptanoids and **3**–**9** are diarylheptanoid monogluco‐ sides (Fig. 2) [5].

**Figure 2.** Structures of **1–9** and their derivatives

Figure 2

Compound **1** was isolated as a viscous syrup, [α]D +1.7˚ (MeOH). HREIMS of **1** showed an [M]<sup>+</sup> peak at *m/z* 332.1623, corresponding the empirical molecular formula of C19H24O5, which was also deduced by analysis of its 13C NMR and DEPT spectral data. The IR spec‐ trum suggested the presence of hydroxy groups (3347 cm-1) and aromatic rings (1611 and 1515 cm-1). The UV spectrum showed an absorption maximum due to substituted aromatic rings (281.4 nm). The planar structure of **1** was assigned as 3,5-dihydroxy-1-(3,4-dihydroxy‐ phenyl)-7-(4-hydroxyphenyl)heptane by analysis of the 1D (1 H and 13C) and 2D (1 H-<sup>1</sup> H CO‐ SY, HMQC, and HMBC) spectra. The absolute configuration of the 3,5-dihydroxy moieties of the new diarylheptanoids were determined by applying the CD exciton chirality method to acyclic 1,3-dibenzoates [6]. The trimethyl derivative (**1a**) was converted to the correspond‐ ing 3,5-bis(*p*-bromobenzoate) (**1b**) and its CD spectrum exhibited positive (237.4 nm, Δε +29.9) and negative (253.3 nm, Δε –20.0) Cotton effects, which were consistent with a nega‐ tive chirality. Thus, the absolute configurations were determined as 3*R* and 5*R* (Fig. 3). The

5

structure of **1** was shown to be (3*R*,5*R*)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydrox‐ yphenyl)heptane. In the same way, the structure of **2** was elucidated as (3*R*,5*R*)-3,5-dihy‐ droxy-1,7-bis(3,4-dihydroxyphenyl)heptane.

**Figure 3.** Determination of the absolute configurations at C-3 and C-5 of **1**

Compounds **3**–**9** are diarylheptanoid monoglucosides. Enzymatic hydrolysis of **3**–**9** with naringinase gave the diarylheptanoid derivatives and D-glucose. Identification of D-glucose, including its absolute configuration, was carried out by direct HPLC analysis of the hydro‐ lysates. In the HMBC spectra, a long-range correlation was observed from each anomeric proton to the C-3 carbon in **3** and **5**–**9**, and to the C-5 carbon in **4**.

Diarylheptanoids are known to occur in only a limited number species of higher plants be‐ longing to the families Zingiberaceae [7–10], Betulaceae [11], and Aceraceae [12]. This is the first isolation of diarylheptanoids from a plant of the family Taccaceae.

## **4. Ergostane glucosides**

Compounds **10**–**21** are new ergostane glucosides (Fig. 4) [13–15]. Taccasterosides A–C (**10**– **12**) are novel bisdesmosideic oligoglucosides of (24*R*,25*S*)-3β-hydroxyergost-5-ene-26-oic acid (**10a**), whereas **13**–**20** are those of (24*S*,25*R*)-ergost-5-ene-3β,26-diol (**10b**). Compound **21** is an ergostane glucoside with the six-membered lactone on the side chain of the aglycone.

Taccasteroside A (**10**) was obtained as an amorphous solid. Acid hydrolysis of **10** with 1 M HCl in dixane/H2O gave D-glucose and a C28-sterol as the aglycone (**10a**). The structure of **10a**, except for the absolute configurations at C-24 and C-25, was identified as 3β-hydroxyer‐ gost-5-en-26-oic acid by analysis of its <sup>1</sup> H, 13C, and 2D NMR spectra. In order to determine the absolute configuration at C-25, **10a** was reduced with LiAlH4 to (24*R*,25*S*)-ergost-5 ene-3β,26-diol (**10b**). Then, **10b** was converted to the diastereomeric pairs of (*R*)-MTPA (**10a-R**) and (*S*)-MTPA (**10a-S**) esters with respect to the C-26 primary hydroxy group next to the

S6:

HO HO HO

S5:

S4:

H NMR coupling patterns of the H2-26 protons

O HO

O HO

O HO HO <sup>O</sup> HO

HO <sup>O</sup> HO

> HO HO HO

HO HO O

H OR2

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71

O HO

O HO O O

O O

O HO <sup>O</sup> HO O

O OH

HO HO HO

HO <sup>O</sup> HO

O HO

O HO

HO HO HO

HO HO HO

HO HO HO

6

C-25 chiral center and the differences in the 1

O HO

> HO <sup>O</sup> HO

HO HO O

HO HO O

O HO

O HO

HO HO O

<sup>O</sup> <sup>O</sup> <sup>H</sup>

O HO

> O O

> > O O

O O

1''

H O OR

R1O

H H H H

R1 Glc Glc H Glc Glc Glc Glc S6

R2 S1 S2 S2 S4 S3 S5 S6 S4

Phytochemicals of the Chinese Herbal Medicine *Tacca chantrieri* Rhizomes

26

<sup>25</sup> <sup>24</sup>

HO <sup>O</sup> HO

> O HO

1''''' 1'''' 1'''

1''''''' 1''''''

H H H

> **21** R S4

O HO

> HO <sup>O</sup> HO

O HO

> HO HO HO

O HO

RO

O

O HO

1' 3

HO HO HO H H H H

> **10 11 12**

R S1 S2 S3

H

O HO

HO

O HO

> HO HO HO

O HO HO <sup>O</sup> HO

HO <sup>O</sup> HO

HO <sup>O</sup> HO

O HO

HO HO HO

HO HO HO

Glc: <sup>O</sup>

HO HO HO

HO HO HO

HO HO HO

Figure 4

**Figure 4.** Structures of **10–21**

S2:

S1:

S3:

Phytochemicals of the Chinese Herbal Medicine *Tacca chantrieri* Rhizomes http://dx.doi.org/10.5772/53668 71

Figure 4 **Figure 4.** Structures of **10–21**

structure of **1** was shown to be (3*R*,5*R*)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydrox‐ yphenyl)heptane. In the same way, the structure of **2** was elucidated as (3*R*,5*R*)-3,5-dihy‐

Compounds **3**–**9** are diarylheptanoid monoglucosides. Enzymatic hydrolysis of **3**–**9** with naringinase gave the diarylheptanoid derivatives and D-glucose. Identification of D-glucose, including its absolute configuration, was carried out by direct HPLC analysis of the hydro‐ lysates. In the HMBC spectra, a long-range correlation was observed from each anomeric

Diarylheptanoids are known to occur in only a limited number species of higher plants be‐ longing to the families Zingiberaceae [7–10], Betulaceae [11], and Aceraceae [12]. This is the

Compounds **10**–**21** are new ergostane glucosides (Fig. 4) [13–15]. Taccasterosides A–C (**10**– **12**) are novel bisdesmosideic oligoglucosides of (24*R*,25*S*)-3β-hydroxyergost-5-ene-26-oic acid (**10a**), whereas **13**–**20** are those of (24*S*,25*R*)-ergost-5-ene-3β,26-diol (**10b**). Compound **21** is an ergostane glucoside with the six-membered lactone on the side chain of the aglycone.

Taccasteroside A (**10**) was obtained as an amorphous solid. Acid hydrolysis of **10** with 1 M HCl in dixane/H2O gave D-glucose and a C28-sterol as the aglycone (**10a**). The structure of **10a**, except for the absolute configurations at C-24 and C-25, was identified as 3β-hydroxyer‐

H, 13C, and 2D NMR spectra. In order to determine

droxy-1,7-bis(3,4-dihydroxyphenyl)heptane.

70 Alternative Medicine

**Figure 3.** Determination of the absolute configurations at C-3 and C-5 of **1**

proton to the C-3 carbon in **3** and **5**–**9**, and to the C-5 carbon in **4**.

**4. Ergostane glucosides**

gost-5-en-26-oic acid by analysis of its <sup>1</sup>

first isolation of diarylheptanoids from a plant of the family Taccaceae.

the absolute configuration at C-25, **10a** was reduced with LiAlH4 to (24*R*,25*S*)-ergost-5 ene-3β,26-diol (**10b**). Then, **10b** was converted to the diastereomeric pairs of (*R*)-MTPA (**10a-R**) and (*S*)-MTPA (**10a-S**) esters with respect to the C-26 primary hydroxy group next to the C-25 chiral center and the differences in the 1 H NMR coupling patterns of the H2-26 protons

6

were inspected. The H2-26 protons of **10a-R** were observed as a doublet-like signal at δ 4.20 (*J* = 6.3 Hz), whereas those of **10a-S** were observed as a doublet of doublets at δ 4.30 (*J* = 10.8, 6.6 Hz) and 4.09 (*J* = 10.8, 7.2 Hz). Application of these spectral data to the empirical rule reported by Yasuhara et al. [17] allowed us to confirm that the C-25 configuration was exclu‐ sively *S*. The configuration of C-24 position and other steroidal skeleton were established by the following chemical transformations. Compound **10b** was treated with *p*-toluenesulfonyl chloride to give the 26-*O*-tosylate of **10b** (**10b-T**), which was then reduced with LiAlH4, af‐ fording (24*R*)-ergost-5-ene-3β-ol, that is, campesterol. The structure of **10a** was determined as (24*R*,25*S*)-3β-hydroxyergost-5-en-26-oic acid (Fig. 5).

Reagents and conditions: a, LiAlH4, THF, 0 ºC, 5 h; b, (*R*)-MTPA or (*S*)-MTPA, EDC·HCl, 4-DMAP, CH2Cl2, r.t.,12 h; c, *p*-TsCl, pyridine, r.t., 6 h; d, LiAlH4, THF, 0 ºC, 5 h

#### Figure 5 **Figure 5.** Chemical transformations of **10a**

The severe overlap of the proton signals for the sugar moieties in **10** excluded the possibility of complete assignment in a straightforward way by conventional 2D NMR methods such as the 1 H-1 H COSY, 2D TOCSY, and HSQC spectroscopy. The exact structures of the sugar moieties and their linkage positions of the aglycone were resolved by detailed analysis of the 1D TOCSY and 2D NMR spectra. The <sup>1</sup> H NMR subspectra of individual monosaccharide units were ob‐ tained by using selective irradiation of easily identifiable anomeric proton signals, as well as ir‐ radiation of other nonoverlapping proton signals in a series of 1D TOCSY experiments [17–19]. Subsequent analysis of the <sup>1</sup> H-1 H COSY spectrum resulted in the sequential assignment of all the proton resonances due to the seven glucosyl units, including identification of their multip‐ let patterns and coupling constants. The HSQC and HSQC-TOCSY spectra correlated the pro‐ ton resonances to those of the corresponding one-bond coupled carbons, leading to unambiguous assignments of the carbon shifts. The carbon chemical shifts thus assigned were compared with those of the reference methyl α-D- and β-D-glucosides [20], taking into account the known effects of *O*-glycosylation shifts. The comparison indicated that **10** contained three terminal β-D-glucopyranosyl moieties (Glc′, Glc′′′′, Glc′′′′′′′), three C-4 substituted β-D-glu‐ copyranosyl moieties (Glc′′′, Glc′′′′, Gl*p*′′′′′′), and a C-2 and C-6 disubstituted β-D-glucopyra‐ nosyl moiety (Glc′′). The β-orientations of the anomeric centers of all the glucosyl moieties were supported by the relatively large *J* values of their anomeric protons (7.7–8.4 Hz).

7

In the HMBC spectrum, the anomeric proton of the terminal glucosyl unit (Glc′) at δ 5.07 exhibited a long-range correlation with C-3 of the aglycone at δ 78.2, indicating that one glu‐ cosyl unit was attached to the C-3 hydroxy group of the aglycone. Consequently, an oligo‐ glucoside composed of six glucosyl units was presumed to be linkage with the C-26 carboxy group of the aglycone. Further HMBC correlations from H-1 of Glc′′ at δ 6.30 to C-26 of the aglycone at δ 175.2, H-1 of Glc′′′ at δ 5.20 to C-2 of Glc′′ at δ 82.9, H-1 of Glc′′′′′′′ at δ 5.17 to C-4 of Glc′′′′′′ at δ 80.9, H-1 of Glc′′′′ at δ 5.16 to C-4 of Glc′′′ at δ 81.5, H-1 of Glc′′′′′ at δ 5.13 to C-4 of Glc′′′′ at δ 80.9, and H-1 of Glc′′′′′′ at δ 4.93 to C-6 of Glc′′ at δ 69.2 confirmed the hexaglucoside sequence as Glc-(1→4)-Glc-(1→4)-Glc-(1→2)-[Glc-(1→4)-Glc-(1→6)]-Glc, which was attached to C-26 of the aglycone (Fig. 6). Accordingly, the structure of **10** was elu‐ cidated as (24*R*,25*S*)-3β-[(β-D-glucopyranosyl)oxy]-ergost-5-en-26-oic acid *O*-β-D-glucopyr‐

anosyl-(1→4)-*O*-β-D-glucopyranosyl-(1→4)-*O*-β-D-glucopyranosyl-(1→2)-*O*-[*O*-β-D-

8

glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl ester.

In the same way, the structures of **11**–**20** were elucidated as shown in Fig. 4.

C O

H1''''''' H1''''''

H 5.17 H 4.93

H

C O

HO

H1''''' H1'''' H1'''

H 5.13 H 5.16 H 5.20

H H

OH

C 80.9 C 81.5

OH

HO <sup>O</sup>

C 80.9

HO <sup>O</sup>

O

HO

O

HO

HO HO

HO

HO HO HO

HO

O C

Phytochemicals of the Chinese Herbal Medicine *Tacca chantrieri* Rhizomes

H

O

C 175.2

H 6.30

C26 H

O

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73

HO HO

C 69.2

HO <sup>O</sup>

C O

C 82.9

HO

OH

H H

<sup>C</sup> <sup>O</sup>

O

H1''

O

C 78.2

O

HO

H1'

H 5.07

C3 H

Figure 6

HO HO

HO

**Figure 6.** HMBC correlations of the sugar moieties of **10**

8

In the HMBC spectrum, the anomeric proton of the terminal glucosyl unit (Glc′) at δ 5.07 exhibited a long-range correlation with C-3 of the aglycone at δ 78.2, indicating that one glu‐ cosyl unit was attached to the C-3 hydroxy group of the aglycone. Consequently, an oligo‐ glucoside composed of six glucosyl units was presumed to be linkage with the C-26 carboxy group of the aglycone. Further HMBC correlations from H-1 of Glc′′ at δ 6.30 to C-26 of the aglycone at δ 175.2, H-1 of Glc′′′ at δ 5.20 to C-2 of Glc′′ at δ 82.9, H-1 of Glc′′′′′′′ at δ 5.17 to C-4 of Glc′′′′′′ at δ 80.9, H-1 of Glc′′′′ at δ 5.16 to C-4 of Glc′′′ at δ 81.5, H-1 of Glc′′′′′ at δ 5.13 to C-4 of Glc′′′′ at δ 80.9, and H-1 of Glc′′′′′′ at δ 4.93 to C-6 of Glc′′ at δ 69.2 confirmed the hexaglucoside sequence as Glc-(1→4)-Glc-(1→4)-Glc-(1→2)-[Glc-(1→4)-Glc-(1→6)]-Glc, which was attached to C-26 of the aglycone (Fig. 6). Accordingly, the structure of **10** was elu‐ cidated as (24*R*,25*S*)-3β-[(β-D-glucopyranosyl)oxy]-ergost-5-en-26-oic acid *O*-β-D-glucopyr‐ anosyl-(1→4)-*O*-β-D-glucopyranosyl-(1→4)-*O*-β-D-glucopyranosyl-(1→2)-*O*-[*O*-β-Dglucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)]-β-D-glucopyranosyl ester.

In the same way, the structures of **11**–**20** were elucidated as shown in Fig. 4.

were inspected. The H2-26 protons of **10a-R** were observed as a doublet-like signal at δ 4.20 (*J* = 6.3 Hz), whereas those of **10a-S** were observed as a doublet of doublets at δ 4.30 (*J* = 10.8, 6.6 Hz) and 4.09 (*J* = 10.8, 7.2 Hz). Application of these spectral data to the empirical rule reported by Yasuhara et al. [17] allowed us to confirm that the C-25 configuration was exclu‐ sively *S*. The configuration of C-24 position and other steroidal skeleton were established by the following chemical transformations. Compound **10b** was treated with *p*-toluenesulfonyl chloride to give the 26-*O*-tosylate of **10b** (**10b-T**), which was then reduced with LiAlH4, af‐ fording (24*R*)-ergost-5-ene-3β-ol, that is, campesterol. The structure of **10a** was determined

d

O-(*S*)-MTPA H

OTs H **10b 10b-T** Campesterol

**10b-S**

H NMR subspectra of individual monosaccharide units were ob‐

H COSY spectrum resulted in the sequential assignment of all

as (24*R*,25*S*)-3β-hydroxyergost-5-en-26-oic acid (Fig. 5).

b

CH2Cl2, r.t.,12 h; c, *p*-TsCl, pyridine, r.t., 6 h; d, LiAlH4, THF, 0 ºC, 5 h

H-1

**10b-R**

OH H

c

Reagents and conditions: a, LiAlH4, THF, 0 ºC, 5 h; b, (*R*)-MTPA or (*S*)-MTPA, EDC·HCl, 4-DMAP,

The severe overlap of the proton signals for the sugar moieties in **10** excluded the possibility of complete assignment in a straightforward way by conventional 2D NMR methods such as the

tained by using selective irradiation of easily identifiable anomeric proton signals, as well as ir‐ radiation of other nonoverlapping proton signals in a series of 1D TOCSY experiments [17–19].

the proton resonances due to the seven glucosyl units, including identification of their multip‐ let patterns and coupling constants. The HSQC and HSQC-TOCSY spectra correlated the pro‐ ton resonances to those of the corresponding one-bond coupled carbons, leading to unambiguous assignments of the carbon shifts. The carbon chemical shifts thus assigned were compared with those of the reference methyl α-D- and β-D-glucosides [20], taking into account the known effects of *O*-glycosylation shifts. The comparison indicated that **10** contained three terminal β-D-glucopyranosyl moieties (Glc′, Glc′′′′, Glc′′′′′′′), three C-4 substituted β-D-glu‐ copyranosyl moieties (Glc′′′, Glc′′′′, Gl*p*′′′′′′), and a C-2 and C-6 disubstituted β-D-glucopyra‐ nosyl moiety (Glc′′). The β-orientations of the anomeric centers of all the glucosyl moieties

were supported by the relatively large *J* values of their anomeric protons (7.7–8.4 Hz).

H COSY, 2D TOCSY, and HSQC spectroscopy. The exact structures of the sugar moieties and their linkage positions of the aglycone were resolved by detailed analysis of the 1D TOCSY

+

O-(*R*)-MTPA H

a

O OH H

**10a**

24 25 26

72 Alternative Medicine

Figure 5

and 2D NMR spectra. The <sup>1</sup>

Subsequent analysis of the <sup>1</sup>

1 H-1

**Figure 5.** Chemical transformations of **10a**

Figure 6 **Figure 6.** HMBC correlations of the sugar moieties of **10**

7

Phytosterols and their monoglucosides such as campesterol, stigmasterol, and β-sitosterol, and their 3-*O*-glucoside, widely occur in the plant kingdom. However, **10**–**20** are the first representatives of oligoglucosides of a phytosterol derivative to have sugar moieties with a total of four to seven glucose units. The bisdesmosidic nature of these structures, except for **15**, is also notable.

## **5. Withanolide glucosides**

Compounds **22** and **23** are withanolide glucosides, named chantriolides A and B (Fig. 7) [21]. Chantriolides A and B were found to be minor components relative to the other secon‐ dary metabolites concomitantly isolated from *T. chantrieri*. However, it is notable that witha‐ nolides, which have been isolated almost exclusively from plants of the family Solanaceae previously [22, 23], have now been found in a species of the family Taccaceae in the study.

Figure 7 **Figure 7.** Structures of **22** and **23**

## **6. Other glycosides**

Spirostan glucosides (**24**–**28**), furostan glycosides (**29**–**32**), pseudofurostan glycosides (**33**– **37**), pregnane glycosides (**38**–**40**), and a phenolic glucoside (**41**) were also isolated from *T. chantrieri* rhizomes (Fig. 8) [15, 24–26].

The known naturally occurring 22,26-hydroxyfurostan glycosides exclusively exist in the form of glycoside, bearing a monosaccharide at C-26 [27]. The monosaccharide among the furostan glycosides reported thus far is limited to β-d-glucopyranose, except for one furo‐ stan glycoside from *Dracaena afromontana*, which has an α-l-rhamnopyranosyl group at C-26 [28]. Compound **31** is distinctive in carrying a diglucosyl group, *O*-glucosyl-(1→6)-glucosyl, in place of a monoglucosyl unit at C-26.

9

Compounds **33** is the corresponding Δ20(22)-furostan glycoside of **29**. This was confirmed by the fact that the peracetate (**33a**) of **33** agreed with the product (**29a**) obtained by treatment of **29** with Ac2O in pyridine at 110 °C for 2.5 h, during which dehydration at C-20 and C-22, as well as the introduction of an acetyl group to all the hydroxy groups of the sugar moiet‐

The structure of **38**, including the absolute configuration at C-25, was found by the follow‐ ing chemical conversion. When the C-20 and C-22 bond of 33a was oxidatively cleaved by treating it with CrO3 in AcOH at room temperature for 2 h, the resultant product was com‐

10

pletely consistent with the peracetyl derivative of **38 (38a)** (Fig. 9).

O

O

<sup>3</sup> R1O

R2 H OH H OH H

R2

24

Figure 8

**Figure 8.** Structures of **24–41**

RO

RO

R1O

H H H H

> **33 34 35**

H H H H

> **38 39**

R S1 S2 S3

HO HO HO

> R S1 S3

HO HO HO

H H H H

R1 S1 S1 S4 S3 S3

O

O

O O

<sup>O</sup> <sup>O</sup> HO

H

25

RO

H H H O

H

O

O

22 26

R2 H H Glc H

<sup>O</sup> <sup>O</sup> HO

MeO H

OH 22 23

R1 S1 S2 S1 S3

Phytochemicals of the Chinese Herbal Medicine *Tacca chantrieri* Rhizomes

http://dx.doi.org/10.5772/53668

75

O O OH

**40** R S1

20 22

<sup>O</sup> <sup>O</sup> HO

H

RO

H H H H

H H H H

HO HO R2O

> HO HO HO

> > R S1 S3

**36 37**

ies, occurred (Fig. 9).

10

Compounds **33** is the corresponding Δ20(22)-furostan glycoside of **29**. This was confirmed by the fact that the peracetate (**33a**) of **33** agreed with the product (**29a**) obtained by treatment of **29** with Ac2O in pyridine at 110 °C for 2.5 h, during which dehydration at C-20 and C-22, as well as the introduction of an acetyl group to all the hydroxy groups of the sugar moiet‐ ies, occurred (Fig. 9).

The structure of **38**, including the absolute configuration at C-25, was found by the follow‐ ing chemical conversion. When the C-20 and C-22 bond of 33a was oxidatively cleaved by treating it with CrO3 in AcOH at room temperature for 2 h, the resultant product was com‐ pletely consistent with the peracetyl derivative of **38 (38a)** (Fig. 9).

Figure 8 **Figure 8.** Structures of **24–41**

9

Phytosterols and their monoglucosides such as campesterol, stigmasterol, and β-sitosterol, and their 3-*O*-glucoside, widely occur in the plant kingdom. However, **10**–**20** are the first representatives of oligoglucosides of a phytosterol derivative to have sugar moieties with a total of four to seven glucose units. The bisdesmosidic nature of these structures, except for

Compounds **22** and **23** are withanolide glucosides, named chantriolides A and B (Fig. 7) [21]. Chantriolides A and B were found to be minor components relative to the other secon‐ dary metabolites concomitantly isolated from *T. chantrieri*. However, it is notable that witha‐ nolides, which have been isolated almost exclusively from plants of the family Solanaceae previously [22, 23], have now been found in a species of the family Taccaceae in the study.

O

O

OH HO OH

OH

R =O -H, -OH

O O H R

**22 23**

Spirostan glucosides (**24**–**28**), furostan glycosides (**29**–**32**), pseudofurostan glycosides (**33**– **37**), pregnane glycosides (**38**–**40**), and a phenolic glucoside (**41**) were also isolated from *T.*

The known naturally occurring 22,26-hydroxyfurostan glycosides exclusively exist in the form of glycoside, bearing a monosaccharide at C-26 [27]. The monosaccharide among the furostan glycosides reported thus far is limited to β-d-glucopyranose, except for one furo‐ stan glycoside from *Dracaena afromontana*, which has an α-l-rhamnopyranosyl group at C-26 [28]. Compound **31** is distinctive in carrying a diglucosyl group, *O*-glucosyl-(1→6)-glucosyl,

**15**, is also notable.

74 Alternative Medicine

**5. Withanolide glucosides**

Figure 7

**Figure 7.** Structures of **22** and **23**

**6. Other glycosides**

*chantrieri* rhizomes (Fig. 8) [15, 24–26].

in place of a monoglucosyl unit at C-26.

H H

O

O

O H HO O

O

O

Me

Me

**Figure 8.** Continued.

Figure 8. Continued.

A few compounds related to **38** and **39** have been isolated [29-31]; however, their C-25 con‐ figuration is not clearly presented in all the reports. In this investigation, we unequivocally determined the C-25 configuration of **38** to be *S* by a chemical correlation method. Com‐ pounds **38** and **39** could be defined as pregnane glycosides rather than furostan glycosides.

11

Figure 9.

**7. Biological activity**

concentration of 10 μg/mL.

RO

H H H H

> **29a 32a**

AcO AcO AcO

**29 32**

O

R S1 S2 <sup>O</sup> <sup>O</sup> OAc

MeO H

Ac2O / pyridine

RO

AcO O AcO

O OAc AcO

S1: S2:

<sup>O</sup> <sup>O</sup> Me OAc

Ac2O / pyridine 110 ºC

AcO AcO AcO

**Figure 9.** Chemical correlations of the furostan glycosides

**7.1. Cytotoxic activity against HL-60 cells**

AcO Me

H H H H

**33a 35a**

O O

O OAc AcO

R S1 S2

The isolated compounds were evaluated for their cytotoxic activity against HL-60 human promyelocytic leukemia cells by a modified MTT assay method [32]. Diarylheptanoids (**1** and **2**), diarylheptanoid glucosides (**3**, **4**, **6**, and **7**), and spirostan glycosides (**24** and **28**) showed moderate cytotoxicity (IC50 1.8–6.4 μg/mL) against HL-60 cells. Compounds **5**, **8**–**23**, **25**–**27**, and **29**–**41** did not show apparent cytotoxic activity against HL-60 cells at a sample

**7.2. Cytotoxic activity and structure–activity relationships of diarylheptanoids and**

The diarylheptanoids and some derivatives, including **9b** prepared by treatment of **9** with CH2N2, were evaluated for their cytotoxic activities against HL-60 cells, HSC-2 human oral squamous carcinoma cells, and normal human gingival fibroblasts (HGF) (Table 1). The dia‐ rylheptanoids **1**, **2**, and **7a**, and the diarylheptanoid glucosides **3**, **4**, **6**, and **7**, each of which has three or four phenolic hydroxy groups, showed moderate cytotoxic activity against HL-60 cells with IC50 values ranging 1.8 to 6.4 μg/mL, while those possessing two phenolic hydroxy groups (**5**, **5a**, **8**, **8a**, **9**, and **9a**) did not exhibit apparent cytotoxic activity even at a sample concentration of 10 μg/mL. Notably, the diarylheptanoids whose phenolic hydroxy

**diarylheptanoid glucosides against HL-60 cells, HSC-2 cells, and HGF**

AcO AcO AcO

**33 35**

O

20 22

<sup>O</sup> <sup>O</sup> OAc

Ac2O / pyridine

H

RO

AcO O AcO

O OAc AcO

AcO Me

CrO3 / AcOH

Phytochemicals of the Chinese Herbal Medicine *Tacca chantrieri* Rhizomes

AcO Me

O O

O OAc AcO H H H H

**38a 39a**

R S1 S2

AcO AcO AcO

**38 39**

http://dx.doi.org/10.5772/53668

O

O OAc

O O

Ac2O / pyridine

O

H

77

12

Phytochemicals of the Chinese Herbal Medicine *Tacca chantrieri* Rhizomes http://dx.doi.org/10.5772/53668 77

**Figure 9.** Chemical correlations of the furostan glycosides

## **7. Biological activity**

Figure 9.

11

Figure 8. Continued.

**Figure 8.** Continued.

O O

HO <sup>O</sup> Me

S3:

S4:

O O

O OH HO

<sup>O</sup> <sup>O</sup> Me HO

O OH

HO O HO

O OH HO

HO O HO

O OH HO

HO Me

HO HO HO

MeO

O OH

**41**

A few compounds related to **38** and **39** have been isolated [29-31]; however, their C-25 con‐ figuration is not clearly presented in all the reports. In this investigation, we unequivocally determined the C-25 configuration of **38** to be *S* by a chemical correlation method. Com‐ pounds **38** and **39** could be defined as pregnane glycosides rather than furostan glycosides.

O COOH

O OH HO

> O O

O OH HO

O

HO HO O

HO O HO

O OH HO

<sup>O</sup> Me

<sup>O</sup> <sup>O</sup> Me HO

HO

HO HO HO

S1:

76 Alternative Medicine

S2:

HO HO HO

HO Me

HO O O

Me O

O OH HO

HO Me

MeO HO MeO

#### **7.1. Cytotoxic activity against HL-60 cells**

The isolated compounds were evaluated for their cytotoxic activity against HL-60 human promyelocytic leukemia cells by a modified MTT assay method [32]. Diarylheptanoids (**1** and **2**), diarylheptanoid glucosides (**3**, **4**, **6**, and **7**), and spirostan glycosides (**24** and **28**) showed moderate cytotoxicity (IC50 1.8–6.4 μg/mL) against HL-60 cells. Compounds **5**, **8**–**23**, **25**–**27**, and **29**–**41** did not show apparent cytotoxic activity against HL-60 cells at a sample concentration of 10 μg/mL.

#### **7.2. Cytotoxic activity and structure–activity relationships of diarylheptanoids and diarylheptanoid glucosides against HL-60 cells, HSC-2 cells, and HGF**

The diarylheptanoids and some derivatives, including **9b** prepared by treatment of **9** with CH2N2, were evaluated for their cytotoxic activities against HL-60 cells, HSC-2 human oral squamous carcinoma cells, and normal human gingival fibroblasts (HGF) (Table 1). The dia‐ rylheptanoids **1**, **2**, and **7a**, and the diarylheptanoid glucosides **3**, **4**, **6**, and **7**, each of which has three or four phenolic hydroxy groups, showed moderate cytotoxic activity against HL-60 cells with IC50 values ranging 1.8 to 6.4 μg/mL, while those possessing two phenolic hydroxy groups (**5**, **5a**, **8**, **8a**, **9**, and **9a**) did not exhibit apparent cytotoxic activity even at a sample concentration of 10 μg/mL. Notably, the diarylheptanoids whose phenolic hydroxy

12

groups were all masked with methyl groups (**1a**, **2a**, and **9b**) were also cytotoxic. These ob‐ servations suggest that the number of phenolic hydroxy groups contributes to the resultant cytotoxicity. Compounds **1a**, **2a**, and **9b** showed considerable cytotoxic activity against HSC-2 cells, whereas they had little effect on normal HGF.

**7.4. Panel screening in the Japanese Foundation for Cancer Research 39 cell line assay**

CAR-4 (GI50 39 μM), and stomach MKN-7 (GI50 34 μM) were relatively sensitive to **2**.

pounds may be possible leads for new anticancer drugs.

**8. Conclusion**

**Acknowledgements**

cells and HGF.

**Author details**

Akihito Yokosuka and Yoshihiro Mimaki

Diarylheptanoid **2** and spirostan glycosides **24**, which showed significant cytotoxic activity against HL-60 cells, were subjected to the Japanese Foundation for Cancer Research 39 cell line assay [33]. Subsequent evaluation of **2** and **24** showed that the mean concentration re‐ quired for achieving GI50 levels against the panel of cells were 87 μM and 1.8 μM, respective‐ ly. Although **2** and **24** exhibited no significant differential cellar sensitivity, some cell lines such as colon cancer HCT-116 (GI50 25 μM), ovarian cancer OVCAR-3 (GI50 36 μM), OV‐

Phytochemicals of the Chinese Herbal Medicine *Tacca chantrieri* Rhizomes

http://dx.doi.org/10.5772/53668

79

Our systematic chemical investigations of *T. chantrieri* rhizomes revealed that this plant con‐ tains a variety of secondary metabolites, namely, diarylheptanoids, diarylheptanoid gluco‐ sides, steroidal glycosides with the aglycone structures of ergostane, withanolide, spirostan, furostan, pseudofurostan, and pregnane, as well as a phenolic glucoside. Some diarylhepta‐ noids and steroidal glycosides showed cytotoxicity against human cancer cells. These com‐

On the other hand, a number of researchers have reported biological activities of diarylhep‐ tanoids and steroidal glycosides other than cytotoxicity. It has been reported that curcumi‐ noids, well-known diarylheptanoid derivatives, showed antioxidant [34, 35], antiinflammatory [35, 36], estrogenic [37, 38], and anticancer [39] effects. Steroidal glycosides have been shown to have antidiabetic [40, 41], antitumor [42], antitussive [43], antiherpes vi‐ rus [44], and platelet aggregation inhibitory [45] activities. *T. chantrieri* rhizomes could be

We are grateful to Dr. Hiroshi Sakagami for evaluating the cytotoxic activities against HSC-2

Tokyo University of Pharmacy and Life Sciences, School of Pharmacy, Tokyo, Japan

applied to treating a wide variety of ailments as an alternative herbal medicine.


a Key: HL-60 (human promyelocytic leukemia cells); HSC-2 (human oral squamous carcinoma cells); and HGF (normal human gingival fibroblasts). bnot determined.

**Table 1.** Cytotoxic activities of compounds **1**-**9** and their derivates (**1a**, **4a**, **5a**, **7a**-**9a**, and **9b**), and etopside against HL-60 cells, HSC-2 cells, and HGFa

#### **7.3. Cytotoxic activity and structure–activity relationships of steroidal glycosides against HL-60 cells**

Spirostan glycosides (**24** and **28**) showed moderate cytotoxicity (IC50 1.9 and 1.8 μg/mL) against HL-60 cells. Compounds **25** and **27**, the corresponding C-24 hydroxy derivatives of **24** and **28**, and **26**, the analogue of **24** without the terminal rhamnosyl group linked to C-2 of the inner glucosyl residue, did not show any cytotoxic activity at a sample concentration of 10 μg/mL. Furostan glycosides (**29**–**32**), pseudofurostan glycosides (**33**–**37**), and pregnane glycosides (**38**–**40**) also did not show cytotoxic activity. These data suggest that the struc‐ tures of both the aglycone and sugar moieties contribute to the cytotoxicity.

#### **7.4. Panel screening in the Japanese Foundation for Cancer Research 39 cell line assay**

Diarylheptanoid **2** and spirostan glycosides **24**, which showed significant cytotoxic activity against HL-60 cells, were subjected to the Japanese Foundation for Cancer Research 39 cell line assay [33]. Subsequent evaluation of **2** and **24** showed that the mean concentration re‐ quired for achieving GI50 levels against the panel of cells were 87 μM and 1.8 μM, respective‐ ly. Although **2** and **24** exhibited no significant differential cellar sensitivity, some cell lines such as colon cancer HCT-116 (GI50 25 μM), ovarian cancer OVCAR-3 (GI50 36 μM), OV‐ CAR-4 (GI50 39 μM), and stomach MKN-7 (GI50 34 μM) were relatively sensitive to **2**.

## **8. Conclusion**

groups were all masked with methyl groups (**1a**, **2a**, and **9b**) were also cytotoxic. These ob‐ servations suggest that the number of phenolic hydroxy groups contributes to the resultant cytotoxicity. Compounds **1a**, **2a**, and **9b** showed considerable cytotoxic activity against

Key: HL-60 (human promyelocytic leukemia cells); HSC-2 (human oral squamous carcinoma cells); and HGF (normal

**Table 1.** Cytotoxic activities of compounds **1**-**9** and their derivates (**1a**, **4a**, **5a**, **7a**-**9a**, and **9b**), and etopside against

**7.3. Cytotoxic activity and structure–activity relationships of steroidal glycosides against**

Spirostan glycosides (**24** and **28**) showed moderate cytotoxicity (IC50 1.9 and 1.8 μg/mL) against HL-60 cells. Compounds **25** and **27**, the corresponding C-24 hydroxy derivatives of **24** and **28**, and **26**, the analogue of **24** without the terminal rhamnosyl group linked to C-2 of the inner glucosyl residue, did not show any cytotoxic activity at a sample concentration of 10 μg/mL. Furostan glycosides (**29**–**32**), pseudofurostan glycosides (**33**–**37**), and pregnane glycosides (**38**–**40**) also did not show cytotoxic activity. These data suggest that the struc‐

tures of both the aglycone and sugar moieties contribute to the cytotoxicity.

HSC-2 cells, whereas they had little effect on normal HGF.

a

human gingival fibroblasts). bnot determined.

HL-60 cells, HSC-2 cells, and HGFa

**HL-60 cells**

78 Alternative Medicine

Our systematic chemical investigations of *T. chantrieri* rhizomes revealed that this plant con‐ tains a variety of secondary metabolites, namely, diarylheptanoids, diarylheptanoid gluco‐ sides, steroidal glycosides with the aglycone structures of ergostane, withanolide, spirostan, furostan, pseudofurostan, and pregnane, as well as a phenolic glucoside. Some diarylhepta‐ noids and steroidal glycosides showed cytotoxicity against human cancer cells. These com‐ pounds may be possible leads for new anticancer drugs.

On the other hand, a number of researchers have reported biological activities of diarylhep‐ tanoids and steroidal glycosides other than cytotoxicity. It has been reported that curcumi‐ noids, well-known diarylheptanoid derivatives, showed antioxidant [34, 35], antiinflammatory [35, 36], estrogenic [37, 38], and anticancer [39] effects. Steroidal glycosides have been shown to have antidiabetic [40, 41], antitumor [42], antitussive [43], antiherpes vi‐ rus [44], and platelet aggregation inhibitory [45] activities. *T. chantrieri* rhizomes could be applied to treating a wide variety of ailments as an alternative herbal medicine.

## **Acknowledgements**

We are grateful to Dr. Hiroshi Sakagami for evaluating the cytotoxic activities against HSC-2 cells and HGF.

## **Author details**

Akihito Yokosuka and Yoshihiro Mimaki

Tokyo University of Pharmacy and Life Sciences, School of Pharmacy, Tokyo, Japan

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**Chapter 5**

**Application of Saponin-Containing**

**Plants in Foods and Cosmetics**

Yukiyoshi Tamura, Masazumi Miyakoshi and

Additional information is available at the end of the chapter

Saponins are a class of natural products which are structurally constructed of aglycone (tri‐ terpene or steroid) and sugars (hexose and/or uronic acid). The name 'saponin' comes from soap as its containing plants agitated in water form soapy lather. Saponins are widely dis‐ tributed in many plants and are relatively widespread in our foodstuffs and herbal prepara‐ tions. Saponins traditionally used as a natural detergent. In addition to this physical property, plant-derived triterpenoid and steroidal saponins have historically received a number of industrial and commercial applications ranging from their use as sources of raw materials for the production of steroid hormones in the pharmaceutical industry, to their use as food additives and as ingredients in photographic emulsions, fire extinguishers and other industrial applications which take advantage of their generally non-ionic surfactant proper‐ ties [1-3]. They also exhibit a variety of biological activities, and have been investigated to‐ ward the development of new natural medicines and prove the efficacy of traditional herbal medicines [4]. Other interesting biological applications for various specific saponins include their uses as anti-inflammatory [5], hypocholesterolemic [6] and immune-stimulating [7]

As to the application of saponins to foods and cosmetics, it is indispensable that sufficient amounts of plant resources are available, and that the content of saponins must be high. Fur‐ thermore, a plant must have a long history of human use as foodstuffs or ingredients of cos‐

The saponins of Quillaja bark and licorice root are widely utilized in the world. The *Quillaja saponaria* (Rosaceae) tree has remained of special interest, because of its bark containing 9-10

and reproduction in any medium, provided the original work is properly cited.

© 2012 Tamura et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

whose properties are widely recognized and commercially utilized.

metics, and their safety should be officially guaranteed.

Masaji Yamamoto

**1. Introduction**

http://dx.doi.org/10.5772/53333

**Chapter 5**
