Phytochemicals of the Chinese Herbal Medicine Tacca chantrieri Rhizomes

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.


Introduction
The family Taccaceae is composed of two genera, Tacca and Schizocapsa, and about 10 species, 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 characteristic components of the herb [3], but there has been only one report of the secondary metabolites 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.

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/H 2 O gradients,
Diarylheptanoids are known to occur in only a limited number species of higher plants belonging to the families Zingiberaceae [7][8][9][10], Betulaceae [11], and Aceraceae [12]. This is the first isolation of diarylheptanoids from a plant of the family Taccaceae.
Taccasteroside A (10) was obtained as an amorphous solid. Acid hydrolysis of 10 with 1 M HCl in dixane/H 2 O gave D-glucose and a C 28 -sterol as the aglycone (10a). The structure of 10a, except for the absolute configurations at C-24 and C-25, was identified as 3β-hydroxyergost-5-en-26-oic acid by analysis of its 1 H, 13 C, and 2D NMR spectra. In order to determine the absolute configuration at C-25, 10a was reduced with LiAlH 4 to (24R,25S)-ergost-5ene-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 H 2 -26 protons   . 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 exclusively 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 LiAlH 4 , affording (24R)-ergost-5-ene-3β-ol, that is, campesterol. The structure of 10a was determined as (24R,25S)-3β-hydroxyergost-5-en-26-oic acid (Fig. 5). 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 1 H NMR subspectra of individual monosaccharide units were obtained by using selective irradiation of easily identifiable anomeric proton signals, as well as irradiation of other nonoverlapping proton signals in a series of 1D TOCSY experiments [17][18][19]. Subsequent analysis of the 1 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 multiplet patterns and coupling constants. The HSQC and HSQC-TOCSY spectra correlated the proton 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-glucopyranosyl moieties (Glc′′′, Glc′′′′, Glp′′′′′′), and a C-2 and C-6 disubstituted β-D-glucopyranosyl 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).

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 secondary metabolites concomitantly isolated from T. chantrieri. However, it is notable that withanolides, 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.
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 furostan 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.

Alternative Medicine
Compounds 33 is the corresponding Δ 20 (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 Ac 2 O 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 moieties, occurred (Fig. 9).
The structure of 38, including the absolute configuration at C-25, was found by the following chemical conversion. When the C-20 and C-22 bond of 33a was oxidatively cleaved by treating it with CrO 3 in AcOH at room temperature for 2 h, the resultant product was completely consistent with the peracetyl derivative of 38 (38a) (Fig. 9).  A few compounds related to 38 and 39 have been isolated [29][30][31]; however, their C-25 configuration 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. Compounds 38 and 39 could be defined as pregnane glycosides rather than furostan glycosides. groups were all masked with methyl groups (1a, 2a, and 9b) were also cytotoxic. These observations 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. a Key: HL-60 (human promyelocytic leukemia cells); HSC-2 (human oral squamous carcinoma cells); and HGF (normal human gingival fibroblasts). b not 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 HGF a

Cytotoxic activity and structure-activity relationships of steroidal glycosides against HL-60 cells
Spirostan glycosides (24 and 28) showed moderate cytotoxicity (IC 50 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

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 required for achieving GI 50 levels against the panel of cells were 87 μM and 1.8 μM, respectively. Although 2 and 24 exhibited no significant differential cellar sensitivity, some cell lines such as colon cancer HCT-116 (GI 50 25 μM), ovarian cancer OVCAR-3 (GI 50 36 μM), OV-CAR-4 (GI 50 39 μM), and stomach MKN-7 (GI 50 34 μM) were relatively sensitive to 2.

Conclusion
Our systematic chemical investigations of T. chantrieri rhizomes revealed that this plant contains a variety of secondary metabolites, namely, diarylheptanoids, diarylheptanoid glucosides, steroidal glycosides with the aglycone structures of ergostane, withanolide, spirostan, furostan, pseudofurostan, and pregnane, as well as a phenolic glucoside. Some diarylheptanoids and steroidal glycosides showed cytotoxicity against human cancer cells. These compounds may be possible leads for new anticancer drugs.