Taxonomic reconsideration of Disporum luzoniense ( Liliaceae s . l . ) using flavonoid characters

Phytochemical characters of the plants that have been treated as Disporum luzoniense (Merrill & Merritt 1910) or Disporopsis fuscopicta (Jessop 1979) in the Philippines were compared with those of Disporum kawakamii and Disporopsis fuscopicta in Taiwan. The present phytochemical study revealed that Disporum kawakamii had luteolin, apigenin and chrysoeriol as free state, while these flavonoids were not detected in the Philippine plant and Disporopsis fuscopicta from Taiwan. Moreover, flavone O-glycosides were isolated from Disporum kawakamii, while flavone C-glycoside was isolated from the Philippine plant and Disporopsis fuscopicta from Taiwan. In conclusion, the present study suggests that the Philippine plant is chemotaxonomically related to Disporopsis and this agrees with the taxonomic treatment of Jessop (1979).

Flavonoid compounds based on a fifteen-carbon skeleton consist of two phenyl rings (A-and B-rings) connected by a threecarbon bridge (C-ring).In most cases, the flavonoids are present as glycosides in flowers, leaves, stems or roots.They were divided into several classes, i.e., flavones, flavonols, flavanones, anthocyanins, etc. Numerous kinds of flavonoids have been found in plants by the combination of additional hydroxyl, methoxyl, methyl and/or glycosyl groups.At the present, more than 7 000 kinds of flavonoids have been reported as natural products (Andersen & Markham 2006).These flavonoids have frequently been used as chemotaxonomic markers (e.g.Iwashina et al. 1995, Yamazaki et al. 2007).
The aim of the present study is to assess the taxonomic status of the plant previously labelled as Disporum luzoniense from the Philippines by comparing the phytochemical characters with morphological and cytological observation and to compare this plant with specimens labelled as Disporum and Disporopsis from Taiwan.

Plant materials
Plant materials from the experimental greenhouse of Tsukuba Botanical Garden, National Museum of Nature and Science were used for morphological, phytochemical and cytological study.Voucher specimens are deposited in the herbarium of the National Museum of Nature and Science (TNS).

Morphological characters
The following morphological characters of the specimens were observed in this study: presence/absence of corona inside of perianth and presence/absence of rhizomes.
One of two plants collected from Taiwan was morphologically identified as Disporum kawakamii Hayata in having an umbel type of inflorescence with few flowers (Fig. 1a), no corona inside the perianth and very thin or no rhizomes at all (Fig. 1d, Ying 2000); and another was identified as Disporopsis fuscopicta Hance var.arisanensis (Hayata) S.S.Ying in having an axillary inflorescence (Fig. 1c), well-developed rhizomes (Fig. 1f), and presence of a corona inside the perianth (Fig. 2, Ying 2000).
The floral and rhizome morphologies of the plant from the Philippines were similar to those of Disporopsis fuscopicta var.arisanensis, and were also consistent with the description of Disporum luzoniense.

Chromosome study
Root tips were harvested, pretreated in 2 mM 8-hydroxyquinoline at 20 °C for 2h, and fixed in acetic ethanol (1 : 3) at 4 °C for 2h.The fixed root tips were macerated in 2 : 1 mixture of 1N hydrochloric acid and 45 % acetic acid at 60 °C for 10 seconds.Somatic chromosomes at mitotic metaphase were stained in 2 % aceto-orcein at 20 °C for 2h, and spread by the standard squash method.
Abstract Phytochemical characters of the plants that have been treated as Disporum luzoniense (Merrill & Merritt 1910) or Disporopsis fuscopicta (Jessop 1979) in the Philippines were compared with those of Disporum kawakamii and Disporopsis fuscopicta in Taiwan.The present phytochemical study revealed that Disporum kawakamii had luteolin, apigenin and chrysoeriol as free state, while these flavonoids were not detected in the Philippine plant and Disporopsis fuscopicta from Taiwan.Moreover, flavone O-glycosides were isolated from Disporum kawakamii, while flavone C-glycoside was isolated from the Philippine plant and Disporopsis fuscopicta from Taiwan.In conclusion, the present study suggests that the Philippine plant is chemotaxonomically related to Disporopsis and this agrees with the taxonomic treatment of Jessop (1979).

Identification of flavonoids
The isolated compounds were identified by UV spectral survey according to Mabry et al. (1970), LC-MS, acid hydrolysis (in 12 % aq.HCl, 100 °C, 30 min), and direct TLC and HPLC comparisons with authentic specimens.TLC, UV, LC-MS and HPLC data of the isolated compounds were as follows. Luteolin
Previously Kumar & Brandham (1980) detected the chromosome number of 2n = 40 for a plant collected from the type locality of Disporum luzoniense, and then cytotaxonomically supported Jessop (1979).Also the present cytological investigation upholds Jessop's taxonomic treatment (1979) and also agrees with the findings of Kumar & Brandham (1980).

c b a
Furthermore two minor flavonoids were inferred as apigenin (2) and chrysoeriol (3) by HPLC comparisons with authentic samples, but the two minor flavonoids could not be completely identified for lack of enough samples.
On the other hand, only one flavone C-glycoside (8) was isolated from the Philippine plant and Disporopsis fuscopicta var.arisanensis in Taiwan (Table 2), but any flavone O-glycosides and flavone aglycones were not isolated from the two plants differing from that of Disporum kawakamii.
The present phytochemical analysis reveals that the flavonoid composition of the plant labelled as Disporum luzoniense is completely consistent with Disporopsis fuscopicta var.arisanensis but distinctly different from those of Disporum kawakamii and the three Disporum species reported by Williams et al. (1993).In conclusion, the phytochemical study suggests that the Philippine plant that was previously identified as Disporum luzoniense is actually related to Disporopsis.Our phytochemical study result agrees with the taxonomic study of Jessop (1979) and Kumar & Brandham (1980).

Table 1
Comparison of morphological and cytological characters of plant materials studied.