Isolation and Identification of a Novel Chlorophenol from a Cell Suspension Culture of Helichrysum aureonitens

proximately 600 species in Africa, of which 244 are growing in South Africa. Helichrysum species are often used to treat respiratory conditions and tuberculosis. Several Helichrysum species such as H. odoratissimum, H. melanacme and H. caespititium have been reported to have activity against Mycobacterium tuberculosis. The medicinal properties of this genus are mainly attributed to the presence of flavonoids, but the genus is rich in several other interesting compounds (coumarins, phenolic acids, antioxidant micronutrients, e.g. Cu, Mn, Zn) with proven medicinal characteristics. Polyacetylene compounds are present in many plants including the Helichrysum genus and display important biological activities. H. aureonitens SCH. Bip is a hairy perennial herb which grows mostly in the Kwazulu-Natal province of South Africa and is widely used in folk medicine. Previously 3,5,7-trihydroxyflavone (galangin) with antiviral, antifungal and antibacterial activity was isolated from H. aureonitens. In our preliminary experiments with the cell suspension cultures of H. aureonitens to optimize the conditions for the production of phenolic compounds e.g. galangin, it was found that compound 1 existed as major metabolite. In this article we report the isolation and identification of 1 from a cell suspension culture of H. aureonitens and its significance in the biosynthesis of other polyacetylenic compounds in the Helichrysum genus. The results of antitubercolusis activity and toxicity of a crude extract and the chloronated phenol isolated from H. aureonitens cell suspension cultures are also discussed.


Results and Discussion
Compound 1 was isolated as a semi-solid from the nonpolar part of the ethanolic extract of H. aureonitens cell suspension cultures.HR-ESI-MS (negative mode) gave 213.0181 (MϪ1) ϩ corresponding to a molecular formula of C 13 H 7 OCl. 1 1).Compound 1 has not been isolated previously.
The methyl ether derivative (2) of compound 1 was isolated from the roots of H. coriaceum 14) and the other two unusual chlorophenol acetylenes, helitenuin and helitenuone from H. tenuifolium, which are related to 1. 15) It is proposed that fatty acids are the precursors in the biosynthetic pathway of the isolated acetylenes. 16,17)Based on that, 1 is proposed to be an intermediate in the acetylene biosynthetic pathway and the substrate for both helitenuin and helitenuone.
Compound 1 was also detected in the aerial parts of intact plants and tissue cultured plants of H. aureonitens by LC-MS analysis, but at very low concentrations (data not shown).This indicates the higher induction of the polyacetylene biosynthesis enzyme under the cell suspension conditions.This response of the cells in the culture could have been driven by the exogenously applied plant growth regulators through upregulating the involved genes in 1 production biosynthetic pathway.Bohlmann et al. 14,15) proposed the presence of 1 in the roots of H. coriaceum and H. tenuifolium (which normally grow under darkness) and in our experiment 1 was produced in the dark grown cells of H. aureonitens suspension cultures.It might therefore be that dark conditions are necessary for the induction of 1, but more experimentation is needed to confirm this.This result was confirmed in another experiment in which 1 could not be detected after 72 h exposure of cell cultures to UV light (data not shown).
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanol extracts of cell suspension cultures against M. tuberculosis H37Rv was found to be 1000 mg/ml and 2000 mg/ml respectively.The antimycobacterial activity of the crude extract of H. aureonitens cell suspension cultures was found to be rather weak as compared to the other ethanol extract of the aerial parts of the plants whereas MIC observed against H37Rv was found to be 200 mg/ml (unpublished data) and 1 did not show antituberculosis activity at the highest concentration (200 mg/ml) tested.The MIC for the positive control "isoniazid" was found to be 0.2 mg/ml.
The anticancer activity of acetylenic compounds has already been reported. 10)Polyacetylenes found in ginseng 18) and other medicinal plants 19) have been reported to exhibit anticancer activity.There are also some reports on the anticancer activities of Helichrysum genus.The extracts from H. pallassi, H. armenium, H. plicatum have been reported to have a considerable inhibition on DNA topoisomerase I, which represent a major class of anticancer drugs. 20)The ethanol extract of cell suspension cultures and 1 were evaluated in vitro for their cytotoxicity against the monkey kidney Vero (Vero cells) and human prostate epithelial carcinoma (DU145) cell lines. 1 was about 8 times more toxic (IC 50 value, 1.51 mg/ml) than the crude ethanol extract (12.11 mg/ ml) on a Vero cell line.The crude extract and 1 showed similar toxicity on a prostate cancer cell line with IC 50 values of 3.52 and 2.14 mg/ml respectively.This indicates that other toxic compounds could be present in the extract.These results also showed that the anticancer activity of the crude extract and compound 1 was found to be significant and comparable with that of the positive control tested in the present study.The IC 50 values of the positive control "Zearalenone" were found to be 1.63 and 0.53 mg/ml against Vero and DU145 cell lines respectively.Compound 1 warrants further investigation for its potential as an anticancer drug.

Experimental
Establishment of Callus and Cell Suspension Cultures Callus tissues were induced from young leaves of H. aureonitens plants.The leaves were surface sterilized (70% ethanol for 30 s, 0.5% sodium hypochlorite for 30 min) and inoculated onto the culture medium with the inner surface in contact with the medium.These explants grew on Murashige and Skoog (MS) medium 21) supplemented with 3% sucrose (w/v), 5.37 mM naphtaleneacetic acid (NAA), 0.85 mM 6-benzyladenine (BA) on 0.30% agar; the pH was adjusted to 5.70 prior to autoclaving for 20 min at 1.05 kg/cm 2 and 121 °C.The cultures were incubated in a growth chamber at 25 °C in the darkness.After 4-6 weeks, the callus tissues formed, mainly from the surface of leaves and were then subcultured to obtain friable callus.Cell suspension cultures were established from selected H. aureonitens CALLI.Callus pieces (0.1 g) were inoculated into 100 ml flasks containing 20 ml MS medium supplemented with 3% sucrose, and similar concentrations of NAA and BA to the callus induction experiment, and incubated in the dark at 25 °C on a rotary shaker at 120 rpm.Cell cultures were passed through a stainless steel sieve (1 mm 2 ) every 2 weeks for 6 months in order to obtain a fine cell suspension culture.For the cell suspension subculturing, 0.20 g of cells (fresh mass) were inocu-lated into 20 ml of the above-mentioned liquid medium in 100 ml flasks.
Extraction and Isolation of 1 Cells were filtrated from the medium and around 100 g fresh cells were used for extraction.The extraction was carried out with 95% EtOH followed by Sephadex LH-20 column chromatography, using ethanol as eluent.The fractions that contained the impure 1, were further purified on another Sephadex column under the same conditions to yield 20 mg pure compound.