OBJECTIVES

1. Identification of flavonoid profiles of Acacia koa (koa).
2. Determination of the effects of flavonoids of A. koa in disease resistance.
3. Characterization of neutraceutical properties of phyllodes of A. koa.

APPROACH

1. Identification of flavonoid profiles of A. koa. From at least five healthy koa trees, basal bark and phyllodes will be collected. Subsequent to collection, all samples will be freeze-dried and stored at -20C in order to preserve chemical composition for further preparations. Flavonoid contents will be analyzed using methods described by Johnson et al. (1997).

2. Determination of the flavonoids of A. koa in disease resistance. The experiments of this objective will be designed to identify and monitor biochemicals, which promote healthy growth and production of koa. A bioassay will be performed to identify chemical compounds that inhibit growth of Fusarium and Cylindrocladium (Calonectria) isolates from wilt-symptomatic koa. The crude extracts of juvenile sapwood and HPLC-purified fractions from both resistant and sensitive accessions of koa will be assayed for antifungal activity. A Fusarium isolate collection taken from individual koa trees showing symptoms of wilt disease will be used for this assay.

3. Characterization of neutraceutical properties of the phyllodes of A. koa. The experiments of this objective will be designed to identify biochemicals with potential human health benefits. Collections will be made from 3-5 year-old koa trees established as germplasms on the islands of Oahu, Maui and Hawaii. Mature green phyllodes will be collected. Subsequent to collection, all samples will be freeze-dried and stored at -20C in order to preserve chemical composition for further preparations. The crude extracts and total purified phenolics from mature green phyllodes will be assayed for antioxidant activities. To verify that koa phyllodes are safe for human consumption, standardized in vitro assays will be performed to determine toxic effects of crude phyllode extracts on human hepatic cell lines. HepG2 cell line, a human hepatocellular carcinoma cell line, will be purchased from American Tissue Culture Collections.

In a preliminary trial, we used HPLC protocols to differentiate phytochemical extractions from three koa heartwood samples: (i) light blond, (ii) medium brown, and (iii) dark red. In this trial, polyphenolic profiles from each of the koa wood samples were generated at 280 nm. We found that as wood color progresses from light to dark, the profile changes markedly. Two peaks appear consistently throughout the three profiles at 8.9 min and 12.0 min. However, they increase in peak height and area as wood color intensifies, with the 8.9 min peak doubling in peak height from light to medium, and by 9.2 fold, from light to dark. These are likely candidate marker peaks for potential wood color prediction. The remainder of the peaks observed in the light and medium koa extracts are in relatively trace amounts. In contrast, the profile of the dark koa extract shows 5 major peaks including 8.9 and 12.0 min, but also including major peaks at 16.2, 31.8 and 38.6 min. Consequently, the latter three peaks may prove to be specific to dark wood color. This preliminary trial demonstrates that our HPLC protocol is very useful in characterizing wood color in koa and should be useful for characterizing sapwood phytochemical profiles. In a follow up study, ten samples of koa were supplied by James Leary in the form of shavings from the softwood and heartwood portions of five koa branches representing 5 different wood color intensities. These samples were collected from native stands on the island of Kauai. The polyphenolic contents corresponding to wood color was not evident in these samples. There could be a number of reasons for this, including differences in the age of the wood samples this time compared with those samples analyzed last time, or differences in the wood curing/drying process. One exception is the peak eluting at 36 minutes. This peak generally appeared to increase with a darkening of wood color, and so may be a chemical marker in these samples. Sapwood samples did not exhibit any significant polyphenolic content when subjected to HPLC at 280nm (not shown). The same heartwood samples were subjected to HPLC analysis with UV detection set at 365nm, specific for flavonol compounds. From these results there is no discernible pattern relating flavonol content with wood color. Sapwood samples were also ana;yzed for polyphenolic content and flavonol content. These data are not shown. No discernible peaks were observed in the sapwood samples; either polyphenolic or flavonol. Conclusions: The results of this study show that relating polyphenolic content and flavonol content may not be as straightforward as first thought. There are a number of wood preparation methodologies that need standardizing in order to make this comparison more valid. The samples from the earlier study, where a correlation was easily demonstrable were much older than the samples seen here. It is also possible that polyphenolics and flavonols are not correlated to wood color at all. Other compound classes may be better markers.

Koa wilt is a serious epidemic infecting many of the native koa forests and plantations in Hawaii. Fusarium oxysporum f. sp. koae as the main causal organism. Infection by this pathogen causes a vascular wilt disease that can be lethal to both young seedlings and mature trees Several other Fusarium species (i.e. F. solani) have also been routinely isolated from diseased koa seedlings and trees as well and may also contribute to the disease etiology. Furthermore, other pathogenic fungi, such as Calonectria ilicicola (= C.crotalaria; anamorph = Cylindrocladium parasiticum) and C. indusiata (= C. thea, anamorph = Cylindrocladium theae) which cause collar root rot and shoot blight, may also contribute to koa wilt. Field trials using different koa families suggest that genetic disposition can confer resistance to this disease in nature. In order for the koa forest industry to survive, it will be necessary to select for resistant accessions to be used in plantations. Juvenile koa are most susceptible to mortality from Fusarium wilt (Anderson 2002). Thus resistant accessions may have anti-fungal compounds accumulated within the young sapwood. Most recently, our team has identified seed families that appear to confer disease resistance. For this next fiscal year we will be focusing our attention ongenerating new information related to this success.