No abstract

This research describes the identification of three new high molecular weight polysaccharide preparations isolated from food-grade microalgae that are potent activators of human monocytes/macrophages: "Immulina" from Spirulina platensis, "Immunon" from Aphanizomenon flos-aquae, and "Immurella" from Chlorella pyrenoidosa. These polysaccharides are structurally complex and have estimated molecular weights above ten million daltons. All three polysaccharides are highly water soluble and comprise between 0.5 % and 2.0 % of microalgal dry weight. Immunostimulatory activity was measured using a transcription factor-based bioassay for nuclear factor kappa B (NF-kappa B) activation in THP-1 human monocytes/macrophages. Using this system the EC(50) values for these microalgal polysaccharides are between 20 and 110 ng/ml (about 10pM). THP-1 activation was confirmed by measuring immune cytokine mRNA induction using reverse transcriptase-polymerase chain reaction (RT-PCR). Each polysaccharide substantially increased mRNA levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). These polysaccharides are between one hundred and one thousand times more active for in vitro monocyte activation than polysaccharide preparations that are currently used clinically for cancer immunotherapy.

Our continuing effort in antifungal natural product discovery has led to the identification of five 6-acetylenic acids with chain lengths from C 16 to C 20 : 6-hexadecynoic acid (compound 1), 6-heptadecynoic acid (compound 2), 6-octadecynoic acid (compound 3), 6-nonadecynoic acid (compound 4), and 6-icosynoic acid (compound 5) from the plant Sommera sabiceoides.

(+)-Deoxoartemisinin (2), a new and more active antimalarial agent, was successfully prepared from artemisinin in one step using NaBH4 and BF3.Et2O in THF. (-)-Deoxodeoxyartemisinin (5), a potential metabolite of deoxoartemisinin, was also prepared either from 2 or from artemisinic acid. 2 shows 8-fold increased antimalarial activity in vitro against chloroquine-resistant malaria as compared to artemisinin (1). Compound 2 possesses superior in vivo antimalarial activity to 1.

Artemisinin (qinghaosu), a sesquiterpenoid lactone peroxide constituent of annual wormwood (Artemisia annuaL. # ARTAN) that is used as an antimalarial drug, was tested for phytotoxic properties. It inhibited germination of lettuce (Lactuca sativaL.) and annual wormwood, and growth of roots and shoots of lettuce, redroot pigweed (Amaranthus retroflexusL. # AMARE), pitted morningglory (Ipomoea lacunosaL. # IPOLA), annual wormwood, and common purslane (Portulaca oleraceaL. # POROL) was inhibited at 33 μM. No effects of 33 μM artemisinin were detected on growth of velvetleaf (Abutilon theophrastiMedik. # ABUTH) and grain sorghum [Sorghum bicolor(L.) Moench.]. Chlorophyll content was not affected in lettuce, and chlorosis was not observed in any species tested. The probable biosynthetic precursors of artemisinin, arteannuin B and qinghao acid, had no effect on growth or chlorophyll content of lettuce; however, they inhibited lettuce seed germination. Artemisinin and cinmethylin {exo-1-methyl-4-(1-methylethyl)-2-[(2-methylphenyl)methoxy]-7-oxabicyclo [2.2.1] heptane} were equally effective in reducing growth of lettuce; however, cinmethylin had no effect on germination. Respiration of lettuce roots or cotyledons was not inhibited by artemisinin. Artemisinin only marginally increased the mitotic index of lettuce root tips at 33 μM. At the ultrastructural level, however, chromosomes were less condensed during mitosis in artemisinin-treated than control meristematic cells. The growth-inhibiting ability of artemisinin could not be reduced by feeding the plants with hydrolyzed protein or treatment with putrescine. Artemisinin is a selective phytotoxin that reduces growth by a mechanism other than mitotic disruption or inhibition of protein synthesis.

Fatty acid synthase (FAS) has been identified as a potential antifungal target. FAS prepared from Saccharomyces cerevisiae was employed for bioactivity-guided fractionation of Chlorophora tinctoria,Paspalum conjugatum, Symphonia globulifera, Buchenavia parviflora, and Miconia pilgeriana. Thirteen compounds (1-13), including three new natural products (1, 4, 12), were isolated and their structures identified by spectroscopic interpretation. They represented five chemotypes, namely, isoflavones, flavones, biflavonoids, hydrolyzable tannin-related derivatives, and triterpenoids. 3'-Formylgenistein (1) and ellagic acid 4-O-alpha-l-rhamnopyranoside (9) were the most potent compounds against FAS, with IC(50) values of 2.3 and 7.5 microg/mL, respectively. Furthermore, 43 (14-56) analogues of the five chemotypes from our natural product repository and commercial sources were tested for their FAS inhibitory activity. Structure-activity relationships for some chemotypes were investigated. All these compounds were further evaluated for antifungal activity against Candida albicans and Cryptococcus neoformans. Although there were several antifungal compounds in the set, correlation between the FAS inhibitory activity and antifungal activity could not be defined.

Assay-guided fractionation of the ethanol extract of Tovomita krukovii resulted in the identification of four new xanthones (1 - 4) and ten known compounds (5 - 14). The structures of compounds 1 - 14 were determined by spectral data to be 3,5-dihydroxy-4-methoxyxanthone (1), 1,3,5,7-tetrahydroxy-8-isoprenylxanthone (2), 1,3,5-trihydroxy-8-isoprenylxanthone (3), 1,5,7-trihydroxy-8-isoprenylxanthone (4), 1,3,7-trihydroxy-2-isoprenylxanthone (5), 1,5-dihydroxyxanthone (6), 1,6-dihydroxy-5-methoxyxanthone (7), 1,3,5-trihydroxyxanthone (8), 1,3,6-trihydroxy-5-methoxyxanthone (9), 1,6-dihydroxy-3,5-dimethoxyxanthone (10), 1,3,7-trihydroxyxanthone (11), 3-geranyl-2,4,6-trihydroxybenzophenone (12), betulinic acid (13), and 3,4-dihydroxybenzoic acid (14). Compounds 2, 3, 12 and 13 showed inhibitory effects against Candida albicans secreted aspartic proteases (SAP) with IC50 values of 15 microg/ml, 25 microg/ml, 40 microg/ml, and 6.5 microg/ml, respectively, while the other compounds were inactive. In addition, compound 12 showed activity against C. albicans, C. neoformans, S. aureus and methicillin resistant S. aureus (MRS).

HMBC spectroscopy optimized for small couplings was employed to determine the four‐bond and two‐bond proton carbon correlations on the aromatic rings of ellagic acid derivatives. Complete 13C NMR assignments of 3′‐O‐methyl‐3,4‐methylenedioxyellagic acid 4′‐O‐β‐D‐glucopyranoside (1), 3,3′‐di‐O‐methylellagic acid 4′‐O‐β‐D‐xylopyranoside (2), 3,3′,4‐tri‐O‐methylellagic acid 4′‐O‐β‐D‐glucopyranoside (3) and ellagic acid (4) were achieved using this technique. This study indicates that optimization of the delay time in the HMBC spectrum is crucial in assigning the 13C NMR signals of phenolic compounds with highly oxygenated quaternary carbons. Copyright © 1999 John Wiley & Sons, Ltd.