Lessons from Exploring Chemical Space and Chemical Diversity of Propolis Components.

Propolis is a natural resinous material produced by bees and has been used in folk medicines since ancient times. Due to it possessing a broad spectrum of biological activities, it has gained significant scientific and commercial interest over the last two decades. As a result of searching 122 publications reported up to the end of 2019, we assembled a unique compound database consisting of 578 components isolated from both honey bee propolis and stingless bee propolis, and analyzed the chemical space and chemical diversity of these compounds. The results demonstrated that both honey bee propolis and stingless bee propolis are valuable sources for pharmaceutical and nutraceutical development.

Natural products isolated from Tetragonula carbonaria cerumen modulate free radical-scavenging and 5-lipoxygenase activities in vitro.

BACKGROUND: Propolis and cerumen are plant-derived products found in honeybees and stingless bees, respectively. Although propolis is an ancient folk medicine, the bioactivities of cerumen obtained from Australian native stingless bees (Tetragonula carbonaria) have not been widely studied. Therefore, we investigated selected anti-oxidant and anti-inflammatory properties of T. carbonaria cerumen. METHODS: A methanolic extract was prepared from the combined cerumen of 40 T. carbonaria hives, and HPLC was used to screen for chemical constituents that scavenged 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH). The ability of cerumen extracts to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) and to interfere with leukotriene B4 (LTB4) production in ionomycin-stimulated human neutrophils was also examined. RESULTS: The extract dose-dependently scavenged DPPH (EC50 = 27.0 ± 2.3 µg/mL); and inhibited the 5-lipoxygenase (5-LOX)-mediated oxidation of linoleic acid (IC50 = 67.1 ± 9.6 µg/mL). Pre-treatment of isolated human neutrophils with the methanolic cerumen extract additionally inhibited the ionomycin-stimulated production of LTB4 from these cells (IC50 = 13.3 ± 5.3 µg/mL). Following multi-solvent extraction, the free radical-scavenging and 5-LOX-inhibiting activities of the initial cerumen extract were retained in a polar, methanol-water extract, which contained gallic acid and a range of flavonone and phenolic natural products. CONCLUSIONS: The findings identify free radical scavenging activity, and interference by extracts of T. carbonaria cerumen in 5-LOX-LTB4 signaling. Further investigation is needed to determine whether the extracts will provide therapeutic benefits for medical conditions in which oxidative stress and inflammation are implicated, including cardiovascular disease and impaired wound healing.

Investigation of long chain omega-3 PUFAs on arterial blood pressure, vascular reactivity and survival in angiotensin II-infused Apolipoprotein E-knockout mice.

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease. Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) decrease inflammation and oxidative stress in an angiotensin II-infused apolipoprotein E-knockout (ApoE(-/-)) mouse model of AAA. This study investigated the effects of LC n-3 PUFAs on blood pressure and vascular reactivity in fourteen angiotensin II-infused ApoE(-/-) male mice. Blood pressure was obtained using a non-invasive tail cuff method and whole blood was collected by cardiac puncture. Vascular reactivity of the thoracic aorta was assessed using wire myography and activation of endothelial nitric oxide synthase (eNOS) was determined by immunohistochemistry. A high LC n-3 PUFA diet increased the omega-3 index and reduced the n-6 to n-3 PUFA ratio. At day 10 post-infusion with angiotensin II, there was no difference in systolic blood pressure or diastolic blood pressure in mice fed the high or low n-3 PUFA diets. The high LC n-3 PUFA diet resulted in a non-significant trend for delay in time to death from abdominal aortic rupture. Vascular reactivity and eNOS activation remained unchanged in mice fed the high compared to the low LC n-3 PUFA diet. This study argues against direct improvement in vascular reactivity in ApoE(-/-) mice that were supplemented with n-3 PUFA for 8 weeks prior to infusion with angiotensin II.

Chemical constituents of kino extract from Corymbia torelliana.

Seven flavanones were identified from kino exudate of Corymbia torelliana by spectroscopic and spectrometric methods including UV, 1D and 2D NMR and UPLC-HR-MS. The study identified seven molecules, namely 3,4',5,7-tetrahydroxyflavanone (1), 3',4',5,7-tetrahydroxyflavanone (2), 4',5,7-trihydroxyflavanone (3), 3,4',5-trihydroxy-7-methoxyflavanone (4), (+)-(2S)-4',5,7-trihydroxy-6-methylflavanone (5), 4',5,7-trihydroxy-6,8-dimethylflavanone (6) and 4',5-dihydroxy-7-methoxyflavanone (7) from this eucalypt species. This is the first report of these natural products from C. torelliana kino exudate.

Omega-3 fatty acids modulate Weibel-Palade body degranulation and actin cytoskeleton rearrangement in PMA-stimulated human umbilical vein endothelial cells.

Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF) in cytoplasmic Weibel-Palade bodies (WPBs). We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA), and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.

Dihydroxyacetone in the Floral Nectar of Ericomyrtus serpyllifolia (Turcz.) Rye (Myrtaceae) and Verticordia chrysantha Endl. (Myrtaceae) Demonstrates That This Precursor to Bioactive Honey Is Not Restricted to the Genus Leptospermum (Myrtaceae).

Ma̅nuka honey is known for its strong bioactivity, which arises from the autocatalytic conversion of 1,3-dihydroxyacetone (dihydroxyacetone, DHA) in the floral nectar of Leptospermum scoparium (Myrtaceae) to the non-peroxide antibacterial compound methylglyoxal during honey maturation. DHA is also a minor constituent of the nectar of several other Leptospermum species. This study used high-performance liquid chromatography to test whether DHA was present in the floral nectar of five species in other genera of the family Myrtaceae: Ericomyrtus serpyllifolia (Turcz.) Rye, Chamelaucium sp. Bendering (T.J. Alford 110), Kunzea pulchella (Lindl.) A.S. George, Verticordia chrysantha Endl., and Verticordia picta Endl. DHA was found in the floral nectar of two of the five species: E. serpyllifolia and V. chrysantha. The average amount of DHA detected was 0.08 and 0.64 µg per flower, respectively. These findings suggest that the accumulation of DHA in floral nectar is a shared trait among several genera within the family Myrtaceae. Consequently, non-peroxide-based bioactive honey may be sourced from floral nectar outside the genus Leptospermum.

Chemical Characterization, α-Glucosidase, α-Amylase and Lipase Inhibitory Properties of the Australian Honey Bee Propolis.

The use of functional foods and nutraceuticals as a complementary therapy for the prevention and management of type 2 diabetes and obesity has steadily increased over the past few decades. With the aim of exploring the therapeutic potentials of Australian propolis, this study reports the chemical and biological investigation of a propolis sample collected in the Queensland state of Australia which exhibited a potent activity in an in vitro α-glucosidase inhibitory screening. The chemical investigation of the propolis resulted in the identification of six known prenylated flavonoids including propolins C, D, F, G, H, and solophenol D. These compounds potently inhibited the α-glucosidase and two other enzymes associated with diabetes and obesity, α-amylase, and lipase on in vitro and in silico assays. These findings suggest that this propolis is a potential source for the development of a functional food to prevent type 2 diabetes and obesity. The chemical analysis revealed that this propolis possessed a chemical fingerprint relatively similar to the Pacific propolis found in Okinawa (South of Japan), Taiwan, and the Solomon Islands. This is the first time the Pacific propolis has been identified in Australia.

Sugar and dihydroxyacetone ratios in floral nectar suggest continuous exudation and reabsorption in Leptospermum polygalifolium Salisb.

Leptospermum polygalifolium Salisb. can accumulate high concentrations of dihydroxyacetone (DHA), precursor of the antimicrobial compound methylglyoxal found in honey obtained from floral nectar of Leptospermum spp. Floral nectar dynamics over flower lifespan depends on internal and external factors that invariably impact nectar quality. Current models to estimate nectar quality in Leptospermum spp. overlook time of day, daily (24 h), and long-term dynamics of nectar exudation and accumulation over flower lifespan. To explain the dynamics of nectar quality over flower lifespan, accumulated nectar from flowers of different ages was collected from two L. polygalifolium clones, and then re-collected 24 h later from the same flowers. High-Performance Liquid Chromatography was used to quantify DHA amount and total equivalents of glucose + fructose (Tsugar) per flower in the nectar. DHA and Tsugar amount per flower differed with flower age and between clones. In accumulated nectar, the amount of DHA and Tsugar per flower rose to a broad peak post-anthesis before decreasing. Immediately after peaking DHA declined more quickly than Tsugar in accumulated nectar due to a greater decrease in the exudation of DHA than for Tsugar. The DHA : Tsugar ratios in accumulated nectar and in nectar exuded over the next 24 h were similar and decreased with flower age, indicating that exudation and reabsorption occurred concomitantly across flower development. Hence there is a balance between exudation and reabsorption. A quantitative model suggested that flowers have the potential to exude more DHA and Tsugar than actually accumulated.

Quality assessment and chemical diversity of Australian propolis from Apis mellifera bees.

The propolis industry is well established in European, South American and East Asian countries. Within Australia, this industry is beginning to emerge with a few small-scale producers. To contribute to the development of the Australian propolis industry, the present study aimed to examine the quality and chemical diversity of propolis collected from various regions across Australia. The results of testing 158 samples indicated that Australian propolis had pure resin yielding from 2 to 81% by weight, total phenolic content and total flavonoid content in one gram of dry extract ranging from a few up to 181 mg of gallic acid equivalent and 145 mg of quercetin equivalent, respectively. Some Australian propolis showed more potent antioxidant activity than the well-known Brazilian green, Brazilian red, and Uruguayan and New Zealand poplar-type propolis in an in vitro DPPH assay. In addition, an HPLC-UV analysis resulted in the identification of 16 Australian propolis types which can be considered as high-grade propolis owing to their high total phenolic content. Chemometric analysis of their 1H NMR spectra revealed that propolis originating from the eastern and western coasts of Australia could be significantly discriminated based on their chemical composition.

Anti-Fibrotic Potential of Tomentosenol A, a Constituent of Cerumen from the Australian Native Stingless Bee, Tetragonula carbonaria.

Bioactivity-guided fractionation was used to isolate two compounds, tomentosenol A (1) and torellianone A (2), from a cerumen extract from Tetragonula carbonaria. The anti-fibrotic activity of these compounds was examined using human cultured neonatal foreskin fibroblasts (NFF) and immortalised keratinocytes (HaCaTs). Tomentosenol A (1), inhibited NFF and HaCaT cell proliferation and prevented NFF and HaCaT scratch wound repopulation at 12.5-25 µM concentrations. These inhibitory effects were associated with reduced cell viability, determined by tetrazolium dye (MTT) and sulforhodamine B (SRB) assays. Compound 1 further inhibited transforming growth factor-ß1 (TGF-ß1)-stimulated, NFF-myofibroblast differentiation and soluble collagen production; and was an effective scavenger of the model oxidant, 2,2-diphenyl-1-picrylhydrazyl (DPPH·), with an EC50 value of 44.7 ± 3.1 µM. These findings reveal significant anti-fibrotic potential for cerumen-derived tomentosenol A (1).

Use of kinetic data to model potential antioxidant activity: Radical scavenging capacity of Australian Eucalyptus honeys.

Antioxidant activity of honeys may be beneficial in wound healing processes by protecting cells against lipid oxidation. The DPPH assay assesses the efficacy of antioxidant molecules to reduce DPPH• to DPPHH. Studies determining EC50 are limited by single time-point determinations of antioxidant effect and can miss vital information about the rate of antioxidant response. Acquisition of kinetic data allows determination of the radical scavenging capacity (RSC) of honeys. The purpose of this study was to determine the RSC of 53 honeys from 16 species of Australian Eucalyptus trees and four samples of New Zealand manuka (Leptospermum scoparium) honey. Whereas honeys could not be differentiated based on EC50 values, significant differences were observed for RSC, supporting collection of kinetic data for honey analysis. The greatest RSC was observed for New Zealand manuka (4.6 ± 0.3 × 10-5 mg.mL-1.min-1), grey ironbark (E. paniculate; 3.4 ± 0.2 × 10-5 mg.mL-1.min-1) and river red gum honeys (E. camaldulensis; 3.2 ± 0.2 × 10-5 mg.mL-1.min-1).

Floral attraction and flower visitors of a subcanopy, tropical rainforest tree, Fontainea picrosperma.

Flowering plants in tropical rainforests rely heavily on pollen vectors for successful reproduction. Research into pollination systems in tropical rainforests is dominated by canopy species, while subcanopy plant-pollinator interactions remain under-represented. The microclimate beneath the rainforest canopy is characterized by low light levels and is markedly different from the canopy environment that receives more light energy.We studied the floral attractants and floral visitors of a dioecious, subcanopy tree, Fontainea picrosperma (Euphorbiaceae), in the Wet Tropics bioregion of northern Queensland, Australia.We found that wind pollination is rare and male and female flowers do not produce nectar. Female flowers are likely pollinated due to their perceptual similarity to pollen-offering male flowers. Female flowers had the same scent profile as male flowers, and floral scent was an important floral attractant that acted to regulate pollinator behavior. The two most abundant scent compounds present in the floral bouquet were benzyl alcohol and 4-oxoisophorone. These compounds are ubiquitous in nature and are known to attract a wide variety of insects. Both day-time and night-time pollinators contributed to successful pollen deposition on the stigma, and diurnal flower visitors were identified from several orders of insects including beetles, flies, predatory wasps, and thrips. Fontainea picrosperma is therefore likely to be pollinated by a diverse array of small insects.Synthesis. Our data indicate that F. picrosperma has a generalist, entomophilous pollination syndrome. The rainforest subcanopy is a distinctive environment characterized by low light levels, low or turbulent wind speeds, and relatively high humidity. Female flowers of F. picrosperma exhibit cost-saving strategies by not producing nectar and mimicking the smell of reward-offering male flowers. Insects opportunistically forage on or inhabit flowers, and pollination occurs from a pool of small insects with low energy requirements that are found beneath the rainforest canopy.

Relationships between Nut Size, Kernel Quality, Nutritional Composition and Levels of Outcrossing in Three Macadamia Cultivars.

Tree nuts play an important role in healthy diets, but their economic value and nutritional quality may be affected by their size and paternity. We assessed relationships between nut size and kernel recovery, the incidence of whole kernels, fatty acid composition and mineral nutrient concentrations in three macadamia cultivars, "Daddow", "816" and "A4". We determined to what extent differences in nut size and quality were the result of different levels of cross- or self-paternity. Small nuts of all cultivars had lower kernel recovery than large nuts, and small nuts provided lower incidence of whole kernels in "Daddow" and "A4". Small kernels had a lower relative abundance of the saturated fatty acid, palmitic acid, in all cultivars and higher relative abundance of the unsaturated fatty acid, oleic acid, in "Daddow" and "A4". Small kernels had higher concentrations of many essential nutrients such as nitrogen and calcium, although potassium concentrations were lower in small kernels. Most nuts arose from cross-pollination. Therefore, nut size and kernel quality were not related to different levels of cross- and self-paternity. Identified cross-paternity was 88%, 78% and 90%, and identified self-paternity was 3%, 2% and 0%, for "Daddow", "816" and "A4", respectively. Small macadamia kernels are at least as nutritious as large macadamia kernels. High levels of cross-paternity confirmed that many macadamia cultivars are predominantly outcrossing. Macadamia growers may need to closely inter-plant cultivars and manage beehives to maximise cross-pollination.

The occurrence of bioactive micromonosporae in aquatic habitats of the Sunshine Coast in Australia.

Screening strategies based on the ecological knowledge of antibiotic producing microorganisms and their roles in the natural environment are being increasingly employed in the search for novel antibiotic agents. Micromonosporae are common inhabitants of aquatic habitats and have proved to be a continuing source of novel bioactive compounds including antibacterial and antitumor agents. The ecological distribution and frequency of bioactive micromonosporae in Sunshine Coast region aquatic habitats were studied through a range of selective isolation procedures designed to negatively select against the isolation of unwanted microbial taxa commonly associated with marine environments. It was revealed that bioactive compound producing species of micromonosporae were present in the aquatic habitats of the Sunshine Coast region in Australia.

Dihydroxyacetone Production in the Nectar of Australian Leptospermum Is Species Dependent.

This study is the first large-scale survey of the presence of dihydroxyacetone (DHA) in the nectar of the Australian Leptospermum tree species. The work undertaken supports the growing global demand for bioactive Leptospermum honey. Leptospermum honey derived from L. scoparium in New Zealand, also referred to as Ma̅nuka honey, has a reputation for wound-healing and antimicrobial properties, which is based on its methylglyoxal (MGO) content. High-DHA nectar correlates to high-MGO honey, but not all Leptospermum species produce DHA in their nectar. This study investigates 55 of the 84 Leptospermum species native to Australia for their DHA-producing capability, with the DHA to total sugar (DHA:Tsugar) ratio of nectar samples determined by HPLC-PDA. DHA:Tsugar ranged from nondetectable in L. laevigatum, L. coriaceum, and L. trinervium to >16 000 mg/kg in L. speciosum and L. whitei. High-DHA Leptospermum species were identified for beekeepers to target for honey production and plantation development.

Antibacterial Properties of Flavonoids from Kino of the Eucalypt Tree, Corymbia torelliana.

Traditional medicine and ecological cues can both help to reveal bioactive natural compounds. Indigenous Australians have long used kino from trunks of the eucalypt tree, Corymbia citriodora, in traditional medicine. A closely related eucalypt, C. torelliana, produces a fruit resin with antimicrobial properties that is highly attractive to stingless bees. We tested the antimicrobial activity of extracts from kino of C. citriodora, C. torelliana × C. citriodora, and C. torelliana against three Gram-negative and two Gram-positive bacteria and the unicellular fungus, Candida albicans. All extracts were active against all microbes, with the highest activity observed against P. aeruginosa. We tested the activity of seven flavonoids from the kino of C. torelliana against P. aeruginosa and S. aureus. All flavonoids were active against P. aeruginosa, and one compound, (+)-(2S)-4',5,7-trihydroxy-6-methylflavanone, was active against S. aureus. Another compound, 4',5,7-trihydroxy-6,8-dimethylflavanone, greatly increased biofilm formation by both P. aeruginosa and S. aureus. The presence or absence of methyl groups at positions 6 and 8 in the flavonoid A ring determined their anti-Staphylococcus and biofilm-stimulating activity. One of the most abundant and active compounds, 3,4',5,7-tetrahydroxyflavanone, was tested further against P. aeruginosa and was found to be bacteriostatic at its minimum inhibitory concentration of 200 µg/mL. This flavanonol reduced adhesion of P. aeruginosa cells while inducing no cytotoxic effects in Vero cells. This study demonstrated the antimicrobial properties of flavonoids in eucalypt kino and highlighted that traditional medicinal knowledge and ecological cues can reveal valuable natural compounds.

Quantitation of Dihydroxyacetone in Australian Leptospermum Nectar via High-Performance Liquid Chromatography.

The nonperoxide antibacterial activity of New Zealand ma̅nuka honey originates from dihydroxyacetone (DHA) within Leptospermum scoparium nectar. This study determined if DHA was present within the nectar of four Australian Leptospermum species: L. laevigatum, L. polygalifolium, L. trinervium, and L. whitei. A rapid and convenient new method was developed, which quantitated DHA/sugar ratios (ppm). The DHA and sugars were derivatized with o-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride and analyzed via RP-HPLC with diode array detection at two wavelengths (200 and 243 nm). DHA was detected in all L. whitei and L. polygalifolium samples, where DHA/sugar ratios ranged from 10169 to 24199 ppm and from 9321 to 20174 ppm, respectively. DHA was undetected in any of the L. laevigatum and L. trinervium samples, and nectar activity was <100 ppm. The results of this study have implications for the Australian beekeeping industry, as the findings indicated that not all species of Leptospermum will produce active honey.

Moderate Dietary Supplementation with Omega-3 Fatty Acids Does Not Impact Plasma Von Willebrand Factor Profile in Mildly Hypertensive Subjects.

Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) have blood pressure lowering and antithrombotic effects, which may benefit hypertensive patients. Increased plasma concentration of von Willebrand factor (vWF), a procoagulant glycoprotein, has been identified in patients with severe hypertension, with some, but not all studies showing an increase with mild hypertension. In this study, we determined the plasma concentration, multimer distribution, and collagen binding activity of vWF in subjects with mild hypertension and determined whether these parameters might improve after dietary supplementation with moderate amounts of LC n-3 PUFAs. Hypertensive and normotensive subjects were randomized to 12-week treatment with LC n-3 PUFAs (2.52 g/day) or placebo (canola oil). Home blood pressure measurements were recorded daily, and blood samples were collected every 3 weeks. LC n-3 PUFAs increased the n-3 index to cardioprotective levels (>8%). Plasma concentration, multimer distribution, and collagen binding activity of vWF were not reduced by LC n-3 PUFA treatment. We conclude that, at the concentration and duration used in this study, benefits of LC n-3 PUFAs in subjects with mild hypertension are not associated with a direct effect on vWF concentration or function. This trial is registered with the Australian New Zealand Clinical Trials Registry ACTRN12610000713099.

Cardiovascular responsiveness to sympathoexcitatory stress in subjects with and without mild hypertension.

PURPOSE: This study compared blood pressure, heart rate variability (HRV) and forearm blood flow, at rest and in response to sympathoexcitatory stressors between normotensive and mildly hypertensive participants. METHODS: Participants aged 30-79 years with normal blood pressure (n = 49) or mild hypertension (n = 17), with no history of taking antihypertensive medication, were recruited. Participants completed a cold pressor test (CPT) followed by an ischaemic handgrip test (IHGT). Blood pressure, HRV, forearm blood flow and vascular resistance were measured at rest and in response to each test. RESULTS: The CPT and IHGT evoked greater increases in mean arterial blood pressure in hypertensive participants (CPT: 10 ± 2 mmHg, IHGT: 9 ± 1 mmHg) compared with normotensive participants (CPT: 5 ± 1 mmHg, IHGT: 3 ± 1 mmHg; P < 0.05). Resting high frequency power, which is a parameter of HRV associated with parasympathetic cardiac modulation, was lower in hypertensive participants (hypertensive: 31.73 ± 4.07 nu; normotensive: 42.08 ± 2.22 nu; P = 0.026) and was negatively correlated with systolic blood pressure (r = -0.272, P = 0.03) and mean arterial pressure across all participants (r = -0.258, P < 0.05). There were no differences in HRV or forearm blood flow responses to the CPT or IHGT between groups. CONCLUSION: This study demonstrated that sympathoexcitatory stress evoked by the CPT and IHGT induces an augmented blood pressure response in individuals with mild hypertension, which supports the notion that autonomic dysfunction is likely to contribute to the pathogenesis of hypertension. It remains to be determined whether the hypertensive response is mediated through alterations in cardiac activity, peripheral vascular resistance or both.