Mustafa Kassim

Ellagic acid, phenolic acids, and flavonoids in Malaysian honey extracts demonstrate in vitro anti-inflammatory activity

Natural honey has been used in traditional medicine of different cultures throughout the world. This study looked into the extraction of Malaysian honey and the evaluation of the anti-inflammatory activity of these extracts. It was hypothesized that honey extracts contain varying amounts of phenolic compounds and that they possess different in vitro anti-inflammatory activities. Honey extracts were analyzed using liquid chromatography-mass spectrometry to identify and compare phenolic compounds, whereas high-performance liquid chromatography was used for their quantification. Subsequently, honey methanol extract (HME) and honey ethyl acetate extract (HEAE) were tested in vitro for their effect on nitric oxide production in stimulated macrophages. The extracts were also tested for their effects on tumor necrosis factor-α (TNF) cytotoxicity in L929 cells. The major phenolics in the extracts were ellagic, gallic, and ferulic acids; myricetin; chlorogenic acid; and caffeic acid. Other compounds found in lower concentrations were hesperetin, p-coumaric acid, chrysin, quercetin, luteolin, and kaempferol. Ellagic acid was the most abundant of the phenolic compounds recorded, with mean concentrations of 3295.83 and 626.74 μg/100 g of honey in HME and HEAE, respectively. The median maximal effective concentrations for in vitro nitric oxide inhibition by HEAE and HME were calculated to be 37.5 and 271.7 μg/mL, respectively. The median maximal effective concentrations for protection from TNF cytotoxicity by HEAE and HME were 168.1 and 235.4 μg/mL, respectively. In conclusion, HEAE exhibited greater activity in vitro, whereas HME contained a higher concentration of phenolic compounds per 100 g of honey.

Gelam honey inhibits lipopolysaccharide-induced endotoxemia in rats through the induction of heme oxygenase-1 and the inhibition of cytokines, nitric oxide, and high-mobility group protein B1.

Malaysian Gelam honey has anti-inflammatory and antibacterial properties, a high antioxidant capacity, and free radical-scavenging activity. Lipopolysaccharide (LPS) stimulates immune cells to sequentially release early pro- and anti-inflammatory cytokines and induces the synthesis of several related enzymes. The aim of this study was to investigate the effect of the intravenous injection of honey in rats with LPS-induced endotoxemia. The results showed that after 4h of treatment, honey reduced cytokine (tumor necrosis factor-α, interleukins 1β, and 10) and NO levels and increased heme oxygenase-1 levels. After 24h, a decrease in cytokines and NO and an increase in HO-1 were seen in all groups, whereas a reduction in HMGB1 occurred only in the honey-treated groups. These results support the further examination of honey as a natural compound for the treatment of a wide range of inflammatory diseases.

Gelam Honey Has a Protective Effect against Lipopolysaccharide (LPS)-Induced Organ Failure

Gelam honey exerts anti-inflammatory and antioxidant activities and is thought to have potent effects in reducing infections and healing wounds. The aim of this study was to investigate the effects of intravenously-injected Gelam honey in protecting organs from lethal doses of lipopolysaccharide (LPS). Six groups of rabbits (N = 6) were used in this study. Two groups acted as controls and received only saline and no LPS injections. For the test groups, 1 mL honey (500 mg/kg in saline) was intravenously injected into two groups (treated), while saline (1 mL) was injected into the other two groups (untreated); after 1 h, all four test groups were intravenously-injected with LPS (0.5 mg/kg). Eight hours after the LPS injection, blood and organs were collected from three groups (one from each treatment stream) and blood parameters were measured and biochemical tests, histopathology, and myeloperoxidase assessment were performed. For survival rate tests, rabbits from the remaining three groups were monitored over a 2-week period. Treatment with honey showed protective effects on organs through the improvement of organ blood parameters, reduced infiltration of neutrophils, and decreased myeloperoxidase activity. Honey-treated rabbits also showed reduced mortality after LPS injection compared with untreated rabbits. Honey may have a therapeutic effect in protecting organs during inflammatory diseases.

CAFFEIC ACID PHENETHYL ESTER (CAPE): SCAVENGER OF PEROXYNITRITE IN VITRO AND IN SEPSIS MODELS

ABSTRACT Excessive free radical production by immune cells has been linked to cell death and tissue injury during sepsis. Peroxynitrite is a short-lived oxidant and a potent inducer of cell death that has been identified in several pathological conditions. Caffeic acid phenethyl ester (CAPE) is an active component of honeybee products and exhibits antioxidant, anti-inflammatory, and immunomodulatory activities. The present study examined the ability of CAPE to scavenge peroxynitrite in RAW 264.7 murine macrophages stimulated with lipopolysaccharide/interferon-&ggr; that was used as an in vitro model. Conversion of 123-dihydrorhodamine to its oxidation product 123-rhodamine was used to measure peroxynitrite production. Two mouse models of sepsis (endotoxemia and cecal ligation and puncture) were used as in vivo models. The level of serum 3-nitrotyrosine was used as an in vivo marker of peroxynitrite. The results demonstrated that CAPE significantly improved the viability of lipopolysaccharide/interferon-&ggr;–treated RAW 264.7 cells and significantly inhibited nitric oxide production, with effects similar to those observed with an inhibitor of inducible nitric oxide synthase (1400W). In addition, CAPE exclusively inhibited the synthesis of peroxynitrite from the artificial substrate SIN-1 and directly prevented the peroxynitrite-mediated conversion of dihydrorhodamine-123 to its fluorescent oxidation product rhodamine-123. In both sepsis models, CAPE inhibited cellular peroxynitrite synthesis, as evidenced by the absence of serum 3-nitrotyrosine, an in vivo marker of peroxynitrite. Thus, CAPE attenuates the inflammatory responses that lead to cell damage and, potentially, cell death through suppression of the production of cytotoxic molecules such as nitric oxide and peroxynitrite. These observations provide evidence of the therapeutic potential of CAPE treatment for a wide range of inflammatory disorders.

Honey as an immunomodulator during sepsis in animal model

Malaysian honey (Gelam) has antibacterial activity and it also has a high antioxidant capacity and free radical scavenger activities. Honey extracts showed potent activity against TNFI± in L929 cell and NO in RAW 264.7 macrophage as well as inhibitory effects on the prostaglandin E2 and nitric oxide (NO) in inflammatory tissues of rat. Sepsis is mediated in part by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (for example, TNF, IL-1) cytokines and inducible enzymes such as inducible nitric oxide (iNOS) synthase and heme oxygenase 1 (HO-1) and late such as high-mobility group box 1 (HMGB1).

Gelam Honey Scavenges Peroxynitrite During the Immune Response

Monocytes and macrophages are part of the first-line defense against bacterial, fungal, and viral infections during host immune responses; they express high levels of proinflammatory cytokines and cytotoxic molecules, including nitric oxide, reactive oxygen species, and their reaction product peroxynitrite. Peroxynitrite is a short-lived oxidant and a potent inducer of cell death. Honey, in addition to its well-known sweetening properties, is a natural antioxidant that has been used since ancient times in traditional medicine. We examined the ability of Gelam honey, derived from the Gelam tree (Melaleuca spp.), to scavenge peroxynitrite during immune responses mounted in the murine macrophage cell line RAW 264.7 when stimulated with lipopolysaccharide/interferon-γ (LPS/IFN-γ) and in LPS-treated rats. Gelam honey significantly improved the viability of LPS/IFN-γ-treated RAW 264.7 cells and inhibited nitric oxide production—similar to the effects observed with an inhibitor of inducible nitric oxide synthase (1400W). Furthermore, honey, but not 1400W, inhibited peroxynitrite production from the synthetic substrate 3-morpholinosydnonimine (SIN-1) and prevented the peroxynitrite-mediated conversion of dihydrorhodamine 123 to its fluorescent oxidation product rhodamine 123. Honey inhibited peroxynitrite synthesis in LPS-treated rats. Thus, honey may attenuate inflammatory responses that lead to cell damage and death, suggesting its therapeutic uses for several inflammatory disorders.

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.

Antifilarial activity of caffeic acid phenethyl ester on Brugia pahangi in vitro and in vivo

Abstract Lymphatic filariasis (LF) is a vector borne disease caused by parasitic worms such as Wuchereria bancrofti, Brugia malayi and B. timori, which are transmitted by mosquitoes. Current therapeutics to treat LF are mainly microfilarcidal, and lack activity against adult worms. This set back, poses a challenge for the control and elimination of filariasis. Thus, in this study the activities of caffeic acid phenethyl ester (CAPE) against the filarial worm B. pahangi and its bacterial endosymbiont, Wolbachia were evaluated. Different concentrations (2, 5, 10, 15, 20 μg/ml) of CAPE were used to assess its effects on motility, viability and microfilarial (mf) production of B. pahangi in vitro. Anti-Wolbachial activity of CAPE was measured in worms by quantification of Wolbachial wsp gene copy number using real-time polymerase chain reaction. Our findings show that CAPE was found to significantly reduce adult worm motility, viability, and mf release both in vitro and in vivo. 20 μg/ml of CAPE halts the release of mf in vitro by day 6 of post treatment. Also, the number of adult worms recovered in vivo were reduced significantly during and after treatment with 50 mg/kg of CAPE relative to control drugs, diethylcarbamazine and doxycycline. Real time PCR based on the Wolbachia ftsZ gene revealed a significant reduction in Wolbachia copy number upon treatment. Anti-Wolbachia and antifilarial properties of CAPE require further investigation as an alternative strategy to treat LF.