Multi-Endpoint Toxicological Assessment of Chrysin Loaded Oil-in-Water Emulsion System in Different Biological Models.

Chrysin is hypothesized to possess the ability to prevent different illnesses, such as diabetes, cancer, and neurodegenerative disorders. Nonetheless, chrysin has a low solubility under physiological conditions, resulting in limited bioavailability. In a previous study, we utilized an oil-in-water emulsion system (chrysin-ES or chrysin-NE) to encapsulate chrysin, thereby increasing its bioaccessibility and preserving its antioxidant and anti-Alzheimer's properties. To promote the chrysin-ES as a supplementary and functional food, it was obligatory to carry out a safety assessment. Cytotoxicity testing showed that chrysin-ES was harmless, with no killing effect on 3T3-L1 (adipocytes), RAW 264.7 (macrophages), HEK293 (kidney cells), and LX-2 (hepatic stellate cells). The acute toxicity evaluation demonstrated that the 50% lethal dose (LD50) for chrysin-ES was greater than 2000 mg/kg BW. Genotoxicity assessments found that chrysin-ES did not induce DNA mutations in vitro or in vivo. Furthermore, chrysin and chrysin-ES exhibited anti-mutagenic properties against PhIP-induced and IQ-induced mutagenesis in the Ames test, while they inhibited urethane-, ethyl methanesulfonate-, mitomycin C-, and N-nitrosomethylurea-mediated mutations in Drosophila. The present study illustrates the safety and anti-genotoxicity properties of chrysin-ES, allowing for the further development of chrysin-based food supplements and nutraceuticals.

Development of Chrysin Loaded Oil-in-Water Nanoemulsion for Improving Bioaccessibility.

Chrysin (5,7-dihydroxyflavone) is a remarkable flavonoid exhibiting many health-promoting activities, such as antioxidant, anti-inflammatory, and anti-Alzheimer's disease (AD). Nevertheless, chrysin has been addressed regarding its limited applications, due to low bioaccessibility. Therefore, to improve chrysin bioaccessibility, a colloidal delivery system involving nanoemulsion was developed as chrysin nanoemulsion (chrysin-NE) using an oil-in-water system. Our results show that chrysin can be loaded by approximately 174.21 µg/g nanoemulsion (100.29 ± 0.53% w/w) when medium chain triglyceride (MCT) oil was used as an oil phase. The nanocolloidal size, polydispersity index, and surface charge of chrysin-NE were approximately 161 nm, 0.21, and -32 mV, respectively. These properties were stable for at least five weeks at room temperature. Furthermore, in vitro chrysin bioactivities regarding antioxidant and anti-AD were maintained as pure chrysin, suggesting that multistep formulation could not affect chrysin properties. Interestingly, the developed chrysin-NE was more tolerant of gastrointestinal digestion and significantly absorbed by the human intestinal cells (Caco-2) than pure chrysin. These findings demonstrate that the encapsulation of chrysin using oil-in-water nanoemulsion could enhance the bioaccessibility of chrysin, which might be subsequently applied to food and nutraceutical industries.

Bioassay-guided study of the anti-inflammatory effect of Anoectochilus burmannicus ethanolic extract in RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Anoectochilus species is a small terrestrial orchid found in tropical and subtropical rain forest. These orchids are traditionally used extensively in China, Taiwan, and Vietnam due to their medicinal properties and therapeutic benefits. They are employed for treatment in different systems, such as stomach disorders, chest pain, arthritis, tumor, piles, boils, menstrual disorders, and inflammation. Aqueous extract of Anoectochilus burmannicus (AB) has been previously reported to exhibit anti-inflammatory activities, however there is a lack of evidence regarding its bioactive compounds and the mechanism of its actions. AIM OF THE STUDY: The objectives of this study were to identify the anti-inflammatory compound(s) in an ethanolic extract of AB and to determine its anti-inflammatory mechanisms in LPS-stimulated macrophages and also its safety. MATERIALS AND METHODS: The ethanolic extract of AB (ABE) was prepared and subsequently subjected to polarity-dependent extraction using n-hexane and ethyl acetate, which would result in isolation of the n-hexane (ABH), ethyl acetate (ABEA), and residue or aqueous (ABA) fractions. The AB fractions were investigated to determine total phenolic and flavonoid content, antioxidant capacity, toxicity, and safety in RAW 264.7 macrophages, human PBMCs, and RBCs. After extraction anti-inflammation screening of each extract was performed by nitric oxide (NO) production assay. The active fractions were further examined for their effect on proinflammatory mediators. In addition, kinsenoside content in the active fractions was identified using LC-MS/MS. Cellular toxicity and genotoxicity of AB were also tested using the wing spot test in Drosophila melanogaster. RESULTS: The data showed that ABEA had the highest phenolic content and level of antioxidant activities. ABE, ABEA, and ABA, but not ABH, significantly inhibited the LPS-stimulated NO production in the macrophages. Both ABEA and ABA reduced LPS-mediated expression of TNF-α, IL-6, iNOS, and COX-2 at both mRNA and protein levels. Besides, only ABEA notably diminished the LPS-stimulated p65 phosphorylation required for nuclear translocation and transcriptional activation of the nuclear factor-κB (NF-κB). Interestingly, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed ABA contained a high level of kinsenoside, a likely anti-inflammatory compound, while ABE and ABEA might require other compounds in combination with kinsenoside for the inhibition of inflammation. It was shown that all active fractions were neither cytotoxic nor genotoxic. CONCLUSION: Our study demonstrated that the hydrophilic fractions of AB exhibit anti-inflammatory activity in LPS-stimulated macrophages. The mechanism used by the AB involves the scavenging of free radicals and the reduction of proinflammatory mediators, including IL-1ß, IL-6, TNF-α, NO, iNOS and COX-2. The anti-inflammatory action of AB involves the suppression of the NF-κB signaling pathway by some unknown component(s) present in ABEA. This study found that kinsenoside is a major active compound in ABA which could be used as a biomarker for the quality control of the plant extraction. This study provides convincing significant information in vitro regarding the anti-inflammatory mechanism and preliminary evidence of the safety of Anoectochilus burmanicus. Therefore, the knowledge acquired from this study would provide supportive evidence for the development and standardization of the use of the extract of this plant as alternative medicine or functional food to prevent or treat non-communicable chronic diseases related to chronic inflammation.

Health-promoting bioactivity and in vivo genotoxicity evaluation of a hemiepiphyte fig, Ficus dubia.

Ficus species have been used as a typical component in food and folk medicine in Asia for centuries. However, little is known regarding the bioactivity and genotoxicity of the recently identified Ficus dubia (FD), an indigenous plant of the tropical evergreen rain forest. FD is unique from other Ficus species because of its highly sought-after red-brown latex. Antioxidant properties together with phenolic and flavonoid contents of FD were elucidated. Health-promoting characteristics were examined by studying the inhibition of enzymes as a drug target for diabetes, hypertension, Alzheimer's disease, and obesity, together with anticancer ability against human colorectal adenocarcinoma, human hepatocellular carcinoma, human ovarian carcinoma, human prostate adenocarcinoma, and human lung carcinoma. Besides, FD genotoxicity was tested using the Drosophila wing spot test. Results showed that both FD root and latex exhibited antioxidant activity due to the presence of phenolics and flavonoids, specifically caffeic acid and cyanidin. The ethanolic fraction of FD root demonstrated a potent antidiabetic mechanism underlying α-glucosidase inhibitory activity similar to acarbose. This fraction also suppressed lung and ovarian cancer growth, possibly by G1 and G2/M arrest, respectively. All tested fractions lacked mutagenicity in vivo. Results indicated that FD can be developed as novel antidiabetic compounds; however, its bioactive compounds should be further identified.

Safety assessment of Plukenetia volubilis (Inca peanut) seeds, leaves, and their products.

Plukenetia volubilis or Inca peanut is a promising plant with high economic value. Its seeds can be pressed for oil production or roasted and served as a snack, while the dried leaves can be used to make a kind of tea. Although the oil from the cold-pressed seeds has been proven to be safe for human consumption, little information is known about the other parts of the plant regarding safety. Thus, the aim of this study was to investigate the naturally occurring phytotoxins, including saponins, total alkaloids, and lectins in fresh and roasted Inca peanut seeds and leaves. In addition, cytotoxicity on several normal cell types including human peripheral blood mononuclear cells, human embryonic kidney cells, human hepatic stellate cells, and mouse fibroblasts as well as in vivo mutagenic properties was studied. This study showed that fresh Inca peanut seeds and leaves contain saponins, alkaloids, and lectins. However, roasting enables the reduction in alkaloids, saponins, and possibly lectins, suggesting that these phytotoxins become unstable under heat. Furthermore, Inca peanut seeds and leaves, especially after roasting, are safe to a variety of normal cell lines and do not induce DNA mutations in Drosophila expressing high biotransformation system. In conclusion, the data in this study indicated that high and chronic consumption of fresh seeds and leaves should be avoided. Heat processing should be applied before the consumption of Inca peanut seeds and leaves in order to reduce phytotoxins and potential health risks.

Anti-inflammatory and anti-insulin resistance activities of aqueous extract from Anoectochilus burmannicus.

This study investigated biological activities including antioxidative stress, anti-inflammation, and anti-insulin resistance of Anoectochilus burmannicus aqueous extract (ABE). The results showed abilities of ABE to scavenging DPPH and ABTS free radicals in a dose-dependent manner. Besides, ABE significantly reduced nitric oxide (NO) production in the lipopolysaccharide (LPS)-treated RAW 264.7 via inhibition of mRNA and protein expressions of nitric oxide synthase (iNOS). The LPS-induced mRNA expressions of cyclooxygenase-2 (COX-2) and interleukin 1ß (IL-1ß) were suppressed by ABE. Moreover, ABE exerted anti-insulin resistance activity as it significantly improved the glucose uptake in tumor necrosis factor (TNF)-α treated 3T3-L1 adipocytes. In addition, ABE at the concentration of up to 200 µg/mL was not toxic to human peripheral blood mononuclear cells (PBMCs) and did not induce mutations. Finally, the results of our study suggest the potential use of A. burmannicus as anti-inflammatory, anti-insulin resistance agents, or food supplement for prevention of chronic diseases.

Kuguacin J isolated from bitter melon leaves modulates paclitaxel sensitivity in drug-resistant human ovarian cancer cells.

We previously reported the multidrug resistance-reversing ability of kuguacin J (KJ) in cervical cancer cells via the inhibition of P-glycoprotein (P-gp) function. This study investigated whether KJ could promote cisplatin- and paclitaxel (PTX)-induced cancer cell death in drug-resistance human ovarian cancer cells (SKOV3). Cytotoxicity testing showed that SKOV3 was more resistant to cisplatin and PTX compared to drug-sensitive human ovarian cancer cells (A2780). The cytotoxicity of PTX was significantly increased in SKOV3 cells when co-treated with KJ. We found that enhancement of PTX toxicity in the cells was not related to P-gp inhibition. To elucidate the mechanism by which KJ increases PTX sensitivity, the expression of cell death involving proteins was analyzed by Western blot analysis. The results showed that PTX treatment increased the level of an anti-apoptotic protein, survivin, which may be involved in drug resistance in SKOV3. The co-treatment with PTX and KJ dramatically decreased the level of survivin and markedly induced cleavage of PARP and caspase-3, which are apoptotic-induced molecules. These findings may support the use of KJ as an effective chemosensitizer in combination with conventional chemotherapy to promote PTX sensitization in ovarian cancer patients.