Q-TOF LC-MS compounds evaluation of propolis extract derived from Malaysian stingless bees, Tetrigona apicalis, and their bioactivities in breast cancer cell, MCF7.

Propolis is known to exhibit various phytochemical compounds that aid in several biological activities. The current study investigates the phytochemical compounds of ethanolic extract of propolis of Tetrigona apicalis (EEP) using Q-TOF LC-MS, its antioxidant properties using DPPH and ABTS+ radical scavenging assays, total phenolic (TPC) and flavonoid content (TFC), using Folin-Ciocalteu and Aluminium Chloride method, respectively, as well as proapoptotic effects, based on the selected IC50 of the cytotoxic study conducted for EEP using annexin V-FITC assay. Terpene and polyphenol were among of 17 identified compounds. The EC50 of EEP for DPPH and ABTS+ was 1.78 mg/mL and 1.68 mg/mL, while the EEP exhibited TPC and TFC values of 31.99 mgGAE/g and 66.4 mgQCE/g, respectively in which the parameters were strongly correlated. The IC50 of EEP effectively induces apoptosis in MCF7 cells. In conclusion, EEP possessed important phytochemical compounds that work excellently as antioxidants and anticancer agents.

Osteoprotective Effects in Postmenopausal Osteoporosis Rat Model: Oral Tocotrienol vs. Intraosseous Injection of Tocotrienol-Poly Lactic-Co-Glycolic Acid Combination.

Osteoporosis, the most common bone disease, is associated with compromised bone strength and increased risk of fracture. Previous studies have shown that oxidative stress contributes to the progression of osteoporosis. Specifically, for postmenopausal osteoporosis, the reduction in estrogen levels leads to increased oxidative stress in bone remodeling. Tocotrienol, a member of vitamin E that exhibits antioxidant activities, has shown potential as an agent for the treatment of osteoporosis. Most studies on the osteoprotective effects of tocotrienols had used the oral form of tocotrienols, despite their low bioavailability due the lack of transfer proteins and high metabolism in the liver. Several bone studies have utilized tocotrienol combined with a nanocarrier to produce a controlled release of tocotrienol particles into the system. However, the potential of delivering tocotrienol-nanocarrier combination through the intraosseous route has never been explored. In this study, tocotrienol was combined with a nanocarrier, poly lactic-co-glycolic acid (PLGA), and injected intraosseously into the bones of ovariectomized rats to produce targeted and controlled delivery of tocotrienol into the bone microenvironment. This new form of tocotrienol delivery was compared with the conventional oral delivery in terms of their effects on bone parameters. Forty Sprague-Dawley rats were divided into five groups. The first group was sham operated, while other groups were ovariectomized (OVX). Following 2 months, the right tibiae of all the rats were drilled at the metaphysis region to provide access for intraosseous injection. The estrogen group (OVX + ESTO) and tocotrienol group (OVX + TTO) were given daily oral gavages of Premarin (64.5 mg/kg) and annatto-tocotrienol (60 mg/kg), respectively. The locally administered tocotrienol group (OVX + TTL) was given a single intraosseous injection of tocotrienol-PLGA combination. After 8 weeks of treatment, both OVX + TTO and OVX + TTL groups have significantly lower bone markers and higher bone mineral content than the OVX group. In terms of bone microarchitecture, both groups demonstrated significantly higher trabecular separation and connectivity density than the OVX group (p < 0.05). Both groups also showed improvement in bone strength by the significantly higher stress, strain, stiffness, and Young's modulus parameters. In conclusion, daily oral tocotrienol and one-time intraosseous injection of tocotrienol-PLGA combination were equally effective in offering protection against ovariectomy-induced bone changes.

Potential Antioxidant and Anti-Inflammatory Effects of Spilanthes acmella and Its Health Beneficial Effects: A Review.

Oxidative stress and inflammation are two common risk factors of various life-threatening disease pathogenesis. In recent years, medicinal plants that possess antioxidant and anti-inflammatory activities were extensively studied for their potential role in treating and preventing diseases. Spilanthes acmella (S. acmella), which has been traditionally used to treat toothache in Malaysia, contains various active metabolites responsible for its anti-inflammatory, antiseptic, and anesthetic bioactivities. These bioactivities were attributed to bioactive compounds, such as phenolic, flavonoids, and alkamides. The review focused on the summarization of in vitro and in vivo experimental reports on the antioxidant and anti-inflammatory actions of S. acmella, as well as how they contributed to potential health benefits in lowering the risk of diseases that were related to oxidative stress. The molecular mechanism of S. acmella in reducing oxidative stress and inflammatory targets, such as inducible nitric oxide synthase (iNOS), transcription factors of the nuclear factor-κB family (NF-κB), cyclooxygenase-2 (COX-2), and mitogen-activated protein kinase (MAPK) signaling pathways were discussed. Besides, the antioxidant potential of S. acmella was measured by total phenolic content (TPC), total flavonid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and superoxide anion radical scavenging (SOD) and thiobarbituric acid reactive substance (TBARS) assays. This review revealed that S. acmella might have a potential role as a reservoir of bioactive agents contributing to the observed antioxidant, anti-inflammatory, and health beneficial effects.

Biocompatible magnesium-doped biphasic calcium phosphate for bone regeneration.

Autologous bone grafting remains the gold standard for almost all bone void-filling orthopedic surgery. However, autologous bone grafting has several limitations, thus scientists are trying to identify an ideal synthetic material as an alternative bone graft substitute. Magnesium-doped biphasic calcium phosphate (Mg-BCP) has recently been in the spotlight and is considered to be a potential bone substitute. The Mg-BCP is a mixture of two bioceramics, that is, hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP), doped with Mg2+ , and can be synthesized through chemical wet-precipitation, sol-gel, single diffusion gel, and solid state reactions. Regardless of the synthesis routes, it is found that the Mg2+ preferentially accommodates in ß-TCP lattice instead of the HA lattice. The addition of Mg2+ to BCP leads to desirable physicochemical properties and is found to enhance the apatite-forming ability as compared to pristine BCP. In vitro results suggest that the Mg-BCP is bioactive and not toxic to cells. Implantation of Mg-BCP in in vivo models further affirmed its biocompatibility and efficacy as a bone substitute. However, like the other bioceramics, the optimum physicochemical properties of the Mg-BCP scaffold have yet to be determined. Further investigations are required regarding Mg-BCP applications in bone tissue engineering.

GC-MS Evaluation, Antioxidant Content, and Cytotoxic Activity of Propolis Extract from Peninsular Malaysian Stingless Bees, Tetrigona Apicalis.

INTRODUCTION: Propolis has been used traditionally in several countries for treating various diseases as it possessed healing properties including antioxidant and anticancer qualities. In Peninsular Malaysia, Tetrigona apicalis is one of the species of stingless bees mainly found in virgin jungle reserves which largely contribute to propolis production. Therefore, this study is designed to evaluate the phytochemical contents, antioxidant properties, and the cytotoxic effect of ethanolic crude of propolis extract against MCF7 and MCF 10A cell lines. METHOD: The ethanolic extract of propolis (EEP) was extracted using 80% ethanol. Identification of phytochemical contents and antioxidant properties of EEP was analysed by gas chromatography-mass spectrometry (GC-MS) and using 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) method, respectively. The EEP cytotoxic activity was evaluated on MCF7 and MCF 10A using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Phytochemical contents of EEP demonstrated 28 compounds in which caryophyllene (99%), ß-amyrin (96%), α-amyrin (93%), and caryophyllene oxide (93%) were the main compounds. The percentage of ABTS+ scavenging activity of EEP showed an inhibition of 9.5% with half-inhibitory concentration (IC50) value of 1.68 mg/mL. The EEP reduced MCF7 cells viability at IC50 value of 62.24 µg/mL, 44.15 µg/mL, and 32.70 µg/mL at 24, 48, and 72 hours, respectively. The IC50 value of MCF 10A was 49.55 µg/mL, 56.05 µg/mL, and 72.10 µg/mL at 24, 48, and 72 hours, respectively. The EEP cytotoxic effect of T. apicalis was more selective towards MCF7 at 72-hour incubation with a selectivity index (SI) of 2.20. CONCLUSION: The EEP has been shown to have antioxidants and potential bioactive compounds and inhibited proliferation of the MCF7 cells. Further studies on the EEP role in the apoptosis pathway and its screening towards other cell lines will be evaluated.

Experimental fracture protocols in assessments of potential agents for osteoporotic fracture healing using rodent models.

Osteoporosis may cause bone fracture even under slight trauma. Osteoporotic fracture has become a major public health problem but until today, the treatments available are not satisfactory. Many pre-clinical testings on animals were done to find new agents that can be sourced from natural products and synthetic drugs for osteoporotic fracture healing. Animal models are more appropriate for fracture healing study than human subject due to several reasons including the ethical issues involved. The bones of rodents are similar to human in term of their morphological change and response to therapy. Small rodents such as rats and mice are suitable animal models for fracture healing studies as they have a similar bone remodeling system to human. To date, there is no specific guideline to carry out fracture healing studies in animal models for the evaluation of new agents. This paper highlights the protocols of various fracture and fixation methods for experimental osteoporotic fracture healing using rodent models.

The effects of alpha-tocopherol supplementation on fracture healing in a postmenopausal osteoporotic rat model.

OBJECTIVE: Osteoporosis increases the risk of bone fractures and may impair fracture healing. The aim of this study was to investigate whether alpha-tocopherol can improve the late-phase fracture healing of osteoporotic bones in ovariectomized rats. METHOD: In total, 24 female Sprague-Dawley rats were divided into three groups. The first group was sham-operated, and the other two groups were ovariectomized. After two months, the right femora of the rats were fractured under anesthesia and internally repaired with K-wires. The sham-operated and ovariectomized control rat groups were administered olive oil (a vehicle), whereas 60 mg/kg of alpha-tocopherol was administered via oral gavage to the alpha-tocopherol group for six days per week over the course of 8 weeks. The rats were sacrificed, and the femora were dissected out. Computed tomography scans and X-rays were performed to assess fracture healing and callus staging, followed by the assessment of callus strengths through the biomechanical testing of the bones. RESULTS: Significantly higher callus volume and callus staging were observed in the ovariectomized control group compared with the sham-operated and alpha-tocopherol groups. The ovariectomized control group also had significantly lower fracture healing scores than the sham-operated group. There were no differences between the alpha-tocopherol and sham-operated groups with respect to the above parameters. The healed femora of the ovariectomized control group demonstrated significantly lower load and strain parameters than the healed femora of the sham-operated group. Alpha-tocopherol supplementation was not able to restore these biomechanical properties. CONCLUSION: Alpha-tocopherol supplementation appeared to promote bone fracture healing in osteoporotic rats but failed to restore the strength of the fractured bone.

The effects of alpha-tocopherol supplementation on fracture healing in a postmenopausal osteoporotic rat model

OBJECTIVE: Osteoporosis increases the risk of bone fractures and may impair fracture healing. The aim of this study was to investigate whether alpha-tocopherol can improve the late-phase fracture healing of osteoporotic bones in ovariectomized rats. METHOD: In total, 24 female Sprague-Dawley rats were divided into three groups. The first group was sham-operated, and the other two groups were ovariectomized. After two months, the right femora of the rats were fractured under anesthesia and internally repaired with K-wires. The sham-operated and ovariectomized control rat groups were administered olive oil (a vehicle), whereas 60 mg/kg of alpha-tocopherol was administered via oral gavage to the alpha-tocopherol group for six days per week over the course of 8 weeks. The rats were sacrificed, and the femora were dissected out. Computed tomography scans and X-rays were performed to assess fracture healing and callus staging, followed by the assessment of callus strengths through the biomechanical testing of the bones. RESULTS: Significantly higher callus volume and callus staging were observed in the ovariectomized control group compared with the sham-operated and alpha-tocopherol groups. The ovariectomized control group also had significantly lower fracture healing scores than the sham-operated group. There were no differences between the alpha-tocopherol and sham-operated groups with respect to the above parameters. The healed femora of the ovariectomized control group demonstrated significantly lower load and strain parameters than the healed femora of the sham-operated group. Alpha-tocopherol supplementation was not able to restore these biomechanical properties. CONCLUSION: Alpha-tocopherol supplementation appeared to promote bone fracture healing in osteoporotic rats but failed to restore the strength of the fractured bone.

Tocotrienol supplementation improves late-phase fracture healing compared to alpha-tocopherol in a rat model of postmenopausal osteoporosis: a biomechanical evaluation.

This study investigated the effects of α-tocopherol and palm oil tocotrienol supplementations on bone fracture healing in postmenopausal osteoporosis rats. 32 female Sprague-Dawley rats were divided into four groups. The first group was sham operated (SO), while the others were ovariectomised. After 2 months, the right femora were fractured under anesthesia and fixed with K-wire. The SO and ovariectomised-control rats (OVXC) were given olive oil (vehicle), while both the alpha-tocopherol (ATF) and tocotrienol-enriched fraction (TEF) groups were given alpha-tocopherol and tocotrienol-enriched fraction, respectively, at the dose of 60 mg/kg via oral gavages 6 days per week for 8 weeks. The rats were then euthanized and the femora dissected out for bone biomechanical testing to assess their strength. The callous of the TEF group had significantly higher stress parameter than the SO and OVXC groups. Only the SO group showed significantly higher strain parameter compared to the other treatment groups. The load parameter of the OVXC and ATF groups was significantly lower than the SO group. There was no significant difference in the Young's modulus between the groups. In conclusion, tocotrienol is better than α-tocopherol in improving the biomechanical properties of the fracture callous in postmenopausal osteoporosis rat model.

Effects of α-tocopherol on the early phase of osteoporotic fracture healing.

Fracture healing is a complex process, which is more complicated if the bone is osteoporotic. One of the vitamin E isomers, α-tocopherol, has been found to prevent osteoporosis and improve bone fracture healing but its role in the healing of osteoporotic fractures is still unclear. We carried out a study on the effects of α-tocopherol supplementation on osteoporotic fracture healing using an ovariectomized rat model, whereby we focused on the early phase of fracture healing, that is, the phase with excessive production of free radicals. Twenty-four female Sprague-Dawley rats were divided into three groups: sham-operated (SO), ovariectomized-control (OVC), and ovariectomized + α-tocopherol supplementation (ATF) groups. The right femora of all the rats were fractured at mid-diaphysis and K-wires were inserted for internal fixation. After 2 weeks of treatment, the rats were euthanized and the femora were dissected out for measurement of callous volume by CT-scan and radiological staging of callous formation and fracture healing. The oxidative parameters of the fractured femora were also measured. The results showed that the callous volume and callous staging were not different between the groups. However, the fracture healing stage of the OVC group was lower than the SO group, while α-tocopherol supplementation in the ATF group had improved the healing until it was comparable to the SO group. The activities of the anti-oxidatant enzymes, superoxide dismutase, and glutathione peroxidase in the ATF group were found to be significantly higher than in the OVC group. In conclusion, α-tocopherol improved fracture healing but had no effect on the callous volume and staging. The improvement in fracture healing may be due to the increased activities of the anti-oxidatant enzymes in the bone during the early phase of fracture healing of osteoporotic bone.

Effects of calcium supplements on fracture healing in a rat osteoporotic model.

Fracture healing is a complex process, which is further complicated if the bone is osteoporotic. Calcium is one of the important minerals in bone and has been found to prevent osteoporosis but its role in fracture healing of osteoporotic bone is still unclear. We carried out a study on the effects of calcium supplementation on the late phase healing of fractured osteoporotic bone using an ovariectomized rat model. Twenty-four female Sprague-Dawley rats were divided into three groups: sham-operated (SO), ovariectomized-control (OVXC), and ovariectomized + calcium supplements (Ca). The right femurs of all the rats were fractured at mid-epiphysis and a K-wire was inserted for internal fixation. After 2 months of treatment, the rats were sacrificed and the femora were dissected out for radiological and biomechanical assessment. As expected, osteoporosis resulted in impaired healing as shown by the poor radiological and biomechanical properties of the OVXC group. CT scans showed significantly lower callus volumes in the SO and Ca groups compared to the OVXC group. Radiological scoring of fracture healing and callus staging of the SO and Ca groups were better than the OVXC group. However, the biomechanical parameters of the Ca group were significantly lower than the SO group and similar to the OVXC group. Therefore, calcium supplements may appear to improve fracture healing of osteoporotic bone but failed to improve strength.