Structural Elucidation of Garcipaucinones A and B From Garcinia paucinervis Using Quantum Chemical Calculations.

Two tocotrienol derivatives, garcipaucinones A (1) and B (2), and a biosynthetically related known analogue (3) were isolated from the fruit of Garcinia paucinervis. Their structures including absolute configurations were unequivocally determined by spectroscopic methods complemented with electronic circular dichroism (ECD) calculations and gauge-independent atomic orbital (GIAO) NMR calculations. Compounds 1 and 2 are the first naturally occurring tocotrienol derivatives with a 3,10-dioxatricyclo-[7.3.1.02,7]tridecane skeleton incorporating an unusual γ-pyrone motif. A reasonable biosynthetic pathway for formation of the two compounds is proposed. The antiproliferative and anti-inflammatory activities of compounds 1 and 2 were also evaluated.

Efficient Semi-Synthesis of Natural δ-( R)-Tocotrienols from a Renewable Vegetal Source.

Recent studies have highlighted the biological potential of tocotrienols, a vitamin E subfamily. The major natural sources of tocotrienols are complex mixtures requiring particularly challenging purification processes. The present study describes efficient semi-synthetic strategies toward relevant δ-( R)-tocotrienol derivatives, using as a starting material δ-( R)-garcinoic acid, the major vitamin E derivative isolated from Garcinia kola nuts, a renewable vegetal source.

Current technologies in the extraction, enrichment and analytical detection of tocopherols and tocotrienols: A review.

During the past few years the scientific and medical community has been confronted with a continual interest in vitamin E with the interest prompted by new discoveries. Tocopherols and tocotrienols, commonly known as vitamin E, are extremely invaluable compounds and have various nutritional functionalities and benefits to human health. Great deals of research projects have been launched in order to develop effective methods for the extraction of vitamin E. By and large, three distinct extractive methods are usually employed: supercritical fluid extraction (SFE), molecular distillation, and adsorption methods. These methods are sensitive to different experimental conditions, such as pressure, temperature, and flow rate with noticeable effects on the efficiency of the extraction and enrichment of vitamin E. This review has covered the most commonly adapted extraction methods and has probed into the extraction yields under variable operational parameters.

The Effect of Pressure and Solvent on the Supercritical Fluid Chromatography Separation of Tocol Analogs in Palm Oil.

There are six tocol analogs present in palm oil, namely α-tocopherol (α-T), α-tocomonoenol (α-T1), α-tocotrienol (α-T3), γ-tocotrienol (γ-T3), ß-tocotrioenol (ß-T3) and δ-tocotrienol (δ-T3). These analogs were difficult to separate chromatographically due to their similar structures, physical and chemical properties. This paper reports on the effect of pressure and injection solvent on the separation of the tocol analogs in palm oil. Supercritical CO2 modified with ethanol was used as the mobile phase. Both total elution time and resolution of the tocol analogs decreased with increased pressure. Ethanol as an injection solvent resulted in peak broadening of the analogs within the entire pressure range studied. Solvents with an eluent strength of 3.4 or less were more suitable for use as injecting solvents.

Quantification of tocopherols and tocotrienols in soybean oil by supercritical-fluid chromatography coupled to high-resolution mass spectrometry.

For the most effective analytical strategies, development and validation include optimization of such analytical variables as resolution, detectability, sensitivity, simplicity, cost effectiveness, flexibility, and speed. However, other aspects concerning operator safety and environmental impact are not considered at the same level. The result has been many unintended negative effects of analytical methods developed to investigate different kinds of sample, especially hydrophobic compounds that generate a large amount of chemical waste and have a strong negative environmental impact. In this context, quantification of tocopherols and tocotrienols, i.e. the vitamin E family, is usually achieved by normal-phase liquid chromatography using large volumes of toxic organic solvents, or reversed-phase liquid chromatography using a high percentage of methanol for elution. We propose here a "greener" analytical strategy, including the hyphenation of supercritical-fluid chromatography, using CO2 and ethanol as mobile phase, NH2 as stationary phase, and mass spectrometry for the detection and quantification of vitamin E congeners in soybean oil. An atmospheric-pressure photoionization (APPI) source seemed significantly more sensitive and robust than electrospray or atmospheric-pressure chemical ionization (APCI). This method led to shortened analysis time (less than 5 min) and was revealed to be as sensitive as more traditional approaches, with limits of detection and quantification in the tens of µg L(-1).

Protective effects of tocotrienols against lipid-induced nephropathy in experimental type-2 diabetic rats by modulation in TGF-ß expression.

Dyslipidemia is common in patients with diabetes mellitus (DM) and is considered a risk factor for the progression of diabetic nephropathy (DN). Hyperlipidemia and hyperglycemia act synergistically to induce renal injury. The present study was designed to investigate the protective effects of tocotrienols as tocotrienol-rich fraction (TRF) extracted from palm (PO) and rice bran oils (RBO) against lipid induced nephropathy in type-2 diabetic rats and its probable molecular mechanism. Male Wistar rats (175-200 g) were divided into four groups. The first group served as diabetic control, while the second and third groups received PO-TRF and RBO-TRF, respectively by gavage over a period of sixteen weeks post-induction of diabetes. The fourth group comprised of age-matched rats that served as normal control. The effects of TRF on serum lipid profile, oxidative stress markers, expression of TGF-ß, fibronectin and collagen type IV were analyzed in the kidney of diabetic rats. Treatment with PO-TRF and RBO-TRF significantly improved glycemic status, serum lipid profile and renal function in type-2 diabetic rats. In addition, TRF supplementation down-regulated the expression of TGF-ß, fibronectin and collagen type IV in the kidney of diabetic rats. Transforming growth factor-ß (TGF-ß) plays a critical role in progression of DN, but its modulation by tocotrienols in DN remains unexplored. TRF ameliorated lipid induced nephropathy in type-2 diabetes by its hypoglycemic, hypolipidemic and antioxidant activities as well as by modulation of TGF-ß to prevent increased expression of collagen type IV and fibrinogen. We finally propose a mechanism for the expression of molecular markers that are significant in the events leading to diabetic nephropathy and its modulation by tocotrienols/TRF.

Antiangiogenic tocotrienol derivatives from Garcinia amplexicaulis.

Phytochemical investigation of a dichloromethane extract from Garcinia amplexicaulis stem bark led to the isolation of four new tocotrienols (1-4); two known tocotrienols, two triterpenes, and a xanthone were also isolated. Their structures were mainly established using NMR and MS methods. The main compounds isolated, δ-amplexichromanol (1) and γ-amplexichromanol (2), were evaluated on VEGF-induced angiogenesis using a Matrigel assay. Compounds 1 and 2 inhibited in vitro angiogenesis of VEGF-induced human primary endothelial cells in the low nanomolar range. Their capacity to inhibit VEGF-induced proliferation of endothelial cells partially explained this activity, although δ-amplexichromanol (1) also prevented adhesion and migration processes.

Antidiabetic effects of a standardized Egyptian rice bran extract.

An extract was prepared from Egyptian stabilized rice bran and standardized to contain 2% γ-oryzanol in addition to its content of other bioactives, notably tocotrienol and policosanol. The standardized extract was found to have a concentration-dependent effect on insulin release in vitro, which, however, is not mediated by γ-tocotrienol in rice bran (detected by HPLC) as could have been expected. Policosanol and γ-oryzanol have insulinotropic effects. The in vitro data of rice bran directly translate into in vivo data of rats by using a glucose tolerance test (increase in plasma insulin). Tocotrienols are well known for their apoptotic effect on tumor cells; nevertheless, an attempt was made to study glucose uptake in HEP-G2 cells, which needs to induce an insulin-resistant state by TNF-α. The Egyptian rice bran extract has an antidiabetic effect. γ-Oryzanol, which is a possible precursor of the insulinotropic compound ferulic acid, is a candidate for this effect. Therefore, it is reasonable to assume that the prevalence of diabetes or at least a prediabetic (type 2) situation can be ameliorated by the investigated rice bran extract. The potential usefulness of the extract as a nutraceutical is currently undergoing more thorough investigations.

Development and validation of an HPLC method for the simultaneous determination of tocopherols, tocotrienols and carotenoids in cereals after solid-phase extraction.

The increasing interest in antioxidant properties of cereal and cereal-based products has prompted the development of a simple and reliable HPLC method for the simultaneous determination of important phytochemicals like tocopherols (T), tocotrienols (T3) and carotenoids. Separation was carried out on a Nucleosil 100 C(18) column, 5 µm (250 mm × 4.6 mm) thermostated at 25 °C, using a linear gradient elution system starting with methanol and ending with a mixture of methanol-isopropanol-acetonitrile. All separated compounds including the internal standard (α-tocopherol acetate) were eluted within 16 min and detected by dual detection: fluorescence for tocopherols and tocotrienols at 290 nm excitation and 320 nm emission and UV-vis photodiode array detection for lutein and ß-carotene at 450 nm. Detection limits ranged from 0.2 µg/g (ß-carotene) to 1.60 µg/g (α-tocopherol). The intra- and inter-assay coefficients of variation were calculated by using cereals with different levels of lipophilic antioxidants. The extraction method involved sample saponification and clean-up by solid-phase extraction (SPE). The extraction recoveries obtained using OASIS HLB SPE cartridges and dichloromethane as eluent were in the range of 90.2-110.1%, with RSD lower than 10%. The method was successfully applied to cereals: durum wheat, bread wheat, rice, barley, oat, rye, corn and triticale.

'Tocol-omic' diversity in wild barley, short communication.

Hordeum spontaneum, wild barley, is the direct progenitor of domestic barley, Hordeum vulgare, an economically important ingredient of animal feed, beer, soy sauce, and more recently, of nutraceuticals. Domestic barley has also been used in the past as a medicine. Barley is a rich source of tocotrienols, with α-tocotrienol being the most prevalent. Wild barley seeds were harvested from ecogeographically diverse areas across the Fertile Crescent, and the tocopherol (α-δ) and tocotrienol (α-δ) contents were determined. Diversity differences in individual and total 'tocol' values were significant between and within specific countries, and were significantly correlated with temperature. Wild barley may be used in the future to improve functional qualities of domestic barley. 'Tocolome' and 'tocolomics' are proposed to encompass all tocols and potentially synergy-enhancing 'entourage' compounds that may occur in tocols' 'metabolomic neighborhoods', aiding the standardized manufacture of complex barley derivatives for nutraceutical and pharmaceutical functions.

Dietary Annatto-Extracted Tocotrienol Reduces Inflammation and Oxidative Stress, and Improves Macronutrient Metabolism in Obese Mice: A Metabolic Profiling Study.

Obesity and its related complications are a world-wide health problem. Dietary tocotrienols (TT) have been shown to improve obesity-associated metabolic disorders, such as hypercholesterolemia, hyperglycemia, and gut dysbiosis. This study examined the hypothesis that the antioxidant capacity of TT alters metabolites of oxidative stress and improves systemic metabolism. C57BL/6J mice were fed either a high-fat diet (HFD control) or HFD supplemented with 800 mg annatto-extracted TT/kg (HFD+TT800) for 14 weeks. Sera from obese mice were examined by non-targeted metabolite analysis using UHPLC/MS. Compared to the HFD group, the HFD+TT800 group had higher levels of serum metabolites, essential amino acids (lysine and methionine), sphingomyelins, phosphatidylcholine, lysophospholipids, and vitamins (pantothenate, pyridoxamine, pyridoxal, and retinol). TT-treated mice had lowered levels of serum metabolites, dicarboxylic fatty acids, and inflammatory/oxidative stress markers (trimethylamine N-oxide, kynurenate, 12,13-DiHOME, and 13-HODE + 9-HODE) compared to the control. The results suggest that TT supplementation lowered inflammation and oxidative stress (oxidized glutathione and GSH/GSSH) and improved macronutrient metabolism (carbohydrates) in obese mice. Thus, TT actions on metabolites were beneficial in reducing obesity-associated hypercholesterolemia/hyperglycemia. The effects of a non-toxic dose of TT in mice support the potential for clinical applications in obesity and metabolic disease.

Acylphloroglucinol and tocotrienol derivatives from the fruits of Garcinia paucinervis.

Three undescribed polycyclic polyprenylated acylphloroglucinols (PPAPs) and three tocotrienols derivatives, named as paucinochymol A-F (1-3 and 10-12), together with six known PPAPs, were isolated from the fruits of Garcinia paucinervis. Their structures and absolute configurations were determined by extensive NMR analysis and electronic circular dichroism (ECD) calculation methods. Paucinochymol A (1) is the first compound of this type featuring a ω-isogeranyl with tetrahydrofuran unit at C-1. Paucinochymols D and E (4-5) belong to rare tocotrienol with one glorious macrocyclic and an ortho-quinone moiety, respectively. The antiproliferative and anti-inflammatory activities of all isolates were tested. Four PPAPs exhibited weak inhibitory activities against three human cancer cell lines (HepG2, T98, MCF-7) with IC50 values ranging from 10.0 to 16.0 µM. Paucinochymol D (10) displayed moderate inhibitory effects against nitric oxide (NO) production with the IC50 value of 19.8 µM.

Safety and Neuroprotective Efficacy of Palm Oil and Tocotrienol-Rich Fraction from Palm Oil: A Systematic Review.

BACKGROUND: Several natural products have been reported to elicit beneficial effects against neurodegenerative disorders due to their vitamin E contents. However, the neuroprotective efficacy of palm oil or its tocotrienol-rich fraction (TRF) from the pre-clinical cell and animal studies have not been systematically reviewed. METHODS: The protocol for this systematic review was registered in "PROSPERO" (CRD42019150408). This review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The Medical Subject Heading (MeSH) descriptors of PubMed with Boolean operators were used to construct keywords, including ("Palm Oil"[Mesh]) AND "Nervous System"[Mesh], ("Palm Oil"[Mesh]) AND "Neurodegenerative Diseases"[Mesh], ("Palm Oil"[Mesh]) AND "Brain"[Mesh], and ("Palm Oil"[Mesh]) AND "Cognition"[Mesh], to retrieve the pertinent records from PubMed, Scopus, Web of Science and ScienceDirect from 1990 to 2019, while bibliographies, ProQuest and Google Scholar were searched to ensure a comprehensive identification of relevant articles. Two independent investigators were involved at every stage of the systematic review, while discrepancies were resolved through discussion with a third investigator. RESULTS: All of the 18 included studies in this review (10 animal and eight cell studies) showed that palm oil and TRF enhanced the cognitive performance of healthy animals. In diabetes-induced rats, TRF and α-tocotrienol enhanced cognitive function and exerted antioxidant, anti-apoptotic and anti-inflammatory activities, while in a transgenic Alzheimer's disease (AD) animal model, TRF enhanced the cognitive function and reduced the deposition of ß-amyloid by altering the expression of several genes related to AD and neuroprotection. In cell studies, simultaneous treatment with α-tocotrienols and neurotoxins improved the redox status in neuronal cells better than ϒ- and δ-tocotrienols. Both pre-treatment and post-treatment with α-tocotrienol relative to oxidative insults were able to enhance the survival of neuronal cells via increased antioxidant responses. CONCLUSIONS: Palm oil and its TRF enhanced the cognitive functions of healthy animals, while TRF and α-tocotrienol enhanced the cognitive performance with attenuation of oxidative stress, neuroinflammation and apoptosis in diabetes-induced or transgenic AD animal models. In cell studies, TRF and α-tocotrienol exerted prophylactic neuroprotective effects, while α-tocotrienol exerted therapeutic neuroprotective effects that were superior to those of ϒ- and δ-tocotrienol isomers.

A tocotrienol quinone dimer and xanthones from the leaf extract of Garcinia nigrolineata.

Four new compounds (1-4) together with six known compounds (5-10) were isolated from the leaf extract of Garcinia nigrolineata. Compound 4 is a rare tocotrienol quinone dimer. The structures were elucidated based on NMR and MS data. All isolated compounds were evaluated for their antibacterial activities.

Lipid Characterization of Chinese Wild Hazelnuts (Corylus mandshurica Maxim.).

To develop and use the hazelnut, the main composition, and the physicochemical characteristics, fatty acid compositions, triacylglycerol (TAG) distribution and tocol contents of Hazelnut (Corylus mandshurica Maxim.) which growing in Changbai mountain of Jilin province (HO1) and Anshan city of Liaoning province (HO2) in China were investigated, and the comparative study between the two hazelnut oils and American hazelnut / Turkish hazelnut were also explored. The content of crude lipid and protein in HO1 and HO2 were approximately 54% and 17%, 55% and 16%, respectively. The two hazelnut oils were abundant in unsaturated fatty acids, with the primary unsaturated fatty acids were oleic acid (78%-80%) and linoleic acid (14-16%), which accounted for above 90% of the oils. Therefore, both of the hazelnut oils were important sources of essential fatty acid. In addition, the main saturated fatty acid of the two hazelnut oils were palmitic acid (3%) and stearic acid (1-2%). The main triacylglycerols (TGA) profile were dioleolinolein (OOL), oleodilinolein (OLL) and triolein (OOO). The contents of tocol were 574.44 µg/g, 647.49 µg/g oil in HO1 and HO2, respectively, both of them were higher than that of grape seed oils (454 µg/g), olive oils (209 µg/g) and walnut oils (255 µg/g). The total phytosterol contents were over 2000µg/g and ß-sitosterol was the most predominant sterol in two oils.

Some Strategies for Utilization of Rice Bran Functional Lipids and Phytochemicals.

Rice bran contains a great amount of functional lipids and phytochemicals including γ-oryzanols, tocotrienols, and tocopherols. However, utilization of those compounds is limited and needs some proven guidelines for better implementation. We introduce some effective strategies for the utilization of rice functional lipids, including an introduction of pigmented rice varieties for better bioactive compounds, biofortification of rice tocotrienols, plasma technology for improving rice phytochemicals, supercritical CO2 extraction of high quality rice bran oil, and an example on the development of tocotrienol-fortified foods.

Effects of Extraction Methods on Phytochemicals of Rice Bran Oils Produced from Colored Rice.

Rice bran oil (RBO) especially from colored rice is rich in phytochemicals and has become popular in food, cosmetic, nutraceutical and pharmaceutical applications owing to its offering health benefits. This study determined the contents of phytochemicals including oryzanols, phytosterols, tocopherols (Toc) and tocotrienols (T3) in RBOs extracted using different methods namely cold-press extraction (CPE), solvent extraction (SE) and supercritical CO2 extraction (SC-CO2). Two colored rice, Red Jasmine rice (RJM, red rice) and Hom-nin rice (HN, black rice), were studied in comparison with the popular Thai fragrant rice Khao Dawk Mali 105 (KDML 105, white rice). RBOs were found to be the rich source of oryzanols, phytosterols, Toc and T3. Rice varieties had a greater effect on the phytochemicals concentrations than extraction methods. HN rice showed the significantly highest concentration of all phytochemicals, followed by RJM and KDML 105 rice, indicating that colored rice contained high concentration of phytochemicals in the oil than non-colored rice. The RBO samples extracted by the CPE method had a greater concentration of the phytochemicals than those extracted by the SC-CO2 and SE methods, respectively. In terms of phytochemical contents, HN rice extracted using CPE method was found to be the best.

Effect of tocotrienol from Bixa orellana (annatto) on bone microstructure, calcium content, and biomechanical strength in a model of male osteoporosis induced by buserelin.

BACKGROUND: Patients receiving androgen deprivation therapy experience secondary hypogonadism, associated bone loss, and increased fracture risk. It has been shown that tocotrienol from Bixa orellana (annatto) prevents skeletal microstructural changes in rats experiencing primary hypogonadism. However, its potential in preventing bone loss due to androgen deprivation therapy has not been tested. This study aimed to evaluate the skeletal protective effects of annatto tocotrienol using a buserelin-induced osteoporotic rat model. METHODS: Forty-six male Sprague Dawley rats aged 3 months were randomized into six groups. The baseline control (n=6) was sacrificed at the onset of the study. The normal control (n=8) received corn oil (the vehicle of tocotrienol) orally daily and normal saline (the vehicle of buserelin) subcutaneously daily. The buserelin control (n=8) received corn oil orally daily and subcutaneous buserelin injection (75 µg/kg) daily. The calcium control (n=8) was supplemented with 1% calcium in drinking water and daily subcutaneous buserelin injection (75 µg/kg). The remaining rats were given daily oral annatto tocotrienol at 60 mg/kg (n=8) or 100 mg/kg (n=8) plus daily subcutaneous buserelin injection (75 µg/kg) (n=8). At the end of the experiment, the rats were euthanized and their blood, tibia, and femur were harvested. Structural changes of the tibial trabecular and cortical bone were examined using X-ray micro-computed tomography. Femoral bone calcium content and biomechanical strength were also evaluated. RESULTS: Annatto tocotrienol at 60 and 100 mg/kg significantly prevented the deterioration of trabecular bone and cortical thickness in buserelin-treated rats (P<0.05). Both doses of annatto tocotrienol also improved femoral biomechanical strength and bone calcium content in buserelin-treated rats (P<0.05). The effects of annatto tocotrienol were comparable to calcium supplementation. CONCLUSION: Annatto tocotrienol supplementation is effective in preventing degeneration of the bone induced by buserelin. Therefore, it is a potential antiosteoporotic agent for men receiving androgen deprivation therapy.

Effects of tocotrienol from Bixa orellana (annatto) on bone histomorphometry in a male osteoporosis model induced by buserelin.

INTRODUCTION: Osteoporosis is a debilitating skeletal side effect of androgen deprivation therapy based on gonadotropin-releasing hormone (GnRH) agonist in men. Tocotrienol from Bixa orellana (annatto) has been demonstrated to offer protection against osteoporosis by exerting anabolic effects on bone. Thus, it may prevent osteoporosis among GnRH agonist users. OBJECTIVE: This study aimed to determine the effectiveness of annatto-tocotrienol on the bone turnover markers and bone histomorphometry in a model of male osteoporosis induced by buserelin (a GnRH agonist). METHODS: Forty-six three-months-old male Sprague-Dawley rats (three months old; 300-350 g) were randomly divided into six groups. The baseline control group (n = 6) was sacrificed at the onset of the study. The normal control group (n = 8) received corn oil (the vehicle of tocotrienol) orally daily and normal saline (the vehicle of buserelin) subcutaneously daily. The buserelin control (n = 8) received corn oil orally daily and subcutaneous buserelin injection 75 µg/kg/day daily. The calcium control (n = 8) received 1% calcium in drinking water and subcutaneous buserelin injection 75 µg/kg/day. The remaining rats were treated with two different treatments, i.e., (1) oral annatto tocotrienol at 60 mg/kg/day plus subcutaneous buserelin injection 75 µg/kg/day (n = 8); (2) oral annatto tocotrienol at 100 mg/kg/day plus subcutaneous buserelin injection 75 µg/kg/day (n = 8). The rats were injected with calcein twice before being sacrificed to label the bones. The rats were euthanized, and their blood and right femur were harvested at the end of the treatment for bone turnover markers and bone histomorphometry examination. RESULTS: Both serum osteocalcin and C-telopeptide of type 1 collagen were not significantly different between treated groups and buserelin control (P > 0.05). The buserelin control group had a significantly lower bone volume and higher eroded surface compared with the normal control group (P < 0.05). Both groups treated with annatto tocotrienol (60 mg/kg/day and 100 mg/kg/day) had significantly higher bone volume, trabecular thickness and osteoblast number, as well as a significantly lower single-labelled surface compared with the buserelin control (P < 0.05). Only rats treated with annatto tocotrienol 60 mg/kg/day had a significantly higher double-labelled surface compared with buserelin control (P < 0.05). CONCLUSION: Annatto tocotrienol can prevent trabecular bone loss by increasing the mineralising surface and osteoblasts number. Thus, it has a potential role in preventing bone loss in men using GnRH agonist.

Annatto-derived tocotrienol stimulates osteogenic activity in preosteoblastic MC3T3-E1 cells: a temporal sequential study.

PURPOSE: Annatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis. However, detailed studies of the effects of AnTT on preosteoblastic cells were limited. This study was conducted to investigate the osteogenic effect of AnTT on preosteoblast MC3T3-E1 cells in a time-dependent manner. MATERIALS AND METHODS: Murine MC3T3-E1 preosteoblastic cells were cultured in the different concentrations of AnTT (0.001-1 µg/mL) up to 24 days. Expression of osteoblastic differentiation markers was measured by qPCR (osterix [OSX], collagen 1 alpha 1 [COL1α1], alkaline phosphatase [ALP], and osteocalcin [OCN]) and by fluorometric assay for ALP activity. Detection of collagen and mineralized nodules was done via Direct Red staining and Alizarin Red staining, respectively. RESULTS: The results showed that osteoblastic differentiation-related genes, such as OSX, COL1α1, ALP, and OCN, were significantly increased in the AnTT-treated groups compared to the vehicle group in a time-dependent manner (P<0.05). Type 1 collagen level was increased from day 3 to day 15 in the AnTT-treated groups, while ALP activity was increased from day 9 to day 21 in the AnTT-treated groups (P<0.05). Enhanced mineralization was observed in the AnTT-treated groups via increasing Alizarin Red staining from day 3 to day 21 (P<0.05). CONCLUSION: Our results suggest that AnTT enhances the osteogenic activity by promoting the bone formation-related genes and proteins in a temporal and sequential manner.