Numerous bioactive compounds have cytotoxic properties towards cancer cells. However, most studies have used single compounds when bioactives may target different pathways and exert greater cytotoxic effects when used in combination. Therefore, the objective of this study was to determine the anti-proliferative effect of γ-tocotrienol (γ-T3) and 6-gingerol (6G) in combination by evaluating apoptosis and active caspase-3 in HT-29 and SW837 colorectal cancer cells. MTS assays were performed to determine the anti-proliferative and cytotoxicity effect of γ-T3 (0-150 µg/mL) and 6G (0-300 µg/mL) on the cells. The half maximal inhibitory concentration (IC50) value of 6G+ γ-T3 for HT-29 was 105 + 67 µg/mL and for SW837 it was 70 + 20 µg/mL. Apoptosis, active caspase-3 and annexin V FITC assays were performed after 24 h of treatment using flow cytometry. These bioactives in combination showed synergistic effect on HT-29 (CI: 0.89 ± 0.02,) and SW837 (CI: 0.79 ± 0.10) apoptosis was increased by 21.2% in HT-29 and 55.4% in SW837 (p < 0.05) after 24 h treatment, while normal hepatic WRL-68 cells were unaffected. Increased apoptosis by the combined treatments was also observed morphologically, with effects like cell shrinkage and pyknosis. In conclusion, although further studies need to be done, γ-T3 and 6G when used in combination act synergistically increasing cytotoxicity and apoptosis in cancer cells.
Despite the progress in colon cancer treatment, relapse is still a major obstacle. Hence, new drugs or drug combinations are required in the battle against colon cancer. α-Mangostin and betulinic acid (BA) are cytotoxic compounds that work by inducing the mitochondrial apoptosis pathway, and cisplatin is one of the most potent broad spectrum anti-tumor agents. This study aims to investigate the enhancement of BA cytotoxicity by α-mangostin, and the cytoprotection effect of α-mangostin and BA on cisplatin-induced cytotoxicity on HCT 116 human colorectal carcinoma cells. Cytotoxicity was investigated by the XTT cell proliferation test, and the apoptotic effects were investigated on early and late markers including caspases-3/7, mitochondrial membrane potential, cytoplasmic shrinkage, and chromatin condensation. The effect of α-mangostin on four signalling pathways was also investigated by the luciferase assay. α-Mangostin and BA were more cytotoxic to the colon cancer cells than to the normal colonic cells, and both compounds showed a cytoprotective effect against cisplatin-induced cytotoxicity. On the other hand, α-mangostin enhanced the cytotoxic and apoptotic effects of BA. Combination therapy hits multiple targets, which may improve the overall response to the treatment, and may reduce the likelihood of developing drug resistance by the tumor cells. Therefore, α-mangostin and BA may provide a novel combination for the treatment of colorectal carcinoma. The cytoprotective effect of the compounds against cisplatin-induced cytotoxicity may find applications as chemopreventive agents against carcinogens, irradiation and oxidative stress, or to neutralize cisplatin side effects.
The present study investigates the involvement of the l-arginine-Nitric Oxide-cGMP-K⁺ ATP pathways responsible for the action of anti-allodynic and antihyperalgesic activities of zerumbone in chronic constriction injury (CCI) induced neuropathic pain in mice. The role of l-arginine-NO-cGMP-K⁺ was assessed by the von Frey and the Randall-Selitto tests. Both allodynia and hyperalgesia assessments were carried out on the 14th day post CCI, 30 min after treatments were given for each respective pathway. Anti-allodynic and antihyperalgesic effects of zerumbone (10 mg/kg, i.p) were significantly reversed by the pre-treatment of l-arginine (10 mg/kg), 1H [1,2,4]Oxadiazole[4,3a]quinoxalin-1-one (ODQ), a soluble guanosyl cyclase blocker (2 mg/kg i.p.) and glibenclamide (ATP-sensitive potassium channel blocker) (10 mg/kg i.p.) (p < 0.05). Taken together, these results indicate that systemic administration of zerumbone produces significant anti-allodynic and antihyperalgesic activities in neuropathic pain in mice possibly due to involvement of the l-arginine-NO-cGMP-PKG-K⁺ ATP channel pathways in CCI model.
Elaeis guineensis Jacq (Arecaceae) is one of the plants that are central to the lives of traditional societies in West Africa. It has been reported as a traditional folkloric medicine for a variety of ailments. The plant leaves are also used in some parts of Africa for wound healing, but there are no scientific reports on any wound healing activity of the plant.
The aim of this research was to identify the volatile metabolites produced in different organs (leaves, stem and roots) of Polygonum minus, an important essential oil producing crop in Malaysia. Two methods of extraction have been applied: Solid Phase Microextraction (SPME) and hydrodistillation coupled with Gas Chromatography-Mass Spectrometry (GC-MS). Approximately, 77 metabolites have been identified and aliphatic compounds contribute significantly towards the aroma and flavour of this plant. Two main aliphatic compounds: decanal and dodecanal were found to be the major contributor. Terpenoid metabolites were identified abundantly in leaves but not in the stem and root of this plant. Further studies on antioxidant, total phenolic content, anticholinesterase and antimicrobial activities were determined in the essential oil and five different extracts. The plant showed the highest DPPH radical scavenging activity in polar (ethanol) extract for all the tissues tested. For anti-acetylcholinesterase activity, leaf in aqueous extract and methanol extract showed the best acetylcholinesterase inhibitory activities. However, in microbial activity, the non-polar extracts (n-hexane) showed high antimicrobial activity against Methicillin-resistant Staphylococcus aureus (MRSA) compared to polar extracts. This study could provide the first step in the phytochemical profiles of volatile compounds and explore the additional value of pharmacology properties of this essential oil producing crop Polygonum minus.
This project studied the effect of vermicompost application on the composition of bioactive anthocyanin and phenolic compounds, and the antioxidant activity of Clinacanthus nutans. The correlation between the bioactive constituents and antioxidant capacity was also evaluated. In this project, a field study was conducted using a randomized complete block design (RCBD) with four treatment groups, including control plants (CC), plants supplied with chemical fertilizer (CF), plants supplied with vermicompost (VC), and plants supplied with mixed fertilizer (MF). The leaves of C. nutans from all treatment groups were harvested, subjected to solvent extraction, and used for quantification of total anthocyanin content (TAC), total phenolic content (TPC), and total flavonoid content (TFC). The initial antioxidant activity of the extracts was evaluated using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, as well as after two and four weeks of storage at -20 °C and 4 °C. Data analysis showed that CC plants contained the highest TAC (2180.14 ± 338.43 µg/g dry weight) and TFC (276.25 ± 3.09 mg QE/g dry weight). On the other hand, CF plants showed the highest TPC (181.53 ± 35.58 mg GAE/g dry weight). Moreover, we found that CC plants had the highest antioxidant potential against DPPH radicals whereas MF plants showed the lowest antioxidant potential. After four weeks of extract storage at -20 °C and 4 °C, the TPC, TFC, TAC, and antioxidant potential of the extracts decreased. Extracts from VC showed the lowest percentage of total phenolic and total flavonoid loss after extract storage at -20 °C and 4 °C compared with other plant extracts. At this juncture, it could be deduced that the application of vermicompost had little effect on the expression of phenolics, flavonoids, or anthocyanin in C. nutans. However, the extract from plants treated with vermicompost (VC and MF) showed better stability compared with CC and CF after extract storage at different temperatures.
The effects of different drying methods (freeze drying, vacuum oven drying, and shade drying) on the phytochemical constituents associated with the antioxidant activities of Z. officinale var. rubrum Theilade were evaluated to determine the optimal drying process for these rhizomes. Total flavonoid content (TFC), total phenolic content (TPC), and polyphenol oxidase (PPO) activity were measured using the spectrophotometric method. Individual phenolic acids and flavonoids, 6- and 8-gingerol and shogaol were identified by ultra-high performance liquid chromatography method. Ferric reducing antioxidant potential (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were used for the evaluation of antioxidant activities. The highest reduction in moisture content was observed after freeze drying (82.97%), followed by vacuum oven drying (80.43%) and shade drying (72.65%). The highest TPC, TFC, and 6- and 8-shogaol contents were observed in samples dried by the vacuum oven drying method compared to other drying methods. The highest content of 6- and 8-gingerol was observed after freeze drying, followed by vacuum oven drying and shade drying methods. Fresh samples had the highest PPO activity and lowest content of flavonoid and phenolic acid compounds compared to dried samples. Rhizomes dried by the vacuum oven drying method represent the highest DPPH (52.9%) and FRAP activities (566.5 μM of Fe (II)/g DM), followed by freeze drying (48.3% and 527.1 μM of Fe (II)/g DM, respectively) and shade drying methods (37.64% and 471.8 μM of Fe (II)/g DM, respectively) with IC50 values of 27.2, 29.1, and 34.8 μg/mL, respectively. Negative and significant correlations were observed between PPO and antioxidant activity of rhizomes. Vacuum oven dried rhizomes can be utilized as an ingredient for the development of value-added food products as they contain high contents of phytochemicals with valuable antioxidant potential.
Varied pharmacological responses have been reported for mitragynine in the literature, but no supportive scientific explanations have been given for this. These studies have been undertaken without a sufficient understanding of the physicochemical properties of mitragynine. In this work a UV spectrophotometer approach and HPLC-UV method were employed to ascertain the physicochemical properties of mitragynine. The pKa of mitragynine measured by conventional UV (8.11 ± 0.11) was in agreement with the microplate reader determination (8.08 ± 0.04). Mitragynine is a lipophilic alkaloid, as indicated by a logP value of 1.73. Mitragynine had poor solubility in water and basic media, and conversely in acidic environments, but it is acid labile. In an in vitro dissolution the total drug release was higher for the simulated gastric fluid but was prolonged and incomplete for the simulated intestinal fluid. The hydrophobicity, poor water solubility, high variability of drug release in simulated biological fluids and acid degradable characteristics of mitragynine probably explain the large variability of its pharmacological responses reported in the literature. The determined physicochemical properties of mitragynine will provide a basis for developing a suitable formulation to further improve its solubility, stability and oral absorption for better assessment of this compound in preclinical studies.
Ultrasound-assisted extraction (UAE) with ethanol was used to extract the compounds responsible for the antioxidant activities of Misai Kucing (Orthosiphon stamineus). Response surface methodology (RSM) was used to optimize four independent variables: ethanol concentration (%), amplitude (%), duty cycle (W/s) and extraction time (min). Antioxidant compounds were determined by total phenolic content and total flavonoid content to be 1.4 g gallic acid equivalent/100 g DW and 45 g catechin equivalent/100 g DW, respectively. Antioxidant activities were evaluated using the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) radical scavenging capacity assay and the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging capacity assay to be 1,961.3 and 2,423.3 µmol Trolox Equivalent Antioxidant Capacity (TEAC)/100 g DW, respectively. Based on the optimal conditions, experimental values were reported to be close to the predicted value by RSM modeling (p>0.05), indicating the suitability of UAE for extracting the antioxidants of Misai Kucing. Rosmarinic acid, kaempferol-rutinoside and sinesetine were identified by high performance liquid chromatography-mass spectrometry.
The main objective of the current work was to evaluate the suitability of ultrasound-assisted extraction (UAE) for the recovery of oil from papaya seed as compared to conventional extraction techniques (i.e., Soxhlet extraction (SXE) and solvent extraction (SE)). In the present study, the recovery yield, fatty acid composition and triacylglycerol profile of papaya seed oil obtained from different extraction methods and conditions were compared. Results indicated that both solvent extraction (SE, 12 h/25 °C) and ultrasound-assisted extraction (UAE) methods recovered relatively high yields (79.1% and 76.1% of total oil content, respectively). Analysis of fatty acid composition revealed that the predominant fatty acids in papaya seed oil were oleic (18:1, 70.5%-74.7%), palmitic (16:0, 14.9%-17.9%), stearic (18:0, 4.50%-5.25%), and linoleic acid (18:2, 3.63%-4.6%). Moreover, the most abundant triacylglycerols of papaya seed oil were triolein (OOO), palmitoyl diolein (POO) and stearoyl oleoyl linolein (SOL). In this study, ultrasound-assisted extraction (UAE) significantly (p < 0.05) influenced the triacylglycerol profile of papaya seed oil, but no significant differences were observed in the fatty acid composition of papaya seed oil extracted by different extraction methods (SXE, SE and UAE) and conditions.
Honey is a good source of several important chemical compounds and antioxidants and is harvested throughout the year. However, no study has determined how their contents change over the years. The aim of the present research was to investigate the changes in the phenolics, flavonoids and antioxidant properties, as well as other physicochemical properties, of Malaysian acacia honey collected during different months during a two year period. The DPPH (1,1-diphenyl-2-picrylhydrazyl) and FRAP (ferric reducing antioxidant power) methods were used to determine the total antioxidant activity of the honey samples. Generally, honey samples collected in the beginning and the middle of the year tended to have higher sugar content, which may be attributed to its high acidic nature and low moisture content. There was a gradual increase in the phenolic content of the acacia honey samples collected between September 2010 and December 2010. The honey sample collected at the beginning of the year (January) showed the highest color intensity and was dark amber in color. It also contained the highest concentration of phenolic compounds (341.67 ± 2.94 mg(gallic acid)/kg), the highest flavonoid content (113.06 ± 6.18 mg(catechin)/kg) and the highest percentage of DPPH inhibition and the highest FRAP value, confirming its high antioxidant potential. There was a positive correlation between DPPH and total phenolic content, suggesting that phenolic compounds are the strongest contributing factor to the radical scavenging activity of Malaysian acacia honeys. Overall, our results indicated that there were significant seasonal variations in the antioxidant potentials of honey over the two year period and the time of honey collection affects its physicochemical properties. Therefore, acacia honey from Malaysia should ideally be collected during the dry season, particularly in the months of January, May and June.
The air-dried powdered stem bark of Calophyllum nodusum (Guttiferea) collected from Sandakan (Sabah, Malaysia), was extracted sequentially with hexane, chloroform and methanol. The solvents were removed by rotary evaporator to give dark viscous extracts. Detailed and repeated chromatographic separation of the extracts lead to isolation of two new xanthones, identified as nodusuxanthone and trapezifolixanthone A. Other common terpenoids such as betulinic acid, lupeol, stigmasterol and friedelin were also isolated from the extracts and identified. The structures of the compounds were established by detailed spectral analysis and comparison with previously reported data.
A detailed chemical study on the ethyl acetate and methanol extracts of the stem bark of Garcinia mangostana resulted in the successful isolation of one new prenylated xanthone, mangaxanthone B (1), one new benzophenone, mangaphenone (2), and two known xanthones, mangostanin (3) and mangostenol (4). The structures of these compounds were elucidated through analysis of their spectroscopic data obtained using 1D and 2D NMR and MS techniques.
Extraction and chromatographic separation of the extracts of dried stem barks of Glycosmis macrantha lead to isolation of two new acridone alkaloids, macranthanine and 7-hydroxynoracronycine, and a known acridone, atalaphyllidine. The structures of these alkaloids were determined by detailed spectral analysis and also by comparison with reported data.
Preliminary investigations on 14 plant extracts (obtained by ethanolic and aqueous extraction) identified those having high antioxidant and a significant total phenolic content. Antihyperglycemic, α-amylase and α-glucosidase inhibition activities were also observed. A correlation between the antihyperglycemic activity, total phenolic content and antioxidant (DPPH scavenging) activity was established. To further substantiate these findings, the possibility of tannins binding non-specifically to enzymes and thus contributing to the antihyperglycemic activity was also investigated. Our study clearly indicated that the antihyperglycemic activity observed in the plant extracts was indeed not due to non-specific tannin absorption.
Leaves of Centella asiatica (Centella) were analysed for their triterpene composition and bioactivity such as collagen enhancement, antioxidant, anticellulite and UV protection capacity properties. Triterpenes of Centella were measured using HPLC-PAD on an Excil ODS 5 mm (C18) column for the simultaneous determination of asiatic acid, madecassic acid, asiaticoside and madecassoside. Centella was found to contain significant amounts of madecassoside (3.10 ± 4.58 mg/mL) and asiaticoside (1.97 ± 2.65 mg/mL), but was low in asiatic and madecassic acid. The highest collagen synthesis was found at 50 mg/mL of Centella extracts. The antioxidant activity of Centella (84%) was compared to grape seed extract (83%) and Vitamin C (88%). Its lipolytic activity was observed by the release of glycerol (115.9 µmol/L) at 0.02% concentration. Centella extracts exhibited similar UV protection effect to OMC at 10% concentration. In view of these results, the potential application of Centella in food and pharmaceutical industries is now widely open.
Naturally occurring anthraquinones, damnacanthal (1) and nordamnacanthal (2) were synthesized with modified reaction steps and investigated for their cytotoxicity against the MCF-7 and K-562 cancer cell lines, respectively. Intermediate analogues 2-bromomethyl-1,3-dimethoxyanthraquinone (5, IC50 = 5.70 ± 0.21 and 8.50 ± 1.18 mg/mL), 2-hydroxymethyl-1,3-dimethoxyanthraquinone (6, IC50 = 12.10 ± 0.14 and 14.00 ± 2.13), 2-formyl-1,3-dimethoxyantharquinone (7, IC50 = 13.10 ± 1.02 and 14.80 ± 0.74), 1,3-dimethoxy-2-methylanthraquinone (4, IC50 = 9.40 ± 3.51 and 28.40 ± 2.33), and 1,3-dihydroxy-2-methylanthraquinone (3, IC50 = 25.60 ± 0.42 and 28.40 ± 0.79) also exhibited moderate cytotoxicity against MCF-7 and K-562 cancer cell lines, respectively. Other structurally related compounds like 1,3-dihydroxyanthraquinone (13a, IC50 = 19.70 ± 0.35 and 14.50 ± 1.28), 1,3-dimethoxyanthraquinone (13b, IC50 = 6.50 ± 0.66 and 5.90 ± 0.95) were also showed good cytotoxicity. The target compound damnacanthal (1) was found to be the most cytotoxic against the MCF-7 and K-562 cancer cell lines, with IC50 values of 3.80 ± 0.57 and 5.50 ± 1.26, respectively. The structures of all compounds were elucidated with the help of detailed spectroscopic techniques.
Plant bioactives -gingerol (GING), epigallocatechin gallate (EGCG) and asiaticoside (AS) and vitamin E, such as tocotrienol-rich fraction (TRF), have been reported to possess anticancer activity. In this study, we investigated the apoptotic properties of these bioactive compounds alone or in combination on glioma cancer cells. TRF, GING, EGCG and AS were tested for cytotoxicity on glioma cell lines 1321N1 (Grade II), SW1783 (Grade III) and LN18 (Grade IV) in culture by the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) assay. With the exception of AS, combinations of two compounds were tested, and the interactions of each combination were evaluated by the combination index (CI) using an isobologram. Different grades of glioma cancer cells showed different cytotoxic responses to the compounds, where in 1321N1 and LN18 cells, the combination of EGCG + GING exhibited a synergistic effect with CI = 0.77 and CI = 0.55, respectively. In contrast, all combinations tested (TRF + GING, TRF + EGCG and EGCG + GING) were found to be antagonistic on SW1783 with CI values of 1.29, 1.39 and 1.39, respectively. Combined EGCG + GING induced apoptosis in both 1321N1 and LN18 cells, as evidenced by Annexin-V FITC/PI staining and increased active caspase-3. Our current data suggests that the combination of EGCG + GING synergistically induced apoptosis and inhibits the proliferation 1321N1 and LN18 cells, but not SW1783 cells, which may be due to their different genetic profiles.
There has been a growing interest in naturally occurring compounds from traditional medicine with anti-cancer potential. Nigella sativa (black seed) is one of the most widely studied plants. This annual herb grows in countries bordering the Mediterranean Sea and India. Thymoquinone (TQ) is an active ingredient isolated from Nigella sativa. The anti-cancer effect of TQ, via the induction of apoptosis resulting from mitochondrial dysfunction, was assessed in an acute lymphocyte leukemic cell line (CEMss) with an IC50 of 1.5 µg/mL. A significant increase in chromatin condensation in the cell nucleus was observed using fluorescence analysis. The apoptosis was then confirmed by Annexin V and an increased number of cellular DNA breaks in treated cells were observed as a DNA ladder. Treatment of CEMss cells with TQ encouraged apoptosis with cell death-transducing signals by a down-regulation of Bcl-2 and up-regulation of Bax. Moreover, the significant generation of cellular ROS, HSP70 and activation of caspases 3 and 8 were also observed in the treated cells. The mitochondrial apoptosis was clearly associated with the S phase cell cycle arrest. In conclusion, the results from the current study indicated that TQ could be a promising agent for the treatment of leukemia.
The complexation reactions between the macrocyclic ionophore, p-isopropylcalixarene and Cs+ cation were studied in dimethylsulfoxide-acetonitrile (DMSO-AN) binary non-aqueous solvents at different temperatures using a conductometry method. The conductance data show that the stoichiometry of the (p-isopropylcalix-arene·Cs)+ complex in all binary mixed solvents is 1:1. The stability of the complexes is affected by the composition of the binary solvent media and a non-linear behavior was observed for changes of log K(f) of the complex versus the composition of the binary mixed solvents. The thermodynamic parameters (DH°(c) and DS°(c)) for formation of (p-isopropyl-calixarene·Cs)+ complex were obtained from temperature dependence of the stability constant and the obtained results show that the (p-isopropylcalixarene·Cs)+ complex is enthalpy destabilized, but entropy stabilized, and the values of the mentioned parameters are affected strongly by the nature and composition of the binary mixed solvents.