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.
BACKGROUND: Star fruit (Averrhoa carambola) is a well-known plant in Malaysia which bears a great significance in traditional medicine.
OBJECTIVES: This study aimed to evaluate the antihyperlipidemic effect, antioxidant potential and cytotoxicity of aqueous and methanolic extracts of ripe and unripe fruits, leaves and stem of A. carambola.
METHODS: Antihyperlipidemic activity was assessed in poloxamer-407 (P-407) induced acute hyperlipidemic rat's model. The antioxidant activity was assessed in vitro using 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging, 1-diphenyl-2-dipicrylhydrazyl radical scavenging (DPPH) and ferric reducing antioxidant power (FRAP) assays. In addition, cytotoxicity of A. carambola extracts was assessed using MTS assay on four leukemic cell lines (human colon cancer, human promyeloid leukemia, erythroid leukemia, acute myeloid leukemia) and one normal cell (human umbilical vein endothelial cells).
RESULTS: Methanolic extract of leaves had the most potent antihyperlipidemic activity in P-407 model, whereby it significantly reduced serum levels of total cholesterol (P<0.01), triglycerides (P<0.01), low-density lipoprotein (P<0.05), verylow- density lipoprotein (P<0.01) and atherogenic index (P<0.01). On the other hand, methanolic extracts of A. carambola stem and leaves showed the strongest antioxidant activity. Total phenolic and flavonoid contents of the extracts exhibited significant correlations with antioxidant but not with antihyperlipidemic activities. All plant parts showed no cytotoxic effect on the selected cancer or normal cell lines.
CONCLUSION: Antihyperlipidemic activity of different parts of A. carambola is greatly affected by extraction solvents used. Methanolic extract of A. carambola leaves exhibited higher antihyperlipidemic and antioxidant potentials compared to other parts of the plant.
Continuing our interest in the Uncaria genus, the phytochemistry and the in-vitro α-glucosidase inhibitory activities of Malaysian Uncaria cordata var. ferruginea were investigated. The phytochemical study of this plant, which employed various chromatographic techniques including recycling preparative HPLC, led to the isolation of ten compounds with diverse structures comprising three phenolic acids, two coumarins, three flavonoids, a terpene and an iridoid glycoside. These constituents were identified as 2-hydroxybenzoic acid or salicylic acid (1), 2,4-dihydroxybenzoic acid (2), 3,4-dihydroxybenzoic acid (3), scopoletin or 7-hydroxy-6-methoxy-coumarin (4), 3,4-dihydroxy-7-methoxycoumarin (5), quercetin (6), kaempferol (7), taxifolin (8), loganin (9) and β-sitosterol (10). Structure elucidation of the compounds was accomplished with the aid of 1D and 2D Nuclear Magnetic Resonance (NMR) spectral data and Ultraviolet-Visible (UV-Vis), Fourier Transform Infrared (FTIR) spectroscopy and mass spectrometry (MS). In the α-glucosidase inhibitory assay, the crude methanolic extract of the stems of the plant and its acetone fraction exhibited strong α-glucosidase inhibition activity of 87.7% and 89.2%, respectively, while its DCM fraction exhibited only moderate inhibition (75.3%) at a concentration of 1 mg/mL. The IC50 values of both fractions were found to be significantly lower than the standard acarbose suggesting the presence of potential α-glucosidase inhibitors. Selected compounds isolated from the active fractions were then subjected to α-glucosidase assay in which 2,4-dihydroxybenzoic acid and quercetin showed strong inhibitory effects against the enzyme with IC50 values of 549 and 556 μg/mL compared to acarbose (IC50 580 μg/mL) while loganin and scopoletin only showed weak α-glucosidase inhibition of 44.9% and 34.5%, respectively. This is the first report of the isolation of 2-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid and loganin from the genus and the first report of the α-glucosidase inhibitory potential of 2,4-dihydroxybenzoic acid.
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya infection in humans. Despite the widespread distribution of CHIKV, no antiviral medication or vaccine is available against this virus. Therefore, it is crucial to find an effective compound to combat CHIKV. We aimed to predict the possible interactions between non-structural protein 3 (nsP) of CHIKV as one of the most important viral elements in CHIKV intracellular replication and 3 potential flavonoids using a computational approach. The 3-dimensional structure of nsP3 was retrieved from the Protein Data Bank, prepared and, using AutoDock Vina, docked with baicalin, naringenin and quercetagetin as ligands. The first-rated ligand with the strongest binding affinity towards the targeted protein was determined based on the minimum binding energy. Further analysis was conducted to identify both the active site of the protein that reacts with the tested ligands and all of the existing intermolecular bonds. Compared to the other ligands, baicalin was identified as the most potential inhibitor of viral activity by showing the best binding affinity (-9.8 kcal/mol). Baicalin can be considered a good candidate for further evaluation as a potentially efficient antiviral against CHIKV.
Clinacanthus nutans is widely grown in tropical Asia and locally known "belalai gajah" or Sabah snake grass. It has been used as a natural product to treat skin rashes, snake bites, lesion caused by herpes, diabetes, fever, and cancer. Therefore, the objectives of this research are to determine the maximum yield and time of exhaustive flavonoids extraction using Peleg's model and to evaluate potential of antiproliferative activity on human lung cancer cell (A549). The extraction process was carried out on fresh and dried leaves at 28 to 30°C with liquid-to-solid ratio of 10 mL/g for 72 hrs. The extracts were collected intermittently analysed using mathematical Peleg's model and RP-HPLC. The highest amount of flavonoids was used to evaluate the inhibitory concentration (IC50) via 2D cell culture of A549. Based on the results obtained, the predicted maximum extract density was observed at 29.20 ± 14.54 hrs of extraction (texhaustive). However, the exhaustive time of extraction to acquire maximum flavonoids content exhibited approximately 10 hrs earlier. Therefore, 18 hrs of extraction time was chosen to acquire high content of flavonoids. The best antiproliferative effect (IC50) on A549 cell line was observed at 138.82 ± 0.60 µg/mL. In conclusion, the flavonoids content in Clinacanthus nutans water extract possesses potential antiproliferative properties against A549, suggesting an alternative approach for cancer treatment.
This study involves adaptation of bulk or sequential technique to load multiple flavonoids in a single phytosome, which can be termed as "flavonosome". Three widely established and therapeutically valuable flavonoids, such as quercetin (Q), kaempferol (K), and apigenin (A), were quantified in the ethyl acetate fraction of Moringa oleifera leaves extract and were commercially obtained and incorporated in a single flavonosome (QKA-phosphatidylcholine) through four different methods of synthesis - bulk (M1) and serialized (M2) co-sonication and bulk (M3) and sequential (M4) co-loading. The study also established an optimal formulation method based on screening the synthesized flavonosomes with respect to their size, charge, polydispersity index, morphology, drug-carrier interaction, antioxidant potential through in vitro 1,1-diphenyl-2-picrylhydrazyl kinetics, and cytotoxicity evaluation against human hepatoma cell line (HepaRG). Furthermore, entrapment and loading efficiency of flavonoids in the optimal flavonosome have been identified. Among the four synthesis methods, sequential loading technique has been optimized as the best method for the synthesis of QKA-phosphatidylcholine flavonosome, which revealed an average diameter of 375.93±33.61 nm, with a zeta potential of -39.07±3.55 mV, and the entrapment efficiency was >98% for all the flavonoids, whereas the drug-loading capacity of Q, K, and A was 31.63%±0.17%, 34.51%±2.07%, and 31.79%±0.01%, respectively. The in vitro 1,1-diphenyl-2-picrylhydrazyl kinetics of the flavonoids indirectly depicts the release kinetic behavior of the flavonoids from the carrier. The QKA-loaded flavonosome had no indication of toxicity toward human hepatoma cell line as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide result, wherein even at the higher concentration of 200 µg/mL, the flavonosomes exert >85% of cell viability. These results suggest that sequential loading technique may be a promising nanodrug delivery system for loading multiflavonoids in a single entity with sustained activity as an antioxidant, hepatoprotective, and hepatosupplement candidate.
Colorectal cancer (CRC) is the third most common type of cancer in the world, causing thousands of deaths annually. Although chemotherapy is known to be an effective treatment to combat colon cancer, it produces severe side effects. Natural products, on the other hand, appear to generate fewer side effects than do chemotherapeutic drugs. Flavonoids are polyphenolic compounds found in various fruits and vegetables known to possess antioxidant activities, and the literature shows that several of these flavonoids have anti-CRC propertiesFlavonoids are classified into five main subclasses: flavonols, flavanones, flavones, flavan-3-ols, and flavanonols. Of these subclasses, the flavanonols have a minimum effect against CRC, whereas the flavones play an important role. The main targets for the inhibitory effect of flavonoids on CRC signaling pathways are caspase; nuclear factor kappa B; mitogen-activated protein kinase/p38; matrix metalloproteinase (MMP)-2, MMP-7, and MMP-9; p53; β-catenin; cyclin-dependent kinase (CDK)2 and CDK4; and cyclins A, B, D, and E. In this review article, we summarize the in vitro and in vivo studies that have been performed since 2000 on the anti-CRC properties of flavonoids. We also describe the signaling pathways affected by flavonoids that have been found to be involved in CRC. Some flavonoids have the potential to be an effective alternative to chemotherapeutic drugs in the treatment of colon cancer; well-controlled clinical studies should, however, be conducted to support this proposal.
Artonin E is a prenylated flavonoid isolated from the stem bark of Artocarpus elasticus Reinw.(Moraceae). This study aimed to investigate the apoptotic mechanisms induced by artonin E in a metastatic human ovarian cancer cell line SKOV-3 in vitro. MTT assay, clonogenic assay, acridine orange and propidium iodide double staining, cell cycle and annexin V analyses were performed to explore the mode of artonin E-induced cell death at different time points. DNA laddering, activation of caspases-3, -8, and -9, multi-parametric cytotoxicity-3 analysis by high-content screening, measurement of reactive oxygen species generation, and Western blot were employed to study the pathways involved in the apoptosis. MTT results showed that artonin E inhibited the growth of SKOV-3 cells, with IC50 values of 6.5±0.5 μg/mL after 72 h treatment, and showed less toxicity toward a normal human ovarian cell line T1074, with IC50 value of 32.5±0.5 μg/mL. Results showed that artonin E induced apoptosis and cell cycle arrest at the S phase. This compound also promoted the activation of caspases-3, -8, and -9. Further investigation into the depletion of mitochondrial membrane potential and release of cytochrome c revealed that artonin E treatment induced apoptosis via regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The expression levels of survivin and HSP70 proteins were also down regulated in SKOV-3 cells treated with artonin E. We propose that artonin E induced an antiproliferative effect that led to S phase cell cycle arrest and apoptosis through dysregulation of mitochondrial pathways, particularly the pro- and anti-apoptosis signaling pathways.
ETHNOPHARMACOLOGICAL RELEVANCE: Lippia nodiflora has been traditionally used in the Ayurvedic, Unani, and Sidha systems, as well as Traditional Chinese Medicine (TCM) for the treatment of knee joint pain, lithiasis, diuresis, urinary disorder and swelling.
AIM OF THE STUDY: The present study aims to investigate the antihyperuricemic effect of the L. nodiflora methanol extract, fractions, and chemical constituents and their mechanism of action in the rat model.
MATERIALS AND METHODS: The mechanisms were investigated by performing xanthine oxidase inhibitory, uricosuric, and liver xanthine oxidase/xanthine dehydrogenase (XOD/XDH) inhibitory studies in potassium oxonate- and hypoxanthine-induced hyperuricemic rats. The plant safety profile was determined using acute toxicity study. The molecular docking of the active compound to the xanthine oxidase was simulated using computer aided molecular modeling analysis.
RESULTS: Oral administration of methanol extract showed a dose-dependent reduction effect on the serum uric acid level of hyperuricemic rats. F3 was the most potent fraction in lowering the serum uric acid level of hyperuricemic rats. Bioactivity-guided purification of F3 afforded two phenylethanoid glycosides, arenarioside (1) and verbascoside (2) and three flavonoids, 6-hydroxyluteolin (3), 6-hydroxyluteolin-7-O-glycoside (4), and nodifloretin (5). The highest serum uric acid reduction effect was exhibited by 3 (66.94%) in hyperuricemic rats, followed by 5 (55.97%), 4 (49.16%), 2 (29.03%), and 1 (22.08%) at 0.2 mmol/kg. Dose-response investigation on 3 at doses of 0.05, 0.1, and 0.3 mmol/kg produced a significant dose-dependent reduction on the serum uric acid level of hyperuricemic rats. Repeated administration of F3 or 3 to the hyperuricemic rats for 10 continuous days resulted in a significant and progressive serum uric acid lowering effect in hyperuricemic rats. In contrast, methanol extract and F3 did not reduce serum uric acid level of normoruricemic rats. In addition, F4 significantly increased the uric acid excretion of hyperuricemic rats at 200mg/kg. No toxic effect was observed in rats administered with 5000 mg/kg of methanol extract or F3.
CONCLUSION: The potential application of L. nodiflora against hyperuricemia in the animal in accordance with its traditional uses has been demonstrated in the present study for the first time. The antihyperuricemic effect possessed by L. nodiflora was contributed mainly by liver XOD/XDH inhibitory activities and partially by uricosuric effect. Flavonoids mainly accountable for the uric acid lowering effect of L. nodiflora through the inhibition of XOD/XDH activities.
KEYWORDS: Antihyperuricemic; Hypoxanthine-induced hyperuricemic rat; Lippia nodiflora; Liver xanthine oxidase and xanthine dehydrogenase; Serum uric acid; Uric acid excretion
Diverse plants species in the forest remain under-utilised and they are mainly consumed only by local people. However, increasing issues in food security prompted the present study, which explores the nutritional and antioxidant aspects of Malaysian under-utilised vegetables. The studied vegetables were Paku Nyai (Stenochlaena palustris), Cemperai (Champereia manillana), Maman Pasir (Cleome viscose), Dudung (Erechtites valerianifolia) and Semambuk (Ardisia pendula).
Melicope lunu-ankenda leaves are used to treat diabetes in folklore medicinal practices in India and Malaysia. Here we report the isolation of an O-prenylated flavonoid (3,5,4'-trihydroxy-8,3'-dimethoxy-7-(3-methylbut-2-enoxy)flavone; OPF) from the leaves of M. lunu-ankenda and its antidiabetes activity against type-2 diabetes mellitus (T2DM).
The study investigated the changes in the metabolite, antioxidant and α-glucosidase inhibitory activities of Phyllanthus niruri after three drying treatments: air, freeze and oven dryings. Water extracts and extracts obtained using different solvent ratios of ethanol and methanol (50, 70, 80 and 100%) were compared. The relationships among the antioxidant, α-glucosidase inhibitory activity and metabolite levels of the extracts were evaluated using partial least-square analysis (PLS). The solvent selectivity was assessed based on the phytochemical constituents present in the extract and their concentrations quantitatively analyzed using high performance liquid chromatography. The freeze-dried P. niruri samples that were extracted with the mixture of ethanol or methanol with low ratio of water showed higher biological activity values compared with the other extracts. The PLS results for the ethanolic with different ratio and water extracts demonstrated that phenolic acids (chlorogenic acid and ellagic acid) and flavonoids were highly linked to strong α-glucosidase inhibitory and antioxidant activities.
Glycyrrhiza glabra L. (Fabaceae), commonly known as 'liquorice', is a well-known medicinal plant. Roots of this plant have long been used as a sweetening and flavouring agent in food and pharmaceutical products, and also as a traditional remedy for cough, upper and lower respiratory ailments, kidney stones, hepatitis C, skin disorder, cardiovascular diseases, diabetes, gastrointestinal ulcers and stomach ache. Previous pharmacological and clinical studies have revealed its antitussive, antiinflammatory, antiviral, antimicrobial, antioxidant, immunomodulatory, hepatoprotective and cardioprotective properties. While glycyrrhizin, a sweet-tasting triterpene saponin, is the principal bioactive compound, several bioactive flavonoids and isoflavonoids are also present in the roots of this plant. In the present study, the cytotoxicity of the methanol extracts of nine samples of the roots of G. glabra, collected from various geographical origins, was assessed against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cell lines using the in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide cell toxicity/viability assay. Considerable variations in levels of cytotoxicity were observed among various samples of G. glabra.
In this phyto-pharmacological screening of Pistia stratiotes L leaf and root extracts each separately in two different solvents demonstrated its potential medicinal value. Apparent antioxidant value is demonstrated by DPPH, Nitric oxide scavenging and Ferric ion reducing method. Additionally, total flavonoid and phenolic compounds were measured. The leaf methanolic extract scavenged both nitric oxide (NO) and DPPH radical with a dose dependent manner. But the pet ether fraction of root was found to have highest efficacy in Fe(3±) reducing power assay. Flavonoid was found to contain highest in the pet ether fraction of root (411.35mg/g) in terms of quercetin equivalent, similarly highest amount (34.96mg/g) of total phenolic compounds (assayed as gallic acid equivalents) were found to contain in the same fraction. The methanolic fractions appeared less cytotoxic compared to pet ether extracts. The plant extracts caused a dose dependent decrease in faecal droppings in both castor oil and magnesium sulphate induced diarrhea, where as leaf extracts in each solvent appeared most effective. Also, the plant extracts showed anthelmintic activity in earthworm by inducing paralysis and death in a dose dependent manner. At highest doses (50 mg/ml) all fractions were almost effective as the positive control piperazine citrate (10 mg/ml). Thus, besides this cytotoxic effect it's traditional claim for therapeutic use can never be overlooked.
Vernonia amygdalina is a strong natural antioxidant that possessed various medicinal properties. In this study, the spray-dried water extract of V. amygdalina was evaluated for its in vitro antioxidant capacity and in vivo hepatoprotective effect against alcoholic-mediated liver damage. Total phenolic and flavonoid content of spray-dried V. amygdalina water extract were determined. Liver enzyme profiles, liver antioxidant level and nitric oxide level were evaluated in alcohol-induced liver injured mice or co-supplement with spray-dried V. amydalina. Water extract of spray-dried V. amygalina that contained phenolic content of 24.8±1.5 mg/g gallic acid equivalent and total flavonoid content of 25.7±1.3 mg/g catechin equivalent was able to inhibit 50% of xanthine and tyrosinase oxidation at 170 μg/ml and 2 mg/mL, respectively. On the other hand, extracts at both 10 and 50 mg/kg body weight were able to reduce the levels of Alanine transaminase (ALT), Alkaline phosphatase (ALP), Aspartate transaminase (AST), triglyceride and total bilirubin content inthe alcohol-mediated liver injury in mice. Furthermore, it also helped to increase levels of Superoxide dismutase (SOD), Ferric reducing ability of plasma (FRAP) and reduce the levels of Nitric oxide (NO) and Malondialdehyde (MDA) in the liver of the treated mice. These resultssuggestedthat water extract of spray-dried V. amygdalina exhibited liver protective effect, which could be contributed by its antioxidant properties.
Strobilanthes crispus is a well-known herb in Malaysia with various pharmaceutical properties. S. crispus is known to contain several biologically active chemical constituents which are responsible for its pharmaceutical quality.
Staphylococci are facultative anaerobes, perfectly spherical un-encapsulated cocci, with a diameter not exceeding 1 micrometer in diameter. Staphylococcus aureus are generally harmless and remain confined to the skin unless they burrow deep into the body, causing life-threatening infections in bones, joints, bloodstream, heart valves and lungs. Among the 20 medically important staphylococci species, Staphylococcus aureus is one of the emerging human pathogens. Streptomycin had its highest potency against Staphylococcus infections despite the likelihood of getting a resistant type of staphylococcus strains. Methicillin-resistant S. aureus (MRSA) is the persister type of Staphylococcus aureus and was evolved after decades of antibiotic misuse. Inadequate penetration of the antibiotic is one of the principal factors related to success/failure of the therapy. The active drug needs to reach the bacteria at concentrations necessary to kill or suppress the pathogen's growth. In turn the effectiveness of the treatment relied on the physical properties of Staphylococcus aureus. Thus understanding the cell integrity, shape and roughness is crucial to the overall influence of the therapeutic agent on S. aureus of different origins. Hence our experiments were designed to clarify ultrastructural changes of S. aureus treated with streptomycin (synthetic compound) in comparison to artonin E (natural compound). In addition to the standard in vitro microbial techniques, we used transmission electron microscopy to study the disrupted cell architecture under antibacterial regimen and we correlate this with scanning electron microscopy (SEM) to compare results of both techniques.
Flavokawain B (FKB) is a naturally occurring chalcone that can be isolated through the root extracts of the kava-kava plant (Piper methysticum). It can also be synthesized chemically to increase the yield. This compound is a promising candidate as a biological agent, as it is reported to be involved in a wide range of biological activities. Furthermore, FKB was reported to have antitumorigenic effects in several cancer cell lines in vitro. However, the in vivo antitumor effects of FKB have not been reported on yet. Breast cancer is one of the major causes of cancer-related deaths in the world today. Any potential treatment should not only impede the growth of the tumor, but also modulate the immune system efficiently and inhibit the formation of secondary tumors. As presented in our study, FKB induced apoptosis in 4T1 tumors in vivo, as evidenced by the terminal deoxynucleotidyl transferase dUTP nick end labeling and hematoxylin and eosin staining of the tumor. FKB also regulated the immune system by increasing both helper and cytolytic T-cell and natural killer cell populations. In addition, FKB also enhanced the levels of interleukin 2 and interferon gamma but suppressed interleukin 1B. Apart from that, FKB was also found to inhibit metastasis, as evaluated by clonogenic assay, bone marrow smearing assay, real-time polymerase chain reaction, Western blot, and proteome profiler analysis. All in all, FKB may serve as a promising anticancer agent, especially in treating breast cancer.
Diabetes mellitus (DM) is a metabolic diseases characterized by hyperglycemia due to insufficient or inefficient insulin secretory response. This chronic disease is a global problem and there is a need for greater emphasis on therapeutic strategies in the health system. Phytochemicals such as flavonoids have recently attracted attention as source materials for the development of new antidiabetic drugs or alternative therapy for the management of diabetes and its related complications. The antidiabetic potential of flavonoids are mainly through their modulatory effects on glucose transporter by enhancing GLUT-2 expression in pancreatic β cells and increasing expression and promoting translocation of GLUT-4 via PI3K/AKT, CAP/Cb1/TC10 and AMPK pathways. This review highlights the recent findings on beneficial effects of flavonoids in the management of diabetes with particular emphasis on the investigations that explore the role of these compounds in modulating glucose transporter proteins at cellular and molecular level.