Activity-guided isolation and identification of free radical-scavenging components from an aqueous extract of Coleus aromaticus

An activity-directed fractionation and purification process was used to identify the DPPH (l,l-diphenyl-2-picrylhydrazyl) free radical-scavenging components of Coleus aromaticus Benth. Fresh leaves of C. aromaticus were extracted with water and then separated into hexane, ethyl acetate, and water fractions. Among these, only the ethyl acetate phase showed strong DPPH radical-scavenging activity in vitro, when compared with water and hexane phases. The ethyl acetate fraction was then subjected to separation and purification using Sephadex LH-20 chromatography. Three compounds showing strong DPPH radical-scavenging activity were shown, by spectral methods (¹H NMR, ¹³C NMR, and MS) and by comparison with literature values, to be rosmarinic acid, chlorogenic acid and caffeic acid. In addition, HPLC identification and quantification of isolated compounds were also performed. Rosmarinic acid was found as a major component and principally responsible for the radical-scavenging activity of C. aromaticus.     

Antioxidant activity of Rhizoma Smilacis Glabrae extracts and its key constituent-astilbin

Rhizoma Smilacis Glabrae is widely consumed by Chinese as functional food and in folk medicine for its medicinal properties. In this study, methanol and water extracts of Rhizoma Smilacis Glabrae were prepared. The water extract was further divided into polysaccharide and supernatant fractions. Constituents in different extracts were analysed by capillary electrophoresis, and levels of total phenolics were also determined using the Folin-Ciocalteu method. Astilbin, the main constituent in the herb, was isolated and purified. Different antioxidant tests were employed to evaluate the antioxidant activities of the extracts and the isolated astilbin, and the results were compared with two commonly used synthetic antioxidants-butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Methanol, water extract and supernatant fraction showed concentration dependent antioxidant activity while polysaccharide didn’t show any antioxidant activity. Purified astilbin showed the strongest antioxidant activity in comparison to any other extracts.     

Antioxidant and free radical scavenging activities of phenols from onion (Allium cepa)

Four (red, violet, white and green) varieties of Allium cepa were studied for their total phenolic contents (TPC), antioxidant (AOA) and free radical scavenging activities (FRSA). The TPC varied from 4.6 to 74.1 mg/g GAE, AOA varied from 13.6% to 84.1% and FRSA showed wide range in terms of IC₅₀ (inhibitory concentration) from 0.1 to 15.2 mg/ml, EC₅₀ (efficient concentration) from 4.3 to 660.8 mg/mg and ARP (antiradical power) from 0.15 to 23.2. The outer dry layers of red and violet varieties showed better inhibition of lipid peroxidation assayed by ammonium thiocyanate than α-tocopherol. The non-site-specific inhibition of hydroxyl radical induced deoxyribose degradation was also higher in the outer dry layers of red and violet varieties than in their middle and inner layers. The outer layers were also potential inhibitors of nitroblue tetrazolium chloride (NBT) reduction caused by superoxide anions. On the other hand the ferrous ion chelating capacity of the red and violet varieties was highest in the inner layers. Specific phenolic composition performed through HPLC and LC–MS/MS showed the presence of gallic acid, ferulic acid, protocatechuic acid, quercetin, and kaempferol. The unutilised outer layers of the red variety were a rich source of quercetin (5110 μg/g) with high AOA, FRSA and also showed significant protection of DNA damage caused by free radicals.     

Antioxidant activity of Nyctanthes arbor-tristis leaf extract

Nyctanthes arbor-tristis (Harsingar) leaf extracts are extensively used in Indian traditional medicine. The acetone-soluble fraction of its ethyl acetate extract showed impressive antioxidant activity as revealed by several in vitro experiments, e.g., DPPH, hydroxyl and superoxide radicals, as well as H₂O₂ scavenging assays. Moreover, its preventive capacity against Fe(II)-induced lipid peroxidation of liposomes and γ-ray-induced DNA damage also confirmed this. The strong reducing power and high phenolics and flavonoids contents could be responsible for the antioxidant activity.     

Antioxidant and free radical scavenging activities of wild bitter melon (Momordica charantia Linn. var. abbreviata Ser.) in Taiwan

Momordica charantia Linn. var. abbreviata Ser. (Cucurbitaceae), also known as “Shan Ku Gua”, is a wild variety of bitter melon (BM) in Taiwan. The size of its fruits is only about one-fifth of the commonly seen BM. It is commonly consumed as vegetable and also used as a popular folk medicine. In this study, the antioxidant and free radical scavenging activities of BM aqueous (BM-H₂O) and ethanol (BM-EtOH) extracts were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), metal chelation, cytochrome c and xanthine oxidase inhibition (XOI) assays, as well as FeCl₂-ascorbic acid induced lipid peroxidation (thiobarbituric acid reactive substances, TBARS) assay in rat liver homogenates in vitro. Total flavonoid and phenol contents of BM extracts were also analyzed. Results showed that both BM-H₂O (IC₅₀=129.94 μg/ml) and BM-EtOH (IC₅₀=156.78 μg/ml) possess potent DPPH radical scavenging activity, which was better than vitamin E (IC₅₀=172.21 μg/ml). These extracts also showed better iron chelating activity than vitamin E. However, they were weaker than vitamin E in free radical scavenging, xanthine oxidase inhibitory and anti-lipid peroxidation activities. With the exception of XOI activity [IC₅₀=7.90 μg/ml (BM-H₂O) vs. 7.69 μg/ml (BM-EtOH)], BM-H₂O showed a lower IC₅₀ value in free radical scavenging [IC₅₀=6.15 μg/ml (BM-H₂O) vs. 7.08 μg/ml (BM-EtOH)] and anti-lipid peroxidation [IC₅₀=53.72 μg/ml (BM-H₂O) vs. 88.51 μg/ml (BM-EtOH) for liver; 82.53 μg/ml (BM-H₂O) vs. 91.83 μg/ml (BM-EtOH) for brain] activities than BM-EtOH. Both BM extracts showed a weak anti-lipid peroxidation activity in plasma. BM-H₂O (62.0 mg/g) possessed a significant higher concentration of total flavonoids than BM-EtOH (44.0 mg/g), but was lower in the total phenol content (BM-H₂O: 51.6 mg/g vs. BM-EtOH: 68.8 mg/g). In conclusion, BM extracts possess potent antioxidant and free radical scavenging activities. These antioxidant activities could have contributed, at least partly, to the therapeutic benefits of the certain traditional claims of wild BM.     

Antioxidant properties of aqueous extracts from Terminalia catappa leaves

Aqueous extracts were prepared from green, yellow fallen and red fallen leaves of Terminalia catappa L. (Combretaceae) and their antioxidant activity, scavenging and chelating abilities were evaluated. Aqueous extracts from three different leaves showed high antioxidant activities and moderate scavenging abilities on hydroxyl radicals at 1 mg/ml. EC₅₀ values in antioxidant activity were 0.549-0.557 mg/ml whereas those in scavenging ability on hydroxyl radicals were 0.631-0.686 mg/ml for aqueous extracts prepared from three leaves with boiling water for 3 min. EC50 values in reducing power were 0.15-0.23 mg/ml. EC50 values in scavenging abilities on superoxide anion and 1,1-diphenyl-2-picrylhydryl radicals were 0.36-0.44 and 10.4-35.3 mg/ml, respectively. EC50 values in chelating abilities on ferrous and cupric ions were 0.41-2.50 and 8.96-9.89 mg/ml, respectively. Based on the results obtained, the aqueous extracts displayed higher antioxidant properties. Six phenolic compounds identified in the aqueous were p-hydroxybenzoic acid, 4-hydroxyphenylpropionic acid, m-coumaric acid, 3,4-dihydroxybenzoic acid, p-coumaric acid and gallic acid. Therefore, the aqueous extracts from three different leaves of T. catappa might be a potential antioxidant supplement for application in food products or as a drink.     

Antioxidant and free radical scavenging activities of polyphenol-enriched curry leaf (Murraya koenigii L.) extracts

The in vitro antioxidant properties of different extracts (water, alcohol, alcohol:water, hexane or chloroform extract) of curry leaves (Murraya koenigii L.) were evaluated using various assays. The alcohol:water (1:1) extract of curry leaves (AWEC) showed the highest antioxidant and free radical scavenging activity. It inhibited membrane lipid peroxidation by 76%, at 50 μg/ml, scavenged 93% of superoxides at 200 μg/3 ml and scavenged approximately 90% of hydroxyl and 1,1-diphenyl-2-picrylhydrazayl radicals at 4–5-fold lower concentrations compared to the other tested extracts. In addition, the alcohol:water extract reduced cytochrome c and ferric ion levels, chelated ferrous ions and inhibited ferrous sulfate:ascorbate-induced fragmentation and sugar oxidation of DNA. These results establish the antioxidant potential of AWEC, which could be used as natural antioxidant source.     

Antioxidant activities of Salvia miltiorrhiza and Panax notoginseng

Traditional Chinese medicinal herb Salvia miltiorrhiza (SM) and Panax notoginseng (PN) have been widely used for the prevention and treatment of vascular diseases in the clinics. To better understand their mechanisms of pharmacological actions, the in vitro antioxidant activities of extract of Salvia miltiorrhiza (ESM) and extract of Panax notoginseng (EPN) were evaluated with different antioxidant testing systems. Their activities of scavenging superoxide anion radicals, DPPH radicals, hydroxyl radicals, and hydrogen peroxide, chelating Ferrous ion, and ferric ion reducing power were assessed. The results showed that the mechanisms of their antioxidant effects were distinct and diverse. ESM possessed strong reducing power and high scavenging activities against free radicals including superoxide anion, hydroxyl and DPPH radicals, but a weaker scavenging activity for hydrogen peroxide. ferrous ion chelating activity of ESM was undetectable at the tested concentrations. In contrary, EPN exhibited strong ferrous ion chelating activity and high scavenging activities against hydrogen peroxide, hydroxyl radicals, and a weak activity against superoxide anion and DPPH free radicals. EPN did not show any ferric ion reducing power. Since their antioxidant mechanisms are complementary, the combined use of ESM and EPN might be even more beneficial. These antioxidant properties of SM and PN are likely part of the reasons that they are effective in the prevention and treatment of vascular diseases.     

Antioxidant properties of Agaricus blazei, Agrocybe cylindracea, and Boletus edulis

Three mushrooms are currently available in Taiwan, including Agaricus blazei, Agrocybe cylindracea, and Boletus edulis. Their ethanolic and hot water extracts were prepared and antioxidant properties studied. Ethanolic extracts from three mushrooms were more effective than hot water extracts in antioxidant activity using the conjugated diene method and scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals whereas hot water extracts were more effective in reducing power, scavenging ability on hydroxyl radials and chelating ability on ferrous ions as evidenced by their lower EC₅₀ values. Overall, for both extracts, B. edulis was more effective among antioxidant properties assayed. Naturally occurring antioxidant components including total tocopherols (3.18-6.18 mg/g) and total phenols (5.67-5.81 mg/g) were found in the extracts and their contents were associated (r=0.636-0.907) with EC₅₀ value of antioxidant properties. Based on the results obtained, both extracts from these three mushrooms were effective in antioxidant properties.     

Antioxidant activity of ethanolic extract of Cortex fraxini and use in peanut oil

Cortex fraxini was extracted with 95% ethanol to obtain a crude antioxidant extract. The antioxidant activity was evaluated using the linoleic acid peroxidation method and the free radical scavenging assays, namely 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. Cortex fraxini extract (CFE) showed high inhibition of peroxidation of linoleic acid when compared with butylated hydroxytoluene (BHT). CFE also exhibited excellent scavenging activity on DPPH and hydroxyl radicals. Total antioxidant activity was measured by the reduction of Mo(VI) to Mo(V) by the extract, and subsequent formation of a green phosphate/Mo(V) complex at acid pH. CFE had significant total antioxidant activity and the effects were increased with increasing reaction time. The total phenolic content of the sample, analyzed by using Folin–Ciocalteu’s reagent, was 91.33 mg/g dry weight expressed as pyrocatechol equivalents. Then the suitability of CFE as an antioxidant was determined in peanut oil, and the decrease of lipid oxidation was monitored using thiobarbituric acid-reactive substances (TBARS) assay. CFE treatment significantly (P < 0.05) reduced lipid oxidation in peanut oil compared to the control. No significant differences (P = 0.05) in lipid oxidation were detected between CFE antioxidant and BHT antioxidant samples.     

Antioxidant properties of three extracts from Pleurotus citrinopileatus

Pleurotus citrinopileatus Sing. (Lentinaceae) was successfully cultivated and commercially available in Taiwan. The ethanolic, cold and hot water extracts were prepared from P. citrinopileatus fruit bodies, mycelia and fermentation filtrate and their antioxidant properties studied. For three samples, ethanolic extracts exhibited higher antioxidant activities than water extracts. Reducing powers of three extracts from fruit bodies were effective and 1.03-1.10 at 5 mg/ml. With regard to EC₅₀ values of scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl radicals, the effectiveness was in a descending order: ethanolic>hot water>cold water extracts. Scavenging abilities of water extracts from three samples on hydroxyl radicals were 53.4-80.1% at 20 mg/ml. Chelating abilities of cold and hot water extracts on ferrous ions were higher than those of ethanolic extracts. Contents of total phenols were in the descending order: fruit bodies (8.62-12.38 mg/g)>mycelia (5.84-7.85 mg/g)>filtrate (4.80-5.57 mg/g). Overall, three extracts from fruit bodies were more effective in antioxidant properties assayed than those from mycelia and filtrate. Ethanolic extracts were more effective in antioxidant properties assayed, except for scavenging abilities on hydroxyl radicals.     

Antioxidant activity of microwave-assisted extract of Buddleia officinalis and its major active component

Buddleia officinalis Maxim, commonly used as rice dye for festivals, was extracted with ethanol using microwave-assisted extraction and Soxhlet extraction. The antioxidant activities of microwave-assisted extract of B. officialis (MEB) and Soxhlet extract of B. officianils (SEB) at the optimum extraction conditions were evaluated and compared with synthetic antioxidant butylated hydroxytoluene (BHT) employing DPPH free radical assay, ABTS assay, total antioxidant activity and reducing power. MEB and SEB had stronger antioxidant activities than BHT in all assays except reducing power, and the effects decreased as follows: MEB > SEB > BHT. The total phenolic contents of MEB and SEB reached 113.56 mg/g and 100.94 mg/g dry weight of extract, respectively, expressed as pyrocatechol equivalents, while the total flavonoids contents were 75.33 mg/g and 62.56 mg/g dry weight of extract, respectively, expressed as catechin equivalents (P < 0.05). Higher phenolic and flavonoids compounds may be major contributors to their higher antioxidant activities. Following activity-oriented separation, luteolin was isolated as an active principle, which exhibited excellent free radical scavenging activities with DPPH IC₅₀ 3.09 μg/ml and ABTS IC₅₀ 2.20 μg/ml.     

Antioxidant activity and phenolic composition of sumac (Rhus coriaria L.) extracts

Antioxidant activity and phenolic compounds of sumac extracts were investigated. Sumac was extracted in methanol and subjected to solvent–solvent partitioning to yield two fractions as ethyl acetate and aqueous. Methanol extract was further fractioned over Sephadex LH-20 column. Antioxidant activity of extracts and fractions were screened using ferric thiocyanate and DPPH radical scavenging methods. Phenolic composition of active fraction(s) was determined by HPLC–MS systems. Those fractions which exhibited strong antioxidant activity were rich in anthocyanins and hydrolysable tannins. While gallic acid was the main phenolic acid in the extracts, anthocyanin fraction contained cyanidin, peonidin, pelargonidin, petunidin, and delphinidin glucosides and coumarates. Pentagalloyl glucose was abundant in the hydrolysable tannin fraction. Effective scavenging concentration (EC₅₀) on DPPH radical was 0.70 μg/mL both in ethyl acetate and tannin fractions, and 5.33 μg/mL in anthocyanin rich fraction. Same extracts and fractions showed moderate lipid peroxidation inhibition effect compared with the synthetic antioxidants. The findings demonstrate that sumac can be used as a natural antioxidant.     

Antioxidant activity and hepatoprotective potential of Phyllanthus niruri

Antioxidant activity and hepatoprotective potential of Phyllanthus niruri, a widely used medicinal plant, were investigated. Methanolic and aqueous extract of leaves and fruits of P. niruri showed inhibition of membrane lipid peroxidation (LPO), scavenging of 1,1-diphenyl-2picrylhydrazyl (DPPH) radical and inhibition of reactive oxygen species (ROS) in vitro. Antioxidant activity of the extracts were also demonstrable in vivo by the inhibition of the carbon tetrachloride (CCl₄) – induced formation of lipid peroxides in the liver of rats by pretreatment with the extracts. CCl₄ – induced hepatotoxicity in rats, as judged by the raised serum enzymes, glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT), was prevented by pretreatment with the extracts, demonstrating the hepatoprotective action of P. niruri.     

Antioxidant properties of hot water extracts from Agrocybe cylindracea

Agrocybe cylindracea (DC: Fr.) Mre. was available in the form of fruit bodies, mycelia and fermentation filtrate. From these three forms, hot-water extracts were prepared and their antioxidant properties were studied. Antioxidant activities of hot-water extracts from fruit bodies, mycelia and filtrate were 63.6%, 81.6% and 56.8% at 20 mg ml⁻¹, respectively. EC₅₀ values in reducing power were 2.72, 3.97 and 3.09 mg ml⁻¹ whereas those in scavenging abilities of 1,1-diphenyl-2-picrylhydrazyl radicals were 0.62, 1.66 and 0.82 mg ml⁻¹ for fruit bodies, mycelia and filtrate, respectively. At 20 mg ml⁻¹, the scavenging abilities of hydroxyl radicals were 80.1%, 57.0% and 54.3% for fruit bodies, mycelia and filtrate, respectively. With regard to EC₅₀ values in chelating abilities on ferrous ions, the hot-water extract from filtrate was better than that from mycelia. Total phenols were the major naturally occurring antioxidant components found in hot-water extracts and in the range of 23.74–30.16 mg g⁻¹. From EC₅₀ values obtained, it can be concluded that hot-water extracts from three forms of A. cylindracea were good in antioxidant properties.     

Antioxidant properties of polysaccharides from Ganoderma tsugae

Ganoderma tsugae Murrill (Ganodermataceae) were available in the form of mature and baby Ling chih, mycelia and fermentation filtrate. From these four forms, hot water extracted and hot alkali extracted polysaccharides were prepared and their antioxidant properties were studied. Polysaccharides showed good antioxidant activity as evidenced by their particularly low EC₅₀ values (<0.1 mg/ml). At 20 mg/ml, both extracted polysaccharides from mycelia showed reducing powers of 0.41–0.52 whereas reducing powers of other polysaccharides were in the range of 0.87 to 1.14. At 20 mg/ml, scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl radicals increased to 93.7–100%, except for that of the hot water extracted polysaccharide, from filtrate, being 74.9%. At 20 mg/ml, scavenging abilities of both extracted polysaccharides from mycelia on hydroxyl radicals were 13.9 and 24.4%, respectively whereas scavenging abilities of the other polysaccharides were in the range of 39.0–55.2%. At 10 mg/ml, the chelating abilities of polysaccharides from mature and baby Ling chih, mycelia and filtrate were 93.9–100%, 97.6–100%, 85.1–88.0% and 51.2%, respectively. Overall, both extracts of polysaccharides possessed good antioxidant properties except for scavenging ability on hydroxyl radicals and can be developed as a new dietary supplement and functional food.