Identification of phenolics in the fruit of emblica (Phyllanthus emblica L.) and their antioxidant activities

An activity-directed fractionation and purification process was used to identify the antioxidative components of emblica fruit. Dried fruit of emblica was extracted with methanol and then partitioned by ethyl ether, ethyl acetate, butanol and water. The ethyl acetate fraction showed the strongest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity among four fractions. The ethyl acetate fraction was then subjected to separation and purification using Sephadex LH-20 chromatography and reverse-phase high-performance liquid chromatography (HPLC). Six compounds were identified to be geraniin (1), quercetin 3-β-d-glucopyranoside (2), kaempferol 3-β-d-glucopyranoside (3), isocorilagin (4), quercetin (5), and kaempferol (6), respectively, by spectral methods, ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy, ultraviolet–visible (UV–Vis) spectrophotometry and mass spectroscopy (MS), and comparison with literatures. Compounds 2–4 and 6 were identified from emblica fruit for the first time. Furthermore, the antioxidant activities of purified compounds were screened for their antioxidative potential using lipid peroxidation and DPPH systems. All the purified compounds showed strong antioxidant and radical scavenging activities. Amongst, geraniin showed the highest antioxidant activity (4.7 and 65.7 μM of IC₅₀ values for DPPH and lipid peroxidation assay, respectively) than other purified compounds.     

Antioxidant and antiproliferative capacities of phenolics purified from Phyllanthus emblica L. fruit

Phyllanthus emblica is a widely distributed tropical medicinal plant with good therapeutic properties. Gallic acid, ellagic acid, mucic acid 1,4-lactone 3-O-gallate, isocorilagin, chebulanin, chebulagic acid and mallotusinin were isolated and purified from this plant. The antioxidant activities of these compounds were evaluated by in vitro models of scavenging superoxide anion radicals, DPPH radicals and ABTS radicals, chelating ability of ferrous ion and inhibition capability of Fe (II)-induced lipid peroxidation, respectively. The results revealed that all the tested phenolics showed strong radical scavenging activity, good potency to chelate Fe²⁺ and good inhibition ability of lipid peroxidation. Amongst mallotusinin and mucic acid 1,4-lactone 3-O-gallate were reported for the first time to have antioxidant activity. In addition, the in vitro antiproliferative activities of those phenolics against MCF-7 breast cancer cell lines were assessed. Although the growth inhibition values induced by different phenolics on MCF-7 cell line yielded different effects, all the tested phenolics had apparent capacities of inhibiting the survival of MCF-7 human cancer cell line.     

Immunomodulatory and anticancer activities of phenolics from Garcinia mangostana fruit pericarp

The methanolic extract of Garciniamangostana fruit pericarp was partitioned into butanol and water fractions in this work. Three major phenolics were purified and identified as P₁ [1,3,6,7-tetrahydroxy-2,8-(3-methyl-2-butenyl) xanthone], P₂ [1,3,6-trihydroxy-7-methoxy-2,8-(3-methyl-2-butenyl) xanthone] and P₃ (epicatechin). Strong antioxidant activities were detected for P₁–P₃. In vitro cell proliferation trials indicated that P₁ and P₃ exhibited good immunomodulatory activities when 7.5 μg/ml was used. Furthermore, P₁ and P₃ showed good cytotoxicities against human breast cancer cells (MCF-7) and human colon cancer cells (LOVO). P₁ exhibited the maximal cytotoxicity of 73.06% against MCF-7 cells and of 46.27% against LOVO cells when 62.5 μg/ml was used. The cytotoxicities of P₁, P₂, P₃ and paclitaxel against normal embryonic lung fibroblast cells (HELF) were in a decreasing order: paclitaxel > P₃ > P₁ > P_2. These results suggested that P₁ and P₃ could be used as a potential anticancer agent.     

Structural identification of isomallotusinin and other phenolics in Phyllanthus emblica L. fruit hull

The air-dried fruit hull of Phyllanthus emblica L. was extracted with 95% ethanol, and then the extract was partitioned by diethyl ether and ethyl acetate (EA). The EA fraction was then subjected to separation and purification using silica gel and Sephadex LH-20 column chromatography repeatedly to obtain five hydrolysable tannins. They were identified as mucic acid 1,4-lactone 3-o-gallate (C1), isocorilagin (C2), chebulanin (C3), chebulagic acid (C4) and isomallotusinin (C5) using mass spectrometry and nuclear magnetic resonance (NMR) spectrometry. Isomallotusinin and chebulanin were identified from emblica dried fruit hull for the first time, and isomallotusinin was the first time identified from Phyllanthus. Furthermore, the antioxidant abilities of these hydrolysable tannins were investigated using DPPH and ABTS⁺ radical scavenging systems. All hydrolysable tannins showed strong DPPH and ABTS⁺ radical scavenging activities. Isomallotusinin and chebulagic acid exhibited the highest antioxidant activity compared to other purified compounds tested.     

Identification of bioactive compounds in Phyllenthus emblica L. fruit and their free radical scavenging activities

Emblica has been used as an important traditional herbal medicine in southeast Asia since ancient times. In this study, the air-dried hulls of emblica fruit were extracted with 95% ethanol, and then the extract was partitioned by diethyl ether and ethyl acetate (EA). The EA fraction was purified by silica gel column and thin layer chromatography (TLC) to obtain six compounds. They were identified as cinnamic acid (C1), quercetin (C2), 5-hydroxymethylfurfural (C3), gallic acid (C4), β-daucosterol (C5) and ellagic acid (C6) using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Cinnamic acid and 5-hydroxymethylfurfural were identified as components of emblica fruit for the first time. The DPPH and ABTS⁺ radical scavenging activities of components were evaluated. All the compounds showed significant DPPH and ABTS⁺ radical scavenging activity except for cinnamic acid. Gallic acid showed the highest DPPH radical scavenging activity while ellagic acid showed the highest ABTS⁺ scavenging activity amongst all the compounds tested.     

Phenolics from hull of Garcinia mangostana fruit and their antioxidant activities

The air-dried fruit hulls of Garcinia mangostana Linn. were extracted with 70% MeOH, and then partitioned into the n-BuOH fractions. Furthermore, three major phenolic components related to their antioxidant activities were purified by silica gel column chromatography and Sephadex LH-20 and then identified as P₁ (1,3,6,7-tetrahydroxy-2,8-(3-methyl-2-butenyl)), P₂ [1,3,6-trihydroxy-7-methoxy-2,8-(3-methyl-2-butenyl) xanthone] and P₃ (epicatechin) using UV–visible spectrophotometry, IR spectrophotometry and NMR spectroscopy, respectively. The antioxidant activities of three major phenolics were evaluated using different tests, including the free radical scavenging capability and total antioxidant activity in a linoleic acid peroxidation. These three phenolic compounds exhibited different antioxidant activities in these antioxidant tests. The hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capabilities and the activity against linoleic acid peroxidation of P₁ were greater than those of P₂ and P₃, while the superoxide anion radical scavenging activity of P₃ was greater than that of P₁, but was close to that of P₂ or α-tocopherol. An activity-guided isolation and purification process was used to identify the components showing the strong DPPH radical scavenging activity from G. mangostana Linn.     

Volatile compounds from pitanga fruit (Eugenia uniflora L.)

Extracts from pitanga leaves are considered to be effective against many diseases, and are therefore used in popular Brazilian medicines. In this study, the volatile constituents of pitanga fruits (Eugenia uniflora L.) were trapped on to Porapak-Q and eluted with ethyl acetate, and the chemical composition of the extract was analyzed by gas chromatography and gas chromatography/mass spectrometry. Fifty-four compounds were detected, and twenty-nine of those were identified by close matches with standard MS spectra. Monoterpenes (75.3% in mass) were found to comprise the largest class of the pitanga fruit volatiles, including trans-β-ocimene (36.2%), cis-ocimene (13.4%), the isomeric β-ocimene (15.4%) and β-pinene (10.3%). Several known therapeutic constituents of pitanga leaf extract, such as selina-1,3,7(11)-trien-8-one (the major constituent) were also found to be present in the fruit volatile extract, suggesting that the fruit may display therapeutic properties similar to those of the leaf extract.     

Free radical scavenging activities and bioactive substances of Jerusalem artichoke (Helianthus tuberosus L.) leaves

The total phenolic content and radical scavenging activities of Jerusalem artichoke (Helianthus tuberosus L.) leaves were investigated. Results indicated that the ethyl acetate fraction contained the highest total phenolic content (266.69 ± 2.51 mg GAE/g dry extract) accompanied with strongest free radical scavenging abilities. Following an in vitro radical scavenging activity-guide fractionation procedure, six phenolic compounds which strongly quenched free radicals were separated from ethyl acetate fraction. Among them, 3-O-caffeoylquinic acid and 1,5-dicaffeoylquinic acid played a dominant role due to their strong free radical scavenging abilities and their high contents. The content of 3-O-caffeoylquinic acid in n-butanol fraction was 74.58 ± 1.05 mg/g, while 1,5-dicaffeoylquinic acid in ethyl acetate fraction was 104.51 ± 2.86 mg/g. The results imply that the leaves of Jerusalem artichoke might be a potential source of natural antioxidants.     

Chemical and antioxidative assessment of dietary turnip (Brassica rapa var. rapa L.)

The phenolic compounds and organic acids of turnip (Brassica rapa var. rapa L.) edible parts (leaves and stems, flower buds and roots) were determined by HPLC–DAD and HPLC–UV, respectively. The results revealed a profile composed of 14 phenolics (3-p-coumaroylquinic, caffeic, ferulic and sinapic acids, kaempferol 3-O-sophoroside-7-O-glucoside, kaempferol 3-O-sophoroside-7-O-sophoroside, kaempferol 3-O-(feruloyl/caffeoyl)-sophoroside-7-O-glucoside, kaempferol 3,7-O-diglucoside, isorhamnetin 3,7-O-diglucoside, kaempferol 3-O-sophoroside, 1,2-disinapoylgentiobiose, 1,2′-disinapoyl-2-feruloylgentiobiose, kaempferol 3-O-glucoside and isorhamnetin 3-O-glucoside) and six organic acids (aconitic, citric, ketoglutaric, malic, shikimic and fumaric acids). The quantification of the identified compounds showed kaempferol 3-O-sophoroside-7-O-glucoside, kaempferol 3-O-(feruloyl/caffeoyl)-sophoroside-7-O-glucoside, isorhamnetin 3,7-O-diglucoside and isorhamnetin 3-O-glucoside as the main phenolics, and malic acid as the organic acid present in highest amounts. A screening of the antioxidative potential was also performed by means of the DPPH radical scavenging assay. Turnip flower buds exhibited the strongest antioxidant capacity.     

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.     

Antioxidative activities of chromatographic fractions obtained from root,fruit and leaf of Mengkudu (Morinda citrifolia L.)

Crude extracts of root, leaf and fruit of Morinda citrifolia were fractionated on a Sephadex LH-20 column with ethanol as eluate. Based on UV absorption intensity of phenolic compound (725 mm) the Sephadex LH-20 column was able to separate fruit, leaf and root extracts into six, five and five fractions, respectively. The results showed that all the fractions tested exhibited considerably high antioxidative activity in the ferric thiocyanate assay and thiobarbituric acid test and the activities of some of the fractions were as good as those of either tocopherol or BHT. The fractions from different parts of the plants were found to contain different amounts of total phenolic compounds, which, interestingly, do not correspond to the antioxidative activity measured. This is probably due to the presence of different phenolics in the samples, with different antioxidative activities which involves various mechanisms inhibiting the oxidation process. The study suggested that root, leaf and fruit of M. citrifolia might contribute significantly to exogenous antioxidant which is crucial in combating oxidative stress.     

Antioxidant properties of extracts from juemingzi (Cassia tora L.) evaluated in vitro

Methanol extract from juemingzi (Cassia tora L.) was fractionated by liquid-liquid partition using ethyl acetate, n-butanol and water, respectively. The antioxidant activity of three different fractions was evaluated using different antioxidant tests, including inhibition of peroxidation of linoleic acid, reducing power, free-radical scavenging and ferrous ions chelating activity. In the above four assays, all the fractions showed antioxidant potential to varying degrees. Among these fractions, ethyl acetate fraction exhibited more antioxidant potency than other fractions. Furthermore, ethyl acetate fraction was found to be more effective in protecting LDL against oxidation in a concentration-dependent manner. The data suggest that juemingzi especially ethyl acetate-soluble fraction may have a preventive effect against atherosclerosis by inhibiting LDL oxidation.     

Improved loquat (Eriobotrya japonica Lindl.) cultivars: Variation of phenolics and antioxidative potential

Leaves and fruits (peel and flesh) of six improved cultivars (‘Mizuho’, ‘Néctar de Cristal’, ‘Mizauto’, ‘Mizumo’, ‘Centenária’ and ‘NE-3’) of loquat (Eriobotrya japonica Lindl.) were studied for their phenolic composition and antioxidant capacity. The analysis by HPLC-DAD-ESI-MS/MS allowed the identification of 18 compounds (8 hydroxycinnamic acid derivatives and 10 flavonoid glycosides). The quantification of the identified compounds revealed distinct profiles amongst the three analysed materials. Loquat leaves exhibited the lowest amounts of phenolics. 3- And 5-caffeoylquinic, and 5-feruloylquinic acids were the major compounds. Generally, ‘Mizauto’ cultivar presented the highest phenolic content. All loquat materials exhibited DDPH scavenging capacity, in a concentration-dependent manner, the leaves being the most active one. This effect seems to be related to the flavonoid content.     

Antioxidant properties of Phyllanthus amarus extracts as affected by different drying methods

The total phenolic content (TPC) and antioxidant activity of fresh and dried Phyllanthus amarus plant materials were evaluated using the Folin-Ciocalteau method, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and ferric reducing antioxidant power (FRAP) assays. Different drying treatments led to significant reduction (P<0.05) in antioxidant properties of P. amarus methanolic extracts, with microwave drying causing the highest decrease in TPC and antioxidant activity exhibited by the reduction in both radical scavenging activity and FRAP. On the other hand, boiling water extracts appeared to exhibit significantly stronger antioxidant potentials (P<0.05) even in dried plant materials due to greater solubility of compounds, breakdown of cellular constituents as well as hydrolysis of tannins. Its strong free radical scavenging activity suggests that it has great potential in the food industry as functional food ingredient.     

Flavonoid glycosides from Pouteria obovata (R. Br.) fruit flour

Three unknown dihydroflavanol glycosides: 2R,3R-4′O-methyl dihydrokaempferol 7-O-[3″-O-acetyl]-β-d-glucopyranoside (1), 2R,3R-4′-O-methyl dihydrokaempferol 7-O-β-d-β-l-xylopyranosyl-(1″′ → 6″)-[3″-O-acetyl]-β-d-glucopyranoside (2), 2R,3R-4′-O-methyl dihydrokaempferol 3-O-β-d-β-l-xylopyranosyl-(1″′ → 6″)-[3″-O-acetyl]-β-d-glucopyranoside (3), together with gallic acid (4) were isolated from the n-butanol fraction of Pouteria obovata fruit flour by chromatographic methods and their structures were elucidated on the basis of CD, UV, MS, monodimensional NMR (¹H and ¹³C) and bidimensional NMR (COSY, HSQC and HMBC). The quantitative analysis of flavonoids and phenols were also reported. Total phenolic amount (51.1 ± 14.1 mg GAE/1000 g; p < 0.0006) and flavonoid content (153.2 ± 3.5 mg CE/100 g; p < 0.004) were detected spectrophotometrically.     

Antiradical activity of different parts of Walnut (Juglans regia L.) fruit as a function of genotype

The objective of this work was to analyse phenolic compounds and antiradical capacity of different parts of walnut fruit among six genotypes of Juglans regia L. Therefore, total phenolic and flavonoid content were determined and methanolic extracts of walnut genotypes were considered by the reducing power, DPPH (2,2-diphenyl-1-picrylhydrazyl), superoxide anion and nitric oxide radical scavenging. Significant differences were found in phenolic content and radical scavenging capacity of different parts of fruits and among various genotypes. High correlation coefficient (R² = 0.81) was observed between phenol content and radical scavenging activity, but this was not always true (R² = 0.01). These results demonstrated that walnut genotypes have different phenolic compounds and phenolic compounds have different radical scavenging power. The differences of phenolic compounds were confirmed by using high performance liquid chromatography (HPLC).     

Insecticidal activities of constituents identified in the essential oil from leaves of Chamaecyparis obtusa against Callosobruchus chinensis (L.) and Sitophilus oryzae (L.)

Analysis by gas chromatography-mass spectrometry led to the identification of 20 volatiles from the steam distilled oil of the leaves from Chamaecyparis obtusa Siebold et Zuccarni. Seven constituents of the oil were tested for contact and fumigant activity against adults of Callosobruchus chinensis (L.) and Sitophilus oryzae (L.). Responses varied with compound and dose rather than increasing exposure time beyond 1 or 2 days. In the impregnated-paper test with the constituents [bornyl acetate, (+)-limonene, myrcene, α-phellandrene, α-pinene, sabinene and terpinolene], at 0.1 mg/cm², bornyl acetate, α-phellandrene and terpinolene caused 97%, 97% and 87% mortality, respectively, against C. chinensis adults 1 day after treatment. At 0.05 mg/cm², moderate activity was achieved with bornyl acetate (56% mortality), α-phellandrene (75%) and terpinolene (55%). Against S. oryzae adults, at 0.26 mg/cm², terpinolene caused 93% mortality 2 days after treatment, whereas 80% mortality 4 days after treatment was observed with bornyl acetate and α-phellandrene. The toxicity of these compounds was significantly decreased at 0.18 mg/cm². In a fumigation test with S. oryzae adults, bornyl acetate and terpinolene were much more effective in sealed containers than in open ones, indicating that the insecticidal activity of these compounds was largely attributable to fumigant action. These naturally occurring materials derived from leaves of Chamaecyparis could be useful for managing populations of C. chinensis and S. oryzae.     

Chemical and biological variability of hot pepper fruits (Capsicum annuum var. acuminatum L.) in relation to maturity stage

The aim of the present work was to evaluate the chemical composition and the radical-scavenging and antioxidant activities of hot pepper fruits (Capsicum annuum L. var. acuminatum) at three maturity stages (small green, green and red). GC–MS analysis of n-hexane and chloroform fractions showed a different composition between the three stages of ripening. The first stage of maturation (small green) showed the highest radical-scavenging activity (IC₅₀ of 129 μg/ml). Using the bovine brain peroxidation assay, the methanolic extract of green pepper showed significant antioxidant activity (IC₅₀ of 522 μg/ml). Addition of methanolic extract of red and green pepper inhibited oxidation of linoleic acid. Methanolic extract of red pepper showed greater antioxidative potency than the others (IC₅₀ of 3 μg/ml). The different composition of lipophilic compounds and the various amount of phenolics, showed in the three stage of ripening of C. annuum var. acuminatum fruits, modifies the antioxidant activity.     

Antioxidant, antifungal and antiviral activities of chitosan from the larvae of housefly, Musca domestica L.

Antioxidant activity of the chitosan from the larvae of Musca domestica L. was evaluated in two different reactive oxygen species assays, and inhibitory effects against seven fungi were also tested. The results showed that the chitosan had scavenging activity for hydroxyl and superoxide radicals which were similar to that of ascorbic acid. Also the chitosan exhibited excellent antifungal activity, especially in the low concentration, it could significantly inhibit the growth of Rhizopus stolonifer. Besides, antiviral results demonstrated that the chitosan could effectively inhibit the infection of AcMNPV and BmNPV. These results suggested that the chitosan from the larvae of housefly could be effectively used as a natural antioxidant to protect the human body from free radicals and retard the progress of many chronic diseases. Furthermore, the chitosan with antiviral and antifungal activity might provide useful information for antiviral breeding technology of economic insect and development of plant pathological control.