Optimization of extraction method for LC–MS based determination of phenolic acid profiles in different Impatiens species

The main aim of presented study was the comparison of various extraction methods for the quantitative and qualitative analysis (LC-ESI–MS/MS) of phenolic acids present in extracts obtained from leaves, flowers, and roots of Impatiens glandulifera. The accelerated solvent extraction (ASE) at three temperature ranges (80° C, 100° C, and 120° C), ultrasound assisted extraction (USAE) at 60° C, and traditional extraction in Soxhlet apparatus were used. Taking into account the extraction yield, and the diversity of the individual compounds, ultrasound assisted extraction proved to be the most efficient method, and it was used to determine the content of phenolic acids in leaves of four other Impatiens species, including I. balsamina, I. noli-tangere, I. parviflora, and I. walleriana. Eleven phenolic acids were identified in all examined species. These were protocatechuic, gentisic, 4- hydroxybenzoic, vanillic, trans-caffeic, syringic, trans-p-coumaric, trans- and cis-ferulic, salicylic, and 3-hydroxycinnamic acids. In the extract from the leaves of I. balsamina and I. walleriana, gallic and cis-p-coumaric acids were found additionally. The most abundant compounds in all examined extracts were protocatechuic and 3-hydroxycinnamic acids. The latest acid was found in the highest yield in I. noli-tangere (266.12 μg/g DW). In the leaves of I. glandulifera a great amount of 4-hydroxybenzoic (41.44 μg/g DW), vanillic (61.50 μg/g DW), and trans-p-coumaric (58.42 μg/g DW) acids was also observed. Our results indicate that protocatechuic, 4-hydroxybenzoic, vanillic, trans-p-coumaric, trans-ferulic, and 3-hydroxycinnamic acids were most characteristic of Impatiens species.Additionally, various phenolic-rich extracts from leaves, flowers, and roots of Impatiens glandulifera were tested for antioxidant activity. The highest antiradical activity was detected for roots using Soxhlet extraction (EC50 = 0.055 mg [DE/ml]).The study demonstrated that members of the genus Impatiens, and in particular Impatiens glandulifera, and Impatiens noli-tangere, contain significant amounts of phenolic acids. In addition, extracts from various parts of I. glandulifera could be interesting as novel sources of natural antioxidants.     

Total phenolics,flavonoids and antioxidant properties of three Ceropegia species from Western Ghats of India

The aim of present work was to assess the total phenolic content (TPC), total flavonoid content (TFC), phenolic compounds and antioxidant properties of various extracts of three Ceropegia spp.: Ceropegia spiralis, Ceropegia panchganiensis and Ceropegia evansii from Western Ghats of India. TPC of the samples varied from 0.3 ± 0.2 to 28.5 ± 0.3 mg TAE/g FW, whereas, TFC of the samples ranged between 0.1 ± 0.1 and 15.3 ± 0.3 mg RE/g FW. The major phenolic compounds identified were gallic acid, vanillin, cathechol and ferulic acid. All the extracts possess 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP) as well as metal chelating ability and this was also supported by significant correlation with TPC and TFC. To the best of our knowledge, this is the first paper presenting comprehensive data on TPC, TFC, phenolic profile and antioxidant properties of the Ceropegia spp.     

Determination of phenolic compounds and diterpenes in roots of Salvia miltiorrhiza and Salvia przewalskii by two LC–MS tools: Multi-stage and high resolution tandem mass spectrometry with assessment of antioxidant capacity

Comparative phytochemical analyses of hydroalcoholic (50% EtOH) extracts from roots of S. miltiorrhiza (SM) and S. przewalskii (SP) were performed using two complementary LC–MS systems: the first system HPLC-DAD-MSⁿ an ion trap mass spectrometer and the second system consisted high resolution MS/MS Orbitrap mass spectrometer. The individual compounds were identified using a previously published approach via comparison of the exact molecular masses, mass spectra and retention times to those of standard compounds, online available databases and literature data. Moreover, the determination of antioxidative activities of extracts by DPPH and FRAP methods was carried out. Analysis allowed to identify 39 chemical compounds in extracts from both species. Extract from root of SP differs from SM in the presence of several metabolites such as: przewalskinic acid and their derivatives, przewaquinone C, przewaquinonate A, glycosides of rosmarinic acid, methyltanshinonate, whereas tanshinones, salvianolic acids and lithospermic acids occurred in both species. Moreover, it was shown that hydroalcoholic extract from roots of SM exerted stronger antioxidant properties in a FRAP test (max. 323.92 μM Fe²⁺/L) and in DPPH test (max. 78.64 nM TE) in comparison with SP extract.     

Phenolic compounds composition and physiological attributes of Matricaria chamomilla grown in copper excess

Four-week old plants of chamomile (Matricaria chamomilla) cultivated in nutrient solution were exposed to copper (3, 60 and 120 μM) for 10 days. At 120 μM, Cu decreased dry mass production, water, chlorophyll and nitrogen content in both the leaf rosettes and roots. Five phenolic acids were detected in methanol extracts of the leaf rosettes (protocatechuic, p-hydroxybenzoic, vanillic, chlorogenic and salicylic acid) and six additional compounds (gentisic, syringic, caffeic, sinapic and o-/p-coumaric acid) were released after acid hydrolysis. Most of the 11 phenolic acids detected increased in 60 μM Cu but in the 120 μM treatment their contents were lower or not significantly different from the control. Among coumarin-related compounds, (Z)- and (E)-2-ß-d-glucopyranosyloxy-4-methoxycinnamic acids increased in 60 and 120 μM Cu while herniarin rose in the 3 and 60 μM Cu by the end of the experiment. The amounts of umbelliferone were not affected by any of the doses tested. These facts in relation to antioxidative properties of phenolic metabolites are also discussed. The malondialdehyde content of the leaf rosettes was not affected by exposure of plants to 120 μM Cu in a time-course experiment but in the roots a sharp increase was observed after 24 and 48 h of treatment. At 120 μM, Cu stimulated a 9-fold higher K⁺ loss than the 60 μM treatment while at the lowest concentration it stimulated potassium uptake. Cu accumulation in the roots was 3-, 49- and 71-fold higher than that in the leaf rosettes in the 3, 60, and 120 μM Cu treatments, respectively. Results suggest that 120 μM Cu dose is limiting for chamomile growth under the conditions of present research.     

Acridone alkaloids from Vepris verdoorniana (Excell & Mendonça) Mziray (Rutaceae)

Two new acridone alkaloids, verdoocridone A (1) and B (4), together with fifteen known compounds were isolated from methanol extracts of the roots and leaves of Vepris verdoorniana. The structures of all compounds were determined by comprehensive spectroscopic analyses (1D and 2D NMR, EI- and ESI–MS). The ¹³C NMR values of 1,2,3,5-tetramethoxy-N-methylacridone (2) and 5-methoxyaborinine (3) are also reported. The crude extracts and compounds (1-6) were tested for their antimicrobial activity. The test delivered moderate activities for crude extracts and compounds 1, 5 and 6 against the bacterium Staphylococcus aureus and the fungi Mucor meihei and Candida albicans with MIC values between 115 and 180 μg/mL for extracts and between 21.3 and 29.4 μM for compounds, compared to gentamycin with 0.2 μM and nystatin with 5.2 μM against both fungi. The determination of the radical scanvenging activity using 1,1-dephenyl-2-picrylhydrazyl (DPPH) assay gave moderate antioxidant values for all tested compounds, with IC₅₀ between 0.29 and 0.41 μM, compared to the standard 3-t-butyl-4-hydroxyanisole (BHA) displaying 0.03 μM.     

Isolation of antioxidant constituents from Combretum apiculatum subsp. apiculatum

Species of the family Combretaceae are used extensively in traditional medicine against inflammation and infections, and although antibacterial activity has been reported in non-polar extracts, further rationale for the widespread use of the Combretaceae is expected to exist. Methanol extracts of leaves of ten different Combretum species were evaluated for antioxidant activity by spraying TLC chromatograms of each leaf extract with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Compounds with antioxidant activity were detected by bleaching of the purple DPPH colour. Leaf extracts of Combretum apiculatum subsp. apiculatum had the most antioxidant compounds. This species was consequently selected for phytochemical investigation. A DPPH assay-directed fractionation of the leaf extracts of C. apiculatum led to the isolation of four antioxidant compounds from the ethyl acetate and butanol soluble fractions. The structures of the compounds were determined by spectroscopic analyses (¹H-NMR, ¹³C-NMR and MS) and identified as: cardamonin (1), pinocembrin (2), quercetrin (3) and kaempferol (4). In a quantitative antioxidant assay, the more polar fractions (ethyl acetate and butanol) obtained by solvent–solvent fractionation had the highest antioxidant activity among the solvent fractions obtained from C. apiculatum, with EC₅₀ values of 3.91 ± 0.02 and 2.44 ± 0.02 μg/ml respectively. Of the four isolated compounds, quercetrin (4) and kaempferol (3) had the strongest antioxidant activity, with EC₅₀ values of 11.81 ± 85 and 47.36 ± 0.03 μM respectively. Cardamonin (1) and pinocembrin (2) did not demonstrate strong activity. L-ascorbic acid was used as standard antioxidant agent (EC₅₀ = 13.37 ± 0.20 μM or 2.35 μg/ml). The cytotoxicity of cardamonin and pinocembrin was evaluated on Vero kidney cells using the MTT (3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide) assay with berberine as positive control. At concentrations higher than 50 μg/ml of cardamonin or pinocembrin, the cells were not viable. Cardamonin was more toxic (LC₅₀ = 1.97 μg/ml) than pinocembrin (LC₅₀ = 29.47 μg/ml) and even the positive control, berberine (LC₅₀ = 12.35 μg/ml).     

Studies on the accumulation of phenolic acids and flavonoids in different in vitro culture systems of Schisandra chinensis (Turcz.) Baill. using a DAD-HPLC method

Different in vitro culture systems of the East-Asian origin medicinal plant species − Schisandra chinensis, were tested in order to investigate their potential for the accumulation of two groups of phenolic compounds. In vitro cultures were maintained on the Murashige and Skoog (MS) medium supplemented with 3 mg/l BA and 1 mg/l NAA in an agar system (30- and 60-day growth cycles), and also in two different liquid systems: stationary and agitated. Stationary liquid cultures were grown in batch (30- and 60-day growth cycles) and fed-batch modes. Of the twenty compounds, seven free phenolic acids and of the eleven compounds, five flavonoids were quantified in methanolic extracts from lyophilized biomass and in the growth media using the RP-HPLC-DAD method. For comparison purposes, phytochemical analyses of leaf and fruit extracts from the parent plant were also conducted. The estimated compounds were not detected in the growth media. The highest total amounts of phenolic acids (71.48 mg/100 g DW) and flavonoids (29.36 mg/100 g DW) were found in extracts from the biomass of agar cultures harvested after 30 days of cultivation. The main metabolites in all the tested systems were: protocatechuic acid (max. 35.69 mg/100 g DW), chlorogenic acid (max. 13.05 mg/100 g DW), and quercitrin (max. 27.43 mg/100 g DW).     

Deciphering the CNS anti-depressant,antioxidant and cytotoxic profiling of methanol and aqueous extracts of Trametes versicolor and molecular interactions of its phenolic compounds

The present study sought to evaluate the central nervous system (CNS) depressant, antioxidant, and cytotoxicity activity of methanol and aqueous extract of Trametes versicolor (METV and AETV). The CNS activity was assessed by the open field, hole-cross, forced swimming, thiopental sodium-induced sleeping time, hole-board, and rotarod tests in Swiss albino mice. For both extracts, a substantial decrease in locomotion was observed in open field and hole-cross tests. In addition, the molecular docking study has been implemented through Maestro V11.1. The higher dose of METV (400 mg/kg) and the lower dose of AETV (200 mg/kg) exhibited a significant decrease in immobility time in forced swimming test and increased prolongation of sleep in thiopental sodium-induced sleeping time test, respectively. In contrast, a moderate finding was observed for the hole-board and rotarod tests. Additionally, a significant DPPH scavenging assay and a high toxicity effect in brine shrimp lethality assay were observed. Besides, five phenolic compounds, namely baicalin, quercetin, catechin, p-hydroxybenzoic acid, and quinic acid, were used for the molecular docking study, whereas catechin demonstrated the highest binding affinity towards the targets. The findings conclude that the T. versicolor could be an alternative source for CNS anti-depressant and antioxidant activity.     

Antioxidative effect of Iranian Pulicaria gnaphalodes L. extracts in soybean oil

This study was aimed to evaluate antioxidative activities of the ethanol, methanol and water extracts of Pulicaria gnaphalodes in vegetable oil during the storage period. Different concentrations (0, 200, 400 and 800 ppm) of ethanol, methanol and water extracts and beta-hydroxy toluene (BHT; 100, 200 ppm) were added to soybean oil and incubated for 35 days at 65 °C. Peroxide values (PVs) and thiobarbituric acid-reactive substance (TBARS) levels were measured every week during the period of the study. Moreover, antioxidant capacities of the extracts were determined using DPPH and β-carotene–linoleic acid methods. Values were compared among groups in each incubation time points using ANOVA. Results showed that DPPH and β-carotene–linoleic acid assay findings on the P. gnaphalodes extracts were comparable to those found on BHT. Moreover, during incubation time, P. gnaphalodes extracts lowered PVs and TBARS levels when compared to the control (p < 0.001). In this respect, water extract was more potent than the ethanol and methanol extracts. It seems that water extract of P. gnaphalodes is a potent antioxidant which makes it as a potential antioxidant for oil and oily products during storage.     

New phenolic glycosides with cyclooxygenase inhibition from the roots of Tecoma mollis

Three new phenolic glycosides (1–3) together with nine known ones were isolated from the roots of Tecoma mollis using DPPH radical scavenging bioassay-guided chromatographic separation. The structures of the new compounds were established using extensive spectroscopic data and HR-MS. The antioxidant, COX-2 inhibition, and cytotoxic activities were evaluated for the isolated compounds. Compound 4 displayed the strongest radical scavenging activity relative to ascorbic acid with IC₅₀ 8.7 μM. Compounds 5, 6, and 10 showed promising COX-2 inhibitory action, IC₅₀ values of 11.3 μM, 9.4 μM, and 13.4 μM, respectively. All compounds exhibited weak cytotoxic activity against Hela and A549 cancer cell lines.     

Chemical constituents from Tribulus terrestris and screening of their antioxidant activity

Two oligosaccharides (1, 2) and a stereoisomer of di-p-coumaroylquinic acid (3) were isolated from the aerial parts of Tribulus terrestris along with five known compounds (4–8). The structures of the compounds were established as O-β-d-fructofuranosyl-(2 → 6)-α-d-glucopyranosyl-(1 → 6)-β-d-fructofuranosyl-(2 → 6)-β-d-fructofuranosyl-(2 → 1)-α-d-glucopyranosyl-(6 → 2)-β-d-fructofuranoside (1), O-α-d-glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-(1 → 2)-β-d-fructofuranoside (2), 4,5-di-p-cis-coumaroylquinic acid (3) by different spectroscopic methods including 1D NMR (¹H, ¹³C and DEPT) and 2D NMR (COSY, TOCSY, HMQC and HMBC) experiments as well as ESI-MS analysis. This is the first report for the complete NMR spectral data of the known 4,5-di-p-trans-coumaroylquinic acid (4).The antioxidant activity represented as DPPH free radical scavenging activity was investigated revealing that the di-p-coumaroylquinic acid derivatives possess potent antioxidant activity so considered the major constituents contributing to the antioxidant effect of the plant.     

Chemistry,antioxidant and antimicrobial potential of nutmeg (Myristica fragrans Houtt)

Antioxidant and antimicrobial activities of nutmeg (Myristica fragrans Houtt) seed extracts were evaluated. Seeds were extracted with acetone, ethanol, methanol, butanol and water. All the extracts have shown significant antioxidant and antimicrobial activities against the tested microorganisms. Among all extracts, acetone extract has shown the highest antioxidant activity. The acetone extract showed 93.12 ± 1.48 mg gallic acid equivalents (GAE)/100 g dry weight total phenolic content, DPPH scavenging activity of 63.04 ± 1.56%, chelating activity of 64.11 ± 2.21% and 74.36 ± 1.94% inhibition of β-carotene bleaching, at 1 mg/mL extract concentration. Out of all extracts, acetone extract was able to exert antimicrobial activity against all tested bacteria and fungi. Acetone extract has shown the strongest antibacterial and antifungal activity with Staphylococcus aureus (13.8 ± 0.42 mm) and Aspergillus niger (14.4 ± 0.37 mm), respectively. GC–MS analysis of acetone extract has revealed the presence of 32 compounds of extract representing 99.49%. Sabinene (28.61%) has shown the highest occurrence in the extract. β-Pinene (10.26), α-pinene (9.72), myristicin (4.30%), isoeugenol (2.72%), p-cymene (1.81%), carvacrol (1.54%), eugenol (0.89%) and β-caryophellene (0.82%) were reported as possible contributor for antioxidant and antimicrobial activity of nutmeg.     

Isolation and characterization of ellagitannins as the major polyphenolic components of Longan (Dimocarpus longan Lour) seeds

Longan (Dimocarpus longan Lour, syn. Euphoria longan Lam.) represents an important fruit in Northern Thailand and has significant economic impact. The fruit is either consumed fresh or as commercially prepared dried and canned products. The canning industry in Thailand produces considerable quantities of waste products, in particular Longan seeds. Because these seeds may be an exploitable source of natural phenolic antioxidants, it was of interest to identify, purify and quantitate the major potential antioxidant phenolics contained therein. The polyphenolic fraction from ground Longan seeds was obtained by extraction with methanol after delipidation with hexane. The hexane extract contained predominantly long-chain fatty acids with major contributions from palmitic (35%) and oleic (28%) acids. The polyphenolic fraction (80.90 g/kg dry weight) was dominated by ellagic acid (25.84 g/kg) and the known ellagitannins corilagin (13.31 g/kg), chebulagic acid (13.06 g/kg), ellagic acid 4-O-α-l-arabinofuranoside (9.93 g/kg), isomallotinic acid (8.56 g/kg) and geraniin (5.79 g/kg). Structure elucidation was performed with mass spectrometry and complete assignment of ¹H and ¹³C NMR signals. The methanol extracts exhibited strong antioxidant capacities with an IC₅₀ of 154 μg/ml for reactive oxygen species attack on salicylic acid and 78 μg/ml for inhibition of xanthine oxidase in the hypoxanthine/xanthine oxidase assay. The extracts were less effective in the 2-deoxyguanosine assay (IC₅₀ = 2.46 mg/ml), indicating that gallates along with ellagic acid and its congeners exert their potential antioxidant effects predominantly by precipitation of proteins such as xanthine oxidase. This was confirmed for the pure compounds gallic acid, methyl gallate, ellagic acid and corilagin.     

Phenolic acid content,antioxidant and cytotoxic activities of four Kalanchoë species

Phenolic acid composition, antioxidant, and cytotoxic activities in leaves of four Kalanchoe (Crassulaceae) species were evaluated. Determination of phenolic acid contents were conducted by an optimized LC–ESI-MS/MS method. The results show that Kalanchoe daigremontiana Raym.-Hamet & H. Perrier (using ASE extraction) and Kalanchoe pinnata (Lam.) Pers. contain the highest amounts of phenolic acids, while Kalanchoe nyikae Engl. the lowest ones. Among phenolic acids ferulic, caffeic and protocatechuic acids were occurring in the highest quantities in the analysed species. The greatest amounts of ferulic and protocatechuic acids were found in K. daigremontiana and K. pinnata. Moreover, the antiradical and cytotoxic activities of Kalanchoe extracts were investigated. All tested extracts possessed antioxidant activity. The obtained IC₅₀ values (μg/mL) ranged from 49.9 μg/mL to 1410 μg/mL, indicating a large variation of the activity of the analysed extracts. Cytotoxicity assays revealed dose-dependent effects in the cells lines tested. Only K. pinnata extract showed a high cytotoxicity against the H-9 human T cell line. Other extracts (K. daigremontiana, Kalanchoe milloti, K. nyikae) showed more pronounced cytotoxicity towards J45.01 cells (human acute lymphoblastic leukaemia T cells).The present study demonstrated that Kalanchoe extracts have significant antioxidant and cytotoxic effects. This suggests that these species can be used as new sources of natural antioxidants and potential anticancer compounds.     

Cytoprotection by almond skin extracts or catechins of hepatocyte cytotoxicity induced by hydroperoxide (oxidative stress model) versus glyoxal or methylglyoxal (carbonylation model)

Oxidative and carbonyl stress are detrimental in the pathogenesis of diabetic complications, as well as in other chronic diseases. However, this process may be decreased by dietary bioactive compounds. Almond skin is an abundant source of bioactive compounds and antioxidants, including polyphenolic flavonoids, which may contribute to the decrease in oxidative and carbonyl stress. In this study, four Almond Skin Extracts (ASEI, ASEII, ASEIII, and ASEIV) were prepared by different methods and evaluated for their antioxidant activity. The order of the polyphenol content (total μM gallic acid equivalents) of the four extracts was found to be, in decreasing order of effectiveness: ASEI > ASEIII > ASEIV > ASEII. The order of Ferric-reducing antioxidant power (FRAP, μM FeSO₄/g) value, in decreasing order was ASEI (216) > ASEIII (176) > ASEIV (89) > ASEII (85). The order of ASE effectiveness for decreasing protein carbonyation induced by the copper Fenton reaction was ASEI > ASEIV > ASEII > ASEIII. The order of antioxidant effectiveness for inhibiting tertiary-butyl hydroperoxide (TBH) induced microsomal lipid peroxidation was ASEI > ASEIV > ASEII, ASEIII. Also, the order of ASE effectiveness for inhibiting TBH induced hepatocyte cell death was: ASEIII, ASEIV > ASEI, ASEII. Catechin also protected hepatocytes from TBH induced hepatocyte, lipid peroxidation and cytotoxicity. In a cell free model, equimolar concentrations of catechin or epicatechin rescued serum albumin from protein carbonylation induced by methylglyoxal (MGO). Catechin, epicatechin and ASEI all decreased gloxal induced hepatocyte cell death and reactive oxygen species (ROS) formation in GSH-depleted hepatocytes. Catechin and epicatechin protected against GO or MGO induced hepatocyte cell death, protein carbonylation and ROS formation. Catechin was more effective than epicatechin. Our results suggest that (a) bioactive almond skin constituents in the non-lipophilic polyphenol extract were the most effective at protecting hepatocytes against hydroperoxide induced hepatocyte oxidative stress and in protecting against dicarbonyl induced cytotoxicity; (b) catechins, the major polyphenol in the extract, were also effective at preventing GO or MGO cytotoxicity likely by trapping GO and MGO and/or rescuing hepatocytes from protein carbonylation.     

Antioxidative and cytotoxic potential of some Chenopodium L. species growing in Poland

The cytotoxic and antioxidant properties of lipophilic compounds extracted from different parts of four Chenopodium L. (Chenopodium album, Chenopodium hybridum, Chenopodium rubrum and Chenopodium urbicum) species were evaluated. The highest phenolic content was found in herb and seeds of all examined plants. Large amounts of free polyphenols were observed in herb extracts of C. album (3.36 mg/g DW), seeds of C. urbicum (3.87 mg/g DW) and roots of C. urbicum (1.52 mg/g DW). The cytotoxic activities of the extracts were assessed against human lung carcinoma A-549 and ovarian carcinoma TOV-112D and normal human fibroblast cell lines. Our study demonstrated that the extracts from the herb of C. rubrum and C. urbicum had the best antioxidant effect of all the extracts analyzed. Most of the extracts tested exhibited low cytotoxicity. However, the extracts from herb and seeds of C. album and C. hybridum showed the significant antiproliferative effect on the TOV-112 cell line.It can be concluded that antioxidant activity and phenolic composition differ mainly between plant parts and are quite similar between the plants, when the same plant part is analyzed. Thus, the Chenopodium extracts could be used as a readily accessible source of natural antioxidants, and may be used in the pharmaceutical industry and for food supplements production.     

Antimycobacterial activity of ellagitannin and ellagic acid derivate rich crude extracts and fractions of five selected species of Terminalia used for treatment of infectious diseases in African traditional medicine

Ten crude extracts and their solvent partition fractions from five species of Terminalia collected in Tanzania were assessed for antimycobacterial effects using Mycobacterium smegmatis ATCC 14468 as a model organism. We report here, for the first time, on antimycobacterial effects of root and stem bark extracts of Terminalia sambesiaca and Terminalia kaiserana as well as of fruit extracts of Terminalia stenostachya and leaf extracts of Terminalia spinosa. T. sambesiaca gave the best effects of all the investigated species in terms of the sizes of the inhibitory zones of root and stem bark extracts. A crude methanol root extract of T. sambesiaca gave lower MIC values (1250 μg/ml) than its aqueous and butanol soluble fractions (MIC 2500 μg/ml). Our preliminary HPLC–DAD data indicates that methanol and aqueous extracts of T. sambesiaca roots are rich in ellagitannins and ellagic acid glycosides. Particularly, one polar ellagitannin at t_R 10.3–10.9 min dominates the extracts quantitatively and thus may be responsible for their good antimycobacterial effects. In contrast to the more polar fractions, a chloroform soluble fraction of the roots of T. sambesiaca was devoid of antimycobacterial activity. Also crude methanol and aqueous extracts of the stem bark of T. sambesiaca gave promising antimycobacterial effects (MIC 1250 μg/ml). All fractions of T. kaiserana roots, except from the aqueous insoluble gave good antimycobacterial effects (MIC 1250 μg/ml) and the aqueous extract showed the best effects of the fractions in terms of the size of inhibition zones. These results justify the uses of hot water decoctions of the roots of T. kaiserana for treatment of cough, one of the symptoms of TB. According to HPLC–DAD data methanol extracts of T. kaiserana roots and their aqueous fractions are rich in polar ellagitannins and ellagic acid glycosides. Quantitatively, the ellagitannins dominate these extracts and therefore the good antimycobacterial activity of the methanol and aqueous extracts is assumed to be due to these compounds. Sephadex LH-20 CC fractions of a methanol extract of the roots of T. kaiserana inhibited the growth of M. smegmatis, giving MIC values of 1000 μg/ml. Ellagic acid glycosides in these fractions must be responsible for their good antimycobacterial effects since they are present in high concentrations. Good antimycobacterial effects were also obtained with a root extract of Terminalia sericea, and especially the butanol soluble fraction was a good inhibitor of the growth of M. smegmatis (MIC 1562 μg/ml). Our preliminary HPLC–DAD results show that the roots of T. sericea are rich in ellagitannins, ellagic acid glycosides and at least one stilbene compound. Extracts of the fruits of T. stenostachya gave good antimycobacterial effects, butanol extracts being the most active. Also the leaves of T. stenostachya, and especially the butanol soluble extracts, give good antimycobacterial effects. Our HPLC–DAD data indicate that T. stenostachya leaves contain large quantities of gallic acid, ellagitannins and ellagic acid glycosides. Our results indicate that many of the investigated species of Terminalia might contain leads for development of anti-TB drugs. Standardized extracts of T. sambesiaca, T. kaiserana and T. sericea roots could be used as easily available and cheap medicines for treatment of TB in remote regions of East and South Africa.     

Phytochemical and antioxidant properties of unconventional leafy vegetables consumed in southern Africa

Indigenous leafy vegetables possess high horticultural potential based on their long utilisation history by local communities across Africa. Phytochemical and antioxidant properties of 50% aqueous methanol and water extracts of three indigenous as well as two commercial leafy vegetables commonly consumed in southern Africa were evaluated. The total extractable phenolic content was highest for Amarathus dubius (5.16 ± 0.12 mg GAE/g DW) followed by Cleome gynandra (3.94 ± 0.09 mg GAE/g DW). Total flavonoid concentration was highest for A. dubius (3.89 ± 0.28 mg CE/g DW) followed by C. gynandra (2.19 ± 0.11 mg CE/g DW) and Cucurbita maxima (1.55 ± 0.04 mg CE/g DW). No proanthocyanidins were detected in C. maxima and Brassica napus cv Covo whereas low concentrations were recorded in other vegetables. Total saponins were variable across the evaluated extracts, with the highest concentrations recorded for B. napus cv Covo (83.2 ± 16.58 mg DE/g DW). Total iridoid content was highest for C. gynandra (9.14 ± 0.20 mg HE/g DW). More potent DPPH radical scavenging activities were exhibited by 50% aqueous methanol extracts compared to water extracts. A similar trend was observed in the ferric-reducing antioxidant power assay. The antioxidant activity based on the rate of β-carotene bleaching was higher for water extracts compared to 50% aqueous methanol extracts. The indigenous vegetables evaluated in this study had higher levels of phytochemicals and also exhibited more potent antioxidant activity compared to the commercial varieties. These findings not only suggest the importance of the indigenous vegetables in a healthy diet, but also provide a motivation for exploring their horticultural potential.     

Rheediinosides A and B,two antiproliferative and antioxidant triterpene saponins from Entada rheedii

Two triterpenoid saponins have been isolated from the seed kernels of Entada rheedii. Their structures have been established using 1D- and 2D-NMR and mass spectrometry as 3-O-β-d-xylopyranosyl-(1 → 3)-O-α-l-arabinopyranosyl-(1 → 6)-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside A, 1) and 3-O-β-d-glucopyranosyl-(1 → 3)-O-[β-d-xylopyranosyl-(1 → 3)-α-l-arabinopyranosyl-(1 → 6)]-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside B, 2). Compounds 1 and 2 were tested for their antiproliferative activity against T98G, A431, PC3 and B16-F1 cell lines, and further for their antioxidant properties. Moderate cytotoxic potency and antioxidant properties were found for these compounds whereas Rheediinoside B was in all assays more active than Rheediinoside A.     

Filamentous fungi from Plantago lanceolata L. leaves: Contribution to the pattern and stability of bioactive metabolites

The aim of this study was to test contribution of plant-associated microorganism (PAMs) to metabolite stability/instability in a medicinal plant matrix.Therefore, PAM strains were isolated and identified based on relevant DNA sequences from Plantago lanceolata leaves. Sterile water extracts of P. lanceolata were incubated with the isolated strains and antioxidants (ascorbic acid (AA), and EDTA) for 15 days, and changes in the concentrations of chief bioactive constituents (aucubin, catalpol, acteoside (=verbascoside)) were quantified by capillary electrophoresis. Phenolic breakdown-products were identified by GC–MS.PAMs were identified from the genera Epicoccum, Bipolaris, Cladosporium, Leptosphaerulina, Aspergillus, Eurotium and Penicillium (pathongens, endophytes, and other species). Some fungi caused significant decomposition of the chief constituents (p < 0.001). Surprisingly, some strains inhibited breakdown of acteoside (p < 0.001). Meanwhile, concentration of several phenolic acids increased in fungi-infested extracts (p < 0.001). Gentisic acid, 4-hydroxyphenyl acetic acid, 4-hydroxybenzoic acid and hydroxytyrosol were only present when the extract was infested with a PAM. The products are powerful antioxidants and chelators. Concentrations of phenolic acids influenced acteoside stability significantly (p < 0.01), as shown by basic data-mining techniques. AA and EDTA also significantly inhibited acteoside breakdown in sterile model solutions (p < 0.05).Our results suggest that the phenolic acid mixture (produced during the fungal proliferation) protected acteoside from breakdown, possibly via its antioxidant activity and metal complexing ability. It was shown that PAMs can increase or decrease the stability of chief metabolites in herbal matrices, and can significantly alter the chemical pattern of the plant matrix.