Effects of culture medium and auxins on growth of adventitious root cultures of Cuphea aequipetala Cav. and their ability to produce antioxidant compounds

Cuphea aequipetala Cav. (Lythraceae), a species highly valued for its medicinal properties, is threatened in the wild. To provide an alternative source of material for production of bioactive compounds, we established adventitious root cultures of C. aequipetala and determined their phenolic compounds contents and antioxidant activity. Cultures were initiated from root tips of in vitro C. aequipetala plantlets and were grown in B5 or SH culture medium containing either indole butyric acid (IBA) or α-naphthalene acetic acid at 0, 5 or 10 µM. The maximum root biomass (1.6 g/L dry mass (DM) per L medium) was recorded after 14 days of growth in B5 + 5 µM IBA. Roots in B5 medium remained green, whereas they tended to oxidize in SH medium. The highest contents of total phenolic compounds (9.1 ± 0.1 µg gallic acid equivalents/g DM) and flavonoids (37.5 ± 0.7 µg quercetin equivalents/g DM) were in roots grown in B5 + 5 µM IBA after 14 days of growth. Root cultures accumulated mainly flavan-3-ols, whereas roots or leaves from whole plants accumulated mainly flavonols. We analyzed the antioxidant properties of root extracts using in vitro assays. Roots grown in B5 medium showed stronger free-radical scavenging activity than that of roots grown in SH medium. Our results show that adventitious root cultures of C. aequipetala are a promising system for research on antioxidant compounds biosynthesis and for scaled-up production of useful biological materials.     

In vitro cytotoxic and in vivo antitumoral activities of some aminomethyl derivatives of 2,4‐dihydro‐3H‐1,2,4‐triazole‐3‐thiones—Evaluation of their acetylcholinesterase and carbonic anhydrase enzymes inhibition profiles

The 1,2,4‐triazole and its derivatives were reported to exhibit various pharmacological activities such as antimicrobial, analgesic, anti‐inflammatory, antitumoural, cytotoxic, and antioxidant properties. In this study, a series of triazole compounds (M1‐M10) were evaluated for some biological activities. In vitro qualifications of these compounds on acetylcholinesterase (AChE) and human carbonic anhydrase enzyme activities were performed. Also, their antitumoral activities in human colon cancer (HT29) cell line cultures were examined. In addition, colon cancer experimentation was induced in rats by an in vivo method, and the in vivo anticancer effects of triazole derivatives were investigated. Also, the effects of these derivatives in levels of antioxidant vitamin A, vitamin E, and MDA were studied in rat liver and blood samples. Most of the compounds were found to exhibit significant antioxidant and antitumoral activities. All the compounds had cytotoxic activities on HT29 cell lines with their IC₅₀ values lower than 10 µM concentrations. The low IC ₅₀ values of the compounds are M1 (3.88 µM), M2 (2.18 µM), M3 (4.2 µM), M4 (2.58 µM), M5 (2.88 µM), M6 (2.37 µM), M7 (3.49 µM), M8 (4.01 µM), M9 (8.90 µM), and M10 (3.12 µM).     

In vitro propagation,clonal fidelity and phytochemical analysis of <Emphasis Type="Italic">Rhodiola imbricata</Emphasis> Edgew: a rare trans-Himalayan medicinal plant

The present study focuses on development of a micropropagation protocol for true to type plants of Rhodiola imbricata, an endangered medicinal plant found in trans-Himalayan Leh-Ladakh region of India. It also aims at analyzing the pharmaceutically important secondary metabolites and antioxidant potential of in vitro and in vivo plants. Various cytokinins and auxins were tested for shoot proliferation and in vitro rooting of the microshoots, respectively. Random primers were used for checking genetic uniformity at different stages of micropropagation. Pharmaceutically important secondary metabolites of R. imbricata such as Rosavin, total polyphenols and free radical scavenging activity were analyzed by HPLC. Among different cytokinins used, BAP (5 µM) and TDZ (1 µM) were found to perform better in terms of shoot proliferation, shoot length and number of leaves as compared to other concentrations. For rooting of microshoots, a lower concentration of NAA (0.5 µM) yielded more efficient rooting of micro shoots (17.33 roots per micro shoot). In vitro rooted microshoots were hardened and showed 60% survival rate. The content of gallic acid, chlorogenic acid and 4-hydroxybenzoic acid was higher in the in vivo plant. The amount of ferulic acid was higher in the in vitro raised plant when compared to field grown plant. Furthermore, caffeic acid and p-coumaric acid were higher in the in vitro raised plants as compared to field grown plants. This work will facilitate in conservation of this endangered herb and provide necessary plant materials for various biotechnological and pharmaceutical applications.     

Plant growth regulator induced phytochemical and antioxidant variations in micropropagated and acclimatized Eucomis autumnalis subspecies autumnalis (Asparagaceae)

Eucomis species is a valuable plant with both medicinal and horticultural potential. The current study evaluated the role of plant growth regulator (PGR) on growth, phytochemicals, and antioxidant activity in Eucomis autumnalis subspecies autumnalis. Five cytokinins including topolins and benzyladenine (BA) at 2 µM in combination with varying (0–15 µM) concentrations of naphthalene acetic acid (NAA) were tested. In vitro regenerants were acclimatized in the greenhouse for 4 months. Highest number of shoots (9 shoots/explant) was observed with 15 µM NAA alone or when combined with BA. Acclimatized plants derived from the 15 µM NAA treatment had the highest number of roots, largest leaf area and biggest bulbs. While applied PGRs increased the iridoids and condensed tannins in the in vitro regenerants, total phenolics and flavonoids were higher in the PGR-free treatment. Among the in vitro regenerants, 5 µM NAA and 2 µM BA treatments produced the best antioxidant activity in the DPPH (55 %) and beta-carotene (88 %) test systems, respectively. A remarkable carry-over effect of the PGR was conspicuous in the phytochemical levels and antioxidant activity of the 4-month-old plants. In addition to the optimized micropropagation protocols, the current findings present a promising potential for manipulating the type and concentration of applied PGRs to improve phytochemical production and hence medicinal value in E. autumnalis subspecies autumnalis.     

Antioxidant and antitumor activities of the polysaccharide from seed cake of Camellia oleifera Abel

To explore biomedical potential of the polysaccharide from seed cake of Camellia oleifera Abel, we investigated antioxidant and antitumor capacities of the polymer. The results showed that the polysaccharide is capable of scavenging both superoxide anion and hydroxyl radicals in vitro. The highest scavenging rate of superoxide anion and hydroxyl radicals is 85% and 76%, respectively. Using the model animal, Caenorhabditis elegans, we further show that the polysaccharide can increase antioxidant enzyme activity, decrease lipid peroxidation level, and reduce paraquat-induced oxidative damage at a polysaccharide concentration more than 50mg/l. We also revealed that the polysaccharide has some ferric chelating ability and strong in vivo antitumor activity. The antitumor rate against Sarcoma180 solid tumor grown in BALB/C mice reached 85.6% at the highest dose of 40×20mg/kgdays.     

Genetic stability and phytochemical profiling of the in vitro regenerated plants of <Emphasis Type="Italic">Angelica glauca</Emphasis> Edgew.: an endangered medicinal plant of Himalaya

The present study describes the first successful report on in vitro propagation through direct organogenesis for multiple shoot induction of Angelica glauca. Rhizomes were used as explant, and maximum shoot multiplication was observed on MS medium supplemented with 6-Benzylaminopurine 8.0 µM and Indole-3-acetic acid 0.1 µM. Roots were observed within 14 days in the MS medium enriched with 0.5 µM IAA and 0.1 µM Naphthalene acetic acid (NAA) with an average production of 4.2 roots per shoot. Rooted plantlets were successfully hardened under greenhouse conditions and subsequently established in field, with a recorded survival rate of 72% after 45 days. The total phenolic content showed significant difference (p?<?0.05) between in vitro raised plants (5.87 mM AAE/ g DW) and control (2.36 mM AAE/ g DW). Antioxidant activity, calculated through two in vitro assays, i.e. 1,1-diphenyl-2 picrylhydrazyl (DPPH) radical scavenging and Ferric Reducing Antioxidant Power (FRAP) assays revealed higher antioxidant activity in in vitro grown plants in comparison to control plants. Essential oil constituent’s analysis was also carried out in control and in vitro raised plants. Thirty-one compounds were identified in the oil samples through Gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) analysis also identified 31 compounds in the essential oil, representing 98.1–98.7% of total oil compositions. The major components of the essential oils were (Z)-ligustilide (51.1–51.5%), (Z)-butylidene phthalide (31.2–31.6%), (E)-butylidene phthalide (2.6–2.9%) and (E)-ligustilide (2.1–1.8%). Genetic stability of in vitro raised plants, evaluated using 20 Inter Simple Sequence Repeats primers, proved true to typeness of in vitro raised plants.     

Structure-antioxidant activity relationships of flavonoids isolated from the resinous exudate of Heliotropium sinuatum

Relationships between the structural characteristics of flavonoids isolated from the resinous exudate of Heliotropium sinuatum and their antioxidant activity were studied. Radical formation energies, DeltaH of dehydrogenation and spin densities were calculated using DFT methods (B3LYP/6-31G*). Results show that studied flavonoids can be divided into two sets according to their activity. It has been found that antioxidant activity depends both on substitution pattern of hydroxyl groups of the flavonoid skeleton and the presence of an unsaturation at the C2-C3 bond. A good tendency between DeltaH of dehydrogenation and antioxidant activity was established.     

In Vitro Anticancer Activity of Staphyloxanthin Pigment Extracted from <Emphasis Type="Italic">Staphylococcus gallinarum</Emphasis> KX912244, a Gut Microbe of <Emphasis Type="Italic">Bombyx mori</Emphasis>

The present study reports the in vitro biological nature of the pigment produced by Staphylococcus gallinarum KX912244, isolated as the gut microflora bacterium of the insect Bombyx mori. The purified pigment was characterized as Staphyloxanthin based on bio-physical characterization techniques like Fourier transform infrared spectroscopy, high performance liquid chromatography, Proton nuclear magnetic resonance spectroscopy (¹H NMR), Liquid chromatography-Mass spectroscopy and Gas chromatography-Mass spectroscopy. The Staphyloxanthin pigment presented considerable biological properties including in vitro antimicrobial activity against pathogens Staphylococcus aureus, Escherichia coli and Candida albicans; in vitro antioxidant activity by % DPPH free radical scavenging activity showing IC₅₀ value of 54.22 µg/mL; DNA damage protection activity against reactive oxygen species and anticancer activity evaluated by cytotoxicity assay against 4 different cancer cell lines like the Dalton’s lymphoma ascites with IC₅₀ value 6.20?±?0.02 µg/mL, Ehrlich ascites carcinoma having IC₅₀ value 6.48?±?0.15 µg/mL, Adenocarcinomic human alveolar basal epithelial cells (A549 Lung carcinoma) bearing IC₅₀ value 7.23?±?0.11 µg/mL and Mus mucus skin melanoma (B16F10) showing IC₅₀ value 6.58?±?0.38 µg/mL and less cytotoxicity towards non-cancerous human fibroblast cell lines (NIH3T3) with IC₅₀ value of 52.24 µg/mL. The present study results suggest that Staphyloxanthin acts as a potential therapeutic agent especially due to its anticancer property.     

Synthesis and antitumour activity of novel diterpenequinone salvicine and the analogs.

A novel diterpenequinone named salvicine (4), structurally modified derivative of a natural product, and a series of the novel analogs have been prepared. Most of the analogs were found to be potently active against tumor cell lines in vitro. Further study on 4 in vivo demonstrated that it possessed a significant antineoplastic activity against murine S-180 Sarcoma and Lewis lung cancer, and human lung adenocarcinoma xenografts A-549 and LAX-83. The preclinical studies of 4 are now under way.     

Variation in antioxidant properties and phenolics concentration in different organs of wild growing and greenhouse cultivated Castilleja tenuiflora Benth.

The content of total phenolic compounds and flavonoids was determined in methanol extracts of root, stem, leaves, and inflorescences from wild growing and greenhouse cultivated plants of Castilleja tenuiflora. The antioxidant activity in each extract was evaluated using three in vitro models: scavenging of free radicals with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), and reducing power by the phosphomolybdenum assay. Both, antioxidant activity and phytochemicals content were influenced significantly (P < 0.05) by the source of the plant material and the organ. Cultivated plants had the highest content of phenolic compounds (37.95 mg gallic acid equiv. g^?1 dry weight, P < 0.05) and the strongest antioxidant activity. Total phenolic compounds content correlated significantly with the antioxidant activity for all studied plant material and organs (P < 0.05). TLC profile using DPPH as a detection reagent indicated that the phenylethanoids verbascoside and isoverbascoside are the main contributors to the free-radical scavenging of C. tenuiflora. Cultivated plants of C. tenuiflora are an alternative source of natural antioxidants to wild growing plants. The antioxidant properties of C. tenuiflora may be associated with its traditional use to treat conditions consistent with radical-related diseases (e.g. inflammation, tumors).     

Role of lipophilicity and hydrogen peroxide formation in the cytotoxicity of flavonols.

We compared the lipophilicity and toxicity of the four flavonols, galangin, kaempferol, quercetin and myricetin, which respectively have no, one, two and three hydroxyl groups on the B-ring. The lipophilicity was in the order of myricetin < quercetin < kaempferol < galangin. The cytotoxicity determined by a colony-formation assay with Chinese hamster lung fibroblast V79 cells was in the order of quercetin < kaempferol < galangin < myricetin. Apart from myricetin, the order of lipophilicity was the same as that of cytotoxicity, implying that the cytotoxicity was attributable to the lipophilicity. The cytotoxicity of myricetin was attributable to the hydrogen peroxide formed by autoxidation.     

Flavonoids from the exudate of Acacia neovernicosa

2′,4′-Dihydroxychalcone, 4′-hydroxy-2′-methoxychalcone and 2′,4′-dihydroxy-3′-methoxychalcone were isolated and characterized from the resinous exudate produced by Acacia neovernicosa. Smaller amounts of isoliquiritigenin, pinocembrin and chrysin were also found and identified by their chromatographic properties and UV spectra. The material of one collection contained galangin, 3-methylkaempferol and 3,3′ -dimethylquercetin.     

In vitro propagation and bioactive compound accumulation in regenerated shoots of <Emphasis Type="Italic">Dioscorea birmanica</Emphasis> Prain &amp; Burkill

Dioscorea birmanica Prain & Burkill is a Thai medicinal plant, which is often used with other medicinal plants for the treatment of cancers, AIDS, and septicemia diseases. Large numbers of this desirable plant can be produced using the plant tissue culture techniques. The objectives of this study were to investigate techniques of in vitro propagation and to examine the bioactive compounds: diosgenin-3-O-α-l-rhamnopyranosyl (1 → 2)–β-d-glucopyranoside (DBS1) content, total phenolic content, and antioxidant activity of the regenerated shoots compared to those of rhizomes growing in the field. For shoot induction, the highest numbers of shoots (2.8 ± 0.5) and nodes per shoot (5.7 ± 0.8) occurred after the single-nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with 2 mg/l BA (6-benzyladenine) for 4 weeks. Shoot multiplication was achieved on MS medium supplemented with 0.01 % activated charcoal (AC) and 2 mg/l BA in combination with 0.1 mg/l IAA or 0.2 mg/l NAA. The regenerated shoots were rooted on ½ MS medium supplemented with 0.01 % AC, 2 mg/l BA and 4 mg/l NAA for 8 weeks. The survival percentage was 71.88 and small rhizomes developed after transplanting for 4–6 weeks. The quantities of 0.37 ± 0.03 % (w/w) DBS1, 44.24 ± 8.47 mg GAE/g dry extract total phenolic and DPPH radical scavenging assay with EC₅₀ value of 53.67 ± 4.16 µg/ml were determined from the regenerated shoots, while 3.27 ± 0.04 % (w/w) DBS1, 259.67 ± 7.34 mg GAE/g dry extract total phenolic and DPPH radical scavenging assay with EC₅₀ value of 11.42 ± 3.28 µg/ml were found in the mother rhizomes.     

Enhancement of total phenolic and flavonoids extraction from <Emphasis Type="Italic">Rosmarinus officinalis</Emphasis> L using electromagnetic induction heating (EMIH) process

Electromagnetic induction heating (EMIH) assisted extraction of phenolic compounds from Rosmarinus officinalis L, and the antioxidant and antimicrobial activities of the plant extract were examined in this study. The extraction yield acquired with this process was found to be 25.1?±?2%, with maximum amounts of phenolic compounds: 127.87?±?2.1 mg Gallic acid equivalents per g dry weight and total flavonoids contents 14.48?±?1.5 mg quercetin equivalents per g dry weight, under optimum extraction conditions (extraction time 2 h, ratio of raw material to liquid 1:2 and 0% of NaCl). The antioxidant activity was assessed by the 1,1-diphenyl-2-picrylhydrazyl (DPPH); 2, 2′-azinobis (3-ethylbenzothiazoline-6- sulfonic acid) radical cation (ABTS⁺) and ferric reducing power (FRAP) methods. The results indicate the extract derived through EMIH showed a strong antioxidant ability (89.25%; EC₅₀ of 0.0148 µg/mL). Besides, the antimicrobial bioassay demonstrated that the extract possessed a good antimicrobial activity against all tested fungi and bacteria.     

Effects of synthetic and naturally occurring flavonoids on metabolic activation of benzo[a]pyrene in hamster embryo cell cultures.

Biochanin A, an isoflavone, has previously been shown to inhibit the metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) to metabolites that bind to DNA in hamster embryo cells and are mutagenic in Chinese hamster V79 cells. To determine the structural features required for this activity and to attempt to find more effective inhibitors, a series of synthetic and naturally occurring flavonids were tested for their ability to modulate B[a]P metabolism in hamster embryo cell cultures. The observed structure-activity relationships indicate that the structural features of flavonoids important for effective inhibition of B[a]P metabolism in hamster embryo cells are the presence of two hydroxyl, two methoxyl, or methyl and hydroxyl substituents at the 5- and 7-positions and a 2,3-double bond. Flavones are slightly better inhibitors of B[a]P metabolism than the corresponding isoflavones. A substituent at the 4'-position is not essential for inhibition of B bdP metabolism. The presence of a hydroxyl group at position 3 slightly enhances activity. Apigenin, acacetin and kaempferide are effective inhibitors of B[a]P-induced mutagenesis in a hamster embryo cell-mediated V79 cell mutation assay. However, apigenin is cytotoxic at the inhibitory dose, whereas acacetin and kaempferide are not. These results suggest that acacetin and kaempferide are promising candidates for in vivo testing as potential chemopreventive agents.     

Callus and cell suspension culture of <Emphasis Type="Italic">Viola odorata</Emphasis> as in vitro production platforms of known and novel cyclotides

Callus from Opuntia streptacantha (cv. Tuna loca), Opuntia megacantha (cv. Rubí reina), and Opuntia ficus-indica (cv. Rojo vigor) were exposed to jasmonic acid (JA) and abiotic stress (drought and UV light) to improve the metabolite production. The callus growth curves, phenolic acids and flavonoids content, antioxidant activity and phenylalanine ammonia lyase (PAL) activity were analyzed under normal and stress conditions. In O. streptacantha callus, the phenolics concentration increased 1.6 to 3 times times in presence of 5% PEG or after irradiation with UV light for 240 min, respectively, while flavonoids triplicate with UV light. A significant increase in antioxidant activity was observed in calli from the three Opuntia species in media with 50 µM JA. The relationships between metabolites/PAL activity, and metabolites/antioxidant activity were analyzed using a surface response methodology. Results showed that PAL activity, induced with PEG and UV, correlated with flavonoids content in O. megacantha and O. ficus-indica calli; PAL activity was related to both flavonoids and phenolics compounds in O. ficus-indica and O. megacantha calli exposed to JA, but only to flavonoids in O. streptacantha callus. In general, the JA stimulated simultaneously the metabolic pathways for phenolics and flavonoids synthesis, while abiotic stress induced mainly flavonoids route. As the stressed Opuntia calli exhibited as high antioxidant activity as cladodes, they are a promising system for research on antioxidant biosynthesis and/or to identify new compounds with antioxidant properties.     

Antioxidant properties of complexes of flavonoids with metal ions

The formation of complexes of metal ions with the flavonoids quercetin (L1), rutin (L2), galangin (L3) and catechin (L4) has been investigated by UV-visible spectroscopy. The antioxidant activities of the compounds were evaluated by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicalscavenging method. In this work, we have shown that the complexed flavonoids are much more effective free radical scavengers than the free flavonoids. We suggest that the higher antioxidant activity of the complexes is due to the acquisition of additional superoxide dismutating centers. Radical scavenging activities of the compounds were also investigated from an electrochemical point of view. There is a relationship between the logarithm of the antioxidant activity (represented by EC50) and the oxidation potential. The synergic effect of the complexes and ascorbic acid were studied by [13C]-NMR analyses. The results show that ascorbic acid can protect flavonoids from oxidative degradation, and reveal antioxidant synergies between ascorbic acid and the compounds.     

Investigation of in vitro and in vivo antioxidant potential of secoisolariciresinol diglucoside

The present study was designed to evaluate the in vitro and in vivo ameliorative antioxidant potential of secoisolariciresinol diglucoside (SDG). In vitro antioxidant activity of synthetic SDG was carried out using DPPH, reducing power potency, and DNA protection assays. Wistar albino rats weighing 180–220 g were used for in vivo studies and liver damage was induced in the experimental animals by a single intraperitoneal (I.P.) injection of CCl₄ (2 g/kg b.w.). Intoxicated animals were treated orally with synthetic SDG at (12.5 and 25 mg/kg b.w.) and Silymarin (25 mg/kg) for 14 consecutive days. The levels of catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), and lipid peroxidase (LPO) were measured in liver and kidney homogenates. The synthetic SDG exerts high in vitro antioxidant potency as it could scavenge DPPH at a IC₅₀ value of 78.9 μg/ml and has dose-dependent reducing power potency and protected DNA at 0.5 mg/ml concentration. Oral administration of synthetic SDG at 12.5 and 25 mg/kg b.w. showed significant protection compared to Silymarin (25 mg/kg) and the activities of CAT, SOD, and POX were markedly increased (P < 0.05), whereas LPO significantly decreased (P < 0.001) in a dose-dependent manner in liver and kidney in both pre- and post-treatment groups when compared to toxin-treated group. The results of in vitro and in vivo investigations revealed that synthetic SDG at 25 mg/kg b.w. is associated with beneficial changes in hepatic enzyme activities and thereby plays a key role in the prevention of oxidative damage in immunologic system.     

Variations in the chemical profile and biological activities of licorice (Glycyrrhiza glabra L.), as influenced by harvest times

This study investigates the variations in the chemical profile, free radical scavenging, antioxidant and gastroprotective activities of licorice extracts (LE) from plants harvested during the months of February to November. Correlations between biological properties and the chemical composition of LE were also investigated. The results showed that the total contents of phenols, flavonoids and tannins in LE varied at different harvest times. Liquiritin and glycyrrhizin, the major components of LE, varied in the range of 28.65–62.80 and 41.84–114.33 mg g^?1, respectively. The relative proportion of glycyrrhizin derivative (3), glabridin (4), glabrene (5) and liquiritigenin derivative (6), varied in the range of 0.88–11.38 %, 1.86–10.03 %, 1.80–18.40 % and 5.53–16.31 %, respectively. These fluctuations correlated positively with changes in the antioxidant and free radical scavenging activities of licorice. In general, the samples from May and November showed the most favorable free radical scavenging and antioxidant effects, whereas the best gastroprotective effect was in May. Liquiritin and glycyrrhizin, the major constituents in the February and May LE, appeared to contribute to the superoxide radical scavenging and gastroprotective effects. Glabridin and glabrene, the compounds with the highest relative proportion in the November LE, accounted for the antioxidant and DPPH scavenging activities of licorice. It is concluded that the chemical profile of licorice quantitatively varied at different harvest times and these fluctuations determined changes in its bioactivities. These data could pave the way to optimize harvesting protocols for licorice in relation with its health-promoting properties.