Antiproliferative and apoptotic activities of extracts of Asclepias subulata

Abstract

Context: Asclepias subulata Decne. (Apocynaceae) is a shrub used in the Mexican traditional medicine for the treatment of cancer.

Objective: The objective of this study was to evaluate the antiproliferative activity of methanol extract of aerial parts of A. subulata and its fractions against different cancer cell lines. Additionally, we analyzed the mechanism of action of the active fractions.

Materials and methods: Methanol extract fractions were prepared by serial extraction with n-hexane, ethyl acetate, and ethanol. The antiproliferative activity of methanol extract and its fractions was evaluated, against several murine (M12.C3.F6, RAW 264.7, and L929) and human (HeLa, A549, PC-3, LS 180, and ARPE-19) cell lines by the MTT assay, using concentrations of 0.4–400?µg/mL for 48?h. Ethanol and residual fractions were separated using silica gel column. Apoptosis induction of cancer cells was evaluated by Annexin and JC-1 staining using flow cytometry.

Results: Methanol extract and its fractions showed antiproliferative activity against all human cancer cell lines tested. Methanol extract had the highest antiproliferative activity on A549 and HeLa cells (IC₅₀ values?<?0.4 and 8.7?µg/mL, respectively). Ethanol and residual fractions exerted significant antiproliferative effect on A549 (IC₅₀?<?0.4?µg/mL) and PC3 cells (IC₅₀ 1.4 and 5.1?µg/mL). Apoptotic assays showed that CEF7, CEF9, CRF6, and CRF5 fractions induced mitochondrial depolarization in A549 cells, 70, 73, 77, and 80%, respectively. Those fractions triggered the apoptosis mitochondrial pathway.

Conclusion: Our data show that A. subulata extracts have potent antiproliferative properties on human cancer cell lines. This plant should be considered an important source of potent anticancer compounds.     

Antioxidant and cytotoxic potential of a new thienyl derivative from Tagetes erecta roots

Context: The search for newer compounds against pathogenic species continues unabated due to drug resistance. Traditionally, Tagetes erecta Linn. (Compositae) has been used for the treatment of various parasitic and microbial diseases.

Objective: To evaluate the antioxidant activity of the ethanol extract of Tagetes erecta roots and its cytotoxicity against prostate and HeLa cancer cell lines followed by activity-guided isolation.

Materials and Methods: The antioxidant screening was carried out using diphenylpicrylhydrazyl (DPPH) radical scavenging assay with serial concentrations ranging from 2 to 100 µg/mL, and cytotoxicity was evaluated against prostate (PC-3) and HeLa cell lines using microculture tetrazolium test (MTT) assay with concentrations ranging from 500 to 1.89 µg/mL. Isolation of the ethanol extract was carried out using column chromatography whereby 21 isolates were obtained (T₁-T₂₁), and the most active isolate was subjected for characterization using ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR), and mass spectroscopic techniques.

Results: The ethanol extract scavenged DPPH free radicals thereby exhibiting antioxidant activity with an IC₅₀ of 35.9 µg/mL. In addition, the extract conferred noticeable cytotoxicity against the HeLa (LD₅₀ of 164.28 µg/mL) and PC-3 cell lines (LD₅₀ of 407.3 µg/mL). Among all the isolates, T₃ showed antioxidant activity with IC₅₀ of 11.56 µg/mL and cytotoxicity with LD₅₀ of 12.5 µg/mL against HeLa and 30.25 µg/mL against PC-3 cell lines and was characterized as 2-ethynyl-5-(thiophen-2-yl) thiophene.

Discussion: The new thienyl compound (T₃) exhibited profound antioxidant activity and cytotoxicity at relatively lower concentrations than the extract.

Conclusion: The observations provide support for the ethnobotanical use of the plant.     

Evaluation of antiurolithiatic and antioxidant potential of Lepidagathis prostrata: A Pashanbhed plant

Context: Oxidative stress acts as an essential mediator in the pathophysiology of urolithiasis. Lepidagathis prostrata Dalz. (Acanthaceae) is a Pashanbhed plant that is recommended for the management of urolithiasis; however, no scientific validation has been reported.

Objectives: To evaluate the antiurolithiatic and antioxidant potential of L. prostrata.

Materials and methods: Methanol extract (LPM) and fractions; petroleum ether (LPPE), ethyl acetate (LPEA), n-butanol (LPBU) and aqueous (LPAQ) were prepared. In vitro antiurolithiatic activity was evaluated by the capacity to inhibit calcium oxalate (CaOx) nucleation and aggregation at different concentrations of extract/fractions (0.04–3?mg/mL) for 30?min. Total phenol and flavonoid content and antioxidant potential were determined. A validated HPTLC method was performed to quantify lupeol and β-sitosterol.

Results: LPEA exhibited the highest dose-dependent inhibition of CaOx nucleation (IC₅₀: 336.23?±?30.79?µg/mL) and aggregation (IC₅₀: 149.63?±?10.31?µg/mL), which was significantly (p?<?0.05) better than standard Cystone®. The polar LPBU fraction was enriched with phenols (47.34?±?0.19?mg GAE/g) and flavonoids (20.38?±?0.05?mg QE/g), which correlates with its highest antioxidant potential in DPPH, ABTS, nitric oxide scavenging and iron chelating activities (IC₅₀: 1.18–87.34?µg/mL). To our knowledge, this is the first study reporting the presence of lupeol and β-sitosterol in L. prostrata.

Conclusion: The antiurolithiatic activity of L. prostrata is probably mediated through the inhibition of CaOx crystallization. In addition to its free radical scavenging and antioxidant activities, it would act as an excellent agent for the prevention of urolithiasis.     

Chemical profile and biological activities of Veronica thymoides subsp. pseudocinerea

Abstract

Context: In Turkey, Veronica species (Plantaginaceae) have been used as a diuretic and for wound healing in traditional medicine.

Objective: To examine the fatty acid and essential oil profiles, the antioxidant, anticholinesterase, antimicrobial, and DNA damage effects of Veronica thymoides P.H. Davis subsp. pseudocinerea M.A. Fischer as a potential source of natural active compounds.

Materials and methods: GC/MS was used to analyze essential oil and fatty acid obtained from whole plant. The antioxidant activity was evaluated by the β-carotene-linoleic acid test system, DPPH-free and ABTS cation radicals scavenging, and cupric reducing antioxidant capacity assays. The anticholinesterase and antimicrobial activities were determined by Ellman and broth macrodillution methods, respectively. The effect of the methanol extract on DNA cleavage was investigated.

Results: Hexatriacontene (21.0%) was found to be the main constituent in essential oil, and linoleic acid (25.2%) and palmitic acid (20.6%) in fatty acid. Methanol extract demonstrated the best IC₅₀ values in lipid peroxidation (49.81?±?0.31?µg/ml) and DPPH-free radical scavenging activity (15.32?±?0.17?µg/ml). Methanol and water extracts possessed strong ABTS cation radical scavenging activity with IC₅₀ values 9.15?±?0.28 and 8.90?±?0.14?µg/ml, respectively. The acetone extract exhibited moderate butyrylcholinesterase inhibitory activity. The highest antimicrobial activity was determined in methanol extract against Escherichia coli with 31.25?µg/ml MIC value. Inhibition of methanol extract on plasmid DNA cleavage by OH radicals was found to be 93.32% at 500?µg/ml.

Conclusion: The methanol extract having strong antioxidant and DNA damage effects could be investigated phytochemically to find natural active compounds.     

Anti-inflammatory,anti-cholinergic and cytotoxic effects of Sida rhombifolia

Context: Sida (Malvaceae) has been used as a traditional remedy for the treatment of diarrhoea, malarial, gastrointestinal dysentery, fevers, asthma and inflammation.

Objectives: This study evaluates the anti-inflammatory, cytotoxic and anti-cholinergic activities of Sida rhombifolia Linn. whole plant for the first time.

Materials and methods: S. rhombifolia whole plant was extracted by n-hexane, ethyl acetate and methanol using Soxhlet apparatus. The plant extracts were evaluated for their antioxidant (DPPH, FIC and FRAP), anti-inflammatory (NO and protein denaturation inhibitions), cytotoxic (MTT) and anti-cholinesterase (AChE) properties in a range of concentrations to obtain IC₅₀ values. GC-MS analysis was carried out on the n-hexane extract.

Results and discussion: The ethyl acetate extract exhibited the most significant antioxidant activities by scavenging DPPH radicals and ferrous ions with EC₅₀ of 380.5 and 263.4?μg/mL, respectively. In contrast, the n-hexane extract showed the strongest anti-inflammatory activity with IC₅₀ of 52.16 and 146.03?μg/mL for NO and protein denaturation inhibition assays, respectively. The same extract also revealed the strongest effects in anti-cholinesterase and cytotoxic tests at the concentration of 100?μg/mL, AChE enzyme inhibition was 58.55% and human cancer cells, SNU-1 and Hep G2 inhibition was 68.52% and 47.82%, respectively. The phytochemicals present in the n-hexane extract are palmitic acid, linoleic acid and γ-sitosterol.

Conclusions: The present study revealed that the n-hexane extract possessed relatively high pharmacological activities in anti-inflammation, cytotoxicity and anti-cholinesterase assays. Thus, further work on the detail mechanism of the bioactive phytochemicals which contribute to the biological properties are strongly recommended.     

Antioxidative and cardiovascular-protective activities of metabolite usnic acid and psoromic acid produced by lichen species Usnea complanata under submerged fermentation

Context: Lichens have been used for various purposes such as dyes, perfumes and remedies in folk medicine indicating the pharmaceutical potential of lichens.

Objective: Lichen growth in nature is very slow. To overcome this major drawback, we standardized the culture media to culture the lichen Usnea complanata (Müll.Arg.) Motyka (Parmeliaceae) for (1) in vitro synthesis of natural lichen substances, and (2) determination of antioxidative and cardiovascular-protective activity of usnic acid and psoromic acid.

Materials and methods: Lichen U. complanata has been cultured in fermentor under submerged condition. Antioxidative and cardiovascular-protective activity of the extract and the purified lichen substances usnic and psoromic acid have been determined.

Results: Except methanol, all other extracts exhibited antioxidative action in terms of free radical scavenging activity (FRSA) with a half-inhibiting concentration (IC₅₀) value of 22.86 to 25.0 µg/mL, nitric oxide radical scavenging activity (NORSA) 141.3 to 149.1 µg/mL and for lipid peroxidation inhibition (LPI) 125 to 157.9 µg/mL. Usnic acid or psoromic acid showed antioxidative action with IC₅₀ values ranging from 0.174 to 0.271?mg/mL. Methanol and ethyl acetate extract showed hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) inhibition of 65.18 to 74.81%. Only 43.47% inhibition of angiotensin converting enzyme (ACE) was shown by methanol extract. Usnic acid showed noncompetitive type of HMGR inhibition and uncompetitive type of ACE inhibition. Psoromic acid exhibited competitive type of HMGR inhibition and mixed type of ACE inhibition.

Discussion: U. complanata showed both cardiovascular-protective and antioxidant properties.

The lichen species U. complanata may be a natural bioresource for possible pharmaceutical applications.     

Antidiabetic and antioxidant potency evaluation of different fractions obtained from Cucumis prophetarum fruit

Abstract

Context: Cucumis prophetarum Linn. (Cucurbitaceae) fruit is used for inflammatory-related problems and is proved to be possessing anticancer and hepatoprotective effects.

Objective: The present investigation was to study the effect of different fractions of C. prophetarum on antidiabetic and antioxidant activity.

Materials and methods: Aqueous crude extract (CE) of C. prophetarum fruits was fractionated into water soluble fraction 1 (F1), chloroform fraction 2 (F2), basic fraction 3 (F3), and neutral fraction 4 (F4) by acid–base extraction. CE and its fractions at different doses (0.02–0.1?mg/mL) were subjected to antidiabetic (α-amylase and α-glucosidase inhibition assays) and antioxidant (DPPH, superoxide radical scavenging (SO) and metal chelation) evaluation.

Results: F1 exhibited effective antidiabetic activity (p?<?0.05) with an IC₅₀ value of 20.6 and 59.9?µg/mL. The activity decreased in the order of CE?>?F4?>F3?>?F2, according to α-amylase assay, which were the same, with the exception of the rank order of F4 and CE, as the α-glucosidase assay. Furthermore, F1 (IC₅₀?=?73?µg/mL) showed better reducing ability than CE?>F4?>F2?>?F3 (IC₅₀?=?78–272?µg/mL), according to the DPPH assay. In SO and metal chelation assays, F1 showed the highest activity (IC₅₀?=?101 and 147?µg/mL), respectively; the activity decreased in the order of CE?>F4?>F3?>?F2 (IC₅₀?=?126–469?µg/mL) for SO and 194–944?µg/mL for metal chelation assay.

Conclusion: The results indicate that F1 possesses potent in vitro antidiabetic and antioxidant activities.     

Biological activity of HPLC-characterized ethanol extract from the aerial parts of Haplophyllum tuberculatum

Context: The search for new sources of natural antioxidants from plant material may have beneficial therapeutic potential for those diseases associated with oxidative stress. The medicinal plant Haplophyllum tuberculatum (Forsskal) A. Juss. (Rutaceae) contains phenolic compounds as main phytochemicals; however, there are no reports on its antioxidant properties.

Objective: To evaluate antioxidant and cytoprotective potential of ethanol extract of Haplophyllum tuberculatum aerial parts.

Materials and methods: Total phenol content was determined using Folin–Ciocalteu reagent; antiradical activity was measured using ORAC assay and the analysis of the major polyphenols was carried out using a HPLC-MS method. The antioxidant and cytoprotective effect were also investigated by the MTT assay and DCFH–DA method. The human astrocytoma U373-MG cell line was pretreated with ethanol extract (from 0.025 to 250?µg/mL) for 24?h, prior to 1?mM H₂O₂ exposure (30?min).

Results and conclusion: Total phenol content was 46.2?mg gallic acid/g sample and ORAC value was 1.283?µmol TE/mg sample. Chemical constituents were methoxyflavones, flavonols (mainly quercetin derivatives), cinnamic acids and benzoic acids. In cell system model of oxidative stress, pretreatments with ethanol extract at the concentrations of 2.5, 0.25 and 0.025?µg/mL significantly attenuated H₂O₂-induced loss in viability by 13.5, 17 and 20.5%, respectively. Furthermore, these ethanol extract concentrations markedly inhibited intracellular ROS production with IC₅₀ 0.026?µg/mL. These findings demonstrate the beneficial properties of ethanol extract of Haplophyllum tuberculatum aerial parts, rich in phenolic compounds, as antioxidant and radical scavenger ameliorating ROS-related processes and diseases such as several neurodegenerative disorders.     

Antiproliferative and antioxidant activities of Juglans regia fruit extracts

Context: Cancer chemopreventive action of walnut [Juglans regia L. (Juglandaceae)] has been explored.

Objective: This study evaluated antiproliferative and antioxidant activities of walnut.

Materials and methods: Various fractions of walnut extract have been screened for antiproliferative activity against human cancer cell lines using the MTT assay. All these fractions have also been evaluated for total phenolic content, antioxidant activity, and reducing power capacity.

Results and discussion: Chloroform and ethyl acetate fractions exhibited a high level of antiproliferation against HepG-2, liver cancer cell line (IC₅₀?=?9 and 15 µg/mL, respectively).

Conclusion: Exhibiting high phenolic content, antioxidant activity, and potent antiproliferative activity, walnut may act as a cancer chemopreventive agent.     

Pharmacological evaluation for anti-asthmatic and anti-inflammatory potential of Woodfordia fruticosa flower extracts

Context: Woodfordia fruticosa Kurz. (Lythraceae) flowers are ethnopharmacologically acclaimed in the Indian medicinal system to treat asthma.

Objective: To evaluate W. fruticosa flower extracts for anti-asthmatic effect.

Materials and methods: Ethyl acetate, acetone, methanol, and hydro-alcohol extracts of W. fruticosa flowers were obtained successively and standardized. Ability of extracts to stabilize free radicals and compound-48/80-induced mast cell degranulation was evaluated. In vitro anti-inflammatory potential of extracts at 100 and 200?µg/ml by membrane stabilization and in vivo inhibition of rat paw edema up to 5?h (100 and 200?mg/ml; p.o.) was evaluated. In vitro bronchorelaxant effect was examined against histamine- and acetylcholine (1?µg/ml; independently)-induced guinea pig tracheal contraction. Extracts were evaluated for bronchoprotection (in vivo) ability against 0.1% histamine- and 2% acetylcholine-induced bronchospasm in guinea pigs at 100 and 200?mg/ml; p.o.

Results: Standardization studies revealed that the methanol extract exhibited highest polyphenolic (62.66 GAE), and flavonoid (6.32 RE) content and HPLC fingerprinting confirmed the presence of gallic acid (R_t 1.383). IC₅₀ values for DPPH scavenging and metal chelation by the methanol extract were 40.42 and 31.50?µg/ml. Methanol and ethyl acetate extracts at 100?µg/ml exhibited 06.52 and 07.12% of histamine release. Methanol, ethyl acetate, and hydro alcohol extracts at 200?mg/kg demonstrated 32.73, 29.83, 26.75% and 32.46, 9.38, 26.75% inhibition of egg albumin and carrageenan-induced inflammation, respectively. Methanol extract exhibited 100% bronchorelaxation and 48.83% bronchoprotection.

Conclusion: Woodfordia fruticosa flower (WFF) extracts exhibited anti-asthmatic effect by demonstrating bronchoprotection, bronchorelaxation, anti-inflammatory, antioxidant, and mast cell stabilization ability.     

Evaluation of the antimutagenic,antigenotoxic, and antioxidant activities of Eriobotrya japonica leaves

Abstract

Context: The leaves of Eriobotrya japonica (Thunb.) Lindl. (Rosaceae) are used in traditional medicine to treat inflammatory diseases. However, information about the antigenotoxic and antioxidant properties of its leaves remains to be elucidated.

Objective: The objective of this work was to evaluate the mutagenic/antimutagenic, genotoxic/antigenotoxic, and antioxidant potentials of aqueous and total oligomers flavonoid (TOF) extracts from E. japonica.

Materials and methods: The mutagenic/antimutagenic and genotoxic/antigenotoxic potentials of extracts (50, 250, and 500?µg/plate) were evaluated, respectively, by the Ames test with 48?h incubation and the SOS chromotest test with 2?h incubation. The antioxidant capacity of these extracts (ranging from 50 to 700?µg/mL) was tested using xanthine/xanthine oxidase and the deoxyribose assays.

Results: Eriobotrya japonica extracts showed neither mutagenic nor genotoxic effect. The highest protective effect against methyl methanesulfonate and 2-aminoanthracene was obtained in the presence of aqueous extract, with IC₅₀ values of 80 and 140?µg/plate, respectively, against S. typhimurium TA104. Moreover, this extract (500?µg/plate) was also able to reduce significantly the genotoxicity induced by nitrofurantoin and aflatoxin B1 with IC₅₀ values of 140 and 240?µg/assay, respectively. Likewise, aqueous and TOF extracts inhibited xanthine oxidase and superoxide anion formation with IC₅₀ values ranging from 45 to 95 and from 70 to 90?µg/mL, respectively. However, TOF extract is more efficient in inhibiting hydroxyl radical and chelating iron ion with IC₅₀ values of 140 and 400?µg/mL, respectively, when compared with the aqueous extract.

Conclusion: Eriobotrya japonica prevents the genotoxicity of some carcinogenic substances probably thanks to its antioxidant capacities.     

Antioxidant,anti-inflammatory and anti-septic potential of phenolic acids and flavonoid fractions isolated from Lolium multiflorum

Context: Interest has recently renewed in using Lolium multiflorum Lam. (Poaceae) (called Italian ryegrass; IRG) silage as an antioxidant and anti-inflammatory diet.

Objective: This study investigated the antioxidant, anti-inflammatory and anti-septic potential of IRG silage and identified the primary components in IRG active fractions.

Materials and methods: Total 16 fractions were separated from the chloroform-soluble extract of IRG aerial part using Sephadex LH-20 column before HPLC analysis. Antioxidant and anti-inflammatory activities of the fractions at doses of 0–100?μg/mL were investigated using various cell-free and cell-mediated assay systems. To explore anti-septic effect of IRG fractions, female ICR and BALB/c mice orally received 40?mg/kg of phenolic acid and flavonoid-rich active fractions F₇ and F₈ every other day for 10 days, respectively, followed by LPS challenge.

Results: The active fractions showed greater antioxidant and anti-inflammatory potential compared with other fractions. IC₅₀ values of F₇ and F₈ to reduce LPS-stimulated NO and TNF-α production were around 15 and 30?μg/mL, respectively. Comparison of retention times with authentic compounds through HPLC analysis revealed the presence of caffeic acid, ferulic acid, myricetin and kaempferol in the fractions as primary components. These fractions inhibited LPS-stimulated MAPK and NF-κB activation. Supplementation with F₇ or F₈ improved the survival rates of mice to 70 and 60%, respectively, in LPS-injected mice and reduced near completely serum TNF-α and IL-6 levels.

Discussion and conclusion: This study highlights antioxidant, anti-inflammatory and anti-septic activities of IRG active fractions, eventually suggesting their usefulness in preventing oxidative damage and inflammatory disorders.     

Antioxidant activity against H2O2-induced cytotoxicity of the ethanol extract and compounds from Pyrola decorate leaves

Context: The leaves of Pyrola decorate H. Andr (Pyrolaceae), known as Luxiancao, have long been used for treating kidney deficiency, gastric haemorrhage and rheumatic arthritic diseases in traditional Chinese medicine.

Objective: The phytochemicals and antioxidant capacities in vitro of P. decorate leaves were investigated.

Materials and methods: Ethanol, petroleum ether, acetidin, n-butyl alcohol and aqueous extracts of Pyrola decorate leaves were prepared by solvent sequential process, and then isolated and purified to obtain phytochemicals. Cell viability was measured by MTT assay. PC12 cells were pretreated for 24?h with different extractions of P. decorate leaves at concentrations of 0.1, 0.5, 1, 5 and 10?mg/mL, then H₂O₂ of 0.4?mM was added in all samples for an additional 2?h. The antioxidant capacities of betulin, ursolic acid and monotropein were determined in PC12 cells against H₂O₂ induced cytotoxicity in vitro as well.

Results: Nine compounds (1–9) were isolated and structurally determined by spectroscopic methods, especially 2D NMR analyses. Ethanol extract treated groups showed inhibitory activity with IC₅₀ value of 10.83?mg/mL. Betulin, ursolic acid and monotropein were isolated from P. decorate, and demonstrated with IC₅₀ values of 6.88, 6.15 and 6.13?μg/mL, respectively.

Discussion and conclusions: In conclusion, Pyrola decorate is a potential antioxidative natural plant and worth testing for further pharmacological investigation in the treatment of oxidative stress related neurological disease.     

HPTLC analysis,antioxidant, anti-inflammatory and antiproliferative activities of Arisaema tortuosum tuber extract

Context: Oxidative stress and inflammation are related to several chronic diseases including cancer and atherosclerosis. Arisaema tortuosum (Wall.) Schott (Araceae) is an Indian folk medicinal herb traditionally used for treatment of various diseases related to inflammation and stress.

Objective: This study was carried out for HPTLC analysis and evaluation of antioxidant, anti-inflammatory and antiproliferative activities of a methanol extract of A. tortuosum tuber.

Materials and methods: The antioxidant activities of methanol extract of A. tortuosum tuber (1?mg/mL) were evaluated by DPPH, ABTS and FRAP assays and anti-inflammatory effects by diene-conjugate and β-glucuronidase assays, with in vitro tumor growth inhibition on HeLa cancer cells. The results for antioxidant and anti-inflammatory effects were compared using Trolox and salicylic acid as reference compounds, respectively.

Results: The TLC and HPTLC analysis showed the presence of quercetin, rutin, luteolin and lectin (R_f values 0.97, 0.53, 0.59 and 1.58, respectively). The methanol fraction of tuber exhibit higher activity in each antioxidant system with a special attention for DPPH (IC₅₀?=?852?μg/mL), ABTS (IC₅₀?=?532?μg/mL), and FRAP (IC₅₀?=?458?μg/mL), as compared with Trolox as standard, with a remarkable amount of phenolics (86.2?mg/100?g) and flavonoids (175.5?mg/100?g), along with potent anti-inflammatory activity indicated by diene-conjugate (86.20%) and β-glucuronidase (92.92%) inhibition, as compared with salicylic acid as reference compound. The antiproliferative activity at 100?mg/mL was 88% inhibition with HeLa cells. The inhibition of HeLa cell proliferation was greatest (p?A. tortuosum tuber extract treatments and least with the 25?mg/mL dose.

Discussion and conclusion: Our results suggested that A. tortuosum tuber might be used as a promising and potent antioxidant, anti-inflammatory, and antiproliferative agent and might be used for standardization of potential drug after successful isolation and characterization of bioactive compounds.     

Bioassay-guided isolation of antioxidant agents with analgesic properties from flowers of Tagetes patula

Context: Tagetes patula L. is one of the French marigold group of the Asteraceae family. It is recognized in folklore for its medicinal and pesticidal properties.

Objective: In search of more effective, but non-toxic compounds with antioxidative potential led to the bioassay guided isolation studies on the extracts of T. patula.

Materials and methods: The bioassay on Tagetes patula flowers were carried out guided by in vitro antioxidant activity using DPPH assay. A minor but proven plant constituent methyl protocatechuate (1) was isolated by column chromatography, while patuletin (2) and patulitrin (3) obtained in bulk by employing solvent partition of methanol extract. Derivatization of patuletin into benzoyl, cinnamoyl and methyl was conducted to establish the structure activity relationship (SAR). Analgesic activity of compound 2 was evaluated using acetic acid-induced writhing test and hot-plate test in mice. The toxicity of methanol extract and compound 2 were also determined.

Results: Polar extracts, fractions and phases demonstrated better antioxidant activity. The synthetic methyl protocatechuate (1) showed IC₅₀ value of 2.8?±?0.2?μg/mL, whereas patuletin (2) (IC₅₀?=?4.3?±?0.25 µg/mL) was comparable to quercetin and rutin but significantly better than patulitrin (3) (IC₅₀?=?10.17?±?1.16 µg/mL). Toxicity test for the methanol extract and compound 2 did not elicit any behavioral changes or cause mortality in mice. Compound 2 also demonstrated mild analgesic property.

Discussion and conclusion: These findings demonstrate that the plant polar extracts and fractions possess significant antioxidant property with non-toxic effect. Compound 1 is a genuine plant constituent of T. patula.     

Bioactive potential of endophytic Myrothecium sp. isolate M1-CA-102, associated with Calophyllum apetalum

Context: Endophytes colonizing medicinal plants are diverse, constituting a rich bioresource for novel natural products.

Objective: Myrothecium sp. isolate M1-CA-102 was the most promising among the 16 Myrothecium isolates screened. The bioactive potential of the crude extract from the Calophyllum apetalum Willd. endophytic Myrothecium sp. (Alb. &; Schwein.) Ditmar (Incertae sedis) isolate M1-CA-102 and its thin layer chromatography (TLC) fractions were screened based on antioxidant, anti-inflammatory, antimicrobial activities, and cytotoxicity.

Materials and methods: The antioxidant activity was measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical scavenging capacities. Further, 15-lipoxygenase (15-LOX) and human cyclooxygenase-2 (COX-2) inhibition were assessed at different concentrations (25, 50, and 100?μg/mL for the crude extract, 5, 25, and 50?μg/mL for the TLC fractions). DNA-nicking assay as an indicator of the capacity of extracts to scavenge hydroxyl radical was recorded at a concentration of 50?μg/mL. Cell cytotoxicity was recorded by colorimetric 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Antibacterial (Bacillus subtilis) and anti-Candida (Candida albicans) assays were performed by the microdilution method.

Results: The DPPH and ABTS IC₅₀ values of M1-CA-102 extract were 10 and 6?μg/mL compared with 6.1 and 7.03?μg/mL for the positive control quercetin. The cytotoxicity IC₅₀ value of M1-CA-102 extract was 37?μg/mL, while the M-I TLC fraction was 21?μg/mL. The M1-CA-102 extract gave an IC₅₀ value of 58 and 8?μg/mL for 15-LOX and COX-2, respectively. The MIC values for antimicrobial activity for M1-CA-102 extract ranged from 35 to 54?μg/mL, while for the TLC fractions, it ranged from 91 to 515?μg/mL.

Conclusion: The results indicate that Myrothecium M1-CA-102 isolated from C. apetalum is a potential source of natural metabolites of pharmaceutical importance.     

An unprecedented antioxidative isopimarane norditerpenoid from bivalve clam,Paphia malabarica with anti-cyclooxygenase and lipoxygenase potential

Context: The yellow-foot bivalve clam, Paphia malabarica Chemnitz (Veneridae) is distributed in the southwest coastal regions of India. The ethyl acetate-methanol extract of this species exhibited significant antioxidant and anti-inflammatory activities.

Objectives: To purify and characterize the bioactive compound from P. malabarica along with in vitro assays.

Materials and methods: The edible portion of P. malabarica was freeze dried (1.20?kg, yield 20.0%) and extracted with ethyl acetate and methanol (1:1 v/v, 500?mL ×3) by sonication (8?h). The antioxidant activity against DPPH/ABTS^+?and anti-inflammatory potential against cyclooxygenase-1,2 (COX-1, 2)/5-lipoxygenase (5-LOX) enzymes were carried out with varying concentrations (0.25–2.00?mg/mL) to determine the IC₅₀ values. The crude extract was chromatographically fractionated and the fraction showing greater potential was further fractionated to yield the pure compound, which was characterized by extensive NMR, IR and mass spectroscopic analyses.

Results and discussion: The fractionation of crude extract of P. malabarica was followed by structural characterization of the new rearranged isopimarane derivative, 18 (4 → 14), 19 (4 → 8)-bis-abeo C₁₉ norditerpenoid. The isopimarane derivative displayed comparable antioxidant activity with α-tocopherol (IC₅₀ DPPH scavenging activity ～0.6?mg/mL), whereas anti-inflammatory (anti-5-LOX) effect of the title compound was significantly greater (IC₅₀ 0.75?mg/mL) than ibuprofen (IC₅₀ 0.93?mg/mL). In addition, the greater selectivity index (anti-COX-1_IC50/anti-COX-2_IC50 0.85) explained the lesser side effects of the isopimarane norditerpenoid than the nonsteroidal anti-inflammatory drug-based therapies.

Conclusions: The isopimarane derivative isolated from P. malabrica can be a natural substitute to commercial drugs in future.     

Antioxidant Potential of an Extract of Calendula officinalis. Flowers in Vitro. and in Vivo.

Abstract

An extract of Calendula officinalis. Linn. (Compositae) was evaluated for its antioxidant potential in vitro. and in vivo.. Calendula officinalis. extract was found to scavenge superoxide radicals generated by photoreduction of riboflavin and hydroxyl radicals generated by Fenton reaction and inhibited in vitro. lipid peroxidation. Concentrations needed for 50% inhibition (IC₅₀) were 500, 480, and 2000 µg/mL, respectively. Extract scavenged ABTS radicals and DPPH radicals and IC₅₀ were 6.5 and 100 µg/mL, respectively. IC₅₀ values were compared with that of ginger extract, which is a standard antioxidant extract. The drug also scavenged nitric oxide, and the IC₅₀ was found to be 575 µg/mL. Extract also produced dose-dependent scavenging of nitric oxide in culture. The oral administration of Calendula. extract inhibited superoxide generation in macrophages in vivo. by 12.6% and 38.7% at doses of 100 and 250 mg/kg b.wt. Oral administration of Calendula officinalis. to mice for 1 month significantly increased catalase activity. The extract produced significant increase in glutathione levels in blood and liver. Glutathione reductase was found to be increased, whereas glutathione peroxidase was found to be decreased after administration of Calendula. extract. These results indicated Calendula officinalis. has significant antioxidant activity in vitro. and in vivo..     

Antitumor Activity of Extracts from Peperomia elongata.

Abstract

The cytotoxic potential of ethanol extracts from Peperomia elongata. H. B. & K. (Piperaceae) were evaluated against human cancer cell lines by the MTT method. The samples considered cytotoxic were tested for antimitotic activity with the sea urchin egg development test and for hemolytic activity using mice erythrocytes. The extracts from leaves (hexane), stems (ethanol, hexane, hexane:AcOEt, AcOEt, and MeOH:H₂O insoluble), and roots (R4) presented potential cytotoxic action. The stems extracts showed the highest toxicity in all tumor cell lines tested, with an IC₅₀ ≤ 9.0 µg/mL for ethanol extract, IC₅₀ ≤ 11.6 µg/mL for MeOH:H₂O insoluble, IC₅₀ ≤ 7.3 µg/mL for hexane extract, IC₅₀ ≤ 11.4 µg/mL for hexane: AcOEt, and IC₅₀ ≤ 16.2 µg/mL for AcOEt extract. All extracts considered cytotoxic for tumoral cell lines presented antimitotic activity. The samples from roots (R4) and stems (ethanol, MeOH:H₂O insoluble, and hexane extract from leaves) were found to possess lytic activity in mice erythrocytes but in higher doses (> 125 µg/mL). Further studies for the isolation and identification of the active principles of these extracts should be undertaken.     

Radical scavenging,prolyl endopeptidase inhibitory,and antimicrobial potential of a cultured Himalayan lichen Cetrelia olivetorum

Context: Lichens are source of natural bioactive compounds which are traditionally used to cure a variety of ailments.

Objective: The objective of this study is to assess free radical scavenging, prolyl endopeptidase inhibitory (PEPI), and antimicrobial potential of a high altitude lichen species Cetrelia olivetorum (Nyl.) W. L. Culb. & C. F. Culb (Parmeliaceae).

Materials and methods: Lichen C. olivetorum has been cultured in vitro, and optimized culture conditions were implemented in bioreactor to obtain high quantity of biomass for the study of radical scavenging, PEPI, and antimicrobial activities. Radical scavenging activity of methanol extract of Cetrelia olivetorum (MECO) was tested at 100?µg/mL, PEPI activity at 25 and 50?µg/mL, and antimicrobial activity at 5, 25, 50, and 100?µg/mL conc. All the biological activities of natural thallus extract and its derived culture extract were evaluated spectrophotometrically.

Results: Murashige and Skoog medium supplemented with 3% glucose and 100?ppb indole-3-butyric acid (IBA) supported biomass growth at flask level and yielded 5.095?g biomass in bioreactor. MECO of both the cultured and the natural lichen exhibited half inhibiting concentration (IC₅₀) for radical scavenging activities in the range of 50–60?µg/mL, whereas the IC₅₀ value of standard antioxidants was found to be in the range of 12–29?µg/mL. The IC₅₀ value of lichen extract for PEPI activity was 144–288?µg/mL, whereas the IC₅₀ value of standard prolyl endopeptidase inhibitor, Z-pro-prolinal, was 57.73?µg/mL. As far as the antimicrobial activity of MECO is concerned, minimum inhibitory concentration (MIC) value of lichen extracts against tested microorganisms was obtained in the range of 50–104?µg/mL and found to be more effective than commercially available standard erythromycin.

Discussion: Murashige and Skoog medium containing IBA was found to be suitable for maximum biomass production of C. olivetorum under bioreactor conditions. The cultured lichen biomass extract also showed antioxidant, PEPI, and antimicrobial potential.

Conclusion: The present study indicates therapeutic potential of Himalayan lichen C. olivetorum against neurodegenerative diseases owing to its radical scavenging, PEPI, and antimicrobial activities. Further, the result encourages its commercial exploitation through mass culture for production of its bioactive components and their use in pharmaceutical and nutraceutical industries.