Application of Box-Behnken design for ultrasound-assisted extraction and recycling preparative HPLC for isolation of anthraquinones from Cassia singueana.

INTRODUCTION: Cassia singueana Del. (Fabaceae) is a rare medicinal plant used in the traditional medicine preparations to treat various ailments. The root of C. singueana is a rich source of anthraquinones that possess anticancer, antibacterial and antifungal properties. OBJECTIVE: The objective of this study was to develop an ultrasound-assisted extraction (UAE) method for achieving a high extraction yield of anthraquinones using the response surface methodology (RSM), Box-Behnken design (BBD), and a recycling preparative high-performance liquid chromatography (HPLC) protocol for isolation of anthraquinones from C. singueana. METHODOLOGY: Optimisation of UAE was performed using the Box-Behnken experimental design. Recycling preparative HPLC was employed to isolate anthraquinones from the root extract of C. singueana. RESULTS: The BBD was well-described by a quadratic polynomial model (R2  = 0.9751). The predicted optimal UAE conditions for a high extraction yield were obtained at: extraction time 25.00 min, temperature 50°C and solvent-sample ratio of 10 mL/g. Under the predicted conditions, the experimental value (1.65 ± 0.07%) closely agreed to the predicted yield (1.64%). The obtained crude extract of C. singueana root was subsequently purified to afford eight anthraquinones. CONCLUSION: The extraction protocol described here is suitable for large-scale extraction of anthraquinones from plant extracts.

Chemical profiling and biological properties of Neolitsea kedahense Gamble essential oils.

Hydrodistillation of the fresh stem and leaf of Neolitsea kedahense Gamble, collected from Gunung Jerai, Malaysia followed by the GC-FID and GC-MS analysis revealed the detection of a total of 47 constituents of which 28 (86.4%) from the stem and 31 (96.4%) constituents from the leaf. δ-Cadinene (17.4%), 1-epi-cubenol (11.8%), cyperotundone (9.0%), cis-cadin-4-en-7-ol (7.7%), τ-cadinol (7.1%) and α-cadinol (7.1%) were the principle constituents in the stem oil, whereas ß-caryophyllene (18.9%), bicyclogermacrene (18.6%) and trans-muurola-4(14),5-diene (9.8%) were the major constituents in the leaf oil. Among the identified constituents, three constituents namely 7-epi-α-selinene, junenol and cis-cadin-4-en-7-ol have not been found previously from Neolitsea oils. The stem and leaf oils were screened for their α-glucosidase inhibitory and antibacterial activities. Both oils displayed potential α-glucosidase inhibitory activity, while the stem oil possessed weak antibacterial activity against Bacillus subtilis.

Tyrosinase inhibition, anti-acetylcholinesterase, and antimicrobial activities of the phytochemicals from Gynotroches axillaris Blume.

The leaves of Gynotroches axillaris were chemically and biologically studied. Sequential extraction of the leaves using petroleum ether, chloroform, and methanol afforded three extracts. Purification of pet. ether extract yielded, squalene and ß-amyrin palmitate as the major compounds, together with palmitic acid and myristic acid as the minor components. The methanol extract yielded two flavonoids, quercitrin and epicatechin. The isolated compounds were characterized by MS, IR and NMR (1D and 2D). Anti-acetyl cholinesterase screening using TLC bio-autography assay showed that palmitic acid and myristic acid were the strongest inhibition with detection limit 1.14 and 1.28 µ/g/ 5 µL respectively. Antibacterial against Gram-positive and negative and antifungal activities exhibited that ß-amyrin palmitate was the strongest (450-225 µ/mL) against all the tested microbes. The tyrosinase inhibition assay of extracts and the pure compounds were screened against tyrosinase enzyme. The inhibition percentage (I%) of methanol extract against tyrosinase enzyme was stronger than the other extracts with value 68.4%. Quercitrin (59%) was found to be the highest in the tyrosinase inhibition activity amongst the pure compounds. To the best of our knowledge, this is first report on the phytochemicals, tyrosinase inhibition, anti-acetycholinesterase and antimicrobial activities of the leaves of G. axillaris.

Chemical Composition, Antibacterial and α-Glucosidase Inhibitory Activities of the Essential Oils of Neolitsea coccinea (Lauraceae).

The chemical composition, antibacterial and α-glucosidase inhibitory activities of the essential oils obtained from hydrodistillation of the fresh stem and leaf of Neolitsea coccinea B. C. Stone are reported for the first time. GC and GC-MS analysis revealed the presence of 42 volatile compounds from the stem and leaf oils, accounting for 84.9% and 90.4%, respectively of the identified components. The principle compounds in the stem oil were δ-cadinene (21.2%), 1-epicubenol (11.3%) and cyperotundone (10.7%), while the main compounds in the leaf oil were selin-ll-en-4-α-ol (26.8%), bicyclogermacrene (12.6%), γ-eudesmol (7.1%), germacrene D (6.1%) and globulol (5.9%). The leaf oil demonstrated moderate to weak antibacterial activity towards Bacillus subtilis and Staphylococcus aureus with MIC values of 250 µg/mL and 500 µg/mL, respectively, whereas the stem oil posessed weak antibacterial activity against B. subtilis with a MIC value of 500 µg/mL. The stem and leaf oils showed significant α-glucosidase inhibitory activity with IC(50) values of 32.2 ± 0.8 µg/mL and 70.9 ± 1.1 µg/mL, respectively.

Antioxidant and α-Glucosidase Inhibitory Constituents from Hornstedtia Species of Malaysia.

Seven compounds were isolated from the n-hexane and chloroform extracts of the flowers and leaves of four Hornstedtia species and their structures were identified using spectroscopic techniques as 3,7,4'-trimethylkaempferol (1), 3,7-dimethylkaempferol (2), 7,4'-dimethylkaempferol (3), 3,5-dimethylkaempferol (4), 3-methylkaempferol (5), stigmast-4-en-3-one (6), and 6-hydroxy-stigmast-4-en-3-one (7). Compounds 1 to 7 were isolated from these species for the first time. They were assayed for free radical scavenging and α-glucosidase inhibition activities. The DPPH assay showed that 3-methylkaempferol (5) was the most potent antioxidant agent with an IC50 value 78.6 µM, followed by 7,4'-dimethylkaempferol (3) (IC50 = 86.1 µM). For α-glucosidase inhibition activity, 3-methylkaempferol (5) exhibited significant inhibitory activity with an IC50 value 21.0 µM. The present study revealed that Hornstedtia species have potential activities as antioxidant and α-glucosidase inhibitors.

Antioxidant and Anti-inflammatory Activities of Essential Oil and Extracts of Piper miniatum.

The chemical composition of the essential oil and antioxidant and anti-inflammatory activities of the extracts from Piper miniatum were determined. GC and GC-MS analysis of the essential oil resulted in the identification of 64 components, accounting for 89.2% of the total. The major components were caryophyllene oxide (20.3%) and α-cubebene (10.4%). The antioxidant activity was evaluated by ß-carotene/linoleic acid bleaching, DPPH radical scavenging and total phenolic content. In the ß-carotene assay, the n-hexane extract showed the highest inhibition activity with 42.7%, while the oil gave 91.3%. The essential oil and extracts were tested for anti-inflammatory activity by using the TPA-induced mouse ear edema model and lipoxygenase assays. The essential oil exhibited significant activity in both models as an anti-inflammatory agent. The n-hexane extract showed strong activity with inhibition of 85.9% in the TPA-induced mouse ear edema model, while the chloroform extract showed the highest activity with 94.2% in the lipoxygenase assay.

Chemical compositions and antimicrobial activity of the essential oils of Hornstedtia havilandii (Zingiberaceae).

The chemical compositions and antimicrobial activity of the fresh rhizome and flower oils of Hornstedtia havilandii were studied. The components present were analyzed using GC and GC-MS. A total of forty-eight constituents were successfully identified from the flower and rhizome oils, representing 93.4% and 89.6%, respectively. The most abundant components of the flower oil were beta-pinene, (19.5%), beta-elemene, (10.0%), beta-cubebene, (6.2%), alpha-pinene, (5.6%), gamma-cadinene, (4.3%) and germacrene D, (3.3%), while alpha-copaene, (10.2%), beta-selinene, (8.4%), beta-elemene, (7.0%), gamma-cadinene, (6.9%), beta-cubebene, (5.3%) and germacrene D, (5.3%) were found as the main components of the rhizome oil. The essential oils were tested for antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and yeasts. Both oils showed activity against the Gram-positive bacterium, Staphylococcus aureus, with moderate MIC values of 112.5 micro/mL, however, for Gram-negative bacteria, the flower oil showed weak antimicrobial activity with a MIC value of 225 microg/mL. Only very weak activity was shown against the yeasts Candida albicans and C. glabrata, with MIC values of 900-1800 microg/mL.

Potent microbial and tyrosinase inhibitors from stem bark of Bauhinia rufescens (Fabaceae).

The stem bark extracts of Bauhinia rufescens Lam. (Fabaceae) yielded 6-methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin, alpha-amyrin acetate, beta-sitosterol 3-O-beta-D-xylopyranoside, 4-(2'-Hydroxyphenethyl)-5-methoxy-2-methylphenol, menisdaurin and sequoyitol. Their structures were determined using spectroscopic methods and comparisons with the literature data. For the antimicrobial assay Gram-positive and Gram-negative bacterial and fungal strains were tested, while the tyrosinase inhibition assay utilized L-DOPA as a substrate for the tyrosinase enzyme. 6-Methoxy-7-methyl-8-hydroxydibenz[b,f]oxepin, a-amyrin acetate, beta-sitosterol 3-O-D-xylopyranoside, menisdaurin and sequoyitol showed weak to moderate activities with minimum inhibition concentration (MIC) values in the range of 112.5-900 microg/mL against all bacterial strains, while the MIC values for the fungal strains were in the range of 28.1-450 microg/mL. In the tyrosinase inhibition assay, a-amyrin acetate was found to be moderately active against tyrosinase with an inhibition of 62% at 0.1 mg/mL. This activity was lower than that of the positive control, kojic acid (85%).

Essential oils of Alpinia rafflesiana and their antimicrobial activities.

The essential oils from the leaves, pseudostems, rhizomes and fruits of Alpinia rafflesiana were isolated by hydrodistillation. The oils were analysed by capillary GC and GC-MS. The most abundant components in the leaf oil were trans-caryophyllene (32.61%), caryophyllene oxide (8.67%), (2E,6Z)-farnesol (4.91%) and alpha-terpineol (4.25%), while 1,8-cineole (32.25%), myrcene (13.63%), alpha-terpineol (9.90%) and trans-caryophyllene (9.80%) were the main constituents in the pseudostem oil. The rhizome constituted of tetracosane (42.61%), tau-cadinol (7.46%), alpha-terpineol (6.71%) were the major components, whereas tetracosane (13.39%), (2E,6E)-farnesol (7.31%), alpha-terpineol (8.51%) and caryophyllene oxide (8.05%) were the main components in the fruit oil. Antimicrobial assay revealed that all the essential oils showed moderate to weak inhibition against the tested microorganisms. The leaf oil was the most active and inhibited both S. aureus and E. coli with MIC values of 7.81 microg/mL and 15.6 microg/mL, respectively.

Chemical constituents of the leaf of Alpinia mutica Roxb.

Hydrodistillation of the fresh leaves of Alpinia mutica afforded 0.005% colourless essential oil. GC and GC-MS analysis revealed the presence of 33 components accounting for 92.9% of the total oil, dominated by 20 sesquiterpenes (76.7%) and 10 monoterpenes (8.3%). The major constituent was found to be ß-sesquiphellandrene which was 29.2% of the total oil. Soxhlet extraction, followed by repeated column chromatography of the dried leaves yielded two phenolic compounds, identified as 5,6-dehydrokawain and aniba dimer A, together with one amide assigned as auranamide. The structures of these compounds were determined by using spectroscopic analysis. Antibacterial screening of the essential oil, the crude and isolated compounds showed weak to moderate inhibitory activity.

Pharmacological mechanisms underlying gastroprotective activities of the fractions obtained from Polygonum minus in Sprague Dawley rats.

The leaves of Polygonum minus were fractionated using an eluting solvent to evaluate the pharmacological mechanisms underlying the anti-ulcerogenic activity of P. minus. Different P. minus fractions were obtained and evaluated for their ulcer preventing capabilities using the ethanol induction method. In this study, Sprague Dawley rats weighing 150-200 g were used. Different parameters were estimated to identify the active fraction underlying the mechanism of the gastroprotective action of P. minus: the gastric mucus barrier, as well as superoxide dismutase, total hexosamine, and prostaglandin synthesis. Amongst the five fractions from the ethanolic extract of P. minus, the ethyl acetate:methanol 1:1 v/v fraction (F2) significantly (p < 0.005) exhibited better inhibition of ulcer lesions in a dose-dependent manner. In addition, rats pre-treated with F2 showed a significant elevation in superoxide dismutase (SOD), hexosamine and PGE2 levels in the stomach wall mucosa in a dose-dependent matter. Based on these results, the ethyl acetate:methanol 1:1 v/v fraction was considered to be the best fraction for mucous protection in the ethanol induction model. The mechanisms underlying this protection were attributed to the synthesis of antioxidants and PGE2.

Chemical compositions, antioxidant and antimicrobial activity of the essential oils of Piper officinarum (Piperaceae).

This study was designed to investigate the antioxidant and antimicrobial activities of the essential oils from Piper officinarum C. DC. GC and GC/MS analysis of the leaf and stem oils showed forty one components, representing 85.6% and 93.0% of the oil, respectively. The most abundant components in the leaf oil were beta-caryophyllene (11.2%), alpha-pinene (9.3%), sabinene (7.6%), beta-selinene (5.3%) and limonene (4.6%), while beta-caryophyllene (10.9%), alpha-phellandrene (9.3%), linalool (6.9%), limonene (6.7%) and alpha-pinene (5.0%) were the main components of the stem oil. The antioxidant activities were determined by using complementary tests: namely beta-carotene-linoleic acid, DPPH radical scavenging and total phenolic assays. The stems oil showed weak activity (IC50 = 777.4 microg/mL) in the DPPH system, but showed moderate lipid peroxidation inhibition in the beta-carotene-linoleic acid system (88.9 +/- 0.35%) compared with BHT (95.5 +/- 0.30%). Both oils showed weak activity against P. aeruginosa and E. coli with M IC values of 250 microg/mL.

Chemical compositions, antioxidant and antimicrobial activities of essential oils of Piper caninum Blume.

Chemical composition, antioxidant and antimicrobial activities of the fresh leaves and stems oils of Piper caninum were investigated. A total of forty eight constituents were identified in the leaves (77.9%) and stems (87.0%) oil which were characterized by high proportions of phenylpropanoid, safrole with 17.1% for leaves and 25.5% for stems oil. Antioxidant activities were evaluated by using ß-carotene/linoleic acid bleaching, DPPH radical scavenging and total phenolic content. Stems oil showed the highest inhibitory activity towards lipid peroxidation (114.9 ± 0.9%), compared to BHT (95.5 ± 0.5%), while leaves oil showed significant total phenolic content (27.4 ± 0.5 mg GA/g) equivalent to gallic acid. However, the essential oils showed weak activity towards DPPH free-radical scavenging. Evaluation of antimicrobial activity revealed that both oils exhibited strong activity against all bacteria strains with MIC values in the range 62.5 to 250 µg/mL, but weak activity against fungal strains. These findings suggest that the essential oils can be used as antioxidant and antimicrobial agents for therapeutic, nutraceutical industries and food manufactures.

Anti-inflammatory activities of cucurbitacin E isolated from Citrullus lanatus var. citroides: role of reactive nitrogen species and cyclooxygenase enzyme inhibition.

The in vivo and in vitro mechanistic anti-inflammatory actions of cucurbitacin E (CE) (Citrullus lanatus var. citroides) were examined. The results showed that LPS/INF-γ increased NO production in RAW264.7 macrophages, whereas L-NAME and CE curtailed it. CE did not reveal any cytotoxicity on RAW264.7 and WRL-68 cells. CE inhibited both COX enzymes with more selectivity toward COX-2. Intraperitoneal injection of CE significantly suppressed carrageenan-induced rat's paw edema. ORAC and FRAP assays showed that CE is not a potent ROS scavenger. It could be concluded that CE is potentially useful in treating inflammation through the inhibition of COX and RNS but not ROS.

A new prenylated dihydrochalcone from the leaves of Artocarpus lowii.

A new prenylated dihydrochalcone, 2',4'-dihydroxy-4-methoxy-3'-prenyldihydrochalcone (1), along with two known compounds, 2',4',4-trihydroxy-3'-prenylchalcone (2) and 2',4-dihydroxy-3',4'-(2,2-dimethylchromene)chalcone (3) were isolated from the leaves of Artocarpus lowii. The structures of 1-3 were elucidated by spectroscopic methods and by comparison with data reported in the literature. Compounds 1-3 showed strong free radical scavenging activity towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) measured by electron spin resonance (ESR) spectrometry.

Inhibitory effects of compounds from Zingiberaceae species on platelet activating factor receptor binding.

Ten compounds isolated from Alpinia mutica Roxb., Curcuma xanthorrhiza Roxb. and Kaempferia rotunda Linn. (Family: Zingiberaceae) were investigated for their platelet-activating factor (PAF) antagonistic activities on rabbit platelets using 3H-PAF as a ligand. Among them, four compounds showed significant inhibitory effects. Alpinetin and 5,6-dehydrokawain isolated from A. mutica exhibited IC50 values of 41.6 and 59.3 microM, respectively. The IC50 values of 3-deacetylcrotepoxide and 2-hydroxy-4,4',6'-trimethoxychalcone from K. rotunda were 45.6 and 57.4 microM, respectively. 1-Methoxy-2-methyl-5-(1',5'-dimethylhex-4'-enyl)-benzene, synthesized by methylation of xanthorrhizol which was obtained from C. xanthorrhiza, showed an IC50 value of 40.9 microM. The results indicated that these compounds were relatively strong PAF receptor binding inhibitors.