An efficient microwave-assisted extraction process of stevioside and rebaudioside-A from Stevia rebaudiana (Bertoni).

INTRODUCTION: Stevioside and rebaudioside-A are major low-calorie diterpene steviol glycosides in the leaves of Stevia rebaudiana. They are widely used as natural sweeteners for diabetic patients, but the long extraction procedures required and the optimisation of product yield present challenging problems. OBJECTIVE: To develop a rapid and effective methodology for the extraction of stevioside and rebaudioside-A from S. rebaudiana leaves and to compare yields using different extraction techniques. METHODOLOGY: Dried and powdered leaves of S. rebaudiana were extracted by conventional, ultrasound and microwave-assisted extraction techniques using methanol, ethanol and water as single solvents as well as in binary mixtures. Conventional cold extraction was performed at 25 degrees C for 12 h while ultrasound extraction was carried out at temperature of 35 +/- 5 degrees C for 30 min. Microwave-assisted extraction (MAE) was carried out at a power level of 80 W for 1 min at 50 degrees C. RESULTS: MAE yielded 8.64 and 2.34% of stevioside and rebaudioside-A, respectively, while conventional and ultrasound techniques yielded 6.54 and 1.20%, and 4.20 and 1.98% of stevioside and rebaudioside-A, respectively. CONCLUSION: A rapid and efficient method has been developed for the extraction of stevioside and rebaudioside-A in optimum yields using MAE procedure. This method has the advantage of rapid extraction and fast screening of a large number of S. rebaudiana samples for assessment of planting material. MAE saves considerable time, energy and has implications in the quality assessment of stevioside and rebaudioside-A prior to their industrial production from the leaves of S. rebaudiana.

Validated high-performance thin-layer chromatography method for steviol glycosides in Stevia rebaudiana.

A high-performance thin-layer chromatographic (HPTLC) method was developed and validated as per ICH (International Conferences on Harmonization) guidelines for simultaneous quantification of three steviol glycosides, i.e. steviolbioside, stevioside and rebaudioside-A in Stevia rebaudiana leaves. For achieving good separation, mobile phase of ethyl acetate-ethanol-water (80:20:12, v/v/v) on pre-coated silica gel 60 F254 HPTLC plates were used. The densitometric quantification of steviol glycosides was carried out at lambda=510 nm in reflection-absorption mode after spraying with acetic anhydride:sulphuric acid:ethanol reagent. The calibration curves were linear in the range of 160-960 ng/spot for steviolbioside, 1-6 microg/spot for stevioside and 0.5-3 microg/spot for rebaudioside-A with good correlation coefficients (0.998-0.999). The method was found to be reproducible for quantitative analysis of steviol glycosides in S. rebaudiana leaves collected from ten different locations and will serve as a quality control indicator to monitor the commercial production of stevioside and its allied molecules during different stages of its processing.

Insecticidal activity of Valeriana jatamansi (Valerianaceae) against mosquitoes.

A root extract of Valeriana jatamansi (code BAL-O) exhibited larvicidal and adulticidal activity against different mosquito species. The median lethal concentration (LC50) of BAL-O against larvae of Anopheles stephensi, Anopheles culicifacies, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus were 68.1, 42.8, 51.2, 53.8, and 80.6 mg/liter, respectively. The LC50 and the 90% lethal concentration against adult An. stephensi, An. culicifacies, Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus were 0.14, 0.16, 0.09, 0.08, and 0.17 and 0.24, 0.34, 0.25, 0.21, and 0.28 mg/cm2, respectively. The median knock-down time and 90% knock-down time of the fraction were 13, 13, 12, 13, and 18 and 24, 25, 21, 20, and 42 min against An. stephensi, An. culicifacies, Ae. aegypti, An. albopictus and Cx. quinquefasciatus, respectively, using 0.28 mg/cm2 impregnated papers. The median effective time and 90% effective time against An. stephensi at 4 degrees C were 46.6 and 8.7 days, and at 29 degrees C, 25.5 and 5.6 days, respectively. Gas chromatographic-mass spectrometric analysis of BAL-O showed 2-butanone,4-(2,6,6-trimethyl-2-cyclohexen-l-yl) (10.11%), patchouli alcohol (8.55%), cubenol (5.97%), caryophyllene oxide (5.46%), cadinol (5.23%), and aristolene (5.19%).

Variability of volatile constituents in Artemisia maritima in western Himalaya.

Chemical composition of the essential oil of Artemisia maritima, collected from three different high altitude locations in western Himalaya was studied by gas-chromatography mass spectrometry. Twenty-five constituents were identified in the oil distilled from the sample from Pooh, of which 1,8-cineole (23.8%) and chrysanthenone (17.54%) were the major constituents. Twenty volatile constituents were identified from the sample collected from Rhongtong pass, of which chrysanthenone (38.1%) and 1,8-cineole (37.3%) were the major constituents. In the oil distilled from the sample collected from Lahaul-Spiti 28 constituents were identified, of which 1,8-cineole (44.22%), camphor (9.16%) and borneol (10.94%) were the major constituents. In this sample chrysanthenone was present in very low percentage.

A new phthalic acid ester from Ajuga bracteosa.

A new phthalic acid ester 1,2-benzenedicarboxylic acid bis(2S-methyl heptyl) ester (1) was isolated from the hexane extract of the whole plant of Ajuga bracteosa. In addition, chloroform and methanol extracts yielded neo-clerodane diterpene ajugarin-I and two iridoid glycosides, reptoside and 8-O-acetyl harpagide. The structures of all the compounds were confirmed by extensive spectroscopic analysis. From the two oily fractions nine and six volatile constituents respectively were identified by GCMS. Linalyl acetate was found to be common in both the oil fractions. This plant is a new source of linalyl acetate, a valuable perfumery compound.

A new alkylated benzoquinone from rhizomes of Iris kumaonensis.

A novel alkylated unsaturated p-benzoquinone designated as 3-[(z)-12'-heptadecenyl]-2-hydroxy-5-methoxy-1,4-benzoquinone was isolated from hexane extract of the rhizomes of Iris kumaonensis and it's structure was confirmed by extensive spectroscopic analysis, IR, MS, HREIMS, 1D, 2D NMR and comparison with the literature data of known compounds.

Congenital cataract: etiology and morphology.

In a study of congenital cataract in northern India, 76 patients (146 eyes) were evaluated for morphological patterns and etiology. Partial cataracts were found to be three times more common than total cataracts, the lamellar types being the most common among the partial cataracts. Hereditary cataracts had a 19.7% incidence. The majority (46.05%) of cases of congenital cataract were of idiopathic origin. A few cases were associated with metabolic disorders (Marfan's and Marchand's). Strabismus and nystagmus were the most common associated ocular abnormalities (28.94% and 15.79%), and mental retardation was the most commonly associated systemic abnormality. Rubella cataract was seen far less frequently than expected.

Microwave-assisted rapid synthesis of methyl 2,4,5-trimethoxyphenylpropionate, a metabolite of Cordia alliodora.

Microwave assisted condensation of asaronaldehyde (2) with malonic acid in piperidine-AcOH provides 2,4,5-trimethoxycinnamic acid (3) in 87% yield within 4 min, which upon further reduction with PdCl2- HCOOH-aq. NaOH gives 3-(2,4,5-trimethoxy)phenyl propionic acid (4) in 88% yield within 3 min. Esterification of 4 with MeOH-H+ gives methyl 2,4,5-trimethoxyphenylpropionate (1), a metabolite of Cordia alliodora, in 94% yield within 3 min (overall 69% yield).

Composition and antimicrobial activity of the essential oil of Heracleum thomsonii (Clarke) from the cold desert of the western Himalayas.

Volatile oil composition of hydro-distilled (HD) and supercritical carbon dioxide (SC-CO(2)) essential oil of freshly collected aerial parts of Heracleum thomsonii (Umbeliferae) from the western Himalayas was studied by GC-FID and GC-MS. Results revealed qualitative and quantitative dissimilarity in the composition of hydro-distilled and SC-CO(2) extracted oils. Nineteen constituents, which accounted for 89.32% of total constituents in HD oil, represented by limonene (4.31%), (Z)-ß-ocimene (3.69%), terpinolene (22.24%), neryl acetate (36.19%), nerol (9.51%) and p-cymene-8-ol (2.61%) were identified. In SC-CO(2) extracted oil, 24 constituents representing 89.95% of total constituents were identified. Terpinolene (5.08%), germacrene D (2.17%), neryl acetate (51.62%), nerol (9.78%), geranyl acetate (2.06%), α-bisabolol (2.48%) and 1-nonadecanol (4.96%) were the dominating constituents. In vitro antimicrobial activity of hydro-distilled oil was conducted against microrobial strains including two Gram-positive (Staphylococcus aureus and Bacillus subtilis) and five Gram-negative (Burkholderia cepacia, Escherichia coli, Enterobacter cloacae, Klebseilla pneumoniae and Pseudomonas aeruginosa) bacteria as well as seven fungi (Candida albicans, Issatchenkia orientalis, Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, Aspergillus sydowii and Trichophyton rubrum) using broth microdilution method. The results of bioassay showed that the oil exhibited moderate to high antimicrobial activity against fungi C. albicans (MIC 625 µg ml(-1)), A. parasiticus (MIC 312.5 µg ml(-1)), A. sydowii (MIC 312.5 µg ml(-1)), T. rubrum (MIC 625 µg ml(-1)), Gram-positive bacteria B. subtilis (MIC 625 µg ml(-1)) and Gram-negative bacteria P. aeruginosa (MIC 312.5 µg ml(-1)).

Synthesis of novel acetates of beta-caryophyllene under solvent-free Lewis acid catalysis.

Acylation of beta-caryophyllene, a sesquiterpene hydrocarbon with acetic anhydride, was carried out under mild catalytic conditions using Lewis acids such as BF3.Et2O, ZnCl2, FeCl3, I2 and AlCl3 as catalysts. Among these, BF3.Et2O was found to catalyse the reaction most efficiently, yielding three acylated products in 75% yield under solvent-free conditions. The structures were confirmed by NMR and MS techniques.

Volatile constituents of Plectranthus rugosus leaf oil.

The essential oil of Plectranthus rugosus (Labiatae) was investigated by GLC, NMR and mass spectrometry. Main constituents are caryophyllene, germacrene D, alpha-phellandrene, alpha-pinene, caryophyllene oxide, alpha-cadinol, delta-cadinene, limonene, beta-phellandrene, myrcene, and p-cymene.

Constituents of Juniperus recurva var. squamata Oil.

The essential oil of leaves and twigs of JUNIPERUS RECURVA VAR. SQUAMATA (Cupressaceae) was investigated by GLC, MS, and NMR spectroscopy. Main constituents were monoterpene hydrocarbons (68%); in addition, about 12% of sesquiterpene hydrocarbons, 9% of oxygenated sesquiterpenes, and 5% of oxygenated monoterpenes were identified. 4beta-Hydroxygermacra-1(10),5-diene ( 28), beta-oplopenone ( 29A), oplopanone ( 34), 8-acetoxy elemol ( 35), and manool ( 36) were found as trace components.

Volatile Constituents of Artemisia vestita Oil1.

The essential oil of ARTEMISIA VESTITA (Compositae) was investigated by GLC, NMR, and mass spectrometry. Main constituents are alpha-, beta-, gamma-himachalene, caryophyllene, germacrene D, himachalol, allohimachalol, alpha-, gamma-atlantone, 1,8-cineole, yomogi alcohol, artemisia, and santolina alcohol and their acetates, and the thujones and thujanols. The characteristic oder of the oil is determined by the freshly smelling, 1, 8-cineole and thujone, and by the woody and sweet note of himachalol and atlantone.