Vijay K. Kaul

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.

Simultaneous determination of sugars and picrosides in Picrorhiza species using ultrasonic extraction and high-performance liquid chromatography with evaporative light scattering detection☆

Sugars play a critical role in regulating overall cellular metabolism in high altitude growing plants. These plants are shown to have high levels of sugars to enhance their tolerance to abiotic stresses such as drought and freezing temperature. In the present study, a simple, sensitive, selective and reliable HPLC method based on ultrasonic extraction and evaporative light scattering detection (ELSD) has been developed and validated for the simultaneous determination of important sugars (xylose, xylitol, mannitol, glucose and sucrose) and picrosides (picroside-I and picroside-II) in two species Picrorhiza kurroa and P. scrophulariiflora. The analysis was carried out on a Zorbax amino column (250 mm x 4.6 mm i.d., 5 microm) with isocratic elution of acetonitrile:water (78:22, v/v). The method was validated for accuracy, precision, limit of detection and quantification according to International Conference on Harmonization (ICH) guidelines. The drift tube temperature of the ELSD system was set to 81 degrees C and nitrogen flow rate was 2.0 standard liter per minute (SLM). The regression equation revealed a good linear relationship (r(2)=0.9997+/-0.0012) within test ranges. The limit of detection and quantification for seven analytes in ELSD were less than 0.98 and 2.95 microg, respectively. The method showed good reproducibility for the quantification of seven analytes in Picrorhiza species with intra- and inter-day variation of less than 2.0%.

Reversed phase-HPLC for rapid determination of polyphenols in flowers of rose species*

A rapid, simple, sensitive, robust, and improved HPLC method was developed and validated for determination of 10 polyphenols, namely gallic acid, catechin, epicatechin, rutin, m-coumaric acid, quercitrin, myricetin, quercetin, apigenin, and kaempferol in fresh flowers of Rosa bourboniana and R. brunonii and in both fresh flowers and marc (left after industrial distillation of rose oil) of R. damascena. Six polyphenols, gallic acid, rutin, quercitrin, myricetin, quercetin, and kaempferol, were detected and quantified in all extracts. The chromatographic separation of 10 polyphenols was achieved in less than 16 min by RP-HPLC (Phenomenex, Luna C18 (2) column, 5 microm, 250 mm x 4.6 mm) using linear gradient elution of water and acetonitrile (0.02% trifluroacetic acid) with a flow rate of 1 mL/min at lambda 280 nm. Standard calibration curves were linear in the range of 0.39-500 microg/mL. Good results were achieved with respect to repeatability (RSD <3%) and recovery (98.6-100.8%). The method was validated for linearity, accuracy, repeatability, LOD, and LOQ.

A comprehensive analysis of fifteen genes of steviol glycosides biosynthesis pathway in Stevia rebaudiana (Bertoni).

Stevia [Stevia rebuaidana (Bertoni); family: Asteraceae] is known to yield diterpenoid steviol glycosides (SGs), which are about 300 times sweeter than sugar. The present work analyzed the expression of various genes of the SGs biosynthesis pathway in different organs of the plant in relation to the SGs content. Of the various genes of the pathway, SrDXS, SrDXR, SrCPPS, SrKS, SrKO and three glucosyltransferases namely SrUGT85C2, SrUGT74G1 and SrUGT76G1 were reported from stevia. Here, we report cloning of seven additional full-length cDNA sequences namely, SrMCT, SrCMK, SrMDS, SrHDS, SrHDR, SrIDI and SrGGDPS followed by expression analysis of all the fifteen genes vis-à-vis SGs content analysis. SGs content was highest in the leaf at 3rd node position (node position with reference to the apical leaf as the first leaf) as compared to the leaves at other node positions. Except for SrDXR and SrKO, gene expression was maximum in leaf at 1st node and minimum in leaf at 5th node. The expression of SrKO was highest in leaf at 3rd node while in case of SrDXR expression showed an increase up to 3rd leaf and decrease thereafter. SGs accumulated maximum in leaf tissue followed by stem and root, and similar was the pattern of expression of all the fifteen genes. The genes responded to the modulators of the terpenopids biosynthesis. Gibberellin (GA(3)) treatment up-regulated the expression of SrMCT, SrCMK, SrMDS and SrUGT74G1, whereas methyl jasmonate and kinetin treatment down-regulated the expression of all the fifteen genes of the pathway.

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.

Chemical composition and antibacterial activity of essential oils of Lantana camara, Ageratum houstonianum and Eupatorium adenophorum.

Essential oils have applications in folk medicine, food preservation, and as feed additives. The essential oils of Lantana camara Linn. (Verbenaceae), Ageratum houstonianum Mill. (Asteraceae) and Eupatorium adenophorum Spreng. (Asteraceae) were analyzed by Gas chromatography-mass spectrometry (GCMS). In L. camara oil, of the total identified (83.91%) volatile constituents, five constituents [3,7,11-trimethyl-1,6,10-dodecatriene (28.86%), β-caryophyllene (12.28%), zingiberene (7.63%), γ-curcumene (7.50%) and α-humulene (3.99%)] represented the major ones. In A. houstonianum oil, among the total identified volatile constituents (94.51%), three [precocene-II (52.64%), precocene-I (22.45%) and β-caryophyllene (9.66%)] represented the major ones. In E. adenophorum oil, of the total identified volatile constituents (84.95%), six [1-napthalenol (17.50%), α-bisabolol (9.53%), bornyl acetate (8.98%), β-bisabolene (6.16%), germacrene-D (5.74%) and α- phellandrene (3.85%)] represented the major ones. The antibacterial activity expressed as Minimum Bactericidal Concentration (MBC) (μg/mL) was determined by the broth dilution method. The essential oil of E. adenophorum had antibacterial activity against Arthrobacter protophormiae, Escherichia coli, Micrococcus luteus, Rhodococcus rhodochrous, and Staphylococcus aureus with MBC values of 200, 100, 100, 12.5, and 200, respectively. The essential oil of A. houstonianum showed antibacterial activity against M. luteus and R. rhodochrous with MBC of 100 and 12.5, but not against A. protophormiae, E. coli, and S. aureus. The essential oil of L. camara showed antibacterial activity against A. protophormiae, M. luteus, R. rhodochrous and S. aureus with MBC of 50, 25, 12.5, and 200, respectively, but not against E. coli. MBC was lowest for R. rhodochrous for all the three essential oils.

The sesquiterpene fraction of the essential oil of Artemisia laciniata Willd.

The sesquiterpene fraction of the essential oil from Artemisia laciniata Willd. (Compositae) from the Kashmir region of India was analysed and found to contain mainly oxygenated derivatives. The main constituent, about 4% of the total oil, is silphiperfol-5-en-3-ol A (29). Besides many other minor compounds possessing this rare skeleton, the biogenetically related presilphiperfolan-9a-ol (17, ca. 2%) was isolated. Almost all the other compounds represent the davanones (21, 27, 32) and their oxygenated derivatives (23, 36, 37, 40). Most important are the bicyclic artedouglasia oxides (13, 15, 24, 30) and the laciniatafuranones (12, 14, 16, 19, 20). Five of the eight possible diastereoisomers of this novel compound were isolated and stereochemically assigned by extensive 1 H-NMR studies and molecular modelling considerations. Two compounds, the 3-hydroxyisodavonone (23) and its 1,2-dehydro derivative (37) show a unique keto enol moiety. Presilphiperfolan-9-ol (17, woody, sweet) and silphiperfol-5-en-3-ol A (29, woody, ambergris) contribute remarkably to the interesting odour of this Artemisia oil.

Constituents of the essential oil from the fruits of Zanthoxylum rhetsoides Drake from Vietnam and from the aerial parts of Zanthoxylum alatum Roxb. from India

The essential oil from the fruits of Zanthoxylum rhetsoides Drake (syn. Z. myriacanthum Wall. ex Hook. f.) of north-west Vietnamese origin consists of 78% of monoterpene hydrocarbons, 19% of oxygenated monoterpenes, 1% of n-alcohols and only 1% of sesquiterpenes. The essential oil from aerial parts of Zanthoxylum alatum Roxb. (syn. Z. armatum DC., Z. planispinium Sieb. et Zucc.) collected in north-west India contains 33% of monoterpene hydrocarbons. The main constituents are 1,8-cineole (15.7%, 18), linalol (18.8%, 28) and undecan-2-one (17.0%, 60). Copyright

Quantification of Picroside‐I and Picroside‐II in Picrorhiza kurroa by HPTLC

Abstract A new high performance thin layer chromatography (HPTLC) method for the simultaneous quantification of picroside‐I and picroside‐II in P. kurroa is described. Separation of picroside‐I and picroside‐II was achieved by mobile phase of CHCl3:MeOH (82:18, v/v) on precoated silica gel 60 F254 aluminum plate. The densitometric determination of picrosides was carried out at 290 nm, in absorption‐reflection mode. The calibration curves were linear in the range of (2–5 µg). The method is simple, specific, rapid, and reliable for simultaneous determination of P‐I and P‐II in P. kurroa. The proposed method was applied for accurate quantification of large number of samples collected from different altitudes of western Himalaya.

Domestication of Wild Marigold (Tagetes minuta L.) as a Potential Economic Crop in Western Himalaya and North Indian Plains

Tagetes minuta L, syn.T. glandulifera Schrank grows wild in western Himalaya and is one of the important source of essential oils. The crop has been domesticated by our Institute not only in sub-temperate but also in subtropical zones and farmers have opted Tagetes minuta as an essential oil crop in their cropping system. Four cropping systems have been evolved based on variable sowing and harvesting times yielding varying quality of essential oil. All the four crop practices can be practised in sub-temperate regions while one crop practice is recommended for subtropical regions. Tagetes oil contains three major constituents of monoterpene ketones namely ocimenones E&Z, tagetones E&Z and dihydrotagetone and a hydrocarbon ocimene which are important base materials for synthesizing new aroma molecules.Tagetes oil of desired constituents can be produced by adopting selective crop practice based on experimental findings of present investigation. Ocimene rich crop can be harvested in winter (December–January), dihydrotagetone rich crop in autumn (October–November), tagetone rich crop in summer (June) and ocimenone rich crop can be harvested in short duration autumn–winter (December–January) and summer crops.