HPLC-DAD-MS/MS profiling of antioxidant flavonoid glycosides in sea buckthorn (Hippophae rhamnoides L.) seeds.

This study was aimed at the chemical profiling of flavonoid glycosides in antioxidant (AO) fractions of sea buckthorn (Hippophae rhamnoides) seed. Seed fractions were evaluated for their DPPH, ABTS, superoxide and hydroxyl radical scavenging, ferric reduction, ferrous chelation and xanthine oxidase inhibitory capacities. HPLC-DAD-ESI-MS/MS analytical conditions for the profiling of seed flavonoids were optimized and the AO-rich fraction was analysed. Quercetin-3-O-rutinoside (5.9%), isorhamnetin-3-O-rutinoside (4.9%) and isorhamnetin-3-O-sophroside-7-O-rhamnoside (3.7%) were found as the major flavonoid glycosides in the fraction. Significant amounts of isorhamnetin-3-O-glucoside (2.8%), 3-O-sophroside-7-O-rhamnosides of quercetin (2.4%) and kaempherol (1.3%), and 3-O-glucoside-7-O-rhamnosides of quercetin (1.1%) and isorhamnetin (1.1%) along with their free forms: isorhamnetin (2.7%), quercetin (1.1%) and kaempherol (0.6%) were also found in the fraction. The identification of flavonoids as the major less polar AO phenolics in the seeds was rationalized by demonstrating the high AO activity of isorhamnetin, quercetin, kaempherol and quercetin-3-O-rutinoside.

RP-HPLC-DAD method for the estimation of embelin as marker in Embelia ribes and its polyherbal formulations.

Evidence-based herbal products with assured quality are assuming importance for complementary and alternative medicine. Traditional medicines by and large are not standardized and validated to meet the new requirements. In the present study, marker (embelin)-based standardization of a major medicinal plant, Embelia ribes and its polyherbal formulations was attempted. Conditions for the quantitative extraction of the marker compound embelin from E. ribes fruits and herbal formulations were also optimized. Reversed-phase high-performance liquid chromatography, coupled with diode array detection (RP-HPLC-DAD) for the quantification of embelin was developed and validated. Satisfactory results were obtained with respect to linearity (15-250 µg/mL), LOD (3.97 µg/mL), LOQ (13.2 µg/mL), recovery (99.4-103.8%) and precision (1.43-2.87%). The applicability of the method was demonstrated with selected phytopharmaceuticals. The present method was sensitive, accurate, simple and reproducible and therefore can be recommended for marker-based standardization, and quality assurance of E. ribes herbal formulations.

Simultaneous estimation of phenolic acids in sea buckthorn (Hippophaë rhamnoides) using RP-HPLC with DAD.

A RP-HPLC-DAD method was developed and validated for the simultaneous analysis of nine phenolic acids including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid, p-coumaric acid, cinnamic acid, caffiec acid and ferulic acid in sea buckthorn (SB) (Hippophaë rhamnoides) berries and leaves. The method was validated in terms of linearity, LOD, precision, accuracy and recovery and found to be satisfactory. Phenolic acid derivatives in anatomical parts of SB berries and leaves were separated into free phenolic acids, phenolic acids bound as esters and phenolic acids bound as glycosides and profiled in HPLC. Berry pulp contained a total of 1068 mg/kg phenolic acids, of which 58.8% was derived from phenolic glycosides. Free phenolic acids and phenolic acid esters constituted 20.0% and 21.2%, respectively, of total phenolic acids in SB berry pulp. The total phenolic acid content in seed kernel (5741 mg/kg) was higher than that in berry pulp and seed coat (Table 2). Phenolic acids liberated from soluble esters constituted the major fraction of phenolic acids (57.3% of total phenolic acids) in seed kernel. 8.4% and 34.3% of total phenolic acids in seed kernel were, respectively contributed by free and phenolic acids liberated from glycosidic bonds. The total soluble phenolic acids content in seed coat (448 mg/kg) was lower than that in seed kernel and pulp (Table 2). Proportion of free phenolic acids in total phenolic acids in seed coat was higher than that in seed kernel and pulp. Phenolic acids bound as esters and glycosides, respectively contributed 49.1% and 20.3% of total phenolic acids in seed coat. The major fraction (approximately 70%) of phenolic acids in SB berries was found to be concentrated in the seeds. Gallic acid was the predominant phenolic acid both in free and bound forms in SB berry parts and leaves.

HPTLC fingerprinting and quantification of lignans as markers in sesame oil and its polyherbal formulations.

Sesamum (Sesamum indicum) seed and its oil have been in use in Indian traditional medicine, 'Ayurveda' since antiquity. However, there has been no attempt to standardize the polyherbal formulations containing sesamum oil as the main ingredient in terms of its active principle or marker compound. Biologically active lignans in sesamum oil are identified as the marker compound for the oil and its formulations. In this report, a simple, rapid and sensitive HPTLC method is described for the first time to identify and quantify sesamin and sesamolin, the major lignans of the sesamum oil and the method was applied to polyherbal formulations containing the oil for their quantitative estimation. The method was validated in terms of its calibration curve, limits of detection and quantification, precision, accuracy and robustness following standard protocols. The method thus developed was applied to sesamum oil and its commercial herbal formulations to quantify sesamin and sesamolin. The method for fingerprints of the formulations in the form of densitogram following charring of the chromatographic plate was also developed that could be useful for marker-based quality assurance of the polyherbal products containing sesamum oil.

Semi-preparative HPLC preparation and HPTLC quantification of tetrahydroamentoflavone as marker in Semecarpus anacardium and its polyherbal formulations.

Application of modern scientific knowledge coupled with sensitive analytical technique is important for the quality evaluation and standardization of polyherbal formulations. Semecarpus anacardium, an important medicinal plant with wide medicinal properties, is frequently used in a large number of traditional herbal preparations. Tetrahydroamentoflavone (THA), a major bioactive biflavonoid was selected as a chemical marker of S. anacardium and RP-semi-preparative HPLC conditions were optimized for the isolation of tetrahydroamentoflavone. HPTLC analytical method was developed for the fingerprinting of S. anacardium flavonoids and quantification of tetrahydroamentoflavone. The method was validated in terms of their linearity, LOD, LOQ, precision and accuracy and compared with RP-HPLC-DAD method. The methods were demonstrated for the chemical fingerprinting of S. anacardium plant parts and some commercial polyherbal formulations and the amount of tetrahydroamentoflavone was quantified. HPTLC analysis showed that S. anacardium seed contained approximately 10 g kg(-1) of tetrahydroamentoflavone. The methods were able to identify and quantify tetrahydroamentoflavone from complex mixtures of phytochemicals and could be extended to the marker-based standardization of polyherbal formulations, containing S. anacardium.

An ecofriendly approach to process rice bran for high quality rice bran oil using supercritical carbon dioxide for nutraceutical applications.

An integrated approach to extraction and refining of RBO using supercritical carbon dioxide (SC-CO2) in order to preserve the nutritionally important phytochemicals is reported here. Process variables such as pressure, temperature, time, solvent flow rate and packing material on extraction yield and quality of RBO were investigated using a pilot model SC-CO2 extraction system. Three isobaric (350, 425 and 500 bar), three isothermal temperatures (50, 60 and 70 degrees C), three extraction times (0.5, 1 and 1.5h), at 40/min CO2 flow rate and three packing materials (pebbles, glass beads and structured SS rings) were employed. The RBO yield with SC-CO2 extraction increased with temperature and time under isobaric conditions. At the 60 degrees C isotherm, an increase in the RBO yield was obtained with an increase in the pressure and time. The RBO yield increased significantly with structured SS rings used as packing material. The RBO extracted with SC-CO2 had negligible phosphatides, wax and prooxidant metals (Fe and Cu) and was far superior in color quality when compared with RBO extracted with hexane. At the optimum condition of extraction at 500 bar, 60 degrees C for 1.5h, with structured SS rings used as packing material, the yield of RBO was comparable with that of hexane extraction (22.5%). The phytochemical contents of the RBO under the optimum conditions were in the range of tocols, 1500-1800 ppm; sterols, 15,350-19,120 ppm and oryzanol 5800-11,110 ppm.

Lipase catalyzed interesterification of palm stearin and rice bran oil blends for preparation of zero trans shortening with bioactive phytochemicals.

Palm stearin (PS) and rice bran oil (RBO) blends of varying proportions were subjected to enzymatic interesterification (EIE) using a 1,3 specific immobilized lipase. The interesterified blends were evaluated for their physicochemical characteristics and bioactive phytochemical contents using differential scanning calorimeter (DSC), X-ray diffraction (XRD), gas chromatography (GC) and high performance liquid chromatography (HPLC). The blends of PS and RBO in different proportions (40:60, 50:50, 60:40 and 70:30) had saturated fatty acid content and unsaturated fatty acid content in the range of 37.6-52.0% and 48.0-62.4%, respectively. The blends 40:60, 50:50 and 60:40 showed a considerable reduction in their highest melt peak temperature (TP) and solid fat content (SFC) on EIE. The interesterified blends retained bioactive phytochemicals like tocols (839-1172 ppm), sterols (4318-9647 ppm), oryzanols (3000-6800ppm) and carotene (121-180 ppm). XRD studies demonstrated that the interesterified blends contained beta and beta' polymorphic forms.

Antiradical efficacy of phytochemical extracts from defatted rice bran.

The phytochemical compounds oryzanols, tocopherols, tocotrienols and ferulic acid were identified in the crude methanolic extracts (CME) of defatted rice bran (DRB) by HPLC. Enrichment of antioxidants in CME was achieved by sequential extraction and fractionation resulting in three enriched fractions viz. acetone extract (AE), acetone extract-lipophilic fraction (AE-LP) and acetone extract-polar fraction (AE-PP). The scavenging effects of DRB extracts and their phytochemical constituents against DPPH and superoxide radicals were investigated. The EC(50) (g antioxidant/kg DPPH) values of CME, AE, AE-LP, AE-PP, oryzanols, ferulic acid, tocols (Tmix), tricin, beta-sitosterol, BHT and TBHQ were 1,977, 1,945, 7,985, 1,072, 972, 174, 164, 3,947, 21,416, 1,120 and 61, respectively. Tricin and beta-sitosterol isolated from CME were identified with the help of spectral data. The DRB extracts and their phytochemical constituents when assayed by cytochrome c and NBT methods showed positive superoxide radical scavenging effects. The order of efficacies of the extracts was AE-PP>AE>CME>AE-LP in both assays, but the activities were higher for the former method. The DPPH as well as superoxide scavenging activities of AE, AE-LP and AE-PP could largely be attributed to the levels of total phenols (TPC) and ferulic acid in it.

Phytochemical characterization of defatted rice bran and optimization of a process for their extraction and enrichment.

The aim of this study was to characterize the defatted rice bran (DRB) employing HPLC for identifying the major phytochemicals in DRB and to examine its commercial potential as a source of bioactive phytochemicals leading to value addition of DRB otherwise used as cattle feed. Various solvent extracts showed the presence of oryzanols, tocols, and ferulic acid. Methanol was the most effective extractant under the optimized conditions of a material-solvent ratio of 1:15 (wt./vol.) and a time of extraction of 10h. The yields of total phenols, oryzanols and ferulic acid from DRB with methanol were 2204, 316, and 233 ppm, respectively. Enrichment of antioxidants in the crude methanolic extract (CME) was achieved by sequential extraction and fractionation, resulting in three enriched fractions, viz., acetone extract (AE), acetone extract-lipophilic fraction (AE-LP) and acetone extract-polar fraction (AE-PP). While AE-LP was enriched in oryzanols and tocols by about 65 times, AE-PP was enriched in ferulic acid by 70 times as compared to their contents in DRB.

Purification and characterization of soluble peroxidase from oil palm (Elaeis guineensis Jacq) leaf.

Soluble peroxidase (POD) from oil palm leaf was purified by (NH(4))(2)SO(4) precipitation, anion exchange chromatography and molecular exclusion chromatography. The purification grade obtained was 429 yielding 54% of the enzyme activity. Electrophoresis of purified enzyme under denatured conditions revealed M(r) of 48+/-2 kDa. It has an optimum pH of 5 and it exhibited very high pH and thermal stabilities. K(m) for guaiacol, ABTS and pyrogallol were 3.96, 1 and 0.84 mM, respectively. Immunocytochemical localization studies showed that soluble POD was mainly located in the vascular bundles and epidermis of leaf.

Influence of stage of lactation on the triacylglycerol composition of buffalo milk fat.

Milk fats obtained from colostrum and early, middle and late lactation samples of buffalo milk were analyzed for their triacylglycerol (TG) compositions. Each milk fat was first separated by thin layer chromatography (TLC) into high, medium and low molecular weight TG. The TG fractions thus obtained were further segregated by argentation TLC, according to their degree of unsaturation into saturated, trans-monoene, cis-monoene, diene and polyene species. With progressive lactation, the major changes from colostral fat were an increase in lower fatty acids and decline in oleic acid. This caused, in turn, marked variations in saturated TG and diene TG and, to a smaller extent, in polyene TG. Monoene TG, both cis and trans, remained practically constant throughout. These trends were largely reversed toward the end of lactation.

Effect of polymerization on antioxidant and xanthine oxidase inhibitory potential of sea buckthorn (H. rhamnoides) proanthocyanidins.

Inhibitory potential of sea buckthorn (Hippophae rhamnoides L) seed proanthocyanidins against oxidative stress and xanthine oxidase activity was evaluated. Composition of antioxidant proanthocyanidins was profiled by analyzing the cleavage products obtained by the acid catalyzed hydrolysis in the presence of phloroglucinol. Catechin, epicatechin, gallocatechin, and epigallocatechin were found as the extension and terminal subunits of proanthocyanidins with an average degree of polymerization (ADP) of 14.7. Seed proanthocyanidins showed considerably high antioxidant and xanthine oxidase inhibitory potentials. Antioxidant and xanthine oxidase inhibitory capacity evaluation of proanthocyanidin fractions with varying ADP showed that proanthocyanidins with lower molecular size were more effective as superoxide anion (ADP ≤ 4.2) and hydroxyl radical (ADP ≤ 5.9) scavengers and xanthine oxidase (ADP ≤ 3.1) inhibitors. ADP of the studied proanthocyanidin fractions did not show significant influence on their DPPH and ABTS radical scavenging and ferric reduction capacities.

Effect of saponification on composition of unsaponifiable matter in rice bran oil.

Rice Bran Oil contains a variety of Unsaponifiable Constituents (USC) that are presumed to contribute to the high value of Unsaponifable Matter (USM). The objectives of the present study were to identify and quantify the constituents in USM. The changes that the unsaponifiables undergo during saponification were also quantitatively investigated. While analyzing the percentage of all constituents, the percentage of sterol get increased from 22.46 to 23.77 in USM of crude rice bran oil (CRBO) and 33.42 to 36.79 in USM of refined rice bran oil (RRBO). Oryzanol that comprised 34% of the unsaponifiable in the crude oil by direct estimation was almost eliminated in USM and same in refined oil. The results also revealed the presence of four additional classes of compounds that were quantified in USM (policosanol, fatty aldehydes, triterpene alcohols and potassium salt of oryzanols). Among the four classes of compounds, policosanol contributed high percentage in USM, (43.39% in CRBO and 28.46% in RRBO). Fatty aldehydes, triterpene alcohols and potassium salt of oryzanols together contributed 27.68% and 25.13% of USM from CRBO and RRBO respectively. The HPTLC method employed here thus, accounted for 96.75% by wt of the USM of CRBO and 92.00% by wt of the USM of RRBO.

Sea buckthorn (Hippophae rhamnoides) proanthocyanidins inhibit in vitro enzymatic hydrolysis of protein.

Interactions of phenolics with other food constituents and digestive enzymes are likely to have interference with the digestion and bioavailability of food and phenolics. In this study the effect of sea buckthorn proanthocyanidins on in vitro digestion of protein was evaluated. Optimization of the extraction conditions showed that maximum recovery of sea buckthorn proanthocyanidins was obtained with acidified acetone; water mixture (60% to 70%, v/v). Crude proanthocyanidin extracts thus prepared were purified using sephadex gel column chromatography and their average degree of polymerization and the effects on enzymatic hydrolysis of bovine serum albumin as influenced by their protein precipitation capacities were studied. Average degree of polymerization of proanthocyanidins in berry pulp, kernel, seed coat, and leaves was 7.4, 5.6, 8.2, and 10.6, respectively. The EC50 values for the protein precipitation by the PA of berry pulp, kernel seed coat, and leaves were 44.2, 44.1, 65.8, and 39.8 µg, respectively. Relative enzymatic hydrolysis of the protein-proanthocyanidin complexes was 44.1% to 60.3% for pepsin and 57.5% to 67.7% for trypsin. Interactions of sea buckthorn proanthocyanidins with food proteins and digestive enzymes might alter the protein digestibility and phenolic bioavailabilty.

Tetrahydroamentoflavone (THA) from Semecarpus anacardium as a potent inhibitor of xanthine oxidase.

ETHNOPHARMACOLOGICAL RELEVANCE: Seed of Semecarpus anacardium L. is widely used in Indian traditional medicine; Ayurveda and Sidha, for treatment of inflammatory disorders and gout. AIM OF THE STUDY: The present study was aimed at isolation of a compound for its potential to inhibit xanthine oxidase (XO), over expression of which lead to inflammation and gout. MATERIALS AND METHODS: Activity guided fractionation of S. anacardium seed was conducted using liquid-liquid partition and preparative HPLC. The fractions were evaluated for their XO inhibition and antioxidant activity. The ethyl acetate fraction with the highest XO activity yielded a biflavonoid compound tetrahydroamentoflavone (THA). Lineweaver-Burk (LB) plot for the XO inhibition of THA and allopurinol was constructed from the kinetic data. RESULTS: IC50 values of THA and allopurinol for XO inhibition were 92 and 100 nM respectively and their corresponding values for K(i) were 0.982 and 0.612 µM respectively. CONCLUSION: THA was a potent XO inhibitor which could be considered as a drug candidate or chemopreventive agent, after establishing its pharmacological and clinical evaluation. The study results appear to support the claim of the traditional medicine with respect to the efficacy of S. anacardium seed against inflammation and gout.

In vitro antioxidant activities of the methanol extract and its different solvent fractions obtained from the fruit pericarp of Terminalia bellerica.

Terminalia bellerica has been used as a traditional medicine in a variety of ailments including anaemia, asthma, cancer, inflammation, rheumatism and hypertension. In this study, the free radical scavenging and antioxidant activities of methanol extract (ME) and its different solvent fractions (namely hexane (HE), ethyl acetate (EA), butanol (BL) and water (WA)) of the T. bellerica fruit pericarp were evaluated and compared with standard antioxidant compounds like gallic acid (GA), catechin and ascorbic acid. Among the different fractions tested, the EA fraction exhibited higher antioxidant and radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide and hydroxyl radicals than the other fractions, which may be attributed to its higher phenolic and flavonoid content, since a linear relation was observed between the phenolic content and the antioxidant parameters. The HPTLC analysis of the EA fraction revealed that it mainly contains GA and ferulic acid (FA) as major phenolics, and the higher antioxidant activities of EA fraction may be due to the presence of these compounds.

Characterization of sn-2 alk-1'-enyl ethers of glycerol from rice bran oil.

Ether lipids have biological applications which would dissipated as an important constituent in cell membranes. These are mostly found in animal tissues and rare in plant origin. Alk-1'-enyl ethers are class of ether lipid forming aldehydes on cleavage of ether bonds. The present study enrolled the presence of aldehyde in unsaponifiable matter of rice bran oil (RBO) and hence the identification of source of aldehydes in RBO was conducted. With respect to the earlier reports the investigation turned to major lipid constituents such as triacylglycerols, diacylglycerols etc. Using the column chomatographic method lipid fractions are separated, recolumned, purified and analyzed by spectrochemical methods such as FT-IR, (1)HNMR, (13)CNMR, Mass spectrometry and confirmed the presence of ether lipids. The sn-2 position was confirmed by enzymatic hydrolysis using pancreatic lipase. Moreover the formation of aldehyde from these ether lipids was also confirmed by spectrometric methods.

Anti-atherogenic effects of seabuckthorn (Hippophaea rhamnoides) seed oil.

Seabuckthorn (SBT) seed oil is a rich source of unsaturated fatty acids, phytosterols, carotenoids and flavonoids, which are known to have significant anti-atherogenic and cardioprotective activity. The anti-atherogenic activity of supercritical CO(2) extracted SBT seed oil was evaluated in white albino rabbits fed on high cholesterol diet for 60 days. The study was performed on 20 male healthy rabbits divided into four groups of 5 animals each. Group I - control, group II - SBT seed oil, group III - cholesterol (1%) for 60 days, group IV - cholesterol+SBT seed oil. After 30 days of high cholesterol diet, group IV rabbits received 1 ml of SBT seed oil daily for 30 days. Blood total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C) and triglyceride (TG) levels were measured before and after the administration of SBT seed oil. The vasorelaxant activity of the seed oil was studied in vitro using aortic ring model technique and changes in isometric force were recorded using a polygraphic recording system. Accumulation of cholesterol in the aorta was studied using Sudan-IV staining technique. SBT seed oil feeding to normal rabbits for 18 days caused a significant decline in plasma cholesterol, LDL-C, atherogenic index (AI) and LDL/HDL ratio. The HDL-C levels, HDL-C/TC ratio (HTR) and vasorelaxant activity of the aorta were significantly increased. In cholesterol-fed animals the TC, TG, LDL-C and AI were significantly increased and showed a decline following seed oil administration. The increase in HDL-C was more marked in seed oil treated hypercholesterolemic animals. The acetylcholine-induced vasorelaxant activity was significantly decreased in cholesterol-fed animals and could be restored to that of normal values by seed oil administration. These observations suggest that supercritical CO(2) extracted SBT seed oil has significant anti-atherogenic and cardioprotective activity.

Triacylglycerol composition of buffalo milk fat.

Buffalo milk fat was first separated by thin layer chromatography (t.l.c.) into high (HMT), medium(MMT) and low (LMT) molecular weight triacylglycerols. The triacylglycerol (TG) fractions thus obtained were further resolved by argentation t.l.c. int saturated, trans-monoene, cis-monoene, diene and polyene TG species. Whereas 18:1 was concentrated in HMT, 6:0, 8:0 and 10:0 tended to be located in MMT. LMT was unique in containing almost all 4: 0. HMT, MMT and LMT showed great variations in their levels of different TG species. The distribution of fatty acids among the TG species was found to be characteristic of each TG species.

Impact of vitamin A supplementation through different dosages of red palm oil and retinol palmitate on preschool children.

Red palm oil (5 ml and 10 ml), ground nut oil fortified with 400 and 800 retinol equivalent retinol palmitate, and ground nut oil (5 and 10 ml), were administered to six groups of preschool children (four experimental and two control groups) in randomly assigned balwadis of Ramanathapuram District of Tamil Nadu for a period of 7 months, to monitor the difference in the efficacy of the mode of supplementation and the optimum dose for improving vitamin A status. Results show that red palm oil groups recorded more gain in retinol and beta-carotene levels compared to other dosage groups, and that administration of 10 ml did not offer any substantial improvement over the 5-ml daily dose.