Eugene Kokkalou

Simultaneous reversed-phase high-performance liquid chromatographic method for the determination of diosmin, hesperidin and naringin in different citrus fruit juices and pharmaceutical formulations

Diosmin, hesperidin and naringin are flavonoid glycosides that occur naturally in citrus fruits. They exert a variety of pharmacological properties such as anti-inflammatory, antioxidant and free radical scavenging and antiulcer effects and also inhibit selected cytochrome P-450 enzymes resulting in drug interactions. A reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of diosmin, hesperidin and naringin in different citrus fruit juices and pharmaceutical preparations. Diosmin, hesperidin, naringin and the internal standard rhoifolin were separated using tetrahydrofuran/water/acetic acid (21:77:2, v/v/v) as the mobile phase at 34 degrees C, using a C8 reversed-phase column. The method was linear in the 0.25-20.0 microg/ml concentration range for all three flavonoid glycosides (r>0.999). The method has been successfully applied to the determination of all three flavonoid glycosides in several samples of different citrus fruit juices sold in Greece and for the determination of diosmin and hesperidin in pharmaceutical preparations.

Essential oil variation of Lavandula stoechas L. ssp. stoechas growing wild in Crete (Greece)

Abstract The essential oils from four wild populations of Lavandula stoechas L. ssp. stoechas of Crete, Greece, have been analysed at the full bloom stage. The main essential oil constituents were α-pinene,1,8-cineole, fenchone, camphor and myrtenyl acetate, all products of different biogenetic routes. A considerable quantitative variation of the essential oils occurred among the different populations, three of them being fenchone/camphor type and one 1,8-cineole/fenchone type. The variation in the quantitative essential oil composition between leaves and inflorescences was also significant. In all cases, inflorescences contained more fenchone, myrtenyl acetate and α-pinene, while leaves contained more 1,8-cineole and camphor. Additionally, the inflorescences produced notably larger essential oil amounts than the leaves.

The phytochemical analysis and antioxidant activity assessment of orange peel (Citrus sinensis) cultivated in Greece-Crete indicates a new commercial source of hesperidin

The flavonoid content of several methanolic extract fractions of Navel orange peel (flavedo and albedo of Citrus sinensis) cultivated in Crete (Greece) was first analysed phytochemically and then assessed for its antioxidant activity in vitro. The chemical structures of the constituents fractionated were originally determined by comparing their retention times and the obtained UV spectral data with the available bibliographic data and further verified by detailed LC-DAD-MS (ESI+) analysis. The main flavonoid groups found within the fractions examined were polymethoxylated flavones, O-glycosylated flavones, C-glycosylated flavones, O-glycosylated flavonols, O-glycosylated flavanones and phenolic acids along with their ester derivatives. In addition, the quantitative HPLC analysis confirmed that hesperidin is the major flavonoid glycoside found in the orange peel. Interestingly enough, its quantity at 48 mg/g of dry peel permits the commercial use of orange peel as a source for the production of hesperidin. The antioxidant activity of the orange peel methanolic extract fractions was evaluated by applying two complementary methodologies, DPPH(*) assay and the Co(II)/EDTA-induced luminol chemiluminescence approach. Overall, the results have shown that orange peel methanolic extracts possess moderate antioxidant activity as compared with the activity seen in tests where the corresponding aglycones, diosmetin and hesperetin were assessed in different ratios.

LC-DAD-MS (ESI+) Analysis and Antioxidant Capacity of Crocus sativus Petal Extracts

In this study, various fractions isolated from the petals of Crocus sativus were assessed at first for their phenolic content both qualitatively and quantitatively and secondly for their antioxidant activity. The phytochemical analysis was carried out by LC-DAD-MS (ESI (+)) whereas the antioxidant potential was evaluated by applying two methodologies, the DPPH. radical scavenging activity test and the Co(II)-induced luminol chemiluminescence procedure. According to data obtained from these antioxidant tests, the diethyl ether, ethyl acetate and aqueous fractions demonstrated the strongest antioxidant capacity. Interestingly, the major constituents identified in these fractions correspond to kaempferol, quercetin, naringenin and some flavanone and flavanol derivatives glycosylated and esterified with phenylpropanoic acids. In addition, the presence of some nitrogen-containing substances, as well as other phenolics and phenylpropanoic derivatives was also traced. The identification and structural elucidation of all substances isolated in this study was achieved by both comparing available literature data and by proposed fragmentation mechanisms based on evaluating the LC-DAD-MS (ESI (+)) experimental data. The quantitative analysis data obtained thus far have shown that Crocus sativus petals are a rich source of flavonoids. Such a fact suggests that the good antioxidant capacity detected in the various fractions of Crocus sativus petals could be attributed to the presence of flavonoids, since it is already known that these molecules exert antioxidant capability. The latter, along with the use of Crocus sativus in food and pharmaceutical industry is discussed.

Dissolution rate and stability study of flavanone aglycones, naringenin and hesperetin, by drug delivery systems based on polyvinylpyrrolidone (PVP) nanodispersions

Objective: To study the dissolution behavior, the release mechanism and the stability of nanodispersion system of aglycones with PVP. Methods: The nanodispersion system of polyvinylpyrrolidone (PVP)/naringenin–hesperetin was prepared using the solvent evaporation method. The chemical stability (compatibility) of naringenin and hesperetin in the prepared dispersions was studied under accelerated conditions for 3 months. The evaluation of physical stability was performed by X-ray diffraction analysis (XRD) and by comparing the dissolution profile before and after storage at high temperature and moisture (40ºC, RH 75%). Results: The dissolution rate of naringenin and hesperetin released was dramatically increased in the nanodispersion system of PVP/naringenin–hesperetin (80/20, w/w). The release mechanism of both flavanone aglycones was better described by the diffusion model (Higuchi model). Also it was found that the rate-limiting step that controlled the release of naringenin and hesperetin in the nanodispersion system was dissolution of the carrier (PVP). Conclusions: During accelerated degradation analysis, for 3 months at high temperature and moisture, PVP nanodispersion system showed enhanced chemical compatibility and physical stability. The physical evaluation (obtained from XRD analysis) of PVP/naringenin–hesperetin (80/20, w/w) in the selected storage conditions did not show any crystallization of flavanone aglycones in the PVP nanodispersion system or any change in their release profile.

Chemical Variability of Flowers, Leaves, and Peels Oils of Four Sour Orange Provenances

Abstract Peel, leaf, and flower oils belonging to Citrus aurantium L. var amara (sour orange) of four provenances were obtained from sour orange trees submitted to the same pedoclimatic and horticultural conditions. Their Chemical composition was investigated by GC-FID and GC-MS. Remarkable differences were found between the constituent percentages of the different provenances. This Chemical variability was characterized by a limonene chemotype distinguished in peel oils, a linalool/linalyl acetate chemotype observed for petitgrain and a linalool/linalyl one associated with β-pinene for neroli oils.

Phytochemical analysis and antioxidant activity of Lycium barbarum (Goji) cultivated in Greece

Abstract Context: The fruit of Lycium barbarum L. (Solanaceae), known as goji berry, has been exploited for a long time in traditional Chinese medicine. In recent decades, it has received much attention as one of the trendiest functional foods with a wide array of pharmacological activities in Western diets. Objective: In this study the phenolic profile and potential antioxidant capacity of Lycium barbarum cultivated in Crete (Greece) were investigated. Materials and methods: The berries were defatted with hexane and then extracted with dichloromethane and methanol using a Soxhlet apparatus. Furthermore, the methanol extract was fractionated with ethyl acetate and butanol. All fractions/extracts were tested for their antioxidant activity (DPPH, FRAP, chemiluminescence). Folin–Ciocalteu and LC-DAD-MS analyses were utilized for the identification of the phenolic compounds. Results: The total phenolic content ranged from 14.13 ± 0.40 (water fraction) to 109.72 ± 4.09 (ethyl acetate fraction) mg gallic acid equivalent/g dry extract. Ethyl acetate extract exhibited the highest scavenging activities determined as EC50 (4.73 ± 0.20 mg/mL) and IC50 (0.47 ± 0.001 mg/mL) using DPPH and chemiluminescence assays. Seventeen phenolic compounds, including cinnamoylquinic acids and derivatives, hydrocinnamic acids and flavonoid derivatives, were tentatively identified. To the best of our knowledge, quercetin 3-O-hexose coumaric ester and quercetin 3-O-hexose-O-hexose-O-rhamnose are reported for the first time in goji berry fruits. Discussion and conclusion: The results of this study suggest that consumption of goji berry fruits could serve as a potential source of natural antioxidant compounds and that goji berry phenolic extracts could be exploited for nutritional pharmaceutical purposes.

Phytochemical analysis with the antioxidant and aldose reductase inhibitory capacities of Tephrosia humilis aerial parts’ extracts

Abstract The aerial parts of Tephrosia humilis were tested about their antioxidant potential, their ability to inhibit the aldose/aldehyde reductase enzymes and their phenolic content. The plant material was exhaustively extracted with petroleum ether, dichloromethane and methanol, consecutively. The concentrated methanol extract was re-extracted, successively, with diethyl ether, ethyl acetate and n-butanol. All extracts showed significant antioxidant capacity, but the most effective was the ethyl acetate extract. As about the aldose reductase inhibition, all fractions, except the aqueous, were strong inhibitors of the enzyme, with the n-butanolic and ethyl acetate fractions to inhibit the enzyme above 75%. These findings provide support to the ethnopharmacological usage of the plant as antioxidant and validate its potential to act against the long-term diabetic complications. The phytochemical analysis showed the presence of 1,4-dihydroxy-3,4-(epoxyethano)-5-cyclohexene(1), cleroindicin E(2), lupeol(3), methyl p-coumarate(4), methyl 4-hydroxybenzoate(5), prunin(6), 5,7,2ʹ,5ʹ-tetrahydroxyflavanone 7-rutinoside(7), protocatechuic acid(8), luteolin 7-glucoside(9), apigenin(10), naringin(11), rhoifolin(12) and luteolin 7-glucuronate(13). Graphical Abstract

GC-MS Analysis of Volatile Constituents of Cornus mas Fruits and Pulp

Cornus mas is a tree bearing edible fruits, known for its traditional homemade beverages and preserves and for its usage as a traditional medicinal plant. There are no references cited in the literature for the volatile components of mature fruits and their pulp (product that is commercially available). The aim of the present study was the identification of the volatile constituents of Cornus mas pulp and mature fruits by GC-MS using capillary columns of different polarity and selectivity. The analysis showed that Cornus mas pulp is rich in fatty acid methyl/ethyl esters, while mature fruits have higher content of alcohols, aldehydes and terpenes. Additionally, the use of two or more capillary columns of different polarity and selectivity could be the key to a much more integrated analysis and a much more accurate identification of the components of natural complex mixtures such as volatile constituents of Cornus mas or other plants.

Antioxidant and aldose reductase inhibitory capacities of Tephrosia humilis aerial parts' extracts

Tephrosia humilis (Leguminosae), is a tropical shrub endemic in central Africa. Species of this genus are used in traditional remedies as antimicrobial, tonic, diuretic, anthelminthic, blood purifier etc [1,2]. Phytochemical analysis of other Tephrosia species resulted in the isolation of coumarins, and several flavonoids [3–9]. The aim of this study was the evaluation of the antioxidant capacity of several plant extracts of Tephrosia humilis (DPPH assay and Co(II)/EDTA -induced luminol chemiluminescence test), as well as their ability to inhibit the aldose reductase enzyme (AR, ALR2, E.C. 1.1.1.21), indicating a potential of this plant to act against the long term diabetic complications [10,11]. After defatting, plant material was extracted in a soxhlet apparatus with methanol and the dry remaining was partitioned with solvents of increasing polarity, giving five different fractions (diethyl ether, ethyl acetate, ethyl acetate residue, n-butanol, water). Antioxidant results according to both tests proved the good antioxidant capacity of the ethyl acetate fraction in comparison to the standards used (trolox and quercetin) [12]. While according to the CL test all other fractions showed low hydroxyl radical scavenging ability, DPPH test results proved that the n-butanolic and ethyl acetate residue fractions are also effective radical scavengers. As about the ALR2 inhibition, all fractions, except the aqueous, were strong inhibitors at the concentration of 25µg/ml in comparison to sorbinil. The n-butanolic and the ethyl acetate fractions inhibited the enzyme above 75%. This research work proves in vitro the good antioxidant capacity of Tephrosia humilis extracts, as well as their strong inhibitory activity against ALR2 enzyme. References: 1. Bashir, A.K. et al. (1992) Fitoterapia 63: 371. 2. Kole, RK et al. (1992)J. Agric. Food Chem. 40: 1208–1210. 3. Ahmad, S. (1986) Phytochem. 25: 955–958. 4. Menichini, F. et al. (1982) Planta Med. 45: 243. 5. Rajani, P. et al. (1988) Phytochem. 27: 648–649. 6. Rao, E.V. et al. (1984) Phytochem. 23: 1493–1501. 7. Venkata, R. et al. (1979) Phytochem. 18: 1581–1582. 8. Hussaini, F.A. et al. (1986) Planta Med. 52: 220–221. 9. Gupta, R.K. et al. (1980) Phytochem. 19: 1264. 10. Nicolaou, I. et al. (2004)J. Med. Chem. 47: 2706–2709. 11. Matthew, J. et al. (2002)J Amer Med Assoc 288: 2579–2582. 12. Termentzi, A. et al. (2006) Food Chem. 98: 599–608