RESUMEN

The aim of this study is the development of validated HPTLC method for the quantification of vitexin from Passiflora foetida commercial herbal formulations. The developed method was validated, in accordance with ICH guidelines for precision, accuracy, specificity and robustness. The plate was developed using ethyl acetate:methanol:water:formic acid 30:4:2:1(%, v/v/v/v) on 20 × 10 cm glass coated silica gel 60 F254 plates and the developed plate was scanned and quantified densitometrically at λ = 340 nm. Linear regression analysis revealed a good linear relationship between peak area and amount of vitexin in the range of 100-700 ng/spot. The amount of vitexin in nine commercial herbal formulations was successfully quantified by the developed HPTLC method. The developed and validated high performance thin layer chromatographic method offers a new sensitive and reliable tool for quantification of vitexinin in various herbal formulations containing Passiflora foetida.

RESUMEN

A new rapid, simple, sensitive and high performance thin layer chromatography (HPTLC) has been established for the simultaneous determination of ascorbic acid and gallic acid in the freeze-dried pomegranate fruit juice and herbal formulation. HPTLC method was carried out using ethyl acetate: acetone: water: formic acid, 10:6:2:2 (%, v/v/v/v)) on 20 × 10 cm glass coated silica gel 60 F254 plates and scanned at 254 nm for ascorbic acid and gallic acid. Ascorbic acid and gallic acid in the freeze-dried pomegranate fruit juice were identified by comparing their single spot at Rf = 0.54 ±â€¯0.02 and Rf = 0.83 ±â€¯0.01 respectively. The value of regression equation (r2 ≥ 0.9992) revealed a good linear relationship between peak area and amount of ascorbic acid and gallic acid in the range of 100-800 ng/band. The method was validated for precision, accuracy, robustness LOD and LOQ. The method proposed can be useful for routine determination of ascorbic acid and gallic acid in various crude as well as herbal formulations as a quality control tool.

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Pomegranate is a well known fruit for its unique flavor, taste and health benefits. The medicinal properties of this fruits directly associated with the phenolic content present, with great anti-oxidant potential. The research is intended to develop matrix solid phase dispersion method (MSPD) and HPLC quantification of four major anti-oxidant marker constituents (vitamin C, gallic acid, rutin & ellagic acid) in pomegranate molasses samples. The effects of several important experimental parameters like type of dispersant, sample-dispersant ratio, solvents and its volume, time of extraction were investigated. A C18 column with the specification (5 µm, 250 × 4.0 mm) was used for the separation. A gradient flow of mobile phase was selected after many trials containing 0.1%, v/v solution of orthophosphoric acid and acetonitrile. The flow rate was 1.0 mL/min; and the chromatograms were recorded at 254 nm. The validation parameters, like linearity (r2 = 0.9985, 0.9965, 0.9925 & 0.9986), accuracy (100.3, 99.5, 100.9 & 101.9%), intra-day precision (%RSD = 1.09, 1.02, 1.26 & 0.97), inter-day precision (%RSD = 1.32, 0.83, 1.07, & 1.15) LOD (0.07, 4.50, 0.45 & 0.40 µg/mL), LOQ (0.095, 9.50, 0.85 & 9.5 µg/mL) and robustness (% RSD = 0.92, 0.76, 0.81 & 0.83) respectively for vitamin C, gallic acid, rutin & ellagic acid, were found satisfactory as per ICH guidelines.

RESUMEN

The essential oils (EOs) of Anthemis melampodina (Am) and Anthemis scrobicularis (As) (Asteraceae) were extracted from the aerial parts of the plants by hydrodistillation, and their chemical compositions were analyzed using GC-FID and GC-MS. Fifty-six components representing 85.5% of the oil composition of Anthemis melampdina were identified, and the major components were α-pinene (17.1%) and ß-eudesmol (13.8%). Forty-one components representing 86% of the oil composition of Anthemis scrobicularis were identified, and the major component was ß-eudesmol (12.8%). Laboratory bioassays were conducted to determine repellency of Am and As EOs against the yellow fever mosquito Aedes aegypti L. and the lone star tick Amblyomma americanum L. The minimum effective doses (MEDs) of the Am and As EOs against mosquitoes were 0.187 ±â€¯0.000 and 0.312 ±â€¯0.063 mg/cm2 respectively, which were significantly higher than that of DEET (0.023 ±â€¯0.000 mg/cm2) in human-based repellent bioassays. The As EO was more repellent than Am EO against nymphal ticks but was less effective than DEET in vertical paper bioassays.

RESUMEN

The essential oil (EO) of the aerial parts of Rhanterium epapposum Oliv. (Asteraceae), was obtained by hydrodistillation. The oil was subsequently analyzed by both GC-FID and GC-MS, simultaneously. Forty-five components representing 99.2% of the oil composition were identified. The most abundant compounds were camphene (38.5%), myrcene (17.5%), limonene (10.1%) and α-pinene (8.7%). Referring to the ethnobotanical utilization, an insecticidal assay was performed, where the oil repelled the yellow fever mosquito Aedes aegypti L. at a minimum effective dose (MED of 0.035 ± 0.010 mg/cm2) compared to the positive control DEET (MED of 0.015 ± 0.004 mg/cm2). Additionally, the in vitro antimicrobial activity against a panel of pathogens was determined using a microdilution method. The acetyl- and butyrylcholine esterase inhibitory activities were measured using the colorimetric Ellman method. The bioassay results showed that the oil was rather moderate in antimicrobial and cholinesterase inhibitions when compared to the standard compounds.

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Two simple, precise and stability-indicating densitometric HPTLC method were developed and validated for qualitative and quantitative analysis of Coenzyme Q10 in pharmaceutical formulations using normal-phase (Method I) and reversed phase (Method II) silica gel TLC plates. Both methods were developed and validated with 10×20 cm glass-backed plates coated with 0.2 mm layers of either silica gel 60 F254 (E-Merck, Germany) using hexane-ethyl acetate (8.5:1.5 v/v) as developing system (Method I) or RP-18 silica gel 60 F254 (E-Merck, Germany) using methanol-acetone (4:6 v/v) as mobile phase (Method II). Both analyses were scanned with a densitometer at 282 nm. Linearity was found in the ranges 50-800 ng/spot (r(2)=0.9989) and 50-800 ng/spot (r(2)=0.9987) for Method I and Method II respectively. Stability of Coenzyme Q10 was explored by the two methods using acid, base, hydrogen peroxide, temperature and different solvents. Due to the efficiency of the method in separating Coenzyme Q10 from other ingredients including its degradation products, it can be applied for quality control, standardization of different pharmaceutical formulations and stability study.

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Phytochemical study of the CH(2)Cl(2) soluble fraction of the aerial parts of R. natalensis resulted in the isolation and identification of six flavonoid derivatives, ß-amyrin and ß-sitosterol glucoside (daucosterol). The isolated compounds were identified utilizing physical, chemical and different spectral methods including UV, 1D- 2D-NMR and MS. The compounds were identified as four flavanones; 7-O-methyl hesperetin (1), 7-O-methyl naringenin (4), (-)-homoeriodictyol (eriodictyonone) (5), eriodictyol-7-methyl ether (6) and two flavones; 7-O-methyl isokaemferide (2) and genkwanin (3). The isolated compounds as well as some available standards representing structurally similar flavones and flavanones were used to study their antioxidant potential using DPPH and try to explore the impact of structures on the antioxidant activity. In other assays flavanones were less active than flavones as antioxidant due to lack of the C-4 carbonyl group in conjugation with 2,3-double bond. However, in DPPH assay based on the ability of molecules to donate hydrogen flavanones were found more active than flavones.

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Phytochemical study of the aerial parts of Ficus palmata utilizing liquid-liquid fractionation and different chromatographic techniques resulted in the isolation of a new isomer of psoralenoside namely, trans-psoralenoside (5) in addition to, one triterpene: germanicol acetate (1), two furanocoumarins: psoralene (2), bergapten (3), one aromatic acid vanillic acid (4) and the flavone glycoside rutin (6). Structures of the isolated compounds were established through physical, 1D- and 2D-NMR and MS data. The total extract and fractions of the plant were examined in vivo for its possible effects as hepatoprotective, nephroprotective, antiulcer and anticoagulant activities in comparison with standard drugs. Hepatoprotective activity was assessed via serum biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and total bilirubin. Tissue parameters such as non-protein sulfhydryl groups (NP-SH), malonaldehyde (MDA) and total protein (TP) were also measured. In addition to tissue parameters, nephroprotective effect was evaluated by measuring the serum levels of sodium, potassium, creatinine and urea. Histopathological study for both liver and kidney cells was also conducted. Antiulcer activity was explored by observing stomach lesions after treatment with ethanol. Whole blood clotting time (CT) was taken as a measure for the anticoagulant activity of the extract. Antioxidant activity of the total extract and fractions of the plant was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and ascorbic acid as standard.

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Phytochemical study of the aerial parts of Ficus cordata utilizing liquid-liquid fractionation and different chromatographic techniques resulted in the isolation of four furanocoumarins: psoralene (1), hydroxy isoimperatorin (2), oxypeucedanin hydrate (3) and dorsteniol (4), the flavone glycoside rutin (5), b-sitosterol and sucrose. Structures of the isolated compounds were established through physical, 1D- and 2D-NMR and MS data. The total extract of the plant was examined in vivo for its possible effects as hepatoprotective, nephroprotective, antiulcer and anticoagulant in comparison with standard drugs. Hepatoprotective activitys were accessed via serum biochemical parameters including aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and total bilirubin. Tissue parameters such as non-protein sulfhydryl groups (NP-SH), malonaldehyde (MDA) and total protein (TP) were also measured. In addition to tissue parameters, nephroprotective effect was evaluated by measuring the serum levels of sodium, potassium, creatinine and urea. Histopathological study for both liver and kidney cells was also conducted. Antiulcer activity was explored by observing stomach lesions after treatment with ethanol. Whole blood clotting time (CT) was taken as measure for the anticoagulant activity of the extract. All the studied parameters indicated that the total extract of Ficus cordata at 500mg/kg possess moderate hepatoprotective effect, good protection against ethanol induced ulcer and weak nephroprotective effect. The CT was about one quarter of that of warfarin.

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Different fraction obtained from the aerial parts of Juniperus phoenicea showed significant activity as hepatoprotective when investigated against carbon tetrachloride induced liver injury. The hepatoprotective activity was evaluated through the quantification of biochemical parameters and confirmed using histopathology study. Phytochemical investigation of the petroleum ether, chloroform and methanol fractions utilizing different chromatographic techniques resulted in the isolation of five known diterpenoids namely: 13-epicupressic acid (1), imbricatolic acid (2), 7α-hydroxysandaracopimaric acid (3), 3ß-hydroxysandaracopimaric acid (4), isopimaric acid (5), four flavonoid derivatives: cupressuflavone (6), hinokiflavone (7), hypolaetin-7-O-ß-xylopyranoside (9), (-) catechin (10), inaddition to sucrose (8). Both physical and spectral data were used for structure determination and all isolates were evaluated for their hepatoprotective activity. Compounds 2 and 6 were effective, however; 7 was the most active. Hepatoprotective activity of 7 is comparable with the standard drug silymarin in reducing the elevated liver enzymes and restoring normal appearance of hepatocytes. Hepatoprotective effect of combination of 6, 7 and silymarin with the diterpene sugiol was also explored.

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Two new flavane gallates were isolated from the leaves of Plicosepalus curviflorus. The structure of the new compounds was established as 2S,3R-3,3',4',5,7-pentahydroxyflavane-5-O-gallate (1) and 2S,3R-3,3',4',5,5',7-hexahydroxyflavane-4',5-di-O-gallate (2), respectively. In addition, seven known compounds (-)-catechin (3), quercetin (4), lupeol (5), ß-sitosterol (6), pomolic acid (7), ß-sitosterol 3-O-ß-d-glucopyranoside (8) and 4-methoxycinnamic acid (9) were reported for the first time from the genus Plicosepalus. Compounds 1, 2 and 3 were investigated for their hypoglycemic activity and showed significant hypoglycemic activity in Swiss Albino mice.

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The petroleum ether fraction of Juniperus procera showed significant activity as hepatoprotective when investigated against carbon tetrachloride induced liver injury. The hepatoprotective activity was evaluated through the quantification of biochemical parameters and confirmed using histopathology analysis. Phytochemical investigation of the petroleum ether fraction utilizing different chromatographic techniques resulted in the isolation of six known diterpenoids namely: 4-epi-abietol (1), ferruginol (2), hinokiol (3), sugiol (4), Z-communic acid (5) and hinokiol-1-one 3ß,12-dihydroxyabieta-8,11,13-triene-1-one) (6), in addition to the sesquiterpene 8α-acetoxyelemol (7). Both physical and spectral data were used for structure determination and all isolates were evaluated for their hepatoprotective activity. Compounds 1 and 4 were the most effective in reducing the elevated liver enzymes as indication for liver protection.

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Total alcohol and successive extracts (diethyl ether, chloroform, ethyl acetate and n-butanol) of Covolvulus hystrix Vahl were screened for their antioxidant activity using free radical 2-2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox as reference substance. The tested extracts showed variable antioxidant activities. The highest antioxidant activity was achieved in total alcohol (48.32%) followed by ethyl acetate (37.8%) and n-butanol (32.96%) extracts. From the ethyl acetate extract four compounds were isolated and identified as quercetin, quercetin 3-0-rutinoside and quercetin-7-O-rhamnoside in addition to ferulic acid using different spectroscopic analysis such as, 1HNMR, 13CNMR, COSY, HMBC and HMQC.

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The effect of ethanol extract from aerial parts of Cleome droserifolia was investigated against carbon tetrachloride induced liver injury. The hepatoprotective activity was evaluated through the quantification of biochemical parameters and confirmed using histopathology analysis. Efficient hepatoprotective effect was achieved by crude extract, fractions and some pure compounds. The phytochemical studies showed that the petroleum ether fraction afforded two known guaiane sesquiterpenes buchariol (1) and teucladiol (2) in addition to daucosterol (beta-sitosterol glucoside) (3). The CHCl(3) fraction afforded three known flavonoid derivatives; 5,3'-dihydroxy-3,6,7,4',5'-pentamethoxyflavone (4), 5'-hydroxy-3,6,7,3',4',5'-hexamethoxyflavone (5) and luteolin (6) and a known dolabellane diterpene (1R,2R,3E,7E,11R,12S)-2-O-acetyl-16-O-(3-hydroxy-3-methylglutaryl)-dolabella-3,7-dien-2,16,18-triol (7). The active parts of the MeOH fraction afforded the previously unreported 3'-methoxy-3,5,4'-trihydroxy flavone-7-neohesperidoside (8) and a known megastigmane norterpene; (6S,9R)-roseoside (9).

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