Development and validation of UPLC-PDA method for concurrent analysis of bergenin and menisdaurin in aerial parts of Flueggea virosa (Roxb. ex Willd.).

Bergenin and menisdaurin are biologically active components which are found in plant Flueggea virosa (Phyllanthaceae). Bergenin has pharmacological actions such as chemopreventive and antihepatotoxic while menisdaurin has an anti-viral activity which needs its evaluation by an analytical method (UPLC-PDA method) that can be applied to the quality control of pharmaceutical preparations. The developed UPLC-PDA method was applied for identification and quantification of standards bergenin and menisdaurin in the methanol extract of F. virosa (FVME). The analysis was carried out using Eclipse C18 (4.6 × 100 mm, 3.5 µm) UPLC column. The optimized chromatographic condition was achieved at 0.16 mL/min flow rate using gradient system with acetonitrile and water as mobile phase. Both biomarkers were measured at λmax 235 nm in PDA detector at ambient temperature. The developed method furnished sharp and intense peaks of menisdaurin and bergenin at Rt = 2.723 and 3.068 min, respectively along with r2 > 0.99 for both. The recoveries of bergenin and menisdaurin were found in the range of 99.37-101.49% and 98.20-100.08%, respectively. With other validation data, including precision, specificity, accuracy, and robustness, this method demonstrated excellent reliability and sensitivity. The separation parameters i.e. retention, separation, and resolution factors for resolved standards (bergenin and menisdaurin) were >1, which showed good separation. The quantity of bergenin and menisdaurin in the FVME sample was found as 15.16 and 3.28% w/w, respectively. The developed UPLC-PDA method could be conveniently adopted for the routine quality control analysis.

In vitro evaluation of novel antiviral activities of 60 medicinal plants extracts against hepatitis B virus.

Currently, >35 Saudi Arabian medicinal plants are traditionally used for various liver disorders without a scientific rationale. This is the first experimental evaluation of the anti-hepatitis B virus (HBV) potential of the total ethanolic and sequential organic extracts of 60 candidate medicinal plants. The extracts were tested for toxicity on HepG2.2.15 cells and cytotoxicity concentration (CC50) values were determined. The extracts were further investigated on HepG2.2.15 cells for anti-HBV activities by analyzing the inhibition of HBsAg and HBeAg production in the culture supernatants, and their half maximal inhibitory concentration (IC50) and therapeutic index (TI) values were determined. Of the screened plants, Guiera senegalensis (dichloromethane extract, IC50=10.65), Pulicaria crispa (ethyl acetate extract, IC50=14.45), Coccinea grandis (total ethanol extract, IC50=31.57), Fumaria parviflora (hexane extract, IC50=35.44), Capparis decidua (aqueous extract, IC50=66.82), Corallocarpus epigeus (total ethanol extract, IC50=71.9), Indigofera caerulea (methanol extract, IC50=73.21), Abutilon figarianum (dichloromethane extract, IC50=99.76) and Acacia oerfota (total ethanol extract, IC50=101.46) demonstrated novel anti-HBV activities in a time- and dose-dependent manner. Further qualitative phytochemical analysis of the active extracts revealed the presence of alkaloids, tannins, flavonoids and saponins, which are attributed to antiviral efficacies. In conclusion, P. crispa, G. senegalensis and F. parviflora had the most promising anti-HBV potentials, including those of C. decidua, C. epigeus, A. figarianum, A. oerfota and I. caerulea with marked activities. However, a detailed phytochemical study of these extracts is essential to isolate the active principle(s) responsible for their novel anti-HBV potential.

Rhanterium epapposum Oliv. essential oil: Chemical composition and antimicrobial, insect-repellent and anticholinesterase activities.

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.

Inter-species comparative antioxidant assay and HPTLC analysis of sakuranetin in the chloroform and ethanol extracts of aerial parts of Rhus retinorrhoea and Rhus tripartita.

CONTEXT: Extensive research on Rhus (Anacardiaceae) shows their antioxidant potential, which warrants further evaluation of its other species. OBJECTIVE: To perform a comparative antioxidant assay on extracts of R. retinorrhoea and R. tripartita, including sakuranetin quantification by a validated HPTLC method. MATERIALS AND METHODS: In vitro antioxidant assay was performed on chloroform and ethanol extracts of R. retinorrhoea Steud. ex Oliv. (RRCE and RREE) and R. tripartita (Ucria) Grande (RTCE and RTEE) by DPPH radical scavenging (at 31.25, 62.5, 125, 250 and 500 µg/mL concentrations) and ß-carotene-linoleic acid bleaching methods at 500 µg/mL concentration. Densitometric HPTLC method was developed and validated using toluene: ethyl acetate: methanol (8:2:0.2; v/v/v) as mobile phase, executed on glass-backed silica gel F254 plate and scanned at 292 nm. RESULTS: Antioxidant activity of Rhus extracts tested by the two methods (DPPH/BCB) was found in order of RTEE > RREE > RTCE > RRCE with IC50 118.67/256.26, 315.75/82.35, 827.92/380.0 and 443.69/292.75, respectively. Scanning of the HPTLC plate provided an intense peak of sakuranetin at Rf = 0.59. The estimated sakuranetin content in the dry weight of the extracts was highest in RREE (27.95 µg/mg) followed by RRCE (25.22 µg/mg), RTEE (0.487 µg/mg) and RTCE (0.0 µg/mg). Presence of sakuranetin in RREE, RRCE and RTEE supported the highest antioxidant property of the two Rhus species. Nonetheless, low sakuratenin in R. tripartita indicated the presence of other bioactive constituents responsible for synergistic antioxidant activity. CONCLUSION: The developed HPTLC method therefore guarantees its application in quality control of commercialized herbal drugs and formulations containing sakuranetin.

Studies on the Red Sea Sponge Haliclona sp. for its Chemical and Cytotoxic Properties.

BACKGROUND: A great number of novel compounds with rich chemical diversity and significant bioactivity have been reported from Red Sea sponges. OBJECTIVE: To isolate, identify, and evaluate the cytotoxic activity of the chemical constituents of a sponge belonging to genus Haliclona collected from the Eastern coast of the Red Sea. MATERIALS AND METHODS: The total ethanolic extract of the titled sponge was subjected to intensive chromatographic fractionation and purification guided by cytotoxic bioassay toward various cancer cell lines. The structures of the isolated compounds were elucidated using spectroscopic techniques including one-dimension and two-dimension nuclear magnetic resonance, mass spectrometry, ultraviolet, and infrared data, as well as comparison with the reported spectral data for the known compounds. X-ray single-crystal structure determination was performed to determine the absolute configuration of compound 4. The screening of antiproliferative activity of the compounds was carried on three tumor cell lines, namely the human cervical cancer (HeLa), human hepatocellular carcinoma (HepG2), and human medulloblastoma (Daoy) cells using MTT assay. RESULTS: This investigation resulted in the isolation of a new indole alkaloid, 1-(1H-indol-3-yloxy) propan-2-ol (1), with the previously synthesized pyrrolidine alkaloid, (2R, 3S, 4R, 5R) pyrrolidine-(1-hydroxyethyl)-3,4-diol hydrochloride (4), isolated here from a natural source for the first time. In addition, six known compounds tetillapyrone (2), nortetillapyrone (3), 2-methyl maleimide-5-oxime (5), maleimide-5-oxime (6), 5-(hydroxymethyl) dihydrofuran-2 (3H)-one (7), and ergosta-5,24 (28)-dien-3-ol (8) were also identified. Most of the isolated compounds exhibited weak cytotoxic activity against HepG-2, Daoy, and HeLa cancer cell lines. CONCLUSION: This is the first report of the occurrence of the indole and pyrrolidine alkaloids, 1-(1H-indol-2-yloxy) propan-2-ol (1), and the - (1-hydroxyethyl)-3,4-diol hydrochloride (4), in the Red Sea Haliclona sp. SUMMARY: From the Red Sea Haliclona sp. two alkaloids with indole and pyrrolidine nuclei, 1-(1H-indol-2-yloxy) propan-2-ol-(1) and pyrrolidine-(1-hydroxyethyl)-3,4-diol hydrochloride (4) were isolated and fully characterized; in addition to six known compounds (2, 3, 5-8)The absolute configuration and the three-dimension stereo-molecular structure of compound 4 were determined by X-ray crystallographyThe different extracts and isolated compounds showed weak cytotoxic activity against HepG-2, Daoy, and HeLa cancer cell lines.

Densitometric validation and analysis of biomarker ß-amyrin in different Acacia species (leaves) grown in Kingdom of Saudi Arabia by high performance thin-layer chromatography.

Biomarker ß-amyrin was analyzed in the leaves of four different Acacia species (A. salicina, A. loreta, A. hamulosa and A. tortilis) grown in Kingdom of Saudi Arabia by a validated HPTLC method. The chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates using solvents toluene: methanol (9:1, v/v) as mobile phase. The developed TLC plate was derivatized with anisaldehyde and scanned at 520 nm. A sharp peak of ß-amyrin was found at Rf=0.58±0.01. The r2 and the linear regression equation for ß-amyrin was found to be 0.991 and 19.913X+107.803, respectively in the concentration range of 100-800 ng. The percentage of ß-amyrin was found to be maximum 2.70% w/w in A. tortilis, 1.85% w/w in A. loreta and 1.80% w/w in A. hamulosa while it was totally absent in A. salicina. This study conceives maiden reporting of quantification of ß-amyrin in four different species of Acacia by validated HPTLC method. The developed method for the analysis of ß-amyrin was proved to be reproducible by statistical analysis hence it can be employed for further analysis of ß-amyrin in plasma, other biological fluids and in finished products available in the market.

Isolation and characterization of a new oxygenated homoditerpenoid from leaves of Centaurothamnus maximus with antimicrobial potential.

A new bioactive oxygenated homoditerpenic compound along with one known compound from the antimicrobial active ethanol extract of leaves of an endemic plant Centaurothamnus maximus was isolated. The n -hexane, dichloromethane, ethyl acetate and ethanol fractions of C. maximus leaves were evaluated for their antimicrobial potential by using standard agar well diffusion method against various microorganisms viz. B. subtilis, S. aureus, E. coli, P. aeruginosa, C. albicans and M. smegmatis. The results revealed that only ethanol extract was active against all microbes except the fungus C. albicans. A new compound 2α, 3α-dihydroxy-8α-methoxy-15-hydroxy-methylene- pimar-5,9 (11)-diene (CM-1) was isolated along with a known compound α-D-xylose (CM-2) from ethanol extract by reverse phase (RP-18) column chromatography and 1D and 2D NMR (DEPT, COSY, HMBC and HSQC) aided by EIMS mass and IR spectra were used to establish the structure. CM-1 was found to be active against B. subtilis, S. aureus and M. smegmatis (P>0.005) at MIC 20 µg/ml. Findings of this study may provide a lead for synthesis of more potent antimicrobial agents to serve the humanity against multidrug-resistant bacterial infections.

A novel ß-lactam derivative, albactam from the flowers of Albizia lebbeck with platelets anti-aggregatory activity in vitro.

A novel ß-lactam derivative, albactam, was isolated from the alcoholic extract of the flowers of Albizia lebbeck. It showed a significant anti-aggregatory activity against adenosine diphosphate and arachidonic acid induced guinea-pigs' platelets aggregation in vitro. Six more known compounds were also isolated and fully characterized by measuring 1D and 2D NMR, two of them are the triterpenes ß-amyrin and 11α, 12α-oxidotaraxerol, two ceramide derivatives and two flavonoids, kampferol 3-O-rutinoside and rutin.

Molecular mechanisms that underlie the sexual stimulant actions of Avicennia marina (Forssk.) Vierh. and Crocus sativus L.

The effects of extracts and sub-fractions of Avicennia marina, Crocus sativus and sildenafil on the sexual behavior of male rats and their effects on the intracavernosal pressure (I.CV), intracavernosal cyclic GMP and dihydrotestosterone plasma level were examined. The sexual behavior was followed for four hours using infra-red video cameras to quantify the effects on various male sexual behaviors. The results revealed that the active sub-fraction in case of A. marina was the hexane fraction of the chloroform extracts (C/H) whereas that of C. sativus was the hexane fraction of the alcoholic extract (A/H). (C/H), (A/H) and sildenafil significantly increased the total sexual stimulation index from 53.8±2.7 (control) to 406±7.8, 225±4 and 401±30.1, respectively (P<0.001, N=6). They significantly increased the index of successful mounting and ejaculation from 2.6±0.5 (control) to 40±2.7, 21±2.3 and 18±1.7, respectively (P<0.01, N=6). They significantly increased the cyclic GMP level from 0.94±0.07 (control) to 3.1±0.13, 1.59±0.11 and 3.66±0.19 ng/mg wet tissue, respectively (P<0.05, N=7). They did not affect dihydrotestosterone plasma level. (C/H), (A/H) and sildenafil increased the (I.CV) pressure by 4.8±0.3, 1.4±0.8 and 4.2±0.9 mmHg. The (C/H) seemed to be more active than sildenafil and twice active than (A/H). Both extracts and sildenafil acted via an increase in cyclic GMP.

Quantification of glycyrrhizin in anti-stress herbal formulations by validate HPTLC method: a rational paradigm towards quality control of herbals.

In the present study an analytical method of high-performance thin-layer chromatography (HPTLC) has been developed for quantification of glycyrrhizin for marketed antistressliquorice root capsules (LRC) and herbal tea (HT). Chromatography was performed by using mobile phase ethyl acetate (EA): glacial acetic acid (GAA): Methanol (MeOH): water (H(2)O) in proportion of 6:2:2:1, v/v/v/v. The developed plate was scanned and quantified densitometrically at absorption maxima 254nm. The method was validated for various analytical parameters viz. precision, accuracy, recovery, robustness, specificity, detection and quantification limits. The developed system was found to give compact spot for glycyrrhizin (R(f)= 0.33± 0.001). The linearity relationship was described by the equation Y=6.841X+ 70.428. The limit of detection (34 ng band(-1)), limit of quantification (101 ng band(-1)), recovery (99.4-99.8%), and precision (<1.84% and <1.62%; intraday and interday, respectively) were found satisfactory for glycyrrhizin. Linearity range for glycyrrhizin was 100-600ng (r(2)=0.998). The amount of glycyrrhizin was estimated by comparing the peak area of standard and the same was present in crude extract. The content of glycyrrhizin was estimated as 11.4% and 4.7% w/w in sample LRC and HT, respectively. The proposed method will be useful to quantify the therapeutic dose of glycyrrhizin in herbal formulations as well as in bulk drug.

Phytochemical and pharmacological study of Ficus palmata growing in Saudi Arabia.

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.

Phytochemical and pharmacological study of Ficus cordata growing in Saudi Arabia.

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.

Scropolioside-D2 and harpagoside-B: two new iridoid glycosides from Scrophularia deserti and their antidiabetic and antiinflammatory activity.

Five iridoid glycosides, including the two new compounds scropolioside-D(2) (1) and harpagoside-B (2), were isolated from the aerial parts of Scrophularia deserti DEL (Scrophulariaceae). Their structures were elucidated on the basis of spectral data to be 6-O-[2",4"-di-O-acetyl-3"-O-trans-cinnamoyl)-alpha-L-rhamnopyranosyl]-8 alpha-hydroxymethyl-1 alpha,5 beta,6 alpha,7 alpha,9 beta-pentahydro-7(8)-epoxy-2-oxaind-3-ene-1-O-beta-D-glucopyranoside-6'-O-acetate (1) and 5-O-beta-hydroxy-8-O-beta-trans-cinnamoyl-8 alpha-methyl-1,6,7,9-tetrahydro-2-oxaind-3-ene-1-O-beta-D-glucopyranoside (2), respectively. In addition, three more iridoid glycosides, scropolioside-D (3), koelzioside (4), and 8-O-acetyl-harpagide (5), were also isolated and characterized from this source. The biological activity and the structure activity relationship of the compounds were also studied, and scropolioside-D (3) and harpagoside-B (2) were found to possess significant antidiabetic and antiinflammatory activity, respectively.