Nasir A. Siddiqui

Ameliorative effect of methanol extract of Rumex vesicarius on CCl4-induced liver damage in Wistar albino rats

Abstract Context: Rumex vesicarius L. (Polygonaceae), an edible plant, is reported to have many bioactive phytochemicals, especially flavonoids and anthraquinones with antioxidant and detoxifying properties. Objective: This study evaluated the methanolic extract of R. vasicarius (MERV) for hepatoprotective activity in rats against CCl4-induced liver damage. Materials and methods: The whole plant extract was prepared and investigated for its hepatoprotective activity. Rats were pretreated with MERV (100 and 200 mg/kg, p.o.) for 7 d prior to the induction of liver damage by CCl4. Animals were then sacrificed 24 h after CCl4 administration for the biochemical (AST, ALT, and ALP activity in serum; lipid peroxidation (LPO) and glutathione (GSH) levels in liver tissue) and histological analyses. Results: CCl4-induced hepatotoxicity was confirmed by an increase (p < 0.05) in serum AST (4.55-fold), ALT (3.51-fold), and ALP (1.82-fold) activities. CCl4-induced hepatotoxicity was also manifested by an increase (p < 0.05) in LPO (3.88-fold) and depletion of reduced glutathione (3.14-fold) activity in liver tissue. The multiple dose MERV administration at 200 mg/kg showed promising hepatoprotective activity as evident from significant decrease levels of serum AST (230.01 ± 13.21), serum ALT (82.15 ± 5.01), serum ALP (504.75 ± 19.72), hepatic LPO (3.38 ± 0.33), and increased levels of hepatic glutathione (0.34 ± 0.04) towards near normal. Further, biochemical results were confirmed by histopathological changes as compared with CCl4-intoxicated rats. Discussion and conclusion: The results obtained from this study indicate hepatoprotective activity of Rumex plant against CCl4-induced liver toxicity; hence, it can be used as a hepatoprotective agent.

Stability-indicating densitometric high-performance thin-layer chromatographic method for the quantitative analysis of biomarker naringin in the leaves and stems of Rumex vesicarius L.

A simple, sensitive, and stability-indicating high-performance thinlayer chromatography (HPTLC)-densitometric method was developed for the quantification of biomarker naringin in the methanol extracts of stems and leaves of Rumex vesicarius. Chromatography was performed on glass-backed silica gel 60 F254 high-performance thin-layer chromatography (HPTLC) plates with ethyl acetate- glacial acetic acid-MeOH-H2O (30:10:5:1, v/v) as mobile phase. Scanning and quantification were done at 275 nm. The system was found to give compact spot for naringin at RF = 0.46 ± 0.001. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.998 with respect to area in the concentration range of 100–1000 ng. The regression equation of standard was found to be Y = 3.438X + 38.485. Naringin was subjected to acid and alkali hydrolysis, peroxide oxidation, photodegradation, dry heat, moist heat, and ultraviolet (UV) treatment. The drug undergoes complete degradation under acidic treatment and mild degradation under basic and hydrogen peroxide treatment. The degraded products were well-separated from the pure drug. The statistical analysis proves that the developed method for quantification of naringin is reproducible and selective. Due to the ability of the method in separating naringin from other constituents including its degradation products, it can be employed as stability-indicating method for in-process as well as finished products in the market. It is for the first time that authors are reporting a complete stability-indicating densitometric HPTLC method for the estimation of biomarker naringin in the leaves and stems of R. vesicarius L.

Simultaneous Quantification of Biomarkers Bergenin and Menisdaurin in the Methanol Extract of Aerial Parts of Flueggea virosa by Validated HPTLC Densitometric Method

A simple, sensitive high-performance thin-layer chromatography (HPTLC) method was developed for the simultaneous quantification of biomarker bergenin and menisdaurin in the methanol extracts of aerial parts of Flueggea virosa (FVME). Chromatography was performed on glass-backed silica gel 60F254 HPTLC plates using dichloromethane: methanol as mobile phase. Scanning and quantification was done at UV absorption maxima of 260 nm. The system was found to give compact spot for bergenin and menisdaurin at Rf = 0.29 ± 0.01 and 0.16 ± 0.01, respectively. The linearity ranges for bergenin and menisdaurin were found to be the same (100-800 ng/spot) with correlation coefficients (R(2) values) of 0.997 and 0.999, respectively. The limit of detection for bergenin and menisdaurin was found to be 27 and 36.2 ng/band, respectively, while the limit of quantification was found to be 81 and 108 ng/band, respectively. Intra- and interday precisions (n = 6) for bergenin and menisdaurin were found to be 1.41-1.71 and 1.65-1.87%, and 1.68-1.89 and 1.75-1.93%, respectively. The percent recoveries were found to be 98.7-99.4 and 99.5-99.9%, respectively, for bergenin and menisdaurin. The percentage of bergenin and menisdaurin was found to be 15.25 and 4.22% (w/w), respectively, in FVME. The developed method permitted the simultaneous quantification of bergenin and menisdaurin and showed good resolution and separation from other constituents of extract; hence, the method can be used to standardize herbal formulations as well as bulk drugs for bergenin and menisdaurin.

Antioxidant potential of Rumex vesicarius L.: in vitro approach.

OBJECTIVE To assess in-vitro antioxidant activity of different fraction and perform high performance thin layer chromatography fingerprint analysis of most active fraction of Rumex vesicarius L. (R. vesicarius). METHODS In the present study, acetone, ethyl acetate, n-butanol, and methanol extracts of R. vesicarius were evaluated for radical scavenging activity by studying the inhibition of the level of lipid peroxidation induced by Fe(++)/ascorbate, DNA sugar damage, scavenging of hydrogen peroxide, diphenylphosphine DPPH radical scavenging activity, total phenolic content, total flavonoids content and total proanthocyanidin. High performance thin layer chromatography finger print profiling of R. vesicarius L. was also done. RESULTS Lipid peroxidation induced by the iron/ascorbate system, hydrogen peroxide, diphenylphosphine and DNA sugar damage were inhibited by the addition of different extract of R. vesicarius. Among them, methanolic extract showed maximum efficacy. The methanolic extract showed the highest total phenolic, total flavonoids and total proanthocyanidin contents. CONCLUSIONS The results suggest that the extracts can be a vital source of phytochemical antioxidants.

A stability-indicating assay of biomarker bergenin in the aerial parts of Flueggea virosa by a validated high-performance thin-layer chromatographic-densitometric method

A simple, sensitive, and stability-indicating thin-layer chromatographic (TLC)—densitometric method was developed for the assay of biomarker bergenin in the methanol extract of aerial parts of Flueggea virosa (FVME). Chromatography was performed on glass-backed silica gel 60 F254 high-performance thin-layer chromatographic (HPTLC) plates with dichloromethane–methanol (8.5:1.5, v/v) as the mobile phase. Scanning and quantification were done at 220 nm. The system was found to give compact spot for bergenin at RF = 0.29 ± 0.01. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.998 with respect to area in the concentration range of 100–800 ng. The regression equation of bergenin was found to be Y = 8.708X + 51.017. The limit of detection (LOD) and limit of quantification (LOQ) for bergenin were found to be 17.5 and 53 ng band−1, respectively. The percentage of bergenin was found to be 15.5% w/w in the FVME. Bergenin was subjected to acid and alkali hydrolysis, peroxide oxidation, photodegradation, dry heat, moist heat, and ultraviolet (UV) treatment for its stability studies. Treatment of bergenin with acid showed 21.25% degradation resulting in the formation of three degraded products at RF 0.01, 0.11, and 0.48, respectively. Bergenin showed 100% degradation with the formation of one degraded product appearing at RF 0.03 under alkali hydrolysis. Under hydrogen peroxide treatment, bergenin showed 17.76% degradation and the resultant degraded products appeared at RF 0.01 and 0.07. On treatment with dry heat, moist heat, photochemical, and UV (254 nm) light, bergenin showed no degradation products which suggested its stability under these conditions. The degraded products were found to be well separated from the pure drug. In this study, we report for the first time the quantification of bergenin in F. virosa by a validated HPTLC method. The stability study performed in this experiment may help in selecting or rejecting the in-process procedural conditions for making the formulation more efficacious and safe.

Comparative study of antioxidant activity and validated RP-HPTLC analysis of rutin in the leaves of different Acacia species grown in Saudi Arabia

The present study assessed the comparative antioxidant potential of the ethanol extract (EE) of leaves of four Acacia species (Acacia salicina, AS; Acacia laeta, AL; Acacia hamulosa AH; and Acacia tortilis, AT) grown in Saudi Arabia, including RP-HPTLC quantification of antioxidant biomarker rutin. In vitro DPPH radical scavenging and β-carotene-linoleic acid bleaching assays showed the promising antioxidant activities of Acacia extracts: ASEE (IC50: 60.39 and 324.65 μg/ml) >ALEE (IC50: 217.06 and 423.36 μg/ml) >ATEE (IC50: 250.13 and 747.50 μg/ml) >AHEE (IC50: 255.83 and 417.28 μg/ml). This was comparable to rutin tested at 500 μg/ml. Further, a RP- HPTLC densitometric method was developed (acetonitrile:water; 6:4; v/v) using glass-backed RP-18 silica gel F254 plate, and scanned at UV max 254 nm. The method was validated as per the ICH guidelines. Analysis of the validated RP-HPTLC displayed an intense peak (Rf = 0.65 ± 0.004) of rutin that was estimated (μg/mg dry weight) to be highest in ASEE (10.42), followed by ALEE (2.67), AHEE (1.36) and ATEE (0.31). Taken together, presence of rutin strongly supported the high antioxidant property of the tested Acacia species, especially Acacia salicina. The developed RP-HPTLC method therefore, affirms its application in the quality control of commercialized herbal drugs or formulation containing rutin.

Simultaneous separation of antihyperlipidemic drugs by green ultrahigh-performance liquid chromatography–diode array detector method: Improving the health of liquid chromatography

Statins in combination with fibrates show beneficial effects on the lipoprotein profile of patients because they have positive complimentary effects on lipid profile. A new green ultrahigh-performance liquid chromatography-diode array detector method for simultaneous analysis of simvastatin (SMV) and fenofibrate (FNF) in standard form, marketed formulations, and self-emulsifying drug delivery system formulations was developed and validated in the present investigation. The method utilized C18 as stationary phase and a combination of methanol:water (8:2) as an eluent. It was found that selected eluent provided short run time (2.5 minutes), better peak symmetry and satisfactory values of other chromatographic parameters such as resolution (Rs=2.325), capacity factor (k, 3.0 and 4.2 for SMV and FNF, respectively), selectivity (α =1.4), and number of theoretical plates (N, 4265 and 5285 for SMV and FNF, respectively). An excellent linear relationship (r2 0.998 and 0.997 for SMV and FNF, respectively) was observed for linear regression data for the calibration plots. The developed system was validated for accuracy, precision, robustness (˃ 2% for both drugs) and recovery (98-102% for both drugs). Results obtained from the statistical treatment of the values obtained for different parameters proved that the method is suitable, reproducible, and selective for the simultaneous analysis of SMV and FNF in bulk, marketed, and self-emulsifying drug delivery system formulations. The replacement of commonly applied toxic solvents with innocuous and environmentally benign solvents provides a better option than the more toxic processes in drug analysis.

New constituents triterpene ester and sugar derivatives from Panax ginseng Meyer and their evaluation of antioxidant activities

Panax ginseng C. A. Meyer (Araliaceae), is a well-known herb and used in the old established system of Oriental remedy, especially in Japan, China and Korea. Four new compounds characterized as (cis)- 7β,11α,19,21-tetra-O-decanoyl-18, 22β-dihydroxy-dammar-1-en-3-one (1), 3β,4α,12β-trihydroxystigmast-5-en-21-yl octadecan-9′,12′-dienoate (2), dammar-12, 24-dien-3α, 6β, 15α-triol-3α-D-arabinopyranosyl-6β-L-arabinopyranoside (3) and dammar-24-en-3α, 6β, 16α, 20β-tetraol-3α-D-arabinopyranosyl-6β-D-arabinopyranoside (4) were isolated and established from the ethyl acetate and butanol extracts of the roots of P. ginseng. Their structures were established on the basis of spectral data and chemical reactions. Natural compounds indicative a great reservoir of materials and compounds with evolved biological activity, including antioxidant. Compounds 1–4 were investigated in vitro for antioxidant potential using ferric reducing antioxidant power (FRAP), the Nitric oxide (NO) scavenging activity, reducing power, phosphomolybdenum and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging actions, and the decision showed the compounds 3and 4 have probablyessential antioxidant properties than the compounds 1and 2 presented weak activity.

Concurrent analysis of bioactive triterpenes oleanolic acid and β-amyrin in antioxidant active fractions of Hibiscus calyphyllus, Hibiscus deflersii and Hibiscus micranthus grown in Saudi Arabia by applying validated HPTLC method

In this study, we developed a validated HPTLC method for concurrent analysis of two natural antioxidant triterpenes, oleanolic acid (OA) and β-amyrin (BA) in the biologically active fractions (petroleum ether, toluene, chloroform, ethyl acetate and n-butanol) of aerial parts of three Hibiscus species (H. calyphyllus, H. deflersii and H. micranthus). The chromatography was conducted on normal HPTLC (ready to use glass-plate coated with silica gel 60 F254) plate with chloroform and methanol (97:3, V/V) used as mobile phase. The derivatization of the developed plate was done with p-anisaldehyde and scanned at λmax = 575 nm. Well resolved and intense peaks of OA and BA were obtained at Rf = 0.36 and 0.57, respectively. The linear regression equation/correlation coefficient (r2) for OA and BA were Y = 6.65x + 553.35/0.994 and Y = 9.177x + 637.23/0.998, respectively in the linearity range of 100–1200 ng/spot indicated good linear relationship. The low values of %RSD for intra-day/inter-day precision of OA (1.45–1.61/1.38–1.59) and BA (1.52–1.57/1.50–1.53) suggested that the method was precise. The recovery/RSD (%) values for OA and BA were found to be 99.21–99.62/1.39–1.95 and 98.75–99.70/1.56–1.80, respectively assures the reasonably good accuracy of the proposed method. Fifteen samples were analyzed to check the content of OA and BA by using the developed HPTLC methods. The content of OA in different samples were found to be 3.87 (HmP) > 1.212 (HcP) > 0.673 (HdC) > 0.493 (HdP) > 0.168 (HdE) > 0.059 (HcC) > 0.015 (HcE) > 0.008 (HmT) µg/mg of the dried weight of extract. However the content of BA was found as: 2.293 (HmP) > 1.852 (HdT) > 0.345 (HdC) > 0.172 (HmT) > 0.041 (HdE) > 0.008 (HcC) µg/mg of the dried weight of extract. Some Hibiscus species fractions exhibited good antioxidant potential like: HcE (IC50 = 17.6 ± 1.8) > HdB (IC50 = 32.16 ± 0.9) > HmP (IC50 = 80.4 ± 4.5) > HmT (IC50 = 99.7 ± 8.2) when compared with ascorbic acid (IC50 = 14.2 ± 0.5), while other fractions exhibited only mild antioxidant capability. The developed HPTLC method can be further exploited for analysis of these markers in the quality assessment of raw material as well as herbal formulations available in the market.

Development and Validation of a High-Performance Thin-Layer Chromatographic Method for the Determination of Biomarker β-Amyrin in the Leaves of Different Ficus Species

A simple and sensitive high-performance thin-layer chromatographic (HPTLC) method was developed for the evaluation of biomarker β-amyrin in the leaves of five different species of genus Ficus (Ficus carica, Ficus nitida, Ficus ingens, Ficus palmata, and Ficus vasta) grown in the Kingdom of Saudi Arabia. Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with solvents toluene-methanol (9:1, v/v) as the mobile phase. After development, the HPTLC plate was derivatized with p-anisaldehyde reagent to give well-resolved and compact spot of β-amyrin. Scanning and quantification were done at 550 nm. The system was found to give compact spot for β-amyrin at RF = 0.58. The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.998 with respect to area in the concentration range of 100–900 ng. The regression equation for β-amyrin standard was found to be Y = 5.835X + 87. The precisions (n = 6) for β-amyrin were found to be 1.64–1.77% and 1.68–1.84%, respectively, for intra-day and inter-day batches, and the recovery values were found to be 97.6–98.3%. β-Amyrin was found to be present in three species, i.e., F. carica (0.29% , w/w), F. nitida (0.54% w/w), and F. palmata (0.31%, w/w), while it was absent in F. vasta and F. ingens. The statistical analysis proves that the developed method for the quantification of β-amyrin is reproducible; hence, it can be employed for the determination of β-amyrin in plasma and other biological fluids as well as in finished products available in the market.