Perwez Alam

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.

Quantification of bioactive marker β-amyrin by validated high-performance thin-layer chromatographic- densitometric method in different species of Maytenus grown in Saudi Arabia

A simple and sensitive high-performance thin-layer chromatographic (HPTLC)-densitometric method was developed and validated for quantification of β-amyrin in the crude extracts of two species of Maytenus (Maytenus obscura and Maytenus parviflora) grown in Saudi Arabia. HPTLC-densitometry was performed on glass-backed silica gel 60 F254 TLC plates with the binary mobile phase hexane-ethyl acetate (3:1, v / v). The developed plate was derivatized with p-anisaldehyde, then scanned and quantified densitometrically at 550 nm. The system was found to give a compact spot for β-amyrin at RF value of 0.38 ± 0.01. The method was found to be satisfactory in terms of sensitivity, accuracy, precision, and recovery. The content of β-amyrin was estimated as 0.42% ± 0.01% and 0.88% ± 0.01% w / w in M. obscura and M. parviflora, respectively. The developed HPTLC technique can be very useful for the quantification of β-amyrin present in various medicinal plants.

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.

Therapeutic efficacy of ethanolic extract of Aerva javanica aerial parts in the amelioration of CCl4-induced hepatotoxicity and oxidative damage in rats.

Background Liver diseases, the fifth most common cause of global death, can be metabolic, toxin-induced, or infective. Though approximately 35 Saudi medicinal plants are traditionally used to treat liver disorders, the hepatoprotective potential of Aerva javanica has not been explored. Objective To investigate the antioxidative and hepatoprotective effect of Aerva javanica. Design Total ethanol extract of A. javanica aerial parts was prepared and tested on DCFH-toxicated HepG2 cells ex vivo, and in CCl4-injured Wistar rats in vivo. MTT assay was used to determine cell viability and the serum biochemical markers of liver injury as well as histopathology was performed. In vitro 1,1-diphenyl-2-picrylhydrazyl and β-carotene free-radical scavenging assay and phytochemical screening of the extract were done. Furthermore, A. javanica total extract was standardized and validated by high-performance thin layer chromatographic method. Results MTT assay showed that, while DCFH-injured cells were recovered to ~56.7% by 100 µg/ml of the extract, a 200 µg/ml dose resulted in hepatocytes recovery by ~90.2%. Oral administration of the extract (100 and 200 mg/kg.bw/day) significantly normalized the serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, gamma-glutamyl transferase, alkaline phosphatase, bilirubin, cholesterol, high-density lipoprotein, low-density lipoprotein, very-low-density lipoprotein, triglyceride, and malondialdehyde levels, including tissue nonprotein sulfhydryl and total protein in CCl4-injured rats. In addition, the histopathology of dissected liver also revealed that A. javanica cured the tissue lesion compared to silymarin treatment. In vitro assays revealed strong free-radical scavenging ability of the extract and presence of alkaloids, flavonoids, tannins, sterols, and saponins where rutin, a well-known antioxidant flavonoid, was identified. Conclusions Our findings demonstrate the potential of A. javanica in the attenuation of ex vivo and in vivo hepatotoxicity and oxidative damage. This further suggests its therapeutic value in various liver diseases. However, isolations of the active principles, their mechanisms of action, and other therapeutic contributions remain to be addressed.

Anti-inflammatory activity and qualitative analysis of different extracts of Maytenus obscura (A. Rich.) Cuf. by high performance thin layer chromatography method

OBJECTIVE To perform aqueous ethanol soluble fraction (AESF) and dichloromethane extract of aerial parts of Maytenus obscura (A. Rich.) Cuf. using high performance thin layer chromatography (HPTLC) and to test anti-inflammatory activity of these extracts. METHODS HPTLC studies were carried out using CAMAG HPTLC system equipped with Linomat IV applicator, TLC scanner 3, Reprostar 3, CAMAG ADC 2 and WIN CATS-4 software were used. The anti-inflammatory activity was tested by injecting different groups of rats (6 each) with formalin in hind paw and measuring the edema volume before and 1 h later formalin injection. Control group received saline i.p. The extracts treatment was injected i.p. in doses of 100 and 200 mg/kg 1 h before formalin administration. Indomethacin (30 mg/kg) was used as standard. RESULTS The results of preliminary phytochemical studies confirmed the presence of protein, lipid, carbohydrate, phenol, flavonoid, saponin, triterpenoid, alkaloid and anthraquinone in both extracts. Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with the green solvents toluene: ethyacetate: glacial acetic acid (5:3:0.2, v/v/v) as mobile phase. HPTLC finger printing of AESF revealed major eight peaks with Rf values in the range of 0.28 to 0.80 and the dichloromethane revealed major 11 peaks with Rf values in the range of 0.12 to 0.76. The purity of sample was confirmed by comparing the absorption spectra at start, middle and end position of the band. Treatment of rats (i.p.) with AESF and dichloromethane in doses of 100 and 200 mg/kg inhibited singnificantly (P<0.05, n=6) formalin-induced inflammation by 50%, 55.9%, 45.5%, and 51.4%, respectively. CONCLUSIONS HPTLC finger printing of AESF and dichloromethane of Maytenus obscura revealed eight major spots for alcoholic extracts and nine major spots for dichloromethane extracts. These HPTLC profiles may be of great usefulness in the quality control of herbal products containing these extracts. The anti-inflammatory activity of both extracts also revealed the medicinal importance of these extracts. The plant can be further explored for the isolation of phytoconstituents having anti-inflammatory activity.

Theoretical and experimental study on lipophilicity and wound healing activity of ginger compounds

OBJECTIVE To correlate the chromatographic and computational method to calculate lipophilicity of selected ginger compounds and to observe the effects of log P on wound healing. METHODS Mixtures of acetonitrile and water with acetonitrile content between 95% and 50% v/v in 5% increments were kept separately in 10 different chromatographic chambers, saturated with solvent for 2 h. Spots were observed under UV light at λ=254 nm p-anisaldehyde used as a spraying reagent. Theoretical calculation was done using the Alogps 2.1 online program at www.vcclab.org/lab/alogps. For percentage wound contraction, five groups of animal (mice) (25-30 g) of either sex were selected. Wound were created on dorsal surface of animals using toothed forceps, scalpel and pointed scissors. The wound areas were calculated using vernier caliper. After making wound mice were orally administered 35 mg/kg 6-shogoal, 6-gingerol, 8-gingerol and 10-gingerol respectively. Group E as the control group received tap water. RESULTS The lipophilicity values determined in thin layer chromatography were correlated with the theoretically calculated various log P by linear regression analysis. Significant correlations were found between log P values calculated by software program and the experimental reversed-phase thin-layer chromatography data. Order of wound healing property of ginger compounds is directly dependent on lipophilicity i.e. more lipophilic compound has highest activity. CONCLUSIONS Experimentally determined lipophilicity (R MO) values were correlated with log P determined by softwares and found satisfactory. Lipophilicity (R MO) is a useful parameter for the determination and prediction of biological activity of ginger compounds.

Quantitative analysis of rutin, quercetin, naringenin, and gallic acid by validated RP- and NP-HPTLC methods for quality control of anti-HBV active extract of Guiera senegalensis

Abstract Context: Guiera senegalensis J.F. Gmel (Combretaceae) is a folk medicinal plant used in various metabolic and infectious diseases. In addition to its antiviral activities against herpes and fowlpox, the anti-HBV efficacy is very recently reported. Objective: To develop and validate simple, sensitive RP-/NP-HPTLC methods for quantitative determination of biomarkers rutin, quercetin, naringenin, and gallic acid in the anti-HBV active G. senegalensis leaves ethanol-extract. Materials and methods: RP-HPTLC (rutin & quercetin; phase- acetonitrile:water, 4:6) and NP-HPTLC (naringenin & gallic acid; phase- toluene:ethyl acetate:formic acid, 6:4:0.8) were performed on glass-backed silica gel plates 60F254-RP18 and 60F254, respectively. The methods were validated according to the ICH guidelines. Results: Well-separated and compact spots (Rf) of rutin (0.52 ± 0.006), quercetin (0.23 ± 0.005), naringenin (0.56 ± 0.009) and gallic acid (0.28 ± 0.006) were detected. The regression equations (Y) were 12.434x + 443.49, 10.08x + 216.85, 11.253x + 973.52 and 11.082x + 446.41 whereas the coefficient correlations (r2) were 0.997 ± 0.0004, 0.9982 ± 0.0001, 0.9974 ± 0.0004 and 0.9981 ± 0.0001, respectively. The linearity ranges (ng/spot) were 200–1400 (RP-HPTLC) and 100–1200 (NP-HPTLC). The LOD/LOQ (ng/band) were 33.03/100.1 (rutin), 9.67/29.31 (quercetin), 35.574/107.8 (naringenin), and 12.32/37.35 (gallic acid). Gallic acid (7.01 μg/mg) was the most abundant biomarker compared to rutin (2.42 μg/mg), quercetin (1.53 μg/mg) and naringenin (0.14 μg/mg) in the extract. Conclusion: The validated NP-/RP-HPTLC methods were simple, accurate, and sensitive for separating and quantifying antiviral biomarkers in G. senegalensis, and endorsed its anti-HBV activity. The developed methods could be further employed in the standardization and quality-control of herbal formulations.

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.

HPTLC finger print and anti-inflammatory activity of ethanolic extract of different Maytenus species grown in Kingdom of Saudi Arabia

Objective To evaluate and compare the anti-inflammatory activity and to develop HPTLC fingerprint profile of ethanolic extract of Maytenus obscura (M. obscura) and Maytenus parviflora (M. parviflora).

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.