Amukkara curanam, a Siddha formulation, currently used in all types of gastric disorders, rheumatic pain, insomnia and sexual insufficiency, was investigated for the estimation of the marker compounds, withaferine A and piperine contents in a prepared standard formulation and a commercial formulation by using HPTLC method of analysis. The two formulations were subjected to methanol, ethyl acetate and chloroform extractions by using Soxhhlet apparatus The chromatogram was developed using chloroform: methanol (8.5:1.5 v/v) and toluene: ethyl acetate (7:3 v/v) as mobile phases for the estimation of withferine A and piperine respectively. The detection and quantification were performed at a wavelength of 220 nm for withaferine A and 254 nm for piperine. The linear regression analysis of calibration plots of withferine A and piperine exhibited linear relationship in the range of 5 – 15 µg and 50 – 150 ng respectively, while the % recovery was found to be 94.52% w/w of withaferine A and 98.73%w/w of piperine, thus proving the accuracy and precision of the analysis. Methanol and ethyl acetate were found to be the suitable solvents for the extraction of withaferin A and piperine respectively. The withaferine A content in standard formulation was found to be much higher in all the three extracts than that of the commercial sample. However, the piperine content in all the three extracts of standard formulation was slightly lower than the respective extracts of commercial formulation. The proposed HPTLC method was found to be rapid, simple and accurate for quantitative estimation of withferine A and piperine in different formulation extracts.
HPTLC; Amukkara Curanam (AKC); Withaferine A; Piperine; Standard Formulation (SF); Commercial Formulation (CF)
The aim of the present study was to standardize Brahmi vati (BV) by simultaneous quantitative estimation of Bacoside A3 and Piperine adopting HPLC–UV method. BV very important Ayurvedic polyherbo formulation used to treat epilepsy and mental disorders containing thirty eight ingredients including Bacopa monnieri L. and Piper longum L.
Materials and methods
An HPLC–UV method was developed for the standardization of BV in light of simultaneous quantitative estimation of Bacoside A3 and Piperine, the major constituents of B. monnieri L. and P. longum L. respectively. The developed method was validated on parameters including linearity, precision, accuracy and robustness.
The HPLC analysis showed significant increase in amount of Bacoside A3 and Piperine in the in-house sample of BV when compared with all three different marketed samples of the same. Results showed variations in the amount of Bacoside A3 and Piperine in different samples which indicate non-uniformity in their quality which will lead to difference in their therapeutic effects.
The outcome of the present investigation underlines the importance of standardization of Ayurvedic formulations. The developed method may be further used to standardize other samples of BV or other formulations containing Bacoside A3 and Piperine.
Brahmi vati; Bacoside A3; Piperine; HPLC
To investigate the effects of piperine, a major pungent alkaloid present in Piper nigrum and Piper longum, on the tumor growth and metastasis of mouse 4T1 mammary carcinoma in vitro and in vivo, and elucidate the underlying mechanisms.
Methods: Growth of 4T1 cells was assessed using MTT assay. Apoptosis and cell cycle of 4T1 cells were evaluated with flow cytometry, and the related proteins were examined using Western blotting. Real-time quantitative PCR was applied to detect the expression of matrix metalloproteinases (MMPs). A highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model was used to evaluate the in vivo antitumor activity. Piperine was injected into tumors every 3 d for 3 times.
Results: Piperine (35–280 μmol/L) inhibited the growth of 4T1 cells in time- and dose-dependent manners (the IC50 values were 105±1.08 and 78.52±1.06 μmol/L, respectively, at 48 and 72 h). Treatment of 4T1 cells with piperine (70–280 μmol/L) dose-dependently induced apoptosis of 4T1 cells, accompanying activation of caspase 3. The cells treated with piperine (140 and 280 μmol/L) significantly increased the percentage of cells in G2/M phase with a reduction in the expression of cyclin B1. Piperine (140 and 280 μmol/L) significantly decreased the expression of MMP-9 and MMP-13, and inhibited 4T1 cell migration in vitro. Injection of piperine (2.5 and 5 mg/kg) dose-dependently suppressed the primary 4T1 tumor growth and injection of piperine (5 mg/kg) significantly inhibited the lung metastasis.
Conclusion: These results demonstrated that piperine is an effective antitumor compound in vitro and in vivo, and has the potential to be developed as a new anticancer drug.
anticancer drug; piperine; 4T1 breast cancer; apoptosis; cell cycle; metastasis; MMP-9; MMP-13
A simple, selective, precise, and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for analysis of cefpodoxime proxetil both in bulk and in pharmaceutical formulation has been developed and validated.
Materials and Methods:
The method employed HPTLC aluminum plates precoated with silica gel 60 RP-18 F254 as the stationary phase. The solvent system consisted of toluene:methanol:chloroform (4:2:4 v/v). The system was found to give compact spot for cefpodoxime proxetil (Rf value of 0.55 ± 0.02). Densitometric analysis of cefpodoxime proxetil was carried out in the absorbance mode at 289 nm.
The linear regression analysis data for the calibration plots showed good linear relationship, with r2 = 0.998 ± 0.0015 with respect to peak area in the concentration range of 100–600 ng per spot. The mean value±SD of slope and intercept were 3.38 ± 1.47 and 986.9 ± 108.78 with respect to peak area. The method was validated for precision, recovery, and robustness. The limits of detection and quantification were 3.99 and 12.39 ng per spot, respectively. Cefpodoxime proxetil was subjected to acid and alkali hydrolysis, oxidation, and thermal degradation. The drug undergoes degradation under acidic and basic conditions, indicating that the drug is susceptible to both acid and base. The degraded product was well resolved from the pure drug, with significantly different Rf value. Statistical analysis proves that the method is repeatable, selective, and accurate for the estimation of the investigated drug.
The proposed HPTLC method can be applied for identification and quantitative determination of cefpodoxime proxetil in both bulk drug and pharmaceutical formulation.
Cefpodoxime proxetil; degradation; High performance thil layer chromatography (HPTLC); stability; validation
PURPOSE: Piperine, a major alkaloid of black pepper (Piper nigrum) and long pepper (Piper longum), was shown to have anti-inflammatory activity through the suppression of cyclooxygenase (COX)-2 gene expression and enzyme activity. It is also reported to exhibit anti-platelet activity, but the mechanism underlying this action remains unknown. In this study, we investigated a putative anti-platelet aggregation mechanism involving arachidonic acid (AA) metabolism and how this compares with the mechanism by which it inhibits macrophage inflammatory responses; METHODS: Rabbit platelets and murine macrophage RAW264.7 cells were treated with piperine, and the effect of piperine on the activity of AA-metabolizing enzymes, including cytosolic phospholipase A2 (cPLA2), COX-1, COX-2, and thromboxane A2 (TXA2) synthase, as well as its effect on AA liberation from the plasma membrane components, were assessed using isotopic labeling methods and enzyme immunoassay kit; RESULTS: Piperine significantly suppressed AA liberation by attenuating cPLA2 activity in collagen-stimulated platelets. It also significantly inhibited the activity of TXA2 synthase, but not of COX-1, in platelets. These results suggest that piperine inhibits platelet aggregation by attenuating cPLA2 and TXA2 synthase activities, rather than through the inhibition of COX-1 activity. On the other hand, piperine significantly inhibited lipopolysaccharide-induced generation of prostaglandin (PG)E2 and PGD2 in RAW264.7 cells by suppressing the activity of COX-2, without effect on cPLA2; CONCLUSION: Our findings indicate that piperine inhibits platelet aggregation and macrophage inflammatory response by different mechanisms.
pepper; piperine; platelet aggregation; arachidonic acid; cyclooxygenase; phospholipase A2; thromboxane A2 synthase; prostaglandins
A sensitive, selective, precise and stability-indicating high-performance thin-layer chromatographic (HPTLC) method for analysis of repaglinide both as a bulk drug and in nanoemulsion formulation was developed and validated. The method employed TLC aluminum plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of chloroform/methanol/ammonia/glacial acetic acid (7.5:1.5:0.9:0.1, v/v/v/v). This system was found to give compact spots for repaglinide (Rf value of 0.38 ± 0.02). Repaglinide was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment. Also, the degraded products were well separated from the pure drug. Densitometric analysis of repaglinide was carried out in the absorbance mode at 240 nm. The linear regression data for the calibration plots showed good linear relationship with r2= 0.998 ± 0.032 in the concentration range of 50-800 ng. The method was validated for precision, accuracy as recovery, robustness and specificity. The limits of detection and quantitation were 0.023 and 0.069 ng per spot, respectively. The drug undergoes degradation under acidic and basic conditions, oxidation and dry heat treatment. All the peaks of the degraded product were resolved from the standard drug with significantly different Rf values. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability-indicating one. Moreover, the proposed HPTLC method was utilized to investigate the degradation kinetics in 1M NaOH.
Degradation; high-performance thin-layer chromatography; repaglinide; stability-indicating
A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the simultaneous estimation of valsartan and hydrochlorothiazide in combined dosage forms. The stationary phase used was precoated silica gel 60F254. The mobile phase used was a mixture of chloroform: methanol: toluene: glacial acetic acid (6:2:1:0.1 v/v/v/v). The detection of spots were carried out at 260 nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 300 to 800 ng/spot for valsartan and 100 to 600 ng/spot for hydrochlorothiazide. The limit of detection and the limit of quantification for the valsartan were found to be 100 and 300 ng/spot respectively and for hydrochlorothiazide 30 and 100 ng/spot respectively. The proposed method can be successfully used to determine the drug content of marketed formulation.
Valsartan; hydrochlorothiazide; simultaneous estimation; HPTLC
Mucuna pruriens Linn. is an important medicinal plant used for treatment of Parkinson's disease and many others in ancient Indian medical system. L-DOPA extracted from seeds of Mucuna is a constituent of more than 200 indigenous drug formulations and is more effective as drug than the synthetic counterpart. A densitometric high performance thin-layer chromatographic (HPTLC) method was developed for quantification of L-DOPA content present in the seeds extract. The method involves separation of L-DOPA on precoated silica gel 60 GF254 HPTLC plates using a solvent system of n-butanol-acetic-acid-water (4:1:1, v/v) as the mobile phase. Quantification was done at 280 nm using absorbance reflectance mode. Linearity was found in the concentration range of 100 to 1000 ng/spot with the correlation coefficient value of 0.9980. The method was validated for accuracy, precision and repeatability. Mean recovery was 100.89%. The LOD and LOQ for L-DOPA determination were found to be 3.41 ng/spot and 10.35 ng/spot respectively. The proposed HPTLC method was found to be precise, specific and accurate for quantitative determination of L-DOPA. It can be used for rapid screening of large germplasm collections of Mucuna pruriens for L-DOPA content. The method was used to study variation in fifteen accessions of Mucuna germplasm collected from different geographical regions.
Germplasm; high performance thin layer chromatography; L-DOPA; linearity; Mucuna pruriens
A simple, selective, precise high-performance thin-layer chromatographic method for simultaneous determination of amlodipine besylate and metoprolol succinate in bulk and pharmaceutical combined dosage form was developed and validated. The method employed HPTLC aluminum plates precoated with silica gel 60F-254 (10×10) as the stationary phase. The solvent system consisted of toluene:ethyl acetate:methanol:triethylamine (4:1:1:0.4 v/v/v). The system was found to give a compact spot for amlodipine besylate (Rf = 0.39±0.02) and metoprolol succinate (Rf = 0.59±0.02). Densitometric analysis of amlodipine besylate and metoprolol succinate was carried out in the absorbance mode at 254 nm. Linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.9990±0.0013 with respect to peak area in the concentration range 400-1400 ng per spot for amlodipine besylate and r2 = 0.9993±0.0013 with respect to peak area in the concentration range 3800–13300 ng per spot for metoprolol succinate. The method was validated for precision, recovery and robustness. The limits of detection and quantitation were 39.99 and 121.20 ng per spot for amlodipine besylate and 234.31 and 710.03 ng per spot for metoprolol succinate, respectively. Statistical analysis proved that the method is selective, precise and accurate for the estimation of amlodipine and metoprolol.
Amlodipine besylate; HPTLC; metoprolol succinate; pharmaceutical formulation
A simple, precise, accurate, and rapid high-performance thin layer chromatographic method has been developed and validated for the simultaneous quantitation of flunarizine dihydrochloride and propranolol hydrochloride in a combined capsule dosage form. The method was carried out on precoated silica gel 60 F254 TLC aluminum plate, (20×10 cm2). The solvent system was ethyl acetate:methanol:glacial acetic acid in the proportion of 8:1:1, (v/v/v). Rf value for flunarizine dihydrochloride and propranolol hydrochloride was found to be 0.62±0.02 and 0.18±0.02, respectively. The linearity regression analysis for calibration showed 0.999 and 0.999 for flunarizine dihydrochloride and propranolol hydrochloride with respect to peak area and height in the concentration range of 50-350 ng/spot and 500-3500 ng/spot, respectively. Accuracy of recovery studies was found to be 98-100.28 and 99.11-99.45% for flunarizine dihydrochloride and propranolol hydrochloride, respectively. The amounts of drug in marketed formulation were 100.5 and 101.25% of flunarizine dihydrochloride and propranolol hydrochloride, respectively. The method developed can be used for routine analysis in bulk drug and capsule dosage form.
Flunarizine dihydrochloride and propranolol hydrochloride; high performance thin layer chromatography; validation
A high performance thin layer chromatographic (HPTLC) method was developed for simultaneous estimation of stevioside and rebaudioside A in Stevia rebaudiana samples collected from different regions of Indian subcontinent.
Materials and Methods:
The separation was achieved by using acetone: ethyl acetate: water (5:4:1, v/v/v) as the solvent system on precoated silica gel 60 F254 TLC plates. The densitometric quantification of stevia glycosides was carried out at wavelength 360 nm in absorption mode after spraying with anisaldehyde sulphuric acid as detecting reagent.
The well resolved peaks for stevioside and rebaudioside A were observed at Rf values 0.31± 0.02 and 0.21± 0.02 respectively. The calibration curves were found linear with a wide range of concentration 100 - 2000 ng spot-1 with good correlation coefficient 0.996 and 0.991 for stevioside and rebaudioside A, respectively.
The proposed method was validated as per the ICH (International Conferences on Harmonization) guidelines and found simple, sensitive, economic, reproducible, robust and accurate for quantitative analysis of stevia glycosides, which can be applied for quality control of stevia as well as to check.
Densitometry; HPTLC; rebaudioside A; Stevia rebaudiana; stevioside
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.
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.
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.
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.
Maytenus obscura; Phytochemical screening; Finger print; Anti-inflammatory; HPTLC
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).
Preliminary phytochemical screening was done and 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. 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 after formalin injection. Control group received saline i.p. The extract treatment was injected i.p with doses of 200 and 400 mg/kg 1 h before formalin administration. Indomethacin (30 mg/kg) was used as standard.
Treatment of rats (i.p.) with M. obscura and M. parviflora in doses of 200 and 400 mg/kg inhibited significantly (P<0.05) formalin-induced inflammation by 55.9%, 63.2% and 77.9%, 82.4%, respectively. Preliminary phytochemical studies were done which confirmed the presence of protein, lipid, carbohydrate, phenol, flavonoid, saponin, triterpenoid, alkaloid and anthraquinone. Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with the solvent system: Toluene: ethylacetate: glacial acetic acid (5:2:0.1, v/v/v) as mobile phase. HPTLC finger printing of M. obscura revealed major 8 peaks with Rf values in the range of 0.27 to 0.77 and the M. parviflora revealed maximum 9 peaks with Rf values in the range of 0.17 to 0.76. The purity of sample was confirmed by comparing the absorption spectra at start, middle and end position of the band.
HPTLC of M. parviflora revealed 8 major spots and 9 spots for M. obscura. HPTLC finger printing of ethanolic extract of M. obscura and M. parviflora may become potential tool for checking authenticity of these species. It may help in quality control against adulterant and act as a biochemical marker for these medicinally important plants in the pharmaceutical industry and plant systematic studies. The anti-inflammatory potential of these plants also reveals its medicinal importance. It might be further explored for the isolation of phytoconstituents having anti-inflammatory potential.
Maytenus obscura; Maytenus parviflora; Phytochemical screening; Finger print; Standardization; HPTLC
Some studies showed that piperine (the alkaloid of piper nigrum) can change the activities of microsomal enzymes. Midazolam concentration is applied as a probe to determine the CYP3A enzyme activity. This study was done to determine piperine pretreatment role on midazolam plasma concentration.
Twenty healthy volunteers (14 men and 6 women) received oral dose of piperine (15 mg) or placebo for three days as pretreatment and midazolam (10 mg) on fourth day of study and the blood samples were taken at 0.5, 2.5 and 5 h after midazolam administration. The midazolam plasma levels were assayed using HPLC method (C18 analytical column, 75:25 methanol:water as mobile phase, UV detector at 242 nm wavelength and diazepam as internal standard). Data were fit in a “one-compartment PK model” using P-Pharm 1.5 software and analyzed under statistical tests.
The mean ±SD of the age and body mass index were 24.3 ± 1.83 years (range: 21–28 years) and 23.46± 2.85, respectively. The duration of sedation in piperine receiving group was greater that the placebo group (188±59 vs. 102±43 min, p<0.0001). Half-life and clearance of midazolam were higher in piperine pretreatment group compared to placebo [1.88±0.03 vs. 1.71± 0.04 h (p<0.0001) and 33.62 ± 0.4 vs. 37.09 ± 1.07 ml/min (p<0.0001), respectively].
According to the results, piperine can significantly increases half-life and decreases clearance of midazolam compared to placebo. It is suggested that piperine can demonstrate those effects by inhibition CYP3A4 enzyme activity in liver microsomal system.
Piperine; Midazolam; CYP3A; Clearance; Half-life; Microsomal hepatic metabolism; HPLC
A Simple, rapid and economical procedure for estimation of piperine by UV Spectrophotometer in different piper species was developed and is described. The method is based on method is based on the identification of piperine by TLC and on the ultra violet absorbance maxima in alcohol at 328 nm.
Active components of complementary/alternative medicines and natural supplements are often anionic compounds and flavonoids. As such, organic anion transporters (OATs) may play a key role in their pharmacokinetic and pharmacological profiles, and represent sites for adverse drug-drug interactions. Therefore, we assessed the inhibitory effects of nine natural products, including flavonoids (catechin and epicatechin), chlorogenic acids (1,3- and 1,5-dicaffeoylquinic acid), phenolic acids (ginkgolic acids (13 : 0), (15 : 1), and (17 : 1)), and the organic acids ursolic acid and 18β-glycyrrhetinic acid, on the transport activity of the human OATs, hOAT1 (SLC22A6), hOAT3 (SLC22A8), and hOAT4 (SLC22A11). Four compounds, 1,3- and 1,5-dicaffeoylquinic acid, ginkgolic acid (17 : 1), and 18β-glycyrrhetinic acid, significantly inhibited hOAT1-mediated transport (50 μM inhibitor versus 1 μM substrate). Five compounds, 1,3- and 1,5-dicaffeoylquinic acid, ginkgolic acids (15 : 1) and (17 : 1), and epicatechin, significantly inhibited hOAT3 transport under similar conditions. Only catechin inhibited hOAT4. Dose-dependency studies were conducted for 1,3-dicaffeoylquinic acid and 18β-glycyrrhetinic acid on hOAT1, and IC50 values were estimated as 1.2 ± 0.4 μM and 2.7 ± 0.2 μM, respectively. These data suggest that 1,3-dicaffeoylquinic acid and 18β-glycyrrhetinic acid may cause significant hOAT1-mediated DDIs in vivo; potential should be considered for safety issues during use and in future drug development.
Piperine, a major constituent of Piper nigrum (Black pepper), is one of the well known components in many Ayurvedic
formulations. Piperine is most studied bioenhancer because it inhibits drug metabolizing enzymes in rodents and increases plasma
concentrations of several drugs, including P-glycoprotein substrates. However, there areno evidences on piperine-iron conjugate to
inhibit human CYP450 3A4. We therefore investigated the influence of piperine-Fe conjugate to study the metabolism of iron with
CYP450 3A4. Our in silico results showed that Piperine when conjugated with iron, inhibited activity of CYP450 3A4. This
improved the binding of piperine-Fe conjugate with CYP450 3A4 and increased bioavailability.
Bioavailability; metabolic enzymes; Cytochrome P450 isoenzymes; piperine-iron conjugate; docking scores
A simple, precise, sensitive, rapid and reproducible HPTLC method for the simultaneous estimation of the rabeprazole and itopride hydrochloride in tablets was developed and validated. This method involves separation of the components by TLC on precoated silica gel G60F254 plate with solvent system of n-butanol, toluene and ammonia (8.5:0.5:1 v/v/v) and detection was carried out densitometrically using a UV detector at 288 nm in absorbance mode. This system was found to give compact spots for rabeprazole (Rf value of 0.23 0.02) and for itopride hydrochloride (Rf value of 0.75±0.02). Linearity was found to be in the range of 40-200 ng/spot and 300-1500 ng/spot for rabeprazole and itopride hydrochloride. The limit of detection and limit of quantification for rabeprazole were 10 and 20 ng/spot and for itopride hydrochloride were 50 and 100 ng/spot, respectively. The method was found to be beneficial for the routine analysis of combined dosage form.
Simultaneous estimation; HPTLC; rabeprazole and itopride
Duloxetine hydrochloride is a potent dual reuptake inhibitor of serotonin and norepinephrine used to treat major depressive disorders. The present work describes a simple, precise and accurate HPTLC method for its estimation as bulk and in tablet dosage form. The chromatographic separation was carried out on precoated silica gel 60 F254 aluminium plates using mixture of chloroform:methanol (8:1 v/v) as mobile phase and densitometric evaluation of spots was carried out at 235 nm using Camag TLC Scanner-3 with win CAT 1.3.4 version software. The experimental parameters like band size of the spot applied, chamber saturation time, solvent front migration, slit width etc. were critically studied and optimum conditions were evolved. The drug was satisfactorily resolved with Rf value 0.11±0.01. The accuracy and reliability of the proposed method was ascertained by evaluating various validation parameters like linearity (40-200 ng/spot), precision (intra-day RSD 0.46-0.75%, inter-day RSD 0.46-1.59%), accuracy (98.72±0.20) and specificity according to ICH guidelines. The proposed method can analyse ten or more formulation units simultaneously on a single plate and provides a faster and cost-effective quality control tool for routine analysis of duloxetine hydrochloride as bulk drug and in tablet formulation.
Duloxetine hydrochloride; HPTLC; densitometric estimation; method development and validation
Rotaviruses are the single most important cause of severe diarrhea in young children worldwide. The developments of specific, potent and accessible antiviral treatments that restrain rotavirus infection remain important to control rotavirus disease.
150 plant extracts with nutritional applications were screened in vitro on MA-104 cells for their antiviral activity against rhesus rotavirus (RRV). One extract (Aspalathus linearis (Burm.f.) R.Dahlgren) was also tested for its effect on the loss of transepithelial resistance (TER) of Caco-2 cells caused by simian rotavirus (SA-11) infection.
Aqueous extracts of Nelumbo nucifera Gaertn. fruit, Urtica dioica L. root, Aspalathus linearis (Burm.f.) R.Dahlgren leaves, Glycyrrhiza glabra L. root and Olea europaea L. leaves were found to have strong significant antiviral activity with a 50% inhibitory concentration (IC50) < 300 μg/ml. The pure compound 18ß-glycyrrhetinic acid from Glycyrrhiza glabra was found to have the strongest antiviral activity (IC50 46 μM), followed by luteolin and vitexin from Aspalathus linearis (IC50 respectively 116 μM and 129 μM) and apigenin-7-O-glucoside from Melissa officinalis (IC50 150 μM). A combination of Glycyrrhiza glabra L. + Nelumbo nucifera Gaertn. and Urtica dioica L. + Nelumbo nucifera Gaertn. showed synergy in their anti-viral activities. Aspalathus linearis (Burm.f.) R.Dahlgren showed no positive effect on the maintenance of the TER.
These results indicate that nutritional intervention with extracts of Nelumbo nucifera Gaertn., Aspalathus linearis (Burm.f.) R.Dahlgren, Urtica dioica L., Glycyrrhiza glabra L. and Olea europaea L. might be useful in the treatment of diarrhea caused by rotavirus infection.
Rotavirus; Antiviral activity; Transepithelial resistance; Plant extracts
A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the estimation of sumatriptan in tablet dosage forms. The stationary phase used was precoated silica gel 60F254. The mobile phase used was a mixture of methanol:water:glacial acetic acid (4.0:8.0:0.1, v/v/v). The detection of spots was carried out at 230 nm. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 200 to 800 ng/spot. The limit of detection and the limit of quantification for the sumatriptan were found to be 63.87 and 193.54 ng/spot, respectively. The proposed method can be successfully used to determine the drug content of marketed formulation.
HPTLC; sumatriptan; validation
Glycyrrhiza glabra, commonly known as licorice, is a popular herbal supplement used for the treatment of chronic inflammatory conditions and as sweetener in the food industry. This species contains a myriad of phytochemicals including the major saponin glycoside glycyrrhizin (G) of Glycyrrhetinic acid (GA) aglycone. In this study, 2D-ROESY NMR technique was successfully applied for distinguishing 18α and 18β glycyrrhetinic acid (GA). ROESY spectra acquired from G. glabra, Glycyrrhiza uralensis and Glycyrrhiza inflata crude extracts revealed the presence of G in its β-form. Anti-inflammatory activity of four Glycyrrhiza species, G, glabra, G. uralensis, G. inflata, and G. echinata roots was assessed against COX-1 inhibition revealing that phenolics rather than glycyrrhizin are biologically active in this assay. G. inflata exhibits a strong cytotoxic effect against PC3 and HT29 cells lines, whereas other species are inactive. This study presents an effective NMR method for G isomer assignment in licorice extracts that does not require any preliminary chromatography or any other purification step.
G. glabra; G. inflata; G. uralensis; Glycyrrhizin; Licorice; ROESY
Emblica (Phyllanthus emblica L.), an euphorbiaceous plant, is widely distributed in subtropical and tropical areas of India, China and Indonesia. The fruits possess antimicrobial, antioxidant, anti-inflammatory, analgesic and antipyretic properties. In the current article a new, simple, sensitive, selective, precise, and robust high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the determination of gallic acid in dried fruit powder of Phyllanthus emblica.
Materials and Methods:
The quantitative determination of gallic acid was performed on TLC aluminium plates pre-coated with silica gel 60F-254 as the stationary phase. The linear ascending development was carried out in a twin trough glass chamber saturated with a mobile phase consisting of toluene: ethyl acetate: formic acid: methanol (3:3:0.8:0.2) at room temperature (25 ± 2°C). Camag TLC scanner III was used for spectrodensitometric scanning and analysis, in the absorbance mode, at 278 nm.
The linear regression analysis data for the calibration plots showed good linear relationship with r2 = 0.99977 in the concentration range of 40 – 240 ng spot—1, with respect to the peak area. According to the guidelines of the International Conference on Harmonization (ICH), the method was validated for precision, accuracy, and recovery.
Statistical analysis of the data showed that the method was reproducible and selective for the estimation of gallic acid.
Phyllanthus emblica; HPTLC fingerprinting; gallic acid
Andrographolide and wedelolactone are active components of Andrographis paniculata and Eclipta alba, respectively. The extracts of these plants are used in many traditional hepatoprotective formulations. An attempt has been made to develop an accurate, precise and specific HPTLC method to quantify simultaneously both these chemical markers of diversified chemical structures in different dosage forms like tablet and syrup. Precoated silica 60F254 plates with toluene:acetone:formic acid (9:6:1) as mobile phase and detection wavelength of 254 nm were used. The method was validated in terms of linearity, accuracy, precision and specificity. The calibration curve was found to be linear between 200 to 400 ng/spot for andrographolide and 100 to 200 ng/spot for wedelolactone. The limit of detection and the limit of quantification for andrographolide were 26.16 and 79.28 ng/spot, respectively and for wedelolactone 5.06 and 15.32 ng/spot, respectively.
Andrographolide; wedelolactone; Andrographis paniculata; Eclipta alba; hepatoprotective agents; HPTLC
Effects of varying temperatures with constant pressure of solvent on extraction efficiency of two chemically different alkaloids were studied. Camptothecin (CPT) from stem of Nothapodytes nimmoniana (Grah.) Mabb. and piperine from the fruits of Piper nigrum L. were extracted using Accelerated Solvent Extractor (ASE). Three cycles of extraction for a particular sample cell at a given temperature assured complete extraction. CPT and piperine were determined and quantified by using a simple and efficient UFLC-PDA (245 and 343 nm) method. Temperature increased efficiency of extraction to yield higher amount of CPT, whereas temperature had diminutive effect on yield of piperine. Maximum yield for CPT was achieved at 80°C and for piperine at 40°C. Thus, the study determines compound specific extraction of CPT from N. nimmoniana and piperine from P. nigrum using ASE method. The present study indicates the use of this method for simple, fast, and accurate extraction of the compound of interest.