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1.  Standardization of a Siddha Formulation Amukkara Curanam by HPTLC 
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.
PMCID: PMC2816538  PMID: 20161928
HPTLC; Amukkara Curanam (AKC); Withaferine A; Piperine; Standard Formulation (SF); Commercial Formulation (CF)
2.  HPLC analysis and standardization of Brahmi vati – An Ayurvedic poly-herbal formulation 
Objectives
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.
Results
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.
Conclusion
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.
doi:10.1016/j.jyp.2013.09.001
PMCID: PMC3812882  PMID: 24396246
Brahmi vati; Bacoside A3; Piperine; HPLC
3.  A novel high-performance liquid chromatography-electron spray ionization-mass spectrometry method for simultaneous determination of guggulsterones, piperine and gallic acid in Triphala guggulu 
Pharmacognosy Magazine  2015;11(Suppl 1):S66-S72.
“Triphalaguggulu” is an important Ayurvedic formulation comprising of Guggulu, that is, Commiphora wightii (Arn.) Bhandari as a base wherein powdered fruits of triphala, that is, Phyllanthus emblica L., Terminalia bellirica (Gaertn.) Roxb and Terminalia chebula Retz, along with powdered fruit of Piper longum L. are compounded. This polyherbal preparation has been strongly recommended in chronic inflammation, piles, and fistula. However, due to the complexity of compound formulation standardization of commercial products is challenging. In the present communication marker-based standardization of “Triphalaguggulu” preparation using gallic acid (for triphala), piperine (for P. longum L.) and guggulsterones (for guggulu) is reported. These compounds of diverse chemistry were successfully separated on a Waters HR-C18 column by isocratic elution with methanol and water (80:20 v/v) as mobile phase at the flow rate of 1.0 mL/min coupled with photodiode array detector. These optimal chromatographic conditions were used for simultaneous quantification of gallic acid, guggulsterones (E and Z) and piperine in commercial samples by high-performance liquid chromatography-electron spray ionization-mass spectrometry and method was validated as per ICH guidelines.
doi:10.4103/0973-1296.157696
PMCID: PMC4461971  PMID: 26109777
Gallic acid; guggulsterones; high-performance liquid chromatography-electron spray ionization-mass spectrometry; piperine; Triphalaguggulu
4.  Validated stability-indicating high-performance thin-layer chromatographic method for estimation of cefpodoxime proxetil in bulk and in pharmaceutical formulation according to International conference on harmonization guidelines 
Aim:
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.
Results:
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.
Conclusion:
The proposed HPTLC method can be applied for identification and quantitative determination of cefpodoxime proxetil in both bulk drug and pharmaceutical formulation.
doi:10.4103/0975-7406.94809
PMCID: PMC3341712  PMID: 22557919
Cefpodoxime proxetil; degradation; High performance thil layer chromatography (HPTLC); stability; validation
5.  Piperine suppresses tumor growth and metastasis in vitro and in vivo in a 4T1 murine breast cancer model 
Acta Pharmacologica Sinica  2012;33(4):523-530.
Aim:
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:
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:
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:
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.
doi:10.1038/aps.2011.209
PMCID: PMC4003369  PMID: 22388073
anticancer drug; piperine; 4T1 breast cancer; apoptosis; cell cycle; metastasis; MMP-9; MMP-13
6.  Stability-indicating assay of repaglinide in bulk and optimized nanoemulsion by validated high performance thin layer chromatography technique 
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.
doi:10.4103/0975-7406.116800
PMCID: PMC3778587  PMID: 24082694
Degradation; high-performance thin-layer chromatography; repaglinide; stability-indicating
7.  Piperine Inhibits the Activities of Platelet Cytosolic Phospholipase A2 and Thromboxane A2 Synthase without Affecting Cyclooxygenase-1 Activity: Different Mechanisms of Action Are Involved in the Inhibition of Platelet Aggregation and Macrophage Inflammatory Response 
Nutrients  2014;6(8):3336-3352.
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.
doi:10.3390/nu6083336
PMCID: PMC4145312  PMID: 25153972
pepper; piperine; platelet aggregation; arachidonic acid; cyclooxygenase; phospholipase A2; thromboxane A2 synthase; prostaglandins
8.  HPTLC Method for the Simultaneous Estimation of Valsartan and Hydrochlorothiazide in Tablet Dosage Form 
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.
doi:10.4103/0250-474X.51967
PMCID: PMC2810056  PMID: 20177464
Valsartan; hydrochlorothiazide; simultaneous estimation; HPTLC
9.  Ultra-low Flow Liquid Chromatography Assay with Ultraviolet (UV) Detection for Piperine Quantitation in Human Plasma 
A robust and sensitive ultra-low flow liquid chromatography (UFLC) method that can reproducibly, at reasonable cost, detect low concentrations of piperine from human plasma is necessary. Piperine in plasma was separated and quantified by a gradient method using ultraviolet detection at a maximal absorbance wavelength of 340 nm. An aliquot was injected onto a reversed-phase column Waters SymmetryShield, 2.1 × 100 mm, 3.5 μm, C18 column, attached to a Waters absorbosphere, 4.6 × 30 mm, C18 guard column and eluted with a mobile phase containing a mixture of acetonitrile/water/ acetic acid (25:74.9:0.1, v/v/v) on line A and acetonitrile/acetic acid (99.9:0.1, v/v) on line B. The flow rate was 0.3 mL/min. The gradient method consisted of an opening condition of 20% pump B, with a linear increase to 37% pump B over 8 min, then a linear increase to 100% pump B at 11 min, 2 min at 100% pump B, and then a return to the opening condition (20% pump B) via a linear gradient over 2 min, followed by 5 min re-equilibration at opening conditions. The total run time was 20 min for each sample. All samples were processed protected from ambient light to avoid isomerization of piperine. The plasma assay was linear with R = 0.9995, with a lower limit of detection [signal-to-noise (S/N) > 5:1] of 100 pg of piperine loaded into the analytical system with acceptable accuracy and precision. Extraction recoveries of piperine from human plasma were 88% for quality control high (QCH), 93% for quality control medium (QCM), and 90% for quality control low (QCL), and the matrix effect was <12%. Piperine was quantifiable from a 50 mg oral dose given to human volunteers. A UFLC method for the rapid assay of human plasma with sensitivity to detect as low as 5 ng/mL piperine was developed. The method sensitivity equals that of liquid chromatography/tandem mass spectrometry (LC/MSMS) methods with much less cost.
doi:10.1021/jf100657r
PMCID: PMC4508855  PMID: 20465211
Liquid chromatography assay; piperine; natural compounds; bioavailability
10.  Bio-enhancing Effect of Piperine with Metformin on Lowering Blood Glucose Level in Alloxan Induced Diabetic Mice 
Pharmacognosy Research  2016;8(1):56-60.
Background:
Diabetes mellitus is the most rampant metabolic pandemic of the 21st century. Piperine, the chief alkaloid of Piper nigrum (black pepper) is widely used in alternative and complementary therapies has been extensively studied for its bio-enhancing property.
Objective:
To evaluate the bio-enhancing effect of piperine with metformin in lowering blood glucose levels in alloxan-induced diabetic mice.
Materials and Methods:
Piperine was isolated from an extract of fruits of P. nigrum. Alloxan-induced (150 mg/kg intraperitoneal) diabetic mice were divided into four groups. Group I (control 2% gum acacia 2 g/100 mL), Group II (metformin 250 mg/kg), Group III (metformin and piperine 250 mg/kg + 10 mg/kg), and Group IV (metformin and piperine 125 mg/kg + 10 mg/kg). All the drugs were administered orally once daily for 28 days. Blood glucose levels were estimated at day 0, day 14, and end of the study (day 28).
Results:
The combination of piperine with therapeutic dose of metformin (10 mg/kg + 250 mg/kg) showed significantly more lowering of blood glucose level as compared to metformin alone on both 14th and 28th day (P < 0.05). Piperine in combination with sub-therapeutic dose of metformin (10 mg/kg + 125 mg/kg) showed significantly more lowering of blood glucose as compared to control group and also showed greater lowering of blood glucose as compared to metformin (250 mg/kg) alone.
Conclusion:
Piperine has the potential to be used as a bio-enhancing agent in combination with metformin which can help reduce the dose of metformin and its adverse effects.
SUMMARY
Piperine is known for its bioenhancing property. This study evaluates the effect of piperine in combination with oral antidiabetic drug metformin. Drugs were administered for 28 days in alloxan induced diabetic mice and blood glucose lowering effect was seen. Results showed significantly better effect of combination of piperine with therapeutic dose of metformin in comparison to metformin alone. Piperine in combination with subtherapeutic dose of metformin also showed better effect than therapeutic dose of metformin. Piperine, thus shows potential to be used as bioenhancer in combination with metformin.
doi:10.4103/0974-8490.171096
PMCID: PMC4753761  PMID: 26941537
Bio-enhancing effect; Diabetes; Metformin; Piperine
11.  Quantitative Determination of L-DOPA in Seeds of Mucuna Pruriens Germplasm by High Performance Thin Layer Chromatography 
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.
doi:10.4103/0250-474X.95651
PMCID: PMC3374567  PMID: 22707835
Germplasm; high performance thin layer chromatography; L-DOPA; linearity; Mucuna pruriens
12.  Development and Validation of HPTLC Method for Simultaneous Determination of Amlodipine Besylate and Metoprolol Succinate in Bulk and Tablets 
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.
doi:10.4103/0250-474X.103849
PMCID: PMC3546332  PMID: 23325996
Amlodipine besylate; HPTLC; metoprolol succinate; pharmaceutical formulation
13.  Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed 
Objective:
The present study was used to design simple, accurate and sensitive reversed phase-high-performance liquid chromatography RP-HPLC and high-performance thin-layer chromatography (HPTLC) methods for the development of quantification of khellin present in the seeds of Ammi visnaga.
Materials and Methods:
RP-HPLC analysis was performed on a C18 column with methanol: Water (75: 25, v/v) as a mobile phase. The HPTLC method involved densitometric evaluation of khellin after resolving it on silica gel plate using ethyl acetate: Toluene: Formic acid (5.5:4.0:0.5, v/v/v) as a mobile phase.
Results:
The developed HPLC and HPTLC methods were validated for precision (interday, intraday and intersystem), robustness and accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response was linear in both HPLC and HPTLC methods with the concentration range of 10–80 μg/mL in HPLC and 25–1,000 ng/spot in HPTLC for khellin. The % relative standard deviation values for method precision was found to be 0.63–1.97%, 0.62–2.05% in HPLC and HPTLC for khellin respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.53% in HPLC and 100.08% in HPTLC for khellin.
Conclusions:
The developed HPLC and HPTLC methods for the quantification of khellin were found simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of A. visnaga and several formulations containing it as an ingredient.
doi:10.4103/0975-7406.168033
PMCID: PMC4678975  PMID: 26681890
Khellin; method development; validation; high-performance liquid chromatography; high-performance thin-layer chromatography
14.  Development and Validation of a HPTLC Method for Simultaneous Quantitation of Flunarizine Dihydrochloride and Propranolol Hydrochloride in Capsule Dosage Form 
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.
doi:10.4103/0250-474X.117418
PMCID: PMC3783757  PMID: 24082355
Flunarizine dihydrochloride and propranolol hydrochloride; high performance thin layer chromatography; validation
15.  A High-Performance Thin Layer Chromatography (HPTLC) Method for Simultaneous Determination of Diphenhydramine Hydrochloride and Naproxen Sodium in Tablets 
A rapid and simple high-performance thin layer chromatography (HPTLC) method with densitometry at 230 nm was developed and validated for simultaneous determination of diphenhydramine hydrochloride (DPH) and naproxen sodium (NPS) from pharmaceutical preparation. The separation was carried out on aluminum plates precoated with silica gel 60 F254 using mobile phase toluene:methanol:glacial acetic acid (7.5:1:0.2, v/v/v). The linearity range lies between 200 and 1200 ng/band for DPH and 1760 and 10,560 ng/band for NPS with correlation coefficients of 0.994 and 0.995, respectively. The Rf value for DPH is 0.20 ± 0.05 and for NPS is 0.61 ± 0.06. % Recoveries of DPH and NPS was in the range of 99.70%–99.95% and 99.63%–99.95%, respectively. Limit of detection value for DPH was 13.21 ng/band and for NPS was 8.03 ng/band. Limit of quantitation value for DPH was 40.06 ng/band and for NPS was 24.34 ng/band. The developed method was validated as per ICH guidelines. In stability testing, DPH was found unstable to acid and alkaline hydrolysis, and DPH and NPS were found unstable to oxidation, whereas both the drugs were stable to neutral and photodegradation. The proposed method was successfully applied for the routine quantitative analysis of dosage form containing DPH and NPS.
doi:10.4137/ACI.S31506
PMCID: PMC4670047  PMID: 26692760
diphenhydramine hydrochloride; naproxen sodium; HPTLC; validation
16.  In vitro cytotoxic and in silico activity of piperine isolated from Piper nigrum fruits Linn 
Background
Piper nigrum [Piperaceae], commonly known as black pepper is used as medicine fairly throughout the greater part of India and as a spice globally.
Purpose
To isolate piperine and evaluate in vitro cytotoxic [antiproliferative] activity and in silico method.
Methods
Piperine was isolated from the fruits of P.nigrum. Piperine was characterized by UV,IR, 1H-NMR, 13C-NMR and Mass spectrum. Standardization of piperine was done also by HPTLC fingerprinting. In vitro cytotoxic activity was done using HeLa cell lines by MTT assay at different concentrations ranging from 20 to 100 μg/ml in triplicate and in silico docking studies using enzyme EGFR tyrosine kinase.
Results
Fingerprinting of isolated piperine were done by HPTLC method. The IC50 value was found to be 61.94 ± 0.054 μg/ml in in vitro cytotoxic activity in HeLa Cell lines. Piperine was subjected to molecular docking studies for the inhibition of the enzyme EGFR tyrosine kinase, which is one of the targets for inhibition of cancer cells. It has shown −7.6 kJ mol−1 binding and 7.06 kJ mol−1 docking energy with two hydrogen bonds.
Conclusion
piperine has shown to possess in vitro cytotoxic activity and in silico studies.
doi:10.1186/s40203-015-0013-2
PMCID: PMC4731375  PMID: 26820894
In vitro cytotoxic activity; In silico docking studies; Isolation; Piperine; Piper nigrum
17.  Simultaneous quantification of stevioside and rebaudioside A in different stevia samples collected from the Indian subcontinent 
Background:
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.
Results:
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.
Conclusions:
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.
doi:10.4103/0975-7406.103236
PMCID: PMC3523521  PMID: 23248559
Densitometry; HPTLC; rebaudioside A; Stevia rebaudiana; stevioside
18.  Bergenin determination in different extracts by high-performance thin-layer chromatographic densitometry 
Aim:
Bergenin is an active secondary metabolite, found in Bergenia ligulata, family Saxifragaceae, which is an important medicinal plant used in the traditional system of medicine. It is distributed throughout the South and East Asia and some European countries, usually growing on high altitude in the Himalayan region and known as Pashanbhed (meaning “to break the stone”). The rhizome of B. ligulata has been used since long time in different traditional formulations of kidney and liver disorders. Due to its exhaustive use in the traditional system, it is commonly adulterated with the rhizome of other plants which do not contain its chemical marker bergenin. Hence, we developed high-performance thin-layer chromatographic (HPTLC) method for quantification of bergenin in B. ligulata which can be used for its quality control.
Materials and Methods:
A sensitive HPTLC method has been developed for the estimation of bergenin in different extracts of B. ligulata and its traditional formulations. Precoated HPTLC silica gel plates were used as stationary phase, and chloroform: methanol: acetic acid (8:1:1, v/v/v) was used as mobile phase.
Results:
The Rf value of bergenin was found to be 0.28 ± 0.03. Detection and quantification were performed by densitometry at 276 nm. The calibration plot was linear in the range of 200–5000 ng of bergenin with the correlation coefficient of (r2) 0.999, which confirms good linearity. The content of bergenin in methanolic and acetone extracts was found to be 5.51 ± 0.14 and 5.76 ± 0.16, respectively.
Conclusion:
The method can be applied for quality control and standardization of B. ligulata and its traditional formulations as well as for checking the presence of adulterants.
doi:10.4103/0975-7406.168024
PMCID: PMC4678973  PMID: 26681881
Bergenin; high- performance thin- layer chromatographic; quality control
19.  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.
doi:10.1016/S2221-1691(14)60224-0
PMCID: PMC3819484  PMID: 25182287
Maytenus obscura; Phytochemical screening; Finger print; Anti-inflammatory; HPTLC
20.  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).
Methods
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.
Results
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.
Conclusions
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.
doi:10.1016/S2222-1808(13)60082-1
PMCID: PMC4027328
Maytenus obscura; Maytenus parviflora; Phytochemical screening; Finger print; Standardization; HPTLC
21.  Modulation by glycyrrhetinic acid derivatives of TPA-induced mouse ear oedema. 
British Journal of Pharmacology  1989;96(1):204-210.
1. The anti-inflammatory effects of glycyrrhetinic acid and its derivatives on TPA (12-O-tetradecanoylphorbol-13-acetate)-induced mouse ear oedema were studied. The mechanisms of TPA-induced ear oedema were first investigated with respect to the chemical mediators. 2. The formation of ear oedema reached a maximum 5 h after TPA application (2 micrograms per ear) and the prostaglandin E2 (PGE2) production of mouse ear increased with the oedema formation. 3. TPA-induced ear oedema was prevented by actinomycin D and cycloheximide (0.1 mg per ear, respectively) when applied during 60 min after TPA treatment. 4. Of glycyrrhetinic acid derivatives examined, dihemiphthalate derivatives (IIe, IIe', IIIa, IIIa', IVa, IVa') most strongly inhibited ear oedema on both topical (ID50, 1.6 mg per ear for IIe, 2.0 mg per ear for IIIa and 1.6 mg per ear for IVa) and oral (ID50, 88 mg kg-1 for IIe', 130 mg kg-1 for IIIa' and 92 mg kg-1 for IVa') administration. 5. Glycyrrhetinic acid (Ia) and its derivatives applied 30 min before TPA treatment were much more effective in inhibiting oedema than when applied 30 min after TPA. A dihemiphthalate of triterpenoid compound IVa completely inhibited oedema, even when applied 3 h before TPA treatment. 6. Glycyrrhetinic acid (Ia) and deoxoglycyrrhetol (IIa), the parent compounds, produced little inhibition by oral administration at less than 200 mg kg-1. 7. These results suggest that the dihemiphthalate derivatives of triterpenes derived from glycyrrhetinic acid by chemical modification are useful for the treatment of skin inflammation by both topical and oral application.
PMCID: PMC1854326  PMID: 2924072
22.  The effect of piperine on midazolam plasma concentration in healthy volunteers, a research on the CYP3A-involving metabolism 
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.
doi:10.1186/2008-2231-22-8
PMCID: PMC3904487  PMID: 24398010
Piperine; Midazolam; CYP3A; Clearance; Half-life; Microsomal hepatic metabolism; HPLC
23.  Simultaneous HPTLC Determination of Rabeprazole and Itopride Hydrochloride From Their Combined Dosage Form 
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.
doi:10.4103/0250-474X.43004
PMCID: PMC2792527  PMID: 20046748
Simultaneous estimation; HPTLC; rabeprazole and itopride
24.  Simultaneous Estimation of Aloe Emodin and Emodin from Rheum emodi, Cassia alata and Aloes by HPTLC 
A simple, precise, specific, accurate high performance thin layer chromatography method was developed for simultaneous estimation of aloe emodin and emodin from medicinal plants like Rheum emodi (Rhubarb), Barbados aloes (dried juice of Aloe barbadensis leaf) and Cassia alata (Candle bush). Thin layer chromatographic aluminum plates pre-coated with silica gel 60 F254 was used as the stationary phase for chromatographic separation of the drugs. Toluene:ethyl acetate:formic acid (10:2:1 v/v/v) was selected as mobile phase and analysis was carried out in absorbance mode at iso-absorptive wavelength of 263 nm. This method shows good resolution for both drugs with retention factor 0.37±0.03 and 0.55±0.03 for aloe emodin and emodin, respectively. The regression analysis data indicated good linear relationship for the calibration plots for aloe emodin and emodin in the range of 300 - 800 ng/spot and 150 - 400 ng/spot and regression coefficient was 0.9993 and 0.9994, respectively. Validation of the method was performed according to International Conference on Harmonisation guidelines for following parameters: Accuracy, precision, limit of detection, linearity, limit of quantification, robustness and specificity. In conclusion, the developed method was found to be rapid, simple, reliable and specific for the identification and quantitation of these anthraquinones in medicinal plants and marketed formulations.
PMCID: PMC4778241  PMID: 26997709
High performance thin layer chromatography; aloe emodin; emodin; quantification; anthraquinones
25.  Development and validation of a HPTLC method for Estimation of Duloxetine Hydrochloride in Bulk Drug and in Tablet Dosage Form 
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.
doi:10.4103/0250-474X.41463
PMCID: PMC2792489  PMID: 20046720
Duloxetine hydrochloride; HPTLC; densitometric estimation; method development and validation

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