Application of the "Method Operable Design Region" (MODR) approach for the development of a UHPLC method for the assay and purity determination of risperidone in risperidone drug substance and other formulations.

To understand the role of analytics in drug development, regulatory bodies also started using the approach of Quality by Design (QbD) during analytical method developments. The present study deals with the development of the "Method Operable Design Region" for assay and purity determination of risperidone in risperidone drug substance and formulations usingy UHPLC. Five different column chemistries, five different pH buffers, oven temperatures from 25 to 45°C, and different organic modifier composition, column lengths and flow rates were studied and statistically evaluated using Fusion QbD software. The final method parameters were selected by performing multivariable changes in a single run and evaluated using the Monte Carlo simulation approach. The uniqueness of this method is that it is mass compatible, a total of 10 peaks are separated within a short run time of 12.0 min and it uses a "Platforming approach", which means the use of a single method for testing the drug substance, different strengths of a drug product and different formulations. The same method can be also used for the determination of the preservative (benzoic acid) in risperidone 1 mg/ml oral solution. The use of the QbD approach is aligned with the US Pharmacopeia <1220>, BP supplementary chapter 2022 and the International Conference on Harmonization Q14 guidelines for life cycle management of analytical methods.

A liquid chromatography-tandem mass spectrometry method for the determination of apixaban in human plasma and its application to pharmacokinetics studies in the Indian population.

Apixaban is a novel oral anticoagulant intended to treat and prevent blood clots and to prevent strokes in patients with nonvalvular atrial fibrillation. The development and validation of a fast, selective, accurate, and precise method using high-performance liquid chromatography tandem mass spectrometry is described for the estimation of apixaban in human plasma, with apixaban 13CD3 as an internal standard (IS). Using a reverse phase Gemini C18 column (50 mm × 4.6 mm, 3 µm) and a mixture of acetonitrile (2 mM) and ammonium formate buffer (50 : 50 v/v) as the mobile phase, chromatographic separation was achieved following extraction via a solid-phase extraction process. To track multiple reaction monitoring transitions set at 460/443 (m/z) and 464/447 (m/z) for apixaban and apixaban 13CD3, respectively, liquid chromatography coupled with triple quadrupole mass spectrometry was employed. A concentration linearity range between 1.01 and 280.00 ng mL-1 was validated with regression ≥0.99, and the method was successfully applied to apixaban pharmacokinetics analysis. At a flow rate of 1.0 mL min-1, the run time was around 1.8 min, which is short. With an extraction recovery of >73% for both apixaban and apixaban 13CD3, the method was sensitive, with a limit of quantitation of 1.01 ng mL-1. The inter-day/between-run precision ranged from 1.21% to 3.21%, while the accuracy ranged from 96.5% to 102%. For pharmacokinetics analysis, the validated method was applied. The percentage difference between findings from samples that were reanalyzed and samples that were initially analyzed was within ±20%. With high-quality assay specificity and accuracy in relation to apixaban analysis in human plasma under the experimental conditions used, the method provided is accurate.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of free and total dabigatran in human plasma and its application to a pharmacokinetic study.

A high-performance liquid chromatography-tandem mass spectrometric method for the determination of free and total dabigatran in human plasma has been developed and validated using a stable labeled internal standard (IS) as dabigatran D4. The extraction of analyte and IS was accomplished by the solid-phase extraction technique. Chromatographic separations were achieved using Peerless basic C8 (150 × 4.6) mm, 5 µ column eluted at a flow rate of 1 mL/min with mobile phase Acetonitrile: 5 mM ammonium formate: Methanol and 0.2% formic acid (30:20:50, v/v/v). The run time of the method was about 2.5 min with elution times of dabigatran and dabigatran D4 at around 1.2 min. The multiple reaction monitoring transitions (Q1/Q3) were set at 472/289, 172 (m/z) for dabigatran, and 476/293 (m/z) for dabigatran D4. The calibration curves were linear (r2 ≥0.99) over the range of 1.04-406.49 ng/mL. The presented method was successfully employed in the analysis of pharmacokinetic studies with the added advantage of demonstrating the effect of co-administration of dabigatran with the proton pump inhibitor pantoprazole on bioavailability and pharmacokinetic characteristics. Re-analysis of incurred sample resulted in >98% compliance indicating good assay precision of target analytes.

Development of LC-MS/MS method for simultaneous determination of Canagliflozin and Metformin in human plasma and its pharmacokinetic application in Indian population under fast and fed conditions.

A novel, selective and sensitive method is developed for simultaneous estimation of canagliflozin and metformin and successfully applied to fast and fed pharmacokinetic studies in healthy Indian volunteers. The current study reports the development, optimization, and validation of liquid chromatography-mass spectrometry (LC-MS/MS) method for simultaneous quantification of canagliflozin and metformin in human plasma using deuterated canagliflozin D4 and metformin D6 as an internal standard (IS). The solid-phase extraction technique was employed where strata X polymeric reverse phase (30 mg-1 cc) SPE cartridges were used for the extraction of analytes and IS from plasma. The ACE 5 C18 column (50 × 4.6 mm, 5µ) was used to chromatograph the prepared samples. The mobile phase consisted of methanol and 5 mM ammonium trifluoroacetate in water, pH 5 (50:50, v/v) at a flow rate of 0.8 mL/min. Detection was performed by positive ion Turbo ion spray in Multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 461.9 â†’ m/z 191.1 and m/z 461.9 â†’ m/z 267.2, for quantification of canagliflozin. The response of canagliflozin fragments m/z 461.9 â†’ m/z 191.1 and m/z 461.9 â†’ m/z 267.2 was combined. Also, for metformin transitions were monitored at m/z 130.0 â†’ m/z 71.1. Full validation of the method was performed according to the United States Food and Drugs Administration (USFDA) guidelines. Linearity was in the range of 24.95-2806.55 ng/mL for canagliflozin and 24.99-3400.72 ng/mL for metformin. The mean extraction recovery of canagliflozin, canagliflozin D4, metformin, and metformin D6 was 77.240, 84.663, 66.747, and 67.449, respectively across four QC levels. This rapid method with the run time of 2.80 min allows the analysis of more than 400 plasma samples per day.

Liquid chromatography-tandem mass spectrometry method for determination of rivaroxaban in human plasma and its application to a pharmacokinetic study.

A high-performance liquid chromatography tandem mass spectrometric method for the determination of Rivaroxaban in human plasma has been developed and validated using Rivaroxaban D4 as an internal standard. The extraction of analyte and internal standard was accomplished by solid phase extraction technique. The method has been validated over a concentration range of 5.96-801 ng/mL. Chromatographic separations were achieved using Gemini C18, 150 mm × 4.6 mm, 5 µm, column eluted at flow rate of 1.5 mL/min with mobile phase (acetonitrile: ammonium acetate buffer (80:20 v/v)). The overall run time of method was about 1.8 min with elution times of Rivaroxaban and its internal standard Rivaroxaban D4 at around 1.18 min. The multiple reaction monitoring transitions were set at 436/145 (m/z) and 440/145 (m/z) for Rivaroxaban and Rivaroxaban D4, respectively. The calibration curves were linear (r2 ≥ 0.99) over the range of 5.96-801 ng/mL with lower limit of quantitation validated at 5.96 ng/mL. Extraction recoveries were >88% for both rivaroxaban and its stable labeled internal standard rivaroxaban D4. The inter-day/between run precisions were ranged from 1.08% to 3.75%, while accuracy ranged from 96.3% to 102%. The presented method was used in pharmacokinetic study in healthy volunteers. Results of incurred sample reanalysis were within the acceptance range of ±20% of original value, for 98.3% of samples reanalyzed. This indicated good assay precision of target analytes in their real matrix at the employed experimental conditions. The applicability of the assay for the determination of the pharmacokinetic parameters was demonstrated.

Development of simultaneous determination of empagliflozin and metformin in human plasma using liquid chromatography-mass spectrometry and application to pharmacokinetics.

A rapid and sensitive liquid chromatography-mass spectrometry method was developed, optimized, and validated for simultaneous quantification of empagliflozin and metformin in human plasma using empagliflozin D4and metformin D6 as an internal standard. Analytes and internal standard were extracted from plasma by optimized solid-phase extraction technique using Strata X polymeric reverse phase (30 mg-1cc) solid-phase extraction cartridges. The prepared samples were chromatographed on Orosil C18 column (150 × 4.6 mm, 3 µ). Separation was done by pumping isocratic mobile phase consisting of methanol and 10 mM ammonium trifluoroacetate (90:10, v/v) in positive ion mode at a flow rate of 0.8 mL/min. The API 3200 liquid chromatography-mass spectrometry system having turbo ion spray as an ion source coupled with Shimadzu Prominence ultrafast liquid chromatography system was operated under the selected reaction monitoring mode. Turbo ion spray ionization was used for mass transition of m/z 468.070/355.100 and m/z 130.072/71.200 for empagliflozin and metformin, respectively. A method was successfully validated for concentration range of 10.09-5013.46 ng/mL for both the analytes and according to the United States Food and Drugs Administration guidelines. The linearity was found to be in the range of 10.09-403.46 ng/mL for empagliflozin and 25.44-5013.46 ng/mL for metformin. The limit of quantification was found to be 10.09 ng/mL for empagliflozin and 25.44 ng/mL for metformin. Intra- and inter-day/between batch precision determination for empagliflozin and metformin, expressed as coefficient of variation were within the acceptance limits and ranged below 13.16%. A short run time of 3.3 min allows analysis of more than 400 plasma samples per day. The developed method was successfully applied to fasting pharmacokinetic study in healthy human volunteers. Results of incurred sample re-analysis were within the acceptance range of ±20% of original value, for 97.2% of samples reanalyzed for empagliflozin and 100% of samples reanalyzed for metformin.

High- performance liquid chromatography method applied to investigate mechanism, kinetics, isotherm, and thermodynamics of bile acid adsorption onto bile acid sequestrants.

The purpose of the study was to develop and validate a high-performance liquid chromatography (HPLC) method which can be further applied to understand the mechanism, kinetics, isotherm, and thermodynamics of bile acid adsorption onto bile acid sequestrants. To investigate these properties a HPLC method was developed using peerless C-8 (150 x 4.6 mm, 5 µm) column with a detection wavelength of 200 nm and run time of about 12.5 min. Bile salts glycocholic (GC), glycochenodeoxycholic (GCDC), and taurodeoxycholic acid (TDC), were used and colesevelam hydrochloride was employed as the bile acid sequestrant. The calibration range was found linear from 10 to 6500 mgL-1 for GC and GCDC and 4to 2400 mg L-1 for TDC. The precision was less than 8.8% and accuracy was found well within the range of 85 to 115%. On treating the data with various established models, it was known that, the adsorption kinetics followed the pseudo second order equation indicating chemisorption mechanism. Equilibrium isotherms revealed that the linear form of Langmuir model was the best fit. The separation factor (RL) calculated revealed that the reaction is favorable and reversible. The positive value of heat of sorption (B) calculated from Temkin model indicated towards the exothermic nature of adsorption. The adsorption energy (E) calculated from Dubinin-Kaganer-Radushkevich model was found to be greater than 8 KJmol-1 conforming chemisorption mechanism. The Gibbs free energy calculated established the affinity of bile salts as TDC > GCDC > GC.

Ex-vivo intestinal absorption study of boswellic acid, cyclodextrin complexes and poloxamer solid dispersions using everted gut sac technique.

Acetyl- Keto-ß-boswellic acid (AKBA) is a pentacyclic triterpenic acid found in gum resin of Boswellia serrata. Even though it is shown to have anti-inflammatory activity, its bioavailability gets limited due to its poor aqueous solubility and permeability. The present study, hence, deals in enhancement of the intestinal absorption of AKBA from total boswellic acid fraction (TA fraction) using cyclodextrin (CD) and poloxamer solid dispersion (PXM SDs) formulations. Absorption studies were performed using the everted gut sac model prepared from rat jejunum. The glucose uptake assay was performed to show viability of gut sac tissue. The apparent permeability (Papp) value of AKBA from TA fraction was 1.08 ± 0.17 × 10-6 which was found to be increased by 10-14 fold with CD complex and SD formulations. The intestinal absorption studies showed highest absorption of AKBA from HP-ß-CD complex and PXM 407 SD as compared to that from TA fraction. From this study, it can be concluded that HP-ß-CD and PXM 407 effectively enhanced intestinal absorption through improved solubility, highlighting their role as efficient drug delivery agents and bioavailability enhancers.

Study of degradation behavior of besifloxacin, characterization of its degradation products by LC-ESI-QTOF-MS and their in silico toxicity prediction.

Knowledge and understanding of the stability profile of a drug is important as it affects its safety and efficacy. In the present work, besifloxacin, a new, fourth-generation fluoroquinolone antibiotic, was subjected to different forced-degradation conditions as per International Conference on Harmonization (ICH) guidelines such as hydrolysis (acid, base and neutral), oxidation, thermal and photolysis. The drug degraded under acidic, basic, oxidative and photolytic conditions while it was found to be stable under dry heat and neutral hydrolytic conditions. In total, five degradation products (DPs) were formed under different conditions-DP1 and DP2 (photolysis), DP3 (oxidation), DP4 (acidic), DP3 and DP5 (basic). The chromatographic separation of besifloxacin and its degradation products was achieved on a Sunfire C18 (250 mm × 4.6 mm, 5 µm) column with 0.1% aqueous formic acid-acetonitrile as a mobile phase. The gradient RP-HPLC method was developed and validated as per ICH guidelines. The degradation products were characterized with the help of LC-ESI-QTOF mass spectrometric studies and the most likely degradation pathway of the drug was proposed. In silico toxicity assessment of the drug and its degradation products was carried out, which indicated that DP3 and DP4 carry a mutagenicity alert.

A novel analytical approach towards in-vitro bile acid binding studies to Colesevelam Hydrochloride tablets: An ultra-high performance liquid chromatography tandem mass spectrometric method.

Colesevelam hydrochloride is a bile acid sequestrant used as a low density lipoprotein (LDL) reducing agent in hyperlipidemia with an additional advantage to improve glycemic control in type 2 diabetes patients. The objective of the study was to develop and validate a liquid chromatography tandem mass spectroscopic method for the simultaneous in-vitro estimation of bile acid salts of Glycocholic acid (GC), Glycochenodeoxycholic acid (GCDC) and Taurodeoxycholic acid (TDC) and its application in performing in-vitro binding study with Colesevelam Hydrochloride tablets. The method was developed using C-18 (50 x 4.6 mm, 3 µm) column with detection on negative ion mode and acquisition time of 3.5 min. The calibration range was linear from 0.0002 mM to 0.0065 mM for GC, 0.0002 mM to 0.0065 mM for GCDC and 0.0001 mM to 0.0021 mM for TDC. The precision was less than 3.0% and accuracy was found well within the range of 85 to 115%. The validated method was further applied to conduct in-vitro equilibrium binding study. The data was subjected to Langmuir isotherm and affinity constant (k1) and capacity constant (k2) were calculated.

In vitro Inhibitory Effect of Gymnema sylvestre Extracts and Total Gymnemic Acids Fraction on Select Cytochrome P450 Activities in Rat Liver Microsomes.

BACKGROUND AND OBJECTIVES: Gymnema sylvestre R. Br. is a well-known Indian medicinal herb. Gymnemic acids are pentacyclic triterpenes saponins and active phytoconstituents of Gymnema sylvestre. The study aimed at evaluation of the in vitro rat liver cytochrome P450 (CYP) inhibition potential of extracts and total gymnemic acid (TA)-enriched fractions from G. sylvestre. METHODS: Standardization of G. sylvestre [ethanolic (EL), hydroethanolic (HE), total acid of ethanolic (TAE), total acid of hydroethanolic (TAHE) and total acid of aqueous (TAAQ) extract] was done with respect to deacyl gymnemic acid (DAGA), using reverse phase-high performance liquid chromatography (RP-HPLC). Total triterpenoid content was determined by vanillin perchloric acid assay. RESULTS: Total triterpene content was found to be the highest in TAAQ (59.86 ± 0.005% w/w) and TAE (49.77 ± 0.009% w/w). TAAQ showed IC50 ≤ 50 µg/ml for all selected CYP activities. Testosterone 6ß-hydroxylation was strongly inhibited by TAE (IC50: 15.48 ± 2.13 µg/ml) and was moderately by TAAQ and EL with IC50 ≥ 50 µg/ml. Flurbiprofen 4'-hydroxylation was subject to strong, weak and moderate inhibition by TAAQ (IC50: 34.67 ± 1.38 µg/ml), TAE (IC50: ≥ 50 µg/ml) and EL (IC50: > 50 µg/ml), respectively. Dextromethorphan O-demethylation was inhibited by TAHE and TAAQ. CONCLUSIONS: In vitro inhibition studies suggested that TA strongly inhibits activity of selected CYP. This inhibition may possibly be due to triterpenoids and gymnemic acids that have been reported to be present in it. Data also suggest a potential for possible in vivo herb-drug interactions involving G. sylvestre and other medications that are metabolized by the same CYP.

In vivo pharmacokinetic interaction by ethanolic extract of Gymnema sylvestre with CYP2C9 (Tolbutamide), CYP3A4 (Amlodipine) and CYP1A2 (Phenacetin) in rats.

Gymnema sylvestre (GS) is a medicinal herb used for diabetes mellitus (DM). Herbs are gaining popularity as medicines in DM for its safety purpose. The aim of the present study was to evaluate in vivo pharmacokinetic (PK) interaction between allopathic drugs tolbutamide (TOLBU), amlodipine (AMLO), and phenacetin (PHENA) at low (L) and high (H) doses with ethanolic extract (EL) from GS. EL was extracted and subjected to TLC, total triterpenoid content (19.76 ± 0.02 W/W) and sterol content (0.1837 ± 0.0046 W/W) estimation followed by identification of phytoconstituents using HRLC-MS and GC-MS. PK interaction study with CYP2C9, CYP3A4 and CYP1A2 enzymes were assessed using TOLBU, AMLO and PHENA respectively to index cytochrome (CYP) mediated interaction in rats after concomitant administration of EL extract (400 mg/kg) from GS for 7 days. The rats were divided into four groups for each PK study where, group I and II were positive control for low and high dose of test drugs (CYP substrates) while group II and IV were orally administered EL. The PK study result of PHENA indicated that area under the plasma concentration-time curve (AUC0-24) was significantly (P < 0.0001) increased by 1.4 (L) and 1.3-fold (H), plasma concentration (Cmax) was significantly (P < 0.001) increased by 1.6 (L) and 1.4-fold (H). Whereas for TOLBU; clearance rate (CL) was significantly (P < 0.0001) decreased by 2.4 (L) and 2.3-fold (H), Cmax, was significantly (P < 0.001) decreased by 26.5% (L) and 50.4% (H) and AUC0-24 was significantly (P < 0.0001) decreased by 59.8% (L) and 57.5% (H). Thus, EL is seen to be interacting with CYP1A2 by inhibiting its metabolic activity. HRLC-MS and GC-MS helped identify the presence of gymnemic acid (GA), triterpenoids and steroids in EL which could be the reason for PK interaction of CYP1A2 and CYP2C9. Also, in silico structure based site of metabolism study showed Fe accessibility and intrinsic activity for GA-IV, GA-VI, GA-VII and GA-X with CYP2C9. PK parameters of AMLO were not significantly affected by pre-treatment of EL. Thereby our findings indicate that co-administration of GS with drugs that are metabolized by CYP2C9 and CYP1A2 could lead to potential HDI.

Swertisin rich fraction from Enicostema littorale ameliorates hyperglycemia and hyperlipidemia in high-fat fed diet and low dose streptozotacin induced type 2 diabetes mellitus in rats.

INTRODUCTION: Enicostema littorale blume (A. Raynal) is a traditional Indian plant belongs to the Gentianaceae family. A lot of research has been done on this plant for its antidiabetic activity. However, there are no reports on flavonoids from E. littorale for its antidiabetic activity and their mechanism of action. Thus, the aim of this study is to evaluate the antidiabetic activity of Swertisin rich flavonoid fraction (SRF) from Enicostema littorale blume and their mechanism of action. MATERIALS & METHODS: Type 2 Diabetes Mellitus rat model was established by inducing insulin resistance using high fat diet and low dose of streptozotacin injection and was authenticated by HOMA index. The antidiabetic effect of SRF was evaluated on diabetic rats to investigate its long term effects on fasting blood glucose, OGTT, weight of rats, insulin, liver profile, lipid profile, kidney profile, histopathology of liver and pancreas. In addition, antioxidant activity by lipid peroxidation and catalase assay, ex vivo assays and hepatic glycogen content were performed to determine its effect on glycogenesis and hepatic glucose production. Furthermore, the mechanism of action of SRF was evaluated by Real time PCR and the mRNA expression was quantified for Glucokinase (GCK), Insulin receptor substrate (IRS-1), Glucose transporter-2 (GLUT-2) and Glucose transporter-4 (GLUT-4) genes. RESULTS: Treatment of diabetic rats with SRF demonstrated significant (p<0.0001) dose dependant hypoglycemic activity as compared to positive control metformin group. A decrease in liver, lipid and kidney function tests was seen as compared to diabetic control indicating normalization of organ function tests. Also, antioxidant activity showed significant decrease in malondialdehyde (MDA) content in liver (p<0.001) as compared to pancreas and increased catalytic activity in liver, kidney, spleen and pancreas. The hepatic glycogen content was significantly (p<0.001) increased in SRF treated rats indicating its inhibition of hepatic glucose production. Furthermore, ex vivo assays showed the significant (p<0.05) increase in glucose uptake by diaphragm. The mRNA expression for GCK, IRS-1, GLUT-2 and GLUT-4 genes showed significant up regulation as compared to diabetic control indicating its mechanism via insulin signalling pathway. CONCLUSION: The studies suggest that SRF ameliorates the insulin resistance by increasing glucose uptake and sensitizing cells towards insulin via IRS1/PI3K/Akt2 pathway.

Flavonoids from Enicostema littorale blume enhances glucose uptake of cells in insulin resistant human liver cancer (HepG2) cell line via IRS-1/PI3K/Akt pathway.

Diabetes mellitus has spread over the world with 347 million people affected. Insulin resistance is a main pathogenic event in Type 2 Diabetes Mellitus (T2DM) leading to a reduction in glucose uptake by peripheral tissue and increased hepatic glucose output. In this study, we have isolated four flavonoid rich fractions fraction A (FA), fraction B (FB), fraction C (FC) and fraction D (FD) from Enicostema littorale. All the fractions were preliminary screened for TLC fingerprinting, total flavonoid content. Total eight flavonoids were identified by LC/MS. Insulin resistant HepG2 (IR/HepG2) model was established by inducing insulin resistance in HepG2 cells to investigate the effect of these fractions on IR/HepG2 cell line for their glucose uptake. The results showed the significant dose dependant increase in glucose uptake of cells treated with FD. It showed significant activity at a concentration of 10µg/ml. The LC/MS results of FD demonstrated the presence of C-glycoside Swertisin which could be responsible for the effect. Further, to investigate the mechanism of action, gene expression for insulin receptor substrate 1 (IRS-1), protein kinase B (Akt-2) and glucose transporter 4 (GLUT-4) genes were evaluated by real time PCR. A significant upregulation of these genes was observed in FD treated samples, thereby indicating the enhancement of glucose uptake rate of cells via IRS-1/PI3K/Akt pathway.

Validated high performance thin layer chromatography method for the quantification of bioactive marker compounds in Draksharishta, an ayurvedic polyherbal formulation

ABSTRACT Draksharishta is an ayurvedic polyherbal formulation with Draksha (Vitis vinifera L., Vitaceae) as chief ingredient prescribed for digestive impairment, respiratory disorders and weakness. These herbal medicines containing biologically active compounds play a significant role. Therefore it is necessary to carry out the chemical standardization of bioactive marker compounds present in the polyherbal ayurvedic formulation like Draksharishta. The aim of the present work was to develop and validate a HPTLC method for determination of gallic acid, catechin and resveratrol in commercially available marketed and in-house prepared formulations of Draksharishta. This is the first report of quantification of bioactive marker compound resveratrol using HPTLC in Draksharishta. The method employed silica gel precoated thin layer chromatography plates with F254 as the stationary phase. The respective mobile phases were used to develop the plates which separated bands according to the marker compound. Camag scanner V was used for densitometric scanning. Further, the method was validated according to International Conference of Harmonization (ICH) guidelines. The Rƒ values of the three marker compounds were measured. Correlation coefficients were calculated from the standard graph of linearity. Accuracy, precision and recovery were all within the required limits. The developed HPTLC methods for bioactive marker compounds present in in-house and marketed formulations were found to be simple, accurate, precise and robust.

Evaluation of Skin Anti-aging Potential of Citrus reticulata Blanco Peel.

BACKGROUND: The peel of Citrus reticulata Blanco is traditionally used as tonic, stomachic, astringent, and carminative. It is also useful in skin care. OBJECTIVE: To study the anti-aging potential of alcoholic extracts of C. reticulata Blanco peel using in vitro antioxidant and anti-enzyme assays. MATERIALS AND METHODS: Plant extracts were obtained by Soxhlation (CR HAE- Hot Alcoholic Extract of Citrus reticulata) and maceration method (CR CAE- Cold Alcoholic Extract of Citrus reticulata). Qualitative and quantitative phytochemical analysis was performed. Further, in vitro antioxidant, anti-enzyme, and gas chromatography-mass spectrometry (GC-MS) analyses were performed. RESULTS: Total phenolic and flavonoid contents of CR HAE were found to be higher than CR CAE. EC50 value of CR HAE and CR CAE for 1,1-Diphenyl-2-picrylhydrazyl, Superoxide anion, and 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assays were 250.33 ± 40.16 µg/ml and 254.73 ± 15.78 µg/ml, 221.27 ± 11.25 µg/ml and 354.20 ± 23.79 µg/ml, and 59.16 ± 2.17 µg/ml and 59.12 ± 6.21 µg/ml, respectively. Oxygen radical absorbance capacity values for CR HAE and CR CAE were found to be 1243 and 1063 µmoles 6-hydroxy-2,5,7,8-tetra methylchromane-2-carboxylic acid equivalent/g of substance, respectively. Anti-collagenase and anti-elastase activities were evaluated for both CR HAE and CR CAE. EC50 values of CR HAE and CR CAE for anti-collagenase and anti-elastase were 329.33 ± 6.38 µg/ml, 466.93 ± 8.04 µg/ml and 3.22 ± 0.24 mg/ml, 5.09 ± 0.30 mg/ml, respectively. CR HAE exhibited stronger anti-collagenase and anti-elastase activity than CR CAE. GC-MS analysis of CR HAE was carried out because CR HAE exhibited higher antioxidant and anti-enzyme potential than CR CAE. CONCLUSION: C. reticulata peel can be utilized in anti-wrinkle skin care formulations. SUMMARY: Skin anti-aging potential of Citrus reticulata Blanco peel was evaluated throughIn vitro antioxidant and anti-enzyme assaysTwo types of extraction were performed and extracts were subjected to qualitative and quantitative phytochemical analysis. Extract obtained by Soxhlation (CR HAE) showed higher total phenolic and flavonoid contents than extract obtained by maceration (CR CAE)CR HAE demonstrated strong DPPH and Superoxide free radical scavenging activity whereas, ABTS scavenging activity of both the extracts were found to be similar. Oxygen Radical Absorbance Capacity (ORAC) of CR HAE was found to be more; indicating its strong antioxidant potentialIn vitro collagenase and elastase enzyme inhibition activities were evaluated for both the extracts and CR HAE showed strong anti-collagenase and antielastase potential indicating its anti-aging abilityGC-MS analysis of CR HAE revealed the presence of various compounds mainly including Polymethoxyflavones. CR HAE exhibited promising antioxidant and anti-enzymatic activity and can be used as a potent antiwrinkle agent in anti-aging skin care formulations. Abbreviation Used: ECM: Extracellular matrix, UV: Ultra violet, ROS: Reactive Oxygen Species, MMP: Matrix metalloproteinase, Chc: Clostridium histolyticum collagenase, DPPH: 2, 2-diphenyl-1-picrylhydrazyl, GC-MS: Gas Chromatography-Mass Spectroscopy, RT: Room Temperature, µg GAE/ mg: Microgram Gallic acid equivalent / milligram, W/V: Weight by Volume, µg QE/ mg: Microgram Quercetin equivalent / milligram, CR HAE: Hot Alcoholic Extract of Citrus reticulata Blanco, CR CAE: Cold Alcoholic Extract of Citrus reticulata Blanco, EC50: Half Maximal Effective Concentration, PMS NADH: Phenazine methosulfate nicotinamide adenine dinucleotide, NBT: Nitroblue tetrazolium, DMSO: Dimethyl sulfoxide, APS: Ammonium Persulphate, AAPH: 2,2 -azobis(2-amidino-propane) dihydrochloride, TROLOX: (±) 6-hydroxy-2,5,7,8-tetramethyl chromane-2-carboxylic acid, ORAC: Oxygen Radical Absorbance Capacity, FALGPA: N-[3-(2-Furyl) acryloyl)]-Leu-Gly-Pro-Ala, SANA: Succinyl-Ala-Ala-Ala-p-nitroanilide, Rf: Retardation Factor, MSD: Mass Selective Detector.

Determination of Quality Standards for Draksharishta, a Polyherbal Ayurvedic Formulation.

Draksharishta is an ayurvedic polyherbal formulation is prescribed for digestive impairment, respiratory disorders and weakness. Though the formula composition and therapeutic claims of draksharishta are part of the Ayurvedic Formulary of India, the scientific methods for its quality and safety evaluation are yet to be documented. The current work is an attempt to evaluate the quality parameters of draksharishta which has been checked vis a vis herbs used in the formulation by modern scientific control procedures like macroscopic and microscopic study, physico-chemical analysis, preliminary phytochemical analysis, thin layer chromatography and high performance thin layer chromatography to fix the quality standard of this formulation with reference to two marketed formulations i.e. M1 and M2, respectively. The quality control parameters were within the limit as per the Ayurvedic Pharmacopeia of India which signifies good quality and purity of the plant materials. Thin layer chromatography profiles showed the presence of gallic acid, catechin and resveratrol and further it was confirmed by HPTLC fingerprints. The results obtained can be used by pharmaceutical companies as quality control parameters in order to have a proper quality check during processing.

In Vitro CYP2D Inhibitory Effect and Influence on Pharmacokinetics and Pharmacodynamic Parameters of Metoprolol Succinate by Terminalia arjuna in Rats.

BACKGROUND: Terminalia arjuna Wight & Arn. (Combretaceae) is a tree having an extensive medicinal potential in cardiovascular disorders. T. arjuna bark extract has been reported to play a significant role as a cardiac stimulant for its beneficial effects in angina. Herb - drug interactions (HDI) are one of the most important clinical concerns in the concomitant consumption of herbs and prescription drugs. Our study was to investigate the in vitro CYP2D inhibition potential of Terminalia arjuna (T. arjuna) extracts in rat liver microsomes and to study the influence of aqueous bark extract of T. arjuna on the oral pharmacokinetics and pharmacodynamics of metoprolol succinate in rats. METHODS: The CYP2D inhibition potential of herbal extracts of T. arjuna was investigated in rat liver microsomes. Pharmacokinetic-pharmacodynamic interaction of aqueous extract of T. arjuna with metoprolol succinate was investigated in rats. RESULTS: The ethyl acetate, alcoholic & aqueous bark extracts of T. arjuna showed potent reversible non-competitive inhibition CYP2D enzyme in rat liver microsomes with IC50 values less than 40 µg/mL. Arjunic acid, arjunetin and arjungenin did not show significant inhibition of CYP2D enzyme in rat liver microsomes. Pharmacokinetic studies showed that aqueous bark extract of T. arjuna led to a significant reduction (P < 0.05) in AUC0-24h and Cmax of metoprolol succinate in rats, when co-administered. Pharmacodynamic studies reveal a significant reduction in therapeutic activity of metoprolol succinate on co-administration with aqueous bark extract of T. arjuna. CONCLUSION: Based on our in vitro and in vivo findings and until further clinical drug interaction experiments are conducted, the co-administration of drugs, especially those primarily cleared via CYP2D catalyzed metabolism, with T. arjuna extracts should be done with caution.

In vitro assessment of CYP1A2 and 2C9 inhibition potential of Withania somnifera and Centella asiatica in human liver microsomes.

BACKGROUND: Several herbal drugs and allopathic medicines when co-administered can lead to severe herb-drug interactions. Hence, this study was undertaken in order to assess the in vitro inhibition potential of Withania somnifera and Centella asiatica with cytochrome P450 (CYP) 1A2 and 2C9 enzyme using human liver microsomes. METHODS: Inhibitory potential of crude extracts of both the medicinal plants along with their principal phytoconstituents were investigated using selective probe substrate technique. IC50, Ki values and mode of inhibition were determined. RESULTS: The results of the study revealed that W. somnifera showed no significant interaction with both the isoforms of CYP. However, ethanolic extract of C. asiatica significantly inhibited both CYP1A2 (IC50 value - 42.23±3.65 µg/mL/Ki value - 14.93±4.59 µg/mL) and 2C9 enzyme (IC50 value - 48.41±4.64 µg/mL/Ki value - 23.89±3.14 µg/mL) in a competitive manner. The flavonoids, quercetin and kaempferol showed potent (IC50 values less than 10 µM) inhibition of CYP1A2 activity with no significant inhibition of CYP2C9 enzyme. CONCLUSIONS: Thus, these findings of the study might be helpful for safe and effective use of C. asiatica in clinical practice. However, its in vivo interaction study in humans is still warranted.

Preparation and characterization of standardized pomegranate extract-phospholipid complex as an effective drug delivery tool.

Punicalagins, a pair of anomeric ellagitannins, present in Punica granatum (Pomegranates) are known to possess excellent antioxidant activity in vitro, but poor oral bioavailability. The reasons cited for poor bioavailability are their large molecular size, poor lipophilicity, and degradation by colonic microflora into less active metabolites. The objective of the present research work was to complex the standardized pomegranate extract (SPE) with phospholipid to formulate standardized pomegranate extract-phospholipid complex (SPEPC), characterize it and check its permeability through an ex vivo everted gut sac experiment. SPEPC was prepared by mixing SPE (30% punicalagins) and soya phosphatidylcholine (PC) in 1:1 v/v mixture of methanol and dioxane and spray-drying the mixture. The complex was characterized by infrared spectroscopy, differential scanning calorimetry, X-ray diffraction, and scanning electron microscopy. It was evaluated for its octanol solubility, dissolution, and permeability by everted the gut sac technique. The characterization methods confirmed the formation of complex. Increased n-octanol solubility of the complex proved its increased lipophilicity. Dissolution studies revealed that the phospholipid covering may prevent the punicalagins to be released in gastro-intestinal tract, thus preventing their colonic microbial degradation. SPEPC showed better apparent permeability than SPE in an everted gut sac technique. Hence, it could be concluded that phospholipid complex of SPE may be of potential use in increasing the permeability and hence the bioavailability of punicalagins.