Purpose Zein/phospholipid composite nanoparticles (CNPs) were developed as a delivery platform for gallic acid (GA), a polyphenolic compound with reported preclinical antifibrotic activities. However, the therapeutic applicability of GA is hampered owing to its low bioavailability and rapid clearance. Accordingly, we developed GA-loaded CNPs. The effect of their size, surface charge and targeting strategies was investigated and optimized, with the aim of enhancing their ability to deliver GA to the activated hepatic stellate cells (aHSCs) in order to suppress hepatic fibrosis progression. Methods Different CNP systems were prepared and characterized with regard to their particle size, zeta potential, and GA entrapment efficiency (EE%). Also, they were statistically optimized via response surface methodology. The optimized systems were investigated with regard to their in vitro GA release, in vitro efficacy on aHSCs, and in vivo biodistribution in healthy rats. Results The GA-loaded cationic CNPs coupled with vitamin A (GA-CACNP/VA; 192 nm) showed high GA EE% (60% w/w), highest cellular internalization via active targeting, and more selective hepatic distribution, relative to free GA solution, GA-loaded anionic, and GA-loaded cationic systems. Furthermore, GA-CACNP/VA markedly triggered the apoptosis of aHSCs, repressed collagen deposition, and inhibited HSCs' activation to a lesser extent. Conclusion The GA-CACNP/VA was shown to be a promising candidate for specific and controlled delivery of GA to aHSCs, which may provide an effective antifibrotic therapeutic approach.

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Zein/Phospholipid Composite Nanoparticles for Successful Delivery of Gallic Acid into aHSCs: Influence of Size, Surface Charge, and Vitamin A Coupling

Gallic acid (GA) is a polyphenolic compound with proven efficacy against hepatic fibrosis in experimental animals. However, it suffers from poor bioavailability and rapid clearance that hinders its clinical investigation. Accordingly, we designed and optimized reverse micelle-loaded lipid nanocapsules (RMLNC) using Box-Behnken design that can deliver GA directly into activated-hepatic stellate cells (aHSCs) aiming to suppress hepatic fibrosis progression. GA-RMLNC was prepared using soft energy, solvent free phase inversion temperature method. Effects of formulation variables on particle size, zeta potential, entrapment efficiency (EE%) and GA release were studied. In-vivo biodistribution of GA-RMLNC in rats and in-vitro activities on aHSCs were also explored. Nano-sized GA-RMLNCs (30.35 ± 2.34 nm) were formulated with high GA-EE% (63.95 ± 2.98% w/w) and physical stability (9 months). The formulated system showed burst GA release in the first 2 h followed by sustained release profile. In-vivo biodistribution imaging revealed that RMLNC-loaded with rhodamine-B accumulated mainly in rats’ livers. Relative to GA; GA-RMLNC displayed higher anti-proliferative activities, effective internalization into aHSCs, marked down-regulation in pro-fibrogenic biomarkers’ expressions and elevated HSCs’ apoptosis. These findings emphasize the promising application of RMLNC as a delivery system in hepatic fibrosis treatment, where successful delivery of GA into aHSCs was ensured via increased cellular uptake and antifibrotic activities.

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Impact of Reverse Micelle Loaded Lipid Nanocapsules on the Delivery of Gallic Acid into Activated Hepatic Stellate Cells: A Promising Therapeutic Approach for Hepatic Fibrosis

Embelin, a naturally occurring benzoquinone, is obtained from fruits of Embelia tsjeriam cottam, which plays a vital role in the Ayurvedic system of medicine. An approach for the stress degradation was successfully applied for the development of stability indicating HPTLC method for the determination of Embelin. It was spotted on the plates precoated with silica gel 60 F254 and developed using toluene: ethyl acetate: formic acid, (6:3.5:0.5 v/v/v) as mobile phase. Densitometry analysis was carried out at 291 nm. The method showed high sensitivity with good linearity over the concentration range of 200-1000ng/spot with correlation coefficient value 0.997. The accuracy of the method was established based on the recovery studies. The LOD and LOQ were 6.09ng/band and 18.48ng/band respectively. The system showed a peak for Embelin at Rf of 0.68 ± 0.02.The aim of our study was to observe the effect of various stress conditions on this potential drug candidate. Stress testing of Embelin was carried out according to the International Conference of Harmonization (ICH) guideline Q1A (R2). Embelin was subjected to stress conditions of acid, alkaline hydrolysis, oxidation, photolysis and thermal degradation. A significant degradation was found to occur by acid and oxidation hydrolysis and to a lesser extent under thermal stress and alkaline hydrolysis; and much lesser with photolytic stress. Stress degradation studies on embelin provide an insight into its stability.

Development and validation of stability indicating hptlc method for estimation of embelin in embelia tsjeriam cottam

Rp-hplc method for determination of piperine, guggulusterone and embelin in ayurvedic formulation kaishoraguggulu

Embelin, is an active phytoconstituent obtained from fruits of Embelia tsjeriam cottam, commonly known as Vidanga. A simple stability indicating HPLC method for the estimation of Embelin has been developed and validated as per ICH Q2A(R1). Sample was eluted using HiQsil C 8 (4.6mm×250mm) column. The mobile phase consisted of Methanol: Acetonitrile: 1% O-phospheric acid in water in the ratio of 70:15:15 v/v/v which was sonicated to degas and delivered at a flow rate of 1ml/min at ambient temperature. The retention time of Embelin was 6.05 ± 0.2 minutes. Studies were performed using an HPLC system equipped with a UV detector; the response was monitored at 291 nm. A good linear relationship over the range of 2–10 µg/ml concentrations with correlation coefficient value of 0.999 was obtained. The accuracy of the method is indicated by good recovery in the range of 98.9-101.2 % and precision less than 2% RSD. The limit of detection and limit of quantification were found to be 0.47 and 1.44µg/ml respectively. Embelin was subjected to stress conditions as per ICH Q2A(R2) and a significant degradation was found to occur by acid hydrolysis, oxidation and thermal stress. Stress degradation studies on embelin provide an insight into its stability.

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Development and validation of Stability Indicating HPLC method for estimation of Embelin in Embelia tsjeriam cottam(Vidanga)

Objectives: A new simple spectrophotometric method for simultaneous estimation of Embelin and Gallic Acid in Vidang, Harade and Vidangadi Churna was investigated. The λmax of Embelin and Gallic Acid were found to be 341 and 258 nm. Method: The graphical absorbance ratio method was used for simultaneous estimation of Embelin and Gallic acid in Vidangadi Churna, and the Isoabsorbtive point for simultaneous estimation of both the drug were found to be 307.5 nm. The method obeys Beers law in the concentration range from 2-12 μg/ml. The correlation coefficient was found to be 0.9973 for Embelin and 0.998 for Gallic Acid. The method was validated as per ICH guidelines. Results: The result of estimation of Embelin in Vidang, Gallic acid in Harade and both in Vidangadi Churna were found to be 1.05 percent, 1.04 percent, and 1.85. The proposed method was validated for linearity, accuracy and application for assay as per ICH guidelines. Conclusion: The study showed good reproducibility and recovery with percent RSD less than 2. So, the proposed method was found to be simple, specific, precise, and accurate. Hence it can be applied for simultaneous estimation of Embelin and Gallic Acid in various Ayurvedic Churna formulations. Key Word : Embelin, Vidang. Gallic Acid, Harade, Vidangadi Churna.

Standardization, development and validation of spectrophotometric method for simultaneous estimation of embelin and gallic acid as individual and in combination in ayurvedic churna formulation

Rakesh K Patel

Papers

Standardization, development and validation of spectrophotometric method for simultaneous estimation of embelin and gallic acid as individual and in combination in ayurvedic churna formulation

Standardization, development and validation of spectrophotometric method for estimation of piperine in ayurvedic vati formulation