Analytical method development, validation and forced degradation studies for rutin, quercetin, curcumin, and piperine by RP-UFLC method.

SIGNIFICANCE: Curcumin, rutin, and quercetin are well-known flavonoids and piperine is an alkaloid, commonly used as spices and traditionally used to treat a variety of conditions. In the current scenario, the stability problems of phytoconstituents are a major problem for regulators and because of the complex nature of the components of plant extracts. OBJECTIVE: A simple, fast, and sensitive ultra-force reverse phase liquid chromatography (RP-UFLC) has been developed, validated, and studied for degradation studies. METHODS: Seven different plant extracts were quantified and the stability of the constituents was estimated by forced degradation studies. The separation of the phytoconstituents was performed on a Phenomenex C18 column with a mobile phase of 80% acetonitrile and 20% (25 mM) ammonium acetate (pH 3) at a flow rate of 1 mL min-1 detected at 380 nm. RESULTS: The results of the study showed that the method developed was linear with a range of correlation coefficient 0.994-0.999. The specificity, precision, and accuracy were well within the limits. Quantification showed that a maximum content of curcumin (3.61%, w/w) was found in the extract of Curcuma longa L extract, piperine in Piper nigrum L (13.92%, w/w), rutin in Glycyrrhiza glabra L (15.19%, w/w), and quercetin in Camellia sinensis L (0.36%, w/w). Forced degradation studies have shown that rutin was very stable in acidic media (6.65%, w/w) and curcumin was less stable in alkaline media (100%, w/w). CONCLUSION: The method developed was simple, fast, accurate, sensitive, and applicable for the determination of phytoconstituents in natural extracts and herbal formulations, individually or in combination and can be used as a quality control tool.

Formulation and characterization of chitosan encapsulated phytoconstituents of curcumin and rutin nanoparticles.

Curcumin and Rutin are natural polyphenolic molecules exhibits several pharmacological actives like antibacterial, anticancer, antioxidant, chemo-preventive and anti-inflammatory properties. However till date, no studies have been reported on their combination efficacy, especially in treating multi-drug resistance of cancers because of their poor solubility and bioavailability. Hence in the present study, an attempt has been made to load both these drugs into a single nanoparticlulate system to enhance their bioavailability and efficacy. This novel formulation was prepared by solvent evaporation technique and was evaluated for particle size and shape using Zeta Sizer, Scanning Electron Microscopy (SEM) and Fourier Transform Infra Red (FT-IR) Spectroscopy. The optimized formulation was further subjected to in vitro and in vivo evaluations. The prepared nanoparticles were in the size range of 25-100nm and the release profile was found to be Non -Fickian transport. In-vivo pharmacokinetic studies were carried in rabbits and the pharmacokinetic profile was studied. The results indicate that oral bioavailability of Curcumin and Rutin has been increased to 3.06 and 4.24 folds respectively when compared to their pure drugs. This data suggest that the present novel nanoparticles loaded with these combinational drugs may have better therapeutic potential in treating drug resistant cancers.

Screening of siddha medicinal plants for its in-vitro acetylcholinesterase and butyrylcholinesterase inhibitory activity.

BACKGROUND: The plants selected for the study were traditionally used in siddha system of medicine in neurological disorders. AIM: The aim of the following study isto screen the plant species for both acetylcholinesterase (AchE) and butyrylcholinesterase (BuchE) inhibition by in-vitro Ellman's method and a thin layer chromatography bioautographic assay for newer drug candidates for the treatment of Alzheimer's disease. MATERIALS AND METHODS: Ellman's colorimetric method was performed in a 96 well micro plate for cholinesterases inhibition using galantamine as standard drug. RESULTS: Present studies confirmed that out of all the tested extracts Hemidesmus indicus R.Br (HI) showed considerable IC50 values for AchE (28.40 ± 0.92 µg/mL) and BuchE (43.47 ± 0.64 µg/mL) inhibition which indicates that HI extract has considerable specificity toward AchE and BuchE compared with all the tested extracts and the activity was followed by Vernonia anthelmintica (VA) Willd and Saussurea lappa Clarke (SL). The bioautograms also confirmed the activity potent extracts. CONCLUSION: Besides various bioactivities HI, VA and SL exhibited considerable cholinesterases inhibition making it to consider these species for further investigation of new compounds.

Development and validation of simultaneous estimation method for curcumin and piperine by RP-UFLC.

Curcumin and piperine are proven for their potent medicinal benefits to treat various diseases and they are most commonly used combination in various Indian systems of medicine such as Ayurveda, Siddha and Unani. The objective of the present work is to develop a simultaneous estimation of curcumin and piperine by reverse phase Ultra-fast liquid chromatographic (RP-UFLC) method. The chromatographic separation was performed on a C8 column (250 x 4.6 mm, 5µ i.d.) stationary phase using a mobile phase of 25mM potassium dihydrogen ortho phosphate buffer (pH 3.5) and acetonitrile (30: 70 v/v) at a flow rate of lml/min at detection wave length of 280nm. The calibration curve was plotted in the concentration range of 0-2200ng/ml and found to be linear for both curcumin (r(2)=0.996) and piperine (r(2)=0.999). The method was validated for parameters such as accuracy, sensitivity, precision, linearity, specificity, ruggedness and robustness as per ICH guidelines. The developed simple, precise and specific method can be used as a quality control tool for qualitative and quantitative estimation of curcumin and piperine in various food products, herbal medicines and nutraceuticals.

Calotropis gigantiea (L.) R. Br (Apocynaceae): a phytochemical and pharmacological review.

ETHNOPHARMACOLOGICAL RELEVANCE: Calotropis gigantiea (L.) R. Br (Apocynaceae) commonly called as "crown flower" or "giant milk weed" is a well-known weed to many cultures for treating various disorders related to central nervous system, skin diseases, digestive system, respiratory system, reproductive system etc. Indigenous groups made the plant as a part of their lives since they use the fruit fibre to make ropes, household items, for weaving clothes and flowers for garlands apart from usage for various indications. The study aims at far-reaching review on phytochemistry, pharmacological activities, ethnopharmacology, intellectual property transfer on pharmacological therapies, toxicity which aids to provide scientific evidence for the ethnobotanical claims and to identify gaps required to be conducted as a future research prerequisite. MATERIALS AND METHODS: A systematic literature search was performed using different databases such as Scopus, Science direct, PubMed and Sciverse with no timeline limit set during the search. All the available abstracts and full text articles were included in the systematic review. RESULTS: Most of the folkloric uses were validated by the scientific studies such as analgesic, anti-arthritic, anti-asthmatic, anti-bacterial, anti-convulsant, anti-pyretic, central nervous system disorders, contraceptive, anti-ulcer and wound healing. In addition other studies such as anti-diabetic, anti-diarrhoeal, anti-helminthic, anti-histamine, anti-inflammatory, anti-microbial, anti-oxidant, cardio-protective studies, cytotoxicity, hepatoprotectivity, fibrinolytic, mosquitocidal, nerve muscle activity, vasodilation and skeletal muscle activities were also reported for the plant. Isolated compounds such as calotropin, frugoside and 4'-O-ß-D-glucopyranosyl frugoside were tested for the cytotoxicity efficacy against both human and rat cell lines out of which calotropin showed potent activity (IC50-15 ng/ml). However there were no clinical trials reported on the plant which is one of the major lacunas. CONCLUSIONS: This review article explores the ethnopharmacological, pharmacological activities phytochemistry and intellectual rights of Cg which gives the evidence of a potent and commercial drug which up on further research leads to the most viable drug for variety of treatments. However there is further need for in-vivo studies and clinical trials on isolated phytoconstituents which will help to commercialise.

Effects of kindling and irradiation on neuronal density in the rat dentate gyrus.

Low-dose radiosurgery is presently in use as a treatment modality for focal epilepsy, but the mechanisms underlying the associated changes in seizure expression are poorly understood. We investigated whether total and parvalbumin expressing (PV+) neuronal densities within the hippocampus and amygdala are affected by analogous focal irradiation in amygdala-kindled rats. Adult rats were kindled by electrical stimulation through 10 stage 5 seizures. The kindled amygdala was then focally irradiated at 18 or 25 Gy, and generalized seizure thresholds were subsequently monitored for approximately 6 months. Histological and immunohistochemical assays of total and PV+ neuronal densities were performed bilaterally throughout the hippocampus and within the basolateral amygdala. PV+ neuronal densities were unaffected by kindling or irradiation in these regions. Kindling selectively reduced neuronal densities in the dentate granule cell layer, and medial CA3 pyramidal cell layer. Irradiation at 25 Gy, but not at 18 Gy, prevented or reversed this kindling-associated reduction in density.