Formulation, biopharmaceutical evaluation and in-vitro screening of polyherbal phytosomes for breast cancer therapy.

Saudi Arabia has a rich culture of folk medicines and three such common herbs used by Saudi people for therapy of breast cancer are Turmeric (Kurkum) Curcuma longa, Chamomile (Babunaj) Matricaria chamomilla, and Aswaghantha (Aswaghadh) Withania somnifera. Hence, the present study aims to develop a polyherbal phytosome formulation by thin film hydration technique with a synergistic anti-cancer effect for the treatment of breast cancer. The phytosomes were standardized for their phytoconstituents by HPTLC and showed the best optimal properties with a mean vesicle diameter of less than 200 nm, zeta potential in the range of -24.43 to -35.70 mV, and relatively integrated structure with fairly uniform size on TEM. The in vitro MTT assay on MCF-7 breast cancer cell lines and MDA MB 231 breast adenocarcinoma cell lines was carried out. MTT assay on MCF-7 breast cancer cell lines indicated that plant extract-loaded phytosomes exhibited enhanced cytotoxic effects at IC50 values. of 55, 50, 45, 52, 42, 44, and 20 µg/mL compared to the extracts of C. longa, M. chamomilla, W. somnifera, and their combined extracts (80, 82, 74, 60, 70, 60, and 35 µg/mL respectively). Moreover, intracellular reactive oxygen species production was found to be higher for phytosomes treated cells at respective IC50 concentrations when compared to extracts. Overall, the developed polyherbal phytosomes were found to be effective and afford synergistic effects for breast cancer therapy.

Ziziphus mauritiana Lam attenuates inflammation via downregulating NFκB pathway in LPS-stimulated RAW 264.7 macrophages & OVA-induced airway inflammation in mice models.

ETHNOPHARMACOLOGICAL RELEVANCE: Ziziphus mauritiana Lam leaves were utilized in treating asthma, diabetes, inflammation, and hepatic diseases in Indian traditional medicine. The leaves were used as an edible vegetables in rural parts of India. AIM OF THE STUDY: The aim is to prove the anti-inflammatory activity of Ziziphus mauritiana Lam leaves against LPS-stimulated RAW 264.7 macrophages and OVA-induced airway inflammation in mice through its attenuation mechanism in the NFκB signalling pathway. MATERIALS AND METHODS: Terpenoids present in MEZ were quantified using U(H)PLC analysis. MEZ at 50 and 100 µg/mL were tested against LPS stimulated RAW 264.7 macrophages. The concentration of NO, ROS, and cytokines was quantified from the cell culture supernatants. OVA-induced asthma in mice was adopted for screening airway inflammation. MEZ at 250 and 500 mg/kg was tested for airway hyperresponsiveness, leukocyte counting, pro-inflammatory cytokines (IL-4, IL-5, IL-13 and TNF-α), lung histopathology, and various inflammatory gene expressions in lungs for NFκB signalling pathway in asthma. RESULTS: Terpenoids like betulin, betulinic acid, oleanolic acid, and ursolic acid were quantified from U(H)PLC analysis. MEZ at higher doses reduced the NO, ROS, and pro-inflammatory cytokines in LPS stimulated RAW 264.7 macrophages. MEZ at 500 mg/kg significantly reduced AHR and also decreased total and differential leukocytes. MEZ also reduced the expressions of ICAM, VCAM, and Muc5C genes. Histopathological analysis revealed MEZ significantly reduced the leukocyte infiltration and mucus hypersecretion in the lungs. MEZ suppressed lung inflammation by inhibition of p65 mediated IκB-α translocation in the NFκB signalling pathway. CONCLUSION: From these findings, MEZ significantly reduced airway inflammation by inhibiting NFκB mediated inflammatory pathway. Hence, this study proved that Ziziphus mauritiana Lam has anti-asthmatic potential in Indian traditional medicine.

Standardization, in-silico and in-vivo safety assessment of methanol extract of Ziziphus mauritiana Lam leaves.

Ziziphus mauritana Lam leaves were used to treat asthma, diabetes, pain, and inflammation in the Indian traditional system of medicine. The leaves of the Ziziphus mauritiana Lam were consumed as a vegetable in Indonesia and India. The present study aims to predict the pharmacokinetic properties of flavonoids identified & quantified through U(H)PLC and to evaluate the safety of methanol extract of Ziziphus mauritana Lam leaves (MEZ) in rats. A U(H)PLC-ESI-QTOF-MS/MS was performed to identify flavonoids present in MEZ and quantified using U(H)PLC method. The in-silico ADME properties of the flavonoids were analyzed using Schrodinger Maestro software. The acute oral toxicity study was performed by administering a single dose of MEZ (5000 mg/kg) in female rats and observed for 14 days. The sub-chronic studies were carried out by oral administration of MEZ at 500, 750, and 1000 mg/kg daily for 90 days. The changes in hematological parameters, clinical biochemistry, and histopathology were observed after the treatment period. Eight flavonoids rutin, kaempferol, luteolin, myricetin, catechin, and apigenin were identified from were identified in UPLC-QTOF-MS/MS analysis. These results showed the highest amount of luteolin (5.41 µg/ml) and kaempferol (4.02 µg/ml) present in MEZ. No signs of toxicity or mortality were observed in acute toxicity studies. In the sub-chronic studies, data showed that MEZ does not produce any changes in hematological and clinical biochemical parameters compared to control rats. MEZ (1000 mg/kg) significantly (p < 0.05) reduced total cholesterol, triglycerides, in male rats, which was more prominent on day 90. The histopathological analysis also revealed no changes in the vital organs. These results conclude that MEZ was considered safe and well-tolerated in rats.

Combinatorial analysis of quercetin and resveratrol by HPTLC in Sesbania grandiflora/phyto-based nanoformulations.

A sensitive HPTLC method was developed for the simultaneous estimation of quercetin (QUR) and resveratrol (RES). The chromatographic separation was achieved using mobile phase toluene:chloroform:ethyl acetate:formic acid (3:2:4.9:0.1% v/v) and densitometric scan performed at 280 nm. The developed method was linear at 2-10 µg/mL with correlation coefficient of 0.9907 (QUR) and 0.9917 (RES). The method was validated for its precision, specificity, detection and quantification limits and % RSD was found to be less than 4.0%. The developed HPTLC method was evaluated in QUR and RES-loaded nanoformulation and Sesbania grandiflora leaf extract. The amount of QUR and RES present in the SG leaf extract was found to be 26.13 ± 0.7 µg/mg and 4.31 ± 0.8 µg/mg, respectively. The pH-dependent stability of RES has checked using the developed method. The above-developed method can be used to check the QUR/RES content in herbal/pharmaceutical formulation with scope towards industries.

Myxopyrum serratulum ameliorates airway inflammation in LPS-stimulated RAW 264.7 macrophages and OVA-induced murine model of allergic asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Myxopyrum serratulum A. W. Hill. (Oleaceae) is a traditionally used Indian medicinal plant for the treatment of cough, asthma and many other inflammatory diseases. AIM OF THE STUDY: In this study, the protective effects of M. serratulum on airway inflammation was investigated in ovalbumin (OVA)-induced murine model of allergic asthma and lipopolysaccharide (LPS)-stimulated inflammation in RAW 264.7 murine macrophages, and the possible mechanisms were elucidated. MATERIALS AND METHODS: The phytochemicals present in the methanolic leaf extract of M. serratulum (MEMS) were identified by reverse phase high performance liquid chromatography (RP-HPLC) analysis. In vitro anti-inflammatory activity of MEMS were evaluated by estimating the levels of nitric oxide (NO), reactive oxygen species (ROS) and cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, IFN-γ, TNF-α, G-CSF and GM-CSF) in LPS-stimulated RAW 264.7 macrophages. In vivo anti-asthmatic activity of MEMS was studied using OVA-induced murine model. Airway hyperresponsiveness (AHR), was measured; total and differential cell counts, eosinophil peroxidase (EPO), prostaglandin E2 (PGE2), NO, ROS, and cytokines (IL-4, IL-5 and IL-13), were estimated in bronchoalveolar lavage fluid (BALF). Serum total IgE level was measured; and the histopathological changes of lung tissues were observed. The expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in lung tissue homogenates were detected by Western blot. RESULTS: The chromatographic analysis of MEMS identified the presence of gallic acid, protocatechuic acid, catechin, ellagic acid, rutin, p-coumaric acid, quercetin, naringenin and apigenin. MEMS (125 and 250 µg/mL) dose-dependently reduced the levels of NO, ROS and pro-inflammatory cytokines in LPS-stimulated RAW 264.7 macrophages. MEMS (200 and 400 mg/kg, p.o.) significantly (p < 0.05) alleviated AHR; number of inflammatory cells, EPO, PGE2, NO, ROS, and cytokines (IL-4, IL-5 and IL-13) in BALF; serum total IgE and the histopathological changes associated with lung inflammation. Western blot studies showed that MEMS substantially suppressed COX-2 and iNOS protein expressions in the lung tissues of OVA-sensitized/challenged mice. CONCLUSIONS: The present study corroborates for the first time the ameliorative effects of MEMS on airway inflammation by reducing the levels of oxidative stress, pro-inflammatory cytokines and inhibiting COX-2, iNOS protein expressions, thereby validating the ethnopharmacological uses of M. serratulum.

HPLC-ESI-QqQ based standardization, mutagenic and genotoxic potential of methanol extract of Ziziphus mauritiana Lam leaves.

BACKGROUND: The leaves of Ziziphus mauritiana Lam have been an integral part of the traditional system of medicine for the treatment of inflammation, wounds, fever, asthma and liver disorders. The leaves are utilised as an edible vegetable in rural parts of India and Indonesia. Despite its pharmacological significance, Ziziphus mauritiana Lam lacks scientific evidence on its mutagenic and genotoxic potential. RATIONALE: The aim of the present work is to identify bioactive compounds present in the methanol extract of Ziziphus mauritiana Lam leaves (MEZ) using HPLC-ESI-QqQ and to evaluate its mutagenic and genotoxic potential. METHODS: The phytochemical standardization of the MEZ was done using HPLC-ESI-QqQ. The mutagenic and genotoxic potential of MEZ was tested using bacterial reverse mutation (Ames test), chromosomal aberration, and micronucleus tests. The Ames test was performed in Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537, and the genotoxic potential was tested in in-vitro using chromosome aberration assay with Chinese hamster ovary (CHO) cells and in-vivo micronucleus test using mouse bone marrow cells. RESULTS: Fifteen phytochemical compounds were identified in HPLC-ESI- QqQ. It was observed from the Ames test that MEZ did not induce gene mutations in the S. typhimurium in the presence or absence of S9 activation. Similarly, no significant increase in the number of structural aberrations was observed in CHO cells with or without S9 activation. The oral administration of MEZ at a dose of up to 2000 mg/kg caused no significant increase in the number of micronucleated polychromatic erythrocytes or in the mean ratio of polychromatic to total erythrocytes. CONCLUSION: The findings of the present study confirm that MEZ is not-mutagenic and non-genotoxic in the presence or absence of the exogenous metabolizing system.

Design and development of artemisinin and dexamethasone loaded topical nanodispersion for the effective treatment of age-related macular degeneration.

Age-related macular degeneration (AMD) is a disease affecting the macula by the new blood vessels formation. AMD is widely treated with a combination of anti-angiogenic and anti-vascular endothelial growth factor (VEGF) agents. The topical administration of nanodispersions showed enhanced ocular residence time with controlled and prolonged drug delivery to the disease site at the back of the eye. In the present study we developed and characterized nanodispersion containing anti-angiogenic (artemisinin) and anti-VEGF agent (dexamethasone) for the topical ocular administration in order to obtain a required drug concentration in the posterior part of the eye. The nanodispersions were prepared with varying concentration of polymer, polyvinyl pyrrolidone K90 and polymeric surfactant, Poloxamer 407. The nanodispersions were found to be smooth and spherical in shape with a size range of 12-26 nm. In-vitro drug release studies showed the 90-101% of artemisinin and 55-103% of dexamethasone release from the nanodispersions. The blank formulation with a high concentration of polymer and polymeric surfactant showed an acceptable level of haemolysis and DNA damage. The chorioallantoic membrane assay suggested that the nanodispersion possess good anti-angiogenic effect. Hence the formulated artemisinin and dexamethasone nanodispersion may have the great potential for the AMD treatment.

Evaluation of antidiabetic activity of bud and flower of Avaram Senna (Cassia auriculata L.) In high fat diet and streptozotocin induced diabetic rats.

Type 2 Diabetes Mellitus (T2DM) is very common metabolic disorder affecting people of all age groups. The change in life style and environmental factors are the considerable factors which are involved in the development of the disorder. The different parts of medicinal plants vary in their composition of bioactive compounds. There are reports on antidiabetic activity of C. auriculata L. flower and leaves. Traditionally bud of C. auriculata L. is used to treat diabetes rather than flower. This study aims to explore the antidiabetic efficiency of bud and flower and to identify the differential composition of phycompounds present in bud and flower parts of C. auriculata L. The compounds present in the bud and flower parts were identified using LC-ESI/MS analysis. Antidiabetic activity of C. auriculata L. bud and flower parts was studied in high fat diet (HFD) and streptozotocin (STZ) induced diabetic rats. During which parameters such as feed intake, water intake, and body weight were monitored. After 21 days of the study, blood parameters like insulin, glucose, lipid profile, hepatic function test, renal function test and oxidative stress markers were analysed. Real time PCR was done to monitor the expression of IRS2 and GRIA2 genes. The LC-ESI/MS analysis showed the presence of various phenolics and flavonoid compounds specific to bud and flower parts. The antidiabetic activity results showed that the animal treated with C. auriculata L. bud ethanol extract (CABE500) could better reverse and control the progression of the disease compared to the flower ethanol extract. The gene expression studies revealed that regulation of IRS2 gene occurred in bud but not in flower extract treated animal livers and no differential expression of GRIA2 gene in all the experimental groups. C. auriculata L. bud extract can potentially better control the diabetes compared to the flower extract.

Novel nano therapeutic materials for the effective treatment of rheumatoid arthritis-recent insights.

Rheumatoid arthritis (RA) is the most common complex multifactorial joint related autoimmune inflammatory disease with unknown etiology accomplished with increased cardiovascular risks. RA is characterized by the clinical findings of synovial inflammation, autoantibody production, and cartilage/bone destruction, cardiovascular, pulmonary and skeletal disorders. Pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and IL-10 were responsible for the induction of inflammation in RA patients. Drawbacks such as poor efficacy, higher doses, frequent administration, low responsiveness, and higher cost and serious side effects were associated with the conventional dosage forms for RA treatment. Nanomedicines were recently gaining more interest towards the treatment of RA, and researchers were also focusing towards the development of various anti-inflammatory drug loaded nanoformulations with an aid to both actively/passively targeting the inflamed site to afford an effective treatment regimen for RA. Alterations in the surface area and nanoscale size of the nanoformulations elicit beneficial physical and chemical properties for better pharmacological activities. These drug loaded nanoformulations may enhances the solubility of poorly water soluble drugs, improves the bioavailability, affords targetability and may improve the therapeutic activity. In this regimen, the present review focus towards the novel nanoparticulate formulations (nanoparticles, nanoemulsions, solid lipid nanoparticles, nanomicelles, and nanocapsules) utilized for the treatment of RA. The recent advancements such as siRNA, peptide and targeted based nanoparticulate systems for RA treatment were also discussed. Special emphasis was provided regarding the pathophysiology, prevalence and symptoms towards the development of RA.

Chitosan stabilized camptothecin nanoemulsions: Development, evaluation and biodistribution in preclinical breast cancer animal mode.

Clinical use of camptothecin (CPT) is hindered due to its poor water and oil solubility, active lactone ring instability and non-targeted toxicity. Recently we reported formulation of camptothecin microemulsions with increased solubility for the improved treatment of breast cancer. In this research chitosan stabilized camptothecin nanoemulsions (CHI-CPT-NEs) were formulated improve the cancer targeting efficiency of CPT. The developed NEs were characterized for their droplet size distribution, stability in plasma and evaluated for in-vitro drug release, in-vivo targeting potential, in-vitro hemolytic potential, cytotoxicity, genotoxicity and in-vivo biodistribution. The CHI-CPT-NEs showed uniform droplet size distribution, extended drug release (61.65±1.57% at 24h), tolerable hemolytic potential (16.4±1.4%), significant cytotoxicity (178±4.3ng/ml) against MCF-7 cancer cells and low DNA damage to lymphocytes. In-vivo biodistribution study conducted in 4T1-breast tumor xenograft BALB/c mice showed that 2495.22±174.66ng/gm of camptothecin was passively targeted to breast cancer by CHI-CPT-NEs compared to the non-stabilized nanoemulsion (1677.58±134.21ng/gm). Thus, passive targeting of developed CHI-CPT-NEs may provide a promising approach for the efficient breast cancer therapy.

Physicochemical characteristics and antioxidant activity of Prunus cerasoides D. Don gum exudates.

The physicochemical properties and antioxidant activity of Prunus cerasoides D. Don gum exudates was investigated in this study. The total carbohydrate and protein content were found to be 73.72±2.44% and 2.33±1.25%, respectively. Analysis of monosaccharide composition by HPLC-RI system after acid hydrolysis of the gum showed the presence of arabinose, galactose, glucose, rhamnose and xylose. The molecular weight of the gum was also found to be 5.55×10(5)Da. FTIR and DSC studies showed characteristics typical of a natural polysaccharide. The viscosity of 2% aqueous solution of the gum exhibited non-Newtonian type of flow and the gum was also found to show pH dependent swelling. Determination of the angle of repose, Carr's index and Hausner ratio indicate the gum possess fairly good powder flow property. The antioxidant properties of the gum were evaluated by determining DPPH and hydroxyl scavenging activities, reducing power and total phenolic contents which showed the gum possess antioxidant property.