Raghuram Kandimalla

Chloramphenicol encapsulated in poly-ε-caprolactone–pluronic composite: nanoparticles for treatment of MRSA-infected burn wounds

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) infection has increased precipitously over the past several decades, with far-reaching health care and societal costs. MRSA infections in the context of burn wounds lead to invasive disease that could potentially cause mortality. Chloramphenicol is a well-known broad-spectrum bacteriostatic antibiotic that has been used since 1949, but due to its hydrophobicity, poor penetration in skin, fast degradation, and toxicity, its application has been hindered. Furthermore, it has been demonstrated that old antibiotics such as chloramphenicol remained active against a large number of currently prevalent resistant bacterial isolates due to their low-level use in the past. Recently, the novel nanoparticulate drug-delivery system has been used and reported to be exceptionally useful for topical therapeutics, due to its distinctive physical characteristics such as a high surface-to-volume ratio and minuscule size. It helps to achieve better hydrophilicity, bioavailability, and controlled delivery with enhanced therapeutic index, which has resulted in decreased toxicity levels compared to the crude drug. Here, we report a novel chloramphenicol loaded with poly(ε-caprolactone) (PCL)-pluronic composite nanoparticles (CAM-PCL-P NPs), physicochemical characterizations, and its bioactivity evaluation in a MRSA-infected burn-wound animal model. CAM-PCL-P NPs could encapsulate 98.3% of the drug in the nanoparticles and release 81% of the encapsulated drug over 36 days with a time to 50% drug release of 72 hours (51%). Nanoparticle suspensions maintained the initial properties with respect to size and encapsulation efficiency, even after 6 months of storage at 4°C and 25°C, respectively (P>0.05). Significant reduction in the level of toxicity was observed for CAM-PCL-P NPs compared with that of free drug as confirmed from hemolytic activity against human blood erythrocytes and cytotoxicity assay against an MCF-7 breast cancer cell line. In vitro antibacterial activities were performed by zone of inhibition, minimum inhibitory concentrations, minimum bacterial concentration, and time-kill assays, which showed that CAM-PCL-P NPs exhibited significantly enhanced anti-MRSA activity against ten clinical isolates of MRSA strains. The augmented activity of CAM-PCL-P NPs was further tested on a MRSA-infected burn-wound animal model and achieved quicker efficacy in MRSA clearance and improved the survival rate compared with free-chloramphenicol treatment. Thus, we propose CAM-PCL-P NPs as a promising novel antimicrobial candidate that may have a good potential for preclinical applications.

Potential of silk fibroin/chondrocyte constructs of muga silkworm Antheraea assamensis for cartilage tissue engineering

Articular cartilage damage represents one of the most perplexing clinical problems of musculoskeletal therapeutics due to its limited self-repair and regenerative capabilities. In this study, 3D porous silk fibroin scaffolds derived from non-mulberry muga silkworm Antheraea assamensis were fabricated and examined for their ability to support cartilage tissue engineering. Additionally, Bombyx mori and Philosamia ricini silk fibroin scaffolds were utilized for comparative studies. Herein, the fabricated scaffolds were thoroughly characterized and compared for cartilaginous tissue formation within the silk fibroin scaffolds seeded with primary porcine chondrocytes and cultured in vitro for 2 weeks. Surface morphology and structural conformation studies revealed the highly interconnected porous structure (pore size 80-150 μm) with enhanced stability within their structure. The fabricated scaffolds demonstrated improved mechanical properties and were followed-up with sequential experiments to reveal improved thermal and degradation properties. Silk fibroin scaffolds of A. assamensis and P. ricini supported better chondrocyte attachment and proliferation as indicated by metabolic activities and fluorescence microscopic studies. Biochemical analysis demonstrated significantly higher production of sulphated glycosaminoglycans (sGAGs) and type II collagen in A. assamensis silk fibroin scaffolds followed by P. ricini and B. mori scaffolds (p < 0.001). Furthermore, histochemistry and immunohistochemical studies indicated enhanced accumulation of sGAGs and expression of collagen II. Moreover, the scaffolds in a subcutaneous model of rat demonstrated in vivo biocompatibility after 8 weeks of implantation. Taken together, these results demonstrate the positive attributes from the non-mulberry silk fibroin scaffold of A. assamensis and suggest its suitability as a promising scaffold for chondrocyte based cartilage repair.

Fiber from ramie plant (Boehmeria nivea): A novel suture biomaterial

The quest for developing an ideal suture material prompted our interest to develop a novel suture with advantageous characters to market available ones. From natural origin only silk, cotton and linen fibers are presently available in market as non-absorbable suture biomaterials. In this study, we have developed a novel, cost-effective, and biocompatible suture biomaterial from ramie plant, Boehmeria nivea fiber. Field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and thermo gravimetric analysis (TGA) results revealed the physicochemical properties of raw and degummed ramie fiber, where the former one showed desirable characteristics for suture preparation. The braided multifilament ramie suture prepared from degummed fiber exhibited excellent tensile strength. The suture found to be biocompatible towards human erythrocytes and nontoxic to mammalian cells. The fabricated ramie suture exhibited significant antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus; which can be attributed to the inherent bacteriostatic ability of ramie plant fiber. In vivo wound closure efficacy was evaluated in adult male wister rats by suturing the superficial wound incisions. Within seven days of surgery the wound got completely healed leaving no rash and scar. The role of the ramie suture in complete wound healing was supported by the reduced levels of serum inflammatory mediators. Histopathology studies confirmed the wound healing ability of ramie suture, as rapid synthesis of collagen, connective tissue and other skin adnexal structures were observed within seven days of surgery. Tensile properties, biocompatibility and wound closure efficacy of the ramie suture were comparable with market available BMSF suture. The outcome of this study can drive tremendous possibility for the utilization of ramie plant fiber for various biomedical applications.

Penicillin impregnation on oxygen plasma surface functionalized chitosan/Antheraea assama silk fibroin: Studies of antibacterial activity and antithrombogenic property

Low temperature plasma can effectively tailor the surface properties of natural polymeric biomaterials according to the need for various biomedical applications. Non-mulberry silk, Antheraea assama silk fibroin (AASF) is a natural polymer having excellent biocompatibility and mechanical strength yet unlike mulberry silk, Bombyx mori silk fibroin, has drawn less interest in biomedical research. In the quest for developing as potential biomaterial, surface functionalization of plasma induced chitosan (Cs) grafted AASF ((AASF/O2-CS)g/O2) yarn is carried out using oxygen (O2) plasma. The (AASF/O2-CS)g/O2 yarn exhibits enhanced antithrombogenic property as well as antimicrobial activity against Gram positive (Bacillus subtilis) and Gram negative (Escherichia coli) bacteria as compared to AASF yarn. Moreover, impregnation of antibiotic drug (penicillin G sodium salt, PEN) on (AASF/O2-CS)g/O2 yarn further improves the observed properties. In-vitro hemolysis assay reveals that O2 plasma treatment and subsequent impregnation of PEN do not affect the hemocompatibility of AASF yarn. The present research findings demonstrate that plasma induced grafting of Cs followed by penicillin impregnation could significantly improve the potential applicability of AASF in the field of surgical research.

Antidiabetic and Antilipidemic Effect of Musa balbisiana Root Extract: A Potent Agent for Glucose Homeostasis in Streptozotocin-Induced Diabetic Rat

Folklore studies have revealed that Musa balbisiana Colla (MB; Family: Musaceae) has high medicinal properties. The purpose of the present study is to evaluate antihyperglycemic, and antioxidant activity of MB extracts in streptozotocin (STZ) induced diabetic rats. In vitro antioxidant and antidiabetic activity of MB extracts, i.e., root extract (RE), shoot extract and inflorescence extract were determined by using various methods viz 1,-1-diphenyl-2-picrylhydrazyl (DPPH) and a method to assess their possible effect on glucose diffusion across gastrointestinal tract and identify bioactive compound of potent extract. In vivo antilipidemic and antidiabetic activity was evaluated by administrating oral dose of RE for 15 days on STZ- induced diabetic rat. RE showed highest antioxidant activity by scavenging DPPH radical (IC50 32.96 μg/ml) and inhibit 30% glucose movement in vitro. The methanol extract of root showed the presence of calyx [4] arene category of the compound. Furthermore, RE treated rat revealed a reduction in fasting blood glucose (62.5%), serum total cholesterol (36.2%), triglyceride (54.5%), and low-density lipoprotein (50.94%) after 15 days as compared to STZ treated animal. There was an initiation of regenerative structures of the affected organs after 15 days of RE treatment. Histopathological observations clearly differentiate the structural changes in pancreas, liver, and kidney of STZ and RE treated group. The presence of calyx [4] arene class of compound may be responsible for its antioxidant and antidiabetic properties by absorbing glucose in vivo.

Anticancer Activity of Garcinia morella on T-Cell Murine Lymphoma Via Apoptotic Induction

Traditional knowledge (TK) based medicines have gained worldwide attention and presently the scientific community is focussing on proper pharmacological validation and identification of lead compounds for the treatment of various diseases. The North East region of India is the home of valuable traditional herbal remedies. Garcinia morella Desr. (Guttiferae) is one such medicinal plant used by traditional healers for the treatment of inflammatory disorders. The present study was aimed to evaluate the antioxidant and anticancer activity of methanol extracts of the leaf, bark and fruit of G. morella (GM) in different in vitro and in vivo experimental conditions. The results of this study showed that GM methanol extracts possessed in vitro antioxidant and anticancer properties, where the fruit extract (GF) showed maximum activity. The anticancer activity was further confirmed by the results of in vivo administration of GF (200 mg/kg) for ten days to Dalton’s lymphoma (DLA) induced mice. GF extract significantly increased the mean survival time (MST) of the animals, decreased the tumor volume and restored the hematological and biochemical parameters. The present study for the first time reported the anticancer property of GF on DLA. Further from the experiments conducted to elucidate the mechanism of action of GF on DLA, it can be concluded that GF exerts its anticancer effect through induction of caspases and DNA fragmentation that ultimately leads to apoptosis. However, further experimentation is required to elucidate the active principle and validate these findings in various in vivo settings.

Chemical Composition and Anti-Candidiasis Mediated Wound Healing Property of Cymbopogon nardus Essential Oil on Chronic Diabetic Wounds

Poor wound healing is one of the major complication of diabetic patients which arises due to different factors like hyperglycemia, oxidative stress, vascular insufficiency and microbial infections. Candidiasis of diabetic wounds is a difficult to treat condition and potentially can lead to organ amputation. There are a few number of medications available in market to treat this chronic condition; which demands for alternative treatment options. In traditional system of medicine like Ayurveda, essential oil extracted from leaves of Cymbopogon nardus L. (Poaceae) has been using for the treatment of microbial infections, inflammation and pain. In this regard, we have evaluated anti-Candida and anti-inflammatory activity mediated wound healing property of C. nardus essential oil (EO-CN) on candidiasis of diabetic wounds. EO-CN was obtained through hydro-distillation and subjected to Gas chromatography–mass spectroscopy (GC–MS) analysis for chemical profiling. Anti-Candida activity of EO-CN was tested against Candida albicans, C. glabrata and C. tropicalis by in vitro zone of inhibition and minimum inhibitory concentration (MIC) assays. Anti-candidiasis ability of EO-CN was evaluated on C. albicans infected diabetic wounds of mice through measuring candida load on the 7th, 14th, and 21st day of treatment. Further progression in wound healing was confirmed by measuring the inflammatory marker levels and histopathology of wounded tissues on last day of EO-CN treatment. A total of 95 compounds were identified through GC–MS analysis, with major compounds like citral, 2,6-octadienal-, 3,7-dimethyl-, geranyl acetate, citronellal, geraniol, and citronellol. In vitro test results demonstrated strong anti-Candida activity of EO-CN with a MIC value of 25 μg/ml against C. albicans, 50 μg/ml against C. glabrata and C. tropicalis. EO-CN treatment resulted in significant reduction of candida load on diabetic wounds. Acceleration in wound healing was indicated by declined levels of inflammatory cytokines at wounded area in EO-CN treated animals compared to non-treated group, which was further confirmed by histopathological examination. This study suggests that through significant anti-Candida and anti-inflammatory activity, EO-CN attenuates the growth of the fungus on diabetic wounds and simultaneously reduces the inflammation which leads to acceleration of the wound healing process.

Protective Effect of Bioactivity Guided Fractions of Ziziphus jujuba Mill. Root Bark against Hepatic Injury and Chronic Inflammation via Inhibiting Inflammatory Markers and Oxidative Stress

The tribal communities of North Eastern India rely on herbal medicine to cure various disease conditions. Ziziphus jujuba Mill. (Rhamnaceae) is one of such medicinal plants used for curing liver ailments, insomnia, anemia, diarrhea, diabetic complications, cancer, and loss of appetite. The present study was aimed to describe the protective ability of Z. jujuba root bark (ZJRB) against hepatic injury and chronic inflammation. Bioactivity guided fractionation of Z. jujuba methanol extract (ZJME) was performed using different solvents of increasing polarity viz. hexane (ZJHF), chloroform (ZJCF), ethyl acetate (ZJEAF), water (ZJWF), and residue (ZJMR). In vitro antioxidant results revealed that both ZJME and ZJWF possess strong antioxidant activity among all the fractions and mother extract tested. Further, ZJME and ZJWF showed significant protection against CCl4 intoxicated HepG2 cell lines by means of increased cell viability and decreased LDH levels compared to control group. ZJME at 200, 400 mg/kg and ZJWF at 50, 100 mg/kg inhibited the lipid peroxidation and significantly restored the liver function markers (AST, ALT, ALP, LDH, SOD, and CAT) and cytokine levels (TNF-α, Il-1β, and Il-10) in CCl4 induced acute liver damage in rats. All the results were comparable with standard drug silymarin which was further confirmed by histopathology analysis of liver. Similarly, inflammation and increase inflammatory cytokines levels of carrageenan induced paw edema in rats have been refurbished to normal levels on par with the standard drug indomethacin. ZJWF demonstrated potent response than ZJME in all the biological tests conducted. The results of the study signify the ability of ZJRB as good therapeutic agent for liver toxicity and chronic inflammation.

Dual delivery of chloramphenicol and essential oil by poly-ε-caprolactone–Pluronic nanocapsules to treat MRSA-Candida co-infected chronic burn wounds

Methicillin-resistant Staphylococcus aureus (MRSA) and Candida co-infections in the context of burn wounds, leads to morbidity and mortality. Eradication of such difficult to treat complex infection needs concomitant effective topical administration of an antibiotic–antifungal cocktail. Application of the broad-spectrum antibiotic chloramphenicol (CAM) has been hindered owing to its hydrophobicity, poor dermal penetration, rapid degradation and toxicity issues. But, due to its lesser uses in the past, CAM continues to be active against a majority of currently predominant resistant bacterial strains. Additionally, the essential oil of Cymbopogon flexuous possesses strong, broad spectrum antimicrobial property and also acts as a synergistic enhancer when used in combination with antibiotics. But due to its highly volatile nature and instability in free form, topical administration has not been possible. Keeping this in mind, we co-encapsulate CAM with lemongrass essential oil in PCL–Pluronic composite nanocapsules (CAM–LEO–PCL–P NCs) and were successful in overcoming the limitations in drug administration and achieved a significant reduction in cytotoxicity than that of the free counterparts with increased therapeutic index. CAM–LEO–PCL–P NCs exhibited significantly enhanced in vitro antimicrobial activity against 22 microbial pathogens including ten clinical MRSA isolates and three Candida species. This therapeutic nanocapsule was able to penetrate into burn wounds and demonstrated pronounced wound healing ability against MRSA-Candida co-infected burn wounds of mice by significant reduction of the pathogen burden. It also helps in increased cellular proliferation and collagen synthesis in the wounded area with the reduction of inflammatory cytokines. We propose CAM–LEO–PCL–P NCs as a potential candidate to treat chronic wound infections.

Bioactive Fraction of Annona reticulata Bark (or) Ziziphus jujuba Root Bark along with Insulin Attenuates Painful Diabetic Neuropathy through Inhibiting NF-κB Inflammatory Cascade

The present study explains the neuroprotective ability of bioactive fractions of Annona reticulata bark (ARB) and Ziziphus jujuba root bark (ZJ) along with insulin against diabetic neuropathy. By using different solvents of increasing polarity ARB and ZJ were undergone for bioactive guided fractionation. The neuroprotective ability of the all the plant fractions were tested against H2O2 induced toxicity in SHSY5Y neuroblastoma cell lines and DRG neuronal cells. Among all the fractions tested, the methanol extract of ARB and ZJ (ARBME and ZJME) and its water fractions (ARBWF and ZJWF) exhibited significant neuroprotection against H2O2 induced toxicity in SHSY5Y cells and DRG neuronal cells. Further both the active fractions were tested against streptozotocin (55 mg/kg i.p.) induced diabetic neuropathy in male Wistar rats. Body weight changes, blood glucose levels and pain threshold through hot plate, tail immersion, cold plate and Randall-Sillitto methods were measured throughout the study at weekly interval. After completion of the drug treatment period, all the animals were sacrificed to measure the sciatic nerve lipid peroxidation, antioxidative enzyme levels (SOD, catalase, and GSH) and cytokine levels (IL-1β, IL-6, IL-10, TNF-α, iNOS, and NFκB) through ELISA and western blotting analysis. Results of this study explain that ARBME, ZJME, ARBWF, and ZJWF along with insulin potentially attenuate the thermal, mechanical hyperalgesia and cold allodynia in diabetic neuropathic rats, where insulin treatment alone failed to diminish the same. Reduction of sciatic nerve oxidative stress, NF-κB and iNOS mediated inflammatory cascade and normalization of abnormal cytokine release confirms the possible mechanism of action. The present study confirms the neuroprotective ability of ARB and ZJ against painful diabetic neuropathy through inhibiting oxidative stress and NF-κB inflammatory cascade.