ABSTRACT
Currently, bone tuberculosis (TB) treatment largely involves lifelong drug prescriptions and surgical intervention, resulting in poor quality of life for patients. Therefore, the fabrication of injectable scaffolds to form a solid framework around the defective bone region is gaining importance over the extensive use of antimicrobial inhibitors. Herein, we synthesized a novel bone-adhesive and thermoresponsive hydrogel via conjugation of poly(N-isopropylacrylamide-co-glycidyl methacrylate) (PNIPAM-co-GMA) and cysteine (CYS). Thiolation of the polymer enables chemical cross-linking with the bone glycoprotein, enhancing bone adhesion and permitting control of scaffold retention time. The PNIPAM-co-GMA-CYS hydrogel shows higher cross-linking behavior at 37 °C, forms a strong gel in 260 s, and has 151 kPa adhesion strength on cortical bone. The lead compounds 5-methyl-5H-[1,2,4]triazino[5,6-b]indole-3-thiol (MTIT) and N-tert-butyl-4-methyl-6-(5-methyl-5H-[1,2,4]triazino[5,6-b]indol-3-ylthio)pyrimidin-2-amine (TMTIPA) were identified by a high-throughput screening method. Effective MTIT and TMTIPA are encapsulated in bone-adhesive hydrogel separately, and both have a high release rate above >70% in 180 h. The MTIT- and TMTIPA-loaded PNIPAM-co-GMA-CYS showed an excellent bactericidal effect, reducing the relative intracellular bacterial survival in macrophages. Furthermore, the as-synthesized hydrogel has outstanding mechanical and biocompatibility properties to become a bone-replacing material and provide support to promote bone repair. This work presents a novel bone-adhesive PNIPAM-co-GMA-CYS for the sustained release of lead compounds toward promising alternative bone TB treatment.
Subject(s)
Hydrogels , Tuberculosis , Humans , Hydrogels/chemistry , Quality of Life , Polymers , OsteoblastsABSTRACT
Bacterial polysaccharides can be easily modified to offer dual stimuli-responsive drug delivery systems with double targeting potential. In this research work, bacterial polysaccharides hyaluronic acid (HA) were functionalized with α-tocopherol polyethylene glycol succinate (TPGS) and cholic acid (CA) to form multifunctional polysaccharides nanoconjugates (TPGS-HA-CA). Smart nanoconjugates were synthesized by forming a redox-responsive disulfide bond, and it is composed of double targeting ligands. Doxorubicin (DOX) encapsulated smart nanoconjugates were exhibited an average size of 200 nm with a uniform core-shell structure. It serves the pH-responsive side chain modulation of TPGS-HA-CA, which affords a high degree of swelling at acidic pH. Under the pH 5.0 it shows 57% of release due to the side chain modulation of C-H/N-H. Polysaccharides nanoconjugates exhibited the double stimuli-responsive drug delivery by rapid disassembly of disulfide linkage, which exhibited 72% drug release (pH 5.0+GSH 10 mM). In cytotoxic studies, DOX@TPGS-HA-CA exhibited a higher cytotoxic effect compared to DOX. Hyaluronic acid functionalization with CA, TPGS increases cell internalization, and dual stimuli activity promotes more cell death. Overall, multifunctional polysaccharides hydrogel nanoconjugates is a prospective material that has great potential for targeting breast cancer therapy.
Subject(s)
Antineoplastic Agents , Nanoconjugates , Humans , Nanoconjugates/chemistry , Hyaluronic Acid/chemistry , Antineoplastic Agents/therapeutic use , Doxorubicin , Drug Delivery Systems , Disulfides , Drug Liberation , Hydrogen-Ion ConcentrationABSTRACT
The shape and responsiveness of nanoengineered delivery carriers are crucial characteristics for the rapid and efficient delivery of therapeutics. We report on a novel type of micrometer-sized hydrogel particles of controlled shape with dual pH and redox sensitivity for intracellular delivery of anticancer drugs and phototherapy. The cubical HA-DOP-CS-PEG networks with disulfide links are obtained by cross-linking HA-DOP-CS-PEG with cystamine. The pH-triggered hydrogel swelling/shrinkage was not only affords effective doxorubicin release. It also actively provides the endosomal/lysosomal escape, redox-triggered drug release. The hydrogels degrade rapidly to low molecular weight chains in the presence of the typical intracellular concentration of glutathione. Drug-loaded cube particles found to be 12% more cytotoxic. ICG and DOX-loaded hydrogel cubes demonstrate 90% cytotoxicity when incubated with MCF-7 cancer cells for 24 and 48 h, respectively. This approach integrates the advantages of pH sensitivity, enzymatic degradation, and shape-regulated internalization for novel types of "intelligent" three-dimensional networks with programmable behavior for controlled delivery of therapeutics.
Subject(s)
Antineoplastic Agents , Breast Neoplasms , Antineoplastic Agents/chemistry , Doxorubicin/chemistry , Doxorubicin/pharmacology , Drug Carriers/chemistry , Drug Delivery Systems/methods , Drug Liberation , Female , Humans , Hydrogels/chemistry , Hydrogen-Ion Concentration , PhototherapyABSTRACT
Bioresources are attaining much importance in the discovery of drugs and delivering agents. In particular, marine sponges are of great interest due to their metabolites production for the survival in risky environment. The incorporation of silver nanoparticles with marine sponge derived metabolites was reported for the first time. In this work, a facile material has been generated of great efficacy in solving environmental and health issues, as a recipe of silver and marine sponge Clathria frondifera, named as Ag Fortified Sponge spheres (AFS). AFS spheres were successfully synthesized after method optimization, using the various extracts of marine sponge Clathria frondifera as effective reducing agent in Ag (I) to Ag (0) reduction. Bioactive material from marine sponge and AgNP from the reduction of AgNO3 solution stablishing one another and thus AFS spheres were attaining long lifetime along with enhanced antimicrobial activity. The characterization of synthesized AFS and other AgNPs (1-4) has done using FT-IR, PXRD, FESEM, TEM, and UV-vis data. The presence of functional groups such as, Ag-O, and Ag-C stretching bonds in the AFS compounds indicated that it is composed of silver oxides and organo-silver, respectively. The synthesized Ag NPs were found to be spherical like structure with an average size of â¼20 nm. The cytotoxic response of AFS was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and morphological changes. AFS are exact spherical, micro sized and effective in inhibiting the growth of both gram positive and gram-negative bacteria. Anticancer studies were also carried out and ensued with excellent activity in the HELA cells with potential application in the medical industry.
Subject(s)
Metal Nanoparticles , Porifera , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , HeLa Cells , Humans , Metal Nanoparticles/chemistry , Metal Nanoparticles/toxicity , Microbial Sensitivity Tests , Microspheres , Porifera/metabolism , Silver/pharmacology , Spectroscopy, Fourier Transform InfraredABSTRACT
One of the very reliable, attractive, and cheapest techniques for synthesizing nanofibers for biomedical applications is electrospinning. Here, we have created a novel nanofibrous composite coated Ti plate to mimic an Extra Cellular Matrix (ECM) of native bone in order to enhance the bone tissue regeneration. An electrospun fibrous composite was obtained by the combination of minerals (Zn, Mg, Si) substituted hydroxyapatite (MHAP)/Polyethylene Glycol (PEG)/Cissus quadrangularis (CQ) extract. Fibrous composite's functionality, phase characteristics, and morphology were evaluated by FT-IR, XRD, and SEM techniques, respectively. The average fiber diameter of MHAP/PVA had decreased from ~274 to ~255 nm after incorporating PEG polymer. That further increased from ~255 to ~275 nm after adding CQ extract. Besides the bioactivity in SBF solution, the degradable nature was confirmed by immersing the fibrous composite in Tris-HCL solution. The degradable studies evaluate that the composite was degraded depending on time, and it degrades about 9.42% after 7 days of immersion. Osteoblasts like MG-63 cells differentiation, proliferation, and calcium deposition were also determined. These results show that this new fibrous composite exhibits advanced osteoblasts properties. Thus, we concluded that this new fibrous scaffold coated Ti implant could act as a better implant to mimic ECM of bone structure and to improve osteogenesis during bone regeneration.
Subject(s)
Nanofibers , Titanium , Bone Regeneration , Bone and Bones , Cell Proliferation , Durapatite , Osteoblasts , Spectroscopy, Fourier Transform Infrared , Tissue ScaffoldsABSTRACT
Here, the retinal targeting SA-g-AA coated multilamellar liposomes carrier synthesized to deliver the bioactive agents into the retinal region of the eye. The multilayered targeting macromolecules of liposomes prepared using a layer-by-layer assembly. The curcumin (CUR) and Rhodamine B (RhB) dyes loaded in a multilamellar vesicle (MLV) were synthesised by the lipid film hydration method. The sodium alginate grafted acrylic acid (SA-g-AA) conjugated with riboflavin (RB) was coated over MLV by O/W emulsion method followed by ionotropic gelation. FT-IR and 1H NMR spectroscopy techniques used to analyse the structural features of the MLV-SA-g-AA-RB. The results of DLS and TEM revealed that the carrier could be of uniform spheres, with a low polydispersity index, and outstanding performance in phrases of dye encapsulation and extended-release ability. An MTT assay investigated cell viability against Fibroblast WS1, and human embryonic stem cells-derived retinal pigment epithelial cells (hESC-RPE) implied that the carrier is of excellent biocompatibility. Retina targeting nature of the system confirmed via cellular uptake results revealed that the increases the dye concentration in the cells. Overall, the outcomes suggested that carriers could lead to the improvement of a feasible two photoreceptors targeting drug carriers, and it has the potential to deliver the multidrug in the retinal region of the eye.
Subject(s)
Alginates , Liposomes , Acrylates , Drug Carriers , Humans , Photoreceptor Cells, Vertebrate , Spectroscopy, Fourier Transform InfraredABSTRACT
Nanofibers based transdermal drug delivery is a promising platform, and it effectively delivers the drug to tumor sites. The objective of the study was to fabricate stimuli-responsive polymeric nanofibers encapsulated with an active targeting micellar system for in situ drug delivery. Stimuli-responsive core-shell nanofibers release thedrug at target sites with minimum side effects to the other organs, decrease the drug administration concentration. Initially, we prepared CA conjugated PCPP polymeric micelles loaded with PTX. Then, core-shell nanofibers were prepared using PHM with coaxial electrospinning and distinct core-shell nanofibers formation confirm by SEM and TEM. Nanofibers showed a homogenous distribution of micelles inside the fiber mesh, diffusion, and erosion processes lead to a controlled release of PTX.In vitro drug release and swelling, revealed the pH based sustained release of the drug for 180 h from the nanofibers mat. Functional and stimuli-responsive nanofibers highly absorb H+ ions and repulsion of cations promoting maximum swelling to release more drugs in acidic pH. An increased transportation rate of 70% drug release through epidermis for 120 h. Nanofibers effectively internalize to the skin, and it confirmed by confocal microscopy. MCF-7 cells grown and spread over the nanofibers, which show the biocompatibility of nanofibers. Compared to PTX, drug-loaded nanofibers exhibited higher cytotoxicity for 8 days which was confirmed by the flow cytometry. These promising results confirm, the novel stimuli-responsive core-shell nanofibers actively target breast cancer cells and lead the way to safe cancer therapy.
Subject(s)
Drug Carriers/pharmacokinetics , Epidermis/metabolism , Micelles , Nanofibers/chemistry , Paclitaxel/pharmacokinetics , Animals , Breast Neoplasms/drug therapy , Cell Survival , Cholic Acid/chemistry , Delayed-Action Preparations , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Liberation , Fibroblasts/metabolism , Humans , Hydrogen-Ion Concentration , MCF-7 Cells , Mice , Microscopy, Electrochemical, Scanning , Paclitaxel/administration & dosage , Polymers/chemistry , Psyllium/chemistry , Subcutaneous Absorption , SwineABSTRACT
Polysaccharides are increasingly used as biodegradable nanocarrier to selectively deliver therapeutic agents to specific cells. In this study, maleate gellan gum (MA-GG) formed by addition of free radical polymerizable groups, which can be polymerized presence of acetone to design biodegradable three-dimensional networks, were synthesized by esterification. Natural silk sericin was grafted over the maleate gellan gum surface. Maleate Gellan Gum- Silk Sericin-Chitosan (MA-GG-SS-CS) nanocomposites loaded with rifampicin (RF) and pyrazinamide (PZA) to overcome the problems associated with Tuberculosis (TB) therapy. The pH responsive behavior of gellan gum nanocomposites was reposed by silk sericin and exhibited sustained release of 79% RF and 82% PZA for 120â¯h at pHâ¯4.0. The designed formulations shows higher antimycobacterial activity and rapid delivery of drugs at TB infected macrophage. Nanomaterial effectively aggregated and internalized into the bacterial cells and MH-S cells. Dual drug release inside the cells makes damage in the cell membrane. Green nanocomposites studies pave the way for important use of macromolecules in pulmonary delivery TB drugs.
Subject(s)
Anti-Bacterial Agents/pharmacology , Chitosan/chemistry , Mycobacterium tuberculosis/drug effects , Nanocomposites/chemistry , Polysaccharides, Bacterial/chemistry , Sericins/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Biological Transport , Cell Line , Humans , Hydrogen-Ion Concentration , Maleates/chemistry , Materials Testing , Mycobacterium tuberculosis/cytologyABSTRACT
Polysaccharides protein complex offers a green alternative to synthetic polymers in the drug delivery system. Sericin (SC), a natural protein, in combination with rice bran albumin (RBA) and gellan gum (GG) forms a green based protein polysaccharide complex. The sericin functionalized gellan gum-rice bran (SC-GG-RBA) nanocomposites were characterized by different characterization techniques. It shows their prominent ability in balancing the biocompatibility, stability, biodegradability, and functionality of nanocarriers. The nanocomposites exhibited spherical shape with core protein-polysaccharide structures, and the average size was about 218â¯nm. High amount of Doxorubicin (DOX) was encapsulated into SC-GG-RBA nanocomposites in order to investigate the effective drug release in acidic tumor environment. DOX of 84% was released in vitro condition after 120â¯h in pHâ¯4.0. DOX loaded green nanocomposites shows IC50 5⯵g/mL which was very low compared to free DOX of 9⯵g/mL after treatment with MCF-7 cells. Only 42% of cells were survived after treatment with green nanocomposites. This was due to the effective uptake of nanomaterial by cancer cells and direct release of DOX in cytoplasmic region. Such high performance green nanocomposites have great potential in expanding the utilization of biomaterial from natural resources and development of sensible application in biomedical field.
Subject(s)
Albumins/chemistry , Drug Carriers/chemistry , Nanostructures/chemistry , Oryza/chemistry , Polysaccharides, Bacterial/chemistry , Sericins/chemistry , Biological Transport , Doxorubicin/chemistry , Doxorubicin/metabolism , Drug Liberation , Humans , MCF-7 Cells , Models, Molecular , Molecular ConformationABSTRACT
Recently, drug functionalized biodegradable polymers have been appreciated to be imperative to fabricate multi-drug delivery nanosystems for sustainable drug release. In this work, amphiphilic chitosan-grafted-(cetyl alcohol-maleic anhydride-pyrazinamide) (CS-g-(CA-MA-PZA)) was synthesized by multi-step reactions. The incorporation of rifampicin (RF) and entrapment of silver nanoparticles (Ag NPs) on CS-g-(CA-MA-PZA) polymer was carried out by dialysis technique. From the FT-IR experiment, the polymer modification, incorporation of drugs and the entrapment of Ag NPs on micelles were confirmed. The surface morphology of Ag NPs, polymeric system and drug loaded micelles was described by SEM, TEM and AFM techniques. In addition, the controlled release behaviour of CS-g-(CA-MA-PZA) micelles was studied by UV-Vis spectroscopy. In vitro cell viability, cell apoptosis and cellular uptake experiments shows that multi-drug delivery system could enhance the biocompatibility and higher the cytotoxicity effect on the cells. Since the prepared amphiphilic polymeric micelles exhibit spotty features and the system is a promising strategy for a novel candidate for immediate therapeutically effects for alveolar macrophages.
Subject(s)
Antitubercular Agents/administration & dosage , Chitosan , Drug Carriers , Drug Delivery Systems , Metal Nanoparticles , Polymers , Silver , Antitubercular Agents/chemistry , Apoptosis , Cell Line , Chitosan/chemistry , Drug Carriers/chemistry , Drug Compounding , Dynamic Light Scattering , Fatty Alcohols/chemistry , Humans , Maleic Anhydrides/chemistry , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Micelles , Microscopy, Atomic Force , Polymers/chemistry , Silver/chemistry , Spectroscopy, Fourier Transform Infrared , X-Ray DiffractionABSTRACT
Combinations of natural bee wax flavones chrysin with a chemo drug have been exhibiting high potential with reduced adverse effect. To extend the synergistic effect of chrysin and improve the MLNPs (Multi Layer Nanoparticles) performance in drug release, layer-by-layer of poly [di(sodium carboxyphenoxy)phosphazene] (PDCPP) and poly (diallyldimethyl ammonium chloride) (PDADMAC) deposited on the CaCO3 nanoparticles (CCNPs) surface. The results suggest spherical MLNPs of 237â¯nm are formed with high drug loading content with enhanced cellular uptake. Under acidic conditions, multi layer structure effectively controls burst release, providing sustained drug release for long period. The combined effect of chrysin and cisplatin improved the cytotoxic potential of MLNPs at 25⯵g.mL-1 concentration. Angiogenesis inhibitor chrysin activates reactive oxygen species (ROS) production and eventually leads to mitochondrial dysfunction. Furthermore, significant decreases of buccal pouch carcinoma in hamster model. Dual drug loaded MLNPs achieves 92% regressions of tumor volume as compared to cisplatin alone loaded MLNPs. In addition, Histopathology studies demonstrated the biocompatible effect of MLNPs on vital organs. This work provides a simple method to formulate multiple drugs in single nanosystem with high therapeutic efficacy on oral cancer.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Carcinoma/drug therapy , Delayed-Action Preparations/chemistry , Mouth Neoplasms/drug therapy , Nanoparticles/chemistry , Animals , Carcinoma/metabolism , Cell Line, Tumor , Cisplatin/chemistry , Cisplatin/pharmacology , Drug Carriers/chemistry , Drug Delivery Systems/methods , Drug Liberation/drug effects , Drug Synergism , Flavonoids/chemistry , Flavonoids/pharmacology , Humans , KB Cells , Male , Mesocricetus , Mitochondrial Diseases/drug therapy , Mitochondrial Diseases/metabolism , Mouth Neoplasms/metabolism , Polyethylenes/chemistry , Quaternary Ammonium Compounds/chemistry , Reactive Oxygen Species/metabolismABSTRACT
Natural rubber Latex (Lax) is a colloidal dispersion of polymer particles in liquid and shows good biodegradable, biocompatibility, and non-toxicity. Natural polymers are the most important materials used in food packaging, micro/nano-drug delivery, tissue engineering, agriculture, and coating. In the present study, natural compounds extracted from plant Lax were designed to function as drug carriers using various surfactants via emulation and solvent evaporation method. Calotropis gigantea belongs to the family Apocynaceae and has received considerable attention in modern medicine, ayurvedeic, siddha, and traditional medicine. Since, we were isolated biodegradable, non-toxic, and biocompatible materials as latex from Calotropis gigantea plant. The Lax was separated as per their solubility nature and it was designed as a carrier using surfactant namely; Sorbitanmonolaurate (Span-20), sodium lauryl sulfate (SLS), and cetyltrimethylammonium bromide (CTAB). The isolated compounds from Lax of Calotropis gigantea were analyzed using high-performance liquid chromatography. To confirm the encapsulation efficiency and in vitro drug release of the carriers, doxorubicin (DOX) was used as a model natural drug. The hybrid nanocarriers were successfully synthesized through simple solvent evaporation using three surfactants, and the morphology was characterized by SEM and TEM technique. The functionality and crystalline nature of the nanocarriers were confirmed using FTIR and XRD, respectively. Within 90min, the maximum amount of DOX was encapsulated in the carriers, and prolonged cumulative drug release by the nanocarriers was observed. The formulated natural carriers were found to have potentially effective cytotoxic effects on lung cancer cells.
Subject(s)
Calotropis/chemistry , Delayed-Action Preparations/chemistry , Doxorubicin/chemistry , Drug Carriers/chemistry , Latex/chemistry , Nanoparticles/chemistry , A549 Cells , Cell Line, Tumor , Cetrimonium , Cetrimonium Compounds/chemistry , Chemistry, Pharmaceutical/methods , Delayed-Action Preparations/pharmacology , Doxorubicin/pharmacology , Hexoses/chemistry , Humans , Lung Neoplasms/drug therapy , Plant Extracts/chemistry , Sodium Dodecyl Sulfate/chemistry , SolubilityABSTRACT
Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3%) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA3 [5 mg/l]) and higher direct somatic embryogenic frequency (89.6%). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA3 (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.
Subject(s)
Plant Somatic Embryogenesis Techniques , Podophyllotoxin/biosynthesis , Podophyllum/embryology , Podophyllum/metabolism , 2,4-Dichlorophenoxyacetic Acid/pharmacology , Endangered Species , Plant Shoots , Regeneration , Seeds/growth & developmentABSTRACT
Now-a-days synthesis and characterization of silver nanoparticles (AgNPs) through biological entity is quite interesting to employ AgNPs for various biomedical applications in general and treatment of cancer in particular. This paper presents the green synthesis of AgNPs using leaf extract of Podophyllum hexandrum Royle and optimized with various parameters such as pH, temperature, reaction time, volume of extract and metal ion concentration for synthesis of AgNPs. TEM, XRD and FTIR were adopted for characterization. The synthesized nanoparticles were found to be spherical shaped with average size of 14 nm. Effects of AgNPs were analyzed against human cervical carcinoma cells by MTT Assay, quantification of ROS, RT-PCR and western blotting techniques. The overall result indicates that AgNPs can selectively inhibit the cellular mechanism of HeLa by DNA damage and caspase mediated cell death. This biological procedure for synthesis of AgNPs and selective inhibition of cancerous cells gives an alternative avenue to treat human cancer effectively.
Subject(s)
Caspases/metabolism , Plant Extracts/chemistry , Podophyllum/chemistry , Silver/chemistry , Apoptosis/drug effects , Blotting, Western , Cell Line, Tumor , DNA Damage/drug effects , Female , Humans , Hydrogen-Ion Concentration , Metal Nanoparticles/adverse effects , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Microscopy, Electron, Transmission , Polymerase Chain Reaction , Spectroscopy, Fourier Transform InfraredABSTRACT
Withanolide is one of the most extensively exploited steroidal lactones, which are biosynthesized in Withania somnifera. Its production from cell suspension culture was analyzed to defeat limitations coupled with its regular supply from the plant organs. In order to optimize the different factors for sustainable production of withanolides and biomass accumulations, different concentrations of auxins or cytokinins and their combinations, carbon sources, agitation speed, organic additives and seaweed extracts was studied in cell suspension culture. Maximum biomass accumulation (16.72 g fresh weight [FW] and 4.18 g dry weight [DW]) and withanolides production (withanolide A 7.21 mg/g DW, withanolide B 4.23 mg/g DW, withaferin A 3.88 mg/g DW and withanone 6.72 mg/g DW) were achieved in the treatment of Gracilaria edulis extract at 40 % level. Organic additive L-glutamine at 200 mg/l in combination with picloram (1 mg/l) and KN (0.5 mg/l) promoted growth characteristics (11.87 g FW and 2.96 g DW) and withanolides synthesis (withanolide A 5.04 mg/g DW, withanolide B 2.59 mg/g DW, withaferin A 2.36 mg/g DW and withanone 4.32 mg/g DW). Sucrose at 5 % level revolved out to be a superior carbon source yielded highest withanolides production (withanolide A 2.88 mg/g DW, withanolide B 1.48 mg/g DW, withaferin A 1.35 mg/g DW and withanone 2.47 mg/g DW), whereas biomass (7.28 g FW and 1.82 g DW) was gratefully increased at 2 % level of sucrose in cell suspension culture. This optimized protocol can be utilized for large scale cultivation of W. somnifera cells in industrial bioreactors for mass synthesis of major withanolides.
Subject(s)
Biomass , Cell Culture Techniques/methods , Withania/cytology , Withania/growth & development , Withanolides/metabolism , Carbon/metabolism , Cells, Cultured , Culture Media , Withania/metabolismABSTRACT
This study optimized carbon sources in half MS liquid medium for maximum biomass accumulation and withanolides production in hairy root culture of Withania somnifera. The highest production of withaferin A and withanone was achieved when sucrose and sucrose+glucose were used individually as carbon sources. The hairy root suspension culture supplemented with a lower level of sucrose (2%) favored hairy root biomass accumulation (1.41 g DW) followed by sucrose+glucose (2+1) when compared with other carbon sources in half MS liquid medium after 40 days of culture. The hairy roots grown on sucrose (4%) enriched half MS liquid medium stimulated higher production of withaferin A (2.21 mg/g DW) and withanone (2.41 mg/g DW) on the 40th day of culture, followed by sucrose+glucose (4+1%) compared with glucose, fructose, maltose and other combinations tested.
Subject(s)
Antineoplastic Agents, Phytogenic/biosynthesis , Plant Roots/growth & development , Plant Roots/metabolism , Triterpenes/metabolism , Withania/chemistry , Withanolides/metabolism , Biomass , Carbohydrates/pharmacology , Carbon/metabolism , Chromatography, High Pressure Liquid , Culture Media , Sucrose/metabolism , Triterpenes/chemistry , Withanolides/chemistryABSTRACT
Adventitious root cultures derived from leaf derived callus of Withania somnifera (L.) Dunal were treated with methyl jasmonate and salicylic acid independently. Biomass accumulation, culture age, elicitation period, and culture duration were optimized for higher withanolides production in the two best-responding varieties collected from Kolli hills (Eastern Ghats) and Cumbum (Western Ghats) of Tamil Nadu, India. Between the two elicitors, salicylic acid (SA) improved the production of major withanolides (withanolide A, withanolide B, withaferin A, and withanone) as well as minor constituents (12-deoxy withastramonolide, withanoside V, and withanoside IV) in the Kolli hills variety. Treatment of root biomass (11.70 g FW) on 30-day-old adventitious root cultures with 150 µM SA for 4 h elicitor exposure period resulted in the production of 64.65 mg g(-l) dry weight (DW) withanolide A (48-fold), 33.74 mg g(-l) DW withanolide B (29-fold), 17.47 mg g(-l) DW withaferin A (20-fold), 42.88 mg g(-l) DW withanone (37-fold), 5.34 mg g(-l) DW 12-deoxy withastramonolide (nine fold), 7.23 mg g(-l) DW withanoside V (seven fold), and 9.45 mg g(-l) DW withanoside IV (nine fold) after 10 days of elicitation (40th day of culture) when compared to untreated cultures. This is the first report on the use of elicitation strategy on the significant improvement in withanolides production in the adventitious root cultures of W. somnifera.