Novel Methods to Generate Active Ingredients-Enriched Ashwagandha Leaves and Extracts.
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ABSTRACT:
SUBMITTER: Kaul SC
PROVIDER: S-EPMC5147857 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Insomnia is the most common sleep complaint which occurs due to difficulty in falling asleep or maintaining it. Most of currently available drugs for insomnia develop dependency and/or adverse effects. Hence natural therapies could be an alternative choice of treatment for insomnia. The root or whole plant extract of Ashwagandha (Withania somnifera) has been used to induce sleep in Indian system of traditional home medicine, Ayurveda. However, its active somnogenic components remain unidentified. We investigated the effect of various components of Ashwagandha leaf on sleep regulation by oral administration in mice. We found that the alcoholic extract that contained high amount of active withanolides was ineffective to induce sleep in mice. However, the water extract which contain triethylene glycol as a major component induced significant amount of non-rapid eye movement sleep with slight change in rapid eye movement sleep. Commercially available triethylene glycol also increased non-rapid eye movement sleep in mice in a dose-dependent (10-30 mg/mouse) manner. These results clearly demonstrated that triethylene glycol is an active sleep-inducing component of Ashwagandha leaves and could potentially be useful for insomnia therapy.
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Project description:Alzheimer's Disease (AD) is the most common form of dementia that is associated with extracellular amyloid beta (Aβ) plaque formation. Genetic, environmental, and nutrition factors have been suggested as contributors to oxidative stress and neuroinflammation events that are connected to AD etiology, and secondary metabolites, such as triterpenes, have shown promising results in AD prevention. In this work, the neuroprotective and anti-inflammatory potential of an olive leaves fraction enriched in triterpenoid compounds obtained using supercritical fluid extraction (SFE) and dynamic adsorption/desorption using sea sand as adsorbent has been performed. In addition, a comprehensive lipidomics study of the response of SH-SY5Y neuroblastoma cell line to this fraction was carried out using advanced analytical methodologies, namely, charged-surface hybrid chromatography-quadrupole-time of flight mass spectrometry (CSH-Q-TOF MS/MS). The use of freely available lipidomic annotation tools and databases, and stringent cut-off filters allowed the annotation of more than 250 intracellular lipids. Advanced bioinformatics and statistical tools showed a number of phosphatidylcholines and phosphatidylethanolamines significantly increased, which could explain the protection against the cell death caused by Aβ1-42. Moreover, several triacylglycerols were found decreased. These results suggest triterpenoids from olive leaves as good neuroprotective candidates, and open a new gate for future experiments using in vivo models to corroborate this hypothesis.
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Project description:Milk phospholipids (MPLs) have been used as ingredients for food fortification, such as bakery products, yogurt, and infant formula, because of their technical and nutritional functionalities. Starting from either buttermilk or beta serum as the original source, this review assessed four typical extraction processes and estimated that the life-cycle carbon footprints (CFs) of MPLs were 87.40, 170.59, 159.07, and 101.05 kg CO2/kg MPLs for membrane separation process, supercritical fluid extraction (SFE) by CO2 and dimethyl ether (DME), SFE by DME, and organic solvent extraction, respectively. Regardless of the MPL content of the final products, membrane separation remains the most efficient way to concentrate MPLs, yielding an 11.1-20.0% dry matter purity. Both SFE and solvent extraction processes are effective at purifying MPLs to relatively higher purity (76.8-88.0% w/w).
| S-EPMC7143133 | biostudies-literature
Project description:Extraction of valuable bioactive compounds from olive leaves is a hot topic and the use of sustainable and green technologies is mandatory in terms of circular economy. In this way, the use of fermentation technologies showed very interesting results in terms of phenolic compound recovery. Because of that in this work the use of solid state fermentations, as valuable tool to improve the phenolic extraction has been checked. Aspergillus oryzae (in mycelium and spore form), Aspergillus awamori and Aspergillus niger were used as fermentation microrganisms. Phenolic compounds were determined by HPLC-ESI-TOF-MS and, to our knowledge, new compounds have been tentatively identified in olive leaves. Fermentation using mycelium of Aspergillus awamori, Aspergillus niger and Aspergillus oryzae were effective to increase both hydroxytyrosol and elenolic acid derivatives whereas the use of spores of Aspergillus oryzae caused a loss of hydroxytyrosoyl derivatives, contrary the content of elenolic derivatives are comparable with the other fermentation treatments and higher than control. The proposed fermentation processes using the mycelium of Aspergillus awamori, Aspergillus niger and Aspergillus oryzae lead to an increase the hydroxytyrosyl and elenolic acid derivatives and could be used at industrial scale to obtain enriched extracts.
| S-EPMC9496001 | biostudies-literature
Project description:Pluripotency can be induced from different cell types using different methods. Several studies have reported that iPSC retain an epigenetic memory from the tissue from which they were derived. However, the influence of the method used for reprogramming in the final pluripotent state has not been investigated yet. Therefore, we have taken advantage of germline stem cells (GSCs) that can be converted into germline pluripotent stem cells (gPSCs) using specific culture conditions or into induced pluripotent stem cells (iPSCs) after the forced expression of four transcription factors. Here, we compare the gene expression profile, imprinting methylation pattern and differentiation potential between GSC-derived iPSCs and gPSCs. Our analysis demonstrates that both reprogramming methods induce equivalent levels of pluripotency and that a donor GSC transcriptional memory is retained only at early steps after reprogramming. In summary, our results show that different reprogramming methods generate undistinguishable pluripotent states and exclude a method-dependent epigenetic memory. Cell Culture. All the cell lines used in this study were derived from OG2 mice. Unless otherwise noted, all cell culture reagents were purchased from Gibco or PAA. Neonatal GSCs derived from 10 days postpartum (d.p.p) murine testis and adult GSCs derived from 35 d.p.p murine testis were derived as previously described and cultured on irradiated C3H feeder cells in a medium composed of StemPro-34 SFM (including supplement), N2 supplement (1:100), 1 % heat-inactivated fetal calf serum (FCS), 5 mg ml-1 bovine serum albumin (BSA) fraction V, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 2 mM Lâglutamine, 0.1 mM non-essential amino acids (NEAA), 0.1 mM βâmercaptoethanol, MEM vitamins (1:100), 6 mg ml-1 Dâ(+)âglucose (Sigma), 30 mg ml-1 pyruvic acid, 0.1 % DLâlactic acid (Sigma), 60 ng ml-1 progesterone (Sigma), 30 ng ml-1 βâestradiol (Sigma), 15 ng ml-1 mouse EGF (Peprotech), 10 ng ml-1 human bFGF (Peprotech), 10 ng ml-1 human GDNF (Peprotech), and 1000 U ml-1 mouse LIF (inâhouse preparation). For maintenance culture, GSCs were passaged at a 1:2âratio once a week after digestion with 0.05 % trypsin/EDTA. In addition, ESCs, iPSC and gPSC were cultured on irradiated C3H feeders in the following medium: Knockout DMEM, 20% Knockout Serum Replacement, penicillin, streptomycin, Lâglutamine, NEAA, βâmercaptoethanol, and LIF. Generation of pluripotent cell lines. iPSC were generated using tetracycline-inducible lentiviruses. For the lentiviral production, 293T cells were plated at 50 % confluency in 10-cm dishes one day prior to transfection. The next day, the lentiviral vector (3 µg) together with the packaging plasmids psPAX2 (2 µg) and pMD2.G (1 µg) (Addgene plasmids #12260 and #12259, respectively) were transfected using FuGENE 6 reagent (Roche) according to the manufacturerâs protocol. After 48 h, viral-containing supernatants were filtered (0.45 µm), concentrated around 30-fold by centrifugation (26,000 à g, 2 h, 4 °C) and stored at -80 °C. For the generation of iPSCs, a single-cell suspension of 50,000 GSCs was infected overnight with five tetracycline-inducible lentiviruses (5â20 µl concentrated supernatant each) encoding for Oct4, Sox2, Klf4, c-Myc, and the reverse tetracycline-transactivator M2-rtTA (Addgene plasmids #20323, #20326, #20322, #20324, and #20342) in the presence of 4 µg ml-1 polybrene (Sigma). One week after the infection, the expression of the viral transgenes was induced by adding 4 µg ml-1 doxycycline (Sigma) to the medium. MEFs-derived iPSCs were generated using the same protocol. gPSC were generated following a previous reported protocol. Briefly, GSCs were seeded at a density of approx. 1000 cells cm-2 on feeder cells and were not splitted until gPSC colonies were observed. Both iPSC and gPSC colonies were identified by Oct4-GFP expression, manually picked and expanded under ESC conditions. In vitro differentiation of pluripotent cells. Embryoid body (EB) formation was induced in suspension culture adopting the hanging drop method (600 cells in 20 µl) for 5 days in ESC medium without LIF. EBs were seeded on gelatine-coated plates and further cultured for 14 days in the same medium. Chimera formation. Aggregations of putative pluripotent cells with denuded 8-cell stage embryos were performed as previously reported. After overnight incubation at 37 °C and 5 % CO2, 11â14 aggregates were transferred into the uterine horns of 2.5 d.p.c pseudo-pregnant surrogate mothers (mated with vasectomized males). The chimeric pups were obtained and dissected at 14.5 d.p.c. Microarray gene expression analysis. Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 hours of in-vitro transcription incorporating biotin-labelled nucleotides. Hybridizations onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturerâs recommendations. Two replicates were hybridized from each sample. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Samples were hybridised as biological replicates. 26 samples were analyzed. iPSC[GSCneonatal]#1: iPSC day 10 from GSC, clone 1, 2 biological rep iPSC[GSCneonatal]#2: iPSC day 10 from GSC, clone 2, 2 biological rep iPSC[GSCadult]#1: iPSC day 40 from GSC, clone 1, 2 biological rep iPSC[GSCadult]#2: iPSC day 40 from GSC, clone 2, 2 biological rep gPSC[GSCneonatal]#1: gPSC day 10 from GSC, clone 1, 2 biological rep gPSC[GSCneonatal]#2: gPSC day 10 from GSC, clone 2, 2 biological rep gPSC[GSCadult]#1: gPSC day 40 from GSC, clone 1, 2 biological rep gPSC[GSCadult]#2: gPSC day 40 from GSC, clone 2, 2 biological rep ESC: OG2 ESCs, 2 biological rep iPSC[MEF]: iPSC (Tet-4F) from OG2 MEFs, 2 biological rep GSC[neonatal]: GSC day 10, 2 biological rep GSC[adult]: GSC day 40, 2 biological rep MEF: Mouse embryonic fibroblasts, 2 biological rep
2015-12-17 | E-GEOD-42993 | biostudies-arrayexpress
Project description:Pluripotency can be induced from different cell types using different methods. Several studies have reported that iPSC retain an epigenetic memory from the tissue from which they were derived. However, the influence of the method used for reprogramming in the final pluripotent state has not been investigated yet. Therefore, we have taken advantage of germline stem cells (GSCs) that can be converted into germline pluripotent stem cells (gPSCs) using specific culture conditions or into induced pluripotent stem cells (iPSCs) after the forced expression of four transcription factors. Here, we compare the gene expression profile, imprinting methylation pattern and differentiation potential between GSC-derived iPSCs and gPSCs. Our analysis demonstrates that both reprogramming methods induce equivalent levels of pluripotency and that a donor GSC transcriptional memory is retained only at early steps after reprogramming. In summary, our results show that different reprogramming methods generate undistinguishable pluripotent states and exclude a method-dependent epigenetic memory. Cell Culture. All the cell lines used in this study were derived from OG2 mice. Unless otherwise noted, all cell culture reagents were purchased from Gibco or PAA. Neonatal GSCs derived from 10 days postpartum (d.p.p) murine testis and adult GSCs derived from 35 d.p.p murine testis were derived as previously described and cultured on irradiated C3H feeder cells in a medium composed of StemPro-34 SFM (including supplement), N2 supplement (1:100), 1 % heat-inactivated fetal calf serum (FCS), 5 mg ml-1 bovine serum albumin (BSA) fraction V, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin, 2 mM L‑glutamine, 0.1 mM non-essential amino acids (NEAA), 0.1 mM β‑mercaptoethanol, MEM vitamins (1:100), 6 mg ml-1 D‑(+)‑glucose (Sigma), 30 mg ml-1 pyruvic acid, 0.1 % DL‑lactic acid (Sigma), 60 ng ml-1 progesterone (Sigma), 30 ng ml-1 β‑estradiol (Sigma), 15 ng ml-1 mouse EGF (Peprotech), 10 ng ml-1 human bFGF (Peprotech), 10 ng ml-1 human GDNF (Peprotech), and 1000 U ml-1 mouse LIF (in‑house preparation). For maintenance culture, GSCs were passaged at a 1:2‑ratio once a week after digestion with 0.05 % trypsin/EDTA. In addition, ESCs, iPSC and gPSC were cultured on irradiated C3H feeders in the following medium: Knockout DMEM, 20% Knockout Serum Replacement, penicillin, streptomycin, L‑glutamine, NEAA, β‑mercaptoethanol, and LIF. Generation of pluripotent cell lines. iPSC were generated using tetracycline-inducible lentiviruses. For the lentiviral production, 293T cells were plated at 50 % confluency in 10-cm dishes one day prior to transfection. The next day, the lentiviral vector (3 µg) together with the packaging plasmids psPAX2 (2 µg) and pMD2.G (1 µg) (Addgene plasmids #12260 and #12259, respectively) were transfected using FuGENE 6 reagent (Roche) according to the manufacturer’s protocol. After 48 h, viral-containing supernatants were filtered (0.45 µm), concentrated around 30-fold by centrifugation (26,000 × g, 2 h, 4 °C) and stored at -80 °C. For the generation of iPSCs, a single-cell suspension of 50,000 GSCs was infected overnight with five tetracycline-inducible lentiviruses (5–20 µl concentrated supernatant each) encoding for Oct4, Sox2, Klf4, c-Myc, and the reverse tetracycline-transactivator M2-rtTA (Addgene plasmids #20323, #20326, #20322, #20324, and #20342) in the presence of 4 µg ml-1 polybrene (Sigma). One week after the infection, the expression of the viral transgenes was induced by adding 4 µg ml-1 doxycycline (Sigma) to the medium. MEFs-derived iPSCs were generated using the same protocol. gPSC were generated following a previous reported protocol. Briefly, GSCs were seeded at a density of approx. 1000 cells cm-2 on feeder cells and were not splitted until gPSC colonies were observed. Both iPSC and gPSC colonies were identified by Oct4-GFP expression, manually picked and expanded under ESC conditions. In vitro differentiation of pluripotent cells. Embryoid body (EB) formation was induced in suspension culture adopting the hanging drop method (600 cells in 20 µl) for 5 days in ESC medium without LIF. EBs were seeded on gelatine-coated plates and further cultured for 14 days in the same medium. Chimera formation. Aggregations of putative pluripotent cells with denuded 8-cell stage embryos were performed as previously reported. After overnight incubation at 37 °C and 5 % CO2, 11–14 aggregates were transferred into the uterine horns of 2.5 d.p.c pseudo-pregnant surrogate mothers (mated with vasectomized males). The chimeric pups were obtained and dissected at 14.5 d.p.c. Microarray gene expression analysis. Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 hours of in-vitro transcription incorporating biotin-labelled nucleotides. Hybridizations onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturer’s recommendations. Two replicates were hybridized from each sample. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor.
2015-12-17 | GSE42993 | GEO
Project description:BackgroundA new model of care is required to meet the changing needs of people living with HIV (PLWH), particularly in low and middle-income countries, where prevalence is highest. We evaluated a palliative care intervention for PLWH in Mombasa, Kenya. Although we found no effect on pain (primary outcome), there was a positive effect on mental health (secondary outcome) in the intervention group. To inform replication and implementation, we have determined the active ingredients of the intervention and their mechanisms of action.MethodsWe conducted a randomised controlled trial (RCT) with qualitative exit interviews in HIV clinic attenders. The intervention was delivered over 5 months, with a minimum of 7 clinical contacts. Longitudinal quantitative data on components of care received were analysed using area under the curve and logistic regression. Qualitative data were analysed using inductive and deductive thematic analysis.ResultsQuantitative data analysis identified that intervention patients received more weak opioid, laxatives, discussion about spiritual worries, emotional support from staff for themselves and their families, time to talk about worries, discussion about future and planning ahead. Qualitative data analysis found that patients reported that having time to talk, appropriate pain medication and effective health education was of therapeutic value for their psychological well-being. Integration of mixed method findings suggest that positive effect in quantitative measures of mental health and well-being are attributable to the active ingredients of: appropriate medication, effective health education and counselling, and having time to talk in clinical encounters. Mechanisms of action include symptom relief, improved understanding of illness and treatment, and support focused on articulated concerns.ConclusionsRoutine care must provide opportunities and means for existing clinical staff to make routine appointments more person-centred. This approach enabled staff to identify and manage multidimensional problems and provide tailored health education and counselling.Trial registrationClinicalTrials.gov ( NCT01608802 ). Registered 12th May 2012.
| S-EPMC5761128 | biostudies-literature
Project description:The information of protein targets and small molecule has been highly valued by biomedical and pharmaceutical research. Several protein target databases are available online for FDA-approved drugs as well as the promising precursors that have largely facilitated the mechanistic study and subsequent research for drug discovery. However, those related resources regarding to herbal active ingredients, although being unusually valued as a precious resource for new drug development, is rarely found. In this article, a comprehensive and fully curated database for Herb Ingredients' Targets (HIT, http://lifecenter.sgst.cn/hit/) has been constructed to complement above resources. Those herbal ingredients with protein target information were carefully curated. The molecular target information involves those proteins being directly/indirectly activated/inhibited, protein binders and enzymes whose substrates or products are those compounds. Those up/down regulated genes are also included under the treatment of individual ingredients. In addition, the experimental condition, observed bioactivity and various references are provided as well for user's reference. Derived from more than 3250 literatures, it currently contains 5208 entries about 1301 known protein targets (221 of them are described as direct targets) affected by 586 herbal compounds from more than 1300 reputable Chinese herbs, overlapping with 280 therapeutic targets from Therapeutic Targets Database (TTD), and 445 protein targets from DrugBank corresponding to 1488 drug agents. The database can be queried via keyword search or similarity search. Crosslinks have been made to TTD, DrugBank, KEGG, PDB, Uniprot, Pfam, NCBI, TCM-ID and other databases.
| S-EPMC3013727 | biostudies-literature
Project description:Mulberry (Morus alba L.) leaves have long been considered beneficial in traditional Chinese medicine to treat infectious and internal diseases. Recently studies have discovered that the mulberry leaf's total flavonoids (MLF) display excellent hypoglycemia properties. However, the active ingredients and their molecular mechanisms are still uncharacterized. In this study, we explored the hypoglycemic effects of MLF and mulberry leaf polysaccharides (MLP) on ob/ob mice, an animal model of type 2 diabetes mellitus (T2DM), compared with Ramulus Mori (Sangzhi) alkaloid (RMA). Network pharmacology was employed to identify the potential available targets and active compounds of MLF and RMA against hyperglycemia. Molecular docking, an insulin-resistant cell model and qPCR were employed to verify the antidiabetic activity of the critical compounds and the gene expression profiles of the top molecular targets. Here, the results showed that MLF and MLP improved glucose uptake in insulin-resistant hepatocytes. MLF, MLP and RMA alleviated insulin resistance and glucose intolerance in ob/ob mice. Unlike MLF and MLP, RMA administration did not influence the accumulation of intrahepatic lipids. Network pharmacology analysis revealed that morusin, kuwanon C and morusyunnansin L are the main active compounds of MLF and that they amend insulin resistance and glycemia via the PI3K- Akt signaling pathway, lipid and atherosclerosis pathways, and the AGE-RAGE signaling pathway. Moreover, 1-deoxynojirimycin (DNJ), fagomine (FA), and N-methyl-1-deoxynojirimycin are the primary active ingredients of RMA and target carbohydrate metabolism and regulate alpha-glucosidase activity to produce a potent anti-diabetic effect. The molecular docking results indicated that morusin, kuwanon C and morusyunnansin L are the critical bioactive compounds of MLF. They had high affinities with the key targets adenosine A1 receptor (ADORA1), AKT serine/threonine kinase 1 (AKT1), peroxisome proliferator-activated receptor gamma (PPARγ), and glycogen synthase kinase 3 beta (GSK3β), which play crucial roles in the MLF-mediated glucose-lowering effect. Additionally, morusin plays a role in amending insulin resistance of hepatocytes by repressing the expression of the ADORA1 and PPARG genes. Our results shed light on the mechanism behind the glucose-lowering effects of MLF, suggesting that morusin, kuwanon C, and morusyunnansin L might be promising drug leads for the management of T2DM.
| S-EPMC9581293 | biostudies-literature
Project description:BackgroundLonicera japonica Thunb. is a plant used in traditional Chinese medicine known for its anti-inflammatory, anti-oxidative, anti-carcinogenic, and antiviral pharmacological properties. The major active secondary metabolites of this plant are chlorogenic acid (CGA) and luteoloside. While the biosynthetic pathways of these metabolites are relatively well known, the genetic information available for this species, especially the biosynthetic pathways of its active ingredients, is limited.Methodology/principal findingsWe obtained one million reads (average length of 400 bp) in a whole sequence run using a Roche/454 GS FLX titanium platform. Altogether, 85.69% of the unigenes covering the entire life cycle of the plant were annotated and 325 unigenes were assigned to secondary metabolic pathways. Moreover, 2039 unigenes were predicted as transcription factors. Nearly all of the possible enzymes involved in the biosynthesis of CGA and luteoloside were discovered in L. japonica. Three hydroxycinnamoyl transferase genes, including two hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase genes and one hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) gene featuring high similarity to known genes from other species, were cloned. The HCT gene was discovered for the first time in L. japonica. In addition, 188 candidate cytochrome P450 unigenes and 245 glycosyltransferase unigenes were found in the expressed sequence tag (EST) dataset.ConclusionThis study provides a high quality EST database for L. japonica by 454 pyrosequencing. Based on the EST annotation, a set of putative genes involved in CGA and luteoloside biosynthetic pathways were discovered. The database serves as an important source of public information on genetic markers, gene expression, genomics, and functional genomics in L. japonica.
| S-EPMC3636143 | biostudies-literature