Development and validation of HPLC method for simultaneous determination of Leflunomide and folic acid in the nanoparticulate system by reversed-phase HPLC.

OBJECTIVE: The main objective of this study was to develop a highly sensitive, accurate, and reproducible analytical method for the simultaneous detection of LEF and FA in polymeric nanocarriers. SIGNIFICANCE: Leflunomide (LEF), is widely employed in the treatment of rheumatoid arthritis (RA). However, long-term delivery of the drug is associated with systemic side effects. Therefore, folate (FA) conjugated LEF nanocarriers were fabricated for targeting the nanocarriers toward activated macrophages. HPLC is considered one of the most sensitive and precise analytical techniques for the simultaneous detection and estimation of different components in a particular sample. METHODS: Analysis was performed on HPLC (Shimadzu 10 A), having a reversed-phase C-18 column (Beckmen, 250 X 4.6 mm, 5 µm) equipped with a photodiode detector set at a wavelength of 260 nm (LEF) and 285 nm (Folic acid). The isocratic mobile phase was composed of acetonitrile, water, and trimethylamine in a ratio of 65:35:0.5 at pH 4. Rapid analysis of both agents was performed, with a total run time of 10 min (FA = 2.1 ± 0.1 min, LEF = 5.9 ± 1 min) at a 1 mL/min flow rate. RESULTS: The assay demonstrated good linearity of 0.9989 of 0.9997 for LEF and FA respectively with a recovery in the range of 95-100%. The method also depicted good specificity, and intra and inter-day precision based on relative standard deviation (RSD) values. CONCLUSIONS: The study concludes, that the developed method was helpful in the detection and quantitation of lower values of both agents from polymeric nanocarriers.

HighlightsOptimization and validation of the RP-HPLC method were performed for the simultaneous detection of LEF and FA.Validation was performed on the basis of linearity, accuracy, precision, LOD, LOQ, and robustness in accordance with ICH criteria.Validated analytical procedure was employed for the simultaneous detection of LEF and FA from polymeric nanocarriers.The proposed analytical method is reliable, fast, robust, and can be successfully applied for quantification of % EE, and % DL in polymeric nanocarriers.

Structural characterization and antileishmanial activity of newly synthesized organo-bismuth(V) carboxylates: experimental and molecular docking studies.

In a quest to discover new formulations for the treatment of various parasitic diseases, a series of heteroleptic triorganobismuth(V) biscarboxylates of type [BiR3(O2CR')2], where R=C6H5 for 1-4 and p-CH3C6H4 for 5-8, were synthesized, characterized and evaluated for their biological potential against L. tropica. All the synthesized complexes were fully characterized by elemental analysis, FT-IR, multinuclear (1H and 13C) NMR spectroscopy and X-ray crystallography. The crystal structures for [BiPh3(O2CC6H4(o-Br))2] (1), [BiPh3(O2CC2H2C6H4)2] (2), [BiPh3(O2CC6H4(m-NO2))2] (3) and [BiPh3(O2CC6H4(2-OH,3-CH3))2] (4) were determined and found to have a distorted pentagonal bipyramidal molecular geometry with seven coordinated bismuth center for 1-3 and for 4 distorted octahedral geometry, respectively. All the synthesized complexes demonstrated a moderate to significant activity against leishmania parasites. A broad analytical approach was followed to testify the stability for (1-8) in solid state as well as in solution and in leishmanial culture M199, ensuring them to be stable enough to exert a significant antileishmanial effect with promising results. Cytotoxicity profile suggests that tris(tolyl) derivatives show lower toxicity against isolated lymphocytes with higher antileishmanial potential. Molecular docking studies were carried out to reveal the binding modes for (1-8) targeting the active site of trypanothione reductase (TR) (PDB ID: 4APN) and Trypanothione Synthetase-Amidase structure (PDB ID 2vob).

Dibutylstannanediyl (2Z,2'Z)-bis(4-(benzylamino)-4-oxobut-2-enoate inhibits prostate cancer progression by activating p38 MAPK/PPARα/SMAD4 signaling.

Organotin (IV) compounds are a focus of research for potential use in cancer chemotherapy. Here, we established anticancer profile of dibutyltin (IV) carboxylate derivatives in prostate cancer (PCa) model. We determined cytotoxicity of a library of dibutyltin (IV) carboxylate derivatives and observed that dibutylstannanediyl (2Z,2'Z)-bis(4-(benzylamino)-4-oxobut-2-enoate (Ch-620; 10 µM) was minimally toxic to normal fibroblasts. Ch-620 (1-1.25 µM) inhibited proliferation of PCa and melanoma cells on short- and long-term exposures with induction of cell cycle arrest. Ch-620 treatment increased population of apoptotic cells, as assessed by flow cytometry, and activated caspase 3. Proteomics showed activation of PPARα, with repression of SMAD4 and integrin ß5 (ITGB5) in Ch-620-treated PCa cells. Further analysis demonstrated that Ch-620 resulted in phosphorylation of p38 MAPK, upregulation of PPARα and decreased expression of SMAD4 and ITGB5 with reduced migration of PCa cells. In vivo studies in PC3M grafted athymic nude mice showed that Ch-620 (5 µg/week; 7 weeks) treatment reduced tumor growth as opposed to untreated controls. Immunoblot analysis of tumors demonstrated upregulated p-p38 MAPK and PPARα, followed by a decline in SMAD4 and ITGB5. Immunohistochemistry reinforced these results with increased caspase 3 and p-p38 MAPK and diminished Ki67 staining in Ch-620 treated animals. Taken together, our data indicate that Ch-620 inhibited proliferation of PCa through modulation of MAPK/PPARα/SMAD4 signaling. Organotin (IV) carboxylate compounds; specifically Ch-620 can be a potential anticancer agent for the treatment of PCa subject to detailed pre-clinical and clinical investigations. This unlocks prospects for the development of new tin-based drugs in cancer therapeutics.

An Extensive Pharmacological Evaluation of New Anti-Cancer Triterpenoid (Nummularic Acid) from Ipomoea batatas through In Vitro, In Silico, and In Vivo Studies.

Prostate cancer (PCa) is the most common cancer in men, accounting for approximately 10% of all new cases in the United States. Plant-derived bioactive compounds, such as pentacyclic triterpenoids (PTs), have the ability to inhibit PCa cell proliferation. We isolated and characterized nummularic acid (NA), a potent PT, as a major chemical constituent of Ipomoea batatas, a medicinal food plant used in ethnomedicine for centuries. In the current study, in vitro antiproliferative potential against PCa cells (DU145 and PC3) via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay; Western blot protein expression analysis; absorption, distribution, metabolism, excretion (ADME); pharmacokinetic prediction studies; and bisphenol A (BPA)-induced prostate inhibition in Sprague Dawley rats were conducted to gauge the anti-cancer ability of NA. Significant (p < 0.05 and p < 0.01) time- and dose-dependent reductions in proliferation of PCa cells, reduced migration, invasion, and increased apoptotic cell population were recorded after NA treatment (3−50 µM). After 72 h of treatment, NA displayed significant IC50 of 21.18 ± 3.43 µM against DU145 and 24.21 ± 3.38 µM against PC3 cells in comparison to the controls cabazitaxel (9.56 ± 1.45 µM and 12.78 ± 2.67 µM) and doxorubicin (10.98 ± 2.71 µM and 15.97 ± 2.77 µM). Further deep mechanistic studies reveal that NA treatment considerably increased the cleavage of caspases and downstream PARP, upregulated BAX and P53, and downregulated BCL-2 and NF-κB, inducing apoptosis in PCa cells. Pharmacokinetic and ADME characterization indicate that NA has a favorable physicochemical nature, with high gastrointestinal absorption, low blood−brain barrier permeability, no hepatotoxicity, and cytochrome inhibition. BPA-induced perturbations of prostate glands in Sprague Dawley rats show a potential increase (0.478 ± 0.28 g) in prostate weight compared to the control (0.385 ± 0.13 g). Multi-dose treatment with NA (10 mg/kg) significantly reduced the prostate size (0.409 ± 0.21 g) in comparison to the control. NA-treated groups exhibited substantial restoration of hematological and histological parameters, reinstatement of serum hormones, and suppression of inflammatory markers. This multifaceted analysis suggests that NA, as a novel small molecule with a strong pharmacokinetic and pharmacological profile, has the potential to induce apoptosis and death in PCa cells.

Ameliorative Effect of Structurally Divergent Oleanane Triterpenoid, 3-Epifriedelinol from Ipomoea batatas against BPA-Induced Gonadotoxicity by Targeting PARP and NF-κB Signaling in Rats.

The pentacyclic triterpenoids (PTs) of plant origin are reputed to restrain prostate cancer (PCa) cell proliferation. This study aims to assess 3-epifriedelinol (EFD) isolated from aerial part of Ipomoea batatas against PCa and its potential mechanism, in vitro and in vivo. Molecular docking affirms good binding affinity of the compound with target proteins exhibiting binding energy of −7.9 Kcal/mol with BAX, −8.1 Kcal/mol (BCL-2), −1.9 Kcal/mol (NF-κB) and −8.5 Kcal/mol with P53. In the MTT assay, EFD treatment (3−50 µM) showed a significant (p < 0.05 and p < 0.01) dose and time dependent drop in the proliferative graph of DU145 and PC3, and an upsurge in apoptotic cell population. EFD displayed substantial IC50 against DU145 (32.32 ± 3.72 µM) and PC3 (35.22 ± 3.47 µM). According to Western blots, EFD administration significantly enhanced the cleavage of caspases and PARP, elevated BAX and P53 and decreased BCL-2 and NF-κB expression, thereby triggering apoptosis in PCa cells. When male Sprague Dawley rats were intoxicated with Bisphenol A (BPA), an apparent increase in prostate mass (0.478 ± 0.08 g) in comparison to control (0.385 ± 0.03 g) indicates prostatitis. Multidose treatment of EFD (10 mg/kg) significantly reduced prostate size (0.404 ± 0.05 g). EFD exhibited substantial curative potential in vivo, as hematological, hormonal and histopathological parameters have been significantly improved. Reduced peroxidation (TBARS), and suppression of inflammatory markers i.e., NO, IL-6 and TNF-α, signposts substantial antiinflammatory potential of the compound. Overall, EFD has shown better binding affinity with target molecules, acceptable ADMET profile, potent antiproliferative and apoptotic nature and significant reduction in inflamed prostate mass of rats. The present study demonstrates acceptable physicochemical and pharmacokinetic properties of the compound with excellent drugable nature, hence EFD in the form of standardized formulation can be developed as primary or adjuvant therapy against PCa and toxins-induced gonadotoxicity.

Phytochemical analysis and comprehensive evaluation of pharmacological potential of Artemisia brevifolia Wall. ex DC.

Multitude of diseases and side effects from conventional drugs have surged the use of herbal remedies. Thus, the current study aimed to appraise various pharmacological attributes of Artemisia brevifolia Wall. ex DC. Extracts prepared by successive solvent extraction were subjected to phytochemical and multimode antioxidant assays. Various polyphenolics and artemisinin derivatives were detected and quantified using RP-HPLC analysis. Compounds present in methanol (M) and distilled water (DW) extracts were identified using high resolution mass spectrometry (HRMS). Extracts were pharmacologically evaluated for their antibacterial, antifungal, antimalarial, antileishmanial and antidiabetic potentials. Moreover, cytotoxicity against Artemiasalina, human cancer cell lines and isolated lymphocytes was assessed. Genotoxicity was evaluated using comet, micronucleus and chromosomal aberration assays. Lastly, anti-inflammatory potential was determined through a series of in vitro and in vivo assays using BALB/c mice. Maximum extract recovery (5.95% w/w) was obtained by DW extract. Highest phenolics and flavonoids content, total antioxidant capacity, total reduction potential, percentfree radical scavenging, ß-carotene scavenging and iron chelating activities were exhibited by M extract. RP-HPLC analysis revealed significant amounts of various polyphenolic compounds (vanillic acid, syringic acid, emodin and luteolin), artemisinin, dihydro artemisinin, artesunate and artemether in ethyl acetate (EA) extract. Total 40 compounds were detected through HRMS. A noteworthy antimicrobial activity (MIC 22.22 µg/ml) was exhibited by EA extract against A. fumigatus and several bacterial strains. Maximum antimalarial, antileishmanial, brine shrimp lethality and cytotoxic potential against cancer cells was manifested by EA extract. None of the extracts exhibited genotoxicity and toxicity against isolated lymphocytes. Highest α-amylase and α-glucosidase inhibition capacities were demonstrated by DW extract. Various in-vivo anti-inflammatory models revealed significant (p < 0.05) anti-inflammatory potential of M and DW extracts. In conclusion, present findings divulged theremarkable pharmacological potential of A. brevifolia and endorse its richness in artemisinin.

Withaferin A: From Ancient Remedy to Potential Drug Candidate.

Withaferin A (WA) is a pivotal withanolide that has conquered a conspicuous place in research, owning to its multidimensional biological properties. It is an abundant constituent in Withania somnifera Dunal. (Ashwagandha, WS) that is one of the prehistoric pivotal remedies in Ayurveda. This article reviews the literature about the pharmacological profile of WA with special emphasis on its anticancer aspect. We reviewed research publications concerning WA through four databases and provided a descriptive analysis of literature without statistical or qualitative analysis. WA has been found as an effective remedy with multifaceted mechanisms and a broad spectrum of pharmacological profiles. It has anticancer, anti-inflammatory, antiherpetic, antifibrotic, antiplatelet, profibrinolytic, immunosuppressive, antipigmentation, antileishmanial, and healing potentials. Evidence for wide pharmacological actions of WA has been established by both in vivo and in vitro studies. Further, the scientific literature accentuates the role of WA harboring a variable therapeutic spectrum for integrative cancer chemoprevention and cure. WA is a modern drug from traditional medicine that is necessary to be advanced to clinical trials for advocating its utility as a commercial drug.

Profiling of Antifungal Activities and In Silico Studies of Natural Polyphenols from Some Plants.

A worldwide increase in the incidence of fungal infections, emergence of new fungal strains, and antifungal resistance to commercially available antibiotics indicate the need to investigate new treatment options for fungal diseases. Therefore, the interest in exploring the antifungal activity of medicinal plants has now been increased to discover phyto-therapeutics in replacement to conventional antifungal drugs. The study was conducted to explore and identify the mechanism of action of antifungal agents of edible plants, including Cinnamomum zeylanicum, Cinnamomum tamala, Amomum subulatum, Trigonella foenumgraecum, Mentha piperita, Coriandrum sativum, Lactuca sativa, and Brassica oleraceae var. italica. The antifungal potential was assessed via the disc diffusion method and, subsequently, the extracts were assessed for phytochemicals and total antioxidant activity. Potent polyphenols were detected using high-performance liquid chromatography (HPLC) and antifungal mechanism of action was evaluated in silico. Cinnamomum zeylanicum exhibited antifungal activity against all the tested strains while all plant extracts showed antifungal activity against Fusarium solani. Rutin, kaempferol, and quercetin were identified as common polyphenols. In silico studies showed that rutin displayed the greatest affinity with binding pocket of fungal 14-alpha demethylase and nucleoside diphosphokinase with the binding affinity (Kd, -9.4 and -8.9, respectively), as compared to terbinafine. Results indicated that Cinnamomum zeylanicum and Cinnamomum tamala exert their antifungal effect possibly due to kaempferol and rutin, respectively, or possibly by inhibition of nucleoside diphosphokinase (NDK) and 14-alpha demethylase (CYP51), while Amomum subulatum and Trigonella foenum graecum might exhibit antifungal potential due to quercetin. Overall, the study demonstrates that plant-derived products have a high potential to control fungal infections.

Caralluma tuberculata N.E.Br Manifests Extraction Medium Reliant Disparity in Phytochemical and Pharmacological Analysis.

Solubility of phytoconstituents depends on the polarity of the extraction medium used, which might result in the different pharmacological responses of extracts. In line with this, ethnomedicinally important food plant (i.e., Caralluma tuberculata extracts) have been made in fourteen distinct solvent systems that were then analyzed phytochemically via total phenolic amount estimation, total flavonoid amount estimation, and HPLC detection and quantification of the selected polyphenols. Test extracts were then subjected to a battery of in vitro assays i.e., antioxidants (DDPH scavenging, antioxidant capacity, and reducing power estimation), antimicrobial (antibacterial, antifungal, and antileishmanial), cytotoxic (brine shrimps, THP-1 human leukemia cell lines and normal lymphocytes), and protein kinase inhibition assays. Maximum phenolic and flavonoid contents were computed in distilled water-acetone and acetone extracts (i.e., 16 ± 1 µg/mg extract and 8 ± 0.4/mg extract, respectively). HPLC-DAD quantified rutin (0.58 µg/mg extract) and gallic acid (0.4 µg/mg extract) in methanol-ethyl acetate and methanol extracts, respectively. Water-acetone extract exhibited the highest DPPH scavenging of 36 ± 1%. Total reducing potential of 76.0 ± 1 µg/mg extract was shown by ethanol chloroform while maximum total antioxidant capacity was depicted by the acetone extract (92.21 ± 0.70 µg/mg extract). Maximal antifungal effect against Mucor sp., antileishmanial, brine shrimp cytotoxicity, THP-1 cell line cytotoxicity, and protein kinase inhibitory activities were shown by ethyl acetate-methanol (MIC: 50 µg/disc), n-hexane (IC50: 120.8 ± 3.7 µg/mL), ethyl acetate (LD50: 29.94 ± 1.6 µg/mL), distilled water-acetone (IC50: 118 ± 3.4 µg/mL) and methanol-chloroform (ZOI: 19 ± 1 mm) extracts, respectively. Our findings show the dependency of phytochemicals and bioactivities on the polarity of the extraction solvent and our preliminary screening suggests the C. tuberculata extract formulations to be tested and used in different ailments, however, detailed studies remain necessary for corroboration with our results.

HPLC-DAD Based Polyphenolic Profiling and Evaluation of Pharmacological Attributes of Putranjiva roxburghii Wall.

The current study was intended to explore the phytochemical profiling and therapeutic activities of Putranjiva roxburghii Wall. Crude extracts of different plant parts were subjected to the determination of antioxidant, antimicrobial, antidiabetic, cytotoxic, and protein kinase inhibitory potential by using solvents of varying polarity ranges. Maximum phenolic content was notified in distilled water extracts of the stem (DW-S) and leaf (DW-L) while the highest flavonoid content was obtained in ethyl acetate leaf (EA-L) extract. HPLC-DAD analysis confirmed the presence of various polyphenols, quantified in the range of 0.02 ± 0.36 to 2.05 ± 0.18 µg/mg extract. Maximum DPPH scavenging activity was expressed by methanolic extract of the stem (MeOH-S). The highest antioxidant capacity and reducing power was shown by MeOH-S and leaf methanolic extract (MeOH-L), respectively. Proficient antibacterial activity was shown by EA-L extract against Bacillus subtilis and Escherichia coli. Remarkable α-amylase and α-glucosidase inhibition potential was expressed by ethyl acetate fruit (EA-F) and n-Hexane leaf (nH-L) extracts, respectively. In case of brine shrimp lethality assay, 41.67% of the extracts (LC50 < 50 µg/mL) were considered as extremely cytotoxic. The test extracts also showed mild antifungal and protein kinase inhibition activities. The present study explores the therapeutic potential of P. roxburghii and calls for subsequent studies to isolate new bioactive leads through bioactivity-guided isolation.

The triphenyltin carboxylate derivative triphenylstannyl 2-(benzylcarbamoyl)benzoate impedes prostate cancer progression via modulation of Akt/FOXO3a signaling.

Prostate cancer (PCa) incidence is surging in United States and other parts of the world. Synthetic and natural compounds have been explored as potential modulators of PI3K/Akt signaling that is known to drive PCa growth. Here, we evaluated the efficacy of a series of triphenyltin (IV) carboxylate derivatives against PCa. From this library, triphenylstannyl 2-(benzylcarbamoyl)benzoate (Ch-319) resulted in reduced viability and induction of cell cycle arrest in PTEN-/- PC3M and PTEN+/- DU145 cells. In parallel, downregulation of PI3K p85/p110 subunits, dephosphorylation of Akt-1 and increase in FOXO3a expression were observed. In silico studies indicated binding interactions of Ch-319 within the ATP binding site of Akt-1 at Met281, Phe442 and Glu234 residues. Elevated po-pulation of apoptotic cells, activation of Bax and reduced Bcl2 expression indicated apoptosis by Ch-319. Pre-sensitization of PCa cells with Ch-319 augmented the effect of cabazitaxel, a commonly used taxane in patients with castration-resistant PCa. Next, in a prostate-specific PTENp-/- mice, Ch-319 showed reduced weights of genitourinary apparatus as compared to DMSO treated controls. Histological studies indicated absence of neoplastic foci in Ch-319 treated prostates. Consistently, dephosphorylation of Akt-1, reduced expression of PRAS40 and androgen receptor and increase in FOXO3a were observed in treated group. Notably, no overt organ toxicity was noted in Ch-319 treated animals. Our studies identify Akt/FOXO3a signaling as a target of triphenyltin (IV) carboxylate Ch-319 and provide a molecular basis of its growth inhibitory effect in PCa cells. We propose that Ch-319 has the potential to be developed as an anticancer agent against PCa.

Development, optimisation, and evaluation of nanoencapsulated diacerein emulgel for potential use in osteoarthritis.

AIM: This study aimed to utilise the proficient function of diacerein (DCR) and anti-inflammatory polymers to develop sustained release nanoencapsulated emulgel for potential use in osteoarthritis (OA). METHODS: Chitosan (CHS) and chondroitin sulphate (CS) were employed as natural anti-inflammatory polymers to encapsulate nanoformulation of DCR. Optimised nanoformulation was prepared and characterised by investigating impact of polymers and surfactant on particle size, PDI, and encapsulation efficiency (EE). Afterwards, nanoemulgel of optimised DCR-NPs was formulated and evaluated for transdermal application. RESULTS: Optimised nanoformulation depicted spherical shape with particle size of 320 nm having PDI and EE of 0.3 ± 0.07 and 82 ± 4% (w/w), respectively. DCR-nanoemulgel depicts sustained action of drug up to 96 h with enhanced permeation activity and non-irritancy index. CONCLUSIONS: The elaborated nanoemulgel sustained release of drug having superior penetration properties with provision of enhanced therapeutic effect owing to the presence of CHS, CS, and Argan oil possessing indelible anti-inflammatory attributes.

Withametelin: a biologically active withanolide in cancer, inflammation, pain and depression.

Withanolides are natural medicinal agents whose safety and therapeutic profiles make them valuable to mankind. Among multiple withanolides, withametelin is underexplored. The present study was aimed to create a general biological profile of isolated withametelin from Datura innoxia Mill. targeting different biological models. In-silico studies include drug-likeliness, pharmacokinetics, toxicity, molecular targets and cytotoxicity to cancer cell lines predictions. In silico directed preliminary in-vitro evaluation comprised of cancer/normal cell cytotoxicity, DPPH and protein kinase inhibition assays while in-vivo bioactivities include antiinflammatory, analgesic, antidepressant and anticoagulant assays. Pharmacological findings were strengthened by molecular docking studies to check interactions with various proteins and to propose the future path of studies. Results indicated compliance with Lipinski drug-likeliness rule (score -0.55). ADMET prediction showed strong plasma protein binding, GI absorption (Caco-2 cells permeability = 46.74 nm/s), blood brain barrier penetration (Cbrain/Cblood = 0.31), efflux by P-glycoprotein, metabolism by CYP1A2, CYP2C19 and CYP3A4, medium hERG inhibition and non-carcinogenicity in rodents. Predicted molecular targets included mainly receptors (glucocorticoid, kappa opioid, delta opioid, adrenergic and dopamine), oxidoreductase (arachidonate 5-lipoxygenase and cyclooxygenase-2), enzymes (HMG-CoA reductase) and kinase (NFκb). Withametelin was more cytotoxic to cancer cells (DU145 IC50 7.67 ± 0.54 µM) than normal lymphocytes (IC50 33.55 ± 1.31 µM). It also showed good antioxidant and protein kinase inhibition potentials. Furthermore, withametelin (20 mg/kg) significantly reduced inflammatory paw edema (68.94 ± 5.55%), heat-induced pain (78.94 ± 6.87%) and immobility time (50%) in animals. Molecular docking showed hydrogen bonding interactions (binding energies: -11.3 to -7.8 kcal/mol) with arachidonate 5 lipoxygenase, NFκb and glucocorticoid receptor. Withametelin has potential for advance investigations for its cytotoxic, anti-inflammatory, analgesic and antidepressant activities.

Ipomoea batatas L. Lam. ameliorates acute and chronic inflammations by suppressing inflammatory mediators, a comprehensive exploration using in vitro and in vivo models.

BACKGROUND: Ipomoea batatas L. Lam. is a functional food and belongs to family Convolvulaceae. It is used as an antiinflammatory, aphrodisiac, antiasthmatic, anticonvalescent, antitumor, antanemic and antidiabetic agent by local communities. This study has been planned to evaluate its antiinflammatory and antiarthritic potentials. METHODS: Dry powder of I. batatas tuber and roots were extracted with ethyl acetate (IPT-EA, IPR-EA) and methanol (IPT-M, IPR-M), respectively. These extracts were tested for total phenolic and flavonoid contents (TPC and TFC), HPLC finger printing, multidimensional in vitro and in vivo antioxidant potential and albumin denaturation inhibition. Carrageenan-induced paw edema, croton oil-induced ear and anal edema inhibition and Complete Freund's Adjuvant (CFA)-induced antiarthritic assays were executed at a dose of 300 mg/kg body weight on Sprague-Dawley rats. Serum levels of interleukins IL-1ß and IL-6 and nitric oxide (NO) were assessed to measure the inhibition of inflammation. RESULTS: Maximal TPC (319.81 ± 14.20 µg GAE/mg dry extract) and TFC (208.77 ± 9.09 µg QE/mg DE) were estimated in IPR-EA extract. IPT-EA and IPR-EA yielded the maximum amounts of rutin (7.3 ± 1.12 and 4.5 ± 0.55), caffeic acid (1.60 ± 0.25 and 2.17 ± 0.26) and myricetin (2.7 ± 0.14 and 1.01 ± 0.08 µg/mg DE), respectively in HPLC-DAD analysis. All extracts showed dose dependent response in in vitro antioxidant assays. Best inhibition (76.92 ± 3.07%) of albumin denaturation was shown by IPT-EA in comparison to ibuprofen (79.48 ± 4.71%). IPR-EA exhibited highest edema inhibition in models of carrageenan-induced paw edema (79.11 ± 5.47%) and croton oil-induced ear and anal edema (72.01 ± 7.80% and 70.80 ± 4.94%, respectively). Significant inhibition of CFA-induced arthritic edema and arthritic score were observed by IPR-EA as compared to ibuprofen. Suppression of pro-inflammatory cytokines (IL-1ß, IL-6) and NO levels was shown by IPR-EA and IPT-EA, respectively. CONCLUSION: These results depict that richness of polyphenols and phytoconstituents in I. batatas ameliorates oxidative stress and inflammation of acute and chronic nature. Dose dependent antioxidant potential and inhibition of inflammatory edema, pro-inflammatory cytokines and hematological, biochemical and histological changes prove I. batatas therapeutic potential as an antiinflammatory and antiarthritic agent.

Report- Antioxidant, cytotoxicity, protein kinase inhibition and antibacterial activities of Fragaria x ananassa leaves.

Fragaria × ananassa leaves extracts prepared in different solvents were subject for antioxidative, cytotoxicity, protein kinase inhibition and antibacterial activities. The extracts showed varying activities depending upon solvent used for extraction. Combined effect of methanol and ethyl acetate showed maximum antioxidant and reducing power potential (207.65±6µg AAE/mg and 88.58±20µg AAE/mg, respectively). Maximum DPPH (2,2-diphenyl-1-picryl hydrazyl) free radical scavenging activity was calculated by when methanol: chloroform and acetate fractions were used (87.68% and 86.88% inhibition, respectively). Total phenolics varied from 186 to 1.91µg AAE/mg while total flavonoids also significantly varied among the extracts. The extracts also showed significant activities against brine shrimp larvae and bacterial strains tested. The study concludes that Fragaria × ananassa leaves can be a good source for isolation of active phytochemicals to be used in different industries.

Polarity based characterization of biologically active extracts of Ajuga bracteosa Wall. ex Benth. and RP-HPLC analysis.

BACKGROUND: The concept of botanical therapeutics has revitalized due to wide importance of plant derived pharmaceuticals. Therefore, the ameliorative characteristics of Ajuga bracteosa were studied. METHODS: Total phenolic content, flavonoid content, antioxidant capacity, reducing power and free-radical scavenging activity were determined colorimetrically. Specific polyphenols were quantified by RP-HPLC analysis. Preliminary cytotoxicity was tested using brine shrimp lethality assay while antiproliferative activity against THP-1 and Hep-G2 cell lines was determined by MTT and SRB protocols respectively. Antileishmanial potential was assessed via MTT colorimetric method. To investigate antidiabetic prospect, α-amylase inhibition assay was adopted whereas disc diffusion method was used to detect likely protein kinase inhibitory, antibacterial and antifungal activities. RESULTS: Among fifteen different extracts, maximum total phenolic content (10.75 ± 0.70 µg GAE/mg DW), total reducing power (23.90 ± 0.70 µg AAE/mg DW) and total antioxidant capacity (11.30 ± 0.80 µg AAE/mg DW) were exhibited by methanol extract with superlative percent extract recovery (17.50 ± 0.80% w/w). Chloroform-methanol extract demonstrated maximum flavonoid content (4.10 ± 0.40 µg QE/mg DW) and ethanol extract exhibited greatest radical scavenging activity (IC50 14.40 ± 0.20 µg/ml). RP-HPLC based quantification confirmed polyphenols such as pyrocatechol, gallic acid, resorcinol, catechin, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, vanillic acid, coumarin, sinapinic acid, trans-cinnamic acid, rutin, quercetin and kaempferol. The brine shrimp lethality assay ranked 78.60% extracts as cytotoxic (LC50 ≤ 250 µg/ml) whereas significant THP-1 inhibition was shown by methanol-acetone extract (IC50 4.70 ± 0.43 µg/ml). The antiproliferative activity against Hep-G2 hepatoma cancer cell line was demonstrated by n-hexane, ethylacetate and methanol-distilled water (IC50 8.65-8.95 µg/ml) extracts. Methanol extract displayed prominent protein kinase inhibitory activity (MIC 12.5 µg/disc) while n-hexane extract revealed remarkable antileishmanial activity (IC50 4.69 ± 0.01 µg/ml). The antidiabetic potential was confirmed by n-hexane extract (44.70 ± 0.30% α-amylase inhibition at 200 µg/ml concentration) while a moderate antibacterial and antifungal activities were unveiled. CONCLUSION: The variation in biological spectrum resulted due to use of multiple solvent systems for extraction. We also deduce that the valuable information gathered can be utilized for discovery of anticancer, antileishmanial, antioxidant and antidiabetic bioactive lead candidates.

Rol genes enhance the biosynthesis of antioxidants in Artemisia carvifolia Buch.

BACKGROUND: The secondary metabolites of the Artemisia genus are well known for their important therapeutic properties. This genus is one of the valuable sources of flavonoids and other polyphenols, but due to the low contents of these important metabolites, there is a need to either enhance their concentration in the original plant or seek alternative sources for them. The aim of the current study was to detect and enhance the yield of antioxidant compounds of Artemisia carvifolia Buch. HPLC analysis was performed to detect the antioxidants. With the aim of increasing flavonoid content, Rol gene transgenics of A. carvifolia were established. Two genes of the flavonoid biosynthetic pathway, phenylalanine ammonia-lyase and chalcone synthase, were studied by real time qPCR. Antioxidant potential was determined by performing different antioxidant assays. RESULTS: HPLC analysis of wild-type A. carvifolia revealed the presence of flavonoids such as caffeic acid (30 µg/g DW), quercetin (10 µg/g DW), isoquercetin (400 µg/g DW) and rutin (300 µg/g DW). Compared to the untransformed plants, flavonoid levels increased 1.9-6-fold and 1.6-4-fold in rol B and rol C transgenics, respectively. RT qPCR analysis showed a variable expression of the flavonoid biosynthetic genes, including those encoding phenylalanine ammonia-lyase and chalcone synthase, which were found to be relatively more expressed in transformed than wild-type plants, thus correlating with the metabolite concentration. Methanolic extracts of transgenics showed higher antioxidant capacity, reducing power, and protection against free radical-induced DNA damage. Among the transgenic plants, those harboring rol B were slightly more active than the rol C-transformants. CONCLUSION: As well as demonstrating the effectiveness of rol genes in inducing plant secondary metabolism, this study provides insight into the molecular dynamics of the flavonoid accumulation pattern, which correlated with the expression of biosynthetic genes.

Hedera nepalensis K. Koch: A Novel Source of Natural Cancer Chemopreventive and Anticancerous Compounds.

Traditional medicinal plants are often used for both the prevention and the treatment of local diseases. Taking into consideration the medicinal importance of Hedera nepalensis within local Pakistani traditions, the present study was undertaken to analyze the in vitro cancer chemopreventive and cytotoxic properties of the plant. The in vitro cancer chemopreventive testing was performed using nitrite assay, NFκB assay, aromatase assay, and quinone reductase 1 (QR1) assay. The cytotoxic potential was evaluated on three cancer-cell lines: MCF-7, MDA-MB-231, and HeLa using sulforhodamine B (SRB) assay. The results of cancer chemopreventive assays show that n-hexane and ethyl acetate fractions of tested plant have promising cancer chemopreventive potential. Lupeol isolated from n-hexane as well as ethyl acetate fraction showed lowest IC50 (0.20 ± 1.9 µM) in NFκB assay. Crude extract and its fractions inhibited the growth of three cancer cell lines by more than 60%, IC50 value of lupeol varied from 2.32 to 10.2 µM. HPLC-DAD-based quantification of lupeol in different plant tissues demonstrated that leaves of H. nepalensis are a rich source of lupeol (0.196 mg/100 mg dry weight). Our data have shown that H. nepalensis harbors cancer chemopreventive and cytotoxic agents.

Antioxidant, anticancer and antibacterial potential of Zakhm-e-hayat rhizomes crude extract and fractions.

Bergenia ciliata (locally known as Zakhm-e-hayat; wound healer) is commonly employed for wound healing, curing diarrhea and vomiting, fever, cough and pulmonary affections. Local community uses this plant as tea decoction with table salt. B. ciliata crude extract and its fractions were subjected to antibacterial, antioxidant effects as well as determination of total flavonoids and phenolics, DNA damage and anticancerous activities following standard protocols. Increased percentage inhibition of free radical in DPPH assay as well as elevated phenolic and flavonoid contents revealed antioxidant potential of this potent herb. Ethyl acetate and aqueous extracts showed IC(50) of 0.7 and 0.3 mg/ml respectively, against H157 cell line. Antibacterial analysis showed MIC 0.4-10mg/ml for crude extract and fractions. The results obtained conclude that extracts of B. ciliata contain remedial latent and can be used as possible source for drug development by pharmaceutical industries.

Extraction optimization of medicinally important metabolites from Datura innoxia Mill.: an in vitro biological and phytochemical investigation.

BACKGROUND: The present study aims to probe the impact of polarity dependent extraction efficiency variation on pharmacological spectrum of Datura innoxia Mill. in order to reconnoiter its underexplored therapeutic potential. METHODS: A range of solvent extracts was subjected to phytochemical and biological assays to find the most proficient solvent system and plant part for each type of bioactivity. Total phenolic and flavonoid contents were determined colorimetrically and specific polyphenols were quantified by HPLC-DAD analysis. The samples were biologically evaluated by employing multimode antioxidant, cytotoxic, protein kinase inhibition and antimicrobial assays. RESULTS: Among all the solvents used, maximum percent extract recovery (33.28 %) was obtained in aqueous leaf extract. The highest amount of gallic acid equivalent phenolic and quercetin equivalent flavonoid content was obtained in the distilled water and ethyl acetate-ethanol extracts of leaf i.e., 29.91 ± 0.12 and 15.68 ± 0.18 mg/g dry weight (DW) respectively. Reverse phase HPLC-DAD based quantification revealed the presence of significant amounts of catechin, caffiec acid, apigenin and rutin ranging from 0.16 to 5.41 mg/g DW. Highest DPPH radical scavenging activity (IC50 = 16.14 µg/ml) was displayed by the ethyl acetate-acetone stem extract. Maximum total antioxidant capacity and reducing power potential were recorded in the aqueous leaf and ethyl acetate stem extracts i.e., 46.98 ± 0.24 and 15.35 ± 0.61 mg ascorbic acid equivalent/g DW respectively. Cytotoxicity against brine shrimps categorized 25 % of the leaf, 16 % of the stem and 8.3 % of the fruit extracts as highly potent (LC50 ≤ 100 µg/ml). Significant cytotoxicity against human leukemia (THP-1) cell line was exhibited by the chloroform and n-hexane fruit extracts with IC50 4.52 and 3.49 µg/ml respectively. Ethyl acetate and methanol-chloroform extracts of leaf and stem exhibited conspicuous protein kinase inhibitory activity against Streptomyces 85E strain with 22 mm bald phenotype. A noteworthy antimicrobial activity was exhibited by leaf extracts against Micrococcus luteus and n-hexane fruit extract against Aspergillus niger (MIC 3.70 and 12.5 µg/ml respectively). CONCLUSION: Multiple solvent system is a crucial variable to retrieve pharmacological potential of medicinal plants and D. innoxia can be envisaged as a novel source of natural antioxidants, antimicrobials and anticancer compounds.