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   2018| July-September  | Volume 14 | Issue 57  
    Online since September 10, 2018

 
 
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ORIGINAL ARTICLES
Evaluation of antiangiogenic potential of Psidium guajava leaves using In-Ovo chick chorioallantoic membrane assay
S Latha, P Yamini, Rajani Mathur
July-September 2018, 14(57):284-293
DOI:10.4103/pm.pm_133_18  
Background: Angiogenesis is the process of formation of new blood vessels from the existing one. Pathological angiogenesis is widely implicated in many diseases, including cancer, diabetic neuropathy, retinopathy, obesity, and arthritis. Objective: The present study was aimed to evaluate the in vitro antioxidant and in ovo antiangiogenic activity of aqueous extract of Psidium guajava leaves (AEPG). Materials and Methods: Psidium guajava commonly known as guava reported to contain polyphenols and flavonoids such as gallic acid, epigallocatechin, catechin, rutin, and quercetin in glycosidic forms in its leaves. The antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), nitric oxide, hydrogen peroxide, hydroxyl, and superoxide radical scavenging assays (RSAs) and antiangiogenic activity was evaluated using vascular endothelial growth factor (VEGF)-induced chick chorioallantoic membrane (CAM).The correlation between the antioxidant and antiangiogenic activity was correlated with total phenolic content (TPC) of AEPG. Results: The TPC of AEPG was found to be 493.8 ± 8.9 mg of GAE/g. The total flavonoid content of AEPG was found to be 254.9 ± 13.7 mg of CE/g. In vitro antioxidant activity of AEPG showed IC50 values of 19.4 ± 1.9, 25.5 ± 0.2, 4.9 ± 0.5, 29.9 ± 2.06, 39.5 ± 2.07, and 29.9 ± 0.9 μg/ml, respectively, for DPPH, ABTS, nitric oxide, hydrogen peroxide, hydroxyl, and superoxide RSAs. Significant reduction in angiogenesis in the AEPG treated groups when compared to untreated VEGF groups and the Pearson's correlation coefficient between TPC of AEPG and total length, area, branches of blood vessels and CAM thickness were −0.9261, −0.9807, −0.9637, and −0.9597, respectively. Conclusion: The results revealed potent antiangiogenic activity of AEPG leaves and exhibit significant correlation between the antioxidant and antiangiogenic activity of AEPG and its TPC. Abbreviations used: EGF: Epidermal growth factor; FGF: Fibroblast growth factor; G-CSF: Granulocyte colony stimulating factor; IL: Interleukin; INF: Interferon; MMP: Matrix metalloproteinases; NOS: Nitric oxide synthase; PAF: Platelet-activating factor; PAI: Plasminogen activator inhibitor; PDGF: Platelet-derived growth factor; PG-E: Prostaglandin E; RSA: Radical scavenging assay; TFC: Total flavonoid content; TPC: Total Phenolic content; TIMP: Tissue inhibitors of metalloproteinases; TNF-α: Tumor necrosis factor alpha; VEGF: Vascular endothelial growth factor.
  4,080 170 -
In vitro antioxidant and anticancer activities of leaf extracts of Rhododendron arboreum and Rhododendron campanulatum from Uttarakhand region of India
Sakshi Painuli, Swati Joshi, Anuja Bhardwaj, Ramesh Chand Meena, Kshipra Misra, Nishant Rai, Navin Kumar
July-September 2018, 14(57):294-303
DOI:10.4103/pm.pm_15_18  
Background: Rhododendron arboreum and Rhododendron campanulatum are two important trees of Uttarakhand, known for their ethnopharmacological importance. In the present study, the aqueous and methanolic extracts of leaves of both the plants were analyzed for phytochemical, antioxidant, and antiproliferative activities against cancer cell line and repression of vascular endothelial cell growth factor (VEGF) and hypoxia-inducible factor-1 (HIF-1α) transcription. Materials and Methods: Aqueous and methanolic leaf extracts of both the plants were prepared through pressurized liquid extraction method. Various assays were performed to analyze phytochemical and antioxidant potential. The antiproliferative activity was determined through 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxy anilide salt (XTT) assay. Further, the effect of plant extracts on the modulation of HIF-1α and VEGF mRNA expression was analyzed through quantitative real-time polymerase chain reaction. Results: A significantly high total phenol content, total flavonoid content, free radical scavenging, and reducing power activities were observed in both the plant extracts. High-performance thin-layer chromatography analysis indicated the presence of ascorbic acid, quercetin, gallic acid, and hesperidin in different extracts. XTT reduction assay confirmed the antiproliferative activities of aqueous extracts against cancer cell line, HeLa. Aqueous extract of R. arboreum (RAA), at a concentration of 31.25 μg/ml, inhibited 60.12% and 25.41% proliferation of HeLa and Vero cell lines, respectively. An equal concentration of aqueous extract of R. campanulatum (RCA) inhibited the growth by 48.04% and 15.17% for HeLa and Vero cell lines, respectively. Further, the RAA and RCA have downregulated the expression of two key angiogenic factors responsible for tumor neovascularization, HIF-1α and VEGF to a great extent. Conclusion: The present study affirms that the leaf extracts of R. arboreum and R. campanulatum have enormous potential to be developed as an effective natural antioxidant and anticancer drug. Abbreviations used: PLE: Pressurized liquid extraction; XTT: 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxy anilide salt; TPC: Total phenol content; TFC: Total flavonoid content.
  3,897 185 -
Ultra-high-performance liquid chromatography-based identification and quantification of thymoquinone in Nigella sativa extract from different geographical regions
Niyaz Ahmad, Rizwan Ahmad, Abdullah Al-layly, Hassan Al-shawi, Ahmed Al-ali, Mohd Amir, Ahmed Mostafa
July-September 2018, 14(57):471-480
DOI:10.4103/pm.pm_119_18  
Background: Thymoquinone (THQ) is a major bioactive constituent of traditional medicinal plant such as Black seed (Nigella sativa, Family: Ranunculaceae). Objective: The objective of this study was to identify and quantify THQ in Black seeds from different geographical regions and to develop a new ultra-high-performance liquid chromatography-photodiode array detector (UHPLC-PDA) method. Materials and Methods: Black seeds were collected in the end of autumn season. The mobile phase was used in this study [Acetonitrile (2 mM): Ammonium Formate at 50:50 proportions, with a flow rate of 0.200 mL min-1 and also with Isocratic elution on the Accucoro Vanguish C18 UHPLC column (1.5 μm; 100 × 2.1 mm)]. Results: Retention and total run times were 3.147 and 6.0 min, respectively, with injection volume of 5 μL at 254 nm. The method was validated for linearity (r2 ≥ 0.9987), accuracy (≥92.90%), and precision (coefficient of variance [CV] ≤2.54%) with a calibration curve range of 100.00–2000.00 ng/mL. THQ was degraded under ultra-violet-light, basic, acidic, and oxidation stress conditions during forced degradation studies. Our developed UHPLC method reported the degradation peaks of the final product of THQ. UHPLC-PDA analysis showed large variation (0.01%–3.03% w/w) of THQ in the samples of different regions in respect to their concentration and occurrence of metabolite. Conclusions: Turkey has the maximum quantity of THQ throughout the world. The authors suggested that Turkey is the best region for cultivating Black seed plant of superior variety. One more important study performed, i.e., the gas chromatography and mass spectrometry study, reported THQ (2.863%) to be the primary constituent in the extract of N. sativa. Abbreviations used: THQ: Thymoquinone; UHPLC-PDA: Ultra-high-performance liquid chromatography-photodiode array detector; ACN: Acetonitrile; UV: Ultraviolet; GC-MS: Gas chromatography and mass spectrometry; NSO: Nigella sativa extracted oil; TLC: Thin-layer chromatography; IR: Infrared spectroscopy; DSC: Differential scanning calorimetry; QC: Quality control; HQC: High-quality control; MQC: Middle-quality control' LQC: Low-quality control; LLOQQC: Lower limit of quality control; US-FDA: United States of Food and Drug Administration; PSS: Primary stock solution; SS: System suitability; LOD: Limit of detection; LOQ: Limit of quantification.
  3,432 261 -
Efficacy and safety of herbal formulation rich in standardized fenugreek seed extract as add-on supplementation in patients with type 2 diabetes mellitus on sulfonylurea therapy: A 12-week, randomized, double-blind, placebo-controlled, multi-center study
Amit D Kandhare, Nadeem Rais, Nivedita Moulick, Alaka Deshpande, Prasad Thakurdesai, Sunil Bhaskaran
July-September 2018, 14(57):393-402
DOI:10.4103/pm.pm_260_18  
Background: Type 2 diabetes mellitus (T2DM) is a chronic, complex, and progressive illness that often needs combination therapy for better glycemic control. IDM1, an herbal formulation which is rich in standardized fenugreek seed extract. Aim: The aim of this study is to evaluate the efficacy and safety of add-on therapy of IDM1 in T2DM patients inadequately controlled on sulfonylurea monotherapy. Materials and Methods: In this 12-week, randomized, double-blind, placebo-controlled, multi-centric study, T2DM patients which inadequate glycemic control with background stable dose medication of sulfonylurea was screened (n = 120). The patients were randomized 1:1 to add-on therapy of IDM1 and placebo, 700 mg three times daily for 12 weeks. Results: A total of 119 patients were randomized and included in the efficacy analysis (IDM1, n = 60; placebo, n = 59). At week 12, adjusted fasting plasma glucose (FPG) (20 mg%), postprandial plasma glucose (PPPG) (26 mg%), and glycated hemoglobin (HbA1c) (0.9 mg%) was reduced significantly (P < 0.05) from baseline as compared to placebo group (FPG: 7 mg%; PPPG: 4 mg% and HbA1c: 0.4 mg%). These beneficial effects were seen as early as 1 month after consumption of IDM1 and continued until at least 15 days after withdrawal of IDM1. Hypoglycemic events were mostly mild, and none required emergency treatment. There were no major changes in body weight, hematology, and biochemistry at week 12 as compared to baseline. Overall AEs rates were similar in both groups. Conclusions: IDM1 is a safe, effective, and well-tolerated add-on oral medication therapy that supports healthy blood sugar levels and glycosylated hemoglobin levels in T2DM patients inadequately controlled with a sulfonylurea. Abbreviation used: AEs: Adverse events; ALP: Alkaline phosphatase; ALT: Alanine transaminase; AST: Aspartate transaminase; BMI: Body mass index; BUN: Blood urea nitrogen; FPG: Fasting plasma glucose; GGT: Gamma glutamyl transferase; HbA1c: Glycated hemoglobin; HDL: High-density lipoproteins; LDH: Lactate dehydrogenase; LDL: Low-density lipoproteins.
  3,168 194 -
Metabolic profiling and expression analysis of key genes during leaf maturation of Stevia rebaudiana bertoni
Nazima Nasrullah, Javed Ahmad, Monica Saifi, Umara Rafiqi, Naved Quadri, Irum Gul Shah, Malik Zainul Abdin
July-September 2018, 14(57):327-334
DOI:10.4103/pm.pm_554_17  
Background: Stevia (Stevia rebaudiana) is a plant of nutritional and industrial importance for its diverse steviol glycosides. Stevioside, rebaudioside-A and their aglycon steviol – 200-300 times sweeter than normal sucrose are novel contenders for the development of antidiabetic drugs. Stevia leaf flavor at different harvest stages is a function of the metabolite content, which results from physiological changes during plant growth and development. Objectives: The main purpose of this study was to investigate metabolite changes during plant development using GC-MS metabolic profiling and HPTLC and to analyze expression of key genes of steviol glycoside biosynthetic pathway by qPCR. Material Methods: Metabolite data and gene expression from leaf samples of eight developmental stages underwent a variety of chemometric analyses, to identify the true differences between samples. Results: There was a significant increase of steviol from 0.23% to 6.6%, stevioside from 3.3% to 14.23%, rebaudioside-A from 0.826% to 4.99% and (+)-isomenthol showed decrease in concentration from 16.79% to 5.23% with plant growth. srUGTs, srKO, srKS, srKAH, srUGP1, and srDXR increased whereas expression of (+)-srLMS and srNMD decreased with plant progression. Metabolite and gene correlation analysis revealed the interdependencies of individual metabolites and metabolic pathways genes. Conclusion: These results will help in selecting and utilizing the appropriate traits in Stevia crop. Abbreviations used: SG: Steviol glycosides; PCA: Principal component analysis; VIP: Variable importance in the projection; UGT: Uracil glycosyltransferase.
  2,677 169 -
Antidiabetic, toxicological, and metabolomic profiling of aqueous extract of Cichorium intybus seeds
Kailash Chandra, Washim Khan, Sujata Jetley, Sayeed Ahmad, SK Jain
July-September 2018, 14(57):377-383
DOI:10.4103/pm.pm_583_17  
Background: Cichorium intybus has a wide range of therapeutic applications in Indian traditional systems of medicine, especially in metabolic disorders. Objective: To evaluate the toxicity profile and to investigate the antidiabetic, antihyperlipidemic, and antioxidative efficacy of C. intybus seeds in Wistar rats. Materials and Methods: The aqueous extract of seeds was prepared by decoction, and its quality control analysis was carried out by thin-layer chromatography and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) fingerprinting. Wistar rats were fed with high-fat diet for 5 weeks followed by a single dose of streptozotocin intraperitoneally to induce diabetes. The protective group of rats was given aqueous extract during and after the induction of type 2 diabetes mellitus. Further, repeated dose 28-day (subacute) and repeated dose 90-day (chronic) toxicity studies were conducted as per the OECD guidelines. Results: A total of 18 metabolites have been tentatively identified by UPLC-MS profiling in aqueous extract of C. intybus seeds. No significant changes in mortality and biochemical parameters have been observed during toxicity studies. Moreover, administration of the extract to a protective group of diabetic rats attenuated serum glucose and triglyceride levels by 52.7% and 65.3%, respectively, supported by similar results for parameters related to insulin resistance and oxidative stress. The beneficial effect of extract has also been confirmed through in silico screening. Conclusion: C. intybus can be used as a natural dietary supplement for the prevention and management of diabetes and can be explored to develop a potent phytopharmaceutical for diabetes. Abbreviations used: AECIS: Aqueous extract of Cichorium intybus seeds; HOMA-IR: Homeostatic model assessment of insulin resistance; OGTT: Oral glucose tolerance test; TLC: Thin-layer chromatography; UPLC-MS: Ultra-performance liquid chromatography-mass spectrometry.
  2,492 175 -
Naringenin protects against 1-methyl-4-phenylpyridinium- induced neuroinflammation and resulting reactive oxygen species production in SH-SY5Y cell line: An in Vitro model of parkinson's disease
Sugumar Mani, Sathiya Sekar, Saravana Babu Chidambaram, Murugan Sevanan
July-September 2018, 14(57):458-464
DOI:10.4103/pm.pm_23_18  
Background: Parkinson's disease is a progressive neurodegenerative disorder which affects 1% of the population worldwide. It is well known that 1-methyl-4-phenylpyridinium (MPP+) selectively and potentially inhibit Complex I of the mitochondrial electron transport chain. This inactivation leads to the generation of reactive oxygen species (ROS), which in turn damage neurons. In addition, neuroinflammation plays a major role in neurodegeneration processes. Objective: In the present study, the effect of naringenin (NGN), on MPP+-induced neuroinflammation and ROS generation in SH-SY5Y cells were investigated. Materials and Methods: Cells were pretreated with (0.1% dimethyl sulfoxide) or NGN (25, 50, and 100 μm/mL) for 24 h, and then induced with 1 mM MPP+ for 15 min. Following overnight incubation, cells were harvested for ROS staining, gene expression of apoptotic markers such as B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax), inflammatory markers such as tumor necrosis factor-alpha (TNF-α), and nuclear factor-κB (NF-κB) and protein expression of neuronal markers such as dopamine transporter (DAT), tyrosine hydroxylase (TH), and α-synuclein (SYN). Results: In this study, NGN exhibited oxidative protection by decreasing ROS generation which is evidenced through significant regulation of oxidative stress markers. Likewise, NGN decreased TNF-α, Bax, and NF-κB and increased Bcl-2 gene expressions in MPP+-induced SH-SY5Y cells compared to normal SH-SY5Y cells. Further, NGN significantly and dose-dependently decreased SYN and increased DAT and TH levels in MPP+-induced SH-SY5Y cells in comparison to the normal cells. Conclusion: The results obtained from the present study revealed that NGN has the potential to encounter MPP+-induced dopaminergic degeneration through regulating ROS generation and neuroinflammation. Abbreviations used: TNF-α: Tumor Necrosis Factor alpha; NF-κB: Nuclear factor; SYN-α: synuclein; NGN: Naringenin; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine; PD-Parkinson's disease; DCFH-DA: 2',7' - dichlorofluorescin diacetate; SOD: Superoxide dismutase; NO: Nitric oxide; MDA: Malondialdehyde; DAT: Dopamine transporter; TH: Tyrosine hydroxylase; ANOVA: Analysis of variance; SEM: Standard error of the mean; RT-PCR: Reverse transcriptase-polymerase chain reaction; BSA: Bovine serum albumin; PBS: Phosphate buffered saline; TBST: Tris-Buffered saline with Tween; RNA: Ribonucleic acid.
  2,484 172 -
Chemoprotective potential of zingerone (vanillyl acetone) in cyclophosphamide-induced hepatic toxicity
Bilal Ahmad Mir, Insha Amin, Muneeb U Rehman, Rahil Razak, Aarif Ali, Omer Khalil Baba, Bilques Fatima, Rayeesa Ali, Sheikh Bilal, Showkeen Muzamil, Isharq Hussain, S Mudasir Rashid, Adil Farooq Wali, Manzoor-ur- Rahman Mir
July-September 2018, 14(57):434-439
DOI:10.4103/pm.pm_170_16  
Introduction: Cancer is one of the lethal diseases in the global world. Proliferation of cancer cells is commonly inhibited by chemotherapeutics. Cyclophosphamide (CP) is an alkylating chemotherapeutic agent often used for treatment of various types of cancers, but it is full of side effects which in turn lead to organ toxicity. Zingerone, a polyphenolic alkanone found in ginger, has strong antioxidant potential and causes extensive scavenging of free radicals and offers defense against oxidative stress. Twenty-four adult male Wistar rats were divided into four groups, six rats in each group. Materials and Methods: Group I (control), Group II (CP, 2 mg/kg bwt), Group III (cotreatment with zingerone at the dose of 50 mg/kg bwt and CP at the dose of 2 mg/kg bwt), and Group IV (pretreatment of zingerone at the dose of 50 mg/kg bwt for 7 days and CP at the dose of 2 mg/kg bwt for next 7 days). Results: CP significantly increased the level of hepatic marker enzymes such as alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase, drastically caused alteration in lipid profile and deficiency in antioxidant defense mechanism by decreasing the activities of antioxidant enzymes such as catalase, glutathione, glutathione-S-transferase, and glutathione peroxidase. This was accompanied by subsequent increase in lipid peroxidation, nitrite production, and marked DNA damage. Conclusion: The restoration of hepatic markers, amelioration of lipid profile, and improvement of antioxidant status and DNA damage by pre- and co-treatment with zingerone clearly indicate the ameliorative potential of zingerone against CP-induced organ toxicity and oxidative stress. The protective potential of zingerone may be attributed to its strong antioxidant activity. Abbreviations used: CP: Cyclophosphamide; ALT: Alanine transaminase; AST: Aspartate transaminase; ALP: Alkaline phosphatase; CAT: Catalase; GSH: Reduced glutathione; ROS: Reactive oxygen species; SOD: Superoxide dismutase; LPO: lipid peroxidation; MDA: Malonaldehyde; GPX: Glutathione peroxidase.
  2,334 146 -
Flavonoid and Phenolic Compounds from Carissa macrocarpa: Molecular Docking and Cytotoxicity Studies
Hany E Khalil, Maged E Mohamed, Mohamed A Morsy, Mahmoud Kandeel
July-September 2018, 14(57):304-310
DOI:10.4103/pm.pm_104_18  
Objective: The objective of the study is to investigate the phytochemical contents of the methanol extract of leaves of Carissa macrocarpa, the possible anticancer activities of the isolated compounds through molecular docking approaches as well as the potential cytotoxic activity. Materials and Methods: The methanol extract of the plant was subjected to several chromatographic procedures. In silico studies of the isolated compounds against four anticancer target kinases, namely, protein kinase B (PKB/AKT), phosphatidylinositol 3-kinase, protein kinase C, and rapidly accelerated fibrosarcoma kinase were performed. Potential cytotoxic activity of the isolated compounds was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay against A549 cells. Results: Phytochemical investigation led to the isolation of three known flavonoid compounds: kaempferol 3-O-robinobioside (1), Kaempferol-3-O-α-L-rhamnopyranosyl (1-6)(4``-p-coumaroyl)β-D-galactopyranoside7-O-α-L-rhamnopyranoside (2), and variabiloside E (3) as well as three phenolic compounds: p-coumaric acid (4), salicin (5), and 3,4-dimethylphenol β-gentiobioside (6). In silico studies revealed that three out of the six compounds were strongly bound with one or more of the targets enzymes. Compound 3 showed broad-spectrum binding with the four targets with high docking score. Compounds 1–3 showed IC50comparable to that of positive control, doxorubicin. The rest of the compounds 4–6 showed relatively discrete IC50. Conclusion: The isolated compounds were reported for the first time from this plant. Compounds 1–3 could serve as lead compounds for development of new anticancer drugs. Abbreviation used: PKB/AKT: Protein kinase B; PI3K: Phosphatidylinositol 3-kinase; PKC: Protein kinase C; RAFK: Rapidly accelerated fibrosarcoma kinase; 1H- and 13C-NMR: Proton and carbon-13 nuclear magnetic resonance; SCC: Silica gel column chromatography; RPCC: Reversed-phase silica gel column chromatography; TLC: Thin-layer chromatography; MVD: Molegro Virtual Docker; DMEM: Dulbecco's Modified Eagle's Medium; FBS: Fetal bovine serum; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2H-tetrazolium bromide; DMSO: Dimethyl sulfoxide.
  2,263 172 -
Ethnomedicinal knowledge among the tribes of the little Andaman Island, Andaman and Nicobar Islands, India
M Punnam Chander, Paluru Vijayachari
July-September 2018, 14(57):488-493
DOI:10.4103/pm.pm_585_17  
Background: In Little Andaman Island, Onges is one of the particularly vulnerable Tribal Groups settled in Dugong Creek, whereas the Nicobarese tribe resides at Harminder Bay village. Having abode in the vicinity of the forest, a strong ethnobotanical practice prevails in this Island since ancient time. Objectives: This study was an attempt to document the medicinal plants used in this Island, with a view to preserve the ethnobotanical knowledge and to protect the biodiversity of this area. Materials and Methods: Fieldwork was conducted during December 2012 and December 2014. Data were collected through interview, questionnaire, and group discussion with traditional knowledge practitioners residing in the study site. Data were analyzed to determine the informant consensus factor (ICF), family use value, and UV of the plants. Results: The study documented 42 medicinal plant species belonging to 26 families, which are used for the treatment of at least 25 different ailments. The highest ICF value (0.64) was observed for injuries and trauma. Euphorbiaceae family constituted the highest number of plant species. Plants with the highest UVs were Ocimum tenuiflorum L., Morinda citrifolia L., Zingiber squarrosum Roxb., and Eupatorium odoratum L. The most common growth form observed were shrubs (33.33%). Leaves were found to be the most frequently used plant part. Conclusions: The present study revealed the use of few endemic plants, extensively for medicinal preparation, demonstrating an effective ethnobotanical practice in the study area. In future, these plants can be subjected to bioassay-guided investigation, while other plants can be screened for their bioactivity, to determine their medicinal property. Abbreviations used: UV: Use value; FUV: Family use value; TKP: Traditional knowledge provider; ICF: Informant consent factor.
  2,268 133 -
Angiotensin-converting enzyme inhibitory activity of Senna garrettiana active compounds: Potential markers for standardized herbal medicines
Fameera Madaka, Tossaton Charoonratana
July-September 2018, 14(57):335-339
DOI:10.4103/pm.pm_325_17  
Background: Fifteen medicinal plants used in traditional Thai herbal medicine (HM) for hypertension treatment were previously tested for their inhibitory activity against an angiotensin-converting enzyme (ACE). The ethyl acetate extract from one of these medicinal plants, Senna garrettiana, possessed satisfactory ACE inhibitory (ACEi) activity. Objective: For this study, S. garrettiana extract was further subjected to an isolation process to uncover the active compounds with ACEi activity. Materials and Methods: The ethyl acetate fraction was subjected to isolate and purify by column chromatography accompany with the ACEi assay. The structures of all compounds were elucidated using nuclear magnetic resonance spectroscopy. Results: It was found that S. garrettiana extract possessed two active compounds, piceatannol and betulinic acid, which both had good ACEi activity displaying IC50values of 8.44 μM and 26.77 μM, respectively. Conclusions: These findings show that both compounds can be used as markers for quality control of any standardized HM for hypertension treatment and not just for traditional Thai herbal remedies. Moreover, to the best of our knowledge, this is the first report investigating both compounds for their ACEi activity. Abbreviations used: ACE: Angiotensin-converting enzyme; ACEi: Angiotensin-converting enzyme inhibitory; CH2Cl2: Dichloromethane; DMSO: Dimethyl sulfoxide; HA: Hippuric acid; HCl: Hydrochloric acid; HHL: Hippuryl-L-histidyl-L-leucine; HM: Herbal medicine; HMs: Herbal medicines; HPLC: High-performance liquid chromatography; IC50: The half maximal inhibitory concentration; L-NAME: N (ω)-nitro-L-arginine methyl ester; MeOH: Methanol; NaCl: Sodium chloride; NADPH: Nicotinamide adenine dinucleotide phosphate; NMR: Nuclear magnetic resonance spectroscopy; NO: Nitric oxide; TDR: The Special Programme for Research and Training in Tropical Disease; μL: Microliter; μM: Micromolar.
  2,245 132 -
In Vitro antigastric cancer activity of squalene, a triterpenoid compound isolated from Rhizophora Mucronata mangrove plant leaves against AGS cell line
Thirunavukkarasu Palaniyandi, Asha Sivaji, Ramanathan Thiruganasambandam, Sudhakar Natarajan, Rajeshwari Hari
July-September 2018, 14(57):369-376
DOI:10.4103/pm.pm_577_17  
Background: Mangrove plants have high potential in medical research and they have been used in traditional medicine for any diseases. Squalene, a natural 30-carbon triterpenoid compound, was originally obtained from Shark and for the first time isolated from mangrove plant species. From the historical background, it was known that squalene display protective actions against several diseases and carcinogens. Objective: Herein, we desire to report the in vitro evaluation of squalene on AGS cell line. In the present investigation, after 24 h incubation, the inhibitory effect of squalene was found to have significant activity on AGS cell lines proliferation. Materials and Methods: Mangrove plant isolated squalene compound was treated with gastric adenocarcinoma cancer cell line AGS for 24 h with control. The cells were treated at varying concentration range 10, 30, and 50 μg/mL. The cytotoxicity effect of squalene was studied by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide, mitochondria membrane potential, etc., Results: Among the tested concentrations, squalene at 50 μg/mL showed a higher level of mitochondrial depolarization, DNA fragmentation, and induced apoptosis in AGS cells when compared to control. A similar but lower activity was observed in minimum doses of 20 and 30 μg/mL compared to maximum dose of 50 μg/mL. A 50% and 100% cell viability was observed at 30 and 50 μg/mL, respectively. Likewise, there was a significant reduction in thiobarbituric acid reactive substrate and lipid hydroperoxides levels, while antioxidant enzymes like superoxide dismutase. Catalase, glutathione (GSH) peroxidase, reduced GSH, Vitamin-C (Vit-C), and Vitamin-E were increased on squalene treatment in a dose-dependent manner. Conclusion: On the basis of results, it was concluded that isolated compound (squalene) from Rhizophora mucronata had a potent antigastric carcinogenic effect at 50 μg/mL in AGS cell lines and appeared to be more sensitive toward the AGS cell line. Abbreviations used: PBS: Phosphate-buffered saline; FBS: Fetal bovine serum; DMEM: Dulbecco's modified eagle medium; MTT: 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-tetrazolium bromide; DCFH-DH-7: Diacetyl dichlorofluorescein; Rh 123: Rhodamine 123; EB: Ethidium bromide; AO: Acridine orange; TBA: Thiobarbituric acid; NBT: Nitro blue tetrazolium; PMS: Phenazine methosulfate; DTNB-5: 5-dithiobis 2-nitrobenzoic acid; GSH: Reduced glutathione.
  2,198 167 -
Solvent-based microwave-assisted extraction and identification of bioactive compounds from Sesamum indicum leaves using particle swarm optimization-integrated response surface methodology
Lopamudra Sarma, Sourav Chakraborty, Raj Kumar Duary
July-September 2018, 14(57):275-283
DOI:10.4103/pm.pm_570_17  
Background: Sesamum indicum is a widely cultivated crop with edible seeds which are well known for its rich antioxidant properties. The plant leaves have also been reported to have health functional attributes such as lowering cholesterol level in body, reducing cancer, and acting as antihypertensive resource. Objective: The aim of the study was to optimize the effects of solvent-based microwave-assisted extractions (SMAEs) of phenolic compounds from S. indicum (Sesame) leaves using combined approach of response surface methodology and particle swarm optimization (PSO). Materials and Methods: SMAEs along with response surface model development and its optimization by PSO were done. The optimization parameters such as microwave power (180–900 W), extraction time (1–30 min), solid-liquid ratio (1:10–40 g/mL), and solvent (methanol) concentration (50–70%) were employed to obtain extracts showing higher total phenolic content (TPC) and antioxidant activities by 2,2-diphenyl-1-picrylhydrazyl (DPPH) value. Results: The maximum TPC and DPPH values were 206.14 ± 2.70 mg GAE/100 g and 96.04 ± 1.67% for extracts against 51.38% methanol concentration, 1:23 g/mL solid-liquid ratio, 900 W power level, and 16.95 min of extraction time, respectively. The gas chromatography–mass spectrometry chromatographs showed the presence of various essential phytocomponents such as ascorbic acid, phytol, and flavonol as functional molecules at 26.79, 29.03, and 39.70 min retention time, respectively. The high-performance liquid chromatogram further confirmed the presence of quercetin, chlorogenic acid, and gallic acid at a concentration of 11.25, 4.28, and 1.61 ppm, respectively. Conclusion: The results of this study suggest application of PSO algorithms as an efficient approach to optimize the extraction of bioactive components from Sesame leaves which can be used as a potential source for functional and nutraceutical food development. Abbreviations used: TPC: Total phenolic content; DPPH: 2,2-diphenyl-1-picrylhydrazyl; RSM: Response surface methodology; BBD: Box–Behnken design; BSTFA: N, O-Bis (trimethylsilyl) trifluoroacetamide); LDPE: Low-density polyethylene.
  2,273 77 -
Molecular docking and density function theory studies of compounds from Euphorbia hirta and Bacopa monnieri to zika virus structural and nonstructural proteins
Sangeetha Kothandan, Indu Purushothaman, Stella Puthur George, Sasikala Reddy Purushothaman, Meena Karunakaran Sulochana
July-September 2018, 14(57):481-487
DOI:10.4103/pm.pm_89_17  
Background: Zika virus is an arbovirus belongs to the genus flavivirus and pose a serious global threat. The recent 2015 outbreak in Brazil was associated with a significant increase in microcephaly cases and other neurological complications in newborn babies and WHO declared Zika to be an international public health emergency. Currently, there is no specific treatment or Vaccine available for the Zika virus, and thus due to the unavailability of the antiviral drugs, the need for the identification of novel drugs is paramount. Materials and Methods: The compounds from two medicinal plants (Bacopa monnieri and Euphorbia hirta) were selected for the in silico molecular docking studies against the structural and nonstructural proteins of Zika virus. Quantum–chemical parameters density functional theory and absorption, distribution, metabolism, and excretion-toxicity (ADMET) was performed to identify the drug-likeliness properties. Results: Among the tested compounds, galloylquinic acid, Bacopaside III, and Bacopaside A were identified as leads against multiple targets of Zika virus. The identified compounds also exhibited desirable quantum chemical and ADMET properties. Conclusion: Hence, the compounds hampering the active site of the three different proteins playing a prime role in replication and fusion with desirable pharmacokinetic properties could be suggested for further in vitro and in vivo analysis of Zika virus. Abbreviations used: WHO: World Health Organization; DFT: Density function theory; ADMET: Absorption, distribution, metabolism, and excretion-toxicity; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HIV: Human immunodeficiency virus; HSV: Herpes simplex virus; PDB: Protein data bank; OPLS: Optimized potential for liquid simulations; RMSD: Root-mean-square deviation; IFD XP: Induced fit docking extra precision; XP: Extra precision; PSA: Polar surface area; BBB: Blood brain barrier; A LogP 98: Atom-based Log P98; HOMO: Highest occupied molecular orbital; LUMO: Lowest unoccupied molecular orbital; DS V 4.0: Discovery Studio version 4.0; NS3: Nonstructural protein 3; NS1: Nonstructural protein 1
  2,204 145 -
Cardioprotective effect of oregano oil against doxorubicin-induced myocardial infarction in rats
Damita Lourdes Cota, VP Rasal, Sanjay Mishra, Sushant Shengule
July-September 2018, 14(57):363-368
DOI:10.4103/pm.pm_70_18  
Aim: The objective of the study was to evaluate the cardioprotective effect of oregano oil (O.OIL) in doxorubicin (DOX)-induced myocardial infarction (MI) in rats. Materials and Methods: O.OIL (100 mg/kg and 200 mg/kg, p.o) was administered for 21 days in rats. MI was induced by DOX (5 mg/kg, i.p.) administered on the 7th, 14th, and 21st day of the study to obtain a cumulative dose of 15 mg/kg. On day 22, changes in electrocardiogram (ECG); force of contraction; serum markers: lactate dehydrogenase (LDH), creatine kinase (CK) MB isoenzyme, troponin I; lipid profile: total cholesterol and triglycerides; antioxidant enzymes level: malondialdehyde (MDA) and glutathione (GSH); heart and body weight; and histopathology of heart were determined. Results: Pretreatment with O.OIL significantly protected the myocardium from the toxic effects of DOX by reducing the elevated level of CK-MB, LDH, and troponin I to the normal levels. O.OIL increased the GSH levels and decreased the MDA levels in cardiac tissue. It also restored the changes in ECG and force of contraction and showed significant recovery of heart tissue in histopathological studies. Statistical analysis: All results are expressed as mean ± standard error of the mean. The results were analyzed for statistical significance by one-way ANOVA followed by Dunnet's Multiple Comparison Test using GraphPad version 5.01, P < 0.01 was considered statistically significant. Conclusion: O.OIL (100 and 200 mg/kg p.o.) reduced cardiac complications in DOX-induced MI in rats. Abbreviations used: ECG: Electrocardiogram; O.OIL: Oregano oil; DOX: Doxorubicin; MI: Myocardial infarction.
  2,096 152 -
Bio-based synthesised and characterized monodispersed Curcuma longa silver nanoparticles induces targeted anticancer activity in breast cancer cells
Eram Sheikh, ML B. Bhatt, Madhu Tripathi
July-September 2018, 14(57):340-345
DOI:10.4103/pm.pm_71_18  
Background: Among the various metal nanoparticles, plant extract-based silver nanoparticles are gradually getting preference because the bioreduction potential of plant extracts is much faster for silver ions. The silver nanoparticles have the bactericidal activity and have shown to possess anticancer potential for developing effective and safe novel therapeutic agents. Objectives: The objective of the study was to synthesize Curcuma longa silver nanoparticles for the induction of anticancer activity in breast cancer cell. Materials and Methods: The 20 ml and 10 ml of C. longa stock extract were mixed with 10 ml and 10 μl of AgNO3solution (A) 1 mM and (B) 1M, respectively; reduction process initiates the formation of nanoparticles as detected by color change of AgNO3from brown to dark brown; thereafter, characterization done by ultraviolet–visible spectrophotometer, Scanning electron microscope, and transmission electron microscopy. Results: The synthesized and characterized C. longa silver nanoparticles induce targeted anticancer activity in breast cancer cells. Abbreviations used: μl: Microlitre; μg: Microgram; mM: Milli Molar; M: Molar; rpm: Revolution per minute; IC50: Inhibitory concentration at which 50% cells get died; PBS: Phosphate-buffered saline solution; DMSO: Dimethylsulfoxide; MTT: 3-4,5-dimethyl thiazole-2-yl)-2, 5-diphenyl tetrazolium bromide; DMEM: Modified eagle's media; DPPH-2,2-diphenyl-1 picrylhydrazyl; Abs: Absorbance.
  2,109 124 -
Bioconversion from Scutellaria baicalensis (baicalin) feremted with Leatiporus sulphureus into enriched-baicalein and anti-wrinkle effects
Mi-Young Yun, Eun-Young Won, Jung-Hee Lee, Ju-Im Jung, Hwa-Jung Choi
July-September 2018, 14(57):453-457
DOI:10.4103/pm.pm_32_18  
Background: Ultraviolet (UV) radiation causes photoaging in human skin. Phytochemicals from herbs have been used to treat various skin problems. Bioconversion of phytochemicals can increase efficacy and absorption rate by fermentation. Objective: The aim was to evaluate the anti-wrinkle of enriched-baicalein Scutellaria baicalensis (baicalin) was bioconverted by fermented with Leatiporus sulphureus (enriched-baicalein). Materials and Methods: Bioconversion of baicalein from baicalin were analyzed by high-performance liquid chromatography. The effect of enriched-baicalein on matrix metalloproteinase (MMP)-1 and procollagen production was examined in neonatal human dermal fibroblasts (HDFn) using ELISA, and then the effect of enriched-baicalein on wrinkle formation, skin thickness, and changes in collagen fibers were evaluated in hairless mice by histological analysis of their skin. Results: In vitro enriched-baicalein reduced UVB-induced MMP-1 production and promoted procollagen synthesis. In vivo, enrich-baicalein inhibited UVB-induced wrinkle formation, epidermal thickening, and damage to collagen fiber. Conclusions: These results suggest that enriched-baicalein has the potential to prevent wrinkle formation by inhibition of MMP-1 and promotion of Type-1 procollagen in HDFn, and by inhibition of skin thickness, and changes in collagen fibers in UVB-induced mice. Abbreviations used: MMP-1: Matrix metalloproteinase; HDFn: Neonatal human dermal fibroblasts; HPLC: High performance liquid chromatography; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM: Dulbecco's modified Eagle's medium.
  1,910 176 -
Evaluation of biological activity, toxicity, and phytochemical content of Bowdichia virgilioides (Fabaceae) aqueous extract
Isabela Bacelar Assis, Eliana Maria Mauricio da Rocha, Daniel Silqueira Martins Guimarães, Glaécia Aparecida do Nascimento Pereira, Fabiana Paula Pereira, Jaqueline Maria Siqueira Ferreira, Emiliano O Barreto
July-September 2018, 14(57):403-408
DOI:10.4103/pm.pm_273_17  
Background: Antibiotic resistance is a worldwide problem that poses a serious threat to human health, limiting the therapeutic options for bacterial infections. The spread of falciparum-resistant malaria is also concerning, making the patient treatment an extremely difficult task. Those facts have heightened the interest to find alternate options to treat infections caused by drug-resistant microorganisms. Objective: Considering the importance of the development of new substances with antibacterial and antimalarial properties, the present study aimed to investigate the activity of the aqueous extract of stem bark of Bowdichia virgilioides (AEBv). This plant is commonly used in Brazilian folk medicine to treat a wide range of illnesses, including signs and symptoms associated with malaria. Materials and Methods: The AEBv was assayed for toxicity against two cell lines and Artemia salina larvae. In vitro activity of the extract was screened against a panel of Gram-positive and Gram-negative bacteria, a chloroquine-resistant (W2) and a chloroquine-sensitive (3D7) Plasmodium falciparum strains. The extract was also tested as antimalarial in vivo against Plasmodium berghei. Results: The AEBv presented no significant toxicity and was found to exert in vitro growth inhibitory effect against the tested bacterial species. The lowest minimal inhibitory concentration was reported for Staphylococcus aureus (0.125 mg/ml) followed by Staphylococcus epidermidis and Staphylococcus saprophyticus (0.50 mg/ml). B. virgilioides extract showed weak in vitro antimalarial activity against P. falciparum. A preliminary phytochemical analysis revealed the presence of flavonoids, phenolic groups, terpenoids, saponins, and tannins and the absence of alkaloids. Conclusion: The AEBv showed promising activity against Gram-positive microorganisms. Abbreviation used: AEBv: Aqueous extract of stem bark of Bowdichia virgilioides; FBS: fetal bovine serum; CC50: 50% cytotoxic concentration; LC50: Median lethal concentration; ATCC: American Type Culture Collection; MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentrations; CQR: Chloroquine resistant; CQS: Chloroquine-sensitive; HRP2 Histidine rich protein 2; ELISA: Enzyme linked immunosorbent assay; PBS T: Phosphate buffer saline with 0.05% Tween 20; ANOVA: Analysis of variance; TLC: Thin layer chromatography; Rf: Retention factor; SI: Selectivity índex; MRSA: Methicillin resistant Staphylococcus aureus.
  1,949 124 -
Amelioration of oxidative stress through apoptosis-mediated pathway in colon cancer cells by hexane fraction of Moringa oleifera extract
Liang Jinghua, Sun Linmei, He Ping, Sivapragasam Gothai, Katyakyini Muniandy, S Suresh Kumar, Norhaizan Mohd Esa, Palanisamy Arulselvan
July-September 2018, 14(57):311-318
DOI:10.4103/pm.pm_284_17  
Background: Colorectal cancer (CRC) is one of the most common cancers worldwide and it is predicted that the number of cases will rise to 2.4 million by 2035. Therefore, preventative and therapeutic approaches are essential for the fight against cancer. The management of colon cancer through conventional methods mainly surgery, radiotherapy, and chemotherapy have produced unpredictable complications, especially metastasis and relapse of tumors, leading to loss of patient's life. In the last decade, association between oxidative stress and CRC has been intensively studied and it was found that the CRC has increased levels of oxidative stress markers. Natural dietary supplements provide a better choice for cancer prevention and treatments. Moringa oleifera (MO) generally known as the Miracle tree and most of the parts have high nutritional and medicinal properties; though, the leaves are suggested to have the highest concentration of vitamins, minerals, amino acids, and active compounds. Materials and Methods: The major purpose of the analysis was to investigate the dose-dependent cytotoxic and apoptosis-inducing effects of the hexane fraction of the MO extract on the colon cancer cells, HT-29. The anticancer potential of hexane fraction of MO extract was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and apoptotic mechanistic action by fluorescent staining, caspases, and immunoblot analysis. Results: The hexane fractions of MO extract suppressed the proliferation of HT-29 cells and induced apoptosis through mitochondrial apoptotic pathway. Conclusions: These findings suggested that hexane fraction of the extract has significant cytotoxic and apoptotic activity and therefore it could be discovered as a unique target for anticancer drug development. Abbreviations used: CRC: Colorectal cancer; MO: Moringa oleifera; HT-29: Colon cancer cells; HFMOL: Hexane fraction of Moringa oleifera leaves.
  1,959 76 -
Anti-inflammatory properties of Bixa Orellana leaves extract are associated with suppression of bradykinin-induced endothelial hyperpermeability
Yoke Keong Yong, Muhd Nazrul Somchit, Zuraini Ahmad
July-September 2018, 14(57):352-357
DOI:10.4103/pm.pm_540_17  
Background: Previous study showed that an aqueous extract of Bixa orellana L. (Family of Bixaceae) leaf (AEBO) is capable of inhibiting bradykinin (BK)-induced inflammation in animal models. Objective: This study further investigates the effect of AEBO on BK-induce inflammation in vitro model. Materials and Methods: The endothelial barrier protective effect of AEBO was examined via an in vitro endothelial permeability assay. Human umbilical vein endothelial cell (HUVEC) was first pretreated with AEBO with a concentration range from 0.1, 0.2, and 0.4 mg/mL and then induced with BK. Fluorescein isothiocyanate-conjugated-dextran was used as an indicator of permeability flux. To elucidate its mechanism of action, the phospholipase C (PLC) – nitric oxide (NO) – cyclic guanosine monophosphate (cGMP) signaling pathway and protein kinase C (PKC) activity were evaluated. Results: Pretreatment of AEBO significantly (P < 0.05) suppressed BK-induced HUVEC hyperpermeability and 0.4 mg/mL possessed the maximal inhibitory effect (87%, 70%, and 57% inhibition rate at 5, 15, and 30 min time point, respectively). Moreover, AEBO has presented remarkable IC50 = 0.24 mg/mL for anti-PLC activity, 0.36 mg/mL for anti-NO production, and 0.19 mg/mL for anti-cGMP production. For PKC inhibition, the IC50 (0.42 mg/mL) was slightly higher compared to others. Conclusion: This study provided supportive evidence for the previous study where AEBO exhibited anti-inflammatory activity against BK in vivo. The anti-inflammatory activity of AEBO may partly be associated with the reduction of endothelial hyperpermeability via the suppression of PLC-NO-cGMP signaling and PKC activity. Abbreviations used: AEBO: Aqueous extract of Bixa orellana; BK: Bradykinin; PLC: Phospholipase C; NO: Nitric oxide; cGMP: Cyclic guanosine monophosphate; PKC: Protein kinase C; HUVEC: Human umbilical vein endothelial cell; PBS: Phosphate buffered saline; eNOS: Endothelial nitric oxide synthase.
  1,725 112 -
Identification of phytoconstituents of Memecylon sisparense gamble leaf and evaluation against cisplatin-induced oxidative renal damage in mice
Jaya Lakshmi Uppu, Veerabhadra Swamy Challa, Devender Bhattula, Ganga Modi Naidu Vegi, Malathi Jojula, Asha Syed
July-September 2018, 14(57):384-392
DOI:10.4103/pm.pm_116_18  
Background: Memecylon sisparense Gamble (MSG) belongs to Melastomataceae family, having a wide range of pharmacological activities such as antioxidant, hepatoprotective, and anti-inflammatory. Objective: The present study aimed for the first time toward the identification of biologically active compounds in MSG leaf ethyl acetate extract (MSGLEAE) by gas chromatography–mass spectrometry (GC-MS) analysis and compared with docking studies along with its nephroprotective activity against cisplatin (CP)-induced nephrotoxicity in mice. Materials and Methods: MSGLEAE was subjected to GC-MS analysis and molecular docking studies. Swiss albino male mice were treated with MSGLEAE (250, 500 mg/kg, PO) against CP 12 mg/kg IP evaluated for nephroprotective activity. The changes in renal tissue were assessed from serum biochemical renal toxicity, antioxidant stress markers along with histopathological studies. Results: Out of 41 compounds identified, 20 were found having biological activities such as nephroprotective, hepatoprotective, anticancer, antioxidant, and antimicrobial and inhibition of uric acid production. The nephroprotective active compounds (N,N,O-triacetylhydroxylamine, 2(4H)-benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-, N-{(4-hydroxy-3-methoxyphenyl) methyl}-8-methyl-6-nonenamide) had shown binding energy of −5.27, −5.98, −5.27 (ΔG(Kcal/mol)), respectively, in docking studies. MSGLEAE showed a significant protective effect against CP-induced nephrotoxicity because of the identified compounds by reducing the oxidative stress in renal tissue evident by histopathological studies. Conclusion: This is the first ever report in terms of identification of bioactive constituents in MSGLEAE. Pretreatment has a significant therapeutic benefit during CP therapy by inhibiting oxidative stress, enhancing nephroprotective activity. Abbreviations used: MSGLEAE: Memecylon sisparense Gamble leaf ethyl acetate extract; CP: Cisplatin; BUN: Blood urea nitrogen; SOD: Superoxide dismutase; CAT: Catalase; GSH: Glutathione; NO: Nitric oxide; MDA: Malondialdehyde; IAEC: Institutional Animal Ethics Committee; TCA: Trichloroacetic acid; DTNB: 5,51-dithiobis-2-nitrobenzoic acid; FC: Folin–Ciocalteu reagent; SNP: Sodium nitroprusside; H and E: Hematoxylin and eosin.
  1,671 109 -
Cytotoxicity evaluation of Zingiber zerumbet ethyl acetate extract on K-562, erythroleukemia cell line
Asmah Hamid, Che Yang Aminah Ramly, Saiful Ridzuan Karim, Umi Noraashikin Zainudin, Nor Fadilah Rajab, Khairana Husain, Nurul Farhana Jufri
July-September 2018, 14(57):430-433
DOI:10.4103/pm.pm_436_17  
Background: Zingiber zerumbet locally known as lempoyang is used traditionally to cure swelling, sores, and loss of appetite. Its rhizomes contain many active compounds such as zerumbone and kaempferol that have been shown to exhibit antiproliferative activities on various cancer cell types. However, the effect of this plant on leukemia has not thoroughly been investigated. Objective: This study was conducted to investigate the cytotoxic effects of Z. zerumbet ethyl acetate crude extract on apoptosis-resistant erythroleukemia cell line, K-562. Materials and Methods: The cytotoxic effects and morphological changes of Z. zerumbet ethyl acetate crude extract on apoptosis-resistant erythroleukemia cell line, K-562, were investigated spectrometrically through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and morphologically using the phase-contrast microscopy, respectively. Results: MTT assay demonstrated that this crude extract showed cytotoxic effects on erythroleukemia cell line K-562 in a dose-dependent and time-dependent manner with IC50values of 13.83 ± 1.82 and 10.13 ± 1.67 μg/ml for 48 and 72 h time points, respectively. The morphology of treated cells showed apparent changes of features such as membrane blebbing, cell shrinkage, and formation of apoptotic bodies which were the signs of apoptosis. The changes of cell morphology that resembled apoptotic features were more obvious with increase in treatment time. Conclusions: This finding indicates that ethyl acetate crude extract of Z. zerumbet exhibits cytotoxic effects on K-562 cell line through induction of apoptosis as morphologically seen in the cells. Further investigations are needed to ascertain the possibility of apoptosis as its major event of cell death. Abbreviations used: MTT: 3-(4,5-Dimethylthiazol-2-yI)-2,5-diphenyl tetrazolium bromide; DMSO: Dimethylsulfoxide; h: Hour.
  1,672 98 -
Terminalia chebula: A promising indigenous phytotherapeutics for Alzheimer's disease
Roohollah Sobhani, Sivaprasad Mitra, Kripamoy Aguan
July-September 2018, 14(57):465-470
DOI:10.4103/pm.pm_402_17  
Introduction: Alzheimer's disease (AD) is a progressive neurologic disorder that leads to the irreversible loss of neurons. Amyloid plaques and loss of cholinergic neurons are two main characteristics of AD. In AD, the action of neurotransmitter, acetylcholine, is terminated by acetylcholinesterase (AChE) present in the synaptic cleft, and formation of amyloid plaques in the synapse prevents acetylcholine to reach their receptors on the postsynaptic neurons. Over the years, scientists have discovered various classes of the molecules for treatment of the disease and some are derived from plants such as rivastigmine, huperzine, and galantamine. Numerous plants are being used traditionally as treatment for neurodegenerative diseases such as AD, anti-aging, and preventing dementia. Among these indigenous plants, Terminalia chebula (Tc) is regarded to slow down the aging process and to improve the cognition in the traditional age-old practices. Objective: In this study, we have assayed the methanolic extract of Tc leaves to assess its inhibitory effect on four different pathways related to AD. Materials and Methods: Ellman method was used to measure the effect of Tc on AChE activity. To assess the effect of Tc on amyloid-β (Aβ)-secretion pathway, we measured the amount of Aβ40 in cell culture medium using ELISA Kit. Aβ-aggregation (40 and 42) and disruption of Aβ-aggregates (40 and 42) were monitored using a fluorescence technique, Thioflavin-T, in vitro. Results: Our results show that Tc has significant inhibitory effect on all the above-mentioned assays, with the best IC50 = 10.3 μg/ml for Aβ40-aggregation followed by Aβ42-aggregation (IC50 = 18.6 μg/ml), disruption of Aβ40-aggregates (IC50 = 41.8 μg/ml), disruption of Aβ42-aggregates (IC50 = 132.2 μg/ml), and AChE activity (IC50 = 200 μg/ml). Besides, it showed 21.46% inhibition on Aβ40 level in HEK293 cell line. Abbreviations used: AD: Alzheimer's disease, Tc: Terminalia chebula, AChE: acetylcholinesterase, Aβ: Amyloid-β, APP: Amyloid precursor protein.
  1,577 118 -
Stimulation of murine immune response by Clerodendrum infortunatum
Somit Dutta, Arnab Kumar Chakraborty, Pallab Kar, Priyankar Dey, Arnab Sen, Tapas Kumar Chaudhuri
July-September 2018, 14(57):417-429
DOI:10.4103/pm.pm_549_17  
Background: Clerodendrum infortunatum is a medicinal plant found especially in the Sub-Himalayan regions of West Bengal. Various tribal communities of this region have been using these plants as a source of natural medicine. Objective: Medicinal values of C. infortunatum are well established, but the immunomodulatory properties have never been studied. Therefore, we wanted to investigate various immunomodulatory activities of this plant. Materials and Methods: Several parameters such as plaque-forming cell assay, hemagglutination titer, phagocytic activity of macrophages, and inhibition of lipopolysaccharide (LPS) were performed. Moreover, effects of C. infortunatum on the weight of various organs and total serum protein, albumin, and globulin levels were also determined. The phytochemical fingerprints of C. infortunatum extracts were obtained from Fourier transform infrared and gas chromatography-mass spectrometry analysis. Results: The macrophage stimulation and plaque-forming cell numbers (using sheep red blood cell as antigen) were significantly proliferated and found optimum at 100 and 250 mg/kg doses of leaf and root extracts simultaneously. The extent of stimulation in mural humoral immunity was in the order of leaf > root > stem. The anti-inflammatory activity of C. infortunatum was confirmed by the inhibition of LPS-induced nitric oxide synthesis by macrophages. Conclusion: It can be concluded that 70% hydromethanolic extract of C. infortunatum has profound immunomodulation potentials in the murine model, stimulating both humoral and innate form of the immune system. Abbreviations used: PFC: Plaque-forming cell assay; HA titer: Hemagglutination titer; LPS: Lipopolysaccharide; FTIR: Fourier transform infrared; GC-MS: Gas chromatography-mass spectrometry; SDS: Sodium dodecyl sulfate; sRBC: Sheep red blood cell; PBS: Phosphate buffer saline; PC: Phagocytic capacity; PI: Phagocytic index; NaCl: Sodium chloride; CaCl2: Calcium chloride; NBT: Nitro blue tetrazolium; DMSO: Dimethyl sulfoxide; KOH: Potassium hydroxide; H2SO4: Sulfuric acid; HOCl: Hypochlorous acid; H2O2: Hydrogen peroxide; EDTA: Ethylenediaminetetraacetic acid.
  1,571 114 -
In vitro anticholinesterase and neurotoxicity activities of Ocotea aciphylla fractions
Monique Marylin A de A. Carneiro, Rodrigo Souza Conceição, Isabella Mary Alves Reis, Alessandra Bispo Da Silva, Joana Da Luz Oliveira, Alexsandro Branco, Silvia Lima Costa, Mariana Borges Botura
July-September 2018, 14(57):448-452
DOI:10.4103/pm.pm_366_17  
Background: Ocotea species are known to produce secondary metabolites with a range of biological activities. This study aimed to evaluate the in vitro acetylcholinesterase (AChE) inhibition and neurotoxicity activities of the Ocotea aciphylla leaves. Materials and Methods: The in vitro anticholinesterase effect of crude extracts of O. aciphylla was investigated by means of spectrophotometric microplate assay. The most active extract, aqueous extract (AQE), was fractionated using column chromatography with silica gel as stationary phase to furnish several fractions that were also evaluated for the anticholinesterase effect. The neurotoxicity activity of AQE and active fraction (F9) was investigated in rat adrenal medulla pheochromocytoma strain cultures by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. The chemical characterization of the most active fraction was performed through high-performance liquid chromatography coupled with mass spectrometry multistage (HPLC-MS/MS). Results: Ethanolic extract (EE) and AQE exhibited significant inhibitory effects of the activity of AChE, with inhibitory concentration (IC50) of 443.7 and 412.8 μg/mL, respectively. Among the fractions, F9 was more effective AChE inhibition with IC50of 286.2 μg/mL. In the neurocytotoxicity assays, only the F9, at the highest concentration (500 μg/mL), induced a significant reduction in cell viability. HPLC-MS/MS analysis of the active fraction enabled the characterization of the natural compounds, i.e., procyanidin B-type dimer, propelargonidin dimer, catechin, and methoxy-luteolin-deoxyhexose-hexose. Conclusion: The leaves of O. aciphylla showed in vitro anticholinesterase activity and low neurotoxicity, and these effects might be related to the presence of phenolic compounds. Abbreviation used: AChE: acetylcholinesterase; ACTI: Acetylthiocholine iodide; AD: Alzheimer's disease; AQE: Aqueous extract; BSA: Bovine serum albumin; DMSO: Dimethyl sulfoxide; DTNB: 5,5'-dithiobis (2-nitrobenzoic acid); EAE: Ethyl acetate extract; EE: Ethanolic extract; EtOAc: Ethyl acetate; HE: Hexane extract; Hex: Hexane; HPLC-MS/MS: High-performance liquid chromatography coupled with mass spectrometry multistage; MeOH: Methanol; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PC-12: Rat adrenal medulla pheochromocytoma strain; RPMI: Roswell park memorial institute medium.
  1,562 116 -
Phytochemical investigation and bioactivities of Alternanthera ramosissima (Mart.) Chodat and Hassl.
Sukanya Dej-adisai, Sathianpong Phoopha, Jindaporn Puripattanavong
July-September 2018, 14(57):346-351
DOI:10.4103/pm.pm_478_17  
Background: Since there are many researches of phytochemical and bioactivities from genus Alternanthera but did not have any reports from Alternanthera ramosissima. Objective: The objective of the study is to isolate, identify, and determine the bioactivities of the chemicals in this plant, which has not been reported. Materials and Methods: Bioactivity studies of A. ramosissima ethanol extracts were as cytotoxic activity against human cancer cells (human breast carcinoma cell (MCF-7), human cervical carcinoma cell (KB), human cervix adenocarcinoma cell (HeLa) and human colon adenocarcinoma cell (HT-29)) and anti-inflammation. Chromatographic and spectroscopic techniques were used for the isolation and identification of pure compounds. Results: A. ramosissima extracts had the potential effects of cytotoxic activity against human cancer cells and anti-inflammation. Six compounds were isolated; two mixture compounds as the mixture of β-sitosterol and stigmasterol, the mixture of spinasterol and stigmast-7-en-3 β-ol; and four pure compounds as β-sitosterol-D-glucoside, 7-O-β-D-glucopyranosyl chrysoeriol, 7-O-(6”-O-Acetyl)-β-D-glucopyranosyl chrysoeriol, and kaempferol-3-O-β-rutinoside. The isolated compounds were determined the cytotoxic activity against human cancer cells and anti-inflammatory activity. The results showed that all of them did not have cytotoxic activity against human cancer cells. However, the isolated compounds; 7-O-β-D-glucopyranosyl chrysoeriol and 7-O-(6”-O-Acetyl)-β-D-glucopyranosyl chrysoeriol exhibited the potential effect of anti-inflammation by nitric oxide inhibition with IC50as 25.30 and 39.81 μM, respectively, when compared with the positive standards; indomethacin, caffeic acid phenethyl ester, and L-nitroarginine which showed nitric oxide inhibition with IC50as 50.30, 5.62, and 61.80 μM, respectively. Conclusion: This is the first report of chemicals and bioactivities of A. ramosisima and the first report of cytotoxic and anti-inflammatory activities of isolated compounds; 7-O-β-D-glucopyranosyl chrysoeriol and 7-O-(6”-O-Acetyl-)-β-D-glucopyranosyl chrysoeriol. Abbreviations used: MCF-7: Human breast carcinoma cell, KB: Human cervical carcinoma cell, HeLa: Human cervix adenocarcinoma cell, HT-29: Human colon adenocarcinoma cell, HGF: Human gingival fibroblast cell, IC50: The half maximal inhibitory concentration, DMEM: Dulbecco's Modified Eagle Medium, cDMEM: Completed DMEM medium, DMSO: Dimethyl sulfoxide, SRB: Sulforhodamine B, NO: Nitric oxide, EIMS: Electron ionized mass spectrometry, NMR: Nuclear magnetic resonance, 1H NMR: Proton NMR, 13C NMR: Carbon-13 NMR.
  1,556 120 -
The reactive oxygen species/AMP-activated protein kinase signaling pathway's role in the apoptotic induction of MCF-7 human breast cancer cells caused by the ethanol extract of Citrus Unshiu peel
Min Yeong Kim, Hyun HwangBo, Eun Ok Choi, Da He Kwon, Kyu Im Ahn, Seon Yeong Ji, Jin-Woo Jeong, Su-Hyun Hong, Cheol Park, Yung Hyun Choi
July-September 2018, 14(57):440-447
DOI:10.4103/pm.pm_484_17  
Objective: Citrus unshiu Markovich, which has been used for many different purposes in traditional medicine, has been reported to possess various pharmacological properties; however, its anticancer potentials are relatively unknown. This study aimed to investigate the effect of the ethanol extract of C. unshiu peel (EECU) on MCF-7 human breast cancer cells. Materials and Methods: Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected using DAPI staining and flow cytometry. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS) assay, caspase activity, and Western blotting analysis were used to confirm the basis of apoptosis. Results: Our results demonstrated that the inhibition of MCF-7 cell survival by EECU was associated with the induction of apoptosis. EECU-induced apoptosis resulted in a sequence of events, which began with the increased expression of death receptor-related proteins and a Bax/Bcl-2 expression ratio. This led to the collapse of MMP and the cytosolic release of cytochrome c, which was accompanied by and the activation of caspase-9 and caspase-8 and proteolytic degradation of poly (ADP-ribose) polymerase. EECU also induced apoptosis of MCF-7 cells by stimulating AMP-activated protein kinase (AMPK), through the generation of ROS. However, compound C, a pharmacological inhibitor of AMPK, significantly weakened EECU-induced apoptosis. Furthermore, the activation of AMKP, induction of apoptosis, and reduction of cell viability by EECU were effectively prevented when ROS production was blocked. Conclusions: These results demonstrate that EECU inhibits MCF-7 cell proliferation by activating the intrinsic and extrinsic apoptosis pathways through the ROS-dependent activation of the AMPK pathway. Abbreviations used: ACC: Acetyl-CoA carboxylase; AMPK: 5′-AMP-activated protein kinase; DCF-DA: 2′,7′-dichlorofluorescin diacetate; DMSO: Dimethylsulfoxide; DR: Death receptor; ECL: Enhanced chemiluminescence; EECU: Ethanol extract of Citrus unshiu peel; ELISA: Enzyme-linked immunosorbent assay; FADD: Fas-associated protein with death domain; FasL: Fas ligand; FBS: fetal bovine serum; FITC: Fluorescein isothiocyanate; HRP: Horseradish peroxidase; IETD: Ile-Glu-Thr-Asp; JC: 1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide; LEHD: Leu-Glu-His-Asp; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC: N-acetyl-L-cysteine; PARP: poly (ADP-ribose) polymerase; PBS: Phosphate-buffered saline; PI: Propidium Iodide; pNA: p-nitroaniline; ROS: Reactive oxygen species; SD: Standard deviation; SDS: Sodium-dodecyl sulfate; tBid: Truncated Bid; TRAIL: TNF-related apoptosis-inducing ligand
  1,507 106 -
Phenotype behavioral impairment after the administration of Xylopia Aromatica to male Balb-c mice and cytotoxicity to breast and cancer cell lines
Ivana Barbosa Suffredini, Mateus L B. Paciencia, Bruna V Santana, Humberto Vieira Frias, Cinthia Dos Santos Alves, Ingrit E C. Díaz, Maria Martha Bernardi
July-September 2018, 14(57):409-416
DOI:10.4103/pm.pm_373_17  
Background: Crude extract obtained from the stem of Xylopia aromatica (Annonaceae, EB149) known as pimenta-de-macaco, a traditional Brazilian medicinal and edible plant, showed cytotoxicity against leukemia cell lines. Despite the initial findings, no information regarding its influence on behavioral phenotype (BP) has been previously reported. Objective: The objective was to assess the effect on EB149 on BP in male mice and to perform a bioguide-fractionation aimed at the verification of the cytotoxic potential against breast and prostate cancer cell lines. Materials and Methods: BP was assessed by observation in an open cage and subsequent analysis in an open field (OF) at 15, 30, 60, 120, and 180 min, after intraperitoneal administration of X. aromatica, in a two-stage experimental delineation. Results: EB149 impaired general activity, piloerection, defecation, breathing, auricular and corneal reflexes, tail squeeze, response to touch, hindquarter fall, surface-righting reflex, body tone, and grip reflex in the first stage. In the second stage, general activity, tail squeeze, touch response, and breathing were impaired, and a decrease in locomotion frequency in OF was also observed. All behavioral changes were recovered during the period of experiment in both stages. The LD50 of EB149 was 1.944 g/kg. Two fractions obtained from EB149 named FSIST, which contains sitosterol and stigmasterol as major compounds, and total alkaloid fraction, which contains total alkaloids, showed cytotoxicity against breast and prostate cancer cell lines. Conclusions: The traditional plant X. aromatica EB149 organic extract showed cytotoxicity against breast and prostate cancer cell lines and the recovery of behavioral impairment related to general activity, tail squeeze, touch response, breath, and locomotion frequency, causing no harm to male lab mice. Abbreviations used: MMA/ICMBio/SISBIO: Ministério do Meio Ambiente/Instituto Chico Mendes de Conservação da Biodiversidade/Sistema de Autorização e Informação em Biodiversidade; CGen/IBAMA/MMA: Conselho de Gestão do Patrimônio Genético/Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis/Ministério do Meio Ambiente; AM: Amazonas State; UNIP: Universidade Paulista; g: Gram; mg: Milligram; μg: Microgram; kg: Kilogram; I. P: Intraperitoneal; CEUA/ICS/UNIP: Comissão de êtica no Uso de Animais/Instituto de Ciências da Saúde/Universidade Paulista; BP: Behavioral phenotype; OF: Open-field apparatus; NLD: Nonlethal dose; LD: Lethal dose; ANOVA: analysis of variance; FCHCl3: Fraction chloroform; FBuOH: Fraction buthanol; FH2O: Fraction water; FHEX: Fraction hexane; FDCM: Fraction dichloromethane; FMeOH: Fraction methanol; TLC: Thin-layer chromatography; U. V.: Ultraviolet; TAF: Total alkaloid fraction; nm: Nanometer; CC: Column chromatography; ATLC: Analytical thin-layer chromatography; NMR: Nuclear magnetic resonance apparatus; FSIST: Fraction containing sitosterol and stigmasterol; NC: Naive control; VC: Vehicle control; DIA: Diazepam
  1,501 80 -
Ginsenoyne C, a polyacetylene isolated from Panax Ginseng inhibit inflammatory mediators via regulating extracellular regulated kinases signaling
Sun-Yup Shim, Sang Hyun Sung, Mina Lee
July-September 2018, 14(57):358-362
DOI:10.4103/pm.pm_58_18  
Background: Panax ginseng has been used as a traditional medicine for various pathological conditions. Ginsenoyne C (GSC) is a polyacetylene, minor constituent. Objective: The objective of the present study was to determine the anti-inflammatory activities of GSC for the protection of inflammation. Materials and Methods: The effects of GSC on cell viability and nitric oxide (NO) production in lipopolysaccharide (LPS)–induced RAW 264.7 cells were investigated through MTT assay and the Griess reaction, respectively. The levels of inflammatory cytokines such as interleukin (IL-1β) and IL-6 and inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and pERK were measured using enzyme-linked immunosorbent assay and Western blot analysis, respectively. Results: GSC suppressed NO production in LPS-induced RAW 264.7 cells. GSC suppressed the production of inflammatory mediators through downregulating phosphorylation of extracellular regulated kinases signaling in LPS-induced RAW 264.7. Conclusion: GSC has a potential therapeutic agent for protection of inflammation. Abbreviation used: CCK: Cell counting kit; COX-2: Cyclooxygenase-2; ELISA: Enzyme linked-immunosorbent assay; ERK: Extracellular regulated kinases; FBS: Fetal bovine serum; FEG: Fermented ginseng extract; GSC: Ginsenoyne C; IL: Interleukin; iNOS: Inducible nitric oxide synthase; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein kinases; NO: Nitric oxide; PVDF: Polyvinylidene difluoride; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  1,428 89 -
An aspartic-metalloprotease from an endemic plant tuber (Burnatia enneandra micheli): Purification and biochemical characterization
Ngangoum Eric Serge, Mezajoug Kenfack Laurette Blandine, Sanjit Kumar, Tchiegang Clerge, Mookambeswaran Vijayalakshmi
July-September 2018, 14(57):319-326
DOI:10.4103/pm.pm_555_17  
Background: The objective of this work was to isolate, optimize, and characterize protease from Burnatia enneandra which is an endemic plant found abundantly in the Far-Nord Region of Cameroon. The optimum condition to extract maximum quantity of protease from B. enneandra with respect to pH, the ratio (m/v), and agitation frequency was defined as 5.1%, 4%, and 100 rpm, respectively. Materials and Methods: The enzyme was purified using ammonium sulphate precipitation, double gel filtration chromatography sephadex G200 followed by sephadex G75 and the purified protease was further characterized. With an apparent molecular weight of 23 kDa on SDS-PAGE, the purified protease showed maximum activity at 5.1 and 40°C respectively for pH and temperature. Its activity was enhanced by metal ions such as Ca2+ and Ni2+, while Fe2+and Zn2+ showed significant inhibition. Results: B. enneandra protease activity was not affected by proteases inhibitors such as phenylmethylsulfonyl fluoride, aprotinin, and iodoacetamide but was strongly inhibited by Pepstatin A and ethylenediaminetetraacetic acid which allowed to classify this new protease as aspartic-metalloproteases. Using casein as substrate, protease from B. enneandra had a maximum rate of reaction (Vmax) and Michaelis-Menten constant (Km) of 64.935 (U/mL) and 373.941 (μg/mL), respectively. Abbreviations used: CCD: Central composite design; Km: Michaelis–Menten constant; Vmax: Maximum Velocity; PBD: Plackett–Burman design; PMSF: Phenylmethylsulfonyl floride; AAD: Absolute Average Deviation; AF: Accuracy Factor; BSA : Bovine serum albumin; BF: Bias factor; EDTA: Ethylene diamine tetraacetic acid; RSM: Response surface methodology; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis.
  1,311 48 -
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