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November 2019
Volume 15 | Issue 66 (Supplement)
Page Nos. 371-552

Online since Thursday, November 28, 2019

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ORIGINAL ARTICLES  

Inhibitory effects of fermented soybean tempeh on the anti-adhesive properties of Actinomyces viscosus and plaque growth in vitro Highly accessed article p. 371
Allimalar Sathiaseelan, Chong Seng Shit, Tsun-Thai Chai
DOI:10.4103/pm.pm_29_19  
Background: Tempeh, the Southeast Asian traditional food, has garnered great attention for its antibacterial property against Gram-positive and Gram-negative pathogens, and antidiarrheal effect. We have previously reported the potentiality of tempeh hexane fraction (HXF) in ceasing Actinomyces viscosus biofilm formation in vitro. Objective: Here, we investigated the efficacy of tempeh HXF on other cariogenic virulence traits of A. viscosus such as adhesive properties, acid production, and plaque growth. Materials and Methods: Anti adhesion of HXF was assessed based on its effects on the number of cells adhering to the surface of tooth in sucrose-dependent (SD) and sucrose-independent (SI) medium. The potential of HXF to inhibit the capability of A. viscosus to generate acids was investigated by pH drop assay. The HXF at different concentrations were used to determine the LC50based on brine shrimp lethality assay. Finally, the prospect of HXF as an inhibitor of plaque formation was investigated using artificial saliva-coated denture as an in vitro batch model. Results: HXF significantly decreased colony-forming unit of SD (1.07 log reduction) and SI (0.56 log reduction)-mediated adsorption of bacterial cells onto the tooth surface over 4- and 12-h incubation, respectively. Acid production was reduced after treated with HXF in a dose-dependent manner. Finally, a substantial reduction in plaque coverage area >55% was found on the HXF treated-denture. Conclusion: The anti-biofilm effect of HXF was associated with the suppression of A. viscosus adhesion to tooth surfaces and reduction in acid production. Furthermore, in vitro anti-plaque potential of HXF was demonstrated.
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Selection of suitable reference genes for reverse transcription-quantitative polymerase chain reaction normalization in Artemisia annua L. plants at different stages of growth and development p. 377
Javed Ahmad, Nazima Nasrullah, Naved Quadri, Umara Nissar, Shashi Kumar, Malik Zainul Abdin
DOI:10.4103/pm.pm_600_18  
Background: Artemisinin, an antimalarial compound suggested by the WHO to treat drug-resistant malaria, was obtained from Artemisia annua L. plants. However, due to the low level of artemisinin in the plant causes limitation to its commercialization, so to increase the concentration of artemisinin, two transgenic lines were developed by us, overexpressing the key genes of artemisinin biosynthetic pathway, namely, 3 S-hydroxy-3-methyl glutaryl-CoA reductase (HMGR), amorpha-4,11-diene synthase (ADS) (Trans. 1) and HMGR, ADS, and CYP71AV1 (cytochrome P450 monooxygenase) (Trans. 2). Objectives: Our main aim for this study was to select the suitable reference gene for the normalization of reverse transcription-quantitative polymerase chain reaction ( Reverse transcription RT-qPCR) data in different tissues at various developmental stages in A. annua L. plants. Materials and Methods: Six candidate reference genes, namely; β-actin (ACT), elongation factor 1-alpha (EF1α), TAP-42 interacting protein (TAP42), SAND family protein, β-tubulin, and protein phosphatase 2A (PP2A) for their expression stability in the root, stem, leaf, and flower of A. annua L. plants at vegetative, preflowering, and flowering stages were analyzed using geNorm, NormFinder and BestKeeper, the Excel-based research tools. Results: The genes ACT/PP2A, PP2A/TAP42, and EF1α/PP2A were appropriate as reference genes in the leaf tissues at vegetative, preflowering, and flowering stages, respectively. In addition, EF1α/PP2A genes at vegetative and flowering stage, while EF1α/TAP42 gene at preflowering stage was found suitable reference genes for normalization of expression data in the stem. In the root samples, ACT/EF1α, EF1α/PP2A, and ACT/TAP42 sets were found to be reliable reference genes at vegetative, preflowering, and flowering stages, respectively, whereas, PP2A/TAP42 gene set was found suitable for flower tissues at flowering stage. Conclusion: These results will be helpful in the normalization of expression data in RT-qPCR to find the reliable outcome.
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Modulation of sodium arsenite-induced Toxicity in mice by ethanolic seed extract of Trigonella foenum graecum Highly accessed article p. 386
Surjyo Jyoti Biswas, Goutam Ghosh, Ved Prakash Dubey
DOI:10.4103/pm.pm_518_18  
Background: Trigonella foenum graecum (TG) Linn. (Methi) is widely used as a spice and known for its pharmacological properties. Objective: The current study was conducted to examine the efficacy of TG Linn., family: Fabaceae, against sodium arsenite-induced toxicity in mice. Materials and Methods: Sixty mice (Mus musculus) weighing about 25 g were randomized into six groups; each of ten mice: Group I served as untreated control; Group II received only sodium arsenite (100 ppm) in drinking water for 2 months. The Group III mice fed chronically with sodium arsenite for 2 months as in Group II and then fed a vehicle of 1:20 alcohol to distilled water (1:20) for 15 and 30 days, respectively; Group IV to VI mice were treated as in Group II and then fed with 50, 150, and 250 mg/kg of TG seed extract, once daily for 15 and 30 days. Results: The IC50of the seed extract was 66.78 μg/mL, and it reduced the activities of toxicity marker enzymes such as gamma glutamyl transferase, lactate dehydrogenase, lipid peroxidation, aspartate transaminase, alanine aminotransaminase, acid phosphatase, alkaline phosphatase, and pro-inflammatory cytokines such as tumor necrosis factor-alpha and interleukin-6 (P < 0.05 to P < 0.001) and elevated the activities of catalase, superoxide dismutase, and G6PD (P < 0.05 to P < 0.001), a similar trend was also noted with hematological variables. Further, the normal architecture of the kidney was retained in the TG-fed series than arsenic (As)-treated series. Urinary excretion of As was high in treated groups compared to controls (P < 0.05 to P < 0.001), and 150 mg/kg dose offered better protection than the other two doses. Conclusion: TG seed extract has revealed potent antioxidant properties and consequently can be used as a protective agent in As-induced toxicity.
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Protective Effect of Madhuca longifolia Leaves in 7, 12-Dimethylbenz(a)anthracene Induced Mammary Carcinoma in Sprague Dawley Rat model p. 396
Maheswari Chinnadhurai, Faisal Al-Otaibi, Kenneth Nelson, Geetha Kandasamy, Meena Shanmugam, R Venkatnarayanan
DOI:10.4103/pm.pm_7_19  
Objectives: To evaluate the pretreatment with Madhuca longifolia leaves on 7, 12-Dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in Sprague Dawley rat. Materials and Methods: Thirty female Sprague Dawley rats were divided into five groups, and each group is having six rats. Group I rats received vehicle (1 mL of emulsion of sunflower oil and physiological saline) subcutaneously and 1 mL of 2% dimethyl sulfoxide per orally. Groups II, III, IV, and V were induced mammary carcinogenesis by giving single dose of subcutaneous injection of 25 mg of DMBA. Group III, IV, and V rats were administered with MEML 100, 200 mg/kg and Vincristine 0.5 mg/kg dissolved in 1 ml of 2% dimethylsulfoxide given 1 week before the administration of the carcinogen, respectively, and continued for 16 weeks. At the end of experiment, the animals were sacrificed and biochemical estimations were done in all groups. Mammary tissues in all groups were dissected out and used for histopathological studies. Results: Oral administration of 200 mg/kg of MEML to DMBA-treated rats effectively prevented the tumor incidence, total number of tumors, and tumor volume and brought back the biochemical markers to normal, which was comparable with standard group. In lower dose 100 mg/kg, the effect was very less compared to normal and standard groups. Our data showed that MEML 200 mg/kg significantly restored the breast tissue biochemically and histologically which was comparable with standard. Conclusion: Our results concluded that the leaves of Madhuca longifolia may be used in the treatment of mammary carcinoma.
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Exploration of antioxidant capacity of extracts of Perna viridis, a marine bivalve p. 402
Venkateskumar Krishnamoorthy, Leow Yu Chuen, Vengadan Sivayogi, Sathasivam Kathiresan, Mohd Baidi Bahari, Gunasunderi Raju, Subramani Parasuraman
DOI:10.4103/pm.pm_301_19  
Background: The biopotential of Malaysian green mussels (Perna viridis) has not been fully explored. The aim of the study is to screen the antioxidant capacity of extracts of Malaysian green mussels. Materials and Methods: Mussels were extracted by using solvents such as water, methanol, and ethanol, and methods such as microwave-assisted extraction (MAE) and animal tissue homogenization (ATH) were employed. The percentage yield, total protein content (TPC), total phenolic content, trace element analysis, and biochemical screening of extracts were carried out. The antioxidant capacity of the extracts was assessed by 2,2,-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay, 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) assay, hydrogen peroxide scavenging assay, and ferric-reducing antioxidant power (FRAP) methods. Results: The yield of extracts prepared by MAE methods was higher than the extracts prepared by ATH methods. The total phenolic contents and TPC were higher in the extracts prepared by ATH method than those prepared by MAE methods. Biochemical screening results revealed the presence of alkaloids, phenolic compounds, and saponins. The percentage radical scavenging and inhibitory concentration50(IC50) values of all extracts were found to be lesser than the standard. Results showed that extracts by ATH methods displayed high antioxidant activity than extracts by MAE methods. The methanolic extracts of P. viridis showed better antioxidant capacity than other extracts in DPPH and ABTS assay methods, whereas the ethanolic extracts displayed high antioxidant activity than other extracts in hydrogen peroxide and FRAP assay methods. The antioxidant activity of the extracts could be attributed to the hydrogen-donating ability of bioactive peptides, phenolic compounds, alkaloids, reducing sugars, and trace elements present in such extracts. Conclusion: The results of the present study lay the platform to isolate newer antioxidants from Malaysian green mussels. It is concluded that extensive mechanistic studies are imminent to ascertain the molecular mechanism involved in the antioxidant activity of such extracts.
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Comparative in vitro and in silico characterization of anticancer compounds piceatannol, biochanin-A and resveratrol on breast cancer cells p. 410
Pardhasaradhi Mathi, Neelima Musunuru, Udayapriya Adurthi, Mahendran Botlagunta
DOI:10.4103/pm.pm_146_19  
Background: Biochanin-A and Piceatannol are phytochemical constituents extracted from Sophora interrupta. Although both the compounds were isolated from a single plant, these compounds were not compared against anticancer activity. Objective: A systematic comparative analysis of biochanin-A, piceatannol, and resveratrol was performed to investigate cancer cell viability, motility, metabolic changes in Michigan Cancer Foundation-7 breast cancer cells, and structure compound interaction with the vascular endothelial growth factor (VEGF) receptors were studied. Materials and Methods: Cancer cell viability was studied using 3 (4, 5 dimethyl thiazol 2yl) 2, 5 diphenyltetrazo- lium bromide and acridine orange (AO)/ethidium bromide (EtBr) assay. The wound-healing assay was performed by measuring cell migration from the scratch area. Metabolic changes of the compounds in culture conditions were recorded using Fourier-transform infrared (FT-IR) spectroscopy. Molecular docking and dynamic simulations were performed using Schrödinger software. Results: Our results showed that the half-maximal growth inhibitory concentration for biochanin-A is 150 μM/ml and piceatannol and resveratrol showed 150 μM/ml, which is evident from the uptake of AO and EtBr dyes by live/dead cells. Moreover, drug-treated cells were unable to fill the cleared area from the scratch area, which suggests that all compounds effectively inhibit cell motility. FT-IR fingerprint showed a marked difference in the percentage of transition and dynamic structural changes between untreated and treated samples. Strong hydrogen-bond interaction with VEGF receptor-1 (VEGFR1) and VEGFR2 proteins and their interactions were stable throughout the simulation period. Moreover, these compounds inhibited sprouting of a new blood vessel from the chicken aorta and microvessels formation in the in ovo chorioallantoic membrane assay. Conclusion: Taken together, we conclude that anticancer and anti-angiogenic activity, structure-function relationship of biochanin-A is like well-known anticancer compound resveratrol and its metabolic product piceatannol in breast cancer cells.
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Statistical approach towards optimization of extraction process of karanjin from Pongamia pinnata seeds p. 419
Sartaj Akhtar Ansari, Lubna Abidin, Tinku Gupta, Mohd Mujeeb, Aftab Ahmad, Varish Ahmad, Showkat Rasool Mir
DOI:10.4103/pm.pm_609_18  
Background: Pongamia pinnata is a valuable herb with loads of pharmacological activities owing to its phytochemical profile. Karanjin is one phytocompound found in the seeds of P. pinnata. Optimization of karanjin extraction process becomes a high priority task because of its high significance. Objective: Use of Box–Behnken design for optimization of extraction of karanjin from P. pinnata seeds. Materials and Methods: Design expert software was used for optimization purpose. Extraction temperature, extraction time and solvent-to-drug ratio were taken as input variables which affected the karanjin content. Quantification of karanjin in different extracts was done through high-performance liquid chromatography using methanol and water (80:20% v/v) as mobile phase. Results: Ultrasound-assisted extraction stood out to be the most efficient mode for extraction of karanjin using methanol as solvent. Extraction temperature of 57.85°C, extraction time of 25.45 min, and solvent-to-drug ratio of 86.4709% v/w were established as optimum conditions for extraction of karanjin from P. pinnata seeds. Under such extraction conditions, 8.33%w/w karanjin was extracted. Conclusion: From our study, it was concluded that non-thermal methods are a better choice for extraction of karanjin and methanol is the most efficient solvent for the same. All the three input variables significantly affected karanjin content which was confirmed by model fitting and analysis of regression coefficients. Our research shows the relevance of a statistical approach in phytocompound research area which makes the extraction process cheap and less laborious.
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Metabolic Profile Elucidation of Ventilago calyculata Aqueous Extract Attenuating Sequelae of Aspirin Retarded Wound Healing p. 426
Shweta Kumar, Rajesh Singh Pawar, Deepti Jain
DOI:10.4103/pm.pm_131_19  
Background: Impaired wound healing due to aspirin is a common cause of delay in healing. Ventilago calyculata Tul. (Rhamnaceae) is used extensively in the Indian traditional medicines for skin problems. Objective: The objective of this work was to test the potential of formulations prepared from the extracts of V. calyculata against aspirin-retarded wound healing in rats and investigate the probable mechanism of action. Materials and Methods: The effect of topical administration of fractionates of V. calyculata against aspirin-delayed wound healing was assessed in Wistar albino rats. The chemo-profiling (liquid chromatography-mass spectroscopy [LC-MS]) of the extract with the most potent wound healing activity was carried out. Further, the mechanism of the most potent Ventilago calyculata aqueous extract (VCA) was determined by viability and plasma membrane integrity assays in H2O2-challenged wild type Saccharomyces cerevisiae BY4743) and knock-out strain (Δtrx2) strains. Results: The results of our investigations showed that in excision wound model; all formulations had statistically significant (P < 0.01) wound healing activity compared to the negative control. However, aqueous extract treatment (HF3) exhibited maximum activity, and the chemo-profiling of VCA by LCMS suggested that the potent activity may be due to individual and/or synergistic effect of the identified pharmacologically active phytoconstituents. The study on yeast indicates that VCA was able to act intracellularly also as it was able to overcome the growth inhibitory effect of the H2O2significantly (P < 0.01). Conclusion: We propose that treatment with HF3(VCA) is a therapeutically beneficial method of decelerating wound retardation caused by aspirin intake in patients on long-term aspirin therapy.
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Ameliorative potential of a traditionally used plant Fraxinus micrantha against oxidative stress and paracetamol-induced hepatotoxicity p. 433
Hasandeep Singh, Sarabjit Kaur, Saroj Arora, Balbir Singh
DOI:10.4103/pm.pm_236_19  
Background: Liver disorders are one of the serious health issues. The treatment of liver disorders and their associated complications must be done with care due to the adverse effects of present-day medications. Therefore, there is an urgent need to develop effective and non-toxic herbal drugs for hepatoprotection. Objective: The objective of the present study is to investigate the hepatoprotective potential of traditionally used plant Fraxinus micrantha against paracetamol (PCM)-induced hepatotoxicity in rats. Methods: Hepatotoxicity was induced by a standardized single oral dose of paracetamol (PCM) at 3 g/kg body weight. Standard drug (silymarin 50 mg/kg) and test drugs (chloroform and methanol extracts) were administered orally to rats for 7 days. All the animals were sacrificed on the 8th day, and blood was withdrawn by retro-orbital vein puncture, collected in fresh centrifugation tubes, and centrifuged at 10,000 rpm for 10 min to separate serum for various estimations. Livers were isolated immediately for various biochemical and histopathological studies. Results: Treatment with chloroform extract (200, 400, and 800 mg/kg) and methanol extract (200 and 400 mg/kg) ameliorated the elevated levels of hepatic markers, but a significant reduction in these levels was observed by treatment with methanol extract at 800 mg/kg. In addition, treatment with chloroform and methanol extracts resulted in the amelioration of oxidative stress along with the histopathological changes. High-performance liquid chromatographic analysis of methanol extract of F. micrantha revealed the presence of various polyphenols such as rutin, naringenin, kaempferol, physicion, quercetin, and gallic acid. Conclusion: The results of the present study revealed that the observed hepatoprotective effect of methanol extract of F. micrantha may be due to the presence of various polyphenols and also provides pharmacological evidence for the use of F. micrantha bark in folk medicine for the treatment of liver diseases.
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Antithrombocytopenic potential of bioactivity guided fractions of traditionally used Psidium guajava Linn. leaves in busulfan induced-thrombocytopenic rats p. 440
Adil Ahmad, Mohammad Ibrahim, Karishma Chester, Washim Khan, Sayeed Ahmad, SH Ansari
DOI:10.4103/pm.pm_608_18  
Background: Psidium guajava L. (family: Myrtaceae) is a traditionally used medicinal plant and possesses various therapeutic properties. This study was aimed to explore the antithrombocytopenic potential of bioactive fraction of P. guajava L. Materials and Methodology: Hydroalcoholic extract of dried leaves was prepared through soxhlation, and further, it was fractionated. Hydroalcoholic extract and its fractions were tested for antithrombocytopenic potential in busulfan-treated rats. Bioactive fractions were metabolically characterized through ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) and. Results: A total of 30.3% w/w hydroalcoholic extract was obtained from which 5.7%, 4.2%, 8.6%, and 11.8% w/w of n-hexane, dichloromethane, n-butanol, and aqueous fractions were collected, respectively. n-butanol and aqueous fractions were identified as the potent bioactive fractions as antithrombocytopenic agents. Using toluene:ethyl acetate:formic acid (6:3:1 v/v/v) as a solvent system in TLC, 12 and 9 metabolites were separated in n-butanol and aqueous fractions, respectively, whereas 11 and 12 metabolites were tentatively identified through UPLC-MS in aqueous and n-butanol fractions, respectively. Five metabolites (showing m/z values of 303.05, 302.5, 313.2, 573.51, and 341.32 eluted at 2.80, 3.81, 7.94, 8.55 and 8.69 min, respectively) were found in both aqueous and n-butanol fractions. Conclusion: Chromatographically characterized n-butanol and aqueous fractions of hydroalcoholic extract of P. guajava increased platelet count, and it can be further explored for the development of new phytopharmaceutical.
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The cytoprotective benefits of a turmeric, quercetin, and rosemary blend through activation of the oxidative stress pathway p. 449
Arun Rajgopal, Samantha J Roloff, Charlie R Burns, David J Fast, Jeffrey D Scholten
DOI:10.4103/pm.pm_556_18  
Background: An imbalance between oxidative and reductive processes within cells can result in oxidative stress leading to a decrease in cellular survival. Use of natural products with antioxidant properties may reduce this oxidative stress. Objective: The objective of the study is to determine whether a natural product blend of turmeric, pagoda tree seed pod (quercetin), and rosemary (TQR) extracts can protect human liver cells from oxidative stress-induced cytotoxicity. Materials and Methods: HepG2 cells were treated with a blend of botanical extracts of TQR (1:3:5, w: w:w) and expression of genes downstream of nuclear factor (erythroid-derived 2)-like 2 (NRF2) activation was measured. We also measured the ability of the extract blend to protect DNA and lipids from oxidative stress by measuring via the comet assay and 8-isoprostane production, respectively. Finally, we measured the effect of the extract blend on HepG2 cell protection against oxidative stress-induced cytotoxicity. Results: We provide evidence that the TQR blend activates the NRF2 pathway leading to DNA protection, a decrease in lipid peroxidation, and whole cell protection against oxidative stress. Conclusion: These results suggest that consuming a blend of TQR at the ratio of 1:3:5 could provide benefits against environmental stressors that increase exposure to reactive oxygen species.
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Antidiarrheal potential of Eriosema chinense vogel. against enteropathogenic Escherichia coli-induced infectious diarrhea p. 455
Komal M Parmar, Jayshri R Hirudkar, Dhiraj S Bhagwat, Satyendra Kuldip Prasad
DOI:10.4103/pm.pm_129_19  
Background: The plant Eriosema chinense Vogel (Fabaceae) is mainly found in the Eastern Himalayan regions of India and China, and its roots are used traditionally by the tribal people of Meghalaya (India) in treatment of diarrhea. Objective: The objective of the study was to evaluate the potential of roots from E. chinense against enteropathogenic Escherichia coli (EPEC)-induced infectious diarrhea. Materials and Methods: Ethanolic extract of E. chinense (EEC) roots and its chloroform fraction (CEC) were standardized with eriosematin E using high-performance liquid chromatography. The efficacy of EEC (100 and 200 mg/kg, p.o.) and CEC (50 and 100 mg/kg, p.o.) was evaluated against EPEC-induced infectious diarrhea, where behavioral parameters at the 6th and 24th h followed by determination of water content and density of EPEC in stools along with blood parameters examination. Further, the colonic and small intestinal tissues were subjected to biochemical analysis, antioxidant evaluation, determination of ion concentration, Na+/K+-ATPase activity, and histopathology. Results:The results demonstrated a significant antidiarrheal potential of EEC and CEC at both dose levels; however, EEC at 200 and CEC at 100 mg/kg p.o. were found to be more effective, which also reduced EPEC density in stools and also its water content. The treatment also demonstrated a significant restoration of altered antioxidant and electrolyte status and reactivated Na+/K+-ATPase and prevented epithelial tissue damage. Conclusion: The effect may be attributed to an inhibition in intestinal secretion, nitric oxide production, and reactivation of Na+/K+-ATPase.
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Evaluation of diosgenin content in Costus speciosus germplasm collected from Eastern Ghats of India and identification of elite chemotypes p. 462
Akanksha Srivastava, Manish Kumar, Ankita Misra, Pushpendra Kumar Shukla, Pawan Kumar Agrawal, Sharad Srivastava
DOI:10.4103/pm.pm_72_19  
Background: Costus speciosus known as "insulin plant" for its anti-diabetic potential. It has commercial significance due to the presence of industrially viable metabolite diosgenin. Objective: The identification of elite chemotypes of C. speciosus (Rhizome) through high-performance thin-layer chromatography (HPTLC) from the Eastern Ghats (India). Materials and Methods:A validated HPTLC method for the quantification of diosgenin was developed in accordance with the International Conference on Harmonization Guidelines. Results: In total, 11 populations of species were collected from their natural habitat with all the Global Positioning System (GPS) coordinates. The method was developed on HPTLC pre-coated silica gel 60 F254plates under a binary solvent system of n-hexane and ethyl acetate (7:2 v/v). The linearity was established at concentration range of 0.1–0.9 μg/spot having regression equation, 0.010× + 0.002. The limit of detection and limit of quantification were 0.907 and 2.751 with a regression coefficient of 0.999. The diosgenin content varies significantly (P < 0.05) from 0.002% to 0.076% and NBCS-06 from Patiya, Bhubaneswar, was identified as elite chemotype. Conclusion: The validation data confirm that developed HPTLC method was precise, accurate, robust, reproducible, and reliable in nature. The study resulted in the identification of elite chemotype of C. speciosus through validated HPTLC method from the Eastern Ghats of India. It will promote site-specific commercial cultivation of high metabolite yielding germplasm for good quality raw material to meet the industrial demand and in turn income generation of local inhabitants. The developed method will also aid in the regulation of quality standard and batch consistency of diosgenin-containing formulations in industry.
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Neuroprotective efficacy of polyphenols of marine brown macroalga Ecklonia cava in diabetic peripheral neuropathy p. 468
Suman Samaddar, Raju Koneri
DOI:10.4103/pm.pm_212_19  
Objectives: In this study, the neuroprotective effect of polyphenols isolated from the brown marine macroalga Ecklonia cava (EC) was evaluated in experimental diabetic peripheral neuropathy (DPN). Materials and Methods: The polyphenolic fraction from EC was isolated. DPN was induced in animals by intraperitoneal injection of streptozotocin (45 mg/kg, b. w) and maintained for 6 weeks followed by treatment with EC polyphenols (ECPP) or epalrestat for 30 days. Nerve conduction velocity (NCV) of sciatic nerves and the compound muscle action potential (CMAP) of the gastrocnemius muscle were measured using a non-invasive method followed by neuropathic thermal analgesia and muscular grip strength. Sciatic nerve aldose reductase (AR) activity, intraneural sorbitol accumulation, Na+K+-ATPase activity, production of proinflammatory cytokines (interleukin-6 [IL-6], IL-1 β, and tumor necrosis factor alpha [TNF-α]), and expression of AR and protein kinase C (PKC) were assessed. Results: The ECPP were found to inhibit AR activity as well as their expression in diabetic animals, thereby improving the NCV, CMAP, muscle grip strength, hot plate, and tail-flick response time. Improvements in the sciatic nerve Na+K+-ATPase activity and intraneural accumulation of sorbitol, an index of AR overactivity, were evident with ECPP treatment. The production of proinflammatory cytokines (IL-6, IL-1 β, and TNF-α) and expression of PKC were also diminished. Conclusion: The data suggest that the polyphenols of EC have neuroprotective potential against experimental DPN.
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Evaluation of lipotropic effect of herbal formulation on hepatic fat accumulation in rats fed with methionine-choline deficient diet p. 476
Prasanna Raja Chandrasekaran, Sasikumar Murugan, Edwin Jothie Richard, Bharathi Bethapudi, Divya Purusothaman, Chandrasekaran Chinampudur Velusami, Prashanth D'Souza, Deepak Mundkinajeddu, Muralidhar S Talkad
DOI:10.4103/pm.pm_111_19  
Background: Choline is an essential lipotropic nutrient for regulating fatty acid synthesis and hepatic lipid mobilization. Deficiency of choline causes fatty liver leading to dysregulated liver function. Objective: To investigate the lipotropic activity of the proprietary herbal formulation (PHF) containing Acacia nilotica and Curcuma longa. Materials and Methods: Fatty liver disease was induced in Wistar rats by feeding methionine/choline-deficient (MCD) diet for 4 weeks. Animals were concurrently treated with PHF (at 50, 100, 200, and 400 mg/kg rat body weight/day) for 4 weeks. Methionine/choline-sufficient (MCS) diet-fed rats were used as control. Serum biochemistry and liver parameters were determined at the end of experimental period. Further, anti-lipogenic and lipolytic activity of PHF extract was studied in HepG2 cells. Results: Rats fed with MCD diet, showed significant increase in liver lipids, triglycerides, cholesterol, thiobarbituric acid reactive substance, and serum alanine transaminase (ALT) and decreased serum triglyceride level compared to MCS diet-fed rats indicating significant fat accumulation and liver damage. PHF treatment significantly decreased the liver lipids, triglyceride and serum ALT compared to MCD diet-fed rat. Histological evaluation revealed the restoration of hepatic architecture after PHF treatment. In in vitro studies, the PHF extract decreased the oleic acid induced fat accumulation in HepG2 cells. Conclusion: The study demonstrated the lipotropic effect of PHF evident from decreased fat accumulation and antilipogenic activity. These data suggests that PHF could be a potential supplement for preventing fatty liver.
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Dioscorea villosa Leaf Extract Enhances in vitro Wound Healing and Expression of Extra Cellular Matrix Factors Transforming Growth Factor-Beta 1 and Collagen-1 in L929 Cell Lines p. 483
Murad Alsawalha, Abeer Mohammed Al-Subaie, Reem Yousuf Al-Jindan, Srinivasa Rao Bolla, Mohammed Salahuddin, Vishnu Priya Veeraraghavan, Dwaipayan Sen, Janardhana Papayya Balakrishna, Padma Kanchi Ravi, Joel Palpath Joseph, Aruthra Arumugam Pillai, Shiva Shankar Reddy Gollapalli, Shonima Pala, Fajre Pengateeri, George Dominic, Surapaneni Krishna Mohan
DOI:10.4103/pm.pm_81_19  
Background:Easy availability, relatively low cost with fewer side effects, has made the herbal extracts/fractions/pure compounds as prominent source of medicinally important molecules. Dioscorea villosa L. commonly known as wild yam belongs to the family Dioscoreaceae and has been used in various parts of India to treat joint pain, arthritis, and various other diseases. However, its role in wound healing has not been documented so far. In the current study, the in vitro wound healing capabilities of D. villosa were examined using L929 cells. Materials and Methods:Methanolic extraction of D. villosa leaves was prepared by applying inexpensive maceration method. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the cytotoxicity of D. villosa extract and in vitro wound healing capabilities were investigated by applying scratch assay. The qualitative measurement of different secondary metabolites was determined by standard biochemical assays. Gas chromatography-mass spectrometry (GC-MS) was performed to identify the possible wound healing components present in the methanolic leaf extract of D. villosa, and the antioxidant properties of the plant extract were evaluated by α,α-diphenyl-β-picrylhydrazyl and ferric reducing antioxidant power assays. Furthermore, the possible molecular factors involved in the proliferation and migration of fibroblast in the presence of D. villosa extract was determined by flow cytometry technique. Results:The experiments to analyze the cytotoxic effect of D. villosa on L929 cells revealed that at the highest concentration used, i.e., 500 μg/mL after 48 h of incubation, 96.06% ± 0.42% of the cells were viable. The results of the scratch assay revealed that 125 μg/mL of plant extract induced the migration in 88.58% of fibroblast cells. Through GC-MS analysis, antioxidant and anti-inflammatory molecules such as 1 H-Indole-2,3-dione (Isatin) and Dexamethasone have been identified. In addition, flow cytometry data showed the influence of plant extract on the expression of Collagen-1 and transforming growth factor (TGF)-beta, which play a major role in the wound healing processes. 125 μg/mL of plant extract induced Collagen-1 in 22.18% cells and TGF-beta in 80.77% of cells, respectively. Conclusion:The presence of potent antioxidant and anti-inflammatory molecules and capability to induce the expression of fundamental wound healing molecular factors TGF-beta and collagen-1 in fibroblast cells, endorsed D. villosa as a potential wound healing agent. Abbreviations used:DA: Dioscorea villosa; LDH: Lactate dehydrogenase; ECM: Extra Cellular matrix; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco's Modified Eagle's Medium; FBS: Fetal bovine serum; FITC: Fluorescein isothiocyanate. D-PBS: Dulbecco's phosphate-buffered saline; FACS: Fluorescent activated cell sorter; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; μg: Micrograms; ng: Nanogram; mL: milliliter; SD: Standard Deviation; hEGF: Human epidermal growth factor.
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Curcumin vasorelaxation in uterine artery of goat (Capra hircus) is mediated by differential activation of nitric oxide, prostaglandin I2, soluble guanylyl cyclase, and gap junction communication p. 495
Harithalakshmi Jandhyam, Subas Chandra Parija
DOI:10.4103/pm.pm_188_19  
Background: Curcumin is a principal active constituent of Curcuma longa and has potential therapeutic application in various disease states. In this study, we investigated the mechanism of vasorelaxatory effects of curcumin in middle uterine artery (MUA) of both pregnant (P) and nonpregnant (NP) Capra hircus (Ch). Materials and Methods: The middle uterine arterial rings (MUA) were mounted in an automatic organ bath attached to a PowerLab data acquisition system. We analyzed the effect of curcumin on signaling mediators of endothelium-derived hyperpolarizing factor: nitric oxide (NO), soluble guanylyl cyclase (sGC), prostaglandin I2(PGI2), and myoendothelial gap junctions (MEGJs). Results:The maximal vasorelaxation response to curcumin (1 ρM–100 μM) induced in phenylephrine- precontracted endothelium intact and denuded MUA rings were 42.58%, 25.12% in NP and 55.49%, 12.66%, in P Ch. In the presence of 1H-(1,2,4) oxadiazolo (4,3,-a) quinoxalin-1-one, the maximal curcumin-induced vasorelaxation (CVR) was inhibited to 20.65%, and 15.81% in MUA of NP and P Ch. In the presence of N.-nitro-L-arginine methyl, indomethacin and combination of both CVR were decreased to 20.49%, 12.47%, 12.82% in MUA ring of NP and to 40.60%, 52.55%, 46.53% in MUA ring of P Ch, respectively. The sensitivity of curcumin to MEGJ blockers carbenoxolone, 18β-glycyrrhetinic acid in causing vasorelaxation was 33.18%, 22.61% in MUA rings of NP, and to 15.76%, 18.54% in P Ch. Conclusion: In P Ch, endothelium-dependent vasorelaxation to curcumin is augmented by two-fold as that of MUA of NP. Endothelial vasorelaxation in MUA of NP is mediated through the activation of cyclooxygenase-PGI2-cyclic adenosine monophosphate and endothelial NO synthase (eNOS)-sGC-cyclic guanosine monophosphate (cGMP) signaling with low activation of sGC and MEGJ. In contrast, vasorelaxation to curcumin is mediated through increased activity of sGC and MEGJ with major involvement of eNOS-NO-sGC-cGMP in MUA of P Ch. These studies have strong implications in the therapeutic targeting for hypertensive disorders.
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Inhibition of key digestive enzymes involved in glucose metabolism by biosynthesized zinc oxide nanoparticles from Syzygium cumini (L.): An in vitro and in silico approach p. 502
J Arul Daniel, S Asha Devi
DOI:10.4103/pm.pm_253_19  
Background: In recent years, fusion of nanotechnology and medicine represents one of the major breakthroughs of modern science with the aim of developing nanomaterials for diagnosis, treatment, and overall improving health for the benefit of humankind. Objective: The aim of the study was to synthesize, characterize, and evaluate the antidiabetic effect of zinc oxide nanoparticles (ZnO NPs) from Syzygium cumini (SC). Materials and Methods: ZnO NPs were synthesized using SC seed extract and was characterized by ultraviolet-visible spectroscopy, Fourier transform-infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), and transmission electron microscopy (TEM). Inhibitory potential of the ZnO NPs against α-amylase and α-glucosidase was analyzed in vitro, while AutoDock was used to analyze its potential in silico. Results: The FT-IR analysis suggested that the obtained ZnO NPs have been stabilized through the interactions of phenolic present in the seed extract. XRD pattern analysis confirmed the crystalline nature of the nanoparticles and TEM images revealed the polygonal structure of ZnO NPs of 16.7–22.9 nm in size. Molecular interaction studies of the phenolics from the SC seed extract with α-amylase and α-glucosidase have outlined its high affinity toward it relative to acarbose, while in vitro inhibitory potential studies of the ZnO NPs revealed that it could inhibit the enzymes similar to acarbose. Conclusion: Overall observations imply that phenolics of the SC seed extract helped in synthesis of the NPs and also its role in inhibitory potential which can be used as a potent antidiabetic drug.
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Herniarin, a natural coumarin, inhibits mammary carcinogenesis by modulating liver X receptor-α/β-PI3K-Akt-Maf1 Pathway in sprague-dawley rats p. 510
Pritha Bose, Shakti Prasad Pattanayak
DOI:10.4103/pm.pm_264_19  
Background: The massive tumor burden following breast cancer initiation requires a constant supply of energy achieved by modification of different metabolic pathways. Enhanced lipid synthesis associated with glucose metabolism modification triggered by a battery of signaling events promotes unregulated cell division and growth. Coumarins with their diversified bioactivity are also recognized for their therapeutic efficacy in breast cancer. Objectives: Thus, this study was performed to assess the chemotherapeutic potential of herniarin (HER), a 7-methoxycoumarin derivative against polycyclic aromatic hydrocarbon-induced mammary cancer. Materials and Methods: In silico molecular docking was carried out to identify the interactions of HER with LXR α and β, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), PI3K, and Akt followed by in vitro evaluation of HMGCoAR inhibitory potential of HER. Detailed in vivo studies was then performed in 7,12-dimethylbenz(a) anthracene-induced breast cancer model in Sprague-Dawley (SD) rats using HER at doses 20 mg/kg, b. w. and 40 mg/kg, b. w. along with molecular-level analysis (messenger RNA and proteins) by real-time quantitative polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Results: The docking studies revealed a significant binding affinity of HER to the aforesaid receptors and its HMGCoAR inhibitory potential (IC50 =103.1 nM) validated the docking studies. HER successfully re-established the lipid and lipoprotein levels along with the activities of lipid-metabolizing enzymes and glycolytic enzymes that were altered following cancer induction. The molecular biology evaluations reflected the efficiency of HER as it evidently alleviated the overexpression of HMGCOAR, PI3K, and Akt along with the lipogenic genes fatty acid synthase and acetyl-CoA carboxylase 1 in the carcinoma-developed animals by upregulating LXR (α and β) and Maf1. Conclusion: The bioactive moiety HER efficiently controlled alterations in metabolic pathways, thus balancing energy consumption, attenuating tumor progression through LXR/PI3K/Akt/Maf1 axis in breast cancer in SD rats.
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Cellular protection induced by genistein in mouse and its antioxidant capacity p. 520
Rogelio Paniagua-Pérez, Susana Reyes-Cadena, Carlos Martínez-Canseco, Celia Reyes-Legorreta, Jesús Martínez-Castro, Eduardo O Madrigal-Santillán, José A Morales-González, José M Cristóbal-Luna, Isela Álvarez-González, Eduardo Madrigal-Bujaidar
DOI:10.4103/pm.pm_78_19  
Background: Genistein (GT) is an isoflavone phytoestrogen present in a number of plants. The chemical has been reported to have antioxidant, antigenotoxic, and cancer-preventive qualities; however, no studies against cisplatin (CP) have been reported. Objective: The main objective of the study is to determine the capacity of GT to inhibit the genotoxic and cytotoxic damage induced by CP in mouse, as well as its immunostimulant ability and its capacity to scavenge free radicals. Materials and Methods: We determined the effect of six doses of GT on the rate of sister chromatid exchanges (SCEs) and of micronuclei (MN) in mice administered with 5 mg/kg of CP. Besides, we determined its capacity to increase the amount of lymphocytes in mouse and to reduce oxidation with the 2,2-diphenyl-1-picrylhydrazyl assay. Results: Our results showed that GT (10–60 mg/kg) significantly decreased the frequency of SCE and of MN in mice. Furthermore, we also observed a moderate bone marrow cytotoxic correction of the damage induced by CP, as shown by an improvement in the rate of polychromatic erythrocytes. In addition, with 60 mg/kg, GT increased 69.6% the production of mouse lymphocytes over the control value throughout a 72-h trial. Moreover, the compound also showed a high capacity to trap free radicals (95.25%, with 250 μg/ml). Conclusion: Our results, therefore, established that GT is an effective cellular protective agent against the action of CP.
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Kalanchoe blossfeldiana Extract Induces Cell Cycle Arrest and Necrosis in Human Cervical Cancer Cells p. 527
Justyna Stefanowicz-Hajduk, Magdalena Gucwa, Adam Hajduk, J Renata Ochocka
DOI:10.4103/pm.pm_86_19  
Background: Genus Kalanchoe (Crassulaceae) comprises plants originating from Africa and Asia. Their pharmaceutical preparations are commercially available in the form of anti-inflammatory and antimicrobial medicaments. Recent studies have shown anticancer activity of Kalanchoe plants. However, studies on Kalanchoe blossfeldiana (KB) are extremely limited, and its cytotoxic properties have not yet been evaluated. Objective: The objective of the study is to estimate the cytotoxic activity of KB on human cervical carcinoma (HeLa) cells and determine the mode of cell death. Materials and Methods: The cytotoxic activity of ethanolic extract of leaves of KB was tested on HeLa cells using Real Time xCelligence System (RTCA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death, the generation of reactive oxygen species (ROS), depolarization of the mitochondria, cell cycle arrest, and the activity of caspases-3/7/9 were assessed by flow cytometry or a luminometer. The activity of poly (ADP-ribose) polymerase (PARP), translocation of apoptosis-inducing factor (AIF) in the cells, and expression of 92 genes associated with apoptosis/necrosis were also estimated. Results: According to the results, the IC50values of KB extract were 8.28 ± 0.29 and 9.63 ± 1.07 μg/mL in RTCA and MTT assay, respectively. The results showed a significant increase in the generation of ROS and depolarization of the mitochondria in the dead cell population. Caspases-3/7/9 were not activated during treatment with KB extract. We also found that the pathway of HeLa cell death was induced by tumor necrosis factor-related apoptosis-inducing ligand and was independent of the PARP and AIF. The extract also induced cell cycle arrest in S phase. Conclusions: The results of this study indicate that KB extract induces necrosis in HeLa cells and this process is death receptor-mediated leading to the overproduction of ROS, mitochondrial dysfunction, and cell cycle arrest.
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SUNCA standardized extract from Helianthus annus L exerts enhanced digestive enzyme activity and subsides obesity through inhibition of C/EBP-α and PPAR-γ expression both in in vitro and in vivo model p. 538
K Gouthamchandra, HV Sudeep, Amritharaj , HB Lingaraju, K Shyam Prasad
DOI:10.4103/pm.pm_204_19  
Background: Helianthus annuus L. (sunflower) is well known for its edible oil, sunflower seed has been used as folk remedy for several diseases including cancer. Materials and Methods: In this study, SUNCA, a standardized extract enriched with chlorogenic acid (CGA) from sunflower seed was investigated for in vitro antidiabetic activity using α-amylase and α-glucosidase enzyme inhibition assay. Further, for preventing obesity, we demonstrated anti-adipogenesis in 3T3-L1 cell culture system and on body weight gain and adiposity in rat fed high-fat diet (HFD). Results:SUNCA showed significant inhibitory activity toward digestive enzymes such as α-amylase and α-glucosidase. On the other hand, SUNCA significantly inhibits lipid accumulation during adipogenesis in a dose-dependent manner. The treatment with 100 μg/kg b.w of SUNCA apparently prevented the body weight gain and the increase of triglyceride and total cholesterol level in rat fed an HFD for 6 weeks. Further immunoblotting studies reveal that SUNCA significantly down-regulates the expression of transcription factors peroxisome proliferator-activated receptor gamma and CCAT/enhancer-binding protein alpha, both in adipocytes and adipose tissues. Conclusion: The findings reported here provide the therapeutic potential of SUNCA, a standardized extract from Helanthus annus L.
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Proanthocyanidins rich extract of Calligonum comosum ameliorates doxorubicin-induced immunosuppression and hepatorenal toxicity p. 545
Essam Abdel-Sattar, Doaa H Abdelhady, Emad W Ghazy, Walied Abdo, Zizy I Elbialy, Mustafa Shukry, Engy Abdelhameed Mahrous, Sendker Jandirk
DOI:10.4103/pm.pm_670_18  
Background: Doxorubicin (DOX) is a highly effective chemotherapeutic agent which use has been restricted due to its multi-organ toxicity. Objective: We investigated the possible protective effect of Calligonum comosum extract (CCE) against DOX toxicity while identifying its major phytoconstituents. Materials and Methods: CCE was administered at 100 mg/kg b.w for 2 weeks to rats which have previously received an intraperitoneal injection of DOX (20 mg/kg). Major phytoconstituents of CCE were assessed using high-performance liquid chromatography and UPLC/MS/MS. Results: CCE was rich in phenolic constituents, especially proanthocyanidins, corresponding to a concentration of 167 mg catechin per gram of the extract after hydrolysis. Other constituents identified were procyanidin B1-gallate, procyanidin B2-gallate, quercetin, and kaempferol. Animals that received CCE following DOX injection showed less signs of oxidative stress (indicated by levels of malondialdehyde, superoxide dismutase, and reduced glutathione), DNA fragmentation, and hepato-renal genotoxicity (Comet's assay). When compared to animals that received DOX only, administration of CCE following DOX maintained normal tissue architecture and restored liver and kidney functions to near their normal levels (measured as creatinine, glucose, urea serum concentration and aspartate aminotransferase, lactate dehydrogenase, and alanine transaminase activities). Interestingly, healthy animals receiving CCE showed an increase in total lymphocyte count and phagocytic activity, whereas those receiving CCE following DOX intoxication showed no signs of immunosuppression that was observed in animals receiving DOX only. Conclusion: C. comosum is a promising candidate as a supportive treatment for those receiving DOX as a chemotherapeutic agent due to its ability to ameliorate signs of DOX toxicities.
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