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Apr-Jun 2020
Volume 16 | Issue 69
Page Nos. 207-447

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ORIGINAL ARTICLES  

Effect of phloretin treatment ameliorated the cisplatin-induced nephrotoxicity and oxidative stress in experimental rats p. 207
Yan Zhao, Wei Dai
DOI:10.4103/pm.pm_346_19  
Background: The cisplatin is one of the widely employed platinum-based drugs as the chemotherapeutic substance for the treatment of various cancers such as bladder cancer, prostate cancer, cervix cancer, and lung cancer. Hence, the present work was designed to test the curative efficacy of phloretin against the cisplatin-induced nephrotoxicity in rat model. Materials and Methods: Male Wistar albino rats were divided into five groups and the Group I rats were served as control, Group II rats were injected with cisplatin (8 mg/kg), Group III rats were administered with the low dose of phloretin (25 mg/kg), Group IV rats were administered with the high dose of phloretin (50 mg/kg), and the Group V rats were administered with the standard drug silymarin (50 mg/kg) for 10 days. After the experimental period, the animals have been sacrificed and collected all the samples for further analysis. Results: In the current study, the cisplatin was exhibited the severe renal damage in Wistar albino rats by possessing the various clinical complications such as increased serum urea, proteinuria and creatinine levels, decreased antioxidant enzymes level, increased DNA fragmentation, elevated oxidative stress, and severe injury to renal tissues. In addition, the cisplatin increased the poly (ADP-ribose) polymerase-1 and caspase-3 activity and pro-inflammatory cytokines level such as tumor necrosis factor alpha and interleukin-1 β. Whereas, the treatment with the low dose and high dose of phloretin (25–50 mg/kg) was exhibited a significant (P < 0.05) attenuation against the cisplatin-induced nephrotoxicity in experimental rats. Conclusion: The phloretin treatment significantly protected the kidneys against cisplatin-induced renal damages in rats.
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Fucoxanthin averts isoprenaline hydrochloride-induced myocardial infarction in rats p. 214
Fang Wang, Hao Zhang, Guo Lv, Zhenguo Liu, Xin Zheng, Xianjun Wu
DOI:10.4103/pm.pm_357_19  
Background: In this current investigation, we aimed to assess the efficacy of natural antioxidant fucoxanthin against the myocardial infarction since it can be consumed as a regular diet. Objective: In this scientific investigation, we planned to investigate the curative effectual of fucoxanthin on isoprenaline hydrochloride provoked myocardial infarction on the experimental rats. Materials and Methods: Healthy Wistar albino rats were grouped into control, fucoxanthin alone, myocardial infarction induced, and myocardial infarction induced pretreated with fucoxanthin. The control rats were treated with a standard food diet, whereas fucoxanthin alone group rats were treated with 50 mg/kg/bwt of fucoxanthin along with standard diet. Myocardial infarction-induced groups were treated with 85 mg/kg/bwt of isoprenaline hydrochloride to induce myocardial infarction on the 29th and 30th days of the treatment period. Group IV rats were pretreated with 50 mg/kg/bwt of fucoxanthin from day 1 of experiment and on the 29th and 30th days of treatment period treated with 85 mg/kg/bwt of isoprenaline hydrochloride to induce myocardial infarction. Results: The fucoxanthin possessed effective cardioprotective activity in mycardiac infarction-induced rats. Fucoxanthin treatment reduced the statuses of cardiac troponin T and cardiac troponin I on myocardial infarction provoked rats; also, it exhibited the reduced Thiobarbituric acid reactive substances (TBARS) level in the rats. The antioxidant status such as superoxide dismutase, catalase, and glutathione peroxidase was extensively elevated in the myocardial infarction-induced fucoxanthin pretreated rats. Myocardial infarction-induced fucoxanthin pretreated rats shows decreased statuses of Na+/K+ ATPase and increase on the Na+/K+ ATPase level. Conclusion: Our results confirmed that fucoxanthin is a potent cardioprotective drug which increases the antioxidant levels and decreases the oxidative stress and inflammation during myocardial infarction.
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Identification of anticancer compounds from Linum usitatissimum seed extract and their effect on HeLa cells Highly accessed article p. 221
Anitha Joseph, Sriram Sridharan, Sampathkumar Palanisamy, Sivakumar Ramalingam, Renuka Saravanan
DOI:10.4103/pm.pm_341_19  
Aim: The aim of the study is to analyze the phytoconstituents and to determine the anticancer potential of Linum usitatissimum seed against Human cervical cancer cell line is the correct expansion (HeLa). Materials and Methods: The seeds of L. usitatissimum were extracted with methanol, ethyl acetate, and chloroform by cold maceration method. These extracts were subjected to qualitative phytochemical analysis, free-radical scavenging ability (1,1-diphenyl-2-picrylhydrazyl) assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and flow cytometric analysis for studying their cytotoxic and apoptotic effect on HeLa cell. Ethyl acetate extract of L. usitatissimum seeds was subjected to liquid chromatography–mass spectrometry (LC-MS)/MS analysis for identifying its phytoconstituents. Results: Qualitative analysis of the different extracts of the seed revealed the presence of flavonoids, terpenoids, alkaloids, saponins, glycosides, phenols, steroids, and anthraquinones. Free-radical scavenging activity of chloroform extract was found to be more than methanolic and ethyl acetate extracts. Further, methanol, ethyl acetate, and chloroform extracts exerted cytotoxic effect with an 50% inhibitory concentration value of 21 μg/ml, 17.5 μg/ml, and 20 μg/ml, respectively. Flow cytometric analysis revealed that the number of proapoptotic and late apoptotic cells were found to increase with the increasing concentration of seed extract. LC-MS/MS analysis of ethyl acetate extract revealed the presence of glabranine, protocatechuic acid-O-hexoside, and naringenin. Conclusion: The study demonstrated the anticancer effect of the seeds of L. usitatissimum , and the presence of glabranine and naringenin, well-known anticancer agents, is being reported for the first time in L. usitatissimum seed. The antioxidant and anticancer potential of the seed could be due to the presence of glabranine and naringenin.
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Extraction, isolation and identification of kaempferol 3,7 – Diglucoside in the leaf extracts of Evolvulus alsinoides (Linn.) and its inhibition potency against α-amylase, α-glucosidase, Acetylcholinesterase and amyloid aggregation Highly accessed article p. 227
Pavithra Mettupalayam Kaliyannan Sundaramoorthy, Kannan Kilavan Packiam
DOI:10.4103/pm.pm_583_19  
Background: Evolvulus alsinoides (Linn.) is an eminent kaphahara (balancing kapha ) plant in Ayurvedic medicine commonly used as brain tonic. Objectives: To report the enzyme inhibitory potency of the plant extract and to isolate the compound of significant impact. Materials and Methods: The plant extracts were obtained by serial exhaustive extraction and successive chromatographic separation. Antioxidant and enzyme inhibition assays were carried out to extract the required isolate. Results: The current research identifies for the first time, the presence of Kaempferol 3,7 diglycoside in the leaf extracts by Spectrophotometry, Chromatography,[1]H and[13]C Nuclear Magnetic Resonance, carboxylic and hydroxylic hydrolysis. The isolated and characterized compound possesses enzyme inhibition property towards α-amylase, α-glucosidase and Acetylcholinesterase . Conclusion: The compound protects differentiating neuronal cells, SH-SY5Y from Amyloid β Peptide-induced Injury. Thus, the newly identified compound could serve as a plant-based bioactive in the management of Alzheimer's.
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Fucoxanthin attenuates the lipopolysaccharide-induced sepsis and acute lung injury through the inactivation of nuclear factor-kappa B signaling pathway p. 235
Yun Xiao, Chun Yang, Minna Dong, Jinfeng Zhao, Xia Wang, Mingyao Xiao
DOI:10.4103/pm.pm_487_19  
Objectives: The acute lung injury was a very severe health consequence and it was allied with the highest morbidity as well as mortality rate among the peoples. The acute lung injury can be induced through the various direct or indirect causative factors such as serious septic conditions, inflammation of the lung tissues, swelling of the lungs, injury of inhalation, pulmonary vasculitis, pancreatitis, and severe burnings which can lead to the unrestrained inflammation. Materials and Methods: The sepsis was a major clinical syndrome that can cause by severe infection or injury and it was distinguished by the response to the entire body inflammation. The current research work aimed to assess the curative properties of fucoxanthin against the lipopolysaccharides (LPS)-induced acute lung injury in the experimental animal model. Results: The fucoxanthin treatment followed by the LPS administration was exhibited the marked reduction in the wet-to-dry ratio of the lungs, reduced level of infiltration of inflammatory cells in BALF, reduced the enzymatic action of myeloperoxidase in the lungs, reduced the interleukin-6 (IL-6), IL-1 β, and tumor necrosis factor-alpha levels, possessed the notable suppression and downregulation in the expression of immunoreactivity of inflammatory markers like NF-κB p65 and decreased the oozing of mucus and the inflammatory cellular infiltration in lung histopathology which is stimulated by the LPS administration. Conclusion: The fucoxanthin was effectively attenuated the LPS-stimulated acute lung injury in an animal model. Hence, it can be concluded that the fucoxanthin had potent therapeutic properties against the LPS-stimulated acute lung injury.
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Protective effect of fucoxanthin on ovariectomy-induced osteoporosis in rats p. 242
Lichun Guo, Minyan Dang, Qichun Song, Wenzhi Zhang, Bing Li
DOI:10.4103/pm.pm_340_19  
Background: The necessity for development of antiosteoporotic drug is receiving increased attention because of high mortality and morbidity rates arising globally. The natural herbal-based compounds such as fucoxanthin possessed greater therapeutic potentials in biomedical field. Objective: We planned to investigate the therapeutic effect of fucoxanthin on ovariectomy-stimulated osteoporosis in experimental rats. Hence, the study is conducted to evaluate the protective effect of fucoxanthin against ovariectomy-induced osteoporosis in female Sprague Dawley (SD) rats. Materials and Methods: Healthy adult female SD rats weighing about 230–245 g were randomized into four groups of six animals each. Group I served as sham-operated control. Group II served as model (ovariectomized [OVX]) rats. OVX rats were administered with fucoxanthin at a dose of 20 mg/kg and 40 mg/kg orally for 16 weeks which served as Group III and Group IV, respectively. Results: A significant increase in body weight and decrease in uterine index was observed in OVX rats, whereas treatment with fucoxanthin substantially reverted the body weight and uterine mass. Bone turnover markers (Ca, P, and osteocalcin), levels of estrogen and 1,25-dihydroxycholecalciferol 1,25(OH)2D3, osteoprotegerin and receptor activator of nuclear factor-κB ligand, and inflammatory markers were reverted back to a significant extent by treatment with fucoxanthin. The biomechanical stability of bones was significantly increased with administration of fucoxanthin. The findings were also substantiated by histopathological analysis. Conclusion: Based on the outcome of the results, it can be concluded that fucoxanthin showed better protection against osteoporosis by improving bone mineral content and bone density in addition to biomechanical parameters.
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Protective effect of adropin against high fat diet-induced obese diabetic wistar rats via nuclear factor erythroid 2-related factor 2 pathway p. 250
Fang Luo, Qing-Chu Li, Feng-Jiao Zhang, Lei Li, Li-Ge Song, Yu Mao, Jing Li, Hong-Mei Liu, Feng-Li Li, Ling-Yu Xu, Ya-Jie Huo, Huan-Huan Wang, Zhi-Qiang Kang, Li He
DOI:10.4103/pm.pm_434_19  
Introduction: Liver steatosis (fatty liver) is frequently found during the conditions such as diabetes and obesity. The current experimental study was executed the effect of adropin against high fat diet (HFD)-induced obese diabetic Wistar rats via nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Materials and Methods: Wistar rats were randomly divided into six groups and divided as follows: normal, HFD control, HFD + adropin (2.5, 5 and 10 mg/kg), and HFD + Glibenclamide (2.5 mg/kg), respectively. Lipid and carbohydrate metabolism, hepatic parameters, antioxidant, and proinflammatory cytokines were estimated at the end of the experimental study. Nrf2 transcription and nuclear level were also estimated. In vitro adropin diminished the accumulation of lipid droplets in dose-dependent manner and no effect was observed on the lipolysis. Type II diabetic rats fed with HFD exhibited a marked reduction in hepatic extraction faction and hepatic steatosis after the adropin and glibenclamide treatment. Results: Adropin significantly (P < 0.001) altered the hepatic parameter such as alanine transaminase, aspartate transaminase, alkaline phosphatase; antioxidant parameters such as thiobarbituric acid reactive substances, 8-OhdG, superoxide dismutase, glutathione (GSH) peroxidase, catalase, GSH, GSH reductase, GSH S-transferase; proinflammatory cytokines, namely tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1, respectively. In addition, adropin significantly reduced the nuclear Nrf2 activity at dose-dependent manner. Conclusion: Adropin improved insulin sensitivity, reduced lipogenesis in the adipocytes and also decrease the inflammation through downregulated cytokines (Nrf2 pathway).
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Exploration of aurora B and cyclin-dependent kinase 4 inhibitors isolated from Scorzonera tortuosissima boiss. and their docking studies Highly accessed article p. 258
Ehab M Mostafa
DOI:10.4103/pm.pm_1_20  
Background: Flavonoids are components of the daily human diet (fruits and vegetables), and it has been shown to inhibit several kinase enzymes. Due to its kinase inhibitory activity, they are expected to be of great importance in the discovery of new anticancer drugs. Objectives: The objective was to study the cytotoxicity, kinase inhibitory activity, and docking of the isolated aglycones. Materials and Methods: Ultraviolet, high-performance liquid chromatography (LC), nuclear magnetic resonance, and LC-mass spectrometry were used for the identification of the isolated metabolites. Antiproliferative (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and radiometric protein kinase (PK) assays were used to measure the cytotoxicity and PK inhibitory effect of the isolated flavonoids. The docking study on both Aurora B and cyclin-dependent kinase 4 (CDK4)/CycD1 was performed by molecular operating environment (MOE). Results: Luteolin (1), quercetin (2), myricetin (3), apigenin-7-O -β-D-glucopyranoside (4), and kaempferol-7-O -β-D-glucopyranoside (5) were isolated from Scorzonera tortuosissima . Quercetin and myricetin exhibited the highest cytotoxicity against the Michigan Cancer Foundation-7 (MCF-7) (IC50: 5.56 and 7.14 μM, respectively) and against human hepatocellular carcinoma (HepG2) (IC50: 8.61 and 10.31 μM, respectively), while compounds luteolin and apigenin-7-O-β-D-glucopyranoside showed the least cytotoxicity compared to doxorubicin against to MCF-7 and HepG2 (IC50: 2.24 ± 0.85 and 1.82 ± 0.34 μM, respectively). The radiometric PK assay was applied for measurement of kinase inhibitory activity against Aurora B, CDK4/D1, cancer Osaka thyroid, IGF1-R, and FAK kinases, where the aglycone myricetin showed the highest inhibitory activity against Aurora B (IC50: 2.82 μM) and against CDK4/cyclin D1 (IC50: 3.16 μM), while the isolated glycosides 4 and 5 revealed the lowest activity. Docking of the most active compounds 1, 2, and 3 against Aurora B and CDK4/cyclin D1 confirmed its cytotoxic profile. Conclusion: The isolated flavonoids were firstly isolated from S. tortuosissima . The hypothetical mechanism of cytotoxic activity of 1, 2, and 3 on Aurora B and CDK4/cyclin D1 kinases was estimated by in silico study with these enzymes using MOE program.
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Bioassay-guided isolation, identification, and evaluation of anti-inflammatory activity of β-boswellic alcohol and 3-o-acetyl-11-hydroxy-β-boswellic acid from the leaves of Boswellia serrata p. 264
Tarun Sharma, Snehasis Jana
DOI:10.4103/pm.pm_17_20  
Background: There are no in-depth studies on the extracts of the leaves of Boswellia serrata (BS), which contain many bioactive phytoconstituents and responsible for their anti-inflammatory effects. Objectives: The goal of this research plan was the isolation and purification of most active compounds from the leaves of BS via bioassay-guided fractionation based on anti-inflammatory activity. Materials and Methods: The silica gel column chromatographic techniques with different solvent systems were used for the separation of the constituents of the ethyl acetate-soluble fraction of BS leaves. The structures of the isolated compounds were assigned based on various spectroscopic analysis (high-resolution mass spectrometry,[1]H NMR, and[13]C NMR) and comparison with literature data. The anti-inflammatory activity of all crude extracts, subfractions of ethyl acetate extract, and two isolated new compounds were evaluated in cell-free (cyclooxygenase and 5-lipoxygenase) and cell-based assays (nitrite and tumor necrosis factor-alpha) in RAW264.7. Results: Bioassay-guided fractionation of the extracts of the leaves of BS afforded two ursane-type triterpenoids (compound 5, β-boswellic alcohol, and compound 6, 3-O-acetyl-11-hydroxy-β-boswellic acid) and other ten known triterpene acids. Compound 5 and 6 exhibited strong anti-inflammatory activity with an IC50value of a range 26.5–34.2 μM and 18.3–21.1 μM, respectively, in cell-free and cell-based assays. Conclusion: This is the first report on isolation and identification of β-boswellic alcohol and 3-O-acetyl-11-hydroxy-β-boswellic acid from the leaves of BS, which showed strong anti-inflammatory activity and holds great promise for the treatment of numerous inflammatory diseases.
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Screening of potential inhibitors against flotillin-1 as therapeutics for Alzheimer's disease p. 274
Amit Chaudhary, Ashutosh Mani
DOI:10.4103/pm.pm_514_19  
Background: Alzheimer's disease (AD) is one of the most common neurological disorders occurring in older people. So far, no specific drug is available for the disease, and only palliative medicines are available for the patients. Accumulation of amyloid beta (Aβ) peptides is considered to play a crucial role in the generation of the disease. Aβ peptide is generated by the proteolysis of amyloid precursor protein by two distinct proteases β and γ-secretase. Flotillin-1 (FLOT1) directly binds to the cytoplasmic tail of β-secretase and affects the sorting and recycling of the enzyme. Increased expression of FLOT1 has been correlated with the progression of AD, while FLOT1 knockdown causes a reduction in Aβ production. Thus, FLOT1 is an attractive therapeutic target for the suppression of beta-secretase 1 (BACE-1 ). Objective: This study aims to screen the potential inhibitors against FLOT1 as therapeutics for AD. Materials and Methodology: In this work, protein–protein interactions, tertiary structure prediction, molecular docking, and molecular dynamics (MD) simulation were performed. Results: Tertiary structure prediction of FLOT1 and screening inhibitors against it helped in finding key molecules with potential therapeutic properties. Protein–protein interaction study of FLOT1 deciphered the interactors playing key role in AD. Pharmacokinetic parameters were quantified for each potential inhibitor. The results of MD simulation analysis revealed that the ZINC67911837 had better inhibitory activities with FLOT1. Conclusion: The analysis suggests that the ZINC67911837 compound could be a novel potential inhibitor of FLOT1 to modulate BACE-1 activity and used as a therapeutic agent for the treatment of AD. This study facilitates the initiation of the natural drug discovery process for the treatment of AD patients.
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Charantin relieves pain by inhibiting pro-inflammatory cytokine induction p. 282
Jaewon Shim, Jae Goo Kim, Eun Yeong Lim, Yun Tai Kim
DOI:10.4103/pm.pm_348_19  
Background: The fruits of Momordica charantia, commonly known as bitter melon, have been used as a traditional medicine in several countries. Some studies have reported its pharmacological effects in various disorders. Objectives: Because there have been little reports on charantin's role as an analgesic, we evaluated its pain relief effect to determine if it could be a novel pain killer candidate. Materials and Methods: We established post-operative and neuropathic pain models, which represent acute and chronic pain, respectively. Mechanical withdrawal threshold assay and ultrasonic vocalization analysis were used as behavioral tests. Results: The administration of charantin reduced both the post-operative and neuropathic pain. The application of charantin did not make a difference in the activation of action potentials of dorsal root ganglion (DRG) neurons. However, charantin inhibited the induction of the pro-inflammatory cytokines interleukin IL-12 and IL-1β in DRG neurons. Conclusion: Our findings indicate that charantin seems to relieve pain by inhibiting the inflammatory process rather than by directly influencing the activity of neurons. We conclude that charantin, the commercially available extract from M. charantia, has great efficacy as a novel analgesic compound.
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Identification and quantification bioactive compounds on liquid extract of Capsicum chinense jacq. biquinho variety: Standardization and quality control is frontier to acceptance of herbal products on market p. 288
Lucélia de Sousa Brito, Aline Neves Pereira, Emannuel Ítalo Alves Campos, Andressa Tuane Santana Paz, Mariana Cristina de Morais, Leonardo Luiz Borges, Edemilson Cardoso da Conceição
DOI:10.4103/pm.pm_303_19  
Introduction: The peppers of the Capsicum genus (Solanaceae ) are originating in the Americas and have aroused great interest from cosmetics and food industries because these species are rich in antioxidant compounds. Objectives: The aim of the present study was to develop and characterize the liquid extract of Capsicum chinense Jacq. Biquinho variety standardized in capsanthin. Materials and Methods: The liquid extract of the pepper was obtained by percolation and characterized about the solids content, pH, density and viscosity. Subsequently, the total carotenoids were determined by spectrophotometry in the pepper liquid extract, and an analytical method by high performance liquid chromatography was validated to quantify the capsanthin in the same sample.Results: The extract showed the following physicochemical properties: 19.16% ±1.03% (w/w) to solids content, a pH of 3.7 ± 0.06, density of 1.040 ± 0.0014 g/mL, and a viscosity of 3.22 mPas. The concentration of total carotenoids was 84.403 mg expressed as β-carotene/100 g of the extract. Conclusion: The analytical method developed was considered simple, fast, selective, linear (in the range of 0.48 to 7.2 μg/mL for the liquid extract), precise, exact, and robust, and the found content of capsanthin was 1.13%. The analytical method used for the quantification of capsanthin was validated, proving the simplicity and speed of analysis of the evaluated sample.
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Dendrobium officinale kimura et migo improved dry eye symptoms via promoting tear production in an experimental dry eye rat model p. 294
Qiang Zeng, Wing-Sum Siu, Chun-Hay Ko, Chun-Wai Wong, Clara Bik-San Lau, Zheng-Zhi Wu, Jiang-Miao Hu, Ping-Chung Leung
DOI:10.4103/pm.pm_435_19  
Objectives: To evaluate the ameliorative effect of Dendrobium officinale Kimura et Migo (DO) on a desiccated environment-induced experimental dry eye rat model and elucidate its underlying mechanisms. Materials and Methods: The Sprague-Dawley rats were kept in low-humidity environment and received constant airflow for 8 weeks to establish the experimental dry eye model. DO water extract (DOW, 372 mg/kg/day) was orally administered daily for 8 weeks. Schirmer's test was used to measure the tear fluid production at days 0, 14, 28, 42, and 56. At the end of experiment, lacrimal gland tissues and eyeballs were collected for hematoxylin and eosin staining, PAS staining, and immunohistochemical staining. Inflammatory cytokines in conjunctiva including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and matrix metalloproteinase 9 were measured by real-time PCR. The aquaporin 5 (AQP5) expression in lacrimal gland was also determined using Western blot assay. Results: DOW treatment (DOWT) increased the tear production of rats significantly in the desiccated environment at day 42. Histological analysis revealed that DOW could reverse destruction of conjunctiva and increase goblet cell number and mucin expression in the experimental dry eye rats. In dry eye rats, desiccated environment and constant airflow induced TNF-α and IL-1β production in the conjunctiva, whereas DOWT reversed the upregulation of proinflammatory cytokines. Moreover, DOWT increased the expression of AQP5 at protein level in the lacrimal gland tissues in both desiccated and normal environmental conditions. Conclusion: The present study suggests that DO has therapeutic potential on dry eye symptoms through upregulating AQP5 expression, increasing tear production, inhibiting conjunctiva destruction and inflammation, as well as promoting mucin production.
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D-carvone attenuates biochemical and molecular expression via oncogenic signaling in aryl hydrocarbon-induced hamster mucosal carcinogenesis p. 303
Jingxuan Wang, Yan Hu, Yifan Wang, Yingshun Yang, Song Li, Yujiao Hou, Zhizheng Zhuang, Fan Wu
DOI:10.4103/pm.pm_302_19  
Background: Chemo prevention through nutritional constituents has appeared as an innovative methodology to control the oral cancer incidence. The main target of this research exists to explore the chemopreventive effect of D-carvone by way of biochemical and molecular prototype during 7,12-dimethylbenz[a]anthracene (DMBA)-stimulated hamsters mucosal carcinogenesis. Materials and Methods: Topical application of 0.5% DMBA in liquid paraffin, thrice a week, for 10 weeks well developed oral squamous cell carcinoma in hamsters cheek pouch (HCP). All the same 100% tumor formation was perceived in hamsters induced with DMBA alone, but intragastric administration of D-carvone, at a dose of 10 mg/kg bw, to DMBA-treated hamster totally prevented the formation of oral tumor. Results: D-carvone significantly lessens lipid peroxidation (LPO) by-products and enhanced the status of enzymatic, non-enzymatic antioxidants, and varied the status of Phase I and II xenobiotic enzymes, favoring the secretion of cancer metabolites through downregulation of proliferating cell nuclear antigen and p53 expression during oral tumor hamsters. Conclusion: The nearby study suggests that D-carvone relies on its anti-LPO, antioxidant, xenobiotic metabolic enzymes as well as anti-cell proliferation and induced apoptosis during DMBA-induced hamster oral mucosal carcinogenesis.
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Fucoxanthin inhibits cell proliferation and stimulates apoptosis through downregulation of PI3K/AKT/mTOR signaling pathway in human ovarian cancer cells p. 311
Yan Li, Leisi Tao, Lei Bao, Arunachalam Chinnathambi, Sulaiman Ali Alharbi, Jianying Cui
DOI:10.4103/pm.pm_330_19  
Background: Fucoxanthin is rich in seaweed and considered the most efficient anticancer drug because of its powerful antioxidant properties. The aim of this study was to elucidate the role of fucoxanthin on apoptosis via signaling pathway in ovarian cancer cells. First, fucoxanthin exhibited obvious cytotoxicity against A2780 human ovarian cancer cells. Materials and Methods: We established the action potential of fucoxanthin on cell proliferation in addition to apoptosis in A2780 cells, which were measured using reactive oxygen species (ROS) generation and the mitochondrial membrane potential (MMP). Apoptotic-related morphological alterations were inspected by acridine orange/EtBr dual staining. In addition, we analyzed caspase family protein expression by enzyme-linked immunosorbent assay analysis. Results: Our results established that fucoxanthin stimulates apoptosis as confirmed through reduced cell viability, improved production of ROS, and altered MMP in A2780 cells. Further, the fucoxanthin upregulates the expression of caspase (3, 8, and 9) protein experimentally in A2780 cell. In addition, we uncovered upstream signaling cascade (Akt/mTOR) induced by incubation A2780 cell lines with fucoxanthin that mediated cell apoptosis, migration, and invasion process. Conclusion: These findings suggest that fucoxanthin augments apoptosis; reduces cell proliferation, migration, and invasion; and reveals a potential mechanism of fucoxanthin-mediated Akt/mTOR suppression in human ovarian cancer cell line. Hence, fucoxanthin could be reflected as a potential therapeutic agent for ovarian cancer treatment.
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Phytochemical analysis, antipropulsive and antilymphoma activities of leaves extract of Annona cherimola miller p. 317
Fernando Calzada, Miguel Valdés, Verenice Itzel Merlin, Claudia Velázquez, Elizabeth Barbosa, Marco Chávez-Soto, Emmanuel Pina-Jiménez, Rosa Maria Ordoñez-Razo, Normand García-Hernández
DOI:10.4103/pm.pm_462_19  
Background: Diarrhea and cancer are significant health problems worldwide. Annona cherimola Mill. (Annonaceae) is a fruit tree extensively used in Mexican traditional medicine for the treatment of diarrhea and cancer. Objectives: The present work reports the identification of flavonol glycosides present in the ethanol crude extract using high-performance liquid chromatography with diode array detection (HPLC-DAD). Materials and Methods: The identification of flavonoids was made by comparing their retention times and ultraviolet spectra with those standards. The crude extract, dichloromethane, and aqueous residual (AR) fractions were tested for their antipropulsive and antilymphoma activities. Results: The results revealed high levels of three flavonoid glycosides (rutin, nicotiflorin, and narcissin), especially rutin (24.5 mg/g of extract). The antipropulsive and antilymphoma activities of the leaf extract and subsequent fractions showed that the ethanol extract of the leaves of A. cherimola is cursive and AR fraction exhibited the best biological effects in both assays. Conclusion: The HPLC-DAD method used in this work enables the determination of rutin as a major flavonol glycoside of the leaves of A. cherimola . In addition, it can be suggested that rutin may play an important role in the biological properties of A. cherimola.
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Comparison of anti-inflammatory efficacy between dexamethasone and a standardized herbal formula, PM014, in a cigarette smoke-induced subacute mouse model of chronic obstructive pulmonary disease p. 321
Kwan-Il Kim, Beom-Joon Lee
DOI:10.4103/pm.pm_222_19  
Objective: A standardized herbal formula, PM014, originated from Chungsangboha -tang, which has been used to treat various respiratory diseases, including bronchitis, asthma, and emphysema. Several previous studies have reported that the therapeutic mechanism of PM014 was mediated by an anti-inflammatory effect. Therefore, we compared anti-inflammatory efficacy between PM014 and dexamethasone (DEX) using a mouse model of cigarette smoke (CS)-induced subacute pulmonary inflammation. Materials and Methods: Female C57BL/6 mice were revealed to CS for 2 h/day, three cigarettes per day, 5 days a week for 3 weeks; the control group got no other treatment, while the DEX and PM014 groups received 1 mg/kg of DEX and 100 mg/kg of PM014, respectively (both were orally administered). The histological morphology and average alveolar size were determined by lung histology. The inflammatory cell profiles and protein expressions of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 β, IL-6, and one pro-inflammatory chemokine (monocyte chemoattractant protein 1 [MCP-1]), were measured in bronchoalveolar lavage fluid (BALF). The mRNA expressions of the pro-inflammatory cytokines and chemokine were also measured in lung tissues. Results: Both PM014 and DEX attenuated histological injury and air space enlargement in lung tissue and decreased the number of inflammatory cells in BALF. Both also decreased the mRNA expressions of TNF-α, IL-6, and IL-1 β in lung tissue and reduced the protein expressions of TNF-α, IL-1 β, IL-6, and MCP-1 in BALF. Conclusion: Our results showed that the anti-inflammatory efficacy of PM014 and DEX was equivalent in a subacute mouse model of CS-induced chronic obstructive pulmonary disease.
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Neuroprotective compounds from the embryo of Nelumbo nucifera seeds p. 329
Jin Bae Weon, Choong Je Ma
DOI:10.4103/pm.pm_613_18  
Background: Previous studies have shown the cognitive effect of the embryo of Nelumbo nucifera on scopolamine-induced memory impairment and neuroprotective effect against glutamate-injured neurotoxicity in HT-22 cells. Objectives: The present study was designed for the purpose of evaluating the neuroprotective activity of compounds isolated from the embryo of N. nucifera seeds on glutamate-induced cell death in HT-22 cells. Materials and Methods: We isolated compound from the embryo of N. nucifera using various chromatograms and confirmed chemical structure by various spectroscopy. The neuroprotective effects of the compounds against glutamate-induced cell death in HT-22 cells were investigated using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: We isolated 6 compounds, 1,2,3,4-tetrahydro-7,8-isoquinolinediol (1), 1,2,3,4-tetrahydro-1-[(4-hydroxyphenyl) methyl]-2-methyl-6,7-Isoquinoline diol (2), 1,2,3,4-tetrahydro-1-[(4-hydroxyphenyl) methyl]-2-methyl -7-Isoquinolinol(3),1-(3,4,5-trihydroxyphenyl)-ethanone(4), 1-(2,3,5,6-tetrahydroxyphenyl)-ethanone(5),3-(prop-1-enyl) benzene-1,2,4,5-tetrol (6) from the embryo of N. nucifera . Among these compounds, 1,2,3,4-tetrahydro-7,8-isoquinolinediol and 1,2,3,4-tetrahydro-1-[(4-hydroxyphenyl)methyl]-2-methyl-6,7-Isoquino linediol significantly decreased glutamate-induced cell death in HT-22 cells. In addition, these compounds exacerbated the reactive oxygen species level and intracellular Ca2+ accumulation. Conclusion: The neuroprotective efficacy of 1,2,3,4-tetrahydro-7,8-isoquinolinediol and 1,2,3,4-tetrahydro-1-[(4-hydroxyphenyl) methyl]-2-methyl-6,7-Isoquinolinediolmay be related to their antioxidative effect.
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Anti-aging, anti-inflammatory, and wound-healing activities of edible bird's nest in human skin keratinocytes and fibroblasts p. 336
Eunson Hwang, Sul Woong Park, Jung-Eun Yang
DOI:10.4103/pm.pm_326_19  
Background: The “bird's nest soup” is a kind of luxury or tonic food prepared from edible bird's nest (EBN), which is used as an extremely nutritious medicine to improve health. However, its skin anti-inflammatory and wound-healing effects are still not fully understood. Aim: In this study, the skin-protective effects of two geographical types of EBN (EBN-A and EBN-B) were investigated. Materials and Methods: The anti-aging effect was assessed in ultraviolet B (UVB)-irradiated normal human dermal fibroblasts (NHDFs), while the anti-inflammatory effect was evaluated in tumor necrosis factor alpha/interferon gamma (TNF-α/IFN-γ)-stimulated human skin keratinocytes (HaCaTs). The wound-healing activity was investigated in scratched NHDFs and hyalorunan production was examined in HaCaTs. Results: In this study, EBN showed good efficiency in scavenging free radicals. EBN notably decreased the UVB-induced matrix metalloproteinase-1 expression and promoted procollagen type I synthesis that resulted in the protective effect of EBN against UVB-induced skin damage. Overexpression of thymus- and activation-regulated chemokine and macrophage-derived chemokine induced by TNF-α/IFN-γ was significantly decreased after treatment with EBN at 1–10 μg/ml. The most effective wound healer was EBN-B at 1–10 μg/ml, based on the high expression of hyaluronan that has long been associated with the remodeling extracellular matrix in wound healing. Conclusion: These results indicate that EBNs have the potential to ameliorate UVB-induced skin photo aging and NF-α/IFN-γ-stimulated inflammation as well as wound injuries, resulting in rapid healing effects.
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Effects of the Japanese kampo medicine, Rikkunshito, on gastrointestinal motility functions p. 343
Minwoo Hwang, Donghun Han, Byung Joo Kim
DOI:10.4103/pm.pm_370_19  
Background: Rikkunshito is known as a prokinetic agent for gastrointestinal (GI) diseases. Objectives: The objective was to find the effect of GI motility with Rikkunshito. Materials and Methods: The prokinetic effect of Rikkunshito was investigated by studying the gastric emptying (GE) and intestinal transit rate (ITR) or experimentally induced GI motility dysfunction (GMD). Results: The oral administration of Rikkunshito significantly increased GE and ITR and restored the delayed GE and ITR. Its effect was similar to that of conventional prokinetics, such as domperidone and mosapride. Moreover, we made the mouse GMD models, such as acetic acid or streptozotocin, and ITR had shown a great decrease. This decrease was recovered by Rikkunshito. Conclusion: Rikkunshito revealed its prokinetic effect through an increase of GE and ITR. We found a great possibility of Rikkunshito for the GI control drug.
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Study on the characteristics of pharmacokinetics of different doses of gastrodin in mice p. 347
Xinyu Wang, Xin Liu, Xing Wang, Yu Dong
DOI:10.4103/pm.pm_307_19  
Background: Rhizoma Gastrodiae, the dried rhizome of Gastrodia elata Blume (G. elata) , is famous Chinese herb that belongs to the genus Gastrodia , family Orchidaceae. Gastrodin (GAS) is an effective monomer with one of the major active constituents in Rhizoma Gastrodiae . GAS has a good sedative and sleeping effect and has a mitigating effect on neurasthenia, insomnia, and headache. Moreover, in the circulatory system, it has the effect of reducing peripheral vascular resistance, blood pressure, and so on. Objectives: The objective is to investigate the pharmacokinetic characteristics after the administration of different doses of GAS by using pharmacokinetic method and make a preliminary judgment on whether the intake of GAS in the liver is involved in transporters. Materials and Methods: All healthy mice were randomly divided into three groups according to the drug concentration: low-concentration group (LC group), middle-concentration group, and high-concentration group. Then, each group received different doses of GAS by tail vein injection. The blood samples of different groups were harvested at different time points, and the blood drug concentration was evaluated by high-performance liquid chromatography method. The method was confirmed in terms of the linearity, precision, and accuracy. Results: The results showed that the analytical curve was linear over the concentration range of 4–40 μg/mL. In the intra- and inter-assay, the coefficient of variation was <7.23%. Moreover, the regression equation of the line was “Y = 49.43 X +0.027.” The results suggested that the elimination half-life period and area under the curve were slightly decreased, then non-linearly increased accompanying with increase of the dose of GAS. Conclusion: The results suggested that the pharmacokinetics of different dosage GAS administration were in accordance with the two compartment model in mice.
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Comparative analysis of the chemical constituents from the tuberous root and stem of Pueraria candollei var. mirifica and evaluation of their estrogenic activity p. 353
Witsarut Kraithong, Wipawee Juengsanguanpornsuk, Supaluk Krittanai, Gorawit Yusakul, Waraporn Putalun
DOI:10.4103/pm.pm_442_19  
Background: Pueraria candollei var.mirifica (PC) is a tuberous plant enriched with bioactive phytoestrogens. PC is currently high demanded in the global markets as dietary supplement products, which resulted in a shortage of tuberous root from the natural resource and the field cultivation. Materials and Methods: We compare phytoestrogens contents from the tuberous root and stem of PC and their estrogenic activities on MCF-7 cells proliferation. Results: The root and stem of PC accumulated different contents of phytoestrogens. The highest amount of total isoflavonoids and total chromenes was found in the root bark of PC. However, the stem bark (10 μg/mL crude extract) which contains a high level of chromenes and low amount of isoflavonoids exhibited higher growth stimulation of MCF-7 cells (127.25% ± 4.43% relative proliferative effect [RPE] as compared to estradiol [10−10 M]) than root bark at the same concentration with 116.32% ± 3.59% RPE. After the solubilization of the ethanol extract with 10% (v/v) aqueous ethanol, the soluble fraction of crude extract of stem stimulated proliferation of MCF-7 cells without cell growth suppression effect at high concentration (100 μg/mL) as observed in the insoluble fraction of the extract. Conclusion: We suggested that the soluble part of crude extract might be less toxic than the ethanolic crude extract and its non-polar components. Our study opens the possibility of using the stem of PC as a new alternative source of bioactive phytoestrogens for dietary supplement products.
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Evaluation of wound healing potential of ascorbic acid, castor oil, and gum tragacanth formulation in murine excisional wound model p. 359
Ajay K Sharma, Sandeep Kumar Shukla, Aman Kalonia, Priyanka Shaw, MH Yashavarddhan, Kailash Manda
DOI:10.4103/pm.pm_440_19  
Background: Despite remarkable development in wound healing management, there is no ideal drug available to address diverse complexity in wound care. This necessitates the development of a comprehensive wound healing formulation. Objectives: The objective of this study was to evaluate the potential of ascorbic acid, castor oil, and gum tragacanth formulation, as an effective wound healing composition. Materials and Methods: A formulation containing ascorbic acid (0.5%), tragacanth gum (6%), and castor oil (25%) is prepared and evaluated for its efficacy on full-thickness mouse excisional wound model. Povidone-iodine was used as a positive control for comparative efficacy. Morphological and histopathological studies were carried out for understanding its wound healing potential. Antibacterial action was tested using disk diffusion method. Safety assessment for oral toxicity and eye and skin irritation was investigated in mouse and rabbit models, respectively. Permeability studies of formulation with excised skin have been evaluated by diffusion chamber, and its absorption in skin and serum was measured with high-performance liquid chromatography method. Results: Wound treated twice a day with formulation showed up to 24% recovery after 24 h and full skin restoration on the 9th day compared to the untreated wound. The morphological and histopathological examinations of wound indicated noteworthy improvement in recovery. Comparative efficacy showed 98% wound recovery in formulation-treated groups compared to 89% with commercially available povidone-iodine ointment after the 9th day. Permeability data clearly indicate enhanced skin uptake in time-dependent manner. The formulation is found to be safe and effective as demonstrated by antibacterial nature, permeability, and supportive safety studies. Conclusion: This study elucidates this formulation possesses strong wound healing potential.
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Tillandsia usneoides protects RINm5F cells from streptozotocin-induced apoptosis and stimulates insulin secretion p. 369
Janet Alejandra Espejel-Nava, Francisco Alarcon-Aguilar, María del Carmen Escobar-Villanueva, Alejandra Contreras-Ramos, Miguel Cruz, Elisa Vega-Avila, Clara Ortega-Camarillo
DOI:10.4103/pm.pm_277_19  
Background: Tillandsia usneoides (Bromeliaceous) is traditionally used in Mexico for diabetes treatment. Although a significant hypoglycemic effect has been reported, the participation of insulin in this action has not yet been explored. Objectives: The aim of this research was to determine the hypoglycemic effect of an aqueous extract from T. usneoides in normal and diabetic mice as well as to evaluate the participation of insulin in this effect using an in vitro model. Materials and Methods: Aqueous decoction of T. usneoides (250 mg/kg) was administered in healthy and diabetic mice and glycemia was measured. RINm5F cells were cultured with T. usneoides (0.1 and 1 μg/ml), and cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, trypan blue, and apoptosis; secretion and expression of insulin were quantified by enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction, respectively. Results: T. usneoides decreased blood glucose in healthy mice at 4 and 6 h (96 ± 11.2 and 68.2 ± 1.9 mg/dl, respectively) compared with time zero (138.5 ± 5.0 mg/dl, P < 0.05). In diabetic mice, aqueous extract significantly decreased glycemia at 6 h compared with time zero (212.7 ± 3.5 and 243 ± 5.3 and mg/dl, respectively). Besides, T. usneoides aqueous extract stimulated insulin secretion (20%, P < 0.05) without cause changes in insulin gene expression and protects RINm5F cells from streptozotocin-induced apoptosis. Conclusion: These results suggest that aqueous decoction of T. usneoides stimulates insulin secretion still in the absence of changes in the intracellular concentration of Ca2+ in RINm5F cells.
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Comparison of the content of flavonoids, total phenols, and carotenoids and antioxidant activity in guang Citri reticulatae pericarpium during the aging time p. 375
Guangning Wang, Shuwen Chen, Guangshan Gu, Jieqing Qiu, Yuanling Chen, Zhen Jia, Hongmei Tang
DOI:10.4103/pm.pm_295_19  
Background: Guang Citri reticulatae Pericarpium (GCRP) is a traditional Chinese medicine, widely used in respiratory and digestive diseases. GCRP must be aged by stored for several years before it can be used as medicine. In the aging process, content of composition, antioxidant activity, and color of GCRP have changed. Objectives: The objective of this study was to compare the content of flavonoids, total phenols, and carotenoids and antioxidant activity in GCRP during the aging time and analyze the correlations of the data. Materials and Methods: Forty batches of GCRP in different aging years were used for qualitative and quantitative analysis of flavonoids, total phenols, and carotenoids by ultra-high-performance liquid chromatography (HPLC)–mass spectrometry, HPLC–diode array detector, and ultraviolet methods. 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 1,1-diphenyl-2-trinitrophenylhydrazine methods were used to compare the antioxidant activity of GCRP. Colorimetric analysis method was used to determine the external color of GCRP objectively and quantitatively. SPSS software was used to analyze the correlation of all data. Results: With the increase of aging year, the content of three flavonoids and total phenols increased and the content of carotenoids decreased. The results showed that the antioxidant activity of ether extract increased significantly with aging year and methanol extract decreased. The correlation results showed that the content of carotenoids was more closely related to chromaticity values and antioxidant activity. Conclusion: The result provides more objective and detailed information to analyze the quality of GCRP during the aging time.
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Antiviral assessments of honeybee (Apis mellifera L.) venom p. 382
Sang Mi Han, Se Gun Kim, Hyo Young Kim, Hong Min Choi, Hyo Jung Moon, Soon Ok Woo, Sok Cheon Pak
DOI:10.4103/pm.pm_337_19  
Background: The poultry industry has long been challenged by avian influenza which causes significant economic loss due to decreased egg production and quality. In addition, the notable ability of influenza viruses to develop resistance to conventional antibiotics is one of the biggest tasks that the industry currently faces. Attempts have been made to treat this bird flu with multiple approaches, but effective natural solutions remain elusive. Bee venom (BV) is used for the treatment of various human diseases due to its known anti-inflammatory and antibacterial properties. Recent studies suggest that antimicrobial peptides discovered in BV may be utilized as tools for the design of structurally novel antiviral agents effective against influenza viruses. Materials and Methods: In the present study, we purified BV containing 63.9% ± 5.4% melittin, 10.9% ± 1.6% phospholipase A2, and 2.3% ± 0.3% apamin. BV was evaluated in vitro for its ability to inhibit the binding of H9N2 to the chicken red blood cells. Results: We found that anti-influenza activity of BV is equivalent to that of positive control. However, we observed the neutralization of H9N2 by BV as compared to the virus only group without BV. The hypothesized anti-influenza property of BV was further examined in chicken influenza infection model. The administration of BV through intranasal route resulted in no significant antiviral effect in chickens. Conclusion: This study does not support our hypothesis that BV can reduce the viral activity in chickens.
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Effect of exogenous Ca2+ on growth and accumulation of major components in tissue culture seedlings of Sophora tonkinensis gagnep p. 386
Ying Liang, Lin Xuan Li, Jin Yuan Cai, Chuan Hua Deng, Shuang Shuang Qin, Ming Jie Li, Kun Hua Wei, Zhong Yi Zhang
DOI:10.4103/pm.pm_362_19  PMID:10.4103/pm.pm_362_19
Background: Sophora tonkinensis Gagnep., well-known for its medicinal properties, grows in karst areas of China characterized by calcium (Ca) and drought. Due to excessive excavation, wild S. tonkinensis is almost extinct. Ca regulates the growth and development of plants and also functions in stress response. Objectives: The effects of exogenous Ca2+ on plant height, stem diameter, enzyme activity, endogenous hormone content, and other traits of S. tonkinensis , and accumulation of matrine and oxymatrine in its active constituents were studied. The purpose was to explore the mechanism of adaptation of S. tonkinensis to high Ca, to protect and artificially cultivate S. tonkinensis resources. Materials and Methods: Six concentrations of Ca2+ (0, 1.495, 2.99, 5.98, 8.97, and 11.96 mmol/L) were used. Agronomic traits were measured with a ruler, vernier calipers, and an electronic scale. Physiological and biochemical indices were measured using ultraviolet spectrophotometry. Endogenous hormone contents were determined by enzyme-linked immunosorbent assay. We determined the composition of oxymatrine and matrine using high performance liquid chromatography. Results: As Ca2+ concentration increased, the drying rate of S. tonkinensis decreased and then increased; root length and number, and rooting rate decreased; and the soluble sugar, soluble protein, and chlorophyll content increased and then decreased. reactive oxygen species increased, increasing enzyme activity to resist cell membrane damage under low concentrations of Ca2+ (0–2.99 mmol/L); high concentrations of Ca2+ (5.98–11.96 mmol/L) did more damage to S. tonkinensis , and enzyme activity was not coordinated. Low concentrations of Ca2+ (0–2.99 mmol/L) promoted methyl jasmonate content to reduce cell damage. Matrine and oxymatrine in the whole plant were highest under 5.98 mmol/L Ca2+ treatment, and in stems, leaves, and roots they were highest under 11.96 mmol/L Ca2+ treatment. Conclusion: S. tonkinensis can tolerate exogenous Ca concentrations between 2.99 and 5.98 mmol/L.
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Harnessing multiplex polymerase chain reaction assay for convenient and simultaneous differentiation of testudinis carapax et plastrum from trionycis carapax p. 393
Zhaoqun Jiao, Liqun Chen, Lijuan Xia, Yaya Yang, Yang Zheng, Hongxia Chen, Liwei Wang, Pingtian Yu, Yuping Shen, Huan Yang
DOI:10.4103/pm.pm_198_19  
Background: Testudinis Carapax et Plastrum (TCP, Guijia) and Trionycis Carapax (TC, Biejia), which made from the shell of Chinemys reevesii (CR) and Pelodiscus sinensis (PS), were two kinds of widely used animal-derived Traditional Chinese medicine (TCM). Some cheap substitutes such as the carapace and plastron of Trachemys scripta (TS), Mauremys sinensis (MS), or Apalone ferox (AF) shells obtained from restaurants are sometimes used, which expose the public health to a high risk and cause unfair competitions in the market. Objective: The objective of the study was to develop a multiplex polymerase chain reaction (PCR) approach to simultaneously differentiate five Chelonia species and identify adulteration of two natural products. Materials and Methods: Five novel species-specific primers were designed for CR, TS, MS, PS, and AF, followed by optimization of PCR conditions and validation for specificity and sensitivity. Then, a deliberate mixture was analyzed to verify the capability of adulteration detection. Finally, it was used to examine commercial products. Results: The developed method proved to be highly specific and detection limit was 1 ng for all the species tested. Particularly, it was still applicable and reliable when processed products or deliberate adulteration were examined. Two batches of commercial raw TCP products were identified to be counterfeited by TS using the newly proposed approach. Conclusion: The newly proposed multiplex PCR method showed sufficient merits to be readily employed as a regular means to authenticate edible and medicinal TCM products made of TCP and TC.
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Clinacanthus nutans aqueous extract suppresses the release of histamine and β-Hexosaminidase inin-vitro model of IgE-mediated mast cell degranulation p. 400
Audrey Siew Foong Kow, Lai Yen Fong, Leng Wei Khoo, Ji Wei Tan, Ming-Tatt Lee, Daud Ahmad Israf, Faridah Abas, Khozirah Shaari, Chau Ling Tham
DOI:10.4103/pm.pm_221_19  
Introduction: Clinacanthus nutans (Burm. f.) Lindau, a shrub found in South East Asia, particularly in Malaysia, Thailand, and Indonesia has many potential medicinal uses. It is used as a traditional herbal medicine for treating many diseases including skin rashes. Skin rash often appear in an allergic reaction. Recently, we have shown that C. nutans aqueous extract has the ability to alleviate ovalbumin-induced active systemic anaphylaxis rats from anaphylaxis – the acute form of allergy. This present study is aimed at comparing the ability of ethanolic and aqueous extracts of C. nutans to suppress IgE-mast cell degranulation. Materials and Methods: IgE-presensitized rat basophilic leukemic (RBL-2H3) cells pretreated with C. nutans ethanolic (100% ethanolic, 70% and 50% aqueous ethanolic) or 100% aqueous extracts were challenged with dinitrophenyl-bovine serum albumin to analyze the release of early and late-phase pro-inflammatory mediators. Results: We found that at concentrations of 5 mg/ml and above, C. nutans aqueous extract was able to suppress the levels of β-hexosaminidase and histamine while suppression was not seen in ethanolic extracts pretreated RBL-2H3 cells.Conclusion: Hence, we proposed that C. nutans aqueous extract is more active compared to the ethanolic extracts in suppressing the mediators of IgE-mast cell degranulation.
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A new method for purifying Brazilin from lignum sappan – Cytotoxic and anti-proliferative effects in cell lines and improved survival in mice bearing urinary bladder carcinoma p. 409
Xihua Yang, Lili Zhao, Xuebing Han, Lixia Chen, Yongming Yang, Jing Wang, Lei Yan, Tingting Zhang, Huanhu Zhang
DOI:10.4103/pm.pm_308_19  
Background: Urinary bladder carcinoma is characterized by high post-operative recurrence rates. Sufu'ning lotion, prepared with Lignum Sappan, can reduce the recurrence of urinary bladder carcinoma. Objectives: The present study describes a methodology for extracting and obtaining high purity brazilin from Lignum sappan and evaluates its effects on urinary bladder carcinoma, in vitro and in vivo . Materials and Methods: The brazilin was extracted from Lignum sappan, purified and identified. The purified brazilin was assessed in vitro for its effects on tumor cell viability and growth using human (T24) and mouse urinary bladder (BTT) cancer cell lines. The role of brazilin in the inhibition of the cell cycle was determined by flow cytometry. The in vivo activity of brazilin was assessed based on the survival of tumor-bearing mice. Results: Brazilin was purified of 99.10% purity and was bioactive as it significantly (P < 0.05) inhibited the growth of T24 and BTT cells. The IC50was 10.08 μg/ml for T24 cells and 8.76 μg/ml for BTT cells after 48 h of treatment. Brazilin blocked the cell cycle progression in the G2 phase. Brazilin (100, 200 and 300 mg/kg) significantly (P < 0.05) increased the survival of BTT tumor-bearing T739 mice and T24 tumor-bearing BALB/c nude mice. Conclusion: The methodology described for the extraction of brazilin from Lignum sappan yields high purity and bioactive brazilin. The extracted brazilin exerts marked inhibitory effects on urinary bladder cancer cells in vitro and in vivo .
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Antiprotease activity of indigenous medicinal plants against Pakistani Echis carinatus venom p. 416
Nazia Aslam, Tariq Javed, Sofia Khalid, Nusrat Shaheen, Syeda Fatima, Muhammad Latif, Khurram Afzal, Samina Afzal, Khizar Abbas, Muhammad Arfat Yameen, Yasser M. S A. Al-Kharaman, Muhammad Imran Amirzada, Ryan J R. McCleary, Albert A Rizvanov, Muhammad Hassham Hassan Bin Asad
DOI:10.4103/pm.pm_355_19  
Background: Snakebite is a commonly neglected tropical disease globally. According to the World Health Organization, tens of thousands of deaths have been reported due to the venomous snakes previously. Among such snakes, vipers of the genus Echis are extremely important clinically. In folk medicine, plants are commonly used for the treatment of snakebites even though snake venom antiserum is the only efficacious treatment currently known. Objectives: This study was conducted to investigate the inhibitory potential of extracts from Pakistani medicinal plants against protease enzyme abundant in Echis carinatus venom. Materials and Methods: Organic extracts from different indigenous plant species and parts were used for in vitro determination of their inhibition against protease activity in snake venom. Active methanolic extracts were further fractioned using different solvents, and these fractions were also tested for antiprotease activity. Results: Results of this study show that Calotropis procera (Wild.) R. Br., Matthiola incana (L.) R. Br., and Terminalia arjuna Wight and Arn were able to neutralize the protease enzyme by 63%, 71%, and 66%, respectively. Trichodesma indicum (L.) R. Br. showed 51% inhibition of protease activity. Conclusion: The present study indicated about C. procera (Wild.) R. Br., M. incana (L.) R. Br., and T. arjuna Wight and Arn possessed inhibitor (s) against protease enzyme present in E. carinatus venom and would be worthwhile for development as treatment against envenomation in future.
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Apoptosis induction and reactive oxygen species generation by Artemisia absinthium l. leaf extract in MCF-7 breast carcinoma cells p. 422
Long Li, Jin Wang, Shan Miao, Shanbo Ma, Xiaopeng Shi, Aidong Wen
DOI:10.4103/pm.pm_14_20  
Background: Breast cancer is the commonly occurring cancer among women in both high-resource and low-resource settings, and the primary cause of death among women globally owing to suboptimal anticancer chemotherapy. This reflects the imperative need for better management of breast cancer among women. Therefore, the current study was conducted to evaluate the anticancer properties of Artemisia absinthium L. leaf extract on Michigan Cancer Foundation-7 (MCF-7) breast cancer cells. Materials and Methods: Leaf sample of A. absinthium L. was subjected to the Soxhlet extraction method with ethanol. The extract was concentrated to prepare the crude plant extract, which was tested for anticancer properties. The anticancer activity of extract was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and against (MCF-7) cells. Flow cytometry using propidium iodide staining was used for the determination of cell cycle distribution and DCFHDA staining for the analysis of reactive oxygen species (ROS) production. Results: MTT assay revealed that the leaf extract of A. absinthium L. reduced the cell viability of MCF-7 cancer cells. The IC50of the crude extract was found to be 25 μg/mL. The results indicated that plant extract triggered the production of ROS and significantly reduced the mitochondrial membrane potential (ΔΨm). It also leads to the arrest of MCF-7 cells in sub-G1 stage of cell cycle and eventually induced apoptosis in a concentration-dependent manner as indicated by 4'-6-diamidino-2-phenylindole staining. Moreover, plant extract also reduced the colony-forming potential of MCF-7 cells in a dose-dependent manner. Conclusion: The present study demonstrated that ethanol extract of A. absinthium L. exhibited strong antiproliferative properties against breast cancer cells. Therefore, the extract can be used for the treatment for breast cancer directly or the chemical constituents may be used after the isolation.
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Anti-inflammatory effect of Bougainvillea × buttiana (Var. Orange) extract in experimental Bothrops jararaca envenomation p. 428
Rodolfo Abarca-Vargas, Lluvia Arteaga Figueroa, Ronaldo Zucatelli Mendon, Vera L Petricevich
DOI:10.4103/pm.pm_372_19  
Background: Bougainvillea x buttiana is a plant used in folk Mexican medicine to treat different inflammatory diseases. Objective: In this study, the anti-inflammatory effect of B. x buttiana orange extract (BxbO) was evaluated on edema formation, cytokine production, and lethality in mice in response to venom of Bothrops jararaca (VBj) snake. Materials and Methods: The BxbO extract was tested in vitro to determine its effect on phospholipase A2(PLA2) and in vivo for the formation of edema, the paw edema model was used, as well as the toxicity of the extract and the production of cytokines. Lethality induced by VBj, the survival percentage, was calculated. Results: BxbO extract significantly inhibited in vitro PLA2and in vivo, blocked the edema formation and cytokine production, and prevented lethality induced by VBj. The constituents of BxbO may bind to components of VBj, such as PLA2, thereby blocking the proteolytic action of the venom. In animals treated with BxbO extract injected 1 h after the venom injection, no difference was observed in the cytokine secretion. In contrast, for all mice treated with BxbO extract for 1 h before VBj administration or together with VBj, the pro-inflammatory cytokine secretion in the serum was attenuated and an exacerbated production anti-inflammatory cytokine. In the presence of the BxbO extract injected 1 h before the VBj injection or together with the VBj injection, mortality was significantly lower. Conclusion: Altogether, our results show that BxbO extract can inhibit the local and systemic activities of VBj. However, new studies are still required to identify the interaction mechanisms between bioactive compounds and cellular components.
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Formononetin induces apoptosis of PC-3 human prostate cancer cells via regulating long noncoding RNA H19 and the mitochondrial apoptosis pathway p. 435
Ruyue Wang, Kaiguo Li, Zhaodi Xie, Bailei Wang, Yan Dai, Jian Chen, Yu Ye
DOI:10.4103/pm.pm_320_19  
Background: Prostate cancer is a life-threating disease with high incidence and mortality in male. Formononetin, the main active component of some natural products, has been hypothesized as a promising anticancer agent in previous studies. Objectives: We investigated the toxic effects and potential molecular mechanism of formononetin in PC-3 prostate cancer cells to further understand the pharmacological effects of formononetin and provide more references for intensive research. Materials and Methods: PC-3 cells were incubated with different doses of formononetin for 24 h or 48 h. After that, cell viability was measured by Cell Counting Kit-8, and apoptosis was analyzed by Hoechst 33258 stains. The expression levels of tumor-related factors such as long noncoding RNA (LncRNA) H19, Bax, and Bcl-2 were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot methods. Subsequently, PC-3 cells were infected with a lentiviral vector to overexpress or knock down H19, and then, the expression of insulin-like growth factor-1 receptor (IGF-1R) mRNA was measured by RT-qPCR. Results: Formononetin significantly inhibited the viability of PC-3 cells and promoted apoptosis in a time-dose-dependent manner. We observed that the expressions of lncRNA H19 and Bcl-2 were significantly downregulated compared with the untreated group, while an opposite pattern was observed for Bax. According to the results of gene interaction experiments, IGF-1R may be a downstream target of H19 in PC-3 cells. Conclusion: Our results present evidence that formononetin induced apoptosis of PC-3 cells by regulating lncRNA H19 and the mitochondrial apoptosis pathway. Furthermore, we put forward the hypothesis that formononetin has an interference effect on the H19/IGF-1R pathway, which remains to be further confirmed.
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Cloning, prokaryotic expression, and enzyme activity of a UDP-glucose flavonoid 3-o-glycosyltransferase from mulberry (Morus alba L.) leaves p. 441
Xiaofeng Yu, Jia Liu, Jingqiong Wan, Li Zhao, Yaran Liu, Yuan Wei, Zhen Ouyang
DOI:10.4103/pm.pm_396_19  
Background: Mulberry leaves are traditional Chinese medicines, which have pharmacological activities such as anti-inflammation, anti-oxidation, antitumor, and hypoglycemic. Moreover, flavonol glycosides are one of the main functional ingredients. However, the biosynthetic pathway involved in mulberry flavonol glycosides has not been clear. UDP-glucose flavonoid 3-O -glycosyltransferase (UFGT) is a key enzyme in the biosynthesis pathway of flavonol glycosides, which can glycosylate unstable flavonols to form stable flavonol glycosides. Objectives: In this article, MaUFGT , a cDNA encoding the UFGT from mulberry leaves, was cloned, codon optimized, and expressed in Escherichia coli to study its effect in vitro . Materials and Methods: Mulberry UDP-Glucose flavonoid-3-O-glucanotransferase (MaUFGT) gene was reverse transcription-polymerase chain reaction amplified using the cDNA obtained from young leaves of mulberry, and the full-length MaUFGT gene was synthesized by codon-optimized whole-gene synthetic method. Then, the plasmid pCzn1/MaUFGT was constructed and heterologously expressed in E. coli . After denatured, renatured, and purified, the recombinant protein was used to evaluate its function in vitro by determining the final product by high-performance liquid chromatography. Results: The target protein was in the range of 45–66 KD and mainly present in the form of inclusion bodies. The obtained protein was found to transfer UDP-glucose glycosyl moieties to the 3-hydroxyl group of quercetin or kaempferol to form the corresponding products in vitro . Conclusion: The MaUFGT was preliminarily proved to be involved in flavonoid 3-O -glucoside biosynthesis.
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