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October-December 2018
Volume 14 | Issue 58
Page Nos. 471-658

Online since Wednesday, November 21, 2018

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ORIGINAL ARTICLES  

Lactobacillus plantarum attenuates oxidative stress and liver injury in rats with nonalcoholic steatohepatitis Highly accessed article p. 471
Duangporn Werawatganon, Kanjana Somanawat, Somying Tumwasorn, Naruemon Klaikeaw, Prasong Siriviriyakul
DOI:10.4103/pm.pm_279_18  
Background: Steatohepatitis is a morphological pattern of liver injury that, in non-alcoholic patients, may represent a form of chronic liver disease currently known as non-alcoholic steatohepatitis (NASH). Probiotics, Lactobacillus sp. and Bifidobacterium sp., have been proposed to prevent and treat different inflammatory conditions of the gastrointestinal tract. Objective: To examine the effect of Lactobacillus plantarum (L. plantarum) on the liver damage of non-alcoholic steatohepatitis (NASH) rats. Materials and Methods: Male Sprague-Dawley rats were randomly divided into three groups. Group 1 (control, n = 8) was fed with phosphate-buffered saline (PBS) 1 mL/rat. Group 2 (NASH, n = 8) was fed with 100% fat diet for 6 weeks. Group 3 (NASH + L. plantarum, n = 8) was fed with 100% fat diet plus L. plantarum 1.8 × 109 colony-forming unit/mL was suspended in PBS by gavage twice a day at an interval of 4 h for 6 weeks. All rats were sacrificed to collect blood and liver samples at the end of the treatment period. Results: The levels of hepatic malondialdehyde (MDA) and tumor necrosis factor alpha (TNF-α) were increased while the expression of peroxisome proliferator activated receptors gamma (PPAR-γ) was decreased significantly in the NASH group as compared with the control group. Histopathology from the NASH group showed macrovesicular steatosis, hepatocyte ballooning, and lobular inflammation. The NASH + L. plantarum group had attenuated the levels of MDA and TNF-α, enhanced PPAR-γ expression, and improved the histopathology. Conclusion: L. plantarum treatment can attenuate oxidative stress, inflammation, and improvement of histopathology in rats with NASH. Abbreviations used: NAFLD: Nonalcoholic fatty liver disease; NASH: Nonalcoholic steatohepatitis; TNF-α: Tumor necrosis factor-alpha; MDA: Malondialdehyde; PPARγ: Peroxisome proliferator-activated receptor gamma; TBARS: Thiobarbituric acid-reactive substances; ELISA: Enzyme-linked immunosorbent assay.
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Anticancer potential of Moringa oleifera flower extract in human prostate cancer PC-3 cells via induction of apoptosis and downregulation of AKT pathway Highly accessed article p. 477
Jiechang Ju, Sivapragasam Gothai, Mohadeseh Hasanpourghadi, Anmar A Nasser, Ibrahim Abdel Aziz Ibrahim, Naiyer Shahzad, Ashok Kumar Pandurangan, Katyakyini Muniandy, S Suresh Kumar, Palanisamy Arulselvan
DOI:10.4103/pm.pm_516_17  
Background: Moringa oleifera (MO), or the horseradish tree, is a pantropical species that is known by such regional names as benzolive, drumstick tree, kelor, marango, mlonge, mulangay, nébéday, saijhan, and sajna. Over the past two decades, many reports have appeared in mainstream scientific journals describing its anticancer properties of MO. While much of this recent enthusiasm indeed appears to be justified, it is critical to evaluate therapeutic activity of MO on prostate cancer to separate rigorous scientific evidence from anecdote. MO contained active polyphenols such as ellagic acid, gallate, methyl gallate, catechol, kaempferol quercetin, and their derivatives. Objective: It is the purpose of this series of brief reviews to critically assess the efficacy of MO flower extract as an antiprostate cancer agent. Materials and Methods: The cell viability of the extract was determined using MTT in different time point and cell cycle progression studies was analyzed by flow cytometry. Various markers of apoptosis were evaluated by immunoblotting. Results: We observed that prostate cancer cells treated with MO flower extract caused 50% inhibition at a dose of 22.61 μg/mL and 6.25 μg/mL in PC-3 cells at 24 and 48 h, respectively. MO flower extract induced the accumulation of G1 phase cell cycle arrest and apoptosis by annexin V staining. Further, immunoblot detection of PARP cleavage leads to increase the protein expression of caspase-3 activity and Bax indicates induction of apoptosis. Conclusion: Together, the results suggest for the first time that administration of MO flower extract inhibits prostate cancer progression in PC-3 cells by interfering AKT pathway. Abbreviation used: MO: Moringa oleifera; PARP: Poly (ADP-ribose) polymerase; BAD: Bcl-2-associated death promoter; FACS: Fluorescence-activated cell sorting.
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Antidiabetic potential of the total flavone glycoside from okra fruit in type 2 diabetic rats p. 482
Zhi-Yong Huang, Shan-Shan Jia, An Jia, Jia-Wei Huang, Ke Yuan
DOI:10.4103/pm.pm_517_17  
Background: Okra is a commonly consumed healthy vegetable in many countries due to its wide range of pharmacological effects. The present study was designed to investigate in vivo antidiabetic potential of the total flavone glycoside from okra fruit (TFGO) on type 2 diabetic rats. Materials and Methods: TFGO was obtained by column chromatography on 70% ethanol extract of okra fruit, and high-performance liquid chromatography analysis was carried out on its' three main flavone glycosides. In rats, 2000 mg/kg of TFGO did not show any toxicity on the acute toxicity test. Type 2 diabetic rats were induced by streptozotocin (35 mg/kg; i. p.) after fed with high-fat emulsion for 4 weeks. Rats were divided into six groups: normal control group, diabetic control group, metformin control group (100 mg/kg), and three TFGO groups (100, 200, and 400 mg/kg). The antidiabetic potential of TFGO was measured by comparing body weight, food intake, fasting blood glucose (FBG), oral glucose tolerance test (OGTT), superoxide dismutase (SOD), malonaldehyde, triglycerides (TG), total cholesterol (TC), organ index, and histological section of kidney tissue in rats. Results: The results showed that the administration of TFGO significantly (P < 0.01) decreased the levels of FBG, TG, TC, liver index and increased (P < 0.01) OGTT, SOD levels in diabetic rats compared to diabetic control group rats. Moreover, the lesion of diabetic kidney tissue was recovered obviously. Conclusions: Our findings confirmed that TFGO has significant antidiabetic potential in rats. This study provides a reference point for further investigation and development of TFGO. Abbreviations used: TFGO: Total flavone glycoside from okra fruit; OGTT: Oral glucose tolerance test; SOD: Superoxide dismutase; TG: Triglycerides; TC: Total cholesterol; MDA: Malondialdehyde; FBG: Fasting blood glucose; STZ: Streptozotocin.
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Anti-neuro-inflammatory effects of the bioactive compound capsaicin through the NF-κB signaling pathway in LPS-stimulated BV2 microglial cells p. 489
Qin Zheng, Wenjun Sun, Miao Qu
DOI:10.4103/pm.pm_73_18  
Background: Inflammation in the central nervous system, resulting from a loss of control involving a network of neuronal cells, is foremost contributors to the instigation and advancement of major neurodegenerative diseases. Therefore, therapeutic strategies should restore back to a well-controlled and finely-tuned balance of immune reactions, and protect neurons from inflammatory damage. Objective: The objective of this study is to evaluate the anti-neuroinflammatory potential of Capsaicin in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Materials and Methods: In this present study, we selected Capsaicin and investigated through cell-based-assay systems through the various cellular techniques enzyme-linked immunosorbent, immunoblot and immunofluorescence assays to identify anti-inflammatory effects. Results: We found that capsaicin exhibited highly anti-inflammatory and neuroprotective effects in cell culture experiments, reduced nitric oxide, tumor necrosis factor-α, interleukin-1 β, and interleukin expression from activated BV-2 microglia cells dose-dependently. On the intracellular level, capsaicin inhibited IκB-phosphorylation and subsequently nuclear Factor-κB (NF-κB)-translocation in microglia cells. Further, capsaicin blocked the protein expressions of inducible nitric oxide synthase and cyclooxygenase-2. Further, capsaicin inhibits the increased production of pro-inflammatory responses in LPS-stimulated BV-2 cells by suppressing NF-κB activation. Conclusion: The significant inhibition of neuroinflammatory responses in stimulated microglial cells together indicate that capsaicin is a potential therapeutic agent and could possibly be used in the development of novel drug for the prevention and treatment of neuroinflammatory diseases. Abbreviations used: CNS: Central Nervous System; iNOS: Inducible nitric oxide synthase; COX-2: Cyclooxygenase; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; LPS: Lipopolysaccharide; PGE2: Prostaglandin E2; NO: Nitric oxide; IL-6: Interleukin; IL-1 β-interleukin-1 β; TNF-α: Tumor necrosis factor-α
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Effect of phytohormones on rapid In vitro propagation of dendrobium thyrsiflorum Rchb.f.: An endangered medicinal orchid p. 495
Leimapokpam Tikendra, Thoungamba Amom, Potshangbam Nongdam
DOI:10.4103/pm.pm_155_18  
Background: Dendrobium thyrsiflorum is highly vulnerable to extinction in its natural habitats due to overexploitation for the medicinal and horticultural purposes. The poor seed germination in a natural condition and slow plant production through vegetative approach make in vitro micropropagation techniques an ideal choice for effective plant propagation. There are very limited reports on the in vitro culture of this orchid. Objective: The objective of the study is to establish efficient in vitro regeneration protocols for rapid mass propagation of D. thyrsiflorum. Materials and Methods: Five-month-old undehisced capsule was surface sterilized with 0.4% mercuric chloride. To study the influence of growth regulators on in vitro development of shoots and roots, the seeds obtained from the capsule were inoculated on freshly prepared Mitra medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin (KN), indole -3-butyric acid (IBA), indole-3-acetic acid (IAA), and 1-naphthyl acetic acid (NAA). Results: The KN either singly or with IAA produced more effective shooting than BAP at similar concentration and combination. Maximum shoot production (3.83 ± 0.48) was achieved in medium fortified with 1.0 mg/L KN and 2.5 mg/L IAA. IAA was the most influential among auxins tested in promoting rooting and shooting. Prominent shoot development (3.04 ± 0.73) and maximum root formation (6.52 ± 0.37) were noticed in medium supplemented with 2.0 mg/L IAA. The regenerated plantlets were subsequently hardened with 91% survival rate in greenhouse and field condition. Conclusion: The present investigation revealed the possibility of developing effective and reproducible in vitro regeneration protocols for rapid propagation of D. thyrsiflorum. The established in vitro protocols can be employed for fast regeneration of the orchid for useful phytochemical extraction without depending on the already depleted natural population. Abbreviations used: BAP: 6-benzylaminopurine; KN: Kinetin; IBA: Indole-3-butyric acid; IAA: Indole-3-acetic acid; NAA: 1-naphthyl acetic acid; MT: Mitra medium.
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Berberine protects against chronic social defeat stress-induced depressive-like behaviors with upregulation of neuronal PAS domain protein 4/brain-derived neurotrophic factor signaling pathway p. 501
Zhifang Deng, Yan Peng, Wenqi Gao
DOI:10.4103/pm.pm_268_18  
Objective: Depression imposes a huge health problem in the world. Recent studies report that berberine has neuroprotective effects. This study was investigated to evaluate the effects of berberine on depression and the potential underlying mechanisms. Methods: This experimental study included 48 mice, 8 CD1 mice and 40 C57 mice. Experiments were performed in the Laboratory Animal Center of China Three Gorges University, between May 2016 and February 2017. Chronic social defeat stress (CSDS) was used to build the depressive-like behaviors model. Three behavior tests, quantitative polymerase chain reaction, and Western blot were used to assay the changes in vivo. Results: We found that, in mice model induced by CSDS, berberine treatment increased mobility times in forced swim test and tail suspension test and reduced corticosterone level in serum. Furthermore, berberine increased traveled distance, frequencies in enter the central square, and duration time on the center square in the open field test as well. Further experiments showed that berberine treatment upregulated neuronal PAS domain protein 4 (NPAS4) and brain-derived neurotrophic factor (BDNF) protein/mRNA expressions in hippocampus tissue of CSDS mice. Conclusion: The present results suggested that berberine possessed properties in antidepressive-like behaviors, which might be partly attributed to upregulation of NPAS4 and BDNF expressions in hippocampus. Abbreviations used: CSDS: Chronic social defeat stress; BDNF: Brain-derived neurotrophic factor; NPAS4: Neuronal PAS domain protein 4; FST: Force swim test; TST: Tail suspension test; OFT: Open field test.
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Altered cytochrome P450 profiles by Plumbago indica Linn. and plumbagin after oral administration in mice p. 507
Nadta Sukkasem, Waranya Chatuphonprasert, Kanokwan Jarukamjorn
DOI:10.4103/pm.pm_88_18  
Background: Plumbago indica Linn. and its active constituent, plumbagin, are conventionally used in Thai alternative medicines, but information regarding their effects on cytochrome P450 (CYP450) enzymes is limited. Objective: To establish the effects of P. indica Linn. and plumbagin on CYP450 profiles. Materials and Methods: Adult male mice were orally administered P. indica extract (20, 200, and 1000 mg/kg/day) or plumbagin (1, 5, and 15 mg/kg/day) for 14 days. The levels of hepatic CYP450 mRNA and protein were assessed using reverse transcription/real-time polymerase chain reaction and immunoblotting, respectively, and specific enzyme reactions were performed to determine the enzyme activities. Results: Expression of Cyp1a2 was induced by both P. indica and plumbagin, while P. indica, but not plumbagin, slightly suppressed Cyp2c29 expression. Expression of Cyp2d9 was suppressed by both P. indica and plumbagin. Expression of Cyp2e1 was unchanged, but P. indica at the lowest dose increased Cyp2e1 activity. P. indica and plumbagin dose-dependently suppressed the expression of Cyp3a11/13 and its activity. Conclusions: Modulation of CYP450 profiles by P. indica and plumbagin is a concern as there are risks of drug–herb interaction. Abbreviations used: CYP450: Cytochrome P450; BSA: Bovine serum albumin; GAPDH; Glyceraldehyde 3-phosphate dehydrogenase.
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Polyisoprenoids from Avicennia marina and Avicennia lanata inhibit WiDr cells proliferation p. 513
Didi Nurhadi Illian, Mohammad Basyuni, Ridha Wati, Poppy Anjelisa Zaitun Hasibuan
DOI:10.4103/pm.pm_201_18  
Objectives: The current investigation was conducted to examine the anticancer effect of polyisoprenoids from Avicennia marina and Avicennia lanata leaves in WiDr cells. Selectivity index (SI), cell cycle inhibition, and apoptosis activity were evaluated. Materials and Methods: The anticancer activity of polyisoprenoids from A. marina and A. lanata leaves was determined by observing the activity of these compounds toward WiDr cells using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay. The SI was determined from the IC50 of the polyisoprenoid extract in normal cells (Vero) versus cancer cells (WiDr). Inhibited cell cycle and increased apoptosis were analyzed by flow cytometry. Results: Polyisoprenoid extract from A. marina and A. lanata leaves exhibited anticancer activity against WiDr cells with an IC50 of 154.987 μg/mL and 305.928 μg/mL, respectively. The polyisoprenoid extract from A. marina leaves had an SI value of 5.195 (>3) for categorization as exceptionally selective. Cell cycle analysis revealed that the inhibition occurred in the G0–G1 phase and apoptosis occurred in the early-apoptosis development. Conclusion: Polyisoprenoids from A. marina and A. lanata leaves can be used as anticancer agents against WiDr colon cancer cells. The mechanisms that underlie anticancer activity of the extract were due to by inhibiting of cell cycle and inducing of apoptosis. Abbreviations used: DMSO: Dimethylsulfoxide, MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide, PBS: Phosphate buffer saline, SDS: Sodium dodecyl sulfate.
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Molecular cloning and expression analysis of the circadian clock for Patchoulol Synthase gene in Pogostemon Cablin (Blanco) benth p. 519
Xindan Liu, Menghua Wu, Ying Zhang, Hui Cao
DOI:10.4103/pm.pm_250_18  
Background: The circadian clock for patchoulol synthase (PTS) gene in Pogostemon cablin remains largely unexplored. Objective: The objective of this study is to clone and to investigate the expression of PTS gene from the leaves of P. cablin throughout a day. Materials and Methods: The full-length PTS cDNA was isolated by a combination of reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends in P. cablin. Gene expression profiles across leaves at different time points of P. cablin were evaluated using quantitative RT-PCR. Results: The PTS clone contained a 1659-bp open reading frame coding for 552 amino acids, a 61-bp 5′-untranslated end, and a 219-bp 3′-untranslated sequence. The deduced amino acid sequence was 47%–63% identical with the sequence of other known sesquiterpene cyclases from angiosperms. It was observed that the oscillations of PTS transcript persisted in P. cablin during the daily cycle, with peak expression occurring at 1:00 h, 5:00 h, 7:00 h, 9:00 h, 16:00 h, 19:00 h and 22:00 h. In general, PTS had lower oscillation frequency and transcription abundance at daytime than at night, corresponding with the higher temperature, the lower humidity, and the presence of an ultraviolet index. Conclusion: The PTS gene identified in this study is under clock control, which in turn may provide insight into evolutionary progresses in the green lineage. Abbreviations used: PTS: Patchoulol synthase; EC: Evening complex; FDP: Farnesyl diphosphate; RACE: Rapid amplification of cDNA ends; CMA: China Meteorological Administration.
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Secondary metabolites from Sibiraea angustata p. 525
Yang-Zhirong Hu, Wan-Chang Zhang, Shuo Zhang, Guang-Bo Xie
DOI:10.4103/pm.pm_390_17  
Background: Sibiraea angustata was used as folk medicine in Tibetan-inhabited area of Hengduan Mountains, China. Objective: The secondary metabolites from the leaves and twigs of S. angustata were studied. Materials and Methods: The compounds were isolated and purified using silica gel and Sephadex LH-20 column chromatography. Their structures were identified by spectra analysis. Results: Eight known compounds, including 2 monoterpenoids and 3 triterpenoids, were isolated and identified from S. angustata. Conclusions: Four compounds were isolated from this plant for the first time, and the 13C-nuclear magnetic resonance spectra data and absolute configuration of dihydroneroloxide were given for the first time. Abbreviations used: NMR: Nuclear magnetic resonance; TMS: Tetramethylsilane; ECD: Electronic circular dichroism; CC: Column chromatography; TLC: Thin-layer chromatography; DFT: Density functional theory; TDDFT: Time-dependent density functional theory; PCM: Polarizable continuum model.
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Antioxidant effect of royal jelly on immune status of hyperglycemic rats p. 528
Abdullah G Al-Kushi, Eslam A Header, Naser A ElSawy, Reham A Moustafa, Nehal A A. Alfky
DOI:10.4103/pm.pm_87_18  
Context: Diabetes mellitus and its increasing effect on endocrine functionality have been widely observed. Aims: The present study aims at investigating the histopathological changes of splenic cells in diabetic rats. Subjects and Methods: The experiment was conducted on adult male albino rats. They were categorized into four groups: Group I (negative control; fed with standard basal diet [SBD]), Group II (positive control; fed with SBD), Group III (fed on SBD + dose of 50 mg/kg of body weight royal jelly [RJ]) and Group IV were given orally RJ 100 mg/kg of b.wt. The food intake ratio and body weight gain were monitored on a weekly basis. The blood sample and organ tissue samples were collected at the end of feeding and biochemical and immunological analysis was performed for determining the blood glucose level and immune status of the investigated rats. Superoxide dismutase activity was also determined. Results: The results showed increased antioxidant enzymes and lowered glucose levels in diabetic rats fed with RJ. Conclusion: Histopathology tissues indicated moderate to extreme cellular transformations upon treated rats. The histopathological variations in treated rat cells were found to be evident. For this reason, there is likelihood that RJ may have an effect on splenic tissue repair in diabetic rats. Abbreviations used: RJ: Royal jelly; SBD: Standard basal diet; SPSS: Statistical package for the social science; STZ: Streptozotocin; BWG: Body weight gain; SD: Standard deviation.
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Antioxidant and anti-inflammatory effects of a methanol extract from the marine sponge Hyrtios erectus p. 534
Ramachandran Muthiyan, Nilkamal Mahanta, Balwin Nambikkairaj, Titus Immanuel, Arun Kumar De
DOI:10.4103/pm.pm_133_17  
Background: The marine environment, due to its phenomenal diversity, is a rich natural source of many biologically active compounds. Objective: Marine sponge Hyrtios erectus, collected from North Bay of South Andaman Sea, was screened for potential antioxidant and anti-inflammatory activities. Materials and Methods: The antioxidant activities of the methanol (MeOH) extract of the sponge at different concentrations (0–100 μg/mL) were determined by measuring the free radical-scavenging activities. The anti-inflammatory activities of the extract were determined by measuring the inhibitory effect of the extract on albumin denaturation and inducible nitric oxide (NO) production. Quantitative real-time polymerase chain reaction was used to investigate the effect of the sponge extract on the expression of eight proinflammatory cytokine genes. Results: Our results suggested that the MeOH extract of the sponge exhibited antioxidant activity against 2,2-diphenyl-1-picrylhydrazyl-free radicals, superoxide anions, and hydroxyl radicals. More than 50% inhibition (half inhibitory concentration) was recorded with concentration of 50 μg/mL of the sponge extract. Extract of the sponge at a concentration of 25 μg/mL inhibited NO production by a macrophage cell line in vitro by 91.22% ± 5.78%. The sponge extract induced downregulation of eight proinflammatory cytokine genes in breast cancer Michigan Cancer Foundation-7 cell line. Conclusion: The secondary metabolites present in the MeOH extract of the sponge showed the potential antioxidant and anti-inflammatory activities. Further studies are required to identify the bioactive compounds. Abbreviations used: DPPH: 2,2-diphenyl-1-picrylhydrazyl; DMEM: Dulbecco's modified eagle's medium; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EDTA: Ethylenediaminetetraacetic acid; NBT: Nitroblue tetrazolium; PMS: Phenazine methosulfate; DMSO: Dimethyl sulfoxide.
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Antioxidant and anticomplement compounds isolated from Nitraria sibirica fruit by high-speed counter-current chromatography p. 541
Qian Bo Song, Hua Ding Zhao, Ze Long Fu, Dao Feng Chen, Yan Lu
DOI:10.4103/pm.pm_207_18  
Background: Nitraria sibirica fruit is a kind of folk medicine widely used in Northwest China. It contains diverse kinds of bioactive constituents such as flavonoids, alkaloids, and phenols. Objectives: The research was performed to develop an efficient separation method for the bioactive constituents of N. sibirica fruit and evaluate the isolated compounds bioactivities. Materials and Methods: An optimized method of high-speed counter-current chromatography (HSCCC) combined with preparative high-performance liquid chromatography (HPLC) was established and applied to prepare the bioactive compounds from the ethyl acetate extract of N. sibirica fruit. The antioxidant and anticomplement activities of the isolated fractions and compounds were evaluated in vitro. Results: Ethyl acetate extract was the predominant antioxidant and anticomplement fraction of N. sibirica fruit, which yielded 18 compounds including six phenols, three flavonoids, and five alkaloids. Seven compounds (2, 4, 5, 9, 10, 12, and 17) were first isolated from genus Nitraria. Furthermore, under the optimized chromatography conditions, one anticomplement alkaloid (1) could be achieved directly by one-step HSCCC with high purity (92.34%), the other seventeen compounds were further purified with preparative HPLC. Ten compounds (3, 6, 7, 8, 9, 10, 11, 12, 13, and 14) exhibited significant antioxidant activities. Five compounds (1, 2, 7, 10, and 16) showed anticomplement activities and targeted different components in the complement system. Conclusion: The study provided an efficient method to prepare antioxidant and anticomplement compounds and the activities of the compounds were deeply investigated. Abbreviations used: HSCCC: High-speed counter-current chromatography; DPPH: 1,1-diphenyl-2-picrylhydrazyl; FRAP: Ferric reducing antioxidant power; ABTS: 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid); BBS: Barbiturate buffer solution; ASEA: Anti-sheep erythrocyte antibody; NHS: Normal human serum; 2% SRBC: 2% sheep red blood cells.
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Hepatic immune response to environmental carcinogens p. 548
Kawa Amin, Kosar Muhammad Ali, Amanj Saeed, Heshu Sulaiman Rahman, Jonas Bystrom
DOI:10.4103/pm.pm_242_18  
Aim: Environmental carcinogenic substances contribute to increasing incidence of hepatocellular carcinoma (HCC). We employed a sensitive method for the detection of DNA damage combined with analysis of the immune response to gain better knowledge how environmental carcinogens mediate pathology. Materials and Methods: Rat hepatocytes were isolated and stimulated with carcinogenic substances for the assessment of DNA damage. The mycotoxin aflatoxin B1(AFB1), two heterocyclic amines from the cooking of meat amino-3-methylimidazo[4,5-f] quinoline (IQ) and 3-amino-1-methyl-5H-pyrido-(4,3-b)-indole (TRP-P-2), and protein extract from the fungus Lactarius necator were assayed. Unscheduled DNA synthesis in hepatocytes was measured by the incorporation of radioactive thymidine during DNA repair. Stimulation of hepatocyte/immune cell preparation with the substances and measurement of IFNγ release at different time points determined their ability to induce an inflammatory response. Results: DNA repair in the hepatocytes was induced in response to 10−7 M AFB1 and 10−9 M IQ. TRP-P-2 did not induce DNA repair; however, at 10−4 M, the fungus extract did this. Furthermore, liver-resident immune cells responded with differential production of IFNγ over time in response to stimulation by all the carcinogens, with AFB1 being the most potent. TRP-P-2 showed the most significant reduction in IFNγ response over time. Conclusion: DNA damage in hepatocytes induced by environmental substances was detected at low molecular concentrations. The system did provide novel evidence for hepatic carcinogenicity by the fungus L. necator. Analysis of the response by liver-resident immune cells to the substances suggested that highly mutagenic substances induce prolonged inflammatory response. Abbreviations used: HCC: Hepatocellular carcinoma, AFB1: Aflatoxin B1, IQ: Amino-3-methylimidazo[4,5-f] quinoline, TRP-P-2: 3-amino-1-methyl-5H-pyrido-(4,3-b)-indole, UDS: Unscheduled DNA synthesis, EGTA: Ethylene glycol-bis-(2-aminoethyl ether) N-N-N'-N'-tetraacetic acid, WME: Williams' medium E, 2-AAF: 2-Acetylaminofluorene, HBSS: Hanks' balanced salt solution, CYPs: Cytochrome p450 enzymes.
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Phytochemical standardization of panchavalkala: An ayurvedic formulation and evaluation of its anticancer activity in cervical cancer cell lines p. 554
Shama Aphale, Savita Pandita, Prerna Raina, JN Mishra, Ruchika Kaul-Ghanekar
DOI:10.4103/pm.pm_252_18  
Background: Cervical cancer is the most common malignant disease affecting women worldwide. The currently available therapies for cancer, even though effective, affect the patient's health severely due to the associated side effects. Thus, nowadays, complementary/alternative medicines are being extensively researched upon for their use as an adjunct therapy. Panchavalkala, an Ayurvedic formulation, is traditionally being used as a douche in leukorrhea and other gynecological diseases. Objective: The objective of the study was to phytochemically standardize aqueous extract of Panchavalkala (PVaq) and evaluate its anticancer activity against human cervical cancer cell lines. Materials and Methods: The phytochemical characterization of PVaq was done by liquid chromatography–mass spectrometry (LCMS) technique. The effect of PVaq on the viability of SiHa and HeLa cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide dye assay. The effect of the extract on growth kinetics was evaluated by trypan blue dye exclusion method and soft agar assay. Results: LCMS analysis showed presence of 77 compounds, of which 15 major compounds included proanthocyanidin B1, chlorogenic acid, caffeic acid, epicatechin, leucopelargonidin 3-O-alpha-L-rhamno-beta-D-glucopyranoside, leucocyanidin, naringenin-7-o-glucoside, mesoinositol, catechin, vogelin E, mesoinositol, behenic acid, bergenin, acacetin, and gallic acid. PVaq significantly (P < 0.001) reduced the viability of SiHa and HeLa cells with an IC50 of 125.8 and 96.0 μg/ml, respectively. It also reduced the growth of cervical cancer cells in a dose- and time-dependent manner. Conclusion: This preliminary data suggests that PVaq exhibits potential anticancer activity and warrants further studies for detailed elucidation of its mechanism of action. Abbreviations used: PVaq: Aqueous extract of Panchavalkala; LCMS: Liquid chromatography–mass spectrometry; HPV: Human papillomavirus; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
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Toxicological studies of daphnetin p. 561
Kuang Nanzhen, Wang Jieying, Zou Wenwei, Zeng Xiaoping, Fu Yingyuan
DOI:10.4103/pm.pm_523_17  
Background: Numerous studies have demonstrated that daphnetin (DAP) has anti-inflammatory, antioxidant, antibacterial, antitumor, anti-arthritic, and other extensive pharmacological effects. However, its genotoxicity has rarely been examined. Materials and Methods: The safety of DAP was evaluated by an acute toxicity test using a Salmonella typhimurium/mammalian microsomal enzyme assay (Ames test), bone marrow micronucleus test, acute skin allergy test, and local mucosal irritation test. Results: Mice received a dose of 100 mg/kg body weight through lavage, which is more than 175 times the whole-body effective dose and were continuously observed for 14 d; mice showed no signs of poisoning. In the Ames test, each dose of DAP resulted in numbers of revertant colonies that were <2 two times the number of mutation colonies in the negative control group, indicating a negative result. The micronucleus rate of each DAP dose group was not significantly different from that of the negative control group (P > 0.05) but was significantly different from that of the cyclophosphamide (CTX)-positive control group (P < 0.05). Rabbit skin showed no stimulation reaction to DAP or 10% dimethyl sulfoxide (DMSO), including no erythema, edema, or sensitization phenomena. Rabbits were generally in good condition after dosing the oral mucosa with DAP, and no oral mucosal erythema, erosion, ulcers, or other irritation reactions were observed. Conclusions: Within the range of tested doses, DAP did not cause any observed toxic effects, mutagenic effects, sensitization, or stimulation. Abbreviations used: DAP: Daphnetin, CTX: Cyclophosphamide, DMSO: Dimethyl sulfoxide, CIA: Collagen-induced arthritis, PCB: Polychlorinated biphenyl, NS: Normal saline, PCE: Polychromatic erythrocyte, NCE: Normochromatic erythrocyte; LD50: The median lethal dose.
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Traditional chinese medicine ingredients Rosa damascena and Poria cocos promote phagocytosis and a dendritic cell phenotype in THP-1 cells p. 567
Samantha J Roloff, Jeffrey D Scholten, Jennifer Chuang, Chun Hu, David J Fast
DOI:10.4103/pm.pm_564_17  
Background: Rosa damascena and Poria cocos are ingredients commonly used in Traditional Chinese Medicine. R. damascena is used to promote blood circulation as well as liver and stomach function, while P. cocos is used to eliminate dampness and enhance spleen function. Objective: The objective of the study is to investigate possible mechanisms by which R. damascena and P. cocos may promote immune function. Materials and Methods: Phagocytosis and dendritic cell (DC) surface marker expression assays were used to evaluate the effect of R. damascena and P. cocos extracts on human THP-1 monocytic leukemia cell biology. Results: R. damascena and P. cocos extracts both enhanced phagocytosis of latex beads by THP-1 cells, and when combined, phagocytosis was enhanced to a level greater than what might be expected by adding the individual phagocytosis responses together. In addition, both extracts enhanced maturation of THP-1 cells into a DC phenotype as measured by increased surface expression of the costimulatory molecules CD14, CD40, CD80, and CD86. Conclusion: These results suggest that Rosa damascena and P. cocos may promote monocyte phagocytosis and then stimulate differentiation of the cells into DCs thereby bridging innate and adaptive immune responses. Abbreviations used: AKT: Protein kinase B; AP-1: Activator protein-1; Bcl2: B cell lymphoma-2; CD: Cluster of differentiation; COX-2: Cyclooxygenase-2; DC: Dendritic cell; EGFR: Epidermal growth factor receptor; FOXO1: Forkhead box protein-1; GM-CSF: Granulocyte/macrophage colony stimulating factor; HLA: Human leukocyte antigen; HPLC: High-performance liquid chromatography; IL-1β: Interleukin-1β; IL-4: Interleukin-4; M1: Classically activated macrophage; M2: Alternatively activated macrophage; MAPK: Mitogen-activated protein kinase; NFκB: Nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3: NLR Family Pyrin Domain Containing 3; ϕ: Phagocytic index; p53: Tumor protein p53; PARP: Poly (ADP-ribose) polymerase; PMA: Phorbol 12-myristate 13-acetate; PRR: Pattern recognition receptor; STAT: Signal transducer and activator of transcription SD: Standard deviation; Syk: Spleen tyrosine kinase; TCM: Traditional Chinese Medicine.
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Polyherbal formulation containing antioxidants may serve as a prophylactic measure to diabetic cataract: Preclinical investigations in rat model p. 572
Nilesh M Mahajan, Bhushan B Lokhande, Raju R Thenge, Purushottam S Gangane, Nitin G Dumore
DOI:10.4103/pm.pm_622_17  
Background: Cataract is a major cause of visual impairment in diabetic patients. Due to increasing numbers of type 1 and type 2 diabetics worldwide, the incidence of diabetic cataracts steadily rises. Cataract surgery is the best possible cure for patients suffering from this ailment. However, the elucidation of pathomechanisms to delay or prevent the development of cataract in diabetic patients remains a challenge. Objective: The aim of the present study was to develop a polyherbal eyedrops containing potent antioxidant herbal extract and study the effectiveness as prophylactic treatment against galactose-induced diabetic cataract. Materials and Methods: Formulations were prepared by using extracts of Ginkgo biloba leaves, beet root (Beta vulgaris), and amla (Emblica officinalis) fruits. The viscosity enhancers were used to increase the retention time. Formulations (F1–F5) were prepared by using carboxy methyl cellulose and poly ethylene glycol-400 as thickening agents and propylene glycol as a solubilizer. Preliminary evaluation showed that formulations have passed clarity, sterility, and eye irritancy tests. Viscosity and pH of formulation were within the normal range. Diabetic cataract was induced in Wistar rats by 10% galactose drink (for 30 days) and anticataract activity was evaluated. Formulation was installed in eye as a prophylactic treatment from day 1 of galactose drinking and continued for 30 days. Results: Slit-lamp photography of eyes of rats showed clear lens of rats without any trace of opacity. On the other hand, galactose-treated rats developed dense nuclear opacity in lens as an indication of diabetic cataract. Rats which received prophylactic treatment showed less percent opacity as compared to that of cataract control group and decreased vacuoles. Conclusion: We may conclude that polyherbal formulation containing extracts of Ginkgo biloba leaves, beet root (Beta Vulgaris), and amla (Emblica officinalis) fruits may prevent the development of cataract in diabetic patients. Abbreviations used: ARI: Aldose reductase inhibitors; GPx: Glutathione peroxidase; GR: Glutathione reductase; DTNB: Dithio-bis-nitrobenzoic acid; GSH: Thiol.
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Anti-inflammatory and anti-melanogenic effects of major leaf components of Alpinia zerumbet var. excelsa p. 578
Md Shahinozzaman, Takahiro Ishii, Shinichi Gima, Binh Cao Quan Nguyen, Md Amzad Hossain, Shinkichi Tawata
DOI:10.4103/pm.pm_136_18  
Background: The leaves of Alpinia zerumbet var. excelsa (alpinia) are used to prepare traditional food items and folk medicines. Objective: This study was designed with a view to explore the anti-inflammatory and anti-melanogenic potentials of major components of alpinia leaves. Materials and Methods: Anti-inflammatory effects of leaf-derived compounds were primarily assessed with protein denaturation and proteinase assay. Their inhibitory effects on nitrite accumulation and prostaglandin E2 (PGE2) production in lipopolysaccharide-induced RAW 264.7 cells were also evaluated. For anti-melanogenic assays, all the compounds were tested on α-melanocyte-stimulating hormone-stimulated B16F10 cells and on mushroom tyrosinase in vitro. Their cytotoxicity was evaluated using fibroblast cell line 3T3 L-1 and brine shrimps. Results: Five compounds, 5,6-dehydrokawain, dihydro-5,6-dehydrokawain, (E)-5-methoxy-8-(4-methoxy-2-oxo-2H-pyran-6-yl)-7-phenyl-1-styryl-2-oxabicyclo[4.2.0]oct-4-en-3-one (AS-2), kaempferol 3-O-β-D-glucuronide (KOG), and quercetin 3-O-β-D-glucuronide (QOG) were purified from the leaves. Of which, AS-2 and QOG were purified for the first time. All compounds significantly inhibited albumin denaturation and proteinase activity. AS-2, KOG, and QOG remarkably inhibited nitric oxide formation with IC50 values of 8.2, 13.3, and 12.6 μM, respectively, in RAW 264.7 cells. They also inhibited PGE2 production with IC50 values 19.8-23.7 μM. They showed anti-melanogenic effects reducing tyrosinase activity (IC50 values 29.6-112.5 μM) and melanin formation (IC50 values 30.8–164.4 μM) in B16F10 cells, and inhibiting mushroom tyrosinase (IC50 values 61.5-456.4 μg/ml). Conclusion: Taken together, major components of alpinia leaf could be utilized as a potent herbal drug and food supplement with therapeutic prospects against inflammatory disorders and hyperpigmentation. Abbreviations used: HPLC: High performance liquid chromatography; DK: 5,6-dehydrokawain; DDK: Dihydro-5,6-dehydrokawain; AS-2: (E)-5-methoxy-8-(4-methoxy-2-oxo-2H-pyran-6-yl)-7-phenyl-1-styryl-2-oxabicyclo[4.2.0]oct-4-en-3-one; KOG: Kaempferol 3-O-β-D-glucuronide; QOG: Quercetin 3-O-β-D-glucuronide; PGE2: Prostaglandin E2; PAK1: RAC/CDC42-activated kinase 1; UV: Ultra violet; α-MSH: α-Melanocyte-stimulating hormone; COX-2: Cyclooxygenase-2; iNOS: Inducible Nitric Oxide Synthase; PDGF: Platelet Derived Growth Factor; MITF: Microphthalmia-Associated Transcription Factor; TRP: Tyrosinase Related Protein; ATCC: American Type Culture Collection; DMEM: Dulbecco's Modified Eagle Medium; FBS: Fetal bovine serum; CS: Newborn calf serum; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; DMSO: Dimethyl sulfoxide; NO: Nitric oxide; LPS: Lipopolysaccharide; DMRT: Duncan's multiple range test; SPSS: Statistical Package for the Social Sciences.
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Elemental profiling of smokeless tobacco samples using inductively coupled plasma-mass spectrometry, their chemometric analysis and assessment of health hazards p. 587
Hassan A Alhazmi, Waquar Ahsan, Ibraheem M Attafi, Asaad Khalid, Siddig I Abdelwahab, Mohammad Al Bratty, Shahnaz Sultana
DOI:10.4103/pm.pm_262_18  
Background: Smokeless tobacco (ST), locally called shammah, is a form of tobacco that is widely used in Middle Eastern countries, including Saudi Arabia. Objective: A total of 21 ST samples were collected from the southern province of Jazan for elemental analysis. Materials and Methods: These samples were analyzed by inductively coupled plasma mass spectrometry to determine their element concentrations. Chemometric multivariate analysis such as hierarchical cluster analysis, Pearson correlation analysis, and principal component analysis were performed for better understanding and interpretation of the data. Concentrations obtained were used to determine the users' daily and weekly intake of elements and were compared with the acceptable daily intake and provisional tolerable weekly intake. Results: Metal ions present in maximum concentrations were strontium (11608.71 μg/kg) and manganese (3543.10 μg/kg), whereas those with minimum concentrations were silver (53.90 μg/kg) and chromium (62.33 μg/kg). Conclusion: Although the concentrations of all the elements fell under the safe limit, the concentrations of many toxic elements were significantly high and resulted in various health hazards on the intake of these elements with other sources. Abbreviations used: ST: Smokeless tobacco; EDI: Estimated daily intake; ADI: Acceptable daily intake; PTWI: Provisional tolerable weekly intake; HQ: Hazard Quotient; RfD = Oral reference dose; ICH: International Council for Harmonisation; ICP-MS: Inductively coupled plasma-mass spectroscopy; AAS: Atomic absorption spectroscopy; CTA-OTL-1: Oriental Tobacco Leaves; % R. S. D.: % Relative standard deviation; ppb: Parts per billion; LOD: Limit of detection; SD: Standard Deviation; FAO: Food agriculture organization; WHO: World Health Organization; JECFA: Joint Expert Committee on Food Additives.
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Ultra-high performance liquid chromatography/triple quadrupole mass spectrometry study of the stabilities and transformations of four alisols in Alismatis Rhizoma and proprietary traditional Chinese medicine prescriptions p. 597
Chun-Xin Xiao, Fan He, Miao-Ting Yang, Fan Xia, Wai-Jiao Tang, Ben-Jie Zhou, Hua Zhou
DOI:10.4103/pm.pm_614_17  
Background: Alismatis Rhizoma (AR) is a traditional Chinese medicine that is frequently used for the treatment of hyperlipidemia, hypertension, diabetes, and chronic kidney failure. Active alisols contribute to the beneficial effects of AR and AR products. Objective: In the present study, we investigated the stabilities and transformations of alisol B 23-acetate (B23), alisol A 24-acetate (A24), alisol A (AA), and alisol B (AB) in AR and proprietary traditional Chinese medicine prescriptions. Materials and Methods: We first established an ultra-high performance liquid chromatography/triple quadrupole mass spectrometry method for the analysis of AR products. Next, extracts and concentrates of AR and the proprietary prescriptions were prepared and heated at different temperatures. Finally, the developed method was applied to the analysis of these samples. Results: Our method is sensitive, precise, accurate, and reliable for the analysis of these four compounds. The extraction efficacies of the alisols from decoctions prepared from single herb and from mixtures of herbs showed large differences (P < 0.01). The alisols are highly sensitive to changes in temperature and pH and transform during heating. B23 can be transformed to AA and A24, and AB can be transformed to AA. Conclusion: These results indicate that the content change of aliols may affect the quality and efficacy of AR and AR products. Therefore, temperature and pH should be controlled during the manufacture of AR and AR products. Abbreviations used: AR: Alismatis Rhizoma; P1: Prescription 1; HGQZ: Hugan Qingzhi formula; P2: Prescription 2; AA: Alisol A; A24: Alisol A 24-acetate; AB: Alisol B; B23: Alisol B 23-acetate; UHPLC–QqQ–MS/MS: Ultra-high performance liquid chromatography/triple quadrupole mass spectrometry.
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Isolation, elucidation, and molecular docking studies of active compounds from Phyllanthus niruri with angiotensin-converting enzyme inhibition p. 604
Islamudin Ahmad, Abdul Mun'im, Sri Luliana, Berna Elya, Azminah Azminah, Arry Yanuar, Yudithya Artha, Osamu Negishi
DOI:10.4103/pm.pm_151_18  
Background: Phyllanthus niruri, in Indonesia, is known as “Meniran” has a long history of use in ethnic or traditional medicine worldwide, mainly as an antihypertensive agent. Objective: The present study was designed to isolate and identify active compounds with angiotensin-converting enzyme (ACE) inhibition activity from P. niruri herb and confirm the mechanism of action, affinity, and domain specificity interactions of the isolated compounds. Materials and Methods: Some fractions of P. niruri methanolic extract were subjected to column chromatography and preparative thin-layer chromatography to get active compounds. Structural elucidation was determined via spectroscopic methods. ACE inhibition activity was measured using hippuryl-L-histidyl-L-leucine as a substrate in vitro assay. Furthermore, confirmation of the mechanism of action, affinity, and domain specificity interaction of the isolated compounds on ACE complex macromolecule (protein database id: 1O86) was performed by in silico molecular docking studies. Results: In this work, four active compounds were isolated from aerial part of P. niruri, including hypophyllantin (50% inhibition concentration [IC50] = 0.180 μg/mL), phyllantin (IC50 = 0.140 μg/mL), methyl gallate (IC50 = 0.015 μg/mL), and quercetin 3-O-β-D-glucopyranosyl-(1'''-6'')-α-rhamnoside (IC50 = 0.086 μg/mL). In silico molecular docking method emphasizes ligand-residue interactions, thereby predicting the inhibitory activity of these compounds. After docking to an ACE complex macromolecule, quercetin 3-O-β-D-glucopyranosyl-(1'''-6'')-α-rhamnoside obtained more interactions than lisinopril. Conclusion: The results were obtained from in silico and in vitro experiments and confirm the potential active compound is an ACE inhibitor and a new antihypertensive agent. Abbreviations Used: P. niruri: Phyllanthus niruri; ACE: Angiotensin-converting enzyme; HHL: Hippuryl-L-histidyl-L-leucine; HA: Hippuric acid; PDB: Protein database; IC50: 50% inhibition concentration; FH: N-hexane fraction; FE: Ethyl acetate fraction; TLC: Thin layer chromatography; UV-VIS: Ultraviolet-visible; NMR: nuclear magnetic resonance; FTIR: Fourier–Transform infrared; MS: Mass spectrometry; HMQC: Heteronuclear Multiple-Quantum Correlation; HMBC: Heteronuclear multiple bond correlation; TADOK: Tugas Akhir Mahasiswa Doktor.
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Cytotoxicity and antitumoral activity by apoptosis induction of AC13: A brominated curcumin analogue p. 611
Ana Beatriz Bortolozo Oliveira, Renata Prandini Adum de Matos, Bruna Stuqui, Guilherme Silva Torrezan, Carlos Roberto Polaquini, Luis Octávio Regasini, Marilia de Freitas Calmon, Paula Rahal
DOI:10.4103/pm.pm_272_17  
Background: Natural compounds with therapeutic potential have been explored as antitumoral agents, as curcumin (CUR), a substance which has activity against various tumor types and a tool used to improve the action of these compounds is the production of analogs. Objective: In this study, we investigated the antitumoral activity of AC13, a CUR analog. Materials and Methods: Cytotoxicity of AC13 and CUR for different cancer cell lines was analyzed by MTT assay after 24, and 48 h of exposure and caspases 3 and 7 enzymatic activity in CasKi and human spontaneously transformed immortal keratinocyte cell line cells was analyzed after 24 h of incubation with AC13 or CUR at 50 μM. Results: It was observed significant viability loss only for CasKi cells after incubation with AC13. Hence, it was made a more detailed screening of the cytotoxicity for these cells and nontumoral cells incubated with AC13 or CUR, showing concentration-dependent decrease of cell viability. Posteriorly, AC13 induces increase in the caspases activity in both cell lines, being that for tumor cells this increase was greater than that unleashed by CUR. Conclusion: Therefore, AC13 triggers cell death by apoptosis in CasKi and shows greater effect than CUR for these tumor cells, suggesting to be a promising compound for the treatment of cancer and should be studied more thoroughly. Abbreviations used: CUR: Curcumin; TLC: Thin-layer chromatography; DMSO: Dimethyl sulfoxide; Caski: Human cervical carcinoma cell line; HeLa: Human cervical carcinoma cell line; MDA-MB-231: Human breast adenocarcinoma cell line; 786-O: Human renal cell adenocarcinoma cell line; HaCaT: Human spontaneously transformed immortal keratinocyte cell line; DMEM: Dulbecco Modified Eagle Medium; DOXO: Doxorubicin; CLO: Bisphosphonamidate clodronate; U2-OS: Human osteosarcoma cell line; FLLL32: Cell-permeable analog of curcumin; BDMC-A: Analog of curcumin.
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Ophiopogon japonicus herbal tea ameliorates oxidative stress and extends lifespan in caenorhabditis elegans p. 617
Xuesong Yu, Dake Gao, Bing Qi, Xiaochun Xiao, Xufeng Zhai, Chung Wah Ma, Qiangqiang Wang, Zebo Huang
DOI:10.4103/pm.pm_313_18  
Background: Ophiopogon japonicus is a medicinal and edible plant widely used in China. Despite a long history of O. japonicus tea (OJT) in health promotion, however, the mechanistic studies on its actions are lacking. Objective: This study aims to evaluate the antioxidant activity and longevity-promoting potential of OJT using Caenorhabditis elegans model. Materials and Methods: The antioxidant capacities of OJT were first determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and paraquat survival assay, respectively, and then, further investigated by determining the reactive oxygen species (ROS) level, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity. The lifespan assay and lipofuscin accumulation assay were performed in a similar fashion to the paraquat survival assay but without paraquat exposure. Results: We first show by DPPH scavenging assay that OJT can scavenge free radicals. We then reveal that OJT can not only increase the survival rate of the nematodes but also reduce the endogenous levels of ROS under oxidative stress induced by paraquat. We also show that OJT is capable of increasing the activities of SOD and CAT and reducing the content of MDA in paraquat-exposed C. elegans. Further studies indicate that OJT is able not only to extend the lifespan of the nematodes but also to improve the age-related decline of pharyngeal pumping and reduce accumulation of the age pigment lipofuscin in the animals. Conclusion: Our data demonstrate the antioxidant activity and age-delaying effect of OJT and thus provide an important insight into the potentials of O. japonicus for health promotion. Abbreviations used: CAT: Catalase; MDA: Malondialdehyde; OD: Optical density; OJT: Ophiopogon japonicus tea; ROS: Reactive oxygen species; SOD: Superoxide dismutase.
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Systematic understanding the mechanisms of Tripterygium wilfordii on atherosclerosis and pharmacodynamics research in Apo E-/-mice model p. 624
Jingyan Liang, Lu Chen, Yang Pan, Yayun Qian, Lifu Wei, Yumeng Zhang, Kaiming He, Yanqing Liu, Yingge Wang
DOI:10.4103/pm.pm_556_17  
Background: Atherosclerosis (AS) is a chronic arterial disease and a major cause of vascular death, with multiple pathogenesis including chronic inflammatory. Tripterygium wilfordii (TGW) had a good effect on an anti-inflammatory. At present, more and more researches indicated that TGW could also regulate AS. Objective: The aim of this study is to clarify what the anti-atherosclerotic ingredients are in TGW and whether these ingredients improve AS synergistically. Materials and Methods: First, systematic pharmacology was utilized to predict the active ingredients and potential targets of TGW related to AS. Then, a bioactive compound of triptolide (TPL) and Tripterine (TPR) in TGW were evaluated if they presented the synergistically anti-atherosclerotic effects in Apo E-/-mice fed with a high-fat/high-cholesterol diet. In the experiment, Hematoxylin and Eosin tested the plaque areas; reverse transcriptase- polymerase chain reaction and Western blot analysis detected the matrix metalloprotein 9 (MMP-9), tumor necrosis factor alpha (TNF-α), and NF-κB levels in the aortas. Results: The results shown that there are 17 bioactive compounds with 76 therapeutic proteins were identified. Moreover, TGW exhibits a protective effect on treatment AS likely through regulating multiple pathways including immune response, inflammatory response, and vascular structure improving. Further verified that TPL combined with TPR in TGW had synergistic effect on treatment AS by reducing levels of MMP-9, TNF-α, and NF-κB, might be the important pathway. Conclusion: TGW, synergistic effect of different compounds, could regulate AS by multiple pathways, especially improving immune response, inflammatory response, and vascular structure. The major compounds of Tripterine and Triptolide in TGW had a synergistic effect on anti-AS by suppressing matrix metalloprotein 9, TNF-α, and NF-κB. Abbreviations used: TGW: Tripterygium wilfordii, TRL: Triptolide, TRR: Tripterine, TRLR: TRL plus TRR, NC: Normal control, MC: Model control, MMP-9: Matrix metalloprotein 9; NF-kB, Nuclear factor-kappa B; TNF-a, Tumor necrosis factor alpha, AS: Atherosclerosis, H and E: Hematoxylin and Eosin, ox-LDL: Oxidized low-density lipoprotein, ICAM-1: intercellular adhesion molecule-1, VCAM-1: vascular cell adhesion molecule 1, HIF-1: Hypoxia inducible factor-1, IL-2: Interleukin-2, IFN-γ: Interferon-γ, MCP-1: Monocyte chemotactic protein 1, TCMSP: Traditional chinese medicine systems pharmacology, TCM: Traditional chinese medicine, PerOB: Predict oral bioavailability, PerDL: Predict drug-likeness, HL: Half-life, HFC: High-fat/high-cholesterol diet, T-P: Target-Pathway, KEGG: Kyoto Encyclopedia of Genes and Genomes, DAVID: Database for Annotation, Visualization and Integrated Discovery, ADME: Absorption, distribution, metabolism, excretion, TBST: tris-buffered saline, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, DMSO: Dimethyl sulfoxide, HPLC: High Performance Liquid Chromatography.
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Frankincense administration antagonizes adenine-induced chronic renal failure in rats p. 634
Entsar A Saad, Hussam A El-Gayar, Reda S El-Demerdash, Kholoud H Radwan
DOI:10.4103/pm.pm_271_18  
Background: Chronic renal failure (CRF) treatment through kidney transplantation or dialysis is restricted because of economic and medical resources deficiency. Thus, demand for using dietary supplements that can delete or ameliorate uremia or even to delay the need for dialysis is rising. Objectives: This study is the first one conducted to evaluate the efficacy of frankincense aqueous extract on CRF induced by adenine in rats. Materials and Methods: Forty male Sprague-Dawley rats were divided into four equal groups: control, frankincense, adenine, and frankincense + adenine. Kidney function tests, liver function tests, minerals' levels, antioxidant status, and histopathological alterations were investigated. Results: Results showed significant increases in relative kidney weight, serum level of urea, creatinine, blood urea nitrogen (BUN), uric acid, phosphorous, cholesterol, total bilirubin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in adenine group, as well as significant decreases in body weight, calcium, total protein, and albumin. Significant elevation was also demonstrated in lipid peroxidation marker associated with depletion in activities of superoxide dismutase (SOD) and catalase in tissues of kidney and liver. In addition, there were marked histopathological changes of kidney and liver. Conclusion: Study results demonstrated that co-administration of frankincense aqueous extract with adenine is an effective way to reduce the signs of adenine-induced CRF and have returned them to almost completely normal levels. Abbreviations used: CRF: Chronic renal failure; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; SOD: Superoxide dismutase; CKD: Chronic kidney disease; ROS: Reactive oxygen species; MDA: Malondialdehyde; H and E: Hematoxylin and eosin; BUN: Blood urea nitrogen; PTH: Parathyroid hormone.
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Osthole promote differentiation and inhibit proliferation of osteoblast by activating wnt signaling and endoplasmic reticulum stress p. 641
Suyang Zheng, Yong Ma, Yang Guo, Lining Wang, Yalan Pan
DOI:10.4103/pm.pm_591_17  
Background: Osthole is extracted from Fructus Cnidii and is proved to be effective in the treatment of osteoporosis in rats. However, data are still scarce and the mechanism remains elusive. Objective: To investigate the effect of Osthole on proliferation and differentiation of osteoblast. Materials and Methods: Cells were divided into five groups: control group, β-estradiol group (10−8 M), and Osthole groups (10−6 M, 10−5 M, and 10−4 M). Osteoblast proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity was detected by ALP staining and enzymatic measurement. Mineralization was detected by alizarin-red staining. The level of osteocalcin was measured by enzyme-linked immunosorbent assay (ELISA). The expression of key proteins of Wnt/β-catenin signaling pathway and endoplasmic reticulum stress (ERS) was analyzed by Western blot. Results: Cell proliferation was retarded in moderate-dose and high-dose Osthole group at 1d, 2d and 3d and in low-dose Osthole group at 1d and 2d (P < 0.05). ALP activity was enhanced in high-dose Osthole group from 1d to 3d and in moderate-dose Osthole group at 2d (P < 0.05). Mineralization of bone matrix was promoted in high-dose Osthole group at 21d (P < 0.05). The secretion of osteocalcin was promoted in Osthole groups at 21d (P < 0.05). Expression of CHOP, GRP78, PDI, Wnt1, and β-catenin was upregulated in high-dose Osthole group at 2d, indicating that both of ERS and Wnt/β-catenin signaling pathway were activated. Conclusion: It can be concluded that the effect of Osthole on inhibition of proliferation is relevant with activation of ERS, and activation of Wnt/β-catenin signaling pathway is one of the mechanisms how Osthole promotes osteoblast differentiation. In summary, this study provided more evidence for Osthole as a potential anti-osteoporosis medicine. Abbreviations used: ALP: Alkaline phosphatase; ELISA: Enzyme-linked immunosorbent assay; CCK-8: Cell Counting Kit-8; ERS: endoplasmic reticulum stress; DMEM: Dulbecco's Modified Eagle Medium; α-MEM: α-minimum essential medium; EDTA: trypsin/ethylenediaminetetraacetic acid; DMF: N,N-dimethylformamide; TBS-T: Tris-buffered saline-Tween 20; RIPA: Radio Immuno Precipitation Assay; HRP: Horseradish peroxidase; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; BCA: Bicinchoninic acid; ANOVA: Analysis of variance.
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Development and evaluation of sea buckthorn (Hippophae rhamnoides L.) seed oil nanoemulsion gel for wound healing p. 647
Tanurajvir Kaur, Deepak N Kapoor
DOI:10.4103/pm.pm_375_18  
Background: Sea buckthorn (SBT) seed oil is reported to have significant wound-healing activity. However, the oil presents with problems such as poor skin permeation and retention in the deeper layers of skin, as well as leakage, dripping, and spreading during application. These issues can be overcome by formulating nanoemulsion (NE) gel of SBT seed oil. Objective: The present study involves the development of SBT seed oil NE gels for wound healing. Materials and Methods: The NE formulations were prepared by the spontaneous emulsification method. Based on the results of pseudoternary phase diagrams, different NE formulations were prepared using SBT seed oil, surfactant/cosurfactant, and water. The selected NE formulation NEA1 was used to prepare two NE gels containing 0.5% w/w (NG1) and 1% w/w (NG2) of carbopol 940. Results: The optimized formulation NEA1 was selected on the basis of stability and conductivity studies. The NG2 NE gel was selected as the final formulation on the basis of viscosity, spreadability, extrudability, and texture analysis. This optimized NG2 formulation was further evaluated for in vivo wound-healing activity and ex vivo skin penetration studies. A significant improvement was found in NG2-treated group of animals in comparison with SBT seed oil-treated animals w.r.t wound contraction, hydroxyproline, hexosamine content, breaking strength, tensile strength, and epithelialization time of the wound. The optimized formulation also showed improved antibacterial and antifungal activity. Conclusion: The formulated NE gel of SBT seed oil showed better wound-healing, antibacterial, and antifungal activity in comparison to pure SBT seed oil. Abbreviations used: CPCSEA: Committee for the Purpose of Control and Supervision of Experiments on Animals; DMSO: Dimethyl sulfoxide; FITC: Fluorescein isothiocyanate; FAME: Methyl esters formation of fatty acids; GC: Gas chromatography; IAEC: Institutional Animal Ethics Committee; MIC: Minimum inhibitory concentration; MHA: Mueller-Hinton agar media; MHB: Mueller-Hinton broth; MDS: Mean droplet size; NIPER: National Institute of Pharmaceutical Education and Research; NEs: Nanoemulsions; KOH: Potassium hydroxide; PDI: Polydispersity index; RPMI-1640: Roswell Park Memorial Institute-1640 medium; STZ: Streptozotocin; SBT: Sea buckthorn; Smix: surfactant/cosurfactant; TEA: Triethanolamine; TEM: Transmission electron microscopy; YPD: Yeast extract-peptone-dextrose.
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