Pharmacognosy Research

ORIGINAL ARTICLES
	Chemical characterization and evaluation of antioxidant and antimicrobial activities of Litchi chinensis sonn	p. 1
	Marisa De Oliveira Lopes, Ana Flávia Da Silva, Cláudio Daniel Cerdeira, Ingridy Simone Ribeiro, Isael Aparecido Rosa, Luis Felipe Cunha Dos Reis, Marcelo Aparecido Da Silva, Marcos José Marques, Jorge Kleber Chavasco, Geraldo Alves Da Silva DOI:10.4103/pr.pr_105_18   Background: Litchi chinensis is used in traditional Chinese medicine and by Indian medical system. Ethnopharmacological studies show anti-inflammatory, antidiabetic and analgesic activities, among others. However, there are few studies of antimicrobial activity. This study evaluates antimicrobial, antioxidant, and cytotoxicity properties of the lychee's leaves extract (LE) and fractions. Materials and Methods: Extracts were obtained using an exhaustive extraction method with ethanol: Water (7:3 v/v). Subsequently, LE was concentrated in a rotary evaporator. Finally, LE was dried via lyophilization. Fractions were obtained via the partition process. Bioactivity of the LE and fractions (hexane [Hex], ethyl acetate [EtOAc], n-butanol [BuOH], and aqueous [Aq]) from L. chinensis was evaluated through antimicrobial activity using broth microdilution, antioxidant activity via both 1,1-diphenyl-2-picryl-hidrazila assay and ferric reducing capacity and cytotoxicity through 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. Furthermore, mass spectrometry technique electrospray ionization ion trap mass spectrometry was used to identify the chemical composition of the LE and fractions. Results: Phenolic compounds, such as flavonoids and condensed tannins were the main substances found. Total phenolic and flavonoid contents were higher in EtOAc (541.15 ± 2.4 mg/g and 31.06 ± 0.5 mg/g, respectively). This fraction showed the best results for antioxidant activity (IC50= 3.45 mg/mL) and ferric reducing capacity (20.27% ± 0.11). The LE and fractions showed considerable antimicrobial activity, chiefly against Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Proteus mirabilis, with the minimum inhibitory concentration ranging from 50 to 1560 μg/ml. Conclusion: This study revealed that L. chinensis is a source of bioactive compounds potentially useful for pharmaceutical and food industries. Abbreviations Used: LE: Leaves extract, Hex: Hexane fraction, EtOAc: Ethyl acetate fraction, BuOH: n-butanol fraction, Aq: Aqueous fraction, DPPH: 1,1-diphenyl-2-picryl-hidrazila, MTT: 3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide, ESI-IT-MSn: Electrospray ionization ion trap mass spectrometry, IC50: Median Inhibition Concentration (concentration that reduces the effect by 50%), MIC: Minimum inhibitory concentration, MS/MS: Mass spectrometry, MSn: Tandem mass spectrometry, GAE: Equivalents of gallic acid, QE: Equivalents of quercetin, BHT: Butylated Hydroxy Toluene, UV: Ultraviolet, ATCC: American Type Culture Collection, MHB: Mueller Hinton broth, DMSO: Dimethyl sulfoxide, ANOVA: Analysis of variance, CC50: Cytotoxic concentration 50.
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	Saponin of Momordica cymbalaria exhibits anti-inflammatory activity by suppressing the expression of inflammatory mediators in lipopolysaccharide-stimulated RAW264.7 macrophages	p. 8
	Suman Samaddar, Raju Koneri DOI:10.4103/pr.pr_130_18   Background: Inflammation is an intricate biological process that commonly occurs in response to pathologic stimuli, and natural products have potential in healing inflammation. However, the anti-inflammatory of Momordica cymbalaria is not evaluated yet. Objectives: The anti-inflammatory mechanism of saponin of M. cymbalaria(SMC) was investigated in bacterial lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cell line. Methods: The cytotoxicity of SMC on RAW264.7 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay at 500, 250, 125, 62.5, 31.25, 15.625, 7.812, 3.906, and 1.953 μg/mL. For anti-inflammatory activity, RAW264.7 cells were stimulated with Escherichia coli LPS (1 μg/ml) in the presence or absence of SMC (50 μg/ml) for 16–24 h. Western blotting was carried to comprehend the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS) whereas expressions of the pro-inflammatory cytokines (interleukin [IL]-6, IL-1β, and tumor necrosis factor-alpha [TNF-α]) and prostaglandin E2(PGE2) were studied by enzyme-linked immunosorbent assay (ELISA). NO production was estimated by Griess's method. Salicylic acid, a nonsteroidal anti-inflammatory drug, was used as a standard. Results: The saponin did not exert significant cytotoxicity on RAW264.7 cells. Western blot analysis revealed reduction in COX-2 and iNOS expression on SMC treatment. Production of PGE2, IL-6, IL-1β, and TNF-α was also found to be reduced when analyzed by ELISA. NO levels were also lowered. Conclusions: The findings suggest that the SMC possesses potential anti-inflammatory activity by suppressing the expression of inflammatory mediators, COX-2, iNOS, PGE2, and NO, and the cytokines in LPS-stimulated RAW264.7 cells. Abbreviations Used: IL-6: Interleukin-6; IL-1β: Interleukin-1 beta; TNF-α: Tumor necrosis factor-alpha; NO: Nitric oxide; COX-2: Cycloxygenase-2; LPS: Lipopolysaccharide; HPTLC: High-performance thin layer chromatography; TLC: Thin layer chromatography; HPLC: High-performance liquid chromatography; SMC: Saponin of Momordica cymbalaria; DMSO: Dimethylsulfoxide; EDTA: Ethylenediaminetetraacetate; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBST: Tris buffer saline tween; IgG-HRP: Immunoglobulin G-horse radish peroxidase; H2O2: Hydrogen peroxide; ELISA: Enzyme-linked immunosorbent assay.
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	Screening and validating of 1, 1-diphenyl-2-trinitrophenylhydrazine scavengers from Danshen-Honghua herbal pair	p. 14
	Ya-Li Wang, Shi-Jun Yin, Sheng-Li Huang, Qian Zhang, Hua Chen, Feng-Qing Yang DOI:10.4103/pr.pr_94_18   Background: Salvia miltiorrhiza (Danshen [DS]) and Carthamus tinctorius (Honghua [HH]) are commonly used traditional Chinese medicines for activating blood and removing stasis, and they were usually used as DS-HH (Danshen-Honghua [DH]) herbal pair in clinical applications. Characterizing the antioxidant active ingredients in DH herbal pair may be helpful for understanding their curative effect on cardiovascular diseases. Objective: The objective of the study is to screen the 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) scavenging active compounds in DH herbal pair by spectrum-effect relationship analysis. Materials and Methods: First, the water extracts of DH herbal pair with different ratios (DS: HH = 1:0, 0:1, 1:1, 2:1, 3:1, 5:1, 1:5, 1:3, and 1:2) were prepared. Then, the clearance effects of DH herbal pair and single drugs on DPPH were compared, meanwhile, high performance liquid chromatography was applied for chemical analysis of DH extracts. Finally, DPPH scavengers in DH herbal pair were predicted and identified by spectrum-effect relationship analysis and liquid chromatography-mass spectrometry (LC-MS) analysis. Results: Compared with single drugs, the extracts of herbal pairs had higher clearance rate to DPPH. Eighteen potential active compounds (peaks) in the extract of DH herbal pair were predicted, and 13 of them were tentatively identified by LC-MS analysis. Furthermore, antioxidant activities of eight pure compounds from the DH herbal pair were validated by DPPH radical scavenging assay with Vitamin C as positive control drug. Among them, six compounds including danshensu, protocatechuic acid, coffee acid, chlorogenic acid, rutin, and salvianolic acid A were found to have high antioxidant activity. Conclusion: DH herbal pair showed strong clearance effect on DPPH, and danshensu, protocatechuic acid, coffee acid, chlorogenic acid, rutin and salvianolic acid A are the active components. Abbreviations Used: DS: Danshen, HH: Honghua, DH: Danshen-Honghua, TCMs: Traditional Chinese medicines, LC-MS: Liquid chromatography-mass spectrometry, DPPH: 1, 1-diphenyl-2-trinitrophenylhydrazine, VC: Vitamin C, CCA: Canonical correlation analysis.
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	Mitochondrial nephrotoxicity induced by tacrolimus (FK-506) and modulatory effects of Bacopa monnieri (Farafakh) of Tabuk Region	p. 20
	Atif Abdulwahab A. Oyouni, Shalini Saggu, Ehab Tousson, Anand Mohan, Abdullah Farasani DOI:10.4103/pr.pr_100_18   Background: Tacrolimus is a known immunosuppressive drug used widely for organ transplantation, but its nephrotoxicity mechanism is still unclear. Objectives: The present study investigates the protective efficacy of Bacopa monnieri (BM), against tacrolimus-induced nephrotoxicity in rats. Materials and Methods: Group 1 (control group); administered orally with normal saline for 30 days; Group 2 (BM extract treated group); Group 3 (tacrolimus-treated group); and Group 4; (tacrolimus plus BM extract treated group). Tacrolimus-treated rats received 1 mg/kg body weight of tacrolimus intraperitoneally for 30 days, and BM-pretreated rats were administered with the dose of 200 mg/kg orally by gavage once a day for 30 days. Results: Tacrolimus-induced nephrotoxicity was assessed biochemically and histopathologically. Pretreatment with BM has shown to possess a significant protective effect against tacrolimus-induced kidney functions regarding urea, creatinine, and albumin levels, respectively. The creatinine, mitochondrial lipid peroxidation (thiobarbituric acid reactive substances), and protein carbonyl levels were significantly increased dramatically, and however, the total proteins, albumin, glutathione, superoxide dismutase, and glutathione peroxidase were decreased when pretreated with tacrolimus. The nephroprotective efficacy of the BM extract was further evident by histopathological analysis and DNA fragmentation. Conclusion: The outcome of this study indicates that BM extracts exerted protection against tacrolimus-induced kidney toxicity. Abbreviations Used: ANOVA: Analysis of variance, BM: Bacopa monnieri, BUN: Blood urea nitrogen, DNPH: Dinitrophenylhydrazine, DPPH: 2,2-diphenyl-1-picrylhydrazyl, DSR: Deanship of Scientific Research, EOBPV: Egyptian Organization for Biological Products and Vaccines, GPx: Glutathione peroxidase, GSH: Glutathione, H and E: Hematoxylin and eosin, H2O2: Hydrogenperoxide, IAEC: Institutional Animals Ethics Committee, IC: Inhibitory concentration, Ip: Intraperitoneal, mLPO: Mitochondrial lipid peroxidation, Mn-SOD: Mn-superoxide dismutase, PC: Protein carbonyl, ROS: Reactive oxygen species.
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	Efficacy of salivary and diastase extracts of Piper betle in modulating the cellular stress in placental trophoblast during preeclampsia	p. 25
	Padmini Ekambaram, Chithra Balan DOI:10.4103/pr.pr_112_18   Background: Betle leaf (BL) is an ancient herb with a potential nontoxic natural antioxidant efficacy. Oral intake and chewing betel leaves have an effort on the moving parts of salivary gland and induce salivation and detoxification; hence plays a vital role in oral hygiene. BL produces and activates various salivary enzymes which include α-amylase, lipase, lysozyme, and lactoperoxidase. BL possess antibacterial, antioxidant, anti-diabetic, immunomodulatory, and antihypertensive properties. Hence, it can be effective in the treatment of hypertensive disorder of pregnancy like preeclampsia (PE). Objective: In this context, two extracts of BL (Salivary BL extract [SBLE] and diastase BL extract [DBLE]), proved to impart efficient radical scavenging activity in our previous research work were utilized to analyze their efficacy on the level of 4-hydroxy nonenal (4-HNE), heme oxygenase-1 (HO-1), asymmetric dimethylarginine (ADMA), adiponectin/leptin in the trophoblast isolated from placental tissue of both normotensive and preeclamptic patients. Materials and Methods: Trophoblasts were isolated from both subjects and incubated with different extracts of Piper betle (saliva and diastase) to assess the cellular stress in placental trophoblast during PE. Results: Results demonstrate that extracts of BL exhibit a significant role in regulating 4-HNE, ADMA, leptin, HO-1, and adiponectin in preeclamptic trophoblasts. Still, SBLE showed relatively higher efficiency in defining the level of 4-HNE, ADMA, leptin, HO-1, and adiponectin in preeclamptic trophoblasts than DBLE, suggesting that it may be attributed to the interaction of the phytochemicals present in BL with the components of saliva. Conclusion: Hence, BL may be recommended as the effective natural remedy in the management of pathological complications of PE; which may preclude low birthweight babies and ensure live fetal delivery. Abbreviations Used: 4-HNE: 4-hydroxy-nonenal, ADMA: Asymmetric dimethylarginine, BL: Betel leaf, BLE: Betel leaf extract, CO: Carbon monoxide, DB: Diastase with betel leaf, DBLE: Diastase betel leaf extract, ELISA: Enzyme-linked immunosorbent assay, eNOS: Endothelial nitric oxide synthase, FBS: Fetal bovine serum, GSH: Reduced glutathione, HBSS: Hank's balanced salt solution, HO-1: Heme oxygenase-1, iNOS: Inducible nitric oxide synthase, NO: Nitric oxide, NOS: Nitric oxide synthase, PE: Preeclampsia, ROS: Reactive oxygen species, SB: Saliva with betel leaf, SBLE: Salivary betel leaf extract.
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	Effects of the mint monoterpene (R)-(+)-pulegone evaluated by functional observational battery: A potential short method	p. 31
	Yesica P Zaio, Marco M Mazzotta, Agripina Ramírez Sánchez, Elisa Alejandrina Gomez, María Paula Zunino, Andrés Alberto Ponce DOI:10.4103/pr.pr_115_18   Background: Pulegone (PUL) is one of the major constituents of peppermint and pennyroyal. Objective: The main purpose of this study was to investigate the early effects of toxicity of PUL. Materials and Methods: We assessed single doses of toxicity of PUL (2, 0.3 and 0.05 g/kg body weight), administrated by gavage in C57BL/6 mice, evaluated at 1, 6, and 24 h, their clinical status and behavioral, by functional observational battery (FOB), ambulatory conditions, sperm motility, pathological signs, and organ/body weight (O/Bw). Results: No mortality was registered in this in vivo study of oral acute toxicity, in which histological changes were found in selected organs, and PUL mainly showed that the highest concentration reduced mice locomotor activity with significant differences when compared with data of the FOB. Sperm motility also diminished, and hepatic as well as renal alterations were found without modifications in clinical status and O/Bw. Conclusions: We concluded that PUL could be responsible for these findings and consider that FOB is a useful tool to detect early signs of modifications of physiological and biological parameters in mice. Abbreviations Used: PUL: Pulegone, FOB: Functional observational battery, CNS: Central nervous system, OECD: Organization for Economic Cooperation and Development, ANOVA: Analysis of variance, MNI: Mononuclear cell infiltrate, LD50: Median lethal dose
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	A study of In-vitro hypoglycemic and glucose uptake activity of isolated compound from ethanolic leaf extract of Amaranthus tristis Linn.	p. 37
	T Sundarrajan, V Velmurugan, K Manikandan, D Jothieswari DOI:10.4103/pr.pr_119_18   Background: Natural bioactive compounds have a long history to use effectively in the treatment of Diabetes Mellitus. Amaranthus tristis Linn (Amaranthaceae) have been used for the treatment of various ailments. Objective: The present work was undertaken to study the effect of an isolated bioactive compound from the leaf of Amaranthus tristis Linn on glucose uptake in cell lines like 3T3-L1 cell lines and inhibition of alpha-glucosidase and alpha-amylase enzymes. Materials and Methods: The Ethanolic leaf extract of Amaranthus tristis Linn subjected to preliminary phytochemical screening and isolated compound were tested for its cytotoxicity study by MTT assay. Isolated compound was selected further glucose uptake assay based on cytotoxicity concentration CTC50 value. NMR technique was used to identify the structure of the compound. Results: Phytochemical analysis showed the presence of alkaloids, flavonoids, glycosides. The Isolated compound showed moderate cytotoxic activity to 3T3-L1 cell line. Compounds identified as rutin In in-vitro glucose uptake assay of rutin showed an increase in glucose uptake. Conclusion: A. tristis Linn. showed significant antidiabetic activity in dose-dependent manner. Abbreviations Used: MTT: 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, FBS: Fetal Bovine serum, PBS buffer: Phosphate Buffered Saline, DMEM: Modified Eagle's Medium, EDTA: Ethylenediaminetetraacetic acid, IBMX: 3-isobutyl-1-methylxanthine, TLC: Thin-layer chromatography, FTIR: Fourier-transform infrared spectroscopy, 13CNMR: nuclear magnetic resonance, 1H NMR: Proton nuclear magnetic resonance, GC-MS: Gas chromatography–mass spectrometry, DNSA: Dinitro salicylic acid.
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	Cytotoxicity and apoptosis induction of sea cucumber Holothuria atra extracts	p. 41
	Muhammad Nursid, Endar Marraskuranto, Ekowati Chasanah DOI:10.4103/pr.pr_3_18   Context: Cancer is one of the major causes of death. Sea cucumbers are marine invertebrate that is widely used and utilized in traditional medicine. One of the most studied of sea cucumbers efficacy is its anticancer property. The anticancer property of sea cucumbers is much related to the content of an active compound called saponin. Objective: The objective of this research is to determine the cytotoxicity and apoptosis induction of sea cucumber Holothuria atra ethanol extract and to study its anticancer active compound. Materials and Methods: Sea cucumber of H. atra was taken from Halmahera waters, North Maluku Indonesia. The extraction was conducted by maceration method using ethanol 96% then continued with liquid-liquid partition and separation using C18 column. The investigation of active compound content was conducted by phytochemical and spectroscopic analysis using liquid chromatography-ion trap-time of a flight-mass spectrophotometer (LC-IT-ToF-MS). The cytotoxicity test was performed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method against human breast ductal carcinoma cell (T47D), human breast adenocarcinoma (MCF7), human colorectal adenocarcinoma cell (WiDr), and human cervix adenocarcinoma cell lines (HeLa). The apoptotic induction test was performed using flow cytometry using Fluorescein isothiocyanate (FITC) Annexin-V-Apoptosis and caspase-3 detection using FITC Active Caspase-3 kit. Results: Phytochemical test showed that ethanol extract of H. atra contained alkaloids, flavonoids, steroids-triterpenoids, phenols, and saponins. The extract showed cytotoxic activity against the 4 cell lines with Inhibition concentration 50 values ranging from 9.6 to 14.3 μg/ml. Flow cytometry analysis showed that the T47D cell population underwent apoptosis after treated with ethanol extract. The extract also activated caspase-3 on the T47D cells. The results of LC-IT-ToF-MS analysis showed that the active fraction from C18 column separation contained saponin and identified as Cucumechinol and Philinopgenin B. Conclusions: The results of this study indicated that H. atra active extract has good cytotoxicity and has potential to be developed as an anticancer agent. Abbreviations used: MNPs: Marine natural products; LC-IT-ToF-MS: Liquid chromatography-ion trap-time of flight-mass spectrophotometer; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; T47D: Human breast ductal carcinoma cell; MCF7: Human breast adenocarcinoma cell; WiDr: Human colorectal adenocarcinoma cell; HeLa: Human cervix adenocarcinoma cell; RPMI: Roswell park memorial institute medium; FBS: Fetal bovine serum; FITC: Fluorescein isothiocyanate; PBS: Phosphate buffer saline; BD: Becton Dickinson; IC50: Inhibition concentration 50; PS: Phosphatidylserine; PI: Propidium iodide.
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	Phytochemistry in medicinal species of Solanum L. (Solanaceae)	p. 47
	Laudinéia de Jesus Matias, Juliana Almeida Rocha, Vanessa de Andrade Royo, Elytania Veiga Menezes, Afrânio Farias de Melo Júnior, Dario Alves de Oliveira DOI:10.4103/pr.pr_148_18   Background: The genus Solanum L. is the largest of the family Solanaceae; it has used in food, ornamentation, and medicinal. Objective: This work had as objective to perform the phytochemical screening secondary metabolites: saponins, alkaloids, tannins, flavonoids, and anthraquinones by means of colorimetric and precipitation analyzes of root bark, stem, leaf, and epicarp of the following species of Solanum genus: Solanum agrarium, Solanum lycocarpum, Solanum palinacanthum, Solanum paniculatum, and Solanum stipulaceum. The species are found in areas of the Cerrado Biome of Northern Minas Gerais state for initial evaluation of the possibility of cultivation for medicinal use. Materials and Methods: The following qualitative tests were performed: Liebermann–Burchard reaction for steroids/triterpenoids; foam persistence test for saponins; Bertrand and Dragendorff reagents for alkaloids; reactions with lead neutral acetate at 10% and ferric chloride at 2% for tannins; reactions with ferric chloride at 2% and Shinoda reaction for flavonoids; reaction of Bornträeger and reaction with sodium hydroxide at 0.5% for anthraquinones. Results: Plants of the Solanum L. genus, occurring in Cerrado areas in the city of Montes Claros, have considerable amounts of secondary metabolites, varying between the different species and in different structures analyzed. Conclusion: It is important to continue the study evaluating whether in species from planting occurs production of the secondary metabolites identified in wild plants.Abbreviations Used: FAPEMIG: Fundação de Amparo à Pesquisa do Estado de Minas Gerais, CNPq: Conselho Nacional de Desenvolvimento Científico e Tecnológico, BHCB: Herbarium of the Department of Botany, from Institute of Biological Sciences, Federal University of Minas Gerais.
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	Participation of cytokines, opioid, and serotoninergic systems on antinociceptive and anti-inflammatory activities of Simira grazielae peixoto (Rubiaceae)	p. 51
	Gabriela Carmelinda Martins dos Santos, Mirza Nalesso Gomes Sanches, Douglas Siqueira de Almeida Chaves, Mário Geraldo de Carvalho, Raimundo Braz-Filho, Bruno Guimarães Marinho DOI:10.4103/pr.pr_99_18   Background: Simira grazielae P. is widely found at Brazil. S. grazielae have been used to treat pain and inflammation in the Northeast of Brazil. Objective: This study investigated the mechanisms of the extract and partitions using pharmacological techniques in mice. Materials and Methods: Male Swiss mice (20–22 g) were used in models of pain (acetic acid-induced abdominal writhing, formalin, and tail-flick tests) and inflammation (edema paw and air pouch tests) as well as in model for the evaluation of motor activity (open field test). Furthermore, we evaluate the probable action mechanism of S. grazielae using naloxone, L-nitro-arginine methyl ester, L-arginine, glibenclamide, atropine, 4-chloro-DL-phenylalanine, and ondansetron in tail-flick test. The cytokines production was also evaluated. The methanolic extract from the S. grazielae and its partitions were administered orally at doses of 10–100 mg/kg. Results: Methanolic extract from the wood of S. grazielae (SGM) and its partitions showed antinociceptive properties in models of acute pain (SGM and ethyl acetate partition [SGMAc]) as well as in models of inflammation (dichloromethane partition [SGMD]). Prior administration of ondansetron and naloxone reduced the antinociceptive effect of SGMAc. SGMD reduced the production of tumor necrosis factor-α (TNF-α) induced by carrageenan. Conclusion: The results show that the anti-inflammatory activity showed by SGMD involves to reduction of the TNF-α, and the antinociceptive activity showed by SGMAc has relation to participation of the serotoninergic receptors and opioid system. These evidence justify the popular therapeutic use of this species in the control of pain and inflammation. Abbreviations Used: PCPA: 4-chloro-DL-phenylalanine, SGM: Methanolic extract of Simira grazielae, SGMAc: Ethyl acetate partition, SGMD: Dichloromethane partition, SGMH: Hexane partition, SGMB: Butanol partition, SGMR: Residual partition, 5-HT: Serotonin, TNF-α: Tumor necrosis factor-α, n-C6H14: Hexane, CH2Cl2: Dichloromethane, EtOAc: Ethyl acetate, BuOH: Butanol, TLC: Thin-layer chromatography, HPLC: High-performance liquid chromatography, DAD: Diode array detector, COX-2: Cyclooxygenase-2, PGE2: Prostaglandin E2, eNOS: Endothelial nitric oxide synthase, NO: Nitric oxide, TRPA 1: Transient receptor potential cation, LT: Latency time, IBL: Increase in baseline, BL: Baseline.
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	Assessments of antioxidant, antilipid peroxidation, and In-vitro safety of Derris scandensvine extracts from Southern Thailand	p. 60
	Rawiwan Nooin, Pornsiri Pitchakarn, Chaturong Kanchai, Churdsak Jaikang DOI:10.4103/pr.pr_141_18   Background: Derris scandens (DS) has been used in Thai traditional medicine recipes for musculoskeletal pain relief in Southern Thailand. Changing of terrain and climate affected its phytochemical constituents and led to change antioxidant and toxic properties. Objective: The aim of this study was to study phytochemical contents, antioxidant properties, and toxicity of the DS extracts' in vitro models. Materials and Methods: The DS ethanolic extract (EE) was partially extracted with chloroform (CE), ethyl acetate (EAE), and water (aqueous extract [AE]). Phytochemical contents, antioxidant properties, and toxicity in Caco-2 cells, peripheral blood mononuclear cells (PBMCs), and red blood cells (RBCs) were explored. Results: Genistein and gallic acid were rich in the EE and CE. The CE demonstrated the highest 2,2 − diphenyl − 1 − picrylhydrazyl scavenging activity with half-maximal Radical cavenging concentration (SC50) value – 0.81 ± 0.07 mg/mL. The EE showed the highest 2, 2'−azino − bis (3 − ethylbenzothiazoline − 6 − sulfonic acid) radical scavenging activity (SC50 = 22.05 ± 3.91 μg/mL) and all the extracts strongly inhibited lipid peroxidation. The EE and CE were more toxic than the EAE on Caco-2 cells with IC50 values – 26.45 ± 3.57 and 36.36 ± 6.87 μg/mL, respectively. At high dose, all fractions were not toxic to human's PBMCs, but they slightly induced RBC hemolysis by 3-6 percent. Conclusions: The DS grown in Southern Thailand had antioxidant properties, toxic to Caco-2 cells and not toxic to normal cells. High dose and continuous consumption of the DS must be concerned. Further safety assessment models including animals and humans should be performed to find safety dose of the DS. Abbreviations Used: ABTS: 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), DMEM: Dulbecco's Modified Eagle Medium, DMSO: Dimethyl sulfoxide, DPPH: 2,2-diphenyl-1-picrylhydrazyl, HLM: Human liver microsome, HLM: Human liver microsome, HPLC: High-performance liquid chromatography, IC50: Half inhibitory concentration, MDA: Malondialdehyde, OD: Optical density, PBMC: Peripheral blood mononuclear cell, RBC: Red blood cell, RPMI: Roswell Park Memorial Institute Medium, SC50: Halfmaximal radical scavenging concentration, SRB: Sulforhodamine B assay.
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	Determination and quantification of p-coumaric acid in pineapples (Ananas comosus) extracts using gradient mode RP-HPLC	p. 67
	Wan Mazlina Md Saad, Rasdin Ridwan, Nur Syamimi Md Lasim, Nur Liyana Mohd Rapi, Fatimah Salim DOI:10.4103/pr.pr_154_18   Background: Pineapple (Ananas comosus) is a good source of phenolic compounds such as p-coumaric acid. P-Coumaric acid is a hydroxycinnamic acid known to possess antioxidant, antimicrobial, antimutagenic, and immunoregulatory activities. Objective: The present study aims to determine and quantify the p-coumaric acid in the juice extract and methanol extract of unripe and ripe pineapples by gradient mode reverse-phase high-performance liquid chromatography (RP-HPLC). Materials and Methods: The analysis was carried out using 0.5% phosphoric acid and 100% acetonitrile (can) as mobile phase A and B at gradient elution conditions of 0–30 min from 5% B to 80% B, 30–33 min at 80% B, 33–35 min from 80% B to 5% B, and 35–40 min at 5% B. The chromatography separation was performed using Gemini C18 column at flow rate of 0.8 mL/min and detection was made at 280 nm. Results: P-Coumaric acid was determined at retention time of 16.16 min. Standard curves were linear at R2 = 0.9973 with low limit of detection and limit of quantification being 0.0208 and 0.0694, respectively. P-Coumaric acid showed the highest concentration in juice extract of ripe pineapple, 11.76 μg/mL compared to the methanol extract, 0.03 μg/mL, respectively. P-Coumaric acid concentration in juice extract of unripe pineapple was 0.41 μg/mL. Conclusion: The gradient mode RP-HPLC proved to be efficient for determination and quantification of p-coumaric acid in pineapples extracts. Abbreviations Used: RP-HPLC: Reverse-phase high-performance liquid chromatography; ACN: Acetonitrile; H3PO4: Phosphoric acid; PLPs: Pineapple leaf phenols; HepG2: Hepatoma G2; PLEs: Pineapple leaf extracts; TC: Total cholesterol; TG: Triglyceride; DAD: Diode array detector; UV-Vis: Ultraviolet-visible; dH2O: Distilled water; LOD: Limit of detection; LOQ: Limit of quantification.
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	High-temperature condition increases lignanoid biosynthesis of Schisandra chinensisseeds via reactive oxygen species	p. 72
	Guo Huimin, Wang Jiahui, Gao Huiru, Meng Xiangcai DOI:10.4103/pr.pr_42_18   Introduction: The herbal medicine used in many countries came mainly from the wild in the past; now, declining yield resource and laborious gathering result in prevailing cultivated medicine, with a result of prevailing inferior quality of herbal medicine. The contents of major functional ingredients varies greatly in the fruits of Schisandra chinensis, a herbal medicine in many Asian countries. Materials and Methods: These fruits were placed at 20°C, 35°C, 45°C, and 55°C for 1–6 days, respectively, covered with plastics to prevent cells from anhydration during treating. The contents of H2O2, phenylalanine, and lignanoids and activities of antioxidant enzymes and phenylalanine ammonia lyase (PAL) were monitored. Results: The fresh seeds were exposed to 35°C, 45°C, and 55°C for 1 week; the H2O2 was rose sharply at 1 day and then declined but still with a higher level. The superoxide dismutase, catalase, and peroxidase activities were lowered, with inefficient antioxidant capacity. The PAL activities had a certain degree of high-temperature tolerance, remained largely unchanged at 35°C, but reduced gradually as temperature increased. High temperature activated the glycolytic pathway and rose the phenylalanine contents, which increased sharply at 1 day for 35°C and 45°C and at the 2 days for the 55°C and then maintained a stable level with almost 1–3 times than the 0 day. Conclusions: The increased phenylalanine as substrate accelerated the synthesis of lignanoids; the contents of five lignanoids were increased by as much as 31.2%–81.5%, respectively. Abbreviations Used: ROS: Reactive oxygen species; SOD: Superoxide dismutase; CAT: Catalase; POD: Peroxidase; PAL: Phenylalanine ammonia lyase.
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	Euphorbia hirta methanolic extract displays potential antioxidant activity for the development of local natural products	p. 78
	Aziana Ismail, Maizan Mohamed, Yap Fon Kwei, Khoo Boon Yin DOI:10.4103/pr.pr_113_18   Background: The level of free radicals, which counteract the capability of the antioxidant system in plant products, is often measured in advance for further promising antidisease effect. Objective: In this study, we sought to evaluate the antioxidant activity of local medicinal plants (Angelica keiskei, Annona muricata, Chromolaena odorata, Clinacanthus nutans, Euphorbia hirta, and Leea indica) for their potential of use as distinctive local natural nutraceutical products. Materials and Methods: To recover active compounds, including yield and composition of the plants, the solvent extraction method, the Folin–Ciocalteu method, the aluminum chloride approach, and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay were first performed to evaluate the antioxidant level and capacity of the plant extracts. Results: The aqueous extracts presented the highest yield for all plants, with the highest yield observed in C. nutans. However, the highest total phenolic and flavonoid contents were observed in the methanolic extract of E. hirta rather than in the aqueous extract. The methanolic extract of E. hirta also exhibited the most promising antioxidant activity, with the 50% inhibition concentration (IC50) value of DPPH inhibition at 0.013 mg/mL. Conclusion: High total phenolic and flavonoid contents, as well as low IC50value, suggested that E. hirta methanolic extract is a potential antioxidant agent for the development of local natural products for disease treatment. Abbreviations Used: DPPH: 2,2-diphenyl-1-picrylhydrazyl; DMSO: Dimethyl sulfoxide; RPMI: Roswell Park Memorial Institute; DMEM: Dulbecco's Modified Eagle Medium; GAE: Gallic acid equivalents; QE: quercetin equivalent; SEM: Standard error of the mean; IC50: 50% inhibition concentration; MTT: 3-(4,5-Dimethylthial-2-yl)-2, 5-diphenyltetrazalium bromide.
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	Estimation and comparison of amount of organic acids from dried leaves of Garcinia cambogia, Garcinia indica, Garcinia xanthochymus, and Garcinia morella by high-performance liquid chromatography	p. 86
	Madappa Machamada Bheemaiah, Bopaiah Ajjikuttira Kushalappa DOI:10.4103/pr.pr_159_18   Background: Pharmacological properties of (-)-hydroxycitric acid (HCA) derived from plants are extensively investigated for preventing lipid storage and controlling appetite. The Garcinia species is most sought out species to recover (-)-HCA. Objectives: Organic acids from dried leaves of Garcinia cambogia, Garcinia indica, Garcinia xanthochymus, and Garcinia morella were extracted and analyzed by high-performance liquid chromatography. Materials and Methods: Oven-dried leaves of the four plant samples were subjected to Soxhlet water extraction and further acid extraction. After evaporation, the residue was mixed with 50 ml, 30% orthophosphoric acid. 20 μL of the sample was injected to a reversed-phase C 18 column under gradient elution solvent containing 0.2 M sodium sulfate with a pH of 2.5 adjusted with dilute H2SO4. At a flow rate of 0.5 mL/min, reading was taken using ultraviolet detection at 215 nm. Chromatograms of (-)-HCA, lactone, and citric acid were obtained for standards and the samples. Results: The amount of (-)-HCA, lactone, and citric acid in the dry leaves of G. cambogia was estimated at 7.95%w/w, 3.25%w/w, and 0.13%w/w, respectively. In G. indica, the components were estimated at 5.71%w/w, 3.21%w/w, and 0.07%w/w, respectively. In the dry leaves of G. xanthochymus, it was estimated at 0.02%w/w, 0.06%w/w, and 0.18%w/w. In G. morella, it was estimated at 0.0%w/w, 0.01%w/w, and 0.29%w/w. G. cambogia and G. indica have high amounts of (-)-HCA and lactone in samples of dried leaves. (-)-HCA and lactone are absent in G. morella. Higher amounts of citric acid were found in the leaves of G. morella.Abbreviations Used: HCA: Hydroxycitric acid; HPLC: High-performance liquid chromatography.
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SHORT COMMUNICATIONS
	Ashwagandha reverses the dieldrin-induced cognitive impairment by modulation of oxidative stress in rat brain	p. 92
	Tandra Ghosh, Sanvidhan G Suke, Chandrashekhar Yadav, Rafat Ahmed, Basu Deb Banerjee DOI:10.4103/pr.pr_77_18   Dieldrin (DLD) is an organochlorine pesticide heavily used in agriculture to control pests. Widespread exposures of DLD to human population are likely to contribute in neurological disorders. Withania somnifera (WSF), commonly known as "ashwagandha," is used for its broad spectrum of pharmacological activity. The present study was designed to investigate the effect of WSF (100 mg/kg) on DLD (5 mg/kg)-induced modulation of cognitive function and oxidative stress in male Wistar rats. Cognitive function was measured using step-down latency (SDL) on a continuous avoidance apparatus and transfer latency (TL) on an elevated plus maze. Oxidative stress was estimated by measuring brain malondialdehyde (MDA) level, protein carbonyl (PC), and reduced glutathione (GSH) activity. Significant reduction in both acquisition and retention in SDL was found for the DLD-treated group at the end of the exposure study as compared to the control (P < 0.001). DLD caused a significant prolongation in both acquisition and retention in TL after 28 days of the treatment as compared to the control (P < 0.001). Four-week treatment of WSF antagonized the effect of DLD on SDL and TL at the 29th day. DLD produced a statistically significant increase in the brain MDA and PC levels (P < 0.001), and a significant decrease in the brain GSH activity (P < 0.001). Treatment with WSF attenuated the effect of DLD on MDA, PC, and GSH activities. Thus, the finding of this study suggests that WSF has potential in reversing cognitive dysfunction and oxidative stress induced by toxicants such as DLD in the brain. Abbreviations Used: DLD: Dieldrin; GSH: Reduced glutathione; LD: Lethal dose; LPO: Lipid peroxidation; MDA: Malondialdehyde; OCP: Organochlorine pesticide; PC: Protein carbonyl; SDL: Step-down latency; SFZ: Shock-free zone; TL: Transfer latency; WSF: Withania Somnifera.
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	Suppression of polyps formation by saffron extract in Adenomatous polyposis coliMin/+mice	p. 98
	Kyoko Fujimoto, Tomoe Ohta, Hitomi Yamaguchi, Nguyen Huu Tung, Gen Fujii, Michihiro Mutoh, Takuhiro Uto, Yukihiro Shoyama DOI:10.4103/pr.pr_152_18   Saffron (Crocus sativus L.) has been used both as a food additive for flavoring and coloring and in traditional medicine. Saffron extract and its main component crocin decrease the growth of several types of human cancer, including colorectal cancer in vitro. Numerous polyps develop in the small intestine in the Adenomatous polyposis coli (Apc) deficiency mice. ApcMin/+ mice are models for human familial adenomatous polyposis and human colon cancer patients. In this study, we examined the efficacy of saffron extract added to diet on reducing the polyp density in ApcMin/+ mice. ApcMin/+ mice were either given a placebo or saffron extract (0.1% and 0.5%) diet for 4 weeks. At 12 weeks of age, intestines were analyzed for polyp number in the small intestine. Our analysis confirmed that crocin (1), crocin-2 (2), and crocin-4 (4) are the major compounds in the saffron extract and the content of 1 in the tested saffron extract was 29.2%. Saffron extract decreased the number of intestinal polyps in a concentration-dependent manner in ApcMin/+ mice. Notably, the number of polyps in the distal small intestine of the mice fed with 0.5% saffron extract was significantly decreased compared with the placebo. These results indicate that saffron extract can reduce the polyp number in the ApcMin/+ mice. Abbreviations Used: FAP: Familial adenomatous polyposis; Apc: Adenomatous polyposis coli.
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ERRATUM
	Erratum: Antihyperglycemic potential of saponin-enriched fraction from Pithecellobium dulce benth. seed extract	p. 102
	DOI:10.4103/0974-8490.252573
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