Magister Pharmaceuticae - MPharm / Evaluation of the antinociceptive, anti-inflammatory and antipyretic activities of Ruta graveolens L. in mice and rats FIRDOWS LOONAT M. Pharm. Pharmaceutical Sciences thesis: School of Pharmacy, University of the Western Cape Ruta graveolens (Rutaceae) L. is a medicinal plant that is commonly used to manage and treat essential events such as pain, inflammation and fever. Despite its popularity, particularly as a medicinal plant in the Calvinia district and Bredasdorp region of South Africa, scientific data to substantiate its widespread traditional use and the possible mechanisms of action for this plant species is lacking. Therefore, the objectives of this study were: to scientifically evaluate and validate the antinociceptive, anti-inflammatory and antipyretic activities of Ruta graveolens using the acetic-acid writhing test and hot-plate test, the carrageenan rat paw oedema test, and the E. coli-induced pyrexia test, respectively; to investigate the possible mechanisms of the antinociceptive, anti-inflammatory and antipyretic activities of the plant using interaction studies; to determine some secondary metabolites present in the plant species using standard phytochemical analytical procedures; to characterise the plant species using HPLC techniques; and to determine the safety profile of the plant species using an acute toxicity study.Three percent (3 %) acetic acid (0.25 ml, i.p.) produced a substantial number of writhes in mice. The leaf methanol extract of Ruta graveolens (100 mg/kg, i.p.) significantly reduced the number of writhes induced by 3 % acetic acid (0.25 ml, i.p.). R. graveolens (100 mg/kg,i.p.) produced 54 % inhibition of 3 % acetic acid-induced writhes. Indomethacin (20 mg/kg,i.p.) and paracetamol (500 mg/kg, i.p.) significantly reduced the number of 3 % acetic acidinduced writhes. Indomethacin (20 mg/kg, i.p.) and paracetamol (500 mg/kg, i.p.) produced 57 % and 80 % inhibition of 3 % acetic acid-induced writhes, respectively. R. graveolens (25– 50 mg/kg, i.p. and 200 – 400 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.) did not significantly reduce the number of writhes induced by 3 % acetic acid. However, combined therapy of the leaf methanol extract of R. graveolens (25 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.) significantly reduced the number of 3 % acetic acid-induced writhes. The combined therapy of the lowest and sub-effective doses of the leaf methanol extract of R. graveolens (25 mg/kg, i.p.) and indomethacin (10 mg/kg, i.p.) produced 59 % inhibition of the writhes elicited by 3 % acetic acid. The leaf methanol extract of R. graveolens (50 – 400 mg/kg, i.p.) greatly delayed the reaction time in mice to thermal stimulation produced with hot-plate. 50 – 400 mg/kg (i.p.) of the leaf methanol extract of R. graveolens significantly antagonised rat paw oedema induced by 1 % carrageenan (0.1 ml, subplantar) over the 4 h period of testing. In addition, indomethacin (10 mg/kg, i.p.) significantly antagonised 1 % carrageenan-induced rat paw oedema. R. graveolens (25 mg/kg, i.p.) and indomethacin (2mg/kg, i.p.) given separately did not significantly alter rat paw oedema induced by 1 % carrageenan. However, combined therapy of the leaf methanol extract of R. graveolens (25 mg/kg, i.p.) and indomethacin (2 mg/kg, i.p.) significantly reduced 1 % carrageenan-induced rat paw oedema. The leaf methanol extract of R. graveolens (400 mg/kg, i.p.) significantly reduced the mean rectal temperature of normothermic rats. Ruta graveolens (100 – 400 mg/kg, i.p.) significantly reduced pyrexia induced by E. coli (50 μg/kg, i.m.) over the 5 h period of testing. In addition, pentoxifylline (50 mg/kg, i.p.) significantly reduced E. coliinduced pyrexia. Ruta graveolens (25 – 50 mg/kg, i.p.), paracetamol (500 mg/kg, i.p.) and pentoxifylline (10 mg/kg, i.p.) did not significantly reduce pyrexia induced by E. coli.However, combined therapy of the leaf methanol extract of R. graveolens (25 mg/kg, i.p.)and pentoxifylline (10 mg/kg, i.p.) significantly reduced E. coli (50 μg/kg, i.m.)-induced pyrexia.The phytochemical studies of the powdered leaves of Ruta graveolens indicated the presence of alkaloids, cardiac glycosides, flavonoids, saponins, tannins and triterpene steroids. The HPLC fingerprint indicated characteristic peaks at the following retention times; 1.654 min,2.271 min, 2.403 min, 4.705 min and 7.691 min. The LD50 obtained for Ruta graveolens after oral administration was probably greater than 4000 mg/kg which shows that the plant extract is non-toxic to mice.In conclusion, the data obtained indicate that Ruta graveolens possesses antinociceptive, antiinflammatory and antipyretic activities. Since prostaglandins have been shown to mediate acetic acid-induced writhes, prostaglandins, histamine, serotonin, capsaicin and bradykinin implicated in carrageenan-induced rat paw oedema, and tumor necrosis factor-α (TNF-α) implicated in E.coli-induced pyrexia, it is possible that R. graveolens may be producing its antinociceptive, anti-inflammatory and antipyretic activities by affecting these chemical mediators. The data obtained also justify the use of the plant species by traditional medicine practitioners for the treatment of painful and inflammatory conditions, and pyrexia.

Aims: Cancer is the first cause of death in the Peruvian population; searching alternative treatments of medicinal plants constitute a promissory field to find new anticancer drugs. The main objective in this study was to evaluate the phytochemical screening, total phenolic content, antioxidant and cytotoxic activity of ethanol extract of Chromolaena laevigata (C. laevigata) on human tumor cell lines. Study Design: The fresh leaves of C. laevigata were soaked with ethanol followed by phytochemical screening using standard methods. Place and Duration of Study: Laboratory of Applied Chemistry, Faculty of Pharmacy and Biochemistry, Universidad Nacional San Luis Gonzaga de Ica, Ica, Peru; Laboratory “Abraham Vaisberg Wolach”, Universidad Peruana Cayetano Heredia, Lima, Peru. Methodology: Phytochemical screening was assessed by using chemical reactives. Total phenolic content (TPC) was developed using Folin Ciocalteu reactive and the antioxidant activity was determined against DPPH and ABTS radicals by spectrophotometry. The cytotoxic activity was determined on human tumor cell lines followed as: MCF-7, H-460, HT-29, M-14, K-562 and DU-145. Results: Phytochemical study confirmed flavonoids and phenolic compounds in ethanol extract. TPC resulted 45.21 ± 3.5 mg of gallic acid equivalent/g of dried extract. The highest antioxidant extract for DPPH and ABTS radical scavenging tests were IC50 = 11.66 ± 1.0 μg/mL, IC50= 12.45 ± 0.50 μg/mL respectively. Ethanolic extracts (μg/mL) showed a low cytotoxicity on human tumor cell lines (CI50 > 20 μg/mL) for DU-145, HT-29, MCF-7 and M-14. Whereas, for H-460, and K562 tumor cell lines showed high cytotoxicity. Conclusion: In our findings, C. laevigata demonstrated a high antioxidant and total phenolic content. The ethanol extract exhibited better cytotoxic effect compared with 5-FU. Hence, This medicinal plant could be effective to prevent chronical diseases as cancer and oxidative stress disorders.

Medicinal plants are the focus of intense study, in particular whether their traditional uses are supported by real pharmacological effects, or merely based on folklore. Piper capense L.f. (Piperaceae) is used traditionally for the treatment of infectious diseases, and has the potential to be a source of novel antimicrobial compound(s). Crude solvent extracts (water, methanol, hexane and acetone) and sequentially extracted subfractions of the root-bark of P. capense were prepared, of which the hexane-soluble subfraction MsAsHs was identified as the most promising antimicrobial subfraction. Phytochemical analyses of the various extracts and subfractions using TLC with numerous mobile phases and compound selective visualising reagents revealed the presence of quinones in all of the crude solvent extracts. Alkaloids, lipids/sterols/steroids, phenolic compounds and amino acids/peptides were detected in select subfractions. Gradient reverse phase HPLC analyses using 0.1% formic acid and methanol indicated three major peaks in MsAsHs. IR spectroscopy indicated that carbonyl and hydroxyl functional groups, and aromatic characteristics were present in the major compound present in MsAsHs. Further analysis using targeted LC-MS Q-TOF and quadrupole LC-MS/MS analyses indicated an empirical formula of C11H8O3. This formula was confirmed for the isolated compound by GC-MS (HP5-MS column) that identified the compound as 5-hydroxy-2-methyl-1,4-naphthoquinone (C11H8O3 MW: 188.18) with 98% certainty using the database. Although 5-hydroxy-2-methyl-1,4-naphthoquinone (also known as plumbagin) is well-known, this is the first time that the presence of this compound is reported in the Piper genus. Antimicrobial activities of P. capense root-bark extracts and the subfractions were determined against Gram-negative and Gram-positive bacteria and a yeast strain using the disk diffusion and broth micro-dilution assays. Antimicrobial activity was observed against Gram-positive bacteria, Gramnegative bacteria as well as a yeast strain, indicating broad spectrum activity. The antimicrobial activities of the crude solvent extracts decreased in the order: acetone > methanol > hexane > water. The MsAsHs subfraction demonstrated the highest antimicrobial activity with an MIC of 29 μg/ml against both Staphylococcus aureus (ATCC 12600) and Candida albicans (ATCC 10231). HPLC eluents of this subfraction that were collected in a drop-wise fashion onto silica TLC plates and assayed by bioautography, indicated that the major compound eluting at 13.6 minutes accounted for most of the antimicrobial activity. Antioxidant activity was observed for the crude water extract, crude methanol extract, crude acetone extract, MsAsAs subfraction as well as the MsAsHs subfraction. Cytotoxicity against mammalian cells in culture was observed for the crude methanol extract, crude acetone extract, crude hexane extract and the MsAsHs subfraction when determined using C2C12 cells as well as resting and PHA stimulated lymphocytes. Stability testing of the MsAsHs subfraction revealed that the antimicrobial compounds found in this subfraction appear to be stable up to 30 days at both 25°C and 40°C when assayed against S. aureus. However, when assayed against C. albicans, there was an increase in antifungal activity from 29 μg/ml to < 7 μg/ml after 30 days at both temperatures tested. This study provides scientific support for the ethnomedical use of the rootbark of P. capense as an antimicrobial. To date, the presence of plumbagin has not been reported in any other plant in the Piper genus. Due to the significant cytotoxic activity against mammalian cells reported in the current study and the mechanism of action of plumbagin, the therapeutic potential of P. capense extracts is very limited due to non-selective cytotoxicity, despite its marked antimicrobial activity. / Dissertation (MSc)--University of Pretoria, 2011. / Pharmacology / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22494号 / 農博第2398号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5274(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 栗原 達夫, 教授 加納 健司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Plants synthesize a diverse array of phytochemicals in response to interactions with herbivores, pathogens, and commensal microbes. These phytochemicals may simultaneously enhance crop defense and quality, representing a potential pest management strategy. However, plant chemical responses to different types and levels of biotic interactions remain unclear, particularly in fruit tissues, and the feasibility of inducing these defenses through elicitor application in field environments also requires further examination. Thus, apples were used to 1) examine the impact of distinct communities of biotic interactions among plants, insects, and microbes on fruit phenolic chemistry, and 2) examine the impact of the phytohormones jasmonic acid (JA), salicylic acid (SA), and melatonin (M) on fruit phenolic chemistry and resistance against pests and pathogens. Ultimately, phenolic defenses were induced by fungal damage primarily in ripe pulp tissues, where there was also a positive relationship between fungal endophyte and phenolic diversity, supporting a broad hypothesis that chemical diversity may increase with biotic diversity. Additionally, two compounds were upregulated in response to fungal damage: chlorogenic acid and an unidentified benzoic acid. Elicitor applications did not affect phenolic chemistry, but the combined application of JA-SA analogues had some chemical or physical effect, as this treatment reduced emergence of the insect Rhagoletis pomonella. Thus, fruit induced defenses may be tissue-specific and subject to temporal, environmental, or genotypic variation. Overall, these chapters examined the relationship between biotic interactions and induced fruit chemistry, with the goal of improving understanding of plant-microbe-insect interactions and incorporating these interactions into more sustainable agricultural practices. / Master of Science / Plants may produce a diverse array of defensive phytochemical compounds in response to interactions with herbivores, pathogens, and the microorganisms that reside within plant tissues. These phytochemicals may simultaneously improve crop defenses and quality, representing a potential agricultural management strategy. However, plant chemical responses to different types and levels of biotic interactions are not well-understood, particularly in fruit tissues, and the feasibility of activating these defenses in fruits through the application of phytohormones that regulate defense pathways as a potential management strategy also requires further examination. Thus, apples were used to 1) examine the impact of distinct communities of biotic interactions among plants, insects, and microbes on fruit chemistry, focusing on phenolics, an important class of phytochemical compounds, and 2) examine the impact of the defense-activating phytohormones jasmonic acid (JA), salicylic acid (SA), and melatonin (M) on fruit phenolic chemistry and resistance against pests and pathogens. Ultimately, phenolic defenses were activated by fungal damage primarily in ripe pulp tissues, where there was also a positive relationship between fungal endophyte and phenolic diversity, supporting a broad hypothesis that chemical diversity may increase with biotic diversity. Additionally, two compounds were produced in response to fungal damage: chlorogenic acid and an unidentified benzoic acid. Phytohormone applications did not affect phenolic chemistry, but the application of the combined JA-SA analogues had some chemical or physical effect, as this treatment reduced emergence of the insect Rhagoletis pomonella. Overall, the phytochemical defenses activated by biotic interactions in fruits may occur primarily in certain tissue types, and may also vary due to environmental conditions, time of year, or plant species. These chapters examined the relationship between fruit chemistry and biotic interactions with the goal of improving understanding of plant-microbe-insect interactions and incorporating these interactions into more sustainable agricultural practices.

Thesis (PhD(Agric))--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The development and application of science-based tools for determining optimum fruit maturity and postharvest handling protocols to maintain quality and reduce losses during postharvest handling and marketing is essential to maintain the competitiveness of the emerging pomegranate industry in South Africa. Currently, there are no quality standards for the South African pomegranate industry, neither is there a general consensus on the optimal harvest maturity indices for fruit cultivars. These information are important to ensure the delivery of good quality fruit to consumers, particularly for long supply chains. The overall aims of this study were (a) to develop science-based management tools for determining optimum maturity indices and storage performance of pomegranate fruit cultivars grown in South Africa, and (b) to characterise the physico-chemical and pharmacological properties of selected cultivars relevant to postharvest handling and industrial applications. In Section II, seasonal studies on pomegranate (‘Baghwa’ and ‘Ruby’) fruit growth and the evolution of maturity indices during development were conducted. Significant increases in total soluble solids (TSS), sugars (glucose and fructose) and anthocyanin composition, coupled with significant decline in titratable acidity (TA), organic acids and total phenolics (TP) occurred with advancing fruit maturity. Fruit at advanced maturity stages were characterized by intense pigmentation of peel and aril, which coincided with maximum accumulation of anthocyanins. Among all the major maturity indices investigated, TSS, BrimA and anthocyanins did not show significant (p<0.05) seasonal variability, and strong correlations were found among the indices. In combination, these indices accounted for fruit juice sugar content, acidity and colour and could serve as reliable markers to determine optimal maturity for both pomegranate cultivars. The studies in Section III focused on characterization of postharvest quality including nutritional, medicinal and antioxidant properties of fruit parts. Quality attributes of eight commercial cultivars were analysed by cluster analysis, which enabled the cultivars to be separated into two clusters (cluster 1 = ‘Ruby’, ‘Arakta’ and ‘Ganesh’; cluster 2 = ‘Bhagwa’, ‘Acco’ and ‘Herskawitz’) and two ungrouped cultivars (‘Molla de Elche’ and ‘Wonderful’) based on important quality attributes (size, texture, colour, soluble solids, acidity, juiciness and phenolics). Furthermore, pomegranate fruit peel extracts were studied to highlight their potential for value-adding in pharmaceutical and other industrial applications. The results showed that fruit peels of the investigated cultivars possess strong antibacterial, antioxidant and antityrosinase activities, and hence could be exploited as potential sources of natural antimicrobial and antioxidant agents, as well as a potential tyrosinase inhibitor. The research reported in Section IV investigated the effects of harvest maturity and storage conditions on postharvest quality and nutritional value of ‘Bhagwa’ and ‘Ruby’ cultivars. Fruit harvested at commercial maturity were stored at 5±0.3°C, 7±0.5°C and 10±0.4°C with 92±3% RH and at room temperature (20±2.2°C, 65±5.5% RH) for 16 weeks. Fruit physiological responses and quality were affected by storage condition, with the maximum levels of respiration occurring at higher temperature and extended storage duration. Fruit colour and antioxidant capacity varied slightly among storage temperatures, with total soluble solids and titratable acidity decreasing gradually over time at different temperatures. Considering that fruit stored at 5°C and 92% RH had significantly reduced weight loss, low incidence of physiological disorders and best results in maintaining flavour attributes (TSS and TA, TSS:TA ratio), the investigated cultivars may be stored at 5°C and >92% RH for 8 - 12 weeks. In paper 9 (Section IV), the research investigated the relationships between instrumental and sensory measurements of pomegranate fruit at different harvest maturities during storage and shelf life. Mature ‘Bhagwa’ fruit harvested at different times could not be discriminated by sensory attributes assessed by a trained panel. However, TSS (R2 = 0.677) and juice content (R2 = 0.512) were the two most decisive quality attributes at shelf life related to harvest maturity status. For ‘Ruby’, however, a combination of instrumental and sensory attributes appeared to be influential in discriminating mature fruit harvested at different times, with TSS:TA ratio being the most decisive (R2 = 0.654) in distinguishing different fruit harvests, followed by sweet taste (R2 = 0.474) and hue angle (R2 = 0.431). The results showed that to ensure the best post-storage quality of ‘Bhagwa’, the optimum harvest maturity was between 167 - 175 DAFB (H2 and H3) when fruit reached maximum TSS level (>16°Brix; H3) and juice content (>65 mL/100 g aril; H2). However, for ‘Ruby’, this study indicated that the optimum harvest date was at 143 DAFB (H2) when TSS:TA ratio was >55, which coincided with significantly higher sensory rating for sweet taste after shelf life of fruit at H2 than H1 and H3, respectively. The results from this thesis provide new understanding and better insights on fruit characteristics of major pomegranate cultivars grown in South Africa. Overall, the study provides new knowledge on science-based tools for assessing fruit readiness for harvest as well as storage conditions to maintain fruit postharvest quality and reduce losses. It also provides scientific information on phytochemical contents and antioxidant compounds in fruit to promote value-adding of pomegranate as a good raw material with potential applications in health food products and other industrial applications such as pharmaceuticals and cosmetics. / AFRIKAANSE OPSOMMING; Die ontwikkeling en toepassing van wetenskapgegronde instrumente vir die bepaling van optimale vrugrypheid en naoes-hanteringsprotokolle om gedurende die naoes-hantering en -bemarking van vrugte gehalte te behou en verliese te verminder, is noodsaaklik om die mededingendheid van die ontluikende granaatbedryf in Suid-Afrika te verseker. Tans is daar nie enige gehaltestandaarde vir die Suid-Afrikaanse granaatbedryf óf algemene eenstemmigheid oor die optimale oesrypheidsaanwysers vir vrugtekultivars nie. Hierdie inligting is belangrik om die naoes-lewering van uithalervrugte aan verbruikers te verseker, veral vir lang verskaffingskettings. Die oorkoepelende doelwitte van hierdie studie was (a) om wetenskapgegronde bestuursinstrumente te ontwikkel vir die vasstelling van optimale rypheidsaanwysers en bergingsprestasie van granaatkultivars wat in Suid-Afrika verbou word, en (b) om die fisiko-chemiese eienskappe en farmakologiese kenmerke van gekose kultivars te tipeer. In deel II is seisoenale studies oor granaatgroei en die ontwikkeling van rypheidsaanwysers gedurende groei onderneem. Namate vrugte ryp geword het, is beduidende toenames in totale oplosbare vaste stowwe (TSS), suikers (glukose en fruktose) en antosianien-samestelling opgemerk, sowel as ’n beduidende afname in titreerbare suur (TA), organiese suur en totale fenol (TP). Vrugte in gevorderde stadia van rypheid is gekenmerk deur intense pigmentasie van die skil en aril, wat met maksimum opbou van antosianien verband gehou het. Van ál die belangrike rypheidsaanwysers wat ondersoek is, het TSS, BrimA en antosianien onbeduidende (p<0.05) seisoenale veranderlikheid getoon, en is sterk verbande tussen die aanwysers opgemerk. Gesamentlik sou die aanwysers kon rekenskap gee van sapsuikerinhoud, -suurgehalte én -kleur, en sou dit dus as betroubare rypheidsmerkers kon dien om optimale rypheid vir albei granaatkultivars te bepaal. Die studies in deel III het gekonsentreer op die tipering van die naoes-kenmerke, onder meer die voedings-, medisinale en antioksidant-kenmerke van vrugtedele. Kenmerke van agt kommersiële kultivars is deur middel van groepsontleding bestudeer, waarvolgens die kultivars op grond van belangrike kenmerke (grootte, tekstuur, kleur, oplosbare vaste stowwe, suurgehalte, sappigheid en fenol) in twee groepe (groep 1 = ‘Ruby’, ‘Arakta’ en ‘Ganesh’; groep 2 = ‘Bhagwa’, ‘Acco’ en ‘Herskawitz’) en twee niegegroepeerde kultivars (‘Molla de Elche’ en ‘Wonderful’) ingedeel is. Ten einde die toegevoegde waarde van granaatskille vir farmaseutiese en kosmetiese doeleindes te bevorder, is skilekstrakte ook bestudeer. Die resultate toon dat die vrugteskille van die bestudeerde kultivars oor sterk antibakteriese, antioksidant- en antitirosinase- eienskappe beskik. Daarom kan die skil van die granaatkultivars as moontlike bron van natuurlike antimikrobiese en antioksidant-agense sowel as ’n moontlike tirosinase-inhibitor ontgin word. Die navorsing in deel IV het ondersoek ingestel na die uitwerking van oesrypheid en bergingsomstandighede op die naoes-gehalte en -voedingswaarde van die kultivars ‘Bhagwa’ en ‘Ruby’. Vrugte wat op kommersiële rypheid geoes is, is vir 16 weke by 5±0.3 °C, 7±0.5 °C en 10±0.4 °C met 92±3% RH, sowel as by kamertemperatuur (20±2.2 °C, 65±5.5% RH) geberg. Die bergingsomstandighede het die fisiologiese reaksies en gehalte van die vrugte beïnvloed: Maksimum vlakke van respirasie het teen hoër temperature en met verlengde berging voorgekom. Die kleur en antioksidantvermoë van die vrugte het effens tussen bergingstemperature verskil, en totale oplosbare vaste stowwe en titreerbare suur het mettertyd geleidelik by verskillende temperature afgeneem. Gedagtig daaraan dat die vrugte wat teen 5 °C en 92% RH geberg is beduidend minder gewigsverlies, ’n lae voorkoms van fisiologiese afwykings en die beste resultate in blywende geurkenmerke (TSS en TA, TSS:TA-verhouding) getoon het, kan die bestudeerde kultivars vir 8 tot 12 weke teen 5 °C en >92% RH geberg word (navorsingstuk 8). In navorsingstuk 9 (deel IV) is daar ondersoek ingestel na die verhouding tussen instrument- en sintuiglike metings van granate in verskillende stadia van oesrypheid gedurende berging en raklewe. Geen verskil in sintuiglike kenmerke kon bespeur word by ryp ‘Bhagwa’- vrugte wat op verskillende tye geoes is nie. Tog was TSS (R2 = 0.677) en sapinhoud (R2 = 0.512) die twee bepalendste gehaltekenmerke wat betref oesrypheidstatus gedurende raklewe. By ‘Ruby’ kon ’n kombinasie van instrument- en sintuiglike kenmerke egter wél tussen stadia van oesrypheid onderskei, met die TSS:TA-verhouding die bepalendste (R2 = 0.654) in die onderskeid tussen verskillende vrugteoeste, gevolg deur ’n soet smaak (R2 = 0.474) en skakeringshoek (R2 = 0.431). Die resultate toon dat die beste nabergingsgehalte vir ‘Bhagwa’ verkry word by ’n optimale oesrypheid van 167–175 DAFB (H2 en H3), wanneer vrugte die maksimum TSS-vlak (>16°Brix; H3) en sapinhoud (>65 mL/100 g aril; H2) bereik het. Vir ‘Ruby’ dui hierdie studie op ’n optimale oesdatum van 143 DAFB (H2) met ’n TSS:TA-verhouding van >55, wat verband gehou het met ’n beduidend hoër telling vir soet smaak by H2 eerder as by H1 en H3 ná raklewe. Die resultate van hierdie tesis bied ’n beter begrip van, en insig in, die vrugtekenmerke van granaatkultivars wat in Suid-Afrika verbou word. Oor die algemeen bied die studie wetenskaplike inligting om moontlik die toegevoegde waarde van granate as ’n goeie bron van minerale elemente sowel as farmaseutiese, kosmetiese en antioksidant-verbindings te bevorder. Dit bied ook kennis oor die ontwikkeling van wetenskapgegronde instrumente vir die vasstelling van optimale vrugrypheid en naoes-hanteringsprotokolle om gedurende die naoes-hantering en -bemarking van granate vruggehalte te behou en verliese te verminder.

Os endoparasitas têm demonstrado resistência aos principais anti-helmínticos. Por isso é necessário investigar estratégias sustentáveis, sendo uma das alternativas a utilização de plantas leguminosas (Fabaceae) no controle de nematóides. Objetivou-se avaliar a atividade anti-helmíntica de 17 extratos de plantas no teste de inibição da eclodibilidade dos ovos, teste de inibição de desembainhamento de larvas de Haemonchus contortus e o teste de motilidade do Caenorhabditis elegans, in vitro. As duas melhores plantas foram levadas para teste in vivo (Arachis pintoi cv Amarillo e Caesalpinia echinata). Os extratos foram feitos a partir das folhas de Arachis pintoi cv Amarillo, Caesalpinia echinata, Cajanus cajan, Calopogonium velutinum, Canavalia ensiformis, Centrosema pubencens cv Cardillo, Cratylia mollis, Dipteryx alata, Gliricidia sepium, Leucaena leucocephala cv Cunningham, Leucaena pulverulenta, Mimosa caesalpiniaefolia, Neonotonia wighiti cv Cianova, Noenotonia wighiti cv Tinaroo, Piptadenia colubrina, Stylosanthes guianensis e Styzolobium aterrimun, as quais foram coletadas no campo agrostológico do Instituto de Zootecnia em Nova Odessa/SP. Seus princípios ativos foram extraídos em solução de acetona:água 70:30, solvente retirado por rotaevaporação, seguido por lavagem com diclorometano e liofilizado. Os resultados obtidos para o TIEO, TIDL e para o TMCE foram baseados na concentração letal de 50% (CL50), maior concentração testada 50 mg/mL e a menor 0,0975 mg/mL nos TIEO e TIDL, e TMCE a maior foi 50 mg/mL e a menor 0,039 mg/mL. Em ordem da maior para menor eficácia no TIEO foram para o extrato de P. colubrina (0,10 mg/mL), extrato de C. echinata (0,17 mg/mL) e extrato M. caesalpiniaefolia (0,53 mg/mL). Os resultados do TIDL em ordem da maior para menor eficácia foram para o extrato de S. guianensis (0,51 mg/mL), extrato de A. pintoi cv Amarillo (1,04 mg/mL) e extrato de C. echinata (1,25 mg/mL). O resultado obtido com maior eficiência na CL50 do TMCE foi o extrato de C. echinata (0,49 mg/mL), o extrato de M. caesalpiniaefolia (1,03 mg/mL), extrato de Dipteryx alata (1,50 mg/mL) e extrato de P. colubrina (1,56 mg/mL). Para o experimento in vivo utilizou-se 30 ovinos, PV médio de 32 kg (+/- 0,59), mestiços Dorper/Santa Inês, machos, não castrados. Animais foram divididos em três tratamentos: A. pintoi (A), C. echinata (B) e controle (C) infectados sem tratamento, cada tratamento continha 10 animais. Animais dos tratamentos A e B foram tratados com 10 g da planta moída juntamente no concentrado. Foram colhidos sangue e fezes para análises. Final do experimento os animais foram abatidos e abomaso lavado para a recuperação dos H. contortus. O tratamento A obteve resultado significativo de OPG (1015) quando comparado com OPG dos tratamentos B (1244) e C (1537). O comprimento dos nematóides machos (A = 1,24 cm; B = 1,31 e C = 1,35) e fêmeas (A = 1,83 cm; B = 1,91 e C = 1,97) encontrados em cada tratamento teve diferença significativa, sendo a quantidade de nematóides do tratamento A menores. Conclui-se que o experimento com a planta A. pintoi foi que propiciou redução no OPG e comprimento dos nematóides sugerindo ação anti-helmíntica com potencial de uso no controle sustentável desta verminose / Endoparasites have demonstrated resistance to the main anthelmintics found in the Market and new sustainable strategies are needed to control them. This work aimed to evaluate the anthelmintic activity of 17 extracts of taniniferous plants that belongs to the Fabaceae family by in vitro egg hatch test (EHT), larval exsheathment test (LET) with Haemonchus contortus and larval motility test (LMT) with Caenorhabditis elegans. Arachis pintoi cv Amarillo and Caesalpinia echinata. Arachis pintoi cv Amarillo, Caesalpinia echinata, Cajanus cajan, Calopogonium velutinum, Canavalia ensiformis, Centrosema pubencens cv Cardillo, Cratylia mollis, Dipteryx alata, Gliricidia sepium, Leucaena leucocephala cv Cunningham, Leucaena pulverulenta, Mimosa caesalpiniaefolia, Neonotonia wighiti cv Cianova, Noenotonia wighiti cv Tinaroo, Piptadenia colubrina, Stylosanthes guianensis and Styzolobium aterrimun were collected from Agrostological field from Instituto de Zootecnia (Nova Odessa/SP). The phytochemicals were extracted by maceration in acetone:water (70:30). The acetone was rotoevaporated and the aqueous extract was cleared with dicloromethane. The remaining extract was liofilized and kept frozen until the use in vitro tests. EHT, LET were performed with doses ranging from 50 mg/mL to 0.0975 mg/mL and LMT with doses ranging from 50 mg/mL to 0.039 mg/mL. LC50 were calculated to compare efficacies. The extracts P. colubrina (0.1 mg/mL), C. echinata (0.17 mg/mL) and M. caesalpiniaefolia (0.53 mg/mL) presented the best results in EHT. L. pulverulenta, C. cajan, C. mollis and S. aterrimun were not effective at the higher dose in EHT (50 mg/mL). In the LET the extracts of S. guianensis (0.51mg/mL), A. pintoi cv Amarillo (1.04 mg/mL) and C. echinata (1.25 mg/mL) had the best results. In LMT, the extracts of C. echinata (0.49 mg/mL), M. caesalpiniaefolia (1.03 mg/mL), D. alata (1.5 mg/mL) and P. colubrina (1.56 mg/mL) had the best results. A. pintoi and C. echinata were chosen to be tested in vivo. In order to evaluate the plants over lambs infected with parasites, 30 lambs with average body weight of 32 kg (+/- 0,59 kg), Dorper x Santa Ines, male and not castrated were divided equaly in 3 treatments: (A) A. pintoi 10 g/day, (B) C. echinata 10 g/day and (C) control. The plants were grinded and mixed with concentrate and offered during all experiment. Blood and feces were collected for analysis. At the end of experimental period, animals were slaughtered and H. contortus were recovered from abomasum for counts, measurements and evaluation of female fertility (count of eggs). Treatment (A) with A. pintoi had lower FEC (1015) when compared with (B) C. echinata with FEC of 1244 and (C) Control with FEC of 1537 (P<0.05). The length of male worms (A = 1,24 cm; B = 1,31 cm e C = 1,35 cm) and female (A = 1,83 cm; B = 1,91 cm e C = 1,97 cm) presented differences (P<0.05), as the (A) group present smaller length. It was concluded that A. pintoi cv Amarillo reduced FEC and the size of the worms suggesting potencial anthelmintic effect in the sustainable control of parasites

No presente trabalho, espécimes de Mikania cordifolia coletados em Ribeirão Preto, São Carlos, Campos do Jordão e Monte Verde são analisados comparativamente por métodos cromatográficos (CG e CLAE) para a pesquisa de triterpenóides, lactonas sesquiterpênicas e flavonóides. Todos os espécimes de M. cordifolia analisados apresentaram os triterpenóides -Amirina, -Amirina, Lupeol, Lupenona, Taraxasterol, Pseudotaraxasterol, Acetato de -Amirina, Acetato de -Amirina, Acetato de Lupeol, Acetato de Taraxasterol, Campesterol, Estigmasterol e -Sitosterol, as lactonas sesquiterpênicas 14-hidroxi-15[2-hidroximetilacriloiloxi]-germacra-1(10)E,4Z-11(13)-trien-12,8-olídeo, 14-hidroxi-15[2-hidroxi,2-metilpropanoiloxi]-germacra-1(10)E,4Z-11(13)-trien-12,8-olídeo e 14-hidroxi-15[2,3-epoxi,2-metilpropanoiloxi]-germacra-1(10)E,4Z-11(13)-trien-12,8-olídeo, os flavónóides Quercetina-3-O--galactosídeo e Rhamnazina e o ácido 3,4-diidroxi-benzóico. Apenas os espécimes de Ribeirão Preto e São Carlos apresentaram Friedelina. O extrato aquoso de M. cordifolia foi avaliado para atividade antiofídica, mostrando-se efetivo na diminuição do edema de pata de camundongo induzido por veneno de Bothrops moojeni. / In the present work, specimens of Mikania cordifolia collected at Ribeirão Preto, São Carlos, Campos do Jordão and Monte Verde were analyzed comparatively for triterpenoids, sesquiterpene lactones and flavonoids by chromatographic methods (GC and HPLC). All M. cordifolia specimens analyzed showed the triterpenoids -Amyrin, - Amyrin, Lupeol, Lupenone, Taraxasterol, Pseudotaraxasterol, -Amyrin Acetate, - Amyrin Acetate, Lupeol Acetate, Taraxasterol Acetate, Campesterol, Stigmasterol e -Sitosterol, the sesquiterpene lactones 14-hydroxy-15[2 -hydroxymethylacryloyloxygermacra- 1(10)E,4Z-11(13)-trien-12,8 -olide, 14-hydroxy-15[2 -hydroxy,2 - methylpropanoyloxy]-germacra-1(10)E,4Z-11(13)-trien-12,8 -olide e 14-hydroxy- 15[2 ,3 -epoxy,2 -methylpropanoyloxy]-germacra-1(10)E,4Z-11(13)-trien-12,8 -olide, the flavonoids Quercetin-3-O- -galactoside and Rhamnazyn and the compound 3,4- dihydroxy-benzoic acid. Only specimens collected at Ribeirão Preto and São Carlos showed Friedelin. The aqueous extract of M. cordifolia was evaluated for antiofidic activity and was found effective in reducing mice paw edema induced by venom of Bothrops moojeni

Made available in DSpace on 2015-04-22T22:02:00Z (GMT). No. of bitstreams: 1 Dissertacao - Loretta Carvalho.pdf: 5178043 bytes, checksum: 1cf81d7992561f0b9b701f5344a3a91b (MD5) Previous issue date: 2009-12-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The family Rutaceae presents a great diversity of secondary metabolites with potential biological and/or pharmaceutical, which has attracted interest in the studies of its species. Although the Amazon possess a great diversity of species belonging to this family, there are few studies involving the identification of their secondary metabolites. To contribute to the knowledge of the chemical profile of the family in the region, was selected for this study Zanthoxyllum djalma-batistae (Albuq.) P.G. Waterman and Spathelia excelsa (Krause) R.S. Cowan & Brizicky species. Thus, were collected branches of Z. djalma-batistae and foliar rachis of S. excelsa of individuals occurring in the Ducke Reserve for phytochemical studies. The branches methanolic extract of Z. djalma-batista was submitted to a partition, resulting in 4 organic phases. The hexane and dichloromethane phases, after successive fractioning gave the isolation of the mixture of the triterpenes lupeol and epilupeol. The methanolic extract of the foliar rachis of S. excelsa was fractionated on a column of silica gel yielding 56 fractions. The fraction 43 was subjected to further fractionation in columns of silica gel and Sephadex LH-20 resulting in the isolation of limonoids perforatine and limonine diosphenol, in addition to chromone identified as biflorin. The fractionation of the CH2Cl2 extract was performed on a column of silica gel, resulting in 43 fractions and the fraction 40 with solid was purified in acetone the glabretal triterpene type 3β-angeloyl-21,24-epoxy-7α, 21α,23α,25-tetrahydroxy-4α,4β,8β,10β-tetramethyl-25-dimethyl-14,18-cyclo-5α,13α,14α17α-cholestane. Also the essential oil was obtained for hydrodistillation of the foliar rachis of S. excelsa and on the basis of the analyses for GC/MS were identified 13 volatile components (sesquiterpenes), predominating caryophyllene-epoxide (20.54%), β-bisabolene (9.87%), 1,10-di-epi-cubenol (7.51%) and γ-cadinene (4.68%). / A família Rutaceae apresenta uma grande diversidade de metabólitos secundários, com potencial biológico e/ou farmacêutico, o que tem despertado interesse nos estudos de suas espécies. Apesar da Amazônia possuir uma grande diversidade de espécies pertencentes a esta família, existem poucos trabalhos envolvendo a identificação de seus metabólitos secundários. Visando contribuir para o conhecimento do perfil químico da família na região, selecionou-se para este estudo as espécies Zanthoxyllum djalma-batistae (Albuq.) P.G. Waterman e Spathelia excelsa (Krause) R.S. Cowan & Brizicky. Assim, coletaram-se galhos de Z. djalma-batistae e ráquis foliares de S. excelsa de indivíduos ocorrentes na Reserva Ducke, para estudos fitoquímicos. O extrato metanólico dos galhos de Z. djalma-batistae foi submetido a uma partição, originando 4 fases orgânicas. As fases em hexano e diclorometano, após sucessivos fracionamentos cromatográficos forneceram a mistura dos triterpenos lupeol e epilupeol. O extrato metanólico dos ráquis foliares de S. excelsa foi fracionado em coluna de sílica gel fornecendo 56 frações. A fração 43 foi submetida a novos fracionamentos em colunas de sílica gel e Sephadex LH-20 resultando no isolamento dos limonóides perforatina e limonina diosfenol, além da cromona identificada como biflorina. O fracionamento do extrato em CH2Cl2 dos ráquis foliares foi efetuado em coluna de sílica gel, resultando em 43 frações e da fração 40, contendo sólido, foi purificado em acetona o triterpeno do tipo glabretal 3β-angeloil-21,24-epóxi 7α,21α,23α,25-tetra-hidróxi-4α,4β,8β,10β-tetrametil-25-dimetil-14,18-ciclo-5α,13α,4α,17α-colestano. Também foi obtido óleo essencial, por hidrodestilação dos ráquis foliares de S. excelsa e com base nas análises por CG/EM identificaram-se 13 componentes voláteis (sesquiterpenos), predominando o epóxi-cariofileno (20,54%), β-bisaboleno (9,87%), 1,10-di-epi-cubenol (7,51%) e γ-cadineno (4,68%).

Submitted by Geyciane Santos (geyciane_thamires@hotmail.com) on 2015-06-09T14:25:49Z No. of bitstreams: 1 Dissertacao - Nivea Maria Simões Falcão.pdf: 1851149 bytes, checksum: 9f64cfee0db539642d59a412a4bf9eea (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-06-09T19:30:11Z (GMT) No. of bitstreams: 1 Dissertacao - Nivea Maria Simões Falcão.pdf: 1851149 bytes, checksum: 9f64cfee0db539642d59a412a4bf9eea (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2015-06-09T19:32:50Z (GMT) No. of bitstreams: 1 Dissertacao - Nivea Maria Simões Falcão.pdf: 1851149 bytes, checksum: 9f64cfee0db539642d59a412a4bf9eea (MD5) / Made available in DSpace on 2015-06-09T19:32:50Z (GMT). No. of bitstreams: 1 Dissertacao - Nivea Maria Simões Falcão.pdf: 1851149 bytes, checksum: 9f64cfee0db539642d59a412a4bf9eea (MD5) Previous issue date: 2010-03-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Leishmaniasis is a world public health problem wide considered by the World Health Organization as one of five infectious endemic diseases of major relevancy. The search for leishmanicids agents with fewer side effects is one of the greatest challenges in modern research, and is increasingly related to the uses of medicinal plants and other natural products. The species Caesalpinia ferrea Martius widely distributed in Brazil it is popularly used for many therapeutic purposes, including the treatment of various infections like those caused by leishmaniasis, making it a promising tests of biological activity against leishmania. The objective of this study was to evaluate by bioassays the activity of hexane and methanol extracts against protozoa, analyzing semi-purified fractions obtained from the most active. The fruits of C. ferrea were collected at the National Institute of Amazon Research, V8’s Campus in the City of Manaus - AM and processed in the departments of Natural Products and leishmaniasis and Chagas Diseases at INPA. The studies were performed with the whole fruit and its separate parts, coconut shell and seed. Hexane e methanol crude extracts were prepared from the fruits, coconut shell and seeds, which were tested to evaluate the biological activity in vitro against promastigotes of the stationary phase of the protozoan Leishmania (Viannia) guyanensis. The extracts were analyzed by thin layer chromatography Comparison (CCDC) and Nuclear Magnetic Resonance (NMR) to determine the chromatographic profile. All biological assays were carried out experimentally in vitro in microplates containing 106/mL promastigotes to axenic cultures of L. (V.) guyanensis evaluating the inhibitory activity and stimulating parasite growth. The most active crude extract was submitted to CCDC and fractionation by partition líquid-líquid resulting in semi-purified fractions that were evaluated for their chemical composition. The results showed that the epicarp methanolic extract of C. ferrea, showed anti-leishmanial activity for L. (V.) guyanensis in the concentration of 20 mg/mL/24h. However, contrary to expectations, there was a stimulatory effect on L. (V.) guyanensis by the hexanic extract obtained from the fruit seed of C. ferrea. Chemically analyzing the coconut shell and fruits methanolics extracts of, which showed anti-leishmanial activity, we verified the presence of substances such as chlorophyll, flavonoids, xanthones, tannins, triterpenes and saponins as major constituents, and shows antioxidant activity. The results showed that the methanolics extracts of coconut shell and fruitsof C. ferrea can be considered as an promising alternative in the treatment of cutaneous leishmaniasis and should be undertaken pharmacological and toxicological tests in vivo of extract, making other fractioning in order to isolate the substances responsible for the anti-leishmanial activity. / A leishmaniose é um problema de saúde pública mundial considerada pela Organização Mundial da Saúde como uma das cinco doenças infecto-parasitárias endêmicas de maior relevância. A busca por agentes leishmanicidas com poucos efeitos colaterais é um dos maiores desafios na pesquisa moderna, e cada vez mais está relacionada aos usos de plantas medicinais e outros produtos naturais. A espécie Caesalpinia ferrea Martius largamente distribuída no Brasil, é usada popularmente para muitos fins terapêuticos, inclusive no tratamento de diversas infecções como as causadas pela Leishmaniose, tornando-a promissora para a realização de ensaios de atividade biológica contra a leishmania. O objetivo deste trabalho foi o de avaliar, através de bioensaios, a atividade de extratos hexânicos e metanólicos contra protozoários, analisando também frações semi-purificadas obtidas do extrato de melhor atividade leishmanicida. Os frutos de C. ferrea foram coletados no Instituto Nacional de Pesquisas da Amazônia, Campus do V8 na Cidade de Manaus-AM, e processados nos departamentos de Produtos Naturais e de Leishmaniose e Doença de Chagas no INPA. Os estudos foram realizados com os frutos inteiros e suas partes separadas, epicarpos e sementes. Foram preparados extratos brutos hexânicos, e metanólicos a partir dos frutos inteiros e também das partes separadas, epicarpos e sementes, os quais foram submetidos aos ensaios para avaliação da atividade biológica in vitro contra formas promastigotas da fase estacionária de protozoários da espécie Leishmania (Viannia) guyanensis. Os extratos brutos foram analisados através de Cromatografia em Camada Delgada Comparativa (CCDC) e Ressonância Magnética Nuclear (RMN) para determinação do perfil cromatográfico. Todos os ensaios biológicos foram realizados experimentalmente in vitro, em microplacas contendo 106/mL formas promastigotas de culturas axênicas de L. (V.) guyanensis (MHOM/BR/75/IM 4147) avaliando-se a atividade inibitória ou ativadora de crescimento parasitário. O extrato bruto de melhor atividade biológica foi submetido à CCDC e fracionamento por partição líquido-líquido obtendo-se frações semi-purificadas que foram avaliadas quanto a sua composição química. Os resultados mostraram que o extrato metanólico do epicarpo de C. ferrea, apresentou atividade leishmanicida para L. (V.) guyanensis na concentração de 20 mg/mL/24h. No entanto, ao contrário das expectativas, houve um efeito estimulante em L. (V.) guyanensis pelo extrato hexânico obtido das sementes de frutos de C. ferrea. Analisando quimicamente o extrato metanólico de frutos e epicarpos, os quais demonstraram atividade leishmanicida, verificou-se a presença de substâncias como a clorofila, flavonóides, xantonas, taninos, triterpenos e saponinas como constituintes majoritários, além de apresentar atividade antioxidante. Os resultados obtidos demonstraram que os extratos metanólicos de frutos e epicarpos de C. ferrea podem ser considerados como importantes alternativas no tratamento das leishmanioses, devendo ser realizado testes farmacológicos e toxicológicos in vivo desses extratos, realizando outros fracionamentos a fim de se confirmar alguma substância de melhor atividade leishmanicida.