Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Plant and Pest Science, 2004. / "October 2004" Includes bibliographical references (leaves 223-243).

Thesis (Master of Health Policy and Administration)--Washington State University, May 2006. / Includes bibliographical references (p. 29-36).

Most complementary medicines contain a multitude of chemical components, some of which are claimed to contribute to the biological activity of such products. Use of complementary medicines for preventative and therapeutic purposes is increasing rapidly worldwide. Unfortunately, although control of these products is essential to ensure quality, safety, and efficacy, the quality control of most herbal preparations is currently poor to non-existent, with little or no safety and efficacy data required to support the marketing and use of these products. The objective of this study was therefore to develop suitable analytical methods to qualitatively and quantitatively analyse the relevant components (rutin, isoquercitrin, hyperoside, quercitrin, quercetin, kaempferol, hypericin, pseudohypericin and hyperforin) in St John's Wort dosage forms for quality control purposes. A gradient HPLC method using a Luna 5·mC₁₈(2) 150 x 2.00mm internal diameter (i.d.) column and UV detection, was developed for the separation of six of the relevant flavonoid compounds in St John's Wort, namely rutin, isoquercitrin, hyperoside, quercitrin, quercetin and kaempferol. The development process involved a systematic investigation of gradient conditions, flow rate, and temperature. This method was subsequently applied to assay selected commercially available St John's Wort products. This system provided the necessary accuracy, precision and reproducibility and was associated with several advantages when compared to using standard bore (4.60 mm i.d.) HPLC columns. The method developed is currently the only known method that separates all six relevant flavonoids in a reasonable run time (less than 20 minutes). It is also one of the few methods that has sufficient separation between rutin, isoquercitrin and hyperoside. A qualitative method for the fingerprinting of flavonoid components was also developed, using capillary electrophoresis (CE). CE is a rapidly growing powerful analytical technique for the separation of charged compounds. Micellar electrokinetic chromatography (MEKC) is a very powerful electrophoretic technique that is capable of selectively resolving both neutral and ionic solutes in a single run. A MEKC method suitable for the separation and determination of various flavonoid constituents used as marker compounds in Hypericum perforatum was developed. Investigations into the effect of pH, ionic strength, applied voltage and capillary dimensions on separation were performed. The optimised method was then applied to qualitatively analyse various St John's Wort products on the market. This method was found to be advantageous in that it was simple, cost-effective, required minimal sample preparation and utilised very small quantities of sample. Due to the vast differences in chemical properties between the various marker and active components in St John's Wort, it was necessary to develop separate analytical methods for the flavonoids and for the other three relevant compounds (hypericin, pseudohypericin and hyperforin). An isocratic HPLC method using a Luna 5·mC₁₈(2) 150 x 2.00mm (i.d.) column and UV detection was developed for the separation of hypericin, pseudohypericin and hyperforin. The development process involved a systematic investigation of buffer molarity, mobile phase composition, pH, flow rate, and temperature. This method was subsequently applied to assay selected commercially available St John's Wort products on the South African market. This system also provided the necessary accuracy, precision and reproducibility, as well as the advantages associated with the use of a narrow bore column as opposed to the use of the more commonly used wider bore columns. This method was validated and used to quantitate these three compounds in various commercial St John's Wort products. By applying this method to liquid chromatography – tandem mass spectrometry (LC-MS-MS), qualitative analyses of the same products was performed to obtain confirmation of the quantitative HPLC results. Mass spectrometry is a powerful detection tool that is more selective and specific than many detection systems used with HPLC. Natural medicines usually constitute a multitude of constituents with much potential interference. In this regard LC-MS-MS is a powerful tool, with its ability to unequivocally identify target analytes regardless of the presence of interferences or complex matrices. ESI-MS-MS was used for the qualitative analysis of the content of the naphthodianthrones and hyperforin in the respective tablet products assayed with HPLC. LC-MS-MS analyses were performed in order to identify the constituents and to verify the specificity of the HPLC method. High inter-product and inter-batch variability was observed for all nine compounds assayed. These quantitative results were confirmed with the respective qualitative analyses. This study confirms the need for strict quality control of herbal medicinal products commercially available to consumers.

Hypericum perforatum is a herbal medicine that has been used for centuries for the treatment of depression. Many studies have been conducted in the Northern hemisphere on the efficacy of the HP extracts produced there. These studies include clinical trials and pharmacological investigations using a standardised HP extract or a fraction of the HP extract containing certain compounds, such as hypericin, pseudohypericin, hyperforin and several of the flavonoids thought to be responsible for the antidepressant activity. The mechanism of action of HP and its constituents is still not completely clear and it is speculated that the antidepressant activity is the result of several of the compounds acting synergistically. HP is indigenous to and also cultivated in the Western Cape of South Africa. Extracts from these plants are sold in the local health shops and there are no previous studies evaluating the efficacy of these products. The aim of this thesis is to investigate the antidepressant activity of one of these products and two of its constituents, quercetin and caffeic acid, to gain further insight into their mode of antidepressant action and to compare these results with similar studies which used a standardised extract produced in the northern hemisphere. The first study investigated the effect of HP, quercetin and caffeic acid on pineal metabolism. Changes in the synthesis of melatonin produced by the pineal gland have been implicated in depression. The results showed an increase in the level of melatonin produced in the animals treated with quercetin, which suggests that this compound may mediate antidepressant activity through such a mechanism. There are no previous reports on the in vivo effects of HP or any of its constituents on pineal metabolism. The second study investigated the effect of HP, quercetin and caffeic acid on the activity of the liver enzyme, tryptophan-2,3-dioxygenase (TDO). Inhibition of this enzyme has been shown to increase plasma levels of tryptophan, a precursor of serotonin and thereby result in increased serotonin levels in the brain. Low levels of serotonin in the brain have been implicated in depression. This study revealed significant inhibition of TDO by caffeic acid and this suggests that this constituent of HP could be contributing to its antidepressant activity through such a mechanism. There are no previous reports investigating the in vivo effect of HP or any of its constituents on TDO activity. Modulation of the levels of indoleamines, serotonin (5-HT) and dopamine (DA) as well as the metabolites, 3,4 dihydroxyphenyl acetic acid (DOPAC), 5-hydroxyindole acetic acid (5-HIAA) and homovallinic acid (HVA) in the brain have been implicated in the neuropharmacology of depression. Different studies using enzyme-linked immunosorbant assay (ELISA), high performance liquid chromatography with electrochemical detection (HPLC-ECD) and liquid chromatography-mass spectrometry (LC-MS) were used to determine changes in the levels of these indoleamines brought about after treatment with HP caffeic acid and quercetin. The results of the ELISA study showed significant increases in 5-HT levels in the brains of the animals treated with caffeic acid and quercetin. The results of the HPLC-ECD studies also revealed significant increases in 5-HT levels and a decrease in the turnover of 5-HT in the animals treated with quercetin. A significant increase in DA levels in the animals treated with quercetin was shown in both the HPLC-ECD and LC-MS studies. There was also an increase in DA turnover in the animals treated with HP shown in the HPLC-ECD and LC-MS studies. These results suggest that HP and its constituents, quercetin and caffeic acid mediate their antidepressant effects through serotonergic and dopaminergic neurotransmission. Adaptive changes in the density of b-adrenergic (b-AR), 5-HT2 and N-methyl-D-aspartate (NMDA) receptors have been implicated in depression. Several studies, investigating the effect of treatment with HP and quercetin on these different receptor densities, were undertaken using radioactive binding assays. Treatment with HP resulted in significant down regulation of b-AR and NMDA receptor densities and up-regulation of 5HT2 receptors. The effects on the b-AR and 5-HT2 receptors are similar to the results reported using HP in the Northern hemisphere, but the effect on the NMDA receptors is novel providing insight into the mode of action of HP. Apoptosis of neuronal cells has been implicated in neuro-degenerative and depressive disorders. Detection of apoptosis, using fluorescent microscopy observed through the labelling of DNA strand breaks, showed a decrease in the amount of apoptosis in the animals treated with HP and quercetin. This adds further support for the use of HP as an antidepressant and these results are similar to results reported from the Northern hemisphere. The results of all these studies suggest that the quality of the locally produced tincture is similar in efficacy to that of the standardised product of the Northern hemisphere.

Production of hypericine in explantate cultures of Hypericum perforatum Pavel Sazama Diploma thesis Charles university in Prague, Faculty of Pharmacy in Hradec Králové, Pharmacy Key words: St. John's Wort, precursors, hypericine, flavonoids, acetate, cinnamic acid, cinnamate, tyrosine, shikimic acid The goal of this work was to affect the production of hypericin (naphto-dianthrone), hyperoside and quercitrin (flavonoids) in the suspensional Hypericum perforatum explantate cell cultures. The method of precursor feeding was used. The precursors of naphtodianthrones and flavonoids were added into the medium in final concentrations 10 mg.l-1, 50 mg.l-1 and 100 mg.l-1. Samples were taken after 72 and 168 hours after adding the precursor. Potassium acetate, cinnamic acid, sodium cinnamate, tyrosine and shikimic acid were used as precursors. Cultures were cultivated on the Murashige and Skoog medium with the addition of the growth stimulator α-NAA. Concentration of hypericin, hyperosid and quercitrin was measured by HPLC analysis. The highest influence on the production of hypericin in cultures was detected after adding of tyrosine. The concetration of hypericin in cells raised in this experiment from 0,003% (control culture) to 0,03% (culture with tyrosine concen-tration of 100 mg.l-1). This...

Due to the essential demands for novel anti-tuberculosis treatments for global tuberculosis control, this research investigated the antimycobacterial activity of Hypericum perforatum herb (commonly known as St. John’s wort, SJW), including a SJW methanol extract, purified major bioactive constituents of SJW: hypericin (Hpn), pseudohypericin (Phn) and hyperforin (Hfn). The SJW acidified methanol extract showed bactericidal activity against Mycobacterium JLS at 0.05 mg/ml culture. Purified compounds were tested at similar concentrations contained in the SJW methanol extract treatment. Among three purified bioactive compounds, only Hfn was bactericidal at 12 μg/ml. The other two compounds Phn and Hpn were not inhibitory or bactericidal at concentrations corresponding to the SJW methanol extract treatments. The Polysorbate surfactant Tween 80, which is commonly added to the mycobacterial cultures to prevent cell clumping, was found to have inhibitory effects on the antimycobacterial activities of SJW extract and hyperforin. The addition of Tween 80 (0.05% v/v) increased the minimum bactericidal concentration (MIC) of SJW methanol extract from 0.05 to 0.33mg /ml and from 12 to 80 μg/ml for Hfn. This inhibitory effect of Tween 80 on SJW is opposite to the effect of Tween 80 on the antimycobacterial activity of rifampin and isoniazid. These observations are also in conflict with the existing permeability barrier hypothesis. A hypothesis that hyperforin molecules were sequestered in the core of Tween 80 micelles was given out to explain the repression effect of Tween 80 on hyperforin activity. The effectiveness of Tween 60, Tween 40 and Tween 20 on SJW activity was also tested. Tween 60 and Tween 40 showed the similar dose-dependent inhibitory effect on SJW extract activity with Tween 80, while the inhibitory effect of Tween 20 is much weaker. A preliminary test was performed to detect the activity of SJW acidified MeOH extract and hyperforin on M. tuberculosis H37Rv strain. Results showed the MIC was 0.67mg SJW extract/ml and 200 μg Hfn /ml. In all, M. tuberculosis H37Rv stain is not that sensitive to SJW and hyperforin as other non-pathogenic strains tested in the present and previous studies.

Dissertation (Ph.D.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references.

St. John’s wort extract is composed of a large number of components, about 150, but the most studied and interesting substance are hyperforin and hypericin. The herb is very popular and it is known for its pharmacological efficacy in numerous disorders. It is used for treatment of mild to moderated depression as an alternative to synthetic antidepressants. The St. John’s wort products are available in several preparations, as tablets, capsules, tea, fresh plant juice etc. Hyperforin is the part of St. John’s wort that gives the antidepressant effect through inhibition of reuptake of neurotransmitters such norepinephrine, serotonin, dopamine, GABA and L-glutamate.Since 1999, several case reports and clinical studies have demonstrated the interactions of St. John’s wort extract with other medical products, resulting in a decreased plasma concentration and potential therapeutics failure. The mechanism behind this is an interaction between St. John’s wort with intestinal and hepatic cytochrome P450 and P-glycoprotein, resulting in inducing these enzymes. In vitro investigations indicated that this induction is medicated by an interaction of hyperforin with the pregnane X receptor.Long-term co-medication of St. John’s wort extract reduces the bioavailability of wide variety of drugs, including digoxin, warfarin, cyclosporine, oral contraceptive and theophylline.The object of the present thesis was to investigate and describe the influence of St. John’s wort extract on the pharmacokinetics of the drugs mentioned above and what this pharmacokinetic interaction has for clinical consequences.Seven studies, with different numbers of participants, performed in different countries, were compiled to describe the effect of St. John’s wort on the drugs.The results from these studies showed that St. John’s wort extract affects the metabolism of these drugs by significant reducing theirs pharmacological effect. This decrease of the bioavailability and plasma concentration can be the results of intestinal and hepatic induction of CYP enzymes and p-glycoprotein. From these results I conclude that St. John’s wort should be used with caution and patients receiving these drugs should regularly monitory theirs plasma concentration.

Memoria para optar al título de Químico Farmacéutico / Thymus vulgaris (Tomillo) e Hypericum perforatum (Hierba de San Juan), plantas originarias de Europa, Asia y África del Norte, han sido utilizadas extensamente en afecciones respiratorias y trastornos depresivos, respectivamente. Dado el potencial terapéutico de estas especies y considerando que todas las enfermedades, en mayor o menor medida, están asociadas a estrés oxidativo, en este trabajo se estudió la capacidad antioxidante de extractos hidroalcóholicos de hojas de Thymus vulgaris e Hypericum perforatum. Ambos extractos previnieron, con diferente magnitud, la lipoperoxidación microsómica, previnieron y revirtieron la oxidación de los tioles microsómicos, todos éstos fenómenos inducidos por el sistema pro-oxidante Fe+3/ascorbato. Estos extractos además fueron capaces de quelar iones Cu+2, actividad medida por el cambio del espectro de absorbancia de este ión en presencia de los extractos. Más aún, los extractos testeados inhibieron la GSH-transferasa microsómica, enzima cuya forma activa es el dímero -S-S-. Previamente se determinó la concentración de polifenoles para cada extracto para ser utilizada como marcador de la capacidad antioxidante. Los resultados obtenidos indicarían que las actividades no solo dependen del contenido de polifenoles si no que, más bien, a diferencias en la lipofilicidad de los agentes antioxidantes presentes en ambos extractos, propiedad que favorecería la afinidad por la membrana microsómica. Por otra parte, también es probable que existan diferencias en los potenciales redox de los antioxidantes herbales favoreciendo a aquellos presentes en el extracto de Tomillo. Estas consideraciones se discuten al final del manuscrito en términos de su importancia farmacológica / Thymus vulgaris and Hypericum perforatum, plants native from Europe, Asia and North Africa, have been used extensively in respiratory and depressive disorders, respectively. Considering the therapeutic potential of these species and that all diseases, some extent, are associated to oxidative stress, in this work, we studied the antioxidant capacity of hydroalcoholic extracts from leaves of Hypericum perforatum and Thymus vulgaris leaves. Both extracts, to varying extent prevented microsomal lipid peroxidation, and prevented and reversed the microsomal oxidation of thiols, phenomena induced by the pro-oxidant system Fe+3 / ascorbate. These extracts also chelated Cu+2 ions, activity measure by the change in the absorbance spectrum of this ion in the presence of the herbal extracts. Moreover, the extracts tested inhibited microsomal GSH-transferase, an enzyme whose active form is the dimer-SS-. The polyphenols concentration was determined for each extract to be used as a marker of the antioxidant capacity. The results showed that the antioxidants activities tested not only depend on the content of polyphenols, but also on differences in lipophilicity of the antioxidants present in both extracts, a property that would favor the microsomal membrane affinity. Moreover, it is also likely that there are differences in the redox potential of the antioxidant, in this case this property favor those antioxidants in Thymus vulgaris extract. At the end of the manuscript, the results are discussed from the pharmacological point of view

Made available in DSpace on 2017-07-21T14:13:12Z (GMT). No. of bitstreams: 1 DEBORA.pdf: 1139278 bytes, checksum: 191ff40c879efded12bdb95a873d58d6 (MD5) Previous issue date: 2013-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objective of this study was to test the potential neuroprotective action of Hypericum perforatum alcoholic extract on dopaminergic neurons lesioned in rat by the toxin 6-hydroxydopamine (6-OHDA) as an animal model of PD. The 6-OHDA was stereotaxically infused in rats into the medial forebrain bundle unilaterally to test rotational behavior and bilaterally to other tests. Hypericum perforatum was administered at doses of 100, 200 and 400 mg/kg by gavage for 35 days, starting 28 days before the treatment of the lesion. In the test of rotational behavior, a unilateral lesion with 6-OHDA caused an increase in the number of contralateral rotations and this effect was reversed by treatment with Hypericum perforatum. In the sucrose preference test was significant reduction in depressive-like behavior in animals injured with 6-OHDA-treated and Hypericum perforatum. In the forced swimming test, 6-OHDA animals treated with Hypericum perforatum kept less time immobile when compared to 6-OHDA animals treated with vehicle. In the open field test, we observed a significant reduction of locomotor function in the injured groups with 6-OHDA, however, according to results obtained in the forced swimming test, the motor impairment did not affect the immobility time. In the social recognition test, animals treated with 6-OHDA Hypericum perforatum performed significantly better than animals treated with 6-OHDA vehicle. In the olfactory discrimination test, there was no difference in the ability to discriminate odors, and possibly did not olfaction loss in animals injured with 6-OHDA. These results suggest that Hypericum perforatum reduced the death of dopaminergic neurons. This potential neuroprotection was confirmed by analysis of Immunodetection of proteins (Western Blot) by expression of the enzyme tyrosine hydroxylase (TH). Together, these results characterize the Hypericum perforatum as a possible drug to be used in the treatment of PD. / O objetivo deste estudo foi testar o potencial neuroprotetor do extrato alcoólico do Hypericum perforatum sobre neurônios dopaminérgicos de ratos lesionados pela toxina 6-hidroxidopamina (6-OHDA) como modelo animal de DP. Ratos tiveram 6-OHDA estereotaxicamente infundida no feixe prosencefálico medial, unilateralmente para o teste de comportamento rotatório e bilateralmente para os demais testes. O Hypericum perforatum foi administrado nas doses de 100, 200 e 400 mg/Kg por gavagem durante 35 dias, iniciando–se o tratamento 28 dias antes da lesão. No teste de comportamento rotatório, a lesão unilateral com 6-OHDA causou um aumento no número de rotações contralaterais e esse efeito foi revertido pelo tratamento com Hypericum perforatum. No teste de preferência a sacarose houve redução significativa do comportamento tipo depressivo nos animais lesados com 6-OHDA e tratados com Hypericum perforatum. No teste de natação forçada, os animais 6-OHDA tratados com Hypericum perforatum permaneceram menos tempo imóveis quando comparados aos animais tratados com veículo. No teste do campo aberto, foi observada redução significativa da função locomotora nos grupos lesados com 6-OHDA, no entanto, de acordo com os dados obtidos no teste de natação forçada, o prejuízo motor não influenciou o tempo de imobilidade. No teste de reconhecimento social, animais 6-OHDA tratados com Hypericum perforatum tiveram um desempenho significativamente melhor do que os animais 6-OHDA tratados com veículo. No teste de discriminação olfativa, não houve diferença na capacidade de discriminação de odores, sendo que possivelmente não houve prejuízo na olfação dos animais lesados com 6-OHDA. Esses resultados sugerem que o Hypericum perforatum reduziram a morte dos neurônios dopaminérgicos. Essa possível neuroproteção foi confirmada através da análise de Imunodetecção de proteínas (Western Blot), através da expressão da enzima Tirosina Hidroxilase (TH). Em conjunto, esses resultados caracterizam o Hypericum perforatum como possível fármaco a ser utilizado no tratamento da DP.