A dissertation presented in compliance with the requirements for the Master's Degree in Technology: Chiropractic, Technikon Natal, 2001. / Low back pain is a major health problem worldwide, and considerable amounts of money are spent on a variety of practitioners including medical practitioners, chiropractors, osteopaths, physiotherapists and others. There is a lack of consensus among these groups regarding the most appropriate therapy or management for low back pain. This disparity leads to the meritable conclusion that more research is required to accurately identify solutions for the management of low back pain (Walker, 1997:95-96). / M

Humin substances have been used as folk remedies for the last 3000 years. Recent studies have shown that humates possess anti-inflammatory properties, but the mechanism of how it affects inflammation is still unclear. In this study the anti-inflammatory properties of potassium humate, a water soluble humic acid salt, was investigated on different inflammatory pathways in vitro and in vivo. The effect of potassium humate on human mononuclear lymphocyte proliferation showed that potassium humate stimulated lymphocyte proliferation of resting-, PHA- and PWM-stimulated lymphocytes in vitro from concentrations of 20 to 80 µg/ml, in a dose dependant manner, where a maximum proliferation was observed at 80 µg/ml whereas lymphocyte proliferation decreased at 100 µg/ml. On the contrary potassium humate, at 40 µg/ml, significantly inhibited the supernatant concentrations of the following cytokines; TNF-α, IL-1ß, IL-6 and IL-10 by PHA stimulated lymphocytes. The effect of potassium humate on the alternative as well as the classical complement pathway was investigated in vitro using the haemolytic complement assay. Results indicated that potassium humate inhibits both the alternative and classical complement pathways without affecting the red blood cell membrane stability. Different inflammatory mechanisms were investigated in vivo, using the carrageenan-induced paw oedema model and the delayed type hypersensitivity reaction model. The carrageenan-induced paw oedema model was used to determine the effect of potassium humate on acute inflammation in the hind paw. Carrageenan was injected into the right hind footpad of a rat which caused an increase in paw volume due to oedema, which was measured with a plethysmometer. Potassium humate significantly inhibited the oedema at a dose of 60 mg/kg bodyweight and compared favourably with indomethacin at 10 mg/kg bodyweight. The effect of potassium humate on the delayed type hypersensitivity reaction model was also investigated whereby rats were sensitised with sheep erythrocytes. Potassium humate was administered daily by oral gavage at a dose of 60 mg/kg bodyweight. After 7 days, rats were challenged by injecting sheep erythrocytes into the right hind footpad. The degree of inflammation was determined by measuring the increase of paw volume with a plethysmometer. It was found that potassium humate did not have an anti-inflammatory effect on the delayed type hypersensitivity reaction as opposed to the inhibition caused by dexamethasone at a dose of 30 mg/kg bodyweight. This study showed that potassium humate selectively inhibited the inflammatory pathway of the carrageenan-induced paw oedema as opposed to the delayed type hypersensitivity. The mechanism of the anti-inflammatory property of potassium humate might possibly be due to the inhibition of the complement cascade. This study clearly shows that potassium humate possesses anti-inflammatory properties that can be utilised in the future as a potential treatment for inflammatory disorders associated with the activation of complement. However further investigation in the mechanism by which potassium humate inhibits complement activation needs to be done. / Dissertation (MSc (Pharmacology))--University of Pretoria, 2008. / Pharmacology / unrestricted

Previous studies in the sheep, rabbit, cat and rat have demonstrated the ability of the neuropeptide, arginine vasopressin (AVP), to suppress endotoxin-induced fever when perfused into a discrete brain locus. Fever can also be suppressed if AVP is microinjected into the cerebral ventricles of the rat. The mechanisms by which AVP mediates antipyresis are unknown. Experiments were conducted, therefore, to examine the effect of intracerebroventricular (icv) AVP on an established fever and to assess the mechanism of action using a specific, V₁-receptor antagonist (M-AVP). Studies were also conducted to elucidate the effector mechanisms utilized to accomplish antipyresis induced by icv AVP. Finally, cerebrospinal fluid (CSF) AVP concentrations were measured in febrile and non-febrile rats to determine the role of endogenously released AVP in the CSF during fever. AVP administered icv was shown to have marked antipyretic effects at very low doses. This antipyresis was elicited in rats with an established fever but the peptide had no effect on the temperature of non-febrile rats. Thus AVP both prevented and reversed endotoxin-induced fever. Furthermore, this AVP-induced antipyresis was abolished by pretreatment with the the V₁-antagonist, M-AVP. The antipyretic effects of AVP were, therefore, receptor mediated and likely to be of physiological importance. Efforts to manipulate the endogenous AVP system by icv M-AVP were also attempted. When M-AVP was injected icv, the fever height of endotoxin-treated rats was not different from endotoxin-treated controls. In addition, M-AVP did not influence the magnitude of the antipyresis induced by indomethacin. It has become clear, however, that this method of administering the antagonist is inappropriate to block endogenous AVP effects occurring within the neuropil. Subsequent experiments in another laboratory have shown that M-AVP must be microinjected into the AVP-sensitive brain locus to effectively block endogenous activity. The antipyretic response to icv AVP was further investigated at three ambient temperatures in an attempt to identify the effector mechanisms involved. Responses of non-febrile and febrile rats to icv injections of AVP and sc injections of indomethacin were observed at cold (4°C), neutral (25°C) and warm (32°C) ambient temperatures. As in the previous experiments, AVP at 25°C decreased brain temperatures of febrile but not non-febrile rats. This antipyretic effect was also observed at the warm ambient temperature and during cold exposure. Responses to sc indomethacin were qualitatively similar to icv AVP at neutral and warm temperatures. In the cold, however, indomethacin decreased the brain temperature of both non-febrile and febrile animals, although unlike AVP, brain temperature of non-febrile animals decreased somewhat more than that of febrile animals. These data showed that AVP decreased brain temperature of febrile more so than non-febrile rats at all ambient temperatures and may therefore have been acting partially on febrile set-point. It was possible that AVP affected specific effector mechanisms since antipyretic effects were of different magnitudes at different ambient temperatures. The observation that AVP and indomethacin had qualitatively similar effects on fever at three ambient temperatures suggested that they may act via a common neural pathway. Further analysis of the mechanism of icv AVP-induced antipyresis was conducted at the three ambient temperatures while measuring specific effectors: heat loss and heat production. At 25°C, AVP-induced antipyresis was mediated by tail skin vasodilation while metabolic rate was unaffected. At 4°C, the antipyresis produced by AVP was mediated exclusively by inhibition of heat production since the metabolic rate decreased markedly following AVP. This antipyresis at 4°C was accompanied by cutaneous vasoconstriction. At 32°C, neither vasomotor tone, metabolic rate nor evaporative heat loss could be shown to contribute to the small antipyretic effect elicited by AVP. These data strongly suggest that icv AVP produced antipyresis by affecting the febrile body temperature set-point mechanism since the thermoregulatory strategy to lose heat varied at different ambient temperatures and the decrease in body temperature could not be shown to be due to changes in a single effector mechanism. As an index of endogenous AVP activity, cerebrospinal fluid (CSF) concentrations of AVP were measured in febrile and non-febrile rats in order to determine the role of CSF AVP in fever and antipyresis. The results demonstrated that the AVP release pattern was not altered in endotoxin-treated febrile compared to non-febrile rats. It was concluded that CSF AVP had no role in the febrile process. In summary, icv AVP appears to induce antipyresis by its action on febrile set-point rather than on a specific effector system. This action of AVP is mediated by a V₁-like receptor mechanism which is not affected by endogenous CSF AVP. The neural/neurochemical basis for the thermoregulatory set-point has not been clearly established so the mechanism of action by which AVP affects set-point remains to be determined. These data contribute, however, to the growing body of evidence that AVP is acting centrally as a neurotransmitter or neuromodulator to regulate body temperature during the febrile process. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

Inflammation appears to be an attempt by the organism to re-establish homeostasis as a response to local reactive change in tissues following injury or irritation. This injury or irritation (whether caused by micro-organisms, toxins, antigens, etc.) first leads to an increased passage of fluid through the walls of the microvasculature, followed by stasis of circulation within the affected area. This, in turn, is followed by migration of leukocytes into the area and finally concluded by connective tissue proliferation leading to the deposition of granulation tissue. There are many experimental models employed for the evaluation of drugs with possible anti-inflammatory activity. Since many of these systems employ whole animals, it seems rational that various hematological parameters could be applied. Our purpose was to test such parameters in two selected systems (i.e., adjuvant-induced arthritis and cotton pellet granuloma formation) during drug evaluation. The parameters under investigation included: total white blood cell count, differential leukocyte count, sedimentation rate and serum protein patterns.

It has become apparent in recent years that many centrally active agents possess potential anti-inflammatory capabilities. The central or peripheral mechanisms of action for these agents have not been delineated nor has their clinical efficacy baan established or refuted. The centrally active drugs, cryogenine and tetrabenazine (a benzoquinolizine derivative), previously have been shown in this laboratory to inhibit certain models of induced inflammation. The present study in rats verifies the anti-inflammatory properties of cryogenic and established that a structurally related series of benzoquinolizine derivatives possessed the capability of inhibiting both exudative (carrageenan-induced pedal edema) and proliferative (cotton pellet granuloma) models of inflammation when administered orally.

Serious cardiovascular side effects are reported from synthetic cyclooxygenase-2 (COX-2) selective nonsteroidal anti-inflammatory drugs, the most common medication for rheumatoid arthritis (RA) and osteoarthritis (OA). Natural products from herbal medicine are inspirational source of safe and effective remedy due to its distinguished chemical diversity. Nanomagnetic ligand fishing using enzyme-anchored-magnetic nanoparticles (MNPs) is an advanced selective bioseparation strategy based on macromolecular target-ligand binding, which can screen enzyme inhibitors from complex mixtures. "Snow lotus" herbs have been clinically applied as safe and effective treatment for arthritis throughout centuries in Asia. Some major chemicals from the herbs have been found with anti-COX-2 activities. It is therefore hypothesized that novel and safe COX-2 selective inhibitors can be separated from a most representative snow lotus herb via ligand fishing using COX-2-functionalized MNPs (COX-2-MNPs), and that the efficacy and safety of the screened COX-2 ligands can be verified by subsequent evaluation. Saussurea laniceps Hand.-Mazz. (SL), S. medusa Maxim. (SM) and S. involucrata (Kar. et Kir.) Sch.Bip. (SI) are three authenticated sources of "snow lotus" herbs. An ultra-high performance liquid chromatography hyphenated with diode array detector and quadrupole time of flight-mass spectrometry (UPLC-DAD-QTOF-MS) method was developed to analyze 49 herbal samples for species analysis and overall quality evaluation. With 25 simultaneously identified constituents, of which 12 were quantified, the chemical determination, four-dimensional principle component analysis (4D-PCA), and orthogonal hierarchical cluster analysis (2D-HCA) showed a distinctive bioactive component profile of SL from the other two species, and explained the therapeutic potency of SL. As a result, SL has been chosen as a model herb to screen for novel and safe COX-2 selective inhibitors. With systematic uniform experimental designs and statistical modeling, COX-2-MNPs with high magnetic moments and outstanding enzyme activity have been synthesized. Four COX-2-selective compounds, namely, chlorogenic acid, syringin, umbelliferone, and scopoletin, were separated from the herbal extract using fine-tuned fishing protocol and were identified by UPLC-DAD-QTOF-MS. All the four ligands were proved with evidently lower in vitro and in vivo cardiotoxicity than celecoxib, a known selective COX-2 inhibitor. Some of them exerted potent anti-inflammatory activities on cells, and their optimum combination ratios were investigated. Among the ligands, scopoletin showed most evident therapeutic potential in rats with adjuvant-induced arthritis and anterior cruciate ligament transection (ACLT)-induced OA, respectively, by alleviating clinical statuses, immune responses, and joint pathological features. An equal mixture of scopoletin and syringin brought possible synergistic remedial effects on rat OA. Molecular docking results explained the structure-specific enzyme-binding affinities of the ligands; the ligands' inhibition on COX-2 may involve direct interaction as well as upstream signaling pathways. In conclusion, promising candidates of COX-2 selective inibitors, e.g. scopoletin, have been screened and validated on a nanomagnetic ligand fishing platform using COX-2-MNPs from the extract of SL, a most representative snow lotus herb with distinctive chemical composition and outstanding therapeutic efficacies. The quality evaluation strategy of snow lotus herbs combining chemical determination and multidimensional chemometric analysis can be applied in other multi-original herbal medicines. The nanomagnetic ligand fishing platform of compound bio-separation and multi-model bio-evaluation should be equally valuable for uncovering other therapeutic chemicals in different natural sources.

Obese people with insulin resistance are at high risk of developing disease-related complications like heart attack and stroke. Recently, a significant amount of data has been published linking chronic inflammation with obesity and the etiology of the Metabolic syndrome (MetS). Scientists have found many of the same inflammatory pathways and pro-inflammatory molecules are involved in both conditions. In particular, recent studies have elucidated an important role for the inflammasome in the etiology of these diseases. Interfering with these chronic inflammatory processes may provide a new way to treat obesity. Pilot studies in animals and humans have shown positive outcomes using anti-inflammatory drugs for treatment of both obesity and MetS. One advantage to using anti-inflammatory drugs is that many are already clinically approved with known risk/benefit profiles. Trials to test their efficacy in MetS and obesity are thus feasible. If proven beneficial, these drugs could help treat a huge number of patients who do not currently have other safe options. In this thesis I propose that new drugs targeting the inflammasome components, such as caspase 1, may also show clinical benefit in the treatment of MetS and obesity. Also drugs that reduce activation of a subset of macrophages such as the M1 class may also prove useful in treatment of these conditions.

No / Cryptolepine is the major alkaloid of the West African shrub, Cryptolepis sanguinolenta. Cryptolepine has been shown to inhibit nitric oxide production, and DNA binding of Nuclear Factor-kappa B following inflammatory stimuli in vitro. In order to validate the anti-inflammatory property of this compound in vivo, we investigated its effects on a number of animal models of inflammation. Cryptolepine (10¿40 mg/kg i.p.) produced significant dose-dependent inhibition of the carrageenan-induced rat paw oedema, and carrageenaninduced pleurisy in rats. These effects were compared with those of the non-steroidal anti-inflammatory drug indomethacin (10 mg/kg). At doses of 10¿40 mg/kg i.p., cryptolepine inhibited lipopolysaccharide (LPS)-induced microvascular permeability in mice in a dose-related fashion. Oral administration of up to 40 mg/kg of the compound for four consecutive days did not induce gastric lesion formation in rats. Analgesic activity was also exhibited by cryptolepine through a dose-related (10¿40 mg/kg i.p.) inhibition of writhing induced by i.p. administration of acetic acid in mice. The results of this study reveal that cryptolepine possesses in vivo anti-inflammatory activity.Copyright © 2009 John Wiley & Sons, Ltd.

No / Cryptolepine, an indoloquinoline alkaloid in Cryptolepis sanguinolenta, has anti-inflammatory property. In this study, we aimed to evaluate the effects of cryptolepine on lipopolysaccharide (LPS)- induced neuroinflammation in rat microglia and its potential mechanisms. Microglial activation was induced by stimulation with LPS, and the effects of cryptolepine pretreatment on microglial activation and production of proinflammatory mediators, PGE2/COX-2, microsomal prostaglandin E2 synthase and nitric oxide/iNOS were investigated. We further elucidated the role of Nuclear Factor-kappa B (NF-κB) and the mitogen-activated protein kinases in the antiinflammatory actions of cryptolepine in LPS-stimulated microglia. Our results showed that cryptolepine significantly inhibited LPS-induced production of tumour necrosis factor-alpha (TNFα), interleukin-6 (IL-6), interleukin-1beta (IL-1β), nitric oxide, and PGE2. Protein and mRNA levels of COX-2 and iNOS were also attenuated by cryptolepine. Further experiments on intracellular signalling mechanisms show that IκB-independent inhibition of NF-κB nuclear translocation contributes to the anti-neuroinflammatory actions of cryptolepine. Results also show that cryptolepine inhibited LPS-induced p38 and MAPKAPK2 phosphorylation in the microglia. Cell viability experiments revealed that cryptolepine (2.5 and 5 μM) did not produce cytotoxicity in microglia. Taken together, our results suggest that cryptolepine inhibits LPS-induced microglial inflammation by partial targeting of NF-κB signalling and attenuation of p38/MAPKAPK2.

Lau Kit-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Declaration --- p.vi / Table of contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xv / Chapter Chapter One: --- General Introduction / Chapter 1.1 --- Tea and Ku-Ding-Cha --- p.1 / Chapter 1.2 --- Ligustrum robustum / Chapter 1.2.1 --- The plant --- p.4 / Chapter 1.2.2 --- Chemical constituents --- p.4 / Chapter 1.2.3 --- Pharmacological activities --- p.4 / Chapter 1.2.4 --- Toxicity --- p.5 / Chapter 1.3 --- Objectives and scope of the project --- p.7 / Chapter Chapter Two: --- Antioxidative effect / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Oxidants and antioxidants --- p.8 / Chapter 2.1.2 --- In vitro antioxidative tests / Chapter 2.1.2.1 --- PMS-NADH system --- p.19 / Chapter 2.1.2.2 --- Fe3+/ascorbate/H202 system --- p.19 / Chapter 2.1.2.3 --- Red-blood-cell hemolysis model --- p.20 / Chapter 2.2 --- Objectives --- p.22 / Chapter 2.3 --- Materials and Methods / Chapter 2.3.1 --- Materials / Chapter 2.3.1.1 --- Guizhou Ku-Ding-Cha --- p.23 / Chapter 2.3.1.2 --- Other tea leaves --- p.23 / Chapter 2.3.1.3 --- Animals --- p.23 / Chapter 2.3.1.4 --- Chemicals --- p.24 / Chapter 2.3.2 --- Methods / Chapter 2.3.2.1 --- Aqueous extraction of L. robustum and other tea leaves --- p.25 / Chapter 2.3.2.2 --- Ethanol extraction of L. robustum and fraction separations --- p.25 / Chapter 2.3.2.3 --- Activity-guided purification of L. robustum --- p.26 / Chapter 2.3.2.4 --- Assays for testing antioxidative effect / Chapter 2.3.2.4.1 --- PMS-NADH system --- p.28 / Chapter 2.3.2.4.2 --- Fe3+/ascorbate/H202 system --- p.28 / Chapter 2.3.2.4.3 --- Red-blood-cell hemolysis model --- p.29 / Chapter 2.3.2.5 --- Statistical analysis --- p.29 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Ligustrum robustum and other tea leaves --- p.30 / Chapter 2.4.2 --- Ethanol extract of L. robustum --- p.48 / Chapter 2.4.3 --- Water-soluble and water-insoluble fractions --- p.52 / Chapter 2.4.4 --- "Fractions B1, B2 and B3" --- p.56 / Chapter 2.4.5 --- Sub-fractions B2-1 to B2-16 --- p.61 / Chapter 2.4.6 --- Pure compounds --- p.66 / Chapter 2.4.7 --- Changes in antioxidant effects --- p.72 / Chapter 2.5 --- Discussion / Chapter 2.5.1 --- Antioxidant potency of L. robustum --- p.76 / Chapter 2.5.2 --- Effects of extraction methods on antioxidant activities --- p.78 / Chapter 2.5.3 --- Active antioxidant components of L. robustum --- p.78 / Chapter 2.5.4 --- Structure-activity relationship of glycosides and flavonoid --- p.80 / Chapter 2.5.5 --- Antioxidant mechanism of L. robustum --- p.81 / Chapter 2.5.6 --- Prospects for further investigation --- p.82 / Chapter Chapter Three: --- Anti-inflammatory effect / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Mechanisms and mediators of inflammation --- p.83 / Chapter 3.1.2 --- In vivo anti-inflammatory assays / Chapter 3.1.2.1 --- Acetic acid-induced vascular permeability test --- p.94 / Chapter 3.1.2.2 --- Croton oil-induced ear edema test --- p.94 / Chapter 3.2 --- Objective --- p.96 / Chapter 3.3 --- Materials and Methods / Chapter 3.3.1 --- Materials / Chapter 3.3.1.1 --- Animals --- p.97 / Chapter 3.3.1.2 --- Chemicals --- p.97 / Chapter 3.3.2 --- Methods / Chapter 3.3.2.1 --- Assays for testing anti-inflammatory effect / Chapter 3.3.2.1.1 --- Acetic acid-induced vascular permeability test --- p.98 / Chapter 3.3.2.1.2 --- Croton oil-induced ear edema test --- p.98 / Chapter 3.3.2.2 --- Statistical analysis --- p.99 / Chapter 3.4 --- Results / Chapter 3.4.1 --- Acetic acid-induced vascular permeability test --- p.100 / Chapter 3.4.2 --- Croton oil-induced ear edema test --- p.100 / Chapter 3.5 --- Discussion --- p.103 / Chapter Chapter Four: --- Hepato-protective effect / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Liver structures and functions --- p.105 / Chapter 4.1.2 --- Carbon tetrachloride-induced liver injury --- p.112 / Chapter 4.1.2.1 --- Mechanisms --- p.112 / Chapter 4.1.2.2 --- Hepatic cytotoxicity --- p.112 / Chapter 4.1.2.3 --- Diagnostic methods / Chapter 4.1.2.3.1 --- Liver weight --- p.114 / Chapter 4.1.2.3.2 --- Lipid peroxidation --- p.114 / Chapter 4.1.2.3.3 --- Serum enzyme levels --- p.114 / Chapter 4.1.2.3.4 --- Histopathological observation --- p.115 / Chapter 4.2 --- Objectives --- p.116 / Chapter 4.3 --- Materials and Methods / Chapter 4.3.1 --- Materials / Chapter 4.3.1.1 --- Animals --- p.117 / Chapter 4.3.1.2 --- Chemicals --- p.117 / Chapter 4.3.2 --- Methods / Chapter 4.3.2.1 --- Carbon tetrachloride-induced acute liver injury --- p.118 / Chapter 4.3.2.2 --- Statistical analysis --- p.120 / Chapter 4.4 --- Results / Chapter 4.4.1 --- Preventive effect / Chapter 4.4.1.1 --- Liver weight --- p.121 / Chapter 4.4.1.2 --- Malondialdehyde content --- p.121 / Chapter 4.4.1.3 --- Serum aminotransferse levels --- p.121 / Chapter 4.4.1.4 --- Histopathological observations --- p.122 / Chapter 4.4.2 --- Curative effect / Chapter 4.4.2.1 --- Liver weight --- p.126 / Chapter 4.4.2.2 --- Malondialdehyde content --- p.126 / Chapter 4.4.2.3 --- Serum aminotransferse levels --- p.126 / Chapter 4.4.2.4 --- Histopathological observations --- p.126 / Chapter 4.5 --- Discussion --- p.130 / Chapter Chapter Five: --- Prospects for product development --- p.134 / Chapter Chapter Six: --- Conclusion --- p.136 / Appendices / Appendix A. Procedure for determining the activity of aspartate aminotransferase (AST) --- p.139 / Appendix B. Procedure for determining the activity of alanine aminotransferase (ALT) --- p.140 / Appendix C. Procedure for preparing a calibration curve for the measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities --- p.141 / Appendix D. Procedure for tissue preparation for light microscopic study --- p.143 / References --- p.144