Phytochemistry and medicinal properties of Psidium guajava L. leaves: A review

Psidium guajava L. (Myrtaceae), also known as guava, is a medicinal tree native to tropical America that has been introduced and is widely available in many countries. Almost all plant parts of P. guajava have a long history of being used to treat a variety of ailments, in addition to applications as foods. Guava leaves are used as both medicine and food purposes, and there are numerous scientific reports on their medicinal uses, chemical composition and pharmacological properties. Cancer, blood pressure, diarrhea, bowel irregularities, diabetes, cough, cold, constipation, dysentery, scurvy, weight loss, improves skins tonicity are some of the diseases treated with guava leaves. Polyphenols, flavonoids, saponins, tannins, terpenoids, glycosides, flavones, cardiac glycosides, cardenolides, phlobatanins, steroids and other classes of bioactive compounds have been identified from the leaves. The primary chemical constituents of guava leaves are phenolic compounds, iso-flavonoids, gallic acid, catechin, quercetin, epicathechin, rutin, naringenin, kaempferol, caryophyllene oxide, p-selinene etc. Several studies have demonstrated its pharmacological activities including antioxidant, antimicrobial, antidiabetic, antitumor, anticancer, antidiarrheal, healing, cytotoxic, hepatoprotective, anti-inflammatory, antimalarial/ anti-plasmodial, dental plaque, antiglycative and many more. This review is aimed on compiling all the literature reported on pharmacological activities and phytochemical compositions of guava leaves as a support to the scientific community for further studies and to provide scientific data to validate its traditional uses

Investigation of anti-diabetic properties of Psidium guajava leaf in streptozocin induced diabetic rats.

Doctor of Philosophy in Biochemistry. University of KwaZulu-Natal, Westville 2015.Diabetes mellitus results in chronic hyperglycaemia, leading to defects in carbohydrate, fat and protein metabolism. Diabetes mellitus is also linked with elevated plasma cholesterol and triglyceride levels, which may promote the development of cardiovascular disease. Psidium guajava(PG) leaf is known to have a blood-glucose lowering effect in diabetic rats. The aim of this study was to carry out a phytochemical study of PG leaf extract; investigate its protective effect on pancreas and also its effect on muscle and liver glycogen synthase and phosphorylase activities in streptozotocin induced diabetic male Sprague-Dawley rats; Serum biomarkers of liver and muscle dysfunction such as alanine amino transferase (ALT), aspartate amino transferase (AST) and lactate dehydrogenase (LDH) were also analyzed. The effect of PG on markers of lipid metabolism and on hormone sensitive lipase (HSL) enzyme was also investigated. A single dose of 40 mg/kg body weight of streptozotocin was administered to fasted male Sprague-Dawley rats intraperitoneally for diabetes induction. The aqueous extract of PG leaves was used to treat both normal and diabetic animals (400 mg/kg body weight) for 2 weeks while control animals were treated with the vehicle. After 2 weeks of treatment, PG was shown to enhance lowering of blood glucose in diabetic rats following a glucose load and protected pancreatic tissue from diabetic damage. GC-MS analysis of the aqueous extract of PG indicated the presence of phenolic compounds and triterpenes. In acute study, PG activated Protein kinase B(PKB/Akt) in skeletal muscle of streptozotocin induced diabetic rats. In the sub-chronic study, the treatment of rats with PG extract restored glycogen synthase activity depressed by diabetes and decreased glycogen phosphorylase activity in skeletal muscle. These changes in enzyme activity mirrored those in enzyme expression. It also restored glycogen synthase activity depressed by diabetes which was accompanied by reduced glycogen phosphorylase activity and increased glycogen levels in liver. PG decreased HSL activity in adipose tissue and liver and this was accompanied by reduced levels of serum triglycerides, total cholesterol, LDL-cholesterol, cardiac risk factor, atherogenesis and increased HDL-cholesterol. We conclude that PG has significant antidiabetic and hypolipidemic effects, and that these effects may be associated with the presence of triterpenes and phenolic compounds. PG increased GS activity, glycogen storage and reduced GP activity. It also reduced HSL activity and improved serum lipid profile

Studio in vitro e ex vivo dell'attività antiossidante di Casimiroa spp, Croton lechleri, Ribes nigrum e Boswellia serrata nella prevenzione dell'aterosclerosi

In vitro and ex vivo antioxidant activity of Casimiroa spp, Croton lechleri, Ribes nigrum, and Boswellia serrata in atherosclerosis prevention Oxidative and glycoxidative stress are postulated to be the primary events in the pathogenesis of type 2 diabetes mellitus and its vascular implications. Further, LDL oxidation in the vessel wall plays a key role in atherogenesis, also related to damage from oxygen species (ROS). Moreover, the risk for development of atherosclerosis is by approximately three-fold increased in patients with diabetes. The medicinal plants are widely used in folk medicine for the treatment of cardiovascular diseases and diabetes mellitus. The genus Casimiroa (Rutaceae) includes few species which have their habitat in Central America and Mexico; among these, the most common are Casimiroa edulis Llave et lex. and Casimiroa pubescens Ramirez. The decoction of leaves and seeds are traditionally used for treating hypertension. The sap of Croton lechleri (Euphorbiaceae), a South American tree, is used topically in the treatment of wound healing and orally, in a dilute form, mainly for gastric ulcers and intestinal diseases. The gum resin of Boswellia serrata (Burseraceae), which grows in dry mountainous regions of India, Northern Africa and Middle East, has been traditionally used to treat various chronic inflammatory diseases. Ribes nigrum (Grossulariaceae), a species native to central and northern Europe and northern Asia, is a traditional medicine for the treatment of inflammatory disorders such as rheumatic diseases. The aim of this research was to investigate the antioxidant activity of these medicinal plants by means of several experimental methods in vitro and ex vivo to outline their role in the prevention and/or treatment of cardiovascular diseases related to oxidative stress. The antioxidant activity was evaluated by DPPH method, and ORAC (Oxygen Radical Absorbance Capacity) assay. Also, the total phenolic content (TPC) was determined by the use of Folin-Ciocalteu reagent, and the total flavonoid content (TFC) by complexation with chloride aluminium. The activity of the plant extracts on LDL oxidation was studied by monitoring the formation of conjugated dienes, and the quantification of thiobarbituric acid reactive substances (TBARS). Finally, their inhibitory effect on advanced glycation end products (AGEs) formation were evaluated by means of BSA-glucose/ribose fluorescence assay. In DPPH assay, Croton lechleri sap and blackcurrant (Ribes nigrum) bud extract showed higher scavenging activity in comparison with Casimiroa extracts, whereas in the ORAC assay the Casimiroa leaf extracts showed a high ORAC value and Croton lechleri an activity even higher. In TPC test, Croton lechleri showed the highest value (713.76 ± 32.23 mg GAE/g). In the LDL oxidation assay, the plant extracts exhibited considerable protective effects by prolonging the oxidation lag phase; for example, at the concentration of 0.8 µg/ml Croton lechleri increased the lagtime by 58.6%, and in the antiglycation study all extracts inhibited the AGEs formation significantly in the BSA-glucose model. The results from this research suggest that the medicinal plants Croton lechleri, Casimiroa spp. and Ribes nigrum, even if in different manner, may have implications in the prevention of atherosclerotic vascular diseases, whereas Boswellia serrata showed a minor role

Antiglycation and antioxidant properties of Momordica charantia

Diabetes mellitus is a multifactorial disorder characterised by hyperglycaemia and leads to complications. These complications are caused by advanced glycation endproducts (AGEs) that form through protein glycation as a consequence of hyperglycaemia. A diversity of plants are utilised worldwide as traditional medications for the treatment of diabetes mellitus, particularly in developing nations. A considerable quantity of literature has been published on Momordica charantia (MC) as a potent folk medicine for diabetes mellitus. The aim of this study was to investigate the antiglycation and antioxidant effects of HWE and CWE of MC on protein glycation. To examine whether MC can inhibit oxidative stress in endothelial cells by studying oxidative stress-related transcription factor parameters and their expression. Model proteins such as lysozyme and BSA were glycated using sugars (glucose and methylglyoxal) as glycating agents both in the presence and absence of MC extracts. AGE formation and inhibition were monitored by a number of methods. Furthermore, different antioxidant assays were used to study the antioxidant properties of MC. The inhibitory effects of AGEs, glucose, methylglyoxal and MC extracts on proliferation of cultured BAEC were determined in this research. Western blotting was used to examine the potential modulation of the oxidative stress signalling pathways induced by AGEs. Both extracts of MC inhibited the production of AGEs in a dose-dependent fashion and the HWE exhibited the more potent inhibitory effect on AGEs production. Moreover, parameters of oxidative stress including the expression of oxidative stress-related transcription factors were assessed using real-time PCR to study the protective effects of MC. The expressional inhibition of pro-oxidaive genes and enhancement anti-oidative enzymes could be potent element of vascular complications effect of MC extracts. Thus the aqueous extracts of MC, an edible vegetable, may have therapeutic potential in the management of diabetes mellitus

The use of hair detect (and monitor) chronic hyperglycaemia

Background: Diabetes mellitus is a major public health problem resulting in about 5 million deaths per year. This metabolic disorder is characterized by hyperglycaemia, which results in debilitating and life-threatening complications. It is, therefore, vital for diabetics to monitor and control their blood glucose levels in order to keep them below 7mmol/L while fasting and below 9mmol/L after meals. Chronic estimates of glucose control of 8-12 weeks are obtained using glycated haemoglobin A1 (HbA1c). Non-invasive, less expensive methods of monitoring long term glycaemic control may be useful. Since scalp hair consists of about 80% protein, which is subject to non-enzymatic glycation, and growing hair has a rich blood supply exposing it to free glucose, it is likely that hair can be used as an alternative substrate for monitoring chronic hyperglycaemia. Subjects and Methods: Scalp hair and a blood samples (for HbA1c) were collected from 46 diabetic and 46 healthy control subjects. There were 26 diabetic adults (30-70 years), recruited from the outpatient clinic at Groote Schuur hospital and 20 children (7-18 years) recruited from the diabetic clinic at the Red Cross children's hospital. There were 29 healthy control adults (26-65 years) and 17 children (7- 17 years) recruited from the Groote Schuur and Red Cross hospitals respectively. History of chemical hair treatment was recorded for each participant. Hair samples were washed using 1% sodium dodecyl sulphate and analysed using Fourier transform infrared- attenuated total reflection (ATR-FTIR) spectroscopy. Spectra were analysed using statistical software (SIMCA, Umetrics) to determine whether the hair of diabetics was distinguishable from hair of healthy controls as well as whether spectra correlated with HbA1c levels of participants. Hair amino acid concentrations were also analysed as it is known that circulating amino acid concentrations are altered in people with diabetes. Results and discussion: The Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) models between spectra obtained from hair of diabetic participants and spectraobtained from control hair show good separation and predictive ability. When ATR-FTIR spectra were analysed in four groups: children with natural hair, adults with natural hair, adults with dyed hair and adults with relaxed hair, the models yielded predictive abilities (Q2(cum)) ranging from 0.753 to 0.85 and classification abilities ranging from 97.97 to 100%. This demonstrated excellent separation and predictive ability for controls vs. diabetics. Partial least squares (PLS) revealed a good correlation between hair FTIR spectra and participant HbA1c levels (R2 ranging between 0.8067 and 0.9296). These results demonstrated the possibility to use ATR-FTIR alongside multivariate data analysis to detect hyperglycaemia and monitor blood glucose levels via prediction of HbA1c levels from the hair spectra. Amino acid analysis supported the OPLS-DA classifications, as the largest differences were seen between age and chemically treated hair groups. Amino acid results reinforced the necessity to classify spectra into groups in order to distinguish between hair spectra from diabetics or controls, as well as to predict HbA1c. Twelve amino acids (Asp, Glu, Pro, Gly, Met, Ile, His, Lys, Arg, Amm, Cys, Leu) were significantly different between hair from adults and children, eight amino acids (Ala, Cys, Val, Met, Lys, Amm, Ser, Tyr) were statistically significantly different between natural and dyed hair and only up to four amino acids (Gly, Val, Met, Ile in children or Val, Phe, Amm, Arg in adults) were significantly different between diabetic and control groups. Conclusion: There is a need for non-invasive means of monitoring chronic hyperglycaemia. This study demonstrated the ability to distinguish between the hair of diabetics and controls as well as the ability to predict HbA1c levels from hair using ATR-FTIR. However, factors such as age and chemical treatment, which affect the chemical properties of hair, like amino acid levels, should be considered first. This would lead to promising prospects for long term blood glucose monitoring, due to the ability to estimate hair growth rate, and greater insights into the timing and development of diabetic complications. ATR-FTIR relatively simple to use, requires minimal sample preparation and does not require the use of expensive consumables. This technology could, potentially, be adapted into a primary health point of care or home screening or monitoring device for long-term hyperglycaemia, which would assist in early detection and preventing the progression of debilitating complications

An investigation of the anti-hyperglycaemic, biochemical and molecular effects of 4-hydroxyisoleucine and fenugreek seed extract in comparison to metformin in vitro and in vivo.

Doctoral Degree. University of KwaZulu-Natal, Durban.Type two diabetes mellitus (T2D) is a significant cause of premature death and disability, accompanied with negative socio-economic impacts. This metabolic disorder is characterized by hyperglycaemia and defective insulin signalling. Long-term exposure to hyperglycaemia gives rise to altered fat metabolism and reactive oxygen species (ROS) generation. These precursors are central to the progression of dyslipidaemia and attenuated antioxidant (AO) response and detoxification system, respectively. Diabetic dyslipidaemia and oxidative stress (OS) are risk factors for the onset and progression of cardiovascular disease (CVD) and other diabetic complications. The treatment regimen for T2D comprises self-care and anti-diabetic drugs such as metformin. However, due to the lack of compliance to self-care recommendations and some undesirable side effects of metformin, there is the necessity for alternate therapy. Natural products have been used for the treatment of many disorders, including T2D. Trigonella foenum-graecum commonly known as fenugreek is a plant that possesses anti-diabetic effects. These effects are attributed to its bioactive compound – 4-hydroxyisoleucine (4-OH-lle), which constitutes approximately 80% of the bio-composition of the fenugreek seed. Despite these effects, biochemical and molecular effects of 4-OH-lle on insulin signalling, lipid metabolism, and ROS production is not well-documented. This study investigated the effects of 4-OH-lle in comparison to metformin and fenugreek seed extract (FSE) on hyperglycaemic human hepatoma (HepG2) cells and C57BL/6 male mice. Treatments were conducted under normoglycaemic and hyperglycaemic conditions as follows; control, 4-OH-lle (in vitro: 100ng/ml; in vivo: 100mg/kg Body weight) metformin (in vitro: 20mM; in vivo: 20mg/kg Body weight) and FSE (in vitro: 100ng/ml; in vivo: 100mg/kg Body weight) treatment groups. The experiments included; blood glucose measurements, lipid profile analysis, spectrophotometric assays (in vitro), western blotting for protein expression and qPCR for mRNA expression. First, to validate the effects on insulin signalling and glucose sensing, glucose levels were measured with completion of an oral glucose tolerance test. 4-OH-lle treatment attenuated glucose levels, and elevated the mRNA levels of glycogen synthase (GS) and glucokinase (Gck). This was followed by the investigation of the protein and gene expression of insulin signalling regulators: insulin receptor β (IRβ), insulin receptor substrate 1 (IRS1), phosphorylated protein kinase B (pAkt), phosphorylated glycogen synthase kinase 3α/β (pGSK3α/β) and glucose transport 2 (GLUT2). In in vivo hyperglycaemia, 4-OH-lle increased the expression of the investigated proteins and genes. The results showed that 4-OH-lle was just as potent as MF, and FSE in stimulating the insulin signalling cascade. Second, the effect of 4-OH-lle on dyslipidaemia was investigated by measuring mRNA levels of sterol regulatory binding element 1c (SREBP1c) and fatty acid synthase (FAS) – key factors in fatty acid metabolism. Both genes were up-regulated and correlated with the changes in triglyceride and cholesterol levels. Next the protein expression of proprotein convertase subtilisin-like/kexin type (PCSK9) - a regulator of low density lipoprotein cholesterol (LDLc) and peroxisome proliferator-activated receptor gamma (PPARG) – a regulator of high density lipoprotein (HDLc) was evaluated. The data showed that 4-OH-lle down-regulated protein and mRNA expression of PCSK9 and up-regulated protein expression of PPARG. The reduction in PCSK9 levels correlated with the changes observed in low density lipoprotein receptor (LDLr) and LDLc, whereas the increase in PPARG correlated with the elevated mRNA expression of apolipoprotein A1 (Apo A1) and HDLc. Together these results provide substantial evidence for the regulatory effect of 4-OH-lle, in comparison to metformin, and FSE on PCSK9, PPARG and related lipid factors. Finally, the effect of 4-OH-lle on redox status and AO response was assessed by measuring nuclear factor E2-related factor 2 (Nrf2). In both models, there was an increase in the protein expression of phosphorylated Nrf2 accompanied by an increase in mRNA levels of superoxide dismutase 2 (SOD2) and glutathione peroxidase (GPx), and GSH levels. Mitochondria play a central role in contributing to elevated ROS levels. While nuclear responses like Nrf2 regulate ROS, mitochondria possess their own maintenance proteins. These include mitochondrial Lon protease 1 (LonP1), Sirtuin 3 (SIRT3) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) which play an integral role in combatting OS and mitochondrial dysfunction. The results showed that 4-OH-lle displayed a potent effect in inducing the AO response and increasing mitochondrial regulatory proteins. In conclusion, 4-OH-lle improved the compromised insulin signalling and the altered lipid profile as well as induced the AO response and mitochondrial maintenance proteins, in the presence of elevated glucose. Furthermore, the effect of 4-OH-lle was greater than the first-line drug; metformin and FSE, albeit in cultured human liver cells and a mouse model. Also, the crude seed extract displayed promising effects on all investigated parameters. Considering the active role of chronic hyperglycaemia in the onset and progression of CVD and diabetic complications, 4-OH-lle poses as a highly favourable alternate therapy in the treatment of T2D. Moreover, this has great importance in socio-economically challenged communities where T2D is a common disorder, access to healthcare facilities is limited, and plants serve as sources of easily accessible treatments

Effects of naringenin on metformin disposition in a diabetic rat model.

Doctoral Degree. University of KwaZulu-Natal, Durban.Diabetes mellitus (DM) is one of the largest global health emergencies of the 21st century. It is a major cause of blindness, kidney failure, cardiovascular diseases, lower limb amputation and accounted for 10,7 % of global all-cause mortality among people aged between 20 and 79 years old. Metformin is currently the most widely prescribed anti-diabetic drug. It exists as a hydrophilic cation at physiological pH. As such, membrane transporters play a substantial role in its oral absorption, hepatic uptake, and renal elimination. Among these transporters, organic cation transporters OCT 1 (SLC22A1) and OCT 2 (SLC22A2) are known to be important determinants of the pharmacokinetics of metformin. Naringenin, which is a plant-derived compound found in citrus fruits and vegetables, has been presumed to interact with conventional drugs and influence their disposition by modification of drug-metabolizing enzymes and transporters. The aim of this study was to investigate the effects of naringenin on organic cations transporters OCT1 and OCT2 protein expression and subsequently on metformin disposition in streptozotocin- induced diabetic rats. Methods Forty-nine male Sprague Dawley rats 250–300 g body weight (BW) were randomly divided into 7 experimental groups (n = 7). They were orally treated daily with 3.0 ml/kg body weight (BW) of distilled water (group 1) or 250 mg/kg BW of metformin (groups 3, 6 and 7) or 60 mg/kg BW of naringenin (groups 2, 5 and 7) dissolved in distilled water. Groups 4, 5, 6 and 7 were given a single intraperitoneal injection of 60 mg/kg BW of streptozotocin to induce diabetes. Animal body weights and water intake were recorded daily. Fasting blood glucose (FBG) and glucose tolerance tests (GTT) were subsequently done. Urine samples were collected from rats kept in individual metabolic cages for 24 hours, to determine output, electrolytes, albumin, creatinine and metformin levels. Thereafter, the animals were sacrificed by halothane overdose and blood was collected via cardiac puncture. Liver and kidneys were excised, rinsed in normal saline, blotted dry, weighed, snap frozen in liquid nitrogen and stored at -80°c for analysis of OCT 1 and OCT 2 protein expression by Western blot. OCT 1 and OCT 2 proteins were extracted and separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Then, the gel was blotted electrophoretically onto a nitrocellulose membrane which was then probed with a primary antibody and ultimately an enzyme conjugated secondary antibody and substrate to visualize the bands representing the target proteins. Results Diabetic rats treated with naringenin and metformin either alone or in combination exhibited weight gain, improved creatinine clearance and reduced polydipsia, albuminuria, serum creatinine and blood urea nitrogen compared to untreated diabetic rats. By contrast, metformin with/without naringenin did not significantly ameliorate hyperglycemia in diabetic rats. Treatment with naringenin increased hepatic uptake and renal clearance of metformin in diabetic rats compared to untreated groups. In addition, naringenin significantly increased lactate concentrations and metabolic acidosis in rats treated with metformin compared to those that were not treated with metformin. Furthermore, diabetic rats exhibited lower OCT1 and OCT2 protein expressions but naringenin treatment significantly increased hepatic OCT1 and renal OCT2 protein expressions in the presence of metformin. Conclusion Collectively, our data suggest that metformin disposition could be affected by naringenin through the upregulation of OCT1 and OCT2 protein expressions. Upregulation of OCT1 expression may be associated with metformin-induced lactic acidosis while increased renal OCT2 expression might facilitate metformin excretion and reduce the risk of lactic acid. However, increased renal excretion of metformin by naringenin may not be sufficient to avert metformin-induced lactic acidosis

Monitoring glycation through the intrinsic fluorescence of biological fluorophores

The high blood glucose levels associated with diabetes affect various cells and proteins in the body. In response to high blood glucose the proteins collagen, keratin, and human serum albumin (HSA) experience glycation, and the cofactor reduced nicotinamide adenine dinucleotide (NADH) is also known to be affected in some way. This work aims to establish if the intrinsic fluorescence of these compounds could be used to monitor the impact of glucose, and thus offer an alternative method to monitoring long term glycaemic control. We have studied the evolution of the intrinsic fluorescence of four compounds in response to glucose in vitro using steady state and time-resolved fluorescence spectroscopy techniques. For NADH, although significant changes were seen in the sample when in a phosphate buffered saline (PBS) buffer, no similar changes in fluorescence were observed when a Trizma buffer was used. As such, we conclude that glycation does not have an impact on the intrinsic fluorescence of NADH. Changes in the intrinsic fluorescence of collagen, keratin, and HSA however were observed. For collagen, TRES revealed changes in the intrinsic fluorescence kinetics, caused by both collagen aggregation and glycation. In keratin, the addition of glucose caused an increase in the fluorescence intensity at the characteristic wavelength of 460 nm, due to faster formation of new cross-links, and glucose may also cause the formation of two new fluorescent complexes that emit at longer wavelengths. For HSA, fluorescence intensity decay analysis indicates that glycation can be detected through a decrease in the short lifetime component when decays are fitted to a 2-exponential model, however fitting to a non-Debye model more clearly highlights the impact of glucose. We also studied the intrinsic fluoresce of mouse skin and human fingernails. In an initial pilot study on mouse skin, our results suggested that collagen, keratin, and NADH can be detected from a skin autofluorescence (AF) measurement, and that there may be a relationship between skin AF and blood glucose. Using fingernails, it appeared that although both the peak emission wavelengths, and the fitted exponential parameters indicated that we can detect keratin fluorescence, there was no evidence that glycated keratin can be sensed through a nail clipping. Furthermore, neither our steady state or time resolved analysis exposed a correlation between any of the extracted fluorescence parameters and glycated haemoglobin (HbA1c). In conclusion, monitoring the intrinsic fluorescence of particular biological proteins in vitro suggests that this method could be used as a method to monitor long term glycaemic control in patients with diabetes. Further studies would be required to translate these findings into an in vivo environment.The high blood glucose levels associated with diabetes affect various cells and proteins in the body. In response to high blood glucose the proteins collagen, keratin, and human serum albumin (HSA) experience glycation, and the cofactor reduced nicotinamide adenine dinucleotide (NADH) is also known to be affected in some way. This work aims to establish if the intrinsic fluorescence of these compounds could be used to monitor the impact of glucose, and thus offer an alternative method to monitoring long term glycaemic control. We have studied the evolution of the intrinsic fluorescence of four compounds in response to glucose in vitro using steady state and time-resolved fluorescence spectroscopy techniques. For NADH, although significant changes were seen in the sample when in a phosphate buffered saline (PBS) buffer, no similar changes in fluorescence were observed when a Trizma buffer was used. As such, we conclude that glycation does not have an impact on the intrinsic fluorescence of NADH. Changes in the intrinsic fluorescence of collagen, keratin, and HSA however were observed. For collagen, TRES revealed changes in the intrinsic fluorescence kinetics, caused by both collagen aggregation and glycation. In keratin, the addition of glucose caused an increase in the fluorescence intensity at the characteristic wavelength of 460 nm, due to faster formation of new cross-links, and glucose may also cause the formation of two new fluorescent complexes that emit at longer wavelengths. For HSA, fluorescence intensity decay analysis indicates that glycation can be detected through a decrease in the short lifetime component when decays are fitted to a 2-exponential model, however fitting to a non-Debye model more clearly highlights the impact of glucose. We also studied the intrinsic fluoresce of mouse skin and human fingernails. In an initial pilot study on mouse skin, our results suggested that collagen, keratin, and NADH can be detected from a skin autofluorescence (AF) measurement, and that there may be a relationship between skin AF and blood glucose. Using fingernails, it appeared that although both the peak emission wavelengths, and the fitted exponential parameters indicated that we can detect keratin fluorescence, there was no evidence that glycated keratin can be sensed through a nail clipping. Furthermore, neither our steady state or time resolved analysis exposed a correlation between any of the extracted fluorescence parameters and glycated haemoglobin (HbA1c). In conclusion, monitoring the intrinsic fluorescence of particular biological proteins in vitro suggests that this method could be used as a method to monitor long term glycaemic control in patients with diabetes. Further studies would be required to translate these findings into an in vivo environment

Kinetic Analysis on the Sensitivity of Glucose- or Glyoxal-induced LDL Glycation to the Inhibitory Effect of Psidium Guajava Extract in a Physiomimic System

[[abstract]]Experimentation with a physiomimic system and kinetic analysis exhibited four distinct reaction phases in LDL glycation despite of the type of inducer: glucose or glyoxal. LDL glycation was more sensitive to a status of hyperglycemia (such as 400 mg glucose/100 mL) as evidenced by the reaction order of 0.53. Glucose reacted intensively in the Initial Phase (reaction period 0–2 h) which was identified to result from a parallel mechanism involving both the direct Schiff's product formation and the auto-oxidative cleavages. In contrast, a physiological level of glyoxal revealed merely a reaction order of only 0.09, implicitly indicating a far less sensitive glycation which can be attributed to a mechanism proceeding simply through a molecular Schiff's reaction. On treatment with Psidium guajava L. aqueous extract (PE) (0.01–0.625 mg/mL), a rather unique and significant inhibitory characteristic on LDL glycation was observed with a dose-dependent manner. We attributed such an effect of PE to its distinct abundance of polyphenolic content (165.61 ± 10.39 mg gallic acid equivalent (GAE)/g). Conclusively, PE is an excellent anti-LDL glycative agent whose potential therapeutic uses can be extended to the prevention of a variety of cardiovascular and neurodegenerative diseases associated with glycations