Effect of a nutraceutical treatment on diabetic rats with targeted and CE-MS non-targeted approaches

Despite the introduction of hypoglycemic drugs, diabetes and related complications continue being a major medical problem. Diabetes long-term complications are not only related to the genesis of free radicals due to oxidation of glucose and to the non-enzymatic and progressive glycation of proteins but also to the endothelial dysfunction secondary to persistent hyperglycemia that causes cardiovascular complications. In an experimental model of streptozotocin (STZ) diabetic rats, the effect of five doses of an extract containing both an antioxidant (Rosmarinus officinalis) and folic acid were intragastrically administrated. Urine fingerprints of control and diabetic rats, both with and without treatment, were obtained by capillary electrophoresis with mass spectrometry (CE-TOF-MS). In order to have further biochemical knowledge of the effect, after treatment, rats were killed and plasma glucose, triglycerides, cholesterol, total protein, urea were analysed. Vitamin E in plasma and liver was also measured. Among the changes observed, the reduction in diuresis and plasma triglycerides, together with reduction in 2-aminobutyric, leucine/isoleucine, and dimethylglycine have shown that a short term nutraceutical treatment was able to reduce some of the complications in the STZ diabetic rats. In addition, this CE-MS metabolomic approach has permitted to identify metabolites related to metabolism of arginine, histidine, lysine and glycine in urine that can help monitoring the efficiency of treatments against the deleterious effects of type 1 diabetes.

     

Effect of a nutraceutical treatment on diabetic rats with targeted and CE-MS non-targeted approaches

Despite the introduction of hypoglycemic drugs, diabetes and related complications continue being a major medical problem. Diabetes long-term complications are not only related to the genesis of free radicals due to oxidation of glucose and to the non-enzymatic and progressive glycation of proteins but also to the endothelial dysfunction secondary to persistent hyperglycemia that causes cardiovascular complications. In an experimental model of streptozotocin (STZ) diabetic rats, the effect of five doses of an extract containing both an antioxidant (Rosmarinus officinalis) and folic acid were intragastrically administrated. Urine fingerprints of control and diabetic rats, both with and without treatment, were obtained by capillary electrophoresis with mass spectrometry (CE-TOF-MS). In order to have further biochemical knowledge of the effect, after treatment, rats were killed and plasma glucose, triglycerides, cholesterol, total protein, urea were analysed. Vitamin E in plasma and liver was also measured. Among the changes observed, the reduction in diuresis and plasma triglycerides, together with reduction in 2-aminobutyric, leucine/isoleucine, and dimethylglycine have shown that a short term nutraceutical treatment was able to reduce some of the complications in the STZ diabetic rats. In addition, this CE-MS metabolomic approach has permitted to identify metabolites related to metabolism of arginine, histidine, lysine and glycine in urine that can help monitoring the efficiency of treatments against the deleterious effects of type 1 diabetes.     

Subcellular compartmentation of the diterpene carnosic acid and its derivatives in the leaves of rosemary

The potent antioxidant properties of rosemary (Rosmarinus officinalis) extracts have been attributed to its major diterpene, carnosic acid. Carnosic acid has received considerable attention in food science and biomedicine, but little is known about its function in the plant in vivo. We recently found that highly oxidized diterpenes increase in rosemary plants exposed to drought and high light stress as a result of the antioxidant activity of carnosic acid (S. Munné-Bosch, K. Schwarz, L. Alegre [1999] Plant Physiol 121: 1047-1052). To elucidate the significance of the antioxidant function of carnosic acid in vivo we measured the relative amounts of carnosic acid and its metabolites in different compartments of rosemary leaves. Subcellular localization studies show that carnosic acid protects chloroplasts from oxidative stress in vivo by following a highly regulated compartmentation of oxidation products. Carnosic acid scavenges free radicals within the chloroplasts, giving rise to diterpene alcohols, mainly isorosmanol. This oxidation product is O-methylated within the chloroplasts, and the resulting form, 11,12-di-O-methylisorosmanol, is transferred to the plasma membrane. This appears to represent a mechanism of a way out for free radicals from chloroplasts. Carnosic acid also undergoes direct O-methylation within the chloroplasts, and its derived product, 12-O-methylcarnosic acid, accumulates in the plasma membrane. O-methylated diterpenes do not display antioxidant activity, but they may influence the stability of the plasma membrane. This study shows the relevance of the compartmentation of carnosic acid metabolism to the protection of rosemary plants from oxidative stress in vivo.     

A liquid chromatography-quadrupole time-of-flight (LC-QTOF)-based metabolomic approach reveals new metabolic effects of catechin in rats fed high-fat diets

Unbalanced diets generate oxidative stress commonly associated with the development of diabetes, atherosclerosis, obesity and cancer. Dietary flavonoids have antioxidant properties and may limit this stress and reduce the risk of these diseases. We used a metabolomic approach to study the influence of catechin, a common flavonoid naturally occurring in various fruits, wine or chocolate, on the metabolic changes induced by hyperlipidemic diets. Male Wistar rats ( n = 8/group) were fed during 6 weeks normolipidemic (5% w/w) or hyperlipidemic (15 and 25%) diets with or without catechin supplementation (0.2% w/w). Urines were collected at days 17 and 38 and analyzed by reverse-phase liquid chromatography-mass spectrometry (LC-QTOF). Hyperlipidic diets led to a significant increase of oxidative stress in liver and aorta, upon which catechin had no effect. Multivariate analyses (PCA and PLS-DA) of the urine fingerprints allowed discrimination of the different diets. Variables were then classified according to their dependence on lipid and catechin intake (ANOVA). Nine variables were identified as catechin metabolites of tissular or microbial origin. Around 1000 variables were significantly affected by the lipid content of the diet, and 76 were fully reversed by catechin supplementation. Four variables showing an increase in urinary excretion in rats fed the high-fat diets were identified as deoxycytidine, nicotinic acid, dihydroxyquinoline and pipecolinic acid. After catechin supplementation, the excretion of nicotinic acid was fully restored to the level found in the rats fed the low-fat diet. The physiological significance of these metabolic changes is discussed.     

Metabolic regulatory network alterations in response to acute cold stress and ginsenoside intervention

Acute stress may trigger systemic biochemical and physiological changes in living organisms, leading to a rapid loss of homeostasis, which can be gradually reinstated by self-regulatory mechanisms and/or drug intervention strategy. However, such a sophisticated metabolic regulatory process has so far been poorly understood, especially from a holistic view. Urinary metabolite profiling of Sprague-Dawley rats exposed to cold temperature (-10 degrees C) for 2 h using GC/MS in conjunction with modern multivariate statistical techniques revealed drastic biochemical changes as evidenced by fluctuations of urinary metabolites and demonstrated the protective effect of total ginsenosides (TGs) in ginseng extracts on stressed rats. The metabonomics approach enables us to visualize significant alterations in metabolite expression patterns as a result of stress-induced metabolic responses and post-stress compensation, and drug intervention. Several major metabolic pathways including catecholamines, glucocorticoids, the tricarboxylic acid (TCA) cycle, tryptophan (nicotinate), and gut microbiota metabolites were identified to be involved in metabolic regulation and compensation required to restore homeostasis.     

Therapeutic Applications of Naringenin,a Flavanone Enriched in Citrus Fruits,for Disorders beyond Diabetes

In recent years, there has been an increase in epidemiological studies to highlight the health benefits of plant secondary metabolites. Flavonoids (polyphenolic plant secondary metabolites) are recently emerging as an important source for the discovery of new drugs increasing their pharmaceuticals, nutraceutical and medicinal applications. Naringenin is a flavanone, enriched in citrus fruits, tomatoes, bergamot, etc. which has been evaluated extensively for managing diabetes. However, in addition to this, naringenin had been ascribed to various important biological activities like antioxidant, antiviral, anticancer, anti-inflammatory, antiestrogenic, etc. This article aims at highlighting the therapeutic value of naringenin in managing disorders other than diabetes and its role in regulating gene expression by altering chromatin structure as histone deacetylase inhibitor. The understanding of these phenomena will increase the overall knowledge of the various health-promoting effects of citrus fruits.     

Hypolipidemic effects of Rosmarinus officinalis L

Dyslipidemia is one of the major risk factors for cardiovascular diseases (CVDs). Current strategies are not effective in the management of dyslipidemia. Thus, there is a necessity to find new preventative and therapeutic approaches. In recent years, herbal medicine has drawn great attention regarding the prevention and management of dyslipidemia. Rosmarinus officinalis, commonly known as rosemary, is an evergreen shrub containing several polyphenols. The plant grows in the Mediterranean and South American regions. Rosemary and its main components have antioxidant, anti-inflammatory, and lipid-lowering properties. The present review has focused on in vivo and in vitro studies on the hypolipidemic effects of rosemary and its main constituents as well as their functional mechanisms. Studies have described lipid-scavenging activities of rosemary through its flavonoid contents. Modulating inflammation and oxidative stress have been described as possible mechanisms by which rosemary ameliorates dyslipidemia. However, the exact mechanisms are not fully understood yet. Conducting experimental and clinical trial studies are recommended to confirm the safety and efficacy of rosemary in the prevention and management of dyslipidemia and other cardio-metabolic diseases.     

Synergistic effect of folic acid and vitamin B12 in ameliorating arsenic-induced oxidative damage in pancreatic tissue of rat

The efficacies of two nutritional factors, folic acid and vitamin B12, were assessed in this study against arsenic-induced islet cellular toxicity. Rats were divided into four groups consisting of five rats in each group: Group A, control; Group B, arsenic-treated; Group C, arsenic+folic acid; and Group D, arsenic+folic acid+vitamin B12. The dose of arsenic, folic acid and vitamin B12, respectively, was 3 mg, 36 microg and 0.63 microg kg(-1) body weight day(-1) for 30 days. Results showed that, compared to control group, there was a significant increase in the levels of nitric oxide (NO), malondialdehyde (MDA) and hydroxyl radical (OH-) formation in the pancreatic tissue of arsenic-treated rats, while the activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), and cellular content of antioxidant glutathione (GSH) were low in these animals. The serum level of tumor necrosis factor-alpha (TNF-alpha) and IL-6 was significantly high in these animals. Light microscopic examination showed a marked fall in the number of islet cells. Concomitant administration of either folic acid or folic acid and vitamin B12 with arsenic significantly restored all these parameters. Although folic acid alone could not restore the normal level of TNF-alpha and IL-6, combined folic acid and vitamin B12 could restore it. Folic acid and vitamin B12 combined also could recover islet cell count. These results suggest that folic acid+vitamin B12 are capable of reducing arsenic-induced cellular oxidative and inflammatory toxic changes. Thus, supplement with vitamin B12+folic acid may be predicted as a possible nutritional management strategy against arsenic-induced toxicity.     

Ursodeoxycholic Acid Ameliorates Fructose-Induced Metabolic Syndrome in Rats

The metabolic syndrome (MS) is characterized by insulin resistance, dyslipidemia and hypertension. It is associated with increased risk of cardiovascular diseases and type-2 diabetes. Consumption of fructose is linked to increased prevalence of MS. Ursodeoxycholic acid (UDCA) is a steroid bile acid with antioxidant, anti-inflammatory activities and has been shown to improve insulin resistance. The current study aims to investigate the effect of UDCA (150 mg/kg) on MS induced in rats by fructose administration (10%) in drinking water for 12 weeks. The effects of UDCA were compared to fenofibrate (100 mg/kg), an agonist of PPAR-α receptors. Treatment with UDCA or fenofibrate started from the 6^th week after fructose administration once daily. Fructose administration resulted in significant increase in body weight, elevations of blood glucose, serum insulin, cholesterol, triglycerides, advanced glycation end products (AGEs), uric acid levels, insulin resistance index and blood pressure compared to control rats. Moreover, fructose increased oxidative stress in aortic tissues indicated by significant increases of malondialdehyde (MDA), expression of iNOS and reduction of reduced glutathione (GSH) content. These disturbances were associated with decreased eNOS expression, increased infiltration of leukocytes and loss of aortic vascular elasticity. Treatment with UDCA successfully ameliorated the deleterious effects of fructose. The protective effect of UDCA could be attributed to its ability to decrease uric acid level, improve insulin resistance and diminish oxidative stress in vascular tissues. These results might support possible clinical application of UDCA in MS patients especially those present with liver diseases, taking into account its tolerability and safety. However, further investigations on human subjects are needed before the clinical application of UDCA for this indication.     

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyl-l-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.     

Carbohydrate and amino acid metabolism and oxidative status in Holstein heifers precision-fed diets with different forage to concentrate ratios

Previous work led to the proposal that the precision feeding of a high-concentrate diet may represent a potential method with which to enhance feed efficiency (FE) when rearing dairy heifers. However, the physiological and metabolic mechanisms underlying this approach remain unclear. This study used metabolomics analysis to investigate the changes in plasma metabolites of heifers precision-fed diets containing a wide range of forage to concentrate ratios. Twenty-four half-sib Holstein heifers, with a similar body condition, were randomly assigned into four groups and precision fed with diets containing different proportions of concentrate (20%, 40%, 60% and 80% based on DM). After 28 days of feeding, blood samples were collected 6 h after morning feeding and gas chromatography time-of-flight/MS was used to analyze the plasma samples. Parameters of oxidative status were also determined in the plasma. The FE (after being corrected for gut fill) increased linearly (P < 0.01) with increasing level of dietary concentrate. Significant changes were identified for 38 different metabolites in the plasma of heifers fed different dietary forage to concentrate ratios. The main pathways showing alterations were clustered into those relating to carbohydrate and amino acid metabolism; all of which have been previously associated with FE changes in ruminants. Heifers fed with a high-concentrate diet had higher (P < 0.01) plasma total antioxidant capacity and superoxide dismutase but lower (P ≤ 0.02) hydroxyl radical and hydrogen peroxide than heifers fed with a low-concentrate diet, which might indicate a lower plasma oxidative status in the heifers fed a high-concentrate diet. Thus, heifers fed with a high-concentrate diet had higher FE and antioxidant capacity but a lower plasma oxidative status as well as changed carbohydrate and amino acid metabolism. Our findings provide a better understanding of how forage to concentrate ratios affect FE and metabolism in the precision-fed growing heifers.     

Efficacy of lower doses of vanadium in restoring altered glucose metabolism and antioxidant status in diabetic rat lenses

Vanadium compounds are potent in controlling elevated blood glucose levels in experimentally induced diabetes. However the toxicity associated with vanadium limits its role as therapeutic agent for diabetic treatment. A vanadium compound sodium orthovanadate (SOV) was given to alloxan-induced diabetic Wistar rats in lower doses in combination withTrigonella foenum graecum, a well-known hypoglycemic agent used in traditional Indian medicines. The effect of this combination was studied on lens morphology and glucose metabolism in diabetic rats. Lens, an insulin-independent tissue, was found severely affected in diabetes showing visual signs of cataract. Alterations in the activities of glucose metabolizing enzymes (hexokinase, aldose reductase, sorbitol dehydrogenase, glucose-6-phosphate dehydrogenase) and antioxidant enzymes (glutathione peroxidase, glutathione reductase) besides the levels of related metabolites, [sorbitol, fructose, glucose, thiobarbituric acid reactive species (TBARS) and reduced glutathione (GSH)]were observed in the lenses from diabetic rats and diabetic rats treated with insulin (2 IU/day), SOV (0.6 mg/ml),T. f. graecum seed powder (TSP, 5%) and TSP (5%) in combination with lowered dose of vanadium SOV (0.2 mg/ml), for a period of 3 weeks. The activity of the enzymes, hexokinase, aldose reductase and sorbitol dehydrogenase was significantly increased whereas the activity of glucose-6-phosphate dehydrogenase, glutathione peroxidase and glutathione reductase decreased significantly in lenses from 3 week diabetic rats. Significant increase in accumulation of metabolites, sorbitol, fructose, glucose was found in diabetic lenses. TBARS measure of peroxidation increased whereas the levels of antioxidant GSH decreased significantly in diabetic condition. Insulin restored the levels of altered enzyme activities and metabolites almost to control levels. Sodium orthovanadate (0.6 mg/ml) andTrigonella administered separately to diabetic animals could partially reverse the diabetic changes, metabolic and morphological, while vanadate in lowered dose in combination withTrigonella was found to be the most effective in restoring the altered lens metabolism and morphological appearance in diabetes. It may be concluded that vanadate at lowered doses administered in combination withTrigonella was the most effective in controlling the altered glucose metabolism and antioxidant status in diabetic lenses, these being significant factors involved in the development of diabetic complications, that reflects in the reduced lens opacity     

Physiological investigations on the effect of olive and rosemary leaves extracts in male rats exposed to thioacetamide

Physiologically, it is known that thioacetamide (TAA) toxicity is generally associated with hepatic fibrosis induction, complicated metabolic disorders and health problems. The capability of extracts of olive and rosemary leaves to attenuate the severe physiological disturbances induced by thioacetamide (TAA) intoxication in male rats has been evaluated. Healthy male Wistar rats were used in the present study and were divided randomly into eight groups. Rats of the first group were served as normal control. Rats of the second group were administrated with TAA. Rats of the third, fourth and fifth groups were exposed to TAA plus olive leaves extract, TAA plus rosemary leaves extract and TAA plus olive and rosemary leaves extracts respectively. The sixth, seventh and eighth groups were supplemented with olive leaves extract, rosemary leaves extract, and olive and rosemary leaves extracts respectively. After 12 weeks of experimental treatments, the levels of serum glucose, total protein, albumin and high density lipoprotein cholesterol were significantly decreased, while the levels of triglycerides, cholesterol, low density lipoprotein cholesterol, very low density lipoprotein cholesterol, creatine kinase and lactate dehydrogenase were statistically increased in rats exposed to TAA. Administration of the studied extracts inhibited the hematobiochemical parameters and improved the physiological disturbances induced by TAA intoxication. Additionally, most improvements were noted in rats administrated with rosemary leaves extract followed by olive and rosemary leaves extracts and olive leaves extract. These results suggested that the effect of these extracts might be due to their antioxidant activities against TAA toxicity.     

Granger causality in integrated GC–MS and LC–MS metabolomics data reveals the interface of primary and secondary metabolism

Metabolomics has emerged as a key technique of modern life sciences in recent years. Two major techniques for metabolomics in the last 10 years are gas chromatography coupled to mass spectrometry (GC–MS) and liquid chromatography coupled to mass spectrometry (LC–MS). Each platform has a specific performance detecting subsets of metabolites. GC–MS in combination with derivatisation has a preference for small polar metabolites covering primary metabolism. In contrast, reversed phase LC–MS covers large hydrophobic metabolites predominant in secondary metabolism. Here, we present an integrative metabolomics platform providing a mean to reveal the interaction of primary and secondary metabolism in plants and other organisms. The strategy combines GC–MS and LC–MS analysis of the same sample, a novel alignment tool MetMAX and a statistical toolbox COVAIN for data integration and linkage of Granger Causality with metabolic modelling. For metabolic modelling we have implemented the combined GC–LC–MS metabolomics data covariance matrix and a stoichiometric matrix of the underlying biochemical reaction network. The changes in biochemical regulation are expressed as differential Jacobian matrices. Applying the Granger causality, a subset of secondary metabolites was detected with significant correlations to primary metabolites such as sugars and amino acids. These metabolic subsets were compiled into a stoichiometric matrix N. Using N the inverse calculation of a differential Jacobian J from metabolomics data was possible. Key points of regulation at the interface of primary and secondary metabolism were identified.     

Identification of felodipine metabolites in rat urine

After intragastric administration of 100 mumol kg-1 [14C]felodipine to rats eight urinary metabolites were isolated. Batch extraction at pH 2.2 and semipreparative reversed-phase liquid chromatography were used for trace enrichment of the metabolites. Trimethylsilylation followed by transesterification with diazomethane blocked the carboxylic acid and alcohol groups selectively before gas chromatography/mass spectrometry (GC/MS) in the electron impact (EI) mode. Deuterated derivatives of the metabolites and chemical ionization measurements added complementary structural information. All metabolites reported in this study were formed from oxidized felodipine by ester hydrolysis. Hydroxylation of the pyridine methyl group represented an important metabolic pathway and metabolites oxidized to the corresponding carboxylic acids were detected as well. Lactone formation from hydroxy acid metabolites in urine as a possible analytical artefact is discussed.     

Beyond glucose: metabolic shifts in responses to the effects of the oral glucose tolerance test and the high-fructose diet in rats

High-fructose diet-fed rats as one of the insulin resistant models was used widely for understanding the mechanisms of type 2 diabetes mellitus. Systems-level metabolic profiling of the rat model, however, has not been deciphered clearly. To address this issue, mass spectrometry-based metabolomics was employed to unlock the metabolic snapshots of the oral glucose tolerance test (oGTT) effect in either healthy or diabetic rats, as well as to delineate the metabolic signatures in tissues of rats fed with high-fructose diet. Several differentiating metabolites were highlighted to reveal the metabolic perturbation of the oGTT effects in healthy and diabetic rats, which involved amino acid biosynthesis, polyunsaturated fatty acids, phospholipids and purine metabolism. Surprisingly, the patterns of relationships for the metabolic phenotypes by using data mining revealed that glucose ingestion might induce the healthy group to display its trajectory towards diabetic status, while only a very slight influence was observed on the high-fructose diet-fed rats 120 min after glucose ingestion. The data treatment for liver, skeletal muscle and brain tissues suggested that oxidative stress, such as lipid peroxidation and the declined antioxidant, the elevated amino acids and the perturbation of fatty acids, were caused by the high-fructose diet in liver and skeletal muscle tissues. On the other hand, the up-regulation in purine biosynthesis and the decreased concentrations for amino acids were observed in the cerebral cortex and hippocampus tissues. Collectively, the obtained results might provide a new insight not only for the impairment of glucose tolerance but also for the dietary style in rats.     

Green tea polyphenols modify gut-microbiota dependent metabolisms of energy,bile constituents and micronutrients in female Sprague–Dawley rats

Our recent metagenomics analysis has uncovered remarkable modifying effects of green tea polyphenols (GTP) on gut-microbiota community structure and energy conversion related gene orthologs in rats. How these genomic changes could further influence host health is still unclear. In this work, the alterations of gut-microbiota dependent metabolites were studied in the GTP-treated rats. Six groups of female SD rats (n=12/group) were administered drinking water containing 0%, 0.5%, and 1.5% GTP (wt/vol). Their gut contents were collected at 3 and 6 months and were analyzed via high performance liquid chromatography (HPLC) and gas chromatography (GC)-mass spectrometry (MS). GC–MS based metabolomics analysis captured 2668 feature, and 57 metabolites were imputatively from top 200 differential features identified via NIST fragmentation database. A group of key metabolites were quantitated using standard calibration methods. Compared with control, the elevated components in the GTP-treated groups include niacin (8.61-fold), 3-phenyllactic acid (2.20-fold), galactose (3.13-fold), mannose (2.05-fold), pentadecanoic acid (2.15-fold), lactic acid (2.70-fold), and proline (2.15-fold); the reduced components include cholesterol (0.29-fold), cholic acid (0.62-fold), deoxycholic acid (0.41-fold), trehalose (0.14-fold), glucose (0.46-fold), fructose (0.12-fold), and alanine (0.61-fold). These results were in line with the genomic alterations of gut-microbiome previously discovered by metagenomics analysis. The alterations of these metabolites suggested the reduction of calorific carbohydrates, elevation of vitamin production, decreases of bile constituents, and modified metabolic pattern of amino acids in the GTP-treated animals. Changes in gut-microbiota associated metabolism may be a major contributor to the anti-obesity function of GTP.     

碳源对迷迭香悬浮培养细胞的生长、迷迭香酸积累及抗氧化酶活性的影响

以迷迭香悬浮培养细胞为材料,详细研究了基本培养基中添加蔗糖、麦芽糖和葡萄糖对细胞生长及次生代谢产物积累的影响,同时对不同蔗糖浓度处理的悬浮培养细胞抗氧化酶活性进行了研究。研究结果表明：在不同的糖处理中,30 g·L^-1的蔗糖、70 g·L^-1的麦芽糖及40 g·L^-1的葡萄糖最有利于迷迭香悬浮培养细胞生长。30 g·L^-1蔗糖和70 g·L^-1麦芽糖处理中悬浮培养细胞的生长率分别为74.08%和72.33%,高出40 g·L^-1葡萄糖处理接近3倍之多。30 g·L^-1蔗糖处理的悬浮培养细胞迷迭香酸含量高出70 g·L^-1麦芽糖处理228倍,略低于40 g·L^-1葡萄糖处理。在不同蔗糖的处理中,随着蔗糖浓度的增加,迷迭香酸含量均呈现增加趋势,表明高浓度的蔗糖有利于悬浮培养细胞迷迭香酸的积累。在高浓度的蔗糖处理中,悬浮培养细胞H₂O₂和MDA含量明显增加,同时抗氧化酶SOD、POD及CAT的活性也明显增强,表明高浓度的蔗糖产生了渗透胁迫,这种渗透胁迫虽不利于迷迭香悬浮培养细胞的生长,但有利于次生代谢产物的积累。综合迷迭香悬浮细胞的生长率和迷迭香酸的含量,我们最终得出30 g·L^-1的蔗糖最有利于迷迭香悬浮细胞的培养。     

Rosemary (Rosmarinus officinalis) diterpenes affect lipid polymorphism and fluidity in phospholipid membranes

Rosemary (Rosmarinus officinalis) extracts are widely used in the food, nutraceutical and cosmetic areas. Their major bioactive components have shown antioxidant, antimicrobial, anti-inflammatory, antitumorigenic and chemopreventive activities. In this work, the bioactive compounds deriving from rosemary leaves (carnosol, CAR; carnosic acid, CA; rosmadial, RAL; genkwanin, GW; rosmarinic acid, RA) were isolated and their effects on the phase behaviour of model membranes were studied by several complementary biophysical techniques. All diterpenes studied, and specifically CAR, decreased the hydrophobic interactions between acyl chains, as well as broadened and shifted the phospholipid transition to lower temperatures into dimyristoylphosphatidylcholine (DMPC) membranes. In addition, all diterpenes and genkwanin increased the lipid order of fluid DMPC membranes, exhibiting CAR and RAL the strongest membrane-rigidifying effect. The diterpenoids, especially CA and RAL, promoted the formation of hexagonal-H(II) phases at low temperatures in dielaidoylphosphatidylethanolamine (DEPE) membranes which exhibited a smaller tube-to-tube distance compared to pure phospholipid. These diterpenes were also able of promoting isotropic structures in DEPE membranes which consisted of non-periodically ordered lipid structures as demonstrated by X-ray diffraction. In contrast, minor effects were observed by rosmarinic acid. In conclusion, diterpenes and genkwanin from rosemary show membrane-rigidifying effects which may contribute to their antioxidant capacity through hindering diffusion of free radicals.