Analysis of cardioprotective effects using purified Saliva miltiorrhiza extract on isolated rat hearts

The purpose of the current study is to evaluate the cardioprotective effects of purified Salvia miltiorrhiza extract (PSME) on myocardial ischemia/reperfusion injury in isolated rat hearts. Hearts were excised and perfused at constant flow (7 – 9 ml · min⁻¹) via the aorta. Non-recirculating perfusion with Krebs-Henseleit (KH) solution was maintained at 37°C and continuously gassed with 95% O₂ and 5% CO₂. KH solution with or without PSME (100 mg per liter solution) was used after 30-min zero-flow ischemia for the PSME and control group, respectively. Left ventricular (LV) developed pressure; its derivatives, diastolic pressure, and so on were continuously recorded via a pressure transducer attached to a polyvinylchloride balloon that was placed in the left ventricle through an incision in the left atrium. PSME treated hearts showed significant postischemic contractile function recovery (developed pressure recovered to 44.2 ± 4.9% versus 17.1 ± 5.7%, P<0.05; maximum contraction recovered to 57.2 ± 5.9% versus 15.1 ± 6.3%, P<0.001; maximum relaxation restored to 69.3 ± 7.3% versus 15.4 ± 6.3%, P<0.001 in the PSME and control group, respectively). Significant elevation in end-diastolic pressure, which indicated LV stiffening in PSME hearts might have resulted from the excess high dose of PSME used. Further study will be conducted on the potential therapeutic value with lower dose of PSME on prevention of ischemic heart disease.

Effects of purified herbal extract of Saliva miltiorrhiza on ischemic rat myocardium after acute myocardial infarction

In the current study, we compared purified Salvia miltiorrhiza extract (PSME) with Angiotensin-converting enzyme inhibitor, Ramipril, in in vitro experiments and also in vivo using animal model of myocardial infarction. PSME was found to have a significantly higher trolox equivalent antioxidant capacity which indicated a great capacity for scavenging free radicals. PSME could also prevent pyrogallo red bleaching and DNA damage.

After 2 weeks treatment with PSME or Ramipril, survival rates of rats with experimental myocardial infarction were marginally increased (68.2% and 71.4%) compared with saline (61.5%). The ratios of infarct size to left ventricular size in both PSME-and Ramipril-treated rats were significantly less than that in the saline-treated group. Activity of cardiac antioxidant enzyme superoxide dismutase (SOD) was significant higher while level of Thiobarbituric acid-reactive substances (TBARs) was lower in the PSME treated group. Purified and standardized Chinese herb could provide an alternative regimen for the prevention of ischemic heart disease.

A mechanistic study of the intestinal absorption of crypotanshinone, the major active constituent of Saliva miltiorrhiza

The nature of intestinal absorption of most herbal medicine is unknown. Cryptotanshinone (CTS) is the principal active constituent of the widely used cardiovascular herb Salvia miltiorrhiza (Danshen). We investigated the oral bioavailability of CTS in rats and the mechanism for its intestinal absorption using several in vitro and in vivo models:1) Caco-2 cell monolayers; 2) monolayers of MDCKII cells overexpressing P-glycoprotein
(PgP); and 3) single-pass rat intestinal perfusion with mesenteric vein cannulation. The systemic bioavailabilities of CTS after oral and intraperitoneal administration at 100 mg/kg were 2.05 and 10.60%, respectively. In the perfused rat intestinal model, permeability coefficients based on CTS disappearance from the luminal perfusate (P_lumen) were 6.7- to 10.3-fold higher than permeability coefficients based on drug appearance in venous blood (P_blood). P_blood significantly increased in the presence of the P-gP inhibitor, verapamil. CTS transport across Caco-2 monolayers was pH-, temperature- and ATP-dependent. The transport from the apical (AP) to the basolateral (BL) side was 3- to 9-fold lower than that from the BL to the AP side. Inclusion of verapamil (50 µM) in both AP and BL sides abolished the polarized CTS transport across Caco-2 cells. Moreover, CTS was significantly more permeable in the BL to AP than in the AP to BL direction in MDCKII and MDR1-MDCKII cells. The permeability coefficients in the BL to AP direction were significantly higher in MDCKII cells overexpressing PgP. These findings indicate that CTS is a substrate for PgP that can pump CTS into the luminal side.

Measurement of immunoglobulin A in saliva by particle-enhanced nephelometric immunoassay: sample collection, limits of quantitation, precision, stability and reference range

Background: Total immunoglobulin A in saliva (s-IgA) is normally assayed using an enzyme-linked immunosorbent assay. We have investigated methodological issues relating to the use of particle-enhanced nephelometric immunoassay (PENIA)
to measure s-IgA in whole unstimulated saliva and determine its reference range.

Methods: Whole unstimulated resting saliva was collected to determine sample stability (temperature, time, effect of a protease inhibitor), limit of quantitation (LOQ), assay precision and analytical variation. The reference range for 134 healthy adults was determined.

Results: Linearity was excellent (4–10.3 mg L21, P, 0.001; R2 ¼ 0.997) and without significant bias (mean of 20.7%). The lowest intra- and inter-analytical coefficients of variation were 1.8% and 7.5% and LOQ was 1.4 mg L21. The concentration of s-IgA is stable at room temperature for up to 6 h, at 48C for 48 h, at 248C for two weeks and at 2808C for up to 1.3 yr. There is no evidence that a protease inhibitor increases the stability or that repeated freeze–thawing cycles degrade sample quality. The reference ranges for s-IgA concentration, s-IgA secretion, s-IgA:albumin and s-IgA:osmolality were 15.9–414.5 mg L21, 7.2–234.9 mg min21, 0.4–19 and 0.6–8.9, respectively.

Conclusion: Automated PENIA assay of s-IgA is precise and accurate. High stability of collected saliva samples and the ease and speed of the assay make this an ideal method for use in athletic and military training situations. The convenience of measuring albumin and IgA on the same analytical platform adds to the practicability of the test.

Dietary electrolytes are related to mood

Dietary therapies are routinely recommended to reduce disease risk; however, there is concern they may adversely affect mood. We compared the effect on mood of a low-sodium, high-potassium diet (LNAHK) and a high-calcium diet (HC) with a moderate-sodium, high-potassium, high-calcium Dietary Approaches to Stop Hypertension (DASH)-type diet (OD). We also assessed the relationship between dietary electrolytes and cortisol, a stress hormone and marker of hypothalamic–pituitary–adrenal (HPA) axis activity. In a crossover design, subjects were randomized to two diets for 4 weeks, the OD and either LNAHK or HC, each preceded by a 2-week control diet (CD). Dietary compliance was assessed by 24 h urine collections. Mood was measured weekly by the Profile of Mood States (POMS). Saliva samples were collected to measure cortisol. The change in mood between the preceding CD and the test diet (LNAHK or HC) was compared with the change between the CD and OD. Of the thirty-eight women and fifty-six men (mean age 56·3 (sem 9·8) years) that completed the OD, forty-three completed the LNAHK and forty-eight the HC. There was a greater improvement in depression, tension, vigour and the POMS global score for the LNAHK diet compared to OD (P < 0·05). Higher cortisol levels were weakly associated with greater vigour, lower fatigue, and higher levels of urinary potassium and magnesium (r 0·1–0·2, P < 0·05 for all). In conclusion, a LNAHK diet appeared to have a positive effect on overall mood.

Sour taste preferences of children relates to preference for novel and intense stimuli

Previous research has suggested that some children have a preference for sour tastes. The origin of this preference remains unclear. We investigated whether preference for sour tastes is related to a difference in rated sour intensity due to physiological properties of saliva, or to an overall preference for intense and new stimuli. Eighty-nine children 7–12 years old carried out a rank-order procedure for preference and category scale for perceived intensity for four gelatins (i.e. 0.0 M, 0.02 M, 0.08 M, 0.25 M added citric acid) and four yellow cards that differed in brightness. In addition, we measured their willingness to try a novel candy and their flow and buffering capacity of their saliva. Fifty-eight percent of the children tested preferred one of the two most sour gelatins. These children had a higher preference for the brightest color (P < 0.05) and were more likely to try the candy with the unknown flavor (P < 0.001) than children who did not prefer the most sour gelatins. Preference for sour taste was not related with differences in rated sour intensity, however those who preferred sour taste had a higher salivary flow (P < 0.05). These findings show that a substantial proportion of young children have a preference for extreme sour taste. This appears to be related to the willingness to try unknown foods and preference for intense visual stimuli. Further research is needed to investigate how these findings can be implemented in the promotion of sour-tasting food such as fruit.

Oxidised mannan as a novel adjuvant inducing mucosal IgA production.

Mannan, oxidatively coupled to recombinant protein antigens, has here been tested as a possible adjuvant for the production of antibody on the mucosa. Given intranasally, but not intraperitoneally, mannan markedly enhanced the production of IgA, IgG1 and IgG2a in the serum, and IgA locally in the lung and at remote mucosal sites, including tears, vaginal and salivary secretions. Oxidative coupling was critical to its action, since neither mannan simply mixed with protein nor mannan–protein conjugates which had been reduced by treatment with sodium borohydride, acted as adjuvants. Oxidatively coupled mannan was compared with the widely studied mucosal adjuvant, cholera toxin (CT). The use of oxidised mannan as an adjuvant induced better responses than CT judged by the induction of IgA in serum, vaginal washings and saliva. Thus, oxidised mannan, which is non-toxic and can be administered without injection, is a suitable adjuvant coupled with protective antigens for vaccinating against a number of infections that occur via the mucous membranes.

Copper levels in buccal cells of vineyard workers engaged in various activities

Objectives: Copper-based compounds have been used as agricultural fungicides for many years. Their use in Australia is escalating with increase in the scale of planting and associated pest problems. The objective of this study was to identify viticulture activities associated with high exposure to foliage sprays. It would be determined if occupational exposure of vineyard workers to copper-based sprays was associated with raised body copper levels through analysis of saliva and buccal cells.

Methods: The activities of six vineyard workers from four vineyards in the Yarra Valley Victoria, Australia, were monitored over a period of 2 years. During this period, workers carried out seasonal activities, including fungicide spraying, canopy management, and tractor operation. Saliva and buccal cells from workers were collected and analysed for copper levels that were then correlated with the different types of vineyard activity.

Results: The buccal cells of vineyard workers exposed to copper through seasonal activities including fungicide spraying, canopy management, and tractor operation contained copper levels of 0.87, 1.24, and 0.95 ng Cu per 106 cells, respectively. This was up to 10-fold higher than the copper levels in buccal cells from the control subjects (0.1 ng Cu per 106). Copper levels in buccal cells from workers participating in other viticulture activities such as shoot thinning, bunch counting, and disbudding were not significantly different from those of control subjects. The levels of copper in saliva samples of both workers undertaking any vineyard activity and control subjects were below the level of detection.

Conclusions: Seasonal activities undertaken in vineyards that involved direct contact with copper, in particular canopy management, fungicidal spraying, and tractor operation were associated with high copper levels in buccal cells of workers. This indicates that copper derived from copper-based fungicidal compounds is accumulated within body cells. The lack of detectable copper levels in saliva suggests that the route of transport of copper into buccal cells is not through saliva. The results indicate potential adverse health risks associated with use of copper fungicide. Recommendations are made in relation to the precautions that should be taken in relation to use of copper sprays and to validate buccal cells as an indicator of body copper status.

The effect of an ultra-endurance running race on mucosal and humoral immune function

There are reports of the effect of endurance exercise on mucosal immune function and of the effect of short duration exercise on humoral immune function. However, little is known of the effect of endurance exercise on humoral immune function and the related risk of infection. This study examined the effects of an ultra-endurance running race on salivary immunoglobulin-A (s-IgA), serum IgA, leukocyte subset concentrations and the incidence of upper respiratory tract infections (URTI). 

Considerations for the use of salivary IgA for monitoring mucosal immune function

Introduction: 

Study protocol: the childhood to adolescence transition study (CATS)

Background
Puberty is a multifaceted developmental process that begins in late-childhood with a cascade of endocrine changes that ultimately lead to sexual maturation and reproductive capability. The transition through puberty is marked by an increased risk for the onset of a range of health problems, particularly those related to the control of behaviour and emotion. Early onset puberty is associated with a greater risk of cancers of the reproductive tract and cardiovascular disease. Previous studies have had methodological limitations and have tended to view puberty as a unitary process, with little distinction between adrenarche, gonadarche and linear growth. The Childhood to Adolescence Transition Study (CATS) aims to prospectively examine associations between the timing and stage of the different hormonally-mediated changes, as well as the onset and course of common health and behavioural problems that emerge in the transition from childhood to adolescence. The initial focus of CATS is on adrenarche, the first hormonal process in the pubertal cascade, which begins for most children at around 8 years of age.

Methods/Design
CATS is a longitudinal population-based cohort study. All Grade 3 students (8–9 years of age) from a stratified cluster sample of schools in Melbourne, Australia were invited to take part. In total, 1239 students and a parent/guardian were recruited to participate in the study. Measures are repeated annually and comprise student, parent and teacher questionnaires, and student anthropometric measurements. A saliva sample was collected from students at baseline and will be repeated at later waves, with the primary purpose of measuring hormonal indices of adrenarche and gonadarche.

Discussion
CATS is uniquely placed to capture biological and phenotypic indices of the pubertal process from its earliest manifestations, together with anthropometric measures and assessment of child health and development. The cohort will provide rich detail of the development, lifestyle, external circumstances and health of children during the transition from childhood through to adolescence. Baseline associations between the hormonal measures and measures of mental health and behaviour will initially be examined cross-sectionally, and then in later waves longitudinally. CATS will make a unique contribution to the understanding of adrenarche and puberty in children’s health and development.

Physiological responses to psychological stress: importance of adiposity in men aged 50-70 years

We tested the hypothesis that overweight/obese men aged 50–70 years will have a greater salivary cortisol, salivary alpha amylase and heart rate (HR) responses to psychological stress compared with age matched lean men. Lean (BMIZ20–25 kg/m2; nZ19) and overweight/obese (BMIZ27–35 kg/m2; nZ17) men (50–70 years) were subjected to a well-characterised psychological stress (Trier Social Stress Test, TSST) at 1500 h. Concentrations of cortisol and alpha amylase were measured in saliva samples collected every 7–15 min from 1400 to 1700 h. HR was recorded using electrocardiogram. Body weight, BMI, percentage body fat, resting systolic and diastolic blood pressure and mean arterial pressure were significantly higher (P!0.05) in overweight/obese men compared with lean men. Both groups responded to the TSST with a substantial elevation in salivary cortisol (372%), salivary alpha amylase (123%) and HR (22%). These responses did not differ significantly between the groups (time!treatment interaction for salivary cortisol, salivary alpha amylase and HR; PZ0.187, PZ0.288, PZ0.550, respectively). There were no significant differences between the groups for pretreatment values, peak height, difference between pretreatment values and peak height (reactivity) or area under the curve for salivary cortisol, salivary alpha amylase or HR (PO0.05 for all). The results showed that, for men with a moderate level of overweight/obesity who were otherwise healthy, the response of salivary cortisol, salivary alpha amylase and HR to acute psychological stress was not impaired.

Early protection in sheep against intratypic heterologous challenge with serotype O foot-and-mouth disease virus using high-potency, emergency vaccine

In 2009-2011, spread of a serotype O foot-and-mouth disease virus (FMDV) belonging to the South East Asia topotype led to the culling of over 3.5 million cattle and pigs in Japan and Korea. The O1 Manisa vaccine (belonging to the Middle East-South Asian topotype) was used at high potency in Korea to limit the expansion of the outbreak. However, no data are available on the spread of this virus or the efficacy of the O1 Manisa vaccine against this virus in sheep. In this study, the early protection afforded with a high potency (>6 PD50) FMD O1 Manisa vaccine against challenge with the O/SKR/2010 virus was tested in sheep. Sheep (n=8) were vaccinated 4 days prior to continuous direct-contact challenge with donor sheep. Donor sheep were infected with FMDV O/SKR/2010 by coronary band inoculation 24h prior to contact with the vaccinated animals, or unvaccinated controls (n=4). Three of the four control sheep became infected, two clinically. All eight O1 Manisa vaccinated sheep were protected from clinical disease. None had detectable antibodies to FMDV non-structural proteins (3ABC), no virus was isolated from nasal swabs, saliva or oro-pharyngeal fluid and none became carriers. Using this model of challenge, sheep were protected against infection as early as 4 days post vaccination.

The acute physiological stress response to an emergency alarm and mobilization during the day and at night

The purpose of this study was to investigate the acute physiological stress response to an emergency alarm and mobilization during the day and at night. Sixteen healthy males aged 25 ± 4 years (mean ± SD) spent four consecutive days and nights in a sleep laboratory. This research used a within-participants design with repeated measures for time, alarm condition (alarm or control), and trial (day or night). When an alarm sounded, participants were required to mobilize immediately. Saliva samples for cortisol analysis were collected 0 min, 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min after mobilization, and at corresponding times in control conditions. Heart rate was measured continuously throughout the study. Heart rate was higher in the day (F20,442 = 9.140, P < 0.001) and night (F23,459 = 8.356, P < 0.001) alarm conditions compared to the respective control conditions. There was no difference in saliva cortisol between day alarm and day control conditions. Cortisol was higher (F6,183 = 2.450, P < 0.001) following the night alarm and mobilization compared to the night control condition. The magnitude of difference in cortisol between night control and night alarm conditions was greater (F6,174 = 4.071, P < 0.001) than the magnitude of difference between the day control and day alarm conditions. The augmented heart rate response to the day and night alarms supports previous observations in field settings. Variations in the cortisol responses between conditions across the day and night may relate to differences in participants' ability to interpret the alarm when sleeping versus when awake.