Therapeutic effect of aged garlic extract on gingivitis in dogs.

Periodontal disease is one of the most common dental health problems in dogs. Clinical studies in humans have shown that aged garlic extract (AGE), which contains stable and water-soluble sulfur-containing bioactive compounds, improves the symptoms of periodontal diseases. Our previous study demonstrated that oral administration of AGE in healthy Beagle dogs at 90 mg/kg/day for 12 weeks had no adverse effects such as hemolytic anemia, which is well known to occur as a result of ingestion of Allium species, including onions and garlic, in dogs. However, the therapeutic potential of AGE in canine periodontal disease remains unclear. Accordingly, we investigated the therapeutic effects of AGE in Beagle dogs with mild gingivitis. Feeding 18 mg/kg/day of AGE for 8 weeks resulted in the improvement of gingival index score, level of volatile sulfur compounds in exhaled air, and enzyme activity of periodontal pathogens without any adverse effects on clinical signs and hematological and serum biochemical parameters. Moreover, AGE increased the concentration of salivary cathelicidin, an antimicrobial peptide that contributes to the oral innate immune response. These results suggest that AGE could be a potential therapeutic agent for canine gingivitis.

Acupuncture Treatment Improves Transport Stress in Microminipigs Through the Acupoint in Ears.

BACKGROUND/AIM: This study aimed to investigate the effects of acupuncture treatment through the ear acupoints on transport stress in experimental microminipigs. MATERIALS AND METHODS: Experiment 1: Six animals were equally divided into two groups (Control and Treatment). In the treatment group, before transportation (6 h; vehicle and plane), short, ultrathin circular transdermal needles were applied to locations corresponding to the acupoints on the apical area of both ears. Peripheral blood samples were collected from the cranial vena cava 2 days before and immediately after transportation. Blood stress markers, biochemistry indicators, and oxidative stress levels were examined. Experiment 2 (follow-up study: diarrhea incidence after transportation): Diarrhea incidence after transportation in the control and treatment groups was investigated. RESULTS: Experiment 1: Transport stress induced an increase in blood cortisol, serum amyloid A (SAA), glucose, non-esterified fatty acid, and derivatives of reactive oxygen metabolites (d-ROMs) and decreased the biological antioxidant potential (BAP)/d-ROMs ratio yet did not affect BAP. Acupuncture suppressed the increases in SAA and d-ROMs values and the decrease in BAP/d-ROMs ratio. Experiment 2: The total diarrhea incidence was 25% in the control group, whereas diarrhea was not observed in the treatment group. CONCLUSION: Acupuncture treatment suppresses hypothalamic-pituitary-adrenal function and, as a result, reduces transport stress without affecting the suppression of the central catecholaminergic system. Acupuncture treatment for transport stress can improve animal welfare.

The Long-Term Clinical Course of Canine Degenerative Myelopathy and Therapeutic Potential of Curcumin.

Canine degenerative myelopathy (DM), recognized as a spontaneous model of amyotrophic lateral sclerosis, is known as a late-onset progressive degenerative disease of the spinal cord. Because of the progressive nature of DM, many dogs are elected to be euthanized, resulting in limited information on the end-stage clinical presentation. We investigated the long-term clinical course from diagnosis to natural death to further deepen our understanding of the entire clinical picture of this disease. Because curcumin was administered in some cases, the therapeutic effect of curcumin on DM was also examined. Forty dogs included in this study were client-owned Pembroke Welsh Corgis with a definitive diagnosis of DM by necropsy and histopathology. Dogs were excluded from this study if they died from another disease or were elected to be euthanized. Information on the long-term clinical symptoms of DM was investigated based on a questionnaire, which was collected from the dog owners. Urinary incontinence and respiratory disorder were observed in most dogs, as was respiratory impairment-correlated death. In contrast, signs consistent with brainstem dysfunction were noticed at the terminal stage in a small portion of dogs. Although further studies with more cases are needed, the results of this study suggest that administration of curcumin is effective in slowing the progression of DM.

Effects of dietary feed supplementation of heat-treated Lactobacillus sakei HS-1 on the health status, blood parameters, and fecal microbes of Japanese Black calves.

This study investigated the effect of heat-killed Lactobacillus sakei HS-1 (HK-LS HS-1) on the health and fecal bacteriological change of suckling Japanese Black calves as a supplement in milk replacers. Twelve calves were separated from dams to calf-hatch after calving for milk replacers feeding. They were randomly assigned to an HK-LS HS-1 supplement or a control without HK-LS HS-1 group in milk replacers. HK-LS HS-1 was administered from separation day to 3 weeks. Blood and fecal samples were examined. Two calves with a haptoglobin concentration of >500 µg/ml on day 0 were excluded from the experiment, and 10 calves were finally included. Glucose and vitamin A levels on day 7 were significantly higher (P<0.05) in the supplement group than in the control group. No significant differences were observed in haptoglobin or serum amyloid A between the groups. The number of Escherichia coli in feces was lower in the control group than in the supplement group on day 21 (P=0.06). No difference was observed in the number of bifidobacteria, but that of lactic acid bacteria was significantly higher (P<0.05) in the supplement group on day 21. The number of medications administered was significantly lower (P<0.05) in the supplement group (5.2 ± 3.9) than in the control group (10.6 ± 5.9) during the experimental period. The results indicated that HK-LS HS-1 is potentially beneficial for improving intestinal microbes and reducing the number of medical treatments.

Safety and efficacy of aged garlic extract in dogs: upregulation of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and Nrf2-regulated phase II antioxidant enzymes.

BACKGROUND: Plants of Allium spp., including garlic (A. sativum) and onions (A. cepa), are known to be oxidatively toxic to canine erythrocytes resulting in Heinz body hemolytic anemia in dogs. In humans, these plants have been used as medicinal agents for multiple diseases since ancient times. Especially, fresh garlic extracted over a prolonged period produces less irritative and odorless aged garlic extract (AGE), containing unique and beneficial organosulfur compounds that can help prevent many kinds of diseases. In this study, the safety and efficacy of long-term oral administration of AGE is evaluated in dogs. The objectives are to confirm the safe dosage for long-term use and beneficial functions of AGE for dogs and to plan and design a canine health supplement or a preventive agent for multiple diseases based on the data of this study. RESULTS: Beagles were orally administered AGE (45 or 90 mg/kg body weight once a day) or an equivalent amount of water as control for 12 weeks. In AGE-treated groups, at 12 weeks post-administration at a dose of 90 mg/kg, there were no observable changes in the clinical signs, complete blood count, and serum biochemical parameters. Heinz bodies and eccentrocytes, the markers of oxidative damage in erythrocytes, did not appear in blood smear examination. In order to further evaluate the beneficial effects of AGE on health of dogs, the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) gene (NFE2L2) and Nrf2-regulated phase II antioxidant enzyme genes (NQO1, GCLM, HMOX1, and SOD2) were determined in whole blood between pre- and post-AGE administration. The expression of NFE2L2 gene was significantly upregulated in the AGE-treated groups [45 (p < 0.05) and 90 mg/kg (p < 0.01), 8 weeks] as compared to in the control group. Among the Nrf2-regulated enzymes examined, the expressions of NQO1 [45 (p < 0.05) and 90 mg/kg (p < 0.01), 8 weeks] and GCLM [45 (p < 0.05) and 90 mg/kg (p < 0.01), 12 weeks] genes were significantly upregulated. CONCLUSION: The long-term oral administration of AGE at a dose of 90 mg/kg/day for 12 weeks did not show any adverse effects in dogs. Furthermore, the administration of AGE upregulated the gene expressions of canine Nrf2 and Nrf2-regulated phase II antioxidant enzymes. These results suggest that AGE might safely contribute to the health of dogs provided that the appropriate dosage is used.

Fructo-Oligosaccharide (DFA III) Feed Supplementation for Mitigation of Mycotoxin Exposure in Cattle-Clinical Evaluation by a Urinary Zearalenone Monitoring System.

The potential effect of difructose anhydride III (DFA III) supplementation in cattle feed was evaluated using a previously developed urinary-zearalenone (ZEN) monitoring system. Japanese Black cattle from two beef herds aged 9⁻10 months were used. DFA III was supplemented for two weeks. ZEN concentrations in feed were similar in both herds (0.27 and 0.22 mg/kg in roughage and concentrates, respectively), and below the maximum allowance in Japan. ZEN, α-zearalenol (α-ZOL), and ß-ZOL concentrations in urine were measured using LC/MS/MS the day before DFA III administration, 9 and 14 days thereafter, and 9 days after supplementation ceased. Significant differences in ZEN, α-ZOL, ß-ZOL, and total ZEN were recorded on different sampling dates. The concentration of inorganic phosphate in DFA III-supplemented animals was significantly higher than in controls on day 23 (8.4 vs. 7.7 mg/dL), suggesting a possible role of DFA III in tight junction of intestinal epithelial cells. This is the first evidence that DFA III reduces mycotoxin levels reaching the systemic circulation and excreted in urine. This preventive effect may involve an improved tight-junction-dependent intestinal barrier function. Additionally, our practical approach confirmed that monitoring of urinary mycotoxin is useful for evaluating the effects of dietary supplements to prevent mycotoxin adsorption.

Japanese Black breeding herd exhibiting low blood urea nitrogen: a metabolic profile study examining the effect on reproductive performance.

Ten reared cows of a Japanese Black cattle herd in Kagoshima prefecture, Japan, exhibited extremely low blood urea nitrogen (BUN) concentration (2.6 ± 0.6 mg/dL). Examination of dietary feed nutrition and relevant pastureland soil content suggested a correlation with crude protein (CP) deficiency or unbalanced nutritional dietary feeds. Thirteen months after the introduction of a dietary remedial measure (bean cake supplementation), BUN, total cholesterol and albumin concentration from five of the original 10 cows increased significantly compared with their values of before the dietary remedy. The postpartum day open period was significantly lower after the dietary remedial measure than that before it. The abnormally low BUN levels of the cattle herd may be due to inadequate dietary nutritional content, primarily from the imbalance of total digestible nutrient and CP of the feed and far lower han average CP value. In conclusion, routine examination of serum biochemical parameters in Japanese Black breeding cattle may be a useful strategy for determining subclinical metabolic failure of cattle herds, and consequently, its effect on reproductive performance of the herd.

Metabolic profile of Japanese Black breeding cattle herds: usefulness in selection for nutrient supplementation to enhance reproductive performance and regional differences.

The study aims were (1) to confirm the effects of nutritional improvement in prepartal and postpartal periods, monitored using the serum metabolic profile test (MPT) and reproductive performance, and (2) to clarify regional characteristics of the MPT results within our jurisdiction by using our MPT database. Experiment 1: Among 42 breeding cattle herds in our jurisdiction mainly fed home-pasture roughage, 3 experimental herds showing subnormal blood urea nitrogen (BUN) levels were selected and compared with 1 representative excellent herd. Dietary remedial measures were implemented from feed analysis in each herd. BUN concentration in all 3 herds increased significantly, and open days postpartum in 2 of the herds were significantly reduced, compared with values before dietary supplementation. Experiment 2: Thirty-seven herds within our jurisdiction were grouped into 3 categories (Area 1, 2 and 3) by location and soil condition of the herd pastureland. The MPT and reproductive performance in cows whose blood samples were collected at both prepartum (60-20 days before calving) and postpartum (30-90 days after calving) were compared among the 3 areas. Significant regional differences were found in prepartal albumin, total cholesterol, BUN, and glucose and postpartal BUN, glucose and open days (P<0.05). Overall, the MPT (especially BUN) might be useful for determining the metabolic nutritional status of breeding cattle herds, particularly those fed home-pasture roughage. Additionally, poor/unsatisfactory reproductive performance of beef breeding cattle herds probably reflects inadequate nutritional content of the diet, possibly arising from regional pastureland differences.

Radioprotective effect of alk(en)yl thiosulfates derived from allium vegetables against DNA damage caused by X-ray irradiation in cultured cells: antiradiation potential of onions and garlic.

To evaluate a radioprotective effect of sodium n-propyl thiosulfate (NPTS) and sodium 2-propenyl thiosulfate (2PTS) derived from onions and garlic, respectively, rat hepatoma H4IIE cells and mouse lymphoma L5178Y cells were preincubated with each of these compounds for 48 hours at 37°C before receiving 10 Gy of X-ray irradiation. Cell damage caused by the irradiation was quantified as comet tail moment, which represents the degree of DNA damage. X-ray-induced DNA damage was significantly decreased in both H4IIE and L5178Y cells by micromolar concentrations of NPTS and 2PTS compared with the control without the compounds. The protective effect was more potent with 2PTS than NPTS. Onions and garlic have antiradiation potential.

Sodium 2-propenyl thiosulfate derived from garlic induces phase II detoxification enzymes in rat hepatoma H4IIE cells.

There is evidence that onions and garlic protect against cancer in humans. It has been suggested that this effect is partly due to the organosulfur compounds in Allium vegetables and that these substances act through induction of phase II detoxification enzymes. Here, we hypothesized that alk(en)yl thiosulfates, sodium n-propyl thiosulfate (NPTS), and sodium 2-propenyl thiosulfate (2PTS), which were identified in onions and garlic, respectively, may induce phase II enzymes. Therefore, rat hepatoma cells (H4IIE) were cultured with 1 to 100 micromol/L of NPTS or 2PTS for 48 hours at 37 degrees C; and the activities and messenger RNA (mRNA) expression levels of phase II enzymes in H4IIE cells were investigated. The effects of diallyl trisulfide and tert-butylhydroquinone, known as phase II inducers, were also examined as positive controls and compared with the responses of NPTS and 2PTS. Quinone reductase (QR) activity and mRNA expression levels of QR and epoxide hydrolase 1 were significantly increased by 2PTS (P < .05-.005). In particular, QR activity was increased at a relatively low concentration of 2PTS (10 micromol/L). However, glutathione S-transferase activity and mRNA expression levels of glutathione S-transferase A5 and uridine diphosphate glucuronosyl transferase 1A1 were not changed by 2PTS. In contrast, NPTS did not affect the activities and mRNA expression levels of these phase II enzymes. These results show that 2PTS can induce phase II enzymes, and its inductive effect is comparable or superior to that of diallyl trisulfide and tert-butylhydroquinone.

Clinico-pathological findings in peripartum dairy cows fed anion salts lowering the dietary cation-anion difference: involvement of serum inorganic phosphorus, chloride and plasma estrogen concentrations in milk fever.

In our previous study, it was demonstrated that the administration of anion salts, which slightly lower the dietary cation-anion difference (DCAD), in the prepartum period is safe and effective for preventing milk fever in multiparous cows. In the present study, several clinico-pathological constituents in serum and urine, which might be related to milk fever, were analyzed using stored samples from the previous study to identify clinico-pathological parameters for easily evaluating the efficacy of lowering DCAD and to further investigate the mechanism by which lowering DCAD prevents milk fever. Among the parameters analyzed in the present study, inorganic phosphorus (iP) was involved in milk fever because the serum concentration and urinary excretion of iP were significantly higher in the group of primiparous cows (heifer group), which did not develop hypocalcemia, than those in other groups of multiparous cows. Serum chloride concentrations in the heifer group and the group of multiparous cows fed anion salts (anion group) tended to remain higher than those in other control groups of multiparous cows suggesting that serum chloride concentration may be utilized for evaluating the status of metabolic acidosis and the efficacy of lowerng DCAD in dairy cows fed anion salts. In addition, plasma estradiol-17beta concentration in the heifer group tended to be lower at parturition compared with that in other multiparous groups suggesting that estrogen known as a potent inhibitor of bone resorption may be involved in developing milk fever.

Preventive effect of mildly altering dietary cation-anion difference on milk fever in dairy cows.

In the present study, we examined whether mildly altering dietary cation-anion difference (DCAD) contributes to the prevention of milk fever in dairy cows. Thirty multiparous cows and ten primiparous cows (heifer group) were used in this study and the multiparous cows were randomly divided into three groups of ten animals each (anion, non-anion and control groups). The cows in the anion group were given supplemental salts that slightly lowered DCAD. These salts consisted of 115 g of CaCO3, 42 g of CaHPO4, 65 g of MgSO4 x 7 H2O and 80 g of CaCl2 x 2 H2O as a daily dose for each cow, using a catheter from 21 days before the expected date of parturition until parturition. The cows in the non-anion group were given only the same Ca, Mg and ip supplement but no sulfate and chloride salts as that in the anion group. The cows in the control and heifer groups were not given any additional supplement. The incidence of hypocalcemia in the anion group decreased to approximately half of those in the non-anion and control groups, while the heifer group did not develop hypocalcemia at all. In addition, the number of days spent for the treatment of hypocalcemia and the number of drug bottles (calcium borogluconate solution) used for the treatment decreased to less than half in the anion group compared with those in the non-anion and control groups. At parturition, the serum Ca concentration in the control (6.2 +/- 1.9 mg/dl, mean +/- standard deviation) and non-anion groups (6.4 +/- 1.7 mg/dl) were significantly lower than that in the heifer group (8.3 +/- 0.4 mg/dl), and the level in the anion group was intermediate (7.3 +/- 1.3 mg/dl). The change in ionized Ca concentration was almost the same as that in serum Ca concentration, but only the concentration in the anion group tended to increase slightly from a week before parturition and was significantly higher than that in all other groups three days before parturition. Urinary pH in the anion group was maintained at a mildly acidic level (6.8-7.0) for the last two weeks before parturition, compared with those in the control (7.3-7.5) and non-anion groups (7.9-8.1), and similar to that in the heifer group (6.3-7.3). The urinary Ca excretion was the highest in the anion group among all groups during the prepartum period. There were no specific changes in the excretion of parathyroid hormone and 1,25-dihydroxyvitamin D in all groups of multiparous cows while the levels of these hormones remained low in the heifer group throughout the experimental period. The data in the present study indicates that the administration of anion salts that slightly lowered DCAD in the preparum period was effective for preventing milk fever in multiparous cows. Safe and mild metabolic acidosis induced by the anion salts could be evaluated by urinary pH (6.8-7.0), and might increase the responsiveness to Ca requirement at parturition through some complex mechanisms unrelated to the excretion of Ca-related hormones. In addition, it was clarified that primiparous cows have a high potential to respond to sudden Ca demand unrelated to hormone excretion, and their Ca metabolism was in some respects similar to that in multiparous cows fed anion salts. Therefore, manipulating mildly DCAD is expected to be an effective, safe and natural method for preventing milk fever in dairy cows.

Evaluation of the inhibitory activities of the extracts of Indonesian traditional medicinal plants against Plasmodium falciparum and Babesia gibsoni.

Twenty-four kinds of water extracts derived from 22 plants that are traditionally used for the treatment of malaria on Java Island, Indonesia, were screened for their antibabesial and antimalarial activities. Among the extracts, 8 extracts displayed strong antimalarial activity, with an inhibition range from 89.6 to 100%, and 15 showed strong antibabesial activity, with an inhibition range from 84.2 to 98.1%. The extracts of Achillea millefolium, Baeckea frutenscens, Brucea javanica, Curcuma xanthorrhiza, Strychnos lucida and Swietenia macrophylla showed both strong antibabesial and antimalarial activities. The antimalarial activities paralleled the antibabesial activities, but the converse was not true.

Anti-babesial compounds from Curcuma zedoaria.

Anti-babesial activity was confirmed in an extract of the bark of Curcuma zedoaria. The active ingredients were isolated, and their chemical structures were determined to be zedoalactones A, B, and C based on spectral data. Zedoalactone C is a hitherto unreported compound. The IC50 vales of these active compounds against Babesia gibsoni were compared with a standard drug, diminazene aceturate. The IC50 value of diminazene aceturate was 0.6 microg/mL, while those of zedoalactones A, B, and C were 16.5, 1.6 and 4.2 microg/mL, respectively.

Anti-babesial activity of some central kalimantan plant extracts and active oligostilbenoids from Shorea balangeran.

Bark extracts from a total of 22 species of Central Kalimantan plants were evaluated for their anti-babesial activity against Babesia gibsoni in vitro. Of these plant species, extracts of Calophyllum tetrapterum, Garcinia rigida, Lithocarpus sp., Sandoricum emarginatum, and Shorea balangeran showed more than 90% inhibition of the parasite growth at a test concentration of 1000 microg/mL. Activity-guided fractionation of the bark of S. balangeran (Dipterocarpaceae) led to the reisolation of oligostilbenoids, vaticanol A(1), B(2), and G(3). The structures were determined on the basis of spectral evidence. Compounds 1 and 3 showed complete inhibition on the growth of Babesia gibsoni in vitro at a concentration of 25 microg/mL, and compound 2 at concentration of 50 microg/mL.

Growth inhibitory effect of alk(en)yl thiosulfates derived from onion and garlic in human immortalized and tumor cell lines.

Two alk(en)yl thiosulfates, sodium n-propyl thiosulfate (NPTS) and sodium 2-propenyl thiosulfate (2PTS), are natural constituents of onion and garlic, respectively, which were identified originally as causative agents of onion- and garlic-induced hemolytic anemia in dogs. As a continuation of our studies on the beneficial functions of NPTS and 2PTS, in the present study, we investigated the antitumor effects of these compounds. They were shown to inhibit the in vitro proliferation of three human tumorigenic cell lines, WiDr, 293 and HL-60, in a dose-dependent manner. Overall, NPTS seemed to have weak activity for inhibiting cell growth compared with 2PTS, though not in WiDr cells, which were sensitive to both compounds. NPTS and 2PTS caused oxidative damage to HL-60 cells and induced apoptosis. The extent of apoptosis was approximately proportional to that of the oxidative damage and also to that of the cytotoxicity caused by these compounds. These results suggest that the alk(en)yl thiosulfates have an antitumor effect through the induction of apoptosis initiated by oxidative stress.

Anti-babesial and anti-plasmodial compounds from Phyllanthus niruri.

Bioassay-guided fractionation of boiled aqueous extracts from the whole plant of Phyllanthus niruri led to the isolation of 1-O-galloyl-6-O-luteoyl-alpha-d-glucose (1), with IC(50) values of 4.7 microg/mL against Babesia gibsoni and 1.4 microg/mL against Plasmodium falciparum in vitro. The known compounds beta-glucogallin (2), quercetin 3-O-beta-d-glucopyranosyl-(2-->1)-O-beta-d-xylopyranoside (3), beta-sitosterol, and gallic acid were also isolated. Structures of these compounds were elucidated on the basis of their chemical and spectroscopic data.

Heinz body hemolytic anemia with eccentrocytosis from ingestion of Chinese chive (Allium tuberosum) and garlic (Allium sativum) in a dog.

A 4-year-old, intact male miniature schnauzer was presented with anorexia. The dog had ingested some Chinese steamed dumplings 2 days before, which contained Chinese chive (Allium tuberosum) and garlic (Allium sativum). Hematological examinations revealed severe Heinz body hemolytic anemia with eccentrocytosis and an increased concentration of methemoglobin, which was thought to result from oxidative damage to erythrocytes by constituents in these Allium plants. In this case, eccentrocytosis was a hallmark finding and could be detected easily, suggesting that this hematological abnormality is useful in diagnosing Allium plant-induced hemolysis.

Effects of Central Kalimantan plant extracts on intraerythrocytic Babesia gibsoni in culture.

The inhibitory effects of 45 plant extracts selected from Central Kalimantan, Indonesia on Babesia gibsoni in vitro and their acute toxicity to mice were evaluated. Of these plant extracts studied, Arcangelisia flava, Curcuma zedoaria, Garcinia benthamiana, Lansium domesticum and Peronema canescens were found to have appreciable antibabesial activity with IC50 values from 5.3 to 49.3 microg/ml without acute toxicity in mice at the intraperitoneal dose of 0.7 g/kg of body weight.

Isolation and identification of sodium 2-propenyl thiosulfate from boiled garlic (Allium sativum) that oxidizes canine erythrocytes.

Sodium 2-propenyl thiosulfate was identified in boiled garlic (Allium sativum). When canine erythrocytes were incubated with sodium 2-propenyl thiosulfate, the methemoglobin concentration and Heinz body percentage in erythrocytes were both increased, indicating that the compound induced oxidative damage in canine erythrocytes. It seems that this compound is one of the causative agents of garlic-induced hemolysis in dogs.