Antioxidative activity, polyphenolic content and anti-glycation effect of some Thai medicinal plants traditionally used in diabetic patients.

Ethanolic extracts of 30 Thai medicinal plants, traditionally used as alternative treatments in diabetes, were evaluated for antioxidative activity by the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method. They were evaluated in vitro for oxidative stress by thiobarbituric acid-reactive substance (TBARS) assay in pooled plasma of diabetic patients compared to without treatment of the extracts (control). The extracts were also assayed for protein glycation. The results showed that five plants had strong antioxidant activity: Phyllanthus emblica Linn. (PE), Terminalia chebula Retz. (TC), Morinda citrifolia Linn. (MC), Kaempferia parviflora Wall. (KP) and Houttuynia cordata Thunb.(HC), respectively. Thirty plant extracts were good correlation between total antioxidant activity and antiradical activity by TBARS as well as by glycation (r = 0.856, p<0.01 and r = 0.810, p<0.01). PE had stronger antioxidative activity as well as inhibition of TBARS and glycation than the other plants. The investigation showed that total polyphenol and tannin content of PE and the flavonoid content of HC were the highest. The results imply that these plants are potential sources of natural antioxidants which have free radical scavenging activity and might be used for reducing oxidative stress in diabetes.

Antioxidant activities of polyphenolic compounds isolated from Antidesma thwaitesianum Müll. Arg. seeds and marcs.

Antidesma thwaitesianum Müll. Arg. or mao is widely used as commercial products of juice and wine in Thailand. As a result, waste products from the mao plant, such as mao seeds (MS) and mao marcs (MM), are plentiful. We aimed to purify and analyze polyphenolic content in both MS and MM and to investigate the radical scavenging activities of these polyphenolics against 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2'-Azinobis (3-ethylbenzothiazoline 6-sulphonate) (ABTS) radicals and thiobarbituric acid reactive products (TBARP). The results showed MS and MM to be an abundant source of polyphenols (97.32 to 130 mg gallic acid equivalents [GAE]/g) and proanthocyanidins. The radical scavenging activities of MS/MM against DPPH and ABTS radicals (IC(50) of 0.85 to 1.21 microg/mL) were significantly higher (P < 0.05) than that of standard trolox (IC(50) of 5.05 microg/mL). Activity of MS/MM extracts were 3.74 and 3.80 microg/mL trolox eq/g f.w. for the DPPH and ABTS assays, respectively. The oxidation of erythrocyte membranes using 2-thiobarbituric acid demonstrated that the protective effect of MS/MM on lipid peroxidation is as strong as grape seed proanthocyanidin extract. These findings suggest that polyphenolic compounds and proanthocyanidins isolated from these mao extracts had much higher antioxidant activities than those of standard trolox and exhibited similar antioxidant potential to grape seed proanthocyanidin extract. These findings may also increase value of mao waste products and allow development of commercial health products.

Extract of the seed coat of Tamarindus indica inhibits nitric oxide production by murine macrophages in vitro and in vivo.

The seed coat extract of Tamarindus indica, a polyphenolic flavonoid, has been shown to have antioxidant properties. The present studies investigated the inhibitory effect of the seed coat extract of T. indica on nitric oxide production in vitro using a murine macrophage-like cell line, RAW 264.7, and in vitro and in vivo using freshly isolated B6C3F1 mouse peritoneal macrophages. In vitro exposure of RAW 264.7 cells or peritoneal macrophages to 0.2-200 microg/mL of T. indica extract significantly attenuated (as much as 68%) nitric oxide production induced by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) in a concentration-dependent manner. In vivo administration of T. indica extract (100-500 mg/kg) to B6C3F1 mice dose-dependently suppressed TPA, LPS and/or IFN-gamma induced production of nitric oxide in isolated mouse peritoneal macrophages in the absence of any effect on body weight. Exposure to T. indica extract had no effect on cell viability as assessed by the MTT assay. In B6C3F1 mice, preliminary safety studies demonstrated a decrease in body weight at only the highest dose tested (1000 mg/kg) without alterations in hematology, serum chemistry or selected organ weights or effects on NK cell activity. A significant decrease in body weight was observed in BALB/c mice exposed to concentrations of extract of 250 mg/kg or higher. Oral exposure of BALB/c mice to T. indica extract did not modulate the development of T cell-mediated sensitization to DNFB or HCA as measured by the local lymph node assay, or dermal irritation to nonanoic acid or DNFB. These studies suggest that in mice, T. indica extract at concentrations up to 500 mg/kg may modulate nitric oxide production in the absence of overt acute toxicity.

In-house direct cELISA for determining aflatoxin B1 in Thai corn and peanuts.

To overcome the problem of aflatoxins (AF) in Thai foods, a sensitive in-house direct cELISA using monoclonal antibody (mAb) was established and compared with a commercial ELISA kit and thin-layer chromatography for the determination of AFB(1) levels in corn and peanuts. Among eight in-house mAbs (AF1-8), AF5 was used in the direct cELISA owing to its excellent specificity and sensitivity with the detection limit of 4 microg kg(-1). The recovery of AFB(1) spiked at 5, 10, 20, 40 and 80 microg kg(-1) ranged from 88.1 to 99.5%. Correlation coefficients of the ELISA with the commercial ELISA kit and thin-layer chromatography were 0.912 and 0.802 for corn, and 0.941 and 0.832 for peanuts, respectively (p<0.05). The cost per sample was estimated to be about 16 times lower than the commercial ELISA kit. Subsequently, the in-house direct cELISA was successfully applied to screen the contamination of AFB(1) in Thai corn and peanuts. Mean levels of AFB(1) (per cent positive) were 73 microg kg(-1) (85.7%) in corn and 102 microg kg(-1) (67.9%) in peanuts, for which 46.4% of both foods were above the Thailand regulation limit (20 microg kg(-1)).

Biochemical and hematological manifestations of HIV/AIDS in Chiang Mai, Thailand.

HIV/AIDS is a multifactorial and multi-step disease. No single treatment against AIDS can save a patient. Our last report showed that vitamin A, vitamin E and beta-carotene were decreased while malondialdehyde (MDA) was increased. This report aims to evaluate biochemical and hematological parameters in HIV/AIDS patients in Chiang Mai, Thailand by holistic approaches. Sera from HIV/AIDS patients were examined for sugar, cholesterol, uric acid, total protein, albumin, urea, creatinine, AST, ALT, ALP, total/direct bilirubin, vitamin E, MDA, total antioxidant capacity (TAC), beta-carotene, complete blood cell counts, platelet count, CD4 count, prothrombin time, partial prothrombin time and soluble Fas (sFas). The results found that sFas levels in sera prior to holistic approach was not different from reference values and not significantly correlate with CD4 and absolute lymphocyte count. sFas could not serve as putative marker for CD4 destruction. After 3 months CD4 count, MDA, vitamin E and TAC did not change statistically. This approach had no effect on liver and kidney functions, red blood cell, white blood cell, platelet counts, and blood clotting factors. This presentation may be some alternative approaches to combat HIV infections and AIDS, leading to stabilize or extend survival time which should further be elucidated.

Effects of a ferrate-containing preparation on diverse metabolic processes in yeast.

A plant-sap-derived preparation containing bi- and tervalent ferrate anions was tested on growth, respiration on glucose, and membrane transport of 6-deoxy-D-glucose (6-dGlc) and 2-aminoisobutyric acid (Aib) in several yeast species, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Lodderomyces elongisporus, Rhodotorula gracilis, and Dipodascus magnusii. Growth was enhanced by as much as 65%, respiration was not affected significantly except for a decrease in R. gracilis, transport of 6-dGlc was not affected while that of Aib was increased by up to 45% in R. gracilis and up to 27% in L. elongisporus.

Exposure to N-nitroso compounds in a population of high liver cancer regions in Thailand: volatile nitrosamine (VNA) levels in Thai food.

The recent case-control studies in Thailand indicate that a high incidence of liver cancer in Thailand has not been associated with common risk factors such as hepatitis B infection, aflatoxin intake and alcohol consumption. While the infestation by the liver fluke Opisthorchis viverrini (OV) accounted for the high risk in north-east Thailand, there was no such exposure in the other regions of the country where the incidence of liver cancer is also high. Case-control studies suggest that exposure to exogenous and possibly endogenous nitrosamines in food or tobacco in betel nut and cigarettes may play a role in the development of hepatocellular carcinoma (HCC), while OV infestation and chemical interaction of nitrosamines may also be aetiological factors in the development of cholangiocarcinoma (CCA). Over 1800 samples of fresh and preserved food were systematically collected and tested between 1988 and 1996. All the food items identified by anthropological studies to be consumed frequently in four major regions of Thailand were analysed for volatile nitrosamines using gas chromatography combined with a thermal energy analyser. Relatively high levels of N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP) and N-nitrosopyrrolidine (NPYR) were detected in fermented fish ("Plasalid"). NDMA was also detected at levels ranging from trace amounts to 66.5 microg/kg in several salted and dried fish ("Larb-pla" and "Pla-siu"). NDMA and NPYR were frequently detected in several vegetables, particularly fermented beans ("Tau-chiau") at levels ranging between 1 and 95.1 microg/kg and 0-146 microg/kg, respectively. The possible role of nitrosamines in Thai food in the aetiology of liver cancer (HCC, CCA) is discussed.

Volatile nitrosamines and tobacco-specific nitrosamines in the smoke of Thai cigarettes: a risk factor for lung cancer and a suspected risk factor for liver cancer in Thailand.

In Thailand, smoking of commercial cigarettes and of handmade cigarettes has drastically increased in recent decades. Cancer of the lung and of the upper aero-digestive tract have also increased in Thailand as they have in many other countries. It is our working hypothesis that the increase of primary cancer of the liver, especially of cholangiocarcinoma in the north-eastern provinces of Thailand is associated with the use of tobacco in men infested with the liver fluke Opisthorchis viverrini (OV). Bioassays have shown that volatile nitrosamines and tobacco-specific nitrosamines induce cholangiocarcinoma in laboratory animals and that the hepatocarcinogenic action of nitrosodimethylamine in hamsters is significantly increased by infestation with the liver fluke OV. The endogenous formation of nitrosamines is significantly increased by OV infestation. This report presents analytical data on the concentration of volatile nitrosamines and tobacco-specific nitrosamines in mainstream smoke of nine leading brands of commercially produced Thai cigarettes which represent approximately 85% of the market share in Thailand. Observed ranges (ng/cigarette) were 8.5-31.9 for nitrosodimethylamine, 8.8-49.6 for nitrosopyrrolidine and 4.2-18.9 for nitrosodi-n-butylamine. These values are exceptionally high compared with the smoke of light and blended cigarettes from North America and Western Europe. Among the tobacco-specific nitrosamines, the range was 28-730 for nitrosonornicotine and 16-370 for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. There was a correlation between volatile and tobacco-specific nitrosamines, and tar and nicotine deliveries in the mainstream smoke. The analytical data are in line with the rate for lung cancer and support our working hypothesis that nitrosamines, and especially the tobacco-specific nitrosamines, are associated with the increased risk for primary liver cancer among those Thai people who smoke cigarettes and also carry OV infestation.

Risk factors for lung cancer among Northern Thai women: epidemiological, nutritional, serological, and bacteriological surveys of residents in high- and low-incidence areas.

Lung cancer incidence among Northern Thai women is one of the highest in Asia (an annual age-adjusted incidence rate of 37.4 per 100,000), and the incidence rate significantly differs by geographical districts. Therefore, we conducted a comparative study of women living in the Sarapee area, which showed the highest (crude incidence rate, 40.9), and the Chom Tong area, which had one of the lowest incidence rates (8.5) in Chiang Mai Province, despite the two areas' geographical and cultural closeness. The women in this study were either family members of lung cancer patients or their neighbors. To find clues to the etiology of lung cancer, this study used various epidemiological and biochemical approaches: interviewing on lifestyle factors, duplicate meals, chemical examination of drinking water, biochemical analysis of sera, mutagenicity test of urine, and monitoring of fungi and bacteria in the living environment. We found that tobacco smoking (Khiyo, local cigars) was less frequently observed in Sarapee (high incidence), compared with Chom Tong (low incidence), and that the history of chronic benign respiratory diseases was the most distinct event among women in Sarapee, resulting in a significantly increased percentage of those with a history of both benign respiratory diseases and tobacco smoking. This population revealed increased levels of serum tumor necrosis factor (TNF)-alpha, an endogenous tumor promoter. Furthermore, significantly increased urine mutagenicity was found to be closely associated with history of benign respiratory disease in Sarapee. The fungus which was most commonly found in the air inside houses in Sarapee was identified as Microsporum canis. Additionally, significantly increased serum concentrations of a constituent of the fungus were found in Sarapee women, compared with those in Chom Tong. Our results suggest that tobacco (Khiyo) smoking alone may not be able to explain the very high incidence of female lung cancer in Northern Thailand, and that chronic benign respiratory disease, possibly caused by the infection of fungi such as M. canis, is likely to be involved in the etiology of female lung cancer in North Thailand.

Comparison of diets among elderly female residents in two suburban districts in Chiang Mai Province, Thailand, in dry season--survey on high- and low-risk districts of lung cancer incidence.

By means of duplicate meals, we collected food samples of general female residents, aged 50 to 74 years at two suburban districts in Chiang Mai Province, which are distinguished by very high and low incidence rates of lung cancer. Then, on the basis of analyses of their consumption of foods by food groups, we compared their dietary habits in the dry season of northern Thailand with special reference to the difference in lung cancer incidence. In brief, the following features and difference in their dietary habits were found; 1) Rice, vegetables, and pork were most frequently eaten in both the districts. 2) Consumption of fruits, in both quantity and variety, at a high-risk district was much less than that at a low-risk district. 3) Female residents at a low-risk district consumed more variety of green and yellow vegetables than those at a high-risk district. 4). Potato was not found in food samples of a high-risk district. 5) confectionery was more prevalent in a low-risk district than at a high-risk district.

Developmental toxicity of steviol, a metabolite of stevioside, in the hamster.

The developmental toxicity of steviol, a metabolite of stevioside, was studied in hamsters. Pregnant hamsters were intubated with steviol at dose levels of 0, 0.25, 0.5, 0.75 and 1.0 g/kg BW/day on days 6-10 of gestation. Steviol at doses of 0.75 and 1.0 g/kg BW/day were highly toxic to both dams and fetuses. Significant decrease of maternal body-weight gain during the experimental period (days 6-14) and high percentage of maternal mortality indicated the general toxicity of these two high doses. The number of live fetuses per litter and mean fetal weight also significantly decreased in the steviol-treated animals at doses of 0.75 and 1.0 g/kg BW day. The animals treated with an intermediate dose (0.50 g/kg BW/day) exhibited less signs of maternal and developmental toxicity than the two high doses (0.75 and 1.0 g/kg BW/day). One craniomeningocele was found in a fetus under the maternal toxic condition in steviol-treated at a dose of 0.75 g/kg BW/day. Neither the skeleton nor visceral development of the offspring was affected by steviol treatment except delayed ossification of the xiphoid (bifid) and long bones of the limbs and supernumerary thoracic ribs (14th ribs) tended to be increased at doses of 0.5 to 1.0 g/kg BW/day steviol. No dose-related teratogenesis was detected. From the result of the present study concerning maternal toxic condition and embryotoxicity, an oral dose of 0.25 g steviol/kg BW/day is regarded as having no observable effect. This steviol-treated dose is derived from stevioside 625 mg/kg BW/day which is approximately 80 times higher than the suggested acceptable daily intake of stevioside for humans (7.938 mg/kg BW/day).

Total antioxidant capacity in plasma of HIV-infected patients.

Total antioxidant capacity (TAC) of fasting EDTA plasma of 33 healthy and 64 HIV-infected patients was determined using H(2)O(2)-peroxidase-ABTS technique. The results revealed that the average TAC in HIV-infected patients was significantly lower than those in healthy normal persons. (0.161 +/- 0.097 vs 0.269 +/- 0.081 mmol/L Trolox equivalent, p < 0.05). Total lymphocytes were also counted using Hycel automatic cell counter and absolute CD4 numbers using Coulter CD4 manual kit. It was interesting that CD4 count was not correlated with the clinical symptoms of the patients. This paper suggests that prediction of severity and monitoring of the disease should be performed by determining both total lymphocyte count and total antioxidant capacity.

Assessment of major carcinogenic tobacco-specific N-nitrosamines in Thai cigarettes.

This report presents new findings on the content of cancer-causing tobacco-specific N-nitrosamines (TSNA) in mainstream smoke of nine brands of commercially produced Thai cigarettes, representing about 85% of market share in Thailand. Also tested were two major and popular brands of U.S. cigarettes sold in Thailand, representing about 10% of market share. The cigarettes included filter and nonfilter cigarettes with high, moderate, and low tar and nicotine yields. The observed range for N-nitrosonornicotine (NNN) was from 28 to 730 ng/cigarette and for 4-(methylnitrosamino)-1-(3-pyridyl-1-butanone (NNK) from 16 to 369 ng/cigarette. The relatively highest TSNA values were obtained in filter and nonfilter cigarettes with high tar (22.3-28.1 mg/cigarette) and high nicotine (1.78-2.42 mg/cigarette) deliveries. The results demonstrated that there is a correlation between TSNA and tar and nicotine deliveries in mainstream smoke. The TSNA deliveries, along with the tar and nicotine levels in mainstream smoke depended on the tobacco composition. According to these results, the tar levels alone, while significant, are not a sufficient measure for the biological activity and the carcinogenic potential of cigarettes in Thailand. Consumption of tobacco products nearly quadrupled over the last three decades (1966-1995) in Thailand. Lung cancer is the leading malignancy for men and a common malignancy for women in Thailand. This report provides information that may prove helpful in evaluating the TSNA-carcinogens burden on smokers. Our goal is to offer the scientific basis for voluntary and/or government-regulated reduction of the smoke yields of TSNA in tobacco products in Thailand and in other countries.

Survey of microcystins in environmental water by a highly sensitive immunoassay based on monoclonal antibody.

By using a highly sensitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody, microcystin (MC) concentration was analyzed in environmental water samples (total, 134), collected in 1993-1995 from ponds, lakes, reservoirs, and rivers in Japan, Thailand, Germany, and Portugal. MCs detected in the water samples filtered over a glass filter were designated as free MCs, and those samples that were freeze-thawed twice before the filtration were designated as total MCs. MCs (> 50 pg/ml) were detected in 14 of 24 samples collected from the lakes that were used as recreation and water supply in Japan in different regions. In the MC-positive samples, the concentration of free MCs was only a few percentages of the total MCs, indicating that the most part of MCs found in the water samples was present in algal cells. An additional trial on 33 samples collected continuously from Lake Inbanuma, Japan, during June-September 1994-1995 revealed that the total MCs were in a range of 52-52,000 pg/ml. In Chiang Mai, Thailand, 6 of 10 samples were positive, with the mean and highest of 161 and 354 pg/ml, respectively. In the Frankfurt area. Germany, 4 of 10 and 7 of 8 samples collected in the same lakes for recreation in July 1993 and November-December 1994 showed the presence of MCs, with their mean and highest values of 257 and 407 pg/ml, respectively. Another survey of MCs in dense bloomed samples collected with plankton net revealed a contamination of MCs up to 36,000 pg/ml. In Portugal, 28 of 29 samples from 4 lakes, 20 rivers, and 5 reservoirs were positive for MCs, with the respective means of 13,664, 11,048, and 2,278 pg/ml. These data indicated that MCs contaminate environmental water in ponds, rivers, lakes, and reservoirs worldwide. The present ELISA is considered to be a reliable tool for the mass monitoring and risk assessment of MCs in water supplies.

Effects of stevioside and steviol on intestinal glucose absorption in hamsters.

The effects of stevioside and steviol (a product of enzymatic hydrolysis of stevioside) on intestinal glucose absorption were examined in the hamster jejunum in vitro. By using the jejunal rings technique, we found that stevioside at a high dose of 5 mM had no inhibitory effect on glucose absorption. In contrast, glucose absorption was inhibited 43% by 1 mM steviol. The inhibition of glucose absorption by steviol was related to steviol concentration and incubation time. The inhibitory effect of steviol compared to phlorizin and ouabain was also investigated. Steviol, which caused a decrease in glucose accumulation in the intestinal ring tissues, possibly acts on the brush border membrane as does phlorizin. Furthermore, it was also found that steviol altered the morphology of the intestinal absorptive cells. These results suggest that the possible site of inhibitory action of steviol might be on the mucosal side and/or at the intracellular organelles of intestinal absorptive cells.

Mutagenicity and human chromosomal effect of stevioside, a sweetener from Stevia rebaudiana Bertoni.

Leaves of Stevia rebaudiana Bertoni have been popularly used as a sweetener in foods and beverages for diabetics and obese people due to their potent sweetener stevioside. In this report, stevioside and steviol were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 and for chromosomal effects on cultured human lymphocytes. Stevioside was not mutagenic at concentrations up to 25 mg/plate, but showed direct mutagenicity to only TA98 at 50 mg/plate. However, steviol did not exhibit mutagenicity in either TA98 or TA100, with or without metabolic activation. No significant chromosomal effect of stevioside and steviol was observed in cultured blood lymphocytes from healthy donors (n = 5). This study indicates that stevioside and steviol are neither mutagenic nor clastogenic in vitro at the limited doses; however, in vivo genotoxic tests and long-term effects of stevioside and steviol are yet to be investigated.

Suppressive effect of soybean milk protein on experimentally induced skin tumor in mice.

We studied the effect of soybean milk protein (SMP) in a two-stage carcinogenesis experiment on mouse skin. Mice were given soybean protein isolate (SPI) diet or SPI diet supplemented with SMP. After 4 weeks on the diets, the mice were shaved and a tumor initiator was applied. A tumor promotor was then applied twice a week on the same area of the skin throughout the experiment. After 20 weeks on the treatment, the percentage of tumor-bearing mice and the volume of tumor tended to be lower in the mice on the SMP diet than those on the SPI diet. The number of tumor was also significantly lower in the former group as compared to the latter group. There was no difference in growth between mice of the SPI and SMP groups. The results indicate the safety and the anti-carcinogenic effect of SMP in mice.