A SERS method with attomolar sensitivity: a case study with the flavonoid catechin.

Making good use of interactions between analyte molecules and the metal nanoparticles is key to impact the detection limit in a surface-enhanced Raman scattering (SERS) based detections. SERS was applied to the analysis of catechin and it was found that the relative abundance of catechin in the sample to citrate-capped AgNPs and the aggregation agent NaCl plays a critical role in the quality of detection. At a component volume ratio of 6:2:1 (catechin:AgNPs:NaCl), catechin can be detected at µM levels. When the ratio is 12:2:1, Raman signals are discernible even at the attomolar concentration level (10-18 M). Under these conditions, the SERS mechanisms and the force of laser tweezers function best. The extent of signal enhancement enabled an ultrasensitive and reproducible Raman spectroscopic determination of catechin. Graphical abstract At a component volume ratio of 6:2:1 (catechin:AgNPs:NaCl), catechin was detected at 10-3 M to 10-6 M. When the ratio was 12:2:1, the discernible concentration of catechin was found to reach the attomolar level (10-18 M).

Vaccinia viral protein A27 is anchored to the viral membrane via a cooperative interaction with viral membrane protein A17.

The vaccinia viral protein A27 in mature viruses specifically interacts with heparan sulfate for cell surface attachment. In addition, A27 associates with the viral membrane protein A17 to anchor to the viral membrane; however, the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and the underlying binding mechanism, we expressed and purified the N-terminal (18-50 residues) and C-terminal (162-203 residues) fragments of A17, which are denoted A17-N and A17-C. Through surface plasmon resonance, the binding affinity of A27/A17-N (KA = 3.40 × 10(8) m(-1)) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (KA = 3.40 × 10(5) m(-1)), indicating that A27 prefers to interact with A17-N rather than A17-C. Despite the disordered nature of A17-N, the A27-A17 interaction is mediated by a specific and cooperative binding mechanism that includes two active binding sites, namely (32)SFMPK(36) (denoted as F1 binding) and (20)LDKDLFTEEQ(29) (F2). Further analysis showed that F1 has stronger binding affinity and is more resistant to acidic conditions than is F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of the A17 protein were packaged into mature virus particles at a reduced level, demonstrating that the F1/F2 interaction plays a critical role in vivo. Using these results in combination with site-directed mutagenesis data, we established a computer model to explain the specific A27-A17 binding mechanism.

Induction of amyloid fibrils by the C-terminal fragments of TDP-43 in amyotrophic lateral sclerosis.

TAR DNA-binding protein 43 (TDP-43) has been identified as the major ubiquitinated aggregates in the inclusion bodies in the patients of amyotrophic lateral sclerosis (ALS) since 2006 and become a crucial culprit for ALS and related motor neuron diseases. Recent literature has further indicated that the major components of these aggregates are hyper-phosphorylated TDP-43 C-terminus. In an effort to clarify the conformational and physical properties of its disordered C-terminal domain, we have synthesized several peptide fragments and shown that only D1 within D1-4 can form twisted fibrils with a cross section of approximately 11 nm in width under the incubation of phosphate buffer. In contrast, the D2-4 peptides all formed amorphous aggregates, showing different aggregation propensities. In addition to D1, two pathological mutant peptides, A315T and G294A, can also form fibrils that share similar shape and morphology with neuronal cytoplasmic inclusions. We propose that the residues with this region (287-322), which contains myriads of glycine repeats, may contribute significantly to the fiber formation as well as aggregation propensity. Moreover, from the conformational characterizations of D1, A315T, and G294A with EM, CD, fluorescence, and Raman spectroscopy, we found that all three peptides formed an amyloid structure, providing insights into the nature of its aggregation vis a vis the other fragments in the C-terminus of TDP-43.

The chemical aspects of Raman spectroscopy: Statistical structure-spectrum relationship in the analyses of bioflavonoids.

Raman spectroscopy has been accepted as a useful tool for the characterization of natural products. However, to identify a specific compound in a mixture sample of natural products using Raman spectra alone is highly challenging if not impossible. We demonstrated an effective solution to such issues using a method combining statistical Raman spectroscopy and Mass spectrometry. The method was validated with a successful application to the identification of the major anthocyanin components in a purple yam (Dioscorea purpurea) extract. Of particular interest is that statistical grouping of the bioflavonoid standards that formed the database of this study was found to correspond closely to the conventional chemical classification. An initial theory on the chemical aspects of Raman spectroscopy pertaining to the connectivity of Raman-active functional groups in bioflavonoids was developed based on the statistical correlation between chemical classification and Raman spectroscopy.

Label-free SERS characterization of snake venoms by exploring the cysteine environs with bone-shaped gold nanoparticles.

Identification of snake venoms is a vital step in the treatment of fatal snakebites. In this study, we use the gold-thiolate interaction between a cysteine residue and gold nanoparticles to establish a SERS method for the differentiation of the venoms of Trimeresurus stejnegeri and Bungarus multicinctus. We confirm the preference of gold nanoparticles over silver for the SERS study of snake venoms by a binding experiment that also functions to differentiate the two venom samples by colorimetry and UV-vis spectroscopy. We report the SERS spectra of Trimeresurus stejnegeri and Bungarus multicinctus venoms for the first time. The spectra display distinct SERS signatures of the snake venoms on bone-shaped gold nanoparticles made with a house recipe. These signatures correlate to selected segments of the venom proteins due to the anchoring effect of the gold-cysteine bond. The method is quick as it accomplishes in situ isolation of the structure of interest to avoid tedious purification of the samples. The location of the interactive cysteine residue makes a novel characteristic of proteins in general.

Adsorbate enrichment on a zeolite surface and assembly of a SERS sensor: a case study with silver nanoparticles and the flavonoid catechin.

We have studied the adsorption of silver nanoparticles (AgNPs) and catechin on readily available commercial zeolite beads. Both adsorbates became available on the zeolite and were several fold more concentrated after a simple adsorption process, contributing to a 10-times overall increase in the collision probability between the two adsorbates. We were further able to detect AgNP-induced Surface Enhanced Raman Scattering (SERS) of catechin on the zeolite after sequential depositions of AgNPs and catechin on the zeolite using this process. To demonstrate high reproducibility, 93% of the zeolite sensors assembled this way were tested and proved satisfactory, and gave a distinctive catechin SERS signature. Preparation of the zeolite sensor was extremely easy with a nearly 90% yield.

Simultaneous separation of taxon-specific crystallins from Mule duck and characterization of their enzymatic activities and structures.

Methods to obtain pure proteins in large amounts are indispensible in protein research. We report here a large-scale/simultaneous isolation of taxon-specific crystallins (ɛ- and δ-crystallin) from the eye lenses of Mule duck. We also investigate the compositions, enzymatic activities, and structures of these purified taxon-specific proteins. A relatively mild method of ion-exchange chromatography was developed to fractionate ɛ-crystallin and δ-crystallin in large amount, ca. ∼6.60mg/g-lens and ∼41.0mg/g-lens, respectively. Both crystallins were identified by electrophoresis, HPLC, and MALDI-TOF-MS. ɛ-Crystallin, with native composition of Mr 142kDa, consisted of two subunits of 35kDa and 36kDa, while δ-Crystallin, with native molecular mass of 200kDa, comprised single subunit of Mr ∼50kDa. Both ɛ- and δ-crystallin were tetramers. The former showed lactate dehydrogenase (LDH) activity, while the latter appeared slightly active in an argininosuccinate lyase (ASL) assay. Raman spectroscopic results indicated that the secondary structures of ɛ- and δ-crystallin were predominantly α-helix as evidenced by the vibrational stretching of amide III over 1260cm-1 and amide I at 1255cm-1, in greatly contrast to the anti-parallel ß-sheet of α- and ß-crystallin as demonstrated by amide III at 1238cm-1 and amide I at 1672cm-1. The microenvironments of aromatic amino acids and the status of thiol groups also vary in different crystallins. The compositions, enzyme activities, and structures of the ɛ- and δ-crystalline of Mule duck are different from those of Muscovy duck (Cairina moschata) or Kaiya duck (Anas Platyrhynchos var. domestica), which reflect faithfully species specificity.

Influence of soy aglycon isoflavones on bone-related traits and lens protein characteristics of ovariectomized rats and bioactivity performance of osteoprogenitor cells.

Health benefits of soy isoflavones have attracted the concern of the public and the interest of health-care professionals. In this study, two trials were conducted in characterizing bone-related traits and lens proteins as affected by supplementation of soy aglycon isoflavones (SAI). In trial 1, an in vivo study, 20 Sprague-Dawley rats were ovariectomized (OVX) and randomly distributed into OVX and OVX+SAI (135 mg of SAI/kg of feed; 8.33 mg/kg body weight; 2.5 mg/day) groups. Another group containing 10 rats with a sham operation was control (Sham). The experiment period was 3 months, and the rats were subjected to bone-related traits and lens protein characterization. In trial 2, an in vitro study, osteoprogenitor cells (UMR-106) were divided into SAI-supplemented (0.5 mg of SAI/mL of medium) and unsupplemented groups. Results of the in vivo study indicated that daily BW gains in the OVX and OVX+SAI groups were greater than that of the Sham group (p < 0.05). Bone ash and Ca contents of the Sham and OVX+SAI groups were higher than those of the OVX group (p < 0.05), while bone density, strength, and phosphorus contents among groups varied insignificantly (p > 0.05). When the lens proteins were extracted and analyzed with size-exclusion HPLC, the contents of beta- and gamma-crystallins were lowest in the OVX group and the protein solubility decrease could be recovered by dietary SAI supplementation (shown by OVX+SAI group). Based on Raman spectra of the isolated lens proteins, disulfide bonds were observed more in OVX lens than in the Sham and OVX+SAI lens. Results of in vitro study with osteoprogenitor cells revealed that cell viability, alkaline phosphatase activity, osteocalcin, and Ca contents of the SAI-supplemented group were higher than those of the unsupplemented group (p < 0.05). The likely potency to enhance bone and lens health by SAI supplementation is worth pointing out.

Glass distilling collector applied for HCN recovery from submerged culture broth and fruiting body of Pleurotus eryngii for identification and quantification.

Detection and surveillance of food commodities containing cyanide is a crucial issue of food safety. In this study, five strains of Pleurotus eryngii (P. eryngii) were grown in submerged culture of yeast malt broth (YMB) with the suspected production of HCN. A safety-warranted U-bent glass distilling collector with three enlarged bulbs on each arm was designed to recover the broth vapor. When AgNO(3) solution was used as an absorbent to interact with the vapor, a white precipitate was formed. The precipitate was isolated and identified as AgCN by FT-Raman spectroscopic analysis. When the absorbent was substituted by KOH, after evaporation to dryness, dissolved in D(2)O, and followed by (13)C-NMR analysis, a KCN spectrum was achieved. Formation of AgCN and KCN confirmed HCN production in the broth by P. eryngii. When a sodium picrate solution (1.4%) was used as an absorbent and various authentic KCN solutions were applied for distillation and followed by absorbance determination at 510 nm, a linear dose-dependent relationship was obtained and the procedure was applied for HCN quantification of the marketed P. eryngii mushrooms (fruiting body). As estimated, 67.3% of the products contained HCN less than 1.0 mg/kg, 17.3% between 1.0 and 2.0 mg/kg, and 15.4% higher than 2.0 mg/kg. When the mushrooms were sliced and cooked in water at 95 degrees C for 6 min, 89.1% of the original HCN was lost. When the P. eryngii strains were respectively grown by submerged cultivation in YMB or YMB supplemented with 2.5% glycine for 16 days, HCN content was slightly higher in the latter than in the former for each strain.

Differentiating gastrointestinal stromal tumors from gastric adenocarcinomas and normal mucosae using confocal Raman microspectroscopy.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract, and gastric adenocarcinomas are a common cancer worldwide. To differentiate GISTs from adenocarcinomas is important because the surgical processes for both are different; the former excises the tumor with negative margins, while the latter requires radical gastrectomy with lymph node dissection. Endoscopy with biopsy is used to distinguish GISTs from adenocarcinomas; however, it may cause tumor bleeding in GISTs. We reported here the confocal Raman microspectroscopy as an effective tool to differentiate GISTs, adenocarcinomas, and normal mucosae. Of 119 patients enrolled in this study, 102 patients underwent gastrectomy (40 GISTs and 62 adenocarcinomas), and 17 patients with benign lesions were obtained as normal mucosae. Raman signals were integrated for 100 s for each spot on the specimen, and 5 to 10 spots, depending on the sample size, were chosen for each specimen. There were significant differences among those tissues as evidenced by different Raman signal responding to phospholipids and protein structures. The spectral data were further processed and analyzed by using principal component analysis. A two-dimensional plot demonstrated that GISTs, adenocarcinomas, and normal gastric mucosae could be effectively differentiated from each other.

Positive cyclin T expression as a favorable prognostic factor in treating gastric gastrointestinal stromal tumors.

Positive transcriptional elongation factor b (P-TEFb) contains the catalytic subunit cyclin-dependent kinase 9 (Cdk9) and the regulatory subunit cyclin T. Cyclin T1 and Cdk9 are the key factors of the PTEFb pathways and are overexpressed in the human head and neck carcinoma cell line. However, there have been limited studies regarding the role of cyclin T1 and Cdk9 in gastric gastrointestinal stromal tumors (GISTs). The aim of the present study was to assess the association between cyclin T1 and Cdk9 and their clinical significance in gastric GISTs. A total of 30 gastric GIST patients who underwent either laparoscopic or laparotomic partial gastrectomy were enrolled in the study. The surgical tissue slides were stained with Cdk9 and cyclin T1 antibodies, and the immunohistochemistry scores and disease-free survival (DFS) were analyzed. Ten patients were cyclin T1-positive, and 20 were negative. All 11 patients with recurrent tumors or distant metastases were cyclin T1-negative patients. Old age, large tumor size, a high Ki67 IHC staining score, high mitotic count and negative cyclin T1 staining revealed a worse clinical outcome in univariate analysis. By contrast, the Cdk9 score was not associated with clinical parameters. The Kaplan-Meier survival curve illustrated that the DFS rate of the patients with negative cyclin T1 staining was significantly lower than that of the patients with positive cyclin T1 staining. Positive expression of cyclin T1 was a good prognostic factor in patients with gastric GISTs.

Novel Method for Differentiating Histological Types of Gastric Adenocarcinoma by Using Confocal Raman Microspectroscopy.

Gastric adenocarcinoma, a single heterogeneous disease with multiple epidemiological and histopathological characteristics, accounts for approximately 10% of cancers worldwide. It is categorized into four histological types: papillary adenocarcinoma (PAC), tubular adenocarcinoma (TAC), mucinous adenocarcinoma (MAC), and signet ring cell adenocarcinoma (SRC). Effective differentiation of the four types of adenocarcinoma will greatly improve the treatment of gastric adenocarcinoma to increase its five-year survival rate. We reported here the differentiation of the four histological types of gastric adenocarcinoma from the molecularly structural viewpoint of confocal Raman microspectroscopy. In total, 79 patients underwent laparoscopic or open radical gastrectomy during 2008-2011: 21 for signet ring cell carcinoma, 21 for tubular adenocarcinoma, 14 for papillary adenocarcinoma, 6 for mucinous carcinoma, and 17 for normal gastric mucosas obtained from patients underwent operation for other benign lesions. Clinical data were retrospectively reviewed from medical charts, and Raman data were processed and analyzed by using principal component analysis (PCA) and linear discriminant analysis (LDA). Two-dimensional plots of PCA and LDA clearly demonstrated that the four histological types of gastric adenocarcinoma could be differentiated, and confocal Raman microspectroscopy provides potentially a rapid and effective method for differentiating SRC and MAC from TAC or PAC.

Structural investigation of edible zein films/coatings and directly determining their thickness by FT-Raman spectroscopy.

Near-infrared Fourier transform Raman (FT-Raman) spectroscopy was employed to study the molecular structure of edible zein films/coatings, which were fabricated directly from zein protein. The secondary structure of zein protein was mainly in alpha-helix and remained unaltered during film formation as evidenced by the vibrational modes of amide I at 1656 cm(-1) and amide III at 1274 cm(-1). Raman results indicated that hydrophobic interaction played an important role in the formation of zein film and disulfide bonding might be responsible for the structural stability of zein protein during film formation. To enhance its antimicrobial property, an antimicrobial zein film was manufactured by incorporating zein protein with benzoic acid whose structure was then characterized by FT-Raman. It showed that physical entrapment or hydrophobic interaction was crucial to the incorporation of benzoic acid with zein protein, and the secondary structure of the antimicrobial film was still maintained in alpha-helical form. In addition, FT-Raman exhibits its preference in directly determining the thickness of zein films/coatings. By correlating the Raman intensity ratio of nu(1003) to nu(84) (I(1003/84)) versus the thickness of zein film, a linear relationship with high coefficient (R(2) = 0.9927) was obtained, which was then used pragmatically to determine the thickness of zein coatings on apple. It showed that the FT-Raman result (thickness = 0.27 +/- 0.01 mm) was consistent with that of classical micrometric measurement (thickness = 0.28 +/- 0.02 mm). Consequently, FT-Raman provides a direct, simple, and reagent-free method to characterize the structure and the thickness of zein films/coatings.

Increased cyclin T1 expression as a favorable prognostic factor in treating gastric adenocarcinoma.

The expression of cyclin A, B1, D1 and E in gastric adenocarcinoma is known to be associated with clinical outcome. However, few studies have investigated the role of cyclin T1 and cyclin-dependent kinase 9 (CDK9) in gastric adenocarcinoma. Therefore, this study assessed the clinical significance of cyclin T1 and CDK9 expression in gastric adenocarcinoma. A total of 39 gastric adenocarcinoma patients received either radical total or distal gastrectomy in this study. Surgical tissue slides were stained with CDK9 and cyclin T1 antibodies, and immunohistochemistry scores and disease-free survival (DFS) rates were analyzed. Among the 19 patients with tumor-recurrent or distant metastasis, 16 were recorded as exhibiting low expression of cyclin T1. The remaining three patients exhibited high expression of the antibody. The results of patients with a higher T stage, N stage and tumor grade were less favorable. For patients with adenocarcinoma, the percentage of tissue slides stained with cyclin T1 was significantly higher than for those with normal stomach epithelia. The DFS rates of patients with low expression of cyclin T1 were significantly associated with poorer DFS rates. In conclusion, high expression of cyclin T1 is a favorable prognostic factor in treating patients with stomach adenocarcinoma.

Conformational switch of polyglutamine-expanded huntingtin into benign aggregates leads to neuroprotective effect.

The abundant accumulation of inclusion bodies containing polyglutamine-expanded mutant huntingtin (mHTT) aggregates is considered as the key pathological event in Huntington's disease (HD). Here, we demonstrate that FKBP12, an isomerase that exhibits reduced expression in HD, decreases the amyloidogenicity of mHTT, interrupts its oligomerization process, and structurally promotes the formation of amorphous deposits. By combining fluorescence-activated cell sorting with multiple biophysical techniques, we confirm that FKBP12 reduces the amyloid property of these ultrastructural-distinct mHTT aggregates within cells. Moreover, the neuroprotective effect of FKBP12 is demonstrated in both cellular and nematode models. Finally, we show that FKBP12 also inhibit the fibrillization process of other disease-related and aggregation-prone peptides. Our results suggest a novel function of FKBP12 in ameliorating the proteotoxicity in mHTT, which may shed light on unraveling the roles of FKBP12 in different neurodegenerative diseases and developing possible therapeutic strategies.

Structural analysis of triacylglycerols and edible oils by near-infrared Fourier transform Raman spectroscopy.

Fourier transform Raman spectroscopy was employed for structural analysis of triacylglycerols and edible oils. Raman spectra sensitively reflected structural changes in oils. Even slight structural fluctuation between triacylglycerols and free fatty acids led to obvious differences in Raman bands as shown by C-O-C stretching from 800 to 1000 cm(-1) and the band at 1742 cm(-1). Structural difference in geometric isomers was easily distinguished as proved by C = C stretching at 1655 cm(-1) (cis) shifting to 1668 cm(-1) (trans) and by =C-H in-plane bending at 1266 cm(-1) in cis disappearing in the trans isomer. Raman intensity at 1266, 1302, and 1655 cm(-1) changed concomitantly with the change of double-bond content in oils. It showed that FT-Raman was capable of precisly reflecting the content of double bonds in oils. A linear correlation with high consistency between the Raman intensity ratio (v1655/v1444) and the iodine value was obtained for commercial oils. Based on the results, FT-Raman spectroscopy proved itself a simple and rapid technique for oil analysis since each measurement could be directly completed in 3 min without any sample modifications.

Compositional and conformational analysis of yam proteins by near infrared fourier transform Raman spectroscopy.

Fourier transform (FT)-Raman spectroscopy was employed to study the molecular structure of yam proteins isolated from three commonly consumed yam species including Dioscorea alata L., D. alata L. var. purpurea, and Dioscorea japonica. Although D. alata L. and D. alata L. var. purpurea consisted of similar amino acid residues, they still exhibited significant differences in conformational arrangement. The secondary structure of D. alata L. was mainly an alpha-helix, while D. alata L. var. purpurea was mostly in antiparallel beta-sheets. In contrast, D. japonica, which belongs to a different species, exhibited explicit differences in amino acid compositions and molecular structures of which the conformation was a mixed form of alpha-helices and antiparallel beta-sheets. FT-Raman directly proved the existence of S-S in yam proteins, implying that oligomer formation in yam proteins might be due to disulfide linking of dioscorin (32 kDa). The microenvironment of aromatic amino acids and the state of S-S in yam proteins were also discussed.

Dietary vitamin E supplementation affects tissue lipid peroxidation of hybrid tilapia, Oreochromis niloticus x O. aureus.

A feeding trial was conducted to evaluate the effects of dietary vitamin E contents on the growth, ascorbate induced iron-catalyzed lipid peroxidation in post-mortem muscle and liver tissue, and Raman spectral changes in lens of juvenile hybrid tilapia (Oreochromis niloticus x O. aureus). Experimental fish were fed practical diets supplemented with 0, 50, 100, 200, 450 and 700 mg alpha-tocopheryl acetate/kg diet for 14 weeks. There was no significant difference in weight gain, feed conversion ratio and protein efficiency ratio among fish fed test diets (P>0.05). Protein content of fish fed diet containing the lowest vitamin E level was the lowest (P<0.05) among all groups. No difference was found in other body constituents among test fish (P>0.05). The thiobarbituric acid-reactive substances produced by iron-catalyzed lipid peroxidation in muscle and liver tissue of fish fed the diet without alpha-tocopheryl acetate supplementation were significantly (P<0.05) greater than those from fish fed diets containing higher levels of alpha-tocopheryl acetate. Dietary vitamin E supplementation increased the antioxidant capability of tilapia tissues against lipid peroxidation. Further, dietary vitamin E supplementation also influenced the lens cortical membrane structure of tilapia.

Inactivation of Bacillus stearothermophilus leucine aminopeptidase II by hydrogen peroxide and site-directed mutagenesis of methionine residues on the enzyme.

Leucine aminopeptidases (LAPs) are exopeptidases that remove the N-terminal L-leucine from peptide substrates. Oxidative stability assay showed that the recombinant Bacillus stearothermophilus LAP II (rLAPII) was sensitive to oxidative damage by hydrogen peroxide at the elevated temperature. The H2O2-treated enzyme experienced obvious changes in the secondary structure when the oxidant concentration increased to 300 mM. To investigate the role of methionine residues on the oxidative inactivation, each of the five methionine residues in the rLAPII was replaced with leucine by site-directed mutagenesis. The mutant enzymes with an apparent Mr of approximately 44.5 kDa were overexpressed in Escherichia coli and were purified to homogeneity by nickel-chelate chromatography. The specific activities for Met82Leu, Met88Leu, Met254Leu, and Met382Leu were similar to that of the wild-type enzyme, whereas a reduced activity was observed in Met136Leu. The 50% decrease in the catalytic efficiency (kcat/Km) for Met136Leu was caused by 47% decrease in kcat value. As compared with the wild-type enzyme, all mutant proteins were more sensitive to the oxidant, implying that the methionine residues of B. stearothermophilus LAP II are important for the protection of the enzyme from oxidative inactivation.

Antioxidative and antimutagenic activities of healthy herbal drinks from Chinese medicinal herbs.

Twenty-nine Chinese medicinal herbs and three healthy herbal drinks made of those herbs in a food processing pilot plant were tested for their antioxidative, free radical scavenging, mutagenic and antimutagenic activities. Water extracts of herbs (with few exceptions) and herbal drinks showed free radical scavenging activity. All water extracts of herbs and herbal drinks showed no mutagenicity toward Salmonella typhimurium tester strains TA98 and TA100 used in the Ames mutagenic tests. In the antimutagenic tests, the mutagenic activity of 4-nitroquinoline N-oxide (NQNO) toward S. typhimurium TA98 was markedly inhibited by water extracts of herbs and herbal drinks. Based on the results, it is suggested that the herbal drinks manufactured in pilot-plant scale are safe and can be served as health-promoting drinks for the public.