Chemical and biological assessment of Angelica herbal decoction: Comparison of different preparations during historical applications

The commonly used Angelica herbal decoction today is Danggui Buxue Tang (DBT), which is a dietary supplement in treating menopausal irregularity in women, i.e. to nourish "Qi" and to enrich "Blood". According to historical record, many herbal decoctions were also named DBT, but the most popular formulation of DBT was written in Jin dynasty (1247 AD) of China, which contained Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) with a weight ratio of 5:1. However, at least two other Angelica herbal decoctions recorded as DBT were prescribed in Song (1155 AD) and Qing dynasties (1687 AD). Although AR and ASR are still the major components in the DBT herbal decoctions, they are slightly varied in the herb composition. In order to reveal the efficiency of different Angelica herbal decoctions, the chemical and biological properties of three DBT herbal extracts were compared. Significantly, the highest amounts of AR-derived astragaloside III, astragaloside IV, calycosin and formononetin and ASR-derived ferulic acid were found in DBT described in 1247 AD: this preparation showed stronger activities in osteogenic, estrogenic and erythropoetic effects than the other two DBT. The current results supported the difference of three DBT in chemical and biological properties, which could be a result of different herbal combinations. For the first time, this study supports the popularity of DBT described in 1247 AD.     

Study on the effects of sulfur fumigation on chemical constituents and antioxidant activity of Chrysanthemum morifolium cv. Hang-ju

The traditional after-harvesting drying method of C. morifolium cv. Hang-ju (HJ) is sun drying, but recently sulfur fumigation is increasingly used as a cheap and convenient method. However, the effects of sulfur fumigation on chemical constituents and potential activities of HJ were unknown. A comprehensively comparison of the chemical profiles between non-fumigated HJ (NHJ) and sulfur-fumigated HJ (SHJ) was conducted by HPLC fingerprints analysis and the discrepant peaks were identified or tentatively assigned by HPLC–ESI/MSⁿ. Dramatic chemical changes were found that the contents of 4 flavonoid aglycones remarkably increased while those of 7 glycosides significantly reduced which suggested that sulfur-fumigation induced flavonoid glycosides transformed into aglycons by hydrolysis reaction. A significant loss of hydroxycinnamoylquinic acids showed the sulfur fumigation was a destructive effect on HJ. Principal component analysis (PCA) was employed to rapidly discriminate NHJ and SHJ samples. By ICP-OES analysis, it was found that the residue of sulfur of SHJ were three times higher than NHJ (p < 0.05). The antioxidant activity of NHJ and SHJ were evaluated by DPPH and FRAP assay, and the results showed that NHJ had much stronger antioxidant activities than SCF (p < 0.05). Combining the results of chemical analysis, residue of sulfur and pharmacological evaluation, it showed that the sulfur fumigation was a destructive effect on HJ.     

Tomato ABSCISIC ACID STRESS RIPENING (ASR) Gene Family Revisited

Tomato ABSCISIC ACID RIPENING 1 (ASR1) was the first cloned plant ASR gene. ASR orthologs were then cloned from a large number of monocot, dicot and gymnosperm plants, where they are mostly involved in response to abiotic (drought and salinity) stress and fruit ripening. The tomato genome encodes five ASR genes: ASR1, 2, 3 and 5 encode low-molecular-weight proteins (ca. 110 amino acid residues each), whereas ASR4 encodes a 297-residue polypeptide. Information on the expression of the tomato ASR gene family is scarce. We used quantitative RT-PCR to assay the expression of this gene family in plant development and in response to salt and osmotic stresses. ASR1 and ASR4 were the main expressed genes in all tested organs and conditions, whereas ASR2 and ASR3/5 expression was two to three orders of magnitude lower (with the exception of cotyledons). ASR1 is expressed in all plant tissues tested whereas ASR4 expression is limited to photosynthetic organs and stamens. Essentially, ASR1 accounted for most of ASR gene expression in roots, stems and fruits at all developmental stages, whereas ASR4 was the major gene expressed in cotyledons and young and fully developed leaves. Both ASR1 and ASR4 were expressed in flower organs, with ASR1 expression dominating in stamens and pistils, ASR4 in sepals and petals. Steady-state levels of ASR1 and ASR4 were upregulated in plant vegetative organs following exposure to salt stress, osmotic stress or the plant abiotic stress hormone abscisic acid (ABA). Tomato plants overexpressing ASR1 displayed enhanced survival rates under conditions of water stress, whereas ASR1-antisense plants displayed marginal hypersensitivity to water withholding.     

Study on the destructive effect to inherent quality of Fritillaria thunbergii Miq. (Zhebeimu) by sulfur-fumigated process using chromatographic fingerprinting analysis

The after-harvesting sun-dried processing of Fritillariae thunbergii bulbus (Zhebeimu) was the traditional treatment for commodity. Over recent decades the natural drying process for bulbus of Fritillariae has been replaced by sulfur-fumigation for reducing the drying duration and pest control. We used ultra-performance liquid chromatography coupled with evaporative light scattering detection (UPLC-ELSD) fingerprinting analysis and major alkaloids determination to investigate the potential damaging effect of the sulfur-fumigating process. The experimental conditions were as follows: Chromatography was proceeded on Waters Acquity UPLC BEH C(18) column; the linear gradient elution was conducted with mobile phase prepared from acetonitrile-0.02% triethylamine; the drift tube temperature was set at 40°C with a nitrogen flow-rate of 30psi, and the spray parameter was set 40%. All calibration curves showed good linear regression (R>0.9991) within the tested range. The method was validated for precision, accuracy, limit of detection and quantification. The study also has shown that sulfur-fumigated samples had significant loss of the main active compounds and a more destructive fingerprint profile when compared to the sun-dried samples.     

白芷有效成分提取工艺的优化及其中药材质量研究

目的：优化白芷中药材有效成分的提取工艺;全面系统地考察与研究市场上白芷中药材的质量。方法：以白芷中欧前胡素和异欧前胡素的含量为指标,采用高效液相色谱法测定,利用正交试验,对渗漉法提取白芷有效成分的工艺条件进行优选;按照《中国药典》2005年版一部白芷项下[含量测定]方法检验了62批次市场上销售的白芷药材中香豆素类物质的含量。结果：乙醇渗漉提取法：药材浸泡24h,加80%乙醇8倍量渗漉,欧前胡素和异欧前胡素的平均提取率为78%;市场上销售的白芷药材中香豆素类物质的含量符合药典的要求。结论：应用渗漉法工艺提取白芷中药材有效成分效率高,稳定性好,适合工业化生产;目前市场上流通的白芷中药材的质量可靠。     

Factors That Differentiate the H-bond Strengths of Water Near the Schiff Bases in Bacteriorhodopsin and Anabaena Sensory Rhodopsin

Bacteriorhodopsin (BR) functions as a light-driven proton pump, whereas Anabaena sensory rhodopsin (ASR) is believed to function as a photosensor despite the high similarity in their protein sequences. In Fourier transform infrared (FTIR) spectroscopic studies, the lowest O-D stretch for D₂O was observed at ∼2200 cm⁻¹ in BR but was significantly higher in ASR (>2500 cm⁻¹), which was previously attributed to a water molecule near the Schiff base (W402) that is H-bonded to Asp-85 in BR and Asp-75 in ASR. We investigated the factors that differentiate the lowest O-D stretches of W402 in BR and ASR. Quantum mechanical/molecular mechanical calculations reproduced the H-bond geometries of the crystal structures, and the calculated O-D stretching frequencies were corroborated by the FTIR band assignments. The potential energy profiles indicate that the smaller O-D stretching frequency in BR originates from the significantly higher pK_a(Asp-85) in BR relative to the pK_a(Asp-75) in ASR, which were calculated to be 1.5 and −5.1, respectively. The difference is mostly due to the influences of Ala-53, Arg-82, Glu-194–Glu-204, and Asp-212 on pK_a(Asp-85) in BR and the corresponding residues Ser-47, Arg-72, Ser-188-Asp-198, and Pro-206 on pK_a(Asp-75) in ASR. Because these residues participate in proton transfer pathways in BR but not in ASR, the presence of a strongly H-bonded water molecule near the Schiff base ultimately results from the proton-pumping activity in BR.     

白芷有效成分提取工艺的优化及其中药材质量研究

目的:优化白芷中药材有效成分的提取工艺;全面系统地考察与研究市场上白芷中药材的质量.方法:以白芷中欧前胡素和异欧前胡素的含量为指标,采用高效液相色谱法测定,利用正交试验,对渗漉法提取白芷有效成分的工艺条件进行优选;按照<中国药典>2005年版一部白芷项下[含量测定]方法检验了62批次市场上销售的白芷药材中香豆素类物质的含量.结果:乙醇渗漉提取法:药材浸泡24h,加80%乙醇8倍量渗漉,欧前胡素和异欧前胡素的平均提取率为78%;市场上销售的白芷药材中香豆素类物质的含量符合药典的要求.结论:应用渗漉法工艺提取白芷中药材有效成分效率高,稳定性好,适合工业化生产;目前市场上流通的白芷中药材的质量可靠.     

Danggui Buxue Tang,a Chinese herbal decoction containing Astragali Radix and Angelicae Sinensis Radix,improves mitochrondial bioenergetics in osteoblast

Osteoporosis is the process of bone loss, particular after menopause, when the production of estrogen in women is decreaing. Bioenergetic function is one of the critical roles in bone remodeling. Danggui Buxue Tang (DBT) is an herbal mixture containing Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), and which is consumed for “Qi-invigorating”, i.e., stimulating energy metabolism, as a traditional Chinese medicine (TCM). However, the role of DBT in metabolism of osteoblast has not been examined. Here, we employed a metabolic flux to examine the mitochondrial functions of cultured osteoblast in the presence of herbal extracts, including DBT, ASR, AR, AR + ASR (single mixing of two herbal extracts), as well as DBT_∆cal (a DBT extract depeleting calycosin), to examine their roles in osteoblastic metabolism, e.g. glycolysis and energy kinetics. By revealing the rates of oxygen consumption and extracellular acidification of mitochrondia, the DBT-treated osteoblasts were markedly strengthened with increases of maximal respiration, spare capacity, glycolysis capacity and glycolysis reserve, in comparing to other herbal extracts. In addition, the bioenergetic metabolism was modulated by DBT via the signaling of cellular Ca²⁺ and reactive oxgen species (ROS). Furthermore, DBT affected the morphology of mitochondria, as well as mitochondrial dynamic. Here, we propose that DBT can be regarded as benefit herbal extract in improving osteoblastic metabolism for bone disorders via central energy metabolism and mitochondrial bioenergetics.     

Bioaccessibility and safety assessment of trace elements from decoction of “Zhebawei” herbal medicines by in vitro digestion method

Studies on in vitro bioaccessibility and safety assessment of 6 elements namely Fe, Zn, Cu, Mn, As and Cd in decoction of eight commonly consumed “Zhebawei” herbal medicines were carried out. The method was based on simulation of human digestion in the gastrointestinal tract. Total content and content in extracts from gastrointestinal phases were analyzed for these elements by inductively coupled plasma atomic emission spectrometer (ICP-AES). In decoction of all 8 herbal medicines, it was found that the total content and bioaccessible concentration of these elements varied considerably among the types of herb, and the total content of each element did not show linearity with their bioaccessible content of the corresponding element. There is no risk of adverse health effects resulting from overdose of Fe, Zn, Cu, and Mn for almost all individuals by ingesting recommended maximum daily dose of these eight herbal medicines. In terms of Cd only Ophitopogin japonicum had higher total concentration and bioaccessible content than the value of suggested limit (5.4 μg/d). For total As content, only Scrophularia ningpoensis and Corydalis yanhusuo did not meet the safety standard. However, regarding bioaccessible As contents, none of the eight herbal medicines exceeded the value of the daily permissible intake of As (0.12 mg). The risk of toxicity from an element in herbal medicine might be overestimated when the total concentration but not the bioaccessible content is taken into account. In vitro digestion method is rapid, relatively simple and inexpensive, which could be helpful in conducting experiments about safety assessment of an element in herbal medicines.     

The effect of a special herbal tea on obesity and anovulation in androgen-sterilized rats

A special herbal tea has been used to treat clomiphene-resistant anovulatory disease and obesity effectively, especially in polycystic ovary syndrome (PCOS) cases with hyperinsulinemia. The effect of the herbal tea on obesity and anovulation was investigated in androgen-sterilized rats (ASR). The ASR model was established by subcutaneous injection of 1.25 mg testosterone propionate to Sprague-Dawley female rats at the age of 9 days. Rats were sacrificed around 112 days of age. ASR manifested with PCO, anovulation, high food intake, elevated body weight, and obesity. Immunocytochemistry demonstrated that estrogen receptors (ER) were predominantly distributed in the cytoplasm of neuropeptide Y (NPY)-containing neurons in the preoptic area (POA), and the coexpression was also found in the nuclei and fibers of NPY-synthesizing neurons in the arcuate nucleus (ARC). Compared with that in normal control rats, NPY expression was increased, the numbers of ER in hypothalamic ARC-median eminence (ME) decreased, gonadotropin-releasing hormone (GnRH) levels in ME was decreased, serum estrogen (E2) and leptin were elevated, and follicular stimulating hormone (FSH) and luteinizing hormone (LH) levels were reduced significantly in ASR. Significantly negative correlations between NPY and ER or GnRH, and between leptin and FSH or LH were observed. A positive correlation existed between serum leptin and body weight. These metabolic-endocrine changes in ASR were normalized after feeding the herbal tea. Both obesity and hypogonadotropin were expressed in ASR. The abnormal ovarian hormone milieu (elevated E2 levels) may have enhanced NPY expression and resulted in less GnRH and gonadotropin secretion. The herbal tea reduced body weight and induced ovulation in ASR.     

Interferon-mediated antiviral activities of Angelica tenuissima Nakai and its active components

Angelica tenuissima Nakai is a widely used commodity in traditional medicine. Nevertheless, no study has been conducted on the antiviral and immune-modulatory properties of an aqueous extract of Angelica tenuissima Nakai. In the present study, we evaluated the antiviral activities and the mechanism of action of an aqueous extract of Angelica tenuissima Nakai both in vitro and in vivo. In vitro, an effective dose of Angelica tenuissima Nakai markedly inhibited the replication of Influenza A virus (PR8), Vesicular stomatitis virus (VSV), Herpes simplex virus (HSV), Coxsackie virus, and Enterovirus (EV-71) on epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. Such inhibition can be described by the induction of the antiviral state in cells by antiviral, IFNrelated gene induction and secretion of IFNs and pro-inflammatory cytokines. In vivo, Angelica tenuissima Nakai treated BALB/c mice displayed higher survivability and lower lung viral titers when challenged with lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3, and H9N2). We also found that Angelica tenuissima Nakai can induce the secretion of IL-6, IFN-λ, and local IgA in bronchoalveolar lavage fluid (BALF) of Angelica tenuissima Nakai treated mice, which correlating with the observed prophylactic effects. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we evaluated antiviral properties of ferulic acid. Therefore, an extract of Angelica tenuissima Nakai and its components, including ferulic acid, play roles as immunomodulators and may be potential candidates for novel anti-viral/anti-influenza agents.     

Putative Effect of Aquifer Recharge on the Abundance and Taxonomic Composition of Endemic Microbial Communities

Drought events and the overexploitation of freshwater resources have led to the increased need to manage groundwater reserves. Aquifer storage and recovery (ASR), whereby artificial water is injected into aquifers for storage, is one of the proposed methods by which freshwater supplies can be increased. Microbial clogging following injection, however, is a major issue. Here, during laboratory simulations of ASR, we used flow cytometry and bar-coded pyrosequencing to investigate changes in microbial abundance and community dynamics. Bacterial abundance ranged from 5.0 × 10⁴ to 1.4 × 10⁷ cells ml^-1 before the addition of synthetic wastewater. Following wastewater addition, a 25-fold decrease in abundance was observed, coinciding with a 12-fold increase in viral abundance. Taxa shifted from an overrepresentation of Sphingomonadales, Sphingobacteriales, Rhodospirillales, Caulobacterales, Legionellales, Bacillales, Fusobacteriales and Verrucomicrobiales prior to the addition of synthetic wastewater to Burkholderiales, Actinomycetales, Pseudomonadales, Xanthomonadales, Rhodobacterales, Thizobiales and Thiotrichales following the addition of synthetic wastewater. Furthermore, a significant difference in overall taxonomic composition between the groundwater samples before and after the addition of synthetic wastewater was observed, with water samples exhibiting more similarity to sediment samples after wastewater was added. Collectively, these results suggest that ASR may alter the taxonomic composition of endemic microbial communities and that complete profiles of groundwater properties, including microbial community abundance and composition need to be taken into consideration when selecting aquifers for ASR practices.     

The acoustic startle reflex as a tool for assessment of odor environment effects on affective states in laboratory mice

Apart from self and conspecific odors, odors from other species also influence the affective states in laboratory mice (Mus musculus musculus) in their home cages and during experimental procedures, possibly inducing confusion and inconsistency in experimental data. Thus, it is important to detect the types of animal odors associated with housing, husbandry, and laboratory practice that can arouse different types of affective changes in mice. Here, we aimed to test the effectiveness of the acoustic startle reflex (ASR) in detecting changes in the affective states of laboratory mice due to animal-derived-odor as it has a non-zero baseline, and can be enhanced or attenuated by positive or negative affective shifts, respectively. We used ASR to examine the affective changes in mice that were induced by bedding odors and an alarm pheromone. The odor of bedding obtained from the mice’ home cages significantly attenuated the ASR, suggesting positive affective shifts in the test mice, whereas that from bedding obtained from rat cages significantly enhanced the ASR, suggesting negative affective shifts. No significant changes in ASR were observed in mice presented with the odor of bedding obtained from cages of unfamiliar conspecifics. In contrast, there was significant ASR enhancement in mice exposed to volatile components of alarm pheromones trapped in water, suggesting negative affective shifts. Thus, our findings show that ASR may be a valuable tool in assessing the effects of odors on the affective states in laboratory mice.     

Functional finishing of health care cotton for enhanced efficiency of antibacterial activity by chitosan and herbal nanocomposites

To enhance the efficiency of biological, chemical and physical properties like antibacterial activity, wash durability, air-permeability and biocompatibility of cotton fabric finished with chitosan and herbal nanocomposites. Extracts of Cassia angustifolia and Tamarindus indica with chitosan solution was bulk finished on 40s cotton fabrics. To increase the functional properties, chitosan and herbal extract nanocomposites were finished on to another set of fabrics (nanocomposite finishing). Different functional properties were carried out for both the sets of fabrics and comparatively analyzed. Antibacterial activity, physical properties and biocompatible properties of the finished fabric were determined. Antibacterial activity of nanocomposite finished fabrics showed inhibitory zones of 33 mm for E. coli and 31.6 mm for S. aureus. Nanocomposite finished fabrics showed good durable properties and physical properties than bulk finished fabrics. The study concludes that, nanocomposites could provide better functional properties than the bulk finished fabrics. The nano sized particles in the composites was considered significant for its functional applications in hospital based fabrics to prevent the transmission of nosocomial infections.     

Over-expression of the water and salt stress-regulated Asr1 gene confers an increased salt tolerance

ASR1 is a plant‐specific, highly charged, low molecular weight polypeptide. Purified ASR1 was shown to posses sequence specific Zn²⁺‐dependent DNA binding activity (Kalifa et al. Biochemical Journal 381, 373–378, 2004). Steady‐state levels of tomato Asr1 mRNA and protein are transiently increased following exposure of plants to polyethylene glycol, NaCl or abscisic acid. The biological role of ASR1 could not be deduced from sequence analyses or sequence homologies. Tobacco plants over‐expressing tomato ASR1 have a decreased rate of water loss and improved salt tolerance. Upon exposure to salt, ASR1‐over‐expressing plants accumulate less Na⁺ and proline than wild‐type plants, and also results in increased steady‐state levels of other gene products under non‐stressed plant growth conditions. Therefore, ASR1 is probably involved in the regulation of water‐ or salt‐stress‐modulated gene expression.     

当归饮片蛋白质的提取与活性分析

当归是传统的补血中药,其所含的多糖与小分子已有大量的研究,然而当归中的蛋白质组成与功效仍无人知晓。该研究通过0.05 mol·L-1 Tris-HCl( pH＝8.0)缓冲液浸提和组织匀浆得到当归饮片粗提液,结合硫酸铵沉淀和透析法去除粗提液中的多糖及还原糖等小分子成分,得到当归饮片总蛋白质,并首次对其组成和生物活性进行了研究。结果表明：当归饮片蛋白含量较高,分子量为17.5~90.7 kDa,其中17.5 kDa的蛋白含量最高,达47％。饮片蛋白在pH 5~11范围内较为稳定,pH为3时,仅余少量17.5 kDa的蛋白。当归饮片蛋白质中至少有3种蛋白在80℃内稳定存在,其中热稳定性最好的是17.5 kDa的蛋白,在热处理温度达到100℃时,仍然稳定存在,但随着处理时间的延长,该蛋白有部分单体发生了交联反应。当归饮片蛋白质具有清除DPPH自由基的能力,且该能力随着热处理温度及热处理时间的增加而增加,pH处理会影响该能力,pH为5.0时,清除能力最高,5.0两侧清除能力均下降。此外,当归饮片蛋白质对细胞有很强的选择性,表现为对正常肝细胞L-02有显著的增殖作用(1.0~4.0 mg·mL-1,P<0.01),对白血病细胞K562则表现出显著的抑制作用(0.5~1.5 mg·mL-1,P<0.01),当归饮片蛋白浓度为1.0 mg·mL-1时,可使L-02细胞增值率达550％(P<0.01),而K562细胞的抑制率达18.3％( P<0.01)。综上所述,当归饮片饮片蛋白具有重要的生物活性,可望从中开发出具有保肝作用的药物蛋白。     

Hydrolates: a review on their volatiles composition,biological properties and potential uses

An hydrolate (also known as hydrosol, floral water, aromatic water, or herbal water) is a distillate which is a product of condensation obtained after an essential oil distillation isolation procedure. Hydrolates are colloidal suspensions composed of a continuous phase, the distilled water, and a dispersed phase, the emulsion of essential oil droplets and water-soluble components, namely oxygen-containing compounds. The movement towards a circular economy, has increased the interest in the essential oil industry co-products, such as hydrolates. Hydrolates composition were mostly studied based on their volatile constituents. Moreover, they showed a diverse range of biological properties, with potential application in food, beverages, cosmetic and pharmaceutical industries, as well as in the agroforest sector. Nevertheless, the information is fragmented and dispersed under the different hydrolates designations. In the present review, the main volatile components of 362 hydrolates obtained from 194 species from 50 families is described, and the information on the corresponding essential oil main components is detailed. Additionally, the methodologies of extraction and analysis are surveyed, as well as hydrolates characteristics and traditional uses. This review also describes the different biological properties attributed to hydrolates and, according with these properties, their potential uses.

     

FTIR spectroscopy of the all-trans form of Anabaena sensory rhodopsin at 77 K: hydrogen bond of a water between the Schiff base and Asp75

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria, and is believed to function as a photosensor interacting with a 14 kDa soluble protein. Most of the residues in the retinal binding pocket are similar in ASR except proline 206, where the corresponding amino acid in other archaeal-type rhodopsins is highly conserved aspartate that constitutes the counterion complex of the positively charged protonated Schiff base. The recently determined X-ray crystallographic structure of ASR revealed a water molecule between the Schiff base and Asp75 [Vogeley, L., Sineshchekov, O. A., Trivedi, V. D., Sasaki, J., Spudich, J. L., and Luecke, H. (2004) Science 306, 1390-1393], as well as the case for bacteriorhodopsin (BR), a typical transport rhodopsin working as a proton pump. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all-trans form of ASR at 77 K, and compared the local structure around the chromophore and their structural changes upon retinal photoisomerization with those of BR. The K intermediate minus ASR difference spectra were essentially similar to those for BR, indicating that photoisomerization yields formation of the distorted 13-cis form. In contrast, little amide I bands were observed for ASR. The presence of the proline-specific vibrational bands suggests that peptide backbone alterations are limited to the Pro206 moiety in the K state of ASR. The N-D stretching of the Schiff base is presumably located at 2163 (-) and 2125 (-) cm(-)(1) in ASR, suggesting that the hydrogen bonding strength of the Schiff base in ASR is similar to that in BR. A remarkable difference between ASR and BR was revealed from water bands. Although ASR possesses a bridged water molecule like BR, the O-D stretching of water molecules was observed only in the >2500 cm(-)(1) region for ASR. We interpreted that the weak hydrogen bond of the bridged water between the Schiff base and Asp75 originates from their geometry. Since ASR does not pump protons, our result supports the working hypothesis that the existence of strongly hydrogen bonded water molecules is essential for proton pumping activity in archaeal rhodopsins.