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Determination of Heterocyclic Amines and Acrylamide in Agricultural Products with Liquid Chromatography-Tandem Mass Spectrometry

Heterocyclic amines (HCAs) and acrylamide are unintended hazardous substances generated by heating or processing of foods and are known as carcinogenic and mutagenic agents by the animal experiments. A simple method was established for a rapid and accurate determination of 12 types of HCAs (IQ, MeIQ, Glu-P-1, Glu-P-2, MeIQx, Trp-P-1, Trp-P-2, PhIP, AαC, MeAαC, Harman and Norharman) and acrylamide in three food matrices (non-fat liquid, non-fat solid and fat solid) by isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). In every sample, a mixture of internal standards including IQ-d3, MeIQx-d3, PhIP-d3, Trp-P-2-13C2-15N and MeAαC-d3 was spiked for quantification of HCAs and 13C3-acrylamide was also spiked for the analysis of acrylamide. HCAs and acrylamide in sample were extracted with acetonitrile and water, respectively, and then two solid-phase extraction cartridges, ChemElut: HLB for HCAs and Accucat: HLB for acrylamide, were used for efficiently removing interferences such as pigment, lipid, polar, nonpolar and ionic compounds. Established method was validated in terms of recovery, accuracy, precision, limit of detection, limit of quantitation, and linearity. This method showed good precision (RSD < 20%), accuracy (71.8~119.1%) and recovery (66.0~118.9%). The detection limits were < 3.1 ng/g for all analytes. The correlation coefficients for all the HCAs and acrylamide were > 0.995, showing excellent linearity. These methods for the detection of HCAs and acrylamide by LC-MS/MS were applied to real samples and were successfully used for quantitative monitoring in the total diet study and this can be applied to risk assessment in various food matrices.

INTRODUCTION
As the amount of unintended hazardous substances is increased during food production, processing, and cooking, consumer's interest towards these substances is increasing. Heterocyclic amines (HCAs) and acrylamide could be presented as the unintended harmful substances in cooked foodstuffs (1) by the Maillard reaction. HCAs are known as a mutant substance, which emerges when heating (fried, broiled, or barbecued) foodstuffs rich in amino acids or creatine and are categorized into the amino carboline group or amino-imidazo-azaarene (AIA) group according to their chemical structures (2). The amino carboline group is generally known as pyrolytic HCAs because it is formed during the pyrolysis reaction with amino acids or proteins at > 300 o C (2). Possible amino acids are tryptophan, glutamic acid, and phenylalanine, and possible proteins are globulin, casein, or creatine. AIAs are called thermic HCAs, with an imidazo functional group attached to quinolone, quinoxaline, or pyridine. The main precursors of AIAs are free amino acid, creatine, creatinine, sugar, pyrrole, and pyridine derivatives produced by the dehydration and cyclization reactions (2). Heterocyclic pyridine and pyrazine (IQ-256 K.-J. Lee et al. and IQx-type) are produced by the Maillard reaction of hexose and free amino acid (2), Strecker degradation of free amino acid and additional transformation occurs by aldehyde and creatine, forming imidazoquinoline and imidazo-quinoxaline. The factors affecting the HCA production in the food are their precursors (amino acids, sugars, creatine, and creatinine), temperature, moisture, cooking conditions, and antioxidant. Chemical structures and precursors of Table 1. Chemical structure and precursor of HCAs (2) and acrylamide (21) Amino carboline HCAs including full name and abbreviations are listed in Table 1. Acrylamide is a low-molecular-weight vinyl compound and is a colorless and odorless crystalline substance. It is easily soluble in water and rapidly polymerized by reacting with air (3) and is used to make polyacrylamide and its copolymers are used for the treatment of wastewater and purifying water in industries (4). According to the research of the Swedish National Food Administration, acrylamide was detected in the heat-treated potato products and other baked goods (5). Actually, potato products showed the highest amounts of acrylamide (6). When starchy foods are fried, roasted, or baked at temperatures > 120 o C, the Maillard reaction between asparagines and reducing sugars in the food results in the formation of acrylamide (3).
The carcinogenicity of HCAs was tested by many animal In 1994, acrylamide has been classified as a probable carcinogen to human (Group 2A) by the IARC (9). The potential risk of acrylamide in food to the public health has been considered by many government agencies and national authorities (10) and they performed risk assessments of acrylamide in food and concluded that efforts should be made to reduce the acrylamide levels to as low as possible.
A long-term exposure to acrylamide may damage the nervous system in both humans and animals to a certain extent (11).
In the previous literatures, the extraction and clean-up procedures of HCAs were performed mostly based on the method of Gross (12) and Grüter (13) by using three cartridges: Extrelut, propylsulfonic acid (PRS) and C 18 . After a certain amount of sodium hydroxide is added to meat and seafood, extraction is performed, followed by the adsorption to ChemElut and the eluate flows out to PRS and C 18 in a series to clean up interferences. In other literatures, HCAs are extracted with MCX (14) (Mixed-mode: Cation exchange), SCX (15) (Strong cation exchange)-C 18 , and LiChrolut EN (16). For the detection of HCAs, LC-MS (17), LC-MS/MS (18) or DAD (19,20) are used. In the pretreatment of acrylamide, sample extraction was mostly performed by the liquid-liquid extraction method and several studies also used matrix solid-phase dispersion (21). Mainly, solid-phase extraction (SPE) was used for the elimination of matrices by additional step. Accucat, Oasis HLB, and multi mode (9) were commonly used in acrylamide analysis. The analytical instruments such as GC-MS (21), GC-MS/MS (5) and LC-MS/MS (22) were used for qualification and quantitation.
Isotope dilution can be used efficiently for analyzing organic compounds in various food matrices. It can increase accuracy and precision in the real sample analysis. Calibration curve and concentration calculations are obtained with the ratio between sample and internal standard responses. In the previous analytical methods for HCAs, internal standards have been used such as caffeine, 1-naphthylamine, and L-norleucine (14). 2-Amino-3,4,7,8-tetramethyl-3Himidazo [4,5-f] quinoxaline (4,7,8-TriMeIQx) (19) has also been used as the internal standard for 10 species of HCAs. Isotopically labelled materials such as PhIP-d 3 (18), MeIQx-d 3 (23) and 14 C-IQ (17) have been used as a single internal standard for several analytes. However, this cannot solve the problems such as the matrix effect, low recovery, and errors from calibration because one internal standard cannot represent many different kinds of analytes. Therefore, several isotope-labelled standards are needed to analyse different types of analytes.
As previous analytical methods have been studied mainly for fat foods for HCAs (20) and potatoes for acrylamide (6), they cannot be applied to various food matrices, therefore, it needs a fast and simple extracting and clean-up procedure appropriate for the characteristics and properties of each matrix. In this study, for the analysis of acrylamide and 12 species of HCAs (IQ, MeIQ, Glu-P-1, Glu-P-2, MeIQx, PhIP, Trp-P-1, Trp-P-2, AαC, MeAαC, Harman, and Norharman) using LC-MS/MS in the various agricultural matirces, foodstuffs are divided into three groups: non-fat solid, non-fat liquid, and fat solid. This study aimed to achieve significant accuracy, precision, and recovery by applying different internal standards to each analyte. Moreover, the performance of the established analytical method was evaluated in terms of linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ), and its applicability to real samples was investigated to confirm it as the analytical method of monitoring HCAs and acrylamide for the risk assessment in the total diet study.
Acrylamide standards and isotopically labeled internal standard were purchased from Accu Standard Inc. (St Market, CT, USA) and Cambridge Isotope Laboratories, Inc.
(Tewksbury, MA, USA). Every standard solution was dissolved in water, and the mixture solution was prepared by the dilution. All the standard solutions were stored in a refrigerator (−4 o C) until the analysis. All the HPLC grade solvents for the pretreatment and mobile phase were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bond Elut Accucat cartridge (600 mg, 3 mL) was obtained from Agilent Technology (Santa Clara, CA, USA). Oasis HLB cartridge (200 mg, 6 mL) was purchased from Waters Corporation (Dublin, Ireland). A centrifuge machine was purchased from Hanil Science Industrial (Incheon, Korea).

LC-MS/MS analysis.
The HCA analysis was performed using an Agilent 1260 series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to API 3200 triple quadrupole mass spectrometer (MDS Sciex, Concord, ON, Canada) equipped with electrospray ionization (ESI). All the analytes were separated by an Atlantis T3 column (2.1 × 100 mm, i. d., 3 μm; Waters Corporation, Dublin, Ireland), maintained at 35 o C. The mobile phase was 30 mM ammonium formate (pH 3.7) with water (A) and 100% acetonitrile (B), and gradient condition was as follows: 90% mobile phase A for 2 min; 2~2.5 min held at 87% A; 2.54 min, 87~86% A; 4~7.5 min, 86~66% A; 7.5~8.5 min, 665 0% A; 8.5~11 held at 50% A; 11~13 min, 50~0% A; and then returned to the initial condition for 13~20 min. The flow rate was 0.25 mL/min and 5 μL aliquot of the extracts was injected into the LC-MS/MS. A triple quadrupole mass spectrometer was used to detect the analytes by ESI in the positive mode. For selective and sensitive analysis, multiple reaction monitoring (MRM) mode was used to detect the product ions. The optimized parameters for MS were as  follows: ionspray voltage, 5,500 V at 550 o C; curtain gas, 25 psi; nebulizing gas, 50 psi; and heating gas, 50 psi. The mass conditions are listed in detail in Table 2, and the chromatograms are shown in Fig. 1. The acrylamide analysis was performed by using a Shimadzu 20AD HPLC system (Kyoto, Japan) coupled to API 3200 triple quadrupole mass spectrometer (MDS Sciex, Concord, ON, Canada) equipped with electrospray ionization (ESI). Analytes were separated with matrices using an Aqua C18 column (2 × 250 mm, i. d., 5 μm; Torrance, CA, USA), maintained at 40 o C. The mobile phase was eluted by 0.2% acetic acid and 0.5% methanol in water by the isocratic mode. The flow rate was 0.2 mL/min and 25 μL was injected into the LC-MS/MS. A triple quadrupole mass spectrometer was used to detect the analytes by ESI in the positive mode. For selective and sensitive analysis, MRM mode was used to detect the product ions. The optimized parameters for the MS were as follows: ionspray voltage, 5,000 V at 550 o C; curtain gas, 25 psi; nebulizing gas, 50 psi; and heating gas, 50 psi.

Extraction and clean-up procedures.
For sample pretreatment, samples were classified into non-fat solid, non-fat liquid, and fat solid, according to the contents of fat, which with fat content > 20% were classified as fatty. Three types of samples, rice porridge, apple juice, and peanut butter, were used in the method validation as the matrix.
Heterocyclic amines: The method for twelve HCAs was modified to simplify the procedure described by Gross and Grüter (13).
Non-fat solid (Rice porridge): Rice porridge (5 g) was spiked with 100 ng of the internal standard mixture, 12 mL of 1 M sodium hydroxide, 14 mL of acetonitrile, and 1 mL of methanol. The sample was shaken for 10 min in a mechanical shaker to homogenize it and then separated by centrifugation at 7,000 rpm for 10 min at 25 o C. The supernatant was loaded onto 20 mL ChemElut for 10 min and then eluted with 80 mL of a mixture of methylene chloride:ethyl acetate (3 : 1, v/v). The eluate was evaporated and redissolved in 2 mL water-methanol (9 : 1, v/v), and 0.5 mL of 1 M sodium hydroxide. This reconstituted solution was loaded onto HLB cartridge preconditioned with 1 mL of methanol and distilled water. The HLB cartridge was washed with 2 mL of 2% ammonia solution and eluted by using 3 mL of 2% acetic acid containing 90% methanol. Aliquot (5 μL) of the eluate was injected into LC-MS/MS.
Non-fat liquid (Apple juice): Apple juice (15 g) was spiked with 100 ng of the internal standards, followed by the addition of 0.5 mL of 1 M NaOH. The mixture was shaken for 10 min to homogenize it, and the resulting solution was adsorbed for 10 min after loading onto ChemElut and eluted with 80 mL of dichloromethane and ethyl acetate mixture at a v : v ratio of 3 : 1. This eluate was evapo-rated in a rotary evaporator, and then 2 mL of distilled water : MeOH (9 : 1, v/v) and 0.5 mL of 1 M NaOH were added to the effluent. This reconstituted solution was loaded onto an HLB cartridge, preconditioned with 1 mL of methanol and 1 mL of distilled water. The HLB cartridge was washed with 2 mL of 2% ammonia solution and eluted by 3 mL of 2% acetic acid containing 90% methanol and eluted with 3 mL of 2% acetic acid (90% MeOH) and were transferred into vials for the analysis by LC-MS/MS.
Fat solid (Peanut butter): Peanut butter (4 g) was spiked with 100 ng of the internal standards, followed by the addition of 15 mL of 1 M NaOH and 15 mL of acetonitrile. After, shaking for 10 min, the mixture was centrifuged for 10 min at 7,000 rpm to homogenize the mixture and separate the layers. This procedure was repeated twice, and then the supernatants were evaporated using a rotary evaporator. The residue was adsorbed for 10 min after loading onto ChemElut and eluted with 80 mL of dichloromethane and ethyl acetate (3 : 1 v : v). The eluate was evaporated using a rotary evaporator. Distilled water (2 mL) and MeOH at a ratio 9 : 1 and 0.5 mL of 1 M NaOH were added to the eluate and loaded onto the HLB cartridges conditioned with 1 mL of distilled water and 1 mL of MeOH, washed with 2 mL of 2% ammonia solution, eluted with 3 mL of 2% acetic acid (90% MeOH), and finally transferred to vials for the analysis by LC-MS/MS. Acrylamide: The pretreatment of acrylamide was performed by the FDA method (24) for the three matrices (rice porridge, apple juice and peanut butter).
(a) Crushed sample (1 g) was weighed in a 50 mL polypropylene tube. (b) Internal standard solution ( 13 C 3 -labeled acrylamide, 200 ng/mL, 1 mL) was added to the tube, followed by adding 9 mL of water to the test portion. The tube was shaken by hand or vortexed briefly to disperse test portion in water prior to the mixing. (c) Mixing was performed for 20 min in a rotating shaker. (d) The sample was centrifuged at 3,500 rpm for 20 min. Aliquot (5 mL) was filtered using a 0.45 μm PTFE syringe filter (Millipore, Bedford, MA, USA). (e) OASIS HLB cartridge was conditioned with 3.5 mL methanol, followed by 3.5 mL of water. Methanol and water effluent were discarded. (f) OASIS HLB cartridge was loaded with 1.5 mL extract. The extract was allowed to pass completely through the sorbent material. The column was eluted with 0.5 mL water and discarded. Elution was performed with additional 1.5 mL water and collected for following Accucat cartridge cleanup. (g) The outside of the Accucat SPE cartridge was marked at a height of 1 mL volume above the sorbent bed. Accycat SPE cartridge was conditioned with 2.5 mL methanol, followed by 2.5 mL of water. Methanol and water effluent were discarded. Extract (1.5 mL) collected in step (f) was loaded and eluted to 1 mL mark before collecting the remainder of the eluate, and 2 mL of sample was transferred to an amber auto-sampler vial for LC/MS/MS analysis.

Sample preparation.
Eight types of agricultural products (apple juice, vinegar, flour, cereal, jam, chocolates, cookies, and potato chip) were analysed in this study. Representative foods were collected from 18 mega-markets in nine metropolitan cities (Seoul, Pusan, Incheon, Daegu, Daejeon, Kwangju, Ulsan, Suwon and Changwon) nationwide and made into composites by pooling them for total diet study. Matrix samples for validation were apple juice, peanut butter and rice porridge purchased from the retail markets. All samples were analysed according to the optimized procedure.

RESULTS AND DISCUSSION
Sample pretreatment. Pretreatment method of twelve HCAs was modified to simplify the procedure described by Gross and Grüter (13) and method for acrylamide was followed FDA method (24) for the three matrices (rice porridge, apple juice and peanut butter). In the analysis of HCAs, 1M NaOH was added to adjust pH to alkaline (pH 13.5) and acetonitrile was selected as the extraction solvent, which effectively eliminate lipid. Volume of the NaOH and acetonitrile was different depending on the matrix. For example, in non-fat liquid (apple juice) only 1mL of 1M NaOH was added and there was no need for acetonitrile extraction. In the preliminary study, we performed optimization of appropriate cartridges, elution solvents, and solvent composiotions, which are not shown. Cartridges of HLB, QuEChERS (PSA 400 mg, MgSO 4 1200 mg), QuEChERS (PSA 400 mg, MgSO 4 1200 mg, GCB 400 mg), and Chem Elut were examined and combination of Chem Elut and HLB was used as it gave best result. As the eluent for Chem Elut, methylene chloride:ethylacetate (3 : 1) was selected from the comparison of the several solvent compositions (methylene chloride:ethylacetate, 1 : 0, 3 : 1, 4 : 1, 1 : 1, 1 : 4, 0 : 1). In addition, methanol contents as the eluent of HLB was compared with the 30, 50, 70, 90% methanol containing 2% acetic acid and 90% methanol was selected because it showed best recovery for all analytes In the preliminary study of acrylamide, we firstly examined FDA method which represents for all foods and found that this method can be applied to three matrices (non-fat solid, non-fat liquid and fat solid) without any modifications. HLB clean-up after water extraction was used and followed by Accucat clean-up according to the FDA method through all samples in this study.

Method validation.
To evaluate the effectiveness of the newly developed analytical method fitted to the three matrices, validation was performed using the parameters such as selectivity, recovery, accuracy, precision, linearity, LOD, and LOQ and verified with the criteria assigned in the CODEX guideline CAC/GL 71-2009. Samples fortified with all the analytes at their target concentrations (low, medium and high) were extracted and analyzed for the method verification.
Selectivity was validated by comparing the chromatogram to ensure that no interfering peaks were present at the retention times of 12 HCAs and acrylamide. The representative chromatograms with that standards and internal standards are shown in Fig. 1, indicating good peak separation with no interfering peaks.
Calibration curves for the matrix sample spiked with the standards were obtained by plotting the peak area ratio (y) versus the concentration (x) and verified by the correlation coefficient (R 2 ) calculated by the weighted least-square linear regression method. The range of calibration curve was 10~200 ng and 1~400 ng/mL for the HCAs and acrylamide. The calibration curves (not shown) exhibited good linearity with R 2 > 0.995.
Five replicates of the fortified samples (10, 50 and 100 ng for HCAs and 10, 100 and 500 ng/g for acrylamide) were determined for recovery, accuracy, and intra-day precision. Inter-day precision was analysed on the five different days in the same way. The precision was expressed as the relative standard deviation (RSD, %) and the accuracy was expressed as the recovery (%) of the mean observed concentration to the nominal concentration. Both the precision and accuracy data are summarized in Table 3. Intra-day precisions were in the range 1.6~13.3% RSD for nonfatty matrices and 2.0~19.9% for fatty matrix and these results are well within the acceptance range (RSD < 20%). Interday precisions (not shown in Table) were in the ranges 0.91 2.5% and 2.5~14.8% for nonfat matrices and fat solid, respectively, and are in the acceptable range.
The accuracies were in the ranges 71.8~119.1% and 75.9~115.0% for nonfatty matrices and fatty solid, respectively, and are well within CODEX criteria (70~120%), for which the acceptable range is different depending on the concentrations. Highly reproducible accurate results demonstrate the reliability of this method for residue analyses. Analyte-free matrices were used to evaluate the extraction recovery. Using the peak areas of analytes obtained by diluted standard solutions spiked into extracts after the extraction as A, and the peak areas of the diluted standard solutions spiked before extraction as B, the extraction recoveries were calculated as extraction recovery (%) = B/ A × 100. The extraction recoveries (not shown in Table) were in the range 70.9~118.9% and 72.8~102.6% for nonfatty matrices and for fatty solid, respectively, except for Trp-P-2 (66.0%). The extraction recovery of Trp-P-2 was poor (< 70%); however calibration curves were derived from the spiked matrix and accuracy (93.1%) was within the criteria (70~120%). Therefore, this method can be applicable to real sample analysis.
To determine the LOD and LOQ, 10 ng of HCA and acrylamide standards were spiked into each matrix, and five replicates were analysed. The LOD was calculated as 3.3 times of the standard deviation and 10 times of standard deviation for LOQ. LOD and LOQ values are shown in Table 3. LOD of HCAs was in the range 0.02~1.55 ng/mL, 0.15~2.32 ng/g and 0.23~2.59 ng/g for non-fat liquid, nonfat solid and fat solid, respectively. Even though these matrices are different with those of the literature, LODs are similar level with previous LODs ranged in 0.05~4 ng/g (25), which are appropriate for the HCAs analysis in the total diet study. For acrylamide, LOD was 2.69 ng/mL, 3.07 ng/g and 3.00 ng/g for non-fat liquid, non-fat solid and fat solid, respectively, which are also adequate level compared with the literature data, 0.1~10 ng/g (26,27). Considering all these validation results, these methods for three kinds of matrices are reliable and accurate at a low concentration level (μg/kg) for the quantitation of HCAs and acrylamide in agricultural products.
Application to real samples. To evaluate the applicability of the proposed method, eight types of agricultural products (apple juice, vinegar, flour, cereal, jam, chocolates, cookie and potato chip) purchased from 18 retail markets from nine cities (South Korea) were analysed by the optimized analytical procedures.
In this study, among the 12 HCAs and acrylamide, only harman, norharman and acrylamide were detected from eight samples. Harman and norharman were detected in the range of ND-8.50 ng/g, and concentrations of acrylamide were in the range of ND-369.4 ng/g. These results are similar to or below the reported data (Harman: ND-91.7, norharman: ND-187.4, acrylamide: ND-2,762 ng/g) as shown in Table 4.
In conclusion, analytical methods using the LC-MS/MS MRM mode to detect 12 species of HCAs (IQ, MeIQ, Glu-P-1, Glu-P-2, MeIQx, Trp-P-1, Trp-P-2, PhIP, AaC, MeAaC, Harman, and Norharman) and acrylamide formed mainly by the Maillard reaction were established using six isotope labelled internal standards (IQ-d 3 , MeIQx-d 3 , PhIP-d 3 , Trp-P-2-13 C 2 -15 N, MeAaC-d 3 , and AA-13 C 3 ) in agricultural foods. The established method was successfully evaluated using the parameters according to the CODEX guideline CAC/GL 71-2009 and showed good precision, accuracy and recovery. Detection limits were in the adequate range for all analytes and this method was successfully applied to real samples. It could be used as a promising analytical method to monitor HCAs and acrylamide for the risk assessment in the total diet study and the results of real sample analysis may be used as a preliminary data for the strategy on the reduction of hazardous compounds in the food.


Synthesis of novel MgAl layered double oxide grafted TiO2 cuboids and their photocatalytic activity on CO2 reduction with water vapor

A series of magnesium/aluminum (MgAl) layered double oxide (LDO) grafted TiO2 cuboids (MgAl-LDO/TiO2) with various molar ratios of (Mg + Al) to Ti were synthesized by a combination of hydrothermal and coprecipitation methods, in which the growth of MgAl-LDO platelets was controlled. The MgAl-LDO/TiO2 composite materials were used for photocatalytic CO2 reduction with water vapor under UV light irradiation in a continuous-flow reactor. CO was found to be the main product from CO2. At near room temperature (e.g., 50 °C), MgAl-LDO/TiO2 did not significantly enhance CO2 reduction compared with pure TiO2 cuboids. At a moderately elevated reaction temperature (e.g., 150 °C), the MgAl-LDO/TiO2 sample with an optimum 10 wt.% MgAl-LDO loading demonstrated CO2 reduction activity five times higher than that of bare TiO2 cuboids. The photo-induced electrons on TiO2 may migrate to the MgAl-LDO/TiO2 interfacial sites to promote CO2 reduction. Findings in this work may lead to a new area of hybrid adsorbent/photocatalyst materials that are capable of sequential CO2 capture and photocatalytic conversion.

Introduction
Emission of carbon dioxide (CO 2 ) from consumption of fossil fuels is one of the main causes of global climate change. Photocatalytic reduction of CO 2 with water under sunlight has been considered as a promising way to lower the CO 2 level in the atmosphere, meanwhile producing alternative fuels such as carbon monoxide (CO), methane (CH 4 ) and methanol. 1,2 Among the various photocatalyst materials, TiO 2 is widely studied due to its suitable band positions, environmentally benign nature, low cost and easy availability. [3][4][5][6] However, photocatalytic CO 2 reduction with water using TiO 2 as the photocatalyst typically has low energy conversion efficiency. 1,2,7,8 This is because of the major obstacles including the fast recombination rate of photo-generated electron-hole (e-h) pairs, the wide band gap of TiO 2 (3.2 eV for anatase), and the fast backward reactions. 5,9 Approaches such as loading noble metal or metal oxides and doping with non-metal elements have been applied to improve the CO 2 photoreduction activity of TiO 2 . 1,10,11 In addition to these well-known challenges, there are other factors that hinder the photocatalytic activity but are rarely studied in the literature, such as the weakened CO 2 adsorption on TiO 2 at the solidgas interface in the presence of water vapor and limited desorption of reaction products or intermediates from the catalyst surface. 12,13 As a result, enhancing the CO 2 adsorption on the photocatalyst, a prior step to photoreduction, is important to improve the CO 2 photoreduction efficiency of hydrocarbon fuels.
Our previous studies have reported MgO-TiO 2 composites as hybrid adsorbent/photocatalyst materials for enhanced CO 2 photoreduction. 12,13 MgO was chosen as the CO 2 adsorbent because of its good CO 2 adsorption capability that is boosted in the presence of H 2 O vapor. [14][15][16] We found that MgO-TiO 2 possessed much higher activity and more stable performance than pristine TiO 2 , particularly at a medium temperature range (around 150°C), which may be due to the easier desorption of reaction intermediates at a higher temperature and the enhanced CO 2 adsorption by MgO compared with bare TiO 2 . We also reported that the concentration and dispersion of MgO on the MgO/TiO 2 composite strongly influenced the CO 2 photoreduction activity and 5% MgO was the optimum loading on the TiO 2 surface. However, since both MgO and TiO 2 were in the form of nanoparticles in the MgO/TiO 2 composites, it was difficult to distinguish the two components by microscopic analysis and to correlate the catalytic activity with the materials morphology or structure. In addition, MgO has been reported to have relatively low kinetics in CO 2 adsorption at moderate temperatures (250-400°C) possibly due to the changing of basic sites or -OH groups. 17,18 Hence, other medium temperature CO 2 sorbents with different morphologies and faster adsorption kinetics may be of greater interest to serve as the adsorbent component of the hybrid material for enhanced CO 2 photoreduction.
Layered double hydroxides (LDHs), also known as hydrotalcite-like compounds, and their post-calcination product, layered double oxides (LDOs), have been investigated as CO 2 sorbents. [19][20][21] LDHs can be chemically expressed by gen-  28,29 Hence, MgAl-LDO could be a good candidate as a replacement of MgO to combine with the TiO 2 photocatalyst to improve CO 2 photoreduction operated at a medium temperature (~150°C). The objective of this work is to design a novel hybrid adsorbent/photocatalyst material by grafting MgAl-LDOs (as the CO 2 adsorbent component) onto the surface of micrometer size TiO 2 cuboids (as the photocatalyst component) and to investigate the catalytic activity of CO 2 photoreduction in correlation with the materials properties. To obtain such a hybrid material structure, it is desirable to have micrometer size TiO 2 for MgAl-LDO grafting because MgAl-LDOs are reported to be micrometer or sub-micrometer size platelets. The platelet-shape MgAl-LDOs can be distinct from the micrometer-size TiO 2 cuboids, and thus, the morphology and concentration of the two components can be easily manipulated.

Synthesis of MgAl-LDO grafted TiO 2 cuboids
The MgAl-LDH grafted TiO 2 cuboids were synthesized by a two-step process that has not been reported before. In the first step hydrogenated titanate ĲH 2 Ti 3 O 7 ) nanobelts were produced and in the second step, MgAl-LDH was formed by coprecipitation with the H 2 Ti 3 O 7 nanobelts that were transformed into a cuboid shape along the coprecipitation process. H 2 Ti 3 O 7 nanobelts (micrometers in length) were prepared via a hydrothermal method that has been widely reported in the literature. 30 To find the optimum MgAl-LDO concentration in the composites to obtain the best photocatalytic CO 2 reduction activity, the samples with different mass ratios of MgAl-LDO to TiO 2 were prepared. The samples were denoted as x% MgAl-LDO/TiO 2 , in which x represents the measured weight percentage of MgAl-LDO in the sample. Pure TiO 2 cuboids were also synthesized as the control.

Materials characterization
The crystal structures of the MgAl-LDO/TiO 2 samples were identified by X-ray diffraction (XRD, Scintag XDS 2000) using Cu Kα irradiation at 45 kV and a diffracted beam monochromator at 40 mA. The optical properties were examined by UVvis diffuse reflectance spectroscopy using a UV-vis spectrometer (Ocean Optics) with BaSO 4 as the background. Scanning electron microscopy (SEM) (Hitachi S4800) was used to investigate the catalyst morphology. The dispersion of elements (Mg, Al, Ti, O) on MgAl-LDO/TiO 2 was analyzed by X-ray elemental mapping. The real concentration of MgAl-LDO in the sample was calculated by the difference of the weight of MgAl-LDO/TiO 2 before and after washing and drying with 1 M HCl solution.
The Brunauer-Emmett-Teller (BET) specific surface area of the composites was measured by nitrogen adsorption at 77 K on a surface area and porosity analyzer (Micrometrics ASAP 2020). Before each adsorption measurement, approximately 0.10 g of the sample was degassed at 180°C for 6 hours. The BET surface area was determined by a multipoint BET method using the adsorption data in the relative pressure ĲP/P 0 ) range 0.05-0.3. The thermal stability of the catalyst materials was carried out using a thermogravimetric analyzer (TGA-DAT-2960 SDT) at a heating rate of 20°C min −1 from 25 to 700°C in air.

Measurement of CO 2 photoreduction activity
The photocatalytic reduction of CO 2 with H 2 O vapor was conducted using a home-made quartz tube photoreactor operating in a continuous flow mode, as shown in

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For each test, 100 mg of the catalyst was used and evenly dispersed onto a rectangular glass-fiber filter that was placed alongside the wall of the quartz tube and facing the UV light illumination. The catalyst loading process is also shown in Fig. S1. † CO 2 cylinder gas (99.999%, Praxair) continuously passed through a DI water bubbler bringing a gas mixture of CO 2 + H 2 O (with 2.3 vol.% H 2 O) into the photoreactor. A high gas flow rate was used initially to purge out air inside the reactor for 2 h, which in the meantime ensured CO 2 adsorption reaching equilibrium on the catalyst surface. Then the flow rate was lowered and maintained at 2.0 sccm during photoreaction. A 100 W mercury vapor lamp or a 450 W Xe lamp with a 400 nm UV cut-off filter was used as the UV-vis light and visible light source, respectively, and the light spectra are shown in Fig. S2. † For the mercury vapor lamp, the light intensity was measured to be about 10 mW cm −2 in the UV region (λ < 390 nm, centered at 365 nm). As a reference, the UV intensity in the sunlight (AM1.5G) is around 5 mW cm −2 . Thus, the UV intensity from the mercury vapor lamp applied in this study was equivalent to approximately 2 suns. For the Xe lamp with a UV filter, the light intensity in the visible region (400-700 nm) was about 84 mW cm −2 , again close to 2-sun conditions. To reach and maintain a reaction temperature at 150°C, the optimum temperature found in our previous work for MgO-TiO 2 catalysts, 13 a 250 W infrared lamp was used to heat up the photoreactor and the temperature can be adjusted by varying the distance between the IR lamp and the reactor. The gaseous products in the reactor effluent were continuously analyzed at a 15 min interval using a gas chromatograph (GC, Agilent 7890A) equipped with an automated gas valve and a thermal conductivity detector (TCD) and flame ionization detector (FID). MgAl-LDOs consist of platelets around 2 μm in size. The length of the TiO 2 cuboids is in the micrometer size range with a high aspect ratio. The length of TiO 2 cuboids pretty much agrees with that of the titanate nanowires, suggesting that the cuboids are indeed transformed from nanowires during the second step of materials synthesis. The textural properties are characterized by BET analysis and the results are summarized in Table 1. Bare TiO 2 cuboids displayed a specific surface area of 109 m 2 g −1 and a pore volume of 0.48 cm 3 g −1 , where the pores are probably attributed to inter-nanoparticle spacing. The MgAl-LDO had a specific surface area of 180 m 2 g −1 , greater than that of TiO 2 cuboids, possibly due to the smaller interplanar spacing between the layered oxides, which is also reflected by the smaller pore size. Notably, the 10% MgAl-LDO/TiO 2 sample possessed a high specific surface area (175 m 2 g −1 ) close to the bare MgAl-LDO, although the concentration of MgAl-LDO was not high. This indicates certain interaction between the MgAl-LDH nanoflakes and TiO 2 nanoparticles during the formation of the hybrid material.

Results and discussion
The thermogravimetric analysis (TGA) result of the 10% MgAl-LDH/TiO 2 sample is given in Fig. S3, † which shows two main steps of weight loss which are consistent with the literature. 22 The adsorbed water in LDH was released at a relatively low temperature to 200°C where it was mostly in the form of interlamellar water. Carbonate ions of the LDH sample were decomposed at higher temperatures from 200 to 500°C in parallel with the water loss. The weight loss almost ceased at above 500°C indicating the completion of the decarbonation process, i.e., LDH was fully converted to LDO. Fig. 3 shows the SEM images of MgAl-LDO/TiO 2 samples with different compositions. The lower magnification SEM images in Fig. 3a, c, and e show that the three samples were composed of MgAl-LDOs grafted on micrometer size TiO 2 cuboids. The platelet shape of MgAl-LDOs on the composites after calcination was almost the same as the as-prepared uncalcined composites MgAl-LDHs (SEM images not shown here). In most of the literature reports, the morphology of MgAl-LDH platelets cannot be maintained after calcination. 25,35 The successful grafting of MgAl-LDO platelets on micrometer-size TiO 2 cuboids in this work demonstrates a novel approach in synthesizing such composite materials with the desired morphology. The higher magnification SEM images in Fig. 3b, d, and f show that both the size and the coverage of MgAl-LDO platelets grafted on TiO 2 cuboids increased as the MgAl-LDO loading increased.
The distributions of Mg, Al, Ti and O elements in the MgAl-LDO/TiO 2 composites were analyzed by X-ray elemental mapping, and the results are shown in Fig. 4. The elemental mapping images demonstrated that the cuboid skeletons were mainly composed of the Ti element and the grafted platelets were composed of Mg and Al elements that were evenly distributed on the cuboid surface. The O element is distributed on both the cuboids and the platelets, agreeing with the composition of mixed oxides for this composite material.
Diffuse reflectance UV-vis spectra were recorded to investigate the influence of MgAl-LDO on the optical property of TiO 2 , and the plots of Kubelka-Munk function were made to determine the band gap values, as shown in Fig. 5. The absorption edge of the TiO 2 is around 390 nm, corresponding to a band gap of about 3.2 eV, which agrees with most reported literature data on the band gap of TiO 2 anatase. The MgAl-LDO alone shows no light adsorption in the wavelength range measured. The incorporation of MgAl-LDOs on TiO 2 cuboids leads to a slight blue-shift in the adsorption edge and a reduced absorption in the UV region, probably due to the increased surface roughness resulting in more reflection.
To further confirm possible structural changes, UV/Vis adsorption spectra of H 2 Ti 3 O 7 and 10% MgAl-LDH/TiO 2 were recorded as shown in Fig. S4

Photocatalytic activities of CO 2 reduction
Photocatalytic activity tests were carried out under UV light irradiation at 50°C for 4 h (Fig. 6a) and subsequently at 150°C for another 8 h (Fig. 6b). CO was found to be the major product with a minor CH 4 concentration that was one or two orders of magnitude lower than CO. Thus, only CO production results were shown in Fig. 6 to compare the activities of the different catalysts. Fig. 6a shows that bare TiO 2 cuboids had a CO production rate of around 1.0 μmol g −1 h −1 at 50°C while pure MgAl-LDO had no activity for CO production. The addition of MgAl-LDO on TiO 2 cuboids had no significant improvement in the CO production rate except for the 10% MgAl-LDO/TiO 2 sample that has a doubled activity compared with bare TiO 2 cuboids. The main reason for such insignificant improvement is maybe because MgAl-LDOs has weak CO 2 adsorption at low temperatures. 26 For the following 8 h test under UV light at 150°C (Fig. 6b), the CO production rate of bare TiO 2 cuboids was almost the same as the first 4 hours at 50°C. Pure MgAl-LDOs still showed no activity of CO 2 reduction. Both 8% MgAl-LDO/TiO 2 and 10% MgAl-LDO/TiO 2 exhibited an obvious  View Article Online enhancement in CO production, reaching an average of about 2.8 and 4.3 μmol g −1 h −1 , respectively, compared with about 0.7 μmol g −1 h −1 for bare TiO 2 cuboids at 150°C. In other words, 10% MgAl-LDO/TiO 2 was five times more active than bare TiO 2 cuboids. Moreover, the activities of both 8% MgAl-LDO/TiO 2 and 10% MgAl-LDO/TiO 2 at 150°C were much higher than those at 50°C. In addition, the downward trend in the CO production rate for the 8% MgAl-LDO/TiO 2 sample indicates that it is inferior to the 10% MgAl-LDO/TiO 2 sample in terms of stability. Interestingly, the activity of 12% MgAl-LDO/TiO 2 did not show any enhancement compared with bare TiO 2 even at 50°C or 150°C. The above results indicate that at an optimum MgAl-LDO loading (10% in this study), the CO 2 reduction activity can be significantly increased at a higher temperature (150°C). It also agrees with our hypothesis that the CO 2 adsorption capability of MgAl-LDOs could promote the CO 2 photoreduction capability by TiO 2 . It is likely that the photo-induced electrons on TiO 2 could migrate to the adjacent CO 2 adsorption sites at the interface of TiO 2 and MgAl-LDO and thus promote CO 2 reduction. However, the exact mechanism is not clear so far and will be investigated in our future research. A very high loading of MgAl-LDO (12% in this case) did not promote CO 2 photoreduction or may have detrimental effect, probably because of the following two reasons: (1) the MgAl-LDOs may have covered all the TiO 2 cuboid surfaces (Fig. 3f) so that the contact between TiO 2 and adsorbed CO 2 is limited, and (2) the light absorption capacity is significantly reduced at a high MgAl-LDO loading (Fig. 5). The CO 2 photoreduction with H 2 O by the 10% MgAl-LDO/ TiO 2 sample under visible light was also carried out using a Xe lamp equipped with a 400 nm cut-off filter, and the result is shown in Fig. S5. † At 50°C under visible light, the catalyst exhibited no activity and at 150°C, the CO production rate reached around 1.0 μmol g −1 h −1 . Apparently, the material is more active under UV than under visible light, which agrees with the band gap of the material as shown in Fig. 5.
To examine the cycling capability of the catalyst, an additional experiment was conducted using 10% MgAl-LDO/TiO 2 under the mercury vapor lamp irradiation at 150°C for two  on-and-off cycles for a total period of 12 h. The results are shown in Fig. S6. † The CO production rate was around 4.0 μmol g −1 h −1 when light was on. When the light was switched off, the CO production rate decreased rapidly and became zero within one hour. Turning the light back on obviously re-activated the photoreduction and the CO production rate bounced back to 3.6 μmol g −1 h −1 , almost the same level as in the first cycle. Switching off the light again led to a stop in the photoreduction. This on-and-off cycling test result clearly indicates that CO production was indeed from CO 2 photoreduction with water activated by the UV light irradiation. The result that the CO production rate in the 2nd cycle can be recovered to the similar level in the 1st cycle demonstrates a good cycling capability of the catalyst.
Because the MgAl-LDO may still contain a small amount of carbonate species even after the calcination process, we have conducted another set of experiments to understand the possible interference of carbonate species on the catalyst to the reduction of CO 2 from the gas phase. Tests were carried out by comparing the photocatalytic activity of 10% MgAl-LDO/TiO 2 under two gas environments: (1) helium (He) + H 2 O vapor and (2) CO 2 + H 2 O vapor, and the results are shown in Fig. 6b. There was no CO production in the He + H 2 O atmosphere at 50°C, but when the temperature was increased to 150°C, CO was produced at a rate of about 1.0 μmol g −1 h −1 . This result indicates that carbonates in MgAl-LDOs were stable at low temperatures but were activated at higher temperatures and reduced to CO through photocatalytic reactions. By contrast, when CO 2 was present, the CO production rate was much higher, 1.5 μmol g −1 h −1 at 50°C and 4.3 μmol g −1 h −1 at 150°C. Comparison of these results suggests that carbonate residues in the MgAl-LDO/ TiO 2 composites did not have significant contribution to the CO production and most of these were derived from gasphase CO 2 reduction. To further verify the source of CO production, additional experiments were conducted to measure the photocatalytic activity of H 2 Ti 3 O 7 and TiO 2 cuboids in the He/H 2 O atmosphere, and the result is shown in Fig. S7. † No CO production was observed for H 2 Ti 3 O 7 and TiO 2 cuboids at either 50°C or 150°C. Only 10% MgAl-LDO/TiO 2 showed some CO production at 150°C. This result further proved that the small amount of CO produced in the He/H 2 O atmosphere was attributed to carbonate residues in the MgAl-LDO component of the composite and not from carbon contamination of TiO 2 .
On the other hand, the above interesting finding suggests that if we pre-load carbonates on the composite catalyst by capturing CO 2 first and then exposing the material under UV light or sunlight, it is possible that we can convert the captured CO 2 into CO in the second step, and thus we can separate the CO 2 capture and conversion process. A benefit of doing this is to achieve separation of CO 2 conversion products (e.g., CO) from the bulk CO 2 , an approach advantageous to most CO 2 photoreduction processes reported in the literature where the products are mixed with unreacted CO 2 . We will conduct further research to investigate this novel idea.

Conclusions
This work has demonstrated the successful synthesis of novel hybrid materials, MgAl-LDO platelets grafted on TiO 2 cuboids, with a controlled mass ratio of MgAl-LDO to TiO 2 . The surface coverage of MgAl-LDO on TiO 2 and the reaction temperature are two important parameters that influence the activity of CO 2 photocatalytic reduction to CO. The hybrid MgAl-LDO/TiO 2 showed a CO production rate five times higher than that of bare TiO 2 at the optimum 10 wt.% MgAl-LDO loading and at 150°C. The possible reason for the enhancement is that the grafted MgAl-LDO functions as a CO 2 adsorbent and its CO 2 capture ability increases with increasing temperature (below 200°C) and that the photoinduced electrons generated on TiO 2 may migrate to the interfacial sites and promote CO 2 reduction. At too high a loading of MgAl-LDO when the platelets completely cover the TiO 2 cuboid surface, the light absorption ability is impaired and the contact between TiO 2 and CO 2 is blocked, and thus no improvement in CO 2 photoreduction is observed. The carbonate residues on the MgAl-LDO/TiO 2 have a minor contribution to the CO production under photoirradiation, which inspires a new process of sequential CO 2 capture and photocatalytic conversion to fuels using this novel hybrid material. In this case, the products can be effectively separated from unreacted CO 2 . This interesting idea will be validated in our future work.


extfieldsofstudy:
Materials Science
Medicine

text:
Water-enhanced oxidation of graphite to graphene oxide with controlled species of oxygenated groups

Water-enhanced oxidation of graphite via a modified Hummers method can produce graphene oxide with controlled species of oxygenated groups.

mol −1 to remove the remaining acid and metal species. The resultant GO aqueous dispersion was then stirred overnight to exfoliate it to GO. Successively, the GO dispersion was then centrifuged at 3000 r.p.m. for 20 min twice to remove the unexfoliated particles.
Preparation of GO at 0 o C. Water (0 or 4 mL) was mixed with ice-cooled concentrated sulfuric acid (46 mL, < 5 o C) in a 250 mL flask setting in an ice bath with mechanical agitation at 200 r.p.m. After cooling the mixture to < 5 o C, 1 g of natural graphite powder was added, and then 3 g KMnO 4 was slowly put into the system within 30 min. Successively, the reaction mixture was mechanically stirred (300 r.p.m) for 12 or 48 h under water bath cooling to about 0 o C by refrigerator system. After oxidation, the reaction mixture was poured into 300 mL ice/water mixture to keep the temperature < 10 o C. The resulting solution was stirred for 15 min, and followed by the addition of 5 mL H 2 O 2 . The washing and dialysis processes were the same to those described above. Afterward, the GO dispersion was then centrifuged at 3000 r.p.m. for 20 min 5 times to remove the unexfoliated particles and used for further comparisons. To obtain large-size GO sheets, centrifugation rate of 2000 r.p.m. should be applied to avoid precipitation of lager GO sheets at higher centrifugation rates. The SEM images in Figure S18 demonstrate the large-size GO sheets synthesized at 0 o C for 48 h with adding 4 mL water and finally centrifuged at 2000 r.p.m.

Preparation of GO and rGO Papers.
The GO films were prepared by drying 5 mg mL −1 GO suspensions in plastic containers placed in a glass dryer at room temperature. The thickness of each GO film was controlled to be about 6−7 µm by adjusting the volume of GO suspension and the size of container. rGO films were obtained by chemical reduction of GO films by immersing them in a 1:1 (by volume) mixture of HI solution (57 wt. %) and ethanol sealed in a container at room temperature overnight. The rGO films were then repeatedly washed with water and ethanol, and finally dried at room temperature overnight for characterizations.

Measurements of GO yields.
A certain volume of purified GO aqueous dispersion was freeze-dried for over 48 h, and the weight of dried GO was used to calculated the concentration of GO (C GO in mg mL −1 ). The yield of GO (Y GO ) was calculated by Y GO = (C GO ×V GO /m Gr )  100%, where V GO is the total volume of purified GO dispersion (in mL), m Gr is the weight of feeding graphite powder (in mg).

UV-vis spectral studies on the oxidative solutions.
A certain volume of water (0, 4, 8, or 12 mL) was mixed with ice-cooled concentrated sulfuric acid (46 mL, < 10 o C) in a 250 mL flask setting in an ice bath with mechanical agitation at 200 r.p.m. After cooling down the solution to < 10 o C, 3 g of KMnO 4 was slowly added and stirred for 15 min under ice bath. Successively, the reaction system was transferred to a 40 o C oil bath and the stirring rate was increased to 300 r.p.m. Samples were taken out from the reaction system after heating for 0 or 2 h, and diluted for recording their UV-vis spectra.
Characterizations. X-ray photoelectron spectra (XPS) were carried out by the use of an ESCALAB 250 photoelectron spectrometer (ThermoFisher Scientific) with Al Kα (1486.6 eV) as the X-ray source set at 150 W and a pass energy of 30 eV for high resolution scan. Solid-state 13 C magic-angle spinning (MAS) NMR experiments were conducted on a Bruker Avance-III spectrometer (100.6 MHz 13 C, 400.1 MHz 1 H) and a 4 mm MAS rotor probe. Direct 13 C pulse spectra were acquired using a MAS spinning speed of 10,000 Hz and ~10,000 scans. Raman spectra and optical images were recorded on a LabRAM HR Evolution (Horiba Jobin Yvon) with a 514-nm laser.

Water-Enhanced Oxidation of Graphite to GO at 0 o C
Water-enhanced oxidation can greatly increase the yield of high-quality GO synthesized at 0 o C. For comparison, the GO samples were prepared by oxidation of graphite in the systems with addition of a mL (a = 0 or 4) water for b hours (b =12 or 48), and they were nominated as GO-a-0-bh. The yields of GO-4-0-bh were found to be much higher than those of the corresponding GO-0-0-bh, as reflected by the color difference of their dispersions ( Figure S13a). Actually, the yields of GO-0-0-12h and GO-0-0-48h were measured to be only ~0 and 1 ± 0.3 %. However, the yields of GO-4-0-12h and GO-0-0-48h were dramatically increased to 9 ± 1 and 60 ± 2 % ( Figure   S13b). It should be noted here, the GO-0-0-12h product did not have monolayer GO sheets, composing of partially exfoliated particles. GO-0-0-12h completely precipitated from its dispersion upon keeping undisturbed for several days.
Water-induced increase of the yield of GO-a-0-bh was caused by increasing its oxidation degree. As shown in Figure S14a, the relative content of oxidized carbon atoms increased remarkably with the increase of water volume from 43.7 % for GO-0-0-12h to 47.9 % for GO-4-0-12h, and from 45.5 % for GO-0-0-48h to 53.7 % for GO-4-0-48h. Raman spectral studies indicated that the I D /I G s of GO-a-0-bh samples decreased as 'a' changed from 0 to 4 ( Figure S14b), also reflecting the increase of oxidation degree. SEM images ( Figure S14c) showed that the sizes of GO-4-0-bh sheets are larger than those of GO-0-0-bh counterparts. The largest sheets of GO-4-0bh sheets are larger than 10 µm, while those of GO-0-0-bh are smaller than 7 µm. This is mainly due to that the higher oxidation degrees GO-4-0-bh sheets facilitate the exfoliation from their GrO precursors.
The TGA curves of GO-a-0-bh are shown in Figure S15. Among them, the curve of GO-0-0-12h exhibits two steps of thermal decomposition in the temperature range of 150 to 300 o C, indicating the presence of organosulfate groups. This incomplete hydrolysis of organosulfate was resulted from unexfoliated GrO, because the organosulfate groups in GrO matrix were inaccessible to water. The decomposition of organosulfate are not observed from the other TGA curves, indicating the corresponding GO samples have been well exfoliated and hydrolyzed. These TGA curves also indicate that the contents of functional groups in the GO samples are in the sequence of GO-0-0-12h < GO-4-0-12h ≈ GO-0-0-48h < GO-4-0-48h. XPS and Raman analysis also confirmed this observation.
The XRD pattern ( Figure S17b) of rGO-4-0-48h exhibits single characteristic (002) reflection peak at 2θ = 24.2 o , corresponding to a d-space of 3.68 Å. This d-space is smaller than those of rGO-n (3.71−3.74 Å) because of its lower content of the residual oxygenated groups.
Typical Raman spectrum of rGO-4-0-48h ( Figure S17c) features a stronger Dbands with respect to G-band (I D /I G = 1.72), indicating the restoration of graphitic structures. S2, S3 Furthermore, the G band of rGO-4-0-48h is a combination of a narrow G peak centered at ~1580 cm −1 and a broader blue-shifted peak (G+D'), having a higher intensity ratio of I G /I D' with respect to that of rGO-0. S4 Moreover, the intensity ratio (I 2D /I D+G ) of 2D band (~2690 cm −1 ) to D+G band (~2940 cm −1 ) of rGO-4-0-48h was measured to be 1.31, much higher than that (1.03) of rGO-0. S4 This result implies that the structural integrity of rGO-4-0-48h sheets is better than that of rGO-0 sheets.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

text:
Stereo- and Chemodivergent NHC-Promoted Functionalisation of Arylalkylketenes with Chloral**

Stereo- and chemodivergent enantioselective reaction pathways are observed upon treatment of alkylarylketenes and trichloroacetaldehyde (chloral) with N-heterocyclic carbenes, giving selectively either β-lactones (up to 88:12 dr, up to 94 % ee) or α-chloroesters (up to 94 % ee). Either 2-arylsubstitution or an α-branched iPr alkyl substituent within the ketene favours the chlorination pathway, allowing chloral to be used as an electrophilic chlorinating reagent in asymmetric catalysis.

[c] Determined by chiral HPLC analysis.
[c] Determined byc hiral HPLC analysis. The observed change in reactivity from formal [2+ +2] cycloaddition to chlorination with variation in the ketene structure was also investigated computationally using 1,4-dimethyltria-zol-5-ylidene as am odel NHC catalyst with methyl-2-methylphenylketene and isopropylphenylketene ( Figure 2). Grimme's B3LYP-D3(BJ) functional [21] and the 6-31G(d,p )b asis set [22] were used for geometry optimisation and ZPE calculation, with final energies calculated using the TZVPP basis set. [23] Using these constraints, transition structures for both the formal [2+ +2] cycloaddition and a-chlorination reactions from methyl-2-methylphenylketene and iso-propylphenylketenew ere located (Figure 2). In accordance with the resultso fZ hang et al. [24] the transition states forr eactions of the (E)-enolates were significantly lower in energy than those of the (Z)-enolates (see SI for all calculated transition state structures and energies). Using both of these ketenes, transition states for a-chlorination over the formal [2+ +2] cycloaddition process leading to the b-lactones weref avoureds ignificantly as observed experimentally. For b-lactone formation,t he transition state leadingt ot he synproduct was favoured overt he anti-. [25] In the calculated transition states, the forming CÀCb onds in the formal [2+ +2] cycloaddition are significantly shorter (22,1 .88 ; 23,1 .87 ) than the developing CÀCl bonds (24,2 .24 ; 25,2 .37 ). This is consistent with the electrophilic chlorine in the S N 2-type chlorination transition state being less sterically demanding than the sp 2 -hybridised carbonyl carbon in the formal [2+ +2] cycloaddition reaction. With either a2 -substituent within the aromatic substituent of the alkylarylketene, or ab ranched iso-propyl group, the additional sterice ncumbrance of these substituents disfavours the formal [2+ +2] addition, resulting in the chlorination processb eing preferred.
Building upon this model, the observedc hemodivergent reaction pathwaysa re proposed to arise from initial NHC addition to the ketene, with preferentialo nwards reaction arising from the (E)-azolium enolate 26.S ubsequents tereoselective formal [2+ +2] cycloaddition with chloral generates 28,w ith eliminationo ft he NHC giving the b-lactonea nd completing the catalytic cycle. Alternatively, the use of chloral as an electrophilic chlorinating agent results in the formation of an acyl azolium and enolate ion pair 27 that combined to give the observed a-chloroester.N otably,a ssuming these mechanistic extremes,stereodivergent reaction pathways are observed from the (E)-azolium enolatei ntermediate 26. Reface functionalisation of the enolate derived from sterically nondemanding ketenes (such as 1) leads to the observed b-lactone configuration. Conversely, Si-face functionalisation with ketenes bearing either a2 -substituted aryl unit or an a-branched isopropyls ubstituent providest he configuration observed for the chlorinated esters (Figure 3).

Scheme4.[a]
Yieldo fisolated product. [b] Determined by chiralH PLC analysis. Computational studies on am odel system have allowed the structural parameters that lead to selectivity in these reaction processes to be analysed. Current research from this laboratory is directed toward developing alternative uses of NHCs and other Lewis bases in asymmetric catalysis.

Experimental Section
For general experimental details, full characterisation data, NMR spectra and HPLC traces, see the Supporting Information.
General procedure (1): Lactonisation and chlorination at 0 8 8C To af lame dried Schlenk flask under an argon atmosphere was added NHC precatalyst (0.10 mmol), base (0.09 mmol) and toluene (6 mL) and the mixture stirred for 15 min. The mixture was then cooled to 0 8Ci na nice/H 2 Ob ath followed by addition of a08Cs olution of the requisite ketene (1.00 mmol) in toluene (12 mL), immediately followed by chloral (1.00 mmol). To luene (2 mL) was added to wash residual reactants into solution and the reaction was stirred for the stated time at 0 8Cb efore opening the flask to the air for 30 min and concentration in vacuo. The resulting crude residue was purified by flash silica chromatography (ether:petrol) to provide either the isolated lactone or chlorinated ester as stated.
General procedure (2): Lactonisation and chlorination at 0 8 8C with dropwise ketene addition In instances where ketene dimerization was competitive with lactonisation or chlorination the ketene was added dropwise. To aflame dried Schlenk flask under an argon atmosphere was added NHC precatalyst (0.10 mmol), base (0.09 mmol) and toluene (6 mL) and the mixture stirred for 15 min. The mixture was then cooled to 0 8C in an ice/H 2 Ob ath followed by addition of chloral (1.00 mmol). A 0 8Cs olution of the requisite ketene (1.00 mmol) in toluene (12 mL) was subsequently added over 0.5 h. The reaction was stirred for an additional 3h at 0 8Cb efore opening the flask to the air for 0.5 h and concentration in vacuo. The resulting crude residue with the stated diastereomeric ratio was purified by flash silica chromatography (ether:petrol) to provide either the isolated lactone or chlorinated ester.


s2fieldsofstudy:
Chemistry
Agricultural And Food Sciences

text:
Development of a highly sensitive biotin–streptavidin enzyme-linked immunosorbent assay for detecting diethyl phthalate based on a specific polyclonal antibody

An indirect competitive biotin–streptavidin enzyme-linked immunosorbent assay (BA-ELISA) has been established for the determination of diethyl phthalate (DEP) in wine samples. A highly sensitive and specific polyclonal antibody (pAb-DEP) targeting DEP was prepared. Under optimized conditions, good linearity was achieved within a range of 0.021–9.512 μg·L−1. The limit of detection (IC10) was 0.0079 μg·L−1 and the median inhibitory concentration (IC50) was 0.443 μg·L−1. Besides, the BA-ELISA was highly selective, with low cross-reactivity values with DEP analogs (below 5%). Finally, the concentrations of DEP in wine samples ranged from 5.93 μg·L−1 to 59.15 μg·L−1 by BA-ELISA. Satisfactory recoveries (89.19–112.33%) and variation coefficient values (5.81–9.43%) were successfully obtained. The consistency between the results of BA-ELISA and gas chromatography–mass spectrometry (GC–MS) was 97.48%, which further confirmed that the proposed BA-ELISA immunoassay is reliable, rapid, sensitive, and accurate for monitoring DEP in wine samples.

Introduction
Phthalic acid diesters (PAEs) are a group of compounds that are widely used as additives and plasticizers in various commercial and personal care products to improve flexibility, long durability, and general handling properties (Schettler, 2009). And yet, PAEs are ubiquitous endocrine disrupting chemicals that can cause birth defects, sexual dysfunction, cancers, and possibly heart disease in living species through direct mechanisms or interaction with peroxisome proliferator-activated receptor (Diamanti-Kandarakis et al., 2009). Based on the above potential risks of the health and environment related to PAEs, the Council of the European Union, the US Environmental Protection Agency and many other countries' government departments make policy on regulation of PAEs (110th United States Congress, Consumer Product Safety Improvement Act [CPSIA], 2008;Fontelles & Clarke, 2005;Wu et al., 2012). Moreover, previous studies have shown that PAEs are widely distributed at detectable levels in foodstuff samples (Cao, 2010). Although the PAEs levels in wine samples

Preparation of DEP hapten, immunogen, and coating antigen
Synthesis of DEP hapten DEP molecules do not contain functional groups that can connect with proteins directly. Therefore, DEP hapten must be synthesized firstly as Yanaihara described (Yanaihara, Kato, Nagasawa, & Kodaira, 2002). The results of the hapten synthesis and characterization are given below.

Preparation of immunogen and coating antigen
As a contact portion between hapten and carrier protein, linking arm could become an antigenic determinant to determine the specificity of conjugating with antibody (Goodrow & Hammock, 1998). In order to reduce the specific binding caused by linking arm, different coupling methods are often used for preparing immunogen and coating antigen. In hence, the diazotization method and the glutaraldehyde method were used to couple hapten to the carrier proteins using the terminal amino group on a DEP hapten molecule in this study. UV-vis spectrophotometer was used to identify all conjugates, and then the coupling ratios were estimated based on mole absorbance ε and calculated using the following equation (Liu, Feng, Chen, & Qian, 2000;Monroe, 1984): Preparation of biotinylated pAb-DEP (Bio-pAb-DEP) Rabbit polyclonal anti-DEP antibodies (pAb-DEP) were prepared as follows: two male New Zealand white rabbits were immunized with BSA-DEP through subcutaneous and intramuscular injections with the immunogen. The initial immunization was performed by injecting 1mg of BSA-DEP dissolved in 0.5 mL normal saline and emulsified with 0.5 mL of CFA. Twenty days after the injections, the rabbits were boosted six times at two-week intervals by injecting a solution of 1 mg of the immunogen dissolved in 0.5 mL normal saline and emulsified with 0.5 mL of IFA. The serum titer was determined by ELISA. After antiserum titer outreached 60,000 with immunization for three months, pAb-DEP was separated and purified from rabbit serum through ammonium sulfate precipitation method, and subsequently was dialyzed against PBS for five days, freeze-dried, and stored at -20°C. Bio-pAb-DEP was prepared as follows: 5.0 mg of pAb-DEP was dissolved in CBS at the concentration of 1.0 mg·mL −1 . The above solution was mixed with 1.0 mg·mL −1 BNHS in DMSO in the mass ratio of 1:10. The mixture was stirred for four hours at room temperature and then dialyzed against PBS for three days, and was stored at -20°C.

Heterologous indirect competitive BA-ELISA
Heterologous indirect competitive ELISA, based on the immobilization of coating antigens, was performed in 96-well microplates as follows: the microplates were coated with the coating antigen in 100 μL of CBS overnight at 4°C. After three times washing (200 μL/tube of PBST), the unbound active sites were blocked with 200 μL/tube of blocking reagent, and were incubated at 37°C for 60 min. After a second washing step, the DEP standard or sample (50 μL/tube) and Bio-pAb-DEP (50 μL/tube) were added and the mixture was incubated for 60 min at 37°C. After another washing step to remove unbound Bio-pAb-DEP, SA-HRP (dilution 1:1000, 100 μL/tube) was added and the mixture was incubated for 60 min at 37°C. After an additional five times washing, 100 μL of TMB substrate solution was added. The enzymatic reaction was stopped by adding 50 μL of 2 mol·L −1 sulphuric acid after 15 min sufficient color development. The absorbance of each well was immediately recorded in dual-wavelength mode (450 nm as test and 630 nm as the reference).
The results were represented as inhibition (%). The standard curve of BA-ELISA was determined by plotting inhibition (%) against the logarithm of the standard concentration of DEP and negative control, and the linear range was used for quantification of DEP concentration in the samples. The IC 50 , the concentration at which a compound inhibited a particular phenomenon by 50%, was used to evaluate the sensitivity of the methods. Analogously, the limit of detection (LOD) was evaluated in terms of IC 10 .
where A max was the absorbance in the absence of DEP, A min was the absorbance of the blank sample, and A s was the absorbance of DEP at the standard concentration.

Cross-reactivity (CR)
The specificity of the optimized BA-ELISA assay was evaluated by measuring CR of the Bio-pAb-DEP using a group of DEP structural analogs. The CR values were calculated as follows: Sample preparation All the wine samples were purchased from Auchan (China) investment Co. Ltd. in Shanghai, China. Further details of these samples were provided in the Table 4. Besides, these samples collected in this study were all popular brands that leaded by a wide margin in Shanghai market.
To avoid PAEs contamination, all glassware used in the study were immersed with acetone for at least 30 min, and then were washed with hexane three times. All the blanks, standards, spiked samples, and real samples (100 mL) were undergone similar extraction method as follows: after evaporating through the boiling water bath and cooling to room temperature, all of wine samples were dissolved in 2 mL hexane. After incubation 5 min at 28°C on a rotary shaker (250 rpm), the upper layer was removed and evaporated to dry under nitrogen. The treated sample was divided into two fractions: one reconstituted with 1 mL PBS solution with 5% DMSO (v/v) prior to analysis by BA-ELISA, and the other reconstituted with 1 mL hexane for GC-MS analysis.

Characterization of immunogen, coating antigen, and antibody
To develop a highly sensitive and specific immunoassay, the amino group of DEP was the most available for coupling with the protein. Conjugates of DEP to protein BSA were prepared via diazo coupling to form immunogen BSA-DEP. Meanwhile, the amino group was then coupled with proteins to synthesize OVA-DEP used as the coating antigen through efficient glutaraldehyde method. From the UV spectrum (Figure 1), several characteristic absorption peaks of DEP hapten, protein, and conjugates appeared at 295 nm (for hapten), 227 and 278 nm (for BSA), and 234, 241, and 268 nm (for OVA). However, it was known that the diazo bond has a characteristic UV absorption peak between 300 and 350 nm (Guan et al., 2015). From the UV-vis absorption spectrum of BSA-DEP, in addition to the main absorbance peaks at 222 nm, BSA-DEP had a typical peak at 336 nm which was characteristic of the diazo bond. Besides, OVA-DEP showed obvious absorbance at 210 and 360 nm, and these characteristic peaks of OVA-DEP had obvious shift compared with unconjugated DEP hapten and OVA, indicating that the DEP hapten was successfully conjugated to OVA. The results revealed that the DEP hapten was conjugated into the protein successfully. Moreover, the coupling ratio was calculated using the Equation (1) above. The coupling ratio was 16 for BSA-DEP and was 21 for OVA-DEP.
The immunogen BSA-DEP was injected into New Zealand White rabbits, which improved immunity for 15 weeks, with the highest antibody titer at 1:120,000. The concentration of immunogen, coating antigen, and pAb-DEP was determined by coomassie blue staining, i.e. 2.62 mg·mL −1 , 1.04 mg·mL −1 and 3.60 mg·mL −1 , respectively.

Optimisation of BA-ELISA
To develop a sensitive immunoassay method, several parameters were optimized. First, the concentrations of coating antigen and Bio-pAb-DEP were determined using a checkerboard assay. In this immunoassay, different blocking solutions, such as gelatin, OVA, skimmed milk powder (SMP), PEG 20,000, and polyvinyl alcohol (PVA), were dissolved in PBS and their background values were compared. Otherwise, the effects of different ionic strengths, pH in PBS buffer, concentrations of SA-HRP, incubation time, and solvent matrix effects were determined from the DEP standards and the relative antibody titers in PBS. All determinations were performed repeat eight times and the mean absorbance values were calculated. The IC 50 and the maximum absorbance (A max ) were used to assess the optimum conditions for the assays (Hao et al., 2009).
As the primary influencing factor, the optimum concentrations for Bio-pAb-DEP and the coating antigen were used to improve the sensitivity of the immunoassay. According to checkerboard titration, the optimum reagent concentrations were those that resulted in the maximum absorbance (A 0 ) of approximately 1.0 and the lowest antibody and coating antigen concentrations. The optimal concentrations of OVA-DEP was 8.38 μg·mL −1 and Bio-pAb-DEP was at 1:200 dilution (4.86 μg·mL −1 ) (see Table 1).
Given that blocking is advantageous to eliminate unoccupied sites on the tubes, so different blocking solutions, such as gelatin (0.1%, 0.5%, and 1%), 1% OVA, 1% SMP, 1% PEG20,000, 1% PVA in PBS were compared (shown in Figure 2a). An optimum blocking reagent should achieve the minimal background interference, i.e. the lowest  Figure 2b). A 0max decreased from 1.339 to 0.453 with increasing salt concentration. Salt concentrations lightly affected the sensitivity, with the IC 50 ranging from 0.513 μg·L −1 to 6.846 μg·L −1 . The lowest IC 50 (0.513 μg·L −1 ) was obtained at an ionic strength of 0.10 mol·L −1 . Hence, a salt concentration of 0.10 mol·L −1 was selected for the buffer in the competition assay. The antibody-antigen binding reaction is under a dynamic balance, so this reaction is characterized by weak intermolecular bonds, and is easily affected by pH. So, the pH of optimum assay buffer was adjusted to 5.00-9.00. It was found that the pH had an insignificant effect on the sensitivity of the assay (shown in Figure 2c). The A 0max values decreased with increasing pH, the IC 50 and A 0max varied in the ranges of 0.508-30.794 μg·L −1 and 0.666-1.313 A.U., respectively. The best combination of IC 50 and A 0max (IC 50 = 0.508 μg·L −1 , A 0max = 1.052), was obtained at pH 7.40. Thus, pH 7.40 was used in the competitive immunoassay.
In addition, the dilutions of SA-HRP (500, 1000, 1500, 2000, and 3000) were investigated, and then the dilution of 1000 was determined (IC 50 = 0.496 μg·L −1 , A 0max = 1.050, shown in Figure 2d). Furthermore, immunoassay performance was also determined by different incubation time (incubation times ranging from 15 to 90 min). From Figure 2e, we can see that although A 0max value increased with incubation time increasing, the lowest IC 50 (0.475 μg·L −1 ) was obtained at 60 min. Hence, an incubation time of 60 min was selected for the competitive reaction between antigen and antibody.
Considering that different organic solvents' concentrations make a difference in the matrix effect about resulting interference, so four water miscible organic solvents (DMSO, methanol, acetone, and ethanol) were added into immunoassay system (shown in Figure 2f). These results indicated that lower amounts of organic solvent (<5%) negatively affected the performance of the assay, and the PBS solution containing 5% DMSO (v/v) which obtained the lowest IC 50 value 0.452, was used to improve the analyte solubility in the future assay.
Otherwise, the stability of the proposed BA-ELISA was determined by running the BA-ELISA procedures for eight individual times with seven concentrations of DEP (0.05 μg·L −1 , 0.1 μg·L −1 , 0.5 μg·L −1 , 1 μg·L −1 , 2.5 μg·L −1 , 5 μg·L −1 ). The intra-assay repeatability was estimated after performing over 10 replicates, and inter-assay repeatability was evaluated over several weeks. And the results of variation coefficient (CV) were shown in Table 2. The intra-assay CV was <10.55% and the inter-assay CV was <12.67%, indicating the good stability and reproducibility of the proposed BA-ELISA.

Specificity of BA-ELISA
The specificity of immunoassay can be generally evaluated in the ability of the antibodies to combine with only the target molecule, i.e. CR indirectly. The CR values were evaluated using some similar structure analogs about DEP, such as DMP, DPrP, DBP, DIBP, DEHP, DINP, 4-DENP, and 4-DEAP, were calculated using Equation (3). The chemical structures of these analogs and the CR results were shown in Table 3. Although DEP derivatives 4-DENP and 4-DEAP showed higher CR values (i.e. 14.52% and 15.89%, respectively), 4-DENP and 4-DEAP are not present in wine samples (such as foodstuff, water and soil, etc.). In all cases, there was a low CRs (below 5%) between DEP and other structurally similar compounds, indicated that the pAb-DEP exhibited high affinity and were suitable for the specific determination of DEP at low levels.

Determination of DEP in wine samples and recovery tests
The proposed BA-ELISA was used to detect DEP residues in wine samples collected from Auchan (China) Investment Co. Ltd. in Shanghai, China. DEP was found in all the samples, and the concentrations ranged from 5.93 ± 0.068 μg·L −1 to 59.15 ± 0.099 μg·L −1 ( Table 4). The concentrations of DEP in white liquor were much higher than beer samples. It may be caused by the migration behavior of PAEs during the longer fermentation and storage period. Besides, these samples were also tested on GC-MS to evaluate the precision of BA-ELISA: consistency (linear regression equation Y = 0.81X + 0.76, R 2 = 0.9748, Figure 4) was observed between the two methods. This indicated that the BA-ELISA could offer a practical approach for screening of DEP in real samples. The p value from the paired sample t test for the comparisons of the two methods was 0.048. That is mean, at the 0.05 level, the difference was statistically significant. In a general, the BA-ELISA results were slightly higher than the GC-MS results. The difference may be caused by the nonspecific absorbance of reagents used in the method, including Bio-pAb-DEP and SA-HRP. In addition, pAb had CR for other PAEs present in the samples, which contributed to the BA-ELISA-derived concentrations.
The recovery of the spiked samples and the CV were calculated to evaluate the accuracy and precision of BA-ELISA. Three samples (samples B1 and W1) were spiked with DEP standard concentrations ranging from 0.05 to 100 μg·L -1 before extraction. Moreover, the spiked samples were treated as described before, and then were tested six times using BA-ELISA and GC-MS for comparison. Table 5 showed that the average recoveries of BA-ELISA were ranged from 89.19% to 112.33%, the CV was 5.81-9.43% (below 15%). Meanwhile, GC-MS showed recovery rates of 92.61-107.25% and CVs of 2.69-5.50%.

Conclusions
This study first developed a highly sensitive and effective indirect competitive BA-ELISA for the rapid detection of DEP in wine samples on the basis of specific pAb-DEP. Several physicochemical factors that influenced the performance of proposed BA-ELISA were studied and optimized. Under optimized conditions, the IC 50 value and the LOD of the assay were 0.443 μg·L -1 and 0.0079 μg·L −1 , respectively. This established BA-ELISA could selectively determinate DEP against a number of structural analogs, with negligible CR below 5%. The BA-ELISA was used to detect the presence of DEP in wine samples, and satisfactory recoveries (89.19-112.33%) and CVs (5.81-9.43%) were achieved for DEP from the spiked samples. Meanwhile, compared with GC-MS, a satisfied correlation coefficient of 0.9748 was obtained by practical sample detection. These results confirmed that this method would be a useful option for the sensitive and selective detection of DEP in real wine samples.


CHARACTERIZATION OF ANTIFUNGAL ACTIVITY OF THE VARIOUS ACTIVE CONSTITUENTS OF COLEUS

The aim of this study was to evaluate the anti-fungal effect of different solvent extracts of Coleus forskohlii which contains minor diterpenoids, deacetylforskohlin, 9-deoxyforskohlin, 1,9-deoxyforskohlin, 1,9-dideoxy-7-deacetylforskohlin, and four other diterpenoids, along with alkaloids and volatile oils. The solvent extracts included both polarized and non-polarized solvents like methanol, ethylacetate, benzene, chloroform, water, hexane, acetone and ethanol. As a result of this study, the antifungal effect was best observed with the methanolic extract against Candida albicans in comparison to the other extracts. This study also revealed that apart from forskohlin, which was confirmed by Thin Layer Chromatography, other compounds that were extracted with methanol are required to produce the antifungal effect and can be taken forward to pharmaceutical preparations.

INTRODUCTION
Coleus forskohlii also known as Plectranthus barbatus (Lamiaceae) is one of the most significant potential medicinal crops of the future, as its pharmacopoeial properties have been discovered only recently. The tuberous roots have been identified as a rich drug for glaucoma, congestive, cardiomyopathy, asthma and certain cancers. Forskolin primary mode of action is to increase cyclic adenosine monophosphate (cAMP) and cAMP mediated functions, via activation of the enzyme adenylatecyclase (Metzger,et al 1981).Forskolin has been shown to increase cAMP formation in all eukaryotic cells except sperm, without hormonal activation of adenylatecyclase (Seamon, et al1981).
Forskolin has been shown to enhance lipolysis, regulated by cAMP, also inhibits fat storage. Forskolin is responsible for virtually all pharmacological activities attributed to Coleus forskohlii and the extracts of this constituent have been used in nearly all existing studies though other plant constituents, such as volatile oils and other diterpenoids and coleonols, contribute to the pharmacological activity and adsorption of forskolin.
Candida albicans causes Candidiasis which is an increasingly important disease that has a worldwide distribution due to the fact that it is a frequent opportunistic pathogen in patients. It is a fungus normally present on the skin and in mucous membrane such as vagina, mouth or rectum. The immune system keeps Candida proliferation under control, but when immune responses are weakened, Candida growth can precede unhindered. The uncontrolled growth of Candida is called Candida over growth. Solid media is generally employed for fungal culture, as the broths are not usually recommended except for fungal blood cultures were bi-phasic medium is used. The media commonly employed is Emmon's modification of Sabouraud dextrose agar. The media may be supplemented with antibiotics, such as gentamicin and chloramphenicol to minimize bacterial contamination and cyclohexamide to inhibit saprobic fungi (Jagdishchander, 1999).
The study of Senff et al., (1990) showed the presence of Candida albicans in samples with scalp psoriasis or seborrhoic dermatitis of the scalp (psoriasis capillitii). Yeasts were found in half of the tongue cultures and in 1/8th of the scalp scales. Further proof that Candida is one of the causes of psoriasis was seen in the study analyzed in more than 40,000 patients which showed that patients with atopic dermatitis and psoriasis had elevated levels of Candida in the intestines (Henseler et al., 1997).The analysis showed that Candida colonization in the digestive tract can be one of the causes of psoriasis.

Collection of raw drugs
The plants of Coleus forskohlii was collected randomly from Tamil Nadu Agricultural University (TNAU) Coimbatore, Coimbatore and Salem regions. The taxonomic identities of these plants were confirmed at Botanical survey of India and the voucher specimen numbers of the plants were preserved. Fresh plant material was washed under running tap water, air dried and then homogenized to fine powder and stored in airtight bottles.

Extraction procedure
3g of air-dried powder was extracted in water and all the solvents in Bio Sox apparatus and then kept on a rotary shaker at 190-220 rpm for 2h. It was then filtered through 8 layers of muslin cloth and centrifuged at5000g for 10 min. The supernatant was collected. This procedure was repeated twice. The supernatant collected was pooled together and concentrated by evaporating in a vacuum drier to make the final volume one fourth of the original volume and stored at room temperature in airtight bottles.

Fungal Strains
In vitro antimicrobial activity was examined for aqueous and solvent extracts of Coleus forskohlii. The culture of Candida albicans was obtained from the PSG Institute of Medical Sciences and Research, Coimbatore (ATCC11778) and maintained at 4° C on nutrient agar slants.
The nutrient broth and nutrient agar were prepared as per the standard composition.

Method
Nutrient broth was prepared as per the standard composition and consisted of peptone 5 gm, NaCl 5 gm, Beef extract 3 gm, yeast extract 2 gm, pH 6.8 per 1 litre. Nutrient Agar-Peptone 5 gm, NaCl 5 gm, Beef extract 3 gm, Yeast extract 2 gm, Agar 20 gm, pH6.8 per 1 litre. Nutrient broth prepared for each culture and was sterilized in an autoclave at 1210C for 15 minutes. After sterilization the drug, different concentrations of the extracts were added. Further the cultures Candida sps. was inoculated in the tubes and subcultured weekly. Control sample was prepared along with culture in the absence of the drug. Only medium served as blank.

Media preparation and antifungal activity
The antimicrobial assay was performed by agar well diffusion method for all the extracts. The molten Mueller Hinton agar was inoculated with 100 µl of the inoculum (1 x 10 8 cfu/ml) and poured into the Petri plate (Hi-media). For agar well diffusion method, a well was prepared in the plates with the help of a cork-borer (0.85 cm). Different concentrations of the test compound were introduced into the well. The plates were incubated overnight at 37°C. Microbial growth was determined by measuring the diameter of zone of inhibition. Controls were maintained where pure solvents were used instead of the extract. The result was obtained by measuring the zone diameter. The experiment was done in triplicate and the mean values recorded.

RESULTS AND DISCUSSION
In the present study the different solvent extracts of Coleus forskohlii (methanol, ethyl acetate, benzene, chloroform, water, hexane, ethanol and acetone) at different concentrations 75µg and 150 µg was tested for its antimicrobial property against the growth of Candida albicans. The zone of inhibition observed after 72 hrs showed potent activity at 150µg of the methanol extract (20 mm) and benzene extract (13mm) and water (12mm), whereas with chloroform it was 6mm and other solvents like acetone, ethylacetate , ethanol , hexane did not reveal any zone of inhibition (Table 1 and Figure 1).
The anti microbial activity of plant oils and extracts has been recognized for many years and the significance of the microbial activity of oils and extracts of plants against Candida albicans, Enterococcus faecalis and E. coli has been reported. The oil of lemon grass, oregano and bay leaves were observed to inhibit the growth of most microorganisms (Naveens, 2008). The plant extracts, as methanolic extracts of Peltophorum pterocarpum and Punica granatum exhibited highest antifungal activity against Candida albicans (Duraipanidyan et al., 2006).
The above results imply that the compounds from Coleus forskohlii that was extracted into methanol had the property to inhibit the growth of Candida albicans compared to the other solvents extracts. There was no antagonistic effect on Candida albicans by the Coleus forskohlii extracted in chloroform, petroleum ether and other solvents. However, maximum inhibition was observed with the reference drug clopidogriel, where as DMSO, used as vehicle control showed no inhibitory effect. The studies of screening results show a correlation with the antibiotic uses of the plant and the active compounds present in extracts that may be responsible for the antimicrobial activity.

CONCLUSION
In the present study, methanolic Coleus forskohlii extracts exhibited high level of antifungal activity against Candida albicans.They have an excellent safety profile and are generally without toxicity or side effects at the recommended dosage. In recent years, secondary plant metabolites (phytochemicals), previously with unknown pharmacological activities, have been extensively investigated as a source of medicinal agents. Thus, it is anticipated that phytochemicals with adequate efficacy can be used for the treatment and prevention of various ailments. The potential for developing therapeutic drugs from higher plants appears rewarding as it will lead to the development of a phytomedicine to act against safely in humans. Plant-based drugs have enormous therapeutic potential as they can serve the purpose with lesser side effects that are often associated with synthetic drugs. Continued further exploration of plantderived products is needed today.
Further research is necessary to determine the identity of the compounds from within these plants and also to determine their full spectrum of efficacy. However, the present study of in vitro evaluation of Coleus forskohlii forms a primary platform for further phytochemical and pharmacological studies. Table 1 plus PGE1 or 2-fluoroadenosine: effects of 2',5'dideoxyadenosine and 5'-methylthioadenosine. Biochem. Pharmacol. 31: 3713-3716 Buslau, M., I. Menzel and H. Holzmann, (1990).
Regulation of dog thyroid epithelial cell cycle by forskolin, and adenylate cyclase activator. Exp. Chloroform -5