extfieldsofstudy: Medicine Chemistry

text:
Onion (Allium cepa L.) Skin: A Rich Resource of Biomolecules for the Sustainable Production of Colored Biofunctional Textiles

The aqueous extract of dry onion skin waste from the ‘Dorata di Parma’ cultivar was tested as a new source of biomolecules for the production of colored and biofunctional wool yarns, through environmentally friendly dyeing procedures. Specific attention was paid to the antioxidant and UV protection properties of the resulting textiles. On the basis of spectrophotometric and mass spectrometry analyses, the obtained deep red-brown color was assigned to quercetin and its glycoside derivatives. The Folin–Ciocalteu method revealed good phenol uptakes on the wool fiber (higher than 27% for the textile after the first dyeing cycle), with respect to the original total content estimated in the water extract (78.50 ± 2.49 mg equivalent gallic acid/g onion skin). The manufactured materials showed remarkable antioxidant activity and ability to protect human skin against lipid peroxidation following UV radiation: 7.65 ± 1.43 (FRAP assay) and 13.60 (ORAC assay) mg equivalent trolox/g textile; lipid peroxidation inhibition up to 89.37%. This photoprotective and antioxidant activity were therefore ascribed to the polyphenol pool contained in the outer dried gold skins of onion. It is worth noting that citofluorimetric analysis demonstrated that the aqueous extract does not have a significative influence on cell viability, neither is capable of inducing a proapoptotic effect.

Introduction
Biofunctional textiles, also known as 'biomedical textiles', and most frequently as 'cosmetotextiles' are opportunely engineered materials able to exert one or more beneficial healthy effects on human skin. Such 'intelligent' textiles can be actually regarded as an efficient delivery system for cosmetic or pharmaceutically-relevant substances through their contact with the skin [1].
The use of fabrics and garments as a tool for a powerful delivery of healthy substances on human skin is an old concept belonging to the philosophy of the Ayurvedic medicine, known as Ayurvastra: an ancient discipline elucidating the beneficial properties of extracts of natural herbs [2].
Over the last decade, functional textiles have tremendously evolved thanks to the active cooperation of chemists, physicians, and textile engineers. Indeed, in parallel with the improvement of the already well-established doping and coating procedures, methods based on microencapsulation and grafting technologies have gained increasing popularity in the production process of these products, ensuring enhanced quality of the resulting vehicles [1,[3][4][5].
In recent years, innovative 'biofunctional' applications have been receiving attention in the field of cosmetotextile technology, mostly to meet the needs of people with highly sensitive skin. These kinds of material are indeed becoming diffusely appreciated as an innovative vehicle for the local treatment of skin diseases caused by microbial agents [6,7] or sunlight induced photooxidation [5,8].
Aimed at limiting the toxicity and the high environmental impact of many synthetic molecules often used by the biofunctional textile manufacturers [6], extracts of vegetal and animal origin are becoming increasingly preferred even by big companies, as a natural source of biomolecules for their production. Indeed, it has been widely demonstrated that antioxidants from natural sources can sensitively attenuate the effects of reactive oxygen species (ROS), sometimes even more efficiently than endogenous antioxidants [9,10].
Flavonoids and phenolic acids are among the most important chemical classes exhibiting antioxidant activity, mostly via radical-scavenging mechanisms [8,11]. This evidence along with the their strong interactions with fiber material has fueled the interest by scientists towards the functionalization of textiles by these molecules [8,12,13]. In this context, natural dyes, extracted from various species of wild plants or crops, are particularly appreciated because, in addition to the aforementioned beneficial biofunctional properties, they are also able to confer peculiar soft colors with a unique shade. A precious source of colored bioactive molecules is represented by biomasses, which constitute waste material from agricultural and food processing industries. This practice is in perfect line with the "circular economy" fundamentals [14].
Among others, the dried outer skin of onions (Allium cepa L.), representing the waste of onion consumption and processing, contains a multitude of colored bioactive phenolics, some of which are absent in the edible part of the vegetable [15][16][17]. In depth investigations at a molecular level have revealed that quercetin and its glucosides are the most abundant phenolics in onion outer dry layers, which explains the strong antioxidant effects of the resulting extracts [18][19][20].
In this paper, the waste of onion skin from the 'Dorata di Parma' cultivar was used as a new source of bioactive compounds to produce wool yarn with antioxidant and UV-protection properties. Besides its functional potential, this kind of biomass was chosen by the authors for many other reasons. First of all, onion is the second most important horticultural crop after tomatoes with a worldwide current production of around 98 million tons [21], making its related by-products widespread and easily available. Furthermore, onion waste is an easy handling biomass because of its low weight and dryness state, and has been historically used in Europe and Arab countries for dyeing textiles with deep yellow and orange nuances [22]. The exploitation of this biomass for the production of biofunctional textiles therefore represents an intriguing link between tradition and innovation.

Results and Discussion
Biomolecules from dried onion skins were extracted through a traditional boiling in H 2 O (see Material and Methods section for details) and readily used for dyeing scoured wool skeins in two successive dyeing cycles and using two different percentage (100% and 75%) of onion skin with respect to the dry wool weight (Figure 1). The use of chemicals was deliberately avoided in both the extraction and dyeing processes. Instead, the application of mordants was intentionally circumvented in order to favor the slow release of molecules from the fibers once they are in contact with the human skin. A multi-analytical approach, based on chromatographic techniques and a variety of conventional and reflectance UV-vis spectroscopic methodologies, was utilized to characterize the optical properties, the chemical composition and the biofunctional behavior of the aqueous extract and textiles. Moreover, the water extract was also tested in a cell-based assay to verify its safety and exclude any toxicity, such as the induction of proapoptotic effects.

Optical Properties of Skin Extracts and Cosmeto-Textiles
The absorption spectrum of the aqueous onion skin extract (Figure 2a) shows the typical flavonoid features (Scheme 1) consisting of a band with maximum at 360 nm (Band I), assignable to the cinnamoyl system formed by B and C rings, a weak signal at 290 nm (Band II) attributed to the A and C ring benzoyl moiety and a band centered at 250 nm ascribable to the C ring [23]. A further band is observed at about 495 nm, probably due to the formation of flavonoid aggregates or to flavonoid degradation products. In particular, the absorption profile corresponds well with that of quercetin, as expected being quercetin and their glycoside derivatives the main flavonoid components found in outer dry onion skin [24]. This result is confirmed by the HPLC-MS and MS 2 analysis (Section 2.2). The absorption maxima retrieved on all textile samples are slightly shifted to longer wavelengths with respect to those observed for the aqueous extract (Figure 2b), likely because of a specific interaction of flavonoid dyes with wool. The samples dyed with the original onion skin water extract (T1 and T3) showed a deep red-brown color, whereas those dyed in the residue dyeing solution (T2 and T4) exhibited a pale brown color, as indicated by positive values of a * and b * colorimetric coordinates ( Table 1). The color ( Figure 1) is mainly determined by the tail of the flavonoid absorption Band I, centered at 380 nm, and the bathochromic unidentified component at 510 nm (Figure 2b). Lower absorbance and colorimetric index values are obtained for samples T2 and T4, that were dyed with the residue solution used to prepare samples T1 and T3, respectively, indicating a lower dye uptake on wool fibers. This result was anticipated by the drastic decrease of absorbance detected for the residue dyeing solution (Figure 2a). On the other hand, no sensitive variation of color parameters and absorption features were observed when the onion waste amount was reduced from 100% to 75% with respect to the wool weight. This indicates that the dye accumulation on wool fibers is equivalent. Very interestingly, the spectra of all textile samples, even those dyed in a less concentrated onion skin extract (T3), showed absorption values higher than the untreated wool in all the UV-vis spectral range including the UVa and UVb (Figure 2b). This finding highlights the potential protection function of the prepared textile from the photo-oxidative damage caused by UV radiations on human cell skin as already reported for other natural dyes [25,26].    L * for the lightness and a * and b * for the green-red and blue-yellow color components, respectively.

Metabolite Profiling of Polyphenols in Onion Extracts
Several flavonols have been described in onions, with quercetin derivatives being the most abundant ones [27]. The glycosyl moiety of these quercetin derivatives is almost exclusively glucose, which is linked to the 4 , 3, and/or 7-positions of the aglycone [27]. In particular, quercetin-4 -glucoside and quercetin 3,4 -diglucoside have been frequently found in the literature as the main flavonols present in onion extracts [27]. Derivatives of kaempferol and isorhamnetin have also been detected as minor flavonols [27]. Some studies have reported that small yellow onion bulbs and aerial parts contain several phenolic acids [28]. Red onion anthocyanins are mainly cyanidin glucosides acylated with malonic acid or non-acylated [27]. Some of these compounds present unique structural features, like 4 -glycosylation and unusual substitution patterns of sugar moieties [27].
As regards the extracts from the 'Dorata di Parma' onion skin analyzed in this work, UV-vis data, together with MS and MS 2 , allowed us to identify 11 compounds in the aqueous extract, belonging to phenolic acids and flavonols ( Table 2). The glycosylation sites of quercetin glucosides and diglucosides was tentatively assigned in this study on the basis of the literature data [24,[29][30][31]. Protocatechuic acid was found to be the main phenolic acid, while quercetin-4 -glucoside and quercetin were the major peaks for flavonols ( Figure 3a). The same trend was observed for the chromatograms obtained for the residues after dyeing (Figure 3b). A previous work on onion solid wastes has shown a similar phenolic profile [17].
However, many studies have described quercetin 3,4 -diglucoside and quercetin 4 -glucoside as the main flavonols present in onion extracts [24,[29][30][31]. The different chromatographic profiles obtained in the present work for onion extract and residues after dyeing may be attributed to the hydrolysis of quercetin 3,4 -diglucoside and quercetin 4 -glucoside, due to the high temperature applied during the extraction and dyeing procedures in the aqueous medium. As regards protocatechuic acid, it represents the main oxidation product of quercetin in the presence of air, as previously described in the literature [32], and it is therefore a product of quercetin decomposition under the conditions applied during the extraction. Protocatechuic acid is produced by the oxidation of quercetin during the process of browning [33,34]; it can also be further oxidized to protocatechuquinone [35]. Protocatechuic acid has been found in the outer scales of yellow onion bulbs, where it is known to exert an antifungal activity [33]. It should be specified that the release of quercetin from its glucosides by hydrolysis has been described to proceed faster than quercetin oxidative decomposition in onion solid wastes [17].    Table 2.

Measurement of Total Phenolic Content (TPC)
The amount of total phenolic compounds was determined spectrophotometrically by applying the original Folin-Ciocalteu method with only few modifications [36].
In the present study, the Folin-Ciocalteu method was applied both to the diluted dry onion skin extract and to the hydro-alcoholic solutions containing the differently treated textiles (Table 3). In order to simulate as closely as possible the delivery process of phenols to human skin, the assay for textiles was performed on the intact fiber, thus avoiding aggressive treatments which can favor an extreme and disproportionate biomolecule release. The test was also performed on the untreated wool (UW), and the result obtained was in agreement with the well documented, nonspecific, oxidation ability of the Folin-Ciocalteu reagent towards non-phenolic organic as well as inorganic substances [37,38]. Indeed, it has been demonstrated that reducing agents, including certain amino acids, can interfere with the spectrophotometric assay, leading to an overestimation of the phenolic content [37,38]. We assume this value (referred to as sample UW in Table 3) originated from specific amino acids sequences embedded in the keratin wool [39]. However, this is not a serious concern in the present study, since the same test was performed for comparative purposes on differently treated textiles.
In line with our expectations, the phenol loading abruptly decreased in successive dying cycles (samples T1 and T2, Table 3). This result unambiguously finds its roots in the lower total phenol content in the dying bath after the first dying cycle. Accordingly, we estimated that about 40% (30.85 ± 0.57 mg Eq. GA/g onion skin; with GA standing for gallic acid) of the initial phenol content was still present in solution after the first dipping step. This evidence is further supported by the consistent decrease of absorbance of the flavonoid feature (360 nm) observed in the dyeing solution after the first dyeing step (Figure 2a), that consequently led to a lower dye uptake (Figure 2b) for the sample within it dyed (T2, second dyeing cycle). Interestingly, we found that using a slightly lower amount of onion skin in the preparation of the dying solution (75% of that previously described) did not significantly alter the entity of adsorbed phenolic species onto the wool textile (samples T3 and T4, Table 3). This observation is consistent with the almost identical colorimetric coordinate values (Table 1) and absorption spectra (data not shown) of T1 and T3 samples.

Determination of Total Antioxidant Capacity (TAC) of Onion Skin Extracts and Biofunctional Textiles
A single method to univocally determine the TAC of a plant extract does not actually exist. Moreover, although numerous methods are currently in use for TAC estimation, standardized protocols are still far from being officially approved. The availability of official protocols is furthermore complicated by the common practice of researchers to adapt, case-by-case, procedures already developed by other authors for specific applications. In the study of complex matrices, it is of crucial importance for researchers to take in mind that every method is selective for specific antioxidant families and reactions [40]. Therefore, in order to evaluate the TAC of a phytocomplex, the results from different methods have to be cumulatively considered. In this context, both UV-vis spectrophotometric and spectrofluorimetric-based assays can provide important complementary information. Accordingly, in the present study, TAC was appraised on the same samples by relying upon the ferric reducing antioxidant power (FRAP) spectrophotometric method as well as the spectrofluorimetric-based oxygen radical absorbance capacity (ORAC) assay.
Basically, two different reaction mechanisms can be exploited for TAC measurements: one based on a hydrogen atom transfer (HAT) process, the other on a single electron transfer (ET) [38,41].
It is worth highlighting that some polyphenol sub-families respond better to HAT-based assays, while others respond better to ET-based ones.
ORAC is a 'pure' HAT-based method that measures the ability of an antioxidant (or a mixture of antioxidants) to quench free radicals by means of hydrogen donation. In the ORAC assay, the pool of antioxidants in the phytocomplex and the selected substrate (i.e., fluorescein in the present study) compete for thermally generated peroxyl radicals [37,42]. The peroxyl radical is generated by thermal decomposition of 2,2 -azobis(2-methylpropionamidine)dihydrochloride (AAPH) and preferably abstracts a H · from the antioxidants in the phytocomplex [41]. As a consequence, its reaction with the fluorescein probe occurs later. Peroxyl radicals decrease the fluorescence of the probe, and the decrease constant for its fluorescence decay profile is used to measure the antioxidant capacity of the sample under investigation [37,[41][42][43][44]. Unlike many methods for TAC determinations (including FRAP), the ORAC assay holds the big advantage to mimic physiologically relevant perturbations.
Previous studies by other authors have clearly highlighted a distinct difference in the ORAC values from hydrophilic and lipophilic extracts of fruits and vegetables [43,44]. Most of the time, hydrophilic ORAC (H-ORAC) values were higher than those generated by lipophilic extracts (lipophilic ORAC, L-ORAC) [43,44].
Contrary to ORAC, the FRAP method is exclusively based on an ET mechanism. Indeed, FRAP assay measures the ability of antioxidants to reduce the Fe 3+ -TPTZ (with TPTZ standing for 2,4,6-tris(2-pyridyl)-s-triazine) complex to its ferrous form (Fe 2+ -TPTZ) at low pH [37]. It has been reported that the reducing power of polyphenols measured through the FRAP test is somehow related to the degree of hydroxylation and to the extent of conjugation in their molecular structure [38,45].
As a consequence of its underlying mechanism, FRAP cannot be used for the analysis of compounds acting as radical quenchers via HAT process. However, this is only an apparent drawback of the method, since, in combination with other assays, it can be very useful in distinguishing dominant mechanisms by different antioxidants [37], and, in turn, establishing the molecular species contributing to TAC. Instead, the absence of an oxidable substrate in the FRAP analysis is a serious limitation of the method since it does not allow evaluating the potential protective properties of the antioxidant pool in the extract. From data listed in Table 3, a strict correlation (R 2 = 0.88) between data from the FRAP and ORAC assays readily comes to light. These results unambiguously suggest that the chemical nature of the functional molecules in the onion extract does not influence the degree of adsorption onto the wool fiber. A remarkably significant (R 2 = 0.98) relationship between results from FRAP and Folin-Ciocalteu tests also emerges, thus supporting the opinion of some authors [46] that the former method is actually applicable to measure the total antioxidant concentration of a mixture. On the other hand, although the Folin-Ciocalteu assay is diffusely used to measure the total phenolics in natural products, its basic mechanism relies upon an ET process and, therefore, it can be considered another method for TAC estimation. The conventional Folin-Ciocalteu reagent has proven to be only applicable to water-soluble antioxidants [37,38].
Trying to summarize, on the basis of all the above, one can state that the higher the loading of the phenol constituents onto the textile, the greater the TAC of the adsorbed extract.

Evaluation of TPC on Strip after Percutaneous Absorption
The Folin-Ciocalteu assay was applied for the evaluation of the amount of phenolic species transferred from textiles to skin. Biofunctional textiles containing phenol species were fixed on forearms of two volunteers (named to as A and B) for periods of 12 and 24 h. The outermost layers of the stratum corneum (SC) were then obtained using strips.
The results obtained, expressed as concentration (mg Eq GA) are shown in the plot in Figure 4. The bar couples related to biofunctional textiles (i.e., 3 and 5 in Figure 4) are always higher than those corresponding to controls (i.e., 1, 2, and 4). This result further demonstrates that phenols are able to efficiently pass from textile to skin.

Determination of Lipoperoxidation by 2-thiobarbituric acid (TBA) Assay
Skin lipids can undergo oxidative damage as to UV ray exposure. The entity of lipid peroxidation can be evaluated in several ways, including the quantitative analysis of end products of the photooxidative process. Malonaldehyde bis(dimethyl acetal) (MDA) is one of such end products, and its quantification as thiobarbituric derivative is often used to quantify the extent of lipid peroxidation in an injured tissue [47].
The reaction between TBA and MDA yields an intensely colored chromogen fluorescent red adduct [48] that can be easily analyzed spectrophotometrically.
In this work, the strips were irradiated with UV light spectrally simulating solar radiation, and lipid peroxides (LPOs) removed from the SC were measured by TBA assay. The results obtained with TBA test were expressed as MDA equivalents (µM MDA) using a regression curve for pure MDA-TBA complexes. As indicated by the elevated R 2 value (>0.98), the method is significantly linear within a rather broad concentration range, namely, 0.05-1 µM of MDA.
In Figure 5 the values expressed as concentration of MDA (µM) obtained for strips irradiated with UV radiation are shown. Comparing the results obtained for control-textiles (UW) and biofunctional textiles (T1), a lower concentration of MDA was always detected after the application of the treated textiles.
The results obtained in the present study clearly indicate that a certain quantity of antioxidant has been released from biofunctional textiles to SC. Moreover, these compounds were able to protect skin against lipid peroxidation following UV radiation. This finding agrees with the greatest capacity (highest absorbance values) of dyed yarns to absorb UVa and UVb radiation with respect to untreated wool ( Figure 2b). As evident from data in Table 4, the obtained values of the LPO inhibition (%) were quite similar for both volunteers. Being aware of the limited number of recruited volunteers, we have here highlighted the potential of the produced material as biofunctional textiles.  Table 4. LPO values determined using strip irradiated at 1 × 10 5 µW/cm 2 for 45 min. % LPO inhibition mean and standard deviation are reported. The meaning of P 0 , P x , A 0 , and A x is explained in Section 3.11.

Analysis of Cell Viability and Induction of Apoptosis after Treatment of RAW 264.7 Cells with Onion Aqueous Extract
The safety of the aqueous extract from onion skin was investigated in RAW 264.7 cells, a macrophage cell line commonly used to assess biologically active compounds for their toxicity and/or effects on inflammatory processes and immune responses. Macrophages were untreated or treated for 24 h with a single dose of onion extract, corresponding to 80 µg Eq. of GA/mL. Cell viability and induction of apoptosis were then assessed by citofluorimetric analysis, after staining the cells with a fluorochrome conjugated Annexin V, a protein commonly used to detect apoptotic cells. Annexin V is an endogenous human protein endowed with a high affinity for membrane-bound phosphatidylserine (PS). The number of annexin V binding sites per cell with the onset of apoptosis increases 100-to 1,000-fold during apoptosis. PS exposure on the cell surface occurs before DNA fragmentation, therefore annexin V is a sensitive marker of the early to intermediate phases of apoptosis [49]. The results, shown in Figure 6, demonstrate that, as expected, aqueous extracts of onion skin do not have a significative influence on cell viability and they did not induce a proapoptotic effect, further confirming the safety and the absence of cellular toxic effects of the polyphenolic compounds found in the extract.

Photophysical Measurements
Folin-Ciocalteu as well as FRAP tests were carried out at 25 • C with a Varian Cary 100 (Varian Inc., Palo Alto, CA, USA) dual beam, dual chopper spectrophotometer. Emission intensity for ORAC experiments were determined by a HORIBA Scientific FluoroMax ® -4P spectrofluorimeter (Kyoto, Japan) operated by FluorEssence TM . The instrument provides corrected fluorescence emission spectra taking into account both the monochromator response and detector sensitivity.
Reflectance spectra on solid samples were performed using a portable instrument composed of Avantes parts and equipped with a quartz fiber optic system already described in a previous paper [50]. The reflected light was collected under a 21 • angle respect to the surface, using a 0/0 geometry, thus avoiding the specular reflected light. A 99% Spectralon ® diffuse reflectance standard (Labsphere, North Sutton, NH, USA) was used for calibration. The instrument provides a 8 nm spectral resolution, whereas the fiber optic probe allows a surface area of 2 mm 2 to be analyzed. The reflectance spectra were expressed in terms of pseudoabsorbance, A (λ), according to Equation (1) [51] A (λ) = log [1/(0.01·R(λ))] (1) where R is the measured reflectance at each specific wavelength λ. The color of dyed wool samples was assessed in terms of CIELab values. In the CIELab color space the color is defined by three numerical coordinates: L * for the lightness and a * and b * for the green-red and blue-yellow color components, respectively. The lightness value, L *, ranges from L * = 0 for the darkest black to L * = 100 for the brightest white. The a* coordinate represents the green-red component, with green in the negative direction (a * < 0) and red in the positive direction (a * > 0). The b * coordinate indicates the blue-yellow component, with blue in the negative direction (b * < 0) and yellow in the positive direction (b * > 0). Colorimetric measurements were carried out on a Konica Minolta CM-700d spectrophotometer (Tokyo, Japan) under D65 illuminant and 10 • standard observer. Four measurements were made for each sample recording the percentage reflectance values over the 350-750 nm spectral range.

Extraction of Polyphenols from Dried Outer Onion Skin
A 200 mL volume of H 2 O was added to 2 g of onion skin and the resulting solution was kept under magnetic stirring at 95 • C for 1 h. For an efficient removal of onion skin debris, the extract was filtered through paper filter. Extraction was performed in triplicate. The extracts were stored at −20 • C until further analysis. The same extraction procedure was followed to prepare the dye bath for wool dyeing experiments.

Scouring and Dyeing Procedure
The wool yarn was washed with a Brij ® S10 non-ionic detergent solution (5 gr/L, yarn to liquid ratio 1:100 w/v) at 50 • C for 30 min in order to remove eventual impurities due to the manufacturing processes thus improving its hydrophilicity. The scoured wool was then rinsed several times with H 2 O and air-dried at room temperature for 24 h. This sample will be referred to as untreated wool (UW) along the text.
Wool dyeing was carried out by the exhaustion method using a 1:80 (w/v) yarn to liquid ratio and different percentage of onion skin (100% and 75%) with respect to the dry wool weight. Sample #1 (T1) was obtained by dipping 1 g of scoured wool into 80 mL of onion skin aqueous extract (100% onion skin with respect to the dry wool weight) prepared as described above and previously heated at 40 • C. The temperature was then increased to 95 • C and kept constant for 60 min. The wool was then removed, rinsed several times with H 2 O until the aqueous phase appeared uncolored and air dried in the dark at room temperature. Sample #2 (T2) was obtained by dipping 1 g of scoured wool in the dyeing solution residue previously used for the preparation of T1 and dyed under the same experimental conditions used for T1. Samples #3 (T3) and #4 (T4) were obtained following the same procedure used for T1 and T2 respectively, in a less concentrated onion skin aqueous extract (75% onion skin with respect to the dry wool weight). For each sample, the dyed wool yarn was carefully rolled around a Plexiglas sheet in order to have a 3 × 1 cm skein with a flat and homogeneous surface therefore adequate for in situ reflectance and colorimetric measurements.

HPLC-UV/DAD Analysis of Polyphenols
HPLC analyses were performed on an Agilent Technologies (Waldbronn, Germany) modular model 1100 system, consisting of a vacuum degasser, a quaternary pump, an autosampler, a thermostated column compartment and a diode array detector (DAD). The chromatograms were recorded using an Agilent Chemstation for LC and LC-MS systems (Rev. B.01.03). The HPLC analysis of phenolic acids and flavonols was carried out on an Ascentis Express C 18 column (150 × 4.6 mm I.D., 2.7 µm, Supelco, Bellefonte, PA, USA). The mobile phase was composed of 0.1% HCOOH (v/v) in both (A) H 2 O and (B) ACN. The gradient elution was modified as follows: 0-5 min 3% B, 5-45 min from 3% to 50% B, 45-60 min from 50% to 100% B, 60-80 min 100% B. The post-running time was 10 min. The flow rate was 0.8 mL/min. The column temperature was set at 25 • C. The sample injection volume was 10 µL. The UV/DAD chromatograms were acquired at 254, 330, 350, and 520 nm. Three injections were performed for each sample.

HPLC-ESI-MS and MS 2 Analysis of Polyphenols
HPLC-ESI-MS and MS 2 analyses were carried out using an Agilent Technologies modular 1200 system (Agilent, Waldbronn, Germany), equipped with a vacuum degasser, a binary pump, a thermostated autosampler, a thermostated column compartment and a 6310A ion trap mass analyzer with an ESI ion source. The HPLC column and the applied chromatographic conditions were the same as reported for the HPLC-UV/DAD system. The flow rate was split 2:1 before the ESI source.
The HPLC-ESI-MS system was operated both in the positive and in the negative ion modes. The experimental parameters were set as follows: the capillary voltage was 3.5 kV, the nebulizer (N 2 ) pressure was 32 psi, the drying gas temperature was 350 • C, the drying gas flow was 11 L/min and the skimmer voltage was 40 V. Data were acquired by Agilent 6300 Series Ion Trap LC/MS system software (version 6.2, Agilent Technologies, Waldbronn, Germany). The mass spectrometer was operated in the full-scan mode in the m/z range 100-1000. MS 2 spectra were automatically performed with helium as the collision gas in the m/z range 50-1000 with the SmartFrag function.

Determination of Total Phenol Content (TPC) by the Folin-Ciocalteu Method
The total phenol content (TPC) of each extract was determined in triplicate according to the Folin-Ciocalteu method described in [36] with only few modifications.
The Folin-Ciocalteu reagent was diluted 10-fold with H 2 O. A definite volume of extract (0.1 mL) was mixed with 0.75 mL of the diluted Folin-Ciocalteu reagent and incubated in the dark for 10 min at room temperature. The term 'extract' is related either to the onion extract 4-fold diluted with H 2 O or to the yarn sample (about 2.0 mg) added to a 0.1 mL of H 2 O/MeOH (1:1, v/v) solution. Then, 0.75 mL of 2% sodium carbonate (w/v) aqueous solution were added. The mixture was kept in the dark for 3 h before measuring the absorbance at 765 nm. The content of total phenolics was determined by using a standard curve prepared with gallic acid (GA) solutions previously treated in the same way as for the real samples. Therefore, results were expressed as mg of GA equivalents/g onion skin and as mg of GA equivalents/g textile.
The amount of phenolic species transferred from textiles to skin was also determined by the Folin-Ciocalteu assay. Each penetration area was stripped twice with two different strips which were then combined and immersed into a beaker containing the Folin-Ciocalteu solution.

Determination of the Total Antioxidant Capacity (TAC) by the FRAP Method
The reducing power was determined according to the method described in [36]. The FRAP reagent was prepared from 2.5 mL of a TPTZ solution (10 mM) in HCl (40 mM) and 2.5 mL of a FeCl 3 aqueous solution (20 mM), mixed with 25 mL of NaOAc (300 mM, pH 3.6). For the determination of the antioxidant activity, 1.5 mL of FRAP reagent were mixed with 100 µL of H 2 O and 100 µL of the sample extract. The term 'extract' is related either to the onion extract 12-fold diluted with H 2 O or the yarn sample (about 2.0 mg) or the strip added to a 0.1 mL of H 2 O/MeOH (1:1, v/v) solution. The reaction mixture was allowed to stand for 4 min at room temperature before measuring the absorbance at 593 nm. Analyses were performed in triplicate for each sample. The total antioxidant capacity (TAC) values were determined from a calibration curve prepared with Trolox standard solutions, previously treated by applying the same procedure as for the real sample. The antioxidant capacity of the sample was expressed as mg of Trolox equivalents/g onion skin and as mg of Trolox equivalents/g textile.

Determination of the Oxygen Radical Absorbance Capacity by the ORAC Method
A slightly modified version of the method developed by Ou et al. was applied in this experiment [52,53]. A fluorescein solution (42 nM in PBS) freshly prepared each day was pre-incubated in a warm water bath (37 • C) for 15 min before measurements. AAPH (153 mM) was prepared daily by solubilizing 400 mg of AAPH in 10 mL PBS. In each cuvette, 2.25 mL of fluorescein and either 375 µL of sample (either the onion extract or 2 mg of fabric sample treated with 5 mL of PBS) or the same volume of blank (PBS) or of the standard solution (Trolox, 6, 12.5, 25, and 50 µM) were placed. Then, 375 µL of AAPH were added. The fluorescence was measured immediately after the AAPH addition and measurements were then made every 5 min until the relative fluorescence intensity was less than 5% of the value of the initial reading. The measurements were made in triplicate. Analyses were carried out on a spectrofluorimeter by fixing the excitation and emission wavelength at 493 nm and 515 nm, respectively. Both excitation and emission slits were set at 2.5 nm.
The ORAC final calculations were made by applying a regression equation built up with Trolox (used as standard) concentrations (x-axis) vs AUC (y-axis) values. AUC represents the area under the fluorescence decay curve, calculated as where, f0 is the initial fluorescence intensity before AAPH addition at time 0, and f5, f10, f15 are the fluorescence intensity at time 5, 10, 15 min after the addition of AAPH. All the obtained AUC values were subtracted of that found blank. Results were expressed as mg of Trolox equivalents/g onion skin and mg of Trolox equivalents/g textile.

Evaluation of the Percutaneous Absorption with the 'Ex Vivo' Stripping Method
The experimental protocol for the percutaneous absorption of antioxidant containing biofunctional textiles was conducted with two healthy Caucasian women volunteers (referred to as A and B in the following sections). The two volunteers, both aged 27 years, refrained to use any kind of cosmetics on their harms 14 days prior to the study. The biofunctional textile (wool dyed with onion skin extract, T1) and control textile (UW) were applied on subjects' forearms by maintaining a close contact with the skin for time frames of 12 and 24 h as a bandage application. In order to avoid any passage of functional molecules from the functionalized textile and the compression bandage, a piece of impermeable paper was placed between the two compartments.
The skin penetration behavior was studied through the application of the stripping method as described in [10,54], with only few modifications. Corneofix ® strips were used for the stripping of the stratum corneum (SC) of each forearm. Each strip was applied to the skin surface and a hand pressure was exerted. After few seconds, the strip was removed in one gentle movement.
In order to compare the condition of SC after application of textiles with that of 'untreated' SC, also the stripping of the latter was evaluated and this sample was indicated as the 'control'. Then the strips were submitted to two assays, i.e., Folin-Ciocalteu assay and TBA assay.
The protocol was approved by the Ethics Committee of Umbria Region and the study was conducted in accordance with the standards of Good Clinical Practice for trials of medicinal products in humans. Written informed consent was obtained.

Determination of Lipoperoxidation by Thiobarbituric Acid (TBA) Assay
In this study, the lipid peroxides (LPO) formed in the strip were measured by TBA assay. The strips were irradiated for 45 min using a light source simulating the UV solar radiation (1 × 10 5 µW/cm 2 ). Afterwards, the strips were immersed into a H 2 O/MeOH (1:1, v/v) solution (0.2 mL). Then, 1 mL of a solution made up with 0.4% (w/v) TBA and 15% (w/v) TCA in 100 mL of HCl solution (0.25 M) was added. The mixture was incubated for 1 h in a boiling water bath before measuring the absorbance at 534 nm with a UV-vis spectrophotometer.
Quantification of MDA-TBA complex was appraised by mean of a calibration curve prepared with MDA standard solutions at different concentrations in the same hydro-organic solvent as before.
The effect of phenol-containing biofunctionaltextiles on skin was also evaluated in terms of % LPO inhibition. This quantity was determined by applying the equation where, Ax and Ao represent the concentration values achieved by analyzing the strips submitted or not to UV irradiation, respectively. In this case, the strips removed the SC previously coated with phenol containing biofunctionaltextiles. Instead, Px and Po represent the concentration values achieved by analyzing the strips submitted or not to UV irradiation, respectively. In this case, the strips removed the SC previously coated with untreated wool. Once again, the subscripts "x" and "o" refer to irradiated or not irradiated sample, respectively. A value equal to 0% indicates that the antioxidant does not protect at all the skin against UV irradiation, while, a 100% of LPO inhibition means that the compounds released from textile have a complete antioxidant capacity.

Analysis of Cellular Viability and Apoptosis
RAW 264.7 cells were seeded into a 12 well plate at a density of 1.2 × 10 6 cells/mL. The aqueous extract from onion skin was added to cells for 24 h at a final dilution of 1:10 (v/v), corresponding to a concentration of 80 µg Eq GA/mL. After the treatment, cells were harvested by trypsinization, washed with PBS and stained with Fixable Viability Dye eFluor ® 780 for 30 min. After washing with PBS and Flow Cytometry Staining Buffer, 5 µL of PerCP-eFluor 710-conjugated Annexin-V were added to cells and incubated at room temperature for 15 min. The percentage of PerCP-eFluor 710-positive apoptotic cells was determined by flow cytometry, and analyzed with FloJo software. Each experimental point was performed in triplicate.

Conclusions
In the present paper, we demonstrated that biofunctional wool textiles can be produced with waste material from agricultural and food processing industries. In particular, biofunctional textiles were efficiently realized by employing the dried outer skin of onions. This biomass holds several advantages, including its low weight and dryness state.
For the wool dyeing process, gold onion bulbs from the cultivar 'Dorata di Parma' were selected. Among the reasons accounting for this choice, the well-known rich content in polyphenols of this cultivar as well as the ability of its outer dried skin to confer peculiar soft colors with a unique shade are worth mentioning.
Textiles were produced with environmental-friendly manufacture practices without any addition of mordants. As a result, a material with a peculiar ability to protect skin against lipid peroxidation following UV radiation was manufactured in small scale. In order to draw a firm conclusion about the photoprotective action of the produced materials, additional studies on a larger number of volunteers are strictly required. Nevertheless, the results achieved in our investigation strongly suggest their high potential in protecting skin against lipid peroxidation following UV radiation.
The photoprotective and antioxidant activity of the biofunctional textiles under investigation were ascribed to the polyphenolic pool contained in the outer dried gold skins of onion. This assumption was confirmed by the application of a multi-analytical approach, based on the chromatographic, spectroscopic and spectrometric analysis of the aqueous extracts and the textile samples. Very importantly, citofluorimetric analysis demonstrated the safety and the absence of cellular toxic effects of the polyphenolic compounds present in the extracts.
This study lays the foundation for future investigations where other fibers and various waste material from agricultural and food processing industries will be evaluated for the production of smart textiles. In parallel, new dyeing modalities will be carefully considered with the aim to further enhance the loading and release of biofanctional molecules onto the selected material. Additionally, other properties, such as the antimicrobial activity of specific extracts, will also be deeply investigated.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Price Spread and Marketing Efficiency Measure Analysis of Tea Platation Crop in Mokokchung District, India

India is the second largest producer of tea in the world after China. The country is home to a wide variety of teas including CTC tea, orthodox tea, green tea and organic tea. Unlike many other tea producing and exporting nations, India has a manufacturing base for both CTC and orthodox tea, in addition to green tea. India offers high quality specialty teas, such as Darjeeling, Assam Orthodox and high-range Nilgiri tea, which have a distinct aroma, strength, color and flavor. The tea industry in India is about 172 years old and it is perhaps one of the only industries which have maintained its leadership in terms of production over the past 150 years. Indian tea is among the finest International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 06 (2019) Journal homepage: http://www.ijcmas.com

Introduction
India is the second largest producer of tea in the world after China. The country is home to a wide variety of teas including CTC tea, orthodox tea, green tea and organic tea. Unlike many other tea producing and exporting nations, India has a manufacturing base for both CTC and orthodox tea, in addition to green tea. India offers high quality specialty teas, such as Darjeeling, Assam Orthodox and high-range Nilgiri tea, which have a distinct aroma, strength, color and flavor. The tea industry in India is about 172 years old and it is perhaps one of the only industries which have maintained its leadership in terms of production over the past 150 years. Indian tea is among the finest in the world owing to strong geographical indications, heavy investments in tea processing units, continuous innovation, augmented product mix and strategic market expansion (Tea Board of India, 2018).
Nagaland is emerging as one of the nontraditional tea growing area with a large number of Naga people taking up tea cultivation as an alternate means of livelihood. This has made a significant impact on the pattern of agricultural land use, creation of rural employment opportunities, occupational structure shift and economic benefit to the people engaged in tea cultivation. However, the production of tea in Nagaland is very less as compared to other tea producing states despite its favorable agroclimatic, topographic and natural conditions for tea cultivation. The concept of small tea cultivation in Nagaland is relatively new compared to neighboring states like Assam and most of the tea growers lack technical knowledge of cultivation. The growers mostly rely on factory owners for dispersing their green leaves at a price fixed by the factory owners. In the aspect of marketing, many small tea farmers suffer from obtaining reasonable price for their produce which affects their profitability to a considerable extent .
Tea cultivation in Mokokchung district of Nagaland is being taken up by more farmers every year and factories have been established over the years. However, no study or research has been conducted till now by concerned department regarding marketing pattern aspects of tea in Mokokchung district of Nagaland. Taking all these into consideration the present research is designed in order to know the present scenario, and to address bottle-necks and provide suggestions in enhancing the tea industry in the state of Nagaland in coming days.

Materials and Methods
Mokokchung is a district of Nagaland state in India which is mainly occupied by the Ao Naga tribe. Its headquarters is Mokokchung town. This district covers an area of about 1615 sq km and is bounded by the state of Assam to its north, Wokha district to its west, Tuensang and Longleng district to its east, and Zunheboto district to its south. The selection of villages was done purposively based on the availability of tea growers. From Tuli sub-division, 6 villages were selected namely Anike, Wamaken, Merangkong, Kangtsung, Anakiyimsen and Wamakenyimsen. From each village, the list of farmers who were growing tea for the past 5 to 6 years obtained, from which 15 farmers were randomly selected so that there will be uniformity in their yield. Thus, altogether the total number of respondents were 90. The selected respondents were then categorized into three groups viz; marginal, small and large. Due to wide variation amongst the area under tea garden, the categorization was done by adding and subtracting the mean ± standard deviation of the respondent's tea areas. The categorization was viz; Small: up to 0.70 ha, Medium: 0.71 to 3.20 ha and Large: 3.21 ha and above.
Marketing cost was calculated by estimating the cost incurred in the process of marketing of tea. The cost incurred after harvesting of the crop till it reaches the consumers generally constitutes the marketing cost. It includes transportation cost, handling cost, storage cost, market fees, weighing charges and labor charges for packing, loading and unloading. The marketing cost at various stages of marketing was calculated and finally the total marketing cost was computed (Choudhary et al., 2017).
Marketing margin at any stage of marketing was calculated as follows: Whereas: MM i = Marketing margin of the ith middleman.
SP i = Selling price of the ith middleman. PP i = purchasing price of the ith middleman. MC i = Marketing cost incurred by the ith middleman.
The price spread is the difference between the price paid by the consumer and the price received by the producer. It mainly consists of marketing costs and margins (Dinesh and Sharma, 2019;Jamir and Sharma, 2014). The price spread analysis was carried out by using the formula:

×100
Marketing efficiency is the ratio of market output to the marketing input (Lal et al., 2003;Murry and Sharma, 2016). A detailed study of marketing efficiency on the produce of sampled respondents was determined. Shepherd's method was used to assess the efficiency of the marketing channels which is given by: The process of estimating the marketing pattern and efficiency of tea leaves from producer till consumer is complicated as it involves processing in the middle of the channel, wherein, 3 kg of fresh tea leaves gets processed to only 1 kg of made tea. Despite the limitations, an attempt was made by the researcher to analyze the marketing pattern and efficiency of tea leaves, taking into account the information obtained from sampled respondents, 6 commission agents (1 from each village), 1 processing unit, 6 wholesalers and 6 retailers.

Results and Discussion
The marketing pattern of the sampled tea growers followed two channels. In Channel-I, the sampled farmers sold their produce to commission agents, who in turn sold the purchased tea to factory for processing. Here, the farmers do not incur any expense for marketing. However, the farmers were paid lesser by the commission agent as compared to factory purchase price. The difference in price was Rs 2 per kg of fresh plucked tea leaves. In Channel-II, the farmers sell their produce directly to tea factory at their own expense. Hence, they get better price for their produce as compared to those who sell to commission agents. Similar findings was obtained by Sharma and Singh (2001); Sakhrie and Sharma (2017). Table 1 reveals that the marketing channels it was observed that 64.00 per cent of the sampled respondents sold their produce through channel-I and the remaining 28.90 per cent sold though channel-II. Those respondents who owned transportation facilities sold their produce themselves directly to processing unit, while the rest sold through channel-I, where commission agent was involved. Similar studies were carried out by Sharma and Tungoe (2011). Table 2 reveals that the activity wise marketing cost incurred at various steps of marketing channel was estimated. The total cost incurred for marketing 1 kg of green leaf through Channel-I and Channel-II. The only difference between the two channels is the presence of commission agent in Channel-I, while all the sampled respondents produce were found out to be processed in the same factory. Therefore, marketing cost of processing unit remains same in both the channels. After the green leaf is processed in the processing unit, it is being marketed through wholesalers and retailers, therefore, from the point of processing unit the marketing costs remain same in both the channels. Certain quantity of processed tea leaf from the studied processing unit were also auctioned at Guwahati auction center, however, it has been neglected in the marketing channel by the researcher due to time limitation.
From the table 2 in channel-I, the total cost incurred by the commission agent was Rs 0.83 per kg of green leaf. Adding up all the cost for fuel, power, wages, factory overheads, general overheads, packing, tax and transportation, the total cost incurred by the processing unit for processing 1 kg of green leaf was Rs 4.19/-. The cost incurred by wholesaler and retailer for marketing 1 kg of processed tea leaf were Rs 0.86/-and Rs 1.20/-, respectively. While in Cha -nel-II, the cost incurred by the producer farmer was Rs 0.30/-per kg cost incurred by the processing unit, wholesaler and retailer were Rs 4.19/-, Rs 0.86/-and Rs 1.20/-, respectively. The total marketing cost incurred in Channel-1 was Rs 7.08/-per kg and the total cost incurred in channel-II was Rs 6.55/-, which shows that total marketing cost is more in Channel-I as compared to Channel-II, respectively. Similar studies were carried out by Sharma et al., (2010); Sharma (2013). Table 3 reveals that the marketing margins of commission agents, processing unit, wholesalers and retailers had been calculated considering the value of 1 kg of green leaf till it reaches the consumers in processed form. Marketing margins for both the channels had been calculated separately. In channel 1, marketing margin for commission agent had been estimated as Rs 1 per kg of green leaf. After commission agent, the market intermediaries were same in both the channels. In both the channels, marketing margin obtained by the processing unit was Rs 52.14/-per kg of processed tea leaf. The marketing margin obtained by processing unit was high due to various value addition process carried out during processing. Average marketing margin for the wholesalers were obtained as Rs 16.13/-per kg of processed tea leaf which was higher than the marketing margin obtained by the retailers (Rs 8.67/-per kg). So, the total margin observed in Channel-I was Rs 77.94/-, which was slightly higher than Rs 76.94/-as obtained in Channel-II, respectively. Similar studies were in the line with Yadav et al., (2018).
The price spread analysis of the present study refers to the difference between price paid by the consumer and the net price received by the farmer for an equivalent quantity of tea leaf. The consumer's price for 1 kg of processed tea leaf is Rs 300.00/-which is made from 3 kg of green leaf. So in order to estimate the producer's share of 1 kg of green leaf in consumer's price, the price of processed tea has been divided by 3. The producers share in consumer price was found out to be very less which is because of value addition done in the processing unit. In case of channel-I, producer's share in consumer price was Rs 15.00/-, which is less than Rs 17.00/-as obtained in channel-II, respectively. Similar studies were carried out by Tangjang and Sharma (2018). Table 4 reveals that the marketing efficiency is the ratio of market output to the marketing input. A detailed study of marketing efficiency on the produce of sampled respondents had been determined in this segment. Here, the researcher had used Shepherd's method to assess the efficiency of the two channels. The marketing efficiency in Channel-I was estimated to be 14.10 and in Channel-II it was 15.30. Hence, it can be concluded that Channel-II is more efficient than Channel-I. This shows that as intermediaries increases between producer and consumer, marketing efficiency decreases. Similar studies were carried out by Sharma et al., (2018) (Fig. 1-3).   Based the present study some of the conclusions must be drawn for future guidelines viz., the marketing pattern of the sampled tea growers followed two channels. In Channel-I, sold their produce to commission agents, who in turn sold the purchased tea to processing unit for processing. In Channel-II, the farmers sell their produce directly to tea factory at their own expense. Hence, they get better price for their produce as compared to those who sell through commission agents. The total marketing cost incurred in Channel-I was Rs 7.08/-per kg and the total cost incurred in channel-II was Rs 6.55/-, which showed that total marketing cost was more in Channel-I as compared to Channel-II. The total margin observed in Chanel-I was Rs 77.94/-, which was slightly higher than Rs 76.94/-as obtained in Channel-II. The marketing efficiency in Channel-I was estimated to be 14.1 and in Channel-II it was 15.3.


extfieldsofstudy: Chemistry Medicine

text:
In situ fast analysis of cadmium in rice by diluted acid extraction-anodic stripping voltammetry

In China, the production has not realized intensive cultivation and the problem of cadmium (Cd)-contaminated rice is salient, so it is important to classify rice with different degrees of Cd pollution by rapid detection method in situ. This paper established a method with a combination of dilute acid extraction pretreatment and electrochemical devices. Cd was extracted from rice using 3% HCl for 5 min. A standard curve was obtained based on a certified reference material in the rice matrix with different concentrations of Cd, which was fitted with the Cd concentration (μg kg−1) against the stripping peak current value (μA), and the linear correlation coefficient was 0.9997. To analyze the applicability of the method, three factors including substrate diluents, particle diameter of the sample, and stability towards the method were evaluated. The limit of detection (LOD) was 2.02 μg kg−1, and the repeatability and accuracy were satisfactory. Cd was determined in 142 samples collected from three major grain-producing provinces of China, and the results have good consistence with the microwave digestion-ICP-MS method. The developed method combined dilute acid extraction with a matrix matching standard curve in ASV for the first time, and it was significantly satisfactory for the detection requirements in China.

Introduction
Cadmium (Cd) is a toxic heavy metal that can be accumulated in the body through food, water, and air, and is also associated with the induction of cancer. 1,2 In recent years, with intensied industrialization and urbanization, agricultural soils polluted with Cd were about 2.786 Â 10 9 m 2 in China, 3 and Cd in soil can readily translocate into crops and accumulate in edible grains. 4 Rice, as the most important staple, supports approximately 65% of China's population, 5 so Cd-contaminated rice severely increases the human health risk, and it is extremely urgent to monitor and manage cadmium uxes of rice in agricultural systems. The most common methods for Cd detection in rice are atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma optical emission spectrometry (ICP-OES), and so on. [6][7][8][9] These methods have achieved quick and exact detection, but they are also time-consuming, require sophisticated equipment and are very demanding for the operator, generally in a laboratory setting. Unlike other countries, China's agricultural production is dominated by scattered smallholders, which also increased a security supervision of difficulty. 10 The conicts between small-scale production and large regulation demands are very obvious, so a real-time detection method is necessary when the grain department purchases rice from elds or from grain depots, while the above-mentioned methods are unsuitable for eld measurement.
Electroanalytical methods have been extensively employed in recent years, as they are low cost, highly sensitive, easy to operate and portable. [11][12][13] In particular, anodic stripping voltammetry (ASV) has been proved to be a high-potential and predominant analytical technique, [14][15][16] which is widely applied in the detection of Cd within different complex media, such as tea, 17 edible oil, 18 sh tissue, 19 and soil. 20 There are numerous reports on Cd detection in rice by ASV, which mainly focused on the choice of metal chelating agent and modication of the electrodes. For example, luminol has been used to chelate Cd and Cu ions in rice samples, 21 and a crown ether/Naon-modied screen-printed carbon electrode coated with a bismuth lm was prepared to determinate Cd and Pb ions in rice products. 22 However, the samples which are used for electrochemical detection must be digested completely, so that Cd is released into its ionic form. Conventional digestion methods, such as wet digestion, microwave digestion and dry ashing, need a lot of nitric acid (grade pure), a strong oxidizer, high temperature or pressure, and a long pretreatment time (at least 3 hours). 23 Therefore, the reported methods were suitable for Cd determination in a lab, but could not be applied to detection in the elds. To resolve the conicts between the complicated digestion methods and the need for eld-portable detection equipment, we established a rapid pretreatment method for Cd in rice using diluted acid without microwave 24 or ultrasound 25 assistance, and it could extract Cd from rice at room temperature within 5 min. 26,27 Sequential extraction has been used to identify the chemical forms of Cd in roots, leaf sheaths, and leaves of both types of rice plants, and 0.6 M of HCl has been used to extract cadmium oxalatefrom frozen shoot and root tissues. [28][29][30] However, no studies have been reported on the morphology of Cd in rice grain extracted with diluted acids and its suitability for the detection of electrochemical methods.
The aim of the present work is to develop the in situ fast analysis of cadmium in rice. Dilute acid extraction-ASV was developed based on analyzing the form of Cd in the extract of rice and optimizing the conditions of matrix disturbance, dilute acid type, concentration, sample particle size, and detection stability. The schematic diagram of the working principle of this method is shown in Fig. 1. The method was almost "ready-touse" and can easily be adapted in eld testing, and it will provide an alternative way to monitor Cd-contaminated rice in China.

Experimental
Testing samples

Chemicals and apparatus
All reagents were guaranteed grade. Nitric acid, perchloric acid and hydrochloric acid were purchased from Beijing Chemical Reagent Research Institute. Sodium acetate and potassium chloride were supplied by Tianjin Guangfu Science and Technology Development Co., Ltd. All solutions were prepared with double-distilled water. Cd standard solution (1,000 mg mL À1 , GBW08612) was supplied by the National Institute of Metrology, China. Argon (99.99%) was provided by Beijing North Temperature Gas Factory. A dialysis bag with a molecular weight cutoff of 3500 was purchased from Viskase, USA.
Cd in the extracts was quantied by ICP-MS (7500CX, Agilent, USA) and AAS (ZEEnit 700 P, Analytik Jena, Germany). All voltammetric measurements were conducted by differential pulse anodic stripping voltammetry (HM-5000P, Jiangsu Tianrui, China). The electrode stand consisted of a 3 mm diameter glassy carbon electrode as the working electrode, double-junction Ag/AgCl (3 M KCl, saturated AgCl, and 3 M KCl in the bridge) as the reference electrode, and platinum wire as the auxiliary electrode. A microwave (MARS X, CEM, USA) accelerated reaction system was used for comparison with the sample treatment. A high-speed centrifuge (3-30KS, Sigma Laborzentrifugen GmbH, Germany) was used to separate the extracting solutions.

Sample preparation
Dilute acid extraction. Sample powder (1.00 g) and 10 mL extracting solution (3% HCl, v/v) were transferred into a 50 mL centrifuge tube and then mixed using a vortex mixer for about 5 min. The mixed solution was used for the subsequent voltammetric procedure.
Microwave digestion. Samples of grain powder (0.20 g) were digested using concentrated nitric acid (5 mL, 65%, w/w) in Fig. 1 The schematic diagram of the working principle of this method. a microwave digestion system and then diluted to 25 mL for detection by ICP-MS and AAS. For ASV detection, 0.50 g of grain powder was digested using 7 mL of concentrated nitric acid (65%, w/w). The digestion protocol utilized a 20 min ramp to 180 C followed by a 20 min hold and 30 min cool down. The digested sample solution for ASV detection was heated at 130 C until nearly dry, then ltered and diluted to 5 mL for the voltammetric procedure.

Voltammetric procedure
The same volume of electrolyte solution (59 g of sodium acetate dissolved in 500 mL distilled water) was added into the centrifuge tube containing the mixed solution of dilute acid extraction, and then blended well. To better separate the phases, the mixed solution was centrifuged for 5 min at 10 000 rpm.
The electrode maintenance and analysis parameters were set according to the instructions provided in the electrochemical instrument. The preparation of the glassy carbon surface was carried out as follows: the glassy carbon surface was rst polished with polishing paper (Jiangsu Tianrui, China), and the nal polishing was carried out using aluminium oxide powder (0.3 mm). The electrode should be rinsed with deionized water between sample measurements.
The instrumental parameters used for ASV were: A constant potential (À1.40 V) was applied for 300 s to form the mercury lm. The deposition potential and deposition time were À1.00 V and 110 s respectively. The pre-electrolysis step was followed by stripping from À0.90 V to À0.25 V, which gave a specic stripping current peak in the end.

Development of the method
Development of matrix matching standard curves. Different levels of certied reference materials or reference materials containing Cd were processed as described in the dilute acid extraction and Voltammetry procedure. Matrix matching standard curves were constructed by plotting stripping peak current (mA) against the concentrations of Cd (mg kg À1 ) in the standard samples.
Inuence of key factors on detection results. Several factors can considerably affect the extraction efficiency and voltammetry procedure. Therefore, the effects of type and concentration of the extracting acid, as well as the effects of sample particle size, settlement method, and number of test times should be investigated to obtain the optimized parameters.
Evaluation of the detection Detection limit and quantication limit. The LOD and LOQ of the ASV method were calculated as described in ref. 31. The equation is as follows: The equation is appropriate for the situation when acquiring voltammetric scans of the analyte-free support electrolyte solution or when using instrumentations which automatically subtract the background from the response of any test sample.
Precision and accuracy. The precision of dilute acid extraction-ASV for Cd detection was calculated from 7 consecutive measurements of the sample and expressed as the relative standard deviation (RSD). The accuracy of the method for Cd detection was evaluated using certied reference materials containing Cd at varying concentration levels.

Feasibility of the method
In accordance with the principles of electrochemical measurement, ASV can only be used for testing ions in samples. Whether the speciation of Cd in diluted acid extraction solution could satisfy the requirements of the electrochemical method has become an important basis for the accuracy and stability of the method.
The speciation of heavy metals in the environment (water and soil) and the relationship between bioavailability and toxicity have drawn signicant attention in recent years. [32][33][34] However, the speciation of heavy metals in food clearly varies from that in the environment, and heavy metals in food exist mainly in the form of macromolecular complexes. 35,36 Cd in rice is mainly combined with protein to form Cd-binding protein rich charged amino acids or hydrophobic amino acids. The varying chemical forms of Cd in rice grains were also extracted by sequential extraction, and the extraction solution is made up of the following: (i) deionized water, (ii) 5% NaCl, (iii) 70% ethanol, and (iv) 0.2% NaOH. 37,38 M. H. Zhou et al. 26 previously proposed that an acid reagent at a certain concentration can be used to extract Cd in rice efficiently. They also veried the credibility of Cd extraction using diluted acid by graphite furnace atomic absorption spectrometry (GFAAS). 27 The dissociation of extracted Cd into the ion form under acidic conditions has yet to be conrmed.
The particle radius of Cd 2+ is 0.095 nm, however, the molecular mass of the composition formed from Cd with organics is more than 5.5KD. [37][38][39][40] According to this difference, a dialysis bag with an intercept molecular weight of 3500 (the pore size is about 1.5 nm) was used to explore the speciation of Cd in rice. The diluted acid extraction solutions of three certi-ed reference materials pre-and post-dialysis were all digested via the microwave digestion protocol then analyzed by ICP-MS. The results are shown in Table 1, and aer full dialysis, more than 91% of the Cd could pass through the pores of the dialysis bag. The Cd in rice extracted by diluted HCl mostly was in Cd 2+ form, which could be detected by electrochemistry.
Optimization and development of the method Effects of the type and concentration of acid for extraction. The rst step of the present study was focused on the optimization of extraction conditions. Initially, the suitable conditions for the stripping peak current signal (mA) were investigated. For this study, we prepared extracted Cd from rice with varying types of acid. Table 2 showed the analytical signal varies with the type of acid. A higher signal was obtained when 1% HCl was employed as the extractant.
To evaluate the extraction efficiency at varying concentrations of HCl, the results for Cd in CRM were determined by ICP-MS (Fig. 2). The results showed that the concentrations of Cd in the extract were gradually increased in 0.5-5% HCl (v/v), and when the concentration of HCl was 3% and above, concentrations of Cd in the extract were the most consistent with the certied reference value, X ref . Thus, 3% HCl was selected as the extraction solution concentration. Note that in Fig. 2  Effects of extraction time. Other than the optimized conditions, another factor was the extraction time. In Fig. 3, extraction time was varied from 1 min to 8 min, and all of the results were consistent with the certied reference value, X ref . However, when extraction time was set to 5 min, the test results were closer to the mid-value of the reference value. Thus, 5 min for extracting was more suitable for application.

Development of the calibration curve
To calculate the content of Cd in samples, the standard curves were constructed with the standard solution of Cd and extracted solutions of reference materials containing different levels of Cd (rice matrix). The abscissa of the curve represented the concentration of Cd (mg kg À1 ), whereas the ordinate represented the stripping peak current (mA). The linear equations of two curves were y ¼ 0.0312x À 0.4561 and y ¼ 0.0096x + 0.0033 for matrix matched standard curve and aqueous standard solutions, respectively, and there was a signicant difference between the slopes, indicating that the matrix of rice extract exerted a signicant effect on signals.
Establishing a correct calibration was essential in any analytical methodology to prevent the occurrence of systematic errors and matrix effects in the procedure. A CRM (GBW10045) was used to investigate the applicability of calibration. Satisfactory results were obtained by using a rice matrix matching the standard, rather than the solution standard (Table 3), which suggested that the matrix matching standard curve could reduce the inuence of the rice substrate on the signals.
The anodic stripping voltammetry for the matrix matching calibration curve was measured by adsorbing Cd 2+ at specied concentrations for 110 s at À1.0 V, at rst, then stripping from À0.90 V to À0.25 V with a linear sweep rate of 0.4 V s À1 , which    gave a special stripping peak at À0.590 V in the end (Fig. 4A). For the matrix matching standard curve, it is difficult to nd reference materials with higher concentrations of Cd. The linearity of the calibration plots is veried up to 1220 mg kg À1 total concentration for Cd in rice samples (122 mg L À1 in extract solution), and the correlation coefficients (r 2 ) was 0.9997 (Fig. 4B).

Matrix interferences
Other metal ions and organic compounds present in the sample solution might co-deposit with the target metal ions, and might result in poor peak resolution. To study interferences, earlier studies have investigated the inuence by adding interfering ions, surface-active compounds and EDTA. 41 However, the compounds from the diluted acid extraction solution cannot be simply simulated by adding the chemicals mentioned above. We therefore directly analyse the ASV results of the sample treated by microwave digestion and diluted acid extraction. As can be seen in Fig. 5, there was no signicant difference between the current signals for Cd in the aqueous standard and in the sample digestion solution at the same concentration. The stripping peak of Cd in the sample diluted acid extraction solution was broadened, the stripping current decreased by 65% (from 6.1 mA to 2.1 mA), and the peak potential was shied from À0.62 V to À0.59 V. These changes indicated that the organic compound in the sample diluted acid extraction solution may be adsorbed on the surface of the working electrode, which reduces the efficiency of the electrode to the target analyte. However, there was no interference to ASV detection when the matrix matched calibration was developed.

Selection of sample diluents
The matrix interferences were veried to be signicant, so the effect of different diluents on the determination of Cd content in rice samples should be evaluated. The diluents were prepared in rice extraction without Cd and aqueous solvent diluents (1 : 1, v/v; extraction solution: electrolyte solution). The inuence of two kinds of diluents on the measurement was investigated, and the obtained results are presented in Table 4. The use of aqueous solvent diluents was eliminated as it resulted in noticeably larger values than the reference value, but the blank rice sample was extracted for its satisfactory use for high Cd content samples. This result conrms that the matrix exerts signicant effects on this method. Effect of sample particle size on detection. Sample particle size is another signicant factor affecting the extraction efficiency and speed of analysis. It is also an important indicator of the requirements of sample treatment. Thus, the effects of sample particle size on detection have to be evaluated by ASV. Fig. 6 Comparison of the determination results of samples with different particle sizes by ASV, AAS, and ICP-MS. s1 denotes rice sample 1 and s2 denotes rice sample 2.     6 showed that two rice samples with particle sizes ranging from 0.25 mm to 1 mm were analysed by ASV, AAS, and ICP-MS. The results showed no signicant difference in varying sample particle size (P > 0.05). Therefore, the rice particle size of 1 mm can meet the detection requirements, and this sample processing condition was easily met in the elds.
Effect of different sedimentation patterns on the stability of detection by ASV. The state of the mercury membrane is important for detection using ASV. 42 A complete and uniform mercury membrane directly affects the accuracy and stability of the testing results. However, with the established extraction method, the sample liquid partially contains soluble organic matter and granular material. During voltammetric analysis, the accumulation of stripping and mix scouring processes, and the inuence of some organic impurities or particles in the liquid, can result in loss of mercury lm, and the mercury lms become thinner, rendering them non-detectable. [43][44][45] Therefore, the frequency and sedimentation pattern of the extract need to be investigated.
As can be seen in Fig. 7, the thick black lines correspond to X ref for Cd in GBW10045, and the dashed lines mark the expanded standard uncertainties of the characterization, X ref AE 2u ref ¼ 190 AE 20 mg kg À1 . Sedimentation patterns (centrifugation at 10 000 rpm for 5 min and remaining static for 15 min) showed no difference in detections as the most visible particles in the extractions can sink to the bottom by settling. When using a complete and uniform mercury lm, the method can ensure the accuracy of the 40 samples, therefore ASV based on thin-layer mercury electrodes maintains an excellent stability and is highly applied in the present study. 41,46 Evaluation of the strategy Limits of detection and quantication. The limits of detection and quantication for the proposed methodology were derived from repetitive analysis of the samples. The detection limit for Cd(II) was 2.02 mg kg À1 (0.20 mg L À1 in solution), which was calculated in terms of the 3.29S b /a rule, where S b is the standard deviation corresponding to 20 determinations of blank sample detection, and a is the slope of the calibration plot. The quantication limit was 6.15 mg kg À1 (0.62 mg L À1 in solution), according to 10S b /a. The LODs and LOQs of different methods are shown in Table 5. Even though the ASV approach  had no advantage in terms of LOD and LOQ, it could fully satisfy the national requirements for the detection of Cd in rice.
Precision. When the concentration of Cd in rice was 0.235 mg kg À1 , the precision of dilute acid extraction-ASV (RSD) was 2.16%, obtained by the determination of 7 replicate measurements of the rice sample. This level of precision was satisfactory for this type of procedure.
Accuracy. The accuracy of the method was veried by analysis of the CRMs (GBW(E)100378, GBW10045, and IRMM-804). In Table 6, the results are within the assigned range for the CRMs of rice powder and brown rice our, verifying the accuracy of the proposed method for the determination of Cd in rice.
In total, 142 rice and brown rice samples were collected from three major grain-producing provinces of China, Hunan (n ¼ 70), Guizhou (n ¼ 38) and Jiangxi (n ¼ 34), and the Cd content ranged from 16 to 670 mg kg À1 in the samples. As shown in Fig. 8, there was a good relationship between ICP-MS and ASV, which indicated that the results of ASV were credible in real detection. More specically, the fan diagram showed the distribution of the relative deviation between the ICP-MS and ASV results. The relative deviation below 5% represented the largest portion of total samples (quantity 49, proportion 35%), followed by 5-10% (39, 27%). The relative deviation ranging from 25% to 30% represented the smallest portion of the total samples (4, 3%). In the nal analysis, up to 92% of samples could reach a relative deviation below 20% for two different ways.

Conclusions
We established a rice matrix-matched standard curve coupled with an electrochemical detection method for the determination of Cd in rice and brown rice. Cd was extracted from rice and brown rice using 3% HCl solution. The pretreatment of samples could be completed within 5 min, and then the extracted solution was determined by ASV. The whole procedure was easily accomplished with 15 min. The detection limit of the method was 2.02 mg kg À1 . Precision and accuracy tests indicated that the proposed diluted acid extraction-ASV method could be used in the analysis of Cd in rice samples. Moreover, compared to the previous methods for the detection of Cd in rice and brown rice by ASV, 21,22 the pretreatment method needs less time, less corrosive acid of high concentration, and no instruments for high temperature and pressure. Meanwhile, the device for detection using ASV is portable, and easy and simple to operate, so that it could meet the urgent needs for the rapid determination of Cd, particularly in the eld of grain purchase or supervision in countries with serious Cd contamination.

Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent
Not applicable.

Conflicts of interest
There are no conicts to declare.