extfieldsofstudy: Medicine Chemistry

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Sorption, persistence, and leaching of the allelochemical umbelliferone in soils treated with nanoengineered sorbents

Coumarins represent an important family of allelochemicals with fungicidal, bactericidal, insecticidal, nematicidal, and herbicidal properties. Like for other allelochemicals, the short persistence of coumarins in soils can reduce their biological activity and hamper their application as environmentally friendly agrochemicals. We evaluated the sorption of the coumarin umbelliferone by eight soils and six sorbent materials, and then selected two nanoengineered sorbents, hexadecyltrimethylammonium-modified Arizona montmorillonite (SA-HDTMA) and olive-mill waste biochar (BC), to assess the effect of their addition to two distinct soils on umbelliferone sorption, persistence, and leaching. Umbelliferone was sorbed to a greater extent by the acid soils (A1-A2, Kd > 4.0 L kg−1) than by the alkaline soils (B1-B6, Kd < 0.5 L kg−1). The addition of BC and SA-HDTMA at a rate of 4% to alkaline soil (B2) increased the umbelliferone sorption Kd value from 0.3 to 1.6–2.0 L kg−1, whereas their addition to acid soil (A1) increased the Kd value from 4.6 to 12.2–19.0 L kg−1. Incubation experiments showed that BC had more impact than SA-HDTMA on the persistence of umbelliferone in the soils, increasing its half-life from 0.3-2.5 to 1.2–14.4 days, depending on the soil. Furthermore, the addition of BC to the top 0–5 cm of soil columns reduced leaching of umbelliferone and led to accumulation of umbelliferone residues in the top 0–5 cm soil layer. The addition of nanoengineered materials, such as organoclays and biochars, could thus be a suitable strategy to increase the persistence and reduce the mobility of coumarins in the rhizosphere with the aim of prolonging their biological activity.

Minerals Society webpage 45 . The organo-montmorillonite (SA-HDTMA) was prepared by treating a portion of SAz-1 montmorillonite (4 g) with an aqueous solution (200 mL) containing an amount of hexadecyltrimethylammonium (HDTMA) cation (chloride salt) corresponding to 100% of the cation exchange capacity of SAz-1 (1200 mmol c kg −1 ). It is known that modification of high-charge montmorillonites, such as SAz-1, with HDTMA cations provides these clay minerals with a wide hydrophobic interlayer phase and positively charged ammonium groups, which increase their affinity for organic compounds, including anionic and acid compounds 46,47 . The organic waste (OW) and its biochar (BC) were, respectively, a composted olive-mill waste supplied by an olive-processing factory located in Jaén (Spain) and its pyrolysis product, which was obtained by heating a sample of OW at 400 °C for 4 h under a flow of N 2 at 1.5 L/min 41 . Olive-mill waste, a by-product of the extraction of olive oil, is generated in high quantities in olive oil-producing countries, and has been proposed as a pesticide biosorbent in its raw, composted, and pyrolysed forms 26,41,42 . Prior to use, the OW was air-dried and ground to pass a 2 mm-aperture sieve. For the preparation of BC, we selected a low pyrolysis temperature to increase the fraction of condensed organic matter while maintaining surface functionalities, because these surface characteristics are known to increase the bioavailability of sorbed compounds compared to biochars prepared at high pyrolysis temperatures 28 .
Detailed characterisation of SA-HDTMA and BC. The X-ray diffractogram of SA-HDTMA was recorded using a D-5000 diffractometer (Siemens, Germany) with CuK α radiation. The scanning electron micrograph of BC was obtained using a dual beam Zeis Auriga Scanning Electron Microscope (Microscopy Service, University of Seville). Carbon content measurements were conducted using a Leco Truspec CHNS Micro Analyser (Microanalysis Service, University of Seville). Fourier-transform infrared (FT-IR) spectra were recorded using a Tensor II FT/IR spectrometer (Microanalysis Service, University of Seville) on samples diluted (1%) in KBr disks.
Umbelliferone stability experiments. The impact of light exposure and presence of soluble soil components on the storage stability of umbelliferone was studied for umbelliferone aqueous solutions containing different amounts of methanol, which was used as a stabiliser. High concentrations of alcohol have antimicrobial properties 48 and previous experiments had shown that the addition of methanol helped stabilise aqueous solutions of readily biodegradable organic compounds, presumably by eliminating microbial activity 24,49 . For the stability experiments, 2 mg L −1 umbelliferone solutions (100 mL) were prepared either in distilled water or in a soil extract. The soil extract was obtained by shaking 50 g of B2 soil (the soil in which umbelliferone displayed lower persistence) with distilled water (100 mL) for 24 h, centrifuging, and filtering the supernatant solution with filter paper (Whatman No. 1). Each umbelliferone solution contained different amounts of methanol (1, 10, and 50%) and was either exposed to or protected from light for a period of 8 days. At t = 0, 1, 4, 6 and 8 days the solutions were sampled, filtered, and immediately analysed by high performance liquid chromatography (HPLC) to determine the amount of umbelliferone remaining in solution.
sorption experiments. Sorption of umbelliferone on unamended and amended soil samples and on the sorbent materials was determined by batch sorption experiments using glass centrifuge tubes lined with Teflon caps. In triplicate, 8 mL of a 2 mg L −1 umbelliferone aqueous solution (C ini ) was added to samples of the unamended soils (1 g), sorbent materials (20 mg), or soils amended with selected sorbents (BC and SA-HDTMA) at a rate of 4% (40 mg g −1 ). Assuming a soil bulk density of 1.2 g cm −3 , this rate of BC and SA-HDTMA would be achieved by a field dose of 5 t ha −1 for an incorporation depth of 1 cm, or 50 t ha −1 for an incorporation depth of 10 cm. This range (5-50 t ha −1 ) coincides with that for which organic amendments such as biochars have been reported to have positive effects on crop yields, with appropriate nutrient management 50 . To avoid the degradation of umbelliferone during the sorption experiment, the tubes containing unamended or amended soil samples were pre-sterilised by autoclaving for 20 min at 121 °C and 200 kPa three times on consecutive days, with 24 h incubation periods at 25 °C between successive autoclaving cycles. After adding the umbelliferone solution, which was also subjected to one autoclaving step, the suspensions were shaken in the dark for 24 h, centrifuged, and then 6 mL of supernatant solution was removed, stabilised with 6 mL of methanol, and then filtered and analysed by HPLC in order to determine the solution concentration of umbelliferone in the equilibrated suspensions, C e (mg L −1 ). The amount of umbelliferone sorbed, C s (mg kg −1 ), was calculated from the difference between the initial (C ini ) and the equilibrated (C e ) solution concentration as is the volume of solution and m (kg) is the mass of solid. Initial umbelliferone solutions without solids were also shaken for 24 h and indicated no losses of organic compound by sorption to the tubes or to the filter material. Distribution coefficients, K d (L kg −1 ), were calculated as K d = C s /C e , whereas sorption percentages were calculated as %Ads = [(C ini − C e )/C ini ] × 100.
To confirm the indirect sorption measurements, these were compared with sorbed amounts directly determined by extraction. For this purpose, the 6 mL of supernatant solution removed for the indirect sorption analysis was replaced with 6 mL of methanol to promote desorption of the umbelliferone previously sorbed by the soils or sorbents. The suspensions were shaken for 24 h, centrifuged, filtered, and the concentration of umbelliferone in the methanolic extract (C ext ) was determined by HPLC. The amount of umbelliferone desorbed from the solids upon extraction (mg kg −1 ) was then calculated as C s-dir = (C ext − C e /4) × V/m. Samples of SA-HDTMA and BC (10 mg) were also treated with 8 mL of a concentrated umbelliferone aqueous solution (ca. 200 mg L −1 ) and the sorption products were analysed by FT-IR to get insight into the sorption mechanisms. The suspensions were shaken for 24 h, the supernatant was removed and analysed by HPLC, and the solids (sorbent-umbelliferone complexes) were dried at 60 °C and analysed by FT-IR spectroscopy as samples diluted (1%) in KBr disks.
www.nature.com/scientificreports www.nature.com/scientificreports/ Dissipation experiments. The effect of the addition of SA-HDTMA and BC on the dissipation of umbelliferone in selected soils (A1 and B2) was studied by an incubation experiment conducted in 10 mL-glass centrifuge tubes. Samples of 1 g of the non-sterile soils, either unamended or amended with SA-HDTMA or BC at a rate of 40 mg g −1 soil, were added to the tubes and then treated with an aqueous solution of umbelliferone with appropriate concentration to give an application rate of 2 mg umbelliferone kg −1 soil and a water content of 35% (soil A1) or 30% (soil B2). These water contents were selected for being close to the water holding capacity of each soil. Next, the tubes were closed and incubated in the dark at 25 °C. Separate triplicate tubes were incubated for each sampling time (t = 0 to 11 days), to be destructively removed from the incubator and immediately frozen until analysed. Duplicate sterile treatments were also included to control for abiotic umbelliferone breakdown; these consisted of autoclaved unamended soil samples (20 min at 121 °C and 200 kPa, three times on consecutive days) spiked with a sterilised umbelliferone solution as described above for the non-sterile treatments.
For each sampling time, umbelliferone soil residues were determined by extraction with 5 mL of an 80:20 methanol:aqueous H 3 PO 4 (pH = 2.2) mixture by shaking for 24 h. The suspensions were centrifuged, filtered, and the extracts analysed by HPLC in order to determine the amount of umbelliferone remaining in the soil. Recoveries were >90% of the umbelliferone freshly applied to the unamended or amended soils.
Data obtained in the dissipation experiment were analysed using first-order kinetic model: where C (mg kg −1 ) is the concentration of umbelliferone in soil at a time t (days), C 0 is the concentration at t = 0, and k (day −1 ) is the first-order dissipation rate constant. Half-lives were calculated as t 1/2 = 0.693/k. In a separate incubation experiment, the changes in the basal respiration of the soils upon amendment with SA-HDTMA or BC were also determined. Soil respiration measurements were conducted following the alkali trapping-titrimetric procedure 51 . The method consisted of quantifying the amount of CO 2 released by soil samples during 7 days, under conditions that were representative of those used for the umbelliferone dissipation experiment. In brief, triplicate samples of 20 g of the soils (A1 and B2), either unamended or amended with SA-HDTMA or BC at a rate of 40 mg g −1 and with the water content adjusted to 35% (A1) or 30% (B2), were incubated in Erlenmeyer flasks at 25 °C in the dark. After 7 days, the amount of CO 2 released by the soil samples was determined by back-titration of vials containing 20 mL of 0.2 M NaOH that were placed inside the flasks as CO 2 traps.
Column leaching experiments. Leaching of umbelliferone was studied in glass columns (30 cm length × 3.1 cm internal diameter) hand-packed with soils A1 and B2, either unamended or amended with BC. Glass wool and sea sand (10 g) was placed on the bottom of the columns to avoid losses of soil and contamination of leachates with soil particles. Next, the columns were hand-packed with 20 cm of unamended or BC-amended soil. The BC-amended soil columns were prepared by mixing BC with the upper 0-5 cm of soil at a rate of 40 mg BC g −1 soil. Finally, sea sand (10 g) was placed on the top of the columns to minimise disturbance of the soil surface and also to allow a uniform distribution of the umbelliferone and water subsequently added to the soil columns. All leaching experiments were conducted in triplicate.
The columns were saturated by adding water to their surface (90 mL), and then were allowed to drain for 4 h. The pore volume of the soil columns, calculated by subtracting the amount of water leached from the amount added, ranged between 60 and 64 mL.
Umbelliferone was applied to the surface of the columns at a rate of 2 kg ha −1 as 0.15 mg of compound dissolved in 0.75 mL of methanol, which was allowed to evaporate for 1 h. If we assume a soil bulk density of 1.2 g cm −3 and that umbelliferone is uniformly distributed along the top 0-8 cm soil layer, an application rate of 2 kg ha −1 would yield the initial umbelliferone concentration used in the dissipation experiments (2 mg kg −1 ). The columns were eluted by conducting daily 3 additions of 15 mL of distilled water at 3 h intervals for a total of 12 additions in 4 days. The leachate resulting from each water addition was collected in a glass vial containing 5 mL of methanol, filtered, and analysed by HPLC to determine the concentration of umbelliferone. Both the columns and the vials used to collect the leachates were covered with aluminium foil to protect umbelliferone from light exposure. At the end of the leaching experiment, soil samples were taken from different depths of the columns (0-5, 5-10, 10-15, and 15-20 cm depth) and shaken with an 80:20 methanol:aqueous H 3 PO 4 (pH = 2.2) mixture (100 mL) for 24 h. The suspensions were centrifuged, filtered, and analysed by HPLC to determine the amount of umbelliferone remaining at different depths of the soil columns.
Analysis of umbelliferone. Umbelliferone was determined by HPLC using a Waters 515 HPLC Pump coupled to a Waters 2996 photodiode array detector and a Waters 717plus Autosampler injector. The analysis was carried out using a Kinetex C18 column (150 mm length × 4.6 mm internal diameter and 5 µm particle size), methanol:aqueous H 3 PO 4 pH = 2.2 (65:35) eluent mixture at a flow rate of 1 mL min −1 , 25 µL sample injection volume, and UV detection at 323 nm. Under these conditions, the retention time for umbelliferone was 4.7 min. The limit of quantification, calculated as the concentration resulting in a signal-to-noise ratio of 10:1, was 0.03 mg L −1 . External calibration curves with five standard solutions between 0.1 and 6 mg L −1 prepared in 50:50 methanol:water were used in the calculations. statistical analysis. Statistical analyses were carried out using IBM SPSS Statistics 15.0 for Windows. Standard error was used to describe variability among independent replicate samples. Differences in umbelliferone sorption, dissipation, and leaching data and in soil respiration values were compared using One-Way ANOVA followed by (2019) 9:9764 | https://doi.org/10.1038/s41598-019-46031-z www.nature.com/scientificreports www.nature.com/scientificreports/ post-hoc Tukey's Honestly Significant Difference test for pairwise comparison of treatments. Fitting of dissipation data to first-order kinetics was carried out using Sigma Plot for Windows Version 12.5.

Results and Discussion
storage stability of umbelliferone. Since allelochemicals can display a poor stability in aqueous solution and/or soil extracts, it is important to test their storage stability to avoid analytical artefacts when conducting experiments to assess their behaviour in soils 24,49 . The stability of umbelliferone in different water/methanol mixtures exposed to light and under dark conditions is illustrated in Supplementary Fig. S1a. Under light exposure, the concentration of umbelliferone progressively decreased with time when the amount of methanol in the mixture was ≤10%; however, it remained stable when the amount of methanol was increased to 50% and also in all water/methanol mixtures when these were protected from light.
The stability of umbelliferone in a soil extract containing different amounts of methanol was also studied ( Supplementary Fig. S1b). Under light exposure, the concentration of umbelliferone rapidly decreased in all cases, although increasing the amount of methanol reduced the dissipation of the compound in the soil extract. Protection from light further increased the stability of umbelliferone in the soil extracts; more than 90% of the umbelliferone initially present remained in solution after 8 days when the methanol content was 10 or 50%.
On the basis of the results of the stability experiments, we concluded that umbelliferone aqueous solutions were unstable when exposed to light and to water-extractable soil constituents, presumably because of its susceptibility to photochemical and microbial degradation processes, respectively. Nevertheless, the addition of methanol at a rate ≥ 10% combined with storage in the dark stabilised the compound for at least 8 days ( Supplementary  Fig. S1b). Consequently, all solutions of umbelliferone to be analysed during the sorption and leaching experiments conducted in this work were stabilised with an amount of methanol higher than 10% and kept in the dark to prevent any disappearance of compound during storage (<1 week) prior to analysis.
Umbelliferone sorption on soils and sorbents. Umbelliferone sorption on soils was evaluated using eight pre-sterilised soil samples that covered a range of physicochemical characteristics representative of the Mediterranean area (Supplementary Table S1). Sorption was noticeable only for the acid soils tested (A1 and A2), while all alkaline soils (B1-B6) displayed very low sorption values (%Ads < 5%, K d < 0.5 L kg −1 ) (Fig. 2). The amount of umbelliferone desorbed from soils A1 and A2, by addition of methanol immediately after the 24 h sorption experiment, accounted for more than 90% of the compound depleted from solution during the sorption step. This confirmed that sorption measurement was accurate and that chemical or biological transformation processes did not interfere during the sorption experiment. The phenolic group of umbelliferone (Fig. 1) has a pK a ≈ 7.5, and so should have remained mainly undissociated at the pH of the acid soils (pH = 5.5-6.0), favouring sorption. Conversely, it should have become partially ionised at the pH of the alkaline soils (pH = 8.0-8.6), and at such pH levels umbelliferone anions were probably repelled by the negatively charged surfaces of soil clays and organic matter, thus explaining low sorption values 52,53 .
The results of umbelliferone sorption on different sorbent materials are summarised in Fig. 3. All sorbents, except KGa-2 (pH = 6.6), yielded alkaline pH values ranging from 7.8 to 9.5 (Supplementary Table S2). The affinity of umbelliferone for the sorbents decreased in the following order: SA-HDTMA  BC > OW > SAz-1 ≈ IMt-1 ≈ KGa-2. Thus, the affinity of umbelliferone for the organoclay SA-HDTMA (%Ads = 94 ± 1%) was greater than for the unmodified clay (SAz-1). Similarly, the pyrolysed OW (BC) displayed increased sorption (%Ads = 34 ± 1%) compared to its feedstock (OW) material (%Ads = 7 ± 2%). The sorption percentages for SAz-1, KGa-2 and IMt-1 were very low (<5%). As for the soils, the amount of umbelliferone desorbed from SA-HDTMA and BC upon the addition of methanol immediately after the 24 h sorption experiment accounted for most (>85%) of the compound depleted from solution during the sorption step. The nanoengineered sorbents, SA-HDTMA and BC, were thus those displaying the greatest affinity for umbelliferone, with the organoclay (SA-HDTMA) showing significantly (p < 0.001) greater performance than the biochar (BC) sample at the experimental conditions used. www.nature.com/scientificreports www.nature.com/scientificreports/ Umbelliferone sorption mechanisms on SA-HDTMA and BC. The incorporation of HDTMA cations in the interlayer of SAz-1 is known to increase the organic carbon content and the basal spacing value of the clay, creating a paraffin-like interlayer structure with hydrophobic properties and high affinity for organic compounds 37 . For the SA-HDTMA sample used in this work, the organic carbon content increased from < 0.1% for the untreated clay to 22.1 ± 0.1% for the modified clay, and the clay basal spacing was expanded from 15.2 Å to 23.0 Å (Supplementary Fig. S2). These changes increased the affinity of the clay for umbelliferone (Fig. 3), probably by favouring hydrophobic interactions between the alkyl chains of the organic modifier and the aromatic moieties of the allelochemical (Fig. 1). Furthermore, the HDTMA cations also provide the clay mineral surface with positively charged quaternary ammonium groups (Supplementary Fig. S2) and some of these are known to constitute potential sites for the sorption of organic anions 46,47 . Given that umbelliferone should have largely existed as anionic species at the pH of the equilibrated suspension of SA-HDTMA (pH = 7.9, Supplementary Table S2), electrostatic (ionic) attraction between positively charged quaternary ammonium groups of SA-HDTMA and umbelliferone anions could have contributed to the sorption mechanism. Supplementary Fig. S3 shows the FT-IR spectrum of SA-HDTMA treated with a concentrated umbelliferone solution along with the spectra of the untreated organoclay and pure umbelliferone. The spectra showed two noticeable effects. First, the absence of the O-H stretching vibration band of umbelliferone at 3140 cm −1 in the spectrum of the SA-HDTMA-umbelliferone complex suggested that umbelliferone could have predominated in the sorbed state as ionised species. Second, when comparing the spectra of pure and sorbed umbelliferone, small changes in the position of the C=C stretching vibrations at 1400-1600 cm −1 were observed, suggesting involvement of the aromatic rings of the allelochemical in the interaction with the organoclay. Ionic attraction and aromatic ring-alkyl chain hydrophobic interactions have previously been suggested as cooperative mechanisms driving the sorption of aromatic acids by paraffinic organoclays 46,53 . In the case of the BC sample, it is known that biochars produced at low pyrolysis temperatures (e.g., 400 °C) contain, along with newly-formed condensed carbon and porosity, surface polar functional groups from the feedstock ( Supplementary Fig. S4), which allow both hydrophobic and polar interactions with ionisable organic compounds 33 . Since pyrolysis of biomass leads to a reduction in abundance of surface polar functional groups, the increase in aromaticity and porosity probably explained the high sorption of umbelliferone by the BC sample 54 . Lian and Xing 33 and Kah et al. 55 indicated that electrostatic repulsions, charge assisted H-bonds, cation bridging, and anion-π bonds dominate the interaction of organic acids with carbonaceous sorbents at high pH values, such as that measured in this study for the BC-umbelliferone suspension (pH = 9.5, Supplementary Table S2). Unfortunately, the FT-IR spectra were inconclusive about the relative importance of the above-mentioned interaction mechanisms, because the amount of umbelliferone sorbed in the complex prepared for the FT-IR spectroscopy study was too low (11 mg g −1 ) to produce significant changes in the spectrum of BC (Supplementary Fig. S3).

Effect of SA-HDTMA and BC addition on umbelliferone sorption by selected soils. On the basis
of the results presented in the preceding sections, the nanoengineered sorbents, SA-HDTMA and BC, were selected as soil amendments to evaluate their effect on umbelliferone sorption and persistence in two distinct soils, one acid (A1) and one alkaline (B2). The results of the sorption experiments are given in Table 1.
The addition of BC and SA-HDTMA at a rate of 4% led to an important increase in the sorption of umbelliferone by both soils. The K d value for umbelliferone in unamended soil A1 (4.6 L kg −1 ) increased to 12.2 L kg −1 upon amendment with BC and to 19.0 L kg −1 upon amendment with SA-HDTMA. For soil B2, the low sorption of umbelliferone in the unamended soil (K d = 0.3 L kg −1 ) increased upon the addition of BC and SA-HDTMA, to K d values of 1.6 and 2.0 L kg −1 , respectively. The fact that the SA-HDTMA-amended soils showed higher sorption than the BC-amended soils (Table 1) reflected the sorptive properties of the pure amendments (Fig. 3). Nevertheless, when we compared the K d values obtained for the amended soils with the values predicted assuming linear and independent sorption behaviour for the soil and the amendment in the mixtures (K d-calc ), we concluded that the increase in sorption produced by the amendments was, in general, less than expected from the  www.nature.com/scientificreports www.nature.com/scientificreports/ individual sorption constants (Table 1). This result indicated that the amendments could have lost some of their sorption performance in the presence of the soils. Most likely, the interaction of the amendments with soil constituents resulted in competition with umbelliferone for sorption sites and/or blockage of access to such sorption sites, partially reducing the performance of the amendments as sorbents of umbelliferone 25,47 . Data in Table 1 show that this effect was soil-and amendment-dependent, and more pronounced for SA-HDTMA and for the alkaline soil (B2).
Dissipation of umbelliferone in unamended and amended soils. After 7 days of incubation, the residual percentage of umbelliferone in nonsterilised A1 and B2 soil had decreased to 10 and 0%, respectively, while in the sterilised controls the residual percentages of umbelliferone remained >75% (Fig. 4a). Thus, the degradation of umbelliferone in the soils was fast and mainly microbial-mediated, in agreement with data recently reported by Guo et al. 22 . The values of the first-order dissipation rate constants showed that dissipation was faster in B2 soil (k = 2.092 day −1 ) than in A1 soil (k = 0.273 day −1 ), with half-lives (t 1/2 ) of 0.3 and 2.5 days, respectively. Along with an intrinsic reduced biodegradative activity of the acid soil (A1), supported by a low basal respiration (Supplementary Table S3), sorption (Fig. 2) could have contributed to delay the degradation of umbelliferone in this soil, as sorption processes are known to reduce the bioavailability of organic compounds and protect them from microbial degradation 56 .
The persistence of umbelliferone in soil B2 increased by the addition of both SA-HDTMA (k = 0.812 day −1 ) and BC (k = 0.560 day −1 ). At t = 1 day, umbelliferone had completely dissipated from unamended B2 soil, whereas more than 40% of the amount initially added still remained in SA-HDTMA-and BC-amended soil (Fig. 4b).
From t = 0.4 to 4 days, umbelliferone residues in the soil amended with SA-HDTMA and BC were significantly greater (p < 0.05) than those in unamended soil. Since the effect of SA-HDTMA and BC on the respiration of the soils used in the present study did not suggest an inhibitory effect of the amendments on the soil microbial activity (Supplementary Table S3), the increase in sorption of umbelliferone produced by the amendments (Table 1) probably reduced its availability to microorganisms and increased its persistence in the soil. According to the experimental K d values reported in Table 1, we estimated that at the beginning of the dissipation experiment the amount of umbelliferone sorbed to the solid phase accounted for 50% for the unamended soil B2, and increased to 85 and 87% for BC-amended and SA-HDTMA-amended soil B2, respectively. The limited amount of umbelliferone available in solution in the amended soil probably reduced its availability to microorganisms and increased its persistence in the soil. For soil A1, we observed a significant increase in the persistence of umbelliferone (p < 0.05 for residues between t = 3 and 11 days) only upon amendment with BC (Fig. 4c). According to the (high) K d values reported in Table 1, the percentage of umbelliferone sorbed were estimated to be 93% for unamended soil, 97% for BC-amended soil, and 98% for SA-HDTMA-amended soil. The high fraction of sorbed umbelliferone in the unamended soil probably made the effect of the amendments on the bioavailability of the compound less noticeable than in soil B2.
It is interesting to note that, conversely to what predicted by the K d values reported in Table 1, the increase in persistence of umbelliferone in both soils produced by the addition of BC was greater than that produced by the addition of SA-HDTMA. In this regard, Gámiz et al. 47 monitored the changes in sorption of the phytohormone abscisic acid with time in a soil amended with organoclay and a high pyrolysis temperature apple wood biochar and their results indicated some loss of the sorption capacity with time for the organoclay-amended soil and, conversely, increasing sorption of abscisic acid with time in BC-amended soil. These distinct time-dependent sorption behaviours observed by Gámiz et al. 47 suggested external surface sorption mechanism for the organoclay and slow kinetics (potentially pore diffusion) in BC-amended soil 57,58 . This would also explain the higher potential of BC for protecting umbelliferone from biodegradation in the soils tested in this study, which was particularly noticeable at longer incubation times.
Effect of BC addition on umbelliferone leaching in soils. On the basis of its greater ability to increase the persistence of umbelliferone in the soils used in the incubation experiments, BC was selected as amendment to evaluate its effect on the mobility of umbelliferone in column leaching experiments with the same soils. In agreement with the low K d value measured for soil B2 in the sorption study (Table 1, Fig. 2), umbelliferone was detected only in leachates from unamended B2 soil columns ( Table 2). The total amount of umbelliferone leached www.nature.com/scientificreports www.nature.com/scientificreports/ was low (14%), probably because umbelliferone rapidly degraded during leaching through this soil (Fig. 4a). Umbelliferone was not detected in the leachates from the columns packed with unamended soil A1 or with BC-amended soils (Table 2), for which the compound presented high K d values (Table 1).  www.nature.com/scientificreports www.nature.com/scientificreports/ At the end of the leaching experiment (240 mm of water added within 4 days), the soil in each column was analysed to determine umbelliferone residues at different depths. For both soils, the presence of BC led to an increase in the amount of umbelliferone remaining in the soils and accumulation of the compound in the top 0-5 cm (BC-amended) soil layer (Table 2, Fig. 5). This effect was more pronounced for the acid soil (A1), for which 57% of the applied umbelliferone was recovered from the 0-5 cm layer (Fig. 5). Clearly, sorption of umbelliferone in the BC-amended (0-5 cm) soil layer reduced its leaching and delayed its degradation compared to the unamended soils.

Conclusions
The results of this work show, in summary, that nanoengineered sorbents can be useful as soil additives to modulate the mobility and persistence of allelochemicals in soils, which may be applied to overcome some of the limitations associated with the use of allelochemicals as environmentally friendly crop protection substances. Specifically, we showed how organoclays and biochars can be designed at the nanoscale to increase the sorption and reduce the mobility and degradation of the allelochemical umbelliferone in two distinct soils. The observed effects were both sorbent-and soil-dependent, and particularly pronounced for biochar added to acid soil. Changes in the sorption properties of the sorbents in the presence of soil and sorption evolution with time were identified as two important factors determining their effect on the mobility and persistence of umbelliferone. Controlling these two factors seems important to optimise the performance of organoclays and biochars after their addition to soils, and presumably, to achieve the desired effect on the biological activity of allelochemicals, in the context of taking advantage of their bioactivity for crop protection.

Data Availability
The datasets generated during the current study are available from the corresponding author on reasonable request.  Table 2. Umbelliferone leached, extracted from the soil, and not recovered after the leaching experiment conducted in unamended and BC-amended soil columns (±s.e.m., n = 3).


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

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Evaluation of anti-inflammatory, antinociceptive and biological activities of Cenostigma macrophyllum standardized extracts and determination and quantification of the main metabolites

The stem barks and leaves of Cenostigma macrophyllum are used in Brazilian folk medicines in the treatment of stomach and intestinal diseases. However, there are no reports of chromatographic methods used to evaluate the bioactives of its standardized extracts and for biological evaluation. An analytical method was developed and validated for simultaneous determination and quantification of the bioactive phenolics gallic acid, methyl gallate, ellagic acid and, the biflavonoids agathisflavone and amentoflavone in the leaves and stem bark of C. macrophyllum. HPLC operating conditions were optimized and the parameters such as selectivity, linearity, precision, accuracy, LOD, LOQ and, robustness of the method were also evaluated. Robustness was evaluated using a multivariate optimization technique. Linear relationships within the range of investigated concentrations were observed with their correlation coefficients greater than 0.9991. The method was validated for repeatability (RSD ≤ 2.88%), intermediate precision (RSD ≤ 3.38%) with recovery between 84.12 and 106.64% and the RSD less than 3.40% and proved to be robust. Besides, antioxidant, acetylcholinesterase inhibition, anti-inflammatory and antinociceptive activities of the standardized hydromethanolic extracts of leaves and stem bark of this species were evaluated. The method was successfully applied in the quantification of the gallic acid, methyl gallate, ellagic acid, agathisflavone and amentoflavone of standardized extracts. The results showed the present method developed was simple, sensitive, reproducible, accurate and precise. The standardized hydromethanolic extracts of leaves and stem bark of C. macrophyllum showed antioxidant activity (EC50 69.09 and 83.06 μg mL−1), acetylcholinesterase inhibition (52.23 and 83.36%) and they were able to inhibit the formalin-induced nociception and also reduced the edema formations at 100 mg kg−1 doses. The anti-inflammatory potentials were evaluated by the decrease of the Cg-induced neutrophils migrations at the same doses.

Introduction
Cenostigma macrophyllum Tul. is a tree belonging to the family Leguminoseae. This species is popularly known as "canela-develho", "caneleiro" and "catingueira" and can be found in the semi-arid region of Brazilian northeastern. 1,2 The decoction or infusion of stem barks, leaves and owers of this species are used in folk medicine for the treatment of stomach and intestinal diseases. 3 Cenostigma macrophyllum of the "Caatinga" Brazilian biome was classied until recently as another specie (C. gardnerianum Tul.) but they were considered as synonyms aer the morphological variation analysis. The study was carried out in populations of its habitats that would be consistent with C. gardnerianum, from the old system, however the taxonomic polymorphism in the chemical characteristics demonstrated proposes a reevaluation of the taxonomic equation of this species. 4,5 Previous studies with extracts and fractions of the leaves, stem barks and seeds of a variety of this plant (C. macrophyllum Tul. var. acuminata Teles Freitas) revealed different biological activities. The hydroalcoholic extracts presented antioxidant activity 6 and effect against gastric ulcers induced by ethanol or HCl/ethanol, ischemia-reperfusion and cold restraint stress with lesion inhibitions at 100 or 200 mg kg À1 . In other studies dealing with antinociceptive actions of the ethanol, ethyl acetate extract and emulsion of hexane extract obtained from stem bark in streptozotocin (STZ)-induced diabetic rats showed a pain threshold higher than those in the STZ-vehicle group indicating antinociceptive activity but in this study the chemical composition of the extracts were presumed based on simple thin layer chromatography analysis and no evidences about the phenolics content. [8][9][10] Phytochemical studies with C. macrophyllum have led to the isolation and identication of avonoids, biavonoids, benzoic acid derivatives, peptide derivatives, triterpenoids, steroids, vitamin A and E, fatty acids, coumarin and volatile esters. 3,11,12 However, there are no reports in the literature on the development of a validated chromatographic method for the quantication of active principles present in this species. Thus, in the present study, a HPLC-DAD method was developed to simultaneously detect and quantify ve bioactive compounds ( Fig. 1) in C. macrophyllum standardized extracts. The method was applied in the analysis of leaves and stem bark of the species. These is the rst study of antioxidant activity, acetylcholinesterase inhibition, anti-inammatory and antinociceptive activities of the standardized with chemical markers of hydromethanolic extracts of leaves and stem bark of C. macrophyllum were evaluated.

Instrumentation, materials, reagents and standards
Chromatographic determinations were performed using a Dionex UltiMate 3000 HPLC equipment equipped with a quaternary pump with on-line vacuum degasser of four channels, autosampler, column temperature control and diode array detector (DAD). Chromatographic analysis was carried out on a XBridge, BEH C 18 column (100 Â 3.0 mm i.d., 2.5 mm, Waters) and Acclaim RSLC 120 C 18 column (100 Â 2.1 mm i.d., 5 mm, Thermo Fisher Scientic). Analytical grade MeOH and DCM (Qhemis) and deionized water were used for extraction procedures, while HPLC grade acetonitrile, MeOH, acetic acid and formic acid (TEDIA or J. T. Baker) were used in chromatographic analyses. Water was puried using a NANOpure Diamond system (Barnstead®, Dubuque, Iowa, USA). The sample solution was ltered through a 0.45 mm Millipore membrane before sample injection. Three reference standards, including gallic acid (1), ellagic acid (3) and amentoavone (5) were purchased from the Sigma-Aldrich (St. Louis, Missouri, USA). Methyl gallate (2) and agathisavone (4) were isolated and puried from of the leaves of Schinopsis brasiliensis (Anacardiaceae) 13 and C. macrophyllum, 10 respectively. These compounds were identied by spectroscopic techniques and the purity were determined injecting 20 mL of the appropriate solution (at a concentration of about 0.1 mg mL À1 ) into an analytical HPLC-DAD under the conditions described below. All standards

Extract preparations
The botanical material was dried at room temperature and then powdered. Part of ground powder (10.0 g of the leaves and 10.0 g of the stem bark) were macerated at 40

Chromatographic conditions
Several attempts of optimization of the chromatographic conditions were carried out, modifying solvents, the gradient of elution, ow rate, analytical columns, and column temperature. Aer optimization of the method, the chromatographic separation was performed using an XBridge, BEH C 18 column (100 Â 3.0 mm i.d., 2.5 mm, Waters). The separations were obtained by gradient elution using solvent A (0.2% aqueous formic acid) and solvent B (acetonitrile). The following multi-step linear gradient prole was applied: 0.1% B (4 min), 0.1-8% B (16 min), 8-10% B (20 min), 10-30% B (15 min), 30-100% B (4 min) and 100% B (1 min) in a 60 min. total time analysis. Sample injection volumes were 10 mL, column temperature of 30 C and a 0.6 mL min À1 mobile phase ow rate were set. The column was conditioned at 0.1% B for ve minutes between each analysis. UV spectra were recorded between l ¼ 190 and 400 nm and the chromatographic proles were recorded at l ¼ 254, 265, 290 and 330 nm, but the detection wavelengths were set at l ¼ 265 nm.

Preparation of standard solutions
Five stock solutions of gallic acid (1), methyl gallate (2), ellagic acid (3), agathisavone (4) and amentoavone (5) in MeOH were separately prepared at concentrations of 400 mg L À1 , 400 mg L À1 , 200 mg L À1 , 200 mg L À1 and 24 mg L À1 , respectively. Serial dilutions of each stock solution afforded the working solutions for generating calibration curves and for method validation.

Method validation
Selectivity, linearity, precision, accuracy, limit of detection (LOD), limit of quantication (LOQ) ( Table 1) and robustness were determinated according to the requirements of the Brazilian Government proposed by National Agency of Sanitary Vigilance (ANVISA), 14 International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use -ICH, 15 and suggestions proposed by Ribani et al. 16 The selectivity of the method was evaluated by the spectral homogeneity of the chromatographic peaks, by comparing the retention times of the standards with the chromatographic peaks of the analyzed compounds, by the overlapping of the chromatograms and the UV spectra obtained from the standards and extract. The analysis of the spectral homogeneity of the peaks was performed by observing the spectra recorded in the ascending, apical and descending regions of the peaks, being considered pure when they presented exact overlap (Fig. 2). The standard stock solutions were diluted with methanol to provide solutions with a range of ve different concentrations of the standards were analyzed by three replicate injections (Table 1). Calibration graphs generated by plotting peak areas vs. concentrations were analyzed by linear regression. Linearity was accessed by analyzing regression parameters, by residual plot analysis and by analysis of variance (ANOVA, with p ¼ 95%). The repeatability (intraday precision) and the intermediate precision (interday precision) of the method were determined in three different concentrations. The repeatability was performed on a single day with the same equipment and analyst, the analysis was performed in quadruplicate of the standard solutions in different levels of concentration: low, medium and a RT ¼ retention times in minutes. b In the regression equation y ¼ ax + b, x refers to the concentration (mg mL À1 ) and y is the peak area. c R 2 is the correlation coefficient of the equation. d LOD is the limit of detection. LOQ is the limit of quantication.  (Table 2). Intermediate precision was assessed by performing triplicate analyzes on four non-consecutive days of the standard solutions at the same concentrations used for intraday precision. The relative standard deviation (RSD) was taken as a measure of precision. Accuracy was determined in recovery experiments using both MLCM and MBCM extracts of. The MBCM extract was fortied with gallic acid (1), methyl gallate (2) and ellagic acid (3) and the MLCM extract was fortied with agathisavone (4) and amen-toavone (5) once the biavonoids did not occur in barks of C. macrophyllum. All fortications were performed in triplicate and at three different concentration levels (low, medium and high) for each standard ( Table 3).
The linear regression equation of the calibration curve was used to determine the LOD and LOQ ( Table 1). The LOD and LOQ were calculated based on the standard deviation (SD) of the y-intercept and the slope (s) as 3.3 SD/s and 10 SD/s, respectively. To evaluate the behavior of the analytical system, when small changes were made in the optimal operating conditions, the robustness test was performed. A standard solution of 12.5 mg mL À1 ellagic acid was used in all experiments. This analysis was performed using the previously optimized chromatographic conditions. In the evaluation of the robustness, the following parameters were used: ow (0.55, 0.60 and 0.65 mL min À1 ), percentage of formic acid in water (0.1%, 0.2% and 0.3%) and column temperature (25, 30 and 35 C). The robustness evaluation was performed through a full two-level factorial design and the results interpreted from the analysis of variance (ANOVA). The central point was determined in triplicate to allow an estimation of the error and to verify if the modications cause curvature in the model and the other experiments were performed in duplicate. All experiments were performed randomly. Table 4 shows the experimental matrix.
The results were treated in the STATISTICA 8.0 program with a 95% condence level.
2.7 Biological activity 2.7.1 Antioxidant activity by DPPH quenching. The determination of the antioxidant activity of MLCM and MBCM extracts was carried out as previously reported with few adaptations. 17 A methanolic stock solution of DPPH (Sigma, 40 mg mL À1 ) and solutions with the MLCM and MBCM extracts (50, 100, 150, 200, 250 mg mL À1 ) in methanol were prepared. The absorbance measurements of the reaction mixtures (300 mL of extract solution or standard or methanol for control, and 2.7 mL DPPH stock solution) were performed at 515 nm, immediately aer 30 minutes of incubation of the reaction at room temperature, protected from light, in a spectrophotometer (Cary 50 UV-VIS, Varian, Australia). Quercetin was used as standard 17 and were prepared in the same way as the extracts. All analyzes were performed in triplicates.
The percentage of free radical scavenging (% FRS) was calculated as: The efficient concentration, amount of antioxidant needed to reduce the initial concentration of DPPH by 50% (EC 50 ), was calculated by linear regression, where the abscissa axis represented sample (mg mL À1 ) or positive control concentrations and ordinate the percentage of free radical sequestration (% FRS). When necessary, dilutions were carried out at the concentrations of the samples and the standard to enable the calculation of the EC 50 .
2.7.2 Antioxidant assay using the b-carotene/linolenic acid method. The evaluation of the antioxidant activity using the bcarotene/linolenic acid system was performed according to a method previously described. 18 A solution of 2 mg of b-carotene in 1 mL of CHCl 3 was pipetted into a ask containing 40 mg of linolenic acid and 400 mg of Tween 80. The solvent was removed from the mixture by rotary evaporation and then 200 mL of distilled water was added, stirring to promote aeration and forming an emulsion. A 2.7 mL aliquot of the emulsion was added to a cuvette containing 300 mL of the extracts or standards (50, 100, 150, 200, 250 mg mL À1 in methanol) and the absorbance was measured at 470 nm with immediate reading and 15 minutes intervals during 1 h. The cuvettes were incubated in a water bath at 50 C. The control was prepared with 2.7 mL of the oxidizing medium and 300 mL of methanol. All analyses were performed in triplicates, using quercetin as standard, at the same concentrations of the extracts. Antioxidant activity (AA) was calculated using the following equation: where DR C is the degradation rate of the control (DR C ¼ ln(a/b)/ 60), DR S is the degradation rate in the presence of the sample (DR S ¼ ln(a/b)/60), a is the initial absorbance at time 0, and b is the absorbance at 60 or 30 min. 19 The efficient concentration, amount of antioxidant needed to reduce the percent inhibition of oxidation by 50% (EC 50 ), was calculated by linear regression, where the abscissa axis represented sample (mg mL À1 ) or positive control concentrations and ordinate the percentage of antioxidant activity (% AA). When necessary, dilutions were carried out at the concentrations of the samples and the standard to enable the calculation of the EC 50 . 2.7.3 Evaluation of acetylcholinesterase (AChE) inhibitory activity. The in vitro inhibition of AChE was determined by the spectrophotometric method based on the methodology previously described. 12 Solutions of 10 mg mL À1 of extracts and positive control were prepared in HPLC grade ethanol. To perform the test, 140 mL of phosphate buffer (0.1 M) pH 7.5 with 0.1% bovine serum albumin, 20 mL of the extracts, standard (physostigmine, Sigma $99%, was used as positive control) or ethanol (blank) and 20 mL of AChE 5.0 U mL À1 , were added on a microplate reader, all the experiments were carried out in triplicate. Aer 15 minutes of incubation, 10 mL of 75 mM acetylthiocholine iodide (ACTI) and 10 mL of 10 mM 5-5 0dithiobis-2-nitrobenzoic acid (DTNB) was added. The microplate was then read at 405 nm. The percentage of inhibition was obtained through the equation: where AChI represents the activity obtained in the presence of the inhibitor and AChE in the absence of the inhibitor.
2.7.4 Pharmacological tests 2.7.4.1 Animals. All experiments were performed with male Balb/C mice (bodyweights between 20-25 g) obtained and maintained at our Animal Facility at the Multidisciplinary Health Institute of the Federal University of Bahia. The animals were maintained in a temperature-controlled room (25 AE 2 C) with controlled humidity (50-70%) and a 12 h light/dark cycle. The animals were kept in polypropylene boxes containing wood shavings at the base of the box with free access to food (Labina®, Purina) and ltered water. The mice were homogeneously distributed among the groups. All animals were allowed to acclimatize to the air-conditioned laboratory for at least two hours before the tests, which were performed during the light cycle phase.
2.7.4.2 Acetic acid-induced writhing test. The antinociceptive effect was evaluated in mice using the acid acetic writhing test according to the procedures previously described. 21 Animals were treated subcutaneously (s.c.) with the MLCM and MBCM extracts (100 or 200 mg kg À1 ) 30 min prior to intraperitoneal (i.p.) injection of 1.0% acetic acid (0.1 mL 10 g À1 , Sigma, St. Louis, MO, USA). The negative control received only vehicle (negative control group, s.c.). The mice were placed in separate boxes and, aer the administration of the acetic acid, the number of writhes and stretching movements (contraction of the abdominal musculature and extension of the hind limbs) was counted at 5 min intervals for a period of 30 min. Antinociceptive activity was expressed as the writhing scores over 30 min.
2.7.4.3 Nociception induced by intraplantar injection of formalin. Formalin-induced hypernociception behavior was assessed as described previously. 20 Mice were allowed to acclimate for 15 min in a plastic box and pretreated 30 min prior to the injection with MLCM and MBCM extract (100 mg kg À1 , s.c.), vehicle (s.c.), or morphine (5 mg kg À1 , s.c.). Then, the mice were injected subcutaneously with 20 mL of 1.5% formaldehyde (St. Louis, MO, USA) in 0.9% saline into the plantar right hindpaws. The mice were observed for 30 min aer the formalin injection, and the pain behavior was determined by the number of inches, biting and licking of the injected paw counted during the observation period time. The acute phase (phase 1) was dened as 0-15 min aer injection, and the persistent tonic phase (phase 2) was dened as 15-30 min aer injection.
2.7.4.4 Paw edema induced by intraplantar injection of formalin. Aer the end of the experiment of nociception induced by intraplantar injection of formalin, both posterior paws were removed and weighed using an analytical balance. The contralateral paw was used as a control. The weight of the paw that did not receive the formalin injection was subtracted from the weight of the injected paw to determine edema formation. The results were express in grams (g). Percentage of antiedematogenic effect was also calculated according to the following formula: % AN ¼ [(control group average of the edema À test group average of edema)/(control group average of the edema)] Â 100.

Evaluation of neutrophil migration.
To investigate neutrophil migration to the peritoneal cavity, the MLCM and MBCM extracts (100 mg kg À1 ), were administered subcutaneously 30 min prior to the administration of the inammatory stimulus: an intraperitoneal injection of Carrageenan (Cg -Sigma, St. Louis, MO, USA) at a dose of 500 mg per cavity. 20 The negative control group received the vehicle. The mice were sacriced 4 h aer Cg administration, and the peritoneal cavity cells were harvested by washing the cavity with 3 mL of phosphate-buffered saline (PBS) containing 1 mM EDTA. The recovered volume was similar in all experimental groups and equated to approximately 95% of the injected volume. Total counts were performed in a Newbauer chamber. Differential cell counts (100 cells in total) were performed on a cytocentrifuge (PRESVAC CT12, Curitiba, Brazil) using slides stained with Panoptic. The results are presented as the number of neutrophils per cavity.

Statistical analysis.
In the tests performed with animals the results were presented as the mean AE SD (n ¼ 6 per experiment). Statistical comparisons between groups were rst analyzed using one-way analysis of variance (ANOVA) followed by the Bonferroni test for multiple comparisons in the Graph-Pad PRISM version 5.00 program. Statistical differences were considered to be signicant at p < 0.05. In the assays of antioxidant activity and inhibition of AChE the results presented in this study correspond to the average of three replicates (n ¼ 3) AE SD. For these assays, the results were considered statistically different, with a probability of occurrence of the null hypothesis less than 5% (p < 0.05) applying one-way analysis of variance (ANOVA), followed by the Bonferroni test for multiple comparisons in the Microcal Origin 7.0 soware.

Statement to conrm the experimental protocols
Animal care and research protocols were in accordance with the guidelines and regulations adopted by the Brazilian College of Animal Experimentation (COBEA) and were approved by the Ethical Committee for Animal Research of the University of Uberaba, Brazil (protocol # 0107/2009). The number of animals used was the minimum number necessary to show consistent effects of the drug treatments.

Results and discussion
For the optimization of the chromatographic separation conditions the inuence of the following factors in the chromatographic peak elutions were evaluated: different stationary and mobile phases, changes in the elution program, mobile phase ow, volume injection, wavelength and, column temperature. Two columns, XBridge, BEH C 18 column (100 Â 3.0 mm i.d., 2.5 mm) and Acclaim RSLC 120 C 18 column (100 Â 2.1 mm i.d., 5 mm), were tested for their separation efficiencies. The results showed that the best separation was achieved on an XBridge, BEH C 18 column.
The choice of the best wavelength for the analysis of the phenolics was performed by monitoring the wavelength range of 190 to 400 nm and the UV absorption spectrum of each peak was registered. The wavelength selected for the analyzes was 265 nm, in which good sensitivity was obtained for most of the substances present in the extracts. The mobile phase composed of acetonitrile-water presented the best separation resolution. The presence of acid modiers in the mobile phase suppressed the tailing effect of the peaks and an improvement in the separation was observed (formic acid 0.2% (v/v) and ow-rate of 0.6 mL min À1 ). The separation was better when the column temperature was kept at 30 C rather than other temperatures. Fig. 2 shows the HPLC-DAD chromatograms of MLCM and MBCM extracts and the chromatograms were used to considerate the obtained extracts as standardized.
The identication compounds present in the extracts were carried out by comparison of their retention times and UV spectra with the standards (in the ascending, apical and descending regions of the peaks), conrming the selectivity of the method. Before starting the linearity study, the reproducibility of sample preparation based on the evaluation of retention time and peak area was evaluated for the chromatographic peaks referring to compounds 1-3 present in the MBCM extract (Fig. 2C). 21 The results demonstrated the reproducibility of the sample preparation, since the RSD was less than 2.38% for triplicate analyses. The MBCM extract was analyzed in replicate (n ¼ 10) to evaluate the precision of the injection, which was measured by sequential repetitive injections of the same sample. The analysis of the results of the injection precision showed that the preparation of the samples presented good reproducibility in the optimized method (RSD < 5%, for the peaks referring to 1, 2 and 3).
The linearity was determined by the external standard method. Table 1 describes the retention times, regression equation, the correlation coefficient for triplicate analyses, range, LOD, LOQ and, RSD (%). The ve compounds analyzed showed satisfactory linearity with the correlation coefficient (R 2 ) greater than 0.999 for all compounds. The optimized method presented high sensitivity for all the compounds, which indicated an excellent capacity to respond to distinctly small differences in concentrations. The residual dispersion of all analytes followed a random distribution pattern, which indicates adequacy of the linear models applied to the obtained responses, which conrms the good linear t of the analytical curve. The analysis of variance (ANOVA) allowed evaluating the linearity of the method and the validity of the regression. One way to verify the validity of the regression is by comparing the values of calculated F and tabulated F value. If it is veried that calculated F > tabulated F (at least 10 times greater), there is enough statistical evidence to believe in the existence of a linear relationship between the variables y and x. The values of calculated F were 880.04, 1810.27, 4723.36, 7621.22 and 4738.07 for the data obtained in the construction of the analytical curves of the standards 1-5 respectively, which are much higher than the tabulated F (F 1,13 ¼ 4.67) at a condence level of 95%. Therefore, the linearity of the method is accepted, and the regression can be considered signicant.
Good sensitivity was demonstrated with LODs and LOQs in the range of 0.21-0.56 mg mL À1 and 0.65-1.70 mg mL À1 , respectively for the gallic acid derivatives (1-3) and in the range of 0.44-0.56 mg mL À1 and 1.34-1.70 mg mL À1 , respectively for the bioavonoids 4 and 5, respectively. The values were comparable to those reported in previous analysis for compounds 1, 2, 3 and 4. [22][23][24][25][26][27] The results obtained for repeatability and intermediate precision are also presented in Table 2. For both tests, the RSD was less than 5% for triplicate analyses at the three different concentrations, as recommended by ICH protocols and ANVISA. 14,15 Accuracy was determined in recovery experiments and the results are presented in Table 3. The method proposed in the present study presented a good recovery for all the compounds in the three concentration levels, in the range of 84.12 to 106.64% and with RSD less than 5%, all values being within acceptable limits.
There are few reports of the use of multivariate optimization techniques to determine robustness in validation studies. 28,29 The results for robustness evaluation are presented in the Pareto chart (Fig. 3) and demonstrate that the modications performed in the optimal conditions of the chromatographic method did not present signicant effects for the variables ow, column temperature and percentage of formic acid in water. Thus, the chromatographic method presented robustness and therefore the validation parameters indicate that the conditions used in the quantitative determination were satisfactory.
The presented chromatographic method was successfully applied in the simultaneous determination of 1-5 in the extracts MBCM and MLCM (Table 5). Ellagic acid (3) is the compound present in major concentrations in both extracts and the bioavonoids agathisavone (4) and amentoavone (5) were detected only on leaves which is in accordance with the literature, since 4 and 5 were isolated only from the leaves of C. macrophyllum, 7,11 whereas ellagic acid was isolated from both leaves and stem bark 3,7 and, this is the rst report of gallic acid and methyl gallate in the stem bark of this species. In the previous work 11 from stem bark from a specimen collected in different habitat was isolated bergenin as the main compound of this species. However, it is not detected in both extracts of the specimen studied. Other avonoid derivates also previously isolated from leaves C. macrophyllum were not also detected in the present species collected in different bioma. As pointed previously C. macrophyllum species of the Caatinga biome was classied until recently as another species, C. gardnerianum Tul. and later it was considered a synonym of C. macrophyllum.
The substances quantied in this work present different biological activities [30][31][32][33][34][35] and even if they are not directly responsible for the use of the species in traditional medicine, it is expected with this analytical methodology to contribute to a future application in the quality control of future phytotherapics of this medicinal plant.
The analysis of the HPLC ngerprints of the MLCM and MBCM extracts allows the realization of biological and pharmacological assays in order to investigate the folk medicinal employment of this plant. So, antioxidant, inhibition of AChE, antinociceptive and anti-inammatory activities of the standardized extracts of MBCM and MLCM were evaluated. Table 6 shows the EC 50 values of AA of the extracts and the control obtained by the two known methods. According to literature 36,37 the AA by the DPPH method can be classied as good if the EC 50 of the extract is up to three times the EC 50 of the standard. Following this criterion, the two extracts presented a good antioxidant activity. Regarding the statistical analysis of the results, it was veried that the extract MLCM showed a higher free radical scavenging activity with concerning MBCM extract (p < 0.05). This result can be justied by the presence of the avonoids agathisavone and amentoavone in this extract. The results of the assay using the b-carotene/linolenic acid system indicated that the MLCM extract presented a higher AA when compared to the MBCM extract in the two analyzed times, statistically signicant differences were veried (p < 0.05), which corroborates with the results obtained in the evaluation of the AA by the DPPH method. There was a decrease in activity over time for both extracts and standards, and this result was signicant for the extracts and not signicant for the standards (p < 0.05). The AA of the standards was statistically higher when compared to the extracts. These results encouraged the evaluation of the antinociceptive and anti-inammatory activities of both extracts.
To evaluate the peripheral antinociceptive effect extracts the acetic acid-induced writhing test was performed with 100 and 200 mg kg À1 doses of MBCM and MLCM (Fig. 4). Both 200 mg kg À1 doses resulted in a signicant reduction (P < 0.05) in the  number of writhing episodes compared to the group treated with the vehicle (negative control group). But, at the doses of 100 mg kg À1 only the MBCM extract was able to signicantly reduce (P < 0.05) the number of writhing episodes compared to the group treated with the vehicle. Formalin model was used to conrm the antinociceptive effect of extracts. In this model, the antinociceptive and also a possible anti-inammatory effect were evaluated (Fig. 5). The administration of MBCM and MLCM extracts at 100 mg kg À1 dose 30 min before formalin nociception were able to inhibit the formalin-induced nociception produced during the neurogenic phase in the animal paw (P < 0.05) (Fig. 5, 0-5 min). Morphine showed good activity in reduction of inches compared to negative control (P < 0.05) and the MBCM extract did not present statistical difference in relation to the positive control (morphine, P < 0.05). Flinches in the inammatory phase (Fig. 5, 5-30 min) were signicantly decreased by the two extracts compared to the vehicle-treated group (P < 0.05). The MLCM extract did not present a statistical difference in relation to the positive control (morphine, P < 0.05) in the inammatory phase.
The treatment of mice with the MBCM and MLCM extracts showed a signicant reduction in the edema formation induced by formalin injection in the paws when compared to the vehicle group (P < 0.05), indicating the anti-inammatory activity of the extracts (Fig. 6).
The anti-inammatory potentials of the MBCM and MLCM extracts were also evaluated using the inhibition of Cg-induced neutrophil migration to the peritoneal cavity (Fig. 7). Pretreatment with 100 mg kg À1 (s.c.) with both extracts promoted a decrease in neutrophil migration (P < 0.05) to the peritoneal cavity. Further tests are needed to elucidate the mechanism of action of the extract.
Previously the antinociceptive activity reported in the literature for bergenin isolated from barks of C. macrophyllum, 11 a specimen collected in the Caatinga of Bahia. In the present Table 6 Antioxidant activity by the free radical scavenging method (DPPH) and using the b-carotene/linolenic acid system and inhibition of AChE study, this compound is not responsible for the activity presented by the extract MBCM, since bergenin was not found in this extract. Piaulino et al. 9 investigated the antinociceptive actions of the ethanol extract and ethyl acetate fraction from C. macrophyllum stem bark in streptozotocin-induced diabetic rats and the involvement of opioid and nitrergic mechanisms. The authors showed that analysis by thin layer chromatography using authentic standard and suggested the presence of ellagic acid and valoneic acid dilactone. Thus, the results on antinociceptive activity presented in the present study conrm the results reported in the literature, since the MBCM extract ellagic acid was detected as the main compound. Besides that, the qualitative analysis of the HPLC ngerprints of the MBCM extract showed the presence of ellagic acid derivatives. Besides, the compound 1 that shows recognized antinociceptive activity 38 was also quantied in the present extracts. The MLCM extract also showed antinociceptive activity, in addition to compounds 1 and 3 (the main compounds), this activity is related to the presence of biavonoids 4 and 5. The anti-inammatory activity presented by both extracts is due to the presence of compounds 1, 3, 4 and 5, which have recognized activity. 34,36,39,40 The AChE inhibitory activity of standardized extracts was also evaluated. MBCM extract presented higher AChE inhibitory activity than the MLCM (Table 6) in the two analyzed periods of time and, statistically signicant differences (p < 0.05) between the extracts were observed. The MBCM activity in 60 min test did not show statistically signicant differences in relation to the standard (p < 0.05). This result shows excellent activity of this extract. Previously, from leaves of C. macrophyllum were isolated two peptides inhibitors of AChE 12 but the compounds present in the extracts did not show AChE inhibition.

Conclusions
An HPLC-PAD method was developed to evaluate standardized C. macrophyllum extracts by chromatographic ngerprints and it also permitted to validate a simultaneous analysis gallic acid, methyl gallate, ellagic acid, amentoavone and agathisavone present in the leaves and stem bark of this plant species. The selectivity, correlation coefficients, LODs, LOQs, repeatability, intermediate precision, and accuracy were in good agreement with the requirements for the developed method. The robustness evaluation was performed through a full two-level factorial design and the results showed that the method was robust. The analytical method was successfully applied to determine the ve compounds in extracts being ellagic acid as the major component. The standardized extracts presented expressive antioxidant, AChE inhibitory, antinociceptive and anti-inammatory activities.

Conflicts of interest
There are no conicts to declare. Fig. 6 Effect of MBCM and MLCM extracts on the induction of paw edema in mice pretreated with an intraplantar injection of 1.5% formalin. VH is the vehicle group (negative control). The MBCM and MLCM extracts were tested at dose of 100 mg kg À1 (s.c.). The results are presented as the mean AE SD (n ¼ 8). Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P < 0.05 compared to the vehicle negative control treated group. # P < 0.05 when the vehicle group was compared to the saline group. Fig. 7 Effect of the MBCM and MLCM extracts on neutrophil migration into the peritoneal cavity of mice pretreated subcutaneously 30 min prior to Cg injection (500 mg per cavity) to induce peritonitis. VH is the vehicle group (negative control). The MBCM and MLCM extracts was tested at dose 100 mg kg À1 (s.c.). The results are presented as the mean AE SD (n ¼ 6). Statistical significance was calculated by ANOVA followed by Bonferroni's test. *P < 0.05 compared to the vehicle negative control treated group.


extfieldsofstudy: Chemistry Medicine

text:
Heats of combustion representative of the carbohydrate mass contained in fruits, vegetables, or cereals

Abstract The obtainment of suitable values for metabolizable energy requires the previous knowledge of accurate and precise values of the heat of combustion of the different macronutrients. Thus, in this work, the heats of combustion of six carbohydrates (glucose, fructose, sucrose, maltose, starch, and cellulose) were experimentally measured, and such values were statistically compared with equivalent bibliographic values collected in a parallel work (Heats of combustion of the main carbohydrates in vegetable foods: a bibliographic approach, 2019), proposing, for each carbohydrate, an “overall interval” and an actualized representative value, which were estimated considering jointly the bibliographic and experimental information. Besides, a numerical methodology that used such parameters and the relative content of the different carbohydrates in selected foods was proposed, to estimate the global heat of combustion producible by the carbohydrate mass contained in such foods. The results estimated for 68 foods were globalized to propose the following generalized heats of combustion: (a) for fruits: 3.88 kcal/g, (b) for vegetables: 3.98 kcal/g and, and (c) for cereals: 4.13 kcal/g. These results demonstrated that the use of the Atwater's value (4.2 kcal/g of carbohydrate of vegetable source) involves a clear overestimation of the heat of combustion of the carbohydrate mass contained in vegetable source foods.

first paper of this series (Sánchez-Peña et al., 2017), an exhaustive critical review of the experiments carried out by Atwater to obtain such values was presented, demonstrating that there is a series of weakness in the experimental design that could compromise the validity of such values.
Regarding carbohydrates, Atwater (1900) proposed that the metabolizable energy of carbohydrates (ME c ) can be estimated as follows: ME c = (HC c )A c , being HC c , the heat of combustion produced by each gram of carbohydrate and, A c , the availability coefficient of such macronutrient. Focusing on the A c values characterizing the vegetal source foods, Atwater and Bryant (1900) proposed the following values for such coefficient: for cereals: 0.98, for legumes: 0.97, for vegetables: 0.95, and for fruits: 0.90; although the deficiencies and information absences related to the obtainment procedure of such factors have been recently summarized (Sánchez-Peña et al., 2017), implicitly, such factors are still used. Therefore, even recognizing certain inaccuracy level on the A c factors reported by Atwater and Bryant (1900), it could be expectable that such factors are very close to 1.0; thus, the accuracy and precision level of the ME c value characterizing a given sample must be strongly dependent on the accuracy and precision level of its correspondent HC c value. Therefore, as a first step in the estimation of a suitable ME c value, the establishment of a suitable HC c value is required.
With respect to the HC c value, Atwater and Bryant (1900) proposed that, for vegetal source carbohydrates, HC c = 4.2 kcal/g.
Nevertheless, since such value was proposed considering an arbitrary "averaging procedure" that probably used inaccurate data (Sánchez-Peña et al., 2017), it could be considered necessary to reevaluate such parameter.
With such background, in a parallel work , an exhaustive bibliographic analysis related to the heats of combustion of glucose, fructose, sucrose, maltose, starch, and cellulose was performed. In this analysis, the experimental errors possibly involved in the original sources were taken into account, and following rigorous statistical arguments, the outlier data were eliminated.
In that work , the following information was reported for each carbohydrate: (a) a representative value for the heat of combustion of the respective carbohydrate (which could be considered as the best approximation to the real heat of combus- Therefore, the estimation of the agreement level between such parameters and a set of experimental values currently measured could be considered as necessary. Thus, to reinforce the validity of the previously mentioned bibliographic parameters (first intermediate aim of this work), an experimental procedure to measure the heat of combustion of the carbohydrates of interest was here implemented, where the experimental inaccuracy of the previous works were carefully minimized (see details below); for each carbohydrate, an "overall interval" and an actualized representative value were statistically estimated, considering jointly the bibliographic information collected by Sánchez-Peña (2019) and the experimental data obtained in this work.
Then, a mathematical procedure was here proposed, which allow to estimate a global representative value by mass unit (global value) and a global interval for the heat of combustion by mass unit (global interval) that could be properly used to characterize to the carbohydrate mass contained in a given food; second intermediate goal of this work. In such procedure, the statistically estimated parameters mentioned in the previous paragraph, as well as the contents of the different carbohydrates contained in the considered vegetal products (obtained from the USDA Food Composition Database (National Nutrient Database for Standard Reference, 2016)) were used.
Finally, the global objective of this work was to propose a final representative value and a global interval that could be used to adequately characterize the heat of combustion per unit mass, corresponding to the carbohydrate mass contained in each food group.
For this, the global intervals calculated for each one of the considered foods were grouped, following a procedure proposed here.

| Pretreatment and bromatological characterization
Samples were carefully dried in a vacuum oven (Fisher Scientific; Model 281A) heated at around 65°C. However, since the glucose, fructose, and sucrose samples exhibited a perceptible degradation process during such heating (e.g., browning and/or characteristic odor production), they were dried at vacuum conditions and room temperature. The dried samples were characterized, following the AOAC procedures (to avoid the humidification of samples, a careful handling was performed); the crude protein content was obtained by Kjeldahl method (Tests: 979.09; Official Methods of Analysis. AOAC International. Protein in grains, 2018a), considering a conversion factor of 5.83 g of protein/g of nitrogen (FAO Food and Nutrition, 2002), whereas the lipid content was measured by Soxhlet extraction (Test 920.39; Official Methods of Analysis. AOAC International. Fat (crude) or ether extract in animal feed, 2018b).

| Calorimetric characterization
The heat of combustion measurements were performed using an isoperibolic calorimeter, which included a combustion static bomb (Parr Model 1108; inner volume: 342 cm 3 ); the used calorimeter has been described in more detail elsewhere . The total calorific capacity of the calorimeter was determined by calibrating, using a certified standard of benzoic acid (certified heat of combustion: 6.318 ± 0.001 kcal/g) as the reference material and averaging 10 experimental measurements; benzoic acid samples used in these experiments were carefully handled to avoid any kind of contamination. Calorimetric measurements were performed in triplicate (using the same pattern sample), reporting as the representative value the mean value of the three experiments (HC exp ; expressed in kcal/g). The HC exp value was corrected considering the relative amounts of lipids and proteins contained in the characterized samples, which were determined by means of the above mentioned test. The correction equation used is as follows: In Equation 1, HC is the corrected value of the heat of combustion (expressed in kcal/g), and M T, M l and M p are, respectively, the mass of the tested sample and the masses of the lipids and proteins contained in such sample (expressed in g). Equation 1 considers the heats of combustion reported by Atwater and Bryant (1900), that is, 9.3 kcal/g for the vegetable source lipids and 5.8 kcal/g for proteins contained in cereals.
To validate the respective "bibliographic intervals" with the corrected experimental data (i.e., the respective HC values), as in our previous work , the boxplot (Iglewicz, 2011) and/or the Grubbs' test (Grubbs, 1969;NIST/SEMATECH, 2012) were used to assign, or not, the considered data as "statistically outlier data." Thus, the possible assignation of the experimental data (HC values) as "non-outlier data" (Sánchez-Peña et al., 2019) could be interpreted as a self-validation of the two information sources, the bibliographic, and the experimental one. In that case, the "overall intervals" can be estimated following the same procedure used to determine the correspondent "bibliographic intervals" (Sánchez-Peña et al., 2019), but considering as an additional datum of the each set of data the correspondent HC value. Since our experimental data were very carefully measured, following our parallel work (Sánchez-Peña et al., 2019), they were considered type L-III data. Henceforth, the respective "overall intervals" will be defined by means of their respective extreme values, that is, the HC h i (the highest value of the "overall interval" for the carbohydrate i ) and HC l i (the lowest value of the "overall interval" for the carbohydrate i ) values; from now on, for glucose, i = 1; fructose, i = 2; sucrose, i = 3 ; maltose, i = 4; starch, i = 5; cellulose, i = 6. Besides, the respective actualized representative values were estimated, like the correspondent representative values in our parallel paper (Sánchez-Peña et al., 2019), but considering the correspondent experimental value (HC value) as an additional type L-III datum.
To estimate the HC value of an anhydrous substance (HC anh ), the following equation can be used (Sánchez-Peña et al., 2019): where HC anh and HC hyd are, respectively, the heats of combustion for the anhydrous and hydrated forms of the tested substance, whereas MH and MA are, respectively, the relative masses of the hydrated and anhydrous substance, finally, HH substance is the hydration heat of the tested substance. Due to crystallographic reasons, there are X + 18 g of hydrated substance by each X g of anhydrous substance and 18 g of water, being X the molar mass (expressed in g/mol) of the tested substance. Since the HC exp value for the anhydrous maltose could not be experimentally determined, the equation 2 will be used to estimate the actualized representative value and the "overall interval" for such substance.

| Estimation of the "global interval"
for the heat of combustion and the global heat of combustion of the carbohydrate mass contained in a given food

Composition intervals
In the case of the carbohydrates contained in vegetal source foods, the information required to estimate the experimental interval for the respective compositions (C i and se i values; a pair for each carbohydrate contained in the considered food) was obtained from the USDA Food Composition Database (National Nutrient Database for Standard Reference, 2016), being such intervals estimable as follows (Montgomery, 2001): where h i is the statistical factor assignable to each carbohydrate (1 ≤ i ≤ 6; see definitions above), which depend on the number of data measured (n value), the desired level of distribution coverage (here, a coverage of 90% was selected) and the defined confidence level (in this work, a 90% was chosen, assuming a distribution with two tail) (Montgomery, 2001), C i are the respective mean values for the compositions of each carbohydrate contained in the different foods (expressed in mass percentage; 1 ≤ i ≤ 6; see definitions above), and se i are the respective standard errors of the experimental data used to calculate the corresponding C i values (1 ≤ i ≤ 6; see definitions above). Alternatively, such intervals can be defined by means of their respective extreme values, that is, the values; note that, when se i = 0, or this datum is not

The "overall interval" for the heats of combustion
Equivalently to the indicated intervals in section "Composition intervals", for each carbohydrate, there is an "overall interval" for the respective heats of combustion, which is defined by their respective extreme values, that is, the above defined HC h i and HC l i values (section 2.1.3.).

Estimating the "global interval" for the heat of combustion of the carbohydrates contained in a given food
When the six composition intervals (one for each carbohydrate type; section "Composition intervals") and the six "overall intervals" for the heats of combustion (one for each carbohydrate type; section The "overall interval" for the heats of combustion) are available, it is possible to define an interval (the "global interval") that very probably includes the heat of combustion produced by the carbohydrate mass contained in a given food.
To estimate such "global interval," the following calculation procedure is proposed: a Consider the extreme values of the six composition intervals (one interval for each carbohydrate type; i = 1, 2,…up to 6), that is, the six pairs of values C h i and C l i ; see section "Composition intervals". b Define 64 (=2 6 ) different sets, each one containing six values (one for each carbohydrate type, that is, one for each value of i ), utilizing all the combinations that result from selecting for each value of i , one of the two following values: C h i or C l i . Please consider the following example: C h 1 ,C h 2 ,C l 3 ,C h 4 ,C l 5 ,C h 6 ; in this set, the first term could be: C h 1 or C l 1 , having been selected in this case C h 1 , the second one could be, C h 2 or C l 2 , having been selected for this set C h 2 , and so on up to the last term, C h 6 , which was chosen between C h 6 and C l 6 .
c Consider now the extreme values of the six "overall intervals" for the heats of combustion (one interval for each carbohydrate type, that is, for i = 1, 2,…up to 6), that is, the six pairs of values HC  where C i is the mean percentage composition reported for the respective carbohydrate i in a given food (e.g., those values reported for the USDA Food Composition Database (National Nutrient Database for Standard Reference, 2016)), and HC i is the actualized representative value for the heat of combustion of the corresponding carbohydrate (estimated according to section 2.1.3.).

| Global heat of combustion
However, when the information required to estimate the "global interval" for the heat of combustion of the carbohydrates contained in a given food is available (section "Estimating the "global interval" for the heat of combustion of the carbohydrates contained in a given food"), an alternative option to estimate the global heat of combustion representative of the carbohydrate mass contained in such food (HC f c ), is to average the 4,096 HC f c | x values (section "Estimating the "global interval" for the heat of combustion of the carbohydrates contained in a given food"). Note that, although the HC f c value could a priori be considered equivalent to the HC cinf value, the statistical superiority of the first one is evident; numerical comparisons on this topic will be presented below.
For this study, a computational program was specifically designed to perform the calculation procedures described in sections "Estimating the "global interval" for the heat of combustion of the carbohydrates contained in a given food" and 2.2.2.   Figure 1, the data referring to vegetables were presented in Figure 2 and, finally, the values regarding cereals were showed in Figure 3.

| D ISCUSS I ON
Since the respective HC values could not be statistically classified as "outlier data" (Grubbs,1969;Iglewicz, 2011;NIST/SEMATECH, 2012),  Regarding the global intervals, the first aspect that should be mentioned is related with the corresponding "interval lengths" (e.g., the maximum value minus the minimum one of the respective global interval). About this topic, although the USDA Food Composition Database (National Nutrient Database for Standard Reference, 2016) lists the contents of the different carbohydrates present in numerous foods, for some of them, the reported se values were comparatively high, which produced "interval lengths" that were herein considered too long. In this work, the "interval lengths"> 0.32 kcal/g were considered too long, therefore, the foods that fulfill this condition were not considered in this report. Taking into account only the 68 foods considered in Tables 1-3, only in five of them, the "interval lengths" were in the proposed limit value (0.32 kcal/g or, equivalently, ±4%).
Thus, looking for a estimation of the inaccuracy level expectable to use the HC cinf parameter as a representative value for a given food (its calculation facility makes it a "very attractive" parameter) instead of the correspondent global interval, in Tables 1-3 it can be noticed that, in the vast majority of considered foods (87%), the correspondent global intervals are within the generic interval HC cinf ± 4%; only in nine foods, one of the extremes of the individual interval exceeded to the generic interval, falling out of it around 0.01 kcal/g. Based on the previous results, it could be affirmed that the expectable error to consider as representative of the calorimetric behavior of a given food to its HC cinf value is ≤4%; by its evident calculation facility, at least in some cases, this could be an attractive option to approximate the calorimetric behavior in a given food.
To facilitate the analysis of the global intervals of the samples corresponding to the same type of food (fruits, vegetables, or cereals), Figures 1-3 are presented. Thus, Figure 1 includes the available information for fruits, being there possible to appreciate that the 31 global intervals can be suitably represented by the following globalizing mathematical expression: 3.88 kcal/g ± 4%; such expression is graphically represented by the horizontal lines in Figure 1.  Table 1. The proposed interval to represent the set of global intervals is limited by dotted lines and its central line is represented by the continuous horizontal line (see additional information in text) The previous expression was ex-profeso calculated to "exclude," in both extremes (superior and inferior), less than 2.5% of the "global interval length" (calculated as the arithmetic sum of the 31 "interval lengths").
Per the above, and considering the available data for fruits ( Figure 1), it can here be generalized that: HC fruits c = 3.88kcal/g ± 4% . When comparing this value with the equivalent one proposed by Atwater for vegetable source foods (HC c = 4.2kcal/g), it is evident that an overestimation that ranges between 4% and 11% is involved when Atwater's value is used.
In Figure 2, the available information relative to the considered vegetables is presented. Then, after an equivalent analysis to the previously described, it was found that the mathematical expression suitable for vegetables is as follows: HC vegetables c = 3.98kcal/g ± 5%; in this type of foods, the use of Atwater's value (4.2 kcal/g) involves an overestimation on the heat of combustion value that can become as high as 10%.
Finally, when the available results for cereals are analyzed in Figure 3, it can be proposed that the representative expression for this type of foods is as follows: HC cereals c = 4.13kcal/g ± 3%. As in previous food groups (fruits and vegetables), the use of Atwater's value involves an overestimation of the correspondent heat of combustion value, which, in this specific food group can become as high as 5%.

| CON CLUS IONS
The mutual consistency between the experimental results determined herein and the bibliographic data collected in our parallel work (Sánchez-Peña et al., 2019) allow us to consider them as experimentally validated, allowing its suitable use in later calculations.
In addition, the obtained generalized intervals demonstrate that, in the case of vegetable source foods, the use of Atwater's value  Table 3. The proposed interval to represent the set of global intervals is limited by dotted lines and its central line is represented by the continuous horizontal line (see additional information in text) the heat of combustion of the carbohydrates contained in vegetable source foods, but, besides (and maybe mainly), represents a set of values much more supported/argued from the experimental and statistical point of views.

ACK N OWLED G M ENTS
Authors thank the University of Guadalajara for the financial support, and the students Martínez-Navarro A.G. and Muñiz-Mendoza P. are grateful for their respective Conacyt scholarships.

CO N FLI C T O F I NTE R E S T
Authors declare that they do not have any conflict of interest.

E TH I C A L R E V I E W
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Does not apply.