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Official websites use. Share sensitive information only on official, secure websites. This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The following preliminary phytochemical study of the Bidens pilosa L. The changes that were made in relation to the previous version were updating of the bibliography in the Phytochemical screening section, and comments were also added in the Determination of total phenols and flavonoids and Vegetal material sections. Lastly, Figure 1 was added. Background: Given the chemical richness of medicinal plants Bidens pilosa L. Three extraction processes were carried out, with two solvents of different polarities: hexane and ethanol. The extraction methods that were applied to the leaves of the plants were Soxhlet, ultrasonic bath and maceration, the latter two at room temperature and Soxhlet at the boiling temperature of the solvent. Determination of the total content of phenols and flavonoids is carried out using the Follin-Ciocalteau colorimetric reaction, Quercetin standard, Aluminum Chloride solution measured with a UV-Vis spectrophotometer. The antioxidant activity was performed with the DPPH radical and measured with the same equipment. Finally, the antioxidant activity against the 1. Conclusions: The following preliminary phytochemical study of the Bidens pilosa L. Keywords: extraction, phytochemical study, phenols, flavonoids, Bidens pilosa L, Croton floccosus, Asteraceae, Euphorbiaceae. The biodiversity of Ecuador ranks sixth worldwide for the number of species. Antimicrobial, 6 antioxidant, 7 anti-inflammatory 8 and anticancer 9 , 10 biological activity studies have been carried out on them in America, Asia and Africa. Brian A. Smith found a new species of Croton Euphorbiaceae on the western slopes of the Ecuadorian Andes in , Croton floccosus. This plant is commonly found next to streams and disturbed sites in the provinces of Pichincha and Imbabura in Ecuador. Some to species of Bidens have been described, 12 , 13 including Bidens pilosa , which was identified in by Carl Linnaeus. This is a representative perennial herb, distributed globally in temperate and tropical regions, which has traditionally been used in food and medicine without having any obvious adverse effects. About publications on this species have documented its medical use, 14 and phytochemical studies show that it contains a high number of flavonoids and polyines, which have anticancer, 15 anti-inflammatory, 16 antibacterial 17 biological activity, as well as antifungal, 18 antimalarial, 19 and antioxidant 20 , 21 properties. Bidens pilosa is an erect, strongly branched plant with a strong aromatic odor. It has opposite leaves and a long petiole, whose limbs are generally deeply divided into 3 to 5 segments giving it the appearance of a compound leaf. It also has white ray flowers and numerous yellow tubular flowers. Although this species has been studied to a greater extent than Croton floccosus , we have not found phytochemical studies of these plants in Ecuador, which is why it has important research potential that may allow us to transform traditional knowledge into scientific knowledge. In the following work, a preliminary phytochemical study of the leaves of the species Bidens pilosa and Croton floccosus was carried out using the Tukey test, 22 which is employed in ANOVA in order to compare the means of the values obtained. This study joins the recently published work by Ruiz-Reyes et al. The fresh leaves of the Bidens pilosa L. The leaves that present better conservation in their structure are chosen to clean them of dust and branches, then the petiole of the leaf blade is extracted. The studied leaves were collected from various plants and then mixed to create the sample for analysis. The botanical identification was carried out by the botanists in charge of the herbarium in the botanical garden at the UTM. The vouchers of the specimens were deposited with the following codes Bidens pilosa L. Asteraceae and Croton floccosus Euphorbiaceae. It is shown in the following map of the botanical garden. The collection points of the studied species. For croton floccosus it was two points while for bidens pilosa it was three points. The coordinates of the studied species are placed on the map Figure 1. The chemicals and reagents employed in this study were: Folin-Ciocalteu reagent, 1. All the reagents and solvents were supplied by Sigma-Aldrich and are of an appropriate analytical grade for study. A reference weight was taken every two hours until it remained constant. The sample was pulverized using a blade mill grinder with a sieve so that a more uniform size of the material can be obtained, in order to fractionate the plant tissue and produce a better extraction. The time used for grinding was 5 minutes. The extracts of the crude plants were obtained using the ultrasonic bath, maceration and Soxhlet extraction methods. In the case of Soxhlet extraction, 10 g of plant material were used and placed in the equipment extractor cartridge with mL of ethanol. Between 8 and 10 extractions were performed at the boiling temperature of the solvent, and the solvent was eventually evaporated in order to obtain the extracts. With regard to ultrasonic bath extraction, 10 g of finely divided plant material was placed in a mL beaker containing mL of ethanol. The mixture was sonicated at 50 W for 1 h in an ultrasonic bath and subsequently filtered. The solvent was then evaporated in a vacuum to obtain the corresponding extracts for analysis. In the case of extraction with maceration, 10 g of finely divided plant material was placed in a mL beaker with mL of ethanol. The mixture was macerated for 72 hours and stirred from time to time. It was subsequently filtered and the solvent was evaporated in a vacuum to obtain the corresponding extracts for analysis. Drops of Wagner, Mayer, Bouchardat, and Dragendorff reagents were added to each one. A red-brown precipitate Wagner , a yellowish white precipitate Mayer , a brown precipitate Bouchardat , and an orange-red precipitate Dragendorff indicated the presence of these metabolites. Flavonoids Shinoda test : 1 ml of absolute ethanol and three drops of concentrated hydrochloric acid were added to 10 drops of the extract diluted in isopropyl alcohol. The formation of a red color indicated the presence of aurones and chalcones while the formation of an orange, red, or magenta color formation indicated the presence of isoflavones, flavonols, and flavonoids, respectively. The formation of honeycomb shaped foam indicated the presence of saponins, and tannins: 10 mg of each extract was dissolved in 1 mL of ethanol and extract with 3 mL of boiling distilled water for 15 minutes. Once it had been allowed to stand at room temperature, 0. The precipitation observed was indicative of the presence of tannins. It was considered positive if it turned reddish brown. Determination of the total content of phenols in the extracts was carried out using the Follin-Ciocalteau reaction and by employing gallic acid as the reference phenolic compound. The gallic acid calibration curve was prepared by weighing 2 mg of gallic acid and making it up to a volume of 10 mL with distilled water, which is the stock solution at a concentration of 0. After the absence of light for 30 minutes and at room temperature, we measured the absorbance at nm using the prepared solution as a blank without the analyte. Total flavonoid content was determined using quercetin as standard. The same solution was used as a blank without adding the standard compound. After waiting 15 min for the reaction, the absorbance of the solution was measured at a wavelength of nm in the UV-Vis spectrophotometer. After waiting 15 min, the absorbance of the solution was measured at a wavelength of nm in the UV-Vis spectrophotometer. Both methodologies were described by Ruiz-Reyes. The antioxidant activity was determined using a spectrophotometer and the DPPH molecule as a reagent to generate the free radical following the methodology described by the author himself and other authors. This was then homogenized and left to react for 24 hours in an amber bottle in the dark. A scan was carried out in the spectrophotometer. A maximum absorbance of 0. Methanol 15 mL was added and a scan was performed in the spectrophotometer to verify that it did not have absorbance in the working wavelength range in which the maximum of the sample is found. We took 3. Once the sample and the blank were prepared, the absorbance reading was performed in the Thermo Scientific Genesys UV-Visible spectrophotometer at nM every 10 minutes. A multifactorial statistical analysis of variance ANOVA was carried out for the different species studied, with a maximum order of interaction of 2 for the yield values. This made it possible to determine which of the extraction factors evaluated had a statistically significant effect on the yield. Table 1 presents a summary of the multifactorial arrangement used for the processes of obtaining extracts from Bidens pilosa L. Table 2 shows the analysis of variance for the extraction performance in the case of Bidens pilosa L. The contribution of each factor was measured by eliminating the effects of the other factors. The P-values test the statistical significance of each of the factors. Since the P-value of factor C solvent is less than 0. Figure 2 shows the influence of the factors of the experimental design with respect to the extraction yield for the extracts obtained from Bidens pilosa L. A notable decrease in yields is observed when the extraction process is carried out using hexane. The process that obtained the best yields was the Soxhlet extraction. For each significant factor, multiple range tests provide information regarding which means were significantly different from others. This test showed that there was no significant difference between the means for the drying temperatures of the leaves of Bidens pilosa L. The multiple range test carried out for Bidens pilosa L. It is, however, necessary to mention that the extraction with an ultrasonic bath and Soxhlet led to higher yields than the maceration process, despite the fact that they are not statistically different. Furthermore, Table 3 shows the analysis of variance for the extraction performance of Croton floccosus , which determined that there is a statistically significant difference in the treatments. The contribution of each factor is measured by eliminating the effects of the other factors. It is also necessary to highlight that the interaction between factor B extraction process and C solvent has a significant effect on the extraction process. Figure 5 shows the influence of the factors of the experimental design on the extraction yield. A notable decrease in yields is observed when hexane is used in the extraction process. It will also be observed that there is a difference with respect to the yields obtained as regards the interaction between the extraction method and the solvent B and C , showing that the highest yields are obtained with the Soxhlet extraction processes with ethanol at both drying temperatures. As described above for the temperatures selected in order to dry Bidens pilosa L. It was also noted that the multiple range test carried out for Croton floccosus made it possible to identify that there was a difference between the Soxhlet and maceration process extraction methods Figure 7. The Soxhlet extraction process method obtains higher yields than the maceration and ultrasonic bath extraction methods. Finally, in the case of both plants, the multiple range test showed that there was a difference between the solvents used in the extraction Figure 8. The processes in which hexane was used obtained a low yield when compared to extractions with ethanol. Table 4 details the qualitative results of the phytochemical tests performed on the ethanolic extracts of Bidens pilosa L. Asteraceae and Croton floccosus Euphorbiaceae , obtained with Soxhlet extraction. There is a low presence of flavonoids and saponins, and a relatively abundant presence of phenols and tannins for both species. In addition, a relatively abundant presence of alkaloids is detected for the species Croton floccosus and no reducing sugars are detected, while a low presence of reducing sugars is detected for the species Bidens pilosa L. The equivalent mg of gallic acid corresponding to each gram of dry sample of Bidens pilosa L. Samples that share the same letter are homogeneous. Values are the mean of triplicate determinations. Based on data from Ruiz Reyes et al. Those with the lowest value correspond to the extracts obtained using the maceration method and those with the highest value correspond to the Soxhlet extraction process, with a value of In their research, Singh et al. In their investigation, meanwhile, Falowo et al. The equivalent mg of gallic acid corresponding to each gram of dry Croton floccosus sample were obtained in the same way. These values are presented in Table 6. Those with the lowest value correspond to the extracts obtained using the ultrasonic bath method, and those with the highest value correspond to the extraction process carried out using maceration, with a value of This contrasts strongly with the values obtained for Bidens pilosa L. The equivalent mg of quercetin corresponding to each gram of dry sample of Bidens pilosa L. Samples that shared the same letter are homogeneous. Those with the lowest value correspond to the extracts obtained using the maceration method, and those with the highest value correspond to the extraction process carried out with Soxhlet and the ultrasonic bath. There is statistical homogeneity between the values obtained for both processes, with a value of In their study, Cortes-Rojas et al. These values coincide with those obtained in the present study, with the exception of those for dynamic maceration and microwave. The equivalent mg of quercetin corresponding to each gram of dry Croton floccosus sample were obtained in the same way, as shown in Table 8. As can be seen, there is a great difference between the different treatments. The in vitro antioxidant activity was performed by employing the 2. All the antioxidant activity values for the ethanolic extracts of both species were obtained from the Trolox equivalent calibration curve TEAC. The means of the values were compared using the Tukey test, which showed that there is no marked difference among the processes studied with regard to the TEAC. It was, therefore, established that the drying temperatures do not directly influence the TEAC values for the maceration, ultrasonic bath and Soxhlet extraction processes. The difference between the extract and the standard used in the previous determinations is compared. Furthermore, Singh et al. We are, therefore, of the opinion that the differences between our IC50 values and those reported are related to the time of year and the country in which the plant was collected, along with the conditions under which the plant material was prepared and the method used to obtain the extract of secondary metabolites. The means of the values were compared using the Tukey test. No marked difference among the processes studied was found for the TEAC, and there was statistical homogeneity among them. It was, therefore, established that, as with Bidens pilosa L. It is necessary to mention that, since Croton floccosus is a species that is endemic to Ecuador, there are only a few studies referring to this species. In his study, M. Flores 35 obtained IC50 values of This, therefore, made it possible to establish what is described by Altamirano 36 in his research work on Croton species, which states that the collection carried out to obtain extracts must take place at the beginning of flowering because it is the moment at which these plants contain the greatest amount of active substances. This may, therefore, have influenced the properties of the extract, as may the conditions under which the material was prepared, the extraction and the storage of the extracts. The use of phytochemical tests to identify metabolites, along with their qualitative characterization show that the plants studied have a great variety of chemical compounds, which contain flavonoids, phenols, reducing sugars, saponins and tannins in the case of Bidens pilosa L. It has been shown that the content of total phenols for Bidens pilosa L. With regard to the content of flavonoids, a maximum value of Finally, both plant species showed antioxidant potential, although it is necessary to establish that the Bidens pilosa L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. I appreciate the changes you have made to the manuscript. I am glad that my comments were taken into consideration and now the improved article is better. I have no other questions or comments. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear PhD. Yailet Albernas Carvajal, I am grateful for the analysis of version 2 of the article that you have carried out. The article entitled 'Phytochemical study of the plant species Bidens pilosa L. Asteraceae and Croton floccosus Euphorbiaceae ' is very interesting because it performs a preliminary phytochemical study of the plants Bidens pilosa L. Important information was determined related to the content of secondary metabolites and the response to the DPPH radical reported for the first time in Ecuador, a relevant aspect, which may be of future use for its application in the field of medicine. They show that both plants studied have antioxidant potential, although they state that it is necessary to establish that the species Bidens pilosa L. The methods employed are very well supported with adequate techniques, using modern analytical equipment and techniques. It presents a correct description as to be replicable should any reader need it with any other species. Competing interests: I declare that there is no conflict of interest regarding the validity or importance of the article or the peer review report. I agree with the evaluation issued by the reviewer and, following their suggestion, references 24, 25 and 26 were replaced by others that correspond to the last 3 years. I appreciate the work entitled: Phytochemical study of the plant species Bidens pilosa L. The strengths of the manuscript can be listed below:. The Authors have thoroughly studied two important folk medicine plants originating from two botanical species growing in Ecuador. They investigated total content of phenols and flavonoids as well as the antioxidant activity using DPPH method for comparison of two plants. The choice of materials and methods were appropriate, not counting my minor comments given below. Page 3: in Methods Vegetal material - please write whether the fresh leaves of the studied plant species were collected only from one plant or from several plants, and then mixed to create the sample for analysis? Page 3: in Extraction - please add more details on how the sample was pulverized for example what type of sample mill was used, how long it lasted? I also think that it would be helpful to add a map showing the collection places in Portoviejo Botanical Garden. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Competing interests: I agree with the comments of the arbitration that has been made to our article. The coordinates of the studied species are placed on the map, which is linked here. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Other versions. Phytochemical study of the plant species Bidens pilosa L. Find articles by Enrique Ruiz-Reyes. Find articles by Mayte Ariana Mendoza-Cevallos. Find articles by Ana Patricia Polanco-Moreira. Accepted Oct 11; Collection date Version Changes Revised. Amendments from Version 1 The following preliminary phytochemical study of the Bidens pilosa L. Open in a new tab. Bidens pilosa L. Find articles by Pawel Konieczynski. Competing interests: No competing interests were disclosed. Pawel Konieczynski : Referee. Find articles by Yailet Albernas Carvajal. Yailet Albernas Carvajal : Referee. Find articles by Enrique Ruiz Reyes. PMC Copyright notice. Associated Data. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel. Ethanolic extract of Bidens pilosa L. Ethanol extract of Croton floccosus obtained by Soxhlet.

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