Determination of the chemical profile extracts obtained from Kalanchoe pinnata (Lam.) Pers native of municipality Tabatinga-AM

Kalanchoe pinnata formerly known as Bryophyllum pinnatum , is a herbaceous species native to tropical regions such as Africa. In Brazil, it was successfully introduced and propagated, being commonly used in communities far from large capitals. The juice of the leaves is usually used in the treatment of inflammatory diseases, gastric ulcers, burns, diarrhea , vomiting, insect bites and body aches. In the literature, compounds such as anthocyanins and flavonoids are reported that have different biological activities such as antimicrobial, antioxidant, cytotoxic, antitumor, antiparasitic, antiallergic and hepatoprotective . Based on this information and considering that there are few studies on the chemical compounds of K. pinnata in Amazonas , especially in regions farther from the capital, such as in the municipality of Tabatinga. The present work aimed to determine the chemical profile of the fractions of the crude extract of K. pinnata native to the municipality of Tabatinga AM. For this, plant material was collected from a local producer in the municipality of Tabatinga. Then the leaves were subjected to an extraction by infusion. Soon after, the crude extract obtained was fractionated with hexane, chloroform and ethyl acetate. The resulting fractions were analyzed by High Performance Liquid Chromatography (HPLC) at wavelength λ=254 nm and, using the information available in the literature, it was possible to establish a relationship between the compound quercetin 3O -α-Larabinopyranosyl (1→2)-α-Lrhamnopyranoside , as responsible for the chromatographic peaks observed in the hexane, chloroform, and ethyl acetate fractions. Thus, the present study will serve as a basis for future work on the characterization of chemical compounds present in K. pinnata from the municipality of Tabatinga.


Amazon and its biodiversity
The so-called Brazilian Amazon encompasses the states of Acre, Amapá, Amazonas, Mato Grosso, Pará, Roraima, Rondônia and Tocantins. Together, these states comprise 60% of the national territory, which is the equivalent of 5,000,000 km 2 , 11,000 km of international borders with 12,000,000 hectares of floodplains and 25,000 km of navigable rivers (Higuchi, 2004;Benchimol, 2010).
Regarding the flora of the Brazilian Amazon, the totality of species remains undetermined, however, it is estimated that about 40,000 plant species can be found in the region, 2,500 of which are large and more than 30,000 are medium and small plants. It is worth mentioning that approximately 2,000 species of plants in the region are used as food, in folk medicine and in the production of oils, greases, waxes, among other products that benefit the population (Wright, 2002;Embrapa, 2014).
In the midst of this immense biodiversity, the Amazon has an unexplored potential when it comes to the study of medicinal plants, especially in regions further away from the capital. Medicinal plants are often fundamental in the prevention and treatment of primary diseases in communities, as they are easily accessible and readily available. It is worth mentioning that studies involving these organisms are relevant since science can contribute to the valorization of products or even encourage the commercialization of these plants (Zucchi, et al., 2012;Batista, et al., 2019).
According to the literature, in 2012 the trade in medicinal plants was estimated at US$ 2.2 billion. In 2017, the indication was that the market for plant extracts and natural medicines would move more than US$ 100 billion dollars. In addition, the works are relevant because they allow the expansion of knowledge about the bioactive compounds present in these organisms, encourage the preservation of the flora and the rational use of natural resources (Zucchi, Oliveira Júnior, et al., 2012;Ahn, 2017).

Kalanchoe pinnata ( Lam .) Pers.
Belonging to the Crassulaceae family , the genus Kalanchoe , according to the literature, has approximately 125 species distributed in tropical and subtropical areas, the vast majority being native to Africa. Among the species, Kalanchoe pinnata stands out for being widely used in the treatment of inflammatory diseases, gastric ulcers, burns, diarrhea, vomiting, insect bites and body aches, from the juice of the plant. In the academic field, this organism is the subject of studies because, according to the reports found in the literature, there are compounds that have biological activities such as antimicrobial, antioxidant, cytotoxic, antitumor, antiparasitic, antiallergic, hepatoprotector, among others (Almeida et al., 2000;El Abdellaqui, et al., 2010).
It is worth mentioning that K. pinnata was formerly known as Bryophyllum pinnatum and popularly as saião, leaf-dafortuna, courama, coirama, leaf-of-coast, leaf-of-pirarucu, pirarucu, roda-da-fortuna, leaf thick (Almeida, Silva, et al., 2000;Smith, 2004). It is a perennial herbaceous organism, with little branching that can reach up to 1.5 m in height with the presence of simple, alternate, opposite, succulent, oval leaves with a crenate margin with a maximum length of 20 cm. Its seedlings grow on the margins of its leaves and its development occurs preferentially in regions with a hot and humid climate. Thus, this organism can be found in certain parts of Asia, Australia, the West Indies, the Galapagos Islands and in other countries with tropical and subtropical climates (Lorenzi & Matos, 2008;Kamboj & Saluja, 2009;Snafi, 2013).
In Brazil, this species can be found, preferentially, along the coast between the states of Bahia and São Paulo. Because it is easy to propagate, this plant can be found in the State of Amazonas and its leaves are widely used in inland cities, such as Tabatinga, for the treatment of topical wounds, stomach pain, fever remedy and as a vermifuge (Lisboa et al., 2006;Pedroza, et al., 2017).
According to reports found in the literature, these compounds are obtained from methodologies that involve the preparation of ethanolic, hydroalcoholic or aqueous extracts followed by biological tests. As an example of this, there is the study carried out by PEREZ et al., (1999) who used the ethanolic extract of Kalanchoe sp. to reduce gastric lesions caused by indomethacin. Using the aqueous extract of K. pinnata Sousa and collaborators (2005), Afzal and collaborators (2012) found an anti-inflammatory potential, as there was a reduction in edema in rats, caused by chemical and physical stimuli.
To evaluate the antioxidant potential, Chibli (2013) used the ethanolic extract of K. pinnata, and determined, through the DPPH method, that the EC50 is at least 16.43 µg/ mL. The study also revealed that the ethyl acetate fraction had an EC50 of 9.44 µg/ mL, a value close to that observed for rutin, used as a positive control, which has an EC50 of 8.66 µg/ mL. In a study carried out by de Souza and collaborators (2013), only the aqueous extract of K. pinnata was evaluated, and an IC50 of 98.13 µg/ mL was found. These results are justified by the presence of flavonoids in the crude extract and its fractions.
The literature reports that extracts of K. pinnata present compounds with antimicrobial activity. Biswas and collaborators (2011) observed the juice of K. pinnata was used for the treatment of diarrhea, in this way when elaborating the ethanolic extract of K. pinnata and testing it against the pathogenic bacterium Escherichia coli, which is responsible by infections of the gastrointestinal tract, the ability to inhibit the growth of the pathogen was observed.
In another study, Okwu and Nnamdi, (2011) isolated and identified for the first time the alkaloid ethamino-7-Hex-1-in-5-one phenanthrene from the ethanolic extract of K. pinnata. This compound was able to inhibit the growth of the pathogens Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, Candida albicans and Aspergillus niger.

High performance liquid chromatography (HPLC) as tool for identifying organic compounds
Chromatography is an analytical method used effectively in the separation, identification and determination of chemical components of complex samples such as biological fluids, natural products, river sediments, among others (Skoog, et al., 2010).
Obtaining this profile is considered one of the best strategies for analyzing secondary metabolites and their derivatives.
However, the metabolome of an organism such as a plant is highly complex due to the diversity of chemical structures generated by metabolism, so it is necessary to use complementary approaches in order to differentiate and obtain the largest number of compounds. Even with the great advance of new techniques in liquid chromatography, the identification and resolution of chromatographic peaks is still a difficult task (Klitgaard, et al., 2014;Wehrens, et al., 2013). Therefore, the present study aimed to determine the chemical profile of the fractions of the crude extract of K. pinnata native to the municipality of Tabatinga -AM.

Obtaining plant material, elaboration of the extract and fractionation
The plant material was obtained from a local producer in the city of Tabatinga. The plant was properly stored in paper bags and transported to the Multidisciplinary Laboratory of Chemistry of the Centro de Estudos Superiores de Tabatinga, Universidade do Estado do Amazonas, where it was washed and placed to dry in an oven at 70 ºC. When dry, the weight of 260 grams was recorded on a semi-analytical scale (Shimadzu). After that, the material was stored in glass jars to be transported to the MBT Bioorganic Laboratory for preparation of the extract according to the methodology established by Rodrigues and collaborators (2016), Oliveira and collaborators (2016) with some adaptations. After heating 2 liters of distilled water to a temperature of 100 °C, 260 g of the leaves were infused. Upon reaching room temperature, the experiment was filtered and then the water was removed with the aid of a hot plate. When dry, the sample was resuspended in 500 mL of distilled water and centrifuged to remove suspended solids. After this process, the sample was fractionated by liquid-liquid extraction in a 1:1 proportion with the solvents hexane, chloroform and ethyl acetate. Using a rotary evaporator, the solvent was removed from the fractions and the excess water was removed in a desiccator. Finally, 1 mg of each fraction was weighed for liquid chromatography analysis.

Determination of the chemical profile of extracts by HPLC
Analytical High Performance Liquid Chromatography (HPLC) was performed on a Shimadzu HPLC system (LC-6 AD pump; DGU-20A5 degasser; SPD-20AV UV detector; CBM-20A modular communication) (Columbia, MD, USA) equipped with the Luna C18 column (250 × 10 mm, 5 µm) (Phenomenex -Torrance, CA, USA). All solvents used in the chromatography were HPLC grade and purchased from JT Baker (Phillipsburg, NJ, USA), and the water was purified using a Mili-Q system (Millipore, Bedford, MA, USA). Elution was performed with a gradient of 20-100% B over 25 minutes at a flow rate of 1 ml /min. UV spectra were recorded in the range of 254 nm.

Yield of fractions obtained from K. pinnata extract
After obtaining the crude extract, the yield obtained was 19.15 g, approximately 7.36% of the dry plant material. This amount was resuspended in water and fractionated with various organic solvents. The yield of the fractions obtained is described in Table 1.

Source: Authors.
After obtaining the yield, 1 mg of each fraction was weighed for HPLC analysis. When comparing the yields of the fractions obtained in the present study, with those observed in the work carried out by Ferreira et al. (2014) on K. pinnata, certain similarities are found since, starting from 2.52 kg of crushed fresh inflorescences, infused with distilled water at 50 °C, 90.2 g (3.82%) of lyophilized extract were obtained. The initial extract was then precipitated in ethanol and then partitioned in ethyl acetate and water solvents, yielding the ethyl acetate fractions with a yield of 5.58 g (6.2%) and aqueous with 12.35 g (13, 7 %).
These results show that, although the material used in the authors' research and in the present study integrate different parts of the plant, and consequently present different morphological characteristics, when similar extraction processes are used, it is possible that the yield is similar. Corroborating the studies, Muzitano (2006) using 6.76 kg of fresh leaves, being extracted by infusion with distilled water at 50 °C, obtained the crude extract, being partitioned with CH 2 Cl 2 and NaOH. The residual aqueous phase was partitioned into EtOAc, yielding 423.2 mg (0.006%), 113.0 mg (0.001%) and 1.67 g (0.02%), respectively.
In this way, the extraction method is efficient because it is a simple methodology to obtain the extract, in addition to simulating the way of preparation by the population, which constantly consumes the plant in the form of tea. It is worth mentioning that, after obtaining the extracts, there is the possibility of prospecting their chemical components and evaluating their biological potential (Silva, 2019). Tatsimo (2012) used the whole dry plant, with 1.50 kg of plant material, applying the extraction technique by percolation with methanol, at room temperature, reducing the material to 148 g, this generating the yield fractions of 38.03 g (25.69%) for hexane and 34.03 g (22.99%) for ethyl acetate. In contrast, Lanna and coauthors (2019), Almeida (2019) and Sobreira (2013) used the maceration-type extraction technique. Sobreira (2013), in turn, used ethanol and water, where they compared the hydroethanolic extract (incubated for 7 days) of leaves harvested at two times of the year. The initial extract was dissolved in distilled water and passed through liquid-liquid partition, generating the following fractions, with their respective yields: chloroform with 1.1 g and 4.4 g (5.5 % and 5.6 %), acetate ethyl with 0.74 g and 2.4 g (3.7% and 4%), and aqueous with 13.6 g and 41.6 g (68% and 69.3%). Concluding that, there is little difference between the fractions from samples with weights and collected in different climatic conditions. Similarly, Chibli (2013) and Soares (2017) used the technique by steeping in ethanol and methanol in dry leaves, being kept in contact with the solvent for periods of 24 hours and two weeks. The initial extract was partitioned liquid-liquid in solvents, including ethyl acetate and hexane, obtaining the hexane fractions with 9.11 g and 7.6 g and  Soares (2017) deserves to be highlighted, as it achieved a notably higher yield.
Comparing the methodologies, the present study presented results close to those observed in other studies. The differences in yields can be explained by the use of different extraction techniques and solvents with different degrees of polarity, as well as the temperature and application time (Oliveira et al., 2016) influence the production of extracts and fractions with different levels of concentration and weight (Marques, 2005;Brum et al .,2009).

HPLC analysis of K. pinnata fractions
When performing the HPLC analyses, the chemical profiles of the K. pinnata fractions shown in Figure 1 were determined. As can be seen in the figure above, there is a variety of metabolites in all fractions that occur between 4 and 16 minutes.
The fractions in chloroform and ethyl acetate show similarities with respect to the chromatographic profile at the retention times of 13 minutes. It is worth noting that the fraction in hexane presents a major compound in 13 minutes.
The literature points out that several chemical compounds absorb at a wavelength of 254 nm, such as flavonoids and anthocyanins.
In a study of the ethyl acetate fraction, Gonçalves (2017)

Final Considerations
The extraction method used in the present study proved to be efficient to obtain the extract and its fractions with yields in line with those observed in the literature. After analyzing the data obtained by HPLC, a good separation of the compounds was observed, with major peaks that increase the chance of identification or isolation of the compounds in further studies.
Several methodologies were found to obtain extracts from the leaves of K. pinnata, with aqueous extracts and ethyl acetate being the most used, containing mostly compounds of the flavonoid class. Comparative studies show that these derivatives are detectable in the 254 nm range. In the present study, the retention time of 13 minutes stands out for presenting a large peak in the hexane, chloroform, and ethyl acetate fractions, indicating that it may be the compound quercetin 3 -O -α-Larabinopyranosyl-(1→2)-α-L-rhamnopyranoside because the same was obtained in several studies found in the literature.
Therefore, in the future, the fractions will be analyzed using chromatographic techniques, such as HPLC-MS/MS, to characterize the chemical compounds present in the fractions. When it is not possible to identify the substance by HPLC-MS/MS, the compound will be isolated and sent to NMR for the determination of its structure.