Phytochemical and in vitro antimicrobial and antidiabetical activities of Cordiera sessilis stem barkAbstract
Studies based on medicinal plants have stood out as a complementary treatment intervention to type 2 Diabetes mellitus and its complications. Among the Brazilian medicinal plants, we highlight Cordiera sessilis (Vell.) Kuntze, in turn, has been studied in several chemical and biological studies due to its ethnopharmacological indications. Thus, this study aimed to evaluate the antimicrobial, antioxidant, antiglycation, and digestive enzyme inhibition effects of the fractions obtained from the ethanolic extract of the stem bark of C. sessilis. The results indicated that the dichloromethane, ethyl acetate, and n-butanol were the most promising fractions and therefore their phytochemical constitution was suggested by mass spectrometry. Several bioactive compounds belonging to the six classes were found, with a predominance of compounds from the flavonoid class, whose presence justifies the observed results. Thus, this study presented new results on the biological activities of this plant, contributing to the understanding of the action and effectiveness of its use in the management of diabetes mellitus and its complications.
Key words
antimicrobial; α-amylase; antiglycation; antioxidant; rubiaceae
INTRODUCTION
Type 2 diabetes mellitus (T2DM) affects millions of people around the world (WHO 2017), and is an endocrinopathy characterized by resistance to the action of the hormone insulin and obesity, which generates blood hyperglycemia (Olokoba et al. 2012). According to the literature, hyperlipidemia, production of reactive oxygen species (ROS), formation of advanced glycation end products (AGEs), and impairment of the immune system in the face of infections are also marked factors of T2DM (Al-Maskari et al. 2011, Verhulst et al. 2019).
Research-based medicinal plant products for the development of interventions against T2DM are increasing worldwide (Liyanagamage et al. 2020) because many plant molecules like polyphenols, terpenoids, and glycosides have antidiabetic properties (Salehi et al. 2019). Previous studies described that these secondary metabolites are useful in combating infectious agents, in addition to having antioxidant and antiglycation properties, they can also act as inhibitors of digestive enzymes and thereby assist in the management of postprandial hyperglycemia and hyperlipidemia (Franco et al. 2020b).
Among the numerous plants traditionally used in Brazil to treat numerous disease conditions, we have Cordiera sessilis (Vell.) Kuntze (Alibertia sessilis as a synonym), belongs to the Rubiaceae family, is a native species found in Brazilian Savanah (Cerrado), and is popularly known as “marmelada-de-cachorro” (Brazilian Portuguese) (Zappi 2015). According to Canelhas (2012), the essential oil of C. sessilis stem bark is mainly composed of fatty acids, while wood oil reveals the presence of fatty acids, long-chain alcohol, phenol, and trans-caryophyllene sesquiterpene.
In a study by Aquino et al. (2013), the essential oil of C. sessilis leaves has antimicrobial activities and antioxidants. The leaves of C. sessilis have a high antioxidant capacity and promising activity in inhibiting the growth of oral bacteria (Aquino et al. 2013) and are indicated by popular knowledge in bath forms or plasters to treat skin conditions (Souza et al. 2013). The species found in the Rubiaceae family are capable of inhibiting glycosidic hydrolases (Elya et al. 2012), and the genera Cordiera sp. and its synonym, Alibertia sp. have already been described as containing species with hypoglycemic effects (de Santana Aquino et al. 2020), without cytotoxic, genotoxic and mutagenic effects for human cells (Tolouei et al. 2020). However, there are few studies in the literature indicating antidiabetic properties or enzymatic and glycation inhibitions using C. sessilis.
This study aimed to investigate C. sessilis stem bark fractions against α-amylase, α-glycosidase, lipase, and non-enzymatic glycation. Also, evaluate its capacity to reduce oral bacterial and fungal proliferation, and antioxidant activity and suggest the phytochemical composition of the best fractions.
MATERIALS AND METHODS
Exsiccata and steam bark sample
Approximately 1 kg of C. sessilis steam bark was collected in a rural area in the municipality of Morrinhos - Goiás (Latitude: 17 ° 42’00.9 “S; Longitude: 49 ° 21’48.6” W), in the dry season (October 2015). The exsiccata of C. sessilis used was identified by Professor Dr. Ivan Schiavini da Silva, from the Federal University of Uberlândia, and deposited at the Herbarium Uberlandense, under the code HUFU 74562.
Extraction and partitioning
The stem bark was dried, ground, and macerated in ethanol for 5 weeks. The entire process took place at room temperature. The solvent was removed using rotary evaporation under controlled pressure, and the obtained extract was then lyophilized. Approximately 30 g of the dry extract was resolubilized in 200 ml of methanol-water (9: 1) and subjected to liquid-liquid partition, with the following solvent sequence: hexane (Hex), dichloromethane (DCM), ethyl acetate (Acoet), n-butanol (ButOH) and water (H2O). The fractions were named using these name solvents, but all solvent residues were removed by the lyophilization process.
Phytochemical prospection
Total polyphenols, flavonoids, and condensed tannins contents were determined in fractions of C. sessilis stem bark. The samples were solubilized in methanol (10 mg/mL) and all analyses were performed in triplicate. A blank with methanol alone was measured and subtracted from all values. The phytochemicals were determined using an analytical curve constructed with standards (gallic acid, catechin, and quercetin to polyphenols, flavonoids, and condensed tannins, respectively) at concentrations of 15.62; 31.25; 62.5; 125; 250; 500; 1000, and 2000 μg/mL (Franco et al. 2020b).
Polyphenolic content: the assay was started with the addition of 5 μL of fraction/standard, 25 μL of Folin-Ciocalteu solution, and 195 μL of deionized water in each well. After 6 min of incubation (25 °C), 75 μL of 7% Na2CO3 was added, and the mixture was incubated for 2 h at 25 °C in the absence of light and the absorbance was read at 760 nm (Molecular Devices, Menlo Park, CA, USA). The results were expressed as milligrams of gallic acid equivalents per gram of sample (mg GAE/g).
Condensed tannins content: in each well, 10 µL of fraction/standard was incubated with 200 µL of 4% vanillin methanolic solution and 100 µL of HCl at 25 °C for 15 min. After incubation, the absorbance was read at 500 nm (Molecular Devices, Menlo Park, CA, USA). The results were expressed as milligrams of catechin equivalents per gram of sample (mg CE/g).
Flavonoids content: in each well, 30 µL of fraction/standard was incubated with 180 μL of milli-Q water and 10 μL of 5% NaNO2 aqueous solution at 25 °C for 6 min in the absence of light. Then, 20 μL of 10% AlCl3 aqueous solution was added and the mixture was incubated at 25 °C for 6 min. After incubation, 60 μL of 4% NaOH was added and the absorbance was read at 425 nm (Molecular Devices, Menlo Park, CA, USA). The results were expressed as milligrams of quercetin equivalents per gram of sample (mg QE/g).
Mass spectrometry analysis (HPLC ESI-MS / MS)
The mass spectrometry analysis of DCM, Acoet, and ButOH fractions, the samples that showed the best results, was performed in an Agilent HPLC (High-Performance Liquid Chromatography) model, Infinity 1260, coupled to a high-resolution mass spectrometer (QTOF), model Agilent, model 6520 B, with electrospray ionization source and electron impact energy set to 10–30 eV in negative and positive mode. The chromatographic parameters were: Agilent column model Zorbax, 2.1 mm internal diameter, 5 cm long, particles of 1.8 μm, mobile phase: water acidified with formic acid (0.1%, v/v) (A) and methanol (B), with the following solvent gradient system: 2% B (0 min), 98% B (0-15 min); 100% B (15-17 min); 2% B (17-18 min) and 2% B (18-22 min). The ionization parameters were: the pressure of the 58 psi nebulizer, drying gas at 8L / min, at a temperature of 220 ° C, and capillary energy of 4.5 KVa.
Antioxidant properties
The samples were solubilized in methanol at a concentration of 1 mg/mL for ferric-reducing antioxidant power (FRAP) and at 100 µg/mL for the absorption capacity of oxygen radicals (ORAC) methods. Some samples were serially diluted from a concentration of 10 mg/mL for DPPH IC50 determination. In these methods, ascorbic acid and methanol were used, respectively, as positive and negative controls. All analyses were performed in triplicate and results were expressed as µg/mL of IC50 for DPPH and Trolox equivalents (µmol TE/g) for FRAP and ORAC (Franco et al. 2018).
Oxygen radical absorbance capacity (ORAC): the samples were incubated with 0.085 nM fluorescein at room temperature for 15 min. After incubation, 153 mM 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) was added and the fluorescence (485 nmex/528 nmem) (Perkin-Elmer LS 55, Massachusetts, USA) was measured at 37 °C for 90 min. All reagents were prepared in 75 mM phosphate buffer (pH 7.4) and the loss of fluorescence was measured by calculating the area under the curve.
Iron Reduction Capacity (FRAP): the samples were incubated with 10 volumes of 300 mM sodium acetate buffer (pH 3.6), 1 volume of 10 mM 2,4,6-tri(2pyridyl)-striazine (TPTZ), and 1 volume of 20 mM ferric chloride at 37 °C for 6 min and the absorbance were measured at 593 nm (Molecular Devices, Menlo Park, CA, USA).
Sequestration of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH): the samples were incubated with a methanolic solution of 60 mM DPPH at 30 °C for 20 min, in the absence of light. The reduction in absorbance of the mixture was measured at 517 nm (Molecular Devices, Menlo Park, CA, USA). Scavenging of DPPH radicals was calculated using the following equation DPPH(%) = [(A control − A sample) / (A control)] X 100, where A control is the absorbance value of the DPPH radical and A sample is the absorbance value of each extract/positive control.
Advanced glycation end-products (AGES) formation
The samples were diluted in methanol and serially diluted from a concentration of 15 mg/mL for IC50 determination. Results are presented as percentage (%) of inhibition, calculated using the following equation: GI(%) = [(F control − F sample) / (F control)] X 100, where F control is the fluorescence value of the negative control and F sample is the fluorescence value of each sample. In these methods, quercetin and methanol were used, respectively, as positive and negative controls. All analyses were performed in triplicate (Franco et al. 2019).
Bovine Serum Albumin and Fructose Method (BSA/FRU): the samples were incubated (at 37 °C, for 72 h in the absence of light) with BSA (50 mg/mL) and fructose (1.25 M) both diluted in 200 mM phosphate buffer, pH 7.4 containing 0.02% sodium azide. After incubation, 20% trichloroacetic acid (m/v) was added and the mixture was centrifuged at 10.000 for 10 min. The Pellet was resuspended in phosphate buffer and the fluorescence intensity of glycated albumin was measured (350 nmex/420 nmem) (Perkin-Elmer LS 55, Massachusetts, USA).
Bovine Serum Albumin and Methylglyoxal Method (BSA/MGO): the samples were incubated (at 37 °C, for 72 hours in the absence of light) with BSA (50mg/mL, diluted in 200 mmol/L phosphate buffer, pH 7.4, with 0.02% sodium azide) and methylglyoxal (53.3 mmol/L, diluted in deionized water). After incubation, 20% trichloroacetic acid (m v-1) was added and the mixture was centrifuged at 10.000 xg for 10 minutes. The pellet was resuspended in phosphate buffer and the fluorescence intensity (340 nmex/380 nmem) (Perkin-Elmer LS 55, Massachusetts, USA) was measured.
Arginine and Methylglyoxal Method (Arg/MGO): the samples were incubated (in the absence of light at 37 °C for 72 hours) with arginine (106.6 mmol/L, diluted in 200 mmol/L phosphate buffer, pH 7.4, with 0.02% sodium azide) and methylglyoxal (53.3 mmol/L, diluted in deionized water). After incubation, the fluorescence intensity (340 nmex/380 nmem) (Perkin-Elmer LS 55, Massachusetts, USA) was measured.
Digestive enzimes catalisys
The samples were diluted in methanol and used at a concentration of 10 mg/mL for initial assays and serially diluted for IC50 determination. Acarbose was used as a positive control for α-amylase/α-glucosidase and Orlistat was used for lipase. All analyses were performed in triplicate and results are presented as percentage (%) of inhibition, calculated using the following equation: I (%) = [(A control – A sample) / (A control)] X 100, where A control is the absorbance value of the negative control and A sample is the absorbance value of each extract/positive control (Franco et al. 2020a).
Salivary α-amylase: the samples were incubated (for 30 min at 37 °C) with an α-amylase-enriched fraction (from Human saliva) diluted in 50 mM of 2-(N-morpholino)-ethane sulfonic acid (MES) buffer pH 6.0 (containing 5 mM of calcium chloride, 140 mM of potassium thiocyanate and 300 mM of sodium chloride). The reaction started by adding 2-chloro-4-nitrophenyl-4-β-D-galactopyranosylmaltoside (12 mM) substrate. The absorbance was measured at 37 °C for 3 min and 405 nm (Molecular Devices, Menlo Park, CA, USA).
Intestinal α-glucosidase: the samples were incubated (for 20 min at 37 °C) with an α-glucosidase-enriched fraction (from rat’s intestinal acetone powder) and 1.5 mM reduced glutathione (both diluted in 50 mM phosphate buffer pH 6.8). The reaction started by adding 4-nitrophenyl α-D-glucopyranoside (4 mM) and the absorbance was measured at 405 nm (Molecular Devices, Menlo Park, CA, USA) for 30 min at 37°C.
Pancreatic lipase: the samples were incubated with lipase (from the porcine pancreas) (diluted in 50 mM Tris-HCl buffer pH 8.0, containing 10 mM CaCl2 and 25 mM NaCl) for 20 min at 37 ºC. The reaction started by adding p-nitrophenyl palmitate (0.8 mM) substrate (diluted in 10% isopropanol and 50 mM Tris-HCl buffer, containing 10 mM CaCl2, 25 mM NaCl, 0.5% Triton X-100 and 0.1% gum arabic). The absorbance was measured at 410 nm (Molecular Devices, Menlo Park, CA, USA) for 30 min.
Antimicrobial analysis
The evaluation of the antibacterial activity of the crude extract and partitions was carried out by regulation of the Clinical and Laboratory Standards Institute (CLSI) using the Broth Microdilution method. Aerobic strains were used Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49456), Streptococcus sanguinis (ATCC 10556), and Aggregatibacter actinomycetemcomitans (ATCC 43717) and anaerobic Porphyromonas gingivalis (ATCC 33277), Fusobacterium nucleatum (ATCC 25586) e Actinomyces naeslundii (ATCC 19039). The evaluation of the antifungal activity of the crude extract and partitions was done by the broth microdilution method and by CLSI regulation (2008). Microorganisms were used Candida albicans (ATCC 28366), Candida tropicalis (ATCC 13803), and Candida glabrata (ATCC 15126).
Oral bacteria: soybean tryptone (TSB) was used as a culture medium for aerobic bacteria and anaerobic Schaedler broth supplemented with hemin (5 μg/mL) and menadione (10 μg/ mL). The test was carried out in a 96-well microplate, where the inoculum, broth, and sample were added to each orifice, resulting in a final volume of 100 μL for aerobic bacteria and 200 μL for anaerobic bacteria. The inoculum was prepared and standardized according to the McFarland scale and the volume added in each well for aerobic bacteria was 20 μL and for anaerobic 40 μL. For the test with aerobic bacteria, the microplates were sealed with parafilm and incubated in microaerophilia by the flame/candle system, at 37 ° C, for 24 h. After the incubation period, 30 μL of resazurin indicator (0.01% in water) was added to each well. The anaerobic bacteria were incubated for 72 h in an anaerobic chamber (5-10% H2, 10% CO2, 80-85% N2), at 36 ° C, and resazurin was also used as an indicator after the incubation. The concentrations of the samples tested for aerobic and anaerobic bacteria were from 400 μg / mL to 0.195 μg / mL. Chlorhexidine was used as a positive control for aerobic bacteria, the tested concentrations of this control were 5,900 mg / mL at 0.0115 μg / mL and for anaerobic microorganisms, Metronidazole was used as a positive control, with tested concentrations between 5,900 μg / mL and 0.0115 μg / mL. As a negative control, DMSO (concentration 4% v / v) was used. The test result is expressed in Minimum Inhibitory Concentration (MIC), using the μg / mL unit.
Oral fungi: for the evaluation of antifungal activity, the samples were dissolved in DMSO at a concentration of 192,000 μg / mL (stock solution), dilutions of the stock solution of each sample were carried out in RPMI 1640 culture medium, buffered at pH 7.2, with 0.165 mol / L of 3-N-morpholinopropanesulfonic acid (MOPS) at 12,000 μg / mL. The inoculum was prepared according to the McFarland scale and obtained a value of 6.0106 CFU / mL. Subsequently, dilutions were made in RPMI broth until the inoculum reached 1.2.103 CFU / mL. The test was performed in microdilution plates with 96 wells, in which serial dilutions were made with concentrations ranging from 3,000 μg / mL to 1.46 μg / mL. The culture medium used in the test was the RPMI broth buffered with MOPS (pH 7.2). The volume of 100 μL of the inoculum suspension was placed in each well and the final volume of each orifice was 200 μL. Amphotericin B was used as a positive control and the tested concentrations varied between 16.0 μg / mL and 0.1 μg / mL, for the negative control, DMSO was used, and the tested concentrations were between 10% to 1% (v / v). The result was obtained in Minimum Inhibitory Concentration (MIC) using the μg / mL unit.
Statistical analysis
The statistical analyses and graphics were done using GraphPad Prism 8.0 software. The data were expressed as mean ± standard error of the mean (SEM) and the significance of difference was calculated using one-way ANOVA and Tukey post-test of multiple comparisons. Values of p < 0.05 were considered significant.
RESULTS AND DISCUSSION
In our study, we aimed to evaluate the biological properties of fractions of ethanolic extract from C. sessilis stem bark, analyzing their antioxidant and antiglycation effects and ability to inhibit digestive enzymes related to postprandial hyperglycemia and hyperlipidemia. Also, we analyzed the capacities of these fractions to reduce the proliferation of oral bacteria and fungi and suggested the presence of the main compounds present in subfractions obtained from the DCM, Acoet, and ButOH fractions. Our results indicated that the Acoet fraction demonstrated better antioxidant and antiglycation capacities and good enzymatic inhibition. Besides, the DCM, Acoet, and ButOH fractions stood out for reducing the proliferation of oral microorganisms.
Phytochemical prospection
The results of the phytochemical prospecting are shown in Figure 1. The fractions DCM, Acoet, and ButOH showed the highest concentrations of polyphenols (91.5 ± 5.1, 177.5 ± 9.4, and 114.0 ± 2.8 mg GAE / g, respectively). The Acoet and ButOH fractions had the highest content of flavonoids (44.5 ± 3.8 and 27.1 ± 2.2 mg QE / g), but only the ButOH fraction showed prominence condensed tannins concentration (427.3 ± 22.6 mg CE / g). In the phytochemical prospection, the DCM, Acoet, and ButOH fractions showed a considerable amount of total polyphenols, but Acoet and ButOH concentrated more flavonoids and condensed tannins, respectively.
Phytochemical prospection of the total content of polyphenols (a), flavonoids (b), and condensed tannins (c) using C. sessilis stem bark. Hex: hexane; DCM: dichloromethane; Acoet: ethyl acetate; ButOH: n-butanol; H2O: water. All fractions were diluted in methanol at concentrations of 10 mg / mL. Values expressed as mean ± standard error. Different letters indicate a significant difference (p <0.05).
Chromatographic fractionation and biomolecules suggest
The DCM, Acoet, and ButOH fractions showed promising results in all analyses, therefore, their bioactive compounds were suggested by mass spectrometry. Six metabolites classes were suggested: 1) organic acid; 2) carbohydrate; 3) phenolic acid; 4) iridoid glycoside; 5) flavonoid, and 6) triterpenoid. Table I shows the metabolites and their classes and Table II presents the HPLC-MS-ESI analyses.
Compound annotation in the DCM, Acoet, and ButOH fractions of the ethanolic extract from C. sessilis stem bark using HPLC-ESI-MS analysis.
Mass spectrometry analysis indicated the presence of various compound classes, including carboxylic acids, carbohydrates, phenolic acids, iridoid glycosides, and flavonoids across all fractions. Notably, only the Acoet fraction contained metabolites from triterpenoid class. In the bioactive fractions of C. sessilis, organic acids was represented by quinic acid (1), identified by its molecular ion [M–H]⁻ at m/z 191.0561 (Abu-Reidah et al. 2015, Metlin 2022). Additionally, several phenolic acids derived from quinic acid were identified in these fractions. Precursor ions [M–H]⁻ at m/z 353, m/z 367, and m/z 515 exhibited similar fragmentation patterns, corresponding to caffeoylquinic acids (3, 4, and 5), feruloylquinic acids (8 and 9), and dicaffeoylquinic acids (12, 14, and 16), respectively.
Isomers of each phenolic acid were differentiated by their retention times and MS2 data characteristics (Lin & Harnly 2007, Masike et al. 2017, Ruan et al. 2019). Fragmentation patterns of all phenolic acids showed [M–H–caffeoyl]⁻, [M–H–feruloyl]⁻, and [M–H–2caffeoyl]⁻ ions at m/z 191, indicating the presence of quinic acid within their structures. The MS2 spectra of caffeoylquinic and feruloylquinic acid isomers displayed ions at m/z 179 ([caffeoyl–H]⁻) and m/z 193 ([feruloyl–H]⁻), arising from ester bond cleavage between the caffeoyl/feruloyl groups and quinic acid (Masike et al. 2017, Ruan et al. 2019). Dicaffeoylquinic acid isomers were identified with molecular ions [M–H]⁻ at m/z 515, showing a consistent fragmentation pattern, including ions at m/z 353 ([M–H–caffeoyl]⁻), m/z 191 ([M–H–2caffeoyl]⁻), m/z 179 ([caffeoyl–H]⁻), m/z 173 ([quinic acid–H–H₂O]⁻), m/z 161 ([caffeoyl–H–H₂O]⁻), and m/z 135 ([caffeoyl–H–CO₂]⁻), as with caffeoylquinic acids (Ruan et al. 2019). Caffeoylquinic acids were present across all fractions, while feruloylquinic and dicaffeoylquinic acids were exclusive to the DCM and Acoet fractions. Additionally, p-coumaric acid was identified only in the Acoet fraction, with a molecular ion [M–H]⁻ at m/z 163 and a main fragment at m/z 119 ([M–H–CO₂]⁻) (Metlin 2022).”
Another class identified in this study was the flavonoids, which were categorized into two groups: flavones and flavonols. The flavones included cirsimarin I and II (20 and 25), luteolin (21), apigenin (23), wogonin 7-glucoside (24), and cirsimaritin (27), corresponding to the molecular ions [M + H]⁺ at m / z 477.1390, [M–H]⁻ at m / z 285.0406, [M–H]⁻ at m/z 269.0457, [M + H]⁺ at m/z 447.1287, and [M + H]⁺ at m/z 315.0864, respectively (Aziz et al. 2022, Metlin 2022, Velamuri et al. 2020). The annotated flavonols included kaempferol rhamnoside-hexoside-rhamnoside (13), rutin (15), kaempferol rutinoside (17), and isorhamnetin 3-galactoside/glucoside (22), with molecular ions [M–H]⁻ at m/z 739.2094, [M+H]⁺ at m/z 611.1608, m/z 595.1656, and m/z 479.1187, respectively.
Regarding the MS2 fragmentation pattern of flavonoids, compounds 20, 22, 24 and 25 showed as main fragments the ions at m/z 315, m/z 317, m/z 285 and m/z 315, resulting from the loss of a hexose unit (162 Da) (Abu-Reidah et al. 2015, Li et al. 2021, Gallegos-Olea et al. 1997). Kaempferol rhamnoside-hexoside-rhamnoside (13) was annotated from the molecular ion [M–H]− at m/z 739.2094 and the fragments [M–H–C6H10O4]- at m/z 593 and [M–H–2C6H10O4–C6H10O5]- at m/z 285 are related to the sequential loss of sugar units in the molecule. The presence of the ion at m/z 285 ion in the MS2 spectra indicates the kaempferol aglycone (Li et al. 2021). Compound 15 was annotated as rutin ([M + H]+ at m/z 611.1608) and the fragments [M–H–C6H10O4]+ at m/z 465 and [M–H–C6H10O4–C6H10O5]+ at m/z 303 correspond to the losses of the rhamnosyl (146 Da) and glucoside (162 Da) units, resulting in the quercetin aglycone (ion at m/z 303) (Gallegos-Olea et al. 1997). Kaempferol rutinoside (17) was related to the molecular ion [M + H]+ at m/z 595.1656. The MS2 spectrum showed the fragments [M + H–146]+ at m/z 449 and [M+H–146–162]+ at m/z 287 (kaempferol aglycone), indicating the loss of the rutinoside group (mannosyl and glucoside units, respectively) (Abu-Reidah et al. 2015).
Compounds 21, 23, 27, and 30 showed molecular ions at [M–H]⁻ m/z 285.0406 and m/z 269.0457, and [M + H]⁺ m/z 315.0864 and m/z 285.0758, respectively. MS² fragmentation confirmed these as flavone aglycones, specifically luteolin (21), apigenin (23), cirsimaritin (27), and wogonin (30). The MS² spectrum of luteolin (21) displayed characteristic ions [M–H–C₈H₆O₂]⁻ at m/z 151 and [M–H–C₇H₄O₄]⁻ at m/z 133, consistent with a retro Diels-Alder mechanism occurring in its C ring. Through a similar mechanism, apigenin (23) showed ions [M–H–C₈H₆O]⁻ at m/z 151 and [M–H–C₇H₄O₄]⁻ at m/z 117, confirming two hydroxyl groups in ring B for luteolin and one for apigenin.
Other notable fragments for apigenin include [M–H–CO₂]⁻ at m/z 225 and [M–H–C₇H₄O₂]⁻ at m/z 149 (Aziz et al. 2022, Metlin 2022, Velamuri et al. 2020). Cirsimaritin (27) ([M + H]⁺ at m/z 315.0864) and wogonin (30) ([M+H]⁺ at m/z 285.0758) showed fragments [M + H–15]⁺ at m/z 300 and m/z 270, respectively, indicating the loss of a methyl group (Aziz et al. 2022, Metlin 2022, Xiao et al. 2018). Additional methoxylated flavones (compounds 31, 32, and 33) were also identified based on the loss of methoxyl groups. All flavonoid aglycones were found exclusively in the Acoet fraction, which contained the highest concentration and diversity of flavonoids. In contrast, the ButOH fraction contained only rutin (15) and kaempferol rutinoside (17) among the flavonoids annotated.
Triterpenoids were annotated exclusively in the Acoet fraction, including compounds 34 ([M + H]⁺ at m/z 505.3525), 35 ([M–H]⁻ at m/z 487.3430), 36 ([M + H]⁺ at m/z 487.3418), 37 ([M + H]⁺ at m/z 471.3465), 38 ([M + H]⁺ at m/z 435.3520), and 39 ([M + H]⁺ at m/z 455.3531). Their molecular ions and MS² spectra were consistent with triterpenoids such as betulonic acid and either ursolic or oleanolic acid. The MS² fragmentation patterns for these compounds featured characteristic losses, including successive water losses (18 Da), HCOOH (46 Da), and CO (28 Da) groups, which align with the known fragmentation behavior of triterpenoid structures (Gallegos-Olea et al. 1997, Pham et al. 2022, Räsänen et al. 2019, Yang et al. 2020).
Two iridoid isomers, annotated as loganin I and II (compounds 7 and 11), were identified in the DCM, Acoet, and ButOH fractions. Both compounds showed a molecular ion [M + H]⁺ at m/z 391.1597, with a main fragment at m/z 229 ([M + H–162]⁺) indicating the loss of a glucose unit (Liu et al. 2015). Overall, the dereplication analysis of the C. sessilis fractions highlighted a diversity of phytochemicals, with a notable emphasis on phenolic acids and flavonoids.
Antioxidant activities
Figure 2 shows the antioxidant results obtained for the fractions of C. sessilis stem bark. The samples and controls were diluted, respectively, at 100 µg/mL, 1 mg / mL, and 10 mg/mL for ORAC, FRAP, and DPPH methods. In the ORAC assay, the DCM, Acoet, and ButOH fractions showed high antioxidant activities (1950.8 ± 21.1, 2195.2 ± 90.8, and 1423.2 ± 95.6 µM Trolox eq / g), with values without significant differences when compared to ascorbic acid (2002.1 ± 22.9 µM Trolox eq / g). These same fractions showed prominent results in the IC50 of the DPPH test (DCM: 12.6 ± 1.4, Acoet: 5.8 ± 1.3, ButOH: 13.3 ± 2.0 µg / mL), with values without significant differences when compared to ascorbic acid (2.7 ± 0.8 µg / mL). In the FRAP assay, none of the fractions showed high results, with significant differences when compared to ascorbic acid (1155.0 ± 5.0 µM Trolox eq / g).
Evaluation of antioxidant activities using ORAC (a), FRAP (b), and DPPH (c) methods using C. sessilis stem bark. Hex: hexane; DCM: dichloromethane; Acoet: ethyl acetate; ButOH: n-butanol; H2O: water. All fractions were diluted in methanol at concentrations of 10 mg / mL. Values expressed as mean ± standard error. Different letters indicate a significant difference (p <0.05). In (c) DPPH, values are expressed in µg/mL.
Previous studies have highlighted the antioxidant properties of the classes of compounds identified within our fractions (Bešlo et al. 2023, Cheng et al. 2021, Benali et al. 2022, Santiago et al. 2014). The presence of flavonoids is particularly significant, as these molecules are rich in hydroxyl groups (OH-) within the A, B, and C rings of their structure, making them excellent antioxidants, as well as antiglycation agents and potential enzyme inhibitors (Franco et al. 2019).
The DCM and Acoet fractions showed relevant antioxidant activity in the ORAC method, and the Acoet fraction also stood out in the DPPH test. None of the tested samples showed significant results in the FRAP method. According to Liang & Kitts (2014), the ORAC method evaluates the ability of the samples to transfer hydrogen atoms (scavenger); the FRAP test evaluates the electron transfer mechanism (quenching) and the DPPH, which can detect the two mechanisms of free radical reduction. Thus, the probable antioxidant mechanism of the compounds present in the Acoet fraction is of the scavenger type.
Antiglycation properties
The results of antiglycation properties of the fractions of C. sessilis stem bark are shown in Figure 3. All fractions were serially diluted in methanol from concentrations of 10 mg / mL for IC50 evaluation. The Acoet and ButOH fractions showed considerable antiglycation activities, with values (24.2 ± 5.0, 30.5 ± 4.6 µg / mL, respectively) statistically equal to those obtained for quercetin (7.3 ± 0.7 µg / mL). In the BSA-MGO method, only the Acoet and H2O fractions showed results (254.3 ± 9.2 and 188.9 ± 8.4 µg / mL, respectively) without significant differences when compared to quercetin (246.4 ± 12.7 µg / mL). In the Arg-MGO assay, none of the fractions showed results comparable to quercetin (1155.0 ± 5.0 µM Trolox eq / g) results. Of the suggested compounds in these fractions, the antiglycation activity of quinic acid (Han et al. 2024), rutin (Dubey et al. 2017), luteolin (Fecka et al. 2023), and apigenin (Zhou et al. 2019) are well-documented in the literature.
Evaluation of antiglycation activities using BSA-FRU (a), BSA-MGO (b), and Arg-MGO (c) methods using C. sessilis stem bark. Hex: hexane; DCM: dichloromethane; Acoet: ethyl acetate; ButOH: n-butanol; H2O: water. All fractions were serially diluted in methanol from concentrations of 10 mg / mL for IC50 evaluation. Values expressed as mean ± standard error. Different letters indicate a significant difference (p <0.05).
It’s noted that many polyphenols also have antiglycation activities and can reduce the production of AGEs through different mechanisms. According to Wang et al. (2011), the BSA / FRU model is used to evaluate the sample potential at all stages of protein glycation, while BSA / MGO and ARG / MGO evaluate, respectively, the intermediate stage and the main amino acid in the glycation process of proteins.
Among the possible mechanisms of the compounds present in the Acoet fraction, the best sample in all antiglycation methods, stands out for inhibition of Schiff’s base and Amadori products, in addition to the inhibition of the formation of ROS and carbonyl groups mediated by fructose or methylglyoxal during these processes (Yeh et al. 2017).
Enzimatic inhibitions
Figure 4 shows the results of enzymatic inhibitions obtained for the fractions of C. sessilis stem bark. All fractions were diluted in methanol at the concentrations of 10 mg / mL and serially from 10 mg / mL for the IC50 assay (C) using fractions that showed more than 50% activity. The Acoet and ButOH fractions showed statistically equal effects to the acarbose control in the α-amylase inhibition method when tested at a concentration of 10mg / mL (92.9 ± 0.4, 86.7 ± 0.2% and 99.6 ± 0.02, respectively). Still in this method, the IC50 values for these fractions were statistically equal to that of the control (Acoet: 0.7 ± 0.02, ButOH: 1.0 ± 0.04, and Acarbose: 0.25 ± 0.005 µg / mL). On the other hand, in the lipase enzyme inhibition assay, the H2O fraction was the only one to stand out about the Orlistat control when tested at 10mg / mL (68.1 ± 2.8 and 94.0 ± 3.0%), and in the IC50 assay, this same fraction obtained results without significant differences concerning the control (H2O: 2.5 ± 0.2 and Orlistat: 7.7 ± 0.6 µg / mL). In the α-glucosidase inhibition assay, none of the fractions showed results comparable to acarbose (75.2 ± 1.2%) in 10 mg/mL.
Evaluation of α-amylase (a and d), lipase (b and e), and α- glucosidase (c) enzymes inhibitions using fractions of C. sessilis stem bark. Hex: hexane; DCM: dichloromethane; Acoet: ethyl acetate; ButOH: n-butanol; H2O: water. All fractions were diluted in methanol at the concentrations of 10 mg / mL and serially from 10 mg / mL for the IC50 assay (C) using fractions that showed more than 50% activity. Values expressed as mean ± standard error. Different letters indicate a significant difference (p <0.05).
Enzyme inhibition is also considered an interesting measure for the reduction of hyperglycemia and hyperlipidemia that are marked in T2DM (Tucci et al. 2010). The literature shows that inhibition of carbohydrate digestion by acarbose and lipids by orlistat in the small intestine reduced the risk of T2DM by 25% (Chiasson et al. 2002) and 37% (Torgerson et al. 2004), respectively. In this scenario, the α-amylase and lipase can be strongly inhibited by polyphenols containing hydroxyl and galloyl groups according to Xiao et al. (2013) and Lunagariya et al. (2014). In our study, the Acoet fraction showed the best α-amylase inhibition results, and the H2O fraction showed the best lipase inhibition values. There is limited information regarding the ability of the compounds in our fractions to inhibit the lipase enzyme; however, quinic acid (Han et al. 2024), mannitol (Chukwuma et al. 2019), rutin (Dubey et al. 2017), luteolin (Fecka et al. 2023), apigenin (Zhou et al. 2019), kaempferol derivates (Yin et al. 2018), betulonic acid and ursolic acid and/or oleanolic acid (Silva et al. 2016) have demonstrated inhibitory activity against α-amylase and α-glycosidase enzymes.
Antimicrobial effects
The microbiological results for the fraction of C. sessilis stem bark are shown in Table III. The MIC results were presented as μg/mL of the sample. The samples of C. sessilis stem bark showed satisfactory results in inhibiting the growth of aerobic and oral anaerobic bacteria. The Hex and ButOH fractions showed a MIC of 200 μg/mL, indicating moderate activity against the aerobic S. mitis. The dichloromethane fraction showed a MIC of 200 μg/mL, revealing moderate activity against the aerobic S. sanguinis. The n-butanol fraction showed promising activity against the S. sanguinis strain, as it showed a MIC value of 100 μg/mL The most polar fractions, acetate and n-butanol, showed moderate activity against the anaerobic strain F. nucleatum, exhibiting a MIC of 200 μg/mL. The dichloromethane fraction also showed moderate activity against the anaerobic microorganism A. naeslundii (MIC 200 μg/mL). In antifungal activity, the Acoet and ButOH fractions showed promising activity against the fungi C. albicans and C. tropicalis, with a MIC of 93.75 μg/mL. The H2O fraction expressed MIC values of 187.5 μg/mL against the microorganisms C. albicans and C. tropicalis, these results reveal that the hydromethanolic residue was moderately active in inhibiting the growth of the tested fungi.
The effects of the ButOH fraction against S. sanguinis and the AcOEt and ButOH fractions against C. albicans and C. tropicalis were the most pronounced. However, there is limited information in the literature regarding the effects of the compounds suggested in this work on S. sanguinis and C. tropicalis. Some studies, however, indicate that quinic acid derivatives (Ma & Ma 2015), kaempferol, rutin (Savu & Ștefan 2024), and ursolic and/or oleanolic acid (Stan et al. 2021) exhibit fungicidal activity against C. albicans. Among the complications generated by T2DM, we highlight immune dysfunction and inflammatory processes that affect specific regions of the body, such as the oral cavity, and that can favor the proliferation and action of pathogenic microorganisms. (Verhulst et al. 2019). The S. mutans, S. mitis, S. sanguinis, and A. actinomycetemcomitans bacteria are responsible for several oral diseases, including gingivitis, development of dental plaque, caries, periodontitis, changes in dental restorations and are related to endocarditis, caused by bacteria entering the bloodstream after trauma (Eto et al. 2003, Yamaguchi et al. 2006). The P. gingivalis, F. nucleatum, and A. naeslundii bacteria are responsible for the development of dental biofilm, periodontitis, and acute appendicitis (Castellarin et al. 2012, Dige et al. 2009).
The C. albicans, C. tropicalis, and C. glabrata fungi are responsible for candidiasis, a pathology that affects the skin, oral cavity, esophagus, gastrointestinal tract, sexual organs, and vascular system of human beings (Calderone & Fonzi 2001). Here, the DCM, Acoet, and ButOH fractions showed promising activity against oral bacteria and fungi, possibly because the metabolites found are capable of causing dysfunctions of the cell membrane and cellular appearance, also interrupting cell division, inhibiting bacterial and fungal enzymes, and affecting the establishment of colonies (Guimarães et al. 2019, Mahizan et al. 2019, Yuyama et al. 2020, Manso et al. 2021).
CONCLUSIONS
In summary, after evaluation of fractions of ethanolic extract from C. sessilis stems bark, our results showed that the Acoet has prominent antioxidant and antiglycation potential, as well as being good inhibitors of the α-amylase enzyme and DCM, Acoet, and ButOH fractions have important antibacterial and antifungal capacities when compared to other fractions. Our study contributes to understanding the phytochemical constitution and the pharmacological effects of this plant and open possibilities for further projects using the isolated compounds from these fractions, to assess their biological potentials in vivo models.
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