Flavonoids are presented in many plant extracts, being constantly the focus of pharmacological studies. Despite of their well-described antioxidant activity [
Sepsis is an important health problem and a leading cause of morbidity and mortality in many intensive care units. This condition is characterized by the overproduction of proinflammatory mediators, which frequently occurs after various noxious injuries, especially bacterial infection, as a consequence of abdominal surgery, appendicitis, perforated ulcers, or an ischemic bowel [
A common cause of sepsis is the exposure to lipopolysaccharide (LPS), the structural component of gram-negative bacteria membrane, or to peptidoglycan and lipoteichoic acid, structural components of gram-positive bacteria membrane [
Thus, phytochemical analysis of a flavonoid-rich fraction obtained from
Fresh leaves of
Fresh leaves of
HE was fractionated by degree of polarity using a liquid-liquid partition as follows: 1 g was dissolved in H2O (200 mL), followed by the addition of CHCl3 (200 mL) and vigorous stirring. The mixture was left in a decanting funnel until the formation of two phases, which were separated. The CHCl3 fraction (HE-Chl) was dried under reduced pressure and stored at −20°C. The aqueous fraction was completed with additional 200 mL of ethyl acetate, followed by the same separation procedure, giving rise to an organic fraction (HE-EA), which was evaporated and stored. Subsequently,
In order to determine its chemical composition, the fraction HE-Bu (2 mg) was hydrolyzed in 0.5 mL of 45% formic acid (v/v), 14 h, 100°C [
This was carried out in an Acquity-UPLC system (Waters, MA, USA), composed by a binary pump, sample manager, and column oven. Detection was provided by a photodiode array detector (PDA) and a triple quadrupole, electrospray ionization-mass spectrometry (ESI-MS) Quattro LC (Wates, MA, USA). The separations were developed on Waters BEH-C18 column, with 50 × 2.1 mm length and 1.7
This was carried out in a Varian Saturn 2000 R, equipped with a capillary column DB-225-MS (J&W), 30 m long. The mass spectrometry was consisted of an Ion Trap detector, with electron ionization (EI) operating at 70 eV. Each monosaccharide was previously converted to its alditol acetate and identified using authentic standards.
Male albino Swiss mice (3 months old, weighing 25–30 g) from the Federal University of Paraná were used for biological tests. They were maintained under standard laboratory conditions, at a 12 h light/dark cycle and controlled temperature (
Mice were randomly divided into five groups, containing 10 mice/group: sham operation; CLP plus vehicle (water + ethanol 3%; p.o.); CLP plus HE-BU 75 mg·kg−1 p.o.; CLP plus HE-Bu 150 mg·kg−1 p.o.; CLP plus HE-Bu 300 mg·kg−1, p.o. It was administered 30
For the next experiments, 1 h before the surgery, mice were orally treated with vehicle, HE-Bu (75, 150, or 300 mg·kg−1, p.o.), and after 6 h postoperation, all mice were sacrificed. Their tissues from lungs, small intestines (ileum), and blood were collected and frozen for later use to determine the myeloperoxidase (MPO) activity and investigate cytokine production in serum and iNOS and COX-2 tissue expression. Dexamethasone, a corticosteroid anti-inflammatory drug, was used as control and was administered subcutaneously at dose 0.5 mg/kg.
The MPO activity, assessment of the neutrophil influx, was determined according to established protocols [
Tumor necrosis factor alpha (TNF-
The ileum samples were washed twice with PBS and then homogenated and lysed in extraction buffer (composition in mM: Tris/HCl 20 (pH 7.5; QBiogene), NaCl 150, Na3VO4, sodium pyrophosphate 10, NaF 20, okadaic acid 0.01 (Sigma), a tablet of protease inhibitor (Roche), and 1% Triton X-100 (QBiogen)). Total protein (20
Data were expressed as means ± SE of five or ten mice examined for each group. Statistical error was determined by one-way ANOVA; the post hoc test was Bonferroni’s. Calculations performed with Graphpad Prism 5.0.
It was observed that lethality was markedly delayed in mice orally administered with HE-Bu (see Figure
HE-Bu obtained from
It is believed that sepsis may lead to aberrant host inflammatory responses, causing cell injury and organ dysfunction. Neutrophil infiltration is an important pathophysiologic alteration associated with sepsis. These cells cause directly damage to the tissues by releasing proinflammatory mediators, as cytokines, superoxide-derived free radicals, and lysosomal enzymes, such as MPO, which amplify the systemic inflammatory response and cause multiple organ failure [
Considering that MPO is a lysosomal enzyme, produced by polymorphonuclear leukocytes, and related to the production of hypochlorous acid (powerful oxidant), the effects of HE-Bu treatment on MPO activity were also investigated. CLP surgery markedly increased lung tissue MPO levels compared with sham group (46.9%) (Figure
Several previous studies have demonstrated that some cytokines, especially TNF-
Effect of HE-Bu obtained from
TNF-
Besides the cytokines, the nitric oxide (NO) and eicosanoids are also mediators involved in the excessive proinflammatory response during sepsis. Overproduction of NO by inducible nitric oxide synthase (iNOS) is associated with the septic shock, considered as the main cause of mortality among the septic patients [
Another important product of inflammation from cells of the innate immune system is cyclooxygenase-2 (COX-2). This inducible isoform of the cyclooxygenase enzyme catalyzes the formation of inflammatory prostanoids, such as prostaglandins and thromboxane, which can mediate a significant inflammatory response. Systemic COX-2 is increasingly recognized as an important player in sepsis-induced inflammation. In fact, COX-2-deficient mice are protected from sepsis-induced inflammation and death [
In this study, we examined the effects of HE-Bu, by immunoblotting, on iNOS and COX-2 expression by ileum cells of septic mice. HE-Bu (75, 150, and 300 mg·kg−1) decreased the levels of iNOS by 35.2%, 33.5%, and 75.5% respectively (Figures
HE-Bu from
Considering that the overwhelming inflammatory and immune responses during the early stage of sepsis involve a vast array of mediators, it is critical to control this complex inflammatory cascade and consequently to manage the sepsis effects [
Identification of compounds detected by UHPLC-MS.
Peak | R |
Ion (−) | *Main fragments | Identification |
---|---|---|---|---|
1 | 3.58 | 631 | 479, 316, 287, 169 | Myricetin-hexosyl-gallate |
2 | 3.89 | 479 | 316, 287, 271 | Myricetin-hexoside |
3 | 3.93 | 463 | 300, 271, 255 | Quercetin-hexoside |
4 | 4.05 | 615 | 463, 300, 271, 169 | Quercetin-hexosyl-gallate |
5 | 4.20 | 615 | 463, 300, 271, 169 | Quercetin-hexosyl-gallate |
6 | 4.31 | 449 | 316, 287, 271 | Myricetin-arabinoside |
7 | 4.40 | 463 | 316, 287, 271 | Myricetin-rhamnoside |
8 | 4.55 | 599 | 447, 300, 285, 169, 124 | Quercetin-rhamnosyl-gallate |
9 | 4.70 | 653 | 501, 463, 317, 169, 124 | n.i. |
10 | 4.91 | 539 | 169, 151, 123 | n.i. |
11 | 5.05 | 433 | 301, 271, 255 | Quercetin-arabinoside |
12 | 5.28 | 447 | 301, 271, 255 | Quercetin-rhamnoside |
13 | 6.11 | 609 | 463, 300, 271 | Quercetin-rhamnosyl-hexoside |
*Negative fragment-ions.
n.i.: not identified.
(a) Chromatogram of fraction HE-Bu detected by PDA 360 nm. All peaks had absorbance curves characteristic of flavonols (b). (c) Schematic representation of the homolytic cleavage on CID-MS of peaks 7 (d) and 12 (e) identified as myricetin and quercetin rhamnosides, respectively.
With the aim of identifying more compounds, HE-Bu was acid hydrolyzed, followed by a liquid-liquid partition. The organic layer was analyzed by UHPLC-PDA-MS, which confirmed the presence of quercetin and myricetin, by comparison with authentic material (Sigma). The monosaccharides present in the aqueous phase were analyzed by GC-MS as their alditol acetates, showing the presence of arabinose (20%), rhamnose (28%), galactose (21%), and glucose (31%). Considering the absence of other monosaccharides, the presence of arabinose and rhamnose on the flavonoids could be stated without any misinterpretation, whereas galactose and glucose could not. As stated above, the glycosylation site on flavonols occurring in fruit of
Then, HE-Bu was submitted to UHPLC-PDA-MS analysis obtained in the reversed phase (Figure
Compounds of low abundance were consistent with other glycosides of quercetin and myricetin, containing different monosaccharides (Table
It has been reported that quercetin attenuates lethal systemic inflammation caused by endotoxemia, even if treatment is started after the early TNF response, and also that quercetin is able to inhibit the iNOS and COX-2 gene expressions in macrophages [
In conclusion, we have clearly demonstrated that HE-Bu inhibits important proinflammatory parameters: lethality induced by sepsis, neutrophil influx, proinflammatory cytokines, and iNOS and COX-2 expression. These beneficial effects may be related to the presence of glycosides of quercetin and myricetin in HE-Bu. This work may lead to the confirmation of the pharmacological properties of
Cecal ligation and puncture
Flavonoid-rich fraction
Tumor necrosis factor-
Interleukin-1
Nitric oxide
Inducible nitric oxide synthase
Cyclooxygenase 2
Myeloperoxidase
Ultrahigh performance liquid chromatography-mass spectrometry
Gas chromatography-mass spectrometry.
Y. D. Rattmann and L. M. de Souza contributed equally to this work.
The authors declare no conflict of interest.
The authors would like to thank the Brazilian funding agencies: CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and Fundação Araucária for financial support.