Secretory phospholipases A2 (sPLA2) are proteins with a molecular mass of approximately 13 to 20 kDa. They are widely found in various animals, such as mammals, and in the venom of snakes, and they are almost exclusively calcium dependent. These enzymes have the same structural elements for enzymatic catalysis and have significant structural similarities among themselves [1
]. sPLA2 and the calcium-dependent cytosolic PLA2 have the same enzyme triad, which allows these enzymes to hydrolyze membrane glycerophospholipids to produce arachidonic acid and lysophospholipids. Excessive hydrolysis of membrane phospholipids by activated sPLA2 may lead to altered membrane function, leading to functional membrane failure and cell death. In addition, free fatty acids and lysophospholipid hydrolysis products are precursors of bioactive pro-inflammatory mediators, such as eicosanoids and the platelet-activating factor (PAF) [4
]. In particular, sPLA2 in snake venom is an attractive therapeutic target due to its accessibility, ease of purification and, in the case of sPLA2 in humans, high levels of systemic enzymatic activity, which characterize and contribute to most of the inflammatory disorders, including neurodegenerative diseases. Therefore, the discovery of new inhibitors of sPLA2 is essential to produce new safe and effective drugs with fewer side effects compared to the current therapy [5
Flavonoids are a class of special metabolites from plants, characterized by the flavan nucleus. These compounds are widely distributed in the leaves, seeds, bark, and flowers of plants; in addition, a recent estimate showed that human’s intake ranges from 26 mg to 1 g per day [7
]. Quercetin (Q) is a flavonol widely found in tea (Camellia sinemensis
), wine, beer, fruits, and vegetables [8
]. Additionally, this compound exhibits antioxidant and anti-inflammatory activities [9
]. A methylated derivative of Q shows lipophilicity, which promotes the compound’s access to the cell. Achyrocline satureioides
, Coriandrum sativum
, and Rhamnus petiolaris
are species that show the presence of 3-O
-methylquercetin (3MQ), rhamnetin (Rhm), and rhamnazin (Rhz), respectively, and have anti-inflammatory activities. In all of these cases, there is no evidence of the effect of methylated quercetins, such as Rhm, against inflammation mediated by sPLA2 and the consequent mobilization of arachidonic acid metabolism or the possible side effects, including cytotoxic activities [10
]. Considering the Q activities, this work aimed at evaluating 3MQ, Rhm, Rhz, and Q regarding this anti-inflammatory function, besides analyzing their cytotoxic activities toward the J774 cell lineage and evaluating the creatine kinase (CK) levels. In addition, the methylation of free hydroxyl groups in flavones results in more metabolically-stable derivatives with superior membrane-penetrating properties and, thus, vastly improves bioavailability, which should improve their ability to act inside cells [13
]. The anti-inflammatory effects of Rhm by modulation of kinases, oxidative stress, and suppression of pro-inflammatory mediators have already been observed [11
], however, there are no studies that reveal sPLA2’s inhibitory action of this compound and the relation of the structure and function with other quercetin derivatives. Thus, in this study, the inhibitory potential of Q and their methylated derivatives in vitro and in vivo were evaluated against sPLA2 from Bothrops jararacussu
. To find safe and effective compounds, cytotoxic effects were evaluated using the cell lineage J774 as a model for cytotoxicity and CK levels were assayed as a model of acute toxicity in vivo.
Plant extracts are characterized by the presence of active compounds and studies reveal the PLA2 inhibitory potential of extracts and isolated compounds. Crude aqueous extracts of Casearia sylvestris
, Piper umbellatum
, and Piper peltatum
show this inhibitory activity [15
]. Numerous studies have shown the anti-inflammatory and antioxidant activities of flavonoids and Q is widely distributed in vegetables and fruits [8
]. Its potential and distribution highlight the search for new molecules and the need to understand their structures and functions. The methylation of polyphenolic compounds seems to be the second most significant conjugation reaction from a nutritional and metabolic point of view [21
There are studies that have evaluated the effect of flavonoids and glycosylated flavonoids on the course of the enzymatic and pharmacological activity of secretory phospholipases A2 [5
]. Moreover, these studies deserve further efforts to elucidate the mechanism of action of these types of flavonoids on sPLA2, including studies of the conformational changes of flavonoids that modify their antioxidant properties to pro-oxidants, and these mechanisms should be better understood. The chemical structure of polyphenols gives them the ability to act as free radical scavengers. The type of compound and the degree of methylation influence this capacity, and the number of hydroxyl groups is another parameter that determines the antioxidant activity [22
]. Thus, within molecular logic, and with substantial data showing the antioxidant effects of various flavonoids, it would be assumed that any protective or neutralizing effect of flavonoids on sPLA2 could be associated with the presence of different numbers of OH moieties in the B ring of the flavonols. These characteristics may contribute to flavonoid’s antioxidant activity as well as its toxicity [23
]. Despite the relevant role of sPLA2 in the course of the inflammatory process, there are not methylated flavonoid studies on the enzymatic and pharmacological activity of sPLA2.
Results presented herein about the treatment of sPLA2 samples in the presence of Q and its derivatives clearly showed that it is not only the hydroxyls present in these flavonoids that are capable of neutralizing the pharmacological and enzymatic activities of sPLA2 from Bothrops jararacussu
. The combination of methyl groups, the hydroxyl moiety present in the C-3 of the C ring and C-′4 of the B ring are shown to be essential to inhibit the enzymatic activity of sPLA2, as are the groups responsible for the neutralization of the edematogenic effects induced by this sPLA2 in this study. It can be accounted for because the C and B rings of some flavonoids may play a crucial role in the interaction of these compounds with the catalytic site of sPLA2 [5
]; however, in the case of Rhm, the presence of the OH group in the C ring is crucial to decrease both the edematogenic and enzymatic activities of sPLA2. Besides that, the hydroxyl group present in the C-′4 also shows an enzymatic inhibitory role. In addition, our results showed that the methyl group present in the A ring of Rhm can be responsible to the absence of cytotoxicity and inhibition of high CK levels induced by sPLA2 from B. jararacussu
venom. Once macrophages are essential in the primary response in inflammation [25
], Rhm is revealed to be a compound that inhibits inflammation without impairing the viability of the body´s natural defence.
One important point is that the methylation of flavonoids may increase their hydrophobicity and, thus, increase the strength of the hydrophobic contacts between the protein and the flavonoid [26
]. Studies with quercetrin reveal a high inhibition of sPLA2 from Crotalus durissus terrificus
, possibly due the molecular interactions between the compound and the protein, as hydrogen bonding and hydrophobic interactions. The electrostatic interactions were observed between quercetrin and the amino acid residues Gly 30, Gly 32, His48, and Asp 49 and Ca2+
ion of sPLA2 leading to structural changes on the protein These interactions involves amino acids from de Ca2+
-binding loop (e.g., Gly 30) and catalytic site (e.g., Asp 49 and His 48) [5
]. Thus, besides the essential role of 3-OH to inhibit sPLA2, 4’-OH of Rhm can interact with Gly-30 of the protein leading to a higher decrease of its activity. These data suggest an essential role of hydroxyls on the interactions between the compound and sPLA2 and its following inhibition.
The circular dichroism clearly shows that, in the case of sPLA2, both methylations and the presence of hydroxyls can change the CD spectrum of sPLA2. Q strongly decreased sPLA2 α-helix and shows the higher enzymatic inhibition. Despite Rhz exhibit also a potent inhibition of the enzyme, CD data revels that this compound do not leads strong modifications in sPLA2, conserving the structure. High structure modification is observed in 3MQ, however, this change leads a decrease in the inhibition activity. Rhm interact with the enzyme besides exhibit an inhibitory potential. In this study, the presence of OCH3 on the catechol group increase sPLA2 inhibition, without strongly changing the enzyme structure. Despite the presence of OCH3 on the B ring, leading to a higher inhibition activity than Rhm, this methylation leads to high toxicity and high CK. Thus, 3-OH presence is also associated with sPLA2 inhibition, and methylation on the A ring brings no toxicity and lower CK. Higher interaction with sPLA2 of compounds with an absence of 3-OH was observed in 3MQ in this study and in quercetin [5
]. These data are robust enough to show that methylation in the A ring is important to essentially abolish the cytotoxic and muscle damage effects via a free radical neutralizing-dependent pathway.
All flavonoids tested in this work are methylated derivatives of Q, which occurs naturally and is found in several species of phytochemical interest. In the case of Rhm, there is considerable evidence showing that this particular flavonoid exerts its anti-inflammatory activity through two basic mechanisms: via envelope suppression of free radicals and by pro-inflammatory mediators [27
]. These results show that the myonecrosis induced by several basic sPLA2s, such as sPLA2 from this work, would involve a strong increase in oxidative cellular stress, and the antioxidant capacity of Rhm would be very interesting for treatment of muscular degeneration induced by this protein. In vivo, all quercetins showed co-protection effects, revealing a decrease in paw edema at all times. The co-protection effect of Q was elucidated in another study [28
], and its effect on chronic inflammation was higher than hesperidin (glycosylated and methylated quercetin). As it is known, there is a greater potential for aglycone flavonoids in terms of antioxidant activity in relation to the glycosylated forms [28
]. This lowest potential is due to the hydroxyl substitution at the C-3 position in the C ring with a methylated or glycosylated group as a result of a decrease in OH and the changes in the molecular conformation of hydroxyl group. The flavonols with OH in the C-3 position of the C ring are planar; thus, compounds are able to conjugate and relocate their electrons, as well as increase the radical phenoxyl stability of the flavonoid. Therefore, 3-OH absence results in a twist on the B ring changing the antioxidant potential [24
]. In this study, 3MQ reveals a loss of its sPLA2 inhibition activity; it also shows high cytotoxicity and CK. These data suggest the essential role of methylations in the A ring, the planar conformation’s maintenance due to the presence of 3-OH and catechol group, besides the OH interactions between compound and protein to lead an inhibitory potential plus no toxicity against the immune system.
A decrease in paw edema was observed in Cassia sophera
Linn, and this species has Rhm in its composition. In addition, Coriandrum sativum
L. and Prusmus cerasus
L. revealed the presence of Rhm, highlighting the potential of dietary species in phytochemical studies due to their benefits [11
]. The suppression of the free radicals and pro-inflammatory mediators of Rhm would possibly inhibit of JNK and p38 MAPK activities by decreasing the COX2 pathway [11
]. These pathways are important in the inflammation route, and our work shows that Rhm is beyond this activity; however, Rhm can both modulate and modify the action of sPLA2, thereby decreasing inflammation not only by maintaining inflammatory stimuli, but by also neutralizing enzymatic and pharmacological effects of sPLA2. In addition, in this work, Rhm and Q abolished the inflammatory effect induced by sPLA2 within 10 min after its injection—i.e., even after the onset of inflammatory process—Rhm and Q were effective for edema neutralization (Figure 2
). However, the cytotoxicity results showed that the best choice is Rhm, and the fact that this flavonoid has molecular regions involved in the inactivation of sPLA2 (Figure 2
), reveals that this compound inhibits the action of sPLA2 by neutralizing the protein, itself, and protecting the cell from the effects of free radicals. Studies reveal a protection function of catechol moiety of Q, improving cellular uptake, metabolic stability, and lower toxicity of the methylation in Q. Additionally, structural modifications at the 5 or 7 position preserve most of the antioxidant capacity and this feature is observed in Rhm, once methylation occurs at C-7 [35
Studies reveal that nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs in inflammatory diseases. However, these drugs inhibit cyclooxygenase (COX) and COX-1 inhibition leads to gastrointestinal and renal side effects [36
]. The enzymatic reaction involved in prostaglandin production by activation of COX is a two-step reaction: cyclooxygenase catalyses the formation of PG from arachidonic acid and a subsequent peroxidase reduces hydroperoxides (PGG2) and activates the cyclooxygenase and the peroxidase activity of COX can also generate reactive oxygen species. Thus, there is no total certainty of inhibiting COX-2 without inhibiting COX-1 and there is still a third group of COX (COX-3), which are inhibited by the same COX-1 inhibitory drugs [37
]. It is also important to remember that all COX-2 drugs target the activity of converting arachidonic acid to prostaglandin, but forget that COX is comprised of bifunctional enzymes, where the reactions of bis oxygenase and peroxidase occur at distinct sites, which are structurally and functionally interconnected. NSAIDs bind specifically to the arachidonic acid binding site, but do not inhibit the peroxidase site that remains active, and COX-1 is found in most tissues and is related to the production of prostanoids involved in homeostasis processes in the body [38
]. Thus, by looking at the entire spectrum of COX-2, COX-1, and COX-3, the search for an inhibitory drug with inflammatory activity as a function of the specific inhibition of sPLA2 is complex, since the inhibition is not complete and the production of prostanoids is physiological, and COX-1 can also be modulated.
In turn, in this study it is believed that the structure and molecular conformation of Rhm has anti-inflammatory activity and did not induce toxicity to cell lineages, nor to the experimental model; therefore, it can be considered a prototype anti-inflammatory drug.