Background
Several diseases, such as cancer, vascular diseases (atherosclerosis, stroke, heart failure, and cerebrovascular disease), metabolic diseases (diabetes and metabolic syndrome), neurological diseases (Parkinson’s disease, Alzheimer’s disease, epilepsy and dementia), pulmonary diseases, rheumatoid arthritis, osteoarthritis, muscular dystrophy and chronic fatigue syndrome are associated with inflammation processes. Inflammation is a complex host response to injury, involving the recruitment of leukocytes and extravasation of plasma proteins. This process is coordinated by a range of chemical mediators, such as arachidonic acid metabolites, cytokines and nitric oxide (NO) [
1‐
4].
Traditionally in clinical practice, treatment of inflammatory disorders includes the extensive use of non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. The action of these therapeutic agents ranges from inhibition of enzymes responsible for production of arachidonic acid metabolites to inhibition of cytokines expression. However, these medicines have some adverse effects that limit their use, such as gastrointestinal damage, mainly when administered at high doses for prolonged periods [
5].
In recent years, numerous products derived from plants have been investigated for therapeutic application due to their pharmacological activity on inflammatory processes and other illnesses. Dietary agents can interfere with several cell-signaling pathways and the molecular targets modulated may be the basis for how these compounds not only prevent but also treat the diseases [
5]. Many plants, herbs and spices typically used for food flavoring and nutrition are excellent sources of phenolic compounds, which have been reported to show good antioxidant activity. Frequently the anti-inflammatory activity of natural compounds has been associated to their antioxidant activity, but their capacity to suppress nitric oxide production is also emphasized [
6‐
8].
Ginger (
Zingiber officinale Roscoe) rhizomes and rosemary (
Rosmarinus officinalis L.) leaves are among the most important and extensively used spices worldwide. Extracts or components from ginger and rosemary, such as polyphenolic compounds (6-gingerol and its derivatives for ginger rhizome, as well as carnosic acid and carnosol for rosemary leaves) have received special attention, specifically for their anti-inflammatory, antitumor and antioxidant activities [
7,
9‐
14].
Animal cell culture studies are useful for elucidating the mechanisms of action of plant extracts. But, these evaluations are often limited due to the high hydrophobicity of the plant extracts, their sensitivity to heat, light, oxygen, and their inherent poor bioavailability. In these studies, organic compounds such as ethanol, methanol, ethyl acetate, tetrahydrofuran, dimethylsulfoxide (DMSO), dichloromethane and carboxy methylcellulose are commonly used as vehicles to deliver the liposoluble materials to the cells. DMSO stands out in this type of application, because this polar and aprotic solvent is able to dissolve an enormous range of polar and nonpolar small molecules, being, in addition, miscible with water. Its uses encompass cells, tissue and organ preservation as well as enhancement of pharmaceutical agent penetration.
While plant extracts or their components are recommended for prevention of inflammatory process and other diseases, the use of solvents or detergents to disperse hydrophobic active molecules during cell culture or animal testing has been questioned [
15,
16]. In this sense, the design of adequate systems to protect, carry, deliver and control the release of lipophilic bioactive molecules extracted from plants is of paramount importance to proper analyze their pharmacological effects.
Nanoparticles, such as liposomes or lipid vesicles, have proved themselves as excellent systems for medical applications ranging from diagnostics to controlled drug delivery. Liposomes are able to efficiently incorporate hydrophobic, hydrophilic and amphiphilic molecules, being then useful as vehicles for the administration of species with different characteristics. These vesicles may be obtained reproducibly and with costs relatively low through the use of methodologies which do not require harmful organic solvents, and as a consequence, inherent vehicle toxicity may then be significantly reduced when assessing in vitro and in vivo effects of plant extracts [
17,
18].
Another strategy useful to disperse hydrophobic components is their solubilization with the Pluronic F-68 (PF-68), also known as Lutrol VR F68 or Poloxamer 188, a triblock copolymer composed of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO). PF-68 is a low-foaming, non-ionic surfactant that has multiple functional effects on animal cells [
19,
20], being widely used to protect the cells from shear stress during culture in stirred tanks.
Thus, the purpose of the present study was to evaluate and compare the in vitro effects of ginger and rosemary extracts obtained by supercritical fluid extraction, a technology that renders preparations free of toxic organic solvents, using liposomes, Pluronic-F-68 and DMSO as vehicles. The production of inflammatory mediators, specifically cytokines and nitric oxide, by two different types of macrophage cells was analyzed. To our knowledge, no other study has been carried out to compare the performance of DMSO, Pluronic-F-68 and liposomes as delivery systems for the in vitro evaluation of these plant extracts.
Discussion
Aiming at the determination of an adequate vehicle to deliver the ginger and rosemary extracts to macrophage cells in culture, DMSO, Pluronic F-68 and liposomes were evaluated in this study. In the present study, it was observed that DMSO was the most adequate vehicle for the tested plant extracts. DMSO has been recognized to have the ability to be an effective carrier of small molecules through a variety of barriers. This solvent can be employed to dissolve organic compounds that are required in animal cell culture media due to its excellent solvating power. Its efficiency as a solvent in the study of the antioxidant properties of lipophilic compounds has been also demonstrated [
28].
Probably, the superior effect of DMSO containing-extracts can be attributed to a higher accumulation of the plant compounds in the cells induced by this solvent than by liposomes, which would require adsorption, fusion, endocytosis or leakage of the liposomal contents to cell vicinities, or with Pluronic F-68, which can absorb to the surface of the cells, somehow impairing the diffusion of the extract components to the cell interior. The transport of a particular compound through a given cell membrane is affected mainly by the concentration of the compound in the vehicle, the diffusion coefficient of the compound through the membrane, the partition coefficient of the compound between the membrane and the vehicle, and the thickness of the membrane barrier. DMSO, specially, can have aprotic interactions with intercellular lipids, being able to cause reversible distortion of lipid head groups, which in turn could produce a more permeable packing arrangement [
29].
Moreover, when used as a delivery system, DMSO may alter in vitro ginger and rosemary extracts bioavailability and cytotoxicity. At very small and non-lethal concentrations, DMSO can be an exogenous antioxidant due to its ability to react with free radicals [
30], acting as a radical scavenger that protects cells against compounds that increase intracellular levels of radical oxygen species [
31]. This behavior, despite being desirable, may affect the analysis of potential pharmacological effectiveness of hydrophobic compounds in vitro.
Given that the delivery of plant extracts to cell cultures is generally difficult due to their predominantly hydrophobic nature, organic solvents, serum lipoproteins and surfactants are frequently used to disperse them. Alternatively, plant extracts may also be delivered incorporated in liposomes or in other types of particulate vehicles. Our results also demonstrate that the co-administration of liposomes and plant extracts may cause death of macrophages cells and induction of NO production. It is unclear why the lipid vesicles used in this work were cytotoxic to macrophage cells, but this fact might be related to their lipid composition or to the fact that liposomes can be rapidly phagocytized by macrophages. The lipid vesicles were prepared using DPPC and cholesterol, which could go through oxidation and hydrolysis during cell culture, causing alterations in physicochemical properties of the dispersion such as pH, zeta potential, and phase behavior [
32], which could be detrimental to sensitive cell lines.
Abnormalities in lipid metabolism may cause an overproduction of reactive oxygen species and as a result, damage to cell components, including proteins, carbohydrates, nucleic acids, and lipids, might occur, leading to progressive decline in physiological function and ultimately, to cell death [
33]. Also, when in excess, cholesterol can be a potent inducer of death on macrophage cells in cultured [
34]. In this sense, Takano et al. [
35] reported that liposomes could be cytotoxic to the mouse macrophage-like RAW264.7 cell line. These authors proposed that liposome cytotoxicity could be attributed to generation of reactive oxygen species (ROS), required for the induction of apoptosis, being clearly dependent on the cholesterol concentration in the liposomal formulation.
The search for compounds of natural origin with high anti-inflammatory activity, low toxicity, and low cost is of great interest for the development of new treatments to inflammatory diseases. In the last years, several products derived from plants with therapeutic activity on inflammatory processes have been investigated, and frequently extracts with high antioxidant activity are employed for this purpose. In this context, ginger and rosemary extracts have been traditionally used in the popular medicine for a wide range of health-related problems, including chronic inflammatory diseases.
The majority of phytochemicals with anti-oxidative and anti-inflammatory activities so far identified belong to the vast family of polyphenols. Despite the fact that relatively high concentrations may be required to reach the desired therapeutic effect regarding anti-inflammatory activity, great importance is given to the quality of the extract [
36]. Ideally, these extracts should be obtained from organically grown plants to avoid the presence of deleterious contaminants as pesticides and through technologies that protect the many labile components of these extracts from degradation during processing. However, traditional organic solvent extraction results in the presence of residual solvent and low purity of the extract. Avoidance of those issues is possible with the use of extraction procedures based on supercritical carbon dioxide technology.
In the present study, the effects of ginger and rosemary extracts on murine macrophages and on a macrophage tumor cell line were analyzed. In vitro studies involving animal and tumor cells are a fast and convenient tool for development of new drugs. Macrophages can initiate inflammatory response and these cells have been commonly used as models in studies for elucidating the anti-inflammatory mechanisms of many plants and herbal extracts [
37]. Specifically, the mouse macrophage tumor J774 cell line, first described by Ralph and Nakoinz [
38], consists of adherent slow-migrating monocyte-macrophages with the ability to phagocyte or kill foreign cells. This cell line has been widely used as a model for studying new potential drugs on various aspects of macrophage functions, being an effective model in studies of anti-inflammatory activity [
39].
In this work, it was noticed that J774 macrophages were less sensitive to the cytotoxic effects of Pluronic F68 and DMSO and to high concentrations of ginger and rosemary extracts than the peritoneal macrophages. J774 is a murine macrophage cell line established from a spontaneous tumor developed in a female BALB/c mouse and some reports showed that the J774 cell line is a suitable model for the study of cytotoxicity and anti-inflammatory activity of different compounds to macrophages [
39‐
41]. Tumor-originated cell lines are frequently resistant to a number of distinct external stimuli, what probably explains why the effects observed on the J774 cells were not as strong as the ones observed on the peritoneal macrophages.
Cytokines, including interleukins and tumor necrosis factor-α (TNF-α), as well as reactive oxygen and nitrogen species (ROS and RNS) have been the most studied inflammatory mediators. These substances initiate the inflammatory response, recruit and activate other cells to the site of injury and subsequently resolve the inflammatory process. ROS include hydrogen peroxide (H
2O
2), superoxide anion (O
2●−), hydroxyl radical (OH
●), single oxygen and lipid peroxides, while RNS include nitric oxide (NO) and species derived from NO, such as peroxynitrite (ONOO
−). ROS and RNS at low levels contribute to cell signaling, mitochondrial respiration and biogenesis. However, when not properly controlled, high ROS and RNS levels cause DNA, lipid, and protein damage [
42], and consequently, cell death may occur [
43].
Inflammatory cells produce large amounts of nitric oxide (NO) when properly stimulated. NO is synthesized from L-arginine by a family of enzymes known as the nitric oxide synthases (NOS). In the immune system, the most important isoform is inducible NOS (iNOS). A wide variety of immune stimulants such as certain cytokines (IFN-γ, IL-1 and TNF-α) and/or bacterial endotoxins are potent inducers of iNOS [
44]. Lipopolysaccharide (LPS) is an endotoxin and the major component of the outer membrane of Gram-negative bacteria, known for its various biological activities and strong stimulation of pro-inflammatory responses in macrophages [
45,
46].
Nitric oxide released by iNOS from murine macrophages is cytostatic and cytotoxic for protozoan parasites, fungi and bacteria and despite the induction and activation of NOS along with excessive production of NO are common features of almost all infection-related diseases, acute or chronic inflammation may also result in the same outcomes [
47]. Given that these mediators and cytokines play important roles in the pathogenesis of a vast number of human diseases [
48], selective inhibitors of iNOS may in the future be useful in the treatment of several illnesses. In this regard, plant-derived substances as those evaluated in this work may be potential candidates.
In general, cytokines related with inflammatory response are not constitutively produced or are produced in low levels. However, the presence of appropriate stimuli, such as LPS, induces the production of pro-inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and TNF-α, leading to the initiation of an inflammatory response [
49]. Based on these facts, the development of new biological therapies for inflammatory diseases has generally focused on the blockage of members of the inflammatory cascade, such as cytokines [
50‐
52].
In this study, we have shown that both ginger and rosemary extracts dissolved in DMSO were able to inhibit LPS-induced NO production and were also capable to inhibit the production of TNF-α in murine elicited peritoneal macrophages. TNF-α and NO production were influenced in the same way by plant extracts and this could be explained by the fact that synthesis of NO is closely regulated by stimuli such as cytokine TNF-α. Endotoxins, such as LPS, and inflammatory cytokines, such as TNF-α and IL-1β, have been implicated in the expression of inducible nitric oxide synthase (iNOS), which produces NO in large amounts [
53].
Some previous reports have documented the anti-inflammatory effects of ginger extracts [
7,
54‐
56], but the results vary with cell type and inflammatory stimuli. Hong & Oh [
54] and Shimoda et al [
7] verified that ginger extracts or their constituents affect nitric oxide production by LPS-activated cells in a dose-dependent way for RAW264 cells. On the other hand, according to Jiang et al. [
55], the bioactivity of ginger extracts may not be easily predicted. These authors verified that inhibition of LPS-induced prostaglandin E production in human histiocytes in vitro is possible with crude CH
2Cl
2 organic extracts of ginger, however, despite their components may act at several sites, the extracts were not nearly as effective at inhibiting TNF-α. In contrast, Surh [
56] points that gingerol from the rhizome of ginger can suppress TNF-α production in mice and has potential for the therapy of TNF-related diseases. Lee and collaborators showed that ginger extracts from supercritical fluid extraction demonstrated free radical scavenging ability, reducing power and chelating property in dose-dependent manners [
57].
Similarly, the biological activity of rosemary extracts and their main constituents has been extensively investigated, giving sometimes contradictory results. A methanol extract of rosemary and its hexane fraction reduced nitric oxide generation and blocked TNF-α production in LPS-stimulated RAW 264.7 cells. The inhibitory effects of methanol extract of rosemary re-extracted with others solvents (chloroform, ethyl acetate, n-butanol and water) on LPS-induced NO production was also analyzed. The effects of these extracts were not as effective as those of the methanol extract or of its hexane fraction [
58]. Rosemary extract dissolved in DMSO showed to have a pronounced inhibitory effect on NO production by lipopolysaccharide (LPS)-activated RAW 264.7 macrophages [
59]. On the other hand, no induction of inhibitory effects on the LPS-induced nitrite production by the same cell line was noticed when compared to the reference drug indomethacin [
28]. Conversely, carnosic acid (CA), the main constituent of rosemary extract, inhibited LPS-induced oxidative/nitrosative stress in vivo by decreasing lipid peroxidation, protein carbonylation, and serum levels of nitric oxide [
14]. Since NO has been suggested to play an important role in the physiology of inflammatory diseases, it is possible to infer that some vehicles used to deliver in vitro active compounds may therefore contribute negatively in this treatment.
Corroborating this result, Checker et al. [
60] described the mechanism of anti-inflammatory activity of ursolic acid in activated T cells, B cells and macrophages. This acid is a pentacyclic triterpenoid carboxylic acid with antioxidant and anti-tumor properties and a constituent of rosemary extracts. Treatment of cells with ursolic acid significantly reduced the serum levels of pro-inflammatory cytokines [
60].
Kuo et al. [
61] also reported relevant results to support the potential use of rosemary extract obtained by supercritical carbon dioxide extraction and of its purified fractions as a nutraceutical formulation with inflammatory activity, showing marked suppression of LPS-induced production of NO and TNF-α by RAW 264.7 cells in a dose-dependent manner, particularly regarding carnosic acid. In addition, Tripathi et al. [
62] reported a decrease of almost 10 fold in TNF-α production by macrophages obtained from C57BL/6 mice in the presence of ginger alcoholic extract plus LPS stimulation in comparison to macrophages stimulated with LPS alone. The authors showed that the production of LPS-induced IL-1β was completely inhibited in the presence of ginger alcoholic extract. However, these authors did not evaluate the effects of solvent alone nor of TNF-α / LPS stimulation in tumor cell lines [
62]. Cattaneo and colleagues [
63], reported the major components of the rosemary extract: rosmarinic acid, luteolin, apigenin, carnosol, caffeic acid and scutellarin. Hydroalcoholic extract of
Rosmarinus officinalis reduced the proliferation of the human melanoma A375 cell line by a pro-oxidant activity of the extract. The antiproliferative activity was a property of the whole extract and seems to be resulting from multi-factorial effects of its components [
63].
Our data showed that only the rosemary extract was able to significantly reduce the production of IL-1 in peritoneal macrophages cultured in the presence of LPS/IFN-γ. However, the ginger extract showed higher anti-inflammatory activity on the J774 tumor cell line than the rosemary extract. Thus, differences on NO, TNF-α and IL-1 production and cell proliferation between the macrophage models tested herein were observed. Most likely, these differences may be attributed to the origin of the cells. Tumor cells, such as the J774 cell line, may naturally present more resistance to external stimulus.
At a cellular level, the onset of pro-inflammatory reactions tends to start by release of early-responding cytokines such as IL-1α and -β and TNF-α. IL-1α/β and TNF-α subsequently regulate the expression of a variety of secondary cytokines and chemokines, including IL-6 and IL-8 [
3]. Agents able to suppress TNF-α and IL-1 activity have potential for therapy of these TNF-α and IL-1-associated diseases. Monoclonal antibodies against these cytokines have emerged as an efficient treatment with many clinical benefits in experimental models of some diseases [
64,
65]. However, the costs of antibody-based therapy are usually very high, thus supporting, justifying and stimulating the search for alternative approaches.
Neoplastic cells often over-express pro-inflammatory mediators including proteases, eicosanoids, chemokines and cytokines. Cytokines are major mediators of communication between cells in the inflammatory tumor microenvironment. Several cytokines such as macrophage migratory inhibitory factor (MIF), TNF-α, IL-6, IL-17, IL-12, IL-23, IL-10, and TGF-β have been associated with both experimental and human cancers and can either promote or inhibit tumor development. Then, inflammatory conditions precede development of malignancy in some cancers. In others tumors, oncogenic change drives a tumor-promoting inflammatory milieu [
66].
Also, our results suggest that the lipid vesicles, DMSO and Pluronic, can protect the cells, probably due to changes in the form of absorption, distribution and cellular metabolism of hydrophobic molecules present in the culture medium. These results also can indicate that these vehicles may be rapidly absorbed and prevent the development the free radicals, acting as a radical scavenger that protects cells against compounds that increase intracellular levels of radical oxygen species. However, further assays are necessary to verify this hypothesis.