Anti-Inflammatory Activity of Natural Products.

1. Introduction
Inflammation usually occurs when infectious microorganisms such as bacteria, viruses or fungi invade the body, reside in particular tissues and/or circulate in the blood [1,2,3]. Inflammation may also happen in response to processes such as tissue injury, cell death, cancer, ischemia and degeneration [1,4,5,6,7,8,9]. Mostly, both the innate immune response as well as the adaptive immune response are involved in the formation of inflammation [1,5,9]. The innate immune system is the foremost defense mechanism against invading microorganisms and cancer cells, involving the activity of various cells including macrophages, mast cells and dendritic cells. The adaptive immune systems involves the activity of more specialized cells such as B and T cells who are responsible for eradicating invading pathogens and cancer cells by producing specific receptors and antibodies.
Numerous inflammatory mediators are synthetized and secreted during inflammatory responses of different types. Inflammatory substances are usually divided to two main categories: pro- and anti-inflammatory mediators. Nevertheless, some mediators such as interleukin (IL)-12 possess both pro- and anti-inflammatory properties [10]. Among the inflammatory mediators and cellular pathways that have been extensively studied in association with human pathological conditions are cytokines (e.g., interferons, interleukins and tumor necrosis factor α), chemokines (e.g., monocyte chemoattractant protein 1), eicosanoids (e.g., prostaglandins and leukotrienes) and the potent inflammation-modulating transcription factor nuclear factor κ B.
Tumor necrosis factor (TNF)-α is an important pro-inflammatory cytokine which is secreted from various cells and exerts many cellular effects [11,12]. TNF-α has been associated with multiple illness states in humans, including immune and inflammatory diseases, cancer, psychiatric disorders, among others. Another cytokine which mostly exerts a pro-inflammatory activity is IL-1α [13,14]. It stimulates the secretion of pro-inflammatory cytokines such as IL-1β and TNF-α [13,14]. However, IL-1α has also been associated with anti-inflammatory activity. Similar to IL-1α, IL-6 usually acts as a pro-inflammatory cytokine but it also has some anti-inflammatory effects. As mentioned above, the IL-12 family of cytokines (including IL-12, IL-23, IL-27 and IL-35) possess both pro- and anti-inflammatory functions [10,15,16]. On the other hand, IL-10 is a potent anti-inflammatory cytokine the activity of which impedes the action of many pro-inflammatory mediators [17,18,19]. By weakening and controlling the inflammatory response IL-10 helps to maintain tissue homeostasis and attenuates the damage that may result from an exaggerated inflammatory response [17,18,19].
Prostaglandin (PG) E2 is probably the most studied PG in association with human physiological and pathological conditions [20]. It has various physiological roles including regulation of normal body temperature, gastric mucosal integrity, renal blood flow and the function of female reproductive system. On the other hand, alterations in PGE2 activity are associated with pathological conditions such as inflammatory diseases, abnormal changes in body temperature, colorectal cancer, among others. The pathway of PGs synthesis starts with generation of arachidonic acid from cell membrane phospholipids by phospholipase A2 (PLA2). Then, arachidonic acid is converted to PGs by the enzyme cycloogygenase (COX) [20]. Among the three known COX isoforms (COX-1, COX-2 and COX-3), the inducible enzyme COX-2 is recognized as the most active during inflammatory processes. Leukotrienes (LTs) such as LTB4 were also linked to human illness states including inflammation, asthma and depression [21,22,23]. LTs are produced by the enzyme 5-lipooxygenase (5-LOX) [22]. Another enzyme that is highly associated with inflammatory conditions is nitric oxide synthase (NOS) which produces nitric oxide (NO) [24]. Similar to COX-2, inducible NOS (iNOS) is the most pro-inflammatory NOS isoform.
The transcription factor nuclear factor κ B (NFκB) is a prominent regulator of immune and inflammatory responses and is highly involved in the pathophysiology of cancer [25,26,27]. In mammals, the NFκB machinery comprises several members (e.g., p50 and p65) which regulate both physiological and pathological processes [25,26]. At resting (un-stimulated) conditions NFκB resides in the cytoplasm [26]. Following activation by various infectious/inflammatory/mitogenic stimuli, NFκB proteins translocate to the nucleus and induce transcription of inflammatory-associated genes [26,27].
The practice of using plants, their parts or extracts as anti-inflammatory compounds is known since antiquity. For example, concentrated, viscous aqueous extract of ripe carob (Ceratonia siliqua L.) has been used for decades in Arab folk medicine, especially for treating mouth inflammations [28]. The use of plants or plant products for medicinal purposes was mostly documented in books and, lately, in an enormous number of websites (where the reliability of some of these websites must be examined carefully). In the last decades, hundreds of research and review articles were published regarding the anti-inflammatory activities of plants. In this review we introduce some highlights of the literature published mainly during the last three decades, with a few references to earlier reports.

2. Review Articles of Natural Non-Plant Materials
As mentioned above, dozens of review articles have been published in the last few decades. Interestingly, a notable number of them were published by scholars from India, a country with a well rooted traditional plant medicine and a vast diversity of medicinal plants. Our summary here focuses on some of these reviews, but also includes articles from other parts of the world in order to provide a wider view. This part includes review articles which summarize the anti-inflammatory activities of non-plant natural products which exist in mushrooms and honey. Mushrooms and honey traditional therapies are very well established in most cultures. Moreover, mushrooms/honey mixtures with other plant materials (including various extracts) were used in folk medicines since ancient times.
One of the early articles that introduced the anti-inflammatory activities of mushrooms and some of their compounds was published by Lindequist et al. in 2005 [29]. Four different mushroom species were reviewed: Phellinus linteus that is used in traditional medicines of cultures of East Asia, Ganoderma lucidum (Lingzhi mushroom) which also has a long history of medicinal use in China, the widespread Pleurotus pulmonarius (subtropical forests) and the edible Grifola frondosa. Some biologically active compounds were extracted from each of these mushrooms. For example, eight different triterpenoid ganoderic acids were isolated from G. lucidum, but only four of them exerted anti-inflammatory activity (Figure 1A shows one of these compounds). From G. frondosa, an ergosterol oxidation product active as an anti-inflammatory agent was isolated (Figure 1B).
An excellent, comprehensive review of anti-inflammatory activities of mushrooms was published by Elsayed and his colleagues in 2014 [30]. This article provides detailed, systematic information about a large number of mushroom species, many biologically active compounds, and importantly, suggested mechanisms of action. Among the most established anti-inflammatory effects of mushrooms that were reported in this article were: reduction of IL-1β, IL-6, LTs, PGs and TNF-α levels, and, inhibition of COX-2, iNOS and NFκB activity [30]. The authors state that terpenoids are the largest group of anti-inflammatory compounds in mushrooms and presented some seven-membered, structurally interesting examples of these compounds (such as cyathins and related compounds, Figure 2).
In their article, Elsayed et al. [30] addressed a study by Ngai et al. [31] which reported on the isolation of a 15 amino acids peptide from Agrocybe cylindrace which the authors named “agrocybin”. Ngai et al. [31] reported that “agrocybin” exerted antifungal but not anti-inflammatory activity. However, for the sake of accuracy, it is important to mention that the name agrocybin also refers to a different compound (not a peptide), reported by Rosa and his colleagues [32] who isolated it from another Agrocybe species, A. perfecta. To the best of our knowledge, this compound also named agrocybin is a polyeyne amide [32,33], as shown in Figure 3.
The second source of non-plant, natural material with anti-inflammatory activity is honey. Since it is one of the most ancient nutritious foods and was mentioned in most holy religious texts, honey has been used for medicinal purposes since antiquity. Numerous review articles were published about the anti-inflammatory properties of honey. Almost all of these reviews focus on clinical evidence for the anti-inflammatory activity of honey but lack any reporting of active compounds. Moreover, most of the articles indicate that the precise mechanism underlying the anti-inflammatory activity of honey is unknown, although some present proposed mechanisms [34,35]. Mostly, honey was reported to have anti-inflammatory effects (such as reduction of TNF-α levels, attenuation of COX-2 activity, and inhibition of NFκB translocation to the nucleus) but pro-inflammatory actions were also indicated (e.g., elevation of NO production) [34,35].

3. Review Articles on Natural Plant Materials
Among the different biological activities of natural plant products that have been published until now, anti-inflammation is one of the most reported effects. Table 1 summarizes selected review articles which report on the anti-inflammatory properties of natural plant materials.

4. Active Anti-Inflammatory Plant Extracts, Essential Oils, Juices and Powders
Extracting plant materials is the first major step towards testing the biological activities of this plant. In doing so, there are many advantages and some disadvantages, comparing with isolation of pure active compounds. When a whole extract is used, there is a good chance for synergism between active components that might be lost when each of these components is isolated. Such synergism was discovered in several medicinal tests, including those for anti-inflammatory activity [36,37]. On the contrary, the mixture of different compounds together may also lead to inhibitory effects, namely, that one component may reduce the biological activity of the other. In line with this assumption, some studies have showed that the anti-inflammatory activity of pure compounds (such as amentoflavone, pseudohypericin, and hyperforin, isolated from extracts of Hypericum perforatum) is higher than that of the extracts [38]. In addition to plant extracts, essential oils [39,40], plant juices [41] and plant powders [42] are also widely used for medicinal purposes.
Solvent selection for extraction of plant materials is one of the most important factors in determining the potential activity of the extract, since the solvent polarity determines which compounds will be extracted and which will not. For example, it is unlikely that water (very polar) will extract the active anti-inflammatory compound monoterpene 1,8-cineole (Achillea millefolium) but will easily extract protocatechuic acid (Boswellia dalzielii), and vice versa for n-hexane (non-polar). Thus, in many cases of newly studied plants, various extracts are prepared with solvents that have a wide polarity range. Table 2 summarizes selected research articles which have reported on the anti-inflammatory activity of plant extracts.
There are several worth mentioning points regarding the information presented in Table 2. The plant Corchorus olitorius, known as Mulukhiyah in the Middle East, is one of the most important edible plants in this region. Despite this fact there are relatively very few reported studies regarding the medicinal properties of this plant. A study by Zakaria et al. [43] found that it exerted potent anti-inflammatory and antipyretic effects (Table 2). The title of the article by Islam et al. [44] states that “ethanol” was used to prepare extracts from mango (Mangifera indica) leaves, however, in the “Materials and Methods” section only methanol was mentioned as the extracting solvent. In the study by Li et al. [45] different extracts were prepared from hawthorn fruit (Crataegus pinnatifida Bunge var. typica Schneider). A first extract was prepared using 70% methanol in water. Then, this extract was concentrated and extracted again with each of the following solvents: water, ethyl acetate, n-butanol and dichloromethane. Only the aqueous extract showed a significant anti-inflammatory activity. Of note, the most abundant hawthorn species in eastern Mediterranean region—Crataegus aronia—was never reported, although many of its medicinal activities are well acknowledged. A study by Abu-Gharbieh et al. [46] examined the anti-inflammatory effect of the aqueous extract of Micromeria fruticosa in mice. They reported a prominent reduction in carrageenan-induced paw edema. Moreover, pretreatment with the extract led to a significant decrease in gastric mucosal lesions induced by high-dose indomethacin, attesting for a gastro-protective effect of the extract.
Interestingly, M. fruticosa is one of the most useful herbs in western Asia, especially in the Middle East. Nevertheless, the specific compound(s) that is/are responsible for its anti-inflammatory activity is/are still unknown. Furthermore, M. sylvestris L. is an extensively eaten and widely used plant for medicinal purposes in the east Mediterranean region. A similar Micromeria species is M. nicaeenis. The chemical composition of this plant is unknown and, to the best of our knowledge, its anti-inflammatory activity has not been studied yet.
A study by Walker et al. [101] examined the anti-inflammatory properties of Eriodictyon angustifolium (a North American shrub) and its major active compounds on LPS-induced inflammation in human gingival fibroblasts. The dried leaves of the plant were extracted and the crude extracts were analyzed. Eight active compounds were identified as shown in Figure 4. Some of the extracts showed a profound anti-inflammatory activity. As mentioned above, aqueous extract of ripe carob (Ceratonia siliqua) is among the most used remedies in Arab traditional medicine [28]. A recent study by Lachkar et al. [103] clearly demonstrated that carob exerts prominent anti-inflammatory properties which are comparable to those of the potent anti-inflammatory drug indomethacin. Ripe pods of carob provide food for humans and animals. Ripe pods are traditionally extracted with boiling water after being crushed. The filtered extract is evaporated to viscous, sweet paste. In addition to its nutritional value, this paste has traditional, proven anti-inflammatory qualities, especially regarding mouth inflammations. Thus, it is strange that these qualities are just being studied in the last few years [103,109,110].

5. Selected Reports of Single Natural Products with Anti-Inflammatory Activities
As indicated in the previous section, isolation and testing of a single natural product for biological activities has both advantages and disadvantages. Two major advantages that were not mentioned are: (i) Testing a single active compound enables a thorough elucidation and better understanding of its mechanism of action; and (ii) if a single compound proves efficacious, it is possible to perform slight modifications on its structure or produce synthetic analogues in order to obtain more potent/efficacious compounds. In this regard, half of the the 2015 Nobel Prize in medicine was awarded to Campbell and Omura mainly for the synthesis and discovery of the anti-malarial compound ivermectin, which is the result of a very slight modification (a dihydro derivative) of the natural product avermectin [111].
Table 3 summarizes selected reports of anti-inflammatory activity of pure compounds that have been thoroughly investigated so far. An early study by Gupta et al. [112] reported that ursolic acid and cucurbitacin B did not exhibit anti-inflammatory properties. However, the findings concerning ursolic acid [112] are contradicted by later reports [50,75].
Many studies have presented ursolic acid as one of the major compounds responsible for the anti-inflammatory activities of various plants [119,120]. Moreover, as seen in Figure 5, oleanolic acid (which possesses anti-inflammatory effects, Table 3) and ursolic acid are structural isomers with very small difference in their structures. As for cucurbitacin B, similarly, the findings of Gupta et al. [112] contradict later reports which clearly indicated that the anti-inflammatory activity of Ecballium elaterium (squirting cucumber) [121,122] and Cucurbita andreana [123] is mainly due to this compound.
In a study by Guardia et al. [113] three plant flavonoids—rutin, quercetin and hesperidin—were found to have anti-inflammatory effects. Quercetin is an abundant polyphenol in the plant kingdom. Its structure (with other compounds) is shown in Figure 6. Onions (Allium cepa) contain a high concentration of quercetin and studies confirmed the anti-inflammatory activities of onion juice and extracts [124]. Abutilon indicum also contains high amounts of quercetin and has significant anti-inflammatory activity [94]. Furthermore, garlic contains large amounts of allicin (the structure of which is shown in Figure 6) which exerts potent anti-inflammatory effects [114].
As for the vast majority of natural products, even short term heating of garlic reduces the anti-inflammatory activity of allicin [125]. Another potent anti-inflammatory compound is (−)-myrtenol ([115], Table 3). As seen in Figure 6, it is essentially a mono-oxidized isomer of (−)-α-pinene. Interestingly, the anti-inflammatory activity of (−)-α-pinene is negligible compared with (+)-α-pinene [126], while the anti-inflammatory activity of (+)-myrtenol was never reported. This “enantiomeric selectivity” does not always occur as reported for equal anti-inflammatory activities of the enantiomers shikonin and alkannin found in Alkanna tinctoria [127]. A study by Thao et al. [116] examined the anti-inflammatory properties of different terpenes and polyphenols. Twenty six compounds, some of which were novel, were isolated and tested in this research. The most active anti-inflammatory compound was a derivative of juglone (5-hydroxy-7-methyl-2-methoxy-1,4-naphthaquinone). These results are consistent with previous reports regarding the anti-inflammatory activity of juglone [128].

6. Concluding Remarks
The data summarized in this article suggest that many compounds derived from natural products exert potent anti-inflammatory properties. Although the drugability of pure anti-inflammatory compounds extracted from natural products seems a complicated task, extracts and pure compounds of natural products may still open new venues for therapeutic interventions. Pharmaceutical companies will probably not express high interest and invest hugely in compounds that will be difficult to patent. Nevertheless, if proven efficacious and safe, the use of natural products-derived compounds should be advocated by policy makers and health authorities. Regular consumption of such products may become a successful and safe strategy to treat chronic inflammatory conditions.