Phytochemical investigation and anti-inflammatory potential of Atriplex leucoclada Boiss

Inflammation is a self-defense mechanism that is triggered by pathogens, tissue injury, trauma or dysregulation of the normal physiological processes. Inflammatory response rises from the production of prostaglandins that is synthesized from the unsaturated arachidonic acid via cyclooxygenase (COX) enzymes [1, 2]. Cyclooxygenase enzymes are responsible for the formation of important biological mediators called prostanoids, including prostaglandins, prostacyclin and thromboxane. There are two known isoenzymes: COX-1 and COX-2. COX-1 is constitutively expressed in many tissues and is the predominant form in gastric mucosa and in kidney. COX-2 is not normally expressed in most cells, but elevated levels are found at sites of inflammation. Pharmacological inhibition of COX can provide relief signs of inflammation and pain [3].

Anti-inflammatory drugs include steroids and non-steroid drugs. Steroid drugs have some serious side effects such as osteoporosis and fractures, immunosuppression, myopathy, cardiovascular disease, glaucoma and cataracts, diabetes and hyperglycemia, psychiatric disturbances, gastrointestinal and dermatologic adverse effects. These adverse effects limit their utility and make them less popular to be used in inflammatory diseases compared with nonsteroidal drugs [4]. However, nonsteroidal drugs may have some side effects too such as bronchospasm, renal failure, thrombosis, and gastrointestinal bleeding [5]. To overcome these problems, herbal medicines and phytochemicals have been submitted to studies to identify and develop natural products that can be used as anti-inflammatory agents [6‐8] or as a combinatorial therapy with these synthetic drugs [9].

The Atriplex genus (Amaranthaceae) constitutes herbaceous halophytes that include about 260 species distributed throughout the world, especially in the arid and semi-arid regions of Europe, Asia, Africa, Australia, and North America [10, 11]. Recent studies have shown that some species have high nutritional value and protein content and can be used as cereal grains as A. hortensis seeds [12]. Phytochemical investigations of some Atriplex species revealed various chemical constituents belonging to different chemical classes as: phenolics [13, 14], triterpenes, sterols [15], phytoecdysteroids [16, 17] and triterpene saponins [13, 15, 18, 19]. From biological point of view some species have been reported to have anti-inflammatory [20], antioxidants, anticholinestrase [13], antidiabetic [21], antimicrobial [22], hepatoprotective [23], immunomodulatory [14], analgesic, antipyretic, and, cytotoxic activities [24, 25].

Atriplex leucoclada Boiss. (English name: cut-leaf saltbush, orach, Arabic name: Ragal, رغل), is a low perennial shrub commonly growing in Saudi Arabian desert. This species has an agricultural importance in arid regions. It can adapt high salt habitats via different strategies [13, 26]. Reviewing the relevant literature little research was found discussing the metabolic content and/or the biological activity of A. leucoclada, where, one previous study reported the isolation of five triterpenoidal saponins and highlighted their molluscidal potential [27]. Accordingly, this study was designed to add more research about the phytochemical constituents and anti-inflammatory activity of A. leucoclada.

Reviewing the relevant literature several studies were found reporting diverse chemical structures of the metabolic contents of some Atriplex species. While, in regards of A. leucoclada, previous investigations were not sufficient to describe the chemical profile of the plant. To achieve this purpose, the chemical composition of both defatted methanol (ATD) and hexane (ATH) extracts were investigated. The current findings showed that all purified compounds, except for compound (1), were isolated for the first time from A. leucoclada [46]. Furthermore, compounds (6) and (8) were isolated for the first time from this genus. Compound (1) was previously obtained from other Atriplex species as A. inflata and A. nummularia [16, 17], compounds (3) and (4) were previously reported in A. stocksii [15], compound (7) in A. canescens [47] and compound (9) in A. inflate [46], while compound (2) and (5) were identified by GC–MS only in the methanol extract of A. halimus [48, 49].

Interestingly, this study is the first to report GC–MS investigation of A. leucoclada. The current GC–MS analysis results enabled the tentative qualitative identification of numerous phytochemicals in ATH. The compounds identified have been interpreted as given in Table 1. Oxygenated and non-oxygenated hydrocarbons, alcohols, phenolics, steroidal and terpenoidal compounds were identified. Among the isolated compounds: phytol (21.24%) and cholest-5-en-3-ol (12.50%) are the major detected compounds. These results were similar to those reported for GC–MS analysis for A. lindleyi Moq [50].

Based upon the above recorded results, the anti-inflammatory activity of A. leucoclada may be attributed to its content of palmitic (5) and β-sitosterol (3) that were previously reported to reduce expression of COX- 1 and COX-2 [51‐53]. Moreover, 20-hdroxy ecdysone (1) [54], stigmasterol (4) [55], luteolin (6) [56], β-sitosterol-3-O-β-D-glucopyranoside (7) [57], and isorhamnetin 3-O-β-galactopyranoside (9) [58] were also reported to suppress COX-2 expression. Also, computational study on phytol (2) indicated its efficient interaction with COX-1 and 2 enzymes [59]. Concerning pallidol (8), a resveratrol dimer, it was reported to have weak activity against COX enzymes [60, 61]

In regard of GC–MS results, the high anti-inflammatory activity of the ATH fraction may be attributed to the synergistic effect of certain compounds e.g. 2-hexadecen-1-ol [62] and hexadecanoic acid, methyl ester [63]. Also, ursodeoxycholic acid was reported to show COX-2 inhibition [64]. From another point of view, neophytadiene significantly inhibited NO production and inflammatory cytokines TNF-α, IL-6 and IL-10 both in vitro and in vivo [65, 66], azulene derivative reverses osteoarthritic inflammation through regulation of matrix metalloproteinases and NF-kβ pathway in in vitro and in vivo models [67] and villosin exerted inhibitory effects against NO production with IC₅₀ = 15.5 μM [68].

It is already stated that, computational studies played an effective role in drug development as it can provides a fast, cheap and easy method for expectation of the possible promising bioactive drugs. Herein, we examined the binding affinity of isolated (from ATD extract) as well as GC–MS identified compounds (in ATH extract) in this plant with key anti-inflammatory targets. Analysis of the binding with the two proteins COX-1 and COX-2 revealed variable binding patterns depending on the nature of the compounds. Steroids that constitute 44.44% of isolated compounds from ATD extract and 20.25% of detected compounds in ATH extract exhibited docking score with COX-2 ranged from -8.4 to -7.4 kcal/mol. During this study, we explored some data that can be gleaned from analysis of steroids conformation and interactions at the cyclooxygenase active site. Previous studies that examined the binding of the substrate “arachidonic acid” into COX active site suggested that the active site could be viewed as including three parts; proximal, central, and distal binding pockets and that the distal and proximal binding pockets are important for stabilization of the substrate, while the central part that contains the catalytic Tyr385 is the place where substrate is transformed into PGG₂ [69]. Analysis of our docking results unveiled that steroids occupied the proximal binding pocket and may extends towards the central binding pocket. It was noted that presence of more hydrophilic groups at C-3 may contribute to the increased binding affinity as exemplified in β-sitosterol3-O-β-D-glucopyranoside (-8.4 kcal/mol) with glucosylation at C-3 where it exhibited flat inverted conformation compared to that of β-sitosterol, furthermore, one of the sugar hydroxyl groups formed two hydrogen bonds with His90 and Gln192 in the side pocket, a pocket that was generated in COX-2 due to Ile523 mutation in COX-1 to the smaller Val523 in COX-2 [69] (Fig. 4B), while β-sitosterol (-7.4 kcal/mol) exhibited only covalent bonds with Tyr355 at the constriction located near the entrance of the active site and with His90, Ser353, and His356 (Fig. 4A). Similarly, presence of carbonyl group at C-6 and hydroxyl groups at C-14, C-20, C-22, and C-25 as in the case of 20-hydroxyecdysone (-8.4 kcal/mol) may play a role in improving its binding affinity via formation of two hydrogen bonds between His356 and OH groups at C-20 and C-25, respectively and oxygen of the carbonyl group at C-6 exhibited two hydrogen bonds with Phe 580, and Ser581, in addition to hydrogen bonding between hydroxyl group at C-14 and Gln350 (Fig. 4C). Additionally, the current findings highlighted the flavonoid luteolin that was one of the lead metabolites exhibiting the highest binding affinity to COX-2 (-8.5 kcal/mol); analysis of the binding orientation of luteolin revealed that it formed one hydrogen bond with Trp387 in the catalytic region of the active site and another hydrogen bond with Asn382 in addition to π-π stacking with His388 and three π-alkyl interactions with Ala202, His207, and His386 (Fig. 4D).

The current study presented a detailed chromatographic exploration of A. leucoclada with successful isolation of nine compounds, eight of which were isolated for the first time from this species, moreover, GC–MS analysis identified the non-polar components of the hexane extract of the plant. These findings will add to chemical profile of this species, as well as to Atriplex genus. Additionally, COX-1 and COX-2 inhibitory activity testing noted the n-hexane and defatted methanol extracts for selective inhibitory activity against COX-2 enzyme. The molecular docking studies revealed high binding affinities and good binding interactions of most of the compounds with binding scores ranged from -8.5 to -6.6 kcal/mol for isolated compounds from ATD and -8.3 to -5.1 kcal/mol for compounds identified by GC–MS in ATH. Accordingly, A. leucoclada is suggested as a valuable source of safe anti-inflammatory agents. Future studies are recommended for evaluating the anti-inflammatory potential of the highlighted metabolites whether, alone or in combination with commercially used anti-inflammatory drugs.