Eucalyptus torquata L. flowers: a comprehensive study reporting their metabolites profiling and anti-gouty arthritis potential

Gouty arthritis is one of the most common metabolic disorders affecting people. Plant based drugs can lower the risk of this health disorder. The anti-gouty potential of Eucalyptus torquata flowers methanol extract (ETME) was evaluated in vitro via measuring the inhibitory effects of five pro-inflammatory enzymes; xanthine oxidase (XO), hyaluronidase, lipoxygenase (5-LOX), cyclooxygenases COX-1, and COX-2, in addition to evaluating the inhibition of histamine release, albumin denaturation, membrane stabilization, tyrosinase, and protease inhibitory activities. Also, its antioxidant potential was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assays and ferric reducing power assay (FRAP). HPLC–PDA-MS/MS was used to identify the metabolites in the tested extract. The latter exhibited substantial anti-arthritic properties in all assays with comparable potential to the corresponding reference drugs. HPLC–MS/MS analysis of this bioactive extract tentatively annotated 46 metabolites including phloroglucinols, gallic and ellagic acids derivatives, terpenes, flavonoids, fatty acids, and miscellaneous metabolites. Our study highlights the medicinal importance of E. torquata as an anti-gouty candidate and opens new avenues of gouty management.

www.nature.com/scientificreports/The aim of this study is to evaluate the potential of E. torquata flowers methanol extract in the management of gouty arthritis, by testing its inhibitory activity against several key enzymes related to gout and inflammation.These include xanthine oxidase, hyaluronidase, 5-LOX, COX-1, COX-2, tyrosinase, and protease enzymes.In addition, the potential of E. torquata to affect histamine release, albumin denaturation, and membrane stabilization will be explored.Moreover, HPLC-MS/MS will be used to tentatively annotate the secondary metabolites composition of ETME.

Plant material and extraction
Eucalyptus torquata Luehm.flowers were collected in May 2019 from El-Kobba palace, Cairo, Egypt.Mrs. Therese Labib, Consultant of Plant Taxonomy at the Ministry of Agriculture, Cairo, Egypt authenticated the plant.A voucher specimen (No. 05.06.19.II) was deposited in the Pharmacognosy Department, Faculty of Pharmacy, Cairo University Museum.The air-dried flowers were ground, extracted by maceration in methanol.The combined methanol extract was concentrated under reduced pressure at temperature not exceeding 50 ºC till dryness.Collection of the plant material, complied with the national, institutional, and international legislations and guidelines.

Biological study
In vitro antioxidant activity DPPH radical scavenging, ABTS radical scavenging and FRAP assays were done according to Dudonne et al. 19 method minor modifications.The details are mentioned in the supplementary file.

COX-1, COX-2, and LOX inhibitory activity
The COX inhibitory activity was assayed using Cayman colorimetric COX (ovine) inhibitor screening assay kit, according to the manufacturer's instructions.The selectivity index (S.I.) was then calculated as IC 50 (COX-1)/ IC 50 (COX-2).In vitro 5-lipoxygenase (5-LOX) inhibitory assay was carried out using lipoxygenase inhibitor screening assay kit, according to the manufacturer's instructions.Diclofenac sodium was used as a reference drug.

Albumin denaturation, membrane stabilization and protease inhibitory activities
Albumin denaturation inhibition was evaluated using the previously described method 20 .Membrane stabilization and proteinase inhibitory potentials were evaluated using the previously reported method 21 .Diclofenac was used as the reference drug.All the experiments were performed in triplicate.

Xanthine oxidase (XO) inhibitory activity
The XO inhibitory activity was assayed following the protocol reported in 22 .Detailed procedures are described in the supplementary file.

Tyrosinase inhibitory activity
Tyrosinase inhibition assay was performed with L-DOPA as substrate adopting the previously described method 23 .Detailed procedures are described in the supplementary file.

Hyaluronidase inhibitory activity
Hyaluronidase inhibitory activity of ETME was evaluated spectrophotometrically as reported by Perera et al. 24 with minor modifications.Detailed procedures are described in the supplementary file.
Histamine release inhibitory activity U937 human monocytes (ATCC, Manassas, VA, USA) were used to study the effect of the extract on histamine release.U937 cells (50,000 cells) were plated in a 96-well plate and treated with various concentration (1000-7.81µg/mL) of the extract, in presence or absence of 20 nM phorbol myristate acetate (PMA) for 1 h.The cell culture supernatants collected from either untreated control or treated cultures were centrifuged at 10,000 g for 5 min at 4 °C and assessed for the released histamine by the commercially available ELISA kit.Diclofenac was used as the positive control.

Phytochemical analysis
HPLC-PDA-MS was used to identify the phytoconstituents of ETME using ThermoFinnigan LCQ-Duo ion trap mass spectrometer (ThermoElectron Corporation, Waltham, MA, USA) with an ESI source (ThermoQuest Corporation, Austin, TX, USA) 25,26 .Details of the HPLC system are described in the supplementary file.
www.nature.com/scientificreports/Virtual screening and drug likeness analysis Molecular modeling was done via docking the phytoconstituents of ETME to cyclooxygenase 1 (COX-1, PDB code: 5WBE), cyclooxygenase 2 (COX-2, PDB code: 5IKR), 5-lipoxygenase (5-LOX, PDB code: 3V99), and xanthine oxidase (XO, PDB code: 3NVY) using MOE software (MOE2022.v11.18.1) as previously described by Sobeh et al. 27 to virtually screen the binding mode of the extract's components in the aforementioned enzymes active site.Moreover, drug likeness analysis was performed by calculating the five molecular descriptors associated with drug likeness and oral bioavailability using the QSAR tool of MOE software.

Results
Gouty arthritis is one of the most common metabolic complaints affecting patients worldwide.Our study traced the potential use of E. torquata flowers for the management of hyperuricemia and gout, in correlation with its phytochemical profile.Scheme for extraction, biological evaluation, and chemical analysis of ETME is presented in Fig. 1.

Biological study
In vitro antioxidant activity ETME exhibited potent antioxidant activity in comparison to ascorbic acid, as revealed by three different assays: DPPH, FRAP, and ABTS, Table 1.

In vitro anti-inflammatory and anti-arthritic activities of E. torquata flower methanol extract (ETME)
ETME inhibited COX-1, COX-2 and 5-LOX with comparable potential to the reference drugs.It also showed excellent selectivity towards COX-2 enzyme, Table 1.Anti-gouty arthritic activity of ETME was tested in vitro via evaluating its potential towards albumin denaturation, membrane stabilization, histamine release, and its inhibitory potential against protease, xanthine oxidase, tyrosinase, and hyaluronidase enzymes.ETME showed very promising activity in all tested assays, Table 1.

Phytochemical analysis
The HPLC-MS analysis of ETME allowed the tentative identification of 46 plant metabolites belonging to several phytochemical classes.These include phloroglucinols, gallic and ellagic acid derivatives, terpenes, flavonoid glycoside, fatty acids, and miscellaneous metabolites.Each metabolite observed molecular weight, fragment ions, and its identity is recorded in Table 2. Identification of the peaks were based on comparing their spectral masses with the data reported in the previous literature on Genus Eucalyptus 6,9,16 .
Results revealed the high abundance of phloroglucinols in ETME; 34 phloroglucinols.Eighteen phloroglucinol-sesquiterpene adducts were tentatively identified.Out of which, five signals with fragmentation pattern; m/z 471, m/z 249 and m/z 207 typical to that reported for macrocarpals 16 ; they were annotated as macrocarpals isomers.In addition, ten signals were also detected and identified as euglobals through their intense peak at m/z 249 and less intense peak at m/z 207 as previously reported 16,30 .Moreover, four dimeric phloroglucinols were identified as sideroxylonal A/B/C and grandinal.They were characterized by the presence of molecular ion peak at m/z 499 and daughter ions at m/z 471, m/z 249 and m/z 207.All these metabolites were previously reported in E. sideroxylon leaves and flowers 9,16 .Other phloroglucinols, formylated phloroglucinol compounds (FPC 1-10), were tentatively characterized by comparing their masses with the previously reported data 30,32 .
Nine gallic and ellagic acid derivatives were detected.Fragments of galloyl moiety (m/z 169) and product ions due to its loss [M-H -169] − were detected in the mass chromatogram of the corresponding peaks.Five flavonoid glycosides; kaempferol, quercetin, and isorhamnetin derivatives have been tentatively identified through their fragmentation pattern that showed the presence of m/z 285, 301, and 315, respectively.This fragmentation pattern showed the loss of rhamnose (m/z 146), glucose (m/z 162), rutinoside (m/z 308), and gallate (m/z 152) fragments that appeared independently to be cleaved from the main structure.
Two fatty acids were tentatively identified.Their fragmentation is consistent with the previously reported data 16 .The two fatty acids are hydroxy tetracosanoic acid and hydroxy octadecadienoic acid that showed molecular ion peaks at m/z 383 and m/z 295, respectively.

Virtual screening and drug likeness analysis
We have previously studied the binding affinity of several organic acids, phenolics and flavonoids towards some of the prominent pro-inflammatory enzyme targets by the aid of the molecular docking computational tool 25,27 .Herein, we docked 35 phloroglucinols, the most abundant identified class of compounds, to COX-1, COX-2, 5-LOX, and XO Table 3, so that we could virtually investigate their binding affinity and blocking potential towards these key enzymes mediating the inflammation process.This would help identify the key compounds, which could contribute to the extracts' inhibitory potential against these target proteins and present them as leads for developing novel drug candidates of natural origin.Regarding COX-1 and COX-2 enzymes, it was observed that the docked phloroglucinols showed generally a very good binding affinity to both target enzymes, however they showed much better affinity towards COX-2, which confirms their COX-2/COX-1 selectivity as revealed in the in vitro assays.Sideroxylonal C showed the best affinity (minimum docking score) to COX-1 with a docking score of − 14.23 kcal/mol.However, macrocarpal I showed the highest affinity towards COX-2 with a docking score of − 7.4 kcal/mol and retained a score of − 10.99 kcal/mole towards COX-1, which indicates high selectivity towards COX-2.Docking to 5-LOX revealed appreciable binding affinity of the docked phloroglucinols at the binding site of the target enzyme reflected by the docking score values that ranged from − 18.00 to − 10.67 kcal/ mol.The best binding affinity was shown by sideroxylonal B. As for XO, the binding affinity showed by the phloroglucinol derivatives docked into this target enzyme was not as good as it was in the other three targets, as the docking score ranged only from − 7.87 to − 6.03 kcal/mol.Sideroxylonal B was the best docked compound with the least minimum docking score of − 7.87 kcal/mol.It afforded two hydrogen bonding interactions with Ser876 and Val1011, neither of which was reported by the co-crystallized inhibitor quercetin.In view of these results, we could conclude that the extracts' compounds are most likely targeting COX/LOX enzymatic pathway.
We also investigated the drug likeness potential of the phloroglucinol derivatives identified in the extract by measuring the descriptors associated with drug likeness and oral bioavailability, namely the molecular weight, number of H-bond acceptors (lip_acc), number of H-bond donors (lip_don), and the partition coefficient www.nature.com/scientificreports/(logP(o/w)).Among the investigated compounds, only 8 showed to have drug likeness potential as they obeyed Lipiniski's rule of five, the most common rule used by medicinal chemists to check the oral bioavailability of newly developed drug candidates, Table 4. Interestingly, these compounds included sideroxylonal C and macrocarpal I, which showed the best binding affinity towards COX-1 and COX-2 enzymes, respectively and sideroxylonal B as well that was the best docked compound into both 5-LOX and XO enzymes.These three compounds, thus, can be very promising lead hits for developing novel anti-inflammatory agents of natural origin.

Discussion
Several studies have been conducted to discover plant-derived natural drugs.We herein report the effective role of E. torquata flowers in the treatment of gouty arthritis.A close relationship has been highlighted between uric acid and oxidative stress, where a large amount of reactive oxygen species is produced along with uric acid production.Antioxidants are considered of great importance in the management of hyperuricemia 36 .Also, oxidative stress plays a key role in the pathogenesis of gout and is responsible for a series of inflammatory pathways 37,38 .Comparable antioxidant properties were reported for several Eucalyptus species; E. camaldulensis 39,40 , E. globulus leaves and bark 41,42 and E. sideroxylon bark 43 .In addition, several Myrtaceae plants are rich in polyphenolics such as Eugenia uniflora, Syzygium samarangense, Syzyium jambos, Syzygium aqueum [44][45][46][47] , and Callistemon citrinus 48 .
It is well documented that pro-inflammatory enzymes play an essential role in inflammation pathogenesis through different pathways.So, cyclooxygenases and lipoxygenases inhibition are considered as targets for the management of oxidative stress associated diseases and inflammation that contribute in the amelioration of gouty inflammation 24 .It was reported that the selective COX-2 inhibitors that showed 2-100 fold difference in the concentrations needed to inhibit COX-2 versus COX-1, decreases prostaglandins levels at the inflammatory sites only and has no effect on gastric mucosal prostaglandin levels 49 .Our results are in accordance with that reported about the inhibition potential of several Eucalyptus species oils and extracts against cyclooxygenases and lipoxygenases enzymes [50][51][52] .
Denaturation of tissue proteins is well documented in the pathogenesis of inflammatory events like arthritis.The protection against protein denaturation is one of NSAIDs mechanisms of action.In addition, red blood cell hemolysis inhibition may provide insights into the process of inflammation.Stabilization of the red blood cell membranes may delay or prevent the lysis and the consequent cytoplasmic contents release and the inflammatory response.So, plants that can interfere with these responses could be very useful for developing novel antiarthritic drugs 53 .Natural extracts from other Eucalyptus species produced similar albumin denaturation and cell membrane stabilization activities including E. obliqua leaves 54 , E. sideroxylon bark 9 and E. globulus leaves 55 .
Moreover, during inflammation, leukocytes release lysosomal enzymes, including proteases, triggering further tissue damage and consequent inflammation.Proteinases have been related to arthritic reactions, where a significant level of protection was provided by inhibitors of proteinase enzyme.The observed protease inhibitory potential of ETME is in accordance with the reported data about the proteinase inhibitory activity of other Eucalyptus species 9,56 .
Regarding histamine, it is a very important mediator in inflammatory conditions.The synovial fluids from patients with acute gout have significantly high levels of histamine 57 .In addition, hyaluronidase enhances mast cells degranulation and releases inflammatory mediators leading to several pathological conditions including arthritis 24 .Tyrosinase enzyme, on the other hand, is a multifunctional, widely distributed enzyme in nature, which when accrued in excess, melanin results in hyperpigmentation disorders, Parkinson's disease, and others oxidative stress disorders like arthritis 58 .Interestingly, E. globulus extract was previously reported to inhibit histamine release, hyaluronidase and tyrosinase enzymes, which comes in agreement of our data in this study [59][60][61] .
The oxidation xanthine and hypoxanthine to uric acid is catalyzed by xanthine oxidase 62 .Hyperuricemia is caused by the over expression of xanthine oxidase resulting in high levels of uric acid that contribute to the deposition of monosodium urate in the joint cavity, which in turn activates inflammatory cytokines, leading to gouty arthritis.Therefore, keeping xanthine oxidase and serum uric acid levels within normal range is very important in treating or ameliorating this distressfully agonizing condition 63 .Noteworthy, Eucalyptus deglupta was reported to inhibit xanthine oxidase 64 , which, along with our results, confirm the value of this genus in managing inflammation and gouty arthritis.
The secondary metabolites identified in ETME are responsible for its newly explored anti-hyperuricemic and anti-inflammatory potentials.Ursolic acid, for instance, has reported potent hypouricemic activity and XO inhibitory activities 65 .In addition, several studies have reported flavonoids as potent inhibitors of COX, 5-LOX and XO enzymes [66][67][68] .Furthermore, methyl gallate was reported to possess not only xanthine oxidase inhibitory activity but it also suppressed superoxide anion generated by XO 69 in addition to its potent COX-I, COX-II and 5-LOX inhibitory activities 70,71 .Ellagic acid was also reported to strongly inhibit XO 72 and scavenge DPPH stable radical 73 .Phloroglucinols and their derivatives have been reported for their anti-inflammatory and antioxidant activities [74][75][76] .
The extensively explored E. torquata flowers potential, herein, for the management of gouty arthritis suggests its future deep investigation at in vivo and clinical levels for the discovery of novel gouty arthritis drugs.Further chemical investigation on the plant to isolate its bioactive constituents and drug development is in demand as well.

Conclusion
Eucalyptus torquata flowers extract showed potent in vitro anti-gouty activity with remarkable selective COX-2 inhibitory potential.Several secondary metabolites belonging to different phytochemical classes were identified in the extract.The most abundant was the phloroglucinols.In view of our in-silico results, some of the identified metabolites from E. torquata flowers can be considered as novel leads for the discovery and development of drug candidates of natural origin that could be used to manage and combat gouty arthritis.

Table 1 .
In vitro antioxidant, anti-inflammatory and antiarthritic potentials of E. torquata flower methanol extract (ETME).All experiments were carried out in a triplicate manner and values are expressed as mean ± SD.SI is COX selectivity index calculated as IC 50 (COX-1)/IC 50 (COX-2).

Table 2 .
Secondary metabolites identified in E. torquata flower using HPLC-PDA-MS.

Table 3 .
Docking scores of the identified phloroglucinols from E. torquata flowers.

Table 4 .
Drug likeness analysis of phloroglucinol derivatives identified in E. torquata.