Respiratory disorders are one of the most prevalent diseases in the world. According to the World Health Organization (WHO), 262 million people worldwide suffer from asthma, and this number will increase to 400 million in 2025, where 80% of deaths from asthma occur in developing countries1. Asthma is characterized by bronchoconstriction, mucus hypersecretion, and airway inflammation2. Products from the 5-lipoxygenase (5-LOX) activity, such as leukotrienes B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE), are responsible for triggering bronchoconstriction and excessive mucus secretion in response to inflammation3. One approach used in the treatment of asthma is through the inhibition of 5-LOX activity. Lipoxygenase (LOX) is an oxidative enzyme that contains non-heme iron (Fe) in its active site. This enzyme initiates inflammatory reactions by triggering the formation of proinflammatory mediators known as leukotrienes. Lipoxygenasecatalyzes the addition of oxygen (O2) to poly-unsaturated fatty acids (PUFAs) such as arachidonic acid and linoleic acid4,5.

5-lipoxygenaseis known to have an essential role in acute inflammation and trigger cardiovascular disorders through increased leukocyte chemotaxis, blood vessel inflammation, and increased permeability of the respiratory tract membrane6. 5-lipoxygenase is also known to play an essential role in triggering asthma responses. Activated immune cells will first produce arachidonic acid as a result of a reaction catalyzed by phospholipase A2 in the plasma membrane then, followed by the formation of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) by 5-LOX to produce further leukotrienes, which have strong potential to cause bronchoconstriction via binding to the cysteinyl leukotriene receptor 17,8.

Zileuton (trade name Zyflo) is the only 5-LOX inhibitor available for over 25 years9,10. Along with research assessing the effectiveness of 5-LOX inhibitors in treating asthma, research to find potential 5-LOX inhibitor candidates is ongoing, especially research on natural compounds derived from plant extracts11,12. Coffee is one of the exciting plants to be further developed as a 5-LOX inhibitor. Coffee, a significant agricultural product, yields over seven million tons of green beans annually and is the second most traded commodity worldwide. The two main species cultivated throughout the tropical world are Coffea arabica and Coffea canephora var. Robusta represents 70% and 30%, respectively, in world production13. Coffee has a positive effect on reducing the inflammatory reaction that triggers asthma. Coffee consumption has an inverse relationship with mortality due to respiratory disorders14,15.

Coffee studies in treating asthma have been carried out at the extract level. Coffee extract (CE) provides a weak bronchodilation effect and reduces muscle fatigue in the airways. Coffee extract from Ethiopia, Kenya, and Brazil inhibited LOX activity with EC50 values of 2750 to 2940 µg/mL16. The compound responsible for producing the anti-asthma effect in CE is predicted to be caffeine, the dominant compound in coffee. Chemically, caffeine is similar to theophylline used as an asthma medication. One of the results of caffeine metabolism in the body is also theophylline. From this relationship, it is predicted that caffeine can provide anti-asthma effects like theophylline17. Clinical trials on 55 patients showed that caffeine consumption at doses of less than 5 mg/kg BW could improve lung function for 2 hours after use. Based on the study, caffeine in CE can inhibit the NFκB signaling pathway, vital in producing various proinflammatory cytokines and chemokines (TNF-α and IL-6) by suppressing cyclooxygenase-2 (COX2) expression18. Meanwhile, studies on caffeine activity in inhibiting LOX have never been done.

Although clinically proven, the mechanism of action of CE in the treatment of asthma is still being studied. We tested C. canephora var. Robusta extract (CRE), C. arabica extract (CAE), and caffeine as bioactive compounds on 5-LOX activity in this study. This investigation aims to determine whether CE and caffeine can hinder the creation of leukotrienes that initiate airway inflammation by targeting LOX activity. This study is intended to serve as preliminary research, contributing to our understanding of how caffeine, as the primary component in CE, is involved in anti-asthmatic effects, particularly concerning the suppression of 5-LOX activity.

Materials

The materials and instruments used in this research include soybean 5-LOX (Sigma Aldrich-L7395, US), apigenin (Sigma Aldrich-10798, US), linoleic acid (Sigma Aldrich-1376, US), demineralized water (Brataco, Indonesia), 96% ethanol (Brataco, Indonesia), distilled water (Brataco, Indonesia), Robusta Gold and Arabica Gold coffee powders (commercially available product from Indonesia), microplate reader (BioTek ELX800), and thin-layer chromatography (TLC) scanner (CAMAG).

Methods

Coffee extraction and caffeine isolation

One hundred g of coffee powder (Robusta and Arabica) were extracted with 400 mL of 96% ethanol for 2 hours using the reflux method. The extract was concentrated using a rotary vacuum evaporator at 60±5°C and dried in a vacuum oven for 6 hours to form a thick extract. This extract was used as a sample in the LOX activity assay. The remained thick extract of Robusta coffee was then added with 50 mg of MgO and 300 mL of distilled water, heated for 1 hour at 90±10°C, and filtered. The residue was boiled again for 1 hour with 500 mL of distilled water; this process was repeated two times, and then the obtained extract was filtered using a Buchner funnel. The filtrate obtained was added with 50 mL of 10% H2SO4 and then concentrated until the volume was reduced to 250 mL. The liquid-liquid extraction was added with 250 mL of chloroform into the aqueous filtrate. The chloroform layer was taken and washed with 40 mL of 1% NaOH and shaken with 40 mL of hot water. The transparent-colored chloroform layer is evaporated to obtain a concentrated filtrate. The sublimation was then carried out to the filtrate at a temperature of 180-200°C to obtain caffeine isolate in white needle crystals19. The caffeine isolate was used in the LOX activity assay as a sample.

Examination of isolate purity using TLC-densitometry

The isolate was dissolved in 96% ethanol until a concentration of 1000 µg/mL was obtained and then eluted with the mobile phase n-hexane : ethyl acetate : ethanol (2.5 : 1.5 : 0.4). The spot was compared to standard caffeine. The purity level was assessed from the number of visible spots under UV 254 and 366 nm and the instrument calculations20.

Melting point determination

The melting point of the isolate was determined using the Fisher-Johns melting point apparatus with corrected temperature21. Isolate crystals obtained from the sublimation process were put in the capillary tube. The temperature when the crystals melt for the first time until they melt entirely was observed using a thermometer on the instrument. The melting point was then compared to the caffeine standard.

5-LOX inhibitory activity assay

5-LOX inhibitory activity assay was carried out based on previous studies22-24 with slight modifications. As much as 50 μL of each CRE, CAE, caffeine isolate, and apigenin solution was put into the vial, then added with 1650 μL of 0.2 M borate buffer solution pH 9 and 1000 μL of 300 μM linoleic acid as substrate solution. The mixture was vortexed and then incubated for 10 minutes at 25°C. Then 300 μL of 1000 U/mL 5-LOX solution was added to the mixture, then incubated for 15 minutes at 25°C. After incubation, 1000 μL methanol was added to the mixture and vortexed. The absorbance of the solution was measured using a UV spectrophotometer under 234 nm.

Enzyme kinetics assay

The kinetics of the inhibitory enzyme activity was carried out by varying the concentration of the linoleic acid as substrate (final concentration = 50, 75, 100, and 125 µM) with constant inhibitor concentration (final concentration of caffeine = 18.75 µg/mL). The concentration of caffeine used for the enzyme kinetics assay was determined by the concentration of caffeine that could inhibit 50% of the enzyme activity (IC50). To determine the type of inhibition of 5-LOX activity, an enzyme kinetics assay of 5-LOX inhibition by caffeine was performed using Michaelis–Menten kinetics, as shown in Equations 1to3, in which Vi was the initial velocity of an enzymatic reaction, Vmax was the maximal velocity of the enzymatic reaction, Km was Michaelis constant, and [S] was substrate concentration.

[1]

[2]

= [3]

Data analysis

Statistical analysis was performed using one-way ANOVA, and further multiple comparison between groups was analyzed using the Tukey Post Hoc Method.

The coffee plant contains various bioactive compounds with antioxidant properties, specifically phenolic compounds such as caffeic acid, chlorogenic acid, coumaric acid, ferulic acid, and cinnamic acid25. In addition to phenolic compounds, coffee contains methylxanthine alkaloid compounds such as caffeine, theophylline, and theobromine. Both groups of phenolic compounds and methylxanthine alkaloids have good solubility in organic solvents26. Thus, to produce a yield with a high content of bioactive compounds, the solvent chosen for the extraction of coffee bean powder is ethanol—extraction of C. canephora var. Robusta and C. arabica using ethanol yielded 9.84% and 8.12% samples, respectively. The resulting extract had a dark brown color and emitted a coffee aroma. The subsequent isolation process was performed in CRE following the alkaloid extraction principle, involving adding a base and extraction from the organic solvent layer (chloroform). This process yielded fractions containing multiple compounds. The crude fraction was then subjected to purification through sublimation, producing pure caffeine in the form of needle-like crystals, with a yield of 0.11% relative to the total ground sample (Table I). The purity of the caffeine crystals formed from the sublimation process was confirmed by comparing the melting point of the crystal to caffeine standards, observing the number of spots visible on TLC, and observing the spot purity level by TLC-densitometry.

TableI. Extraction yield.

The melting point test results found that the caffeine crystals isolated from CRE had a narrow melting point range between 234-236°C, indicating a high purity level. The melting point range of isolated caffeine was similar to the caffeine standard (Table II). The chromatographic profile was evaluated using the TLC technique. The isolated compound produced a single spot under UV 254 nm with the same color, Rf, and spectral profile as standard caffeine. Examination of the purity level using TLC-densitometry showed a purity level of 98.6%. The results indicated that the isolated crystal was pure and confirmed as caffeine.

TableII. Evaluation of the melting point range of isolated and standard caffeine.

All CRE, CAE, and caffeine isolate samples were tested for 5-LOX inhibitory activity. The assay was carried out in triplicate. The IC50 values of CRE, CAE, and caffeine against 5-LOX were 32.2 ± 1.4, 42.1 ± 2.3, and 14.3 ± 1.6 µg/mL respectively. The standard compound used as a positive control was apigenin, which showed an IC50 value of 7.4 ± 1.7 µg/mL. In Figure 1, it can be observed that there is a significant difference in activity between CRE, CAE, and caffeine. As a single compound isolated from coffee extract, caffeine demonstrates an inhibitory activity of 2.3 times stronger than CRE and three times stronger than CAE, as indicated by the smaller IC50 values. Interestingly, caffeine's inhibition of 5-LOX activity is not significantly different (ns) compared to apigenin, used as the positive control. CRE's activity is superior to CAE; we hypothesize that this difference is due to variations in caffeine content in the samples. Robusta coffee consistently shows higher caffeine levels in various studies compared to Arabica coffee (±2.54% vs ±1.22%)27,28.

Figure1. Inhibition of 5-LOX activity by CRE, CAE, caffeine, and apigenin represented by the IC50 value.

Data were obtained in the enzyme kinetics assay, as shown in Table III, and the Lineweaver-Burke graph shows the intersection on the x-axis (Figure 2). The results indicate that the type of inhibition is non-competitive. Non-competitive inhibition occurs when the inhibitor (Inh) binds to the enzyme at a different site than the substrate (S). There is no competition between the inhibitor (caffeine) and the substrate in non-competitive inhibition. The presence of non-competitive inhibitors decreases the Vmax value with a relatively stable Km value.

TableIII. Enzyme kinetic measurement data.

Figure2. Enzyme kinetic profile of 5-LOX activity inhibition by inhibitors (caffeine).

Lipoxygenase is a metalloenzyme that has pro-oxidation and pro-inflammatory properties. Lipoxygenase generally catalyzes the oxidation of unsaturated fatty acids. In the human body, LOX metabolizes arachidonic acid to leukotrienes (a potent inflammatory mediator), making LOX a critical enzyme in the inflammatory pathway. Based on the relative oxidation position in the arachidonic acid structure, LOX is classified into 5-LOX, 12-LOX, and 15-LOX29. Inhibition of LOX activity can reduce oxidation and inflammation, which can trigger asthma. The studies for effective LOX inhibitors from plants are still ongoing. Compounds from plants that are known to inhibit LOX activity are phenolics and alkaloids, both of which are found in coffee30. It is known that coffee extract also contains alkaloid compounds of the methylxanthines type: caffeine (1,3,7-trimethyl xanthine), theophylline (3,7-dimethyl xanthine), and theobromine (1,3-dimethyl xanthine). Coffee also has theobromine and theophylline despite concentrations 20 times lower than caffeine31. The caffeine content in one cup of coffee depends on the variety of coffee plants used. The average caffeine content in brewed coffee grounds is 57 mg/100 mL32. Other research stated that coffee contains caffeine ± 1177 mg/g as the main compound33.

Our study proves that caffeine plays a role in inhibiting LOX. The inhibitory potency of caffeine in inhibiting 5-LOX cannot be compared with Zileuton (a standard drug approved by the FDA as a 5-LOX inhibitor) due to access limitation; thus, in this study, apigenin was used as a positive control. However, from other studies, it was known that using a similar colorimetric method, Zileuton showed an IC50 of 2.08 µM or 32 times more potent in inhibiting 5-LOX activity compared to caffeine in this study. Zileuton is the ligand that inhibits iron binding to the LOX and has a weak potential reduction34.

Although caffeine has a weaker 5-LOX inhibitory potency than Zileuton, caffeine in coffee may be used as an alternative in relieving asthma because of its pharmacokinetic profile. A comparative study showed that caffeine has a more rapid onset of action, lower fluctuations in plasma concentrations, a longer half-life, and fewer peripheral side effects compared to other methylxanthines32,35,36. In oral administration, gastrointestinal absorption of caffeine is rapid and complete, achieving almost 100% bioavailability. After reaching the bloodstream, caffeine binds to albumin and is distributed to all tissues by simple diffusion or carrier-mediated transport32,37.

This study's in vitro enzymatic activity assay results might be used as preliminary data to assess the caffeine potency in inhibiting 5-LOX activity. For future research, more selective and stable methods might be developed, one of which is the colorimetric method using thiocyanate ions (SCN-) to form a red ferric thiocyanate (FTC) complex measured at λ 480 nm. This colorimetric method is considered more selective and stable for measuring products formed from the enzymatic activity of 5-LOX38.

Coffee exhibits potential inhibition activity against 5-LOX, with IC50 values of 32.2 ± 1.4 µg/mL for CRE, 42.1 ± 2.3 µg/mL for CAE, 14.3 ± 1.6 µg/mL for caffeine, and 7.4 ± 1.7 µg/mL for apigenin. Further bioactive compound isolation of coffee extract produced caffeine with a more substantial inhibitory potential against 5-LOX. Caffeine inhibits 5-LOX activity in a non-competitive manner.

The authors acknowledge Universitas Indonesia, which has provided research funding through the IRP grant number 018/UN2.F15.D5/HKP.05.00.

Conceptualization: Tegar Achsendo Yuniarta, Rosita Handayani

Data curation: Tegar Achsendo Yuniarta, Rosita Handayani

Formal analysis: Tegar Achsendo Yuniarta, Rosita Handayani

Funding acquisition: Rosita Handayani

Investigation: Tegar Achsendo Yuniarta, Rosita Handayani

Methodology: Tegar Achsendo Yuniarta, Rosita Handayani

Project administration: Rosita Handayani

Resources: Tegar Achsendo Yuniarta, Rosita Handayani

Software: -

Supervision: -

Validation: Tegar Achsendo Yuniarta, Rosita Handayani

Visualization: Tegar Achsendo Yuniarta, Rosita Handayani

Writing - original draft: Tegar Achsendo Yuniarta

Writing - review & editing: Rosita Handayani

None.

The authors declare there is no conflict of interest.

1. Song P, Adeloye D, Salim H, Dos Santos JP, Campbell H, Sheikh A, et al. Global, regional, and national prevalence of asthma in 2019: a systematic analysis and modelling study. J Glob Health. 2022;12:04052. DOI: 10.7189/jogh.12.04052; PMCID: PMC9239324; PMID: 35765786

2. Habib N, Pasha MA, Tang DD. Current Understanding of Asthma Pathogenesis and Biomarkers. Cells. 2022;11(17):2764. DOI: 10.3390/cells11172764; PMCID: PMC9454904; PMID: 36078171

3. Ayola-Serrano NC, Roy N, Fathah Z, Anwar MM, Singh B, Ammar N, et al. The role of 5-lipoxygenase in the pathophysiology of COVID-19 and its therapeutic implications. Inflamm Res. 2021;70(8):877-89. DOI: 10.1007/s00011-021-01473-y; PMCID: PMC8176665; PMID: 34086061

4. Sinha S, Doble M, Manju SL. 5-Lipoxygenase as a drug target: A review on trends in inhibitors structural design, SAR and mechanism based approach. Bioorg Med Chem. 2019;27(17):3745-59. DOI: 10.1016/j.bmc.2019.06.040; PMID: 31331653

5. Mashima R, Okuyama T. The role of lipoxygenases in pathophysiology; new insights and future perspectives. Redox Biol. 2015;6:297–310. DOI: 10.1016/j.redox.2015.08.006; PMCID: PMC4556770; PMID: 26298204

6. Giménez-Bastida JA, González-Sarrías A, Laparra-Llopis JM, Schneider C, Espín JC. Targeting Mammalian 5-Lipoxygenase by Dietary Phenolics as an Anti-Inflammatory Mechanism: A Systematic Review. Int J Mol Sci. 2021;22(15):7937. DOI: 10.3390/ijms22157937; PMCID: PMC8348464; PMID: 34360703

7. Wisastra R, Dekker FJ. Inflammation, Cancer and Oxidative Lipoxygenase Activity are Intimately Linked. Cancers. 2014;6(3):1500-21. DOI: 10.3390/cancers6031500; PMCID: PMC4190552; PMID: 25037020

8. Duroudier NP, Tulah AS, Sayers I. Leukotriene pathway genetics and pharmacogenetics in allergy. Allergy. 2009;64(6):823–39. DOI: 10.1111/j.1398-9995.2009.02015.x; PMID: 19416143

9. Poeckel D, Funk CD. The 5-lipoxygenase/leukotriene pathway in preclinical models of cardiovascular disease. Cardiovasc Res. 2010;86(2):243–53. DOI: 10.1093/cvr/cvq016; PMID: 20093252

10. Rossi A, Pergola C, Koerberle A, Hoffmann M, Dehm F, Bramanti P, et al. The 5-lipoxygenase inhibitor, zileuton, suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages. Br J Pharmacol. 2010;161(3):555-70. DOI: 10.1111/j.1476-5381.2010.00930.x; PMCID: PMC2990155; PMID: 20880396

11. Sharanya CS, Abhithaj J, Arun KG, Eeda KR, Bhat V, Variyar EJ, et al. Lipoxygenase inhibitory synthetic derivatives of methyl gallate regulate gene expressions of COX-2 and cytokines to reduce animal model arthritis. Sci Rep. 2023;13(1):10644. DOI: 10.1038/s41598-023-37613-z; PMCID: PMC10313808; PMID: 37391468

12. Schneider I, Bucar F. Lipoxygenase inhibitors from natural plant sources. Part 1: Medicinal plants with inhibitory activity on arachidonate 5-lipoxygenase and 5-lipoxygenase[sol ]cyclooxygenase. Phytother Res. 2005;19(2):81–102. DOI: 10.1002/ptr.1603; PMID: 15852496

13. Perrois C, Strickler SR, Mathieu G, Lepelley M, Bedon L, Michaux S, et al. Differential regulation of caffeine metabolism in Coffeaarabica (Arabica) and Coffea canephora (Robusta). Planta. 2015;241(1):179–91. DOI: 10.1007/s00425-014-2170-7; PMCID: PMC4282694; PMID: 25249475

14. Bresciani L, Calani L, Bruni R, Brighenti F, Rio DD. Phenolic composition, caffeine content and antioxidant capacity of coffee silverskin. Food Res Int. 2014;61:196–201. DOI: 10.1016/j.foodres.2013.10.047

15. Freedman ND, Park Y, Abnet CC, Hollenbeck AR, Sinha R. Association of coffee drinking with total and cause-specific mortality. N Engl J Med. 2012;366(20):1891–904. DOI: 10.1056/nejmoa1112010; PMCID: PMC3439152; PMID: 22591295

16. Złotek U, Karaś M, Gawlik-Dziki U, Szymanowska U, Baraniak B, Jakubczyk A. Antioxidant activity of the aqueous and methanolic extracts of coffee beans (Coffea arabica L.). Acta Sci Pol Technol Aliment. 2016;15(3):281–8. DOI: 10.17306/j.afs.2016.3.27; PMID: 28071027

17. Welsh EJ, Bara A, Barley E, Cates CJ. Caffeine for asthma. Cochrane Database Syst Rev. 2010;2010(1):CD001112. DOI: 10.1002/14651858.cd001112.pub2; PMCID: PMC7053252; PMID: 20091514

18. Jung S, Kim MH, Park JH, Jeong Y, Ko KS. Cellular Antioxidant and Anti-Inflammatory Effects of Coffee Extracts with Different Roasting Levels. J Med Food. 2017;20(6):626–35. DOI: 10.1089/jmf.2017.3935; PMID: 28581877

19. Ikan R. Natural Products: A Laboratory Guide. Cambridge (US): Academic Press; 1991.

20. Irfanah L, Yuwono M, Primaharinastiti R. Optimization and Prevalidation of TLC-Densitometry Method for Fucoidan Analysis in Sargassum sp. Aqueous Extract. J Farmasi Ilmu Kefarmasian Indones. 2023;10(2):210-6. DOI: 10.20473/jfiki.v10i22023.210-216

21. Young JCOC. True Melting Point Determination. Chem Educator. 2013;18:203-8. DOI: 10.1333/s00897132500a

22. Subardini DS, Elya B, Noviani A. Lipoxygenase inhibitory assay of ethyl acetate fraction from star fruit leaves (Averrhoa Carambola L.) from three regions in West Java. Int J Appl Pharm. 2020;12(Special 1):119-21. DOI: 10.22159/ijap.2020.v12s1.FF025

23. Nabilah, Elya B, Djajadisastra J. Lipoxygenase inhibitory assay of Averrhoa carambola L. Leaves extract. Int J ChemTech Res. 2017;10(1):342–7.

24. Alaba CSM, Chichioco-Hernandez CL. 15-Lipoxygenase inhibition of Commelina benghalensis, Tradescantia fluminensis, Tradescantia zebrina. Asian Pac J Trop Biomed. 2014;4(3):184–8. DOI: 10.1016/s2221-1691(14)60229-x; PMCID: PMC3868787; PMID: 25182435

25. Patay ÉB, Bencsik T, Papp N. Phytochemical overview and medicinal importance of Coffea species from the past until now. Asian Pac J Trop Med. 2016;9(12):1127–35. DOI: 10.1016/j.apjtm.2016.11.008; PMID: 27955739

26. Monteiro JP, Alves MG, Oliveira PF, Silva BM. Structure-Bioactivity Relationships of Methylxanthines: Trying to Make Sense of All the Promises and the Drawbacks. Molecules. 2016;21(8):974. DOI: 10.3390/molecules21080974; PMCID: PMC6273298; PMID: 27472311

27. Park JB, Peters R, Novotny JA. Impact of roasting on javamide-I/-II in Arabica and Robusta coffee beans. Food Chem. 2023;412:135586. DOI: 10.1016/j.foodchem.2023.135586; PMID: 36739725

28. Ky CL, Louarn J, Dussert S, Guyot B, Hamon S, Noirot M. Caffeine, trigonelline, chlorogenic acids and sucrose diversity in wild Coffea arabica L. and C. canephora P. accessions. Food Chem. 2001;75(2):223–30. DOI: 10.1016/s0308-8146(01)00204-7

29. Gawlik-Dziki U, Świeca M, Dziki D, Kowalska I, Pecio Ł, Durak A, et al. Lipoxygenase inhibitors and antioxidants from green coffee—mechanism of action in the light of potential bioaccessibility. Food Res Int. 2014;61:48–55. DOI: 10.1016/j.foodres.2014.05.002

30. Tomy MJ, Dileep K V, Prasanth S, Preethidan DS, Sabu A, Sadasivan C, et al. Cuminaldehyde as a lipoxygenase inhibitor: in vitro and in silico validation. Appl Biochem Biotechnol. 2014;174(1):388–97. DOI: 10.1007/s12010-014-1066-0; PMID: 25080377

31. Purkiewicz A, Pietrzak-Fiećko R, Sörgel F, Kinzig M. Caffeine, Paraxanthine, Theophylline, and Theobromine Content in Human Milk. Nutrients. 2022;14(11):2196. DOI: 10.3390/nu14112196; PMCID: PMC9182860; PMID: 35683994

32. Oñatibia-Astibia A, Martínez-Pinilla E, Franco R. The potential of methylxanthine-based therapies in pediatric respiratory tract diseases. Respir Med. 2016;112:1–9. DOI: 10.1016/j.rmed.2016.01.022; PMID: 26880379

33. Kitzberger CSG, Scholz MB dos S, Benassi M de T. Bioactive compounds content in roasted coffee from traditional and modern Coffea arabica cultivars grown under the same edapho-climatic conditions. Food Res Int. 2014;61:61–6. DOI: 10.1016/j.foodres.2014.04.031

34. Carter GW, Young PR, Albert DH, Bouska J, Dyer R, Bell RL, et al. 5-lipoxygenase inhibitory activity of zileuton. J Pharmacol Exp Ther. 1991;256(3):929–37. PMID: 1848634

35. Picone S, Bedetta M, Paolillo P. Caffeine citrate: when and for how long. A literature review. J Matern Fetal Neonatal Med. 2012;25(Suppl 3):11–4. DOI: 10.3109/14767058.2012.712305; PMID: 23016611

36. Aranda JV, Turmen T. Methylxanthines in apnea of prematurity. Clin Perinatol. 1979;6(1):87–108. PMID: 383366

37. Mukhtar I, Iftikhar A, Imran M, Ijaz MU, Irfan S, Anwar H. The Competitive Absorption by the Gut Microbiome Suggests the First-Order Absorption Kinetics of Caffeine. Dose Response. 2021;19(3):15593258211033111. DOI: 10.1177/15593258211033111; PMCID: PMC8375357; PMID: 34421438

38. Lu W, Zhao X, Xu Z, Dong N, Zou S, Shen X, et al. Development of a new colorimetric assay for lipoxygenase activity. Anal Biochem. 2013;441(2):162–8. DOI: 10.1016/j.ab.2013.06.007; PMID: 23811155