Investigation of the Protective Effects of Graviola (Annona muricata), Moringa (Moringa oleifera Lam.) and Lactobacillus gasseri in a Pseudomonas aeruginosa Fibroblast Wound Model

Yıl 2025, Cilt: 52 Sayı: 4, 769 - 781, 12.12.2025

Özgür Çelebi , Demet Çelebi , Sümeyye Başer , Sıdıka Genç , Ali Taghizadehghalehjoughi

https://doi.org/10.5798/dicletip.1840881

Öz

Objective: This study aimed to investigate the antimicrobial, anti-inflammatory, antioxidant, and anti-apoptotic effects of Graviola (Annona muricata), Moringa oleifera, and Lactobacillus gasseri, alone and in combination, against Pseudomonas aeruginosa in vitro wound infection model.
Methods: Antibacterial activity tests against Pseudomonas aeruginosa was determined using the Kirby-Bauer disk diffusion method, MIC/MBC, and FIC methods. Biofilm inhibition was measured by crystal violet staining. A scratch wound model using fibroblast cell line was infected with Pseudomonas aeruginosa. Following treatment with the extracts, cell viability, oxidative stress, cytokine levels (IL-1beta, IL-10 via ELISA), and apoptotic gene expression (BAX, BCL-2 via qPCR) were evaluated.
Results: The triple combination (Graviola + Moringa + Lactobacillus gasseri) showed the strongest antibacterial activity (23 mm zone; FIC = 0.25) and notable antibiofilm effects. In infected fibroblasts, cell viability increased to with combination treatment, approaching control levels. TOS levels decreased significantly while TAC increased, indicating reduced oxidative stress. IL-1beta levels were significantly lowered, and IL-10 levels were restored. Additionally, BAX gene expression decreased by 47%, and BCL-2 increased by 50% in the triple treatment group, suggesting protection against apoptosis.
Conclusion: The combined application of Graviola, Moringa oleifera, and Lactobacillus gasseri exerts synergistic antimicrobial, anti-inflammatory, antioxidant, and anti-apoptotic effects against Pseudomonas aeruginosa induced wound infection. These results will shed light on the search for alternative natural therapeutic strategies, particularly in the treatment of chronic wounds associated with antibiotic-resistant pathogens.

Anahtar Kelimeler

Antibacterial effect , Apoptosis , Biofilm inhibition , Oxidative stress , Wound model

Etik Beyan

Commercially available bacterial strain was used in this study. Therefore, no ethical committee approval was required.

Graviola (Annona muricata), Moringa (Moringa oleifera Lam.) ve Lactobacillus gasseri'nin Pseudomonas aeruginosa Fibroblast Yara Modelinde Koruyucu Etkilerinin İncelenmesi

Öz

Amaç: Bu çalışmada, Graviola (Annona muricata), Moringa oleifera ve Lactobacillus gasseri'nin tekli ve kombinasyon halinde, Pseudomonas aeruginosa'ya karşı antimikrobiyal, antiinflamatuar, antioksidan ve antiapoptotik etkileri in vitro yara enfeksiyon modelinde araştırılmıştır.
Yöntemler: Pseudomonas aeruginosa'ya karşı antibakteriyel aktivite testleri; Kirby-Bauer disk difüzyon yöntemi, MIC/MBC ve FIC yöntemleri kullanılarak belirlenmiştir. Biyofilm inhibisyonu kristal violet boyama yöntemiyle değerlendirilmiştir. Fibroblast hücre hattı kullanılarak oluşturulan çizik yara modeli Pseudomonas aeruginosa ile enfekte edilmiştir. Tedavi sonrası hücre canlılığı, oksidatif stres, sitokin düzeyleri (IL-1beta, IL-10, ELISA ile) ve apoptotik gen ekspresyonları (BAX, BCL-2, qPCR ile) analiz edilmiştir.
Sonuçlar: Graviola + Moringa + Lactobacillus gasseri üçlü kombinasyonu en güçlü antibakteriyel aktiviteyi (23 mm zon; FIC = 0.25) ve anlamlı biyofilm inhibisyonunu göstermiştir. Enfekte fibroblastlarda, kombinasyon tedavisi ile hücre canlılığı artmış ve kontrol düzeylerine yaklaşmıştır. TOS seviyeleri anlamlı şekilde düşerken, TAC artmıştır; bu durum azalan oksidatif stresi göstermektedir. IL-1beta düzeyleri önemli ölçüde azalmış, IL-10 seviyeleri ise normale dönmüştür. Ayrıca, BAX gen ekspresyonu %47 azalmış, BCL-2 ise %50 artmıştır; bu durum apoptozisin önlendiğini göstermektedir.
Tartışma: Graviola, Moringa oleifera ve Lactobacillus gasseri'nin kombinasyonu, Pseudomonas aeruginosa indüklenmiş yara enfeksiyonuna karşı sinerjik antimikrobiyal, antiinflamatuar, antioksidan ve antiapoptotik etkilere sahiptir. Bu bulgular, özellikle antibiyotik dirençli patojenlerle ilişkili kronik yaraların tedavisinde alternatif doğal bir terapötik strateji arayışına ışık tutacaktır.

Anahtar Kelimeler

Antibakteriyel etki , Apoptozis , Biyofilm inhibisyonu , Oksidatif stres , Yara Modeli.

Kaynakça

• 1.Phan S, Feng CH, Huang R., et al. Relativeabundance and detection of Pseudomonasaeruginosa from chronic wound infections globally.Microorganisms. 2023;11(5):1210.
• 2.Ruffin M, Brochiero E. Repair process impairmentby Pseudomonas aeruginosa in epithelial tissues:Major features and potential therapeutic avenues.Front Cell Infect Microbiol. 2019; 9:182.
• 3.Serra R, Grande R, Butrico L et al. Chronic woundinfections: The role of Pseudomonas aeruginosa andStaphylococcus aureus. Expert Rev Anti-Infect Ther.2015;13(5):605-13.
• 4.Bush LM, Chaparro-Rojas F, Okeh V, Etienne J.Cumulative clinical experience from over a decade of use of levofloxacin in urinary tract infections: Critical appraisal and role in therapy. Infect Drug Resist. 2011; 4:177-89.
• 5. Salem AI, Abd El-Fadil H, Al-Sayed N, Alazzouni AS, El-Nabtity S. Pharmacological activities of Graviola(Annona muricata): A mini-review. J Adv Vet Res.2022;12(6):785-90.
• 6.Moghadamtousi SZ, Rouhollahi E, Hajrezaie M etal. Annona muricata leaves accelerate woundhealing in rats via involvement of Hsp70 andantioxidant defence. Int J Surg. 2015; 18:110-7.
• 7.Choi M, Kang YR, Zu HD et al. Effects of time onphenolics and in vitro bioactivity in autoclaveextraction of Graviola (Annona muricata) leaf.Biotechnol Bioprocess Eng. 2020; 25:9-15.
• 8.Jaya FB, Syamsunarno MRAA, Sahiratmadja E.Moringa oleifera Lam. to accelerate wound healing:A review. J Med Sci. 2022;55(3):278-93
• 9.Waqiah SN, Malaka R, Ako A. Potential of Moringaoleifera Lam leaf extract as wound medicine inlivestock. J Pharm Negat Results. 2023;2210-7.
• 10.Mohammad Shafie N, Raja Shahriman Shah RNI,Krishnan P, Abdul Haleem N, Tan TYC. Scopingreview: Evaluation of Moringa oleifera (Lam.) forpotential wound healing in in vivo studies.Molecules. 2022;27(17):5541.
• 11.Gothai S, Arulselvan P, Tan WS, Fakurazi S.Wound healing properties of ethyl acetate fractionof Moringa oleifera in normal human dermalfibroblasts. J Intercult Ethnopharmacol.2016;5(1):1-6.
• 12.Gebremariam HG, Qazi KR, Somiah T et al.Lactobacillus gasseri suppresses the production ofproinflammatory cytokines in Helicobacter pylori-infected macrophages by inhibiting the expressionof ADAM17. Front Immunol 2019; 10:2326.
• 13.Lenzmeier TD, Mudaliar NS, Stanbro JA et al.Application of Lactobacillus gasseri 63 AMsupernatant to Pseudomonas aeruginosa-infectedwounds prevents sepsis in murine models ofthermal injury and dorsal excision. J Med Microbiol.2019;68(10):1560-72.
• 14.Han N, Jia L, Su Y et al. Lactobacillus reuteriextracts promoted wound healing via PI3K/AKT/β- catenin/TGFβ1 pathway. Stem Cell Res Ther. 2019; 10:1-11.
• 15. Çelebi D, Taghizadehghalehjoughi A, Başer S et al.Effects of boric acid and potassium metaborate oncytokine levels and redox stress parameters in awound model infected with methicillin-resistantStaphylococcus aureus. Mol Med Rep. 2022;26(3):1-11.
• 16.Yeni Y, Taghizadehghalehjoughi A, Genç S et al.Glioblastoma cell-derived exosomes induce celldeath and oxidative stress in primary cultures ofolfactory neurons. Role of redox stress. Mol Biol Rep. 2023;50(5):3999-4009.
• 17.Çiçek B, Hacımuftuoğlu A, Kuzucu M et al.Sorafenib alleviates inflammatory signaling oftumor microenvironment in precancerous lunginjuries. Pharmaceuticals. 2023;16(2):221.
• 18.Spernovasilis N, Psichogiou M, Poulakou G. Skinmanifestations of Pseudomonas aeruginosainfections. Curr Opin Infect Dis. 2021;34(2):72-9.
• 19.Okwu MU, Imade OS, Akpoka OA et al.Comparative evaluation of the antibacterial effectsof Moringa oleifera and Nigella sativa on someclinical bacterial isolates obtained from IgbinedionUniversity, Okada, Nigeria. J Biol Sci. 2020;13(2):20-7.
• 20.Vinca DT, Iqbal M, Triyandi R, Oktarlina RZ.Review: Antibacterial activity of ekstrak daun kelor(Moringa oleifera L.) terhadap bakteriStaphylococcus aureus. MEDULA. 2023;13(4):649-54.
• 21.Pakan P, Indriarini D, Amat AL, Wungouw HP.Topical antibacterial therapy from Moringa oleiferaextract against Staphylococcus epidermidis. Trop JNat Prod Res. 2023;7(10):4182-5.
• 22.De Gregorio PR, Tomás MSJ, Terraf MCL, Nader-Macías MEF. In vitro and in vivo effects of beneficialvaginal lactobacilli on pathogens responsible forurogenital tract infections. J Med Microbiol.2014;63(5):685-96.
• 23.Mursyid M, Annisa R, Zahran I, Langkong J,Kamaruddin I. Antimicrobial activity of Moringaoleifera leaf (Moringa oleifera L.) extract againstStaphylococcus epidermidis. IOP Conf Ser EarthEnviron Sci. 2019;012145.
• 24.Gao X, Wang Z, Li X et al. A new Lactobacillusgasseri strain HMV18 inhibits the growth ofpathogenic bacteria. Food Sci Hum Wellness.2022;11(2):247-54.
• 25.Ishikawa T, Imai S, Nakano T et al. Antibacterialactivity of the probiotic candidate Lactobacillusgasseri against methicillin-resistant Staphylococcusaureus. Asian Pac J Dent. 2020;20(1):1-8.
• 26.Pai BM, Rajesh G, Shenoy R, Rao A. Antimicrobialefficacy of soursop leaf extract (Annona muricata)on oral pathogens: An in vitro study. J Clin Diagn Res. 2016;10(11): ZC01-4.
• 27. Chen J, Guo Q, Chen Q et al. Interleukin 10 inhibits oxidative stress-induced autophagosome formationin hepatic stellate cells by activating the mTOR-STAT3 pathway. Exp Cell Res. 2022;411(2):113001.
• 28.Elmarakby AA, Sullivan JC. Relationship betweenoxidative stress and inflammatory cytokines indiabetic nephropathy. Cardiovasc Ther.2012;30(1):49-58.
• 29.Wood SJ, Goldufsky JW, Bello D, Masood S,Shafikhani SH. Pseudomonas aeruginosa ExoTinduces mitochondrial apoptosis in target host cellsin a manner that depends on its GTPase-activatingprotein (GAP) domain activity. J Biol Chem.2015;290(48):29063-3.