Sajeli Ahil Begum1, Kirti Hira2, Pragya Paramita Pal2, Samrun Nessa2, Onkar P Kulkarni2, Jeyapragash Danaraj3, Ameer Basha Shaik4, Hiroshi Araya5, Yoshinori Fujimoto5. 1. Department of Pharmacy, Birla Institute of Technology and Science Pilani Hyderabad Campus, Jawahar Nagar, Kapra Mandal, Hyderabad, Telangana, 500 078, India. sajeli@hyderabad.bits-pilani.ac.in. 2. Department of Pharmacy, Birla Institute of Technology and Science Pilani Hyderabad Campus, Jawahar Nagar, Kapra Mandal, Hyderabad, Telangana, 500 078, India. 3. Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, 608 502, India. 4. Department of Plant Pathology, College of Agriculture, Professor Jayashankar Telangana State Agricultural University, Rajendra Nagar, Hyderabad, Telangana, 500 030, India. 5. School of Agriculture, Meiji University, Kawasaki, Kanagawa, 214-8571, Japan.
Abstract
OBJECTIVES: The study aimed to explore the anti-inflammatory effect, underlying mechanism, and chemistry of Halodule pinifolia extract. METHODS: The ethyl acetate (EHP) and methanol (MHP) extracts of Halodule pinifolia were screened for pro-inflammatory cytokine inhibition effect under various in vitro (LPSand crystal-induced inflammation) and in vivo models (LPS-induced endotoxaemia model, carrageenan-induced paw oedema model, and oxalate-induced renal nephropathy model of inflammation). The effect of EHP on the expression of inflammatory markers using western blot analysis (in vitro) was investigated. Chemical constituents of bioactive EHP were isolated through chromatography and characterised using NMR spectroscopy. Furthermore, EHP was standardised for rosmarinic acid, vanillic acid, and ethyl protocatechuate using HPLC. Also, total phytosterols, phenolic, and flavonoid content of EHP were determined by UV spectroscopy. KEY FINDINGS: EHP was comparatively more effective than MHP in inhibiting cytokines secretions under LPS-induced in vitro models. Furthermore, EHP was screened under endotoxaemia in vivo model, EHP (250 mg/kg) reduced plasma IL-6, TNF-α, and IL-1β levels by 88.3%, 78.2%, and 74.5%, respectively. In the carrageenan-induced oedema model, EHP (200 mg/kg) reduced paw volume and release of TNF-α (69.3%) and IL-1β (43.1%). EHP (200 mg/kg) further controlled renal nephropathy by inhibiting plasma IL-1β and BUN levels. Also, a significant reduction of mRNA expressions of TNF-α and IL-1β and KIM-1 in renal tissues was observed. Through western blot, EHP was identified to regulate the expression of pro-form as well as mature-form of IL-1β and caspase-1. EHP constituted rosmarinic acid (RA), vanillic acid (VA), ethyl protocatechuate (EP), sitosterol, stigmasterol, campesterol, and dihydrobrassicasterol. It was determined that 4.6 mg/g of RA, 2.92 mg/g of VA, 0.76 mg/g of EP, 21.7 mg/g of total phenolics, 29.8 mg/g of total flavonoids, and 48.2 mg/g of total phytosterols were present in dry EHP. The presence of anti-inflammatory constituents such as RA, VA, and PE in EHP corroborated the in vitro and in vivo anti-inflammatory activity of EHP. CONCLUSION: The anti-inflammatory property of EHP and its action through attenuation of pan-cytokines suggest that it can be developed into an oral pharmaceutical drug.
OBJECTIVES: The study aimed to explore the anti-inflammatory effect, underlying mechanism, and chemistry of Halodule pinifolia extract. METHODS: The ethyl acetate (EHP) and methanol (MHP) extracts of Halodule pinifolia were screened for pro-inflammatory cytokine inhibition effect under various in vitro (LPSand crystal-induced inflammation) and in vivo models (LPS-induced endotoxaemia model, carrageenan-induced paw oedema model, and oxalate-induced renal nephropathy model of inflammation). The effect of EHP on the expression of inflammatory markers using western blot analysis (in vitro) was investigated. Chemical constituents of bioactive EHP were isolated through chromatography and characterised using NMR spectroscopy. Furthermore, EHP was standardised for rosmarinic acid, vanillic acid, and ethyl protocatechuate using HPLC. Also, total phytosterols, phenolic, and flavonoid content of EHP were determined by UV spectroscopy. KEY FINDINGS:EHP was comparatively more effective than MHP in inhibiting cytokines secretions under LPS-induced in vitro models. Furthermore, EHP was screened under endotoxaemia in vivo model, EHP (250 mg/kg) reduced plasma IL-6, TNF-α, and IL-1β levels by 88.3%, 78.2%, and 74.5%, respectively. In the carrageenan-induced oedema model, EHP (200 mg/kg) reduced paw volume and release of TNF-α (69.3%) and IL-1β (43.1%). EHP (200 mg/kg) further controlled renal nephropathy by inhibiting plasma IL-1β and BUN levels. Also, a significant reduction of mRNA expressions of TNF-α and IL-1β and KIM-1 in renal tissues was observed. Through western blot, EHP was identified to regulate the expression of pro-form as well as mature-form of IL-1β and caspase-1. EHP constituted rosmarinic acid (RA), vanillic acid (VA), ethyl protocatechuate (EP), sitosterol, stigmasterol, campesterol, and dihydrobrassicasterol. It was determined that 4.6 mg/g of RA, 2.92 mg/g of VA, 0.76 mg/g of EP, 21.7 mg/g of total phenolics, 29.8 mg/g of total flavonoids, and 48.2 mg/g of total phytosterols were present in dry EHP. The presence of anti-inflammatory constituents such as RA, VA, and PE in EHP corroborated the in vitro and in vivo anti-inflammatory activity of EHP. CONCLUSION: The anti-inflammatory property of EHP and its action through attenuation of pan-cytokines suggest that it can be developed into an oral pharmaceutical drug.