Paper Title
Bark Extracts of Holoptelea Integrifolia on Canine Demodicosis and Anti-Inflammation

Abstract
Holoptelea integrifolia (Roxb.) Planch or Indian elm is a deciduous tree distributed over northern tropical Asia. In Thailand, the fresh stem bark has been traditionally treated on canine demodectic mange (CDM) that caused by parasitic mites. The overgrown mites develop clinical symptoms such as hair loss, skin inflammation and lesion in immature or defective immune dogs. However, intensive research of H. integrifolia bark extracts on CDM has not been reported. The aim of this study was to establish the efficacy of H. integrifolia fresh stem bark extracts on CDM treatment and anti-inflammation. The fresh stem bark was extracted with various solvents such as hexane, dichloromethane, ethyl acetate, ethanol, methanol and water. The crude extracts were analyzed for major active components by thin layer chromatography (TLC) using toluene: ethyl acetate: ethanol (8:6:1) as a mobile phase. Then, 1 mg/mL of all extracts were developed as a spray solution. The developed sprays were preliminary tested in CDM dogs by volunteering owners for 30 days in Lampang province, Thailand. The wound size, hair growth and dog side effects were recorded every week. The anti-inflammatory activity was investigated in RAW 264.7 murine macrophage cell line. The inflammatory genes, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions were evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The triterpenoids, oleanolic acid, lupeol and friedelin were spotted as the phytochemical component in the crude extracts. The hexane extract showed the most effectiveness on treatment in CDM dog. The wound was absolutely healed and the hair was regrown within 2 weeks while other extracts used 3-4 weeks. The wound size was quickly reduced 2.5 cm per week after hexane extract treatment but 0.5-1 cm per week by other extracts. COX-2 and iNOS gene expressions were potently suppressed by hexane extract at 5 μg/μL and all phytochemical compounds at 10 μg/μL. Among them, lupeol exhibited the most effectiveness at the same concentration. This study supports the ability of using H. integrifolia fresh bark extracts and its phytochemical compounds on CDM healing through an anti-inflammatory effect. Keywords - Holoptelea integrifolia, Indian elm, Canine demodectic mange, COX-2, iNOS