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Title: Development of mouthwash containing Hemp extract for mouth ulcer 
เกศกัญญา คงคดี
Neti Waranuch
เนติ วระนุช
Naresuan University
Neti Waranuch
เนติ วระนุช
Keywords: Anti-inflammation
Cannabis sativa L.
Oral ulcer
Wound healing
Issue Date: 2021
Publisher: Naresuan University
Abstract: Oral inflammation and ulceration occur when head and neck cancer patients undergo chemotherapy and radiation treatment. The damaged cells generate reactive oxygen species (ROS) and inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukins; IL-1β, IL-6, IL-8, and prostaglandin E2 (PGE2) in response to the inflammation. The infection from gram-positive bacteria and yeast can occur in the ulcerative area before the stage of wound remodeling. The use of a non-irritating mouthwash is the simplest method, which can do in a routine, to deal with those oral conditions. Recently, the use of plants either for cosmetic or pharmaceutical purposes is popular worldwide. Cannabis sativa L. subsp. sativa (hemp) is one of the interesting plants that contains, a major non-psychoactive compound, cannabidiol (CBD) which demonstrates the anti-inflammatory activity in several models of inflammation.    The objectives of this study were, firstly, to investigate the antioxidation, anti-inflammation, collagen type I production, wound healing, and antimicrobial activities of the hemp extract and CBD. As well, the hemp extract was used as an active ingredient in a mouthwash formulation and the stability of the finished product was investigated. The hemp extract was obtained by ultrasonic-assisted extraction. The significant compound of interest found in this extract was CBD. The IC50 of the hemp extract, which was examined by DPPH radical scavenging assay, was 216.23 ± 5.80 µg/mL. However, the CBD standard showed more potent antioxidant activity than the extract with an IC50 value of 107.67 ± 5.58 µg/mL. The non-cytotoxic concentrations of the hemp extract and the CBD were investigated by MTT assay before measuring other cellular biological activities. The treatment of the hemp extracts 50 µg/mL and CBD standard 1 µg/mL greatly inhibited TNF-α and IL-1β production in LPS-induced inflammation in murine macrophage (RAW 264.7) cells. To further explore the inhibition of PGE2 release, inflammation in human gingival fibroblast (HGF-1) cells was induced by adding IL-1β to the cells. The treatment using the hemp extracts 10 µg/mL greatly inhibited PGE2 production. As well, the hemp extract at 25 µg/mL was the potential concentration that enhanced pro-collagen type I synthesis. The ability of hemp in oral wound healing was then determined using a scratch test assay which showed that the hemp extract (5 µg/mL) increased wound closure by 27.92 ± 1.21%, while the treatment with CBD (0.5 µg/mL) also enhanced wound closure by 33.49 ± 1.67% at 48 hours after wounding. Unfortunately, neither the hemp extract nor the standard CBD showed antimicrobial activity. Based on the results of the cellular biological activity tests that we undertook, the recommended dose of the hemp extract to develop a mouthwash is 5 µg/mL. The stability study of the tested mouthwash indicated chemical stability and stable pH, but the color changed due to the oxidation of the chlorophyll in the plant material. A preservative efficacy test of the mouthwash showed that it was effective against microbial growth during the manufacturing and storage periods. It can be concluded, therefore, that hemp extract provides antioxidation and anti-inflammation protection and induces collagen synthesis, as well as gingival wound healing activity. Also, the biological activities of hemp extract from the inflorescences are influenced by CBD. The results suggest that mouthwash containing hemp extract may have potential in the treatment of oral inflammation and ulcers.
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