Investigating Natural Substances from plants as inhibitors of inflammation in Cultured Human Keratinocytes

  • Rabia Farzand

Student thesis: Doctoral Thesis


Witch hazel (Hamamelis) bark and leaves have been known to ease inflammation. Some studies have demonstrated its effectiveness against dermatitis, but its anti-inflammatory and antihistamine mechanisms and comparative efficacy to a steroid or commonly used antihistamines drugs were unknown. Little is known about the anti-inflammatory properties of Witch hazel in HaCaT cells (cultured human keratinocytes) and mast cells (that release histamine and other allergic substances). In this thesis, the anti-inflammatory activity of Hamamelis (0.25%, 0.5%, and 1%) was investigated in TNF-α activated HaCaT cells using hydrocortisone as a benchmark control for ameliorating inflammation throughout in this thesis.
The cytokines TNF-α, IL-6, IL-1β, IL-4, IL-13, TSLP, and chemokine CCL-17, CCL-5 were measured in activated HaCaT cells using ELISA (enzyme-linked immunosorbent assay). The amounts of nitrite were calculated utilizing a Griess assay. TNF-α produced ROS (reactive oxygen species) in HaCaT cells were analysed using DCFDA (dichlorofluorescein diacetate) assay. Protein concentrations in NF-κB (Nuclear factor kappa B) pathway and MAPK (mitogen-activated protein kinase) pathway were also quantified using ELISA. The effect of NRF2 (nuclear factor erythroid 2–related factor 2) pathway in mediating the anti-inflammatory properties of Hamamelis in HaCaT cells was measured by Western blot examination of HO-1, NQO-1, and ELISA for NRF2. The impact of Hamamelis on mast cells was compared with commonly used antihistamines (chlorpheniramine maleate and loratadine) to assess its role in allergic mediated inflammatory reactions on the skin.
The results confirmed that the Hamamelis reduced NO productivity in TNF-α provoked HaCaT cells. Hamamelis further diminished the release of other pro-inflammatory factors IL-6, TNFα, IL-4, IL-13, IL-1β, TSLP, CCL-17, CCL-5, NO, and ROS. However, it had minimal effect on IL-1β and IL-13 in TNF-α stimulated HaCaT cells. The mechanistic studies concluded that the anti-inflammatory properties of Hamamelis was mediated through the inhibition of the MAPK or NF-κB pathways. Furthermore, the Hamamelis modulated antioxidant proteins HO1 and NQO-1, suggesting that the activity was mediated through NRF2 signaling pathway.
Tea tree oil (TTO) was also considered as a potential natural substance for its anti-inflammatory properties due to its abundance in skin products and history of use on human skin. However, we found that it was not well tolerated by the cultured keratinocytes even at a very low dose. Moreover, TTO did not inhibit the production of proinflammatory cytokine, such as IL-1 β. Due to lack of anti-inflammatory properties and observed cytotoxic effects in HaCaT cells, TTO was not considered for further studies in this thesis.
Considering the safety and anti-inflammatory efficacy of Hamamelis, it was also formulated as a topical ointment for potential application on the skin using conventional pharmacopeial methods. The formulation and excipients were also assessed for cytotoxicity to ensure safety and formulated product was tested for anti-inflammatory markers to ascertain the formulated product's efficacy compared to a typical steroid hydrocortisone as a control. The other formulation components, the olive and coconut oils (virgin, mechanically pressed) were also investigated individually to assess their impact on cell viability and sensitivity to keratinocytes resulting in a synergistic effect against inflammatory cascade. Both oils have been famous for their emollient properties on the skin. Therefore, the coformulation of coconut and olive oils with Hamamelis was deemed beneficial for topical application on the skin.
The thesis provided material evidence confirming Hamamelis prevented TNF-α mediated inflammation through NRF2/ARE (antioxidant response element) antioxidant pathways in HaCaT cells. The antihistaminic effects of the Hamamelis on mast cells also hinted a potential anti-inflammatory role on skin via ameliorating processes triggered by histamine-mediated skin allergy. The formulated topical product of Hamamelis successfully retained its anti- inflammatory properties on a 15-month storage. A comprehensive stability evaluation of the formulated product as per ICH stability guidelines (ICH Q1A), is however, part of future works.
The natural formulation offered a steroid-free natural alternative for topical application in patients with clinical and sub-clinical conditions of skin, offering the benefits of steroid and emollients in a single preparation. Despite a variety of emollients available in the market for soothing inflammatory conditions of the skin such as eczema; there is none available with strong anti-inflammatory and antipruritic properties except topical steroids which are often not preferred due to their obvious side effects and safety concerns over long term use.
It is, therefore, remarkable to conclude that a topical formulation containing natural plant extracts from Hamamelis in combination with olive and coconut oils could be offered in prevention and treating skin inflammation for clinical and subclinical inflammatory conditions of the skin such as eczema or dermatitis. The results from anti-inflammatory screening in cultured human keratinocytes in this thesis are very promising and the product is worth taking forward for a formal clinical trial to confirm its clinical efficacy. Once clinically validated, the formulation can be scaled-up easily for commercial production using conventional manufacturing methods using commercially available “Hamamelis water” that is already compliant to the BPC (British Pharmaceutical Codex) or US NF (United States National Formulary) monographs, that was also investigated in this thesis alongside Hamamelis methanolic extract.
Date of Award30 Nov 2022
Original languageEnglish
SupervisorHamid Merchant (Main Supervisor) & Olumayokun Olajide (Co-Supervisor)

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