Antiseptic, Antiperspirant and Deodorant Diaper for the Frail Older Adult with Urinary Incontinence

Abstract

Introduction: Urinary incontinence is a common complaint among older people which could cause great burden to the carer and healthcare system. The idea of creating a special diaper for the older and frail older people suffering from urinary incontinence to prevent complications arising from over-night and persistent wetting of the perineal region therefore appear appealing and beneficial. Aim of the study: The aim of this s tudy was to perform further laboratory studies to understand the sweat modulation effect of the herbal formula, and to determine whether the herbal formula and the herbal formula containing diaper would cause any skin toxicity. Materials and Methods: In vitro studies including the Acetylcholinesterase (AChE) activity assay, and measurement of chloride efflux by short circuit current assay was performed to understand the sweat modulation effect of the herbal formula. In vitro skin toxicity test was also performed to determine whether the herbal formula and the herbal formula containing diaper would cause any skin toxicity. Results: Our herbal formula containing Cortex Fraxini, Mori Follium and Calamine significantly increased the acetylcholinesterase activity in PC12 cells. This formula also significantly inhibited the UTP-evoked Clsecretion and ACh-induced Cltransport. Further skin toxicity test suggested this formula exerted no significant toxicity to the cells in the skin. Conclusion: These data further supported the positive data observed in two clinical studies previously completed which demonstrated that the herbal formula could reduce sweat secretion, reduce odour, skin irritations and the herbal extract-containing diaper could improve the quality of life of individuals with urinary incontinence. Citation: Wat E, Wang YP, Tsz Chan HY, Wong S, Hung Ko W, et al. Antiseptic, Antiperspirant and Deodorant Diaper for the Frail Older Adult with Urinary Incontinence. J Geriatrics Palliative Care 2019;6(1): 5. J Geriatrics Palliative Care 6(1): 5 (2019) Page 02 ISSN: 2373-1133 herbal extract. Comparing with their control diapers, the majority of volunteers preferred the herbal extract containing diapers due to the less leakage and unpleasant odour. They were also found to have better skin conditions [4]. These clinical studies demonstrated that our herbal extract containing diapers could reduce sweat production and improve the quality of life of the elderly people. Here, we have reported further laboratory studies to understand the underlying mechanism contributing to the observed beneficial effects of the herbal extract in previous clinical studies. We conducted several in vitro studies including the Acetylcholinesterase (AChE) activity assay to understand the anti-cholinergic effects of the herbal extract; and measurement of chloride efflux by short circuit current assay to understand the sweat modulation mechanism of the herbal extract. In vitro skin toxicity test was also performed to determine whether the herbal extract and the herbal extract containing diaper would cause any skin toxicity. Materials and Method Herbal materials authentication and preparation Herbal material authentication: Raw herbal material of Mori Follium and Fraxini Cortex were purchased from a renowned supplier in Hong Kong. Calamine powder (Pharm Grade) were purchased from Wing Hing Chemical Co. Ltd., Hong Kong. Mori Follium and Fraxini were chemically authenticated using Thin Layer Chromatography (TLC) in accordance to Chinese Pharmacopoeia (CP) [12]. Upon chemical authentications, herbarium voucher specimen of Cortex Fraxini and Folium Mori were deposited at the museum of the Institute of Chinese Medicine at the Chinese University of Hong Kong, with voucher specimen number as 20163491 and 2016-3490, respectively. Herbal extract preparation Extraction of Cortex Fraxini and Folium Mori were performed at the Hong Kong Institute of Biotechnology (HKIB) following the traditional practice of herbal extraction in accordance to Good Manufacturing Practice (GMP). Briefly, raw herb was extracted twice by heating under reflux at 100 °C using 10x distilled water for each extraction. The aqueous extracts were combined and filtered. Filtrate was concentrated under reduced pressure at 60 °C. The concentrated extract was then spray dried with no excipient added. The dried extracts were packed in vacuum condition and stored until use. In vitro cell culture experiments Cell culture: The PC12 cell line was obtained from the American Type Culture Collection (USA). Cells were grown and maintained in Roswell Park Memorial Institute medium (RPMI) supplemented with 10 % (v/v) horse serum (Gibco, USA), 5 % (v/v) fetal bovine serum (Gibco, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin in a 10 % CO2 humidified atmosphere at 37 °C. Cells grown to 80 % confluence in T75 culture flasks were trypsinized and seeded into 6-well culture plates for experiment. Inhibition of chloride secretion using the short Circuit current assay was performed using NCl-SG3 cells, the human eccrine sweat gland epithelial cell line. The cell line was kindly provided by Prof. Ko Wing Hung, School of Biomedical Sciences, the Chinese University of Hong Kong. NCL-SG3 cells were maintained in Williams Medium E, supplemented with epidermal growth factor (0.1 %), fetal bovine serum (5 %), Glutamic monosodium salt hydrate (2 mM), hydrocortisone (0.01 %), ITS-liquid supplement (1 %) and penicillin (1 %). Cultures were maintained at 37 oC in a humidified atmosphere of 5 % CO2. Acetylcholinesterase activity assay Acetylcholinesterase assay was performed following the manufacturer’s protocol. Briefly, 6-well plate was coated with 0.1 mg/ ml Poly-L-Lysine overnight. PC12 cells were seeded at 4 x 105 cells/ well. Cells were then treated with various concentrations of herbal extracts (0 1 mg/ml) for 48 hrs. Cells were sonicated with RIPA buffer on ice for 30 minutes (Sigma-Aldrich, St. Louis, MO. USA.), followed by centrifugation at 14,000 x g. Cell lysate were mixed with the reaction mix solution containing acetylthiocholine stock solution, followed by incubation at 37 oC for 1 hr, and absorbance was measured spectrophotometrically at 410 nm. Short Circuit current assay Membranes response to Cltransportation was monitored with Ussing chambers as previously described [13]. Prior to the experiment, NCL-SG3 cells (3 x 105/ 250 μL) were grown on MFTM Membrane Filters for 7 days. Fresh medium was changed every other day to obtain confluent cell monolayer. The integrated monolayer was mounted between two halves of Ussing chambers, which were filled with 7 ml of KrebsHenseleit Solution (KHS) on both sides. The K-H solution contained the following components: 25mM NaHCO3, 117 mM NaCl; 4.7 mM KCl; 2.5 mM CaCl2; 1.2 mM KH2PO4; 1.2 mM MgSO4; and 11 mM D-glucose; its pH was 7.4 when bubbled with 5% CO2/95% O2 and was kept at 37 oC by water jacket before use. Inhibition of chloride secretion on NCl-SG3 was then analyzed. Once the condition had stabilized (20-30 min), pre-incubation of herbal formula (at 1,2 or 3 mg/ml) or KHS (as blank control) on the basolateral side was performed for 10 min. Effect of herbal formula on efflux of Clion induced by UTP or ACh were then investigated by adding 10 μM of UTP or ACh and incubation of 10 min. The short-circuit current was recorded by a short-circuit clamp amplifier and displayed using a chart recorder. And the current inhibited by herbal formula was calculated using the resultant change of current resistance. In vitro skin toxicity test