National Journal of Physiology, Pharmacy and Pharmacology RESEARCH ARTICLE Phytochemical properties of a rare mangrove Aegialitis rotundifolia Roxb. leaf extract and its influence on human dermal fibroblast cell migration using wound scratch model Debjit Ghosh1, Sumanta Mondal2, K Ramakrishna1 1Department of Chemistry, GITAM Institute of Science, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India, 2Department of Pharmaceutical Chemistry, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India Correspondence to: Sumanta Mondal , E-mail: [email protected] Received: October 13, 2018; Accepted: February 16, 2019 ABSTRACT Background: Aegialitis rotundifolia Roxb. is a small mangrove plant traditionally used for curing pain arising from sundry injuries. At present, there are no scientific evidences of its wound healing properties and few reports have claimed that reduction of pain could accelerate the wound healing process. Aims and Objectives: The present work deals with the qualitative and quantitative study of the phytoconstituents present in A. rotundifolia Roxb., ethanolic leaves extract and also evaluate its wound healing properties in vitro. Materials and Methods: The qualitative phytochemical analysis of the extract was performed using preliminary phytochemical tests whereas the quantitative determination of phytochemicals was evaluated spectroscopically using ultraviolet spectroscopy. The in vitro wound healing activity was determined on human dermal fibroblast (HDF) cells using the wound scratch assay. Results: The quantitative estimation of phytochemicals revealed that total phenolics was the most abundantly found phytochemical followed by a total flavonoid, total flavonol, total tannin, total alkaloid, total proanthocyanidin, and total saponin. In the in vitro wound healing assay, the sample was first examined for possible cytotoxicity on HDF cells where the results showed that it was slightly toxic at higher concentration (inhibit cell growth by 50%: 157.41 ± 3.4 µg/mL); thus, a concentration of 40 and 80 µg/mL was taken to carry out the scratch assay. The wound scratch assay revealed significant (P < 0.01) wound healing activity in a dose- dependent manner at 40 and 80 µg/mL concentrations. Conclusion: Thus, the extract proved effective in wound healing which may have been attributed by the presence of several bioactive phytochemicals. KEY WORDS: Aegialitis rotundifolia; Mangrove; Phytochemical Analysis; Cytotoxicity; Wound Scratch Assay INTRODUCTION for the scientists.[1] The treatment of several diseases with the aid of plants and plant products is known as herbal medicine, which Mangroves have long been a source of huge astonishment along is considered as a part of the traditional and folk medicinal with abiding curiosity to the common men and of endless interest system. From the dawn of civilization, several ethnic groups have been using medicinal plants to combat several diseases as Access this article online [2] Website: www.njppp.com Quick Response code they were the only resource available for medication. Wound can be defined as a disruption in the cellular and DOI: 10.5455/njppp.2019.9.1030716022019 anatomical architecture and physiological function of tissue which includes skin, mucus membrane, deeply lying tissues or surface of internal organs ranging from incision, National Journal of Physiology, Pharmacy and Pharmacology Online 2019. © 2019 Sumanta Mondal, et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creative commons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license. 335 National Journal of Physiology, Pharmacy and Pharmacology 2019 | Vol 9 | Issue 4 Ghosh et al. In vitro wound healing activity of Aegialitis rotundifolia leaves laceration, abrasion, puncture, and closed wounds such India), and Thermo Fisher Scientific, Inc., (Waltham, MA, as contusion, hematoma, and crush.[3] Impaired wound USA). All the solvents used were of high purity and HPLC healing is considered a major problem which may be grade. All other chemicals and reagents used in the whole caused by excessive production of reactive oxygen species study were of analytical grade. (ROS), microbial infection and uncontrolled inflammatory responses. On disruption of such tissues, intricate and Collection and Authentication of Plant Materials complex processes are initiated to repair the damaged tissue which involves a complex cascade of cellular events resulting The fresh leaves of A. rotundifolia Roxb. were collected from in reconstitution, resurfacing, and restoration of the tensile healthy fully grown plants from Bichitrapur Mangrove located strength of the injured skin.[4] Wound healing is a complex in Kharibil, Orissa, India (21°34’54.0”N - 87°25’25.4”E). biological process due to which several in vitro and in vivo The plant materials were then authenticated from Botanical methods are available. Among these models, the scratch Survey of India, Central National Herbarium, Botanic Garden, assay has proven to be an important and valuable tool which Howrah, West Bengal, India, and were assigned with a provides first insights to how the natural products positively Voucher no. CNH/Tech.II/2016/11a and specimen no. DG-01. influence the regeneration of new tissues.[5,6] Preparation of Extracts Aegialitis rotundifolia Roxb. (Plumbaginaceae) is a small mangrove tree or shrub which usually grows up to a height The collected plant materials were gently washed in tap water of 2–3 m and is available in shorelines of the Andaman Sea to remove dirt and were shade dried under room temperature and the Bay of Bengal and is endemic to the coastal parts of (24 ± 2°C) for 3–4 weeks. After complete drying, the dried South Asia. In Orissa, it is locally known as Banrua.[7,8] This plant materials were pulverized by using a mechanical grinder mangrove species is reported to produce one of the best quality followed by sieving to obtain a coarse powder. The powdered honey.[9] Conventionally, the leaf is used in the treatment of plant material was then extracted with ethanol (99.9%) using sundry injuries accompanied by pain and inflammation and reflux technique. The crude extract solution obtained was is locally utilized as an anti-acne agent.[10] Further, the leaf of filtered using Whatman No. 42 filter paper after which the the plant is pounded with oil to make a paste which acts as an excess solvents were evaporated by rotary vacuum evaporator antidote for insect bites.[11] According to the present literature, (Evator, Media Instrument Mfg. Co., Mumbai, India) and there have been very few scientific reports of pharmacological concentrated on water bath to obtain A. rotundifolia Roxb., screening conducted such as analgesic, antipyretic,[11] in vitro ethanolic leaves extract (ARELE). The crude ethanol extract antioxidant,[12] antimicrobial,[13,14] anti-inflammatory,[11,15] obtained was stored at 4°C before analysis. in vitro thrombolytic activity,[15] antibacterial,[15] and anticancer activity.[16] Recently, we have reported the presence Qualitative and Quantitative Determination of of gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, Phytochemical Groups rutin, coumarin, and quercetin by performing quantitative high-performance liquid chromatography (HPLC)_ For qualitative phytochemical analysis, preliminary analysis and an organosilicon compound, (-)-spiro[1-[(tert- phytochemical test was conducted on ARELE according to [17-19] Butyldimethylsiloxy)methyl]-3,5,8-trimethyl-bicyclo[4.3.0] the standard procedures. non-2-en-5,7-diol-4,1’-cyclopropane] was detected in gas chromatography-mass spectrometry analysis as the most For the quantitative estimation of phytochemical groups, abundantly found compound.[8] However, there has been the crude extract was screened for total phenolic content (TPC),[20] total flavonoid content,[21] total flavonol content,[22] no scientific study conducted on the wound healing activity [23] [24] of the plant and also its phytochemical properties are also total proanthocyanidin content, total alkaloid content, total saponin content,[25] and total tannin content[26] which were hugely unexplored. Therefore, the present study was designed expressed as gallic acid equivalent (mg/g of extract), quercetin to evaluate the qualitative and quantitative phytochemical equivalent (mg/g of extract), quercetin equivalent (mg/g of analysis and also investigate the wound healing potential extract), catechin equivalent (mg/g of extract), atropine equivalent of A. rotundifolia using the scratch model on human dermal (mg/g of extract), diosgenin equivalents (mg/g of extract), and fibroblast (HDF) cell line. tannic acid equivalent (mg/g of extract), respectively. MATERIALS AND METHODS In vitro Wound Healing Activity Using Scratch Wound Healing Model Chemicals and Reagents Cell culture and treatment Ethanol 99.9% used for extraction was procured from Changshu Hongsheng Fine Chemicals Co. Ltd., China. For Cell lines HDF was procured from National Centre for in vitro wound healing studies, the required chemicals were Cell Science, Pune, and cultured in Ham’s F-12 media procured from HiMedia Laboratories Pvt. Ltd. (Mumbai, supplemented with 10% inactivated fetal bovine serum (FBS),