In-Vitro Anti-Inflammatory Assay Of Crassocephalum Crepidiode

Description

ABSTRACT

Crassocephalum crepidioides is one of the important medicinal plants used in different parts of the world (mostly Asia and African) as medicinal and nutritional herbs. It is a herbaceous perennial belonging to family Asteraceae. It is commonly found in abandoned farmlands, wastage places and backyard gardens that are rich in organic matter. Crassocephalum crepidioides has many nutraceutical potential which  anti-inflammatory activity potential is one of them. This study is aimed at evaluating the wound healing activity of Crassocephalum crepidioides hydroethanolic extract via its antioxidant and anti-inflammation activities and healing capability on a mouse excision wound model. Crassocephalum crepidioides hydroethanolic extract (CCLE) at a dose of 50 mg/kg/day reduced the wound closure time about 3.5 days, compared to vehicle treatment. The granulation tissue on day 7 after surgery from the treated group showed a 2.8-fold decrease in the density of inflammatory cells, 1.9-fold increase in the fibroblast density, and a higher number of blood vessels. Real-time PCR analysis indicated that the mRNA expression level of NF-κB1 and TNF-α mRNA in CCLE-treated wounds decreased by 4.6 and 3.3 times, respectively, while TGF-β1 and VEGF were found to increase by 3.3 and 2.4 times, respectively.

ABBREVIATIONS

CT:             Threshold cycle

CCLE:        Crassocephalum crepidioides leaf hydroethanolic extract

DMSO:       Dimethyl sulfoxide

DPPH:        2,2-Diphenyl-1-picrylhydrazyl

FBS:           Fetal bovine serum

GAPDH:     Glyceraldehyde 3-phosphate dehydrogenase

LPS:            Lipopolysaccharide

MTT:          3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NF-κB1:     Nuclear factor kappa-light-chain-enhancer of activated B cells 1

OD:             Optical density

TGF-β1:      Transforming growth factor 1

TNF-α:        Tumor necrosis factor α

VEGF:        Vascular endothelial growth factors

CHAPTER ONE

1.0                                          INTRODUCTION

1.1                            BACKGROUND OF THE STUDY

Wound healing is a complex process that often is divided into 3 overlap phases: inflammation, proliferation, and remodeling [T. Maver, 2015]. The inflammatory phase starts immediately after injury, together with hemostasis. In this phase, leukocytes are recruited to the wound site to remove alien substances and dead tissue and prevent infection [T. Maver, 2015]. Fibroblasts and keratinocyte proliferation and migration play a key role in the proliferation phase. Fibroblasts are predominant cells at the wound site in this phase, responsible for new matrix and collagen production. The remodeling phase occurs in several months to years, involving change in components of the extracellular matrix at the wound site and restoration of about 80% of tensile strength normal skin [P. K. Ghosh and A. Gaba, 2013].

Many traditional medicinal plants (TMPs) have been discovered and used for wound care for centuries. Recent reports suggested that the wound healing mechanism of TMPs might be due to antioxidant, antimicrobial, and anti-inflammatory activities and stimulation on DNA and protein synthesis, epithelium cell proliferation, collagen formation, and angiogenesis [P. K. Ghosh and A. Gaba, 2013].

Crassocephalum crepidioides (Benth.) S. Moore. (Compositae family) is a TMP found in various parts of Asia, Africa, and Australia [R. Lemmens and N, 2003]. The leaves of crassocephalum crepidioides are traditionally used to treat indigestion, stomachache [Grubben and O. A. Denton, 2014], and wound [D. T. Loi, 2004]. Crassocephalum crepidioides has been reported to have antibacterial, hypoglycemic, antioxidant, anti-inflammatory, antitumor, and antidiabetic activities. Several bioactive compounds have been found in crassocephalum crepidioides leaves, including some phenolic and flavonoid compounds; the essential oil from crassocephalum crepidioides leaves contains β-cubebene, α-farnesene, and α-caryophyllene. Crassocephalum crepidioides leaves has been also used for treating wounds in Vietnam, China, India, and some African countries.

This study is aimed at investigating the wound healing activity of C. crepidioides hydroethanolic leaf extract (CCLE) on a mouse excision wound model, focusing on the wound closure rate, histopathology characteristics, and the mRNA expression level of several genes involved in the wound healing process.

1.2                                   PROBLEM STATEMENT

Wound is one the most popular health challenge which can happen to anyone at any point time and which is mostly caused by accident. Crassocephalum crepidioides has been discovered as an anti-inflammatory agent apart from other health activities it can perform. This study was carriedout to determine the anti-inflammatory potential of Crassocephalum crepidioides.

1.3                                       AIM OF THE STUDY

The main aim of this study is to evaluate the anti-inflammation potential of Crassocephalum crepidioides and its healing capability on a mouse excision wound model.

1.4                               OBJECTIVE OF THE STUDY

At the end of this work, the study will provide proofs of Crassocephalum crepidioides leaf wound healing activity due to its antioxidant, anti-inflammation, fibroblast proliferation, wound contraction, and angiogenesis effects.

1.5                   SCOPE AND LIMITATION OF THE STUDY

Some medicinal plants and herbs can effectively treat and cure multiple health problems. Large no of the drugs are also extracted from herbs and the most important advantages of using herbs are they are being economical, effective and easily available. The World Health Organisation (WHO) has listed 21,000 plants which are used for medicinal purposes around the world, which Crassocephalum crepidioides is one of those herbs. This study was carried out because of the popular use of Crassocephalum crepidioides as an anti-inflammatory agent.

1.6                             SIGNIFICANCE OF THE STUDY

The findings from this study would be useful to medical personel in that it will help them to understand the anti-inflammatory potential of Crassocephalum crepidioides.

1.7                                                  LIMITATION OF STUDY

As we all know that no human effort to achieve a set of goals goes without difficulties, certain constraints were encountered in the course of carrying out this project and they are as follows:-

1. Difficulty in information collection: I found it too difficult in laying hands of useful information regarding this work and this course me to visit different libraries and internet for solution.
2. Financial Constraint: Insufficient fund tends to impede the efficiency of the researcher in sourcing for the relevant materials, literature or information and in the process of data collection.

• Time Constraint: The researcher will simultaneously engage in this study with other academic work. This consequently will cut down on the time devoted for the research work

1.8                                                         PROJECT ORGANISATION

The work is organized as follows: chapter one discuses the introductory part of the work,   chapter two presents the literature review of the study,  chapter three describes the methods applied, chapter four discusses the results of the work, chapter five summarizes the research outcomes and the recommendations.