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      Wound healing properties and kill kinetics of Clerodendron splendens G. Don, a Ghanaian wound healing plant

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          Abstract

          As part of our general objective of investigating indigenous plants used in wound healing in Ghana, we hereby report our findings from some in vitro and in vivo studies related to wound healing activities of Clerodendron splendens G. Don (Verbanaceae). Methanolic extract of the aerial parts of the plant was tested for antimicrobial activity against Gram positive bacteria ( Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, Micrococcus flavus, as well as resistant strains of Staph. aureus SA1199B, RN4220 and XU212), Gram negative bacteria ( Escherichia coli, Pseudomonas aeruginosa, Proteous mirabilis, Klebsiella pneumoniae) and Candida albicans using the micro-well dilution method. Survivor–time studies of the microorganisms, radical scavenging activity using 2,2’-diphenylpicrylhydrazyl (DPPH) and various in vivo wound healing activity studies were also conducted on the extract. The extract exhibited biostatic action against all the test microorganisms with a Minimum Inhibition Concentration (MIC) ranging between 64 and 512 μg/ml and a free radical scavenging property with an IC 50 value of 103.2 μg/ml. The results of the in vivo wound healing tests showed that upon application of C. splendens ointment, there was a reduction in the epithelization period from 26.7 days (control) to 13.6 days along with a marked decrease in the scar area from 54.2 mm 2 (control) to 25.2 mm 2. Significant increase in the tensile strength and hydroxyproline content were also observed as compared to the control and was comparable to nitrofurazone. The above results appear to justify the traditional use of C. splendens in wound healing and treatment of skin infections in Ghana.

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          A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria.

          J Eloff (1998)
          Agar diffusion techniques are used widely to assay plant extracts for antimicrobial activity, but there are problems associated with this technique. A micro-dilution technique was developed using 96-well microplates and tetrazolium salts to indicate bacterial growth. p-Iodonitrotetrazolium violet [0.2 mg/ml] gave better results than tetrazolium red or thiazolyl blue. The method is quick, worked well with Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, and Escherichia coli and with non-aqueous extracts from many different plants. The method gave reproducible results; required only 10-25 microliters of extract to determine minimal inhibitory concentrations, distinguished between microcidal and microstatic effects, and provided a permanent record of the results. Using S. aureus, and a Combretum molle extract, the technique was 32 times more sensitive than agar diffusion techniques and was not sensitive to culture age of the test organism up to 24 hours. The S. aureus culture could be stored up to 10 days in a cold room with little effect on the assay results. This method was useful in screening plants for antimicrobial activity and for the bioassay-guided isolation of antimicrobial compounds from plants. MIC values determined for sulfisoxazole, norfloxacin, gentamicin, and nitrofuratoin were similar to values indicated in the literature but values obtained with trimethroprim and ampicillin were higher with some bacteria.
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            In vitro tests and ethnopharmacological investigations: wound healing as an example.

            In vitro tests are now widely employed in ethnopharmacological research because of ethical reasons and their usefulness in bioactive-guided fractionation and determination of active compounds. For many disease conditions, a variety of in vitro tests can now be employed as the biochemical mechanisms underlying disease and healing processes are understood. Approaches to the in vitro investigations of wound healing processes are exemplified by studies on extracts of Buddleja species and three Ghanaian species Spathodea campanulata, Commelina diffusa and Secamone afzelii. Most studies have been carried out on Buddleja officinalis or Buddleja globosa. The extracts have been shown to have anti-inflammatory and antioxidant properties due to flavonoids, triterpenoids, diterpenoids and caffeic acid derivatives. There appears to a slight effect on proliferation of fibroblasts at lower concentrations, but this was not significant, and higher concentrations appeared to be cytotoxic. Novel findings are the ability of Buddleja globosa leaf extracts to induce differentiation in keratinocytes and to alter the profile of proteins produced by cultured fibroblasts. Extracts also had some effect on lattice contraction. The three Ghanaian species examined show a mixture of antimicrobial and antioxidant activities. The evolution over recent years of tests for wound healing, from in vivo tests to cell-based systems and chemical reactions and on to investigations into effects on secondary messengers and protein expression, is described.
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              In vitro and in vivo wound healing activity of asiaticoside isolated from Centella asiatica.

              The activity of asiaticoside, isolated from Centella asiatica, has been studied in normal as well as delayed-type wound healing. In guinea pig punch wounds topical applications of 0.2% solution of asiaticoside produced 56% increase in hydroxyproline, 57% increase in tensile strength, increased collagen content and better epithelisation. In streptozotocin diabetic rats, where healing is delayed, topical application of 0.4% solution of asiaticoside over punch wounds increased hydroxyproline content, tensile strength, collagen content and epithelisation thereby facilitating the healing. Asiaticoside was active by the oral route also at 1 mg/kg dose in the guinea pig punch wound model. It promoted angiogenesis in the chick chorioallantoic membrane model at 40 microg/disk concentration. These results indicate that asiaticoside exhibits significant wound healing activity in normal as well as delayed healing models and is the main active constituent of Centella asiatica.
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                Author and article information

                Journal
                Pharmacognosy Res
                PR
                Pharmacognosy Research
                Medknow Publications Pvt Ltd (India )
                0976-4836
                0974-8490
                Mar-Apr 2010
                : 2
                : 2
                : 63-68
                Affiliations
                [1] Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana, West Africa
                [1 ] Department of Pharmacognosy, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana, West Africa
                [2 ] Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana, West Africa
                Author notes
                Address for correspondence: Gbedema Y. Stephen, Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana, West Africa. E-mail: sgbedema@ 123456yahoo.com
                Article
                PR-2-63
                10.4103/0974-8490.62948
                3140108
                21808542
                1bb74cf8-ef56-497b-9fd9-dd03505e23f0
                Copyright © Pharmacognosy Research

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 17 February 2010
                : 22 March 2010
                : 04 May 2010
                Categories
                Original Article

                Pharmacology & Pharmaceutical medicine
                wound healing,clerodendron splendens,biostatic,antioxidant,antimicrobial

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