Perspective paperIn vitro tests and ethnopharmacological investigations: Wound healing as an example
Introduction
Over the last 20 years ethnopharmacological studies have increasingly included in vitro bioassays as a replacement for experiments using tissues or whole animals. This is partly due to the ethical and commercial problems of using animals but also the unsuitability of such practices for bioassay-guided fractionation of the compounds responsible for any activity observed (Houghton, 2000). These procedures may be based on cultured cells, enzyme studies or receptor-ligand binding experiments and nowadays also gene-expression-arrays are extensively used, not only in ethnopharmacological research, but also by the pharmaceutical industry. Both constituencies use the techniques for screening large numbers of natural extracts and fractions, but industrial concerns are often highly automated to enable high throughput screening and tend to more often screen synthetic compounds and mixtures.
In spite of the widespread use of in vitro assays, it is important to point out the deficiencies of such an approach. It is very unusual for one in vitro assay alone to represent a disease state and the use of a battery of relevant tests is preferred, since most disease states are complex and several mechanisms are involved, all of which may offer targets for compounds which effect amelioration of the condition. However, even with a variety of relevant tests, it is generally acknowledged that in vitro tests are too reductionist to extrapolate their results to provide evidence for clinical efficacy, and that eventually animal testing and clinical trials have to be performed. Fractionation, which is often carried out when in vitro tests are employed, often results in loss of activity, or less increase per fraction than might be expected. This may be due to decomposition of the active products or to breakup of synergistic relationships.
In spite of these negative aspects, in vitro bioassays have resulted in the discovery of some novel therapeutic agents and are continually revealing compounds from traditional medicines, which help explain their usage. They may be valuable also in providing evidence of the modes of action of materials, which have shown clinical or in vivo activity. This paper seeks to exemplify some of the issues concerning in vitro tests for wound healing by reviewing existing data, and providing novel findings, concerning the leaves and flowers of the genus Buddleja (Loganiaceae) and also illustrates other in vitro tests used to investigate three Ghanaian species.
The leaves of a number of species of Buddleja are reported to be applied topically as a poultice or lotion for healing of wounds (Houghton, 1984). Studies on wound-healing properties have focused on the South American species Buddleja globosa Hope which is endemic to Chile and Argentina, the leaves being used by the indigenous Mapuche for the treatment of ulcers and wounds (Houghton and Manby, 1985). Studies have also been carried out on the Far Eastern Buddleja officinalis L. whose flowers are used to treat sore and damaged eyes, a condition which is similar to skin wounds (Houghton, 1984). The fairly detailed knowledge of the constituents present in Buddleja enabled speculation that the presence of saponins, flavonoids and other phenolics could contribute to wound healing because of their detergent ability to remove grease, dirt and bacteria from tissue and act as antimicrobials (Houghton and Mensah, 1997). However, this speculation had no experimental support at the time because of the comparative lack of in vitro tests related to wound healing.
Interviews with traditional Ashanti healers in Ghana revealed a variety of plants used to treat wounds. Three species were selected on the basis of little previous chemical or biological work having been carried out and these were the stembark of Spathodea campanulata P. Beauv. (Bignoniaceae), the leafy shoots of Secamone afzelii Rhoem. (Asclepiadaceae) and the herb of Commelina diffusa Burn. (Commelinaceae). All three plants are used as poultices of lotions made by steeping the material in hot water or local alcoholic spirits (Abbiw, 1990).
Section snippets
The wound healing process and relevant biological tests
In vivo models of wound healing generally use small rodents such as guinea pigs or rats. The backs are shaved and wounds induced by scarification or burning. Placebo and test solutions are applied to the wound and the times taken for the wounds to reach recognized stages of the wound healing process for test substance and controls are noted (Saha et al., 1997). Another approach is to measure the surface area and tensile strength of the wound (Rashed et al., 2003). However, in many parts of the
Antimicrobial characteristics
Open wounds are particularly prone to infection, especially by bacteria, and also provide an entrypoint for systemic infections. Infected wounds heal less rapidly and also often result in the formation of unpleasant exudates and toxins will be produced with concomitant killing of regenerating cells. Antibacterial and antifungal compounds in a traditional remedy may prevent this occurring and may underlie its use in treating wounds. Probably the best antimicrobial assay to use is the serial
Plant material and preparation of extract
The aqueous extract of fresh leaves of Buddleja globosa used was prepared as described earlier (Mensah et al., 2001) from material lodged as voucher specimen Bg 004 in the herbarium of the Department of Pharmacy, King's College London.
The stock was diluted accordingly in Dulbecco's modified eagle medium (DMEM) or Hank's balanced salt solution (HBSS) and then used for the assay. A fresh sterile solution was used for each experiment.
Induction of differentiation of primary human keratinocytes
NHK were isolated as primary cells from human skin, obtained by
Effect of Buddleja globosa extract on differentiation of NHK keratinocytes
Buddleja globosa extract triggered the differentiation of the NHK cells (see Table 2). The content of the differentiation specific protein involucrin in the NHK was significantly higher than that of the untreated control cells and the NHK cells also formed significantly more cytokeratins K1 and K10 when the extract was added to the medium. In comparison, with the positive control (calcium ionophore A 23187, 13 μg/ml) there was no significant increase observed because of the high variation in the
Conclusions
The results discussed above show that the plant extracts investigated demonstrate polyvalent activity in their potential for helping wounds to heal. Buddleja contains flavonoids, triterpenes and carotenoids which have anti-inflammatory properties, flavonoids and phenylethanoids which are antioxidant and undetermined substances which might cause proliferation of fibroblasts. Secamone afzelii and Spathodea campanulata exhibit both antioxidant and antimicrobial properties. Other tests for
Acknowledgements
A.Y. Mensah was supported by a Commonwealth Scholarship and part of this work was funded by Oxford Natural Products PLC.
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