Rationale for Using Peltophorum Africanum (fabaceae) Extracts in Veterinary Medicine

INTRODUCTION Phytotherapy, the treatment and prevention of disease using medicinal plants, is an ancient tradition that has existed with human habitations. About 80 % of the world's people still depend on the traditional healthcare practices using herbs.


INTRODUCTION
Phytotherapy, the treatment and prevention of disease using medicinal plants, is an ancient tradition that has existed with human habitations.About 80 % of the world's people still depend on the traditional healthcare practices using herbs. 11his is so mainly in rural communities in the developing world where modern drugs may be unaffordable or inaccessible.Disease concepts are largely similar in humans and animals; in many traditional systems, healers of people are often called upon to treat animals and vice versa 28 .Healers frequently use the same herbs, compounds or techniques.
Many pharmacologically active com-pounds have been discovered following ethnobotanical leads 4,11 .As tropical forests still present a great storehouse of medicinal genetic resources, the search for compounds with novel bioactivity from plants continues 32 .
Peltophorum africanum (Sond), commonly called 'weeping wattle' or 'huilboom', is a member of the Fabaceae.It is a deciduous tree growing up to 15 m high with a wide canopy that occurs widely in medium to low altitudes in wooded grassland areas of southern Africa 24 .Whereas the genus is found throughout the tropics, P. africanum is the only member of the genus in southern Africa.The plant has many traditional medicinal uses in humans and animals.The roots and bark are used to treat sore throat, wounds, diarrhoea, dysentery, helminthosis, abdominal pains, ascites, back and joint pains, HIV-AIDS, venereal diseases, infertility, colic and eye infections 12,31,34 .Pastoralists and rural farmers use the root and bark to treat diarrhoea, dysentery and infertility in cattle and to promote well-being and resistance to disease 30,34 .Bark from P. africanum was identified as one of the most important products sold in informal medicinal plant markets in Pretoria 17 .
The phytochemistry of P. africanum has been studied by several authors.A sulphate ester of trans-4-hydroxypipecolic acid has been isolated from the seed 10 .Several condensed flavonoids, a novel cyanomaclurin analogue 1 , profisetinidin-type 4-arylflavan-3-ols and related δ-lactones 2 were found in the heartwood.Mebe and Makuhunga 19 isolated new compounds (bergenin, norbergenin and 11-0(E)-pcoumaroylbergenin from ethanol extracts of the bark.Khattab and Nassar 14 isolated coumarins from the leaves.The chemical structures of the novel compounds isolated were elucidated but the biological activity of isolated compounds has hardly been investigated.Leaf extracts have betaadrenergic activity on the rabbit jejunum, an effect that was blocked by propranolol 20 , and anticestodal activity 21 .Bark acetone extracts of P. africanum had MIC values of 0.02 to 0.08 mg/m towards Staphylococcus aureus 17 in an unpublished M.Sc.thesis of a member of our group.These results further motivated this study.
Use of the bark and root are destructive practices that may lead to destruction of resources and even to plant extinction.Qualitative and quantitative investigation of the bioactive compounds present in the leaves, bark and root merits further study, to determine if there is a rationale in the traditional use of the plant by rural farmers and whether leaves may not be used.Suitable methods of extraction of bioactive compounds, adapted to resources available in rural communities, could be developed for sustainable use of P. africanum extracts in primary health care practices.

Collection, preparation and storage of plant material
Leaves, stem bark and root bark (referred to as leaf, bark and root in this article) were (ethanol extract of the root) to 0.01 (hexane extract of the leaf).The total antibacterial activity (volume to which active compounds present in 1 g plant material can be diluted and still inhibit bacterial growth) was 1263 m /g for ethanol extract of the root against S. aureus , and 800 m /g for acetone extract of the root against P. aeruginosa.There was substantial activity against both Gram-positive and Gram-negative bacteria, with MIC values of 0.08 mg/m for S. aureus and 0.16 mg/m for P. aeruginosa.There is therefore a rationale for the traditional use of root and bark of P. africanum in treating bacterial infection related diseases.
collected in May 2003 from a mature tree growing naturally on the campus of the Faculty of Veterinary Sciences at Onderstepoort.A voucher specimen (PM 001) is stored in the Medicinal Plant Herbarium, Department of Paraclinical Sciences, University of Pretoria.The plant material was dried in the shade at ambient temperature and ground to fine powder in a Macsalab mill (Model 200 LAB), Eriez ® , Bramley.The powdered material was stored separately in the dark in tightly closed glass bottles before analysis.

Extraction
Four solvents with varying polarities 13 were selected to extract a diversity of compounds from the plant material i.e. technical grade ethanol, acetone, dichloromethane and hexane (Merck ® ).One gram of the leaf, bark and root was extracted in 10 m of the respective solvent in a centrifuge tube by vigorously shaking on an orbital shaker (Labotec ® , model 202, South Africa) for 10 minutes.After centrifugation for 10 minutes, the extract was decanted into pre-weighed glass vials.The process was repeated 3 times.The solvent was evaporated in a stream of air at room temperature overnight to determine the mass extracted 5 .
Separated compounds were inspected and marked under visible and ultraviolet light (255 and 360 nm Camac Universal UV lamp).The TLC plates were subsequently sprayed with vanillin spray reagent [0.1 g vanillin (Sigma) in 28 m methanol to which 1 m of concentrated sulphuric acid was carefully added].Corresponding plates were sprayed with p-anisaldehyde spraying agent [1 m p-anisaldehyde (Sigma) in 18 m ethanol to which 1 m concentrated sulphuric acid was carefully added].Thereafter, the plates were heated at 100 °C for a few minutes for optimal colour development.

Polyphenols
Polyphenols were quantified by the Folin-Ciocalteu method, with gallic acid as a standard 27 .

Antioxidant screening
Reaction of 1,1-diphenyl-2-picryl hydrazyl (DPPH), a purple stable free radical with an antioxidant results in decolouration of the free radical.Chromatograms were uniformly sprayed with 0.2 % DPPH in methanol.Antioxidant activity is detected on the chromatogram when the initially purple DPPH background turns yellow in bands where an antioxidant is present 29 .
Quantification of antioxidant activity was by the Trolox equivalent antioxidant capacity (TEAC) assay.The decolourising assay method 26 was used with minor modifications.Free radical was preformed by reacting potassium persulphate (Sigma) with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (Sigma) and was stored at 4 °C before use.The free radical was diluted with absolute ethanol (Merck) to an absorbance of 0.7 ± 0.02 at 734 nm (Beckman).Trolox (6-hydroxy-2, 5,7,8-tetramethylchromane-2-carboxylic acid) (Fluka) is a synthetic water soluble vitamin E analogue.It was prepared fresh in ethanol solution and used as the antioxidant standard.Initially, 4 different concentrations of the Trolox and extracts were made.One m of ABTS was added to 10 µ (in quadruplicate) of each concentration of Trolox and extracts.Only the solutions that gave absorbency of between 20 % and 80 % of the initial absorbency were considered.The TEAC values were measured after 6 minutes.If an extract had equivalent antioxidant activity to Trolox, its TEAC value would be 1 and if the extract was more active its TEAC value would be greater than 1.Re et al. 26 used equivalent mM concentrations of different standards to obtain their results.With unknown compounds in a plant extract this is not possible and we used equivalent mg/m concentrations.By bringing the molecular mass into consideration, we can convert the published data 26 to data comparable with our results.

Antibacterial screening
Acetone was used to dissolve dried extracts to 10 mg/m because of the low toxicity of acetone for the test organisms 5 .A modification of the bioautography procedure described by Begue and Kline 3 was used.TLC plates were sprayed with concentrated broth culture suspensions of actively growing cells of S. aureus (ATCC 29213), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Enterococcus faecalis (ATCC 29219).These are the strains recommended by the National Committee for Clinical Laboratory Standards 22 .Cultures grown overnight were centrifuged and the pellet was resuspended with fresh broth in about 50 % of the original volume to yield a high concentration of the bacteria.The number of bacterial cells in the concentrated bacterial suspensions used for spraying chromatograms cultures was determined by a modified serial dilution technique 16 .The values ranged from 28-50 × 10 16 colony-forming units per m (cfu/m ).The plates were incubated at 38 °C and 100 % relative humidity overnight.The plates were then sprayed with 2 mg/m p-iodonitrotetrazolium (INT).Clear zones on the chromatogram indicated inhibition of bacterial growth after further incubation at 37 °C for 30-120 minutes.
A microplate serial dilution method 6 was used to determine the minimum inhibitory concentrations (MIC) for the test bacteria, i.e. the lowest concentrations of the extract that inhibit bacterial growth.INT was used as growth indicator dye and gentamicin (50 µg/m ) was the positive control.
Total antibacterial activity (TAA) equivalent (m /g) of the plant parts was determined by dividing the quantity of material (mg) extracted from 1 g of dried plant material by the MIC value (mg/m ) 8 .The TAA value is the volume in m to which the extract obtained from 1 g of plant material can be diluted and still inhibits bacterial growth 8 .

RESULTS
Whereas the non-polar extractants extracted a small quantity of compounds from root and bark, ethanol and acetone extracted the largest quantity in total from all plant parts (Fig. 1).There was hardly any extract obtained at the 3rd extraction with hexane and dichloromethane, and only a small quantity with acetone and ethanol, indicating that extraction was exhaustive.
Polyphenol concentrations were high (more than 20 %) in the acetone and ethanol extracts of the root, bark and leaves (Fig. 2).
There were 5-6 antioxidant compounds present in acetone and ethanol extracts of especially root and bark, and the highest antioxidant activity was in the ethanol and acetone extracts of the root and the ethanol extract of the bark (Figs 3, 4).In all the acetone and ethanol extracts, a compound with an Rf value of c. 0.48 (similar to that of catechin) in the EMW TLC system was present.
The antibacterial compounds in root and bark were highly polar and hardly moved from the origin when EMW was used with S. aureus as test organism (results not shown).In all leaf extracts there were 2 or 3 non-polar antibacterial compounds present with Rf values of 0.74, 0.85 and 0.92 (EMW system).These values corresponded to some of the Rf values of antioxidant compounds present in root, bark and leaves (Fig. 3).
The minimum inhibitory concentration (MIC) values of the extracts varied from 0.08 to 2.5 mg/m (Table 1).There was substantial activity against both Grampositive and Gram-negative bacteria, with an MIC value 0.08 mg/m for S. aureus and 0.16 mg/m for P. aeruginosa.The ethanol extract of the root had the same MIC value (0.08 mg/m ) as the dichloromethane extract of the leaf against S. aureus.E. coli was the most resistant against the extracts.
The total antibacterial activity values obtained for the ethanol and acetone extracts of the root and barks were high, varying from 79 to 1263 m /g (Table 2).

DISCUSSION
Pharmacologically active plant-derived compounds have been discovered by following the ethnobotanical use of plants 11 .The ability to isolate compounds depends on the ability to screen using the 'seek and ye shall find' as the operative principle 4 .Use of organic solvents is the most popular method for obtaining plant extracts 13 .There was hardly any extract obtained at the 3rd extraction with hexane and dichloromethane, and only a small quantity with acetone and ethanol.This would imply that the ground particles were small and that the shaking and separation of phases by centrifugation was efficient.Acetone and ethanol extracted the largest quantity in total, from all plant parts.Acetone has already been shown to be a good extractant for a diversity of plant compounds 5 .Extractable compounds from the root and bark were mainly polar, as few compounds were extracted from the root and bark by non-polar solvents.This indicates that P. africanum does not have a high concentration of non-polar compounds in the bark and root.The TLC and bioautography results support this conclusion, because root and bark extracts had low Rf values even in polar solvent systems.
Our results show that P. africanum had several antioxidant compounds especially in the root and bark (Fig. 3).Antioxidants in plants prevent damage caused by free radicals to DNA and other molecules, reduce inflammation, and promote good health and resistance to disease 15,18,25 .In the qualitative antioxidant analysis most of the compounds had a high polarity as would be expected of polyphenols or tannins.The antioxidant activity in P. africanum may be due to polyphenols or tannins as these compounds have been found in its heartwood 1,2,14,19 .The Folin-Ciocalteu method does not differentiate between tannins and many other phenolics that are not tannins 27 .
The TEAC values of 1.32, 0.95 and 0.83 for the ethanol and acetone extracts of the root and acetone extracts of the bark respectively, compare favourably with the value of 1.49 for pure Vitamin C found by Re et al. 26 .
Manana 17 found that the acetone extracts of P. africanum bark had MIC values of 0.02-0.08mg/m against S. aureus.Obi   et al. 23 using disc diffusion method demonstrated antibacterial action of P. africanum extracts against some Gramnegative bacteria (not done for Grampositive).In the present work, we found the best MIC values of 0.08 mg/m were obtained for ethanol extracts of the root and dichloromethane extracts of the leaf against S. aureus.The best values for Gram-negative bacteria were 0.16 mg/m for acetone and ethanol extracts of the bark and root against P. aeruginosa.As Eloff 8 pointed out, when selecting plants for investigation in rural areas, not only MIC values but also the quantity extracted should be incorporated to calculate the total antibacterial activity.Whereas the MIC values for ethanol extract of the root and dichloromethane extract of the leaf against S. aureus were the same (0.08 mg/m ) , extracts from 1 g of plant material could have been diluted to 1263 m in the former and 188 m in the latter and still inhibit growth of S. aureus.This in effect would imply that much less plant material would have to be extracted by acetone compared with the amount of plant material that would be required by extraction with dichloromethane to achieve the same antibacterial activity.If 2 different plant species have different TAA values, this information helps to select the best plant for isolating active compounds and for application in rural areas.The TAA values of 1263 and 800 m /g for ethanol and acetone extracts of the root, respectively, are much higher than those obtained to date from the members of Combretaceae and Celastraceae 7,9 .
The high level of antibacterial and antioxidant activity in acetone and ethanol extracts validates some of the traditional uses of P. africanum in people and animals 12,17,30,34 .The results provide a rationale for the use of the plant in treating bacterial infection-related diseases in cattle by rural farmers in the Madikwe area of South Africa 30 .
The antibacterial activity in the roots and bark are probably largely due to tannins 33 , based on the high polyphenol content of these extracts and the high polarity of the active compounds based on bioautography results.The effect on animal infections may either be directly by inhibiting growth of pathogens or indirectly by stimulating the immune system of the host.The high polarity of the bioactive compounds in the root and bark also means that these compounds may be extractable by the polar solvents available to rural users.
We are in the process of isolating and characterizing the antioxidant and antibacterial compounds from roots and leaves and then testing extracts of crude extracts and isolated compounds on animals to confirm the in vitro results.

Table 2 :
Total antibacterial activity (volume to which active compounds present in 1 g of plant material can be diluted and still inhibit growth) values (m /g) of ethanol (E), acetone (A), dichloromethane (D) and hexane (H) extracts of the bark (B), root (R) and leaf (L) tested on 4 bacteria.