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Follow-up of in vitro activity hit status in corresponding animal models lead status. A variety of test systems should be employed in the in vitro screens because the use of only one bioassay yields an incomplete picture of the effect of the extract on the whole system involved Houghton etal. Many diseases involve more than one factor, and Houghton etal. An assortment of chemicals is present in an extract, and these may each have a different biological or pharmacological activity, together contributing to the overall clinical effect Houghton etal.

It is therefore preferable to use a range of tests for different activities, all related to the particular disease state under investigation. Pharmacological evaluation of medicinal plants followed by bioassay-guided fractionation can lead to the isolation of pure active compounds with potential for commercialization.

An alternative to this route, particularly pertinent to developing countries, is the preparation of standardized, formulated extracts that could contribute to an innovative and successful local pharmaceutical industry Pieters and Vlietinck, A growing number of publications document the use of herbal remedies by smallscale farmers to treat an assortment of livestock diseases, from skin conditions to babesiosis and anaplasmosis Masika, Sonandi, and Van Averbeke, ; Masika, Van Averbeke, and Sonandi, ; Dold and Cocks, ; Van der Merwe, Swan, and Botha, ; Njoroge and Bussmann, ; Luseba and Van der Merwe, Methods of obtaining information range from participatory methods to semistructured interviews, field observations, and questionnaire surveys.

This expanding documentation of plants used in EVM is anticipated to precede increased investigation of these plants for bioactivity, reflecting the situation in human ethnomedicine.

It is generally accepted that a great deal of work remains to be done on recording the uses of plants in EVM. Factors to be taken into consideration by researchers aiming to evaluate the biological activity of herbal EVM remedies include whether the plants are used singly or in combination with other plants, the plant part used, method of preparation, dosage, and the way in which the remedy is applied.

For example, herbal remedies can be prepared from fresh or dry material in the form of infusions, decoctions, pastes, or expressed juices from fresh plants.

Masika, Van Averbeke, and Sonandi stated that the route and method of application of a remedy depends on the perceived cause of the disease condition. Topical applications are commonly used for skin conditions, powders are rubbed into incisions, drops are placed in the ears and eyes, and drenches are popular in treating systemic conditions. In other studies in South Africa, it was noted that plants are generally not processed or mixed with other materials and.

This is in contrast with traditional medicine intended for human use, for which processing milling, extracting, etc. Diagnosis of a disease made by a rural farmer may be inadequate as it is easier to identify symptoms than the actual cause of the disease. Dosages are not precise, contributing to the perception that the remedies are not standardized.

The methodology for validating EVM should be scientifically acceptable but also must take into account the probability that EVM remedies might not work as powerfully as orthodox medicines.

They may not completely eliminate all microorganisms causing a particular disease, but this could allow the bodys immune system to build up immunity against the remaining organisms. It must be kept in mind that ethnoveterinary practices comprise a complex system and isolating one aspect for efficacy studies may not yield the anticipated results, although the system might be adequate for the conditions in the field.

Fundamentally, if a plant-based ethnoveterinary remedy is to be deemed suitable for further development, efficacy and toxicity tests must meet certain standards. Legal constraints must be kept in mind if commercial development is anticipated.

However, this standard is not so rigorous if the validation and understanding of EVM is the aim of the study. Research can be used to select a particular remedy, improve it through pharmacological and toxicological research, and then return it as a valueadded product to the community. In EVM studies, traditional methods of dosage and preparation of remedies are vital components often neglected for the sake of ease of preparation and standardization of laboratory extracts for testing.

For a more true reflection of efficacy of the treatment, it would be advisable to closely follow if possible the preparation and application technique employed by the indigenous user. On the other hand, there is a case to be made for using standard scientific methods to prepare aqueous and organic solvent extracts if extraction of a wide range of chemicals present in the plant is desired for in vitro studies.

Traditional remedies may be composed of single plants or mixtures of plants, and if mixtures are employed it is recommended to test not only the individual component plants, but also the mixture in the correct proportions used traditionally.

Repeatability of activity is important, and together with well-designed screening programs to elucidate activity using complementary techniques, plant material sourced from different areas could be included in a screening program. The accurate identification of plant material is naturally essential, and numbered voucher specimens should be deposited at a reputable herbarium to allow other researchers to verify the identity of the plants as well as to make allowances for possible future taxonomic revisions.

A further limitation in the laboratory testing of EVM remedies is the difficulty in culturing parasitic nematodes and protozoa, for example. Parasitic infestations of intestinal worms are commonly diagnosed and treated by livestock keepers. Some model systems are available, such as the free-living nematode Caenorhabditis elegans, for detecting anthelmintic activity, but sometimes the only true indication of efficacy is activity in an animal model.

It is often not possible to assume efficacy in vivo after achieving good results with in vitro tests. First, it may not be practical to extrapolate the dose from that which is active in vitro to that which would be required to reach adequate plasma concentrations in the target species Houghton etal.

Bioavailability is an essential consideration if the remedy is orally administered. Second, factors such as absorption and metabolism may be responsible for discrepancies between in vitro and in vivo tests Houghton etal. Even if rat models, for example, are used, differences have been noted between intraperitoneal and oral administration.

This can be explained inter alia by metabolic breakdown of compounds in the gastrointestinal tract or by lack of absorption from the gut into the bloodstream Laupattarakasem etal. Absorption and metabolism can be affected by other compounds in the extract that may enhance or inhibit absorption, and other compounds may upregulate metabolic enzymes in the liver.

Houghton et al. Most activity investigations in the published scientific literature concentrate on in vitro studies for practical, economic, and ethical reasons. Therefore, there exists an unavoidable bias toward in vitro tests for evaluation of EVM remedies as this iswhat is reflected in the available literature.

Even in the case of in vivo studies, tests of ethnoveterinary remedies in a laboratory where the animals are given fixed diets and kept under controlled conditions, accurate indications of efficacy of the treatment may not be discovered, while in the real-life situation, conditions under which the animals are kept are more varied.

The reason for which the screening is being undertaken must be kept in mind when designing the study, including preparation of the extract for pharmacological testing. If the purpose is solely to validate the use of a certain remedy, then it is advisable to closely follow the traditional method of preparation as many factors may influence the activity of the resultant mixture.

In addition, the route of administration must somehow be taken into account. In some cases, it is thus possible that in vivo tests are the only mechanism by which the efficacy of an EVM medicine can be verified.

Alternatively, careful design of in vitro screening systems may yield a reasonable idea of the efficacy and nontoxicity of remedies. The selection of a screening system will for the most part depend on the nature of the disease being investigated. For broader screening programs aimed at discovering biological activity in particular plants used in EVM, standardized methods are widely used. Care must be taken to ensure that potentially active compounds are not lost during processing; for example, some constituents may be thermolabile or photosensitive.

Plant extracts may be prepared using fresh material or, more commonly, dried powdered material. The plant material can be extracted using water or organic solvents that vary in polarity. For extraction of hydrophilic compounds, polar solvents such as methanol, ethanol, and ethyl acetate can be used, while if lipophilic compounds are being targeted, more nonpolar solvents such as dichloromethane and hexane may be used. Eloff b examined a spectrum of solvents for their ability to extract antimicrobial compounds from plant material and other factors, including their hazardous nature and ease of removal from the extract.

The aim of the study was to identify a more standardized extraction method, and acetone was highlighted as the solvent with the best rating, followed in order by dichloromethane, methanol, ethanol, and water. However, this may vary with the plant species or plant part under investigation see also Chapter 4. Following extraction, appropriate handling of the extracts is important to avoid decomposition of active compounds or other changes that may affect biological activity.

It is common practice to resuspend dried extract residues in the extracting solvent to a known concentration prior to screening, provided the extract redissolves adequately in the solvent and the solvent is not toxic in the testing system.

In quantifying the extract, some researchers dry down only a small aliquot of the extract to determine the original concentration and then use the remaining intact extract for testing. Dimethyl sulfoxide DMSO is a popular solvent in which to prepare test compound solutions at a stock concentration Cos etal. Acetone was put forward as the solvent of choice for use in antibacterial testing systems as, at the concentrations used in a serial broth microdilution assay, it was found to be nontoxic to various species of bacteria tested Eloff, a.

This was also held to be the case for antifungal assays based on a similar method Masoko, Picard, and Eloff, Regarding storage issues, Cos etal. This is meant to reduce degradation of components. Storage in methylated solvents is not advised because of the possible formation of artifacts.

In the following sections of this chapter, examples of EVM plants that have been screened for biological activity, the methods used to screen them, and indications of activity discovered in the plants are given. Particular emphasis is placed on treatments for those diseases of importance in livestock.

Techniques available for bioassaying. It is commonly found that there is an overlap between veterinary and human medicine in many communities, but the emphasis here is on the former. Antibacterial assays may be broadly divided into agar diffusion, dilution, and bioautography methods.

In agar diffusion, a reservoir containing a known concentration of the test substance is brought into contact with an inoculated medium, and the diameter of the inhibition zone clear zone around the reservoir is measured after incubation. Before incubating, the compounds from the reservoir are commonly allowed to diffuse into the agar medium at a lower temperature for a few hours before inoculation with the test bacteria Cos etal.

The types of reservoirs used can be filter paper disks placed on top of the agar surface or wells punched into the agar, for example. Advantages of the system include small sample requirements and the ability to test up to six extracts per plate against one microorganism Hadacek and Greger, However, a major disadvantage is that this method is not suitable for testing nonpolar samples or samples that are unable to diffuse readily through the agar matrix Cos etal.

With dilution methods, the test sample is mixed with a medium liquid broth or solid agar inoculated with the test microorganism. Growth of the microorganism after incubation can then be monitored in various ways. In agar dilution methods, the minimum inhibitory concentration MIC is the lowest concentration of test compound able to inhibit visible microbial growth. In broth dilution methods, turbidity measured visually or spectrophotometrically and redox indicators commonly a tetrazolium salt, e.

The presence of cidal or static effects of a certain concentration of compound or extract can be determined using broth dilution methods Cos etal. Minimal bactericidal or fungicidal concentrations MBC or MFC, respectively can be detected by plating out samples at inhibitory concentrations onto agar and assessing growth static or no growth cidal after incubation. Dilution methods are useful in testing both polar and nonpolar extracts or compounds.

The microdilution assay, using various growth indicators, including tetrazolium salts, has been successfully used with fastgrowing species of mycobacteria, including Mycobacterium smegmatis, M. For quantifying antibacterial activity, Eloff proposed that the quantity of material extracted from 1 g of dried plant material be divided by the MIC value to give the total activity of the plant.

This measure, in milliliters per gram, indicates the largest volume to which 1 g of the extract containing active compounds can be diluted and still inhibit growth of the bacterial or fungal species under investigation and thus the potency of the extract. Bioautography is a valuable technique that localizes antibacterial or antifungal activity on a thin-layer chromatographic TLC plate.

Components of an extract are. A balance needs to be struck between allowing sufficient time prior to bioautography to pass for the eluting solvent to evaporate completely from the TLC plate, but not too much time for the exposed compounds separated on the TLC plate to decompose as a result of exposure to light and oxygen. In agar overlay bioautography Hamburger and Cordell, ; Rahalison etal.

In a popular method that avoids the difficulties associated with compounds not being able to diffuse into the agar medium from the TLC plate, a suspension of bacteria or fungi in liquid medium is sprayed onto the developed TLC plate.

This is termed direct bioautography Begue and Kline, After the plate is sprayed with a suspension of a tetrazolium salt such as INT the presence of clear zones of inhibition are visualized against a purple background to indicate microbial growth. Bioautography facilitates bioassay-guided fractionation for the isolation of antibacterial or antifungal compounds, but its use is restricted to those microorganisms that are able to grow rapidly on a TLC plate with the limited amount of nutrients available for growth in the medium that adheres to the surface of the TLC plate.

In this regard, using the technique for filamentous fungi is inappropriate. Selection of test bacterial species to use in a screening procedure is dependent on the purpose of the study.

For antifungal screening projects, representatives from the yeasts e. An inoculum size that is too low may give false positive results, while a too large inoculum could increase false negatives Cos etal.

When screened against a panel of 10 bacteria and 5 fungi, extracts of Combretum caffrum, Salix capensis, and Schotia latifolia showed good activity against all the Gram-positive bacteria and some antifungal activity Masika and Afolayan, Most of the extracts were not active against the Gramnegative bacterial species; interestingly, some water extracts actually promoted fungal growth Masika and Afolayan, This may have been due to nutritive sugars, which partition into the aqueous fraction.

The organisms used in this study were selected from those generally associated with infections or disease in humans and animals. Different concentrations of each plant extract were mixed with liquid agar at approximately 60C before being poured into Petri dishes. Solvent was allowed to evaporate overnight from the plates, and bacteria or fungi were inoculated.

It was concluded that the inhibition of growth of Gram-positive bacteria, the Gram-negative Enterobacter cloacae, and several fungal species by water extracts of the plants indicated possible broadspectrum antimicrobial effects of the plants, validating to a degree the traditional use of these plants Masika and Afolayan, Ethnoveterinary plants used to treat infectious diseases in cattle were screened in a broth microdilution assay for antibacterial activity McGaw, Van der Merwe, and Eloff, against the organisms recommended for antibacterial testing by the NCCLS Hexane, methanol, and water extracts were found to be most active against the Gram-positive E.

Gram-positive species are known to be more susceptible to antimicrobials than are Gram-negative bacteria owing to differences in the bacterial cell wall composition Vlietinck etal. Ziziphus mucronata Rhamnaceae demonstrated excellent antibacterial activity in the preliminary assay McGaw, Van der Merwe, and Eloff, , and the antibacterial compounds 2,3-dihydroxyl-upenoic acid and zizyberanalic acid were subsequently isolated from the leaves Moloto, The first compound was very active against Staphylococcus aureus, supporting claims of the efficacy of leaf pastes of Z.

Bizimenyera et al. The root and bark extracts are used by farmers to treat stomach ailments such as diarrhea and dysentery in cattle Bizimenyera etal. Rhizomes and roots of the popular ethnoveterinary plant Gunnera perpensa Gunneraceae are used to treat endometritis and retained placenta in cattle and women Hutchings etal.

Gunnera perpensa rhizome extracts showed only slight activity against several Gram-negative and Gram-positive bacterial species McGaw, Jger, and van Staden, ; McGaw etal. Drewes etal. Noteworthy antifungal activity in several Terminalia species Combretaceae was reported by Masoko, Picard, and Eloff against various morphological forms of fungi, including yeasts Candida albicans and Cryptococcus neoformans , molds Aspergillus fumigatus , and thermally dimorphic fungi Sporothrix schenckii.

These fungal species were carefully selected to represent a spectrum of clinical isolates of the most common and important disease-causing fungi in animals.

From extracts of Terminalia leaves prepared using several organic solvents, the acetone extracts were most active. Certain viruses cause cytopathic effects CPEs or form plaques in lawns of cells, facilitating detection of antiviral effects of a substance. Inhibition of viral replication can also be discovered by monitoring the presence of viral products, such as viral RNA, DNA, or polypeptides.

Virucidal substances inactivate the ability of a virus to be infective extracellularly and find application as broad-spectrum biocides. Antiviral agents are more interesting as candidates for clinical use because they may interfere with some aspect of viral biosynthesis Cos etal.

Vlietinck and Vanden Berghe supplied a useful outline of cell-based assays that can be used for antiviral or virucidal evaluation of pure compounds or plant extracts. Toxicity to the host cell system must be assessed as part of the antiviral investigation. It is essential to gain an indication of cytotoxicity of the test substance as, without this, results do not distinguish between antiviral effect and effect against the host cell system.

The choice of viruses to use in a screening panel should include representatives of DNA viruses and RNA viruses and could include criteria such as their ability to replicate in the same cell culture. In the Phytomedicine Program at the University of Pretoria, we have begun investigating antiviral activity of ethnoveterinary plants against feline herpesvirus type 1 FHV-1 as an enveloped virus relatively sensitive to environmental influences.

Plants with good activity in this preliminary screen are then assayed for activity against more resistant viruses, such as the lumpy skin disease virus. No reports could be found of EVM plants being tested for antiviral activity, although many publications reported on efficacy of ethnobotanically chosen plants against a number of different viruses e.

To screen for antiviral activity, variations on virucidal assays are available in the ethnopharmacological literature, and these mainly focus on inhibition of viral CPE or plaque inhibition. In the virucidal assays, monolayers of the appropriate host cell type are cultured in well microtiter plates.

Following this, the cells are examined using an inverted microscope for signs of damage. Alternatively, a tetrazolium salt or other color indicator of cell viability may be used to detect cytotoxicity compared to untreated cells.

In the antiviral test, serial plant extract dilutions are prepared as for the cytotoxicity assay, but virus is added to the cells. The cultures were incubated for an hour to allow adsorption of viral particles, after which L per well of plant extract dilutions were added to the wells. The plates were incubated for a certain period to allow development of CPEs, if any, and results compared to the controls.

A range of different viruses was used in this method. In an example of a plaque inhibition assay, Zhang et al. After incubating the plates for 1 h to allow adsorption of the virus, the inoculum was aspirated from the cells, and the cultures were overlaid with 0. After 3 days of incubation at 37C, the plates were fixed with formalin, stained with crystal violet, air dried, and the number of plaques counted.

The rhizome extract, prepared using acetone, of the popular ethnoveterinary medicinal plant Elephantorrhiza elephantina was shown to have in vitro anti babesial activity Naidoo etal.

In this test system, Babesia caballi cultures isolated from a horse were incubated in well culture plates with plant extracts at varying concentrations, together with uninfected blood.

Parasite growth inhibition was monitored initially by a change in the color of the culture medium, where inhibited cultures remained bright red while unaffected protozoal cultures turned a dark coffee color. Culture smears were then evaluated using light microscopy to determine the percentage of infected cells. The registered antibabesial drugs diminazene aceturate Berenil and imidocarb diproprionate Forray were included as positive controls.

Acetone extracts of Urginea sanguinea, Rhoicissus tridentata, and Aloe marlothii were not active in this assay Naidoo etal. Another important protozoal disease occurring in domestic livestock and chickens is coccidiosis, which results from infection with Eimeria or Isospora species.

Coccidiosis causes losses worth millions of U. The use of plants to combat coccidiosis is an emerging field of investigation as these remedies may function by mechanisms different from those of conventional therapeutic agents.

In one such study, four plant extracts with reported antioxidant activity were screened for their anticoccidial activity against an artificially induced mixed Eimeria infection in poultry Naidoo et al. Tulbaghia violacea significantly decreased the oocyst production in the birds, and it was concluded that antioxidant-rich plant extracts have potential benefits in treating and possibly preventing coccidial infections Naidoo etal.

The results for extracts of T. The antirickettsial activity of Elephantorrhiza elephantina and Aloe marlothii was evaluated using an in vitro Ehrlichia ruminantium culture system Naidoo, Chikoto, et al. Acetone extracts of the leaves were incubated with E. Elephantorrhiza elephantina and A.

The EC50 and EC90 values for oxytetracycline were 0. Naidoo, Chikoto, et al. Laboratory research on anthelmintic activity of plant extracts is constrained by the expense, ethical issues and time associated with performing in vivo trials, and the difficulties experienced in maintaining parasitic nematodes in culture systems in vitro. A free-living nematode, Caenorhabditis elegans, has been used as a model organism in broad screening studies as it is easier and cheaper than using parasitic nematodes Simpkin and Coles, ; Rasoanaivo and Ratsimamanga-Urverg, Notwithstanding the limitations encountered in extrapolating activity against a freeliving nematode to activity against a parasitic species Geary and Thompson, , most commercially available broad-spectrum anthelmintics demonstrate activity against C.

In vitro screening investigations have revealed that many plant extracts show activity against the free-living C. A rapid inhibition assay is easy and simple to perform and entails incubating varying concentrations of plant extracts with nematodes for a defined period of 2 h and scoring the percentage of paralyzed or dead nematodes in comparison to the untreated control Rasoanaivo and Ratsimamanga-Urverg, Using this assay, several plant species belonging to the family Combretaceae exhibited interesting anthelmintic activity against C.

These studies may constitute a first step in validating the use of these plants in treating worm infestations in animals and in humans. In a more complicated screening system that evaluates the ability of the nematodes to grow and reproduce, plant extracts are incubated with nematodes in appropriate culture medium with bacterial and fungal growth inhibitors in well assay plates for 7 days, after which the percentage of surviving nematodes is.

Plant extracts have also been tested using in vitro assays with parasitic nematode eggs and larvae. Egg hatch and larval development inhibition against the two most important livestock nematode parasites Haemonchus contortus and Trichostronglyus colubriformis by various plant extracts have been reported. In these assays, the nematodes are maintained in monospecifically infected lambs, and eggs are collected from the feces.

Test substances incubated with the freshly collected eggs may inhibit hatching in the aptly termed egg hatch assay Coles etal. The larval development assay Coles etal. The combination of the two assays can provide a practical indication of anthelmintic activity of plant extracts or pure compounds isolated from the extracts.

Peltophorum africanum is a popular plant for use in treating helminthosis, and the acetone extracts of the leaf, bark, and root have been screened for activity against H. The extracts all showed activity in the assays at a concentration of 0.

Further confirmation of nontoxicity and efficacy is required, particularly in vivo. Various animal models have been used to detect anthelmintic effects of plant extracts Kahiya, Mukaratirwa, and Thamsborg, ; Iqbal etal. Research has been undertaken on the repellent and toxic effects of plant extracts against ticks, with promising results thus far. Nchu analyzed the repellent effects of extracts of Allium species, as well as the direct toxicity, against adults of Hyalomma marginatum rufipes.

Acetone extracts of A. Lippia javanica and Tagetes minuta essential oils had a concentration-dependent effect on the ticks Nchu, , and T.

Thembo showed that Senna italica ssp. When S. Take nothing but photograph, leave nothing but footprints. Services We are moving towards online services. Mount Kinabalu Hostel Booking. Adoption Programme. Turtle Nest Adoption Programme. Sabah Parks e-Library. Subscribe to our Newsletter. From the arch at the park entrance, proceed along a wide undulating ridge trail. The trail attractions include scenic views of the west coast and an assortment of local tree species.

Chances of spotting a pitcher plant on this trail is high, so do watch out of them. As one of the shortest routes at the park, it only takes a few minutes to reach the sheltered viewpoint from nearby the Kinabalu Conservation Centre. Is a broad well graded trail offering refreshing views.

This trail may be a mere metres, but do not underestimate it as it is quite steep, crisscrossing into the Kiau View Trail. It also offers opportunities to see birds, pitcher plants and lots of squirrels. Good view of Park Headquarters and Liwagu Valley. Splendid view of Park Headquarters and tree canopy area. What are my options for climbing Mount Kinabalu? When looking for a climb on Mount Kinabalu, you can opt to go with the classic, 2-day climb to the summit and then go back down or you can choose to combine your trek with some other activity.

Mount Kinabalu and Borneo have a wide range of options to spice up your Kota Kinabalu trek. Here you will find all our offers for Mount Kinabalu. There are several combinations when it comes to climbing Mount Kinabalu. If you like going up the mountain and then taking a well-deserved rest, then a good option for you would be to combine your Kota Kinabalu trek with a relaxing bath at the Poring Hotsprings.

For those adventurous trekkers, the climb to the top can be combined with a white-river rafting expedition or, if you prefer hights and adrenaline, you can add one of the breathtaking Via Ferrata trails to your climb. Here you can find all the offers we have for these combinations.

Via Ferrata on Mount Kinabalu, what is it? This road starts at 3, meters and ends at 3, meters above sea level. However, no climbing experience is required for any of the two. Here you will find all our offers including the Via Ferrata trails. Are you brave enough? What does a typical trek to the summit of Mount Kinabalu look like? When it comes to climbing Mount Kota Kinabalu there are several options. The classic trek to the top takes 2 days. Luckily for those planning on trekking in Malasia, you can pretty much visit Sabah at any time of year.

Bear in mind that June to September can be hot. For those interested in climbing Mount Kinabalu, however, we recommend doing it during the dry season, in March and April, when you have higher chances of a clearer view. Anyhow, rain can occur at any time of the year.

The least recommended months are November and December because of the monsoon season. Although the summit can be attempted at any time of the year, if the weather does not permit, Sabah Parks Authority will close the gate to the summit for safety reasons. Choose the time of the year that suits you best and find the perfect trek for you here!

How fit do I need to be to climb Kota Kinabalu?