Analytical studies on the most biologically active extracts of Pterocarpus erinaceus and Daniellia oliveri

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CAPITURE of the most relevant plants based on calculated values

Equipped with the various parameters and values calculated as described above, we have then developed reliable selection criteria to rank the 43 plants put forward by the traditional healers and to select the most promising ones, without, of course, ruling out any of the others (Figure 1.2).
After the RU and the UV for all plants were calculated (the Frequency of Citation or the Fidelity level could also be used instead of the RU), a long-list list of likely candidates was obtained. A cut-off based on an RU (≥2) or a UV (≥ 0.0377) was applied to obtain a short list.
Subsequently, a bibliographic review on the plants with the highest UV (≥ 0.0377) or RU (≥2) was conducted. This resulted in a final list of plants which were (a) frequently used in the Tchamba District to treat (various) fungal diseases, (b) were independently mentioned as medicinal plants in the literature and (c) where still of sufficient novelty to warrant further investigation.
For these plants, we have two alternatives:
– firstly, we can compute the following specific indices in this order: PPV, SU and IUV. Here, the PPV leads us to the most interesting plant part(s) or organs to treat fungal diseases. The SU points to plant parts specifically used to treat one fungal disease and the IUV, as already mentioned, refines the comparison further by comparing the SU of different parts of the same plant. The most interesting plants will be the ones with the highest SU and IUV.
– secondly, we may compute the ICF, SU and IUV. Here, the ICF needs to be calculated for the different types of fungal diseases reported by the TH (Table 3) as a first step. The second step is to focus on fungal diseases that have the highest ICF. And in the third step, we can compute the SU and the IUV for the plant parts (from the short list) recorded to be used for the treatment of the diseases with the highest ICF. And here again, the most interesting plants will be the ones which exhibit the highest SU and therefore IUV.
This method is preferably employed as an ethnobiological recording and pre-screening exercise before more eloquent and expensive analytical and activity screening methods are unleashed.
The use value (UV) and the reported use (RU) were computed for all 43 plant species identified as part of the survey. Eventually, Pterocarpus erinaceus (UV = 0.28), Daniellia oliveri (UV = 0.11), Ficus virosa (UV = 0.05) and Paullinia pinnata (UV = 0.05) yield the highest values among the 43 species and, based on these values, it appears that they are the preferred plants administered by traditional healers to treat fungal diseases (Table 1.1, see also Table 1 in Appendix where the information on the plants recorded is provided). Since the UV values differ considerably, a cut-off of UV at 0.04 was implemented to consider those plants with an UV ≥ 0.04. This gives a short-list of plants namely Allium sativum, Anacardium occidentale, Calotropis procera, Cochlospermum planchonii, Quisqualis indica, Ricinus communis, Desmodium gangeticum, Flueggea virosa, Daniellia oliveri, Pterocarpus erinaceus, Xeroderris stuhlmannii, Milicia excels, Musa sapientum, Piper guineense, Zea mays, Paullinia pinnata, Nicotiana tabacum and Vitex doniana. The bibliographic survey was then performed on plants from the short list (See Table 2 in Appendix, the table provides information gathered during the review exercise).
Surprisingly, this survey revealed that virtually none of the plant species identified have been previously studied in vivo for antifungal activity (except P. erinaceus) (Etuk et al., 2008). Furthermore, the underlying chemical composition and activity of the materials so far is also not well documented. It also appears that none of them, with the notable exceptions of P. erinaceus and Ricinus communis, have been cited previously in other ethnopharmacological studies (Etuk et al., 2008; Manpreet et al., 2012). In contrast, plants such as Allium sativum (the common garlic), Calotropis procera and Anacardium occidentale which have been previously studied extensively did not score highly in the CAPITURE analysis (Khan et al., 2000; Sumbul et al., 2004; de Freitas et al., 2011; Goyal et al., 2013). Running this review has helped to identify a final list of plants as promising candidates and as the most original plants to be analyzed and studied further considering the feeble data of previous biological or chemical studies on them. The plants are P. erinaceus, D. oliveri, Ficus virosa and P. pinnata which also represent the ones mostly used by traditional healers as revealed by the survey. The two options described in the CAPITURE method was then applied to those four plants.
In the case of the first option, after the first two steps above, namely the calculation of Usage Values (UV) and a literature survey for activity and originality, the third step involved the calculation of the more specific and informative PPV, SU and IUV parameters for the plants on the final list. The results obtained for the PPV are summarized in Table 1.2. In the case of P. erinaceus, the bark (0.4) and the sap (0.53) represent the parts most frequently employed by TH to treat fungal infections. Similarly, in the case of D. oliveri, the sap (0.71) and bark (0.28) are most frequently used, whilst for F. virosa and P. pinnata, the roots are the only parts utilized (Table 1.2).
To address the question which particular fungal infections to consider, the SU and IUV were computed. As this calculation places a very narrow focus, i.e. on a specific plant, its specific part(s) and specific infections, it provides highly refined information yet in some cases also fails because of the limited number of records (43) or incomplete or unspecific records available. For instance, a reasonable calculation has not been possible for the roots of F. virosa and P. pinnata because they were not used against a specific fungal disease. The results obtained for the other plant parts are shown in Table 1.3. It becomes immediately apparent that the sap of P. erinaceus is frequently used in the treatment of ringworm, whilst the sap of D. oliveri is utilized in the context of intertrigo.
The other alternative in the CAPITURE method is to compute the ICF which revealed that ringworm (0.59), intertrigo (0.57) and candidiasis (0.41) are the fungal diseases with the highest ICF (Table 1.4). The high value of ICF means that most of the TH agree on a set of plants on the short list, which they use preferably to treat ringworm, intertrigo and candidiasis. Consequently, the SU and IUV of those plants were computed just focusing on their use in the treatment of the two ailments. This way, the results presented in Table 1.3 was obtained.
Eventually, the present study was able to indicate which parts of the plants are used for the treatment of which specific fungal infections. This has culminated in the understanding that 27 the sap of P. erinaceus is the most effective plant material employed against ringworm, whilst the roots of this plant seem to be particularly effective against candidiasis. The sap of D. oliveri appears to be effective against intertrigo, and the roots of this tree bear promise in the treatment of candidiasis.
The identification of these 2 candidates, extracted from 53 testimonies provided by TH from the Tchamba District and narrowed down from 43 individual plant species mentioned in this survey, is the final outcome of the CAPITURE pre-selection exercise. These plants (described in the following lines) materials have therefore been collected and studied in considerably more detail in antimicrobial assays indicative of the relevant biological activity (to be presented in the second chapter).

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Description of Pterocarpus erinaceus Poir

The Greek-Latin name ‘Pterocarpus’ comes from the Greek words pteron (wing in English) and karpos (fruit in English) (referring to the winged membrane that surrounds the fruit of these species) and the Latin word erinaceus means Hedgehog in English (in comparison to the central part of the seed covered with stiff hairs crossed in all directions, like the thorns of the hedgehog). The plant is named Boutô in Tchamba and Barwood, Muninga or vène in English. It is a small tree, 10 to 15 m of height. Its leaves are imparipinnate, alternate and distilled (Figure 1.3). The leaves have a rachis of 15-25 cm long with 4 to 5 pairs of leaflets alternate or sub-opposed. The leaflets are elliptic, 5 to 10 cm long, 3 to 6 cm wide, with a rounded base or short cuneiform, generally emarginated. The yellow flowers are numerous, loose and short panicles. Flowering occurs in March-April, before the foliage. The corolla is short, 10-12 mm. The calyx is pubescent, 5 mm long, with short and obtuse triangular teeth. The fruits are winged, suborbicular, broad from 4 to 6 cm and have in the center, above the seed, numerous rigid, spiny bristles, crossing each other in all directions (Berhaut J, 1976; Tittikpina, 2012).

Table of contents :

Chapter 1: Ethnobotanical study of plants used in the Tchamba district of Togo to treat infectious diseases
1. Introduction
2. Selection of a suitable area for an ethnobotanical survey: the Tchamba District
3. Recording of traditional knowledge and data collection
3.1. Turning testimonies into data
3.2. CAPITURE of the most relevant plants based on calculated values
4. Results
Description of Pterocarpus erinaceus Poir
Description of Daniellia oliveri (Rolfe) Hutch. et Dalz
5. Discussion
6. Conclusions
Chapter 2: Biological activities of Pterocarpus erinaceus and Daniellia oliveri
1. Introduction
2. Collection and preparation of plant material
3. Extraction and fractionation
3.1. Check-up
3.2. Extraction
3.3. Fractionation
4. Bio-assays
4.1. Antibacterial and antifungal tests
4.2. Anti-cancer assay
4.3. Tests against nematodes
4.4. Tests for cytotoxicity
5. Results
5.1. Antibacterial tests
5.2. Anti-fungal tests
5.3. Anti-cancer tests
5.4. Tests against nematodes
5.5. Cytotoxicity tests
6. Discussion
6.1. Antibacterial and cytotoxicity tests
6.2. Antifungal and cytotoxicity results
6.3. Anti-cancer and cytotoxicity tests
6.4. Nematicidal activities
6.5. Cytotoxicity tests
7. Conclusions
Chapter 3: Analytical studies on the most biologically active extracts of Pterocarpus erinaceus and Daniellia oliveri
1. Introduction
2. Extraction
2.1. Maceration
2.3. Other types of conventional methods that could be easily run in developing countries
2.4. Summarised presentation of some of the modern methods of extraction
3. Clean-up: fractionation
4. Qualitative analysis of the cleaned-up extracts
4.1. Gas chromatography (GC)
4.2. Liquid chromatography (LC)
4.3. Mass spectrometry
4.4. GC-MS on the apolar fractions obtained from the parts of P. erinaceus
4.4.4. GC-MS analysis of the apolar fractions obtained from the parts of D. oliveri
4.5. LC-MS on the polar fractions obtained from the trunk barks of P. erinaceus
5. Purification
5.1. Definition and methods
5.2. Purification performed on the roots of Pterocarpus erinaceus
5.3. Purification performed on the trunk barks of P. erinaceus
6. Qualitative analysis of the isolated compounds
6.1. Nuclear Magnetic Resonance
6.2. Mass spectrometry
6.3. Ultraviolet and infrared absorbance
6.4. Melting point
6.5. Results of the purification process run on the roots of P. erinaceus
6.6. Results of the purification process run on the trunk barks of P. erinaceus
7. Discussion
7.1. Pterocarpus erinaceus
7.2. Daniellia oliveri
8. Conclusions
Chapter 4: Nanoparticles of plant powders
1. Introduction
2. Material and methods
2.1. Plant material
2.2. Biological activity against nematode (Steinernema feltiae), bacterium Escherichia coli) and yeast (Saccharomyces cerevisiae)
3. Results
3.1. Homogenized particles of P. erinaceus barks
3.2. Biological Activity of P. erinaceus barks nanomaterial and respective against E. coli and S. feltiae
3.3. Biological Activity of P. erinaceus barks nanomaterial and respective Extracts against E.
4. Discussion
5. Conclusions

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