Phytochemical and Anti-Termite Efficiency Study of Guibourtia tessmanii (harms) J. Léonard (Kévazingo) Bark Extracts from Gabon

The handling and harvesting of the colony of wild termites “Cubitermes sp.” of the genus Isoptera was carried out on the day of the experiments so that the withdrawal from their natural habitat had no significant influence on their mortality. For the anti-termite tests, the worker termites were put in a small empty butter pot with the clods of soil and its perforated lid using a heated needle. The termites were immediately brought to the laboratory of the Iphamétra (Pharmacopoeia Institute of Traditional Medicine) in Libreville (Gabon) for testing.

The bark sampling phase was carried out according to a previously established procedure. The mature Kévazingo bark was collected using a machete on April 8, 2020. The collection of Kévazingo bark in Kango, in the Estuary province (N 00°02’44.5” and E 010°17’23.7”) of size 10 to 15 cm was carried out on two heights on the same tree: at the level of the buttress and six meters from the ground. Because in general, the content of secondary metabolites, also called extractable, decreases with height in the tree (Caron et al., 2013). The bark was cut into small pieces using a pair of scissors. They were then sent to the Multidisciplinary Science Laboratory (LaPluS) of the Ecole Normale Supérieure de Libreville where they were dried in the open air for three weeks.

The extraction of the chemical elements began with the fragmentation and grinding of the bark. The bark was first cut with scissors to facilitate crushing; the fragments obtained were then introduced in small quantities into a Retsch-type mill, with Iphametra. This is necessary to obtain a suitable grain size with a fraction between 1 and 2 mm, thus corresponding to the requirements of ASTM No. 1105 (1996) in terms of grain size to quantify the rate of wood extract. Finally, the powder collected was sieved and stored in the dark in glass jars, closed until the time of chemical tests. The chemical tests began with the determination of the extract levels. We opted for the successive extraction by cold maceration of the bark powders using solvents of increasing polarities in the order of Trichloroethylene, Acetone, ethanol, and Distilled water. The bark powder was placed in a 1/10 ratio (10 g of dry matter in 100 mL of solvent) in a 250 mL glass Erlenmeyer flask, closed with a rubber stopper covered with aluminum foil. The aluminum thus prevents the solvent from coming into contact with the rubber, which prevents possible contamination. In addition, the Erlenmeyer flasks were covered with aluminum foil to prevent degradation of photosensitive molecules. The mixtures were subsequent placed under stirring on a stirrer (of the PIERRON type) for 24 h. After 24 h, the mixtures were separated by vacuum filtration through a Whatman filter in a Buchner type funnel. With regard to the extraction in aqueous medium, the water is evaporated by lyophilization. The extract solutions were then evaporated in flasks (weighed beforehand) using a rotary evaporator, in a 40°C. bath and were then placed in a desiccator until constant mass and weighed again. The extracts are then stored in the refrigerator in closed bottles and covered with aluminum foil for the next tests. We can thus calculate the percentage of extracts relative to the initial mass of bark powder using the following Equation (1):

Where:

R: yield of extracts in%

M_ext: mass of the extract after evaporation in grams

M_ech : anhydrous mass of the bark powder sample in grams.

The reagents used to carry out the phytochemical screening of the extracts were prepared and used according to the protocols described by (Akinjogunla et al., 2010; Badiaga, 2012; Houghton and Raman, 1998). All the different tests were carried out in triplicate.

The protocol described for the anti-termite tests was inspired by those described by (Bédounguidzi et al., 2020; Bopenga Bopenga et al., 2020a, 2020b). Only the acetone, ethanolic and aqueous extracts were tested. Two concentrations were tested against wild termites for the screening tests, mass ratios in percentage of (50:50) and (25:75) (extract: extraction solvent). 70 μL of solutions were impregnated on Whatman filter papers before being exposed to termites. The papers impregnated with the various solutions to be tested were dried in the open air, 27°C/75% relative humidity (RH) for 2 hours. The tests were carried out in Petri dishes (9 cm in diameter) where 15 g of wet sand (1 volume of water for 4 volumes of sand) were placed at the periphery. The Whatman extracts soaked papers were placed on a plastic rack in the middle of the petri dish. Then 20 wild worker termites were added to each test device without any possibility of feeding to check termite survival. For each concentration tested, we prepared three Petri dishes with extract and three Petri dishes with extraction solvent without extract which were used as controls during the test. Petri dishes were stored in the dark at 27°C, 75% RH for 12 days. At the end, the samples were cleaned and air dried. These devices were monitored regularly throughout the trial. The test is then stopped when all of the termites in the boxes tested have died. Mortality and daily survival rates were respectively calculated according to Equation (2) and deduced according to Equation (3). The results of the screening tests were presented in the form of curves of survival rates as a function of day and time of incubation in order to facilitate interpretations.

XLSTAT version 2019 (ADDINSOFT SARL) was used, and the results were known as the main values representing the mean of the repetitions ± SD. The. The values are statistically significant at p < 0.05.

In general, the results obtained in this study concerning the extraction yields indicate that the rate of extractables vary from one solvent to another, in the two samples of Kévazingo bark, the base and therefore the buttress up to 6 m. Acetone extraction rates are higher in samples E₁KC, E₂KC (9.92% and 6.82% respectively). The rates of extracts obtained with trichlorethylene and with water are the lowest regardless of the type of sample. Indeed, for trichlorethylene, the first solvent used during the successive extraction is the least polar solvent and therefore dissolves the phenolic compounds less. Non-polar solvents mainly extracted from nonpolar substances (oils, fats, terpenes) while polar solvents dissolve polar compounds such as polyphenols. The successive extraction, which combines non-polar and polar solvents, allows the extractables to be partitioned into different fractions, facilitating subsequent analyzes and the sum of the extracts with each solvent gives an idea of the overall extract content of the bark. The overall extract rates of the different parts studied vary from 17.11% to 13.42% respectively for the buttress and the height at 6 m (Table 1). The results showed that the content of total extracts varies depending on the diameter and height of the trees (the level of extractables decreases with height and increases with diameter). These results do not differ from most of those obtained by other authors. Indeed, Rather et al. (2017) obtained significantly higher extraction yields for Eucalyptus (Eucalyptus terepticornis) in the wood at the base of the trunk compared to the rest of the tree. In addition, numerous studies carried out on several tropical species have shown very variable extract rates, sometimes with high levels of 20% to 22% for certain woods (Cheumani, 2009; Huang et al., 2009; Mburu et al., 2007; Neya et al., 2004; Sirmah, 2009).

Overall, no significant difference was observed in the results of the phytochemical screening on the E₁KC and E₂KC samples. It appears from Tables 2 and 3 that the Alkaloids, Polyphenols, Tannins, Reducing compounds, Flavonoids and Anthraquinones were identified in the acetone, ethanol, and aqueous extracts of the bark of Guibourtea tessmanii. In addition, Polyphenols were also identified in the trichloroethylene extracts of the bark of the E₁KC sample. Saponins were identified only in aqueous extracts. These compounds are identified in the literature for their antioxidant, antifungal, anti-termite, antibacterial, antimicrobial properties (Bopenga Bopenga et al., 2020a, 2020b; Jeong et al., 2017; Kim et al., 2018; Min et al., 2019; Oliveira et al., 2010; Ohmura, 2000; Wu and Lin, 2001). Sterols and triterpenes identified only in trichloroethylene extracts. Terpene compounds are known in the literature (Andréa et al., 2010; Becker et al., 2005; Bédounguindzi et al., 2020; Bläske et al., 2003; Escalante et al., 2002; Lajide et al., 1995; Park and Shin, 2005; Watanabe et al., 2005; Viegas, 2003) for their antifungal and anti-termite activities. Our phytochemical screening results are in line with those obtained by Sima Obiang et al. (2018) which demonstrated the presence of saponines, tannins, phenols and flavonoids, alkaloids, reducing compounds, anthraquinone, sterols in the bark of barks of Coula edulis Baill, Pseudospondias longifolia Engl and Carapa klaineana Pierre from Gabon. The presence of all classes of compounds in tropical timber has been reported in the literature (Kilic and Niemz, 2012; Masendra et al., 2019), when studying extractables from some tropical species, found lipophilic compounds, mainly fatty acids and hydrophilic compounds (phenolic acids, flavonoids, sterols, stilbenes, and lignans).

The results obtained (Fig. 1 and 2) indicate that the anti-termite activity varies with the different parts of the bark studied (heights), the extraction solvent and the concentration of the extracts used as often reported in the literature (Andréa et al., 2010). Overall, the extracts at concentrations (50/50) exhibit slightly better anti-termite activity compared to (25/75). The strongest anti-termite activities were recorded with the aqueous extract of the E₁KC sample with a survival rate of 0% after 2 days, explaining the concentration of the extracts at this level. Diluting the solutions to a concentration of (25/75) increases the survival rate. These results demonstrate that the control paper impregnated only with water or acetone has no effect on the behavior of termites as shown by the survival rates (termite survival > 12 days with 80%) at the end of the trial. Basically all papers impregnated with extracts showed a protective effect of whatman paper, therefore strong resistance against termites. For each of these extracts, all the termites died before the end of the test. However, the use of less concentrated solutions still resulted in a 0% survival rate, hence anti-termite activity. Previous analyzes, in particular phytochemical screening, have made it possible to identify secondary metabolites such as polyphenols, tannins, flavonoids and anthraquinones, making it possible to explain the good anti-termite activity of these extracts (Jung et al., 2017; Morina et al., 2020). The results are in agreement with the literature which explains that tannins are known to be strongly astringent (Lee et al., 2020; Min et al., 2017; Monteiro et al., 2005; Mun and Darrel, 2017). Ayres et al. (1997) verified that the high mortality of insects treated with condensed and hydrolyzable tannins appears to be due to the toxic properties of these compounds and not to inhibition of digestion. Phytochemical screening revealed the presence of flavonoids in these extracts which are potential termite control agents (Hadi et al., 2020; Manurung et al., 2019; Nam et al., 2018; Simmonds, 2001). Other previous studies have also reported their anti-termite activity (Arsyad et al., 2020; Chen et al., 2004; Morimoto et al., 2006) and have shown that flavonoids such as catechin interact with the ecdysone receptor of termites (Oberdörster et al., 2001). Due to the ability of flavonoids to bind to ecdysone receptors, flavonoids can affect other biological systems in termites (Boué and Raina, 2003). Ragon et al. (2008) hypothesized that termites detected and avoided woods containing the extracts rich in antioxidant molecules because they could interfere with the digestion of lignocellulose by termite symbionts. Anthraquinones are very present in the ethanolic and aqueous extracts for the sample at 6 m and very abundant in the acetone, ethanolic and aqueous extracts for the buttress or certain quinones such as 2-methylanthraquinone are repellent against termites, others such as 7-methyljuglone and its derivatives have anti-termite activities (Arsyad et al., 2019; Hadi et al., 2018; Leyva et al., 1998; Niamké et al., 2012; Thévenon et al., 2001). These results explain the importance of phenolic compounds in the resistance of wood to termites.

Fig. 1. Kinetics of the daily survival rate of termites according to the concentrations of the different extracts of the E₁KC sample. E₁KC: sample 1 Kévazingo from Kango collected at the buttress.

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Fig. 2. Kinetics of the daily survival rate of termites according to the concentrations of the different extracts of the E₂KC sample. E₂KC: sample 2 Kévazingo from Kango collected at 6 m.

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