Bio-guided anti-cariogenic and phytochemical valorization of Guiera senegalensis and Pseudocedrela kotschyi stem extracts

The objective of the present study is to carry out bio-guided phytochemical investigation of Guiera senegalensis and Pseudocedrela kotschyi stem extracts. The two plants are used as toothpicks for oral hygiene. The inhibition test revealed a bacteriostatic effect of hexane extract PK1 of P. kotschyi against Streptococcus mutans ATCC 25175 and Streptococcus salivarius ATCC 20560, two bacterial cariogenic strains, with a decrease in the number of bacterial colonies of 1 Log/control. The aqueous extract GS5 obtained from the stems of G. senegalensis is bactericidal, with total inhibition of S. salivarius ATCC 20560. The antimicrobial effect of the stem extracts from the two plants studied varies according to the plant species and the type of bacterial strain. The phytocompounds 8-Hydroxy-6,7-dimethoxy-3methylisochroman-4-one, 1-(4-hydroxy-3-methoxyphe-nyl) propane-1,2-dione and (4E, 15E) -Nonadeca4,15dien-10-one were isolated, respectively from GS5A extracts of G. senegalensis and PK1 of P. kotschyi by normal column chromatography.

The African continent is endowed with an impressive floristic biodiversity, with a large variety of plants for food and therapeutic needs.This natural floristic richness is only slightly valued chemically and pharmacologically.The bacterial resistance towards synthetic antibiotics is one of the major concerns of the medical research today (Ouelhadj et al., 2017).Yet medicinal plants are interesting alternatives to explore alongside synthetic drugs.That is why, the objective of this study consists of a phytochemical investigation of G. senegalensis and P. kotschyi stem extracts by a bio-guided anti-cariogenic way to find out more new and effective antibacterials.

Plant
The plant parts (leaves, stems) were harvested in July 2016 from Longorola in Sikasso region (11° 11 ' 59 " North,7° 05 ' 49" West) in Southern Mali.The leaves of each plant were identified at Abidjan National Floral Center (CNF) according to herbariums N°s 7569 and 8664, respectively for G. senegalensis and P. kotschyi.The G. senegalensis (GS) and P. kotschyi (PK) stems used for phytochemical and antibacterial analyses were dried under permanent air conditioning (16°C) for 10 days.Then, they were reduced to powders with an electric grinder to obtain the analysis extracts.

Pathogenic bacteria
Two international cariogenic bacterial strains from the American type culture collection, namely Streptococcus mutans (S. mutans ATCC 25175) and Streptococcus salivarius (S. salivarius ATCC 20560) were used for antibacterial tests.

Preparation of extracts
The powder maceration of each plant (200 g) in hexane (600 mL) under agitation at ambient temperature during 48 h, gave dry hexanic extracts from G. senegalensis (GS1) and P. kotschyi (PK1) after filtration and solvent elimination, using a rotary evaporator (Büchi R-210 Rotavapor TM).

Antibacterial power evaluation
The antibacterial potency of the different extracts GS1-GS5 and PK1-PK5 were evaluated according to Yew (2015).The bacterial strains to be tested were cultured for 24 h in culture media specific for streptococci.The initial absorbance (650 nm) was first measured for each tube.Then, 300 µL of the overnight culture was added to each tube, and the mixture was adjusted to an absorbance of 1.0 +/-0.05 at the initial wavelength.After 24 h of culture, the absorbance was determined for each tube to estimate bacterial growth.A series of dilutions of 10 -3 , 10 -4 , 10 -5 , and 10 -6 for the seeded tubes were performed.An aliquot of 50 µL of the dilutions obtained were subsequently spread on the specific agar media.Colony counting was performed between 24 and 72 h after seeding, at which time the averages of the triplicates of each series were calculated.The results obtained were converted into Colony Forming Units per mL of medium (CFU/mL) and expressed in logarithmic decimals (CFU Log/mL).

Aglyconic extracts preparation
The aglyconic extracts were obtained from plant extracts according to Alilou et al. (2014) method, and it showed a better antibacterial profile.
GS5 and PK5 (15 g) were put in touch with HCl (328 mL, 2N) in a flask.The reactional mass was refluxed for 150 min.After filtration and cooling at room temperature, the hydrolyzate was treated with diethyl ether (3 × 100 mL).After decantation, the organic phase was washed with water (3 × 100 mL), then recovered and dried on anhydrous MgSO4 for 60 min.After filtration on Whatman paper and elimination of the solvent with a rotary evaporator to dryness (40°C), GS5A and PK5A aglyconic extracts were provided.

Secondary phytoconstituants fractionation, purification and separation
PK1, GS5A and PK5A extracts were retained for chromatographic fractionation and purification with regard to their better antibacterial profile.Isolation and purification were achieved through an elution series on silica gel in the normal phase (Figures 1 and 2).

Isolated phytoconstituants spectroscopic characterization
Structural elucidation of secondary metabolites has been performed on purified native phytoconstituants.The 1 H and 13 C NMR spectra were obtained on BRÜKER Avance 400 MHz.IR spectra were recorded on Perkin Elmer FT-IR 2000 between 4000 and 500 cm -1 .

Extraction yields
The extraction yield of each plant extract is shown in Table 1.
In Table 1, maceration in water supplied the best extracts yields; which seems to accredit the recurring use of this extraction process in endogenous phytotherapy.However, the latter also shows that the yield may differ from a botanical species in another one.The aglyconic extracts yields from G. senegalensis and P. kotschyi are 0.78 and 0.67%, respectively.

Plant extracts antibacterial profile
The antibacterial profile of ten raw extracts (GS1-GS5, PK1-PK5) has been estimated towards the two pathogenic bacterial strains directly involved in tooth decay.The results are shown in Tables 2 and 3.
Extracts effect was appreciated according to the  (2015).Indeed, an extract is bacteriostatic with regard to a decrease in the number of 1 Log/control colonies.On the other hand, it is bactericidal if the total inhibition of bacterial culture is observed.
The results of the antibacterial tests reveal that, GS1, GS2, GS3, GS4, PK2, PK3, PK4 and PK5 do not exhibit any significant inhibition effect on the growth of S. mutans and S. salivarius at the tested concentrations.PK1 is bacteriostatic towards S. mutans (Table 2).Indeed, this extract inhibits the growth of this bacterial strain by stopping the number of colonies of more than 1 Log/control at concentrations of 0.2 and 0.5 g/L.GS5 inhibits the growth of S. salivarius because no bacterial colony is found on the agar in its presence.GS5 is therefore bactericidal at 0.2 g/L (Table 3).
On the other hand, the latter showed no inhibitive activity against S. mutans (Table 2).GS1 and PK1 extracts caused a decrease in the number of S. salivarius colonies from about 0.75 Log/control to 0.5 g/L.This value close to 1 Log/control could be improved either by increasing the concentration of these extracts, or by isolating the active phytocompounds which they contain.
Phytochemical screening of G. senegalensis and P. kotschyi stems has already been performed (Kadja, 2014).The author reports that these organs contain phytophenols (coumarins, flavonoids, etc.) and terpenes among other identified active second principles.Besides, the beneficial effects of terpenes and polyphenols on oral health are known (Bitty, 1982;Kadja et al., 2011;Atsain et al., 2016).Thus, the antibacterial activities exhibited by hexanic extracts GS1 and PK1 suggest a synergic action due to the existence of terpenic phytoconstituents.As for the aqueous extracts of GS5 and PK5, their manifest antibacterial profile seems to have a correlation with the aforementioned extracts of water-soluble secondary metabolites with antibacterial potential.

Isolated phytocompound structural elucidation
PK1, GS5A and PK5A extracts were chromatographically fractionated on a normal phase silica gel column.Fractionation and purification of GS5A led to the isolation of phytoconstituents A and B, with yields of 43.30 and 2.62%, respectively, with regard to the mass fractions (Figure 1).As for PK1, its fractionation and purification allowed the isolation of phytocompound C, with a yield of 4.92% (Figure 2).The interpretation of IR spectra was done according to the method of Brown et al. (1992) and Robert et al. (2005).

Structure of isolated phytocompound C from PK1 extract
NMR analysis shows that compound C has molecular symmetry.The 13 C NMR and HMBC correlation spectra revealed the presence of 2 primary C which resound in 14.08 ppm; 16 secondary C among which 4 C equivalent type C=C (C4=C5, C15=C16) appearing at 128.07 and   6).Analysis of the IR spectrum showed an absorption band at 3054 cm -1 (C-H asymmetric, valence vibration in CH=CH); 1680 cm -1 (C=C, E configuration, valence vibration); 1709 cm -1 (C=O, valence vibration); and 738 cm -1 (C-H, deformation vibration).The spectral data set confirm the molecular structure of phytocompound C (Figure 5).

Conclusion
In this work, valorization of the extracts of stems for G. senegalensis and P. kotschyi has been undertaken, for two plant species used by the populations in sub-Saharan Africa used like toothbrush for the maintenance of oral health.Biologically, the stem extracts of these plants have exhibited inhibitory effects on both tested cariogenic bacterial strains.These antibacterial effects were owed to the synergic combination of the active secondary metabolites, which they contain.On the one hand, these results support the first intention, the utility of popular use of both plants as toothpicks; on the other hand, it recommends the possibility of the use of these plants in the prevention of dental caries.In the phytochemical plan, three isolated phytoconstituants have been characterized.These are 1-(4'-Hydroxy-3'methoxyphenyl) propane-1, 2-dione, 5-hydroxy-6, 7-dimethoxy-2-methylisochroman-1-one and (E, E)-nonadeca-4, 15-dien-10-one.The study of their anticariogenic potential is in progress.

Table 2 .
Antibacterial profile of plant extracts (in CFU Log/mL) by inhibition of S. mutans ATCC 25175 growth.

Table 3 .
Antibacterial profile of plant extracts (in log CFU/mL) by inhibition of S. salivarius ATCC 20560 growth.

Table 4 .
NMR spectral data of compound A.

Table 5 .
NMR spectral data of compound B.

Table 6 .
NMR spectral data of compound C.