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Anti-caries activity of selected Sudanese medicinal plants with emphasis on Terminalia laxiflora

Abstract

In Sudan, some medicinal plants, such as Acacia seyal, Calotropis procera and Balanites aegyptiaca have been used to prevent or treat oral health problems. The stem and stem bark of Terminalia laxiflora Engl., Combretaceae, are used as antiseptics for mouthwash to prevent gingivitis and thrush in Africa. Methanol and 50% hydroethanolic extracts of 25 plants that are used in traditional Sudanese medicine for several diseases and cavity disorders were screened for anti-cavity activities. T. laxiflora methanolic wood extracts, which exhibited such activity, were investigated. The crude extracts were assayed for their antimicrobial activities against Streptococcus sobrinus in terms of minimum inhibitory concentration and glucosyltransferase inhibition. The active extract of T. laxiflora wood was subsequently fractionated by different chromatographic techniques. Isolated compounds were identified by spectroscopic methods and assessed for S. sobrinus and glucosyltransferase inhibitory effects. Methanolic extracts of Terminalia brownii (bark), T. laxiflora (wood), A. seyal (bark), Persicaria glabra (leaves) and Tamarix nilotica (stem) showed good activities against both S. sobrinus and glucosyltransferase (MIC ≤ 1 mg/ml, IC50 values <50 µg/ml). Over all plant extracts, T. laxiflora demonstrated the good combined activities (MIC 0.5 mg/ml, glucosyltransferase, IC50 10.3 µg/ml); therefore, its methanolic wood extracts were selected for further phytochemical studies. Four constituents were isolated by chromatographic techniques and identified by spectroscopic techniques. Pharmacological evaluation of the obtained compounds showed that flavogallonic acid dilactone had comparatively good antibacterial activity. In the glucosyltransferase inhibitory test, terchebulin displayed potent activity with an IC50 of 7.5 µM. The screening presented in this study showed that methanol extracts of T. laxiflora wood possessed promising anti-cavity effects.

Keywords
Glucosyltransferase; Dental cavity; Antibacterial; Terchebulin

Introduction

Dental caries is defined as an irreversible microbial disease of the calcified tissues of the teeth, characterized by demineralization of the inorganic portion and destruction of the organic substance of the tooth (Rajendran et al., 2009Rajendran, R., Sivapathasundharam, B., Shafer, Hine, Levy, 2009. Shafer's Textbook of Oral Pathology, 6th ed. Elsevier, India, pp. 409.).

Bacterial plaque composed of native oral flora accumulated on dental surfaces embedded in an extracellular polysaccharide (EPS) matrix and is the primary etiologic agent of dental caries (Kolenbrander et al., 2006Kolenbrander, P.E., Palmer, R.J., Rickard, A.H., Jakubovics, N.S., Chalmers, N.I., Diaz, P.I., 2006. Bacterial interactions and successions during plaque development. Periodontology 42, 47-79.; Koo et al., 2009Koo, H., Xiao, J., Klein, M.I., 2009. Extracellular polysaccharides matrix – an often forgotten virulence factor in oral biofilm research. Int. J. Oral Sci. 1, 229-234.). Out of the seven species of mutans streptococci group, Streptococcus sobrinus have been one of the most commonly implicated in the pathogenesis of dental cavity; moreover, it produces exoenzymes named glucosyltransferases (GTF), which play critical roles in the synthesis of glucan and EPS to providing sites on dental surfaces for microbial colonization, in addition to adherent glucan for bacterial coherence (Paes Leme et al., 2006Paes Leme, A.F., Koo, H., Bellato, C.M., Bedi, G., Cury, J.A., 2006. The role of sucrose in cariogenic dental biofilm formation – new insight. J. Dent. Res. 85, 878-887.; Bowen and Koo, 2011Bowen, W.H., Koo, H., 2011. Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res. 45, 69-86.; Nishimura et al., 2012Nishimura, J., Saito, T., Yoneyama, H., Bai, L.L., Okumura, K., Isogai, E., 2012. Biofilm formation by Streptococcus mutans and related bacteria. Adv. Microbiol. 2, 208-215.; Hashizume-Takizawa et al., 2014Hashizume-Takizawa, T., Shinozaki-Kuwahara, N., Tomita, N., Kurita-Ochiai, T., 2014. Establishment of a convenient sandwich-ELISA for direct quantification of glucosyltransferase-I: application for dual diagnosis of dental caries. Monoclon. Antib. Immunodiagn. Immunother. 33, 89-93.).

Medicinal plants have been a great source of novel drug compounds from long time. Plant derived products have made large contributions to the well being of human health. Scientists across the globe have reported antimicrobial properties of several medicinal plants but still a very few of this enormous potential drug has been scientifically screened (Siqueira and Rocas, 2005Siqueira, J.F., Rocas, I.N., 2005. Exploiting molecular methods to explore endodontic infection: Part 2 – redefining the endodontic microbiota. J. Endod. 3, 488-498.; Karuppiah and Rajaram, 2012Karuppiah, P., Rajaram, S., 2012. Antibacterial effect of Allium sativum cloves and Zingiber officinale rhizomes against multiple drug resistant clinical pathogens. Asian Pac. J. Trop. Biomed. 2, 597-601.; Gauniyal and Teotia, 2014Gauniyal, P., Teotia, U.V.S., 2014. Phytochemical screening and antimicrobial activity of some medicinal plants against oral flora. Asian Pac. J. Health Sci. 1, 236-255.). In Sudan, various medicinal plants have been used to prevent or treat oral health problems. This study investigated some plants that are used in traditional Sudanese medicine as mouth detergents such as Acacia seyal, Calotropis procera and Balanites aegyptiaca (El Ghazali et al., 2003El Ghazali, G.B., Abdalla, W.E., Khalid, H.E., Khalafalla, M.M., Hamad, A.A., 2003. Medicinal Plants of Sudan. Part V. Medicinal Plants of Ingassana Area. National Council for Research, Khartoum Press, Khartoum, Sudan.; Khalid et al., 2012Khalid, H.E., Abdalla, W.E., Haider, A., Till, O., Thomas, E., 2012. Gems from traditional north-African medicine: medicinal and aromatic plants from Sudan. Nat. Prod. Bioprospect. 2, 92-103.). Different parts of Terminalia laxiflora Engl., Combretaceae, are used to prevent gingivitis and thrush in Congo; the stems are used as chewing sticks and also macerated stem bark are used as antiseptic to wash mouth (Fasola et al., 2013Fasola, T.R., Oluwole, M.E., Olaniyi, I.F., Adeboye, I.E., 2013. The phytochemical and antimicrobial activities of Terminalia laxiflora Engl. & Diels root bark extract. Nat. Sci. 11, 122-127.).

In the present study, 25 plant species were selected and evaluated for their anti-cariogenic activity in terms of inhibition of S. sobrinus bacterial growth and GTF inhibitory effects by using their methanolic and 50% hydroethanolic extracts. Methanolic extracts of T. laxiflora wood demonstrated significant combined activities; thus, it was further fractionated in order to identify the active compounds responsible for the biological activities.

Materials and methods

Reagents

All materials were purchased from Wako, Japan except p-iodonitrotetrazolium (INT) violet, which was from Sigma-Aldrich Co. Ltd, Japan.

Plant materials and extraction

The plants were collected from Khartoum state (Khartoum, Omdurman and Shambat cities) and Elgadarif state of Sudan. Voucher specimens are deposited in the Horticultural Laboratory, Department of Horticulture, Faculty of Agriculture, University of Khartoum (Table 1).

Table 1
Minimum inhibitory concentration (MIC) and glucosyltransferase (GTF) inhibitory activities of selected Sudanese medicinal plant extracts.

Plant materials were shade dried and powdered before extraction; they were each extracted three times for 12 h, with methanol and 50% hydroethanol. The extracts were filtered and the solvent was removed under reduced pressure using rotary evaporator. The concentrated extracts were then dried with a freeze dryer.

Fractionation, purification and identification of pure compounds from Terminalia laxiflora

Fractionation and isolation of Terminalia laxiflora Engl., Combretaceae, wood were performed by the method described by Muddathir et al. (2013)Muddathir, A.M., Mitsunaga, T., Yamauchi, K., 2013. Anti-acne activity of tannin-related compounds isolated from Terminalia laxiflora. J. Wood Sci. 59, 426-431.. Methanolic crude extracts (5 g) were chromatographed on medium pressure liquid chromatography (MPLC) using octadecyl-silica (ODS) column (YMC-DispoPackAT ODS-25, particle size 25 µm; column size 120 g, (40 mm × 188 mm), Japan), chromatography pump (Co. No. 540 Yamazen, Osaka, Japan) with pressure of 1.2 MPa, UV detector at 280 wavelength (UV-10V Yamazen, Osaka, Japan) and fraction collector (SF-2120, Advantec Tokyo Ltd, Japan).

The column was conditioned with the first eluent used for separation for 30 min with flow rate of 0.5 ml/min. Then water containing increasing proportions of methanol stepwise elution to obtain two fractions (F1 and F2). F1 (0.89 g) was passed through column chromatography (40 mm × 430 mm) on a Sephadex LH-20 (18–111 µm, GE Healthcare Bio-Sciences Corp, Tokyo, Japan) that revealed four subfractions. These four subfractions, F (1a, 1b, 1c and 1d) introduced through preparative high performance liquid chromatography (HPLC) with reversed phase Inertsil ODS-3 column (10 mm × 250 mm, GL Sciences Inc., Tokyo, Japan) and monitored at 280 nm. Solvent system used 10–100% gradient methanol in water with 0.05% TFA programmed for 60 min at a flow rate of 5 ml/min to give compound (1) (14 mg), compound (2) (8.5 mg) and compound (3) (5 mg). F2 (0.50 g) was also subjected to preparative HPLC under the same condition to isolate compound (4) (17.5 mg). The compounds purity was confirmed using analytical HPLC (Shimadzu SIL-20A) with reversed phase Inertsil ODS-3V column (5 µm (4.6 mm × 250 mm), GL Sciences Inc., Tokyo, Japan), flow rate: 1 ml/min, wavelength: 280 nm, gradient program: methanol:0.05% TFA aqueous solution for 60 min (Fig. 1).

Fig. 1
HPLC fingerprint of methanol extract of Terminalia laxiflora and their isolated compounds. (1) Flavogallonic acid dilactone [retention time: 13.6 min], (2) terchebulin [retention time: 17.3 min], (3) gallic acid [retention time: 8.8 min] and (4) ellagic acid [retention time: 26.7 min].

The isolated compounds from T. laxiflora wood methanol extract, exactly compounds (1) and (2) were identified using the 1H,13C nuclear magnetic resonance (NMR). Spectra were recorded in methanol-d 4 with a JEOL EC600 MHz NMR (Tokyo, Japan). Additionally, ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS, Waters Xevo™ QTof MS, Waters, Milford, MA, USA) was performed using a C18 column (2.1 mm × 100 mm, Waters) with MeOH/H2O = 5/95 (30 min), 100/0 (10 min) with a linear gradient as eluent. The UPLC-TOFMS data were collected in negative ionization mode (Fig. 2).

Fig. 2
Mass spectra of flavogallonic acid dilactone (A) [m/z 469] and terchebulin (B) [m/z 1083] by UPLC-TOFMS in negative ionization mode.

Flavogallonic acid dilactone (1)

Tan powder. 1H NMR (in CD3OD): δ 7.26 (s), 7.50 (s). 13C NMR (in CD3OD): δ 108.1 (C-1, 1′), 110.1–114.4 (C-6, 6′), 112.8 (C-5), 113.3 (C-6″), 117.5–120.2 (C-5′, 2″), 124.9 (C-1″), 135.7 (C-2), 136.3 (C-3), 136.5 (C-4), 137.8 (C-2′), 139.2 (C-3′), 143.2 (C-4′), 144.1 (C-3″), 145.9 (C-4″), 147.8 (C-5″), 158.9–160.4 (C-7, 7′), 168.9 (C-7″); UPLC-TOFMS m/z 469 [M−H] (calcd. for C21 H10 O13 470.2963).


Terchebulin (2)

Tan powder. 1H NMR (in CD3OD): δ 3.04 (t, J = 11.6 Hz, one of the H-6″), 4.21 (t, J = 10.3 Hz, H-5″), 4.48 (t, J = 8.9 Hz, one of the H-6″), 4.78 (t, J = 11.0 Hz, H-4″), 4.98 (dd, J = 3.5, 9.7 Hz, H-2″), 5.23 (d, J = 2.8 Hz, H-1″), 5.64 (t, J = 9.6 Hz, H-3″), 6.37 (s, H-B6), 6.42 (s, H-D6), 6.56 (s, H-A2), 6.79 (s, H-C2), 7.48 (s, H-5). 13C NMR (in CD3OD): δ 63.4 (C-6″), 68.5 (C-4″), 69.0 (C-5″), 74.1 (C-3″), 74.2 (C-2″), 90.2 (C-1″), 106.4 (C-B6), 106.5 (C-D6), 106.8 (C-A2), 108.5 (C-C2), 112.0–114.0 (C-A6, B2, 5, 5′, 1, 1′, 2, 2′, 6, 6′), 116.0 (C-C6), 122.2 (C-D1), 123.5 (C-B1, C1), 125.1 (C-A1), 135.9 (C-B4), 136.1 (C-A4), 137.5 (C-C4), 137.6 (C-D4), 138.4 (C-3), 139.1 (C-D3), 140.7 (C-3′), 141.7 (C-D2), 143.4–143.6 (C-A5, B3, C5), 144.5–144.6 (C-A3, B5, C3, D5), 147.4 (C-4′), 150.3 (C-4), 158.3 (C-7′), 159.5 (C-7), 166.9 (C-D7), 167.0 (C-C7), 168.9 (C-A7), 169.5 (C-B7); UPLC-TOFMS m/z 1083.07 [M−H] (calcd. for C48 H28 O30 1084.7179).


Compound (3) and compound (4) were identified by comparing the retention time with standard of the highest grade (purity >97.0%) gallic acid (Nacalai Tesque, Inc., Kyoto, Japan) and ellagic acid (Sigma, Japan) respectively.

Determination of the minimum inhibitory concentration (MIC)

MIC was determined by the broth dilution method according to Iwaki et al. (2006)Iwaki, K., Koya-Miyata, S., Kohno, K., Ushio, S., Fukuda, S., 2006. Antimicrobial activity of Polygonum tinctorium Lour: extract against oral pathogenic bacteria. J. Nat. Med. 60, 121-125.. S. sobrinus 6715 was cultured in a Brain-Heart Infusion Broth. The crude extracts, fractions or pure compounds were tested for antibacterial activity in sterile 96-well plates. The inoculums were prepared by diluting the broth culture to approximately 106 cells/ml. To each well containing sample, 100 µl of microbial inoculums were added, followed by addition of medium to achieve a final volume of 200 µl. The tested sample was prepared in a concentration range of 4000–31 µg/ml using a two-fold dilution method. Solvent and medium controls were included in each test plate. In order to dissolve the sample extracts, 20% dimethyl sulfoxide (DMSO) was used in this study. The final concentration of DMSO alone in the well showed no inhibitory effect on S. sobrinus growth. The experiments were performed in triplicate. Chlorhexidine was included in the assays as positive control. The cultures were incubated for 24 h at 37 °C under anaerobic conditions. Microbial growth was indicated by the addition of 50 µl of (0.2 mg/ml) INT to the culture and incubated at 37 °C for 2 h. The MIC was defined as the lowest concentration that inhibited the color change of INT (Eloff, 2001Eloff, J.N., 2001. Antibacterial activity of Marula (Sclerocarya birrea (A. rich) Hochst. subsp. caffra (Sond.) Kokwaro) (Anacardiaceae) bark and leaves. J. Ethnopharmacol. 76, 305-308.).

Assay for GTF inhibitory activity

Streptococcus sobrinus 6715 was cultured for 20 h at 37 °C in 4 l of Todd Hewitt broth. After centrifugation of the culture at 1300 × g for 10 min at 4 °C, the cells were collected and then extracted with 8 M urea for 1 h while stirring. The crude enzyme solution was dialyzed against 10 mM sodium phosphate buffer (pH 6.0). The crude enzyme solution was stored in a freezer at −80 °C.

GTF were incubated in 300 µl of 0.1 M phosphate buffer (pH 6.0) containing 1% sucrose, 0.5% dextran T-10, in the presence or absence of a sample at 37 °C for 3 h. The volume of the crude GTF solution used in the assay was determined by measured turbidity around 1.0 absorbance at 590 nm (Mitsunaga et al., 1997Mitsunaga, T., Abe, I., Kontani, M., Ono, H., Tanaka, T., 1997. Inhibitory effects of bark proanthocyanidins on the activities of glucosyltransferases of Streptococcus sobrinus. J. Wood Chem. Technol. 17, 327-340.).

= Absorbance of control Absorbance of sample Absorbance of control × 100

Statistical analysis

The inhibitory percentage and IC50 values of GTF were expressed as the mean (mean ± standard deviation). The significant differences between samples were assessed by one-way analysis of variance (ANOVA) followed by pairwise comparison of the mean using Tukey's multiple comparison test. Values were determined to be significant when p was less than 0.05 (p < 0.05).

Results

Twenty-five plant species that are used in traditional Sudanese medicine were investigated. Sixty-two methanolic and 50% hydroethanolic extracts were prepared; and their antibacterial activities against S. sobrinus and GTF enzyme inhibitory activities were investigated.

Antibacterial assay

To evaluate the antibacterial activity of selected Sudanese medicinal plant extracts against S. sobrinus using dilution methods. The MIC were determined in Table 1. Among 62 plant extracts 35 extracts showed antibacterial activity. Also noteworthy the methanolic extracts of Combretaceae family; Terminalia brownii (bark) and T. laxiflora (wood) demonstrated highest antibacterial activity (MIC of 0.5 mg/ml) among them.

GTF enzyme inhibitory activity

Inhibitory effects over 55% on GTF enzyme activity were demonstrated by 18 of the 62 plant extracts at 100 µg/ml (Table 1); of these thirteen extracts exhibited inhibitory activity more than 80%. Table 2 shows the IC50 values of GTF inhibitory activity of nine methanolic extracts ranged between 3.8 and 49.8 µg/ml. A. seyal (bark), T. brownii (bark) and T. laxiflora (wood) display a significant inhibitory activity.

Table 2
IC50 values (µg/ml) obtained by the potent methanolic extracts against glucosyltransferase (GTF) enzyme.

Combined activities of the crude extracts and isolated compounds

Methanolic extracts of T. brownii (bark), T. laxiflora, A. seyal (bark), Persicaria glabra (leaves) and Tamarix nilotica (stems) demonstrated MIC ≤ 1 mg/ml against S. sobrinus and IC50 less than 50 µg/ml against GTF enzyme.

Methanolic wood extracts of T. laxiflora which showed good combined activities (MIC 0.5 mg/ml, GTF, IC50 10.3 µg/ml), were selected for further purification, crude methanolic extracts after subjected to MPLC resulted in two fractions F1 (MIC 0.5 mg/ml, GTF 96.4%) and F2 (MIC 1 mg/ml, GTF 93.1%); moreover purification revealing the presence of flavogallonic acid dilactone (1), terchebulin (2), gallic acid (3), and ellagic acid (4). Antibacterial and GTF inhibitory activities for isolated compounds are shown in Table 3. Flavogallonic acid dilactone (1) demonstrated relatively good antibacterial activity, with MIC of 0.5 mg/ml. Terchebulin (2) and ellagic acid (4) showed moderate antibacterial activity, however, terchebulin (2) displayed potent activity against GTF enzyme.

Table 3
Anti-S. sobrinus and glucosyltransferase (GTF) inhibitory activity of compounds isolated from T. laxiflora wood.

Discussion

Antibacterial and GTF enzyme inhibitory activities of extracts

In this study solvent selection relied on previous studies mentioning that methanol is classified as a polar solvent due to the presence of hydroxyl group. Nevertheless, there is also methyl group presence in methanol, which is sort of non-polar, so it has ability to extract various types of compounds and increase extract's yield. Furthermore, it can give higher concentrations of bioactive molecules from plants such as different classes of phenolic compounds. 50% hydroethanol yield high content of phenolic and flavonoid compounds (Ahmad et al., 2009Ahmad, A., Alkarkhi, A.A., Hena, S., Lim, H.K., 2009. Extraction, separation and identification of chemical ingredients of Elephantopus scaber (L) using factorial design of experiment. Int. J. Chem. 1, 36-49.; Caunii et al., 2012Caunii, A., Pribac, G., Grozea, I., Gaitin, D., Samfira, I., 2012. Design of optimal solvent for extraction of bioactive ingredients from six varieties of Medicago sativa. Chem. Cent. J. 6, 123.; Thanh et al., 2016Thanh, V.N., Christopher, J.S., Michael, C.B., Phuong, D.N., Quan, V., 2016. Impact of different extraction solvents on bioactive compounds and antioxidant capacity from the root of Salacia chinensis L.. J. Food Qual. 2017, 8.). Some studies mentioned that polyphenols, such as flavonoids, phenolic acids and tannins showed anti-enzyme, antibacterial and/or anti-biofilm activities (Gulati et al., 2012Gulati, V., Harding, I.H., Palombo, E.A., 2012. Enzyme inhibitory and antioxidant activities of traditional medicinal plants: potential application in the management of hyperglycemia. BMC Complement. Altern. Med., http://dx.doi.org/10.1186/1472-6882-12-77.
http://dx.doi.org/10.1186/1472-6882-12-7...
; Livia et al., 2016Livia, S., Silvia, F., Katarina, R., Jan, K., Pavel, M., 2016. Antibiofilm activity of plant polyphenols. Molecules 21, 1717.).

The MIC values of the methanol extracts were relatively lower than those of the 50% hydroethanolic extracts, implying more active antibacterial composition in methanol than in 50% hydroethanolic extracts. These findings were similar to those reported by Samuelsen (2000)Samuelsen, A.B., 2000. The traditional uses, chemical constituents and biological activities of Plantago major L. A review. J. Ethnopharmacol. 71, 1-21.. Ncube et al. (2012)Ncube, B., Finnie, J.F., Van Staden, J., 2012. In vitro antimicrobial synergism within plant extract combinations from three South African medicinal bulbs. J. Ethnopharmacol. 139, 81-89. believed that the crude extract will be active when having MIC values less than 8 mg/ml, whilst Gibbons (2005)Gibbons, S., 2005. Plants as a source of bacterial resistance modulators and anti-infective agents. Phytochem. Rev. 4, 63-78. suggested that isolated phytochemicals should demonstrate at least MIC < 1 mg/ml. In this study, MIC values of 0.5 mg/ml were considered an indication of good antibacterial activity.

Zhi et al. (2016)Zhi, R., Lulu, C., Jiyao, L., Yuqing, L., 2016. Inhibition of Streptococcus mutans polysaccharide synthesis by molecules targeting glycosyltransferase activity. J. Oral Microbiol. 8, 31095. stated that inhibition of GTF activity and the consequential polysaccharide synthesis may diminish the virulence of cariogenic biofilms, which could be an alternative strategy to eradicate dental caries. Methanolic extracts of A. seyal (bark), T. brownii (bark) and T. laxiflora (wood) showed significant inhibitory activities on GTF. A previous study mentioned that methanolic extracts of these plants contain condensed and hydrolysable tannins (Muddathir and Mitsunaga, 2013Muddathir, A.M., Mitsunaga, T., 2013. Evaluation of anti-acne activity of selected Sudanese medicinal plants. J. Wood Sci. 59, 73-79.). Yamauchi et al. (2016)Yamauchi, K., Mitsunaga, T., Muddathir, A.M., 2016. Screening for melanogenesis-controlled agents using Sudanese medicinal plants and identification of active compounds in the methanol extract of Terminalia brownii bark. J. Wood Sci. 62, 285-293. reported that T. brownii (bark) contained gallic acid, punicalagin, terchebulin, ellagic acid 4-O-α-L-rhamnopyranoside, ellagic acid and 3,4,3′-tri-O-methylellagic acid. Plant polyphenols shared with catechin-based oligomeric forms (condensed tannins) and/or gallate ester form compounds (hydrolysable tannins) display strong anti-GTF activities (Yanagida et al., 2000Yanagida, A., Kanda, T., Tanabe, M., Matsudaira, F., Oliveira Cordeiro, J.G., 2000. Inhibitory effects of apple polyphenols and related compounds on cariogenic factors of mutans streptococci. J. Agric. Food Chem. 48, 5666-5671.).

Biological activities of compounds isolated from Terminalia laxiflora wood

Our chemical profiling of the methanol extracts of the T. laxiflora wood disclosed the presences of four compounds as we described in experimental part. Flavogallonic acid dilactone (1) showed 1H NMR spectral pattern with two 1H singlets (δ H 7.26 and 7.50). 13C NMR spectrum displayed signals of nine non-oxygenated aromatic carbons (δ C 108.1, 108.1, 110.1, 112.8, 113.3, 114.4, 117.5, 120.2, 124.9) and nine oxygenated carbons (δ C 135.7, 136.3, 136.5, 137.8, 139.2, 143.2, 144.1, 145.9, 147.8) of three aromatic rings. The spectrum showed also signals of two lactonized carbonyl carbons (δ C 158.9 and 160.4) and a carboxylic carbon (δ C 168.9). Data confirmed by UPLC-TOFMS (m/z 469 [M−H]). These spectroscopic data were the same to literature values reported by Orabi et al. (2015)Orabi, M.A.A., Yoshimura, M., Amakura, Y., Hatano, T., 2015. Ellagitannins, gallotannins, and gallo-ellagitannins from the galls of Tamarix aphylla. Fitoterapia 104, 55-63. and Tanaka et al. (1986)Tanaka, T., Nonaka, G., Nishioka, I., 1986. Tannins and related compounds. XLII. Isolation and characterization of four new hydrolysable tannins, terflavins A and B, tegallagin and tercatain from the leaves of Terminalia catappa L.. Chem. Pharm. Bull. 34, 1039-1049.. The chemical structure has been excellently reported by several authors (Grimshaw and Haworth, 1956Grimshaw, J., Haworth, R.D., 1956. Flavogallol. J. Chem. Soc. , 4225-4232.; Tanaka et al., 1986Tanaka, T., Nonaka, G., Nishioka, I., 1986. Tannins and related compounds. XLII. Isolation and characterization of four new hydrolysable tannins, terflavins A and B, tegallagin and tercatain from the leaves of Terminalia catappa L.. Chem. Pharm. Bull. 34, 1039-1049., 1996Tanaka, T., Ueda, N., Shinohara, H., Nonaka, G., Fujioka, T., Mihashi, K., Kouno, I., 1996. C-glycosidic ellagitannin metabolites in the heartwood of Japanese chestnut tree (Castanea crenata Sieb. et Zucc.). Chem. Pharm. Bull. 44, 2236-2242.; Kinjo et al., 2001Kinjo, J., Nagao, T., Tanaka, T., Nonaka, G.I., Okabe, H., 2001. Antiproliferative constituents in the plant 8. Seeds of Rhynchosia volubilis. Biol. Pharm. Bull. 24, 1443-1445.; Hirano et al., 2003Hirano, Y., Kondo, R., Sakai, K., 2003. 5α-Reductase inhibitory tannin-related compound isolated from Shorea laevifolia. J. Wood Sci. 49, 339-342.; Shuaibu et al., 2008Shuaibu, M.N., Wuyep, P.A., Yanagi, T., Hirayama, K., Tanaka, T., Kouno, I., 2008. The use of microfluorometric method for activity-guided isolation of antiplasmodial compound from plant extracts. J. Parasitol. Res. 102, 1119-1127.; Ibrahim et al., 2014Ibrahim, M.A., Mohammed, A., Isah, M.B., Aliyu, A.B., 2014. Anti-trypanosomal activity of African medicinal plants: a review update. J. Ethnopharmacol. 154, 26-54.; Orabi et al., 2015Orabi, M.A.A., Yoshimura, M., Amakura, Y., Hatano, T., 2015. Ellagitannins, gallotannins, and gallo-ellagitannins from the galls of Tamarix aphylla. Fitoterapia 104, 55-63.). Flavogallonic acid dilactone (1) was isolated earlier from Terminalia catappa in free form and as a single-bonded acyl unit on their tannin pyranose cores, such as terflavins A–D (Tanaka et al., 1986Tanaka, T., Nonaka, G., Nishioka, I., 1986. Tannins and related compounds. XLII. Isolation and characterization of four new hydrolysable tannins, terflavins A and B, tegallagin and tercatain from the leaves of Terminalia catappa L.. Chem. Pharm. Bull. 34, 1039-1049.).

The 1H and 13C NMR data of terchebulin (2) were similar to those reported by Lin et al. (1990)Lin, T.C., Nonaka, G.I., Nishioka, I., Ho, F.C., 1990. Tannins and related compounds, CII. Structures of terchebulin, an ellagitannin having a novel tetraphenylcarboxylic acid (terchebulic acid) moiety, and biogenetically related tannins from Terminalia chebula Retz. L.. Chem. Pharm. Bull. 38, 3004-3008.. The UPLC-TOFMS (m/z 1083 [M−H]) data was in agreement with Silva et al. (2000)Silva, O., Gomes, E.T., Wolfender, J.L., Marston, A., Hostettmann, K., 2000. Application of high performance liquid chromatography coupled with ultraviolet spectroscopy and electrospray mass spectrometry to the characterisation of ellagitannins from Terminalia macroptera roots. Pharm. Res. 17, 1396-1401.. Previously Silva et al. (2000)Silva, O., Gomes, E.T., Wolfender, J.L., Marston, A., Hostettmann, K., 2000. Application of high performance liquid chromatography coupled with ultraviolet spectroscopy and electrospray mass spectrometry to the characterisation of ellagitannins from Terminalia macroptera roots. Pharm. Res. 17, 1396-1401. reported that terchebulin was the main compound present in Terminalia macroptera root. Gallic acid (3) and ellagic acid (4) were also detected in T. laxiflora wood extract; these two compounds were isolated from different plants of the genus Terminalia (Silva et al., 2000Silva, O., Gomes, E.T., Wolfender, J.L., Marston, A., Hostettmann, K., 2000. Application of high performance liquid chromatography coupled with ultraviolet spectroscopy and electrospray mass spectrometry to the characterisation of ellagitannins from Terminalia macroptera roots. Pharm. Res. 17, 1396-1401.; Shuaibu et al., 2008Shuaibu, M.N., Wuyep, P.A., Yanagi, T., Hirayama, K., Tanaka, T., Kouno, I., 2008. The use of microfluorometric method for activity-guided isolation of antiplasmodial compound from plant extracts. J. Parasitol. Res. 102, 1119-1127.).

Data in Table 3 showed that flavogallonic acid dilactone (1) exhibited good antibacterial activity (0.5 mg/ml). From previous study, the activity of Propionibacterium acnes was inhibited at concentration 0.25 mg/ml (Muddathir and Mitsunaga, 2013Muddathir, A.M., Mitsunaga, T., 2013. Evaluation of anti-acne activity of selected Sudanese medicinal plants. J. Wood Sci. 59, 73-79.). The MIC for gallic acid (3) has no activity toward S. sobrinus up to 4 mg/ml. Kang et al. (2008)Kang, M.S., Oh, J.S., Kang, I.C., Hong, S.J., Choi, C.H., 2008. Inhibitory effect of methyl gallate and gallic acid on oral bacteria. J. Microbiol. (Seoul, Korea) 46, 744-750. stated that the MIC of gallic acid against S. sobrinus was 8 mg/ml. The MIC of ellagic acid (4) was 1 mg/ml. Sarabhai et al. (2013)Sarabhai, S., Sharma, P., Capalash, N., 2013. Ellagic acid derivatives from Terminalia chebula Retz. downregulate the expression of quorum sensing genes to attenuate Pseudomonas aeruginosa PAO1 virulence. PLoS One 8, 1-12. mentioned that pure ellagic acid did not exert much biofilm inhibiting effect.

According to our findings, ellagic acid (4) showed weak GTF inhibitory activity (IC50 1017.5 µM). Even though Sawamura et al. (1992)Sawamura, S., Tonosaki, Y., Hamada, S., 1992. Inhibitory effect of ellagic acid on glucosyltransferase from mutans streptococci. Biosci. Biotechnol. Biochem. 56, 766-768. suggested that the use of ellagic acid will not affect the ecological balance of oral bacterial flora, but it could be a possible anti-carries agent through its GTF inhibitory action.

Terchebulin (2) exhibited significance activity against GTF (IC50 7.5 µM), when compared to the positive control of chlorhexidine (IC50 5.8 µM). Oolong tea fraction rich in high molecular weight polyphenols inhibited the synthesis of glucan non-competitively (Matsumoto et al., 2003Matsumoto, M., Hamada, S., Ooshima, T., 2003. Molecular analysis of the inhibitory effects of oolong tea polyphenols on glucan-binding domain of recombinant glucosyltransferases from Streptococcus mutans MT8148. FEMS Microbiol. Lett. 228, 73-80.).

Chlorhexidine demonstrated the strongest activity against S. sobrinus and in addition, it showed good activity against GTF (Table 3). Nonetheless, it has been reported that chlorhexidine was found to be a cytotoxic agent to murine fibroblast cell lines, human alveolar bone cells and human osteoblastic cell line. These results approve that chlorhexidine is not cell type specific (Cabral and Fernandes, 2007Cabral, C.T., Fernandes, M.H., 2007. In vitro comparison of chlorhexidine and povidone-iodine on the long-term proliferation and functional activity of human alveolar bone cells. Clin. Oral Invest. 11, 155-164.; Giannelli et al., 2008Giannelli, M., Chellini, F., Margheri, M., Tonelli, P., Tani, A., 2008. Effect of chlorhexidine digluconate on different cell types: a molecular and ultrastructural investigation. Toxicol. In Vitro 22, 308-317.; Faria et al., 2009Faria, G., Cardoso, C.R.B., Larson, R.E., Silva, J.S., Rossi, M.A., 2009. Chlorhexidine-induced apoptosis or necrosis in L929 fibroblasts: a role for endoplasmic reticulum stress. Toxicol. Appl. Pharmacol. 234, 256-265.; Li et al., 2014Li, Y.C., Kuan, Y.H., Lee, T.H., Huang, F.M., Chang, Y.C., 2014. Assessment of the cytotoxicity of chlorhexidine by employing an in vitro mammalian test system. J. Dent. Sci. 9, 130-135.). Toxicity assay with mouse fibroblasts showed that ellagic acid, terchebulin and flavogallonic acid isolated from Terminalia avicennoides stem bark, had IC50 ≥ 1500 µg/ml as well as they did not affect the integrity of human erythrocyte membrane of the human (Shuaibu et al., 2008Shuaibu, M.N., Wuyep, P.A., Yanagi, T., Hirayama, K., Tanaka, T., Kouno, I., 2008. The use of microfluorometric method for activity-guided isolation of antiplasmodial compound from plant extracts. J. Parasitol. Res. 102, 1119-1127.).

Conclusion

In the present study, T. laxiflora wood demonstrated significant anti-cavity activity. These results justify the use of this plant for oral care in traditional African medicine. The promising results of antimicrobial and GTF inhibitory activity shown here, suggests terchebulin (2) and flavogallonic acid dilactone (1) could be considered for further pharmacological studies, evaluating the toxicity and development of a natural anticariogenic agent for dental caries.

Ethical disclosures

Protection of human and animal subjects

The authors declare that no experiments were performed on humans or animals for this study.

Confidentiality of data

The authors declare that they have followed the protocols of their work center on the publication of patient data.

Right to privacy and informed consent

The authors declare that no patient data appear in this article.

Acknowledgements

The authors thank Dr. Ashraf Mohamed from the Faculty of Forestry, Mrs. Hamza Tag EL-Sir, Botanist, Faculty of Agriculture, Department of Botany (Herbarium), University of Khartoum, Sudan for their assistance in the plants identification and authentications. EAMM acknowledge for University of Gifu, Japan.

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Publication Dates

  • Publication in this collection
    Sep-Oct 2017

History

  • Received
    27 Nov 2016
  • Accepted
    19 Apr 2017
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