Trans-resveratrol, piceatannol and gallic acid: Potent polyphenols isolated from Mezoneuron benthamianum effective as anticaries, antioxidant and cytotoxic agents

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Trans-Resveratrol, Piceatannol and Gallic acid: Potent polyphenols isolated from Mezoneuron benthamianum effective as anticaries, antioxidant and cytotoxic agents P.M. Osamudiamen , B.B. Oluremi , O.O. Oderinlo , O.O. Aiyelaagbe PII: DOI: Reference:

S2468-2276(19)30805-1 https://doi.org/10.1016/j.sciaf.2019.e00244 SCIAF 244

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Scientific African

Received date: Revised date: Accepted date:

1 June 2019 8 October 2019 13 November 2019

Please cite this article as: P.M. Osamudiamen , B.B. Oluremi , O.O. Oderinlo , O.O. Aiyelaagbe , Trans-Resveratrol, Piceatannol and Gallic acid: Potent polyphenols isolated from Mezoneuron benthamianum effective as anticaries, antioxidant and cytotoxic agents, Scientific African (2019), doi: https://doi.org/10.1016/j.sciaf.2019.e00244

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Trans-Resveratrol, Piceatannol and Gallic acid: Potent polyphenols isolated from Mezoneuron benthamianum effective as anticaries, antioxidant and cytotoxic agents 1,4

1

P.M. Osamudiamen, 2B.B. Oluremi, 3O. O. Oderinlo and 4O.O. Aiyelaagbe

Department of Chemical Sciences, Bells University of Technology, Sango-Ota, Ogun State 2

Department of Pharmaceutical Microbiology, University of Ibadan, Ibadan, Oyo State 3

Department of Chemistry, Federal University, Utuoke, Bayelsa State

4

Department of Chemistry, University of Ibadan, Oyo State, Nigeria

* Corresponding Author: +234-807-380-9762, [email protected]

ABSTRACT Phytochemical investigation of the ethyl acetate extract of the root of Mezoneuron benthamianum, a shrub commonly used as chewing stick in the southwest of Nigeria, led to the isolation of three compounds characterised as trans-resveratrol, piceatannol and gallic acid by the use of spectroscopic techniques. The anticaries activity of the polyphenols was evaluated against four oral bacteria pathogens (Streptococcus mutans, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli), isolated from clinical samples presented by patients having dental caries, while their antioxidant and cytotoxic activities were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and brine shrimp (artemia salina nauplii) bioassay techniques. The results showed that the isolated compounds exhibited very strong anticaries activity with minimum inhibitory concentration (MIC) ranging from 25 – 300 µg/ml, antioxidant activity with IC50 of 35.81, 30.35 and 11.73 µM, when compared with ascorbic acid (IC50 = 38.20 µM) and cytotoxic activity with LC50 of 7.15, 99.99 and 98.71 µg/ml for resveratrol, piceatannol and gallic acid respectively The isolated compounds demonstrated very strong anticaries, antioxidant and cytotoxic activities. This study further justifies the ethno-medicinal use of M. benthamianum as an effective tool in maintaining oral hygiene. Keywords: Mezoneuron benthamianum, polyphenols, dental caries, resveratrol, piceatannol

Introduction Dental caries or tooth decay is a disease caused by bacteria especially Streptococcus mutans (Ozden et al, 2015) and its symptoms include pain and difficulty with eating. The use of chewing sticks has been documented since ancent times (Osho and Adelani, 2012). Chewing sticks are the common means of preventing dental caries and maintaining oral hygiene in rural communities in Africa and some of these chewing sticks have been shown to demonstrate different degrees of antimicrobial activity against oral microbes. (Ugoji et al, 2000) Mezoneuron benthamianum Baill. is a shrub commonly used as a chewing stick in the southwest of Nigeria (Burkill, 1985). Traditionally, M. benthamianum is reportedly used in antiseptic cleansing and healing of ulcers and in the treating of enteralgia (Dickson et al., 2011a) It is known locally among the Yoruba people of Nigeria as “meyinro” meaning “that which makes the teeth to be strong”. Despite, the importance of M. benthamianum in African traditional medicine, there are few reports of its phytochemical constituents and biological activities especially activities against oral microbes; even though it has the potential of being a source of bioactive constituents which could serve as new leads for the development of drugs against oral infections. Polyphenols are compounds containing phenolic rings in their structure and are found in different forms in nature. They are classified on the basis of the number of phenol rings that they contain and structural elements that are attached to these rings into different groups such as phenolic acids, flavonoids, and stilbenes (Croteau et al, 2000). They function as micronutrients that complement the roles of antioxidants, vitamins and enzymes as a defense against oxidative stress caused by excess reactive oxygen species. Previous work shows the anticaries, antioxidant and cytotoxic activities of the extracts of Mezoneuron benthamianum (Osamudiamen, 2018), we hereby report the isolation and biological activities of the chemical constituents of Mezoneuron benthamianum

2.0

Experimental

2.1

Reagents and Materials

Column chromatography was carried out using Silica gel (60 – 120 and 200 – 400 mesh sizes; Fisher Scientific). Thin Layer Chromatography plates (TLC Silica gel 60 F254) were obtained from Merck, Germany. The Thin Layer Chromatography plates were detected under UV light at 254 nm and 365 nm wavelength respectively and also sprayed with Ceric Ammonium Sulphate solution followed by heating on a TLC heater at about 150 OC for few minutes.

1

H and

13

C

NMR were run on Bruker Avance III 400 MHz and 300 MHz using DMSO-d6 and MeOD as Solvent. Ultraviolet spectroscopy was determined using Labomed Inc. Spectrometer UV-VIS Double PC 8 Auto Cell Scanning Spectrophotometer UV-VIS Double Beam Model UVD-3200. Infra-Red spectroscopy were obtained using Perkin Elmer FT-IR Spectrum Two spectrometer. The mass of the compounds were determined using Agilent 1260 liquid chromatography equipped with a quaternary solvent delivery system, and Triple quad MS system (Agilent, USA) and Agilent technologies 6540 UHD Accurate mass Q-TOF Liquid chromatography-mass spectrometer (Agilent, USA). Brine shrimp (Artemia salina), eggs and salt, were purchased from Essex Marine Aquatics, UK. Mueller-Hilton agar, MacConkey agar and nutrient broth for antimicrobial assay, were all obtained from LAB M, Bury, UK while sterile normal saline was supplied by Dana Ashmina Ltd, Ibadan, Nigeria. Materials used for Brine shrimp cytotoxicity assay. 2.2

Plant collection

The plant samples, M. benthamianum.Baill. root was collected in Ibadan, Oyo State and identified and authenticated at the Department of Botany, University of Ibadan, Ibadan, Nigeria where voucher specimen is being deposited in the herbarium. (No: UIH-22401).

2.3

Extraction of Plant Material

The dried plant material of M. benthamianum (2.74 kg) was extracted by maceration with methanol (10L x 2, for 72 h each) and the extracts were concentrated using rotary evaporator under reduced pressure to give dark brownish extract (200 g). This was then dissolved in aqueous methanol and was further partitioned into hexane, ethyl acetate and aqueous methanol fractions 2.4

Fractionation and Isolation

The ethyl acetate fraction (40 g) was fractionated by column chromatography using silica gel (200-400 mesh size), and eluted with hexane in increasing proportions of ethyl acetate (5% ethyl acetate in 95% n-hexane to 100% ethyl acetate) and thereafter with ethyl acetate in increasing proportions of methanol (5% methanol in 95% ethyl acetate to 100% methanol). Twenty-four fractions were collected in all and pooled together on the basis of TLC analysis to give seven sub-fractions F1 to F7 Fractions F2 (1.5 g) eluted with ethyl acetate: hexane (40:60) was purified by column chromatography using ethyl acetate: hexane (40:60) to afford a creamy amorphous solid, compound 1(800 mg). Fraction F4 (200 mg) eluted with ethyl acetate: hexane (50:50) was further purified by column chromatography using ethyl acetate: hexane (40:60) to afford an orange powdery solid, compound 2 (80 mg) While fraction F5 (80 mg) eluted with ethyl acetate: hexane (50:50) was further purified by column chromatography using ethyl acetate: hexane (40:60) to afford a grey solid, compound 3 (40 mg). These three compounds were subjected to 1D and 2D NMR, FT-IR and Mass spectroscopic techniques to fully characterize them.

2.5

Bioassays

2.5.1

Anticaries Activities

Agar well dilution method was performed to determine anticaries activity of isolated compounds against four clinical bacterial isolates of [Streptococcus mutans (gram positive), Pseudomonas aeruginosa (gram negative), Staphylococcus aureus (gram positive) and Escherichia coli (gram negative)] that were isolated from clinical samples presented by patients having dental caries at the Dental clinic of the University of Ibadan and three typed bacterial strains [S. aureus (ATCC 29213), E. coli (ATCC 35218) and P. aeruginosa (ATCC 27855)]. The inoculum was prepared from overnight grown culture on MHB agar plates by suspending well-isolated colonies of culture in 2 ml sterile saline. 10 mg of the compounds was dissolved in 5 ml of methanol to give 2 mg/ml after which serial dilutions were prepared from the stock solution by adding 2 ml of this solution to 18ml of Nutrient Agar solution to give 0.20 mg/ml further dilutions were carried out to give 0.10, 0.05, 0.025 and 0.0125 mg/ml. All the plates including Streptococcus mutans, Pseudomonas aeruginosa, Staphylococos aureus, and Escherichia coli were incubated at 37oC overnight. The minimum inhibition concentration (MIC) of the isolated compounds was determined from the lowest concentration of samples in the well showing no growth. 2.5.2

In-vitro Antioxidant Assay

The free radical scavenging activity of the compounds was measured by the 2,2-diphenyl-1picrylhydrazyl (DPPH) method described by, Brand-Williams et al., (1995) with slight modifications. Briefly, a 0.1 mM solution of DPPH in methanol was prepared and 1.0 ml of this solution was added to 0.5 ml of the samples dissolved in methanol and using a range of 12.5-200 µg/ml. After 20 min, the absorbance was measured at 517 nm. The DPPH radical scavenging activity was calculated according to the following equation: DPPH scavenging activity (%) = [(Ao - AI)/Ao] x 100

Where Ao is the absorbance of the control and AI is the absorbance in the presence of the test substance. DPPH solution alone served as control (Ao). A graph of % inhibition against concentration was plotted and IC50 determined using the GraphPad Prism 7.0 programme. 2.5.3

Brine Shrimp cytotoxic assay

The brine shrimp lethality test (Meyer et al., 1982) was used to predict the bioactivity of the extracts and isolated compounds. Brine shrimp eggs, Artemia salina were hatched in a vessel containing distilled water to which the salt and eggs were added. The vessel was kept under an inflorescent bulb and facilitated with good aeration for 48 h at room temperature. After hatching, nauplii released from the egg shells were collected at the bright side of the vessel (near the light source) by using micropipette. The stock solution of 1000 ppm was prepared by weighing 20 mg of sample into 2 ml of 1% DMSO in sea water. Serial dilutions of 100 ppm and 10 ppm were prepared by transferring 0.2 ml of the stock solution to 1.8 ml of 1 % DMSO in sea water in another test-tube to give 100 ppm which was also further diluted accordingly to give 10 ppm. This was done in triplicate. After two days (when the shrimp larvae are ready), 4ml of sea water is added to each test tube and 10 shrimps are introduced into the test tube after which the volume is adjusted with sea water to 5 ml /test tube. The test-tubes are placed uncovered, under the lamp and the setup was allowed to stand for 24 h after which the number of survivors are counted and recorded. The results are analysed with Finney computer program for probit analysis to determine the LC50 values at 95% confidence intervals.

3

Results and Discussion

The methanol fractions was partitioned to give 5 g, 70 g and 100 g of hexane, ethyl acetate and methanol extracts respectively.

3.1

Compound 1(trans-Resveratrol) (systematic name: 3,4’,5-trihydroxy stillbene)

3.1.1

Spectra data of Compound 1

creamy powder; m.p. 260 – 262 oC; UV (MeOH) λmax 248, 257, 322, 330 nm; IR (KBr) νmax 3421 (O-H), 1633 (C=C) cm-1, HRESI-MS m/z [M]+ 228.2433; calculated for C14H12O3, 228.0786; 13C NMR (MeOD, 100 MHz ): δ. 159.57 C-3, 5, 158.27 C-4’, 141.32 C-1, 130.39 C-1’, 129.40 C-8, 128.83 C-2’, 6’, 126.95 C-7, 116.48 C-3’, 5’, 105.80 C-2, 6, 102.62 C-4; 1H NMR (MeOD, 400 MHz): δ 7.35 (d, J = 8.6 Hz, 2H, H-3’, 5’), 6.97 (d, J = 16.3 Hz, 1H, H-8), 6.81 (d, J = 16.4 Hz, 1H, H-7), 6.78 (d, J = 8.6 Hz, 2H, 2’, 6’), 6.48 (d, J = 2.1 Hz, 2H, H-2, 6), 6.19 (t, J = 2.1 Hz, 1H, H-4). 3.1.2

Characterisation of Isolated Compound 1

The structure of compound 1 was determined using 1H NMR, 13C NMR, COSY, HMBC, HSQC, Infra-red, EIMS, HRESI-MS data and by comparison with literature. The positive EIMS and HR-EIMS spectra of compound 1 gave the m/z of the (MH)+ ion at 229.0 and 229.0859, suggesting the formula of C14H12O3 (DBE=9). The number of unsaturation could be accounted for as two benzene rings and one alkene group. The 1H NMR spectrum displayed two doublet signals (δ 7.35, d, J = 8.56 Hz, H-2’, H-6’ and 6.78, d, J = 8.6 Hz, H-3’, H-5’) which are indicative of two aromatic ortho-coupled protons. It also showed another set of two doublets peaks (δ 6.97, d, J = 16.3 Hz, H-8 and 6.81, d, J = 16.44 Hz, H-7) which are indicative of coupled trans-olefinic protons. And finally, it gave a doublet and a triplet peak (δ 6.48, d, J = 2.1 Hz, H-1) and (δ 6.19, t, J = 2.1 Hz, H-4) respectively, which suggests a meta-coupled aromatic proton. The

13

C NMR spectrum showed the presence of seven aromatic methine carbons (δ

102.62 C-4, 105.80 C-2 and C-6, 116.48 C-3’ and C-5’, 128.83 C-2’ and C-6’), two olefinic methine carbons (δ 126.95 and 129.40) and five aromatic quaternary carbons (δ 130.39 C-1’, 141.32 C-1, 158.27 C-4’, 159.57 , C-3 and C-5). This accounted for the fourteen carbons. The one-bond proton-carbon observed in HSQC spectrum facilitated the assigning of the protons to

their carbons with the assistance of the long range correlations observed in HMBC and also compared with data obtained from literature (Mattivi et al, 1995).

3.2

Compound 2 (Piceatannol) (Systematic name: 3,3’,4’,5 – tetrahydroxy stillbene)

3.2.1

Spectra data of Compound 2

orange solid; m.p. 226-228 oC; UV (MeOH) λmax 250, 257, 331; IR (KBr) νmax 3511, 3346 (O-H), 2925, 2849, 1633, 1600, 1633 (C=C) cm-1, HRESI-MS m/z [M]+ 244.2472; calculated for C14H12O4, 244.0736;

13

C NMR (DMSO-d6, 75 MHz): δ 139.69 (C-1), 104.64 (C-2,6),

158.93 (C3,5), 102.07 (C-4), 125.91 ( C-7), 118.94 (C-8), 129.07 (C-1’), 113.65 (C-2’), 145.85 (C-3’), 145.97 ( C-4’), 116.02 (C-5’), 128.67 (C-6’); 1H NMR (DMSO-d6, 300 MHz): δ 6.36 (d, J = 1.6 Hz, 1H, H-2,6), 6.10 (s, 1H, H-4), 6.72 (d, J = 3.2 Hz, 1H, H-7), 6.86 (d, J = 8.7 Hz, 1H, H-8), 6.95 (d, J = 1.4 Hz, 1H, H-2’), 6.68 (d, J = 4.7 Hz, 1H, H-5’), 6.82 (s, 1H, H-6’) 3.2.2

Characterisation of Compound 2

The structure of compound 2 was established with the aid of the analysis of its 1H NMR,

13

C

NMR, COSY, HMBC, HSQC, Infra-red, EIMS, HREIMS data and by comparison with literature. The positive EIMS spectrum of compound 2 gave the (M+H)+ ion at 245.1, indicating the molecular formula of C14H12O4 (DBE = 9). The number of unsaturation is accounted for as two benzene ring and one alkene group as in compound 1. The infrared spectrum gave sharp peak at 3511 cm-1 representing a free O-H stretch and a broad peak at 3346cm-1 indicating a hydrogen bonded O-H stretching vibration,

a weak peak at 2925 cm-1 representing an

unsaturated C-H stretching vibration and an absorption peak at 1633 and 1600 representing C=C stretch due to benzene ring and alkene group respectively. The 1H NMR (DMSO-d6) spectrum revealed the presence of a singlet at δ 6.91 (2H, s, H-2 and H-6) indicating aromatic protons. The 13

C spectrum (75 MHz, DMSO-d6) for compound 2 reveals five peaks at δ 167.9 (C-7), 145.9

(C-3 and C-5), 138.4 (C-4), 120.9 (C-1) and 109.2 (C-2 and C-6). HSQC and HMBC assisted in the assigning of the protons to their respective carbons. The structure of compound 2 was thereby confirmed to be piceatannol by comparing with the values obtained in literature (Ferrigni and McLaughlin, 1984; Cardona et al., 1986). 3.3

Compound 3: (Gallic Acid) (Systematic name: 3,4,5-trihydroxy benzoic acid)

3.3.1

Spectra data of compound 3

Grey solid; m.p. 257-259 oC; UV (MeOH) λmax 277, 330; IR (KBr) νmax 3368 (O-H), 3288, 3066, 1701 (C=O) cm-1, HRESI-MS m/z [M]+ 170.0215; calculated for C7H6O5, 170.1195; 13C NMR (DMSO-d6, 75 MHz): δ. 138.44 (C-4), 167.93 (COOH), 120.91 (C-1), 145.85 (C-3,5), 109.18 (C-2,6); 1H NMR (DMSO-d6, 300 MHz): δ 9.08 (s, 1H, H-COOH), 6.91 (s, 2H, H2,6), 3.40 (s, 3H, OH). 3.3.2

Characterisation of Compound 3

The structural elucidation of compound 3 was based on its IR, 1H and 13C NMR spectral data and by comparison with data available in literature. The Infra-red spectrum gave a broad absorption at

3368 and 3288 cm-1 indicating hydrogen bonded O-H stretch, 3066 representing an

unsaturated C-H stretching vibration, 1701 cm-1 indicates a carboxylic carbonyl group and an absorption peak at 1617 cm-1 indicates an aromatic C=C stretch. The 1H NMR (DMSO-d6) spectrum revealed the presence of a singlet at δ 6.91 (2H, s, H-2 and H-6) indicating aromatic protons. The 13C spectrum (75 MHz, DMSO-d6) for compound 3 revealed five peaks at δ 167.9 (C-7), 145.9 (C-3 and C-5), 138.4 (C-4), 120.9 (C-1) and 109.2 (C-2 and C-6). HSQC and HMBC assisted in the assigning of the protons to their respective carbons. The structure of compound 3 was thereby confirmed to be gallic acid by comparing it with the values obtained in literature (Chanwitheesuk et al, 2007).

3.4

Anticaries Assay

The Minimum Inhibitory Concentration of the three isolated compounds (Fig. 2) showed that they were active against the seven strains of microorganisms tested against with resveratrol having the highest activity with an MIC value of 0.025 mg/ml against the seven pathogens and 0.2 µg/ml against P. aeruginosa (ATCC 27855), followed by piceatannol and gallic acid with MIC values in the range of 0.025 – 0.3 and 0.025 – 0.1 mg/ml respectively. Although several compounds have been investigated against dental caries, only a few isolated from plants are available because of effectiveness, stability, odour, taste and economic feasibility. The in vitro anticaries activities of some polyphenols have been investigated against streptococus mutans for example, Xanthorrhizol, a polyphenol isolated from Curcuma xanthorrhiza Roxb., had a concentration of 5 µmol/L completely inhibiting the formation of S.mutans (Murphy, 1999), while malvin isolated from the flowers of Alcea longipedicellata I, Kuwanon G isolated from the root bark of Morus alba L, Guaijaverin isolated from the leaves of Psidium guaiava L and Dihydrobiochanin A isolated from Swartzia polyphylla DC had MIC values of 160, 8, 4000 and 50 µg/ml against S. mutans (Ooshima et al., 1993; Ooshima et al., 1998; Osawa et al., 1992 and Saito 1990) . The anticaries activities of the polyphenols isolated in this study are comparable with the results of polyphenols in previous studies and even had better activity than most polyphenols as resveratrol, picetannol and gallic acid exhibited MIC values of 25, 37.5 and 62.5 µg/ml against S. mutans. These compounds thereby justify the use of M. benthamianum traditionally in maintaining oral hygiene.

3.3

Antioxidant Assay

The result of the antioxidant activity of the isolated compounds is shown in Fig. 3. The result showed that gallic acid exhibited a very strong antioxidant activity with IC50 of 11.73 µM, while resveratrol and piceatannol equally had strong antioxidant activity with IC50 of 35.81 and 30.35

µM respectively. Gallic acid has been established to possess the highest antioxidant potential having reduced six (6) DPPH molecules per radical (Brand-Williams et, al., 1995). Apart from DPPH assay, gallic acid emerged as the strongest antioxidant in other well-known assays as well, such as Trolox equivalent antioxidant capacity (TEAC I-III) assay, Total radical-trapping antioxidant parameter (TRAP) assay, Photochemiluminescence (PCL) assay and Ferric reducing ability of plasma (FRAP) assay. It is found to be a better antioxidant than popular antioxidants like ascorbic acid, Trolox and uric acid (Schlesier et al, 2002). It has been demonstrated as the chief antioxidant component responsible for the antioxidant properties of a number of plant extracts (Veluri et al, 2006; Chia,et al, 2010; Bhadoriya et al, 2012; Palafox-Carlos et al, 2012 and Gonzalez-Abuín et.al, 2014). Similarly, resveratrol and piceatannol have been shown to possess strong antioxidant activities in a number of studies and piceatannol has been shown to possess a stronger antioxidant activity than resveratrol. (He and Yan, 2013). The presence of gallic acid, piceatannol and resveratrol in M. benthamianum is a very good factor responsible for its effectiveness in promoting oral hygiene traditionally.

3.4

Cytotoxic Assay

The brine shrimp cytotoxic assay is a simple bench-top assay that is beneficial in determining the preliminary bioactivities of the isolated compounds based on the LC50 values obtained (Solanki and Selvanayagam, 2013). Compounds having LC50 values < 200 µg/ml are considered active (Anderson et al., 1991, Meyer et al., 1982). The result of the brine shrimp cytotoxic activity of the isolated compounds (Fig. 4.0) revealed that trans-resveratrol has the highest activity with LC50 value of 7.15 µg/ml

Piceatannol and gallic acid had similar LC50 values of 99.99 and

98.71 µg/ml respectively. However, the brine shrimp cytotoxic activities of the polyphenols are comparable to their previously reported values (Ferrigni and McLaughlin, 1984; Wanjala and Majinda, 2001 and Urrea-Bulla et al., 2004).

4.0

Conclusion

This is the first report of the anticaries activities of resveratrol and piceatannol, which are polyphenols isolated from the root of Mezoneuron benthamianum. The polyphenols demonstrated a marked inhibitory activity against the bacterial organisms, in addition to antioxidant and cytotoxic activities. The efficacy of the use of M. benthamianum traditionally in oral hygiene could be attributed to the presence of these bioactive compounds, which have shown to be very potent. This result further justifies the ethno-medicinal use of M. benthamianum as a therapeutic agent in preventing dental caries and maintaining oral hygiene.

Acknowledgement The authors thank the Head of Department of Pharmaceutical Microbiology, for granting the bench space where part of this work was carried out and Mr. Sunday, the technologist for his selfless service. References Anderson, J.E., Goetz, C.M., McLaughlin, J.L., Suffness, M. (1991). A blind comparison of simple bench-top bioassays and human tumor cell cytotoxicities as antitumor prescreens. Phytochemical Analysis 2: 107-111. Bhadoriya, U., Sharma P. and Solanki, S. S.2012. In Vitro Free Radical Scavenging Activity of Gallic Acid Isolated from Caesalpinia Decapetala Wood. Asian Pacific Journal of Tropical Disease S833-S836. Brand-Williams, W. Cuvelier, M.E. Berset, C. 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm. – Wiss. Technology. 28: 25-30 Burkill, H.M. 1985. The Useful Plants of West Tropical Africa 2nd Ed. Royal Botanic Gardens Kew, Vol 1: 91 Cardona, M.L., Fernandez, I.F., Garcia. M.B. and Pedro, J.R. 1986. Synthesis of Natural Polyhydroxystilbenes. Tetrahedron 42.10: 2725 – 2730 Chanwitheesuk, A., Teerawutgulrag, A., Kilburn, J.D. and Rakariyatham, N. 2007. Antimicrobial Gallic acid from Caesalpinia mimosoides Lamk. Food Chemistry. 100:1044-1048.

Chia, Y. C., Rajbanshi, R., Calhoun, C. and Chiu, R. H. 2010. Anti-Neoplastic Effects of Gallic Acid, A major component of Toona sinensis leaf Extract, on Oral Squamous Carcinoma cells. Molecules 15.11:8377-8389. Croteau, R., Kutchan, T.M. and Lewis, N.G. 2000. Natural products (secondary metabolites). Biochemistry & molecular biology of plants. Buchanan B, Gruissem W, Jones R (Eds) American Society of Plant Physiologists, Rockville. 1250 Dickson, R. A., Annan, K. and Komlaga, G. 2011a. Pharmacognostic Standardization of the Leaves and Root Bark of Caesalpinia benthamiana. Pharmacognosy Journal 224 3.24: 29 – 36. Ferrigni N.R. and McLaughlin J.L. 1984. Use of Potato Disc and Brine Shrimp Bioassays to Detect Activity and Isolate Piceatannol as the Antiluekemic Principle from the seeds of Euphorbia lagascae. Journal of Natural Products. 47(2): 347-352 González-Abuín, N., Martínez-Micaelo, N., Margalef, M. Blay, M. Arola-Arnal, A. Muguerza, B. Ardévol A. and Pinent, M. 2014. A Grape seed extract increases active glucagol-like pettide-1 after an oral glucose load in rats. Food and Function 5.9: 2357-2364. He, S., and Yan, X. 2013. From resveratrol to its derivatives: new sources of natural antioxidant. Current medicinal chemistry, 20.8:1005-1017. Laudenbach, J.M and Simon, Z (2014). "Common Dental and Periodontal Diseases: Evaluation and Management". The Medical Clinics of North America. 98 (6): 1239–1260 Mattivi F, Reniero F and Korhammer S. 1995. Isolation, Characterization and Evolution in Red wine Vinification of Resveratrol Monomers. Journal of Agricultural and Food Chemistry 43:1820 - 1823 Meyer, B.N, Ferrigni N.R, Putnam J.E, Jacobsen L.B, Nichols D.E and McLaughlin J.L (1982) Brine Shrimp: A convenient General Bioassay for Active Plant Constituents. Planta Medica 45:31-34 Murphy, C.M. (1999). Plant products as antimicrobial agents. Clin. Microbiol., 12, 564-582. Ooshima, T.; Minami, T.; Aono, W.; Izumitani, A.; Sobue, S.; Fujiwara, T.; Kawabata, S.; Hamada, S. (1993) Oolong tea polyphenols inhibit experimental dental caries in SPF rats infected with mutans streptococci. Caries Res., 27, 124-129. Ooshima, T.; Minami, T.; Matsumoto, M; Fujiwara, T.; Sobue, S.; Hamada, S. (1998) Comparison of the cariostatic effects between regimens to administer oolong tea polyphenols in SPF rats. Caries Res., 32, 75-80.

Osamudiamen P.M. (2018). Chemical Constitutents, Anticaries, Antioxidants and Cytotoxic activities of the Root of Mezoneuron bentamianum BAILL. (Unpublished Doctoral thesis). University of Ibadan, Ibadan, Nigeria. Osawa, K., Yasuda, H.,

Maruyama, T., Morita, H,

Takeya, K., Itokawa, H. (1992)

Isoflavanones from the heartwood of Swartzia polyphylla and their antibacterial activity against cariogenic bacteria.Chem. Pharm. Bull., 40, 2970-2974. Osho A, Adelani O.A. (2012) The antimicrobial effect of some selected Nigerian chewing sticks on clinical isolates of Candida species. Journal of Microbiology Research. ;2(1):1-5. Ozden, F.O., Ozgonenel, O., Ozden, B., Aydogdu, A., 2015. Diagnosis of periodontal diseases using different classification algorithms: a preliminary study. Nigerian Journal of Clinical Practice 18: 416–421. Palafox-Carlos, H., Gil-Chávez, J., Sotelo-Mundo, R., Namiesnik, J., Gorinstein S. and González-Aguilar, G.A. 2012. Antioxidants interactions between Major Phenolic Compounds Found in "Ataulfo" Mango Pulp: Chlorogenic, Gallic, Protocatechuic and Vanillic Acid. Molecules 17.11: 12657-12664. Saito, N. (1990) Anti-caries effects of polyphenol compound from Camellia sinensis. Nichidai Koko Kagaku, 16, 154-163. Schlesier, K., Harwat, M., Böhm, V., and Bitsch, R. (2002). Assessment of antioxidant activity by using different in vitro methods. Free radical research, 36(2), 177-187 Solanki, S.S and Selvanayagam, M. (2013) Phytochemical screening and study of predictive toxicity of certain medicinal plants and extracts using brine shrimp. Herb Tech Ind; 10(1):1-4. Ugoji E, Egwari LO, Obisesan B. (2000)Antibacterial activities of aqueous extracts of ten African chewing sticks on oral pathogens. Nig. Journal of Internal Medicine. ;3(1):7-11. Urrea-Bulla, A and Moreno-Murillo, B (2004). Biological Activity of Phenolic Compounds from Alchornea glandulosa. Fitoterepia 75(3-4):392-394 Veluri, P., Singh, R. P., Liu, Z., Thompson, J. A., Agarwal, R. and Agarwal,C. (2006). Fractionation of grape seed extract and identification of Gallic acid as one of the major active constituents causing growth inhibition and apoptotic death of DU145 human prostate carcinoma cells. Carcinogenesis 27.7: 1445-1453. Wanjala, C. C., and Majinda, R. R. 2001. A new stilbene glycoside from Elephantorrhiza goetzei. Fitoterapia, 72.6: 649-655.

OH

OH

OH

OH

OH

HO

HO

HO

HO HO

resveratrol

HO

Figure 1

Piceatannol

O

Gallic acid

0.3 0.3

0.35

0.3

0.25

P. aeruginosa

0.2

P. aeruginosa* (ATCC 27855)

0.2

S. aureus

0.15

S. aureus* (ATCC 29213) 0.1 0.1 0.1 0.1

MIC mg/mL

S. mutans

E. coli

0.025 0.025

0.025 0.025 0.025 0.025

0.025 0.025 0.025 0.025 0.0375

0.05

0.025 0.025

0.0625

0.1

0 Resveratrol

Piceatannol

Gallic acid

Figure 2: Minimum inhibitory Concentration (MIC) of Isolated Compounds

E. coli* (ATCC 35218)

50

IC50 µM

40 30

38.2

35.81

Resveratrol

30.35

Piceatannol Gallic Acid

20 11.73

Ascorbic Acid

10 0

Isolated Compounds

Figure 3: In-vitro Antioxidant Activity (IC50) of Isolated Compounds from M. benthamianum

120

LC50 µg/mL

100 80

Resverastrol

60

Piceatannol

40

Gallic Acid

20 0

7.15 Isolated Compounds

Fig. 4: Brine shrimp in-vitro cytotoxic activities of isolated compounds from M. benthamianum