New triterpene and new flavone glucoside from Rhynchospora corymbosa (Cyperaceae) with their antimicrobial, tyrosinase and butyrylcholinesterase inhibitory activities

Phytochemistry Letters 16 (2016) 121–128

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New triterpene and new flavone glucoside from Rhynchospora corymbosa (Cyperaceae) with their antimicrobial, tyrosinase and butyrylcholinesterase inhibitory activities Annie Laure Ngankeu Pagninga,b , Jean-de-Dieu Tamokouc , Mehreen Lateefd, Léon Azefack Tapondjoua , Jules-Roger Kuiatec , David Ngnokama,* , Muhammad Shaiq Alib,* a

Laboratory of Environmental and Applied Chemistry, Department of Chemistry, Faculty of Science, University of Dschang, P. O. Box 67 Dschang, Cameroon International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan c Laboratory of Microbiology and Antimicrobial Substances, Department of Biochemistry, Faculty of Science, University of Dschang, P. O. Box 67 Dschang, Cameroon d Pakistan Council of Scientific and Industrial Research Laboratories Complex, Karachi 75280, Pakistan b

A R T I C L E I N F O

Article history: Received 14 November 2015 Received in revised form 15 March 2016 Accepted 24 March 2016 Available online xxx Keywords: Rhynchospora corymbosa Triterpene Flavone glucoside Antimicrobial activity Antityrosinase activity Antibutyrylcholinesterase activity

A B S T R A C T

The CH2Cl2-MeOH (1:1) extract of the whole plant of Rhynchospora corymbosa (L.) Britton, led to the isolation of two new secondary metabolites 1, 2 (1 and 2a) together with nine known compounds 3–11. The flavone named tricin (11) was partially modified by esterification reaction to three semi-synthetic derivatives 11a–11c). Their structures were elucidated on the basis of spectroscopic analysis. The antimicrobial, tyrosinase and butyrylcholinesterase inhibitory activities revealed that, extract and compounds 1, 2a, 6, 11, 11a-c exhibited variable MICs (1–512 mg/mL) and significant antimicrobial activity, depending on the microbial species; Antifungal activity of 11 and 11c was more important than that of nystatine used as reference antifungal drugs. The inhibitory activity of 4 (IC50 = 43.28 mg/mL) was higher than that of the kojic acid (IC50 = 49.62 mg/mL) which is known tyrosinase inhibitor. The extract, compounds 1, 7–10, and 11a-c exhibited significant (p < 0.05) differences in the concentrations of 50% BuChE inhibition (IC50). These results suggest that the, R. corymbosa could be exploited as a potential source of natural antimicrobial, and antibutyrylcholinesterase agents as well as tyrosinase inhibitors. ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General experimental procedures . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Plant Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Extraction and isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. New compounds information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Antimicrobial assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. 3.5.1. Bacterial and fungal strains . . . . . . . . . . . . . . . . . . . . . . . . Preparation of microbial inoculum . . . . . . . . . . . . . . . . . . 3.5.2. Determination of the minimum inhibitory concentrations 3.5.3. In vitro Butyrylcholinesterase (BuChE) inhibition assay . . . . . . . . . 3.6. 3.7. In vitro tyrosinase inhibition assay . . . . . . . . . . . . . . . . . . . . . . . . .

................................................. ................................................. ................................................. ................................................. ................................................. ................................................. ................................................. ................................................. ................................................. ................................................. (MICs), and minimum microbicidal concentrations (MMCs) ................................................. .................................................

* Corresponding authors. E-mail addresses: [email protected], [email protected] (D. Ngnokam), [email protected] (M.S. Ali). http://dx.doi.org/10.1016/j.phytol.2016.03.011 1874-3900/ ã 2016 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

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122 123 126 126 126 126 127 127 127 127 127 127 127

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3.8. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

1. Introduction The genus Rhynchospora Vahl. (Cyperaceae) is widely distributed in the Americas, primarily in warm temperate zones (southeastern United States) and the Neotropics, with about 270 species worldwide (Strong, 2006). Many species of this genus are used as medicinal plants in the different folk medicine (Jimenez et al., 2001; Diallo et al., 2002; Magassouba et al., 2007). Phenolic

compounds such as flavonoids, stilbenoids, coumaran, quinones and sesquiterpenes were found to be the active principles of the studied species of this genus (Harborne et al., 1985; Nyasse et al., 1988; Williams and Harborne,1977; Morimoto et al., 1999; Meng et al., 2001; Yamada et al., 2006; Ito et al., 2012). In the course of our search for bioactive compounds from cameroonian medicinal plants, we carried out the phytochemical investigation of Rhynchospora corymbosa (L.) Britton, and the antimicrobial,

Fig. 1. Chemical structures of compounds 1–11c.

A.L. Ngankeu Pagning et al. / Phytochemistry Letters 16 (2016) 121–128

tyrosinase and butyrylcholinesterase inhibitory activities of the isolated and semi-synthetic compounds as well as the whole plant extract. 2. Results and discussion The CH2Cl2/MeOH (1:1) extract of the whole plant of Rhynchospora corymbosa was subjected to column chromatography over silica gel to afford unreported triterpene oleanane 3-(30 Rhydroxy)-hexadecanoate (1) and semi-synthetic flavone derivative (2a) along with nine known compounds as b-sitosterol (3), glycoside of b-sitosterol (4), oleanolic acid 5, trans-cinnamic acid (6) (Kim et al., 2015), dendrotriol (7) (Shaaban et al., 2011), (24R)24-ethyl-5a-cholestane-3b,5,6b-triol (8) (Taro et al., 1991), glycerol (9) (Uhal and Longmore, 1988), Docosanoic acid 2-hydroxy-1-hydroxymethyl-ethyl ester (10) (Graça and Pereira, 2000; Su et al., 2013), tricin (11) (Bhattacharyya et al., 1978), triacetyl tricin (11a) (Gulati and Venkataraman, 1933), diacetyl tricin (11b) (Anderson and Perkin, 1931), monoacetyl tricin (11c). Their structures (Fig. 1) were elucidated by analysis of their NMR Table 1 1 H- and

13

123

and MS data and by comparison of these data with those reported in the literature. Compound 1 was obtained as a colorless powder with mp 104–105  C, [a]D + 12.1 (c = 2.95  106, CH2Cl2). Its molecular formula C46H80O3, corresponding to 7 of unsaturation was determined from HREIMS at m/z 680.6039 [M]+ (calcd 680.6107 for C46H80O3) and TOFMS at m/z 681.42 [M + H]+. This molecular formula would contained a palmitic acid group, which was confirmed by the presence of the ion fragment at m/z 409 [M-C16H31O3]+ in its EIMS spectrum. The base peak characteristic of oleanane series was depicted at m/z 218 [C16H26]+ (Elias et al., 1997; Ragasa et al., 2012; Fingolo et al., 2013). The IR absorption bands at 1708 and 3449 cm1 indicated the presence of carbonyl and hydroxyl groups, respectively. The triterpenic skeleton of 1 was confirmed by its 1H NMR spectrum on which signals at d 0.85 (Me-29/Me-30), 0.86 (Me-24/Me-28), 0.87 (Me-26), 0.95 (Me-23/Me-25) and 1.11 (Me-27) corresponding to the eight characteristic methyl groups were observed. These resonances along with olefinic proton at d 5.16 (t, J = 3.5 Hz) were agree with the oleanane serie (Mahato and Kundu, 1994). Further 1H NMR

C NMR Spectral Data of 1 in CDCl3, and its HMBC and COSY correlations.

Atom 13

C (125 MHz)

1 2

38.1 23.5

3 4 5 6 7

81.3 37.7 55.2 18.2 32.5

8 9 10 11

39.7 47.1 36.8 23.5

12 13 14 15

121.5 145.2 41.6 26.8

16

26.1

17 18 19 20 21

32.4 47.5 46.7 31.0 34.6

22

36.5

23 24 25 26 27 28 29 30 (C-10 ) or COOCH2COO-

16.7 28.0 16.7 15.5 25.9 28.3 33.3 23.6 172.8 41.5

1

H (500 MHz)

1.60 (2H, m, H-1) 1.51 (1H, m, H-2a) 1.85 (1H, m, H-2b) 4.54 (dd, J = 7.0, 9.0 Hz)

HMBC

COSY

81.3

23.5, 37.6, 55.2, 172.8

0.81 (1H, m, ov. H-5) 1.51 (2H, m, H-6) 1.30 (1H, m, H-7a) 1.50 (1H, m, H-7b) 1.63 (1H, m, H-9) 1.51 (1H, m, H-11a) 1.85 (1H, m, H-11b) 5.16 (t, J = 3.5 Hz)

1.85 $ 5.16 23.5, 41.6, 47.1, 145.2

5.16 $ 1.85

2.48 (dd, H- 2a0 ,J = 3.0, 16.0 Hz) 2.38 (dd, H- 2b0 , J = 9.0, 16.5 Hz)

68.1, 172.8

2.48 $ 3.98 2.38 $ 3.98 2.38 $ 2.48 3.98 $ 2.38 3.98 $ 2.48

0.94 (1H, m, H-15a) 1.85 (1H, m, H-15b) 1.72 (1H, m, H-16a) 1.94 (1H, m, H-16b) 1.94 (1H, m, H-18) 1.62 (1H, m, H-19) 1.11 (1H, m, H-21a) 1.23 (1H, m, H-21b) 1.48 (m) 1.38 (m) 0.95 (s) 0.86 (s) 0.95 (s) 0.87 (s) 1.10 (s) 0.86 (s) 0.85 (s) 0.85 (s)

CHOH-

68.1

3.98 (m, H- 30 )

25.9, 172.8

CH2

37.1

(CH2)n CH3

22.6- 31.9 14.1

2.48 (dd, H- 4a0 , J = 3.0, 16.0 Hz) 2.38 (dd, H- 4b0 , J = 9.0, 16.5 Hz) 1.23 (br s, H-50 to H-150 ) 0.86 (s, H-160 )

25.4

124

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analysis indicates also resonances for two oxymethine protons at d 4.54 (1H, dd, J = 7.0, 9.0 Hz, H-3) and 3.98 (1H, m, H-30 ). Additionally, a broad singlet signal at d 1.23 (H50 -H150 ), the methylene protons at d 2.38 (2H, dd, J = 3.0, 16.0 Hz, H20 a-H40 a) and 2.38 (2H, dd, J = 3.0, 16.0 Hz, H20 b-H40 b) together with one methyl singlet at d 0.86 were deduced from the presence of fatty acid substituent (Furukawa et al., 2002). 13 C NMR spectral analysis (Table 1) gave resonances for 46 carbons including two olefinic carbons at d 121.5 (C-12) and 145.2 (C-13); two oxymethine at d 81.3 (C-3) and 68.1 (C-30 ); one carbonyl ester at d 171.8 (C-10 ); nine methyl at d 16.7 (C-23/C-25); 28.0 (C-24); 15.5 (C-26); 25.9 (C-27); 28.3 (C-28); 33.3 (C-29); 23.6 (C-30) and 14.1 (C-160 ); twenty-three methylene at d 38.1 (C-1); 23.5 (C-2/C-11); 18.2 (C-6); 32.5 (C-7); 26.8 (C-15); 26.1 (C-16); 46.7 (C-19); 34.6 (C-21); 36.5 (C-22); 41.5 (C-20 ); 37.1 (C-40 ); 22.6–31.9 (C-50 -C-150 ); three methine at d 55.2 (C-5); 47.1 (C-9); 47.5 (C-18); six quaternary carbons at d 37.6 (C-4); 36.8 (C-10); 41.6 (C-14); 32.4 (C-17); 31.0 (C-20); 39.7 (C-8). The COSY 1H-1H analysis indicated correlations between H1-11/H-12; H-20 /H-30 ; H-30 /H-40 . The long chain of fatty acid ester substituent was located at C-3 on the basis of long-range HMBC correlation between the oxymethine proton (H-3) and the carbonyl carbon of the ester at d 171.8 (C-10 ). Furthermore, this oxymethine proton H-3 gave longrange correlation with C-23 and C-24. The olefin was assigned to C-12 due to the long-range correlations between the olefinic proton at d 5.16 (H-12) and C-11-C-14 and C-18. The oxymethine at 3.98 was located at C-30 based on long- range correlations between this proton and C-40 and C-10. All long-range correlations observed in HMBC spectrum were consistent with the structure of 1. Horeau’s method (Horeau, 1962, 1977) was applied to 1 in order to determine the configuration at C-30 . A mixture of 1 with an excess of 2-phenylbutyric anhydride and DMAP in chloroform showed an immediate evolution of the optical rotation in the (+) sense, thus including the preferential esterification by the () antipode of the acid. Silica gel column chromatography coupled with an optical rotation detector (Chiral detector: Knauer France, reference: 1000) allowed the isolation of dextrorotatory 2-phenylbutyric acid. According to the Horeau’s method, when (+)-(S)-2-phenylbutyric acid accumulates in the mixture (i.e. when the (+)-(R)-acid is the preferential esterifying acid), the C-30 secondary hydroxyl has the (R) configuration. Therefore, the chemical structure of 1 was determined to be oleanane 3-(30 Rhydroxy)-hexadecanoate. Compound 2a was obtained as yellowish powder from acetone, mp 172–173  C, [a]D  6.8 (c = 2.84  107, CH2Cl2). It reacted positively to FeCl3 reagent, suggesting the presence of phenolic hydroxyl group in the molecule. Its molecular formula established as C33H34O17 by the HREIMS spectrum displayed a molecular ion peak at m/z 702.1801 [M+] (calcd 702.1796 for C33H34O17) and TOFMS spectrum at m/z 703.13 [M + H]+. This formula accounted for 17 degree of unsaturation. The UV [lmax (log e): 278 (4.12) nm] and the IR [(KBr)] nmax spectrum exhibited three significant absorption bands due to the hydroxy group (OH), a,b-unsaturated ketone (C¼O) and aromatic ring (C¼C) at 3699,1660, 1496 cm1 respectively. The 1H-NMR spectrum of 2a exhibited a characteristic proton signal at dH 12.60 corresponding to a chelated hydroxy group at C-5. In addition, five aromatic protons were observed on this spectrum, assignable to H-3 (d 6.56, 1H, s); H-8 (d 6.52, 1H, s); H-60 (d 7.70, 1H, dd, J = 2.5; 9 Hz); H-50 (d 7.13, 1H, d, J = 9 Hz); H-20 (d 7.60, 1H, d, J = 2.5 Hz). This 1H-NMR spectrum (Table 2) also showed the presence of glucose moiety with the anomeric proton at d 5.16 (1H, d, J = 7.9 Hz), a methylene group at d H 4.18 (1H, dd, J = 2.5; 12.5 Hz) and 4.28 (1H, dd, J = 5.5; 12.5 Hz). Other protons (H-200 -H-500 ) of the sugar moiety were observed between 3.94 and 5.31 ppm.

Table 2 1 H- and 13C NMR Spectral Data of 2a in CDCl3, and its HMBC and COSY correlations. Atom 13 C (125 MHz)

1 2 3 4 5 6 7 8 9 10 10 60 50 4' 30 20

1

162.3 105.2 182.5 153.2 132.9 158.9 90.6 153.1 106.2 126.5 125.1 115.4 150.9 140.5 121.6

H (500 MHz)

HMBC

6.56 (s)

106.2, 162.3, 182.5

6.52 (s)

106.2, 132.9, 158.9,

7.70 (dd, J = 2.5, 9 Hz) 121.5, 150.9, 7.13 (d, J = 9 Hz) 126.5, 150.9, 140.5

7.60 (d, J = 2.5 Hz)

COSY

7.70 $ 7.13 7.13 $ 7.70

125.1, 140.5, 162.3, 150.9

400 -O-Glc 100 200 300 400 500

98.3 70.4 72.4 68.1 72.3

5.16 (m) 5.31 (m) 5.31 (m) 5.16(m) 3.94 (m)

150.9, 70.5, 72.4

600

61.8

4.18 (dd, J = 2.5, 12.5 Hz) 4.28 (dd, J = 5.5, 12.5 Hz) 3.95 (s) 3.90 (s) 2.01 (s) 2.01 (s) 2.07(s) 2.04 (s) 2.01 (s)

170.4, 72.3, 68.1

6- OMe 7-OMe 50 -OCOCH3 200 -OCOCH3 300 -OCOCH3 400 -OCOCH3 600 -OCOCH3 50 -OCOCH3 200 -OCOCH3 300 -OCOCH3 400 -OCOCH3 600 -OCOCH3

60.9 56.4 20.7 20.6 20.5 20.5 20.4 170.4 170.1 169.5 169.4 168.7

5.16 $ 5.31 5.31 $ 5.16

3.94 $ 5.16 3.94 $ 4.28 4.18 $ 4.28 4.28 $ 3.94

158.9 132.9 170.4 170.1 169.5 169.4 168.7

The 13C-NMR spectrum (Table 2) showed thirty-three carbon signals among them, fifteen belonging to the flavone skeleton (Harborne and Baxter, 1999). The DEPT 135 spectrum revealed the presence of five acetyl group (CH3CO) between d 20.7 and 20.4 ppm; two methoxy group at d 60.9 (6-OMe) and 56.4 (7OMe) ppm and one methylene at 61.8 (C-600 ). From the DEPT 90 spectrum five sp2 methine carbon were accounted at d 125.1 (C-60 ); 121.6 (C-20 ); 115.4 (C-50 ); 105.3 (C-3); 90.7 (C-8) assigned to the flavone skeleton and five sp3 methine carbon assigned to glucose moiety at d 98.3 (C-100 ); 72.4 (C-300 ); 72.3 (C-500 ); 70.4 (C-200 ) and 68.1 (C-400 ) (Agrawal, 1992). The sugar moiety position at C-40 was confirmed by HMBC correlation observed between H-100 and C-40 . Hence compound 2a was identified as 30 -acetoxy-6,7-dimetoxy-40 (200 ,300 ,400 ,600 -tetraacetylglucopyranosyl)flavone (Fig. 1). The antimicrobial activity of CH2Cl2-MeOH (1:1) extract and isolated compounds from R. corymbosa as well as their novel semisynthetic glycoside flavone derivative were examined by broth micro dilution susceptibility assay against three bacterial and three yeast species selected on the basis of their relevance as human pathogens. The experiments revealed that the CH2Cl2-MeOH (1:1) extract and compounds 1, 2a, 6, 11, 11a-c exhibited variable MICs (1 to 512 mg/mL) and significant antimicrobial activity, depending on the microbial species (Table 3). Compound 11c was the most active compound (i.e. had the lowest MIC values) against bacteria and yeasts (MIC = 1–4 mg/mL) followed in decreasing order by 11

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125

Table 3 Antimicrobial activity (MIC and MMC in mg/mL) of R. corymbosa extracts, isolated compounds and semi-synthetic compounds. Samples

Inhibition parameters

Pseudomonas aeruginosa PA01

Escherichia coli ATCC8739

Shigella flexneri

Cryptococcus neoformans IP Candida albicans ATCC Candida 90526 9002 parapsilosis

CH2Cl2-MeOH (1:1) extract

MIC

128

64

64

512

256

256

MMC MMC/MIC MIC MMC MMC/MIC MIC MMC MMC/MIC MIC MMC MMC/MIC MIC MMC MMC/MIC MIC MMC MMC/MIC MIC MMC MMC/MIC MIC MMC MMC/MIC MIC MMC MMC/MIC MIC MMC MMC/MIC MIC MMC MMC/MIC

256 2 16 16 1 8 8 1 64 64 1 4 4 1 8 16 2 16 16 1 2 4 2 0.5 2 4 2 2 1 / / /

64 1 16 16 1 4 8 2 16 16 1 2 4 2 4 4 1 8 16 2 2 2 1 2 2 1 16 16 1 / / /

64 1 8 16 2 4 4 1 16 16 1 2 2 1 2 2 1 8 8 1 1 2 2 0.50 0.50 1 4 4 1 / / /

512 1 16 32 2 16 16 1 64 64 1 8 16 2 32 32 1 64 64 1 4 4 1 / / / / / / 8 8 1

512 2 16 16 1 8 8 1 32 64 2 4 4 1 16 16 1 64 128 2 2 2 1 / / / / / / 4 4 1

512 2 16 16 1 8 8 1 32 32 1 4 4 1 16 16 1 64 64 1 2 4 2 / / / / / / 8 8 1

1

2a

6

11

11a

11b

11c

Ampicillin

Ciprofloxacin

Nystatin

nd: not determined;/: not tested. 3, 4, 5, 7, 8, 9 and 10 were not active at concentrations up to 256 mg/ml, MIC: minimum inhibitory concentration; MMC: minimum microbicidal concentration.

(MIC = 2–8 mg/mL), 2a (MIC = 4–16 mg/mL), 11a (MIC = 2–32 mg/ mL), 1 (MIC = 8–16 mg/mL), 11b (MIC = 8–64 mg/mL), 6 (MIC = 32– 64 mg/mL), and CH2Cl2-MeOH (1:1) extract (MIC = 64–512 mg/mL). The antifungal activity of compounds 11c and 11 was more important than that of nystatine, used as reference antifungal drugs while the antibacterial activities of compounds 11c, 11, 11a, 2, 11b, and 6 were in some cases equal or higher than those of ciprofloxacin and ampicillin. These findings highlight the good antimicrobial activities of the tested samples and suggest that their administration may represent an alternative treatment for infections caused by the studied microorganisms. No activity was noticed for compounds 3–5, 7–9 and 10 against all the tested microorganisms (results not shown). The lowest MIC value of 1 mg/ mL was recorded on Shigella flexneri with compound 11 while the lowest MMC value of 2 mg/mL was obtained on Shigella flexneri with compounds 11c, 11 and 11a and on Candida albicans and Escherichia coli with compound 11c. However, the highest MIC value 512 mg/mL was obtained on Cryptococcus neoformans with CH2Cl2-MeOH (1:1) extract while the highest MMC value of 512 mg/mL was recorded on Cryptococcus neoformans, Candida albicans and Candida parapsilosis with CH2Cl2-MeOH (1:1) extract. A lower MMC/MIC ( 4) value signifies that a minimum amount of sample is used to kill the microbial species, whereas, a higher MMC/MIC (>4) value signifies the use of comparatively more amount of sample for the control of any microorganism (Djouossi et al., 2015). The findings of the present study suggest that the active samples possess microbicidal activity against the sensitive microorganisms. Tyrosinase inhibitors are chemical agents capable of reducing enzymatic reactions such as food browning and melanisation of

human skin (Yoshimura et al., 2005). Several compounds such as kojic acid and arbutin are used as strong tyrosinase inhibitors but these compounds certain adverse effects including gastrointestinal disturbances and liver damage (Chiari et al., 2011; Souza et al., 2012). Therefore, there is considerable interest in the development of new natural antityrosinase agents. The antityrosinase activity of the CH2Cl2-MeOH (1:1) extract and isolated compounds from R. corymbosa as well as their novel semi-synthetic

Table 4 Butyrylcholinesterase and Tyrosinase inhibition activities (IC50 in mM) of Rhynchospora corymbosa extracts, isolated compounds and semi-synthetic compounds. CH2Cl2-MeOH (1:1) extract

56.39  0.51a

41.90  0.10a

1 3 4 5 6 7 8 9 10 11 11a 11b 11c * Standard inhibitor

NS 94.87  0.23b 43.28  0.52d 121.95  0.91c 96.19  0.31e 88.02  0.39g NS NS NS 92.10  0.61f NS NS NS 49.62  0.31h

66.49  0.12c NS NS NS NS 43.43  0.47e 79.44  0.16d 35.66  0.23h 54.38  0.12b NS 67.36  0.84c 58.91  0.43g 24.25  0.21f 7.83  0.27i

NS = Not Significant; *Eserine and kojic acid were used as standard inhibitors of butyrylcholinesterase and tyrosine kinase respectively; in the same column, IC50 value marked with different superscript letters are significantly different (p < 0.05).

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glycoside flavone derivative was spectrophotometrically measured and the results are presented in Table 4. The CH2Cl2-MeOH (1:1) extract, compounds 3, 5, 4, 6, 11, and 7 exhibited significant (p < 0.05) differences in the concentrations of 50% tyrosinase inhibition (IC50) while non significant inhibitions were noted with compounds 10, 1, 8, 7, 11a-11c, against diphenolase. The most active sample was compound 4 (IC50 = 43.28 mg/mL) followed in decreasing order by CH2Cl2-MeOH (1:1) extract (IC50 = 56.39 mg/ mL), 7 (IC50 = 88.02 mg/ml), 11 (IC50 = 92.10 mg/mL), 3 (IC50 = 94.87 mg/mL), 6 (IC50 = 96.19 mg/mL), and 5 (IC50 = 121.95 mg/mL). Interestingly, the inhibitory activity of compound 4 (IC50 = 43.28 mg/mL) was higher than that of the positive control kojic acid (IC50 = 49.62 mg/mL) which make him as strong candidate to be used in cosmetics. The antityrosinase activities of compound 11 were most important when compared with other flavonoid compounds 2a and 11a-11c. This implies that the number of free OH groups plays an important role in determining the activity. The results are in accordance with other reports in the literature, which showed the importance of hydroxyl groups in tyrosinase inhibitory activity (Neelakantan et al., 1982; Rescigno et al., 2002). Polyphenols such as flavonoid constituents are among the largest groups in tyrosinase inhibitors until now and the structure of flavonoid is compatible with the roles of both substrates and inhibitors of tyrosinase (Chang, 2009). Similar to compounds 6 and 11, some phenolics such as kaempferol and quercetin inhibit tyrosinase activity by their ability to chelate the copper in the active site, leading to irreversible inactivation of tyrosinase (Kubo and Kinst-Hori, 1999; Kim and Uyama, 2005). Cholinesterase inhibitors serve as a strategy for the treatment of Alzheimer’s diseases. These inhibitors promote an increase in the level of acetylcholine in neuronal synaptic area, is considered to play a vital role in the memory disturbances of Alzheimer patients (Mukherjee et al., 2007). Several cholinesterase inhibitors (donepezil, galatamine, tacrin,) are synthetically developed in drug industry. However, the synthetic drugs are known to have limitations due to short half-lifes and unfavorable side effects like gastrointestinal disturbances and hepatotoxicity (Wszelaki et al., 2010). Therefore, the search for novel and safe cholinesterase inhibitors from natural resources with better properties is necessary for the treatment of Alzheimer’s disease. The inhibition effect of the CH2Cl2-MeOH (1:1) extract and compounds from R. corymbosa as well as their semi-synthetic derivatives on butyrylcholinesterase (BuChE) was screened and the results are depicted in Table 4. The CH2Cl2-MeOH (1:1) extract, compounds 1, 7–10, and 11a-c exhibited significant (p < 0.05) differences in the concentrations of 50% BuChE inhibition (IC50) while non significant inhibitions were noted with compounds 3–6, and 11. The most active sample was compound 11c (IC50 = 24.25 mg/mL) followed in decreasing order by 9 (IC50 = 35.66 mg/mL), CH2Cl2-MeOH (1:1) extract (IC50 = 41.90 mg/mL), 7 (IC50 = 43.43 mg/mL), 10 (IC50 = 54.38 mg/mL), 11b (IC50 = 58.91 mg/mL), 1 (IC50 = 66.49 mg/mL), 11a (IC50 = 67.36 mg/mL), and 8 (IC50 = 79.44 mg/mL). The inhibitory activities of all the tested samples (IC50 = 24.25–79.44 mg/mL) were lower than that of eserine (IC50 = 7.83 mg/mL) which is a known BuChE inhibitor. The results of compounds 1 and 11a-c are in accordance with other reports in the literature, which showed butyrylcholinesterase inhibitory activities of triterpenes (Lee et al., 2011) and flavonoids (Atia-tun-Noor et al., 2007). Comparing the butyrylcholinesterase inhibitory activity of compounds 2a, 11-11a-c, it is found that compound 2a with the hydroxyl group at position 5 has no activity as compound 11 which has additional free hydroxyl groups at C-7, C-30 and C-40 . On the contrary, compound 11c with two free hydroxyl groups at C-5 and C-40 showed stronger activity compared with its acetylated derivative 11a and compound 11b which one of the hydroxyl groups was acetylated at C-40 . These results showed that the free

hydroxyl groups both at C-5 and C-40 enhance the inhibitory activity of compound 11c. 3. Experimental 3.1. General experimental procedures The UV spectra were recorded on evolution 300 UV–vis spectrophotometer (Shimadzu, Tokyo, Japan). The IR spectra were taken on a FTIR-8900 Shimadzu spectrophotometer (Shimadzu, Tokyo, Japan). The 1H-NMR and the 13C-NMR spectra were recorded at 75, 100 and 125 MHz on a Bruker AM 300, AM 400 and AM 500 NMR spectrometers, respectively. Chemical shifts are expressed in d (ppm) units relative to tetramethylsilane (TMS) as an internal standard and coupling constants are given in Hz. HR-EI-MS (High Resolution Electron Impact Mass Spectrum) were obtained on Joel JMS 600-H spectrometer. TOFMS spectrum was recorded on QSTAR XL MS/MS System. Melting points were determined on Büchi M-560 melting point apparatus and uncorrected using glass capillary tubes. Optical rotations were measured on a P-2000 polarimeter Jasco (Japan Spectroscopic Co. Ltd., Tokyo, Japan). Column chromatography was performed on silica gel (Kieselgel 60, 70–230 mesh) and different fractions were monitored by TLC, using precoated silica gel F254 aluminum sheets (0.25 mm thickness) and spots were visualized under UV light (254 and 365 nm), further sprayed with cerric sulfate and heat up to 60  C for about 2 min. 3.2. Plant Material The whole plant of R. corymbosa was collected in Mbouda, western region Cameroon, in May 2013. The plant material was identified by Mr. Nana in the Cameroon National Herbarium, Yaoundé (Cameroon) where a voucher specimen (N 47546HNC) is deposited. 3.3. Extraction and isolation The air-dried and powdered plant material (3 Kg) was extracted with CH2Cl2/MeOH (1:1, 4  10 L) at room temperature for 72 h to yield a crude extract (162 g) after evaporation under reduced pressure. A part of this extract (150 g) was subjected to column chromatography over silica gel eluting with gradient of n-hexaneEtOAc and EtOAc-MeOH to afford 50 fractions of 300 mL each. These fractions were combined on the basis of their TLC profiles into 6 major fractions A-F (A: 1-11; B: 12-17; C: 18-29; D: 30-36; E: 37-41; F: 42–50). Fraction A (250 mg) contained mostly fats. Fraction B (150 mg) was chromatographed on a silica gel column, eluting with n-hexane-EtOAc (95–5) to obtain compound 1 (10 mg), gradually with n-hexane-EtOAc (92–8) to afford compound 3 (10 mg) and increase polarity to fifteen per cent EtOAc to yield compound 5 (5 mg). Fraction C (240 mg) was purified with a CH2Cl2/MeOH (99:1) as solvent system to obtain compound 8 (7 mg) and increase to three per cent MeOH to yield compound 7 (12 mg). Fraction D (2 g) was purified over silica gel column chromatography with a gradient of CH2Cl2/MeOH as solvent system to afford three subfractions (D1-D3). Subfraction D3 (170 mg) was further purified by silica gel using n-hexane-EtOAc (80–20) to give compound 11 (80 mg). Subfractions D1 (115 mg) and D2 (100 mg) were also purified by silica gel column chromatography using n-hexane-EtOAc (85–15) and n-hexaneEtOAc (62–38) as eluent to yield respectively compound 10 (7 mg) and compound 6 (5 mg). Fraction E (160 mg) was purified with a mixture of n-hexane-EtOAc (35–75) as solvent system to obtain compound 9 (30 mg). Fraction F (170 mg) was dissolved in pyridine (2 mL); Ac2O (20 mL) was added, and the mixture was stirred at

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room temperature for 24 h. H2O (10 mL) was added to the mixture, which was stirred for 30 min. Extraction with EtOAc and chromatographic purification on silica gel column with n-hexane-Acetone (65:35) as eluent gave compound 2a (10 mg). Compound 11 (55 mg) was also dissolved in pyridine (2 mL); Ac2O (5 mL) was added and the mixture was stirred at room temperature for 24 h. H2O (5 mL) was added to the mixture, which was stirred for 30 min. Extraction with EtOAc and chromatographic purification on silica gel column with CH2Cl2/MeOH (99:1) allowed the isolation of compound 11a (8 mg), gradually with CH2Cl2/ MeOH (97.8:2.2) to obtain compound 11b (7 mg) and CH2Cl2/MeOH (97:3) to give compound 11c (5 mg).

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DMSO, medium and inoculum) and positive control (10% aqueous DMSO, medium, inoculum and water-soluble antibiotics) were included. MICs were assessed visually after the incubation period and were taken as the lowest sample concentration at which there was no growth or virtually no growth. The lowest concentration that yielded no growth after the sub-culturing was taken as the minimum microbicidal concentrations (MMCs). Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) and ampicillin (Sigma-Aldrich, Steinheim, Germany) for bacteria, and nystatin (Sigma-Aldrich, Steinheim, Germany) for yeasts were used as positive controls. The assay was repeated thrice. 3.6. In vitro Butyrylcholinesterase (BuChE) inhibition assay

3.4. New compounds information Oleanane 3-(30 R-hydroxy)-hexadecanoate (1) Colorless powder: mp 104–105  C, [a]D + 12.1 (c = 2.95  106, CH2Cl2); IR nmax (KBr): 1708 and 3449 cm1; 1H- and 13C-NMR data, see Table 1; EIMS, m/z (rel. int): 409(1), 257 (3), 218(90); HREIMS at m/z 680.6039[M+] (Calcd 680.6107 for C46H80O3) and TOFMS at m/z 681.42 [M + H] +. 30 -acetoxy-6,7-dimetoxy-40 (200 ,300 ,400 ,600 -tetraacetylglucopyranosyl)flavone (2a) Yellowish powder: mp 172–173  C, [a]D  6.8 (c = 2.84  107, CH2Cl2); UV [lmax (log e): 278 (4.12) nm]; IR nmax (KBr): at 3699,1660, 1496 cm1; 1H- and 13C-NMR data, see Table 2; EIMS, m/z (rel. int): 702(9), 169 (100), 109 (50), 43(60); HREIMS at m/z 702.1801[M+] (calcd 702.1796 for C33H34O17) and TOFMS spectrum at m/z 703.13 [M + H] +. 3.5. Antimicrobial assay 3.5.1. Bacterial and fungal strains A total of two Gram-negative bacteria (Escherichia coli ATCC8739 and Pseudomonas aeruginosa PA01) and two yeasts (Candida albicans ATCC 9002 and Cryptococcus neoformans IP 90526) both reference (from the American Type Culture Collection) and clinical (from Pasteur Institute Paris, France) strains were tested for their susceptibility to compounds. Also, included were two clinical isolates of Candida parapsilosis and Shigella flexneri obtained from Centre Pasteur (Yaoundé-Cameroon). The bacterial and fungal species were maintained on agar slant at 4  C and grown on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud Dextrose Agar (SDA, Conda) slants respectively 24 h prior to any antimicrobial test. 3.5.2. Preparation of microbial inoculum The inocula of microorganisms were prepared from overnight cultures by picking numerous colonies and suspending them in sterile saline (NaCl) solution (0.90%). Absorbance was read at 530 nm for yeasts or at 600 nm for bacteria and adjusted with the saline solution to match that of a 0.50 McFarland standard solution. From the prepared microbial solutions, other dilutions with saline solution were prepared to give a final concentration of 106 yeast cells/mL and 106 CFU/mL for bacteria (Djouossi et al., 2015). 3.5.3. Determination of the minimum inhibitory concentrations (MICs), and minimum microbicidal concentrations (MMCs) MICs were performed in 96-wells microplate by broth micro dilution as previously described (Tamokou et al., 2009; Djouossi et al., 2015). Stock solutions of the tested samples were prepared in 10% v/v aqueous dimethylsulfoxide (DMSO) solution (Fisher chemicals, Strasbourg, France) and twofold serially diluted in Mueller-Hinton Broth (MHB) for bacteria and Sabouraud Dextrose Broth (SDB) for fungi. For every experiment, a sterility check (10% aqueous DMSO and medium), negative control (10% aqueous

BuChE inhibiting activity was determined by the method of Ellman et al., 1961. Butyrylthiocholine chloride was used as substrate to assay BuChE. The reaction mixture containing 100 mM sodium phosphate buffer (pH 8, 150 mL), DTNB (10 mL), test sample solution (10 mL) and BuChE solution (20 mL) were mixed and incubated for 15 min (25  C). The reaction was then initiated by the addition of butyryl-thiocholine (10 mL). The hydrolysis of butyrylthiocholine was monitored by the formation of yellow 5-thio-2nitrobenzoate anions as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of butyrylthiocholine at a wavelength of 412 nm (15 min). Test samples and the positive control (eserine) were dissolved in EtOH. All the reactions were performed in triplicate in 96-well micro-titre plates in SpectraMax 340 (Molecular Devices, U.S.A.). The percentage (%) inhibition was calculated as follows (E-S)/E  100, where E is the activity of the enzyme without test sample and S is the activity of enzyme with test sample. 3.7. In vitro tyrosinase inhibition assay Tyrosinase inhibition assay was performed using kojic acid as standard inhibitor for tyrosinase in spectraMax 340 microplate reader as previously described (Vaibhav and Lakshaman, 2012) with slight modifications. The compounds were screened for o-diphenolase inhibitory activity of tyrosinase using L-3,4dihydroxyphenylalanine (L-Dopa, Sigma, USA) as substrate. 80 mL of mushroom tyrosinase (28 mM, Sigma, USA) were mixed with 80 mL of kojic acid or sample in 50 mM sodium phosphate buffer (pH 6.8). After 15 min pre-incubation at 37  C, 40 mL of L-DOPA were added to the mixture and incubated further for 30 min at 37  C. Concentration of L-DOPA in the final reaction mixture was 1 mM. The amount of Dopachrome formation was read at 475 nm. The percentage tyrosinase inhibition was calculated as follows: % inhibition = [(A controlA sample)/ Acontrol]  100 where Acontrol is the absorbance of DMSO and Asample is the absorbance of the test reaction mixture containing compounds or kojic acid. The IC50 values of compounds and kojic acid were calculated. 3.8. Statistical analysis Data were analyzed by one-way analysis of variance followed by Waller-Duncan Post Hoc test. The experimental results were expressed as the mean  Standard Deviation (SD). Differences between groups were considered significant when p < 0.05. All analyses were performed using the Statistical Package for Social Sciences (SPSS, version 12.0) software. Acknowledgements This research work was supported under the umbrella of TWASICCBS Postgraduate Fellowship Program. We are grateful to the

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