Phytochemical and antimicrobial study of Pilocarpus pennatifolius Lemaire

Fitoterapia 131 (2018) 1–8

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Phytochemical and antimicrobial study of Pilocarpus pennatifolius Lemaire

T

Gabriele do Carmo, Tanize S. Fernandes, Marcelo Pedroso, Adriano Ferraz, Alexandre T. Neto, ⁎ Ubiratan F. Silva, Marco A. Mostardeiro, Davi F. Back, Ionara I. Dalcol, Ademir F. Morel Department of chemistry, Federal University of Santa Maria, 97105-900, Rio Grande do Sul, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Pilocarpus pennatifolius Rutaceae Chemical constituents Antimicrobial activity Alkaloids Coumarins

The investigation of the crude extract of leaves and bark of Pilocarpus pennatifolius Lemaire allowed isolated of a not yet described coumarin, together with three known coumarins (bergapten, xanthotoxin and dimethyl allyl xanthyletin), and a not yet described imidazole alkaloid. All structures were established by means of spectral analysis, including extensive 2D NMR studies. In addition, the alkaloid had its absolute stereochemistry determined by X-ray diffraction. Meanwhile, extracts and pure compounds were tested against various strains of bacteria and fungi, showing promising antimicrobial activities. We highlight the activities of crude bark methanol extract (CBME), of the leaf basic acetate fraction (LBAcF), and of compound 2 against the Gram negative bacteria Shigella flexneri (MICs = 7.8, 7.8 and 3.12 μg·mL−1, respectively), of compound 5 against the Gram positive Enterococcus fecalis (MIC = 1.56 μg·mL−1), and against two Gram negative bacteria Salmonella enteritidis (MIC = 1.56 μg·mL−1), and Pseudomonas aeruginosa (MIC = 6.25 μg·ml−1). On the other hand, CBME and compounds 3–5 showed excellent activity against the fungus Candida krusei with MICs of 15.6, 1.56, and 3.12 μg·mL−1 respectively, as actives or better than the antifungal standard fluconazole (MIC = 3.12 μg·mL−1).

1. Introduction The Rutaceae family is composed by around 1900 species and 160 genera [1]. It is estimated that approximately 182 species and 29 genera of this family can be found in Brazil [2]. Plants from the Rutaceae family are known for their morphological diversity and for presenting several classes of secondary metabolites such as terpenes, lignans, alkaloids, flavonoids and coumarins [1]. These constituents confer antimicrobial [3–6] antioxidant [7,8], anti-inflammatory [9–11] and antitumor [12] potential to Rutaceae plants. Pilocarpus pennatifolius Lemaire, a species of the Pilocarpus genus, is native from some countries of South America, such as Brazil, Argentina, Uruguay and Paraguay. In Rio Grande do Sul (Brazil), is popularly known as “jaborandi” or “cutia-branca” [13,14]. Generally, species of Pilocarpus genus occur in the form of shrubs or small trees, and can reach up to 2 or 3 m [14]. They are usually found in the American continent, from southern Mexico to southern Brazil and Argentina. According to the literature, several alkaloids have been found in species of Pilocarpus genus: pilocarpine, isopilocarpine, pilosin, anhydropilosine, 3-nor-8-11 dihydropilocarpine, pilosinin, 4,6-dehydro1,2,4,5-tetrahydro-2,5-dioxopyloctypin, 3-(3-methyl-3H-imidazol-4methyl)-1-phenylbut-3-en-1-one, 3-hydroxymethyl-4-(3-methyl-3Himidazol-4)-1-phenyl-butan-1-one, 3-benzoyl-4(3-methyl-3H-

⁎

imidazol-4-methyl) -dihydrofuran-2-one and epiisopiloturine [3,11,15,16]. Amongst these we highlight pilocarpine, an imidazole alkaloid extracted from the leaves and applied in the treatment of glaucoma. In addition, various coumarins have been commonly found: xantyletin, bergapten, 7-hydroxy-3- (1′,1′-dimethyl-allyl)-8-methoxycoumarin, and 3-(1′,1′-dimethyl-allyl)-scopoletin, which present antibacterial and antifungal properties [13]. Although not yet reported in the literature, infusions of leaves, dried leaves and extracts, are used in popular medicine for the treatment of asthma, diabetes, liver diseases and in the form of cosmetic for hair treatment. 2. Material and methods 2.1. Equipment NMR spectra were recorded on a Bruker DPX 400 spectrometer, operating at 400 MHz for 1H and 100 MHz for 13C, and on a Bruker ASCEND 600 spectrometer, operating at 600 MHz for 1H and 150 MHz for 13C in CDCl3 or MeOH-d4, using TMS as internal standard. Melting points were determined on a MQAPF-301 apparatus and are uncorrected. The GC chromatograms were recorded on Varian models 3400 and 3800. The mass spectra were recorded on a Shimadzu spectrometer. Crystallographic data were collected on a Bruker D8 Quest

Corresponding author. E-mail address: [email protected] (A.F. Morel).

https://doi.org/10.1016/j.fitote.2018.09.009 Received 9 July 2018; Received in revised form 12 September 2018; Accepted 17 September 2018 Available online 19 September 2018 0367-326X/ © 2018 Published by Elsevier B.V.

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Scheme 1. Extraction of Pilocarpus pennatifolius leaves.

small quantify. The acid solution of the extract from the stem bark was extracted firstly com ethyl ether (3 × 100 mL), followed by by EtOAc (3 × 100 mL), resulting on the acidic ether fraction (BAEF, 0.4 g), and the acidic acetate fraction (BAAcF, 2.0 g). Continuing, the aqueous solution was basified with NH4OH (aq) (pH 8–9), and extracted with ethyl ether (5 × 100 mL) resulting in the bark basic ether fraction (BBEF, 0.150 g). As this fraction did not give a positive test with the Dragenforff reagent, it was not analyzed. A portion of the leaves acidic ether fraction (LAEF 2.5 g) was applied to the silica gel column (70–230 mesh) as the stationary phase (160 g), eluted initially with n-hexane and increasing amounts of CH2Cl2 (up to 100%), what produced 5 sub fractions: (I, n-hexane), (II, n-hexane - CH2Cl2 10%), (III, n-hexane - CH2Cl2 20%), (IV, n-hexane CH2Cl2 50%), and (V, CH2Cl2). On the sequence, it was eluted with increasing amounts of MeOH (up to 100%), which yielded 7 subfractions: (VI and VII, CH2Cl2-MeOH 1%), (VIII and IX, CH2Cl2-MeOH 5%), (X, CH2Cl2-MeOH 10%), (XII, CH2Cl2-MeOH 50%), and (XII, MeOH). From sub fractions VI and VIII, the position isomers bergapten (1, 5.0 mg) and xanthotoxin (2, 20.0 mg) were isolated, respectively. The bark acidic acetate fraction (BAAcF, 2.0 g) was subjected to column chromatography (CC) using silica gel (70–230 mesh) as stationary phase (160 g) and increasing amounts of CHCl3-MeOH (up to 100%), what formed eleven subfractions: (I, CHCl3), (II, CHCl3-MeOH 3%), (III, CHCl3-MeOH 5%), (IV, CHCl3-MeOH 10%), (V, CHCl3-MeOH 15%), (VI, CHCl3-MeOH 20%), (VII, CHCl3-MeOH 25%), (VIII, CHCl3MeOH 30%), (IX, CHCl3-MeOH 40%), (X, CHCl3-MeOH 50%), and (XI, MeOH). From the subfraction III, dimethyl allyl xanthyletin (3, 12.1 mg) was isolated. Fraction V (51 mg) was purified by preparative thin layer chromatography (PTLC) CH2Cl2 -MeOH 12%, which resulted on the coumarin jaborandine (4, 5.4 mg). The leaves basic acetate fraction (LBAcF, 4.12 g) was applied to the

Photon 100 diffractometer equipped with an Incoatec IμS high brilliance Cu Kα (1.54178 Å) X-ray. Optical rotation measurements were obtained on an automatic Perkin Elmer polarimeter model 341. SpectraMax M2 and SoftMax Pro 5.4.1 spectrophotometers (Molecular Devices Inc., USA) operating at 620 nm were used for the evaluation of the antimicrobial activities. 2.2. Plant material The leaves of Pilocarpus pennatifolius Lemaire were collected in July 2012 and January 2013, within the municipality of Mata (29° 33′ 38″ S, 54° 27′ 19″). The identification of the plant material was performed by Professor Renato Zachia, and a plant exsiccate can be found in the Herbarium from the Botany Department – UFSM, under N. SMDB 2753. 2.3. Extraction and isolation of compounds 1–5 The leaves (2 kg) and stem bark (98 g) (both dried and crushed) were extracted with MeOH in a soxhlet apparatus for 24 h (Schemes 1 and 2). The resulting MeOH extracts were filtered and concentrated in order to obtain the crude MeOH extract from the leaves (CLME, 350 g) and the crude MeOH extract from the stem bark (CBME, 10 g). On the sequence, both extracts were dissolved in H2O and acidified with 2 M HCl (pH 2–3). The acidic solution of the CLME was exhaustively extracted with cyclohexane to eliminate fats and decrease the chlorophyll in the extract. Subsequently, the aqueous residue was thoroughly extracted with Et2O (5 × 300 mL), which resulted on the acidic ether fraction (LAEF, 2.5 g). Continuing, the aqueous solution was basified with NH4OH (aq) (pH 8–9) and extracted with ethyl ether (5 × 300 mL), resulting in the leaf basic ethyl ether fraction (LBEF, 0.2 g), followed by extraction with EtOAc resulting on the basic acetate fraction of leaves (LBAcF, 4.12 g). The LBEF not was analyzed due to 2

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Scheme 2. Extraction of Pilocarpus pennatifolius stem bark.

Coumarins 12 4 3

OCH 3

5

4a

6

2

O

O 1 8a

8

4

O

O 1 8a

3

16 15

4 4

4a

5

6

12

2

O

O 1 8a

8

7

5

6

11

O

8 7

10

OCH 3 12

2

1 17' 17

4a

2 10

O

7

3

11

3

11

10

6

11 10

2

O

13

O

5

4a

O 1 8a

8

7

O

4

3 Alkaloid 1

HO H 13

15 16 15'

13

H3 C

14

13'

14

12

12

14'

H

11 10

O

H H

H

5

6 7 H

O

2

HN

8

4

N3

H

5 Fig. 1. Structures of compounds 1-5 isolated from Pilocarpus pennatifolius Lemaire.

3

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Table 1 NMR spectroscopic data for compound 4 (CDCl3, 400.13/100.6 MHz) and 5 (MeOH-d4, 600/150 MHz). 4

Position

Position

δH (ppm), (J in Hz)

δC (ppm)

1 2 3 4 4a 5

– – 6.19 (d, 6.8) 7.57 (d, 6.8) – 6.76 (s)

– 163.27 112.08 143.42 112.57 97.87

1 2 3 4 5 6

6

–

155.84

6’

7 8

– 7.20 (s)

124.32 123.17

7 8

8a

–

142.87

8’

9 10

– 5.30 (t, 16)

– 87.36

10 11

11 and 11’

3.36 and 3.04 (dd, 16, 8) 5.10 (m) 4.96 (m) 1.78 (bs)

33.39

12

112.73 112.73 16.93

OH 13 14 and 14’ 15 and 15’

12 13 14

16

5 δH (ppm), (J in Hz)

δC (ppm)

– 7.40, s – 6.48, s – 2.39, dd, (8.4, 14.7) 2.26, dd, (6.6, 14.7) 2.92 (m) 4.37, dd (8.1, 9.0) 4.02, dd (5.4, 9.0) – 2.74, dd (2.7, 6.0) 5.27, d (2.7)

– 136.23 – 115 145.2 31.69

4.59 – 7.37, d (7.5) 7.32, dd (7.5, 7.85) 7.25, dd (7.5, 15.0)

– 143.75 126.62 129.43

31.69 36.03 73.93 73.93 180.74 54.42 72.74

126.31

bs = broad signal.

4 3

5

4a

6

H H

11

Fig. 3. X-ray ORTEP drawing of compound 5.

12

10

2

O

O 1 8a

8

7

O H3 C

14

1

HO H

14

13

15 16 15'

12

14'

H

11 10

O

H H

H

2

HN

5 6 7 H

O

2.4. Identification of compounds 1–5

13

8

4

Bergapten (1): white crystals, mp:186.4–188.3 °C [188–190 °C (17)]; IR(KBr)γmax 3002, 2798,1776, 1600, 1450 cm−1; HRESIMS (+): m/z: 217.0480 [M + H]+ (calc. For C12H8O4, 216.0423; C12H8O4 + H+, 217.00456), 1H NMR (400 MHz, CDCl3):δ (ppm): 6.28 (1H, d, J = 9.8 Hz, H-3), 8.16 (1H, d, J = 9.8 Hz, H-4), 7.14 (1H, s, H8), 7.59 (1H, d, J = 2.0 Hz, H-10), 7.01 (1H, d, J = 2.0 Hz, H-11), 4.26 (3H, s, OCH3-12); 13C NMR (100.62 MHz, CDCl3): ?? (ppm): 160.83 (C2), 112.33 (C-3), 138.66 (C-4), 106.20 (C-4a), 149.30 (C-5), 112.48 (C6), 158.10 (C-7), 93.61 (C-8), 152.46 (C-8a), 144.47 (C-10), 104.66 (C11), 59.91(C-12) . Xanthotoxin (2): white crystals, mp:145.4–147.1 °C [147–148 °C (18)]; IR(KBr)γmax 1704, 1680, 1615, 1600, 1587 cm−1; HRESIMS (+): m/z: 217.0521 [M + H]+ calc. For C12H8O4, 216.0423; C12H8O4 + H+,217.1456, 1H NMR (400.13 MHz, CDCl3), ?? (ppm):6.36 (1H, d, J = 9.6 Hz, H-3), 7.75 (1H, d, J = 9.6 Hz, H-4), 7.33(1H, s, H-5), 7.67 (1H, d, J = 2.2 Hz, H-10), 6.80 (1H, d, J = 2.2 Hz, H-11), 4.20 (3H, s, OCH3-12); 13C NMR (100.62 MHz, CDCl3), ?? (ppm): 159.65 (C-2), 114.1 (C-3), 143.53 (C-4), 115.86 (C4a), 112.19 (C-5), 125.43 (C-6), 147.09 (C-7), 132.19 (C-8), 142.41 (C8a), 145.92(C-10), 106.6 (C-11), 60.61(C-12). Dimethyl allyl xanthyletin (3): white crystals, mp: 97.4–98.8 °C [98–99 °C (19)]; IR(KBr)γmax 1720, 1680, 1615, 1600, 1510 cm−1 m/z: 296.1 [M+]; 1H NMR (400 MHz, CDCl3):δ (ppm): 7.01 (1H, s, H-4), 7.45 (1H, s, H-5), 6.68 (1H, s, H-8), 5.65 (1H, d, J = 12 Hz, H-11), 6.32 (1H, d, J = 12 Hz, H-12), 1.46 (6H, s, CH3 H-13 and H-13′), 6.16 (1H, dd, J = 16 Hz, J = 12 Hz, H-15), 5.07 (2H, dd, J = 16 Hz, J = 8 Hz, H16), 1.45 (6H, s, CH3−17 and 17′); 13C NMR (100.62 MHz, CDCl3): ?? (ppm): 159.87 (C-2), 131.81 (C-3), 124.62 (C-4), 113.17 (C-4a), 137.52

COSY HMBC

N3

H

Fig. 2. Some correlations presented in HMBC of the compounds 4 e 5.

silica gel column (70–230 mesh) as the stationary phase (330 g), eluted initially with n-hexane and increasing amounts of EtOAc (up to 100%), resulting in 4 subfractions: (I, n-hexane), (II, n-hexane-EtOAc 20%), (III, n-hexane-EtOAc 50%), and (IV, EtOAc). Afterwards, it was eluted with EtOAc and increasing amounts of MeOH (up to 100%) and resulted in 9 subfractions: (V-IX, EtOAc-MeOH 10%), (X, EtOAc-MeOH 20%), (XI, EtOAc-MeOH 30%), (XII, EtOAc-MeOH 50%), and (XII, MeOH 10%). Subfraction VI (515 mg) was subjected to CC using silica gel and solvent gradient CHCl3-MeOH (100:0 → 0:100), which originated pennatifoline A (5, CHCl3-MeOH 15%, 6.0 mg).

4

5

500 > 500 > 500 > 500 > 500 500

> 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 500

250 250 125 125 250 250

500 500 250 500 500 500 250 62.5 500 250 250

500 250 250 500 250 > 500 250 7,8 500 125 250

250 500 250 500 125 250

CIM

MIC

MLC

LAEF

CLME

> 500 > 500 500 > 500 > 500 > 500 500 > 500 > 500 > 500 500

> 500 > 500 > 500 > 500 > 500 500

MLC

500 250 250 500 250 500 250 7,8 500 62,5 250

250 500 500 500 625 125

MIC

LBAcF

> 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500

> 500 > 500 > 500 > 500 > 500 > 500

MLC

Fractions and pure compounds MIC 50/MLC(μg/mL)

NT 250 500 500 250 250 125 7.8 500 250 250

250 500 250 250 250 250

MIC

CBME

NT > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 500 500

> 500 > 500 > 500 > 500 > 500 > 500

MLC

100 100 50 NT 100 50 50 NT NT 50 NT

50 25 25 25 NT NT

MIC

1

> 100 > 100 > 100 NT > 100 > 100 > 100 NT NT > 100 NT

> 100 100 > 100 100 NT NT

MLC

100 100 50 200 50 100 100 3.12 200 50 50

50 50 25 25 25 50

MIC

2

> 100 > 100 > 100 > 200 > 100 > 100 > 100 > 200 > 200 > 100 > 200

> 100 > 100 > 100 100 > 200 200

MLC

NT 100 25 200 100 12.5 200 100 200 100 100

100 100 100 25 200 200

MIC

3

NT > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 200 > 200

> 200 > 200 > 200 100 > 200 > 200

MLC

NT 200 200 200 100 200 200 200 200 200 200

200 100 200 NT 100 200

MIC

4

NT > 200 > 200 > 200 200 > 200 > 200 > 200 > 200 > 200 > 200

> 200 > 200 > 200 NT > 200 > 200

MLC

NT 100 6.25 200 100 50 100 100 1.56 100 100

25 100 100 50 1.56 200

MIC

5

NT > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200 > 200

> 200 > 200 > 200 100 > 200 > 200

MLC

3.12 12.,5 3.12 6.,25 3.,12 3.12 6.25 1.56 1.56 1.56 6.25

3.12 6.25 1.,56 1.56 3.12 3.,12

MIC

50 200 12.5 100 25 12.5 50 3.12 12.,5 12,5 200

12.5 50 12.,5 1.56 12,5 6.25

MLC

Chloramphenicol

NT 200 50 25 25 200 200 12,5 3,12 200 100

200 100 200 NT 1.56 50

MIC

NT > 200 > 200 200 200 > 200 > 200 200 100 > 200 > 200

> 200 > 200 > 200 NT 12,5 200

MLC

Ampicillin;

CLME: leaves methanol extract; LAEF: acidic ethereal fraction; LBAcF: basic ethyl acetate fraction; CBME: bark methanol extract; CHL: chloramphenicol; AMP: ampicillin; MIC: minimum inhibitory concentration; MLC: minimum lethal concentration; NT: Not Test. In bold type to emphasize better results.

Gram positive bacteria Staphylococcus aureus Bacillus subtilis Bacillus cereus Enterococcus spp. Enterococcus fecalis Staphylococcus epidermidis Gram negative bacteria Bulkhoderia cepacia Escherichia coli Pseudomonas aeruginosa Proteus mirabilis Shigella sonnei Salmonella typhimurium Morganella morganii Shigella flexneri Salmonella enteritidis Enterobacter aerogenes Klebsiella pneumoniae

Microorganismos

Table 2 Antibacterial activity (MIC/MLC μg/mL) for extracts and isolated compounds of Pilocarpus pennatifolius.

G. do Carmo et al.

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Table 3 Antifungal activity (MIC/MLC μg/mL) for extracts and isolated compounds of Pilocarpus pennatifolius. Microrganisms

Fractions and pure compounds MIC 50/MLC(μg/mL) CLME

C. albicans C. tropicalis C. krusei C. parapslosis C. neoformans C. gatti S. cerivisae

LAEF

LBAcF

CBME

1

2

3

4

5

Fluconazole

MIC

MLC

MIC

MLC

MIC

MLC

MIC

MLC

MIC

MLC

MIC

MLC

MIC

MLC

MIC

MLC

MIC

MLC

MIC

MLC

500 250 250 500 125 500 250

> 500 > 500 > 500 > 500 > 500 > 500 > 500

500 250 31.2 500 125 250 250

500 500 > 500 500 500 250 500

500 250 125 500 250 125 500

> 500 500 > 500 > 500 500 500 > 500

250 500 15.6 250 125 125 500

> 500 > 500 > 500 > 500 > 500 250 > 500

50 50 50 100 50 100 50

> 100 > 100 > 100 > 100 > 100 > 100 > 100

50 100 50 50 25 100 100

> 100 > 100 > 100 > 100 > 100 > 100 > 100

50 50 1.56 100 50 50 100

200 200 > 200 200 100 200 200

100 100 1.56 100 50 25 100

200 > 200 200 > 200 > 200 200 > 200

100 100 3.12 100 50 50 100

200 > 200 200 > 200 50 100 200

1.56 100 3.12 1.56 1.56 1.56 3.12

> 200 200 100 12,5 12,5 50 50

CLME: leaves methanol extract; LAEF: acidic ethereal fraction; LBAcF: basic ethyl acetate fraction; CBME: bark methanol extract; MIC: minimum inhibitory concentration; MLC: minimum lethal concentration; NT: Not Test. In bold type to emphasize better results.

Crystal data and further details on the data collection and refinements of the compound 5 are presented in Tables S13–S17.

(C-5), 118.32 (C-6), 154.65 (C-7), 103.65 (C-8), 155.96 (C-8a), 77.42 (C-10), 130.95 (C-11), 121.02 (C-12), 28.23 (C-13 and C-13′), 40.38 (C14), 145.65 (C-15), 112.05 (C-16), 26.15 (C-17 and C-17′). Jaborandine (4): yellow crystals, mp: 100.9–102 °C; IR(KBr)γmax 1700, 1670, 1620, 1602, 1580 cm−1; HRESIMS (+): m/z 229.0872 [M + H]+ (calc. For C14H12O3, 228.0786; C14H12O3 + H+, 229.0865); [α]D25 +4.55° (c 0. 016, CHCl3); 1H NMR (400 MHz, CDCl3):δ (ppm): 6.19 (1H, d, J = 6.8 Hz, H-3), 7.57 (1H, d, J = 6.8 Hz, H-4), 6.76 (1H, s, H-5), 7.20 (1H, s, H-8), 5.30 (1H, t, J = 16 Hz, H-10), 3.36 (1H, dd, J = 16 Hz, J = 8 Hz, H-11), 3.04 (1H, dd, J = 16 Hz, J = 8 Hz, H-11′), 5.10 (1H, m, H-12), 4.96 (1H, m, H-13), 1.78 (3H, s, CH3, H-14); 13C NMR (100.62 MHz, CDCl3):?? (ppm): 163.27 (C-2), 112.08 (C-3), 143.42 (C-4), 112.57 (C-4a), 97.87 (C-5), 155.84 (C-6), 124.32 (C-7), 123.17 (C-8), 142.87 (C-8a), 87.36 (C-10), 33.39 (C-11), 112.73 (C-12 and C-13), 16.93 (C-14). Pennatifoline A (5):white crystals, mp: 194.5 °C – 196.3 °C; IR (KBr)γmax 3435, 2920, 2860, 1644 cm−1; HRESIMS (+): m/z 273.1252 [M + H]+ (calc. For C15H16N2O3, 272. 1161; C15H16N2O3 + H+, 273.1239); [α]D20 -85° (c 0. 002, MeOH); 1H NMR (600 MHz, MeOHd4),?? (ppm): 7.40 (1H, s, H-2), 6.48 (1H, s, H-4), 2.39 (1H, dd, J = 8.4 Hz, 14.7 Hz, H-6), 2.26 (1H, dd, J = 6.6 Hz, 14.7 Hz, H-6′), 2.92 (1H, m, H-7), 4.37 (1H, dd, J = 8.1 Hz, J = 9 Hz, H-8), 4.02 (1H, dd, J = 5.4 Hz, 9 Hz, H-8′), 2.74 (1H, dd, J = 2.7 Hz, 6.0 Hz, H-11), 5.27 (1H, d, J = 2.7 Hz, H-12), 4.59 (OH), 7.37 (2H, d, J = 7.5 Hz, H14,14′), 7.32 (2H, dd, J = 7.5 Hz, J = 15 Hz, H-15,15′), 7.25 (1H, dd, J = 7.5 Hz, J = 15 Hz, H-16); 13C NMR (150 MHz, MeOH-d4),?? (ppm): 136.23 (C-2), 115 (C-4), 145.2 (C-5), 31.69 (C-6), 36.03 (C-7), 73.93 (C-8), 180.14 (C-10), 54.42 (C-11), 72.74 (C-12),143.75 (C-13), 126.62 (C-14), 129.43 (C-15), 126.31 (C-16).

2.6. Antimicrobial activity Antibacterial and antifungal activities were assayed using the broth microdilution method [24]. A collection of twenty-four microorganisms was used, including six Gram-positive bacteria: Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6633); Bacillus cereus (ATCC 33019), Enterococcus spp. (ATCC 6589), Enterococcus fecalis (ATCC 19433), Staphylococcus epidermidis (ATCC 35984), eleven Gram-negative bacteria: Enterobacter aerogenes (ATCC 13048), Shigella sonnei (ATCC 25931), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027), Proteus mirabilis (ATCC 25933), Bulkhoderia cepacia (ATCC 17759), Salmonella typhimurium (ATCC 14028), Shigella flexneri (ATCC 12022), Salmonella enteritidis (ATCC 13076), Morganella morganii (ATCC 25829), Klebsiella pneumoniae (clinical isolate); and seven yeasts: Saccharomyces cerevisiae (ATCC 2601), Candida albicans (ATCC 10231), Candida tropicalis (ATCC 18803), Candida krusei (ATCC 6258), Candida parapsilosis (ATCC 22018), Cryptococcus neoformans (ATCC 28952), and Cryptococcus gattii (ATCC 2601). Standard strains of the microorganisms were obtained from American Type Culture Collection (ATCC), and standard antibiotics chloramphenicol, ampicillin and fluconazole were used in order to control the sensitivity of the microbial test. The minimal inhibitory concentration (MIC) was determined on 96-well culture plates by a micro dilution method using a microorganism suspension at a density of 105 CFU mL−1 with Casein Soy Broth incubated for 24 h at 37 °C for bacteria and Sabouraud Broth incubated for 48 h at 25 °C for yeasts [24]. The cultures that did not present growth were used to inoculate plates again (Casein Soy Broth and Sabouraud), in order to determine the minimal lethal concentration (MLC). Proper blanks were simultaneously assayed, and all samples were tested in triplicate.

2.5. X-ray diffraction of compound 5 Data were collected on a Bruker D8 Quest Photon 100 diffractometer equipped with an Incoatec IμS high brilliance Cu Kα (1.54178 Å) X-ray. The structures were solved by direct methods using SHELXS [20]. Subsequent Fourier difference map analyses yielded the positions of the non-hydrogen atoms. Refinements were carried out with the SHELXL package [20]. All refinements were made by fullmatrix least-squares on F2 with anisotropic displacement parameters for all non–hydrogen atoms. Hydrogen atoms were included in the calculated positions refinement, but the atoms (hydrogen) that were part of special bonds were located in the Fourier map. Drawings were made using ORTEP Windows [21]. The spatial group chosen was P1211 based on systemic absences and data statistics, and confirmed by successful solution and refinement. The final R indices are R1 = 0.0291 (I > 2σ (I)) and wR2 = 0.0787 (all data); goodness of fit = 1.087. The refinement of the Flack [22, 23] parameter revealed an index of 0.07 (4) (crystal of an enantiomerically pure molecule) which allows to assign an absolute configuration related to the atoms C7 S, C11 R and C12 R.

3. Results and discussion The constituents of leaves and stem bark of the species Pilocarpus pennatifolius Lemaire, collected in the state of Rio Grande do Sul, Brazil, were investigated and afforded four coumarins (1–4), and one alkaloid (5) (Fig. 1). After the initial extraction of the plant material, the crude leaves methanolic extract (CLME) and the crude bark methanolic extract (CBME) were obtained. Both were dissolved in water and separated into fractions. First, the aqueous solution of the CLME was acidified and extracted with cyclohexane and ethyl ether successively, what resulted in the acidic cyclohexanic fraction (LAChF) and acidic ether fraction (LAEF). After these steps, the aqueous fraction was basified and extracted with ethyl ether and ethyl acetate successively, what resulted in the basic ether fraction (LBEF) and basic acetate fraction (LBAcF). The 6

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whose structures were proposed by HRMS [27], but were not isolated. Therefore, in this work we present the isolation of one of these isomers, with their physical, 1H and 13C NMR data (Table 1), and the absolute configuration of all stereogenic centers of its structure. Alkaloid 5 is named here as pennatifoline A. Compounds 1–3 were identified by their spectroscopic data and corresponded to those reported in the literature [17–19]. Antibacterial and antifungal assays were performed with the crude leaves methanol extract (CLME), crude bark methanolic extract (CBME), leaves acidic ethereal fraction, (LAEF), and leaves basic acetate fraction (LBAcF), and with the isolated compounds 1–5 (Tables 2 and 3). The extracts and fraction tested showed some activity against the strains of bacteria tested (MICs between 62 and 500 μg.mL−1). The most sensitive was the Gram negative bacteria Shigella flexneri against the CBME, and against the fractions LAEF and LBAcF, with MICs = 7.8 μg. mL−1. From the isolated compounds, 1 and 2 (isolated from the CLME) showed a considerable activity against S. aureus, B. subitilis, B. cereus, Enterococcus spp., E fecalis, and P. aeroginosa (MICs between 25 and 50 μg·mL−1), and 2 showed to be strongly active against the gram negative bacteria Shigella flexnery (MIC = 3.12 μg·mL−1, when compared to the antimicrobial agents chloramphenicol (MIC = 1.56 μg·mL−1), and ampicillin (MIC = 12.5 μg·mL−1). Compound 3, isolated from the CBME, showed good activity against Salmonella typhimurium (MIC = 12.2 μg. mL−1), and a moderate activity against Enterococcus spp. (25.0 μg. mL−1). Compound 5 (isolated from the LCME), showed the best activities against the Gram positive bacteria Enterococcus faecalis (MIC = 1.56 μg·mL−1), compared to the antimicrobial ampicillin (MIC = 1.56 μg·mL−1), and twice as active as the antimicrobial chloramphenicol (MIC = 3.2 μg·mL−1), and against the Gram negative bacteria Salmonella enteritidis (MIC = 1.56 μg·mL−1), compared to the antimicrobial chloramphenicol, and twice as ampicillin (MIC = 3.2 μg·mL−1). In addition, compound 5 shows moderate activities against E. aureus (25 μg·mL−1) and Enterococcus spp. (50 μg·mL−1). The CBME, the fraction LAEF, and the isolated compounds 1–5 presented interesting results as their antifungal activities (Table 3). The fungus C. krusei was the most sensitive front the CBME (MIC = 15.6 μg·mL−1), and front the LAEF (MIC = 31.2 μg·mL−1), while compounds 3 and 4 were the more active components from the CBME (MICs = 1.56 μg·mL−1), twice as much activity as the standard antifungal agent fluconazole (MIC = 3.12 μg·mL−1). Compound 5 was the second most active against C. krusei, with an activity compared to the antifungal agent fluconazole (MIC = 3.12 μg·mL−1).

same procedure was performed with CBME, but the fractions obtained were: bark acidic ether (BAEF), bark acidic acetate (BAAcF) and bark basic ether (BBEF) fractions. Successive purification of the acidic ether fraction (LAEF) obtained from the leaves by repeated chromatography afforded two known coumarins: bergapten (1) and xanthotoxin (2), previously isolated in species from the Rutaceae family, such as Pilocarpus pennatifolius [13,25]. The chromatographic purification of BAAcF, obtained from the crude methanolic extract of the bark, provided the isolation of two coumarins: dimethyl allyl xanthyletin (3), previously described in species of the Rutaceae family, but not reported in Pilocarpus spp. [13,26], and the coumarin 4, not yet described in the literature. 1 H and 13C NMR experiments and high-resolution mass spectrometry analyses allowed the identification of compound 4, [α]D25 +4.55°, that presents molecular formula C14H12O3, HRESIMS (+): m/z 229.0872 [M + H]+. In the 1H and 3C NMR experiments (Table 1) it is possible to find the presence of a lactonic carbonyl (C-2) at δC 163.27, two CH olefins in δH 4.96 (m, H-13) and δC 112.73, δH 5.10 (m, H-12) and δC112.73, four aromatic hydrogens in δH 6.19 (d, J = 6.8 Hz, H-3) and δC 112.08, δH 7.57 (d, J = 6.8 Hz, H-4)/δC143.42, δH 6.76 (s, H-5) and δC 97.81, δH 7.20 (s, H-8) and δC 123.17, one methyl at δH 1.78 (bs, H-14) and δC 16.93, one methine carbon at δH 5.30 (t, J = 16 Hz, H-10) and δC 87.36 and two diastereotopic hydrogens at δH 3.04 e 3.36 (dd, J = 16 Hz, 8 Hz, H-11, H-11′) and δC 33.39. In the COSY experiment (Fig. 2), couplings were observed between H-11and H-10, H-3 and H-4, and between H-14 (CH3) and H-13, and H-13 with H-12. In addition, in the HMBC (Fig. 2) it was possible to observe the correlations of H14 with C-10 and C-12 and with C-13, between H2–11 and C-7, H-3 and H4 with C-2, H-3 and C-4a and H-4 with C-6, what confirms the proposed structure. Furthermore, in the 1D NOESY spectrum, we can observe the correlations of H-12 with H-13, confirming the cis geometry of the olefinic hydrogens. In this work, this compound was named jaborandine (4). Chromatographic purification of the leaves basic acetate fraction (LBAcF) led to the isolation of an imidazole alkaloid (5). The structure of 5, [α]D20-85° (MeOH), was determined based on 1D and 2D NMR analyses and high-resolution mass spectrometry, where its HRESIMS (+) displays [M + H, m/z 273.1225], which in combination with NMR data (Table 1) suggests a C15H16N2O3 molecular formula. 1H and 13C NMR spectra confirmed the presence of the following hydrogens and aromatic carbons: δH 7.37 (d, J = 7.5 Hz, H2–14, 14′) and δC 126.62(C14, 14′), δH 7.32 (dd, J = 7.5 and 15 Hz, H2–15, 15′) and δC 129.43 (C15, 15′, and δH 7.25 (dd, J = 7.5 and 15 Hz, h-16) and δC 126.31 (C-16) which correspond to the aromatic ring monosubstituted. In addition, in the aromatic region, it was possible to identify the presence of the H and C related to the imidazole nucleus: δH 7.40 (s, H-2) and δC 136.23, δH 6.48 (s, H-4) and δC 115. The diastereotopic methylene hydrogens appear at δH 4.37 (dd, J = 8.1; 9.0 Hz, H-8) and 4.02 (dd, J = 5.4; 9.0 Hz, H8′) and δC 73.9, δH 2.39 (dd, J = 14.7; 8.4 Hz, H6) and 2.26 (dd, J = 14.7; 6.6 Hz, H-6′) and δC 31.7. A hydroxyl group can be found in δH 4.59 (s), while the methine H and C appear in δH 2.92 (m, H-7) and δC 36.03, and δH 5.27 (d, J = 2.72 Hz, H-12) and δC 72.7. In the 2D COSY experiment, it was possible to verify the correlations of the H-7 with the diastereotopic hydrogens H2-8, H2-6, and H-11, and the correlations of the aromatic H2-14 WITH H2-15, and H-16 with H2-15. In addition, the correlations observed in HMBC (Fig. 2) between H-14 with C-12 and C-16, H-15 with C-13 and C-14, H-16 with C-14, H6, 6′ with C-7, C11, and C-8, H8,8′ with C-6, and C-10, and H-8′ with C-7, H11 with C-10, C-6 and C-8, and H-12 with C-10, C-14, C-15, C7 and C11, confirmed the proposed structure for alkaloid 5. Furthermore, X-ray diffraction provided the absolute configuration of all stereogenic centers of 5, identified as the alkaloid (7S, 11R, 12S) 2 (3H)-furanone, dihydro-3-(hydroxyphenylmethyl)-4-(1H-imidazol-5-ylmethyl) (Fig. 3). Alkaloids with the same structure were previously identified, in the form of an isomeric mixture, in P. pennatifolius and P. microphyllus,

4. Conclusion In conclusion, five compounds were isolated from the Pilocarpus pennatifolius,collected in Rio Grande do Sul, Brazil, with 3 known coumarins 1–3, one not yet previously isolated coumarin (4), and an alkaloid (5). Their structures and the absolute configuration of the chiral centers of 5 were determined by HRME, 1H and 13C NMR, and by X-ray diffraction experiments. Pilocarpine, a common alkaloid found in the genus Pilocarpus, was not identified in this species. In addition, extracts, fractions, and compounds 1–5 showed promising antimicrobial and antifungal potentials, which indicates that the studied species may be an alternative source in the search for new antimicrobial agents for the treatment of diseases caused by bacteria such as Enterococcus fecalis, a Gram positive bacterium that can cause urinary tract infection and meningitis [28], Shigella flexneri, a Gram negative bacterium that can cause shigellosis, a type of intestinal infection that causes fever, cramps and diarrhea with blood and mucus [29], and Salmonella enteritidis, a Gram negative bacterium that causes gastroenteritis [30], and for diseases caused by the fungus Candida krusei, a fungus that often cause infection in patients with weakened immune systems [31]. 7

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Conflicts of interest The authors declare no conflict of interest. [12]

Acknowledgements The authors thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the financial support. The Bruker X8 Kappa APEX II CCD diffractometer was financed by FINEP (CT/INFRA 2004). We are indebted to Prof. E. M. Flores (UFSM) for the HRMS.

[13] [14] [15]

Appendix A. Supplementary data [16]

CCDC 1812767 contain the supplementary crystallographic data for the 5. These data can be obtained free of charge via http://www.ccdc. cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223–336–033; or e–mail: [email protected].

[17] [18]

[19]

References

[20] [21]

[1] M. Groppo, J.R. Pirani, M.L.F. Salatino, S.R. Blanco, J.A. Kallunki, Phylogeny of Rutaceae based on two noncoding regions from cpDNA, Am. J. Bot. 95 (2008) 985–1005. [2] M.D.F.F. Melo, C.S. Zickel, Os gêneros Zanthoxylum L. e Esenbeckia Kunth (Rutaceae) no Estado de Pernambuco, Brasil, Acta Bot. Bras. 18 (2004) 73–90. [3] R.C. De Souza, J.B. Fernandes, P.C. Vieira, M.F.D.G. Da Silva, M.F.P. Godoy, F.C. Pagnocca, O.C. Bueno, M.J.A. Hebling, J.R. Pirani, A new imidazole alkaloid and other constituents from Pilocarpus grandiflorus and their antifungal activity, Zeitschrift fur Naturforsch. - Sect. B J. Chem. Sci. 60 (2005) 787–791. [4] V. Kuete, J.D. Wansi, A.T. Mbaveng, M.M. Kana Sop, A.T. Tadjong, V.P. Beng, F.X. Etoa, J. Wandji, J.J.M. Meyer, N. Lall, Antimicrobial activity of the methanolic extract and compounds from Teclea afzelii (Rutaceae), South African J. Bot. 74 (2008) 572–576. [5] M. Lv, P. Xu, Y. Tian, J. Liang, Y. Gao, F. Xu, Z. Zhang, J. Sun, Medicinal uses, phytochemistry and pharmacology of the genus Dictamnus (Rutaceae), J. Ethnopharmacol. 171 (2015) 247–263. [6] K.N. Venugopala, V. Rashmi, B. Odhav, Review article review on natural coumarin lead compounds for their pharmacological activity, Biomed. Res. Int. 963248 (2013) 1–14. [7] M.K. Islam, N.N. Biswas, S. Saha, H. Hossain, I.A. Jahan, T.A. Khan, K. Awang, J.A. Shilpi, Antinociceptive and antioxidant activity of zanthoxylum budrunga wall (rutaceae) seeds, Sci. World J. 2014 (2014) 1–7 article ID 869537. [8] N.K. Kassim, M. Rahmani, A. Ismail, M.A. Sukari, G.C.L. Ee, N.M. Nasir, K. Awang, Antioxidant activity-guided separation of coumarins and lignan from Melicope glabra (Rutaceae), Food Chem. 139 (2013) 87–92. [9] J.Y. Cho, T.L. Hwang, T.H. Chang, Y.P. Lim, P.J. Sung, T.H. Lee, J.J. Chen, New coumarins and anti-inflammatory constituents from Zanthoxylum avicennae, Food Chem. 135 (2012) 17–23. [10] Jinous Asgarpanah, Phytochemistry and pharmacological properties of Ruta graveolens L, J. Med. Plant Res. 6 (2012) 3942–3949. [11] V.G. Silva, R.O. Silva, S.R.B. Damasceno, N.S. Carvalho, R.S. Prudeîncio,

[22] [23] [24]

[25]

[26]

[27]

[28]

[29] [30] [31]

8

K.S. Aragão, M.A. Guimarães, S.A. Campos, L.M.C. Véras, M. Godejohann, J.R.S.A. Leite, A.L.R. Barbosa, J.V.R. Medeiros, Anti-inflammatory and antinociceptive activity of epiisopiloturine, an imidazole alkaloid isolated from pilocarpus microphyllus, J. Nat. Prod. 76 (2013) 1071–1077. A. Bisi, C. Cappadone, A. Rampa, G. Farruggia, A. Sargenti, F. Belluti, R.M.C. Di Martino, E. Malucelli, A. Meluzzi, S. Iotti, S. Gobbi, Coumarin derivatives as potential antitumor agents: growth inhibition, apoptosis induction and multidrug resistance reverting activity, Eur. J. Med. Chem. 127 (2017) 577–585. A.P. Santos, P.R.H. Moreno, Pilocarpus spp.: a survey of its chemical constituents and biological activities, Rev. Bras. Ciências Farm. 40 (2004) 116–137. L.A. Souza, K.S.M. Mourão, I.S. Moscheta, S.M. Rosa, Morfologia e anatomia da flor de Pilocarpus pennatifolius Lem. (Rutaceae), Rev. Bras. Bot. 26 (2003) 175–184. I.N. Abreu, P. Mazzafera, M.N. Eberlin, M.A. Zullo, A.C. Sawaya, Characterization of the variation in the imidazole alkaloid profile of Pilocarpus microphyllus in different seasons and parts of the plant by electrospray ionization mass spectrometry fingerprinting and identification of novel alkaloids by tandem mass spe, Rapid Commun. Mass Spectrom. 21 (2007) 1205–1213. S.G. Davies, P.M. Roberts, P.T. Stephenson, H.R. Storr, J.E. Thomson, A practical and scaleable total synthesis of the jaborandi alkaloid (+)-pilocarpine, Tetrahedron 65 (2009) 8283–8296. T. Masuda, M. Takasugi, M. Anetai, Psoralen and others linear furanocoumarins as phytoalexins in Glehnia littoralis, Phytochemistry 47 (1998) 13–16. J.S. Kim, J.C. Kim, S.H. Chim, E.J. Lee, W.Y. Jin, K.H. Bae, K.H. Son, H.P. Kim, S.S. Kang, H.W. Chang, Chemical constituents of the root of Dystaenia takeshimana and their anti-inflamatory activity, Arch. Pharm. Res. 29 (2006) 617–623. M.N.S. Nayar, M.K. Bhan, V. Georg, A new coumarin in Boenninghausenia albiflora, Phytochemistry 12 (1973) 2073–2074. G.M. Sheldrick, A short history of SHELX, Acta Cryst A64 (2008) 112–122. C.L. Barnes, ORTEP -3 for Windows - a version of ORTEP -III with a Graphical User Interface (GUI) by J. Farrugia, J. Appl. Crystallogr. 30 (1997) 568. H.D. Flack, G. Bernardinelli, The use of X-ray crystallography to determine absolute configuration, Chirality 20 (2008) 681–690. H.D. Flack, The use of X-ray crystallography to determine absolute configuration (II), Acta Chim. Slov. 55 (2008) 689–691. NCCL: National Committee for Clinical Laboratory, Manual of antimicrobial susceptibility testing (2005) and NCCLS: National Committee for Clinical Laboratory Standards, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard, 2th ed., 2002 NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087–1898 USA, 2002. A.A.L. Gunatilaka, D.G.I. Kingston, E.M.K. Wijeratne, B.M.R. Bandara, G.A. Hofmann, R.K. Johnson, Biological activity of some coumarins from Sri Lankan rutaceae, J. Nat. Prod. 57 (1994) 518–520. A.L. Anaya, M. Macías-Rubalcava, R. Cruz-Ortega, C. García-Santana, P.N. SánchezMonterrubio, B.E. Hernández-Bautista, R. Mata, Allelochemicals from Stauranthus perforatus, a Rutaceous tree of the Yucatan Peninsula, Mexico, Phytochemistry 66 (2005) 487–494. A.C.H.F. Sawaya, B.G. Vaz, M.N. Eberlin, P. Mazzafera, Screening species of Pilocarpus (Rutaceae) as sources of pilocarpine and other imidazole alkaloids, Genet. Resour. Crop. Evol. 58 (2011) 471–480. L.K. Zou, H.N. Wang, B. Zeng, J.N. Li, X.T. Li, A.Y. Zhang, Q.Q. Xia, Erythromycin resistance and virulence genes in Enterococcus faecalis from swine in China, New Microbiol. 34 (2011) 73–80. M. Ogawa, Y. Handa, H. Ashida, M. Suzuki, C. Sasakawa, The versatility of Shigella effectors, Nat. Rev. Microbiol. 6 (2008) 11–16. R.A. Giannella, Salmonella, Baron's Medical Microbiology, 4th ed., Univ of Texas Medical Branch, 1996ISBN 0-9631172-1-1 via NCBI Bookshelf. Y.H. Samaranayake, L.P. Samaranayake, Candida krusei: biology, epidemiology, pathogenicity and clinical manifestations of an emerging pathogen, J. Med. Microbiol. 41 (1994) 295–310.