Two stilbenes from Indonesian Cassia grandis and their anti bacterial activities

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Research Journal of Chemistry and Environment Vol. 24 (1) January (2020)
Res. J. Chem. Environ.
61
Two stilbenes from Indonesian Cassia grandis and their antibacterial activities
Valentina Adimurti Kusumaningtyas1,2*, Yana Maolana Syah2 and Lia Dewi Juliawaty2
1. Department of Chemistry, Faculty of Science and Informatics, Universitas Jenderal Achmad Yani, Jalan Terusan Jenderal Sudirman, Cimahi 40285, INDONESIA
2. Natural Product Chemistry Group, Faculty of Mathematics and Natural Sciences, Institut Tenologi Bandung, Jalan Ganesha no. 10, Bandung 40132, INDONESIA
*valentina.adimurti@lecture.unjani.ac.id
Abstract
Cassia grandis is an ornamental plant that has been reported to have ethnopharmacological uses to heal wounds, scabies and other skin medications in Indonesian traditional medicine. Phytochemical investigation of the heartwood C. grandis led to the isolation of two known stilbenes: piceatannol 1 and resveratrol 2 isolated from the methanol extract. The structures were established on the basis of chemical evidence and spectroscopic methods, especially NMR as well as by comparing with published data.
The antibacterial activity was carried out by using broth microdilution method and evaluated against two pathogenic bacteria: Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. One of the isolated compounds 1 was reported as secondary metabolite of Indonesian C. grandis. Compound 2 exhibited promising selective antibacterial potent against Escherichia coli ATCC 25922.
Keywords: Cassia grandis, stilbenes, piceatannol, resveratrol, antibacterial.
Introduction
Cassia grandis is a tropical species belonging to Fabaceae (Leguminosae). In Indonesia, this plant is known as “Johar Merah”, which has been reported in Indonesian traditional medicines to heal wounds, scabies and other skin medications1. The species of Cassia are rich source of anthraquinones, stilbenoids and flavonoids2. In the search of new antibacterial compounds from natural sources, we report here in the isolation and structural elucidation of two known stilbenes resveratrol (1) and piceatannol (2) as well as their antibacterial activities.
Material and Methods
General Experimental: 1H and 13C NMR spectra were recorded in CD3OD and (CD3)2CO using residual and deuterated solvent peaks as internal standard on an Agilent DD2 system (1H 500 MHz and 13C 125 MHz) Spectrophotometer. The absorbances for antibacterial activity were determined by Bio-Rad X Mark Microplate Spectrophotometer.
Plant Material: The heartwoods of C. grandis were collected from Cikaret, Bogor, West Java, Indonesia in
December 2011. The plant was identified by Dr. Didik Widyatmoko, M.Sc., Bogoriense Herbarium and a specimen was deposited at Bogoriense Herbarium, Bogor, Indonesia.
Extraction and Isolation: Powdered and dried heartwoods of C. grandis (2.0 kg) were extracted with methanol to give a crude methanol extract (82 g). The methanolic extract (40
g) was subjected to a vacuum liquid chromatography over silica gel 60 GF254 eluted with n-hexane-EtOAc in a polarity gradient manner to give six (I-VI) fractions. Fraction II was purified by centrifugal chromatography eluted with chloroform-EtOAc (8:2) to give compounds 2 (85 mg) and 1 (12 mg). Their structures were elucidated on the basis of NMR data as well as by comparison of their NMR spectral data with reported values3,4.
Resveratrol (1): Yellowish white powders, m.p. 261-263oC;
1H NMR [500 MHz, (CD3)2CO] δ (ppm): 8.47 (s, OH-4'),
8.19 (s, OH-3/5), 7.41 (2H, d, J = 8.6 Hz, H-2'/6'), 7.01 (1H,
d, J = 16.3 Hz, H-8), 6.88 (1H, d, J = 16.3 Hz, H-7), 6.84
(2H, d, J = 8.6 Hz, H-3'/5'), 6.54 (2H, d, J = 2.2 Hz, H-2/6), 6.27 (1H, t, J = 2.2 Hz, H-4). 13C NMR [125 MHz, (CD3)2CO] δ (ppm): 159.6 (C-3/5), 158.1 (C-4'), 140.9 (C-
1), 129.9 (C-8), 129.1 (C-7), 128.7 (C-2'/6'), 126.8 (C-1'),
116.4 (C-3'/5'), 105.7 (C-2/6), 102.7 (C-4).
Piceatannol (2): Yellowish crystals, m.p. 226-227 ºC; 1H NMR [500 MHz, CD3OD] δ (ppm): 6.99 (1H, d, J = 2.1 Hz, H-2'), 6.89 (1H, d, J = 16.3 Hz, H-8), 6.83 (1H, dd, J = 8.3,
2.1 Hz, H-6'), 6.76 (1H, d, J = 16.3 Hz, H-7), 6.74 (1H, d, J
= 8.0 Hz, H-5'), 6.46 (2H, d, J = 2.2 Hz, H-2/6), 6.19 (1H, t, J = 2.2 Hz, H-4). 13C NMR [125 MHz, CD3OD] δ ppm): 159.4 (C-3/5), 146.3 (C-3'), 146.2 (C-4'), 141.2 (C-1), 131.0
(C-1'), 129.6 (C-8), 126.9 (C-7), 120.2 (C-6'), 116.4 (C-5'),
113.8 (C-2'), 105.8 (C-2/6), 102.6 (C-4).
Preparation of bacterial suspension: The bacteria colonies from the fresh agar plate were transferred into a sterile capped glass tube containing sterile broth or saline solution (NaCl 0.9 % b/v) using a sterile loop or cotton swab and mixed well. The suspension was adjusted to achieve a turbidity equivalent to a 0.5 McFarland turbidity standard.
Determination of minimum inhibitory concentration (MIC): The samples were dissolved in dimethyl sulfoxide (DMSO). The samples were prepared to achieve 250 μg/mL for isolated compounds in the first well. Two-fold dilution of samples was performed in 96-wells microplate over the range 1.95 to 250 μg/mL. This was achieved by filling all
Research Journal of Chemistry and Environment Vol. 24 (1) January (2020)
Res. J. Chem. Environ.
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wells with 200 μL of Mueller Hinton Broth (MHB) medium. Then 200 μL of samples was transferred into the first well. Two-fold serial dilution was performed by transfer of 200 μL of the mixture in the first well into the next consecutive well until the end of the row. At the last well 200 μL of the mixture was discharged, so that the total volume solution in each well was 200 μL. Then 10 μL bacterial suspension was transferred into all wells. The final test concentration of bacteria will be approximately 5 x 105 CFU/mL.
The microplate was incubated for 24 h at 37 °C. Bacterial growth was determined using a universal microplate reader. MIC is defined as the lowest concentration at which no visible bacterial growth was observed. The positive controls used in these assays were chloramphenicol.
Determination of minimum bactericidal concentration (MBC): To determine MBC, aliquot removed from wells that showed no bacterial growth was streaked onto MHA plate and incubated under the same conditions. MBC was defined as the lowest concentration at which colonies failed to grow after incubation.
Results and Discussion
13C NMR of compound 1 displayed 10 C-sp2 signals representing 14 carbon atoms, suggesting a stilbene
structure, which was corroborated with the presence of pair of doublets (δH 7.01 and 6.88) for a trans-vinyl (J = 16.3 Hz) group. The observed two oxyaryl signals at δC 159.6 (2C) and 158.1 were indicative for the presence of a 3,5- dihydroxylphenyl and a 4-hydroxylphenyl groups. This was supported by the presence of meta- and double meta-coupled aromatic signals at δH 6.54 (2H) and 6.27 for the 3,5- dihydroxylphenyl group and a pair of ortho-coupled (each 2H, J = 8.6 Hz) at δH 7.41 and 6.84. These NMR data are consistent for compound 1 as 3,5,4’-trihydroxystilbene or resveratrol6,7.
1H and 13C NMR data of compound 2 were very similar to this compound 1. The differences were observed in compound 2 one additional oxyaryl carbon signal as an ortho-dihydroxyl group (δC 146.3 and 146.2) and three proton signals (δH 6.99, 6.83 and 6.74) with multiplicities consistent with a 3,4-dihydroxyl group. Thus, compound 2 is a 3’-hydroxylated derivative of compound 1, or trivially named piceatannol4.
The MIC and MBC results (Table 2) indicate that the isolated compounds had different levels of activity against the bacteria. The inhibitory and bactericidal properties were observed within the range of 12.5 to 125 μg/mL.
Table 1
1H-NMR (500 MHz), 13C-NMR (125 MHz) spectral data of the isolated compounds [in CD3OD (Compound 1) and (CD3)2CO (Compound 2)]
C
Piceatannol (1)
C
Resveratrol (2)
?H (mult., J/Hz) ppm
?C (ppm)
?H (mult., J/Hz) ppm
?C (ppm)
1
-
141,2
1
-
140,9
2/6
6,46(d, 2,2)
105,8
2/6
6,54 (d, 2,2)
105,7
3/5
-
159,4
3/5
-
159,6
4
6,19 (t, 2,2)
102,6
4
6,27 (t, 2,2)
102,7
7
6,76 (d, 16,3)
126,9
7
6,88 (d, 16,3)
129,1
8
6,89 (d, 16,3)
129,6
8
7,01 (d, 16,3)
129,9
1'
-
131,0
1'
-
126,8
2'
6,99 (d, 2,1)
113,8
2'/6'
7,41 (d, 8,6)
128,7
3'
-
146,3
3'/5'
6,84 (d, 8,6)
116,4
4'
-
146,2
4'
-
158,1
5'
6,74 (d, 8,0)
116,4
6'
6,83 (dd, 8,3; 2,1)
120,2
Figure 1: Structures of the isolated compounds
Research Journal of Chemistry and Environment Vol. 24 (1) January (2020)
Res. J. Chem. Environ.
63
Table 2
Antibacterial activity (μg/mL) of the isolated compounds
Bacteria
Piceatannol (1)
Resveratrol (2)
Chloramphenicol
MIC
MBC
MIC
MBC
MIC
MBC
Staphylococcus aureus ATCC 25923
125
125
125
125
25
100
Escherichia coli ATCC 25922
100
125
50
125
12.5
100
Based on the results, it showed that resveratrol (2) exhibited promising selective antibacterial potent against Escherichia coli ATCC 25922 with MIC values of 50 μg/mL. Meanwhile, the addition of a hydroxy group to ring B of piceatannol (1) decreases the MIC and MBC values.
Conclusion
Two stilbenes have been isolated from heartwood C. grandis and were identified as piceatannol (1) and resveratrol (2). Resveratrol was selective antibacterial potent against Escherichia coli ATCC 25922 with MIC values of 50 μg/mL. However, piceatannol showed a weak antibacterial activity.
Acknowledgement
We are thankful to Ministry of Research, Technology and Higher Education Republic Indonesia and LPPM Universitas Jenderal Achmad Yani for financial support.
References
1. Heyne K., Tumbuhan Berguna Indonesia, Jilid I, Yayasan Sarana Wanajaya, Jakarta, 15-17 (1987)
2. Francis and Taylor, Dictionary of Natural Products Data CRC Press 1982-2009, HDS Software Copy right © Hampden Data Services Ltd., London (2009)
3. Kim J.H., Byun J.C., Bandi A.K.R., Hyun C. and Lee N.H., Compounds with elastase inhibition and free radical scavenging activities from Callistemon lanceolatus, Journal of Medicinal Plants Research, 3(11), 914-920 (2009)
4. Park J. and Boo Y.C., Isolation of Resveratrol from Vitis Viniferae Caulis and Its Potent Inhibition of Human Tyrosinase, Evidence-Based Complementary and Alternative Medicine, 1-11 (2013)
5. CLSI (Clinical and Laboratory Standards Institute), Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically approved standard, 9th ed., CLSI Document M07-A9, Wayne, PA, USA (2012)
6. Guiso M., Marra C. and Farina A., A new efficient resveratrol synthesis, Tetrahedron Letters, 43(4), 597–598 (2002)
7. Roat C., Kumar N. and Ramawat K.G., Occurrence of stilbenes in unorganized cultures of Cayratia trifolia (L.) Domin, Journal of Herbal Medicine and Toxicology, 2(1), 11–15 (2008).

Two stilbenes from Indonesian Cassia grandis and their anti bacterial activities

Two stilbenes from Indonesian Cassia grandis and their anti bacterial activities

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