http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2014; 52(9): 1204–1207 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.884607
ORIGINAL ARTICLE
Anti-Propionibacterium acnes assay-guided purification of brazilin and preparation of brazilin rich extract from Caesalpinia sappan heartwood Nilesh Prakash Nirmal1 and Pharkphoom Panichayupakaranant1,2 Pharmaceutical Biology Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
1
Department of Pharmacognosy and Pharmaceutical Botany and 2Phytomedicine and Pharmaceutical Biotechnology Research Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla, Thailand Abstract
Keywords
Context: Caesalpinia sappan L. (Leguminosae or Fabaceae) heartwood has been used as a coloring agent, with antibacterial activity in food, beverages, cosmetics, and garments. Objectives: To purify brazilin from C. sappan heartwood and use it as a standard marker for the preparation and standardization of an active constituent-rich extract. Material and methods: Crude ethanol extracts of C. sappan heartwood (CSE) were fractionated to isolate brazilin by an anti-P. acnes assay-guided isolation. Quantitative analysis was performed by HPLC. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined by the broth microdilution method. Results and discussion: Brazilin isolated from CSE possessed antibacterial activity against P. acnes with MIC and MBC values of 15.6 and 31.2 mg/mL, respectively. Brazilin was, therefore, used as a standard marker for standardization and preparation of a brazilin rich extract (BRE). BRE was prepared from CSE using a simple one-step purification using a macroporous resin column eluted with 35% v/v ethanol. This method increased the brazilin content in the BRE up to 39.9% w/w. The antibacterial activity of the standardized BRE against acne involved bacteria was higher than for the CSE but lower than brazilin. However, for industrial applications, a large-scale one-step preparation of BRE has more advantages than the use of pure brazilin in terms of convenience and a low-cost production process. Therefore, BRE is considered as a potential coloring agent with antibacterial activity which is used for pharmaceutical, cosmetic, and nutraceutical applications.
Antibacterial, acne, HPLC, standardization
Introduction Acne is a chronic inflammatory disease mostly present on the face and commonly found in adolescents. It involves multi-factorial mechanisms that include epidermal hyperproliferation, excess sebum, inflammation, and the presence of Propionibacterium acnes (Heymann, 2006). Propionibacterium acnes is a Gram positive anaerobe normally implicated in the inflammatory phase of acne as it secretes several pro-inflammatory products that play an important role in the development of inflammation. Therefore, any compound inhibiting the growth of P. acnes could be a promising agent for acne control. Caesalpinia sappan L. (Leguminosae or Fabaceae), commonly known as Brazil or Sappan wood, is a plant cultivated in Southeast Asia, Africa, and the Americas. In Thailand, the heartwood has been used as a coloring agent in food,
Correspondence: Pharkphoom Panichayupakaranant, PhD, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand. Tel/Fax: +66 74 428220. E-mail: [email protected]
History Received 27 November 2013 Revised 2 January 2014 Accepted 14 January 2014 Published online 18 June 2014
beverages, cosmetics, and garments. It has been reported that C. sappan wood extract also exhibited better antibacterial activity against P. acnes as compared with other 39 medicinal plants from Indonesia (Batubara et al., 2009). Brazilin has been reported to be the active compound against P. acnes (Batubara et al., 2010), but the MIC and MBC values (0.50 mg/mL) were too high to be the antibacterial active principle. We have therefore reinvestigated the antibacterial activity of the crude extract of C. sappan heartwood and purified the antibacterial constituent again using the antibacterial assay-guided isolation. Brazilin [(6aS,11bR)-7,11b-dihydro-6H-indeno[2,1-c] chromene-3,6a,9,10-tetrol] (Figure 1) is a biologically active compound that has antibacterial, anti-inflammatory, and anti-oxidant activities and has been isolated from C. sappan heartwood (Bae et al., 2005; Batubara et al., 2010; Hu et al., 2009; Xu & Lee, 2004). However, the major limitation of the use of this pure naturally derived compound is its high cost of production, due to the many steps required and time involved for purification. Recently, an herbal extract that was rich in the active constituent was considered to be an alternative source of the natural pure compound (Panichayupakaranant, 2011). Many extracts rich in active constituents, e.g., an extract from Rhinacanthus nasutus (Acanthaceae) leaves rich
DOI: 10.3109/13880209.2014.884607
Antibacterial activities of brazilin and C. sappan extracts
1205
and DMSO was used as a negative control. Propionibacterium acnes were incubated at 37 C under anaerobic conditions for 72 h. Staphylococcus aureus and S. epidermidis were incubated at 37 C for 24 h. The MIC was defined as the lowest concentration of the compound to inhibit the growth of micro-organisms and the MBC was defined as the lowest concentration of the compound required to kill the micro-organisms.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
Isolation of the antibacterial compound
in rhinacanthins (Puttarak et al., 2010) and an extract from Senna alata (Fabaceae) leaves rich in anthraquinones (Sakunpak et al., 2009), showed antimicrobial activity equal to or higher than their pure active compounds. Thus, brazilin could be a useful marker for the preparation and standardization of a brazilin-rich extract (BRE) from a crude C. sappan extract (CSE). Nevertheless, there is no report on the preparation and standardization of any BRE. This study was carried out using a bioassay-guided purification of the anti-P. acnes compound from C. sappan heartwood to develop methods for the preparation and standardization of a BRE. The antibacterial activities of BRE, CSE and pure brazilin against acne involved bacteria were also evaluated.
The dried powder (500 g) was successively extracted with 95% ethanol (3 L 3), under reflux conditions for 1 h, to obtain a dark brown extract (58.5 g) after solvent evaporation under reduced pressure. The crude extract was then subjected to isolate anti P. acnes compound using an antibacterial assay guided isolation. The crude extract (10 g) was fractionation on a silica gel column (7 24 cm) (1 g extract per 50 g silica gel) and eluted with a mixture of hexane and ethyl acetate (6:4, v/v) to give 13 pooled fractions (fractions 1–13). The fractions were then subjected to antibacterial assay. The antibacterial fractions (fractions 5 and 6) were pooled together and further purified using a SephadexÕ LH-20 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) (3 80 cm) using methanol as eluent to give seven pooled fractions (fractions A–G). Fraction E with antibacterial activity was re-chromatographed on a silica gel column (3 25 cm) and eluted with a mixture of hexane and ethyl acetate (7:3, v/v) to obtain the red crystals of CS-1 (640 mg).
Materials and methods
Identification of CS-1
Plant material
Red crystal; 1H NMR (CD3OD, 500 MHz): 2.76 (1H, d, J ¼ 15.8 Hz, H-7), 3.01 (1H, d, J ¼ 15.4 Hz, H-7), 3.68 (1H, d, J ¼ 11.5 Hz, H-6), 3.91 (1H, d, J ¼ 11.5 Hz, H-6), 3.95 (1H, s, H-12), 6.28 (1H, d, J ¼ 2.5 Hz, H-4), 6.46 (1H, dd, J ¼ 8.2, 2.5 Hz, H-2), 6.58 (1H, s, H-11), 6.70 (1H, s, H-8), 7.18 (1H, d, J ¼ 8.2, H-1).13C NMR (CD3OD, 125 MHz) 42.9 (C-7), 51.1 (C-12), 70.8 (C-6), 78.0 (C-6 a), 104.2 (C-4), 109.9 (C-2), 112.4 (C-11), 112.8 (C-8), 115.5 (C-1 a), 131.3 (C-7 a), 132.2 (C-1), 137.4 (C-11 a), 145.3 (C-10), 145.6 (C-9), 155.7 (C-3), 157.8 (C-4 a). Based on these 1H NMR and 13C NMR results, CS-1 was identified as brazilin by comparing with the data reported previously (Batubara et al., 2010).
Figure 1. Chemical structure of brazilin.
Caesalpinia sappan heartwoods were collected from Chonburi province, Thailand, in February 2012. A voucher specimen (specimen no. SKP 098 03 19 01) was authenticated by Associate Professor Pharkphoom Panichayupakaranant, and has been deposited at the herbarium of the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. The plant material was washed and dried at 60 C for 24 h in a hot-air oven, and reduced to powder using a grinder, and the powder was passed through a sieve No. 45. Antibacterial activity assay Propionibacterium acnes (DMST 14916) was obtained from the Department of Medical Science Center, Thailand. Staphylococcus aureus (ATCC 25923), and S. epidermidis (ATCC 14990) were obtained from the Department of Microbiology, Faculty of Medicine, Prince of Songkhla University, Thailand. Brain–heart infusion (BHI) and agar were purchased from Becton Dickinson (Franklin lakes, NJ). The cultures of the anaerobe (P. acnes) and the facultative anaerobic bacteria (S. aureus and S. epidermidis) were for 72 and 24 h, respectively. Several colonies of each culture were suspended in 0.85% sodium chloride and diluted to a turbidity equivalent to the McFarland No. 0.5 standard to yield approximately 108 CFU/mL. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth microdilution assay (NCCLS, 2008). Ampicillin was used as the standard drug
Preparation of a BRE The CSE (25 g) was dissolved in 35% ethanol (3 L) to obtain a clear solution after filtering. The solution was loaded on a DiaionÕ HP-20 column (Shimadzu Company, Tokyo, Japan) (1 g CSE per 60 g resin) and eluted with 35% ethanol. The first 2 L of fraction was discarded and another fraction (4 L) that contained brazilin was collected. The experiment was performed in triplicate. The collected fractions were evaporated to dryness to obtain a reddish extract of BRE (11.96 ± 0.55 g). Quantitative HPLC analysis of brazilin HPLC analysis was carried out using a binary HPLC pump (Waters 1525, Westbrook, CT) with a photodiode array
N. P. Nirmal & P. Panichayupakaranant
Pharm Biol, 2014; 52(9): 1204–1207
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
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Figure 2. HPLC chromatograms of (A) brazilin, (B) BRE, and (C) CSE.
detector and an autosampler (Waters 2707, Delft, Netherland). Separation was achieved at 25 C on an Intersil ODS-3 column (Shimadzu Company, Tokyo, Japan) (5 mm, 6 250 mm). The mobile phase consisted of methanol and 2.5% aqueous acetic acid (30:70, v/v), and was pumped at a flow rate of 1.0 mL/min. The sample injection volume was 20 mL, and the quantitative detection was set at 280 nm. The calibration curve of brazilin was established using authentic
brazilin at the concentration ranges of between 15.6 and 250 mg/mL. Brazilin exhibited a linearity over the evaluated ranges, with the linear equation of Y ¼ 21106X – 38659 (r2 ¼ 0.9999).
Results and discussion Determination of the antibacterial activity of CSE against P. acnes showed that CSE possessed satisfactory antibacterial
Antibacterial activities of brazilin and C. sappan extracts
DOI: 10.3109/13880209.2014.884607
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Table 1. Antibacterial activity of CSE, BRE, and brazilin against acne involved bacteria.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
P. acnes
S. epidermidis
S. aureus
Test sample
MIC (mg/mL)
MBC (mg/mL)
MIC (mg/mL)
MBC (mg/mL)
MIC (mg/mL)
MBC (mg/mL)
CSE BRE Brazilin Ampicillin
62.5 31.2 15.6 0.12
125 62.5 31.2 0.12
125 62.5 31.2 0.12
250 125 62.5 0.12
250 125 62.5 0.12
250 125 62.5 0.12
activity with MIC and MBC values of 31.2 and 125 mg/mL, respectively. The anti-P. acnes activity of CSE was markedly stronger than that previously reported by Batubara et al. (2009). This may be due to the different solvents used for the extraction as well as the quality of the plant raw materials. On the basis of the antibacterial assay-guided isolation, CS-1 that had been purified as the antibacterial constituent of C. sappan heartwood was identified as brazilin by 1H NMR and 13C NMR and compared with the data in the literature (Batubara et al., 2010). Brazilin itself showed a stronger antiP. acnes activity than the CSE, with MIC and MBC values of 15.6 and 31.2 mg/mL, respectively. The anti-P. acnes activity of brazilin was also markedly stronger than that previously reported by Batubara et al. (2010). This may be due to the different strains of P. acnes as well as purity of brazilin used in the assay. In addition, brazilin has been reported to possess other bioactivities involed in acne treatment, including anti-inflammatory (IC50 ¼ 24.3 mM) (Bae et al., 2005) and antioxidatative activities (IC50 ¼ 8.8 mM) (Batubara et al., 2010). Brazilin was therefore used as an indicative marker for the preparation and standardization of the active constituent rich C. sappan extract. BRE was successfully prepared from CSE in one step of DiaionÕ HP-20 chromatography. Ethanol (35% v/v) was the appropriate solvent system used for the separation of brazilin. The yield of BRE was 47.8 ± 2.2% w/w dry weight of CSE. On the basis of quantitative HPLC analysis (Figure 2), this method was capable of increasing the content of brazilin in BRE up to 39.9 ± 0.34% w/w, which was almost two-fold higher than that of CSE (20.0 ± 0.26 % w/w). The BRE used for further evaluation of antibacterial activity was standardized by the HPLC method to contain brazilin content of not less than 39% w/w. The antibacterial activity evaluation of brazilin, BRE, and CSE against acne involved bacteria, including P. acnes, S. aureus and S. epidermidis showed that P. acnes was most susceptible to all tested samples. Amongst these samples, brazilin possessed the strongest antibacterial activity against P. acnes, S. epidermidis, and S. aureus, with MIC/MBC values of 15.6/31.3, 31.3/62.5, and 62.5/62.5 mg/mL, respectively, followed by the BRE and CSE (Table 1). This indicated that brazilin plays an important role in the antibacterial activity of CSE and BRE. This result agrees with the findings of Xu and Lee (2004) and Batubara et al. (2010) who have identified brazilin as the major antibacterial constituent of C. sappan heartwood. However, the previous reports by Batubara et al. (2009, 2010) indicated that brazilin possessed lower anti-P. acnes than the crude ethanol extract of C. sappan wood. In contrast, our study has confirmed that
brazilin was the major antibacterial constituent of C. sappan heartwood, which showed a higher anti-P. acnes activity than CSE as well as itself in the previous report by Batubara et al. (2010). This may be due to different purities of the isolated brazilin used in the antibacterial assay. Although BRE had a lower antibacterial activity than brazilin, it showed higher activity than CSE. Based on the yields of BRE (48% w/w) and brazilin (6.4% w/w), BRE has more advantages than brazilin for industrial applications because of its lower cost of production. BRE is therefore an alternative antibacterial agent, with the anti-oxidative (Batubara et al., 2010) and anti-inflammatory activities (Bae et al., 2005; Hu et al., 2009) of brazilin, from C. sappan heartwood that can be used in the pharmaceutical, cosmetic, and nutraceutical industries.
Acknowledgements This work was supported by Prince of Songkla University, Thailand, under postdoctoral fellowship program. Thanks to Dr. Brian Hodgson for assistance with the English language.
Declaration of interest The authors have declared that there is no conflict of interest.
References Bae IK, Min HY, Han AR, et al. (2005). Suppression of lipopolysacchride-induced expression of inducible nitric oxide synthase by brazilin in RAW 264.7 macrophage cells. Eur J Pharmacol 513: 237–42. Batubara I, Mitsunaga T, Ohashi H. (2009). Screening antiacne potency of Indonesian medicinal plants: Antibacterial, lipase inhibition, and antioxidant activities. J Wood Sci 55:230–5. Batubara I, Mitsunaga T, Ohashi H. (2010). Brazilin from Caesalpinia sappan wood as an antiacne agent. J Wood Sci 56:77–81. Heymann WR. (2006). Toll like receptors in acne vulgaris. J Am Acad Dermatol 55:691–2. Hu CM, Liu YH, Cheah KP, et al. (2009). Heme oxygenase-1 mediates the inhibitory actions of brazilin in RAW264.7 macrophages stimulated with lipopolysaccharide. J Ethnopharmacol 121:79–85. NCCLS. (2008). Performance standards for antimicrobial susceptibility testing; ninth informational supplement, NCCLS documents M100S9, National Committee for Clinical Laboratory Standard, Wayne, 120–6. Panichayupakaranant P. (2011). Quality Control and Standardisation of Herbal Medicines. Songkhla: Leo Design and Print. Puttarak P, Charoonratana T, Panichayupakaranant P. (2010). Antimicrobial activity and stability of rhinacanthins-rich Rhinacanthus nasutus extract. Phytomedicine 17:323–7. Sakunpak A, Sirikatitham A, Panichayupakaranant P. (2009). Preparation of anthraquinone high-yielding Senna alata extract and its stability. Pharm Biol 47:236–41. Xu HX, Lee SF. (2004). The antibacterial principle of Caesalpinia sappan. Phytother Res 18:647–51.
ORIGINAL ARTICLE
Anti-Propionibacterium acnes assay-guided purification of brazilin and preparation of brazilin rich extract from Caesalpinia sappan heartwood Nilesh Prakash Nirmal1 and Pharkphoom Panichayupakaranant1,2 Pharmaceutical Biology Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
1
Department of Pharmacognosy and Pharmaceutical Botany and 2Phytomedicine and Pharmaceutical Biotechnology Research Center, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla, Thailand Abstract
Keywords
Context: Caesalpinia sappan L. (Leguminosae or Fabaceae) heartwood has been used as a coloring agent, with antibacterial activity in food, beverages, cosmetics, and garments. Objectives: To purify brazilin from C. sappan heartwood and use it as a standard marker for the preparation and standardization of an active constituent-rich extract. Material and methods: Crude ethanol extracts of C. sappan heartwood (CSE) were fractionated to isolate brazilin by an anti-P. acnes assay-guided isolation. Quantitative analysis was performed by HPLC. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined by the broth microdilution method. Results and discussion: Brazilin isolated from CSE possessed antibacterial activity against P. acnes with MIC and MBC values of 15.6 and 31.2 mg/mL, respectively. Brazilin was, therefore, used as a standard marker for standardization and preparation of a brazilin rich extract (BRE). BRE was prepared from CSE using a simple one-step purification using a macroporous resin column eluted with 35% v/v ethanol. This method increased the brazilin content in the BRE up to 39.9% w/w. The antibacterial activity of the standardized BRE against acne involved bacteria was higher than for the CSE but lower than brazilin. However, for industrial applications, a large-scale one-step preparation of BRE has more advantages than the use of pure brazilin in terms of convenience and a low-cost production process. Therefore, BRE is considered as a potential coloring agent with antibacterial activity which is used for pharmaceutical, cosmetic, and nutraceutical applications.
Antibacterial, acne, HPLC, standardization
Introduction Acne is a chronic inflammatory disease mostly present on the face and commonly found in adolescents. It involves multi-factorial mechanisms that include epidermal hyperproliferation, excess sebum, inflammation, and the presence of Propionibacterium acnes (Heymann, 2006). Propionibacterium acnes is a Gram positive anaerobe normally implicated in the inflammatory phase of acne as it secretes several pro-inflammatory products that play an important role in the development of inflammation. Therefore, any compound inhibiting the growth of P. acnes could be a promising agent for acne control. Caesalpinia sappan L. (Leguminosae or Fabaceae), commonly known as Brazil or Sappan wood, is a plant cultivated in Southeast Asia, Africa, and the Americas. In Thailand, the heartwood has been used as a coloring agent in food,
Correspondence: Pharkphoom Panichayupakaranant, PhD, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand. Tel/Fax: +66 74 428220. E-mail: [email protected]
History Received 27 November 2013 Revised 2 January 2014 Accepted 14 January 2014 Published online 18 June 2014
beverages, cosmetics, and garments. It has been reported that C. sappan wood extract also exhibited better antibacterial activity against P. acnes as compared with other 39 medicinal plants from Indonesia (Batubara et al., 2009). Brazilin has been reported to be the active compound against P. acnes (Batubara et al., 2010), but the MIC and MBC values (0.50 mg/mL) were too high to be the antibacterial active principle. We have therefore reinvestigated the antibacterial activity of the crude extract of C. sappan heartwood and purified the antibacterial constituent again using the antibacterial assay-guided isolation. Brazilin [(6aS,11bR)-7,11b-dihydro-6H-indeno[2,1-c] chromene-3,6a,9,10-tetrol] (Figure 1) is a biologically active compound that has antibacterial, anti-inflammatory, and anti-oxidant activities and has been isolated from C. sappan heartwood (Bae et al., 2005; Batubara et al., 2010; Hu et al., 2009; Xu & Lee, 2004). However, the major limitation of the use of this pure naturally derived compound is its high cost of production, due to the many steps required and time involved for purification. Recently, an herbal extract that was rich in the active constituent was considered to be an alternative source of the natural pure compound (Panichayupakaranant, 2011). Many extracts rich in active constituents, e.g., an extract from Rhinacanthus nasutus (Acanthaceae) leaves rich
DOI: 10.3109/13880209.2014.884607
Antibacterial activities of brazilin and C. sappan extracts
1205
and DMSO was used as a negative control. Propionibacterium acnes were incubated at 37 C under anaerobic conditions for 72 h. Staphylococcus aureus and S. epidermidis were incubated at 37 C for 24 h. The MIC was defined as the lowest concentration of the compound to inhibit the growth of micro-organisms and the MBC was defined as the lowest concentration of the compound required to kill the micro-organisms.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
Isolation of the antibacterial compound
in rhinacanthins (Puttarak et al., 2010) and an extract from Senna alata (Fabaceae) leaves rich in anthraquinones (Sakunpak et al., 2009), showed antimicrobial activity equal to or higher than their pure active compounds. Thus, brazilin could be a useful marker for the preparation and standardization of a brazilin-rich extract (BRE) from a crude C. sappan extract (CSE). Nevertheless, there is no report on the preparation and standardization of any BRE. This study was carried out using a bioassay-guided purification of the anti-P. acnes compound from C. sappan heartwood to develop methods for the preparation and standardization of a BRE. The antibacterial activities of BRE, CSE and pure brazilin against acne involved bacteria were also evaluated.
The dried powder (500 g) was successively extracted with 95% ethanol (3 L 3), under reflux conditions for 1 h, to obtain a dark brown extract (58.5 g) after solvent evaporation under reduced pressure. The crude extract was then subjected to isolate anti P. acnes compound using an antibacterial assay guided isolation. The crude extract (10 g) was fractionation on a silica gel column (7 24 cm) (1 g extract per 50 g silica gel) and eluted with a mixture of hexane and ethyl acetate (6:4, v/v) to give 13 pooled fractions (fractions 1–13). The fractions were then subjected to antibacterial assay. The antibacterial fractions (fractions 5 and 6) were pooled together and further purified using a SephadexÕ LH-20 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) (3 80 cm) using methanol as eluent to give seven pooled fractions (fractions A–G). Fraction E with antibacterial activity was re-chromatographed on a silica gel column (3 25 cm) and eluted with a mixture of hexane and ethyl acetate (7:3, v/v) to obtain the red crystals of CS-1 (640 mg).
Materials and methods
Identification of CS-1
Plant material
Red crystal; 1H NMR (CD3OD, 500 MHz): 2.76 (1H, d, J ¼ 15.8 Hz, H-7), 3.01 (1H, d, J ¼ 15.4 Hz, H-7), 3.68 (1H, d, J ¼ 11.5 Hz, H-6), 3.91 (1H, d, J ¼ 11.5 Hz, H-6), 3.95 (1H, s, H-12), 6.28 (1H, d, J ¼ 2.5 Hz, H-4), 6.46 (1H, dd, J ¼ 8.2, 2.5 Hz, H-2), 6.58 (1H, s, H-11), 6.70 (1H, s, H-8), 7.18 (1H, d, J ¼ 8.2, H-1).13C NMR (CD3OD, 125 MHz) 42.9 (C-7), 51.1 (C-12), 70.8 (C-6), 78.0 (C-6 a), 104.2 (C-4), 109.9 (C-2), 112.4 (C-11), 112.8 (C-8), 115.5 (C-1 a), 131.3 (C-7 a), 132.2 (C-1), 137.4 (C-11 a), 145.3 (C-10), 145.6 (C-9), 155.7 (C-3), 157.8 (C-4 a). Based on these 1H NMR and 13C NMR results, CS-1 was identified as brazilin by comparing with the data reported previously (Batubara et al., 2010).
Figure 1. Chemical structure of brazilin.
Caesalpinia sappan heartwoods were collected from Chonburi province, Thailand, in February 2012. A voucher specimen (specimen no. SKP 098 03 19 01) was authenticated by Associate Professor Pharkphoom Panichayupakaranant, and has been deposited at the herbarium of the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand. The plant material was washed and dried at 60 C for 24 h in a hot-air oven, and reduced to powder using a grinder, and the powder was passed through a sieve No. 45. Antibacterial activity assay Propionibacterium acnes (DMST 14916) was obtained from the Department of Medical Science Center, Thailand. Staphylococcus aureus (ATCC 25923), and S. epidermidis (ATCC 14990) were obtained from the Department of Microbiology, Faculty of Medicine, Prince of Songkhla University, Thailand. Brain–heart infusion (BHI) and agar were purchased from Becton Dickinson (Franklin lakes, NJ). The cultures of the anaerobe (P. acnes) and the facultative anaerobic bacteria (S. aureus and S. epidermidis) were for 72 and 24 h, respectively. Several colonies of each culture were suspended in 0.85% sodium chloride and diluted to a turbidity equivalent to the McFarland No. 0.5 standard to yield approximately 108 CFU/mL. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth microdilution assay (NCCLS, 2008). Ampicillin was used as the standard drug
Preparation of a BRE The CSE (25 g) was dissolved in 35% ethanol (3 L) to obtain a clear solution after filtering. The solution was loaded on a DiaionÕ HP-20 column (Shimadzu Company, Tokyo, Japan) (1 g CSE per 60 g resin) and eluted with 35% ethanol. The first 2 L of fraction was discarded and another fraction (4 L) that contained brazilin was collected. The experiment was performed in triplicate. The collected fractions were evaporated to dryness to obtain a reddish extract of BRE (11.96 ± 0.55 g). Quantitative HPLC analysis of brazilin HPLC analysis was carried out using a binary HPLC pump (Waters 1525, Westbrook, CT) with a photodiode array
N. P. Nirmal & P. Panichayupakaranant
Pharm Biol, 2014; 52(9): 1204–1207
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
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Figure 2. HPLC chromatograms of (A) brazilin, (B) BRE, and (C) CSE.
detector and an autosampler (Waters 2707, Delft, Netherland). Separation was achieved at 25 C on an Intersil ODS-3 column (Shimadzu Company, Tokyo, Japan) (5 mm, 6 250 mm). The mobile phase consisted of methanol and 2.5% aqueous acetic acid (30:70, v/v), and was pumped at a flow rate of 1.0 mL/min. The sample injection volume was 20 mL, and the quantitative detection was set at 280 nm. The calibration curve of brazilin was established using authentic
brazilin at the concentration ranges of between 15.6 and 250 mg/mL. Brazilin exhibited a linearity over the evaluated ranges, with the linear equation of Y ¼ 21106X – 38659 (r2 ¼ 0.9999).
Results and discussion Determination of the antibacterial activity of CSE against P. acnes showed that CSE possessed satisfactory antibacterial
Antibacterial activities of brazilin and C. sappan extracts
DOI: 10.3109/13880209.2014.884607
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Table 1. Antibacterial activity of CSE, BRE, and brazilin against acne involved bacteria.
Pharmaceutical Biology Downloaded from informahealthcare.com by University of Colorado Libraries on 12/26/14 For personal use only.
P. acnes
S. epidermidis
S. aureus
Test sample
MIC (mg/mL)
MBC (mg/mL)
MIC (mg/mL)
MBC (mg/mL)
MIC (mg/mL)
MBC (mg/mL)
CSE BRE Brazilin Ampicillin
62.5 31.2 15.6 0.12
125 62.5 31.2 0.12
125 62.5 31.2 0.12
250 125 62.5 0.12
250 125 62.5 0.12
250 125 62.5 0.12
activity with MIC and MBC values of 31.2 and 125 mg/mL, respectively. The anti-P. acnes activity of CSE was markedly stronger than that previously reported by Batubara et al. (2009). This may be due to the different solvents used for the extraction as well as the quality of the plant raw materials. On the basis of the antibacterial assay-guided isolation, CS-1 that had been purified as the antibacterial constituent of C. sappan heartwood was identified as brazilin by 1H NMR and 13C NMR and compared with the data in the literature (Batubara et al., 2010). Brazilin itself showed a stronger antiP. acnes activity than the CSE, with MIC and MBC values of 15.6 and 31.2 mg/mL, respectively. The anti-P. acnes activity of brazilin was also markedly stronger than that previously reported by Batubara et al. (2010). This may be due to the different strains of P. acnes as well as purity of brazilin used in the assay. In addition, brazilin has been reported to possess other bioactivities involed in acne treatment, including anti-inflammatory (IC50 ¼ 24.3 mM) (Bae et al., 2005) and antioxidatative activities (IC50 ¼ 8.8 mM) (Batubara et al., 2010). Brazilin was therefore used as an indicative marker for the preparation and standardization of the active constituent rich C. sappan extract. BRE was successfully prepared from CSE in one step of DiaionÕ HP-20 chromatography. Ethanol (35% v/v) was the appropriate solvent system used for the separation of brazilin. The yield of BRE was 47.8 ± 2.2% w/w dry weight of CSE. On the basis of quantitative HPLC analysis (Figure 2), this method was capable of increasing the content of brazilin in BRE up to 39.9 ± 0.34% w/w, which was almost two-fold higher than that of CSE (20.0 ± 0.26 % w/w). The BRE used for further evaluation of antibacterial activity was standardized by the HPLC method to contain brazilin content of not less than 39% w/w. The antibacterial activity evaluation of brazilin, BRE, and CSE against acne involved bacteria, including P. acnes, S. aureus and S. epidermidis showed that P. acnes was most susceptible to all tested samples. Amongst these samples, brazilin possessed the strongest antibacterial activity against P. acnes, S. epidermidis, and S. aureus, with MIC/MBC values of 15.6/31.3, 31.3/62.5, and 62.5/62.5 mg/mL, respectively, followed by the BRE and CSE (Table 1). This indicated that brazilin plays an important role in the antibacterial activity of CSE and BRE. This result agrees with the findings of Xu and Lee (2004) and Batubara et al. (2010) who have identified brazilin as the major antibacterial constituent of C. sappan heartwood. However, the previous reports by Batubara et al. (2009, 2010) indicated that brazilin possessed lower anti-P. acnes than the crude ethanol extract of C. sappan wood. In contrast, our study has confirmed that
brazilin was the major antibacterial constituent of C. sappan heartwood, which showed a higher anti-P. acnes activity than CSE as well as itself in the previous report by Batubara et al. (2010). This may be due to different purities of the isolated brazilin used in the antibacterial assay. Although BRE had a lower antibacterial activity than brazilin, it showed higher activity than CSE. Based on the yields of BRE (48% w/w) and brazilin (6.4% w/w), BRE has more advantages than brazilin for industrial applications because of its lower cost of production. BRE is therefore an alternative antibacterial agent, with the anti-oxidative (Batubara et al., 2010) and anti-inflammatory activities (Bae et al., 2005; Hu et al., 2009) of brazilin, from C. sappan heartwood that can be used in the pharmaceutical, cosmetic, and nutraceutical industries.
Acknowledgements This work was supported by Prince of Songkla University, Thailand, under postdoctoral fellowship program. Thanks to Dr. Brian Hodgson for assistance with the English language.
Declaration of interest The authors have declared that there is no conflict of interest.
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