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IOS Press. Original Research Report. Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L. Ratu Safitria,∗. , Ponis Tarigana, Hans ...
BioFactors 19 (2003) 71–77 IOS Press

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Original Research Report

Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L Ratu Safitria,∗ , Ponis Tarigana , Hans Joachim Freislebenb , Rymond J. Rumampukc and Akira Murakamid a Faculty

of Mathematics and Natural Sciences, Universitas Padjadjaran, Bandung Jawa, Indonesia of Medicine, Universitas Indonesia, Jakarta, Indonesia c Faculty of Mathematics and Natural Sciences, Universitas Negeri Manado, Tondano-Minahasa, Indonesia d Division of Food Science and Biotechnology,Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan b Faculty

Received 5 April 2003 Revised 20 May 2003 Accepted 3 July 2003 Abstract. Two antioxidant compounds were isolated from C. sappan L by multiple steps of column chromatography and thin layer chromatography in succession with superoxide scavenging assay as activity monitor. Structures of the two compounds were determined by spectroscopic methods as 1 ,4 -dihydro-spiro[benzofuran-3(2H),3 -[3H-2]benzopyran]-1 ,6 ,6 ,7 -tetrol (compound 1) and 3-[[4,5-dihydroxy-2(hydroxymethyl) phenyl]-methyl]-2,3-dihydro-3,6-benzofurandiol (compound 2). Characterization of antioxidant properties of these two compounds was done by determining the inhibitory effect on xanthine oxidase activity as well as scavenging effect on superoxide anion and hydroxyl radicals. Our results indicated that compounds 1 and 2 inhibited xanthine oxidase activity and scavenged superoxide anion and hydroxyl radicals. Compounds 1 and 2 possessed similar radical scavenging activities as ascorbic acid, and they were more effective than other well-known antioxidants such as α-tocopherol, β-carotene, and BHT. As inhibitors of free radical formation, compounds 1 and 2 were more effective than all the other antioxidants tested. In conclusion, compounds 1 and 2 can be regarded as primary antioxidants with radical-scavenging and chain-breaking activities as well as secondary antioxidants with inhibitory effect on radical generation. Keywords: Flavonoids, free radicals, antioxidants, xanthine oxidase, Caesalpinia sappan

1. Introduction Caesalpinia sappan L (Indonesian name Secang) is a traditional drink or jamu material in Indonesia. It has also been widely used for treatment of tuberculosis, diarrhea, dysentery, pain, and inflammation [1,2]. A group of phenolic compounds such as homoisoflavonoids and related compounds [3], protosappanin ∗

Address for correpondence: Dr. Ratu Safitri, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jl. Jatinangor km 21, Bandung-Sumedang, Jawa Barat, Indonesia. Tel.: +62 22 7206774; Fax: +62 22 2509013; E-mail: [email protected]. 0951-6433/03/$8.00  2003 – IOS Press. All rights reserved

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R. Safitri et al. / Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L

E-1 and E-2 [4], brazilin and brazilein [5], have been isolated from C. sappan. A previous report showed that two compounds, brazilin and isobrazilin, possessed antioxidant activity [6]. Phenolic compounds such as flavonoids are well known in their role as antioxidants; they exhibit radical scavenging activity and inhibit the enzymes that are responsible for radical production. Antioxidants can influence the oxidation process and by different means. In this regard, antioxidants can be categorized into two main types, namely primary antioxidants and secondary antioxidants [7]. While the former type scavenges reactive oxygen species, the latter type suppresses the formation of reactive oxygen species by inhibition of enzymes or chelating of transition metal ions [8]. In the present study, we report the antioxidant activities of two aromatic compounds 1 and 2, of which chemical structures have previously been established by spectroscopic methods as 1 ,4 -dihydro-spiro[benzofuran-3(2H),3-[3H-2]benzopyran]-1 ,6 ,6 ,7 -tetrol and 3-[[4,5-dihydroxy-2(hydroxymethyl)phenyl]methyl]-2,3-dihydro-3,6-benzofuran-diol, respectively [9,10].

2. Materials and methods All chemicals were purchased from Sigma (Indonesia) and Merck (Indonesia) and were of analytical grade. All assays were done in triplicate with increasing concentrations and at three ranging from 125 to 500 µg/mL. Data were analyzed by Student’s t test and significant differences were determined when p < 0.05. 2.1. Plant material The bark of C. sappan L was collected from Wonolagi forest, located at Wonosari region in the Center of Java, Indonesia. This plant was identified in the Bogoriense Herbarium, Indonesian Institute of Sciences (LIPI), Bogor, Indonesia. 2.2. Extraction and isolation Air-dried powdered bark (1 kg) was extracted with 5 L H 2 O at 96◦ C for 15 minutes. The filtrate was evaporated at 50 ◦ C to obtain an aqueous extract (93.1 g). This extract (15 g) was partitioned with petroleum ether/methanol (MeOH) (5:1; v/v). The MeOH layer was evaporated to obtain a methanolic extract (11.38 g). The methanol extract (10 g) was subjected to silica gel column chromatography and eluted with chloroform (CHCl3 ), ethylacetate (EtOAc), MeOH, and H 2 O in succession to yield 8 fractions. Further separation was done for fraction 5 (3.92 g) by column chromatography using the same stationary and mobile phases as above to give 16 fractions (F5.1–F5.16). The fraction F5.5 (1.43 g) was subjected to silica gel column chromatography using a solvent system of CHCl 3 /MeOH/H2 O (9:3:1; v/v/v) to yield another 5 fractions (F.5.5.1–F.5.5.5). Fraction F.5.5.3 (0.22 g) fractionated by an ODS column using MeOH/H2 O (4:6; v/v) to elute 12 sub-fractions (F.5.5.3.1–F.5.5.3.12). The sub-fractions (F.5.5.3.7 - F.5.5.3.9) were combined and purified by preparative thin layer chromatography on silica gel F254 with CHCl3 /MeOH (9:1; v/v), plus one drop of acetic acid as the mobile phase to obtain compounds 1 and 2.

R. Safitri et al. / Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L

4

HO

4a

3

1a

2 1

6 6a 7

O

O 3

4a

2

1a 1

8

HO

11a

OH 7

6a

7a 8

11a 9

9

11

11 10

6

12

7a

12 HO

4

HO

O

10

OH

OH

OH

OH

1

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2

Fig. 1. The structures of test compounds from Caesalpinia sappan L. The structure of compound 1 is 1 ,4 -dihydrospiro[benzofuran-3(2H),3 -[3H-2]benzopyran]-1 ,6 ,6 ,7 -tetrol and the structure of compound 2 is 3-[[4,5-dihydroxy-2(hydroxymethyl)phenyl]methyl]-2,3-dihydro-3,6-benzofuran-diol [9,10].

2.3. Antioxidant tests 2.3.1. Inhibition of xanthine oxidase assay Xanthine oxidase (XOD) activity was measured by spectrophotometric method as described in [11]. The assay mixture consisted of 1.0 mL test solution, 2.9 mL 1/15 phosphate buffer (pH 7.5) and 0.1 mL of enzyme solution. After incubating the mixture at 25 ◦C for 15 min, the reaction was initiated by adding 2.0 mL of substrate solution. This assay mixture was incubated at 25 ◦ C for 30 min. Then, the reaction was stopped by adding 1.0 mL of sodium dodecyl sulfate (SDS) and the absorbance was read spectrophotometrically at 290 nm. A blank was prepared in the same way, but the enzyme solution was added to the assay mixture after adding 1.0 mL of SDS. Xanthine oxidase inhibitory activity was expressed as the percentage of inhibition estimated from the equation: (1-B/A) × 100, where A is the activity of the enzyme without the test material and B is the activity of the enzyme with the test material. 2.3.2. Superoxide anion radical scavenging activity The superoxide anion radical scavenging activity was measured using a hypoxanthine and xanthine oxidase system (pH 7.4) for generating superoxide radicals as described [12]. XOD solution (500 µL) from butter milk 0.049 U/mL was added to a 500 µL mixture of solution containing NBT (0.24 µM) or phosphate buffered saline (PBS), xanthine (0.4 µM), and a solution of the test substance dissolved in 50 µL of dimethylsulfoxide (DMSO). After incubating at 37 ◦ C for 20 min, the reaction was stopped by the addition of 1 mL of SDS solution. Absorption of the reaction mixture was measured at 560 nm to determine the inhibition of nitotetrazolium blue reduction [12]. 2.3.3. Hydroxyl radical scavenging activity The hydroxyl radical scavenging activity was determined using deoxyribose assay as described [13]. 2-deoxyribose was oxidized and degraded into thiobarbituric acid reactive substances by • OH generated from the Fenton reaction. An aliquot (0.2 mL) of FeSO 4 × 7 H2 O (10 mM) and EDTA (10 mM) was added into a screw-capped test tube and mixed with 0.2 mL of 2-deoxyribose (10 mM) as well as enough

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R. Safitri et al. / Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L Table 1 Inhibition activities of 1,2, and commercial antioxidants Compounds Compound 1 Compound 2 Ascorbic acid α-Tocopherol β-Carotene BHT

500 µg/mL 96.7 100 51.7 29.3 100 74.9

Activities (%) 250 µg/mL 125 µg/mL 81.7 75 98.1 94.3 40.6 21.5 16.5 5.9 61.4 49.6 55.5 34.9

Mean + SD 84.5 ± 9.1 97.5 ± 2.9 37.9 ± 12.5 17.2 ± 10.2 70.3 + 21.9 55.1 ± 9.1

volumes of sample solution and phosphate buffer (0.1 M; pH 7.4) to make up a final volume of 1.8 mL. Finally, 200 µL of a H2 O2 solution (10 mM) was added to the mixture and the reaction mixture was incubated at 37◦ C for 4 hours. After the incubation, 1 mL of TCA (2.8%, w/v) and thiobarbituric acid (1%, w/v) were added and the mixture was boiled for 10 min. After cooling on ice, absorbance at 532 nm of the reaction mixture was measured [13].

3. Results and discussion The heartwood of C. sappan was extracted with methanol (MeOH). Then, the MeOH extract was fractionated by column chromatography gradient elution with chloroform, ethyl acetate, methanol and water. The activity of inhibition of xanthine oxidase and superoxide anion radical and hydroxyl radical scavenging of each fraction was tested and the most active fractions were further purified by silica gel column chromatography to obtain two antioxidant compounds, 1 and 2 (Fig. 1) [9,10]. The structures of 1 and 2 in Fig. 1 show that these compounds contain phenolic hydroxyl groups. The inhibition of xanthine oxidase and the radical scavenging activity of these compounds were compared with four commercial antioxidants, ascorbic acid, α-tocopherol, β -carotene, and butylated hydroxytoluene (BHT). 3.1. Antioxidant activity In all experiments, most compounds exhibited dose-dependent efficacy; exceptions will be discussed. The highest activity was set to 100% and the other activities accordingly as percentages of the highest activity. Of each compound and each assay, mean values and standard deviation (± SD) were calculated. Significance of the differences is only given for XOD inhibition. No significant differences were determined for the two radical scavenging assays, because the efficacy of water- and lipid-soluble antioxidants depends very much on experimental conditions. Hence, the comparison between the various antioxidants used is only valid in the actual experiment. This means, if the same experiment is carried out in aqueous solution and secondly in lipophilic environment, e.g., in organic solvents or in liposomes, activities of ascorbate on the one hand and tocopherol or carotenoids on the other hand will vary considerably. Of course, the natural environment of tocopherol and carotenoids are membrane lipids and there, they will develop maximum activity. Under these conditions, it does not make sense to calculate significant differences. The compounds tested (compounds 1 and 2) phenolic antioxidants (flavonoids) exert intermediate solubility. Their mechanism of action should resemble that of phenolic tocopherol although the latter is more lipophilic.

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Table 2 IC50 Values of 1, 2, and commercial antioxidants for inhibition of xanthine oxidase and radical scavenging activities Compounds Compound 2 Compound 1 Ascorbic acid α Tocopherol β-Carotene BHT

IC50 (µg/mL) Xanthine oxidase inhibition < 50 > 50 > 500 > 500 > 200 > 250

IC50 (µg/mL), superoxide anion radical scavenging < 50 < 50 < 50 > 500 > 250 > 500

IC50 (µg/mL), hydroxyl radical scavenging < 100 < 300 < 150 < 500 > 500 > 500

3.2. Inhibition of xanthine oxidase activity Compounds 1 and 2 (C1, C2) inhibit xanthine oxidase activity (Table 1). The compounds are stronger inhibitors than the commercial antioxidants, carotene, BHT, ascorbate, and tocopherol; compound 2 exhibiting highest inhibition: C2>C1>carotene> BHT>ascorbate>tocopherol. Compound 2 ranges from 94.3 to 100% inhibition (97.5 ± 2.9); compound 1 is 75.0 to 96.7% (84.5 ± 9.1), followed by carotene, 49.6 – 100% (70.3 ± 21.9), BHT 34.9 – 74.9% (55.1 ± 17.2), ascorbate, 21.5 – 51.7% (37.9 ± 12.5), and finally tocopherol, 5.9 – 29.3% (17.2 ± 10.2). Differences between compound 2 and all other substances are significant; compound 1 does not differ significantly only from carotene. IC50 values (Table 2) indicate that compounds 1 and 2 in spite of their flavonoid properties slightly differ as enzyme inhibitors. The number and position of hydroxyl groups may contribute to xanthine oxidase inhibiting capacity [14], but in this case lower capacity may be due to steric hindrance because of the presence of cyclic hemiacetal at C-6 and C-12 of compound 1. Inhibitory activity is furthermore influenced by planarity of the structure. In flavonoids, it is primarily the presence of hydroxyl groups at C-5 and C-7 that is important for effective inhibition whereas hydroxyls at C-6 and C-3 reduce the inhibitory effect. The latter is also decreased in flavonoids by the presence of C-2  , C-3 and C-4 hydroxyl substituents at ring B [14]. It is concluded that the number and position of hydroxyl groups besides other aspects of molecule structure (e.g., planarity, steric hindrance) influence inhibitory activity. The inhibition of xanthine oxidase activity is of medical interest because it may decrease uric acid formation [11]. Thus, compounds 1 and 2 are possibly novel and alternative therapeutics to prevent hyperuricaemia associated with gout. 3.3. Superoxide anion radical scavenging activity Compounds 1 and 2 and ascorbic acid are effective superoxide anion and hydroxyl radical scavengers in our test system (Table 3). Ascorbate and compound 2 exhibit 100% scavenging activity of the superoxide anion radical at each concentration tested (no dose-dependent efficacy in the range from 125 to 500 µg/mL), whereas compound 1 is in the range from 95 to 100% (98.3 ± 2.9). Carotene, 28.4 ± 72.4% (54.8 ± 19.0); BHT, 19.1 – 32.7% (25.0 ± 7.0), and tocopherol, 9.0 – 50.8% (23.7 ± 19.2) are inferior in our test system. The mechanism of hydrogen donation can be compared to that of ascorbic acid. The antioxidant mechanism of ascorbic acid by donation of one electron from ascorbate yields semidehydroascorbyl radical (SDA), which can be further oxidized to dehydroascorbate (DHA) [15].

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R. Safitri et al. / Antioxidant activity in vitro of two aromatic compounds from Caesalpinia sappan L Table 3 • Superoxide anion (O•− 2 ) and hydroxyl radical ( OH) scavenging activities of 1,2, and commercial antioxidants Campounds Radicals Activities (%) Mean + SD 500 µg/mL 250 µg/mL 125 µg/mL Compound 1 O•− 100 100 95 98.3 ± 2.9 2 • OH 68.6 39 36.7 48.1 ± 15.2 100 100 100 100 ± 0 Compound 2 O•− 2 • OH 89 63.8 55.8 69.5 ± 14.9 Ascorbic acid O•− 100 100 100 100 ± 0 2 • OH 90.5 76.9 39.7 69.0 ± 21.5 50.8 11.2 9.0 23.7 ± 19.2 α-Tocopherol O•− 2 • OH 66.2 50.2 41.1 52.1 ± 11.0 β-Carotene O•− 72.4 63.7 28.4 54.8 ± 19.0 2 • OH 37.7 28.7 22.0 29.5 ± 6.5 BHT O•− 32.7 23.3 19.1 25.0 ± 7.0 2 • OH 51.8 47.2 33.3 44.1 ± 8.2

3.4. Hydroxyl radical scavenging activity Compound 2, 55.8 to 89.0% (69.5 ± 14.9) and ascorbate, 39.7– 90.5% (69.0 ± 21.5) are the most potent hydroxyl radical scavengers in our system (Table 3), followed by tocopherol, 41.1 ± 66.2% (52.1 ± 11.0) and compound 1, 36.7 ± 68.6% (48.1 ± 15.2), whereas BHT, 33.3 ± 51.8% (44.1 ± 8.2) and carotene, 22.0– 37.7% (29.5 ± 6.5) show lower activities. Compounds 1 and 2 are strong superoxide anion and hydroxyl radical scavengers, comparable to ascorbate and α-tocopherol, but with higher scavenging activities than β -carotene and BHT. This result can also be explained by the presence of phenolic hydroxyl groups as hydrogen donors. As a result of hydrogen abstraction, a radical species is formed, and in order to fill its empty electron shell compound 1 and 2 can undergo several mesomeric rearrangements. In flavonoids and other phenolic compounds, the antioxidative potency is related to the structure, in particular to electron delocalisation of the aromatic nucleus. The resonance effect of the aromatic nucleus stabilizes the radical formed in these compounds by hydrogen donation. The most likely reaction or rearrangement is donation of a second hydrogen to form an o-quinone [16–19]. As mentioned above, the presence of phenolic hydroxyls, especially in ortho position (catechol group) strengthens the electron donating properties [8]. Compounds with ortho- and para-dihydroxy groups contribute markedly to the antioxidative activity, ortho position having greater activity than para position, whereas meta position has no positive effect on antioxidant activity [20]. Ortho position provides increased stability to the radical form of polyphenols because phenoxyl radical is stabilized by intra-molecular hydrogen bonds [20]. The IC50 values in Table 2 show that at low concentration, 3-[[4,5-dihydroxy-2-(hydroxymethyl) phenyl] methyl]-2,3-dihydro-3,6-benzofuran-diol (compound 2) inhibits XOD activity, i.e., it decreases uric acid production, which automatically also decreases superoxide anion radical production. The IC 50 values also indicate that compounds 1 and 2 can act as scavengers for superoxide anion and hydroxyl radicals, compound 2 being more potent than compound 1. In conclusion, these flavonoids (compounds 1 and 2) can be grouped as primary and secondary antioxidants, because they act as radical scavengers and as inhibitors of xanthine oxidase. Among the commercial antioxidants used for comparison, only β -carotene exerts similar inhibitory properties, although its IC50 is at least four-fold higher. In the superoxide anion radical scavenging test, ascorbate is

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more effective than the other commercial antioxidants and it resembles compounds 1 and 2. Scavenging hydroxyl radical, flavonoid 2 and ascorbate are the most potent, followed by α-tocopherol and compound 1. Especially flavonoid 2 is a very potent antioxidant with a broad spectrum of efficacy and it is worthwhile to be further tested in vivo. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]

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