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Antioxidant Potential of Anthrarobin (1, 2, 10 trihydroxy anthracene) and its Acyl Derivatives.

Byline: Mehreen Lateef, Sobiya Perwaiz, Bina Shaheeen Sidduqui, Abid Azhar, Lubna Iqbal, Kaniz Fizza Azhar, Kauser Sidduiqui and Kanwal Abbassi

Summary: The current study explores the antioxidant potential of anthrarobin and its synthesized derivatives determined by various methods including DPPH radical scavenging ability, reducing power and superoxide scavenging activity. Anthrarobin was acetylated with acetic anhydride in the presence of pyridine to afford anthracen-1, 10-dihydroxy-2-acetate (1), anthracen-1-hydroxy-2, 10- diacetate (2) and anthracen-1, 2, 10-triacetate (3). Anthrarobin exhibited good antioxidant potential with 68 and 78% at 50 M and 100 M concentrations, respectively.

Reducing power of anthrarobin increases with increase in concentration in a dose-dependent manner as could be seen from 37 and 54% activity at the concentration of 50 and 100 M, respectively. 3-Tetra-butyl-4-hydroxyanisole (BHA) used as standard showed 84 and 93% activities at 50 and 100 M, respectively. BHA and anthrarobin were compared to determine that how much superoxide radicals were scavenged in alkaline DMSO by different concentrations.

It was found that 50 M of anthrarobin was needed to scavenge 50% radicals while 12.5 M of BHA scavenged 50% radicals in DMSO. It is concluded that anthrarobin has highest antioxidant potential as compared to its derivatives. It is postulated that position and numbers of hydroxyl group in anthrarobin are responsible for antioxidant potential and the activity decreases with the substitution of acyl groups at various positions in synthesized derivatives.

Keywords: Anthrarobin, antioxidant, 1, 10 dihydoxyanthracen-2-O-acetate, Anthracen-1,2-10-tri-O acetate, Reducing power, Superoxide radical.


Antioxidants are the compounds, which terminate the attack of reactive species like free radicals and prevent aging and diverse diseases associated with oxidative damages in various body systems. Free radicals are generated as a result of normal aerobic cellular metabolism [1]. Due to incorporation of free radicals from environment to living system or excess production during metabolism may have severe consequences and can lead neuro-degeneration. It may also represent imbalanced defense mechanism of antioxidants. Apart from environmental or genetic factors, free radical in form of oxidative stress attack on neural cells and shows disastrous role to neuro- degeneration and may end into a variety of disorders including Alzheimer's disease, Parkinson's disease, aging and different other neural disorders.

Free radicals toxicity subsidizes to DNA and protein injury, inflammation, tissue damage and consequent cellular apoptosis [2, 3]. The antioxidants are now being explored as an influential therapeutic against overproduction of ROS due to their ability to neutralize free radicals by oxidation. Diet is a major source of antioxidants but synthetic antioxidants are also catching attention as potential source of antioxidants over naturally isolated antioxidant compounds may be due to the following reason. Molecule isolated from natural source is that in very small quantity and additional material may be required for further testing or determination of structure if it possess significant biological activity. Therefore, biological evaluation of compounds obtained by synthetic means has attracted attention because these are inexpensive, less tedious and enough quantities for suitable testing can easily be obtained [4].

Anthrarobin is substituted polycyclic hydrocarbon and its derivatives have shown to have diverse activity depending on the nature of functional group attached. In one study, several other natural and synthetic polyphenolic compounds including anthrarobin were found to be effective inhibitor of type I 5-alpha reductase which is involved in production of benign prostatic hyperplastic and prostatic cancer [5].

Anthrarobin at the concentration of 100 M inhibited 4, 5-pyrenequinone reduction (PQR) by 64-92%. This quinone reductase (PQR) and catechol-O-methyltransferase (COMT) activities of M. vanbaalenii PYR-1 may play an important role in the detoxification of PAH catechols [6]. There are very few studies reported based on biological evaluation of synthetic anthrarobin [7-9] including xanthine oxidase inhibitory potential and chodilator activity, but not for their potential antioxidant activity. Therefore in the present study, antioxidant potential of anthrarobin obtained by synthetic mean and its derivatives was explored by various antioxidant methods for the first time in this study.


Materials and Methods

Anthrarobin and the reagents were purchased from Sigma and Aldrich Chemical Co. Column chromatography was performed with chromatography grade silica gel (particle size 35-70 L and pores size 60 A ; Merck) using a 9.5:0.5 hexane-EtOAc gradient mixture. NMR spectra were recorded using Bruker AC 400 and AM 400 spectrometers operating at 400 MHz for 1H NMR. Deutrated solvent (CDCl3) used was purchased from Apollo Scientific Ltd containing more than 99.8% D. The chemical shift values (d) were measured after calibrating the residual solvent peak for deutrated CDCl3 at 7.23 ppm. The EIMS were recorded on a Finnigan-MAT 311 mass spectrometer at 250 0C and 70 eV. Analytical thin layer chromatography was done on pre-coated silica gel plates. Visualization was achieved by ultraviolet lights and by I2 vapors. Yields are given after purification.

Acetylation of Anthrarobin

To a solution of anthrarobin (100 mg) in pyridine (1 mL), acetic anhydride (2 mL) was added and the reaction mixture was kept overnight at room temperature after warming briefly. The mixture was poured into crushed ice and filtered. The residue was washed with H2O, dried over filter paper and the products were passed through silica gel column which was eluted with pet.ether-EtOAc, in increasing order of polarity to obtain mono-, di- and tri-acetyl derivatives (1-3) respectively (Table-1).

Table-1: Anthrarobin and List of Its Synthetic

Compound###R1###R2###R3 Molecular Formula Yield (%)

1###OH###COCH3 OH###C16H12O4###23

2###OH###COCH3 COCH3###C18H14O5###26.5

3###COCH3 COCH3 COCH3###C20H16O6###33.7

Derivatives 1-3.

Characterization of Compounds

All products were synthesized and purified as above and obtained as yellowish brown powders. The structures of compounds were elucidated on the basis of mass and 1D (1H, 13C) and 2D (COSY, HETCOR and HMBC) NMR experiments.

Anthracen-1, 10-dihydroxy-2-acetate (1)

Yield: Yield: 23%, UV (CHCl3) max/nm: 226; IR (CHCl3) max E/cm-1:2925 (OH), 1735 (R- COO-R), 1653-1444 (C=C); 1H NMR (CDCl3) d : 12.75 (1H, s, OH), 8.33 (1H, d, 8.5 Hz H-5), 8.29 (1H, m, H-7), 7.86 (1H,d, 8.7Hz, H-3), 7.81(1H, m, H-6), 7.45 (1H, d, J= 8.7Hz, H-4), 7.23 (s, H-9), 2.38 (3H, s, OAc); EIMS m/z: 268.

Anthracen-1-hydroxy-2, 10-diacetate (2)

Yield: 26.5%, UV (CHCl3) max/ nm: 270; IR (CHCl3) max E/cm-1: 1730 (R- COO-R ), 1651-1440 (C=C); 1H NMR (CDCl3) d: 12.76 (1H, s, OH), 8.29 (1H, dd, 8.6, 1.2 Hz, H-5), 8.24 (1H, m, H-7), 7.91 (1H, dd, 8.6, 1.2 Hz, H-8), 7.66 (1H,d, 8.4 Hz, H-3), 7.37 (1H, m, H-6), 7.35 (1H, s, H-9), 7.24, (1H, d, J= 8.4 Hz, H-4), 2.48 (6H, s, 2xOAc) ; EIMS m/z: 310.

Anthracen-1, 2, 10-triacetate (3)

Yield: 33.7%, UV (CHCl3) max/ nm: 251; IR( CHCl3) max E/cm-1: 1733 (R- COO-R), 1655-1446 (C=C) cm-1, 1H NMR (CDCl3) d: 8.32 (1H,s, H-9). 7.99, (1H, dd, Hz, 9.2, 1.6, H-5), 7.89 (1H, dd, 9.2, 1.2 Hz, H-8), 7.86 (1H,d, 9.2 Hz, H-3), 7.50 (2H, m, H-6,7), 7.33, (1H, d, J= 9.2 Hz, H-4), 2.34, 2.52, 2.61 (each 3H, s, 3xOAc); EIMS m/z: 352.

Following methods are used to evaluate the antioxidant potential of compounds.

DPPH Radical Scavenging Activity

The antioxidant activity was measured by measurement of scavenging ability of the compounds on free radical 2, 2'-diphenyl-1-picryl hydrazyl (DPPH; C18H12N5O6). On reaction with hydrogen donors, the radicals of DPPH were reduced to the resultant colorless hydrazine derivative. The solution of DPPH was prepared in the concentration of 0.3 mM in ethanol, while serial dilution of fractions was made in DMSO or methanol depending upon the best solubility and diluted to obtain final concentrations of 500, 250, 150, 125, 62.5, 31.2, 15.6, 7.8 M.

DPPH solution was added in the volume of 90 l in each of the well of 96-well plate marked for control and test compounds. Then, 10 l of each of the concentrations of the compound was added to the particular well to start the reaction. The contents of the wells were mixed for few seconds and the mixture was kept at incubation for 30 minutes at 37C and the absorbance was recorded at 517 by microtitre plate reader (Spectra max plus 384 Molecular devices USA). The assay was standardized by butylated hydroxyanisole (BHA) prior to testing the compounds. Percent radical scavenging activity was calcin comparison with methanol treated control and IC50 was calculated for each compound by EZ fit software (Perrella Software, USA) [10].


Reduction Capability

Total reduction capability of Anthrarobin and its derivatives were determined by doing some modifications in the previously described method [11]. Phosphate buffer (250 l, 0.2 M, pH 6.6) was mixed with 100 l of each test compound (10-1000 M). Then, 250 l potassium ferricyanide (K3 Fe- (CN)6 1%) was added and the mixture was kept at incubation for twenty minutes at 50 C . After incubation, 250 l trichloro acetic acid (10 %) was added and then followed by centrifugation for ten minutes. The solution's upper portion (250 l) was collected in separate tubes and 250 l deionized water was added to it. Fifty microliters ferric chloride (Fecl3 0.1 %) was finally added to the contents and the absorbance was read at 700 nm on spectrophotometer (Specord 200, Germany). The increase in absorbance with the increase in concentration of compound showed stronger reducing capability.

Superoxide Scavenging Activity By alkaline DMSO method

For this method to be performed [12]. Nitro- blue tetrazolium (NBT) was prepared in the concentration of 1 mg/ml pure DMSO (AVONCHEM). Alkaline DMSO was prepared by adding 0.1ml 5mM NaOH solution in water and 0.9 ml DMSO. Stock solution of 1mM of test compounds was prepared and serial dilutions were made at the time of assay in the range of 7-1000 M in DMSO or methanol depending on the best solubility. Hundred microliters ml NBT (1 mg/ml) was added in each of the tubes. In tube marked as test, compound was added of different known concentrations in the volume of 300 l. Finally, 1 ml alkaline DMSO was added to give total volume 1.4 ml.

After 5 minutes incubation time, test compound may inhibit the purple coloured formazan formation which was generated by reaction of superoxide radicals liberated by alkaline DMSO with NBT. In the tube of control, 0.1 mM of NBT in 100 l solvent (DMSO or methanol) in which compounds were made and 1 ml pure DMSO (not alkaline) were taken. Absorbance was measured at 560 nm against control and the percentage of super oxide radical scavenging by the test compounds were calculated by


Results and Discussion

The mono-, di- and tri- acetyl derivatives of anthrarobin were synthesized by its reaction with acetic anhydride in presence of pyridine by keeping the reaction mixture over night at room temperature. These derivatives were separated by column chromatography as described in experimental part and their structures were confirmed using spectroscopic techniques.

1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), radical scavenging test is used widely for the determination of antioxidant activity by most researchers. The advantage of this spectrophotometric method is that it requires only single which is stable in aqueous or ethanolic solutions and after accepting an electron or hydrogen ion converted into stable diamagnetic molecule. When various concentrations of BHA and anthrarobin scavenged the free radicals, it is discolored from purple into yellow coloured solution.

Anthrarobin (1, 2, 10 trihydroxy anthracene) shows a very significant antioxidant potential at the concentrations used (Fig. 1). Anthrarobin have 68 and 78 % activity at the concentration of 50 M and 100 M, respectively while BHA shows 90 and 94 percent (at 50 and 100 M respectively) activity. Compounds, anthracen-1, 10-dihydroxy-2-acetate (1), anthracen-1-hydroxy-2, 10-diacetate (2) and anthracen-1, 2, 10-triacetate (3) showed 0.5, 3.2 and 6.9 % at 50 M respectively and at 100 M, compound 1,2 and 3 have 0.9, 4.3 and 20.9 % activity.

On the basis of structure activity relationship it is postulated position of hydroxyl group is responsible for radical scavenging activity and substitution of acyl group in case of compound 1,2 and 3 may decrease the affinity of compound to scavenge the free radicals generated by DPPH. Further comparison of radical scavenging activity of anthrarobin and BHA for the determination of fifty percent inhibitory concentration [IC50value] reveals that radical scavenging activity increases with increase in the concentration in a dose-dependent manner from 12.5 M to 100 M concentration range and IC50 value of anthrarobin is found to be 38.7 0.9 M (Fig. 2).

When anthrarobin was incubated with DPPH for 30 minutes, it scavenged the DPPH solution very rapidly. This effect was observed by the decrease in optical density with time (figure 3). The decrease in optical density was due to the change in colour of DPPH solution from purple to yellow. Antioxidant scavenged the radicals by donating hydrogen. The colour of DPPH in control (DMSO), on the other hand, remained unchanged. BHA was used as standard.

The reducing ability of anthrarobin and BHA was determined by the using the method of Oyaizu (1986) based on the principle of conversion of ferric ions into ferrous state. Anthrarobin have 37 and 54 % activity at 50 and 100uM concentration respectively while BHA shows 84 % (50uM) and 93% (100uM) activities in dose dependent manner. On the other hand compound 1, 2 and 3 showed no significant reducing power. It indicates that reducing ability of anthrarobin also increases in dose dependent manner with increase in concentration of a compound like DPPH radical scavenging activity (Fig. 4).

Superoxide radicals are very harmful to the cellular component. The radicals of super oxide were formed by alkaline DMSO which reacts with NBT to produce colored diformazan. Anthrarobin and BHA at various concentrations scavenge superoxide radicals and inhibited the formazzan formation in dose dependent manner (Fig. 5). It is observed that 50 M of anthrarobin were needed to scavenge 50 % radicals while 12.5 M of BHA scavenged 50 % radicals in alkaline DMSO.


On the basis of above mentioned results it is concluded that anthrarobin (1, 2, 10 trihydroxy anthracene) is found to have significant antioxidant activity but none of its derivatives have good potential to scavenge free radicals created by DPPH or alkaline DMSO. The structure activity relationship of these compounds revealed that the significant activities of the anthrarobin may be attributed due to coordinating capabilities of the OH groups in structure. Due to substitution of acyl group instead of OH group in 1, 10 dihydoxyanthracen-2-O-acetate (1) and Anthracen-1, 2-10-tri-O acetate (2), the affinity of compound decreases to scavenge the free radicals generated by DPPH.


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Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Oct 31, 2015
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