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DOI: 10.1055/s-2002-35666
© Georg Thieme Verlag Stuttgart · New York
Antioxidant, Free Radical Scavenging and Anti-Inflammatory Effects of Aloesin Derivatives in Aloe vera
Prof. Akira Yagi
Faculty of Pharmacy and Pharmaceutical Sciences
Fukuyama University
Fukuyama Hiroshima 729-0292 Japan
Email: yagi@fupharm.fukuyama-u.ac.jp
Fax: +81-849/36-2024
Publication History
Received: February 8, 2002
Accepted: June 15, 2002
Publication Date:
26 November 2002 (online)
Abstract
Antioxidant components in Aloe vera were examined for lipid peroxidation using rat liver microsomal and mitochondrial enzymes. Among the aloesin derivatives examined, isorabaichromone showed a potent antioxidative activity. The DPPH radical and superoxide anion scavenging activities were determined. As one of the most potent components, isorabaichromone together with feruloylaloesin and p-coumaroylaloesin showed potent DPPH radical and superoxide anion scavenging activities. Electron spin resonance (ESR) using the spin trapping method suggested that the potent superoxide anion scavenging activity of isorabaichromone may have been due to its caffeoyl group. As A. vera has long been used to promote wound healing, the inhibitory effects of aloesin derivatives for cyclooxygenase (Cox)-2 and thromboxane (Tx) A2 synthase were examined and the participation of p-coumaroyl and feruloyl ester groups in the aloesin skeleton was demonstrated. These findings may explain, at least in part, the wound healing effects of A.vera.
#Abbreviations
ADP:adenosine diphosphate
ASA:ascorbic acid
BHT:butylated hydroxytoluene
BSA:bovine serum albumin
DMPO:5,5-dimethyl-1-pyrroline N-oxide
DPPH:1,1-diphenyl-2-picrylhydrazyl
EDTA:edetic acid
HEPES:N-(2-hydroxyethyl)-piperazine-N-2′-ethane-sulfonic acid
NADH:reduced nicotinamide adenine dinucleotide
NADPH:reduced nicotinamide adenine dinucleotide phosphate
NBT:nitroblue tetrazolium
Pg:prostaglandin
SOD:superoxide dismutase
TBA:thiobarbituric acid
TCA:trichloroacetic acid
XOD:xanthine oxidase
Key words
Aloe vera - Asphodelaceae - isorabaichromone - antioxidants - free radical - ESR - anti-inflammatory
Introduction
In vitro systems undergoing lipid peroxidation were chosen as experimental models to study the effect of aloesin derivatives [1]. As an antioxidant, aloeresin D has been reported using rat brain homogenates [2]. As inhibitory components for mushroom-tyrosinase, aloeresin A and B [3] and iso-aloeresin E [4] were identified in A. arborescens var. natalensis and A. vera, respectively. The results showed a possible candidate with the chromone skeleton for antioxidant activity. Barbaloin from the yellow bitter sap in the leaf epidermis is possibly responsible for the antioxidant activity; however, it was shown to be present in a lower ratio in A. vera gel than in the epidermal tissue by HPLC [5]. The wound healing property of A. vera [6] and the inhibitory effect of aloeresin E against ear-swelling induced by topical use of croton oil in mice [7] were reported. In this in vitro experiment, we evaluated the components of A. vera showing antioxidative effects against Cox-2 and TxA2 synthase, both of which are deeply linked to inflammation [8].
#Materials and Methods
#Chemicals
Aloesin (1), aleresin A (p-coumaroylaloesin, 2), feruloylaloesin (3), isorabaichromone (4), isoaloeresin D (5), isoaloeresin E (6) and 7-methoxy-8-glucosylaloesol (7) (Fig. [1]) were isolated from A. vera and identified by HPLC with the purity of 99 % [9]. The biochemicals were obtained from the following sources: ADP (Sigma Chemicals Co., USA); NADH, NADPH (Oriental Yeast Co., Japan); SOD, BSA (Nacalai Tesque, Japan) and XOD (Wako Pure Chemical Ind., Japan). All chemicals used were of the highest analytical grade available. Instrument: JEOL JES-TE 200 ESR spectrometer. Parameters were as follows: microwave power, 1.00 mV; frequency, 100 KHz; amplitude, 200; response time, 0.1 sec; sweep rate, 4.00 min.
Fig. 1 Chemical structures of compounds 1 - 7 (1 = aloesin, 2 = aloeresin A, 3 = aloeresin B, 4 = isorabaichromone, 5 = isoaloeresin D, 6 = aloeresin E, 7 = 8-C-glucosyl-7-O-methyl-(S)-aloesol ).
Preparation of rat liver microsomes and mitochondria
Male Wistar rats (Japan SLC Inc.) were maintained within the policy on animal care expressed in Fukuyama University guidelines. The livers of the rats weighing 100 - 150 g, were removed and dropped into ice-cold 3 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose and 0.1 mM EDTA. Mitochondria were obtained as a pellet after centrifugation at 15,000 g [10] and then resuspended in 100 mM HEPES buffer (pH 7.4). Submitochondrial particles were prepared by sonication of mitochondrial suspension for 1 min at 4 °C using a Model 450 Sonifier. Microsomes were obtained as a pellet after centrifugation at 105,000 g for 60 min [11].
#NADPH-dependent peroxidation of microsomal lipid
The microsomes (0.2 mg protein) were incubated at 37 °C in 1 ml reaction mixtures containing 0.05 M Tris-HCl (pH 7.5), 2 mM ADP, 0.12 mM FeCl3 and 0.1 mM NADPH. The reaction was initiated by the addition of NADPH. After 15 min, BHT-TBA reagent [90 μl of 2 % BHT and 2 ml of TCA-TBA-HCl (15 % w/v TCA, 0.375 % TBA, 0.25 N HCl)] was added to the reaction mixture. The solution was heated for 15 min at 100 °C. After cooling and centrifugation at 5000 g for 10 min, the absorbance of the supernatant was determined at 535 nm.
#Ascorbate (ASA)-induced microsomal lipid peroxidation
The lipid peroxidation was determined by incubation with rat liver microsomes at 37 °C in 1 ml reaction mixtures containing 20 mM phosphate buffer (pH 6.0), 90 mM KCl, 0.12 mM FeCl3 and 0.5 mM ASA. The reaction was initiated by the addition of ASA. After 15 min, BHT-TBA reagent was added to the reaction mixture to evaluate lipid peroxidation [12].
#NADH-dependent mitochondrial lipid peroxidation
Rat liver submitochondrial particles (0.3 mg protein) were incubated at 37 °C in 1 ml reaction mixtures containing 50 mM HEPES-NaOH (pH 7.0), 2 mM ADP, 2 mM FeCl3, 10 mM rotenone and 0.1 mM NADH. After 5 min, the reaction was terminated [12].
#Ascorbate (ASA)-induced mitochondrial lipid peroxidation
The lipid peroxidation was determined by incubation of rat liver submitochondrial particles at 37 °C in 1 ml reaction mixtures containing 0.05 M HEPES-NaOH buffer (pH 7.4), 0.125 M KCl, 0.1 mM FeSO4 and 0.2 mM ASA. The reaction was initiated by the addition of ASA. After 20 min, BHT-TBA reagent was added to the reaction mixture to evaluate the lipid peroxidation [12].
#Radical scavenging activity on DPPH
The reaction mixture consisted of 1.2 ml of 250 mM acetate buffer (pH 5.5), 0.8 ml of ethanol and 0.6 ml of 0.25 mM DPPH. After incubation of the mixture at 30 °C for 30 min, the reaction was terminated by the addition of 30 μl of 0.3 % BHT. The absorbance of the remaining DPPH was determined colorimetrically at 517 nm. The scavenging activities were expressed as a percentage of absorbance of control DPPH solution [13].
#Assay of superoxide anion
The reaction mixture consisted of 100 mM Na phosphate buffer, (pH 7.8), containing 0.1 mM EDTA, 50 μM NBT, 0.1 % BSA and 5.9 × 10-3 U of XOD in a final volume of 3 ml. After incubation at 37 °C for 20 min, the reaction was terminated by addition of 0.1 ml of 6 mM CuCl2. The absorbance of formazan produced was determined at 560 nm.
#Determination of xanthine oxidase activity
The reaction mixture consisted of 0.1 M phosphate buffer (pH 7.8) containing 0.1 mM EDTA and 5.9 × 10-3 U of XOD in a final volume of 3 ml. After incubation at 37 °C for 20 min, the reaction was terminated by the addition of 0.1 ml of 6 mM CuCl102. The absorbance of the uric acid formed was determined at 290 nm [14].
#Electron spin resonance (ESR) study
Aliquots of 1 μl of the sample were incubated in reaction mixtures containing 1.5 mM hypoxanthine and 0.27 U/ml of XOD at 37 °C for 10 min. Sequential ESR scans were recorded after the spin trapping of superoxide anion in DMPO and compared to the ESR scans from SOD as a positive control at a final concentration of 60 U/ml [15].
#Determination of cyclooxygenase-2 and thromboxane A2 synthase
The reaction mixture was incubated with 0.11 U Cox-2, 1 mM reduced glutathione, 500 μM phenol and 1 μM hematin for 15 min at 37 °C. The reaction was initiated by addition of 0.3 μM arachidonic acid as a substrate in Tris-HCl buffer (pH 7.7) and terminated after 5 min incubation at 37 °C by addition of 1 N HCl. Following centrifugation, substrate conversion to PgE2 was measured using an Amersham enzyme immunoassay kit [16]. The reaction mixture was incubated with 5 μg/ml TxA2 synthase and 0.1 μg PgG2 as substrate in Tris-HCl buffer (pH 7.4) for 30 min at 37 °C. TxB2 produced was quantified by radioimmunoassay [17].
#Results and Discussion
Compound 4 inhibited the production of lipid peroxides induced by microsomal NADPH-oxidation with IC50 of 23 μM, while aloesin and related derivatives showed weak activity in this system (Table [1]). Quercetin and catechin were used as reference compounds and showed activities at IC50 of 21.8 and 14.5 μM, respectively. In ASA-dependent microsomal lipid peroxidation, compound 3 inhibited the lipid peroxides with an IC50 of 85 μM, while aloesin and related derivatives showed weak activity. Barbaloin inhibited the production of lipid peroxides in this system with an IC50 of 64 μM. α-Tocopherol, quercetin and catechin were used as reference compounds and showed activity with IC50 values of 98, 19 and 29.3 μM, respectively.
NADH supports enzymatically induced lipid peroxidation in submitochondrial particles in the presence of an iron chelate. All tested aloesin derivatives showed weak activity that was comparable to those of α-tocopherol and quercetin. In contrast, barbaloin and catechin showed inhibition of lipid peroxidation with IC50 values of 51 and 48.6 μM, respectively. In ASA-dependent mitochondrial lipid peroxidation, compound 4 showed activity in this type of non-enzymatically stimulated lipid peroxidation and complete inhibition was obtained at 30 μM (data are not shown). Compound 3 also showed protection against lipid peroxidation with an IC50 of 95 μM. No significant activity was observed from aloesin and other derivatives, while barbaloin showed protection against lipid peroxidation in this system with an IC50 of 65 μM. Quercetin and catechin showed activity with IC50 values of 13.4 and 12.7 μM, respectively. The radical scavenging activities of the compounds that can be measured as decolorizing activity following the trapping of the unpaired electron of DPPH are shown in Table [1]. Compound 4 which showed anti-oxidative activity against microsomal and non-enzymatic mitochondrial lipid peroxidation was a potent radical scavenger with an IC50 value of 4 μM. Compounds 1, 2 and 3 showed scavenging activity with IC50 values of 20, 26 and 26 μM, respectively, while compounds 5, 6 and 7 had no effect. α-Tocopherol, barbaloin, quercetin and catechin showed activity with IC50 values of 14, 68, 3 and 4.3 μM, respectively. The superoxide anion (O2 -) actively participates in the initiation of lipid peroxidation and several oxidative enzymes, e. g., XOD, produce O2 - radicals as a normal product of the one electron reduction of oxygen, resulting in tissue injury. Compound 4 was also effective in scavenging O2 - generated by the xanthine/XOD system (Table [1]). Compounds 1, 2, 3, 5, 6 and 7 showed weak activity that was comparable to α-tocopherol and barbaloin. Quercetin and catechin showed activity with IC50 values of 53.8 and 0.8 μM, respectively. Only compound 4 inhibited NBT reduction and other compounds had no effect on O2 - generation by XOD, therefore, it must be clarified if the inhibition of NBT reduction was caused by O2 - scavenging activity or XOD inhibition. The effect of compound 4 on XOD activity was determined through uric acid formation and the results showed that it had no effect on XOD activity at a concentration of 100 μM (data are not shown). ESR which detects the unpaired electron present in the free radical, was carried out using the spin trapping technique [18] to confirm the mechanism of action of compound 4 as a potent O2 - scavenger among the tested aloesin derivatives. Compound 4 showed scavenging activity on the superoxide anion generated by the XOD/hypoxanthine system at a concentration of 5.0 μM. The anti-inflammatory activity of A. vera extract demonstrated its superiority as a Cox-2 inhibitor over another anti-inflammatory agent, aspirin, with regard to the eicosanoid cascade [19] and it also acts as a TxA2 synthase inhibitor at the time of injury, penetrates the injured area and relieves pain [20]. Compounds 2 and 3 reduced TxA2 level with IC50 values of 58 and 13.6 μM, respectively (Table [2]). Compounds 1 and 4 showed some inhibitory activities on Cox-2 and TxA2 synthase (data are not shown). Isorabaichromone with a catechol moiety, in which the hydrogen atom of the caffeoly functional group can be readily donated, acts mainly as a radical scavenger and breaks the chain propagation in the chain reaction of lipid peroxidation. The activities of p-coumaroyl- and feruloyl-aloesin on TxA2 together with that of aloesin on Cox-2 may explain, at least in part, the wound healing activity of A. vera.
| IC50 b | ||||||
| Compounda | Lipid peroxidation | Radical scavenging | ||||
| Microsome | Mitochondria | DPPH radical | Superoxide anion | |||
| NADPH- | ASA- | NADH- | ASA- | |||
| dependent | dependent | |||||
| 1 | >100 | >100 | >100 | >100 | 20 ± 0.3 | >100 |
| 2 | >100 | >100 | >100 | >100 | 26 ± 0.9 | >100 |
| 3 | >100 | 85 ± 1.0 | >100 | 95 ± 2.8 | 26 ± 0.2 | >100 |
| 4 | 23.0 ± 0.5 | >100 | >100 | 20 ± 0.2 | 4.0 ± 0.1 | 7.0 ± 0.2 |
| 5 | >100 | >100 | >100 | >100 | >100 | >100 |
| 6 | >100 | >100 | >100 | >100 | >100 | >100 |
| 7 | >100 | >100 | >100 | >100 | >100 | >100 |
| Barbaloin | >100 | 64 ± 1.0 | 51 ± 1.7 | 65 ± 0.1 | 68 ± 0.9 | >100 |
| α-Tocoph. | >100 | 98 ± 1.3 | >100 | >100 | 14 ± 0.2 | >100 |
| Quercetin | 21.8 ± 1.9 | 19.0 ± 0.2 | >100 | 13.4 ± 1.6 | 3.0 ± 0.2 | 53.8 ± 4.2 |
| Catechin | 14.5 ± 1.6 | 29.3 ± 3.1 | 48.6 ± 3.4 | 12.7 ± 1.8 | 4.3 ± 0.3 | 0.8 ± 0.1 |
| a The test samples and positive controls were used at a maximum concentration of 100 μM in DMSO. | ||||||
| b Inhibitory activity is expressed as the mean of 50 % inhibitory concentration of triplicate determinations, obtained by interpolation of concentration-inhibition curves. | ||||||
| IC50 | ||
| Compounda | Cycloxygenase -2 | Thromboxane A2 synthase |
| 1 | >100 | >100 |
| 2 | >100 | 58.0 ± 5.0 |
| 3 | >100 | 13.6 ± 0.42 |
| 4 | >100 | >100 |
| Nimesulide 1-(7-Carboxyheptyl)- imidazol HCl |
2.5 ± 0.4 |
9.0 ± 0.7 |
| a The compounds were tested at an maximum concentration of 100 µM and DMSO was used as a solvent for the assay. | ||
References
- 1 Yagi A, Haraguchi H. Protective effect of terpenoids against oxidative stresses. In: Pandarai SG. editor
Resent research development in phytochemistry . Vol.1 India; Research Signpost 1997: 11-24 - 2 Lee K Y, Weintraub S T, Yu B P. Isolation and identification of a phenolic antioxidant from Aloe barbadensis . Free Radical Biological & Medicines. 2000; 28 261-5
- 3 Yagi A, Kanbara T, Morinobu N. Inhibition of mushroom tyrosinase by Aloe extract. Planta Medica. 1987; 53 515-7
- 4 Okamura N, Hine N, Harada S, Fujioka T, Mihashi K, Yagi A. Three chromone components from Aloe vera leaves. Phytochemistry. 1996; 43 495-8
- 5 Okamura N, Asai M, Hine N, Yagi A. High-performance liquid chromatograpic determination of phenolic compounds in Aloe species. Journal of Chromatography A.. 1996; 746 225-31
- 6 Davis R H. Aspirin and Aloe, Aloe vera - a scientific approach. New York; Vantage Press 1997: 226-62
- 7 Hutter J A, Salman M, Stavinoha W B, Satsangi N, Williams R F, Streeper R T, Weintraub S T. Anti-inflammatory C-glycosyl chromone from Aloe barbadensis . Journal of Natural Products. 1996; 59 541-3
- 8 Reynolds T, Dweck A C. Aloe vera leaf gel: a review update. Journal of Ethnopharmacology. 1999; 68 3-37
- 9 Okamura N, Hine N, Tateyama Y, Nakazawa M, Fujioka T, Mihashi K, Yagi A. Three chromones of Aloe vera leaves. Phytochemistry. 1997; 45 1511-3
- 10 Johnson D, Lardy H. Isolation of the liver and kidney mitochondria. In: Estabrook RW, Pullman M. editors
Methods in Enzymology . New York:; Academic Press 1967 10: 94-6 - 11 Liu G, Zhang T, Wang B, Wang Y. Protective action of seven natural phenolic compounds against peroxidative damage to biomembrane. Biochemical Pharmacology. 1992; 43 147-52
- 12 Wills E D. Lipid peroxidation in microsomes. General consideration. Biochemical Journal. 1969; 113 315-24
- 13 Blois M S. Antioxidant determination by the use of a stable free radical. Nature. 1958; 182 1199-200
- 14 Halliwell B. Use of desferrioxamine as a ”probe” for iron-dependent formation of hydroxyl radicals. Biochemical Pharmacology. 1985; 34 229-33
- 15 Mitsuya K, Mizuta Y, Kohno M, Mori A. The application of ESR spin trapping technique to the evaluation of SOD-like activity of biological substances. Bulletin of Chemical Society of Japan. 1990; 63 187-91
- 16 Riendeau D, Charleson S, Cromlish W, Mancini J A, Wong E, Guay J. Comparison of the cyclooxygenase-1 inhibitory properties of non-steroidal anti-inflammatory drugs and selective Cox-2 inhibitors, using sensitive microsomal and platelet assays. Canadian Journal Physiological Pharmacology. 1997; 75 1088-95
- 17 Borsch-Haubold A G, Pasquet S, Watson S P. Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203 580 and PD 98 059. SB also inhibits thromboxane synthase. Journal Biological Chemistry. 1998; 273 28 766-72
- 18 Janzen E G, Stronks H J, Dubose C M, Poyer J L, McCay P B. Chemistry and biology of spin trapping radicals associated with halocarbon metabolism in vitro and in vivo . Enviromental Health Perspectives. 1985; 64 151-70
- 19 Heggers J P, Roboson M C. Eicosanoids in wound healing. In: Prostaglandins in clinical practice. Watkins WD, Fletcher JR, Stubbs DF, Peterson MB editors New York; Raven Press 1989: 183-94
- 20 Mac Claudy R L, Hing D N, Robson M C, Heggers J P. Frostbite injuries: a rational approach based on the pathophysiology. Journal of Trauma. 1983; 23 143-7
Prof. Akira Yagi
Faculty of Pharmacy and Pharmaceutical Sciences
Fukuyama University
Fukuyama Hiroshima 729-0292 Japan
Email: yagi@fupharm.fukuyama-u.ac.jp
Fax: +81-849/36-2024
References
- 1 Yagi A, Haraguchi H. Protective effect of terpenoids against oxidative stresses. In: Pandarai SG. editor
Resent research development in phytochemistry . Vol.1 India; Research Signpost 1997: 11-24 - 2 Lee K Y, Weintraub S T, Yu B P. Isolation and identification of a phenolic antioxidant from Aloe barbadensis . Free Radical Biological & Medicines. 2000; 28 261-5
- 3 Yagi A, Kanbara T, Morinobu N. Inhibition of mushroom tyrosinase by Aloe extract. Planta Medica. 1987; 53 515-7
- 4 Okamura N, Hine N, Harada S, Fujioka T, Mihashi K, Yagi A. Three chromone components from Aloe vera leaves. Phytochemistry. 1996; 43 495-8
- 5 Okamura N, Asai M, Hine N, Yagi A. High-performance liquid chromatograpic determination of phenolic compounds in Aloe species. Journal of Chromatography A.. 1996; 746 225-31
- 6 Davis R H. Aspirin and Aloe, Aloe vera - a scientific approach. New York; Vantage Press 1997: 226-62
- 7 Hutter J A, Salman M, Stavinoha W B, Satsangi N, Williams R F, Streeper R T, Weintraub S T. Anti-inflammatory C-glycosyl chromone from Aloe barbadensis . Journal of Natural Products. 1996; 59 541-3
- 8 Reynolds T, Dweck A C. Aloe vera leaf gel: a review update. Journal of Ethnopharmacology. 1999; 68 3-37
- 9 Okamura N, Hine N, Tateyama Y, Nakazawa M, Fujioka T, Mihashi K, Yagi A. Three chromones of Aloe vera leaves. Phytochemistry. 1997; 45 1511-3
- 10 Johnson D, Lardy H. Isolation of the liver and kidney mitochondria. In: Estabrook RW, Pullman M. editors
Methods in Enzymology . New York:; Academic Press 1967 10: 94-6 - 11 Liu G, Zhang T, Wang B, Wang Y. Protective action of seven natural phenolic compounds against peroxidative damage to biomembrane. Biochemical Pharmacology. 1992; 43 147-52
- 12 Wills E D. Lipid peroxidation in microsomes. General consideration. Biochemical Journal. 1969; 113 315-24
- 13 Blois M S. Antioxidant determination by the use of a stable free radical. Nature. 1958; 182 1199-200
- 14 Halliwell B. Use of desferrioxamine as a ”probe” for iron-dependent formation of hydroxyl radicals. Biochemical Pharmacology. 1985; 34 229-33
- 15 Mitsuya K, Mizuta Y, Kohno M, Mori A. The application of ESR spin trapping technique to the evaluation of SOD-like activity of biological substances. Bulletin of Chemical Society of Japan. 1990; 63 187-91
- 16 Riendeau D, Charleson S, Cromlish W, Mancini J A, Wong E, Guay J. Comparison of the cyclooxygenase-1 inhibitory properties of non-steroidal anti-inflammatory drugs and selective Cox-2 inhibitors, using sensitive microsomal and platelet assays. Canadian Journal Physiological Pharmacology. 1997; 75 1088-95
- 17 Borsch-Haubold A G, Pasquet S, Watson S P. Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203 580 and PD 98 059. SB also inhibits thromboxane synthase. Journal Biological Chemistry. 1998; 273 28 766-72
- 18 Janzen E G, Stronks H J, Dubose C M, Poyer J L, McCay P B. Chemistry and biology of spin trapping radicals associated with halocarbon metabolism in vitro and in vivo . Enviromental Health Perspectives. 1985; 64 151-70
- 19 Heggers J P, Roboson M C. Eicosanoids in wound healing. In: Prostaglandins in clinical practice. Watkins WD, Fletcher JR, Stubbs DF, Peterson MB editors New York; Raven Press 1989: 183-94
- 20 Mac Claudy R L, Hing D N, Robson M C, Heggers J P. Frostbite injuries: a rational approach based on the pathophysiology. Journal of Trauma. 1983; 23 143-7
Prof. Akira Yagi
Faculty of Pharmacy and Pharmaceutical Sciences
Fukuyama University
Fukuyama Hiroshima 729-0292 Japan
Email: yagi@fupharm.fukuyama-u.ac.jp
Fax: +81-849/36-2024
Fig. 1 Chemical structures of compounds 1 - 7 (1 = aloesin, 2 = aloeresin A, 3 = aloeresin B, 4 = isorabaichromone, 5 = isoaloeresin D, 6 = aloeresin E, 7 = 8-C-glucosyl-7-O-methyl-(S)-aloesol ).