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Original Article
1 In vivo diabetic wound healing effect and HPLC-DAD-MS/MS profiling
2 of the methanol extracts of eight Aloe species
3 qi Abeer M. El Sayeda, Shahira M. Ezzat3 *, M.M. El Nagarb, Seham S. El Hawarya
4 a Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
5 b Department of Pharmacognosy, Faculty of Pharmacy, Damanhour University, Damanhour City, Egypt
ARTICLE INFO
Article history: Received 12 October 2015 Accepted 12 January 2016 Available online xxx
Keywords: Aloe
HPLC-DAD-MS/MS Chemotaxonomic Anti-inflammatory Wound healing
ABSTRACT
Genus Aloe, Xanthorrhoeaceae, is well distributed all over Egypt, and many species have been used as medicinal plants; mainly reported to prevent cardiovascular diseases, cancer and diabetes. This study attempts to analyze the secondary metabolites in the methanol extract of the leaves of eight Aloe species; A. vera (L.) Burm. f.,A. arborescens Mill.,A. eru A. Berger, A. grandidentata Salm-Dyck,A. perfoliata L.,A. brevi-folia Mill., A. saponaria Haw. and A.ferox Mill. growing in Egypt. For this aim HPLC-DAD-MS/MS in negative ion mode was used. Although belonging to the same genus, the composition of each species presented different particularities. Seventy one compounds were identified in the investigated Aloe species, of which cis-p-coumaric acid derivaties, 3,4-O-(E) caffeoylferuloylquinic acid and caffeoyl quinic acid hexoside were the most common phenolic acids identified. Aloeresin E and isoaloeresin D, 2'-O-feruloylaloesin were the common anthraquinones identified. Lucenin II, vicenin II, and orientin were the common identified flavonoids in the investigated Aloe species. 6'-Malonylnataloin, aloe-emodin-8-O-glucoside, flavone-6,8-di-C-glucosides could be considered as chemotaxonomic markers for the investigated Aloe species. The eight Aloe species had significant anti-inflammatory activity, in addition to the significant acceleration of diabetic wound healing in rats following topical application of the methanol extracts of their leaves. This is the first simultaneous characterization and qualitative determination of multiple phenolic compounds in Aloe species from locally grown cultivars in Egypt using HPLC-DAD-MS/MS, which can be applied to standardize the quality of different Aloe species and the future design of nutraceutical and cosmetic preparations.
© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
The genus Aloe L. (family Xanthorrhoeaceae, subfamily Aspho-deloideae) is an Old World genus which comprises more than 400 species, which has centers of diversity in southern and east Africa and Madagascar (Newton, 2004). Aloe is used as a traditional medicine for treatment of different diseases, as ingredients of food products and cosmetics (Eshun and He, 2004). Phenolic compounds including chromones, anthraquinones, and pyrones constitute the major secondary metabolites of Aloe (Conner et al., 1990; Okamura et al., 1998; Duriet al., 2004). Most of these compounds have been reported to possess several biological activities such as antitumor, antidiabetic and antityrosinase, in addition to antiulcer activity (Imanishi et al., 1981; Yagi et al., 1987; Beppu et al., 1993;
* Corresponding author. E-mail: shahira.ezzat@pharma.cu.edu.org (S.M. Ezzat).
Teradaira et al., 1993). Some components isolated from Aloe species were reported to possess anti-inflammatory effect for example; an effective anti-inflammatory cinnamoyl-C-glucosyl-chromone has been isolated from Aloe barbadensis (Hutter et al., 1996). Moreover, the wound-healing properties of A. barbadensis was also reported (Davis et al., 1989).
The phytochemical constituents and bioactivity ofAloe spp. have attracted research interest since the trade in 'drug aloes', prepared from the leaf exudate, expanded rapidly in the nineteenth century (Yeats, 1870). Today, the principal sources of these natural products are wild populations of Aloeferox in South Africa, and Aloe scabrifo-lia, Aloe secundiflora and Aloe turkanensis in east Africa (Oldfield, 2004). In contrast, Aloe vera, the source of the leaf parenchyma known as 'aloe gel', is widely cultivated.
Aloe leaves in addition of being of pharmacological importance, their chemistry have systematic significance, particularly at the infrageneric rank. Secondary metabolites profile was used in the evaluation of infrageneric groups such as series Longistylae, section
http://dx.doi.org/10.1016/j.bjp.2016.01.009
0102-695X/© 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Pachydendron, section Anguialoe and series Purpurascentes SalmDyck (Van Heerden et al., 1996; Reynolds, 1997; Viljoen and Van Wyk, 2001). Phytochemical data may offer a tool to study the complexity of maculate species, which comprise about 40 species so-named because of their conspicuous leaf markings. Although it is widely regarded as a well-supported group, infrageneric boundaries and species delimitation in the maculate complex are problematic (Grace et al., 2008).
For this purpose we have started a detailed screening of eight Aloe species; A. vera (A1), Aloe arborescens (A2), Aloe eru (A3), Aloe grandidentata (A4), Aloe perfoliata (A5), Aloe brevifo-lia (A6), Aloe saponaria (A7) and A. ferox (A8) growing in Egypt; using HPLC coupled to electrospray mass spectrometry (ESI-MS) [HPLC-DAD-MS/MS] with the aim of identifying unique constituents which may be used as markers with possible chemo-taxonomic significance. In addition, to evaluate the diabetic wound healing and anti-inflammatory effects of the eight Aloe species in rats using the diabetic wound model and carrageenan-induced paw edema tests.
Experimental
Plant material
The leaves of the eight Aloe species, Xanthorrhoeaceae, A. vera (L.) Burm. f., A. arborescens Mill., A. eru A. Berger, A. grandidentata Salm-Dyck, A. perfoliata L., A. brevifolia Mill., A. saponaria Haw. and A. ferox Mill. were collected in April 2014 from El-Orman Garden, Giza, Egypt. The plants were authenticated by Dr. Reem Samir Hamdy, Lecturer of Plant Taxonomy, Botany Department, Faculty of Science, Cairo University, Giza, Egypt. Voucher samples no. 2014422 of the plants were deposited at the Museum of the Pharmacognosy Department, Faculty of Pharmacy, Cairo University.
Extraction
The fresh leaves of the eight Aloe species (500 g each) were cut into pieces and extracted with methanol using cold method of extraction (percolation) (4x 2l) till exhaustion. The methanol extract in each case was filtered, distilled and evaporated under reduced pressure to give 12.65, 12.44, 5.21, 20.02, 6.97, 6.94, 3.86 and 3.96 g of A. vera, A. arborescens, A. eru, A. grandidentata, A. perfoliata, A. brevifolia, A. saponaria and A. ferox, respectively.
Qualitative determination of the phenolic contents of Aloe leaves using HPLC-DAD-ESI-MS/MS
Sample preparation
Aloe extracts were dissolved in HPLC grade methanol in a concentration of 10 mg/ml then filtrated through a syringe-filter-membrane. Aliquots of 5 |il were injected into the LC-DAD/MS Dionex Ultimate 3000 HPLC (Germany), used for performing the analyses.
HPLC conditions
The method of El Maggar (2012) was employed. HPLC apparatus (Hewlett-Packard 1100, Waldbronn, Germany) with a quaternary pump and on line degasser was employed for analysis. A thermostatted column compartment with an auto sampler, a photodiode array detector (DAD). The separation was performed on Eclipse XDB C18 column (50 mm x 2.1 mm, 1.8 |im, Agilent Company, USA). 1100 ChemStation software was used. The mobile phase was composed of two solvents, A: methanol and B: 0.2%
formic acid in water. Gradient elution profile was performed: 0 min, A:B 10:90; 36 min, A:B 100:0; 40 min, A:B 100:0. Chromatography was performed at 30 °C with a flow-rate of 0.2ml/min. UV traces were measured at 290, 254 and 350 nm and UV spectra (DAD) were recorded between 190 and 900 nm.
Mass spectrometric conditions
Electrospray ionization (ESI) interfaced Bruker Daltonik Esquire-LC ion trap mass spectrometer (Bremen, Germany) and an Agilent HP1100 HPLC system were used for HPLC-MS analysis. An autosampler and a UV-vis absorbance detector were used. The ionization parameters were as follows: capillary voltage 4000 V, end plate voltage -500 V; nebulizing gas of nitrogen at 35.0 p.s.i.; drying gas of 10 l/min nitrogen at 350 °C. Mass analyzer scanned from 15 to 1000 u. The MS-MS spectra were recorded in auto-MS-MS mode. The fragmentation amplitude was set to 1.0 V. MS2 data. Mass spectra were simultaneously acquired using electrospray ionization in the negative ionization mode.
Chemicals
Dermazine® cream (silver sulfadiazine) was used as a standard wound healing drug. Indomethacin (Indomethacin)®, Eipico, Egyptian Int. Pharmaceutical Industries Co.; Carrageenan and Alloxan (Sigma Co., USA).
Evaluation of the biological activity
Animals
Adult male rats of Sprague-Dawley strain (130-150 g) were obtained from the laboratory animal facility of the National Research Center, Dokki, Giza. Animals were housed in steel cages under standard conditions and fed with standard pellets and water ad libitum. All experimental procedures were conducted in accordance with internationally accepted principles for laboratory animal use and care, and were approved by the Ethics Committee (No. 9-031) in accordance with recommendations for the proper care and use of laboratory animals (NIH Publication No. 80-23; revised 1978).
Evaluation of anti-inflammatory activity
The method of Winter et al. (1962) was adopted, where ten groups of rats (each of six) were used, eight groups served as the test groups, each of them was administered a single oral dose of each of the methanol extract of the eight tested species (A1-A8) in a dose of 250 mg/kg body weight. The dose of the extracts was determined as 250 mg/kg on the basis of a preliminary short-term pilot study with a range of variable doses. The other two groups, one of them received 1 ml saline and served as a negative control group and the other received Indomethacin, 20 mg/kg body weight as a reference drug. Drugs were orally administered 1 h prior to carrageenan injection. Edema was induced in the rat right hind paw by subcutaneous injection of 0.1 ml of 1% carrageenan suspension in saline. Thickness of the right hind paw (mm) was measured immediately before and 1, 2, 3 and 4h post carrageenan injection with a micrometer caliber. The results are considered significant when they are different from zero time at p < 0.05.
In vivo diabetic wound healing study
A previously established chemically induced type II diabetic foot ulcer animal model was employed in this study (Lau et al., 2008). For those diabetic rats (plasma glucose levels >250 mg/dl, samples collected from tail veins), sixty rats were divided into ten groups, a standardized wound area (2 mm x 5 mm skin in full thickness removed) was induced on the dorsal surface of the right hind foot
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of rats under anaesthetization with phenobarbitone. Eight groups were treated topically with each of the methanol extract of each of the eight tested species by topical application on the wounded area. The other two groups act as a negative (untreated group) and a positive control group (treated with Dermazine® cream). The treatment in each group was started at the day of the operation and continued till the 10th day. The wounds were covered with appropriate dressings, which were changed regularly. On changing the dressings, wounds were inspected, measured and photographed. The
grandidentata and A. perfoliata only. Peak 30 (Rt 970.2 s) showed [M-H]- at m/z at 499 in the negative ionization mode. Peak 30 showed daughter ions at m/z 353, m/z 191 (base peak), m/z 179 and m/z 163. This compound was identified as 3-O-(E)-caffeoyl-5-O-p-coumaroylquinic acid (3) (Gouveia and Castilho, 2011), this compound was only detected in A. vera, A. arborescens and A. per-foliata.
Peak 23 (Rt 942.1 s) was identified as caffeoylquinic hexoside (Gobbo-Neto and Lopes, 2008) and peak 16 (Rt 875.2 s) was likely
identified as trans p-coumaric derivative.
Rj=R2=£ -caffeic acid,R3=H; 3,4-di-O-E-caffeoylquinic caid (2)
Rj=£-caffeic acid, R2=ferulic acid,R3=H; 3-O-E-caffeoyl,4-O-feruloylquinic acid (17)
Rj=£-caffeic acid, R2=H,R3=^-coumaric acid; 3-O-E-caffeoyl,5-/> -coumaroylquinic acid (30)
wound areas were measured while animals were under anesthesia on 2nd, 6th and 10th day after surgery. The progressive changes in wound area were measured by a plantimeter, wound contraction were expressed as percentage reduction of original wound size [wound area at day 0 - wound area at day n/wound area at day 0] x 100.
Statistical analysis
The statistical comparison of difference between the control group and the treated groups was carried out using two-way ANOVA followed by Duncan's multiple range test.
Results and discussion
The classes of compounds were recognizable from their characteristic UV spectra, which were identified based on the HPLC-DAD-MS/MS data and subsequent confirmation by comparison with literature data. The chromatographic and spectroscopic data summarized in Table 1 and Fig. 1.
Chromatographic peaks annotation Phenolic acids
Hydroxycinnamic acids such as esters of quinic acid were detected as about 10 compounds in A. eru, A. grandidentata, A. brevi-folia, A. saponaria and A.ferox extracts condensed either with caffeic, ferulic or coumaric acids. The detected mass [M-H]- at m/z 651, 515, 408, 353, 601, 620, 529, 516, 613 and 483 of the peaks, 1 (Rt 118.2 s), 2 (Rt 248 s), 8 (Rt 716 s), 10 (Rt 728 s), 11 (Rt 796 s), 16 (Rt 875.2 s), 17 (Rt 888 s), 23 (Rt 942.1 s), 31 (Rt 976 s), and 38 (Rt 1025), respectively. Those peaks showed the characteristic base peak of quinic acid, ferulic acid, coumaric acid and caffeic acid at m/z 191,193,163 and 179, respectively.
Regarding dicaffeoylquinic acids, peak 2 (Rt 248 s) showed m/z at 515 [M-H]-, this was identified as 3,4-di-O-(E)-caffeoylquinic acid (1). Peak 17 (Rt 888 s) was identified as 3-O-(E)-caffeoyl-4-O-feruloylquinic acid (2), it showed [M-H]- at m/z 529 (C26H26O12) in the negative ionization mode. This parent ion was fragmented in product ions at m/z 367 base peak, produced by the loss of a caffeoyl unit, and m/z 353, produced by the loss of a feruloyl unit. Caffeoylferuloylquinic acid was detected in A. arborescens, A. eru, A.
Polyketides
Mass spectrum of peak 3 (Rt 599 s) showed [M-H]- at m/z 393 which showed a daughter ion at m/z 247 due to the loss of 90 amu as a result of a cross-ring cleavage in the hexosidic part involved in the formation of the ion at 247. This fragmentation was previously reported for deprotonated and protonated flavonoid C-glycosides. Common ions were also noted at m/z 41 and 59 and a neutral loss of 120 amu was observed. This compound was identified as aloesin (4, aloeresin B), which was detected in A. grandidentata and A. perfoliata. This compound was previously reported in leaves of A. castanea (Van Heerden et al., 2000). Aloesin A and B are Aloe resin with no purgative action. Aloesin B is a chromone-C-glucoside, where aloesin A is a p-coumaric acid ester of aloesin B.
The negative mode ESI-MS spectrum of peak 13 (Rt 841.9 s) showed a strong [M-H]- parent ion at m/z 595 corresponding to the caffeoyl ester of 5-hydroxyaloin A. Fragment ions at m/z 433 and 271 were observed in MS2 consistent with the loss of caffeoyl and caffeoyl plus carbohydrate moieties from the parent molecules, respectively. A further fragment ion with m/z 314 corresponds to the loss of ester group together with a four carbon fragment of the carbohydrate moiety. Caffeoyl ester of 5-hydroxyaloin A was noted in A. vera, A. arborescens and A. grandidentata.
Aloeresin C (5) was detected as peak 19 (Rt 915.8 s) showing [M-H]- at m/z 702,541 (703-C6H1005), corresponding to aloeresin A (6), m/z 394 equivalent to aloesin and m/z 232 corresponding to aloesone. Aloeresin C represent the first example of C,O-diglucoside of 5-alkylchromoneaglycone detected only in A. vera, A. arborescens, A. perfoliata, and A. brevifolia.
Mass spectra of peak 29 (Rt 962.6 s) showed [M-H]- at m/z 409.1 was identified as aloenin (7, 4-methoxy-6-(2-6-D-glucopyranosyloxy-4-hydroxy-6-methylphenyl)-2-pyrone). This compound was detected only in A. eru. Aloenin B (8, peak 58, Rt 1290 s), is a bitter glucoside previously isolated from Aloe leaves and found to exhibit an inhibitory action on gastric juice secretion of rats (Hirata and Suga, 1978). This compound was detected in A. vera, A. arborescens, A. eru and A. grandidentata.
The negative mode ESI-MS spectrum of peak 33 (Rt 998 s) showed a strong [M-H]- parent ion at m/z 433. MS2 of the compound yielded a daughter ion at m/z 271 resulted from the loss of a sugar moiety (C6H1105) from the parent molecules. A moderately abundant ion at m/z 313 probably results from the loss of a (C4H8O4) fragment from the [M-H]- parent ion. Compound 33
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Table 1
Peak assignments of metabolites identified in LC/MS spectrum of eight different Aloe species.
No. Rt(s) [M-Hj- MSn ion m/z (-) ppm Tentative identification A, A2 A3 A4 A5 As Ay As
1. 118.2 651 591,501,436, 349,302, ris-p-Coumaric acid derivatives + + - - - + + +
206/489,325, 205,163,119
2. 248 515 464, 382, 301 3,4-di-O-(E)-Caffeoylquinic acid - - - - - - + -
3. 599 393 376,304, 274, 245, 219, 203, Aloesin (aloeresin B) - - - + + - - -
4. 603 439 380,313, 274, 135 Unk - + - - + - - -
5. 672 593 431,258,175 Aloe emodin-diglucoside - + - + - - - +
6. 677 456 394,364,333, 291,223 Unk - - - + + - - -
7. 698 395 350, 274.8, 230, 202/313, 259, 1-Hexanol-pentosylhexoside - - - - + - - -
8. 716 408 315, 252,147/354,303, 269, Caffeoyl ferulic acid derivatives - - - + - - - -
9. 727 475 405,301,367, 286, 224,145 Chrysoeriol-7-O-glycuronyl - - - + - - - -
10. 728 353 248, 209,191,179,173,161, 5-O-Caffeolyquinic acid - - - - - + - -
135,145
11. 796 601 563, 537, 529, 496, 429 Malonyl-3,4-O-dicaffeoyl quinic + - - - - - + -
12. 811.8 609 590,518, 489 Luteolin-6,8-C-diglucoside + + - + - - + +
(Lucenin II)
13. 841.9 595 533.1, 433, 415.6, 294.4,314, 6'-O-Caffeoyl-5-hydroxyaloin A + + - + - - - -
271,145.1
14. 857.8 542 145.4,191.8, 250.0,300.5, Unk + - - - - - - +
360.9,415.1,505, 396
15. 871.2 593 575, 503, 473, 449, 383, 353, Apigenin-6,8-C-diglucoside + + + + - + - +
287 (vicenin II)
16. 875.2 620 583.0, 437.0, 370.8 trans-p-Coumaric derivatives + + - - - - - -
17. 888 529 475,367,192, 145 3-O-(E)-caffeoyl-4-O- - + + + + - - -
feruloylquinic
18. 915.2 667 605,513,439, 394, 285,232 Luteolin-O-xylosylglucoside + + - + + + + -
malonylated
19. 915.8 702 541,395,233 Aloeresin C (isomer) + + - - + + - -
20. 921 739 704,587,577, 550, 453,310, Epi-catechin digalloyl rhamnoside - - - - + + - -
289, 242
21. 923.9 624 584.2,517.0,314 lsorhamnetin-3-O- + + - + - + + -
deoxyhexosyl(1-6)
hexoside
22. 934.8 581 516.2 7-O-Methyl kaempferol dimmer + + - - + - - -
23. 942.1 516 483.9,354.9,210.7,186.7 Caffeoyl quinic acid hexoside + + - - - + - -
24. 945 411 246.8/249,161, 113 Iso pentyldihexose - - + - - - - -
25. 948 445 439.6, 409, 248.8, 271, 269,153 Apigenin-7-O-glycuronyl - - + - - - - -
26. 948.2 579 495.1, 447.4,356.9, 287, 285 Kaempferol-3-O-hexosyl-O- - + + - - - -
pentoside
27. 948.2 447 357.3/405,327,147 Luteolin-8-C-glucoside (orientin) + + - + + - - +
28. 955.6 447.8 429.8, 405, 356.9, 326.9, 271, Luteolin-6-C-glucoside + - - + - - - -
147 (isoorientin)
29. 962.6 409.1 247 Aloenin - - + - - - - -
30. 970.2 499 472.0,436.7,312.5 3-O-Caffeoyl-5-O-coumaroylquinic + + - - + - - -
31. 976 613 570, 508, 457, 391 4-Succinyl-3,4-dicaffeoylquinic - + - - + + + -
32. 998 469 435/395, 298,192,149 Unk - - - + - - - +
33. 998 433 396.6,342.9,313, 269.8, 271, 5-Hydroxyaloin A - - - + + - - -
34. 1011.9 505 448,343.1,342.9, 341.1,172 6'-Malonylnataloin (nataloin) + + + + - + - +
35. 1013.1 343 316,315, 299, 287, 285,236 5,3'-Dihydroxy-6,7,4'-trimethoxy - + + + - + - -
flavones (Eupatorin)
36. 1018.1 463 515.8, 472.5/301 lsoquercetrin - + + - - - - -
37. 1018.4 563.2 505.5, 500, 435, 406, 342.9, Aloinoside A/B + + + + + - - -
328, 290
38. 1025 483 446, 422,325.9/337,319,173, 3,4-Di-O-(E)-p-coumaroylquinic - + - + - - - -
163 acid
39. 1057 577 451, 425, 413, 305, 271, 293.4 Epi (afzelechin) - (epi) - - + + + - - -
gallocatechin
40. 1063 543 500, 462, 361 Unk + + - + - - - -
41. 1081 509 492.6, 387.9, 345.9, 283.8, Unk + + - - - - - -
42. 1084.2 506 396.5, 343.0, 342.9 Unk + + - - - - - -
43. 1096 570 548,510.8, 471.9,433,411, 2'-O-Feruloylaloesin - + + + - + + -
357.5, 320, 248, 203
44. 1114 778 658, 570, 508, 455 Unk - - - + + - - -
45. 1120.5 616 582.7,578.8,516.8, 500.1, 7-O-Methylaloesin-penta acetate + + - - + - + -
393.2,147.1,145.3
46. 1121 583 570, 554, 477, 407, 243, 272 7-Methylether of 2'-feruloylaloesin - - + + - - + -
47. 1143.7 604 567.1,500.3, 421.0 Malonyl-4,5-O-dicaffeoylquinic + + - - - - - -
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Table 1 (Continued)
No. Rt(s) [M-H]- MSn ion m/z (-) ppm Tentative identification A1 A2 A3 A4 A5 A6 A7 A8
48. 1161 629 547, 481, 208,146/537, 425, Glucuronides - - - - - -
378,325, 236,178,146
49. 1164 670 616, 550, 473, 274 Unk - - - - - -
50. 1167 555 538, 512, 410,392,366, 259, Isoaloeresin D - - t - -
193,147, 119
51. 1198 554 - Unk - - - t - - -
52. 1224.4 556 494, 423, 282, 207 Aloeresin - - t - - -
53. 1226 671 615, 581, 481 Unk - - - t - - -
54. 1256.4 418 417.6, 343.2 Nataloin - t - - - -
55. 1258 297 - Veracylglucan A - - - - - - -
56. 1265 885 431,307, 284 Kaempferol di - - - - - -
deoxyhexosylhexoside
57. 1281.6 833 636.2, 713.5, 775.6 Unk t t - - - - -
58. 1290 713 635.9, 564.3, 516.5,343.4 Aloenin B t t - - - -
59. 1292.0 747 482.1 Wighteone-O-diglucoside t t - - - - - -
malonylate
60. 1302.1 417 396.1, 343.1, 296.9 Aloin A t t - - - - -
61. 1318 417 145, 297, 343 Aloin B - - - - - -
62. 1328 432 361,359, 353,340.7, 310, 283, Isovitexin (6-C-glucosyl-apigenin) - - - - - - -
63. 1342 539 432,359, 312, 254,146 6'-O-Coumaroyl aloesin - - - - - - -
64. 1366 297 248.9, 174.9 Hydroxy octadecenic acid - - - - - - -
65. 1396 586.9 587, 518.1, 427, 257, 147 Unk - - - - - - -
66. 1407 540 468, 420, 393,312 Aloeresin A isomer - - - - - - -
67. 1410 329.7 286,214, 147 Trihydroxy octadecenoic acid - - - - - - -
68. 1424 1016 979, 916.9, 859.6, 834.9, 606, Unk - - - - -
69. 1430 657.6 620, 530, 458/533, 480, 391, Unk - - - - - -
70. 1448 869.2 833,717, 650, 586.3, 511.3, Unk t - - - - -
71. 1460 569 426,355, 285, 225,161 Caffeoylester of aloesin - t - - - -
72. 1462 496 418, 225, 397, 285, 248, 145 Unk - - - - - -
73. 1507 580 463,326, 296, 265 Unk - - - - - -
74. 1519 605 562, 425,317, 267,146, 92 Unk - - - - - -
75. 1590 565 534, 488, 343, 241 Trihydroxycinnamic acid - - - t - - - -
derivatives
76. 1600 599 564,512, 443.0 Unk - t - - - -
77. 1611 685 623, 571, 500, 523.3, 539, 521 Aloeresin E t - -
78. 1612 557 505, 444, 392,322, 251 Acetyl dicaffeoylquinic acid - - - t - - -
79. 1645 831.3 813.2,711.2, 669.8, 742, 686, Unk - - - - -
80. 1659.9 416.4 267.8 Barbaloin (10R)/Isobarbaloin (10S) - - - - - -
81. 1666 445 380, 295, 256, 216,145/269 Apigenin-7-O-glycuronyl - - - - - - -
82. 1725 741.6 416, 301, 300/609, 301 Quercetin pentosyl rutinoside - - - - - - -
83. 2083 689.1 854, 603, 505 Unk - - - t - -
84. 2097 675.9 640, 586, 534/640, 506, 497 Unk - - - - t -
85. 2117.8 711.9 675.2, 550.6, 396.9 Quercetin-7-O-hexoside-3-O- - - t - t -
malonylhexoside
86. 2174 431 316.1, 269, 225 Aloe-emodin-8-O-glucoside - t - t
87. 2226 553 408,376, 341,275, 259, 256, 2'-p-Methoxycoumaroylaloresin - - - t - -
233,193, 178,161
88. 2235 552 448.1, 359.8, 145.1 Unk - - - t - - -
89. 2252 715 625.6, 553.6, 455.1,357.6, Unk - t - - - - - -
303.8, 207,129.2
90. 2293 567 528,313, 271, 253,169 Chrysophanol-8-O-(6'-O-galloyl-) - t - - - - -
glucoside
91. 2339 637 579, 504, 359 Unk - - - - - - -
92. 2450 825 788, 669, 626, 581, 514, 452, Aloeresin H tetra-O-methyl ether - - - - - - -
93. 2549 769 730.5, 326.1 Aloeresin H - - - t - - -
94. 2574 612 555, 492.3, 427, 260.4 Unk - t - - -
95. 2603 642, 643 612, 557,513, 446,375, 269, Unk - - - t -
135/550, 501/580,508, 459
96. 2625 809.8 642, 556,515, 448 Unk t - - t - -
97. 2632 444.2 392, 203.7 Unk t - - - - - -
98. 2650 811.8 777.8, 672.4, 573.3, 512.8, Unk - t - - - - -
462.3, 392,341, 269, 135
99. 2712 813.4 529.4, 462.4 Unk - t - - - - -
100. 2768 789.6 672.3, 612.6, 533.9, 467, 398.7, Unk - t - - - - - -
308.8, 272,135, 93
101. 2871 627.8 571.2, 505.8, 444.8,388.7, Pentahydroxyflavonol-O-hexosyl - t - - - - -
135.6/481,319 rhamnoside
102. 2872 532/533 464,388, 171/489, 285 Kaempferol-3-O-malonylhexoside - - - - - -
A1 = Aloe vera, A2 = A. arborences, A3 = A. eru, A4 = A. grandidentata, A5 = A. perfoliata, A6 = A. brevifolia, A7 = A. saponaria, A8 = A.ferox. (+) denotes presence and (-) denotes absence of each compound in examined Aloe species
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Ihi PI il . L -..^AAL ......
I—MOATI000039.D aloel-A.vera: BPC 50.0-1300.0 -All MS
Intens. x106
I—M0ATI000058.D: BPC 50.0-1300.0 +All MS
Intens. x106
0 500 1000 1500 2000 2500 Time [s]
— M0ATI000041.Daloe3-eru: BPC 50.0-1300.0 -All MS
Intens.. x106 0.8
0 500 1000 1500
I—M0ATI000043.Daloe4-grandidentata: BPC 50.0-1300.0 -All MS
Intens. x105~
il t\,»
Time [s]
-M0AT1000044.D aloe5-perfoliata: BPC 50.0-1300.0 -All MS
htens.
x104 6 -
ime [s]
I—M0ATI000045.D aloe 6-brevifolia: BPC 50-1300 -All
Intens.
x104- ~
6420 l-., -—
t „,-^A
Time [s]
I—M0ATI000046.D aloe 7-saponaria: BPC 50-1300 -All MSn
Intens.
x108^ H 6420
— M0ATI000047.D aloe 8-ferox: BPC 50-1300 -All
Fig. 1. HPLC fingerprint of A, Aloe vera; B, A. arborences; C, A. eru, D, A. grandidentata; E, A. perfoliata; F, A. brevifolia; G, A. saponaria, H, A.ferox recorded at 254 nm.
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was identified as 5-hydroxyaloin A (9) (Holzapfel et al., 1997) and this was only detected in A. grandidentata and A. perfoliata.
Peak 34 (Rt 1011.9 s) showed [M-H]- at m/z 505 was identified as 6'-malonylnataloin (10, nataloin), compound considered of great importance in systematic discrimination of different Aloe species. 6-Malonylnataloin is a putative marker for African taxa in the maculate species complex, although it was not detected in Aloe maculata [A. saponaria]; commonly known as the Soap Aloe or Zebra Aloe; which is a Southern African species of Aloe. On the contrary, nataloin has been observed in non-maculate species as diverse in form and infrageneric position as A. vera, A. arborescens, A. eru, A. grandidentata, A. brevifolia and A.ferox; those are related east African species and nataloin may therefore, serve as a phy-tochemical marker for them (Wabuyele, 2006). Comparative data indicated that the anthrone C-glycoside, 6-malonylnataloin (7-hydroxychrysaloin 6-O-malonate) is typical of maculate species in East Africa.
Peak 37 was identified as aloinoside A and/or B (stereoisomers of aloin-11-a-rhamnoside). This was noted at Rt 1018.4 s with a deprotonated mass peak at m/z 563.2. This compound was observed in A. vera, A. arborescens, A. eru, A. grandidentata and A. perfoliata.
ESI-MS spectrum of peak 45 (Rt 1120.5 s) showed [M-H]- at m/z 616 which on further fragmentation yielded ions at m/z 516, 500, 393 and 147. This compound was identified as 7-O-methylaloesin penta acetate and detected in A. vera, A. arborescens, A. perfoliata and A. saponaria.
The negative ESI-MS of peak 46 (Rt 1121 s) showed strong peaks at m/z 583 [M-H]- which yielded a peak at m/z 407 [M-177]- due to the loss of a fragment with mass 177. This could be interpreted as the loss of a (COCH=CHC6H3 (OCH3) OH) fragment from a ferulic acid derivative. This fragmentation is followed by the loss of water from m/z 407. The spectrum showed no peaks corresponding to the subsequent loss of the carbohydrate moiety after the loss of the acyl group. In contrast, the negative ESI-MS of aloesin showed a strong [M-H]- ion and a base peak at m/z 272 corresponding to the loss of carbohydrate moiety. This compound was identified as (E)-2-acetonyl-8-(2'-O-feruloyl)-P-D-glucopyranosyl-7-methoxy-5-methylchromone (11, 7-methyletherof2'-feruloylaloesin) and detected in A. eru, A. grandidentata and A. saponaria. 7-Methylether of 2'-feruloylaloesin was previously reported in the leaves of A. broomii, A. Africana and A. speciosa (Holzapfel et al., 1997).
Peak 50 (Rt 1167 s) revealed a molecular ion peak at m/z 555 [M-H]-, consistent with a molecular weight 556 which was detected in A. eru, A. grandidentata, A. perfoli-ata and A. brevifolia. MS/MS analysis revealed the following fragments at m/z 538 [M-H2O] ([M-18]-), m/z 512 [M-CH3CHO] ([M-44]-), m/z 410 [M-coumaroyl] ([M-146]-), m/z 392 [M-(146 +18)]-,m/z 366 [M-(146 + 44)]-, m/z 259 [M-(133 + 146 + 18)]-, m/z 193[M-(133 +146 +84)-], m/z 147 [coumaroyl]+ and m/z 119 [HOC6H4CHCH]+. This spectra was consistent with the spectrum reported by Lee et al. (2000) for isoaloeresin D (12, 8-C-P-D-[2-O-(E)-coumaroyl]glucopyranosyl-2-[2-hydroxy]-propyl-7-methoxy-5-methyl chromone).
Nataloin was detected as peak 54 (Rt 1256.4 s) with [M-H]-, at m/z 418, this compound was observed in A. vera, A. arborescens and A. grandidentata. Aloin A/B (13, 10-C-P-D-glycopyranoside of
aloe-emodin-anthrone), which in most cases was detected as a mixture of a- and P-stereoisomers (MW 418), was previously isolated from the leaf exudates of the non maculate Kenyan species (Conner et al., 1987). Aloin A was observed in the leaf extract of A. vera, A. arborescens, and A. grandidentata as peak 60 with [M-H]- at m/z 417. Peak 61 (Rt 1318 s) which showed [M-H]- at m/z 417 was identified as aloin B and was detected in A. vera and A. grandidentata.
Peak 63 (Rt 1342 s) was identified as a chromone; 6-O-coumaroyl aloesin having [M-H]- at m/z 539 which was detected only in A. perfoliata. This chromone is a marker metabolite restricted to the section Anguialoe, thus providing chemotaxo-nomic corroboration for the presumed monophyly of the section (All species of Aloe that belong to section Anguialoe share a single, unique apomorphy which is the sessile flowers) (Reynolds, 1985).
Peak 66 (Rt 1407 s) with [M-H]- at m/z 540 consistent with a molecular weight 541 of aloeresin A (6). The daughter fragment at m/z 393 with difference 146 amu was in agreement with the loss of a p-coumaroyl group. In previous work dealing with the mass spectral characterization of plant glycoconjugates, a neutral loss of 120 amu due to a cross-ring cleavage in the hexosidic part for deprotonated and protonated flavonoids C-glycosides was reported (Wang et al., 2003). The latter loss has also been reported for the protonated chromone C-glycosides, aloesin and aloeresin A. Alo-eresin A was detected only in A. perfoliata.
Mass spectra of peak 71 at (Rt 1460 s) showed [M-H]- at m/z 569 which was tentatively interpreted as caffeoyl ester of aloesin. The compound was identified as (E)-2-acetonyl-8-(2'-O-caffeoyl)-P-D-glucopyranosyl-7-methoxy-5-methyl chromone and observed in A. arborescens, A. eru and A. perfoliata. Peak 77 (Rt 1611 s) showed [M-H]- at m/z 685 which was tentatively interpreted as (E)-2-acetonyl-(2',6'-di-O,O-coumaroyl)-P-D-glucopyranosyl-7-hydroxy-5-methyl chromone (aloeresin E, 14). This compound was observed in A. vera, A. arborescens, A. eru, A. grandidentata, A. perfoliata and A. ferox. MS2 showed daughter fragment ions at m/z 539 and 521 corresponded to the loss of acetonyl and coumaroyl plus carbohydrate moieties from the parent molecules, respectively. Mass spectrum of peak 86 (Rt 2174 s) showed [M-H]- at m/z 431 which was tentatively interpreted as aloe-emodin-8-O-glucoside (14). MS2 of the compound showed fragment ions at m/z 316,269, and 225. This was detected in A. vera, A. arborescens, A. eru, A. perfoliata, A. saponaria and A. ferox.
Peak 87 (Rt 2226 s) in the negative ion mode showed a deprotonated parent ion peak at m/z 553 [M-H]-. A daughter ion was obtained in MS2 due to loss of p-methoxy coumaric acid at m/z 376 and the presence of the p-methoxy coumaroyl fragment ion at m/z 161 which were consistent with the previously reported data for 2'-p-methoxy coumaroylaloeresin in leaves of A. excels (Mebe, 1987). This compound was detected in A. eru, A. perfoliata and A. saponaria.
The ESI-MS spectrum of peak 92 (Rt 2450 s) gave deprotonated molecule [M-H]- at m/z 825 which was tentatively interpreted as aloeresin H tetra-O-methylether observed only in A. saponaria. For peak 93 (Rt 2549 s), the ESI-MS spectrum gave deprotonated molecule [M-H]- at m/z 769 which was tentatively interpreted as aloeresin H (C,C-di-glucoside polyketides) and observed only in A. arborescens and A. perfoliata.
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Rj=CH2COCH3, R2=OH,R3=R4=H Aloesin (3)
Rj=CH2CH(OH)CH3, R2=OMe, R3=p-(E)-coumaroyl,R4=H Isoaloeresin D (50) Rj=CH2COCH3, R2=OH,R3=p -(E)-coumaroyl, R4=H Aloe res in A (66)
Rj=CH2COCH3,R2=B-D-Glc,R3=p-(E)-coumaroyl,R4=H Aloeresin C (19)
Rj=CH2COCH3, R2=B-D-Glc,R3=cinnamoyl,R4=H Aloeresin E (77)
Rj=CH2COCH3, R2=OMe,R3=feryolyl, R4=H 7-methylether of 2"-feruloylaloesin (46)
Rj=H,R2=glucopyanoxyl Aloenin A (29)
Rj=B-D-glucosyl, R2=p-£'-coumaroylglucosyl Aloenin B (58)
Rj=R4=H, R2=O, R3=CH2OH, R5=B-D-glucose Rj=R4=R5=H, R2=I3-D-glucose, R3=CH2OH Rj=R4=R5=H, R2=B-D-glucose, R3=CH2OH Rj=OH,R4=R5=H, R2=B-D-glucose, R3=CH2OH Rj=R5=H, R4=OH, R2=6' -malonylglucose, R3=CH3
376 C-glycosylflavonoids
Aloe-emodin-8-O-glucoside (86) Aloin A (10-aH) (60),Aloin B (10-BH(61) Barbaloin (10Í), Isobarbaloin (10S1) (80) 5-Hydroxyaloin A (33) 6,-Malonylnataloin (34)
The occurrence of flavonoids such as isoorientin (15, luteolin-6-C-glucoside) and isovitexin (16, apigenin 6-C-glucoside) was observed in biogeographical trends. Isoorientin is a common constituent of tropical and sub-tropical species of Aloe, whereas isovitexin is restricted to a few southern African species. Isoorientin and isovitexin co occur in the Southern African maculate species; A. parvibracteata, and the disjunct West African maculate species, A. macrocarpa. The presence of isoorientin and isovitexin in maculate species of Aloe was first reported by Grace et al., 2010. The presence of flavonoids in section Pictae is therefore of taxonomic interest.
ESI-MS spectrum of Peak 12 (Rt 811.8 s) showed [M-H]- at m/z 609, its MS/MS spectrum give rise to daughter ions at m/z 590 [(M-H)-18]-, 518 [(M-H)-90]- and a base peak at m/z 489 [(M-H)-120]-. This compound was tentatively characterized as luteolin-6,8-C-diglycoside (17, lucenin II). The MS/MS spectrum of peak 15 (Rt 871.2 s) in negative ion mode with [M-H]- at m/z 593 which produced daughter ions at m/z 575 [(M-H)-18]-, m/z 503 [(M-H)-90]- and a base peak at m/z 473 [M-H-120]-, exhibiting a fragmentation pattern of flavones di-C-glycoside. The ions at m/z 353 [(M-H)-(120 +120)]- and at m/z 383 [(M-H)-(90 + 120)]-indicated the presence of apigenin (MW 270) as aglycone and two
hexose moieties (glucoses). Comparing with MS literature data 398
(Piccinelli et al., 2008), this compound was characterized as 6,8- 399
di-C-glucosylapigenin and also known as vicenin II (18). 400
Peak 27 (Rt 948.2 s), showed ESI-MS spectrum with [M-H]- 401
at m/z 447. Tendam mass of this peak yielded fragment ions at m/z 402
357.3 [(M-H)-90]- and abase peak at 327 [(M-H)-120]-, together 403
with the absence of the loss of 18 amu suggesting that the mono- 404
C-glycosylation is in position 8 (Ferreres et al., 2007). Peak 27 was 405
tentatively characterized as luteolin 8-C-glucoside, also known as 406
orientin (19). This compound was detected in A. vera, A. arborescens, 407
A. grandidentata, A. perfoliata and A. ferox. ESI-MS spectrum of peak 408
28 (Rt 955.6 s) showed a deprotonated molecule [M-H]- at m/z 409
447.8. The spectrum exhibited fragments at m/z356.9 [(M-H)-90]- 410
and a base peak at 326.9 [(M-H)-120]-. For flavones mono-C- 411
hexosides, the position of the sugar residue can be assigned through 412
observation of the abundance of fragment ion [(M-H)-18]. In gen- 413
eral, the fragmentation of the 6-C-isomers is more extensive, giving 414
an ion corresponding to m/z 429 [(M-H)-18]-, probably due to the 415
formation of an additional hydrogen bond between the 2"-hydroxyl 416
group of the sugar and the 5- or 7-hydroxylgroup of the agly- 417
cone, which confers additional rigidity (Abad-Garcia et al., 2009). 418
The abundance of fragment ion at 429.8 suggested the mono-C- 419
glycosylation is in position 6, hence, compound 28 was identified as 420
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luteolin-6-C-glucoside, also known as isoorientin (15). Isoorientin was detected in A. vera and A. grandidentata.
Peak 62 at Rt 1328 s, in which the ESI-MS spectrum showed [M-H]- at m/z 432 was characterized as 6-C-glycosyl apigenin, also known as isovitexin (16). Its MS/MS spectrum gave mass fragmentation at m/z 340.7 and 310. Isovitexin was observed only in A. perfoliata. The main product ions in the negative ionization mode were due to dehydration, cleavage of sugar ring, and the loss of glycosidic methyl group as formaldehyde. In addition, the intensity ratios of these major fragments could be a way to differentiate vitexin from isovitexin (Abad-García et al., 2008). The MS/MS in the negative ionization mode, the product ions obtained with cleavage of sugar ring was proposed as diagnostic ions, since m/z 313 is the base peak of apigenin-8-C-glycoside and m/z 283 is the base peak of apigenin-6-C-glycoside. In contrast, an ion at m/z 361 was only found in apigenin-6-C-glycoside, probably because of the additional hydrogen bond that is required for loss of the extra water (Abad-García et al., 2008).
R1=R3=H, R2=B-D-glucose R1=B-D-glucose, R2=H, R3=OH R1=H, R2=B-D-glucose, R3=OH R1=R2=B-D-glucose, R3=OH R1=R2=B-D-glucose, R3=H
Isovitexin (62) Orientin (27) Isoorientin (28) Lucenin-II(12) Vicenin-II (15)
O-Glycosylflavonoids
Peak 9 showed precursor ions at m/z 475 [M-H]- and MS2 at m/z 301, corresponding to the loss of a glycuronyl unit. A further fragmentation of the precursor ion at m/z 301 resulted in a product ion at m/z 286, produced by the loss of methyl group (El-Hela et al., 2010). Therefore, peak 9 was identified as chrysoeriol-7-O-glycuronyl detected only in A. grandidentata.
MS spectra interpretation allowed the assignment of the compound luteolin-O-xylosylglucoside malonylated as peak 18 and its molecular ion at m/z 667 and further fragmentation of parent ion which gave aglycone fragment at m/z 285 as the most abundant ion in the fragmentation spectrum (Wojakowska et al., 2013) indicating the presence of luteolin. Malonylation increase both the affinity and transportation, of anthocyanins or other flavonoids and facilitate their transport into the vacuole by a defined transporter. It has been suggested that aromatic acylation enhances the color of anthocyanins, whereas aliphatic acylation (usually malonylation) may stabilize flavonoids and increase the resistance of flavonoid glucoside malonates to enzymatic degradation (Luo et al., 2007). Malonylation of flavonoids occurs widely in plants, and malonyltransferases have been characterized from several different species (Yu et al., 2008). Malonylation, but not p-coumaroylation, of flavonoids induces a conformational change that might facilitate their transport and storage in the vacuole, possibly through improved solubility and reaction with other vacuolar components (Matern et al., 1983). Compound 18 was observed in all of the investigated Aloe species except A. eru and A. ferox. The presence of flavonoids in section Pictae is oftaxonomic interest.
Peak 21 (Rt 923.9 s) was identified as isorhamnetin derivative. Isorhamnetin is a methylated derivative of quercetin and it was identified on the basis of the presence of a specific base peak at
m/z 314, in addition to characteristic loss of a methyl from its methoxy group (-15amu). The results were consistent with the loss of a deoxyhexosyl-hexoside moiety (308 amu). Compound 21 was identified as isorhamnetin-3-O-deoxyhexosyl-(1-6) hexoside and was noted in A. vera, A. arborescens, A. grandidentata, A. brevifolia and A. saponaria.
The mass spectrum of peak 25 at (Rt 948 s) showed precursor ion in the negative ionization mode at m/z 445 [M-H]- and a daughter ion peak at m/z 269, corresponding to the loss of a glycuronyl unit. Therefore, this compound was identified as apigenin-7-O-glycuronyl (Surowiec et al., 2007; Petroviciu et al., 2010) noted only in A. eru.
Mass spectrum of peak 26 (Rt 948.2 s) showed precursor ions at m/z 579 [M-H]- could be identified as a kaempferol-hexosyl-pentoside based on the fragment at m/z 447 [(M-H)-132]- and at m/z 285 [(M-H)-132-162]-. Therefore, peak 26 was identified as kaempferol-3-O-hexosyl-pentoside. It is important to note that product ion at m/z 287 showed a fragmentation pattern in agreement to kaempferol (Cuyckens and Claeys, 2004; Vallverdu-Queralt et al., 2010).
Mass spectrum of peak 35 (Rt 1013.1 s) showed [M-H]- at m/z 343, it was identified as dihydroxyl trimethoxyflavone (5,3'-dihydroxy-6,7,4'-trimethoxyflavone) Eupatorin and noted in A. arborescens, A. eru, A. grandidentata and A. brevifolia. MS/MS spectrum gave product ions at m/z 316, 315, 299 and 287, which were in accordance to this compound (Jaffer and Mahmoud, 2011; Akowuah et al., 2012).
Peak 36 (Rt 1018.1 s) showed [M-H]- at m/z 463 consistent with the molecular formula (C21H20O12). MS/MS spectrum of this compound showed a product ion at m/z 301 indicating a quercetin nucleus (Breiter et al., 2011). This peak was compared with authentic standard and identified as quercetin-3-O-glucoside (isoquercetrin) and was noted only in A. arborensis and A. eru.
HPLC analysis of the methanol extract of the leaves of the eight investigated Aloe species revealed apart from the known metabolites; aloesin (4), nataloin (10), aloin A/B (13), aloeresin E (14) and aloeresin H, which are of particular importance in the chemo-taxonomic study of the genus Aloe. Barbaloin/isobarbaloin, which is C-glucoside of aloe emodin anthrone (10-P-D-glucopyranosyl-1,8-dihydroxy-3-hydroxymethyl-9 (10H)-anthracenone) are considered to be the most specific secondary phytoconstituent in Aloe species. Barbaloin has been found to have a strong inhibitory effect on histamine release from mast cells (Patel et al., 2012). Aloeresin H, 8-C-P-D-glucopyranosyl-7-hydroxy-5-methylchromone portion that is common to all aloeresins (A-G) were detected in most of the investigated Aloe species. Aloeresin Hrepresents the first C,C-diglucosylated molecule discovered in Aloe spp. extracts. Although cinnamic acid derivatives are present in Aloe metabolites in the aloin series, aloeresin E (14) and aloeresin F represent the first cinnamic acid derivatives in the aloeresin series of metabolites (Van Heerden et al., 1996). Aloenin B (8), a new bitter glycoside was detected in most of the leaves of the investigated Aloe species. 6'-Malonylnataloin (10), a malony-lated derivative of the rare anthrone nataloin, also was identified in all investigated samples of Aloe. Anthrone C-glycosides are among a suite of chemical constituents of systematic importance in Aloe.
Aloe has long been recognized by different pharmacopeias over the world as a purgative drug (Rowson et al., 1967; Osol et al., 1973). The purgative effects of Aloe leaf (drug aloes) have been attributed to anthraquinone C-glycosides, notably barbaloin, aloin A and B (Steenkamp and Stewart, 2007). Further studies were conducted to investigate the Aloe's constituents responsible for this activity. Anthranol, aloe-emodin, chrysophanic acid (chryso-phanol), aloin (barbaloin) and p-coumaric acid were previously
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Table 2
Antiinflammatory activity of the methanol extract of the eight Aloe species (A1-A8).
Time (h) Zero 1h 2h 3h 4h
Group Paw Paw Edema Edema Paw Edema Paw Paw Edema
diameter diameter thickness thickness diameter thickness diameter diameter thickness
(mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm) (mm)
Control 3.30 ± 0.08 4.41 ± 0.1* 1.11 4.71 ± 0.13* 1.41 4.81 ± 0.12* 1.51 4.85 ± 0.07* 1.55
A. vera 3.33 ± 0.08 4.26 ± 0.10* 0.93 4.10 ± 0.13* 0.77 3.82 ± 0.12* 0.49 3.64 ± 0.08* 0.31
A. arborences 3.49 ± 0.09 4.34 ± 0.09* 0.85 4.21 ± 0.08* 0.72 4.03 ± 0.09* 0.55 3.84 ± 0.09* 0.35
A. eru 3.51 ± 0.10 4.38 ± 0.14* 0.87 4.28 ±0.14* 0.77 4.12 ± 0.14* 0. 61 3.94 ± 0.11* 0.43
A. grandidentata 3.56 ± 0.11 4.16 ± 0.20* 0.6 3.99 ±0.10* 0.43 3.95 ± 0.05* 0.39 3.90 ± 0.7* 0.34
A. perfoliata 3.57 ± 0.1 4.46 ±0.12* 0.89 4.27 ±0.14* 0.70 4.04 ± 0.05* 0.47 3.94 ± 0.05* 0.37
A. brevifolia 3.49 ± 0.06 4.37 ± 09* 0.89 4.30 ± 0.06* 0.81 4.27 ± 0.07* 0.78 4.03 ± 0.6* 0.54
A. saponaria 3.61 ± 0.02 4.51 ± 0.07* 0.90 4.38 ± 0.02* 0.87 4.14 ± 0.08* 0.53 4.08 ± 0.06* 0.47
A. ferox 3.60 ± 0.09 4.49 ± 0.09* 0.89 4.31 ± 0.08* 0.71 4.11 ± 0.09* 0.51 3.98 ± 0.09* 0.38
Indomethacin 3.56 ± 0.08 4.12 ± 0.09* 0.6 3.99 ± 0.06* 0.43 3.92 ± 0.01* 0.36 3.84 ± 0.07* 0.28
* Significantly different from zero time at p < 0.05 (n = 6). The dose of the extracts were 250 mg/kg.
Table 3
Effect of the methanol extract of the eight Aloe species on wound area in diabetic rats.
The tested sample Wound area (cm2)
Zero time Day 2 Day 6 Day 10
Control 1.53 ± 0.003 1.55 ± 0.002 1.59 ± 0.001* 1.60 ± 0.003'
A. vera 1.58 ± 0.001 1.26 ± 0.004* 0.69 ± 0.003* 0.36 ± 0.001*
A. arborences 1.63 ± 0.002 1.23 ± 0.002* 0.67 ± 0.001* 0.46 ± 0.001*
A. eru 1.67 ± 0.002 0.89 ± 0.003* 0.46 ± 0.001* 0.23 ± 0.001*
A. grandidentata 1.71 ± 0.001 1.49 ± 0.002* 1.13 ± 0.002* 0.74 ± 0.001*
A. perfoliata 1.69 ± 0.003 1.41 ± 0.001* 0.86 ± 0.001* 0.53 ± 0.001*
A. brevifolia 1.56 ± 0.001 1.45 ± 0.001* 0.81 ± 0.002* 0.63 ± 0.002*
A. saponaria 1.68 ± 0.002 1.34 ± 0.002* 0.65 ± 0.001* 0.31 ± 0.001*
A. ferox 1.51 ± 0.001 1.37 ± 0.003* 0.98 ± 0.001* 0.47 ± 0.002*
Dermazine® 1.65 ± 0.004 0.72 ± 0.001* 0.43 ± 0.002* 0.13 ± 0.001*
* Significantly different from zero time at p < 0.05 (n = 6).
538 reported in A. vera. A thin layer chromatographic study of22species
539 of Aloe (Meredova et al., 1973) showed that twelve species contain
540 flavonoids, hydroxy anthraquinones and coumarin.
541 Concerning the diabetic wound healing effect of the methanol
542 extracts of the eight investigated Aloe species, results obtained in
543 the present study suggested that treatment of diabetic rats with
544 the methanol extract of each of the eight studied Aloe species may
545 have a beneficial influence on wound healing. It is noteworthy
546 to mention that the eight investigated species showed significant
547 anti-inflammatory activity (Table 2) as their action in reducing the
548 thickness of edema started from the first hour and continued for
549 the four hours. The anti-inflammatory activity of Aloe may be due
550 to the inhibition of the cyclooxygenase pathway and reduction
551 of prostaglandin E2 production this could be attributed to its C-
552 glucosyl chromones (Hutter et al., 1996). The findings of our study
553 confirmed the significant acceleration of diabetic wound healing
554 in rats following topical application of the methanolic extract of
555 the leaves of the various Aloe species. The healing effect on the
556 diabetic wounds was observed for the topical application of the
557 methanol extracts of the eight studied Aloe species (Table 3), how-
558 ever, A. eru showed the highest effect in reducing the wounds
559 area which reached 86.2% by day 10 (Table 4). It was previously
560 reported that the healing properties of Aloe could be attributed to
561 its glucomannan type polysaccharide, which stimulates the activ-
562 ity of fibroblasts and causes proliferation, which in turn increases
563 collagen synthesis (Chithra et al., 1998). The high wound healing
564 activity observed for A. eru could be mainly attributable to its major
565 constituent aloenin, which was reported to significantly promote
566 hair growth and demonstrated recuperative effects on human skin
567 (Wolfson and Gutterman, 2009). Aloenin was detected only in A.
568 eru (Table 1).
Table 4
Percent of change in wound area in diabetic rats treated with methanol extract of the eight Aloe species.
The tested samples % of change in wound area
Day 2 Day 6 Day 10
Control 1.31 3.92 4.6
A. vera 30.30 56.3 77.2
A. arborences 24.5 58.8 71.8
A. eru 46.71 72.9 86.2
A. grandidentata 12.9 33.9 57.9
A. perfoliata 17.2 59.2 68.6
A. brevifolia 7.1 48.1 59.6
A. saponaria 20.2 61.3 81.6
A. ferox 14 35.1 68.9
Dermazine® 56.4 73.9 92.1
Conclusion 569
The occurrence of flavones-C-glycosides, heptaketides (e.g. 570
5-methylchromones) and octaketides (e.g. 1,8-dihydroxy-9- 571
anthrones) in Aloe species is known. The results obtained here 572
showed that these species possess different chemical composition, 573
justifying the importance of studies aiming for the chemical 574
characterization of different Aloe species. The anti inflammatory 575
and wound healing activity of the Aloe species has been attributed 576
to polyphenolic compounds. A. eru showed the highest effect in 577
reducing the wounds area in ten days which is rich in Aloenin. 578
Among the species presented in this paper, flavonoids are found 579
as main constituents specially lucienin II, vicenin II, orientin and 580
isovitexin in A. vera, A. arborescens, A. grandidentata, A. perfoliata, A. 58i
brevifolia and A. saponaria. Heptaketides (5-methylchromones) are 582
Ifll^^^W AllIHI.E IN PRESS
A.M. El Sayed et al. / Revista Brasileira de Farmacognosia xxx (2016)xxx-xxx 11
found as main constituents in all investigated Aloe species except A. vera, A. perfoliata and A. ferox. Polyketides are of chemotaxonomic interest in all Aloe species investigated.
Authors' contributions
AME contributed in collecting the plant material, preparation of some of the methanol extracts, interpretation of the HPLC-MS/MS data and constructing the manuscript. SME contributed in preparation of some of the methanol extracts, interpretation of the LC-MS data, revising and finalizing the manuscript and interpretation of the biological results. MME contributed in performing the HPLC-MS/MS analysis and revising the interpretation of HPLC-MS/MS data. SSE contributed in suggesting the point of the research and supervising the work.
Ethical disclosures
Protection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).
Confidentiality of data. The authors declare that no patient data appear in this article.
Right to privacy and informed consent. The authors declare that no patient data appear in this article.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgment
The authors are deeply thankful to Prof. Dr. Wafaa El Eraky, Department of Pharmacology, National Research Center, Giza, Egypt, for carrying out the biological experiments.
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