Vacillantins A and B, New Anthrone C-glycosides, and a New Dihydroisocoumarin Glucoside from Aloe vacillans and Its Antioxidant Activities

A new dihydroisocoumarin glucoside, vacillanoside (3), and two new anthrone C-glycosides microdantin derivatives; vacillantin A (10) and B (11), together with nine known compounds belonging to the anthraquinone, anthrone and isocoumarin groups were isolated from the leaves of Aloe vacillans. The structures were determined based on spectroscopic evidence including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HRESIMS) data, along with comparisons to reported data. The leaves were used to extract compounds with different solvents. The extracts were tested for antioxidant activity with a variety of in vitro tests including 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate (ABTS•+), ferric reducing antioxidant power assay (FRAP), superoxide, and nitric oxide radical scavenging assays. The dichloromethane fraction was most active, displaying significant free radical scavenging activity. The n-butanol fraction also showed notable activity in all assays. Therefore, these findings support the potential use of A. vacillans leaves as an antioxidant medication due to the presence of polyphenolic compounds.

Compound (3) was isolated as a white amorphous powder. The spectral data of (3) including IR, UV, and NMR were very similar to that of feralolide (2) [20], suggesting a similar dihydorisocoumarin skeleton. HRESIMS showed quasi-molecular ion peaks at m/z 507.1500 [M+H] + (calcd 507.1503 for C 24 K) with 162 amu more than that of 2, suggesting the presence of a monosaccharide moiety. A significant fragment using the high-resolution electron impact mode (HR-EIMS) appeared at 327.0868 corresponding to C 18  reflected the glycosidic nature [26]. Absorption bands at 3451, and 1645 cm −1 were observed in the IR spectrum assigned to OH and C=O, respectively.
The detailed NMR spectral analyses for 3 were also very similar to that of 2, particularly the aglycone that showed slight chemical shift differences due to the glycosylation site of the aglycone (i.e., left-hand side of the molecule). The main difference in the 1 H NMR (Table 1) data between the two compounds was the downfield shift of H-5 and H-7 from δ H 6.20 and δ H 6.19 (2) to δ H 6.51, representing two protons (3), respectively, accompanied by the downfield shift of H-4 from δ H 2.87 in 2 to δ H 2.92 in 3. As expected, C-5 and C-7 in 3 were also downfield shifted by + 0.4 and + 1.4 ppm compared to those in the aglycone (2). Furthermore, the monosaccharide part was identified as glucose by the appearance of a doublet signal H-1" at δ H 4.99 with a large J value (7.2 Hz), indicating its β-configuration. The remaining proton and carbon signals of glucose were assigned carefully by 13 C, DEPT-13 C, and 2D NMR, and were in good agreement with those reported for glucose [27] The above data confirmed that 3 is the glycoside of 2. The glycosylation site was confirmed as C-6 by J 2&3 bond correlations observed in the HMBC experiment from H-1" to C-6; and H-5, H-7 to C-6. Other significant HMBC cross-peaks were observed from H-7 to C-1; H-3 to C-1 and C-4a; H-4 to C-3 and C8a; H-1 to C-3 and C-7 ; H-7 to C-3 , C-2 and C-6 ; and H-5 to C-3 and C-6 ( Figure 2). ECD spectral data of compound 3 were similar to those reported for the known dihydroisocoumarin derivative, feralolide (2) previously isolated from A. ferox [20]. The final structure of 3 was determined as 3,4 dihydro-6-glucopyranozyl-8-hydroxy-3-(2 -acetyl-3 ,5 -dihydroxyphenyl)methyl-1H- [2]benzopyran-1one, isolated for the first time from nature and given the name vacillanoside. Notably, a similar isocoumarin glycoside (feralolide 3 -O-glycosyl) from A. hildebrandtii [27] and A. arborescens [21] was previously reported, but with a different glycosylation position.              [19]. UV and IR spectral data of both compounds showed close similarity; each showed a λ max in the UV spectrum at 209, 244, 300, and 330 nm, and the IR spectrum showed bands at 3400, 1606, 1511, 1240, 1640, and 1720 cm −1 , indicating the presence of hydroxyl groups, aromatic rings, and chelated carbonyl and conjugated ester carbonyl groups. Both compounds gave a positive Molisch's test, reflecting their glycosidic nature [26]. The detailed NMR spectral analyses of 10 and 11 showed a resemblance to those of compounds 8 and 9, identified as microdontin A and B and originally isolated from A. microdonta [23]. The similarities of 10 and 11 in their NMR spectra can be summarized as follows: Both were C-glycosides of an aloe-emodin-9-anthrone derivative with a glucose unit esterified with caffeic acid at C-6 . This structure was confirmed by 1 H-NMR signals for aloe-emodin-9-anthrone observed as a pair of meta-coupled aromatic protons resonating at δ H 6.80 (brs, H-2) and 6.97 (brs, H-4) in (10)  The sugar moiety for both (10) and (11) proved to be β-D-glucopyranosyl connected to the aglycone via a C-C bond (C-glycoside), similar to microdontins A and B (8 and 9). This structure is indicated by the chemical shift of the anomeric proton of the sugar with β-configuration at δ H 3.30 (d, J = 9.5 Hz) in (10) and δ H 3.29 (d, J = 9.4 Hz) in (11) correlated to C-1 carbons at δ C 85.8 and 85.9 ppm, respectively, in the HSQC (Heteronuclear Single-Quantum Correlation) experiment. The remaining sugar signals (H2 -H6 ) were in complete agreement with the signals reported for microdontins A and B [23]. The glycosidation site in 10 and 11 was confirmed at C-10 by significant cross-peaks in the HMBC experiment from H-1 to C-4a and C-5a, and from H-10 to C-4, C-4a, C-5, C-5a and C-1 . Additionally, the methylene protons at C-6 were downfield shifted to δ H 3.83 (dd, J = 11.7, 6.9 Hz, H-6 a) and δ H 4.23 (br d, J = 11.7 Hz, H-6 b) compared to aloins A (6) and B (7), indicating esterification at C-6 . The downfield shift of C-6 from δ C 63.1 ppm in aloin B to δ C 64.4 and 64.5 ppm in 10 and 11, respectively, (around 1.5 ppm) further support C-6 acylation. This result also agrees with HMBC correlations from H-6 to C-9" and from the trans-olefinic protons to C-1", C-2", and C-6" (Figure 2).
A remarkable difference was observed in the aromatic region between microdontin A and B and 10 and 11. The phenolic acid in microdontins A and B was identified as p-hydroxycinnamic acid, but in 10 and 11 was identified as a caffeic acid moiety. This finding was confirmed by three coupled aromatic protons at δ H 6.83 (d, J = 8.2 Hz), δ H 6.97 (br d, J = 8.2 Hz), and δ H 7.09 (br s), forming an ABX system and assigned for H-5", H-6", and H-2". The system was accompanied with a trans-olefinic system H-7" and H-8" [δ H 7.33 (d, J = 15.9 Hz), and δ H 6.06 (d, J = 15.9 Hz)] in 10 and [δ H 7.35 (d, J = 15.9 Hz), and δ H 6.08 (d, J = 15.9 Hz)] in 11. These data matched data reported for caffeic acid [28]. The signal in 13 C-NMR at δ C 168.9 was assigned to C-9" in the two compounds. HMBC correlations established further evidence for C-6 where cross-peaks from H-6 to C-9" and from the trans-olefinic protons to C-1", C-2", and C-6" were observed ( Figure 2).
Overall, the above data prove that compounds 10 and 11 are diasteroisomers and derivatives of microdontins A and B (8 and 9). Isomer A or B was established in a NOESY experiment. Clear cross-peaks were observed from H-10 to H-4, H-5, and H-1 in 11, but not in 10, confirming the α form in the latter. Furthermore, the α orientation of the glucose moiety at C-10 in 11 was confirmed by comparing its ECD spectrum with the related compounds, aloin B, microdontin B, and 10-hydroxy aloin B [29], proving that 11 is the β isomer and 10 the α isomer.
Trivial names, vacillantin A and B, were given to compounds 10 and 11. Notably, for all of the isolated compounds with α orientation at H-10 (6, 8, and 10), the chemical shift of H-4 was more downfield than H-5, while the opposite occurred in the β equivalents (7, 9, and 11) Table 2. Moreover, during RP C 18 HPLC separation using MeOH/H 2 O, the polarity of the α isomer was less than the polarity of the β form.
All tested samples produced concentration-dependent antioxidant effects in the superoxide scavenging assay ( Similarly, the CH 2 Cl 2 fraction produced the highest inhibition activity among the tested samples in the nitric oxide scavenging assay (Table 5) with 80.03 ± 3.43% inhibition at the concentration of 100 µg/mL (IC 50 37.23 ± 3.72) compared to ascorbic acid (89.28 ± 2.02% inhibition, IC 50 22.37 ± 3.82). Conversely, MeOH and EtOAc fractions showed moderate to weak inhibitory actions in all assays.
Aloe-emodin, one of the main anthraquinone compounds isolated from Aloe spp., displayed strong antioxidant activity [38], revealed by its powerful reducing properties and the ability to inhibit the oxidation of linolenic acid [39]. In contrast, aloin, found in most Aloe plants, exhibited very similar properties, inhibiting lipid peroxidation in the cerebral cortex by inactivation of Fe(II)-dependent ascorbate [40].
In addition, the anthrone C-glycoside microdentin A/B isolated from the leaf latex of A. schelpei displayed stronger antioxidant activity compared to aloinoside A/B and aloin A/B using vitamin C as a standard [41].  Statistical significance was calculated using one-way ANOVA with p ≤ 0.05 as the threshold of significance for all parameters, with mean ± SD of three independent measurements. Statistical significance was calculated using one-way ANOVA with p ≤ 0.05 as the threshold of significance for all parameters, with mean ± SD of three independent measurements.

Apparatus and Chemicals
Silica gel (Merck 60 A, 230-400 mesh ASTM, Darmstadt, Germany) was used for column chromatography. Normal and reversed phase silica gel (Merck, Darmstadt, Germany) were used for thin-layer chromatography (TLC). Anthraquinones were detected using a 254/366 nm UV lamp, followed by exposure to concentrated ammonia vapors or by spraying with 10% alcoholic KOH or NaOH. Additionally, compounds were visualized spraying with 15% H 2 SO 4 /ethanol, followed by heating.
HPLC analysis was performed on a Prominence Shimadzu LC Solution (Shimadzu Corporation, Kyoto, Japan) system with an InertSustain ® C 18 analytical column (250 × 10 mm i.d.; 5 µm particle size) and a GL Sciences C 18 preparative column (250 × 20 mm i.d.; 5 µm particle size) protected by a Waters Novapack RP C 18 column guard. A binary LC-10AD pump, inline degasser, auto-sampler, and HP-1040A photodiode array detector coupled to an HP-85 personal computer were used for the analysis. UV-Vis spectra were recorded in the 200-700 nm range.
NMR spectroscopy was performed using deuterated solvents in an UltraShield Plus 500 (Bruker, Billerica, MA, USA) spectrometer operating at 500 MHz for 1 H and 125 MHz for 13 C. Some measurements used a Bruker AV-700 MHz NMR spectrometer (Bruker, Billerica, MA, USA) operating at 700 MHz for 1 H and 175 MHz for 13 C at the College of Pharmacy, King Saud University. Chemical shift values are reported in δ (ppm) relative to an internal standard (TMS) or residual solvent peak, and coupling constants (J) are reported in Hertz (Hz). The standard Bruker pulse program was used for the two-dimensional NMR analyses (COSY, HSQC, HMBC, and NOESY). HRMS was conducted by direct injection using a Thermo Scientific UPLC RS Ultimate 3000 Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (company, city, country) (Mundelein, Illinois 60060 USA) combined with high-performance quadrupole precursor selection with high resolution, accurate-mass (HR/AM) Orbitrap™ detection. Direct infusion of isocratic elution was done using CH 3 CN/MeOH (7:3) as a solvent system with 0.1% formic acid. The experiment time was run for 1 min using nitrogen as the supplementary gas. A scan range from 160-1500 m/z was used. Detection was performed in both positive and negative modes, separately. The instruments were located at Prince Sattam Bin Abdulaziz University, College of Pharmacy. In addition, accurate mass determination was also achieved with a JEOL JMS-700 High-Resolution Mass Spectrophotometer (JEOL USA Inc., Peabody, MA, USA). The electron impact mode with an ionization energy of 70 eV was adopted. A direct probe was used with the following temperature ramp settings: Initial temperature of 50 • C; increasing by 32 • C/min, reaching a final temperature of 350 • C; resolution was adjusted to 10 k. IR spectrum was acquired using a JASCO 320-A spectrometer (JASCO International Co., Ltd., Easton, MD, USA).

Plant Material
The leaves of A. vacillans Forssk were collected in February 2018 in Mahayil Asir, in the southwestern region of Saudi Arabia (latitude: 18 • 13 0.4692" N and longitude: 42 • 30 13.5540" E). The specimens were kindly identified by Dr Raja Krishnan, Botany and Microbiology Department at the College of Science, King Saud University, Riyadh, Saudi Arabia. A voucher specimen (#11965) was submitted to the herbarium of the College of Science, King Saud University.

Extraction and Isolation
The succulent leaves of A. vacillans (4 kg) were chopped into small pieces and extracted in 70% hot methanol several times until exhaustion. The pooled extracts were then concentrated in a rotary evaporator to obtain a dark semi-solid residue (180 g). Total methanolic extract (MeOH) was dispersed in 300 mL of distilled water and successively partitioned with dichloromethane (CH 2 Cl 2 ), ethyl acetate (EtOAc), and n-butanol (n-BuOH). Organic fractions were filtered over anhydrous sodium sulfate and evaporated to dryness to yield fractions A (DCM, 4.0 g), B (EtOAc, 8.5 g), C (n-BuOH, 70 g), and D (aqueous fraction, 90 g). The fractions were monitored on normal and RP C 18 TLC using different solvent systems: n-hexane/EtOAc, CH 2 Cl 2 /MeOH, and CH 2 Cl 2 /MeOH/H 2 O at different ratios. The CH 2 Cl 2 and EtOAc fractions were the richest in secondary metabolites including anthraquinones, triterpenes, and sterols; based on these results, these extracts were chosen for further chromatographic investigation.
Part of the CH 2 Cl 2 fraction (3.5 g) was chromatographed on a silica gel column (100 × 4 cm, 358 g silica) using the n-hexane/EtOAc solvent system, followed by EtOAc/MeOH in gradient elution mode, yielding 251 fractions. Similar fractions, monitored on Kiesel gel 60 F 254 TLC, were combined to give six main fractions (A-F). Direct crystallization of fraction C, eluted by 40% EtOAc/n-hexane, afforded compound 1, aloe-emodin, while compound 2, dihydroisocoumarin, was recovered from fraction E after sub-column treatment, on a silica gel column, using a CH 2 Cl 2 /MeOH solvent system in gradient elution mode.
The EtOAc extract (8 g) was applied on top of a silica gel column (100 × 4 cm, 500 g silica) and eluted with a CH 2 Cl 2 /MeOH mixture of increasing polarity. Eighty sub-fractions were collected and monitored on F 254 TLC using n-hexane/EtOAc, and CHCl 3 /MeOH at different concentrations as well as on RP C 18 TLC using MeOH/H 2 O at different ratios. Based on the results obtained from TLC, similar fractions were combined to yield seven main fractions (I-VII). Two fractions were chosen for further purification by reversed-phase C 18

DPPH Radical Scavenging Assay
The antioxidant activity of the extracts and fractions was determined using DPPH (2,2-diphenyl-1-picrylhydrazyl) based on the method described by [42]. Absorbance was determined after 30 min at 520 nm, and percentage inhibition was obtained with the following the equation: where A t is the absorbance of the extract and A 0 is the absorbance of the control.

ABTS Radical Cation Scavenging Assay
The assay was performed following the procedure described by [43]. The ability of samples to reduce the ABTS free radical (2,20-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) was also estimated using the above formula.

Reducing Power Assay
The reducing power of the extracts was determined using the method adapted by [44]. The antioxidant method (i.e., FRAP) is based on the capability of a test sample to reduce ferric ions (Fe 3+ ) to ferrous ions (Fe 2+ ) by electron donation.

Superoxide Radical Anion Scavenging Assay
Superoxide anion radical scavenging activity was assessed as previously described [45] with slight modification. Superoxide radicals were created by oxidation of NADH in a PMS-NADH system, and antioxidant activity was measured by the extent to which the extract and fractions of A. vacillans reduced nitro blue tetrazolium (NBT). The percentage of superoxide radical scavenging was also calculated using the above formula.

Nitric Oxide Radical Scavenging Assay
The assay was performed as previously described [46]. The free radical scavenging activity of the extract and fractions was determined by evaluating the % inhibition of the nitrite ions generated from the interaction of nitric oxide with oxygen using the same equation above-mentioned.

Statistical Analysis
Analysis of variance (ANOVA) was used to evaluate significance differences, followed by the Student's t-test. Data were expressed as mean ± SD, and the difference was considered significant at p < 0.05 compared to the control. All statistical calculations used OriginLab software (version 8, Northampton, MA, USA) and Microsoft Excel.

Conclusions
In summary, a new dihydroisocoumarin derivative, vacillanoside (3), 3,4 dihydro-6 glucopyranozyl-8-hydroxy-3-(2 -acetyl-3 ,5 -dihydroxyphenyl)-methyl-1H- [2]benzopyran-1-one (6), and two new anthraquinone derivatives, vacillantins A and B (10 and 11) were isolated from the leaves of A. vacillans (Asphodelaceae) together with nine known compounds (1, 2, 4-9, and 12). The structures of these compounds were elucidated through extensive spectroscopic analyses. The total alcohol extract and different fractions were tested for their antioxidant activities in five spectrophotometric assays. The dichloromethane fraction exhibited promising free radical scavenging activity in most of the assays. Our findings add new information to the literature on the structural diversity and pharmacological activities of Aloe species. Our results suggest A. vacillans as a potential source of secondary metabolites with pharmacological and industrial importance. Moreover, these results advocate further investigation of the remaining fractions with the aim of isolating bioactive compounds exhibiting interesting biological capacities.