Chemical Constituents From the Flowers of Aloe arborescens

Abstract

(3R)-3,4-Dihydro-6,8-dihydroxy-3-(2'-acetyl-3'-O-β-d-glucopyranosyl-5'-hydroxyphenyl)methyl-2(1H)-benzopyran-1-one (feralolide-3’-O-β- d -glucopyranoside, 1), feralolide (2), aloe-emodin (3), feroxidin (4), and chlorogenic acid (5) were isolated from the 70% EtOH extract of flowers of Aloe arborescens. Structures of these compounds were elucidated on the basis of physical and spectral data. Absolute structure of 1 was determined on the basis of circular dichroism spectra for the first time.

Various plants of the genus Aloe (Family: Asphodelaceae) are used in traditional medicine for treating burns, wounds, and constipation. ¹ Recently, there is a growing interest on the use of Aloe as functional foods and cosmetics, and most of the scientific studies are focused on the chemical constituents and bioactivities of leaves. However, there are only few studies regarding the flowers of Aloe plants except few studies on phenolic constituents, and antioxidant and anti-proliferative activities. ^2-6 Aloe arborescens Mill. is commonly known as “Kidachi-aloe” in Japan. The leaves of A. arborescens are reported to have chromone or anthrone derivatives ⁷ and antioxidant, anti-tumor, and antifungal activities. ^8-11 In this study, we aimed for the isolation and identification of chemical constituents from the flowers of A. arborescens.

The dried flowers of A. arborescens were extracted with 70% EtOH and the extract was then subjected to repeated column chromatography (CC) on MCI gel CHP20P, Sephadex LH-20, and silica gel to afford totally 5 compounds (1-5). Compound 1 was obtained as a yellow amorphous powder, [α]_D ²¹―78.9. The high resolution fast-atom bombardment mass spectrometry (HR-FAB-MS) of 1 showed the quasi-molecular ion [M―H]- peak at m/z: 505.1327 supporting the formula C₂₄H₂₆O₁₂. The ¹H-and ¹³C-nuclear magnetic resonance (NMR) spectral data of compound 1 were superimposable with that of 3,4-dihydro-6,8-dihydroxy-3-(2'-acetyl-3’-O-β-d-glucopyrano-syl-5'-hydroxy-phenyl) methyl-2(1H)-benzopyran-1-one, previously reported from the leaf exudate of A. hildebrandtii. ¹² However, the absolute configuration at C-3 position was not reported yet. To determine the absolute configuration, we performed the acid hydrolysis of 1 which afforded aglycone (feralolide, 2) ¹³ and d-glucose. Circular dichroism (CD) spectral data of compound 1 and its aglycone (2) were similar to that of previously reported CD data of feralolide from Cape Aloe, ¹³ which revealed that the absolute configuration at C-3 position to be R. On the basis of these data, the structure of 1 was assigned as (3R)−3,4-dihydro-6,8-dihydroxy-3-(2'-acetyl-3'-O-β-d-glucopyranosyl-5'-hydroxyphenyl)methyl-2(1H)-benzopyran-1-one or feralolide-3’-O-β-d-glucopyranoside (Figure 1).

Figure 1

Structures of 1 and 2.

The structures of other compounds were elucidated as feralolide (2), aloe-emodin (3), ¹⁴ feroxidin (4), ¹⁵ and chlorogenic acid (5) ¹⁶ on the basis of NMR spectral data and comparison with reported literature values. Among them, compound 5 was reported previously from the flowers of Aloe vera. ²

Experimental

General Experimental Procedures

Circular dichroism spectra were recorded on a JASCO J-810 spectropolarimeter. Optical rotations were measured with a JASCO DIP-1000KUY polarimeter. 1H-, 13C-NMR spectra were measured on a BRUKER AVANCE 600 spectrometer. Chemical shifts are given in ppm with reference to tetramethylsilane. Mass spectra were recorded on a JEOL JMS 700 MStation mass spectrometer. Column chromatography was carried out with silica gel 60 (0.040-0.063 mm, Merck), MCI gel CHP20P (75-150 µm, Mitsubishi Chemical Industries Co., Ltd.), Sephadex LH-20 (Amersham Pharmacia Biotech), and Chromatorex ODS (30-50 µm, Fuji Silysia Chemical Co., Ltd.). Thin layer chromatography (TLC) was performed on a precoated silica gel 60 F₂₅₄ (0.2 mm, aluminum sheet, Merck).

Plant Material

The dried flowers of A. arborescens were obtained from Hirata Farm Co. Ltd., Gunma, Japan in January 2017.

Extraction and Isolation

The dried flowers of A. arborescens (950 g) were extracted twice with 70% EtOH (9 L) at room temperature for 3 days each time. The filtered extracts were combined and evaporated under reduced pressure to give 224.9 g of extract. The extract was then suspended in water and subjected to MCI gel CHP20P CC and eluted successively with water, 40%, 60%, 80%, and 100% MeOH to give nine fractions (Fr. 1-Fr. 9). Fraction 2 (3.1 g, water eluate) and fraction 3 (5.2 g, water eluate) were combined and subjected to MCI gel CHP20P CC and eluted successively with water, 20% and 100% MeOH to obtain 5 subfractions (2-1 ~ 2-5). Subfraction 2-3 subjected to Sephadex LH-20 CC (water) to obtain three subfractions (2-3-1 ~ 2-3-3). Subfraction 2-3-2 (1269.9 mg) was subjected to Sephadex LH-20 CC (water) to obtain 6 subfractions (2-3-2-1 ~ 2-3-2-6). Subfraction 2-3-2-3 (327.9 mg) was subjected to silica gel CC (CH₂Cl₂:MeOH:H₂O = 7:3:0.5) to obtain compound 5 (22.4 mg). Fraction 4 (6.9 g, 40% MeOH eluate) and fraction 5 (1.0 g, 40% MeOH eluate) were combined and subjected to Sephadex LH-20 CC (50%MeOH) followed by repeated CC on silica gel CC (CH₂Cl₂:MeOH:H₂O = 7:3:0.5) to obtain compounds 1 (29.7 mg) and 4 (1.3 mg). Fraction 6 (2.6 g, 60% MeOH eluate) was subjected to Sephadex LH-20 CC (50% MeOH) followed by repeated CC on silica gel CC (CH₂Cl₂:MeOH:H₂O = 9:1:0.1) to obtain compounds 1 (34.9 mg) and 2 (14.3 mg). Fraction 7 (3.9 g, 80% MeOH eluate) was subjected to Sephadex LH-20 CC (50% MeOH) followed by repeated CC on silica gel CC (Hexane:EtOAc = 1:1) to obtain compound 3 (7.7 mg).

Feralolide-3’-O- β -d-glucopyranoside (1)

Yellow amorphous powder.

[α]_D21: ― 78.9 (c = 0.23, MeOH).

1H-NMR (CD₃OD, 600 MHz): Table 1.

Table 1

NMR Spectroscopic Data for 1 and 2.

Position	1		2
	δ C	δ H, mult. (J in Hz)	δ C	δ H, mult. (J in Hz)
1	171.2		171.3
3	81.3	4.70, m	81.5	4.72, m
4	33.6	2.84, m	33.6	2.87, m
4a	143.2		143.3
5	108.1	6.19, m	108.1	6.20, d (2.2)
6	165.6		165.6
7	102.3	6.19, m	102.3	6.18, d (2.2)
8	166.3		166.3
8a	101.6		101.7
9	39.1	3.02, dd (13.9, 7.4)2.92, dd (13.9, 5.2)	39.7	3.06, dd (13.7, 7.4)2.95, dd (13.7, 5.2)
1'	138.2		139.4
2'	125.3		121.9
3'	158.3		160.2
4'	102.7	6.62, d (2.0)	102.6	6.25, d (2.3)
5'	161.0		161.4
6'	113.5	6.48, d (2.0)	111.6	6.29, d (2.3)
COCH3	207.2		206.8
COCH 3	33.6	2.59, s	33.0	2.55, s
1"	102.2	4.95, d (7.7)
2"	74.9	3.45, m
3"	78.3 a	3.43, m
4"	71.3	3.40, m
5"	78.4 a	3.43, m
6"	62.6	3.90, dd (12.1, 2.1)3.71, dd (12.1, 5.5)

^aAssignments with the same superscript may be interchanged.

13C-NMR (CD₃OD, 150 MHz): Table 1.

HR-FAB-MS m/z: 505.1327 [M-H]- (Calcd. for C₂₄H₂₅O₁₂, 505.1346).

CD (MeOH): Δε (nm) +1.42 (234), – 1.66 (271), +0.32 (298), – 0.85 (318).

Feralolide (2)

White amorphous powder.

[α]D21:―21.4 (c = 0.13, MeOH).

1H-NMR (CD3OD, 600 MHz): Table 1.

13C-NMR (CD3OD, 150 MHz): Table 1.

CD (MeOH): Δε (nm) +0.83 (235), – 2.74 (269), +0.042 (297), – 1.14 (317).

Acid Hydrolysis of 1

Acid hydrolysis was performed by heating a solution of compound 1 (10.3 mg) in 2 M HCl (5 mL) in a sealed tube at 80°C for 3 hours. The reaction mixture was then extracted with EtOAc (5 mL, five times) to separate EtOAc soluble fraction and aqueous fraction. The aqueous fraction was further evaporated to obtain d-glucose (3.7 mg), [α]_D21 +30.4 (c = 0.37, MeOH). Identification of sugars was further supported by co-TLC with authentic samples. The EtOAc soluble fraction was evaporated and subjected to silica gel column (CH₂Cl₂:MeOH:H₂O = 9:1:0.1) to obtain aglycone (3.5 mg) which was identified as feralolide (2).