Objective: The present study aimed to investigate the potential bioactive compounds from Phyllanthus emblica L. fruit. Methods: Acetone extracts (60%) of P emblica fruit were dissolved in water and fractionated sequentially with light petroleum and ethyl acetate (EtOAc). The water fraction was separated using Sephadex LH-20 and the isolated compounds were purified by preparative high-performance liquid chromatography. The structures of the isolated compounds were determined through HR-ESI-MS and spectroscopic methods, including 1D- and 2D-NMR, and the antioxidant abilities of the isolates were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. Results: Three new phenolic compounds, mucic acid 1-ethyl ester 3-O-gallate (1), mucic acid 6-methyl ester 3-O-gallate (2), and mucic acid 6-ethyl ester 2-O-gallate (3), together with 4 known compounds (4-7), were isolated from P emblica fruit. All phenolics exhibited potent antioxidant abilities (DPPH: IC50 7.5-13.2 µM; ABTS: 1.12-2.84 µM Trolox/µM; FRAP: 1.11-2.26 µM Fe2+/µM). Conclusions: Our research contributes to the growing body of evidence demonstrating the benefits of P emblica for health and supports the functional food and nutraceutical applications of the fruit.
Phyllanthus emblica (P emblica) L., family Euphorbiaceae, is widely distributed in the subtropical and tropical areas of Southeast Asia.1 Its fruit, commonly known as Amla and Indian gooseberry, is well accepted by consumers for its sweet and slightly astringent taste2 and various medicinal benefits, including antioxidant,3-5 antimicrobial,6 antidiabetic,7 cardioprotective,8 anticancer,9,10 and anti-inflammatory11 activities. Previous phytochemical studies of P emblica fruit have revealed the presence of ellagitannins, gallotannins, anthocyanins, vitamin C, mucic acids, and phenolic acids.12-14 As a result of our current study aimed at discovering the potential bioactive compounds in P emblica fruit, 7 phenolic compounds (1-7) were isolated and identified, of which 3 were new phenolics, mucic acid 1-ethyl ester 3-O-gallate (1), mucic acid 6-methyl ester 3-O-gallate (2), and mucic acid 6-ethyl ester 2-O-gallate (3). The structure (Figure 1) of all metabolites was determined through HR-ESI-MS and spectroscopic methods, including 1D- and 2D-NMR, as well as comparison with values reported in the literature. In addition, the antioxidant properties of the isolates were assessed, and the results showed that all phenolics exhibited potent antioxidant abilities.
Structures of compounds (1-7) isolated from the fruits of Phyllanthus emblica.
Results and Discussion
Compound 1 was isolated as an amorphous white powder. Its molecular formula of C15H18O12 was assigned according to the HR-ESI-MS ion at m/z 389.0734 [M–H]– (calculated for C15H17O12, 389.0725) and the NMR data (see Table 1), indicating 7 degrees of unsaturation. Detailed analysis of the 1H- and 13C-NMR spectroscopic data of 1 suggested the presence of a galloyl group and a mucic acid (2,3,4,5-tetrahydroxyadipic acid) unit,15,16 which was verified by HSQC, HMBC, and 1H−1H COSY correlations (Figure 2). The HMBC correlations from H-2 and H-3 to C-1 and from H-4 and H-5 to C-6, together with the 1H−1H COSY correlations of H-2/H-3/H-4/H-5 confirmed the presence of a mucic acid unit. The characterization of a galloyl group in the structure was supported by the HMBC correlations from H-2′ (H-6′) in the low field to C-1′, C-3′, C-4′, C-5′, and C-7′. The connection between the mucic acid unit and the galloyl group was determined by the HMBC correlation of the double doublet-like methine proton (H-3) of the mucic acid unit with the carbonyl (C-7′, δC 167.0) of the galloyl group. Moreover, an isolated 1H−1H COSY spin system (H-1′′/H-2′′) was indicative of the existence of an ethyl moiety in the structure. The location of the ethyl moiety was determined to be at C-1 based on the HMBC cross-peak between H-1′′ and the carbon C-1. The acid hydrolysate was identified as mucic acid by direct comparison of the 1H-NMR spectra (see Supplemental Material), specifically the chemical shifts and coupling patterns, with those of the authentic sample.16 As can be seen, the chemical shifts and coupling patterns of the hydrolysate were identical to those of the authentic sample. The specific optical rotation (([α]20 D-0.002 (c 0.2, CH3OH)) indicated that the hydrolysate was a mesomer compound, which was consistent with that of mucic acid. Moreover, mucic acid and its gallate esters have been isolated and identified from P emblica.14-16 Therefore, 1 was tentatively determined as mucic acid 1-ethyl ester 3-O-gallate, as shown in Figure 1.
1H−1H COSY and key HMBC correlations of compounds 1-3.
1H- and 13C-NMR Spectroscopic Data of Compounds 1-3 in CD3OD (δ, ppm; J, Hz).
Position
1
2
3
δH
δC
δH
δC
δH
δC
1
174.4
174.9
173.0
2
4.65(1H, d, J = 1.2 Hz)
70.4
4.65 (1H, d, J = 1.8 Hz)
70.2
5.48 (1H, d, J = 1.8 Hz)
74.2
3
5.44 (1H, dd, J = 1.2, 10.2 Hz)
74.5
5.40 (1H, dd, J = 1.2, 10.2 Hz)
74.4
4.37 (1H, dd, J = 1.8, 9.6 Hz)
71.5
4
4.41 (1H, dd, J = 1.8, 10.2 Hz)
71.0
4.39 (1H, dd, J = 1.8, 10.2 Hz)
71.0
4.02 (1H, dd, J = 1.8, 9.6 Hz)
72.9
5
4.14 (1H, d, J = 1.8 Hz)
71.5
4.22 (1H, d, J = 1.8 Hz)
71.8
4.51 (1H, d, J = 1.8 Hz)
71.8
6
176.6
174.9
175.1
1′
120.9
120.8
121.2
2′
7.08 (1H, s)
110.4
7.06 (1H, s)
110.4
7.19 (1H, s)
110.4
3′
146.5
146.5
146.5
4′
140.1
140.1
140.0
5′
146.5
146.5
146.5
6′
7.08 (1H, s)
110.4
7.06 (1H, s)
110.4
7.19 (1H, s)
110.4
7′
167.0
167.0
167.9
1′′
4.14 (2H, m)
62.5
3.76 (3H, s)
52.6
4.21 (2H, m)
62.3
2′′
1.23 (3H, t, J = 7.2 Hz)
14.4
1.26 (3H, t, J = 7.2 Hz)
14.5
The molecular formula, C14H16O12, of 2, was determined by the HR-ESI-MS ion at m/z 375.0578 [M–H]– (calculated for C14H15O12, 375.0569), which was 14 mass units less than that of 1 and suggested the lack of a methylene group. The 1H- and 13C-NMR data of 2 closely resembled those of 1, except for the absence of an oxygenated methylene. The signals of 2 carbonyls of the mucic acid unit were at δC 174.85 and 174.89, representing the esterification of the carboxyl group at C-6 of the mucic acid unit.13 In the HMBC spectrum (Figure 2), H-3 (δH 5.40) was correlated with C-1 (δC 174.9) and the carbonyl (C-7′, δC 167.0) of the galloyl group, and the new methoxyl protons (δH 3.76) were correlated with C-6 (δC 174.9). The presence of a mucic acid unit in the structure was confirmed by the same method as that for 1. Hence, the structure of 2 was tentatively established as mucic acid 6-methyl ester 3-O-gallate.
Compound 3 had the same molecular formula of C15H18O12 as 1 on the basis of the HR-ESI-MS ion (m/z 389.0735 [M–H]–, calculated for 389.0725). The 1H- and 13C-NMR data of 3 were similar to those of 1, except for the chemical shifts of H-2 (δH 5.48) and H-5 (δH 4.51) of 3, which were deshielded by +1.17 and 0.37 ppm, respectively, and those of H-3 (δH 4.37) and H-4 (δH 4.02), which were shielded by −1.07 and 0.39 ppm, respectively, implying that compound 3 was a regioisomer of 1. This was confirmed by the HMBC cross peak between H-2 and C-7′ (Figure 2), suggesting that the galloyl group was esterified to OH-2 of the mucic acid. The HMBC cross peak from H-1″ to C-6 indicated that the carbonyl carbon at C-6 of the mucic acid was esterified to ethanol. Accordingly, 3 was tentatively determined as mucic acid 6-ethyl ester 2-O-gallate.
On the basis of their spectroscopic data and comparison with those of the literature,13 the structures of the 4 known compounds were elucidated as mucic acid 1,6-dimethyl ester 2-O-gallate (4), mucic acid 1-methyl ester 2-O-gallate (5), mucic acid 1,4-lactone methyl ester 5-O-gallate (6), and mucic acid 6-methyl ester 2-O-gallate (7).
Previous studies showed that organic acid gallates such as 4, 6, and mucic acid 1-ethyl 6-methyl ester 2-O-gallate mucic, isolated from P emblica, exhibited strong antioxidant activities.15 As such, all isolated compounds were tested for their antioxidant effects using DPPH radical scavenging, ABTS radical scavenging, and ferric reducing antioxidant power (FRAP) assay methods. As shown in Table 2, all isolates displayed DPPH free radical scavenging effects with IC50 values from 7.5 to 14.3 µM compared with that of the positive control α-tocopherol (IC50 23.8 µM). Meanwhile, compounds 1-7 exhibited ABTS scavenging activities in the range of 1.56 to 3.12 µM Trolox/µM, as well as ferric reducing power in the range of 1.11 to 2.26 µM Fe2+/µM.
Antioxidant Activities of Compounds (1-7) Isolated From the Fruits of P emblica.
Compounds
DPPH IC50 (μM)
ABTS (μM Trolox/μM)
FRAP (μM Fe2+/μM)
1
7.5 ± 0.6
1.96 ± 0.09
1.84 ± 0.02
2
8.6 ± 0.3
2.24 ± 0.05
1.36 ± 0.09
3
9.5 ± 0.2
2.46 ± 0.18
1.11 ± 0.14
4
8.9 ± 0.4
2.84 ± 0.07
1.41 ± 0.12
5
9.9 ± 0.6
2.64 ± 0.10
1.72 ± 0.08
6
13.2 ± 0.5
1.56 ± 0.09
2.26 ± 0.07
7
10.6 ± 0.6
3.12 ± 0.25
1.26 ± 0.18
α-Tocopherol
23.8 ± 0.3
ND
2.42 ± 0.15
Until now, 12 mucic acid gallates have been isolated from P emblica,15,17 but only one has been identified as mucic acid 3-O-gallate. In the current work, we isolated and identified 2 new methyl and ethyl esters of mucic acid 3-O-gallate, which were proved not to be separation artifacts by analysis of the 60% acetone extract of P emblica fruit through high-performance liquid chromatography (HPLC) and previously reported literature.13,16,17 Thus, our research not only increased the number of mucic acid gallates from P emblica but also enriched the chemical diversity of these natural compounds. In addition, the results of the antioxidant abilities of all isolated compounds indicate that the existence of these compounds may provide a basis for the antioxidant activities of P emblica fruit and will be helpful to understand this important herbal medicine. In conclusion, the current research contributes to the growing body of evidence demonstrating the benefits of P emblica for health and supports the functional food and nutraceutical applications of the fruit.
Experimental
General Experimental Procedures
NMR spectra were recorded on a Bruker Ascend NMR spectrometer operating at 600 MHz for 1H and 150 MHz for 13C. Coupling constants were expressed in Hz, and chemical shifts were referenced to the solvent residual signal of deuterated methanol (CD3OD). The HR-ESI-MS analysis was performed using an AB SCIEX Triple TOF 6600 mass spectrometer. A Shimadzu LC-6AD instrument with a YMC-Pack ODS column (250 mm × 20 mm, 5 cm, YMC Corp) was used for preparative HPLC. Sephadex LH-20 (GE Healthcare) was used for column chromatography. The column fractions were guided by a Waters Arc HPLC system equipped with an ultraviolet detector. DPPH, ABTS, and FRAP assay kits were purchased from Sigma Chemical Co. and Trolox from Macklin Biochemical Technology Co. Acetonitrile used for analytical and preparative HPLC was of chromatographic grade (Merck). Water used in the experiments was produced by a Milli-Q water system (Millipore). All other chemicals and solvents were of analytical grade.
Plant Material
The fruits of P emblica were purchased from Chuxiong, Yunnan Province, in September 2021 and authenticated by Shen Huang, an associate professor at the Zhengzhou University of Light Industry. The voucher specimen (PE2021008) is maintained in the laboratory of flavors and fragrances of Shenzhen Tobacco Industrial Co., Ltd.
Extraction and Isolation
The dry fruits (10 kg) of P emblica were ground and subjected to ultrasound-assisted extraction 3 times with 15 L of 60% acetone for 3 h, each. Then, the 60% acetone extracts were filtered, combined, and concentrated under reduced pressure to obtain the crude residue (3.62 kg). The brown crude extract was dissolved in sterile distilled water (5 L) and extracted sequentially with light petroleum (3 × 5 L) and ethyl acetate (EtOAc, 3 × 5L). The water fraction (655 g) was subjected to chromatography on a Sephadex LH-20 column (150 × 10 cm i.d.), eluting successively with a MeOH/H2O gradient system (0%, 10%, 20%, 30%, 40%, 50%, 75%, and 100%, vol/vol, 1.5 L each), to provide 15 fractions (fractions A–O) according to the HPLC profiles. Fractions E and F were purified repeatedly by reverse-phase preparative HPLC. Ultimately, compounds 1 (13 mg) and 2 (11 mg) were isolated from fraction E using MeCN/H2O (13:87, vol/vol) as the mobile phase at 5 mL/min, as well as 3 (8 mg) using MeCN/H2O (10:90, vol/vol) as the mobile phase. Compound 4 (28 mg) was obtained from fraction F using MeCN/H2O (5:95, vol/vol) as the mobile phase, and 5 (16 mg), 6 (9 mg), and 7 (4 mg) were purified from fraction F using MeCN/H2O (10:90, vol/vol) as the mobile phase. All isolates were dried under reduced pressure, and their spectroscopic data were collected.
Acid Hydrolysis of Compounds 1-3
Each compound (1:4 mg, 2: 3 mg, 3: 3 mg) was dissolved in H2O (1.0 mL) and 2 M aqueous CF3COOH (5.0 mL). The solution was heated in a water bath at 60 °C for 5 h.16 Next, the reaction mixture was subjected to chromatography on a silica gel column with CHCl3–MeOH–H2O (1 : 0 : 0—6 : 4 : 0.2) to obtain mucic acid, which was analyzed by TLC in comparison with an authentic sample.
Antioxidant Assays
The antioxidant effects of compounds 1-7 were examined by DPPH, ABTS, and FRAP assays with reference to described procedures.18
Mucic acid 1-ethyl ester 3-O-gallate (1): White amorphous powder. [α]20 D-10.6 (c 0.2, CH3OH), 1H- and 13C-NMR: see Table 1. HR-ESI-MS m/z 389.0734 [M–H]– (calculated for C15H17O12, 389.0725).
Mucic acid 6-methyl ester 3-O-gallate (2): White amorphous powder. [α]20 D-16.6 (c 0.2, CH3OH), 1H- and 13C-NMR: see Table 1. HR-ESI-MS m/z 375.0578 [M–H]– (calculated for C14H15O12, 375.0569).
Mucic acid 6-ethyl ester 2-O-gallate (3): White amorphous powder. [α]20 D-46.6 (c 0.2, CH3OH), 1H- and 13C-NMR: see Table 1. HR-ESI-MS m/z 389.0375 [M–H]– (calculated for C15H17O12, 389.0725).
Conclusion
Seven phenolic compounds (1-7), that is, 3 new phenolics, mucic acid 1-ethyl ester 3-O-gallate (1), mucic acid 6-methyl ester 3-O-gallate (2), and mucic acid 6-ethyl ester 2-O-gallate (3), were isolated. The structures of all metabolites were determined by HR-ESI-MS, and spectroscopic methods, including 1D- and 2D-NMR, as well as by comparison with values reported in the literature. In addition, the antioxidant properties of the isolates were assessed, and the results of the antioxidant assays showed that all phenolics exhibited potent antioxidant abilities (DPPH: IC50 7.5-13.2 µM; ABTS: 1.12-2.84 µM Trolox/µM; FRAP: 1.11-2.26 µM Fe2+/µM).
Supplemental Material
sj-docx-1-npx-10.1177_1934578X231155717 - Supplemental material for Three New Antioxidative Phenolics From Phyllanthus emblica L. Fruit
Supplemental material, sj-docx-1-npx-10.1177_1934578X231155717 for Three New Antioxidative Phenolics From Phyllanthus emblica L. Fruit by Hai-feng Sun, Qiao Lv, Xiong Ji, Cheng Fang, Jia-xiang Fei, Xian-jun Liu, Jing-xun Liu and Xiao-hui Liu in Natural Product Communications
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the the Foundation of Shenzhen Tobacco Industry Co., Ltd (grant number KJ2022022).
Supplemental Material
Supplemental material for this article is available online.
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