Phenolic Compounds as Potent Free Radical Scavenging and Enzyme Inhibitory Components From the Leaves of Guiera senegalensis

Abstract

Guiera senegalensis J.F. Gmel. (Combretaceae) is commonly used as a traditional medicine in Africa. In our previous study, the hydro-alcoholic extracts of the leaves of G. senegalensis showed potent free radical scavenging and enzyme inhibitory activities. Detailed chemical analysis of the 70% ethanol extract afforded 8 phenolic compounds including myricetin (1), myricitrin (2), quercetin (3), (-)-gallocatechin (4), 1,3,4,5-tetra-O-galloylquinic acid (5), gallic acid (6), methyl gallate (7), and ethyl gallate (8). Structures of these compounds were elucidated on the basis of NMR spectroscopic data. Among these compounds, 1,3,4,5-tetra-O-galloylquinic acid (5) (IC₅₀= 7.21 ±0.21 µM), quercetin (3) (IC₅₀ = 7.71 ± 0.17 µM), and myricitrin (2) (0.25 ± 0.03 µM) showed the most potent free radical scavenging, α-glucosidase inhibitory and pancreatic lipase inhibitory activities, respectively.

Keywords

Sudan free radical scavenging α-glucosidase pancreatic lipase

Guiera senegalensis J.F. Gmel. (Combretaceae) is a shrub commonly distributed in African countries including Senegal, Guinea, Mali,¹ Nigeria,² Burkina Faso,³ and Sudan.⁴ Traditionally, it is used in the treatment of cold, bronchitis, fever,¹ diabetes, vomiting, stomach pain, jaundice, kidney stones, and also as a tonic.^4,5 In our previous study, we analyzed the effect of extraction solvents on the antioxidant and enzyme inhibitory activities for 6 medicinal plants from Sudan and found that the hydro-ethanolic extract of the leaves of G. senegalensis showed potent activities.⁶ In continuation to that, we herein report the isolation and identification of potent bioactive phenolic compounds from the leaves of G. senegalensis.

The leaves of G. senegalensis collected from Kordofan State, Sudan, were extracted twice with 70% EtOH. The combined extract was dried under reduced pressure and then subjected to repeated column chromatography (CC) on MCI gel CHP20P, Sephadex LH20, ODS, and silica gel to isolate 8 pure compounds (1-8). The structures of these compounds were elucidated on the basis of NMR spectral data (see supplementary material) and by comparison with literature values as; myricetin (1),⁷ myricitrin (2),⁷ quercetin (3),⁷ (–)-gallocatechin (4),⁸ 1,3,4,5-tetra-O-galloylquinic acid (5),⁹ gallic acid (6), methyl gallate (7),¹⁰ and ethyl gallate (8) (Figure 1).¹¹ To the best of our knowledge, compounds 2, 4, 7, and 8 were identified for the first time in this plant species.

Figure 1

Structures of compounds isolated from the leaves of G. senegalensis.

All of these isolated compounds were evaluated for their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, α-glucosidase and pancreatic lipase inhibitory activities. The IC₅₀ values of active compounds for all these assays are given in Table 1. All compounds showed potent free radical scavenging activities and among them, compound 5 was the most potent one. Compound 5 has also been reported from other plant species with significant therapeutic activities such as potent anti-asthmatic activity¹² and antiviral activity against HIV.⁹ Regarding enzyme inhibitory activities, compound 3 showed the most potent α-glucosidase inhibitory activity followed by compounds 5 and 1. Similarly, compound 2 showed the potent pancreatic lipase inhibitory activity followed by 5. These results of bioactivities are also similar to previous studies.^13,14 In conclusion, 8 phenolic compounds were isolated from the leaves of G. senegalensis as potent free radical scavenging and enzyme inhibitory components.

Table 1

IC₅₀ (μM)^{^a} Values of Compounds Isolated From G. senegalensis

Compounds	DPPH free radical scavenging activity	α-Glucosidase inhibitory activity	Pancreatic lipase inhibitory activity
1	19.31 ± 0.35	13.48 ± 0.33	8.86 ± 0.14
2	35.29 ± 1.22	NA^b	0.25 ± 0.03
3	23.69 ± 0.73	7.71 ± 0.17	17.64 ± 0.37
4	15.44 ± 0.74	106.15 ± 8.04	NA^b
5	7.21 ± 0.21	8.97 ± 0.95	1.30 ± 0.03
6	21.51 ± 0.65	NA^b	NA^b
7	22.37 ± 0.64	NA^b	NA^b
8	23.06 ± 0.36	NA^b	NA^b
Positive control	43.35 ± 0.27 (Trolox)	561.63 ± 37.3 (Acarbose)	18.33 ± 0.66 (Cetilistat)

^aValues are represented as mean ± SD (n = 3).

^bnot active.

Experimental

General Experimental Procedures

Optical rotations, JASCO DIP-1000KUY polarimeter; NMR, BRUKER AVANCE 600 spectrometer; CC, silica gel 60 (0.040-0.063 mm, Merck KGaA), MCI gel CHP20P (75-150 µm, Mitsubishi Chemical Industries, Japan), Sephadex LH-20 (Amersham Pharmacia Biotech, Tokyo, Japan), and Chromatorex ODS (30-50 µm, Fuji Silysia Chemical Co., Ltd., Aichi, Japan). Thin layer chromatography was performed on a precoated silica gel 60 F₂₅₄ (0.2 mm, aluminum sheet, Merck KGaA, Darmstadt, Germany).

Chemicals

1,1-Diphenyl-2-picrylhydrazyl, enzymes α-glucosidase (from Saccharomyces cerevisiae), and porcine pancreatic lipase were purchased from Sigma Aldrich, Co. (Tokyo, Japan). 6-Hydroxy-2,5,7,8-tetramethylchromancarboxylic acid (Trolox) and acarbose were from Wako Pure Chemical Industries, Ltd (Tokyo, Japan), and 2-(N-morpholino)ethanesulfonic acid buffer was purchased from Dojindo Chemical Research, Kumamoto, Japan. 4-Nitrophenyl-α-d-glucopyranoside was purchased from Nacalai Tesque, Inc., Kyoto, Japan. Cetilistat was obtained from Combi-Blocks, San Diego, USA.

Plant Material

The leaves of G. senegalensis were collected from Kordofan state in March 2017. The plant species was identified by Mr. Yahya Sulieman Mohamed, Taxonomist, Medicinal, Aromatic Plants and Traditional Medicine Research Institute (MAPTRI), Khartoum, Sudan. The voucher specimen of the plant species (Voucher No.: MPRTMI-H G/2/82) was deposited at the Herbarium of MAPTRI, Khartoum, Sudan.

Extraction and Isolation

The leaves of G. senegalensis were shade dried for 2 weeks. The dried leaves (2.6 kg) were extracted twice with 70% EtOH (22 L, 48 hours). The filtered extracts were combined and evaporated under reduced pressure to give 586.2 g of extract. The extract was then subjected to MCI gel CHP20P CC and eluted successively with water, 40%, 70% and 100% MeOH to give 6 fractions (Fr. 1-Fr. 6). Fraction 2 (256.7 g, water eluate), and fraction 3 (46.6 g, water eluate) were combined and subjected to MCI gel CHP20P CC and eluted with water, 10%, 20%, 30%, 40%, 70% and 100% MeOH to obtain 9 fractions (SubFr. 2-1-SubFr. 2-9). The subtractions 2-3 (7.8 g, water eluate) and 2-4 (17.8 g, 10% MeOH eluate) were combined and subjected to Sephadex LH-20 CC (water, 50% and 100% MeOH) to obtain 7 subfractions (SubFr. 2-3-1-SubFr. 2-3-7). Subfraction 2-3-3 (8.3 g, water eluate) was subjected on ODS CC (water, 10%, 20%, 50%, 100% MeOH) to obtain compound 6 (124.4 mg). Subfraction 2-3-5 (2.3 g, 50% MeOH eluate) was also subjected to ODS CC (water, 10%, 20%, 30%, and 50% MeOH) to obtain compound 5 (370 mg). Compound 4 (352.2 mg) was obtained from subfraction 2-3-4 (0.66 g, water eluate) from silica gel CC (CH₂Cl₂:MeOH:H₂O = 8:2:0.1). Also, the subfractions 2-5 (18.5 g, 20% MeOH eluate), 2-6 (13.48 g, 30% MeOH eluate), and 2-7 (9.5 g, 40% MeOH eluate) were combined and subjected to Sephadex LH-20 CC (MeOH) to obtain 4 subfractions (SubFr. 2-5-1-SubFr. 2-5-4). Compound 7 (169.4 mg) was obtained from subfraction 2-5-2 (34.8 g, MeOH eluate) from silica gel CC (CH₂Cl₂:MeOH:H₂O = 7:3:0.5). Subfraction 2-8 (4.2 g) was subjected to silica gel CC (CH₂Cl₂:MeOH:H₂O = 8:2:0.1) to obtain compound 1 (100.6 mg) and 2 (831.1 mg). Fraction 4 (61.4 g, 40% MeOH eluate) was subjected to Sephadex LH-20 CC (MeOH) to obtain 4 subfractions (SubFr. 4-1-SubFr. 4-4). Among them, subfractions 4-1 (3.5 g) and 4-2 (22.3 g) were combined and applied to silica gel CC (CH₂Cl₂:MeOH:H₂O = 9:1:0.1) to obtain compound 8 (736.2 mg). Fraction 5 (48.0 g, 70% MeOH eluate) was subjected to Sephadex LH-20 CC (MeOH) followed by silica gel CC (CH₂Cl₂:MeOH:H₂O = 9:1:0.1) to obtain compound 3 (50.2 mg).

Optical Rotations

Myricitrin (2): [α]_D ²⁶ −103.2^o (c 0.45, MeOH); (−)-Gallocatechin (4): [α]_D ²⁷ -7.3^o (c 0.53, MeOH); 1,3,4,5-Tetra-O-galloylquinic acid (5): [α]_D ²⁷ -43.6^o (c 0.50, MeOH).

Evaluation of Bioactivities

The DPPH radical-scavenging activity, α-glucosidase inhibitory and pancreatic lipase inhibitory activities were performed as reported previously.⁶

Supplemental Material

Supplementary Data - Supplemental material for Phenolic Compounds as Potent Free Radical Scavenging and Enzyme Inhibitory Components From the Leaves of Guiera senegalensis

Supplemental material, Supplementary Data, for Phenolic Compounds as Potent Free Radical Scavenging and Enzyme Inhibitory Components From the Leaves of Guiera senegalensis by Amina Ibrahim Dirar, Anjana Adhikari-Devkota, Md. Mahadi Hassan, Mikiyo Wada, Takashi Watanabe, and Hari Prasad Devkota in Natural Product Communications

Footnotes

Acknowledgments

The authors would like to acknowledge the JSPS RONPAKU Program for fellowship support to Ms Amina Ibrahim Dirar. We are also grateful to Mr Yahya Sulieman Mohamed, Taxonomist, Herbarium, Medicinal, Aromatic Plants and Traditional Medicine Research Institute (MAPTRI) Sudan, for the identification of the plant.

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 in part by Program for Leading Graduate Schools, Health Life Science: Interdisciplinary and Glocal Oriented (HIGO) Program, MEXT, Japan and Program for Building Regional Innovation Ecosystems at Kumamoto University, Japan.

References

Bouchet

Levesque

Blond

Bodo

Pousset

J-L

. 1,3-di-O-galloylquinic acid from Guiera senegalensis. Phytochemistry. 1996;42(1):189-190.doi:10.1016/0031-9422(95)00925-6

Alawa

JP.

Jokthan

GE.

Akut

. Ethnoveterinary medical practice for ruminants in the subhumid zone of northern Nigeria. Prev Vet Med. 2002;54(1):79-90.doi:10.1016/S0167-5877(01)00273-2

Aderbauer

Clausen

P-H.

Kershaw

Melzig

. In vitro and in vivo trypanocidal effect of lipophilic extracts of medicinal plants from Mali and Burkina Faso. J Ethnopharmacol. 2008;119(2):225-231.doi:10.1016/j.jep.2008.06.024

EL-Kamali

. Ethnopharmacology of medicinal plants used in North Kordofan (Western Sudan. Ethnobot Leaflets. 2009;13:203-210.

El Ghazali

GEB.

El Tohami

MS.

El Egami

AA.

Abdalla

WE.

Galal

. Medicinal Plants of the Sudan Part IV. In: Medicinal Plants of North Kordofan. Khartoum: National Council for Research; 1997.

Dirar

AI.

Alsaadi

DHM.

Wada

Mohamed

MA.

Watanabe

Devkota

. Effects of extraction solvents on total phenolic and flavonoid contents and biological activities of extracts from Sudanese medicinal plants. South Afr J Bot. 2019;120:261-267.doi:10.1016/j.sajb.2018.07.003

Markham

KR.

Chari

. Carbon-13 NMR spectroscopy of flavonoids. In: Horborne

JB.

Mabry

TJ.

Mabry

, eds. The flavonoids: advances in research. New York, NY: Chapman and Hall; 1982:19-132.

Davis

AL.

Cai

Davies

AP.

Lewis

. ¹H and ¹³C NMR assignments of some green tea polyphenols. Magn Reson Chem. 1996;34(11):887-890.doi:10.1002/(SICI)1097-458X(199611)34:11<887::AID-OMR995>3.0.CO;2-U

Nishizawa

Yamagishi

Dutschman

et al . Anti-AIDS agents, 1. Isolation and characterization of four new tetragalloylquinic acids as a new class of HIV reverse transcriptase inhibitors from tannic acid. J Nat Prod. 1989;52(4):762-768.doi:10.1021/np50064a016

10.

Kamatham

Kumar

Gudipalli

. Isolation and characterization of gallic acid and methyl gallate from the seed coats of Givotia rottleriformis Griff. and their anti-proliferative effect on human epidermoid carcinoma A431 cells. Toxicol Rep. 2015;2:520-529.doi:10.1016/j.toxrep.2015.03.001

11.

Ooshiro

Hiradate

Kawano

et al . Identification and activity of ethyl gallate as an antimicrobial compound produced by Geranium carolinianum . Weed Biol Manag. 2009;9(2):169-172.doi:10.1111/j.1445-6664.2009.00335.x

12.

Neszmélyi

Kreher

Müller

Dorsch

Wagner

. Tetragalloylquinic acid, the major antiasthmatic principle of Galphimia glauca . Planta Med. 1993;59(2):164-167.doi:10.1055/s-2006-959635

13.

Adhikari-Devkota

Elbashir

SMI.

Watanabe

Devkota

. Chemical constituents from the flowers of Satsuma mandarin and their free radical scavenging and α-glucosidase inhibitory activities. Nat Prod Res. 2019;33(11):1670-1673.doi:10.1080/14786419.2018.1425856

14.

Zhang

Zhao

Hong

Cai

Pang

. Phenolic composition, antioxidant and pancreatic lipase inhibitory activities of Chinese sumac ( Rhus chinensis Mill.) fruits extracted by different solvents and interaction between myricetin-3-O-rhamnoside and quercetin-3-O-rhamnoside. Int J Food Sci Technol. 2018;53(4):1045-1053.doi:10.1111/ijfs.13680

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.11 MB

0.00 MB