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Nugroho, Choi, Seong, Song, Park, and Park: Isolation of Flavonoid Glycosides with Cholinesterase Inhibition Activity and Quantification from Stachys japonica
Original Article

Natural Product Sciences 2018;24(4):259-265.

Published online: 17 December 2018

DOI: https://doi.org/10.20307/nps.2018.24.4.259

Isolation of Flavonoid Glycosides with Cholinesterase Inhibition Activity and Quantification from Stachys japonica

Agung Nugroho1, Jae Sue Choi2, Su Hui Seong2, Byong-Min Song3, Kyoung-Sik Park4, Hee-Juhn Park5, ∗

1Department of Agro-industrial Technology, Lambung Mangkurat University, Banjarbaru 70714, Indonesia

2Department of Food Nutrition, Pukyong National University, Busan 48513, Korea

3Deparment of Forest Science, Sangji University, Wonju 26339, Korea

4Deparment of Oriental Medicine, Sangji University, Wonju 26339, Korea

5Department of Pharmaceutical Engineering, Sangji University, Wonju 26339, Korea

∗Author for correspondence Hee-Juhn Park, Ph.D, Department of Pharmaceutical Engineering, Sangji University, Wonju 26339, Republic of Korea. Tel: +82-33-730-0564; E-mail: hjpark@sangji.ac.kr

Received 14 June 2018       Revised 17 July 2018       Accepted 17 July 2018

Copyright © 2018 The Korean Society of Pharmacognosy

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The three flavone glycosides, 4′-O-methylisoscutellarein 7-O-(6″′-O-acetyl)-β-D-allopyranosyl(1 → 2)-β-D-glucopyranoside (1), isoscutellarein 7-O-(6″′-O-acetyl)-β-D-allopyranosyl(1 → 2)-β-D-glucopyranoside (3), and isoscutellarein 7-O-β-D-allopyranosyl(1 → 2)-β-D-glucopyranoside (4) in addition to a flavonol glycoside, kaempferol 3-O-β-D-glucopyranoside (astragalin, 2), were isolated from Stachys japonica (Lamiaceae). In cholinesterase inhibition assay, compound 1 significantly inhibited aceylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities (IC50 s, 39.94 μ g/ml for AChE and 86.98 μ g/ml for BChE). The content of isolated compounds were evaluated in this plant extract by HPLC analysis. Our experimental results suggest that the flavonoid glycosides of S. japonica could prevent the memory impairment of Alzheimer's disease.

Keywords: Stachys japonica, Lamiaceae, isoscutellarein glycoside, cholinesterase, Alzheimer's disease

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Fig. 1.
Structure of flavonoid glycosides (1–4) isolated from S. japonica and their aglycones (2a and 3a).
nps-24-259f1.tif
Fig. 2.
HPLC chromatogram of standard compounds and 80% MeOH fraction of S. japonica.
nps-24-259f2.tif
Table 1.
1H-NMR data of compounds 1, 3, and 4 isolated from S. japonica (600 MHz, DMSO-d6)
Position 1 3 4
Aglycone-3 6.84 (1H, s) 6.84 (1H, s) 6.83 (1H, s)
5-OH 12.32 (1H, s) 12.36 (1H, s) 12.40 (1H, s)
6 6.71 (1H, s) 6.71 (1H, s) 6.66 (1H, s)
2′ 8.11 (1H, d, J = 9.0 Hz) 8.00 (1H, d, J = 9.0 Hz) 8.00 (1H, d, J = 9.0 Hz)
3′ 7.15 (1H, d, J = 9.0 Hz) 6.96 (1H, d, J = 9.0 Hz) 6.97 (1H, d, J = 9.0 Hz)
5′ 7.15 (1H, d, J = 9.0 Hz) 6.96 (1H, d, J = 9.0 Hz) 6.97 (1H, d, J = 9.0 Hz)
6′ 8.11 (1H, d, J = 9.0 Hz) 8.00 (1H, d, J = 9.0 Hz) 8.00 (1H, d, J = 9.0 Hz)
OMe 3.89 (3H, s) - -
Glucose-1 5.09 (1H, d, J = 7.8 Hz) 5.08 (1H, d, J = 7.8 Hz) 5.11 (1H, d, J = 7.8 Hz)
2 3.60 (1H, m) 3.60 (1H, m) 3.60 (1H, m)
3 3.48 (1H, m) 3.49 (1H, m) 3.49 (1H, m)
4 3.27 (1H, m) 3.27 (1H, m) 3.27 (1H, m)
5 3.55 (1H, m) 3.55 (1H, m) 3.54 (1H, m)
6 3.52 (1H, m) 3.51 (1H, m) 3.49 (1H, m)
  3.76 (1H, m) 3.76 (1H, m) 3.74 (1H, m)
Allose-1 4.93 (1H, d, J = 7.8 Hz) 4.92 (1H, d, J = 7.8 Hz) 4.92 (1H, d, J = 7.8 Hz)
2 3.27 (1H, m) 3.27 (1H, m) 3.27 (1H, m)
3 3.93 (1H, m) 3.93 (1H, m) 3.23 (1H, m)
4 3.43 (1H, m) 3.43 (1H, m) 3.34 (1H, m)
5 3.89 (1H, m) 3.88 (1H, m) 3.64 (1H, m)
6a 4.06 (2H, m) 4.05 (2H, m) 3.41 (1H, m)
6b -   3.55 (1H, m)
OAc 1.89 (3H, s) 1.89 (3H, s) -
Table 2.
13 C-NMR data of compounds 1, 3, and 4 isolated from S. japonica (150 MHz, DMSO-d6)
Position 1 3 4
Isoscutellarein 2 164.1 164.1 164.6
3 103.0 103.1 103.1
4 182.9 182.8 182.8
5 152.7 152.7 152.9
6 100.0 100.0 99.3
7 151.1 151.0 151.7
8 128.0 128.0 127.7
9 144.3 144.2 144.8
10 106.1 106.1 105.7
1′ 123.4 121.7 121.7
2′ 129.0 129.1 129.1
3′ 115.1 116.4 116.5
4′ 163.0 161.9 161.9
5′ 115.1 116.4 116.5
6′ 129.0 129.1 129.1
OMe 56.1 -  
Glc 1 100.6 100.6 100.2
2 83.0 83.1 81.8
3 77.7 77.7 77.6
4 69.8 69.8 69.8
5 76.1 76.1 76.2
6 61.1 61.1 61.1
All 1 103.9 103.2 102.2
2 72.0 72.0 71.5
3 71.3 71.3 72.0
4 67.4 67.3 67.7
5 72.0 72.0 75.0
6 64.0 64.0 61.5
COCH3 20.9 20.9 -
COCH3 170.8 170.8 -
Table 3.
Cholinesterase inhibitory activities of the S. japonica extract, its fractions and isolated compounds
Samples AChEa BChE
Mean ± SEM Mean ± SEM
Aq. MeOH extract 86.09 ± 1.27 172.88 ± 0.71
CHCl3 fraction 68.66 ± 1.32 133.96 ± 2.43
BuOH fraction 74.20 ± 2.20 117.94 ± 7.61
1 39.94 ± 0.76 86.98 ± 1.72
2 66.76 ± 2.82 > 200
3 65.40 ± 0.45 109.76 ± 2.79
4 59.55 ± 2.92 160.84 ± 3.26
2a 32.19 ± 0.82 52.34 ± 1.51
3a 58.19 ± 1.11 79.60 ± 0.28
Apigeninb 12.80 ± 0.37 9.18 ± 0.67

a The values (μ g/ml) indicate 50% cholinesteraseinhibitory effects. These data represent the average values of three repeated experiments.

b Positive control.

Table 4.
Linearity of standard curves and limits of detection and quantification for the standard compounds
Compound t R (min) Calibration equation (linear model)a Linear range (µg/ml) R2b LODc (µg/ml) LOQd (µg/ml)
1 20.11 y = 157.68 x + 113.14 7.81–250.0 0.999 1.18 3.95
2 16.92 y = 378.45 x + 106.78 7.81–250.0 0.999 0.51 1.70
2a 22.10 y = 957.20 x + 158.03 7.81–250.0 0.999 0.15 0.49
3 17.22 y = 278.85 x + 122.48 7.81–250.0 0.999 0.63 2.12
3a 17.98 y = 313.95 x + 86.27 7.81–250.0 0.999 0.68 2.27
4 14.83 y = 95.75 x + 55.73 7.81–250.0 0.999 2.03 6.76

a y, peak area at 254nm; x, concentration of the standard (µg/ml);

b R2, correlation coefficient for 6 data points in the calibration curves (n =4)

c LOD, limit of detection (S/N = 3);

d LOQ, limit of quantification (S/N = 10).

Table 5.
Content of six compounds (mg/g) in the extract and fractions of Stachys
Fraction/ extract Analytes Total
1 2 2a 3 3a 4
80% MeOH fraction 55.96 50.94 2.43 153.54 4.45 53.70 321.03
BuOH fraction 26.76 24.36 1.16 73.43 2.13 25.68 153.54
MeOH extract 3.19 2.90 0.14 8.75 0.25 3.06 18.30
Dry plant material 0.33 0.30 0.01 0.91 0.03 0.32 1.90

The data was present as average of three determinations.

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