Thi Thu Thao Nguyenab,
Vy Anh Tranc,
Thi Hien Trand,
Viet Duc Hoe,
Thi Ha Dof,
Quoc Ky Truongg,
Minh Quan Phamhi and
Thi Hong Van Le
*a
aFaculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, Vietnam. E-mail: levan@ump.edu.vn
bUniversity of Health Sciences, Vietnam National University, Ho Chi Minh City, Vietnam
cDepartment of Material Science, Institute of Applied Technology and Sustainable Development, Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam
dDepartment of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Box 117, Lund, SE-221 00, Sweden
eFaculty of Pharmacy, Hue University of Medicine and Pharmacy, Hue University, 06 Ngo Quyen, Hue City, Vietnam
fDepartment of Medical Plant Chemistry, National Institute of Medical Materials (NIMM), Hanoi, 11022, Vietnam
gFaculty of Pharmacy, Pham Ngoc Thach University of Medicine, Ho Chi Minh City, Vietnam
hInstitute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Hanoi, Vietnam
iGraduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam
First published on 14th April 2025
With the aim of isolating immunomodulatory compounds from the n-butanol extract of Boehmeria nivea leaves, nine megastigmane compounds were identified. Among these, the structure of the new compound 1, namely, “boehmegaside A”, was established using NMR and HR-ESI-MS, and its absolute configurations were established through ECD calculations and DP4+ analysis using DFT-NMR chemical shift calculations. Furthermore, eight of these compounds were discovered for the first time in the Boehmeria genus, marking this the first report on megastigmane compounds isolated from this genus. Regarding their immunomodulatory activity, the isolated megastigmane compounds 3, 2, and 6 exhibited pro-inflammatory cytokine IL-1β secretion inhibitory activity. Compounds 3 and 6 significantly increased anti-inflammatory cytokine IL-10 secretion in LPS-activated RAW264.7 cells. Furthermore, the study on the mechanism of the immunomodulatory and other biological activities of megastigmane compounds through molecular docking simulations revealed that the planar structures of 3, 2, and 6 were critical in their ability to directly suppress TLR4 signalling. Instead, they attached to a nearby smooth area in TLR4. This interference likely disrupted the ability of TLR4 and MD-2 to form their primary contact interface and recognize LPS. These findings highlight the significant role of TLR4 in inflammation and immunity, indicating that these megastigmane compounds may be beneficial in treating various inflammatory disorders associated with immunological issues.
Several methods for the pharmacological screening of medicinal plants with immunomodulatory activity have been used based on immunological factors. Among them, interleukin-1β (IL-1β) and interleukin-10 (IL-10) cytokines are potential targets for immune therapy. IL-1β is a pro-inflammatory cytokine involved in immune responses and inflammation, and it can contribute to carcinogenesis, tumor growth, and invasion.3 IL-10, in contrast, is an important cytokine with anti-inflammatory properties and regulates the functions of various immune cells.4 The role of IL-10 in cancer is complex, with both tumor-promoting and tumor-repressing effects. On the one hand, IL-10 can inhibit the activation of cytotoxic T cells by reducing the expression of major histocompatibility complexes on cancer cells and antigen-presenting cells, thereby impeding the recognition and targeting of cancer cells by antigen-specific T cells. On the other hand, studies have shown that high doses of IL-10 can enhance the proliferation and cytotoxic activity of CD8+ T cells.5
Boehmeria nivea (L.) Gaudich, Urticaceae is a versatile plant that has been utilized for many purposes, including food, animal feed, medicinal applications, and fiber production. The root of this plant is used in traditional Chinese herbal medicine to treat the common cold, edema, fever, fetal irritability, urinary tract infections, nephritis, and abortion risk, and it possesses various pharmacological properties. Its leaves have been reported to have antibiotic and anti-inflammatory effects.6 Numerous studies have shown that the extract of Boehmeria nivea leaves (BNL) exerts many biological activities, such as anti-proliferative, antitumor, anti-inflammatory, antibacterial, antioxidant, glucose-lowering, and anti-hepatitis B virus effects.7 Nevertheless, studies on the chemical components have shown that these leaves contain several common ingredients, including flavonoids, simple phenols, sterols, terpenoids, coumarins, anthraquinones, reducing sugars, fatty acids, and polysaccharides.8 In an ongoing investigation into the immune-inducing activity of some Vietnamese medicinal plants, it was found that a 70% ethanol BNL extract had immunomodulatory activity against IL-1β and IL-10 cytokines production in RAW 264.7 murine macrophages; therefore, this plant was selected for further study. Consequently, this study aimed to investigate the immunomodulatory effects of BNL extracts in a RAW 264.7 macrophage cell line, and then isolate and elucidate the chemical components from the active extract of B. nivea leaves. In our recent publication, we identified 8 compounds belonging to neolignan, phenolic and sugar derivatives from B. nivea, namely glochidioboside, protocatechuic acid, ethyl β-fructopyranoside, glycerol, eugenyl O-β-apiofuranosyl-(1′′ → 6′)-O-β-glucopyranoside, ethyl α-D-glucopyranoside, hydroxytyrosol 4-β-D-glucoside, and cimidahurinine, in the active extract (n-butanol).9
In this article, we report on the immunomodulatory effects of BNL extracts in a RAW 264.7 macrophage cell line, as well as the isolation and structural identification of 9 compounds belonging to megastigmane from bioassay-guided isolation processing of an n-butanol extract. We also investigated the immunomodulatory activity of certain compounds from the leaves of B. nivea on the secretion of the pro-inflammatory cytokine IL-1β and on anti-inflammatory cytokines IL-10 production in RAW 264.7 macrophages.
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Fig. 1 Effects of extracts from BNL on the pro-inflammatory cytokine IL-1 β. Note: (****p < 0.0001) when compared with the LPS-positive control group (n = 5). |
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Fig. 2 Effects of extracts from BNL on the anti-inflammatory cytokine IL-10. Note: (****p < 0.0001, ***p < 0.001, ns > 0.05) when compared with the LPS-positive control group (n = 5). |
The results on IL-1β showed that with the action of LPS, the RAW 264.7 cells increased IL-1β secretion (at 24 h). When treating the RAW264.7 cells with the BNL extracts, three extracts enormously reduced IL-1β to levels ranging from 46.61 to 63.67 pg mL−1, compared to the LPS-positive control group with a concentration of 434.82 pg mL−1 (p < 0.0001).
Furthermore, upon testing against IL-10 cytokines, all the extracts showed enormously increased IL-10 production, with levels ranging from 267.07 to 292.98 pg mL−1 compared to the LPS-positive control group, with a concentration of 109.79 pg mL−1 (p < 0.0001 and p < 0.001). Among them, the polar extract (n-BuOH) showed the most potent immunomodulatory activity through inhibiting IL-1β and stimulating IL-10 production (p < 0.0001 and p < 0.001). Consequently, the n-BuOH extract was chosen to investigate the chemical composition.
Diastereomers | 6R,9R (1a) | 6R,9S (1b) | 6S,9R (1c) | 6S,9S (1d) |
---|---|---|---|---|
mPW1PW91/6-31G+(d,p)/PCM | ||||
DP4+ (H data) | 0.08% | 0.01% | 0.07% | 0.00% |
DP4+ (C data) | 34.22% | 0.00% | 65.78% | 0.00% |
DP4+ (all data) | 37.37% | 0.00% | 62.63% | 0.00% |
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mPW1PW91/6-311G(d,p)/PCM | ||||
DP4+ (H data) | 0.02% | 0.45% | 99.53% | 0.00% |
DP4+ (C data) | 0.03% | 0.03% | 99.93% | 0.00% |
DP4+ (all data) | 0.00% | 0.00% | 100% | 0.00% |
Position | Type | δCa (CD3OD) | δCb (CDCl3) | δHb (m, J Hz, nH) (CDCl3) | HMBCb (CDCl3) | COSYb (CDCl3) |
---|---|---|---|---|---|---|
a 13C-NMR: 150 MHz.b 13C-NMR: 150 MHz, 1H-NMR: 600 MHz. | ||||||
1 | C | 37.2 | 36.2 | — | — | — |
2 | CH2 | 48.4 | 47.2 | 2.35 (d, 16.8 Hz, 1H) | 1, 3, 12 | — |
2.06 (d, 16.8 Hz, 1H) | — | — | ||||
3 | C | 202.1 | 199.7 | — | — | — |
4 | CH | 126.1 | 125.8 | 5.91 (d, 1.5 Hz, 1H) | 2, 6, 13 | — |
5 | C | 166.0 | 162.5 | — | — | — |
6 | CH | 56.8 | 55.3 | 2.53 (d, 8.8 Hz, 1H) | 2, 4, 5, 7, 8, 11, 13 | — |
7 | CH | 128.9 | 128.3 | 5.58 (dd, 15.4, 8.8 Hz, 1H) | 8, 6, 1 | 8, 6 |
8 | CH | 138.4 | 136.2 | 5.66 (dd, 15.4, 7.0 Hz, 1H) | 7, 6, 10 | 7, 9 |
9 | CH | 78.1 | 77.6 | 4.30 (m, 1H) | 8, 7, 10 | 10 |
10 | CH3 | 21.1 | 21.4 | 1.30 (d, 6.4 Hz, 3H) | 8, 9 | — |
11 | CH3 | 27.4 | 27.4 | 0.96 (d, 7.0 Hz, 3H) | 1, 2, 6 | — |
12 | CH3 | 28.0 | 27.8 | 1.03 (s, 3H) | 1, 2, 6 | — |
13 | CH3 | 23.9 | 23.7 | 1.90 (m, 3H) | 5, 4 | — |
1′ | CH | 102.5 | 101.7 | 4.34 (d, 7.8 Hz, 1H) | 9 | 2′ |
2′ | CH | 75.3 | 73.5 | 3.35 (t, 8.4 Hz, 1H) | 1′, 3′ | — |
3′ | CH | 78.0 | 76.3 | 3.48 (m, 1H) | 4′ | — |
4′ | CH | 71.5 | 69.3 | 3.59 (t, 9.6 Hz, 1H) | 3′, 6′ | — |
5′ | CH | 77.0 | 75.7 | 3.21 (m,1H) | — | 4′ |
6′ | CH2 | 62.7 | 61.3 | 3.82 (d, 12.0 Hz, 1H) | — | — |
3.73 (d, 12.0 Hz, 1H) | — | — |
Position | 1![]() |
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4![]() |
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6![]() |
7![]() |
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9![]() |
---|---|---|---|---|---|---|---|---|---|
a Abbreviation Glc = glucopyranoside.b CDCl3, 13C-NMR: 150 MHz, 1H-NMR: 600 MHz.c CD3OD, 13C-NMR: 150 MHz, 1H-NMR: 600 MHz.d CD3OD, 13C-NMR: 200 MHz, 1H-NMR: 800 MHz.e CD3OD, 13C-NMR: 125 MHz, 1H-NMR: 500 MHz. | |||||||||
1 | 36.2, C | 42.4, C | 39.9, C | 35.9, C | 36.7, C | 35.3, C | 45.3, C | 32.9, C | 34.0, C |
2 | 47.2, CH2 | 50.7, CH2 | 54.4, CH2 | 45.7, CH2 | 48.7, CH2 | 44.9, CH2 | 50.1, CH2 | 47.7, CH2 | 54.6, CH2 |
3 | 199.7, C![]() |
201.2, C![]() |
201.9, C![]() |
73.0, CH | 65.4, CH | 72.3, CH | 77.6, CH | 73.1, CH | 209.3, C![]() |
4 | 125.8, CH | 127.2, CH | 127.1, CH | 38.5, CH2 | 42.4, CH2 | 37.6, CH2 | 49.5, CH2 | 46.7, CH2 | 55.9, CH2 |
5 | 162.5, C | 167.2, C | 159.0, C | 67.7, C | 68.9, C | 67.4, C | 83.0, C | 87.7, C | 89.0, C |
6 | 55.3, CH | 80.0, C | 145.0, C | 71.4, C | 71.9, C | 69.6, C | 93.5, C | 153.9, C | 150.7, C |
7 | 128.3, CH | 131.6, CH | 135.2, CH | 125.8, CH | 130.4, CH | 142.1, CH | 127.4, CH | 117.3, CH | 120.5, CH |
8 | 136.2, CH | 135.3, CH | 71.3, CH | 139.1, CH | 136.8, CH | 132.7, CH | 135.1, CH | 86.9, CH | 86.8, CH |
9 | 77.6, CH | 77.3, CH | 79.5, CH | 68.4, CH | 75.5, CH | 197.4, C | 78.9, CH | 69.6, CH | 75.6, CH |
10 | 21.4, CH3 | 21.2, CH3 | 15.4, CH3 | 23.8, CH3 | 23.2, CH3 | 28.3, CH3 | 22.2, CH3 | 17.1, CH3 | 13.7, CH3 |
11 | 27.4, CH3 | 23.4, CH3 | 29.8, CH3 | 25.2, CH3 | 30.9, CH3 | 24.9, CH3 | 26.7, CH3 | 26.0, CH3 | 26.7, CH3 |
12 | 27.8, CH3 | 24.7, CH3 | 29.7, CH3 | 29.8, CH3 | 25.8, CH3 | 29.4, CH3 | 33.6, CH3 | 29.9, CH3 | 29.4, CH3 |
13 | 23.7, CH3 | 19.6, CH3 | 22.8, CH3 | 20.3, CH3 | 21.5, CH3 | 19.8, CH3 | 32.3, CH3 | 26.1, CH3 | 26.9, CH3 |
1′-O-Glc | 101.7, CH | 102.8, CH | 103.0, CH | 102.9, CH | 102.1, CH | 101.5, CH | 103.6, CH | 101.4, CH | 100.7, CH |
2′ | 73.5, CH | 75.3, CH | 75.0, CH | 75.2, CH | 75.9, CH | 75.6, CH | 76.2, CH | 73.8, CH | 73.8, CH |
3′ | 76.3, CH | 78.1, CH | 77.9, CH | 78.2, CH | 78.9, CH | 76.5, CH | 78.9, CH | 76.7, CH | 76.6, CH |
4′ | 69.3, CH | 71.7, CH | 71.6, CH | 71.6, CH | 72.6, CH | 69.5, CH | 72.2, CH | 70.4, CH | 70.5, CH |
5′ | 75.7, CH | 78.0, CH | 78.0, CH | 77.8, CH | 78.9, CH | 73.4, CH | 78.8, CH | 76.6, CH | 76.7, CH |
6′ | 61.3, CH2 | 62.9, CH2 | 62.7, CH2 | 62.7, CH2 | 63.7, CH2 | 61.5, CH2 | 63.5, CH2 | 61.5, CH2 | 61.6, CH2 |
Position | 1![]() |
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9![]() |
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a Abbreviation Glc = glucopyranoside.b CDCl3, 13C-NMR: 150 MHz, 1H-NMR: 600 MHz.c CD3OD, 13C-NMR: 150 MHz, 1H-NMR: 600 MHz.d CD3OD, 13C-NMR: 200 MHz, 1H-NMR: 800 MHz.e CD3OD, 13C-NMR: 125 MHz, 1H-NMR: 500 MHz. | |||||||||
1 | — | — | — | — | — | — | — | — | — |
2 | 2.35 (d, 16.8, 1H) | 2.51 (d, 17.0, 1H) | 2.39 (d, 2.4, 2H) | 1.35 (dd, 13.1, 10.4,1H) | 1.22 (m, 1H) | 1.73 (m, 1H) | 1.76 (ddd, 11.6, 6.0, 2.4, 1H) | 1.27 (m, 1H) | 2.21 (dd, 13.5, 2.0, 1H) |
2.06 (d, 16.8, 1H) | 2.15 (d, 17.0, 1H) | — | 1.72 (m, 1H) | 1.54 (ddd, 12.8, 3.4, 1.8, 1H) | 1.35 (m, 1H) | 1.58 (d, 11.6, 1H) | 1.96 (ddd, 12.7, 4.2, 2.0, 1H) | 2.42 (d, 13.5, 1H) | |
3 | — | — | — | 3.89 (dddd, 10.4, 8.5, 5.0, 3.5, 1H) | 3.74 (m, 1H) | 3.88 (m, 1H) | 4.33 (t, 6.0, 1H) | 4.08 (tt, 11.8, 4.2, 1H) | — |
4 | 5.91 (d, 1.5, 1H) | 5.87 (t, 1.26, 1H) | 5.98 (1H, s) | 1.75 (dd, 14.4, 8.5, 1H) | 1.61 (dd, 14.2, 9.0, 1H) | 2.45 (d, 14.1, 1H) | 1.94 (ddd, 11.6, 6.0, 2.4, 1H) | 1.48 (m, 1H) | 2.56 (dd, 12.9, 2.0, 1H) |
— | — | — | 2.39 (ddd, 14.4, 5.0, 1.7, 1H) | 2.27 (ddd, 14.2, 5.0, 1.8, 1H) | 1.73 (m, 1H) | 1.64 (d, 12, 1H) | 2.36 (ddd, 11.5, 4.2, 2.0. 1H) | 2.84 (dt, 12.9, 1.0, 1H) | |
5 | — | — | — | — | — | — | — | — | — |
6 | 2.53 (d, 8.8, 1H) | — | — | — | — | — | — | — | — |
7 | 5.58 (dd, 15.4, 8.8, 1H) | 5.86 (m, 1H) | 6.12 (d, 9.2, 1H) | 5.91 (dd, 15.4, 1.3, 1H) | 6.05 (dd, 15.5, 1.0, 1H) | 7.0 (dd, 15.6, 3.8, 1H) | 5.79 (d, 16, 1H) | 5.39 (d, 1.0, 1H) | 5.79 (d, 1.2, 1H) |
8 | 5.66 (dd, 15.4, 7.0, 1H) | 5.85 (m, 1H) | 4.95 (1H, dd, 9.2, 4.0) | 5.67 (dd, 15.4, 6.0, 1H) | 5.57 (dd, 15.5, 7.5, 1H) | 6.26 (dd, 15.6, 4.6, 1H) | 5.74 (dd, 16, 6.4, 1H) | 4.59 (dd, 5.3, 1.0, 1H) | 4.86 (dd, 4.4, 1.2, 1H) |
9 | 4.3 (m, 1H) | 4.42 (pd, 6.6, 4.4, 1H) | 3.99 (1H, qd, 6.4, 4.0) | 4.28 (dquin, 6.0, 1.3, 1H) | 4.52 (m, 1H) | — | 4.39 (p, 6.4, 1H) | 3.61 (m, 1H) | 3.94 (qd, 6.4, 4.4, 1H) |
10 | 1.30 (d, 6.4, 3H) | 1.29 (d, 6.6, 3H) | 1.26 (3H, d, 6.4) | 1.22 (d, 6.4, 3H) | 1.27 (d, 6.4, 3H) | 2.28 (d, 4.6, 3H) | 1.29 (d, 6.4, 3H) | 1.09 (d, 6.4, 3H) | 1.16 (d, 6.4, 3H) |
11 | 0.96 (d, 7.0, 3H) | 1.04 (s, 3H) | 1.36 (s, 3H) | 0.96 (s, 3H) | 1.11 (s, 3H) | 0.97 (d, 3.8, 3H) | 1.39 (s, 3H) | 1.13 (s, 3H) | 1.11 (s, 3H) |
12 | 1.03 (s, 3H) | 1.03 (s, 3H) | 1.40 (s, 3H) | 1.14 (s, 3H) | 0.93 (s, 3H) | 1.17 (s, 3H) | 0.84 (s, 3H) | 1.20 (s, 3H) | 1.32 (s, 3H) |
13 | 1.90 (m, 3H) | 1.92 (d, 1.4,3H) | 2.16 (s, 3H) | 1.19 (s, 3H) | 1.24 (s, 3H) | 1.19 (d, 4.4, 3H) | 1.18 (s, 3H) | 1.39 (d, 1.0, 3H) | 1.38 (s, 3H) |
1′-OGlc | 4.34 (d, 7.8, 1H) | 4.34 (d, 7.8, 1H) | 4.40 (d, 7.8, 1H) | 4.33 (d, 7.8, 1H) | 4.27 (d, 7.8, 1H) | 4.39 (d, 7.6, 1H) | 4.35 (d, 7.8, 1H) | 4.37 (d, 7.8, 1H) | 4.33 (d, 7.7, 1H) |
2′ | 3.35 (t, 8.4, 1H) | 3.17 (dd, 9.2, 7.8, 1H) | 3.22 (dd, 9.1, 7.8, 1H) | 3.12 (dd, 9.0, 7.8, 1H) | 3.18 (m, 1H) | 3.31 (m, 1H) | 3.17 (m, 1H) | 3.10 (dd, 9.2, 7.8, 1H) | 3.14 (dd, 9.3, 7.7, 1H) |
3′ | 3.51 (m, 1H) | 3.33 (m, 1H) | 3.40 (m, 1H) | 3.34 (t, 9.0, 1H) | 3.25 (m, 1H) | 3.52 (m, 1H) | 3.32 (m, 1H) | 3.25 (m, 1H) | 3.33 (m, 1H) |
4′ | 3.59 (t, 9.6, 1H) | 3.26 (d, 9.8, 1H) | 3.44 (m, 1H) | 3.28 (m, 1H) | 3.25 (m, 1H) | 3.58 (m, 1H) | 3.30 (m, 1H) | 3.25 (m, 1H) | 3.23 (m, 1H) |
5′ | 3.21 (m, 1H) | 3.23 (m, 1H) | 3.39 (t, 8.7 1H) | 3.26 (m, 1H) | 3.16 (m, 1H) | 3.33 (m, 1H) | 3.18 (m, 1H) | 3.33 (m, 1H) | 3.23 (m, 1H) |
6′ | 3.82 (d, 12.0, 1H) | 3.85 (dd, 11.8, 2.2, 1H) | 3.88 (dd, 11.6, 2.0, 1H) | 3.67 (dd, 12, 5.3, 1H) | 3.63 (dd, 12.0, 6.2, 1H) | 3.83 (s, 2H) | 3.81 (dd, 11.9, 2.4, 1H) | 3.65 (dd, 5.6, 2.2, 1H) | 3.62 (dd, 11.1, 5.2, 1H) |
3.73 (d, 12.0, 1H) | 3.62 (dd, 11.8, 5.5, 1H) | 3.69 (dd, 11.7 5.0, 1H) | 3.84 (dd, 12, 2.3, 1H) | 3.85 (dd, 12.0, 2.4, 1H) | 3.65 (dd, 11.9, 5.3, 1H) | 3.84 (m, 1H) | 3.84 (dd, 11.7, 1.71H) |
Compound 1 was a colorless gum. The molecular formula was determined to be C19H30O7 from the HR-ESI-MS (positive mode) m/z: 393.20361 [M + Na]+ (calcd. for C19H30O7Na, 393.20392). The 1H and 13C-NMR spectra showed a typical pattern of megastigmane glycoside. Spectroscopic data showed that 1 was a megastigmane glycoside with the following data: the 1H-NMR spectrum of 1 showed an olefin proton CCH at δH 5.91 (1H; d; J = 1.5; H-4) and 2 protons of trans olefin (CH
CH) at δH 5.58 (1H; dd; J = 15.4, 9.0; H-7) and 5.66 (1H, dd, J = 15.4, 7.0; H-8); 1 methylene group [δH 2.35 (1H; d; J = 16.8 Hz, H-2a)/2.06 (1H; d; J = 16.8 Hz; H-2b)]; 1 oxymethine group [4.30 (1H; m; H-9)], and 4 methyl groups. Furthermore, the 1H-NMR spectrum displayed the signal of an anomer proton at δH 4.34 (1H, d, J = 7.58 Hz, H-1′), with 2 hydroxy methylene protons and hydroxy methine proton signals located in the range of 3.21–3.82 ppm, suggesting the presence of a sugar unit in a β configuration. The 13C-NMR data (Table 2), assigned with the aid of the HSQC and HMBC spectra, displayed 19 carbon resonances, including 13 carbon resonances of the megastigmane structure [3 quaternary carbons with a carbonyl group at δC 199.7 (C-3); 2 methine olefin groups at δC 128.3 (C-7) and 136.2 (C-8), another olefin carbon of CH
C at 125.8 (C-4); an oxymethine at δC 77.6 (C-9); a methylene at δC 47.2 (C-2); 4 methyl groups at δC 21.4 (C-10), 23.7 (C-13), 27.4 (C-11), 27.8 (C-12)] and 6 carbons of a sugar unit glucopyranose [δC 101.7(C-1′)]. The sugar unit was confirmed as being linked at C-9 by HMBC correlation of the anomeric proton (H-1) with C-9 (δC 77.6).
On the other hand, the monosaccharide composition, sugar moiety linkage, and the attachment site of the sugar moiety to the aglycone were determined by NMR spectroscopic analyses combined with the acid hydrolysis of 1 followed by reduction, per-acetylated derivatization, and GC-MS analysis. The acid hydrolysis revealed the presence of the D-glucose moiety in 1. This was deduced to be the hexose moiety in the D-glucopyranose form based on the corresponding resonances in the 1H and 13C-NMR spectra (Tables 2 and 3) and the correlations observed in the 1H–1H COSY and HMBC spectra (Fig. 3A). Furthermore, the coupling constant of 7.58 Hz between H-1′ and H-2′ indicated the β-configuration at the respective anomeric position of the D-glucopyranose moiety (Fig. 3A). To clarify the stereochemistry of the aglycone moiety of 1, DFT-NMR chemical shift calculations coupled with the DP4+ probability method were applied for four diastereomers: (6R,9R)-1a, (6R,9S)-1b, (6S,9R)-1c, and (6S,9S)-1d (see the ESI†). Briefly, the conformational searches using the molecular mechanics set yielded 6, 4, 6, and 6 main conformers, with Boltzmann distributions > 1%, for 1a–1d, respectively. The main conformers were further DFT optimized, and the harmonic vibrational frequencies were calculated at the B3LYP/6-31G(d) level in the gas phase. Finally, the 1H and 13C chemical shifts of the optimized conformers were calculated using Gaussian 09 with GIAO/mPW1PW91/6-31+G(d,p)/PCM/chloroform and GIAO/mPW1PW91/6-311G(d,p)/PCM/chloroform. Shielding tensors were used for the DP4+ probability calculations. As a result, DP4+ analysis showed that the most probable structure was (6S,9R)-1c (Table 1). Furthermore, the predicted ECD curves were carefully evaluated for the four diastereomers, whereupon it was found that the experimental ECD spectrum of 1 was in good agreement with the predicted ECD curves of (6S,9R)-1c; and (6S,9S)-1d (Fig. 3B). Combining the ECD and DP4+ analyses, 1 was elucidated as (6S,9R)-3-oxo-α-ionol-β-D-glucopyranoside and was named boehmegaside A.
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Fig. 3 HMBC, COSY interactions of 1 (A). Experimental and calculated ECD spectra of compound 1 in MeOH, σ 0.3 eV, and UV shift = +5 nm (B). |
In addition, the 9R configuration was strengthened by the agreement of the chemical shift of C-9 (δC 78.1) with that of (9R)-3-oxo-ionol (δC 77.0), but being quite different from that of (9S)-3-oxo-ionol (δC 74.7) in CD3OD.10,11 Meanwhile, the 6S configuration was confirmed by the fact that the ECD spectrum of 1 [Δε: −28.90 (245 nm), + 1.15 (319 nm)] showed almost the same tendency to the ECD data of (6S,9R)-3-oxo-α-ionol-9-O-β-D-glucopyranosyl (1 → 2)-β-D-glucopyranoside [Δε: −54.11 (243 nm), + 2.34 (310 nm)],12 but opposite to that of the C-6R absolute configuration (the positive Cotton effects at 249 nm of two published isomers (6R,9R) and (6R,9S) 3-oxo-α-ionol-β-D-glucopyranoside).10 Therefore, based on the above argument, the absolute configuration of compound 1 was completely suitable with the above computationally calculated results.
Compound 2 was obtained as a colorless gum, with the molecular formula of C19H30O8 as deduced from the HR-ESI-MS peak at m/z 409.1823 [M + Na]+ (calcd for C19H30O8Na 409.1838). The 1H and 13C-NMR spectra were almost identical to those of 1, showing a typical pattern of megastigmane glycoside, with the only difference being in their chemical shifts observed at C-6 (due to the change from a tertiary carbon to quaternary carbon). The complete planar structure of 2 was determined by 2D NMR analysis (1H–1H COSY, HSQC, and HMBC), and was namely roseoside. The 6S,9R configuration of 2 was determined based on the chemical shifts of the carbon at positions C-7, C-8, and C-9 compared to the published spectroscopic data of the four diastereomers of roseoside in the same ref. 13 Based on the above results, 2 was identified as (6S,9R)-roseoside (Fig. 4).
Compound 3 was isolated as a colorless gum. It had the same molecular formula (C19H30O8) as 2, as deduced by the HR-ESI-MS peak at m/z 409.18714 [M + Na]+(calcd. for C19H30O8Na 409.1838). The 1H and 13C-NMR spectra were almost identical to those of 2, with the only differences being in their chemical shifts observed at C-6 and C-8 due to changes in the double bond and OH group positions. The complete planar structure of 3, was determined as 8 hydroxymegastigmane-4,6-dien-3-on-9-O-β-D-glucopyranoside. The relative configuration of 3 was proposed from a NOESY experiment and by comparison of the observed and reported NMR data.14 In the NOESY spectrum, the correlations between H-9 and H-1′ were tentatively assigned to an α-orientation. In addition, the NOESY spectrum also showed interactions between protons H-8 and H-12, H-8 and H-7, H-8 and H-9; consequently, H-8 was in the same direction as H-7, H-9 and H-12 (α orientation). Accordingly, the structure of 3 was proposed as 8S,9R-megastigman-3-one-4,6-diene-8,9-diol-9-O-β-D-glucopyranoside, also named as akequintoside D (Fig. 4).
Compound 4 was a colorless gum. Its identified molecular formula was C19H32O8 based on the HR-ESI-MS peak at m/z 411.1966 [M + Na]+ (calcd for C19H32O8Na 411.1989) in combination with the NMR data. The 1H and 13C-NMR spectra of 4 displayed typical signals for a megastigmane-type skeleton. The coupling constant (J = 7.8 Hz) of the anomeric proton signal was assigned as β-D-glucopyranose. The position of the sugar unit was confirmed by the HMBC correlation observed for the anomeric proton (H-1) with C-3 (δC 73.0). The complete planar structure of 4 was determined by 2D-NMR analysis (1H–1H COSY, HSQC, and HMBC). In addition, as with the current data, to meet the requirements for the molecular formula and unsaturation, compound 4, needed to contain an epoxy ring, which was suggested to be formed between C-5 (δC 67.7) and C-6 (δC 71.4). Although the stereochemistry at C-5, C-6, and C-9 could not be established from the available data, the similarity between all the NMR data suggested that 4 may have the same stereoisomeric structure as alangionoside E15 (Fig. 4).
Compound 5 was also a colorless gum. It had the same molecular formula as 4 C19H32O8 (M = 388.2019, with unsaturation Δ = 4) according to the Q-Tof-MS spectrum m/z 411.2014 [M + Na]+. Its NMR spectrum was quite similar to that of compound 4, with the only difference being that part of the sugar was linked to the main frame at C-9. With these spectrum data, 5 was identified as 3-hydroxy-5,6-epoxy-β-ionol-9-O-β-D-glucopyranoside. This planar structure was similar to the structure of the two announced compounds staphylionoside H and phlomuroside (two diastereomers only different in the C-9 configuration). When comparing the spectroscopic data of 5 with the spectroscopic data of these two substances, it was found that the chemical shift and peak splitting of 5 were highly similar to those of compound staphylionoside H (9S configuration).16 In addition, the identification with the 9S configuration was strengthened by the agreement of the chemical shift of C-9 with that of (9S)-3-oxo-ionol (δC 74.7), while being quite different from that of (9R)-3-oxo-ionol (δC 77).10,11 From the above arguments, it could be concluded that 5 was staphylionoside H (Fig. 4).
Compound 6 was also a colorless gum. It had the same molecular formula as C19H30O8 and was deduced by the HR-ESI-MS peak at m/z 409.1876 [M + Na]+(calcd for C19H30O8Na 409.1838). The 1H and 13C-NMR spectra were almost identical to those of 4, but with differences in their chemical shifts observed at C-9 due to the change from an oxycarbon to ketone signal. Based on the spectral characteristics and comparison with the prior observed NMR data in the literature,17 the structure of 6 was deduced as 5,6-epoxy-β-ionone-3-O-β-D-glucopyranoside or icariside B2 (Fig. 4).
Three known megastigmane compounds, each containing an epoxy ring, were identified as crotalionoside C (compound 7),18 officinoside B (compound 8),19 and 5,8-epoxymegastigmane-6-en-3-on 9-O-β-D glucopyranoside (compound 9)20 by comparison of their spectroscopic data (HR-MS and NMR) with those reported in the literature for the aforementioned compounds (Fig. 4).
Among the 5 tested compounds (10 μg mL−1), 3 megastigmane compounds, namely akequintoside D (3), (6S,9R)-roseoside (2), and icariside B2 (6), significantly inhibited LPS-induced IL-1β production, with IL-1β concentrations of 72.09, 71.36, and 63.47 pg mL−1 compared to the positive control group treated with 5 ng mL−1 LPS (465.84 pg mL−1). Additionally, the megastigmane compounds 3 and 6 demonstrated significant stimulation of IL-10 production, with IL-10 concentrations of 260.45 and 267.49 pg mL−1, respectively, compared to the positive control group treated with 5 ng mL−1 LPS (105.42 pg mL−1). These results highlight the potential of the isolated compounds, especially the remaining megastigmane compounds, which are considered promising candidates for further exploration for developing immunomodulatory agents.
Lipopolysaccharide (LPS) is a significant component of the outer membrane of Gram-negative bacteria and stimulates macrophages to produce inflammatory factors and nitric oxide (NO), initiating an inflammatory response.24 TLR4, the primary LPS receptor on the surface of monocytes, macrophages, and dendritic cells, facilitates innate immunity.25 It mediates the phagocytic inflammatory response to various microorganisms by activating the NF-κB signaling pathway.26 The recognition of LPS requires the formation of the TLR4-MD-2 complex, which then binds to LPS to mediate signal transduction and stimulate macrophages to produce an inflammatory response.27 Therefore, we speculated that the studied compounds could bind to TLR4-MD-2 instead of LPS, thereby blocking the TLR4-MD-2-mediated NF-κB/MAPK signaling pathway and exerting their immunomodulatory effects (Fig. 5).
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Fig. 5 Mechanism of action of the inhibiting compounds on TLR4, and the diagram of its anti-inflammatory pathway.28 |
Numerous compounds have been reported to interact with TLR4-MD-2, exerting either pro-inflammatory or anti-inflammatory effects.29,30 Thus, molecular docking simulation was performed to clarify the interaction between the studied compounds and TLR4-MD-2. AutoDock4 is among the most popular docking software, with over 6000 citations since 2010.31–33 It is a valuable tool for rapidly predicting the binding affinity of ligands to specific proteins or enzyme targets. Here, AutoDock4 was utilized for searching for the potential mechanisms of the bioactive compounds. The docking scores are shown in Table 5.
Compounds | Docking score (kcal mol−1) | No. of H-bonds | Interacting residues |
---|---|---|---|
Akequintoside D (compound 3) | −11.87 | 3 | Val93, Leu94, Tyr102, Arg337 |
(6S,9R)-roseoside (compound 2) | −10.40 | 3 | Val93, Arg337, Gln339 |
Icariside B2 (compound 6) | −10.75 | 3 | Lys91, Glu92, Val93, Arg337 |
Boehmegaside A (compound 1) | −7.39 | 2 | Val93, His96, Met358, Asn359, Gly361, Asn381 |
Hydroxytyrosol 4-β-D-glucoside | −7.90 | 2 | Val93, Arg337, Met358, Gly361, Arg380 |
According to the ranking criteria of AutoDock4, a more negative docking energy suggests a higher binding affinity of a compound towards the targeted receptor.34 The results obtained in the docking study indicated that akequintoside D (3), (6S,9R)-roseoside (2), and icariside B2 (6) could be assumed as “HITs” based on their docking scores. Considering the compounds suggested to be bioactive in the experiments, the obtained results showed a high correlation. A stereoview of the binding mode of the potential ligands is depicted in Fig. 6.
The active site of TLR4-MD-2 is a pocket mainly composed of TLR4 domain residues and MD-2 domain residues, which have been reported to include the following essential residues: Lys89, Arg90, Val93, Tyr102, Lys125, Lys360 and Arg337.35 It was observed that the potential compounds did not bind to the hydrophobic pocket in MD-2, which is where LPS bridges TLR4 and MD-2 to initiate signal transduction. Instead, they bound to a cleft primarily formed by TLR4. Fig. 6A reveals that residues Arg337, Gln339, Met358, Lys360, and Arg380 from TLR4, along with His96 from MD-2, created a smooth surface. The planar structures of akequintoside D (3), (6S,9R)-roseoside (2) and andicariside B2 (6) fit this shape perfectly and adhered closely to the surface.
Of all the docked results, compound 3 exhibited the highest binding affinity (−11.87 kcal mol−1). Analysis of the binding orientation of 3 showed that Val93 and Leu94 were the key residues that participated in hydrophobic interactions. The interaction was further stabilized through H-bonds with Tyr102 and Arg337. In the docking pose of 2, the hydrophobic pockets formed with this ligand involved residue Val93. The interaction was further strengthened through three conventional hydrogen bonds with Arg337 and Gln339. An array of H-bond interactions was observed to be contributed by Glu92, Val93 and Arg337 for binding with compound 6. Also, the hydrophobic bond was constituted from the interaction with Lys91.
Megastigmane belongs to the family of terpene compounds with a 13-carbon aglycon framework, including a hydroxyl group and chiral carbon positions mainly at C-6 and C-9. In addition, oxidation reactions at the double bonds or ring closures and glycosylation create structural diversity in the megastigmane group. A review publication in 2017 showed that there were about 230 megastigmane compounds that had been isolated and identified in nature from plant sources, such as Vitaceae, Fabaceae, Apocynaceae, and Berberidaceae.36 According to the reference article, the publications related to megastigmane compounds to date have been mainly related to their chemical structures, and rarely associated with their biological effects. This study is the first to report the isolation of megastigman terpenes, specifically a new megastigmane, and eight megastigmane compounds in the genus Boehmeria. For the new megastigmane (6S,9R)-3-oxo-α-ionol-β-D-glucopyranoside, named as boehmegaside A, three isomers of this structural framework have been reported previously as isomers (6R,9R), (6R,9S), and (6S,9S) of 3-oxo-α-ionol-β-D-glucopyranoside.10,37 Also, some epoxy ring structures were isolated, including alangionoside E, staphylionoside H, officinoside B, and akequintoside D, which are considered rare structures. To the best of our knowledge, alangionoside E was only published in Alangium premnifolium (1995)15 and officinoside B was only published in Egyptian Calendula officinalis (2001),19 with no biological testing reports yet. This is the second time staphylionoside H has been published in nature.
Besides, previous publications showed that the isolated megastigmane compound akequintoside D was also only published in Akebia quinata species (2015), showing a moderate IL-6 production inhibitory activity on TNF-α-stimulated MG-63 cells (40% inhibition of the IL-6 concentration) compared to the positive control group TNF-α 10 ng mL−1 (p < 0.001).38 Meanwhile, (6S,9R)-roseoside was shown to enhance insulin release from the INS-1 cell line in in vitro testing.39 Studies have also synthesized isomers of roseoside13 and demonstrated that 4 stereoisomers inhibited leukotriene release from cultured mast cells derived from mouse bone marrow.40 Another publication showed that (6S,9R)-roseoside had anti-inflammatory effects on a model for inhibiting NO release on the RAW 264.7 cell line stimulated with LPS, with IC50 = 7.31 μM (compared to the control hydrocortisone with IC50 = 64.34 μM). Icariside B2 has shown anti-inflammatory effects in in vitro tests (on a model using BV2 glial cells stimulated with LPS) and in vivo (at a dose of 50 mg kg−1, it showed anti-edema effects in mice in a model of acute inflammation in rat paws with carrageenan) by various proven mechanisms, such as inhibiting the generation of NO and prostaglandin E2 by reducing the expression of inducible NO synthase and cyclooxygenase 2, reducing the expression of the cytokines that cause inflammation (TNF-α, IL-6, and IL-1β) and also hinder phosphorylation of the inhibitory protein κBα, and inhibiting COX-2 enzyme with an IC50 value of 7.80 ± 0.26 μM. From the above review, it is evident that the results of immunomodulatory activity studies in in vitro and in silico tests of akequintoside D (3), (6S,9R)-roseoside (2), and icariside B2 (6) were also consistent with the published biological results. These findings demonstrate the potential of B. nivea leaf extracts and isolated compounds, particularly megastigmane, which are considered promising structures for further in-depth exploration. Further studies into the mechanism of the immunomodulatory and other biological activities of B. nivea are warranted.
Sample: The fraction extracts and the isolated compounds were dissolved in DMSO (20 and 10 μg mL−1) by vortexing at 300–500 rpm.
Positive control group: Cells were incubated with the inflammatory inducer LPS 5 ng mL−1 for 24 h.
Control sample: Cells were incubated with 1% DMSO in the medium.
The n-butanol extract (70 g) was subjected to column chromatography (CC) with silica gel and eluted with a gradient mixture of CHCl3–MeOH–H2O solvent (9:
1
:
1–6
:
4
:
1, v/v) to obtain 19 fractions (Bu1–19). Two fractions, Bu9 (2.3 g) and Bu10 (4.5 g), were CC separated with a saturated EtOAc–MeOH solvent system (95
:
5–85
:
15, v/v) to obtain 10 fractions (Bu9.1–9.10) and 15 fractions (Bu10.1–10.15), respectively. Fraction Bu9.6 (445 mg) was separated by silica gel CC with a mixture of CHCl3–MeOH–(Me)2CO–H2O solvent (7
:
3
:
1
:
1, v/v) to obtain compound 3 (60.9 mg). Fraction Bu10.7 (938 mg) was separated by Sephadex LH-20 with 100% MeOH to obtain 6 fractions (Bu10.7.1–10.7.6). Compounds 2 (78.8 mg), 4 (69.0 mg), 5 (9.3 mg), and 7 (9.4 mg) were purified from fraction Bu10.7.2 (690 mg) using silica gel RP-C18 CC eluted with H2O–MeOH (4
:
1–3
:
2, v/v), respectively. Fraction Bu10.10 (189 mg) was isolated by silica gel RP-C18 CC eluted with a gradient mixture of H2O–MeOH (3
:
1–3
:
2, v/v) to obtain 9 (3.6 mg) and 8 (3.8 mg). Fraction Bu14 (2.9 g) was applied to Sephadex LH-20 with 100% MeOH to obtain 6 fractions (Bu14.1–14.6). Fraction Bu14.2 (1.58 g) was isolated by silica gel CC with a mixture of EtOAc–MeOH–H2O solvent (17
:
3
:
4, v/v) to obtain 14 fractions (Bu14.2.1–14.2.14). Fraction Bu14.2.6 (210 mg) was separated by silica gel RP-C18 CC eluted with H2O–MeOH (7
:
3–3
:
2, v/v) to obtain compound 6 (17.2 mg) and compound 1 (8.6 mg).
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra06545j |
This journal is © The Royal Society of Chemistry 2025 |