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Abstract

Objective: This study aimed to isolate and elucidate the structure of water-soluble constituents from Nelumbo nucifera Gaertn. leaves and evaluate their anti-aging effect. Methods: The natural products were isolated from the 60% aqueous acetone extract of the leaves of N. nucifera by column chromatography on MCI gel CHP 20P, YMC-Gel ODS-AQ-HG, and TSK gel Toyopearl HW-40F. Their structures were elucidated using high-resolution-electrospray ionization-mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, and electronic circular dichroism spectroscopy. The anti-aging activity was evaluated in the Caenorhabditis elegans model. Results/Conclusion: One novel flavonoid nuciferanoid (1), together with eleven known compounds, were isolated from the extract of the leaves of N. nucifera. Among them, compound 1 could be a starting point for further development of anti-aging drugs.

Keywords

water-soluble constituents structure determination flavonoids anti-aging activity

Introduction

Nelumbo nucifera Gaertn. (N. nucifera) is a perennial, rhizomatous, and aquatic plant, which was distributed throughout Asia and Australia. Among the different organs of N. nucifera, the leaves and the flowers are commonly used in traditional medicine, while the roots and the seeds provide important nutritional foods.¹ Currently, N. nucifera has acquired enormous significance because of its high content of bioactive secondary metabolites, such as flavonoids and anthocyanins in the flower²; alkaloids, flavonoids, steroids, and polysaccharides in the plumule³; polyphenols, fatty acids, procyanidins, and polysaccharides in the rhizome and seeds^4,5; and phenolic acids, flavonoids, alkaloids, polysaccharides, and terpenoids in the leaves.⁶ In ancient China, the leaves of N. nucifera were known for diuretic and astringent properties and were used to treat fever, sweating, and strangury and as a styptic.⁷ As early as 1991, the leaves of N. nucifera were included in the second list of “food and drugs” by the Ministry of Health of China. Modern medical studies have revealed the extensive pharmacological activities of the leaves, including antioxidant, antimicrobial, anti-obesity, and anti-cardiovascular disease activities. As a part of our study on the hydrophilic bioactive constituents of Chinese medicines,^8,9 we systematically studied the water-soluble constituents of N. nucifera. The water-soluble fraction from 60% aqueous acetone extract of the leaves of N. nucifera was subjected to column chromatography on MCI gel CHP 20P, YMC-Gel ODS-AQ-HG, and TSK gel Toyopearl HW-40F to afford one novel flavonoid (1), together with eleven known compounds (Figure 1) were isolated from N. nucifera. The Caenorhabditis elegans (C. elegans.) model was employed to preliminarily explore the potential of compounds 1, 2, 4, 8, 9, 11, and 12 in anti-aging effects.

Figure 1.

Chemical structures of compounds 1–12.

Results and Discussion

Compound 1 was obtained as a white powder. The electrospray ionization-mass spectrometry (ESI-MS) showed quasi-molecular ion peaks at m/z 459 [M + Na]⁺ and 435 [M - H]⁺, respectively, indicating a molecular weight of 436. According to the high- resolution-electrospray ionization-mass spectrometry (HR-ESI-MS), ¹H, and ¹³C nuclear magnetic resonance (NMR) spectrum data, the molecular formula of 1 was determined to be C₂₀H₂₀O₁₁.

In the ¹H NMR spectrum (Table 1), the signals at δ 6.76 (1H, br d, J = 8.2 Hz, H-6′) and δ 6.90 (1H, d, J = 8.2 Hz, H-5′) and δ 6.88 (1H, d, J = 1.6 Hz, H-2′) defined a 13,4-trisubstituted aromatic ring, which was further confirmed in the ¹³C NMR spectrum by three quaternary aromatic carbons at δ 130.83 (C-1′), 144.12 (C-3′, C-4′) and three methine peaks at δ 114.09 (C-2′), 116.24 (C-5′) and 118.80 (C-6′). The signals at δ 6.15 (1H, s) in the low field of the ¹H NMR spectrum and five quaternary aromatic carbons and one methine peak in the ¹³C NMR spectrum indicated the existence of another aromatic ring. In the high field of ¹H NMR spectrum, one methylene group δ 2.64 (1H, dd, J = 16.4, 4.6 Hz, H-4a), 2.58 (1H, dd, J = 16.4, 6.1 Hz, H-4b), and two methine groups δ 5.05 (1H, d, J = 5.7 Hz, H-2) and 4.21 (1H, m, H-3) indicated the following CH(2)-CH(3)-CH₂(4) fragment by the analysis of ¹H-¹H correlation spectroscopy (¹H-¹H COSY) spectrum and heteronuclear multiple bond coherence (HMBC) spectrum (Figure 2). Further analysis of ¹H-¹H COSY, heteronuclear single quantum coherence and HMBC spectra established the skeleton of catechin, in which HMBC long-range correlations were observed not only between H-2 (δ 5.05), H-3 (δ 4.21), and C-1′ (δ 130.83), but also between H-4a (δ 2.64), H-4b (δ 2.58), and C-5 (δ 158.95), C-9 (δ 152.43), and C-10 (δ 102.37).

Figure 2.

Key COSY, HMBC, and NOESY correlations for compound 1. HMBC, heteronuclear multiple bond coherence; NOESY, nuclear Overhauser effect spectroscopy.

Table 1.

NMR Data for Compound 1.

Position	¹H NMR^a	¹³C NMR^b	HMBC
1
2	5.05 d (5.7)	80.09 d	H-4a/b, H-2′, H-6′
3	4.21 m	66.32 d	H-2, H-4a/b
4a	2.64 dd (16.4, 4.6)	25.05 t	H-2, H-3
4b	2.58 dd (16.4, 6.1)
5		158.95 s	H-4a/b, H-6
6	6.15 s	91.17 d
7		156.93 s	H-6
8		100.77 s	H-6
9		152.43 s	H-2, H-4a/b
10		102.37 s	H-3, H-4a/b, H-6
1′		130.83 s	H-2, H-3, H-5′
2′	6.88 d (1.6)	114.09 d	H-2, H-6′
3′		144.12 s	H-5′
4′		144.12 s	H-2′, H-5′, H-6′
5′	6.90 d (8.2)	116.24 d
6′	6.76 br d (8.2)	118.80 d	H-2, H-2′
1″		110.80 s	H-2″, H-3″
2″	4.51 d (5.4)	83.32 d	H-3″, H-4′a/b
3″	4.22 m	69.39 d	H-2″, H-4′a/b
4″a	3.86 dd (11.8, 4.3)	62.11 t	H-2′, H-3″
4″b	3.75 dd (11.8, 6.6)
5″		174.20 s	H-2″

600 MHz, D₂O; chemical shifts in ppm; coupling constant (J) in Hz.

150 MHz, D₂O; multiplicity was established from the DEPT data.

HMBC, heteronuclear multiple bond coherence; NMR, nuclear magnetic resonance.

In addition, the low field of the ¹³C NMR spectrum still showed the presence of one carbonyl carbon, one quarternary carbon, two methines, and one methylene group. The analysis of ¹H-¹H COSY showed another remaining -CH(O)-CH(O)-CH₂-O- fragment. The HMBC long-range correlations between H-2″ (δ 4.51) and C-1″ (δ 110.80), C-5″ (δ 174.20) confirmed the presence of CH(2″)-C(1″)-COOH(5″) fragment. The methylene group δ 3.86 (1H, dd, J = 11.8, 4.3 Hz, H-4″a), 3.75 (1H, dd, J = 11.8, 6.6 Hz, H-4″b) in ¹H NMR and C-4″ (δ 62.11) in ¹³C NMR suggested the formation of ether ring between C-4″ and C-1″, instead of forming lactone between C-4″ and C-5″. The HMBC long-range correlations between H-6 (δ 6.15) and C-5 (δ 158.95), C-7 (δ 156.3), C-8 (δ 100.77), C-10 (δ 102.37) confirmed the site of C-8 is a quaternary carbon. Therefore, the five-membered heterocyclic ring should be directly connected to the C8.

The relative configuration of epicatechin was deduced from the results of a nuclear Overhauser effect spectroscopy (NOESY) spectrum (Figure S7), in which NOESY signals were observed between H-2 (δ 5.05) and H-3 (δ 4.21), H-2′ (δ 6.88), H-6′ (δ 6.76). Similarly, NOESY signals were observed between H-3″and H-2″ and H-4″a/b, indicating that H-3″ and H-2″ are on the same side of the five-membered heterocyclic ring. The chemical shift of H-2″ is located in a lower field relative to H-3″, which may be caused by intramolecular hydrogen bonding of -COOH and -OH are on the same side. The absolute configuration is determined as (−)-2R, 3R, 1″R, 2″S, 3″S by comparing the calculated electronic circular dichroism (ECD) spectroscopy with the measured circular dichroism (CD) spectroscopy results (Figure S8). Consequently, the chemical structure of 1 was determined as shown in Figure 1, named nuciferanoid.

The eleven known compounds were identified as Quercetin 3-O-β-D-Glucuronide (2),¹⁰ l -Tryptophan (3),¹¹ (+)-Catechin (4),¹² (+)-Gallocatechin (5),¹³ Procyanidin B1 (6),¹⁴ 3,4-Dihydroxyphenylethanol (7),¹⁵ (-)-Epicatechin (8),¹³ Isoquercitrin (9),¹⁶ (+)-Gallocatechin-(4α-8)-(+)-Catechin (10),¹⁷ Procyanidin B3 (11),¹⁴ Astragalin (12),¹⁸ by comparison of the spectral data with those reported in the literature.

The C. elegans. model was employed to explore the potential of compounds 1, 2, 4, 8, 9, 11, and 12 in anti-aging effects. Our results showed that compound 1 increased the mean lifespan relative to a blank control (Figure 3, Table 2, and Table S1).

Figure 3.

Caenorhabditis elegans survival curves of selected compounds (100 μg/ml).

Table 2.

The Mean Lifespan After Treatment of Selected Compounds.

Compound	Mean lifespan / d	Increase in mean lifespan / %
Control	23.90	-
1	24.44	2.26
2	22.94	-
4	23.66	-
8	23.76	-
9	22.19	-
11	22.33	-
12	23.16	-

In summary, compound 1 is a newly discovered compound from N. nucifera. Yosuke Matsuo et al¹⁹ reported that the five-membered heterocyclic ring of compound 1 could be formed through oxidation. We then employed the C. elegans model using the method reported^20,21 to conduct a preliminary screening of the anti-aging effects of compounds 1, 2, 4, 8, 9, 11, and 12. Our findings suggest that compound 1 could increase the mean lifespan of C. elegans by 2.26%. One limitation of the findings was that we only used a preliminary screening model to assess the anti-aging activity of the compounds. Further pharmacodynamic and pharmacological research should be undertaken to explore the anti-aging activity and mechanism of compound 1.

Conclusion

One novel compound 1 and eleven known compounds 2–12 were isolated from N. nucifera leaves. HR-ESI-MS, 1D, and 2D NMR were utilized to elucidate the structure of compound 1. The absolute configurations of compound 1 were determined by comparing the calculated ECD with the measured CD results. Furthermore, compound 1 increased the mean lifespan of C. elegans and showed potential for further development.

Experimental

General Experimental Procedures

Optical rotation data were obtained on a Perkin–Elmer 241 automatic digital polarimeter. ¹H, ¹³C NMR, ¹H-¹H COSY, HSQC and HMBC spectra were recorded on a Bruker DRX-600 spectrometer (¹H 600 MHz and ¹³C 150 MHz). The carbon multiplicities were obtained by the distortionless enhancement by polarization transfer experiment. ESI-MS data was measured on an ESI-MS HP5989A mass spectrometer. TSK gel Toyopearl HW-40F (30-60 μm, Toso Co., Ltd), MCI gel CHP 20P (75-150 μm, Mitsubishi Chemical Industries Co., Ltd) and YMC-Gel ODS-AQ-HG (42-105 μm, YMC Group) were used for column chromatography. Thin layer chromatography was performed using a Kiesel gel 60 F₂₅₄ pre-coated plate. (0.2 mm, Merck). HR-ESI-MS data were measured on AB Qtrap5500.

Plant Material

The 5 kg fresh leaves of N. nucifera were collected in Chongming Island, Shanghai, People's Republic of China in autumn 2021, and certified by Professor T. Guo. A voucher specimen has been deposited in the School of Pharmacy, Fudan University.

Extraction and Isolation

Fresh leaves of N. nucifera (5 kg) were subjected to two extractions using 60% aqueous acetone at room temperature (10 L). The solvent was evaporated under reduced pressure to 1 L and subsequently filtered through cotton and celite. The resulting filtrate was concentrated in vacuo, yielding a black oil, of approximately 100 mL. Water was then supplemented to 300 mL and subjected to chromatography using MCI gel CHP 20P (8 × 60 cm) eluted with methanol (MeOH)/H₂O [300 mL H₂O → 10% MeOH → 30% MeOH → 50% MeOH → 70% MeOH → 100% MeOH] and acetone/H₂O [300 mL 60% acetone → 100% acetone] to obtain fraction 1 and fraction 2. Fraction 1 was further chromatographed on Toyopearl HW-40F (6 × 60 cm), resulting in four sub-fractions 1A-1D [400 mL H₂O → 300 mL 50% MeOH → 300 mL 100% MeOH → 400 mL 60% acetone]. Fraction 1C was then purified using YMC-Gel ODS-AQ-HG (4 × 30 cm) with elution of H₂O → 70% MeOH and middle-pressure liquid chromatography (C₁₈-MPLC) with elution of [H₂O → 50% MeOH], yielding compound 1 (22 mg). Fraction 1A, 1B, and 1D were purified using YMC-Gel ODS-AQ-HG (4 × 30 cm) with elution of H₂O → 70% MeOH, middle-pressure liquid chromatography (C₁₈-MPLC) with elution of H₂O → 50% MeOH, and Toyopearl HW-40F (6 × 60 cm) with elution of [400 mL H₂O → 300 mL 50% MeOH → 300 mL 80% MeOH → 300 mL 100% MeOH]. This process resulted in the isolation of seven chemical constituents: 2 (2.26 g), 3 (22 mg), 4 (19.8 mg), 5 (137.4 mg), 6 (276.4 mg), 7 (8 mg), 8 (47 mg), and 10 (18.9 mg). Fraction 2 was chromatographed on Toyopearl HW-40F (6 × 60 cm) with elution of [400 mL H₂O → 300 mL 50% MeOH → 300 mL 100% MeOH → 400 mL 60% acetone], and further purified using YMC-Gel ODS-AQ-HG (4 × 30 cm) with elution of [H₂O → 70% MeOH], resulting in the isolation of compounds 9 (18 mg), 11 (9.3 mg), and 12 (18 mg). The eluent was collected in test tubes and monitored using thin layer chromatography developed in benzene/formic acid/ethyl acetate (3:2:5).

Nuciferanoid (1)

White amorphous powder; [α]²⁵ _D −34.9 (c 0.10, EtOH); ESI-MS m/z 435 [M - H]⁻; HR-ESI-MS m/z 436.1005 [M]⁺ (calcd for C₂₀H₂₀O₁₁, 436.1011); ¹H and ¹³C NMR, see Table 1.

ECD Calculation

The random conformational searches were performed by Spartan’14 using Molecular Merck force field. The conformers with a Boltzmann population of over 5% were subsequently optimized by using Gaussion09 software at the B3LYP/6-31G (d) level in MeOH using the IEFPCM model. The optimized stable conformers were selected for further ECD calculations at the B3LYP/6-311G (d, p) level in MeOH. The overall ECD data were weighted by Boltzmann distribution, and the ECD curves and enantiomeric ECD curves were produced by SpecDis 1.70.1 software with a half-bandwidth of 0.3 eV, according to the Boltzmann-calculated contribution of each conformer after UV correction.

Anti-Aging Tests

Compounds 1, 2, 4, 8, 9, 11, and 12 extracted from N. nucifera were screened for their anti-aging potential by extending the lifespan of C. elegans, which was acquired from the Shanghai Research Center of the Southern model organisms in China, following the reported protocol.^20,21

Statistical Analysis

Survival curves were plotted and analyzed using GraphPad Prism 9.5.1.

The formula for calculating the mean lifespan of C. elegans is:

\begin{aligned} Mean lifespan = \sum n * d / \sum n, ‘ ‘ n " denotes the number of deaths, and ‘ ‘ d " represents the time of death . \end{aligned}

Supplemental Material

sj-docx-1-npx-10.1177_1934578X241271623 - Supplemental material for Research on Water-Soluble Constituents and Anti-Aging Effect of the Leaves of Nelumbo nucifera Gaertn

Supplemental material, sj-docx-1-npx-10.1177_1934578X241271623 for Research on Water-Soluble Constituents and Anti-Aging Effect of the Leaves of Nelumbo nucifera Gaertn by Rong-Qian Cheng, Heyanhao Zhang and Jun Chang in Natural Product Communications

Footnotes

Acknowledgements

This project was supported financially by Science and Technology Commission of Shanghai Municipality (21S11907400).

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Science and Technology Commission of Shanghai Municipality, (grant number 21S11907400).

ORCID iD

Jun Chang

Supplemental Material

Supplemental material for this article is available online.

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