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Hibiscus sabdariffa L. extract prolongs lifespan and protects against amyloid-β toxicity in Caenorhabditis elegans: involvement of the FoxO and Nrf2 orthologues DAF-16 and SKN-1

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Abstract

Purpose

Hibiscus sabdariffa L. is commonly used as an ingredient for herbal teas and food supplements. Several studies demonstrated the beneficial effects of Hibiscus sabdariffa L. extracts (HSE); however, the bioactive components and their mode of action still remain unclear. Caenorhabditis elegans (C. elegans) was used to study health-related effects and the underlying molecular mechanisms of HSE in this model organism as well as effects of hydroxycitric acid (HCA), a main compound of HSE, and its structural analogue isocitric acid (ICA).

Methods

Survival and locomotion were detected by touch-provoked movement. Thermotolerance was analysed using the nucleic acid stain SYTOX green, and intracellular ROS accumulation was measured via oxidation of H2DCF. Localisation of the transcription factors DAF-16 and SKN-1 was analysed in transgenic strains (DAF-16::GFP, SKN-1::GFP). The involvement of DAF-16 and SKN-1 was further investigated using loss-of-function strains as well as gene silencing by feeding RNAi-inducing bacteria. Protection against amyloid-β toxicity was analysed using a transgenic strain with an inducible expression of human amyloid-β peptides in body wall muscle cells (paralysis assay).

Results

HSE treatment resulted in a prominent extension of lifespan (up to 24%) and a reduction of the age-dependent decline in locomotion. HCA, a main compound of HSE increased lifespan too, but to a lesser extent (6%) while ICA was not effective. HSE and HCA did not modulate resistance against thermal stress conditions and did not exert antioxidative effects: HSE rather increased intracellular ROS levels, suggesting a pro-oxidative effect of the extract in vivo. HSE and HCA increased the nuclear localisation of the pivotal transcription factors DAF-16 and SKN-1 indicating an activation of these factors. Consistent with this result, lifespan prolongation by HSE was dependent on both transcription factors. In addition to the positive effect on lifespan, HSE treatment also elicited a (strong) protection against amyloid-ß induced toxicity in C. elegans in a DAF-16- and SKN-1-dependent manner.

Conclusion

Our results demonstrate that HSE increases lifespan and protects against amyloid-β toxicity in the model organism C. elegans. These effects were mediated, at least in parts via modulation of pathways leading to activation/nuclear localisation of DAF-16 and SKN-1. Since HCA, a main component of HSE causes only minor effects, additional bioactive compounds like flavonoids or anthocyanins as well as synergistic effects of these compounds should be investigated.

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Abbreviations

daf-16 :

Abnormal dauer formation-16 (FoxO orthologue)

DCF:

2′,7′-Dichlorofluorescein

FoxO:

Forkhead box O

GFP:

Green fluorescent protein

HCA:

Hydroxycitric acid

HSE:

Hibiscus sabdariffa L. extract

ICA:

Isocitric acid

lof:

Loss of function

NGM:

Nematode growth medium

ROS:

Reactive oxygen species

skn-1 :

Skinhead-1 (Nrf2-orthologue)

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Acknowledgements

The nematode strains used in this work were provided by the Caenorhabditis Genetics Centre, which is funded by the NIH National Centre for Research Resources (NCRR). This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. We thank Dr. Sebastian Honnen for helpful discussions.

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Author notes

  1. Karoline Koch and Nora Weldle contributed equally.

Authors and Affiliations

  1. Institute of Agricultural and Nutritional Sciences, Biofunctionality of Secondary Plant Compounds, Martin-Luther-University Halle-Wittenberg, Weinbergweg 22, 06120, Halle/Saale, Germany

    Karoline Koch, Nora Weldle, Sabrina Baier, Christian Büchter & Wim Wätjen

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  1. Karoline Koch
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Contributions

NW, KK: performed experiments, KK, CB: supervision of experiments, WW: coordination of experiments, WW, KK: preparation of manuscript.

Corresponding author

Correspondence to Wim Wätjen.

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On behalf of all authors, the corresponding author states that there is no conflict of interest.

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Supplementary material 1 (TIF 898 KB)

Supplemental Fig. 1 a

HSE does not influence pharyngeal pumping of C. elegans (left). 10 nematodes per group were incubated with HSE, HCA, ICA or the respective control for 24 h and pharyngeal pumping of every animal was counted for 15 s and repeated three times, n = 3, one-way ANOVA with Dunnett’s multiple comparisons test, *p ≤ 0,05 vs. control; HSE does not influence bacterial growth of E. coli OP50-1 (right). The OD600 of a growing OP50-1 E. coli culture was photometrically measured at different time points. A freshly prepared OP50-1 E. coli solution with an OD600 of 0.2 was mixed with the HSE stock solution to a final concentration of 1 mg/ml HSE or the equivalent amount H2O and 50 µg/ml streptomycin. Bacteria were allowed to grow for 6 h at 37°C while shaking and an aliquot of the mixture was measured every hour (Synergy MX, BioTek Instruments, Inc.). Simultaneously, a mixture with heat-inactivated OP50-1 E. coli (37°C for 60 min) was measured, since we observed a colour change of the HSE after 1 h of incubation. n = 4 for OP50-1 E. coli, n = 1 for inactivated OP50-1 E. coli. b HSE does not influence growth of C. elegans. In order to investigate if HSE affects larvae growth or c adult body size, body area (left) and length (right) of wild-type nematodes (N2) was measured starting from b L1 or c L4 stage on. Synchronisation was performed according to Stiernagle [19] to obtain L4 larvae or arrested L1 larvae. Animals were incubated in liquid media with different HSE concentrations at 20°C. Images of 10 nematodes were taken daily with a camera (Motic Images Plus 2.0) connected to a stereo microscope (Stemi 2000, Zeiss) and the area and length of the body were measured with ImageJ. Adult nematodes were transferred daily to new incubation media to prevent overcrowding. n = 3 (20 individuals per group), one-way ANOVA with Dunnett’s multiple comparisons test (TIF 1003 KB)

Supplemental Fig. 2

Efficiency of daf-16 and skn-1 knock-down in C. elegans via feeding HT115 E. coli expressing dsRNA for the corresponding gene. a Representative images of TJ356 nematodes (left) expressing DAF-16::GFP and LD001 nematodes (right) expressing SKN-1::GFP. b Efficiency of daf-16 knock-down. TJ356 nematodes were fed with HT115 E. coli expressing daf-16 dsRNA or empty vector for 40 h at 16 °C. Images of single nematodes were taken with a camera connected to a fluorescence microscope equipped with a GFP filter. Fluorescence intensities of the single nematodes were measured with ImageJ. n = 1 (30 individuals), Student’s t-test, *** p ≤ 0.001 vs. empty vector control. c Efficiency of skn-1 knock-down. CL4176 nematodes were fed with HT115 E. coli expressing skn-1 dsRNA or empty vector for 40 h at 16 °C. Subsequently nematodes were transferred to RNAi plates and hatched larvae (left) and non-hatched eggs (right) were counted until the end of the reproductive period. n = 2 (10 individuals per group), Student’s t-test, ** p ≤ 0.01 vs. empty vector control (TIF 2254 KB)

Supplemental Fig. 3

A: HSE (1 mg/ml) increases oxidative stress resistance of C. elegans. L4 larvae (N2) were incubated with 1 mg/ml HSE or the respective control at 20°C for 3 days. Afterwards 30 nematodes of every treatment group were transferred to S-media containing 50 mM paraquat, 50 µg/ml streptomycin and 109 OP50-1 E. coli. The survival of the animals was measured daily by touch-provoked movement. Lost or ruptured animals were censored. n = 5 (30 individuals per group), Kaplan–Meier survival analysis with Log-Rank (Mantel-Cox)-test, * p ≤ 0.05 vs. control. B: Treatment with HSE increases oxidative stress resistance of Δmev-1 C. elegans (TK22): L4 nematodes of the strain TK22 lacking mev-1 were incubated with 1 mg/ml HSE or the respective control at 20°C for 3 days. Afterwards 40 nematodes of every treatment group were transferred to S-media containing 50 mM paraquat, 50 µg/ml streptomycin and 109 OP50-1 E. coli. The survival of the animals was measured daily by touch-provoked movement. Lost or ruptured animals were censored. n = 3 (40 individuals per group), Kaplan–Meier survival analysis with Log-Rank (Mantel-Cox)-test, *** p < 0,001 vs. control. D: HSE treatment decreases lipofuscin accumulation in the upper intestine of C. elegans. L4/young adult nematodes (N2) were treated with 1 mg/ml HSE or the respective control for 3 days at 20°C. Fluorescence intensities of the upper intestine were measured by densitometric analysis, n = 3 (10 individuals/trial), **p ≤ 0.01 one-way ANOVA with Dunnett‘s multiple comparisons test. D: Antioxidant NAC prevents HSE-induced nuclear accumulation of DAF-16 and SKN-1. L4 nematodes of the strain TJ356 (left) or LD001 (right) were incubated with 1 mg/ml HSE, 10 mM NAC, 1 mg/ml HSE and 10 mM NAC or the respective control at 20°C for 1 h and subsequently analysed by means of fluorescence microscopy. Nematodes with nuclear localisation of DAF-16 or SKN-1 in the intestinal cells were counted, n = 3 (30 individuals per group), (TIF 1053 KB)

Supplemental Fig. 4

HSE at a high concentration (1 mg/ml) is not able to protect against Aβ-induced toxicity in C. elegans fed with HT115 E. coli. Eggs of CL4176 nematodes fed with HT115 E. coli expressing daf-16 dsRNA (left), skn-1 dsRNA (right) or empty vector were treated with 1 mg/ml HSE or the solvent control NGMk for 40 h at 16°C before temperature was shifted to 25°C. Paralysis curve of pretreated C. elegans strain CL4176 was measured 26 h after temperature up-shift every other hour. n = 3 (40 individuals per group), Kaplan–Meier survival analysis with Log-Rank (Mantel-Cox)-test, *** p ≤ 0.001 vs. empty vector control (TIF 673 KB)

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Koch, K., Weldle, N., Baier, S. et al. Hibiscus sabdariffa L. extract prolongs lifespan and protects against amyloid-β toxicity in Caenorhabditis elegans: involvement of the FoxO and Nrf2 orthologues DAF-16 and SKN-1. Eur J Nutr 59, 137–150 (2020). https://doi.org/10.1007/s00394-019-01894-w

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