Antiaging, Stress Resistance, and Neuroprotective Efficacies of Cleistocalyx nervosum var. paniala Fruit Extracts Using Caenorhabditis elegans Model

Abstract

Plant parts and their bioactive compounds are widely used by mankind for their health benefits. Cleistocalyx nervosum var. paniala is one berry fruit, native to Thailand, known to exhibit various health benefits in vitro. The present study was focused on analyzing the antiaging, stress resistance, and neuroprotective effects of C. nervosum in model system Caenorhabditis elegans using physiological assays, fluorescent imaging, and qPCR analysis. The results suggest that the fruit extract was able to significantly extend the median and maximum lifespan of the nematode. It could also extend the healthspan by reducing the accumulation of the "age pigment" lipofuscin, inside the nematode along with regulating the expression of col-19, egl-8, egl-30, dgk-1, and goa-1 genes. Further, the extracts upregulated the expression of daf-16 while downregulating the expression of daf-2 and age-1 in wild-type nematodes. Interestingly, it could extend the lifespan in DAF-16 mutants suggesting that the extension of lifespan and healthspan was dependent and independent of DAF-16-mediated pathway. The fruit extract was also observed to reduce the level of Reactive Oxygen Species (ROS) inside the nematode during oxidative stress. The qPCR analysis suggests the involvement of skn-1 and sir-2.1 in initiating stress resistance by activating the antioxidant mechanism. Additionally, the fruit could also elicit neuroprotection as it could extend the median and maximum lifespan of transgenic strain integrated with Aβ. SKN-1 could play a pivotal role in establishing the antiaging, stress resistance, and neuroprotective effect of C. nervosum. Overall, C. nervosum can be used as a nutraceutical in the food industry which could offer potential health benefits.

Figure 1

CMK-P extract can extend the mean and median lifespan of C. elegans. (a) Wild-type nematodes were treated with different concentrations of CMK-P extracts ranging from 1 to 100 μg/ml which could significantly (p < 0.05) extend the maximum lifespan of the nematode. Nematodes used as the control which did not receive any extract treatment survived up to 22 days. DMSO was used as a vehicle control which also survived for 22 days. (b) Wild-type nematodes were treated with different concentrations of CMK-P extracts ranging from 1 to 100 μg/ml which could significantly (p < 0.05) extend the median lifespan of the nematode. (c) Selective doses which showed maximum extension of lifespan were represented. CMK-P extracts at 10, 20, 30, and 40 μg/ml could extend the lifespan of the nematode up to 28, 30, 29, and 29 days, respectively.

Figure 2

LMK-P extract can extend the mean and median lifespan of C. elegans. (a) Wild-type nematodes were treated with different concentrations of LMK-P extracts ranging from 1 to 100 μg/ml which could significantly (p < 0.05) extend the maximum lifespan of the nematode. Nematodes used as the control which did not receive any extract treatment survived up to 22 days. DMSO was used as a vehicle control which also survived for 22 days. (b) Wild-type nematodes were treated with different concentrations of LMK-P extracts ranging from 1 to 100 μg/ml which could significantly (p < 0.05) extend the median lifespan of the nematode. (c) Selective doses which showed maximum extension of lifespan were represented. LMK-P extracts at 10, 20, 30, and 40 μg/ml could extend the lifespan of the nematode up to 27, 30, 29, and 28 days, respectively.

Figure 3

Both CMK-P and LMK-P could extend the healthspan of C. elegans. (a) Pharyngeal pumping assay of wild-type nematodes treated with CMK-P and LMK-P at 20 and 30 μg/ml concentrations. Both the extracts did not show any significant change in the pharyngeal pumping rate when compared to the control. (b) qPCR analysis of col-19, egl-8, egl-30, dgk-1, and goa-1 in wild-type nematodes treated with CMK-P and LMK-P. Both the extracts significantly (p < 0.05) downregulated the expression of col-19, dgk-1, and goa-1 and upregulated the expression of egl-8 and egl-30. (c) Representative image of wild-type nematode with no extract treatment (control) with the level of lipofuscin accumulation. (d) Representative image of wild-type nematode with 20 μg/ml of CMK-P with the level of lipofuscin accumulation. (e) Representative image of wild-type nematode with 30 μg/ml of CMK-P with the level of lipofuscin accumulation. (f) Relative fluorescence intensity comparison of nematodes treated with CMK-P extract at 20 and 30 μg/ml showing significant (p < 0.05) reduction in fluorescence when compared to the control (n = 10). (g) Representative image of wild-type nematode with no extract treatment (control) with the level of lipofuscin accumulation. (h) Representative image of wild-type nematode with 20 μg/ml of LMK-P with the level of lipofuscin accumulation. (e) Representative image of wild-type nematode with 30 μg/ml of LMK-P with the level of lipofuscin accumulation. (f) Relative fluorescence intensity comparison of nematodes treated with LMK-P extract at 20 and 30 μg/ml showing significant (p < 0.05) reduction in fluorescence when compared to the control (n = 10).

Figure 4

C. nervosum extracts are dependent and independent of DAF-16-mediated pathway. (a) qPCR analysis of candidate genes of DAF-16-mediated pathway. Wild-type nematodes treated with CMK-P and LMK-P extracts at 20 and 30 μg/ml showed significant (p < 0.05) upregulation in the expression of daf-16 at selective doses and corresponding significant (p < 0.05) downregulation of daf-2, age-1, and utx-1. (b) CMK-P at 10, 20, 30, and 40 μg/ml could extend the maximum lifespan of daf-16 mutants. (c) Graph showing significant increase in the average maximum survival days of daf-16 mutant nematodes treated with 10, 20, 30, and 40 μg/ml of CMK-P extracts. (d) Graph showing significant increase in the average median survival days of daf-16 mutant nematodes treated with 10, 20, 30, and 40 μg/ml of CMK-P extracts. (e) LMK-P at 10, 20, 30, and 40 μg/ml could extend the maximum lifespan of daf-16 mutants. (f) Graph showing significant increase in the average maximum survival days of daf-16 mutant nematodes treated with 10, 20, 30, and 40 μg/ml of LMK-P extracts. (g) Graph showing significant increase in the average median survival days of daf-16 mutant nematodes treated with 10, 20, 30, and 40 μg/ml of LMK-P extracts.

Figure 5

C. nervosum extracts could activate the antioxidant potential by reducing the level of ROS in C. elegans. (a) Relative fluorescence intensity comparison of nematodes exposed to UV-A for 4 h to induce stress along with co- and posttreatment with CMK-P and LMK-P extract at 20 and 30 μg/ml showing significant (p < 0.05) reduction in fluorescence when compared to control worms exposed to UV-A without any extract treatment (n = 10). (b) Representative image of the negative control worm which was not exposed to UV-A and did not receive any extract treatment. (c) Representative image of the positive control worm which was exposed to UV-A for 4 h but did not receive any extract treatment. (d) Representative image of the worm which was treated with 20 μg/ml of CMK-P along with UV-A exposure for 4 h (cotreatment). (e) Representative image of the worm which was treated with 30 μg/ml of CMK-P along with UV-A exposure for 4 h (cotreatment). (f) Representative image of the worm which was treated with 20 μg/ml of CMK-P after UV-A exposure for 4 h (posttreatment). (g) Representative image of the worm which was treated with 30 μg/ml of CMK-P after UV-A exposure for 4 h (posttreatment). (h) Representative image of the worm which was treated with 20 μg/ml of LMK-P along with UV-A exposure for 4 h (cotreatment). (i) Representative image of worm which was treated with 30 μg/ml of LMK-P along with UV-A exposure for 4 h (cotreatment). (j) Representative image of the worm which was treated with 20 μg/ml of LMK-P after UV-A exposure for 4 h (posttreatment). (k) Representative image of the worm which was treated with 30 μg/ml of LMK-P after UV-A exposure for 4 h (posttreatment).

Figure 6

qPCR analysis of candidate genes skn-1 and sir-2.1 that mediate the antioxidant mechanism in C. elegans. Both skn-1 and sir-2.1 expressed significant (p < 0.05) upregulation in wild-type nematodes when treated with 20 and 30 μg/ml of CMK-P and LMK-P extracts.

Figure 7

C. nervosum extracts could extend the survival of Aβ transgenic strain CL2006. (a) CMK-P at 10, 20, 30, and 40 μg/ml could extend the maximum lifespan of Aβ transgenic strain. (b) Graph showing significant increase in the average maximum survival days of Aβ transgenic strain treated with 10, 20, 30, and 40 μg/ml of CMK-P extracts. (c) Graph showing significant increase in the average median survival days of Aβ transgenic strain treated with 10, 20, 30, and 40 μg/ml of CMK-P extracts. (d) LMK-P at 10, 20, 30, and 40 μg/ml could extend the maximum lifespan of Aβ transgenic strain. (e) Graph showing significant increase in the average maximum survival days of Aβ transgenic strain treated with 10, 20, 30, and 40 μg/ml of LMK-P extracts. (f) Graph showing significant increase in the average median survival days of Aβ transgenic strain treated with 10, 20, 30, and 40 μg/ml of LMK-P extracts.