Withania somnifera root extract (LongeFera^™) confers beneficial effects on health and lifespan of the model worm Caenorhabditis elegans

Plant extract

The hydroalcoholic extract (LongeFera™) of Withania somnifera root was procured from Phytoveda Pvt. Ltd., Mumbai. The roots of W. somnifera were collected from Madhya Pradesh, India. It was authenticated by a taxonomist at the Botanical Survey of India, Jodhpur, and the voucher specimen was deposited there with reference ID: BSI/AZRC/I.12012/Tech/19-20/PI.Id/671. The root material was washed, dried, and then pulverized. The resulting powder was then extracted with ethanol: water (8:2 v/v) at 60°C. A powdered form of this extract was then used for assay purposes. The extract was analyzed as per United States Pharmacopeia (USP) (15) for various quality control parameters. The content of withanosides and withanolides was determined using HPLC. Major phytocompounds identified in the extract are depicted in the supplementary chromatogram (Figure S1). The total withanolides content in the extract was found to be 2.69 ± 0.02%.

The extract powder was divided into two batches. One was suspended in water, and another in DMSO (Merck) for bioassay purposes. While the extract was fully soluble in DMSO, its insoluble fraction from the aqueous suspension was removed by centrifugation (7500 g at 25°C) for 10 minutes. The soluble fraction (supernatant) was filtered through a syringe filter (0.45 µm; Axiva), and the filtrate was stored in a sterile 15 mL glass vial (Borosil) under refrigeration. The solubility of the extract in water was 81.84%.

Test organism

Wild type N2 Bristol strain of C. elegans procured from the Caenorhabditis Genetics Center (Minneapolis, USA) was used as a model organism in this study. Lyophilized E. coli OP50 procured from Biovirid (Netherlands) was used as food for C. elegans while maintaining the worm on NGM agar plates (Nematode Growing Medium). Worm synchronization was done as described in literature (16). Prior to the in vivo assays, worms were kept without food for two days to make them gnotobiotic.

Lifespan assay

Synchronized (L3-L4 stage) gnotobiotic worms were incubated at 22 ± 1°C with different concentrations (5-1000 µg/mL) of the plant extract in 24-well plates (HiMedia). Each well contained 1 mL total volume (995 µL M9 buffer + 5 µL extract). Control wells contained only M9 buffer with worms but no extract. Ten worms were added in each well, and these 24-well plates were monitored over a 12-day period (till all worms died in the control wells) under a microscope (4× objective) for live-dead counting. Appropriate vehicle control containing 0.5%v/v DMSO was also included in the experiment.

Motility assay

Synchronized worms were incubated at 22±1°C with or without extract (600 ppm) in 24-well plates, wherein each well contained approximately 100 worms in 1 mL of M9 buffer. While microscopic observation qualitatively confirmed more agile movement in worm population incubated with extract, quantification of the motility in control vs. experimental wells was achieved employing an automated worm tracker machine, WMicrotracker ARENA (Phylumtech, Argentina). Worm activity counts were recorded once every day till progenies appeared in the extract-containing wells (because the presence of progenies will contribute to higher activity counts).

Metabolic activity assay

Alamar Blue® assay (17,18) was used to quantify the viability or metabolic activity of the worms. Synchronized worms were incubated at 22 ± 1°C with or without extract (600 ppm) in 24-well plates, wherein each well contained approximately 100 worms in M9 buffer. One hundred µL of Alamar Blue® (Thermofisher) was added into each well, making the total volume 1 mL, before incubation started. To quantify the amount of dye reduced, after every 24 h, content from wells was transferred into a separate plastic vial (1.5 mL), followed by centrifugation (13,600 g at 25°C) for 10 min. Then, the supernatant was read at 570 nm (Agilent Cary 60 UV-vis). A total of three wells were set for the control as well as the experimental worm population, and content from one well from the control and experimental group each was used on a particular day. Appropriate abiotic controls (containing the dye and other media components but no worms) were also included in the assay.

Whole Transcriptome Analysis

To unravel the molecular mechanisms through which W. somnifera conferred beneficial effects on the worms with respect to lifespan and fertility, the gene expression pattern of extract-treated worms was compared with that of the control worm population at the whole transcriptome scale. While harvesting the worms for RNA isolation on the seventh day of the experiment (when ~50% of worms in the control population were dead), progenies were separated from the experimental population by resting the tubes containing worms incubated with extract in static condition for a few minutes, allowing settling down of adult worms. Progenies were removed by collecting the upper layer of liquid. This removed liquid was microscopically observed to confirm the presence of largely progeny worms in it, and not the adult ones added at the starting day of the experiment. Following this, worms were washed thrice with sterile M9 buffer before proceeding further. The overall workflow of this whole transcriptome analysis (WTA) aimed at capturing a holistic picture regarding modes of action of the test extract is given in Figure S2, along with full methodological details.

All the raw sequence data were submitted to the Sequence Read Archive. The relevant accession number for the control and experimental worm population is SRX20790765 (https://www.ncbi.nlm.nih.gov/sra/SRX20790765) and SRX20790719 (https://www.ncbi.nlm.nih.gov/sra/SRX20790719) respectively.

Polymerase Chain Reaction (RT-PCR)

Differential expression of the potential hubs found using network analysis of DEG revealed from WTA was further confirmed using PCR. Primer designing for the shortlisted genes was carried out using Primer3 Plus. These primer sequences (Table 1; Figure S3) were checked for their binding exclusivity to the target gene sequence within the whole C. elegans genome. Following RNA extraction, cDNA synthesis was carried out using the synthesis kit SuperScript™ VILO™ (Invitrogen Biosciences). PCR assay employed the gene-specific primers purchased from Sigma-Aldrich. FastStart Essential DNA Green Master mix (Roche, Germany) was used as the reaction mix. The real-time PCR assay was performed on a Quant Studio 5 real-time PCR machine (Thermo Fisher Scientific, USA). The temperature profile followed is given in Table S2.

Statistics

All values reported are derived from three or more independent experiments, wherein each experiment contained three replicates (unless specified otherwise). Statistical significance was assessed using a t-test performed in Microsoft Excel® (Version 2016), and data with p ≤ 0.05 were considered to be significant.

Worms incubated with the root extract exhibited extended lifespans and higher fertility rates than the control population

We incubated worms with the water-soluble fraction of the root extract, or that dissolved in DMSO, and observed the worms over a period of 12 days (till almost all worms in control wells died) for live-dead count, morphology, agility, and whether any progenies were formed. While the comparison between control and experimental wells was made on a daily basis, two time points can particularly be considered important, i.e., the days on which the control population exhibited ~50% and ~100% death. With respect to these two endpoints, while the DMSO-dissolved extract could impart a longevity benefit to the worms at all tested concentrations ≥100 µg/mL, the water-soluble fraction of the extract could do so at ≥250 µg/mL. Additionally, concentrations ≥ 500 µg/mL of both DMSO-solubilized and water-solubilized extract supported worm fertility from day 4 onward, as evident from the appearance of progenies in the extract-containing wells and their absence in control wells (Videos S1–S2). Till day 12, these progenies could be differentiated from the parent worms based on size. Thereafter, almost all the parent worms in control wells were dead, and the size of the surviving parent worms and their progenies in the experimental wells became similar to the extent that they could not be differentiated. The magnitude of survival benefit (i.e., the higher number of surviving worms in extract-containing wells than in control wells) conferred on the worms by DMSO-solubilized extract (Figure 1A) was somewhat higher than that of water-solubilized extract (Figure 1B), except at 750 µg/mL. At the latter concentration, the water-solubilized fraction of the extract performed better (p = 0.0004) than the DMSO-solubilized extract, as per the final-day endpoint. We decided to perform further experiments with water-solubilized fraction only, as water-solubility of any therapeutic preparation is looked at favorably with respect to bioavailability (19).

While the results described in Figure1A-1B demonstrated the beneficial effect of test extract on worm lifespan as well as fertility, to investigate its effect on worm longevity separately from fertility, we repeated this assay using FUdR (5-fluoro-20-deoxyuridine; HiMedia)- pre-treated worms, wherein eggs were allowed to hatch on FUdR-containing plates pre-seeded with E. coli OP50, and then resulting L3-L4 stage worms were washed with M9 buffer before being transferred to a fresh NGM agar plate for further use. FUdR can sterilize the adult nematode worms without affecting their development (20). However, FUdR-pre-treated worms could not benefit from the pro-longevity effect of the water-soluble fraction of the W. somnifera root extract except at 750 µg/mL on day 12 (Figure 1C), and here, too, the effect was much lesser than when FUdR was not used (Figure 1B). These results raise caution regarding the use of FUdR in lifespan assays with C. elegans, as this may lead to false-negative conclusions. Though the practice of using FUdR-treated worms is popular (in order to avoid the laborious separation of offspring from adults during the reproductive period) among the worm researchers studying longevity and aging, our results show that FUdR can prevent the detection of pro-longevity activity in a bioactive extract, even when the said activity is there. This corroborates with the earlier suggestion by Aitlhadj and Stürzenbaum (21) that the effect of FUdR is not neutral and owing to its mechanism of actions (inhibition of DNA and RNA synthesis, death of mitotic cells, and the inhibition of protein synthesis), its inclusion in the assays may result in misinterpretation. Hence, it can be suggested that while investigating any natural product’s biological effect in the C. elegans model, it is more useful to do a wholistic assay assessing multiple parameters like lifespan, health, metabolic activity, fertility, etc., rather than doing an assay exclusively focusing on longevity using additional chemicals like FUdR which may prove to be a confounding factor.

W. somnifera root extract positively affects worm motility and metabolic activity

Worms incubated in the presence of the test extract exhibited more active movement (Figure 2A), which can be considered another indicator of overall good health. This observation matches well with the high fertility of extract-exposed worms described in the preceding section since the egg-laying active state and the defecation motor program are both linked to changes in forward and reverse locomotion (22,23). Automated monitoring of C. elegans’ movement is a useful and faster healthspan-based method to study aging (24). Besides positively impacting worm lifespan, fertility, and motility, the root extract also had a stimulatory effect on worm metabolic activity (Fig. 2B). Worms incubated in the presence of the extract were found to have higher reducing potential (captured in terms of their ability to reduce the dye Alamar blue), which can be taken as an indication of better health, as healthy living cells maintain a reducing state within their cytosol (25).

Overall, the root extract imparted its beneficial effect on multiple health parameters of the worm, i.e., lifespan, fertility, motility, and metabolic activity. Although many natural products have been reported to impart ­pro-longevity effects on different biological model organisms based on lifespan assays, which is a conventional method to monitor aging, it should be noted that a compound or formulation that extends lifespan may not necessarily maintain health (26). Lifespan extension makes sense only if it does not cost on the healthspan front (27). Increased egg laying in extract-exposed older adults reflects an increase in the number of eggs expelled per vulval opening, as well as longer active behavior states. Since the timing of expulsive behaviors (defecation and egg laying) is regulated by sensory mechanisms that detect changes in internal pressure and/or stretch to maintain homeostasis (28), we believe that the test extract had a multifactorial impact on worm physiology at different levels. Feedback of successful egg laying in the presence of the extract might have signaled the germ line to continue the production of oocytes for fertilization. A potential trade-off between reproductive capacity and somatic maintenance in C. elegans has already been mentioned in earlier published literature. Higher levels of progeny production are a biomarker for studying aging and correlate positively with a longer lifespan (29,30). Prolonged reproduction can have a beneficial impact on lifespan.

Since the most promising effect of the root extract on the worm’s overall health (i.e., lifespan and fertility) was observed while using a water-soluble fraction of the extract, and the maximum beneficial effect was observed at 600-750 µg/mL, we decided to investigate the gene expression pattern of worms exposed to the test extract at 600 µg/mL at the whole transcriptome level. Since the biological effect of both these concentrations (600 and 750 µg/mL) was statistically similar (p>0.05), we went ahead with 600 µg/mL. While all experiments presented in Figure 1-2 were performed with gnotobiotic worms facing starvation, we also compared the effect of this root extract on worms fed with their regular lab food, E. coli OP50. Since the worm’s response to the W. somnifera extract was not affected (data not shown) owing to the presence of the bacterial food, we performed the transcriptome study with gnotobiotic worms only to avoid any possible confounding role of bacteria.

W. somnifera root extract exerts its beneficial effect on the nematode worm by triggering differential expression of multiple genes

A whole transcriptome level comparison of the gene expression profile of the extract-treated worms with their extract-non-exposed counterparts revealed all the DEG in the experimental worm population. Keeping the criteria of log FC ≥1.5 and FDR (false discovery rate) ≤0.05, the differentially expressed gene (DEG) count was 85. However, to have higher confidence in our data interpretation, we set more stringent dual criteria of log FC ≥ 2 and FDR ≤ 0.01 to shortlist the genes for further analysis. A total of 16 upregulated and 29 downregulated DEGs passing the said dual criteria are listed in Table 2. These 45 DEG comprise 0.83% of the total C. elegans genome (~5423 genes). Function-wise categorization of these DEG is presented in Figure 3, and the corresponding heat map (Fig. S4) and volcano plot (Fig. S5) are provided in the supplementary file. A detailed discussion on important up/down-regulated genes and how their differential expression would have contributed to the observed results is provided in the supplementary file “Appendix A.”

Since the transcriptome data has identified multiple genes associated with worm exoskeleton components and muscle structure as differently expressed in extract-fed worms, it is clear that the root extract used in the present study has caused restructuring of the exoskeleton in such a way that age-associated development of stiffness in worm skeleton is delayed, and the worm can age in a healthier fashion. We had conducted transcriptome profiling from the worms after seven days of extract exposure, but the classic signs of old age and fertility loss usually observed in worms at this stage of the life cycle did not yet arise in the presence of the extract. Hence, W. somnifera root extract can be concluded to be capable of delaying aging and senescence. Under routine conditions, at day seven of adulthood, sarcopenia is apparent in histologically examined worms, which leads to behavioral change in terms of reduced motor activity. Reduced motility stems from stiffening and thickening of the cuticle, which itself results from unregulated collagen biosynthesis (31), and the root extract used in this study seems to have affected collagen synthesis in such a way that stiffening/ thickening of the cuticle was delayed in the extract-fed worms, and thereby maintaining them in an active motility state for a longer period.

Identifying the most important DEG through network analysis

To identify the proteins with high network centrality, i.e., hub proteins from among the DEG, we generated a protein-protein interaction (PPI) network of upregulated (Fig. 4A) and downregulated (Fig. 5A) genes separately. We arranged members of the PPI network of upregulated genes in decreasing order of node degree (Table S3), and those with a score of ≥5 were subjected to ranking by different cytoHubba methods. Then, we looked for genes that appeared among the top 5 ranked candidates by ≥6 cytoHubba methods, and five such shortlisted genes (Table 3) were further checked for interactions among themselves, followed by cluster analysis (Fig. 4B). The PPI network generated through STRING showed these five important genes to be distributed among three different local network clusters. Since cpg-3 appeared to be part of two different clusters, and both the cpg genes had multiple edges connecting them together (node degree score was 3 for both of them), we selected these two genes (cpg-2 and cpg-3) for further RT-PCR validation. PCR assay did confirm the upregulation of these two proteins in extract-exposed worms (Fig. 6A).

We arranged members of the PPI network for the downregulated genes (Fig. 5A) in decreasing order of node degree (Table S4), and those with a score of ≥5 were subjected to ranking by different cytoHubba methods. Then, we looked for genes that appeared among the top-6 ranked candidates by ≥6 cytoHubba methods, and six such shortlisted genes (Tabl 4) were further checked for interactions among themselves, followed by cluster analysis (Fig. 5B). Four (sqt-1, dpy-4, dpy-13, and col-17) of these six genes belonged to a single cluster (each with a node degree score of 4) and were chosen for further RT-PCR validation. PCR assay did confirm the downregulation of these proteins in extract-exposed worms (Fig. 6B).

Identifying the most important DEG based on their homology with the human genome

To have an empirical idea about the relevance of the results of this study performed in C. elegans, with respect to W. somnifera’s possible beneficial effect in humans, we conducted a co-occurrence analysis between all the 45 DEG in extract-treated C. elegans and the human genome. This analysis resulted in the identification of 16 genes whose counterparts are present in humans (Fig. 7). The presence of multiple genes in the human genome with homology to those expressed differently in extract-exposed worms indicates a good probability of similar effect on humans as observed in C. elegans in this study. Among the DEG, gfat-2 seemed to have the highest similarity with its homolog in humans. The C. elegans gfat-2 (glutamine-fructose 6-phosphate aminotransferase-2) is 88.3% similar to gfat-1 (glutamine-fructose 6-phosphate aminotransferase-1) in amino acid sequence, and the latter is considered as a longevity gene (32). gfat-1 is the key enzyme of the hexosamine pathway and has been mentioned as a regulator of protein quality control and longevity. Increased functionality of gfat-1 induces endoplasmic reticulum-associated protein degradation and autophagy and correlatively extends lifespan and ameliorates a wide spectrum of proteinopathies. It can be said that W. somnifera root extract promotes health and extends the lifespan of C. elegans through endogenous modulation of protein quality control. The upregulation of gfat-1 in our long-lived worms corroborates with the previously published observation that hexosamine pathway metabolites enhance protein quality control and extend life (33). In mammals, too, gfat is the rate-limiting enzyme of the hexosamine pathway. Discussion on important genes listed in Figure-7 other than gfat-2 has been done in preceding sections.