Towards the development of phytoextract based healthy ageing cognitive booster formulation, explored through Caenorhabditis elegans model

Abstract

The positive effect of herbal supplements on aging and age-related disorders has led to the evolution of natural curatives for remedial neurodegenerative diseases in humans. The advancement in aging is exceedingly linked to oxidative stress. Enhanced oxidative stress interrupts health of humans in various ways, necessitating to find stress alleviating herbal resources. Currently, minimal scientifically validated health and cognitive booster resources are available. Therefore, we explored the impact of plant extracts in different combinations on oxidative stress, life span and cognition using the multicellular transgenic humanized C. elegans, and further validated the same in Mus musculus, besides testing their safety and toxicity. In our investigations, the final product-the HACBF (healthy ageing cognitive booster formulation) thus developed was found to reduce major aging biomarkers like lipofuscin, protein carbonyl, lipid levels and enhanced activity of antioxidant enzymes. Further confirmation was done using transgenic worms and RT-PCR. The cognitive boosting activities analyzed in C. elegans and M. musculus model system were found to be at par with donepezil and L-dopa, the two drugs which are commonly used to treat Parkinson's and Alzheimer's diseases. In the transgenic C. elegans model system, the HACBF exhibited reduced aggregation of misfolded disease proteins α-synuclein and increased the health of nicotinic acetylcholine receptor, levels of Acetylcholine and Dopamine contents respectively, the major neurotransmitters responsible for memory, language, learning behavior and movement. Molecular studies clearly indicate that HACBF upregulated major genes responsible for healthy aging and cognitive booster activities in C. elegans and as well as in M. musculus. As such, the present herbal product thus developed may be quite useful for healthy aging and cognitive boosting activities, and more so during this covid-19 pandemic.

Supplementary information: The online version contains supplementary material available at 10.1007/s13237-022-00407-1.

Keywords: Caenorhabditis elegans; Camellia sinensis; Cognition; Cymbopogon khasianus; HACBF (healthy ageing cognitive booster formulation); Longevity; Mus musculus; Ocimum tenuiflorum; Phyto-extract.

Fig. 1

Chemical Finger Prints of Ocimum tenuiflorum, CIM-Agna (A), Cymbopogon khasianus (B) and Camellia sinensis (C) and mixture of ABC

Fig. 2

Analysis of heavy metal toxicity

Fig. 3

Toxicity assay of HACBF in C. elegans. The toxicity assay was performed after 48 h. The 1–4 mg/ml concentrations of plant resource extract were found to be non-toxic after 48 h followed by toxic effects demonstrated in 5 mg/ml. The data was statistically analyzed using unpaired student t-test in Graph Pad Prism 5.03 statistical software

Fig. 4

Effect of lyophilized extract powder as a single acute oral dose at 5, 50, 300, 1000 and 2000 mg/kg body weight in Swiss albino mice, a differential leucocytes counts, b absolute organ weight, c relative organ weight.; Effect of lyophilized extract powder at 0.2, 2, 20 and 200 mg/kg body weight once orally for 28 days in Swiss albino mice. d Differential leucocytes count, e absolute organ weight, f relative organ weight (Mean ± SE, n = 6)

Fig.5

Anti-Inflammatory profile of HACBF against carrageenan-induced acute inflammation in rats

Fig. 6

In vitro antioxidant activity of HACBF

Fig. 7

Lifespan studies in C. elegans. a DCF-DA test in wild-type C. elegans. b Pre-treatment with different doses of HACBF exerts oxidative stress reduction in C. elegans. c Mean lifespan of C. elegans affected with HACBF d Survival curves of animals control and treated with different doses (HACBF treated worms at1mg/ml, 2 mg/ml, 4 mg/ml) of HACBF. e 4 mg/ml HACBF impacted average lifespan by 27%, f HACBF significantly reduced the protein carbonyl level as compared to control

Fig. 8

Measurement of mitochondrial viability by MitoTracker assay. The fluorescence of mitochondrial viability of randomly HACBF treated/untreated worms was captured using Rhodamine filter. a Untreated control (RFU) b Treated with 4 mg/ml HACBF (RFU). c HACBF (4 mg/ml) supplementation reduced the mitochondrial health of treated worms with respect to untreated control

Fig. 9

Effect of HACBF on fat accumulation and relative lipofuscin levels. The age synchronized wildtype worms were grown till their adulthood with or without HACBF treatment (4 mg/ml). a Microphotograph untreated control worms, b microphotograph of day 2 HACBF (4 mg/ml) treated worms. c HACBF (4 mg/ml) supplementation reduced the fat content. d Relative lipofuscin content in untreated control worms, e Lipofuscin content in HACBF (4 mg/ml)) treated worms. f HACBF (4 mg/ml) supplementation reduced lipofuscin level in treated worms with respect to untreated control

Fig. 10

HACBF (4 mg/ml) supplementation elevates the expression of stress responsive genes. The age synchronized worms were grown in the presence or absence of HACBF (4 mg/ml) till their adulthood. The fluorescence of randomly selected treated/untreated SOD-3::GFP transgenic worms were captured using GFP (green fluorescence protein) filter, a Microphotograph captured on day 2 of control SOD-3::GFP worms, b Microphotograph captured on day 2 of HACBF (4 mg/ml) treated SOD-3::GFP worms, c HACBF (4 mg/ml) supplementation increased the SOD-3 expression in treated worms with respect to control; The fluorescence of randomly selected treated/untreated GST-4::GFP transgenic worms were captured using GFP (green fluorescence protein) filter. d Microphotograph captured on day 2 of untreated control GST-4::GFP worms. e Microphotograph captured on day 2 of HACBF (4 mg/ml) treated GST-4::GFP worms. f Quantification of fluorescence level of GST-4::GFP expression presented as mean relative fluorescent unit (RFU)

Fig. 11

HACBF elevates synaptic ACh availability, receptor health and expression of Ach cascade gene. a Aldicarb sensitivity assay with HACBF treatment, b Levamisole assay with HACBF treatment. c Gene expression on treatment with HACBF (4 mg/ml). d HACBF (4 mg/ml) supplementation upregulated expression neuromodulatory gene related to acetylcholine cascade in male and female mice respectively unc-29

Fig. 12

Impact of HACBF on α-syn aggregation C. elegans. a Effect of different doses of HACBF relative α-syn mass b Impact of HACBF on YFP expression with different treatments, c 1-Nonanol assay d Effect of HACBF on body bend. e Effect of HACBF on head thrash in C. elegans. f Effect of HACBF on mRNA expression, g HACBF (4 mg/ml) supplementation upregulated the expression of neuromodulatory gene in male and female mice respectively ubc-12 and pink-1

Fig. 13

HACBF (4 mg/ml) supplementation elevates the expression of stress responsive and longevity promoting genes. a Impact of HACBF on mRNA expression level of sod-3, sod-4, gst-4, gst-7, ctl-2 and jnk-1, b HACBF (4 mg/ml) supplementation upregulated expression stress responsive in male and female mice respectively of ctl-1, sod-2, sod-3 and sod-4. c Effect of HACBF on mRNA expression level of daf-2, daf-16, pha-4, lgg-1, bec-1 and skn-1