Project Description

According to the World Health Organization (WHO), the number of older people aged 60 years and over will reach 1.4 billion by 2030, and this number will continue to grow, reaching an estimated 2.1 billion by 2050 [1][2]. In addition, many geriatric diseases such as diabetes, hypertension, and Alzheimer's disease (AD) are major factors of death and disability in the aging population [1][3]. This situation of global ageing is becoming increasingly serious and poses enormous challenges for families, public health, and the economy.

Figure 1: Percentage of the Population Age 65+, according to the US Census

Healthy aging is people's real needs as claimed by the universal declaration of human rights [4]. Therefore, the development of anti-aging drugs for a long and healthy life, especially in geriatrics, is an important research topic. Up to now, a variety of anti-aging drugs have made significant progress in preclinical research, such as nicotinamide mononucleotide (NMN) [5], rapamycin [6], metformin [7], resveratrol [6], etc., which have significant anti-aging and life-extending pharmacological activities at the model animal level. However, no anti-aging drug has been approved or proven effective in the clinical study phase so far, and even the popular anti-aging drug NMN has been proven ineffective in recent clinical studies [5]. The challenges in this area remain enormous.

In China, pepper is a common food additive and traditional herbal medicine. In people's long-term practice, the efficacy of pepper in the fields of analgesia, anti-inflammatory, antioxidant and antibacterial has been confirmed, and pepper has been included in the Chinese Pharmacopoeia. Studies have shown that the active ingredient of Capsaicin WGX-50 has anti-inflammatory and antioxidant effects similar to those of peppercorns, and is a candidate for anti-Alzheimer's disease (AD) [8], as well as a potential analgesic [9] and cardiac injury-protecting drug [10], which can inhibit neuroinflammation induced by amyloid Aβ peptide and reduce neurological damage caused by Aβ accumulation through a variety of biological pathways [11][12].

As of now, there are very limited studies on the anti-aging properties of the peppermint WGX-50. The earliest evidence from experimental studies [8][13] suggests that peppercornin WGX-50 inhibits aberrant Ca2+ inward flow in rat neurons, and that disturbed calcium ion regulation is a recognised symptom accompanying brain ageing [14]. In a follow-up study, peppercorn WGX-50 was found to inhibit galactosidase (SA-β-gal) activity, down-regulate malondialdehyde (MDA) and reactive oxygen species (ROS), and regulate the expression of the senescence secretory phenotype (SASP) molecule TGF-β1 [15], ultimately delaying skin aging in mice. which ultimately slowed down the aging process of mouse skin. Accordingly, we deduce that the peppermint WGX-50 is likely to have systemic anti-aging pharmacological activities. In the present invention, we employed the model organism nematode (C. elegans) and mice to carry out a systematic study. The results showed that the peppermint WGX-50 has anti-aging and life extension pharmacological effects.

In this study, we integrated the cross fertilization of pharmacology, molecular biology and bioinformatics of Chinese herbal medicine, and screened natural small molecules for Alzheimer's disease from the database of traditional Chinese medicine by using artificial intelligence and computer-aided drug design, and then explored and verified the molecular mechanism of Alzheimer's disease by combining with pharmacy and molecular biology to investigate the mechanism of Alzheimer's disease treatment in vivo, in vitro, and in the cognitive and behavioral aspects of Alzheimer's disease. We investigated the mechanism of the prolongation of healthy life span of peppermint from in vivo, in vitro and in cognitive and behavioral aspects, and provided a theoretical basis for slowing down the deterioration of behavioral and cognitive functions in the elderly.

(1). Lifespan test of the C. elegans:

In the view of the previously described beneficial effects of WGX-50 (Figure 1a) on Alzheimer's disease (AD) [11], pain relief [9], drug-induced heart injury [10] and skin health [15], we sought to investigate its impact on longevity of C. elegans because the underlying mechanism on longevity is uncertain. To investigate whether WGX-50 could prolong lifespan, we conducted a longevity test in C. elegans. Interestingly, WGX-50 significantly prolonged the lifespan of C. elegans and the best lifespan extension effect was observed at a concentration of 50 μM (Figure 1b). Interestingly, in accordance with our study, WGX-50 extended lifespan in w.t nematodes (unpublished data; dose range tested, 3.75 ~ 250 M), in which the dose 15 M increased the median survival to 45% (from 22 to 32 days, Figure 1b) at 20 oC. To this end, we concluded that WGX-50 prolongs lifespan in C. elegans.

Figure 2: WGX-50 extends lifespan in N2 C. elegans. (a): WGX-50 was previously characterized as a novel drug candidate for AD, which is a natural product derived from Zanthoxylum bungeanum Maxim. (Image credit: Royalty-free photo from https://create.vista.com/, slightly changed by the authors). (b): WGX-50 extends lifespan in N2 nematodes at 20 °C (b, n > 280 worms per condition). **** p < 0.0001.

(2). Molecular dynamics methods:

In the domains of biochemistry, molecular biology, drug development, and other sciences, fluorescence kinetics experimentation is a vital tool for researching molecular interactions, reaction kinetics, and molecular motion. The intensity of light emitted by fluorescent molecules is monitored over time to provide information on chemical reaction rate, molecular conformation change, and ion concentration change.

In vitro experiments show that WGX-50 can inhibit the aggregation of Aβ monomers, indicating that peppercorns may have the effect of preventing Alzheimer's disease.

Figure 3: Thioflavin T fluorescence kinetics experiment of WGX-50 (10μM) inhibiting Aβ monomer aggregation within 24 hours (α-M: mangostin, a lead compound against Alzheimer’s disease)

In vitro tests showed that WGX-50 could inhibit the aggregation of Aβ monomers (shown in Figure 3), suggesting that peppermint may have the efficacy of preventing Alzheimer's disease, and the mechanism of action of peppermint was investigated by molecular dynamics simulation. The results of this experiment are a continuation of the applicant's previous anti-Alzheimer's disease candidate WGX-50, which was discovered in the laboratory through the computerised virtual drug screening platform, and was shown to be able to depolymerise Aβ aggregates in vitro (shown in Figure 4) and improve the memory of demented transgenic mice in an extended test. This provides a test basis for the potential of WGX-50 to extend life span and improve cognitive behaviour.

Figure 4: AFM shows the Aβ depolymerization of WGX-50

[1] Newman, A. B., Odden, M. C. & Cauley, J. A. Epidemiology of Aging. in Handbook of Epidemiology 1–37 (Springer New York, 2023). doi:10.1007/978-1-4614-6625-3_79-1.

[2] World Health Organization. Ageing and health. who.int (2022).

[3] Cox, L. S. Therapeutic approaches to treat and prevent age-related diseases through understanding the underlying biological drivers of ageing. J. Econ. Ageing 23, 100423 (2022).

[4] The Lancet Healthy Longevity. Human rights for healthy longevity. Lancet Heal. Longev. 4, e517 (2023).

[5] Damgaard, M. V. & Treebak, J. T. What is really known about the effects of nicotinamide riboside supplementation in humans. Sci. Adv. 9, (2023).

[6] Latorre, E. et al. Small molecule modulation of splicing factor expression is associated with rescue from cellular senescence. BMC Cell Biol. 18, 31 (2017).

[7] Song, J. et al. Metformin prolongs lifespan through remodeling the energy distribution strategy in silkworm, Bombyx mori. Aging (Albany. NY). 11, 240–248 (2019).

[8] Tang, M. et al. A Novel Drug Candidate for Alzheimer’s Disease Treatment: gx-50 Derived from Zanthoxylum Bungeanum. J. Alzheimer’s Dis. 34, 203–213 (2013).

[9] Wang, Z.-Y. et al. Lemairamin, isolated from the Zanthoxylum plants, alleviates pain hypersensitivity via spinal α7 nicotinic acetylcholine receptors. Biochem. Biophys. Res. Commun. 525, 1087–1094 (2020).

[10] Tai, P. et al. WGX50 mitigates doxorubicin-induced cardiotoxicity through inhibition of mitochondrial ROS and ferroptosis. J. Transl. Med. 21, 823 (2023).

[11] Shi, S. et al. Gx-50 Inhibits Neuroinflammation via α7 nAChR Activation of the JAK2/STAT3 and PI3K/AKT Pathways. J. Alzheimer’s Dis. 50, 859–871 (2016).

[12] Shi, S. et al. Gx-50 reduces β-amyloid-induced TNF-α, IL-1β, NO, and PGE 2 expression and inhibits NF-κB signaling in a mouse model of Alzheimer’s disease. Eur. J. Immunol. 46, 665–676 (2016).

[13] Hou, S., Gu, R.-X. & Wei, D.-Q. Inhibition of β-Amyloid Channels with a Drug Candidate wgx-50 Revealed by Molecular Dynamics Simulations. J. Chem. Inf. Model. 57, 2811–2821 (2017).

[14] Thibault, O., Gant, J. C. & Landfield, P. W. Expansion of the calcium hypothesis of brain aging and Alzheimer’s disease: minding the store. Aging Cell 6, 307–317 (2007).

[15] Hou, X. F. et al. Anti-skin aging effect of GX-50. J. Shanghai Jiaotong Univ. (Medical Sci. 33, 145–149 (2013).