Use of spearmint extract for retinal neurotrophy

Abstract

The present invention relates to a method for attenuating retinal cell damage and other neuronal cell damage outside the central nervous system associated with reduced supply of neurotrophin, i.e., nerve growth factor (NGF), induced by glaucoma and other forms of neurodegeneration by orally administering a therapeutically effective amount of an aqueous extract of spearmint to a subject.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 420,064, filed October 27, 2022, entitled "USE OF A SPEARMINT EXTRACT FOR RETINA NEUROTROPHISM," the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Some of the most significant age-related changes in the human body are related to vision (Goodman et al., Life 2023). The prevalence of eye conditions involving the external, anterior, and posterior segments of the eye (eyelids and lacrimal glands, cornea, lens, and retina) increases with age (Goodman et al., Life 2023). Aging is characterized by an increased incidence of dry eye, cataracts, and retinal degenerative eye diseases such as age-related macular degeneration (AMD) and glaucoma. See Nuzzi R, et al. Eye Brain. 2023 Jun 17;13:159-173. Damage to neural tissue outside the brain, such as the retina and peripheral nervous system (PNS), leads to numerous known impairments, including dry eye, age-related macular degeneration, diabetic retinopathy, glaucoma, diabetic neuropathy, neuropathic pain, and other neurodegenerative conditions.

[0003] Neurotrophins are proteins found in the central nervous system (CNS) and PNS that influence neuronal survival and development, including dendritic and synaptic growth, maintenance of target innervation, sustaining cell survival, plasticity mechanisms, axonal pruning, regulating neurotransmitter levels and neuronal excitability, and promoting regeneration and sprouting following neuronal injury. Nerve growth factor (NGF) was the first neurotrophin discovered. Other neurotrophins include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin 4 / 5 (NT4 / 5). Neurotrophins are part of a larger family called neurotrophins. Other neurotrophic factors include neurokines and glial cell line-derived neurotrophic factor.

[0004] NGF plays a very different role in the nervous system compared to BDNF. BDNF is found in high concentrations in the brain and is thought to play a central role in neurogenesis, whereas NGF is concentrated in the peripheral nervous system outside the brain. NGF has been shown to be produced by the iris, ciliary body, lens, vitreous humor, choroid, and retina. NGF and BDNF may have similar effects on neurons, but at different sites due to different expression patterns of their receptors and / or accessory proteins. In addition, BDNF and NGF bind to different receptors in the nervous system. NGF binds to TrkA receptors, while BDNF binds to TrkB receptors.

[0005] The role of NGF in the eye has been extensively studied, and NGF is believed to be important in maintaining homeostasis of the cornea (a densely innervated tissue) in addition to promoting tear production and maintenance, among other protective functions. Studies have demonstrated that NGF is upregulated in corneal cells under pressure and that NGF induces reduced NFKB activation and apoptotic cell death, resulting in enhanced protection of ocular tissue. In 2018, Cenegermin ophthalmic solution 0.002% received initial FDA approval as the first topical recombinant human nerve growth factor (tNGF) therapy for the treatment of neurotrophic keratitis, a neurodegenerative disease of the cornea. Because it has been established that tNGF does not normally cross the BBB, topical administration or intraocular injection has been the only means of ocular NGF therapy (Liu et al., 2014). Additionally, direct injection of NGF is likely to cause severe peripheral side effects in patients (Barker et al., 2020).

[0006] To the best of our knowledge, there is currently no method for safely orally administering plant substances to increase NGF levels in the eye and other areas outside the brain. To date, only one study has examined the effect of oral administration of plant extracts on NGF levels (Kosaka, K., and Yokoi, T. 2003, Biol. Pharm. Bull., 26, 1620-1622). This study, conducted using an in vitro cell culture model, demonstrated that the oily portion of rosemary extract increased NGF expression in glioblastoma cell lines, while the water-soluble portion of the extract had no effect on cellular NGF content, as summarized in Table 1. Despite its name, rosemary actually has a relatively low content of the phenolic compound rosmarinic acid, and instead typically exhibits high levels of other chemical compounds, including limonene and various monoterpenes (Lhaithloul et al. Front Plant Sci. 2023 May 18;14:1155698).

[0007] [Table 1] On the other hand, spearmint has been shown to accumulate high levels of rosmarinic acid in addition to other important polyphenolic compounds, and importantly, the combination with this polyphenol complex is crucial for any therapeutic benefit. For example, polyphenol-rich spearmint extracts have been observed to affect inflammatory parameters differently than rosmarinic acid alone. Indeed, in animal models of inflammation, spearmint extracts performed significantly better than equivalent amounts of rosmarinic acid alone (Fonseca et al., (2017, Anti-inflammatory, Exp Biology)). In this study, the anti-inflammatory effects of spearmint extract were evaluated using a rat paw edema model. Four groups of male Wistar rats were intraperitoneally administered a proprietary spearmint extract containing 15% rosmarinic acid at doses of 10, 30, or 100 mg / kg (n = 18), a rosmarinic acid standard at doses of 15 or 50 mg / kg (n = 10), the anti-inflammatory agent indomethacin at a dose of 10 mg / kg (n = 6), or saline alone as a vehicle control (n = 10). Fifteen minutes later, an intraplantar injection of carrageenan (100 μl, 1%) was administered to induce paw edema in all animals except for four animals in the saline control group (saline-saline group), which received intraplantar injections of saline. Paw volume was assessed by the volume displacement method immediately after carrageenan injection (baseline) and 3 and 6 hours thereafter, as summarized in Figure 1. Paw edema was expressed as the mean percent change in paw volume at 3 and 6 hours compared to baseline.

[0008] As expected for rats in the saline-carrageenan group, paw volume increased by 36% and 46%, respectively, at 3 and 6 hours after injection, significantly higher than that of the saline-saline group at the same time points (p = 0.014 and p = 0.014, respectively). Meanwhile, rats treated with spearmint extract showed limited inflammation, with paw volume increases at 6 hours after injection of 9%, 11%, and -8% in the 10, 30, and 100 mg / kg spearmint groups, respectively, which were significantly lower than that of the saline-carrageenan group at 6 hours (p < 0.05 for all). Additionally, the increase in paw volume at 3 hours for rats treated with 100 mg / kg spearmint extract was also significantly lower than that of the saline-carrageenan group at 3 hours (p = 0.008). Similarly, rats treated with anti-inflammatory agents also had limited inflammation compared to the saline-carrageenan group at 3 and 6 hours post-injection (p = 0.031 and p = 0.005, respectively), with paw volume increases from baseline of 6% and 3%. Animals treated with 15 mg / kg and 50 mg / kg rosmarinic acid showed paw volume increases of 20% and 7% at 3 hours and 10% and 5% at 6 hours, respectively, compared to baseline. These volumes were also significantly lower than the saline-carrageenan group at 3 hours (p = 0.043, 50 mg / kg) and 6 hours (p < 0.05, 15 and 50 mg / kg). When the attenuation of inflammation after treatment with spearmint extract was compared to anti-inflammatory agents, the spearmint extract group performed equally well in attenuating local inflammation. Furthermore, when the 15 mg / kg rosmarinic acid group was compared with the 100 mg / kg spearmint extract group containing the same amount of rosmarinic acid, the animals receiving the spearmint extract injections exhibited significantly less inflammation than the standard rosmarinic acid group at both 3 and 6 hours (p=0.013 and p=0.045, respectively), indicating that the additional active substances in the spearmint extract have an effect on the inflammatory response. In conclusion, these data indicate that the polyphenol-rich spearmint extract has a superior effect than rosmarinic acid alone, indicating a potential synergistic effect between the components present in the spearmint extract.

[0009] [Table 2] Glaucoma is one of the leading causes of blindness worldwide. It is primarily characterized by a progressive optic neuropathy involving chronic axonal damage and loss of retinal ganglion cells (RGCs). The degenerative process is not limited to RGCs and the optic nerve, but extends along the entire visual pathway. Brain changes in glaucoma appear to be related to transsynaptic neurodegenerative processes. See, for example, Nuzzi R, et al. Eye Brain. 2023 Jun 17;13:159-173. Furthermore, the neurodegenerative process is not limited to the primary visual pathway. Patients with glaucoma have shown alterations in association areas and domains related to vision and working memory and attention (Nuzzi et al., Front Neurosci. 2018 May 29;12:363; Nuzzi R, et al. Eye Brain. 2023 Jun 17;13:159-173). Because the complex pathophysiology behind neuronal RGC degeneration encompasses a range of genetic, metabolic, and environmental factors, the path to novel, more effective treatments remains unresolved in glaucoma management. Elevated intraocular pressure (IOP) is one of the most frequent risk factors driving glaucoma progression and RGC neurodegeneration. Therefore, contrast of ocular hypertension is currently the primary target of current pharmaceutical treatments, with significant but not resolving effects. This evidence from clinical settings suggests the importance of additional contributing pathophysiological mechanisms beyond elevated IOP that also drive disease progression or pathogenesis (e.g., normal-tension glaucoma). Summary of the Invention

[0010] Thus, there is a need in the art for effective means of protecting neural tissue outside the brain, particularly neural tissue in the eye or retina of an animal or human. One aspect of the present invention relates to protecting neural tissue outside the brain by modulating neurotrophins to maintain or restore levels found in a healthy state in an animal or human. For example, in at least one embodiment, neurotrophin levels are modulated to within a range considered normal or healthy for an animal or human.

[0011] This Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0012] The present invention, as defined in the appended claims, relates to a spearmint extract and its use for protecting nerve tissue outside the brain in humans and animals. Specifically, orally administered spearmint extract protects nerve cells in the eye and peripheral nervous system from damage, dysfunction, and death, and serves as nutritional support in the management of neurodegenerative conditions in these organs and systems, such as dry eye, AMD, diabetic retinopathy, glaucoma, diabetic neuropathy, and neuropathic pain. A particular aspect of the present invention relates to a method of administering an effective amount of spearmint extract to a subject to regulate levels of a neurotrophin, namely, NGF, which is associated with neurodegeneration. According to at least one embodiment, protection of nerve tissue outside the brain is achieved by regulating neurotrophins to maintain or restore levels found in healthy conditions. For example, in at least one embodiment, neurotrophin levels are regulated to a range considered normal for the subject. In at least one embodiment, administration of the spearmint extract reduces nerve cell loss or, alternatively, restores ganglion cell function to normal as measured by electrophysiological recordings.

[0013] Further, by way of non-limiting example, in at least one embodiment, administration of spearmint extract results in at least a 10% increase in NGF levels, e.g., at least a 25% increase in NGF levels detected in neural tissue outside the brain, e.g., levels detected in tears. Still further, in certain embodiments, administration of spearmint extract results in at least a 50% increase in NGF levels in tissue outside the brain. In other embodiments, total NGF levels may be doubled above baseline levels after administration of spearmint extract. In certain embodiments, spearmint extract was administered before and during elevated intraocular pressure. In certain embodiments, spearmint extract was administered at least once daily for at least 14 days.

[0014] In certain embodiments, the methods of the present invention involve administering a spearmint extract for neuroprotection by administering a composition containing the spearmint extract to a subject, wherein the spearmint extract comprises at least about 5% to about 20% rosmarinic acid by weight. In certain embodiments, the spearmint extract contains two or more polyphenolic compounds, and further comprises about 10% to about 50% total phenolics by weight. According to certain embodiments, the spearmint extract is administered to an animal at a dose corresponding to a human-equivalent dose of about 1 to about 20 mg / kg BW / day. [Brief explanation of the drawings]

[0015] [Figure 1]Figure 1 shows the percent change from baseline in paw volume 3 and 6 hours after intraplantar injection of carrageenan into the paws of animals administered intraperitoneally either three doses of spearmint extract (10, 30, and 100 mg / kg), two doses of RA standard (15 and 50 mg / kg), or vehicle (carrageenan). Saline bars (gray) show the percent change in paw volume of control animals that received intraplantar injections of saline (saline-saline group). Carrageenan (red bars) caused an increase in paw volume that was significantly attenuated by indomethacin, all doses of spearmint extract, and the high dose of RA. The low dose of RA (15 mg / kg) contained the same amount of RA as the 100 mg / kg spearmint extract group. Data are presented as mean ± SEM (n = 4 or 6 per group). *p<0.05 vs. carrageenan; **p<0.01 vs. carrageenan; #p<0.05 vs. RA 15 mg / kg (pairwise comparisons using nonparametric Kruskal-Wallis test followed by Mann-Whitney U test). RA = rosmarinic acid, CGN = carrageenan, IND = indomethacin. Fonseca et al., (2017, Anti-inflammatory, Exp Biology). [Figure 2] Figure 2 is a chart showing IOP values ​​throughout the course of the experiment. MCE injection increased IOP, while SPE or vehicle (placebo) had no effect on IOP elevation. Data are shown as mean ± SEM (n = 10 for each group). Day 0 corresponds to the day of MCE intraocular injection. Healthy controls did not receive MCE injection and had no change in IOP. [Figure 3]Figure 3A shows ERG waveforms (representing retinal activity) recorded under dark-adapted conditions at a light intensity of 10 cd·s / m² in control rats and MCE-injected rats given either vehicle or SPE. Figure 3B shows a graph representing the photoreceptor a-wave amplitude assessed under dark-adapted conditions (light intensity of 10 cd·s / m²) in control rats and MCE-injected rats given either vehicle or SPE. The photoreceptor response to flash light was not affected by MCE, vehicle, or SPE, as evidenced by the lack of change in a-wave amplitude. Data are shown as mean ± SEM (n = 10 for each group). Figure 3C shows a graph representing the post-photoreceptor b-wave amplitude assessed under dark-adapted conditions (light intensity of 10 cd·s / m²) in control rats and MCE-injected rats given either vehicle or SPE. The post-photoreceptor response to flash light was not affected by MCE, vehicle, or SPE, as evidenced by the lack of change in b-wave amplitude. Data are presented as mean ± SEM (n=10 for each group). [Figure 4]Figure 4A shows ERG waveforms recorded under photopic conditions with a stimulus light of 3 cd·s / m² on a rod-saturating background light of 30 cd·s / m² in healthy control rats and MCE-injected rats given either vehicle or SPE. MCE, vehicle, or SPE did not affect photopic b-wave amplitude (reflecting post-photoreceptor activity specific to cone photoreceptors), whereas SPE prevented the MCE-induced decrease in PhNR amplitude. Data are shown as mean ± SEM (n = 10 for each group). **P < 0.01 and ****P < 0.0001 vs. control; §§§P < 0.001 and §§§§P < 0.0001 vs. MCE; ###P < 0.001 vs. low-dose SPE (one-way ANOVA followed by multiple comparison Tukey's test). Figure 4B shows a graph depicting cone photoreceptor-specific post-photoreceptor activity, as reflected by photopic b-wave amplitude, in control rats and rats injected with MCE given either vehicle or SPE. This figure demonstrates that MCE, vehicle, or SPE did not affect activity, as evidenced by no change in b-wave amplitude. Data are presented as mean ± SEM (n = 10 for each group). Figure 4C shows a graph depicting RGC-specific activity, as reflected by photopic PhNR amplitude, in control rats and rats injected with MCE given either vehicle or SPE. SPE dose-dependently attenuated the MCE-induced decrease in PhNR amplitude. Data are presented as mean ± SEM (n = 10 for each group). **P<0.01 and ****P<0.0001 vs. control; §§§P<0.001 and §§§§P<0.0001 vs. vehicle; ###P<0.001 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). [Figure 5]Figure 5A shows PERG charts reflecting specific RGC function, showing two negative peaks (N35 and N95) and a positive peak P50 in healthy control rats and rats injected with MCE given either vehicle or SPE. The figure demonstrates that SPE attenuated the MCE-induced decrease in PERG amplitude. Figure 5B shows a graph representing the mean amplitude of the N35-P50 wave. MCE reduced the amplitude of this wave, and this effect was significantly attenuated by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n = 10 for each group). ***P < 0.001 and ****P < 0.0001 vs. control; §§§§P < 0.0001 vs. vehicle; #P < 0.05 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). Figure 5C shows a graph representing the mean amplitude of the P50-N95 wave. MCE reduced the amplitude of this wave, and this effect was significantly attenuated by SPE in a dose-dependent manner. (Data are shown as mean ± SEM (n = 10 for each group). ***P<0.001 and ****P<0.0001 vs. control; §§§§P<0.0001 vs. vehicle; ###P<0.001 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). [Figure 6] Figure 6A shows images depicting RBPMS staining in the central and peripheral regions of the retina from control rats and rats injected with MCE given either vehicle or SPE. Scale bar: 100 μm. Data are presented as mean ± SEM (n = 10 for each group). SPE prevented the MCE-induced decrease in RGC density, as evidenced by the density of white dots on the images. Figure 6B graphically depicts the analysis of RBPMS-positive cell density collected separately from the peripheral and central regions of the retina. MCE injection reduced RGC density, an effect that was attenuated / prevented by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n = 10 for each group). ***P<0.001 and ****P<0.0001 vs. control; §§P<0.01 and §§§§P<0.0001 vs. vehicle; ###P<0.0001 vs. SPE treatment (one-way ANOVA followed by multiple comparison Tukey's test). [Figure 7] Figure 7A shows Western blots representing the neurotrophins BDNF and NGF from retinal homogenates of healthy control rats or rats that underwent MCE with either vehicle or SPE. SPE attenuated the MCE-induced decrease in the levels of both neurotrophins. β-actin represents the loading control. Figure 7B shows densitometry analysis of NGF levels normalized to the corresponding β-actin density. MCE resulted in a decrease in NGF levels, and this effect was significantly attenuated by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n = 10 for each group). ***P < 0.001 and ****P < 0.0001 vs. control; §§§P < 0.001 and §§§§P < 0.0001 vs. vehicle; ####P < 0.0001 vs. SPE administration (one-way ANOVA followed by multiple comparison Tukey's test). Figure 7C shows densitometry analysis of BDNF levels normalized to the corresponding β-actin density. MCE resulted in a decrease in BDNF levels, and this effect was significantly attenuated by SPE in a dose-dependent manner. Data are shown as mean ± SEM (n = 10 for each group). ***P < 0.001 and ****P < 0.0001 vs. control; §§P < 0.01 and §§§§P < 0.0001 vs. vehicle; ##P < 0.01 vs. SPE treatment (one-way ANOVA followed by multiple comparison Tukey's test). [Figure 8]Figure 8A graphically depicts the levels of the oxidative stress biomarker MDA in healthy control rats or rats undergoing MCE with either vehicle or SPE. MCE increased the levels of MDA. This effect was significantly attenuated by SPE in a dose-dependent manner. (Data are shown as mean ± SEM (n = 10 for each group). *P < 0.05 and ****P < 0.0001 vs. control; §§P < 0.01 and §§§§P < 0.0001 vs. vehicle; #P < 0.05 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). Figure 8B graphically depicts the levels of the oxidative stress biomarker MDA in control rats or rats undergoing MCE with either vehicle or SPE. The graph shows the levels of 8-OHdG. MCE increased the levels of 8-OHdG. This effect was significantly attenuated by SPE in a dose-dependent manner. Data are shown as mean ± SEM (n = 10 for each group). ****P < 0.0001 vs. control; §P < 0.05 and §§§§P < 0.0001 vs. vehicle; ##P < 0.01 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). Figure 8C graphically depicts the levels of the oxidative stress biomarker 4-HNE in control rats or rats that underwent MCE with either vehicle or SPE. MCE increased 4-HNE levels. This effect was attenuated / prevented by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n = 10 for each group). ****P < 0.0001 vs. control; §§§§P < 0.0001 vs. vehicle; ##P < 0.01 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). Figure 8D graphically depicts the levels of the endogenous antioxidant GSH in the retinas of control rats or rats that underwent MCE with either vehicle or SPE. MCE reduced GSH levels. This effect was attenuated / prevented by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n = 10 for each group).****P<0.0001 vs. control; §§P<0.01 and §§§§P<0.0001 vs. vehicle; ##P<0.01 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). Figure 8E shows Western blots representing Nrf2 and HO-1 from retinal homogenates of healthy control rats or rats undergoing MCE with either vehicle or SPE. SPE prevented the MCE-induced upregulation of the cellular antioxidant response mediated by Nrf2 and HO-1. β-actin represents the loading control. Figure 8F shows densitometry analysis of intraretinal levels of Nrf2 normalized to the corresponding β-actin density from healthy control rats or rats undergoing MCE with either vehicle or SPE. MCE resulted in increased levels of Nrf2, an effect that was completely prevented or attenuated by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n = 10 for each group). ****P < 0.0001 vs. control; §§§§P < 0.0001 vs. vehicle; ####P < 0.0001 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). Figure 8G shows densitometry analysis of intraretinal levels of HO-1 normalized to the corresponding β-actin density in healthy control rats or rats that underwent MCE with either vehicle or SPE. MCE resulted in increased levels of HO-1, an effect that was completely prevented or attenuated by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n = 10 for each group). *P<0.05 and ****P<0.0001 vs. control; §§§P<0.001 and §§§§P<0.0001 vs. vehicle; ##P<0.01 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). [Figure 9]Figure 9A shows Western blots representing NF-kB, IL-6, IL-1β, and IL-10 from retinal homogenates of healthy control rats or rats that underwent MCE with either vehicle or SPE. SPE prevented the increase in MCE-induced inflammatory responses mediated by all evaluated compounds. β-actin represents the loading control. Figure 9B shows densitometric analysis of the levels of phosphorylated forms of NF-kB normalized to the corresponding β-actin density in healthy control rats or rats that underwent MCE with either vehicle or SPE. MCE resulted in an increase in the levels of NK-kB, an effect that was completely prevented or attenuated by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n = 10 for each group). ****P<0.0001 vs. control; §§§§P<0.0001 vs. vehicle; ####P<0.01 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). Figure 9C shows densitometry analysis of IL-6 levels normalized to the corresponding β-actin density in healthy control rats or rats that underwent MCE with either vehicle or SPE. MCE resulted in increased levels of IL-6, but this effect was significantly attenuated by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n=10 for each group). *P<0.05 and ****P<0.0001 vs. control; §§§P<0.001 and §§§§P<0.0001 vs. vehicle; ####P<0.0001 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). Figure 9D shows densitometry analysis of IL-1β levels normalized to the corresponding β-actin density in healthy control rats or rats undergoing MCE with either vehicle or SPE. MCE resulted in increased levels of IL-1β, an effect that was completely prevented or greatly attenuated by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n = 10 for each group).***P<0.001 and ****P<0.0001 vs. control; §§§§P<0.0001 vs. vehicle; ##P<0.01 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). Figure 9E shows densitometry analysis of IL-10 levels normalized to the corresponding β-actin density in healthy control rats or rats that underwent MCE with either vehicle or SPE. MCE resulted in a decrease in IL-10 levels, but this effect was completely prevented or greatly attenuated by SPE in a dose-dependent manner. Data are presented as mean ± SEM (n=10 for each group). ***P<0.001 and ****P<0.0001 vs. control; §§§§P<0.0001 vs. vehicle; ##P<0.01 vs. SPE low dose (one-way ANOVA followed by multiple comparison Tukey's test). [Figure 10] Figure 10A is a schematic diagram of the putative pathophysiological mechanism of glaucoma. In glaucoma, several factors combine to lead to an imbalance in RGC metabolic activity, regardless of whether IOP is elevated. Increased oxidative stress and inflammation, two interrelated processes, combined with the resulting decrease in RGC nutrition, lead to a decline in RGC function and viability. Figure 10B is a schematic diagram of the effect of SPE on the pathophysiological mechanism of glaucoma. SPE likely exerts antioxidant effects due to the scavenging properties of its main component, RA, and other spearmint polyphenols, thereby inhibiting the increase in IOP-induced oxidative stress. Inhibition of oxidative processes consequently impacts the inflammatory response, reducing inflammation and thereby attenuating the alterations in RGC metabolism that typically occur in both high- and normal-tension glaucoma. Restoration of metabolic balance is reflected by enhanced RGC neurotrophy, which correlates with the preservation of both cellular activity and viability. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description of the Invention The spearmint extract referred to in this invention is a proprietary ingredient sourced from a patented, non-GMO line of native spearmint (Mentha spicata L.) grown in the United States and developed using traditional plant breeding methods. See U.S. Patent Nos. 9,545,075 and 9,545,076, incorporated herein by reference in their entireties. See also Cirlini M, Mena P, Tassotti M, et al. Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract. Molecules. 2016;21:1007, incorporated herein by reference in its entirety.

[0017] Spearmint extract possesses a distinctive and consistent phenolic fingerprint and contains a combination of potent phenolic compounds, including, but not limited to, RA, salvianolic acid, caffeic acid, caffeic acid, quinic acid, and lithospermic acid. Commercially available spearmint is known to contain polyphenols, including rosmarinic acid, but the levels typically found in native commercial spearmint are significantly lower than those found in patented spearmint extracts. Specifically, these plants have the ability to accumulate rosmarinic acid (RA) at levels exceeding 100 mg / g on a dry weight basis, a significantly higher concentration than previously reported in conventional spearmint plants, where RA typically ranges from 7.1 to 58.5 mg / g (Narasimhamoorthy (2015)). Recognizing that this ingredient is a plant extract with a complex polyphenol profile, researchers have observed unexpected interactions and synergistic effects not present in single molecules alone. For example, some of the observed interactions may enhance or reduce the effects of RA. Therefore, results are unpredictable. In other words, one skilled in the art will understand that results obtained using RA alone in an in vitro model or animal study will not necessarily translate directly to an in vitro model or animal study using spearmint extract.

[0018] Rosmarinic acid (RA) is an important contributor to the antioxidant capacity of spearmint (Fletcher et al. Heat stress reduces the accumulation of rosmarinic acid and the total antioxidant capacity in spearmint (Mentha spicata L). Journal of the Science of Food and Agriculture 85: 2429-2436, 2005). RA is a natural phenolic compound, an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. Its structure consists of a carbonyl group, an unsaturated double bond, and a carboxylic acid between two phenolic rings. RA has demonstrated several biological activities, including anti-inflammatory, antimutagenic, antibacterial, antidepressant, HIV-1 inhibitory, antioxidant, and antiviral properties. These properties make RA an attractive raw material ingredient for the pharmaceutical and cosmetic industries. RA has been used topically in Europe as a nonsteroidal anti-inflammatory drug (Ritschel et al. Percutaneous absorption of rosmarinic acid in the rat. Methods and Findings in Experimental and Clinical Pharmacology 11: 345-352, 1989). Because of its widespread use as a flavoring and preservative in the food industry, RA is considered a safe ingredient for daily consumption (Alkam et al. A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Aβ25-35. Behavioral Brain Research 180: 139-145, 2007).

[0019] Preclinical studies have shown that spearmint extract is effective in reducing markers of oxidative stress and inflammation, promotes neuroprotection of neural tissue, and supports blood-brain barrier function in animal models of cognitive decline or stroke. Furthermore, spearmint extract has been shown to modulate brain neurotransmitters and promote neurogenesis in hippocampal neurons in rats. Additionally, clinical studies have shown that spearmint extract is well tolerated and helps improve cognitive function (working memory and attention) in adults (Falcone PH, Nieman KM, Tribby AC, et al. Nutr Res. 2019;64:24-38., Falcone PH, Tribby AC, Vogel RM, et al. J Int Soc Sports Nutr. 2018;15:58., Herrlinger KA, Nieman KM, Sanoshy KD, et al. Spearmint Extract Improves Working Memory in Men and Women with Age-Associated Memory Impairment. J Altern Complement Med. 2018;24:37-47).

[0020] Conversely, little is known about the effectiveness of spearmint extract in supporting neural health outside the brain, such as in the eye or peripheral nervous tissue. Research focused on age-related macular degeneration (AMD), diabetic retinopathy (DR), and glaucoma suggests that in these ocular conditions, neurodegenerative changes are not limited to retinal neurons but also affect the central nervous system, both structurally and functionally. Nuzzi R and Vitale A. Cerebral Modifications in Glaucoma and Macular Degeneration: Analysis of Current Evidence in Literature and Their Implications on Therapeutic Perspective. Eye and Brain 2021:13 159-173.

[0021] Metabolic stress and bioenergetic failure play a key role in the progression of these neurodegenerative ocular pathologies, leading to neuronal death and dysfunction through the promotion of oxidative stress and inflammation. In this context, the use of spearmint extract, which has been shown to be beneficial for brain health and function, may provide appropriate nutritional support. Furthermore, the levels of neurotrophic factors such as NGF may play an important role in the progression of ocular neurodegenerative pathologies. For example, it has been reported that a lack of neurotrophin support induces the apoptotic death of neurons such as RGCs. Lambuk L, Mohd Lazaldin MA, Ahmad S, et al. Brain-Derived Neurotrophic Factor-Mediated Neuroprotection in Glaucoma: A Review of Current State of the Art. Front Pharmacol. 2022;13:875662; Kimura A, Namekata K, Guo X, Harada C, Harada T. Neuroprotection, Growth Factors, and BDNF-TrkB Signaling in Retinal Degeneration. Int J Mol Sci. 2016;17:1584. Inclusion of spearmint extract in a multi-component mixture demonstrated significant efficacy in blocking oxidative stress, inflammatory processes, and morphofunctional changes resulting from glaucoma pathology in various animal models of glaucoma.

[0022] In the present disclosure, researchers have confirmed the effects of spearmint extract in reducing oxidative stress, markers of inflammation, and its ability to improve the viability and function of ocular neurons. However, they have also newly and unexpectedly identified for the first time that orally administered spearmint extract can improve neuronal health and function outside the brain, i.e., in the retina, through a statistically significant, dose-dependent increase in the tissue supply of neurotrophins (specifically, NGF). In at least one embodiment, a composition containing spearmint extract is administered to a subject in need thereof, wherein the spearmint extract comprises at least about 5% by weight of rosmarinic acid, e.g., at least about 10% by weight, such as at least about 14% by weight or at least about 20% by weight of rosmarinic acid. In at least one embodiment, the rosmarinic acid is present in an amount ranging from about 14.5 to about 17.5% rosmarinic acid.

[0023] According to certain embodiments, the spearmint extract contains one or more, e.g., two or more, polyphenolic compounds, and further comprises at least about 10% by weight, e.g., at least 20% by weight, total phenols. According to at least one embodiment, the spearmint extract contains at least about 50% by weight total phenols. In at least one embodiment, the spearmint extract comprises about 24% to about 37% by weight total phenols. According to certain embodiments, the spearmint extract is administered to a subject at a dose corresponding to a human-equivalent dose of about 1 to about 20 mg / Kg BW / day.

[0024] The present invention, as defined in the appended claims, relates to a spearmint extract and its use for protecting nerve tissue outside the brain in humans and animals. Specifically, orally administered spearmint extract protects nerve cells in the eye and peripheral nervous system from damage, dysfunction, and death, and serves as nutritional support in the management of neurodegenerative conditions in these organs and systems, such as dry eye, AMD, diabetic retinopathy, glaucoma, diabetic neuropathy, and neuropathic pain. A particular aspect of the present invention relates to a method of administering an effective amount of spearmint extract to a subject to regulate the level of a neurotrophin, i.e., NGF, which is associated with neurodegeneration. According to at least one embodiment, protection of nerve tissue outside the brain is achieved by regulating neurotrophins to maintain or restore levels found in the subject under healthy conditions. For example, in at least one embodiment, neurotrophin levels are regulated to a range considered normal for the subject. In at least one embodiment, administration of the spearmint extract reduces neuronal cell loss or, alternatively, restores ganglion cell function to normal as measured by ERG.

[0025] Further, by way of non-limiting example, in at least one embodiment, administration of spearmint extract results in at least a 10% increase in NGF levels, e.g., at least a 25% increase in NGF levels detected in neural tissue outside the brain, e.g., levels detected in tears. Still further, in certain embodiments, administration of spearmint extract results in at least a 50% increase in NGF levels in tissue outside the brain. In other embodiments, total NGF levels may be doubled above baseline levels after administration of spearmint extract. In certain embodiments, spearmint extract was administered before and during the increase in blood pressure. In certain embodiments, spearmint extract was administered at least once daily for at least 14 days.

[0026] In certain embodiments, the methods of the present invention relate to administering a spearmint extract for neuroprotection by administering a composition containing the spearmint extract to a subject in need thereof, wherein the spearmint extract comprises at least about 5% to about 20% by weight of rosmarinic acid. In certain embodiments, the spearmint extract contains two or more polyphenolic compounds, and further comprises about 10% to about 50% by weight of total phenols. According to certain embodiments, the spearmint extract is administered to a subject at a dose corresponding to a human equivalent dose of about 1 to about 20 mg / kg BW / day.

[0027] According to at least one embodiment of the present invention, the nervous tissue is present in the eye or retina of a subject. For example, in certain embodiments, the health and function of the subject's nervous tissue relates to nerve cells within the subject's eye or the subject's peripheral nervous system. By way of non-limiting example, in certain embodiments, a subject in need thereof may be suffering from symptoms of dry eye, age-related macular degeneration, diabetic retinopathy, glaucoma, and other degenerative eye conditions. In at least one embodiment, the health and function of the nervous tissue can be measured by electrophysiological testing, such as, but not limited to, electroretinography (ERG) and electroretinography (PhNR).

[0028] In alternative embodiments, a subject in need thereof may be experiencing symptoms associated with diabetic neuropathy and neuropathic pain, and administering an effective amount of spearmint extract results in a reduction or lessening of the severity of symptoms associated with diabetic neuropathy and neuropathic pain, which may be measured by tests such as, but not limited to, electrophysiological recording, OCT, peripheral nerve pain scale, electromyography, and nerve conduction velocity.

[0029] Example the purpose To determine whether spearmint extract can support the health and function of neurons outside the brain, we conducted a study to examine the effect of oral treatment with two doses of spearmint extract (SPE) in preventing RGC degeneration and dysfunction in a rat model of methylcellulose (MCE)-induced ocular hypertension glaucoma.

[0030] Materials and Methods Dietary Supplement: SPE is a proprietary water-extracted spearmint (Neumentix®) sourced from patented, non-GMO lines of native spearmint (Mentha spicata L.) cultivated in the United States and developed by Kemin using traditional plant breeding methods, as described in U.S. Patent Nos. 9,545,075 and 9,545,076, the entire disclosures of which are incorporated herein by reference. The extract is standardized to 14.5-17.5% rosmarinic acid and 24-37% total phenolics. These proprietary plants have the ability to accumulate greater than 100 mg / g rosmarinic acid (RA) on a dry weight basis, a significantly higher concentration than reported concentrations for conventional spearmint, which range from 7.1 to 58.5 mg / g. Combined with their high RA content, these plants possess a unique phenolic fingerprint. Ultra-high performance liquid chromatography-mass spectrometry of SPE indicates that this product has a distinctive and consistent phenolic fingerprint and contains a combination of potent phenolic compounds, including but not limited to rosmarinic acid, salvianolic acid, caffeic acid, caftaric acid, quinic acid, and lithospermic acid. See Amato R et al. Efficacy of a spearmint (Mentha spicata L.) extract as nutritional support in a rat model of hypertensive glaucoma (in press).

[0031] Dry spearmint extract consists of a blend of polyphenols, including the key molecule rosmarinic acid and its derivatives, along with lesser amounts of salvianolic acid, caffeoylquinic acid, hydroxybenzoic acid, and hydroxycinnamic acid. The phenolic fraction of spearmint extract has been fully characterized by ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS), revealing the presence of a total of 66 compounds in the extract. See Cirlini M, Mena P, Tassotti M, et al. Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract. Molecules. 2016;21:1007. The total amount of the polyphenol blend in the spearmint extract analyzed by UHPLC-ESI-MS was 263 mg / g. Analysis of the extracts along with the percentage of specific polyphenols shows that rosmarinic acid accounts for approximately 88% of the total amount of polyphenols detected, followed by salvianolic acid at 6% and caffeoylquinic acid at 1%, as well as hydroxycinnamic acids (including caftaric acid) at also approximately 1% (Table 3).

[0032] [Table 3] Two doses of SPE were evaluated in the glaucoma animal studies. The doses were calculated by converting the human dose found to have cognitive benefits in clinical trials to account for metabolic differences between the two species and the doses used in previous preclinical studies. - High Dose (SPE-High): 93.0 mg / Kg BW / day (equivalent to a human daily dose of 15 mg / Kg BW, or 900 mg / day for a 60 kg human) - Low Dose (SPE-Low): 46.5 mg / Kg BW / day (equivalent to a human daily dose of 7.5 mg / Kg BW, or 450 mg / day for a 60 kg human) High- and low-dose solutions of SPE were prepared fresh daily in distilled water immediately before treatment. SPE doses were administered by oral gavage daily for 14 days before and 14 days after MCE injection.

[0033] Animal experiments Forty animals were tested in the experiment. The animals were randomly and equally assigned to four groups: one group (10 rats) of healthy controls (Group 1 - Control; rats not receiving MCE injections or nutritional support), and three groups of glaucomatous rats (10 rats / group) randomly assigned to receive MCE injections and oral nutritional support with: vehicle (MCE + Vehicle; Group 2); low-dose SPE (MCE + SPE - Low Dose; Group 3); high-dose SPE (MCE + SPE - High Dose; Group 4). 15 days after MCE administration, the animals were subjected to efficacy evaluation and euthanized.

[0034] MCE-induced ocular hypertension model Induction of intraocular hypertension was performed according to published procedures (Dal Monte et al., 2020). Briefly, 2% wt / vol MCE in sterile saline was prepared to a solution viscosity ranging from 3500 to 5600 cps. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg / kg), and 15 μL of MCE solution was injected into the anterior chamber of both eyes using a Hamilton syringe equipped with an 18G needle. The needle was inserted into the iridocorneal angle approximately 1 mm from the ora serrata and oriented parallel to the iris surface. After slow injection of MCE (duration 1 min), the needle was held in place for 1 min to avoid spillage of MCE.

[0035] Evaluation items IOP measurement To test the effect of treatment on elevated intraocular pressure, a time-dependent IOP profile was constructed for each experimental group. IOP was assessed noninvasively daily using rebound tonometry before and after MCE injection in each group over the course of the study (28 days). Multiple sampling procedures (5–10 times) were performed at the same time each day.

[0036] Electroretinogram recording for assessment of RGC activity The effects of SPE treatment on glaucomatous RGC dysfunction were analyzed using ERG recordings. In particular, two major functional outputs related to RGC activity were analyzed: the photopic negative response (PhNR) and the pattern ERG (PERG). These two procedures provide information on intraretinal responses in competition with global photopic retinal activity (PhNR) and specific RGC activity (PERG). These two parameters were analyzed both by themselves and in relation to overall retinal activity as assessed by dark-adapted ERGs. ERG recordings were performed at the end of the treatment period. After overnight dark adaptation, each rat was anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg / kg) and gently restrained in a custom-made holder that did not obstruct the visual field. Corneal moisture was maintained during the ERG routine by instilling balanced salt solution every 15 min. Dark-adapted ERG responses were obtained after a single 10 cd·s / m² flash light stimulus on a dark background. After 10 minutes of light adaptation, rats were then subjected to photopic ERG recordings using a 3 cd·s / m² stimulus against a 30 cd·s / m² rod-saturated background. Responses to 10 consecutive stimuli with a 3-second interstimulus interval were recorded and averaged. PERG recordings were then performed using pattern stimuli consisting of black and white bars at 0.05 cycles / degree, presented at 98% contrast and reversed at 1 Hz. The pattern stimuli were applied through a light-emitting diode display with an average luminance of 50 cd / m², aligned approximately 20 cm from the corneal surface. A total of 200 signals were averaged. ERG waveforms were analyzed for consistency and subjected to signal processing and noise filtering. In photopic ERG waveforms, the PhNR was identified as the first negative deflection after the b-wave. PERG waveforms were analyzed for both positive (N35-P50) and negative (P50-N95) components. The amplitudes of PhNR and PERG, as well as the latency of PERG, were considered as analytical parameters related to RGC function.

[0037] Immunofluorescence analysis of RGC density The effect of SPE on glaucomatous RGC loss was assessed by analyzing RGC density using immunofluorescence. Briefly, rats were euthanized immediately after ERG recording, and the retinas were dissected free from other ocular tissues using a microsurgical procedure. Isolated retinas were immersion-fixed in 4% wt / vol paraformaldehyde and stored at 4°C. Retinas were subjected to whole-mount immunostaining for mRNA processing factors (RBPMS), an RNA-binding protein widely established as an RGC-specific marker. After processing, retinas were analyzed using an epifluorescence microscope equipped with a motorized stage for organotypic whole-mount reconstruction. The resulting images were automatically analyzed for RBPMS-positive cell density after sampling four radially opposed images at two different radial eccentricities (0.5 mm = central, 4 mm = peripheral from the optic nerve head) to obtain the average RGC density in the periphery and center of the retina.

[0038] Oxidative stress and inflammation The effectiveness of SPE in contrasting glaucoma-induced oxidative stress was evaluated using both Western blot analysis for typical markers of endogenous antioxidant responses and a colorimetric assay to assess retinal oxidative status. Specifically, Western blot analysis was performed to assess the protein levels of erythroid transcription factor 2-related transcription factor 2 (Nrf2), a transcription factor sensitive to ROS, and heme oxygenase-1 (HO-1), one of the antioxidant enzymes involved in the defense response against oxidative stress. Additionally, the levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and 8-hydroxy-deoxyguanosine (8-OH-dG), as markers of oxidative stress, and glutathione (GSH), a major antioxidant in the retina, were assessed using specific kits. The efficacy of SPE in blocking the inflammatory process was assessed by Western blot analysis for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), a key transcriptional regulator of pro-inflammatory factors, and interleukin-1β (IL-1β) and IL-6 as relative pro-inflammatory cytokines. In addition, the levels of IL-10, a key mediator of inflammation resolution through the primary suppression of pro-inflammatory cytokines, were also assessed.

[0039] Neurotrophins Neurotrophins play an important role in neuronal survival. Under healthy conditions, RGCs can receive neurotrophin support from Müller cells or through retrograde axonal transport from the brain. Neurotrophin deficiency is associated with glaucoma. To evaluate the effectiveness of SPE in restoring neuronal nutrition, we assessed retinal levels of NGF and BDNF by Western blot analysis.

[0040] Data analysis A priori power analysis was performed in accordance with the 3R principles for the ethical use of animals in scientific research (G *GPower 3.0.10 (www.gpower.hhu.de). Sample size was calculated considering an α of 0.05, an effect size of at least 0.6 (high enough to assess significant differences between groups), and a statistical power of at least 0.80. Animal treatments were performed in an unblinded manner. Researchers performing routine ERG, immunohistochemistry, Western blot, and oxidative stress marker measurements were blinded to the treatment groups. Blinding was achieved by assigning numeric code identifiers to samples. Unblinding was performed after data collection. For data analysis of IOP and ERG results, the mean value of both eyes was obtained as the representative measurement for each rat. Data were analyzed using the Shapiro-Wilk test to confirm normal distribution. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test in Prism 8.0.2 (GraphPad Software, Inc., San Diego, CA, USA). Data are presented as mean ± SEM for the reported n values. Differences at p<0.05 were considered statistically significant.

[0041] result SPE has no effect on MCE-induced intraocular hypertension The IOP profiles of MCE-treated rats, assessed by rebound tonometry, revealed a significant increase in IOP levels, reaching a peak of approximately 34 mmHg within 24 hours after MCE injection (Figure 2). Thereafter, IOP gradually decreased over time, although it remained elevated compared to controls. MCE rats treated with low or high doses of SPE exhibited IOP profiles similar to those of untreated MCE rats, with no significant differences in either the initial IOP peak or the subsequent gradual decline.

[0042] SPE treatment has beneficial effects on RGC-related ERG parameters in a dose-dependent manner During the ERG routine performed by the researchers, scotopic ERGs were performed at the end of the experimental protocol to obtain global photoreceptor activity, reflected by the scotopic a-wave, and global post-photoreceptor activity, reflected by the scotopic b-wave. Both scotopic ERG parameters were unaffected by MCE injection or treatment with either dose of SPE (Figures 3A, 3B, and 3C). Thus, the light-adapted b-wave, reflecting cone-specific global post-photoreceptor activity, showed no change in amplitude in any of the experimental groups. In contrast, PhNR amplitude, reflecting RGC-specific activity, was significantly reduced in untreated MCE rats compared with controls (Figures 4A, 4B, and 4C). The reduction in PhNR amplitude was attenuated dose-dependently after SPE treatment. Definitive confirmation of RGC functional changes in untreated MCE rats was obtained by evaluation of PERG responses, whose amplitudes were halved compared with controls in both the N35-P50 and P50-N95 components. Treatment with SPE resulted in significant preservation of PERG amplitudes for both N35-P50 and P50-N95 components (Figures 5A, 5B, and 5C). Together, these data suggest that treatment with SPE significantly prevents RGC-specific functional changes following MCE-induced intraocular hypertension in a dose-dependent manner.

[0043] SPE preserves RGC density after MCE-induced IOP elevation Immunostaining for RBPMS in whole-mount retinas revealed typical differences in RGC density between the peripheral and central parts of the retina. Intracameral injection of MCE proportionally reduced RGC density in both the central and peripheral parts of the retina. Treatment with SPE resulted in a dose-dependent maintenance of RGC density (Figures 6A and 6B). The beneficial effects of treatment were proportionally equivalent in both parts of the retina, with the high dose maintaining RGC density in the central retina at control levels.

[0044] Treatment with SPE promotes the maintenance of neurotrophy under MCE-induced glaucomatous stress Neurotrophy was assessed by analyzing the protein levels of BDNF and NGF, critical neurotrophins involved in maintaining RGC viability. As demonstrated by Western blot analysis, retinal levels of both BDNF and NGF were significantly reduced in untreated MCE mice. Rats receiving both doses of SPE exhibited higher protein levels of both neurotrophins in a dose-dependent manner compared to untreated MCE mice (Figures 7A, 7B, and 7C).

[0045] SPE blocks MCE-induced oxidative stress process The effect of SPE in preventing MCE-induced oxidative stress was analyzed at different levels of the oxidative process. In particular, Western blot and colorimetric analysis were performed to assess the levels of oxidation-derived products such as MDA, 8-OH-dG, and 4-HNE, the depletion of the endogenous antioxidant GSH, and the activation status of the endogenous antioxidant response, with NRF2 as the master transcriptional regulator and HO-1 as one of the major induced endogenous antioxidants (Figures 8A-G). As demonstrated by colorimetric assay, the levels of MDA, 8-OH-dG, and 4-HNE in the retinas of untreated MCE rats were significantly increased compared to controls, resulting in a significant depletion of GSH. The increase in oxidation products was reflected in the induction of the endogenous antioxidant response, as demonstrated by an increase in NRF2 and subsequent increase in HO-1 protein levels. Treatment with SPE caused a dose-dependent inhibition of oxidative stress-related phenomena. Indeed, the levels of oxidative stress products in treated retinas were significantly lower than those in controls, consistent with the higher levels of GSH. The reduction in oxidative stress was also reflected by SPE-induced protection from MCE-induced increases in both Nrf2 and HO-1.

[0046] Treatment with SPE blocks MCE-induced inflammatory mechanisms The inflammatory process involved in glaucomatous RGC degeneration was analyzed by examining the levels of activated (phosphorylated) NF-kB, one of the key transcriptional regulators of the proinflammatory response, and the associated levels of pro- and anti-inflammatory cytokines (Figures 9A-9E). Western blot analysis revealed that phosphorylated NF-kB was increased in the retinas of untreated MCE mice, which was accompanied by increased protein levels of the pro-inflammatory cytokines IL-6 and IL-1β and a significant decrease in the anti-inflammatory cytokine IL-10. Treatment with SPE dose-dependently inhibited the pro-inflammatory process, as demonstrated by a significant decrease in pNF-kB and subsequent decreases in the levels of IL-6 and IL-1β and an increase in the anti-inflammatory cytokine IL-10.

[0047] Scientific Implications Both low- and high-dose SPE-exposed animals showed reduced neuronal cell (RGC) loss and attenuated functional impairment as a result of damage induced by stressors such as elevated IOP. Importantly, the preservation of neuronal function and density was dose-dependent. Examination of the possible underlying mechanisms of neuroprotection revealed significant dose-dependent reductions in markers of oxidative stress and inflammation, as well as the important novel finding that oral supplementation with a natural plant extract resulted in increased levels of the neurotrophin NGF under glaucoma conditions, a model of neuronal stress and damage (Figures 10A and 10B).

[0048] Glaucoma provides a model of neurodegeneration and is understood as a multifactorial condition influenced by elevated IOP and other mechanisms, such as oxidative stress, inflammation, and lack of neurotrophic support. Recent advances in understanding neurodegenerative phenomena in glaucoma point to the central role of metabolic imbalances in retinal neurons (RGCs) as early alterations that promote neuronal complications and cell death in the pre-disease phase. Guymer C, Wood JPM, Chidlow G, Casson RJ. Neuroprotection in glaucoma: recent advances and clinical translation. Clin Exp Ophthalmol. 2019;47:88-105. These metabolic changes in neurons result in mitochondrial dysfunction accompanied by increased oxidative stress, activated inflammatory processes, decreased neurotrophin levels, and consequent cell damage and death. As shown in this experiment, the oxidative, inflammatory, and neurotrophic damage to neurons typical of glaucoma pathology is well reproduced by the MCE model of glaucoma. Our results demonstrate that oral SPE is effective in preventing neuronal degeneration and dysfunction outside the brain by not only reducing markers of oxidative stress and inflammation but also attenuating the decline in the production or availability of neurotrophins, particularly NGF. This novel finding is particularly significant. Indeed, insufficient neurotrophin supply to RGCs is a common mechanism in various retinal neurodegenerative diseases, and the use of neurotrophins to prevent retinal degeneration has been widely discussed. Furthermore, neurotrophins are key for neuronal survival and synaptic plasticity in the optical system and are involved in learning and memory processes. Metabolic and neurotrophic imbalances in retinal tissue can be associated with morphological and functional changes in many brain regions along and outside the visual pathway. The effectiveness of oral SPE in maintaining neurotrophin levels under conditions of neuronal stress may further expand the suitability of this ingredient for nutritional support specific to ocular disorders, in which neurotrophic changes typically drive neurovascular breakout.

[0049] conclusion Using an animal model of glaucoma, the researchers were able to examine neuronal activity outside the brain. The retina is a thin tissue covering the back of the eyeball, composed of 10 layers of neurons connected to each other by synapses. Retinal ganglion cells (RGCs) constitute one of the outermost layers and are the retina's main output neurons, sending their axons to the brain via the optic nerve. The retina provides an ideal structure for examining neuronal function outside the brain due to its easy accessibility. In glaucoma, RGCs degenerate, leading to a decline in visual ability and ultimately blindness. One possible cause of glaucoma is elevated intraocular pressure. Therefore, animal models of elevated intraocular pressure provide a way to examine neuronal degeneration outside the eye, possible underlying mechanisms of degeneration, along with potential therapeutic interventions to halt or slow neuronal loss outside the brain.

[0050] In this experiment, animals were orally administered a unique spearmint aqueous extract containing high levels of key polyphenols to evaluate the effect of spearmint on retinal neuronal degeneration. The researchers artificially elevated intraocular pressure to induce rapid degeneration of retinal neurons. The increase in intraocular pressure is shown in Figure 2. The researchers then assessed the health of the neurons by measuring their electrical activity, which indicates how well the neurons communicate with each other. Neuronal electrical activity can be measured using a technique known as electroretinogram (ERG). If neurons are damaged, the ERG will exhibit different electrical activity compared to healthy, functioning neurons. Additionally, the researchers simply counted the number of neurons that remained viable in the retina after exposure to elevated intraocular pressure (IOP).

[0051] We found that RGC neurons degenerated in animals exposed to elevated intraocular pressure compared with control animals. We found that administering spearmint extract to animals before and during the pressure increase dose-dependently reduced the amount of neuronal death, as seen in Figures 6A and 6B. Additionally, when we examined the electrical activity of retinal neurons, we found that photoreceptors remained intact, but RGC neurons were dysfunctional. Again, oral administration of spearmint extract dose-dependently reduced RGC dysfunction, as seen in the PhNR and PERG results in Figures 4C, 5B, and 5C. To understand the underlying mechanisms that may protect RGC neurons in this glaucoma animal model, we assessed neurotrophin levels 15 days after IOP elevation. We found that NGF levels were decreased under conditions of intraocular hypertension, and that spearmint extract administration resulted in a dose-dependent increase in neurotrophin levels (Figures 7A and 7B).

[0052] The researchers found that, surprisingly and unexpectedly, the benefits in this study support low doses, produce novel results not seen before, and have overall benefits (complementary interventions) that aid in the management of neurodegeneration. Benefits, including increased NGF levels, preservation of retinal ganglion neuron density and functionality, and reduced markers of oxidative stress and inflammation, may be seen alone or in combination with other supplemental ingredients, in a variety of models (combinations), and in a variety of animals.

[0053] While the present invention has been described with reference to particular compositions, theories of operation, and the like, it will be apparent to those skilled in the art that the invention is not intended to be limited to such exemplary embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit of the present invention as defined in the appended claims. All such obvious modifications and variations are intended to be included within the scope of the present invention as defined in the appended claims. The claims are intended to encompass the claimed components and steps in any order that is effective to meet the intended purpose, unless the context clearly indicates otherwise.

[0054] It is also understood that minor dosage and formulation variations of the compositions and ranges expressed herein may be made and still fall within the scope and spirit of the invention. It should also be understood that the formulations and processes illustrated in the accompanying drawings and described in the following specification are merely exemplary embodiments of the inventive concepts defined in the appended claims. Accordingly, specific dimensions and other physical characteristics relating to the embodiments disclosed herein should not be considered as limiting, unless expressly stated otherwise in the claims. Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intermediate value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intermediate value within that stated range, is encompassed within the scope of the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the scope of the disclosure, except where any limit in the stated range is specifically excluded. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the scope of the disclosure. All ranges and parameters disclosed herein, including but not limited to percentages, parts, and ratios, are understood to encompass all subranges contemplated and incorporated therein, and all numbers between the endpoints. For example, a range stated as "1 to 10" should be considered to include any subrange beginning at the minimum value of 1 or more and ending at the maximum value of 10 or less (e.g., 1 to 6.1, or 2.3 to 9.4), and each integer subsumed within that range (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Throughout this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. As used herein, all combinations of method or process steps may be performed in any order unless otherwise specified or unless the contrary is clearly implied by the context in which the combination is recited.

[0055] To the extent the term "includes," "including," "have," or "having" is used herein or in the claims, it is intended to be inclusive, similar to the interpretation of "comprising" when that term is used as a transitional phrase in the claims. Furthermore, to the extent the term "or" is used (e.g., A or B), it is intended to mean either "A" or "B," or both "A" and "B." Where applicant intends to indicate "A or B, but not both," the term "A or B, but not both" or similar construction would be used. Thus, the use of the term "or" herein is inclusive, not exclusive. Also, to the extent the term "in" or "into" is used herein or in the claims, it is intended to mean "on" or "onto." In this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0056] The foregoing description has been presented for purposes of illustration and description. It is not intended to be an exhaustive list or to limit the invention to the precise form disclosed. It is intended that other alternative processes and methods apparent to those skilled in the art be considered within the scope of the present invention. The description is merely an example of an embodiment. It is understood that any other modifications, substitutions, and / or additions may be made and are within the intended spirit and scope of the present disclosure. From the foregoing, it can be seen that the exemplary aspects of the present disclosure achieve at least all of their intended objectives.

Claims

1. A method for protecting neural tissue outside the brain in animals and humans, comprising orally administering to a subject in need thereof an effective amount of a spearmint extract containing rosmarinic acid and two or more polyphenols sufficient to regulate the level of nerve growth factor (NGF) present in the neural tissue, wherein the extract is water-soluble and contains at least 5% by weight of rosmarinic acid and at least 10% by weight of total polyphenols.

2. 10. The method of claim 1, wherein the neural tissue is present in the eye or retina of a subject, and the effective amount is sufficient to attenuate damage to retinal cells.

3. 10. The method of claim 1, wherein the spearmint extract comprises at least 10% by weight of rosmarinic acid.

4. 10. The method of claim 1, wherein the spearmint extract contains at least 14% by weight of rosmarinic acid.

5. 5. The method of claim 4, wherein the spearmint extract comprises from about 14.5% to about 17.5% rosmarinic acid.

6. 10. The method of claim 1, wherein the spearmint extract comprises at least 20% by weight of total phenolics.

7. 10. The method of claim 1, wherein the spearmint extract comprises from about 24% to about 37% total phenolics by weight.

8. 10. The method of claim 1, wherein the spearmint extract is administered to the animal at a dose corresponding to a human equivalent dose range of about 1 to 20 mg / kg BW / day.

9. 1. A method for promoting neuronal health and function outside the brain, comprising orally administering to a subject in need thereof an effective amount of a spearmint extract containing rosmarinic acid and two or more polyphenols sufficient to regulate the level of NGF present in the neural tissue, thereby maintaining or restoring the viability and function of the neural cells, wherein the extract is water-soluble and contains at least 5% by weight of rosmarinic acid and at least 10% by weight of total polyphenols.

10. 10. The method of claim 9, wherein the neural tissue is present in the eye or retina of the subject.

11. 10. The method of claim 9, wherein the nervous tissue health and function relates to nerve cells in the subject's eye or nerve cells in the subject's peripheral nervous system.

12. 10. The method of claim 9, wherein the subject in need thereof may be suffering from symptoms of dry eye, age-related macular degeneration, diabetic retinopathy, glaucoma, and other degenerative eye conditions.

13. 10. The method of claim 9, wherein the health and function of the neural tissue can be measured by electrophysiological tests such as, but not limited to, electroretinogram (ERG) recordings, including dark-adapted and light-adapted ERG, and PERG.

14. 10. The method of claim 9, wherein the nerve tissue health and function relates to improving the peripheral nervous system and reducing symptoms associated with diabetic neuropathy and neuropathic pain.

15. 10. The method of claim 9, wherein the subject in need thereof is experiencing symptoms associated with diabetic neuropathy and neuropathic pain that can be measured by tests such as, but not limited to, electrophysiological recording, OCT, peripheral nerve pain scale, electromyography, and nerve conduction velocity.

16. 10. The method of claim 9, wherein the spearmint extract is administered to the subject at a dose corresponding to a human equivalent dose range of about 1 to 20 mg / kg BW / day.

17. A method for regulating NGF present in neural tissue outside the brain, comprising orally administering to a subject in need thereof an effective amount of a spearmint extract containing rosmarinic acid and two or more polyphenols sufficient to restore the level of NGF present in said neural tissue to a normal range, said extract being water-soluble and containing at least 5% by weight of rosmarinic acid and at least 10% by weight of total polyphenols.

18. 18. The method of claim 17, wherein the neural tissue is present in the eye or retina of the subject.

19. 18. The method of claim 17, wherein the subject in need thereof may be suffering from symptoms of dry eye, age-related macular degeneration, diabetic retinopathy, glaucoma, and other degenerative eye conditions.

20. 18. The method of claim 17, wherein the spearmint extract is administered to the subject at a dose corresponding to a human equivalent dose range of about 1 to 20 mg / kg BW / day.

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