CN117304054A

CN117304054A - Enrichment method of caffeoylspermidine compound, anti-aging property and method for inhibiting histone deacetylase

Info

Publication number: CN117304054A
Application number: CN202310067460.5A
Authority: CN
Inventors: 杜军; 陈亮; 甘小娜; 张璐; 李波; 张雪; 杨彬睿; 李峰; 朱喆思
Original assignee: Access Business Group International LLC
Current assignee: Access Business Group International LLC
Priority date: 2023-01-12
Filing date: 2023-01-12
Publication date: 2023-12-29

Abstract

A process for preparing a powder of a caffeoylspermidine compound is provided. Methods of enriching lycium ruthenicum fruit to provide an extract as a product are also provided. Methods of inhibiting histone deacetylase are also provided.

Description

Enrichment method of caffeoylspermidine compound, anti-aging property and method for inhibiting histone deacetylase

Technical Field

The present application relates to methods of enriching compounds, as well as anti-aging properties and inhibition of the compounds, and compositions comprising the compounds.

Background

Spermidine is the most abundant polyamine in most different human tissues. Spermidine is produced by amino acid metabolism and includes essential cellular functions including regulation of cell growth, proliferation, tissue regeneration, DNA and RNA stabilization, enzymatic regulation (enzymatic modulation), translational regulation, and autophagy. In addition, spermidine exhibits anti-inflammatory and antioxidant properties, enhances mitochondrial metabolic function and respiration, promotes chaperone (chaperone) activity, and improves protein homeostasis. During normal aging, intracellular spermidine concentrations decrease. In contrast, the administration of spermidine is associated with an increase in survival of yeasts, helminths, drosophila and human immune cells, and with a decrease in death rate associated with the age of the mice. The very high concentration of spermidine in semen prevents cell senescence and confers long-term survival to germ cell lines. Spermidine homeostasis is affected by nutrient uptake, intestinal origin, endogenous biosynthesis, degradation and the active transport system between compartments.

Many of the anti-aging properties of spermidine are causally related to the ability of spermidine to ensure protein homeostasis by stimulating cytoprotective giant autophagy. Spermidine induces autophagy by inhibiting several acetyltransferases. Maintaining optimal spermidine concentrations in humans for optimal autophagy levels for healthy aging is an ongoing topic of research. Age-induced decline in spermidine may involve a change in one or several different factors that determine systemic availability of spermidine. The bioavailability of spermidine is determined by the different sources of this polyamine: (i) cellular biosynthesis, (ii) production of intestinal microorganisms, and (iii) nutrient supply, and (iv) catabolism and (v) urine excretion.

Lycium ruthenicum (Lycium ruthenicum) is a flowering plant commonly known as "Russian Lycium ruthenicum". Lycium ruthenicum is a member of the Solanaceae family, distributed in the middle asia, southern Russia, northwest China, northern India and Pakistan. Lycium ruthenicum grows in the saline-alkali deserts, sandy lands and roadsides of 400-3000 m in the northwest of China and in the middle asia.

Caenorhabditis elegans (Caenorhabditis elegans, "c.elegans") is an independently living nematode of about 1mm in length that lives in a mild soil environment. Caenorhabditis elegans has been the model organism for studying senescence. For example, inhibition of the insulin-like growth factor signaling pathway may increase adult life by three times, while glucose feeding may promote oxidative stress and reduce adult life by half. Also, induction of insulin/IGF-1 receptor degradation later in life may significantly extend the life expectancy of worms. Furthermore, caenorhabditis elegans exposed to 5mM lithium chloride (LiCl) showed a longer lifetime. When exposed to 10 μm LiCl, a reduction in mortality was observed. However, no mortality reduction was observed with exposure to 1. Mu.M caenorhabditis elegans.

The Kaplan-Meier estimator is a non-parametric statistic used to estimate the survival function from the lifecycle data. In the study, kaplan-Meier estimates are typically used to measure the proportion of patients who survive a certain period of time after treatment.

Histone deacetylases ("HDACs") are a class of enzymes that remove acetyl groups from epsilon-N-acetyllysine amino acids on histones (o=c-CH ₃ ) Thereby allowing the histones to more tightly encapsulate the DNA. DNA is wrapped around the histone and expressedAcetylation and deacetylation regulation. Because of the amine groups present on the lysines and arginines of the histone tails, the histone tails are typically positively charged, which facilitates interaction and binding of the histone tails with negatively charged phosphate groups on the DNA backbone. Acetylation generally occurs in cells, neutralizing the positive charge on the histone by turning the amine into an amide, and reducing the ability of the histone to bind to DNA. This reduced binding allows chromatin expansion, allowing gene transcription to occur. However, histone deacetylases remove these acetyl groups, increasing the positive charge of the histone tail and promoting high affinity binding between the histone and the DNA backbone. Increased DNA binding concentrates the DNA structure, thereby preventing transcription.

Histone deacetylase inhibitors ("HDACi") have long been used in psychiatry and neurology as mood stabilizers and antiepileptics, such as valproic acid. Recently, HDACi has been studied as an alleviation or treatment agent of neurodegenerative diseases. In addition, there has been a recent effort to develop HDACi for cancer therapy. Vorinostat (Vorinostat) was approved by the FDA in 2006 for the treatment of cutaneous clinical manifestations in patients with Cutaneous T Cell Lymphoma (CTCL) who failed prior therapy. The second HDACi, istodax, was approved for CTCL patients in 2009. HDACi also has an effect on non-histone proteins associated with acetylation, and can alter the degree of acetylation of these molecules, increasing or inhibiting their activity.

There are four subtypes (I, II, III and IV) of HDAC. Class I mediates most of the cellular deacetylation activity and consists of HDAC1, HDAC2, HDAC3 and HDAC 8.

HDAC1 and HDAC2 tend to be overexpressed in colon, breast, prostate, thyroid, cervical, gastric, hepatocellular, pancreatic, endometrial, and lung cancers. It is reported that there is a negative correlation with overall survival in colorectal, hepatocellular, pancreatic, endometrial, and lung cancers. Furthermore, associations with advanced disease, metastasis and invasive disease have been found in hepatocellular, colorectal, prostate, NSCLC and breast cancers. HDAC1 and HDAC2 play a role in transcription and promotion of cancer progression pathways. HDAC1 and HDAC2 overexpression results in cell proliferation, migration, angiogenesis, increased invasion and decreased apoptosis. HDAC1 is known to promote proliferation of breast cancer by inhibiting ER transcription. Deletions of HDAC1 and HDAC2 lead to G1 phase or G2/M phase arrest and reduce breast cancer proliferation.

HDAC3 plays a role in regulating site-specific transcription. HDAC3 is a component of the NCOR/SMRT inhibition complex that is recruited by nuclear hormone receptors and other site-specific DNA binding factors to inhibit expression of individual genes.

HDAC8 is associated with a number of disease states, including acute myelogenous leukemia, and with actin cytoskeleton in smooth muscle cells. Sirnas targeting HDAC8 showed anticancer effects. Inhibition of HDAC 8-induced apoptosis has been observed in T cell lymphomas. Furthermore, HDAC8 enzymes are involved in the pathogenesis of neuroblastomas.

An effective method of enriching dicaffeoylspermidine derivatives would represent a beneficial contribution to the art. Furthermore, derivatives exhibiting anti-aging properties would represent a beneficial contribution to the art. Furthermore, it has been demonstrated that derivatives inhibiting histone deacetylase would represent a useful contribution to the art.

Disclosure of Invention

In one example, the present disclosure provides a composition comprising an extract of lycium ruthenicum fruit and an additive. The extract may comprise a compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) spermidine, and combinations thereof. The composition can increase the survival rate of caenorhabditis elegans. The composition can increase the survival rate of caenorhabditis elegans by at least 10%. The extract may comprise at least 15 wt% N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine. Extract can be packagedContaining at least 20% by weight of N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine. The extract may comprise at least 25 wt% N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine. The composition can inhibit histone deacetylase activity. The histone deacetylase can be selected from the group consisting of HDAC1, HDAC2, HDAC3, HDAC8, and combinations thereof. The histone deacetylase can be HDAC1, wherein activity is inhibited by at least 50%. The extract may comprise at least one HDAC8 specific inhibitor. The at least one specific inhibitor may comprise N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine. N (N) ¹ -dihydrocaffeoyl-N ¹⁰ The caffeoylspermidine may have an IC of about 41.75. Mu. Mol/L ₅₀ Values. The at least one specific inhibitor may comprise N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine. N (N) ¹ -caffeoyl-N ¹⁰ The dihydrocaffeoylspermidine may have an IC of about 62.52. Mu. Mol/L ₅₀ Values. The extract may be the product of a process for enriching from dried lycium ruthenicum fruit, the process comprising: maintaining the fruit in 75% ethanol in water at reflux for at least two hours to provide a mixture of extraction compounds dissolved in ethanol and water; submitting the mixture to ion exchange resin chromatography to provide an extract solution dissolved in an eluting solvent, wherein the resin used for chromatography is a weak cation exchange resin; and concentrating the extract solution under reduced pressure to provide an extract. The method may further comprise: after submission and before concentration: washing the eluting solvent with saturated sodium bicarbonate solution; loading an eluting solvent onto a nonpolar microporous adsorbent resin; eluting the extract with 95% ethanol to obtain ethanol solution of the extract. The yield of the extract may be from about 1.10% to about 1.45% of the dry lycium ruthenicum fruit mass. The retaining may include: maintaining the fruit at reflux in a volume of 75% aqueous ethanol for two hours to provide a mixture of extracted compounds dissolved in ethanol and water; pouring the mixture from the fruit; adding a second volume of 75% aqueous ethanol; maintaining the fruit at reflux in the second volume for two hours to provide a second mixture of extracted compounds dissolved in ethanol and water; pouring the second mixture from the fruit A material; the first mixture is combined with the second mixture. The method may further comprise freeze drying the extract after concentrating. The composition can be used for neuroprotection. The composition can be used for treating and/or preventing Alzheimer's disease. The composition can be used for anti-aging treatment. The composition can be used for topical administration. The composition can be used as cosmetic. The composition can be used as a nutritional supplement.

In another example, the present disclosure provides a method of producing a caffeoylspermidine powder comprising: heating the dried lycium ruthenicum fruit in 75% ethanol water solution at reflux for at least two hours to provide a mixture of extraction compounds dissolved in ethanol and water; submitting the mixture to ion exchange resin chromatography to provide a solution of a powder of a caffeoylspermidine compound dissolved in an eluting solvent, wherein the resin used in the chromatography is a weak cation exchange resin; and concentrating the solution under reduced pressure to obtain a powder of the caffeoylspermidine compound. The caffeoylspermidine compound powder may comprise a compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine and combinations thereof. The method may further comprise: after submission and before concentration: washing the eluting solvent with saturated sodium bicarbonate solution; loading an eluting solvent onto a nonpolar microporous adsorbent resin; eluting the caffeoylspermidine compound powder with 95% ethanol to obtain ethanol solution of caffeoylspermidine compound powder. Heating may include: maintaining the fruit at reflux in a volume of 75% aqueous ethanol for two hours to provide a first mixture of extraction compounds dissolved in ethanol and water; pouring the first mixture from the fruit; adding a second volume of 75% aqueous ethanol; maintaining the fruit at reflux in the second volume for two hours to provide a second mixture of extracted compounds dissolved in ethanol and water; pouring the second mixture from the fruit; the first mixture is combined with the second mixture. The method may further comprise freeze-drying the powder of the caffeoylspermidine compound after concentrating.

In yet another example, the present disclosure provides a method of enriching for caffeoylspermidine compounds extracted from dried lycium ruthenicum fruit, comprising submitting a mixture of caffeoylspermidine compounds to ion exchange resin chromatography to provide a solution of the compounds in an eluting solvent, wherein the resin used in the chromatography is a weak cation exchange resin.

In yet another example, the present disclosure provides a method of inhibiting histone deacetylase comprising administering a composition comprising a dry lycium ruthenicum extract and an additive. The histone deacetylase can be HDAC1, HDAC2, HDAC3, HDAC8 or a combination thereof. Histone deacetylases can be specifically inhibited.

In yet another example, the present disclosure provides a composition comprising i) a compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof; and ii) an additive. The composition can be used for neuroprotection. The composition can be used for treating and/or preventing Alzheimer's disease. The composition can be used for anti-aging treatment. The composition can be used for topical administration. The composition can be used as cosmetic. The composition can be used as a nutritional supplement.

In yet another example, the present disclosure provides a supplement to spermidine comprising: i) A compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine,N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof; and ii) an additive. The compound is at least partially hydrolyzable in vivo to provide spermidine.

Further areas of applicability will become apparent from the description provided herein. It should be understood that the description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.

Drawings

In order that the present disclosure may be well understood, a description thereof in various forms will now be given by way of example with reference to the accompanying drawings. The components in the drawings are not necessarily to scale. Furthermore, in the drawings, like reference numerals designate corresponding parts throughout the different views.

FIG. 1 illustrates purified high content caffeoylspermidine, N, prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -high performance liquid chromatography chromatograms of uv detection of caffeoylspermidine ("AA 001");

FIG. 2 illustrates the purification of high content caffeoylspermidine, N, prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -a profile of the results of positive ion mass spectrometry ("MS") performed on caffeoylspermidine ("AA 001");

FIG. 3 illustrates purified high content caffeoylspermidine, N prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine ("AA 001") ¹ H nuclear magnetic resonance ("NMR") spectroscopy;

FIG. 4 illustrates purified high content caffeoylspermidine, N prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine ("AA 001") ¹³ C nuclear magnetic resonance ("NMR") spectroscopy;

fig. 5 shows an HPLC chromatogram of a 75% ethanol extract from lycium ruthenicum fruit prepared according to the principles of the present disclosure.

FIG. 6 illustrates an N-enriched preparation made in accordance with the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-pyri-dine)Pyranoglucosyl) -caffeoylspermidine and N ¹ -caffeoyl-N ¹⁰ HPLC chromatogram of the extracted component of dihydrocaffeoylspermidine ("mix");

FIG. 7 shows N prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ HPLC chromatogram of caffeoylspermidine;

FIG. 8 shows an HPLC chromatogram of an extracted component that does not include caffeoylspermidine prepared according to the principles of the present disclosure;

FIG. 9 shows N prepared according to the principles of the present disclosure with 50 μM and 100 μM ¹ Hydrogen caffeoyl-N ¹⁰ Survival of caenorhabditis elegans (top or middle line) treated with caffeoylspermidine over time compared to control (bottom line);

FIG. 10 illustrates N prepared according to principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine ("AA 001"), N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine ("AA 002") and N ¹ -caffeoyl-N ¹⁰ HPLC chromatogram of dihydrocaffeoylspermidine ("AA 003");

fig. 11 shows an HDAC1 activity inhibition assay demonstrating N-enriched prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine and N ¹ -caffeoyl-N ¹⁰ -inhibition of the extracted portion of lycium ruthenicum fruit by dihydrocaffeoylspermidine (MixD), comprising comparison with HDAC inhibitor trichostatin A ("TSA") as positive control;

fig. 12 shows an HDAC2 activity inhibition assay demonstrating N-enriched prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine and N ¹ -caffeoyl-N ¹⁰ Inhibition of the extract fraction of Lycium ruthenicum fruit of Dihydrocaffeoylspermidine (MixD), including with as positive controlThe HDAC inhibitor trichostatin a ("TSA");

fig. 13 shows an HDAC3 activity inhibition assay demonstrating N-enriched prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine and N ¹ -caffeoyl-N ¹⁰ -inhibition of the extracted portion of lycium ruthenicum fruit by dihydrocaffeoylspermidine (MixD), comprising comparison with HDAC inhibitor trichostatin A ("TSA") as positive control;

fig. 14 shows an HDAC8 activity inhibition assay demonstrating N-enriched prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine and N ¹ -caffeoyl-N ¹⁰ -inhibition of the extracted portion of lycium ruthenicum fruit by dihydrocaffeoylspermidine (MixD), comprising comparison with HDAC inhibitor trichostatin A ("TSA") as positive control;

Fig. 15 shows a generic HDAC inhibition assay demonstrating N-enriched prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine and N ¹ -caffeoyl-N ¹⁰ -inhibition of the extracted portion of lycium ruthenicum fruit by dihydrocaffeoylspermidine (MixD), comprising comparison with HDAC inhibitor trichostatin A ("TSA") as positive control;

FIGS. 16A and 16B illustrate N prepared according to principles of the present disclosure, respectively ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine (AA 001) and N ¹ -caffeoyl-N ¹⁰ HPLC chromatogram of dihydrocaffeoylspermidine (AA 003);

FIG. 17 shows a schematic diagram illustrating N prepared according to principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ Modeling of ligand-receptor interactions and docking postures of caffeoylspermidine (AA 001) with HDAC 8;

FIG. 18 shows a display of N prepared according to principles of the present disclosure ¹ -caffeoyl-N ¹⁰ Modeling of ligand-receptor interactions and docking postures of dihydrocaffeoylspermidine (AA 003) with HDAC 8;

fig. 19 shows modeling showing ligand-receptor interactions and docking postures of trichostatin a (TSA) with HDAC8 prepared according to principles of the present disclosure;

FIG. 20 shows an HDAC8 activity inhibition assay demonstrating N prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -inhibition of caffeoylspermidine (AA 001) at various concentrations, including comparison with HDAC inhibitors as positive control (TSA);

FIG. 21 shows an HDAC8 activity inhibition assay demonstrating N prepared according to the principles of the present disclosure ¹ -caffeoyl-N ¹⁰ -inhibition of dihydrocaffeoylspermidine (AA 003) at different concentrations, including comparison with HDAC inhibitors as positive control (TSA);

FIG. 22 shows N prepared according to principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -a graph of half maximal inhibitory concentration of caffeoylspermidine (AA 001) inhibiting HDAC 8;

FIG. 23 shows N prepared according to the principles of the present disclosure ¹ -caffeoyl-N ¹⁰ -a plot of half maximal inhibitory concentration of dihydrocaffeoylspermidine (AA 003) inhibiting HDAC 8;

FIG. 24 shows N prepared according to the principles of the present disclosure ¹ -dihydrocaffeoyl-N ¹⁰ -a graph of the effect of caffeoylspermidine (AA 001) on OP50 growth;

FIG. 25 shows N prepared according to the principles of the present disclosure for control (DMSO), or for a concentration of 50 μM or 100 μM ¹ -dihydrocaffeoyl-N ¹⁰ -a graph of the number of nematodes of caffeoylspermidine (AA 001);

FIG. 26 shows N prepared according to the principles of the present disclosure for control (DMSO), or for a concentration of 50 μM or 100 μM ¹ -dihydrocaffeoyl-N ¹⁰ -a plot of the number of offspring of nematodes of caffeoylspermidine (AA 001);

FIG. 27 shows N prepared according to the principles of the present disclosure for control (DMSO), or for a concentration of 50 μM or 100 μM ¹ -dihydrocaffeoyl-N ¹⁰ -body flexural frequency plot of nematodes of caffeoylspermidine (AA 001) within 30 seconds;

FIG. 28 shows N prepared according to the principles of the present disclosure for control (DMSO), or for a concentration of 50 μM or 100 μM ¹ -dihydrocaffeoyl-N ¹⁰ -a frequency plot of pharyngeal pumping (pharyngeal pumps) of nematodes of caffeoylspermidine (AA 001) within 30 seconds; and

FIG. 29 shows N prepared according to the principles of the present disclosure for control (DMSO), or for a concentration of 50 μM or 100 μM ¹ -dihydrocaffeoyl-N ¹⁰ -graph of percentage of movement of the aerial worms at day 9, day 12, day 15 and day 18 from time zero of caffeoylspermidine (AA 001).

The drawings described herein are for illustration purposes only and are not intended to limit the scope of the present disclosure in any way.

Detailed Description

The following description is merely exemplary in nature and is not intended to limit the present disclosure, application, or uses. It should be understood that throughout the drawings, corresponding reference numerals indicate like or corresponding parts and features.

Where reference numerals are added to elements of each figure, although the same elements are shown on different figures, it should be noted that the same elements have the same numerals.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "plurality" is defined by the applicant in the broadest sense to mean more than one number, superseding any other implicit definition or limitation above or below, unless the applicant explicitly asserts the contrary. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

As used herein, the terms "comprising," "including," "having," "can," "containing," and variations thereof are intended to be open-ended terms, or words that do not exclude the possibility of additional acts or structures. Other examples, "comprising," "consisting of," and "consisting essentially of," are also contemplated by this specification, whether or not explicitly stated, examples or elements are presented herein.

In describing elements of the present disclosure, the terms 1 st, 2 nd, first, second, A, B, (a), (b), etc. may be used herein. These terms are only used to distinguish one element from another element, but do not limit the corresponding element, regardless of the nature or order of the corresponding elements.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Terms defined in the general dictionary should be interpreted to have the same meaning as the contextual meaning of the related art.

As used herein, the term "about" when used in the context of the listed values or ranges means a variation of ±15% or less of the value. For example, values differing by + -15%, + -14%, + -10% or + -5%, etc., will satisfy the definition of "about" unless defined narrower in a particular case.

The term "N", as used herein ¹ -dihydrocaffeoyl-N ¹⁰ The term "caffeoylspermidine" and "AA001" refer to chemical abstracts ("CAS") accession number 121850-61-1, molecular formula C ₂₅ H ₃₃ N ₃ O ₆ A compound of the lycium ruthenicum fruit having the structural formula:

N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine and AA001 may also be referred to as "N ¹ Hydrogen caffeoyl-N ¹⁰ -caffeoylspermidine. "

The term "N", as used herein ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine "and" AA002 "are represented by formula C ₃₁ H ₄₃ N ₃ O ₁ A compound of the lycium ruthenicum fruit having the structural formula:

the term "N", as used herein ¹ -caffeoyl-N ¹⁰ Dihydrocaffeoylspermidine and "AA003" are those having CAS registry number 1807893-35-1 and molecular formula C ₂₅ H ₃₃ N ₃ O, a compound of lycium ruthenicum fruit with the following structural formula:

the term "N", as used herein ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine "means a compound of formula C ₃₁ H ₄₃ N ₃ O ₁₁ A compound of the lycium ruthenicum fruit having the structural formula:

the term "N", as used herein ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine "refers to a compound of formula C ₂₅ H ₃₅ N ₃ O ₆ A compound of the lycium ruthenicum fruit having the structural formula:

the term "N", as used herein ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine "refers to a compound of formula C ₂₅ H ₃₁ N ₃ O ₆ A compound of the lycium ruthenicum fruit having the structural formula:

as used herein, the term "specific inhibitor" refers to an enzyme inhibitor that exerts a specific effect on a particular enzyme, rather than affecting all enzymes in the same way.

As used herein, the abbreviation "HDAC" refers to histone deacetylases, an enzyme that removes acetyl groups from epsilon-N-acetyllysine amino acids on histones (o=c-CH ₃ ) Thereby allowing the histones to more tightly encapsulate the DNA.

As used herein, the abbreviation "HDAC1" refers to histone deacetylase 1.

As used herein, the abbreviation "HDAC2" refers to histone deacetylase 2.

As used herein, the abbreviation "HDAC3" refers to histone deacetylase 3.

As used herein, the abbreviation "HDAC8" refers to histone deacetylase 8.

In one example, a composition comprising the lycium ruthenicum fruit extract of the present disclosure and an additive can increase the survival rate of caenorhabditis elegans. In certain examples, the composition may increase the survival rate of caenorhabditis elegans by at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 11%, or at least about 12%, or at least about 13%, or at least about 14%, or at least about 15%, or at least about 16%, or at least about 17%, or at least about 18%, or at least about 19%, or at least about 20%, or at least about 21%, or at least about 22%, or at least about 22.23%, or at least about 23%, or at least about 24%, or at least about 25%, or at least about 26%, or at least about 27%, or at least about 28%, or at least about 29%, or at least about 30%, or at least about 31%, or at least about 32%, or at least about 33%, or at least about 34%, or at least about 34.86%, or at least about 35%.

In one example, the extract of lycium ruthenicum fruit may include a compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof. In certain examples, the extract may comprise at least about 0.5 wt% N ¹ -dihydrocaffeoyl-N ¹⁰ At least about 1.0 wt.%, or at least about 1.5 wt.%, or at least about 2.0 wt.%, or at least about 2.5 wt.%, or at least about 3.0 wt.%, or at least about 3.5 wt.%, or at least about 4.0 wt.%, or at least about 4.5 wt.%, or at least about 5.0 wt.%, or at least about 5.5 wt.%, or at least about 6.0 wt.%, or at least about 6.5 wt.%, or at least about 7.0 wt.%, or at least about 7.5 wt.%, or at least about 8.0 wt.%, or at least about 8.5 wt.%, or at least about 9.0 wt.%, or at least about 9.5 wt.%, or at least about 10.0 wt.%, or at least about 10.5 wt.%, or at least about 11.0 wt%, or at least about 11.5 wt%, or at least about 12.0 wt%, or at least about 12.5 wt%, or at least about 13.0 wt%, or at least about 13.5 wt%, or at least about 14.0 wt%, or at least about 14.5 wt%, or at least about 15.0 wt%, or at least about 16.0 wt%, or at least about 16.5 wt%, or at least about 17.0 wt%, or at least about 17.5 wt%, or at least about 18.0 wt%, or at least about 18.5 wt%, or at least about 19.0 wt%, or at least about 19.5 wt%, or at least about 20.0 wt%, or at least about 20.5 wt%, Or at least about 21.0 wt%, or at least about 21.5 wt%, or at least about 22.0 wt%, or at least about 22.5 wt%, or at least about 23.0 wt%, or at least about 23.5 wt%, or at least about 24.0 wt%, or at least about 24.5 wt%, or at least about 25.0 wt%, or at least about 25.5 wt%, or at least about 26.0 wt%, or at least about 26.5 wt%, or at least about 27.0 wt%, or at least about 27.5 wt%, or at least about 28.0 wt%, or at least about 28.5 wt%, or at least about 29.0 wt%.

In one example, the extract is the product of a process for concentrating from dried lycium ruthenicum fruit. In certain examples, the method comprises: maintaining the fruit in 75% ethanol in water at reflux for at least two hours to provide a mixture of extracted compounds dissolved in ethanol and water; submitting the mixture to ion exchange resin chromatography to provide an extract solution dissolved in an eluting solvent, wherein the resin used for chromatography is a weak cation exchange resin; and concentrating the extract solution under reduced pressure to obtain an extract. In other examples, the method further comprises, after submitting and before concentrating: washing the eluting solvent with saturated sodium bicarbonate solution; loading an eluting solvent onto a nonpolar microporous adsorbent resin; and eluting the extract with 95% ethanol to obtain an ethanol solution of the extract. In other examples, the maintaining may include: maintaining the fruit at reflux in a volume of 75% aqueous ethanol for two hours to provide a mixture of extracted compounds dissolved in ethanol and water; pouring the mixture from the fruit; adding a second volume of 75% aqueous ethanol; maintaining the fruit at reflux in the second volume for two hours to provide a second mixture of extracted compounds dissolved in ethanol and water; pouring the second mixture from the fruit; the first mixture is combined with the second mixture. In other examples, the method further comprises freeze drying the extract after concentrating.

In one example, the compositions of the present disclosure may be used for neuroprotection. In another example, the compositions of the present disclosure may be used to treat and/or prevent alzheimer's disease. In yet another example, the compositions of the present disclosure may be used in anti-aging treatment. In yet another example, the composition may be for topical administration. In yet another example, the composition may be used as a cosmetic. In yet another example, the composition may be used as a nutritional supplement.

In other examples, the yield of the extract is from about 1.10% to about 1.45% of the dry lycium ruthenicum fruit mass. In other examples, the yield of the extract is about 1.10%, or about 1.11%, or about 1.12%, or about 1.13%, or about 1.14%, or about 1.15%, or about 1.16%, or about 1.17%, or about 1.18%, or about 1.19%, or about 1.20%, or about 1.21%, or about 1.22%, or about 1.23%, or about 1.24%, or about 1.25%, or about 1.26%, or about 1.27%, or about 1.28%, or about 1.29%, or about 1.30%, or about 1.31%, or about 1.32%, or about 1.33%, or about 1.34%, or about 1.35%, or about 1.36%, or about 1.37%, or about 1.38%, or about 1.39%, or about 1.40%, or about 1.41%, or about 1.42%, or about 1.43%, or about 1.44% to about 1.45%; or about 1.10% to about 1.11%, or to about 1.12%, or to about 1.13%, or to about 1.14%, or to about 1.15%, or to about 1.16%, or to about 1.17%, or to about 1.18%, or to about 1.19%, or to about 1.20%, or to about 1.21%, or to about 1.22%, or to about 1.23%, or to about 1.24%, or to about 1.25%, or to about 1.26%, or to about 1.27%, or to about 1.28%, or to about 1.29%, or to about 1.30%, or to about 1.31%, or to about 1.32%, or to about 1.33%, or to about 1.34%, or to about 1.35%, or to about 1.36%, or to about 1.37%, or to about 1.38%, or to about 1.39%, or to about 1.40%, or to about 1.41%, or to about 1.42%, or to about 1.43%, or to about 1.44%, or to about 1.45%. Or one of the above minimum values to one of the above maximum values.

In one example, suitable dosage forms of the compositions of the present disclosure may include tablets, capsules, solutions, suspensions, powders, chewing gums, and candies. Examples of compositions may include supplements. Sublingual delivery systems may include, but are not limited to, sublingual and sublingual dissolvable tablets, drops and beverages. Edible films, hydrophilic polymers, orally dissolvable films or orally dissolvable strips may be used. Other useful delivery systems may include oral or nasal sprays or inhalers, and the like. Other useful delivery systems may include topical compositions, such as patches or cosmetics. Examples of compositions may include additives such as excipients or carriers.

In one example, for oral administration, the extract of the present disclosure may be combined with one or more solid inactive ingredients to prepare tablets, capsules, pills, powders, granules, or other suitable solid dosage forms of the composition. For example, the extract may be combined with at least one excipient selected from the group consisting of fillers, binders, humectants, disintegrants, solution retarders, absorption promoters, wetting agents, absorbents, or lubricants. Other useful excipients may include magnesium stearate, calcium stearate, mannitol, xylitol, sweeteners, starches, carboxymethylcellulose, microcrystalline cellulose, colloidal silicon dioxide, gelatin, silicon dioxide and the like.

In certain examples, for preparing a composition from a compound or extract of the present disclosure, an acceptable carrier may be a solid or a liquid. Solid form preparations suitable for oral administration may include powders, tablets, pills, cachets, lozenges, dragees, capsules, cachets, suppositories, and dispersible granules. The solid carrier may be one or more substances which may also be used as diluents, flavouring agents, solubilising agents, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

In certain examples of the extracts of the present disclosure in formulations including compositions in powder form, the carrier may be a finely divided solid that is admixed with the same finely divided extract. In other examples of the extracts of the present disclosure in the preparation of compositions, including tablet forms, the extracts may be mixed with a carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.

In certain examples, compositions suitable for topical administration in the mouth may include lozenges comprising the extract or compound of the present disclosure in a flavored base (typically sucrose and gum arabic or tragacanth); a lozenge comprising an extract or compound of the disclosure in an inert base (e.g., gelatin and glycerin or sucrose and acacia); and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The term "formulation" may be intended to include the formulation of the extract or compound of the present disclosure with an encapsulating material as a carrier, providing a capsule in which the extract or compound is surrounded by the carrier, with or without other carriers, and thus, is associated with the extract or compound.

In certain examples of the extract of the present disclosure in a formulation comprising a composition in the form of a powder or tablet, the powder or tablet may comprise the extract or compound of the present disclosure in an amount of about 5 wt%, or about 10 wt%, or about 15 wt%, or about 20 wt%, or about 25 wt%, or about 30 wt%, or about 35 wt%, or about 40 wt%, or about 45 wt%, or about 50 wt%, or about 55 wt%, or about 60 wt%, or about 65 wt%, to about 70 wt%; or about 10 wt% to about 15 wt%, or to about 20 wt%, or to about 25 wt%, or to about 30 wt%, or to about 35 wt%, or to about 40 wt%, or to about 45 wt%, or to about 50 wt%, or to about 55 wt%, or to about 60 wt%, or to about 65 wt%; or from any one of the above minimum values to any one of the above maximum values. Examples of suitable carriers may include microcrystalline cellulose, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, and cocoa butter. Examples of other excipients may include magnesium stearate, stearic acid, talc and silica.

In certain examples, a binder may be included in the composition. Examples of binders may include disaccharides such as sucrose or lactose; polysaccharides and derivatives thereof, such as starch, cellulose, modified cellulose (e.g. microcrystalline cellulose or cellulose ethers, such as hydroxypropyl cellulose); sugar alcohols such as xylitol, sorbitol or mannitol; proteins, such as gelatin; synthetic polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG).

In certain examples, a humectant may be included in the composition. Examples of humectants may include propylene glycol, hexylene glycol, butylene glycol, aloe vera gel, alpha hydroxy acids such as lactic acid, egg yolk or egg white, glyceryl triacetate, honey, lithium chloride, molasses, polymeric polyols such as polydextrose, quillaja, sodium hexametaphosphate E452i, urea or castor oil.

In certain examples, a disintegrant may be included in the composition. Examples of the disintegrant may include crosslinked polymers such as crosslinked polyvinylpyrrolidone (crospovidone) or crosslinked sodium carboxymethylcellulose (croscarmellose sodium), or modified starches such as sodium starch glycolate.

In certain examples, a wetting agent may be included in the composition. Examples of the wetting agent may include benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, poloxamer 188, poloxamer 407, polyethylene glycol 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium lauryl sulfate, sorbitan monooleate, or sorbitan monostearate.

In certain examples, a lubricant may be included in the composition. Examples of lubricants may include talc, silica gel or fat, such as vegetable stearin, magnesium stearate or stearic acid.

In certain examples, a sweetener may be included in the composition. Examples of sweeteners may include sugar, corn syrup, aspartame, sucralose, acesulfame potassium, saccharin or xylitol.

In one example, examples of liquid formulations of the extract or compound may include compositions in combination with a carrier, such as a solution, suspension, or emulsion, wherein the carrier may be a fluid, such as a solvent or an emulsifier. In certain examples, the liquid formulation may be water or a water-propylene glycol solution. In other examples, parenteral injection liquid formulations may be formulated as solutions in aqueous polyethylene glycol solutions. Thus, the extracts or compounds of the present disclosure may be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion) and may be presented in unit doses or ampoules, pre-filled syringes, small volume infusions, or in multi-dose containers with added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the extract or compound of the present disclosure may be in powder form, obtained by sterile isolation of sterile solids or by lyophilization from solution for reconstitution with a suitable vehicle, such as sterile, pyrogen-free water, prior to use.

In certain examples, aqueous solutions suitable for oral use may be prepared by dissolving the extracts or compounds of the present disclosure in water and adding suitable colorants, flavors, stabilizers, and thickeners as desired. Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in water with viscous substances, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose or other well known suspending agents.

In certain examples, an emulsifier may be included in a composition that provides for combining two or more immiscible fluids in a single mixture and maintaining the mixture stable. Examples of emulsifiers may include acacia, carbomer copolymer, carbomer interpolymer, cholesterol, coconut oil, diethylene glycol stearate, ethylene glycol stearate, glycerol distearate, glycerol monolinoleate, glycerol monoleate, glycerol monostearate, lanolin alcohol, lecithin, mono and diglycerides, poloxamer, polyoxyethylene 50 stearate, polyoxyethylene 10 oil ether, polyoxyethylene 20 cetyl stearyl ether, polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil, polyoxyethylene 40 stearate, polyoxyethylene lauryl ether, polyoxyethylene stearyl ether, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol monostearate, sodium cetostearyl sulfate (sodium cetostearyl sulfate), sodium lauryl sulfate, sodium stearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, stearic acid, wax.

The solution or suspension may be applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or nebulizer. The compositions may be provided in single or multiple dose form. In compositions intended for administration to the respiratory tract, including intranasal compositions, the extract or compound may have a small particle size, e.g., on the order of 5 microns or less. Such particle sizes may be obtained by means known in the art, for example by micronization.

The formulation may preferably be in unit dosage form. In such forms, the formulation may be subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form may be a packaged formulation, the package containing discrete amounts of the formulation, such as packaged tablets, capsules and powders in vials or ampoules. Furthermore, the unit dosage form itself may be a capsule, tablet, cachet or lozenge, or the unit dosage form may be the appropriate number of any such unit dosage forms in packaged form.

For more details on formulations and administration techniques, please refer to the latest edition Remington' sPharmaceutical Sciences (Mack Publishing co., easton, pa.).

In one example, the cosmetic and/or topical composition comprising the extract or compound of the present disclosure may be in the form of an ointment, cream, lotion, gel, paste, or glycerogelatin, or other form of transdermal delivery system, such as Sample preparation of Pharmaceutical Dosage Forms (b.nickel ed., springer 2011); v. allen, jr., et al, ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems 272 (9 th edition, lippincott Williams & Wilkins 2011), each incorporated herein by reference. Transdermal formulations may be formed from ointments, creams or gels that have been combined with permeation enhancers, and are designed to systematically deliver the extracts or compounds of the present disclosure. In certain examples, a topical composition comprising an extract or compound of the present disclosure may be a prophylactic (e.g., chemo-prophylactic) or protective (e.g., cytoprotective) composition that is administered prior to any sign or symptom of a disorder in a particular individual.

In certain examples, a chemical permeation enhancer may facilitate permeation of an extract or compound of the present disclosure through the skin by increasing the partition of the extract or compound into the stratum corneum barrier domain (barrier domain), increasing the diffusivity of the extract or compound into the stratum corneum barrier domain, or a combination of both. Examples of the permeation enhancer may include alcohols (e.g., ethanol, amyl alcohol, benzyl alcohol, lauryl alcohol, propylene glycol, and glycerin); fatty acids (e.g., linoleic acid, oleic acid, and lauric acid); amines; esters (such as ethyl acetate); amides, hydrocarbons, surfactants, terpenes, sulfoxides (e.g., dimethyl sulfoxide); and phospholipids (e.g., lecithin). See Thong et al Percutaneous penetration enhancers: an overview,20Skin Pharmacol.Physiol.272 (2007); s. Paudel et al Challenges and opportunities in dermal/transdermal delivery,1Ther Deliv.109 (2010); each of which is incorporated herein by reference.

In certain examples, the ointments may be semi-solid formulations, including ointment bases with the extracts or compounds of the present disclosure incorporated or fused therein (in other words, formulations that melt together with the other components of the formulation and cool under continuous agitation to form a coagulum). The ointment base may be of the following form: oleaginous or hydrocarbon bases (e.g., petrolatum or petrolatum/wax combination); an absorbent matrix that allows for incorporation of an aqueous solution resulting in the formation of a water-in-oil emulsion (e.g., hydrophilic petroleum) or that allows for incorporation of additional amounts of an aqueous solution (e.g., lanolin); a water-removable matrix, which is an oil-in-water emulsion, which may be diluted with water or an aqueous solution (e.g., a hydrophilic ointment); or a water-soluble matrix free of oily components (e.g., a polyethylene glycol ("PEG") formulation in which PEG having an average molecular weight below 600 is conjugated to PEG having an average molecular weight above 1,000); etc.

In some examples, the paste may be a formulation containing a greater proportion of solid material, such that the formulation is harder than an ointment.

In certain examples, glycerogelatin may be a formulation comprising gelatin, glycerol, water, and an extract or compound of the present disclosure.

In certain examples, the cream may be a semi-solid formulation comprising the extract or compound of the present disclosure dissolved or dispersed in a water-in-oil emulsion or an oil-in-water emulsion or another type of water-washable matrix. In general, creams differ from ointments in that they may be water-soluble, whereas ointments contain more oil and may be more greasy or viscous than creams and may take longer to be absorbed by the skin than creams.

In certain examples, lotions can be suspensions of solid materials in aqueous vehicles and can have less greasy character and increased spreadability on large areas of skin as compared to ointments, creams and gels.

In certain examples, the gel may be a semi-solid system comprising a dispersion of an extract or compound of the present disclosure in an aqueous liquid vehicle that becomes gelatinous by the addition of a gelling agent. Examples of gelling agents may include synthetic macromolecules (e.g., carbomer polymers), cellulose derivatives (e.g., carboxymethyl cellulose and/or hydroxypropyl methylcellulose), and natural gums (e.g., tragacanth, carrageenan, and the like). The gel formulation may be in the form of a single phase gel in which the extract or compound of the present disclosure is uniformly dispersed throughout the liquid vehicle with no visible boundaries, or a dual phase gel in which the flocculant or different small particles of the extract or compound of the present disclosure are dispersed in the liquid vehicle.

In one example, the present disclosure provides a method of preparing a powder of a caffeoylspermidine compound, comprising: reflux heating dry lycium ruthenicum fruit in 75% aqueous ethanol for at least two hours to provide a mixture of extraction compounds dissolved in ethanol and water; submitting the mixture to ion exchange resin chromatography to obtain a solution of caffeoylspermidine compound powder dissolved in an eluting solvent, wherein the resin used for chromatography is a weak cation exchange resin; concentrating the solution under reduced pressure to obtain caffeoylspermidine compound powder. In certain examples, the coffee imide compound powder may include a compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof. In other examples, the method may further comprise: after submission and before concentration: washing the eluting solvent with saturated sodium bicarbonate solution; loading an eluting solvent onto a nonpolar microporous adsorbent resin; eluting the caffeoylspermidine compound powder with 95% ethanol to obtain ethanol solution of caffeoylspermidine compound powder. In other examples, heating may include: maintaining the fruit at reflux in a volume of 75% aqueous ethanol for two hours to provide a first mixture of extraction compounds dissolved in ethanol and water; pouring the first mixture from the fruit; adding a second volume of 75% aqueous ethanol; maintaining the fruit at reflux in the second volume for two hours to provide a second mixture of extracted compounds dissolved in ethanol and water; pouring the second mixture from the fruit; the first mixture is combined with the second mixture. In other examples, the method may further comprise freeze-drying the powder of the caffeoylspermidine compound after concentrating.

In one example, the present disclosure provides a method of enriching for caffeoylspermidine compounds extracted from dried lycium ruthenicum fruit comprising submitting a mixture of caffeoylspermidine compounds to ion exchange resin chromatography to provide a solution of the compounds in an eluting solvent, wherein the resin used in the chromatography is a weak cation exchange resin.

In one example, the present disclosure provides a method of inhibiting histone deacetylase comprising administering a composition comprising a dried lycium ruthenicum fruit extract. In certain examples, the histone deacetylase can be HDAC1, HDAC2, HDAC3, HDAC8, or a combination thereof. In other examples, histone deacetylase can be specifically inhibited.

In one example, the present disclosure provides a composition comprising: i) A compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine、N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof; and ii) an additive. In certain examples, the compositions are useful for neuroprotection. In other examples, the compositions are useful for treating and/or preventing alzheimer's disease. In other examples, the composition may be used in anti-aging treatment. In other examples, the compositions may be used for topical administration. In other examples, the compositions may be used as cosmetics. In other examples, the compositions may be used as nutritional supplements.

In one example, the present disclosure provides a supplement to spermidine comprising: i) A compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof; and ii) an additive. In certain examples, the compound may be at least partially hydrolyzed in vivo to provide spermidine.

The above compositions and methods may be better understood in conjunction with the following examples. Further, the following non-limiting examples are illustrative. The illustrated methods are applicable to other examples of the composition of matter to be screened and evaluated of the present disclosure. Procedures described as general methods describe methods that are believed to be generally effective for screening and evaluating compositions of matter. However, those of skill in the art will understand that for any given example of the disclosure, it may be necessary to change the procedure, e.g., change the order or steps and/or the chemical reagents used

Examples

1. The caffeoylspermidine derivative AA001 is enriched from Lycium ruthenicum fruit.

A. Is extracted from Lycium ruthenicum fruit by hot reflux.

500 grams of dry lycium ruthenicum fruit was added to a round bottom flask and a volume of a 75% aqueous ethanol mixture was added to achieve a 1:10 mass of lycium ruthenicum fruit to ethanol-water mixture: volume ratio. The ethanol-water mixture was heated to reflux and maintained at reflux for two hours. The ethanol-water-extract mixture was decanted from the lycium ruthenicum fruit and an equal volume of a 75% ethanol in water mixture was added to the lycium ruthenicum fruit. The ethanol-water mixture was heated to reflux and maintained at reflux for an additional two hours, after which the ethanol-water-extract mixture was again decanted from the lycium ruthenicum fruit. The ethanol-water-extracts were combined and concentrated under vacuum.

B. Enrichment of the concentrated extract.

The concentrated extract was submitted to ion exchange resin chromatography. Weak cation exchange resin LK152 is used as the ion exchange resin. The extract was loaded at a ratio of 1:2 resin volume to dried lycium ruthenicum mass and a loading flow rate of 1.0 bed volume ("BV")/h. The extract was eluted through the resin using 50%0.2M aqueous HCl and 50% ethanol in an eluent volume of 3-5BV at an eluent flow rate of 2.0 BV/h.

C. And (5) post-treatment of eluent.

The pH of the eluate was adjusted to 4-5 by washing with saturated sodium bicarbonate solution. The eluate was loaded onto D101 macroporous resin to remove sodium salts, and then high content caffeoylspermidine extract ("mix") powder was collected with 95% ethanol eluate. Subsequently, the ethanol was removed under reduced pressure and the high content MixD powder was freeze dried.

Final mass of dried lycium ruthenicum and high content MixD powder: the mass ratio is about 100:1. the final yield was calculated by dividing the mass of the high-content MixD powder by the mass of the Lycium ruthenicum fruit and multiplying the quotient by 100%. High content MixD powder N ¹ -dihydrocaffeoyl-N ¹⁰ The mass percentage of the caffeoylspermidine (AA 001) is more than 15 percent. About 2.5mg N/g lycium ruthenicum fruit ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine. Table 1 illustrates the black colorFour test results for enriching caffeoylspermidine compounds in dried fruits of Lycium ruthenicum.

TABLE 1

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FIG. 1 is a schematic diagram showing purified high content caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ HPLC-UV chromatogram of caffeoylspermidine ("AA 001"). Fig. 2 shows the results of positive ion mass spectrometry analysis on AA001 obtained as a product of experiment No. 1. FIG. 3 shows purified AA001 ¹ H NMR spectrum. FIG. 4 shows purified AA001 ¹³ C NMR spectrum.

Figures 5-8 show HPLC chromatograms of fractions enriched for lycium ruthenicum fruits according to the enrichment procedure described herein. Figure 5 shows an HPLC chromatogram of an ethanol-water-extract mixture resulting from hot reflux extraction of lycium ruthenicum fruit. Fig. 6 shows an HPLC chromatogram of enriched high content caffeoylspermidine powder (mix) obtained as a product of experiment No. 1 after the enrichment procedure described herein. FIG. 7 shows N ¹ -dihydrocaffeoyl-N ¹⁰ HPLC chromatogram of caffeoylspermidine. Figure 8 shows HPLC chromatograms of fractions from the enrichment procedure described herein, excluding caffeoylspermidine.

2. By N ¹ -dihydrocaffeoyl-N ¹⁰ Treatment of caenorhabditis elegans with caffeoylspermidine

With 50. Mu.M and 100. Mu.M N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine solution treatment of caenorhabditis elegans. Longevity assays were tested by measuring the number of caenorhabditis elegans that die. Surviving caenorhabditis elegans were counted until all nematodes died. FIG. 9 shows the survival rate of caenorhabditis elegans over time (top or middle) compared to the control group (bottom line) A wire). As shown in Table 2, the mean life span and median survival were calculated using the Kaplan-Meier survival assay. The treatment result of caenorhabditis elegans shows that N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine has statistically significant anti-aging and life-prolonging properties.

TABLE 2

Data are expressed as mean ± SEM. * P <0.001 compared to DMSO group.

3. Inhibition of histone deacetylase by caffeoylspermidine compounds.

High levels of caffeoylspermidine powder (MixD) were dissolved in DMSO at a final concentration of 25mmol AA001 per liter DMSO. The DMSO solution was diluted with water to six different concentrations: 8mmol/L, 4mmol/L, 2mmol/L, 1mmol/L, 0.5mmol/L and 0.25mmol/L, respectively. MixD powder includes N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine (AA 001), N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine (AA 002) and N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine (AA 003) which is set forth in table 3 below. Fig. 10 shows HPLC chromatograms of AA001, AA002 and AA 003.

TABLE 3 Table 3

5. Mu.L of diluted DMSO solution was added to 100. Mu.L of the reaction system to achieve final concentrations of 400. Mu. Mol/L, 200. Mu. Mol/L, 100. Mu. Mol/L, 50. Mu. Mol/L, 25. Mu. Mol/L and 12.5. Mu. Mol/L according to the instructions of the HDAC1/2/3/8 fluorescence detection kit (BPS) manufacturer. The inhibition of HDAC1/2/3/8 activity was examined by the extract. Trichostatin a ("TSA"), an HDAC inhibitor, was used as a positive control.

Fig. 11 shows the relative percentage of activity of HDAC1 at different concentrations of mix powder in DMSO and water. The activity of HDAC1 was inhibited by at least 50%. Fig. 12 shows the relative percentage of activity of HDAC2 at different concentrations of mix powder in DMSO and water. Fig. 13 shows the relative percentage of activity of HDAC3 at high levels of mix powder at different concentrations in DMSO and water. Fig. 14 shows the relative percentage of activity of HDAC8 at different concentrations of mix powder in DMSO and water. According to fig. 11-14, the mix d powder resulted in a decrease in the percentage of activity of each of HDAC1, HDAC2, HDAC3 and HDAC 8.

2. Mu.L of diluted DMS solution was added to 50. Mu.L of the reaction system to achieve final concentrations of 400. Mu. Mol/L, 200. Mu. Mol/L, 100. Mu. Mol/L, 50. Mu. Mol/L, 25. Mu. Mol/L and 12.5. Mu. Mol/L according to the instructions of the manufacturer of the HDAC inhibitor drug screening kit (Abcam). HDAC activity inhibition was detected by the extract. TSA was used as a positive control. Fig. 15 shows inhibition of HDAC activity by the mix d powder.

Modeling of the docking and ligand-receptor interactions between hdac8 and AA001 and AA 003.

Using Meastro12.8%Software, 2021) for molecular docking. The crystals of HDAC8 (PDB ID:1T 64) were downloaded from the protein database (www.rcsb.org) and used for docking. The chemical structures of AA001 and AA003 as shown in fig. 16 were downloaded from PubChem or drawn using ChemDraw 19.0Suite (PerkinElmer, USA), assigned the appropriate two-dimensional orientation, and the three-dimensional structure was prepared by LigPrep Wizard in Meastro 12.8. Ligand status was generated at a target pH of 7.4±0.5. Other parameters retain default settings. Proteins were prepared using Protein Preparation Wizard by adding hydrogen and missing residues, removing selected water molecules and cofactors. Epik was used to generate het states at pH 7.4±0.5. Prior to ligand docking, the pre-treated proteins and ligands were minimized using the OPLS4 force field. The receptor grid was generated by creating an artificial bond between two TSA ligands and defining a merged ligand using the 3DBuilder tool. The size of the lattice is set at the centroid of the defined ligand, the length is no more than +. >Ligand docking was performed at SP (standard precision) using Glide. In the course of docking, a maximum of ten conformational isomers are considered for each ligand. The conformation with the lowest Glide score is selected to analyze the interaction between the target receptor and the ligand. FIG. 17 shows AA001 docking to HDAC8 with Glide at a Glide score of-6.794 kcal/mol. FIG. 18 shows AA003 docked with Glide to HDAC8 at a Glide score of-6.969 kcal/mol. FIG. 19 shows trichostatin A (TSA) docked with Glide to HDAC8 at a Glide score of-8.837 kcal/mol.

HDAC8 inhibition assay and HDAC8 half maximal inhibition concentration assay.

AA001 and AA003 were dissolved in DMSO at a final concentration of 50mmol/L, respectively, and then diluted with water to 6 different concentrations: 8mmol/L, 4mmol/L, 2mmol/L, 1mmol/L, 0.5mmol/L and 0.25mmol/L, respectively. 5. Mu.L of diluted AA001 or AA003 was added to 100. Mu.L of the reaction system to achieve final concentrations of 400. Mu. Mol/L, 200. Mu. Mol/L, 100. Mu. Mol/L, 50. Mu. Mol/L, 25. Mu. Mol/L and 12.5. Mu. Mol/L, respectively, according to the instructions of the manufacturer of the HDAC8 fluorometric kit (BPS, catalog number 50068). Inhibition of HDAC8 by AA001 and AA003 was examined and shown in fig. 20 and 21, respectively. The half maximal inhibitory concentrations of AA001 and AA003 were calculated as shown in fig. 22 and 23, respectively. Fig. 22 and 23 show that AA001 and AA003 are specific inhibitors of HDAC 8. Half maximal Inhibitory Concentration (IC) of AA001 on HDAC8 ₅₀ ) About 41.75. Mu. Mol/L, half maximal Inhibitory Concentration (IC) of AA003 on HDAC8 ₅₀ ) About 65.52. Mu. Mol/L.

6. Consideration of potential false positives in aging.

According to fig. 9, aa001 may have an anti-aging effect. However, there are several reasons that figure 9 may have false positive results. Thus, the effect of AA001 on OP50 growth was studied to determine whether AA001 might extend caenorhabditis elegans longevity by limiting diet. The results indicate that AA001 did not affect the growth of OP50, indicating that the anti-aging effect of AA001 was not caused by dietary restrictions, as shown in figure 24.

Avoiding the use of AA001 compounds may also lead to false positive results. However, as can be seen from figure 25, there was no significant difference in the number of caenorhabditis elegans distributed over the control (DMSO), 50 μm and 100 μm samples, indicating that the nematodes have no apparent chemotaxis or avoidance of AA 001. In addition, AA001 did not have a nematode reproduction reducing effect, as shown in fig. 26. Thus, FIGS. 24-26 demonstrate that AA001 has anti-aging effects because of its pharmacodynamic efficacy.

7. Health life in anti-aging studies.

Health life may be an important research direction in aging research. The frequency of body bending by caenorhabditis elegans was recorded within 30 seconds. As shown in fig. 27, the older the nematode, the lower its movement track. There was a significant difference in body bending from day 6 after time zero.

The pharyngeal pumping frequency of caenorhabditis elegans was also recorded. As shown in fig. 28, the frequency decreases with nematode aging. As shown in fig. 28, the frequency of the nematode group treated with AA001 was higher from day 12 after time zero than the control (DMSO) group.

Nematode movements were calculated on days 9, 12, 15 and 18, as shown in figure 29. Group a of fig. 29 shows nematodes that can spontaneously move independently. Group B shows those nematodes that began to move after contact. Group C shows those nematodes that did not exhibit movement even after contact. More nematodes occupied the group represented by group a, and less nematodes occupied the group treated with AA 001. The data shown in FIGS. 27-29 demonstrate that treatment of caenorhabditis elegans with AA001 improves the healthy life of caenorhabditis elegans.

Although the present disclosure has been described with reference to the embodiments and the drawings, the present disclosure is not limited thereto, but various modifications and changes may be made by those skilled in the art to which the present disclosure pertains without departing from the spirit and scope of the present disclosure.

The subject matter of the present disclosure may also relate to, among other things, the following:

the first aspect relates to a composition comprising an extract of lycium ruthenicum fruit and an additive.

A second aspect relates to the composition of aspect 1, wherein the extract comprises a compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof.

A third aspect relates to the composition of any preceding aspect, wherein the composition increases the survival rate of caenorhabditis elegans ("c.

A fourth aspect relates to the composition of aspect 3, wherein the composition increases the survival rate of caenorhabditis elegans by at least 10%.

A fifth aspect relates to the composition of any preceding aspect, wherein the extract comprises at least 10% by weight N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine.

A sixth aspect relates to the composition of any preceding aspect, wherein the extract comprises at least 25% by weight N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine.

A seventh aspect relates to the composition of any preceding aspect, wherein the extract comprises at least 45 wt% N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine.

An eighth aspect relates to the composition of any preceding aspect, wherein the composition inhibits histone deacetylase activity.

A ninth aspect relates to the composition of aspect 8, wherein the histone deacetylase is selected from the group consisting of HDAC1, HDAC2, HDAC3, HDAC8, and combinations thereof.

A tenth aspect relates to the composition of aspects 7 or 8, wherein the histone deacetylase is HDAC1, and wherein activity is inhibited by at least 50%.

An eleventh aspect relates to the composition of any preceding aspect, wherein the extract comprises at least one specific inhibitor of HDAC 8.

The twelfth aspect relates to the composition of aspect 11, wherein the at least one specific inhibitor comprises N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine.

A thirteenth aspect relates to the composition of aspect 12, wherein N ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine IC ₅₀ The value was about 41.75. Mu. Mol/L.

A fourteenth aspect relates to the composition of aspect 11, wherein said at least one specific inhibitor comprises N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine.

The fifteenth aspect relates to the composition of aspect 14, wherein N ¹ -caffeoyl-N ¹⁰ IC of dihydrocaffeoylspermidine ₅₀ The value was about 62.52. Mu. Mol/L.

A sixteenth aspect relates to the composition of any preceding aspect, wherein the extract is the product of a process for enriching from dry lycium ruthenicum fruit, the process comprising: maintaining the fruit in 75% ethanol in water at reflux for at least two hours to provide a mixture of extracted compounds dissolved in ethanol and water; submitting the mixture to ion exchange resin chromatography to provide an extract solution dissolved in an eluting solvent, wherein the resin used for chromatography is a weak cation exchange resin; concentrating the extract solution under reduced pressure to obtain extract.

A seventeenth aspect relates to the composition of aspect 16, wherein the method further comprises, after submitting and before concentrating: washing the eluting solvent with saturated sodium bicarbonate solution; loading an eluting solvent onto a nonpolar microporous adsorbent resin; and eluting the extract with 95% ethanol to obtain an ethanol solution of the extract.

An eighteenth aspect relates to the composition of aspects 16 or 17, wherein the extract is produced in a yield of about 1.10% to about 1.45% of the mass of dry lycium ruthenicum fruit.

A nineteenth aspect relates to the composition of aspects 16 through 18, wherein the maintaining comprises: maintaining the fruit at reflux in a volume of 75% aqueous ethanol for two hours to provide a mixture of extracted compounds dissolved in ethanol and water; pouring the mixture from the fruit; adding a second volume of 75% aqueous ethanol; maintaining the fruit at reflux in the second volume for two hours to provide a second mixture of extracted compounds dissolved in ethanol and water; pouring the second mixture from the fruit; the first mixture is combined with the second mixture.

A twentieth aspect relates to the composition of aspects 16 to 19, wherein the method further comprises freeze-drying the extract after concentrating.

A twenty-first aspect relates to the use of a composition of any of the preceding aspects for neuroprotection.

A twenty-second aspect relates to the use of the composition of aspects 1 to 20 for the treatment and/or prevention of alzheimer's disease.

A twenty-third aspect relates to the use of the composition of aspects 1 to 20 for anti-aging treatment.

A twenty-fourth aspect relates to the use of the composition of aspects 1 to 20 for topical administration.

A twenty-fifth aspect relates to the use of the composition of aspects 1 to 20 as a cosmetic.

A twenty-sixth aspect relates to the use of the composition of aspects 1 to 20 as a nutritional supplement.

A twenty-seventh aspect relates to a method for producing a coffee imide compound powder, comprising: reflux heating dry lycium ruthenicum fruit in 75% aqueous ethanol for at least two hours to provide a mixture of extraction compounds dissolved in ethanol and water; submitting the mixture to ion exchange resin chromatography to obtain a solution of caffeoylspermidine compound powder dissolved in an eluting solvent, wherein the resin used for chromatography is a weak cation exchange resin; concentrating the solution under reduced pressure to obtain caffeoylspermidine compound powder.

A twenty-eighth aspect is directed to the method of aspect 27, wherein the caffeoylspermidine compound powder comprises a compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-pyri-dine)Pyranoglucosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof.

A twenty-ninth aspect relates to the method of aspect 27 or 28, further comprising: after submission and before concentration: washing the eluting solvent with saturated sodium bicarbonate solution; loading an eluting solvent onto a nonpolar microporous adsorbent resin; eluting the caffeoylspermidine compound powder with 95% ethanol to obtain ethanol solution of caffeoylspermidine compound powder.

A thirty-third aspect relates to the method of aspects 27-29, wherein the heating comprises: maintaining the fruit at reflux in a volume of 75% aqueous ethanol for two hours to provide a first mixture of extraction compounds dissolved in ethanol and water; pouring the first mixture from the fruit; adding a second volume of 75% aqueous ethanol; maintaining the fruit at reflux in the second volume for two hours to provide a second mixture of extracted compounds dissolved in ethanol and water; pouring the second mixture from the fruit; the first mixture is combined with the second mixture.

A thirty-first aspect relates to the method of aspects 27-30, further comprising freeze-drying the caffeoylspermidine compound powder after concentrating.

A thirty-second aspect relates to a method of enriching for caffeoylspermidine compounds extracted from dried lycium ruthenicum fruit comprising submitting a mixture of caffeoylspermidine compounds to ion exchange resin chromatography to provide a solution of the compounds in an eluting solvent, wherein the resin used in the chromatography is a weak cation exchange resin.

A thirty-third aspect relates to a method of inhibiting histone deacetylase comprising administering a composition comprising a dried lycium ruthenicum fruit extract.

A thirty-fourth aspect relates to the method of aspect 33, wherein the histone deacetylase is HDAC1, HDAC2, HDAC3, HDAC8, or a combination thereof.

A thirty-fifth aspect relates to the method of aspects 33 or 34, wherein the histone deacetylase is specifically inhibited.

A thirty-sixth aspect relates to a composition comprising: i) A compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof; and ii) an additive.

A thirty-seventh aspect relates to the use of the composition of aspect 36 for neuroprotection.

A thirty-eighth aspect relates to the use of the composition of aspect 36 for the treatment and/or prevention of alzheimer's disease.

A thirty-ninth aspect relates to the use of the composition of aspect 36 for anti-aging treatment.

A fortieth aspect relates to the use of the composition of aspect 36 for topical administration.

A fortieth aspect relates to the use of the composition of aspect 36 as a cosmetic.

A fortieth aspect relates to the use of the composition of aspect 36 as a nutritional supplement.

A forty-third aspect relates to a supplement of arginine comprising: i) A compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof; and ii) an additive.

A forty-fourth aspect relates to the supplement of aspect 43, wherein the compound is at least partially hydrolyzed in vivo to provide spermidine.

In addition to the features mentioned in each of the independent aspects enumerated above, some examples may display optional features mentioned in the dependent aspects and/or disclosed in the description above and shown in the drawings, alone or in combination.

Claims

1. A method for preparing a powder of a caffeoylspermidine compound, comprising:

reflux heating dry lycium ruthenicum fruit in 75% aqueous ethanol for at least two hours to provide a mixture of extraction compounds dissolved in ethanol and water;

submitting the mixture to ion exchange resin chromatography to obtain a solution of caffeoylspermidine compound powder dissolved in an eluting solvent, wherein the resin used for chromatography is a weak cation exchange resin; and

the solution was concentrated under reduced pressure to obtain a powder of caffeoylspermidine compound.

2. The method of claim 1, wherein the caffeoylspermidine compound powder comprises a compound selected from the group consisting of: n (N) ¹ -dihydrocaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ -dihydrocaffeoyl-N ¹⁰ - (4-beta-D-glucopyranosyl) -caffeoylspermidine, N ¹ -caffeoyl-N ¹⁰ -dihydrocaffeoylspermidine, N ¹ - (4-beta-D-glucopyranosyl) -dihydrochalcaffeoyl-N ¹⁰ -caffeoylspermidine, N ¹ ,N ¹⁰ -bis- (dihydrocaffeoyl) -spermidine, N ¹ ,N ¹⁰ -bis- (caffeoyl) -spermidine and combinations thereof.

3. The method of claim 1, further comprising: after submission and before concentration:

washing the eluting solvent with saturated sodium bicarbonate solution;

loading an eluting solvent onto a nonpolar microporous adsorbent resin; and

eluting the caffeoylspermidine compound powder with 95% ethanol to obtain ethanol solution of caffeoylspermidine compound powder.

4. The method of claim 1, wherein the heating comprises:

maintaining the fruit at reflux in a volume of 75% aqueous ethanol for two hours to provide a first mixture of extraction compounds dissolved in ethanol and water;

pouring the first mixture from the fruit;

adding a second volume of 75% aqueous ethanol;

maintaining the fruit at reflux in the second volume for two hours to provide a second mixture of extracted compounds dissolved in ethanol and water;

pouring the second mixture from the fruit; and

the first mixture is combined with the second mixture.

5. The method of claim 1, further comprising freeze-drying the caffeoylspermidine compound powder after concentrating.

6. A method of enriching for a caffeoylspermidine compound extracted from dried lycium ruthenicum fruit comprising submitting a mixture of caffeoylspermidine compounds to ion exchange resin chromatography to provide a solution of the compound in an eluting solvent, wherein the resin used in the chromatography is a weak cation exchange resin.

7. A method of inhibiting histone deacetylase comprising administering a composition comprising a dried lycium ruthenicum fruit extract.

8. The method of claim 7, wherein the histone deacetylase is HDAC1, HDAC2, HDAC3, HDAC8, or a combination thereof.

9. The method of claim 7, wherein the histone deacetylase is specifically inhibited.