LYCIUM BARBARUM EXTRACTS, RESULTING COMPOSITIONS, METHODS OF MAKING AND METHODS OF USING THE SAME

FIELD OF THE INVENTION

The disclosure relates to generally a composition having pharmaceutical or functional properties, comprising ingredients derived from a berry. More particularly, the disclosed subject matter relates to a Lycium barbarum extract, a resulting composition, methods of making the same, and methods of using the same, for example, as pharmaceutical compositions, functional compositions, and dietary supplements.

BACKGROUND

Skeletal muscle is the most abundant tissue in mammal's body, accounting for approximately 40-50 % of the body mass. In addition to its primary role in posture and movement, skeletal muscle is also an extremely important metabolic organ in maintaining normal whole-body energy homeostasis.

Skeletal muscle is composed of distinct muscle fiber subtypes defined by myosin heavy chain (MyHC) isoforms and metabolic activity. Different muscle fiber subtypes differentially impact muscle metabolism and function. In mammals, skeletal muscle fibers are classified as type I (slow oxidative muscle fibers), type IIa (fast oxidative muscle fibers), type IIx/d (intermediate muscle fibers), and type IIb (fast glycolytic muscle fibers). Type I and type IIA are rich in mitochondria and myoglobin, and have high oxidative capacity and good endurance. In contrast, type IIB has lower endurance and less mitochondria and generate ATP primarily through glycolysis. The types and composition of muscle fibers directly affect the skeletal muscle fitness and the body's energy metabolism. Many diseases, such as muscle atrophy, diabetes, obesity, liver metabolism disorder and aging, are closely related to the muscle fiber types and energy metabolism.

Exercise is effective in improving muscle endurance and performance. It can significantly increase the proportion of slow oxidative fibers, and enhance mitochondrial biogenesis. Although how exercise promotes muscle fitness remains unclear, many studies have found that some signal molecules are involved in this process. Exercise can increase the level of AMP and NAD+ to activate the metabolic sensors AMPK (AMP-activated protein kinase) and SIRT1 (Sirtuin 1), then activate PGC1 by post-translational modifications. PGC1a/β function as transcription co-activators of the nuclear receptor PPAR (peroxisome proliferator-activated receptor δ) and ERRα/γ (estrogen-related receptor a/γ), which have been identified that contribute to exercise-induced muscle fiber type switching and endurance.

In consideration of the benefic effects of exercise on skeletal muscle fitness, some muscle-targeted “exercise mimetics” drugs were begun to be developed. These compounds mimic exercise by activating some of the key regulators involved in the metabolic remodeling of skeletal muscle, such as AMPK activator, Sirt1 activator, or PPAR ligand. Although the synthetic exercise mimetics have the potential to improve the muscle fitness and endurance, the safety and health of the drug must be considered.

SUMMARY

The present disclosure provides a Lycium barbarum extract, a composition comprising such a Lycium barbarum extract, methods of making the same, and methods of using the same.

The invention proves that such a composition comprising a Lycium barbarum extract increases skeletal muscle weight, increases the proportion or content of the skeletal muscle oxidative muscle fibers, increases the aerobic metabolism level of skeletal muscles, reduces glycolysis level, increases myoglobin content, improves aerobic endurance, and provides resistance to exercise fatigue.

Through a series of experiments, the inventors found that long-term consumption of a water extract of Lycium barbarum significantly increases the muscle weight of mice, increases the proportion of oxidative muscle fibers, increases aerobic metabolism capacity, and improves exercise capacity. Through in vitro experiments by treating C2C12 myoblasts with the water extract of Lycium barbarum, it was found that the water extract of Lycium barbarum increases the expression of oxidative muscle fiber-related genes, increases the level of aerobic metabolism, and reduces the level of glycolysis. Further research found that activation of the ERRγ pathway is involved in mediating the important effect of the Lycium barbarum water extract on promoting transition of muscle fiber.

Therefore, the object of the present invention is to provide an important effect of Lycium barbarum water extract by increasing the ratio of oxidative muscle fibers and increasing aerobic metabolism of skeletal muscle by activating the ERRγ pathway. The Lycium barbarum water extract and related compositions have potential application value in at least the following six aspects: 1. The extract and the related compositions have potential application value of Lycium barbarum water extract in functional foods that enhance aerobic endurance exercise or functional foods that are simulated by exercise. 2. Lycium barbarum water extract has potential preventive and protective effects on astronaut's muscle tissue atrophy. 3. Lycium barbarum water extract has potential preventive and protective effects on muscle atrophy of patients with chronic bed. 4. Lycium barbarum water extract has potential preventive and protective effects on muscle atrophy in the elderly. 5. Lycium barbarum water extract has potential therapeutic effect on improving metabolic abnormalities in obese people. 6. Lycium barbarum water extract has potential application value in increasing the level of aerobic metabolism in athletes' skeletal muscle, improving aerobic endurance, and resisting sports fatigue.

In accordance with some embodiments, the present disclosure provides a composition for promoting skeletal muscle oxidative muscle fiber generation. The composition comprises Lycium barbarum, a Lycium barbarum water extract, or a combination thereof. In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients selected from the group consisting of a solvent, a co-solvent, an emulsifier, a preservative, a buffer, a protein powder, and a combination thereof.

In some embodiments, the composition comprises a Lycium barbarum water extract, and a Lycium barbarum water extract comprises polysaccharide, Lycium barbarum flavone, carotenoid, polyphenols and Lycium barbarum pigment. Each of these ingredients may have only one, or two or more of the same type. For example, the composition may include two or more types of polysaccharides, two or more types of Lycium barbarum flavones, two or more types of carotenoids, two or more types of polyphenols, and/or two or more types of Lycium barbarum pigments.

In some embodiments, in the Lycium barbarum extract, polysaccharide is present in a range of from about 10.0 wt. % to about 70.0 wt. % (e.g., about 50 wt. % to about 70 wt. %), Lycium barbarum flavone is a range of from about 0.1 wt. % to about 5.0 wt. %, carotenoid is a range of from about 0.1 wt. % to about 3.0 wt. %, polyphenol is a range of from about 0.1 wt. % to about 8.0 wt. %, and Lycium barbarum pigment is a range of from about 0.1 wt. % to about 8.0 wt. %, based on the total dry weight of the extract. The dry weight is equivalent weight corresponding to the extract in a dry powder (without water). The extract may contain other residues of a very small amount. The extract in the form of a dry powder can be mixed with water to provide an extract in the form of an aqueous solution having a selected concentration as described herein.

In some embodiments, the Lycium barbarum extract is in a powder form. In some embodiments, the Lycium barbarum extract is in an aqueous liquid form having a concentration, for example, in a range of from 0.1 g/mL to 5 g/mL. In some embodiments, the composition is a pharmaceutical composition including an effective amount of ingredients for promoting skeletal muscle oxidative muscle fiber generation, a functional composition, a type of food, or a dietary supplement. The composition may be in any suitable form, for example, tablet, drink (e.g., sports drink), candy, or snack bar.

In another aspect, the present disclosure provides a method of making the composition as described above. Such a method includes making an aqueous extract of Lycium barbarum by extracting Lycium barbarum berries in water. The method includes steps of making a Lycium barbarum extract. In some embodiments, the steps of making the Lycium barbarum extract include steps as described herein. For example, the steps of making the Lycium barbarum extract include: soaking dried Lycium barbarum berries in water, crushing the

Lycium barbarum berries to provide a soaked berry powder, decocting the soaked berry powder in boiling water twice to obtain a decoction, distilling the decoction under vacuum after a filtration to obtain a concentrate, and lyophilizing the concentrate to obtain a dry powder as the Lycium barbarum extract. The method may further comprise formulating the composition by adding one or more ingredients into the aqueous or dried powder extract of Lycium barbarum. The method may further comprise dissolving the Lycium barbarum extract in water to obtain an aqueous liquid.

In another aspect, the present disclosure provides a method of using the composition as described above. Such a method includes administering or taking a suitable amount of the composition into a human subject. In some embodiments, the composition is used to increase the weight of skeletal muscle, and/or increase the proportion of skeletal muscle oxidative muscle fibers. In some embodiments, the composition may also be used to improve the level of aerobic metabolism, and/or reduce glycolysis level. In some embodiments, the composition may also be used to increase the content of myoglobin, improve exercise endurance, and/or combat sports fatigue, and/or in exercise simulation. In some embodiments, the composition is used to activate the ERRγ pathway, and/or regulate muscle fiber types by activating the ERRγ pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not necessarily to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Like reference numerals denote like features throughout specification and drawings.

FIG. 1A shows the body weights of the mice in the control group, and the mice in the experimental group administrated with the Lycium barbarum (LB) water extract.

FIGS. 1B-1C show the weights of the tibialis anterior muscle and gastrocnemius muscle of the control group, and the experimental group with the Lycium barbarum water extract.

FIG. 2 shows the exhausted running distance of the mice in the control group, and the mice in the experimental group with the Lycium barbarum water extract.

FIG. 3 shows the lactic acid content in the serum of the mice in the control group and in the experimental group with the Lycium barbarum water extract.

FIG. 4 shows the immunofluorescence staining and statistical results of different types of muscle fibers in the tibialis anterior and soleus muscles of the mice in the control group and the experimental group with water extract of Lycium barbarum. Type I (green), type IIa (blue), and IIb Type (red).

FIG. 5 shows the expression levels of myosin heavy chain isoforms in the tibialis anterior muscles of the mice in the control group and in the experimental group with the Lycium barbarum water extract.

FIG. 6 shows the expression level of myoglobin in the tibialis anterior muscle of the mice in the control group and in the experimental group with the Lycium barbarum water extract.

FIG. 7 shows the number of mitochondrial DNA copies in the tibialis anterior muscles of the mice in the control group and in the experimental group with water extracts of Lycium barbarum.

FIG. 8 shows the expression levels of muscle fiber-related genes in the myotubes treated with the water extract of Lycium barbarum and those in the control group.

FIG. 9 shows the maximal oxygen consumption, carbon dioxide exhalation, respiratory entropy, and energy exchange rate of the mice in the control group and the experimental group with the water extract of Lycium barbarum.

FIG. 10 shows the changes in aerobic metabolism levels and related gene expression levels of myoblasts treated with aqueous extracts of Lycium barbarum and myoblasts in the control group.

FIG. 11 shows the changes in cell aerobic respiration levels and the expression levels of related genes in myoblasts treated with aqueous extracts of Lycium barbarum and the myoblasts in the control group.

FIG. 12 shows the changes in cell glycolysis levels and the expression levels of related genes in myoblasts treated with aqueous extracts of Lycium barbarum and the myoblasts in the control group.

FIG. 13 shows the expression level of ERRγ in the tibialis anterior muscles of the experimental group mice treated with the Lycium barbarum water extract and those in the control group mice.

FIG. 14 shows the expression level of ERRγ in myotubes treated with the Lycium barbarum water extract and myotubes of the control group.

FIG. 15 shows the expression level of Esrrg, the expression level of skeletal muscle fiber-related genes, and the expression level of Atp5b as a key gene of ATP, in myotubes treated with water extract of Lycium barbarum and those in the control group after the Esrrg gene was disturbed.

FIG. 16 shows the morphology of the C2C12 myotubes treated with different concentrations (1.0, 2.0, 4.0 and 8.0 mg/mL) of LB extract and the RNA levels of Esrrg, Mef2a and Myh2 in the C2C12 myotubes treated with different doses of LB extract and determined by qPCR.

DETAILED DESCRIPTION

For purposes of the description hereinafter, it is to be understood that the embodiments described below may assume alternative variations and embodiments. It is also to be understood that the specific articles, compositions, and/or processes described herein are exemplary and should not be considered as limiting.

In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a nano structure” or an ingredient is a reference to one or more of such structures and equivalents thereof known to those skilled in the art, and so forth. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. As used herein, “about X” (where X is a numerical value) preferably refers to ±10 % of the recited value, inclusive. For example, the phrase “about 8” preferably refers to a value of 7.2 to 8.8, inclusive; as another example, the phrase “about 8%” preferably (but not always) refers to a value of 7.2% to 8.8%, inclusive. Where present, all ranges are inclusive and combinable. For example, when a range of “1 to 5” is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, “2-5”, and the like. In addition, when a list of alternatives is positively provided, such listing can be interpreted to mean that any of the alternatives may be excluded, e.g., by a negative limitation in the claims. For example, when a range of “1 to 5” is recited, the recited range may be construed as including situations whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a recitation of “1 to 5” may be construed as “1 and 3-5, but not 2”, or simply “wherein 2 is not included.” It is intended that any component, element, attribute, or step that is positively recited herein may be explicitly excluded in the claims, whether such components, elements, attributes, or steps are listed as alternatives or whether they are recited in isolation.

As described above, the synthetic exercise mimetics have the potential to improve the muscle fitness and endurance, but the synthetic mimetics may have the safety and health issues needed to be considered.

Therefore, discovery of natural medicine edible plants that have mimetic effects of exercise has great importance and significance.

The present disclosure provides compositions and methods related to the application of Lycium barbarum (LB) extract in promoting the generation of skeletal muscle oxidative muscle fibers, and belongs to the field of natural product treatment.

The present disclosure provides a composition and a method for application of Lycium barbarum extract in promoting skeletal muscle oxidative muscle fiber generation. In the present invention, the water extract of Lycium barbarum was used to administer mice. Compared with the control group, the weight of the tibialis anterior muscle and gastrocnemius muscle of the mice in the administration group significantly increased, the proportion of oxidative muscle fibers increased, and the content of myoglobin increased, aerobic metabolism capacity increased, exercise endurance capacity, and average exhaustion running distance increased by about 40%. In the in vitro experiment, C2C12 myoblasts were treated with water extract of Lycium barbarum. Compared with the control group, the water extract of Lycium barbarum can increase the expression of oxidative muscle fiber-related genes, increase the level of aerobic metabolism, and reduce the level of glycolysis. Furthermore, by interfering with the expression of ERRγ (Estrogen receptor-related receptor-γ) gene, it was proved that the water extract of Lycium barbarum promotes the generation of oxidative muscle fibers by activating the ERRγ pathway. The present invention proves that Lycium barbarum has the effect of promoting the generation of oxidative muscle fibers of skeletal muscle, and Lycium barbarum has the potential to be applied to sports food or exercise simulation food for enhancing aerobic endurance.

Lycium barbarum berries are used for extraction. In the present disclosure, Lycium barbarum (or L. barbarum) extract can be used as a dietary natural “exercise mimetic” agent for the people lacking or unable to achieve adequate physical exercise. Lycium barbarum extract has effect on skeletal muscle fiber type switch and energy metabolism in vitro and in vivo.

Unless expressly indicated otherwise, references to a “composition” made herein are understood to encompass a composition in any suitable form, including but not limited to a solid, a liquid, and a paste. Unless expressly indicated otherwise, references to a “water extract” or “extract” are understood to encompass an extract of Lycium barbarum in any suitable form, including but not limited to a solid form such as a dry powder, a paste, and a liquid form. For example, as described herein, dried berries of Lycium barbarum can be extracted using the method descried herein using boiling water to obtain an aqueous extract, which is dried to provide an extract in the dry powder form. The term “water extract” may refer to an extract in powder form. The dry powder is then dissolved in water to make an aqueous extract of a suitable concentration. The weight percentages of ingredients in an extract are the contents of the ingredients in an extract of dry powder form. Unless expressly indicated otherwise, a concentration of an aqueous extract is defined by the mass of a dry weight of the extract (the equivalent mass of an extract in dry powder form) and the volume of the aqueous extract. The terms “weight” and “mass” herein are used interchangeably.

In the embodiment of the present disclosure, the components of the water extract of Lycium barbarum mainly include water-soluble ingredients such as polysaccharides, Lycium barbarum flavones, carotenoids, polyphenols, and Lycium barbarum pigments. The Lycium barbarum polysaccharides may be in a range of from about 10.0 wt. % to about 70.0 wt. %, and the molecular weight may be in a range between 4.50×10³D to 6.05×10⁵D. For example, the polysaccharides may be in a range of from about 20.0 wt. % to about 70.0 wt. %, from about 30.0 wt. % to about 70.0 wt. %, or from about 40.0 wt. % to about 70.0 wt. %. In some embodiments, the polysaccharides is more preferably from about 50.0 wt. % to about 70.0 wt. %. In some embodiments, the content of arabinose, galacturonic acid and galactose is not less than 60 % in sugar composition. In some embodiments, the content of polysaccharide was 54 % and the molecular weight was between 6.50×10³D to 5.05×10⁵D.

In addition to polysaccharide, each other ingredient may be in a suitable range. For example, in some embodiments, Lycium barbarum flavone has a content in a range of from about 0.1 wt. % to about 5.0 wt. % (e.g., 0.5-5%, 1-5%, 2-5%, or 3-5%), carotenoid has a content in a range of from about 0.1 wt. % to about 3.0 wt. % (e.g., 0.2-3%, 0.5-3%, or 1-3 %), polyphenol has a content in a range of from about 0.1 wt. % to about 8.0 wt. % (e.g., 0.5-8%, 1-8%, 2-8%, 4-8%, 5-8% or any other suitable range), and Lycium barbarum pigment has a content in a range of from about 0.1 wt. % to about 8.0 wt. % (e.g., 0.1-7%, 0.1-6%, 0.1-5%, 0.1-4%, or any other suitable range). These weight percentages are relative to the total weight of the extract in dry powder form.

In the embodiment of the present invention, the aqueous extract of Lycium barbarum is administered by gavage or orally, but it is not limited thereto. Any form of administration of having the composition into stomach may be suitable.

In some embodiments of the present invention, the dosage of the Lycium barbarum water extract is 2.5 g/Kg (the dry weight of the exemplary extract/the body weight of animals such as mice). With reference to the equivalent dose ratio between humans and animals in “Pharmacological Experiment Methodology” edited by Professor Xu Shuyun, it was calculated that this dose was equivalent to 19.39 g/70 Kg in humans. The ratio of a dosage in mice (D₁) to that in humans (D₂) is calculated based on an equation: D₁=D₂*9.023. The administered dose does not limit the scope of the invention in any way. For example, the dosage may be in a range of from about 10.0 g/70 Kg to about 40.0 g/70 Kg. In these dosages, the mass in grams refers to the equivalent dry weight of the corresponding extract, and the mass in Kg refers to body weight of a human subject.

The water extract of Lycium barbarum provided by the invention is homologous in medicine and food, and can be used as a healthy food. Food or health food preparations are not particularly limited. For example, it can be made into tablets, drinks, candies, and the like. Each food preparation may include other ingredients used in the art in addition to the Lycium barbarum water extract. The other ingredients can be selected by those skilled in the art considering the specific formulation or purpose used.

In some embodiments, the composition is a food or health product including sports drinks, protein powder and the like.

The features and effects of the present invention will be explained through

Preparation Examples and Test Examples. However, the following preparation examples and test examples are for illustration only, and do not limit the scope of the present invention.

EXAMPLES

Unless indicated otherwise, the water used herein is double-distilled water (pH=7).

All animal experiments with mice were performed in C57BL/6. The care and handling of animals was performed in accordance with the guidelines of the Animal Ethics Committee.

1. Preparation of Lycium barbarum Extract:

Lycium barbarum extract was prepared in the following general procedures: Lycium barbarum berries were cultivated in and obtained from Zhongning County, Yinchuan, Ningxia, China. The dried berries were soaked in double-distilled water (pH=7) at room temperature for 2 hours after being washed 3-5 times, then crushed. The soaked berries powder were added 5-8 times neutral water, mixed uniformly and decocted at a boiling temperature twice, with a period of time for decocting of 2.0 h and 1.5 h, respectively. The combined concentrated decoctions were filtered by a hollow fiber membrane. The above filtrates were merged and evaporated under a vacuum at 30-55° C. to remove water and obtain the concentrate. The resulting concentrate was lyophilized into a powder and stored in a desiccator, and it was used for the following experiments at suitable concentration.

In the experiments, an exemplary resulting Lycium barbarum extract (Experimental Example 1) was used. Such exemplary extract is an aqueous extract solution, and has a concentration of 1 g/mL (the equivalent mass of dry powder or dry weight of the extract/the volume of the extract). This concentration is for illustration only. The aqueous extract may be adjusted to have any suitable concentration, for example, in a range of from about 0.1 g/mL to 5 g/mL (the dry weight of the exemplary extract/the volume of extract). Such an aqueous extract may be diluted for administration in some embodiments. The aqueous extract may be further diluted for experiments with cells.

The Lycium barbarum extracts provided in the present disclosure include mainly water-soluble Lycium barbarum polysaccharides, Lycium barbarum flavonoids, carotenoids, polyphenols, and pigment. In the exemplary extract used, polysaccharide is present in a range of from about 50.0 wt. % to about 70.0 wt. % (e.g., about 54%-56% or 54%), Lycium barbarum flavone is a range of from about 0.1 wt. % to about 5.0 wt. %, carotenoid is a range of from about 0.1 wt. % to about 3.0 wt. %, polyphenol is a range of from about 0.1 wt. % to about 8.0 wt. %, and Lycium barbarum pigment is a range of from about 0.1 wt. % to about 8.0 wt. %, based on the total equivalent weight of the extract in dry powder form.

2. Cell Culture and Treatment:

Mouse C2C12 cells were cultured in growth medium consisting of Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 4.5 g/liter glucose, 10% fetal bovine serum (FBS), 1% antibiotic/antimycotic, and 1% gentamycin at 37° C. in a 5% CO₂atmosphere.

3. Grip Strength

Treadmill Test: The treadmill test was performed using an Exer3/6 (Columbus Instruments). Mice were acclimated to treadmill running three times (every other day) before the test was performed. Mice ran on the treadmill at 15° downhill, starting at a speed of 10 m/min. After 3 min, the speed was increased by 1 m/min to a final speed of 20 m/min. Exhaustion was defined as the inability of the animal to remain on the treadmill despite electrical prodding.

4. Measurement of Lactate Release:

After treatment the Lycium barbarum extract for 12 or 24 h, the conditioned media of C2C12 cells were collected, and the amount of lactate released into the medium was determined.

5. Immunofluorescence Staining of Frozen Sections:

Prior to immunofluorescent staining, the frozen sections of muscle were fixed in 4% paraformaldehyde for 20 min, washed three times with PBS, and permeabilized with 0.2% Triton X-100/PBS (PBST) for 10 min. After washing with PBS, the cells were blocked by incubating with 5% BSA at room temperature for 2 h. Cells were immunostained by incubating with the first antibody overnight at 4° C. After washing, immunoreactive proteins were visualized following incubation with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Zhongshanjinqiao Corp.). Nuclei were stained with Hoechst (Santa Cruz). The cells were then observed using a confocal laserscanning microscope (LSM750) (Carl Zeiss).

6. Real-Time qPCR:

Total RNA was extracted using TRIzol reagent according to the manufacturer's (Invitrogen) protocol. RNA samples were then reverse transcribed using M-MuLV reverse transcriptase, and mRNA levels were measured by performing RT-qPCR on a 7500 Real-Time PCR System (Applied Biosystems). The samples were heated to 95° C. for 2 min and subjected to 40 cycles of amplification (1 min at 94° C., 1 min at 58° C., and 1 min at 72° C.), followed by 10 min at 72° C. for the final extension.

7. Western Blotting:

Cells were lysed in radio immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 7.4, 5 mM EDTA and protease inhibitor cocktail solution (Roche)), and lysates were cleared by centrifugation. For each sample, 40 mg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. Membranes were blocked in 5% skim milk powder in TBST (0.1% Tween-20) for 1 h and incubated with the following primary antibodies overnight at 4° C.; anti-iNOS (1:500, Santa Cruz); anti-p-ERK, anti-ERK, anti-p-AKT, anti-AKT, anti-p-AMPKα/β, and anti-AMPKα/β (1:1000, Cell Signaling); anti-HIF-1α (1:200, Santa Cruz); anti-Nrf2 (1:200, Santa Cruz); anti-HSP60, anti-HSP70, and anti-HSP90 (1:500, Santa Cruz); anti-LC3 (1:1000, MBL) or anti-actin (1:2000, Sungene Biotech). After washing in TBST, the membranes were incubated for 1 h at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody (Zhongshanjinqiao Corp.) at a 1:2000 dilution and then washed three times with TBST. Each membrane was placed into ECL solution (Thermo), and signals were subsequently detected using a Molecular Imager ChemiDoc XRS+(BioRad) and analyzed using Image Lab 4.0.1.

8. Metabolic Measurement:

Metabolic measurements were obtained by using an Oxymax Lab Animal Monitoring System (Columbus Instruments, Columbus, Ohio). The system was calibrated against a standard gas mixture to measure O₂consumed (VO₂, ml/kg/h) and CO₂generated (VCO₂, ml/kg/h). These measurements were taken on animals that had received Lycium barbarum for 2 months. The first 12 h were a period of adaptation for the animals, and then metabolic rate (VO₂) and food intake were evaluated for a 24-h period. Energy expenditure (or heat production) was calculated as the product of the calorific value of oxygen (3.815_1.232_respiratory quotient) and the volume of O₂consumed.

9. XF24 Oxygen Consumption Assay and Oxygen Consumption Rate:

Oxygen consumption was measured using the XF24 Extracellular Flux Analyser from Seahorse Bioscience. For this, 2 h before the analysis, control, overexpressing C2C12 myoblasts on 24-well XF24 V28 cell culture microplate (Seahorse Bioscience) were pretreated compound C. One hour before the experiment, cells were washed and incubated in 630 ml of non-buffered (without sodium carbonate) DMEM (4.5 g l_1 glucose) pH 7.4 at 37° C. in a non-CO₂incubator. Five replicates per cell type were included in the experiment, and four wells evenly distributed within the plate were used for correction of temperature variations. During the time course of the experiment, oxygen concentration was measured over time periods of 2 min at 6-min intervals, consisting of a 2 min of mixing period and a 4 min waiting period. OCR over the 2 min measurement period was calculated using the Fixed Delta technique for determining the slope.

10. ATP Production Assay/Statistical Analysis

The results are presented as the mean±standard error of the mean (SEM) of at least 3 independent experiments. The statistical significance of the difference between two means was calculated using Student's t-test, and differences between young and old groups were calculated by performing a 2-way ANOVA analysis. For all analyses, p<0.05 was considered statistically significant, and significance levels are indicated as follows: ***, p<0.001; **, p<0.01; *, p<0.05.

Experimental Example 2

Lycium barbarum Extracts (LB) Increases the Weight of Skeletal Muscles and Enhance Exercise Capacity

Male C57BL/6 mice of about 8 weeks of age purchased from Weitong Lihua Company were randomly divided into a control group and a Lycium barbarum administration group. In the Lycium barbarum group (“LB”), Lycium barbarum extract was administered by gavage to 2.5 g/kg daily, and physiological saline was used as a control group (“Ctrl”). The mice were given Lycium barbarum for 4 months. After the Lycium barbarum extract was administered to the stomach, the mice in the control group and the Lycium barbarum group were weighed respectively. The mice were anesthetized with ether, blood was taken, then the neck was sacrificed, and the anterior tibialis and gastrocnemius muscles of the legs of the mice were dissected and weighted.

As shown in FIGS. 1A-1C, the water extract of Lycium barbarum had no significant change in mouse weight (FIG. 1A), but it could significantly increase the weight of the tibialis anterior and gastrocnemius muscles of mice (FIGS. 1B-1C).

The running experiment was used to test whether the extract of Lycium barbarum could improve the exercise ability of mice. The mouse running instrument was set to rotate the conveyor belt, that is, the running speed of the mouse and the intensity of electrical stimulation. The inclination of the running platform of the mouse was set to 15 degrees. The mouse was placed in the running channel, first equilibrated for 3 minutes at a speed of 10 m/min, then accelerated to 30 m/min at an acceleration of 2 m/min within 10 minutes, and then moved at a uniform speed at this speed. The mouse's continuous activity was taken in the electric shock zone or the second half of the running platform connected to the electric shock zone as the standard for the initial fatigue of the mouse. If the mouse stayed in the electric shock zone under the 2.5 mA current stimulation, the mouse was completely fatigued. The total distance that the mice ran was recorded at the time of complete fatigue, and this was taken as the mouse's exhausted exercise capacity.

As shown in FIG. 2, water extract of Lycium barbarum can significantly increase the running distance of mice in exercise experiments by about 40%, which improves the exercise ability of mice. Lycium barbarum extracts relieve muscle fatigue and enhance endurance.

The effect of Lycium barbarum extracts (LB) on skeletal muscles was evaluated in male C57BL/6J mice that were administrated a dose of 2.5 g/kg/day of LB (normal saline as control) through intragastric administration for 4 months. The mice in LB group had no change of the body weight compared to the control group (FIG. 1A), but they showed obvious change in muscle mass (FIGS. 1B-1C). LB significantly increased the weight of tibialis antrior (TA) and gastronemius (GAS) (FIGS. 1B-1C).

In order to investigate the effect of LB on improving muscle function, the exercise capacity of the mice was tested. The exhausting running distance of the mice in LB group is much longer than the control group (FIG. 2). These findings suggest that endurance based on oxidative fiber type was increased by LB. Meanwhile, the decreased lactic acid level in serum also supported that LB relieved the muscle fatigue (FIG. 3). Therefore, LB feeding in mice for long time relieved the muscle fatigue and enhanced endurance, and this function may be related to the change of muscle fiber types.

Experimental Example 3
LB (Lycium barbarum extract) Promoted Muscle Glycolytic-to-Oxidative Fiber Type Switch In Vivo and In Vitro

To further validate the shift in muscle fiber types following LB feeding, type I (oxidative), type IIa (oxidative) and type IIb (glycolytic) fibers were stained by IF on the frozen sections of the muscles (FIG. 4). In TA muscles, there was almost no type I fibers, the ratio of type IIa fibers was increased significantly, with corresponding the ratio of type IIb was decreased (FIG. 4A), which indicated that LB promoted glycolytic-to-oxidative fiber type switch in vivo.

At the same time, qPCR was used to determine the expression of mRNA characteristic of type IIa and type IIb fibers. It revealed an increase in mRNA expression of Myh2, a marker of type IIa fibers (FIG. 5B), and no change in the transcript levels of Myh4, which is a marker of type IIb fibers (FIG. 5C). Because oxidative fibers are rich in mitochondria and myoglobin, increased myoglobin expression and mtDNA content in TA muscles after LB treatment also confirmed that LB enhanced endurance through promoting glycolytic-to-oxidative fiber type switch (FIGS. 6-7). However, in soleus muscles, there was no significant change in the proportion of three types of muscle fibers due to the extremely small number of type IIb fibers (FIG. 4B).

In differentiated myotube, the expression of markers of type IIa and type IIb fibers was also evaluated in vitro. Consistent with the results in vivo, the mRNA level of the markers of oxidative fiber (Myh7 and Myh2) was induced by LB in a dose dependent manner, and the level of Myh4 had no change (FIG. 8). Therefore, LB promoted muscle glycolytic-to-oxidative fiber type switch in vivo and in vitro.

Experimental Example 4
LB Enhanced the Oxidative Capacity In Vivo

Distinct muscle fiber subtypes are closely related to metabolic activity, type I and type IIA are rich in mitochondria and myoglobin, and have high oxidative capacity.

To further validate the enhanced oxidative capacity in the mice from LB group, the inventors measured energy influx and consumption in the whole bodies of both control and LB group mice with metabolic-chamber analysis. The mice in LB group had a significant overall increase in O₂consumption (FIG. 9A), and energy expenditure (EE) (FIG. 9B), suggesting that LB increased the energy expenditure in mice under normal physiological conditions. But there is no significant difference of CO₂production between control and LB group (FIG. 9C), with corresponding a decrease in respiration quotient (RQ) in LB group (FIG. 9D). These data indicate the mice in LB group had an increase in oxidative respiration and was more likely to generate energy using fat. These results were correlated with increased oxidative fiber type.

Next, the levels of key genes about energy production in skeletal muscles were detected. LB decreased the expression of Pfk1, and had no obvious effect on Hk2 expression in TA muscles, which are two key enzymes in glycolysis (FIG. 10A). Meanwhile, LB up-regulated the level of aerobic metabolism related gene Atp5b and fatty acid oxidation related gene Cpt1 in TA muscles (FIGS. 10C-10D). Meanwhile, LB up-regulated the level of aerobic metabolism related gene Cox5b, Cox7a (Cytochrome c oxidase subunits) and Cytc (Cytochrome C), suggesting that aerobic metabolism was induced by LB (FIGS. 10B-10C). Increased Atp5b (ATP synthetase 5b) level indicated ATP level was induced by LB (FIG. 10C). The inventors found that LB treatment didn't change the expression of pyruvate dehydrogenase kinase 4 (Pdk4), showing that the increase of ATP synthesis is not realized by the increase of glucose and protein decomposition (FIG. 10D). Fatty acid oxidation related gene cpt1 was significantly induced by LB in TA muscles, indicating that LB increases ATP by promoting fatty acid metabolism (FIG. 10D). These data also support that the TA muscle of the mice in LB group have a higher oxidative capacity than the control group.

Experimental Example 5
LB Promoted Aerobic Respiration in C2C12 Myoblasts

To further assess the effect of LB extract on mitochondrial function in C2C12 myoblasts, cellular bioenergetic profile was analyzed using a Seahorse XF24 Analyzer. As shown in FIG. 11A, 2 mg/mL of LB treated myoblasts had an increase in oxygen consumption rate (OCR). The increased basal respiration resulted from an elevation in proton leak and mitochondrial ATP generation (FIG. 11A), suggesting that LB promoted both heat production and ATP production.

Furthermore, after addition of an uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), 2 mg/mL of LB treated myoblasts showed higher maximal respiration and spare respiratory capacity (FIG. 11A), indicating better respiration potential. Consistent with increased ATP generation in C2C12 cells, total ATP level detected using kit was also increased by more than 2 times (FIG. 11A). Meanwhile, the transcriptional levels of the central components of the electron transport chain in mitochondria were measured. LB dose dependently increased the expression of cytochrome c (Cytc), the subunits of cytochrome c oxidase (Cox2 and Cox5b) and ATP synthase (Atp5b) (FIG. 11B-11E), further supporting the increased ATP generation. Furthermore, LB treated myoblasts exhibited a lower extracellular acidification rate (ECAR) as measured by a Seahorse XF24 Analyzer, indicating decreased glycolytic flux and attenuated glycolytic capacity (FIGS. 12A). Consistent with the decreased glycolytic flux, LB treated myoblasts showed lower mRNA level of hexokinase 2 (Hk2), a key enzyme in the first step in glucose metabolism pathways, with no change of the level of phosphofructokinase 1 (Pfk1) (FIG. 12B, 12C). Therefore, LB promoted aerobic respiration and inhibited glycolysis in C2C12 myoblasts.

Experimental Example 6
LB Modulated Muscle Fiber Type Switch in ERRγ Dependent Manner

Next, the inventors investigated the molecular mechanism behind how LB modulated muscle fiber type switch. The inventors first assessed the expression of the known key regulators involved in the metabolic remodeling of skeletal muscle, including AMPK, PGC-1, Sirt1 and PPAR. The inventors found LB increase the transcriptional level of Esrrg both in vitro and in vivo (FIGS. 13-14). To confirm that LB modulates muscle fiber type and metabolic type switch by regulating ERR pathway, the expression of Esrrg was knocked down using siRNA in C2C12 myotubes. Deficiency of ERRγ inhibited LB induced the mRNA levels of the markers of oxidative fibers (Myh7 and Myh2) and ATP synthase (Atp5b) (FIG. 15), demonstrating that LB increased the ratio of oxidative fiber types and enhanced the aerobic respiration in an ERRγ dependent manner.

To test whether LB showed hormetic effects, high doses of LB extract were used to treat C2C12 myotubes. The inventors found that when the concentration of LB reached 8 mg/mL, many cells died. Although LB at a concentration of 4 mg/mL also increased the level of the gene (Esrrg and Mef2a) expression, the extent of this upregulation was significantly lower than that induced by the 2 mg/mL extract (FIG. 16). Therefore, the beneficial function of LB on C2C12 cells is dose-dependent only in a certain concentration range (approximately 0˜2 mg/mL, for example, 0.1˜2 mg/mL or 0.5˜2 mg/mL).

As reported earlier, exercise was considered an effective way to improve muscle mass and function. Endurance exercise increases the muscle mass, reduces the fat mass, promotes fiber type transformation toward the slow-twitch, stimulates mitochondrial biogenesis and fatty acid oxidation, promotes O₂utilization, CO₂and heat production and resulting in a more oxidative phenotype and improved endurance capacity of the muscles. Thus, exercise triggers a metabolic and structural remodeling in skeletal muscle, thus leading to improved skeletal muscle performance. In recent years, some natural compounds defined as “exercise mimetics” was reported to produce metabolic effects similar to those induced by exercise. Although these functional natural compounds cannot fully replace the benefits of exercise, they have the potential to offer at least some of the benefits of exercise. Lycium barbarum extract can increase muscle mass, reduce fat mass, promotes fiber type switch, stimulate mitochondrial biogenesis and fatty acid oxidation, promotes O₂utilization, ATP and heat production resulting in a more oxidative phenotype and enhanced endurance of the muscles. So it is extremely useful as an “exercise mimetic”.

As a Chinese herbal medicine and functional food, Lycium barbarum extract with “exercise mimetic” effect may be applied to many fields. For example, for endurance athletes, Lycium barbarum may be beneficial to increase the proportion of oxidative muscle fibers. For people lacking or unable to achieve adequate physical exercise, Lycium barbarum extract may be able to prevent muscle loss and promote muscle metabolism to a certain extent. Moreover, loss of skeletal muscle mass is one of the most widespread and deleterious processes in human aging, which compromises the quality of life. Aging muscle atrophy results from atrophy of type II myofibers. In the present disclosure, the inventors found that Lycium barbarum can increase the type IIa fibers and the muscle mass. At the same time, mitochondria play a pivotal role in skeletal muscle homeostasis and aging-related defects in mitochondrial energy metabolism have been proposed to be involved in aging muscle atrophy, while the aerobic respiration and energy production was improved by Lycium barbarum treatment. Therefore, Lycium barbarum extract could maintain functional skeletal muscle mass by up-regulating mitochondrial metabolism and fiber-type transformation, and it may contribute to ameliorate aging muscle atrophy.

Although the subject matter has been described in terms of exemplary embodiments, it is not limited thereto. Rather, the appended claims should be construed broadly, to include other variants and embodiments, which may be made by those skilled in the art.

LYCIUM BARBARUM EXTRACTS, RESULTING COMPOSITIONS, METHODS OF MAKING AND METHODS OF USING THE SAME

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