TECHNICAL FIELD

The phytochemical content of various extracts of the Ononis natrix plant, which is also known as semisk, kayi§kiran, and dkuzgani in Turkey, was determined by our previous analyzes. It has been determined that it is quite rich in terms of quercetin, apigenin and luteolin phenolic compounds. Studies conducted with various animal and in-vitro cell culture models of these three phenolic compounds in the literature have shown that each of them inhibits protein degradation in skeletal muscle that develops due to various reasons. However, there is no study showing that 0. natrix plant extracts itself prevent protein breakdown in the muscles. For this reason, in a study conducted by us, the effect of 0. natrix extracts on atrophy in the skeletal muscle cell culture model has tested: Morphological and molecular analyzes showed that 0. natrix extracts significantly reduced protein degradation in skeletal muscle cells where protein degradation was induced. All these findings show that the extracts of the 0. natrix plant have the potential to be an important preventive supplement or additive in all health problems that require the preservation of muscle function. Therefore, it is anticipated that Onanis natrix plant extracts can be used as a supplement to reduce the loss of skeletal muscle functions in cases where the protein content of skeletal muscle is degraded by aging or secondary to chronic diseases.

BACKGROUND

Currently, there are drugs under development to prevent skeletal muscle atrophy, but the expected results from these drug candidates have not yet been achieved in animal models or human clinical trials. Thus, there is no drug currently in use. The main reasons for this situation are: Some of the drugs target hormones that regulate protein synthesis or degradation pathways, but these drugs have broad side-effect profiles because these hormones also have other roles.

Drugs targeting regulators of protein degradation or synthesis pathways may show side effects as they exhibit affinity for other molecules besides their target.

Most of the drugs tested did not achieve sufficient efficacy in animal models, or sufficient human clinical trials have not yet been conducted for those with good efficacy in animal models.

The current approach to improve the clinical picture of people with skeletal muscle atrophy only includes resistance exercise and a protein-rich diet.

Health problems where skeletal muscle atrophy is observed in humans and preventive practices.

Skeletal muscle is a dynamic organ that constitutes approximately 40% of the total body weight and contains 50% of the total protein amount of the body. The main function of skeletal muscle is to produce movement by contraction. At the same time, it maintains the posture of the body by resisting gravity. It provides the stability of the skeleton by preventing the extra movement of bones and joints, thus preventing the skeletal structure from being deformed and damaged. It also has a critical effect on the body's energy and protein metabolism. Skeletal muscle is the main tissue for glucose uptake and storage. It also acts as a reservoir for the amino acids it stores in the form of protein. In case of need in any part of the body, skeletal muscle tries to meet this need by releasing amino acids.

In people who take bed rest for a long period because of various injuries, shrinkage of skeletal muscles occurs due to inactivity and disuse of skeletal muscles. This is because the body maintains enough muscle tissue to meet the movement it produces. It breaks down skeletal muscle protein content to reduce muscle mass. The damage can generally be reversed with appropriate exercises. A similar situation occurs when fasting for a long time. Although the body's fat stores are primarily used, skeletal muscle proteins are degraded to meet both the energy and amino acid needs of the body. The damage can be reversed with a diet that contains the basic nutrients that the body needs.

Synthesis and degradation rates of skeletal muscle proteins are usually in balance. The body chooses one of the ways of synthesis or degradation according to changing conditions and balances the muscle mass according to the need.

There is a balance between protein synthesis and degradation in skeletal muscle. According to the changing environmental conditions, the body chooses one of the ways of synthesis or destruction, and thus it can balance the muscle mass according to the need. However, some physiological processes or acute and chronic diseases cause the balance shifts in the direction of deterioration. The balance shift to the direction of destruction causes muscle loss that cannot be overcame only by consuming the needed nutrients or by exercise. This condition, which is characterized by progressive protein degradation in the muscles and therefore a continuous decrease in muscle mass, is known as muscle atrophy. Muscle atrophy can cause partial or complete loss of function of the muscle, resulting in a serious loss of strength in the body. Atrophy may cause individuals to be unable to perform their daily tasks, and in severe cases may result in the death of the individual. The main conditions in which skeletal muscle atrophy is observed are described below:

• Age-related sarcopenia. It occurs in approximately 20 % of older adults. It is a condition in which the protein mass in the muscles gradually decreases with aging. Several factors may induce protein breakdown during sarcopenia including a decrease in the number of nerve cells that stimulate the muscles, a reduction in the vascular supply that feeds the muscles, a decrease in physical activity, and a decrease in growth hormones. Sarcopenia significantly reduces the quality of life of elderly individuals. Individuals who affected by sarcopeni may need support to walk, have difficulty climbing stairs, and experience repeated falls.

• Cancer cachexia. It is progressive skeletal muscle atrophy associated with cancer. It occurs in approximately 35% of cancer patients and is responsible for the death of approximately 20% of affected patients. In addition, it reduces the response of patients to treatment or decreases their quality of life.

• Neurodegenerative diseases such as amyotrophic lateral sclerosis. In neurodegenerative diseases, the main damage does not occur in skeletal muscle cells. Since nerve cells are damaged, contraction command cannot be transmitted to skeletal muscle cells. In the absence of a stimulus, the protein contents are constantly degraded, as the muscles are immobilized.

• Myopathies such as Duchenne muscular dystrophy. The primary cause of muscle wasting observed in myopathies is a defect that interferes with the normal function of skeletal muscle cells. Deficiency or dysfunction of structural or metabolic proteins of muscle cells causes permanent damage to muscle cells. This damage increases with age, resulting in a low quality of life and premature death of affected individuals.

• Psychological disorders such as anorexia. These types of eating disorders cause systemic damage to the body, as patients cannot provide the basic nutritional components that the body needs. One of the most affected tissues is skeletal muscle tissue. Since there is no sufficient nutritional support around muscle cells, a continuous protein breakdown occurs, and muscle mass decreases as the disease progresses.

Although its incidence is very high in humans, it has significant effects on human health and quality of life and also causes severe economic burdens on the health system, there is no clinically approved drug targeting skeletal muscle atrophy yet. Currently, drug candidates targeting skeletal muscle atrophy are under development. The drugs under development are mainly aimed at suppressing the protein degradation pathways or stimulating the protein synthesis pathways. Many of these drug candidates are currently being tested in animal models and human clinic trials. Current drug candidates under development or testing for efficacy are given below.

Anti-inflammatory drug candidates: Thalidomide (a TNFa cytokine inhibitor.)

Xilonix (other name is MABpl . It is an antibody that targets the cytokine IL1a.)

ALD518 (An antibody that targets the IL6 cytokine.)

Ruxolitinib (It is a molecule that inhibits the effect of IL6 cytokine in cells.) Meloxicam (it is a COX2 enzyme blocker.)

Celebrex (a COX2 enzyme blocker.)

MSTN/ActRII signaling pathway inhibitors (MSTN or myostalin is a type of autocrine hormone that inhibits muscle cell growth):

LY2495655 and REGN1033 (The antibodies targeting myostatin hormone.) ACE-031 and ACE-083 (activin I IB (ActRIIB) receptor blockers that regulate the functioning of myostatin hormone.)

NGM120 (An antibody that targets GFD15, a growth and differentiation hormone.)

Drugs that stimulate protein synthesis:

LGD-4033, MK0773 and Enobosarm (Tissue-specific, non-steroidal, selective androgen receptor modulators. They are called SARMS. SARM, selective androgen receptor modulators)

Anamorelin and SLIN11031 (Synthesic compounds that stimulate the ghrelin hormone pathway. Ghrelin is a hormone that stimulates hunger.)

Apart from pharmacologically developed drugs, some plant-derived polyphenolic compounds have also been found to inhibit protein breakdown in skeletal muscle. Among them, the Matrine compound, the main alkaloid of the plant Sophora flavesces, used in traditional Chinese medicine, has been approved by the Chinese Food and Drug Administration (CFDA) for use in patients with cancer cachexia.

DESCRIPTION OF THE INVENTION

The invention in question eliminates the disadvantages described in the state of the art and meets the needs. Our polyphenolic compound content analysis has revealed that various extracts of Ononis natrix plant are quite rich in terms of quercetin, apigenin and luteolin. For each of these compounds, there are studies in the literature showing that they prevent skeletal muscle atrophy. We have not detected any other plant containing all three polyphenolic compounds in the literature. Today, each of these polyphenolic compounds is available to people as a supplement under different brands. However, the plant extracts have not yet been commercialized as a supplement by any company. We also determined that when skeletal muscle cells were treated with Onanis natrix plant extracts under atrophy conditions, they were less affected by atrophy morphologically and molecularly than cells without plant extracts. This finding indicates that the plant extracts of Onanis natrix can be used by humans as a supplement against skeletal muscle atrophy.

Certain properties make Onanis natrix plant a potential medicinal supplement, such as being a natural source with rich polyphenolic contents that prevent protein breakdown in skeletal muscle, easy to produce and prepare its own extracts, and the extracts itself have been shown to prevent skeletal muscle atrophy.

Brief Descrption Of The Drawings

Figure 1: Representative schematic view of the subject invention.

DETAILED DESCRIPTION

Basic explanations of the invention;

1 ) In the present invention, the name "Onanis natrix” refers to all subspecies of the genus Onanis natrix belonging to the Fabaceae family.

2) In the present invention, the whole plant or individual parts such as root, stem and leaves can be used as raw material to prepare the extracts. The fresh or dried form of the whole plant or its parts can be used to prepare the extracts.

3) In the present invention, the extracts can be prepared by all known protocols used to prepare extracts from plants. Conventional organic solvents or water can be used to prepare the extracts. 4) In the present invention, the extracts of Onanis natrix are prepared as an orally consumable supplement, either on their own or in complex with other supplements, in tablet or liquid form. For oral administration, Ononis natrix plant extracts can be readily formulated in combination with a well- known pharmaceutically acceptable carrier. Such carriers mediate the preparation of the extract as oral tablets, pills, capsules, syrups or various suspension compositions. Oral supplements may require mixing the extract with solid excipients to bring them into tablet or pill form. Fillers in the form of sugars such as lactose, sucrose, mannitol or sorbitol or cellulose-based substances such as cornstarch, wheat starch, rice starch, gelatin and methylcellulose can be used as suitable excipients. Solutions in syrup or suspension form can be prepared by dissolving the extract in a suitable lipophilic solvent, in particular sesame oil, ethyl oleate or a triglyceride.

We determined the phytochemical content of ethyl acetate, methanol and water extracts of Onanis natrix hispanica. In addition, the efficacy of the water extract to inhibit skeletal muscle atrophy was tested on C2C12 murine myoblast cells. Relevant analyzes and results are detailed below.

• Determination of phenolic content of the extracts

Phenolic content of ethyl acetate, methanol and water extracts of Onanis natrix hispanica was determined. While preparing the ethyl acetate and methanol extracts, the air-dried aerial part of the plant were macerated for 24 hours at room temperature (25 ± 1 °C) using 200 ml of the corresponding solvent. The extracts were concentrated under vacuum at 40°C with a rotary evaporator. To obtain the water extract, the powdered samples were boiled with 250 ml of distilled water for 30 minutes. The aqueous extract was then filtered and lyophilized (-80°C, 48 hours).

The phenolic compound content of the extracts was evaluated by RP-HPLC (Shimadzu Scientific Instruments, Tokyo, Japan). Compounds were identified and quantified using the LC-10ADvp pump, Diode Array Detector, CTO-1 OAvp column heater, SCL-10Avp system controller, DGU-14A degasser, and SIL-1 OADvp auto- sampler (Shimadzu Scientific Instruments, Columbia, MD). Separation was performed on an Agilent R Eclipse XDB C-18 reverse-phase column (250 x 4.6 mm length, 5-pm particle size) at 40°C. Gallic acid, protocatechic acid, (+)-catechin, p- hydroxybenzoic acid, chlorogenic acid, caffeic acid, (-)-epicatechin, syringic acid, vanillin, p-coumaric acid, ferulic acid, sinapic acid, benzoic acid, o- Coumaric acid, rutin, hesperidin, rosmarinic acid, eriodictyol, trans-cinnamic acid, quercetin, luteolin, kaempferol and apigenin were used as standards. Identification and quantification were performed by comparison with standards. The amount of each phenolic compound was expressed as micrograms per gram of extract using external calibration curves obtained for each phenolic standard.

We detected 1356 pg/g, 1006 pg/g and 850 pg/g, quercetin, luteolin and apigenin in the water extracts of Onanis natrix hispanica, respectively. In addition, 756 pg/g and 4136 pg/g, luteolin and apigenin in the ethyl acetate extract, respectively; 908 pg/g and 3314 pg/g, luteolin and apigenin were determined in the methanol extract, respectively.

• Testing the efficacy of extracts in preventing skeletal muscle atrophy Atrophy inhibition potentials of 400 pl/ml and 600 pl/ml concentrations of O. natrix water extract were investigated in differentiated C2C12 cells in which atrophy was induced using dexamethasone at 100pM concentration.

C2C12 myoblast cells were grown in DMEM (Dulbecco's modified Eagle medium) medium supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin and 100pg/ml streptomycin. When the proliferating cells reached 70-80% confluency, the medium used for growth was replaced with DMEM medium supplemented with 2% horse serum, 100U/ml penicillin, and 100pg/ml streptomycin to stimulate differentiation. The proliferation and differentiation processes were carried out in a 37°C incubator containing 5% CO2. Cells stimulated to differentiation were kept in medium containing 100pM dexamethasone and 400 - 600pg/ml water extract of Onanis natrix for 2 days from the 3rd day of differentiation.

LADD staining of differentiated C2C12 cells was performed to measure myotube diameter. After washing the cells with 1XPBS (phosphate-buffered saline) buffer, they were kept for 10 minutes in 70% ethanol to fix the cells. After removal of ethanol, cells were incubated with LADD dye for 1.5 minutes. The LADD dye was removed by 3 consecutive washes of distilled water. After the cells were dried at room temperature, they were visualized under a light microscope. qRT-PCR method was used to determine how Onanis natrix plant extracts affect the mRNA expressions of MAFbx and MuRF1 genes, which are markers of protein degradation pathway in muscle cells. Total RNA from cells was isolated using trizol reagent according to the manufacturer's instructions. Total RNA from cells was isolated using trizol reagent according to the manufacturer's instructions. The cDNA library from total RNA was constructed using the reverse transcriptase enzyme with oligo-dT primers targeting only mRNAs. Quantitative real-time PCR was performed using the iTaq universal SYBR green mix. The qRT-PCR reaction was performed in a total volume of 10pl using 5pl master mix, 2pl cDNA, 2pl nuclease free water and 0.5pl of each primer. 5'-CTCAGAGAGGCAGATTCGCA-3' and 5'- GGTGACCCCATACTGCTCTC-3' primers were used for the MAFbx gene. 5'- GAGGGCCATTGACTTTGGGA-3' and 5'-TCTTTACCCTCTGTGGTCACG-3' primers were used for the MuRF1 gene. 5'-TCCCAGCTTAGGTTCATCAGG-3' and 5'-CCCAATACGGCCAAATCCGT-3' primers were used for the GAPDH gene. qRT- PCR reactions were performed with an initial denaturation of 30 seconds at 95°C and 5 seconds at 95°C and 25 seconds at 57°C for 40 cycles.