Naturally occurring sesquiterpene lactone that prolongs lifespan on an in vivo model of caenorhabditis elegans: germinolide

Abstract

The present invention relates to a compound that has effects such as preventing, reversing, stopping or slowing aging, and a drug / food supplement comprising this compound.

Description

[0001] NATURALLY OCCURRING SESQUITERPENE LACTONE THAT PROLONGS LIFESPAN ON AN IN VIVO MODEL OF CAENORHABDITIS ELEGANS:

[0002] GERMINOLIDE

[0003] Technical Field

[0004] The present invention relates to a compound that has effects such as preventing, reversing, stopping or slowing down aging, and a drug / food supplement comprising this compound.

[0005] Background

[0006] Aging is a complex process that starts from birth. The risk of developing diseases and the mortality rate increase with aging. The aging process has more than one mechanism, hence more than one distinctive feature. While the preparations used affect the aging process in different ways, a compound / intervention that covers all mechanisms (which stops, slows down, prevents or reverses aging) has not been found yet.

[0007] Popular preparations, which are widely used in the healthcare sector worldwide, such as resveratrol, pycnogenol or quercetin, whose natural active compounds are phenolic substances, exhibit the activity of prolonging the lifespan. For example, metformin is a drug in the class of senomorphics (transforming aged cells), while quercetin is used for its senolytic effect (selectively killing aged cells). Resveratrol acts on sirtuins. Pycnogenol, another preparation in use, is not a pure compound, but a mixture. Apart from these, the possibility of targeting a mechanism and providing a more comprehensive scheme of action than existing treatments is important.

[0008] The patent document No. CN113116871 A relates to anti-aging stilbene compounds and especially their applications in prolonging the lifespan of nematodes.

[0009] In the patent document No. CN111759837A, the use of Chrysanthemum lactone compound as an anti-stress drug or a life-prolonging drug for Caenorhabditis elegans species is described. In the patent document No. CN111388460A, the invention relates to the anti-aging application of hematoxylin A, an active ingredient of traditional Chinese medicine.

[0010] The patent document No. EP1675557A1 describes a dermatological or cosmetic composition comprising at least one sesquiterpene lactone selected from vulgarin, 4-epivulgarin, 2,3- dihydrovulgarin and their salts and one or more vehicles or excipients as active ingredient.

[0011] However, there is a need for alternative new compounds that stop, slow down, prevent or reverse aging through known or new mechanisms (through unknown targets).

[0012] Brief Summary and Objects of the Invention

[0013] Upon the demonstration of a very high lifespan prolonging effect of the pure molecule with terpene structure isolated from the Inula germanica plant with the invention on an in vivo model of Caenorhabditis elegans (C. elegans), this compound and its derivatives with a sesquiterpene lactone skeleton structure, which have an anti-aging effect, that is, reduce or stop aging, are synthesized.

[0014] The invention also aims to clarify the action pathway of this compound.

[0015] Definitions of Drawings Illustrating the Invention

[0016] Figure 1: Germinolide1HNMR spectrum (CDCh)

[0017] Figure 2: Germinolide APT spectrum (CDCh)

[0018] Figure 3: Germinolide COSY spectrum (CDCh)

[0019] Figure 4: Germinolide HSQC spectrum (CDCh)

[0020] Figure 5: Germinolide HMBC spectrum (CDCh)

[0021] Figure 6: Germinolide NOESY spectrum (CDCh)

[0022] Figure 7: Germinolide UV-Vis spectrum (MeOH)

[0023] UV (MeOH) km ax (log a) nm: 206 (1.73), 283 (sh) (0.46), 323 (0.40) nm

[0024] Figure 8: Germinolide IR spectrum IR vmax (NaCl) cm'1: 2924, 2855, 1756, 1714, 1666, 1455, 1383, 1303, 1232, 1149, 1065, 1040, 1019, 977,854, 817, 787, 735, 707, 621, 567, 509 cm'1

[0025] Figure 9: Germinolide HRMS m / z [M+Na] 385.16104 (calculated: 385.1627) (Error rate: 4 PPm)

[0026] Figure 10: Time (minutes) vs. Life (%) Values of Germinolide and Control Groups

[0027] Detailed Description of the Invention

[0028] The present invention relates to an active and novel compound of Formula (I) naturally obtained from the Inula germanica species, whose structure was clarified, or a pharmaceutically acceptable derivative thereof.

[0029] Germinolide

[0030] Formula (I)

[0031] The compound of the invention or a pharmaceutically acceptable derivative thereof can be used as a drug to prevent, reverse, stop or slow down aging. Pharmaceutical composition comprising the compound of the invention and food supplement or drug comprising this composition can be obtained.

[0032] It is very important to collect the medicinal plant, which is the natural source used in obtaining the compound subject to the invention, at the right time, to dry it under the right conditions, to store it until the start of the processes, and to choose the extraction as maceration, in terms of richness in sesquiterpene lactone. The chromatographic methods chosen for the purification of the obtained dry extract and the solvent solubility and systems used in these methods, and the adsorbents used in column chromatography provide serious contributions to obtaining the pure compound in high yield. The synthesis method of the compound of the invention generally comprises the following steps:

[0033] • Drying of Inula germanica L. plant,

[0034] • Macerating it in organic solvent,

[0035] • Removal of organic solvent from the environment.

[0036] The method of preparation of the compound of the invention is described in detail below.

[0037] 1-Drying: Inula germanica L. plant, which was collected from nature, was dried in the open air and in the shade. It should be collected while the plant is flowered.

[0038] 2-Maceration: In order to obtain the pure compound optimally, the extraction step, which is the first step in the isolation of the compound, should be carried out with a short-term maceration, which is a special method. Sesquiterpene lactones are very unstable structures in terms of their chemical structure. For this reason, the maceration method should be performed at room temperature (between 18-25 °C). During the removal of the obtained extract from its solvent, high temperature should not be applied (over 60 °C) and it should be protected from light. It is aimed to isolate and clarify the structures of the secondary compounds, which are found especially in the glandular hairs of the above-ground parts and secreted by the plant for self-defense. For extraction, after drying 4 kg of plants, they were macerated 3 times for 15 minutes with dichloromethane organic solvent (chloroform can also be used instead of this solvent) without grinding (may be performed 3-5 times at intervals of 15-25 minutes). Extraction can be performed by continuous extraction with a soxhlet apparatus using dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol and n-butanol solvents, as well as using the supercritical carbon dioxide extraction method. Afterwards, the dichloromethane solvent was completely concentrated from the coarse extract with a rotary evaporator and was removed at low temperature (30-60 °C) and in vacuum.

[0039] 3-Removal of chlorophylls: In order to remove the chlorophylls in the obtained dry extract, the dry extract was dissolved with 60% ethanol: water, an organic solvent mixture, and then exhausted with petroleum ether, toluene, dichloromethane and ethyl acetate using liquid- liquid extraction method. Removal of chlorophylls can also be performed with the help of activated charcoal. The di chloromethane extract obtained was completely dried with a rotary evaporator again and removed from the organic solvent. During isolation, this step contributes to purification.

[0040] 4-Chromatographic analyses: In order to purify the natural compounds, fractionation and isolation studies were carried out on dry di chloromethane extract together with the chromatographic analyses (Sephadex LH-20 column chromatography, thin layer and preparative thin layer chromatography). Methods using different adsorbent and mobile phases as chromatographic methods in purification steps may be used (Stationary phase for column chromatography: Silica gel, polyamide; mobile phase: Petroleum ether, cyclohexane, ether, dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol; stationary phase for preparative HPLC method: Phenyl-hexyl, C-18; mobile phase: acetonitrile, distilled water, methanol, acetic acid, trifluoroacetic acid, tetrahydrofuran, hexane, ethyl acetate; or chromatotron).

[0041] As a result of these studies, Ig (65-88) (2) coded germinolide substance was obtained in a pure and dry form. When clarified by spectral analyses, it was determined that it is a sesquiterpene lactone derivative compound with melampolide structure, which is not available in the literature. The selection of the stationary and mobile phases used to obtain the pure active compound from the plant in chromatographic analyses is very important.

[0042] 5-Spectral analysis: The obtained pure compound was subjected to various spectral analyses for the clarification of the structure (Figures 1-9). It was observed that the molecular structure clarified in the light of all these spectral analysis data is a sesquiterpene lactone derivative that is not available in the scientific literature. NMR spectra constitute the most important part in the structure determination of the active pure compound.

[0043] 6-Biological activity: It is the step that plays an important role in determining the activity of the compound obtained from a natural source. Since the plant is known to be used in chronic diseases such as diabetes, this new compound, along with other isolated compounds, was also tested in the model organism Caenorhabditis elegans (C. elegans) and was found to prolong the lifespan of the organism more than 3 times (Figure 10). The thermotolerance method used helps to screen for the possible effects of the tested substance on the lifespan of the worms within hours. It is possible that the molecule, which exhibits high activity on an in vivo model, becomes active in various metabolic pathways in humans. In the experiment, C. elegans nematodes were stored at room temperature. Synchronized eggs were followed up to adulthood using OP50-1 strain E. coli bacteria on NGM (Nematode Growth Medium) as a food source. Afterwards, the organisms were mixed with E. coli bacteria and transferred to the experimental petri dishes to which the invention material was added, at a ratio of 40 individuals per petri dish. After exposure at room temperature for 24 hours, the organisms were followed up by capturing high-resolution images every 20 minutes in a preheated incubator at 35 °C. The experiment was continued until all individuals died. The data obtained were compared with the data of the control group, which underwent the same processes and was not treated with the natural compound, and a significant difference was found (p < 0.05). As shown in Table 1, 125 organisms in the control group and 117 organisms in the Germinolide group were included in the experiment and the experiment was planned in triplicate. The experimental process was accelerated under the heat stress of organisms and images were captured with a high-resolution scanner every 20 minutes. Those who did not appear to move in two consecutive images were considered dead. As a result of the statistical study carried out with the obtained data, it was determined that there was a significant difference between the groups, and it was observed that the germinolide substance had a strong life-prolonging effect.

[0044] Table 1: Concentration of Substances Used in Experimental Groups and Number of Organisms

[0045] In Table 2, the % mortality values of both experimental groups calculated by the Kaplan- Meier method are shown against time. When the control group reached the 100% death point at the 100th minute, the corresponding value of the germinolide group was calculated as 4.30%. The death of the whole experimental group was observed as of the 260th minute.

[0046] Table 2: % Mortality Calculated by Kaplan-Meier Method

Claims

CLAIMS1. The compound of Formula (I) or a pharmaceutically acceptable derivative thereof.Formula (I)2. A compound according to claim 1 or a pharmaceutically acceptable derivative thereof, for use as a drug in the prevention, reversal, stopping or slowing of aging.

3. A pharmaceutical composition, characterized in that it comprises the compound according to Claim 1.

4. A pharmaceutical composition according to claim 3, characterized in that it comprises a pharmaceutically acceptable excipient in addition to the compound of Formula (I).

5. A drug, characterized in that it comprises the composition according to Claim 3 or 4.

6. A food supplement, characterized in that it comprises the composition according to Claim 3 or 4.

7. A synthesis method of the compound according to claim 1, characterized in that it comprises the following process steps: a) Drying of Inula germanica L. plant, b) Macerating it in organic solvent, c) Removal of organic solvent from the environment.

8. A method according to claim 7, characterized in that it comprises the process step of removing the chlorophylls in the dry extract from the environment.

9. A method according to claim 8, characterized in that it comprises the process step of applying chromatographic analyses to obtain the pure compound.

10. A method according to claim 7, characterized in that the maceration is performed in the range of 18-25 °C.

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