A method for the synthesis of the isorhamnetin metabolite

Abstract

The present invention relates to a method for the synthesis of isorhamnetin metabolite from the quercetin molecule and the isorhamnetin metabolite synthesized by this method. In this method, isorhamnetin is synthesised from quercetin molecule by microbial transformation of Aspergillus flavus. The method of the present invention provides the synthesis of isorhamnetin with 18.20% yield in a short time and at low cost.

Description

[0001] A METHOD FOR THE SYNTHESIS OF THE ISORHAMNETIN METABOLITE

[0002] Technical Field of the Invention

[0003] The present invention relates to a method for the synthesis of isorhamnetin metabolite from the quercetin molecule and the isorhamnetin metabolite synthesized by this method. In this method, isorhamnetin is synthesised from quercetin molecule by microbial transformation of Aspergillus flavus (ATCC 9807). The method of the present invention can be carried out in a short time and at low cost.

[0004] State of the art

[0005] Polyphenolic compounds are secondary metabolites with important physiological and morphological effects in plants, which are found in many plant foods containing flavonoids, phenolic acids, lignans and stilbenes, as well as more than 8000 varieties in nature [1]. These compounds basically have an aromatic ring bearing one or more hydroxyl groups. There are also forms containing sugar groups such as monosaccharide and disaccharide in their structures. Polyphenolic compounds are of great importance especially due to their phytoalexin, antifeedant, antioxidant, cardioprotective biological activities [2], In fact, epidemiological studies also confirm that moderate and long-term intake of foods rich in polyphenols can prevent the development of cancer and chronic diseases such as cardiovascular, neurodegenerative, type 2 diabetes and obesity [3] and polyphenols provide a vital function in protecting the organism from external stimuli and eliminating reactive oxygen species (ROS), which are the triggers of many diseases. In addition to their pharmacological effects, these compounds have many different uses such as their protective properties against UV rays, as attractants for pollinators and as colouring dyes [4],

[0006] Flavonoids, which are secondary metabolites with a polyphenolic structure, are molecules of natural origin occurring in different parts of many plants. Flavonoids are recognised as an indispensable component in various nutraceutical, pharmaceutical, medical and cosmetic applications due to their anti-oxidative, anti-inflammatory, anti- mutagenic and anti-carcinogenic properties together with their capacity to modulate cellular enzyme function. Although they have polyphenolic structure, due to the differences in their structures, diversity is observed in the biological activities of flavonoids and they are also used in the treatment of cardiovascular system disorders, chronic diseases and cancer diseases in terms of their biological activities. However, flavonoids are usually linked to sugar groups by glycosidic bonds and glycoside forms containing water-soluble sugar groups are difficult to be absorbed in the gastrointestinal tract (Gl tract) due to their low pharmacological activity, resulting in low bioavailability in the body [5]. To solve this problem, most studies in the state of the art show that bioavailability of flavonoids increases when they are converted into simple phenolic acids and absorbed by the intestinal microbiota through enzymatic degradation, hydrolysis, reduction, dehydroxylation and other reactions [6]-

[0007] Quercetin, a flavonoid derivative with a flavonol structure, is yellow in color and is in aglycone form that does not contain a sugar group. Quercetin is found in various plants such as onions, green and black tea, cherries, legumes, and is commercially obtained from green-blue algae. It is important for human health like other flavonoids due to its antioxidant, anti-tumor, anti-inflammatory, antihypertensive, antiobesity, anticholesterolemic and vasodilator effects [7], In addition to the effects of quercetin, isorhamnetin, the secondary metabolite of quercetin, also provides positive effects on human health [8], but the secondary metabolite of isorhamnetin is less common in nature than quercetin. Although this secondary metabolite has effects such as cardiovascular and cerebrovascular protection, anti-tumour, anti-inflammatory, antioxidation, organ protection, obesity prevention, etc. [9], flavonoids pose a problem for dosage formulations due to their solubility problem and unstable structure although they show strong biological activity. At the same time, the bioavailability of flavonoids is quite low when used internally. For this reason, in the state of the art, enzymatic, chemical or chemoenzymatic modification methods can be used against these problems encountered with flavonoids. However, since these methods work with living structures, there is a risk of contamination, there is a need for regular analyses in production processes in order to ensure the same yield of metabolites on a regular basis, as well as the growth cycles of living structures when the incubation process is started, the aforementioned methods are disadvantageous due to the necessity of the same period. In the state of the art, the methods by which isorhamnetin compound is obtained are chemical synthesis and isolation from extracts of plant origin. In chemical synthesis, the synthesis is completed in a plurality of steps. In this case, toxic solvents are generally used

[0010] . In studies carried out with isolation methods, the efficiency is quite low. In the study conducted by Cao et al.

[0011] , 11.82 mg isorhamnetin was isolated from 700 mg extract with a yield of 1 ,69%.

[0008] A study carried out by Chatla et al.

[0012] , which is in the state of the art, relates to obtaining the campferol metabolite from quercetin by microbial transformation. In the abovementioned study, microbial transformation of quercetin is carried out using potato dextrose broth and Aspergillus niger is used as the transformation strain. The fermentation process takes place under aerobic conditions at 37 °C, pH 6, but there is no description of the method efficiency.

[0009] Due to the limitations and inadequacies of the solutions in the state of the art, the low bioavailability of flavonoids, the synthesis of flavonoid metabolites in low yields or the use of toxic chemicals during the synthesis phase, the high risk of contamination in the methods in the state of the art, the need for regular analyses in the production processes to ensure the same yield of metabolites and the associated time loss and cost increase, it has become necessary to develop a method for the synthesis of isorhamnetin.

[0010] Brief Description and Objects of the Invention

[0011] The invention describes a method for the synthesis of isorhamnetin metabolite from the quercetin molecule and the isorhamnetin metabolite synthesized by this method. In this method, isorhamnetin is synthesised from quercetin molecule by microbial transformation of Aspergillus flavus. The method of the present invention can be carried out in a short time and at low cost.

[0012] The object of the present invention is to synthesize the secondary metabolite of isorhamnetin. In the invention, isorhamnetin metabolite is synthesized from the quercetin molecule, a flavonoid, by microbial transformation.

[0013] Another object of the invention is to synthesize the secondary metabolite of isorhamnetin in high yield, in a short time and at low cost. With the optimized parameters in the method of the present invention, the efficiency of the method is 18.20%. Since the abovementioned method is based on microbial transformation of Aspergillus flavus, the synthesis of isorhamnetin metabolite occurs in a short time. In addition, with the help of the shortening of the process time and optimum parameters compared to the methods in the current technique, the method of the present invention can be carried out in a short time with low cost.

[0014] Description of the Figures

[0015] Figure 1. TLC image of Quercetin-A flavus transformation extracts and metabolite (Rf: 0.46) at 365 nm (F15Q day 4: Aspergillus flavus day 4 transformation extract F15 day 4: A. flavus day 4 blind extract; F15Q day 8: A. flavus day 8 transformation extract F15 day 8: A. flavus day 8 blind extract; F15Q day 12: A. flavus day 12 transformation extract F15 day 12: A. flavus day 12 blind extract)

[0016] Figure 2. Metabolite mass spectrum of isorhamnetin

[0017] Figure 3. A. f / avus-quercetin microbial transformation time-dependent isorhamnetin change graph

[0018] Detailed Description of the Invention

[0019] The present invention relates to a method for the synthesis of isorhamnetin metabolite from the quercetin molecule and the isorhamnetin metabolite synthesized by this method. In this method, isorhamnetin is synthesised from quercetin molecule by microbial transformation of Aspergillus flavus. The method of the present invention provides the synthesis of isorhamnetin with 18.20% yield in a short time and at low cost.

[0020] Synthesis method of isorhamnetin metabolite comprises the following process steps; i. sowing the microorganism first on slant agar and then on a-medium liquid media, ii. preparing the blank medium containing only medium and microorganisms, iii. adding the medium, microorganism and quercetin into the conical flask, iv. autoclaving the prepared conical flasks, v. sowing the activated microorganism in solid medium into autoclaved flasks, vi. leaving the conical flasks prepared in (iv) for pre-incubation in the shaking incubator, vii. adding quercetin to the flasks taken from the incubator in the sterile cabinet, viii. adding the solvent used to dissolve quercetin to the blank media prepared in (ii), ix. incubating the flasks prepared in step (vii) in a shaking incubator, x. at the end of the 4th, 8th and 12th days, collecting the samples from the incubating flasks under sterile conditions, xi. adding solvent to the collected samples in sterile environment, xii. liquid-liquid extraction by using liquid-liquid phase separation to the samples to which solvent is added, xiii. removing the added solvent from the extracted samples (xi) and removing the solvent from the extract by evaporation method.

[0021] In said method, the solvent mentioned in the process step (viii) is acetone, and the solvent mentioned in the process step (xi) is ethyl acetate.

[0022] In another embodiment of the present invention, synthesis method of isorhamnetin metabolite comprises the following process steps; i. sowing the microorganism first on slant agar and then on a-medium liquid media, ii. preparing the blank medium containing only medium and microorganisms, iii. adding the medium, microorganism and quercetin into the conical flask, iv. autoclaving the prepared conical flasks, v. inoculating the microorganism activated in solid medium into the medium with 25-30oC, 6±0.5 pH conditions in autoclaved flasks, vi. pre-incubation of the flasks prepared in shaking incubator (iv) for 3 days, vii. adding quercetin to the flasks taken from the incubator in the sterile cabinet, viii. adding solvent to the blank media prepared in (ii) with a final concentration of 5mg / mL in order to dissolve quercetin, ix. incubation of the media in the flasks prepared in step (vii) in a shaking incubator, x. at the end of the 4th, 8th and 12th days, collecting the samples from the incubating flasks under sterile conditions, xi. adding solvent to the collected samples in a sterile environment with a media:solvent ratio of 1 :1 , xii. liquid-liquid extraction by using liquid-liquid phase separation to the samples to which solvent is added, xiii. removing the solvent from the samples at the end of the extraction process and removing the solvent from the extract by evaporation method.

[0023] The microorganism mentioned in the method of the present invention is Aspergillus flavus. Additionally, in the abovementioned method, a blank medium was prepared in order to understand whether the transformation occurred due to environmental conditions, the microorganism itself, or quercetin as targeted. In the abovementioned method, the solvent mentioned in the process step (viii) is acetone, and the solvent mentioned in the process step (xi) is ethyl acetate.

[0024] In the present invention, Aspergillus flavus taken from the culture collection was first planted on slanted agar and then on a-medium liquid media. The contents of these media are listed in Tables 1 and 2. The medium used within the scope of experimental studies was sterilized in an autoclave at 1.1 Atm and 121 °C for 1 hour before microorganism cultivation. Table 2. Slant agar medium components

[0025] For each microorganism, a so-called blank medium containing only the medium and the microorganism was prepared. For the substrate (quercetin), the medium was prepared as containing microorganisms and substrate (5 mg / mL) in a conical flask. The flasks, whose preliminary preparations were completed in this way, were autoclaved at 1.1 Atm and 121 °C for 1 hour, and after the autoclaving process, microorganisms previously activated in solid medium were inoculated into the flasks brought to room temperature in a sterile cabin environment. The flasks containing the microorganism (fungus) cultivated in this way were pre-incubated for three days in a shaking incubator environment set at 120 rpm at 25°C. After the microorganisms are sufficiently grown, the substrate is added in a sterile container and the solvent (ethyl acetate) used in the dissolution of the substrate is added to the blind flasks at a ratio of ethyl acetate :medi urn of 1 :1. At the end of 4, 8, 12 days, 1 -2 mL samples were taken from the flasks in which the transformation process continued in the shaker incubator and the time-dependent growth processes of microbial transformation metabolites were analysed. These samples were subjected to 1 :1 extraction with ethyl acetate 3 times and then the ethyl acetate portion was removed and the solvent was evaporated. Ethyl acetate extracts were checked for the presence of metabolites using the TLC (Thin Layer Chromatography) system and the presence of metabolites was evaluated at 245 and 365 nm under UV light after dragging on the TLC plate in the TLC system with mobile phase ethyl acetate: n-hexane: formic acid (3:1 :0.5).

[0026] The analysis conditions of Liquid Chromatography / Mass Spectrometry-Mass Spectrometry (SK / KS-KS) was determined as follows, 15 cm, RP-C18, 3um column; 10%-100% 20 min; flow rate: 0.7; At room temperature; the mobile phase A: MeOH (methanol): water: formic acid (10:89:1 ) B: MeOH: water: formic acid (89:10:1 ). The Liquid Chromatography (LC) section consists of two dual pumps, degassing unit, column oven, autosampler and detector. Mass analyses were performed on an ion trap time-of-flight mass spectrometer (IT-TOF) using electron spray ionisation (ESI) positive and negative mode. Analyses were performed using a 150 x 2 mm, 2 pm column. Acetonitrile water mixture containing 0.1% formic acid as the mobile phase was sent to the system at a flow rate of 0.25 mL / min and the column oven temperature was optimised as 40 °C, CDL temperature as 200 °C, heat block temperature as 200 °C and nebuliser gas flow as 1.5 L / min for samples sent in 2 pL injection volume. Quantification studies were also carried out with a detector using UV absorbers at a wavelength of 254 nm. The mass spectrum of isorhamnetin is [M- H]- 314.8, as seen in Figure 2. In Figure 3, two different electron volts (15 and 50 eV) are given. Since these spectra are KS-KS, they provide information about the mass fragments of the metabolite. Unlike quercetin, isorhamnetin has methoxy at the 3' position in its structure. Among the KS-KS fragments of the A ring, m / z 151 and m / z 107 are characteristic for isorhamnetin and these peaks are observed in the spectra.

[0027] During the Aspergillus f / avus-quercetin transformation process, transformation samples were collected at three different time periods (days four, eight and twelfth) and liquid-liquid extraction was performed with ethyl acetate solvent. While the qualitative presence of isorhamnetin in the extracts previously analysed in the ITK system was determined, SK-KS device was used for quantitative quantification. Within the scope of the quantification study, firstly the calibration curves of isorhamnetin were created. The amounts of metabolites in the transformation extracts on days four, eight and twelve were analyzed. The time-dependent change graph of isorhamnetin is shown in Figure 3. According to the data obtained, the highest conversion efficiency of isorhamnetin in the determined time periods was calculated as 18.20% on the fourth day. As indicated in Figure 3, there is an inversely proportional change in the amount of conversion metabolite with time.

[0028] REFERENCES

[0029] [1] Balasundram N, Sundram K, Samman S. Phenolic compounds in plants and agriindustrial by-products: Antioxidant activity, occurrence, and potential uses Food Chemistry. 2006 ;99(1 ):191 -203. DOI: 10.1016 / j.foodchem.2005.07.042.

[0030] [2] Otr^ba, M., Kosmider, L., & Rzepecka-Stojko, A. (2021 , February 3). Polyphenols’ cardioprotective potential: Review of rat fibroblasts as well as rat and human cardiomyocyte cell lines research. Molecules (Basel, Switzerland). https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC7913231 /

[0031] [3] Bie, J., Sepodes, B., Fernandes, P. C. B., & Ribeiro, M. H. L. (2023, January 5).

[0032] Polyphenols in health and disease: Gut microbiota, bioaccessibility, and bioavailability. MDPI. https: / / www.mdpi.com / 2673-

[0033] 6918 / 3 / 1 / 5#:~:text=EDidemioloqical%20studies%20confirm%20that%20moderat e,tvpe%202%20diabetes%2C%20and%20obesity.

[0034] [4] VI;, I. I. I. (n.d.). A critical review of methods for characterisation of polyphenolic compounds in fruits and vegetables. Food chemistry. https: / / Dubmed.ncbi.nlm.nih.qov / 25213963 /

[0035] [5] Flavonoids. Linus Pauling Institute. (2023, January 3). https: / / lpi.oregonstate.edu / mic / dietary- factors / phytochemicals / flavonoids#:~:text=ln%20general%2C%20the%20bioav ailability%20of,absorbed%20(8%2C%2022).

[0036] [6] Men, W., Chen, Y., Li, Y., Yang, Q., Weng, X., Gong, Z. (2015). Research progress of biotransformation on effective ingredients of Chinese medicine via intestinal bacteria. Chin. J. Exp. Tradit. Med. Formulae, 21 (2), 229-234

[0037] [7] Guan, C.J., Ji, Y.J., Hu, J.L., Hu, C.N., Yang, F„ Yang, G.E. (2017).

[0038] Biotransformation of Rutin Using Crude Enzyme from Rhodopseudomonas palustris. Curr. Microbiol., 74 (4), 431 -436.

[0039] [8] Jiang, H., Yamashita, Y., Nakamura, A., Croft, K., & Ashida, H. (2019, February

[0040] 25). Quercetin and its metabolite isorhamnetin promote glucose uptake through different signalling pathways in myotubes. Scientific reports. httDs: / / www.ncbi.nlm.nih.qov / Dmc / articles / PMC6389993 /

[0041] [9] Gong G;Guan YY;Zhang ZL;Rahman K;Wang SJ;Zhou S;Luan X;Zhang H; (n.d.).

[0042] Isorhamnetin: A review of Pharmacological Effects. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. httDs: / / Dubmed.ncbi.nlm.nih.qov / 32502837 /

[0043]

[0010] Banerji, A. (2017). Bioinspired syntheses of partially methylated flavonoids™ untapped source of bioactivities. Journal of Exploratory Research in Pharmacology, 2(1 ), 16-20.

[0044]

[0011] Cao, X., Wei, Y., & Ito, Y. (2008). Preparative isolation of isorhamnetin from Stigma maydis using high speed countercurrent chromatography. Journal of Liquid Chromatography & Related Technologies®, 32(2), 273-280.

[0045]

[0012] Biotransformation of quercetin by aspergillus niger from ALLIUM CEPA: International Journal of Pharmaceutical Sciences and Research. IJPSR. (2021 , May 23). https: / / ijpsr.com / bft-article / biotransformation-of-quercetin-by- aspergillus-niger-from-allium-cepa /

Claims

CLAIMS1. Synthesis method of isorhamnetin metabolite, characterized by comprising the process steps of; i. sowing the microorganism first on slant agar and then on a-medium liquid media, ii. preparing the blank medium containing only medium and microorganisms, iii. adding the medium, microorganism and quercetin into the conical flask, iv. autoclaving the prepared conical flasks, v. sowing the activated microorganism in solid medium into autoclaved flasks, vi. leaving the conical flasks prepared in (iv) for pre-incubation in the shaking incubator, vii. adding quercetin to the flasks taken from the incubator in the sterile cabinet, viii. adding the solvent used to dissolve quercetin to the blank media prepared in (ii), ix. incubating the flasks prepared in step (vii) in a shaking incubator, x. at the end of the 4th, 8th and 12th days, collecting the samples from the incubating flasks under sterile conditions, xi. adding solvent to the collected samples in sterile environment, xii. liquid-liquid extraction by using liquid-liquid phase separation to the samples to which solvent is added, xiii. removing the added solvent from the extracted samples (xi) and removing the solvent from the extract by evaporation method.

2. A method according to claim 1 , characterized by comprising the process steps of; i. sowing the microorganism first on slant agar and then on a-medium liquid media, ii. preparing the blank medium containing only medium and microorganisms, iii. adding the medium, microorganism and quercetin into the conical flask, iv. autoclaving the prepared conical flasks,v. inoculating the microorganism activated in solid medium into the medium with 25-30oC, 6±0.5 pH conditions in autoclaved flasks, vi. pre-incubation of the flasks prepared in shaking incubator (iv) for 3 days, vii. adding quercetin to the flasks taken from the incubator in the sterile cabinet, viii. adding solvent to the blank media prepared in (ii) with a final concentration of 5mg / mL in order to dissolve quercetin, ix. incubation of the media in the flasks prepared in step (vii) in a shaking incubator, x. at the end of the 4th, 8th and 12th days, collecting the samples from the incubating flasks under sterile conditions, xi. adding solvent to the collected samples in a sterile environment with a media:solvent ratio of 1 :1 , xii. liquid-liquid extraction by using liquid-liquid phase separation to the samples to which solvent is added, xiii. removing the solvent from the samples at the end of the extraction process and removing the solvent from the extract by evaporation method.

3. A method according to claim 1 or 2, wherein said microorganism is Aspergillus flavus.

4. A method according to claim 1 or 2, wherein the solvent mentioned in process step (viii) is acetone.

5. A method according to claim 1 or 2, wherein the solvent mentioned in process step (xi) is ethyl acetate.

6. The metabolite of isorhamnetin synthesised by a method according to any one of the preceding claims.

Images