Quality Control and Biological Activity Studies on Commercially Available Hyssopi Herba Drug

Project no. 26315 · Status: Kapanmış (Yayın bekleniyor) · 2023

Summary

Hyssopus officinalis, which is a member of the Lamiaceae family, a rich family in terms of medicinal and aromatic plants, naturally spreads in the region from the Caspian Sea to the Black Sea and in the Asian continent. (Kulak, 2022). The plant has stimulant, carminative and stomachic (Baytop, 1984) as well as worm-reducing, diuretic and antibacterial properties. (Güler,2007).  Studies have reported that Hyssopi herba has anticonvulsant effects (Yang et al., 2008), anti-inflammatory and immune modulatory effects, antimicrobial, antibacterial, antifungal, antiviral (effective against HIV virus), antispasmodic, antidiabetic, cytotoxic, antioxidant (Mićović et al., 2022), antituberculosis, anthelmintic (HILAL et al.1978) effects. There are not enough studies on hyssop, which is used among the public, and when various monographs such as EMA, ESCOP, WHO and FFD, especially the Turkish and European Pharmacopoeia, were examined, no data about the plant was found. In order to eliminate this deficiency, the quality control methods in the European Pharmacopoeia and the Turkish Pharmacopoeia, such as loss on drying, total ash, sulphate ash and ash insoluble in hydrochloric acid, will be tested.As a result of metabolic events in the body or due to environmental factors (radiation, smoking, various drugs, environmental pollution), reactive oxygen radicals are formed, which cause a condition called oxidative damage. Reactive oxygen radicals refer to a group of toxic substances that have an important effect on the development of a wide variety of health problems, from infectious diseases to cancer, from metabolic diseases to Alzheimer's. Substances that protect the body by preventing the harmful effects of these substances are called antioxidants. (Yang ve ark., 2008 )  In the studies, quercetin, apigenin, diosmin, chlorogenic acid, rosmarinic acid and syringic acid were detected in Hyssopi herba extracts (Mićović et al., 2022). Due to the antioxidant effect of flavonoids and other phenolic compounds, antioxidant activity studies, total phenolic substance, total flavonoid analyses and HPLC-DAD quantification of apigenin, one of the major components, are planned on the extracts to be prepared from the plant. A limited number of apigenin quantification studies have been found on the drug (Proestos, 2005).Alzheimer's disease, the most common form of dementia in the elderly, is a disease characterized by disorders in the CNS and cognitive functions and can result in death. Studies have determined that it occurs as a result of structural abnormalities and a decrease in the neurotransmitter substance acetylcholine. (Kennedy and Scholey, 2022). Acetylcholine level is increased by inhibition of acetylcholinesterase (Abdurqadir, 2016; Aynacı, Arslan, Sarı, & Yaşar, 2014; Diken, 2019). With the increase in the elderly population in the world and in Turkey, Alzheimer's disease and related mortality rates are increasing. Although there is no definitive treatment, current treatments have side effects.There are a few studies on acetylcholinesterase inhibition in the essential oil and leaf extract of the plant (Perry N, 1996), but no study has been found on the herb sample. In addition, it is aimed to determine the amount of apigenin, which is a major compound in our drug and is stated to be a strong antioxidant effective in delaying the onset or slowing the progression of Alzheimer's disease (Salehi et al., 2019).Data obtained from various quality control studies planned to be conducted on drugs to be prepared from this plant, which is used among the public but is not included in various monographs such as EMA, ESCOP, WHO and FFD, especially in the Turkish and European Pharmacopoeia, can be used in the preparation of monographs for drugs to be obtained from the plant in the future. In addition, due to the limited number of antioxidant activity studies and anticholinesterase activity studies on the plant, it has the potential to contribute to the literature.Within the scope of the thesis study, Hyssopi herba drug obtained from the market will be used. Loss on drying, total ash, sulfate ash and ash insoluble in hydrochloric acid tests will be applied on Hyssopi herba drug (Turkish Pharmacopoeia). In addition, methanol extracts prepared from powdered Hyssopi herba drug will constitute the study material.Preparation of Methanol Extract: Approximately 10 g of drug will be weighed and extraction will be done with approximately 100 ml of methanol in an ultrasonic bath for 30 minutes. After filtration, the process will be repeated 3 times, the filtrates will be combined, the solvent will be distilled to dryness under low pressure and the % yield will be calculated. If necessary, modifications may be made for optimization (Akyıldız Zİ 2019).Total Phenol Quantification: Total phenolic content in all extracts will be determined according to the Folin-Ciocalteu method (McDonald, Prenzler, Antalovich, Robards, 2001). In the method based on the reduction of the Folin-Ciocalteu reagent by the phenolic compounds in the sample in a basic environment and their own oxidation, the intensity of the blue color formed by the reduced Folin reagent is directly proportional to the concentration of phenolic substances. The sample solution and 10% Folin Solution are mixed, 1M Sodium carbonate is added after 30 minutes and kept in the dark for one hour, then the absorbances of the samples at 760 nm wavelength will be read against the blank solution. The total phenolic content of the samples will be calculated from the standard curve equation created with the gallic acid solution to be prepared at different concentrations.Determination of Total Flavonoid Quantity: Total flavonoid quantity determination of the drugs to be prepared (Chang, Yang, Wen & Chern, 2002) will be carried out using the spectrophotometric method. The method is based on the principle that aluminum chloride forms stable complexes with the C-4 keto group and C-3 or C-5 hydroxyl groups of flavone and flavonol structures in acidic medium. The sample solution is mixed with 10% aluminum chloride, 1 M potassium acetate and kept for 30 min. The colored complex formed at 415 nm wavelength gives maximum absorbance at 415 nm. Total flavonoid quantities corresponding to the absorbance values obtained from the standard curve equation created with the quercetin solution to be prepared at different concentrations will be calculated.Determination of Apigenin: For the routine determination of the major phenolics found in the plant, the HPLC-DAD method will be used (Brantner and Males, 1999). The routine content of methanol extracts prepared from different parts of the plant will be analyzed using a reverse-phase column as the stationary phase and a water-phosphoric acid-acetonitrile gradient elution system as the mobile phase. If necessary, modifications may be made for the optimization of the method during the analysis. In addition, parameters such as linearity, selectivity, decision and determination limits, precision, realism and robustness will be examined for the validation of the analytical method. Apigenin analysis will be performed by purchasing services from EGE-MATAL.Anticholinesterase Activity Determination: For the inhibition of acetylcholinesterase and butyrylcholinesterase enzymes, in vitro Ellman method (Bozkurt, Coban, Kaya, Onur, & Unver-Somer, 2017; Ellman, Courtney, Andres Jr, & Featherstone, 1961) based microplate assay will be applied. In the microplate assay, thiocholine formed as a result of the interaction of enzymes (acetylcholinesterase and butyrylcholinesterase) with substrates (acetylthiocholine and butyrylthiocholine) reacts with Ellman's reagent 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) and forms 2-nitrobenzoate-5-mercaptothiocholine and yellow 5-thio-2-nitrobenzoate. After preparing the solutions of the extracts at appropriate concentrations, the absorbances of the 5-thio-2-nitrobenzoate compound formed as a result of the reaction will be measured using a microplate reader device at 405 nm wavelength and IC50 values will be calculated using the Graph-Pad Prism 5.0 program. The effectiveness of the extracts against oxidative damage will be examined in terms of cell viability in the hydrogen peroxide-induced oxidative stress model (Loubidi M. et al., 2018).It is planned to transform the obtained results into papers and articles in scientific congresses.The results obtained may have the potential to be used in the preparation of monographs on drugs prepared from the plant in the future.

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