STUDYING THE CHEMICAL COMPOSITION OF THE INTRODUCED Ginkgo biloba

УДК 615.322:543.664.8

Abstract

The aim of the research is to study the polyphenol content of G.biloba and to create new medicinal food additives based on the results of experimental research.

In this article, the total polyphenols of the leaves of a five-year-old Ginkgo biloba tree introduced into the climate of Uzbekistan were isolated by the extraction method (4.31g). It was found that the plant retains more polyphenol compounds during the fruiting period than during the dormant period. The total amount of biologically active substances obtained was divided into fractions, and the main part in terms of quantity was gallic acid, which was identified to determine the second polyphenol. When the IR spectrum, NMR spectra of this unknown polyphenol were obtained and analyzed, it was experimentally proven that it is a rutin flavonoid. It has been determined that the leaves of the plant are particularly rich in flavonoids during the fruiting period.This plant can be used to treat neurasthenia and normalize human cognitive function by improving microcirculation in the brain. It has been scientifically substantiated that Ginkgo biloba can be used to improve memory by enhancing microcirculation in the central nervous system. We have obtained new food additives under the names “ALINAB” and “ASNABALI” based on the leaves of this medicinal plant.

Аннотация

Цель исследования – изучение содержания полифенолов в G. biloba и создание новых лечебных пищевых добавок на основе результатов экспериментальных исследований.

В данной статье методом экстракции были выделены суммарные полифенолы из листьев пятилетнего дерева Ginkgo biloba, интродуцированного в климатических условиях Узбекистана (4,31 г). Установлено, что в период плодоношения растение сохраняет большее количество полифенольных соединений по сравнению с периодом покоя. Полученная общая масса биологически активных веществ была разделена на фракции, при этом основную часть по количеству составляла галловая кислота, которая была идентифицирована для определения второго полифенольного соединения. После получения и анализа ИК-спектра, а также спектров ЯМР данного неизвестного полифенола было экспериментально доказано, что он представляет собой флавоноид рутин. Установлено, что листья растения особенно богаты флавоноидами в период плодоношения. Данное растение может использоваться для лечения неврастении и нормализации когнитивных функций человека за счёт улучшения микроциркуляции в головном мозге. Научно обосновано, что Ginkgo biloba может применяться для улучшения памяти посредством усиления микроциркуляции в центральной нервной системе. На основе листьев этого лекарственного растения нами были получены новые пищевые добавки под названиями «ALINAB» и «ASNABALI».

Keywords: Polyphenols, gallic acid, rutin, spectrometer, absorption spectrum, neurasthenia, cognitive, food additive.

Ключевые слова: полифенолы, галловая кислота, рутин, спектрометр, спектр поглощения, неврастения, когнитивные функции, пищевая добавка

Introduction

The healing effect of medicinal plants certainly depends on their chemical composition, and the chemical composition of the plant is determined by the climate and soil composition in which it grows. Enriching the reserves of medicinal plants around the world and analyzing their chemical composition using modern physicochemical methods, and producing new natural medicines based on the results obtained, remains one of the promising research directions today. By studying the polyphenol content of the Ginkgo biloba tree introduced to the climate of Uzbekistan, and identifying the extracted flavonoids, it is possible to determine changes in the chemical composition of the plant bark.

It is known that flavonoids and their derivatives play an important role in plant life, for example, protecting against various stresses, attracting pollinators, and at the same time regulating the activity of phytohormones. These biologically active compounds are important secondary metabolites that are beneficial to the body. Ginkgo biloba L. is a well-known relict tree, also known as a “living fossil”[1]. Flavonoids present in Ginkgo leaves are known to have antioxidant, biological anti-stress activities and therapeutic effects for various neurological diseases. In today, research on flavonoids has mainly focused on their isolation, pharmacological effects, and analysis of components Although flavonoids are known as secondary metabolites of plants, they play a role in plant survival. They protect plants from pathogenic insects and fungi, act as antioxidants, and are active as antimicrobial agents [2].

Ginkgo biloba L. is a protected relict species of the Ginkgo family, valued for its decorative, economic and medicinal properties. Flavonoids, the main medicinal components of G. biloba, have anti-inflammatory, anticancer and antioxidant activities. Since they selectively inhibit xanthine oxidase and platelet-activating factor receptors, it is effective in the treatment of Alzheimer's and hypertension [3]. In the selection of G. biloba, tree varieties rich in flavonoids are usually selected and cultivated. Although this species originated in China, it is now widely distributed throughout the world. Ginkgo is characterized by its remarkable resistance to various abiotic and biotic stresses. Proof of its resistance is the fact that it survived the nuclear bombing of Hiroshima and was the first to sprout [4]. Because it stores specific metabolites, it is one of the main factors that helps it adapt perfectly to different environmental conditions [5].  Before considering the effect of various polyphenols on diseases of the central nervous system, it is necessary to pay attention to their passage through the blood-brain barrier. It is known that the organs of the central nervous system are in the highest place among all organs in terms of their energy requirements, and the blood-brain barrier of metabolism is of great importance in meeting this need and excreting the resulting products [6].  Flavonoids are angioprotectors, increasing the strength of capillary vessels and improving blood circulation [7].

The chemical composition of the leaves of a plant introduced to the climate of Europe has been studied. Scientists analyzed the leaves of Ginkgo trees growing in a city park in Koprivnica, Croatia, during the growing season (from May to November). The study was conducted in 2022, and all the trees were 30 years old, about 8 m high and located 6-8 m apart. Five pieces were collected for analysis each month. According to the results of the chemical analysis, the total amount of polyphenols in the leaves increased significantly from May to November: gallic acid content relative to dry bark mass increased from 15.15 ± 0.14 mg to 45.18 ± 0.42 mg. The total flavonoid content reached a maximum in August and amounted to 5.87 ± 0.18 mg gallic acid in the dried sample. The total polyphenol content also increased significantly from May to November: from 15.15 ± 0.14 mg to 45.18 ± 0.42 mg/gallic acid. The total flavonoid content reached its highest level in August and amounted to 5.87 ± 0.18 mg gallic acid per gram of dry mass [8]. Flavonoids serve as biomarkers of the external environment, i.e., factors such as light, drought, temperature and nutrient availability play a decisive role in the synthesis and accumulation of flavonoids in plant bark. Therefore, the time of natural leaf collection has a significant impact on the composition of phytochemicals [9].

Methods and materials. To isolate the polyphenol content of Ginkgo biloba leaves introduced to the climate of the Fergana Valley of Uzbekistan using a special extraction method and to study the polyphenol content. Identification of substances using modern physical and chemical methods. Determination of the concentration of polyphenols using high-performance liquid chromatography and identification of substances by the spectrum using Nuclear Magnetic Resonance and Proton Magnetic Resonance equipment.

1000 g of plant raw materials were taken and extracted in chloroform (8-10 mm grinding degree), (1:6 ratio) for 2 hours, 3 times repeated, at 45⁰C, in a refluxing water bath. The extracts were filtered and the raw materials were dried at room temperature until the chloroform odor disappeared (48 hours). Then, the raw material was extracted 3 times in 70% aqueous acetone (1:6 ratio) at 45⁰C for 2 hours. The extracts were filtered, acetone was evaporated under vacuum at 35-400C, and the aqueous phase was separated. The aqueous phase was extracted with ethyl acetate (1:4 ratio) to obtain the ethyl acetate fraction. This fraction was dried over anhydrous Na₂SO₄ and filtered, and the ethyl acetate concentrate was extracted by rotary evaporation. The concentrate was precipitated with chloroform at a ratio of 1:4, and a total of 4.31g of the plant dry weight of the polyphenols was isolated. In order to separate the total of polyphenols into individual compounds, various chemical decompositions (stepwise and acidic hydrolysis and methylation reactions) were carried out. To separate polyphenols into individual compounds using column chromatography, a special silica gel of the brand LH 254 64/100 (Czechoslovakia) was used. The polyphenols were collected, dissolved in ethanol, mixed with 15 g of silica gel (64/100) and dried at room temperature. 70 g of silica gel was mixed with petroleum ether, placed in a 4.5x150 cm column and the adsorbent was compacted by gently tapping the walls of the column. Then 10 g of silica gel soaked in the substance was placed on the column and the column was washed several times with petroleum ether.

Results and discussion

NMR spectra were recorded on a Bruker AM 400 (1H 600 MHz and 13C 100 MHz, tetramethylsilane was used as an internal standard, the signals of which were taken as 0 (d-scale)) in a solution of deuteroacetone and its mixture with heavy water (1:1), and IR spectra were recorded on an "IRTracer-100" (Shimadzu Germany) spectrometer. Below is the IR spectrum of the Rutin substance isolated from the bark of the G. biloba plant, which was introduced by us for the first time:

Figure 1. Spectrum of rutin polyphenol isolated from Ginkgo biloba leaves

IR spectroscopy was used to identify the flavonoid rutin. The IR spectrum of the substance was obtained in the 400-4000 cm-1 region on vaseline oil and KBr tablets on the devices "Bruker Optics" Ettlingen (Germany) and "Perkin-Elmer" (USA) in the IR spectrum. We can see that the valence vibration characteristic of the C-H bond in the rutin molecule at 3334 cm^-1 and the high-intensity C-O-C valence vibrations at 1052 cm^-1 coincide very well. The results of quantum chemical calculations were carried out in the Gaussian 09W program using the DFT/B3LY hybrid method 6 – 316. The optimized structure of rutin by this method is given below

Figure 2. Molecular structure of rutin optimized by the hybrid DFT/B3LY method 6 – 316

The results of the comparative analysis are presented in the following table:

Table 1. Results of quantum chemical calculated and experimentally determined IR-spectroscopic analysis of rutin

The above table shows the peak in the C=C region at 1363 cm^-1, which is characteristic of the 1-2-3 aromatic rings of the rutin molecule, and the vibrations in the region at 899 cm^-1, which is characteristic of the –CH₃ group in the 5-ring.

To fully identify the isolated rutin substance, an NMR spectrum was obtained, and the following spectrogram is given below:

a)

b)

Figure 2. NMR spectrum of rutin polyphenol extracted from Ginkgo biloba leaves. a) Expanded ¹³C-NMR region (60–80 ppm) b) Full ¹³C-NMR spectrum (0–180 ppm)

The following absorption peaks can be seen in the above spectrum. NMR ¹³C (150 MHz, DMSO-d6+SSl4, d, m.d.): 156.77 (C-2), 133.49 (C-3), 177.29 (C-4), 161.27 (C-5), 98.56 (C-6), 164.04 (C-7), 93.35 (C-8), 156.38 (C-9), 103.93 (C-10), 121.04 (C-1`), 116.28 (C-2`), 144.44 (C-3`), 148.21 (C-4`), 115.01 (C-5`), 121.47 (C-6`), 102.07 (C-1``), 73.85 (C-2``), 76.45 (C-3``), 69.43 (C-4``), 75.63 (C-5``), 66.52 (C-6``), 100.55 (C-1``), 70.17 (C-2``), 70.58 (C-3``), 71.96 (C-4``), 67.89 (C-5``), 17.52 (C-6``).

Table 2. Analysis of the results obtained in the NMR ¹³C spectrum

In the above spectrogram, the peaks in the range of 6.5–8 ppm correspond to protons in the aromatic rings, while the signals in the range of 3–5.5 ppm represent glucoside protons, confirming the presence of a glucoside moiety in the molecule.

Conclusion

In conclusion, we conducted a study of the polyphenol content of the leaves of a five-year-old Ginkgo biloba tree introduced to the sunny climate of Uzbekistan (in the Andijan region) using modern physicochemical methods. The samples were studied in two different seasons, namely during fruiting and fruiting. A total of 4.31g polyphenols were isolated from the dried leaves. The most significant component in terms of quantity was gallic acid, which was identified to determine the second polyphenol. When the IR spectrum, 13C NMR spectra of this unknown polyphenol were obtained and analyzed, it was experimentally proven that it is a rutin flavonoid. Thus, Ginkgo biloba has successfully adapted to the climate of Uzbekistan and has a rich polyphenol content. A reserve of this medicinal plant has been created in Uzbekistan.

The extract of this medicinal plant not only improves blood circulation in arterial and venous blood vessels, but also improves microcirculation (blood circulation in the wrapper of capillary blood vessels of the brain) and prevents the formation of blood clots in the blood [10].

Therefore, it can be concluded that its use in diseases of the nervous system, cognitive function disorders (memory loss) gives effective results. We have developed new food additives based on the leaves of this medicinal plant under the names “ALINAB” and “ASNABALI”.

References:

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