MASS-CHROMATOGRAPHIC ANALYSIS OF METABOLITES OF THE FUNGUS Trichoderma asperellum

ABSTRACT

To determine the qualitative and quantitative composition of the secondary metabolites obtained by cultivation in different nutrient media of the local micromicrosite Trichoderma asperellum fungus, preparative and columnar chromatographic methods were used. In addition, the lipid criteria of Trichoderma asperellum extracts were indicated using gas chromatography-mass spectrometry (GX-MS) and their physichemical magnitudes were determined. Chloroform:methanol 9:1 was found to have high amounts of metabolites such as 3,4,5 trimethoxybenzaldehyde, Oleic Acid, and Diisooctyl phthalate. As a result of the study, it turned out that the isolated metabolites are preservative, low-molecular, and most of them are compounds close to terpinoids, while many of these metabolites embody a number of useful and important functions.

АННОТАЦИЯ

В статье определен качественный и количественный состав вторичных метаболитов, полученных при культивировании в различных питательных средах локального микромикросайта гриба Trichoderma asperellum, использовались препаративный и колоночной методы хроматографии. Кроме того, липидные критерии Trichоderma экстракты аспереллума были выявлены с помощью газовой хроматографии-масс. Также были определены спектрометрия (GX-MS) и их физико-химические величины. Хлороформ: метанол было обнаружено, что 9:1 содержит большое количество метаболитов, таких как 3,4,5 триметоксибензальдегид, олеиновая кислота и диизооктилфталат. В результате, в ходе исследования выяснилось, что выделенные метаболиты являются консервантами, низкомолекулярные, и большинство из них являются соединениями, близкими к терпиноидам, в то время как многие из этих метаболитов воплощают в себе ряд полезных и важных функций.

Keywords: Secondary metabolites, extraction, micromycetes, Trichoderma fungi, biologically active, Trichoderma asperellum.

Ключевые слова: Вторичные метаболиты, извлечение, микромицеты, грибы рода Trichoderma, биологически активный, Trichoderma asperellum.

Introduction

Today, the field of study of various fungi is developing, due to the fact that most of the metabolites (substances) extracted from the microorganism are becoming more and more practical. For this reason, it is important to determine in what structure and nature the metabolites being isolated are in different spectroscopic or other methods. In this experiment, it was revealed that the most important of these identified volatile metabolite compounds is 2H-Piran-2-on, which is a compound that is used against mycotoxins [1] and is also known to have antifungal properties [2,3], (which is also known to be a growth-enhancing substance in plants such as wheat and tomatoes [4]. The presence of some metabolites from Trichoderma sp volatile secondary metabolites: 3-methyl-heptadekanol, methyl cyclohexane, 6-nonylene alcohol, methyl-cyclopentane, 2-methyl heptadecanol, N-methylpyrrolidine, dermadine, ketotriol was detected by GC-MS/MS assay analysis. In addition, palmitic acid, such as 3-(2'-hydroxypropyl)-4-(hexa-2'-4-dyneyl)-2-(5H)-furanone and 3-(propenone)-4-(hexa-2'-4'-dineyl)-2-(5H)-furanone, have been found to be antifungal properties [5]. Overall, it can be seen that the T. asperellum strain forms volatile secondary metabolites that perform many important functions. Substances known to suppress the growth of T. asperellum Pythium myriotylum by more than 60% have been noted to be highly produced by cell-wall-disrupting enzymes T. asperellum isolates and to show high antagonistic activity against Fusarium oxysporum [6].

Polycyclic aromatic hydrocarbons (PAH) are considered to be among the pollutants that are highly toxic, immobilized and of high risk to the environment due to their bioaccumulative properties. Synthetic PAHs, such as phenanthren, pyrene, and benzo[a]pyrene, are commonly used in pesticides. The recent benefits of the bioremediation property of T. asperellum H15 were discovered by Zafra et al. T. asperellum fungus has been shown to be an effective PAH biodegrade in soil due to its high resistance to high levels of H15 PAH strains, which are found to be effective PAH biodegradable in soils [7].

In addition, GC-MS analysis is also used to investigate polar, non-volatile metabolites, but chemical derivation is required, which reduces the polarity of the metabolites and ultimately makes them more volatile for proper separation in the GC column. While the above method is generally useful for the detection of primary metabolites of fungus, the method used for the analysis of secondary metabolites that do not have volatile properties is the reverse phase (RP) LC-MS, which is LC-ESI (electrospray ionization)-HRMS (high-resolution MS), which now allows for large-scale, unbiased coverage of secondary metabolites [8]. Most of the substances isolated from T. asperellum are known to be etheric and contain terpenoid groups, many of which have been found to have antioxidant, anti-inflammatory properties [9, 10, 11]. Benzeneethanol, a 4-hydroxy- metabolite nematic, antibacterial (Manilal et al., 2016). Metabolites of 9,12-Octadecadienoic acid (Z,Z)-, methyl ester, Octadecane, 3-ethyl-5-(2-ethylbutyl), Octadecanoic acid, 11-Octadecenoic acid, methyl ester, 9,12-Octadecadienoic acid (Z,Z)-, metabolites, have been found to be cancer-preventing, hepatoprotectin, nematicide compounds. Trichoderma turcumi micromycetes contain anyllic terpenoids, tetracyclic diterpenes and terpenes, tetracyclic diterpenes, and triterpene viridinni and ichiga oladi [12]. Using the OSMAC method, biologically active chromone derivatives were isolated from the Trichoderma harzianum M10 isolate by screening for metabolites. Pure crystals of 5-hydroxy-2,3-dimethyl-7-methoxychromone ( 1 ) were isolated from the T. harzianum M10 strain, initially in pure form and under special nutrient conditions (potato dextrose broth, PDB) and when induced in the conditions (light and shaking), and its structure was studied by X-ray and spectroscopic studies. This substance showed a significant antibiotic effect against Rhizoctonia solani and was found to significantly reduce the viability of human colorectal cancer cells in a concentration-dependent manner [13].

Material and methods

The strain of T. asperellum isolated from nature was grown in agar Mandels medium (in a test tube) for 6 days and used as an equating material from its suspension at a concentration of 106 spores/ml. Microscopic fungus of T. asperellum modified Mandels were grown in 500 ml Erlenmeyer tubes with a volume of 500 mL in 250 ml of nutrient medium, in a shaker at a speed of 180 revolutions/min (IKA® KS 130 shakers) for 14 days at a temperature of 24-26°C and separated from their cultured liquid biomass by filtration. The pH of the extracted cultured liquid was equivalent to 2-2.2 with 2 n HCl and the cultured liquid was extracted 3 times in the separation vine in an ethyl acetate ratio of 3:1. Water layer is singled out. The extraction sum was dried at a temperature of 40°C under vacuum conditions (rotary spar). With gas chromatography of YL 6900 GX/MS (30 m × 0.25 mm internal diameter, 0.25 μm film thickness) equipped with the DB-5MS column, YL 6900 GX/MS gas chromatography detected unknown volatile substances in the mass spectrometric detector. The components were identified from the mass spectra of each component after comparing it with available spectral data from NIST 2017.

The next step is a fractionation process involving solvents and for this the obtained extraction mass prepared for separation by column chromatography method.

Results and discussion

The fungal biomass obtained as a result of the above process and placed in the chromatographic column was first fractionated in n-hexane and the following result was obtained when mass chromatogram analysis of the obtained fraction was obtained. When the further fractionation process was carried out in a chloroform:methanol ratio system with a ratio of 9:1, it was found that the chromatogram of the fraction was as follows (Fig. 1).

Figure 1. Gaseous liquid chromatogram of chloroform:methanol 9:1 fraction in solvent system

Known volatile metabalites on the GLX chromatogram of the resulting fraction were identified in the MC database (Table 1).

Table 1.

Metabolites detected from fraction in chloroform: methanol 9:1 solvent system

Some of the substances isolated from this chloroform:methanol 9:1 system turned out to be amorphous substances with a white color.

Conclusion

As a conclusion, it can be noted that the identified compounds are elements of group C and H, as well as lower molecular substances, and many of them are compounds with a close structure to terpenoids. 9,12-Octadecadienoic acid (Z, Z)-, methyl ester, Octadecane, 3-ethyl-5-(2-ethylbutyl), Octadecanoic acid, cis-13-Octadecenoic acid 11-Octadecenoic acid, methyl ester, 9,12-Octadecadienoic acid (Z, Z). Substances with effective properties have been identified, such as: antibacterial, anti-ulcerative, anti-ulcer, hypocholesterolemic, cancer prevention, hepatoprotectin, nematicide, antieczemic, 5-alpha reductase inhibitor.

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