DETERMINATION OF AMINO ACID AND VITAMIN CONTENT OF Phlomoides nuda AND Phlomoides speciosa (LAMIACEAE) SPECIES

ABSTRACT

The article presents the results of the study of the surface part of the plant Phlomoides nuda and Phlomoides speciosa by physico-chemical methods. The qualitative and quantitative composition of vitamins and amino acids was determined. Glutamine (2.05 mg/g), proline (1.95 mg/g), histidine (1.90 mg/g) in Phlomoides nuda plant and asparagine (4.38 mg/g), glycine (4.38 mg/g) in Phlomoides speciosa plant, 32mg/g), significant amounts of tyrosine (3.94 mg/g) were shown.

АННОТАЦИЯ

В статье представлены результаты исследования надземной части растений Phlomoides nuda и Phlomoides speciosa физико-химическими методами. Определен качественный и количественный состав витаминов и аминокислот. Глутамин (2,05 мг/г), пролин (1,95 мг/г), гистидин (1,90 мг/г) в растении Phlomoides nuda и аспарагин (4,38 мг/г), глицин (4,38 мг/г) в растении Phlomoides speciosa, 32 мг/г. г) были показаны значительные количества тирозина (3,94 мг/г).

Keywords: Phlomoides nuda, Phlomoides speciosa, amino acid, vitamin, Chromatography

Ключевые слова: Phlomoides nuda, Phlomoides speciosa, аминокислота, витамин, хроматография.

Introduction. In recent years, the scope of research on the taxonomy, geography, ecology and protection of the species of Phlomoides Moench, distributed not only in the flora of Uzbekistan, but also in the Fergana Valley, as well as the study of their chemical composition [1]. In particular, Phlomoides nuda (Regel) Adylov, Kamelin & Makhmedov, distributed in the Fergana valley (Figure 1). In an in vitro experiment, it was proved that the drug has a hepatoprotective effect, which affects membrane recovery in a concentration-dependent manner, and that it contains biologically active substances that have the ability to prevent hepatitis B and hypertension [2].

In addition, for the first time, β-sitosterol as a biologically active substance was isolated from the Phlomoides nuda plant, and a qualitative reaction specific to the flavonoids of the species was conducted [3].

In 2018-2020, the composition of macro and microelements of Phlomoides speciosa, Phlomoides isochila, Phlomoides nuda plants collected from the steppes of Namangan region (Uzbekistan) was experimentally studied at the ‘Ecology and Biotechnology’ scientific laboratory of the Nuclear Physics Institute of of the Academy of Sciences of the Republic of Uzbekistan [4].

In subsequent studies, the substances contained in the essential oil of Phomoides speciosa (Rupr.) Adylov, Kamelin & Machmedov (Figure 2) [5] and the quantitative composition of chemical elements of Phlomoides canescens [6] and the carbohydrate content of this species were studied [7].

Today, research and chemical analyzes have not been carried out on the study of amino acid and vitamin content of Phlomoides nuda and Phlomoides speciosa plants. The implementation of these analyses, the obtained results and the isolated amino acids, vitamins in the food and pharmaceutical industry, the preparation of various nutritional products and vitamin preparations useful for human health, and their application in practice are of urgent scientific and practical importance.

1. Phlomoides nuda (Regel) Adylov, Kamelin & Machmedov in Opred. Rast. Sred. Azii 9: 97 (1987). 

Life form. Hemicryptophyte (Partial rosette plants), polycarpic.

Phenology. Flowering April – June, fruiting June – July.

Habitat. On rocky, gravel and clay slopes in the foothills and in the lower mountain belt, 8.00-1.800 m a.s.l.

Note. This species differs from the Phlomoides nuda species by its less radical leaves. Intermediate forms are also known in nature. Both species are sympathetic, possibly Phlomoides integior – Phlomoides nuda. Molecular research on these species is needed.

Conservation. Unprotected.

General distribution. Middle Asia: Tajikistan, Kyrgyzstan and Uzbekistan.

Figure 1. А) Description of the species Phlomoides nuda in nature (foto by Gulomov Rustam); В) Species ecology; С) Distribution GIS map

2. Phlomoides speciosa (Rupr.) Adylov, Kamelin & Machmedov in Opred. Rast. Sred. Azii 9: 95 (1987).

Life form. Hemicryptophyte (Partial rosette plants), polycarpic.

Phenology. Flowering April – July, fruiting June – August.

Habitat. On stony, gravelly and fine-grained slopes from foothills to the upper belt of mountains. Among thickets of bushes or in walnut forests, in fescue-wormwood steppes, on river terraces, 6.00-3.200 m a.s.l.

Note. There are hybridization processes between Phlomoides cephalarifolia, Phlomoides gymnocalyx and Phlomoides sarawschaniaca species.

Conservation. The species population is widespread in all regions and does not require conservation.

General distribution and ecology. Middle Asia: Afghanistan, Kazakhstan, Kyrgyzstan, Pakistan, Tajikistan, Turkmenistan, Uzbekistan, China (Xinjiang).

Figure 2. А) Description of the species Phlomoides speciosa in nature (foto by Gulomov Rustam); В) Species ecology; С) Distribution GIS map

Materials and methods

Amino acid identification experiment. Phenylthio-carbomyl-derivatives (FTK) of amino acids were obtained by the method of Steven A., Cohen Daviel as a result of the reaction of the hydrolysis product of the isolated protein and peptide fraction [8] with phenylthioisocyanate [8]. FTK (phenylthiocarbomoyl) derivatives of amino acids were identified based on high performance liquid chromatography analysis. The detection process was carried out under the following equipment and conditions: Agilent Technologies 1200 with DAD detector, 75x4.6 mm Discovery HSC18 column. Solution A: 0.14 M CH₃COONa + 0.05% TEA pH=6.4, Solution B: CH₃CN. B: Flow rate 1.2 ml/min, absorption band 269nm. Gradient %/min: 1-6%/0-2.5min; 6-30%/2.51-40min; 30-60% / 40.1-45 min; 60-60%/45.1-50min; 60-0%/50.1-55min.

Vitamin identification experiment. Working solutions of water-soluble vitamins with a concentration of 1 mg/ml were prepared. For this, 50.0 mg of each vitamin standard was taken on an analytical balance and dissolved in 40% ethanol in a 50 ml volumetric flask and filled with the standard.

Chromatographic conditions:

• Chromatograph Agilent-1200 (equipped with an autodoser)
• Colonna eclipse XDB C 18 (obraschenno-faznyy), 5 μm, 4.6 x150mm
• Diode matrix detector (DAD), identified in 204 nm, 254 nm, 290 nm fields.
• Flow rate 1ml/min
• Eluent - acetate buffer: acetonitrile: 0-5 min 96:4, 6-8 min 90:10, 9-15 min 80:20, 15-17 min 96:4, thermostat temperature 250C - 5 μl injected amount.

First, working standard solutions and then prepared working solutions were introduced into the chromatograph.

Results and discussions

Quantitative composition of amino acids was determined by high-performance liquid chromatography (Table 1). Aspartic and glutamic acids are also present; it was found that there is almost a complete set of natural amino acids. The total amount of amino acids was 24.72 mg/g. The largest quantitative components corresponded to glutamine (2.05 mg/g), proline (1.95 mg/g), histidine (1.90 mg/g).

Table 1.

Results of determination of amino acids in Phlomoides nuda
and Phlomoides speciosa

Quantitative composition of amino acids was determined by high-performance liquid chromatography (Table 2). Aspartic and glutamic acids are also present, it has been found that there is almost a complete set of natural amino acids. The total amount of amino acids was 25.45 mg/g. The largest quantitative components are asparagine (4.38 mg/g), glycine (4.32 mg/g), tyrosine (3.94 mg/g). It can be seen that there is a significant difference between the two types of studied plants.

Quantitative composition of vitamins was determined by the method of high-performance liquid chromatography (Figure 5). The study of water-soluble vitamins showed that the largest amount of vitamins in Phlomoides nuda was contributed by thiamine (B₁). Its amount was 1,877 mg/g.

The study of water-soluble vitamins of the plant Phlomoides speciosa showed that the largest amount of vitamins corresponded to the contribution of ascorbic acid (C). Its amount was 2.0784 mg/g. The difference between the two types is significant, especially in vitamins B₂ and B₉. Both types of vitamins B₁, B₆ and C can be indicated as the main components.

Table 2.

Percentage of vitamins extracted from Phlomoides nuda (A) and Phlomoides speciosa (B) plants

Conclusion. Amino acid and vitamin content of Phlomoides nuda and Phlomoides speciosa plants were studied. The total amount of amino acids is close in both species. But there is a significant difference in the amount of the main components. The difference in the composition of vitamins between the two species is significant, especially in vitamins B₂ and B₉. Both types of vitamins B₁, B₆ and C can be indicated as the main components.

References:

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