PHYTOCHEMICAL ANALYSIS OF STEMS AND LEAVES OF Phlomoides anisochila (Pazij & Vved.) Salmaki AND Phlomoides sogdiana (Pazij & Vved.) Salmaki (Lamiaceae), SPECIES DISTRIBUTED IN THE FLORA OF UZBEKISTAN

The research was carried out within the framework of the applied research project AL–9224104319 “Development of a digital platform for the flora of the Fergana Valley as a biodiversity-threatened region” (2025–2027), implemented at Namangan State University

ABSTRACT

Species of the genus Phlomoides are widely used in medicine for the prevention and treatment of many diseases. Phlomoides Moench species. they are used in the preparation of anti-inflammatory, antioxidant, immunomodulatory, antibacterial, antiviral, antiallergic drugs. The genus is 150-170 species in the world, and 43 species in Uzbekistan. Despite the many medicinal properties, the chemical composition of Phlomoides species distributed in Uzbekistan has been little studied. In our studies, the protein content, amino acid composition, quantitative indicators and concentration of vitamins of the species were determined. Quantitative determination of total protein was carried out by the Kjeldahl method.  Phenylthiocarbomyl (FTC) synthesis of free amino acids was carried out by the method of Steven, Cohen David. The results showed that P. sogdiana has the highest protein content and amino acid concentration, and the amount of B vitamins is the highest in P. sogdiana 32.802821 mg/g and in P. anisochila 19.484143 mg/g. In summary, it was found that the chemical composition of P. sogdiana, P. anisochila species distributed in Uzbekistan has a high concentration of amino acids, proteins, and vitamins.

АННОТАЦИЯ

Виды рода Phlomoides широко используются в медицине для профилактики и лечения многих заболеваний. Виды Phlomoides Moench применяются при создании противовоспалительных, антиоксидантных, иммуномодулирующих, антибактериальных, противовирусных и антиаллергических препаратов. Род включает 150–170 видов в мире и 43 вида в Узбекистане. Несмотря на многочисленные лечебные свойства, химический состав видов Phlomoides, распространённых в Узбекистане, изучен недостаточно. В наших исследованиях были определены содержание белка, аминокислотный состав, количественные показатели и концентрация витаминов видов. Количественное определение общего белка проводилось методом Кьельдаля. Синтез свободных аминокислот в форме фенилтиокарбамила (FTC) выполнялся по методу Стивена и Коэна Дэвида. Результаты показали, что вид P. sogdiana обладает самым высоким содержанием белка и концентрацией аминокислот. Количество витаминов группы B также оказалось максимальным у P. sogdiana — 32.802821 мг/г, а у P. anisochila — 19.484143 мг/г. В целом установлено, что виды P. sogdiana и P. anisochila, распространённые в Узбекистане, имеют высокую концентрацию аминокислот, белков и витаминов.

Keywords: Phlomoides sogdiana, Phlomoides anisochila, endemic species, aminoacids, proteins, vitamin composition.

Ключевые слова: Phlomoides sogdiana, Phlomoides anisochila, эндемичные виды, аминокислоты, белки, витаминный состав.

Introduction. Genus Phlomoides one of the largest polymorphic genera belonging to the Lamiaceae family, the range of species of the genus extends to Central Europe and the Russian Far East, and the main centers of species diversity are China, Southern Europe and Central Asia, the mountainous regions of Iran (Iran and Afghanistan) and the Mediterranean regions. There are 150-170 species of the genus in the world, 67 species in Central Asia, 43 species in the flora of Uzbekistan. It was also determined that there are 36 species in Kyrgyzstan, 27 species in Tajikistan, 12 species in Turkmenistan, and 30 species in Kazakhstan [2, 3, 4, 6, 7, 9, 10, 11].

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 [5]. In particular, Phlomoides nuda (Regel) Adylov, Kamelin & Makhmedov, distributed in the Fergana valley. 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 [13, 8]. 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 [13, 8].

Today, research and chemical analyzes have not been carried out on the study of amino acid and vitamin content of Phlomoides anisochila and Phlomoides sogdiana 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.

Materials and Methods

Plant materials. Our research was aimed at determining the chemical composition of species that are endemic to the flora of our country. The object of the study is the species Phlomoides anisochila (Pazij & Vved.) Salmaki, Phlomoides sogdiana (Pazij & Vved.) Salmaki belonging to the genus Phlomoides. Plant samples were collected in the Farish district of the Jizzakh region (Figure 1).

Figure 1. Distribution of P. anisochila and P. sogdiana in the Nurata mountain range

Phlomoides anisochila (Pazij & Vved) Salmaki is an extremely rare endemic plant of the Nurata ridge. A perennial, heavily pubescent herbaceous plant up to 20 cm tall. There are three stems, branched from below, covered with thick hairs. The leaf blade is short, thick and long-haired. The leaves on the stem are diamond-shaped-ovate, the edges are serrated, densely pubescent, the veins are thick, and in the upper part of the stem the leaves are without bandages. The bract leaves are located in the axils of the petals in the form of a ring.

The bract leaves of the flower are lanceolate, fluffy. The flower cup is bell-shaped, about 20 mm long, the tube is densely hairy. The flower crown is yellow, 4 mm long, the tube length is 15 mm, hairless, the back is short, densely hairy, the lower lip of the crown is twice as long as the upper one. Blooms in June-July, bears fruit in August. In the central part of the Nurata ridge of the Jizzakh region, it occurs on dry slopes with gravel and stones, singly and among small shrubs.

Phlomoides sogdiana (Pazii & Vved.) Salmaki. It is a perennial herbaceous plant with turnip-shaped main and lateral roots. The stem is erect, branched, 23-25 cm long, densely covered with glandular hairs. The basal leaves are dark green, egg-shaped, the leaves on the stem are arranged in 2, ovate with very short bandages or without bandages. The flowers are located in the axils of the petals. The stem is branched into 3-5 branches, the lateral branches are slightly recumbent, and the middle ones grow in an upright position.

There are 2-4 rings on the branch, 3-10 flowers on the ring, 14-32 flowers on the branch and 50-60 flowers on the plant. The flower cup is tubular-bell-shaped. This species is widespread in the Turkestan, Zeravshan, Nurata, Hissar, Molguzar ranges and on the Surkhan-Sherabad plain of the Western Pamirs. This species is distributed only in the areas of the Western Pamir Alai belonging to the Republic of Uzbekistan, is considered one of the national endemics of Uzbekistan [12].

Methods and Reagents. The chemical composition of plant leaves was determined at the Institute of Bioorganic Chemistry of the Academy of Sciences of the Republic of Uzbekistan. Samples were taken from the leaves and stems of plants in different seasons of 2022. The collected samples were dried at room temperature. Then the dried mass was crushed. The crushed mass was weighed on analytical scales, extracted and degreased with white spirit, hexane or chloroform for degreasing. Quantitative determination of total protein was carried out by the Kjeldahl method. This method is based on the calculation of the total protein content by determining the nitrogen content. The mass fraction of nitrogen (X) in the analyzed sample is calculated by the formula as a percentage of the sample mass to volume after titration of the amount of ammonia passed through dilute sulfuric acid.

Phenylthiocarbomyl (FTC) synthesis of free amino acids was carried out by the method of Steven, Cohen Daviel (Steven, 1988.). Phenylthiocarbomyl (FTC) and its derivative (compound) of free amino acids were analyzed on the basis of high-performance liquid chromatography (HPLC). Proteins and peptides contained in the aqueous extracts of the samples were precipitated. 1 ml of the supernatant was taken and 1 ml of 20% Trichloroethanoic acid was added to it. After 10 minutes, it was centrifuged at 8000 rpm for 15 minutes and 0.1 ml of the residual liquid was dried in a lyophilizer.

Identification of amino acids Phenylthiocarbamyl FTK was carried out on an Agilent Technologies 1200 chromatograph on a Discovery HS C₁₈ 75x4.6 mm column. The following mixtures were used: 0.14M CH₃COO Na + 0.05% Triethanolamine TEA pH 6.4, B:CH₃CN. The flow rate is 1.2 ml per minute, absorption is 269 nm. Gradient %B/min: 1-6%/0-2.5 min; 6-30%/2.51-40 min; 30-60%/40.1-45 min; 60-60%/45.1-50 min; 60-0%/50.1-55 min.

High-performance liquid chromatography was carried out on an Agilent-1200 brand device, an Agilent C18 column of 5mkm.4.6x250 mm. The process was carried out once, using a mixture of 0.1% trifluoroacetic acid and acetonitrile (70:30) as the mobile phase. The volumetric flow rate of the eluent is 1.0 ml/min, the volume of the injected sample is 10 µl. The length is 254, 290 nm.

The analysis of high-performance liquid chromatography HPLC of water-soluble vitamins is carried out on an Agilent Technologies 1200 chromatograph on an Exlipse XDB C18 column (reversed-phase), 3.5 microns, 4,6x150mm. Diode-matrix detector (DAD), 254, 290 nm. Solution A: 0.5% acetic acid, pH 1.7: B:CH3CN (acetonitrile). The flow rate is 1 ml/min. Gradient %B/min: 0-5 min/96:4%, 6-8 min/90:30%, 9-15 min/80:20%, 15-17 min/96:4%. The thermostat is 25⁰C.

Results and Discussion

Species of the genus Phlomoides are distinguished by their rich chemical composition. Including: flavonoids, phenylpropanoids, phenol carboxylic acids, neolignans, iridoids, higher fatty acids, nitrogenous compounds, steroids, hydrocarbons, carotenoids, triterpenoids, diterpenes, carbohydrates, ascorbic acid, proteins, trace elements, etc.

Proteins regulate, coordinate and integrate various chemical changes throughout the living organism. But protein cannot be postponed “for later”, its reserves must be constantly replenished. Protein deficiency, in turn, leads to tissue loss. Plants are of great importance in replenishing the place of proteins.

As can be seen from the diagram, the total percentage of proteins is slightly higher in P. sogdiana (Figure 2).

Figure 2. Total protein content (%) in stems and leaves of P. anisochila and P. sogdiana

Species of the genus Phlomoides are rich in various amino acids. Amino acids are very important for restoring the activity of the body and its normal functioning. 10 of the identified amino acids are essential amino acids (threonine, arginine, valine, methionine, isoleucine, leucine, histidine, tryptophan, phenylalanine, lysine), which make up 46.48% of the total amino acids in P. anisochila and 41.07% in P. sogdiana (Table 1).

Table 1.

Amino acid content in leaves and stems of P. anisochila and P. sogdiana

Also among the identified amino acids, tyrosine was 7.472434 mg/g in P. anisochila species, asparagine was 7.006748 mg/g in P. sogdiana species. On the contrary, it was found that the amino acid phenylalanine is 0.660396 mg/g in P. anisochila, the amino acid serine (0.904018 mg/g) is the lowest in P. sogdiana, and aspartic acid is not found in both species (Figure 3).

Figure 3. Amino acid chromatography of leaves and stems of P. anisochila and P. sogdiana

The number of amino acids in the two studied species differs somewhat. According to the chemical structure of P. anisochila, the number of amino acids is as follows: aromatic > basic diaminoacids > aliphatic amino acids > sulfur-containing > iminoacids > acidic dicarboxylic amino acids, and in P. sogdiana: sulfur-containing amino acids > basic diaminoacids > aromatic amino acids > aliphatic > iminoacids > acidic, it was found that the diacarbon decreases in the order of amino acids.

Vitamins are biologically active organic substances necessary for the vital activity of the body. These are bridges that ensure the interaction of the enzyme and the substrate catalyzed by it. They play an important role in metabolism, the processes of assimilation and use of all nutrients by the body, the protective functions of various organs and other vital processes.

As a result of the research, the amount of B vitamins, ascorbic and nicotinic acids in the stems and leaves of plants was determined (Figure 4).

Figure 4. Vitamin chromatography of leaves and stems of P. anisochila and P. sogdiana

Plants are the main source of vitamins for the human body and animals. The human body assimilates vitamins directly from plant products or by accumulating them in the body of animals as a result of consuming plants and taking these animal products [1]. As shown in the table, it was found that the amount of vitamins belonging to group B (B1, B2, B6, B9, B12) has a number of species differences. The highest concentration of vitamin B1 was found in P. anisochila (1,023,151 mg/g). On the other hand, the vitamin B2 content was higher in P. sogdiana (Table 2).

Table 2

Vitamin content in leaves and stems of Phlomoides anisochila and Phlomoides sogdiana

The total amount of B vitamins was highest in P. sogdiana, 32.802821 mg/g and 19.484143 mg/g in P. anisochila.

Сonclusion. As a result of our research, the amount of proteins, amino acids and vitamins of P. anisochila, P. sogdiana species distributed in the Nurota mountain ridge was determined for the first time. The obtained results showed that the amount of proteins in the 1.008 g sample of P. sogdiana is 12.153%, in the 1.020 g sample of P. anisochila it is 11.152%. When 20 amino acids in these species were studied, their sum was the highest in P. sogdiana stems and leaves at 53.08358 mg/g, while the percentage of non-exchangeable amino acids was found to be higher in P. anisochila stems and leaves at 46.48%. It was found that the content of vitamins differed in both species and was relatively higher in P. sogdiana.

Acknowledgements

The authors wish to express their gratitude to  the laboratory “Chemistry of proteins and peptides” of the Institute of Bioorganic Chemistry of the Academy of Sciences of the Republic of Uzbekistan.

References:

1. Derkach T, Tarasenko H. 2021. Chemistry of medicinal plants as an integral part of ecological education. E3S Web of Conferences 280, 11015. https://doi.org/10.1051/e3sconf/202128011015
2. Gulomov R.K. Distribution of the genus Phlomoides Moench in the Fergana Valley (taxonomy, geography, ecology and conservation): author’s abstract of scientific paper, PhD –Tashkent: 2022– 43 p
3. Gulomov R.К., Batoshov A.R. Morphological Phylogeny of the Species Phlomoides Moench (Lamiaceae) Distributed in the Fergana Valley // International Journal of Virology and Molecular Biology. – USA, 2022. – № 11 (1). – P. 9-15. https://doi.org/10.5923/j.ijvmb.20221101.03
4. Gulomov, R., Rakhimova, K., Batoshov, A., Komilov, D. (2023). Bioclimatic modeling of the species Phlomoides canescens (Lamiaceae). The Scientific Temper, 14(4):1060-1065. https://doi.10.58414/SCIENTIFICTEMPER. 2023.14.4.01 
5. Khaydarova D.R. Lamiaceae obtaining medicinal food additives from certain types of herbs. author’s abstract of scientific paper, PhD. - Andijan. 2022. – 46 p.
6. Lazkov GA. 2016. Family Labiatae Juss. in flora of Kyrgyzstan. – Pocheon, Republic of Korea. P. 176-215.
7. Nowak et al., 2020. Illustrated Flora of Tajikistan and adjacent areas. Polish Botanical Society Al. Ujazdowskie 4, 00-478 Warsaw, Poland. 475-478 p.
8. Rahimova H.R., Gulomov R.K., Ibragimov A. DETERMINATION OF AMINO ACID AND VITAMIN CONTENT OF Phlomoides nuda AND Phlomoides speciosa (LAMIACEAE) SPECIES // Universum: химия и биология: электрон. научн. журн. 2023. 4(106). URL: https://7universum.com/ru/nature/ archive/item/15224
9. Salmaki Y, Zarre Sh, Ryding O, Lindqvist Ch, Scheuner A, Bräuchler Ch & Heubl G. 2012. Phylogeny of the tribe Phlomideae (Lamioideae: Lamiaceae) with special focus on Eremostachys and Phlomoides: New insights from nuclear and chloroplast sequences. Taxon 61 (1). February: 161–179. https://doi.doi.org/10. 1002/tax.611012 
10. Tojibаev KS, Kаrimov FI, Hoshimov HR, Gulomov R, Lаzkov GА, Jаng C-G, Gil H-Y, Jаng J-E, Bаtoshov АR, Iskаndаrov А, Choi HJ (2023) Importаnt plаnt аreаs (IPАs) in the Fergаnа Vаlley (Centrаl Аsiа): Th Bozbu-Too Ungortepа mаssif. Nаture Conservаtion 51: 13–70. https://doi.org/10.3897/nаtureconservаtion. 51.94477 
11. Tojibaev KS, Hoshimov HR, Gulomov RR, Sheraliev OK, Karimov FI, Kor L, Batoshov AR, Baasanmunkh S, Jang JE, Jung J, Gil H-Y and Choi HJ (2025) Conservation priorities in Central Asia: the Shorsuv Massif IPA and its role in the Fergana Valley’s biodiversity. Front. Conserv. Sci. 6:1629529. https://doi:10.3389/fcosc.2025.1629529
12. Turginov OT., Ghulomov RK. 2017. Species of Phlomoides Moench (Lamiaceae) distributed in Uzbekicton flora // News of Uzbekicton National University. - Tashkent. No. 3/1. - B. 36-41.
13. Yulbarsova M., Khaydarova D.R., Siddikov G.U., Abdullaev Sh.V. Phlomoides nuda as a source of β-sitosterol. Journal of Chemistry of Goods and Traditional Medicine. Volume 1, Issue 4, 2022.