IN VITRO PROPAGATION AND ANALYSIS OF BIOLOGICALLY ACTIVE COMPOUNDS OF Nitraria schoberi L. DISTRIBUTED IN THE OROLQUM REGION

ABSTRACT

Nitraria schoberi L., a perennial halophytic plant adapted to the saline conditions of the Aralkum region, is considered a promising source for ecological and pharmaceutical research due to its synthesis of bioactive metabolites [1]. In this study, N. schoberi seeds were propagated in vitro on DKW nutrient medium, cultivated under different salinity stress conditions, and the composition of biologically active compounds was determined using HPLC profiling. The HPLC results were analyzed based on retention times and peak areas, confirming the presence of phenolic, flavonoid, and alkaloid fractions. The results demonstrated that N. schoberi L. from the Aralkum region possesses a distinct metabolic profile, indicating its potential as a valuable natural raw material for pharmaceutical, biotechnological, and environmental research applications [2].

АННОТАЦИЯ

Nitraria schoberi L., многолетнее галофитное растение, адаптированное к засолённым условиям региона Аралькум, считается перспективным источником для экологических и фармацевтических исследований благодаря синтезу биоактивных метаболитов [1]. В данном исследовании семена N. schoberi были размножены in vitro на питательной среде DKW, выращены при различных уровнях солевого стресса, а состав биологически активных веществ был определён методом ВЭЖХ (HPLC). Результаты ВЭЖХ анализировались по времени удерживания и площади пиков, что подтвердило наличие фенольных, флавоноидных и алкалоидных фракций. Полученные данные показали, что N. schoberi L. из региона Аралькум имеет уникальный метаболический профиль и может рассматриваться как перспективное природное сырьё для фармацевтики, биотехнологии и экологических исследований [2].

Keywords: Nitraria schoberi, halophytic plants, Aralkum, biologically active compounds, HPLC, salinity stress, in vitro.

Ключевые слова: Nitraria schoberi, галофитные растения, Аралькум, биологически активные вещества, ВЭЖХ (HPLC), солевой стресс, in vitro.

Introduction. The metabolic profile of plants growing in saline environments is of significant ecological and pharmaceutical importance [1]. The Aralkum region is characterized by a high level of soil salinization; therefore, halophytic plants dominate the flora and exhibit specific adaptive mechanisms for the synthesis of bioactive metabolites [2]. Nitraria schoberi L. is a perennial shrub-like halophyte rich in phenolic compounds, flavonoids, alkaloids, and triterpenoids [3]. However, the HPLC profiling of N. schoberi seeds from the Aralkum region, their response to salinity stress, and the composition of their bioactive metabolites have not been thoroughly investigated in previous studies [4,5].

The aim of this study is to propagate Nitraria schoberi L. seeds from the Aralkum region under modern in vitro conditions and to systematically investigate their bioactive metabolite composition and response to salinity stress under controlled biotechnological laboratory conditions.

Materials and Methods

Seed sterilization: Seeds of N. schoberi collected from the Aralkum region were placed in sterile Petri dishes and subjected to surface sterilization using 5% sodium hypochlorite solution for 10 minutes. Subsequently, the material was treated with 70% ethanol for 1–2 minutes, followed by immersion in 30% H₂O₂ for 15 minutes. The seeds were then rinsed with sterile distilled water for 10 minutes and incubated at 23 ± 2 °C for 2 days. A second sterilization cycle was performed using 30% H₂O₂ for 15 minutes, followed by washing with sterile water for 10 minutes [1].

In vitro propagation: Prior to culture initiation, seeds were cold-treated at 20 °C for 21 days and subsequently soaked in a 2 mg/L GA₃ solution for 48 hours. Driver & Kuniyuki (DKW) medium was used as the basal culture medium, containing sucrose (30 g/L), agar (7.5 g/L), and activated charcoal (2 g/L), with pH adjusted to 5.6–5.8 [2]. A total of 100 seeds were inoculated per treatment, with four independent replicates.

Salinity stress treatment: Salinity stress was induced by supplementing DKW medium with NaCl at concentrations ranging from 50 to 900 mM. Additionally, chloride-based (NaCl, MgCl₂), sulfate-based (MgSO₄, CaSO₄, Na₂SO₄), and carbonate-based (NaHCO₃, Na₂CO₃) salts were applied. Cultures were incubated at 26 ± 2 °C under a 16 h photoperiod with a light intensity of 2000 lux for 21 days [3].

Extraction and HPLC analysis: Harvested plant biomass was extracted with 80% methanol for 48 hours. HPLC analysis was performed using a C18 column with a mobile phase of water:methanol (70:30), a flow rate of 1 mL/min, and an injection volume of 10 mL. Detection was carried out using a DAD detector at 254 nm and a FLD detector with excitation/emission wavelengths of 230/300 nm, 350 nm, and 500 nm. Data analysis was based on retention time and peak area. Method validation included LOD, LOQ, and triplicate replications [4].

Statistical analysis: Data were analyzed using one-way ANOVA followed by Tukey’s post hoc test at a significance level of p < 0.05. Results are presented as mean ± standard deviation (SD) [5,6].

Results and Discussion

HPLC analysis of Nitraria schoberi L. revealed distinct profiles of bioactive metabolites based on retention times. The results demonstrated that a peak at a retention time of 2.976 min, detected by the DAD detector, corresponded to a phenolic compound accounting for 19.17% of the total peak area. In addition, peaks observed at 3.188 and 3.365 min were identified as flavonoid and an additional phenolic fraction, with relative peak areas of 10.94% and 5.95%, respectively [2,3].

The dominant component was detected at a retention time of 3.737 min, representing 63.94% of the total peak area and indicating a complex mixture of phenolic and flavonoid fractions [4]. Using the FLD detector, a flavonoid fraction was identified at 4.127 min with a relative peak area of 47.97%, suggesting enhanced accumulation under salinity stress conditions [5]. Furthermore, additional peaks were observed at 4.528 min (phenolic fraction, 18.67%), 5.034 min (flavonoid fraction, 4.03%), and 6.356 min (alkaloid fraction, 47.15%).

Overall, the obtained results indicate that salinity stress significantly enhances the biosynthesis of bioactive metabolites in N. schoberi, particularly phenolic compounds, flavonoids, and alkaloids, highlighting its adaptive metabolic response and potential pharmacological value under extreme environmental conditions.

Figure 1. Chromatographic analysis results of Nitraria schoberi L. plant extract

Conclusion. Nitraria schoberi L. enhances the synthesis of bioactive metabolites under salinity stress in the Aralkum region. HPLC profiling, analyzed in detail through retention times and peak areas, confirmed that phenolic, flavonoid, and alkaloid fractions are the dominant components. These findings suggest that this species represents a promising natural raw material for pharmaceutical and biotechnological applications due to its adaptive metabolic response to extreme saline environments.

References:

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