SOME BIOCHEMICAL INDICATORS OF LAVENDER (Lavandula angustifolia) AND ROSEMARY (Rosmarinus officinalis) PLANTS GROWN UNDER THE CONDITIONS OF SYRDARYA REGION

ABSTRACT

The article presents the results of a study on the content of the amino acid proline and the activity of the enzyme peroxidase in the leaves of Lavandula angustifolia and Rosmarinus officinalis grown under the agro-climatic conditions of the Syrdarya region. The amount of proline was determined spectrophotometrically using the ninhydrin reagent according to the method of Bates et al. (1973). In the lower leaf parts of rosemary, a higher absorption value was recorded — 0.255 Abs, corresponding to 26.3 µmol/L, while in the upper leaf parts of lavender, the absorption value was relatively lower — 0.105 Abs, corresponding to 10.82 µmol/L. The peroxidase activity in lavender leaves was 0.5616 U/ml, and in rosemary leaves — 0.2304 U/ml. The obtained data may be important for assessing the ecological adaptability of lavender, as well as for the selection of stress-resistant varieties and the development of innovative agro-technologies.

АННОТАЦИЯ

В статье представлены результаты исследования содержания аминокислоты пролина и активности фермента пероксидазы в листьях Lavandula angustifolia и Rosmarinus officinalis, выращенных в агроклиматических условиях Сырдарьинской области. Количество пролина определяли спектрофотометрически с использованием реактива нингидрина по методу Bates и соавт. (1973). В нижних частях листьев розмарина было зарегистрировано более высокое значение абсорбции — 0,255 Abs, что соответствует 26,3 мкмоль/л, тогда как в верхних частях листьев лаванды значение абсорбции было относительно ниже — 0,105 Abs, что соответствует 10,82 мкмоль/л. Активность пероксидазы в листьях лаванды составила 0,5616 Ед/мл, а в листьях розмарина — 0,2304 Ед/мл. Полученные данные могут быть важны для оценки экологической адаптированности лаванды, а также для селекции стрессоустойчивых сортов и разработки инновационных агротехнологи.

Keywords: proline, lavender, spectrophotometer, stress, innovation, peroxidase, amino acid, antioxidant. 

Ключевые слова: пролин, лаванда, спектрофотометр, стресс, инновации, пероксидаза, аминокислота, антиоксидант.

Introduction. In order to implement the Decree of the President of the Republic of Uzbekistan No. PQ-251 dated 20.05.2022 “On measures to organize the widespread use, cultivation, and processing of medicinal plants for medical purposes” [8], in recent years, the study of plant tolerance to abiotic stress factors (drought, salinity, temperature fluctuations) has become one of the urgent research directions in biochemistry and physiology. The stress response of plant cells is a multi-component process involving osmolytes (proline, glycine betaine, sugars) and antioxidant enzymes (peroxidase, catalase, superoxide dismutase) [2,3,5]. Lavender (Lavandula angustifolia Mill.) and rosemary (Rosmarinus officinalis L.) are medicinal essential oil plants, and their biological activity largely depends on high levels of antioxidant and antimicrobial compounds [7]. One of the main factors determining the stress tolerance of these plants is the accumulation of proline and the activity of the enzyme peroxidase.

Proline is an amino acid that rapidly accumulates in plant cells under water deficiency or oxidative stress [3]. It enhances plant adaptability by maintaining osmotic balance within the cell, scavenging free radicals, and protecting proteins [2,4]. At the same time, peroxidase enzymes (POD) decompose hydrogen peroxide and reduce the harmful effects of reactive oxygen species (ROS) [6]. Thus, studying changes in proline content and peroxidase activity in lavender and rosemary plants serves as an important physiological indicator for assessing their stress tolerance. This research is aimed at analyzing the biochemical defense systems of these plants and identifying their mechanisms of adaptation to stress conditions. The proline amino acid content and peroxidase enzyme activity of Lavandula angustifolia and Rosmarinus officinalis species grown under the arid and hot agroclimatic conditions of the Syrdarya region were studied for the first time in a comprehensive manner. Previously, the physiological adaptation parameters of these two medicinal plants had not been analyzed for the territory of Uzbekistan. New diagnostic criteria for the ecological adaptability of medicinal plants were proposed. Based on the obtained results, the proline content and peroxidase activity can be recommended as integral biochemical indicators of ecological adaptability. This approach opens a new direction in the selection of stress-tolerant varieties and in agrobiotechnological breeding processes.

Main part.

1. The Role of Proline in Plants

Proline is an osmolyte amino acid in plants that, under stress conditions, helps retain water in cells, maintains membrane stability, and protects proteins from denaturation [2,3]. It is synthesized through the glutamate pathway, involving the enzyme Δ¹-pyrroline-5-carboxylate synthase (P5CS) [4]. Under stress, the activity of the ProDH enzyme decreases, resulting in proline accumulation in the cells [4]. This process enhances the physiological adaptation mechanism of the plant.

In lavender and rosemary plants, proline accumulation was observed under water deficit and high temperature conditions [7]. The increase in proline levels allows plants to maintain leaf turgor, stabilize the photosynthesis process, and protect against oxidative stress [6]. The amount of proline is commonly determined using the ninhydrin-based spectrophotometric method developed by Bates and coauthors (1973) [1]. In this method, proline reacts with ninhydrin to form a red-violet complex, the intensity of which is measured at 520 nm [1].

2.Physiological significance of the peroxidase enzyme

Peroxidase is an antioxidant enzyme that decomposes hydrogen peroxide and protects plant cells from oxidative stress [6]. It neutralizes reactive oxygen species (ROS), prevents lipid peroxidation, and maintains the integrity of cell membranes.

Peroxidase enzyme activity in lavender and rosemary plants increases under drought and heat stress conditions [7]. The increase in this enzyme’s activity occurs in parallel with proline levels, indicating that they are complementary protective mechanisms. In a study by Ben Ahmed and coauthors (2010), it was found that peroxidase activity increased in olive trees under water deficit conditions [6]. A similar mechanism is observed in lavender and rosemary plants, indicating the similarity of their adaptation systems to abiotic stress.

3. Interaction of proline and peroxidase in the antioxidant system of lavender and rosemary

The defense system of lavender and rosemary plants operates through a balance between antioxidant enzymes and osmoprotectants [7]. Proline and peroxidase act synergistically: one maintains osmotic balance, while the other preserves redox balance. Studies conducted by Uzun and Çelik (2019) showed that these plant extracts have high levels of proline and peroxidase activity, which enhances their antioxidant and antimicrobial properties [7]. For this reason, lavender and rosemary extracts are widely used in pharmaceuticals and cosmetics as natural antioxidants

Materials and Methods. For the experiment, fresh leaves of lavender and rosemary grown in the Sirdarya region were collected.

Table 1.

Experimental data

1. Proline-L 100 mg (0.1 g), 100 ml distilled water.

2. Preparation of ninhydrin solution: Ninhydrin 1.25 g, 30 ml acetic acid (CH₃COOH), 6M H₃PO₄ (8.2 ml acid, diluted with water to 20 ml).3. Into the test tube, 2 ml of proline, 2 ml of ninhydrin solution, and 2 ml of acetic acid were added; for the control (0), 2 ml of acetic acid, 2 ml of ninhydrin, and 2 ml of distilled water were used. After that, the tubes were placed in a water bath at 100 °C for 1 hour and then cooled for 10 minutes.4. 4 ml of toluene was added to the test tube and mixed thoroughly. 5. The spectrophotometer was zeroed at 520 nm, and then the absorbance (Abs) values were recorded.6. 0.5 g of the sample is ground in 10 ml of 3% sulfosalicylic acid, then filtered or centrifuged.7. Boiled for 30 minutes together with the control (0). Then, measured at 520 nm using a spectrophotometer..8.The Beer–Lambert law formula: A=ɛ*c*l A-absorbance  ɛ- (epsilon) – molar absorption coefficient (L.mol^-1.cm^-1) c- concentration of the substance (mol/L)l-Path length of the light (usually in cm, e.g., 1 cm)

Sample =1.082*10^-5 mol/L

=2.6289*10^-5 mol/L

c=10.82µmol/Lavander

c=26.3µmol/L Rosmary

Table 2.

Values

Based on the obtained results, a calibration curve was plotted (Figure 1).

Figure 1. Graph

The graph shows a linear relationship between the optical density and concentration of proline standard solutions. Figure 1. Proline calibration curve.

y = 34,254x- 0.2756 R² = 0.97

(Graph: x – proline concentration (µg/ml); y – absorbance (520 nm))

Peroxidase activity

Phosphate buffer pH 5.4, guaiacol 164 µl, H₂O₂ 0.3 %, 100 ml distilled water, plant extract.First, prepare the phosphate buffer (100 ml): NaH₂PO₄ — 1.773 g, Na₂HPO₄ — 0.0331 g, pH 5.4.To prepare guaiacol, take 164 µl (dissolved in ethanol) and dilute in 25 ml distilled water.Plant extract: 0.5 g of plant material is ground in 10 ml in a mortar, left for 10 minutes, then centrifuged at 4000 rpm.On the spectrophotometer: 0.5 ml guaiacol, 1.5 ml buffer, 0.5 ml extract, 0.5 ml distilled water in a cuvette.Control: 0.5 ml guaiacol, 1.5 ml buffer, 0.5 ml extract, 0.5 ml H₂O₂.Measurements are taken every 10 seconds at a wavelength of 470 nm for up to 60 seconds.

A=(D₂-D₁)VV₂*

A- Activity (in relative units per 1 g of raw mass or per 1 unit of protein)

D₁- Optical density at the beginning of the experiment (first measurement)

D₂- Optical density at the end of the experiment

t 1-t 2- Time at the beginning and end of the experiment (in seconds)

m- Sample mass

V- Total extraction volume, cm³

V₁- Volume taken for the reaction, cm³

V₂- Total volume in the cuvette, cm³

60- Coefficient for conversion to minutes

Table 3.

Values

Note: * – statistical significance level compared to the control p<0.05, ** – p<0.01 (n=3–4)

A=(0.061-0.022)=0.039*3 =0.5616

Simplified form

A=

⊿D- Change in adsorption D2-D1

⊿t- Reaction time t 2-t1

Vreaction — total reaction volume: 3 mL

Vsample — volume of enzyme used: 0.5 mL

m — sample mass: 0.5 g

A===0.5616U/mL

A===0.2304U/mL

In the lavender extract, the optical density value increased from 0.022 to 0.061 over a period of 10 to 60 seconds. The increase was almost linear, indicating that the reaction kinetics were stable. In the rosemary extract, the value rose from 0.045 to 0.061, but the increase slowed down at 60 seconds, suggesting that the process may have stabilized. According to the results, the proline content in rosemary leaves is 2.43 times higher than in lavender. This indicates that rosemary is better adapted to the climatic conditions of the Syrdarya region. The accumulation of proline is one of the protective mechanisms of plant cells against water loss. The peroxidase activity was measured as 0.5616 U/mL in lavender and 0.2304 U/mL in rosemary leaves. Therefore, the higher proline content in rosemary confirms its greater tolerance to stress conditions. In contrast, the lower proline level in lavender suggests that this plant is more sensitive under the conditions of the Syrdarya region. These findings are of practical importance for cultivating medicinal plants adapted to local climates and for monitoring their biochemical composition. In addition, the study revealed that the activity of the peroxidase (POD) enzyme was 0.5616 U/mg in lavender leaves and 0.2304 U/mg in rosemary leaves. Peroxidases are among the key components of the plant’s antioxidant defense system, as they decompose H₂O₂ and other reactive oxygen species.[10,12] according to the data, peroxidase activity varies depending on the plant’s stress adaptation strategy: in some species, activity increases under stress, while in others, it may relatively decrease. In rosemary, the higher proline content and relatively lower peroxidase activity indicate that the mechanisms of protection against oxidative stress in this species are mainly carried out through osmotic pathways (i.e., via proline accumulation). As noted by Vodiasova and colleagues [15], changes in peroxidase activity are directly related to the plant’s climatic conditions and the type of environmental stress. Therefore, under the hot and arid climate of the Syrdarya region, the higher tolerance of rosemary compared to lavender, achieved through the proline accumulation mechanism, is scientifically substantiated. Overall, these results are consistent with many previously published studies[9,10,11,12,13,14,15] and are in agreement with them, having practical significance for understanding the adaptation of medicinal plants to local climatic conditions and for regulating their biochemical parameters.

Conclusion. The conducted analyses showed that lavender (Lavandula angustifolia Mill.) and rosemary (Rosmarinus officinalis L.) plants enhance their physiological and biochemical response mechanisms under abiotic stress conditions. In particular, under water deficiency or high temperature, the amount of proline in cells increases, which plays an important role in stabilizing osmotic pressure, protecting cell membranes, and reducing oxidative stress. Proline also acts as an antioxidant within plant cells, mitigating the harmful effects of reactive oxygen species. At the same time, an increase in peroxidase enzyme activity indicates the strengthening of the plant’s antioxidant defense system. This enzyme reduces lipid peroxidation, thereby maintaining the stability of cellular structures. Experimental results confirm that lavender and rosemary possess adaptive physiological mechanisms to cope with drought stress, enhancing their tolerance through proline synthesis and peroxidase activity. Therefore, in addition to their medicinal properties, these plants can serve as model species for scientific studies focusing on growth stability under biotic and abiotic stress conditions. In the future, investigating the molecular pathways of proline biosynthesis and identifying the genetic regulation of antioxidant enzyme systems in lavender and rosemary will remain a promising area of research.

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