Evaluation of Histochemical Profile and Numerical Indicators of Salvia dumetorum, Growing in Central Kazakhstan

Yana K. Levaya¹, Gayane A. Atazhanova¹*, Margarita Y. Ishmuratova²,

Mussa E. Zholdasbayev¹, Aigul T. Medeshova¹

¹School of Pharmacy, Karaganda Medical University, Gogol 40, Karaganda, Kazakhstan.

²Department of Botany, Karaganda Buketov University, Universitetskaya 28, Karaganda, Kazakhstan.

*Corresponding Author E-mail: yaninka_25@mail.ru

ABSTRACT:

Salvia dumetorum (L.) Andrz. ex Besser is a promising medicinal plat used in traditional medicine for treating various diseases. In this study, we continue investigation of S. dumetorum in Central Kazakhstan in terms of histochemical profile and numerical parameters of raw material. Histochemical profile was assessed using histochemical tests of cross sections of leaves, petioles, stems and sepals of S. dumetorum in Strauss-Fleming solution and were examined under light microscope, in order to determine secondary metabolites, such as essential oil, phenolic acids, flavonoids, sesquiterpene lactones, polysaccharides and alkaloids. Numerical parameters were assessed using methods described in State Pharmacopoeia of the Republic of Kazakhstan. Determination of quantitative content of heavy metals was carried out using the inductively coupled plasma atomic emission spectrometry (ICP-AES) method. The aim of the study was to determine histochemical profile and numerical parameters of S. dumetorum raw material. Histochemical analysis of raw material demonstrated that S. dumetorum contain high amount of phenolic compounds, flavonoids and essential oil. However, sesquiterpene lactones, polysaccharides and alkaloids were not detected in raw material. Results of assessing numerical parameters showed that all parameters: foreign impurities 1.58±0.11%, total ash 12±0.22%, ash insoluble in 10% HCl 1.66±0.23%, moisture content 6.5±0.17%, extractives 64.17±0,31% meet the requirements of regulatory documents. For the first time, the localization of secondary metabolites was studied using histochemical tests in the tissues of S. dumetorum growing in Central Kazakhstan. The analysis of raw material quality indicators showed that S. dumetorum match with the requirements of State Pharmacopoeia of the Republic of Kazakhstan. This species has biological activity and significant thickets in nature, successfully grown in culture, which confirms the prospect of its practical use as a medicinal plant. The data obtained will be used to confirm the authenticity, identification and standardization of the S. dumetorum and in the technological process for the production of extraction preparations.

KEYWORDS: Flavonoids, Medicinal plant raw material, Phenolic compounds, Plant structure, Quality control, Salvia dumetorum, Secondary metabolites.

INTRODUCTION:

Recently, non-pharmacopoeial plant species have a great interest in the development of herbal medicines. Plants of the genus Sage (Salvia L., family Lamiaceae) include almost 900 species, which are widely used in folk medicine in many countries¹. Currently, in the State Pharmacopoeia of the Republic of Kazakhstan (SPhRK)², only one species of sage (Salvia officinalis L.) is included, while other species have potential for practical application in medicine and pharmacy³. As an alternative plant to S. officinallis, we are studying S. dumetorum (symonym Salvia stepposa Des.-Shost.), from the family Lamiaceae, which is widespread in the territory of the Republic of Kazakhstan⁴ and has significant raw material resources.

Among the Lamiaceae plant group, Salvia is one of the most sought after plants used in traditional medicine for treating various diseases. The aerial part of Salvia has been used for treatment inflammation, wound healing, ulcers, tremor, rheumatism, dyspepsia and others depending on Salvia species⁵. Different content of chemical constituents responsible for the biological effect of this plant. The main chemical constituents of genus Salvia are phenolic compounds and flavonoids. S. officinalis essential oil contains predominantly α-thujone (12.6–42.6%), β-thujone (1.3–13.8%), camphor (12.4–33.3%), and 1,8-cineole (4.2–9.9%)⁶, while S. dumetorum essential oil contains extremely small amounts of them⁷. Despite the different chemical composition of the essential oil of these two species, S. dumetorum can be considered as a potential source of phenolic acids and flavonoids.

Medicinal plant raw materials, which are used today in medicine, must be high quality and meet the requirements of regulatory documentation. In order to standardize the medicinal plant materials, it is necessary to determine main biologically active substances and quality control indicators of raw materials. Considering that there are no approved quality parameters for S. dumetorum, the first priority is to establish numerical parameters, as well as the authenticity of the plant. Today, there are a number of methods that make it possible to assess whether a drug belongs to a particular group in a classification based on the content of biologically active substances. One of these methods is histochemical analysis, which allows to identify the presence and localization in plant tissues of secondary metabolites – alkaloids, terpenoids, tannins, flavonoids, anthracene derivatives, and etc. using the colour-stain reaction technique and photographic recording⁸. These studies include preparation of fixed variably stained specimens and then the examination under the microscopic devices⁹.

The main objective of our research is to search raw plant material growing on the territory of the Republic of Kazakhstan, which can serve as an alternative medicinal raw material of SPhRK. To our knowledge, the pharmacognostic analysis including histochemical studies on S. dumetorum is still limited. Therefore, the study of the localization of secondary metabolites in the organs and tissues of the aerial parts of S. dumetorum is an important stage of ongoing research.

MATERIALS AND METHODS:

Plant material:

The object of study is an aerial part of S. dumetorum, which consist of stems 35-50cm long, petioles, leaves and inflorescences collected in June-August 2021 in the vicinity of the Karaganda region (49.88898°N; 73.15569°E) (Figure 1). Species identity was confirmed by members of the Department of Botany. S. dumetorum herbarium specimen number QAR00068 collected - Karkaraly mountains, Karkaraly district, Karaganda region, 25 June 1988 and was deposited at the herbarium fund (Index Herbarium – QAR), Department of Botany, Karaganda Buketov University, Karaganda, Kazakhstan.

Figure 1. Aerial part of Salvia stepposa Des.-Shost growing in Central Kazakhstan

Histochemical characterization:

Fragments of freshly collected aerial part of S. dumetorum were fixed in a mixture consisting of 70% alcohol, glycerine and distilled water in a ratio of 1:1:1 (Strauss-Fleming solution) for 7 days¹⁰. Histochemical examination was carried out on transverse sections of the stem, leaves, petiole and sepal. The following secretory structures of S. dumetorum were studied using specific reagents. For determination of essential oil 1% alcohol solution of methylene blue was used¹¹. Determination of starch, was carried out by submerging the section for 10 min in Lugol's solution. Highlighting of phenolic compounds was assessed using 10% solution of potassium dichromate for 30 min, washed twice in distilled water to remove excess reagent and mounted in glyceringelatin¹². Determination of flavonoids was performed using 10% ferric chloride solution for 30 min and washed twice in distilled water to remove excess of ferric chloride. The section was treated with Dragendorff reagent for 20 min, rinsed in 5% sodium nitrite and mounted in distilled water in order to determine alkaloids⁹. Sesquiterpene lactones and polysaccharides identification was carried out using concentrated sulfuric acid and vanillin solution, 10% alcohol solution of thymol and concentrated sulfuric acid, respectively¹³. A sign of the localization of substances is a change in the colour of the studying plant part. The positive control was performed according to the reference of the respective author.

Image recording of micro specimens were taken using a Altami BIO light microscope (Russia) coupled with a digital eyepiece USB camera UCMOS03100KPA and E Plan 4×/0.10lens. Photo processing was carried out in the Altami Studio 4.0 and Paint 10.1programs.

Numerical indicators:

Measurement of physical and chemical indicators: determination of impurity content, total ash, ash insoluble in 10-% hydrochloric acid, moisture content, extractives was done according to the SPhRK and Pharmacopoeia of the Eurasian Economic Union^2,14.

Determination of impurity content:

500 g of raw materials were weighed, laid out in a thin layer, and the raw materials were examined with a magnifying glass for the presence of foreign organs and components. Foreign impurities were separated, weighed, and the impurity content was calculated as a percentage dividing mass of separated foreign impurities by mass of raw materials, measured in grams.

Determination of total ash:

The porcelain crucible was heated to red heat (550–650 °C) for 30 minutes until a constant mass was achieved, then cooled in a desiccator and accurately weighed. 1.00 g of dried plant material was placed in a prepared crucible, evenly distributing the sample to be analysed along the bottom of the crucible. The test sample was dried in a crucible at 100–105°C for 1 hour and burned until constant weight was reached in a muffle furnace at a temperature of 600±25°C. The crucible was then cooled in a desiccator and weighed. Calcination was repeated until the mass of the ash residue remained constant.

The percentage of total ash in dried plant materials was calculated dividing mass of ash by mass of plant materials, measured in grams.

Determination of ash insoluble in 10-% hydrochloric acid:

15ml of water and 10ml of hydrochloric acid were added to the crucible containing the residue after determining the total ash, the crucible was covered with a watch glass and the mixture was boiled for 10minutes. After cooling, the contents of the crucible were filtered through an ashless filter, transferring the sediment onto it and washing the watch glass with hot water. The filter with the precipitate was washed with hot water until the wash water was neutral on universal indicator paper, transferred to the same crucible, dried and calcined at red heat (550–650°C), cooled in a desiccator and weighed.

The content of ash, insoluble in 10% hydrochloric acid, in the raw material in percent was calculated dividing the difference in mass of ash and ash from the filter by the mass of raw materials, measured in grams.

Determination of moisture content:

In a flat-bottomed cup, 0.50g of the test extract was quickly weighed and dried in an oven at 100-105℃ for 3 hours until constant weight was achieved, cooled in a desiccator over phosphorus (V) oxide P and then weighed. The weight loss during drying of raw materials as a percentage was calculated dividing mass of raw materials before and after drying, measured in grams.

Determination of extractives:

A sample of crushed raw material weighing 3g was placed in a flask with a ground section and 50ml of extractant (1:1 ethanol:water v/v) was added, the flask was capped, weighed to the nearest 0.01g and left for 1 hour. Then the flask was heated, maintaining a low boil for 2hours. Next, the flask was cooled and weighed with a stopper, the loss in mass was replenished with the extractant. The contents of the flask were thoroughly shaken and filtered through a paper filter into a dry flask. 25ml of the filtrate was evaporated to dryness in a water bath in a dried and accurately weighed porcelain dish. The dry residue was dried in an oven at a temperature of (102.5± 2.5℃) to constant weight, then cooled in a desiccator for 30min and weighed.

The content of extractive substances as a percentage in terms of absolutely dry raw materials was calculated using the formula:

m*200*100

x = -----------------

m₁*(100-W),

where

m – mass of dry residue, g;

m1 – mass of raw materials, g;

W –moisture content, %.

Determination of quantitative content of heavy metals:

The analysis was carried out on the basis of the accredited chemical analytical laboratory of Azimut-geology LLP using the inductively coupled plasma atomic emission spectrometry (ICP-AES) method, calculations were made using an approximate quantitative method. Regulatory document on the method for determining MVI KZ07.00.01378-2016, protocol No. 1132-SB dated November 4, 2020.

Statistical analysis:

All experiments were conducted three times. The results were mean± SD of three parallel measurements. All statistical comparisons were made by means of Student’s t – test, values of p<0.05 were regarded as significant. Statistical analysis of the results was obtained was carried out by the method of the smallest squares according to the SPhRK. For the calculations and statistical analysis of the obtained data Microsoft Office Excel 7.0 and Statistica 6.0 were used.

Ethical approval: The research conducted is not related to either human or animal use.

RESULTS AND DISCUSSION:

Assessing the quality, safety and effectiveness of pharmaceutical products are one of the important tasks facing scientists today, which are directly related to the quality and authenticity of raw material. Macroscopic and microscopic methods are the traditional methods for botanical authentication of raw materials. Previously, we carried out a macroscopic and microscopic analysis of S. dumetorum, during which diagnostic features characteristic of this type of raw material were identified¹⁵. The next stage of quality assessment is the identification of secondary metabolites necessary for further standardization of pharmaceutical products.

Histochemical profile:

Histochemical analysis of medicinal plants and raw materials is intended to study the localization of various chemicals substances and the products of their metabolism in tissues. The study revealed characteristic staining of cells upon interaction of reagents with detected substances. The results of the histochemical analysis in the studied samples are presented in Table 1.

Treatment of micro preparations of a leaf, stem, sepal and petiole with a solution of methylene blue solution showed that no specific blue staining of surface preparations was observed, although accumulation of essential oil in individual cells and tissues was noted. The main site of essential oil localization oil glands, trichomes, and non-specialized epidermal cells, which was previously described for many representatives of the Lamiaceae family¹⁶ (Figure 2). Previously, we isolated the essential oil of S. dumetorum and studied its chemical composition using GC-MS⁷.

Figure 2. The result of histochemical reactions with methylene blue (magnitude × 10). (A) cross section of a leaf. (B) cross section of a stem. (C) inflorescence. (D) cross section of a petiole

After treating a microsamples of the test material with a 1% alcoholic FeCl₃ solution, intensive staining of palisade mesophyll, epidermis, and angular collenchyma of the leaf; epidermis, bark parenchyma, and vascular-conducting bundles of the stem; chlorenchyma and vascular bundles of the leaf petiole; veins and epidermis of sepal was observed (Figure 3).

Figure 3. The result of histochemical reactions with a 1% alcohol solution of FeCl3 (magnitude × 10). (A) cross section of a leaf. (B) cross section of a stem. (C) inflorescence. (D) cross section of a petiole

Table 1. Histochemical analysis of the aerial part of Salvia stepposa Des. -Shost.

Detectable secondary metabolites	Result of interaction with the reagent	Result of testing micro specimens
Detectable secondary metabolites	Result of interaction with the reagent	Cross section of stem	Cross section of a leaf	Cross section of petiole	Surface of inflorescences
Essential oil	Blue	+	+	+	+
Sesquiterpene lactones	Raspberry-violet	-	-	-	-
Flavonoids	Black-blue-green	+	+	+	+
Phenolic compounds	Brown, yellow	+	+	+	+
Polysaccharides	Orange-red	-	-	-	-
Starch	Blue	-	-	-	-
Inulin	Spherocrystals of spherical shape	-	-	-	-
Alkaloids	Black	-	-	-	-

*Note: – negative reaction; + positive reaction

The presence of phenolic acids was confirmed by intense yellow-brown staining in all the studied organs of S. dumetorum, indicating the presence of phenolic compounds in all cells. However, the staining of micro preparations was uneven, which allows us to judge about the different degree of accumulation of phenolic compounds in cells. Thus, the areas with maximum accumulation of phenolic compounds are leaf sclerenchyma and chlorenchyma, bark parenchyma and conductive zone of the stem, conductive zone, and mechanical tissues of the leaf petiole (Figure 4). Staining of microscopic preparations with vanillin solution in sulfuric acid, thymol, Dragendorf's reagent and Lugol's solution did not show characteristic staining, which may indicate the absence of accumulation of sesquiterpene lactones, polysaccharides, starch and alkaloids.

Figure 4. The result of histochemical reactions with a 10% alcohol solution of potassium dichromate (magnitude × 10). (A) cross section of a leaf. (B) cross section of a stem. (C) inflorescence. (D) cross section of a petiole

Previous findings showed that in S. aegyptiaca were detected polysaccharides in both types of capitate trichomes, essential oil in petal trichomes, lipids and terpenes in both type of trichomes¹⁷. Comparing to other studies regarding Salvia phenolic substances detected by ferric chloride, peltate trichrome were found in S. aurea¹⁸ and S. blepharophylla ¹⁹and were not found in S. aegyptiaca histochemical test, but were detected using spectrophotometric method¹⁷. In Salvia aurea, peltate and two types of capitate trichomes were identified, each demonstrating a specific specialization in the secretion process. Histochemical analysis revealed the presence of polysaccharides, polyphenols, proteins, and essential oil in all types of trichomes, indicating a complex and heterogeneous composition of the secreted material. Particularly noteworthy is the presence of phenolic compounds, detected by ferric chloride reaction specifically in the peltate trichomes, confirming their active role in the accumulation of biologically active substances¹⁸. Histochemical research of S. dolomitica²⁰ leaves showed that the secretion produced by peltate trichomes is chemically complex and functionally diverse. The predominance of hydrophobic compounds suggests a protective role against desiccation and herbivory, while the presence of hydrophilic substances such as polysaccharides and phenolic compounds indicates additional functions related to defense and interaction with the environment. Capitate trichomes, although less abundant, also contributed to the secretion profile, with histochemical tests revealing the presence of lipophilic and phenolic compounds, albeit in lower concentrations compared to peltate trichomes. Overall, it can be seen that depending on the species of Salvia, differences in the amount, localization and type of various compounds can be observed.

Determination of numerical indicators and quantitative content of heavy metals of S. dumetorum leaves:

During the study, were determined organoleptic characteristics of S. dumetorum: the smell of the dried raw material is aromatic, especially when ground, the taste is bitter-spicy, slightly astringent.

In the study are determined: foreign impurities, total ash, ash insoluble in 10% HCl, moisture content and extractives. Due to the fact that numerical quality indicators for S. dumetorum leaves have not yet been developed, we selected for comparison the quality indicators of raw materials from the SPhRK pharmacopoeial article “Salvia officinalis leaves”. The results of measuring the numerical indicators of raw materials are presented in Table 2.

The elemental composition of raw material plays an important role, since plants are easily contaminated during the growing process, therefore, the limits of elemental composition must be clearly defined for medicinal plants. Ignoring the control of elements in medicinal plant raw material, due to their accumulation in the human body, can subsequently lead to disorders associated with the brain, digestion, kidneys, liver, pancreas, reproductive system and central nervous system. In addition, it can lead to the development of serious illnesses if exposure is high and repeated. Therefore, it is necessary to analyze the maximum content of metals in pharmaceutical products^21-23. In this regard, a quantitative determination of heavy metals in the S. dumetorum leaves was carried out using the ICP-AES method, the results of the determination are presented in Table 3.

Table 3. Content of heavy metals in the Salvia stepposa Des.-Shost. leaves

S. No.	Determined component	Component content, mg/kg
1	Arsenic (As)	<0,1
2	Cadmium (Cd)	<0,5
3	Cooper (Cu)	12,7
4	Iron (Fe)	762,6
5	Nickel (Ni)	1,6
6	Lead (Pb)	1,6
7	Zinc (Zn)	37,9

When assessing the basic numerical parameters, we established, the content of heavy metals allows us to conclude that samples of S. dumetorum, collected in the vicinity of the Karaganda region, fully comply with the requirements of SPhRK.

CONCLUSION:

In the course of the study, the secretory structures of S. dumetorum growing in Kazakhstan were studied for the first time using light microscopy in combination with histochemical tests. As a result of histochemical tests carried out on transverse sections of the leaf, petiole, stem and sepal of S. dumetorum essential oils, phenolic acids, flavonoids were found and their localization was established:

· Essential oils – in essential oil glands and capitate hairs;

· Phenolic acids – in the sclerenchyma and chlorenchyma of the leaf, the cortex and conductive zone of the stem, the conductive zone and mechanical tissue of the petiole;

· Flavonoids – in the leaf mesophyll; angular collenchyma, epidermis, chlorenchyma and vascular bundles of the stem; chlorenchyma and vascular bundles of the petiole.

Sesquiterpene lactones, polysaccharides, starch, inulin and alkaloids were not detected in aerial part of S. dumetorum. Based on the results of the study, an assessment of the histochemical profile and numerical indicators it can be concluded that S. dumetorum, collected in the vicinity of the Karaganda region, comply with the requirements of the SPhRK. The data obtained are important in identifying raw materials and determining quality parameters and can also serve as the basis for the subsequent development of regulatory documents for S. dumetorum.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

This work was supported by the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan under Grant No. АР23488250.

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Received on 18.03.2025 Revised on 12.07.2025

Accepted on 30.09.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):6077-6083.

DOI: 10.52711/0974-360X.2025.00879

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Raw material	Foreign impurities, %	Total ash, %	Ash insoluble in 10% HCl, %	Moisture content, %	Extractives,%
Salvia stepposa Des.-Shost. leaves	1.58	12	1.66	6.5	64.17
Salvia officinalis leaves	no more 2%	no more 12	no more 3	no more 14	no more 30