Pharmacognostic Standardization of Stevia rebaudiana and its HPLC Quantification for Stevioside as Sweetening Compound

Introduction

The perennial shrub Stevia rebaudiana (Bertoni) is indigenous to South America, particularly Paraguay and Brazil, and has gained global recognition for its sweet-tasting leaves, which are widely used as a natural, non-caloric sweetener. In the United States, Stevia was first sold as a nutritional supplement before being authorized as a food ingredient in 2008. Right now, it is widely employed in nations such as Japan, South Korea, Malaysia, Russia, Israel, and Latin America, where its inherent sweetness is appreciated. Health benefits such as blood sugar control and diabetes, weight management, blood pressure reduction, anti-inflammatory, and antioxidant effects make it an appealing alternative to artificial sweeteners.¹

The remarkable sweetness of Stevia is attributed to diterpene glycosides, primarily stevioside and rebaudioside, which are estimated to be 200-300 times sweeter than sucrose. Stevia's potential is increased by its presence of bioactive phytochemicals, such as flavonoids and phenolic compounds, which have antihypertensive, antihyperglycemic, and antioxidant effects in addition to its sweetening capabilities.² This dual role as a natural sweetener and therapeutic agent makes S. rebaudiana valuable in the food, beverage, and pharmaceutical industries.

Ensuring Stevia’s quality, purity, and safety for commercial and medicinal use requires comprehensive pharmacognostic analysis. Macroscopic studies focus on physical attributes like leaf size, shape, color, and venation, while microscopic examinations reveal internal structures such as vascular bundles, trichomes, and stomata. These characteristics are vital for accurate species identification and differentiation from other plants, reducing the risk of adulteration and ensuring the authenticity of raw materials.

Phytochemical screening is equally important, enabling the identification and quantification of bioactive constituents. Such assessments are crucial for standardizing Stevia extracts, ensuring consistency and efficacy in various applications.³ Additionally, microscopic evaluations assist in detecting impurities or foreign substances, further safeguarding product quality.

As global demand for natural and non-caloric sweeteners continues to rise, the pharmacognostic and phytochemical characterization of S. rebaudiana is essential for its reliable use. These evaluations support the development of standardized formulations and reinforce their therapeutic potential, ensuring consumer safety. A thorough understanding of Stevia’s morphological, microscopic, and phytochemical properties is thus critical for its practical application in the food and pharmaceutical sectors. This study introduces a novel integrated approach to the pharmacognostic and analytical evaluation of S. rebaudiana leaves. Unlike previous research emphasi-zing phytochemical screening and stevioside quantification in isolation, this work integrates macroscopic, microscopic, and powder microscopy analysis with an HPLC-based quantification method.

Materials & Methods

The source of stevioside was Yucca Enterprises in Mumbai. Analytical-grade chemicals, solvents, and reagents were used in the investigation.

Plant Collection and Authentication

The plant of Stevia rebaudiana was gathered from the Rani Chennamma College of Pharmacy's medicinal garden in Belagavi, Karnataka. The Indian Council of Medical Research (ICMR), located in Belagavi, Karnataka, India, was later authenticated by Dr. Harsha Hegede Taxonomist.

Macroscopic (Organoleptic) Evaluation

The macroscopic evaluation involved studying the organoleptic and morphological characteristics of the S. rebaudiana leaves. This included observing size, shape, color, venation, margin, apex, texture, and lamina.

Microscopic Evaluation

Microscopic analysis offers a thorough examination of the plant material, allowing the identification of organized drugs based on their histological characteristics. Enlarging minute structures provides comprehensive information on crude medicine and helps validate the structural characteristics of the plant material being studied.

Qualitative Microscopy

Transverse Section of the Leaves

To prepare thin transverse sections, mature S. rebaudiana leaves were collected, washed thoroughly with water, and sectioned from the middle portion of the lamina. These thin sections were kept in water to preserve their moistness. To make interior structures more visible, staining agents such as safranin, Phloroglucinol, and diluted hydrochloric acid (HCl) were applied.^3,4

Powder Microscopy

A few drops of chloral hydrate were applied to a glass slide containing a small amount of leaf powder. After that, the slide was heated to prevent the chloral hydrate from evaporating. A coverslip was carefully placed to avoid air bubbles, and excess chloral hydrate was removed using blotting paper. The sample was stained with Phloroglucinol and concentrated HCl to confirm lignified tissues. A mixture of Phloroglucinol and concentrated HCl (1:1 ratio) was applied on a separate slide, and the Preparation was examined under the microscope.^3,4

Quantitative Microscopy

Quantitative microscopy is crucial for identifying, characterizing, and standardizing crude drugs and determining their cellular content. The quality and purity of the plant material can be assessed using several crucial metrics.

Determination of Stomatal Number and Stomatal Index

Procedure: The middle part of the leaf was boiled for five minutes in a chloral hydrate solution to clarify it. Glycerine water was used to properly peel and mount the upper and lower epidermis on a glass slide. The number of stomata in various leaf sections was counted while the slide was inspected under a microscope. The average number of stomata was computed. The following formula was used to determine the stomatal index:⁵

Stomata Index = S × 100/E + S

S = number of stomata per unit area

E = number of ordinary epidermal cells (including trichomes) in the same unit area.

Determination of Vein Islet and Vein Termination Number

Procedure: Several leaves were heated in a chloral hydrate solution to clarify the leaf structure. After cleaning, they were temporarily mounted using a glycerine solution and stained with safranin. The vein islets and vein terminals within a designated square area were counted. The cleaned leaf sections were then examined under a microscope, and the vein islet and vein terminal data were recorded as numbers per square millimeter.⁵

Determination of Palisade Ratio

Procedure: A leaf slice that was 2 mm thick was boiled for five minutes in a chloral hydrate solution to clarify it. The leaf was mounted, and the epidermal cells were traced after clarity. The number of palisade cells beneath four epidermal cells was counted while focused on the palisade layer. The palisade ratio was calculated using the average count after this process was repeated at various leaf sections.⁵

Physical Evaluation

The quality and purity of the drug were evaluated by analyzing the leaf's physicochemical constants.^3,6,7

Ash values

Ash content is critical for determining the quality and purity of powdered crude drugs. As assessed by the residue left after combustion, the ash concentration reveals the presence of inorganic salts found naturally in the drug or introduced as an adulterant.

Total Ash Determination

Procedure: The entire quantity of inorganic residue that remains after burning. This comprises non-physiological ash, which is the residue of foreign substances sticking to the plant surface, as well as physiological ash, which comes from the plant tissue itself. This process involved heating a crucible to 450˚C for 15 minutes in a muffle furnace and cooling it for an hour in a desiccator. To remove all moisture and completely burn the plant material, a 3.0 g volume of powdered material was placed in the crucible and gradually heated. The temperature gradually increased to 600˚C until all of the carbon was evaporated, leaving behind a carbon-free residue that looked like grey or white ash. After that, the crucible was taken out with crucible tongs and allowed to cool in a desiccator; using a formula, the percentage of ash content was determined.^3,6,7

Determining Water-Soluble Ash

Procedure: After transferring the crucible ash contents from Total Ash into a beaker, 25 ml of water were added, and the mixture was heated for five minutes. After passing a solution through ash-free filter paper, the remaining substance and the paper were dried in an oven. After being squeezed into the crucible, the ash-free filter paper containing the residue was heated to 450˚C until completely removed. After reweighing the crucible, the variations were recorded using a formula.^3,6,7

Determining acid-insoluble ash

Procedure: After being separated from the entire ash, the ash was put into a beaker with 25 ml of diluted hydrochloric acid and allowed to boil for five minutes. An ash-free filter paper and a sintered crucible were used to capture the insoluble material. Hot water was used to repeatedly wash the crucible and beaker through the filter paper until no more acid was present. After being moved into a crucible, the filter paper was burned in a muffle furnace at 500ºC until all the carbon was gone. After cooling in a desiccator, the crucible and its contents were weighed. Concerning the air-dried material, the quantity of acid-insoluble ash was figured.^3,6,7

Determination of loss on drying

Procedure: The mass was lost due to heat expressed as a percentage w/w. A 10 g sample that had been accurately weighed was put onto a Petri dish and heated to 105˚C for five hours in a hot air oven. Loss on the specimen’s death was calculated from the initial volume.^3,6,7

Determination of Foreign Organic Matters

Procedure: The method involved weighing 500g of the sample, spreading it thinly on a white tray, examining it with the naked eye, and manually separating as much of the foreign organic matter as possible. Weighing the foreign organic matter allowed us to calculate its percentage based on the weight of the drugs consumed^{.3 6,7}

Chemical Evaluation (Phytochemical Screening)

Method for Preparation of aqueous leaf extract

Procedure: A standardized process was used to prepare the aqueous leaf extract. Fresh leaves were cleaned with tap water before being rinsed with distilled water to remove dust. After being cleaned, the leaves were shredded and milled into a fine powder after being air-dried for four to six days. The powdered leaves were kept in a container with the appropriate label for further inspection. Twenty grams of leaf powder was weighed for extraction and put into a conical flask with a label that held 100 ml of sterile, double-distilled water. After 20 minutes of heating at 60˚C, the mixture was left to cool for another 60 minutes; the cooled mixture was first filtered through muslin cloth and then through Whatman filter paper no.1. The final filtrate was collected and kept at 4˚C for future use.⁸

Qualitative Chemical Investigation of Aqueous Extract^{7, 9}

Qualitative chemical tests were performed on the aqueous extract of S. rebaudiana to identify the presence of various phytoconstituents. These tests followed standard procedures to evaluate the extract for its chemical composition and confirm the presence of bioactive compounds.

Carbohydrates Test

Molisch's Test: After adding a few drops of an alcohol-based alpha-naphthol solution to the aqueous extract, 1-2 ml of concentrated sulfuric acid solution were gently poured along the test tube's sides. Carbohydrates were present when a violet ring formed at the intersection of the two liquids.

Fehling’s Test: One milliliter of Fehling's A and Fehling's B solutions were combined and heated for a minute. After adding an equivalent volume of the test solution, the heat was for five to ten minutes in a boiling water bath. The presence of reducing sugars was verified by forming a yellow precipitate followed by a brick-red precipitate.

Proteins Test

Biuret Test: Add a few drops of 1% copper sulfate (CuSO₄) solution and 4% sodium hydroxide (NaOH) solution to 3 ml of the aqueous extract. The appearance of a violet or pink tinge indicated the presence of protein.

Millon’s Test: Millon's reagent is mixed with 3 ml of an extract to produce a white precipitate. When the precipitate gets warm, it either dissolves or turns brick red or red.

Steroid Test

Salkowski Reaction: 2ml of concentrated sulfuric acid (H₂SO₄), chloroform, and extract were added. The presence of steroids was shown by the greenish-yellow fluorescence in the acid layer and the red coloration in the chloroform layer.

Glycosides Test

Keller-Killiani Test: One drop of 5% ferric chloride (FeCl₃), glacial acetic acid, and concentrated H₂SO₄ were carefully applied along the test tube's side to two milliliters of the extract. The presence of glycosides was demonstrated by the change of a bluish green in the upper layer and a reddish-brown colour at the intersection of the two liquids.

Legal Test: The aqueous extract was treated with 1 ml of pyridine and 1 ml of sodium nitroprusside. The appearance of a pink-to-red tint indicated the presence of glycosides.

Flavonoid Test

Shinoda Test: 5 mL of 95% ethanol, a few drops of strong hydrochloric acid (HCl), and 0.5 g of magnesium turnings were mixed into the dry powder or extract. Flavonoids were detected when the coloration turned orange, pink, red, or purple.

Ferric Chloride Test: The extract was treated with a few drops of ferric chloride solution. The appearance of a green tint indicated the presence of flavonoids.

Alkaloids Test

Wagner’s Test: Wagner's reagent and 2-3 ml of the filtrate were mixed. The development of a reddish-brown precipitate indicated the presence of alkaloids.

Tannic Acid Test: When a tannic acid solution was added to the extract, a buff-colored precipitate formed, confirming the presence of alkaloids.

Tannins and Phenolic Compounds Test: Two to three milliliters of the aqueous extract were treated with a few drops of the following reagents:

Gelatin Solution: Tannins and phenolic compounds were present when a white precipitate formed.

Lead Acetate Solution: The development of white precipitate confirmed the presence of tannins and phenolic compounds.

HPLC analysis

Chromatographic conditions:

The chromatographic conditions utilized in this study were rigorously established through trial and error and remained consistent across all experiments. High-performance liquid chromatography (HPLC) was performed with a slight modification of the existing methodology by applying an AGILENT 1100 Gradient System with a Diode Array Detector (DAD) and Chem Station software. A column with a 4.6 mm inner diameter and 250 mm length, packed with 5.0 μm RP C-18 stationary phase, was employed. The mobile phase comprised acetonitrile (ACN) and 0.1% orthophosphoric acid (PA) with a pH of 3.0, delivered at a ratio of 90:10, respectively. Analyses were detected at a 210 nm wavelength. A steady flow rate of 0.7 ml/min was maintained, and the system temperature was set to 25ºC. Samples at 20 μl were injected into the system for analysis.^10-13

Preparation of stock and standard solution

For standard solutions, preparation, a series of dilutions were performed using a stock solution of 10 mg of stevioside in 10 milliliters of methanol (MeOH) of HPLC quality, yielding 1000 μg/ml (stock-1).¹³ From stock-1, successive dilutions were made: Methanol and 0.1 ml of stock-1 were combined to produce a 10 μg/ml stevioside concentration. To reach stevioside concentrations of 20 μg/ml, 30 μg/ml, 40 μg/ml, and 50 μg/ml, methanol was mixed with 0.2 ml, 0.3 ml, 0.4 ml, and 0.5 ml of stock-1.

These prepared standard solutions served as reference points for calibrating the analytical method and quantifying the concentration of stevioside in the samples under investigation.

Analysis of content in the extract

A 1000 μg/ml concentration was obtained by dissolving the extracted sample, which weighed 10 mg, in 10 ml of methanol. 10 mg of the extract was weighed using an analytical balance and transferred into a clean, dry glass container. 10 ml of methanol was added to a glass container containing the extract. The solution was prepared by gently swirling or stirring until the extract was completely dissolved.¹⁴ Once the extract was dissolved, the resulting solution was ready for further analysis.

Results

Collection and authentication of plant

A specimen of Stevia rebaudiana (Bert.) Bertoni, belonging to the Asteraceae family, has been documented and placed in herbaria with the accession number RMRC-1774.

Macroscopic (Organoleptic) Evaluation:

The morphological and organoleptic properties of S. rebaudiana leaves are unique. They have a distinct sweet smell that reflects their high sweetness content and are green in color. Stevioside and rebaudioside are two compounds that give stevia its distinctively sweet flavor. The leaves have a smooth texture and are usually 5-6 cm long. Oval-elliptical in shape, the leaves have saw-toothed edges and serrated margins. Furthermore, as is typical of dicot plants, the venation is reticulate, meaning the leaf veins create a net-like pattern. The macroscopic characteristics of S. rebaudiana, including its organoleptic properties, morphological features, and leaf measurements, were documented and observed, as shown in Figure 1. These characteristics provide valuable insights into the plant's physical appearance and sensory qualities, helping with its identification.

Microscopic Analysis

Qualitative Evaluation

The transverse section (T.S.) of S. rebaudiana leaves revealed a detailed anatomical structure with key components such as the epidermis, vascular bundles, xylem, phloem, and starch grains. The section, taken from the midrib region, exhibited distinct characteristics, as illustrated in Figure 2. The lower epidermis, forming the bottom layer, is covered with a waxy cuticle that reduces water loss and provides protection. At the same time, the upper epidermis also features a similar cuticle. The vascular bundle, arranged in a cylindrical pattern, consists of the xylem and phloem, facilitating the transport of water, minerals, and nutrients. Two types of glandular trichomes were observed: a longer, conical biseriate trichome with 8-10 cells and an elongated tip and a shorter, thinner trichome comprising 3-6 cells.

Anomocytic stomata, also known as Ranunculaceous or irregular stomata, were identified on the abaxial surface. Additionally, collenchyma cells provide mechanical support, while the mesophyll layer, located between the thin upper and lower epidermis, comprises palisade and spongy parenchyma cells. This anatomical study underscores the unique and intricate structures in S. rebaudiana leaves.

Powder Microscopy: The powder microscopy of S. rebaudiana leaf revealed several diagnostic characteristics vital for identifying the plant's anatomy. The study highlighted the presence of the epidermis, starch grains, and components of the vascular bundle, including the xylem and phloem. Structural fibers and portions of parenchyma tissues were also observed, contributing to the plant's mechanical support and storage functions. Notably, two types of non-glandular (vector) trichomes were identified. The shorter trichomes were thinner, uniseriate, and were of three to six cells, whereas the longer trichomes were conical, erect, and uniseriate, with eight to ten cells. These diagnostic features, detailed in Figure 2, provide valuable insight into the structural attributes of S. rebaudiana leaves.

Quantitative Evaluation

These parameters are essential for quantifying crude drugs, particularly when the sample is left. The findings are presented in Table 1.

Physicochemical Evaluation

The physicochemical parameters of the sample were analyzed, with the outcomes presented as mean values along with standard deviations (% w/w). Loss on drying measures the free water content in the sample, a critical factor in determining moisture levels that can affect the stability and overall quality of the substance. Table 1 provides specifics of the analysis's conclusions. These parameters are important in assessing the sample's quality, purity, and composition.

Chemical Evaluation (Phytochemical Test)

The Chemical Evaluation of the aqueous extract of S. rebaudiana leaves showed the presence of several important bioactive substances. Carbohydrates were confirmed through positive Molisch’s and Fehling's tests, while proteins were detected using Biuret and Million's tests. The presence of glycosides was confirmed by the Kellar Killani, Legal's, and Borntrager’s tests. Flavonoids were identified through positive Shinoda and Ferric chloride tests, and alkaloids were detected with Wagner’s and Tannic acid tests. Additionally, tannins and phenolic compounds were present as positive results in the Gelatin and Lead acetate tests indicated. However, no steroids were found, as shown by negative results in Salkowski and Libermann's reactions. These results imply that a range of bioactive substances, such as proteins, carbohydrates, glycosides, flavonoids, alkaloids, and tannins, are present in S. rebaudiana leaves and may be responsible for their nutritional and medicinal qualities.

HPLC analysis

Linearity

The calibration curve was obtained by analyzing the five standard solution concentrations ranging from 10-50 μg/ml. A graph between the concentrations versus peak area gives slope, intercept, and coefficient of correlation (r2) Figure 3 y = 143.76x - 131.32 = 0.999

The analysis of the Stevia extract revealed a retention time of 5.165 minutes. The peak exhibited an area of 1501 mAu units and a height of 61.61 mAu. The resolution was determined to be 0.86, indicating the degree of separation between the peaks as illustrated in Figure 4b. The concentration of the stevioside in the sample was calculated to be 11.35 μg/ml.

Discussion

The current study on S. rebaudiana aimed to investigate various macroscopic, microscopic, physi-cochemical, and phytochemical characteristics to standardize the plant material for further medicinal use. The macroscopic features of the leaves, including their shape, size, and color, were consistent with previous reports, confirming the plant's identity. The leaf's morphological structure, with its characteristic midrib, serrate margins, and granular upper surface, aligns with descriptions found in the literature, suggesting its authenticity.¹⁵ Microscopic analysis revealed significant anatomical features, such as the presence of xylem, phloem, vascular bundles, and starch grains, typical of Stevia leaves. The powder microscopy further supported these findings, identifying key diagnostic characteristics, including the non-glandular trichomes in the epidermis. Similar observations were reported by, confirming these characteristics.¹⁶

The quality control of Stevia leaf samples relies on microscopic and physicochemical analyses to ensure purity and authenticity. The stomatal index and vein islet number help distinguish Stevia from adulterants, while physicochemical parameters like total ash (5-12%), acid-insoluble ash (≤1-2%), and water-soluble ash (1-5%). Foreign organic matter (<2%) ensures minimal impurities, and moisture content (≤10%) prevents microbial degradation. These findings are consistent with earlier studies that highlight how crucial it is to regulate herbal raw materials to preserve their stability and effectiveness.¹⁷

Numerous secondary metabolites with pharmacological properties, including proteins, carbohydrates, glycosides, flavonoids, alkaloids, and tannins, were found by phytochemical screening. However, the absence of steroids in the aqueous extract suggests that Stevia may not possess certain expected bioactivities, aligning with its established use primarily for its sweetening properties rather than steroid-related therapeutic benefits.¹⁸ The calibration curve analysis demonstrated high precision in quantifying the bioactive compounds within the extract, with the concentration of the active compound calculated at 11.35 μg/ml, which should be compared with commercially available Stevia products to ensure standardization and quality control. The results of this study provide an in-depth understanding of S. rebaudiana's chemical composition, aiding in its standardization and enhancing its potential for use in both pharmaceutical and food industries. The consistent findings across various evaluations confirm the plant’s suitability for further medicinal research and applications.

Conclusion

In conclusion, this study’s comprehensive evaluation of Stevia rebaudiana has provided valuable insights into its macroscopic, microscopic, physicochemical, and phytochemical characteristics. The morphological and anatomical features observed in macroscopic and microscopic evaluations confirm the plant's authenticity and quality. The physicochemical characteristics, including moisture content and ash values, further emphasize the purity and stability of the plant material, making it suitable for use in pharmaceutical and medicinal formulations. The phytochemical screening revealed a range of bioactive components, highlighting the plant's medicinal potential, including proteins, carbohydrates, glycosides, flavonoids, alkaloids, and tannins. Moreover, the quantification of bioactive compounds via calibration curve analysis indicates that S. rebaudiana holds promise as a vital source of natural compounds for potential medical uses in the future. These findings support the standardization and quality control of S. rebaudiana for both medicinal and commercial purposes, ensuring its efficacy and safety in use.

Conflict of interest

The authors state that there is no conflict of interest with the publishing of this study.

Acknowledgment

For authenticating the S. rebaudiana plant samples and providing invaluable advice during the study, I am grateful to Dr. Harsh Hegde, Taxonomist, ICMR, Belagavi, Karnataka. Additionally, I express my sincere gratitude to Rani Chennamma College of Pharmacy in Belagavi, Karnataka, for providing the required facilities, resources, and assistance.