BIOCHEMICAL CHARACTERIZATION OF STEVIA ESSENTIAL OIL THROUGH DIFFERENT DRYING METHODS AND SOLVENTS

Essential extracts from plants have been used as a food and medicinal cure since ancient times. Some plants have a good portion of carbohydrates e


INTRODUCTION
Stevia rebaudiana Bertoni is a tiny perennial shrub native to Paraguay, Brazil, and Argentina in the Asteraceae (Compositae) family.For ages, indigenous peoples have utilized the leaves of this plant in remedies and to sweeten drinks such as mate i.e., a green herbal tea.(Samuel et al., 2018).Stevia, a natural sweetener has 200 species of herbs and shrubs from the Asteraceae family (Composite), however only S. rebaudiana has the highest level of sweetness among these species (Cosson et al., 2019).Stevia i.e., a perennial shrub has been successfully acclimatized to environmental conditions in several parts of Pakistan, particularly Punjab province, being a production pocket of 1.6-1.8tons per acre (Chugtai et al., 2019).Currently, stevia cultivation in Pakistan is significantly being focused to constrict the use of conventional sweeteners i.e., sucrose from Sugar cane and Beetroot because most serious health problems, such as diabetes and obesity, are becoming prevalent.However, sugar consumption cannot be eliminated from our daily lives since it is a necessary component of our food.In these circumstances, stevia with fewer negative impacts on human health is being explored.Moreover, the stevia extracts such as Stevia oil is gradually gaining limelight in medicinal and therapeutic fields.Nevertheless, in Pakistan no study has been carried out so far for its biochemical characterization in terms of nutritional and compositional factors.Biochemical Composition of Stevia: Stevia leaves are high in a variety of compounds.There are nine essential amino acids among the chemicals: glutamate, aspartate, methionine, tyrosine, proline, alanine, isoleucine, lysine, and serine.(Mlambo et al., 2022).It is a nutrient-dense herb that contains a significant amount of protein, fibre, amino acids, sugars, lipids, essential oils, ascorbic acid, -carotene, riboflavin, thiamine, minerals (chromium, cobalt, magnesium, iron, potassium, phosphorus), and bioactive compounds such as austroinulin, sterebins A-H, nilacin, rebaudi oxide (Elhassaneen, 2019).Stevia was initially studied because of the steviosides, but in recent years, many research has concentrated on other useful components such as diterpene, triterpenes, sterols, pigments, and others, which account for 80%-90% of the dry leaves' composition, Carvacrol (isomer monoterpene phenol), caryophyllene (bicyclic sesquiterpene), caryophyllene oxide, and aromatic chemicals such as spatulenola, kardinola, -pinene, limonen, and iso-pinokarveola that demonstrate antioxidant, anti-inflammatory, and anti-bacterial action were identified in leaf extracts.(Putnik et al 2020).The major glycosides of stevia plants are stevioside and rebaudioside A (Bergs et al., 2012).The steviol (chemical backbone structure) of all diterpenoid glycosides isolated from Stevia rebaudiana is identical, with minor changes in the quantity of carbohydrates residues.Because of the expanding diabetic and obese populations in Europe they use sugar in food and drinks, this herbal sweetener has a high market potential (Ismail et al., 2020).Therefore, biochemical characterization of well-adapted and well-cultivated Stevia is urgently required to build a better nutritional framework that can help to explore its significant role in medical and nutritive fields.As a result, researchers have focused their attention on the antioxidant potential of essential oils.Medicinal Properties of Stevia: The leaves of S. rebaudiana have several medical applications, including antibacterial, antiviral, antifungal, antihypertensive, anti-hyperglycaemic, anti-tumor, antiinflammatory, anti-diarrheal, anti-human rotavirus activity, anti-HIV, hepatoprotective, and immune modulatory actions (Elhassaneen, 2019).Stevia extracts have been shown to offer significant health benefits, making them useful in the treatment of a wide range of chronic and nonchronic disorders, including diabetes, cardiovascular disease, cancer, renal disease, obesity, inflammatory bowel disease, and dental caries (Putnik et al., 2020).Furthermore, due to their benign properties, antioxidant chemicals found in edible plants have recently been advocated as food additives.
Stevia has been utilized as a non-caloric sweetener in dietary supplements.An unpleasant taste is also identified in human's herbal tea, natural sugar, and ayurvedic health system as anti-diabetic, anti-obesity, and anti-cholesterol.The antediluvian ayurvedic system of medicine provides several pasts on the use of S. rebaudiana .S. rebaudiana leaves have been recommended as a treatment for a variety of chronic and non-chronic disorders, including kidney disease, diabetes, cancer, obesity, inflammatory bowel disease, cardiovascular disease, and dental caries (Kumar et al., 2021).Although sweeteners that are artificial and noncaloric can be used to sweeten foods and beverages but cause serious health problems.As a result, it might be argued that using a natural, safe sweetener like stevia is still the best option, as it has gotten a lot of attention and acceptance from both scientists and consumers.Extraction Methods of Essential Oils (EO): In order for the extracts and essential oils to be utilized safely by humans, the least toxic solvent and the safest extraction procedure are required for optimal phytochemical separation of natural sources of highactivity antioxidants to replace dangerous synthetic antioxidants.Supercritical fluid methods, solvents extraction, enzymatic extractions, ultrasound and microwaves are used as extraction procedures, which are followed by solvent-liquid-liquid extraction, purification using columns, ion exchange, nanofiltration, ultra-membranes crystallization, and fractional distillation (Rao et al., 2019).Despite significant improvements in extraction, producing lowimpurity products that are scalable and need the least amount of solvents remains a challenge.Stevia has sparked economic and scientific attention due to its sweet and medicinal characteristics.This plant requires extremely little water i.e., 5 % of that required by sugarcane, that is now a big issue.The chemical makeup of stevia leaves varies depending on geographical location and cultivar (Khiraoui, 2017).The chemical composition of stevia active components is also affected by the drying and processing method (Gasmalla., 2014).Many studies have shown that stevia is the most common sugar substitute.Industrial plant biomass could also be a source of an essential oil (EO) that could be further useful in the development of new pesticidesEmerging technologies such as ultrasound-assisted crystallization, colloidal gas aphrons, isoelectric solubilization-precipitation, hydro distillation and pressured microwave-assisted extraction are currently being used (Alvarez et al., 2019;Yuan et al., 2019).

MATERIAL AND METHODS
Procurement of Plant Material: Stevia plants of variety i.e., Honey Stevia were grown in pots at plant propagation unit at Horticulture research area Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi.The leaves from these plants were used in this study.The obtained leaves were taken to department laboratory for cleaning purpose where they were thoroughly washed by a disinfectant i.e., 70 % ethanol available in the laboratory so that any foreign matter, microbes, dust or any fibrous material was cleaned.Fresh green leaves were harvested from the whole plant by cutting the plant 5-10 cm from upper parts so that they can be subjected to drying process.Drying and Grinding: The leaves were divided into two parts.Half of the leaves were dried in open air at 20-22℃ for 8 days and rest were subjected to oven dry at 50℃ for 5hrs.Once dried, leaves were grinded to powdered form by using a mortar and pestle or a lab grinding mill as shown in figure 1.The powdered leaves were packed in an airtight container for further oil extraction and chemical characterization.All the drying and grinding procedure took place in Department of Horticulture, PMAS-AAUR, Rawalpind The intended product was received after intervals and then filtered properly for further analysis as shown in figure .HPLC Analysis: In HPLC, a sample mixture or analyte in a solvent i.e., oil extract also known as the mobile phase was pumped through a column with chromatographic packing material at high pressure i.e., stationary phase.A moving carrier gas stream of helium or nitrogen transported the sample.The interaction between the stationary phase, the molecules being analyzed, and the solvent, or solvents utilized, affected retention time of sample.Because the analytes had distinct polarity, the sample interacted with the two phases at different rates as it went through the column.Analytes with the least interaction with the stationary phase and the most interaction with the mobile phase escaped the column first.HPLC is capable of separating and identifying chemicals in any material that can be dissolved in a liquid at trace quantities as low as parts per trillion.Triplicate analysis were performed on all samples under the completely randomized design.A mean and standard deviation were calculated for the composition analysis.While the difference between the mean values were determined by using LSD (least significant difference) at 5%.

Comparison of ethanol and hexane after air drying:
Table 1 depicts the concentration of different biochemical compounds extracted as a result of air drying method by using ethanol hexane as a solvent.10 µl sample was subjected for HPLC analysis for 30 min run time, with column temperature 50 ℃ with flow 1.0ml/min.Both solvents used in the study resulted in significant difference in the chemical composition of stevia leaves obtained by both air drying method.By using ethanol solvent highest concentration of Rebaudioside D (1127.47 m/z) was obtained followed by Rebaudioside E (965.42 m/z) at par with Rebaudioside A. While the lowest concentration was found in steviol bioside (317.07 m/z).By using hexane solvent, Rebaudioside D was found maximum (1120.47m/z) while the lowest content was observed in Steviol bioside (315.1104m/z).

Fig. 1 :
Fig. 1: Drying and grinding process of stevia leaves Working of Soxhlet Apparatus: Soxhlet apparatus was used for the extraction procedure.Two extraction solvents i.e., ethanol and hexane were subjected into the Soxhlet apparatus each time for oven and air-dried leaves.10g of powdered leaves were placed in a thimble and sunk in the condensate solvent from the boiling container in an extraction chamber.The solvent reached its maximum level during heating i.e., 80℃ in the thimble during heating, and then trilled into the boiling flask via the syphon duct, extracting the bioactive components into the solvent reservoir.The menstruum i.e., solvents (ethanol and hexane) were present in a flask with a circular bottom.Through the side tube, the vapours reached the condenser, where they condensed and dripped into the extractor.It percolated through the medication, dissolving the soluble components before falling into the flask.The entire extract drained out into the flask through the syphon tube when the level of menstruum in the extractor climbed over the syphon mark.The procedure was repeated for a total of 4-5 cycles until the

Figure 4 :
Figure 4: Comparison of concentration by using two different solventsComparison of ethanol and hexane after oven drying:The results regarding the impact of oven drying method by using ethanol and hexane as a solvent on the Composition of stevia leaves are shown in table 2. 10 µl sample was subjected for HPLC analysis for 30 min run time, with column temperature

Figure. 5 :
Figure.5: Comparison of concentration by using two different solventsComparison of ethanol and hexane for fresh leaves: 10 µl sample was subjected for HPLC analysis for 30 min run time, with column temperature 50 ℃ with flow 1.0ml/min.Very slight difference in concentrations were observed by using ethanol and hexane.The proximate chemical composition of fresh Stevia rebaudiana leaf extracted by using ethanol and hexane solvent is shown in Table3.The results

Table 1 :
Concentration of compounds obtained by using ethanol and hexane as extraction solvent after air drying method

Table 2 :
Concentration of compounds obtained by using ethanol and hexane as extraction solvent after oven drying method

Table 3 :
Concentration of compounds obtained by using ethanol and hexane as extraction solvent for fresh leaves Figure.6: Comparison of concentration by using two different solvents