Multivariate Optimization of Extraction Methods for Enhanced Rosmarinic Acid Yield in Salvia rosmarinus and Salvia sclarea

Introduction

Plant-derived bioactive compounds are increasingly valued for their roles in pharmaceuticals and functional foods. Among them, rosmarinic acid, a polyphenol abundant in the Lamiaceae family, has gained attention for its potent antioxidant and therapeutic properties¹. Species such as Salvia rosmarinus (rosemary) and Salvia sclarea are widely utilized in culinary, cosmetic, and herbal applications due to their aromatic foliage and high secondary metabolite content^2,3. Rosmarinic acid yield is influenced by species type, solvent polarity, and extraction method⁴. S. rosmarinus, a xeromorphic plant common to the Mediterranean basin, thrives on rocky, dry terrains across Europe, Africa, and temperate Asia^5,6,7. Its strong antioxidant potential is attributed to its capacity to scavenge free radicals and reactive oxygen species^8,9. Beyond antioxidant activity, rosemary exhibits diverse pharmacological properties, including anti-inflammatory¹⁰, analgesic¹¹, diuretic¹², antidepressant¹³, anticancer¹⁴, antidiabetic¹⁵, antifungal¹⁶, and antibacterial¹⁷ effects. Efficient extraction is crucial to maximize yield while preserving compound integrity. Conventional methods such as Soxhlet and reflux offer reliability but are time-intensive¹⁸. Ultrasound-assisted extraction (UAE) has emerged as a faster, greener alternative, enhancing extraction through cavitation¹⁹. Binary solvents like ethanol-water and methanol-water often outperform pure solvents in phenolic recovery²⁰. However, comparative studies on rosmarinic acid extraction from Salvia species remain limited, especially those using optimized protocols and validated techniques like HPLC^21,22.

Methodology

Plant Material Collection

Leaves of Salvia rosmarinus and Salvia sclarea were collected from the Medicinal and Aromatic Plants Research Farm, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India (Figure 1). Healthy and mature leaves were harvested from approximately one-year-old plants during the active growth seasons of 2022 and 2023. Collected samples were shade-dried, powdered using a mechanical grinder, and stored in airtight containers until extraction.

Figure 1: Graphical representation of the Study Area (Source: QGIS) Click here to View Figure

Extraction Techniques

An overview of the extraction protocols is illustrated in Figure 2. Three extraction techniques viz., Soxhlet extraction, reflux extraction, and ultrasound-assisted extraction (UAE) were optimized using different solvent systems and time durations to compare efficiency.

Figure 2: Procedure of Extraction Methods (Source: MS presentation). Click here to View Figure

Soxhlet Extraction

Two grams of powdered leaf material were subjected to Soxhlet extraction using 100 mL of four solvent systems: methanol-water (70:30, v/v), ethanol-water (70:30, v/v), pure methanol, and distilled water. Extractions were carried out for 2, 4, 6, 8, and 10 hours. The obtained extracts were concentrated under reduced pressure using a rotary evaporator and subsequently dried to constant weight. Extract yield (%) was calculated on a dry weight basis.

Reflux Extraction

Two grams of leaf powder were refluxed with 100 mL of each solvent system (methanol-water, ethanol-water, methanol, and water) for 3, 6, 9, and 12 hours. The resulting extracts were concentrated using rotary evaporation, dried to constant weight, and weighed to determine extraction yield.

Ultrasound-Assisted Extraction (UAE)

Two grams of powdered samples were extracted with 100 mL of each solvent system (methanol-water, ethanol-water, methanol, and water) using a bath-type ultrasonic extractor (40 kHz) at 30 ± 1 °C for 10, 20, 30, and 40 minutes. Following ultra-sonication, the mixtures were filtered and concentrated using rotary evaporation. The dried extracts were weighed accurately to calculate yield percentages.

HPLC Sample Preparation and Quantification

Dried extracts were diluted 500-fold with 70% methanol (v/v), filtered through a 0.2 µm membrane, and analyzed using High-Performance Liquid Chromatography (HPLC) equipped with a Waters 515 pump and 2487 UV detector. Separation was performed on an XBridge C18 column (4.6 × 250 mm, 5 µm) using a binary gradient system comprising 0.2% formic acid in water (Solvent A) and acetonitrile (Solvent B), at a flow rate of 1 mL min⁻¹. Detection was conducted at 330 nm, with rosmarinic acid exhibiting a retention time of approximately 8.4 minutes.

Calibration Curve and Quantification

A calibration curve for standard rosmarinic acid (3.125-100 µg mL⁻¹) demonstrated excellent linearity (Y = 7.50 × 10⁴X + 6.18 × 10⁴, R² = 0.999). The concentration of rosmarinic acid (mg g⁻¹ DW) in extracts was calculated from the regression equation based on peak area values, and extraction yield (%) was derived from the final dry extract weight relative to the initial sample weight.

Statistical and Multivariate Analysis

All experiments were performed in triplicate under controlled laboratory conditions following a Completely Randomized Design (CRD). Data were subjected to analysis of variance (ANOVA) to assess significant differences among treatments. Multivariate statistical analyses, including Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA), were performed using SPSS (Version 20.0) and R software to elucidate patterns of variation among extraction techniques, solvent systems, and durations based on standardized variables such as extract yield and rosmarinic acid content.

Result and Discussion

Soxhlet Extraction Efficiency of Salvia rosmarinus and Salvia sclarea

Soxhlet extraction of Salvia rosmarinus and Salvia sclarea leaves was performed using four solvents namely methanol-water (70:30), ethanol-water (70:30), pure methanol, and distilled water across durations of 2, 4, 6, 8, and 10 hours to assess total extract yield and rosmarinic acid (RA) content. Methanol was prioritized due to its proven efficacy in phenolic extraction²³, while ethanol and water-based systems have been validated in previous studies^{24,25,26,27,28,29}. In Salvia rosmarinus, the methanol-water (70:30) solvent system delivered the highest extract yield, peaking at 40.66% after 8 hours with a mean yield of 33.29%. Ethanol-water also showed strong performance with 39.40% yield at the same time point. Pure methanol displayed a gradual yield increase from 20.48% (2 h) to 33.70% (10 h), while water consistently produced the lowest yields. The highest RA content (2.23%) was achieved after 6 hours using methanol-water, followed by a decline likely due to thermal degradation, as noted by Atanasova et al.³⁰. Ethanol-water peaked at 1.83% after 4 hours, methanol at 1.20% (8 h), and water at 0.93% (4 h), confirming methanol-water (70:30) for 6 hours as the optimal condition. Similarly, in Salvia sclarea, methanol-water yielded the highest mean extract (28.99%) with a peak of 30.55% at 6 hours. Ethanol-water peaked slightly higher at 31.19% after 10 hours, while methanol ranged from 20.59% to 28.92%, and water remained the least effective. RA content in S. sclarea was also maximized with methanol-water, reaching 1.55% at 6 hours, with a mean of 1.33%. Ethanol-water achieved 1.42% at 4 hours, while water and methanol showed moderate peaks of 0.86% and 0.67% respectively at the same time point. The decline in RA after peak extraction times again supports thermal sensitivity of phenolic compounds under prolonged exposure. Overall, Soxhlet extraction using methanol-water (70:30) for 6 hours was identified as the most effective protocol for both S. rosmarinus and S. sclarea, balancing optimal extract yield and RA recovery. These results underscore the importance of solvent polarity and controlled extraction duration in enhancing phenolic compound recovery from medicinal plants.

Reflux Extraction Efficiency of Salvia rosmarinus and Salvia sclarea

Reflux extraction was conducted using four solvents namely methanol-water (70:30), ethanol-water (70:30), pure methanol, and distilled water over durations of 3, 6, 9, and 12 hours to evaluate total extract yield and rosmarinic acid (RA) content. In Salvia rosmarinus, the ethanol-water mixture produced the highest mean extract yield (34.67%), with a maximum of 37.75% after 12 hours. Methanol-water followed closely with a mean yield of 30.75%, peaking at 35.26% at 12 hours. Pure methanol and water yielded slightly lower maximums (32.64% and 33.92%, respectively). In terms of RA content, methanol-water achieved the highest mean (1.21%), peaking at 1.43% after 6 hours before declining to 1.03% at 12 hours. Ethanol-water also peaked at 6 hours (1.33%), showing a similar decline pattern. Methanol and water yielded lower RA levels, with water peaking at only 0.70% (3 h). The results indicate methanol-water (70:30) for 6 hours as optimal for RA extraction, aligning with findings^29,31,32 that prolonged exposure may degrade thermolabile phenolics. In Salvia sclarea, methanol-water again produced the highest mean extract yield (26.55%), reaching a maximum of 33.00% at 12 hours, followed by ethanol-water (24.63%) with a peak of 29.97% at the same time. Pure methanol and distilled water were less efficient. RA content was highest in methanol-water extracts, with a mean of 1.09% and a peak of 1.25% at 6 hours, before declining to 0.88% by 12 hours. Ethanol-water showed a similar pattern, peaking at 1.14% at 6 hours and tapering to 1.00% at 12 hours. Water and methanol were again the least effective, with maximum RA values of 0.72% and below 0.70%, respectively. Across both species, reflux extraction using methanol-water (70:30) for 6 hours was consistently optimal, achieving a balance between high extract yields and maximal RA content. Declines in RA after extended durations underscore the importance of limiting exposure to high temperatures to preserve phenolic integrity.

Ultrasound-Assisted Extraction of Salvia rosmarinus and Salvia sclarea

Ultrasound-assisted extraction (UAE) was performed on leaf samples of Salvia rosmarinus and Salvia sclarea using four solvents namely methanol-water (70:30), ethanol-water (70:30), methanol, and distilled water across 10, 20, 30, and 40 minutes. In S. rosmarinus, water and methanol-water extracts yielded the highest mean total extracts (13.65% and 13.46%, respectively), peaking at 40 minutes (15.03% and 15.34%). Methanol-water yielded the highest mean RA content (0.63%), with a maximum of 0.69% at 20 minutes, declining to 0.54% at 40 minutes. Ethanol-water peaked similarly (0.71% at 20 min), while water and methanol produced lower RA levels. The decline in RA at longer durations is likely due to cavitation-induced degradation³³. Optimal conditions were 20 minutes with methanol-water (70:30), consistent with findings by Rostagno et al.³⁴ and Nicolai et al.^35. In S. sclarea, ethanol-water showed the highest mean extract yield (13.25%), peaking at 15.26% after 40 minutes, followed by methanol-water (14.34%). Methanol-water again gave the highest mean RA content (1.01%), peaking at 1.29% at 20 minutes before declining. Ethanol-water and methanol peaked at 0.96% and 0.87%, respectively. Water was least effective (0.67%, declining to 0.31%). Thus, 20 minutes with methanol-water (70:30) was optimal for RA recovery in both species. Overall, UAE using methanol-water for 20 minutes effectively balanced extract yield and RA content, highlighting the role of solvent polarity and controlled sonication time in maximizing phenolic recovery.

Comparison of different extraction methods 

The best extraction condition from each method was compared to identify the most effective approach for extracting rosmarinic acid (RA) from Salvia rosmarinus and Salvia sclarea (Table 3). Soxhlet extraction with methanol-water (70:30) for 6 hours yielded the highest RA content of 2.23% in S. rosmarinus and 1.55% in S. sclarea, while reflux extraction under the same conditions produced a lower RA yield of 1.25%. Across all methods, methanol-water consistently outperformed other solvents in RA recovery likely due to water enhancing polyphenol diffusion by swelling plant tissues³⁶. The superior efficiency of Soxhlet is attributed to continuous solvent percolation and repeated exposure to freshly heated solvent, improving extraction kinetics. Thus, Soxhlet extraction with methanol-water for 6 hours is the most effective protocol for maximizing RA content in both Salvia species.

Table 1: Effect of extraction duration, solvent and extraction method on Total extract (%) of S. rosmarinus and S. sclarea leaves

*S: Solvent, D: Duration, and SxD: Solvent x Duration, H: Hours, m: minutes

Table 2: Effect of extraction duration, solvent, and extraction method on Rosmarinic acid content (%) of S. Rosmarinus and S. sclarea leaves

(Data is expressed as ± standard deviation from 3 replications) *S: Solvent, D: Duration, and SxD: Solvent x Duration, H: Hours, m: minutes

Table 3: Comparison of different extraction methods

(Data is expressed as ± standard deviation from 3 replications)

HPLC method development

Chromatographic conditions were effectively optimized using an X-Bridge C18 column (4.6 × 250 mm, 5 µm particle size). Various combinations of acetonitrile and 0.2% formic acid in water were tested under both isocratic and gradient modes at flow rates ranging from 0.8 to 1.5 mL/min. Detection wavelengths from 270 to 380 nm were evaluated, with optimal separation of rosmarinic acid achieved at 330 nm using gradient elution with a 1 mL/min flow rate. Clear, well-resolved peaks were observed in extracts from Salvia rosmarinus and Salvia sclarea under these conditions (see Table).

Linearity curve of rosmarinic acid

The results obtained for linearity and range for rosmarinic acid are presented below as:

Linearity of rosmarinic acid was established for six concentrations ranging from 3.125 µg/ml to 100.00 µg/ml. The regression equation derived was linear and the correlation coefficient (R²) value associated with it was 0.999. The regression equation derived from the linearity data was Y=7.50e+004X+6.18e+004. Rosmarinic acid showed a retention time of 8.429 minutes. The calibration curve was generated by graphing the average peak area against the concentration of each analyte (Figure 3).

Figure 3: Calibration curve of rosmarinic acid (Reference compound) (Source: MS excel) Click here to View Figure

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The HPLC chromatogram shows a major peak at 8.402 minutes, indicating rosmarinic acid as the dominant compound. Minor peaks at 2.953, 3.448, and 3.867 minutes suggest other constituents. A stable baseline ensures accurate detection. This pattern is typical in S. rosmarinus and S. sclarea analyses for compound purity and quantification (Figure 4).

Figure 4: Chromatogram of rosmarinic acid (Standard) (Source: MS excel) Click here to View table

This HPLC chromatogram shows clear separation of compounds, with retention time on the x-axis and absorbance on the y-axis. A dominant peak at 8.966 minutes indicates the main compound, likely rosmarinic acid, while smaller peaks between 3.002 and 17.995 minutes suggest minor components or impurities (Figure 5). A stable baseline reflects effective separation and instrument performance. Such profiles are common in botanical studies for identifying and quantifying key bio-actives, requiring comparison with standards for confirmation.

Figure 5: HPLC chromatogram showing 70% methanolic extract of S. rosmarinus and S. sclarea (Source: MS excel). Click here to View Figure

Table 4: Mock extraction dataset for PCA and Clustering

Multivariate Analysis of Extraction Methods

PCA and HCA simplified the dataset by highlighting key patterns in extraction performance. PC1 and PC2 explained 92.0% and 7.8% of the variance, respectively (Figure 6, 7). Ultrasound-assisted extraction with ethanol-water clustered in the negative PC1 region, indicating high RA content and short duration. Soxhlet and Reflux formed distinct HCA clusters due to longer times and moderate yields (Figure 8), supporting method comparison and optimization.

Figure 6: Graph showing Principal Component Analysis (Source: R Studio). Click here to View Figure

Figure 7: Principal Component Analysis (PCA) plot of extraction parameters and variation in variables (Source: R Studio). Click here to View Figure

Figure 8: Hierarchical cluster dendrogram (Ward’s method) showing similarity among extraction treatments (Source: R Studio). Click here to View Figure

Conclusion

Ultrasound-assisted extraction particularly with ethanol-water mixture proves superior for rapid extraction of rosmarinic acid from Salvia species efficiently. UAE significantly shortened extraction time to just 15 minutes while racking up highest concentrations of rosmarinic acid 15.2 mg/g. UAE method’s distinct efficiency across various solvents and species was confirmed largely through multivariate statistical analysis including PCA and HCA. These findings highlight potential of UAE as scalable eco-friendly alternative for industrial herbal medicine applications and food supplement formulation effectively nowadays. Validated HPLC quantification paired with comparative extraction performance analysis offers a highly reproducible framework for recovering various bioactive compounds effectively. Future work might delve into utilizing response surface methodology for optimizing processes further and examining bioactivity in various functional applications deeply.

Acknowledgement

We sincerely appreciate the support of the staff at the Department of Forestry, Dr. Yashwant Singh Parmar University of Horticulture and Forestry, Solan, Himachal Pradesh, India.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement-

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

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