Isolation and Characterization of Saponins from Asparagus racemosus: A HPTLC and LC-MS Study

INTRODUCTION

The Indian System of Medicine (ISM) relies heavily on plant-based medicine and has been practiced for thousands of years. It encompasses several traditional systems including Ayurveda, Unani, Siddha, Homeopathy, Yoga and Naturopathy. Ayurveda is one of the oldest and most purified system of medicine. There are some prominent ayurvedic herb which has unique property and health benefits. One of the most important plants in this tradition is Shatavari (Asparagus racemosus) which is widely used especially in Ayurveda (Selvaraj et al., 2019). In Ayurvedic medicine, Shatavari is classified as a Rasayana, a rejuvenating tonic that promotes longevity and vitality (Akhtar et al., 2024).

Shatavari (Asparagus racemosus) is a therapeutic herb which belongs to the family Liliaceae (Arya et al., 2018). It is an important monocot that grows in tropical as well as subtropical regions of Asia, Sri Lanka and India (Prabhakaran et al., 2015). It is highly valued in Ayurveda for its medicinal properties, particularly in supporting female reproductive health. Its various medicinal properties are reported in various Pharmacopoeias (Kumar et al., 2020). The roots of the plant are used in different formulations (Raval et al., 2012). The pharmacological properties depend on the phyto-active constituent present in the plant (Raval et al., 2012; Prabhakaran et al., 2015). It has been used as a medicine in lactating mothers, bleeding through urethra, epilepsy and cough condition. It is rich in phytochemicals like steroidal saponin (Shatavarin-1 to 6) (Selvaraj et al., 2019), carboxylic acid, isoflavones, polycyclic hydrocarbons, furan compound, flavonoids, carbohydrates, a trace of minerals, sterols, kaempferol and some miscellaneous constituents (Pandiyan et al., 2022). It is valued for its diabetes, jaundice and immunomodulatory properties (Akhtar et al., 2024). A major objective of the study was to assess the Hydroalcoholic root extract of Asparagus racemosus by phytochemical screening using different methods such as HPTLC and LCMS (Vijayalakshmi et al., 2015).

MATERIALS AND METHODS

Collection and authentication of Plant material

The fresh plant (Asparagus racemosus) was collected from herbal garden from Lucknow, Uttar Pradesh, India in the month of July 2023. A Herbarium Voucher Specimen of selected plant was prepared and authenticated by Dr. Sunita Garg, former head of RHMD at CSIR-NIScPR, New Delhi (NIScPR/RHMD/Consult/2023/4577-89).

Pharmacognostical studies

The technique of standardizing the crude drugs by several parameters. The parameters include morphology (colour, shape, size, fracture, odour, and taste) (Ahmad et al., 2017; Brahma et al., 2022) and microscopy (Transverse section of the drug) (Prabhakaran et al., 2015; Unadhkat et al., 2022; Singh et al., 2017).

Physicochemical Parameters

This parameter includes Ash Value (Goel et al., 2022; Mandal et al., 2017), Extractive Value Prabhakaran et al., 2015; Siraj et al., 2020; Loss on Drying Tripathi et al., 2015; Goel et al., 2022).

Phytochemical Parameter

This Parameter is used to assess primary and secondary metabolites (Hassan et al., 2022; Irawan et al., 2018). A plant extract is prepared by specific extraction method (Ajuru et al., 2017). The crude extracts is used to identify the presence of different types of components (Karunarathne et al., 2019; Ahmad et al., 2017).

Preparation of Plant Extract

The roots were washed with distilled water, shade-dried, and powdered (Sharma et al., 2017). The powdered drug was extracted with a water-ethanol mixture using a Soxhlet apparatus until a clear siphon liquid was obtained (Basoudan et al., 2024; Ahmad et al., 2017). The filtrate was concentrated, dried, weighed, and subsequently used for phytochemical screening (Prabhakaran et al., 2015; Alqethami et al., 2021).

Saponin Estimation Procedure

2 g of extract in 50 mL of Petroleum ether was heated in a water bath at 40ºC for 5 min with intermittent shaking. The petroleum ether layer was filtered, and the process was repeated three additional times. The petroleum ether was discarded and the marc was preserved. The obtained marc was extracted with methanol (60 mL) under mild heating, repeating the process four times. The methanolic layer was filtered and transferred into another beaker.150 mL of dry acetone was added to the methanolic layer which was combined and concentrated. Saponins formed the precipitate. Filter the saponin and calculate its percentage (Kumaran et al., 2015, Ma et al., 2022).

Extraction of Saponin

Isolation of saponin was performed by liquid-liquid extraction. The SRF were isolated from extract. Procedure: The SRF were segmented into Aqueous phase from the organic phase using LLE (Chua et al., 2019). 5 g dried extract of Asparagus racemosus was weighed, dissolved in 60 mL of hot water and fully extracted with 20 mL ethyl acetate. The organic layer was abstracted and 20 mL of fresh ethyl acetate was added and extracted again. The extraction was redone for six times. All the organic layers as well as aqueous layer were collaborated and evaporated up to dryness and dried in an oven at 65ºC (Segaran et al., 2020).

Chromatographic Analysis

HPTLC: It is a modern form of TLC that provides accuracy and reproducibility. HPTLC is an advance technology based on the principle of TLC which requires less and provide better resolution. It is widely used for the qualitative as well as quantitative analysis of complex mixtures in food, herbal products, forensics and pharmaceuticals (Vyas et al., 2023). The study provides TLC fingerprint profiles and estimation study of biomarkers (Mehta et al., 2021). The major advantage of HPTLC is that it can examine samples together using small amount of mobile phase (Champati et al., 2022). Procedure: The HPTLC was performed on a 10×10 cm pre-activated HPTLC silica gel 60 F254 plate (Hazra et al., 2019). Samples are applied on the plate with an automatic TLC applicator (Mohan et al., 2024). The plates were developed using ethyl acetate, ethanol, and water at a ratio of 8:2:1.2 as a solvent system. The scanning was done on CAMAG scanner II (Bhargava et al., 2021; Abraham et al., 2020). The chromatographic finger print was developed for detection of Phytoconstituents and R_f values were calculated (Prabhakaran et al., 2015).

LCMS Analysis

LCMS is an analytical tool for the identification of wide variety of non-volatile or volatile organic compounds. (Swati et al., 2023; Vijaylakshmi et al., 2024). It is highly sophisticated instrument used for the detection of low and high molecular weight compounds (Patil et al., 2014). The Phyto-active components present in the SRF of the roots of Asparagus racemosus were identified by LCMS method. The analysis was performed using TOF/Q-TOF Mass Spectrometer system which is equipped with a Dual AJS ESI source (Sawada et al., 2013). The gradient elution was operated for 30 min (stop time) at a flow rate of 100.000 mL/min². The full scan mass spectra were obtained within the range of m/z, amu 100-3000 at 1.00 scan rate (Irawan et al., 2018). The valve switch time was 1 which was enabled with 10 µL injection volume (Marr et al., 2021).

The mobile phase consists of Channel A and Channel B. Channel A contains water with 0.1% formic acid, while Channel B comprises Acetonitrile (ACN) with 10% water and formic acid. Both solvents were employed in the analysis to ensure effective separation and ionization of analytes.

Ethical Statement

The present study involved phytochemical, isolation and analytical characterization of saponins from Asparagus racemosus roots using HPTLC and LC-MS techniques. No human participants or animals were used in the above study. All procedures were performed in accordance with standard laboratory practices and institutional guidelines.

Statistical Analysis

All experiments were performed in triplicate, and the results were expressed as mean ± SD. HPTLC densitometric data were analyzed using appropriate chromatographic software. LC-MS data were evaluated based on molecular ion intensity and fragmentation patterns for qualitative confirmation.

RESULTS

Pharmacogenetic studies

Morphological Characters

The morphological characters were examined by organoleptic method using fresh roots of Asparagus racemosus Willd (Table 1).

Microscopical Characters

The T.S. of Asparagus racemosus (Shatavari) (Figure 1) root shows an epidermis with unicellular root hairs, followed by an outer cortex of 6-7 layers of thick-walled polygonal cells and an inner cortex of 16-22 layers of thin-walled oval to polygonal cells. A single-layered thin-walled endodermis surrounds the centrally located, radially arranged vascular bundles (xylem and phloem) and a large pith.

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Physico-chemical Parameters

All the Physico-chemical parameters and their values are mentioned below (Table 2).

Preliminary Phytochemical Screening

The Phytochemical screening of the hydro-alcoholic extract of the roots of Asparagus racemosus (Table 3).

Saponin Estimation

The percentage of Saponin content:

= 0.40/2×100

= 20%

High-Performance Thin Layer Chromatography

The HPTLC profile of the samples revealed the presence of Seventeen peaks. The chromatogram showed the presence of Shatavarin IV in both the standard and sample tracks, as shown by the violet zones observed during daylight mode (Figure 2). The mobile phase consisted of a mixture of ethyl acetate, ethanol, and water at a ratio of 8:2:1.2 provided satisfactory separation with a distinct R_f value. The bands that were separated based on their chemical properties were seen in the range of R_f 0.01 to 0.96 in the extract. Among these bands, the one with the R_f value of 0.60 indicated the existence of a specific compound, Shatavarin IV as shown in Figures 3.

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Figure 3 (a) shows a single, sharp peak at a R_f value of approximately 0.60 for standard Shatavarin-IV which confirms its purity, specificity, and reproducibility under the selected chromatographic conditions.

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Figure (b) reveals multiple well-resolved peaks across the R_f range of 0.05-0.90 in the saponin-rich extract, reflecting the complex saponin composition of A. racemosus roots. Notably, a prominent peak appears at a R_f value close to that of the Shatavarin-IV standard (~0.60), which indicates the presence of Shatavarin-IV.

Liquid Chromatogram Mass Spectroscopy

Data Interpretation and Identification of Compounds

The chromatograms and mass spectra were processed by MZ mine 2.5 software by Agilent. The continuous data which was acquired in positive mode was processed. KEGG was the online data base search method which is used to identify the compounds. Other data base such as MoNA and PubChem were used for search of compounds. The fragments of accurate masses were obtained in the same ionization mode, which is matched with the database by setting a tolerance of 5 ppm. The identified metabolites were then exported in a sheet containing the active molecule name, molecular mass, retention time, peak area, peak height. and m/z ratio. After database matching the complex and adduct search were performed, followed by the peak list which is directly exported from MZ mine.

LCMS Reports

The LC-MS Chromatogram obtained from the Saponin rich fractions of Hydroalcoholic extracts of roots of Asparagus racemosus Willd was identified and mass spectrum of Shatavarin-IV was shown in Figure 4. It was noticed that different peaks were obtained at different retention times. The retention time of the major compound was mentioned in Table 4.

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DISCUSSION

The study successfully established a systematic approach for the isolation and characterization of saponins from A. racemosus roots. The Morphological and physicochemical evaluations confirmed the authenticity and quality of the plant material, while HPTLC and LC-MS analyses enabled reliable identification of saponins, particularly Shatavarin-IV.

CONCLUSION

The present study was assigned to evaluate different Pharmacognostic parameters and physicochemical properties of crude drug. The phytochemical screening of plant metabolites were performed which helped in the Authentication of A. racemosus Willd. Various chemical components were identified via HPTLC and LCMS methods. The HPLC and LC-MS analysis reported details of the separation and identification of the Compounds within a sample. The HPLC data signifies Retention time and the LC-MS data signifies mass-to-charge ratio (MS data). The results of the study revealed the presence of saponin compounds like Sarasapogenin, Asparanin B, and Kaempferol-3-o-pentoside.

ACKNOWLEDGEMENT

The authors are grateful to the Teerthanker Mahaveer College of Pharmacy for the opportunity.

ABBREVIATIONS

T.S

Transverse section

SRF

Saponin rich fractions

HPTLC

High-Performance Thin Layer Chromatography

LC-MS

Liquid Chromatography Mass Spectrometry

KEGG

Kyoto Encyclopedia of Genes and Genomes

ISM

Indian System of Medicine

LOD

Loss on drying

LLE

Liquid-liquid extraction

ESI

Electro spray ionization

FA

Formic Acid

ACN

Acetonitrile

R_f

Retention factor

m/z

Mass-to-charge ratio

TOF/Q-TOF

Time-of-Flight/Quadrupole Time-of-Flight

amu

Atomic Mass Unit

TLC

Thin-Layer Chromatography

SD

Standard deviation

AU

Absorbance units

CSIR-NIScPR

Council of Scientific and Industrial Research - National Institute of Science Communication and Policy Research

RHMD

Raw Herbarium Materials and Dispensary

MoNA

Mass Bank of North America.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

FUNDING

This work did not receive funding from any sources.

AUTHORS CONTRIBUTION

The authors collectively contributed to the conceptualization, experimental design, data acquisition, analysis, and interpretation of the study.

SUMMARY

Saponins were isolated and characterization was done using HPTLC and LC-MS techniques. HPTLC analysis provided a characteristic fingerprint profile for saponin-rich fractions, enabling qualitative assessment and standardization. Further LC-MS analysis confirmed the presence of major steroidal saponins, particularly Shatavarin derivatives, based on molecular ion peaks and fragmentation patterns. The integrated analytical approach ensures reliable identification, purity assessment, and quality control of A. racemosus saponins.