Toxicological evaluation of Asparagus racemosus – based low-alcohol nutraceutical beverage: acute and subacute safety assessment in mice†

Divya Choudhary*^a, S. N. Naik^a, Vidushi Tyagi^bc, Anirban Pal^bc and P. Hariprasad^d
aSupercritical Extraction Laboratory, Centre, for Rural Development and Technology, Indian Institute of Technology Delhi, New Delhi-110016, India. E-mail: divyabt14@gmail.com; naiksn@gmail.com; Tel: +91 7303196165
^bBioprospection and Product Development Division, Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow 226015, India. E-mail: itishatyagi2@gmail.com; drapaul@gmail.com
^cAcademy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India
^dEnvironmental Biotechnology Laboratory, Centre for Rural Development and Technology, Indian Institute of Technology Delhi, New Delhi 110016, India. E-mail: phimprovement.iitd@gmail.com

First published on 1st July 2025

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Sustainability spotlight The developed Shatavari-based low-alcoholic Nutra-beverage (SLANB) promotes sustainable health solutions by utilizing Asparagus racemosus, a medicinal plant known for its antioxidant, anti-inflammatory, and adaptogenic properties. Fermentation significantly enhanced the bioavailability and functional potential of its key bioactive compounds. LC-MS analysis confirmed the presence of 10 distinct bioactive metabolites essential for health benefits, while GC-MS profiling revealed natural aroma and ethanol compounds after 28 days of fermentation. Toxicological evaluations demonstrated proven safety: an acute toxicity study using a 1.5 ml/100 g BW dose in mice showed no mortality or adverse effects over 14 days, and a 28 days subacute study across three dose levels confirmed no physiological toxicity. This eco-friendly innovation supports the development of a safe, functional nutraceutical beverage and validates SLANB as a non-toxic, plant-based formulation with potential applications in medicinal and wellness-oriented food systems.

1 Introduction

Indian traditional herbs have increasingly garnered international interest due to their rich ethnopharmacological heritage and widespread use in traditional medicine systems such as Ayurveda, Siddha, and Unani. This global recognition is largely attributed to their diverse therapeutic potentials and centuries-long historical application.¹ One such herb is Shatavari (Asparagus racemosus Willd), a climbing perennial plant belonging to the Asparagaceae family. It is widely distributed across tropical and subtropical regions, including Asia, Africa, and Australia, with its origins traced to the Himalayan regions of Nepal and India.² In Indian traditional medicine, especially in the northern and southern states, the air-dried roots of Shatavari have been extensively utilized as a general health tonic and for the management of various reproductive and gastrointestinal disorders.

Shatavari is known for its inclusion in nutrient-enriched functional beverages due to its wealth of secondary metabolites, including steroidal saponins, flavonoids, polyphenols, and alkaloids, which are associated with numerous health benefits such as antioxidant, anti-inflammatory, adaptogenic, and immunomodulatory effects.^3,4 The conversion of Shatavari roots into shelf-stable and value-added formulations not only prolongs their usability but also enhances the economic returns for producers and industries focused on functional and nutraceutical food products. One of the sustainable and efficient approaches to achieving this transformation is bioprocessing through fermentation, which has been increasingly recognized for its ability to improve nutritional quality, safety, and organoleptic properties of food products.⁵ Fermentation facilitates the breakdown of complex phytochemicals into bioavailable forms, potentially enhancing the pharmacological efficacy of herbal components.

Numerous studies have highlighted the pharmacological significance of Shatavari, attributing to it a broad spectrum of therapeutic properties including aphrodisiac, galactagogue, diuretic, antispasmodic, and adaptogenic effects.⁶ These therapeutic potentials have led to its inclusion in more than 64 traditional Ayurvedic formulations such as Shatavari Kalpa, Phalaghrita, and Vishnu Taila, which are employed for the management of reproductive, digestive, and nervous system disorders in both men and women.

Artificial Neural Network (ANN) coupled with Genetic Algorithm (GA) was successfully utilized to model and optimize fermentation parameters for the development of a low-alcohol Shatavari-based Nutra-beverage. The ANN-GA model effectively predicted the fermentation outcomes, including ethanol yield and antioxidant activity, thus providing a computationally robust tool for process optimization. Specifically, the model predicted and experimental outcomes aligned closely, with ethanol yield reaching 3.21 g L⁻¹ and antioxidant activity recorded at 421.47 μg L⁻¹, indicating enhanced functional properties during fermentation.⁷ This demonstrates the potential of ANN-GA in improving biotechnological applications in the food and nutraceutical sectors.⁸

Despite the promising pharmacological attributes of Shatavari, safety concerns associated with herbal and alternative medicines have gained attention, particularly with growing evidence of toxicity and adverse effects in some Ayurvedic formulations. Such safety risks are often linked to contamination, adulteration, or incorrect dosage, emphasizing the importance of rigorous toxicological evaluation.^9,10 Currently, limited data exist regarding the acute and sub-acute toxicity of Shatavari-based nutraceutical beverages, thereby necessitating systematic in vivo studies to evaluate their safety profiles before commercialization. Ensuring the toxicological safety of these preparations is critical for consumer health and regulatory compliance.

To address these concerns, our study includes acute and sub-acute toxicity assessments in animal models, alongside sensory evaluation protocols to determine consumer acceptance of the developed product.

2 Material and methods

2.1 Chemicals used

Biochemical Diagnostic kits such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), total and high-density lipoprotein (HDL) cholesterol, triglycerides and creatinine were obtained from Agappe (India). E-Merck India Ltd supplied other Guaranteed Reagent (GR) chemicals utilized in the experiment.

2.2 Animals

Animal studies were conducted with the approval from CPCSEA, Government of India through the Institutional Animal Ethics Committee under protocol (CIMAP/IAEC/2020–23/23). The study was conducted on Swiss albino mice, weighing 20–25 g. The animals were kept at 25 ± 2 °C with controlled humidity and a 12 hours light/dark cycle. Standard mice feed and water were provided ad libitum.

2.3 Preparation of shatavari-based beverage

Fresh Shatavari roots (Asparagus racemosus) were procured from the CSIR–Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India. The roots were thoroughly washed, chopped, and dried in a hot air oven for five days, then ground into powder. A slurry was prepared by mixing 500 g of root powder with 1 L of distilled water. Enzymatic hydrolysis was carried out by adding α-amylase (25 U g⁻¹) and incubating at high temperature for 1 hour, followed by saccharification using glucoamylase (78 U g⁻¹) at 50 °C for 2 hours with continuous stirring.¹¹

For fermentation, Saccharomyces cerevisiae NCIM 2428 was cultured in YEPD medium and incubated at 150 rpm for 24 hours. The activated inoculum was transferred to sterile Shatavari juice (pH 3.4) in 3-L Erlenmeyer flasks. Sucrose was added to increase sugar content to 190 g L⁻¹ (as glucose), and fermentation parameters such as temperature (28–35 °C), pH (4–6), and inoculum concentration (1–5%, v/v) were optimized according to experimental design.¹²

Fermentation was carried out under sterile conditions in a 3-L BioFlo/CelliGen 115 bioreactor with a working volume of 2 L and agitation set at 150 rpm. Upon completion, the fermented product was centrifuged at 4000 rpm for 10 minutes, and the resulting supernatant was stored at 20 °C in the dark for 90 days.¹³ The preparation process of the Shatavari-based low-alcohol Nutra-beverage is illustrated in Fig. 1.

2.4 Sensory evaluation

2.5 Volatile compounds by GC-MS

The volatile compounds in Nutra-beverage samples were analyzed using GC-MS following a procedure described.¹⁵ In a 20 ml glass vial, 2 g of the sample and 10 ml of the internal standard (4-methyl-2-pentanol at 80.2 g ml⁻¹ in methanol) were combined. Peak detection and integration of peak area were performed using TraceFinder 4.1 (Thermo Scientific) with the total ion chromatogram (TIC). The analysis was conducted using an Agilent 5977B EI/CI MSD with a column oven temperature of 80 °C, injection temperature of 260 °C, split injection mode, linear velocity flow control mode, pressure of 81.9 kPa, total flow of 16.3 ml min⁻¹, and column flow of 1.21 ml min⁻¹. The hold time was 2 min at 80 °C and 18 min at 280 °C.

2.6 Untargeted compounds and high resolution LC-ESI/MS (LC-MS)

The validated sample analysis was confirmed using the LC-ESI/MS (LC-MS) Waters Alliance e2695/HPLC-TQD Mass Spectrometer instrument. This analysis was conducted at the Central Drug Research Institute (CDRI), Lucknow, as detailed.¹⁶ The chromatographic conditions were as follows: Waters Alliance 2695 HPLC pump initial conditions, solvents: Degasser normal stroke volume 130.0. Final flow setting being held at the end of run A% 0.0H₂O, B% 5.0 CAN, C% 0.0 MEOH, D% 95.0 0.1% FA. Flow (ml min⁻¹) 0.600, flow ramp 1.00, stop time (mins) 40.0, column temperature (°C) 30.0, column temperature limit (°C) 5.0, min pressure (Bar) 0.0, max pressure (Bar) 300.0, pre-column volume (μl) 0.00, column position no change pre-column volume 0.00. Waters Alliance 2695 Autosampler initial conditions: needle depth (mm) 0.00, sample temperature (°C) 20.0, sample temperature limit (°C) 5.0, purge loop volumes 0.00, sample run injection parameter, injection volume (μl) – 2.00.

2.7 In vivo acute oral toxicity

Acute oral toxicity of nutraceutical beverage was conducted according to the Organization for Economic Cooperation and Development (OECD) test guideline no. 423.¹⁷ Briefly, 12 mice were taken and divided into two groups comprising 6 mice (3 males and 3 females) in each group. Animals were acclimatized for 7 days prior to experimentation. Group 1 mice were given sterile water and group 2 mice were given Nutraceutical Beverage (single dose at 1.5 ml/100 g of body weight). On 7th day, mice were euthanized and blood was collected from retro orbital plexus for haematological parameters and serum was isolated to study biochemical parameters. Change in body weight, weight of vital organs like liver, spleen, kidney, heart, and lungs were also recorded.¹⁸

2.8 In vivo sub-acute oral toxicity

Sub-acute oral toxicity of Nutra-Beverage was conducted according to the Organization for Economic Co-operation and Development (OECD) test guideline no. 407.¹⁹ Briefly, 24 mice were taken and divided into four groups comprising 6 mice (3 males and 3 females) in each group. Animals were acclimatized for 7 days prior to experimentation. Group 1 mice were given sterile water and mice in group 2, 3 and 4 were given different volumes (at 0.375 ml, 0.75 ml and 1.5 ml/100 g body weight for 28 days) of Nutra-Beverage (7 days a week for 28 days). On day 28th, mice were euthanized by cervical dislocation and blood was collected for haematological parameters and serum was isolated to study biochemical parameters. Change in body weight, weights of vital organs like liver, spleen, kidney, heart, and lungs were also recorded.

2.9 Statistically analysis

The statistical analyses were conducted using analysis of variance (ANOVA) to assess the data. For the in vivo experiment, a two-way ANOVA was employed to evaluate the effects of multiple factors. Windows-based GraphPad Prism 6.04 was used for the analysis.

3 Result and discussion

3.1 Sensory evaluation of shatavari based low alcohol nutra-beverage

Hedonic data analysis shows the frequency with which tasters identified each attribute in the samples during sensory evaluation, resulting in the contingency in Table 1. The section provides data on the sensory acceptability of the developed product among the target group of consumers. Hedonic analysis's tasters' ratings for each fermented product's texture, fragrance, taste, color, flavor, and aftertaste are shown in Table 1.

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The texture, aroma, taste, colour, flavour, and aftertaste of four distinct samples were evaluated, and their ratings are presented in Table 1. The results revealed that Sample 1 had a texture rating of 6, making it moderately likeable, while Sample 2 received a slightly higher rating of 7. Sample 3 achieved the highest texture rating of 8, indicating it was the most preferred among all samples. In contrast, Sample 4 received a lower rating of 4, suggesting it was the least liked.

In terms of aroma, Sample 3 was again the most favoured. Regarding taste, Sample 3 received the highest overall score of 16, making it the most satisfying in terms of flavour. Although Samples 1, 2, and 4 were acceptable, they did not surpass Sample 3 in any category.

The flavour and aftertaste of Sample 3 (14th–21st day of fermentation) were also well-received, though no other sample matched its high ratings. Similarly, in terms of colour, Sample 3 was considered the best overall. Fig. 3 presents a radar chart illustrating the sensory acceptability of the samples from the 0th to the 28th day of fermentation.

3.2 Dynamic of volatile compounds (GC-MS) during fermentation

The GC-MS method enabled the identification of components in the Shatavari-based low-alcohol Nutra-beverage with a confidence level exceeding 72%. These included three alcohols (phenylethyl alcohol, propargyl alcohol, and 1-butanol), one ketone (acetoin), one fatty acid (2-hydroxyoctanoic acid), four carboxylic acids (ethyl acetate, succinic acid, oxalic acid, and acetic acid), two alkanes (pentane and hexane), and one ether (ethyl ether), as presented in Table 2.

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During the fermentation process, the composition of volatile compounds varied over different days. Alcoholic compounds were notably detected on the 7th day of fermentation. Phenylethyl alcohol appeared with a peak area of 0.50 and a retention time of 31.65 minutes, but was not detected on subsequent days. Similarly, propargyl alcohol was detected on the 7th day with a peak area of 0.85 and a retention time of 14.52 minutes. 1-Butanol was observed at a retention time of 12.59 minutes on both the 7th and 21st days, with peak areas of 2.58 and 32.61, respectively. The ketone compound acetoin was identified with a retention time of 12.36 minutes. The corresponding chromatogram peaks are shown in ESI Fig. S1.†

GC-MS analysis revealed that the number and relative abundance of volatile compounds were influenced by storage duration. In the freshly fermented Shatavari beverage (Day 7, D7), 11 volatile components were identified, comprising a total area percentage of 21.25%. During storage at 32 °C, the number of volatiles varied: 12 components on Day 14 (D14), 10 on Day 21 (D21), and 8 on Day 28 (D28). Interestingly, the total area percentage of volatile compounds increased to 20.57%, 111.16%, and 56.28% on D14, D21, and D28, respectively.

These findings suggest that volatile compounds peak around the 14th day of fermentation, contributing to enhanced aroma. After the 21st day, the levels begin to decline, likely due to the release of CO₂, which may reduce the concentration of aroma-active compounds. The fruity and floral aroma in fermented products such as wine is typically associated with esters, which are synthesized through esterification of alcohols with fatty acids, or via alcohol acetyltransferase-mediated reactions involving acetyl-CoA and higher alcohols.²⁰

While the fermented Shatavari beverage contained fewer esters than the fresh beverage, ester content increased after 7–14 days at 32 °C compared to day 0. Alcohols in the beverage are primarily derived from alcoholic fermentation, amino acid metabolism, and the oxidation of linolenic acid degradation products,²¹ all of which contribute desirable aromas.²² The alcohols detected during storage included phenylethyl alcohol, propargyl alcohol, and 1-butanol (Table 2). The highest total alcohol area percentage (32.61%) was observed on the 21st day, while lower values were recorded on the 7th and 14th days, at 3.18% and 0.85%, respectively. The higher alcohol content in stored fermented Shatavari beverages, compared to freshly fermented ones, could be attributed to continued yeast activity at elevated temperatures, which promotes the conversion of residual sugars into alcohol. These alcohols impart fruity, floral, and characteristic alcoholic aromas to the beverage.

Acids and ketones serve as both primary metabolites and precursors to aromatic compounds. Secondary metabolites such as esters and lactones are formed through the interaction of volatiles—including alcohols and fatty acids—during fermentation.²³ Although present in small amounts, these additional volatile compounds may play a significant role in the aroma and flavor profile of the fermented Shatavari beverage.

3.3 Untargeted compounds detected during fermentation using LC-ESI/MS (LC-MS) Waters Alliance e2695/HPLC-TQD mass spectrometer

The Shatavari nutraceutical beverage was utilized for a safety evaluation study. To ensure its quality, reproducible LC-ESI/MS (LC-MS) analysis was conducted using reverse-phase separation with a methanol–water mobile phase (detailed methodology is provided in the Experimental section). Partial untargeted compounds identified in the fermented Shatavari beverage are listed in Table 3. Phenolic compounds such as gallic acid and caffeic acid, which readily react with oxygen, function as antioxidants. In fermented foods, the growth and reproduction of microbial colonies are closely associated with fermentation duration. As fermentation progresses, colony development typically follows four phases: lag phase, logarithmic (exponential) phase, stationary phase, and stable (decline) phase. Chromatographic peaks observed from day 0 to day 28 of fermentation confirmed the presence of major compounds, as illustrated in ESI Fig. S2.†

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The composition of the colony will change accordingly,²⁴ leading to changes in mycelium color, colony vitality, texture characteristics of the product, sensory acceptance, and product quality. The longer the fermentation time, the darker the color of the FAR fermented Shatavari beverage, due to the oxidation of polyphenol and flavonoid compounds, resulting in color browning. Other factors, such as light and temperature, might contribute to the degradation of phenolic compounds.²⁵ When these substances are oxidized, they form brown pigments that may eventually precipitate.²⁶

The five signature compounds were identified by LC-ESI/MS (LC-MS) analysis using Waters Alliance e2695/HPLC-TQD Mass spectrometer instrument, namely Chalconaringenin 2′-rhamnosyl-(1->4)-glucoside, Shatavarin I, Shatavarin VIII, Shatavarin V, Shatavarin IX, Asparoside B, Quercetin, Lysyl-Asparagine compounds. To further investigate the effect of fermentation, representative metabolites were selected for relative abundance analysis in this study. The data from Table 3 regarding the relative abundance of specific compounds in the Shatavari-based nutra-beverage are refined as a Shatavarin VIII is observed to be present in relatively high abundance within the nutra-beverage formulation. Both Shatavarin I and Shatavarin V are found in equal abundance levels, suggesting a balanced presence of these saponins. Asparoside B emerges as the most abundant compound, highlighting its significant contribution to the beverage's composition. Conversely, the compound Chalconaringenin 2′-rhamnosyl-(1->4)-glucoside is noted for its low abundance in the formulation, all of which are shown in Fig. 4.

3.4 Acute oral toxicity in mice

Mice treated with nutraceutical beverages (at 1.5 ml/100 g of body weight) showed no observable changes when compared with control. No instances of illness or fatality were recorded over the entire study duration. The experimental and control groups had comparable weight alterations (Table 4).

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Moreover, no significant difference was observed in the mean values of absolute and relative weights of essential organs, including the kidney, liver, lung, heart, and spleen, in the treatment group compared to the control group (see Fig. 5).

Nutraceutical beverage at a single dose of 1.5 ml/100 g body weight did not induce any significant change in haematological parameters [RBC count, WBC count and Haemoglobin (Hb)] in either of the sex studied (Table 4). Creatinine, SGOT, SGPT, total cholesterol, triglycerides, bilirubin, and alkaline phosphatase did not vary significantly either.

3.5 Sub-acute oral toxicity in mice

In sub-acute oral toxicity studies, there was no significant difference in change in body weight in group 2 and 3, while change in body weight in group 4 was found to be decreased when compared with control that implies the high antioxidant nature of nutraceutical that helps in reducing the body weight (Table 5).

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Moreover, the absolute and relative organ weights of kidney, lung, heart, liver, and spleen in group 2, 3, and 4 was comparable to the control group (see Fig. 6). Additionally, the haematological parameters including RBC count, WBC count and Haemoglobin (Hb) also remained within normal ranges across all three dosage of 0.375 ml, 0.75 ml and 1.5 ml/100 g body weight. Furthermore, there was no significant changes were observed in biochemical parameters such as serum creatinine, SGOT, SGPT, total cholesterol, triglycerides, bilirubin, and Alkaline phosphatase (ALP).

3.6 In-life observation

The animals were checked daily for mortality and any signs of illness. A general clinical examination was conducted before and after the start of the experiment, including observations of changes in the skin, mucous membranes, eyes, and any secretions or excretions. Additionally, responses such as tearing, raised hair, and respiratory patterns were noted. Changes in gait, posture, and response to handling were also observed.²⁷

3.7 Hematological and biochemical analysis

Neubauer's chamber was utilized to measure the total count of red blood cells (RBC) and white blood cells (WBC). Serum biochemical markers, including creatinine, SGOT, SGPT, total cholesterol, triglycerides, bilirubin, and alkaline phosphatase, were assessed utilizing kits from Agappe (India). The outcomes of the hedonic analysis consist of the evaluators' scores for each fermented product for texture, scent, taste, color, flavor, and aftertaste. The test substance, a nutraceutical beverage, was administered orally to Swiss albino mice in acute (1.5 ml/100 g body weight as a single dose) and sub-acute doses of 0.375 ml, 0.75 ml, and 1.5 ml/100 g body weight once daily for 28 days to investigate oral toxicity, enabling data extrapolation to human patients.²⁸

No one died or got sick throughout the experiment. At 0.375 ml and 0.75 ml/100 g of BW, acute and sub-acute testing showed no significant changes in body weight, however group 4 (1.5 ml/100 g of BW) showed a reduction compared to control. This impact could be due to the high antioxidant nature of nutraceutical beverage that helps in losing body weight. No alterations were noted in the assessment of vital organ weights, both absolutely and relatively. The hematological parameters exhibited negligible alterations. SGOT, serum creatinine, total cholesterol, triglycerides, bilirubin, and ALP all showed no significant increases (Table 5). This is the first report on the safety profile of chemically characterized Nutraceutical beverage, an herbal formulation of much use in Ayurvedic Medicine. A parallel investigation with Bacopa monnieri leaf enriched extract at acute and sub-acute levels in Sprague-Dawley rats found that 500 mg kg⁻¹ body weight for 14 days and 85, 210, and 500 mg kg⁻¹ for 90 days were well tolerated.

However, there is no report on the toxicity profile of Nutraceutical beverage. We have used Nutraceutical beverage, which is chemically defined and chemical constituents for the safety profiling. Nutraceutical beverage did not affect morbidity and mortality in our investigation, including body weight, important organ weight, and main biochemical indicators. Our data indicate that a single oral dose of Nutraceutical beverage at 1.5 ml g⁻¹ of body weight is well tolerated in both male and female Swiss albino mice, as well as doses of 0.375 ml, 0.75 ml, and 1.5 ml g⁻¹ of body weight administered once daily for 28 days.

3.8 Statistical analysis

The evaluation of relative organ weight showed no significant differences between the control and beverage-treated groups, indicating that the beverage consumption did not affect the proportional weight of key organs. This finding suggests that the beverage does not induce adverse effects on organ size relative to body weight, supporting its safety for consumption all shown in ESI Tables T6–T8.†

Similarly, the analysis of absolute organ weight demonstrated no significant variations between the control and beverage groups. This further confirms that the beverage does not interfere with the normal growth or development of individual organs, underscoring its non-toxic nature in terms of organ-specific impacts.

In terms of body weight, no significant differences were observed between the control and beverage groups, highlighting that the beverage had no detrimental effect on overall body growth or weight regulation. This outcome suggests that the beverage is metabolically neutral, with no apparent influence on weight gain or loss during the study period.

Overall, these findings collectively indicate the safety of the beverage, as evidenced by the absence of adverse effects on relative and absolute organ weights as well as body weight in comparison to the control group. This provides a strong basis for considering the beverage as a safe functional product for further development and consumption.

4 Conclusion

Shatavari has a variety of pharmacological effects and holds promise as both a functional food and a medicinal agent. Fermentation is typically used for making Shatavari drinks. Shatavari-based low-alcohol nutraceutical drinks (SLANB) have not been tested for toxicity. In this study, we evaluated the sensory, volatile/untargeted compounds and toxicological characteristics of SLANB for the first time. SLANB was administered over a 4 weeks (28 days) period, and its texture, aroma, taste, colour, flavour, and aftertaste were evaluated by 20 panellists using a 9-point hedonic scale. Sample 3 received the highest rating, indicating that it was the most well-liked product among the samples tested. It was also discovered that volatile and untargeted compounds present in Shatavari root were identified during shelf life analysis. This study sheds light on the chemical and biochemical properties of SLANB and its fermentation process. The toxicological properties of SLANB were evaluated in group 2 mice at a dosage of 1.5 ml/100 g body weight, and no fatalities or toxicity were observed in the acute study, indicating that SLANB may be regarded as non-toxic at the administered dose. In the 28 days sub-acute toxicity study, mice were fed orally with SLANB at different doses showed no adverse effects on mortality, body weight, relative and absolute organ weights, or haematology. SLANB had no harmful effects in both sexes in the sub-acute toxicity investigation. These findings indicate safe SLANB usage as a functional food and medicine.

Ethical statement

Animal experiments were conducted under the protocol (CIMAP/IAEC/2020–23/22) approved by Institutional Animal Ethics Committee of CSIR-CIMAP, Lucknow, India.

Data availability

Data will be made available on reasonable request.

Author contributions

Divya Choudhary: conceptualization, methodology, data curation, formal analysis, writing – original draft, writing – review & editing. Satyanarayan Naik: supervision, writing – review & editing, resources, project administration. Vidushi Tyagi and Anirban Pal: conceptualization, supervision, writing – review & editing, resources, experiment, and project administration. Hariprasad P.: supervision.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

We are grateful to the Central Institute of Medicinal and Aromatic Plants (CIMAP), located in Lucknow, Uttar Pradesh, for arranging the raw material for the study. We are also grateful to the Indian Institute of Technology Delhi (IITD), which provided research space and the necessary facilities. Both institutions are our premier research institutions.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5fb00109a

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