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Bioactive Properties of Phytochemicals from Selected Microgreens: A Comparative Study on Solvent-specific Extraction

S
S. Arya Krishnan1,2,*
P
P.K. Binsi3
P
P. Viji3
U
U. Parvathy3
C
C.G. Joshy3
1Kerala University of Fisheries and Ocean Studies, Kochi-682 506, Kerala, India.
2Department of Food Technology, Amal Jyothi College of Engineering, Kanjirappally-686 518, Kerala, India.
3Central Institute of Fisheries Technology, Kochi-682 506, Kerala, India.

ABSTRACT

Background: This study evaluated the extraction efficiency of water, ethanol and acetone for recovering bioactive compounds from selected microgreen species.

Methods: Green gram, broccoli, radish, mustard and cabbage microgreens were extracted using water, ethanol and acetone. The extracts were analysed for phytochemical composition, antioxidant and anti-inflammatory activities.

Result: Acetone extracts showed the highest phenolic and flavonoid contents, with radish microgreens exhibiting the highest total phenolic content (141 mg GAE/100 g). Green gram microgreens showed the highest antioxidant activity, while ethanolic extracts exhibited the strongest anti-inflammatory activity (IC50 = 0.04 µg/mL). Water and ethanol extracts showed higher FRAP values. Overall, microgreens were rich in bioactive phytochemicals.

INTRODUCTION

Microgreens are young leafy vegetables rich in phytonutrients and bioactive compounds. Their high nutrient density imparts antioxidant, anti-inflammatory, anticancer, antidiabetic and antibacterial properties, making them valuable for food and pharmaceutical applications (Xiao et al., 2015). Their ease of cultivation and low input requirements also make them suitable for space food production (Renna et al., 2018). Compared with mature plants, microgreens contain higher levels of nutrients and beneficial phytochemicals with lower antinutritional compounds, while being environmentally friendly due to cultivation without pesticides or fertilizers (Kyriacou et al., 2016; Saini et al., 2017; Marchioni et al., 2021; Bhatt et al., 2023). Microgreens, rich in phytochemicals with antioxidant, anti-inflammatory and antimicrobial properties, have emerged as promising functional foods (Nawaz et al., 2020; Sanyukta et al., 2023). Moreover, nearly 80% of the global population relies on plant-based remedies for primary healthcare (Sharma et al., 2021).
       
Extraction is a technique used to isolate bio active components from raw bio mass based on their solubility in two immiscible liquids. The separation of target components can be accomplished using solvents that maximize biological activity (Chang et al., 2002; Ajanal et al., 2012; Kumar et al., 2019). Solvents such as methanol, ethanol, acetone or water are commonly used for the extraction of bioactive compounds, depending on the component of interest and its solubility (Mahdi-Pour  et al., 2012). In addition, the polarity of the solvent significantly affects the yield and bioavailability of phytochemical constituents, making it necessary to use a combination of solvents rather than relying solely on a single one (Nawaz et al., 2020). 
       
Cultivation practices and light conditions significantly influence the quality of microgreens, while appropriate washing and drying treatments ensure their microbiological safety after harvest. Low-intensity light has been reported to preserve vitamin C in Chinese kale microgreens (Noichinda et al., 2007), whereas continuous light enhances tocopherol and carotenoid contents in baby leaf spinach. Several studies have also investigated the phytochemical composition of microgreens belonging to the families Asteraceae, Apiaceae, Amaryllidaceae, Amaranthaceae, Brassicaceae, Cucurbitaceae and Fabaceae (Marchioni et al., 2021; Tomas et al., 2021; Ghoora et al., 2020; Mlinarić et al., 2020; Wojdyło et al., 2020; Altuner, 2021).
       
Despite extensive studies on the nutritional and phytochemical composition of microgreens, comparative evaluation of different extraction solvents remains limited. Therefore, this study investigated the efficiency of water, ethanol and acetone for extracting bioactive compounds and evaluated their phytochemical composition, antioxidant and anti-inflammatory activities.

MATERIALS AND METHODS

Materials
 
Five microgreen samples such as green gram (Vigna radiata), broccoli (Brassica oleracea var. Italica), radish (Raphanus sativus), mustard (Brassica nigra) and cabbage (Brassica oleracea var. Capitata) were procured in fresh form from a commercial market. The experimental work was carried out at the Central Institute of Fisheries Technology (CIFT), Kochi, Kerala, India, during the period 2023-2024. All reagents and chemicals were supplied by Sigma-Aldrich (Steinheim, Germany).
 
Preparation of microgreen extracts
 
Water, 80% ethanol and acetone extracts were prepared to evaluate the phytochemical content. Microgreens were washed, vacuum-dried (40°C, 12 h) and extracted (1:5, w/v) at 40°C for 48 h. Fresh samples were used for chlorophyll and antinutritional analyses. The extracts were re-extracted, filtered (Whatman No. 1), concentrated to one-tenth of the original volume using a rotary evaporator and stored at 4°C until analysis.
 
Estimation of total phenols and flavonoid
 
Total phenolic content (TPC) was determined using the modified folin-ciocalteu method. Absorbance was measured at 765 nm and results were expressed as mg gallic acid equivalents (GAE) using gallic acid as the standard.
       
Total flavonoid content (TFC) was determined by the aluminium chloride colorimetric method (Kapewangolo et al., 2013). Absorbance was recorded at 510 nm and results were expressed as mg quercetin equivalents (QE) per 100 mg of sample.
 
Estimation of chlorophyll
 
Chlorophyll a, chlorophyll b, total chlorophyll and total carotenoid contents were determined spectrophotometrically using acetone extracts following the method described by Nurhidayati et al., (2012). The absorbance of the extracts was measured at 646.6, 663.6 and 470 nm using a UV-visible spectrophotometer. Chlorophyll a and chlorophyll b contents were calculated using the following equations:
 
Chla= (12.25 x A663.6) - (2.79 x A646.6)       ....(1)
    
Chlb= (21.3 x A646.6) - (5.1 x A663.6)       ....(2)
      
TChl = Chla + Chlb        ....(3)
 
             ....(4)
 
Estimation of antinutritional factors
 
The anti-nutritional factors, including oxalate, phytate and tannin were analysed in the microgreen samples. Oxalate, phytic acid and tannin levels were determined following previously established methodologies (Krisna and Ranjhan, 1980; Makkar and Goodchild, 1996).
 
Estimation of antioxidant activity
 
The antioxidant capacity of microgreen extracts was evaluated using three assays.
 
DPPH free radical scavenging assay
 
Ethanol, water and acetone extracts were tested for DPPH scavenging activity. Extracts at varying concentrations 40 to 90 µg/mL were mixed with two mL of 0.004% of DPPH solution. The mixture was vortexed and incubated in dark for 30 minutes. Absorbance was measured at 517 nm using a UV visible spectrophotometer, with absolute ethanol as the blank. The radical scavenging activity was calculated using the following formula:
  
     .....(5)
 
Where,
A0= The absorbance of control.
A= The absorbance of the sample.
 
Ferric reducing antioxidant power (FRAP)
 
FRAP assay was performed for all the three microgreen extracts following the standard protocol (Kowitcharoen et al., 2021). A mixture of 3 mL of FRAP reagent and 100 µL microgreen extract was vortexed and absorbance was recorded at 593 nm against a blank. Ferrous sulphate (100µmol/L) was used as the standard. The results were expressed as Ferrous equivalents (µmol FE/g) using the formula (Benzie and Strain, 1999).
     .....(6)

Metal chelating assay
 
Metal chelation was assessed by mixing 0.2 mL microgreen extract with 0.1 mM FeSO4 0.2 mL and 0.25 mM ferrozine (0.4 mL). After 10 minutes incubation, the absorbance was measured at 562 nm. The percentage of metal chelation was calculated using:
  
      .....(7)
 
Where,
Acontrol= The absorbance of control.
Asample= The absorbance of the microgreen extracts (Chew  et al., 2009).
 
Estimation of anti-inflammatory activity
 
Anti-inflammatory activity was assessed using the albumin denaturation assay (Gunathilake et al., 2018). The reaction mixture consisted of 0.2 mL of 1% egg albumin, 4.78 mL of phosphate- buffered saline (PBS, pH 6.4) and 0.02 mL extract. The mixture was incubated in a water bath at 37°C for 15 min, followed by heating at 70°C for 5 min. After cooling turbidity was measured at 660 nm in a UV visible spectrophotometer. 
       
% inhibition of protein denaturation was calculated as: 
 
     .....(8)
 
Statistical analysis
 
All experiments were performed in triplicate (n = 3) and the data were expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) using IBM SPSS Statistics software, version 29.2. Significant differences among the means were further evaluated using Tukey’s post-hoc test at a significance level of p<0.05.

RESULTS AND DISCUSSION

Estimation of total phenols and flavanoid
 
Phenolic compounds are important plant secondary metabolites involved in growth and defence. Among the tested solvents, acetone extracts exhibited the highest total phenolic and flavonoid contents. Radish microgreens showed the highest phenolic content, followed by green gram, broccoli, mustard and cabbage. Cabbage consistently exhibited the lowest phenolic content across all extracts. Similarly, acetone extracts contained the highest flavonoid levels, with green gram microgreens recording the highest flavonoid content (16.26±0.03 mg QE/100 mg) and cabbage water extracts the lowest (1.34±0.03 mg QE/100 mg). These findings are consistent with previous reports (Ghoora et al., 2020, Kowitcharoen et al., 2021) (Table 1 and 2).

Table 1: Total phenols mg GAE/100 g.



Table 2: Total flavonoid concentration mg QE/100 mg.


 
Estimation of chlorophyll and total carotenoids
 
Chlorophyll content is an important quality attribute influencing consumer acceptance of microgreens (Wojdyło et al., 2020). Total chlorophyll content ranged from 10.63 to 97.63 µg/mL, with cabbage microgreens exhibiting the highest content, followed by mustard, green gram, broccoli and radish. In all samples, chlorophyll a was higher than chlorophyll b, consistent with previous findings (Ghoora et al., 2020) (Table 3).

Table 3: Chlorophyll and total carotenoids chlorophyll estimation.


 
Antinutritional factors
 
Microgreens contained measurable levels of antinutritional factors, including oxalates, phytates and tannins. Oxalates may contribute to kidney stone formation by binding calcium, while phytates can reduce mineral bioavailability by chelating essential minerals (Muhammad et al., 2011). Phytate content was highest in radish microgreens (4.64 mg/100 g) and lowest in cabbage microgreens (1.26 mg/100 g) (Table 4). Tannin content was highest in green gram microgreens, followed by mustard, radish, cabbage and broccoli. Although tannins may reduce nutrient absorption through protein binding and digestive enzyme inhibition, the phytate and tannin levels observed in this study were within safe limits and consistent with previous reports (Ee et al., 2013; Alam et al., 2013).

Table 4: Antinutritional compounds.


 
Determination of antioxidant activity
 
The antioxidant potential of microgreens was assessed using DPPH free radical scavenging, metal chelating and FRAP assays.
 
DPPH free radical scavenging assay
 
Acetone extracts exhibited the highest DPPH radical scavenging activity, with green gram microgreens showing the greatest antioxidant potential (IC50 = 0.90 µg/mL). The DPPH assay measures the ability of antioxidants to reduce the stable DPPH radical through hydrogen donation (Alam et al., 2013). Ethanolic extracts exhibited moderate radical scavenging activity, whereas water extracts showed comparatively lower activity. The DPPH scavenging activities of water, acetone and ethanol extracts are presented in Fig 1-3.

Fig 1: DPPH assay-water extract.



Fig 2: DPPH assay acetone extract.



Fig 3: DPPH assay ethanol extract.


 
Metal chelating assay
 
Metal chelation is an important antioxidant mechanism that prevents oxidative damage by binding metal ions. Among the tested samples, acetone extracts of green gram microgreens exhibited the highest metal chelating activity (IC50 = 1.04 µg/mL), followed by broccoli (1.76 µg/mL), radish (2.89 µg/mL), mustard (3.49 µg/mL) and cabbage (3.55 µg/mL) (Fig 4). A similar trend was observed for ethanolic extracts, although higher concentrations (100-900 µg/mL) were required (Fig 5). In contrast, water extracts showed significantly lower chelating activity, with broccoli exhibiting the highest activity (IC50 = 2.28 mg/mL) and cabbage the lowest (IC50 = 4.65 mg/mL) (Fig 6). The reduced activity of water extracts may be attributed to differences in solubility and the stability of bioactive compounds.

Fig 4: Metal chelating assay-acetone extract.



​

Fig 5: Metal chelating assay-ethanol extract.



​

Fig 6: Metal chelating assay-water extract.


 
FRAP assay
 
The FRAP assay measures antioxidant capacity based on the reduction of Fe+  to Fe+. Among the tested extracts, water extracts of broccoli microgreens exhibited the highest ferric reducing power (40.84 μg FE/g). Overall, water extracts showed higher FRAP values than ethanol and acetone extracts across all microgreen samples. These findings are consistent with previous studies on microgreens and chia microgreens (Mlinaric et al., 2020). The results are presented in Table 5.

Table 5: FRAP assay.


 
Anti-inflammatory property
 
Effect on albumin denaturation
 
Inflammation is a protective response to tissue injury, although excessive lysosomal enzyme release can aggravate tissue damage (Okoli et al., 2008). All microgreen extracts exhibited anti-inflammatory activity by inhibiting albumin denaturation. Among the ethanolic extracts, green gram showed the highest activity (IC50 = 0.04 µg/mL), followed by mustard, broccoli, radish and cabbage. In acetone extracts, mustard exhibited the highest activity (IC50 = 0.99 µg/mL), whereas broccoli showed the highest activity among water extracts (IC50 = 1.01 mg/mL). The IC50 values for ethanolic and acetone extracts are expressed in µg/mL, whereas those for water extracts are expressed in mg/mL, reflecting the respective concentration ranges required to achieve 50% inhibition. Overall, water extracts exhibited lower anti-inflammatory activity than ethanolic and acetone extracts. These findings are consistent with previous reports on medicinal plant extracts (Ondua et al., 2018) (Table 6).

​

Table 6: Anti-inflammatory properties.



Comparison of extraction solvents
 
Solvent polarity plays a key role in extraction efficiency. Water is an effective green solvent for hydrophilic compounds, whereas ethanol (GRAS) is widely used for extracting polyphenols and triterpenes. Acetone, with intermediate polarity and a low boiling point, efficiently extracts both polar and non-polar phytochemicals, resulting in higher bioactive compound recovery (Bitwell et al., 2023; Kumar et al., 2023). Extraction enhances the bioavailability of plant bioactive compounds, supporting their application in functional foods and nutraceuticals (Kumar et al., 2023; Veiga et al., 2020). Although acetone extracts contained the highest levels of phytochemicals, water and ethanol extracts also showed considerable extraction potential. The higher phenolic and flavonoid contents found in acetone extracts were generally associated with stronger DPPH and metal-chelating activities. In contrast, the higher FRAP values observed in water and ethanol extracts may be due to differences in the types of phytochemicals extracted and their interaction with the specific assay conditions. However, the study had some limitations, including the absence of individual compound identification using HPLC/LC-MS, the use of a single extraction cycle and the lack of in vivo validation.

CONCLUSION

The present study demonstrated that acetone extracts of microgreens contained the highest levels of phytochemicals, antioxidants and anti-inflammatory compounds, while water and ethanol extracts exhibited significantly higher FRAP values. Among the extracts, ethanolic green gram microgreens showed the strongest anti-inflammatory activity (IC50 = 0.04 µg/mL), likely due to their phenolic acids and flavonoids, which modulate inflammatory pathways. Acetone extracts of green gram microgreens also exhibited superior metal chelating and DPPH radical scavenging activities, reflecting the ability of phytochemicals to chelate metal ions and inhibit oxidative reactions. Overall, the findings confirm that microgreens are rich sources of carotenoids, flavonoids, phenolics and chlorophyll, highlighting their potential as functional foods and promising ingredients for drug delivery applications.

ACKNOWLEDGEMENT

The authors sincerely acknowledge the Director, ICAR-Central Institute of Fisheries Technology (CIFT), Kochi, for providing the laboratory facilities and technical support required to carry out this research. The authors also thank Kerala University of Fisheries and Ocean Studies (KUFOS) for the academic support extended during the course of this study.
 
Author contributions
 
Arya Krishnan S: Conceptualization, methodology, investigation, laboratory experiments, data curation, formal analysis, interpretation of results, manuscript preparation, writing-original draft and correspondence. Viji P.: Experimental  design, supervision, methodology, manuscript review and editing. Parvathy U.: Laboratory support, data validation, review and editing. Joshy C.G.: Statistical analysis, validation, manuscript review. Binsi P.K.: Conceptualization, supervision, project administration, critical review, editing and final approval of the manuscript.
 
Funding
 
The authors declare that this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
 
Ethical approval
 
This study did not involve human participants or experimental animals. Therefore, ethical approval was not required.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this manuscript.

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    Full Research Article

    Bioactive Properties of Phytochemicals from Selected Microgreens: A Comparative Study on Solvent-specific Extraction

    S
    S. Arya Krishnan1,2,*
    P
    P.K. Binsi3
    P
    P. Viji3
    U
    U. Parvathy3
    C
    C.G. Joshy3
    1Kerala University of Fisheries and Ocean Studies, Kochi-682 506, Kerala, India.
    2Department of Food Technology, Amal Jyothi College of Engineering, Kanjirappally-686 518, Kerala, India.
    3Central Institute of Fisheries Technology, Kochi-682 506, Kerala, India.

    ABSTRACT

    Background: This study evaluated the extraction efficiency of water, ethanol and acetone for recovering bioactive compounds from selected microgreen species.

    Methods: Green gram, broccoli, radish, mustard and cabbage microgreens were extracted using water, ethanol and acetone. The extracts were analysed for phytochemical composition, antioxidant and anti-inflammatory activities.

    Result: Acetone extracts showed the highest phenolic and flavonoid contents, with radish microgreens exhibiting the highest total phenolic content (141 mg GAE/100 g). Green gram microgreens showed the highest antioxidant activity, while ethanolic extracts exhibited the strongest anti-inflammatory activity (IC50 = 0.04 µg/mL). Water and ethanol extracts showed higher FRAP values. Overall, microgreens were rich in bioactive phytochemicals.

    INTRODUCTION

    Microgreens are young leafy vegetables rich in phytonutrients and bioactive compounds. Their high nutrient density imparts antioxidant, anti-inflammatory, anticancer, antidiabetic and antibacterial properties, making them valuable for food and pharmaceutical applications (Xiao et al., 2015). Their ease of cultivation and low input requirements also make them suitable for space food production (Renna et al., 2018). Compared with mature plants, microgreens contain higher levels of nutrients and beneficial phytochemicals with lower antinutritional compounds, while being environmentally friendly due to cultivation without pesticides or fertilizers (Kyriacou et al., 2016; Saini et al., 2017; Marchioni et al., 2021; Bhatt et al., 2023). Microgreens, rich in phytochemicals with antioxidant, anti-inflammatory and antimicrobial properties, have emerged as promising functional foods (Nawaz et al., 2020; Sanyukta et al., 2023). Moreover, nearly 80% of the global population relies on plant-based remedies for primary healthcare (Sharma et al., 2021).
           
    Extraction is a technique used to isolate bio active components from raw bio mass based on their solubility in two immiscible liquids. The separation of target components can be accomplished using solvents that maximize biological activity (Chang et al., 2002; Ajanal et al., 2012; Kumar et al., 2019). Solvents such as methanol, ethanol, acetone or water are commonly used for the extraction of bioactive compounds, depending on the component of interest and its solubility (Mahdi-Pour  et al., 2012). In addition, the polarity of the solvent significantly affects the yield and bioavailability of phytochemical constituents, making it necessary to use a combination of solvents rather than relying solely on a single one (Nawaz et al., 2020). 
           
    Cultivation practices and light conditions significantly influence the quality of microgreens, while appropriate washing and drying treatments ensure their microbiological safety after harvest. Low-intensity light has been reported to preserve vitamin C in Chinese kale microgreens (Noichinda et al., 2007), whereas continuous light enhances tocopherol and carotenoid contents in baby leaf spinach. Several studies have also investigated the phytochemical composition of microgreens belonging to the families Asteraceae, Apiaceae, Amaryllidaceae, Amaranthaceae, Brassicaceae, Cucurbitaceae and Fabaceae (Marchioni et al., 2021; Tomas et al., 2021; Ghoora et al., 2020; Mlinarić et al., 2020; Wojdyło et al., 2020; Altuner, 2021).
           
    Despite extensive studies on the nutritional and phytochemical composition of microgreens, comparative evaluation of different extraction solvents remains limited. Therefore, this study investigated the efficiency of water, ethanol and acetone for extracting bioactive compounds and evaluated their phytochemical composition, antioxidant and anti-inflammatory activities.

    MATERIALS AND METHODS

    Materials
     
    Five microgreen samples such as green gram (Vigna radiata), broccoli (Brassica oleracea var. Italica), radish (Raphanus sativus), mustard (Brassica nigra) and cabbage (Brassica oleracea var. Capitata) were procured in fresh form from a commercial market. The experimental work was carried out at the Central Institute of Fisheries Technology (CIFT), Kochi, Kerala, India, during the period 2023-2024. All reagents and chemicals were supplied by Sigma-Aldrich (Steinheim, Germany).
     
    Preparation of microgreen extracts
     
    Water, 80% ethanol and acetone extracts were prepared to evaluate the phytochemical content. Microgreens were washed, vacuum-dried (40°C, 12 h) and extracted (1:5, w/v) at 40°C for 48 h. Fresh samples were used for chlorophyll and antinutritional analyses. The extracts were re-extracted, filtered (Whatman No. 1), concentrated to one-tenth of the original volume using a rotary evaporator and stored at 4°C until analysis.
     
    Estimation of total phenols and flavonoid
     
    Total phenolic content (TPC) was determined using the modified folin-ciocalteu method. Absorbance was measured at 765 nm and results were expressed as mg gallic acid equivalents (GAE) using gallic acid as the standard.
           
    Total flavonoid content (TFC) was determined by the aluminium chloride colorimetric method (Kapewangolo et al., 2013). Absorbance was recorded at 510 nm and results were expressed as mg quercetin equivalents (QE) per 100 mg of sample.
     
    Estimation of chlorophyll
     
    Chlorophyll a, chlorophyll b, total chlorophyll and total carotenoid contents were determined spectrophotometrically using acetone extracts following the method described by Nurhidayati et al., (2012). The absorbance of the extracts was measured at 646.6, 663.6 and 470 nm using a UV-visible spectrophotometer. Chlorophyll a and chlorophyll b contents were calculated using the following equations:
     
    Chla= (12.25 x A663.6) - (2.79 x A646.6)       ....(1)
        
    Chlb= (21.3 x A646.6) - (5.1 x A663.6)       ....(2)
          
    TChl = Chla + Chlb        ....(3)
     
                 ....(4)
     
    Estimation of antinutritional factors
     
    The anti-nutritional factors, including oxalate, phytate and tannin were analysed in the microgreen samples. Oxalate, phytic acid and tannin levels were determined following previously established methodologies (Krisna and Ranjhan, 1980; Makkar and Goodchild, 1996).
     
    Estimation of antioxidant activity
     
    The antioxidant capacity of microgreen extracts was evaluated using three assays.
     
    DPPH free radical scavenging assay
     
    Ethanol, water and acetone extracts were tested for DPPH scavenging activity. Extracts at varying concentrations 40 to 90 µg/mL were mixed with two mL of 0.004% of DPPH solution. The mixture was vortexed and incubated in dark for 30 minutes. Absorbance was measured at 517 nm using a UV visible spectrophotometer, with absolute ethanol as the blank. The radical scavenging activity was calculated using the following formula:
      
         .....(5)
     
    Where,
    A0= The absorbance of control.
    A= The absorbance of the sample.
     
    Ferric reducing antioxidant power (FRAP)
     
    FRAP assay was performed for all the three microgreen extracts following the standard protocol (Kowitcharoen et al., 2021). A mixture of 3 mL of FRAP reagent and 100 µL microgreen extract was vortexed and absorbance was recorded at 593 nm against a blank. Ferrous sulphate (100µmol/L) was used as the standard. The results were expressed as Ferrous equivalents (µmol FE/g) using the formula (Benzie and Strain, 1999).
         .....(6)

    Metal chelating assay
     
    Metal chelation was assessed by mixing 0.2 mL microgreen extract with 0.1 mM FeSO4 0.2 mL and 0.25 mM ferrozine (0.4 mL). After 10 minutes incubation, the absorbance was measured at 562 nm. The percentage of metal chelation was calculated using:
      
          .....(7)
     
    Where,
    Acontrol= The absorbance of control.
    Asample= The absorbance of the microgreen extracts (Chew  et al., 2009).
     
    Estimation of anti-inflammatory activity
     
    Anti-inflammatory activity was assessed using the albumin denaturation assay (Gunathilake et al., 2018). The reaction mixture consisted of 0.2 mL of 1% egg albumin, 4.78 mL of phosphate- buffered saline (PBS, pH 6.4) and 0.02 mL extract. The mixture was incubated in a water bath at 37°C for 15 min, followed by heating at 70°C for 5 min. After cooling turbidity was measured at 660 nm in a UV visible spectrophotometer. 
           
    % inhibition of protein denaturation was calculated as: 
     
         .....(8)
     
    Statistical analysis
     
    All experiments were performed in triplicate (n = 3) and the data were expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA) using IBM SPSS Statistics software, version 29.2. Significant differences among the means were further evaluated using Tukey’s post-hoc test at a significance level of p<0.05.

    RESULTS AND DISCUSSION

    Estimation of total phenols and flavanoid
     
    Phenolic compounds are important plant secondary metabolites involved in growth and defence. Among the tested solvents, acetone extracts exhibited the highest total phenolic and flavonoid contents. Radish microgreens showed the highest phenolic content, followed by green gram, broccoli, mustard and cabbage. Cabbage consistently exhibited the lowest phenolic content across all extracts. Similarly, acetone extracts contained the highest flavonoid levels, with green gram microgreens recording the highest flavonoid content (16.26±0.03 mg QE/100 mg) and cabbage water extracts the lowest (1.34±0.03 mg QE/100 mg). These findings are consistent with previous reports (Ghoora et al., 2020, Kowitcharoen et al., 2021) (Table 1 and 2).

    Table 1: Total phenols mg GAE/100 g.



    Table 2: Total flavonoid concentration mg QE/100 mg.


     
    Estimation of chlorophyll and total carotenoids
     
    Chlorophyll content is an important quality attribute influencing consumer acceptance of microgreens (Wojdyło et al., 2020). Total chlorophyll content ranged from 10.63 to 97.63 µg/mL, with cabbage microgreens exhibiting the highest content, followed by mustard, green gram, broccoli and radish. In all samples, chlorophyll a was higher than chlorophyll b, consistent with previous findings (Ghoora et al., 2020) (Table 3).

    Table 3: Chlorophyll and total carotenoids chlorophyll estimation.


     
    Antinutritional factors
     
    Microgreens contained measurable levels of antinutritional factors, including oxalates, phytates and tannins. Oxalates may contribute to kidney stone formation by binding calcium, while phytates can reduce mineral bioavailability by chelating essential minerals (Muhammad et al., 2011). Phytate content was highest in radish microgreens (4.64 mg/100 g) and lowest in cabbage microgreens (1.26 mg/100 g) (Table 4). Tannin content was highest in green gram microgreens, followed by mustard, radish, cabbage and broccoli. Although tannins may reduce nutrient absorption through protein binding and digestive enzyme inhibition, the phytate and tannin levels observed in this study were within safe limits and consistent with previous reports (Ee et al., 2013; Alam et al., 2013).

    Table 4: Antinutritional compounds.


     
    Determination of antioxidant activity
     
    The antioxidant potential of microgreens was assessed using DPPH free radical scavenging, metal chelating and FRAP assays.
     
    DPPH free radical scavenging assay
     
    Acetone extracts exhibited the highest DPPH radical scavenging activity, with green gram microgreens showing the greatest antioxidant potential (IC50 = 0.90 µg/mL). The DPPH assay measures the ability of antioxidants to reduce the stable DPPH radical through hydrogen donation (Alam et al., 2013). Ethanolic extracts exhibited moderate radical scavenging activity, whereas water extracts showed comparatively lower activity. The DPPH scavenging activities of water, acetone and ethanol extracts are presented in Fig 1-3.

    Fig 1: DPPH assay-water extract.



    Fig 2: DPPH assay acetone extract.



    Fig 3: DPPH assay ethanol extract.


     
    Metal chelating assay
     
    Metal chelation is an important antioxidant mechanism that prevents oxidative damage by binding metal ions. Among the tested samples, acetone extracts of green gram microgreens exhibited the highest metal chelating activity (IC50 = 1.04 µg/mL), followed by broccoli (1.76 µg/mL), radish (2.89 µg/mL), mustard (3.49 µg/mL) and cabbage (3.55 µg/mL) (Fig 4). A similar trend was observed for ethanolic extracts, although higher concentrations (100-900 µg/mL) were required (Fig 5). In contrast, water extracts showed significantly lower chelating activity, with broccoli exhibiting the highest activity (IC50 = 2.28 mg/mL) and cabbage the lowest (IC50 = 4.65 mg/mL) (Fig 6). The reduced activity of water extracts may be attributed to differences in solubility and the stability of bioactive compounds.

    Fig 4: Metal chelating assay-acetone extract.



    ​

    Fig 5: Metal chelating assay-ethanol extract.



    ​

    Fig 6: Metal chelating assay-water extract.


     
    FRAP assay
     
    The FRAP assay measures antioxidant capacity based on the reduction of Fe+  to Fe+. Among the tested extracts, water extracts of broccoli microgreens exhibited the highest ferric reducing power (40.84 μg FE/g). Overall, water extracts showed higher FRAP values than ethanol and acetone extracts across all microgreen samples. These findings are consistent with previous studies on microgreens and chia microgreens (Mlinaric et al., 2020). The results are presented in Table 5.

    Table 5: FRAP assay.


     
    Anti-inflammatory property
     
    Effect on albumin denaturation
     
    Inflammation is a protective response to tissue injury, although excessive lysosomal enzyme release can aggravate tissue damage (Okoli et al., 2008). All microgreen extracts exhibited anti-inflammatory activity by inhibiting albumin denaturation. Among the ethanolic extracts, green gram showed the highest activity (IC50 = 0.04 µg/mL), followed by mustard, broccoli, radish and cabbage. In acetone extracts, mustard exhibited the highest activity (IC50 = 0.99 µg/mL), whereas broccoli showed the highest activity among water extracts (IC50 = 1.01 mg/mL). The IC50 values for ethanolic and acetone extracts are expressed in µg/mL, whereas those for water extracts are expressed in mg/mL, reflecting the respective concentration ranges required to achieve 50% inhibition. Overall, water extracts exhibited lower anti-inflammatory activity than ethanolic and acetone extracts. These findings are consistent with previous reports on medicinal plant extracts (Ondua et al., 2018) (Table 6).

    ​

    Table 6: Anti-inflammatory properties.



    Comparison of extraction solvents
     
    Solvent polarity plays a key role in extraction efficiency. Water is an effective green solvent for hydrophilic compounds, whereas ethanol (GRAS) is widely used for extracting polyphenols and triterpenes. Acetone, with intermediate polarity and a low boiling point, efficiently extracts both polar and non-polar phytochemicals, resulting in higher bioactive compound recovery (Bitwell et al., 2023; Kumar et al., 2023). Extraction enhances the bioavailability of plant bioactive compounds, supporting their application in functional foods and nutraceuticals (Kumar et al., 2023; Veiga et al., 2020). Although acetone extracts contained the highest levels of phytochemicals, water and ethanol extracts also showed considerable extraction potential. The higher phenolic and flavonoid contents found in acetone extracts were generally associated with stronger DPPH and metal-chelating activities. In contrast, the higher FRAP values observed in water and ethanol extracts may be due to differences in the types of phytochemicals extracted and their interaction with the specific assay conditions. However, the study had some limitations, including the absence of individual compound identification using HPLC/LC-MS, the use of a single extraction cycle and the lack of in vivo validation.

    CONCLUSION

    The present study demonstrated that acetone extracts of microgreens contained the highest levels of phytochemicals, antioxidants and anti-inflammatory compounds, while water and ethanol extracts exhibited significantly higher FRAP values. Among the extracts, ethanolic green gram microgreens showed the strongest anti-inflammatory activity (IC50 = 0.04 µg/mL), likely due to their phenolic acids and flavonoids, which modulate inflammatory pathways. Acetone extracts of green gram microgreens also exhibited superior metal chelating and DPPH radical scavenging activities, reflecting the ability of phytochemicals to chelate metal ions and inhibit oxidative reactions. Overall, the findings confirm that microgreens are rich sources of carotenoids, flavonoids, phenolics and chlorophyll, highlighting their potential as functional foods and promising ingredients for drug delivery applications.

    ACKNOWLEDGEMENT

    The authors sincerely acknowledge the Director, ICAR-Central Institute of Fisheries Technology (CIFT), Kochi, for providing the laboratory facilities and technical support required to carry out this research. The authors also thank Kerala University of Fisheries and Ocean Studies (KUFOS) for the academic support extended during the course of this study.
     
    Author contributions
     
    Arya Krishnan S: Conceptualization, methodology, investigation, laboratory experiments, data curation, formal analysis, interpretation of results, manuscript preparation, writing-original draft and correspondence. Viji P.: Experimental  design, supervision, methodology, manuscript review and editing. Parvathy U.: Laboratory support, data validation, review and editing. Joshy C.G.: Statistical analysis, validation, manuscript review. Binsi P.K.: Conceptualization, supervision, project administration, critical review, editing and final approval of the manuscript.
     
    Funding
     
    The authors declare that this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
     
    Ethical approval
     
    This study did not involve human participants or experimental animals. Therefore, ethical approval was not required.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this manuscript.

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