BIBECHANA Vol. 21, No. 3, December 2024, 241-253 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar Phytochemical analysis and digestive enzymes inhibition study of Beta vulgaris Sabina Khatri, Akash Budha Magar, Tinky Sharma, Khaga Raj Sharma∗ Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu ∗Corresponding author. Email: khaga.sharma@cdc.tu.edu.np Abstract Beta vulgaris is an annual crop grown for its edible roots and leaves. It is traditionally used for the treatment of diabetes, cancer, obesity, heart problems, kidney problems, and liver diseases. The present work is centered on the phytochemical analysis and assessment of antioxidant, antimicrobial, and antidiabetic activities, and toxicity in the root and leaf extracts and solvent fractions. TPC and TFC were measured using the Folin-Ciocalteu phenol reagent method and AlCl3 colorimetric method respectively. Antioxidant and antidiabetic activity were measured with DPPH assay and α-glucosidase enzyme inhibition assay. Antimicrobial activity was determined with the agar disc diffusion method and brine shrimp assay was performed to measure toxicity. Phytochemical analysis revealed the presence of phenols, flavonoids, glycosides, saponins, tannins, terpenoids, and alkaloids. The ethyl acetate fraction of the root contained the highest amount of phenolics with 84.35 ± 0.94 mg GAE/g. Total flavonoid content was found highest in the hexane fraction of root at 150.48 ± 1.10 mg QE/g. The ethyl acetate fraction of the root displayed an IC50 of 3.92 ± 0.06 µg/mL in the DPPH assay. The plant extracts and fractions possessed weak α-glucosidase enzyme inhibition activity. They were inactive against bacterial species of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, and fungal species of Fusarium solani. Toxicity assay found the plant to be non-toxic against the brine shrimp nauplii with the lowest LC50 value being 1166.36 ± 100.21 µg/mL for the hexane fraction of leaf. The study finds B. vulgaris to be rich in phytochemicals and antioxidant activity with weak α-glucosidase enzyme inhibition activity. It is non-toxic to brine shrimp larvae. Keywords Beta vulgaris, α-glucosidase, DPPH, antidiabetic, antimicrobial, toxicity. Article information Manuscript received: March 20, 2024; Revised: April 16, 2024; Accepted: April 17, 2024 DOI https://doi.org/10.3126/bibechana.v21i3.63978 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 241 http://nepjol.info/index.php/BIBECHANA khaga.sharma@cdc.tu.edu.np https://doi.org/10.3126/bibechana.v21i3.63978 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 242 1 Introduction Humans have been using plants as both food and medicine. Dietary plants contain many important secondary metabolites besides basic nutrients. The secondary metabolites in such food act as func- tional components that can prevent or treat dis- eases [1]. Many plants used in traditional medicines are also consumed as spices, vegetables, and fruits. They are known as functional foods and defined as ‘Foods or dietary components that may provide a health benefit beyond basic nutrition’ [2]. The func- tional food market has grown due to an increase in the health consciousness among the modern popu- lation. Medicinal or functional food plays a sup- porting role in modern medicine in the treatment of illness. So, it is essential to explore the phyto- chemicals and biological activities in our fruits and vegetables and assess their medicinal value. Beta vulgaris subsp. vulgaris, commonly known as beetroot is an annual crop plant from the Chenopodiaceae family [3]. Its wild forms are dis- tributed from Europe, and North Africa to West- ern Asia whereas cultivated beetroots are grown worldwide and commonly consumed in our daily diets [4]. B. vulgaris is rich in minerals and vita- mins [5]. Proximate analysis by Kale et al., (2018) has revealed the composition of the plant as 1.35 ± 0.2% proteins, 7.59 ± 0.4% carbohydrates, 0.3 ± 0.1% fats, 1.9 ± 0.2% dietary fibers, 1.4 ± 0.2% ash, and 87.4 ± 0.3% moisture [6]. Alkaloids, terpenoids, steroids, flavonoids, tannins, saponins, and glycosides in root extracts and flavonoids, tan- nins, oxalate, anthocyanins, saponins, phenolics, carotenoids, and phytate in leaf extracts of B. vul- garis has been detected in previous studies [7]. The plant is used in traditional medicine for the treat- ment of diabetes, cancer, obesity, heart, kidney, and liver diseases, and it also improves the immune system and hematopoietic system [8–10]. B. vul- garis has also been used for dandruff, diminished libido, constipation, and gastrointestinal and mus- culoskeletal pain [11]. In addition to traditional uses, the plant has displayed remarkable biologi- cal activities in different scientific studies. Rehman et al., (2021) observed significant protein denatu- ration inhibitory activity, acetylcholinesterase in- hibitory activity, and red blood cell stabilizing ac- tivity in root and leaf extract of B. vulgaris [12]. Mzoughi et al., (2019) have revealed the antioxi- dant and antidiabetic potential of the plant [13]. The roots of the plant displayed anticancer and an- tibacterial activities in a study by El-Beltagi et al., (2018) [14]. The plant has also gained significant attraction in scientific research because of its high nitrate (NO3 -) content that promotes health aids for cardiac ailments through endogenic nitric ox- ide (NO) synthesis [15]. It is widely consumed and utilized to develop food coloring agents [16]. B. vul- garis is a source of a class of bioactive compound, ‘betaine’ that shows a hepatoprotective effect by increasing the expression of the quinone reductase enzyme [17]. Beetin 27 (BE27) is a protein isolated from B. vulgaris that contains antiviral and cyto- toxic activities [18]. Additionally, aqueous extract of the plant has been used in the biosynthesis of ZnO nanoparticles that display antimicrobial, an- ticancer, and antidiabetic activity [19]. Due to its high nutritional and medicinal properties, B. vul- garis has attracted many scholars over the years and there have been many studies regarding its nu- tritional and pharmaceutical activities. The present study adds brine shrimp toxicity assay and com- parative phytochemical and biological analysis of the plant using methanol extract and hexane, ethyl acetate, dichloromethane, and aqueous fraction of root and leaf of B. vulgaris to the ongoing research on the plant. The findings of the present study may guide future work regarding the isolation, purifi- cation, and characterization of active metabolites from B. vulgaris. Diabetes mellitus is a chronic metabolic dis- order characterized by high levels of blood sugar and caused by insulin deficiency or insulin resis- tance [20]. Synthetic antidiabetic drugs are asso- ciated with adverse effects including hypoglycemic coma, weight gain, and kidney and liver diseases [21]. So, the World Health Organization (WHO) recommends the treatment of diabetes using medic- inal plants that are cheap, effective, and contain fewer side effects [22]. The antidiabetic potential of many fruits and vegetables remains unexplored. So, there is a need to study antidiabetic activity present in our food plants to lower the risk of dia- betes with appropriate diets and possible discovery of noble antidiabetic compounds. Overproduction of highly reactive oxygen and nitrogen species during various metabolic processes in our bodies causes oxidative stress [23]. Dur- ing oxidative stress, reactive oxygen and nitrogen species oxidize and damage important biomolecules like lipids, proteins, DNA, and RNA [24]. This leads to chronic diseases and disorders including cancer, diabetes mellitus, cataract, cardiovascular disease, neurodegenerative diseases, asthma, and rheumatoid arthritis [23]. Antioxidants are bioac- tive compounds that are involved in preventing or postponing the oxidation of particles or molecules and protecting our health and well-being [25]. An- tioxidants may be enzymatic or non-enzymatic and endogenous or exogenous. The correlation between the intake of antioxidant-rich pomegranate juice and its positive impact on health has been observed in previous studies [26]. Antioxidants in foods as- sist in preventing oxidative stress-related diabetes, obesity, hypertension, cardiovascular and chronic Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 243 inflammation disease, and display neuroprotective and anticancer activity [27]. Foodborne diseases and infections cause about 600 million hospital cases and 420,000 deaths each year [28]. Most of such infections are caused by the growth of pathogenic bacteria and fungi in foods. In addition to health risks, food spoilage is asso- ciated with environmental and resource costs [29]. Synthetic preservatives like benzoates, caffeine, sac- charin, and sorbic acid are known to produce side effects such as asthma, heart defects, diabetes, and dermatitis [30]. So, there is an urgent need for an- timicrobial agents of natural origin that are effec- tive, safe for human consumption and display mini- mal side effects. Many edible plants display signifi- cant antimicrobial activity and they are considered as sources of inhibitory substance against foodborne pathogens [31]. Traditional medicines of natural origin are widely regarded as safer alternatives to modern syn- thetic drugs. A previous study reported that the fa- talities caused by adverse reactions to modern phar- maceuticals were more than 100,000 whereas herbal medicines were responsible for less than 24 deaths each year [32]. Nevertheless, evaluation of toxic and other side effects is incorporated in the assessment of medicinal properties of plants for their potential toxicity and bioactivity, but the safety assessment of edible and cultivated plants is generally ignored as they are considered safe for human consumption. An adequate study of medicinal food requires an assessment of its cytotoxic activity to increase con- fidence in human consumption and potential phar- maceutical developments. Figure 1: Photographs of fresh plant sample, herbarium, sample size collection, and drying of plant samples. 2 Materials and Methods 2.1 Chemicals and reagents The 4-nitrophenyl -D-glucopyranoside (CAS NO: 3767-28-0), α-glucosidase enzyme (CAS NO: 9001- 42-7), and quercetin (CAS NO: 117-39-5), were pro- cured from Sigma-Aldrich (Germany). DPPH was purchased from Hi-media (India) and gallic acid was purchased from Molychem (India). Analytical grade (extra pure) organic solvents like methanol, hexane, ethyl acetate, DCM, DMSO, and other chemicals used in the experimentation were pur- chased from Merck and Fischer Scientific (India). 2.2 Equipment The equipment used during the study were mortar and pestle, glassware, an electric grinder, a weigh- ing scale (Pioneer, DHAUS), a rotary evaporator (Buchi RE111), a hot air oven (Griffin-Grundy), a microplate reader (Synergy LX, Bio Tek, Instru- ments, Inc., USA), water-bath (Clifton), pipettes, vials, micropipettes (Erba BIHOT). 2.3 Plant collection and identification Leaf and root samples of the plant were harvested from the cultivated site in Bhaktapur, Nepal in Au- gust 2021. The altitude and coordinates of the col- lection site are given in Table 1. The plant was iden- tified by the Central Department of Botany, Trib- huvan University, Kathmandu, Nepal as herbarium sample TUCH-210075. Photographs of plant sam- ples are given in Figure 1. Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 244 Table 1: Traditional medicinal uses of the plant Name of the plant Identification no. Altitude/plant growing Plant parts Medicinal uses and health ben- efits Obesity, diabetes, and cardiovascu- lar disease [8, 9] Beta vulgaris TUCH-210075 Altitude 1401 m Roots Cancer, heart dis- ease [10] 85° 25’ 48” E, 27° 40’ 14” N. Leaves Dandruff, di- minished libido, constipation, gas- trointestinal, and musculoskeletal pain [11] Hypertension and endothelial func- tion [4] Food coloring [16] 2.4 Extraction and fractionation Plant samples were cleaned, shade-dried to a con- stant weight, and ground to powder. The cold percolation method was used for the extraction of phytochemicals. 250 g root powder and 250 g leaf powder were soaked in reagent grade methanol (500 mL) in separate conical flasks at a temperature of 21 0C for 72 hours. The flasks were shaken vigor- ously after every 24-hour interval. Then, the con- tents were filtered with a clean muslin followed by the Whatman-1 filter paper. A rotatory evapora- tor was used to concentrate the filtrate at reduced pressure and 40 0C temperature. Solvent fractions of methanol extract were iso- lated using hexane, dichloromethane, ethyl acetate, and water. 20 g methanol extract was dissolved in 50 mL of distilled water in a separating funnel and an equal volume of hexane was added. The con- tents were vigorously shaken and then left undis- turbed until clear layers of hexane at the top and water at the bottom were formed. The hexane layer was concentrated in the rotary evaporator at re- duced pressure and 40 C temperature. The aqueous layer was subjected to further fractionation with dichloromethane and ethyl acetate. 2.5 Phytochemical analysis The qualitative and preliminary analysis of the plant extracts was performed according to the stan- dard procedures as described by Savithramma et al., (2011) [33]. 2.6 Total phenolic content (TPC) The Folin-Ciocalteu reagent method as described by Slinkard et al., (1977) was employed to mea- sure the TPC [34]. 20 µL gallic acid solution (10 to 80 µg/mL in methanol) and 20 µL of each ex- tract and fraction (500 µg/mL in 50% DMSO) were loaded in triplicates in a 96-well plate. To each bore, 100 µL of Folin-Ciocalteu reagent and 80 µL of Na2CO3 solutions were added and the 96-well plate was placed in the dark for 30 minutes. Then, a mi- croplate reader was used to measure the absorbance at 765 nm. A regression equation obtained from the absorbance versus concentrations of the gallic acid curve was used to calculate TPC. It was expressed as milligrams of gallic acid equivalent per gram (mg GAE/g) of extract and fraction. 2.7 Total flavonoid content (TFC) The AlCl3 colorimetric method as described by Marinova et al., (2005) was used to estimate the to- tal phenolic content [35]. 130 µL standard quercetin solution (10 to 80 µg/mL in methanol) and 20 µL of 500 µg/mL plant extracts and fractions in 50% DMSO were loaded in a 96-well plate. It was fol- lowed by adding 110 µL of deionized water to the bores containing plant extracts and fractions. Sub- sequently, each bore received 60 µL of ethanol, 5 µL of AlCl3, and 5 µL of CH3COOK. Then, the 96-well plate was placed in the dark. A microplate reader was used to measure the absorbance at 415 nm. A standard absorbance versus concentration of quercetin curve was constructed and TFC was calculated using its regression equation. It was ex- pressed as milligrams of quercetin equivalent per Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 245 gram (mg QE/g) of extract and fraction. 2.8 Antioxidant assay The DPPH assay was used to measure the antiox- idant activities [36]. 100 µL of plant extracts and fractions at concentrations 1.5625, 3.125, 6.25, 12.5, 25, and 50 µg/mL in 50% DMSO were loaded to the bores of a 96-well plate in triplicates. To each bore, 100 µL DPPH solution (0.1 mM in methanol) was added and the 96-well plate was placed in the dark. After 30 minutes, a microplate reader was used to measure the absorbance at 517 nm. Quercetin and 50% DMSO were used as the standard and control respectively. The following formula was used to cal- culate the percentage of radical scavenging Percentage scavenging = A1 −A2 A1 × 100 where A1=Absorbance of control A2=Absorbance of sample GraphPad Prism 9 software was used to calculate the IC50 (half maximal inhibitory concentration) values of extracts and fractions. 2.9 Antidiabetic assay The α-glucosidase enzyme inhibition assay was used to measure the in-vitro antidiabetic activities [37]. 20 µL each of 0.5 unit/mL α-glucosidase enzyme and 500 µg/mL plant extracts were premixed in- side the bores of a 96-well plate. Then, 120 µL of potassium phosphate buffer (pH 6.8) and 40 µL of pNPG substrate were added. After 15 minutes of incubation at 37 °C, the absorbance of the reaction mixture was measured at 405 nm. The reaction mixture with the volume of plant extract replaced by an equal volume of buffer solution was used as a control. The following formula was used to calcu- late the percentage of enzyme inhibition Percentage inhibition = A1 −A2 A1 × 100 where A1=Absorbance of control A2=Absorbance of sample 2.10 Antibacterial assay The agar disc diffusion method with slight modi- fications was used to measure antibacterial activ- ity [38]. The list of pathogenic bacteria, type, and ATCC numbers are given in Table 2. Bacterial broth cultures were prepared in nutrient broth me- dia and incubated overnight. Then, the inoculum (0.5 McFarland standards) was transferred to steril- ized Muller-Hinton Agar (MHA) plates using ster- ile cotton swabs. Discs impregnated with 50 µL of plant extracts and fractions (25 mg/mL) were placed on the surface of MHA plates using sterilized forceps. Once all discs were in place, the plates were covered with lids, inverted, and then incubated at 35 C for 18 hours. Then, zones of inhibition (ZOI) were measured. Ampicillin (1 mg/mL) and 100% DMSO were used as standard and control respec- tively. Table 2: Names of the bacteria, types, and ATCC number Bacteria Type ATCC Klebsiella pneumoniae Gram-negative 700603 Escherichia coli Gram-negative 25922 Bacillus subtilis Gram-positive 35021 Staphylococcus aureus Gram-positive 25923 2.11 Antifungal assay The antifungal activities in extracts and fractions were measured using the disc diffusion method [38]. The fungal species Fusarium solani (ATCC 11712) was used as a test organism. The broth culture of the test organism was prepared in nutrient broth media and incubated overnight and then the in- oculum (0.5 McFarland standards) was spread on potato dextrose agar (PDA) plates. Discs impreg- nated with 50 µL of 25 mg/mL plant extract and fractions were placed on the surface of the PDA plate. The plate was covered with lids, inverted, and then incubated at 35 0C for 18 hours. ZOIs were measured after incubation. Cycloheximide (20 mg/mL) and 100% DMSO were used as standard and control respectively. 2.12 Toxicity The brine shrimp lethality assay was used to mea- sure toxicity [39]. 2 mL of each of the plant extracts and fractions at concentrations 10, 100, and 1000 µg/mL in methanol were added to different test tubes in triplicates. 2 mL of methanol was used as the control. The solvent was evaporated to dryness Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 246 using a water bath. After this, artificial seawater (5 mL) was used to redissolve the leftover residue in each test tube. Then, 10 matured brine shrimp larvae were transferred to each test tube. The num- bers of surviving brine shrimp nauplii in each test tube were counted after 24 hours. The concentra- tion of plant extract and fraction that kills half of the test organisms (LC50) was calculated from the percentage mortality versus concentration curve. 2.13 Statistical analysis All the tests were performed in triplicates and the results are expressed as mean ± SE. The results are compared using one-way ANOVA followed by Tukey’s test with the help of SPSS version 29 soft- ware. The values with p < 0.05 were considered statistically different. 3 Results 3.1 Phytochemical Phytochemical analy- sis The preliminary phytochemical analysis found phe- nols, flavonoids, glycosides, saponins, tannins, ter- penoids, alkaloids, and carbohydrates in both root and leaf extracts of B. vulgaris (Table 3). Protein was detected only in the root extract of B. vulgaris. Table 3: Phytochemical constituents in plant extracts. Group of Compounds BRC BLC Alkaloids + + Phenols + + Flavonoids + + Glycosides + + Tannins + + Terpenoids + + Saponins + + Reducing Sugars + + Proteins + - BRC: Crude extract of roots, BLC: Crude extract of leaves. ‘+‘ present, ‘-‘ absent. BRC: crude extract of root, BRH: hexane fraction of root, BRD: dichloromethane fraction of root, BRE: ethyl acetate fraction of root, BRA: aqueous fraction of root, BLC: crude extract of leaf, BLH: hexane fraction of leaf, BLD: dichloromethane fraction of leaf, BLE: ethyl acetate fraction of leaf, BLA: aqueous fraction of leaf. # positive standard, Nd: values not determined. Values are the mean ± SE (n=3). Values followed by the different letters in the same columns are not significantly different at p < 0.05. Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 247 3.2 Total phenolic and flavonoid content B. vulgaris contained a significant amount of phe- nolics and flavonoids, and the observed results are presented in Table 4. The descending order of phe- nolic content was 84.35 ± 0.94 (BRE) > 83.39 ± 0.35 (BLC) > 71.51 ± 0.66 (BLD) > 42.04 ± 1.14 (BRD) > 39.73±0.28 (BLE) > 34.53±0.18 (BRH) > 33.04 ± 1.58 (BRA) > 26.51 ± 1.19 (BLH) > 25.75 ± 0.27 (BRC) > 15.87 ± 2.28 mg GAE/g (BLA). Similarly, the descending order of TFC was found as 150.48±1.10 (BRH) > 63.46±0.94 (BRA) > 39.39 ± 0.84 (BLD) > 24.27 ± 0.70 (BRE) > 19.34±0.93 (BRD) > 9.16±0.44 (BLC) > 7.94±0.18 (BRC) > 6.31±0.22 (BLE) > 2.49±0.13 (BLH) > 2.40± 0.06 mg QE/g (BLA). (a) (b) Figure 2: a) Correlation between total phenolic content and IC50 in DPPH assay. b) Correlation between total phenolic content and percentage of α-glucosidase inhibition. 3.3 Antioxidant potential IC50 value of ethyl acetate and dichloromethane fractions of leaves and ethyl acetate and hexane fractions of roots were determined as they dis- played the highest radical scavenging activity dur- ing screening. These fractions displayed remarkable antioxidant activity. The highest activity was ob- served in BRE with half maximal inhibitory con- centration of 3.92 ± 0.06 µg/mL followed by BLD (6.07 ± 0.16 µg/mL), BRH (15.5 ± 1.02 µg/mL), and BLE (30.41 ± 0.85 µg/mL) (Table 4). The IC50 of quercetin was 3.9 ± 0.07 µg/mL. Figure 3: Percentage radical scavenging against concentrations (µg/mL) of plant extracts, solvent frac- tions, and quercetin. Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 248 3.4 Antidiabetic potential The percentage of α-glucosidase enzyme inhibition for plant extracts and fractions at concentrations of 500 µg/mL is given in Table 4. The descending order of percentage enzyme inhibition is 35.36 ± 0.29 (BRH) > 32.16 ± 0.88 (BLA) > 29.29 ± 0.29 (BRE) > 26.64 ± 1.15 (BLE) > 24.66 ± 0.72 (BLH) > 21.01 ± 0.58 (BLD) > 17.93 ± 0.57 (BRA) > 14.07 ± 0.90 (BRD) > 10.31 ± 0.38 (BLC) > 5.35 ± 0.19 (BRC). 3.5 Antimicrobial activity Plant extracts and fractions did not display signifi- cant ZOI against bacterium and fungal strains used in the study. Photographs of Petri plates are given in Figure 4. Ampicillin used as a standard was found to be most potent against K. pneumoniae with ZOI 34 mm, followed by B. subtilis (32 mm), S. aureus (23 mm), and E. coli (23 mm). Cyclohex- imide used as standard in antifungal assay displayed a ZOI of 15 mm against F. solani (Table 5). Figure 4: Antimicrobial activities of plant extract and fractions against bacterial and fungal strains (a. Fusarium solani, b. Escherichia coli, c. Bacillus subtilis c. Staphylococcus aureus, e. Klebsiella pneumoniae.) Table 5: Antimicrobial activity (ZOI) shown by the extract and solvent fractions. Plant extracts K. pneumoniae S. aureus B. subtilis E. coli F. solani #Ampicillin 34 23 32 23 Nd #Cycloheximide Nd Nd Nd Nd 15 # positive standard, Nd: values not determined. 3.6 Toxicity against Brine shrimp nauplii The toxicities of plant extracts and fractions were measured in terms of their ability to kill brine shrimp nauplii and the results are given in Table 6. The plant extracts and fractions did not display significant toxicities in the assay. All the LC50 val- ues were well over 1000 µg/mL. The ascending or- der of LC50 is 1166.36 ± 100.21 (BLH) < 1195.83 ± 115.34 (BLE) < 1573.90 ± 145.26 (BLC) < 1807.78 ± 270.77 (BRC) < 1906.72 ± 285.36 (BRE) < 2337.71 ± 570.09 (BRD) < 2711.11 ± 170.05 (BLD) < 4079.84 ± 809.06 (BRA) < 5287.16 ± 393.51 (BRH) < 5692.20 ± 381.99 g/mL (BLA). Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 249 Table 6: Median lethal concentration (LC50) for plant extract and solvent fractions in brine shrimp lethality assay. Plant extracts LC50 (µg/mL) BRC 1807.78 ± 270.77a BRH 5287.16 ± 393.51b BRD 2337.71 ± 570.09a,c BRE 1906.72 ± 285.36a BRA 4079.84 ± 809.06b,c BLC 1573.90 ± 145.26a BLH 1166.36 ± 100.21a BLD 2711.11 ± 170.05a,c BLE 1195.83 ± 115.34a BLA 5692.20 ± 381.99b Values are the mean ± SE (n=3). Values followed by the different letters are not significantly different at p < 0.05. Figure 5: Percentage mortality versus concentrations (µg/mL) of plant extracts and solvent fractions. 4 Discusion The present study detected the presence of pheno- lics, flavonoids, glycosides, tannins, alkaloids, and carbohydrates in the methanol extract of the root. All these secondary metabolites are also reported in the literature [40, 41]. However, Ahmad et al., (2013) have reported the absence of proteins and saponins in root extract [40]. Extracts and frac- tions of B. vulgaris displayed a significant amount of phenolics and flavonoids in the present study. The amount of TPC and TFC in different plant extracts were significantly different from each other at p < 0.05. The crude extract of the root displayed higher TPC than the crude extract of the leaf, but TFC was found to be higher in the crude extract of the leaf than in the root. The slightly higher con- centration of flavonoids in leaf extract is due to the accumulation of flavonoids in leaves [42]. The high- est amount of phenolics was recorded for the ethyl acetate fraction of the root whereas the hexane frac- tion of the root contained the most flavonoid con- tent in the present study. Observed TPC of 25.75 ± 0.27 mg GAE/g for BRC was found intermedia- tory between previously reported values of 11.23 ± 0.13 mg GAE/g by Ahmad et al., (2013) and 39.75 ± 2.32 mg GAE/g by Edziri et al., (2019) [40, 43]. Similarly, the TFC of 7.94 ± 0.18 mg QE/g was found comparable to 6.41 mg QE/g as reported by Odoh et al., (2012) and lower than 20.73 ± 1.25 mg Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 250 QE/g as reported by Edziri et al., (2019) [43, 44]. The number and concentrations of phytochemicals in fruits and vegetables depend upon factors such as cultivar variation, agronomic practices, environ- mental stress, food processing, and storage meth- ods [45]. Appropriate measures should be taken to preserve the pharmacological activities of fruits and vegetables during the processing, storing, and cooking stages. Phenolic compounds exert health-promoting ef- fects and therapeutic activities such as antioxidant, antidiabetic, antimicrobial, anticancer, and anal- gesic functions [46]. These compounds are synthe- sized from shikimate pathways. The structural fea- ture of phenolics consists of one or more hydroxyl groups with carbon skeletons [47]. They mainly ex- ert their pharmaceutical effect due to their antiox- idant activity. The current investigation found a strong negative correlation (r = -0.804, p < 0.003) between total phenolic content and the IC50 value in the DPPH assay. The correlation is graphi- cally presented as a linear regression plot in Fig- ure 2(a). Such correlations are also reported in the literature [48]. Flavonoids are a type of phenolic compound with significant pharmacological impor- tance. They are effective against oxidative stress, bacteria, viruses, malaria, and HIV, and are respon- sible for natural pigmentation and flavor in many fruits and beverages [49]. The presence of vari- ous plant secondary metabolites and the high con- centrations of phenolics and flavonoids indicate the high potential of B. vulgaris in pharmacological re- search. The ethyl acetate, hexane and dichloromethane fractions of roots and leaves of B. vulgaris displayed high antioxidant activities. The percentage of free radical scavenging increased with the increase in the concentrations of plant fractions (Figure 3). An- tioxidant activities in the ethyl acetate fraction of the root and dichloromethane fraction of the leaf were similar to that of standard quercetin at p < 0.05 indicating the possibility of the presence of an active compound. The high antioxidant activity in B. vulgaris also makes it an ideal natural food preservative. The antioxidants in fruits and veg- etables are mainly due to polyphenols, carotenoids, or vitamins [50]. Further work involving GC-MS and LC-MS analysis could identify specific active principals responsible for bioactivity. Previously, Edziri et al., (2019) have reported IC50 values in ABTS and DPPH assay of methanol extract of root as 359.65 and 254.76 µg/mL respectively [43]. The DPPH IC50 value of 3.92 ± 0.06 µg/mL for BRE was comparable to the IC50 values of 2.32 ± 0.72 µg/mL and 2.20 ± 0.72 µg/mL for root and leaf extracts reported in the literature [12]. α-Glucosidase inhibitors competitively bind with α-glucosidase enzyme and reduce the rate of disaccharide hydrolysis and subsequent glucose ab- sorption in the blood [51]. Thus,α-glucosidase en- zyme inhibition assay was used in the present study to measure antidiabetic activity. The plant extracts and fractions displayed weak inhibition activities in the present study. However, a previous study by Mzoughi et al. (2019) has reported the IC50 val- ues for ethanolic leaf extract as 0.13 mg/mL and 1.03 mg/mL in α-glucosidase inhibition assay and -amylase inhibition assay respectively [13]. Varia- tions in IC50 values may have arisen due to the dif- ferences in environmental stress, cultivar variation, or extraction solvent. Mechanism of antidiabetic activity in plants includes blocking of liver gluco- neogenesis, stimulation of insulin secretion and sen- sitivity, inhibition of glucagon secretion, prevention of glucose absorption in the intestine, and reabsorp- tion of glucose in the kidneys [52]. A previous study has found an increase in the number of β-cells of the islet of Langerhans in streptozotocin-induced diabetic rats on oral administration of 2 g/kg B. vulgaris extract for 28 days [53]. This indicates an- tidiabetic activity in the plant may involve stimula- tion of insulin secretion and inhibition of digestive enzymes. The α-glucosidase inhibition assay dis- played a mild positive correlation (r = 0.413, p < 0.024) with total flavonoid content. The graphical representation of such correlation is given in Figure 2(b). The leaf extracts and fractions lacked signifi- cant zones of inhibition against bacterium and fun- gal species at a concentration of 25 mg/mL. The findings in this study agreed with the observations made by Rauha et al., (2000) in which they reported the absence of activity against C. albicans, A. niger, E. coli, and S. aureus for 500 µg of 80% methanol extract of B. vulgaris tuber [54]. Another study reported ZOI of 2 mm against S. aureus for leaf aqueous extract while methanol extract was found to be inactive [55]. Ethanolic extract of the roots displayed ZOIs of 12.54 ± 0.35, 8.37 ± 0.21, and 0 mm against S. aureus, B. cereus, and A. niger re- spectively in the study conducted by El-Beltagi et al., (2018) [14]. Thus, there are differences in the antibacterial activity reported in various studies. These differences may be due to variations in the number and concentration of secondary metabolites caused by genetic and environmental factors, the solvent used for extractions, concentrations used, and bacterial susceptibility. The cytotoxic activity in plant extracts and frac- tions was measured using a brine shrimp lethality assay. The assay is a reliable method for estimat- ing preliminary cytotoxic activity. Lagarto Parra et al., (2005) have reported a significant correlation between the brine shrimp assay and oral lethality in mice [56]. In the present study, all LC50 val- ues were higher than 1000 µg/mL. So, the plant is Sabina Khatri et al./ BIBECHANA 21 (2024) 241-253 251 relatively safe for human consumption, a fact evi- dent from its worldwide cultivation and inclusion in kitchens. Observed percentage mortality increased with the increase in concentration of plant extracts as shown by Figure 5. 5 Conclusion The medicinal food B. vulgaris contains impor- tant phytochemicals such as alkaloids, phenols, flavonoids, glycosides, tannins, terpenoids, and saponins. The extracts and fractions of the plant possess high concentrations of phenolics and flavonoids and display significant antioxidant activ- ities. Thus, the consumption of the plant could reduce the risk of chronic diseases associated with oxidative stress. The plant displayed weak α- glucosidase inhibition activity that was significantly correlated with total flavonoid content. The plant is found to be inactive against F. solani, K. pneu- moniae, E. coli, B. subtilis, and S. aureus. It is not toxic to the brine shrimp nauplii. Thus, the inclu- sion of B. vulgaris in the diet will have potential pharmacological benefits due to the presence of im- portant phytochemicals. Further isolation, purifi- cation, and characterization of active metabolites coupled with in-vivo studies may lead to increased use of the plant as a medicinal food. 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Comparative study of the assay of Artemia salina l. and the estimate of the medium lethal dose (LD50 value) in mice, to deter- mine oral acute toxicity of plant extracts. Phytomedicine, 8(5):395–400, 2001. Introduction Materials and Methods Chemicals and reagents Equipment Plant collection and identification Extraction and fractionation Phytochemical analysis Total phenolic content (TPC) Total flavonoid content (TFC) Antioxidant assay Antidiabetic assay Antibacterial assay Antifungal assay Toxicity Statistical analysis Results Phytochemical Phytochemical analysis Total phenolic and flavonoid content Antioxidant potential Antidiabetic potential Antimicrobial activity Toxicity against Brine shrimp nauplii Discusion Conclusion