Edible Potential of Allophylus villosus and Mycetia sinensis for Sustainable Functional Food Development

TL;DR

This study explores two wild plants used by the Chakma community in Bangladesh for their nutritional value and safety as potential functional foods.

Contribution

The study provides a novel evaluation of the nutritional and toxicological profiles of Allophylus villosus and Mycetia sinensis for sustainable food development.

Findings

Both plants showed high protein and fiber content with safe levels of anti-nutritional factors.

Ethanol extracts were moderately toxic, while other extracts were non-toxic and showed strong cell compatibility.

Heavy metal content was below detectable limits, indicating safety for consumption.

Abstract

The Chakma indigenous community in Bangladesh traditionally uses Allophylus villosus and Mycetia sinensis as wild food plants with therapeutic benefits against diabetes, pain, and other ailments. This study evaluates their nutritional properties, anti‐nutritional factors, heavy metal content, and cytotoxicity to assess their potential as functional foods and pharmacological agents. Leaves of
A. villosus
and aerial parts of
M. sinensis
were shade‐dried, powdered, and sequentially extracted with n‐hexane, ethyl acetate, and ethanol. Macronutrient profiling revealed high protein (12.72717% ± 0.00010% in
A. villosus
), dietary fiber (37.19% ± 0.06% in
M. sinensis
), and safe levels of phytic and oxalic acids. Inductively coupled plasma mass spectrometry analysis confirmed that toxic heavy metals were below detectable limits across all solvent extracts. Cytotoxicity was assessed using brine shrimp lethality bioassays, which categorized ethanol extracts as moderately toxic (LC50: 174.5–331.9 μg/mL), while n‐hexane and ethyl acetate fractions were non‐toxic. Vero cell line assays demonstrated > 95% viability, indicating strong cytocompatibility. Statistical analyses included triplicate experiments, with results expressed as mean ± SD and significance evaluated by one‐way ANOVA followed by Tukey's HSD post hoc test. Overall,
A. villosus
and
M. sinensis
may have potential as safe, nutrient‐rich candidates for future functional food and ethno‐pharmacological applications, pending further in vivo validation. Their sustainable use could contribute to improved dietary diversity, plant‐based health solutions, and biodiversity preservation, aligning with broader goals of nutrition and ecological resilience.

Nutritional assessment and toxicological evaluation of M. sinesis and A. villosus.

Figures

Leaves and herbarium sheet of Allophylus villosus.

Leaf and herbarium sheet of Mycetia sinensis.

Map showing the location of the collected plants. (A) Rangamati District, (B) Rangamati Sadar Upazila, (C) Sapchari union [image source: Banglapedia and Wikipedia].

A schematic workflow summarizing the evaluation of Allophylus villosus and Mycetia sinensis for their functional food potential.

Standard curve for carbohydrate determination.

Standard curve for phytic acid determination.

Standard curve for oxalic acid determination.

Trinocular microscopic analysis of solvent‐specific extracts of M. sinensis and A. villosus.

Comparative bar chart of carbohydrate and fiber contents (% dry matter) in wild food plants from Bangladesh, India, Ethiopia, Botswana, and global Amaranthus. Bars show midpoint values with ranges where available, highlighting fiber‐dominant Bangladeshi species, carbohydrate‐rich Moringa, energy‐dense Amaranthus grains, and balanced Indian and Ethiopian WFPs.

Tables

TABLE 1. Detection of extractable heavy metals and toxicity in six fractioned extracts.

Serial no.	Element	LOD (mg/kg)	LOQ	Reporting limit (mg/kg)	Result (mg/kg)
1	Aluminum (Al)	0.81	53.90	2.69	Not detected
2	Antimony (Sb)	0.12	7.79	0.39	Not detected
3	Arsenic (As)	0.01	0.61	0.03	Not detected
4	Barium (Ba)	1.19	79.53	3.98	Not detected
5	Boron (B)	0.80	53.42	2.67	Not detected
6	Cadmium (Cd)	0.01	0.60	0.03	Not detected
7	Cobalt (Co)	0.12	7.79	0.39	Not detected
8	Copper (Cu)	1.04	69.49	3.47	Not detected
9	Lead (Pb)	0.12	8.10	0.40	Not detected
10	Manganese (Mn)	0.77	51.41	2.57	Not detected
11	Mercury (Hg)	0.01	0.97	0.05	Not detected
12	Nickel (Ni)	1.01	67.47	3.37	Not detected
13	Selenium (Se)	0.09	6.02	0.30	Not detected
14	Strontium (Sr)	0.94	62.47	3.12	Not detected
15	Tin (Sn)	0.10	6.93	0.35	Not detected
16	Zinc (Zn)	1.05	70.18	3.51	Not detected
17	Chromium+3	0.00	0.15	0.01	Not detected
18	Chromium+6	0.002	0.15	0.01	Not detected

TABLE 2. LC50 values of extracts from Allophylus villosus and Mycetia sinensis.

Test sample	LC50 (μg/mL) mean ± SEM; SD; N = 10	Toxicity profile
Vincristine sulfate	1.0528 ± 0.09; ±0.284	Highly toxic
A. villosus , n‐Hexane	533.911 ± 0.1; ±0.316	Less toxic
A. villosus , ethyl acetate	1043.403 ± 0.11; ±0.348	Non‐toxic
A. villosus , ethanol	174.541 ± 0.103; ±0.326	Toxic
M. sinensis , n‐hexane	707.504 ± 0.104; ±0.329	Less toxic
M. sinensis , ethyl acetate	5268.151 ± 0.11; ±0.348	Non‐toxic
M. sinensis , ethanol	331.852 ± 0.104; ±0.329	Toxic

TABLE 3. Cell survival rate on Vero cells for extracts of Allophylus villosus and Mycetia sinensis.

Sample ID	Survival rate (%)
Vero	No cytotoxicity observed
Solvent−	100%
Solvent+	> 95%
A_N	> 95%
A_EA	> 95%
A_E	> 95%
M_N	> 95%
M_EA	> 95%
M_E	> 95%

TABLE 4. Summary of nutritional composition, anti‐nutritional content, and toxicity profile of Allophylus villosus and Mycetia sinensis.

Component	Allophylus villosus	Mycetia sinensis
Carbohydrate (mg/g)	0.4843 ± 0.0056	0.1386 ± 0.0032
Crude fat (%)	0.7783 ± 0.0063	1.23 ± 0.04
Protein (%)	12.72717 ± 0.00010	5.8653 ± 0.0073
Total energy (kcal/100 g)	58.11 ± 0.06	3.51 ± 0.04
Dietary fiber (%)	22.81 ± 0.11	37.19 ± 0.06
Moisture content (%)	6.224 ± 0.006	6.23 ± 0.06
Ash content (%)	6.631 ± 0.001	13.002 ± 0.014
Phytic acid (mg/kg)	124.18 ± 0.52	99.32 ± 1.08
Oxalic acid (mg/kg)	178.03 ± 0.06	198.34 ± 0.44
Heavy metals (ICP‐MS)	Within safe regulatory limits	Within safe regulatory limits
Ethyl acetate toxicity (LC50 μg/mL)	1043.403 (non‐toxic)	5268.151 (non‐toxic)
Ethanol toxicity (LC50 μg/mL)	174.541 (toxic)	331.852 (toxic)
n‐hexane toxicity (LC50 μg/mL)	533.911 (less toxic)	707.504 (less toxic)
Vero cell survival (%)	> 95%	> 95%

TABLE 5. Carbohydrate and fiber contents (% DM) of wild food plants across regions, highlighting contrasting nutritional strategies and their roles in food security and health.

Group/species	Carbohydrate (% DM, midpoint)	Range (min–max)	Fiber (% DM, midpoint)	Range (min–max)	Key nutritional note	References
Allophylus villosus (Bangladesh)	0.52	—	24.3	—	Extremely low‐carbohydrate, fiber‐dominant profile	—
Mycetia sinensis (Bangladesh)	0.15	—	39.7	—	Virtually carbohydrate‐free, exceptionally high fiber	—
Moringa oleifera leaves (Bangladesh)	51.5	47–56	7.8	6.0–9.6	Carbohydrate‐rich, moderate fiber, widely used functional food	Sultana 2020
Moringa oleifera leaves (Botswana)	46.5	—	9.1	—	Similar to Bangladesh Moringa, energy‐dense with moderate fiber	Masitlha et al. 2024
Amaranthus grains (global)	64	51–77	12.15	9.8–14.5	Energy‐dense cereal alternative, balanced with moderate fiber	Manyelo et al. 2020
Amaranthus leaves (global)	5.7	1.3–10.1	2.35	1.7–3.0	Low carbohydrate and fiber, mainly valued for micronutrients	Manyelo et al. 2020
Indian WFPs (10 species, average)	10.9	4.5–21.3	9.4	3.5–12.2	Nutrient‐balanced: moderate carbohydrate and fiber, often high protein	Seal et al. 2023
Ethiopian WFPs (average)	52.5	40–65	14	8–20	Mid‐range carbohydrate and fiber, reflecting biodiversity breadth	Rumicha et al. 2025

Full text

Introduction

1

Ethnomedicinal wild food plants are naturally growing plants that are not cultivated and are mostly utilized by local communities for nutritional and medicinal needs. These plants often play a crucial role in local diets and traditional healthcare systems, contributing to a deep understanding of the interactions between humans and plants (Asfaw et al. 2023; De Medeiros et al. 2021; Ghanimi et al. 2022; Jalali et al. 2024). Wild food plants (WFPs) are usually consumed during food shortages, economic hardship, severe weather conditions, droughts, famines, and political instability, serving as unconventional food sources that help meet nutritional needs and mitigate vulnerability to malnutrition and disease (Asfaw et al. 2023; Jalali et al. 2024). Many of these plants also possess therapeutic properties, supporting their role in traditional medicine and healthcare. Their ability to satiate hunger, provide essential nutrients, and support health underlines their significance in food security and sustainable nutrition strategies, particularly for vulnerable communities (Azam et al. 2014). Beyond nutrition, WFPs contribute to cultural heritage, ecological resilience, and local livelihoods. They preserve traditional knowledge, strengthen social connections, and generate income. At the same time, their role in cultural ecosystems helps mitigate environmental stress such as drought, soil degradation, biodiversity loss, and pollution. As accessible and cost‐effective food sources, sustainable utilization of WFPs supports both ecological conservation and community well‐being (Jalali et al. 2024). However, the decline of WFPs poses significant risks to biodiversity, food security, and cultural heritage, driven by climate change, habitat degradation, and modern agricultural practices. Urbanization and globalization further disrupt the intergenerational transmission of indigenous ecological knowledge, impeding conservation efforts. Historically, 10,000 plant species were used for food, but this has decreased to a few dozen, endangering agricultural diversity (Ghanimi et al. 2022; Jalali et al. 2024). Preserving and promoting traditional plant knowledge is vital for sustaining biodiversity, securing local food sources, and maintaining cultural traditions.

Medicinal plants, likewise, have been used for centuries to enhance flavor, improve digestion, and promote overall health, with a growing interest in nutraceuticals that provide disease‐preventing benefits. Studies highlight their role as functional foods that can improve human health (Rahmatullah et al. 2009). For example, Oats (Grundy et al. 2018) and Beans (Bell et al. 2024) can lower cholesterol, while Allium vegetables may reduce the risk of stomach cancer (Nicastro et al. 2015). Certain herbs can also help manage benign prostatic hyperplasia and diabetes (Rahmatullah et al. 2009). Low‐glycemic plant sources enhance weight regulation and mitigate obesity‐related pathologies (Landry et al. 2021), while secondary metabolites such as flavonoids and alkaloids demonstrate therapeutic efficacy against chronic conditions (Riaz et al. 2023). Dietary fiber from plants modulates gut microbiota composition and immune responses (Samtiya et al. 2021), and ash fortification augments antioxidant capacity, as observed in Moringa oleifera (Ukom et al. 2020). Together, ethnomedicinal WFPs and medicinal plants illustrate the intertwined roles of biodiversity, cultural heritage, and scientific innovation in advancing food security, public health, and sustainable development.

WFPs are generally consumed for nutrition and survival, whereas medicinal plants are used specifically to treat illnesses and physiological conditions. Despite the growing recognition of WFPs as nutraceutical resources, concerns around safety remain under examined. Although approximately 80% of the global population relies on plant‐based remedies, nearly 150,000 plant species are known to contain toxic compounds with adverse effects ranging from mutagenic and carcinogenic activity to organ‐specific toxicity (Anywar et al. 2021; Mugale et al. 2024). Given this, rigorous toxicological validation is needed to ensure the safe use of WFPs, particularly those used in vulnerable populations with limited access to formal healthcare systems.

This study addresses a significant gap in existing research by evaluating the nutritional composition, anti‐nutritional factors, and toxicity profiles of two wild food plants, Allophylus villosus and Mycetia sinensis, which are traditionally consumed and medically employed by the Chakma community in Bangladesh. A. villosus (Roxb.) Blume, a shrub from the Sapindaceae family, and M. sinensis (Hemsl.) Craib, a member of the Rubiaceae family, are used by the Chakma community as wild food plants, forming part of their diet and traditional ethnomedicine system for treating diabetes, as well as various types of pain and inflammation (Azam et al. 2024, 2025). Additionally, A. villosus has been ethnomedicinally used to treat menorrhagia in women, dysentery in cattle, debility in the young, and jaundice, while M. sinensis has been used for uncontrolled urination, weakness in adults, and meho in young individuals (Azam et al. 2024). To our knowledge, this is the first integrated analysis using standard nutritional assays, inductively coupled plasma mass spectrometry (ICP‐MS), brine shrimp lethality bioassays, and Vero cell line viability tests to validate their ethno‐pharmacological applications. By bridging indigenous knowledge with evidence‐based safety assessments, we aim to position these species as viable candidates for functional food development and plant‐derived therapeutics aligned with the UN Sustainable Development Goals.

Methods and Materials

2

Plant Species Collection

2.1

The leaves of A. villosus shown at Figure 1 and aerial parts of M. sinensis shown at Figure 2 were collected from the wet tropical biome of Sapchari Union, Rangamati Sadar Upazila, Rangamati Hill Tracts District, Bangladesh, during the rainy season of 2019, 2020, and 2021, with guidance of Chakma folk medicinal practitioners. The collection site, located at latitude 22°61′12″ and longitude 92°14′, is about 3 km from Furomon hill in Rangamati, bordered by Sukarchari to the north and Kawkhali Upazila to the south. Rangamati Sadar Upazila spans 546 km^2^ and comprises seven unions: Jibtali, Sapchari, Kutuk Chari, Banduk Bhanga, Balukhali, and Mogban. The demonstrated Figure 3 shows the location of the plant collection area. However, A. villosus , commonly known as “tin shingha pada,” and M. sinensis , commonly referred to as “Soidemi” by the Chakma community.

Leaves and herbarium sheet of Allophylus villosus.

Leaf and herbarium sheet of Mycetia sinensis.

Map showing the location of the collected plants. (A) Rangamati District, (B) Rangamati Sadar Upazila, (C) Sapchari union [image source: Banglapedia and Wikipedia].

A total of 6 kg in 2019, 4.5 kg in 2020 and 8 kg in 2021 of M. sinensis aerial parts and 8 kg in 2019, 7.5 kg in 2020 and 9 kg in 2021 of A. villosus leaves were gathered, washed, cleaned, drained, and shade‐dried (protected from direct sunlight) at the Laboratory of Pharmaceutical Biotechnology and Bioinformatics, Jashore University of Science and Technology. Herbarium sheets were prepared for identification and certification, authenticating in 2019 M. sinensis (Hemsl.) Craib (accession number: 54906) and A. villosus (Roxb.) Blume (accession number: 54909), by Dr. Mahbuba Sultana, Senior Scientific Officer and Associate Professor (Botany) at the Bangladesh National Herbarium, Mirpur, Dhaka, Bangladesh.

Plant Sample Preparation

2.1.1

The dried aerial parts and leaves were ground into a fine powder using a capacitor start motor (Wuhu Motor, China) at the Laboratory of Pharmaceutical Biotechnology & Bioinformatics, Jashore University of Science and Technology. The resulting powders, 570, 310, and 900 g of M. sinensis and 800, 750, and 85 g of A. villosus, were stored in separate glass jars thrice for further research. The process was performed in triplicate (2019–2021), yielding ~90 g dried material per kg of A. villosus and ~100 g dried material per kg of M. sinensis.

Nutrient Analysis

2.2

Total Carbohydrate Determination

2.2.1

Carbohydrate content was assessed using freshly prepared 2 g of marketed Anthrone reagent, ACS grade 97% (≤ 0.75 at 425 nm in sulfuric acid) mixed with 1 L concentrated 98% H_2_SO_4_ (Shakappa et al. 2022). A 200‐μg glucose solution served as the stock and blank for spectrometric analysis. For the standard curve, stock solutions of 10, 20, 40, 60, 80, and 100 μg were diluted to 1 mL with distilled water in separate test tubes. To prepare plant samples, 5 g of dried A. villosus and M. sinensis powder were dissolved in two different test tubes with 40 mL of hot water for 30 min. A 1‐mL aliquot from each sample and standard was mixed with 5 mL Anthrone reagent, capped, vortexed, and heated at 90°C for 17 min in a boiling water bath. After cooling to room temperature, spectrometric measurements were taken at 620 nm against a blank (distilled water and Anthrone reagent), with carbohydrate content determined using the calibration curve equation (Y = mx + C). All tests were performed in triplicate, and the average weights were reported in the results.

Total Fat Determination

2.2.2

Total fat content in M. sinensis and A. villosus was determined using Soxhlet extraction with n‐hexane. The dried powders were weighed (Ws; M. sinensis : 3.52 g, A. villosus : 5.775 g) and placed in extraction thimbles, sealed with fat‐free cotton, and immersed in 150 mL n‐hexane for M. sinensis and 250 mL n‐hexane for A. villosus . Soxhlet extraction was conducted at 60°C for 210 min, with solvent reduction cycles lasting in a rotary evaporator in 70°C for 5 min. After extraction, beakers were dried at 105°C for 60 min and cooled in a desiccator and weighed, W0. After transferring samples to the fat transfer beaker and weighing (We = 49.435 g for M. sinensis , 45.84 g for A. villosus ) (Hyvönen 1996). All tests were performed in triplicate, and the average weights were reported in the results. Crude fat percentage was calculated using the formula:

$$[eqn]$$

Total Crude Protein Determination

2.2.3

Total nitrogen (crude protein) was determined using the Kjeldahl method (Beljkaš et al. 2010). A 40% sodium hydroxide solution was prepared for distillation; while a 4% boric acid‐based titration solution was mixed and stored. Indicator solutions were made using methyl red and bromocresol green, in 100 mL of ethanol in separate flasks, then mixed with 1:5 ratios for titration. For digestion, 98 g K_2_SO_4_ and 2 g CuSO_4_·5H_2_O were finely blended, and 1.5 g of this mixture was added to the digestion tubes containing the samples (M. sinensis, 1.045 g; A. villosus, 1.1 g). Digestion was performed with 13 mL concentrated H_2_SO_4_ (98%, 0.1142 N) in a block heater at 380°C under a fume hood until the solution became transparent. For distillation, the digested samples were transferred to a distillation unit with 60 mL of distilled water and 40 mL of 40% NaOH. The distillate was collected in an Erlenmeyer flask containing 10 mL of indicator solution and titrated with 0.1142 N H_2_SO_4_ until a color change (purple to red) indicated complete conversion of nitrogen to ammonia. Blanks were prepared using 1.5 g of the K_2_SO_4_–CuSO_4_ mixture and 20 mL concentrated H_2_SO_4_. All analyses were performed in triplicate, and mean values were reported in the results.

Nitrogen percentage was calculated using:

$$[eqn]$$

where Va is the volume of acid used for sample titration, Vb is the volume for the blank, N is the normality of the acid, and W is the sample weight.

Total crude protein was determined using:

$$[eqn]$$

where F = 6.25 for all forages.

Total Crude Fiber Determination

2.2.4

Crude fiber content was determined using the acid‐alkali digestion method (Monro 1991). Solutions of 0.255 N 1.25% H_2_SO_4_ and 0.313 N 1.25% NaOH were prepared, and filter bags were labeled and weighed. Well‐ground samples (*W_1_ * =  M. sinensis : 2.015 g, A. villosus : 2.01 g) were placed in filter bags, sealed, and pre‐treated with 100 mL petroleum ether for fat extraction. Samples were then processed in a fiber analyzer, where they were digested in 1900–2000 mL of 1.25% H_2_SO_4_ solution for 40 min, followed by repeated rinses with warm water and 95% acetone. After drying at 105°C ± 2°C, cooled in a desiccator until room temperature, and weighed (W _ 2 _ = 36.74 g for M. sinensis , 37.45 g for A. villosus ). Samples were ashed at 600°C ± 15°C for 2 h, cooled in a desiccator until room temperature, and weighed (W _ 3 _ = 35.99 g for M. sinensis, 36.99 g for A. villosus). All tests were performed in triplicate, and the average weights were reported in the results. Crude fiber content was calculated using:

$$[eqn]$$

Energy Determination

2.2.5

One gram of protein provides four calories, 1 g of carbohydrate provides four calories, and 1 g of fat provides nine calories (Murphy 2013). The Association of Official Agricultural Chemists (AOAC) and the USDA have approved the 4‐4‐9 method for energy determination (Stilinović et al. 2020). All tests were performed in triplicate, and the average weights were reported in the results.

The formula is expressed as:

$$[eqn]$$

Ash

2.2.6

Empty porcelain crucibles were dried in a hot air oven at 105°C for 2 h and cooled in desiccators to room temperature before weighed (W 0; 39.275 and 39.165 g). M. sinensis (Ws; 3.075 g) and A. villosus (Ws; 3.845 g) samples were placed in separate pre‐weighed dried crucibles and dried again at 550°C overnight. After cooling in desiccators to room temperature, the final weights were recorded (Wc; M. sinensis : 39.675 g, A. villosus : 39.42 g) (Harris and Marshall 2017). All tests were performed in triplicate, and the average weights were reported in the results. Ash content was calculated using:

$$[eqn]$$

Moisture

2.2.7

Empty porcelain crucibles (39.16 g for M. sinensis, 37.03 g for A. villosus) were dried at 105°C in a hot air oven for 60 min and cooled in desiccators to room temperature according to AOAC International (2005), method 2001.12. Samples (Ws; M. sinensis : 2.18 g, A. villosus : 3.25 g) were placed in crucibles and weighed initially (W _ 0 _ = 41.34 g for M. sinensis , 40.25 g for A. villosus ). After placing the crucibles were drying at 105°C for 5 h with a dry and cool cycle before getting a constant weight. Final weights were recorded (W _ 1 _ = 41.2 g for M. sinensis , 40.05 g for A. villosus ) (Ahn et al. 2014). All tests were performed in triplicate, and the average weights were reported in the results. Moisture content was calculated using:

$$[eqn]$$

Anti‐Nutrient Analysis

2.3

Total Phytic Acid Determination

2.3.1

Phytate content in M. sinensis and A. villosus was determined using crude powder samples. Each 0.50 g sample was mixed with 10 mL of 2.4% HCl in 25‐mL Falcon tubes, shaken at 300 rpm at a flat shaker for 16 h at room temperature, and centrifuged at 3000 rpm at 10°C for 20 min. The filtered supernatant was mixed with 1 g NaCl, shaken at 300 rpm for 20 min, and allowed to settle at 40°C for 60 min. For final analysis, 3 mL of diluted sample was mixed with 1 mL of Wade reagent, centrifuged at 3000 rpm for 10 min at 10°C, and analyzed spectrophotometrically at 500 nm. A phytate standard stock solution (5–60 mg/mL, 320 mg/L sodium phytate) and deionized water were used to generate the calibration curve (Y = mx + C), with sodium phytate expressed in mg/mL per gram of dried powder (Park et al. 2006).

Total Oxalic Acid Determination

2.3.2

Oxalic acid content in M. sinensis and A. villosus was determined using a standardized extraction process. One gram of dried powder from each plant was boiled with 150 mL of water containing 27.5 mL of 6 M HCl and two drops of octanol for 25 min in a thermostable beaker. After cooling, the mixture was diluted to 250 mL and filtered using 41 no. 125‐mm Whatman filter paper. A 10‐mL aliquot of the filtrate was evaporated at 40°C–45°C in a vacuum oven and re‐dissolved in 10 mL of 0.01 M H_2_SO_4_. Oxalic acid standard solutions (0.5–3 mg/mL) were prepared for the calibration curve, and 1 mL of each sample extract was mixed with 9 mL of 0.5 mM K_2_Cr_2_O_7_ and 0.25 mM MnCl_2_ blank solution for spectrophotometric analysis. Oxalic acid concentration (mg/mL) was determined from the calibration curve equation (Y = mx + C) and expressed in terms of oxalic acid equivalent per gram of dried powder for both plants (Dominik 2000).

Inductively Coupled Plasma Mass Spectrometry (ICP‐MS) Analysis

2.4

Extract Preparation

2.4.1

The dried aerial parts of M. sinensis and leaves of A. villosus were ground into fine powder using a capacitor start motor (Wuhu Motor, China) at the Laboratory of Pharmaceutical Biotechnology and Bioinformatics, Jashore University of Science and Technology. Coarse powders ( M. sinensis : 750 g, A. villosus : 600 g; values represent the average weight obtained from triplicate experiments) were macerated in hexane (nonpolar), ethyl acetate (semi‐polar), and 98% ethanol (polar) over 8 days, with thrice daily stirring at room temperature. Each jar contained 150 g of powder, with M. sinensis processed in five jars and A. villosus in four jars. Maceration with 300 mL n‐hexane (Daejung Chemicals, Lot no. H3243SH1) was followed by filtration using 110‐mm Whatman filter paper (GE Healthcare) and concentration via rotary evaporation (RE‐100 PRO, DLAB Scientific Inc., China) at 40°C with 40 rpm. Residues were dried at room temperature and macerated with ethyl acetate (400 mL per jar, Daejung Chemicals, Lot no. E3180TF1) at room temperature and extracted with a rotary evaporator as per the previous procedure. Again, residues were dried at room temperature for further maceration with 98% ethanol (EMSURE, Merck KGaA), subsequently filtered and concentrated. The final extracts, n‐hexane, ethyl acetate, and ethanol, were stored in labeled glass vials: M. sinensis (M_N, M_EA, M_E; respectively yields 13, 28, 18 g) and A. villosus (A_N, A_EA, A_E; respectively yields 11, 34, 26 g), maintained at 4°C for experimental use.

Total Lead and Cadmium Determination

2.4.2

A 1 mg sample of each extract (M_N, M_EA, M_E, A_N, A_EA, A_E) was placed in microwave combustion unit tubes, labeled accordingly, and analyzed using the BS EN 16711‐1:2015 method for lead and cadmium detection. Under a fume hood, 9 mL of HNO_3_ and 3 mL of HCl were added to each sample. The tubes were positioned on a rotating plate and processed in a microwave. After cooling to room temperature, the combusted samples were transferred to 100‐mL volumetric flasks containing grade‐1 water, mixed thoroughly, and filtered through a 0.45‐μm filter. ICP‐MS analysis was performed using an Agilent 7700 ICP‐MS with plasma gas flow (15 L/min), auxiliary gas (argon) flow (1 L/min), carrier gas flow (1 L/min), nebulizer pump speed (0.2 rpm), sample uptake time (40 s), and stabilization time (50 s). A full quant table was used for precise measurement. Reference standard of 1000 ppm lead and cadmium purchased from Merck KGaA, Darmstadt, Germany. Total lead and cadmium concentrations (mg/kg) were calculated using:

$$[eqn]$$

where W = sample weight (g), V = final volume (100 mL), C = sample concentration from the instrument (ppb), and Df = dilution factor (if applicable).

Extractable Heavy Metal Determination

2.4.3

Heavy metal detection in M. sinensis and A. villosus extracts followed the BS EN 16711‐2:2015 European standard method. A 2‐mg test sample was incubated at 37°C ± 2°C with agitation at 60 ± 7 rpm in a shaker water bath for 60 ± 5 min. The samples were filtered through a 0.45‐μm nylon filter into ICP‐MS tubes with 100 μL of 65% HNO_3_ added using a pasteurized pipette (five drops). ICP‐MS analysis was conducted using an Agilent 7700 model with plasma gas flow (15 L/min), auxiliary argon gas flow (1 L/min), carrier gas flow (1 L/min), nebulizer pump speed (0.2 rpm), sample uptake time (40 s), and stabilization time (50 s). A full quant table was applied for precise measurement alongside commercially available heavy metals with 1000 ppm of each metal as blanks and standards. Heavy metal concentration (mg/kg) was calculated using:

$$[eqn]$$

where Cᵦₗ = blank solution concentration (μg/L), Cₛₘₚ = sample solution concentration (μg/L), V = final sample volume (mL), W = sample weight (g), and Df = dilution factor (if applicable).

Toxicity Determination

2.4.4

Heavy metal detection in M. sinensis and A. villosus extracts followed the BS EN 71‐3:2019 European standard method. Each fractioned extract was treated with 25 mL of 0.07 ± 0.005 N HCl solutions and incubated in a water bath at 37°C ± 2°C with agitation at 60 ± 7 rpm for 60 ± 5 min, without shaking. After incubation, the samples were filtered through a 0.45‐μm nylon filter, yielding 10 mL of filtrate for ICP‐MS analysis. The Agilent 7700 ICP‐MS was used with plasma gas flow (15 L/min), auxiliary argon gas flow (1 L/min), carrier gas flow (1 L/min), nebulizer pump speed (0.2 rpm), sample uptake time (40 s), and stabilization time (50 s). A full quant table was applied for precise measurement.

Cytotoxic Bioassay

2.5

Brine Shrimp Lethality Bioassay

2.5.1

Artemia salina nauplii were hatched in simulated seawater 1 L of 1 M (pH 8.5) sodium chloride solution with continuous oxygen supply for 2 days (Karim et al. 2020). Vincristine sulfate (10 mg) was used as the standard at varying concentrations (15.625–500 μg/mL), while six fractioned extracts were prepared using 1% DMSO (1 mg/mL) diluted in 5 mL of simulated seawater. Each solution was transferred into pre‐marked vials containing 10 live nauplii and incubated at room temperature for 24 h. The number of surviving nauplii was visually counted, and the lethal concentration (LC_50_) was calculated using LdP Line software (Ehab Soft). A control solution of 1% DMSO in simulated seawater was included for comparison.

Cell Line Cytotoxicity Bioassay

2.5.2

The cytotoxic potential of fractionated extracts from M. sinensis and A. villosus was evaluated using the Trypan Blue Exclusion Method (Strober 2015). Three solvent fractions (n‐hexane, ethyl acetate, and ethanol) were prepared from each plant species. For cytotoxicity assessment, 1 mg of each fraction was dissolved in 1% (v/v) dimethyl sulfoxide (DMSO) to obtain a stock concentration of 1 mg/mL. The extract solutions were designated as M_N, M_EA, M_E (M. sinensis) and A_N, A_EA, A_E (A. villosus). All solutions were sterile‐filtered and transported at 10°C maintained temperature to the Centre for Advanced Research in Sciences (CARS), University of Dhaka, for analysis. Dimethyl sulfoxide (1% v/v), used as the extraction vehicle, served as the negative (vehicle) control, while untreated cells maintained in complete culture medium were used as an additional baseline control. No chemical cytotoxic agent was included as a positive control, as the study focused on evaluating the safety profile of the plant extracts on non‐cancerous cells.

The Vero cell line, derived from African green monkey kidney epithelial cells, was cultured in sterile flasks with DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 1% penicillin–streptomycin, 0.2% gentamycin (1:1), and 10% fetal bovine serum (FBS). Cells were incubated at 37°C in a humidified incubator with pH 7.4, in 5% CO_2_. Subculturing was performed by washing cells with phosphate‐buffered saline (PBS), detaching them at 37°C for 2–4 min, and replenishing with fresh DMEM after 48 h of incubation.

For cytotoxicity evaluation, Vero cells were seeded into 48‐well plates at a density of 3.0 × 10^4^ cells per well in 200 μL of complete medium and incubated for 24 h to allow attachment. Subsequently, 50 μL of sterile‐filtered extract solution was added to each well. Cells treated with 1% DMSO served as the vehicle control, while untreated cells served as the baseline control. All treatments were performed in duplicate.

After 48 h of exposure, cells were harvested and washed three times with phosphate‐buffered saline. A 1‐mL aliquot of the resulting cell suspension was used for viability assessment.

All procedures were carried out under aseptic conditions in a biological safety cabinet (NU‐400E, Nuaire, USA). Cell counting was performed using a hemocytometer, and cell viability was assessed by mixing the cell suspension with trypan blue dye in a 1:1 ratio. Viable and non‐viable cells were counted under an inverted trinocular light microscope equipped with a camera (Optika, Italy), following the in‐house protocol at CARS (Nesa et al. 2021; Yu et al. 2018). Cell survival was expressed as the percentage of viable cells relative to the vehicle‐treated control.

Statistical Analysis

2.6

Before analysis, assumptions of normality and homogeneity of variances were assessed using the Shapiro–Wilk and Levene's tests, respectively. Differences among treatment groups were evaluated using one‐way ANOVA. Where significant differences were observed, Tukey's HSD post hoc test was applied to identify pairwise differences. A 95% confidence interval was used throughout. LC_50_ values were calculated using a linear dose–response model via LDPLINE software (https://www.ehabsoft.com/ldpline/), while standard curves and coefficients of determination (R ^2^) were generated in Microsoft Excel. All experiments were performed in triplicate, and results are presented as mean ± SD.

The total methodology can be represented by a schematic overview in Figure 4 of the analytical pipeline assessing the nutritional, toxicological, and pharmacological viability of ethnomedicinal wild food plants, A. villosus and M. sinensis.

A schematic workflow summarizing the evaluation of Allophylus villosus and Mycetia sinensis for their functional food potential.

Results

3

Nutritional Composition

3.1

The nutritional analysis of the dried crude powders of A. villosus and M. sinensis was conducted using standard estimation methods. The results are summarized as follows:

Carbohydrate Content

3.1.1

Using a calibration curve (Y = 0.0294X + 0.0374; R ^2^ = 0.9825), as shown in Figure 5, carbohydrate content was found to be 0.1386 ± 0.0032 mg/g in M. sinensis and 0.4843 ± 0.0056 mg/g in A. villosus .

Standard curve for carbohydrate determination.

Crude Fat

3.1.2

Determined using the fat estimation formula, crude fat content was 1.23% ± 0.04% in M. sinensis and 0.7783% ± 0.0063% in A. villosus .

Nitrogen and Protein Content

3.1.3

Titration volumes were 1.2 mL for M. sinensis and 2.1 mL for A. villosus , with a blank volume of 0.5 mL. Nitrogen equivalent percentages were 0.9378% in M. sinensis and 2.03634% in A. villosus . Using the crude protein equation, protein content was 5.8653% ± 0.0073% in M. sinensis and 12.72717% ± 0.00010% in A. villosus .

Total Energy

3.1.4

Energy value was calculated using Atwater conversion factors [4 × carbohydrate +4 × protein +9 × fat] (USDA). Results are expressed as mean ± SD, with uncertainty estimated by error propagation from triplicate measurements. Total energy content was 3.51 ± 0.04 kcal/100 g for M. sinensis and 58.11 ± 0.06 kcal/100 g for A. villosus .

Dietary Fiber

3.1.5

Determined via organic residue digestion, dietary fiber content was 37.19% ± 0.06% in M. sinensis and 22.81% ± 0.11% in A. villosus .

Moisture Content

3.1.6

Moisture levels were 6.23% ± 0.06% in M. sinensis and 6.224% ± 0.006% in A. villosus .

Ash Content

3.1.7

Ash content was recorded at 13.002% ± 0.014% in M. sinensis and 6.631% ± 0.001% in A. villosus .

Anti‐Nutrient Content

3.2

The total phytic acid and oxalic acid content in the dried crude powders of M. sinensis and A. villosus was estimated using calibration curves, with the results detailed below:

Phytic Acid Content

3.2.1

Using the calibration equation Y = 0.0042X + 0.014 (R ^2^ = 0.9969), as shown in Figure 6, the phytic acid equivalent was found to be 99.32 ± 1.08 in M. sinensis and 124.18 ± 0.52 mg/kg in A. villosus .

Standard curve for phytic acid determination.

Oxalic Acid Content

3.2.2

Based on the calibration equation Y = 0.21262X + 0.0216 (R ^2^ = 0.9845), as shown in Figure 7, the oxalic acid equivalent was determined to be 198.34 ± 0.44 mg/kg in M. sinensis and 178.03 ± 0.06 mg/kg in A. villosus .

Standard curve for oxalic acid determination.

Heavy Metal and Toxicity Assessment

3.3

Heavy metal detection and toxicity assessment of fractioned extracts (M_N, M_EA, M_E, A_N, A_EA, A_E) from M. sinensis and A. villosus were performed using ICP‐MS analysis following European standard methods:

• BS EN 16711‐1 (ICP‐MS): used to detect lead and cadmium.
• BS EN 16711‐2:2015 (ICP‐MS): applied for extractable heavy metal analysis.
• BS EN 71‐3:2019 + A1:2021 (ICP‐MS): used for toxicity evaluation.

All fractioned extracts tested negative for significant elemental contamination. The results are summarized in Table 1 and confirm the absence of harmful heavy metals in the extracts, supporting their safety for potential pharmaceutical and nutritional applications.

Brine Shrimp Lethality Bioassay

3.3.1

The cytotoxicity of different extracts from M. sinensis and A. villosus, as shown in Table 2, was evaluated using the brine shrimp lethality bioassay. LC_50_ values were determined for six fractioned extracts and compared with vincristine sulfate (LC_50_ = 1.0528 ± 0.09 μg/mL), which served as the reference standard.

LC_50_ values for M. sinensis extracts were:

• n‐Hexane (M_N): 707.504 ± 0.104 μg/mL (less toxic).
• Ethyl acetate (M_EA): 5268.151 ± 0.11 μg/mL (non‐toxic).
• Ethanol (M_E): 331.852 ± 0.104 μg/mL (toxic).

LC_50_ values for A. villosus extracts were:

• n‐Hexane (A_N): 533.911 ± 0.1 μg/mL (less toxic).
• Ethyl acetate (A_EA): 1043.403 ± 0.11 μg/mL (non‐toxic).
• Ethanol (A_E): 174.541 ± 0.103 μg/mL (toxic).

The experimental extracts exhibited negligible larvicidal activity compared to vincristine sulfate, which was categorized as highly toxic (LC_50_ < 20 μg/mL).

Values represent the mean of triplicate studies (mean ± SEM), with N = 10. LC_50_ classification:

• Highly toxic: < 20 μg/mL.
• Toxic: 20–500 μg/mL.
• Less toxic: 500–1000 μg/mL.
• Non‐toxic: > 1000 μg/mL.

Cytotoxicity Assessment on Vero Cells

3.3.2

The cell viability and cytotoxic efficacy of fractioned extracts from M. sinensis and A. villosus were evaluated using an in vitro cultured Vero cell line (African green monkey kidney cells) after 24 h of exposure. Extracts (M_N, M_EA, M_E, A_N, A_EA, A_E) showed no cytotoxicity, with Vero cell survival rates exceeding 95%, whereas the 1% DMSO‐containing solvent exhibited a 100% survival rate.

Although ethanol and ethyl acetate extracts of M. sinensis and A. villosus exhibited morphological alterations under microscopic evaluation shown in Figure 8, suggestive of solvent‐dependent deposition and potential cellular stress, these changes did not correspond to significant cytotoxicity in Vero cell assays. All extracts showed greater than 95% cell survival, indicating no acute toxicity under the experimental conditions (see Table 3). This suggests that observed structural effects may reflect non‐lethal cellular responses, residue accumulation, or extract‐specific compound solubility, rather than direct cytotoxic action.

Trinocular microscopic analysis of solvent‐specific extracts of M. sinensis and A. villosus.

Discussion

4

Our study on the nutritional and toxicological evaluation of A. villosus and M. sinensis, two ethnomedicinal wild food plants (WFPs) utilized by the Chakma community of Bangladesh, provides crucial insights into their potential as functional food sources. The study comprehensively assessed the nutritional composition, anti‐nutritional factors, and toxicity profiles using standard analytical methods. To effectively profile heavy metals, we employed inductively coupled plasma mass spectrometry (ICP‐MS). Additionally, we conducted brine shrimp lethality assays to evaluate acute toxicity along with Vero cell viability tests to determine cytocompatibility. These methodologies are consistent with the established practices for thorough toxicological profiling of medicinal plants, ensuring a robust evaluation of their safety and efficacy (Filipiak‐Szok et al. 2015; Ogbole et al. 2017).

A comprehensive analysis revealed that both A. villosus and M. sinensis contain essential macronutrients, emphasizing their potential as ethnomedicinal wild food plants. Carbohydrate levels were extremely low, measured at 0.1386 ± 0.0032 mg/g in M. sinensis and 0.4843 ± 0.0056 mg/g in A. villosus . Crude fat content was modest (1.23% ± 0.04% and 0.7783% ± 0.0063%, respectively), while protein levels reached 5.87% ± 0.01% in M. sinensis and 12.73% ± 0.0001% in A. villosus , contributing to total energy values of 3.51 ± 0.04 kcal/100 g and 58.11 ± 0.06 kcal/100 g. Dietary fiber analysis confirmed their fiber‐rich nature, with M. sinensis containing 37.19% ± 0.06% and A. villosus 22.81% ± 0.11%. Moisture content was comparable between the two species (6.23% ± 0.06% and 6.22% ± 0.006%), while ash content was notably higher in M. sinensis (13.00% ± 0.01%) than in A. villosus (6.63% ± 0.001%). Taken together, these findings highlight the nutritional value of both species. A. villosus is particularly rich in protein and energy, whereas M. sinensis stands out for its exceptionally high dietary fiber. This fiber‐dominant profile distinguishes them from many commonly consumed WFPs and positions them as functional foods with potential roles in digestive health, glycemic regulation, and chronic disease prevention. Table 4 summarizes a structured comparison of A. villosus and M. sinensis, highlighting their nutritional and toxicological properties. This overview provides valuable insights into their macronutrient composition, anti‐nutritional factors, and safety profile, thereby supporting their potential applications as functional food sources.

By contrast, Moringa oleifera leaves are carbohydrate‐rich and moderately fibrous. In Bangladesh, reported values range from 47% to 56% dry matter (DM) carbohydrate and 6%–9.6% DM fiber (Sultana 2020), while leaves grown in Gaborone, Botswana, averaged 46.5% carbohydrate and 9.1% fiber (Masitlha et al. 2024). Similarly, Amaranthus grains are energy‐dense, with carbohydrate levels ranging from 51% to 77% DM and fiber contents of 9.8%–14.5% DM. In contrast, Amaranthus leaves contain only 1.3%–10% DM carbohydrate and 1.7%–3% DM fiber (Manyelo et al. 2020), resembling the Bangladeshi species M. sinensis and A. villosus in their low carbohydrate content, but differing in their relatively low fiber. This limits their functional food potential compared to the fiber‐dominant Bangladeshi wild food plants (WFPs).

These findings resonate with the Ethiopian review by Rumicha et al. (2025), which documented 679 wild food plants (WFPs) consumed across diverse ecological zones, many contributing fruits, leaves, and roots to traditional diets. Ethiopian WFPs typically fall within mid‐range carbohydrate values (40%–65% DM) and moderate fiber levels (8%–20% DM), reflecting a balance between energy provision and digestive health. They also align with the study by Seal et al. (2023) in Meghalaya, India, which reported high protein and mineral content in species such as Nasturtium indicum and Spinacia oleracea , alongside carbohydrate levels of 45%–60% DM and fiber contents of 10%–13% DM. These Indian WFPs exemplify nutrient balance, combining energy, protein, and fiber in proportions that support both dietary diversity and functional health benefits.

Values are expressed on a dry matter (DM) basis. Midpoints were calculated as the arithmetic mean of reported minimum and maximum values ([min + max]/2). Where only a single value was available, that value was used directly as the midpoint. Ranges represent the lowest and highest values documented across studies. This approach standardizes heterogeneous datasets to enable cross‐regional comparison of wild food plants.

Collectively, the comparative evidence, as shown in Table 5 and Figure 9, underlines the diversity of nutritional strategies among WFPs. While Ethiopian and Indian species often provide balanced macronutrient profiles, Bangladeshi species such as A. villosus and M. sinensis stand out for their fiber‐dominant composition. This contrast emphasizes the need to incorporate region‐specific WFPs into food security and nutrition policies. Carbohydrate‐rich species provide caloric sufficiency, while fiber‐rich species improve functional health outcomes. Together, these plants demonstrate that WFPs are not merely supplementary foods but nutrient‐dense resources that can surpass conventional crops in both diversity and functional value.

Comparative bar chart of carbohydrate and fiber contents (% dry matter) in wild food plants from Bangladesh, India, Ethiopia, Botswana, and global Amaranthus. Bars show midpoint values with ranges where available, highlighting fiber‐dominant Bangladeshi species, carbohydrate‐rich Moringa, energy‐dense Amaranthus grains, and balanced Indian and Ethiopian WFPs.

Although A. villosus and M. sinensis have favorable macronutrient profiles, their dietary benefits are limited by the presence of anti‐nutritional compounds such as phytic acid (124.18 ± 0.52 mg/kg for A. villosus and 99.32 ± 1.08 mg/kg for M. sinensis ) and oxalic acid (178.03 ± 0.06 mg/kg for A. villosus and 198.34 ± 0.44 mg/kg for M. sinensis ). These compounds can inhibit the absorption of essential minerals and may pose health risks, such as mineral deficiencies and urinary stone formation, if these foods are consumed regularly without proper processing (Akter et al. 2020; Lee et al. 2023). These findings highlight the importance of considering anti‐nutritional factors in dietary planning and functional food research.

Beyond nutrition and anti‐nutrition, A. villosus and M. sinensis exhibited significant pharmacological properties. Phytochemical screening confirmed the presence of phenolics and flavonoids, while in vivo assays demonstrated glucose‐lowering and analgesic effects. In silico docking studies further identified bioactive compounds with strong affinities for metabolic and pain‐related enzymes, supporting their antidiabetic and anti‐inflammatory potential (Azam et al. 2024, 2025). These results extend the Ethiopian emphasis on ethnomedicine, where species such as Centella asiatica and Withania somnifera are valued for therapeutic properties, and complement the Indian validation of nutraceutical safety (Rumicha et al. 2025; Seal et al. 2023). Together, these studies highlight WFPs as promising candidates for functional food development and drug discovery, bridging traditional knowledge with modern pharmacological science.

Safety assessments of A. villosus and M. sinensis revealed no detectable heavy metals through ICP‐MS analysis referenced with 1000 ppm pure heavy metal, while brine shrimp lethality and Vero cell viability assays confirmed minimal cytotoxicity (> 95% survival). Although ethanol extracts exhibited moderate toxicity in brine shrimp assays, they remained non‐toxic to mammalian cells, highlighting solvent‐specific bioactivity rather than inherent risk. This is consistent with previous studies showing that ethanol efficiently extracts bioactive secondary metabolites—such as alkaloids, flavonoids, and saponins—that often induce acute toxicity in Artemia salina models (Meyer et al. 1982; Ekalu et al. 2021; Othman et al. 2020). These results are consistent with the Indian study, which found that antinutrients such as oxalate, phytate, tannin, and saponin were present in safe concentrations, while heavy metals were at levels below those considered harmful (Seal et al. 2023). In Ethiopia (Rumicha et al. 2025), emphasized the need for more systematic toxicological characterization, highlighting how the studies from Bangladesh and India provide critical assurance of safety. Collectively, these findings establish WFPs as safe dietary components when consumed traditionally or with appropriate processing.

Beyond safety, the Allophylus and Mycetia genera hold significant promise for functional food development and pharmaceutical applications due to their rich phytochemical profiles and proven bioactivity. Allophylus species, including A. cobbe and A. serratus , are known for their flavonoids, sesquiterpenes, and fatty acids, contributing to their traditional use in wound healing, ulcer treatment, and bone fracture recovery. Their edible fruits serve as valuable nutritional resources, offering antioxidant and tonic benefits. Despite extensive ethnomedicinal applications, research has primarily focused on leaves and fruits, while stems and roots remain underexplored (Chavan and Gaikwad 2016). Meanwhile, the Mycetia genus, particularly M. longifolia and M. cauliflora , contains alkaloids, flavonoids, tannins, glycosides, and polyphenols, supporting their traditional use in treating pain, inflammation, ulcers, and wounds. Experimental studies confirm their therapeutic properties, with M. cauliflora demonstrating anti‐inflammatory effects through PDK1 modulation in the NF‐κB signaling pathway. Systematic phytochemical analysis and clinical investigations could unlock new drug development avenues, particularly for cancer, cardiovascular disorders, diabetes, and inflammatory diseases (Jain et al. 2015; Jeong et al. 2019).

Substantially, the comparative evidence from Ethiopia, India, and Bangladesh illustrates a complementary framework for understanding the role of wild food plants. Ethiopia demonstrates the breadth of biodiversity and ecological adaptation, India provides depth through nutritional and toxicological validation, and Bangladesh integrates nutritional, toxicological, and pharmacological insights, bridging ethnomedicine with modern drug discovery. This unified perspective confirms that wild food plants are nutrient‐rich, climate‐resilient, and pharmacologically promising, yet remain underutilized in mainstream food systems. By placing A. villosus and M. sinensis within this broader global context, the study emphasizes the potential of localized ethnomedicinal plants to be recognized as safe and functional foods with therapeutic benefits. The convergence of evidence from Ethiopia, India, and Bangladesh strengthens the case for systematic documentation, toxicological validation, and pharmacological investigation of wild food plants across diverse regions. Such integration promotes their sustainable use for nutrition and health, reinforcing their importance as essential resources for both traditional communities and contemporary health systems.

Future Directions and Limitations

5

Wild food plants (WFPs) provide essential nutrients, reliable accessibility, and economic benefits, thereby playing a crucial role in ensuring food security. Their harvesting supports livelihoods, while their rich composition of carbohydrates, proteins, fats, minerals, and vitamins helps combat “hidden hunger.” Many WFPs possess medicinal properties, making them valuable for nutraceutical development. Despite their important nutritional profiles, their use is limited by anti‐nutritional factors and potential toxicity. Ensuring their sustainable utilization is vital for biodiversity conservation, food security, and public health (Tadesse et al. 2024). In recent decades, WFPs have regained attention for their nutritional and medicinal benefits, enhancing dietary diversity (Satter et al. 2016). The Food and Agriculture Organization (FAO) estimates that one billion people worldwide rely on wild foods for daily nutrition (Paul et al. 2020). Additionally, promoting wild food plants enhances dietary diversity, supports agriculture, strengthens local economies, enriches ecological knowledge, and preserves cultural heritage (Leisembi et al. 2024).

The findings from this study promote A. villosus and M. sinensis as promising candidates for functional food and medicinal applications. Future research should focus on optimizing processing techniques to enhance their bioavailability and minimize anti‐nutritional factors such as phytic and oxalic acids. Advanced in vivo and human clinical trials are necessary to validate their therapeutic efficacy. Additionally, standardized extraction methods and formulation strategies should be developed to ensure consistent health benefits. Computational modeling, such as functional food modeling and epigenomic insights, and pharmacokinetics studies, could further refine the understanding of their interactions with biological systems, enabling the design of more effective plant‐based therapeutics. Given their ethnonutritional significance, the incorporation of A. villosus and M. sinensis into sustainable food systems may contribute meaningfully to advancing key UN Sustainable Development Goals (SDGs). They contribute to SDG 2 (Zero Hunger) by enhancing food security, support SDG 3 (Good Health and Well‐Being) through their medicinal properties, promote biodiversity conservation in alignment with SDG 12 (Responsible Consumption and Production), and aid in preserving traditional plant knowledge and ecosystems, aligning with SDG 15 (Life on Land).

Despite their promising attributes, several limitations must be addressed before the widespread application of A. villosus and M. sinensis. The study primarily relied on in vitro, in silico, and animal model analyses, necessitating further clinical validation in human subjects. The presence of anti‐nutritional factors may limit direct dietary incorporation without appropriate processing. Bioactivity assessments were conducted on fractioned extracts, which may differ from the effects of whole‐plant formulations. Additionally, standardization of active compounds and dosage optimization is required to ensure consistent pharmacological outcomes. Environmental and agricultural factors influencing phytochemical composition need further investigation to maintain reproducibility across different regions. While molecular docking and computational simulations indicate strong interactions with metabolic enzymes, long‐term safety assessments are critical to rule out potential side effects. By addressing these challenges, A. villosus and M. sinensis can be effectively harnessed for plant‐based functional foods, medicinal formulations, and sustainable health interventions, supporting both traditional medicine and modern therapeutic advancements.

Conclusion

6

This study provides a comprehensive evaluation of A. villosus and M. sinensis, highlighting their nutritional composition and ethno‐pharmacological relevance. Nutritional profiling revealed essential macronutrients (proteins, carbohydrates, dietary fiber, and crude fat within expected ranges), while the presence of anti‐nutritional factors such as phytic and oxalic acids emphasizes the necessity for proper processing before consumption. Toxicity assessments, including ICP‐MS, brine shrimp lethality bioassays, and Vero cell line testing, confirmed that concentrations of toxic heavy metals were within safe regulatory limits and demonstrated high biocompatibility, with cell viability exceeding 95%. Notably, ethanol extracts exhibited moderate toxicity in brine shrimp assays, yet cellular assays suggested no acute cytotoxicity, indicating solvent‐dependent effects. Microscopic analysis revealed changes in morphology that were linked to the deposition of specific compounds. Although the precise mechanisms remain uncertain, these observations highlight the importance of solvent selection and extraction techniques in medicinal plant research. Taken together, the findings suggest that A. villosus and M. sinensis may have potential as functional food ingredients, though further in vivo studies on nutritional bioavailability and long‐term safety are required before definitive claims can be made. Their integration into sustainable food systems could contribute to global goals by enhancing dietary diversity, supporting safe plant‐based health solutions, and preserving biodiversity and traditional knowledge. Overall, this study bridges traditional plant knowledge with contemporary toxicological validation, positioning these species as promising candidates for future sustainable nutrition and ethno‐pharmacological applications.

Author Contributions

Md Nasir Ahmed: visualization, writing – original draft, writing – review and editing. Md. Nazmul Hasan: supervision, formal analysis, writing – review and editing, funding acquisition. Md. Nur Kabidul Azam: conceptualization, visualization, methodology, validation, investigation, writing – original draft, writing – review and editing, resources, data curation. Md. Rafiqul Islam: analysis, writing: original draft and review. Md. Iqbal Hossain: data curation and formal analysis. Mohammad Shahedur Rahman: supervision, formal analysis, validation, writing: review and editing.

Funding

This work was supported by the University Grant Commission (Grant: BMK/Scholarship/Research/College‐5/Ph.D/2018/710). The project was also supported by a grant from the Jashore University of Science and Technology (Grant: JUST/Research Cell‐112/FoBST‐05/2022‐23), and the Ministry of Science and Technology, Bangladesh (Grant: MoST/2020‐21/BS‐278).

Conflicts of Interest

The authors declare no conflicts of interest.