Green Synthesis of Magnesium Nanoparticles From Native Neotropical Savannah Plants For Scalable Nanophytocosmetic Formulations

Summary

This work describes a protocol for the green synthesis of magnesium nanoparticles using Stryphnodendron adstringens plant extract, a species from the Native Neotropical Savannah, followed by their incorporation and evaluation in a cosmetic serum formulation.

Abstract

This study describes a protocol for the green synthesis of magnesium-based nanoparticles using ethanol extracts of Stryphnodendron adstringens, a plant species native to the Neotropical savanna, and their incorporation into nanophytocosmetic formulations. The procedure includes collection and preparation of plant material, followed by ethanol extraction, in which phytochemicals act as reducing and stabilizing agents for magnesium ions. Nanoparticles are synthesized under controlled conditions using magnesium chloride and plant extracts, enabling an environmentally friendly process without the use of toxic reducing agents. Nanoparticle properties are characterized using dynamic light scattering and atomic force microscopy to assess size distribution, morphology, and colloidal behavior. The protocol further describes the incorporation of the synthesized nanoparticles into a cosmetic serum formulation. Stability is evaluated under controlled storage conditions by monitoring organoleptic properties, pH, and viscosity. This method provides a reproducible and sustainable approach for producing plant-mediated magnesium nanoparticles and assessing their application in cosmetic formulations.

Introduction

The Cerrado phytogeographic domain, which comprises the largest Neotropical savanna formation in South America, represents the most biodiverse tropical savanna region in the world. This ecosystem harbors thousands of plant species rich in secondary metabolites such as flavonoids, polyphenols, terpenes, and phenolic acids, many of which exhibit documented antioxidant, anti-inflammatory, and photoprotective activities of interest to the cosmetic and pharmaceutical industries^1,2,3,4. These phytochemicals are widely incorporated as high-value active ingredients in functional cosmetic formulations. In addition to their biological activity, the hydroxyl and carbonyl functional groups present in many polyphenols and flavonoids enable them to act as natural reducing and stabilizing agents for metal ions in solution^5,6. This chemical property has been increasingly explored in green nanotechnology for the synthesis of metal nanoparticles, representing a promising and underexplored opportunity for developing sustainable cosmetic formulations derived from Neotropical biodiversity^2,3,4,5,6.

Traditionally, nanoparticles (NPs) have been produced through top-down approaches, in which bulk materials are broken down into nanosized particles using physical methods such as laser ablation or sputtering, and bottom-up approaches, in which NPs are built from smaller units using chemical or biological routes⁷. While chemical and physical methods are effective, they often rely on toxic reagents, high energy consumption, and complex equipment. This has led to the rise of green chemistry approaches, which emphasize sustainability, eco-friendliness, and safety in nanomaterial production⁶.

Within this framework, green synthesis of NPs has gained significant attention. The capacity of plant phytochemicals to act simultaneously as reducing, stabilizing, and capping agents, enabling the efficient synthesis of metal and metal oxide NPs, combined with the limitations of conventional methods, which require specialized equipment, extreme conditions, and generate toxic waste, justifies the development of a green synthesis protocol that operates under moderate conditions, is scalable, and produces no hazardous byproducts^6,7,8,9. Among the metal oxide NPs explored through this strategy, magnesium nanoparticles (MgNPs) present significant advantages over silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnO NPs), since MgNPs show no tendency toward bioaccumulation, exhibit lower cytotoxicity in human cells, and are recognized by the United States Food and Drug Administration (FDA) as generally safe substances for human applications^10,11. Additionally, MgNPs synthesized using Cerrado plant extracts are expected to combine the antioxidant and antibacterial properties of magnesium with the bioactive phytochemicals present in the extract, thereby enhancing their cosmetic functional value compared to conventionally synthesized counterparts¹⁰.

In the cosmetic industry, the application of nanotechnology has revolutionized product development by enhancing the delivery, stability, and efficacy of active ingredients¹¹. NPs can penetrate deeper into the skin, provide controlled release, and protect sensitive compounds from degradation, making them highly valuable for formulations targeting skin aging, pigmentation, acne, and sun protection¹⁰. Despite these documented advantages, the green synthesis of MgNPs using native plants from Neotropical savannas as reducing agents for cosmetic applications remains largely unexplored and lacks methodological standardization, in contrast to what has been widely reported for plants from Asia, Africa, and Europe^11,12,13,14. Most existing studies pay limited attention to the incorporation of NPs into functional cosmetic formulations and to their stability under controlled storage conditions^11,14,15. The present protocol helps fill this methodological gap by integrating, in a single, reproducible methodology, the green synthesis of MgNPs from Neotropical flora, their incorporation into a nanophytocosmetic facial serum, and the comprehensive evaluation of their physicochemical and functional stability.

The overall goal of this protocol is to describe a standardized, reproducible, and scalable methodology for the green synthesis of MgNPs using Stryphnodendron adstringens, a plant native to the Cerrado Neotropical savannas, as both a reducing and stabilizing agent, thereby leveraging its intrinsic bioactive properties in a facial serum formulation. The development of MgNPs synthesized from S. adstringens for topical human applications constitutes a novel nanoformulation currently protected under Brazilian patent application (BR 10 2025 015416 1). The proposed experimental design represents a sustainable and biocompatible approach to NP production, avoiding the use of toxic reducing agents commonly employed in conventional NP synthesis, while facilitating the incorporation of phytochemicals with potential biological activity. This protocol is particularly suitable for researchers seeking environmentally friendly methods for nanoparticle fabrication for cosmetic, pharmaceutical, or biomedical applications, especially when plant extracts rich in flavonoids and polyphenols are available. Since these compounds act as natural reducing and stabilizing agents, the protocol can be readily adapted to other plant species with comparable phytochemical profiles, allowing its application to native flora from different biogeographic regions based on the local availability of plant material.

Protocol

1. Plant material

1. Selection and documentation
   1. Select healthy S. adstringens plants growing in representative areas of the natural environment for bark collection. Record the sampling location, collection date, geographic coordinates using a GPS device, and local environmental conditions at the time of collection.
   2. Perform botanical identification using specialized taxonomic literature for Neotropical Savannah species. Confirm the identification by consulting a qualified taxonomist¹⁷.
   3. Collect and separate representative plant material for herbarium documentation, including vegetative and reproductive structures (e.g., leaves, flowers, fruits, and small branches).
      NOTE: Collect herbarium material separately from the bark used for experimental procedures. Press and dry the plant material using standard herbarium techniques, mount voucher specimens, and deposit them in a recognized herbarium to ensure traceability.
2. Bark collection
   1. Collect the bark from the trunk of the S. adstringens tree at BBCH (Biologische Bundesanstalt, Bundessortenamt, and Chemische Industrie) stages 31–39, corresponding to the vegetative growth phase as described in previous phenological studies^16,17.
      NOTE: Store the collected material in a dark bag to prevent the degradation of secondary metabolites and to prevent the degradation of secondary metabolites.
3. Moisture determination
   1. Weigh the bark samples using an analytical balance. Divide the material into three replicates and record the initial weights.
   2. Place the bark samples in an oven at 105 °C. Dry the samples until they reach a constant weight.
      NOTE: Constant weight is achieved when two consecutive measurements differ by no more than 1 mg, indicating complete moisture removal.
   3. Remove the samples from the oven, allow them to cool to room temperature, and record the final weight. Calculate moisture content and dry matter using the following equations:
      
      Dry matter (%) = 100 - Humidity (%)
      Where:
      W1 = weight of the empty container
      W2 = weight of container + sample before drying
      W3 = weight of container + sample after drying

2. Extract preparation

NOTE: All procedures involving hazardous materials were conducted in accordance with internationally recognized chemical safety guidelines¹⁸.

1. Rinse the freshly collected bark with running water to remove soil, dust, and surface impurities.
2. Place the bark samples in an oven at 36 °C for 72 h. Grind the dried bark using a knife mill to obtain a fine powder.
3. Weigh 200 g of powdered bark and transfer it to an Erlenmeyer flask. Add 1000 mL of 98% ethanol.
   CAUTION: Ethanol is highly flammable. Handle away from ignition sources and work in a well-ventilated area.
4. Seal the flask and keep it at room temperature for 72 h, protected from light. Agitate the mixture once every 24 h.
5. Filter the extract first through a metal mesh and then through cotton using a funnel to remove residual debris.

3. Evaluation of antioxidant activity

1. Prepare a 100 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) solution in methanol.
   CAUTION: Methanol is toxic and highly flammable. Handle in a fume hood and wear appropriate personal protective equipment. DPPH is irritant and may be harmful if inhaled or ingested. Avoid direct contact and inhalation.
   NOTE: Protect the solution from light by performing the assay under low-light conditions and caution with the handling of methanol and DPPH, always perform them with gloves.
2. Dilute the ethanolic S. adstringens bark extract in methanol to obtain concentrations ranging from 0.1% to 5% (v/v). Prepare vitamin C solutions at the same concentrations as a positive control.
3. Dispense 100 µL of DPPH solution into each well of a 96-well plate.
4. Add 50 µL of extract or vitamin C solution to each well.
   NOTE: Use DPPH + methanol as the negative control and methanol alone as the blank. Perform all measurements in quadruplicate.
5. Incubate the plate for 30 min at room temperature in the dark.
6. Measure absorbance at 515 nm using a spectrophotometer.
7. Calculate radical scavenging activity using the following equation:
   
   Where:
   ABS control = absorbance of DPPH + methanol
   ABS sample = absorbance of DPPH + extract

4. Cell viability analysis

1. Culture normal human dermal fibroblasts (NHDF) in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Maintain cells at 37 °C with 5% CO₂.
2. Seed cells into 96-well plates at 3 × 10⁴ cells/well and incubate for 24 h.
3. Replace the medium with 100 µL of fresh medium and add 50 µL of extract solution. Incubate for 24 h.
4. Remove the medium and add 10 µL of MTT solution to each well. Incubate for 4 h to allow formazan formation.
   CAUTION: MTT reagent is toxic and should be handled with gloves and appropriate protective equipment. Avoid skin and eye contact.
5. Add 60 µL of solubilization solution (HCl in isopropanol) to dissolve the crystals.
   CAUTION: Hydrochloric acid is corrosive and can cause severe burns. Handle with appropriate protective equipment and work in a fume hood. Isopropanol is flammable and should be handled away from ignition sources in a well-ventilated area.
6. Measure absorbance at 540 nm using a spectrophotometer.
   NOTE: Cell viability assays were conducted with treated and untreated control groups, with each condition analyzed in quadruplicate wells.
7. Calculate cell viability using:
   Cell Viability (%) = (ABS Sample Extract/ABS Cell Control) x 100
   Where:
   ABS Cell Control = absorbance of untreated control wells containing cells and culture medium only.
   ABS Sample Extract = absorbance of wells containing cells treated with the plant extract.

5. Green synthesis of magnesium nanoparticles

1. Add 9890 µL of distilled water to a sterile reaction tube.
2. Add 100 µL of plant extract.
3. Add 10 µL of 1 M MgCl₂ to obtain a final concentration of 10 mM.
   CAUTION: Magnesium chloride may cause irritation. Handle with appropriate protective equipment and avoid contact with eyes and skin.
   NOTE: The base reaction consists of 1% (v/v) extract + 10 mM MgCl₂ in 10 mL total volume. To optimize NP formation, the extract concentration may be adjusted while maintaining the final volume and MgCl₂ concentration.
4. Mix thoroughly to homogenize the solution.
5. Incubate at 75 °C for 18 h.
   CAUTION: Elevated temperatures may cause burns. Use appropriate protective equipment when handling heated samples.
   NOTE: A color change may indicate nanoparticle formation.
6. Allow the mixture to cool to room temperature before characterization.

6. Nanoparticle characterization

1. Dynamic light scattering (DLS)
   1. Transfer ~750 µL of nanoparticle suspension into a measurement cell.
   2. Place the cell into the instrument and perform measurements at 25 °C.
   3. Acquire particle size data using an appropriate detection mode.
   4. Analyze each sample in triplicate with five sub-runs per measurement.
2. Atomic force microscopy (AFM)
   1. Deposit 10 µL of nanoparticle suspension onto freshly cleaved mica.
   2. Dry the sample in a desiccator for 2 h.
   3. Gently rinse the mica sheet three times with 20 µL of filtered distilled water. Return the sample to the desiccator and allow it to dry at room temperature for an additional 2 h.
      NOTE: Rinsing removes excess salts and improves image quality.
   4. Configure the atomic force microscope by performing instrument setup and laser alignment according to the manufacturer’s instructions. Acquire surface images of the NPs with a scan area of 500 × 500 nm.
      NOTE: AFM is used to qualitatively assess surface roughness and visualize the morphological features of the MgNPs synthesized from S. adstringens, generating high-resolution three-dimensional surface images.

7. Formulation of nano-phytocosmetic

1. Heated phase
   1. Weigh 29.7 g of purified water, 0.50 g of hydroxyethylcellulose (HEC), and 3 g of glycerin into separate containers.
   2. Place the water in a magnetic stirrer and heat to 70–75 °C with gentle stirring.
   3. Add HEC gradually to the heated water while stirring to avoid clumping.
      NOTE: Hydration may require 15–30 min.
   4. Add glycerin and mix until homogeneous.
2. Cool-down phase
   1. In separate beakers, weigh 30 g of purified water, 3 g niacinamide, 33.3 g of MgNPs synthesized from S. adstringens, 0.3 g of low molecular weight hyaluronic acid, and 0.2 g of benzyl alcohol and dehydroacetic acid to form the cool-down phase.
   2. Dissolve niacinamide completely in water at room temperature.
   3. Add hyaluronic acid and mix until uniform.
   4. Add S. adstringens MgNPs and mix gently.
   5. Add preservative and mix.
3. Mixing
   1. Cool the heated phase to ~40 °C.
   2. Add the cool-down phase slowly to the heated phase while stirring.
   3. Measure pH of the formulation. For serum formulations containing niacinamide, a target pH of 5.0–6.5 is appropriate.
      NOTE: Niacinamide is stable around neutral to slightly acidic pH. Use small amounts of citric acid or sodium hydroxide to adjust pH if necessary.

8. Cosmetic stability evaluation

1. Preliminary stability (12 days)
   1. Prepare four replicate samples of the cosmetic serum in sealed containers.
   2. Subject the samples to six consecutive freeze–thaw cycles. Store the samples at 50 °C for 24 h and then at −5 °C for 24 h, completing one full cycle every 48 h.
   3. After each cycle, record organoleptic characteristics by visually inspecting the formulation for changes in appearance, color, odor, and phase separation.
   4. Measure pH of the four samples at room temperature, using a calibrated digital pH meter. Determine viscosity using a rotational viscometer according to the equipment manufacturer’s instructions.
      NOTE: Variations ≤10% are acceptable. Changes exceeding this threshold may indicate formulation instability and should be further investigated. If the formulation passes the preliminary study, conduct an accelerated stability assay.
2. Accelerated stability (90 days)
   1. Prepare eight samples of the cosmetic formulation. Store four samples at 50 °C and four samples at 4 °C.
   2. Evaluate at 0, 1, 7, 15, 30, 60, and 90 days.
   3. Evaluate and record organoleptic characteristics by visually inspecting the formulation for changes in appearance, color, odor, and phase separation.
   4. Measure pH of the samples at room temperature, using a calibrated digital pH meter. Determine viscosity using a rotational viscometer according to the equipment manufacturer’s instructions.
      NOTE: The stability assessment was conducted in accordance with guidelines established by the Brazilian Health Surveillance Agency¹⁹. In the United States, stability testing follows the FDA recommendations²⁰. In the European Union, stability evaluation complies with Regulation (EC) No 1223/2009²¹, which mandates inclusion of a Cosmetic Product Safety Report (CPSR). Annex I of this regulation requires documented stability data to support the safety and shelf life of the cosmetic formulation, with testing generally conducted in accordance with the harmonized guideline ISO/TR 18811:2018²², "Guidelines on stability testing of cosmetic products”.

Results

The drying process of plant bark is a crucial step in preserving bioactive compounds and ensuring their applicability in the food, pharmaceutical, and cosmeceutical industries. Conversely, inadequate drying can lead to excessive darkening, loss of aroma, microbial contamination, and degradation of bioactive compounds, thereby compromising the quality of the raw bark. Table 1 summarizes the criteria used to distinguish properly and improperly dried plant material.

Together, the...

Discussion

The protocol presented here describes a reproducible workflow for the green synthesis of MgNPs using S. adstringens extract and their incorporation into a nanophytocosmetic serum formulation. Several steps are critical to ensure reliable results. First, the collection and documentation of plant material must be carefully standardized, including accurate recording of geographic coordinates, environmental conditions, and phenological stage, since variability in plant physiology and secondary metabolite content can...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well plates	Sigma-Aldrich	M0812	Cell assays
Benzyl alcohol & dehydroacetic acid	Sigma-Aldrich	1,09,626	Preservative
Centrifuge tubes (15 mL / 50 mL)	Falcon (Corning)	430766	Sample handling
Citric acid	Sigma-Aldrich	C0759	pH adjustment
CO2 incubator	ThermoFisher Scientific	3110	CO2 incubator used for cell culture
Cotton-filter material	Local supplier	LH R/260	Filtration
Desiccator	merck	BAF424002141	Drying
DPPH (2,2-diphenyl-1-picrylhydrazyl)	Sigma-Aldrich	D9132	Antioxidant assay
Drying oven	NovaÉtica	400/5NDR	Oven for drying of plant material
Easy oven	SolidSteel	SSE-85L	Oven used for cosmetic stability assays
Erlenmeyer flasks 1000 mL	VIDROLABOR	76200B01000	Extraction
Ethanol (98%)	Dinamica-Quimica contemporanea	P.10.0051.015.36	Extraction solvent
Fetal bovine serum (FBS)	Gibco (Thermo Fisher)	ES-009-C	Supplement
Freezer	Gelopar	GTPC-575	Freezer used for stability assays
Glycerin	Bottica botanika	TBV084.003904.086	Humectant
Hyaluronic acid (low molecular weight)	Bottica botanika	J201220136	Hydrating agent
Hydrochloric acid (HCl)	Sigma-Aldrich	1,00,317	MTT solubilization
Hydroxyethylcellulose (HEC)	Bottica botanika	AH230641697	Thickener
Isopropanol	Sigma-Aldrich	100995	MTT solubilization
Knife Mill	7LAB	920Willye 4×4 Blades	Grinding leaves, roots, seed and tubers
Magnesium chloride (MgCl2)	Sigma-Aldrich	1,01,872	Nanoparticle synthesis
Magnetic stirrer–hot plate	IKA	5030000	Mixing
Methanol	Sigma-Aldrich	900641	DPPH assay
Mica sheets	ThermoFisher Scientific	NC9655733	AFM preparation
Microplate reader	ThermoFisher Scientific	Multiskan GO 1510-03581	Microplate reader
MTT reagent	Sigma-Aldrich	CT01-5	Cell viability assay
NHDF cells	BCRJ	0089	Human dermal fibroblasts
Niacinamide	Bottica botanika	211847	Active ingredient
Oven	termo scientific	PR305225M	Drying
Penicillin–streptomycin	Gibco (Thermo Fisher)	15140122	Antibiotic
pHmeter	Digimed	DM-220	Used for monotoring pH in stability assays
Pipettes and tips	Eppendorf	30078551	Liquid handling
Purified / distilled water	Merck (Milli-Q)	ZIX7010T0C	General use
Refrigerator	Metalfrio	VB40W	Refrigerator used for stability assays
Rotary viscometer	Novotest	VISC-5SA	Used for measuring visosity in stability assays
RPMI-1640 medium	Gibco (Thermo Fisher)	11875093	Cell culture
Scale	BEL Engeneering	HPBG-2285	Scale for weighing materials
Scanning Probe Microscope	SHIMADZU	SPM-9700HT	AFM, Scanning probe microscope
Sodium hydroxide (NaOH)	Dinamica-Quimica contemporanea	P.10.0594.024.00	pH adjustment
Spectrophotometer	ThermoFisher Scientific	A51119700C	Absorbance
Vitamin C (ascorbic acid)	Sigma-Aldrich	PHR1008	Positive control
Zetasizer Pro	Malvern Panalytical	Zetasizer Pro	For dynamic light scattering and zeta-size