Multi-Target Anti-Aging Mechanisms of Multani Mitti (Fuller’s Earth): Integrating Enzyme Inhibition and Molecular Docking for Cosmeceuticals

Abstract

The growing demand for natural anti-aging ingredients necessitates scientific validation of traditional cosmetic materials. Multani Mitti (MM), a clay widely used in South Asian traditional skincare, lacks comprehensive chemical and biological characterization. This study employed a multi-analytical approach to investigate MM’s anti-aging potential through chemical analysis, enzyme inhibition studies, and in silico evaluations. Five commercial MM samples were pooled and analyzed using instrumental neutron activation analysis (INAA) and Gas Chromatography–Mass Spectrometry (GC-MS). INAA revealed silicon as the predominant inorganic constituent (169.3742 mg/g), while GC-MS identified 13 bioactive compounds, with Beta-sitosterol (15.45% area), Docosanamide (12.36% area), and Cyclohexasiloxane (9.80% area) being the most abundant. MM demonstrated significant enzyme inhibition against key aging-related enzymes, with notably strong effects on hyaluronidase (IC₅₀: 18 μg/mL) and tyrosinase (IC₅₀: 27 μg/mL), outperforming standard inhibitors. The antioxidant activity showed moderate effectiveness (IC₅₀: 31.938 μg/mL) compared to ascorbic acid (IC₅₀: 8.5 μg/mL). Molecular docking studies of identified compounds against hyaluronidase (PDB: 1FCV) and tyrosinase (PDB: 3NQ1) revealed Beta-sitosterol and Benzyl-piperazine-carboxamide as the most promising candidates, showing strong binding affinities (−8.5 and −8.6 kcal/mol, respectively) and favorable ADMET profiles. This comprehensive characterization provides the first scientific evidence supporting MM’s traditional use in skincare and identifies specific compounds that may contribute to its anti-aging properties, warranting further investigation for modern cosmetic applications.

1. Introduction

The skin, particularly facial skin, serves as a significant reflection of an individual’s overall health. Maintaining a clear and radiant complexion requires consistent skincare practices [1]. Historically, people have been conscious of their skincare routines, adapting them to their specific skin types. Even today, natural remedies remain popular, especially in rural and hilly regions where people continue to use plant extracts such as Aloe vera, tulsi, rose, and neem for their cosmetic benefits [2]. In many parts of Asia, particularly in Pakistan, India, Bangladesh, and Nepal, a unique clay known as MM has long been employed as a cost-effective and efficient skincare solution. Originating from Multan city in Pakistan, MM is prized for its purifying and beautifying properties, with particular emphasis on its anti-aging effects. This clay has been incorporated into skincare routines as a natural solution for maintaining youthful skin, commonly used in face masks [3,4,5]. The powdered form and its application as a face mask are illustrated in Figure 1. MM, also known as ‘Fuller’s Earth’, has been revered for its healing properties for centuries. Scientifically, it is composed of minerals such as montmorillonite, smectite, and bentonite clay [6]. The historical use of clay for therapeutic purposes dates back to ancient Egyptian, Mesopotamian, Chinese, and Indian civilizations, underscoring its enduring significance. Additionally, deposits of Fuller’s Earth are found in various regions globally, including the Americas, France, and Mexico [7,8,9]. The term ‘Fuller’s Earth’ originates from its historical use in the textile industry, where it played a crucial role in cleaning and thickening woolen fabrics—a process known as ‘fulling’. The individuals who specialized in this task were referred to as ‘fullers’, which gave rise to the name ‘Fuller’s Earth’ [10,11].

The mineral-rich composition of clay provides exceptional absorbent properties, making it versatile for medical, dietary, and cosmetic applications. Among clays, MM stands out for its remarkable ability to purify the skin, making it particularly beneficial for oily and sensitive skin types [12]. Due to its superior absorbent properties, MM effectively removes dead skin cells and excess oil from the skin and scalp. These properties, combined with its anti-inflammatory qualities, make it an excellent choice for both skincare routines and hair care. Furthermore, it tones the skin, minimizes the appearance of wrinkles, and serves as an effective deep pore cleanser, leaving the complexion refreshed and radiant [13,14,15,16].

Beyond its cleansing abilities for skin and hair care application in Pakistan, the practice of geophagy—specifically the consumption of MM clay—is widespread among women and children. Pregnant women are often advised to ingest this clay to address deficiencies in essential minerals [4]. Research has also explored developing a reagent for latent fingerprint visualization using MM [10]. Due to its myriad benefits for both skin and hair care—as well as its perceived health advantages—MM has become a highly sought-after cosmetic product. It is commonly marketed as an affordable and readily available cosmetic ingredient with a long history of traditional topical use in Ayurvedic practices, supported by widespread consumer acceptance in South Asian markets [17,18,19,20]. However, comprehensive scientific safety evaluations remain limited. In 2020, the market value of MM was estimated at USD 25.3 million, with a projected compound annual growth rate (CAGR) of 6.3% from 2022 to 2028. This progression reflects an increasing demand for natural beauty products and underscores MM’s reputation for enhancing skin complexion while minimizing blemishes, solidifying its significance in the skincare industry [21,22].

Despite its widespread use in cosmetics and traditional medicine practices, the biological mechanisms underlying its effects on the skin remain largely unexplored. As skincare solutions increasingly prioritize antioxidant, anti-aging, and antimicrobial properties, this research aims to evaluate the antioxidant and enzyme inhibitory activities of MM and identify its potential therapeutic applications in the management of aging-related conditions. Previous studies have indicated that MM lacks significant antimicrobial activity [23], prompting us to shift our focus towards its potential antioxidant and anti-aging properties.

Skin aging is closely associated with the activity of enzymes such as elastase, collagenase, hyaluronidase, and tyrosinase [24]. Elastase and collagenase degrade elastin and collagen, respectively, which are vital structural proteins that maintain skin elasticity and firmness; their overactivity leads to wrinkles and loss of skin integrity [25]. Hyaluronidase breaks down hyaluronic acid, a key molecule responsible for skin hydration and volume, contributing to dryness and sagging when excessively active [26]. Tyrosinase catalyzes melanin production, and its overexpression can cause hyperpigmentation and uneven skin tone [27]. Inhibiting these enzymes can, therefore, slow down the degradation of skin matrix components and regulate pigmentation, making enzyme inhibitors promising targets for anti-aging therapies and cosmetic applications [28].

While a variety of well-established inhibitors—such as EGCG for collagenase and elastase, tannic acid for hyaluronidase, and kojic acid for tyrosinase—are routinely used in laboratory assays and cosmetic formulations, there remains significant interest in identifying new, naturally derived alternatives [29,30]. Given Multani Mitti’s widespread use in traditional skincare, our study aimed to systematically evaluate its potential as a natural source of enzyme inhibitors relevant to skin aging. For comparative purposes and to validate our experimental approach, these known inhibitors were included as positive controls in all assays. This allowed us to directly benchmark the inhibitory effects of MM clay against industry standards, highlighting its promise as a multifunctional, natural ingredient for anti-aging cosmetic applications.

Building on the importance of enzyme inhibition in skin aging and the search for natural alternatives, this study begins with a comprehensive chemical analysis to elucidate the complete organic and inorganic composition of MM powder using Gas Chromatography–Mass Spectrometry (GC-MS) and instrumental neutron activation analysis (INNA). We will then evaluate the inhibitory activity of these compounds against enzymes linked to the aging process of the skin—namely, elastase, collagenase, hyaluronidase, and tyrosinase—through both in vitro assays and in silico molecular modeling techniques. These methods not only allow for active pinpoint compounds but also provide valuable insights into their underlying molecular mechanisms. The outcomes of this research will contribute to understanding MM’s potential as a cosmeceutical agent that could guide the development of innovative antioxidant and anti-aging skincare products.

2. Materials and Methods

2.1. Sample Collection

The soil sample was procured in a dried powdered form from 5 different well-known ISO-certified cosmetics brands suppliers (https://www.daraz.pk/tag/multani-mitti/; accessed on 8 March 2024, https://saeedghani.pk/products/multani-mud-powder-100gm?_pos=1&_sid=622ee6225&_ss=r; accessed on 8 March 2024, https://karachipansar.pk/product/multani-mitti; accessed on 8 March 2024, https://pansarionline.pk/natural-multani-mud/; accessed on 8 March 2024, and https://somethingorganic.pk/product/multani-mitti-powder/; accessed on 8 March 2024) of Pakistan. They represent the actual products consumers use in cosmetic applications, making our findings directly relevant to real-world usage. It was packaged and marketed as MM powder, one of the top-selling products in this category. All these were mixed to make a single sample for a further overall investigation. These commercial samples were pooled to minimize batch-to-batch variability and obtain a representative composite reflecting the general MM composition available in the market. This approach follows established analytical chemistry practices for improving detection limits in GC-MS analysis and aligns with our primary objective to evaluate the collective anti-aging potential of MM as an ingredient class rather than promote specific brands.

2.2. INAA Methodology

The INAA was performed using the protocol adapted from Waheed et al. [31]. The sample preparation involved placing it in a pre-cleaned polyethylene capsule before irradiation in a Miniature Neutrons Source Reactor (MNSR). After irradiation and appropriate cooling, the sample underwent gamma-ray spectrometry with a high-purity germanium detector. Data acquisition and analysis utilized specialized software tools, ensuring appropriate corrections were made. The results were computed based on dry weight as the reference, with error propagation addressing various uncertainties.

2.3. GCMS Analysis

The analysis of the MM followed the outlined by Profumo et al. [32], with necessary adjustments for its inorganic nature. The dried powder was suspended separately in methanol and dichloromethane (DCM) to extract any polar, non-polar, and medium-polarity organic compounds. After vortexing and centrifugation, the supernatant was analyzed using an Agilent GC 7890A coupled with an Agilent MS 5975C (Seoul, Republic of Korea). Compounds were separated using a DB-5MS column (30 m × 0.25 mm, 0.25 μm film thickness), with helium as the carrier gas at a flow rate of 1 mL/min. The injector was set to 275 °C, with an oven temperature starting at 65 °C, held for 2 min, then increased by 7 °C per minute until reaching 280 °C for an additional minute. The total analysis duration was 40 min. The mass spectrometer was configured to scan within a mass range of 35–500 m/z, with both the ion source and transfer line temperatures maintained at 250 °C. The GC-MS data were analyzed using the National Institute of Standards and Technology (NIST) library to identify organic compounds present in the MM powder. The identification of the organic compounds was based on the relative abundance and retention times of the detected compounds, offering detailed insight into the organic composition of the MM sample.

2.4. Antioxidant Assay

The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay was performed according to the method outlined by Brand-Williams et al. [33], with adaptations for soil samples. The MM sample was prepared by thoroughly mixing 5 g of air-dried powder with 50 mL of 80% methanol and agitating the mixture for 2 h. The solution was centrifuged at 4000 rpm for 15 min and filtered through a 0.45 μm membrane filter. Antioxidant potential was evaluated using a modified DPPH radical scavenging assay. The test compound was serially diluted in methanol to achieve concentrations ranging from 10 to 100 μg/mL (10, 20, 40, 80, and 100 μg/mL). Each dilution (100 μL) was combined with 3.9 mL of 0.1 mM DPPH methanolic solution. Following thorough homogenization, the reaction mixture was incubated at ambient temperature under light-protected conditions for 30 min. Subsequently, the absorbance was measured spectrophotometrically at λ = 517 nm. Ascorbic acid, serving as a positive control, was prepared at concentrations ranging from 1 to 10 μg/mL. The percentage of inhibition was calculated using the following formula:

Inhibition % = A_control − A_sample/A_control × 100

(1)

A_control = absorbance of the DPPH blank solution only.

As_ample = absorbance of the DPPH test solutions combined with soil samples at various concentrations, adjusted for their individual absorption levels.

The radical scavenging activity was expressed as a percentage, and the half-maximal inhibitory concentration (IC₅₀) was determined. To ensure reproducibility, all experiments were conducted in triplicate.

Ascorbic acid was utilized as a positive control to evaluate and compare the antioxidant activity of the soil samples. A stock solution of ascorbic acid was prepared in 96% ethanol at a concentration of 100 μg/mL. From this stock solution, a series of dilutions was conducted to achieve final concentrations of 10, 20, 40, 80, and 100 μg/mL of ascorbic acid.

The assay was performed as described above for the soil sample, ensuring that the concentration ranges of ascorbic acid were comparable to those of the soil sample to facilitate meaningful analysis.

2.5. Collagenase Enzyme Inhibitory Assay

The collagenase inhibitory assay was conducted following the procedure outlined by Elgamal et al. [34], with minor modifications. The MM solution (30 μL) was prepared in various concentrations ranging from 15.63 to 1000 μg/mL. To each concentration, 10 μL of collagenase enzyme from Clostridium histolyticum (0.1 mg/mL) was added. The mixture was then combined with 60 μL of tricine buffer, which consisted of 50 mM tricine, 10 mM calcium chloride, and 400 mM sodium chloride at pH 7.5. The mixture was incubated for 20 min at 37 °C.

The assay preparation began with a blank solution comprising 10 μL of the enzymatic component, 80 μL of phosphate buffer, and 30 μL of MM solution. Subsequently, all reaction mixtures, except for the blank, were supplemented with 20 μL of N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA) substrate at a concentration of 1 mM. Following a 15 min incubation period, spectrophotometric measurements were conducted at 335 nm utilizing a microplate reader.

For comparative analysis, EGCG served as the positive control, while a mixture of 10 μL of enzyme and 90 μL phosphate buffer constituted the negative control. The extent of collagenase inhibition was quantified and expressed as a percentage, calculated using Equation (1).

2.6. Elastase Enzyme Inhibitory Assay

The elastase inhibitory assay was conducted following the procedure practiced by Elgamal et al. [34], with minor modifications. Porcine pancreatic elastase (10 μL) was combined with 25 μL of MM solution at concentrations ranging from 15.63 to 1000 μg/mL and Tris-HCl buffer (100 mM, pH 8.0). The mixture was incubated at 37 °C for 5 min. Subsequently, 20 μL of substrate solution (4.4 mM succinyl-Ala-Ala-Ala-p-nitroanilide in Tris-HCl buffer) was added to initiate the reaction. Absorbance was recorded at 410 nm.

EGCG served as the positive control, and distilled water was used as the negative control. The percentage of elastase inhibition was measured using Equation (1).

2.7. Hyaluronidase Enzyme Inhibitory Assay

The hyaluronidase inhibitory potential of MM powder was evaluated using a modified version of the protocol described by Sklirou et al. [35], with minor adjustments. The assay involved preparing a concentration gradient of MM solution (25–250 μg/mL, a low concentration in comparison to collagenase and elastase because of higher sensitivity) in 10% DMSO. The enzymatic reaction was initiated by combining 50 μL of this solution with 10 μL of hyaluronidase enzyme, followed by a 10 min incubation at 37 °C. Subsequently, 20 μL of calcium chloride (12.5 mM) was added, and the mixture was further incubated for an additional 10 min at 37 °C.

Afterwards, 50 μL sodium hyaluronate was introduced, followed by a 40 min incubation at 37 °C. Color development was achieved through the sequential addition of 10 μL sodium hydroxide (0.9 M) and 20 μL sodium borate (0.2 M), followed by heating at 100 °C for 3 min. The chromogenic reaction was completed by adding 50 μL of p-dimethylaminobenzaldehyde (PDMAB, 67 mM). Absorbance measurements were taken at 585 nm, with tannic acid serving as the reference standard and distilled water used as the negative control. The percentage of hyaluronidase inhibition was calculated using Equation (1), providing a quantitative assessment of the inhibitory activity of MM powder.

2.8. Tyrosinase Enzyme Inhibitory Assay

The evaluation of tyrosinase inhibitory activity was conducted using a modified protocol based on the methodology outlined by Fikry et al. [36]. The experimental procedure involved preparing MM solutions across a concentration gradient of 25–250 μg/mL, a range selected based on preliminary optimization and established protocols to accurately capture enzyme sensitivity. A reaction mixture was formulated by combining 20 μL of the prepared MM solution with 10 μL of mushroom tyrosinase (50 units/mL) in an aqueous medium, supplemented with 80 μL of phosphate buffer (pH 6.8). This mixture underwent a 5 min incubation at 37 °C.

Following this initial incubation, 90 μL of L-DOPA solution (2 mg/mL) was added to the reaction vessel, initiating the enzymatic conversion of L-DOPA to dopachrome. The reaction was allowed to progress for an additional 20 min at 37 °C. Quantification of the resultant dopachrome was achieved through spectrophotometric analysis at 475 nm. To ensure experimental validity, a phosphate buffer was used as a blank control, while kojic acid served as the positive control. The extent of tyrosinase inhibition was expressed as a percentage and calculated using Equation (1), providing a quantitative measure of the inhibitory potential of MM powder on tyrosinase activity.

2.9. Statistical Analysis

Experimental rigor was ensured by conducting all assays in triplicate, with each replicate treated as an independent experiment. The resultant data are presented as mean values ± standard deviations (SDs), providing a measure of statistical dispersion. Statistical analyses and graphical representations were performed using GraphPad Prism software (version 10).

To elucidate the relationship between inhibitor concentration and biological response, dose–response curves were constructed. These curves facilitated the determination of the half-maximal inhibitory concentration (IC₅₀), a key parameter quantifying the effectiveness of each tested compound in inhibiting the target enzyme activity.

2.10. Molecular Docking Studies

This study involves the molecular docking of compounds part of this MM Clay Powder based on the results of in vitro enzymatic assays with low IC₅₀, i.e., against two target proteins: tyrosinase and hyaluronidase. Molecular docking studies were conducted using PyRx 0.8 software to evaluate the binding patterns and ligand–receptor interactions of these compounds with the target proteins to understand their potential role in delaying aging.

• Collection and Optimization of Active Compounds

The MM Clay Powder showed significant inhibition against the tyrosinase and hyaluronidase enzymes. To identify the key inhibitory compounds, molecular docking studies were conducted to assess the binding affinity and interactions of these compounds with the amino acids in the active sites of these enzymes. Approximately 56 organic and metallic compounds identified by GC-MS and INNA analysis were employed as ligands to explore their potential binding interactions with the active site amino acids of the selected proteins. The chemical structures of these compounds were retrieved in .sdf format from the PubChem database [37] (https://pubchem.ncbi.nlm.nih.gov; accessed on 3 May 2024). The energy of these compounds was minimized before the molecular docking study. These molecular docking studies aimed to gain insights into the possible therapeutic role of Fuller’s Earth components in the management of aging.

• Retrieval and Preparation of Receptor Proteins

Three-dimensional (3D) structural models of tyrosinase and hyaluronidase were obtained from the Protein Data Bank (PDB) for the in silico molecular docking analyses. The specific structures utilized were tyrosinase (PDB identifier: 3NQ1) and hyaluronidase (PDB identifier: 1FCV), both retrieved in .pdb format from the PDB repository (https://www.rcsb.org; accessed on 7 May 2024). Prior to docking simulations, the protein structures underwent a series of preparatory steps. These included the removal of co-crystallized ligands and solvent molecules, the addition of hydrogen atoms to account for physiological pH, and the implementation of 3D protonation. Subsequently, energy minimization was performed to optimize the protein conformations, ensuring their suitability for docking analyses. The exploration of potential anti-aging drug candidates was facilitated through molecular docking studies.

• Molecular Docking

PyRx software, as described by Dallakyan et al. [38], was employed to simulate the binding interactions between the ligands of interest and the active sites of the prepared receptor proteins. Visualization of the interactions between the receptor proteins and key active compounds was performed using Discovery Studio 21.1.0.0 software [39].

• Drug Scanning through Pharmacokinetics Parameters

Evaluating druggability is essential for assessing the potential of a compound as a drug candidate. The druggability of the top compounds was analyzed using SwissADME [40]. Additionally, the ADMETlab 2.0 online server was employed to evaluate pharmacokinetic and pharmacodynamic properties, including absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling. Key parameters such as blood–brain barrier permeability, carcinogenicity, skin sensitization, Ame’s toxicity, and Caco-2 permeability were examined to predict the clinical potential of the compounds. Furthermore, the compounds were assessed against Lipinski’s Rule of Five (Ro5), which specifies that a drug candidate should have a molecular mass under 500 g/mol, no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, a logP value of 5 or less, and a molecular refractivity index between 40 and 130 [41]. Compounds that met these criteria were considered suitable for further development as potential drug candidates.

3. Results

3.1. INAA Analysis

A total of twenty essential major, minor, and trace elements (Si, Al, Mn, Zn, Ca, V, Rb, Cr, Ba, Co, Sr, Cs, Sn, Fe, Mo, K, Mg, Na, Ti, and Se) were determined in MM clay using the INAA technique. The results of the INAA analysis are presented in Table 1, with concentrations reported on a dry weight basis as averages of multiple determinations. The composition of MM clay reveals the following order for major elements: Si > Al > Mn > Zn > Ca > V > Rb > Cr > Ba. This is followed by minor elements Co and Sr and trace levels of Cs, Sn, Fe, Mo, K, Na, Mg, Ti, and Se. Silicon exhibited the highest concentration at 169.3742 mg/g, while selenium was found to have the lowest concentration at 0.1355 mg/g.

The precision of the INAA results varies among the different elements. Uncertainty values indicate good precision for several elements, with relative uncertainties of less than 10% for Si, Al, Mn, Ca, Rb, Co, Fe, Mo, K, Mg, Na, and Ti. Conversely, higher relative uncertainties were observed for Zn, V, Ba, Sr, Cs, Sn, and Se; these ranged from approximately 11% to 22%. Detection limits for the elements also varied significantly: they ranged from as low as 0.098 mg/g for Fe to as high as 110 mg/g for Na. This variation in detection limits reflects the differing sensitivities of the INAA technique across various elements. Each of these elements plays a specific biological role. For instance, silicon is important for connective tissue health, manganese acts as an antioxidant, zinc is crucial for immune function, and iron is essential for oxygen transport. The presence and concentrations of these elements in MM clay could potentially influence its applications and effects in various contexts [42,43,44,45].

Table 1. Instrumental neutron activation analysis (INAA) of MM clay samples: elemental composition and concentrations.

N°	Element	Symbol	PubChem ID/CID	Concentration (mg/g)	Uncertainty (±)	Detection Limit	Biological Role
01	Silicon	Si	5461123	169.3742	20.5	10.0	Connective tissue health [42]
02	Aluminum	Al	5359268	118.2931	18.0	8.5	Cellular transport [46]
03	Manganese	Mn	23930	123.6701	13.0	3.05	Antioxidant [43]
04	Zinc	Zn	23994	130.1224	22.5	19.0	Immune function [44]
05	Calcium	Ca	5460341	96.7853	15.0	7.0	Bone structure, signaling [47]
06	Vanadium	V	23990	91.4083	22	33.5	Insulin mimetic [48]
07	Rubidium	Rb	5357696	79.5790	6.50	15.5	Potassium substitute [49]
08	Chromium	Cr	23976	75.2774	9.8	5.85	Glucose metabolism [50]
09	Barium	Ba	5355457	72.5890	17	85	Bone density [51]
10	Cobalt	Co	23974	14.6253	1.5	1.12	Vitamin B12 component [52]
11	Strontium	Sr	5359327	14.6253	2.3	29.5	Bone formation [51]
12	Cesium	Cs	5354618	4.8661	0.85	0.91	Cellular fluid balance [53]
13	Tin	Sn	5352426	2.9842	0.66	0.72	Metabolic activity [54]
14	Iron	Fe	23925	1.9465	0.15	0.098	Oxygen transport [45]
15	Molybdenum	Mo	23932	1.5324	0.22	2.00	Enzyme cofactor [55]
16	Potassium	K	5462222	1.2958	0.22	0.19	Electrolyte balance [56]
17	Magnesium	Mg	5462224	0.3414	0.045	0.22	Enzyme cofactor [57]
18	Sodium	Na	5360545	0.2839	0.025	110	Nerve function [58]
19	Titanium	Ti	23963	0.2645	0.042	0.27	Biocompatible metal [59]
20	Selenium	Se	6326970	0.1355	0.031	0.27	Antioxidant [60]

Values expressed in mg/g unless otherwise specified.

Table 1. Instrumental neutron activation analysis (INAA) of MM clay samples: elemental composition and concentrations.

N°	Element	Symbol	PubChem ID/CID	Concentration (mg/g)	Uncertainty (±)	Detection Limit	Biological Role
01	Silicon	Si	5461123	169.3742	20.5	10.0	Connective tissue health [42]
02	Aluminum	Al	5359268	118.2931	18.0	8.5	Cellular transport [46]
03	Manganese	Mn	23930	123.6701	13.0	3.05	Antioxidant [43]
04	Zinc	Zn	23994	130.1224	22.5	19.0	Immune function [44]
05	Calcium	Ca	5460341	96.7853	15.0	7.0	Bone structure, signaling [47]
06	Vanadium	V	23990	91.4083	22	33.5	Insulin mimetic [48]
07	Rubidium	Rb	5357696	79.5790	6.50	15.5	Potassium substitute [49]
08	Chromium	Cr	23976	75.2774	9.8	5.85	Glucose metabolism [50]
09	Barium	Ba	5355457	72.5890	17	85	Bone density [51]
10	Cobalt	Co	23974	14.6253	1.5	1.12	Vitamin B12 component [52]
11	Strontium	Sr	5359327	14.6253	2.3	29.5	Bone formation [51]
12	Cesium	Cs	5354618	4.8661	0.85	0.91	Cellular fluid balance [53]
13	Tin	Sn	5352426	2.9842	0.66	0.72	Metabolic activity [54]
14	Iron	Fe	23925	1.9465	0.15	0.098	Oxygen transport [45]
15	Molybdenum	Mo	23932	1.5324	0.22	2.00	Enzyme cofactor [55]
16	Potassium	K	5462222	1.2958	0.22	0.19	Electrolyte balance [56]
17	Magnesium	Mg	5462224	0.3414	0.045	0.22	Enzyme cofactor [57]
18	Sodium	Na	5360545	0.2839	0.025	110	Nerve function [58]
19	Titanium	Ti	23963	0.2645	0.042	0.27	Biocompatible metal [59]
20	Selenium	Se	6326970	0.1355	0.031	0.27	Antioxidant [60]

Values expressed in mg/g unless otherwise specified.

3.2. GCMS Analysis

The evaluation of the chemical structure and composition of clay samples can reveal various biological potentials of different clay types used in skincare and medicinal applications. To the best of our knowledge, a comprehensive GC-MS-based metabolic characterization revealing the presence of various bioactive compounds in MM, a clay used extensively in traditional skincare, has not yet been reported. Therefore, we conducted a GC-MS analysis for this study.

In the MM sample, 13 peaks were identified, each corresponding to bioactive compounds (shown in Figure 2). These peaks were analyzed by comparing their retention times, molecular weights, and molecular formulas to those of known compounds listed in the NIST library (see

Table 2. Bioactive compounds identified in MM clay by GC-MS analysis.

Peak	RT (Min)	SI (%)	Compound Name	PubChem ID	Molecular Formula	Molecular Weight (g/mol)
1	08.120	83	3-Heptanol, 1-Octanol	957	C8H18O	130
2	10.060	83	Cyclohexasiloxane	10911	C12H36O6Si6	444.92
3	15.929	93	Nonadecane	15979	C19H40	268.5
4	15.995	90	Pentacosane	12406	C25H52	352.7
5	16.060	91	Heptacosane	11636	C27H56	380.7
6	17.020	92	Nonacosane	12409	C29H60	408.8
7	17.130	92	Tetrapentacontane	545963	C54H108Br2	917.2
8	23.975	82	Oleth-2	13387455	C22H44O3	356.6
9	24.170	94	Docosanamide	76468	C22H45NO	339.6
10	25.705	94	Benzyl-piperazine-carboxamide	455963	C37H48N4O4	612.8
11	26.580	75	Bis(2-ethylhexyl) phthalate	8343	C24H38O4	390.6
12	28.650	83	Ganaxolone	6918305	C22H36O2	332.5
13	36.110	77	Beta-sitosterol	222284	C29H50O	414.7

). The analysis revealed a diverse array of compounds, including alcohols (e.g., 3-Heptanol, 1-Octanol), silicon-containing compounds (e.g., Cyclohexasiloxane), alkanes (e.g., Nonadecane, Pentacosane, Heptacosane), amides (e.g., Docosanamide), steroids (e.g., Ganaxolone, Beta-sitosterol), and plasticizers (e.g., bis(2-ethylhexyl) phthalate). The highest concentration was observed for Beta-sitosterol (15.45% area), followed by Docosanamide (12.36% area) and Cyclohexasiloxane (9.80% area). This diverse chemical profile aligns with the reported skincare benefits of MM, providing a scientific basis for understanding its effects on skin health. However, the presence of these compounds in MM clay is intriguing, as some may have originated from natural processes while others could be anthropogenic additions. For instance, long-chain alkanes (Nonadecane through Tetrapentacontane) are commonly found in soil environments and could result from microbial degradation of organic matter. Beta-sitosterol, a plant-derived sterol, might have accumulated in the clay through natural depositional processes. The presence of Cyclohexasiloxane and bis(2-ethylhexyl) phthalate suggests potential contamination or deliberate addition during processing, though their source remains uncertain. This diverse chemical profile provides initial insights into MM’s composition, warranting further investigation into the origins and safety of these compounds in skincare applications.

3.3. Antioxidant Assay

The DPPH free radical scavenging assay was employed to assess the antioxidant activity of MM powder, with ascorbic acid serving as a positive control. The results revealed that MM powder exhibited moderate antioxidant activity, with an IC₅₀ value of 31.938 μg/mL. In comparison, ascorbic acid demonstrated superior antioxidant capacity, with a lower IC₅₀ value of 8.5 μg/mL (see

Table 3. IC₅₀ values of MM powder and ascorbic acid in the DPPH radical scavenging assay.

Sample	IC50
MM Powder (Fuller’s Earth)	31.938 μg/mL
Ascorbic Acid	8.5 μg/mL

). The concentration-dependent increase in DPPH scavenging activity is evident in Figure 3, where both MM powder and ascorbic acid display a typical dose–response curve. Although MM possesses some free radical scavenging ability, its potency is notably lower than that of ascorbic acid, which is a well-known antioxidant. The reduced antioxidant efficacy of MM powder may be attributed to its inorganic nature, which limits its ability to donate electrons or hydrogen atoms to neutralize free radicals as effectively as organic antioxidants [61]. Despite its moderate antioxidant activity, the potential of MM powder for skincare applications remains of interest, particularly when considering its other beneficial properties in conjunction with its antioxidant capacity.

3.4. Enzyme Inhibition Activity

MM was evaluated for its in vitro inhibitory activity against four enzymes involved in skin aging and remodeling: collagenase, hyaluronidase, tyrosinase, and elastase [34]. As illustrated in Figure 4, MM demonstrates varying degrees of inhibition across these enzymes. For collagenase inhibition, MM exhibited moderate inhibitory activity with an IC₅₀ of 38 μg/mL, which was slightly less potent than the reference inhibitor EGCG (IC₅₀ 34 μg/mL); it demonstrated strong inhibition of hyaluronidase with an IC₅₀ of 18 μg/mL, surpassing the efficacy of tannic acid (IC₅₀ 28 μg/mL). Notably, in the tyrosinase assay, MM displayed superior inhibition with an IC₅₀ of 27 μg/mL, outperforming kojic acid (IC₅₀ 36 μg/mL). Lastly, for elastase, MM showed comparable inhibition to EGCG, with an IC₅₀ of 35 μg/mL for MM and 34 μg/mL for EGCG. These results suggest that MM possesses significant enzyme inhibitory activities, particularly against hyaluronidase and tyrosinase, indicating its strong potential in skincare applications aimed at combating signs of aging and hyperpigmentation. Its performance was especially impressive in inhibiting hyaluronidase and tyrosinase, where it outperformed the standard inhibitors. While MM showed moderate to good inhibition across all tested enzymes, its inorganic nature does not appear to hinder its efficacy, as evidenced by its superior performance in some assays compared to organic reference compounds.

3.5. Molecular Docking Studies

Given the strong inhibitory activity of MM powder against tyrosinase and hyaluronidase enzymes, molecular docking studies were conducted to identify the key inhibitory compounds present in MM and asses their binding affinity and interactions with the amino acids in the active sites of these enzymes. The 3D structures of tyrosinase and hyaluronidase enzymes were retrieved from the PDB database and used as the receptor or target proteins. Approximately 33 organic and metallic compounds identified by GC-MS and INNA analysis were employed as ligands to explore their potential binding interactions with the amino acids in the active sites of the selected proteins. These molecular docking studies aimed to gain insights into the possible therapeutic role of MM components in the management of skin aging and related conditions. Among these various organic and metallic compounds, silicon-containing compounds were unable to bind with the receptors.

3.5.1. Interaction Analysis

The PyRx virtual screening tool was utilized to conduct docking studies, while BIOVIA Discovery Studio was employed to visualize the 2D interactions between ligands and receptors [62]. Leading drug candidates were selected based on their binding affinities and the amino acid involved in interactions. PyRx facilitated the evaluation of the ligand occupancy within the binding pocket of the target proteins through conformation scores. From the initial set of compounds, the top five compounds were individually selected for docking against each receptor protein based on their minimum binding scores and interacting amino acids (see

Table 4. Binding scores of the top five compounds with tyrosinase (3NQ1) and hyaluronidase (1FCV) protein receptors.

Name	PubChem ID	Binding Affinity (kcal/mol) with 3NQ1	Interacting Amino Acids with 3NQ1	Binding Affinity (kcal/mol) with 1FCV	Interacting Amino Acids with 1FCV
Benzyl-piperazine-carboxamide	455963	−8.6	PheB:258, AspA:36, ProB:51, LysA:30, ProA:145, ThrA:137, GluA:31, ProB:52 and LeuA:27	−8.1	ArgA:229, AsnA:231, AlaA:185, LeuA:192, ProA:194 and ThrA:193
Bis(2-ethylhexyl) phthalate	8343	−8.0	AsnB:249, GlnB:242, HisB:279, IleB:243 and LysB:281	−5.9	TyrA:168, AlaA:185, ArgA:244, SerA:225, TyrA:190 and ArgA:116
Heptacosane	11636	−8.0	PheB:124, AspB:123 and LysB:150	−4.6	TyrA:227, TrpA:301, TrpA:267, TrA:184, TyrA:55 and AspA:111
Ganaxolone	6918305	−7.2	PheA:262, ProA:273, ValA:276, MetA:266, ProA:67, TrpA:68, GluA:71 and ValA:276	−7.4	SerA:304, AspA:111, AspA:305, Tyra:55, AspA:56, PheA:112, GluA:113, SerA:303 and AspA:305
Beta-sitosterol	222284	−7.1	AsnA:249, PheA:262, MetA:277, tyrA:250, GluA:274, ProA:273, ValA:276, ProA:67, GluA:71, TyrA:72, trpA:269, TrpA:68, ThrA:272, ArgA:70, AspA:275 and MetA:266	−8.5	LysA:45, PheA:20, ProA:100, IleA:99, IleA:99, ProA:105 and AspA:101

).

3.5.2. Anti-Tyrosinase Activity

Among these compounds, Benzyl-piperazine-carboxamide exhibited significant interactions with specific amino acids of the tyrosinase protein, including PheB:258, AspA:36, ProB:51, LysA:30, ProA:145, ThrA:137, GluA:31, ProB:52, and LeuA:27, demonstrating a binding S-score of −8.6 kcal/mol (see Figure 5). The complex revealed a conventional hydrogen bond with LysA:30, ProB:51, and GluA:31, as well as Pi–sigma interactions with ProA:145, ProB:52, and LeuA:27. These Pi–sigma interactions, which encompass Pi–alkyl and Pi–sulfur interactions, facilitate the drug’s intercalation into the receptor’s binding pocket by promoting charge transfer [63].

Similarly, the second-best compound, Bis(2-ethylhexyl) phthalate (S-score of −8.0 kcal/mol), exhibited interactions with the amino acids AsnB:249, GlnB:242, HisB:279, IleB:243, LysB:281 at the binding site of tyrosinase (see Figure 6). The molecular docking results revealed diverse interaction patterns between various compounds and 3NQ1. Particularly, ProA:67 and GluA:71 emerged as common interacting residues for both Ganaxolone and Beta-sitosterol, suggesting their significance in the binding pocket. TrpA:68 also appeared in multiple complexes, interacting with both compounds. Notably, different compounds showed distinct binding preferences: benzyl-piperazine-carboxamide primarily interacted with proline residues (ProB:51, ProA:145, ProB:52) and lysine (LysA:30), while bis(2-ethylhexyl) phthalate formed interactions with asparagine and glutamine residues (AsnB:249, GlnB:242). Heptacosane displayed a simpler interaction pattern, primarily engaging with PheB:124, AspB:123, and LysB:150. Beta-sitosterol demonstrated the most extensive interaction network, involving multiple amino acid types including phenylalanine, methionine, and tyrosine residues. The presence of ValA:276 in both Ganaxolone and Beta-sitosterol complexes suggests its potential importance in ligand binding. The diversity of interacting residues across different compounds indicates a versatile binding site capable of accommodating various molecular structures through multiple interaction points (see Figure 7).

3.5.3. Anti-Hyaluronidase Activity

For the hyaluronidase target protein, the top compound, Beta-sitosterol, exhibited a binding affinity of −8.5 kcal/mol and interacted with the amino acids LysA:45, PheA:20, ProA:100, IleA:99, IleA:99, ProA:105, and AspA:101 within the active pocket (see Figure 8). Similarly, the second-best compound, Benzyl-piperazine-carboxamide, demonstrated interaction with residues ArgA:229, AsnA:231, AlaA:185, LeuA:192, ProA:194, and ThrA:193 at the active site (see Figure 9). This complex also revealed four conventional hydrogen bonds with ArgA:229, AsnA:231, LeuA:192, and ThrA:193. The remaining compounds exhibited strong binding interactions with the amino acids of the hyaluronidase active site (see Figure 10).

Among the top-selected compounds, Benzyl-piperazine-carboxamide and Beta-sitosterol demonstrated strong interactions with both receptor proteins, specifically tyrosinase and hyaluronidase.

3.5.4. Druggability Analyses

The selected compounds were evaluated for their drugability using Lipinski’s Rule of Five (Ro5), which serves as a guideline to determine their potential as active drugs in humans [64]. All compounds demonstrated general adherence to Ro5 parameters, with minor variations noted in

Table 5. Molecular and drug-likeness properties of the top five compounds evaluated through Lipinski’s Rule of Five (3NQ1 and 1FCV).

Molecular Properties
Ligand	Molecular Mass (≤500 Dalton)	Hydrogen Bond Donor (≤5)	Hydrogen Bond Acceptor (≤10)	No. of Rotatable Bonds (≤10)	LogP (≤5)	Refractivity (40–130)	Violations
Benzyl-piperazine-carboxamide	612.80	4	6	14	3.65	184.83	0
Bis(2-ethylhexyl) phthalate	390.6	0	4	16	4.77	116.30	1
Heptacosane	380.73	0	0	24	7.32	130.1	1
Ganaxolone	332.52	1	2	1	3.29	100.29	0
Beta-sitosterol	414.71	1	1	6	5.05	133.23	0

. Further analysis using ADMETLab 2.0 revealed comprehensive absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles [65]. The ADMET profiling showed that Beta-sitosterol and Benzyl-piperazine-carboxamide exhibited particularly favorable characteristics, with Beta-sitosterol demonstrating moderate plasma protein binding (86.9%), acceptable hepatotoxicity (0.573), and Pfizer Rule compliance. Both compounds showed suitable Caco-2 permeability values (−5.122 and −5.246, respectively), though skin sensitization predictions suggested careful consideration for topical formulation (Table 5 and

Table 6. ADMET-related drug-like parameters of the best-selected compounds.

Compounds
	Benzyl-piperazine-carboxamide	Bis(2-ethylhexyl) phthalate	Heptacosane	Ganaxolone	Beta-sitosterol
Absorption and Distribution
BBB	−−−	+++	−−−	+++	−−−
HIA	−−−	−−−	−−−	−−−	−−−
Caco-2 Permeability	−5.246	−4.918	−5.074	−4.806	−5.122
Volume of Distribution (Vd)	0.001	0.94	3.009	0.205	−0.244
Plasma Protein Binding (PPB)	95.1%	98.7%	105.5%	67.4%	86.9%
Excretion
CLplasma	4.928	5.79	4.545	16.978	13.205
T1/2	0.865	0.324	3.031	0.97	0.541
Toxicity
Skin Sensitization	0.807	0.938	0.998	0.992	0.99
Carcinogenicity	0.13	0.328	0.164	0.983	0.688
Human Hepatotoxicity	0.916	0.086	0.473	0.854	0.573
AMES Toxicity	0.129	0.018	0.012	0.183	0.139
Drug-Likeness Rules
Acute Aquatic Toxicity Rule	0	0	1	2	1
Skin Sensitization Rule	1	0	0	2	0
Lipinski Rule	Yes	Yes	Yes	Yes	Yes
Pfizer Rule	Yes	No	No	No	Yes

BBB: blood–brain barrier; HIA: Human Intestinal Absorption; CLplasma: plasma clearance; T1/2: half-life. For classification endpoints, prediction probability values are represented by the following symbols: 0–0.1 (−−−) and 0.9–1.0 (+++).

).

Molecular docking analysis revealed that Beta-sitosterol and Benzyl-piperazine-carboxamide demonstrated the strongest binding interactions with both target proteins. Beta-sitosterol showed a high binding affinity with 1FCV (−8.5 kcal/mol) and formed multiple stable interactions (sixteen interactions with 3NQ1 and seven with 1FCV). Similarly, Benzyl-piperazine-carboxamide exhibited strong binding energies (−8.6 kcal/mol with 3NQ1 and −8.1 kcal/mol with 1FCV) with stable amino acid interactions. While other compounds showed promising binding energies, their ADMET profiles were less favorable. Based on the combined evaluation of the molecular docking results, drug-likeness parameters, and ADMET profiles, Beta-sitosterol and Benzyl-piperazine-carboxamide emerged as the most promising compounds from the MM clay sample, warranting further experimental validation for therapeutic applications. Beta-sitosterol is a phytosterol already used in anti-aging studies.

4. Discussion

In the contemporary industrial era, characterized by vehicular emissions, environmental pollutants, and UV radiation, there is an increasing desire among individuals to maintain clear, youthful-looking skin [66]. Skin aging is an inherent and inevitable process that affects various regions of the body, with the skin being particularly noticeable. The effects and perception of skin aging can vary between individuals and across cultures. Societal emphasis on preserving a youthful appearance can greatly influence psychological well-being and self-esteem on a global scale. To counteract the signs of aging and promote a more youthful appearance, numerous anti-aging products and treatments have been developed [67]. While synthetic cosmeceuticals offer several benefits, they may also present side effects and can be costly. As a result, there has been a surge in popularity for natural or homemade products as cosmeceuticals, primarily due to their accessibility and perceived lack of side effects. This trend has significantly contributed to the growth of the aging-related cosmeceuticals market, which now has become one of the largest consumer markets [68].

MM, a clay-based product widely used in South Asia and other regions for cosmetic purposes, has garnered considerable attention in recent years [69]. Despite its extensive use in traditional practices, the scientific basis for its efficacy and safety has remained largely unexplored. This study aims to bridge this knowledge gap by conducting a comprehensive analysis of MM’s composition and potential biological activities.

INNA analysis was employed to investigate the inorganic constituents of MM, revealing its metallic element composition [70]. This analysis is crucial for understanding the clay’s absorption properties and potential interactions with skin. Notably, the presence of elements, such as silicon (Si) and tin (Sn), was confirmed; however, their direct involvement in the biological activities of the clay remains unclear.

GC-MS analysis of MM revealed a complex composition of 13 distinct organic compounds. Key findings include silicon-containing compounds (Cyclohexasiloxane), which may contribute to texture and moisture retention [71]; organic acids and alcohols that could provide pH balancing, exfoliating, and antioxidant properties [72]; alkanes, like Nonadecane, Pentacosane, Heptacosane, which may impart sensory qualities [73]; and amides that could contribute to emollient effects. Additionally, the analysis detected steroids, like Ganaxolone and a phytosterol (Beta-sitosterol) compound, which may come from plants decomposed over time or added intentionally to make this product more effective [74]. The detection of bis(2-ethylhexyl) phthalate, a common plasticizer, raises concerns regarding potential contamination sources or intentional additions [75].

The study evaluated the potential anti-aging properties of MM through in vitro enzyme inhibition assays targeting key enzymes involved in skin aging: collagenase, elastase, hyaluronidase, and tyrosinase [30]. The results demonstrated promising enzyme inhibition activity against all four enzymes, with the strongest activity observed against hyaluronidase, followed by tyrosinase and elastase, while the least activity was noted against collagenase. These findings suggest that MM may have significant potential in mitigating various aspects of skin aging.

Molecular docking studies were conducted to elucidate the potential mechanisms of action of the compounds identified in MM. Interestingly, the inorganic components, including Si and Sn, could not be docked with the target proteins, suggesting that their direct involvement in enzyme inhibition is unlikely. In contrast, the organic constituents demonstrated significant binding affinities, often surpassing those of known inhibitors, such as kojic acid and EGCG. Notably, Benzyl-piperazine-carboxamide and Beta-sitosterol exhibited strong interactions with both tyrosinase and hyaluronidase receptor proteins.

Despite these promising findings regarding enzyme inhibition, MM demonstrated only moderate antioxidant activity in the DPPH assay. From our INAA elemental analysis, several minerals are known to contribute significantly to antioxidant activity. Manganese (123.6701 mg/g) serves as a direct antioxidant cofactor and is essential for superoxide dismutase enzyme function, which neutralizes reactive oxygen species [76]. Selenium (0.1355 mg/g), though present in lower concentrations, is a critical component of glutathione peroxidase, a major cellular antioxidant enzyme [77]. Zinc (130.1224 mg/g) also plays a crucial role in antioxidant defense systems by supporting catalase and superoxide dismutase activities, while chromium (75.2774 mg/g) contributes to cellular protection against oxidative stress [78,79].

Among the organic compounds identified through GC-MS analysis, Beta-sitosterol (36.110 min RT, 77% SI) emerges as the primary contributor to antioxidant activity. This phytosterol is well-documented for its potent free radical scavenging properties and ability to inhibit lipid peroxidation [80]. Docosanamide (24.170 min RT, 94% SI) also contributes to the antioxidant profile through its fatty acid amide structure, which can neutralize free radicals [81]. Additionally, Benzyl-piperazine-carboxamide (25.705 min RT, 94% SI) may contribute to the overall antioxidant capacity through its aromatic ring system that can stabilize free radicals [82]. This result, combined with a previously reported lack of significant antimicrobial activity [23], suggests that while MM possesses certain beneficial properties, it may not serve as a comprehensive solution for all skincare needs.

The diverse chemical profile of MM aligns with its reported skincare benefits, providing a scientific basis for understanding its effects on skin health. Its primary advantages appear to be related to oil absorption, improvement of skin texture, and mild anti-aging effects. The clay’s ability to absorb excess oil and impurities from the skin surface, combined with its gentle exfoliating properties, likely contributes to the perceived ‘glowing’ effect reported by users [83].

While Multani Mitti (MM) exhibits moderate antioxidant activity—likely attributable to bioactive compounds, such as Beta-sitosterol, and essential inorganic elements, like manganese and selenium—its antimicrobial properties appear limited based on our findings [84,85]. This suggests that, although MM contributes beneficially to skin health through enzyme inhibition and antioxidant effects, it may not fully address all the factors involved in skin aging and microbial protection. To develop a more comprehensive cosmeceutical formulation, supplementation with well-characterized antioxidants (e.g., vitamin C at 0.5–2%, ferulic acid at 0.1–1%) and antimicrobial agents (such as tea tree oil at 0.5–2%, zinc oxide at 1–5%) could be advantageous [86,87]. These compounds have documented efficacy at these concentrations in enhancing skin protection and complementing clay-based ingredients. Notably, traditional and homemade skincare practices often combine MM with plant powders, like turmeric or neem, which provide additional antioxidant and antimicrobial benefits [88]. Incorporating such synergistic actives can optimize MM’s overall efficacy, creating multifunctional formulations that leverage both its traditional appeal and scientifically supported enhancements.

The organic compounds identified in this study that exhibited strong enzyme inhibition have the potential to be developed into individual anti-aging products. However, the holistic effects of MM, including its oil absorption and skin-brightening properties, suggest that complete powder may offer unique benefits that extend beyond those of its individual components.

This study provides valuable insights into the composition and potential mechanisms of action of MM in skincare. While it demonstrates promising anti-aging properties, particularly through enzyme inhibition, its moderate antioxidant activity and lack of antimicrobial effects indicate areas for improvement. Future research should focus on optimizing MM-based formulations by incorporating additional beneficial compounds to create a more comprehensive, natural, and cost-effective skincare solution. Such an approach could leverage the traditional benefits of MM while addressing its limitations, potentially resulting in an enhanced cosmeceutical product with minimal side effects.

5. Conclusions

The use of MM in cosmeceuticals has gained significant popularity due to its beneficial properties. Our study demonstrated that this clay exhibits moderate antioxidant activity (IC₅₀: 31.938 μg/mL) and significant inhibitory effects on hyaluronidase (IC₅₀: 18 μg/mL) and tyrosinase (IC₅₀: 27 μg/mL), outperforming standard inhibitors. It also showed moderate activity against collagenase and elastase—enzymes crucial in skin aging. These effects are likely attributed to synergistic actions of identified components, particularly Beta-sitosterol and Benzyl-piperazine-carboxamide, which demonstrated strong binding affinities (−8.5 to −8.6 kcal/mol) to target enzymes. ADMET analysis confirmed favorable safety profiles for these compounds. Our findings support MM’s incorporation into anti-aging formulations, with future research focusing on the in vivo validation of topical applications and toxicity profiling.