INTRODUCTION:

Like in many other countries, Thai fruit juice industries are generated a substantial amount of waste annually, primarily consisting of by-products such as seeds, skins, pomace, and solid residues.

These by-products are rich in bioactive compounds like phenolic compounds, pectin, antioxidants, dietary fibre, and other essential nutrients, which have been found to possess various health benefits such as antioxidant, anti-inflammatory, antibacterial, antifungal, and anticancer properties. Researchers have been focusing on extracting and recovering these valuable bioactive components from fruit juice industry waste to minimize environmental impact, reduce economic losses, and create new sources of functional ingredients for various industries including food, pharmaceutical, cosmetic, and textile^1-8. Plant polyphenols are common bioactive metabolites with highly variation in chemical structures and biological activities. The fruit and vegetable wastes from industry utilization are often the source of polyphenols^9-11. Plant ingredients are the primary source of bioactive compounds utilized in the cosmetics and pharmaceutical sectors. Recently, cosmetic products derived from plant origin has been greater focus, which is applied for skin care and dermatological disorder relief^12-17. Plant extracts are commonly applied as the ingredients of skin care cosmetics, which are contained high content of bioactive compounds with human skin protection and treatment. The variations of plant extraction are depended overall or parts of plant material and appropriately solvent used. The various factors of plant extract are affected the amount of ingredient and biological activities within natural cosmetic formulae, which are including cultivation, harvesting, post-harvesting and extraction conditions. The secondary metabolites are major bioactive compounds that are mostly considered on application of plant extract, such as phenolics, flavonoids, tannins, terpenes, saponins, alkaloids, steroids, vitamins and volatile oils^18,19.

Antidesma thwaitesianum, in Thai known as "Mamao," is tropical plant within the Phyllanthaceae family, and is prominently cultivated in South-East Asia especially in the North-Eastern region of Thailand²⁰. Almost parts of this plant are medicinal uses in treatment of various diseases and their wide range of biological activities such as antioxidant, antidiabetic, antimicrobial and anti-inflammation had been reported. The red coloured fruits are naturally consumed, and its taste are exhibiting a delightful balance of sour and sweet flavours. There are also processed into various products such as jams, wines, and juices. Polyphenols and flavonoids are main bioactive in A. thwaitesianum fruits including anthocyanin, catechin, gallic acid, quercetin, rutin, terpene, alkaloids, luteolin, tannin, sterols, and saponins, have been reported^21-25. Recently, the photo-protective and anti-inflammation properties of A. thwaitesianum fruit extract on UVB-induce keratinocyte damage had been also reported. These protective effects are associated with antioxidant and anti-inflammation properties from various phenolics, including cyanidin, ferulic acid, caffeic acid, vanillic acid, and protocatechuic acid, that are contained in A. thwaitesianum fruit extract²⁵. Nowadays, A. thwaitesianum fruit juice is become the favourited local products in the North-Eastern of Thailand as nutraceutical product for healthy consumers, which is yielded high content of several bioactive such as phenolics, ascorbic acid, anthocyanins, and flavonoids ^21,22,26. While pomace including seeds and marcs are considered as abundant source of polyphenols and proanthocyanins²⁷. Thus, the pomace from A. thwaitesianum juice processing industries is the valuable and attractive source of antioxidant and bioactive compounds for ingredient skin care products. Therefore, other biological activities were sparely reported. Hence, we were conducted to screen active ingredients, such as phenolic, flavonoid and tannin contents and to evaluate in vitro biological activities of Mamao pomace extract for skin care and treatment including antioxidant, anti-microbial, anti-inflammation, anti-tyrosinase, anti-elastase, cytotoxicity, and wound-healing properties.

MATERIALS AND METHODS:

Pomace preparation and extraction:

The Mamao (A. thwaitesianum) fruits (3kg) were purchased from local markets, Sakon Nakhon, North-Eastern of Thailand during December 2023 to February 2024. The fruits were small rounded in bushy. The purplish black coloured ripe fruits (~2 kg) were selected, pooled together, cleaned and squeezed. After juicing process, the remained pomace was filtered by kitchen sieve, air-dried, and desiccant dried with hot-air oven at 50°C. The dried pomace (~500g) was grinded in powder form with herbal grinder machine. The pomace powder (100g) was macerated with 95% ethanol (500mL/L) within 72h. The ethanol was evaporated from Mamao pomace extract (MPE) by rotary evaporation under vacuum. This extraction was run in triplicate experiment and yield of MPE was calculated.

Phytochemical content:

Total phenolic content:

The MPE was quantitated for total phenolic content (TPC) by colorimetric assay. MPE (20mg) was dissolved in ethanol (RCI Labscan, Thailand) and concentration was 0.2mg/ml. 1.0ml of extract or standard solution was mixed with 0.3ml of saturated sodium bicarbonate (RCI Labscan, Thailand), and 0.1 ml of Folin-Ciocalteu reagent (Loba, Chemie, India), respectively. The volume of mixture was adjusted by distilled water (4.6ml) and incubated at room temperature in the dark for 1 h. Absorbance of TPC was determined by using a UV-visible spectrophotometer at 765nm. Gallic acid (Sigma-Aldrich, USA) was diluted within dimethylsulfoxide, DMSO (RCI Labscan, Thailand) for standard curve plotting (0.00012, 0.00024, 0.00049, 0.00098, 0.00195 and 0.00391mg/ml). TPC was reported as mg of gallic acid equivalent (GAE) per gram²⁸.

Total flavonoid content:

The MPE was quantitated for total flavonoid content (TFC) by colorimetric assay. 50ml of MPE (2.0mg/ml) in ethanol or standard solution was deposited in a 96-well microplate and each well was contained aluminum chloride (Loba Chemie, India), ethanol, and sodium acetate (10: 96: 10). The mixture was incubated at room temperature in the dark for 40min and absorbance of TFC was measured at 415nm. Quercetin (HWI Analytik GmbH, Germany) was used to standard curve and TFC was reported as mg of quercetin equivalent (QE) per gram²⁹.

Total tannin content:

Total tannin content (TTC) of MPE was analysed by the spectrophotometric-based method (lmax = 760nm). This analytical approach involved the use of Folin-Denis reagent (Sigma Alrich, USA) according with the establishment of the Association of Official Analytical Chemists, AOAC (2005) 952.03. A standard curve was performed by utilizing of the range of concentrations of tannic acid (Fluka, Switzerland) and TTC was represneted as mg of Tannic acid equivalent (TE) per gram³⁰.

Evaluations of antioxidant activity:

The MPE was diluted using absolute ethanol, which was adjusted to 0.001, 0.01, 0.1, 1.0, and 10mg/ml. A range of antioxidant assays, such as the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging assays, and the ferrous iron-ferrozine complex method and ferric iron-thiocyanate complex method, were included in this study. The methods were monitored the reduction of DPPH radical, the reduction of NO radical from Griess reagent (Sigma-Aldrich, USA), the ability of metal chelation, and inhibition of lipid peroxidation, respectively. Micro-titer plate reader (BIO-RAD, USA) was employed in order to monitor the absorbance of their color of mixture at maximum wavelength (lmax). Antioxidant activity from each assay was reported a 50% inhibitory concentration (IC₅₀) of MPE, which was calculated from triple measurements. Positive control of DPPH and NO radical scavenging assays was vitamin C, ascorbic acid (Sigma-Aldrich, USA). While, positive control of ferric iron-thiocyanate complex method or inhibition of lipid peroxidation was vitamin E, a-tocopherol (Sigma-Aldrich, Germany). The metal chelating agent, ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, USA) was positive control for ferrous iron-ferrozine complex method^31-33.

Anti-microbial assay:

The assay was carried out according by Kirby-Bauer method. Common skin pathogens, Staphylococcus aureus, and Cutibacterium acnes as bacterial pathogens; and Candida albicans and Malassezia furfur as yeast pathogens were included in this study. Bacterial and yeast media were used brain heart infusion, BHI (HiMedia Laboratories, India), and Sabaurad dextrose agar (HiMedia Laboratories, India), respectively. The testing disc, a 6mm filter paper disc (MachereyNagel, Germany) was applied for different concentrations (0.05, 0.5 and 5.0mg) of MPE, while negative control was ethanol contained disc. Positive control discs (Oxoid, UK) were included erythromycin (0.015mg), clindamycin (0.002mg), fluconazole (0.025mg), and ketoconazole (0.2mg) disc for S. aureus, Cu. acnes, C. albicans and M. furfur, respectively. The diameter of the inhibition zone (mm) surrounding disc was determined and done in triplicated³⁴.

Enzyme inhibitory assays:

Tyrosinase inhibitory assay:

The inhibitory effect of MPE on tyrosinase was evaluated through a dopachrome-based assay method, with tyrosine (Sigma-Aldrich, Germany) using as the substrate. Specifically, 50ml of MPE at varying concentrations (ranging from 0.001 to 10mg/ml) was dissolved in DMSO, along with 50ml of 100 units of mushroom tyrosinase solution (Sigma, Germany) in 0.1 M phosphate buffer, 50ml of tyrosine solution (1.0 mg/ml) in 0.1M phosphate buffer, and 50ml of 0.1M phosphate buffer was dispensed into individual wells of a 96-well plate. The concoction was incubated at 37±2 °C for 60min, and the absorbance at 450nm was determined. A range of 0.001–10mg/ml of kojic acid kojic acid (Sigma-Aldrich, Germany) was employed as the positive control. Conversely, the blank of sample contained solution was designated as the negative control. Every measurement was run in triplicate. The determination of the MPE concentration was elicited 50% inhibition (IC₅₀), which was derived from the graphical representation correlating the tyrosinase inhibition (%) with the concentrations of the MPE^35,36.

Elastase inhibitory assay:

The evaluation of elastase inhibitory activity was monitored the production of p-nitroaniline, which was hydrolyzed from the substrate, N-succinyl-Ala-AlaAla-p-nitroanilide (Sigma-Aldrich, USA), by elastase from porcine pancreas, type I (Sigma-Aldrich, USA) enzyme. Briefly, the 130ml of 1.015mM N-succinyl-Ala-Ala-Ala-p-nitroanilide in 0.1M Tris-Cl buffer, pH 8.0 was filled with 10ml of MPE (0.0075, 0.075, 0.75, 7.5 and 75 mg/ml) dissolved in 10% DMSO in an individual wells of a 96-well plate. The mixture in microplate was pre incubated at room temperature for 5min, and 15ml of elastase solution (0.5U/ml) was added. After beginning of the reaction, the microplate was stored at room temperature for 30min, the absorbance of the product, p-nitroaniline was measured at 410nm by microplate reader. The determination of the MPE concentration was elicited IC₅₀, which was derived from the graphical representation correlating the elastase inhibition (%) with the concentrations of the MPE. Epigallocatechin gallate, EGCG (0.001, 0.01, 0.1, 1.0 and 10.0mg/ml) dissolved in in 0.1 M Tris-Cl buffer, pH 8.0 was positive control³⁶.

In vitro anti-inflammatory assays:

Albumin degradation test:

The MPE and diclofenac diethyl ammonium (Sigma-Aldrich, Germany) were solubilized in 10% (v/v) DMSO and in distilled water, respectively. Sample solution was centrifuged at 150rpm and its supernatant was serial diluted at 0.625, 1.25, 2.5, 5.0 and 10.0 mg/ml. The range of sample or control was performed with 0.2% albumin solution at 70±2°C for 5min. Anti-inflammatory activity of MPE or diclofenac diethyl ammonium was monitored measured the reduction of albumin absorbance at 278nm by UV-visible spectrophotometer. Result was represented as IC₅₀ of MPE compared with diclofenac diethyl ammonium as positive control³⁷.

Reduction of no production from inflammatory cells:

Macrophage cells are important inflammatory cells and play a role of inflammation via the mediators and cytokines production including NO. In this study, anti-inflammatory activity of MPE was monitored the reduction of NO releasing from activated macrophages in present of the extract. Mouse macrophage cell (RAW264.7) was cultured in Dulbecco ‘s modified Eagle ‘s medium, DMEM (Invitrogen, USA), containing foetal bovine serum, FBS (10%) and penicillin/streptomycin (1%) at appropriated condition, which was deposited to a 24-well plate and cell volume was adjusted to 1×10⁵ cells with 500μl of medium/well. The cell suspension was incubated with MPE or control for 1h and activated with lipopolysaccharide, LPS (Sigma, USA) for 24h. The supernatant from treated cell suspension was transferred to a 96-well plate and Griess reagent (Sigma-Aldrich, USA) was added. The reduction of NO production was performed by colorimetric method at 540nm. Result was represented as IC₅₀ of MPE compared with triamcinolone acetonide as positive control³⁸.

In vitro cytotoxicity test:

Normal human skin fibroblasts were obtained from Chiang Mai University, Thailand. The cell culture was cultivated in a 75-cm² flask (Nunc, Denmark) using DMEM supplemented and culture condition as explained in anti-inflammatory assay with some modification. The cells were harvested using 0.25% (w/v) trypsin and 0.06mM EDTA in phosphate buffer saline (PBS), followed by resuspension in complete DMEM and cell counting using a hemacytometer. The experimental procedures were carried out in triplicate. The MPE was determined for cell toxicity against human skin fibroblasts by the sulforhodamine B colorimetric (SRB) assay. The cells were seeded at a density of 1.0×10⁴ cells per well in 96-well plates and incubated overnight in an environment of 5% CO₂ at 37 °C for facilitating of cellular adhesion. Subsequently, the cells were subjected to MPE (0.001 to 10mg/ml) for 24 h. Cell viability of cell culture against MPE and ascorbic acid (control) is given as cell viability (%)³⁹.

Wound healing test:

This assay was conducted after cytotoxicity test on human skin fibroblasts and maximum concentration of MPE without toxicity was used on wound healing³⁵. The cluster of treated-human skin fibroblast cells were scratched, and the gap was lined on cell cluster. The wound healing property was monitored the reduction of gap length among cell cluster, which was monitored by high resolution of microscope (´400) within 0, 6, 24 and 48h. Wound healing property of MPE was represented as the reduction of gap length (%) compared with controls. Ascorbic acid (1 mg/ml) was used as positive control on wound healing activation, whereas DMEM and DMEM contained with 10%DMSO (10%DMSO) were used as negative controls⁴⁰.

RESULT:

The Mamao pomace extract (MPE) was viscous liquid with purplish red colored, and the yield of extraction was about 5%. The TPC and TTC of MPE were 23.77±0.37mg GAE/g and 166.73±2.7mg TE/g, respectively, therefore, TFC was undetectable. The MPE exhibited DPPH scavenging activity (IC50 = 0.12±0.02 mg/ml) and poorly chelated metal ion and inhibited lipid peroxidation. While it was unable to scavenge NO radical and inhibiting tyrosinase and elastase enzymes (Table 1). The graphs with equations between sample concentration and antioxidant activity of four assays were depicted in Fig. 1. Only 5.0mg of MPE slightly inhibited Cu. acnes when compared with 0.002mg of clindamycin (Fig. 2). Their inhibition zones were 10.06±0.04 and 44.31±0.45mm, respectively (Table 2). The MPE was non-toxic on human skin fibroblasts like ascorbic acid (Fig. 3), up to 1.0mg/ml (Table 3). The MPE (0.1mg/ml) was inhibited of NO production from LPS-induced macrophage comparable triamcinolone acetonide (18.94±1.96 and 26.44±1.18%, respectively), which was exhibited anti-inflammatory activity (Table 4). Therefore, The MPE was lack of associated to inhibition of albumin degradation and unable to determined anti-inflammatory activity (Fig. 4) when compared with diclofenac (IC₅₀ = 0.47±0.01mg/ml). For wound healing property, MPE (1.0mg/ml) and ascorbic acid (1.0mg/ml) were reduced the gap length of skin fibroblast cluster when compared with 10% v/v DMSO in DMEM and DMEM, respectively, after treatment within 6, 24 and 48h (Fig. 5). After 48 h, MPE and ascorbic acid were reduced 84.97±3.34 and 87.51±2.27% of the gap length of skin fibroblast cluster, respectively (Table 5).

Table 1. Phytochemical contents and biological activities of MPE

Sample	Assay
Sample	TPC	TFC	TTC	DPPH*	NO*	LPI*	MC*	TYI*	ELI*
(Units)	(mg GAE/g)	(mg QE/g)	(mg TE/g)	(mg/ml)	(mg/ml)	(mg/ml)	(mg/ml)	(mg/ml)	(mg/ml)
MPE	23.77±0.37	ND	166.73±2.7	0.12±0.02	ND	>1,000	>1,000	ND	ND
Ascorbic acid	-	-	-	0.01±0.0	0.13±0.03	-	-	-	-
α-Tocopherol	-	-	-	-	-	0.005±0.0	-	-	-
EDTA	-	-	-	-	-	-	0.02±0.01	-	-
Kojic acid	-	-	-	-	-	-	-	0.003±0.0	-
EGCG	-	-	-	-	-	-	-	-	0.01±0.0

*Biological activities are given as 50% inhibition, IC₅₀ (mg/ml). MPE = Mamao pomace extract; EGCG = epigallocatechin gallate; DPPH = 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity; NO = nitric oxide scavenging activity; LPI = inhibition of lipid peroxidation; MC= metal chelating activity; TYI = -tyrosinase inhibitory activity; ELI = elastase inhibitory activity; EDTA = ethylenediaminetetraacetic acid; ND = Not determined

Table 2. Anti-microbial activity of MPE against skin pathogens by Kirby-Bauer method

Pathogen	Test/Control	Concentration (mg)	Diameter of inhibition zone (mm)
Pathogen	Test/Control	Concentration (mg)	Plate I	Plate II	Plate III	Mean ± SD
S. aureus	MPE	5.0	ND	ND	ND	ND
		0.5	ND	ND	ND	ND
		0.05	ND	ND	ND	ND
	Ethanol	10.0	ND	ND	ND	ND
	Erythromycin	0.015	24.15	22.24	23.48	23.29±0.97
Cu. acnes	MPE	5.0	9.53	10.21	10.44	10.06±0.04
		0.5	ND	ND	ND	ND
		0.05	ND	ND	ND	ND
	Ethanol	10.0	ND	ND	ND	ND
	Clindamycin	0.002	44.25	44.78	43.89	44.31±0.45
C. albicans	MPE	5.0	ND	ND	ND	ND
		0.5	ND	ND	ND	ND
		0.05	ND	ND	ND	ND
	Ethanol	10.0	ND	ND	ND	ND
	Fluconazole	0.025	26.28	27.45	26.78	26.84±0.59
M. furfur	MPE	5.0	ND	ND	ND	ND
		0.5	ND	ND	ND	ND
		0.05	ND	ND	ND	ND
	Ethanol	10.0	ND	ND	ND	ND
	Ketoconazole	0.2	37.16	38.77	36.80	37.58±1.05

ND = not determined

Table 3. Cell viability (%) of human skin fibroblasts after treatment with MPE and ascorbic acid

Sample (mg/ml)	Cell viability (%)
Sample (mg/ml)	0.0001	0.001	0.01	0.1	1.0
MPE	94.93±3.40	93.78±2.99	92.73±4.08	88.34±5.73	84.68±1.19
Ascorbic acid	94.89±1.53	93.38±6.53	91.87±5.11	87.39±4.47	82.54±1.16

Table 4. Inhibition of NO production from LPS-induced macrophages by MPE and control

Sample (mg/ml)	Inhibition of NO production (%)
Sample (mg/ml)	0.0001	0.001	0.01	0.1	1.0
MPE	17.80±1.45	18.18±1.75	18.56±2.27	18.94±1.96	13.64±1.75*
Triamcinolone acetonide	21.03±3.20	23.56±1.18	25.00±1.18	26.44±1.18	30.41±3.41

* This result was interfered from MPE color

Figure 3: A) Human skin fibroblasts (´400) and B) Cell viability (SRB) staining after treated with MPE were like ascorbic treated cells and interpreted as non-cytotoxicity

Figure 4: The negative correlation graphs between MPE and inhibition of albumin degradation compared with control

Figure 5 The reduction of gap length on treated human skin fibroblasts compared with controls after 6, 24 and 48 h

DISCUSSION:

The A. thwaitesianum seed and marcs had contained the high content of the polyphenols and proanthocyanins. The solvents had included acetone, ethyl acetate, and methanol in a range of concentrations from 40% to 70% ²⁷. As our results, A. thwaitesianum pomace was the by-product from fruit industries, which was seed and marcs contained. We were considered that ethanol is less toxicity for A. thwaitesianum pomace extraction, which is commonly use in the preparation of Thai medical plants. Since, proanthocyanins, also known as condensed tannins, are polyphenolic compounds found in various plants like wine, chocolate, and fruits, with potential health promoting properties, such as anti-oxidation and anti-cancer properties^41,42. The MPE was also contained polyphenols and tannins as previous study reported, however, there were in lower amounts²⁷. However, the flavonoid of MPE was undetectable, there was implied that flavonoids were linked together as oligomeric or polymeric forms²⁷. The MPE was exhibited DPPH scavenging activity, therefore it was poorly chelated metal ion and inhibited lipid peroxidation. Our results were contradicted with previous study that A. thwaitesianum seed and marcs were strong antioxidant in polar and non-polar environments²⁷. It was due to the conditions of solvent extraction and resulted to low amount of bioactive content. The MPE was slightly inhibited Cu. acnes and unable to inhibit other testing pathogens. While the A. thwaitesianum juice had exhibited anti-microbial activity against various food-borne pathogens and spoilages. Thus, the part of plant extract is important on anti-microbial activity and bioactive variation of A. thwaitesianum⁴³. Since, MPE was unable to scavenge NO radicals, therefore it was indirectly inhibited NO radicals by the reduced of NO production from LPS-induced macrophages. This anti-inflammatory activity was like pomegranate peel⁴⁴, berry seed⁴⁵, grape seed⁴⁶, citrus pomace⁴⁷, and pear pomace⁴⁸. In addition, MPE (1.0mg/ml) was represented wound healing property by the gap reduction of human skin fibroblasts after treatment for 48h, and this property was nearly vitamin C in same concentration. As our result, polyphenols and tannins contained in MPE may be responsible for wound healing property⁴⁹. The MPE was also lack of cytotoxicity against human skin fibroblasts, which may be due to the use of ethanol on extraction that was safer than other solvents. Thus, the MPE was possessed antioxidant, inhibition of C. acnes (the causative agent for acne vulgaris), anti-inflammation and wound healing properties. Hence, the MPE was prepared from A. thwaitesianum pomace, the waste from fruit product industries, it was possible to sustainable source as cosmetic ingredients for skin care products.

CONCLUSION:

The MPE, an ethanolic extract of Mamao (A. thwaitesianum Müll. Arg.) fruit pomace, was a sustainable source for skin cosmetic ingredient through antioxidant activity including DPPH radical scavenging, inhibition of lipid peroxidation and metal chelating activities; anti-microbial activity against Cu. acnes; anti-inflammation by reduced NO production from LPS-induced macrophages; and wound healing activity on human skin fibroblast without cytotoxicity.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We express our sincere gratitude to Suan Sunandha Rajabhat University in Bangkok, Thailand for their generous research funding and technical assistance. Our appreciation also extends to the Asst. Prof. Dr. Pimporn Thongmuang, Vice President for Samut Songkhram Campus, Suan Sunandha Rajabhat University, provided invaluable support in the identification of herbal specimens and the collection of application data in Thai Traditional Medicine.

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Received on 09.01.2025 Revised on 03.07.2025

Accepted on 06.10.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5749-5757.

DOI: 10.52711/0974-360X.2025.00829

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sample/Control	The reduction of gap length on human skin fibroblast (%)
Sample/Control	6 h	24 h	48 h
MPE (1.0 mg/ml)	8.63±0.39	50.59±0.47	84.97
10% v/v DMSO (control for MPE)	8.31±0.57	15.32±0.48	30.32±4.51
Ascorbic acid (1.0 mg/ml)	18.47±1.68	58.87±4.43	87.51±2.27
DMEM (control for ascorbic acid)	7.59±0.64	10.28±0.66	43.57±3.58